Antioxidant and Antimicrobial Activities of Different Extracts of Callus and Intact Plant (Seeds) of Fenugreek(Trigonellafoenum-graecum)

Abstract

The present study aimed to investigate antioxidant and antimicrobial activities of calls and seeds extracts of fenugreek (Trigonella foenum-graecum). Palnt tissue culture technique was employed to induce callus from hypocotyls and cotyledons explants of T f0enum- graecum by culturing them in MS medium supplemented with different concentrations and combinations of plant growth regulators. Induced callus and seeds of fenugreek were investigated for their in vitro antioxidant activity using DPPH method and antimicrobial activity against four standard bacteria (Bacillus subtilus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli) and two fungi (Aspergillus niger and Candida albicans) using paper disc diffusion method. In order to induce the callus, the explants were cultured in MS medium supplemented with two auxins, 2,4-D and NAA separately with different concentrations, and(O.5 mg/1) of kinetin was used in combination with different concentrations of 2,4-D and NAA hormones. Callus was successfully induced in all concentrations and combinations. No callus formation was observed in the absence of plant growth regulators. Hypocotyls explants were much better in inducing callus than cotyledons explants. The best callus formation was observed at sixth week of cultivation in most growth regulators. The highest mean of callus index was recorded (3.5O:tO.l5) with 100% of callusing by MS medium supplemented with 4.0mg/1 NAA+O.5 Kin from hypocotyls segments compared to other treatments. In the case of callus induction from cotyledons segments the highest mean of callus index was 2.4l:kO.18 with 100% of callusing by MS medium supplemented with 1.0mg/1 2,4-D. Hypocotyls and cotyledons derived calluses and seeds were extracted by petroleum ether and methanol by Soxhlet(continuous extraction method). The extracts were investigated for their antimicrobial and antioxidant activities. The petroleum ether extract of Tfoenum-graecum seeds showed highest antimicrobial activity compared to other extracts with maximum zone of inhibition 2010.88 mm against Aspergillus niger by concentration 250 mg/ml. Petroleum ether extracts for both hypocotyls and cotyledons derived calluses were ineffective against all tested microorganisms. Methanolic extracts of hypocotyls and cotyledons derived calluses showed antibacterial activity against S. aureus by concentration 250mg/ml with inhibition zone ll:h0.00mm, l2i0.57mm respectively. Inhibition gone l0:l:0.00mm was recorded against E. coli for both methanolic extracts of hypocotyls and cotyledons derived callus by concentration 250mg/ml. Methanolic extract of seeds was ineffective against all tested microorganisms except weak effect 8i0.00 rnm against C. albicans. Petroleum ether extract of T.f0enum- graecum seeds by 250mg/ml showed activity higer than Ampicillin/Sulbactam (20 pg/disc) and Ciprofloxacin (5 pg/disc) against S.aureus, also methanolic extracts of hypocotyls and cotyledons derived calluses by 250mg/ml showed activity equal to Ampicillin/Sulbactam (20 pg/disc )and Ciprofloxacin (5 pg/disc) against S. aureus. Phytochemical screening for both seeds and callus extracts indicated the presence of various secondary metabolites like alkaloids, flavonoids, tannins, phenols, saponins, terpenoids and steroids. Antioxidant activity was evaluated for methanolic extracts of callus and seeds and petroleum ether extract of seeds of fenugreek. The highest free radicals scavenging activity was obtained from cotyledons derived callus (91 .5:hO.l6%) followed by hypocotyls derived callus (85.46i0.29%) and methanolic extract of seeds (80.53:l:0.0l%). Petroleum ether extract of seeds demonstrated weak antioxidant activity with inhibition percentage 24.56:h0.l2%. Significant differences(p<0.05) were detected among different antioxidant potential of Tl foenum-graecum callus al1d seeds extracts. IC50 values of DPPH scavenging capacity of methanolic extracts were evaluted in descending order, plant seeds(l.l185mg/g)>hypocotyls derived callus(O.7l59mg/g)> cotyledons derived callus (O.49l4mg/g)>ascorbic acid (0.l874mg/g). A lower value of IC50 indicates a higher antioxidant activity. Highest phenolic content was observed in callus of cotyledons (4l2.087 mg/l), compared to callus of hypocotyls (2ll.l937 mg/l) and methanolic extract of seeds(l24.84 mg/l) calculated as mg/l gallic acid equivalent of phenols. Methanolic extract of seeds showed highest level of flavonoid content (424.951 mg/l), compared to callus of cotyledons (2l7.285 mg/l) and callus of hypocotyls (95.92 mg/1) calculated as mg/l quercetin equivalent of flavonoids. Tarmins content were detected only in methanolic extract of seeds (1l6.259 mg/l calculated as tannic acid equivalent of tannins).