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Evaluating the anti Mycobacterium tuberculosis activity of Alpinia galanga (L.) Willd. axenically under reducing oxygen conditions and in intracellular assays

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In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic conditions) and intracellular assays. Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50 and 100 mug/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv. 1 acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not demonstrate activity under the defined assay parameters. A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may be utilized in combination with the conventional TB therapy for enhanced efficacy.
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R E S E A R C H A R T I C L E Open Access
Evaluating the anti Mycobacterium tuberculosis
activity of Alpinia galanga (L.) Willd. axenically
under reducing oxygen conditions and in
intracellular assays
Pooja Gupta
1
, Purva Bhatter
1
, Desiree Dsouza
1
, Monica Tolani
1
, Poonam Daswani
1
, Pundarikakshudu Tetali
2
and Tannaz Birdi
1*
Abstract
Background: In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB
continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to
expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia
galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was
undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic
conditions) and intracellular assays.
Methods: Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50
and 100 μg/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against
Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The
activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under
intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv.
Results: 1´ acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and
ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not
demonstrate activity under the defined assay parameters.
Conclusion: A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was
observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating
bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may
be utilized in combination with the conventional TB therapy for enhanced efficacy.
Keywords: Mycobacterium tuberculosis, Medicinal plants, Alpinia galanga, Anaerobic assay, Intracellular assays
Background
Tuberculosis (TB) is an infectious bacterial disease caused
by Mycobacterium tuberculosis (M.tb). It commonly af-
fects the lungs but may also affect other parts of the body
viz., the brain, spine and the kidneys. Not every individual
infected with M.tb presents with symptoms, referred to as
latent TB (LTBI). As a result, two TB-related conditions
exist: active TB disease and latent TB infection (LTBI) [1].
Whilst individuals with LTBI are asymptomatic and non
infectious, they are at a risk of progression to active dis-
ease. In LTBI, due to quantitative metabolic shutdown, the
dormant bacilli fail to respond to drug therapies which
target multiplying bacteria. Thus identification and treat-
ment of LTBI is equally important to ensure complete
elimination of TB.
The steadily increasing bacterial resistance to existing
drugs is a serious problem [2,3], resulting in the urgent need
for development of new TB drugs and shorter treatment
* Correspondence: fmr@fmrindia.org
1
The Foundation for Medical Research, 84-A, R.G. Thadani Marg, Worli,
Mumbai 400018, Maharashtra, India
Full list of author information is available at the end of the article
© 2014 Gupta et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Gupta et al. BMC Complementary and Alternative Medicine 2014, 14:84
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regimens. This has led to the search for new classes of
antimicrobial agents. Unlike synthetic drugs, antimicro-
bials of plant origin are found to exhibit fewer side effects
and have the therapeutic potential to treat many infectious
diseases [4,5]. Development of easier, rapid and safer
screening techniques has intensified the search of chem-
ical entities from botanicals and other natural resources
for activity against Mycobacteria species [6]. In vitro in-
hibitory activity of crude extracts and/or pure active com-
pounds extracted from plants against M.tb and its related
species has been extensively reported [7-10].
Alpinia galangal (L.) Willd., family Zingiberaceae com-
monly referred to as galangal, is widely cultivated in
South-east Asian countries such as Philippines, Indonesia,
Thailand, India, and China [11]. It is extensively used in
diets as well as in the traditional systems of medicine viz.,
Thai, Ayurveda, Unani and Chinese folk medicine [12].
Galangal has been known for its use as anti-inflammatory,
antipyretic, emmenagogue, carminative, abortifacient and
aphrodisiac and is used in the treatment of various dis-
eases such as renal calculus, diabetes, heart diseases, bron-
chitis, rheumatism, chronic enteritis and kidney disorders
[12,13]. Among other components, it is reported to con-
tain tannins, glycosides, essential oils, phenol, carbohy-
drates and monoterpenes. Antimicrobial activity of
galangal [13] and the Alpinia species [14] has also been
reported earlier. Additionally 1´ acetoxychavicol acetate, a
phenylpropanoid, isolated from A. galanga and A. nigra is
specifically known to possess antituberculous activity [14].
Crude extract of A. galanga has been demonstrated to
have an activity similar to that of isoniazid [15]. However,
Soundhari and Rajarajan [16] have demonstrated the ac-
tivity of galangal in isoniazid resistant clinical strains in-
vitro. The possible reason for this discrepancy needs to be
elucidated. Phongpaichit et al. evaluated the antimycobac-
terial activity of extracts from galangal and suggested its
use as self-medication for treatment of TB in AIDS pa-
tients in Thailand [14]. Although there are reports of ac-
tivity of A. galanga on axenic aerobic growth of M.tb, to
our knowledge, there are no studies reporting its activity
against M.tb under intracellular conditions and reduced
oxygen concentrations. It is important that the antimyco-
bacterial activity of plants be measured under hypoxic
conditions since it is a model for non replicating and dor-
mant bacilli. Besides, the intracellular environment in
which the TB bacterium resides is anaerobic and is char-
acterized by the switch from aerobic/microaerophilic to
anaerobic respiratory pathways by utilisation of lipids as a
carbon source [17].
Methods
Plant material
Alpinia galanga was selected for the present study on
the basis of its broad antibacterial properties [11,12].
The plant was sourced from Kerala Agricultural Univer-
sity and cultivated at Naoroji Godrej Centre for Plant Re-
search (NGCPR). The plant material was authenticated by
Dr. P. Tetali, a taxonomist at NGCPR. A voucher speci-
men of the plant has been deposited at Botanical Survey
of India (BSI), Western Center, Pune, India, under the
herbarium number 131745.
Extract preparation
Coarsely powdered plant material (rhizomes) was se-
quentially extracted [18] with acetone, ethanol and dis-
tilled water using the Soxhlet apparatus. 300 ml of
respective solvent was continuously refluxed with 25 g
of plant material for a period of 24-30 hours for efficient
extraction of the phytoconstituents. Post ethanol extrac-
tion and evaporation of the solvent, the aqueous extract
was prepared by boiling the plant material until the vol-
ume of water was reduced to 25%. The aqueous extract
was lyophilized (Thermo Fisher Scientific, USA) and the
acetone and ethanolic extracts were allowed to air dry.
The percent yields of the acetone, aqueous and ethanolic
extracts were 2.92, 23.6 and 6.84 (w/w) respectively. For
the assays, the extracts were reconstituted at 20 mg/ml
concentration in dimethyl sulfoxide (DMSO, SD fine
Chemicals, India), filtered through 0.2 μm, 25 mm
DMSO resistant Acrodisc syringe filters (Pall Corpor-
ation, USA) and stored at -20°C for up to 2 weeks.
Phytochemistry
The extracts of A. galanga were subjected to phytochem-
ical fingerprinting using High Performance Thin Layer
chromatography (HPTLC). The extracts were spotted on
pre-coated Silica gel G60 F254 TLC plates (Merck,
Germany) along with the reference standard viz., 1´ acet-
oxychavicol acetate (Natural Remedies, Bangalore, India)
using Linomat V Automatic Sample Spotter (CAMAG,
Switzerland), run in a twin trough TLC chamber,dried
and visualized in CAMAG TLC visualizerpre and post
derivatization with anisaldehyde-sulpuric acid. The mobile
phase used was Toluene: Acetone (7:3).
Bacterial culture
The reference M.tb laboratory strain H37Rv, susceptible
to the first line drugs, along with three drug susceptible
and three multi drug resistant clinical isolates [3] were
used for MABA and intracellular assay. The details of
the isolates used for the study are presented in Table 1.
MABA
The assay was performed as reported by Collins and
Franzblau [19] and Webster et al. [20]. Briefly, 100 μlof
0.5 × 10
6
/ml of the M.tb strains (Viable M.tb, VMTB)
were cultured in 7H9 medium (supplemented with ADC
and 0.5% glycerol) in the presence of the plant extracts
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(1, 10, 25, 50 and 100 μg/ml) in a Nuncflat bottom 96
well plate (Nunclon, Denmark). The controls maintained
for all the tested strains included: medium, DMSO (at a
volume that is used for the highest concentration of
plant extract), 1:100 VMTB and 2 μg/ml Rifampicin
(RIF) (Sigma-Aldrich, USA). To check the interaction of
the plant extracts with Alamar Blue, additionally wells
with plant extracts and media were also maintained. The
plates were incubated at 37°C for seven days. Post incu-
bation, 10 μl of Alamar Blue dye (Invitrogen, USA) [5%
(v/v)] diluted 1:1 in 7H9 medium (supplemented with
ADC and 0.5% glycerol) was added and the plates were
reincubated for 30 hours. The Optical Density (O.D.) of
the wells was measured at 600 nm and 570 nm in an
ELISA reader (Thermo Fisher Scientific, USA), and the
percent reduction of Alamar Blue dye was calculated as
per the manufacturers instructions. Use of percent re-
duction to screen plant extracts allowed identification of
those extracts with marginal activity (not resulting in
99% kill). Triplicate wells were maintained for each vari-
able in every assay and all the assays were performed
thrice and the data was analyzed as Mean ± SD.
The results were interpreted based on the percent re-
duction of the dye which is directly proportional to the
bacterial growth. The extracts were considered to be ac-
tive if the percent reduction value of Alamar Blue dye was
less than that observed for the 1:100 VMTB control [21].
Internal Quality Control was performed by comparing
the results of MABA to the Bactec MGIT960 system
(BD, USA). As the aqueous extract was found to be inef-
fective, only acetone and ethanolic extracts of A. galanga
were tested against H37Rv and the six clinical isolates.
Intracellular assay
The human lung carcinoma epithelial cell line, A549
(NCCS, Pune, India), was grown in DMEM (GibcoBRL,
UK) supplemented with 10% fetal calf serum (FCS) and
4μg/ml of gentamycin. 10
6
cells/well were seeded in a
96 well plate and were infected with H37Rv, drug sus-
ceptible and drug resistant strains at a multiplicity of in-
fection (MOI) of 1:1 for 6 hrs. Post infection, the cells
were treated with amikacin [22] (standardized to 50 μg/ml)
for 2 hrs to kill the remaining extracellular bacteria. The
excess amikacin was washed off and the infected cells
were incubated with the plant extracts at a concentration
of 25 μg/ml and 100 μg/ml overnight in DMEM with 5%
FCS. On the following day the plant extracts were washed
off and the cells maintained in DMEM with 3% FCS. The
gradual reduction in FCS concentration ensured that the
cell line did not over grow. On 0, 3rd, 5th, 7th and 10th
day, post infection, the cells were lysed with 0.1% sodium
dodecyl sulphate (SDS) to release the intracellular bac-
teria. The lysate was ten fold serially diluted with PBS and
10 μl of the two highest dilutions were spotted onto Mid-
dlebrook 7H11 (MB7H11) agar plates supplemented with
OADC (Becton Dickinson, USA) and 0.5% glycerol. The
plates were incubated at 37°C for three weeks and the Col-
ony Forming Units (CFUs) enumerated.
Double stimulus
The intracellular assays were repeated using the above
mentioned protocol with the following modification.
The infected cells were given a second stimulus of the
plant extracts (25 μg/ml) on the fifth day post infection.
This was done to evaluate if a double stimulus of the
plant extracts would augment the intracellular killing of
the bacteria.
The inhibition of bacterial growth was represented as
percent inhibition calculated using the following formula:
%inhibition ¼ððgrowth under control conditions
growth under experimental
conditionsÞ=growth under
control conditionsÞ100
Table 1 Characteristics of strains used for testing the antimycobacterial activity of A.galanga
Sr. no Strain ID Drug susceptibility profile* rpoβ
Ψ
genotype katG
Ψ
genotype inhA promoter genotype
1 H37Rv Susceptible Wild type Wild type Wild type
2 S1 Susceptible Wild type Wild type Wild type
3 S2 Susceptible Wild type Wild type Wild type
4 S3 Susceptible Wild type Wild type Wild type
5 R1 Resistant to HR D516V mixed S315T1/T2 A16G mixed
6 R2 Resistant to HERZ D516V mixed S315T1/T2 A16G mixed
7 R3 Resistant to HERZ S531L S315T1/T2 mixed Wild type
Antimycobacterial activity of A.galanga under anaerobic conditions was tested using only H37Rv.
H-isoniazid, E-Ethambutol, R-Rifampicin, Z-Pyrazinamide.
S- Drug susceptible strain.
R- Drug resistant strain.
*Phenotypic drug resistance was ascertained using the BACTEC MGIT 960 system.
Ψ
Genotypic drug resistance was ascertained using the GenoType MTBDRplus assay (Hain Lifescience, Germany). Mixed indicates heteroresistance (presence of wild
type and mutant bands).
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Axenic assay under differential oxygen concentration
The assay was carried out using only the reference M.tb
laboratory strain H37Rv, susceptible to the first line
drugs.
Ten ml per tube of Middlebrook 7H9 broth, supple-
mented with ADC and 0.5% glycerol, was aliquoted and
bacterial suspension containing 10
4
CFU/ml was inocu-
lated into the tubes. Since significant intracellular activ-
ity was observed with higher concentration of plant
extracts, 100 μg/ml of plant extract was used as the final
concentration per tube. Positive and negative growth
controls along with a rifampicin (1 μg/ml) control were
maintained. The above set up in triplicate was subjected
to differentially reducing oxygen concentration viz., aer-
obic, microaerophilic and anaerobic conditions. The
microaerophilic conditions were obtained using the can-
dle jar method [23]. An anaerobic jar with a gas pack
(HiMedia, India) was used to create anaerobic condi-
tions. The change in colour of the indicator tablets, pro-
vided by the manufacturer, from pink to purple was
indicative of anaerobic conditions. To ensure that the
bacterium had completed sufficient number of replica-
tion cycles, the sets were incubated for a period of
10 days. Post incubation each of the tubes were vortex
mixed, serially diluted tenfold and 10 μl of this dilution
was spotted on MB7H11 agar plate supplemented with
OADC and 0.5% glycerol to enumerate the CFUs.
Results
Phytochemical fingerprinting
The chromatogram of the HPTLC fingerprinting of
the three extracts scanned at 366 nm post derivatiza-
tion with anisaldehyde sulphuric acid has been pre-
sented in Figure 1. As can be seen from this Figure 1
acetoxychavicol acetate, the reference standard used,
was found to be present in all the extracts with an R
f
value of 0.67.
MABA
The antibiotic susceptibility profile of the isolates per-
formed using MABA was in concordance with the
MGIT960 assay.
It was observed that the lower concentrations (1 and
10 μg/ml) of the three A. galanga extracts (acetone,
aqueous and ethanolic) did not exhibit any antibacter-
ial activity. However significant activity was observed
at the higher concentrations (25, 50 and 100 μg/ml).
The acetone and ethanolic extracts were found to be
the most effective against all the isolates (Table 2) un-
like the aqueous extract which did not show any sig-
nificant activity. The higher concentration (100 μg/ml)
of the extracts showed increased inhibition of all the
strains.
The acetone and ethanolic extract of A. galanga were
also tested against H37Rv and the clinical isolates in the
Figure 1 HPTLC fingerprinting of the extracts of Alpinia galanga.Profile of acetone extract (A); ethanolic extract (B); aqueous extract (C);
scanned at 366 nm post derivatization with anisaldehyde sulphuric acid. R Reference compound 1´ acetoxychavicol acetate, Ac Acetone
extract, Eth Ethanolic extract, Aq Aqueous extract.
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MGIT960 system. The activity recorded was concordant
with the MABA results.
Intracellular assays
The preliminary results of the intracellular assay per-
formed at a concentration of 25 μg/ml showed that the
aqueous extract was ineffective against all the strains
tested. The acetone extract was the most efficacious with
more than 80% inhibition against 5/7 isolates (Table 3).
The ethanolic extract showed more than 80% inhibition
against 4/7 isolates (Table 3).
Considering the significant activity demonstrated by
the acetone and ethanolic extracts, the assay protocol
was modified to introduce a second dose of plant extract
at the same concentration on the fifth day post infection.
Significant inhibition of bacterial growth was observed
in all 7 strains after the double stimulus. Both the
extracts also exhibited increased percent inhibition
(90%) in the intracellular assays with the double stimu-
lus (Table 3).
A higher concentration (100 μg/ml) of the plant extracts
(acetone and ethanolic) was also tested and showed aug-
mented bacterial kill in all 7 strains (Table 3).
Axenic assay under hypoxic conditions
The activity of the plant extracts was tested under re-
duced oxygen concentrations to mimic conditions for
studying the effect of plant extracts on latent bacteria
and to dissect the intracellular environment. Hence the
assay was restricted to H37Rv only. Significant inhibition
of the bacteria was observed with acetone and ethanolic
extracts when compared to the 1:100 VMTB growth
control (Figure 2).
Discussion
There has been no anti-TB drug introduced in the past
30 years and the rapid acquisition of drug resistance to
the existing drugs necessitates development of new, ef-
fective and affordable anti-TB drugs [24]. Plant-derived
antimycobacterial compounds belong to an exceptionally
wide diversity of classes, including terpenoids, alkaloids,
peptides, phenolics and coumarins. Hence medicinal
plants remain an important resource to find new thera-
peutic agents [25]. The advantages of using antimicrobial
compounds from medicinal plants include fewer side ef-
fects, better patient acceptance due to long history of
Table 2 Antimycobacterial activity of different extracts of A. galanga using microplate alamar blue assay
Strains 1: 100 VMTB VMTB A. galanga extracts
Acetone (μg/ml) Ethanolic (μg/ml)
25 50 100 25 50 100
% reduction of Alamar Blue dye
H37Rv 21.71 ± 2.84 66.05 ± 5.03 56.76 ± 9.51 25.10 ± 11.15 18.51 ± 3.84 57.27 ± 6.11 14.13 ± 6.46 12.88 ± 7.10
S1 21.09 ± 3.42 56.27 ± 2.30 53.84 ± 2.47 48.93 ± 11.10 21.16 ± 6.22 47.75 ± 13.04 35.60 ± 2.62 16.63 ± 4.40
S2 22.33 ± 7.00 59.17 ± 4.93 52.78 ± 6.74 42.90 ± 12.23 15.23 ± 6.17 41.45 ± 5.70 14.31 ± 6.64 14.87 ± 7.91
S3 19.83 ± 1.80 59.74 ± 10.58 55.58 ± 17.71 52.89 ± 16.92 18.21 ± 2.29 42.05 ± 6.60 32.15 ± 7.40 18.55 ± 4.40
R1 14.40 ± 5.71 51.08 ± 11.91 34.01 ± 6.32 25.57 ± 6.85 21.24 ± 5.62 23.39 ± 3.53 16.89 ± 1.10 13.08 ± 5.78
R2 22.11 ± 2.47 60.93 ± 9.00 53.38 ± 8.37 36.98 ± 8.84 20.04 ± 1.22 36.49 ± 4.06 19.74 ± 4.38 15.74 ± 3.86
R3 16.73 ± 4.14 50.50 ± 4.04 37.10 ± 5.03 30.48 ± 8.23 13.95 ± 2.06 42.59 ± 7.22 14.25 ± 3.88 12.25 ± 2.87
Values in bold indicate those parameters for which the percent reduction in plant extract containing wells was less than that for the 1:100 VMTB control.
Table 3 Percent inhibition of bacterial growth using intracellular (A549) assays
Sr. no. Strains A. galanga extracts
Acetone (μg/ml) Ethanolic (μg/ml)
25 100 25 100
Single stimulus Double stimulus Single stimulus Double stimulus
1 H37Rv 78.25 ± 6.14 94.58 ± 2.98 98.06 ± 0.24 78.41 ± 5.00 98.06 ± 0.24 98.06 ± 0.24
2 S1 83.34 ± 7.76 99.42 ± 0.50 99.37 ± 0.05 86.20 ± 12.04 98.88 ± 1.92 99.37 ± 0.05
3 S2 75.62 ± 2.23 94.47 ± 3.88 99.21 ± 0.02 73.09 ± 3.41 98.86 ± 0.44 99.21 ± 0.02
4 S3 96.41 ± 0.76 97.69 ± 1.25 95.18 ± 1.51 83.11 ± 2.33 100 ± 0 95.20 ± 1.82
5 R1 86.70 ± 5.11 95.00 ± 4.69 94.60 ± 1.30 82.21 ± 12.17 90.89 ± 6.43 97.74 ± 1.11
6 R2 89.33 ± 2.67 98.00 ± 0.00 93.56 ± 2.48 85.33 ± 5.81 93.78 ± 0.20 99.08 ± 0.52
7 R3 91.11 ± 1.92 94.36 ± 2.67 100 ± 0 57.93 ± 8.36 100 ± 0 100 ± 0
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use, reduced costs and cultivability rendering them re-
newable in nature [26].
A. galanga is a known wide spectrum antibacterial
agent. Soundhari and Rajarajan [16] have reported the
antimycobacterial activity of galangal against isoniazid
resistant isolates at minimum inhibitory concentration
(MIC) of 250 μg/ml. In our study, ethanolic extract of A.
galanga was found to be bactericidal to M.tb under
axenic aerobic conditions at 50-100 μg/ml. The variation
in the active concentrations could be due to differences
in the method of extraction and the assay used. The
antimycobacterial activity of the essential oils from A.
galanga has been reported since 1957 [27]. The phenolic
components of the oils have been reported to act as
membrane permeabilisers [28]. In addition, it has been
suggested that low oxygen levels can enhance the activ-
ity of essential oils. Diminished oxygen supply leads to
fewer oxidative changes in the essential oils and/or that
cells obtaining energy via anaerobic metabolism are
more sensitive to the toxic action of essential oils [28].
The enhanced activity of the extracts under anaerobic
conditions could thus be attributed to this. The ability of
the A. galanga extracts to remain active under hypoxic
conditions could further be explored for the treatment
of LTBI, where the bacteria are in a non-replicating and
dormant state and remain unaffected by the conven-
tional TB antibiotics.
Latha et al. [29] have demonstrated the plasmid curing
based activity of crude acetone extract of A. galanga.
The principal compound responsible for the activity was
identified as 1´ acetoxychavicol acetate. The activity of
this compound offers new perspectives to control the
replication and thus exhibits its potential to disrupt plas-
mid replication and re-sensitize bacteria to antibiotics.
Though M.tb may not contain plasmids, it is possible
that 1´ acetoxychavicol acetate (also detected in the
extracts used in the study) may act on the genome of
M.tb to render the bacterium sensitive to antibiotics.
Antimicrobials from plants have been found to be en-
hancers in that though they may not have any antimicro-
bial properties alone, but when consumed in tandem
with conventional drugs they may enhance the effect of
that drug [30]. Studies on synergism between known
antimicrobial agents and bioactive plant extracts have
also been demonstrated [31-33].
The acetone and ethanolic extracts were found to
show antimycobacterial activity under intracellular con-
ditions. It appears that the A. galanga extracts are able
to penetrate into the cells (A549) and act on the bacter-
ium residing intracellularly, unlike the action of some
antibiotics viz gentamycin which cannot penetrate the
cells and exert their action [34].
Conclusions
In conclusion, the antimycobacterial activity of A. galanga
observed under aerobic and anaerobic axenic condi-
tions and in the intracellular assay system could be
due to different phytoactive components acting with
varied mode of action(s). Furthermore, in depth stud-
ies to determine the active component(s) could lead to
potential formulations that serve not only as adjunct
to current therapy but as options in emerging clinical
drug resistance.
The use of A. galanga in diet and traditional medicines
has been extensively reported, its regular intake within
feasible limits could act as an adjunct to the ongoing
conventional TB therapy. Additionally the promising ac-
tivity of A. galanga under microaerophilic and anaerobic
conditions could also be developed further as a treat-
ment for latent infection in TB-endemic regions where
more than one third of the population act as reservoirs
of dormant/non replicating M.tb [35].
Athough the results from the present study are indica-
tive that A. galanga has promising antimycobacterial ac-
tivity, studies using more isolates/strains of M.tb are
needed.
Figure 2 Effect of the Alpinia galanga extracts on H37Rv growth under hypoxic conditions. The figure represents percent reduction in
growth of H37Rv by acetone and ethanolic extracts of A. galanga under reducing oxygen concentration. Rifampicin was used as a positive
control for inhibition of growth.
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Competing interest
The authors declare that they have no competing interests.
Authorscontributions
PG and PB have conducted the intracellular and axenic anaerobic assays,
analyzed the results and drafted the manuscript. DD has conducted the
MABA and edited the manuscript. MT has assisted in the MABA and edited
the manuscript. PD has undertaken the phytochemistry of the plant extracts.
PT has sourced, cultivated and authenticated the plant material. TB is
responsible for the study. All authors read and approved the final
manuscript.
Acknowledgement
The authors would like to acknowledge the contribution of the field staff for
collection of samples. We are thankful to the patients for their consent to be
a part of the study. The technical support of Mr. Dipen Desai for the
intracellular assays is appreciated. We would also like to acknowledge the
assistance of Anchrom Enterprises (India) Private Limited, Mumbai, for the
phytochemical analysis.
Source of funding
The present study was supported by a Department of Biotechnology,
Government of India, Virtual Center of Excellence Grant no.: BT/01/COE/05/06/01.
Author details
1
The Foundation for Medical Research, 84-A, R.G. Thadani Marg, Worli,
Mumbai 400018, Maharashtra, India.
2
Naoroji Godrej Centre for Plant
Research, Lawkim Motors Group Campus, Shindewadi, Shirwal, Satara,
Maharashtra 412801, India.
Received: 19 November 2013 Accepted: 24 February 2014
Published: 4 March 2014
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doi:10.1186/1472-6882-14-84
Cite this article as: Gupta et al.:Evaluating the anti Mycobacterium
tuberculosis activity of Alpinia galanga (L.) Willd. axenically under
reducing oxygen conditions and in intracellular assays. BMC
Complementary and Alternative Medicine 2014 14:84.
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... Further testing of semi-purified fractions of these two extracts showed significant inhibition of clinical MDR strains of MTb. Gupta et al. (2014) investigated antimycobacterial activity of Alpinia galanga (L.) Willd. under intracellular and in axenic (aerobic and anaerobic) conditions. ...
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