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International Journal of Pharmaceutical Studies and Research

E-ISSN 2229-4619

Research Article

INVESTIGATING THE MECHANISM OF ACTION OF


TERPENOIDS AND THE EFFECT OF INTERFERING
SUBSTANCES ON AN INDIAN MEDICINAL PLANT
EXTRACT DEMONSTRATING ANTIBACTERIAL
ACTIVITY
Jasmine Ra*, Selvakumar BNb, Daisy Pc1

Address for Correspondence


a

Department of Biotechnology, Bishop Heber College, Tiruchirappalli, India


b
Department of Microbiology, CSI Hospital, Tiruchirappalli, India
c
Department of Biotechnology, Holy Cross College, Tiruchirappalli, India
E-mail: jasmine_selvakumar@yahoo.com

ABSTRACT
Elephantopus scaber, an Indian medicinal plant, reported to possess potent bactericidal activity against several drugresistant bacteria was investigated for their mode of action.By performing salt tolerance assays, it was found that the
extract of the whole plant of Elephantopus scaber, compromised the integrity of the cytoplasmic membrane of
Staphylococcus aureus, leading to a decrease in ability to exclude NaCl. The bactericidal action of the Elephantopus
scaber extract was concluded to be due to its membrane-active properties. The effect of contaminants on the efficacy of
this extract was also investigated. Organic contaminants (bakers yeast and skim milk powder) decreased the efficacy
of all extracts investigated, while hard water had no effect. Greater understanding of the biocidal properties of the plant
extracts investigated may determine if they have medical, industrial or environmental applications. Since Elephantopus
scaber possessed antibacterial activity, the experiment was further carried out to isolate and identify the putative
antibacterial compounds based on bioassay-guided fractionation. Bioactivity-guided fractionation of the acetone
extract of Elephantopus scaber (ES) yielded a new terpenoid compound (already reported) as, 6-[1-(10,13-dimethyl4,5,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[]phenanthren-17-yl)ethyl ]-3-methyl-3,6-dihydro-2 H2-pyranone. The active compound was purified by repeat column and structure was determined on the basis of
chemical and physiochemical evidence.
KEYWORDS: Elephantopus scaber; terpenoid; salt tolerance; antibacterial.

INTRODUCTION
Antibiotic resistance has increased rapidly during
the last decade, creating a serious threat to the
treatment of infectious diseases. Drug resistance
is one of the most serious global threats to the
treatment of infectious diseases (1,2). In addition
to resulting in significant increases in costs and
toxicity of newer drugs, antibiotic resistance is
eroding our therapeutic armamentarium.
Resistant strains of bacteria are continuing to
increase, both in number and in variety, but not
significantly different newer antibiotics are yet
available. Treatment of infections caused by
these resistant bacteria has become very difficult.
Since they are resistant to many antibiotics,
therapeutic options have become limited.
Therefore, alternative methods of treatment are
sought after. For over several years medicinal
plants have served as the models for many
clinically proven drugs, and are now being reassessed as antimicrobial agents. Literally
thousands of plant species have been tested
against hundreds of bacterial strains in vitro and
many medicinal plants are active against a wide

IJPSR/Vol. II/ Issue II/April-June, 2011/19-24

range of gram-positive and gram-negative


bacteria. However very few of these medicinal
plant extracts have been tested against resistant
bacteria. For a long period of time, plants have
been a valuable source of natural products for
maintaining human health, especially in the last
decade, with more intensive studies for natural
therapies. Plants produce a wide array of organic
compounds, usually secondary metabolites,
which in addition to imparting characteristic
odour, pigment and flavour properties,
sometimes exhibit antimicrobial action [3]. The
extraction, and possible subsequent therapeutic
application, of these biologically active
phytochemicals is not a recent development, with
many plant-derived antimicrobials undergoing
clinical trials for human use [3]. Studies
conducted worldwide, for example, the
nvestigation of traditional African [4,5,6],
Caribbean [7] and Indian [8,9,10] medicinal
plants, function not only to scientifically validate
purported properties of traditional medicinal
plants, but also in the identification of possible
sources of effective drugs. It follows, therefore,

International Journal of Pharmaceutical Studies and Research

that as plant products become more widely


exploited as a source of antimicrobial agents,
those products in-turn require rigorous
characterization, both in identification of active
constituents, and in the study of mechanisms by
which they exert their antimicrobial activity.
However, few studies have included further
investigations of the biological action of
antimicrobial plant extracts. The current study
was designed to characterise selected Indian
medicinal
plant
extracts,
previously
demonstrated to exhibit antibacterial activity
against drug-resistant Gram positive and
negative bacteria [10]. In addition, we recently
identified the active component of the
Elephantopus scaber extract as a terpenoid, a
class of antimicrobial phytochemicals whose
mechanism of action is membrane disruption.
The experiments described here were specifically
undertaken to investigate (i) the mode of
antibacterial action of the Elephantopus scaber
extract and (ii) the effect of contaminants on the
activity of this medicinal plant extracts.
MATERIALS AND METHODS
Plant material
Elephantopus scaber Linn. is a small herb, which
grows in the wild throughout the tropical regions
of the world. The major phytochemical
constituents of the plant are elephantopin,
triterpenes, stigmasterol, epofriedelinol and
lupeol [11,12]. The plant has been used in the
Indian system of medicine as analgesic, diuretic,
astringent and antiemetic. The leaves of the
plant were known to be used for bronchitis,
small pox, and diarrhea and as a brain tonic [13].
Recently, it has been shown to possess antiinflammatory and antitumour activity in animal
models [14], and also found to have antibacterial
activity against a few standard bacterial strains.
Extract Preparation
Elephantopus
scaber
plants
were
collected from Kerala and authenticated
at the Department of Botany of the
College. The voucher specimen is
available
at
the
Department
of
Biotechnology, Holy Cross College,
Trichy-2. The air-dried plants were
powdered and 1kg was extracted using
methanol, acetone and hexane in a
soxhlet apparatus and were evaporated to
dryness under reduced pressure in rotary

IJPSR/Vol. II/ Issue II/April-June, 2011/19-24

E-ISSN 2229-4619

evaporator. The yields of the acetone, hexane


and methanol extracts were 12.1 gm %, 10.9 gm
% and 14.3 gm% respectively. The dry
residues of the crude extracts obtained
was stored for further use. For
convenience the methanol, acetone and
hexane extracts of E.scaber were named
ESM, ESA and ESH respectively.
Fractionation of the crude extract with
promising results.
Fractionation of the crude extract was based on
bioactivity. The most bioactive crude extract
was chromatographed on a silica gel column.
Initial elution with discontinuous gradient of
ethyl acetate and hexane, then with acetone and
ethyl acetate, with acetone and chloroform and
finally with chloroform and hexane yielded 17
fractions (F1-17). The fractions F1-5, F6-8, F9-11,
F12-15 and F16-17 were combined according to their
Rf values into five fractions finally and were
named as F1, F2, F3, F4 and F5 respectively.
Test Organisms
Urinary isolates from symptomatic Urinary tract
infected patients attending or admitted to CSI
Mission General Hospital in Tiruchirappalli,
South India, from October 2005-March 2006,
were identified by conventional methods.
Clinical isolates were included for the study.
The bacterial strains were grown and maintained
on Nutrient Agar slants.
Salt tolerance assay
The salt tolerance assay (15) involved
investigating the ability of S. aureus cells treated
with the E.scaber extract to grow on NA
supplemented with NaCl. Triplicate suspensions
of bacteria were prepared as described above and
treated with extract at 0.5 minimum inhibitory
concentration (MIC) (1: 4000 dilution of extract
in sterilised water), 1 MIC (1: 2000), and 2
MIC (1: 1000). MIC was determined as outlined
below.
After 30 min, samples were removed, serially
diluted, plated onto NA or NA supplemented
with 50 mg/ml and 70 mg/ml NaCl and
incubated at 37 C for 24 h. The mean number of
colony-forming units/ml on the NANaCl plates
were reported as a percentage of those on the
NA-only plate. Controls consisted of untreated
cells incubated under the same conditions.
Effect of interfering substances on biocide
activity

International Journal of Pharmaceutical Studies and Research

E-ISSN 2229-4619

To assess the effect of contaminants and cations


(hard water) on the efficacy of the studied
extracts [16], skim milk powder (Bonlac Foods,
Melbourne, Australia) and bakers yeast
(Defiance Milling Co., UK) were added to NB to
a final concentration of 10% (w/v) and 5% (w/v),
respectively. The effect of hard water was
investigated using NB prepared in distilled water
to which CaCl2 (0.304 mg/ml) and MgCl2
(0.065 mg/ml) [17] had been added. Plant extract
was added to sterile tubes containing NB (or NB
in hard water) and serial 2-fold dilutions were
carried out. The interfering substances were
added to the tubes along with bacterial culture
(10 l of an overnight NB culture) and these
were incubated at 37 C for 24 h. The minimum
bactericidal concentration was then determined.
NB-only cultures served as controls. The assay
was carried out in triplicate and any effect of the
contaminants was observed as an increase in
MBC compared to the control.
Determination of minimum inhibitory
concentration
(MIC)
and
minimum
bactericidal concentration (MBC) of extracts
MICs were determined using a broth dilution
method where doubling dilutions of extract in
NB (in a final volume of 2 ml) were inoculated
with 10 l of an overnight NB culture. Following
incubation at 37 C for 24 h, the MIC was
recorded as the lowest dilution that inhibited
bacterial growth. To determine MBCs, the tubes
were sampled, inoculated onto NA, and
incubated for a further 24 h. The MBC was
recorded as the lowest concentration that
prevented the recovery of viable bacteria.

RESULTS
Determination of the mechanism of
antibacterial action
Investigations of the mechanism of action were
carried out on the acetone extract of E. scaber
as this was the most potent of the extracts
determined from preliminary screening of Indian
medicinal plants. The activity of the extract and
the chemical nature of its active component also
suggested that it might be membrane-active.
Sub-lethal injury of bacterial cell membranes can
alter their permeability and affect the ability to
adequately osmoregulate or exclude toxic
materials. The loss of salt tolerance has been
used in previous studies to reveal membrane
damage in sub-lethally injured bacteria [15]. The
salt tolerance assay was used to investigate the
membrane-active nature of the E.scaber
extract. Fig.1 illustrates the relationship between
the salt tolerance of S. aureus and the
concentration of E.scaber extract to which the
bacteria were exposed. The results are reported
as the percentage decrease in CFU/ml on the
NANaCl plates compared to the NA-only
control plates. Treatment of S. aureus with the
E.scaber extract at MIC level or greater
reduced the ability of the survivors to form
colonies on the NaCl containing media.
Exposure to sub-MIC levels did not affect
survival as there was no significant difference in
survival on NANaCl plates between bacteria
not exposed to extract and those exposed to 0.5
MIC levels of extract. This decreased ability to
exclude salt (at or above the MIC) indicated a
loss of membrane fidelity in the presence of the
E.scaber extract.
Table 1: Effect of a few interfering substances on the efficiency of Elephantopus scaber extracts on
the growth of S.aureus

Interfering substances
Hard water(0.304g/l CaCl2)
Skim milk powder(10%w/v)
Bakers yeast (10%w/v)

ESM*
25.0
50.0
25.0

Minimal Bactericidal concentration(mg/ml)


ESA*
ESH*
12.5
100.0
12.5
100.0
12.5
200.0

*ESM- Elephantopus scaber methanol extract, *ESA- Elephantopus scaber acetone extract, *ESH- Elephantopus scaber hexane extract

Table 2: Effect of a few interfering substances on the efficiency of Elephantopus scaber extracts on
the growth of E.coli
Interfering substances
Hard water(0.304g/l CaCl2)
Skim milk powder(10%w/v)
Bakers yeast (10%w/v)

ESM*
50.0
50.0
25.0

Minimal Bactericidal concentration(mg/ml)


ESA*
ESH*
25.0
200.0
12.5
100.0
25.0
200.0

*ESM- Elephantopus scaber methanol extract, *ESA- Elephantopus scaber acetone extract, *ESH- Elephantopus scaber hexane extract

IJPSR/Vol. II/ Issue II/April-June, 2011/19-24

International Journal of Pharmaceutical Studies and Research

E-ISSN 2229-4619

SALT TOLERANCE ASSAY

L
M
/
U
F
C
F
O
R
E
B
M
U
N

100
90
L
C 80
A 70
N
% 60
0
5 50
+
A 40
N
30
20
10
0
CONTROL 0.5XMIC

1.0XMIC

2.0XMIC

Fig 1 : Proportion of S.aureus cells able to form colonies on NA and NA supplemented with
50mg/ml NaCl after 30 minutes of treatment with E scaber acetone extract at 0.5x, 1x and 2x the
minimum inhibitory concentration (MIC). Control cells were untreated.
Effect of interfering substances
The bactericidal efficacy of all four plant extracts
in the presence of various interfering substances
was investigated. The MBC rather than MIC was
determined for several reasons. In terms of
practicality, the turbidity of the bakers yeast and
skim milk powder added to NB meant that it was
not possible to visually distinguish the
concentration at which growth was inhibited.
Secondly, it has been suggested that the
determination of the MBC of a biocidal
compound
is
more
valuable
to
its
characterisation than its MIC [18]. The MBC of
all extracts increased in the presence of skim
milk powder and bakers yeast but was unaltered
in NB prepared in hard water (Table 1&2).
Specifically, the addition of skim milk powder
and bakers yeast typically caused a four- and
two-fold increase in MBC, respectively.
DISCUSSION
Understanding the mechanisms of biocidal
action and the factors influencing their activity,
such as the effect of interfering substances, have
become key issues for the better use of biocides
[18]. The membrane disruption seen could still

IJPSR/Vol. II/ Issue II/April-June, 2011/19-24

be a function of the biocide-induced autolytic


events described above [19, 20, 18]. Regardless
of the sequence of events, however, the results
indicate that membrane damage is occurring in
the presence of the E.scaber extract.
Demonstrating loss of salt tolerance has been
utilised in other studies to investigate the mode
of action of antibacterial agents against S. aureus
[15]. Overall, the results obtained from these
investigations suggested membrane-activity as a
plausible mode of action for the E.scaber
extract, supporting the finding that the active
component of this extract is a terpenoid. There
are numerous reasons why assessing the effect of
contaminants on biocidal agents is important to
their characterisation. The first is the basic
requirement to determine the effect potentially
interfering compounds may have on efficacy of
the antimicrobial product. That is, when the
extracts are exposed to material commonly found
in non-sterile environments, an assessment needs
to be made of whether they will retain the same
or a similar level of antibacterial action. This
applies both to the use of biocides in stand-alone
applications, and when they are used in

International Journal of Pharmaceutical Studies and Research

conjunction with other products. For example,


incorporation of biocides into pharmaceutical or
general
household
formulations
would
necessitate a study of how this would effect their
antimicrobial activity [15]. Loss of efficacy not
only impacts on the immediate potency of a
biocide but also has implications for increased
resistance. The use of biocides at sub-effective
concentrations can confer bacterial resistance to
third-party therapeutic agents, such as antibiotics
[21]. Therefore, while the loss of efficacy due to
the presence of interfering substances may be the
primary result, future problems of resistance to
the biocide itself as well as to other antimicrobial
agents may arise. The current study indicated
efficacy of extracts was not affected by the
presence of hard water, but was compromised by
the presence of organic matter. As recognized by
Hammer et al. (1999), this data may be useful in
assessing the potential commercial applications
of the extracts. Organic matter is able to
compromise the activity of antimicrobial agents
by several modes [15]. Adherence of organic
matter to the bacterial cell may act to prevent
exposure to the antibacterial substance, thereby
decreasing the level of activity [22, 23]. A
similar effect is also proposed for the interaction
between organic mater and the antimicrobial
compound. This results in the usually active
substance being less readily absorbed by the
bacteria, and therefore less effective 16]. The
data presented in this paper provide important
information about the biocide properties of the
Indian medicinal plants extracts. In addition to
further studies, such as the identification of the
active components of all extracts, these data will
help to determine if these extracts will have
useful medical, industrial or environmental
applications. Further investigation of other
factors or conditions that may affect efficacy of
the extracts, such as pH and temperature, is also
warranted.
REFERENCES
1. Picazo JJ, Betriu C, Rodriguez-Avial I,

2.

Culebras E, Gomez M, Lopez F; Grupo VIRA,


2006. [Antimicrobial resistance surveillance:
VIRA
STUDY
2006]
Enferm Infecc Microbiol Clin. 24(10):617-28.
Kiffer CR, Mendes C, Oplustil CP, Sampaio
JL, 2007. Antibiotic resistance and trend of
urinary pathogens in general outpatients from a
major urbancity. Int Braz J Urol33(1):42-9.

IJPSR/Vol. II/ Issue II/April-June, 2011/19-24

3.
4.

5.

6.

7.

8.

9.

10.

11.

12.
13.

14.

15.

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Cowan, M. M., 1999. Plant products as


antimicrobial agents. Clin. Microbiol. Rev., 12,
564582.
Kudi, A. C., Umoh, J. U., Eduvie, L. O. and
Gefu, J., 1999. Screening of some Nigerian
medicinal plants for antibacterial activity. J.
Ethnopharmacol., 67, 225228.
Okeke, M. I., Iroegbu, C. U., Eze, E. N., Okoli,
A. S. and Esimone C. O., 2001. Evaluation of
extracts of the root of Landolphia owerrience
for antibacterial activity. J. Ethnopharmacol.,
78,119127.
Yff, B. T. S., Lindsey, K. L., Taylor, M. B.,
Erasmus D. G. and Jager A. K., 2002. The
pharmacological screening of Pentansia
prunelloidesa and the isolation of the
antibacterial compound palmitic acid. J.
Ethnopharmacol., 79, 101107.
Chariandy, C. M., Seaforth, C. E., Phelps, R.
H., Pollard, G. V. and Khambay, B. P., 1999.
Screening of medicinal plants from Trinidad
and Tobago for antimicrobial and insecticidal
properties. J. Ethnopharmacol., 64, 265270.
Perumal Samy, R., Ignacimuthu, S. and Raja,
D. P., 1999. Preliminary screening of
ethnomedicinal plants from India. J.
Ethnopharmacol., 66, 235240.
Ahmad, I. and Beg, A. Z., 2001. Antimicrobial
and phytochemical studies on 45 Indian
medicinal plants against multi-drug resistant
human pathogens. J. Ethnopharmacol., 74,
113123.
Jasmine R, Selvakumar BN and Daisy P,
2007.Saponins from Eugenia jambolan with
Antibacterial Activity Against Beta-Lactamase
Producing
Methicillin
Resistant
Staphylococcus aureus. Research Journal of
Medicinal Plant 1(1): 1-6.
Rastogi R.P., Mehrotra B.
N. (1990):
Compendium of Indian Medicinal Plants.
Vol.1, New Delhi, Central Drug Research,
Lucknow and National Institute of Science
Communication.
Kritikar KR, Basu B.D.(1991) In : Indian
Medicinal Plants. Vol.2 2nd Edn.,Allahabad
Publ. Lalit Mohan Basu.
Sankar V, Kalirajan R, Sweetlin Vivian Sales
F, Raghuraman S (2001): Antiinflammatory
activity of Elephantopus scaber in albino rats.
Indian J. Pharm. Sci.2001; 63 : 523-5.
Reico MC (1989): A review of some
antimicrobial compounds
isolated from
medicinal plants reported in the literature
1978 1988. Phytotherap. Res.3: 117-125.
Carson, C. F., Mee, B. J. and Riley, T. V.,
2002. Mechanism of action of Melaleuca
alternifolia (tee tree) oil on Staphylococcus
aureus determined by time-kill, lysis, leakage,
and salt tolerance assays and electron
microscopy. Antimicrob. Agents Chemother.,
46, 19141920.

International Journal of Pharmaceutical Studies and Research

16. Hammer, K. A., Carson, C. F. and Riley, T. V.,

17.
18.
19.

20.
21.

22.

23.

1999. Influence of organic matter, cations and


surfactants on the antimicrobial activity of
Melaleuca alternifolia (tea tree) oil in vitro. J.
Appl. Microbiol.,86, 446452.
Graham BM, 1978. The development of
Australian legislation for disinfectants. Aust. J.
Hosp.Pharm., 8, 149155.
Maillard, J.Y., 2002. Bacterial target sites for
biocide action. J. Appl. Microbiol. Symposium
Supplement,92, 16S27S.
19. Dodd, C. E. R., Sharman, R. L.,
Bloomfield, S. F., Booth, I. R. and Stewart, G.
S. A. B., 1997. Inimical processes: bacterial
self-destruction and sublethal injury. Trends
Food Sci. Tech., 8,238241.
20. Denyer, S. P. and Stewart, G. S. A. B.,
1998. Mechanism of action of disinfectants.
Int. Biodeter.Biodegr., 41, 261268.
Maria-Litran, T., Alison, D. G. and Gilbert, P.,
2000. Expression of the multiple resistance
operon (mar) during growth of Escherichia
coli as a biofilm. J. Appl. Microbiol., 88, 243
247.
Gorman, S. P. Scott, E. M. and Russell, A. D.,
1980. A review. Antimicrobial activity, uses
and mechanisms of action of glutaraldehyde. J.
Appl. Bacteriol., 48, 161190.
Russell, A. D., Hugo, W. B. and Ayliffe, G. A.
J., 1992. Principles and Practice of
Disinfection, Preservation and Sterilisation,
2nd edn, Oxford, UK: Blackwell Scientific
Publications.

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