Abstract: Two clerodane diterpines viz., Kolavenic acid (1) and 16-oxo-cleroda-3, 13(14) E-diene-15-oic acid (2) were isolated from the petroleum ether (C2H5-O-C2H5) extract of the seed of Polyalthia longifolia and were screened against fourteen pathogenic bacteria for their antibacterial activities. The test materials exhibited strong activities against most of the test bacteria. The minimum inhibitory concentration (MIC) of the compound (1) and (2) was determined against Bacillus cereus, Bacillus subtilis, Escherichia coli, Shigella flexneriae and Shigella boydii which were 64, 64, 32, 16 and 32μg ml-1 for compound (1) and 16, 32, 8, 4 and 16 μg ml-1 for compound (2), respectively. The cytotoxic activity of the compound (1) and (2) was determined by brine shrimp lethality bioassay. Both the compounds showed significant cytotoxic activities and LC50 values of Kolavenic acid (1) and 16-oxo-cleroda-3, 13(14)E-diene-15-oic acid (2) were 3.16 and 2.52μg ml-1, respectively.
Introduction
Polyalthia longifolia (Family Annonaceae) is a tree, which is widely distributed in Bangladesh, Srilanka and throughout the hotter parts of India (Hooker and Clarke, 1875). In India, the seeds of this plant were used as febrifuge (Raghunathan and Mitra, 1982). The bark is also used as a febrifuge in the Balasore district of Orissa (Kirtikar, 1993). From the literature survey it is revealed that most of the plants of annonaceae family contain antitumor and anticancer principles (Chakrabarti and Mukherjee, 1968; Yamaguchi et al., 1964). The extract of stem bark and the alkaloids isolated from this extract were found to demonstrate a good antibacterial and antifungal activities (Hasan et al., 1988b). Its aqueous extract stimulates the isolated ileum and uterus, depresses heart, lowers the blood pressure and respiration in experimental animals (Achari and Lal, 1952). The crude extracts of the seeds of this plant also showed remarkable antibacterial activities (Sayeed et al., 1995). Two compounds, viz., Kolavenic acid (1) and 16-oxo-cleroda-3, 13(14) E-diene-15-oic acid (2) have been isolated and characterized from the petroleum ether (C2H5-O-C2H5) extract of Polyalthia longifolia seeds (Islam, 1995).
As a part of our continuing search for antimicrobial agents from the medicinal plants of Bangladesh, we have investigated the antimicrobial activity of the above two clerodane diterpines and report the result of such examination. In this paper we also report the cytotoxicity of the two clerodane diterpines.
Materials and Methods
Plant material: After identification of the plant Polyalthia longifolia by Department of Botany, University of Rajshahi, about 700gm of matured, healthy seeds were collected from the campus of Rajshahi University. The seeds were cleaned, washed with water and dried. Finally, the seeds were ground to course powder by a grinder.
Extraction and isolation of the compounds: The powder materials were extracted in a soxhlet apparatus with petroleum ether, (40-60)0C (Morrison and Boyd, 1994). The petroleum ether (C2H5-O-C2H5) was then subjected to vacuum liquid chromatography followed by preparative thin layer chromatography (Egon and Stahl, 1969) to obtain two pure compounds which were identified as Kolavenic acid (1) and 16-oxo-cleroda-3,13 (14) E-diene-15-oic acid (2) by extensive spectroscopic {UV (Beckman DU-64); IR (Erkinelmer); EIMS; 1H-NMR 500 MHZ; 13C-NMR 50 MHZ} analysis [Islam, 1995].
Antibacterial screening: Fourteen pathogenic bacteria (four Gram positive and ten Gram negative) were selected for the test which were collected from the Department of Microbiology, University of Dhaka, Dhaka, Bangladesh. Nutrient agar was used as a bacteriological media. The isolated compounds (1) and (2) were dissolved separately in ethyl acetate (CH3COOC2H5) to get a concentration of 100 and 200μg 10 μl-1. Then in vitro antimicrobial activity of the compound (1) and (2) was carried out by the standard disc diffusion method (Berghe and Vlietuck, 1991) against selected organisms. The diameter of the zone of inhibition produced by the compound (1) and (2) were compared with those of a standard antibiotic (Kanamycin 30μg disc-1).
Antifungal screening: Seven pathogenic fungi were selected for the test and collected from the Department of Botany, University of Rajshahi, Bangladesh. Potato Dextrose Agar (PDA) was used as a fungicidal media. The compound (1) and (2) were dissolved separately in sufficient volume of methanol (CH3OH) to get a concentration of 200μg disc-1. Then in vitro antifungal activities of the compound (1) and (2) were performed by disc diffusion method (Bauer et al., 1966). Clotrimazole (30μg disc-1) was used as a standard disc.
Minimum inhibitory concentration (MIC): The MIC values of the compound (1) and (2) were determined against two Gram-positive (Bacillus subtilis and Bacillus cereus) and three Gram-negative (Escherichia coli, Shigella flexneriae and Shigella boydii) bacteria. The test were carried out by serial dilution technique (Reiner, 1982). Nutrient agar and nutrient broth were used as bacteriological media.
Cytotoxic evaluation: The cytotoxic effect of compound (1) and (2) were evaluated by LC50 of brine shrimp lethality test (Mayer et al., 1982 and Persoone, 1980). The compound (1) and (2) were dissolved in dimethylsulphoxide (DMSO) separately and six graded doses 1, 2, 4, 8 and 16 μg ml-1 respectively were used for 5 ml sea water containing 10 brine shrimp nauplii in each group. The number of survivors was counted after 24 hours and LC50 value was determined by Probit analysis (Gujrati, 1998 and Finney, 1947).
Results and Discussion
The antibacterial activities of compound (1) and (2) isolated from Polyalthia longifolia seed against fourteen pathogenic bacteria are presented in Table 1. The concentration of the compound (1) and (2) were taken 100 and 200μg disc-1. All Gram positive and Gram negative bacteria showed remarkable sensitivity towards the compound (1) and (2) except Salmonella typhi-A and Klebsiella species. Bacillus subtilis, Bacillus cereus and Bacillus megaterium (Gram positive) and Escherichia coli, Shigella flexneriae, and Shigella boydii (Gram negative) bacteria were excellently sensitive to compound (1) and (2). The antifungal activities of the compound (1) and (2) are presented in Table 2. It is revealed that the compounds showed significant antifungal activity against seven pathogenic fungi.
Minimum inhibitory concentration (MIC) of compound (1) and (2) is presented in Table 3. Both the compounds showed significant activity against Bacillus subtilis, Bacillus cereus, Escherichia coli, Shigella flexneriae and Shigella boydii. As evident from the Table 4 the compounds, (1) and (2) showed promising cytotoxic activity when performed brine shrimp bioassay. After 24 hrs of observation, the number of survived nauplii was counted in both experimental and control. In control group the nauplii remained unchanged but in experimental groups, the percentage of mortality of brine shrimp nauplii was calculated for each concentration and the rate of mortality was found to be increased with increase in the concentration of samples (Table 4).
The 50% mortality (LC50) of the compound (1) and (2) was 2.95 and 2.28μg ml-1 and 95 % confidence limits were 1.55-5.61 and 1.16-4.49, respectively.
| Table 1: | Antibacterial activities of compound (1) and (2) isolated form Polyalthia longifolia seeds |
| Compound (1) = Kolavenic acid (1) Compound (2) = 16-oxo-cleroda-3, 13 (14) E-diene-15-oic acid (2) SK = Standard Kanamycin |
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| Table 2: | Antifungal activities of the compound (1) and (2) isolated form Polyalthia longifolia seeds |
| Compound (1) = Kolavenic acid (1) Compound (2) = 16-oxo-cleroda-3, 13 (14) E-diene-15-oic acid (2) SC = Standard Clotrimazole |
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| Table 3: | The MIC values of the compound (1) and compound (2) against test organisms |
| Compound (1) = Kolavenic acid (1) Compound (2) = 16-oxo-cleroda-3, 13 (14) E-diene-15-oic acid (2) |
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| Table 4: | Cytotoxicity of compound (1) and Compound (2) by brine shrimp lethality bioassay |
A regression equation of compound (1) and (2), Y=4.53+1.38X and Y=4.49+1.42X and χ2 value 0.470 and 0.109, respectively are observed from the probit analyses which were compared with gallic acid (Saker et al., 1998) as a standard one. An evaluation of cytotoxicity is also an important study for possible clinical use i.e., indicative of wide range of pharmaceutical activities of the drugs (Mayer et al., 1982). In that sense, the mortality rate of the compounds with highest concentration suggest that the drug can be used at higher doses and also suitable for further clinical trial.
In conclusion, this study reports for the first time, the antibacterial, antifungal activity and cytotoxicity of the compound (1) and (2) isolated from the Polyalthia longifolia seed. However, further and specific studies are needed to better evaluate the potential effectiveness of the isolated the compounds from the Polyalthia longifolia seed as an antimicrobial agent.
Acknowledgment
The authors wish to thank Dr. Abdur Rashid, the NCI Frederick, Cancer Research and Development Center, Frederick MD, 21702-1201 USA, for spectroscopic analysis of this research samples. The authors would like to thank the Department of Microbiology, University of Dhaka, Bangladesh for the supply of test organisms. We wish to thank Chairman, Department of Pharmacy, University of Rajshahi for providing lab facilities during the research.