Abstract: Two flavonoidal compounds, pectolinarigenin (CA-2) and hispidulin (CA-3) isolated from the stem and root of the plant Clerodendrum indicum. The petroleum ether, chloroform and ethyl acetate extracts of the same portion of the plant were screened against twelve pathogenic bacteria for their antibacterial activities. The test materials found to be significant in vitro antibacterial activities against almost all gram positive and gram negative bacteria. The zones of inhibition produced by the test materials were found to be between 6 mm and 22 mm. The MIC values of the isolated compounds were also determined against four test organisms for CA-2 and two test organisms for CA-3. The values were found to be between 64 μg/ml and 128 μg/ml.
Introduction
Verbenaceae is a large plant family consisting of trees, shrub and herbs (Trease and Evan, 1983). Modern research carried on the Verbenaceous plants revealed that most of the plants belonging to this family are medicinally important as they contain biologically active compounds. Clerodendrum indicum Linn, locally known as Bamanhatti or Vamot in Bangladesh, belongs to the family Verbenaceae. it has considerable reputation for its medicinal values as traditional medicine. The root is considered useful in asthma, cough and scrofulous affections, the resin is employed in Syphilitic rheumatism and the juice of leaves is used with ghee as an application in hepatic eruptions and pemphigus (Kirtikar and Basu, 1994; Watt, 1972).
Although C. indicum is locally used for the above conditions, no antibacterial study of this plant has previously been reported. As a part of continuing search for novel antibacterial principles from the medicinal plants of Bangladesh, we studied C. indicum and herein report the result of in vitro antibacterial investigation.
Materials and Methods
Plant materials: Matured stem and root were collected from botanical garden, Rajshahi University, Rajshahi, Bangladesh and Sericulture Institute, Rajshahi, Bangladesh during the month of November-December, 1997. The plant was taxonomically identified by Professor A.T.M. Nadiruzzaman, Department of Botany, Rajshahi University, Bangladesh as well as the Bangladesh National Herbarium, Dhaka, Bangladesh where a voucher specimen is kept.
Extraction, isolation and characterization: The stem and root were dried in an oven at 45°C, crushed and then extracted separately in soxhlet apparatus using ethanol (95%) for 72 hrs at 70°C. The concentrated ethanolic extracts were diluted with distilled water and solvent-solvent partitioning were successfully carried out by Kupchan method (Grode et al., 1983) using petroleum ether, chloroform and ethyl acetate (Rahman, 1999). Each of the extract was concentrated at reduced pressure and appropriate temperature using rotary evaporator and thus ready for antibacterial screening.
The compound CA-2 was isolated from the petroleum ether and chloroform extract by column chromatography (Beckett and Stenlake, 1986) followed by TLC and PTLC (Egon, 1969). The compound CA-3 was isolated from chloroform extract by column chromatography (Beckett and Stenlake, 1986) followed by TLC and PTLC (Egon, 1969).
These compounds were characterized on the basis of their UV, IR, NMR, MASS and NOE data and comparison with reported data (Hase et al., 1995). Then CA-2 and CA-3 were subjected to antibacterial screening.
Antibacterial Screening: Twelve pathogenic bacteria (six gram positive and six gram negative) were selected for the test and collected from the Department of Microbiology, Dhaka University, Dhaka, Bangladesh. Nutrient agar was used as bacteriological medium. The petroleum ether, chloroform and ethyl acetate extracts were dissolved separately in sufficient amount of methanol to get a concentration of 400 μg per 10 μl. Compound CA-2 and CA-3 were also dissolved separately in methanol in the same way to get a concentration of 200 μg per 10 μl. Then in vitro antibacterial activity of these samples were carried out by the standard disc diffusion method (Barry, 1980; Berghe and Vlietnck, 1991; Bauer et al., 1966; Rios et al., 1988) against selected test organisms. The diameter of zone of inhibition produced by the extracts and flavonoids was then compared with those produced by the standard antibiotic (Kanamycin 30 μg/disc). The experiment was carried out in duplicate to minimize the error.
Minimum Inhibitory Concentration (MIC): The MIC value of the compound CA-2 was determined against two gram positive (Bacillus subtilis and Sarcina lutea) and two gram negative (E. coli and Shigella dysenteriae) bacteria. To determine the MIC value of CA-3, Bacillus subtilis and Shigella dysenteriae were taken as test organisms. The test was carried out by serial tube dilution technique (Reiner, 1982). Nutrient agar and nutrient broth were used as bacteriological media.
Results and Discussion
In antibacterial screening, it was found that all the three extracts showed a significant antibacterial activity. The isolated compounds (CA-2 and CA-3) also exhibited moderate activity against almost all test organisms (Table 1).
The zone of inhibition of petroleum ether extract (stern), chloroform extract (stem), chloroform extract (root) and ethyl acetate extract (root) were found to be 11-22 mm, 15-21 mm, 9-15 mm and 15-22 mm at a concentration of 400 μg/disc, respectively. The compound CA-2 and CA-3 produced zone of inhibition between 6 and 12 mm and 9 and 12 mm at a concentration of 200 pgldisc, respectively (Table 1).
The MIC values of CA-2 were 64 pglml against B. subtilis and E. coli and 128 μg/ml against Samna lutea and Shigella dysenteriae. For the compound CA-3, the MIC value was 128 μg/ml against B. subtilis and Shigella dysenteriae (Table 2).
From the experimental results it is evident that both the crude extracts and the flavonoidal compounds, CA-2 and CA-3, showed significant activity against the bacteria tested. Chloroform extract of stem displayed more activity than that of root against most of the test bacteria and in particular against Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Shigella shiga and Shigella dysenteriae. It was also found that petroleum ether extract of stem was totally inactive against Steptococcus-β-haemoliticus, whereas others are moderate active. Ethyl acetate extract of root showed the highest activity against most of the test bacteria and in particular against Shigella boydii, Shigella dysenteriae and Bacillus subtilis. The extracts of root part exhibited more prominent antibacterial activity than that of stem part.
| Table 1: | Antibacterial activities of extracts, CA-2, CA-3 and Kanamycin standard |
| Table 2: | The MIC values of the isolated compounds against test organisms |
The significant inhibitory activity of the extracts of root towards Shigella boydii, Shigella dysentariae, Shigella shiga, Bacillus subtilis and Bacillus cereus might indicate the traditional use of Clerodendrum indicum in different types of dysentery and diarrhoea.
The flavonoid, CA-2 showed more inhibitory activity against most of gram positive bacteria than the flavonoid, CA-3. On the other hand CA-3 exhibited more inhibitory activity against most of the gram negative bacteria than the flavonoid, CA-2. From the MIC values of CA-2 and CA-3 (Table 2), CA-2 was found to be more potent antibacterial compound against Bacillus subtilis and E. coli possessing less MIC value than CA-3 against Bacillus subtilis.
Acknowledgments
The authors would like to thank the Department of Microbiology, University of Dhaka, Dhaka, Bangladesh for the supply of test organisms. We wish to thank Dr. Naoki Sugimoto, National Institute of Health Sciences, Tokyo, Japan and Dr. Md. Abdur Rashid, Associate Professor, Department of Pharmacy, University of Dhaka, Bangladesh, for their assistance in the characterization of the flavonoids. We are also indebted to Professor A.T.M. Nadiruzzaman, Department of Botany, Rajshahi University, Bangladesh and the Bangladesh National Herbarium for the identification of the plant.