Evaluation of
Antibacterial Activity of the Stem Bark of Alstonia
scholaris Linn. R.Br.
Harsha Leena K.*, Kambhoja S. and Vishesh Upadhyay.
Department of Pharmacognosy, The Oxford College of Pharmacy, Hongasandra, Bangalore- 560068, Karnataka.
ABSTRACT:
Alstonia scholaris Linn. R. Br. also called as Devil’s tree
and Dita bark belongs to the family Apocyanaceae. It is a large, ever green tree, found almost
throughout India up to an altitude, 600m. The bark was collected and
authenticated by Dr. Madhava Chetty,
Sri Venkateshwara University, Tirupathi.
The antibacterial activity of the stem bark of Alstonia
scholaris Linn. R. Br. was investigated as there
is a necessity for the search of new antibiotics as the infectious diseases
continue to be the major concern for health institutions, pharmaceutical
companies and Governments all over the world, especially with the current
increasing trends of multidrug resistance among emerging – reemerging bacterial
pathogens to the available modern drug or antibiotics. The plants are the
cheapest and safe alternative source of antimicrobials. The powder of the dried
stem bark was extracted with different solvents in increasing order of the
polarity by soxhelation. Antibacterial activity was
tested against both Gram positive (Staphylococcus aureus,
Bacillus subtilis) and Gram negative (Pseudomonas
aeruginosa, Escherichia coli) organisms
using agar disc diffusion method. The methanol and benzene extracts exhibited
significant antibacterial activity against Gram negative bacteria and ethanol
extract against Gram positive bacteria. Chloroform and acetone extracts
exhibited less activity, while petroleum ether and aqueous extracts showed no
zone of inhibition. The results were
compared with the standard drug, Ampicillin.
KEYWORDS: Alstonia scholaris Linn. R. Br., antibacterial studies, agar
disc diffusion method, Ampicillin.
INTRODUCTION:
Alstonia scholaris
Linn. R. Br. (Apocyanaceae), popularly known as
Saptaparni1 is used in traditional medicine as a tonic, antiperiodic, anthelminthic,
stimulant, carminative, stomachic and expectorant. The bark is a bitter tonic
and febrifuge, useful for the treatment of malaria, diarrhea and desentery. The decoction of the bark is used to treat
asthma, hypertension, lung cancer and pneumonia2, 3.
The literature suggests that the bark mainly contains
alkaloids, steroids and triterpenoids4. The methanolic
extract of the bark showed anti stress, antioxidant and nootropic
activities5. The butanolic fraction of the
methanolic extract of the stem bark was reported to
show the antibacterial activity6. The ethanolic
and aqueous extracts were reported to promote wound healing activity7
where as the ethanolic extract possessed antioxidant
activity8.
Hence the
present work is done to investigate the antibacterial activity of different
extracts of the bark of Alstonia scholaris Linn.
R. Br
MATERIALS AND METHODS:
COLLECTION OF PLANT MATERIAL:
The fresh bark of Alstonia scholaris Linn. R. Br. was collected in
the month of June 2010 and authenticated by Dr. Madhava
Chetty, Sri Venkateshwara
University, Tirupathi.
PREPARATION OF EXTRACTS:
The stem bark of Alstonia scholaris Linn. R. Br. was dried in
shade for two weeks and powdered. The coarse powder (400gm) was subjected to soxhlet extraction with 1.5 ltrs
of various solvents viz., petroleum ether, benzene, chloroform, acetone, methanol,
ethanol and distilled water in the ascending order of polarity. Each time
before extraction with next solvent, the marc was air dried and the extract was
concentrated by distilling the solvent under reduced pressure using rotary
flash evaporator.
The extracts obtained were suspended in dimethyl sulphoxide (DMSO) to
prepare different concentrations ranging from 200 µg/ml to 1200µg/ml and used
for screening the antibacterial activity.
PHYTOCHEMICAL SCREENING:
All the extracts were screened for the presence of
alkaloids, carbohydrates, cardiac glycosides, saponins,
steroids, tannins and triterpenoids.
MICRO-ORGANISMS USED:
Two gram positive bacterial spp. viz., Staphylococcus aureus,
Bacillus subtilis
and two gram negative bacterial spp. viz., Pseudomonas
aeruginosa, Escherichia
coli were used for the antibacterial screening.
PREPARATION OF MEDIA:
The medium was prepared by dissolving nutrient Agar (HiMedia Laboratories Pvt. Ltd.) in distilled water and autoclaving at 1210 C for 15
minutes. It is used for preliminary antibacterial study.
PREPARATION OF INOCULUM:
Stock cultures of Staphylococcus
aureus, Bacillus
subtilis, Pseudomonas
aeruginosa and Escherichia coli were maintained at 40 C on slopes of
nutrient agar. Active cultures for experiment were prepared by transferring a loopful of microorganisms from stock cultures to test tubes
of nutrient broth and incubated for 24 hours at 370 C. The cultures
were diluted with fresh nutrient broth.
ANTIBACTERIAL SUSCEPTIBILITY
TEST:
The disc diffusion method was used to screen the
antibacterial activity9. The nutrient agar plates were prepared by
pouring 100 ml of molten media in to sterile petriplates.
The plates were allowed to solidify and inoculum
suspension was spreaded uniformly with glass
spreader. Different extracts were loaded on 6mm sterile discs till saturation.
The loaded discs were placed on surface of medium and compound was allowed to
diffuse for 5 minutes. The plates were kept for incubation at 370C
for 24 hours. Zone of inhibition formed around the disc was measured
with transparent ruler in mm. These studies were performed in triplicate by
using standard drug, Ampicillin. MIC values were also
determined for microorganisms. MIC was defined as the lowest concentration of
extract that inhibited the visible growth on agar.
CHEMICALS USED:
·
Ampicillin (standard drug)
·
Dimethyl sulphoxide (DMSO)
·
Nutrient
Agar medium
STATISTICAL ANALYSIS:
The values are represented as mean ± standard error of
mean (SEM) for triplicate set of experiments.
RESULTS
AND DISCUSSION:
Preliminary phytochemical screening of the stem bark of
Alstonia scholaris Linn.
R. Br. showed the presence of alkaloids, carbohydrates, cardiac glycosides, saponins, steroids, tannins and triterpenoids.
Results of preliminary phytochemical screening are tabulated in table 1.
The zone of inhibition of different extracts and
standard were recorded in table 2 to 5. Among all the extracts, methanol and
benzene extracts exhibited significant antibacterial activity against gram
negative bacteria and ethanol extract against gram positive bacteria.
Chloroform and acetone extracts exhibited less activity, while petroleum ether
and aqueous extracts showed no zone of inhibition. The antibacterial potency of
the stem bark of Alstonia scholaris Linn.
R. Br. maybe attributed to single or the combined effect of the phytoconstituents present in the bark.
Table 1: Preliminary
phytochemical evaluation of the stem bark of Alstonia scholaris Linn. R. Br.
|
Phytoconstituents
|
Petroleum ether extract |
Benzene extract |
Chloroform extract |
Acetone extract |
Methanol extract
|
Ethanol extract |
Aqueous extract |
|
Alkaloids |
- |
- |
+ |
+ |
+ |
+ |
+ |
|
Carbohydrates |
- |
- |
- |
- |
- |
- |
+ |
|
Cardiac glycosides |
- |
- |
+ |
+ |
+ |
+ |
+ |
|
Saponins
|
- |
- |
- |
- |
- |
- |
+ |
|
Steroids |
+ |
+ |
+ |
+ |
+ |
+ |
- |
|
Tannins |
- |
- |
- |
- |
- |
+ |
+ |
|
Triterpenoids |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
‘+’ : Present; ‘-’ : Absent
Table 2: Antibacterial
activity of different extracts of the stem bark of Alstonia scholaris Linn. R. Br. against gram
positive bacteria.
|
|
Zone of inhibition (mm) |
|||||||||||
|
Gram positive bacteria |
||||||||||||
|
Staphylococcus
aureus |
Bacillus
subtilis |
|||||||||||
|
Concentration of extract (µg/ml) |
200 |
400 |
600 |
800 |
1000 |
1200 |
200 |
400 |
600 |
800 |
1000 |
1200 |
|
Benzene extract |
0±0 |
8.3± 0.88 |
7.6± 0.88 |
7.83± 0.44 |
10.16± 0.44 |
10.3± 0.88 |
0±0 |
0±0 |
7.6± 0.3 |
8± 0.577 |
10.6± 0.88 |
10± 0.577 |
|
Chloroform extract |
0±0 |
0±0 |
0±0 |
7.3± 0.6 |
8± 0.577 |
10± 0.557 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
|
Acetone extract |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
7.6± 0.88 |
8.3± 0.3 |
8± 0.577 |
|
Methanol extract |
0±0 |
0±0 |
0±0 |
0±0 |
11.6± 0.88 |
16.3± 1.453 |
0±0 |
0±0 |
0±0 |
0±0 |
8.3± 1.453 |
10.3± 1.453 |
|
Ethanol extract |
7.3± 0.6 |
8.3± 0.3 |
8± 0.577 |
11± 0.577 |
11.6± 0.3 |
14.3± 0.3 |
7.3± 0.6 |
8.3± 0.88 |
9.83± 1.66 |
10.16± 0.7265 |
12.5± 0.288 |
15.6 0.88 |
Table 3: Antibacterial
activity of different extracts of the stem bark of Alstonia scholaris Linn. R. Br. against gram
negative bacteria.
|
|
Zone of inhibition (mm) |
|||||||||||
|
Gram negative bacteria |
||||||||||||
|
Pseudomonas
aeruginosa |
Escherichia
coli |
|||||||||||
|
Concentration of extract (µg/ml) |
200 |
400 |
600 |
800 |
1000 |
1200 |
200 |
400 |
600 |
800 |
1000 |
1200 |
|
Benzene extract |
0±0 |
7.6± |
7.3± 0.6 |
10± 0.577 |
11.6± 0.88 |
12.3± 1.453 |
0±0 |
7.6± 0.3 |
8.3± 0.3 |
8± 1.154 |
10± 1.154 |
16.3± 1.453 |
|
Chloroform extract |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
|
Acetone extract |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
|
Methanol extract |
7.6± |
7.3± 0.6 |
8± 0.577 |
9.6± 0.88 |
10± 0.577 |
16.3± 1.453 |
0±0 |
7.6± 0.88 |
12± 0.577 |
14± 0.577 |
13.3± 0.66 |
15.6± 0.3 |
|
Ethanol extract |
0±0 |
7.83± 0.44 |
7.6± 0.60 |
8.16± 0.726 |
8± 0.577 |
10.16± 0.44 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
0±0 |
Table 4: Antibacterial
activity of the standard drug, Ampicillin.
|
|
Zone of inhibition (mm) |
||||
|
Gram positive bacteria |
Gram negative bacteria |
||||
|
Staphylococcus
aureus |
Bacillus
subtilis |
Pseudomonas
aeruginosa |
Escherichia
coli |
||
|
Ampicillin |
20µg/ml |
15.6±0.88 |
10.3±1.453 |
28.3±0.3 |
37.6±0.3 |
|
40µg/ml |
14.3±2.027 |
14.16±0.7265s |
31.6±0.88 |
42.6±1.20 |
|
|
DMSO (control) |
0 |
0 |
0 |
0 |
|
Table 5: Minimum inhibitory
concentration (MIC) values of different extracts of the stem bark of Alstonia scholaris Linn.
R. Br.
|
|
MIC value (in µg/disc) against |
|||
|
Gram positive bacteria |
Gram negative bacteria |
|||
|
Staphylococcus
aureus |
Bacillus
subtilis |
Pseudomonas
aeruginosa |
Escherichia
coli |
|
|
Benzene extract |
400 |
600 |
400 |
400 |
|
Chloroform extract |
800 |
- |
- |
- |
|
Acetone extract |
- |
800 |
- |
- |
|
Methanol extract |
1000 |
1000 |
200 |
400 |
|
Ethanol extract |
200 |
200 |
400 |
- |
CONCLUSION:
The findings in the present study offer a scientific
support to the use of stem bark of Alstonia scholaris Linn. R. Br. as an antibacterial in new drugs
for therapy as it showed significant antibacterial activity compared to
standard drug, Ampicillin.
ACKNOWLEDGEMENT:
The authors are thankful to the Chairman, Director and Principal,
The Oxford College of Pharmacy, Bangalore for providing necessary facilities
and support to conduct this work and Dr. Madhava Chetty, Sri Venkateshwara
University, for providing the authenticated drug material.
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Received on 01.11.2010
Accepted on 03.12.2010
© A&V Publication all right reserved
Research Journal of Pharmacognosy and
Phytochemistry. 3(4): July- August 2011, 180-183