Inhibition fo the growth of Mycobacterium bovis by vegetable extracts diversified with hydrazine

RAMALLO, IA; DE LA IGLESIA, A; MORBIDONI, HR; FURLAN, RLE

Rosario

Congreso; 2° Rerunión Internacional de Ciencias Farmacéuticas; 2012

RICIFA

Introduction Tuberculosis caused by Mycobacterium_tuberculosis (MTb) affects 20.5million people worldwide.1 Increasing resistence of MTb strains to the first-line drugs rifampin and isoniazid (INH) evidence the need for more effective molecules. Isoniazid, like the majority of active-against-MTb compounds, has in its structure the acylhydrazide group.2 Acylhydrazide-portion is in a very little proportion on nature scaffolds (70% of them contain it), is the optimum access to the incorporation of N-N through hydrazine(N2H4.H2O).3 In this work we modified with N2H4.H2O extracts of Sonchus_oleraceus, Brassica_rapa and Solanum_diflorum. The activity of the native(unmodified) and the modified extracts was studied in microplate and bioauthographic assay modality. Materials -Extracts hydrazinolysis: Ethanol solution of extract(2% wt/vol) and N2H4.H2O(1% wt/vol) was refluxed 4hs. -Stearyl hydrazide: Methyl stearate and N2H4.H2O reacted in MeOH 24hs (eq.ratio 1:10) -LDH: Linoleyl chloride and N2H4.H2O (eq. ratio 1:1.1) reacted in CHCl3 (0°C, 2hs). For mixture of linoleyl/oleyl/stearyl hydrazides an eq.ratio 1:10 (linoleyl chloride:N2H4.H2O) was used. -Linoleyl INH: INH and linoleyl chloride (eq.ratio 1:1) reacted in CHCl3 (2 hs, RT) -MTb bioauthography: an agarized culture medium containing MTb was distributed on the TLC plate. After solidification, the plates were incubated. Once plates were revelated white inhibition zones appeared against blue background. Results In all the cases (except for methanol S. oleraceus extract) the MICs values of the microplate assay found for modified extracts were lesser than the respective native controls. We can highlight the modified B. rapa extract whose MIC was more than 30times lower than the value of the native extract (TableI/II). To know how many compounds are responsible of the activity, we performed TLC coupled with MTb-bioautography. Modified extracts of the three species assayed showed halos that were absent in the controls. The most interesting were those observed for modified hexane B. rapa extract. By bioguided fractionation of B. rapa we identified linoleyl_diacylhydrazide (LDH) a novel structure generated by the reaction. The MIC values for linoleic acid, LDH and related structures synthetized are in TableIII. This results validate LDH as the most interesting compound: MIC=12.5μg/mL(22.46μM). How, structures containing INH portion and fatty chains are active againts MTb,5 we synthesized linoleyl derivative of INH. The MIC value was