Friday, February 17, 2012

Antibacterial activity of Vallarai Chooranam against Human Pathogens

International Journal of PharmTech Research
CODEN (USA): IJPRIF        ISSN : 0974-4304
                                                                                                              Vol.4, No.1, pp 162-168,          Jan-Mar 2012
Antibacterial activity of Vallarai Chooranam
against Human Pathogens
, S.Sivakumar
, K.Sangeetha
Department of Biotechnology, Faculty of Science and Humanities, SRM University,
Department of Gunapadam , National institute of Siddha, Tambaram Sanitorium,
*Corres. Author:
Mobile : +919841404193
Abstract:Vallarai chooranam is a polyherb,comprised of 10 different herbs,used for treatment of diabetics,urinary
tract infection,leucorrhea,veneral disesase and also to improve memory power.The present investigation was
carried out to study the unexplored area of the Vallarai  chooranam towards their antibacterial activity against both
Gram positive and Gram negative organisms by Disc diffusion method.Aqueous and solvent extracts of the
chooranam were tested against selected human pathogens viz. S.aureus, P.aeruginosa, B.subtilis ,K.pneumoniae
and E.coli.   B o t h   t h e   e x t r a c t s   w e r e   f o u n d   t o   b e   m o r e   e f f e c t i v e   a g a i n s t   a l l   t h e   t e s t   p a t h o g e n s .B.Subtilis was more
susceptible to the aqueous extracts among the tested organisms.The results of antibacterial activity revealed that
both the extracts exhibited good inhibitory activity against all test pathogens.The presence of phytochemicals in the
formulation were also assayed.The results showed positive response for significant secondary metabolites.
Key words: Polyherbal, antibacterial,Qualitative analysis,Aqueous extract,Solvent extract,Vallarai Choornam.
Herbal medicines derived from plant extracts
are being increasingly utilized to treat a wide variety of
clinical diseases, mainly in developing countries, for
p r i m a r y   h e a l t h   c a r e   b e c a u s e   o f   b e t t e r   c u l t u r a l
acceptability, better compatibility with the human
body and fewer side effects. Nowadays multiple drug
resistance has developed due to the indiscriminate use
of commercial antimicrobial drugs commonly used in
the treatment of infectious disease
. In addition to this
problem, antibiotics are sometimes associated with
adverse effects on the host including hypersensitivity,
immune-suppression and allergic reactions
It has been reported that there has been an
alarming increase in number of diseases and disorders
caused by synthetic drugs prompting a switch over to
traditional herbal medicine
.The earliest record of
human civilization and culture of China, Egypt,
Assyria, and Indies valley reveals that the elders and
w i s e   m e n   o f   t h o s e   t i m e s   u s e d   h e r b s   t o   t r e a t   v a r i o u s
diseases. Many of the indigenous medicinal plants are
used as spices for cooking
.The ancient use of plants
for healing purposes forms the origin of much of
modern medicine. Many traditional drugs originate
from plant sources: a century ago, most of the effective
drugs were plant based
It is estimated that there are 250,000 to 500,000
species of higher plants on earth. But relatively smallT.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 163
percentage (5-15%) has been systematically
investigated for the presence of bioactive compounds
Microorganisms are the causative agents of almost all
kinds of acute and chronic diseases.
Plants based antimicrobials have enormous therapeutic
potential. They are effective in the treatment of
infectious diseases while simultaneously mitigating
m a n y   o f   t h e   s i d e   e f f e c t s   t h a t   a r e   o f t e n   a s s o c i a t e d   w i t h
synthetic antimicrobials.The use of plant extracts with
known antimicrobial properties, can be of great
significance in therapeutic treatments.Although
hundreds of plant species have been tested for
antimicrobial properties, the vast majority of them
have not been adequately evaluated
.Many efforts have
been made to discover new antimicrobial compounds
from various kinds of sources such as micro-organisms,
animals,and plants.One of such resources is folk
medicines. Systematic screening of them results in the
discovery of novel effective compounds
increasing prevalence of multidrug resistant strains of
bacteria and the recent appearance of strains with
reduced susceptibility to antibiotics raises the specter
of untreatable bacterial infections and adds urgency to
the search for new infection-fighting
.Considering the vast potentiality of plants
as sources for antimicrobial drugs with reference to
antibacterial agent, a systematic investigation was
undertaken to screen the polyherbal formulation
Vallarai chooranam for its antibacterial activity
against selected human pathogens.
 This polyherbal formulation is a composition
of 10 different herbs viz.,(In tamil) Vallarai, Jaadhikai,
asikkai,Kadukkai,Nellikai,Thaantrikkai.(Table 1).The
powder form of this sidhha  Chooranam is used to treat
diabetics,Urinary tract infection,Leucorrhea,Veneral
disesase and also used to improve memory power and
blood purification
.The use of alchocol or water as
solvent  is efficient in extracting a wide variety of
active components. Therefore, we analysed the
Vallarai chooranam for the presence of
phytochemicals and evaluated the antimicrobial
activity against selected human pathogens.
Materials and Methods:
Preparation of Formulation:
The ingredients were procured from reputed
commercial Siddha supplier (Dr.Sivakumar and
Dr.Juliet, Selaiyur.Chennai) and authenticated [Table
1]. All the ingredients were shade dried, powdered and
mixed thoroughly in same proportion.The mixture was
further boiled in distilled water at 100ºC for 60
minutes and filtered.The filtrate was evaporated to
dryness, used for subsequent experiments and they are
designated as Chooranam since they are comprised of
multiple herbs.
Solvent extract preparation:
1 0   g r a m s   o f   a i r   d r i e d Vallarai chooranam
powder was extracted with 100ml of Organic solvent
(Ethanol) and kept on rotary shaker at 190-220 rpm for
24 hours.The supernatant was collected and solvent
was evaporated to make the final volume one – fourth
of the original volume and stored at 4
C in air tight
Aqueous extract preparation:
The aqueous extract is prepared by soaking
100grams of Vallarai chooranam powder in 200 ml of
distilled water for 12hours.The extracts were filtered
using Whatman filter paper (125 mm).
Table 1:Polyherbal formulation of Vallarai chooranam
S.No Siddha Name Botanical Name Quantity Family Name
1 Vallarai Centella asiatica 50gms Mackinlayaceae
2 Jaadhikai Myristica fragrans 10gms Myristicaceae
3 Jaadhipathiri Myristica fragrans 10gms Myristicaceae
4 Lawangam Syzgium aromaticum 10gms Myrtaceae
5 Elakkai Eletteria cardamomum 10gms Zingiberaceae
6 Thaaleesa pathiri Taxus beccata 10gms Taxaceae
7 Maasikkai Quercus infectoris 10gms Fagaceae
8 Nellikai Emblica officinalis 10gms Phyllanthaceae
9 Kadukkai Terminalia chebula 10gms Combretaceae
10 Thaantrikkai Terminalia belerica 10gms CombretaceaeT.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 164
Phytochemical screening :
The qualitative tests were carried out in both
the extract of Vallarai chooranam using standard
.Both the extacts were analysed for the
presence of significant secondary metabolites viz
Alkaloids, Tannins, Flavonoids ,Cardiac glycosides,
Steroids and Saponins.
Growth and Maintenance of Test
Microorganism for Antimicrobial Studies:
Bacterial cultures of Bacillus subtilis (B.
subtilis), Escherichia coli (E. coli), Pseudomonas
aeruginosa (P.aeruginosa),Staphylococcus aureus (S.
aureus) and Klebsiella pneumoniae
(K.pneumoniae)were obtained from  Department of
Microbiology, SRM Medical College, India, and the
studies were performed at Department of
Biotechnology,FSH,SRM University.The bacteria
were maintained on Nutrient Agar(NA) slants at
4°C.For further study, cultures have been grown in
Nutrient Broth (NB) for 24hrs as overnight cultures.
Disc diffusion Method:
 The antibacterial assay of aqueous and
ethanolic extracts was performed by Disc diffusion
. T h e   N u t r i e n t   a g a r   m e d i a   ( 2 0 m l )   w a s   p o u r e d
into sterilized petri dishes and left to solidify at room
temperature. The overnight bacterial cultures have
b e e n   s p r e a d   p l a t e d   o n   t h e s e   p e t r i d i s h e s   u s i n g   s t e r i l e   L
rod.Taking the crude extract concentration as
100%,different concentration of aqueous and ethanolic
extracts was prepared viz., 20%,40%, 60%, and 80%.
W h a t t m a n ’ s   N o . 1   f i l t e r   p a p e r   d i s c s   ( 3 m m )   w e r e
soaked in 0.1 ml of Ethanol extract of varying
concentrations  from 20% -80%.The similar procedure
was carried out for aqueous extract with varying
concentration from 20% -80% simultaneously.
T h e   f i l t e r   p a p e r   d i s c s   w e r e   p l a c e d
equidistantly on inoculated media and diffusion of
solution was allowed to occur for 30 minutes at room
t e m p e r a t u r e .   P l a t e s   w e r e   i n c u b a t e d   a t   3 7˚C   f o r   2 4
hours. The average zone of inhibition was recorded.
Sterile distilled water and Ethanol were maintained as
control. The diameters of the inhibition zones were
measured in mm.
Table 2 :Antibacterial screening of the extract showing the range of zone of inhibition (mm)
Table 3 :Zone of inhibition of Ethanolic extract (mm)
 Zone of Inhibition (mm) by varying concentration(%)
Test Organism 20% 40% 60% 80%
E.coli 3.12±0.12 6.22±0.15 8.27±0.13 11.22±0.12
S.aureus 6.53±0.11 10.16±0.10 11.13±0.16 12.15±0.19
P.aeruginosa 2.32±0.10 3.85±0.17 5.61±0.13 7.16±0.18
B.subtilis 7.21±0.13 9.45±0.20 11.33±0.19 14.33±0.16
K.pneumoniae 4.33±0.20 6.27±0.14 9.13±0.10 12.33±0.12
E.coli 3 to 11 2 to 11
S.aureus 6 to 12 3 to 10
  P.aeruginosa 2 to 7 2  to  6
B.subtilis 7 to 14 7  to 14
K.pneumoniae 4 to 12 4 to   9T.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 165
Ethanolic extract Zone of inhibition
20 40 60 80
P e r c e n ta g e   o f   e th a n o l i c   Ex t r a c t
Inhibition (mm)
E . c o l i S . a u r e  u s P . a e  ru  g i  n  o s a
B . s u b t i l i s K . p n e  u m o n i a e
Graph 1:
Table 4 : Zone of inhibition by Aqueous extract (mm)
 Zone of Inhibition (mm) by varying concentration(%)
Test Organism 20% 40% 60% 80%
E.coli 2.75± 0.11 4.51±0.14 6.13±0.13 11.24±0.16
S.aureus 3.87 ± 0.14 5.31±0.15 9.19±0.11 10.16±0.17
P.aeruginosa 2.37 ± 0.20 4.22±0.12 5.31±0.12 6.15±0.18
B.subtilis 7.62±0.15 8.32±0.12 10.14±0.14 14.31±0.20
K.pneumoniae 4.53±0.14 5.12±0.15 7.16±0.13 9.56±0.15
Aqueous  extrac t  Zone  of  inhibition
20 40 60 80
Percentage of Aqueous Extract
Inhibition in mm
E.coli S.aureus P.aeruginosa
B.subtilis K.pneumoniaeT.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 166
Comparision of Antibacterial activity of Ethanolic and
Aqueous extract
20E 20A 40E 40A 60E 60A 80E 80A
Percentage of Extracts %
E.coli S.aureus P.aeruginosa
Table 5: Phytochemical screening results of
Vallarai Chooranam
Compound Result
Alkaloids +
Cardiac glycosides +
Flavonoids +
Steroids +
Saponins +
Tannins +
+  =  Positive
Results :
Antimicrobial activity results obtained in the
present study revealed  that the tested extracts posses
potential antibacterial activity against B. subtilis, S.
aureus,E. coli,K.pneumoniae ,P.auregenosa (Table
2).The diameters of the inhibition zones against all the
tested bacteria were measured in mm. The results
showed that increase in concentration of extract
increased the zone of inhibition .When tested by the
disc diffusion method, the Ethanolic extracts of
Vallarai Chooranam showed significant activity
against S.aureus, K.pneumoniae  around 12mm.The
zone of inhibition for E.coli was observed as 11
mm.The highest antibacterial  activity of 14 mm was
observed in B. subtilis and least activity was  recorded
in P.auergenosa  o f   7 m m.The range of zone of
inhibition by ethanolic extracts against pathogens were
in the following order of higher to
lower,viz.,B.subtilis,S.aureus, E.coli, K.pneumoniae,
The antimicrobial results for aqueous extract
of Vallarai Chooranam showed maximum activity
against B. subtilis o f     1 4   m m   a n d     l e a s t   a c t i v i t y
observed in P.aueregenosa of 6 mm .Inhibitory
activity against E.coli. S.aureus, K.pneumoniae was
around 9-11mm ( T a b l e   4   &   G r a p h   2 ) . T h e   r e s u l t s
elucidated that the activity was higher against Gram
+ve strains than Gram –ve pathogens.In classifying
the antibacterial activity as Gram-Positive or Gramnegative, it would generally be expected that a much
greater number would be active against Gram-positive
than Gram-negative bacteria
, however, in our study
b o t h   G r a m   p o s i t i v e   a n d   G r a m   n e g a t i v e   b a c t e r i a   w e r e
inhibited by the extracts of the Vallarai Chooranam.
On comparison of both the extracts the activity against
pathogens showed similar results with effective and
significant inhibitory action(Graph 3).The aqueous and
s o l v e n t     e x t r a c t s   w e r e   u s e d   t o   i d e n t i f y   t h e   p r e s e n c e   o f
various phytochemicals .Standard test for Flavonoids,
Alkaloids, Saponins, Cardiac glycosides, Tannins and
Steroids showed positive response(Table 5 ).
Ayurveda is a traditional Indian Medicinal
System practiced for thousands of years. Considerable
research on pharmacognosy, chemistry, pharmacology
and clinical therapeutics has been carried out on
ayurvedic medicinal plants. The polyherbal
formulations described in Ayurveda have been the
basis of treatment of various human diseases.
Biological evaluation of herbal formulations based on
their  medicinal uses forms the basis for development
of new drugs from plants
. Historically, plants have
provided a source of inspiration for novel drug
compounds,as plant derived medicine have made large
contribution to human health and well-being.
The combination of multiple herbs has
increased the efficacy of the Vallarai Chooranam to a
greater extent, since they possess medicinal values
exerted by the presence of phytochemicals.Flavonoids
a r e   k n o w n   t o   b e   s y n t h e s i z e d   b y   p l a n t s   i n   r e s p o n s e   t o
microbial infection.Hence it should not be surprising
that they have been found to be effective as
antibacterial substances against a wide array of
infectious agents
.Tannins (commonly referred to as
tannic acid) are also known as antimicrobial
agents.They are water-soluble polyphenols and
precipitated proteins present in many plant foods.T.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 167
Tannins have been reported to prevent the
development of microorganisms by precipitating
microbial protein.The growth of many fungi, yeasts,
bacteria, and viruses were inhibited by this Tannins.
According to World Health Report on
infectious diseases 2000, overcoming antibiotic
r e s i s t a n c e   i s   t h e   m a j o r   i s s u e   o f   t h e   W H O   f o r   t h e   n e x t
millennium.Hence,the last decade witnessed an
increase in the investigations on plants as a source of
human disease management
and not many reports are
available on the exploitation of plants for
the management of plant diseases
.This is mainly due
to lack of information on the screening and evaluation
of diverse plants for their antibacterial
potential.Therefore, in the present investigation of
Vallarai Chooranam,a multiherbal formulation was
evaluated for its antibacterial potential for the first
time against selected human pathogenic bacteria which
are known to cause many infectious diseases.
In the present investigation Vallarai
Chooranam was evaluated for its antibacterial potential
for the first time against selected human pathogenic
bacteria which are known to cause many infectious
diseases and also the phytoanalysis revealed the
presence of medicinally active constituents. The
antibacterial activity of this polyherbal formulation
would help for development of a new alternative
medicine system which has no side effects. Both the
extracts of this formulation possess a broad spectrum
of activity and open the possibility of finding new
clinically effective antimicrobial compounds.Hence
the study has provided  biochemical basis for
ethanopharmacological claims of the Chooaranam in
treatment and prevention of various diseases and
1) Ahmad I, Mehmood Z, Mohammad F.Screening
of some Indian medicinal plants for their
antimicrobial properties. J Ethnopharmacol 62:
183-193, 1998. 3
2) C l a r k   A M   N a t u r a l   p r o d u c t s   a s   r e s o u r c e   f o r   n e w
drugs. Pharm Res 13: 1133-1141
3) Ghule ST, Patil DK. Kisan World. 2001; 28: 33-
4) Okwu DE , Phytochemicals and vitamin content
of indigenous spices of South   Eastern Nigeria.
J. Sustain. Agric. Environ. 6: 30-34.
5) Andrew Vickers, Herbal medicine:  W e s t   J   M e d .
2001 August; 175(2): 125–128.
6) Taylor, L. (2000), Plant Based Drugs and
Medicine, Raintee Nutrition Inc.,pp. 1-5.
7) B a l a n d r i n ,   M . F . ,   J . A .   K l o c k e ,   E . S .   W u r t e l e   a n d
2006. Phytochemical analysis and   antibacterial
W.H. Bollinger, 1985. My. Science., 1: 72-78.
Science, 228: 1154-1160
8) Tomoko.N. Takashi A., Hiromo T., Yuka I.,
(2002): Antibacterial activity of    extracts
preparated from tropical and subtropical plants
on methicillin-resistant Staphylococcus aureus. J.
Health Sci., 48: 273–276
9) Sieradzki K., Roberts R.B.., . (1999):
Thedevelopment of vancomycin resistance  ina
patient with methicillin-resistantStaphylococcus
aureus infection. N. Engl.J. Med., 340: 517–523.
10) Winston, D., Maimes, S., Adaptogens: Herbs
F o r   S t r e n g t h ,   S t a m i n a ,   a n d   S t r e s s   R e l i e f ,   2 0 0 7 ,
pp. 226-7
11) Harborne JB;Phytochemical Methods: A Guide
To Modern Technique of Plant Analysis.3rd
ed .London, England: Chapman and Hall;
12) H . O .   E d e o g a ,   D .   E .   O k w u ,     P h y t o c h e m i c a l
constituents of some Nigerian medicinal Plants;
African Journal of Biotechnology Vol. 4 (7), pp.
685-688 July 2005
13) Sofowora AE (1993). Medicinal Plants and
Traditional Medicines inAfrica.. 2nd edition.
Spectrum Books, Ibadan, Nigeria. p. 289.
14) Trease E.G., Evans W.C 1978. Pharmacognosy.
11th Edition, Balliere Tindall, London. 115-222.
15) Harborne JB;Phytochemical Methods: A Guide
To Modern Technique of Plant Analysis.3rd
ed .London, England: Chapman and Hall;
16) Bauer, A.W., Kirby, W.M.M., Sherris, J.C.,
Turck, M., 1966. Antibiotic susceptibility testing
by a standardized single disk method. Am. J.
Clin. Pathol. 45: 493-496.
17) McCutcheon AR, Roberts TE, Gibbons E, Ellis
S M ,   B a b i u k   L A ,   H a n c o c k   R E W ,   T o w e r s   G H N
(1995). Antiviral screening of British Columbian
medicinal plants. J. Ethnopharmacol. 49: 101-
110.T.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 168
18) Krishnan Kannabiran; Antibacterial activity of
saponin isolated from the leaves of  Solanum
trilobatum Linn; Journal of Pharmacy
Research, Vol 2, No 2 (2009)
19) Jasmine. R, Daisy.P and Selvekumar. B.N., 2007.
In vitro Efficacy of Flavonoids from Eugenia
jambolana Seeds Against ESL-Producing
Multidrug-Resistant Enteric Bacteria. Research
Journal of Microbiology. 2 (4):369–374.
20) P r a s a d ,   N .   R . , e t   a l . ,   2 0 0 8 .   P r e l i m i n a r y
phytochemical screening and antimicrobial
activity of Samanea saman . Journal of
Medicinal Plants Research. 2: 268-270.
21) Aiyelaagbe et al. 2000. Fitoterapia. 72: 544–546.
22) Satish S., Raveesha K. A. and Janardhana G. R.
1999. Lett. Applied Microbiol.28: 145–147.

No comments: