Evaluation of Cytotoxicity of normal Vero and Anticancer Activity of Human Breast Cancer Cell Lines by Aqueous Unripe Fruit Extract of Solanum torvum
D. Shanthi, R. Saravanan
Post Graduate and Research Department of Zoology, Dr Ambedkar Government Arts College,
Vyasarpadi, Chennai 600039, Tamil Nadu.
*Corresponding Author E-mail: rsaravanan51283@yahoo.com
ABSTRACT:
In the present study aqueous extract of Solanum torvum unripe fruits was used to evaluate its cytotoxic effect and anticancer activity through in vitro studies by 3-(4, 5 dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide- MTT assay) on Normal VERO cell line and MCF-7 (Human breast adenocarcinoma cell line). Aqueous extracts of Solanum torvum unripe fruits was found to be effective in the prevention of cell proliferation by breast adenocarcinoma cell lines at 1000 µg/ml from the results obtained during 24 hours of incubation.
KEYWORDS: Unripe fruits of Solanum torvum, Normal VERO cell line, MCF-7 (Human breast adenocarcinoma cell line), MTT assay.
INTRODUCTION:
Cancer is still a growing health problem world-wide characterized by the irregular proliferation of the cells, as a cell progresses from normal to cancerous tissue, the biological imperative to survive and perpetuate drives fundamental changes in cells behaviour2. Cancer risk can be reduced by avoidance of exposure to harmful biological, chemical and physical agents, in addition to the habitual consumption of cancer protective foods.
Breast cancer is generally treated in recent days by radiotherapy, hormonal or chemotherapy. In most of the case, the patients experience adverse side effects from the therapy. Intensive radio or chemotherapeutical agents usually results in nausea and failure of bone marrow function3. Cancer cells are resistant to action of chemical drugs4.
Bioassay and animal studies indicates that many phytochemicals in the plants are potential antioxidants and possess anticancer properties5. In this scenario, secondary metabolites from herbals may be the best remedy and alternative for curing cancer6
Solanum torvum (S.torvum) have revealed cytotoxic activities, antimicrobial, anti-viral activity, anti- inflammatory, anti-tumour (anticancer) activity7,8. Anticancer phenolic compounds have also been isolated from leaves and seeds of this plant9. Extracts of the aerial parts of S.torvum was found to be extremely effective in the prevention of cell proliferation of the mammary gland breast adenocarcinoma cell lines10.
The fruit of S.torvum is edible and nutritive, It is commonly known as Sundaikai in Tamil. We have chosen to study the cytotoxic and anticancer properties of the unripe fruit in the present study. Normal VERO cell line was used to evaluate its cytotoxic effect. Breast cancer line (MCF7- Human breast adenocarcinoma cells) was used in the study to find out the anticancer activity.
MATERIALS AND METHODS:
Collection and Identification of Plant Material:
Unripe fruits of Solanum torvum used for the study were collected from in and around Kancheepuram District, Tamil Nadu. Fresh plant specimen collected was authenticated by Dr. P. Jayaraman, Director, Plant Anatomy Research Center, Tambaram, Chennai. Registration No. (PARC/2018/3855).
Processing and Preservation of Plant Materials:
The plants were freshly collected, the leaves and fruits were separated from the stem. Fruits were washed with running tap water and rinsed in distilled water. The fruits were chopped into small pieces and were shade dried for two weeks for complete dryness. The dried fruits were powdered, using mechanical grinder. They were ground well to fine powder and then transferred into airtight containers until further use.
Preparation of Aqueous Extract: Cold Water Extraction:
5gm of the dried unripe fruit powder samples was soaked and dissolved in 50ml of distilled water in a 250 ml conical flask. The flask was plugged with cotton wool and aluminium foil and was placed in a shaker for 24 hrs. The filtrate was concentrated in a Soxhlet apparatus to get the crude extract. The extract was filtered using Whatman filter paper No 1. The filtered extracts in the form of concentrated paste were used for the study.
Obtaining Cell lines, its sub culture and maintenance:
Normal VERO cell line and Cancer cell line type MCF-7 (Human breast adenocarcinoma cells) were obtained from National Centre for Cell Sciences (NCCS), Pune. The cell lines procured were maintained at Life Teck Research Centre, Arumbakkam, Chennai, Tamil Nadu
The cells were maintained in Minimal Essential Media (MEM) and was supplemented with 10 % Foetal Bovine Serum (FBS), Penicillin (100U/ml) and Streptomycin (100µg/ml) in a humidified atmosphere of 5% CO2 at 370C.
Cell viability and Antiproliferative studies:
In vitro studies: Cytotoxicity analysis of Normal Vero cell line and Anticancer activity of the aqueous unripe fruit extracts of Solanum torvum on MCF-7 (Breast cancer cell line) was performed based on the principle of MTT assay.11
Preparation of two-fold serial dilutions of the leaf extracts for cytotoxicity and anticancer analysis: Weighed extracts of Solanum torvum unripe fruits were separately dissolved in distilled dimethyl sulfoxide (DMSO) and the volume was made up with Dulbecco’s modified eagle medium (DMEM) supplemented with 2% inactivated FBS to obtain a stock solution of 1 mg/ml-1 concentration and sterilized by filtration.
Incubation of Vero and MCF7 cell lines:
Cells (1 × 105/well) were plated in 24-well plates and incubated in 370C with 5% CO2 condition. After the cell reaches the confluence, the prepared extract was added and incubated for 24 hrs. After incubation, the sample was removed from the well and washed with phosphate-buffered saline (pH 7.4) 100µl/well without serum. 5 mg/ml of 0.5% MTT was added and incubated for 4 hours. After incubation, 1ml of DMSO was added in all the wells. The absorbance at 570nm was measured with UV- spectrophotometer using DMSO as blank in triplicates. The average absorbance of the triplicates are represented in the table. Measurements were performed and the concentration required for a 50% inhibition (IC50) was determined.
The % cell viability was calculated using the following formula:
% Cell viability = A570 of treated cells/A570 of control cells × 100
Morphological studies of the cells:
The unripe fruit aqueous extract treated cell lines (Normal Vero and MCF7) were observed and photographed under inverted animal cell culture microscope (Labovert-FS) under 10 x objective.
RESULTS:
Cytotoxicity of aqueous unripe fruit extract of Solanum torvum on normal Vero cell line:
The maximum cell viability of 86.75% was observed in 7.8µg/ml of plant extract at 24 hrs. The minimum cell viability of 51.42% was observed in 1000 µg/ml concentration of plant extract at 24 hrs. The IC50 was recorded at 1000µg/ml at 24hrs (Table 1; Fig 1).
Table 1: Cytotoxicity of aqueous unripe fruit extract of Solanum torvum on normal Vero cell line
|
Concentration (µg/ml) |
Absorbance (Average O.D of three replicates) |
Cell viability (%) |
|
1000 |
0.559 |
51.42 |
|
500 |
0.600 |
55.19 |
|
250 |
0.664 |
61.08 |
|
125 |
0.719 |
66.14 |
|
62.5 |
0.787 |
72.40 |
|
31.2 |
0.843 |
77.55 |
|
15.6 |
0.891 |
81.96 |
|
7.8 |
0.943 |
86.75 |
|
Cell control |
1.087 |
100 |
a. Control cells (Vero cell line) without extract treated
b. Vero cell line treated with extract and appearance of cells at 1000 µg/ml (IC50)
Fig 1: Morphology of Normal Vero cell line treated with aqueous unripe fruit extract of Solanum torvum at 24 hours of incubation
Anticancer activity of aqueous unripe fruit extract of Solanum torvum on MCF7 human breast cancer cell line:
The maximum cell viability of 74.26% was observed in 7.8µg/ml of fruit extract at 24 hrs. The minimum cell viability of 25.38% was observed in 1000µg/ml concentration of fruit extract at 24 hrs. The IC50 was reported in 62.5µg/ml at 24 hrs. (Table 2; Fig 2).
Table 2: Anticancer activity of aqueous unripe fruit extract of Solanum torvum on MCF7 human breast cancer cell line
|
Concentration (µg/ml) |
Absorbance (Average O.D of three replicates) |
Cell viability (%) |
|
1000 |
0.216 |
25.38 |
|
500 |
0.269 |
31.60 |
|
250 |
0.335 |
39.36 |
|
125 |
0.399 |
46.88 |
|
62.5 |
0.458 |
53.81 |
|
31.2 |
0.521 |
61.22 |
|
15.6 |
0.585 |
68.74 |
|
7.8 |
0.632 |
74.26 |
|
Cell control |
0.851 |
100 |
Fig 2 : Morphology of MCF 7 cancer cell line treated with aqueous unripe fruit extract of Solanum torvum at 24 hours of incubation
a. Control cells (MCF7cell line) without extract treatment
b. MCF7 cell line treatment with extract and appearance of cells at 62.5 µg/ml (IC50)
c. MCF7 cell line treatment with extract and appearance of cells at 1000 µg/ml with minimal cell viability
DISCUSSION:
Phytochemicals are increasingly used in treatment of cancer because of their availability and potential anti-cancer activity when compared with chemotherapy12,13,14.
The cytotoxicity analysis of aqueous extract of S. torvum unripe fruit on Vero cell line revealed that the extract is devoid of toxic effect, while the extracts were effective in reducing the cell viability of MCF-7 cell line in the present study at 1000µg/ml. The cytotoxic action indicates that they probably contain secondary metabolites or novel compounds that may inhibit cellular division15,16. A number of species of the genus Solanum have been shown to contain steroidal glycoalkaloids and steroidal saponins with significant cytotoxic and antitumour activities17.
Thus the cytotoxic and anticancer activity of S. torvum can be attributed to steroidal alkaloids and steroidal saponins substances present in unripe fruit extracts in the present study. Similar results were observed in cancer cells treated with ethanolic leaf extract and ethanolic fruit extract of Solanum paniculatum18.
Morphological changes are a consequence of characteristic, molecular and biochemical change occurring in cancer cells19. The control Vero cells and MCF7 cells without extract treatment did not show any morphological changes, whereas a monolayer cell destruction was observed in both cell lines treated with the extracts. The polygonal cells shows shrinkage. The increase in cell shrinkage is in a dose-dependent manner which can be attributed to the growth inhibitory effect of the aqueous extract of Solanum. torvum unripe fruit20.
The extract if tested or administered in human may prevent cell proliferation by directly combining with cell receptors or enzymes and by initiating cell arrest or cell apoptosis. Plant derived chemicals coupled with chemotherapy has gained much importance now-a-days in alleviating the proliferation of various carcinomas with less side effects21
Earlier reports on various cell lines are in support with the present study. The apoptotic activity of Kydia calycina methanolic leaf extract was evaluated for ROS generation and caspase-3 activity on different cell lines. The results showed that the extract has significantly increased the ROS production and caspase-3 levels in the cell cultures in a dose dependent manner22. Methanolic extract of Ficus racemosa show cytotoxic activity against HL-60 and HepG2 cell line with profound IC50 values and shown negligible toxicity against normal cell line HEK-293T23. Anticancer activity of Maytenus emarginata leaves and stems: were assessed by using antimitotic activity by Allium cepa method, seed germination assay by green gram seeds and brine shrimp lethality test using Artemia salina eggs showed cytotoxic effects of the extracts studied24. In vitro anticancer activity of ethanolic and ethyl acetoacetate extracts of sweet cherry, Prunus avium was evaluated. against human breast cancer cell line MCF-7 by MTT assay. The bioactive molecules exhibited potential capability to inhibit the cancer cell when compared with standard drug doxorubicin and expression of cell growth inhibition25.
Many natural products from marine sources are also endowed with promising anti-cancer activity, thus representing invaluable leads in the plans for drug discovery26. It also reported that bioactive compounds have been isolated from Moringa oleifera and used extensively for treatment of cancer27. Ethyl acetate leaf extract of Azima tetracantha has been shown to have pronounced anticancer potential against MCF-7 cell lines while compared to that of the stem extract28. Petroleum ether and chloroform extracts of Barleria gibsoni leaves showed significant ant proliferative effects against MDA MB 4355 (Human breast cancer) by SRB assay method due to presence of phytoconstituents present in the plant29. Anticancer activity of Maytenus emarginata on Ehrlich’s Ascites Carcinoma treated mice proved to have an effect in cancer cells30.
The treatment or chemotherapy given for cancer patient is costly so the treatment should be non-toxic and economic. India is having an unexplored data of hidden treasure of medicinal plants. There are anticancer medicinal plants abundantly present in India. Bioactive constituents can be isolated and patented31. The potential uses of large number of herbal drugs are limited due to their poor absorption and poor bioavailability after oral administration. Phytosome technology has been effectively used to enhance the bioavailability of many popular herbal extracts including milk thistle, ginkgo biloba, grape seed, green tea, hawthorn and ginseng can be developed for various therapeutic uses or dietary suppliments32,33. Similar method of phytosome technology can be adopted to deliver the bioactive constituents present in the unripe fruit of Solanum torvum used in the present study since this fruit is nutritive and edible in nature.
CONCLUSION:
Herbals are natural store house of phytochemicals with immense therapeutic quality. Proper understanding of synergistic interactions of various constituents of anticancer herbs, would help in formulating and designing drug to attack the cancerous cells without harming the normal cells of the body.
ACKNOWLEDGEMENT:
The authors are grateful to Tamil Nadu State Council for Higher Education, Chennai for the grant of financial assistance under Minor Research Project Scheme. We thank Dr. (Mrs). I. Seethalakshmi, Director, Life Teck Research Centre, Arumbakkam, Chennai for the laboratory assistance rendered in procurement and maintenance of cell lines.
CONFLICT OF INTEREST:
None declared.
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Received on 10.04.2020 Modified on 13.07.2020
Accepted on 28.09.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(7):3504-3508.
DOI: 10.52711/0974-360X.2021.00607