Effect of Three Plants Extracts on Some Bacterial Strains and Culex pipiens L. Stages

MATERIALS AND METHODS

This study was conducted in the year 2010.

Organisms:

The aforementioned bacterial strains were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. These strains were subculture on nutrient agar and stored at 4°C. All cultivations of the strains of E. coli, Salmonella typhimurium, B. cereus and aureus sub sp. aureus were at 37°C for 24 h and at 55°C for 48 h for B. stearothermophilus using nutrient broth.

Insects: Insects and second instar larvae were obtained from culture of Culex, reared at laboratory, maintained on pigeon blood and 10% sucrose solution. The experiment was carried out at the Faculty of Meteorology, Environment and Arid land Agriculture, King Abdul-Aziz University, Jeddah, Saudi Arabia. Larvae were reared in tap water.

Plant aqueous extracts: The cold extraction was done at room temperature after milling the tested material by mixing them with distilled water at room temperature to give high concentrations. The concentrate extract was then sterilized by filtration (Sartorious membrane filters, 0.2 μm, Germany). The sterilized extracts were diluted by the growth medium to advise level. Placing a certain weight of herbs in cloth bags did the hot extraction. The weight of herbs was selected to give the required expected concentrations. The bags containing herbs were submerged in the nutrient both medium (or distilled water in case of agar diffusion procedure) and autoclaved at 121°C for 15 min. The test of kill curve was done in these previous medium where hot and cold herbs extracts were placed.

Plant acetonic extracts: Plant materials of Calotropis procera, Acacia nilotica and Cassia senna were collected from different parts in Saudi Arabia. Calotropis procera, Cassia senna leaves and Acacia nilotica seeds were air- dried for 48 h, ground to fine parts and extracted with acetone at ambient temperature. A gentle warming to 35-40°C was sometimes found necessary. The mixture was stirred for 30 min by magnetic stirrer and left 24 h. Then, it was condensed in a vacuum rotary evaporator from solvent in a water bath at 55°C according to Severini et al. (1993). The extracts were then freeze dried using a Labconco Freeze Dryer-18 model 75018 for 48-72 h. Stock solution was prepared from the lyophilized residue.

Antibacterial activity
Agar diffusion: Agar diffusion has been conducted by using the method of Davidson and Parish, (1989). It has often been referred as the disc assay. The antimicrobial compound is applied to agar plate, using an impregnated filter paper disk. The study discs were sterilized separately and impregnated under sterile condition when cold extraction was done at room temperature. However, the filter paper discs were impregnated and sterilized during the hot extraction at 121°C/15 min.

Inhibition or kill curve: This test demonstrated by Schoenknecht et al. (1995) involves inoculation of one of the tested bacteria into the liquid medium, addition of one of the tested herbs, followed by incubation and periodic sampling to determine growth or survival.

Test procedure towards insects: Stock solutions of the three plant acetonic extracts were prepared by dissolving the extracted powder in warm distilled water (at 0.5 g/100 mL water). Different concentrations of 100, 200, 300, 400 and 500 ppm were prepared from stock solution. Twenty freshly laid eggs or ten second instar larvae were transferred from the culture into plastic cups (8 cm diameter, 10 cm deep), each containing 30 mL of desired concentration. Treatments were triplicated and control had only distilled water. Larvae were fed ad libitum and kept under laboratory conditions. Egg hatchability was determined at 4 and 7 days after treatment. Larval mortalities were counted at 2, 4 and 10 days after treatment. Percentage of successful pupation and adult emergence were determined by monitoring on daily basis until all adults in the control have emerged.

Statistical analysis: Data were analyzed using maximum likelihood procedures and values of LC₅₀ were calculated according to Finney (1971). Data were corrected for control mortality (Abbott, 1925). Data of egg hatchability were analyzed by analysis of variance. If significant differences (p<0.05) occurred, means were separated by Duncan’s multiple range test.

RESULTS AND DISCUSSION

Table 1 shows the inhibitory or bactericidal effect of Calotropis procera, Acacia nilotica and Cassia senna using the disc assay. Different concentrations of tested materials were used (1, 5 and 10% as aqueous extracts). The results of the 1% concentration were presented in Table 1. Samples with no inhibitory effect in Table 1, did not show any further response when their concentration were raised. It can be seen that hot extraction give higher inhibition when compared with the cold extraction. Senna has completely inhibited Staphylococcus aureus sub sp. aureus, Salmonella typhimurium and Bacillus stearothermophilus. This is in agreement with Liu and Nakano (1996). This is in contradiction with Holzminden (1982) who found that Salmonella strains were less inhibited than Staphylococcus strains.

Figure 1 shows the effect of Cassia senna and Acacia nilotica on Staphylococcus aureus sub sp. aureus. It is clear that Acacia nilotica has bactericidal effect. Bactericidal effect was achieved after 8 and 18 h incubation for Acacia nilotica and Cassia senna, respectively.

Figure 2 illustrates the effect of Cassia senna and Acacia nilotica also on Salmonella typhimurium. Both of them have bactericidal effect after incubation time of 8 and 12 h for Cassia senna and Acacia nilotica, respectively.

Table 1:	Antibacterial effect of some herbs extracts (hot and cold extraction) at 1% concentration on some food born pathogens and spoilage microorganisms using disc assay
1: Hot extraction, -: No inhibition, ++: Slight inhibition; 2: Cold extraction, +: Very slight inhibition and +++: Complete inhibition

Fig. 1:	Effect of C. senna and A. nilotica on Staphylococcus aureus sub sp. aureus

Fig. 2:	Effect of C. senna and A. nilotica sage on Salmonella typhimurium

It is clear that Cassia senna has more bactericidal activity against Salmonella typhimurium (G^-) than Staphylococcus (G⁺). This is in contradiction with (Imelouane et al., 2009).

Figure 3 shows that senna has bactericidal effect on Bacillus stearothermophilus after incubation time of 25 h. From Fig. 1-3, it clear that Cassia senna has more bactericidal activity against Salmonella typhimurium (G^-) than Staphylococcus aureus sub sp. aureus and Bacillus stearothermophilus (G⁺). This is in contradiction with Imelouane et al. (2009).

The mortality (%) of C. pipiens larvae treated with three plant extracts in acetone and their LC₅₀ values and 95% confidence limits at 2, 4 and 10 days after treatment are shown in Table 2 and 3. The mortality (%) of C. pipiens larvae treated with plant extracts in acetone is given in Table 2. Data showed that larvae suffered up to 93.0 and 82.8% mortality after 10 days of exposure to 500 ppm A. nilotica and C. senna extracts, respectively. However, the lowest A. nilotica concentration caused 45% mortality after 2 days of treatment. C. procera extracts caused the lowest mortalities, while highest concentration (500 ppm) caused 70.7% mortality after 10 days of treatment. LC_50s and 95% Confidence Limits (CL) for each plant are given in Table 3. Data showed a significant differences. Acute and chronic LC₅₀ (2 and 10 day of mortality) for second instar larvae were 212.1 and 144.2, 301.5 and 173.1 and 466.7 and 251.7 for A. nilotica, C. senna and C. procera, respectively. A. nilotica was significantly more toxic at all exposure times than C. senna and C. procera.

Fig. 3:	Effect of C. senna on Bacillus stearothermophilus

Table 2:	Mortality percentage of Culex pipiens larvae reared in media containing acetonic plant extracts

Table 3:	LC50 values and 95% confidence limits for Culex pipiens larvae in media containing acetonic plant extracts

Egg hatchablity was significantly lower (p<0.05) in all extracts than control (Table 4). At 100 ppm concentration, A. nilotica had the most severe effect on egg hatching rate which was reduced by about 30% at their highest concentration (500 ppm).

Table 4:	Egg hatchability percentage of Culex in media containing acetonic plant extracts
Means followed by the same letter are not significantly different at 5% level, Duncan multiple test

Table 5:	Successful pupation and adult emergence of Culex pipiens larvae reared in media containing acetonic plant extracts

The three plant extracts reduced egg hatchability by about 79, 57 and 36%, for A. nilotica, C. senna and C. procera, respectively.

The effect of the three plant materials on growth and development of C. pipiens, larvae to adulthood are given in Table 5. There was considerable reduction in the percentage of larvae undergoing successful pupation in all treatments compared with control. No further larval development took place beyond the second instar in the A. nilotica 500 ppm concentration. On the other hand all plant extracts had an evident inhibitory effect even at their lowest concentrations (100 ppm) where the successful pupation were only 20.3, 39.3 and 68.1 for A. nilotica, C. senna and C. procera, respectively. Complete suppression of adult emergence was evident in the 500 ppm concentration of A. nilotica and C. senna. The adult emerging percentages from the 100 ppm treatments were 9.40, 14.78 and 40.0% for A. nilotica, C. senna and C. procera, respectively, compared with control.

Considerable biological activity related to toxicity and hindrance of growth and development of the larvae of C. pipiens has been observed in this study. From the three plant extracts, A. nilotica was found to cause higher rate of mortality compared to other plant extracts. In agreement with the present study, the activity of A. nilotica seeds extracts has been attributed in part to the saponins and alkaloids in the seeds (Duru and Onyedineke, 2010). C. senna and C. procera exhibited a relatively mild acute effect on mosquito larvae especially in its lower concentrations. However, its chronic toxicity was almost high above 200 ppm.

CONCLUSION

The Cassia senna has faster bactericidal activity against Salmonella typhimurium than Staphylococcus aureus sub sp. aureus and Bacillus stearothermophilus. The extracted plant materials specially A. nilotica and C. senna have toxic, growth and development-retarding influence on C. pipiens mosquitoes. Moreover, application of these materials is not likely to leave harmful residues in the environment since they are naturally occurring among the local flora.