In-vitro Cytotoxic Activity of Plant Saponin Extracts on Breast Cancer Cell-Line

 

Anjali Soni*, Patel Femida, Preeti Sharma

Department of Biotechnology, Veer Narmad South Gujarat University, Udhana-magdalla Road, Surat, 395007, Gujarat, India.

 

ABSTRACT:

The aim of present work is to study anticancer and antioxidant activity of saponins present in Guar (Cyamopsis tetragonoloba) and Soybean (Glycine max) seeds. Seeds were finely powdered and methanolic extracts were prepared by Soxhlet method to study the antioxidant activity. Saponin extracts were prepared to study their effect on cancer cell line. The antioxidant activity was determined by ABTS radical scavenging assay and anticancer activity by MTT and SRB methods on MCF-7 breast cancer cell-line. Maximum 55.29 at 200 ug/ml and 65.18 at 100 ug/ml percentage inhibitions of ABTS radical was obtained by Guar and Soybean seed methanolic extracts respectively. The IC₅₀ value of Guar saponin extract on MCF-7 cell line was found to be 12.4 and 13.75 µg/ml and Soybean saponin extract 6.875 and 7.5 µg/ml using MTT and SRB methods respectively. Result suggests that both plants methanolic extract possess appreciable antioxidant activities. Soybean saponin seed extracts exhibit anticancer activity at very low concentration which can be investigated further to determine its potential use in cancer therapy..

 

 

 

INTRODUCTION:

Cancer is a general term applied to series of malignant diseases that may affect different parts of body. These diseases are characterized by a rapid and uncontrolled formation of abnormal cells, which may mass together to form a growth or tumor, or proliferate throughout the body, initiating abnormal growth at other sites. If the process is not arrested, it may progress until it causes the death of the organism^{1, 2}.

 

The main forms of treatment for cancer in humans are surgery, radiation and drugs (cancer chemotherapeutic agents).Cancer chemotherapeutic agents can often provide temporary relief of symptoms, prolongation of life but occasionally cures. In recent years, a lot of effort has been applied to the synthesis of potential anticancer drugs. Many hundreds of chemical variants of known class of cancer chemotherapeutic agents have been synthesized but have a more side effects. A successful anticancer drug should kill or incapacitate cancer cells without causing excessive damage to normal cells^{3, 4}.

 

Chemotherapy, being a major treatment modality used for the control advanced stages of malignancies and as a prophylactic against possible metastasis, exhibits severe toxicity on normal tissues^{5, 6}.

 

Plants have been used for treating various diseases of human beings and animals since time immemorial. They maintain the health and vitality of individuals, and also cure diseases, including cancer without causing toxicity. More than 50% of all modern drugs in clinical use are of natural products, many of which have the ability to control cancer cells⁷.

 

According to the estimates of the WHO, more than 80% of people in developing countries depend on traditional medicine for their primary health needs^{8, 9}.

 

Most of the medicinal plants have versatile immunomodulatory and antioxidant properties which show protective effect against breast cancer. Many investigations reported that antioxidant property is due to presence of active phyto-constituents such as vitamins (e.g., A, C, E and K), carotenoids, terpenoids, flavonoids (e.g., anthocyanins, catechins, flavones, flavonones and isoflavones), polyphenols (e.g., gallic acid, ellagic acid and tannins), enzymes (e.g., superoxide dismutase, catalase and glutathione peroxidase), minerals (e.g., copper, manganese, zinc and iodine), alkaloids, poly-saccharides, saponins, lignins, xanthones and certain pigments. Guar, Cyamopsis tetragonoloba L., is a drought-tolerant summer annual legume native to India and Pakistan. Guar contains many important nutrients and phytochemicals such as saponin and flavonoids and is well-known traditional plant used in folklore medicine. Many researchers have shown the relationship between legume consumption and health benefits, such as protection from cardiovascular disease, breast cancer, colon cancer, other cancers, diabetes, anti-inflammatory, antiarthritis, anti-oxidant and laxatives effect. It contain 5-13%  triterpenoid saponins^{10, 11}.

 

Soybean (Glycine max. L.) contains several anticarcinogens such as phenolic acids, flavonoids, saponin and isoflavonoids that help in exhibiting their cancer preventive function. Soy isoflavones (genistein, daidzein and glycitein) have antioxidant and anticancer activity by different mechanism including anti-estrogenic and anti-aromatase activity. Saponins are among several plant compounds which have beneficial effects. Various biological effects of saponins are antibacterial, antiprotozoal and anticancer activities. Saponins are secondary metabolites of glycosidic nature widely distributed in higher plants. Saponins occur constitutively in a great many plants species, in both wild plants and cultivated crops. In cultivated crops the triterpenoid saponins are generally predominant, while steroid saponins are common in plants used as herbs or for their health-promoting properties¹².

 

Many saponins are known to be antimicrobial, to inhibit mould and to protect plants from insect attack. Present study aimed to report the anticancer activity of Guar and Soybean plants seed extract having a rich content of saponin¹³.

 

MATERIALS AND METHODS:

Collection of Plant material:

Seeds of Cyamopsis tetragonoloba (Guar) and Glycine max (Soybean) were purchased from seed shop in Gujarat, India and authenticated by botanist in the department. Seeds were finely powdered and used for the preparation of different extracts.

 

Preparation of Extracts:

Methanolic extraction:

To prepare methanolic extracts of both seeds Soxhlet extraction was done. For this powdered seed samples (5g) were extracted with methanol (100 ml) using Soxhlet apparatus. Methanol was evaporated and the residue was dissolved in known volume of methanol to obtain stock solution of seed extracts.

 

Seed Saponin determination:

Seed saponins were extracted using Obadoni and Ochuko (2001) method¹⁴. 20 g of samples powder was put into a conical flask and 100 ml of 20% aqueous ethanol were added. The samples were heated over a hot water bath for 4 h with continuous stirring at about 55°C. The mixture was filtered and the residue re-extracted with another 200 ml 20% ethanol. The combined extracts were reduced to 40 ml over water bath at about 90°C. The concentrate was transferred into a 250 ml separating funnel and 20 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. 60 ml of n-butanol was added. The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a waterbath. After evaporation the samples were dried in the oven to a constant weight and the saponin content was calculated as percentage.

 

% Saponin = [final weight of sample/initial weight of extracts] × 100

 

Procurement of MCF-7 Breast Cancer Cell line:

MCF-7 (Human Breast Adeno carcinoma cell line) was procured from National Centre for Cell Sciences (NCCS), Pune, India. Cell line was maintained with regular subculturing at the interval of 3 days using Minimum Essential Medium (MEM) complete medium supplemented with 10% Fetal Bovine Serum (FBS) and Penicillin(100U)-Streptomycin(100 µg/ml) antibiotics. Incubation was carried out at 37°C with an atmosphere of 5% CO₂.

 

Dilution of drugs:

Dilution of known drug and plant extracts was prepared. The stock solution of anticancer drug Doxorubicin having final concentration 1 mg/ml was prepared in serum free MEM media. The IC₅₀ of doxorubicin was found to be 10 μg/ml, so stock was further diluted to different concentrations ranging from 5, 10 to 15 μg/ml. Stock solutions having final concentration 1mg/ml of saponin extract of Guar and Soybean were prepared in serum-free MEM medium. The samples were syringe filtered using 0.22 µM membrane filters to remove contaminants. Range of dilutions (6.25, 12.50 and 25.00 μg/ml) of extracts was prepared in sterile conditions in laminar flow hood by adding calculated amounts of MEM to the stock solution.

 

Evaluation of Antioxidant activity:

ABTS radical scavenging assay:

ABTS (2, 2-azinobis 3-ethylbenzothiazoline-6-sulfonic acid) assay measures the relative stability of antioxidant to scavenge the ABTS generated in aqueous phase, as compared with the standard (ascorbic acid or BHT). The ABTS is generated by reacting a strong oxidizing agent (e.g., potassium permanganate or potassium persulfate) with the ABTS salt. Reduction of blue-green ABTS radical coloured solution by hydrogen-donating antioxidant is measured by the suppression of its characteristic log wave (734 nm) absorption spectrum.

 

ABTS assay was performed according to Re et al. The stock solution was prepared by mixing equal volumes of 7 mM ABTS solution and 2.45 mM potassium persulfate solution followed by incubation for 16 h at room temperature in the dark to yield a dark-colored solution containing ABTS•+ radicals. Working solution was prepared freshly before each assay by diluting the stock solution by mixing of stock solution to 50% methanol for an initial absorbance of about 0.700 (±0.02) at 745 nm, with temperature control set at 30°C. Free radical scavenging activity was assessed by mixing 300 μl of different fractions (25-500 μg/ml for standard BHT and 25, 50, 100 and 200 μg/ml for plant extracts) with 3.0 ml of ABTS working standard. The decrease in absorbance was measured after mixing the solution. Data for each assay was recorded in triplicate. BHT was used as positive control. The scavenging activity was estimated based on the percentage of ABTS radicals scavenged by the following formula:

 

% Scavenging = [(A₀ − As) /A₀] × 100

 

Where; A₀ is absorption of control; A_S is absorption of tested extract solution.

 

In- vitro Studies:

The cells were then subjected to in-vitro analysis. Cell-based cytotoxicity assays are often used for screening of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death. The cytotoxic assays used in present study are MTT and SRB.

 

MTT assay:

This is based on the quantitative measurement of extracellular reduction of the tetrazolium dye 3-[4,5-dimethylethiazole -2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) to insoluble formazan crystals by metabolically active cells.This reaction is mediated by mitochondrial enzyme lactate dehydrogenase. When dissolved in a appropriate solvent, this formazan crystals exhibit purple color, the intensity of which is propotional to the number of viable cells. Briefly, 100 µl suspension of MCF-7 cells were cultured in 96-well plates at a density of 5 × 10³ to 10⁴ cells per well for required period of time.After the incubation period, the cells were exposed to 100 µl of various concentration (6.25, 12.5 and 25.0 µg/ml) of plant extracts. In addition, negative/ vehicle control and positive control (Doxorubicin) were also used for comparison. After the completion of desired treatment, 10 µl MTT reagent (HiMedia) of concentration 5mg/ml prepared in Phosphate Buffered Saline (PBS) was added to each well and further incubated for 4 h at 37˚C. Finally, the water-insoluble formazan crystals that formed during MTT cleavage in actively metabolizing cells were dissolved in 100 µl of solubilisation solution. The optical density was read by ELISA reader at a wavelength of 490 nm. Results were generated from two independent experiments and each experiment was performed in triplicates.

 

SRB assay:

The Sulphorhdamine B assay is used for determination of cell proliferation and cytotoxicity based on the measurement of cellular protein content.  SRB is a bright-pink aminoxanthene dye with two sulfonic groups that binds to basic amino acid residues in proteins of trichloroacetic acid(  TCA) fixed cells under mild acidic conditions. Under basic condition, dye can be extracted out from the cells and solubilized for colorimetric assay. Procedure was carried out according to SRB HiMedia kit protocol. 100 µl cell suspension with density 5×10³ to 10⁴ per well was cultured in 96 well plate for required period of time. After incubation, 100µl plant extract( drug) in a concentration of 6.25, 12.5, 25.0 µg/ml were added to each test well in triplicates and incubated at 37º C in a 5% CO₂ environment for 24 hours. Following treatment,  cells were fixed by adding 25µl of cold fixative solution on top of culture medium and incubated at -4ºC for 2 hours. The supernatant was then discarded and the plates were washed three times with washing solution to remove traces of fixative and dried. 50µl of Sulphorhodamine B staining solution was added to each well and incubated for 30 min at room temperature. Unbound SRB was removed by washing three times with washing solution. Then, the plates were air-dried and 100µl Solublization solution was added to extract the incorporated stain. The plates were shaken gently for 10 minutes and the absorbance was read on an ELISA reader at 490nm as primary wavelength and at 630nm as reference wavelength in ELISA Reader. Cell viability and Cytotoxicity was determined in both MTT and SRB assays using following formula:

 

                           (Absorbance) _test  –(Absorbance) _blank

Cell viability (%) = -------------------------------------× 100

                                         Average of control

 

Cytotoxicity (%) = 100 - % cell viability

 

 

Statistical analysis:

Data were expressed as mean ± SD. Statistical comparisons of the results were made using one way analysis of variance (ANOVA). Differences between the means of control and treated cells were examined using paired sample t-test when F value was significant. P value <0.05 was considered significant.

 

RESULTS AND DISCUSSION:

Many medicinal plants possess the ability to prevent and even stop the progress of cancer. They possess some chemicals which have the ability to modify the physiology of cells hence acting as anti-cancer compound. In our present study we had tested one phytochemical which is present in some plants in high quantities called Saponins. For our work we had tested commonly occuring saponin rich seeds of Guar and Soybean. Dried seeds were purchased, finely powdered in blender and stored in air tight container for further use. The yield of methanolic extract prepared by Soxhlet method for Guar and Soybean seeds was 1254 and 20.1 mg/ml respectively. Saponins concentration determined by Obadoni and Ochuko (2001)¹⁴protocol in Guar and Soybean seeds was found to be 468 and 14 mg/ml. Previous studies have reported that the concentration of  crude saponin in the Guar was 4.8 ± 0.6% ¹⁵ and 13% dry matter Guar¹⁶. Several studies reported that saponin concentrations are affected by plant species and plant variety¹⁷, degree of maturity, growing environment, agronomic factors such as climate and soil, cultivation year, location grown, season and extraction method^{18, 19}.

 

Figure 1: Free radical scavenging activity of Soybean and Guar seed extracts by ABTS assay

 

ABTS Radical Scavenging Activity:

ABTS is direct and sensitive method for antioxidant screening of plant extracts. Present study has investigated the scavenging activity of methanolic extract of Guar and Soybean seeds and expressed in percentage of inhibition of ABTS free radicals using BHT (Butyl-hydroxy toluene) as standard reference compound. Methanol extract of both the seeds showed significant free radical activity generated by ABTS. Scavenging activity was tested with extract concentrations ranging from 25 to 200 ug/ml of and the results obtained are shown in Table1. 55.29% inhibition at 200 ug/ml in case of Guar and 65.18% at 100 ug/ml for Soybean seeds crude methanolic extract was obtained in ABTS radical scavenging assay performed to determine the antioxidant activity of extracts. In the etiology of cancer, free radicals are one of the major factors necessary to cause DNA mutation, which in turn triggers the initiation stage of carcinogenesis. Exogenous antioxidants from natural sources can improve the function of the endogenous antioxidant system which is responsible for preventing free radicals in the body^20-21. More than 50% inhibition free radical is very significant and indicates the presence of various phytochemicals in crude extract of both Guar and Soybean responsible for antioxidant activity.

 

 

 

Table 1: Results of ABTS Radical Scavenging Assay

Concentration (µg/ml)	% Inhibition
	Std. BHT	Guar seed extract	Soybean seed extract
25	22.86	50	59.21
50	44.02	53.43	61.60
100	54.94	54.26	65.18
200	60.75	55.29	63.28

 

In-vitro Cytotoxicity Studies:

The cytotoxic activity of saponin extracts of Guar, Soybean seeds and standard anticancer drug Doxorubicin was assessed against human breast adenocarcinoma cell line (MCF-7) using cytotoxicity assays viz. MTT and SRB. Cell line was maintained in complete MEM medium. For cytotoxicity assays cells were properly harvested by washing and trypsinization. Centrifugation was carried out at 1000 rpm for 10 minutes. Pellet was suspended in sterile medium and cell density was maintained upto 1×10⁶ cells/ ml. Concentration of plant extracts taken were 25, 12.5 and 6.25 µg/ml. OD values were taken in triplicates for each concentration and Cell viability, % inhibition and IC₅₀ was calculated. The results of MTT and SRB assays are shown in Table 2 and 3 respectively. The half-maximal inhibitory concentration is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. The results showed that the inhibiting function of extract is dose dependent.

 

 

 

Table 2: Results of MTT Cytotoxicity assay on MCF-7 Cell line

S.No.	Saponin Extract (seeds)	Conc. (µg/ml)	O.D± S.D (490 nm)	Cell viability (%)	Cytotoxicity (%)	IC50 (µg/ml)
1		Control	0.240±0.10	-	-	-
2	Guar	6.25	O.566±0.10	60.79	39.21
		12.5	0.387±0.14	26.13	73.87	12
		25	0.301±0.14	16.37	83.63
3	Soybean	6.25	0.533±0.38	53.27	46.73
		12.5	0.361±0.09	28.71	71.29	6.9
		25	0.221±0.42	4.57	95.43
4	Doxorubicin	5	1.226±0.32	52.17	47.83
		10	1.146±0.70	48.24	51.76	7.5
		15	0.594±0.31	8.56	91.44

 

 

As the concentration of the extract is increased there is increase in percentage cytotoxicity. It is interesting that the IC₅₀ value obtained for Soybean saponin extracts was less (6.9 ug/ml) than the value obtained for standard drug Doxorubicin (7.5 ug/ml) presently used in the treatment of breast cancer. The value for Guar saponin extract is also found to be close to standard drug which reveal its probable use to cancer therapy. The saponin extract of Soybean has the potential to kill cancer cells in less concentration in comparison to that required for standard drug Doxorubicin. Another common cytotoxicity assay Sulphorhdamine B (SRB) was performed which was based on different principle of inhibiting the growth of cancer cells. Results obtained were in compliance with MTT assay results. Here also the IC₅₀ value of Soybean extract was found to be low (7.5 ug/ml) in comparison to Doxorubicin (8 ug/ml). The value obtained for Guar saponin extract was also near to what we have got for our standard drug. Literature studied has revealed that not much work has been reported on plants selected for present studies. Moustafa et al.²² have screened plants to 74 families in Egypt for their cytotoxicity against MCF-7 and other common human cancer cell lines. Remarkable results have been obtained on few plants at very low extract concentration (100ppm). Dharshini et al.²³ 2014 had studied the cytotoxic and immunomodulatory effect of raw and roasted soybean seeds and had reported low IC₅₀ value of extract on MCF-7 cell line both by MTT and SRB cytotoxicity assays. No reports of the work done specially on Saponins were found in the literature. The importance of Saponins²⁴ as cytotoxic components has been mentioned in several reviews in literature so initial attempt to screen two commonly occurring plants seed saponin has been undertaken.

 

 

 

Table 3: Results of SRB Cytotoxicity assay on MCF-7 Cell line

S.No.	Saponin Extract (seeds)	Conc. (µg/ml)	O.D± S.D (490 nm)	Cell viability (%)	Cytotoxicity (%)	IC50 (µg/ml)
1		Control	0.133±0.03	-	-	-
2	Guar	6.25	0.548±0.12	26.93	73.07	13.75
		12.5	0.294±0.04	4.8	95.22
		25	0.339±0.01	14.27	85.73
3	Soybean	6.25	0.497±0.02	20.43	79.57	7.5
		12.5	0.293±0.00	2.27	97.73
		25	0.370±0.07	4	96
4	Doxorubicin	5	0.545±0.02	54.93	45.07	8
		10	0.494±0.11	48.13	51.87
		15	0.381±0.00	33.07	66.93

 

CONCLUSION:

Cyamopsis tetragonoloba (Guar) and Glycine max (Soybean) are commonly occurring leguminous crops used by mankind as food material. These plants are found to be rich in pytochemicals which have role as antioxidants. Their seeds were found to store a very important category of phytochemicals called Saponins. Importance of Saponins as cytotoxic agents has been reported by various research groups. Taking that lead present study has been conducted on these two plants to know their antioxidant and anticancer properties. Preliminary study has shown that Soybean methanolic crude and saponin extracts have great potential to be used as antioxidant and anticancer compounds. Further detailed study on these plants is required to be conducted.

 

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Received on 29.11.2016          Modified on 17.12.2016

Accepted on 16.01.2017      ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2017; 9(1): 17-22.

DOI: 10.5958/0975-4385.2017.00003.6