Marathe et al.

Annals of Clinical Microbiology and Antimicrobials 2013, 12:26

http://www.ann-clinmicrob.com/content/12/1/26

RESEARCH Open Access

In vitro antibacterial activity of Tabernaemontana

alternifolia (Roxb) stem bark aqueous extracts
against clinical isolates of methicillin resistant
Staphylococcus aureus
Nachiket P Marathe1†, Mandar H Rasane1†, Himanshu Kumar1, Ankur A Patwardhan2, Yogesh S Shouche1*
and Sham S Diwanay3*

Abstract
Background: The rise of antibiotic resistance among methicillin resistant Staphylococcus aureus (MRSA), have
caused concerns for the treatment of MRSA infections. Hence, search for an alternative therapy for these infections
is inevitable. Folk Indian medicine refers to the use of leaf and stem bark powder of Tabernaemontana alternifolia
(Roxb) in treatment of skin infections, but no scientific report establishes its antibacterial activity.
Methods: Direct aqueous extracts and sequential aqueous extracts of the stem bark of T. alternifolia (using
petroleum ether and ethyl acetate as other solvents) were prepared by soxhlet extraction. The antibiotic sensitivity
profiles of the clinical isolates were determined against 18 antibiotics using disc diffusion method. The isolates were
identified by 16S rRNA gene sequencing. The methicillin resistance among S. aureus (MRSA) was confirmed by PCR
amplification of mecA gene. The disc diffusion method was used to determine the antibacterial activity of the
extracts. The micro-dilution method was used to determine the minimum inhibitory concentration (MIC) of the
extract against the test organism. To further evaluate the therapeutic potential of the extract, cell cytotoxicity was
checked on Vero cells by MTT assay. Chemical profiling of the extract was done by HPTLC method.
Results: The aqueous extracts of T. alternifolia stem bark exhibited antibacterial activity against Gram-positive
microorganisms, particularly against clinical isolates of MRSA and vancomycin resistant S. aureus (VRSA). The
minimum inhibitory concentration (MIC) of extract against the isolates ranged from 600–800 μg/ml. The extract did
not exhibit cytotoxic activity against Vero cells even at the concentration of 4 mg/ml. The chemical profiling
revealed presence of alkaloids, flavonoids, coumarins, saponins and steroids. Petroleum ether and ethyl acetate
extracts did not exhibit antibacterial activity.
Conclusion: Our results offer a scientific basis for the traditional use of T. alternifolia in the treatment of skin
infections, showing that the plant extract has an enormous potential as a prospective alternative therapy against
MRSA skin infections. The present study lays the basis for future studies, to validate the possible use of T. alternifolia
as a candidate in the treatment of MRSA infections.
Keywords: Tabernaemontana alternifolia (Roxb), Anti-MRSA, Cytotoxicity, Plant extract, Antimicrobial

* Correspondence: yogesh@nccs.res.in; diwanay@rediffmail.com

†
Equal contributors
1
Microbial Culture Collection, National Centre for Cell Science, Ganeshkhind,
Pune 411007, India
3
Department of Microbiology, Abasaheb Garware College, Karve Road,
Pune 411004, India
Full list of author information is available at the end of the article

© 2013 Marathe 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 cited.
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Introduction identity was validated at the Department of Biodiversity,

Infectious diseases are one of the world’s leading causes of Abasaheb Garware College, Pune.
premature deaths [1]. Antibiotics which are widely used
for the treatment of infectious diseases are under constant Preparation of extracts
threat due to the emergence of antibiotic resistant patho- The aqueous extracts of stem bark were prepared as
gens such as methicillin resistant Staphylococcus aureus follows: Direct aqueous extract (DAE): The stem bark was
(MRSA), multidrug resistant Pseudomonas aeruginosa, dried at 55°C overnight. Twenty gram of dried stem bark
vancomycin resistant Enterococcus (VRE) and multidrug was subjected to hot extraction using soxhlet appar-
resistant Mycobacterium tuberculosis [2-6]. Among these, atus with 200 ml of distilled water as solvent for
MRSA has emerged as agents causing increasing threat of 6 hours at 100°C (boiling water).
nosocomial infections, more people have died of MRSA Sequential aqueous extract (SAE): Twenty gram of
infection in US hospitals than of HIV/AIDS and tubercu- dried stem bark was subjected to hot extraction using
losis combined during 2007–2008 [7,8]. Hence, search for soxhlet apparatus first with 200 ml petroleum ether as a
novel antimicrobial compounds or alternative therapy for solvent for 2 hours followed with ethyl acetate for
these infections is inevitable. 2 hours and finally with distilled water for 6 hours at
Plant based medicines are widely used and form an 100°C.
integral part of primary health care in many developing The crude extracts were concentrated at 55°C in a
countries across the globe [9-12]. Recently plants have clean and sterile glass petri plate. Final solution of
been explored to obtain crude natural extracts for testing 100 mg/ml was prepared in sterile distilled water for
and further refinement to develop effective antimicrobial aqueous extracts and in dimethyl sulfoxide (DMSO) for
drugs. In India there are different systems of medicine ethyl acetate and petroleum ether extracts. The extracts
practiced like Ayurveda, Siddha, Unani, Amchi and local were filter sterilized using Millipore 0.22 μm filter and
health traditions, which utilize a large number of plants or stored at 4°C.
herbs for the treatment of human diseases [13].
Many plant species widely explored for antimicrobial Test organisms and antibiotic sensitivity determination
compounds fall into the family Apocynaceae. Within this Clinical isolates from skin and soft tissue infections were
family, the genus Tabernaemontana is well documented obtained from Deenanath Mangeshkar Hospital, Pune.
for its biological activities such as antioxidant [14], These clinical isolates were collected by the hospital as a
anticancer [15-17], antifertility/contraceptive [18,19], anti- part of the standard practice and made available for the
pyretic and anti-inflammatory [17,20], anti-mycobacterial study. The type strains of Bacillus subtilis (ATCC 6633),
[14], and antimicrobial agent [21-23]. Staphylococcus aureus (ATCC 6538P), S. epidermidis
T. alternifolia (Roxb), also known as Ervatamia heyneana (ATCC 12228), Escherichia coli (ATCC 8739), methicillin
(Wall) or T. heyneana (Wall) is endemic to India and found resistant S. aureus (ATCC 43300) were obtained from
in Goa, Karnataka, Kerala, Maharashtra and Tamil Nadu NCIM, National Chemical Laboratory, Pune.
states [24]. Traditionally a therapeutic preparation made Antibiotic sensitivity pattern of clinical isolates was
from leaf and stem powder of T. alternifolia (Roxb) in determined by standard disc diffusion method according
combination with stem bark of Ficus racemosa, Ficus to the standards prescribed by Clinical and Laboratory
benghalensis, Madhuca longifolia and Strychnos nux- Standards Institute (CLSI) (former NCCLS) [30].
vomica is used to treat skin infections [25]. The other
plants, except T. alternifolia, used in this preparation have DNA extraction and PCR
reported antibacterial activity, but despite the traditional The genomic DNA extraction was carried out from freshly
use of T. alternifolia in treatment of skin infections, no grown bacterial cultures using Qiagen blood and tissue
scientific report has focused on establishment of the anti- DNA extraction kit (Qiagen, Madison USA), following the
microbial activity of the plant against pathogen [26-29]. We manufacturer’s instructions. The isolates were identified by
therefore, under took this study to evaluate the antimicro- 16S rRNA gene sequencing method as described earlier
bial potential of the extracts of stem bark of T. alternifolia [31]. PCR amplification of 16S rRNA gene was done using
against pathogens causing skin infections, especially MRSA primers 27F 5′-AGAGTTTGATCATGGCTCAG-3′ and
and VRSA. 1488R 5′-CGGTTACCTTGTTACGACTTCACC-3′. PCR
amplification involved a GeneAmp PCR system 9700
Methods (Applied Biosystems Inc. USA). The PCR for mecA gene
Collection of plant material was carried out as described earlier [32]. The DNA
Stem bark was collected in sterile plastic bags from sequencing was done using BigDye 3.1 sequencing termin-
Tamhini Ghat, a part of Western Ghats (18°28′21″ N, ator kit and ABI 3730xl DNA analyzer (Applied Biosystems
73°25′07″ E) near Pune, Maharashtra, India. The plant Inc. USA).
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Antibacterial activity using disc diffusion method different dilutions of DAE as well as SAE. Various dilu-
Antibacterial activity of the extract was determined as per tions of the extracts were prepared from the stock solution
CLSI guidelines (formerly NCCLS guidelines) [30]. Briefly, (100 mg/ml), and incorporated in cell culture (final con-
Whatmann filter paper no. 1 discs were prepared (diameter centrations ranging from 1 to 4 mg/ml), in quadruplicate.
6 mm). Discs were impregnated with 20 μl of 100 mg/ml Cell viability was determined after 24 hour incubation, by
extract per disc and dried at room temperature. Bacterial adding tetrazolium salt (Sigma Aldrich, USA) and reading
suspensions were prepared by suspending overnight grown the absorbance at 570 nm with a ELISA plate reader
culture in sterile normal saline. The turbidity of bacterial Spectra MAX250 (Molecular Devices, USA). Tetrazolium
suspensions was adjusted to 2x106 cfu/ml and 100 μl of salts are cleaved to formazan dye by cellular enzymes (only
suspension was spread on Muller-Hinton agar plate in the viable cells). The level of absorbance directly corre-
(HiMedia Laboratories, India). The discs impregnated lates to the metabolically active cells.
with extract were placed on plate. The plates were incu-
bated at 37°C for 24 hours and the zones of inhibition Chemo-profiling of the extract
were measured. Ciprofloxacin discs (5 μg/disc) (HiMedia Chemical analysis of SAE was done by HPTLC (Camag,
Laboratories, India) were used as a positive control for Switzerland) Sequentially: Applicator – Linomat IV,
growth inhibition. Scanner III; Plate was developed in a twin tray chamber.
Solvent system and spraying reagents were used as
Determination of minimum inhibitory concentration described earlier [36].
Minimum inhibitory concentration (MIC) was determined
by the microdilution broth method [33]. The plant Results
extracts were serially diluted with Mueller–Hinton broth Identification and antibiotic sensitivity pattern of
(HiMedia laboratories, India) to obtain the desired con- pathogenic isolates
centrations of 0.1 to 2 mg/ml. Bacterial suspensions were A total of 14 isolates were obtained from patients with skin
prepared in the similar manner as described in disc diffu- infections. Based on 16S rRNA gene sequence, the isolates
sion assay. One hundred microliter of the suspension was were identified as Pseudomonas aeruginosa (DMH 1),
added to serially diluted extract. The inoculated test tubes Staphylococcus aureus (DMH 2 to DMH 8 and DMH 10 to
were incubated at 37°C under aerobic conditions. Cipro- DMH 14), and Escherichia coli (DMH 9). The 16S rRNA
floxacin (HiMedia Laboratories, India) was used as a posi- gene sequences of isolates are deposited at GenBank under
tive control for growth inhibition, in concentration accession numbers HM559231 to HM559244. The drug
ranging from 0.05 μg/ml to 10 μg/ml. Turbidity was sensitivity pattern, represented in Table 1 revealed that all
checked after 24 hours of incubation. The lowest concen- the isolates were resistant to at least 4 different antibiotics,
tration of the extract that produced no visible growth showing that all isolates are multi-drug resistant. Eleven
when compared to the control (tube containing no out of twelve S. aureus strains were methicillin resistant
inoculum) was considered as MIC. (MRSA). The presence of mecA gene in the isolates, as
demonstrated by PCR and sequencing, confirmed the
Stability of antibacterial activity methicillin resistant nature of the isolates which was earlier
At a regular interval of three days, the antibacterial observed by disc diffusion assay. The mecA gene is the
activity of the extract was checked against MRSA (DMH4) structural gene present in MRSA for penicillin binding
isolate by well diffusion method for a period of 2 months protein 2a, which confers resistance to most of the βeta-
[34]. Briefly, wells of diameter 8 mm were bored in the pre lactam antibiotics [32]. Four of the MRSA isolates were also
seeded Mueller–Hinton agar plates using a cork borer and resistant to vancomycin, Table 1, thus making them VRSA.
100 μl of SAE (20 mg/ml) was added in the well. The
plates were incubated at 37°C and the zones of inhibition Antibacterial activity and MIC of the aqueous extract
were measured after 24 hours. The zone of inhibition after against bacterial pathogen
every 3 days was compared with the zone of inhibition on Both the hot aqueous extracts (DAE and SAE) exhibited
day 1 (first reading) to check the stability of the antibacter- antibacterial activity against Gram positive organisms
ial activity of the extract. such as B. subtilis, S. epidermidis, S. aureus and MRSA
but did not exhibit any antibacterial activity against
Cell cytotoxicity of the aqueous extracts Gram negative organisms like E. coli and P. aeruginosa,
Cytotoxicity of extract on Vero cells was measured by represented in Table 2. This observation is in accordance
microculture tetrazolium (MTT) assay [35]. For the with the antimicrobial activity obtained for other plant
assays, 96-well microplate was seeded with 100 μl medium species from the same genus, for eg., methanolic extracts
containing 10,000 Vero cells in suspension. After 24 hr of T. chippi stem bark has reported antimicrobial activity
incubation and attachment, the cells were treated with against Gram positive organisms and very weak activity
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Table 1 Antibiotic sensitivity patterns of the test organisms

Antibiotics Isolates
DMH1 DMH2 DMH3 DMH4 DMH5 DMH6 DMH7 DMH8 DMH9 DMH10 DMH11 DMH12 DMH13 DMH14
Ampicillin R S R R R R R R R R R R R R
Cefuroxime R R R R R S R R R S R R R R
Cephadroxil R R R R R R R R R S S S S S
Augmentin R S R R R R R R R R R R R R
Penicillin R S R R R R R R R R R R R R
Azithromycin S R S R R S S S S S S R R S
Erythromycin R R S R R S S S R S S R IR S
Cefoperazone S S IR R S S IR S IR IR IR R S S
Clarithromycin R R S R R S S S R S S R IR S
Ciprofloxacin S S R R IR S S S R S IR R R R
Gatifloxacin S S S S S S S S R S S S S S
Aztreonam R R R R R R R R R R R R R R
Vancomycin R S R S S S S S R R S R S R
Doxycycline R S S S S S S S R S S S S S
Hydrochloride
Norfloxacin S S IR IR IR S S S R S IR IR R R
Ofloxacin S S S S S S S S R S S S S S
Sparfloxacin S R S S S S S S R S S S S S
Methicillin R S R R R R R R R R R R R R
Legend: S sensitive, IR intermediate resistant, R resistant.
DMH 1: Pseudomonas aeruginosa, DMH 2 to DMH 8: Staphylococcus aureus, DMH 9: Escherichia coli, DMH 10 to DMH 14: Staphylococcus aureus.

Table 2 Zones of inhibition and MIC values for T. alternifolia stem bark aqueous extracts against the test organisms
Zone of inhibition (mm) Minimum inhibitory concentration (μg/ml)
Isolate
DAE SAE Ciprofloxacin (5 μg/disc) DAE SAE Ciprofloxacin
DMH 2 16 17 27 600 600 0.25
DMH 3 16 16 11 700 650 2
DMH 4 14 14 13 800 800 2
DMH 5 18 19 15 600 600 1.5
DMH 6 13 14 22 800 800 0.25
DMH 7 15 15 21 700 650 0.25
DMH 8 14 14 24 800 800 0.125
DMH 10 15 15 20 700 650 0.25
DMH 11 16 16 16 700 700 1.4
DMH 12 17 17 13 600 600 2.5
DMH 13 16 16 12 700 700 3
DMH 14 14 14 11 800 800 2
ATCC 6633 16 17 25 600 600 0.05
ATCC6538P 17 16 22 600 600 0.15
ATCC12228 17 16 24 600 600 0.1
ATCC 43300 17 17 22 600 600 0.15
Legend: All DMH isolates are Staphylococcus aureus among which DMH 3,10,12,14 are VRSA and all others except DMH2 are MRSA. ATCC 6633: Bacillus subtilis,
ATCC 6538P: Staphylococcus aureus, ATCC 12228: Staphylococcus epidermidis, ATCC 43300: MRSA.
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Figure 1 Consistent antimicrobial activity of SAE (sequential aqueous extract) against DMH 4 (MRSA). The X axis represents the number
of days after preparation of extract and Y axis represents the zone of inhibition (in mm) by well diffusion method.

against gram negative organisms [37]. Similarly T. angulate 4 mg/ml after 24 hrs of incubation. The SAE exhibited
and T. stapfiana (Britten) have antimicrobial activity against less effect as compared to DAE (Figure 2). Three inde-
S. aureus [22,23]. pendent experiments were carried out and statistical
The values for minimum inhibitory concentrations significance was evaluated by Turkey test which is a
(MIC) for DAE and SAE against the test organisms are single-step multiple comparison statistical test.
represented in Table 2. The MIC of extracts against MRSA
isolates fall in the range of 600–800 μg/ml as determined Chemo-profiling of aqueous extract
by broth dilution method. The antibacterial activity of the HPTLC revealed presence of alkaloids, flavonoids, couma-
extract was stable even after 2 months as suggested by rins, saponins and steroids in the extract, however amides
consistent zone of inhibition by well diffusion method and phenolics were absent. The number of spots and the
(Figure 1). Petroleum ether and ethyl acetate extracts did Rf values for each compound are represented in Table 3.
not exhibit any antibacterial activity. The MIC for cipro-
floxacin against MRSA isolates ranged from 0.125 μg/ml Discussion
to 3 μg/ml (Table 2). Plant derived medicines have been a part of traditional
health-care in most parts of the world and the anti-
Cell cytotoxicity of the aqueous extracts microbial property of plant-derived compounds is well-
The extracts did not exhibit any significant cytotoxicity documented [38]. For the first time here, we established
against Vero cell line at concentration ranging from 2 to the antimicrobial activity of crude aqueous extract of

Figure 2 Effect of DAE and SAE on Vero cell viability. The effect of DAE and SAE on cell viability were significantly different at p < 0.05 level.
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Table 3 Chemo profile of sequential aqueous extract of T. alternifolia stem bark performed by HPTLC method
Compound Number of spots observed Rf values
Alkaloids 2 0.77, 0.87
Flavanoids 11 0.03. 0.07, 0.35, 0.43, 0.5, 0.55, 0.6, 0.64, 0.68, 0.75, 0.8
Saponins 18 0.11, 0.17, 0.23, 0.25, 0.27, 0.34, 0.41, 0.45, 0.51, 0.53, 0.58, 0.63, 0.68, 0.84, 0.74, 0.56, 0.78, 0.84
Steroids 7 0.02, 0.04, 0.06, 0.15, 0.58, 0.78, 0.84
Coumarins 5 0.04, 0.07, 0.16, 0.57, 0.64

T. alternifolia against MRSA and VRSA. A study conducted isolation of the active principle. Alkaloids, flavonoids,
by Duraipandiyan et al. in 2006, showed that the methanol coumarins, saponins and steroids are the compounds of
and hexane extract of stem bark of T. alternifolia did not plant origin known to have antibacterial activity. These
exhibit any antibacterial activity [25]. These observations compounds were detected in the SAE [40-44]. Hence fur-
related to activity may be attributed to the fact that different ther objective is to identify a potential lead compound,
compounds from the plant material get extracted in which can be developed into a candidate anti-infective
solvents of different polarities. Moreover, the fact that the drug, in particular for treatment of infections by
antimicrobial activity of both the SAE and DAE was similar multidrug resistant pathogens like MRSA and VRSA.
(as indicated by the MIC values) demonstrates that the In the past few decades, MRSA have caused a
active principle in the plant stem bark was not extracted in major problem with nosocomial infections throughout the
less polar solvents like hexane, ether, methanol, ethyl acet- world [7]. In the developed countries, fluoroquinolones
ate but got extracted in distilled water. During the (ciprofloxacin and ofloxacin) are recommended for serious
extraction process the active principle within the extract infections associated with Staphylococci [45] but vanco-
survived boiling at 100°C for 6 hours suggesting that it is mycin still remains the drug of choice for most MRSA-
heat stable. Such a heat stable active principle has been associated diseases [46]. In this study four MRSA isolates
previously reported from other plants [39,40]. were resistant to vancomycin, five MRSA isolates were
Several studies have focused on establishment of anti- resistant and two were intermediately resistant to cipro-
bacterial activity of the plant extracts but have not floxacin respectively, further emphasizing the difficulties
focused on showing the time dependent stability of its in treatment of MRSA infections with antibiotics (Table 1).
activity [22,23,39]. Here we report that the activity of the Moreover, in the last 2 decades, the number of new anti-
extract is stable over a period of 2 months after extrac- microbial drugs that reached the marketplace has fallen to
tion. This indicates that the active principle present in less than half the previous level [47]. Hence, more efforts
the aqueous extract of T. alternifolia stem bark has a should be directed towards the screening for new anti-
long shelf life in crude preparation. For a crude extract microbial agents. The anti-MRSA activity exhibited by T.
to be used for topical applications it is mandatory that alternifolia stem bark extracts seems a promising step
the extract does not exhibit any cytotoxic activity. towards research for finding a new therapeutic agent
Although anticancer alkaloid camptothecin has been against MRSA. However, extensive research needs to be
isolated from T. alternifolia; aqueous extracts in this carried out on this aspect of T. alternifolia.
study did not show presence of camptothecin (data not
shown) [16]. The aqueous extracts did not exhibit any
Conclusion
significant cytotoxic effect against Vero cell line (Figure 2);
The aqueous extract of T. alternifolia has antibacterial
this is consistent with the absence of camptothecin in the
activity against MRSA and VRSA but does not retain
extracts. On exposure to SAE, the viable cell counts of
any cytotoxicity, which further validates the use of the
Vero cells were observed to be higher as compared to con-
plant in traditional medicine. The present study lays the
trol, suggesting that SAE may have a probable positive ef-
basis for future research, to validate the possible use of
fect on cell proliferation. The toxic compound/s present
T. alternifolia as a candidate in the treatment of MRSA
in the stem bark were probably extracted in less polar sol-
infections.
vents during sequential extraction, thus, explaining the
observed positive effect on cell proliferation only on SAE Competing interests
treatment. The authors declare that they have no competing interests.
The antimicrobial activity of the crude extract may be
attributed to a specific compound or a combination of Authors’ contributions
compounds. The knowledge about the chemical profile MR and NM contributed equally towards the completion of manuscript. NM,
MR, HK performed the experiments and analyzed the data. NM, MR, AP, SD
of the extract helps in explaining the observed activity and YS designed the study. NM and MR wrote the manuscript and SD, HK,
and designing experiments for activity fractionation for AP and YS edited it. All authors read and approved the final manuscript.
Marathe et al. Annals of Clinical Microbiology and Antimicrobials 2013, 12:26 Page 7 of 8
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Acknowledgements Tabernaemontana pandacaqui Poir. J Ethnopharmacol 2003,

We wish to thank the Head of Department of Pathology, Dr. Kishor Bhingare 84:31–35.
and Head of Microbiology, Dr. Sampada Patwardhan from Deenanath 21. Beek TAV, Kuijlaars FLC, Thomassen PM, Verpoorter R, Svendsen B:
Mangeshkar Hospital, Pune for providing cultures and supporting this work. Antimicrobially active alkaloids from Tabernaemontana pachysiphon.
Nachiket Marathe is grateful to Council of Scientific and Industrial Research Phytochemistry 1984, 23(8):1771–1778.
(CSIR), New Delhi, for funding. 22. Suffredini IB, Bacchi EM, Sakuda TK, Ohara MT, Younes RN, Varella AD:
Antibacterial activity of Apocynaceae extracts and MIC of
Author details Tabernaemontana angulata stem organic extract. Braz J Pharm Sci 2002,
1
Microbial Culture Collection, National Centre for Cell Science, Ganeshkhind, 38(1):89–94.
Pune 411007, India. 2Department of Biodiversity, Abasaheb Garware College, 23. Ruttoh EK, Bii C, Tarus PK, Machocho A, Karimi LK, Okemo P: Antifungal
Karve Road, Pune 411004, India. 3Department of Microbiology, Abasaheb activity of Tabernaemontana stapfiana Britten (Apocynaceae) organic
Garware College, Karve Road, Pune 411004, India. extracts. Pharmacognosy Res 2009, 1(6):387–391.
24. Yadav SR, Sardesai MM: Flora of Kolhapur District. Kolhapur, India: Shivaji
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Published: 25 September 2013 25. Duraipandiyan V, Ayyanar M, Ignacimuthu S: Antimicrobial activity of some
ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India.
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doi:10.1186/1476-0711-12-26
Cite this article as: Marathe et al.: In vitro antibacterial activity of
Tabernaemontana alternifolia (Roxb) stem bark aqueous extracts against
clinical isolates of methicillin resistant Staphylococcus aureus. Annals of
Clinical Microbiology and Antimicrobials 2013 12:26.

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