In-vitro Cytotoxic and Antioxidant Activity of Bauhinia racemosa Root

P. Aravanan¹*, S Jayakumari²

¹School of Pharmaceutical Sciences, Vel’s Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai.

²Department of Pharmacognosy, School of Pharmaceutical Sciences, Vels University, Chennai - 600117.

*Corresponding Author E-mail: p_aravanan2011@yahoo.com

ABSTRACT:

Objective: To evaluate cytotoxic and antioxidant activity of the plant Bauhinia racemosa root by in-vitro cell line analysis. Methods: MCF-7 cell lines were used for the study to find the inhibitory concentration of the extract. DPPH and NO scavenging assays were performed to study the antioxidant nature of the extract. Results and Conclusion: In MCF-7 cell line cytotoxicity effect was observed in tested sample concentrations in 48 hours treatment, it also revealed that increased concentration of drug shown increased cytotoxicity over the MCF-7 cell lines. DPPH and NO scavenging Assay revealed a concentration dependent radical scavenging capacity of the tested sample concentrations of Bauhinia racemosa root spectrophotometrically.

KEYWORDS: Bauhinia racemosa, root extract, in-vitro cytotoxicity, MCF-7, DPPH and NO.

INTRODUCTION:

The plant based drugs have the added advantage of being simple, effective, and offering a broad spectrum activity with an emphasis on the preventive action of drugs. Large scale use of medicinal plants and herbs in preparation of such formulations is increasing both in the developed as well as developing countries due to growing concern about the adverse effects of chemical and synthetic substances. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions, and to defend against attack from predators such as fungi and herbivorous mammals¹. Many of these phytochemicals have beneficial effects on long-term health when consumed by humans, and can be used to effectively treat human diseases. At least 12,000 such compounds have been isolated so far; a number estimated to be less than 10% of the total.^2,3 Chemical compounds in plants mediate their effects on the human body through processes identical to those already well understood for the chemical compounds in conventional drugs; thus herbal medicines do not differ greatly from conventional drugs in terms of how they work.

This enables herbal medicines to be as effective as conventional medicines, but also gives them the same potential to cause harmful side effects.^4,5

Bauhinia is a genus of more than 200 species of flowering plants of subfamily, Caesalpiniaceae. Many species are widely planted in the tropics as orchid trees, particularly in northern India, Vietnam and south eastern China. This particular species racemosa is widely distributed throughout India, ascending to an altitude of 1,650m from sea level in the western Himalayas, and in Ceylon, China and Timor. Numerous types of biological activities are attributed to bauhinia species. B. racemosa is the most important species used to treat many ailments in traditional system of medicine.^6,7

MATERIALS AND METHODS:

Extraction:

Extraction was done by using cold percolation method using the roots of the plant Bauhinia racemosa. The extract was subjected to preliminary phytochemical; fluorescence analysis and loss on drying were done as per standard protocol. Aqueous, Ethanolic, Chloroform, Ethyl acetate and Petroleum ether extracts were used for the cytotoxicity and in-vitro antioxidant assays.^8,9

Anti- Cancer Activity of Bauhinia racemosa Roots^10,11

Preparation of cell suspension:

A subculture of MCF-7 cells in Dulbecco’s Modified Eagle’s Medium (DMEM) was Trypsinized separately, after discarding the culture medium. To the disaggregated cells in the flask 25mL of DMEM with 10% FCS was added. The cells suspended in the medium by gentle passage with the pipette and the homogenized.

Seeding of cells:

One mL of the Homogenized cell suspension was added to each well of a 24 well culture plate along with different concentration of samples (ABAE) (0 to 1000 µg/mL) and incubated at 37°C in a humidified CO₂ incubator with 5% CO₂. After 48 hrs incubation the cells were observed under an inverted tissue culture microscope. With 80% confluence of cells cytotoxicity assay was carried out.

Cytotoxicity assay^12,13

The assay was carried out using (3- (4, 5-Dimethyl thiazol-2yl)-2, 5- diphenyl tetrazolium bromide (MTT). MTT is cleaved by Mitochondrial Succinate dehydrogenase and Reductase of viable cells, yielding a measurable purple product Formazan. This Formazan production is directly proportional to the viable cell number and inversely proportional to the degree of cytotoxicity. After 48 h incubation the wells were added with MTT and left for 3 h in room temperature. All wells were removed the content using pipette and 100µl SDS in DMSO were added to dissolve the Formazan crystals, absorbance’s were read in Read Well Touch micro plate reader at 570nm.

Anti- oxidant Activity of Bauhinia racemosa Roots:

DPPH Radical Scavenging Assay^14,15

The estimation of free radical scavenging capacity of the plant extract was performed using the stable DPPH radical with an absorption maximum at 515nm. A solution of DPPH prepared by dissolving 2.4mg of radical in 100ml of Methanol. 5ml Test sample was added to 3.995ml of Methanolic DPPH and shaken vigorously to be kept for 30 mins at room temperature. Absorbance of the reaction mixture was measured at 515nm spectrophotometrically in triplicate and their average is considered. A blank and standard (trolox) are also measured. The %RAS is calculated using the following formula % = [(AB-AA)/AB] x 100 whereas AA is Absorbance of Antioxidant at t = 30min, AB is absorbance of blank at t=0 mins.

Nitric Oxide Radical Scavenging Assay:¹⁶

The standard Gallic acid and the extracts were diluted accordingly. These were mixed with equal volumes of Griess reagent freshly prepared by mixing 1% Sulphanilamide in 2.5% Phosphoric acid and 0.1% Naphthylethylene diamine dihydrochloridein 2.5%, Phosphoric Acid. 0.5mL of 10mM Sodium Nitroprusside in Phosphate buffered saline was mixed with 1ml of different concentrations of extracts respectively and incubated at 25⁰C for 180 mins. Their absorbances were measured using 546nm. The percentage inhibition was determined using the formula % = [(AB-AA)/AB] x 100 whereas AA is Absorbance of Antioxidant at t=30min, AB is absorbance of blank at t=0 mins.

RESULTS AND DISCUSSION:

The in-vitro cytotoxicity activity studies were proved that MCF-7 cell lines were triggered significantly with the increasing of sample concentration. In MCF-7 cell line cytotoxicity effect was observed in tested sample concentrations in 48 hours treatment, it also revealed that increased concentration of drug shown increased cytotoxicity over the MCF-7 cell lines. The IC₅₀ were calculated as 5.846µg/ml, 6.639µg/ml, 12.57µg/ml, 23.63µg/ml and 6.654µg/ml against MCF-7cell lines respectively for Aqueous, Chloroform, Ethylacetate, Ethanolic and Petroleum ether extracts and results were presented in Table No. 1.

Table No. 1: MTT Assay of Bauhinia rascemosa root extract

Sample Concentrations (µg/ml)	Cell Viability (%)
Sample Concentrations (µg/ml)	ABAE	ABCE	ABEA	ABEE	ABPE
1000.000	19.76±0.02	18.54±0.01	40.64±0.01	56.75±0.01	43.18±0.02
500.000	27.04±0.01	25.38± .01	42.08±0.02	57.85±0.02	43.73±0.01
250.000	30.66±0.01	28.75±0.01	44.45±0.01	60.45±0.01	45.45±0.01
125.000	34.25±0.02	32.13±0.02	48.87±0.02	61.58±0.01	47.72±0.01
62.500	36.63±0.01	34.37 ± 0.01	54.73±0.01	69.10±0.01	51.09±0.01
31.250	37.10±0.01	34.79±0.02	64.75±0.01	81.71±0.01	56.51±0.01
15.630	39.85±0.01	37.370±0.02	66.27±0.01	84.96±0.01	58.15±0.01
7.810	51.67±0.02	48.47± 0.02	74.76±0.02	85.69±0.01	65.61±0.01
3.910	56.71±0.01	70.05±0 .01	77.74±0.01	86.44±0.01	77.57±0.01
1.000	100.000	100.000	100.000	100.000	100.000
IC50	5.846 µg/ml	6.639 µg/ml	12.57 µg/ml	23.63 µg/ml	6.654 µg/ml

Table No. 2: DPPH free radical scavenging assay of Bauhinia rascemosa root extract

Sample Concentrations (µg/ml)	% Inhibition
Sample Concentrations (µg/ml)	ABAE	ABCE	ABEA	ABEE	ABPE
100	12.13±0.04	25.29667±0.02	22.16333±0.04	16.63±0.02	39.93±0 .03
200	24.41±0.02	49.09667±0.01	48.84333±0.02	34.42±0.01	57.74±0.02
300	43.33±0.02	71.65334±0.01	61.10667±0.01	58.85±0.02	76.64±0.02
400	52.20±0.01	82.19334±0.01	71.070±0.03	74.40±0.02	88.86±0.02
500	75.53±0.01	93.30334±0.01	83.350±0.04	81.08±0.02	94.42±0.01
EC50	354.59 µg/ml	281.55 µg/ml	249.31 µg/ml	215.26 µg/ml	146.01 µg/ml

Table No. 3: NO Scavenging Activity of Bauhinia rascemosa root extract

Sample Concentrations(µg/ml)	% Inhibition
Sample Concentrations(µg/ml)	AE	CE	EA	EE	PE
100	17.03±0.01	17.02±0.01	47.85±0.01	34.01±0.01	69.13±0.01
200	29.77±0.01	40.41±0.01	55.31±0.01	41.48±0.01	75.51±0.01
300	42.57±0.01	67.03±0.01	67.02±0.01	50.93±0.01	80.83±0.01
400	59.57±0.01	86.15±0.01	75.53±0.01	62.77±0.01	93.60±0.01
500	77.67±0.01	95.73±0.01	88.28±0.01	75.51±0.01	98.92±0.01
EC50	330.91 µg/ml	271.56 µg/ml	133.66 µg/ml	244.498 µg/ml	100 µg/ml

The extract exhibited dose dependent or concentration dependent cytotoxicity & free radical scavenging action. IC 50 was calculated from MTT assay and expressed in terms of percentage inhibition of cancer cells. DPPH and NO free radical scavenging assay indicated that antioxidant activity of various extracts was concentration dependent and the EC₅₀ was also determined. On the basis of the results obtained in the present study, we conclude that the root extract of B. racemosa has significant amounts of Antioxidant and Cytotoxicity activity. Further studies are needed to isolate the exact active component, which are responsible for the antioxidant and anticancer activities.

CONCLUSION:

Free radicals are chemical molecules that can exist separately with one or more unpaired electrons. The propagation of free radicals can brings about thousands of reactions and thus makes excessive tissue damage, lipid, protein, DNA are all susceptible to attack free radicals^17,18. Antioxidants may offers resistant against oxidative stress by scavenging the free radicals, inhibiting lipid peroxidation etc. The results obtained from the present studies are clearly indicated that the roots extract of B. racemosa had powerful antioxidant activity against various antioxidant systems in In-vitro.

The In-vitro cytotoxic study produced considerable cell viability which indicates cytotoxic potential of our extracts. The IC₅₀ determined establishes the safety and effective quotients of our extracts. Methanolic extract showed more cell viability among others.

The content of Flavonoidal compounds in the extract may be contributing factor towards antioxidant action. Flavonoids, Phenolics, Tannins are well known for their cytotoxicity. Thus, these ingredients may contribute the free radical scavenging potential as well as the cytotoxic potential of B. racemosa extract.

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Received on 25.02.2020 Modified on 10.05.2020

Research J. Pharm. and Tech. 2021; 14(6):3333-3336.

DOI: 10.52711/0974-360X.2021.00579