Bioactivity of Hemidesmus indicus (L.) on Human Pathogenic Bacteria and Culex qinquifasciatus (Diptera: Culicidae)

MATERIALS AND METHODS

Plant materials: Hemidesmus indicus is widely seen in the tropical and sub tropical regions of the world and all over the India during the year of January, 2009. Healthy rapidly growing plant roots were collected from the tropical areas of Kanyakumari District (South India) also in and around the College campus Malankara Catholic College in Kaliakkavilai (Kanyakumari District). Then using the Herbarium of TBGRI (Tropical Botanical Garden Research Institute), Trivandrum and the flora of the presidency of Madras identified this specimen.

Preparation of solvent root extraction: The collected roots were thoroughly washed with water. Then, it was dried in shade for 20 days. The shade-dried roots were finely powdered. The powdered plant materials were stored at room temperature for extraction. The dried 25 g powdered root and soaked separately in 100 mL of petroleum ether, chloroform, methanol, ethanol and aqueous by increasing order of their polarity (Kokate, 1999). Each solvents were kept in separate flasks with powdered sample were kept in a rotating shaker for 3 days. The extracts were filtered through cheese cloth and the extracts were reduced to half of its original volume. The organic solvents were concentrated in vacuum using a rotary evaporator, while aqueous extract was dried using water bath.

Test strains for in vitro antimicrobial activity: The test microorganisms like Esherichia coli (MTCC 443), Klebsiella pneumoniae (MTCC 109), Proteus mirabilis (MTCC 1429), Pseudomonas aeruginosa (MTCC 1688), Salmonella paratyphi (MTCC 735), Salmonella typhii (MTCC 733), Salmonella typhimurium (MTCC 2957), Shigella boydii (MTCC 1457), Shigella sonnnei (MTCC 2957) Stapylococcus aureus (MTCC 737), Streptococcus faecalis (MTCC 459) and authentically identified clinical isolates of Citrobacter sp., were obtained from culture repository of Best Biotech culture collection, Bangalore, India. The organisms were inoculated into NB (Nutrient Broth) medium (0.5 Peptone, 0.5 Sodium Chloride, 0.15% Yeast extract; pH 7.4) and incubated at 37°C for overnight. The bacterial cells were harvested by centrifuging at 5000 rpm for 15 min. The pellet formed was washed twice with PBS (Phosphate Buffer Saline) (10 mM Sodium Chloride, pH 7.4) and the cells were counted by haemocytometer. The bacterial cells were diluted to approximately 105 CFU mLG1 before use.

Determination of antibacterial activity: The antimicrobial activity of the root extracts was determined using agar well diffusion method by following the published procedure with slight modification. Nutrient agar was inoculated with the given microorganisms by spreading the bacterial inoculums on the media. Wells (8 mm diameter) were punched in the agar and filled with plant extracts. Control wells containing antibiotic solution chloromphenicol of Hi-Media Laboratories were filled for comparative efficacy. The plates were incubated at 37°C for 18 h and measuring the diameter of the zone of inhibition assessed the antibacterial activity. The relative antibacterial potency of the given preparation was calculated by comparing its zone of inhibition with that of the standard drug chloromphenicol.

Statistical analysis: The resultant clear zones around the discs were measured in mm. Clear zones of inhibition indicated the antibacterial activity of Hemidesmus indicus root extracts. Data of three independent experiments represented by six replicates were maintained. The means were analyzed by one way Analysis of Variance (ANOVA).

Collection of Culex quinquefasciatus mosquito larvae: C. quinquefasciatus mosquito larvae were collected from water stagnated area, and identified in Zonal Entomological Research Laboratory. They were then maintained under suitable temperature and humidity.

Larvicidal effect of Hemidesmus indicus: Twenty larvae of the C. quinquefasciatus were placed in each of the three 150 mL sterile beaker containing 90 mL of water. After that the mosquito larvae were introduced in to the beaker to this 10 mL of aqueous root extracts of H. indicus, was added in each of the beakers, separately. Then the beaker containing the larvae were kept in the growth room maintained at room temperature. Counting the number of dead larvae each day up to three days monitored the larvicidal effects of the extracts. Each test was repeated thrice, the percentages of larval mortality and standard error were calculated for each concentration of aqueous extracts of all the three plants.

RESULTS

Antimicrobial activity: H. indicus extracts (1 mg mL^-1) inhibited the growth of S. aureus and K. pneumonia (13.70 mm) and P. aeruginosa (15.54 mm). Remaining other microbes such as bengalensis. Typhimurium, S. boydii and S. aureus were also showed a similar order of antimicrobial activity against the tested organisms. Among the tested pathogens Proteus mirabilis showed H. indicus extracts exhibited moderate inhibition with the MIC ranging from 6.20 to 10.65 mg against tested bacterial pathogens (Table 1). Standard antibiotics amphicillin, tetracycline and chloromphenicol exhibited marked inhibition with the MIC values ranging from 9.5-21.6 mg mL^-1.

Table 1:	Antibacterial activity of five different extracts of H. indicus (Linn.) root on human pathogenic organisms
M: Methanol, E: Ethanol, C: Chloroform, PE: Petroleum ether, AQ: Aquoeus CHL: Chloromphenicol (10 μg disc-1 as a positive reference standard) Values are mean inhibition zone (mm)±S.D of six replicates.- : No activity observed, s: 5% level of significant, is: Insignificant

Six different solvent extracts of Hemidesmus indicus (L.) roots were assessed for antimicrobial activity by using the agar well diffusion method by measuring the diameter of growth inhibition zones with 100 μL concentrations (Table 1). The results showed that among the four solvent extracts (viz., petroleum ether, chloroform, methanol and ethanol) methanol and ethanol showed significant result of antimicrobial activity. When compared to both, chloroform showed minimum level of inhibition. Eventhough, antimicrobial activity was not observed in case of petroleum ether extracts against this experimental tested all pathogens. Citrobactor species having maximum MIC shown in Ethanol extract compared with methanol treated bacteria. Among the 12 bacterial species maximum inhibition zone was 16.00±0.18, 10.65±0.19 and 16.3±0.20 observed the following bacteria such as E. coli, P. mirabilis and S. typhimurium respectively. For instance, Ethanol treated bacterial organisms expressed Minimum Inhibition.

Among the two methanol and ethanol extracts, methanol extracts recorded significant antibacterial activity followed by extract with ethanol. Furthermore, Salmonella typhii found highly susceptible to methanol extract, where as Proteus mirabilis was less susceptible to both methanol and ethanol extracts. However, P. aeruginosa was found highly susceptible to ethanol extract. Methanol extract exhibited similar antibacterial activity against E. coli, P. aeruginosa, S. paratyphi and S. sonnei where it was around 16 and 14 mm zone of inhibition. Similar result was absorbed for ethanol against Citrobacter sp., S. paratyphi, S. typhimurium, S. aureus and S. faecalis.

The chloroform extract showed highest activity against K. pneumonia. Inhibition zone of 10 mm and above was observed in S. typhii, S. faecalis and Citrobacter sp. Antibacterial activity of aqueous extract varied greatly among the different test pathogenic bacteria. Moreover, maximum antibacterial activity was observed against S. sonnei followed by S. typhii, even though antibacterial activity was observed against other pathogenic bacteria also it was not found significant. Inhibition zone more than 10 mm noted against Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium and Streptococcus faecalis. Both methanol and ethanol showed significant antibacterial activity against Escherichia coli and P. aeruginosa compared to chloromphenicol. The inhibition zone of methanol extracts of Citrobacter sp. was equal to that of the chloromphenicol.

Table 2:	Mortality rate of Culex quinquefasciatus mosquito larvae at different concentrations of aqueous extracts of Hemidesmus indicus root

Larvicidal activity: The larvicidal activity of aqueous extracts of H. indicus root against the larvae C. quinquefasciatus mosquito was given in Table 2. The larvicidal activity on aqueous extract of H. indicus roots showed 28, 55 and 65% of death with the use of 1, 2 and 3% concentrations, respectively, after 3 days. The third day 4% concentration revealed mortality rate was higher (95%) when compared to other treated larvae. Finally, 100% mortality has been observed on higher percentage (5%). Among the three days experiment the optimum level of mortality seen on the third concentration such as 36. 2, 54.3 and 69.7.

DISCUSSION

In the recent years development of multi drug resistance in the pathogenic bacteria and Pseudomonas aeruginosa compared to chloromphenicol, where as against other test pathogens it was not significant. The overall results clearly showed the extract of this medicinal plant root was detected with inhibition zone size ranged from 10.65 to 17.69 for methanol, 0.9 to 16.30 for Ethanol and 0.7 to 12.50 mm for Chloroform. Some other problems such as toxicity of certain antimicrobial drugs on the host tissue triggered interest in searching of new antibacterial drugs of plant origin. Hence the present investigation reveals the screening and scientific evaluation of plants and suggests that this plant could be exploited in the management of diseases caused by these bacteria in human systems (Das et al., 2003). It is interesting to note that antibacterial activity was highly pronounced in solvent extract compared to aqueous extract of H. indicus (Gayathri and Kannabiran, 2009). It is also important to note that susceptibility of the pathogens was varied to solvent extracts and aqueous extract (Wiesman and Chapagain, 2006) If a new plant substance, which has never been used before and about which nothing is known has been developed then the requirements are the same as those required for a new synthetics. This kind of similar results has been observed on another plants based experiment by Aqil et al. (2006), Joseph and Sujatha (2011). Again, Khanna and Kannabiran (2008) demonstrated the time kill assay with most promising fractions of this plant extracts concentration-dependent killing of MRSA within 9-12 h of incubation also accompanying with Pure saponin extract exhibited remarkable antimicrobial activity against Staphylococcus aureus, Salmonella typhi, Klebsiella pneumoniae and fungus like Aspergillus flavus, Aspergillus fumigatus and Aspergillus niger. Anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of ethanolic extracts of four medicinal plants namely Acorus calamus (Rhizome) H. indicus (Stem), H. antidysenterica (bark) and Plumbago zeylanica (root), were detected with inhibition zone size ranged from 11 to 44 mm and Minimum Inhibitory Concentration (MIC) varied from 0.32 to 3.25 mg mL^-1.

It was reviewed that the methanolic extracts of H. indicus possessed inhibitory activity against S. typhimurium, E. coli and S. flexneri in vitro cultures by agar well diffusion method (Das et al., 2003; Das and Devaraj, 2006). For instance, Anoop et al. (2003), Anoop and Jegadeesan (2003) have been established the helicobactericidal activity of various extracts against Helicobacter pylori, which is comparable to standard antibacterial. The comparative efficacy with chloromphenicol is also highly encouraging. E. coli and P. aeruginosa are two important pathogens causing urinary track infection. Both these microorganisms were highly susceptible to methanol and ethanol extract of H. indicus compared to chloromphenicol. Thus, the present study records the scientific validation of this plant for use as an antibacterial agent.

The current investigation also gives importance for the larvicidal effect of H. indicus of aqueous extract. A commercial saponin mixture extracted from Q. saponaria showed increasing toxicity in Anopheles aegypti and Culex pipiens when both saponin concentration and the duration of the experiment were increased (Pelah et al., 2002). Aluminium chloride, known for its phenolic complexing activity, obtained from alder leaf also reported to have the larvicidal activity against A. aegypti (David et al., 2000). Monoterpene hydrocarbons showed a marked mosquito larvicidal activity against C. pipiens which is obtained from the fresh leaves of Anthemis melampodina and Pluchea dioscoridis (Massoud et al., 2001). A piperidine alkaloid from Piper longum fruit was found to be active against mosquito larvae of C. pipiens (Lee, 2000). Similarly an alkaloid derived from the tropical vine Triphyophyllum peltatum (Dioncophyllaceae), was found to have larvicidal activity against the malaria vector Anopheles stephensi (Francois et al., 1996). The comparative study of larvicidal activity of root extract of H. indicus leaves extracts of G. sylvestre and E. prostrata was established against C. quinquefasciatus mosquito (Gopiesh and Kannabiran, 2007). Here, this result showed that the plant H. indicus having higher larvicidal activity, so it is suggested that the aqueous extract of this medicinal plant can be used as eco-friendly and sustainable insecticide to control mosquito. Several reports are available in support of antibacterial activity of several phytochemicals present in plant extracts (Slobodnikov et al., 2004; Chacha et al., 2005; Divysree and Cherupally, 2010). Antibacterial activity of tannins and saponins isolated from plant species are well documented (Mandal et al., 2005). However, further studies are needed to evaluate the antibacterial activity of isolated phytochemicals such as tannins and saponins from these plants against pathogenic bacterial strains. In conclusion, aqueous extracts of the roots of H. indicus exhibited significant antibacterial activity against the tested bacterial strains. Presence of tannins and saponins in higher concentration than the other phytochemicals suggest that these phytochemicals could be responsible for the antibacterial activity (Chakradhar et al., 2005). However, further studies are needed to establish that these plant extracts could form effective antimicrobial therapy against common bacterial diseases.

When the mosquito larvae were treated with various concentration and different treatment periods as a result of this minimum mortality rate has been observed on lowest concentration (1 mg mL^-1) 24.5% followed by medium level of mortality was observed on optimum level of concentration 3 mg mL^-1 (Table 2). When the concentration increased accompanied with prolonged exposure periods all the larvae were died, since this Table 2 clearly showed peak level concentration was 5 mg mL^-1 because this concentration was most probably suitable for control the mosquito larva (Culex quinquefasciatus) in our environment.

ACKNOWLEDGEMENT

The authors gratefully acknowledged to our Malankara Catholic College Correspondent Fr. Premkumar (M.S.W) given encouragement and support for preparation of this research manuscript. The second author was very grateful to Department of Biotechnology, Annai Velankanni College and Tholayavattam for providing facilities of this research.