Gutterman, Jordan U. (Houston, TX, US)
Plaque It!
Sponsored by: Flash of Genius |
What is claimed is:
1. A mixture comprising one or more isolated triterpene glycosides characterized by the following properties: a) isolatable from the tissues of
2. The mixture of claim 1, wherein said mixture induces cytotoxicity in said Jurkat cell with an IC
3. The mixture of claim 1, wherein said apoptosis is induced when administered to said Jurkat cell at a concentration of from about 100 to about 400 ng/ml.
4. The mixture of claim 3, wherein said apoptosis is induced when administered to said Jurkat cell at a concentration of from about 200 to about 400 ng/ml.
5. The mixture of claim 1, wherein said apoptosis is measured by reorganization of plasma membrane of said Jurkat cell by annexin binding.
6. A mixture comprising one or more isolated triterpene glycosides characterized by the following properties: a) isolatable from the tissues of
7. A mixture comprising one or more isolated triterpene glycosides characterized by the following properties: a) isolatable from the tissues of
8. The mixture of claim 7, wherein said caspase activity is from about 0.3 to about 1.6 fluorescence units/minutes/mg.
9. A mixture comprising one or more isolated triterpene glycosides characterized by the following properties: a) isolatable from the tissues of
10. A mixture comprising one or more isolated triterpene glycosides characterized by the following properties: a) isolatable from the tissues of
11. A mixture comprising one or more isolated triterpene glycosides characterized by the following properties: a) isolatable from the tissues of
12. A mixture comprising one or more isolated triterpene glycosides characterized by the following properties: a) isolatable from the tissues of
13. A nutraceutical composition comprising the composition of any one of claims
14. The nutraceutical composition of claim 13, wherein said pharmacologically acceptable medium is a buffer, a solvent, a diluent, an inert carrier, an oil, a creme, or an edible material.
15. A nutraceutical composition comprising dried and ground
16. The nutraceutical composition of claim 15, wherein said pharmacologically acceptable medium is a buffer, a solvent, a diluent, an inert carrier, an oil, a creme, or an edible material.
17. The nutraceutical composition of claim 16, wherein said nutraceutical composition comprises a tablet.
18. The nutraceutical composition of claim 16, wherein said nutraceutical composition comprises a capsule.
19. The nutraceutical composition of claim 16, wherein said nutraceutical composition comprises an ointment.
20. A nutraceutical composition comprising dried and ground
21. The nutraceutical composition of claim 20, wherein said pharmacologically acceptable medium is a buffer, a solvent, a diluent, an inert carrier, an oil, a creme, or an edible material.
22. The nutraceutical composition of claim 21, wherein said nutraceutical composition comprises a tablet.
23. The nutraceutical composition of claim 21, wherein said nutraceutical composition comprises a capsule.
24. The nutraceutical composition of claim 21, wherein said nutraceutical composition comprises an ointment.
25. A process for preparing a composition comprising a mixture of one or more isolated triterpene glycosides, comprising: a) obtaining tissue from an
26. The process of claim 25, wherein the tissue comprises a pod.
27. The process of claim 25, wherein the tissue comprises a root.
28. The process of claim 25, wherein the tissue comprises a seedling.
29. The process of claim 25, wherein the solvent is methanol, ethanol, isopropyl alcohol, dichloromethane, chloroform, ethyl acetate, water, glycerol or a mixture thereof.
30. The process of claim 25, further comprising isolating said composition from plant bagasse by filtration after said extracting.
31. The process of claim 25, further comprising defatting with an organic solvent prior to extracting.
32. The process of claim 31, wherein said organic solvent is hexane, dichloromethane, chloroform, ethyl acetate or a mixture thereof.
33. The process of claim 25, wherein said obtaining comprises isolating at least one triterpene glycoside chromatographically.
34. The process of claim 33, wherein said triterpene glycoside is isolated by elution with methanol, acetonitrile, water, or a mixture thereof.
35. The process of claim 33, wherein said composition is isolated using liquid chromatography.
36. The process of claim 25, further comprising evaporating said solvent after said extracting.
37. A method of preparing an isolated triterpene glycoside composition comprising: a) preparing a tissue culture comprising cells of an
38. The method of claim 37, wherein said tissue culture comprises a hairy root culture.
39. The method of claim 38, wherein said tissue culture is prepared by infecting said
40. The method of claim 38, wherein said tissue culture comprises from about 3% to about 4% sucrose by weight.
41. The method of claim 38, wherein said solvent is methanol, ethanol, isopropyl alcohol, dichloromethane, chloroform, ethyl acetate, water or a mixture thereof.
42. The method of claim 38, further comprising filtering plant bagasse from said triterpene glycoside composition after said extracting.
43. The method of claim 38, further comprising isolating said triterpene glycoside composition by liquid chromatography after said extracting.
44. The method of claim 38, further comprising evaporating the solvent after said extracting.
45. A triterpene glycoside prepared by the process of any one of claims
46. A hairy root tissue culture comprising cells of an
47. The tissue culture of claim 46, wherein said tissue culture medium comprises from about 3% to about 4% sucrose by weight.
48. A method of continually harvesting an
49. The method of claim 48, wherein said growth system is an aeroponic system.
50. The method of claim 49, wherein said tissue is root tissue.
51. A method of inhibiting the initiation and promotion of mammalian epithelial cells to a premalignant or malignant state in a mammal comprising administering to said mammal a therapeutically effective amount of the nutraceutical composition of claim 13.
52. The method of claim 51, wherein said epithelial cell is a skin cell, a colon cell, a uterine cell, an ovarian cell, a pancreatic cell, a prostate cell, a renal cell, a lung cell, a bladder cell or a breast cell.
53. The method of claim 51, wherein said mammal is a human.
54. The method of claim 51, wherein said administering is oral.
55. The method of claim 51, wherein said administering is topical.
56. A method of inducing apoptosis in a malignant mammalian cell in a mammal comprising administering to said mammal a therapeutically effective amount of the nutraceutical composition of claim 13.
57. The method of claim 56, wherein said cell is a skin cell, a colon cell, a uterine cell, an ovarian cell, a pancreatic cell, a prostate cell, a renal cell, a lung cell, a bladder cell or a breast cell.
58. The method of claim 56, wherein said mammal is a human.
59. The method of claim 56, wherein said administering is oral.
60. The method of claim 56, wherein said administering is topical.
61. A method of preventing the abnormal proliferation of mammalian epithelial cells in a mammal comprising administering to said mammal a therapeutically effective amount of the nutraceutical composition of claim 13.
62. The method of claim 61, wherein said epithelial cells are crypt cells.
63. The method of claim 61, wherein said epithelial cells are colon cells.
64. The method of claim 61, wherein said mammal is a human.
65. The method of claim 61, wherein said administering is oral.
66. A method of treating a mammal for inflammation comprising administering to said mammal a therapeutically effective amount of the nutraceutical composition of claim 13.
67. The method of claim 66, wherein said mammal is a human.
68. A composition comprising a triterpene moiety attached to a monoterpene moiety having the molecular formula:
69. The composition of claim 68, wherein R
70. The composition of claim 69, wherein the sugar is selected from the group consisting of glucose, fucose, rhamnose, arabinose, xylose, quinovose, maltose, glucuronic acid, ribose, N-acetyl glucosamine, and galactose.
71. The composition of claim 70, further comprising a monoterpene moiety attached to the sugar.
72. The composition of claim 71, wherein R
73. The composition of claim 72, wherein R
74. The composition of claim 68, wherein R
75. The composition of claim 74, wherein R
76. The composition of claim 75, wherein R
77. The composition of claim 76, wherein at least one sugar is methylated.
78. The composition of claim 68, wherein R
79. The composition of claim 68, wherein the triterpene moiety is oleanolic acid instead of acacic acid.
80. A composition comprising a triterpene glycoside having the molecular formula:
81. The composition of claim 80, having the molecular formula:
82. A composition comprising a triterpene glycoside having the molecular formula:
83. The composition of claim 82, having the molecular formula:
84. A composition comprising a triterpene glycoside having the molecular formula:
85. A composition of claim 84, having the molecular formula:
86. A composition comprising a triterpene moiety, an oligosaccharide and three monoterpene units.
87. The composition of claim 86, wherein the triterpene moiety is acacic acid or oleanolic acid.
88. A pharmaceutical composition comprising the composition of any one of claims
89. The pharmaceutical composition of claim 88, wherein said pharmacologically acceptable medium is a buffer, a solvent, a diluent, an inert carrier, an oil, a creme, or an edible material.
90. The pharmaceutical composition of claim 88, wherein said pharmaceutical composition further comprises a targeting agent.
91. The pharmaceutical composition of claim 90, wherein said targeting agent directs delivery of said pharmaceutical composition to an epithelial cell.
92. The pharmaceutical composition of claim 91, wherein said targeting agent comprises an antibody which binds to the epithelial cell.
93. The pharmaceutical composition of claim 88, wherein said pharmaceutical composition comprises at least a second composition that can kill an epithelial cell.
94. A method of inhibiting the initiation and promotion of a mammalian epithelial cell to a premalignant or malignant state in a mammal comprising administering to said mammal a therapeutically effective amount of the pharmaceutical composition of claim 88.
95. The method of claim 94, wherein said epithelial cell is a skin cell, a colon cell, a uterine cell, an ovarian cell, a pancreatic cell, a lung cell, a bladder cell, a prostate cell, a renal cell, or a breast cell.
96. The method of claim 94, wherein said mammal is a human.
97. The method of claim 94, wherein said administering is oral.
98. The method of claim 94, wherein said administering is topical.
99. The method of claim 94, wherein said administering is via intratumoral injection.
100. The method of claim 94, wherein said administering is intravenous.
101. The method of claim 94, wherein said administering comprises inhaling an aerosol.
102. The method of claim 94, further comprising irradiating said epithelial cell.
103. The method of claim 102, wherein said epithelial cell is irradiated with X-ray radiation, UV-radiation, γ-radiation, or microwave radiation.
104. A method of inducing apoptosis in a malignant mammalian cell in a mammal comprising administering to said mammal a therapeutically effective amount of the pharmaceutical composition of claim 88.
105. The method of claim 104, wherein said cell is a skin cell, a colon cell, a uterine cell, an ovarian cell, a pancreatic cell, a lung cell, a bladder cell, a prostate cell, a renal cell, or a breast cell.
106. A method of preventing the abnormal proliferation of a mammalian epithelial cell in a mammal comprising administering to said mammal a therapeutically effective amount of the pharmaceutical composition of claim 88.
107. The method of claim 106, wherein said epithelial cell is a crypt cell.
108. The method of claim 106, wherein said epithelial cell is a colon cell.
109. The method of claim 106, wherein said mammal is a human.
110. The method of claim 106, wherein said administering is oral.
111. The method of claim 106, wherein said administering is intravenous.
112. The method of claim 106, wherein said administering is intratumoral.
113. The method of claim 106, further comprising irradiating said epithelial cell.
114. A method of treating a mammal for inflammation comprising administering to said mammal a therapeutically effective amount of the pharmaceutical composition of claim 88.
115. The method of claim 114, wherein said mammal is a human.
116. The method of claim 114, wherein said administering is oral.
117. The method of claim 114, wherein said administering is topical.
118. A method of regulating angiogenesis in a mammal comprising administering to said mammal a therapeutically effective amount of the pharmaceutical composition of claim 88.
119. The method of claim 118, wherein said mammal is a human.
[0001] The present application is a continuation-in-part of co-pending U.S. patent application Ser. No. 60/099,066, filed Sep. 3, 1998, and a continuation-in-part of U.S. patent application Ser. No. 60/085,997, filed May 19, 1998. The entire text of each of the above-referenced disclosures is specifically incorporated by reference herein without disclaimer.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the field of medicine. More specifically, the invention relates to methods of obtaining novel plant compounds having therapeutic uses in mammals.
[0004] 2. Description of Related Art
[0005] Plants are valuable sources for the identification of novel biologically active molecules. One diverse class of molecules which has been identified in plants is the class of saponins. Saponins are high molecular weight compounds comprising glycosides with a sugar moiety linked to a triterpene or steroid aglycone. Triterpene saponins particularly have been the subject of much interest because of their biological properties.
[0006] Pharmacological and biological properties of triterpene saponins from different plant species have been studied, including fungicidal, anti-viral, anti-mutagenic, spermicidal or contraceptive, cardiovascular, and anti-inflammatory activities (Hostettmann et al., 1995). Saponins are known to form complexes with cholesterol by binding plasma lipids, thereby altering cholesterol metabolism (Oakenfull et al., 1983). Triterpene glycosides given in feed also have been shown to decrease the amount of cholesterol in the blood and tissues of experimental animals (Cheeke, 1971). Saponins have been found to be constituents of many folk medicine remedies and some of the more recently developed plant drugs.
[0007] The triterpene glycyrrhetinic acid, and certain derivatives thereof, are known to have anti-ulcer, anti-inflammatory, anti-allergic, anti-hepatitis and antiviral actions. For instance, certain glycyrrhetinic acid derivatives can prevent or heal gastric ulcers (Doll et al., 1962). Among such compounds known in the art are carbenoxolone (U.S. Pat. No. 3,070,623), glycyrrhetinic acid ester derivatives having substituents at the 3′ position (U.S. Pat. No. 3,070,624), amino acid salts of glycyrrhetinic acid (Japanese Patent Publication JP-A-44-32798), amide derivatives of glycyrrhetinic acid (Belgian Pat. No. 753773), and amide derivatives of 11-deoxoglycyrrhetinic acid (British Pat. No. 1346871). Glycyrrhetinic acid has been shown to inhibit enzymes involved in leukotriene biosynthesis, including 5-lipoxygenase activity, and this is thought to be responsible for the reported anti-inflammatory activity (Inoue et al., 1986).
[0008] Betulinic acid, a pentacyclic triterpene, is reported to be a selective inhibitor of human melanoma tumor growth in nude mouse xenograft models and was shown to cause cytotoxicity by inducing apoptosis (Pisha et al., 1995). A triterpene saponin from a Chinese medicinal plant in the Cucurbitaceae family has demonstrated anti-tumor activity (Kong et al., 1993). Monoglycosides of triterpenes have been shown to exhibit potent and selective cytotoxicity against MOLT-4 human leukemia cells (Kasiwada et al., 1992) and certain triterpene glycosides of the Iridaceae family inhibited the growth of tumors and increased the life span of mice implanted with Ehrlich ascites carcinoma (Nagamoto et al., 1988). A saponin preparation from the plant
[0009] Genistein, a naturally occurring isoflavonoid isolated from soy products, is a tyrosine kinase inhibitor that has been shown to inhibit the proliferation of estrogen-positive and estrogen-negative breast cancer cell lines (Akiyama et al., 1987). Inositol hexaphosphate (phytic acid), which is abundant in the plant kingdom and is a natural dietary ingredient of cereals and legumes, has been shown to cause terminal differentiation of a colon carcinoma cell line. Phytic acid also exhibits anti-tumor activity against experimental colon and mammary carcinogenesis in vivo (Yang et al., 1995). Some triterpene aglycones also have been demonstrated to have cytotoxic or cytostatic properties, i.e., stem bark from the plant
[0010] While the previous reports have identified triterpene compounds which have any of a number of uses, there still is a great need in the art for the identification of novel biologically active triterpene compounds. Many of these compounds are toxic to normal mammalian cells. Still further, the biological activities of previously identified triterpenes vary widely and many posses limited or varying degrees of efficacy in the treatment of any given human or mammalian condition. The great diversity of different triterpenes which have been identified and the great range of differences and unpredictability in the biological activities observed among even closely related triterpene compounds, underscores the difficulties which have been encountered in obtaining triterpenes which are potential therapeutic agents. Achieving the difficult goal of identifying novel triterpenes with beneficial biological activities could provide entirely new avenues of treatment for a diverse set of human ailments in which therapeutic options currently are limited.
SUMMARY OF THE INVENTION
[0011] The present invention relates to the novel use of
[0012] In one embodiment the present invention provides novel saponin compounds and mixtures thereof which may be isolated from the species
[0013] For preferred embodiments where the saponin contains a triterpene moiety, this triterpene moiety is typically an acacic or oleanolic acid or other structurally similar triterpenoid moiety. The triterpene or triterpene glycoside compositions may also typically comprise a monoterpene moiety or moieties and one of skill in the art will appreciate that the saponin compositions described herein may be further substituted with other chemical functionalities. Thus, the saponin compounds disclosed herein may comprise a triterpene moiety attached to at least one, and preferably two, three, or more, monoterpene moieties. When more than one monoterpene moiety is present, these moieties may each be attached (i) directly to the triterpene moiety, (ii) to a sugar, or other linking group, which is attached to the triterpene moiety, or (iii) to a monoterpene moiety which is attached to the triterpene moiety directly or through a sugar or other linking groups. Linking groups include sugars, acyl, amide, alkoxy, ketyl, alkyl, alkylene and other similar chemical moieties which would be apparent to one of skill in the art. The triterpene glycosides disclosed herein typically have a molecular weight in the range of 1800 to 2600 amu, or from at least 1800, 1900, 2000, 2100 amu to about 2200, 2300, 2400 or 2600 amu.
[0014] An important aspect of the invention provides the isolation of a mixture comprising one or more isolated saponins or triterpene glycosides that may be characterized by the following properties: a) isolatable from the tissues of
[0015] In particular embodiments of the invention, the triterpene composition may be characterized by the following properties: ability to induce cytotoxicity in a Jurkat cell with an IC
[0016] In still other embodiments of the invention, the apoptosis is measured by the reorganization of plasma membrane of a Jurkat cell by annexin binding. This may be measured by flow cytometry and the apotosis induced may be from 16-18%.
[0017] Another embodiments of the invention encompasses a mixture comprising one or more isolated triterpene glycosides characterized by the following properties: a) isolatable from the tissues of
[0018] Still other embodiments of the invention encompasses a mixture comprising one or more isolated triterpene glycosides characterized by the following properties: a) isolatable from the tissues of
[0019] In still other embodiments of the invention, the mixture comprising one or more isolated triterpene glycosides may be characterized by the following properties: a) isolatable from the tissues of
[0020] In further embodiments of the invention, the mixture comprising one or more isolated triterpene glycosides may be characterized by the following properties: a) isolatable from the tissues of
[0021] In yet other embodiments of the invention, the mixture comprising one or more isolated triterpene glycosides may be characterized by the following properties: a) isolatable from the tissues of
[0022] In still other embodiments of the invention, the mixture comprising one or more isolated triterpene glycosides may be characterized by the following properties: a) isolatable from the tissues of
[0023] An important aspect of the invention provides a nutraceutical composition comprising a triterpene glycoside composition in a pharmacologically acceptable medium such as a buffer, a solvent, a diluent, an inert carrier, an oil, a creme, or an edible material. In one embodiment of the invention, the nutraceutical composition may comprise dried and ground
[0024] In another aspect, the invention provides a process for preparing a composition comprising a mixture of one or more isolated triterpene glycosides, comprising: a) obtaining tissue from an
[0025] This process may include additional steps. For example, the process may further comprise isolating the composition from plant bagasse by filtration after the extracting. In a further embodiment, the process further includes the step of defatting the plant tissue with an organic solvent prior to extracting. The organic solvent may be any solvent suitable for defatting, such as hexane, dichloromethane, chloroform, ethyl acetate or mixtures thereof. In another embodiment, the process of isolation further comprises evaporating the solvent after the extracting.
[0026] This process may also comprise obtaining the mixture of the triterpene compositions by chromatographically isolating at least triterpene glycoside composition. Exemplary chromatographic techniques include liquid chromatography, MPLC, or BPLC. Although solvents which may be employed for the chromatographic isolation would be apparent to one of skill in the art, exemplary solvents include methanol, acetonitrile, water, and mixture.
[0027] In yet another aspect, the invention provides a process for preparing a composition comprising a mixture of one or more isolated triterpene glycosides, comprising: a) preparing a tissue culture comprising cells of an
[0028] In another aspect of the invention, the method further comprises additional steps, such as filtering plant bagasse from the triterpene mixture composition, isolating the triterpene mixture composition by liquid chromatography, and/or evaporating the solvent after the extracting step.
[0029] One aspect describes a method of continually propagating the tissues of an
[0030] Another aspect of the invention describes a method of continually harvesting an
[0031] An important aspect of this invention is a method of inhibiting the initiation and promotion of mammalian epithelial cells to a premalignant or malignant state comprising administering to a the mammalian cell a therapeutically effective amount of the nutraceutical compositions described above. In one embodiment, the epithelial cell is a skin cell, a colon cell, a uterine cell, an ovarian cell, a pancreatic cell, a prostate cell, a renal cell, a lung cell, a bladder cell or a breast cell. In a related embodiment, the mammal is a human. In yet another related embodiment, the mode of administering the nutraceutical is oral. In yet another related embodiment of the invention, the mode of administering the nutraceutical is topical.
[0032] The invention also encompasses a method of inducing apoptosis in a malignant mammalian cell, comprising administering to the cell a therapeutically effective amount of a nutraceutical composition described above. In one embodiment, the cell is a skin cell, a colon cell, a uterine cell, an ovarian cell, a pancreatic cell, a prostate cell, a renal cell, a lung cell, a bladder cell or a breast cell. In a related embodiment, the mammal is a human. In yet another related embodiment, the mode of administering the nutraceutical is oral. In an alternative embodiment, the mode of administering the nutraceutical is topical.
[0033] The invention also encompasses a method of preventing the abnormal proliferation of mammalian epithelial cells in vitro or in a mammal comprising administering to the mammalian cell or mammal a therapeutically effective amount of the nutraceutical compositions described above. In one aspect of the invention, the epithelial cells are crypt cells. In another aspect of the invention the epithelial cells are colon cells. In a related embodiment of the invention, the mammal is a human. In yet another related embodiment of the invention, the mode of administering the nutraceutical for in vivo application is oral.
[0034] The invention also contemplates a method of treating a mammal for inflammation, comprising administering to the mammal a therapeutically effective amount of the nutraceutical compositions described above. In a related embodiment of the invention, the mammal is a human.
[0035] The invention also comprises a purified triterpene compound comprising a triterpene moiety attached to a monoterpene moiety having the molecular formula:
[0036] or a pharmaceutical formulation thereof, wherein a) R
[0037] wherein R5 is selected from the group consisting of hydrogen, hydroxyl, C1-C5 alkyl, C1-C5 alkylene, C1-C5 alkyl carbonyl, a sugar, C1-C5 alkyl ester, and a monoterpene group.
[0038] In one embodiment of the invention, R
[0039] In one embodiment of the invention, R
[0040] Another embodiment of the invention describes a composition comprising a triterpene glycoside having the molecular formula:
[0041] or a pharmaceutical formulation thereof, wherein a) R
[0042] or a pharmaceutical formulation thereof is described.
[0043] Another aspect of the invention describes the purification of a composition comprising a triterpene glycoside having the molecular formula:
[0044] or a pharmaceutical formulation thereof wherein, a) R
[0045] or a pharmaceutical formulation thereof.
[0046] Yet another aspect of the invention describes the purification of a composition comprising a triterpene glycoside having the molecular formula:
[0047] or a pharmaceutical formulation thereof, wherein, a) R
[0048] Another aspect of the invention relates to a composition comprising a triterpene moiety, an oligosaccharide and three monoterpene units. In one embodiment the triterpene moiety is acacic acid or oleanolic acid.
[0049] An important aspect of the invention contemplates pharmaceutical preparations of the compounds purified and characterized. In one embodiment the pharmaceutical preparation is in a pharmacologically acceptable medium comprising a buffer, a solvent, a diluent, an inert carrier, an oil, a creme, or an edible material. In some aspects of the invention, the pharmaceutical composition is contemplated to further comprises a targeting agent. In related aspects of the invention, the targeting agent can direct the delivery of the pharmaceutical composition to an epithelial cell. In a related embodiment of the invention, the targeting agent comprises an antibody which binds to the epithelial cell.
[0050] In certain embodiments of the invention, the pharmaceutical composition comprises at least a second composition that can kill an epithelial cell.
[0051] The compounds of this invention have shown chemoprotective effects in mice exposed to the carcinogen DMBA. The invention therefore provides a method of inhibiting the initiation and promotion of a mammalian epithelial cell to a premalignant or malignant state in a mammal comprising administering to the mammal a therapeutically effective amount of the pharmaceutical compositions described above. In one embodiment of the invention, the epithelial cell is a skin cell, a colon cell, a uterine cell, an ovarian cell, a pancreatic cell, a lung cell, a bladder cell, a prostate cell, a renal cell, or a breast cell. In a related embodiment of the invention, the mammal is a human. In yet another related embodiment of the invention, the mode of administering the pharmaceutical is oral. In still another alternative embodiment of the invention, the mode of administering the pharmaceutical is topical. In still other alternative embodiment of the invention, the mode of administering the pharmaceutical is by intratumoral injection. In still another alternative embodiment of the invention, the mode of administering the pharmaceutical is intravenous. In still further alternative embodiments of the invention, the mode of administering the pharmaceutical comprises inhaling an aerosol.
[0052] The invention also contemplates the use of the pharmaceutical preparations of the invention in combination with other therapies. In one embodiment the other therapy comprises irradiating the epithelial cell with X-ray radiation, UV-radiation, γ-radiation, or microwave radiation.
[0053] The invention also envisions a method of inducing apoptosis in a malignant mammalian cell in a mammal comprising administering to the mammal a therapeutically effective amount of the pharmaceutical compositions described herein. In one embodiment of the invention, the cell is a skin cell, a colon cell, a uterine cell, an ovarian cell, a pancreatic cell, a lung cell, a bladder cell, a prostate cell, a renal cell, or a breast cell.
[0054] In one important aspect the invention provides a method of preventing the abnormal proliferation of a mammalian epithelial cell in a mammal comprising administering to the mammal a therapeutically effective amount of the pharmaceutical compositions described above. In one embodiment the epithelial cell is a crypt cell. In another embodiment of the invention, the epithelial cell is a colon cell. In a related embodiment of the invention, the mammal is a human. In yet another related embodiment of the invention, the mode of administering the pharmaceutical is oral. In still another alternative embodiment of the invention, the mode of administering the pharmaceutical is topical. In still other alternative embodiment of the invention, the mode of administering the pharmaceutical is by intratumoral injection. In still another alternative embodiment of the invention, the mode of administering the pharmaceutical is intravenous. In still further alternative embodiments of the invention, the mode of administering the pharmaceutical comprises inhaling an aerosol. The invention also contemplates the use of the pharmaceutical preparations of the invention in combination with other therapies. In one embodiment the other therapy comprises irradiating the epithelial cell with X-ray radiation, UV-radiation, γ-radiation, or microwave radiation.
[0055] The invention also contemplates a method of treating a mammal for inflammation comprising administering to the mammal a therapeutically effective amount of the pharmaceutical compositions of the triterpene compounds described herein. In a related embodiment of the invention, the mammal is a human. In yet another related embodiment of the invention, the mode of administering the pharmaceutical is oral. In still another alternative embodiment of the invention, the mode of administering the pharmaceutical is topical. In still further alternative embodiments of the invention, the mode of administering the pharmaceutical comprises inhaling an aerosol.
[0056] Another important aspect of this invention is a method of regulating angiogenesis in a mammal comprising administering to the mammal a therapeutically effective amount of the pharmaceutical compositions described. The method may be when the mammal is a human.
[0057] Although several of the methods describe herein are in vivo methods it is contemplated that in vivo the triterpene glycoside compounds will exhibit similar effects.
[0058] In addition to providing methods of preventing or treating cancer with the compounds of the invention, the inventors have provided a number of other uses for the compounds of the invention. In particular, the compounds of the invention may be used as solvents, antioxidants, anti-fungal and anti-viral agents, piscicides or molluscicides, contraceptives, antihelmintics, angiogenesis regulators, UV-protectants, expectorants, diuretics, anti-inflammatory agents, regulators of cholesterol metabolism, cardiovascular effectors, anti-ulcer agents, analgesics, sedatives, immunomodulators, antipyretics, as agents for decreasing capillary fragility, as agents to combat the effects of aging, as agents for increasing skin collagen, as agents for enhancing penile function and as agents for improving cognition and memory
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein:
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DETAILED DESCRIPTION OF THE INVENTION
[0110] The present invention seeks to overcome limitations in the prior art by providing novel biologically active triterpene glycoside compositions. In particular, the present inventors have identified and purified triterpene compounds from
[0111] The triterpene compounds of the invention were identified from a targeted screening of 60 plant extracts from selected leguminous species native to arid and semi-arid regions. Of the initial screening, one extract, designated UA-BRF-004-DELEP-F001 and isolated from
[0112] Further testing of the more purified extract further elucidated the biological activities of the extract. The purified extract demonstrated enhanced anti-tumor activity relative to the crude extract, in concentrations that exhibited little or no toxicity to normal human cells. The extract was still further shown to have a chemoprotective effect in mice exposed to carcinogens.
[0113] The plant from which the extract was isolated,
[0114] The present invention relates to the novel use of
[0115] II. Purification and Identification of the Triterpenes of the Invention
[0116] An important aspect in the use of plant extracts as pharmaceutical preparations is the characterization and determination of the individual active constituents. Such also is the case for triterpene saponin preparations, which often require sophisticated techniques for the isolation, structure elucidation and analysis of their components and glycosides. When biological testing of the pure compounds is to be performed, it is necessary to isolate them in sufficient quantity and purity.
[0117] Since triterpenes and other related saponins have relatively large molecular weights and are of high polarity, their isolation can be challenging. A problem involved in the isolation of pure saponins is the presence of complex mixtures of closely related compounds, differing subtly either in the nature of the aglycone or the sugar part (nature, number, positions and chirality of attachment of the monosaccharides). Difficulties also are encountered with labile substituents such as esters. For example, the major genuine soybean saponin, a γ-pyrone derivative (BOA), is only extracted by aqueous ethanol at room temperature. Extraction with heating (80° C.) leads to fission of the ester moiety and formation of soyasaponin I (Bb) (Kudou et al., 1992). In plants, saponins are accompanied by very polar substances, such as saccharides and coloring matter, including phenolic compounds and the like, are not easily crystallized, and can be hygroscopic, making it even more difficult to obtain crystals.
[0118] Characterization of pure saponins also is challenging because of the lack of crystalline material. Melting points are imprecise and often occur with decomposition. Therefore, determinations of sample purity will not generally be made only based on the melting point, optical rotation value or another physical constant. A better test of the purity of a saponin can be obtained by TLC or HPLC examination—if possible by co-chromatography with an authentic sample. The coloration of spots on TLC plates after spraying with suitable reagents is an additional indicator of potential individual components. For example, one of the triterpene glycosides of the invention, D1, has a HPLC retention time of 15.2 minutes. This is different from another related compound, elliptoside E, isolated from
[0119] (i) Chemical Purifications
[0120] Chemical purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of a plant extract into the triterpene glycoside compounds described herein. Having generally separated the compounds of the invention from plant material, the triterpene glycosides of interest may be further purified using the techniques described herein, for example, chromatographic techniques, to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure triterpene glycoside composition are specifically disclosed herein below.
[0121] Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of triterpene glycosides from plant material. In a preferred embodiment of the invention, the triterpene glycosides are purified from a plant of the family Leguminosae, or more preferably from the genus Acacia, and most preferably from the species
[0122] Generally, “isolated” will refer to an organic molecule or group of similar molecules that have been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which triterpene glycosides form the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the molecules in the composition.
[0123] There is no general requirement that the triterpene compositions of the invention always be isolated and provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. For example, the inventors envision the use of dried
[0124] (ii) Extraction and Preliminary Purification
[0125] Extraction procedures should be as mild as possible because certain saponins can undergo transformations including enzymatic hydrolysis during water extraction, esterification of acidic saponins during alcohol treatment, hydrolysis of labile ester groups and transacylation. Therefore, care should be taken to follow the individual steps in an isolation procedure, for example, in thin layer chromatography.
[0126] Although numerous variations are possible, current general procedures for obtaining crude saponin mixtures typically include extraction with methanol, ethanol, water or aqueous alcohol; a defatting step, generally with petroleum ether, performed before the extraction step or on the extract itself; dissolution or suspension of the extract in water; shaking or washing the solution or suspension with n-butanol saturated with water; and precipitation (optional) of saponins with diethyl ether or acetone. A dialysis step also can be included in order to remove small water-soluble molecules such as sugars (see, for example, Zhou et al., 1981; Massiot et al., 1988).
[0127] The most efficient extraction of dry plant material is achieved with methanol or aqueous methanol. Methanol is also used for fresh plant material. Although water is typically a less efficient extraction solvent for saponins (unless specifically water-soluble glycosides are desired) it has the advantages of being easily lyophilized and giving a cleaner extract. Depending on the proportion of water used for extraction, either monodesmosidic or bidesmosidic saponins may be obtained (Domon and Hostettmann, 1984; Kawamura et al., 1988). Fresh vegetable material contains active enzymes (esterases) which, when homogenized with a solvent, are able to convert bidesmosides into mono-desmosides. Even dry material may contain esterases which are activated in the presence of water. In the case of momordin I (a monodesmosidic oleanolic acid saponin) it was found that conversion to momordin II (the corresponding bidesmoside) takes place in water and in 30% and 60% methanol solutions, but not in 80% and 100% methanol solutions. On the contrary, homogenates of the fresh roots in methanol retained enzyme activity. However, the enzymes could be inactivated by first soaking the fresh roots in 4% hydrochloric acid and the bidesmoside was then shown to be the major component. It is, therefore, clear that the correct choice of extraction procedure is an extremely important first step.
[0128] Methods typically used to purify proteins, such as dialysis, ion-exchange chromatography and size-exclusion chromatography, are useful in partially separating saponins in aqueous solution from non-saponin components, but are generally ineffective in separating individual saponins because of the tendency of saponins to form mixed micelles. Hence, effective separation typically requires the use of organic solvents or solvent/water systems that solubilize the amphiphilic saponins as monomers so that the formation of mixed micelles does not interfere with separation.
[0129] A common problem observed for furostanol saponins is the formation of 22-OCH
[0130] (iii) Thin-Layer Chromatography (TLC)
[0131] The qualitative analysis of triterpene saponins by TLC is of great importance for all aspects of saponin investigations. TLC plates (usually silica gel) can handle both pure saponins and crude extracts, are inexpensive, rapid to use and require no specialized equipment. A number of visualization reagents are available for spraying onto the plates (Table 2). Methods of preparation of the most common reagents are as follows:
[0132] Vanillin-sulfuric acid (Godin reagent). A 1% solution of vanillin in ethanol is mixed in a 1:1 ratio with a 3% solution of perchloric acid in water and sprayed onto the TLC plate. This is followed by a 10% solution of sulfuric acid in ethanol and heating at 110° C.
[0133] Liebermann-Burchard reagent. Concentrated sulfuric acid (1 ml) is mixed with acetic anhydride (20 ml) and chloroform (50 ml). Heating at 85-90° C. gives the required coloration on the TLC plate.
[0134] Antimony(III) chloride. The TLC plate is sprayed with a 10% solution of antimony chloride in chloroform and heated to 100° C.
[0135] Anisaldehyde-sulfuric acid. Anisaldehyde (0.5 ml) is mixed with glacial acetic acid (10 ml), methanol (85 ml) and concentrated sulfuric acid (5 ml). This solution is sprayed onto the TLC plate, which is then heated at 100° C.
[0136] Spraying with vanillin-sulfuric acid in the presence of ethanol and perchloric acid, for example, gives a blue or violet coloration with triterpene saponins. With anisaldehyde-sulfuric acid, a blue or violet-blue coloration is produced on heating the TLC plate. Spraying TLC plates with a solution of cerium sulphate in sulfuric acid gives violet-red, blue or green fluorescent zones under 365 nm UV light (Kitagawa et al., 1984b). In some cases, simply spraying the plates with water is sufficient to reveal the saponins present. Additional spray reagents may be found in, for example, Stahl (1969).
[0137] The most frequently used solvent for TLC is chloroform-methanol-water (65:35:10), but other solvents are also useful. The solvent n-butanol-ethanol-ammonia (7:2:5) is especially useful for glycosides containing uronic acid residues; i.e., for very polar mixtures. Other widely used solvents include n-butanol-acetic acid-water (4:1:5; upper layer) or chloroform-methanol-acetic acid-water (60:32:12:8).
[0138] Systems employed for the TLC of glycoalkaloids typically include ethyl acetate-pyridine-water (30:10:30; upper phase). Visualization is with steroid reagents (anisaldehyde-sulfuric acid) or with alkaloid reagents (Dragendorff reagent, cerium(IV) sulphate). Other TLC solvents and visualization reagents are given by Jadhav et al. (1981) and Baerheim Svendsen and Verpoorte (1983).
[0139] Numerous quantitative determinations are possible with TLC. For example, the density of spots obtained with a suitable spray reagent can be measured directly using a densitometer. Alternatively, quantitative determinations are possible by carrying out TLC separations, scraping the relevant band off the plates (located, for example, with iodine vapor), eluting the saponin and measuring the UV absorbance after addition of a suitable reagent (e.g., concentrated sulfuric acid).
[0140] Reversed-phase TLC plates are commercially available and provide an excellent analytical method for saponins which is complementary to TLC on silica gel plates. Almost exclusive use of methanol-water and acetonitrile-water mixtures is made for developing reversed-phase plates (for example, Merck RP-8 or RP-18 HPTLC plates). Alternatively, DIOL HPTLC glass-backed plates may be used. These can be used with normal silica gel TLC-type solvents or with methanol-water and acetonitrile-water solvents, as for RP-TLC.
[0141] Exemplary reagents for TLC detection and for the spectrophotometric and colorimetric determination of saponins are listed below, in Table 2.
[0142] 1. Centrifugal Thin-Layer Chromatography (CTLC)
[0143] The CTLC technique is a planar method related to preparative thin-layer chromatography (TLC) but without the need to scrape bands off the TLC plate (Hostettmann et al., 1980). CTLC relies on the action of a centrifugal force to accelerate mobile phase flow across a circular TLC plate. The plate, coated with a suitable sorbent (1, 2 or 4 mm thickness), is rotated at approximately 800 r.p.m. by an electric motor, while sample introduction occurs at the center and eluent is pumped across the sorbent. Solvent elution produces concentric bands across the plate. These are spun off at the edges and collected for TLC analysis. Separations of 50-500 mg of a mixture on a 2 mm sorbent layer are possible.
[0144] A combination of CTLC with chloroform-methanol-water (100:30:3) and column chromatography has been described for the isolation of ginsenosides (Hostettmann et al., 1980). Saponins also have been obtained with chloroform-methanol-water mixtures on silica gel plates. Two protoprimulagenin A glycosides from
[0145] A Hitachi centrifugal liquid chromatograph, model CLC-5, has been described for use in separation of saponins. Chromatography is carried out with this machine on silica gel plates with the eluent chloroform-methanol-water (7:3:1 (lower phase)→65:35:10 (lower phase)). Using this technique a total of 1 g of semi-purified saponin fraction was chromatographed on the circular plate (Kitagawa et al., 1988; Taniyama et al., 1988).
[0146] (iv) Open-Column Chromatography
[0147] A number of the classical solvent systems employed for the silica gel column chromatography of saponins have previously been described and may be found in, for example, Woitke et al., 1970 and Adler and Hiller, 1985. Open-column chromatography is often used as a first fractionation step for a crude saponin mixture, but in certain cases may yield pure products. In general, though, the resolution is not high and complex mixtures are only partially separated. Other problems are the loss of material because of irreversible adsorption and the length of time required to perform the separations.
[0148] Silica gel chromatography with chloroform-methanol-water eluents is one of the most widely applicable techniques. When a biphasic system is used, the water-saturated chloroform phase is the eluent. Thus, a gradient of chloroform-methanol-water (e.g., 65:35:5→65:40:10) can be employed for the initial separation of a methanol extract of plant tissue on silica gel. Further chromatography on low-pressure columns can be used to yield, for example, a monodesmosidic molluscicidal saponin, while a bidesmosidic saponin can be obtained by silica gel column chromatography with a solvent system such as acetone-n-propanol-water (35:35:5) (Borel et al, 1987).
[0149] A complex mixture of triterpene glycosides has been isolated from the corms of
[0150] Extensive use of silica gel chromatography has also enabled the separation of the dammarane glycosides actinostemmosides A-D from
[0151] Certain ester saponins have been chromatographed on silica gel impregnated with 2% boric acid (Srivastava and Kulshreshtha, 1986; 1988).
[0152] As an addition to normal silica gel, coarse RP sorbents are now employed in the open-column chromatography of saponins. As long as the granulometry is not too fine and the columns not too long, gravity-fed columns are quite suitable. RP chromatography is generally introduced after an initial silica gel separation step and enables a change in selectivity for the substances being separated. Another possibility is to introduce the reversed-phase separation after a DCCC step (Higuchi et al., 1988).
[0153] 1. Open-Column Chromatography with Polymeric Sorbents
[0154] The use of dextran supports, as found in Sephadex column packings, has been current practice for a number of years. Sephadex LH-20 finds the most frequent application but the ‘G’ series of polymers is not without interest.
[0155] In recent work on the isolation of saponins, a new generation of polymers has been exploited, particularly in Japan. Diaion HP-20 (Mitsubishi Chemical Industries, Tokyo), for example, is a highly porous polymer which is widely used for the initial purification steps.
[0156] Typically, the polymeric supports are washed with water after loading the sample in order to elute monosaccharides, small charged molecules such as amino acids, and other highly water-soluble substances. Elution with a methanol-water gradient (or with methanol alone) is then commenced to obtain the saponin fractions. Other chromatographic techniques are employed for the isolation of pure saponins.
[0157] Elution of HP-20 gels with acetone-water mixtures has also been reported. For example, in the isolation of bidesmosidic glycosides of quillaic acid from the tuber of
[0158] For the isolation of fibrinolytic saponins from the seeds of
[0159] (v) Medium-Pressure Liquid Chromatography (MPLC)
[0160] When relatively large amounts of pure saponins are required, MPLC is very useful. Unlike commercially available LPLC equipment, gram quantities of sample can be loaded onto the columns, while separations are run at pressures of up to 40 bar. The granulometry of the support normally lies in the 25-40 μm range and separations are rapid, requiring considerably less time than open-column chromatography. A direct transposition of separation conditions from analytical HPLC to MPLC can be achieved on reversed-phase supports, thus facilitating the choice of solvent (Hostettmann et al., 1986).
[0161] As an example, molluscicidal saponins from
[0162] The isolation of saponins also can be achieved by combination of MPLC, for example using a LiChroprep RP-8 (25-40 μm, 46×2.6 cm) column with methanol-water mixtures in combination with rotation locular countercurrent chromatography (RLCC) (Dorsaz and Hostettmann, 1986). Another MPLC technique uses axially compressed (Jobin-Yvon) columns (Elias et al., 1991).
[0163] Examples of support-solvent combinations which are useful in the separation of triterpenes from plant extracts are given in Table 1, below.
TABLE 1 Applications of MPLC in the Separation of Triterpene Saponins Plant Support Solvent Reference Silica gel CHCl Gunzinger et al., 1986 (6:4:1) C-8 MeOH—H Gunzinger et al., 1986 C-8 MeOH—H Chemli et al., 1987 73:27) Silica gel CHCl Vidal-Ollivier et al., (61:32:5) 1989 C-18 MeOH—H Vidal-Ollivier et al., 80:20) 1989 Silica gel CH Hamburger and (80:20:2) Hostettmann, 1986 C-8 MeOH—H Hamburger and Hostettmann, 1986 C-8 MeOH H Borel and Hostettmann, 1987 C-8 MeOH—H Gafner et al., 1985 C-8 MeOH—H Dorsaz et al.