Micropropagation, bioactive and phytochemical studies of potent herb Eclipta alba L.

Abstract

The use of natural products as such or in the form of isolated compounds continues to be popular and accepted practice throughout the world. Even in the ongoing 1 st decade of 21 sl century many of the new drugs launched to the market have natural product origin which points out the significance of these aquatic and terrestrial resource.s in the ongoing drug discovery efforts. Despite the advancement of technology, development of many new strategies of drug discovery and development, control and treatment of many diseases, complete cure from tuberculosis and cancer is not a reality. Plants have always been important among the terrestrial natural resources in this respect and today several phytochemical terchniques are available for analysis of crude plant extracts. Most of the success stories are linked to the selection of the plants based on their traditional and local use. This attempt has been made to investigate Eclipta alba (L.), Fagonia cretica (L.) and Lantana camara (Fruit) for their possible anti-tuberculosis and estrogen mediated anti-cancer potential. The folk and traditional medicinal uses of these plants suggest their anti-bacterial and anti -cancer potential. The presented work was aimed at the justification of their traditional use through their screening for possible anti-mycobacterial and anti-cancer (estrogenic/anti-estrogenic) effects alongwith the phytochemical investigation of potent herblherbs. Crude extract of each plant was fractionated along a gradient of polarity (Hexane-Ethyl acetate-Methanol) and the subsequent fractions in each case were subjected to the Microplate Alamar Blue Assay (anti-TB assay) and Ishikawa assay. Only crude extract of Eclipta alba showed significant anti-estrogenic activity (53%) with minute cytotoxicity (12%) against human endometrial cancer cell line. No estrogenic/anti-estrogenic/cytotoxic activity could be observed in case of crude extract of Fagonia cretica. Though anti-estrogenic activity of fractions F3 and F4 of Eclipta alba was comparable with the standard (tamoxifen) but these fractions were cytotoxic (>90% cytotoxic activity) to the cells. Fractoin F6 had significant anti-estrogenic activity without any cytotoxicity. This fraction showed dose dependent anti-estrogenic activity with ICso 13.42flg/ml. The fraction F6 of Eclipta alba was subjected to FCPC and HPLC for further isolation. Isolated peaks were characterized by GCIMS. Major compounds identified include several esters and methyl esters of fatty acids including hexadecanoic acid methyl ester, octadecanoic acid methyl ester, 1, 2- benzene dicarboxylic acid mono (2-ethylhexyl) ester, 1, 2-benzenedicarboxylic acid di -isooctyl ester, benzene propanoic acid, 3, 5-bis (1 , I-dimethyl)-4-hydroxy-methyl ester, butenedioic acid (Z) - monodecyl ester and propanoic acid pentadecyl ester. Some other impOliant constituents including coumarin, l-ethynyl-3, 5-dimethyladamantane and 3, 4-dichlorobenzenethiol were also detected but data obtained from GCIMS run showed <90% matching with the standard. Wedelolactone, a known iso lated compound from Eclipta alba, was also investigated for its estrogeni c, antiestrogenic and cytotoxic effects on human endometrial carcinomatic cell line.The anti-estrogenic activity (inhibition of AP in 89% endometrial cancer cells) was comparable with the positive control, tamoxifen (inhibition of AP in 95% of the cells). Keeping in view their reported anti-microbial potential, Eclipta alba, Lantana camara (fruit) and Fagonia cretica were also tested for their inhibitory effects on Mycobacterium tuberculosis. Crude extracts of Fagonic cretica and Lantana camara showed marginal to medium anti-TB activity. Active fractions of both Lantana camara (LC-E and LC-F) and Fagonia cretica (FC-4) had MIC>lOOllg/ml (considered high). Furthermore, these fractions were cytotoxic to the Vero cells so these plants were not fo llowed for further studies. Roots of Eclipta alba are traditionally used as anti-bacterial remedy while stem is famous for its use against tuberculosis, both above ground (leaves along with shoot) and below ground (roots) portions were evaluated alongwith the whole plant for anti-TB potential. Fractions ER-l, ER-3, ER-5 and ER-6 of root of Eclipta alba significantly inhibited the Mycobacterium tuberculosis with MICs 62.65 f,lg/ml , 80.18f,lg/ml, 63.96 f,lg/ml and 120.91Ilg/ml respectively. Phytochemical investigation of the active fraction ER-3 resulted in the detection of docosane as the main constituent. Fraction EA-3 of above ground portion and fraction, EW-l of whole plant (Eclipta alba) also exhibited significant inhibition of Mycobacterium tuberculosis with MICs 58.55 f,lg/ml and 62.88 ~l g/ ml respectively . Continuously increasing medicinal importance of Eclipta alba and its distribution in Pakistan mostly in those areas which are the victim of war on terrorism has put its existence to high risks. Tissue culture technique is important in this respect because artificial propagation can be ensured throughout the year. Since there is no report on in vitro regeneration of Eclipta alba growing in Pakistan so in this study several protocols for in vitro propagation of this highly important herb have been successfully developed. Conclus ively, this is the first ever report which suggests the anti-uterus cancer potential of Eclipta alba, wedelolactone and Lantana camara (Fruit). No previous report of anti-TB potential of Eclipta alba, Fagonia cretica and fruit of Lantana camara against Mycobacterium tuberculosis is avail abl e. In addition, from fraction ER-3 (root of Eclipta alba) having si gnificant inhibition of Mycobacterium tuberculosis, docosane was isolated as the major constituent. Furtherfore, phytochemical investigation of the active fraction F6 having significant antiestrogenic activity has revealed several esters and methyl esters of fatty acids alongwith some other important compounds not reported before.