Study of the potential antibacterial activity of plant extracts of Adina cordifolia using the thiazolyl blue tetrazolium bromide (MTT) assay

Introduction

Numerous plants provide a variety of therapeutic compounds that are useful for both medical and agricultural purposes. Given that many people worldwide rely on traditional plant-based treatments due to the scarcity and high cost of contemporary medications, plants offer enormous promise as sources of medicinal and pharmacopoeial drugs [1] . Many herbal remedies that have been made possible by these medicinal plants are essential for the creation of novel medications that are used to cure and prevent illness [20] . The deciduous tree known as haldu (Adina cordifolia) is native to South Asia and can be found from eastern India and Sri Lanka to southern China and Vietnam. It is a member of the Rubiaceae family, subfamily Cinchonoideae. It thrives in regions as high as 900 meters above sea level south of the Himalayas and is dispersed over the majority of India's deciduous woods. According to www.fes.org.in, A. cordifolia is in danger of being extinct. Oriental medicine has long employed A. cordifolia as an essential ingredient in a variety of antiseptic and antipyretic medicines [5] . The bark is hot, bitter, and aromatic; it is used to cure liver problems and is strengthening, healing, and aphrodisiac in nature. According to Chadha (1985), the roots are used as an astringent for diarrhea. Due to its active ingredients' intriguing H+/K+-ATPase inhibitory actions, A. cordifolia was also investigated for its ability to treat ulcers [9] . According to Rokade and Pawar (2013), four chemicals that were recovered from a strain of Adina cordifolia were identified as stigmasta-5, α-amyrin, octacosanol, 22-diene-3P-O-αrhamnopyranosyl-(1-4)-P-dixylopyranoside and naringenin-7-methyl ether-4'-O-αrhamnopyranoside. The greatest technique for protecting the endangered Adina cordifolia in the wild and quickly multiplying plant populations is through apical bud in vitro propagation. According to Raypa et al. (2013), Adina cordifolia was highly well established in vitro when MS medium was added with either 0.5 mg/L NAA or 2 mg/L BAP. The main compounds identified in the leaf and bark extracts of Mitragyna parvifolia (Rubiaceae) were butyric acid, 2-ethylhexyl ester (19.36%), 4-methylmannose (53.13%), mitraphylline (21.59%), isomitraphylline (3.37%), 1,2-hydrazinedicarboxylic acid, diethyl ester (3.50%), α,αdimethylmuconic acid (22.97%), isobutanoic acid, 2-ethylhexyl ester (43.83%), α-Dglucopyranoside, α-D-glucopyranosyl (27.21%).

M. parvifolia leaves include compounds such as the pentacyclic oxindole alkaloid mitraphylline, which has been shown to have anti-inflammatory and anti-proliferative qualities [22] .

Materials and Methods

Materials

The leaves and roots of Adina cordifolia were collected from Agro-forestry Research Centre (AFRC) Haldi (29. 06441° N, 79.82281° E) G. B. Pant University of Agriculture and Technology, Pantnagar in the month of March.

Experimental work 2.2.1 Preparation of plant material

Leaves and roots were washed thoroughly and oven dried at 40 °C. Then dried samples were grounded to make powder in mechanical grinder and stored in air tight container.

Preparation of samples

Plant extracts were prepared in five different solvents viz chloroform, ethyl acetate, acetone, methanol and water. Powdered plant material was used for respective solvent (w:v) (1:4) extraction. The extracts were filtered; evaporated and dried respective extracts were used for experimentation.

Statistical analysis

All the analyses were performed in triplicates and results were reported as means ± standard deviation (SD). The significance of differences among treatment means were determined by one way analysis of variance (ANOVA) with a significant level of (p=0.05).

Antibacterial assay in different leaf and root extracts against selected human pathogens 2.3.1 Test microorganisms and growth conditions

The animal pathogenic bacterial strains isolated from faecal matter.All bacterial strains were grown in nutrient agar (NA) plates at 37 °C. Glycerol stocks were also prepared for each culture and stored at -20 °C. Every culture was streaked on the NA plate each time before experiment and for cell viability assay each bacterial culture was grown on nutrient broth (NB).

Pathogenic bacterial strains 1. Salmonella typhimurium 52 2. Escherichia coli 13

Determination of antibacterial assay via MTT assay

The antibacterial assay was determined by MTTtetrazolium-based colorimetric assay [13] with slight modifications. In this assay the reduction of the tetrazolium salt MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide) to blue formazan product due to mitochondrial enzymes was measured spectrophotometrically. MTT dye of 5 mg/mL was prepared in phosphate saline buffer (1X). Each extract starting from 1 mg was serially diluted and volume was made up to 100µl with methanol in each well of a 96-well microplate. Last well was used as methanol blank without any extract in it. The bacterial cell suspension (100 µl) was plated at a density of approximate 1.5 ×10 8 cells per well with help of Mc-Farland standard and incubation was done for 24h, followed by adding 20 µl (5 mg/mL) of MTT dye to each well. Excess MTT was discarded after one hour incubation at 37 °C and the intracellular purple insoluble formazan was solubilized by adding 50 µl/well DMSO. After shaking on orbital shaker for 20 mins the optical density (OD) was measured at 570 nm in microplate reader. Gentamycin (1mg/mL) was used as a positive control prepared in phosphate saline buffer (1X) to calculate the cytotoxicity. All tests and analyses were run in triplicate and mean values were recorded. Phosphate saline buffer (1X, g/L) was prepared in desired volume containing Cacl 2 anhydrous (0.10 g), KCl (0.20 g), KH 2 PO 4 (0.20 g), MgCl 2 .6H 2 O (0.10 g), NaCl (8.00 g), Na 2 HPO 4 (1.15 g) anhydrous and the pH was adjusted at 7.4. In the present study, leaf and root extracts (obtained in chloroform, ethyl acetate, acetone, methanol and water) of Adina cordifolia were assayed to check cell viability of six gram negative human pathogenic strains i.e., Salmonela typhimurium 52, Salmonela typhimurium 149, Salmonela typhimurium ATCC 51812, Salmonela entiritidis ATCC 13076, E. coli ATCC-25922, and E coli strain-13 by the method of Mosmann (1983) using gentamycin as a positive control with some modification. Cell viability was determined by using tetrazolium salt, MTT (3-(4, 5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) based colorimetric assay. MTT is the commonly applied method for evaluation of cell viability and cytotoxicity for screening the drugs. The MTT assay based on the reduction of MTT (yellow colored) depends upon cellular metabolic activities due to NAD (P)H-dependent cellular oxidoreductase enzymes [3] . This method uses colorimeter to determine the cell viability [16] . In MTT assay, the linear relationship between metabolically active cells and the colour produced is established, thus allowing an accurate quantification of changes in the rate of cell death or proliferation [3] .

Results

Antibacterial assay for cytotoxicity of various extracts of leaf and root of Adina cordifolia against Salmonella typhaemurium 52

Salmonella typhaemurium is a gram negative bacteria causing gastroenteritis, poisoning and typhoid fever [10] .The increasing effectiveness of plant extracts were determined on the basis of viability percentage of the cells. The extracts concentration were 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL. Cell viability percentage decrease was observed as the concentration of plant extracts were increased. Salmonella typhaemurium 52 was treated with the all five (chloroform, ethyl acetate, acetone, methanol and water) leaf extracts of Adina cordifolia and antibiotic, gentamycin was taken as positive control. Among all the leaf extracts, acetone extract showed good cytotoxicity against S. typhaemurium 52. In leaf acetone extract, only 52.47% viable cells were found at 1000 µg/mL concentration (i.e. mortality percentage was 47.53%). In leaf methanol, water, ethyl acetate and chloroform showed 61.24%, 64.99%, 65.34% and 69% viable cells respectively at 1000 µg/mL concentration Fig. 1) i.e. mortality percentage was 38.76%, 35.01%, 34.66% and 31% in leaf methanol, water, ethyl acetate and chloroform extracts respectively. Gentamycin showed 28.71% viable cells in 1000 µg/mL concentration (i.e. mortality percentage was 71.29%). Single compound 7-hydroxycoumarin which is key component in root (not found in leaf except only trace amount found in leaf water extract) showed 47% viable cells at 1000 µg/mL concentration i.e. mortality percentage was 53%. In root extracts of Adina cordifolia, root ethyl acetate extract showed maximum cytotoxicity against S. typhaemurium 52. In root ethyl acetate extract, only 56.4% viable cells were found at 1000 µg/mL concentration (Fig. 2 (i.e. mortality percentage was 43.6%). In root extracts, chloroform, acetone, methanol and water showed 56.40%, 59.86%, 65.05% and 79.75% viable cells respectively at 1000 µg/mL concentration i.e. mortality percentage was 43.6%, 40.14%, 34.95% and 20.25% in chloroform, acetone, methanol and water extracts respectively. The result showed clearly that different extracts of Adina cordifolia irrespective of the plant parts and solvent used, were able to inhibit bacterial growth to a significant level. Thus the use of plants like Adina cordifolia for prevention and cure of different bacterial ailments might reduce the dependence on synthetic drugs (antibiotics), as well as adverse effects associated with them.

Antibacterial assay for cytotoxicity of various extracts of leaf and root of Adina cordifolia against E coli strain-13

E. coli also reported to cause severe cases including bloody diarrhea, dehydration and kidney failure [6] . E coli strain-13 was treated with the all five leaf extracts of Adina cordifolia. Among all the leaf extracts, water extract showed good cytotoxicity against E. coli strain-13. In leaf water extract, only 47.84% viable cells were found at 1000 µg/mL concentration (i.e. 52.16% cells had been killed by extract).

In leaf extracts, ethyl acetate, chloroform, acetone and methanol showed 54.5%, 56.34%, 60.70% and 63.23%

viable cells respectively at 1000 µg/mL (Fig. 3) i.e. mortality percentage was 45.5%, 43.66%, 39.3% and 36.77% in ethyl acetate, chloroform, acetone and methanol leaf extracts respectively. Antibiotic, gentamycin was taken as positive control. Gentamycin showed 28.9% viable cells in 1000 µg/mL concentration i.e. gentamycin had highest mortality percentage (71.1%). Single compound 7-hydroxycoumarin which is key component in root showed 45.59% viable cells at 1000 µg/mL concentration (i.e. 54.41% cells had been killed by compound).

In root extracts of Adina cordifolia, root ethyl acetate extract showed maximum cytotoxicity against E coli strain-13. In root ethyl acetate extract, only 45.07% viable cells were found at 1000 µg/mL concentration (i.e. 54.93% cells had been killed by extract). In root extracts, water, acetone, methanol and chloroform showed 47.72%, 48.67%, 49.42% and 69.35% viable cells respectively at 1000 µg/mL concentration (Fig. 4.) i.e. mortality percentage was 52.28%, 51.33%, 50.58% and 30.65% in water, acetone, methanol and chloroform root extracts respectively (Fig. 3 and Fig. 4).

The result showed clearly that different extracts of Adina cordifolia irrespective of the plant parts and solvent used, were able to inhibit bacterial growth to a significant level. Diffusion methods (agar disk diffusion and agar well diffusion) have been widely used to screen the antimicrobial activity of extracts and their main compounds however, these tests do not permit the quantification of their bioactivity in terms of minimum inhibitory concentration (MIC), since they are qualitative tests [19] .

Conclusion

Adina cordifolia extracts possess unique bioactive components, such as 7-hydroxycoumarin, which have been shown to have good bactericidal and inhibitory effect against bacterial strains.

Acknowledgement

Authors are grateful to Dept. of Biochemistry, GBPUA&T, Pantnagar to carry out this research work. Our sincere thanks to DSTFIST for providing equipment facility and Directorate of Experiment Station (DES) Pantnagar for utilization of infrastructure.

Competing Interests

Authors have declared that no competing interests exist.

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