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.

 

REFERENCES:

1.        K. M. Nadkarni, “Indian Materia Medica.” Vol I, Popular Prakashan, Bombay (1989), p. 254.

2.        R. Chopra, S. Nayar and I. Chopra, Glossary of Indian Medicinal Plants.” 3rd Edn, Council of Scientific and Industrial Reaserch, New Delhi (1992), p. 94.

3.        P. D. Sethi and D. Charegaonkar, “Identification of Drugs in Pharmaceutical Formulations by Thin Layer Chromatography,” 2nd Edn, CBS Publishers and Distributors, New Delhi (1991), p. 1.

4.        H. Wagner and S. bladt, “Plant drug Analysis a TLC Atlas,” 2nd Edn, CBS Publishers and Distributors, New Delhi (1995), p. 178.

5.        K. Khandelwal, “Practical pharmacognosy,” 2nd Edn, Nirali Prakashan, Pune (2000), 149.

6.        J. P. Duguid, B. P. Marmion and  R. H. A. Swain, “Mackie and Mc Carteney Medical Microbiology, Microbial infections, Vol.I, 13th Edn., ELBS Churchill Livingstone, Edinburgh (1985), p. 59.

7.        W. Hewitt and S. Vincent, “Theory and Application of Microbiological Assay,” Academic Press, Inc., California (1989), p. 246.

8.        D. Chamundeeswari, E. Sukumar, J. Vasantha, S. Gopalkrishanan, B. Barik and A. Patra,  Indian J. Pharm. Sci., 56, 669 (2003).

 

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