Study of Antimicrobial Activity of Canarium strictum Gum
Resin
P.B. Suruse *,
N.J. Duragkar, U.D. Shivhare
and S.B. Bodele
Sharad Pawar College of Pharmacy, Wanadongri,
Hingna Road, Nagpur-441100 (M. S.) INDIA.
ABSTRACT:
Canarium
strictum Roxb. (Burseraceae) is
used in the traditional Ayurvedic medicine under the name Raladhupa
and black dammar resin. The isolation of compound A and compound B were carried
out from chloroform extract by counter current distribution method. The purity
of each compound was estimated by using Thin Layer Chromatography. The preliminary phytochemical
screening of each compounds showed the presence of triterpenoid.
The compound A and Compound B were investigated for
antimicrobial (antibacterial and antifungal) activity by using cup plate method or diffusion agar method. The results obtained shows
that compound A and compound B possess broad-spectrum antimicrobial activity at
concentration of 100µg/ml. The inhibitory effect of each compound is very close
and identical in magnitude for Gram- positive, Gram-negative bacteria and
fungi.
KEYWORDS: Canarium strictum, Raladhupa, Black dammar resin, Antimicrobial activity.
INTRODUCTION:
Canarium
strictum Roxb. (Burseraceae),
known as Kala Dammar in Ayurveda. The gum resin used
with gingelly oil in rheumatic pains. The gum resin
used as plaster and ointment, and as a substitute for burgundy pitch in making
plasters etc. It was useful as an ointment in chronic skin diseases such as
psoriasis and pityriasis. Decoction or powder of the
resin was given orally as a remedy for rheumatism, cough, fever, epilepsy,
asthma, syphilis, blood impurities, various poisons, hernia, chronic skin
diseases, haemorrage and to improve complexion1.
The phytochemical investigation and knowledge of the
biological activities and or chemical constituents of plant is desirable not
only for the discovery of new therapeutic agents but because such information
may be of value in disclosing new sources of such economic material for the
synthesis of complex chemical substance. Also a novel chemical structure
isolated from the plant source often prompts the chemist to a successful series
of modified semi synthetic compounds, which may have some or more potent
medicinal and economical value. Sometimes derivatives made from isolated
compounds have more potent activity than parent molecule2. Herein,
we have attempted extraction, isolation and identification of antimicrobial triterpenoids from Canarium strictum gum resin.
MATERIAL AND METHODS:
The Canarium strictum gum resin was gifted by Rajesh
Chemicals Private Limited Mumbai and all the chemicals used in the study were
of Analytical Reagent grade.
Isolation of triterpenoids
from Canarium strictum:
Canarium strictum gum resin was defatted with petroleum ether at 60-80oC
and extraction of defatted material by using chloroform form a resinous
extract. After concentrating that extract fractionation was done by using
acetone and methanol (1:1) formation of two compounds. Compound A is insoluble in acetone and methanol, which is settled
at bottom and after recrystallization formation of
cream colored compound. Compound B
is soluble in acetone and methanol, which is separated by
concentrating the solvent; it gives yellow colored crystalline compound. Thin
Layer Chromatography (TLC) checked the purity of each compound and Rf values were noted3-4. The
results of TLC are shown in Table 1.
Table 1: Thin Layer
Chromatography study of compound A and compound B
Test compound |
Mobile phase |
No. of spots |
Rf value |
Compound A |
Ethyl acetate : Acetone
(4:6) |
1 |
0.60 |
Compound B |
Ethyl acetate : Acetone
(4:6) |
1 |
0.75 |
Preliminary phytochemical
screening of isolated compounds:5
The preliminary phytochemical screening for the detection of various
chemical constituents and their results are shown in Table 2.
Screening of antimicrobial activity by cup plate method
or diffusion agar method:6-8
The antimicrobial activity
of compound A and compound B have
been studied against bacteria, Gram-negative E. coli and K. aerogens
and Gram-positive S. aureus and B. substilis
and fungi, C. albicans and A. niger by agar plate method. For antibacterial activity
ciprofloxacin was used as standard, while for antifungal activity clotrimazol was used as the standards. This method depends
on the diffusion of drug from cup through the solidified agar layer of a petridish to an extent such that growth of the inoculated
microorganism is prevented entirely in a circular area “zone” around the cup
containing the solution of the compound under test. The medium was sterilized by autoclaving at 15 lb pressure for 30
min. One loopful of the stock culture was inoculated
at 10 ml of agar slant previously in sterilized test tubes, and incubated at 37oC
for 24 hr and 20oC for 48 hr to 7 days respectively for bacteria and
fungi. About 3 ml of distilled water was added to the test tube and a suspension
of the culture was obtained by shaking for few minutes. The solutions of all compounds were made by dissolve in minimum
amount of ethanol, and volume was making up with sterilized water to produce a
concentration of 100µg/ml.
Procedure:
All the operations were
carried out under aseptic conditions. Respective sterile medium was melted on
water bath and kept at 45oC in constant temperature water bath. In
each sterile petridish 25 ml of molten medium was
added and 107/ml of subcultured organism
under study were inoculated. The culture and agar
medium were mixed and allow solidifying. Four cups of 8 mm diameter was then
made with the help of sterile stainless steel cork borer. Two drops of test
solution was added to each cup. Solution was allowed to diffuse in the medium
for 2 hr by keeping the petridish at room temperature
and then incubated for about 24 hr at 37oC (for bacteria) and 48 hr
to 7 days at 20oC (for fungi). The results are shown in Figure 1-4
and Table 3.
Table 2: Preliminary phytochemical screening of compound A and compound B
Tests |
Test performed and
reagents |
Compounds |
|
Compound
A |
Compound B |
||
Test for Sterols |
Salkowaski Reaction |
+ |
+ |
Liebermann’s Reaction |
+ |
+ |
|
Liebermann- Burchard Reaction |
+ |
+ |
|
Test for triterpenoids |
Salkowski test |
+ |
+ |
Table 3: Antimicrobial
activity data of compound A and compound B
Test Compound |
Zone of inhibition (mm) |
|||||
Antibacterial Activity |
Antifungal Activity |
|||||
E. coli |
K. aerogens |
S. aureus |
B. substilis |
C. albicans |
A. niger |
|
Compound A |
28 (0.82) |
18 (0.82) |
26 (0.74) |
17 (0.59) |
22 (1.04) |
21 (0.84) |
Compound B |
21 (0.61) |
20 (0.90) |
22 (0.63) |
21 (0.72) |
23 (1.1) |
19 (0.76) |
Standard |
34 |
22 |
35 |
29 |
23 |
24 |
(Activity index) = Inhibition zone of
the sample / Inhibition zone of the standard; For antibacterial activity : Standard =
Ciprofloxacin; For antifungal activity : Standard = Clotrimazol
RESULTS
AND DISSCUSSION:
The work presented here deals with extraction,
isolation and evaluation of antimicrobial activity of compounds from Canarium strictum gum
resin. Soxhlet extraction of Canarium strictum gum resin with petroleum ether
60-80°C and chloroform were carried out. Isolation of compound A and compound B
were carried out by counter current distribution phenomenon. The preliminary phytochemical screening of the compounds obtained was done
in view to know the various classes of chemical constituents
i.e. primary and secondary metabolites. Compound A and compound B answered
positive salkowski test for triterpenoids.
The antimicrobial activity of Compound A and Compound B were done by using cup
plate technique (Diffusion Agar Method) and results tabulated accordingly. The
results obtained shows that compound A and compound B possess broad-spectrum
antimicrobial activity at concentration of 100µg/ml. The inhibitory effect of
isolated compounds is very close and identical in magnitude for Gram positive,
Gram-negative bacteria and fungi. In future, by isolating the various classes
of secondary metabolites by different separation technique such as preparative
TLC, column chromatography from these extracts and evaluating the
pharmacological activities of the major phytoconstituents
obtained from each extracts. So, in future isolation of such active principles
in pure form and its development can lead to new potent antimicrobial
compounds.
Further study on spectral analysis and
structural elucidation of compound A and compound
B is in progress.
ACKNOWLEDGEMENT:
Authors are greatly thankful to the
management of Sharad Pawar
College of Pharmacy, Wanadongri, Nagpur
for providing free access to their facilities to carry out research work.
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Received
on 27.04.2010
Accepted on 11.09.2010
© A&V Publication all right reserved
Research Journal of Pharmacognosy and Phytochemistry.
2(6): Nov. - Dec. 2010, 435-437