Evaluation of Antimicrobial activity of Tephrosia procumbens Buch –Ham
Faheem I. Patwekar1*, Sanjeev
Heroor2, Mohsina F. Patwekar1 and
Muhammad Asif3
1Luqman College of Pharmacy, Gulbarga.
2HKES’s
Society’s College of Pharmacy, Gulbarga.
3Allana College of Pharmacy, Pune.
ABSTRACT:
In the present study, the
entire plant of Tephrosia procumbens Buch-Ham commonly known as Vempalli,
Indigo sauvage (Fam-Fabaceae/
Leguminosae) was investigated for the phytochemical screening and antimicrobial property against
certain microorganisms using disc diffusion method. The entire plant of Tephrosia procumbens
were collected, shade dried at room temperature, pulverized and extracted with
95% ethanol in soxhlet extractor to get total ethanolic extract which is further fractionated with the
solvents of different polarities. Total aqueous extract was also obtained by
macerating the shade dried material with 3% of chloroform water I.P. All the
extracts and fractions were subjected for Preliminary Phytochemical
screening, which has shown the presence of Carbohydrates, Steroids and Flavonoids and evaluation of antimicrobial property by
using disc diffusion method on various pathogenic forms of microorganisms
including gram positive, gram negative and fungi. It was found that total ethanolic and total aqueous extract of entire plant of Tephrosia procumbens significantly inhibit the growth of
microorganisms as compared to standard drugs Gentamycin
and streptomycin (Hi-Media Lab. Mumbai).
KEYWORDS: Tephrosia procumbens, Antimicrobial
activity, .
INTRODUCTION:
Tephrosia procumbens Buch-Ham (Fam-Fabaceae/ Leguminosae) [1] commonly known as Vempalli, Indigo sauvage is a
small slender, annual, perennial,
diffused, deep rooted herb with white reddish or pinkish flowers, distributed
throughout the world[1-4]. In India it is found in Punjab and
Rajasthan to West Bengal in Chota Nagpur and in
Peninsular India. It is also found near Khailasa
hills Vishakhapatnam[5]
The other species of Tephrosia viz. T. candida T. villosa, T. lanceolata, T. maxima, T. purpuria
is known for the flavonoidal content and few
other are known for antibacterial properties. [6-8]. A great number of plant medicine contain flavonoids,
which have been reported by many authors as having antibacterial,
anti-inflammatory, antiallergic, antimutagenic,
antiviral, antineoplastic, antithrombotic and vasodilator
actions. [9]
Literature survey revealed that the plant Tephrosia procumbens is
not extensively investigated for its chemical constituents and biological
properties therefore an effort has been made in this direction.
Table 1: Preliminary phytochemical
screening of different extracts and fractions of Tephrosia procumbens Buch-Ham
|
Phytoconstituents |
Total Ethanolic |
Pet. ether fraction |
Butanolic fraction |
Ethyl acetate fraction |
Total aqueous |
|
Carbohydrates |
++ |
- |
+ |
- |
++ |
|
Flavonoids |
++ |
- |
+ |
+++ |
++ |
|
Proteins |
- |
- |
- |
- |
- |
|
Alkaloids |
- |
- |
- |
- |
- |
|
Steroids |
+ |
+++ |
+ |
- |
+ |
|
Saponines |
- |
- |
- |
- |
- |
|
Tannins |
- |
- |
- |
- |
- |
“+++”:
High concentration; “++”: Medium
concentration; “+”: Low
concentration; “-”: constituents
not detected.
Table 2: Antimicrobial activity of
different extracts and fractions of Tephrosia procumbens Buch-Ham
|
Sample |
Concentration |
Mean of diameter of inhibition zone (in
mm) |
||
|
Gram Positive |
Gram negative |
Fungi |
||
|
B.
subtilis |
E.
coli |
C.
utilis |
||
|
Total
ethanolic Extract |
1mg/disc |
22 |
20 |
21 |
|
Pet.ether
fraction |
1mg/disc |
12 |
11 |
11 |
|
n-Butanolic fraction |
1mg/disc |
16 |
15 |
14 |
|
Ethyl
acetate fraction |
1mg/disc |
18 |
17 |
17 |
|
Tatal
aqueous extract |
1mg/disc |
19 |
18 |
19 |
|
DMF |
- |
- |
- |
- |
|
Gentamycin |
10µg/disc |
26 |
27 |
- |
|
Streptomycin |
10µg/disc |
25 |
27 |
- |
|
Flucanazole |
10µg/disc |
- |
- |
24 |
“-” :not applicable , DMF: Dimethy formamide
MATERIALS AND METHODS:
Plant
material collection:
The plant Tephrosia procumbens Buch-Ham
(Fam-Fabaceae/ Leguminosae)
were collected from the rural areas of Dharwad
district in the month of October and were authenticated by Dr. G. R. Hegde, Prof and Head, P.G. Department of studies min Botany
Karnataka University Dharwad India.
Preparation
of extracts:
The entire plant of Tephrosia procumbens were washed with clean water
and shade dried at room temperature, pulverized and exhaustively extracted with
95% ethanol in soxhlet apparatus.
The ethanolic
extract were concentrated in rotary flash evaporator under reduced pressure to
1/10th volume. The concentrated ethanolic
extract was then fractionated with petroleum ether (40-600C), n-Butanol and ethyl acetate in succession. An aqueous extract
was also prepared by maceration method using chloroform-water I.P. for seven days with
occasional shaking. The aqueous extract was concentrated in a rotary flash
evaporator. The residue was dried in a desiccator
over sodium sulfite. Part of the dried
extract is subjected for phytochemical screening.
Phytochemical screening:10-11
The ethanolic
extract and its various fractions that is petroleum ether(40-60ºC), n-Butanol, ethyl acetate and total aqueous extract were
subjected to qualitative chemical analysis to observe the presence and absence
of different phytoconstituents viz. alkaloids,
glycosides,
saponines, flavonoids, steroids
tannins etc. Remaining part of the above extracts were
further dried in a desiccator over sodium sulphite to make a powder. The powder was then autoclaved
at 121ºC and 15lbs pressure. This powder was dissolved in 5% w/v DMSO for
evaluating the antimicrobial activity.
Test
organisms:
For evaluation of antimicrobial activity
three bacterial species each of Gram positive (Bacillus subtilis NCIM 2106), Gram
negative (Escherichia coli NCIM 2065) and fungi (Candida utilis NCIM 3055) were used for
screening of antimicrobial property. The microbial strains were obtained from
National Chemical Laboratory, Pune, India.
ANTIMICROBIAL
ACTIVITY:12-15
Antimicrobial activity
of crude extracts were investigated against bacterial and fungal strains
by disc diffusion method (Murray et.al.1995). Bacteria were cultured overnight
at 37 ºC in Mueller Hinton Broth (MHB) and fungus at 28 ºC for 72hours in
Potato Dextrose Broth (PDB) and used as inoculums. Final inoculums using 100µl
of suspension containing 108 CFU/ml of bacteria, 104 spore/ml of fungus spread
on Mueller Hinton Broth (MHB) and Potato Dextrose Broth (PDB) medium
respectively. The disc (6mm in diameter) was impregnated with 10µl of 100mg/ml
(1mg/disc) extract placed on seeded agar. Gentamycin
(10µg/disc) and streptomycin (10µg/disc) were used as a positive control for
bacteria and Flucanazole (10µg/disc) for fungus. The
test plates were incubated at 37 ºC for 24hours for bacteria and 28 ºC for
72hours for fungi depending on the incubation time required for the visible
growth. MIC values were also studied for microorganisms, which were determined
as sensitive to the extract in disc diffusion assay. The MIC was defined as the
lowest concentration of extract that inhibit visible growth on agar.
Statistical
analysis:
Values are represented as the mean for
triplicate set of experiments.
RESULTS AND DISCUSSION:
Preliminary phytochemical
screening of Tephrosia procumbens showed
the presence of Flavonoids, steroids and
carbohydrates mainly in the total ethanolic and total
aqueous extract. Flavonoids were absent in petroleum
ether fraction, but more concentration of steroid was found in petroleum ether fraction while the more
concentration of Flavonoids was found in ethyl
acetate fraction. The results of preliminary phytochemical
screening are tabulated in Table-1.
The disc diffusion method was used to
determine inhibition zone of Tephrosia procumbens extracts
and various fractions. The plant showed significant antibacterial and
antifungal activity against almost all the pathogenic organisms tested, as
tabulated in Table-2.
Total ethanolic,
total aqueous extract has shown stronger and broader spectrum of antimicrobial
property as compared to vehicle dimethyl formamide (DMF). The activity was found proportionate with
the extract concentration. MIC of this extract is summarized in Table-3.
Amongst the total ethanolic and total aqueous extract, total ethanolic has shown MIC values of more than 50µg/disc and
amongst the fractions of total ethanolic extract, ethyl acetate fraction has shown better
MIC values, while its petroleum ether fraction has shown very low MIC values of
25µg/disc.
Table 3; MIC
values (µg/disc) of different extracts and fractions of Tephrosia procumbens against certain
microorganisms
|
Test sample |
MIC values ( in µg/disc )
against |
||
|
|
Gram positive |
Gram negative |
Fungi |
|
B. subtilis |
E. coli. |
C. utillis |
|
|
Total
ethanolic extract |
>200 |
200 |
>200 |
|
Pet.
ether fraction |
100 |
25 |
100 |
|
n-Butanolic fraction |
100 |
50 |
100 |
|
Ethyl
acetate fraction |
100 |
100 |
50 |
|
Total
aqueous extract |
100 |
200 |
200 |
CONCLUSION:
Total
ethanolic, total aqueous extract of Tephrosia procumbens Buch-Ham has shown stronger and broader spectrum of
antimicrobial property.
So
we conclude that the antimicrobial property is may be due to the presence of flavonoids present in the extract.
ACKNOWLEDGEMENT:
We are very thankful to Prof. Syed Sanaullah, Principal, Luqman College of Pharmacy, Gulbarga
for providing necessary facilities for carrying out the research work.
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Received
on 07.03.2010
Accepted on 22.04.2010
© A&V Publication all right reserved
Research Journal of Pharmacognosy and Phytochemistry.
2(3): May-June 2010, 238-240