O R I G I N A L A RTICL E ASIAN JOURNAL OF MEDICAL SCIENCES

Antimycobacterial activity of certain mangrove

plants against multi-drug resistant Mycobacterium
tuberculosis
Amudha1, Prabuseenivasan2, Vanaja Kumar2
1
Department of Biotechnology, Jaya College of Arts and Science, Tirunindravur, Chennai, India, 2Department of Bacteriology, National Institute
for Research in Tuberculosis (ICMR), Chetput, Chennai, Tamil Nadu, India

Submitted: 24‑11‑2013 Revised: 29‑11‑2013 Published: 10‑03‑2014

ABSTRACT
Objectives: The present study was conducted aiming to identify a potent mangrove plant Access this article online
against Mycobacterium tuberculosis using rapid method, luciferase reporter phage (LRP)
Website:
assay. Methods: Seven mangrove plants viz., Ceriops decandra, Aegiceras corniculatum, http://nepjol.info/index.php/AJMS
Excoecaria agollacha,Avicennia officinalis, Rhizophora mucronata, Suaeda monoica and
Sesuvium portulacastrum were collected from Pichavaram mangrove forest in the east coast
of, Tamil Nadu. Hexane and methanol extracts were obtained and antimycobacterial activity
was done by LRP assay at the concentration of 500 µg/ml. Mycobacterial strains viz. reference
strain M. tuberculosis H37Rv and two clinical isolates of M. tuberculosis, one sensitive and
the other resistant to Streptomycin, Isoniazid, Rifampicin and Ethambutol used for this study.
Results: Methanol extract of E. agollacha showed maximum antimycobacterial activity followed
by A. corniculatum and A. officinalis at 500 µg/ml concentration. The remaining four plants
(Suaeda monoica, Sesuvium portulacastrum, R. mucronata and C. decandra) showed less or no
activity. However, hexane extracts of all the tested plants failed to inhibit the mycobacterial
strains. Conclusions: Among the seven plants tested, E. agollacha was found to have better anti-
TB activity. Further studies based on bioassay guided fractionation and phytochemical analysis
may useful to identify the potent antimycobacterial molecule(s) from this mangrove plant.
Key words: Antimycobacterial activity, Mangrove plants, MDR tuberculosis and LRP assay

INTRODUCTION development of new scaffolds of drugs.5 The primary

benefits of using plant derived medicines are that they are
relatively safer than synthetic derivatives, offering profound
Mycobacterium tuberculosis is a highly infectious disease that spreads
therapeutic benefits and more affordable treatment. India
through air. The rate of mortality caused by tuberculosis is
is one of the few countries in the world that has unique
much higher than any other bacterial infections; around 30%
wealth of medicinal plants and vast traditional knowledge
of the AIDS related deaths are due to tuberculosis.1 India is
of use of herbal medicines for cure of various diseases.
the highest tuberculosis burden country accounting for one
Like other terrestrial plants, many mangrove plants have
fifth (21%) of the global incidence.2 At present, because of
ethnopharmacological relevance and have also been
uncontrolled application of anti-tuberculosis drugs, there is
exploited by the local people in the search for remedies
an increasing prevalence of both MDR and XDR strains.3
for various ailments.
So there is an urgent need to discover new antibiotics against
drug resistance M. tuberculosis. Flora of mangrove forests is unique from others in that
their habitat extends along the border where the fresh and
Nearly 80% of the world population depends upon sea water merge. Therefore, unlike common terrestrial
medicines from plant sources.4 It is already proved that plants, they can withstand high salt concentration, can
plants and other natural products is template for the remain submerged in water, and maintain an efficient

Address for Correspondence:

N. Amudha, Department of Biotechnology, Jaya College of Arts and Science, Tirunindravur, Chennai-602024.
E-mail: [email protected] © Copyright AJMS

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 Amudha, et al.: Antimycobacterial activity of certain mangrove plants

nutrient retention mechanism.6 However, only a few of the Institute for Research in Tuberculosis (NIRT- ICMR),
mangrove plants are investigated for their antimycobacterial Chennai, Tamil Nadu, India.
activity, due to lack of adequate information available about
their uses and the difficulty in screening methodology of Antimycobacterial activity using Luciferase Reporter
atimycobacterial activity. Plants often produce secondary Phage assay
metabolites under stressful conditions, as the mangrove Luciferase reporter phage PhAE129, a D29 derived
plants, facing various ecological and environmental stresses, mycobacteriophage, constructed in the laboratory of W.R.
biosynthesis a wide range secondary metabolites of Jacobswas used7 in this study and it was propagated with
potential medicinal importance. In this regard, the present M. smegmatis mc2 155 to get high titer by harvesting with
study was aimed to screen some medicinally important Mycobacteriophage buffer (MP) from lacey plates and
mangrove plant against M. tuberculosis using rapid method stored at 4ºC until use.
luciferase reporter phage (LRP) assay.
Luciferase reporter phage (LRP) assay was carried out using
standard protocol.8 Hundred microliter bacterial suspensions
MATERIALS AND METHODS equivalent to McFarland #2 standard were added to 400 μl of
G7H9 with and without the test compound. For each sample,
Collection of mangrove plants two sample-free controls and single drug concentration
Based on the traditional knowledge and literature survey, (500μg/ml) were prepared, and incubated for 72 h at 37°C.
seven mangrove plants were collected from Pichavaram After incubation, 50 μl of the high-titer phage phAE129
mangrove forest, east coast, Tamil Nadu (Table 1). All the and 40 μl of 0.1 M CaCl2 were added to all the vials and
collected plants were identified and authenticated in CAS incubated at 37°C for another 4 h. After incubation, 100 ml
in Marine Biology, Annamalai University, Chidambaram, of the mixture was transferred from each tube into a star
Tamil Nadu. The collected specimens were shade dried and tube and equal amount of working D-luciferin (0.3 mM in
powdered individually using electric blender. The powdered 0.05 M sodium citrate buffer, pH 4.5) solution was added.
materials were sequentially extracted with solvent (1:5 w/v) The relative light unit (RLU) was measured after 10s of
such as hexane and methanol. Each extract was filtered integration in the luminometer (Monolight 2010). The
and condensed using rotary vacuum evaporator to obtain percentage reduction in RLU was calculated for each test
solvent free residue and these crude extracts were stored sample and compared with the control. Antimycobacterial
in –20ºC until use. activity is indicated by fifty percent reduction in relative light
units (RLU) in the presence of compound in comparison
Mycobacterial strains with compound free control.
Three Mycobacterial strains, viz., standard strain of M.
tuberculosis H37Rv, clinical isolates of M. tuberculosis strains Phytochemical screening
resistant to first line antiTB drugs, Streptomycin(S), Phytochemical screening for the active crude extract of
Isoniazid (H), Rifampicin(R) and Ethambutol(E) and E. agollacha was performed by standard protocol to find
sensitive to SHRE were used for this study. These strains out the presence of phyto-constituents viz., Alkaloids,
were grown and maintained on Lowenstein Jensen (L-J) flavonoids, glycosides, phytosterol, tannins, saponins and
medium in the Department of Bacteriology, National volatile oils.9, 10

Table 1: Selected mangrove plants and their

properties (Bandaranayke, 2002)
RESULTS
S. No. Botanical name Common Medicinal use/ In the present study, two solvent extracts of seven
name properties
mangrove plants were screened for antimycobacterial
Aegiceras Asthma, Diabetes
1
corniculatum
Narikkandal
and Rheumatism
activity against standard strain and two clinical isolates
Avicennia Diuretic, Hepatitis, of tuberculosis. The results are presented in Table 2.
2 Venkandal,
officinalis and Leprosy The methanolic extract of E. agollacha showed maximum
Ceriops Hepatitis, and
3
decandra
Chiru kandal
Ulcers
antimycobacterial activity against standard strain of M.
Excoecaria Leprosy and tuberculosis H37Rv (88.95%), clinical isolate resistant to first
4 Thillai
agollacha, Epilepsy line drugs (70.02 %) and the sensitive strain of M. tuberculosis
Rhizophora
Elephantiasis, (82.54%) followed by A. corniculatum and A. officinalisat
5 Surapunnai Hepatitis and
mucronata
Febrifuge
at 500µg/ml concentration. In other hand C. decandra,
6 Suaeda monoica Umarichedi Hepatitis R. mucronata, S. monoica and S. portulacastrum exhibited weak
7 Sesuvium Vangaravasi Hepatitis or no activity against all three tested strains of M. tuberculosis
portulacastrum
when compared with sample free controls.

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 Amudha, et al.: Antimycobacterial activity of certain mangrove plants

Table 2: Percentage reduction in RLU by selected mangrove plants against standard strain and clinical
isolates of M. tuberculosis at 500µg/ml concentration
Mangrove M. tuberculosis H37Rv M. tuberculosis isolates M. tuberculosis isolates
plants (Standard strain) (Resistant to SHRE) (Sensitive to SHRE)
Hexane Methanol Hexane Methanol Hexane Methanol
A. corniculatum 0.00 66.63 34.36 54.70 15.16 64.33
A. officinalis 43.03 61.39 11.77 51.04 47.58 73.08
C. decandra 0.00 21.05 0.97 28.59 0.00 33.97
E. agollacha 43.94 88.95 43.33 70.02 41.10 82.54
R. mucronata 0.00 0.00 20.27 32.83 11.12 24.46
S. monoica 0.00 46.42 17.30 42.50 46.52 41.81
S. portulacastrum 0.00 43.95 0.00 38.42 17.36 49.23

The preliminary phytochemical screening of active Table 3: Preliminary phytochemical screening of

methanolic extract of E. agollacha is presented in Table 3. active methanolic extract of E. agallocha
The findings revealed that the plant contains alkaloids, S. No. Phyto‑constituents Methanol extract
flavonoids, phytosterols, tannins and absence of glycosides, 1 Alkaloids +
saponins and essential oils. The same observation was made 2 Glycosides ‑
by Dhyanithi et al., 201211 and they found the methanol 3 Flavonoids +
extract of E. agollacha showsignificant antibacterial activity 4 phytosterols +
5 Saponins +
against fish pathogen, Aeromonas hydrophila. 6 Tannins +
7 Essential oils ‑

DISCUSSION
E. agallocha will be worth pursuing and may lead to potential
The antimycobaterial activity exhibited by the mangrove anti-TB agents.
plant extracts could be due to the presence of phytochemicals
such as, alkaloids, tannins and flavonoids present in the CONCLUSIONS
plants. Extracts and products of mangrove plants are used in
traditional medicine to treat various diseases for centuries.12 Among the seven selected plants, E. agollacha was found
The present study indicates that when compared with to have better anti-TB activity. Further studies based on
hexane extracts, the crude methanol extracts showed better bioassay guided fractionation and phytochemical analysis
inhibition activity against all tested mycobacteria indicating may lead to potent antimycobacterial molecule(s) from this
that active ingredients in the plant could be extracted in to mangrove plant.
methanol. However some plants extracts did not exhibit
any activity. This is probably because accumulation of
intracellular active ingredients is low or the concentration ACKNOWLEDGEMENT
of the compound used may not be sufficient.
Authors are grateful to local people of Pitchavaram
Some of the mangrove plants have been screened earlier for providing the information about mangrove plants
for antiviral, antibacterial, antiulcer and anti-inflammatory and sample collection. The authors are also thankful
activity.13 In this study, E. agallocha showed maximum to Director, NIRT for providing facilities to carry out
antimycobacterial activity among the tested plants. In antimycobacterial activity.
traditional Thai medicine, the bark and wood of E. agallocha
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Authors Contribution:
Am – Designed the experiments performed the laboratory tests and drafted the manuscript; SP – Analysed the data and drafted the manuscript; VK – Designed
the study and reviewed the manuscript.

Source of Support: Nil, Conflict of Interest: None declared.

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