Isolation of Solasodine from the Unripe Fruits of Solanum xanthocarpum Schrad and Wendl. (Solanaceae) and it’s Anti Cancer Activity against HeLa and U937 Cell Lines.
1Sarthak Bhattacharya, 2Seema Kohli, 3Amar Singh Chaudhary. 1PhD Research Scholar (Pharmacy) under Uttarakhand Technical University, Dehradun, India. 2Department of Pharmacy, Kalaniketan Polytechnic College, Jabalpur, India. 3College of Pharmacy, Bramhanand Group of Institutions, Bulandsahar, India.
Abstract as a precursor for synthesis of steroidal drugs. Solasodine showed selective cytotoxicity against cervical cancer cell line Recent advancements have shown that solasodine, a nitrogen (HeLa) and human myeloid leukemia cell line (U937). This containing steroidal glycoalkaloid (aglycone) obtained from compound has been isolated from the Solanaceae family by different parts of the genus Solanum (Solanaceae). Solanum different techniques but has never previously been isolated xanthocarpum, Solanum khasianum are the main plant by this technique. More over this compound is shown to be sources for solasodine. Solasodine has various therapeutic a cytotoxic drug. effects on different body systems. Earlier solasodine was used in the drug industries for the synthesis 16-DPA, a novel precursor for anti-fertility and anti-inflammatory agents. Several Introduction studies and articles recently published clearly indicate that therapeutic potential of solasodine should be explored to see Plant materials containing saponins have long been more multiple effects on different types of ailments. Solasodine, used in many parts of the world for their various the principal aglycone is being isolated by different techniques. pharmacological properties. Saponins as glycosides In the present study, it is done by two phase system containing are hydrolyzed by acids to give aglycone (sapogenin) aqueous (aq.) mineral acid- organic aq. immiscible solvent and various sugars. According to the structure of the mixture less than 100°C using single step in which direct acid aglycones or sapogenins, two kinds of sapogenins are hydrolysis-extraction of the glycosides of the plant material is recognized mainly i.e. steroidal and non-steroidal. being carried out and aglycone solasodine is obtained by alkali Phytochemical surveys have shown their presence treatment after the hydrolysis of the glycoside. The present in many monocotyledonous families, particularly process can be applied to obtain solasodine from various Dioscoreaceae, Amaryllidaceae and Liliaceae. In the solasodine bearing plants of Solanaceae family. It is a suitable dicotyledonous, the families include Leguiminosae and method for continuous production of solasodine which serves Solanaceae. Steroidal saponins are of great importance and interest owing to their relationship with such compounds as sex hormones; cortisone etc. steroidal saponins are of two types i.e. nitrogen containing and non-nitrogen containing. Nitrogen containing steroidal saponins is known as steroidal glycoalkaloids. Steroidal Key words: Solasodine, Solanaceae, Steroidal Glycoalkaloid, glycoalkaloids are of particular interest in drug industries, Solasonine, Direct Acid Hydrolysis Extraction, Anticancer as they are used as a starting material for the synthesis Activities, HeLa cell line and U937 cell line. of ant fertility and anti-inflammatory steroidal drugs.1
Address for Correspondence: Sarthak Bhattacharya, Guru Ramdas Khalsa Institute of Science & Technology (Pharmacy), Barela, Jabalpur 483001 M P. E-mail: [email protected] Phone: + 91 9748295438 14 25 Fax: + 91 761 2410036. 199 Sarthak Bhattacharya
Solasodine, nitrogen containing steroidal glycoalkaloid Experimental obtained from different parts of the genus Solanum (Solanaceae) has different therapeutic effects. Earlier Plant material solasodine was used in the drug industries for the synthesis 16-DPA, a novel precursor for anti-fertility and anti-inflammatory steroidal drugs. Solasodine has Collection of plant material biological activities such as antifungal, insect growth regulation and enzyme inhibition property,2 it heals The fresh unripe berries of Solanum xanthocarpum Schrad herpes skin lesions,3 inhibits Trypanosoma cruzi growth in and Wendl. (Solanaceae) (Fig. 01) were collected in the vitro, inhibits proliferation of murine spleen cell cultures months of July-September from local areas of Jabalpur to mobilize gucocorticoids from the adrenals of rats and (near the road side of the college) and authenticated exhibit an antihepatotoxic effect on mice. Solasodine by Dr. O. P. Chaubey, Senior Scientist-State Forest has an antiarthero-sclerotic effect on cholesterol fed Research Institute (SFRI), Jabalpur, India. A voucher rabbits.4, 5 It is having hypotensive effect on cat and specimen (Voucher Specimen no.00010) was deposited anti spermatogenic effect on dogs.6 Solasodine acts in Department of Pharmacognosy, GRKIST (Pharmacy) on female genital tract system including in gestation, and Plant Taxonomy -SFRI, Jabalpur, (M.P.) for future pregnancy and in male, act on testes androgen production reference. The part of plant was shade dried at room followed by low level of sialic acid and epididymides and temperature. The dried berries were reduced to a coarse shrunken leydig cell nuclei.7, 8, 9 Solasodine inhibits mouse powder, using a grinder and passed through sieve no. 40 lymphocyte proliferation induced by phytohemagglutinin for isolation purpose. in a dose-dependent manner. Thus, solasodine appears to have immunosuppressive activity. Solasodine is used as a potential ecdysone antagonist.10 Solasodine has antiurolithiatic and natriuretic activity.11 It shows cytotoxic activities in vitro against variety of cancer cell lines,12 antiproliferative activities against human colon (HT29) and liver (HepG2) cancer cells. According to Chemical Land Inc. Solasodine is a natural precursor to prepare steroidal hormones and drugs which used for contraceptives, arthritis and behavioral disorders. Solasodine earlier was used as starting material to prepare steroid compounds which present glucocorticoid-like effects. Solasodine shows DNA-damaging activity with MIC values of 62.5 and 125 µg.mL-1 respectively against Staphylococcus aureus for antibacterial activity.14 Solasodine is also having analgesic and antinociceptive activity.15
Solasodine, the principal aglycone is being isolated by different techniques. It is done by two phase system containing aq. mineral acid- organic aq. immiscible Fig. 01 Solanum xanthocarpum Schrad & Wendl. solvent mixture less than 1000C. In the present study, in a single step the direct acid hydrolysis of the glycosides of the plant material is being carried out and aglycone Extraction and isolation of Solasodine solasodine is obtained by alkali treatment after the hydrolysis of the glycoside Solasonine. Thus the present Fresh green –yellow unripe fruits of Solanum process is suitable for continuous production of aglycone xanthocarpum Schrad & Wendl.(Solanaceae) were (alkaloid) solasodine from various solasodine bearing collected. Sap portion next to seeds were retained and plants of Solanaceae family. Solasodine is being prepared dried at room temperature and then reduced to coarse from finely ground form of fruits of Solanum xanthocarpum powder, were transferred to 5 liters round bottom flask. Schrad and Wendl. (Solanaceae) using aq. HCl-Toluene The powdered mass was defatted with petroleum ether to mixture followed by alkalinization. yield greenish yellow oil, which is rejected as it is devoid
Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 3, July 2013 pp 199 -213 200 Isolation of Solasodine from the Unripe Fruits of Solanum xanthocarpum .... of glycoalkaloid. Defatted material was then refluxed with Source : Steroidal alkaloid isolated from various 32% v/v Conc. HCl, water and toluene in 1:2:3 ratios for Solanum species 5 hours and pH was recorded 3-4. The reaction mixture was subsequently alkalinized with 40% w/v NaOH, pH Crystal structure: Hexagonal plate from MeOH or by was recorded 9-10 and again refluxed under stirring for 2 sublimation in high vaccum hrs. After reflux two phases were found- one is upper pale yellow toluene layer and the other was lower dark brown aq. layer. The upper toluene layer was siphoned out and Physical properties of Solasodine lower aqueous layer was extracted with toluene 3 times. The whole toluene layer was kept in refrigerator for seeing Melting Point : 198-2000c the presence of solasodine. But for direct crystallization, 25 0 0 the toluene extract was acidified with 25% v/v acetic acid Optical rotation: [α] D =-98 , [α]D= -113 (c 0.14 and stirred well for I hr. On standing two phases were in MeOH) formed, upper pale yellow toluene layer and lower acetic acid layer. The process was repeated for second time too. pKb value : 6.30 The pH was recorded was 3.5.Finally acetic acid layer was alkalinized with 25% v/v NH4OH slowly, pH was UV max (in MeOH): 206 nm checked 10. The mixture was briefly heated and then cooled at room temperature. The colorless solasodine Solubility : freely soluble in benzene, pyridine and was precipitated and filtered off and washed with cold chloroform. Insoluble in ether, water and also finally dried in air (colorless crystalline powder, 2.1 grams). Solubility at 300c (mg/ml) : MeOH=9.5, 95% EtOH=5.0, Acetone=1.5, n-hexane= < 1.0 water=<1.0. Chemical Profile of Solasodine IR (KBr) : 3400(3346), 2950(2931), 2880(2758), 1733 Drug Name: Solasodine (Fig. 02) (1733), 1693 (1592) cm-1.
+ Scientific Name: (3β, 22α, 25R) - Spirosol-5-en-3- HRMS-ESI : m/z [M + H] calculated for C27H43NO2: ol, Solasod-5-en-3β-ol, Δ-5-20βF; 22αF, 25αF, 27- 413.63; found: 413.63. a2aspirosan-3β-ol Phytochemical Screening
Preliminary phytochemical analysis was carried out to identify the phytoconstituents such as tannins, alkaloid, steroid, phenols, terpenoid, flavonoid and anthraquinones. Various screening test of the extract was carried out for estimation of plant constituents. (Table No. 01a)
Table No. 01a
Extract Positive Tests for Fig. 02 Solasodine. Ethanolic extracts Flavonoids, saponins, steroids, Other name: Solacarpidine, Solanidine-S, Pura alkaloids, phytosterols, fixed puridine oils and fats Toluene+ Conc. Flavonoids, saponins, 5% mix. steroids, glycosides, phenolic Molecular Formula: C27H43NO2 compounds, fixed oils and fats Molecular Weight: 413.63 Da
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Rf Value determination: (Table No. 01b) (cell counting) and the proliferation of cells (cell culture assays). It can also be used to determine cytotoxicity of Table No. 01b potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth. Color of spot No. of Color of Rf after spray MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, spot the spot value Iodine vapor 5-diphenyltetrazolium bromide, a yellow tetrazole), is 1 (Test) No color Orange to red 0.6 reduced to purple formazan in living cells. A solubilization solution (usually dimethyl sulfoxide, an acidified ethanol 2 (Test) No color Orange to red 0.6 solution, or a solution of the detergent sodium dodecyl 3 (Std.) No color Orange to red 0.6 sulfate in diluted HCl) is added to dissolve the insoluble purple formazan product into a colored solution. The Solvent System: Toluene: Ethyl Acetate: Diethyl Amine absorbance of this colored solution can be quantified by (7:2:1) measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. The absorption Developing Reagent: Dragendroff’s Reagent followed maximum is dependent on the solvent employed. by 10% Sulphuric acid in MeOH.
Cell lines, Chemicals and Biochemicals
He La and U 937 cell lines are purchased from National Centre for Cell Sciences (NCCS), Pune, India and the experiment was performed in Indian Institute of Chemical Biology (CSIR-Govt. of India). MTT assay of Solasodine
MTT (Roche applied sciences), DMEM (Dulbecco’s MTT assay was done to evaluate cell viability. The cells Modified Eagles medium, high glucose), DMEM were plated in 96 well plates and treated with or without (Dulbecco’s Modified Eagles medium, low glucose), different concentrations of drug for 24 h. Four hours after FBS (Fetal Bovine Serum) (Bioclot) were purchased. the addition of MTT, cells were lysed and formazan was Doxorubicin and ARA-C were supplied by local drug solubilized with acidic isopropanol and the absorbance of distributor. Unless stated otherwise, all other reagents the solution was measured at 595 nm using an ELISA reader. were from Sigma-Aldrich.
Solasodine was isolated from the Unripe Fruits of Assessment of cell morphology Solanum xanthocarpum Schrad and Wendl (Family: Solanaceae) using direct acid hydrolysis of the glycosides Cells (3 × 104/well) grown in 6-well plates in DMEM of the plant material. The assays were performed supplemented with 10% FBS for 24 hours were treated according to the standard procedure. with or without derivatives. Morphological changes were observed with an inverted phase contrast microscope (Model: OLYMPUS 1X 70, Olympus Optical Co. Ltd., MTT assay of solasodine Sibuya-ku, Tokyo, Japan) and images were acquired. Standard drug used was Doxorubicin.
Introduction about MTT Assay Fluorescence microscopy The MTT assay is colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes For the detection of nuclear damage or chromatin (XTT, MTS, and WSTs) to formazan dyes, giving a purple condensation, the treated and untreated cells were color. A main application allows assessing the viability stained with 10 µg.mL-1 of DAPI. (Fig. 03a, 03b and 03c)
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The IC50 value of solasodine was calculated. (Table no. 02) Cell Growth Inhibition Assay of Solasodine
Table No. 02 Cell growth inhibition studies of solasodine were carried out according to Sur et al.16 The cells were resuspended in 5 IC50 values of the compound “Solasodine” on HeLa a concentration of 2*10 /ml in RPMI and the cells were cell lines plated in the multiple sterile plate. The drug “Solasodine” and a standard anticancer drug “ARA-C” at different concentrations were added to the cells of human myeloid Cell Lines IC 50 leukemia cell lines (U937) separately and incubated for 24 hrs at 370c in CO incubator. Viable cells were judged HeLa 12.17±3.3 2 by trypan blue exclusion and counted by haemocytometer.
Fig. 03 a. MTT assay Fig. 03 b. MTT assay
Fig. 03 c. Morphological changes seen under light microscope in 20X: Nuclear changes seen under fluorescence microscopy after DAPI staining respectively.
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The result is shown in the table number 3. The test drug inhibition at 40 µg.mL-1 compared to ARA-C. (Table Solasodine showed greater percentage of inhibition than no. 03) the standard drug ARA-C. Solasodine showed 94.96%
Table No. 03: Cell growth Inhibition Studies of Solasodine against U937 cell lines
Drug or Control No of Cancer Cells(*104) Average Cancer Cells(*104) % Inhibition= (C-A or T/C)*100 C1 168 C2 140 159 100 C3 200 C4 128 A1 100 A2 96 79 50.31 A3 68 A4 52 T1(0.05µl) 36 T2 124 77 51.57 T3 88 T4 64 T1(1.00µl) 24 T2 148 84 47.16 T3 100 T4 64 T1(2.50µl) 32 T2 48 43 72.95 T3 24 T4 64 T1(5.00µl) 12 T2 28 40 74.84 T3 68 T4 52 T1(10.0µl) 96 T2 20 62 61.00 T3 84 T4 48 T1(20.0µl) 32 T2 12 20 87.42 T3 16 T4 20 T1(40.0µl) 4
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T2 18 8 94.96 T3 12 T4 8 T1(60.0µl) No Cancer Cells - - T2 No Cancer Cells - - T1(80.0µl) No Cancer Cells - - T2 No Cancer Cells - - T1(100.0µl) No Cancer Cells - - T2 No Cancer Cells - -
C1, C2, C3, C4 are the controls (without drug). A1, A2, A3, A4 are the standard anticancer drug “ARA-C”. T1, T2, T3, T4 are the test drug “Solasodine” at different concentrations.
Result solasodine glycosides particularly tomatidenol, diosgenin. The unsaponifible matter of fruit contains two sterols one The Rf value of isolated compound solasodine was 0.6, M.P of which is carpesterol. Solasodine, one of the aglycone (198-200°c), Physical characteristics (colorless crystalline (alkaloid) is obtained from different species of Solanaceae Solid) and FTIR IR V max (3400, 2950,2880,1733,1693 by different methods. The developed process is applicable cm-1) along with Mass and NMR data. Data indicated that to all solasodine bearing Solanaceae plants. the isolated compound was solasodine. With respect to the mode of action of solasodine, The in vitro anticancer activities showed appreciable it is assumed that it is cytotoxic substance but exact result. The IC50 value was 12.17 ± 3.3 on HeLa cell line mechanism is not clear till date. More over the and showed better efficacy in TTM assay study compared compound’s activities on cell lines He La and U 937 to standard drug “Doxorubicin” at concentration of strongly support that it is having excellent cytotoxic 20µM. The drug solasodine showed 94.96% inhibition effects and it is effective against cervix cancer and at 40 µg.mL-1 in cell growth inhibition studies on U937 leukemia (blood cancer) ( superficially no correlation is human myeloid leukemia cell line compared to standard being established between cervix and leukemia). Keeping drug “ARA-C”. in mind the compound’s anticancer activity of solasodine, we have further designed nanoparticle formulation to target it’s effect on cancerous cells inherited with other Discussion & Conclusion advantages of nanoformulations.
Solanum xanthocarpum Schrad & Wendl (Solanaceae) is widely used traditional Indian medicine for the treatment Statistical analysis of respiratory disorders like bronchial asthma, cough and bronchitis. It facilitates the treatment of gastrointestinal Statistical calculations were carried out with the SPSS disorders like constipation and flatulence, because of its 10.0 for Windows software package (Statistica). laxative and carminative properties. It is one of the chief ingredients in Dashamoola Rasayanam, an Ayurvedic preparation for the treatment of respiratory ailments. Supporting information According to Ayurveda this amazing herb has multiple health benefits. It is bitter, appetizer, laxative, stomachic, Whole plant and fruits of Solanum xanthocarpum Schrad pains, piles (specially bleeding piles), fever, urinary and and Wendl (Family: Solanaceae), 1H and 13C NMR and heart diseases. Plant contains alkaloids, sterols, saponins, Mass spectra of compound Solasodine are available as fatty acids and amino acids. The plant is richer in Supporting Information.
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Fig. 1S: Mass Spectra of Solasodine
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Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 3, July 2013 pp 199 -213 208 Isolation of Solasodine from the Unripe Fruits of Solanum xanthocarpum ....
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Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 3, July 2013 pp 199 -213 210 Isolation of Solasodine from the Unripe Fruits of Solanum xanthocarpum ....
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Fig. 2S: 1H and 13C NMR of Solasodine
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6. Keeler RF, Young S, Brown D (1976) Spina bifida, exencephaly, Acknowledgement and cranial bleb produced in hamsters by the Solanum alkaloid solasodine. J. Res Commun Chem Pathol Pharmacol, 13 (4), The authors are thankful to Author is thankful to Dr. 723-30. Rajan V, Ex-Director, IICB(CSIR),Kolkata and Registrar , 7. Shashkina LF et al. (1983). Effect of solasodine on the fetus and NIPER, Kolkata and Dr. Ashish Banerjee, Professor, NIPER, offspring in experiments on rats. J KHIM FARM ZH, 17 (1), 114-6. 8. Dixit VP and, Gupta RS (1982). Antispermatogenic/ Kolkata for Mass and NMR studies of the compound and Dr. antiandrogenic properties of solasodine (C27H43O2N) obtained R.C. Pandey, Principal, Kalaniketan Polytechnic College, from Solanum xanthocarpum berries on the male genital tract Jabalpur (M.P.) for providing necessary facilities to carry of dog (Canis familaris). A histopathological approach. J Int J out this research work. The authors will remain grateful Androl, 5(3), 295–307. 9. Baehr V and Haensel R (1982). Immunomodulating properties to Dr. Krishna Das Saha, Scientist, Indian Institute of of 5, 20alpha(r)-dihydroxy-6alpha, 7alpha-epoxy-1-oxo-(5alpha)- Chemical Biology (IICB), Kolkata for in vitro anti cancer witha-2-, 24-dienolide and solasodine. J Planta Med, 44 (1), 32-3. activities of the drug. 10. Cambie RC, Potter GJ. (1979). Utilization of solasodine as a potential ecdysone antagonist. Chem NZ, 43 (3), 91-4. 11. Vina B Patela, Isverbhai S Rathodb, Jaymin M Patela, Maitreyee R Brahmbhatta.(2010) Anti-urolithiatic and natriuretic activity References of steroidal constituents of Solanum xanthocarpum. Der Pharma Chemica.2 (1), 173-176. 12. Takanori Nakamura, Chieko KomorI, Yun-yun Lee, Fumio 1. Trease and Evans. Pharmacognosy 12th Edition Bailliere Tindall Hashimoto, Shoji Yahara, Toshihiro Nohara, Akio Ejima (1996). 1983 475-483. Cytotoxic Activities of Solanum Steroidal Glycosides. Biol. Phar. 2. Genjiro Kusano, Akira Takahashi, Kazuhiro Sugiyama, Shigeo Bull, 19, 564-66. Nozoe (1987). Antifungal Properties of Solanum Alkaloids. J 13. Timtec LLC, Newyork, USA. Chem. Pharm Bull, 35, 4862-67. 14. Srinivas Koduru, D.S. Grierson, M. van de Venter and A.J. Afolayan 3. Chataing, B., (1997).Chemical basis for the biological activity of (2009) Anticancer Activity of Steroid Alkaloids Isolated Imexon and related Cyanaziridines. J Rev. Faculdad de Farmacia from Solanum aculeastrum J Pharm Bio March, 45(8) , 613-618. ULA, 32, 18-25. 15. J. Niño, Y. M. Correa, O. M. Mosquera. (2009). Biological activities 4. W.D.B. Claringbold, J.D. Few, Catherine J. Brace, J.H. Renwick. of steroidal alkaloids isolated from Solanum leucocarpum. J Pharm (1980). The disposition and metabolism of [3H]-solasodine in man Bio March, 47(3), 255-259. and Syrian hamster. J. Steroid Biochem, 13, 889-95. 16. Pratima Sur, S.P. Chatterjee, P. Roy, B. Sur (1995). 5-Nitrofuran 5. Dixit VP, Varma M, Mathur NT, Mathur R, Sharma S. (1992). derivatives of fatty acid hydrazides induce differentiation in human Hypocholesterolaemic and antiatherosclerotic effects of solasodine myeloid leukaemic cell lines. J.Cancer Lett, 94, 27-32. (C27H42O2N) in cholesterol fed rabbits. J. Phytotheray Res, 6(5), 270-273.
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