Biological Characterization of Crude Extract & Pure Compound Isolated from Swertia chirata Ham

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Bioassays of the crude and pure extracts: Brine shrimp lethality bioassay is a recent development in the bioassay for bioactive compounds, which indicates cytotoxicity as well as a wide range of pharmacological activities (e.g. anticancer, antiviral, pesticidal, AIDS etc.) of the compounds. Bioactive compounds are almost always toxic in high doses. Pharmacology is simply toxicology at a lower dose or toxicology is simple pharmacology at a higher dose. Brine shrimp lethality bioassay is a bench top bioassay method for evaluating anticancer, antimicrobial and pharmacological activities natural products. Natural product extracts, fractions or pure compounds can be tested for their bioactivity by this method. Here in vivo lethality of a simple zoological organism (brine shrimp nauplii) is used as a convenient monitor for screening a fractionation in the discovery of new bioactive natural products. Generally the median effective dose (ED₅₀) values for cytotoxicities are one tenth (1/10) of median lethal dose (LC₅₀) values in the brine shrimp test.

3. Results and Discussion

Test organisms used for the study: Fourteen pathogenic bacteria (five gram positive and nine gram negative) were selected for the test (Table 2). These organisms were available in the microbiological research laboratory of Biochemistry and Molecular Biology department, University of Rajshahi, Bangladesh. The pure cultures were collected from the Microbiological Laboratory of the Institute of Nutrition and food Science (INFS) and Department of Microbiology, University of Dhaka, Bangladesh.

Table 2. List of the test pathogenic bacteria

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Antibacterial activity of test sample: The antibacterial activities of the crude extract and the pure compound X-1 were tested. The results obtained are shown in Table 3 and Table 4. The inhibitory activities are shown in Figure 1, 2 and 3.

Antibacterial activity of rectified spirit extract: Antibacterial activity of rectified spirit extract was tested against eight bacteria at concentrations of 30 μg/disc and 90 μg/disc. Standard antibiotic disc of chloramphenicol (30 μg/disc) was used for comparison. The results obtained were shown in Table 3 and Figure 1, 2 and 3. The produced zone of inhibition for rectified spirit extract against Staphylococcus aureus, Bacillus megaterium and Escherichia coli were 10 mm, 9 mm and 9 mm at 30 μg/disc dose respectively. At 90 μg/disc dose, the produced zone of inhibition against the same bacteria was 17 mm, 14 mm and 13 mm respectively. It was evident that the antibacterial activity of rectified spirit extract against the above bacteria showed decrease dose dependency.

Table 3. In vitro antibacterial activity test of the crude extract of S. chirata

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Table 4. Antibacterial activity of pure compound X-1

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Antibacterial activity of pure compound X-1

The pure compound X-1 was screened for their antibacterial activities against 12 pathogenic bacteria, 6 Gram-positive and 6 Gram-negative, by disc diffusion method at a concentration of 200μg/disc. The results obtained were shown in Table 4 compared with those for a standard antibiotic Kanamycin. Pure compound X-1 showed significant activity against Bacillus megaterium (13 mm), Bacillus subtilis (11 mm), Salmonella typhi-A (12 mm), Shigella flexeneriae (12 mm) and Klebsiella sp. (13 mm). A little activity of pure compound X-1 against Staphylo coccus aureus, Sarcina lutea and Shigella dysenteriae was observed.

Minimum inhibitory concentration (MIC) of the test sample

Minimum Inhibitory Concentration (MIC) of rectified spirit extract was determined by serial dilution technique ^[13] against two Gram-positive bacteria Staphylococcus aureus and Sarcina lutea and two Gramnegative bacteria Escherichia coli and Salmonella typhi-A. The MIC value of pure compound X-1 was determined against a Gram- positive bacterium (Bacillus megaterium) and a Gram-negative bacterium (Salmonella typhi-A) These values are shown in (Table 5 and Table 6).

MIC of the crude extract

For the Rectified spirit extract the growth was observed in the test tube containing 128 μg/ml of extract against Escherichia coli, Sarcina lutea, Bacillus megaterium and for Staphylococcus aureus in the test tube containing 64 μg/ml and Salmonella typhi-A containing 132 μg/ml of rectified spirit extract. So the MIC values of Rectified spirit extract for Sarcina lutea, Escherichia coli, Bacillus megaterium were 128 μg/ml and for Staphylococcus aureus was 64 μg/ml and Salmonela typhi-A was 132 μg/ml (Table 5).

MIC of pure compound X-1

The Minimum Inhibitory Concentrations of the pure compound X-1 was determined against Bacillus megaterium and Salmonella typhi-A. The MIC of the pure compound X-1 against Bacillus megaterium and Salmonella typhi-A were 128 μg/ml and 132 μg/ml respectively when tested in nutrient broth medium. The results are shown in (Table 6).

Table 5. Minimum Inhibitory Concentration (μg/ml) values of rectified spirit extract of S. chirata

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Table 6. Minimum inhibitory concentration (MIC) of pure compound X-1

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Figure 1. Antibacterial activity of Rectified spirit extract against Bacillus megaterium

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Figure 2. Antibacterial activity of Rectified spirit extract against Escherichia coli

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Figure 3. Antibacterial activity of Rectified spirit extract against Staphylococcus aureus

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S₁ = 30 µg/disc

S₂ = 90 µg/disc

SC = Solvent control

S = Standard (Chloramphenicol 30 µg/disc)

Brine shrimp mortality of test sample

Brine shrimp mortality test is a recent development in the bioassay for the bioactive compounds ^[9]. Bioactive compounds are almost always toxic in high dose. There is a positive correlation between brine shrimp toxicity and cytotoxicity ^{[7, 10, 12]}. The crude rectified spirit extract and pure compound X-1 showed positive result in brine shrimp lethality bioassay. The results were shown in (Table 7 and Table 8).

Brine shrimp mortality of rectified spirit extract

To determine the cytotoxic effect of rectified spirit extract, the lethal concentration, LC₅₀ (concentration at which 50% mortality of the nauplii occurred) was measured and found to be 80.50 μg/ml. This was obtained from a plot of percentage of mortality versus log of concentration on the graph, which produced approximate linear correlation between them (Figure 4). The result of brine shrimp lethality of rectified spirit extract was given in the (Table 7).

Table 7. Results of the Rectified spirit extract on brine shrimp lethality bioassay

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Brine shrimp mortality by pure compound of X-1

To determine the cytotoxic effect of pure compound X-1 medium lethal concentration (LC₅₀) of brine shrimp lethality was measured and it was found to be 10 μg/ml, which was obtained from a plot of percentage of mortality versus log of concentration (μg/ml) on the graph. This afforded an approximate linear correlation between them (Figure 5). The results of brine shrimp mortality of pure compound X-1 are shown in the (Table 8).

Table 8. Results of the pure compound of X-1 on brine shrimp lethality bioassay

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The pure compound X-1 was found to show significant activity against the brine shrimp nauplii. In this bioassay, the mortality rate of brine shrimp is found to increase with the increasing concentration of the compound. So it was observed that there existed a positive correlation between brine shrimp toxicity and cytotoxicity. The very low value of LC₅₀ (10.10 μg/ml) indicated the high cytotoxic effect of the pure compound X-1.

Figure 4. Determination of LC₅₀ of Rectified spirit extract against brine shrimp nauplii.

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Figure 5. Determination of LC₅₀ of the pure compound X-1 against brine shrimp nauplii.

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