Ethnobotanical
Leaflets 12: 1192-97. 2008.
Antimicrobial Activity of Aqueous
Extracts of Bark, Root, Leaves and Fruits of Terminalia arjuna Wight & Arn.
S. Ramya1, T. Kalaivani1, C. Rajasekaran1, P. Jepachanderamohan2,
N.Alaguchamy2, M. Kalayansundaram2 and R.
Jayakumararaj3
1School of
Biotechnology, Chemical and Biomedical Engineering VIT University, Vellore, India 632014
2Department of
Zoology, Raja Duraisingam Government Arts College, Sivagangai, India 630561
3Department of
Botany, RD Government Arts College, Sivagangai, India 630561
Issued
15 December 2008
ABSTRACT
The
present study was carried out to evaluate the antibacterial activity of bark,
stem, root, leaf and fruit extracts from Terminalia arjuna on selected Gram positive and
Gram negative bacterial strains. Aqueous extracts were used to evaluate the
antibacterial activity. Phytochemical extracts from
different parts of the plant exhibited significant anti-bacterial activity
against tested microbial strains; however, inhibitory activities of the
extracts were plant part and test organism dependent. Phytochemical
extracts limited the growth of both Gram-positive and Gram-negative bacterial
species tested however, Micrococcus luteus was less sensitive to the aqueous extracts.
The results show that antimicrobial activity of phytochemical
extracts of T. arjuna
were concentration dependent (1.0 mg/disc and 5.0 mg/disc) on the
bacterial strains tested. Further, the results depicts that bark extracts of T. arjuna could
be used as a potential source of antimicrobial agents against the bacterial
strains tested.
KEY WORDS: Terminalia arjuna; Medicinal
Plant; Antibacterial Activity; Disc Diffusion Assay; Bark/ Stem, Leaf/ Fruit
extracts.
INTRODUCTION
In India, medicinal plants form the backbone of several
indigenous traditional systems of medicine. Pharmacological studies have
acknowledged the value of medicinal plants as potential source of bioactive
compounds (Prusti et al., 2008). Phytochemicals from
medicinal plants serve as lead compounds in drug discovery and design (Ebi and Ofoefule, 2000).
Medicinal plants are rich source of novel drugs that forms the ingredients in
traditional systems of medicine, modern medicines, nutraceuticals,
food supplements, folk medicines, pharmaceutical intermediates, bioactive
principles and lead compounds in synthetic drugs (Ncube,
2008).
WHO, report depicts that more than
80% of world’s population rely on plants based products to meet their health
care needs. Nearly, 25 to 45% of modern prescriptions contain plant derived
lead molecules as a basic source in drug formulations. The value of plant
based prescribed drugs in 1990 was estimated at $15.5 billon which has been
on the raise since then. Furthermore, about 42% of 25 top selling drugs
marketed world wide are either directly obtained from natural sources or
entities derived from plant products (Ramya et al., 2008). Overexploitation of
selected medicinal plant species has led to significant reduction in number
of plants in the wild. Nevertheless, ruthless hunting has resulted in
inclusion of their name in the red data book (Ahmedullah
and Nayar 1999).
In recent years, multiple drug
resistance in both human and plant pathogens has been developed due to
indiscriminate use of synthetic drugs especially in the developing countries
(Hart and Karriuri, 1998). Thus, a diverse arsenal
of new antibacterial agents is urgently needed to combat the diminishing
efficacy of existing antibiotics (Chopra et
al., 1997). To this emerging problem, phytochemicals
obtained from medicinal plants are the sole remedy. This drives the need to
screen medicinal plants for novel bioactive compounds as plant based drugs
are biodegradable, safe and have fewer side effects (Ramya
et al., 2008). The demanding
healthcare needs and the ability to cure disease with fewer side effects are
the driving force behind the resurge of interest world over in the hunt for
elite indigenous germplasm of pharmacological
prominence.
Terminalia arjuna Wight & Arn.,
(family: Combretaceae) popularly known as ‘Arjuna’ (Dwivedi and Udupa, 1989) is a deciduous tree common throughout India. The tree normally grows to a height of 60-90 feet. Traditionally, it has been used as a cardiotonic and has been indicated for derangement of
three humours viz., vata,
pitta and kapha in Ayurveda. Bark of T.
arjuna has been widely used in traditional
system of medicine for a variety of purposes. Ancient Indian physicians used
the powdered bark of T. arjuna for alleviating "hritshool"
(angina) and other cardiovascular conditions (Dwivedi,
2007). Bark powder used as astringent and diuretic finds mention in works of Carak. Bark of T. arjuna has been attributed to possess cardio
protective properties as described by Vagbhatta, in
‘Astang Hridayam’ as
early as 500 CE (Dwivedi, 2007). As of now, in many
of the traditional medicine, alcoholic extract of bark (asava)
is administered with butter (ghrita) or boiled milk
(kshirpak) (Warrier et al., 1996).
Phytochemical
extracts from Terminalia
species have been known for their antioxidant and antimicrobial properties.
They are used in the management of cardiovascular diseases, myocardial
infarction, degenerative neurological diseases, cancer, amyloidosis,
acute pancreatitis, arthritis, atherosclerosis,
inflammatory bowel disease, diabetes, senile dementia, retinal degeneration
and senile cataract particularly in humans owing to their antioxidation
potential (Dwivedi, 2007). Further, it has been
reported that bark of T. arjuna exhibits antioxidant activity only in direct
aqueous extract as determined in vitro by DPPH radical scavenging and deoxyribose damage protection assay and on lipid peroxidation. Ram et
al. (1997) reported that ethanolic extract of T. arjuna bark
at a concentration of 100-500mg/kg significantly reduces total and LDL
cholesterol levels in hypercholesterolaemic
rabbits. Similarly, it has been reported that bark aqueous extract of T. arjuna at
a concentration of 50 mg/kg prevented the rise in liver injury enzymes, SGPT,
ALP and TBARS and increased the levels of SOD, CAT,
and GSH and the results were comparable to
vitamin C group mice (Manna et al., 2006).
Recently,
Devi et al.
(2007), evaluated the effect of methanolic extract
of T. arjuna
on diclofenac sodium induced gastric ulcer in
experimental rats and concluded that extracts of T. arjuna act as gastroprotective
agent due to its free radical scavenging activity and cytoprotective
nature. Studies have shown that bark of T.
arjuna contains glycosides, flavonoids,
tannins and minerals. Flavonoids have been reported
to exert antioxidant, anti-inflammatory and lipid lowering effects while
glycosides are cardiotonic, thus making T. arjuna
unique amongst the medicinal plants (Dwivedi,
2007). Major phytochemicals present in different
parts of T. arjuna
is listed in Table 1 (Dwivedi, 2007).
However,
only little work has been carried out with the phytochemical
extracts as far as the antimicrobial an activity is concerned. Valsaraj et al.
(1997) reviewed the antibacterial activity of some of the Indian medicinal
plants. In a study, Perumalsamy et al., (1998) showed that aqueous
extracts bark of T. arjuna
holds significant antibacterial activity against Escherichia coli, Klebsiella aerogenes, Proteus vulgaris, and Pseudomonas aerogenes.
Recently, Singh et al., (2008) indicated the presence of antibacterial
principles in the bark of T. arjuna with arjunetin
particularly exhibiting selectively higher activity against S. epidermidis. However, not much of work has been done on the
antibacterial activity of root, leaves and fruits
of T. arjuna.
In the present work, we describe antibacterial
activity of aqueous extracts of bark, root, leaves and fruits of T. arjuna.
MATERIALS AND METHODS
Collection of Plant Material
Mature
bark, root, leaves and fruits of T. arjuna were collected from the wild in Vellore District, Tamilnadu, India during Apr – Jun 2008. The Flora of Presidency of Madras (Gamble, 1935) and The Flora of Tamil Nadu Carnatic (Matthew, 1983) were
used for identification and authentication of the plants. Collected material
was washed thoroughly in running tap water, rinsed in distilled water and
shade dried in open air and ground to powder.
Preparation of Phytochemical
Extracts
The
powder was extracted by maceration in double distilled water. The plant
extracts were concentrated using rotary evaporator (Buchi, Switzerland) and stored at 4℃ until used in the
assay.
Test Organisms
Eight
strains of Gram-positive bacteria - Micrococcus glutamicus, Lactobacillus bulgaris, Streptococcus faecalis,
Staphylococcus aureus, Bacillus stearothermophilus,
Staphylococcus pyogenes, Micrococcus luteus, Bacillus cereus and two strains of Gram
negative bacteria - Escherichia coli and Pseudomonas aeruginosa were used to evaluate the
antibacterial activity (Table 2). All bacterial cultures were maintained in
NA slants/ plates; stored at 4℃ and periodically sub-cultured.
Antimicrobial Activity Test
Antimicrobial
activity was tested using a modified disc diffusion assay (DDA) method
originally described by Bauer (1966) and Ncube et al (2008). Plant extracts were dissolved in 20% DMSO
treated water. The inoculums for each microorganism were prepared from broth
cultures (105 CFU/ml). A loop of culture from the NA slant stock
was cultured in LB medium overnight and spread with a sterile swab into
Petri-plates. Sterile disc (6 mm dia, Hi-media, Mumbai, India) impregnated with the plant extracts (1.0 mg/disc and
5.0 mg/disc) were placed on the cultured plates and incubated for 24 h at 37℃. The solvent
loaded disc without extracts in it served as control in the study. The
results were recorded by measuring the zones of growth inhibition. Clear
inhibition zones around discs indicated the presence of antimicrobial
activity. All data on antimicrobial activity were average of triplicate.
RESULTS AND DISCUSSION
Plants are known to have beneficial therapeutic effects
documented in Traditional Indian System of Medicine. Though bioactive
products of Arjuna have been used in treatment of
various aliments since time immemorial, role of phytochemical
in inhibition of growth of microorganisms has gained less prominence (Sasidharan et al.,
1998). In the present study, aqueous extracts of bark/ stem, root, leaves and
fruits of T. arjuna
were tested against selected Gram positive and Gram negative bacterial
species (Table 3). Different extracts of T.
arjuna exhibited significant anti-bacterial
activity against all test organisms. Bark extracts limited the growth of both
Gram-positive and Gram-negative bacterial species tested. However, inhibitory
role of leaf extracts was organism dependent. Aqueous extracts of leaves and
the fruits were active towards the Gram negative strains and less active towards
the Gram positive stains used in the study (Table 3). Among the different
microorganisms tested maximum inhibition was found in E. coli followed by P. aeruginosa, B.
cereus, B. subtilis and L. bulgaris. However,
M. glutamicus
and M. luteus
remained less sensitive to aqueous extracts of T. arjuna. Of the different extracts
tested, bark extract exhibited significantly higher activity towards all the
strains expect M. glutamicus.
However, leaf and fruit extracts were not active towards any of the Gram
positive strains used in the study.
CONCLUSION
Arjuna, the versatile traditional medicinal plant of India, is the rich source of bioactive compounds with diverse
chemical structure. As of now, little work has been done on the biological
activity and plausible medicinal applications of the phytochemical
compounds and hence extensive investigation is needed to exploit the
bioactive principles of Arjuna for therapeutic
utility. In the present study antibacterial activity of T. arjuna extracts towards drug
resistant/ clinically significant microbes has been
investigated. Further investigations may lead to development of new
antibiotic (s) of high potency.
ACKNOWLEDGEMENTS
The
authors are thankful to VIT Management for their constant support and
encouragements. Thanks are due to Prof. Lazar Mathew for his valuable
comments and suggestions to carry out this research successfully.
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Table 1. Major
chemical constituent in different parts of T. arjuna.
Compound
|
Stem/ bark
|
Root
|
Leaf/fruit
|
Triterpenoids
|
Arjunin, Arjunic acid, Arjunolic acid, Arjungenin, Terminic acid
|
Arjunic acid,
Arjunolic acid,
Oleanolic acid,
Terminic acid
|
|
Glycosides
|
Arjunetin,
Arjunaphthanoloside, Arjunoside I, II and Terminoside A
|
Arjunoside I-IV
Glucopyranoside
|
|
Sitosterol
|
Sitosterol
|
Sitosterol
|
|
Flavonoids
|
Arjunolone, Arjunone , Bicalein
Luteolin, Gallic acid, Ethyl gallate
Kempferol, Proanthocyanidins,
Quercetin, Pelorgonidin,
|
|
Luteolin
|
Tannins
|
Pyrocatechols, Casuarinin, Casurin,
Punicallin, Punicalagin, Castalagin,
Terchebulin, Terflavin C,
|
|
|
Trace elements
|
Calcium, Aluminium,
Magnesium,
Silica, Zinc, Copper
|
|
|
(Source: Dwivedi S (2007), Journal of Ethnopharmacology,
114:114-129).
Table 2.
Bacterial strains used in the present study.
S. No
|
Bacterial strain
|
Gram (+/-)
|
1
|
Escherichia coli
|
−
|
2
|
Pseudomonas aeruginosa
|
−
|
3
|
Bacillus cereus
|
+
|
4
|
Bacillus subtilis
|
+
|
5
|
Lactobacillus bulgaris
|
+
|
6
|
Micrococcus glutamicus
|
+
|
7
|
Micrococcus luteus
|
+
|
8
|
Staphylococcus aureus
|
+
|
9
|
Staphylococcus pyogenes
|
+
|
10
|
Streptococcus faecalis
|
+
|
Table 3.
Anti-microbial activity of T. arjuna aqueous extracts.
Organism
|
Stem/ bark
|
Root
|
Leaf/ fruit
|
1 mg/ml
|
5 mg/ml
|
1 mg/ml
|
5 mg/ml
|
1 mg/ml
|
5 mg/ml
|
Escherichia coli
|
+
|
++
|
+
|
++
|
+
|
++
|
Pseudomonas aeruginosa
|
+
|
++
|
+
|
+
|
+
|
++
|
Bacillus cereus
|
+
|
++
|
+
|
+
|
−
|
−
|
Bacillus subtilis
|
+
|
++
|
+
|
+
|
−
|
−
|
Lactobacillus bulgaris
|
+
|
++
|
+
|
+
|
−
|
−
|
Micrococcus glutamicus
|
−
|
−
|
−
|
−
|
−
|
−
|
Micrococcus luteus
|
−
|
+
|
−
|
−
|
−
|
−
|
Staphylococcus aureus
|
−
|
+
|
−
|
+
|
−
|
−
|
Staphylococcus pyogenes
|
−
|
+
|
−
|
+
|
−
|
−
|
Streptococcus faecalis
|
+
|
++
|
+
|
+
|
−
|
−
|
(Growth analysis: ++
=more; + =less; − = no)
|