Micropropagation and Antimicrobial activity of Brucea mollis Wall. ex Kurz- an endangered plant of Northeast India.

Abstract

Brucea Mollis Wall. ex Kurz belonging to the family Simaroubaceae is an endangered medicinal plant confined to Northeastern region of India. In addition to India, it is distributed in Bhutan, China, Bangladesh, Cambodia, Myanmar, Laos, Indonesia, Malaysia, Sri Lanka, Philippines, Nepal, Vietnam and Thailand. In India, it is distributed in West Bengal, Sikkim, Arunachal Pradesh, Assam, Nagaland, Manipur and Meghalaya. The plant is popularly known as koinine and has been used in traditional healthcare practices against malaria in Karbi Anglong District of Assam. Its fruit and root decoction has been used by the local people of Karbi Anglong District to cure malaria. Besides, the plant is also reported to have anticancer, antitumor, cardiovascular and diuretic activity. Despite its uses in traditional healthcare practices, the population of B. Mollis has been diminishing in alarming rate due to several anthropogenic activities such as habitat fragmentation and over-exploitation. The species has been listed as endangered during the CAMP survey in 2003. In B. Mollis, it has been reported that the pollination, as well as a seed dispersal mechanism, is ineffective. As a result, the plant becomes rare in its natural habitat. Therefore, there is a need to develop an efficient protocol for its mass propagation as a part of conservation initiatives. Further, no work has been carried out on the antimicrobial activity of B. Mollis. The present study was focused to establish a micropropagation protocol and to determine its antimicrobial activity. For micropropagation, leaf, BAP (2 mg/L) + NAA (0.3 mg/L) + IBA (1 mg/L) in internode callus in MS media. Though the maximum number of shoots initiation occurred from the leaf callus, the percentage of shoot regeneration was found to be 73.33 11.54 %. While the internode callus showed 100% with maximum shoot initiation 7.60 0.40. In case of B5 media, the maximum number of shoots (5.53 1.10) with 60% shoot initiation occurred from the leaf callus at BAP (2 mg/L) + NAA (0.5 mg/L) + Kinetin (3 mg/L) concentration. There was no significance in the number of leaves in all the concentrations of growth regulators used in both the media. The in vitro raised shootlets were transferred for rooting in half-strength MS and B5 media supplemented with IBA and NAA at 0.5-4 mg/L concentration. It was observed that MS media was more effective than B5 media. The shoots initiated 100% root initiation at 0.5, 1, 2 and 3 mg/L concentration at IBA and NAA respectively in 21-44 days. Among all the concentrations studied, IBA 3 mg/L concentration was found to be the optimum concentration for a percentage of root initiation, time duration, root number and root length. Though NAA could enhance 100% root initiation, IBA was found to be more effective in the parameters that evaluated. The maximum number of roots (26.46 1.62) initiated at IBA 3 mg/L as compared to NAA 3 mg/L which showed maximum 19.80 1.40 in root number. The maximum root length (1.82 0.20 cm) was obtained in 30 days at IBA 3 mg/L as compared to 1.68 0.20 cm which was maximum in NAA 3 mg/L concentration. In the case of B5 media, similar response exhibited among the concentrations in IBA and NAA. IBA was found to be more effective than NAA in the parameters studied. Though similar kind of response in terms of percentage of root initiation could be observed between IBA and NAA, the former is more effective in terms of the parameters studied. The healthy plantlets with well-developed roots were transferred to soil ripe for acclimatization. The plantlets were covered with polybags for a week to maintain humidity after which the polybags were perforated to lower down the atmospheric humidity. During alternate days, the plantlets were sprinkled with water. The covered polybags were gradually perforated to lower down the atmospheric humidity. When the plantlets began to grow new leaves, they were transferred to soil rite: garden soil: farmyard manure in the ratio of 1:1:1. About 60 % of plantlets survived under natural environment. For the antimicrobial activity of B. Mollis, fresh leaves and callus derived from the leaves were taken as the plant sample for preparing crude extract. Water, methanol and ethanol were taken as the solvents for preparing the extract. The microbial strains B. subtilis MTCC441, S. aureus MTCC3160, P. aeruginosa MTCC424 and S. marcescens MTCC2645 were taken as the bacterial strains and A. fumigatus MTCC2550, A. niger MTCC282 were the two fungal strains used in the study. The antimicrobial activity was studied by disc diffusion method. Both leaf and callus derived from the leaf extracts showed antimicrobial activity against the microbial strains in methanol and ethanol extracts. The water extracts did not show any zone inhibition. Two positive control Ceftadizime (30 g/disc) and Amphotericin B (20 g/disc) were taken for antibacterial and antifungal activity respectively and DMSO was taken as the negative control. In general, the callus extracts showed better activity than the leaf extracts against all the bacterial strains. The highest zone inhibition 17.33 0.33 mm was observed against B. subtilis in callus ethanol extract as compared with positive control 12.33 0.88 mm. With increasing concentration of the extract, it was found to have more potential against the tested bacterial and fungal strains. In case of fungal strains, the leaf ethanol extract showed better effect than callus ethanol extract against A. niger with 18.33 0.33 mm inhibition at 20 mg/ml concentration compared with positive control Amphotericin B (15.66 0.33 mm). While the methanol leaf extract was more effective against A. fumigatus with 17.66 0.33 mm inhibition than the leaf ethanol extract. The minimum inhibitory concentration was determined using the microdilution method and was found to be effective. The MIC of ethanol callus extract was found to be lowest (0.156 mg/ml) against B. subtilis, S. aureus and A. fumigatus. On the other hand, the lowest MIC at 0.156 mg/ml was recorded against A. niger in the methanol leaf extract. In this investigation, micropropagation protocol for B. Mollis was standardized in two different media with different explants through indirect organogenesis. Among the three explants used in the study, the highest potential for organogenic differentiation from callus could be observed from internode explants on MS media. The protocol thus established will facilitate for mass propagation and conservation of the plant. Further, the determination of antimicrobial activities of the plant against the microorganisms has strengthened the potentiality of the plant in the healthcare sector, suggesting for further analyses of the plant.