Assessment of the Activity of Selected Indian Medicinal Plants against Mycobacterium tuberculosis: A Preliminary Screening Using the Microplate Alamar Blue Assay

Abstract

Aim: Identification of anti-Mycobacterium tuberculosis agents of plant origin, against
sensitive and multidrug resistant (MDR) strains.
Study Design: Assessing anti-M. tuberculosis activity of five Indian medicinal plants,
which have been reported in traditional literature for various uses including respiratory
ailments.
Place and Duration of Study: Mumbai, India; May 2009 – December 2011.
Methodology of Study: The reference strain (H37Rv), three susceptible and three MDR
clinical isolates of M. tuberculosis were used. Acetone, ethanol and aqueous extracts
(prepared sequentially) of Acorus calamus L. (rhizome), Andrographis paniculata Nees.
(leaf), Ocimum sanctum L. (leaf), Piper nigrum L. (seed) and Pueraria tuberosa DC.
(tuber) were tested at 1, 10 and 100 μg/ml using the Microplate Alamar Blue Assay. The
active extracts were assessed for cytotoxicity on the human lung epithelial cell line (A549)
using the neutral red assay and a phytochemical analysis was made using High
Performance Thin Layer Chromatography (HPTLC).
Results: Among the plants tested, the acetone extract of P. nigrum appears promising. It
was effective against H37Rv, all susceptible isolates and one MDR isolate at 100 μg/ml.
The ethanol extract caused some inhibition of growth, though less than the cut-off of 99%.
A combination of acetone and ethanol extracts at 50 μg/ml each was effective against all
isolates tested. The known active phytoconstituent of P. nigrum, piperine (also an efflux
pump inhibitor), was effective against H37Rv in the presence of suboptimal concentration
of Rifampicin, but not against the clinical isolates tested. Presence of piperine in the
acetone and ethanol extracts was confirmed by HPTLC. Extracts of P. nigrum and
piperine were not cytotoxic to the A549 cell line.
Conclusion: Amongst the five plants tested, P. nigrum was active. The acetone extract
may have active components in addition to piperine. It is possible that the class and
expression of efflux pumps in H37Rv is different from that in the clinical isolates, and
hence piperine did not inhibit these isolates. Thus, it is necessary to screen clinical
isolates in addition to reference strains. The observation of the increased efficacy of the
combination of acetone and ethanol extracts is interesting.

Full text

____________________________________________________________________________________________
*Corresponding author: Email: fmr@fmrindia.org; fmrmum@gmail.com;
European Journal of Medicinal Plants
2(4): 308-323, 2012
SCIENCEDOMAIN international
www.sciencedomain.org
Assessment of the Activity of Selected Indian
Medicinal Plants against Mycobacterium
tuberculosis: A Preliminary Screening Using
the Microplate Alamar Blue Assay
Tannaz Birdi1*, Desiree D’souza1, Monica Tolani1,
Poonam Daswani1, Vinita Nair1, Pundarikakshudu Tetali2,
Juan Carlos Toro3and Sven Hoffner3
1The Foundation for Medical Research, 84-A, R.G. Thadani Marg, Worli, Mumbai - 400018,
Maharashtra, India.
2Naoroji Godrej Centre for Plant Research, Lawkim Motors Group Campus, Shindewadi,
Shirwal, Satara - 412801, Maharashtra, India.
3Department for Preparedness, Swedish Institute for Communicable Disease Control, SE
17182, Stockholm, Sweden.
Authors’ contributions
This work was carried out in collaboration between all authors. TB was responsible for the
study. DD was involved in co-ordination of the study, carrying out the assays, preparation
and editing of the manuscript. MT was involved in carrying out the assays, undertaking the
literature survey, preparation and editing of the manuscript. PD was involved in preparation
of extracts, undertaking the literature survey, preparation and editing of the manuscript. VN
was involved in carrying out the assays. PT collected and authenticated the plant material,
and obtained the herbarium numbers. JCT was responsible for carrying out the external
quality control of the MABA, editing the manuscript. SH was In charge of WHO
Supranational Reference Laboratory for TB at Swedish Institute for Communicable Disease
Control, editing the manuscript. All authors read and approved the final version of the
manuscript.
Received 12th June 2012
Accepted 17th August 2012
Published 12th October 2012
ABSTRACT
Aim: Identification of anti-Mycobacterium tuberculosis agents of plant origin, against
sensitive and multidrug resistant (MDR) strains.
Research Article

European Journal of Medicinal Plants, 2(4): 308-323, 2012
309
Study Design: Assessing anti-M. tuberculosis activity of five Indian medicinal plants,
which have been reported in traditional literature for various uses including respiratory
ailments.
Place and Duration of Study: Mumbai, India; May 2009 – December 2011.
Methodology of Study: The reference strain (H37Rv), three susceptible and three MDR
clinical isolates of M. tuberculosis were used. Acetone, ethanol and aqueous extracts
(prepared sequentially) of Acorus calamus L. (rhizome), Andrographis paniculata Nees.
(leaf), Ocimum sanctum L. (leaf), Piper nigrum L. (seed) and Pueraria tuberosa DC.
(tuber) were tested at 1, 10 and 100 µg/ml using the Microplate Alamar Blue Assay. The
active extracts were assessed for cytotoxicity on the human lung epithelial cell line (A549)
using the neutral red assay and a phytochemical analysis was made using High
Performance Thin Layer Chromatography (HPTLC).
Results: Among the plants tested, the acetone extract of P. nigrum appears promising. It
was effective against H37Rv, all susceptible isolates and one MDR isolate at 100 µg/ml.
The ethanol extract caused some inhibition of growth, though less than the cut-off of 99%.
A combination of acetone and ethanol extracts at 50 µg/ml each was effective against all
isolates tested. The known active phytoconstituent of P. nigrum, piperine (also an efflux
pump inhibitor), was effective against H37Rv in the presence of suboptimal concentration
of Rifampicin, but not against the clinical isolates tested. Presence of piperine in the
acetone and ethanol extracts was confirmed by HPTLC. Extracts of P. nigrum and
piperine were not cytotoxic to the A549 cell line.
Conclusion: Amongst the five plants tested, P. nigrum was active. The acetone extract
may have active components in addition to piperine. It is possible that the class and
expression of efflux pumps in H37Rv is different from that in the clinical isolates, and
hence piperine did not inhibit these isolates. Thus, it is necessary to screen clinical
isolates in addition to reference strains. The observation of the increased efficacy of the
combination of acetone and ethanol extracts is interesting.
Keywords: Traditional medicine; anti-Mycobacterium tuberculosis; microplate alamar blue
assay; cytotoxicity; HPTLC; Piper nigrum.
1. INTRODUCTION
Tuberculosis (TB) caused by M. tuberculosis is a highly infectious disease, and the morbidity
and mortality due to TB continue to be a cause of concern. Due to the global emergence of
multidrug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis
(Singh, 2007) and more recently the reports of totally drug resistant TB (Udwadia et al.,
2012; Velayati et al., 2009), there is an urgent need to develop new drugs and strategies to
fight TB (Souza, 2006). The last few decades have witnessed a substantial increase in the
investigation of medicinal plants for their biological efficacy in treatment of various disorders.
In the field of anti-TB agents, a number of studies on potential medicinal plants have been
reported from various parts of the world (Gautam et al., 2007; Green et al., 2010;
Lakshmanan et al., 2011; Webster et al., 2010).
Over the years, a number of improved and high throughput techniques towards screening of
anti-mycobacterial agents have been developed (Nayyar and Jain, 2005). Several methods
exist for testing the anti-M. tuberculosis potential of plant extracts, such as fluorescence
based testing on the Bactec MGIT960 system, use of redox indicator dyes such as Alamar
Blue or Resazurin and MTT, using colony forming units (CFU) on solid agar plates. The CFU
assay is too time consuming and tedious for use as a screening protocol, while assays using

European Journal of Medicinal Plants, 2(4): 308-323, 2012
310
indicator dyes are rapid and efficient. Techniques such as the agar diffusion and broth
dilution method have been used, but they too have limitations (Gautam et al., 2007). The
Microplate Alamar Blue Assay (MABA) is a colorimetric oxidation-reduction based assay. It
is a non-radiometric, rapid, high-throughput and comparatively low cost assay producing
results with a high degree of confidence (Collins and Franzblau, 1997; Kumar et al., 2005).
Moreover, this technique has been used by a number of researchers for testing anti-
mycobacterial activity of several plants (Camacho-Corona et al., 2008; Webster et al., 2010).
Hence, in the present study five Indian medicinal plants viz., Acorus calamus L. (rhizomes),
Andrographis paniculata Nees. (leaves), Ocimum sanctum L. (leaves), Piper nigrum L.
(seeds) (along with piperine) and Pueraria tuberosa DC. (tubers) were studied for their anti-
M. tuberculosis potential using the MABA.
The primary reasons for choosing these medicinal plants, besides their known antibacterial
properties, were their use in respiratory ailments as cited in traditional literature. Use of
these plants for respiratory disorders in reported ethnobotanical surveys were also
documented (Table 1). All plants selected are native plants for which cultivation practices
have been developed. Sustainable harvesting is possible for all plants except A. calamus
and P. tuberosa.In vitro screening of organic (acetone and ethanol) and aqueous extracts of
the selected plants against M. tuberculosis reference strain H37Rv and drug susceptible and
MDR clinical isolates using the MABA was undertaken. Thus, in all, 15 extracts were
studied. This preliminary study was intentionally restricted to testing crude extracts since it
has often been noticed that crude extracts are more efficacious than the isolated compounds
(Ginsburg and Deharo, 2011; Houghton, 2000; Sood et al., 2012). Anti-M. tuberculosis
activity in the absence of cytotoxicity would confirm their anti-M. tuberculosis potential.
Hence, cytotoxicity of these plant extracts was also carried out using A549, a human lung
epithelial cell line, since M. tuberculosis is an intracellular pathogen.
2. MATERIALS AND METHODS
2.1 Plants Used and Preparation of Extracts
The plant material used for the present study was collected and authenticated by Dr. P.
Tetali, Naoroji Godrej Centre for Plant Research (NGCPR, Shirwal, Maharashtra). Voucher
specimens of the plants were deposited at the Botanical Survey of India (BSI), Western
Center, Pune, India (A. calamus,A. paniculata,O. sanctum,P. nigrum); or at NGCPR (P.
tuberosa). The area of collection and the herbarium numbers of the plants have been
presented in Table 2.
Extracts were prepared in a sequential manner using acetone, ethanol and distilled water as
solvents from 25g of shade dried and coarsely powdered plant material using the Soxhlet
apparatus. For 25g of plant material 300ml of respective solvent was used and refluxed for a
period of 24-30 hours. Continuous refluxing ensured that efficient extraction of the phyto-
constituents in their respective solvents was achieved. The aqueous extract was prepared
by boiling the plant material post ethanol extraction and evaporation of the solvent until the
volume of water was reduced to 25% (Thakkur, 1976).

European Journal of Medicinal Plants, 2(4): 308-323, 2012
311
Table 1. Basis of selection of plants for the study
Botanical name
(Family)
Common
Name
Selection criteria
Traditional usea
Ethnobotanical surveya
Antibacterialb
Acorus calamus
Linn.
(Araceae)
Sweet Flag
(Vekhand)
Rhizome: Cough and
bronchitis (Sharma et al.,
2000-2002)
Dried rhizome powder:
Tuberculosis, expectorant,
chest congestion (Motley,
1994)
Aqueous root extract: anti-TB
activity (Chopra et al., 1957)
Andrographis
paniculata Nees.
(Acanthaceae)
Kalmegh
Whole plant: Bronchitis
(Sharma et al., 2000-2002)
No reference found for use in
India
Ethanol extract of aerial part:
Antibacterial activityc(Mishra
et al., 2009)
Ocimum
sanctum Linn.
(Lamiaceae)
Holy Basil
(Tulsi)
Part used unspecified:
Bronchitis and asthma
(Sharma et al., 2000-2002)
Leaves crushed with onion
bulbs: Cold and cough (Muthu
et al., 2006)
Leaf extract: anti-TB activity
(Farivar et al., 2006)
Piper nigrum
Linn.
(Piperaceae)
Black pepper
(Kali Mirch)
Fruit: Cough (Sharma et al.,
2000-2002)
Seeds: Throat infection
(Ignacimuthu et al., 2006)
Aqueous and various organic
extracts of the fruit:
Antibacterial activityc(Khan
and Siddiqui, 2007).
Pueraria
tuberosa DC.
(Fabaceae)
Bidaarikand
Tuberous root: Tuberculosis
(Billore et al., 2004);
No reference found for use in
India
Ethyl acetate extract of root
tubers: Antibacterial activityc
(Venkata Ratnam and
Venkata Raju, 2009)
aSelected references only for respiratory ailments
bSelected references only
cActivity reported against bacteria other than M. tuberculosis

European Journal of Medicinal Plants, 2(4): 308-323, 2012
312
The aqueous extract was then lyophilized (Thermo Fisher Scientific, USA) and the acetone
and ethanol extracts were allowed to air dry. The yield of the extracts thus obtained is
presented in Table 2. For the assays, the extracts were reconstituted at 20 mg/ml
concentration in dimethyl sulfoxide (DMSO), filtered through 0.2 µm, 25 mm DMSO resistant
Acrodisc syringe filters (Pall Corporation, USA) and stored at -20ºC for up to 2 weeks.
Table 2. Details of the plants used for the study
Botanical name
(Herbarium no.)
Area of
collection
Part used
Percentage Yield (w/w)a
Acetone
Ethanol
Aqueous
A. calamus Linn.
(BSI-131716)
Shindewadi,
Satara district,
Maharashtra
Rhizomes
10.00
7.04
10.80
A. paniculata Nees.
(BSI-131744)
Shindewadi,
Satara district,
Maharashtra
Leaves
6.28
6.00
7.21
O. sanctum Linn.
(BSI-131712)
Shindewadi,
Satara district,
Maharashtra
Leaves
6.00
3.62
24.00
P. nigrum Linn.
(BSI-131714)
Kottayam,
Kerala
Seeds
3.16
6.64
22.64
P. tuberosa DC.
(NGCPR-670)
Mulshi, Pune
district,
Maharashtra
Tubers
6.72
5.92
22.83
aPercentage yield after sequential extraction using Acetone, Ethanol and Water
2.2 M. tuberculosis Strains and Culture Conditions
The M. tuberculosis reference strain H37Rv; three susceptible (S1, S2 and S3) and three
MDR (MDR1, MDR2 and MDR3) clinical isolates were used to test the anti-M. tuberculosis
activity of the extracts using the Microplate Alamar Blue Assay (MABA). The strains were
selected from among those collected for a community based study on MDRTB transmission
in Mumbai, during 2004-2007. The patients sampled were new pulmonary TB patients who
accessed the Revised National Tuberculosis Programme for diagnosis and treatment of TB.
The drug susceptibility profile was determined during the earlier study (D’souza et al., 2009).
The MDR isolates were included to investigate possible cross resistance with anti-TB drugs.
All strains were inoculated into Middlebrook 7H9 medium (7H9) [Becton and Dickinson,
USA] with added ADC (Albumin Dextrose Catalase) supplement (BD, USA) and incubated
for 6 days at 37ºC in a shaker incubator. Twenty-four hours prior to the experiment the
culture was centrifuged and 10 ml of fresh medium was added. At the Foundation for
Medical Research (FMR) the number of Acid Fast Bacilli (AFB) was estimated
microscopically and the density of the culture was adjusted to 0.5 x 106/ml. At the Swedish
Institute for Communicable Disease Control (SMI), the inocula were prepared with the
standard method used for drug susceptibility testing in the Bactec MGIT960 system (Becton
and Dickinson, USA) according to the recommended procedures from the manufacturer.

European Journal of Medicinal Plants, 2(4): 308-323, 2012
313
2.3 Microplate Alamar Blue Assay
The MABA was carried out as reported before (Collins and Franzblau, 1997; Webster et al.,
2010). Briefly, 100 µl of 0.5 x 106/ml of the M. tuberculosis (MTB) were cultured in 7H9
medium in the presence of the plant extracts (1, 10 and 100 µg/ml) in a Nunc™ flat bottom
96 well plate (Nunclon, Denmark). Standard piperine (Sigma-Aldrich, USA) was tested at
0.25, 2.5 and 25 µg/ml. The controls maintained for all the tested strains included: medium,
DMSO (at a volume that would reflect that used for the highest concentration of plant extract
tested i.e. 5 µl DMSO per ml of medium), 1:100 MTB and 2 µg/ml Rifampicin (RIF) (Sigma-
Aldrich, USA). In case of the susceptible strains an additional suboptimal RIF (a
concentration that allowed a percent reduction of Alamar Blue similar to that of the MTB)
control was also maintained. The plant extracts were tested in both absence and presence
of RIF (suboptimal concentration for susceptible isolates and 2 µg/ml for MDR isolates).
Additionally, wells with plant extracts and medium but no bacteria were maintained to check
the interaction of the extract with Alamar Blue. The plates were incubated at 37ºC for 7 days,
after which 10 µl of Alamar Blue dye (Invitrogen, USA) [5% (v/v)] diluted 1:1 in 7H9 medium
was added and incubated for 30 hours. The Optical Density (O.D.) of the wells was
measured at 600 nm and 570 nm on an ELISA reader (Thermo Fisher Scientific, USA), and
the percent reduction of Alamar Blue dye was calculated as per the manufacturer’s
instructions. Use of percent reduction to identify active plant extracts allowed identification of
those extracts with marginal activity (not resulting in 99% kill). This permitted identification of
extracts which could be tested in combination in cases where the extract demonstrated
some activity when used individually at 100 µg/ml. Extracts thus identified were further
tested at 25 µg/ml of each extract or 50 µg/ml of each extract. Overall, for P. nigrum
(acetone and ethanol extracts) the concentrations tested were 1, 10, 25, 50 and 100 µg/ml.
Triplicate wells were maintained for each variable in every assay and all the assays were
performed thrice. In cases where the growth was insufficient in the controls, the assay was
repeated.
2.3.1 Interpretation of the MABA results
Interpretations were based on the percent reduction of the dye which is directly proportional
to the bacterial growth. The extracts were considered active if the percent reduction value of
Alamar Blue dye was less than that observed for the 1:100 MTB control (Clinical and
Laboratory Standards Institute, 2011).
2.4 Internal Quality Control for the MABA
The results of the MABA were compared with the Bactec MGIT960 system (BD, USA). The
extracts of P. nigrum and piperine could not be tested as they auto fluoresced. Of the
remaining four plants, extracts of two plants were randomly chosen to be tested against
H37Rv and all the clinical isolates.
2.5 External Quality Control for the MABA
Extracts which showed activity at the FMR along with two other randomly chosen extracts
were sent to the WHO Supranational Reference Laboratory for TB at SMI for testing against
H37Rv using an identical protocol of MABA.

European Journal of Medicinal Plants, 2(4): 308-323, 2012
314
2.6 Cytotoxicity Testing of Efficacious Plant Extracts
The cytotoxicity of the active plant extracts was carried out using A549 cell line (National
Center for Cell Sciences, Pune, India) by the neutral red uptake assay (Parish and
Mullbacher, 1983). Briefly, appropriate dilutions of the plant extract stock (1, 10 and 100
µg/ml) prepared in RPMI medium (GIBCO BRL, UK) supplemented with 10% FCS (Biowest,
South America) were incubated overnight onto a 24 hr culture of A549 cells and then
subjected to the Neutral red assay. Wells with DMSO (at a volume that would reflect that
used for the highest concentration of plant extract tested i.e. 5 µl DMSO per ml of medium)
and without the plant extract were used as controls. The O.D. was measured at 540 nm
(reference 630 nm) on an ELISA reader (Labsystems, Finland). The percent viability was
calculated with respect to the DMSO control using the following formula: percent viability =
[test/control] x 100. Triplicate wells were maintained for each variable in every assay and all
the assays were performed thrice.
2.7 Phytochemical Profile of Efficacious Plant Extracts
High performance thin layer chromatography (HPTLC) fingerprinting of plant extracts
showing activity in the MABA was carried out on pre-coated Silica gel G60 F254 TLC plates
(Merck, Germany). The extracts were spotted along with appropriate reference standards
using Linomat V Automatic Sample Spotter (CAMAG, Switzerland), run in a ‘twin trough TLC
chamber’, dried and visualized in ‘CAMAG TLC visualizer’.
3. RESULTS
3.1 Anti-M. tuberculosis Activity of the Plant Extracts Using MABA
Crude plant extracts did not show enhanced reduction of Alamar Blue as compared to the
medium control. The range for the medium control was 7.5-10.9 percent reduction and for
the plant extracts 7.4-12.2 percent reduction. In four of the five plants tested viz. A. calamus,
A. paniculata,O. sanctum and P. tuberosa, no anti-M. tuberculosis activity was noted with
any extract against the strains included in the present study (data not shown). We observed
that the acetone extract of P. nigrum at 100 µg/ml showed activity against H37Rv, all three
susceptible isolates and one of the three MDR isolates (MDR3) (Table 3). However, no
activity was seen at 1 and 10 µg/ml. Though the ethanol extract (100 µg/ml) of P. nigrum
showed a drop in reduction of Alamar Blue compared to the MTB control, it was not less
than that of the 1:100 MTB control indicating that this extract had some degree of anti-M.
tuberculosis activity. The aqueous extract did not show any anti-M. tuberculosis action.
Thus, of the 15 extracts tested, the acetone extract of P. nigrum was found to have anti-M.
tuberculosis activity.

European Journal of Medicinal Plants, 2(4): 308-323, 2012
315
Table 3. Percent reduction of Alamar Blue dye obtained for P. nigrum extracts at 100 µg/ml. Values are mean ± standard deviation of 3
independent experiments. Controls (DMSO, Rifampicin and suboptimal Rifampicin, where applicable) were within permissible range
(data not shown)
Strain
Test parameter
1:100 MTB control
MTB control
MTB + P. nigrum extractsa
Aqueous
Acetone
Ethanol
H37Rv
Extract
26.22 ± 1.47
70.68 ± 3.44
70.09 ± 10.09
25.73 ± 4.74
65.47 ± 13.85
Extract + sub optimal RIFb
72.87 ± 12.30
24.33 ± 4.35
66.04 ± 4.53
S1
Extract
20.91 ± 3.14
54.60 ± 1.10
62.35 ± 3.85
18.14 ± 1.95
49.81 ± 6.05
Extract + sub optimal RIFb
48.82 ± 2.97
20.10 ± 5.76
40.04 ± 2.81
S2
Extract
16.83 ± 4.57
57.61 ± 5.55
58.48 ± 13.81
16.37 ± 7.17
37.19 ± 15.14
Extract + sub optimal RIFb
38.45 ± 5.83
15.91 ± 5.84
34.13 ± 11.27
S3
Extract
22.89 ± 3.68
65.15 ± 1.70
50.08 ± 12.13
19.63 ± 1.43
49.21 ± 8.02
Extract + sub optimal RIFb
43.56 ± 16.55
17.02 ± 1.43
45.51 ± 9.49
MDR1
Extract
20.78 ± 1.98
68.90 ± 10.72
58.91 ± 11.81
32.13 ± 4.96
46.31 ± 13.26
Extract + RIF
42.17 ± 11.29
33.96 ± 7.46
30.03 ± 7.42
MDR2
Extract
18.93 ± 5.97
60.92 ± 7.40
49.31 ± 8.77
38.87 ± 9.52
40.66 ± 2.18
Extract + RIF
48.23 ± 1.57
36.87 ± 7.78
46.10 ± 3.85
MDR3
Extract
16.20 ± 1.57
53.36 ± 2.93
43.87 ± 5.88
18.75 ± 4.78
33.73 ± 12.72
Extract + RIF
38.17 ± 6.49
15.56 ± 2.94
31.67 ± 13.18
aCells shaded in grey indicate those parameters for which the percent reduction in test containing wells was less than that for the 1:100 MTB control.
bSuboptimal concentration of RIF used for H37Rv and sensitive strains 1, 2 and 3 was 0.0125, 0.08, 0.02 and 0.125 µg/ml respectively

European Journal of Medicinal Plants, 2(4): 308-323, 2012
316
3.2 Anti-M. tuberculosis Activity of a Mixture of Acetone and Ethanol Extracts
of P. nigrum
As the ethanol extract of P. nigrum showed marginal activity at 100 µg/ml, its combination
with the acetone extract was tested for anti-M. tuberculosis activity. The mixture of these
extracts was tested at 25 µg/ml each and 50 µg/ml each. It was seen that these mixtures
showed inhibitory activity against H37Rv, all the susceptible isolates and two of the MDR
isolates (MDR1 and MDR3), despite the lack of activity when used individually at 50 µg/ml
(Table 4). In the case of MDR2, although the percent reduction of Alamar Blue was
marginally higher than that of the 1:100 MTB, it could be considered as effective against the
isolate. Studying the mechanism by which this mixture was active across the sensitive and
MDR strains would be useful in developing anti-M. tuberculosis agents that have the
potential to treat MDRTB.
Table 4. Percent reduction of Alamar Blue dye obtained for a mixture of P. nigrum
extracts. Values are presented as mean ± standard deviation of 3 independent
experiments. The remaining controls (DMSO, Rifampicin) were within permissible
range (data not shown)
Strain
1:100 MTB
control
MTB control
MTB + P. nigrum extractsa
Acetone
50 µg/ml
Ethanol
50 µg/ml
Acetone 50 µg/ml
+ Ethanol 50 µg/ml
H37Rv
19.89 ± 0.04
67.23 ± 3.77
21.53 ± 4.83
51.45 ± 1.74
17.39 ± 4.15
S1
18.95 ± 7.23
53.11 ± 3.17
22.33 ± 2.18
67.75 ± 18.94
15.82 ± 9.44
S2
15.17 ± 7.71
61.91 ± 6.72
36.62 ± 6.18
59.34 ± 5.34
15.87 ± 6.88
S3
20.34 ± 1.36
51.81 ± 6.79
28.95 ± 1.12
36.26 ± 5.24
18.96 ± 0.64
MDR1
22.98 ± 3.21
55.51 ± 4.42
35.60 ± 3.91
36.36 ± 7.21
14.66 ± 7.16
MDR2
16.72 ± 0.40
56.13 ± 1.84
32.51 ± 4.38
52.21 ± 2.36
20.55 ± 1.54
MDR3
22.10 ± 1.15
54.88 ± 9.43
25.99 ± 4.24
36.91 ± 1.96
22.18 ± 2.42
aCells shaded in grey indicate those parameters for which the percent reduction in test containing
wells was less than that for the 1:100 MTB control.
3.3 Anti-M. tuberculosis Activity of Piperine
As piperine is one of the active constituents of P. nigrum (Madhavi et al., 2009), this
compound was also tested for its activity. Due to the limited solubility of piperine in DMSO,
the highest concentration tested was 25 µg/ml. It was observed that piperine was active
against H37Rv at 25 µg/ml but only in the presence of a suboptimal concentration of RIF. It
was ineffective against all other strains tested (Table 5)
3.4 Internal Quality Control of the MABA
The acetone extracts of A. paniculata and A. calamus were tested against H37Rv and the
clinical isolates in the MGIT960 system. They did not show any activity and were thus
concordant with the MABA results.

European Journal of Medicinal Plants, 2(4): 308-323, 2012
317
Table 5. Percent reduction of Alamar Blue dye obtained for piperine at 25 µg/ml.
Values are presented as mean ± standard deviation of 3 independent experiments.
The remaining controls (DMSO, Rifampicin and suboptimal Rifampicin, where
applicable) were within permissible range (data not shown)
Strain
1:100 MTB
control
MTB control
MTB + piperine a
MTB + piperine + RIF
(or sub optimal RIFb)a
H37Rv
21.71 ± 2.84
66.05 ± 5.03
59.93 ± 8.45
22.12 ± 2.14
S1
21.73 ± 2.33
54.68 ± 8.29
61.46 ± 6.53
45.52 ± 12.07
S2
17.92 ± 6.05
55.48 ± 5.86
45.48 ± 2.86
32.05 ± 7.28
S3
25.08 ± 1.43
66.55 ± 2.19
50.78 ± 14.37
38.97 ± 5.67
MDR1
19.95 ± 3.10
55.41 ± 13.72
31.37 ± 5.42
28.58 ± 5.84
MDR2
18.93 ± 5.97
60.92 ± 7.40
42.75 ± 7.43
44.16 ± 7.85
MDR3
16.20 ± 1.57
53.36 ± 2.93
29.72 ± 9.75
27.75 ± 5.76
aCells shaded in grey indicate those parameters for which the percent reduction in test containing
wells was less than that for the 1:100 MTB control.
bSuboptimal concentration of RIF used for H37Rv and sensitive strains 1, 2 and 3 was 0.0125, 0.08,
0.02 and 0.125 µg/ml respectively.
3.5 External Quality Control of the MABA
The results with acetone extract of A. calamus,O. sanctum and P. nigrum and the ethanol
extract of P. nigrum tested against H37Rv at SMI were in concordance with the results
obtained by FMR. The results for P. nigrum are depicted in Fig. 1.
Fig. 1. External quality control by the MABA performed at the Swedish Institute for
Communicable Disease Control, Karolinska. The extracts were tested against H37Rv
(n=1)
MTB = M. tuberculosis; # Percent reduction of Alamar Blue of the tests is less than that of the 1:100
MTB control.
M. tuberculosis growth controls; M. tuberculosis + Plant extract
# # #

European Journal of Medicinal Plants, 2(4): 308-323, 2012
318
3.6 Cytotoxicity of P. nigrum Extracts
The percent viability of A549 cells in presence of the highest concentrations of acetone (100
µg/ml), ethanol (100 µg/ml) extracts and mixture of acetone and ethanol extracts of P.
nigrum (50 µg/ml each) was 90.49 ± 6.75, 97.58 ± 18.57 and 116.67 ± 13.82 respectively.
The percent viability of A549 cells in presence of piperine (25 µg/ml) was 111.51 ± 23.85.
3.7 Phytochemical Profile of Acetone and Ethanol Extract of P. nigrum
The HPTLC profile and the chromatogram of the acetone and ethanol extract of P. nigrum
extracts scanned at 254nm and 366nm is depicted in Fig. 2. The chromatogram was
obtained using n-Hexane: Ethyl acetate: Acetic acid (5:5:0.05) as the mobile phase. Piperine
(Sigma-Aldrich) was used as the reference standard. The Rf of piperine was noted to be
0.21.
(i) (ii)
Fig. 2. HPTLC fingerprinting of P. nigrum scanned at (i) 254nm and (ii) 366nm
Lane A corresponds to the reference compound piperine, lane B and lane C correspond to the acetone
and ethanol extracts respectively.
4. DISCUSSION
In the era of emerging drug resistance in infectious diseases, use of medicinal plants as an
alternative therapy has been proposed (Oluyege et al., 2010; Sibanda and Okoh, 2007;

European Journal of Medicinal Plants, 2(4): 308-323, 2012
319
Singh et al., 2010). There are limited reports on the anti-TB activity of Indian medicinal plants
(Gautam et al., 2007). We used the MABA to screen for potential anti-M. tuberculosis activity
of five Indian medicinal plants which have been in use for various respiratory ailments viz.,
A. calamus, A. paniculata,O. sanctum,P. nigrum and P. tuberosa. Amongst these, only P.
nigrum showed anti-M. tuberculosis activity. The external and internal validation of the
results strengthens our observations.
Amongst the Piper spp, P. imperiale (Diaz et al., 2012), P. longum (Singh et al., 2011), P.
sarmentosum (Hussain et al., 2008), P. betle (Gupta and Viswanathan, 1956) and P. cubeba
(Grange and Davey, 1990) have been reported to have anti-mycobacterial activity. P. nigrum
has also been shown to have anti-mycobacterial activity, though the plant part used was
unspecified (Grange and Davey, 1990).
In our study, the acetone extract of P. nigrum at 100 µg/ml demonstrated inhibitory activity
against H37Rv, all three susceptible isolates and one of three MDR isolates. The
combination of the acetone extract of P. nigrum and suboptimal rifampicin was not more
effective than the plant extract alone. Although the ethanol extract of P. nigrum showed only
marginal activity, a mixture of the acetone and ethanol extracts (each at 25 and 50 µg/ml)
were tested to check for possible anti-M. tuberculosis activity. These mixtures showed
inhibitory activity against H37Rv and all the clinical isolates tested, despite the lack of activity
when used individually at 50 µg/ml. This finding has two-fold value since it enabled the use
of lower concentrations of the active extract and demonstrated that non active extracts may
have the potential to show activity if used in combination. The efficacy of the extracts could
be due to the interplay between the different active constituents present, leading to better
activity. It has been demonstrated that different constituents of crude extracts act through
different mechanisms (Mavar-Manga et al., 2008) or act synergistically (Birdi et al., 2010;
Ncube et al., 2012).
The activity of the extracts of P. nigrum could be due to piperine, a principle active
component of the plant (Ahmad et al., 2012; Madhavi et al., 2009). It has also been shown to
inhibit the M. tuberculosis efflux pump Rv1258c (Sharma et al., 2010). The presence of
piperine in the acetone and ethanol extracts was confirmed by HPTLC. In our study, piperine
(sourced from Sigma-Aldrich) was active only against H37Rv at 25 µg/ml in the presence of
suboptimal concentration of rifampicin. It is possible that the level of expression of Rv1258c
in clinical isolates is altered as compared to H37Rv, resulting in the lack of bactericidal
activity when using a combination of piperine and rifampicin. Though Phongpaichit et al.
(2006) reported that piperine isolated from the fruit of P. chaba exhibits anti-mycobacterial
activity, the lack of activity in our study could be due to the difference in the test strains
screened. The active component and target of P. nigrum thus need to be identified. Based
on the results obtained in this study, it is possible that the target molecules of the active
component(s) of P. nigrum may be different from those of standard anti-TB drugs. A similar
conclusion with respect to possible mechanism of action was reached by Lakshmanan et al.,
(2011) for the active molecule of Kaempferia galanga.
The P. nigrum results reiterate the necessity of screening plant extracts against multiple
clinical strains with different clinical profiles and not only against H37Rv. A Mexican study on
plants against drug resistant TB also stated that testing only a laboratory reference strain like
H37Rv would be insufficient as an indicator of activity against clinical isolates, since the
activity might differ against susceptible and resistant strains (Camacho-Corona et al., 2008).

European Journal of Medicinal Plants, 2(4): 308-323, 2012
320
Chopra et al. (1957) reported anti-mycobacterial activity of the essential oil of A. calamus
rhizome and Webster et al. (2010) showed that the aqueous extract of the root of this plant
was active against M. tuberculosis H37Ra. The lack of activity in our study can be attributed
to the different plant part (rhizome), extract used and the test strains screened. Anti-
mycobacterial activity of O. sanctum was reported by Reddi et al. (1986) and Farivar et al.
(2006). However, the above factors, as well as the difference in the concentration of the
extract (50-100 mg/ml) could explain the absence of activity of O. sanctum in this study.
Additionally, this variation could be due to the sequential extraction procedure used by us.
The aqueous extract prepared directly could possibly have contained two or more
compounds that could have had anti-M. tuberculosis activity.
M. tuberculosis is an intracellular pathogen capable of infecting and replicating in epithelial
cells (Castro-Garza et al., 2002). A549, a human lung epithelial cell line, was thus selected
to test the cytotoxicity of the five plants. Hence in the absence of cytotoxicity, P. nigrum
extracts seem promising for intracellular testing of newer anti-M. tuberculosis agents of plant
origin.
5. CONCLUSION
In conclusion, the present study identifies P. nigrum as a promising anti-M. tuberculosis plant
active against both drug sensitive and resistant strains. The study also highlights the
combined activity of the ethanol and acetone extracts. Further studies to identify
constituent(s) of P. nigrum (in addition to piperine) responsible for its anti-M. tuberculosis
activity are being undertaken, along with the exploration of mechanisms of action of the plant
and the possible interactions between P. nigrum and presently available anti-TB drugs.
ACKNOWLEDGEMENTS
This work was supported by a Department of Biotechnology, Government of India, Virtual
Center of Excellence Grant no.: BT/01/COE/05/06/01. We acknowledge the assistance of
Anchrom Enterprises (India) Private Limited, Mumbai, for help in the HPTLC analysis and
Mr. D. Desai for technical support with the assays. We thank Mr. S. Bagwe for technical
assistance in the laboratory.
COMPETING INTERESTS
Authors have declared that no competing interests exist.
REFERENCES
Ahmad, N., Fazal, H., Abbasi, B.H., Farooq, S., Ali, M., Khan, M.A. (2012). Biological role of
P. nigrum L. (Black pepper): A Review. Asian Pacific Journal of Tropical Biomedicine,
1-10.
Billore, K.V., Yelne, M.B., Dennis, T.J., Chaudhari, B.G. (2004). Database on medicinal
plants used in ayurveda, Vol. 6. Central Council for Research in Ayurveda and Siddha.
New Delhi.
Birdi, T., Daswani, P., Brijesh, S., Tetali, P., Natu, A., Antia, N. (2010). Newer insights into
the mechanism of action of Psidium guajava L. leaves in infectious diarrhoea. BMC
Complement. Altern. Med., 10, 3.

European Journal of Medicinal Plants, 2(4): 308-323, 2012
321
Camacho-Corona, M.R., Ramírez-Cabrera, M.A., González-Santiago, O., Garza-González,
E., de Paz Palacios, I., Luna-Herrera, J. (2008). Activity against drug resistant-
tuberculosis strains of plants used in Mexican traditional medicine to treat tuberculosis
and other respiratory diseases. Phytother. Res., 22, 82-85.
Castro-Garza, J., King, C.H., Swords, W.E., Quinn, F.D. (2002). Demonstration of spread of
Mycobacterium tuberculosis bacilli in A549 epithelial cell monolayer. FEMS Microbiol.
Lett., 212, 145-149.
Chopra, L.C., Khajuria, B.N., Chopra, C.L. (1957). Antibacterial properties of volatile
principles from Alpinia galanga and Acorus calamus. Antibiot. Chemother., 7, 378-383.
Clinical and Laboratory Standards Institute (CLSI). (2011). Susceptibility testing of
Mycobacteria, Nocardiae and other aerobic Actinomycetes; Approved Standard –
Second Edition. CLSI document MM24-A2, Clinical and Laboratory Standards
Institute, Wayne, Pennsylvania, USA.
Collins, L.A., Franzblau, S.G. (1997). Microplate Alamar Blue assay versus BACTEC 460 for
high-throughput screening of compounds against Mycobacterium tuberculosis and
Mycobacterium avium. Antimicrob. Agents Chemother., 41, 1004-1009.
Diaz, L.E., Munoz, D.R., Prieto, R.E., Cuervo, S.A., Gonzalez, D.L., Guzman, J.D., Bhakta,
S. (2012). Antioxidant, antitubercular and cytotoxic activities of Piper imperiale.
Molecules, 17, 4142-4157.
D'souza, D.T.B., Mistry, N.F., Vira, T.S., Dholakia, Y., Hoffner, S., Pasvol, G., Nicol, M.,
Wilkinson, R.J. (2009). High levels of multidrug resistant tuberculosis in new and
treatment-failure patients from the Revised National Tuberculosis Control Programme
in an urban metropolis (Mumbai) in Western India. BMC Public Health, 9, 211.
Farivar, T.N., Fard, A.H.M, Zahedani, S.S., Naderi, M., Moud, B.S. (2006). Anti tuberculosis
effect of Ocimum sanctum extracts in in vitro and macrophage culture. J. Med. Sci., 6,
348-351.
Gautam, R., Saklani, A., Jachak, S.M. (2007). Indian medicinal plants as a source of
antimycobacterial agents. J Ethnopharmacol., 110, 200-234.
Ginsburg, H., Deharo, E. (2011). A call for using natural compounds in the development of
new antimalarial treatments- an introduction. Malar J., 10, S1.
Grange, J.M., Davey, R.W. (1990). Detection of antituberculous activity in plant extracts. J.
Appl. Bacteriol., 68, 587-591.
Green, E., Samie, A., Obi, C.L., Bessong, P.O., Ndip, R.N. (2010). Inhibitory properties of
selected South African medicinal plants against Mycobacterium tuberculosis. J.
Ethnopharmacol., 130, 151-157.
Gupta, K.C., Viswananthan, R. (1956). Antitubercular substances from plants: A preliminary
study. Antibiot. Chemother., 6, 194-195.
Houghton, P.J. (2000). Use of small scale bioassays in the discovery of novel drugs from
natural sources. Phytother. Res., 14, 419–423.
Hussain, K., Ismail, Z., Sadikun, A., Ibrahim, P. (2008). Analysis of proteins,
polysaccharides, glycosaponins contents of Piper sarmentosum Roxb. and anti-TB
evaluation for bio-enhancing/ interaction effects of leaf extracts with isoniazid (INH).
Nat. Prod. Rad., 7, 402-408.
Ignacimuthu, S., Ayyanar, M., Sankara Sivaraman, K. (2006). Ethnobotanical investigations
among tribes in Madurai District of Tamil Nadu (India). J. Ethnobiol. Ethnomed., 2, 25.
Khan, M., Siddiqui, M. (2007). Antimicrobial activity of Piper fruits. Nat. Prod. Rad., 6, 111-
113.
Kumar, M., Khan, I.A., Verma, V., Kalyan, N., Qazi, G.N. (2005). Rapid, inexpensive MIC
determination of Mycobacterium tuberculosis isolates by using microplate nitrate
reductase assay. Diagn. Microbiol. Infect. Dis., 53, 121-124.

European Journal of Medicinal Plants, 2(4): 308-323, 2012
322
Lakshmanan, D., Werngren, J., Jose, L., Suja, K.P., Nair, M.S., Varma, R.L., Mundayoor, S.,
Hoffner, S., Kumar, R.A. (2011). Ethyl p-methoxycinnamate isolated from a traditional
anti-tuberculosis medicinal herb inhibits drug resistant strains of Mycobacterium
tuberculosis in vitro. Fitoterapia, 82, 757-761.
Madhavi, B.B., Nath, A.R., Banji, D., Madhu, M.N., Ramalingam, R., Swetha, D. (2009).
Extraction, identification, formulation and evaluation of Piperine in alginate beads. Int.
J. Pharm. Pharmaceut. Sci., 1, 156-161.
Mavar-Manga, H., Haddad, M., Pieters, L., Baccelli, C., Penge, A., Quetin-Leclercq, J.
(2008). Anti-inflammatory compounds from leaves and bark of Alchornea cordifolia
(Schumach. & Thonn.) Mull. Arg. J. Ethnopharmacol., 115, 25-29.
Mishra, U.S., Mishra, A., Kumari, R., Murthy, P.N., Naik, B.S. (2009). Antibacterial activity of
ethanol extracts of Andrographis paniculata. Indian J. Pharm. Sci., 71, 436-438.
Motley, T.J. (1994). The ethnobotany of sweet flag, Acorus calamus (Araceae). Econ. Bot.,
48, 397-412.
Muthu, C., Ayyanar, M., Raja, N., Ignacimuthu, S. (2006). Medicinal plants used by
traditional healers in Kancheepuram District of Tamil Nadu, India. J. Ethnobiol.
Ethnomed., 2, 43.
Nayyar, A., Jain, R. (2005). Recent advances in new structural classes of anti-tuberculosis
agents. Curr. Med. Chem., 12, 1873-1886.
Ncube, B., Finnie, J.F., Van Staden, J. (2012). In vitro antimicrobial synergisms within plant
extract combinations from three South African medicinal bulbs. J. Ethnopharmacol., 6,
81-89.
Oluyege, J.O., Adelabu, D.M. (2010). Microbial contamination of some Hawked herbal
products in Ado-Ekiti, Nigeria. Continental J. Microbiology, 4, 8-14.
Parish, C.R., Mullbacher, A. (1983). Automated colorimetric assay for T cell cytotoxicity. J.
Immunol. Methods., 58, 225-237.
Phongpaichit, S., Vuddhakul, V., Subhadhirasakul, S., Wattanapiromsakul, C. (2006).
Evaluation of the antimycobacterial activity of extracts from plants used as self-
medication by AIDS patients in Thailand. Pharm. Biol., 44, 71-75.
Reddi, G.S., Shukla, N.P., Singh, K.V. (1986). Chemotherapy of tuberculosis - antitubercular
activity of O. sanctum leafy extract. Fitoterapia, 57, 114-116.
Sharma, P.C., Yelne, M.B., Dennis, T.J. (2000–2002). Database on medicinal plants used in
ayurveda, Vols. 1–5. Central Council for Research in Ayurveda and Siddha, New
Delhi.
Sharma, S., Kumar, M., Sharma, S., Nargotra, A., Koul, S., Khan, I.A. (2010). Piperine as an
inhibitor of Rv1258c, a putative multidrug efflux pump of Mycobacterium tuberculosis.
J. Antimicrob. Chemother., 65, 1694-1701.
Sibanda, T., Okoh, A.I. (2007). The challenges of overcoming antibiotic resistance: Plant
extracts as potential sources of antimicrobial and resistance modifying agents. Afr. J.
Biotechnol., 6, 2886-2896.
Singh, C.,Singh, S.K., Nath, G., Rai, N.P. (2011). Anti-mycobacterial activity
of Piper longum L. fruit extracts against multi drug resistant mycobacterium spp.
International Journal of Phytomedicine, 3, 353-361.
Singh, M., Khatoon, S., Singh, S., Kumar, V., Rawat, A.K., Mehrotra, S. (2010). Antimicrobial
screening of ethnobotanically important stem bark of medicinal plants.
Pharmacognosy Res., 2, 254-257.
Singh, M.M. (2007). XDR-TB- danger ahead. Indian J. Tuberc., 54, 1-2.
Sood, R., Swarup, D., Bhatia, S., Kulkarni, D.D., Dey, S., Saini, M., Dubey, S.C. (2012).
Antiviral activity of crude extracts of Eugenia jambolana Lam. against highly
pathogenic avian influenza (H5N1) virus. Indian J Exp Biol., 50, 179-86.

European Journal of Medicinal Plants, 2(4): 308-323, 2012
323
Souza, M.V.N. (2006). Current status and future prospects for new therapies for pulmonary
tuberculosis. Curr. Opin. Pulm. Med., 12, 167-171.
Thakkur, C.G. (1976). Introduction to ayurveda: Basic Indian medicine. 2nd edition.
Jamnagar: Gulakunverba Ayurvedic Society.
Udwadia, Z.F., Amale, R.A., Ajbani, K.K., Rodrigues, C. (2012). Totally drug-resistant
tuberculosis in India. Clin. Infect. Dis., 54, 579-581.
Velayati, A.A., Masjedi, M.R., Farnia, P., Tabarsi, P., Ghanavei, J., Ziazarifi, A.H., Hoffner, S.
(2009). Emergence of new forms of totally drug-resistant tuberculosis bacilli: super
extensively drug-resistant tuberculosis or totally drug-resistant strains in Iran. Chest,
136, 420-425.
Venkata Ratnam, K., Venkata Raju, R.R. (2009). Preliminary phytochemical and
antimicrobial properties of Pueraria tuberosa (Willd.) DC: A potential medicinal plant.
Ethnobotanical Leaflets., 13, 1051-1059.
Webster, D., Lee, T.D., Moore, J., Manning, T., Kunimoto, D., LeBlanc, D., Johnson, J.A.,
Gray, C.A. (2010). Antimycobacterial screening of traditional medicinal plants using
microplate resazurin assay. Can. J. Microbiol., 56, 487-494.
_________________________________________________________________________
© 2012 Birdi et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.