In Vitro Anti-Mycobacterial And Bioactive Compounds Of Pluchea indica (L.) Less. Against Surrogate Tuberculosis Organisms
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Date
2016-07
Authors
Jamaludin, Azirah Inez
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Abstract
Pluchea indica (L.) Less. has been used widely in traditional medicine as a
remedy of a variety of illnesses including symptoms of tuberculosis (TB) and thus,
could provide an alternative source of anti-TB compounds. Preliminary in vitro
screening of three different P. indica partitions, using tetrazolium microdilution assay
showed that leaf and stem n-hexane and leaf chloroform partitions exhibited inhibitory
activity against Mycobacterium smegmatis, M. fortuitum and M. kansasii with
minimum inhibitory concentrations (MICs) of 200 - 3200 μg/mL. The highest activity
was shown by leaf n-hexane against M. kansasii with MIC of 200 μg/mL. The leaf
n-hexane partition was selected for further bioassay guided fractionation process using
thin layer and column chromatographic techniques, which yielded eight fractions
(Fractions A - H). These fractions exhibited activity against M. kansasii with MICs in
the range of 12.5 – 800 μg/mL. The highly active fractions (A, C, D, and E) also
inhibited M. tuberculosis H37Ra with MICs in the range of 50 – 100 μg/mL.
Phytochemical identifications of the most active fraction, Fraction C using gas
chromatography-mass spectrometry produced 10 identified compounds, of which,
n-hexadecanoic acid, 9,12-octadecadienoic acid (Z,Z)-, and octadecanoic acid were
the major compounds. Screening of these compounds against M. tuberculosis H37Ra
showed that their activities were lower than the activity of their original Fraction C.
Thus, Fraction C was further assessed on its interactions with four first-line anti-TB
drugs against M. kansasii and M. tuberculosis H37Ra. One synergistic interaction was
produced by the combination with rifampicin against M. tuberculosis H37Ra with
fractional inhibitory concentration index (FICI) value of 0.375. An antagonistic
interaction was observed in the combination with ethambutol against M. kansasii with
FICI value of 4.125. Further study on the killing rate of the combination of Fraction C
with rifampicin at their MIC values showed a bactericidal activity (95 % killing rate)
towards the growth of M. tuberculosis H37Ra, whereas, Fraction C alone was
bacteriostatic (67 % killing rate). Observation of the ultra-structure of the treated
M. tuberculosis H37Ra cells under transmission electron microscope showed that
Fraction C caused general disintegration of the cells particularly, the cell wall. The
cytotoxicity study of Fraction C on nasopharyngeal epithelial cell line (NP69) showed
that Fraction C was toxic with low IC50 value of 2.68 ± 0.1 μg/mL but the effects could
be dose-dependent or time-dependent. The findings of this study gave a scientific basis
to the traditional use of P. indica for symptoms of TB and this plant could be a potential
source of anti-TB compounds worthy of further investigation.
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Keywords
The traditional use of P. indica for symptoms of TB and this plant could be , a potential source of anti-TB compounds.