Title:
PROCESS TO OBTAIN AN EXTRACT FROM SOLANUM GENUS PLANTS
Kind Code:
A1


Abstract:
The present invention relates to an efficient process to obtain a Saponin and Sapogenin rich extract from fruits of Sonalum genus plants. The process presents a rational consumption of raw materials and energy resources, including polarity modifying agents which facilitate extraction and purification processes, which in turn increases its efficiency. The obtained Solasodine glycosides have biological antitumor and antibiotic activity, and can be used as precursors for the synthesis of pharmaceuticals, as active ingredients in medicinal compositions, cosmetics, veterinary products and bioproducts.



Inventors:
Schafer Elejalde, German Alfredo (Medellin, CO)
Palacio González, Guillermo León (Medellin, CO)
Aristizábal Alzate, Carlos Esteban (Medellin, CO)
Application Number:
14/660007
Publication Date:
06/02/2016
Filing Date:
03/17/2015
Assignee:
BIOPROCOL, BIOPROCESOS DE COLOMBIA SAS (Envigado, CO)
Primary Class:
Other Classes:
536/5
International Classes:
A01N45/00; A01N65/38; A61K8/63; A61K8/97; A61K31/706; A61K36/81; A61Q19/00; C07J71/00
View Patent Images:
Related US Applications:



Primary Examiner:
BERRY, LAYLA D
Attorney, Agent or Firm:
Sonapat LLC (Rockville, MD, US)
Claims:
1. A process for obtaining a solid extract rich in Solasodine glycosides from Solanum genus plants comprising: a) grind the plant fruits in a hydroalcoholic solution, leave it to rest during 36 to 120 hours and filter in order to separate the solid material from the hydroalcoholic extract; b) add a weak acid to the solid obtained in a), leave it to rest during 36 to 120 hours and then filter in order to separate the acidic extract; c) mix the extract obtained in a) with the acidic extract obtained in b); basify until a pH between 10 and 12 is reached to obtain a brown-colored solid and then add enough water to the obtained solid until it turns white; d) recover the solid obtained in c) via filtration, dissolve it in a hot hydroalcoholic solution and concentrate; e) add the solution obtained in d) in a polarity modifying agent until a solid Solasodine glucoside-rich extract is obtained.

2. A process according to claim 1, wherein a further step is executed by re-dissolving the precipitate obtained in e) in a hot solution of ethanol, isopropanol, butanol, isobutanol, or mixtures thereof, filtrating and crystalizing.

3. A process according to claim 1, wherein the Solanum genus plants fruits are selected from the group that consists of Solanum agrarium, Solanum atropurpureum, Solanum betaceum, Solanum quitoense, Solanum crinitum, Solanum hirtum, Solanum lycocarpum, Solanum lycopersicum, Solanum macranthum, Solanum mammosum, Solanum melongena, Solanum nigrum, Solanum psychotrioides, Solanum sycophanta and S. xanthocarpum.

4. A process according to claim 1, wherein the hydroalcoholic solution has a concentration range between 50% and 96% (v/v) of ethanol, propanol, butanol, isobutanol and mixtures thereof.

5. A process according to claim 1, wherein the fruit and hydroalcoholic solution weight ratio from stage a) is 1:2.

6. A process according to claim 1, wherein the polarity modifying agent added in stage d) is selected from the group consisting of water, saline solution, aluminum sulfate, alum stone, aluminum polychloride, and mixtures thereof.

7. A process according to claim 1, wherein the solid extract obtained in e) contains alkaloidal saponins such as Chaconine, Solamargine, Solasonine, Solanidine, Solanine, Sycophantine and Tomatine.

8. A solid extract obtained from Solanum genus plants, through a process according to claim 1.

9. A solid extract obtained from Solanum genus plants that comprise alkaloidal saponins, such as Chaconine, Solamargine, Solasonine, Solanidine, Solanine, Sycophantine and Tomatine.

10. A composition comprising a solid extract according to claim 8, along with a vehicle, adjuvants and/or acceptable excipients.

11. The use of a solid extract according to claim 8, for the synthesis of steroidal hormones, anti-cancer and anti-tumor products.

12. The use of a solid extract according to claim 8 as a biocidal agent for the inactivation of the Herpes Simplex virus, treatment of Leishmaniasis and skin ulcers caused by said disease, treatment of Schistosomiasis and inhibition of fungal growth.

13. The use of a composition that comprises one or more solid extracts according to claim 8, as an active ingredient in revitalizing, hypoallergenic, moisturizing, clarifying, anti-aging and other conditioning and skin care cosmetic formulations.

14. The use of a composition comprising at least one solid extract according to claim 8, for controlling harmful organisms for agriculture, animal breeding and human health.

15. The use of a composition according to claim 10, to treat chronic skin diseases, such as ulcers caused by Leishmanisis, Dermatophytosis, dermatitis, non-melanoma skin cancer and other related disorders.

Description:

FIELD OF THE INVENTION

The present invention relates to an efficient process to obtain a saponin and sapogenin-rich extract from Solanum genus plant fruits. The resulting dry extract is a mixture of Solasodine glucosides with an even particle size distribution and high purity, for several applications in the pharmaceutical, cosmetic, nutraceutic, agricultural and veterinary industries.

DESCRIPTION OF THE ART

There is permanent need in seeking new compounds to satisfy the demand for pharmaceutical, cosmetic and agricultural products, which has turned researchers and industries worldwide to develop bioprospecting, the study and sustainable use of plant biodiversity. In plants and vegetables, bioactive compounds are usually found in very low quantities, which demands an ongoing development of more efficient and profitable extraction processes (1), so that its realization and scaling is feasible, the environment is protected, the mass and energy resources are streamlined and humans and animals can take advantage of the benefits that biodiversity can offer.

Saponins are natural glycosides that are widely distributed in the plant kingdom, given they have been detected in more than 500 genera of plants, including the Solanum genus. These compounds are attributed with phytoprotection functions in plants because of their fungicidal, antibiotic, insecticidal and molluscicidal properties (2-5).

According to the nature of the aglycone skeleton of these glycosides, these can be classified into three groups: steroidal, triterpene and glycoalkaloids, where in the latter case, Solasodine glycosides are found. Among the saponins that can be found in plants of the Solanum genus, the Solasonine (I), the Solamargine (II), the Solanine (III), Chaconine (IV) Tomatine (V) and the Sycophantine (VI) are found, whose structures are illustrated below:

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Saponins present in the plants of the Solanum genus are useful as intermediates in the synthesis of contraceptive agents, corticosteroids, sex hormones and have also been proven effective in the treatment of various cancers in humans, showing high selectivity for tumor cells (5-9). As antibiotics, these glycosides can inhibit the growth of Gram positive bacteria (10-11) and have antiparasitic activity against microorganisms, such as Leishmania amazonenzis and several species of Schistosoma genus causing leishmaniasis and schistosomiasis (12,13).

It has been found that certain plant extracts of Solanum genus rich in saponins, have anti-inflammatory, hypotensive, hypoallergenic, antihistamine and antifungal activity against fungi and yeast that cause dermatiphytosis (3,12), and can be employed for the treatment of skin ulcerations caused by L. braziliensis, diabetes and cholesterol problems (14).

It has further been found that the hydroalcoholic extracts of plants of the Solanum genus, exhibit molluscicidal activity against Bulinus camerunensis, and B. truncates and L. cubensis (4, 5, 15-18).

Several processes have been described for obtaining saponins from plants of the Solanum genus, such as S. sodomaem, S. incanum, S. nigrum and S. robustum. In its vast majority, the processes described are based on crushed or ground dry fruits and extractions are performed using various solvents and purification steps. Thus for example, in U.S. Pat. No. 3,960,839 and U.S. Pat. No. 7,479,290, processes are mentioned wherein a first extraction takes place by mixing the crushed and milled dry fruits with methanol/water solutions, using a Soxhlet apparatus.

In the disclosed processes, a second extraction stage is also included by using pH 3-5 acetic acid or formic acid solutions, in which the residual plant material from the first extraction or the dry hydroalcoholic extract obtained was immersed, as mentioned in U.S. Pat. No. 7,479,290.

Other prior art documents mention the addition of strong bases (alkali, ammonium) to solutions resulting from the hydroalcoholic and acid extraction in order to precipitate the crude Solasodine glycosides. Thus, for example, the processes described in U.S. Pat. No. 7,479,290, U.S. Pat. No. 7,078,063 and EP00200229, concentrated or diluted ammonia is added until a pH of 8 to 10 is reached and the formation of precipitate is induced by heating which can be purified by silica gel chromatography, liquid-liquid extraction and/or crystallization processes. The final products are presented as dried extracts rich in Solasodine glycosides with purity greater than 65%.

Although several processes with different solvents and conditions tried for the extraction and purification of Saponins from Solanum genus plants can be found in prior art, the efficiency of the processes described is still very low and the purity of the obtained extracts cannot reach higher values in excess of 80%.

The process of the present invention involves easier, more selective and more efficient extraction and purification steps in the process of obtaining Solasodine glycosides from Solanum genus plants, using low toxicity solvents. By the process of the invention, an optimum and rational consumption of raw materials and energy resources is presented, given the fruits of Solanum spp can be in any stage of maturation and there is no need to undergo initial drying stages. A further advantage of the claimed process is that the glycosides extraction is performed at room temperature and its purification does not require complete evaporation of solvents, thus obtaining an extract having purity in excess of 92%.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides a process for obtaining dry extracts rich in Solasodine glycosides from Solanum genus plants, comprising initial stages of extraction with a hydroalcoholic solution and an acid solution, followed by alkalinization and precipitation of the glycosides. Subsequently, a polarity modifying agent is added to purify and obtain the dry extract.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. HPLC-UV chromatographic profile of the dry glycoside rich extract obtained from Solanum mammosum in Example 1.

FIG. 2. HPLC-DAD-MS chromatographic profile of the dry extract obtained in Example 1.

FIG. 3. Fragmentation profile of the compound with retention time (Rt=26.5 min) of the dry extract obtained in Example 1.

FIG. 4. Fragmentation profile of the compound with retention time (Rt=27.5 min) of the dry extract obtained in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an improved process for using fresh fruits of Solanum genus plants, a dry standardized extract containing Solasodine glycosides with purity greater than 92%. By the process of the invention, dried extracts rich in saponins and Solasodine glycosides comprising between 50% and 99% of Solasonine and Solamargine are obtained.

Initially, the fruits of one or several Solanum genus plants, undergo an extraction process using a hydroalcoholic solution of 50% to 90% (v/v) in a 1:1, 1:2 or 1:3 ratio, preferably 1:2. After a period of time between 36 and 120 hours, the liquid phase of the solid is filtered off. The obtained solid phase is then subject to extraction using an aqueous acid solution of 5% to 10% (v/v) until a pH value between 3 and 5 is reached, and is then allowed to stand for 36 to 120 hours, is further filtered to recover the liquid phase, and the solid phase is discarded.

Furthermore, the recovered hydroalcoholic and acidic solutions from the previous stages are mixed and insoluble impurities are removed by centrifugation or decantation, wherein later a strong base is added until a pH between 10 and 12 is reached with constant stirring to obtain a precipitate, which is further separated and sufficiently washed with water until it becomes reddish brown solid (precipitate A). Precipitate A can be redissolved in a acetic acid solution (10-15%) for qualitative tests in order to determine the presence of Solasodine glycosides.

Precipitate A is dissolved in a hydroalcoholic solution until a minimal presence of solid material in suspension is noticed. Subsequently, this solution is heated to a temperature between 45° C. and 55° C., after reaching said temperature, the solution is filtered while still hot to remove insoluble impurities and then allowed to stand until it reaches room temperature. The solid phase of the liquid is then removed by filtration and the solid is discarded. A polarity modifying agent is added to the obtained solution in order to form two phases (solid+liquid), wherein the solid (precipitate B) is recovered.

Finally, the obtained precipitate B is re-dissolved in a hydroalcoholic solution (90% to 96% v/v), which is heated until the solution reaches the saturation point and is then allowed to cool to room temperature, in order to allow the formation of crystals, which can be further separated by filtration. The retained product is dried to obtain a dry extract rich in Solasodine glycosides.

The obtained dry extract by carrying out the process of the invention is a fine white or off white color powder/crystals, which its composition comprises at least one compound selected from Solamargine, Solasonine, Chaconine, Solanidine, Solanine, Sycophantine, Tomatine, Solasodine mono and/or diglycosides with a purity exceeding 92%.

Identification of compounds present in the extract obtained by the process of the present invention may be performed according to conventional analytical methods.

In a preferred embodiment of the invention, the plant material used in the initial stage are fruits of plants of the Solanum genus in different stages of maturity, which should be shredded and/or chopped before being processed. Preferably, the fruits of plants of the Solanum genus are selected from the group consisting of: Solanum agrarium, Solanum atropurpureum, Solanum betaceum, Solanum quitoense, Solanum crinitum, Solanum hirtum, Solanum lycocarpum, Solanum lycopersicum, Solanum macranthum, Solanum mammosum, Solanum melongena, Solanum nigrum, Solanum psychotrioides, Solanum sycophanta and Solanum xanthocarpum.

For the purposes of this invention, the hydroalcoholic solutions used in the process are a water/alcohol mixture in concentrations ranging between 50% and 96% (v/v), where the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, isobutanol and mixtures thereof. The aqueous acidic solution is a mixture of water and an organic acid (e.g. acetic acid) at a concentration ranging between 10 to 15% (v/v). The used base for alkalizing and increasing pH is selected from the group of alkali metal hydroxides or ammonium hydroxide. The polarity modifiers are selected from the group consisting of water, sodium and potassium saline solution, aluminum sulfate, alum stone and aluminum polychloride.

In a further embodiment of the invention, the extract obtained is merged in an acceptable carrier, which is defined as one or more components and/or excipients intended to shape and/or give consistency to either a pharmaceutical, cosmetic, nutraceutic, veterinary or agrochemical composition, in such way that it generates stable and safe dosage compositions and/or forms.

The processed and obtained extract from plants of the Solanum genus by the process of the present invention can be used as precursor in the synthesis of various pharmaceuticals and medications, as the active ingredient in dermatological, cosmoceutic and/or cosmetic topical compositions, veterinary products and to manufacture bioproducts for pest control affecting the agricultural and agroindustrial activity. The obtained extract was shown to have molluscicidal activity against A. fulica snails.

In a preferred additional embodiment, the present invention relates to the use of the solid extract derived from plants of the Solanum genus, as a functional ingredient in cosmetic composition to condition and to protect the skin, and as the active ingredient to manufacture cosmoceutic and dermatological products to care and to treat chronic skin conditions.

In a further preferred embodiment, the invention relates to the use of the obtained solid extract from Solanum genus plants, as a precursor in the synthesis of corticosteroids, steroid hormones, antibiotics and anticancer drugs.

In a further preferred embodiment, the invention relates to the use of the obtained solid extract, as a biocide agent to control organisms affecting productivity in several crops with economic and social relevance.

The present invention will be presented in detail through the following examples, which are only provided for illustrative purposes and not intended to limit the scope of the present invention.

EXAMPLES

Example 1

Obtaining a Dry Extract from Solanum mammosum

Approximately 6 kilograms of fresh and ripe fruits of S. mammosum were ground in 20 liters of an ethanol-water (70% v/v) solution. The immersed plant material in the hydroalcoholic solution was stirred and left for digestion during 80 hours at room temperature. Once the digestion time was completed, the plant material was separated from the hydroalcoholic solution by filtration. The trapped plant material in the filter medium was mixed with a solution of 12% v/v acetic acid until a pH of 4 was reached, followed by stirring at room temperature. After 120 hours of acid digestion passed, the solution was filtered, the plant material was discarded and the supernatant was stored in a glass container for further processing.

The hydroalcoholic and acid solutions obtained in the preceding phases were mixed and homogenized at room temperature. The insoluble contaminating material was separated by centrifugation at 4000 rpm for 10 min. The obtained solution after centrifugation was mixed and stirred at room temperature with sufficient sodium hydroxide (10% v/v) until a pH value of 10 was reached. A brown precipitate was obtained, which was mixed and stirred with sufficient water at room temperature. Once the separation of a liquid and solid phase (precipitate) was clearly defined, the latter was separated from the liquid by decantation. This process was repeated 5 times, obtaining a brown precipitate (precipitate A).

Precipitate A was mixed with sufficient ethyl alcohol (96% V/V) until complete dissolution was reached, with a minimal presence of solid material in suspension. The obtained solution was heated and stirred until a temperature of 50° C. Immediately, the solution was hot filtered to remove unwanted insoluble impurities.

The obtained liquid in the previous step was mixed and stirred at room temperature with a polarity modifying solvent, a NaCl 10% w/v salt solution. The insoluble white solid (precipitate B) obtained after the addition of the polarity modifying solvent in the medium was recovered by filtration. The retained solid was re-dissolved in a 96% (v/v) hydroalcoholic solution and concentrated by solvent evaporation.

After turbidity of the solution was observed, the solution was left at rest to obtain the glycosides of interest via crystallization. The obtained crystals are separated by filtration, dried at room temperature and ground to a fine white powder, with high purity and rich in Solasodine glycosides, which was stored in containers and appropriate conditions to ensure stability. 15.5 grams of dry extract was obtained, giving a yield of 0.26%. Table 1 shows the yield and purity of the dry extract obtained is displayed.

TABLE 1
% YieldPurity (HPLC-MS Analysis)
(Wet base)% Solasonine% Solamargine% Total Purity
0.2640.358.098.3

Example 2

Qualitative and Quantitative Identification of the Compounds within the Extract

In order to determine the presence of the Solasodine glycosides, 100 mg of precipitate A from Example 1 was weighed and dissolved in 15 mL of a 10% acetic acid solution. The solution was divided into three fractions of 5 mL and 5 drops of Dragendorff, Mayer and Wagner reagents were added to each fraction (19, 20). The results are illustrated in Table 2.

TABLE 2
REAGENTOBSERVATIONRESULT
DragendorffFormation of an orange-reddishPositive for
colored precipitateSolasodine
glycosides
MayerFormation of a cream coloredPositive for
precipitateSolasodine
glycosides
WagnerFormation of a brown-reddishPositive for
color precipitateSolasodine
glycosides

With the use of High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS), the major components of the extract obtained in Example 1 were identified. Identification and quantification of the purity of the Solasonine sample was carried out in a HPLC chromatograph with an Agilent 1100 quaternary pump, UV detector and RP-18 250 mm×4.6 mm×5.0 um Alltech® column. The mobile phase used was acetonitrile/phosphate buffer 36.5:63.5 with an isocratic mode of 1.0 mL/min. A Solasonine standard of 95.18% purity (Chengdu Biopurify Phytochemicals LTD) was used.

The identification and estimation of the compounds was supplemented with HPLC (MS) in an Agilent 1200 liquid chromatograph with a RP-18 250 mm×4.6 mm×5.0 um Alltech column. The mobile phase used was a mixture of water/acetonitrile/Isopropanol 94.5:5:0.5 with an isocratic flow of 1.0 mL/min. The mass detector was configured in positive ionization mode with a mass range from 100-1500 m/z.

FIG. 1 illustrates the HPLC-UV chromatographic profile of the sample. One of the peaks shown in this figure corresponds to Solasonine, which has an area ratio of 40.3% in the retention time (Rt)=16.705 min. The other peak with retention time (Rt)=20.222 min and an area ratio of 58.0%, corresponds to Solamargine. FIG. 2 shows the chromatographic profile by HPLC-DAD-MS.

The first compound (Rt=26.5 min) corresponds to Solasonine, while the second compound (Rt=27.5 min) was identified as Solamargine, based on analysis of fragmentation profiles illustrated in FIG. 3 and FIG. 4, respectively.

Example 3

Preparing Cosmetic Compositions

Approximately 100 mg of each dry extract obtained using the present invention from S. melongena, S. quitoense y S. lycopersicum fruits, are mixed and crushed until obtaining a white, crystalline, homogeneous consistent and even particle sized powder.

The obtained powder is incorporated with suitable vehicles and excipients until obtaining a composition that can be liquid, semisolid or solid at a concentration between 0.0001% and 1.0%. The composition(s) can be used as depigmenting, sunscreen, anti-inflammatory, antibacterial and anti-fungal agents, to treat chronic skin conditions, such as non melanoma skin cancer, leishmaniasis and other disorders caused by adverse environmental factors, pollution and solar radiation.

Example 4

Evaluation Test of Acute Dermal Toxicity

Acute Dermal Toxicity (TDA) was determined following the protocol indicated in international guide OECD 402, in order to establish possible adverse or harmful effects that could result, in Wistar rats, from topical application of a Solanum genus plant dry extract, obtained according to Example 1. For testing, 13 healthy adult females and with intact skin were used, which were divided into three groups (Table 3).

The first group (Group A) 20 mg of a semisolid formulation containing the extract obtained in Example 1 at a concentration of 0.01% w/v was applied, in a shaved skin area of 10 cm2, while the second group (group B) underwent 20 mg of the same semisolid formulation, but at a concentration of 0.05% w/v. The third group was not applied any formulation and was taken as control. The results are illustrated in Table 3.

TABLE 3
AnimalsAnimals
NumberwithAnimalskilled
oftoxicityfoundduring
Groupanimalssignsdeadthe test
A5000
B5000
Control3000

None of the animals tested in the assay showed signs of pain or suffering, and there was no evidence of clinical signs or changes in normal behavior, confirming that the composition containing the extract rich in glycosides of Solasodine does not cause systemic toxicity or other adverse effects.

Example 5

Biological Activity Assay

The molluscicidal activity of a formulation for controlling Giant African Snail (A. fulica) was determined. The formula comprises several Solanum genus fruit extracts according to Example 1.

The assay was conducted in La Mesa, Támesis, Antioquia, Colombia, wherein A. fulica snails having sizes between 4 and 7 cm in length were collected. Nine of those collected snails underwent product spraying, at a concentration of 50 ppm. Another nine snails were sprayed with deionized water and left under supervision. They were taken as control.

After a period of between 3 and 5 minutes, the immediate reactions of snails, including a slime overproduction and the destruction of the membranes were observed. After 12 hours, the snails sprayed with the product showed no signs of life, and snails sprayed with deionized water and used as control, did not show any adverse reaction.

REFERENCES

  • 1. Lapkin, A., Adou, E., Mlambo, B., Chemat, S., Suberu, J., Collis, A., Clark, A., Barker, G. 2014. Integrating medicinal plants extraction into a high-value biorefinery: An example of Artemisia annua L. C. R. Chimie. 17. 232-241.
  • 2. Francis, G., Kerem, Z., Makkar, H. P. S., Becker, K. 2002. The biological action of saponins in animal systems: a review. Br J Nutr. 88: 587-605.
  • 3. Sparg, S. G., Light, M. E, Van Staden, J. 2004. Biological activities and distribution of plant Saponins J. □Ethnopharmacol. 94, 219-243.
  • 4. Silva, T. M., Batista, M., Camara, C. A., Agra, M. F. 2005. Molluscicidal activity of some Brazilian Solanum spp. (Solanaceae) against Biomphalaria glabrata. Annals of Tropical Medicine and Parasitology, 99, 419-425.
  • 5. Hostettmann, K. & Marston, A. 1995. Saponins Chemistry and Pharmacology of Natural Products. Cambridge University Press, pp. 564.
  • 6. Vincken, J. P., Heng, L., Groot, A., Gruppen, H. 2007. Saponins, classification and occurrence in the plant kingdom. Phytochemistry. 68, 275-297.
  • 7. Amir, M. & Kumar, S. 2004. Possible industrial applications of genus Solanum in twenty first century. A review. J. Sci. Ind. Res, 63, 117-124.
  • 8. Cham, B. E. 2008. The Eggplant Cancer Cure: A treatment for skin cancer and new hope for other cancers from Nature's Pharmacy. Smart Publications, pp. 122.
  • 9. Chase, T. 2011. Curaderm BEC5 for Skin Cancers, Is It? An Overview. Journal of Cancer Therapy, 2, 728-745.
  • 10. Kannabiran, K., Mohankumar, T., Gunaseker, V. 2009. Evaluation of Antimicrobial Activity of Saponin Isolated From Solanum Xanthocarpum and Centella asiática. IJNES, 3 (1), 25-28.
  • 11. Azimi, H., Fallah-Tafti, M., Khakshur, A., Abdollahi, M. 2012. A review of phytotherapy of acne vulgaris: Perspective of nepharmacological treatments. Fitoterapia. 83, 1306-1317.
  • 12. Tiossi, R. F., Da Costa, J. C., Miranda, M. A. Praça, F. S., McChesney, J. D., Bentley, M. V., Bastos, J. K. 2014. In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits. Eur J Pharm Biopharm. [Online early access]. DOI: http://dx.doi.org/10.1016/j.ejpb.2014.01.010
  • 13. Miranda, M. A., Magalhães, L. G., Tiossi, R. F., Kuehn, C. C., Oliveira, L. G., Rodrigues, V., McChesney, J. D., Bastos, J. K. 2012. Evaluation of the schistosomicidal activity of the steroidal alkaloids from Solanum lycocarpum fruits. Parasitol Res. 111(1): 257-62.
  • 14. Vieira, G., Ferreira, P. M., Matos, L. G., Ferreira, E. C., Rodovalho, W., Ferri, P. H., Ferreira, H. D., Costa, E. A. 2003. Anti-inflammatory effect of Solanum lycocarpum fruits. Phytother. Res. 17, 892-896.
  • 15. Larhsini, M., Sebbane, R., Kchakech, H., Markouk, M., Bekkouche, K., Abbad, A., El Abdouni, M., Sterner, O. 2010. Screening of some Moroccan plant extracts for molluscicidal activity. Asian J. Exp. Biol. Sci. 4, 964-967.
  • 16. Perez, M. P. 1998. Actividad molusquicida del Paraiso (Melia azedarach L.) (Meliaceae) sobre Lymnaea cubensis, molusco vector de Fasciolosis. Rev. Saúde Pública. 32 (3), 262-266.
  • 17. Ndamukong, K. J., Ntonifor, N. N., Mbuh, J., Atemnkeng, A. F., Akam, M. T. 2006. Molluscicidal activity of some Cameroonian plants on Bulinus species. East African Medical Journal. 83, 102-109.
  • 18. Hammami, H., Mezghani-Jarraya, R., Damak, M., Ayadi, A. 2011. Molluscicidal activity of various solvent extracts from Solanum nigrum var. villosum l. aerial parts against Galba truncatula. Parasite. 18, 63-70.
  • 19. Prakash, S. & Jain, A. 2011. Antifungal activity and preliminary phytochemical studies of leaf extract of Solanum nigrum linn. Int J Pharm Pharm Sci. 3 (4), 352-355.
  • 20. Sayyed, M., Khan, M., Devanna, N., Syed, Y. H., Ansari, J. A. 2013. Pharmacognostical and phytochemical investigations of the whole plant of Swertia chirata and Hemidesmus indicus. J. Pharm. Bio. Sci. 4, 141-145

It should be understood that the present invention is not limited to the described and illustrated embodiments, for as will be apparent to one skilled in the art, variations and possible modifications that do not depart from the spirit of the invention, which is only found defined by the following claims: