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Comparative antimycobacterial activity of some Indonesian medicinal plants

against multi-drug resistant Mycobacterium tuberculosis

Article in Journal of Applied Pharmaceutical Science · January 2015

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Journal of Applied Pharmaceutical Science Vol. 5 (01), pp. 019-022, January, 2015
Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2015.50104
ISSN 2231-3354

Comparative antimycobacterial activity of some Indonesian medicinal

plants against multi-drug resistant Mycobacterium tuberculosis
Maksum Radji1*, Marita Kurniati1, Ariyani Kiranasari2
1
Laboratory of Microbiology and Biotechnology, Faculty of Pharmacy, University of Indonesia, Depok, 16424, Indonesia.
2
Department of Clinical Microbiology, Medical Faculty, University of Indonesia, Jakarta 10320, Indonesia.

ARTICLE INFO ABSTRACT

Article history: This study has been carried out to compare antimycobacterial activity of five selected Indonesian endogenous
Received on: 24/09/2014 medicinal plants of Andrographis paniculata, Annona muricata, Centella asiatica, Pluchea indica, and Rhoeo
Revised on: 14/10/2014 spathacea against clinical isolate of multi-drug resistant (MDR) Mycobacterium tuberculosis. The aqueous
Accepted on: 22/11/2014 extracts of leaves of Andrographis paniculata, Annona muricata, Centella asiatica, Pluchea indica, and Rhoeo
Available online: 30/01/2015 spathacea were obtained by maceration, and the phytochemical constituents of each extract were screened.
Antimycobacterial activity of aqueous plant extracts were determined by proportion methods using Lowenstein
Key words: Jensen (L-J) medium. Our study exhibited that all extracts of five selected plants showed inhibited activity
Antimycobacterial activity, against Mycobacterium tuberculosis H37Rv strain and multi drug resistant (MDR) strain. The proportion
MDR tuberculosis, medicinal inhibition of aqueous extract of Andrographis paniculata, Annona muricata, Centella asiatica, Pluchea indica,
plants, Indonesia. and Rhoeo spathacea, against Mycobacterium tuberculosis H37Rv strain were 100%, 82.1%, 78.5%, 100%, and
100% respectively, whereas against MDR strain were 93.7%, 50.0%, 50.0%, 100%, and 100% respectively. The
phytochemical analysis showed that the extracts were predominantly contains flavonoids, alkaloids, saponins,
tannins and glycosides. Pluchea indica, and Rhoeo spathacea showed good antimycobacterial activity against
MDR strains and could be useful as complementary alternative therapy in combating the emergence of MDR
strains of Mycobacterium tuberculosis.

INTRODUCTION Tuberculosis generally is asymptomatic disease and effort

to control tuberculosis is still faced with many problems, especially
Tuberculosis (TB) is still one of the most serious public with the increasing spread of multi drug-resistant (MDR) and
health problems in the world, including Indonesia. In 2012, extensively drug-resistant (XDR) tuberculosis. According to a
WHO estimated that 8.6 million incident cases of tuberculosis WHO report in 2012 of TB morbidity caused by MDR-TB is about
worldwide. Most of the TB cases occur in Asia (58%) and 450.000 people, with a high mortality rate of around 170.000 deaths
African region (27%). Smaller proportions of cases occur in the from MDR-TB (WHO, 2013).
Eastern Mediterranean region (8%), European region (4%) and The rapid increasing of the incidence of MDR and XDR
the America regions (3%) (WHO, 2013). M. tuberculosis strains in the world, and no anti-tuberculosis agent
Indonesia is the fourth largest country in incident has been introduced in the past 30 years (Gautam et al., 2007),
cases of TB after India, China, and South Africa. The incidence necessitated the urgent need to search for new anti-tuberculosis.
of TB cases in Indonesia is estimated at about 0.5 million in Medicinal plants offer a great hope to overcome these needs. Herbal
2012. According to reports from the World Health Organization medicines have been used to treat several diseases by traditional
(WHO) in 2012, the prevalence of tuberculosis in Indonesia is practitioner over the world.
estimated at 297 per 100,000 populations, the incidence of 185 In the past few years, a large number of plant
per 100,000 populations and the mortality rate 27 per 100,000 species have been studied for their activity against
populations (WHO, 2013). Mycobacterium tuberculosis (Newton et al., 2002;; Copp,
2003; Okunade et al., 2004; Pauli et al., 2005; Billo et al.,
.

* Corresponding Author
Email: [email protected] 2005; Soejarto et al., 2005; Mmushi et al., 2010; Luo et al., 2011).

© 2015 Maksum Radji et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlike
Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
020 Radji et al. / Journal of Applied Pharmaceutical Science 5 (01); 2015: 019-022

More recently, several studies have also demonstrated significant Medicine University of Indonesia. MDR strain of M. tuberculosis
activity of several compounds derived from plants against MDR was resistant to rifampicin and isoniazid.
strains of Mycobacterium tuberculosis (Camacho-Corona et al.,
2008; Gordien et al., 2009; Gupta et al., 2010; Hannan et al., Antimycobacterial activity assay
2011; Antony et al., 2012; Shukla et al., 2013; Singh et al., 2013; The assessment of the antimycobacterial activity of the
Muthuswamy et al., 2013). extracts was performed using Lowenstein–Jensen (L-J) proportion
In Indonesia, there are several indigenous plants that are methods (Gupta et al., 2010), with slight modification. The
reportedly used traditionally to treat tuberculosis and respiratory inoculum of M. tuberculosis standard strain and MDR strain were
infections. Therefore, the purpose of this study was to compare the prepared from Lowenstein-Jensen (L-J) slants. The inoculums
in vitro activity of antimycobactrial activity of Andrographis were prepared by 10-fold dilution steps of the mycobacterial
paniculata, Annona muricata, Centella asiatica, Pluchea indica, suspension of a 1.0 McFarland standard, to produce the dilution
and Rhoeo spathacea against multi-drug resistant (MDR) of 10-1, 10-2, 10-3, 10-4 and 10-5. The 10-5 of mycobacterial dilution
Mycobacterium tuberculosis. was used for inoculation.
A quantity of 2.5 g of dried extract was dissolved in
MATERIALS AND METHODS minimum amount of DMSO and diluted using a 50.0 ml
volumetric flask with distilled water to make a 50 mg/ml stock
Plant collection
solution, and sterilized by filtration through a 0.2 µm membrane
Five medicinal plants were selected in this study. The
filter (Whatman, USA). Three different concentrations of 1.25
selection of plants was based on their traditional use for the
mg/ml, 2.5 mg/ml, and 5.0 mg/ml of each extracts were prepared.
treatment of tuberculosis and asthma by the local traditional
To make 1.25 mg/ml extract solution, 1.25 ml of stock solution (50
practitioners. The parts of selected plants of Andrographis
mg/ml) was added aseptically to 48.75 ml of sterile LJ medium,
paniculata, Annona muricata, Centella asiatica, Pluchea indica,
and shaken gently until mix thoroughly. Then 7 ml of the medium
and Rhoeo spathacea were obtained from Research Institute for
was dispensed into Mc.Cartney sterile bottles and then inspissated
Medicinal and Aromatic Plants, Bogor, and identified at Research
to coagulate at 85°C for 45 minutes.
Center for Biology, Indonesian Institute of Sciences, Bogor.
Similarly, to make 2.5 mg/ml extract solution, and 5.0
The dried leaves of Annona muricata, Pluchea indica,
mg/ml extract solution, 2.5 ml and 5.0 ml of stock solution (50
Rhoeo spathacea, and herbs of Andrographis paniculata, and
mg/ml) was added aseptically to 47.5 ml and 45 ml of sterile
Centella asiatica were cut to very small pieces and crushed into a
Lowenstein Jensen (L-J) medium, respectively. Then 7 ml of each
fine powder using an electric grinder and stored in airtight
medium was dispensed into Mc.Cartney sterile bottles and then
containers in a dark place to prevent oxidation until the extraction
inspissated to coagulate at 85°C for 45 minutes. The L-J slants
stage.
were left at room temperature for 24 h, the caps of Mc.Cartney
bottles being loosely closed in order to evaporate a part of the
Extraction
humidity. The control medium without extract and control drug
The aqueous extracts of each plant materials from each
(rifampicin, 40 μg/ml) were prepared at the same time as the
species were obtained by maceration. Each finely powder of each
extract containing media. Then all the L-J slants were inoculated
plant materials were soaked in 5 L distilled water for 24 hours.
with 0.1 ml of mycobacterial suspension of the chosen dilutions of
The maceration was repeated 3 times to exhaustively extract the
10-5 and incubated at 37°C for 4 weeks. Reading was taken
plant material. Extracts were filtered and concentrated using a
weekly. For comparison, extract free control L-J slants were used.
rotary evaporator (Buchi, Switzerland) in a water bath set at 70°C.
The dried extracts obtained from each plant were packed in glass
RESULTS
bottles with proper labeling and the amount of extracts obtained
was quantified. The extracts were stored in a refrigerator at 4o until The in vitro antimycobacterial activity of 5 selected
use. plants against M. tuberculosis H37Rv and MDR were assayed by
Lowenstein–Jensen (L-J) proportion method. All extracts of five
Preliminary analysis of phytochemicals selected plants showed inhibited activity against Mycobacterium
Each of plant extracts were screened for the presence of tuberculosis H37Rv strain and multi drug resistant (MDR) strain.
alkaloids, saponins, tannins, glycosides, and flavonoids using the The proportion inhibition of 5 mg/ml of aqueous extract of
standard procedures as described previously (Harborne, 1987). Andrographis paniculata, Annona muricata, Centella asiatica,
Pluchea indica, and Rhoeo spathacea, against Mycobacterium
Mycobacterial strains tuberculosis H37Rv strain were 100%, 82.1%, 78.5%, 100%,
The Standard strain of Mycobacterium tuberculosis and 100% respectively, whereas against MDR strain were
H37Rv and isolated multi drug resistant (MDR) strain were 93.7%, 50.0%, 50.0%, 100%, and 100% respectively, as shown in
provided by Department of Clinical Microbiology, Faculty of Table 1.
 Radji et al. / Journal of Applied Pharmaceutical Science 5 (01); 2015: 019-022 021

Table 1: Antimycobacterial activity of aqueous plant extracts against Mycobacterium tuberculosis H37Rv and MDR strains.
L-J proportion methods
Inhibition rate (%)
Plant Part Mycobacterial Strain
Plant extracts (mg/ml) Rifampicin μg/ml
1.25 2.50 5.0 40
Andrographis paniculata (Burm. F) Ness. Herbs H37Rv 3.5 35.7 100.0 100.0
MDR 6.2 56.2 93.7 0
Annona muricata Linn. Leaves H37Rv 7.1 64.3 82.1 100.0
MDR 0 6.2 50.0 0
Centella asiatica (L.) Urb. Herbs H37Rv 3.5 57.1 78.5 100.0
MDR 6.2 12.5 50.0 0
Pluchea indica (L.) Less. Leaves H37Rv 3.5 50.0 100.0 100.0
MDR 6.2 59.3 100.0 0
Rhoeo spathacea (L. Her) Hance. Leaves H37Rv 32.1 100.0 100.0 100.0
MDR 28.6 100.0 100.0 0

Table 2: Phytochemicals analysis of plant extracts.

Plant Alkaloids Flavonoids Saponins Tannins Glycosides
Pluchea indica (L.) Less. + + + + +
Annona muricata Linn. + + + + -
Rhoeo spathacea (L. Her) Hance. + - + + +
Andrographis paniculata (Burm. F) Ness. + + + + +
Centella asiatica (L.) Urb. + + + + +

The aqueous extract of Rhoeo spathacea exhibited potent In this study we found that Rhoeo spathacea was the potent
antimycobacterial activity. The result of phytochemical analysis bactericidal. The proportion of inhibition of aqueous extract (2.5
of extracts showed that extracts predominantly contains mg/ml) of Rhoeo spathacea was 100% against M. tuberculosis
flavonoids, alkaloids, saponins, tannins and glycosides as shown in H37Rv and MDR strain. To our knowledge the study of
Table 2. Alkaloids, saponins and tannins were present in all plant antimycobacterial activity of Rhoeo spathacea and Pluchea indica
extracts. extracts against MDR strain of M. tuberculosis are reported for the
first time. Regarding to the bioactive constituents of plant
DISCUSSION extracts, we found that the plant extracts predominantly contains
flavonoids, alkaloids, saponins, tannins and glycosides. Alkaloids,
The emergence of multi drug resistant (MDR), extreme
saponins and tannins were present in all plant extracts. It has been
drug resistant (XDR) and recently reported total drug resistant
proposed that the antimycobacterial activity of plant extracts may
(TDR) strains of Mycobacterium tuberculosis (Pandya et al.,
be due to the main bioactive components in the extracts including
2012), highlighted an urgent need to find a new compounds
flavonoids, alkaloids, tannins, saponins, and polyphenols (Arya,
derived from herbal medicines. Present study demonstrated that
2011; McCarthy and Mahony, 2011). Previous study of some
aqueous extracts of Andrographis paniculata, Pluchea indica, and
flavonoids isolated from Dorstenia barteri showed
Rhoeo spathacea, showed to have antimycobacterial activity
antimycobacterial activities. Isobachalcone, kanzanol C, 4-
against M. tuberculosis H37Rv and MDR strains. In Indonesia,
hydroxylonchocarpin, stipulin, and amentoflavone exhibited
stems, leaves and flowers of Andrographis paniculata were used
potential activities at MIC < 10 μg/ml against M. tuberculosis
traditionally for the treatment of respiratory tract infection
(Kuete et al., 2010). Plant terpenoids have been reported to have
including tuberculosis and the aqueous extracts of Andrographis
antimycobacterial activity. Sesquiterpene, longifolene, totarol and
paniculata has been used as alternative medicine for tuberculosis
transcommunic acid, obtained from Juniperus communis showed
in primary health care in Surabaya, East Java (Sulistiawati et al.,
the highest activity against Mycobacterium tuberculosis H37Rv,
2010). In our study we found that aqueous extract of
whereas longifolene and totarol also showed activity against
Andrographis paniculata showed good activity against M.
rifampicin-resistant variants (Gordien et al., 2009). Moreover,
tuberculosis H37Rv strain. In the previous study indicated that the
alkaloids extracted from several plants have also been reported to
administration of aqueous Andrographis paniculata extracts could
exhibit antimycobacterial activity (Copp, 2003). Those various
minimize the hepatotoxic effect of rifampicin in rats
findings indicate that the presence of active compounds such as
(Muthulingam, 2012). These results support the use of
alkaloids, polyphenols, flavonoids, and terpenoids in medicinal
Andrographis paniculata plants in traditional medicine and
plants were considerably responsible for their activity against
complementary medicine. The aqueous extracts of Pluchea indica
Mycobacterium tuberculosis.
and Rhoeo spathacea demonstrated bactericidal effect
against M. tuberculosis H37Rv and MDR strain.
CONCLUSION
Our result consistent with other study that extract
of Pluchea indica showed antimycobacterial activity against M. Our findings showed that Pluchea indica, and Rhoeo
tuberculosis H37Rv standard strain (Mohamad et al., 2010). spathacea demonstrated promising antimycobacterial activity
022 Radji et al. / Journal of Applied Pharmaceutical Science 5 (01); 2015: 019-022

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