Abstract: A terpenoid, 3-oxo-(20S, 24S)-epoxydammarane-19,25-diacetate (CP-1) isolated from the chloroform extract of barks of Caesalpinia pulcherrima was characterized on the basis of spectral data coupled with chemical evidence. The compound showed significant antibacterial activity against a number of pathogenic bacteria and prominent antifungal activity against a few fungi. The minimum inhibitory concentration (MIC) values against Bacillus cereus and Shigella dysenteriae were 16 μg ml-1 and 32 μg ml-1 respectively. The compound was found cytotoxic against brine shrimp and LC50 value was 8.41 μg ml-1. These findings suggested for further clinical trial.
INTRODUCTION
Bangladesh has a rich plant kingdom and some of which are good source of herbal medicines. The most important development of medicine in the present century is the introduction of chemotherapeutic agents such as sulfadrugs, antibiotics and also of other synthetic drugs. Inspite of these, interest (Hese et al., 1966 and Dreyer, 1969) in the chemistry and pharmacology of medicinal plants has increased considerably in resent years. Morphine was isolated from opium by Sertuner (Burger, 1960) and ephedrine (Hese et al., 1966) from Ephedra vulgaris. Many other chemical compounds have since then been isolated and their chemistry and pharmacological action were studied.
Plants belonging to the family Leguminoceae have wide folkloric medicinal uses. Caesalpinia pulcherrima Swartz. vernacularly known as Radha chura in Bangladesh, belonging to the family Leguminoceae is widely distributed in Bangladesh and India (Kirtikar and Basu, 1994). Leaves, flowers, bark and seeds are largely used in Indian medicine (Watt). In Indo China, the plant is considered as a tonic, stimulant and emmenagogue. The bark is used as an abortifacient and an infusion of leaves is used as aborticient, antiperiodic and cathartic as well as infusion of flowers is pectorial and febrifuge (Kirtikar and Basu, 1994). It is obvious that there are some sorts of pharmaceutically active principles in this plant, which may exhibit physiological functions after administration. The physiologically active principles of the plant are of great importance from the medicinal point of view. Therefore, isolation of physiologically active constituents from this plant was an interesting investigation to us.
In this paper, isolation, characterization and antimicrobial action as well as cytrotoxicity of a compound isolated from the chloroform extract of barks of Caesalpinia pulcherrima Stwartz have been reported.
MATERIALS AND METHODS
Collection of the materials: Barks of Caesalpinia pulcherrima were collected from the rural area of the district of Gaibandha in Bangladesh after identification of the plant by Bangladesh National Herberium, Dhaka.
Extraction and isolation: The powdered plant barks (ca. 835 gm) were extracted with chloroform at room temperature after washing the materials with petroleum ether to remove the fatty and waxy matters. The chloroform extract was concentrated by a vacuum rotary evaporator under reduced pressure and was subjected to column chromatography over silica gel followed by TLC and preparative TLC. A pure, white solid compound, (CP-1), m.p. 134-137°C, was obtained.
Spectral characterization: IRvmax: 2918.1, 2850.6, 1728, 1380.9 and 1056.9 cm-1, 1H NMR: δ 4.21 (1H, d, J=11.7 Hz, Ha-19), 4.03 (1H, d, J= 11.7 Hz, Hb-19), 3.89 (1H, dd, J=9.0 Hz, H-24), 1.94 (3H, s, OCOCH3), 1.93 (3H, s, OCOCH3), 1.44 (3H, s, H-26), 1.42 (3H, s, H-27), 1.14 (3H, s, H-21), 7.21 (3H, s, H-29), 7.26 (3H, s, H-30) and 0.89 (3H, s, H-28). 13C NMR: δ 34.20 (C-1), 33.80 (C-2), 216.70 (C-3), 45.80 (C-4), 52.31 (C-5), 19.60 (C-6), 34.40 (C-7), 40.00 (C-8), 51.70 (C-9), 38.61 (C-10), 23.10 (C-11), 25.80 (C-12), 43.01 (C-13), 49.80 (C-14), 31.70 (C-15), 27.01 (C-16), 49.40 (C-17), 16.50 (C-18), 64.50 (C-19), 86.70 (C-20), 27.40 (C-21), 34.60 (C-22), 26.71 (C-23), 84.81 (C-24), 82.80 (C-25), 22.00 (C-26), 22.50 (C-27), 29.40 (C-28), 19.30 (C-29), 15.41 (C-30), 171.20 (OCOCH3), 170.50 (OCOCH3), 20.90 (OCOCH3), 21.80 (OCOCH3).
Antibacterial screening: In vitro antibacterial activity of the isolated compound, CP-1, was studied against five gram-positive and nine gram-negative bacterial strains by the standard disc-diffusion method (Barry , 1980; Buer et al., 1966; Berghe and Vlientinck, 1991). Nutrient agar was the bacteriological medium. Compound, CP-1, was dissolved in sufficient volume of methanol to get a concentration of 200 μg per 10 μl. Diameters of zones of inhibition produced by the isolated agent were compared with those produced by the standard antibiotic (Kanamycin, 30 μg disc-1). The experiment was performed in duplicate to minimize the error.
Minimum Inhibitory Concentration (MIC): The MIC value of the compound, CP-1, was determined against one gram-positive (Bacillus cereus) and one gram-negative (Shigella dysenteriae) bacteria. The test was carried out by serial dilution technique (Reiner, 1982). Nutrient agar and nutrient broth were used as bacteriological media.
Antifungal screening: Seven pathogenic fungi were used for the antifungal test. Potato Dextrose agar was used as fungicidal medium. Compound, CP-1, was dissolved in sufficient volume of methanol to get a concentration of 200 μg per 10 μl. The in vitro antifungal activity of the compound was performed by disc diffusion method (Beur, 1966). Clotrimazole was used as a standard.
Cytotoxic evaluation: The cytotoxic effect of compound CP-1 was evaluated by LC50 of brine shrimp lethality test (Mayer et al., 1982 and Persoone, 1980). The compound was dissolved in dimethylsulphoxide (DMSO) separately and five graded doses 5, 10, 20, 40 and 80 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 h and LC50 value was determined by Probit analysis (Finney, 1947). The experiment was carried out quadruplicate and the mean LC50 value was recorded.
RESULTS AND DISCUSSION
The compound, CP-1, isolated from the chloroform extract of Caesalpinia pulcherrima was white powder, m.p. 134-137°C. IR spectrum showed a strong absorption band at 1728 cm-1 which indicated the presence of ester CO and ketonic CO groups. The spectrum also revealed a C-H streching vibration (in CH3) at 2918.1 and 1380.9. 1H NMR spectrum showed signals of nine methyl protons at δ 1.93 (3H, s, OCOCH3), 1.94 (3H, s, OCOCH3), 1.44 (3H, s, H-26), 1.42 (3H, s, H-27), 1.14 (3H, s, H-21), 7.21 (3H, s, H-29), 7.26 (3H, s, H-30), 0.89 (3H, s, H-28), two doublets at δ 4.21 (1H, d, J =11.7 Hz, Ha-19) and δ 4.03 (1H, d, J= 11.7 Hz, Hb-19) and a double doublet at δ 3.89 (1H, dd, J=9.0 Hz, H-24). 13C NMR spectrum showed signals at δ 86.70 and δ 82.80 for C-20 and C-25 respectively. It displayed signals at δ 84.81 for oxygenated methyl carbon (C-2) and at δ 216.70 was discerned for carbonyl carbon (C-3). The presence of oxygenated carbon resonances at δ 86.7 (s) and δ 82.8 (d) suggested the presence of a 20,25-trisubstituted tetrahydrofuranyl system as ring E in Fig. 1.
Comparison of 13CNMR resonances associated with the oxygenated carbons of the tetrahydrofuranyl unit (ring E) in compound, CP-1, with those of 3-oxo-(20S, 24S)-epoxydammarane-19,25-diacetate established the configuration of C-20 and C-24 as 20S and 24S in Fig. 1.
13C NMR data of the compound, CP-1, closely correspond with the same carbon resonances of 3-oxo-(20S, 24S)-epoxydammarane-19,25-diacetate (Das, 1999). On the basis of physical and chemical characteristics as well as IR, 1H NMR and 13C NMR spectral data the compound, CP-1, was finally suggested as 3-oxo-(20S, 24S)-epoxydammarane-19,25-diacetate. This is the first report of isolation of the compound from this plant.
Results of antibacterial and antifungal activities are presented in Table 1 and 2. Compound, CP-1, showed significant antibacterial activity against all the fourteen bacteria tested. The concentration of the compound was taken 200 μg disc-1. Zones of inhibition produced by the compound were in between 15 mm and 24 mm.
The compound, CP-1, showed antifungal activity against all the fungi tested except Pigment yeast and Rhizopus oligasporum and produced the zone of inhibition in between 7 mm and 10 mm (Table 2).
The minimum inhibitory concentration (MIC) of the compound, CP-1, against Bacillus cereus and Shigella dysenteriae was determined and the values were 16 μg ml-1 and 32 μg ml-1 respectively (Table 3).
| Fig. 1: | Structure of compound, CP-1 |
| Table 1: | Antibacterial activity of compound, CP-1 |
| Table 2: | Antifungal activity of compound, CP-1 |
| Table 3: | The minimum inhibitory concentration (MIC) of the compound, CP-1, against test organisms |
The cytotoxicity of the compound was bioassayed against brine shrimp nauplii and the results were shown in Table 4. The 50% mortality concentration (LC50) of the compound, CP-1, was 8.42 μg ml-1 and 95% confidence limits were 3.95-17.91. A regression equation of the compound Y=3.67+1.44X and χ2 value 0.81 are observed from the probit analysis which were compared with the results reported for Kolavenic acid and Clerodane diterpine (Islam et al., 2001); Isoflavone (M. Shah Alam Bhuyan et al., 2003); Triterpenoid (Rahman et al., 2002) and galic acid (Saker et al., 1998).
In conclusion, the present study reports here the characterization, antibacterial and antifungal activities and cytotoxicity of the compound isolated from Caesalpinia pulcherrima Stwartz. This compound may be used as a versatile compound to the development of a potential antimicrobial and cytotoxicological agent.
| Table 4: | Cytotoxicity of the compound, CP-1, by brine shrimp lethality bioassay |
| Clerodane diterpine=16-oxo-cleroda-3,13(14)E-diene-15-oic
acid; Isoflavone = 5-methoxy-4-hydroxy-2,2-dimethylpyrano(3,4,7,8) isoflavone; Triterpenoid = Bet-20(29)-en-3-ol-28-oic acid; |
|
ACKNOWLEDGEMENT
Authors would like to thank Prof. A.T.M. Nadiruzzaaman, Department of Botany, University of Rajshahi, Rajshahi-6205 and Bangladesh National Herbarium, Dhaka, Bangladesh, for identification of the plant. The authors also wish to thank the Institute of Nutrition and Food Science, Dhaka University, Bangladesh and ICDDRB, Dhaka Bangladesh for supplying the test organisms.