Title:
COMPOSITION COMPRISING XANTHOCERAS SORBIFOLIA EXTRACTS, COMPOUNDS ISOLATED FROM SAME, METHODS FOR PREPARING SAME AND USES THEREOF
Kind Code:
A1


Abstract:
This invention provides compositions, methods and process of producing extracts from Xanthoceras sorbifolia. The extract comprises alkaloids, coumarins, saccharides, proteins, polysaccharides, glycosides, saponins, tannins, acid, flavonoids and others. The composition can be used for treating cancer, arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder, impotence and premature ejaculation; for preventing cerebral aging; for improving memory, cerebral functions; or for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease or other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder. This invention provides compounds comprising at least one sugar, a triterpene, such as Sapogenin, and at least one side chains at Carbon 21 and 22, such as Angeloyl groups. The compounds of the present have various pharmaceutical and therapeutic applications.



Inventors:
Chan, Pui-kwong (Sugarland, TX, US)
Mak, May Sung (Hong Kong, CN)
Wang, Yun (Dunedin, NZ)
Application Number:
13/718575
Publication Date:
07/25/2013
Filing Date:
12/18/2012
Assignee:
PACIFIC ARROW LIMITED (HONG KONG, CN)
Primary Class:
International Classes:
C07H15/256; A61K31/704
View Patent Images:



Primary Examiner:
OLSON, ERIC
Attorney, Agent or Firm:
Law Offices of Albert Wai-Kit Chan, PLLC (Whitestone, NY, US)
Claims:
1. 1-206. (canceled)

207. A method for inhibiting cancer cell growth in a subject, comprising administering to the subject an effective amount of saponins from Wenguanguo or Xanthoceras Sorbifolia, wherein the cancer is selected from the group consisting of breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer, bone cancer, colon, cervix, lung and brain cancer.

208. The method of claim 207, wherein the saponins comprise: embedded image wherein R1, R2, R3 and R4 are individually attached an angeloyl, acetyl or hydrogen; wherein R5 are sugar moieties/alduronic acid of glucose, galactose and arabinose.

209. The method of claim 207, wherein the saponins comprise: embedded image wherein R1, R2, R3 and R4 are individually attached an angeloyl, acetyl or hydrogen; wherein R5 are sugar moieties/alduronic acid of glucose, galactose and arabinose.

210. The method of claim 207, wherein the saponins comprise: text missing or illegible when filed wherein R1, R2 are individually attached an angeloyl; R4 is H; wherein R5 are sugar moieties/alduronic acid of glucose, galactose and arabinose.

211. The method of claim 207, wherein the saponins comprise: embedded image wherein R1, R2 are individually attached an angeloyl, acetyl or H.

212. The method of claim 207, wherein the saponins comprise: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene, embedded image

213. The method of claim 207, wherein the cancer is breast cancer.

214. The method of claim 207, wherein the cancer is leukocyte cancer.

215. The method of claim 207, wherein the cancer is liver cancer.

216. The method of claim 207, wherein the cancer is ovary cancer.

217. The method of claim 207, wherein the cancer is prostate cancer.

218. The method of claim 207, wherein the cancer is bone cancer.

219. The method of claim 207, wherein the cancer is brain cancer.

220. The method of claim 207, wherein the cancer is colon cancer.

221. The method of claim 207, wherein the cancer is cervix cancer.

222. The method of claim 207, wherein the cancer is lung cancer.

223. The method of claim 207, wherein the saponins are in a pharmaceutically suitable carrier.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application (1) is a continuation of U.S. Ser. No. 12/392,795, Filed Feb. 25, 2009 which is a continuation of U.S. Ser. No. 10/906,303, Filed Feb. 14, 2005, which is a Continuation-In-Part application of International Application No. PCT/US04/43465, Filed Dec. 23, 2004, which is a Continuation-In-Part application of PCT/US04/33359, Filed Oct. 8, 2004, which claims benefit of U.S. Ser. Nos. 60/509,851, Filed Oct. 9, 2003 and U.S. Ser. No. 60/532,101, Filed Dec. 23, 2003; and (2) claims benefit of U.S. Ser. Nos. 60/617,379, Filed Oct. 8, 2004; 60/613,811, Filed Sep. 27, 2004; and 60/607,858, Filed Sep. 7, 2004; (3) claims benefit of U.S. Ser. No. 10/471,384, Filed Sep. 4, 2003, National Stage of Intl App'l No. PCT/IB02/04750, Filed Aug. 28, 2002, which is a continuation-in-part, of U.S. Ser. No. 09/944,805, Filed Aug. 31, 2001, now U.S. Pat. No. 6,616,943, issued Sep. 9, 2003. The contents of these preceding applications are hereby incorporated in their entireties by reference into this application.

Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

This invention relates to extracts from a plant called Wenguanguo (Xanthoceras Sorbifolia Bunge), their usages and functions, and methods of their preparation.

BACKGROUND OF THE INVENTION

Wenguanguo is a species of the sapindaceae family. Its scientific name is Xanthoceras sorbifolia Bunge. Wenguanguo is the common Chinese name; others are Wenguanngguo, Wenguanmu, Wenguanhua, Xilacedeng and xilasendeng. Goldenhorn and Yellowhorn are its common English names. Wenguanguo is grown in Liaoning, Jilin, Hebei, Shandong, Jiangsu, Henan, Shanxi, Shaanxi, Gansu, Ningxia and Inner Mongolia, China. Its seeds, leaves and flowers are edible and its seeds have been used as a folk medicine to treat enuresis for centuries. Its branches and woods are also used as a folk medicine.

Chinese patent applications CN 1092991A and CN 1092992A discussed the methods for producing a medicine from Wenguanguo kernel powder for curing enuresis and enhancing cerebral functions. Chinese patent CN 1052636C discussed a method for producing a medicine with ethanol extract from the Wenguanguo kernel powder for curing enuresis and enhancing cerebral functions. Journal of Shenyang University of Pharmacy (2001), 18(1), 53-56 disclosed the n-butanol extract from the wood of Wenguanguo, which has anti-inflammatory effect.

United States Patent Application Publication No. 20030096030 discussed the extracts from the husks of Wenguanguo which are Bunkankasaponin A. B. C. D and two sterols for preventing cerebral aging, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence, and increasing the body's ability to resist the activity of glycosuria.

United States Patent Application Publication No. 20030082293 disclosed the extracts Bunkankasaponin A. B. C. D, crude fats, crude protein and sugars from the shell of Wenguanguo.

U.S. Pat. No. 6,616,943, issued on Sep. 9, 2003, discussed the composition comprising Wenguanguo combined extracts and the methods for preparing them and uses thereof. The methods for preparing the combined extract from the husks comprise the following steps: extracting Wenguanguo husks with an organic solvent (e.g. ethanol) to form an organic (e.g. ethanol) extract; removing the organic solvent (e.g. ethanol) from the organic (e.g. ethanol) extract to form aqueous extracts; and drying and sterilizing the aqueous extracts to form the combined extracts. The combined extracts contain saponins, saccharides, proteins and others. The extracts can be used for producing medicines or health foods for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunction. The medicines or health foods further comprise Vitamin B, Vitamin D, K, anti-oxidant, Cordyceps or its extracts, gingko or its extracts, Echinacea or its extracts, Huperzine A, folic acid, amino acids, creatine, fiber supplement or a combination thereof.

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(4)1387-1394 (1985) described a study on the constituent of Xanthoceras sorbifolia Bunge. See Section V. Saponins from the Fruits of Xanthoceras sorbifolia. Four new saponins were isolated from the fruits of Xanthoceras sorbifolia Bunge. The structures of these saponins are bunkankasaponins A, B, C and D:

  • (1) 22-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin
  • (2) 22-O-acetyl-21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin
  • (3) 28-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin
  • (4) 28-O-acetyl-21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(3)1043-1048 (1985) described studies on the constituent of Xanthoceras sorbifolia Bunge. See Section IV. Structures of the Miner Prosapogenin. The prosapogenins from the partial hydrilyzate of fruit saponin of Xanthoceras sorbifolia were examinated, and are characterized as:

  • 16-O-acetyl-21-O-(3,4-di-O-angeloyl-β-D-fucopyranosyl)protoaecigenin
  • 22-O-acetyl-21-O-(3,4-di-O-angeloyl-β-D-fucopyranosyl)protoaecigenin 3-O-β-D-glucuronopyranoside

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(1)127-134 (1985) describe studies on the constituent of Xanthoceras sorbifolia Bunge. See Section III. Minor Prosapogenins aponins from the Fruits of Xanthoceras sorbifolia Bunge. The structure of 3 minor prosapogenins, obtained by acid hydrolysis of the crude saponin faction, were characterized as:

  • 21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyltheasapogenol B
  • 21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyltheasapogenol B
  • 21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-22-O-acetylprotoaescigenin

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull 33(4)1387-1394 (1985) described a study on the constituent of Xanthoceras sorbifolia Bunge. See Section II. Major Sapogenol and prosapogenin from the Fruits of Xanthoceras sorbifolia.

Laurence Voutquenne, Cecile Kokougan. Catherine Lavaud, Isabelle Pouny, Marc Litaudon. Triterpenoid saponins and Acylated prosapogenins from Harpullia austro-calcdonica. Phytochemistry 59 (2002) 825-832

Zhong Jaing, Jean-francois Gallard, Marie-Therese Adeline, Vincent Dumontet, Mai Van Tri, Thierry Sevenet, and Mary Pais Six Triterpennoid Saponins from Maesa laxiflora. J. Nat. Prod. 1999, 62, 873-876

Young Seo, John M. Berger, Jennine Hoch, Kim M Neddermann, Isia Bursuker, Steven W. Mamber and David G. Kingston. A new Triterpene Saponin from Pittosporum viridiflorum from the Madagascar Rainforest. J. Nat. Prod. 2002, 65, 65-68

Xiu-Wei Yang, Jing Zhao, Xue-Hui Lui, Chao-Mei Ma, Masao Hattori, and Li He Zhang Anti-HIV-1 Protease Triterpenoid Saponins from the Seeds of Aesculus chinensis. J. Nat. Prod. 1999 62, 1510-1513

Yi Lu, Tatsuya Umeda, Akihito Yagi, Kanzo Sakata, Tirthankar Chaudhuri, D. K. Ganguly, Secion Sarma. Triterpenoid Saponins from the roots of the tea plant (Camellia sinensis var. Assamica). Phytochchemistry 53 (2000) 941-946

Sandra Apers, Tess E. De Bruyne, Magda Claeys, Arnold J. Viletinck, Luc A. C. Pieters. New acylated triterpenoid saponins from Maesa laceceolata. Phytochemistry 52 (1999) 1121-1131

Ilaria D'Acquarica, Maria Cristina, Di Giovanni, Francesco Gasparrini, Domenico Misiti, Claudio D′ Arrigo, Nicolina Fagnano, Decimo Guarnieri, Giovanni Iacono, Giuseppe Bifulco and Raffaele Riccio. Isolation and structure elucidation of four new triterpenoid estersaponins from fruits of the Pittosporumtobira AIT.

Tetrahedron 58 (2002) 10127-10136

Cancer cells are defined by two heritable properties: (1) they reproduce in defiance of normal restraints on cell division; and (2) they invade and colonize territories normally reserved for other cells.

Cancers require mutations of many genes to develop, and they are classified according to the tissue and cell type from which they arise. Cancers arising from epithelial cells are named carcinomas; those arising from connective tissue or muscle cells are named sarcomas. In addition, there are cancers called leukemias, which are derived from hemopaietic cells; and cancers derived from cells of the nervous system.

Cancers originating from different types of cells are, in general, very different diseases. Each cancer has characteristics that reflect its origin. Even when a cancer has metastasized and proliferated out of control, its origins can be traced back to a single, primary tumor. Therefore it is important to develop drugs against target cells with a specified character.

Ovarian cancer is the 5th leading cause of cancer death in women and the leading cause of death from gynecologic. In the United States, females have a 1.4 to 2.5% (1 out of 40-60 women) lifelong chance of developing ovarian cancer. Older women are at highest risk. More than half of the deaths from ovarian cancer occur in women between 55 and 74 years of age and approximately one quarter of ovarian cancer deaths occur in women between 35 and 54 years of age.

Ovarian cancer is disproportionately deadly for a number of reasons. First, symptoms are vague and non-specific, so women and their physicians frequently attribute them to more common conditions. By the time the cancer is diagnosed, the tumor has often spread beyond the ovaries. Also, ovarian cancers shed malignant cells that frequently implant on the uterus, bladder, bowel, and lining of the bowel wall (omentum). These cells can begin forming new tumor growths before cancer is even suspected. Second, because no cost-effective screening test for ovarian cancer exists, more than 50 percent of women with ovarian cancer are diagnosed in the advanced stages of the disease.

This invention provides compounds or compositions extracted from Xanthoceras Sorbifolia or synthesized which have substantial potency against ovarian cancer.

SUMMARY OF THE INVENTION

In accordance with these and other objects of the invention, a brief summary of the present invention is presented. Some simplifications and omission may be made in the following summary, which is intended to highlight and introduce some aspects of the present invention, but not to limit its scope. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the invention concepts will follow in later sections.

The invention provides a compound comprising the following structure, with the formula of C57H88O23 and the name of 3-O-β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α, 21β,22α,28-hexahydroxyolean-12-ene, also known as Xanifolia-Y This compound was isolated from Xanthoceras sorbifolia.

embedded image

This invention provides a compound comprising the following structure, with the formula of C65H100O27 and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,1 6α,2β,22α,28-pentahydroxyolean-12-ene, also known as Xanifolia-Y1

embedded image

The above compounds (Y and Y1) have anti-cancer effect. They inhibit the growth of human ovarian cancer (FIG. 3, 4).

These compounds (Y and Y1) are two of the active components identified from extracts of Xanthoceras sorbifolia by methods of FPLC and HPLC as shown in FIG. 5, 6, 7.

The compound Y is purified with procedure as described in this application (FIG. 7A). The purified compound Y shows 10 times higher potency (IC50=1.5 ug/ml) than the original extract (IC50=25 ug/ml) (FIG. 8). The compound Y has a high selectivity toward ovarian cancer (FIG. 9).

The compound Y shows inhibitory activity toward the following human cancer cells (eleven human cancer cell lines were tested in this study) with a higher potency toward ovarian carcinoma (comparison of activities is presented in FIG. 10 and Table 3.1).

This invention provides the extract of Xanthoceras Sorbifolia against cancer growth. The cancer includes, but is not limited to ovary cancer, bladder cancer, prostate cancer, leukocytes cancer, and bone cancer.

The compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized chemically, or extracted from other biological sources.

This invention is related to the use of extracts of Wenguanguo. Extracts from husks, leaves, branches or stems, and fruit-stems, roots and barks of the Wenguanguo can be combined and this invention discloses methods of their preparation.

The extracts contain saponins, saccharides, proteins, glycosides, flavonoids, curmarin extracts, alkaloid extracts, organic acid extracts, tannin and others.

This invention provides the extract of Xanthoceras Sorbifolia for preventing cerebral aging; for improving memory; for improving cerebral functions; for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence, Alzheimer's disease, brain trauma, or other diseases caused by cerebral dysfunctions.

Wenguanguo extracts may be used for accelerating the growth of bladder, for suppressing deep sleep, for increasing alertness in a sleeping subject, for modulating the release, breakdown and uptake of Antidieuretic hormone (ADH) and its receptors, for modulating the secretion, breakdown and uptake of Adrenocorticotropic hormone (ACTH) and its receptors, for modulating the release, breakdown and uptake of 5-hydroxytryptamine and its receptors, for modulating the release, breakdown and uptake of Acetycholine (Ach) and its receptors, for modulating the release, breakdown and uptake of Adrenaline (AD) and its receptors, for modulating the release, breakdown and uptake of Dopamine (DA) and its receptors, for modulating the release, breakdown and uptake of Norepinephrine (NE) and its receptors, for preventing sleep paralysis, for modulating the formation, release, breakdown and activity of neuropeptides and their receptors, for curing cancer, including but not limited to breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer and brain cancer, and for improving the functions of the lung and the bladder.

This invention provides a compound comprising a sugar, a triterpene or Sapogenin, and side chain at Carbon 21 and/or 22 including Angeloyl groups, operatively linked to form a biologically active compound. In an embodiment, the compound comprises one or more sugars.

This invention provides a salt of the above-described compounds.

This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier(s).

This invention provides a method for isolating compounds from Xanthoceras Sorbifolia comprising steps of: extracting Xanthoceras Sorbifolia powder with an appropriate amount of one or more organic solvents for an appropriate amount of time to form an organic extract; collecting the organic extract; refluxing the organic extract to form a second extract; removing the organic solvent from the second extract; drying and sterilizing the second extract to form a Xanthoceras Sorbifolia extract powder; fractionating the extract powder to obtain one or more components of the extract powder; identifying the bioactive components of the extract powder; purifying one or more bioactive components of the extract powder with FPLC to obtain one or more fraction of the bioactive component; and isolating the pure compound with preparative HPLC.

This invention provides a compound having a Structure verified by NMR spectral data derived from proton NMR, Carbon NMR, 2D NMR of the Heteronuclear Multiple Quantum Correlation (HMQC), Heteronuclear Multiple Bond Correlation (HMBC), and COSY, and Mass spectral data derived from WADLI-TOF and ESI-MS.

This invention provides the chemical features of a compound and its derivatives which are effective against cancer. The compounds or compositions of the present invention regulate the receptors or components of cell such as G-protein receptor, Fas protein, receptor Tyrosine Kinases, Mitogen, mitogen receptor. The compound inhibits cellular pathways include TGF Beta-smad, FGF, TGF-beta and TGF-alpha, ras-GTPase-MAP kinase, jun-fos, Src-fyn, Jak-Jnk-STAT, BMP, Wnt, myc-cell proliferation, etc. The mutation of cancer cell causes the cell-death program to become inactive, allowing cells to divide indefinitely. The Xanthoceras Sorbifolia derived compound and/or composition regulates the components and receptors and re-activates the cell death program.

Abnormal changes in components' activities in pathways cause the cells to fail to stop proliferating so as to form cancer. The pathways include TGF Beta-smad, FGF, TGF-beta and TGF-alpha, ras-GTPase-MAP kinase, jun-fos, Src-fyn, Jak-Jnk-STAT, BMP, Wnt, myc-cell proliferation, etc. The mutation of cancer cell causes the cell-death program to become inactive, allowing cells to divide indefinitely. The Xanthoceras Sorbifolia derived compound and/or composition regulates the components and receptors and re-activates the cell death program.

In accordance with these and other objects of the invention, a brief summary of the present invention is presented. Some simplifications and omission may be made in the following summary, which is intended to highlight and introduce some aspects of the present invention, but not to limit its scope. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the inventive concepts will follow in later sections.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure Compound Y with the formula of C57H88O23 and the chemical name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-1 2-ene.

FIG. 2 shows the structure of Compound Y1 with the formula of C65H100O27 and the chemical name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,1 6α,21β,22α,28-pentahydroxyolean-12-ene.

FIG. 3 shows the anticancer activity of purified Compound Y. The periment was performed on ovarian cancer cells (OCAR-3) and the inhibition activity was determined by MTT assay (for details, refer to Experiment 3). Abscissa: Concentration (ug/ml). Ordinate: % Cell Growth. The IC50 is approximately 1 ug/ml. A: Point scale. B: Linear scale.

FIG. 4 shows the inhibition of the purified Compound Y1 and Compound Y2 on Ovarian cancer cells' growth.

FIG. 5 shows the results of the screening of cell growth activity of fractions obtained after FPLC chromatography. The assay was conducted with bladder cells. The fractions obtained from FPLC as shown in FIG. 20 were used. As shown in this figure, that different components of Xanthoceras Sorbifolia extracts cause either growth or inhibition effects on cells. Only fraction 5962 (Fraction Y) causes cell inhibition. Fractions 610 and 1116 cause minor stimulation of cell growth. Abscissa: concentration (ug/ml). Ordinate: % Cell Growth (determined by MTT assay).

FIG. 6 shows the separation of the components of Xanthoceras Sorbifolia extract by HPLC with a μbondapak C18 column. Details of experiment was presented in Experiment 2.

FIG. 7 shows HPLC profile of Fraction Y with 45% Acetonitrile isocratic elution in a preparative C18 column (Delta Pak C18). Under these conditions, fractions Y, Y1 and Y2 are well separated from each other and they are collected individually.

FIG. 7A shows the purity of the collected Compound Y by HPLC using 45% acetonitrile isocratic elution in a preparative C18 column.

FIG. 8 shows the a growth curve of ovarian cancer cells after treatment with the crude extract of Xanthoceras Sorbifolia as determined by MTT assay. This is a preliminary study on the sensitivity of extract of Xanthoceras Sorbifolia on cancer cell. Cell lines from 11 different human organs were employed. With the crude extract, this figure shows the most sensitive cancer cells are Ovary cancer cells. Activities on other cancer cells were represented in FIGS. 10A-D.

FIG. 9 shows the comparison of potency of Compound Y between ovarian cancer cells and cervical cancer cells. Ovarian cancer cells are much more sensitive to Compound Y than the cervical cancer cells. The IC50 for Compound Y in ovary cells is about 1.5. This result confirms that the activity of compound Y is more selective toward ovary cancer.

FIGS. 10A-D show the growth curves of cancer cells derived from different human organs as determined by MTT assay. After treatment with the extract of Xanthoceras Sorbifolia, growth curves of different cell lines were presented and their sensitivities (base on IC50 values) were determined.

10A: Sensitive: bladder and bone.

10B: Semi-sensitive: leukocyte and liver.

10C: Marginal sensitive: prostate, breast and brain.

10D: Least sensitive: colon, cervix and lung.

FIG. 11 shows the spectrum of proton NMR of Compound Y.

FIG. 12 shows 2D NMR (HMQC) results of Compound Y. Also see Table 5.2 for the listed chemical shift data.

FIG. 13 shows 2D NMR (HMBC) results of Compound Y. Also see Table 5.3 for the listed chemical shift data.

FIG. 14 shows the Mass spectrum of compound Y with MALDI-TOF (high mass): Y+Matrix (CHCA)+Angiotensin 1 “two point calibration”.

FIG. 15 shows the Mass spectrum of compound Y with ESI-MS.

FIG. 16 shows the Proton NMR spectrum of Compound Y1.

FIG. 17 shows the 2D NMR (HMQC) results of Compound Y1. Also see the chemical shift data from Table 6.2.

FIG. 18 shows the 2D NMR (HMBC) results of Y1. Also see the chemical shift data from Table 6.3.

FIG. 19 shows COSY-NMR profile of Y1 with chemical shift data from Table 6.4.

FIG. 20 shows the elution profile of an extract of Xanthoceras Sorbifolia in FPLC with 10-80% gradient. Ordinate: Optical density (at 245 nm). Abscissa: Fractions (5 ml/fraction).

FIG. 21 shows the Proton-NMR spectra of compound R1.

FIG. 22 shows the 2D NMR (HMQC) spectra of compound R1.

FIG. 23 shows the 2D NMR (HMBC) spectra of compound R1.

FIG. 24 shows the 2D NMR (COSY) spectra of compound R1.

FIG. 25 shows the C13 NMR spectra of compound R1.

FIG. 26 shows the chemical structure of Compound R1: 3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl -3β,21β,22α,28-tetrahydroxyolean-12-ene

FIG. 27 shows the chemical structure of Compound O54.

FIG. 28 shows the Proton-NMR spectra of compound O54.

FIG. 29 shows the 2D NMR (HMQC) spectra of compound O54.

FIG. 30 shows the 2D NMR (HMBC) spectra of compound O54.

FIG. 31 shows one of the four possible chemical structures of Y1. A: structure Y1-1.

FIG. 32 shows one of the four possible chemical structures of Y1. B: structure Y1-2.

FIG. 33 shows one of the four possible chemical structures of Y1. C: structure Y1-3.

FIG. 34 shows one of the four possible chemical structures of Y1. D: structure Y1-4. R1=A or B or C; R2=A or B or C; R3=A or B or C.

R1R2R3
1AAA
2AAB
3AAC
4ABA
5ABB
6ABC
7ACA
8ACB
9ACC
10BAA
11BAB
12BAC
13BBA
14BBB
15BBC
16BCA
17BCB
18BCC
19CAA
20CAB
21CAC
22CBA
23CBB
24CBC
25CCA
26CCB
27CCC

FIG. 35 shows the chemical structure of Y-a.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C;

Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 36 shows the chemical structure of Y-b.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C;

Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 37 shows the chemical structure of Y-c.

FIG. 38 shows the chemical structure of Y1-a.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C;

Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 39 shows the chemical structure of Y1-b.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C;

Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 40 shows the chemical structure of Y1-c.

R5=B or C or S1 (see note 1); R1=A or B or C; R4=B or C;

Note 1: A=angeloy, B=acetyl, C=H, S1=chain with one or more sugar such as D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D-glucuronic acid, D-galacturonic acid and their derivatives.

FIG. 41 shows the absorption spectrum of Xanthoceras Sorbifolia extract. Abscissa: Wavelength in nm. Ordinate: Optical Density. The extract has three absorption maximum at 207 nm, 278 nm and 500 nm.

FIG. 42 shows elution profile of Fraction 5962 with 64% acetonitrile isocratic elution. Two major FPLC fractions X and Y are separated. Ordinate: optical density (254 nm). Abscissa: fraction Number (1 ml/fraction).

FIG. 43 shows the comparison of inhibition activity in bladder cells by Fractions X (2021) and Y (2728). Only Fraction Y has inhibition activity.

FIG. 44 shows HPLC profile of Fraction Y with 45% Acetonitrile isocratic elution. Two major and 2-3 minor compounds were identified.

FIG. 45 shows purification of Fraction R from with FPLC.

FIG. 46 shows the HPLC analysis of fractions #9, #10 and #11 obtained from FPLC.

FIG. 47 shows purification of component-R with HPLC (Delta-Pak C18). A: Extract from fraction #10 of FPLC (iso-30) was further separated by HPLC. B: Rechromatogram of the major component under same condition as described in A.

FIG. 48. Fractionation of Fraction-O from FPLC with HPLC with 20% acetonitrile isocratic elution (iso-20).

FIG. 49. Rechromatography of O28 and O34 (from iso-20).

FIG. 50. Rechromatography of O54 (from iso-20).

FIG. 51 shows the proton NMR spectrum of Y2.

FIG. 52 shows the 2D NMR spectrum of Y2 (HMQC).

FIG. 53 shows the proton NMR spectrum of Y4.

FIG. 54 shows the 2D NMR (HMQC) spectrum of Y4.

FIG. 55 shows the proton NMR spectrum of O28.

FIG. 56 shows the 2D NMR (HMQC).

FIG. 57 shows the proton NMR spectrum of O34.

FIG. 58 shows the 2D NMR (HMQC) spectrum of O34.

FIG. 59 shows the effects of the extract X and Y on the quantity of urine in mice after 10 days of administration of X and Y.

FIGS. 60(a) and (b) show the water maze learning effect of plant extract administration of aging mice for 9 days.

FIGS. 61(a) and (b) show the result of water maze learning of 3 days injected pentobarbital.

FIG. 62 shows the sleep cycle of a typical person.

FIG. 63. Table 15A-1 shows results of urine volume with water load after administration extract for 25 days. FIG. 63A shows the urine volume with water load after administration of FS(X) and FS(Y) extract for 25 days.

FIG. 64. Table 15A-2 shows results of discharging urine speed with water load after administration extract for 25 days. FIG. 64A shows the discharging urine speed with water load after administration of FS(X) and FS(Y) extract for 25 days.

FIG. 65. Table 15A-3 shows results of urine specific gravity and pH with water load after administration extract for 25 days.

FIG. 66. Table 15A-4 shows concentration of Na+, K+ and Cl− in urine with water load after administration extract for 25 days.

FIG. 67 shows the structure of the compound.

FIG. 68 shows the structure of the compound.

FIG. 69 shows the structure of the compound.

FIG. 70 shows the structure of the compound.

FIG. 71 shows the structure of the compound.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a compound comprising the following structure, with the formula of C57H88O23 and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α, 21β,22α,28-hexahydroxyolean-12-ene, also known as Xanifolia-Y This compound was isolated from Xanthoceras sorbifolia

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This compound belongs to saponins consist of a triterpene, sugar moiety and angeloyl groups links to the backbone. The angeloyl groups linked to the C21 and C22 positions. This compound has the anti-cancer activity.

The assignment of this structure is supported by the spectral data (1D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS). Accordingly, this compound has the characteristic property as shown in FIGS. 11-15 or Table 5.1.

This invention provides another compound comprising the following structure, with the formula of C65H100O27 and the name of 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,1 6α,21β,22α,28-pentahydroxyolean-12-ene, also known as Xanifolia-Y1

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This compound belongs to saponins consist of a triterpene, sugar moiety connected to the backbone. A sugar that linked to the C21 position has two angeloyl groups attached. This compound has anti-cancer activity.

The assignment of this structure is supported by the spectral data (1D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS). Accordingly, this compound has the characteristic property as shown in FIGS. 16-19 or Table 6.1.

This invention provides evidence to show that the extract of Xanthoceras Sorbifolia contains anticancer activity. The experiments for determining the anti-cancer activity employed human cells lines derived from eleven human organs (HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary)). Among the 11 cell lines studies, their sensitivity toward Xanthoceras Sorbifolia extract can be divided into four groups: (A) most sensitive: Ovary (FIG. 8); (B) Sensitive: bladder, bone, prostate, and leukocyte, (C) marginal sensitive: liver, breast, and brain; and (D) lease sensitive: colon, cervix, and lung. (FIG. 10A-D). Their IC50 values are listed in Table 3.1.

TABLE 3.1
IC50 values of Xanthoceras Sorbifolia Extract
Determined in Different Cancer Cells
Cancer cellsIC50 determined by
from different organsMTT assay (ug/ml)
Ovary (most sensitive)15-15
Bladder (sensitive)45-50
Bone40-55
Prostate40-50
Leukocyte45-50
Liver (marginal sensitive)45-65
Breast65
Brain70-85
Colon (least sensitive)90
Cervix115
Lung110

In order to identify the active compounds of Xanthoceras Sorbifolia, the extract from Xanthoceras Sorbifolia were separated by chromatography comprising FPLC (Fast Protein Liquid Chromatography) and HPLC (High Preferment Liquid Chromatography). Multiple fractions were obtained by FPLC procedures (FIG. 20) and HPLC (FIG. 6).

Analysis of the components of Xanthoceras Sorbifolia by HPLC shows that the extract comprises 26 identifiable fractions (named a to z) as shown in FIG. 6.

Anti-cancer activities of these fractions were determined by the MTT assay. Only fraction Ys has the anti-cancer activity (FIG. 5). Fraction Ys were further separated into 4 components (FIG. 7). The compounds Y and Y1 are the active components currently isolated from Xanthoceras Sorbifolia as shown in FIG. 3-4.

The invention tested the inhibition effects of ovarian cancer cells with the MTT assay, and the compound Y shows 10 times higher potency (IC50=1.5 ug/ml) (FIG. 3) than the original crude extract as shown in FIG. 8 (IC50=25 ug/ml).

The selectivity of compound Y was tested, and it has been found that compound Y has a much higher potency toward ovarian cancer cells as compared to the cervical cancer cells (FIG. 9).

The compounds Y1 and Y2 have anti-cancer activity as shown in FIG. 4.

This invention provides the detail isolation procedures for the active compounds of the present invention.

This invention provides the spectral data evidence (1D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, ESI-MS) in supporting the assigned structures.

This invention provides a salt of the above-described compounds.

This invention provides a composition comprising the above-described compounds and a suitable carrier.

This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier.

This invention provides an anti-ovarian cancer agents and composition comprising the above-described composition.

This invention provides the compositions against cancer growth. The cancer includes, but is not limited to bladder cancer, bone cancer, and ovary cancer.

This invention provides a composition comprising the above compounds and their derivatives for inhibition of tumour growth.

The following methods and materials were used in the examples and/or experiments described in this application.

Cells.

Human cancer cell lines were obtained from American Type Culture Collection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary). Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI-1640; HCT-116, U2OS in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% CO2 humidified incubator at 37° C.

MTT Assay.

The procedure for MTT assay followed the method described in (Carmichael et al., 1987) with only minor modifications. Cells were seeded into a 96-wells plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2, U2OS), or 40,000/well (K562), for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U2OS, and 96 hours for MCF-7). After the drug-treatment, MTT (0.5 mg/ml) was added to cultures for an hour. The formation of formazan (product of the reduction of tetrazolium by viable cells) was dissolved with DMSO and the O.D. at 490 nm was measured by an ELISA reader. The MTT level of cells before drug-treatment was also measured (T0). The % cell-growth (% G) is calculated as:


% G=(TD−T0/TC−T0)×100 (1),

where TC or TD represent O.D. readings of control or drug-treated cells. When T0>TD, then the cytotoxicity (LC) expressed as % of the control is calculated as:


% LC=(TD−T0/T0)×100 (2).

In addition to the compounds Y and Y1, other compounds from the extract including R1 and O54, were also purified and the structure were determined by 1D H-NMR, C13-NMR, 2D NMR (HMQC, HMBC, COSY); MS (MALDI-TOF).

The Structure of Compound R1 shown below and in FIG. 26, has a chemical formula of C65H106O29 and chemical name of

3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranos yl-3β, 21β,22α,28-tetrahydroxyolean-12-ene, also known as Xanifolia-R1.

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The assignment of this structure is supported by the spectral data (1D H-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS). Accordingly, this compound has the characteristic property as shown in FIGS. 21-25 or Table 8.1

Compound O54

This invention provides a compound O54 with formula of C60H100O28 and the structure was determined by 1D NMR, 2D NMR, MS).

The Structure of Compound O54 (also shown in FIG. 27):

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The name of Compound O54 is The chemical name of compound O54 is: 3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-t etrahydroxyolean-12-ene

The assignment of this structure is supported by the spectral data (1D H-NMR, 2D NMR (HMBC, HMQC). Accordingly, this compound has the characteristic property as shown in FIGS. 28-30 and table 9.1.

In other embodiments, the structures of the compounds are as follows:

Structure 1 as shown in FIG. 31
Structure 2 as shown in FIG. 32
Structure 3 as shown in FIG. 33
Structure 4 as shown in FIG. 34
Structure Y-a as shown in FIG. 35
Structure Y-b as shown in FIG. 36
Structure Y-c as shown in FIG. 37
Structure Y1-a as shown in FIG. 38
Structure Y1-b as shown in FIG. 39
Structure Y1-c as shown in FIG. 40

This invention provides a compound comprising a sugar, a triterpene or Sapogenin, and a side chain at Carbon 21 and 22 or Angeloyl groups. In an embodiment, the compound comprises two or more sugars.

This invention provides a salt of the above-described compounds.

This invention provides a composition comprising the above-described compounds and a suitable carrier.

This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier.

This invention provides an anti-ovarian cancer agents and composition comprising the above-described composition.

This invention provides the compositions against cancer growth. The cancer includes, but is not limited to bladder cancer, bone cancer, and ovary cancer.

This invention provides composition comprising the above compounds and their derivatives to inhibit tumour growth.

This invention provides composition comprising the above compounds and their derivatives to cure human immunodeficiency virus (HIV) or Severe Acute Respiratory Syndrome (SARS) or flux disease or inhibit virus activities.

This invention provides medicine or health food for improving the sensory stretch receptor in the bladder wall, inhibiting AChE or use as an anti-inflammatory agent.

This invention provides a method for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation.

This invention provide methods for inhibiting tumor cell growth or to treat patients with HIV or SARS, or inhibit virus activities, or for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation comprising contacting an amount of the compound is a triterpene or sapongenin with any two of angeloyl group or tigloyl group or senecioyl group or their combinations attach to carbon 21 and 22, or any two of angeloyl group or tigloyl group or senecioyl group or their combinations attached to a sugar moiety which bonds to carbon 21 or 22.

Wenguanguo is a species of the sapindaceae family. Its scientific name is Xanthoceras sorbifolia Bunge. Wenguanguo is the common Chinese name; others are Wenguannguo, Wenguanmu, Wenguanhua, and Xilacedeng. This plant can grow up to 8 meters in height. It features odd pinnately compound leaf, eraceme with white flowers, capsules with thick and woody husks. Wenguanguo is grown in Liaoning, Jilin, Hebei, Shandong, Jiangsu, Henan, Shanxi, Shaanxi, Gansu, Ningxia and Inner Mongolia, China. Its seeds are edible and have been used as a folk medicine to treat enuresis for centuries. Its branches and woods are also used as a folk medicine.

This invention is a further description of the extracts from Wenguanguo, their uses and methods for preparation. This invention provides the extracts that can prevent enuresis by improving patients' cerebral functions so that patients can be more aware of the signals sent from the bladder and wake up from deep sleep. When the bladder is full of urine, the smooth muscle of the bladder is extended, which produces a signal up to the cerebral cortex and cerebellum through the pelvic nerve and the sacral spinal cord. The response of the cerebral cortex and cerebellum to the signal is to make the bladder sustain contracted but the sphincter relaxed. The urine is then discharged. When the bladder is filled with urine via the urethra during sleep, the detrusor stretches, allowing the bladder to expand. As the bladder starts to accumulate urine, it will stimulate the stretch receptors in the bladder that will generate signals continually to the brain according to the amount of urine accumulated in the bladder. When the bladder is full enough with urine, then the intra-vesicle has accumulated enough pressure for the brain to recognize and wake the person to urinate. If the signal is not strong enough to wake the sleeping person or blocked due to impairment of cerebral function, then enuresis occurs. This particular plant extract can cure enuresis by improving cerebral functions.

The sensory stretch receptors are located within the bladder wall and help with assessing the degree of bladder fullness. This information is transmitted up to the spinal cord and then via the spinothalamic tracts to the central nervous system. The extracts of Wenguanguo make the central nervous system more aware of the signal.

When the bladder becomes contracted under stress and nervousness, the capacity of the urinary bladder will be reduced and then the frequent micturition occurs. The extracts of Wenguanguo can relax the bladder for storing more urine.

The capacity of the urinary bladder is reduced because of aging, and this may even happen to middle-aged people. They suffer from experience of early detrusor contraction due to a sense of urgency to empty the bladder at low urine level. The extracts of Wenguanguo can help relax the detrusor and therefore the bladder capacity increases and urinary frequency decreases.

Patients with detrusor overactivity, detrusor instability, detrusor hyper-reflexia or uninhibited bladder have early, forceful detrusor contractions before the bladder is full. This creates urgency and frequency urinary discharge. The extract of Wenguanguo relaxes the patient's detrusor. The bladder becomes stable and can store a full amount of urine.

The smooth muscle of the urinary bladder has two functions: When the bladder is relaxed, the urine is stored. When it is contracted, the urine will be discharged. The sensory stretch receptors are located within the bladder wall to assess the bladder's fullness. This information is transmitted up the spinal cord via the spinothalamic tracts to the nervous system. The brain generates inhibitory signals when detrusor relaxation is desired. But the brain generates excitatory signal when detrusor contraction is desired. The extracts of Wenguanguo can relax the bladder tissue by inhibiting Acetylcholinesterase, AchE. The inhibiting effect can be maintained for a long period of time. The extracts of Wenguanguo are a good AChE inhibitor that can cure the diseases caused by deficiency of Acetylcholine, ACh.

Antidiuretic hormone (ADH) is stored in the posterior pituitary gland in the brain. It is the primary regulator of body water. ADH acts on the kidneys to increase or decrease total body water. This has an effect on the volume of urine generated by the kidney. The release of ADH is controlled by the cells of osmoreceptors and baroreceptors. Osmoreceptors are the specialized cell hypothalamus. These cells sense the concentration of particles in the blood. When the concentration of particles is higher, more ADH will be released by the pituitary. This stimulates retention of water to dilute body fluids. When the concentration is lower, less ADH will be released by the pituitary. Baroreceptors are located in the right atria and great veins and carotid sinus the specialized area in the heart that sense blood volume and blood pressure. The heart will generate signals to the hypothalamus and pituitary to release more ADH when blood volume or blood pressure is low and vice versa. The extracts of Wenguanguo can regulate the release of ADH which will reduce the volume of urine produced by the body.

This invention relates to the flavone extracts from Wenguanguo husks and fruit-stems, and methods of their preparation. The methods for preparing the extracts from Wenguanguo husks and fruit-stems comprise the following steps: extracting Wenguanguo powder made from husk and fruit-stem with ethanol 3-4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extracts; drying the aqueous extracts to form the flavone extracts that is yellow powder.

This invention provides a composition comprising extracts from husks and fruit-stems which are flavonols, flavanols, dihydroflavonols, phenoloids, and others.

This invention relates to the crude flavone extracts from Wenguanguo leaves that includes a water-soluble flavone extracts and a water-insoluble flavone extract and methods of their preparation. The methods for preparing the extracts from Wenguanguo leaves comprise the following steps: extracting Wenguanguo powder made from the leaves with ethanol for 3 times to form an ethanol extract; concentrating the ethanol extract to form a concentrated condensed extracts; extracting the concentrated extract with hot water to from an aqueous extracts and a water-insoluble extract; drying the aqueous extracts and the water-insoluble extract to form a water-soluble flavone extracts and a water-insoluble flavone extract. This invention provides a composition comprising the crude extracts from leaf which are flavonols, flavanols, dihydroflavonols, phenoloids and others.

This invention relates to the flavone extracts from Wenguanguo branches or stems and methods of their preparation. The methods for preparing the extract from branches or stem comprise the following steps: extracting Wenguanguo powder made from the branches or stems with ethanol for 4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extracts; drying the aqueous extracts to form flavone extracts which is a yellowish powder.

This invention provides a composition comprising extracts from Wenguanguo branches and stems which are flavonols, flavanols, dihydroflavonols, phenoloids and others.

This invention relates to the flavone extracts from Wenguanguo kernels and methods of their preparation. The methods for preparing the extract from kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with ethanol to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extract; drying the aqueous extracts to form a flavone extracts that is a yellow powder.

This invention provides a composition comprising extracts from kernel which are flavonols, flavanols, dihydroflavonols, proteins, phenoloids, and others.

This invention relates to the flavone extract from Wenguanguo root, and methods of their preparation. The methods for preparing the flavone extract from Wenguanguo root comprise the following steps: extracting Wenguanguo powder made from root with ethanol 3-4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extract; drying the aqueous extracts to form the flavone extracts which is a yellow powder.

This invention provides a composition comprising extracts from roots of Wenguanguo which are flavonols, flavanols, dihydroflavonols, phenoloids and others.

This invention relates to the flavone extracts from Wenguanguo barks, and methods of their preparation. The methods for preparing the bark extracts from Wenguanguo barks comprise the following steps: extracting Wenguanguo powder made from the barks with ethanol 3-4 times to form an ethanol extract; removing the ethanol from the ethanol extract to form an aqueous extract; drying the aqueous extracts to form the flavone extracts which is a yellowish powder.

This invention provides an extract composition from Wenguanguo barks comprising flavonols, flavanols, dihydroflavonols, phenoloids and others.

This invention is related to the combined extracts from Wenguanguo husks or fruit-stems and method of their preparation. The methods for preparing the extract from the husks or fruit-stems comprise the following steps: extracting Wenguanguo powder made from the husks or fruit-stems with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the organic extract to from an aqueous extracts; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the combined extracts from the husks or fruit-stems of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo leaves and method of their preparation. The methods for preparing the extracts from the leaves comprise the following steps: extracting Wenguanguo powder made from leaves with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the second extract to an aqueous extract; extracting the aqueous extract with ether and water to form an second aqueous extract; extracting the second aqueous extract with n-butanol to form a n-butanol extract; removing the n-butanol from the n-butanol extract to form a third aqueous extract; drying and sterilizing the third aqueous extract to form the combined extracts.

This invention provides a composition comprising the organic extracts from the leaves of the Wenguanguo. The organic extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo branches or stems and method of their preparation. The methods for preparing the extracts from the branches or stems comprise the following steps: extracting Wenguanguo powder made from the branches or stems with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the second extract to an aqueous extract; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the organic extracts from the branches, and stems and of the Wenguanguo. The organic extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo kernels and method of their preparation. The methods for preparing the extracts from Wenguanguo kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the second extract to an aqueous extract; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the organic extracts from the kernels of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo roots and method of their preparation. The methods for preparing the extracts from Wenguanguo roots comprise the following steps: extracting Wenguanguo powder made from the roots with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the organic extract to from an aqueous extracts; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the combined extracts from the roots of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo barks and method of their preparation. The methods for preparing the extracts from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder made from the barks with an organic solvent (ethanol, methanol and others) to form an organic extract; removing the organic solvent from the organic extract to from an aqueous extract; drying and sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the combined extracts from the barks of the Wenguanguo. The combined extracts comprise saponins, saccharides, proteins and others.

This invention provides the crude saponins from the husks or fruit-stems or seed's shell of Wenguanguo. The methods for preparing the crude saponins from Wenguanguo husks or fruit-stems comprise the following steps: extracting Wenguanguo powder of the husks or fruit-stems with an organic solvent (ethanol, methanol and others) at ratio of 1:2 for 4-5 times, 20-35 hours for each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the leaves of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the leaves comprise the following steps: extracting Wenguanguo powder of the leaves with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form a second extract; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the branches and stems of Wenguanguo. The methods for preparing the crude saponins from the branches or stems comprise the following steps: extracting Wenguanguo powder of the branches or stems with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the kernels of Wenguanguo. The methods for preparing the crude saponins from Wenguanguo kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract for 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; Extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.

This invention provides the crude saponins from the roots of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from Wenguanguo roots comprise the following steps: extracting Wenguanguo powder of the roots with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts contain saponins.

This invention provides the crude saponins from the barks of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the barks comprise the following steps: extracting Wenguanguo powder of the barks with an organic solvent (ethanol, methanol and others) at a ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.

This invention provides a process of producing a coumarin extract from the husks or fruit-stems of Wenguanguo and their applications. The methods for preparing the coumarin extracts from husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks or fruit-stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the husks or fruit-stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the leaves of Wenguanguo and their applications. The methods for preparing the coumarin extracts from leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the leaves of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the coumarin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder branches or stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the extract comprising crude coumarins.

This invention provides a composition comprising the coumarin extracts from the branches and stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides, saccharides, proteins and others.

This invention provides the crude saponins from the leaves of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the leaves comprise the following steps: extracting Wenguanguo powder of the leaves with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form a second extract; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the branches and stems of Wenguanguo. The methods for preparing the crude saponins from the branches or stems comprise the following steps: extracting Wenguanguo powder of the branches or stems with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extract comprises saponins.

This invention provides the crude saponins from the kernels of Wenguanguo. The methods for preparing the crude saponins from Wenguanguo kernels comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract for 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; Extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.

This invention provides the crude saponins from the roots of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from Wenguanguo roots comprise the following steps: extracting Wenguanguo powder of the roots with an organic solvent (ethanol, methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts contain saponins.

This invention provides the crude saponins from the barks of Wenguanguo and method for their preparation. The methods for preparing the crude saponins from the barks comprise the following steps: extracting Wenguanguo powder of the barks with an organic solvent (ethanol, methanol and others) at a ratio of 1:2, 4-5 times, 20-35 hours each time to form an organic extract; collect and reflux the organic extract 2-3 times at 80° C. to form second extracts; resolve the second extracts in water to form an aqueous solution; extract the aqueous solution by n-butanol to form a n-butanol extracts; chromatograph the n-butanol extracts to form the crude saponins. The crude extracts comprise saponins.

This invention provides a process of producing a coumarin extract from the husks or fruit-stems of Wenguanguo and their applications. The methods for preparing the coumarin extracts from husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks or fruit-stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the husks or fruit-stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the leaves of Wenguanguo and their applications. The methods for preparing the coumarin extracts from leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the leaves of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the coumarin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder branches or stems with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the extract comprising crude coumarins.

This invention provides a composition comprising the coumarin extracts from the branches and stems of Wenguanguo. The extract comprises coumarins, coumaric glycosides, saccharides, proteins and others.

This invention provides a process of producing a coumarin extract from the kernels of Wenguanguo and their applications. The methods for preparing the coumarin extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form an ether extract; neutralizing the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the kernels of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the roots of Wenguanguo and their applications. The methods for preparing the coumarin extract from roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the root with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the roots of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the barks of Wenguanguo and their applications. The methods for preparing the coumarin extract from barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the bark with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extract from the barks of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing an aqueous extract from the husks or fruit-stems of Wenguanguo and their applications. The method for preparing the water extracts from the husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husk or fruit-stem with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the husks or fruit-stems of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the leaves of Wenguanguo and their applications. The method for preparing the water extracts from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from leaves of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the branches or stems of Wenguanguo and their applications. The method for preparing the water extracts from branches or stems of Wenguanguo comprise the following steps: extracting the Wenguanguo powder of the branches or stems with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the branches or stems of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the kernels of Wenguanguo and their applications. The method for preparing the water extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from kernels of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the roots of Wenguanguo and their applications. The method for preparing the water extracts from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the roots with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the roots of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the barks of Wenguanguo and their applications. The method for preparing the water extracts from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extracts from the barks of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an alkaloid extract from the husks of Wenguanguo and their applications. The methods for preparing the alkaloid extracts from the husks and fruit-stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the husks or fruit-stems with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the husks or fruit-stems of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the leaves of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extract from the leaves of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the extracts containing alkaloids from branches or stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the branches or stems with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the extract containing crude alkaloids from the branches or stems of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the kernels of Wenguanguo and their applications. The methods for preparing the alkaloid extract from kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at ratio of 1:6 for 3-4 times, 10-15 hours for each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extract from the kernels of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the roots of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the Wenguanguo roots with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the roots of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the barks of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the barks of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing extract containing organic acids from husks and fruit-stems and their applications. The methods for preparing the extracts containing organic acids from the husks or fruit-stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the husks and or fruit-stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acids from the husks of Wenguanguo. The extract comprising aromatic organic acids, fatty organic acids, terpenoid organic acids and others

This invention provides a process of producing extract contains organic acids from leaf and their applications. The methods for preparing the extracts containing organic acids from the leaves of Wenguanguo comprise the following steps: extract Wenguanguo powder of the leaves with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids extract from the leaves of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organic acids from branches and stems and their applications. The methods for preparing the extracts comprising organic acids from the branches or stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the branches or stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the crude organic acids extract from the branches and stems of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract comprise organic acids from kernels and their applications. The methods for preparing the extracts comprising organic acids from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acids extract from the kernels of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organic acids from the roots of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the roots of Wenguanguo comprise the following steps: extract Wenguanguo powder of the roots with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids from the roots of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract comprising organic acids from barks of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the barks of Wenguanguo comprise the following steps: extract Wenguanguo powder of the bark with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids from the barks of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides two methods of producing a tannin extract from Wenguanguo husks and fruit-stems and its usage. The first method for preparing the tannin extract from the husks or fruit-stems of Wenguanguo comprises the following steps: extracting Wenguanguo powder of husks and or fruit-stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract. The second method for preparing the tannin extracts from the husks and or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks and or fruit-stems with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the husks or fruit-stems of Wenguanguo. The extracts are comprised of tannins and others.

This invention provides two methods of producing a tannin extract from Wenguanguo leaves and its usage. The first method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.

The second method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract from the leaves of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo branches and stems and its usage. The first method for preparing the extracts comprising tannins from branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of branches or stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.

The second method for preparing the tannin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the branches or stems and with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins. This invention provides a composition comprising the tannin extract from the branch or stem of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo kernels and its usage. The first method for preparing the tannin extract from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the extract comprising tannins.

The second method for preparing the extracts containing tannins from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract from kernels of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo roots and its usage. The first method for preparing the tannin extract from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of roots with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The method-2 for preparing the tannin extract from the root of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the root with a solvent of acetone-water at a ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the roots of Wenguanguo. The extracts comprise tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo barks and its usage. The method-1 for preparing the tannin extract from the barks of Wenguanguo comprises the following steps: extracting Wenguanguo powder of barks with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The second method for preparing the tannin extract from the barks of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the barks with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the barks of Wenguanguo. The extracts comprise tannins and others.

This invention provides a method for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation.

According to the theory of traditional Chinese medicine, enuresis, frequent micturition and urinary incontinence are caused by “deficiency in kidney (“shen”)”. Therefore, they are treated by using Chinese herbs which can tone the kidney, such as Ginseng Bajitian, Roucongrong Duzhong and Cordyceps. These tonifying herbs can strengthen function of the kidney and regulate water metabolism of human's body through the “kidney pathway” that will help with curing the enuresis, frequent micturition and urinary incontinence.

The Wenguanguo extracts of the present invention can also be used to treat the enuresis, frequent micturition and urinary incontinence. However, the Wenguanguo extracts cure the enuresis, frequent micturition and urinary incontinence through the “bladder pathway” to regulate water metabolism of human's body and urination. The Wenguanguo extracts of the present invention stimulate the growth of the bladder. See FIG. 10A. The Wenguanguo extracts of the present invention increase the capacity of bladder and function of bladder controlling the urination. See Experiment 15 and Experiment 15A. In another aspect of the present invention, Wenguanguo extracts, when used with the “kidney pathway” herbs to treat the enuresis, frequent micturition and urinary incontinence, will strengthen both the pathways of kidney and bladder, and then will produce better treatment results.

This invention provides the medicines or health foods which further comprise Vitamin B, Vitamin D, Vitamin K, grape seed extract and other antioxidants, Cordyceps or its extract, gingko or its extract, Panax ginseng and P. quinquefolium or their extracts, Huangpi (Clausena lansium) or its extracts, Echinacea or its extract, St John's Wort (Hypericum perforatum) or its extract, Gegen (Pueraria lobata) or its extract, Tianma (Gastrodia elata) or its extract, Armillariella mellea or its extract, Danshen (Salvia miltiorrhiza) or its extract, Sanqi (Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice), Huanqi (Hedysarum polybotrys) or its extract, D ihuang (Rehmannia glutinosa) or its extract, Danggui (Angelica sinensis), Yuanzhi (Polygala tenuifoila) or its extract, Lingzhi (Ganoderma spp.) or its extracts, Fuling (Poria cocos) or its extract, enokitake (Flammulina velutipes) or its extract, Can Cao (Glycyrrhiza uralensis Fisch) or its extract, Huperzine A, Lacithin, Metrifonate, Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof.

This invention provides a process of producing a coumarin extract from the kernels of Wenguanguo and their applications. The methods for preparing the coumarin extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and drying them to form the kernel powder; extracting the kernel powder with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form an ether extract; neutralizing the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the kernels of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the roots of Wenguanguo and their applications. The methods for preparing the coumarin extract from roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the root with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extracts from the roots of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract from the barks of Wenguanguo and their applications. The methods for preparing the coumarin extract from barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the bark with 0.5% NaOH solution to form an aqueous extract; collect and extract the aqueous extract by ether to form a ether extract; neutralize the ether extract with HCL to form a neutralized ether extract; concentrate and acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extract from the barks of Wenguanguo. The extract comprises coumarins, coumaric glycosides and others.

This invention provides a process of producing an aqueous extract from the husks or fruit-stems of Wenguanguo and their applications. The method for preparing the water extracts from the husks or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husk or fruit-stem with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the husks or fruit-stems of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the leaves of Wenguanguo and their applications. The method for preparing the water extracts from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from leaves of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the branches or stems of Wenguanguo and their applications. The method for preparing the water extracts from branches or stems of Wenguanguo comprise the following steps: extracting the Wenguanguo powder of the branches or stems with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the branches or stems of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the kernels of Wenguanguo and their applications. The method for preparing the water extracts from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from kernels of Wenguanguo. The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the roots of Wenguanguo and their applications. The method for preparing the water extracts from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the roots with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extract from the roots of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract from the barks of Wenguanguo and their applications. The method for preparing the water extracts from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at room temperature for 24 hours to form an aqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hours to form a second water extract; filtering the second water extract to from a filtered extract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extracts from the barks of Wenguanguo The aqueous extract comprises sugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an alkaloid extract from the husks of Wenguanguo and their applications. The methods for preparing the alkaloid extracts from the husks and fruit-stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the husks or fruit-stems with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the husks or fruit-stems of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the leaves of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extract from the leaves of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the branches and stems of Wenguanguo and their applications. The methods for preparing the extracts containing alkaloids from branches or stems of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the branches or stems with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the extract containing crude alkaloids from the branches or stems of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the kernels of Wenguanguo and their applications. The methods for preparing the alkaloid extract from kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with water at ratio of 1:6 for 3-4 times, 10-15 hours for each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; the toluol extract flows through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extract from the kernels of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the roots of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the roots of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the Wenguanguo roots with water at a ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collecting and alkalifying the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extracting the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the roots of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing an alkaloid extract from the barks of Wenguanguo and their applications. The methods for preparing the alkaloid extract from the barks of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the barks with water at ratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueous extract; collect and alkalify the aqueous extract with NaOH to form a alkalified aqueous extract with pH 10-12; extract the alkalified aqueous extract by toluol to form a toluol extract; flow the toluol extract through 2% of dicarboxyl solution with pH 5-7 to form a dicarboxyl solution; concentrate the dicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extract from the barks of Wenguanguo. The extract comprises alkaloids and others.

This invention provides a process of producing extract containing organic acids from husks and fruit-stems and their applications. The methods for preparing the extracts containing organic acids from the husks or fruit-stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the husks and or fruit-stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acids from the husks of Wenguanguo. The extract comprising aromatic organic acids, fatty organic acids, terpenoid organic acids and others

This invention provides a process of producing extract contains organic acids from leaf and their applications. The methods for preparing the extracts containing organic acids from the leaves of Wenguanguo comprise the following steps: extract Wenguanguo powder of the leaves with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids extract from the leaves of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organic acids from branches and stems and their applications. The methods for preparing the extracts comprising organic acids from the branches or stems of Wenguanguo comprise the following steps: extract Wenguanguo powder of the branches or stems with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the crude organic acids extract from the branches and stems of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract comprise organic acids from kernels and their applications. The methods for preparing the extracts comprising organic acids from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grounding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acids extract from the kernels of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organic acids from the roots of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the roots of Wenguanguo comprise the following steps: extract Wenguanguo powder of the roots with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids from the roots of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract comprising organic acids from barks of Wenguanguo and their applications. The methods for preparing the extracts containing organic acids from the barks of Wenguanguo comprise the following steps: extract Wenguanguo powder of the bark with 10% HCL to form an acid solution; extract the acid solution by an organic solvent (ether or benzol) to form organic extract; extract the organic extract by 5-10% NaHCO3 solution to form a NaHCO3 extract; acidize and filter the NaHCO3 extract to form a deposit matter; extract the deposit matter by an organic solvent to form the second organic extract; remove the organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprising crude organic acids from the barks of Wenguanguo. The extract comprises aromatic organic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides two methods of producing a tannin extract from Wenguanguo husks and fruit-stems and its usage. The first method for preparing the tannin extract from the husks or fruit-stems of Wenguanguo comprises the following steps: extracting Wenguanguo powder of husks and or fruit-stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract. The second method for preparing the tannin extracts from the husks and or fruit-stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the husks and or fruit-stems with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the husks or fruit-stems of Wenguanguo. The extracts are comprised of tannins and others.

This invention provides two methods of producing a tannin extract from Wenguanguo leaves and its usage. The first method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.

The second method for preparing the tannin extract from the leaves of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the leaves with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract from the leaves of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo branches and stems and its usage. The first method for preparing the extracts comprising tannins from branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of branches or stems with 95% ethanol to form an ethanol extract; concentrate the ethanol extract with decompression a to form the tannin extract.

The second method for preparing the tannin extract from the branches or stems of Wenguanguo comprise the following steps: extracting Wenguanguo powder of the branches or stems and with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extract the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins. This invention provides a composition comprising the tannin extract from the branch or stem of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo kernels and its usage. The first method for preparing the tannin extract from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the extract comprising tannins.

The second method for preparing the extracts containing tannins from the kernels of Wenguanguo comprise the following steps: removing oil by pressing the kernels to form kernel cakes; grinding and extracting the kernel cakes with n-hexane to from n-hexane extract; removing the n-hexane from the n-hexane extract and dry them to form the kernel powder; extracting the kernel powder with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract from kernels of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo roots and its usage. The first method for preparing the tannin extract from the roots of Wenguanguo comprises the following steps: extracting Wenguanguo powder of roots with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The method-2 for preparing the tannin extract from the root of Wenguanguo comprises the following steps: extracting Wenguanguo powder of the root with a solvent of acetone-water at a ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the roots of Wenguanguo. The extracts comprise tannins and others.

This invention provides two methods of producing tannin extract from Wenguanguo barks and its usage. The method-1 for preparing the tannin extract from the barks of Wenguanguo comprises the following steps: extracting Wenguanguo powder of barks with 95% ethanol to form an ethanol extract; concentrating the ethanol extract with decompression to form the tannin extract. The second method for preparing the tannin extract from the barks of Wenguanguo comprising the following steps: extracting Wenguanguo powder of the barks with a solvent of acetone-water at ratio of 1:1 for 2-7 days to form an acetone-water extract; removing acetone from the acetone-water extract at 50° C. to form a concentrated extract; filtering the concentrated extract to form a filtered extract; extracting the filtered extract with ether to form an aqueous extract; extracting the aqueous extract with ethyl acetate and n-butanol to form ethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extracts from the barks of Wenguanguo. The extracts comprise tannins and others.

This invention provides a method for preventing cerebral aging, improving memory, improving cerebral functions and curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer's disease, autism, brain trauma, Parkinson's disease and other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder and treating impotence and premature ejaculation.

According to the theory of traditional Chinese medicine, enuresis, frequent micturition and urinary incontinence are caused by “deficiency in kidney (“shen”)”. Therefore, they are treated by using Chinese herbs which can tone the kidney, such as Ginseng Bajitian, Roucongrong Duzhong and Cordyceps. These tonifying herbs can strengthen function of the kidney and regulate water metabolism of human's body through the “kidney pathway” that will help with curing the enuresis, frequent micturition and urinary incontinence.

The Wenguanguo extracts of the present invention can also be used to treat the enuresis, frequent micturition and urinary incontinence. However, the Wenguanguo extracts cure the enuresis, frequent micturition and urinary incontinence through the “bladder pathway” to regulate water metabolism of human's body and urination. The Wenguanguo extracts of the present invention stimulate the growth of the bladder. See FIG. 10A. The Wenguanguo extracts of the present invention increase the capacity of bladder and function of bladder controlling the urination. See Experiment 15 and Experiment 15A. In another aspect of the present invention, Wenguanguo extracts, when used with the “kidney pathway” herbs to treat the enuresis, frequent micturition and urinary incontinence, will strengthen both the pathways of kidney and bladder, and then will produce better treatment results.

This invention provides the medicines or health foods which further comprise Vitamin B, Vitamin D, Vitamin K, grape seed extract and other antioxidants, Cordyceps or its extract, gingko or its extract, Panax ginseng and P. quinquefolium or their extracts, Huangpi (Clausena lansium) or its extracts, Echinacea or its extract, St John's Wort (Hypericum perforatum) or its extract, Gegen (Pueraria lobata) or its extract, Tianma (Gastrodia elata) or its extract, Armillariella mellea or its extract, Danshen (Salvia miltiorrhiza) or its extract, Sanqi (Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice), Huanqi (Hedysarum polybotrys) or its extract, D ihuang (Rehmannia glutinosa) or its extract, Danggui (Angelica sinensis), Yuanzhi (Polygala tenuifoila) or its extract, Lingzhi (Ganoderma spp.) or its extracts, Fuling (Poria cocos) or its extract, enokitake (Flammulina velutipes) or its extract, Gan Cao (Glycyrrhiza uralensis Fisch) or its extract, Huperzine A, Lacithin, Metrifonate, Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof.

There are many different periods of sleep a person goes through. These include Slow-Wave-Sleep 1 (SWS 1), Slow-Wave-Sleep 2 (SWS 2), Slow-Wave-Sleep 3 (SWS 3) Slow-Wave-Sleep 4 (SWS 4) and Rapid Eye Movement (REM). SWS 1 and SWS 2 are both periods of light sleep where it is relatively easy to wake someone up. Light sleep is usually more frequent in the second half of sleep. SWS 3 and SWS 4 are both periods of deep sleep, where it is difficult to wake the sleeper. Deep sleep is more frequent in the first half of sleep and each period will get shorter each time afterward. REM is a period of sleep in which people have their most vivid dreams. The wave patterns are similar to the patterns in which a person is awake. However, it is difficult to wake someone up who is in this state of sleep. The sleep cycle of a typical person can be described as follows:

SWS1, SWS2, SWS3, SWS4, SWS3, SWS2, REM, SWS1, SWS2, SWS3, SWS4, SWS3, SWS2, REM. (See FIG. 62)

However, the above sequence may not be in a fixed order. The sleep status may shift from SWS 4 to SWS1 or wake up because the body turns from one side to the other. It may shift to the SWS2 status after the movement. The interval between two REM is about 90 min. For healthy people, SWS1 will occupy about 5% of sleep, SWS2 will occupy about 50% of sleep, SWS3 will occupy about 10% of sleep, SWS4 will occupy about 10% of sleep and REM will occupy about 25% of sleep. Since a person in SWS1 and SWS2 can easily be woken, a healthy person has enough opportunities to wake up to urinate. However if a person's sleeping status is mostly in SWS4, he has less chance of waking up when the bladder is full. It is difficult for him to break through the barriers of deep sleep. Then enuresis occurs. This invention relates to a plant extract, including Wenguanguo, for preventing enuresis.

This invention provides the extract of Wenguanguo for inhibiting the uptake of 5-hydroxytryptamine (5HT) in a subject.

5-HT controls and modulates a sleep factor that sustains and increases deep sleep. Inhibiting the uptake of 5HT will decrease deep sleep. People who spend too much time in SWS 3 and SWS 4 are unable to awaken from their sleep when their bladder is full because their sleep is too deep. This is the reason that enuresis often occurs during SWS 3 and SWS 4.

This invention provides the extract of Wenguanguo for increasing the activity of Dopamine in a subject thereby making the central nerve system of said subject alert.

This invention provides the extract of Wenguanguo for increasing the secretion of antidiuretic hormone (ADH) in a subject, which reduces urine in said subject.

This invention provides the extract of Wenguanguo for modulating the release, breakdown and uptake of Acetylcholine (Ach) and its receptors in a subject. The said extracts of this invention inhibits the deep sleep created by 5HT and increase REM sleep.

This invention provides the extract of Wenguanguo for preventing sleep paralysis in a subject.

This invention provides the extract of Wenguanguo for providing alertness to a sleeping subject.

This invention provides the extract for helping the growth of the bladder and sphincter.

An immature bladder and sphincter cannot control the process and action of urination. By accelerating the growth of the bladder and the sphincter, this problem will be overcome, and enuresis will not occur.

This invention provides the extract of Wenguanguo against cancer growth. The cancer includes, but is not limited to bladder cancer, cervix cancer, prostate cancer, lung cancer, breast cancer, leukocytes cancer, colon cancer, liver cancer, bone cancer, skin cancer, brain cancer, and ovary cancer

This invention provides the extract of Wenguanguo inhibit tumor activities.

This invention provides the pathways interacted by compounds isolated from Xanthoceras Sorbifolia. In an embodiment, a compound has the formula C57H88O23 and the chemical name 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-1 2-ene, designated herein as “Structure Y”, and derivative compounds which are effective against cancer. In another embodiment, the compounds of the present invention comprise the chemical structures designated herein as “Structure Y1”, “Structure R1”, “Structure 1 to 4”, “Structure Y-a to Y-c” and “Structure Y1-a to Y1-c”, “Structure Y1-1 to Y1-4” and their derivatives. See FIG. 31-40.

They regulate the receptors or components of cells. The compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized.

The compounds of the present invention have structures as shown below: Structure Y (also shown in FIG. 1)

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3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean -12-ene

Structure 1 as shown in FIG. 31
Structure 2 as shown in FIG. 32
Structure 3 as shown in FIG. 33
Structure 4 as shown in FIG. 34
Structure Y-a as shown in FIG. 35
Structure Y-b as shown in FIG. 36
Structure Y-c as shown in FIG. 37
Structure Y1 (also shown in FIG. 2).

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3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β ,16α,21β,22α,28-pentahydroxyolean-12-ene

Structure Y1-a as shown in FIG. 38
Structure Y1-b as shown in FIG. 39
Structure Y1-c as shown in FIG. 40.

This invention further provides a compound comprising the following structure:

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Structure R1: 3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(16)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl- 3β,21β,22α,28-tetrahydroxyolean-12-ene

This invention further provides a compound comprising the following structure:

The structure of compound O54 is presented in the following figure.

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The chemical name of compound O54 is:

O54: 3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-t etrahydroxyolean-12-ene.

There are many components and pathways monitoring cell proliferation.

The Xanthoceras Sorbifolia compound or its derivatives work in the Wnt (Wingless-type MMTV integration site family member) signaling pathway. The Wnt signaling pathway is evolutionarily conserved and controls many events during the embryogenesis. This pathway regulates cell morphology, proliferation, motility and as well as cell apoptosis. It also plays an important role during tumorigenesis. The Wnt pathway has also been observed as inappropriately activated in several different types of cancers in humans.

In the nucleus, the target genes for Wnt signaling are normally kept silent by an inhibitory complex of gene regulatory proteins, e.g. the Groucho corepressor protein bound to the gene regulatory protein LEF-I/TCF. In the absence of a Wnt signal, some β-cartenin is bound to the cytosolic tail of cadherin proteins, and any cytosolic β-cartenin that becomes bound by the APC-axin-GSK-3β will trigger its ubiquitylation and degradation in proteasomes. The result is the decrease of intracellular amount of β-cartenin. However, when the Wnt binding to Frizzled (a seven transmembrane receptor) and LRP (Low density lipoprotein Receptor) activates Dishevelled (a cytoplasmic signaling protein) by a mechanism, this leads to the inactivation of GSK-β3 in the degradation complex by a mechanism which requires casein kinase I, as well as casein kinase II. The activity of the multiprotein complex of β-catenin-axin-adenomatous-polyposis coli (APC)-glycogen synthase kinase (GSK)-3β, which targets β-catenin by phosphorylation for degradation by the proteasome, is then inhibited by Dsh/Dvl (Dishevelled, dsh homolog 1). This then inhibits priming of β-catenin, and indirectly prevents the GSK-3β phosphorylation of β-catenin. When stimulated by Wnt, Dvl recruits the GSK-3 binding protein, GBP, to the multiprotein complex of β-catenin-axin-adenomatous-polyposis coli (APC)-glycogen synthase kinase (GSK)-3β. GBP then titrates GSK-β from axin, and in this way, phosphorylation of β-catenin is inhibited. Then, axin is sequestrated by LRP at the cell membrane. The result of all of this is an accumulation of cytosolic β-catenin. In the nucleus, β-catenin binds to LEF-I/TCF, displaces Groucho, and acts a co-activator to stimulate the transcription of Wnt target genes.

Xanthoceras Sorbifolia compositions regulate the components related to Wnt pathways or its receptors, thereby stopping the proliferation of cancer cells.

The compound or its derivatives work in the Mitogens, Ras and a MAP (Mitogen activation protein) kinase pathway. Mitogens stimulate cell division. The binding of mitogens to cell-surface receptors leads to the activation of Ras and a MAP kinase cascade. One effect of this pathway is the increased production of the gene regulatory protein Myc. Myc increases the transcription of several genes, including the gene encoding cyclin D and a subunit of the SCF ubiquitin ligase. The resulting increase in G1-Cdk and G1/S-Cdk activities promotes Rb phosphyorylation and activation of the gene regulatory protein E2F, resulting in S-phase entry, in which G1-Cdk activity initiates Rb phosphorylation, in turn inactivating Rb and freeing E2F to activate the transcription of S-phase genes including the genes for a G1/S-cyclin (cyclin E) and S-cyclin (cyclin A). The resulting appearance of G1/S-Cdk and S-Cdk further enhances Rb phosphorylation, forming a positive feedback loop, and the E2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop. Myc may also promote E2F activity directly by stimulating the transcription of the E2F gene. The result is the increased transcription of genes entry into S phase. However if this pathway is overactive, it will cause cancer cell growth.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the Ras-MAP kinase cascade so that the pathway is not overactive.

The compound or its derivatives work in Ras-dependent or Myc pathway. Sometimes the mutation of amino acid in Ras causes the protein to become permanently overactive, stimulating the Ras-dependent signal pathways overactive in absence of mitogenic stimulation. Similarly, mutations that cause an overexpression of Myc promote excessive cell growth, which in turn promotes the development of cancer.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components of the Ras-dependent or Myc pathway to make sure it is not overactive.

The compound or its derivatives reactivate the abnormal cell checkpoint mechanism. Inside the cell, there is a checkpoint mechanism which detects abnormal mitogenic stimulation and causes abnormally overactive cells to go into apoptosis. However this mechanism is not active in cancer cells due to mutations in the genes that encode essential components of the checkpoint responses. If the mutation happens in the checkpoint mechanism, the cancer cell will grow and divide endlessly.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia reactivate the checkpoint mechanism to stop the cancer cell growth.

The compound or its derivatives affect the extracellular growth signaling pathways. The extracellular growth factors that stimulate cell growth are bound to receptors on the cell surface and activate intracellular signaling pathways. It activates the enzyme PI3-kinase, which promotes protein synthesis, at least partly through the activation of EIF4e and phosphorylated S6 kinase, resulting in increased mRNA translation and then a stimulation of cell growth.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor relate to extracellular growth. It binds the receptor of ovarian cancer cells so as to stop the cancer cell growth.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components relating to Ras and MAP Kinase, which ceases ovarian cancer cell growth.

The compound or its derivatives affect the intracellular mechanism. Cell division is also controlled by an intracellular mechanism that can limit cell proliferation. In normal cells, the Myc protein acts in the nucleus as a signal for cell proliferation. Large quantities of Myc can cause the cell to proliferate in excess and form a tumor.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of the Myc cell's proliferation to stop the tumor cells from dividing.

The compound or its derivatives affect the TGF-alpha signaling pathway. TGF-alpha is produced by keratincytes, macrophages, hepatocytes, and platelets. Its synthesis is stimulated by the infection by viruses. TGF-Alpha induces the long term proliferation of murine and chicken immature hematopoietic progenitor cell such as BFU-E without causing differentiation. It also induces the terminal differentiation of BFU-Ecell into erythrocytes. TGF-Alpha stimulates the proliferation of cultured endothelial cells. It plays an importance role in the vascularisation of tumor tissues.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-alpha to suppress ovarian cancer and bladder cancer cell growth.

The compound or its derivative compounds affect the TGF-beta signaling pathway. TGF-beta regulates growth and proliferation of cells, blocking growth of many cell types. There are two TGF-beta receptors: Type 1 and Type 2. They are serine-threonine kinases that signal through the SMAD (Protein named after the first two identified, Sma in C. elegans and Mad in Drosophila) family of transcriptional regulators. The TGF-beta pathway and mutation in SMADs are associated with cancer in humans.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-beta to suppress the ovarian cancer and bladder cancer cell growth.

The compound or its derivatives reactivate the cell functions which are damaged by DNA viruses. DNA tumor viruses cause cancer by interfering with cell cycle control Rb protein and the p53 protein. Mutation in p53 gene will allow cancer cells to survive and proliferate despite DNA damage. The papillomanius uses the proteins E6 and E7 to release the p53 and Rb respectively. This action activates mutated cells, allowing them to survive and then divide and accumulate. The accumulation of damaged cells can lead to cancer.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the proteins E6 and E7 and release the proteins Rb and p53, which will prevent abnormal cells from dividing. It also regulates or reacts with the protein, causing the cancer cells to die.

The compound or its derivatives affect the p53 signaling pathway. p53 helps multi-cellular organisms cope safely with DNA damage and other stressful cellular events, stopping cell proliferation in circumstances where it would be dangerous. Cancer cells tend to contain large quantities of mutant p53 protein, suggesting that the genetic accidents they undergo or the stresses of growth in an inappropriate environment created the signals that normally activate the p53 protein. Thus, the loss of p53 activity can be extremely dangerous in relation to cancer because it allows mutant cells to continue through the cell cycle. It also allows them to escape apoptosis. So, if their DNA is damaged, some cells will die but the cells which survive will carry on dividing without pausing to repair the damage. This may cause the cells to die, or they could survive and proliferate with a corrupted genome, which could lead to loss of both tumor suppressor genes and the activation of oncogenes, for example by gene amplification. Gene amplification could enable cells to develop resistance against therapeutic drugs.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components and receptor of the p53 pathway, which stops the cancer cells from dividing.

The compound or its derivatives affect the cell suicide signaling pathway. All cells with a nucleus contain various inactive procaspases, awaiting a signal before destroying the cell. Each suicide protease is made as an inactive proenzyme called procaspase. It is usually activated by proteolytic cleavage by another member of the caspase family. Two of the cleaved fragments come together to form the active part of the caspase, and the active enzyme is thought to be a tetramer of two of these two parts. Each activated caspase molecule can cleave many procaspase molecules, which in turn activates more molecules. Through a chain reaction or cascade, this leads to the explosive action of a large number of procaspase molecules. Then, some of the activated procaspases cleave a number of key proteins in the cell, including specific cytosolic proteins and nuclear-lamins leading to the controlled death of the cell.

Activating the death receptor on the outside of the cell can also trigger inactive procaspases. For example, killer lymphocytes can cause apoptosis by producing the protein Fas on the surface of the targeted cell. These clusters of Fas protein then recruit intracellular adaptor proteins that bind and aggregate procaspase-8 molecules. These then cleave and activate one another. The activated caspase-8 molecules then activate downstream procaspases to induce apoptosis.

However in cancer cells, the signal to destroy the cell is blocked, due to gene mutation. This means that the cancer cells continue to divide, thereby causing a tumor.

Compounds or compositions derived from the plant Xanthoceras Sorbifolia unblock the suicide signals, allowing cancer cells to destroy themselves.

Structure showed in FIG. 67.

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above-described compound, wherein R1, R2, R3, R4 are short aliphatic chain and R5 contains an oxyl group; and a pharmaceutically acceptable carrier effective to inhibit growth of said tumor cells.

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above-described compounds.

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the compound comprising: a sugar; a triterpene or Sapogenin; side chain at Carbon 21 and 22 or Angeloyl groups, operatively linked form the compound; and a pharmaceutically acceptable carrier.

Structure showed in FIG. 67.

This invention provides a method for inhibiting tumor cell growth in a subject comprising administering to the above-described subject, wherein R1, R2, R3, R4 are short aliphatic chain and R5 contains an oxyl group; effective to inhibit growth of said tumor cells and a pharmaceutically acceptable carrier.

This invention provides a method of for inhibiting tumor cell wherein R1=R2=R3=R4=CH3 and R5 contains an oxyl bond.

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above-described compounds.

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This invention provides a compound consist of a triterpene or sapongenin, sugar moiety connected to the backbone. A sugar was linked the C21 position where two angeloyl groups were attached. This compound has the anti-cancer activity

This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the compound is a triterpene or sapongenin with any two of angeloyl group or tigloyl group or senecioyl group or their combinations attach to carbon 21 and 22, or any two of angloyl group or tigloyl group or senecioyl group or their combinations attached to a sugar moiety which bonds to carbon 21 or 22.

The structures of this invention or its derivative can be synthesis or from biological sources.

This invention will be better understood from the examples which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS

Experiment 1

Herb Extraction

(a) extracting Xanthoceras Sorbifolia powder of husks or branches or stems or leaves or kernels or roots or barks with organic solvent at ratio of 1:2 for 4-5 times for 20-35 hours for each time to form an organic extract; (b) collecting the organic extract; (c) refluxing the organic extract for 2-3 times at 80° C. to form second extracts; (d) removing the organic solvent from the second extract; and (e) Drying and sterilizing the extract to form a Xanthoceras Sorbifolia extract powder.

Experiment 2

Analysis of Xanthoceras Sorbifolia Extract Components by HPLC Chromatography

Methods

HPLC.

A C-18 reverse phase μbondapak column (Water P/N 27324) was equilibrated with 10% acetonitrile, 0.005% Trifluoroacetic acid (equilibration solution). An extract of Xanthoceras Sorbifolia prepared using the methods of the present invention was dissolved in equilibration solution (1 mg/ml) before being applied onto the column. 20 ug of samples was applied into column. Elution conditions: Fractions were eluted (flow rate 0.5 ml/min.) with acetonitrile (concentration gradient from 10% to 80% in 70 min) and then remains at 80% for 10 min (70-80 min). The acetonitrile concentration then decreased to 10% (80-85 min) and remained at 10% for 25 min (85-110 min). The fractions were monitored at 207 nm and recorded in chart with a chart speed of 0.25 cm/min and with a OD full scale of 0.128.

Instruments.

Waters Model 510 Solvent Delivery System; Waters 484 tunable Absorbance Detector; Waters 745/745B Data Module

Absorbance Analysis.

The absorption profile of Xanthoceras Sorbifolia extract at various wavelengths was determined. An extract of Xanthoceras Sorbifolia of the present invention was dissolved in 10% acetonitrile/TFA and scanned at 200-700 nm with a spectrophotometer [Spectronic Ins. Model Gene Sys2].

Results

HPLC.

About 60-70 peaks can be accounted for in the profile. Among them four are major peaks, 10 are medium size and the rest are small fractions. The major peaks are labelled with a to z following increased concentration of acetonitrile elution. See FIG. 6.

Absorption Maximum.

Three absorption maximum were identified for Xanthoceras Sorbifolia plant extract; 207 nm, 278 nm and 500 nm. See FIG. 41.

Experiment 3

Screening of Cytotoxicity of Xanthoceras Sorbifolia Extract with Cancer Cells Derived from Different Human Organs using MTT Assay

Methods and Materials

Cells.

Human cancer cell lines were obtained from American Type Culture Collection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary). Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI-1640; HCT-116, U2OS in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% CO2 humidified incubator at 37° C.

MTT Assay.

The procedure for MTT assay followed the method described in (Carmichael et al., 1987) with only minor modifications. Cells were seeded into a 96-wells plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2, U2OS), or 40,000/well (K562), for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U2OS, and 96 hours for MCF-7). After the drug-treatment, MTT (0.5 mg/ml) was added to cultures for an hour. The formation of formazan (product of the reduction of tetrazolium by viable cells) was dissolved with DMSO and the O.D. at 490 nm was measured by an ELISA reader [Dynatech. Model MR700]. The MTT level of cells before drug-treatment was also measured (T0). The % cell-growth (% G) is calculated as:


% G=(TD−T0/TC−T0)×100 (1)

where TC or TD represent O.D. readings of control or drug-treated cells. When T0>TD, then the cytotoxicity (LC) expressed as % of the control is calculated as:


% LC=(TD−T0/T0)×100.

Results.

Among the 10 cell lines studies, their sensitivity toward Xanthoceras Sorbifolia extract can be divided into four groups (most sensitive: Ovary. Sensitive: bladder, bone, prostate, and leukocyte, marginal sensitive: liver, breast, and brain; and lease sensitive: colon, cervix, and lung) (FIG. 8, 10A-D). Their IC50 values are listed in Table 3.1.

TABLE 3.1
IC50 values of Xanthoceras Sorbifolia Extract
Determined in Different Cancer Cells
Cancer cellsIC50 determined by
from different organsMTT assay (ug/ml)
Ovary (most sensitive)15-15
Bladder (sensitive)45-50
Bone40-55
Prostate40-50
Leukocyte45-50
Liver (marginal sensitive)45-65
Breast65
Brain70-85
Colon (least sensitive)90
Cervix115
Lung110

Xanthoceras Sorbifolia plant extract stimulate cell growth of bladder, bone and lung cells. See FIGS. 10A, 10D.

To invest Among these cell line studied, it was found that low concentrations of the igate the growth and inhibition components of the Xanthoceras Sorbifolia plant extract, the plant extract was fractionated. FIG. 5 shows the results of the screening of cell growth activity of fractions obtained after FPLC chromatography. The assay was conducted with bladder cells. The fractions obtained from FPLC as shown in FIG. 20 were used. As shown in this figure, that different components of Xanthoceras Sorbifolia extracts cause either growth or inhibition effects on cells. Only fraction 5962 (Fraction Y) causes cell inhibition. Fractions 610 and 1116 cause minor stimulation of cell growth. Abscissa: concentration (ug/ml). Ordinate: % Cell Growth (determined by MTT assay).

Experiment 4

Purification of Inhibition Components in the Xanthoceras Sorbifolia Extract

(A) Fractionation of Xanthoceras Sorbifolia Extracts Components with FPLC

Methods

Column.

Octadecyl functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 10% acetonitrile—0.005% TFA.

    • Sample loading: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA.
    • Gradient elution: 10-80% acetonitrile in a total volume of 500 ml.
    • Monitor absorption wavelength: at 254 nm.
    • Fraction Collector: 5 ml/fractions (collect from 10% to 72% acetonitrile, total 90 fractions)
    • Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results.

The elution profile shows 4-5 broad fractions. See FIG. 20. These fractions were analyzed with HPLC. Specific components, i.e., a-z as specified in FIG. 6, are then assigned in the FPLC fractions.

FPLC fractions are grouped into 7 pools and analyzed for cell growth activity with bladder cells with MTT assay. It was found only one pool (#5962) contains inhibition activity. See FIG. 5.

(B) Fractionation of Fraction #5962 with FPLC by a C18 Open Column with 64% Acetonitrile Isocratic Elution

Methods

Column.

Octadecyl-functionalized silica gel; 50 ml; 2 cm×28 cm; equilibrated with 64% acetonitrile—0.005% TFA.

    • Sample loading: 0.2 ml, with concentration: 1-2 mg/ml in 65% acetonitrile/TFA.
    • Elution: 64% acetonitrile isocratic.
    • Monitor absorption wavelength: at 254 nm.
    • Fraction Collector: 1 ml fraction (collect the first 90 fractions)
    • Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results.

Fraction 5962 was further separated with an open ODS-C18 column using isocratic 64% acetonitrile elution. Two major fractions, i.e., X and Y, were collected. See FIG. 42. MTT assay showed that only the Y fraction has the inhibition activity. See FIG. 43.

(C) Analysis of Fraction Y with HPLC

Methods

Column.

Waters μ-bondapak C18 (3.9 mm×300 cm).

    • Elution: 35% or 45% isocratic elution.
    • Flow rate: 0.5 ml/min; monitored at 207 nm with O.D. Scale of 0.128; chart speed: 0.25 cm/min.

Results.

On 45% isocratic analysis, Three fractions of Y were obtained (FIG. 44).

(D) Final Isolation of Active Y Component with Preparative HPLC

Methods

Column: A preparative HPLC column (Waters Delta Pak C18-300A);

Elution: 45% acetonitrile isocratic elution with flow rate of 1 ml/min.

Monitor at 207 nm;

Fractions (designated as Y1, Y2, etc.) were collected and lyophilized.

Results.

Final separation of Y fractions was achieved by HPLC with a preparative column (FIG. 7). These fractions (compound Y1, Y2, Y3 and Y4) including the major fraction Y3 (designated as compound Y) were collected. Re-chromatography of the Compound Y showed a single peak in HPLC with a C18 reverse phase column. See FIG. 7A.

(E) Appearance and Solubility

The pure Compound Y is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.

(F) Inhibition Analysis of Compound Y with MTT Assay

Inhibition analysis of Compound Y was determined with MTT assay. The results indicated that (a) Compound Y has activity against ovarian cancer cells (OCAR-3) with IC50 value of 1.5 ug/ml which is 10-15 times more potent than the unpurified extract. See FIG. 3; and FIG. 8.

(b) Compound Y maintains its selectivity against ovarian cancer cells versus cervical cancer cells (HeLa). See FIG. 9.

Experiment 5

Determination of the Chemical Structure of Compound Y of Xanthoceras Sorbifolia Extract

Methods

NMR Analysis.

The pure compound Y of Xanthoceras Sorbifolia were dissolved in pyridine-D5 with 0.05% v/v TMS. All NMR spectra were acquired using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe (1H/13C/15N/31P) at 298 K. The numbers of scans for 1D 1H spectra were 16 to 128, depending on the sample concentration. 2D HMQC spectra were recorded with spectral widths of 6000×24,000 Hz and data points of 2024×256 for t2 and t1 dimensions, respectively. The numbers of scans were 4 to 128. 2D HMBC were acquired with spectral widths of 6000×30,000 Hz and data points of 2024×512 for t2 and t1 dimensions, respectively. The numbers of scans were 64. The 2D data were zero-filled in t1 dimension to double the data points, multiplied by cosine-square-bell window functions in both t1 and t2 dimensions, and Fourier-transformed using software XWIN-NMR. The final real matrix sizes of these 2D spectra are 2048×256 and 2048×512 data points (F2×F1) for HMQC and HMBC, respectively.

Mass Spectral Analysis.

The mass of samples was analyzed by (A) MALDI-TOF Mass Spectrometry and by (B) ESI-MS Mass spectrometry. (A) Samples for MALDI-TOF were first dissolved in acetonitrile, then mixed with the matrix CHCA (Alpha-cyano-4-hydroxycinnamic acid, 10 mg CHCA/mL in 50:50 water/acetonitrile and 0.1% TFA in final concentration). The molecular weight was determined by the high resolution mass spectroscope analysis with standards. (B) For ESI, the sample was analyzed with LCQ DECA XP Plus machine made by Thermo Finnigan. It is ionized with ESI source and the solvent for the compound is acetonitrile.

Results.

The profile of the proton NMR is presented in FIG. 11. The 2D NMR profiles of HMQC and HMBC are shown in FIGS. 12 and 13, respectively.

Table 5.1 summarizes the 2D NMR chemical shift data and shows the assignment of functional groups derived from these chemical shifts. Based on these data and analysis, the structure of compound Y is assigned and shown below.

Structure of Compound Y:

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The chemical name of compound Y is: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-1 2-ene.

TABLE 5.1
13C and 1H NMR Data for Compound Y (in Pyridine-d5)a
PositionCHKey HMBC correlations
138.70.83, 1.40C-3, C-5, C-9
226.41.81, 2.14
389.63.25, 1H, dd, 12.0/4.0 HzC-23, C-24, GlcA C-1′
439.4
555.30.78
618.51.55, 1.59C-8, C-10
736.52.00, 2.10C-5, C-9
841.2
947.03.06C-7, C-8, C-12,
C-14, C-26
1037.2
1123.71.74, 1.89
12125.25.49, 1H, br sC-9, C-11, C-14, C-18
13143.4
1447.5
1567.34.21C-8, C-27
1673.64.45C-14, C-15, C-18
1748.3
1840.83.07C-12, C-13, C-14,
C-16, C-19, C-20, C-28,
1946.81.41, 1.69
2036.2
2179.36.71, 1H, d, 10 HzC-20, C-22, C-29, C-30,
21-O-Ang C-1″″
2273.56.32, 1H, d, 10 HzC-16, C-17, C-21, C-28,
22-O-Ang C-1″″
2327.71.26, 3H, sC-3, C-4, C-5, C-24
2416.51.16, 3H, sC-3, C-4, C-5, C-23
2516.00.81, 3H, sC-1, C-5, C-9, C-10
2617.30.99, 3H, sC-7, C-8, C-9, C-14
2721.01.85, 3H, sC-8, C-13, C-14, C-15
2862.93.50, 1H, d, 11.0 Hz, 3.76, C-16, C-17, C-18, C-22
1H, d, 11.0 Hz,
2929.21.09, 3H, sC-19, C-20, C-21, C-30
3020.01.32, 3H, sC-19, C-20, C-21, C-29
GlcA
1′104.94.89, 1H, d, 7.8 HzC-3
2′79.14.38GlcA C-1′, C-3′, Gal C-1″
3′86.14.20GlcA C-2′, C-4′,
Ara C-1′″
4′71.54.42GlcA C-3′, C-5′, C-6′
5′78.04.52GlcA C-4′, C-6′
6′171.9
Gal
1″104.65.32, 1H, d, 7.7 HzGlcA C-2′
2″73.64.42Gal C-1″, C-3″
3″74.94.10Gal C-2″
4″69.54.56Gal C-2″, C-3″
5″76.43.94Gal C-4″, C-6″
6″61.64.43, 4.52Gal C-4″, C-5″
Ara-f
1′″110.66.03. 1H, br sGlcA C-3′, Ara C-2′″,
C-4′″
2′″83.44.94Ara C-3′″
3′″78.34.78Ara C-2′″
4′″85.24.82Ara C-5′″
5′″62.24.12, 4.28Ara C-3′″
21-O-Ang
1″″167.7
2″″129.6
3″″137.25.96, 1H, dq, 7.0/1.5 HzAng C-1″″, C-4″″, C-5″″
4″″15.52.10, 3H, dq, 7.0/1.5 Hz Ang C-2″″, C-3″″
5″″20.82.00, 3 H, sAng C-1″″, C-2″″, C-3″″
22-O-Ang
1″″167.9
2″″129.8
3″″136.35.78, 1H, dq, 7.0/1.5 HzAng C-1″″, C-4″″, C-5″″
4″″15.51.93, 3H, dq, 7.0/1.5 Hz Ang C-2″″, C-3″″
5″″20.51.74, 3 H, sAng C-1″″, C-2″″, C-3″″
aThe data were assigned based on HMQC and HMBC correlations.

FIGS. 14 and 15 show the mass spectrum of Compound Y as determined by MALDI-TOF and ESI-MS techniques. Based on these data, the mass of compound Y is 1140.57 which agrees with the theoretical mass of the compound Y.

Conclusion

The active compound Y isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with three sugars and biangeloyl groups attached to the C21 and C22 positions of the backbone. The formula of Y is C57H88O23, and the chemical name of Compound Y is: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-1 2-ene.

Experiment 6

Determination of the Chemical Structure of Compound Y1 of Xanthoceras Sorbifolia Extract

Methods

The method for NMR and MS analysis for compound Y1 are same as described in Experiment 5.

Results.

The profile of the H-NMR of Y1 is presented in FIG. 16. The 2D NMR profiles of HMQC, HMBC and COSY are shown in FIGS. 17, 18 and 19, respectively.

Table 6.1 summarizes the 1D and 2D NMR chemical shift data and shows the assignment of functional groups derived from these chemical shifts.

TABLE 6.1
13C and 1H NMR Data for Compound Y1 (in Pyridine-d5)a
PositionCH
138.60.85, 1.33
226.31.86, 2.10
389.73.25, 1H, dd
439.5
555.50.75
618.31.40, 1.43
733.11.20, 1.50
840.0
946.71.69
1036.5
1122.52.30
12123.65.36, 1H, br s
13143.5
1441.8
1534.71.53, 1.73
1668.54.45
1748.2
1839.93.04
1947.61.30, 3.05
2036.7
2185.35.05, 1H, d
2273.86.17, 1H, d
2327.71.29, 3H, s
2416.51.16, 3H, s
2515.50.81, 3H, s
2617.10.82, 3H, s
2720.61.83, 3H, s
2863.73.42, 1H, d, 3.60, 1H, d
2929.91.42, 3H, s
3019.91.37, 3H, s
GlcA
1105.0 4.88, 1H, d
279.04.37
386.04.20
471.64.43
578.04.50
6171.8
Gal
1104.5 5.31, 1H, d
273.54.43
374.94.10
469.54.57
576.33.95
661.14.44, 4.53
Ara-f
1110.9 6.04. 1H, br s
283.34.95
378.34.78
485.24.82
562.04.13, 4.31
21-O-Rha
1105.1 4.92, 1H, d
270.54.25
374.05.59
471.55.70
568.53.89
617.61.18, 3H, d
Rh-3-Ang
1167.2
2127.9
3138.7 5.92, 1H, q
415.72.02, 3H, d
520.61.92, 3H, s
Rh-4-Ang
1167.2
2128.0
3137.9 5.87, 1H, q
415.51.96, 3H, d
519.81.85, 3H, s
22-O-Ac
1171.4
221.82.31, 3H, s

Based on these data and analysis, the structure of compound Y1 is assigned and shown below.

embedded image

The chemical name of Y1 is: 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,1 6α,21β,22α,28-pentahydroxyolean-12-ene.

Conclusion.

Based on the chemical shift analysis, the active compound Y1 isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with four sugars and biangeloyl groups attached to the sugar moiety. The formula of Y1 is C65H100O27,

Results of Y2 Analysis

The profile of the proton NMR of Y2 is presented in FIG. 51.
The profiles of 2D NMR (HMQC) of Y2 is presented in FIG. 52.

Results of Y4 Analysis

The profile of the proton NMR of Y4 is presented in FIG. 53.
The profiles of 2D NMR (HMQC) of Y4 is presented in FIG. 54.

Experiment 7

Acid and Alkaline and Enzyme Hydrolysis of Compound Y

Removal of Sugars from Compound Y

Acid Hydrolysis of compound Y generates a compound with the following structure, designated herein as Y-c: (FIG. 37).

embedded image

Methods:

5 mg of compound Y is dissolved in 3 ml of MeOH and then treated with 3 ml of 3N HCl. Hydrolysis of saponins will be conducted under reflux for 4 hr. After hydrolysis, the solution will be neutralized with 5% Na2CO3 and extracted with Ethyl acetate three times to afford an aqueous layer and an organic layer, containing sugars and aglycon, respectively. Aglycon from the organic layer will be further purified on Silica gel chromatography in (CHCl3:MeOH, 1:9) or with C18 ODS HPLC chromatography. About 2 mg of compound with the above structure Y-c can be obtained.

Method reference: Essentials of Carbohydrate Chemistry. By John F. Robyt, (Springer, 1998).

Partial Removal of Sugars from Compound Y.

The linkage of oligosaccharide can be cleaved by partial acid hydrolysis and by specific enzyme hydrolysis. For example, the 1→4 linkage of arabinofuranosyl can be removed by α-amylase. Other enzymes such as β-amylase, isoamylase, glucose oxidase, mannanse and pullulanase can be used to cleave individual saccharide in saponins.

Compound Structure showed in FIG. 70.
Compound structure showed in FIG. 69.
Compound Structure showed in FIG. 68.

Experiment 8

Purification of Component R from Xanthoceras Sorbifolia Extract

(A) Fractionation of Xanthoceras Sorbifolia Extracts Components with FPLC

Methods

Column: Octadecyl functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 10% acetonitrile—0.005% TFA.

Sample loading: volume: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA.

Gradient elution: 10-80% acetonitrile in a total volume of 500 ml.

Monitored at 254 nm.

Fraction Collector: 5 ml/fraction.

Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results

The elution profile shows 4-5 broad fractions (FIG. 20). These fractions were analyzed with HPLC. By comparison with the profiles of the original sample, specific component, in this case the R component, is identified and then collected for further purification.

Fractionation of R with FPLC with 30% Acetonitrile Isocratic Elution

Methods

Column: Octadecyl-functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 30% acetonitrile—0.005% TFA.

Sample loading: 0.2 ml, with concentration: 1-2 mg/ml.

Elution: 30% acetonitrile isocratic. Monitor absorption wavelength: at 254 nm.

Fraction Collector: 5 ml/fraction.

Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results

Fraction No. 39-41 from gradient elution of FPLC were pooled and further purified with an open ODS-C18 column with isocratic 30% acetonitrile elution. Six identifiable fractions in two groups were collected. See FIG. 45. Fractions 6-13 were further characterized with HPLC.

Analysis and Isolation of R with HPLC

Methods

Column: Waters μ-bondapak C18 (3.9×300 nm) and Waters DeltaPak C18 (7.8 mm×30 cm).
Elution: Gradient (10-80%) and 30% isocratic elution.
Flow rate: 0.5 ml/min; monitored at 207 nm; with attenuation 0.128; chart speed: 0.25 cm/min.

Results

On HPLC gradient elution analysis, Fractions #9-11 contain a major component with a few minor components. See FIG. 46. These components were further separated into 4-5 components with the 30% acetonitrile isocratic elution in a DeltaPak column. The fraction designated herein as “R1”, is the major component. See FIG. 47A. The pure R1 was subsequently collected from the column elution. See FIG. 47B. Appearance and solubility.

The pure R1 is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.

Determination of the chemical structure of R1 isolated from Xanthoceras Sorbifolia extract

Methods

The NMR and MS Analysis of R1 are same as those described in Experiment 5.

Results

The proton NMR profile of pure R1 is presented in FIG. 21. The 2D NMR (HMQC) spectra of R1 are presented in FIG. 22. The 2D NMR (HMBC) spectra of R1 are presented in FIG. 23. The 2D COSY spectrum is presented in FIG. 24. The Carbon 13 NMR spectrum is presented in FIG. 25.

Based on all the data presented above, Table 8.1 summarizes the results of the structural analysis and the assignment of the functional groups of compound R1.

TABLE 8.1
13C and 1H NMR Data for R1 (in Pyridine-d5)a
Position CHKey HMBC correlations
138.61.01, 1.63C-3, C-25
226.41.89, 2.33C-3
389.13.26, 1H, dd, 12.0/4.2 HzC-23, C-24, Glc′ C-1
439.2
555.50.69, 1H, d, 11.4 HzC-4, C-6, C-7, C-9,
C-10, C-23, C-24, C-25
618.31.30, 1.39C-5, C-8, C-10
732.51.41C-6, C-26
840.1
947.71.61C-1, C-5, C-8, C-10,
C-11, C-14, C-25, C-26
1036.7
1123.71.90, 2.00C-8, C-12, C-14
12123.55.35, 1H, br sC-9, C-14, C-18
13142.9
1441.9
1525.71.88, 1.90
1618.01.95, 2.29C-14, C-17, C-18
1743.1
1841.62.60, dd, 12.0/2.4 HzC-19
1946.31.28, 2.11C-18, C-20, C-29, C-30
2036.1
2176.53.73, 1H, d, 9.6 HzC-20, C-22, C-29, C-30
2275.14.31, 1H, d, 9.6 HzC-16, C-17, C-21
2327.91.20, 3H, sC-3, C-4, C-5, C-24
2416.60.95, 3H, sC-3, C-4, C-5, C-23
2515.70.95, 3H, sC-1, C-5, C-9, C-10
2616.71.07, 3H, sC-7, C-8, C-9, C-14
2726.11.25, 3H, sC-8, C-13, C-14
2875.84.10, 2H, br sC-16, C-17, C-18,
C-22, Glc′″ C-1
2930.21.22, 3H, sC-19, C-20, C-21, C-30
3019.51.26, 3H, sC-19, C-20, C-21, C-29
3-Glc′
1106.54.84, 1H, d, 7.2 HzC-3, Glc′ C-5
273.23.99
379.15.90, 1H, t, 9.6 HzAng C-1, Glc′ C-2, C-4
469.44.26Glc′ C-6
576.43.95Glc′ C-1, C-3
669.84.40, 4.83Glc′ C-4, Glc″ C-1
Ang
1167.9
2128.7
3136.75.80, 1H, ddd, 6.6/0.6 HzAng C-1, C-4, C-5
415.71.93, 3H, dd, 6.6/0.6 HzAng C-2, C-3
520.61.81, 3H, sAng C-1, C-2, C-3
Glc″
1105.45.09, 1H, d, 7.8 HzGlc′ C-6, Glc″ C-5
274.94.05Glc″ C-1
378.2b4.22Glc″ C-5
471.4c4.24Glc″ C-6
578.3d3.90
662.4e4.40, 4.51Glc″ C-4
28-Glc″′
1103.54.72, 1H, d, 7.2 HzC-28
275.34.22Glc″′ C-1, C-3, Rha C-1
379.84.25
471.64.20
576.64.13Glc″′ C-3
670.04.67, 2H, d, 10 HzGlc″′ C-1, Glc″′ C-4, C-5
Rha
1100.76.52, 1H, br sGlc″′ C-2, Rha C-3, C-5
272.3f 4.70, d, 3.0 HzRha C-4
372.3f 4.63, dd,Rha C-2
474.14.35Rha C-2, C-5
569.14.79
618.71.82, 3H, d, 6.6 Hz Rha C-4, C-5
Glc″″
1105.55.01, 1H, d, 7.8 HzGlc″′ C-6, Glc″″ C-5
274.94.05Glc″″ C-1
378.2b 4.22Glc″″ C-5
471.4c 4.24Glc″″ C-6
578.3d 3.90
662.5e4.40, 4.51Glc″″ C-4
aThe data were assigned based on COSY, HMQC and HMBC correlations.
b, c, d, fThe data with the same labels in each column were overlapped.
eThe data with the same labels in each column may be interchanged.

Conclusion

Based on the chemical shift analysis, the compound R1 isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with five sugars and one angeloyl group attached to the sugar moiety. The chemical structure of R1 is:

embedded image

The formula of compound R1 is C65H106O29, and the chemical name of R1 is: 3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl -3β,21β,22α,28-tetrahydroxyolean-12-ene

Experiment 9

Purification of Component-O from Xanthoceras Sorbifolia Extract

(A) Fractionation of Xanthoceras Sorbifolia Extracts Components with FPLC

Methods:

Column: Octadecyl functionalized silica gel; column dimension: 2 cm×28 cm; equilibrated with 10% acetonitrile—0.005% TFA.

Sample loading: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA.

Gradient elution: 10-80% acetonitrile in a total volume of 500 ml.

Monitor absorption wavelength: at 254 nm.

Fraction Collector: 5 ml/fraction.

Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results.

The elution profile shows 4-5 broad fractions (FIG. 20). These fractions were analyzed with HPLC. By comparison with the profiles of the original sample, specific component, in this case the component-O, is identified (#28-30) and were collected for further purification.

(B) Purification of Component-O with HPLC with 20% Acetonitrile Isocratic Elution

Methods.

Column: A preparative HPLC column (Waters Delta Pak C18-300A);
Samples: Fraction #28-30 from the gradient elution of FPLC were pooled and applied into the HPLC.
Elution: 20% acetonitrile isocratic with flow rate of 1 ml/min. Fractions were collected.

Monitored at 207 nm;

Fractions of interested were collected and lyophilized.

Results.

Sixteen identifiable fractions were observed in the elution profiles (FIG. 48). Fractions 28, 34 and 54 were further characterized with HPLC using same condition (FIGS. 49 and 50). As show in these figures, a single peak elution of fractions 28, 34 and 54 was obtained, indicating that they are homogeneous fractions (pure). These purified components are named as Compound O28, O34 and O54, respectively.

Appearance and solubility: The purified O-23 and O-34 are light yellow amorphous powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine. The purified O-54 is white amorphous powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.

(C) Structure Analysis of Compound O54

Methods:

The NMR and MS analysis of O54 are same as those described in Experiment 5.

Results

The profile of the proton NMR of compound O54 is presented in FIG. 28. The 2D NMR (HMQC) spectra of O54 is presented in FIG. 29. The 2D NMR (HMBC) spectra of O54 are presented in FIG. 30.

Based on all the data presented above, Table 9.1 summarizes the results of the structural analysis and the assignment of the functional groups of compound O54.

TABLE 9.1
13C and 1H NMR data for 054 (in Pyridine-d5)a
PositionCHKey HMBC correlations
138.61.03, 1.60C-3, C-25
226.31.90, 2.40C-3
389.13.30, 1H, dd, 12.0/4.8 HzC-23, C-24, Glc′ C-1
439.3
555.60.70, 1H, d, 12.0 HzC-4, C-6, C-7, C-9,
C-10, C-23, C-24, C-25
618.51.30, 1.40C-5, C-8, C-10
732.51.40C-6, C-26
840.2
947.81.59C-1, C-5, C-8, C-10,
C-11, C-14, C-25, C-26
1036.6
1123.61.90, 1.95C-8, C-12, C-14
12123.55.34, 1H, br sC-9, C-14, C-18
13142.8
1441.9
1525.81.90
1618.11.90, 2.31C-14, C-17, C-18
1743.2
1841.72.58, dd, 13.2/2.0 HzC-19
1946.51.28, 2.08 (t, 1H, t,C-18, C-20, C-29, C-30
13.2 Hz)
2036.2
2176.63.73, 1H, d, 10.2 HzC-20, C-22, C-29, C-30
2275.14.31, 1H, d, 10.2 HzC-16, C-17, C-21
2327.91.22, 3H, sC-3, C-4, C-5, C-24
2416.80.98, 3H, sC-3, C-4, C-5, C-23
2515.80.95, 3H, sC-1, C-5, C-9, C-10
2616.91.07, 3H, sC-7, C-8, C-9, C-14
2726.11.26, 3H, sC-8, C-13, C-14
2875.94.10, 2H, br sC-16, C-17, C-18,
C-22, Glc′″ C-1
2930.11.24, 3H, sC-19, C-20, C-21, C-30
3019.61.26, 3H, sC-19, C-20, C-21, C-29
3-Glc′
1106.54.86, 1H, d, 7.8 HzC-3, Glc′ C-5
274.93.99
378.2b4.20
470.84.36
576.53.95
670.54.40, 4.88 (d, 1H, 9.6 Hz)Glc″ C-1
Glc″
1105.55.12, 1H, d, 7.8 HzGlc′ C-6, Glc″ C-5
275.0c4.03Glc″ C-1
378.2b4.20
471.5d4.20
578.3e3.91
662.4f4.40, 4.48Glc″ C-4, C-5
28-Glc′″
1103.64.72, 1H, d, 7.2 HzC-28
275.34.22Glc′″ C-1, C-3, Rha C-1
379.84.21
471.64.19
576.84.14Glc′″ C-3
670.34.67, 2H, d, 10.2 HzGlc″″ C-1, Glc′″ C-4,
C-5
Rha
1100.66.51, 1H, br sGlc′″ C-2, Rha C-3, C-5
272.3g4.72, d, 3.0 HzRha C-4
372.3g4.61, dd, 10.8/3.6 Hz,Rha C-2
474.14.36Rha C-2, C-5
569.24.77
618.61.80, 3H, d, 6.0 HzRha C-4, C-5
Glc″″
1105.65.00, 1H, d, 7.8 HzGlc′″ C-6, Glc″″ C-5
275.0c4.03Glc″″ C-1
378.2b4.20
471.5d4.20
578.3e3.91
662.5f4.40, 4.48Glc″″ C-4, C-5
aThe data were assigned based on COSY, HMQC and HMBC correlations.
b,c,d,e,gThe data with the same labels in each column were overlapped.
fThe data with the same labels in each column may be interchanged.

Conclusion

Based on the chemical shift analysis, the compound O54 isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with five sugars. The chemical structure of Compound O54 is:

The structure of compound O54 is presented in the following figure.

embedded image

The formula of compound O54 is C60H100O28, and the chemical name of O54 is: The chemical name of compound O54 is: 3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-t etrahydroxyolean-12-ene.

Results of O28 Analysis

The profile of the proton NMR of O28 is presented in FIG. 55.

The profiles of 2D NMR (HMQC) of O28 is presented in FIG. 56.

Results of O34 Analysis

The profile of the proton NMR of O34 is presented in FIG. 57.

The profiles of 2D NMR (HMQC) of O34 is presented in FIG. 58.

Experiment 11

Extracts X and Y from the Wenguanguo Plant (Xanthoceras Sorbifolia) Improve Learning in Normal Aging Mice

Extracts X and Y are Different Wenguanguo Extracts

Aging male mice at 16 months of age weighing 35-55 gm were trained in a SMG-2 filled with water 11 cm deep (25-26° C.). The SMG-2 had a start point, 4 blind terminals, the escape platform and their routes. The mice were trained to find the escape platform, and the escape latencies from the water and error frequencies were recorded. After training 3 times, the mice which escaped from the water in 2 minutes were selected for the test. The selected aging mice were divided into 9 groups of 11: 1) control: receiving normal saline (NS); 2) Positive control: receiving Xi-en-kai 0.9 g/kg; 3) X-I group: receiving 100 mg/kg4; 4) X-II group: receiving 200 mg/kg; 5) X-III group: receiving 400 mg/kg; 6) Y-I group: receiving 125 mg/kg; 7) Y-II group: receiving 250 mg/kg; 8) Y-III group: receiving 500 mg/kg and 9) model group.

All the drugs were received through oral administration, 20 ml/kg, 3, 6 and 9 days before the water maze test. The escape latencies (EL) from the water and error frequencies were recorded. All data were analyzed with t-test.

11.1. After 3 days of administration of extracts X and Y from Wenguanguo Plant, hereinafter as “X” and “Y”, respectively, the escape latency in the water maze by the group 9 mice receiving 500 mg/kg of Y decreased significantly compared with the control (P<0.05). The other dosage treatments showed improvement too but not significant ones. See Table 11.1.

TABLE 11.1
The Learning Effects of Plant Extracts after Administration in Aging Mice for 3 days
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
control2.12 ± 2.273.62 ± 1.631.37 ± 2.412.68 ± 2.529.81 ± 3.4264.12 ± 24.8 
positive1.09 ± 1.57 2.0 ± 0.89 2.0 ± 1.26 2.0 ± 2.647.09 ± 2.3039.45 ± 16.32
X I1.18 ± 1.4 3.36 ± 2.652.27 ± 2.140.81 ± 1.077.63 ± 4.4751.72 ± 17.23
X II 2.2 ± 1.61 2.5 ± 1.51 2.0 ± 1.69 2.8 ± 1.03 9.5 ± 4.03 50.3 ± 20.84
X III1.45 ± 2.332.72 ± 2.642.09 ± 2.111.81 ± 1.948.09 ± 4.6746.91 ± 19.18
Y I1.36 ± 1.91 3.0 ± 1.943.45 ± 3.2 1.09 ± 1.448.18 ± 3.7846.36 ± 22.33
Y II1.63 ± 1.803.81 ± 1.942.36 ± 1.12 .36 ± 1.5010.18 ± 3.02 48.36 ± 20.61
Y III2.18 ± 3.341.63 ± 1.211.54 ± 1.291.81 ± 1.407.18 ± 4.30 41.45 ± 16.48*
P < 0.05*

11.2. After 6 days of administration of X and Y, the error frequency in the water maze in the mice with all dosage treatments decreased significantly (P<0.05, P<0.01). The escape latency in the water maze by the group 9 mice receiving 500 mg/kg of Y decreased significantly (P<0.05). See Table 11.2.

TABLE 11.2
The Learning Effects of Plant Extract after Administration in aging mice for 6 days
BlindBlindBlindBlindError
GroupTerminalTerminalTerminalTerminalFrequencyEL(sec)
Control2.74 ± 2.98 4.0 ± 2.673.26 ± 3.02  3.0 ± 2.6712.9 ± 4.20 60.58 ± 24.6 
positive1.64 ± 2.063.73 ± 2.051.18 ± 1.47  2.0 ± 1.908.55 ± 4.61* 38.64 ± 13.68
X I 1.7 ± 1.49 3.1 ± 2.021.8 ± 2.10 1.9 ± 1.668.5 ± 1.65*47.71 ± 1.13 
X II1.18 ± 1.66 3.5 ± 2.022.0 ± 1.731.73 ± 2.058.45 ± 3.14* 49.01 ± 3.29 
X III1.09 ± 1.303.55 ± 2.071.91 ± 1.58 1.55 ± 1.448.0 ± 2.32*46.36 ± 13.31
Y I 1.0 ± 1.673.09 ± 1.582.64 ± 1.8 1.54 ± 2.028.36 ± 3.07* 57.27 ± 19.88
Y II1.36 ± 1.503.36 ± 2.062.0 ± 1.671.18 ± 1.177.91 ± 3.05**47.55 ± 22.93
Y III 1.2 ± 1.14 4.1 ± 1.792.5 ± 2.84 1.4 ± 1.909.0 ± 3.23* 39.9 ± 8.56*
P < 0.05*
P < 0.01**

11.3. After 9 days of administration of X and Y, the error frequency in the water maze in the mice with all dosage treatments decreased significantly (P<0.05, P<0.01). The escape latency in the water maze by the group 9 mice receiving 500 mg/kg of Y decreased significantly (P<0.05). See Table 11.3. FIG. 60a, 60b.

TABLE 11.3
The Learning Effects of Plant Extracts after Administration in aging mice for 9 days
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
Control2.36 ± 1.652.64 ± 1.692.86 ± 2.541.5 ± 2.189.71 ± 3.52 59.71 ± 29.42
positive1.64 ± 1.802.18 ± 1.541.55 ± 1.371.64 ± 2.20 7.0 ± 2.19*33.36 ± 10.87
X I 1.1 ± 1.29 2.5 ± 2.37 1.9 ± 0.741.0 ± 1.156.5 ± 3.27*40.8 ± 20.4
X II1.18 ± 1.172.18 ± 1.601.36 ± 1.362.18 ± 1.25 6.91 ± 3.27* 42.73 ± 15.82
X III 1.0 ± 1.25 1.9 ± 1.79 1.3 ± 1.252.2 ± 1.166.4 ± 2.84* 35.1 ± 11.76*
Y I1.82 ± 1.331.64 ± 1.691.82 ± 1.331.82 ± 1.47 7.09 ± 2.47* 42.09 ± 20.93
Y II 1.2 ± 1.32 1.9 ± 1.37 2.6 ± 1.581.2 ± 1.327.1 ± 1.52* 34.4 ± 13.47*
Y III 0.8 ± 1.03 2.5 ± 1.43 1.8 ± 1.402.0 ± 1.707.0 ± 1.41* 31.9 ± 9.87**
P < 0.05*
P < 0.01*

The results indicated that the extracts X and Y had positive effects on improving acquisition and retention of the tested aging mice. In addition, the effects increased with the period of receiving the extracts of X and Y prolonged

Experiment 12

Effects of Extracts X and Y on Improving Impairment Induced by Pentobarbital Sodium in Water Maze Learning

12.1 After 10 days of administration of the extracts X and Y, the administrated mice were injected with pentobarbital sodium to induce amnesia.

After 1 day administration of pentobarbital sodium, the results of water maze learning showed that the time spent searching the terminal platform in the water maze by the mice receiving 100 mg/kg of X, and 125 mg, 250 mg/kg and 500 mg/kg of Y decreased significantly (P<0.05).

Error frequency made in the water maze by the mice receiving 500 mg/kg of Y decreased significantly (P<0.05). See Table 12.1.

TABLE 12.1
Results of Water Maze Learning (First Day after Injection with Pentobarbital)
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
control0.92 ± 0.76 1.46 ± 0.97 1.85 ± 1.07 1.23 ± 0.73 5.38 ± 2.33 63.0 ± 25.31
positive0.69 ± 0.70 1.3 ± 0.951.1 ± 0.741.1 ± 0.744.1 ± 1.85 36.5 ± 15.76**
X I0.5 ± 0.531.7 ± 0.821.2 ± 0.920.9 ± 0.324.2 ± 1.62 42.2 ± 18.83*
X II0.9 ± 0.881.4 ± 0.701.6 ± 1.351.1 ± 0.885.0 ± 2.4953.8 ± 16.10
X III0.9 ± 0.741.7 ± 0.821.8 ± 0.420.9 ± 0.575.4 ± 1.5858.1 ± 16.11
Y I1.0 ± 0.891.36 ± 0.81 1.27 ± 1.01 0.73 ± 0.65 4.09 ± 2.02 42.73 ± 16.17*
Y II0.9 ± 0.74 1.7 ± 0.82* 1.0 ± 0.82* 0.6 ± 0.70*4.2 ± 1.87 38.4 ± 15.19**
Y III0.6 ± 0.700.8 ± 0.631.4 ± 1.350.8 ± 0.63 3.6 ± 1.26* 38.5 ± 13.81**
P < 0.05*
P < 0.01*

12.2. After two days of injected of pentobarbital sodium, the time spent searching the terminal platform and the error frequency made in the water maze by all groups of mice receiving X and Y decreased significantly (P<0.05). See Table 12.2.

TABLE 12.2
Results of Water Maze Learning (Second Day after Injection with Pentobarbital)
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
control1.36 ± 0.50 1.86 ± 0.53 1.29 ± 1.07 1.14 ± 0.66 5.64 ± 1.91 59.5 ± 34.95 
positive0.8 ± 0.63 1.8 ± 0.421.1 ± 0.740.6 ± 0.704.3 ± 0.95*34.7 ± 11.45*
X I0.9 ± 0.57* 1.0 ± 0.67**1.2 ± 0.790.8 ± 0.633.9 ± 1.73*34.5 ± 12.67*
X II0.8 ± 0.79*1.5 ± 0.711.1 ± 0.88 0.5 ± 0.53*3.8 ± 1.40*35.9 ± 12.70*
X III1.0 ± 1.05  1.3 ± 0.48*1.3 ± 0.82 0.5 ± 0.53*4.1 ± 1.79*36.1 ± 11.10*
Y I1.09 ± 0.94 1.45 ± 0.52 0.91 ± 0.83 0.73 ± 0.65 4.18 ± 1.08* 36.64 ± 14.38* 
Y II0.9 ± 0.57*1.3 ± 0.821.1 ± 0.880.8 ± 0.634.1 ± 1.45*35.5 ± 14.27*
Y III0.8 ± 0.63* 0.9 ± 0.74**0.9 ± 0.570.9 ± 0.57 3.4 ± 1.43**32.1 ± 13.12*
P < 0.05*
P < 0.01*

12.3. After three days of administration of pentobarbital sodium, the time spent searching the terminal platform in the water maze by all groups of mice receiving X and Y decreased significantly (P<0.05). The error frequency made in the water maze by the mice receiving 250 mg/kg and 500 mg/kg of Y decreased significantly (P<0.05). See Table 12.3, FIG. 61a and FIG. 61b

TABLE 12.3
Results of Water Maze Learning (Third day after Injection with Pentobarbital)
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
control0.85 ± 0.99 1.0 ± 0.581.46 ± 1.05 0.62 ± 0.65 3.92 ± 1.75 48.92 ± 23.49 
positive0.4 ± 0.520.7 ± 0.480.9 ± 0.740.6 ± 0.70 2.6 ± 0.97*28.4 ± 13.78
X I0.6 ± 0.700.8 ± 0.63 0.6 ± 0.70*0.8 ± 0.922.8 ± 1.69 28.0 ± 17.10*
X II0.4 ± 0.521.0 ± 0.471.0 ± 0.820.6 ± 0.843.0 ± 1.25 32.0 ± 12.36*
X III0.5 ± 0.710.8 ± 0.631.0 ± 0.820.9 ± 0.743.2 ± 1.23 31.8 ± 12.48*
Y I0.5 ± 0.530.7 ± 0.481.0 ± 0.670.6 ± 0.522.8 ± 0.6331.0 ± 9.66*
Y II0.5 ± 0.530.6 ± 0.520.8 ± 0.420.6 ± 0.52 2.5 ± 0.53*29.0 ± 7.80*
Y III0.3 ± 0.481.0 ± 0.470.9 ± 0.570.4 ± 0.52 2.6 ± 0.84*30.2 ± 7.54*
P < 0.05*

The results indicated that the extracts X and Y had distinct positive effects on improving spatial learning and retention impairment induced by pentobarbital sodium.

Experiment 13

Effects of Extracts X and Y on Improving Impairment Induced by Scopolamine Hydrobromide in Passive Avoidance

ICR mice weighing 16-20 gm were trained in a STT-2. A mouse was placed on the platform and the SDL were recorded. When the mouse stepped down and all four feet were on the grid, it received electric shock (36 V) immediately, and the EL was recorded. The mice with SDL and EL within 2-60 seconds were selected for the test. The selected aging mice were divided into 9 groups. Each group had 5 male and 5 female mice. All the drugs were received with oral administration, 20 ml/kg, 3, 6 and 9 days before the platform training. The SDL, El and error frequency (the times of receiving electric shocks) made in 5 minutes were recorded. After 10 days of administration all the groups of mice received Scopolamine hydrobromide by injection, 3 mg/kg. After 30 minutes of administration of Scopolamine hydrobromide the mice were trained on the platform and the training was repeated the next day. The performances of the mice in the training were recorded. The SDL, EL and error frequency were recorded. All data were analyzed with t-test.

13.1. After 9 days of administration of X and Y, the results of passive avoidance in aging mice showed that the EL and the error frequency made by the mice received the X and Yin all doses reduced. See Table 13.1.

TABLE 13.1
Passive Avoidance in Aging Mice after 9 days of Administration
GroupEL (sec)SDL (min)Error Frequency (in 5 min)
control6.8 ± 5.93131.5 ± 106.502.5 ± 1.47
positive5.3 ± 3.74148.6 ± 81.26 1.7 ± 1.25
X I7.0 ± 5.46143.3 ± 19.77 1.8 ± 0.92
X II6.2 ± 5.71141.1 ± 109.892.0 ± 1.25
X III4.9 ± 3.70145.7 ± 107.0 1.9 ± 1.52
Y I5.1 ± 3.95153.0 ± 123.521.6 ± 1.33
Y II3.9 ± 2.77162.7 ± 108.921.8 ± 1.48
Y III5.7 ± 3.12159.4 ± 83.20 1.9 ± 0.93

13.2. After 10 days of administration of the X and Y, the tested mice were administrated with scopolamine. The results of passive avoidance in aging mice showed that the error frequency made in the passive avoidance by the mice receiving 400 mg/kg of X, 250 mg/kg and 500 mg/kg of Y reduced significantly (P<0.05, P<0.01). See Table 13.2.

TABLE 13.2
Effects of plant extract X and Y on improving
impairment induced by Scopolamine
GroupEL (sec)Error Frequency (5 min)
control3.4 ± 3.030.9 ± 1.29
model4.4 ± 4.091.6 ± 1.35
positive3.1 ± 1.20 0.4 ± 0.52*
X I3.1 ± 2.080.8 ± 1.03
X II3.7 ± 3.060.9 ± 1.52
X III2.8 ± 1.48 0.3 ± 0.48**
Y I3.2 ± 2.490.7 ± 1.06
Y II2.5 ± 0.97 0.5 ± 0.71*
Y III2.5 ± 0.71 0.5 ± 0.71*
*p < 0.05
**p < 0.01

The results of passive avoidance test impaired mice by scopolamine showed that the error frequency made by the mice receiving X and Y in all doses reduced significantly (P<0.05). The SDL prolonged significantly in mice receiving 250 mg/kg of Y.

The results indicated that the extracts X and Y had distinct positive effects on improving learning and retention impairment induced by scopolamine. See Table 3.3.

TABLE 13.3
Effects of plant extract X and Y on improving
impairment induced by Scopolamine.
GroupSDL (sec)Error Frequency (5 min)
control230.4 ± 96.61 0.7 ± 1.06 
model216.2 ± 100.771.5 ± 1.35 
positive286.0 ± 34.38*0.4 ± 0.70*
X I245.7 ± 114.480.4 ± 0.84*
X II260.4 ± 87.14 0.4 ± 0.84*
X III266.8 ± 65.64 0.5 ± 0.71*
Y I252.7 ± 101.110.4 ± 0.84*
Y II285.8 ± 29.21*0.4 ± 0.70*
Y III277.4 ± 47.62 0.4 ± 0.70*
*p < 0.05

The results indicated that the extracts X and Y had distinct positive effects on improving learning and retention impairment induced by scopolamine hydrobromide.

Experiment 14

Effects of Extracts X and Y on Improving Impairment Induced by NaNO2 in Water Maze Learning

ICR male mice weighing 16-19 gm were trained in a SMG-2 filled with water 11 cm deep (25-26° C.). The SMG-2 has a start point, 4 blind terminals, the escape platform and their routes. The mice were trained to find the escape platform, and the escape latencies from the water and error frequencies were recorded. After training, the mice which escaped from the water within 1 minute were selected for the test. The selected aging mice were divided into 9 groups of 11 mice. All the drugs were received with oral administration, 20 ml/kg, 3, 6 and 9 days before the water maze test. After 10 days of administration all the groups of mice were received with NaNO2 by injection, 120 mg/kg. After 24 hours of administration of NaNO2 the mice were trained to find the escape platform, and the escape latencies from the water and error frequencies made in 2 minutes were recorded. The administration of pentobarbital sodium continued for 3 days and the performances of the mice in water maze test were recorded. The escape latencies (EL) from the water and errors frequencies were recorded. All data were analyzed with t-test.

14.1. After 3 days of administration of X and Y the escape latency from the water maze and error frequency by the mice receiving X and Y reduced, although not significantly. See Table 14.1.

TABLE 14.1
Effects of Extracts X and Y on Water Maze Learning in Mice after 3 Days of Administration
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
control1.05 ± 0.491.82 ± 0.661.09 ± 0.750.86 ± 0.474.82 ± 0.9648.27 ± 21.47
positive0.91 ± 0.301.27 ± 0.650.82 ± 0.600.64 ± 0.50 3.64 ± 0.92**36.27 ± 11.83
X I1.36 ± 0.811.73 ± 0.791.09 ± 0.830.55 ± 0.524.73 ± 2.0537.82 ± 14.24
X II0.91 ± 0.301.82 ± 0.401.09 ± 0.941.00 ± 0.894.91 ± 1.5136.46 ± 11.97
X III1.09 ± 0.541.45 ± 0.520.91 ± 0.700.45 ± 0.523.91 ± 0.7036.46 ± 11.78
Y I1.55 ± 0.521.82 ± 0.40 1.0 ± 0.890.45 ± 0.694.82 ± 1.3341.46 ± 16.37
Y II0.91 ± 0.301.18 ± 0.601.27 ± 1.100.73 ± 0.794.09 ± 2.2136.82 ± 20.61
Y III0.91 ± 0.301.55 ± 0.820.45 ± 0.520.82 ± 0.403.73 ± 1.2737.55 ± 13.85
*P < 0.05
**P < 0.01

14.2. After 6 days of administration of X and Y the escape latency from the water maze by the mice receiving 400 mg/kg of X and 500 mg/kg of Y reduced significantly compared with the control (P<0.01). See Table 14.2.

TABLE 14.2
Effects of Extracts X and Y on Water Maze Learning in Mice after 6 Days of Administration
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
control0.95 ± 0.381.09 ± 0.430.77 ± 0.610.91 ± 0.533.82 ± 0.8042.96 ± 13.48
positive0.73 ± 0.651.09 ± 0.300.55 ± 0.520.82 ± 0.40 3.18 ± 0.75*32.91 ± 7.15*
X I0.73 ± 0.651.27 ± 0.470.73 ± 0.650.73 ± 0.653.45 ± 1.2137.18 ± 7.65 
X II0.91 ± 0.301.00 ± 0.450.64 ± 0.920.91 ± 0.543.45 ± 1.2137.73 ± 13.26
X III0.91 ± 0.301.09 ± 0.540.91 ± 0.830.82 ± 0.753.73 ± 1.19 31.09 ± 8.15**
Y I0.91 ± 0.30 1.0 ± 0.450.82 ± 0.400.55 ± 0.523.36 ± 0.6735.82 ± 9.93 
Y II0.91 ± 0.541.09 ± 0.541.00 ± 0.770.64 ± 0.673.64 ± 1.1235.09 ± 12.13
Y III0.82 ± 0.401.09 ± 0.540.82 ± 0.600.55 ± 0.523.27 ± 1.10 31.73 ± 8.36**
*P < 0.05
**P < 0.01

14.3. After 9 days of administration of X and Y, the escape latency from the water maze by the mice receiving 250 mg/kg of X, 250 mg and 500 mg/kg of Y reduced significantly compared with the control (P<0.05). See Table 14.3.

TABLE 14.3
Effects of Extracts X and Y on Water Maze Learning
in Mice after 9 Days of Administration.
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
control0.59 ± 0.731.14 ± 0.640.55 ± 0.67 1.0 ± 0.763.27 ± 1.3239.27 ± 15.52
positive0.55 ± 0.521.00 ± 0.000.27 ± 0.650.91 ± 0.542.73 ± 0.6527.64 ± 6.96*
X I0.45 ± 0.521.27 ± 0.470.73 ± 0.650.55 ± 0.523.00 ± 0.7733.55 ± 9.59 
X II0.45 ± 0.520.91 ± 0.700.55 ± 0.690.82 ± 0.892.73 ± 0.9028.00 ± 9.53*
X III0.45 ± 0.521.09 ± 0.700.82 ± 0.750.45 ± 0.522.82 ± 1.2529.45 ± 8.49 
Y I0.91 ± 0.700.91 ± 0.540.45 ± 0.520.55 ± 0.522.82 ± 0.9832.00 ± 9.49 
Y II0.64 ± 0.500.82 ± 0.750.64 ± 0.670.82 ± 0.602.91 ± 1.3026.36 ± 9.82*
Y III0.73 ± 1.010.91 ± 0.300.45 ± 0.690.55 ± 0.692.64 ± 1.1228.09 ± 9.26*
*P < 0.05

14.4. After 10 days of administration of X and Y, the error frequency made in the water maze by the mice receiving 250 mg and 500 mg/kg of Y, the escape latency from the water maze by the mice receiving 500 mg/kg of Y reduced significantly compared with the control (P<0.05, P<0.01). See Table 14.4.

TABLE 14.4
Effects of Extracts X and Y on Water Maze Learning in Mice after 10 Days of Administration
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
control0.64 ± 0.731.14 ± 0.560.64 ± 0.660.91 ± 0.683.32 ± 0.9536.73 ± 13.02
positive0.45 ± 0.690.82 ± 0.400.55 ± 0.520.82 ± 0.40 2.64 ± 0.81* 29.0 ± 10.10
X I0.36 ± 0.670.91 ± 0.700.91 ± 0.830.64 ± 0.812.82 ± 1.0833.09 ± 13.96
X II0.73 ± 0.790.82 ± 0.400.55 ± 0.690.73 ± 0.472.82 ± 0.7528.91 ± 13.34
X III0.91 ± 0.700.91 ± 0.540.73 ± 0.650.36 ± 0.672.91 ± 0.9432.45 ± 13.57
Y I0.73 ± 0.790.91 ± 0.300.36 ± 0.500.73 ± 0.652.73 ± 0.9029.55 ± 13.87
Y II0.55 ± 0.520.64 ± 0.500.45 ± 0.520.64 ± 0.67 2.27 ± 0.79**30.36 ± 12.30
Y III0.45 ± 0.691.09 ± 0.540.27 ± 0.650.55 ± 0.52 2.36 ± 1.21* 25.64 ± 11.02*
*P < 0.05
**P < 0.01

The results indicated that the extracts X and Y had distinct positive effects on improving the learning and retention in mice in a water maze. In addition, the effects increased with the period of receiving the extracts of X and Y prolonged.

14.5. After 10 days of receiving X and Y, the mice were administrated with Na NO2 after the test. The results of treatment with X and Y to prevent impairments induced by Na NO2 in water maze learning in aging mice showed that error frequency made by the mice receiving 100 mg/ka and 200 mg/kg of X, and Y of all doses reduced significantly (P<0.05). See Table 14.5. It indicated the extracts X and Y had distinct positive effects on preventing the impairments induced by Na NO2. See Table 14.5.

TABLE 14.5
Effects of Extract X and Y on preventing Impairments Induced by Na NO2
BlindBlindBlindBlindError
GroupTerminal 1Terminal 2Terminal 3Terminal 4FrequencyEL(sec)
Control0.27 ± 0.470.91 ± 0.540.55 ± 0.521.09 ± 0.542.82 ± 0.75 30.91 ± 12.36
Model1.18 ± 0.750.91 ± 0.300.45 ± 0.520.73 ± 0.653.27 ± 1.01 36.45 ± 16.89
Positive0.45 ± 0.520.91 ± 0.300.55 ± 0.520.45 ± 0.522.36 ± 0.81*32.00 ± 15.83
X I0.55 ± 0.520.82 ± 0.600.18 ± 0.400.82 ± 0.602.36 ± 0.81*29.09 ± 13.80
X II0.18 ± 0.400.82 ± 0.600.55 ± 0.520.82 ± 0.752.36 ± 0.92*25.82 ± 10.82
X III0.45 ± 0.520.64 ± 0.500.82 ± 0.400.91 ± 0.702.82 ± 1.33 31.09 ± 11.76
Y I0.27 ± 0.470.91 ± 0.300.36 ± 0.500.73 ± 0.472.27 ± 0.79*27.00 ± 10.73
Y II0.45 ± 0.520.64 ± 0.500.36 ± 0.500.91 ± 0.302.36 ± 0.81*25.82 ± 11.43
Y III0.64 ± 0.500.82 ± 0.400.36 ± 0.500.64 ± 0.502.45 ± 0.82*25.09 ± 9.67 
*P < 0.05
**P < 0.01

Experiment 15

Effects of Wenguanguo (Xanthoceras sorbifolia) Extracts on Urination in Mice

As used herein, Extracts (or Fractions) X and Y are different extracts of Wenguanguo plant or Xanthoceras sorbifolia.

Methods of Experiment.

One hundred twelve male ICR mice weighing 18-22 gm were divided into 8 groups of 14: 1, control: receiving normal saline (NS); 2, DCT group: receiving DCT 33.4 mg/kg; 3, X-I group: receiving 100 mg/kg4; 4, X-II group: receiving 200 mg/kg; 5, X-III receiving 400 mg/kg; 6, Y-I group: receiving 125 mg/kg; 7, Y-II group: receiving 250 mg/kg and 8, Y-III group: receiving 500 mg/kg.

All the drugs were received with oral administration, 20 ml/kg, once a day for 3 days. After the last administration, the mouse was placed on a filter paper. The filter paper was on the bottom of a 500 ml beak. The quantity of urine was measured at 30, 60, 120, 180, 240, 300, and 360 minutes by weighing the filter paper with the electronic analytical scale. All data were analyzed with t-test.

Results.

After 3 days of administration of X and Y the quantity of urine discharged at 30 minute by the mice receiving 400 mg/kg of X decreased significantly (P<0.01) compared with the mice receiving normal saline. The quantity of urine discharged at 60 minute by the mice receiving 600 mg/kg of Y decreased significantly compared with the mice receiving normal saline (P<0.01).

The quantity of urine discharged at 180 minutes by the mice receiving 200 mg/kg of X, 125 mg and 500 mg/kg of Y decreased significantly compared with the mice receiving normal saline (P<0.01). See Table 15.1.

TABLE 15.1
Effects of Extracts X and Y on the Quantity of Urine in Mice
after 3 Days of Administration
Group30 min60 min120 min
Control0.267 ± 0.1050.367 ± 0.1620.382 ± 0.109
Positive0.348 ± 0.06* 0.471 ± 0.169**0.574 ± 0.249
X I0.304 ± 0.0720.274 ± 0.0760.323 ± 0.173
X II0.341 ± 0.1070.323 ± 0.1020.404 ± 0.138
X III 0.155 ± 0.056** 0.200 ± 0.140**0.455 ± 0.211
Y I0.216 ± 0.1300.309 ± 0.0930.341 ± 0.061
Y II0.278 ± 0.0630.278 ± 0.1190.437 ± 0.112
Y III0.227 ± 0.058 0.235 ± 0.035**0.425 ± 0.133
Compared with the control group: P < 0.05* P < 0.01**
Group180 min240 min300 min360 min
Control0.191 ± 0.0800.161 ± 0.0830.116 ± 0.06 0.103 ± 0.057
Positive0.272 ± 0.1310.182 ± 0.0960.110 ± 0.0510.085 ± 0.031
X I0.184 ± 0.1050.154 ± 0.0930.124 ± 0.0910.102 ± 0.064
X II 0.336 ± 0.103**0.163 ± 0.10 0.107 ± 0.0760.106 ± 0.075
X III0.207 ± 0.1120.204 ± 0.0880.150 ± 0.0660.116 ± 0.077
Y I 0.367 ± 0.104**0.171 ± 0.085 0.173 ± 0.068*0.093 ± 0.053
Y II0.275 ± 0.2060.145 ± 0.0290.109 ± 0.0360.106 ± 0.045
Y III 0.319 ± 0.086** 0.264 ± 0.114**0.152 ± 0.0840.135 ± 0.051
Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantity of urine after 3 days of administration of X and Y.

After 5 days of administration of X and Y, the quantity of urine discharged at 30 minute by the mice receiving 400 mg/kg of X and 500 mg/kg of Y decreased, but not significantly compared with the mice receiving normal saline. The quantity of urine discharged at 4 hour by the mice receiving 400 mg/kg of X and Y in all doses increased significantly compared with the mice receiving normal saline (P<0.05, P<0.01). See Table 15.2.

TABLE 15.2
Effects of Extracts X and Y on the Quantity of Urine in Mice after 5 Days of
Administration
Group30 min60 min120 min
Control0.327 ± 0.1480.330 ± 0.1940.291 ± 0.146
Positive 0.524 ± 0.206** 0.478 ± 0.185* 0.472 ± 0.292*
X I0.382 ± 0.1380.251 ± 0.0710.265 ± 0.172
X II0.348 ± 0.1440.324 ± 0.1130.277 ± 0.131
X III0.245 ± 0.1670.236 ± 0.1290.251 ± 0.142
Y I0.331 ± 0.0980.340 ± 0.1330.291 ± 0.081
Y II0.357 ± 0.1300.290 ± 0.1450.327 ± 0.157
Y III0.230 ± 0.1210.307 ± 0.0820.363 ± 0.100
Group180 min240 min300 min360 min
Control0.186 ± 0.0860.117 ± 0.0690.105 ± 0.06 0.104 ± 0.08 
Positive0.214 ± 0.1510.110 ± 0.0450.126 ± 0.0560.112 ± 0.065
X I0.188 ± 0.0970.175 ± 0.088 0.177 ± 0.102*0.133 ± 0.092
X II0.258 ± 0.1430.150 ± 0.077 0.167 ± 0.097*0.130 ± 0.094
X III0.226 ± 0.107 0.233 ± 0.132**0.120 ± 0.0590.125 ± 0.048
Y I0.273 ± 0.156 0.215 ± 0.095**0.166 ± 0.1510.116 ± 0.068
Y II0.181 ± 0.088 0.181 ± 0.089*0.151 ± 0.1040.101 ± 0.042
Y III0.193 ± 0.09  0.217 ± 0.092**0.112 ± 0.0560.117 ± 0.043
Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantity of urine after 5 days of administration of X and Y.

After 7 days of administration of X and Y, the quantity of urine was measured at 30, 60, 120, 180, 240, 300, and 360 minutes. The quantity of urine discharged at 30 minute by the mice receiving 200, 400 mg/kg of X and 250, 500 mg/kg of Y decreased significantly (P<0.05) but increased at 240 minutes compared with the mice receiving normal saline. See Table 15.3.

TABLE 15.3
Effects of Extracts X and Y on the Quantity of Urine in Mice after 7 Days of
Administration
Group30 min60 min120 min
Control0.252 ± 0.1420.347 ± 0.1590.430 ± 0.192
Positive 0.434 ± 0.230* 0.606 ± 0.214**0.590 ± 0.333
X I0.301 ± 0.1520.314 ± 0.1490.342 ± 0.186
X II0.291 ± 0.1610.332 ± 0.135 0.285 ± 0.173*
X III0.212 ± 0.1130.260 ± 0.103 0.309 ± 0.117*
Y I0.254 ± 0.1750.283 ± 0.1370.313 ± 0.178
Y II0.261 ± 0.1890.292 ± 0.129 0.300 ± 0.128*
Y III0.246 ± 0.1700.268 ± 0.240 0.281 ± 0.146*
Group180 min240 min300 min360 min
Control0.285 ± 0.1360.155 ± 0.1190.122 ± 0.0710.111 ± 0.061
Positive0.314 ± 0.119 0.279 ± 0.192*0.163 ± 0.0870.148 ± 0.071
X I0.267 ± 0.1790.200 ± 0.1140.176 ± 0.1470.157 ± 0.077
X II0.250 ± 0.1160.203 ± 0.134 0.180 ± 0.079*0.129 ± 0.085
X III0.293 ± 0.142 0.250 ± 0.116* 0.194 ± 0.104*0.151 ± 0.076
Y I0.310 ± 0.1680.248 ± 0.1780.155 ± 0.1080.113 ± 0.05 
Y II0.334 ± 0.2080.259 ± 0.205 0.205 ± 0.109* 0.188 ± 0.113*
Y III0.267 ± 0.1330.212 ± 0.125 0.205 ± 0.119* 0.169 ± 0.073*
Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantity of urine after 7 days of administration of X and Y.

After 10 days of administration of X and Y, the quantity of urine discharged at 120 minutes by the mice receiving 200, 400 mg/kg of X and 250, 500 mg/kg of Y decreased significantly (P<0.05) compared with the mice receiving normal saline. See FIG. 59.

TABLE 15.4
Effects of Extracts X and Y on the Quantity of Urine in Mice after 10 Days of
Administration
Group30 min60 min120 min
Control0.292 ± 0.1840.323 ± 0.1580.418 ± 0.221 
Positive0.374 ± 0.159 0.432 ± 0.163* 0.643 ± 0.181**
X I0.306 ± 0.1240.317 ± 0.0880.339 ± 0.145 
X II0.292 ± 0.0820.343 ± 0.1200.279 ± 0.118*
X III0.266 ± 0.1160.348 ± 0.1610.274 ± 0.111*
Y I0.273 ± 0.1170.331 ± 0.1030.406 ± 0.175 
Y II0.289 ± 0.1260.344 ± 0.1470.254 ± 0.102*
Y III0.227 ± 0.1290.322 ± 0.1620.255 ± 0.124*
Group180 min240 min300 min360 min
Control0.203 ± 0.0870.144 ± 0.0980.108 ± 0.0740.091 ± 0.060
Positive0.253 ± 0.1160.147 ± 0.0670.095 ± 0.0940.068 ± 0.049
X I0.249 ± 0.0940.172 ± 0.0930.120 ± 0.0580.093 ± 0.050
X II0.225 ± 0.0740.163 ± 0.0510.116 ± 0.0520.093 ± 0.051
X III0.247 ± 0.1040.186 ± 0.1020.121 ± 0.0530.098 ± 0.065
Y I0.243 ± 0.1010.171 ± 0.0980.126 ± 0.0860.098 ± 0.058
Y II0.229 ± 0.0970.164 ± 0.0910.124 ± 0.0940.111 ± 0.067
Y III0.213 ± 0.1020.170 ± 0.0810.121 ± 0.0590.095 ± 0.045
Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantity of urine after 10 days of administration of X and Y.

Conclusion.

The results indicated that the extract X and Y can regulate the quantity of urine after 3-10 days of administration of X and Y.

Experiment 15A

Antidiuresis Test of Rat in Metabolism Cage

The antidiuresis tests using Xanthoceras Sorbifolia Extract FS(X) and Xanthoceras Sorbifolia saponin extract FS(Y) were carried out in rats. Note: Extracts FS(X) and FS(Y) similar to Extracts X and Y respectively. FS(Y) and FS(X) are crude saponin extracts and the “FS(Y)” and “FS(X)” are the extract names that were used in the experiments.

SD rats were treated with FS(X) by oral administration, daily at the doses of 100, 200, 400 mg/kg for 25 days. The experiment included a negative control group and positive control. Collecting urine in Standard metabolism cage and observation indicators, including urine volume, urine Na+, K+, Cl− content, pH and osmotic pressure. The results obtained were as follows: (1) Extract FS(X) has dose-dependant antidiuresis effect. There is significant antidiuresis at all the time phases for 200 and 400 mg/kg. However, by urination rate, the 400 mg/kg dosage group has a more significant effect in that it can delay urination output by two hours. In addition, there is no change in the impact on total urine volume output. (2) The extract FS(X) increased the concentration of ions in urine after drug treatment. However, there is no apparent dose dependency. (3) FS (X) and FS (Y) have no significant impact on the pH value and osmotic pressure, but have slight impact on the specific gravity.

Objective.

Standard metabolism cage method was carried out in rats to research the antidiuresis of FS(X) and FS(Y).

Test Drug.

FS(X) is compositions of Xanthoceras Sorbifolia extract. FS(Y) is compositions of Xanthoceras Sorbifolia extract. The test drug is a suspension formulated with 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na).

Test Animals.

Male SD rats, initial weight 150˜200 g, 100 individuals. The test animals are raised in cages (volume: 20×30×45 cm), and each cage has 5 animals. The basal feed is the full-rate pellet feed for experimental rat, self-made by the Experimental Animal Center. The cage bottom padding is wood shaving and chaff, dried before use. After the padding replacement that is made every other day on average, the cages are disinfected before reuse. Laboratory temperature 23±2° C., humidity 40˜70%, with air-conditioning, exhaust and ventilation equipment, natural lighting and a light-shade cycle of about 12 hours.

Animal Screening.

The animals are placed individually in the cage for adaptation once a day for 6˜10 hours per day for 2 days. Before placing them into the case, press the lower abdomen gently to discharge the remaining urine, inject 38° C. distilled water into the stomach at the volume of 25 ml/kg as the water load. Collect the urine in the metabolism cage within 2 hours after stomach injection; collect the remaining urine by pressing the lower abdomen gently immediately before taking them out of the cage. Animals whose urine volume attains above 40% of the injection volume will be qualified ones.

Test Instruments.

Standard metabolism cage, Automatic urine analyzer (Miditron Junior II), Urine osmotic pressure tester, and Urine ion tester (EL-ISE, Beckman).

Methods

Test Groups.

There are 3 groups for the FS (X), i.e., for 100, 200, 400 mg/kg/day, respectively. There is a dosage group for FS (Y): 400 mg/kg/day; once a day, fed at 30 min after administration, for 25 days. Control group were fed with 0.5% CMC-Na into the stomach every day. The positive control drug is pituitrin, injected into the abdominal cavity at the rate of 0.25 u/kg before the animals are placed in the cage. Each group includes 10 animals.

Urine Collection and Observation Indicators.

For the test groups, the animals that have experienced the administration period are on diet for 18 hours before urine collection without prohibiting drinking. After pressing their lower abdomen gently to discharge the remaining urine, apply the water load of 38° C. distilled water at the volume of 50 ml/kg, and place them in the cage for urine collection. Collect the urine volume and times at 0.25 hr, 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr and 6 hr after water feeding, when the animals are taken out of the cage at 6 hr, press their lower abdomen gently to collect the remaining urine in the bladder. Conduct the routine urine examination (pH, erythrocyte, leucocyte, protein, etc); measure urine Na+, K+, Cl− concentrations and urine osmotic pressure for the urine samples.

Data Processing.

Urination speed, Relative urine volume, Urine Na+, K+, Cl− content data are shown in X±Sd, and are compared with the control group and subject to a student t-test.

Results

Impact on Urine Volume.

FS (X) has dose-dependant antidiuresis effects. There is a significant antidiuresis effect at all the time phases using dosages of 200 and 400 mg/kg. See Table 15A-1, FIGS. 63 and 63A. However, by urination discharge rate, the 400 mg/kg group has a more significant effect in reducing the urine output during the first 2 hrs as compared with the control. See Table 15A-2, FIGS. 64 and 64A. The FS (Y) 400 mg/kg group has an equivalent drug efficacy to FX (X) 400 mg/kg during the first 2 hours of experiment. In addition, there is no apparent dose dependency in the impact on total urine volume during the whole experiment. The 400 mg/kg groups of FS(X) can significantly reduce the urine volume during the first 6 hours after the drug-treatment which is more effective than the positive control drug (pituitrin).

Impact on Na+, K+, Cl− ions in urine.

By reducing the urine volume, the test drug has increased the concentration of ions in urine to different extent. However, there is no apparent dose dependency. By ionic, there is almost no impact on Na+ and Cl− ions, there is a certain discharge promotion action on K+ ions. The concentration and discharge amount of all ions in urine from the positive drug have risen apparently.

Impact on Urine pH and Osmotic Pressure.

FS (X) and FS (Y) have no significant impact on the pH value and osmotic pressure, but have slight impact on the specific gravity.

FIGS. 63 and 63A, Table 15A-1, shows results of the urine volume with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days. FIGS. 64 and 64A, Table 15A-2, shows results of discharging urine speed with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days. FIG. 65, Table 15A-3, shows results of urine specific gravity and pH with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days. FIG. 66, Table 15A-4, Concentration of N+, K+ and Cl− in urine with water load after FS(X) Xanthoceras Sorbifolia Extract administration for 25 days.

Although the present invention has been described in detail with particular reference to preferred embodiments thereof, it should be understood that the invention is capable of other different embodiments, and its details are capable of modifications in various obvious aspects. As is readily apparent to those skilled in the art, variations and modifications can be affected while remaining within the spirit and scope of the invention. Accordingly, the foregoing disclosure, description, and figures are for illustrative purpose only, and do not in any way limit the invention which is defined only by the claims.

TABLE 5.2
Chemical Shift Data of 2D NMR chemical shift of HMQC
analysis of compound Y
DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,
EXPNO = 5, PROCNO = 1
F1PLO = 144.360 ppm, F1PHI = 10.797 ppm,
F2PLO = 7.966 ppm, F2PHI = 0.417 ppm
MI = 1.00 cm, MAXI = 10000.00 cm, PC = 1.400
ADDRESSFREQUENCY
row[Hz]F1[PPM]F1
#col[Hz]F2[PPM]F2INTENSITY
114820698.986137.1608
3843574.3775.95572.00
215220564.195136.2676
4013471.3375.78402.67
315720465.209135.6117
2204533.7797.554345.24
422318893.424125.1963
4313295.2615.49066.22
523418649.311123.5787
2584311.8207.1845100.00
631516736.119110.9011
3763620.2896.03227.49
735315834.069104.9237
4932934.5504.88963.98
835515778.398104.5548
4493192.3875.31922.99
935515778.398104.5548
4922936.4144.89271.18
1045113524.78889.6213
6601951.8273.25222.41
1145113524.78889.6213
6631934.4013.22312.60
1247312994.27486.1059
5632520.9874.20051.93
1347912861.93385.2290
5002891.9334.81863.96
1449112583.00883.3807
4872967.0124.94376.95
1552311826.20478.3658
3074025.4646.70733.92
1652311826.20478.3658
3094011.8016.68464.78
1752311826.20478.3658
5452627.4924.37801.99
1852911690.02077.4633
5042866.1644.77574.60
1953211624.01677.0260
5302713.9604.52212.32
2053211624.01677.0260
5322703.5634.50472.51
2153511536.33976.4450
5902363.6783.93843.34
2254511299.47574.8754
5732461.3874.10122.35
2354511299.47574.8754
5762447.1794.07752.10
2455511063.55473.3121
5412653.6934.42166.62
2556710795.11371.5333
5372673.0424.45391.84
2656710795.11371.5333
5392662.6834.43662.82
2756710795.11371.5333
5412650.9334.41701.72
2857910495.72569.5494
5272734.0374.55555.06
2959410156.36367.3006
5632523.6514.20507.18
306229486.03762.8588
6082256.6903.76012.91
316229486.03762.8588
6112241.3363.73462.67
326229486.03762.8588
6352100.1993.49942.96
336229486.03762.8588
6372086.7563.47703.42
346279381.43962.1656
5522586.9674.31053.58
356279381.43962.1656
5552568.3344.27943.42
366279381.43962.1656
5682494.5464.15652.54
376279381.43962.1656
5712474.5594.12322.51
386309297.80961.6115
5312709.7344.51502.61
396309297.80961.6115
5392660.7344.43342.66
406708349.67655.3287
911480.2830.80032.14
416708349.67655.3287
913465.7380.77602.18
427267038.86046.6427
6791839.6593.06532.51
437267038.86046.6427
8191016.6271.69391.42
447267038.86046.6427
848848.5111.41382.49
457646151.76940.7644
6791841.1453.06786.40
467646151.76940.7644
6821821.5923.03521.04
477775836.72738.6768
850837.8691.39612.15
487775836.72738.6768
853818.8611.36442.05
497775836.72738.6768
907500.2560.83351.64
507915512.02236.5251
7751277.3402.12832.38
517915512.02236.5251
7781258.9292.09771.38
527915512.02236.5251
7851218.8922.03091.38
537915512.02236.5251
7881201.8472.00251.09
548374417.03829.2693
881655.9731.093023.74
558484174.67927.6633
864757.2061.261720.53
568484174.67927.6633
872709.0601.18153.17
578563984.14926.4008
7741281.3392.13501.66
588563984.14926.4008
7781259.7512.09901.59
598563984.14926.4008
8041107.0621.84461.02
608563984.14926.4008
8071089.2191.81491.21
618733578.06823.7099
7991138.1371.89641.68
628733578.06823.7099
8141046.1851.74321.26
638913142.83720.8259
7881200.6552.000613.95
648913142.83720.8259
8041107.0461.844628.24
658943086.14720.4502
7881200.2751.99993.08
668943086.14720.4502
8041106.8031.84421.17
678943086.14720.4502
8151041.7581.735827.19
688943086.14720.4502
858789.8041.31601.16
698973015.33719.9810
858790.2921.316833.47
709062802.85418.5730
830953.6521.58901.35
719062802.85418.5730
834931.2081.55161.89
729142613.99517.3215
892592.6630.987512.71
739192490.08216.5004
875693.4451.15548.90
749192490.08216.5004
880660.3431.10031.76
759252342.84015.5247
7781258.3452.09675.45
769252342.84015.5247
7821237.1222.06135.31
779252342.84015.5247
7911183.4101.97182.33
789252342.84015.5247
7951159.7321.93242.38
799252342.84015.5247
907503.2600.83856.27
809252342.84015.5247
912474.6990.79107.15

TABLE 5.3
Chemical Shift Data of 2D NMR chemical shift of HMBC analysis of Y
DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,
EXPNO = 6, PROCNO = 1
F1PLO = 178.339 ppm, F1PHI = 10.721 ppm, F2PLO = 6.881 ppm,
F2PHI = 0.573 ppm
MI = 1.00 cm, MAXI = 10000.00 cm, PC = 1.400
ADDRESSFREQUENCY
row[Hz]F1[PPM]F1
#col[Hz]F2[PPM]F2INTENSITY
112326590.750176.2058
895573.2760.95523.11
214525939.373171.8894
5312710.6864.51662.92
314525939.373171.8894
5392662.0334.43552.54
416625312.006167.7321
3084016.4376.69233.45
516625312.006167.7321
3463793.8476.321414.90
616625312.006167.7321
3483785.3226.307212.47
716625312.006167.7321
3853564.4435.93921.47
816625312.006167.7321
4003477.9485.79504.16
916625312.006167.7321
4033458.5525.76273.96
1016625312.006167.7321
7811243.9832.07281.17
1116625312.006167.7321
7881200.4472.000210.44
1216625312.006167.7321
7931171.9921.95283.35
1316625312.006167.7321
8151041.3361.735133.24
1429021640.068143.3997
6791839.9703.06585.67
1529021640.068143.3997
7881199.6091.99881.03
1629021640.068143.3997
8041107.2221.844933.90
1729021640.068143.3997
8131053.9181.75612.30
1829021640.068143.3997
848848.1551.41321.25
1932220697.354137.1527
7801246.5052.077031.46
2032220697.354137.1527
7881200.0751.999644.60
2132220697.354137.1527
7931170.3771.95013.79
2232220697.354137.1527
8151040.8601.73434.31
2332720566.367136.2847
7801246.6292.07723.06
2432720566.367136.2847
7881201.1922.00155.78
2532720566.367136.2847
7931170.7481.950747.25
2632720566.367136.2847
8151041.0671.734658.19
2736519434.006128.7811
7801245.8612.075916.02
2836519434.006128.7811
7881200.4422.000233.73
2936519434.006128.7811
7931171.2041.951546.70
3036519434.006128.7811
8151040.9401.734480.60
3138418893.113125.1968
6791839.5473.06519.80
3238418893.113125.1968
7881200.7292.00072.34
3338418893.113125.1968
8011124.9421.87441.93
3438418893.113125.1968
8031113.9601.85611.36
3538418893.113125.1968
8091077.5561.79543.32
3638418893.113125.1968
8131051.6941.75243.14
3738418893.113125.1968
848847.4991.41211.45
3845716738.236110.9173
3613707.3316.17726.44
3945716738.236110.9173
3903534.1695.88876.75
4045716738.236110.9173
4862972.8034.95341.24
4145716738.236110.9173
4882962.2234.93571.40
4245716738.236110.9173
5632520.5594.199812.21
4348815822.760104.8508
5312712.0554.51891.39
4448815822.760104.8508
5382668.8874.44704.68
4548815822.760104.8508
5452627.7094.378314.61
4648815822.760104.8508
6601952.4743.25332.34
4748815822.760104.8508
6621941.1013.23433.52
4848815822.760104.8508
6641928.9303.21402.41
4948915777.470104.5507
5382669.1014.447312.69
5048915777.470104.5507
5462622.9414.37046.06
5148915777.470104.5507
5902363.6953.93843.08
5248915777.470104.5507
6601953.4433.25492.02
5348915777.470104.5507
6621940.9133.23401.47
5456613527.92089.6439
4922938.1984.895711.15
5556613527.92089.6439
8051101.8521.83591.93
5656613527.92089.6439
851830.5521.38395.09
5756613527.92089.6439
864757.3741.262053.64
5856613527.92089.6439
874696.1101.159939.08
5956613527.92089.6439
880658.8361.09784.62
6056613527.92089.6439
911480.4320.80051.38
6156613527.92089.6439
913466.5060.77731.40
6258412992.84286.0981
3763621.4876.03425.29
6358412992.84286.0981
5312708.5644.51311.17
6458412992.84286.0981
5392662.6484.43664.18
6558412992.84286.0981
5452625.9474.37546.92
6658412992.84286.0981
864757.0401.26141.36
6758812860.94185.2241
3703655.0446.09012.76
6858812860.94185.2241
3763621.6096.034410.88
6958812860.94185.2241
5542574.2944.28931.21
7058812860.94185.2241
864756.2271.26001.05
7159812585.61483.3996
3763621.2686.03381.92
7259812585.61483.3996
4753038.9015.06351.23
7359812585.61483.3996
5002890.9844.81701.60
7459812585.61483.3996
5052863.2334.77083.39
7559812585.61483.3996
881655.1561.09161.11
7662311822.78478.3447
2954093.8186.82122.30
7762311822.78478.3447
3213940.8036.56632.25
7862311822.78478.3447
3483785.1206.306918.28
7962311822.78478.3447
4343277.0405.46031.13
8062311822.78478.3447
4483194.9185.32347.26
8162311822.78478.3447
5632521.1714.20087.36
8262311822.78478.3447
845867.2711.44511.26
8362311822.78478.3447
849844.6691.407410.09
8462311822.78478.3447
858790.4481.317126.63
8562311822.78478.3447
881656.4341.093851.16
8663011619.67476.9987
3483785.0856.30681.21
8763011619.67476.9987
5392661.9364.43546.49
8863011619.67476.9987
5522584.6074.30651.49
8963011619.67476.9987
858790.9781.31792.74
9063011619.67476.9987
881655.9771.09305.21
9164111304.49974.9102
3074021.3286.70041.03
9264111304.49974.9102
5262736.0134.55884.14
9364111304.49974.9102
5382670.6084.44989.80
9464111304.49974.9102
881656.5371.09391.61
9565011028.92573.0841
3084015.9486.691516.79
9665011028.92573.0841
3463793.4596.320713.94
9765011028.92573.0841
5262736.7614.56017.46
9865011028.92573.0841
5742454.4104.08964.70
9965011028.92573.0841
6102245.8563.742111.08
10065011028.92573.0841
6372088.8953.48065.03
10165011028.92573.0841
6791839.5103.06508.83
10265011028.92573.0841
848850.2261.41671.52
10365011028.92573.0841
881656.9861.09471.80
10465810790.32971.5030
3084016.4776.69231.26
10565810790.32971.5030
5312708.8404.51352.58
10665810790.32971.5030
5642518.2034.19595.49
10765810790.32971.5030
6092253.8413.75541.07
10865810790.32971.5030
881655.6771.09251.26
10966810496.01569.5527
5302715.3704.52441.73
11066810496.01569.5527
5332699.7604.49841.85
11166810496.01569.5527
5402658.7084.43003.19
11266810496.01569.5527
5902363.3923.93794.52
11366810496.01569.5527
8041107.6021.84551.00
11466810496.01569.5527
881656.5081.09391.01
11568010155.36467.2954
5412651.0544.41722.15
11668010155.36467.2954
8041107.2281.844932.23
11768010155.36467.2954
881656.5451.09391.00
1187039486.47062.8629
3463793.1696.32039.40
1197039486.47062.8629
3483784.8966.30659.63
1207039486.47062.8629
6791839.3463.06483.08
1217039486.47062.8629
863758.2651.26341.07
1227069376.97662.1373
3473790.7486.31621.61
1237069376.97662.1373
5052862.4564.76952.79
1247069376.97662.1373
881655.3991.09201.01
1257099296.04261.6010
5902363.6843.93847.53
1267418346.30755.3075
7771264.8022.10743.77
1277418346.30755.3075
852825.9081.37613.30
1287418346.30755.3075
864757.4011.262035.15
1297418346.30755.3075
874696.3411.160325.71
1307418346.30755.3075
881658.1991.09673.92
1317418346.30755.3075
891595.6470.99251.15
1327418346.30755.3075
909489.5180.815623.33
1337817163.19147.4675
3463793.1816.32031.69
1347817163.19147.4675
4313294.4775.48935.30
1357817163.19147.4675
5392661.4764.43463.10
1367817163.19147.4675
5412647.9664.41213.42
1377817163.19147.4675
5642517.9844.19551.14
1387817163.19147.4675
6791839.4423.06493.64
1397817163.19147.4675
8041107.1641.844837.94
1407817163.19147.4675
8221002.0931.66971.62
1417817163.19147.4675
849845.0581.40812.98
1427817163.19147.4675
858790.2031.31674.12
1437817163.19147.4675
874695.4411.15881.07
1447817163.19147.4675
881656.2561.09356.01
1457817163.19147.4675
891594.6950.990939.60
1467817163.19147.4675
909489.6130.81583.90
1477867041.05146.6581
4323290.7625.48312.39
1487867041.05146.6581
6691898.1913.16281.21
1497867041.05146.6581
6771850.6533.08362.37
1507867041.05146.6581
6801835.9483.05912.84
1517867041.05146.6581
6821821.5183.03512.66
1527867041.05146.6581
6891782.4322.96992.02
1537867041.05146.6581
7761274.2142.12312.05
1547867041.05146.6581
8001129.5761.88211.51
1557867041.05146.6581
8041106.9391.84446.33
1567867041.05146.6581
8081082.7871.80423.27
1577867041.05146.6581
8111065.1781.77483.51
1587867041.05146.6581
829959.5081.59883.10
1597867041.05146.6581
838906.8371.51101.42
1607867041.05146.6581
848847.2801.41181.15
1617867041.05146.6581
858790.5301.317249.82
1627867041.05146.6581
874696.7701.16101.14
1637867041.05146.6581
881656.4431.093870.13
1647867041.05146.6581
891594.5320.990618.99
1657867041.05146.6581
909489.3630.815421.10
1668146218.37741.2066
4313293.8855.48831.45
1678146218.37741.2066
5612531.5744.21823.03
1688146218.37741.2066
5642517.8674.19533.26
1698146218.37741.2066
6352096.9443.49402.64
1708146218.37741.2066
6781846.0793.07602.51
1718146218.37741.2066
7761274.4842.12361.75
1728146218.37741.2066
7831228.1442.04641.41
1738146218.37741.2066
7861211.7262.01901.51
1748146218.37741.2066
7991136.1421.89311.43
1758146218.37741.2066
8041107.2041.844867.13
1768146218.37741.2066
8191018.3011.69674.37
1778146218.37741.2066
8221001.4891.66874.96
1788146218.37741.2066
833939.6951.56573.66
1798146218.37741.2066
864756.2711.26012.02
1808146218.37741.2066
874695.9631.15962.98
1818146218.37741.2066
880659.0721.09821.66
1828146218.37741.2066
891594.2870.990249.75
1838146218.37741.2066
895572.7240.95432.16
1848146218.37741.2066
909489.8110.81611.62
1858235950.42139.4310
6621940.8053.23381.07
1868235950.42139.4310
7761271.0112.11781.62
1878235950.42139.4310
8041107.5871.84552.29
1888235950.42139.4310
831947.7261.57911.04
1898235950.42139.4310
858790.5201.31721.45
1908235950.42139.4310
864757.0901.261554.14
1918235950.42139.4310
874696.0481.159872.55
1928235950.42139.4310
880658.5031.09729.62
1938235950.42139.4310
891594.5300.99062.75
1948235950.42139.4310
911479.4320.79887.32
1958235950.42139.4310
913465.7760.77617.03
1968275834.79738.6648
8041106.6871.84402.69
1978275834.79738.6648
8191017.8031.69591.66
1988275834.79738.6648
8221001.7081.66911.68
1998275834.79738.6648
841889.9851.48291.01
2008275834.79738.6648
858789.6211.31571.33
2018275834.79738.6648
864758.1641.26333.94
2028275834.79738.6648
874696.1371.15996.09
2038275834.79738.6648
881656.9051.09452.43
2048275834.79738.6648
892593.6060.98911.90
2058275834.79738.6648
909489.2450.815251.13
2068305743.32938.0586
8041108.0311.84621.40
2078305743.32938.0586
858790.5511.31721.95
2088305743.32938.0586
864757.0911.26152.85
2098305743.32938.0586
874696.0521.15983.54
2108305743.32938.0586
881655.6201.09242.41
2118305743.32938.0586
891594.7620.99103.06
2128305743.32938.0586
909489.4740.815610.76
2138345623.89637.2672
8041107.4331.84521.25
2148345623.89637.2672
8221002.0391.66961.43
2158345623.89637.2672
833939.6201.56561.01
2168345623.89637.2672
858790.6031.31734.03
2178345623.89637.2672
864756.7681.26091.89
2188345623.89637.2672
874696.4441.16042.15
2198345623.89637.2672
881656.0111.09314.39
2208345623.89637.2672
891594.7760.99104.19
2218345623.89637.2672
909489.2720.815210.53
2228375533.83536.6704
7771268.6812.11391.72
2238375533.83536.6704
8041108.2821.84661.01
2248375533.83536.6704
8111067.1201.77811.70
2258375533.83536.6704
8191017.4071.69524.37
2268375533.83536.6704
8221001.7301.66914.67
2278375533.83536.6704
832943.7621.57252.78
2288375533.83536.6704
849841.9341.40282.09
2298375533.83536.6704
858790.6301.31748.57
2308375533.83536.6704
864757.9631.26292.10
2318375533.83536.6704
874695.2451.15841.83
2328375533.83536.6704
881656.3511.093611.03
2338375533.83536.6704
891594.6920.990925.74
2348375533.83536.6704
909489.1390.815053.53
2358375533.83536.6704
913467.8930.77966.55
2368405450.41936.1177
3074023.4486.70405.35
2378405450.41936.1177
3094013.8666.68805.05
2388405450.41936.1177
3463794.5466.32261.25
2398405450.41936.1177
6791839.2423.06463.88
2408405450.41936.1177
849842.8301.40436.34
2418405450.41936.1177
858790.2371.316788.69
2428405450.41936.1177
862767.1251.27821.79
2438405450.41936.1177
864755.0411.25811.99
2448405450.41936.1177
874695.4411.15882.24
2458405450.41936.1177
881656.0601.093184.89
2468405450.41936.1177
891594.6750.99097.64
2478405450.41936.1177
909489.3000.81538.88
2488754418.23829.2778
3074022.7786.70288.21
2498754418.23829.2778
3094014.3856.68897.83
2508754418.23829.2778
6791841.5763.06851.85
2518754418.23829.2778
849844.6691.40741.65
2528754418.23829.2778
858790.4921.3171100.00
2538754418.23829.2778
863763.8111.27273.72
2548754418.23829.2778
870718.8851.197822.51
2558754418.23829.2778
875691.9031.15294.90
2568754418.23829.2778
878670.1471.11661.92
2578754418.23829.2778
882648.4701.08051.77
2588754418.23829.2778
892593.3010.988619.66
2598834176.08227.6732
6621941.3003.23461.71
2608834176.08227.6732
853819.3361.365215.64
2618834176.08227.6732
857794.8011.32434.59
2628834176.08227.6732
863763.3741.27201.70
2638834176.08227.6732
874696.0581.159889.76
2648834176.08227.6732
878671.8611.11952.96
2658834176.08227.6732
881657.1981.09507.96
2668834176.08227.6732
911479.4870.79892.54
2678834176.08227.6732
913466.6450.77752.27
2689033588.77523.7813
4313296.1175.49212.85
2699033588.77523.7813
7911182.7561.97071.05
2709033588.77523.7813
8011127.8861.87931.45
2719033588.77523.7813
8041108.5401.84711.43
2729033588.77523.7813
8111064.1281.77311.37
2739033588.77523.7813
8211006.1001.67643.82
2749033588.77523.7813
874696.6491.16082.42
2759033588.77523.7813
881655.7571.09262.43
2769173174.63221.0370
3074021.4196.70061.44
2779173174.63221.0370
3843571.4355.95082.36
2789173174.63221.0370
4033463.3315.77071.11
2799173174.63221.0370
5612531.8504.21865.21
2809173174.63221.0370
5642517.7004.19505.22
2819173174.63221.0370
7781262.7482.10403.52
2829173174.63221.0370
7931170.0551.949622.51
2839173174.63221.0370
8001133.2751.88832.77
2849173174.63221.0370
8051100.4191.83353.08
2859173174.63221.0370
8091079.4271.79862.91
2869173174.63221.0370
8151043.1181.738124.13
2879173174.63221.0370
827975.0201.62462.05
2889173174.63221.0370
848851.2421.41841.60
2899173174.63221.0370
864757.2671.26181.67
2909173174.63221.0370
869727.3061.21191.80
2919173174.63221.0370
881656.4071.09376.11
2929223013.42719.9687
3094014.7436.689513.55
2939223013.42719.9687
4013472.3745.78572.11
2949223013.42719.9687
6791841.8753.06907.26
2959223013.42719.9687
7771263.9522.10601.96
2969223013.42719.9687
7941165.7251.94241.57
2979223013.42719.9687
7991137.9401.89612.13
2989223013.42719.9687
8041104.4561.84033.32
2999223013.42719.9687
8151043.0701.73801.92
3009223013.42719.9687
826975.6881.62573.29
3019223013.42719.9687
847852.6761.420719.79
3029223013.42719.9687
863759.5531.26563.39
3039223013.42719.9687
869726.6471.210819.23
3049223013.42719.9687
881656.1581.093372.09
3059292803.19018.5756
3084021.1886.70021.12
3069292803.19018.5756
823997.1551.66151.03
3079292803.19018.5756
847852.4091.42032.46
3089292803.19018.5756
864756.4171.26042.78
3099292803.19018.5756
869728.7271.21422.15
3109292803.19018.5756
881657.3451.09535.08
3119292803.19018.5756
902531.5830.88571.25
3129292803.19018.5756
911478.0220.79654.88
3139292803.19018.5756
913466.4940.77735.42
3149362611.79317.3073
7771266.0052.10941.92
3159362611.79317.3073
7831228.4912.04692.67
3169362611.79317.3073
7861211.7852.01912.98
3179362611.79317.3073
8191018.3011.69675.90
3189362611.79317.3073
8221001.8601.66937.01
3199362611.79317.3073
864757.4181.26206.44
3209362611.79317.3073
869725.7531.20931.48
3219362611.79317.3073
872707.2641.17851.56
3229362611.79317.3073
881654.7821.091017.83
3239362611.79317.3073
902531.7930.886119.86
3249362611.79317.3073
909490.1940.81682.94
3259362611.79317.3073
920427.6490.71261.52
3269402489.58616.4974
6621941.8193.23552.46
3279402489.58616.4974
6641929.1723.21441.76
3289402489.58616.4974
8221001.0101.66791.20
3299402489.58616.4974
864757.0211.261466.00
3309402489.58616.4974
871712.6221.187410.06
3319402489.58616.4974
880660.6181.10072.75
3329402489.58616.4974
885632.3871.053718.76
3339402489.58616.4974
891594.2440.99013.19
3349402489.58616.4974
899548.1420.91331.83
3359402489.58616.4974
911478.6150.79758.03
3369402489.58616.4974
913466.4290.777210.10
3379402489.58616.4974
920426.4620.71061.57
3389452348.37415.5617
3833576.6125.95942.25
3399452348.37415.5617
4023464.8395.77321.77
3409452348.37415.5617
7691311.1562.18479.92
3419452348.37415.5617
7831233.2082.05484.90
3429452348.37415.5617
7911184.0391.97298.41
3439452348.37415.5617
8041106.4291.84365.06
3449452348.37415.5617
8191018.3651.69683.91
3459452348.37415.5617
8221001.8341.66935.15
3469452348.37415.5617
864756.7411.26096.60
3479452348.37415.5617
872710.4171.18371.20
3489452348.37415.5617
881655.0391.09142.79
3499452348.37415.5617
885632.4861.05392.24
3509452348.37415.5617
899550.1470.916719.61
3519452348.37415.5617
906510.5180.85063.78
3529452348.37415.5617
911480.3010.80038.74
3539452348.37415.5617
914464.3620.77378.49
3549452348.37415.5617
920426.5380.710718.76

TABLE 5.5
Chemical Shift of the Proton NMR of compound Y of Xanthoceras Sorbifolia Extract
#ADDRESS
[Hz][PPM]FREQUENCYINTENSITY
15031.95309.5248.84680.46
25430.05222.2648.7015134.66
35840.95132.1738.55130.48
48204.94613.9017.68780.26
58568.14534.2637.555188.28
68943.74451.9197.41790.33
79209.74393.6027.32070.80
89575.24313.4887.1872180.00
99952.14230.8467.04950.87
1010277.24159.5856.93081.02
1110886.84025.9226.70815.68
1210933.64015.6776.69106.19
1311939.83795.0846.32356.26
1411986.73784.8036.30635.85
1512576.03655.5876.09101.47
1612728.03622.2806.03556.92
1712880.53588.8445.97981.87
1812914.23581.4505.96754.48
1912946.53574.3665.95574.59
2012979.53567.1305.94362.00
2113382.63478.7545.79642.41
2213415.63471.5395.78445.03
2313447.33464.5685.77275.15
2413479.13457.5985.76112.66
2514218.83295.4325.490914.00
2614655.93199.6035.331228.97
2714691.23191.8755.318427.63
2815715.32967.3594.94435.64
2915826.32943.0284.90374.82
3015860.62935.5044.89125.15
3116047.02894.6324.82314.20
3216149.62872.1314.78563.79
3316171.92867.2424.77754.42
3416774.02735.2494.55755.92
3516856.42717.1874.52744.26
3616883.52711.2354.51754.56
3716903.52706.8544.51025.89
3816930.62700.9144.50034.68
3917021.22681.0464.46722.99
4017056.92673.2284.45428.41
4117099.12663.9744.43888.87
4217135.62655.9764.42548.11
4317200.42641.7714.40181.88
4417232.62634.7064.39002.90
4517271.02626.2884.37604.33
4617308.82618.0014.36222.85
4717401.82597.6074.32820.83
4817459.12585.043 4.30733.41
4917512.62573.3284.28774.04
5017596.42554.9474.25710.89
5117628.22547.9684.24550.80
5217666.82539.5184.23141.30
5317723.02527.1844.21096.71
5417745.92522.1724.20257.98
5517791.22512.2484.18602.28
5617874.12494.0714.15572.90
5717895.02489.4774.14803.09
5817927.12482.4544.13632.64
5917948.82477.6834.12842.51
6018034.12458.9934.09723.27
6118064.62452.3134.08612.96
6218076.52449.7024.08172.96
6318118.32440.538 4.06650.72
64 18156.42432.1804.05260.89
6518196.32423.4324.03800.44
6618432.12371.735 3.95182.34
6718459.72365.6843.94184.30
6818487.42359.6003.93162.24
6918962.42255.4663.75813.86
7019011.02244.8163.74044.38
7119669.32100.4853.49994.39
7219717.62089.8983.48224.16
7320365.21947.9203.24572.42
7420401.11940.0543.23262.35
7520418.91936.1613.22612.38
7620751.31863.2903.10470.70
7720815.81849.1523.08113.20
7820857.91839.9193.065711.24
7920924.81825.2363.04121.13
8023431.31275.7302.12573.95
8123491.31262.5732.10375.36
8223538.71252.1872.086415.56
8323570.51245.2032.074815.89
8423696.11217.6732.02892.98
8523770.11201.4632.001927.17
8623827.61188.8491.98091.36
8723887.41175.7341.959015.22
8823919.51168.7001.947315.28
8924010.21148.8111.91420.49
9024104.21128.2071.87981.77
9124197.71107.7161.845728.22
9224263.81093.2281.82163.27
9324321.81080.5031.80042.93
9424373.51069.1661.78152.83
9524497.01042.0941.736426.10
9624598.41019.8711.69933.17
9724626.11013.7931.68923.14
9824676.11002.8261.67091.84
9924931.2946.9001.5777 2.14
10024983.0935.5331.55883.02
10125226.8882.1031.46980.25
10225370.4850.6081.41734.11
10325412.0841.4921.40214.88
10425499.7822.2701.37014.07
10525556.8809.7461.3492 2.62
10625639.9791.5271.318931.95
10725717.9774.4181.29042.84
10825790.4758.5391.263922.85
10926011.7710.0181.18304.66
11026070.7697.0821.1615 24.95
11126249.2657.9531.096331.39
11226536.4594.9810.9914 25.26
11326610.9578.6570.96420.97
11426914.0512.1960.85342.04
11527012.2490.6760.817625.88
11627118.1467.4630.7789 3.69
11727226.4443.7150.73931.07
11828513.4161.5540.26920.89
11928539.8155.7770.25961.54

TABLE 6.2
HMQC of Y1 peaklist
ADDRESSFREQUENCY
row[Hz]F1[PPM]F1
##col[Hz]F2[PPM]F2INTENSITY
1131516740.193 110.9281
3753624.4456.039130.72
235215855.943 105.0686
4902950.4204.916120.34
345013537.57589.7061
4902950.4204.916120.34
4479 12863.84485.2416
476 3032.063 5.052112.42
5479 12863.84485.2416
500 2892.3604.819317.76
6491 12577.80683.3462
487 2969.0874.947225.72
7522 11857.07078.5703
545 2627.024 4.3772 13.55
8529 11685.55377.4337
504 2870.087 4.782216.93
9535 11537.10476.4500
589 2370.150 3.949215.06
10550 11191.63374.1608
421 3355.3205.590712.94
11550 11191.63374.1608
423 3342.8075.5699 15.14
12569 10733.31671.1238
410 3421.2955.7006 24.93
13579 10497.79369.5631
526 2740.4804.566226.50
14586 10332.95168.4708
537 2674.0564.4556 14.53
15586 10332.95168.4708
596 2330.159 3.882611.00
16617 9621.36463.7555
643 2051.8863.418912.23
17630 9299.90861.6254
529 2718.381 4.529413.25
18630 9299.90861.6254
5392665.3654.441114.42
19669 8391.97955.6090
529 2718.381 4.529413.25
20669 8391.97955.6090
539 2665.3654.441114.42
21720 7193.97247.6705
682 1821.9913.035810.83
227257055.42746.7524
820 1015.280 1.6917 8.84
237705994.78239.7241
682 1822.2413.036321.78
247785810.32838.5019
682 1822.2413.036321.78
258035229.78434.6549
836 918.974 1.53129.20
268134978.48332.9897
866 743.550 1.23899.04
278334517.22529.9332
847852.8601.421164.24
288474185.85027.7373
861 773.417 1.288774.68
298504113.89127.2605
806 1096.5821.8271 80.21
308504113.89127.2605
861 773.3921.2886 31.46
318853291.88421.8135
756 1389.0882.3145100.00
328933110.64120.6125
796 1153.0931.9213 89.09
338933110.64120.6125
8031110.425 1.8502 50.66
348972998.50519.8694
803 1111.1011.851345.64
358972998.50519.8694
853 820.1951.366678.07
369192497.78316.5514
874 699.2171.1651 72.04
379192497.78316.5514
910 483.491 0.805637.18
389262336.72515.4842
794 1165.0591.9412 17.70
39926 2336.72515.4842
910484.392 0.807131.53

TABLE 6.2A
2D NMR (HMQC) data of Y1
Assignmentw1w2Data Height
 1C-H110.9046.03824704052
 2C-H 105.0234.91415964054
 3C-H 104.6155.33911824459
 4C-H89.8233.243 7951700
 5C-H89.7123.222 7911944
 6C-H85.5285.038 10494958
 7C-H85.4175.054 8839478
 8C-H85.3384.831 10297378
 9C-H85.248 4.817 12857784
10C-H83.397 4.94819288902
11C-H78.6554.3817900396
12C-H78.602 4.3668972991
13C-H 77.4204.78413438428
14C-H 77.0074.4977206874
15C-H76.5703.95112216028
16C-H74.9944.09211486882
17C-H74.7904.10612904558
18C-H74.2205.59310130728
19C-H74.0625.5749532875
20C-H73.8566.17311098625
21C-H73.8026.15610393206
22C-H73.350 4.46811007188
23C-H 73.2774.4467281630
24C-H71.5774.4538645994
25C-H71.2195.70124595648
26C-H70.6113.6159076031
27C-H70.0674.2569261139
28C-H69.6164.56718994736
29C-H68.7713.8958451744
30C-H68.5434.45512573076
31C-H63.6793.6048239119
32C-H63.7813.4157458621
33C-H 63.862 3.3937841054
34C-H62.1924.31210795595
35C-H 62.1724.2878408334
36C-H62.2664.1627944312
37C-H62.0714.13110031945
38C-H61.8114.52912476046
39C-H61.7934.44011880108
40C-H61.7054.4638055856
41C-H47.6633.0347772454
42C-H46.7401.6938583785
43C-H39.7953.03418038864
44C-H38.4531.3377912356
45C-H34.666 1.5677317314
46C-H32.884 1.2647887187
47C-H29.9541.42158253544
48C-H29.6321.24216326471
49C-H29.3181.1179248448
50C-H27.7231.29247380240
51C-H 27.2511.83159260700
52C-H21.8352.31768135008
53C-H20.604 1.91959543524
54C-H20.3771.85266987324
55C-H19.8501.36968067232
56C-H16.5471.16754857956
57C-H16.2230.80725159514
58C-H15.8902.02814256620
59C-H15.6741.97812750140
60C-H15.6931.94010562185
61C-H15.3780.75816911768
62C-H15.1490.79912756197
63C-H138.6705.9858925989
64C-H137.8375.8816871625
65C-H123.4205.36515877691

TABLE 6.3
2D NMR (HMBC) data of Y1 peaklist
ADDRESSFREQUENCY
row[Hz]F1[PPM]F1
#col[Hz]F2[PPM]F2INTENSITY
114825871.928171.4391
3623702.4906.169211.03
214825871.928171.4391
7561390.0512.316193.44
316925232.139167.1995
7961153.4591.921928.04
416925232.139167.1995
8031112.0881.853029.81
529321555.236142.8347
8061097.3511.828425.74
631420935.686138.7293
7861215.6272.025533.12
731420935.686138.7293
7961153.008 1.921246.88
831820813.068137.9168
7861213.9512.02279.66
931820813.068 137.9168
7921175.438 1.958529.47
1031820813.068 137.9168
8031110.584 1.850546.19
1136919321.684128.0342
7851218.1002.029613.34
1236919321.684128.0342
7921175.050 1.957928.29
1336919321.684128.0342
7961153.691 1.922326.99
1436919321.684128.0342
8031111.953 1.852865.83
1548615874.223105.1898
4773026.433 5.042716.48
1648615874.223105.1898
5452628.1844.37919.96
1748615874.223105.1898
5572559.578 4.264810.46
1856513533.44789.6787
4912942.427 4.902710.78
1956513533.44789.6787
861774.469 1.290441.26
2056513533.44789.6787
874696.834 1.161136.06
2158712903.25685.5028
3623702.844 6.169820.78
2258712903.25685.5028
3753625.743 6.041312.97
2358712903.25685.5028
847852.970 1.421235.15
2458712903.25685.5028
853821.094 1.368130.07
2562211874.16478.6836
4473202.899 5.33679.13
2662211874.16478.6836
5632519.490 4.19809.75
2764711136.59473.7961
4763029.853 5.048419.44
2864711136.59473.7961
6252159.425 3.59819.88
2966010736.22971.1431
873702.447 1.170430.15
3066810508.08369.6313
873702.134 1.169911.82
3167310374.06068.7432
873702.5391.170641.29
327099310.579 61.6961
5882374.061 3.95579.48
337408380.91855.5357
861775.074 1.291442.71
347408380.918 55.5357
874696.643 1.160830.33
357408380.918 55.5357
913469.0720.781621.32
367817183.86447.6035
847853.277 1.421743.72
377817183.86447.6035
853821.6881.369135.29
387867044.03446.6769
848851.746 1.41929.09
397867044.03446.6769
880661.983 1.10308.36
407867044.034 46.6769
913469.396 0.782127.00
418136236.90141.3285
8061097.285 1.828338.52
428136236.90141.3285
910485.0260.808243.99
438225962.60739.5109
8061097.6241.828932.36
448225962.60739.5109
861774.567 1.290641.54
458225962.60739.5109
874696.338 1.160363.83
468225962.60739.5109
910484.561 0.807432.10
478285792.66638.3848
853821.024 1.36809.58
488285792.66638.3848
913469.203 0.781839.07
498325676.14537.6127
847852.6431.420742.80
508325676.14537.6127
853821.900 1.369553.76
518325676.14537.6127
912471.1570.785110.57
528375531.66136.6553
853822.1751.36998.78
538375531.66136.6553
913468.5200.780745.56
548475226.12434.6307
8051099.0401.831227.35
558564961.12632.8747
910484.6320.807532.47
568714515.25029.9201
4773025.946 5.04197.66
578714515.25029.9201
837913.407 1.52198.91
588714515.25029.9201
853821.483 1.368852.28
598834184.06727.7255
7951160.8191.93428.46
608834184.06727.7255
850836.9921.394611.68
618834184.06727.7255
874696.591 1.160777.80
629133294.60421.8315
7451453.3622.421616.90
639133294.60421.8315
7671324.1462.206315.41
649232998.09919.8668
4773025.4085.041012.57
659232998.09919.8668
6831819.2403.03139.99
669232998.09919.8668
842883.1901.471618.02
679232998.09919.8668
847853.1561.421551.49
689232998.09919.8668
864757.915 1.262916.19
699232998.09919.8668
879666.3281.110310.30
709402480.24916.4352
861775.278 1.291842.66
719402480.24916.4352
884636.584 1.060714.60
729402480.24916.4352
900544.7360.907712.91
739402480.24916.4352
921420.806 0.701212.28
749462313.56815.3307
902531.2010.885114.80
759462313.56815.3307
923407.732 0.679412.79

TABLE 6.4
2D NMR (COSY) data of Y1.
Assignmentw1w2Data Height
 1H-H6.7046.3229224233
 2H-H6.6786.3729898637
 3H-H6.4194.45620015776
 4H-H6.3686.68111333652
 5H-H6.3206.70611307411
 6H-H6.1715.044134583264
 7H-H6.0434.95212934876
 8H-H5.9802.03080531096
 9H-H5.9802.021105609248
10H-H5.8811.96381350000
11H-H5.8811.95275881512
12H-H5.7015.700215204304
13H-H5.7005.59018340750
14H-H5.6995.57515251625
15H-H5.5825.70124882492
16H-H5.5834.93032200604
17H-H5.5834.26954166860
18H-H5.5824.260100437000
19H-H5.5824.25150904084
20H-H5.3354.47854908116
21H-H5.3354.46248000284
22H-H5.3344.10020768318
23H-H5.0486.17489161792
24H-H4.9494.79211766272
25H-H4.9504.7829033592
26H-H4.9305.58317147768
27H-H4.9294.26930517720
28H-H4.9274.25128975240
29H-H4.9014.39027806448
30H-H4.9004.37334077108
31H-H4.8314.79099849200
32H-H4.8204.3107275091
33H-H4.8194.2967797132
34H-H4.8224.15713857122
35H-H4.8214.13818211468
36H-H4.7944.95330630578
37H-H4.7874.82786722272
38H-H4.5684.10917535982
39H-H4.5674.09315653847
40H-H4.5424.46632239438
41H-H4.5224.453145233152
42H-H4.5174.2057769614
43H-H4.5233.95273572400
44H-H4.5074.43611835048
45H-H4.4685.33779724560
46H-H4.4684.09983781344
47H-H4.4613.95444029948
48H-H4.4546.5289453154
49H-H4.4546.41879266688
50H-H4.4504.38113566321
51H-H4.4494.3218581714
52H-H4.4444.21429697092
53H-H4.4454.20523505830
54H-H4.4434.19523169768
55H-H4.3824.90275968808
56H-H4.3804.21434194940
57H-H4.3794.20434462264
58H-H4.3804.19534413552
59H-H4.3024.82316076232
60H-H4.3024.148221634448
61H-H4.2625.58280620088
62H-H4.2614.93059383108
63H-H4.2034.90012911051
64H-H4.2044.43320195584
65H-H4.1504.83217371252
66H-H4.1534.81918323128
67H-H4.1494.305216562864
68H-H4.0995.33725030224
69H-H4.1004.57136121208
70H-H4.1014.47268659520
71H-H4.0954.46036864516
72H-H4.0284.4687964866
73H-H4.0284.4588388422
74H-H3.9614.46341897776
75H-H3.9534.53934935532
76H-H3.9524.52350083884
77H-H3.9534.44724637258
78H-H3.8931.16259572844
79H-H3.8871.17595668312
80H-H3.7663.51611119055
81H-H3.7443.4946884830
82H-H3.5983.413146852352
83H-H3.5983.404128194976
84H-H3.5173.76710865892
85H-H3.5083.7599535602
86H-H3.4803.7418054603
87H-H3.4753.7346268863
88H-H3.4093.599107289744
89H-H3.4063.592110786000
90H-H3.2292.10912003553
91H-H2.1101.8388593652
92 92H-H2.1071.82211474128
93H-H2.1071.81110279236
94H-H2.1051.7948116300
95H-H2.0295.99247626580
96H-H2.0275.98447768532
97H-H2.0265.97251874608
98H-H2.0302.1187536440
99H-H2.0271.91989540200
100H-H 2.0245.96441580316
101H-H 1.9595.89149355596
102H-H 1.9555.88246234528
103H-H 1.9565.87051174608
104H-H 1.9541.84877092128
105H-H 1.9232.03054674196
106H-H 1.9172.02054160620
107H-H 1.8481.95862600468
108H-H 1.8341.75616637570
109H-H 1.8291.73226336268
110H-H 1.8281.71115651960
111H-H 1.8351.67725025304
112H-H 1.8301.553106561752
113H-H 1.8281.53692597192
114H-H 1.8203.23115325426
115H-H 1.8193.22013169861
116H-H 1.8182.11414925469
117H-H 1.8182.10616020545
118H-H 1.7421.84824286072
119H-H 1.7451.82735995120
120H-H 1.6691.82714851102
121H-H 1.5461.83496039856
122H-H 1.5431.81972780528
123H-H 1.5451.2707218780
124H-H 1.5461.25536723868
125H-H 1.5441.24122604678
126H-H 1.4761.2716846019
127H-H 1.4771.25510952480
128H-H 1.4711.2428182750
129H-H 1.4740.7277531610
130H-H 1.3480.81223199942
131H-H 1.3480.79627081584
132H-H 1.3440.78422663138
133H-H 1.2601.54012084268
134H-H 1.2631.48914416738
135H-H 1.2631.47417048864
136H-H 1.2670.73616594936
137H-H 1.2660.71916949332
138H-H 1.2521.55813171360
139H-H 1.2501.52519080828
140H-H 1.2370.83711506187
141H-H 1.1673.90186146744
142H-H 1.1673.87780709560
143H-H 0.8401.2348462904
144H-H 0.7981.35139178912
145H-H 0.7981.34232789168
146H-H 0.7301.48211378742
147H-H 0.7311.27011791943
148H-H 0.7291.25515935769
149H-H 4.4604.53192968608
150H-H 4.2034.38245014720
151H-H 4.2034.37332816148
152H-H 1.7951.6754628933

TABLE 6.5
Proton NMR chemical shift data of Y1
FREQUENCY
#ADDRESS[Hz][PPM]INTENSITY
15803.65312.0078.85101.05
26201.85224.7018.7055180.00
36517.25155.5588.59030.09
46612.75134.6268.55541.05
58992.94612.7997.68590.41
69356.84533.0297.553062.60
79733.14450.5147.41550.40
89952.74402.3837.33530.13
99996.24392.8327.31940.90
1010361.54312.7467.1860134.51
1110641.74251.3277.08370.05
1210739.14229.9807.04810.87
1312483.63847.5216.41080.10
1412504.03843.0346.40330.10
1513117.13708.6306.17940.31
1613161.73698.8436.16310.31
1713503.53623.9056.03820.38
1813617.23598.9885.99670.08
1913649.93591.8115.98480.19
2013679.23585.4045.97410.18
2113712.93577.9985.96170.08
2213886.83539.8845.89820.09
2313920.03532.5945.88610.19
2413952.53525.4795.87420.17
2513983.63518.6585.86290.06
2614413.73424.3645.70580.36
2714428.73421.0805.70030.36
2814720.03357.2065.59390.19
2914735.63353.8015.58820.18
3014766.43347.0425.57690.20
3114781.93343.6525.57130.17
3215028.73289.5335.48110.05
3315343.43220.5435.36610.33
3415404.63207.1315.34380.25
3515439.63199.4615.33100.25
3616193.83034.1115.05550.14
3716238.83024.2405.03910.15
3816381.22993.0144.98700.17
3916518.32962.9674.93700.12
4016553.72955.2094.92400.12
4116599.32945.1984.90740.08
4216633.52937.7004.89490.08
4316843.22891.7364.81830.18
4416905.32878.1174.79560.17
4516927.02873.3704.78770.19
4617523.62742.5664.56970.33
4717586.32728.8184.54680.18
4817617.62721.9654.53540.23
4917635.42718.0664.52890.31
5017665.92711.3714.51770.32
5117716.22700.3494.49940.21
5217760.52690.6324.48320.20
5317810.92679.5884.46480.55
5417835.72674.1414.45570.72
5517857.92669.2674.44760.54
5618007.82636.4094.39280.17
5718046.22627.9864.37880.26
5818083.22619.8864.36530.19
5918189.92596.4934.32630.11
6018233.62586.9124.31040.27
6118277.82577.2094.29420.31
6218331.52565.4434.27460.21
6318367.62557.5424.26140.23
6418412.42547.7214.24510.19
6518458.82537.5404.22810.10
6618491.92530.2834.21600.21
6718532.62521.3594.20110.28
6818567.22513.7704.18850.14
6918645.42496.6254.15990.25
7018666.52491.9974.15220.25
7118699.92484.6864.14000.24
7218721.52479.9434.13210.19
7318778.72467.4134.11130.20
7418792.82464.3054.10610.20
7518821.92457.9394.09550.19
7618835.72454.9094.09040.18
7719186.22378.0633.96240.17
7819213.72372.0293.95230.29
7919239.62366.3503.94290.16
8019338.72344.6353.90670.08
8119366.72338.4903.89640.22
8219396.92331.8723.88540.21
8319428.22325.0073.87400.06
8420015.22196.3243.65960.05
8520048.62188.9943.64740.08
8620131.12170.9003.61720.13
8720161.72164.2113.60610.28
8820208.12154.0403.58910.31
8920679.12050.7653.41700.24
9020727.62040.1353.39930.23
9120782.42028.1193.37930.08
9221179.21941.1233.23430.14
9321213.61933.5793.22180.13
9421586.41851.8623.08560.07
9521649.41838.0493.06260.09
9621715.81823.4903.03830.67
9721747.51816.5273.02670.23
9823153.91508.1992.51300.06
9923693.51389.9162.31592.02
10024231.41271.9712.11940.22
10124282.21260.8312.10080.22
10224341.31247.8782.07920.16
10324413.61232.0242.05280.15
10424464.01220.9892.03440.83
10524468.71219.9632.03270.83
10624496.71213.8272.02250.82
10724540.51204.2122.00650.24
10824614.21188.0661.97960.24
10924658.31178.3911.96350.88
11024686.01172.3111.95330.86
11124690.91171.2491.95160.85
11224774.51152.9081.92101.65
11324824.71141.9051.90270.22
11424966.51110.8151.85092.07
11525021.91098.6791.83062.09
11625154.11069.6891.78230.22
11725213.51056.6761.76070.20
11825280.41042.0101.73620.33
11925347.21027.3631.71180.24
12025396.21016.6041.69390.38
12125591.3973.8391.62260.16
12225626.4966.1491.60980.22
12325673.7955.7851.59260.16
12425759.1937.0611.56140.45
12525837.0919.9681.53290.32
12625932.6899.0231.49800.22
12725995.9885.1301.47480.28
12826142.6852.9671.42121.52
12926284.2821.9361.36951.99
13026355.3806.3541.34360.49
13126405.2795.4141.32530.53
13226437.0788.4231.31370.61
13326495.4775.6201.29241.83
13426547.6764.1861.27330.62
13526578.6757.4001.26200.75
13626635.0745.0181.24140.66
13726710.2728.5291.21390.35
13826742.2721.5151.20220.34
13926774.1714.5321.19060.38
14026818.0704.9091.17451.47
14126852.6697.3291.16192.39
14226939.0678.3821.13030.28
14326967.1672.2261.12010.28
14426987.0667.8511.11280.31
14527035.8657.1441.09490.25
14627059.9651.8631.08610.21
14727091.3644.9921.07470.18
14827116.3639.5141.06560.13
14927306.1597.8910.99620.21
15027331.8592.2710.98690.20
15127362.1585.6220.97580.18
15227426.7571.4570.95220.07
15327465.2563.0250.93810.12
15427550.1544.3950.90710.06
15527604.4532.5070.88730.09
15627723.7506.3470.84370.23
15727778.4494.3490.82370.48
15827825.7483.9750.80641.88
15927892.5469.3350.78201.79
16027999.1445.9680.74310.28
16128054.1433.9170.72300.23
16228134.1416.3750.69380.08
16328228.5395.6780.65930.07
16428262.5388.2090.64680.09

TABLE 8.2
2D NMR (HMQC) of R1 peaklist.
ADDRESSFREQUENCY
row[Hz] F1[PPM] F1
#col[Hz] F2[PPM] F2INTENSITY
115120624.836136.6695
3973494.3325.82234.88
215720461.906135.5898
2204534.4697.555467.68
323418646.779123.5620
2584313.1937.1867100.00
423418646.779123.5620
4453213.8635.35509.18
534316069.902106.4864
4962913.3674.85437.58
634316069.902106.4864
4982904.2024.83907.61
735015895.188105.3287
4713059.8915.09857.99
835015895.188105.3287
4733050.6425.08309.11
935015895.188105.3287
4813004.4275.00608.79
1036315605.384103.4083
5102835.5414.72467.96
1138015192.972100.6755
3263912.2066.518616.30
1245413447.55289.1095
6561974.6103.29014.35
1345413447.55289.1095
6591958.0173.26254.45
1451512025.11679.6838
5612533.0494.22064.85
1551811934.44279.0830
3893540.9385.90004.52
1652411802.26878.2071
5622525.1834.207512.80
1752411802.26878.2071
5942340.8803.900411.92
1853411563.50176.6250
5712474.8834.12374.43
1953411563.50176.6250
5882372.5433.95323.52
2053411563.50176.6250
6102246.1653.74265.94
2153411563.50176.6250
6122234.3623.72296.60
2254011437.31275.7888
5742457.3474.094520.96
2354511309.47274.9417
5532580.6284.29995.29
2454511309.47274.9417
5552569.2644.28106.15
2554511309.47274.9417
5612533.9664.22223.23
2654511309.47274.9417
5822411.4574.01809.38
2755111175.79374.0558
5502597.5704.32813.74
2855111175.79374.0558
5522585.9254.30877.04
2955111175.79374.0558
5542574.5154.28973.84
3055611047.37873.2049
5862386.1663.97595.90
3156210907.15972.2758
5112826.2034.709115.78
3256210907.15972.2758
5202777.0204.62715.35
3356710784.60071.4636
5622525.0384.207312.61
3456710784.60071.4636
5672497.6644.16174.33
3557710543.52669.8662
4972908.5304.84634.00
3657710543.52669.8662
5002892.5604.81963.60
3757710543.52669.8662
5142810.2064.68245.98
3857710543.52669.8662
5162796.4134.65945.96
3957710543.52669.8662
5482609.4044.34786.72
406249434.66162.5183
5322705.0294.507211.62
416249434.66162.5183
5342689.7274.48178.01
426249434.66162.5183
5472616.5774.35987.54
436698381.18055.5375
919431.6890.71934.80
446698381.18055.5375
922414.8810.69135.21
457197203.83147.7358
827972.0571.61973.32
467286986.54246.2960
7771265.4172.10853.85
477286986.54246.2960
859784.6261.30743.19
487286986.54246.2960
862764.9701.27463.45
497596273.78841.5729
7241575.1882.62463.64
507596273.78841.5729
7281556.0332.59273.54
518174904.79132.5014
849844.8681.40779.21
528314556.85130.1957
868732.2761.220140.19
538464212.87027.9164
870719.1701.198345.73
548583932.40826.0579
865749.6751.249145.69
558933102.79820.5605
8081085.9041.809437.60
569002935.04419.4489
864755.8571.259442.87
579052821.32218.6953
8081086.3811.810225.73
589182523.12116.7193
883644.7701.074336.59
599182523.12116.7193
895571.3190.951943.39
609242368.33315.6936
7941163.3351.938421.68
619242368.33315.6936
895573.0500.954845.90

TABLE 8.3
HMBC R1 peaklist
DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,
EXPNO = 26, PROCNO = 1 F1PLO = 173.650 ppm, F1PHI = 11.502 ppm,
F2PLO = 6.832 ppm, F2PHI = 0.515 ppm MI = 5.00 cm,
MAXI = 10000.00 cm, PC = 3.000
ADDRESSFREQUENCY
row[Hz] F1[PPM] F1
#col[Hz] F2[PPM] F2INTENSITY
116625330.719167.8561
3893542.3725.902411.00
216625330.719167.8561
8081085.5721.808837.94
329321569.176142.9299
865750.0041.249739.69
432520624.461136.6697
7951160.8261.934254.15
532520624.461136.6697
8081085.1911.808267.95
636619424.748128.7197
7951161.3411.935154.29
736619424.748128.7197
8081085.2931.8083100.00
847916074.168106.5168
5872382.3963.969617.03
948515911.199105.4369
5822411.5704.018213.64
1049515610.658103.4453
5592544.1124.23917.40
1149515610.658103.4453
5742457.7174.09517.72
1250915196.733100.7024
3113999.5666.664210.46
1350915196.733100.7024
3413825.9736.374910.97
1456813447.73989.1126
4972908.0184.845413.78
1556813447.73989.1126
870718.4591.197152.71
1656813447.73989.1126
896570.2710.950233.62
1761612037.52179.7676
5602540.1874.232510.39
1861612037.52179.7676
5672496.4234.159612.77
1962011934.37579.0841
5572555.6704.258316.69
2062011934.37579.0841
5862384.2363.972718.17
2162411794.32978.1561
5612532.4654.219629.22
2262411794.32978.1561
5812415.6754.025113.90
2363211561.07876.6104
5542575.3634.291111.27
2463211561.07876.6104
861774.2141.29008.95
2563211561.07876.6104
864757.6311.262431.05
2663211561.07876.6104
868733.0171.221452.22
2764011319.14775.0073
5622529.4984.214714.09
2864011319.14775.0073
5742457.9794.095512.03
2964011319.14775.0073
6112238.4403.72977.35
3064511173.48974.0421
5112826.3314.70935.08
3164511173.48974.0421
8071087.7421.812427.89
3265011046.94573.2035
3893541.0205.900114.57
3365510904.05872.2566
3263913.5366.520823.27
3465510904.05872.2566
5522586.7684.31019.96
3565510904.05872.2566
5632521.9034.20216.06
3665910780.20471.4359
5322702.9004.50366.93
3765910780.20471.4359
5342692.1274.48578.19
3865910780.20471.4359
5632520.5334.199848.21
3966710532.28769.7931
4723054.4955.089511.46
4066710532.28769.7931
4803007.6395.011418.84
4167110425.30169.0841
3263913.4956.520832.61
4267110425.30169.0841
5522586.0544.30897.93
4367110425.30169.0841
8071088.3131.813458.15
447059430.49262.4919
5602539.8354.23195.37
457408372.17555.4789
870718.6601.197438.08
467408372.17555.4789
895571.4070.952144.50
477807199.30747.7068
883644.6281.074127.47
487807199.30747.7068
895572.4470.953831.41
497886981.52346.2636
864757.7671.262651.63
507886981.52346.2636
868732.9601.221378.63
518046495.94143.0459
5742458.0274.09568.82
528046495.94143.0459
861774.8791.29118.89
538046495.94143.0459
867737.9661.22966.55
548106313.90641.8396
4453213.4545.35438.33
558106313.90641.8396
7551397.4642.32855.40
568106313.90641.8396
865749.8941.249542.24
578106313.90641.8396
883644.6201.074152.86
588146189.25641.0136
865749.2871.24856.31
598146189.25641.0136
883643.3621.07208.62
608176120.84940.5603
865749.1591.248315.69
618176120.84940.5603
883644.2861.073517.94
628176120.84940.5603
896570.1890.95016.27
638196053.88840.1166
865749.6051.249043.77
648196053.88840.1166
883644.3971.073747.98
658196053.88840.1166
896570.0670.94998.91
668245921.43039.2388
864754.1331.25665.80
678245921.43039.2388
870718.2641.196870.97
688245921.43039.2388
896569.4490.948865.30
698245921.43039.2388
920427.4510.71226.70
708245921.43039.2388
922414.5580.69077.41
718275818.31638.5556
870718.1611.19668.71
728275818.31638.5556
895572.9600.954735.78
738295749.30838.0983
896570.0600.94988.61
748375529.91636.6444
829959.4371.59865.22
758375529.91636.6444
864755.9291.259512.15
768375529.91636.6444
868732.6181.22078.77
778375529.91636.6444
895572.3690.953751.81
788375529.91636.6444
920427.3310.71205.36
798375529.91636.6444
922414.5710.69086.76
808405443.60536.0725
7771264.8912.10765.25
818405443.60536.0725
864757.3331.261968.88
828405443.60536.0725
868732.7761.221083.66
838405443.60536.0725
895571.9880.953111.13
848584903.62032.4942
883644.4231.073838.16
858704556.86230.1964
6112240.9763.73408.07
868704556.86230.1964
857794.9871.324618.78
878704556.86230.1964
864756.7931.261079.89
888704556.86230.1964
878670.3101.116918.43
898724487.07929.7340
864756.1251.259916.38
908744422.44529.3057
863758.3841.26368.16
918774356.19328.8667
864755.0841.25818.79
928774356.19328.8667
896569.8980.94966.22
938824211.26727.9063
860779.2121.298319.55
948824211.26727.9063
864754.5741.25735.59
958824211.26727.9063
881654.9941.091419.01
968824211.26727.9063
896570.1620.950087.84
978913938.16526.0966
854812.1191.353219.50
988913938.16526.0966
865751.8281.252710.44
998913938.16526.0966
876686.0031.143017.89
1008913938.16526.0966
896569.8520.94955.20
1018933876.63025.6888
865749.8741.249540.70
1028933876.63025.6888
876686.4001.14375.01
1039193095.31520.5114
3983488.1425.81207.58
1049193095.31520.5114
7971150.5111.91709.43
1059193095.31520.5114
8181022.9041.70449.18
1069193095.31520.5114
868732.8551.22115.53
1079252937.15819.4633
6112240.9103.733913.27
1089252937.15819.4633
7771265.3812.108411.35
1099252937.15819.4633
7801248.2502.07995.80
1109252937.15819.4633
853818.8561.364423.23
1119252937.15819.4633
868733.3551.221989.32
1129252937.15819.4633
875693.4731.155520.36
1139322710.75617.9630
5542574.8804.29036.54
1149322710.75617.9630
849843.9161.40625.24
1159322710.75617.9630
868732.6741.22086.51
1169392527.76516.7504
829960.8741.60106.70
1179392527.76516.7504
870718.6861.197583.68
1189392527.76516.7504
885631.2871.051921.97
1199392527.76516.7504
893583.0380.971514.30
1209392527.76516.7504
906506.6730.844222.28
1219392527.76516.7504
920426.0440.70997.71
1229392527.76516.7504
922414.8830.691310.74
1239442376.34015.7470
7841224.5702.04049.28
1249442376.34015.7470
8061096.6591.82738.54
1259442376.34015.7470
870718.3781.19706.60
1269442376.34015.7470
885633.3811.055419.99
1279442376.34015.7470
906509.4520.848920.05
1289442376.34015.7470
920426.9880.71158.96
1299442376.34015.7470
922415.1070.691710.34

TABLE 8.4
2D NMR (cosy) of R1 peak list
Assignmentw1w2Data Height
16.5216.522257252096
26.5224.7117840252
36.1290.9593905950
45.8985.90132367934
55.9024.84710836326
65.9024.25810138593
75.8994.1279360363
85.9063.97516282743
95.8211.8107215536
105.8175.82231028110
115.8171.94443526536
125.8161.92944815556
135.3555.355178856592
145.0945.102109124320
155.0945.085116649128
165.0924.20515712139
175.0934.04838195456
185.0115.021112612352
195.0105.004121053544
205.0084.21014216868
215.0114.02138155584
224.8475.9135875469
234.8455.9004787344
244.8455.8897478266
254.8414.855118994368
264.8364.82347239140
274.8364.36992117104
284.8314.1349652155
294.8344.1167044446
304.8443.98744245188
314.8453.96244209660
324.7984.79512493983
334.7954.62115346245
344.7961.82157265952
354.7961.80975309312
364.7924.31221750568
374.7534.36024472454
384.7254.73372665064
394.7214.71999191696
404.7274.22650380324
414.7096.52322903380
424.7024.62920594276
434.7074.61219038358
444.7064.32611671381
454.6704.68834985344
464.6694.66040038520
474.6694.35594428136
484.6703.9588587019
494.6723.9417812038
504.6204.8008615562
514.6194.71121326744
524.6184.63329866052
534.6194.60744064012
544.6214.31223669784
554.5844.35527276566
564.5664.36523402122
574.4924.51355339136
584.4914.48271439344
594.4964.370164934224
604.4954.360174062528
614.4993.91217609992
624.4983.89617924064
634.3774.34924720128
644.3654.84397382152
654.3644.829127752656
664.3564.68067867680
674.3554.66781378376
684.3614.491211941920
694.3744.39338442384
704.3644.37227706436
714.3584.33531327086
724.3644.13714876345
734.3634.11313081764
744.3553.96010728486
754.3573.9409552408
764.3713.91320295026
774.3723.89517021364
784.3094.79925905518
794.3094.61943016120
804.3064.31056206892
814.2943.74280581928
824.2953.731101483432
834.2804.3549482896
844.2515.91517374606
854.2505.90110701927
864.2515.88717140758
874.2534.25932444480
884.2584.12531047850
894.2573.9765638105
904.2294.73034642484
914.2075.09213039638
924.2095.01211226875
934.2174.17933544654
944.2094.05828007252
954.2074.04746281960
964.2114.02033876260
974.2074.00521146824
984.2103.90480923600
994.2044.3659066176
1004.2024.03240055832
1014.1673.95118178542
1024.1504.20911484229
1034.1265.9126719130
1044.1225.8907671593
1054.1264.3789045784
1064.1274.27334269312
1074.1194.25832734018
1084.1334.24634046856
1094.1314.14018941800
1104.1164.35511770206
1114.0924.096279326848
1124.0435.09584940104
1134.0354.20850268828
1144.0424.18315655260
1154.0434.06426875002
1164.0195.01577663464
1174.0154.2419036684
1184.0274.22611811450
1194.0164.03719835598
1203.9735.91524059896
1213.9745.90120609464
1223.9735.88722835978
1233.9754.85337923332
1243.9773.99518827896
1253.9494.6825809583
1263.9504.37110668304
1273.9534.34815916606
1283.9494.17518189762
1293.9484.16130027716
1303.9534.14919945408
1313.9403.96614571032
1323.9034.50510048077
1333.9034.48814760281
1343.9034.38719898852
1353.9124.34216459215
1363.9044.23912064081
1373.9074.214108106008
1383.8984.36432756190
1393.9004.19927604272
1403.9013.91743085776
1413.8973.88838415896
1423.7354.30093317560
1433.7364.29098467152
1443.7363.74865327612
1453.7353.72550388764
1463.2703.28317369858
1473.2693.26722139430
1483.2712.3545853844
1493.2712.3427970904
1503.2711.9125107326
1513.2711.8965093256
1522.6022.6315486520
1532.6002.5985094291
1542.6012.5907143968
1552.6022.13214357642
1562.6022.10926185532
1572.6012.08614739034
1582.6031.28914662965
1592.3413.2875980422
1602.3423.2645054491
1612.3342.3605303651
1622.3361.9326500135
1632.3341.8727760766
1642.3252.34113008529
1652.3252.31511101831
1662.3281.9236345431
1672.3261.8959356538
1682.2892.29414262412
1692.1862.1934223454
1702.1032.62030164346
1712.1032.59827444406
1722.1012.1097249900
1732.1051.30722762886
1742.1021.30018897670
1752.1031.27918250726
1762.0702.0784104108
1771.9671.96913308390
1781.9771.6097894784
1791.9561.90911551598
1801.9611.87410608534
1811.9651.6255946444
1821.9315.83439974876
1831.9315.82442157648
1841.9325.81240474008
1851.9315.79830554264
1861.9312.32012384753
1871.9311.942228192416
1881.9311.81059524928
1891.9132.33913306613
1901.9022.0016772636
1911.8953.2879744711
1921.8963.2649357915
1931.8972.3628772959
1941.8861.97316266749
1951.8851.8855269322
1961.8821.61615011650
1971.8831.2428077304
1981.8781.2198767326
1991.8185.8403029832
2001.8055.8293553817
2011.8045.8083418502
2021.8054.82222320414
2031.8054.81319315348
2041.8064.79727156710
2051.8064.78523402710
2061.8064.77422620674
2071.8064.32215274810
2081.8064.31211189881
2091.8064.30217586586
2101.8021.94342426312
2111.8041.93230620986
2121.8041.8101349507072
2131.7761.7788674281
2141.7031.7105954890
2151.6961.7029409535
2161.6131.9118734371
2171.6111.63517314310
2181.6091.61029144662
2191.6091.59916356248
2201.6131.0329463035
2211.6141.0177358422
2221.6130.9919572058
2231.4061.3039782705
2241.3951.40670684776
2251.3931.38522170084
2261.3901.29311202919
2271.3071.3187420988
2281.2852.60928951250
2291.2842.13316431712
2301.2852.11023106420
2311.2832.08314481833
2321.2791.40711364364
2331.2881.3846974150
2341.2861.29918845462
2351.2880.71221876642
2361.2880.69627076318
2371.2511.260768046912
2381.2211.9275903658
2391.2171.2231002405376
2401.1881.197664805376
2411.1681.17116019256
2421.1371.14530540644
2431.1131.12037708240
2441.0701.077775147520
2451.0581.05817850510
2461.0042.3495175155
2471.0031.9166850918
2481.0031.8925963474
2491.0051.63021215516
2501.0051.61023547650
2511.0001.00825525576
2521.0160.95933680632
2530.9850.99911598959
2540.9630.98513491636
2550.9440.9531674528384
2560.9120.91221859108
2570.8720.87812589871
2580.6970.71628731108
2590.6990.69323421302
2600.6941.2977870457
2610.6921.2898126754

TABLE 8.5
R1-proton-NMR-peaklist
FREQUENCY
#ADDRESS[Hz][PPM]INTENSITY
113957.63913.9346.521520.81
215614.03550.8105.91647.03
315656.83541.4085.900814.16
415699.73532.0165.88517.43
515839.63501.3505.83402.56
615872.23494.2045.82216.47
715905.13486.9925.81016.47
815932.83480.9165.80002.49
917152.33213.5545.354512.90
1017853.43059.8485.098417.52
1117888.83052.0935.085518.03
1218074.93011.2865.017516.80
1318110.23003.5445.004617.26
1418524.32912.7514.853315.71
1518558.42905.2824.840820.27
1618597.32896.7494.826612.17
1718638.62887.7094.81165.88
1818667.22881.4214.80116.29
1918680.92878.4274.79616.65
2018709.42872.1874.78575.58
2118857.72839.6724.731513.15
2218891.02832.3564.719315.61
2318902.52829.8444.715113.51
2418918.12826.4314.709513.25
2518996.62809.2084.68088.60
2619038.52800.0324.66559.32
2719136.82778.4714.62967.89
2819151.72775.2084.62416.99
2919179.02769.2204.61418.39
3019194.12765.9074.60867.85
3119474.82704.3694.506116.52
3219515.82695.3854.491114.33
3319529.02692.5034.486320.98
3419797.22633.6994.388310.45
3519819.72628.7544.380117.64
3619850.72621.9684.368826.32
3719872.32617.2284.360927.93
3819897.22611.7714.351817.82
3919930.62604.4384.339611.93
4019963.72597.1914.32758.30
4120006.62587.7914.311814.07
4220031.72582.2894.302711.75
4320048.82578.5454.29648.57
4420076.52572.4674.286311.71
4520107.92565.5844.27487.33
4620151.62556.0014.258914.11
4720174.32551.0244.25066.12
4820196.32546.1964.242513.56
4920214.92542.1284.235816.39
5020247.52534.9684.223826.96
5120285.92526.5554.209839.00
5220305.42522.2714.202724.70
5320319.52519.1904.197523.22
5420346.42513.2984.18777.91
5520358.92510.5464.18317.69
5620379.82505.9594.17558.17
5720422.02496.7194.160110.22
5820461.62488.0434.14565.87
5920487.52482.3524.13627.27
6020514.72476.4024.12627.42
6120558.82466.7274.11017.60
6220596.22458.5284.096531.95
6320697.72436.2664.05948.35
6420735.22428.0564.045713.12
65 20773.12419.7404.031813.77
6620811.22411.3854.017912.76
6720848.02403.3224.00457.29
6820890.72393.9713.98897.87
6920932.72384.7493.973510.17
7020970.32376.4993.959812.68
7121017.72366.1083.94256.44
7221123.52342.9213.903816.37
7321559.52247.3273.744511.72
7421604.42237.4963.728211.07
7522810.21973.1413.28776.60
7622829.61968.8903.28066.56
7722863.11961.5423.26846.70
7822883.01957.1803.26116.59
7924645.41570.7922.61734.82
8024691.21560.7562.60065.30
8125359.91414.1472.35635.53
8225407.31403.7612.339010.56
8325464.11391.3012.31826.50
8425529.51376.9642.29432.87
8525976.11279.0562.13124.43
8626038.81265.3172.10839.07
8726101.11251.6492.08554.68
8826348.41197.4331.99522.18
8926423.11181.0671.96796.86
9026495.71165.1471.941440.57
9126523.11159.1381.931435.58
9226528.41157.9841.929536.42
9326565.01149.9531.916114.14
9426612.01139.6411.898912.71
9526673.01126.2831.87669.66
9626829.51091.9571.819448.60
9726857.01085.9291.8094113.02
9827349.4977.9781.629511.86
9927403.6966.0991.609712.07
10027963.7843.3131.405122.40
10128018.6831.2751.38518.46
10228254.5779.5461.298911.85
10328298.3769.9481.282912.55
10428317.6765.7171.275913.04
10528356.2757.2691.261886.45
10628387.8750.3281.250281.24
10728464.0733.6331.222488.26
10828533.4 718.4141.197073.80
10928677.9686.7391.14433.89
11028745.9671.8201.11943.59
11128867.8645.0981.074974.04
11228987.7618.8041.03114.13
11329051.5604.8261.00788.69
11429106.8592.7030.98764.79
11529207.1570.7110.9509114.09
11629320.8545.7790.90943.45
117 29410.0526.2250.87681.79
11829492.8508.0680.84661.41
119 29857.9428.0300.71329.46
12029911.7416.2330.69359.30

TABLE 8.6
13C NMR of R1 peak list
DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,
EXPNO = 30, PROCNO = 1
F1 = 176.659 ppm, F2 = 6.843 ppm,
MI = 0.06 cm, MAXI = 10000.00 cm, PC = 1.400
FREQUENCY
#ADDRESS[Hz][PPM]INTENSITY
15310.9 25331.195167.85590.12
28227.322634.219149.98451.57
38251.822611.535149.834211.79
48281.1 22584.428149.654612.50
58310.4 22557.355149.475212.30
69381.7 21566.594142.91000.11
710403.120622.062136.65110.14
810577.320460.975135.58361.08
910596.820442.969135.46435.78
1010623.620418.133135.29986.16
1110650.420393.346135.13556.01
1211696.919425.617128.72290.14
1312537.918647.846123.56902.11
1412557.418629.844123.449710.62
1512584.318604.896123.284411.25
1612611.318580.010123.119510.98
1713444.217809.705118.01510.11
1814249.217065.322113.08250.15
1915319.016075.968106.52660.21
2015492.315915.713105.46470.24
2115509.015900.229105.36210.25
2215823.215609.646103.43650.18
2316269.015197.378100.70470.16
2418158.113450.41389.12850.12
2519683.712039.60479.77980.13
2619797.311934.50279.08340.14
2719929.211812.53378.27520.23
2819945.511797.52778.17570.42
2920197.311564.58976.63220.15
3020211.911551.14276.54310.16
3120226.211537.92876.45550.14
3220335.911436.43475.78300.10
3320410.211367.75375.32790.13
3420473.611309.06974.93900.38
3520617.211176.29574.05920.13
3620755.111048.80573.21440.15
3720910.010905.52272.26490.21
3821019.310804.42071.59500.15
3921047.510778.39171.42250.37
4021283.210560.45369.97830.12
4121320.710525.73969.74830.12
4221375.210475.33969.41430.14
4321430.810423.88269.07330.13
4422496.29438.66362.54480.19
4522510.29425.67662.45880.20
4623646.38375.03955.49680.13
4724915.87201.02847.71730.13
4825152.56982.20446.26720.11
4925671.36502.36643.08760.12
5025868.36320.20241.88050.10
5125921.26271.27341.55630.11
5226160.16050.41840.09280.10
5326300.35920.72439.23340.13
5426396.65831.66738.64330.10
5526722.25530.54936.64790.11
5626820.55439.67936.04580.13
5727398.14905.47532.50590.10
5827774.84557.16230.19780.23
5928148.74211.39127.90660.23
6028389.53988.64526.43060.10
6128448.23934.38526.07100.17
6228512.93874.57125.67470.10
6328828.23583.03023.74280.11
6429348.83101.52220.55210.23
6529529.32934.59119.44590.22
6629650.22822.79818.70510.26
6729719.52758.71218.28050.11
6829766.22715.50817.99420.10
6929970.42526.66416.74280.24
7029985.52512.76516.65070.17
7130140.72369.19215.69930.22

TABLE 9.2
2D NMR (HMQC) chemical shift list of O54.
Assignmentw1w2Data Height
1123.5405.33710801059
2 106.6794.8757546160
3 106.7014.8608527622
4 105.1895.12911004760
5105.3824.99711249292
6105.1735.1147915383
7103.3754.7259431190
8 100.6636.51318941426
988.8733.3295975702
1088.8503.3075246604
11 79.7674.2107199634
12 79.7084.1842885672
13 78.2034.21323826162
14 78.1963.90713786228
15 76.8184.1486859174
16 76.4903.9445773632
17 76.6423.7497515808
1876.6283.7308437212
1975.8424.08922251034
2075.3184.2412654032
2175.3304.2139853961
2275.1034.2997580958
2375.1694.2796376272
24 74.9864.02511823145
25 75.1684.00310800605
26 74.0844.3304901724
27 74.0984.3097830222
28 74.2284.2913975150
29 72.2814.71117310028
30 72.2304.6258709239
31 72.2064.6067361591
32 71.4794.22014407576
33 71.4814.20815081282
3471.5814.1694582404
35 71.6134.1508419769
36 71.6284.1215804582
37 71.6374.0997950110
38 71.6674.0803119152
39 70.3054.8907622650
40 70.2984.8687628303
4170.2654.3144762902
42 69.9834.6766227999
4369.9184.6496037791
4470.1054.3419963877
4569.1294.7834704838
4662.5544.50214998714
4762.5324.47714154374
4862.5484.36211342701
4962.5374.3448091356
5056.1593.6971838600
5156.0813.7582144782
5255.7783.6861783080
5355.5900.7175572202
5455.5880.6946671325
5547.7321.6084217994
5647.7851.5953965686
5746.2382.1311734693
5846.2712.1054919338
5946.2992.0722490009
6046.2791.3044826865
6146.2761.2724573323
6241.6122.6054436446
6341.5972.5724591152
6438.7421.6434256302
6538.7301.6123985649
6638.6411.0313538408
6732.5081.41310863677
6830.1891.22058134804
6927.9841.24255524444
7026.4952.4203113032
7126.4682.3923183732
7225.8781.9173435953
7325.8801.8801979946
7426.0661.24263345236
7525.7151.2125054462
7623.8141.9882104036
7723.7051.9593237644
7823.6591.9002754050
7923.6951.8653108091
8019.4161.25740949520
8118.6921.80230867330
8218.3441.3893540974
8318.3081.4136204910
8417.9112.3303260134
8517.9422.3084212457
8617.9141.9362468661
8717.9431.9043914254
8816.8250.98048392540
8916.6401.07252924908
9015.7010.94760375744

TABLE 9.3
2D NMR (HMBC) chemical shift list of O54.
DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,
EXPNO = 37, PROCNO = 1
F1PLO = 149.426 ppm, F1PHI = 11.307 ppm,
F2PLO = 6.861 ppm, F2PHI = 0.388 ppm
MI = 2.00 cm, MAXI = 10000.00 cm, PC = 3.000
ADDRESSFREQUENCY
row[Hz]F1[PPM]F1
#col[Hz]F2[PPM]F2INTENSITY
129421551.752142.8145
7281553.2882.58813.15
229421551.752142.8145
7911185.3451.97503.03
329421551.752142.8145
8011127.8531.87933.00
429421551.752142.8145
866744.8361.241141.49
539218639.387123.5154
7281554.3132.58982.70
639218639.387123.5154
7301540.2252.56642.11
739218639.387123.5154
7941164.9011.94102.94
839218639.387123.5154
8011127.0691.87793.61
947816106.854106.7334
5852391.7233.985120.41
1047816106.854106.7334
6531992.5073.32003.17
1148515890.850105.3020
4942924.2884.87253.34
1248515890.850105.3020
5162797.7924.66173.27
1348515890.850105.3020
5472614.9034.35705.99
1448515890.850105.3020
5832405.2774.007711.83
1549515600.868103.3804
5602539.0464.2306 4.55
1649515600.868103.3804
5742456.564 4.0932 3.63
1750915198.148100.7118
3123996.5106.659111.15
1850915198.148100.7118
3413824.2676.3721 12.11
1950915198.148100.7118
5602539.4214.23122.82
2057013411.60988.8731
4952921.8184.8684 18.81
2157013411.60988.8731
7971147.1421.9114 2.73
2257013411.60988.8731
827975.2601.6250 6.25
2357013411.60988.8731
866746.1251.2432 69.46
2457013411.60988.8731
892589.8460.982848.36
2561612031.10079.7251
5612533.0724.2207 10.23
2661612031.10079.7251
5682492.0764.1524 12.78
2762411792.17478.1418
4693075.6305.1247 3.71
2862411792.17478.1418
4813002.8445.0034 2.57
2962411792.17478.1418
5342690.7324.4834 2.54
3062411792.17478.1418
5472617.2074.3608 2.56
3162411792.17478.1418
5612532.7654.2201 29.07
3262411792.17478.1418
5732460.2864.0994 15.44
33624 11792.17478.1418
5802419.8464.0320 13.41
3462411792.17478.1418
5852390.3433.9828 8.30
3562411792.17478.1418
868732.4421.2204 3.23
3663211558.91976.5961
5532577.6974.2950 11.58
3763211558.91976.5961
5632521.8894.20203.02
3863211558.91976.5961
5732462.2464.1026 13.81
39632 11558.91976.5961
861772.9531.2879 9.11
4063211558.91976.5961
864755.6501.2591 33.06
41632 11558.91976.5961
868732.1701.220053.66
4263911373.42475.3669
3263911.8986.5181 5.00
4363911373.42475.3669
5632523.6304.2049 25.40
4463911373.42475.3669
5742456.0454.0923 5.16
45639 11373.42475.3669
6112240.8653.7338 5.57
4663911373.42475.3669
8081082.2991.8034 2.40
4763911373.42475.3669
864756.6081.2607 2.78
48639 11373.42475.3669
868732.3701.2203 5.47
4964611168.37774.0082
5112828.3444.71278.44
5064611168.37774.0082
5202772.9744.6204 6.66
5164611168.37774.0082
5632524.2844.2060 5.35
5264611168.37774.0082
8081082.3951.8035 49.69
5365510899.10672.2238
3263911.6796.5177 25.27
5465510899.10672.2238
4982902.6624.8365 2.42
5565510899.10672.2238
5112829.4144.7144 5.76
5665510899.10672.2238
5232756.3764.5927 2.33
5765510899.10672.2238
5322703.5754.5048 2.40
5865510899.10672.2238
5522588.3134.3127 9.99
5965510899.10672.2238
5632523.2404.20437.35
6065510899.10672.2238
8081083.9331.8061 2.26
6165910785.63771.4719
3263912.1336.5185 2.28
6265910785.63771.4719
4942925.9994.8754 2.36
6365910785.63771.4719
5162800.1774.6657 3.39
6465910785.63771.4719
5322702.4934.5030 7.49
6565910785.63771.4719
5342691.6984.48507.86
6665910785.63771.4719
5472614.9804.3571 2.35
6765910785.63771.4719
5622525.2014.2075 46.85
6865910785.63771.4719
5842398.0953.9958 2.02
6966510604.59970.2723
3263911.9566.5182 2.12
7066510604.59970.2723
4693075.2525.124118.68
7166510604.59970.2723
4813002.0485.0021 3.29
7266510604.59970.2723
5622525.1384.2074 3.03
7366510604.59970.2723
5732460.7944.1002 6.46
7466510604.59970.2723
8081081.9341.8027 3.10
7566610552.99069.9303
3263912.4596.5190 2.10
7666610552.99069.9303
4683077.1875.1273 13.23
7766610552.99069.9303
4813002.4025.0027 10.72
7866610552.99069.9303
5632522.1294.2024 3.33
7966610552.99069.9303
5732461.2694.1010 4.47
8066610552.99069.9303
8081081.7231.8024 2.72
8167110429.13669.1095
3263911.6396.5177 38.49
8267110429.13669.1095
5522589.0154.3139 8.97
8367110429.13669.1095
8081081.9871.8028 63.77
84705 9425.53762.4591
5602542.2064.2359 3.27
857059425.53762.4591
5622526.3044.2094 2.08
867408376.88455.5101
827971.2391.61833.45
877408376.88455.5101
849843.5611.40563.43
887408376.88455.5101
866746.2921.243548.00
89740 8376.88455.5101
892590.1310.9833 31.95
907408376.88455.5101
896567.0500.9448 25.60
917807196.90847.6909
4473202.461 5.3360 4.85
92780 7196.90847.6909
864756.1271.25992.83
937807196.90847.6909
868731.6581.21913.78
947807196.90847.6909
883642.2291.0701 28.25
95780 7196.90847.6909
896567.1660.9450 32.38
967807196.90847.6909
922414.8950.6913 2.37
977886979.99646.2535
7301540.0552.56612.37
987886979.99646.2535
864755.6791.2591 58.65
997886979.99646.2535
868731.8921.2195 85.12
1008046505.57243.1097
5532577.6544.29494.16
1018046505.57243.1097
5742455.3434.09114.70
1028046505.572 43.1097
7551393.9632.32273.13
1038046505.57243.1097
858789.6031.31573.35
1048046505.57243.1097
861773.4351.28879.90
1058046505.57243.1097
867737.0641.22819.41
1068046505.57243.1097
883642.7551.07102.74
1078046505.57243.1097
892589.0370.98152.09
1088106311.92341.8265
4473202.2955.33578.24
1098106311.92341.8265
7551393.6602.32216.43
1108106311.92341.8265
830830955.9291.59282.54
111810 6311.92341.8265
866744.3691.240345.72
1128106311.92341.8265
869724.7411.20763.80
113810 6311.92341.8265
883642.2711.070253.84
1148106311.92341.8265
892589.7340.98262.36
1158146190.62241.0227
4473204.1695.33892.27
1168146190.62241.0227
866744.9671.24134.20
1178146190.62241.0227
883641.2911.0685 13.51
118814 6190.62241.0227
892590.2660.9835 3.54
1198176122.21240.5694
849842.5411.4039 2.02
1208176122.21240.5694
866744.0981.239810.92
1218176122.21240.5694
883641.8441.0695 19.65
1228176122.21240.5694
892590.0100.9831 4.84
1238196054.44840.1203
8001128.6531.88062.01
1248196054.44840.1203
830955.8741.5927 5.24
125819 6054.44840.1203
849842.4381.4037 7.20
1268196054.44840.1203
866744.3381.240239.00
1278196054.44840.1203
883642.0471.0698 49.93
1288196054.44840.1203
892589.7240.9826 6.64
1298196054.44840.1203
896566.6540.9442 2.44
1308235927.00239.2758
866746.0661.2431 48.54
1318235927.00239.2758
883642.1121.0699 7.22
1328235927.00239.2758
892589.8160.9828 60.87
1338235927.00239.2758
896566.6310.94415.86
1348235927.00239.2758
922415.4730.69237.24
1358275833.93338.6590
827970.3351.61683.11
1368275833.93338.6590
865746.8251.244410.44
1378275833.93338.6590
883642.0351.0698 3.42
1388275833.93338.6590
892588.9570.981311.20
1398275833.93338.6590
896567.1240.945055.77
1408305737.57038.0205
864753.9681.2563 3.79
1418305737.57038.0205
868731.4831.21884.47
1428305737.57038.0205
883641.8751.06952.21
1438305737.57038.0205
892589.2720.98194.70
1448305737.57038.0205
896567.2060.945111.09
1458335652.81937.4589
865751.9421.25295.17
1468335652.81937.4589
868732.1931.22005.78
1478335652.81937.4589
883642.3131.07022.64
1488335652.81937.4589
892590.1890.98342.92
1498335652.81937.4589
896567.1960.945111.22
1508375532.75736.6633
7471441.0472.40112.33
1518375532.75736.6633
830956.2661.59346.70
152837 5532.75736.6633
849840.7911.4009 7.18
1538375532.75736.6633
864754.4171.257010.60
1548375532.75736.6633
868733.7681.2226 10.09
1558375532.75736.6633
883642.2201.0701 2.62
1568375532.75736.6633
896567.0410.944859.49
157837 5532.75736.6633
922415.5100.6923 7.27
158840 5447.50236.0983
6112241.4693.73483.44
1598405447.50236.0983
7751277.7362.1290 2.18
1608405447.50236.0983
7781261.8982.1026 5.77
1618405447.50236.0983
7801246.4962.0769 2.77
162840 5447.50236.0983
861771.3931.2853 7.30
1638405447.50236.0983
864755.5131.258985.50
164840 5447.50236.0983
868731.6841.219194.68
1658405447.50236.0983
883642.2111.0701 2.09
1668405447.50236.0983
896566.7700.9444 11.25
1678584901.02932.4771
859783.8071.3060 2.31
168858 4901.02932.4771
883642.0141.0697 38.68
1698584901.02932.4771
922416.8740.6946 3.35
170870 4554.42830.1803
6112241.5993.7350 9.79
1718704554.42830.1803
7771263.2282.10483.21
172870 4554.42830.1803
857793.8841.3228 18.60
1738704554.42830.1803
864755.2001.2583 93.90
1748704554.42830.1803
879668.6411.1141 19.16
175870 4554.42830.1803
892590.2000.9834 3.11
1768814218.92127.9570
855807.2621.3451 16.41
1778814218.92127.9570
864755.8031.2593 4.75
1788814218.92127.9570
876683.0311.1381 15.67
1798814218.92127.9570
892589.9180.982973.26
1808814218.92127.9570
922416.4460.69392.87
181891 3935.92126.0817
8021120.1761.8665 2.34
182891 3935.92126.0817
855806.4801.3438 19.11
1838913935.92126.0817
866745.9231.24298.92
1848913935.92126.0817
868731.9891.21975.17
1858913935.92126.0817
877680.8511.134418.56
1868913935.92126.0817
892589.6870.9825 4.65
1878933878.91325.7039
7911185.6081.9755 2.05
1888933878.91325.7039
8101072.6051.7872 2.02
1898933878.91325.7039
855805.9701.34294.93
1908933878.91325.7039
866744.2481.240144.75
1918933878.91325.7039
877680.8941.13454.45
1928933878.91325.7039
892590.1700.98343.96
1939252932.82919.4346
6112240.9543.733915.68
1949252932.82919.4346
7751278.7532.13074.57
1959252932.82919.4346
7781262.3722.103411.84
1969252932.82919.4346
7801246.3922.07686.85
1979252932.82919.4346
854816.7341.360922.66
1989252932.82919.4346
868732.1451.219993.22
1999252932.82919.4346
875691.4161.152121.54
2009332709.26017.9531
5532577.9184.295410.65
2019332709.26017.9531
5742456.9694.09392.40
202933 2709.26017.9531
7281553.8122.58902.47
2039332709.26017.9531
7861214.0602.02292.44
2049332709.26017.9531
865746.7591.2443 4.64
205933 2709.26017.9531
868731.9181.2195 5.50
206938 2536.52016.8085
6531992.0543.31922.61
2079382536.52016.8085
829958.4761.5970 2.70
208938 2536.52016.8085
866745.9041.2428 52.11
2099382536.52016.8085
882650.6731.0842 17.18
210938 2536.52016.8085
895574.7360.9576 3.28
211938 2536.52016.8085
903526.0410.8765 16.84
2129382536.52016.8085
922416.2800.69368.32
2139442375.48715.7414
830955.7131.59245.34
214944 2375.48715.7414
866745.6841.24256.94
215944 2375.48715.7414
886627.5761.0457 21.31
2169442375.48715.7414
892588.7740.98103.00
2179442375.48715.7414
907503.8190.8395 21.33
218944 2375.48715.7414
922415.3740.692110.43

TABLE 9.5
Proton NMR peak list of O54.
DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan,
EXPNO = 35, PROCNO = 1
F1 = 10.000 ppm, F2 = 0.000 ppm,
MI = 2.00 cm, MAXI = 10000.00 cm, PC = 1.000
FREQUENCY
#ADDRESS[Hz][PPM]INTENSITY
17886.35225.5098.7069180.00
211038.44534.4387.555472.73
312044.34313.9117.1879160.90
413878.4 3911.8196.518014.53
517112.8 3202.7195.33649.02
617674.0 3079.6805.131410.52
717709.4 3071.9385.118510.66
818004.6 3007.2155.010710.57
918039.9 2999.4804.997810.78
1018330.8 2935.7014.89156.88
1118377.3 2925.5094.874516.06
1218411.1 2918.0994.862210.01
1318591.4 2878.5544.79633.55
1418621.6 2871.9414.78534.53
1518660.9 2863.3254.77093.56
1618768.9 2839.6574.73158.48
1718801.9 2832.4164.719415.95
1818929.2 2804.4964.67295.72
1918974.3 2794.6084.65646.90
2019049.2 2778.1914.62915.41
2119063.7 2775.0074.62384.83
2219091.3 2768.9554.61375.79
2319106.0 2765.7374.60835.24
2419395.7 2702.2284.502511.91
2519447.9 2690.7904.483516.03
2619708.9 2633.5614.38816.86
2719731.8 2628.5524.37988.37
2819761.7 2621.9894.368816.78
2919784.1 2617.0714.360618.07
3019814.8 2610.3504.349412.73
3119834.7 2605.9764.342113.55
3219863.8 2599.6124.33159.52
3319909.8 2589.5234.314713.96
3419947.5 2581.2494.300910.27
3519990.6 2571.8094.28527.86
3620116.2 2544.2754.23936.06
3720155.1 2535.7504.225129.53
3820188.0 2528.5274.213136.53
3920218.4 2521.8594.202022.79
4020258.52513.0724.187310.10
41 20308.52502.1114.16917.17
42 20349.02493.2284.154310.94
4320384.72485.4124.14127.98
4420454.42470.1364.11588.00
4520496.12460.9794.1005 11.54
4620522.92455.1084.090820.70
4720619.42433.9534.05555.10
4820655.02426.1514.04257.46
4920704.62415.2714.02446.32
5020741.82407.1194.01088.93
5120763.72402.3124.00286.78
5220800.32394.2903.98947.59
5320840.02385.5873.97494.23
5420899.52372.5343.95325.04
5520961.52358.9533.93055.93
5621011.82347.9173.91228.88
5721462.42249.1353.74768.28
5821507.42239.2683.73117.60
5922606.31998.3483.32974.57
6022625.11994.2423.32284.57
6122658.91986.8193.31054.74
6222678.21982.5983.30344.55
6324608.61559.3752.59833.63
6424657.01548.7682.58063.98
6525113.61448.6582.41383.70
6625160.11438.4612.39684.03
6725341.71398.6622.33053.88
6825394.71387.0362.31114.43
6925900.21276.2072.12643.15
7025962.81262.4842.10366.25
7126025.41248.7722.08073.23
7226432.41159.5301.93208.83
7326518.71140.6101.900511.87
7426686.81103.7711.83912.00
7526772.71084.9271.807728.83
7626800.21078.9041.797728.10
7727239.8982.5351.63714.71
7827287.4972.0991.61978.43
7927366.6954.7361.59084.35
8027864.2845.6311.409014.94
8128172.8777.9691.29639.42
8228216.7768.3631.28039.50
8328235.2764.2881.27359.84
8428276.7755.2061.258358.74
8528321.2745.4471.242194.67
8628380.5732.4441.220458.53
8728793.1641.9781.069750.29
8828906.6617.1141.02825.31
8929031.6589.696 0.982656.49
9029134.1567.2280.945150.09
9129765.3428.8520.71466.67
9229818.8417.1280.69506.36

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