International Journal of Phytomedicine 3 (2011) 353-361

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Original Research Article

ISSN: 0975-0185

Anti-mycobacterial activity of Piper longum L. fruit extracts against multi

drug resistant Mycobacterium Spp.
Chandan Singh1, Santosh K. Singh1*, Gopal Nath2 and N.P. Rai3
*Corresponding author:
Santosh K. Singh
1

Centre of Experimental Medicine

and Surgery, Institute of Medical
Sciences, Banaras Hindu
University, Varanasi 221005 India
2

Department of Microbiology,
Institute of Medical Sciences,
Banaras Hindu University,
Varanasi 221005 India
3

Department of Kayachikitsa,
Faculty of Ayurveda, Institute of
Medical Sciences, Banaras Hindu
University, Varanasi 221005 India

Introduction

Abstract
A long tradition of using pepper as to fight against several ailments by
the local tribal people is still in the practice, in many parts of the rural
India. So utilizing this tribal knowledge base for this highly medicinal
plant, an attempt was made to isolate some novel natural bioactive
compounds with potential activity against multidrug resistant (MDR)
Mycobacterium. A bioassay guided fractionation of Pippali (Piper
longum L.) was performed in five different organic solvents and their
activities were monitored against different pathogenic bacteria
including MDR Mycobacterium. Different fractions were screened for
the bioactivity against Mycobacterium, and the structure of bioactive
compound was characterized with H1 and C13 NMR. An ethyl acetate
fraction of Pippali extract was found active against M. smegmatis
(3000g ml-1) and M. tuberculosis (39 g ml-1). It also shows very
significant activity against other bacterial strains like E.coli (152 g ml1
), Staphylococcus aureus (14 g ml-1), Salmonella typhi (180 g ml-1),
Enterococcus faecalis (15 g ml-1), and Pseudomonas aeruginosa (52
g ml-1). This fraction of ethyl acetate was then purified and
characterized as piperine [5-(1, 3-benzodioxol-5-yl)-1-piperidin-1ylpenta-2,4-dien-1-one], a well known alkaloid from this plant.
Bioactivity guided fractionation concludes that Piperine is the only
active ingredients in various fractions of fruit extract evaluated for
antibacterial activity. Fraction having piperine has significant activity
against multi drug resistant strains of Mycobacterium spp. than other
purified fractions of fruit extract. The current finding encourages us to
develop new alternative medicine that includes piperine alone and/or in
combination with other drugs to fight against the drug resistance among
Mycobacterial strains.
Keywords: Piper longum, piperine, bioassay guided fractionation,
NMR,
Mycobacterium
tuberculosis.
Natural bioactive compounds are the ultimate
source for innovative therapeutic agents to cope
with communicable and non communicable

Singh et al. International Journal of Phytomedicine 3 (2011) 353-361

the phytochemicals can be of great significance in
therapeutic treatments and could be helpful to
cure the problem of these multi-drug resistant
microorganisms. World Health Organization has
also advocated that the medicinal plants would be
the best source for obtaining a variety of drugs
for malaria, tuberculosis and immunecompromised patients [16]. Several secondary
metabolites of plant origin that include a wide
chemistry like alkaloids and flavonoids has
already been in use as anti-viral, anti-bacterial,
anti-amoebal and anti-cancer agents [17-20].
In this context, we have selected a well known
spice plant Piper longum, to screen and isolate
new bioactive metabolites that are active against
multidrug resistance Mycobacterium. Piper sp.
are distributed widely in the tropical and
subtropical regions of the world, have multiple
applications in different folk medicines, including
Indian system of medicine Ayurveda and have
been reported to possess numerous biological
activities [21-25]. P. longum used as
bioavailability enhancer [26], digestive and in the
treatment of bronchitis [27] and as effective
hepatoprotective agent [28-29]. The main
objective of this study was to screen extracts
from fruits of Piper longum plant and isolate
bioactive compounds with potential activity
against multidrug resistance strains of
Mycobacterium.

diseases and other ailments [1]. Ethno

pharmacologists, botanists, microbiologists and
natural product chemists are searching novel
bioactive metabolites which could be developed
as new pro-drugs for treatment of infectious
diseases and other biomedical challenges
including drug resistance among various
infectious microbes [2]. The genetic ability of
pathogenic bacteria to develop resistance against
commonly used antibiotics is a major medical
problem and challenge worldwide, posing a big
threat to human society [3-5]. Medicinal plants
that have a well defined ethanobotanical history
as well as tribal and folk medicinal uses are of
great concern among the natural product
researchers, for getting new bioactive leads [6-7].
Many efforts have been made to discover new
antimicrobial compounds from various kinds of
sources including microbes and plants.
Systematic screening of them may result in the
discovery of novel effective compounds [8] with
potential of new drug design and development.
Antimicrobials of plant origin are efficient in the
treatment of infectious diseases, while
simultaneously mitigating many of the side
effects that are often associated with synthetic
antimicrobials [9]. A wide variety of plant
secondary metabolites have been identified as
active principles for the treatment of various
ailments [10-11]. A number of studies have been
conducted for the selection of the crude plant
extracts in a therapeutic treatment of bacterial
infections [12-14]. Many plant extracts have
shown the ability to overcome drug resistance in
many pathogenic micro-organisms, which led the
researchers to isolate active principles and
investigate mechanisms. The discovery and
development of structurally novel chemical
entities to control the multi-drug resistant
pathogenic bacteria is desperately desired by the
pharmaceutical industries and drug developers
that are looking towards the underexplored
natural sources for developing the front line
drugs. Many drug leads isolated from plants are
active against a wide range of microbes including
plant and human pathogens also rectify this
approach [15]. The use of the plant extracts and

Materials and Methods

Plant materials and microbes
Healthy plants of Piper longum L. selected and
fruits were randomly collected from herbal
garden of faculty of Ayurveda, Banaras Hindu
University Varanasi. Plant specimens were
identified by Prof. A.K. Singh, Department of
Dravya Guna, Banaras Hindu University BHU).
A voucher specimen (CEMS/PL-054/SKS) was
also deposited in the Centre of Experimental
medicine and Surgery, BHU. Samples were
brought to the laboratory and used to prepare
extracts within 48h of collection. The following
bacteria were used for anti-bacterial bioassay
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Singh et al. International Journal of Phytomedicine 3 (2011) 353-361

besides Mycobacterium tuberculosis GN/mt-75,
M. smegmatis GN/ms-43, Escherichia coli
GN/ec-98, Staphylococcus aureus GN/sa-16,
Pseudomonas aeruginosa GN/pa-40, Salmonella
typhi GN/st-56, Enterococcus faecalis GN/ef-32.
All cultures were obtained from culture collection
of Department of Microbiology, Institute of
Medical Sciences, Banaras Hindu University
India. All these strains were isolated and cultured
on synthetic media, from the samples collected
from the infected subjects in the OPD (outpatient
department).

Isolation and
compound

identification

of

bioactive

Since the ethyl acetate extract showed promising

antibacterial activity in initial screening, it was
subjected to column chromatography over silica
gel (60-120 mesh) and eluted using 500ml of
ethyl acetate:methanol:water (10:1:0.1 v/v). Total
22 fractions were collected and fraction PE-014
was showed maximum activity in preliminary
bioassay. A HPLC study has been performed of
this bioactive fraction with authentic reference
standard of Piperine. Separation was achieved
using a C18 column (250 4.6 mm, 5 ID),
(Merck, India). The solvent system consisted of
50mM potassium dihydrogen orthophosphate (pH
adjusted to 3.5 using orthophosphoric acid):
acetonitrile (40:60 v/v) was pumped isocratically
at a flow rate of 0.6 ml/min. The detection was
carried out using a diode array detector. The final
structure determination of the bioactive fraction
was performed by C13 and H1 NMR.

Instruments
Column chromatography was carried out by
using silica gel G of 60-120 mesh size
(Qualigens, India). TLC analysis was done with
Merck 0.25mm (silica gel G, 13% Calcium
sulphate as binder) ready to use glass plates of
510 cm. Melting point was recorded with a
Stuart-SMP 10 melting point apparatus in open
capillaries. HPLC was performed on a Shimadzu
LC 10. Infra red spectra were recorded in KBr
(4000-350 cm-1) with Shimazdu 8201 PC, while
UV spectra was recorded with a dual beam
spectrophotometer Jasco 7800, Japan. NMR
spectra were recorded in CDCl3 and DMSO- d6
solvent by using TMS as internal standard on a
Buker DRX -300 Mz FTNMR.

Anti-bacterial Assay
Anti-bacterial assay was performed by using the
modified
Kirby-Bauer
disk
diffusion
susceptibility method [30]. The bacterial strains
were suspended in 4ml of normal saline (0.85 %)
and the density of suspension was adjusted to
approximately 108 CFU ml-1 using 0.5 M barium
sulphate buffer as the turbidity standard. The
surface of the sterile 3.8% mueller hinton agar
(Himedia, India) in petri dishes was dried and the
test strain was inoculated with a sterile swab to
obtain a homogenous bacterial lawn. The
sterilized 6mm discs (Himedia) containing 10l
of fraction was placed onto the agar, and the
inhibition zones was measured (in mm) after
incubation for 18 hours at 37C. DMSO was
taken as positive control for organic extracts.
Different organic solvent extracts (1% v/v) in
DMSO did not affect the growth of
microorganisms in accordance with our control
experiments.

Preparation of extract and fractions

The collected fruits were shade dried at room
temperature and ground in a pulvelizer. 2-3 kg of
dried fine powder was thus obtained, which was
extracted with 2L of methanol for 2-3 times. The
total of 6L of extracted methanol was then
evaporated to dryness with a rotary evaporator,
which yield 500-600g of dry extract. It was
further partitioned in petroleum ether (37g),
hexane (58g), chloroform (45g), ethyl acetate
(100g), methanol (35g). The fractions were
filtered through buchner funnel and the filtrates
were evaporated to dryness under reduced
pressure using rotary evaporator at 35C. All the
dried fractions were stored in airtight vials at 4C
for subsequent use.
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Singh et al. International Journal of Phytomedicine 3 (2011) 353-361

In vitro anti-mycobacterial activity against M.
smegmatis

Results and Discussion

Minimum inhibitory concentration (MIC) was

performed according to the standard reference
method [31]. Different dilutions ranging from 510 mg ml-1 were prepared in DMSO and added
into muller hinton broth medium. The bacterial
suspension was prepared in normal saline and
match with Mc farland turbidity standard to
obtain concentration 106 CFU ml-1. This
suspension was added to fractions and incubated
at 37C for 72h. 500l of suspension from each
of the broth which contains different
concentration of compound was spread on plate
and incubated at 37C to observe the bacterial
growth. The lowest concentration of compound
that showed no visible growth in media was
considered as MIC.

Petroleum ether, hexane, chloroform, ethyl

acetate and methanol extracts were screened
against an array of pathogenic bacteria
representing both gram positive and negative
strains, including Mycobacterium. Among all the
organic extracts the ethyl acetate extract showed
notable activity against test microbes. The disk
diffusion assay showed a wide range of growth
inhibition of different organic solvent extracts
against test bacterial strains. Table 1 showed the
antibacterial activity of different fractions. The
petroleum ether fraction was found ineffective
against bacterial strains, while hexane extract was
least inhibitory (6.331.52) among all the
extracts against all test strains (Table 1).
Hexane fraction is showing highest activity
against E. faecalis (12.662.08). Ethyl acetate
fraction of Piper longum fruits was showing
highest activity against all the tested bacterial
strains together with Mycobacterium. S. aureus
(24.331.52) and P. aerugenosa (22.661.52)
were the highly sensitive strains towards ethyl
acetate fraction with average inhibition zones
24.33 mm and 22.66 mm respectively. For
methanolic fraction E. fecalis (21.001.00) was
the highly sensitive strain while E.coli
(9.331.15) strain was the least sensitive with
inhibition zones 21 and 9.33mm respectively
(Table 1). Hexane fraction was showing no
inhibitory effect towards M. smegmatis. These
findings revealed that each fraction was
inhibitory to all tested gram positive and gram
negative bacterial strain except petroleum ether.
Ethyl acetate, methanol and chloroform fractions
were having potent inhibitory property against
bacteria, supported the view that most of the
antimicrobial active fractions have been dissolved
in these high polar solvents. After successful
characterization of piperine from fruit extract of
Piper longum, an anti-mycobacterial activity
bioassay was performed against two multidrug
resistant strains Mycobacterium tuberculosis and

In vitro anti-mycobacterial activity with M.

tuberculosis
For bioactive compound, stock solution of 10 mg
ml-1 in DMSO was prepared and stored at room
temperature. Compound concentrations of 5, 2.5,
1.25, 0.625, 0.312, 0.156, 0.078, 0.039 and
0.01g ml-1 were made accordingly by adding
required volume of stock solution respectively to
15ml of lowenstein jensen (LJ) medium in Mc
cartney bottles. The molten media at 75C poured
and slanted till the media solidify at room temp.
The whole media were incubated at 37C for 24
hours as a check of sterility before inoculation.
Cells from 3 week old culture were first scraped
into a bottle containing saline and glass beads, to
obtain a homogenous suspension of M.
tuberculosis as inoculum. The bottle was
vortexed and kept standing for 20 min. the
supernatant was then transferred into another
sterile tube and turbidity was matched with Mc
farland standard. 150 l of this inoculum was
added to control as well as compound treated
media. The bottles were incubated at 37C and
bacterial growth was checked after four weeks.
The lowest concentration of compound that
showed no visible growth in media was
considered as MIC.
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Singh et al. International Journal of Phytomedicine 3 (2011) 353-361

Table 1 Antibacterial activity of fruit extracts of Piper longum extracted in different organic solvent.

Zone of inhibition (in mm), (meanSD), n=3

Chloroform
Ethyl
acetate Methanol
fraction
fraction
fraction

Bacterial strains

Hexane
fraction

E .coli GN/ec-98

6.331.52

11.331.15

14.330.57

9.331.15

S. aureus GN/sa-16
P. aeruginosa GN/pa-40
S. typhi GN/st-56
E. faecalis GN/ef-32
M. smegmatis GN/ms-43

10.661.52
12.001.00
10.330.57
12.662.08
-

20.332.08
20.661.52
11.661.52
19.002.64
12.330.57

24.331.52
22.661.52
19.331.15
21.661.52
15.661.52

16.661.52
15.660.57
10.660.57
21.001.00
11.66 0.57

Table 2 Minimum inhibitory concentration (MICs) of piperine from fruit extracts against different
bacteria.
MICs (g ml-1)
152
14
52
180
15
3000
39

Tested Organisms
Escherichia coli GN/ec-98
Staphylococcus aureus GN/sa-16
Pseudomonas aeruginosa GN/pa-40
Salmonella typhi GN/st-56
E. faecalis GN/ef-32
Mycobacterium smegmatis GN/ms-43
Mycobacterium tuberculosis GN/mt-75

susceptible to the piperine, rather than the M.

smegmetis,
which
atypical
strain
of
Mycobacterium (Table 2). Pure crystals of
piperine obtained by purification of fraction PE014, were also assay on the plate for inhibition
measurements
against
Mycobacterium
tuberculosis, and observed significant inhibition
of the drug resistance M. tuberculosis strain. Thus
after this assessment the ethyl acetate fraction
was consider for further purification and
characterization study.
Further column
purification of the active fraction was performed,
and concentrated in vacuo. It was then resolved
on TLC plate together with the reference standard
of piperine. The bioactive fraction (PE-014)
extracted in ethyl acetate was then further
purified
by
adopting
bioassay
guided

M. smegmatis, together with some other

pathogenic bacteria to assess the wide spectrum
bioactivity.
The
minimum
inhibitory
concentration of piperine was observed against
Staphylococcus aureus (14 g ml-1), while
significant inhibitory activity was also found
against E. faecalis (15 g ml-1), Pseudomonas
aeruginosa (52 g ml-1), apart from
Mycobacterium (Table 2).
Interesting differences were observed in the
inhibition concentration against two strains of
Mycobacterium, the piperine from extract was
more efficient against M. tuberculosis (39 g ml1
), while against M. smegmetis it was around
3000 g ml-1, however this difference at this level
is beyond any prediction. This seems that multi
drug resistance M. tuberculosis strain is more
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Singh et al. International Journal of Phytomedicine 3 (2011) 353-361

fractionation, followed by HPLC and identified
as piperine C17H19NO3 after comparison with
authentic reference standard (Figure 1and 2).

At Rf value 0.26, a fraction PE-014 and standard

piperine shows equal bands, this confirms the
presence of piperine in the fraction PE-014, from
piper fruits. It was observed that the purified subfraction PE-014 contains piperine crystals
(8g/mL) as most active ingredient. It was also
widely available in the literature that piper fruit
contains this alkaloid which is responsible for the
pungency of this spice. After identification of this
compound, further spectroscopic confirmation
was performed by doing NMR, IR and other
methods. The H1 NMR of the piperine represent
1.48 (4H, m, C-3and C-5), 1.65 (2H, m, C4), 3.57 (4H, m, C-2and C-6), 5.95 (2H, s,
OCH2O), 6.52 (1H, d, J=16 Hz, C-2), 6.82 (2H,
m, C-3 and C4), 7.71 (1H, m, C-5), 7.23 (1H, br,
s, C-2), 6.99 (1H, m, C-6), 6.77 (1H, m, C-5)
(Table 3).

Figure 1 the molecular of pipreine crystals obtained from

Piper longum, and its chemical properties.

Table 3 H1 NMR data for the bioactive piperine

from the fruits of Piper longum.
H
1.48
1.65
3.57
5.95
6.52
6.77
6.82
6.99
7.23
7.71

H
4H
2H
4H
2H
1H
1H
2H
1H
1H
1H

Position
m
m
m
s
d
m
m
m
br, s
m

HMBC
C-3 and C-5
C-4
C-2 and C-6
OCH2O
C-2
C-5
C-3 and C-4
C-6
C-2
C-5

While C13 NMR of this extracted piperine

represent 23.44, 24.52 and 25.25 (C-3, C-4,
C-5), 41.95 and 45.64 (C-2, C-6), 100.19
(OCH2O), 104.48 (C-2)107.31(C-3), 119.24 (C2), 121.34 (C-5), 124.32 (C-6), 129.74 (C-1),
136.83 (C-4), 141.02 (C-5), 147.00 (C-3),
147.08 (C-4), 163.97 (C-1) (Table 4).
Figure 2 HPLC chromatogram of fraction PE-014 shows
peak on retention time 9.52, whichcorrespond to the
authentic reference standard of piperine.

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Singh et al. International Journal of Phytomedicine 3 (2011) 353-361

Table 4 C13 NMR data for the bioactive piperine
from the fruits of Piper longum.
13

C
23.44
24.52
25.25
41.95
45.64
100.19
104.48
107.31
119.24
121.34
124.32
129.74
136.83
141.02
147
147.08
163.97

molecule having maximum antimycobacterial

potential in Piper longum fruit. The interesting
part of this work is the finding related to the
activity of piperine against the multidrug resistant
strains of Mycobacterium, especially M.
tuberculosis. This report is first of its kind, and
has significant biomedical importance in
treatment of tuberculosis. This finding also
encouraged us to develop new alternative therapy
that includes piperine alone or with the
combination of available drugs to cope with
increasing drug resistant among many strains of
Mycobacterium specifically. In our further study
we will study the performance of several drug
combinations with piperine and evaluate its
potential in treatment of tuberculosis.

HMBC
C-3
C-4
C-5
C-2
C-6
OCH2O
C-2
C-3
C-2
C-5
C-6
C-1
C-4
C-5
C-3
C-4
C-1

Acknowledgements
Authors are thankful to the Professor-in-charge,
Centre of Experimental Medicine and Surgery
(CEMS) for providing necessary facilities to
conduct this work.

The IR spectra represent (KBr) 2934, 1634 (CO),

1611.9, 1583.5, 1490, 1443.5, 1364.3, 1251.3,
1194.3, 1131.4, 1026.3, 998.1, 926.4, 847.9,
805.9, 701.3, 608.0 cm-1. Even the compound is
known from the plant since long back, but this is
first ever report about its potential activity against
multidrug resistant Mycobacterium strain. Many
reports available about the anti-bacterial potential
of piperine and sometime even the same
compound (piperine) has been isolated from other
plant sources like Ludwigia hyssopifolia L. and
reported to be active against several pathogenic
bacteria [32-33].

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