Int J Pharm Pharm Sci, Vol 12, Issue 1, 31-35Original Article

CYTOTOXICITY STUDIES OF AN ETHNOBOTANICALLY SELECTED SOLANUM PUBESCENS WILLD. AGAINST SELECTED HUMAN CANCER CELL LINES

SUDHAMA V. N.1,2, M. RAMAKRISHNAN1,3*

1Research and Development Centre, Bharathiar University, Coimbatore, Tamil Nadu, India, 2Department of Botany, Indavara Dodda Siddalinge Gowda Government College, Chikkamagaluru, Karnataka, India, 3Department of Environmental Studies, School of Engineering, Presidency University, Bangalore, Karnataka, India
Email: maniramiyer@yahoo.com

Received: 04 Sep 2019, Revised and Accepted: 21 Nov 2019

ABSTRACT

Objective: The present study was aimed to determine the cytotoxicity concentration (CTC50) of different extracts made from the leaf and stem bark of an ethno botanically selected S. pubescens against Human liver carcinoma (Hep G2), Human colon carcinoma (CaCo2) and Human breast cancer (T-47 D) cell lines.

Methods: Ethnobotanical survey was done through interviewing traditional medicinal practitioners then a potential herbal plant was selected after a thorough literature survey and its identity was confirmed. The soxhlet extraction method was adopted using five different solvents from leaf and stem bark powders of the study plant and the CTC50 of all the extracts were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay method.

Results: Among the different extracts, CTC50 values were significant for stem bark extracts made from methanol (24.51±0.08 µg/ml) against Hep G2, while leaf chloroform extract was promising (57.15±1.75 µg/ml) against CaCo2 and n-hexane extract of leaf exhibited significant value (20.27±1.52 µg/ml) against T-47 D cancer cell lines.

Conclusion: The major findings of the present study clearly provides evidence that the leaf and stem bark of S. pubescens possesses the potential anticancer bioactive compound solasodine.

Keywords: S. pubescens, Cytotoxic activity, MTT assay, Hep G2, CaCo2, T-47 D

© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2020v12i1.35594. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps

INTRODUCTION

Cancer is all concerning uncontrolled cell growth [1], it is escalating at an alarming rate causing a threat to human life and sustainability. Strategies for predictable cancer cure by chemotherapy and radiation therapy decrease the bulk of tumor cells but a population of cancer stem cells remains [2] and plant-based anticancer drug discovery gain importance due to the limited success of clinical therapies using synthetic anticancer drugs together with their immense side effects [3, 4]. Many plant-derived compounds "phytochemicals” have been acknowledged to have anti-tumor properties, for example, induction of apoptosis and inhibition of cell proliferation resulting in reducing the risk of cancer [5, 6]. Still many of the potential herbs which are used by the traditional medicinal practitioners to treat several diseases are yet to be studied for their cytotoxic studies based on their traditional medicinal use. Some of the villagers in the Chitradurga district used to approach traditional medicinal practitioners for various ailments and get treated. This indicates that documentation of the indigenous knowledge, conservation, domestication and better management of key medicinal plants is necessary to ensure intergenerational benefits from the herbal medicines [7]. Cytotoxic studies of different extracts from the species of Solanum like s. pseudocapsicum [8], Solanum nigrum [9, 10], S. trilobatum [3], S. anguivi [11], S. torvum [12, 13], S. erianthum [14] and S. lycopersicum [15] were reported. Researches on the selection of medicinal plants for its phytochemical extraction and their cytotoxicity studies should begin with the ethnobotanical survey-based approach with traditional medical practitioner's knowledge. The genus Solanum of Solanaceae members found to have rich steroidal glycoalkaloids which are an important group of plant secondary metabolites isolated from more than 350 Solanum species [16, 17]. But, comprehensive phytochemicals extraction and their cytotoxic activity studies were not reported in S. pubescens Willd. Hence, in the present study, S. pubescens, an ethnobotanically important plant was selected for the extraction of phytochemicals and investigation of their cytotoxicity against Hep G2, CaCo2 and T-47 D cell lines were made to identify a potent medicinal plant for anticancer drug molecules.

MATERIALS AND METHODS

The ethnobotanical survey, plant material selection and extraction of phytochemicals

To select a herbal medicinal plant with anti-cancerous properties, an ethnobotanical survey of medicinal plants used by the traditional medicinal practitioners was conducted in six taluks of Chitradurga district, Karnataka, India (latitude 14 ° 13' 48" N and longitude 76 ° 24' 0" E) during September 2014 to August 2016. In this survey, a standard questionnaire was framed and used to interview traditional medicinal practitioners [18]. Details of the plants used to cure various diseases were documented and the plants were identified by using valid literature and illustrations [19]. Based on the traditional medicinal use and the research reports, the study plant S. pubescens was collected from the study area and the voucher specimen number is IDSGH43. The plant was identified and confirmed by referring Phytographia [20] and further authenticated by Prof. L. Rajanna, Chairperson, Department of Botany, Bangalore University, Bangalore, Karnataka, India. The plant herbarium was deposited and maintained in the department of Botany, Indavara Dodda Siddalinge Gowda Government College, Chikkamagaluru, Karnataka, India (fig. 1).

The matured leaf and stem bark of S. pubescens was dried, powdered and soxhlet extractions for each sample were made separately using five different solvents and samples were labelled as leaf ethyl acetate extract, stem bark ethyl acetate extract, leaf n-hexane extract, stem bark n-hexane extract, leaf methanol extract, stem bark methanol extract, leaf hydro alcohol extract, stem bark hydro alcohol extract, leaf chloroform extract and stem bark chloroform extract [21].

Fig. 1: A. Habit of S. pubescens, the source of leaf material, B. The stem with bark

Preparation of test solution

For cytotoxicity studies, 10 mg of test substances were separately dissolved and volume was made up with Dulbecco’s Modified Eagles Media–High Glucose (DMEM-HG) supplemented with 2% inactivated Fetal Bovine Serum (FBS) to obtain a stock solution of 1 mg/ml for all the cell lines and sterilized by 0.22µ syringe filtration. Serial two-fold dilutions were prepared from this for carrying out cytotoxic studies.

Chemicals

Ethyl acetate, n-Hexane, Methanol, Chloroform, Dimethyl sulfoxide (DMSO), Propanaol were obtained from E Merck Ltd., Mumbai, India. MTT, FBS, Phosphate Buffered Saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM) and Trypsin were obtained from Sigma Aldrich Co. St Louis, USA. EDTA, Glucose and antibiotics from Hi-Media Laboratories Ltd., Mumbai.

Cell line and culture medium

Hep G2, CaCo2 and T-47 D cell lines were procured from National Centre for Cell Sciences (NCCS), Pune, India. Stock cells were cultured in their respective media viz., DMEM-HG supplemented with 10% inactivated FBS, penicillin (100 IU/ml), streptomycin (100 µg/ml) and amphotericin B (5 µg/ml) in a humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with Trypsin Phosphate Versene Glucose (TPVG) solution [0.2% trypsin, 0.02% ethylene diamine tetraacetic acid (EDTA), 0.05% glucose in PBS]. The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 well microtiter plates (Tarsons India Pvt. Ltd., Kolkata, India).

Cytotoxicity studies

In all the cell lines, the monolayer cell culture was trypsinized and the cell count was adjusted to 100,000 cells/ml using respective media viz., DMEM-HG containing 10% FBS. To each well of the 96 well microtiter plate, 0.1 ml of the diluted cell suspension was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, monolayer washed once with medium and 100 µl of different test concentrations of test substances were added on to the partial monolayer in microtiter plates. The plates were then incubated at 37 °C for 72 h in a 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval [22].

Cell viability assay

After 72 h incubation, the drug solutions in the wells were discarded and 50 µl of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37 °C in a 5% CO2 atmosphere. The supernatant was removed and 100 µl of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the standard formula and the concentration of test substances needed to inhibit cell growth by 50% (CTC50) was generated from the dose-response curves for each cell line. DMSO was used as control. Trials were conducted with three replicates for each concentration and data were expressed as mean±Standard Deviation (SD).

RESULTS AND DISCUSSION

Selection of S. pubescens was accomplished through an ethnobotanical survey, a herbal medicinal plant used by the traditional medicinal practitioners to treat cancer. In the survey, a sum of 190 plant species belonging to 156 genera and 75 families were screened from six taluks of Chitradurga district whereas in the literature cytotoxicity study of S. pubescens is not yet reported. Hence, in the present investigation, MTT assay-based cytotoxicity was determined in all the ten different extracts against Hep G2 cell line with the concentrations of test solutions ranging from 1000-7.8 µg/ml. Best four extracts that exhibited 50% viability of cell lines are 24.51±0.08, 44.92±0.04, 48.85±0.41 and 49.03±0.17 µg/ml in stem bark methanol, stem bark hydro alcohol, leaf chloroform and leaf n-hexane extracts, respectively (table 1 and fig. 2 A, B). In the Hep G2 cell line, the cytotoxic study showed that the stem bark is found to be the good source of the anti-cancer drug for human liver carcinoma and the methanol is a suitable solvent to extract the phytochemicals. Further, cytotoxic studies on CaCo2 and T-47 D cell lines exhibited the CTC50 values 57.15±1.75 and 20.27±1.52 µg/ml respectively and both the fractions were extracted using chloroform from leaves of the study plant. The present study findings indicate that the leaf should be the source of herbal raw material to investigate the anticancerous drugs against human colon and breast cancer (table 1 and fig. 2 C-F).

Table 1: Cytotoxic properties of leaf and stem bark extracts of S. pubescens against HepG2, CaCo2, T-47 D cell lines

S. No. Name of the solvent used Cytotoxicity concentration (µg/ml)
Hep G2 CaCo2 T-47 D
Leaf Stem bark Leaf Stem bark Leaf Stem bark
1. Ethyl acetate 159.73±2.01 187.88±0.86 ND ND ND ND
2. n-hexane 49.03±0.17 >1000 61.03±0.12 ND 26.67±1.70 ND
3. Methanol 116.21±0.58 24.51±0.08 ND 83.68±0.33 ND 112.29±4.28
4. Hydo alcohol 105.40±0.13 44.92±0.04 ND 77.50±0.62 ND 94.93±0.55
5. Chloroform 48.85±0.41 184.55±0.66 57.15±1.75 ND 20.27±1.52 ND

*CTC50 expressed as mean±SD, n=3, ND=Not detected

The ethnobotanical survey in the study area revealed the traditional knowledge from medicinal practitioners about the preferential use of medicinal plants for the treatment of various diseases and S. pubescens for cancer. According to WHO (World Health Organisation), cancer is a leading cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018 and some of the most common causes of cancer death are cancers of colorectal (8,62,000 deaths), liver (7,82,000 deaths) and breast (6,27,000 deaths). The limited success of allopathic anticancer drugs and their massive side effects led to the move towards the development of plant-based anticancer drug discovery [3, 4].

Fig. 2: A-F. Shows the cytotoxic inhibitory effect on cell viability by the different extracts made from leaf and stem bark of S. pubescens on different human cancer cell lines. A-Control for HepG2, B-stem bark hydro alcohol 7.8 µg/ml (CTC50 44.92±0.04 µg/ml), C-Control for CaCo2 cell line, D-leaf chloroform 15.6 µg/ml (CTC50 57.15±1.75 µg/ml), E-Control for T-47D cell line, F-leaf chloroform 15.6 µg/ml (CTC50 20.27±1.52 µg/ml)

Over the past decades, there has been a resurgence of interest in the investigation of herbs as a source of potential anticancer drugs. Investigations on solasodine and its concentration in S. pubescens were made and found that the leaf and stem bark are the right sources of raw material for the cancer drug material [21]. In the present study, all the ten extracts were tested for their cytotoxic activity and the extracts like stem bark methanol, stem bark hydro alcohol, leaf chloroform and leaf n-hexane are noteworthy as they could exhibit promising anti-proliferative effects and the CTC50 values are quite evident (24.51±0.08, 44.92±0.04, 48.85±0.41 and 49.03±0.17 µg/ml) on Hep G2 cell line (table 1 and fig. 2 A, B). These results authenticate that the stem bark and leaf are found to be a good source of herbal drug raw material against human liver carcinoma. Liver cancer is a progressively increasing tumor with high mortality, not responsive to the cytotoxic agents used in chemotherapy and existing effective therapies have harmful side effects [23, 24]. Several Solanum species were extensively screened for its potential cytotoxic and anti-proliferative activity of phytochemicals extracted from the leaf, stem, fruit, seed and aerial parts against Hep G2 [3, 8, 9, 11, 14, 23] and found that the CTC50/IC50 values reported are from 7.04±1.08 to 225±15.2 µg/ml with the concentration ranging from 5 to 1000 µg/ml. In the present study, different extracts’ test solutions concentrations used were 7.8 to 1000 µg/ml and the CTC50 values are between 24.51±0.08 and 49.03±0.17 µg/ml. In the Hep G2 cell line, the cytotoxicity result findings conclude that the methanolic extracts from stem bark induced a significant 50% antiproliferative effect.

Further, the extracts were evaluated for cytotoxic studies on CaCo2 and T-47 D cell lines and the significant CTC50 values recorded are 57.15±1.75 and 20.27±1.52 µg/ml respectively and both the fractions were extracted using chloroform from leaves of the study plant (table 1 and fig. 2 C-F). Similar cytotoxic activity reports were found from the fruit extracts of S. aculeastum [25] and leaf extracts of S. nigrum [10] and the CTC50/IC50 values are 24.40±1.13 µg/ml for CaCo2 and 0.948 mg/ml for Human Colorectal Carcinoma cell lines, respectively. Also, a report on cell viability assay of leaf and fruit extracts from S. erianthum on human breast cancer cell line (MCF-7) was found [14]. In S. pubescens Willd., research reports have been made on pharmacological studies like antidiabetic activity [26], anti-inflammatory activity [27], antidiarrheal activity [28], antinociceptive activity [29], anticonvulsant and sedative effects [30] and antibacterial activity [31, 32] but reports on anticancer activity of phytochemical extracts from its leaf and stem bark extracts are not accessible. Hence, the present study was focused on the extraction of phytochemicals using five different solvents, tested their potent cytotoxic activity on human liver, colon and breast cancer cell lines and revealed the suitable solvents and plant parts that substantiate the herbal drug encounter for cancer therapy from S. pubescens. A thought-provoking fact was found reported [33] on solasodine and other related alkaloids that trigger against the cancer cell and death by the lysosomal-mitochondrial mediated apoptosis pathway and the death receptor-mediated apoptosis pathway. Furthermore, the carbohydrate moiety of these steroidal alkaloids has anti-metastatic properties and plays a key role in expressing antineoplastic actions and also a class of clinically important multi-drug-resistance with cancer cells. Antiproliferative and cytotoxic effects of phytochemcials extracted from leaf of S. trilobatum were reported from a locally available plant Madhavaram, Tamil Nadu, India, its vital that such locally available plants belong to Solanaceae are rich in bioactive compounds for many diseases and hence their therapeutic potential need to be thoroughly investigated [34, 35].

CONCLUSION

Cytotoxicity studies of leaf and stem bark extracts of S. pubescens Willd. on Hep G2, CaCo2 and T-47 D cell lines are a first of its kind report, the findings have inferred that the stem bark (methanolic extract) is found to be the source of raw material for anti-cancer drug for human liver carcinoma, the leaf (chloroform extract) is against both human colon and breast cancer and it is evident that solasodine is the vital anti-carcinogenic compound as reported in our previous investigation. Moreover, the solasodine of S. pubescens Willd. is a major source of anti-proliferative compounds that is to be further investigated on the line of isolation, purification, characterization and testing its individual anti-carcinogenic potential at the molecular level.

ACKNOWLEDGEMENT

The authors are thankful to the P. G. Department of Botany, I.D. S. G. Government College, Chikkamagaluru, Karnataka State, India for providing the facilities for performing the extraction work.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

The first author has planned the research work, designed and carried out all the experiments, data compilations, statistical analysis, and preparation of the manuscript under the guidance of the second author. The second author has made critical observations in the manuscript and developed the final publishable format.

CONFLICT OF INTERESTS

The authors declare that there is no conflict of interest

REFERENCES

  1. Aliomrani M, Jafarian A, Zolfaghari B. Phytochemical screening and cytotoxic evaluation of Euphorbia turcomanica on hela and HT-29 tumor cell lines. Adv Biomed Res 2017;6:68.

  2. Sara Soltanian, Mahboubeh Sheikhbahaei, Neda Mohamadi. Cytotoxicity evaluation of methanol extracts of some medicinal plants on P19 embryonal carcinoma cells. J Appl Pharm Sci 2017;7:142-9.

  3. Kanchana A, Balakrishna M. Anti-cancer effect of saponins isolated from Solanum trilobatum leaf extract and induction of apoptosis in human larynx cancer cell lines. Int J Pharm Pharm Sci 2011;3:356-64.

  4. David J Newman, Gordon M Cragg, Kenneth M Sander. The influence of natural products upon drug discovery. Nat Prod Rep 2000;17:215-34.

  5. Amin A, Gali-Muhtasib H, Ocker M, Schneider Stock R. Overview of major classes of plant-derived anticancer drugs. Int J Biomed Sci 2009;5:1-11.

  6. Tan G, Gyllenhaal C, Soejarto DD. Biodiversity as a source of anticancer drugs. Curr Drug Targets 2006;7:265-77.

  7. Caleb Mutwiwa, Brian Rotich, Matthaeus Kauti, Jafford Rithaa. Ethnobotanical survey of medicinal plants in mwala sub-county, machakos County, Kenya. J Med Plants Res 2018;4:110-9.

  8. Pottekkad Vijayan, Preethi Vijayaraj, Prashanth Haranahalli Chandrappa Setty, Raghu Chandrashekhar Hariharpura, Ashok Godavarthi, Shrishailappa Badami, et al. The cytotoxic activity of the total alkaloids isolated from different parts of Solanum pseudocapsicum. Biol Pharm Bull 2004;27:528-30.

  9. Shokrzadeh M, Azadbakht M, Ahangar N, Hashemi A, Saeedi Saravi SS. Cytotoxicity of hydro-alcoholic extracts of Cucurbita pepo and Solanum nigrum on HepG2 and CT26 cancer cell lines. Pharmacogn Mag 2010;6:176-9.

  10. Chen Jei Tai, Chien Kai Wang, Cheng Jeng Tai, Yi Feng Lin, Chi Shian Lin, Jiun Yu Jian, et al. Aqueous extract of Solanum nigrum leaves induces autophagy and enhances cytotoxicity of cisplatin, doxorubicin, docetaxel, and 5-fluorouracil in human colorectal carcinoma cells. J Evidence Based Complementary Altern Med 2013. http://doi.org/10.1155/514719.

  11. Gandhiappan J, Rengasamy R. Antiproliferative activity of Solanum anguivi against cancer cell lines. Pharm Lett 2012;4:875-80.

  12. Swayamsiddha Panigrahi, Meenakshi Sundaram Muthuraman, Ravichandran Natesan, Brindha Pemiah. Anticancer activity of ethanolic extract of solanum torvum sw. Int J Pharm Pharm Sci 2014;6:93-8.

  13. Joseph M Nguta, Regina Appiah Opong, Alexander K Nyarko, Dorothy Yeboah Manu, Phyllis GA Addo, Isaac Otchere, et al. Antimycobacterial and cytotoxic activity of selected medicinal plant extracts. J Ethnopharmacol 2016;182:10-5.

  14. Radhika Mahadev, Ramakrishnaiah H, Krishna V, Deepalakshmi AP, Naveen Kumar N. Cytotoxic activity of methanolic extracts of Solanum erianthum D. DON. Int J Pharm Pharm Sci 2015;7:106-8.

  15. Homaira Afreen, Toufiq-Ul Amin, Siddiqul Islam, Salma Parvin, Rayhanus Salam. In vitro phytochemical analysis and cytotoxic assay of leaves of Solanum lycopersicum linn by brine shrimp bioassay. J Innovations Pharm Biol Sci 2016;3:81-5.

  16. Kanika Patel, Ravi B Singh, Dinesh K Patel. Medicinal significance, pharmacological activities, and analytical aspects of solasodine: a concise report of current scientific literature. J Acute Dis 2013;2:92-8.

  17. Fang Sun, Shengyu Li, Dajun He, Gang Cao, Xiuzhen Ni, Guihua Tai, et al. Effects of glycoalkaloids from Solanum plants on cucumber root growth. Phytochemistry 2010;71:1534-8.

  18. Jain SK. Methods and approaches in ethnobotany. Society of Ethnobotany, Lucknow, India; 1986.

  19. Gamble JS, Fisher CEC. Flora of the presidency of madras, Reprinted Edition. I-III, Botanical Survey of India, Calcutta, India; 1957.

  20. Phytographia: 5. t. 3. Hook. F., Flora British India 4:230, Gamble, 2, 936;1883; 1794.

  21. Sudhama VN, Ramakrishnan M. Screening, detection and quantification of solasodine in Solanum pubescens willd by reverse phase-high performance liquid chromatography method. Int J Green Pharm 2019;13:343-7.

  22. Francis Denizot, Rita Lang. Rapid colorimetric assay for cell growth and survival modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986;89:271-7.

  23. Mostafa I Fekry, Shahira M Ezzat, Maha M Salama, Ohoud Y Alshehri, Ahmed M Al-Abd. Bioactive glycoalkaloides isolated from Solanum melongena fruit peels with potential anticancer properties against hepatocellular carcinoma cells. Sci Rep 2019;9:1746.

  24. Mothanna AQ, Rozita R, Yeap SK, Omar AR, Ali AM, Alitheen NB. Selective cytotoxicity of goniothalamin against hepatoblastoma HepG-2 cells. Molecul 2011;16:2944-59.

  25. Trevor Burger, Tsholofelo Mokoka, Gerda Fouche, Paul Steenkamp, Vanessa Steenkamp, Werner Cordier. Solamargine, a bioactive steroidal alkaloid isolated from Solanum aculeastrum induces non-selective cytotoxicity and P-glycoprotein inhibition. BMC Complementary Altern Med 2018;18:2-11.

  26. Hemamalini K, Ramya Krishna V, Bhargav A, Vasireddy U. Hepato protective activity of Tabebuia rosea and Solanum pubescens against paracetamol induced hepatotoxicity in rats. Asian J Pharm Clin Res 2012;5:153-6.

  27. Niyogi P, Raju NJ, Reddy PG, Rao BG. Formulation and evaluation of anti-inflammatory activity of Solanum pubescens wild extracts gel on albino wister rats. Int J Pharm 2012;2:484-90.

  28. Hemamalini K, Bhargav A. Antidiarrheal activity of methanolic extracts of leaves of Solanum pubescens willd. Int J Pharmacol Toxicol 2013;3:43-5.

  29. Sumalatha P, Hemamalini K, Shwetha R, Uma Vasi Reddy. Antinociceptive screening of methanol extract of Solanum Pubescens. Int J Pharm 2013;4:149-51.

  30. Suvarchala Kiranmai A, Hemamalini K, Uma Vasireddy. Anticonvulsant and sedative effects of leaf extract of Solanum pubescens. Int J Pharm Sci Res 2013;4:1424-7.

  31. Haseebur Rahman, Riaz Mahmood, Nazneen Rahman, Mir Haris. Antibacterial activity of Solanum pubescens-an ethnomedicinal plant from south western region of Andhra Pradesh. Int J Appl Sci Biotechnol 2014;2:501-9.

  32. Sudhama VN, Ramakrishnan M. Phytochemical extractions, qualitative analysis, and evaluation of antimicrobial activity in the leaf and stem bark of solanum pubescens willd. Asian J Pharm Clin Res 2019;12:280-4.

  33. Cham BE. Solasodine, solamargine and mixtures of solasodine rhamnosides: pathway to expansive clinical anticancer therapies. Int J Clin Med 2017;8:692-713.

  34. Priya G, Chellaram C. Antiproliferative effect of ethanolic leaf extract of Solanum trilobatum on Hep2 cancer cell lines. Asian J Pharm Clin Res 2014;7:58-61.

  35. Lawrence Kalita, Biswajit Dash, Uttam Borah, Juman Deka, Suvakanta Dash. Preliminary phytochemical analysis and antimicrobial activity ethanolic extracts of dried fruits of Solanum torvum (family-solanaceae). Int J Curr Pharm Res 2017;9:123-6.