Evaluation of Anticancer Activity of Withania somnifera L. and Tribulus terrestris L. on Human Breast Cancer Cells In vitro

 

Latifa Nasser A. Abdulqawi1*, Syed Atheruddin Quadri2, Sehbanul Islam3,4,

Manas Kumar Santra3

1Department of Zoology, Maulana Azad Collage, Dr Babasaheb Ambedkar Marathwada University,

Aurangabad - 431004, Maharashtra, India.

2Department of Zoology, Maulana Azad Collage, Dr. Rafiq Zakaria Campus,

Aurangabad -  431001, Maharashtra, India.

3National Centre for Cell Science, NCCS Complex, Ganeshkhind Road, Pune - 411007, Maharashtra, India.

4Department of Biotechnology, Savitribai Phule Pune University,

Ganeshkhind Road, Pune - 411007, Maharashtra India.

*Corresponding Author E-mail: latifaabdulqawi@gmail.com, dratheruddin.qs@gmail.com, sehbanalig@gmail.com, manas@nccs.res.in

 

ABSTRACT:

Tribulus terrestris and Withania somnifera have been used in Indian medicine for a long time to treat a variety of diseases. The Present study evaluates anticancer activities of methanolic and aqueous extracts of W. somnifera and T. terrestris against breast cancer cell lines (MCF-7) were tested using MTT assay In-vitro, concentrations ranging of extracts from 2.5µg/ml to 25µg/ml.The results showed that all the plant extracts inhibited breast cancer cells' growth in dose-dependent. Methanolic and aqueous extracts of W. somnifera showing higher anticancer activity whit IC50 14.20 and 17.00µg/ml, respectively. The results of this study give direction for more pharmacological evaluation of these plants and isolation of their active compounds that may be tested and developed into new drugs for the treatment of the cancer.

 

KEYWORDS: Anticancer, MTT assay, MCF-7, Withania somnifera, Tribulus terrestris.

 

 

INTRODUCTION: 

The loss of normal cell growth control is one of the main causes of cancer development, accompanied by angiogenesis, metastasis, and apoptosis1,2,3. Cancer is caused by both external factors (tobacco, radiation, chemicals, and infectious organisms) and internal factors (inherited mutations, immune conditions, hormones, and mutations that occur from metabolism). Cancer still remains an aggressive killer around the world. Due to a lack of widespread and systematic methods of early detection4,5. Though chemotherapy is now used as a standard method of treatment; but has not succeeded in fulfilling expectations despite the considerable cost of their development, so searches have increased for anticancer agents from natural products6,7,8.

 

 

 

Over the past 30 years, natural products have gained growing attention for their potential as novel preventive and therapeutic agents for cancer5. The antitumor activity of medicinal plants is currently being researched extensively that selectively and locally inhibit tumor growth without harming healthy tissues9. More than 50% of all modern drugs are obtained naturally, many of such drugs have the ability to control cancer cells8,10. According to a WHO report, the growing demand for herbal medicines is expected to be 5 trillion by the year 2050 in the international market11.

 

With 1, 67 million new cases diagnosed with cancer in 2012, breast cancer is the most common cancer among women worldwide, accounting for 25 per cent of all cancers12. In recent years, it has become clear that breast cancer is a collection of molecularly distinct tumors arising from breast epithelial cells, rather than a single disease. Whit regard breast cancer, MCF-7 cells represent a very important candidate as they are used ubiquitously in researches13. So, in this study, we have investigated in vitro anticancer activities againstbreast cancer cell lines (MCF-7) of Withaniasomnifera L. Dunal(roots) Tribulus terrestris L. (fruits).

 

MATERIALS AND METHODS:

Plant Materials:

W. somnifera (roots) and T. terrestris (fruits) were bought from the local vendor of a traditional medicinal product at Aurangabad Maharashtra, India. And they were authenticated14,15 .The roots and fruits of the plants were cleaned and crushed to a fine powder.

 

Preparation of plant extracts:

Methanol and aqueous extracts of W.somnifera (roots) and T.terrestris (fruits) were prepared as described in14, 16.

 

Anticancer activity:

Cell culture:

MCF-7 breast cancer cells were provided by National Center for Cell Science (NCCS), Pune. Cells were grown in Eagles Minimum Essential Medium (EMEM) with 10% FBS at 37°C, 5% CO2 and 95% air in a humid incubator.  Monolayer cells were detached using trypsin/EDTA (ethylenediamine tetra-acetic) then reseeded. The medium is changed twice a week17.

 

MTT assay:

Viable cells were calculated using a hemocytometer, and plated in 96-well microtiter plates were (5×103 cell/well) and incubated for 24 h. Culture medium was used to dilute extracts stock solution (50mg/ml in methanol or water). The required final extracts concentrations were 2.5, 5, 10, 15 and 25μg/ml. The medium was replaced by a fresh medium containing final concentrations of extracts. Cells were re- incubated at 37°C, 5 % CO2 for a further 48h, then 20μl of MTT reagent (5mg/ml in PBS) was added to each well. After incubated for an additional, 4h, the medium containing MTT was discarded, and plates were washed twice with HBSS and replaced by100μl of DMSO to dissolve MTT (Formazan crystals) and left for 10min. at room temperature. Finally, the absorbance was measured at 570nm using a microplate reader18. The cell inhibition % was calculated as the following.

 

Cell Inhibition %  = 100 - Absorbance (sample)/ Absorbance (control)] x100.

 

Statistical analysis:

The IC50 values were calculated by linear regression using Excel Microsoft.

 

RESULTS:

The effect of crude methanolic and aqueous extracts of Tribulus terrestris, and Withania somnifera plants in growth inhibition MCF-7 cells line (breast cancer) for various concentrations (2.5 - 25µg/ml) was evaluated by MTT assay in vitro. The results showed that all the plant extracts inhibited breast cancer cells' growth in a dose-dependent manner (Table 1) (Figures 1 and 2). The highest percentages of inhibition were 69.92% and 66.68% for methanol and aqueous extracts of W. sominifera at 25µg/ml concentration. The IC50 of methanolic and aqueous extracts of W. somnifera 14.20   and 17.00µg/ml, respectively. The percentage of dead cells at 25µg/ml concentration was 55.29% and 51.41% for methanolic and aqueous extracts of T. terrestris with IC50 21.60, 24.21µg/ml, respectively.

 

Table 1: Percentage of cells growth inhibition and IC50 values of extracts of W. Somnifera and T. terrestris against MCF-7 cells line by MTT assay.

Concentrations (µg/ml)

Plants

Withania somnifera

Tribulus terrestris

Extracts (% of growth inhibition)

Methanol

Aqueous

Methanol

Aqueous

2.5

20.98

25.91

22.24

11.11

5

33.35

28.18

25.3

15.64

10

41.98

35.33

35.05

27.81

15

57.22

43.7

39.14

32.12

25

69.92

66.68

55.29

51.41

IC50

 

14.20

17.00

21.60

24.21

 

Figure 1: Anticancer activity of methanolic and aqueous extracts of W. somnifera against MCF-7 breast cancer cells line

 

Figure 2: Anticancer activity of methanolic and aqueous extracts of T. terrestris against MCF-7breast cancer cells line.

 

DISCUSSION:

The results in Table 1 revealed that the methanol and aqueous extracts of W. sominifera showed strong anticancer activity against MCF-7 cell line (IC50 14.20   and 17.00µg/ml.On the report of the American Cancer Institute (USNCI), the curds extract of plants are considered powerful anticancer factors when their IC50 values are below 20μg/ml19.  Nemaet al.,20 conclude that leaves extract of W. somnifera showed efficient cytotoxicity on MCF-7 than PA-1 and A-459 cancer cell lines with IC50 10, 13, and 11µg, respectively. And, an aqueous extract of W. somniferaroot was showed anticancer activity with IC50 0.19mg/ml on MDA MB 231 breast cancer cells21. Also, earlier studies indicated that leaf extract of W. Somnifera has also shown to inhibit the growth against different human cancer cell lines; human breast (MCF-7), colon (HCT-116), lung (NCI-H460), and central nervous system (SF-268)22. Almehdaret al.,23 reported the methanolic petroleum ether, chloroform, and n-butanol extracts of W. somnifera showed strong activity on MCF7 with IC50 16.5, 8.03, 8.23, and 8.44μg/ml, respectively. According to Fouchéet al.,24 the total growth inhibition of methanolic extract of W. somnifera for the breast MCF7, melanoma UACC62, and renal TK10 cancer cell lines were 14.32μg/ml, 7.52μg/ml and 18.87μg/ml, respectively. Anti-cancer activity of W. somnifera is probably due to the action of its major constituents that are Withaferin A and withanolide D25,26, which inhibits RNA and protein production; may lead to increased tumor cell death25. Furthermore, the presence of steroidal alkaloids in the Solanaceae family has been linked to cytotoxicity23.

 

Also, Methanolic and aqueous extracts of T. terrestris, were active against the growth inhibition of the MCF-7 cell line (55.29% and 51.41%) at 25μg/ml concentration with IC50 22.10 and 20.49µg (Figure 2). Our results obtained from the cytotoxicity study of extracts of T. terrestris used MTT assay were in agreement with data from previous studies, that showed T. terrestris extracts have a pronounced dose-dependent an inhibitory effect on viability in human breast cancer (MCF7)27,28,29. The calculated IC50 values vary widely between studies29,30, 31. This high variability is most likely due to differences in the chemical nature and composition of active saponins in T. terrestrisfrom different geographical areas, as well as differences in the extent and mechanism of their action, plant part studied and stage of plant development32. Saponins are among several plant compounds which have beneficial effects. Various biological effects of saponins are antibacterial, antiprotozoal and anticancer activities10. According to33 Tribulus terrestris (fruits) hasflavanoids and saponin compounds which has potentantiyeastactivity, antioxidant activity and anticancer activity.

 

In conclusion, plants remain a prime source of cancer therapy drugs and can provide leads for the development of new anticancer agents through isolates active compounds from plants and tested their activity against different cancer cell lines in vivo and in vitroin future works.

 

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Received on 04.08.2021            Modified on 15.03.2022

Accepted on 27.08.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(7):3079-3082.

DOI: 10.52711/0974-360X.2023.00506