CHAPTER 8 ANTIBACTERIAL ACTIVITY OF THE CRUDE ETHANOLIC EXTRACT AND THE ISOLATED COMPOUNDS FROM THE STEM OF COSTUS IGNEUS

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1 CHAPTER 8 ANTIBACTERIAL ACTIVITY OF THE CRUDE ETHANOLIC EXTRACT AND THE ISOLATED COMPOUNDS FROM THE STEM OF COSTUS IGNEUS 8.1 INTRODUCTION Medicinal plants are the backbone of traditional medicine and the antibacterial activity of plant extract is due to different chemical agent in the extract, which was classified as active antimicrobial compound (Doughari et al., 2007; Kumar et al., 2009; Ignacimuthu et al., 2009; Adegoke and Adebayo-tayo, 2009). In recent years, pharmaceutical companies have been doing phytochemical research and investing billions of dollars in developing natural remedies to produce drugs in affordable price to general population (Doughari, 2006). The rising incidence in multidrug resistance amongst pathogenic microbes has further necessitated the need to search for fewer antibiotic sources from plants (Mohamed Khadeer Ahamed et al., 2007). Costus igneus is traditionally used in India to control diabetes which is also known as fiery costus or spiral flag or insulin plant are rich in protein(18%), iron(40mg) and antioxidant components such as ascorbic acid, β-carotene, α- tocopherol, glutathione, phenols, flavonoids, steroids, alkaloids and terpenoids (Devi and Urooj, 2008; Devi and Urooj, 2010). Gallium (Ga), a trivalent semi-metal that is chemically similar to Fe 3+, is taken up some bacteria (Bernstein, 1998). The ability of gallium to inhibit growth of intracellular bacteria by interfering with bacterial iron metabolism was established in vitro by incorporating gallium nitrate in culture media with Mycobacterium spp. (Olakanmi et al., 2000). Those investigators further demonstrated that the antimicrobial effect was due to the Gallium moiety of the Gallium nitrate, not the nitrate. The potential use of gallium as a chemoprophylactic and chemotherapeutic agent for the control of infection (Bernstein et al., 2000). The antimicrobial effects of gallium were completely abolished when media were supplemented with excess iron, providing evidence that gallium nitrate interferes with 138

2 iron dependent mechanisms that are crucial to the growth and survival of Rhodococcus equi (Boland and Meijer, 2000). 8.2 SPECIFIC AIM The purpose of this study was to examine the antibacterial activity of the crude ethanolic extracts and the isolated compounds (Lupeol and Stigmasterol) toward urinary tract infection causing pathogens using disc diffusion method. 8.3 MATERIALS AND METHODS Collection of plant material and test organisms The stem of Costus igneus used in this experiment were collected from the nursery of the Periyar Maniammai University, Vallam, Thanjavur and identified at Rapinat herbarium, St. Joseph College, Tiruchirapalli, Tamil Nadu, India. To examine the antibacterial activity of each plant extract and the isolated compounds, five strains [Escherichia coli (MTCC 25922), Enterococcus aerogenes (MTCC 29212). Pseudomonas aeruginosa (MTCC 27853), Staphylococcus aureus (MTCC 25923) and Proteus vulgaris (MTCC 7299)] were prepared as test organisms. All the strains were procured from the Microbial Type Culture and Collection (MTCC) at Chandigarh, India. Bacterial strains were cultivated at 37ºC and maintained on nutrient agar (Difco, USA) slant at for 4ºC Preparation of ethanol extracts Extraction procedure was carried out as described in chapter VI Antibacterial activity of crude ethanolic extract and the isolated compound (disc diffusion method) Antibacterial activity of crude ethanolic extract and the isolated compound were determined using the disc diffusion method (Ignacimuthu et al., 2009). The petridishes (diameter 60 mm) was prepared with Muller Hinton Agar and inoculated with test organisms. Sterile disc of six millimeter width were impregnated with 10 µl of crude ethanolic extract of Costus igneus at various concentrations of mg/ml 139

3 and for isolated compound mg/ml respectively. Prepared discs were placed onto the top layer of the agar plates and left for 30 minute at room temperature for compound diffusion. Negative control was prepared using the respective solvent and chloramphenicol (10 µg/disc) was used as a positive control. The dishes were incubated for 24 h at 37ºC and the zone of inhibition was recorded in millimeters and the experiment was repeated twice Minimum inhibitory concentration (MIC) for crude extract and isolated compound A broth dilution technique was used to determine the MIC values of crude ethanolic extract and isolated compound from the stem of Costus igneus. The crude ethanolic extract and isolated compound was dissolved in 2% dimethyl sulfoxide (DMSO) aqueous solution. A volume of 0.1 ml from each serial dilution of crude extract concentration ( µg/ml) and for isolated compound ( µg/ml) was added into tubes and were inoculated with 0.1 ml of overnight cultured bacterial strains ( 10 8 cfu/ml) and incubated at 31ºC for 48 h. Chloramphenicol was used as a positive control. The MIC value was the lowest concentration of compound that showed no growth after 48h of incubation in comparison with the control tube, which included 9.8 ml of nutrient broth and 0.1 ml bacterial strains in addition to 0.1 ml of test compound concentration (un incubated). 8.4 RESULTS ND DISCUSSIONS Antibacterial activity of crude extracts and isolated compounds The crude extract and isolated compounds from the stem of Costus igneus were tested against the UTI causing pathogenic microbes viz. Escherichia coli a most common bacteria of which virulent strains can cause gastroenteritis, urinary tract infections, neonatal meningitis; Staphylococcus aureus which cause septicemia and endocarditis in immune compromised patients; Pseudomonas aeruginosa which infects the pulmonary tract, urinary tract, burns and wounds (Nascimento et al., 2000). The results of the antimicrobial activity of crude extracts and isolated 140

4 compounds were tested against UTI by disk diffusion method are shown in (Tables 8.1 and 8.2). The crude extracts showed growth inhibitory activity against Escherichia coli and Enterococcus aerogenes (15 mm), Pseudomonas aeruginosa and Staphylococcus aureus (14 mm) at concentration 250 mg/ml (Figure 8.1). At concentration 200 mg/ml, the crude extracts exhibited the antibacterial activity all the five bacteria, but was more susceptible against Escherichia coli (14 mm), Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus aerogenes (13 mm). However, the crude extract showed better inhibitory actions against urinary tract infection causing pathogens at a concentration 150, 200 and 250 mg/ml than at lower concentration. As the concentration of extracts increased from mg/ml, the inhibitory actions of the plant extracts increased towards all the strains used in this study. Agar disc diffusion method was used to evaluate the antibacterial activity of isolated compounds by measuring the inhibition zone against the test microorganisms. Lupeol (4 mg/disc) exhibited the prominent antibacterial activity all the five bacteria but was more susceptible against Escherichia coli and Enterococcus aerogenes (22 mm) and Pseudomonas aeruginosa (20 mm) whereas for stigmasterol (4 mg/disc) was more susceptible against Enterococcus aerogenes (22 mm), Staphylococcus aureus (20 mm) and Pseudomonas aeruginosa (19 mm). At lower concentrations (0.50 mg/disc), Lupeol showed maximum inhibitory activity against Escherichia coli (15 mm), Pseudomonas aeruginosa (16 mm) followed by Staphylococcus aureus and Enterococcus aerogenes (11 mm) (Figure 8.2). whereas, Stigmasterol (0.5 mg/discs) showed activity against Enterococcus aerogenes (16 mm), Escherichia coli (14 mm), Proteus vulgaris (13 mm) (Figure 8.3). All compound concentrations were active against all tested pathogens. Among all, Lupeol exhibited more pronounced activity. The Bioactive compound isolated from stem bark of petroleum extract of Grewia titiaefolae demonstracted significant antibacterial activity against gram negative bacteria Pseudomonas aeruginosa and Klebsiella pneumonia (Mohamed Khadeer Ahamed et al., 2007). The extracts of medicinal plants and gallium nitrate 141

5 showed optimum activity against all tested urinary tract infection pathogens. However, result of disc diffusion method as indicated in (Table 8.2) revealed that gallium nitrate represent significant activity against the selected organism. Table 8.1 Antibacterial activity of ethanolic extracts of stem of Costus igneus Plant extracts Costus igneus (stems) Gallium nitrate Concentration (mg/ml) µl/disc 20 µl/disc Escherichia Coli 10± ± ± ± ± ± ± 0.81 Organisms/Zone of inhibition (mm±sd) Pseudomonas aeroginosa 8± ± ± ± ± ±0.3 22±0.21 Staphylococcus aureus 8 ± ± ± ± ± ± ±0.40 Enterococcus aerogenes 8 ± ± ± ± ± ± ± 0.21 Proteus vulgaris 7± ±0.3 11± ± ± ± ±0.21 Chloramphenicol 10 µl/disc 22± ± ± ± ± 0.81 DMSO 10 µl/disc Values showed the diameter of inhibitory zone (mm)±sd for each four replicates. The mean values are significant different at p<0.05 level. DMSO denotes dimethyl sulfoxide. Table 8.2 Antibacterial activity of isolated compound from the stem of Costus igneus Isolated Compound Concentration (mg\ml) Escherichia Coli Organisms\Zone of inhibition (mm) Pseudomonas aeroginosa Staphylococcus aureus Enterococcus aerogenes Proteus vulgaris Lupeol Stigmasterol Gallium nitrate Chloramphenicol DMSO µl/disc 10 µl/disc 10 µl/disc 21± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Values showed the diameter of inhibitory zone (mm)±sd for each four replicates. The mean values are significant different at p<0.05 level. DMSO denotes dimethyl sulfoxide 18 ± ± ± ± ± ± ± ± ± ±

6 8.4.2 Determination of minimum inhibitory concentration (MIC) of crude extract and isolated compounds In view of the results obtained by the disc diffusion method, the minimum inhibitory concentration (MIC) of crude extract and active compounds from Costus igenus were determined by broth dilution technique (Tables 8.3 and 8.4). The highest MIC value for crude extract (25 µg/ml) was observed against Staphylococcus aureus, while Proteus vulgaris (25 µg/ml) and the lowest MIC value for crude extract (3.12 µg/ml) was observed against Escherichia coli and Pseudomonas aeruginosa (6.25 µg/ml). Lupeol and Stigmasterol showed antibacterial activity against the five urinary tract infection causing pathogens used. Lupeol was generally the most effective of the two compounds (Table 8.4). Both compounds were compared with standard antibiotic chloramphenicol. The lowest MIC of Lupeol (15.6 µg/ml) were recorded against Escherichia coli and Enterococcus aerogenes followed by Pseudomonas aeruginosa and Staphylococcus aureus, the MIC value (31.2 µg/ml). The highest MIC of Lupeol (62.5 µg/ml) was observed against Proteus vulgaris. The lowest MIC value for Stignasterol (15.6 µg/ml) were recorded against Staphylococcus aureus, whereas Pseudomonas aeruginosa and Enterococcus aerogenes exhibited the lowest MIC (31.2 µg/ml). The standard chloramphenicol was active against all reference bacteria (MIC range µg/ml; zone of inhibition range: mm). Among all Lupeol demonstracted good antibacterial activity against all the tested pathogens. Table 8.3 Minimal Inhibitory Concentration of ethanolic extract of Costus igneus Microorganisms MIC values (µg/ml) Ethanol extracts Chloramphenicol Gallium nitrate Escherichia coli Pseudomonas aeroginosa Staphylococcus aureus Enterococcus aerogenes Proteus vulgaris

7 Table 8.4 Minimal Inhibitory Concentration of isolated compound from the stem of Costus igneus Microorganisms MIC values (µg/ml) Lupeol Stigmasterol Chloramphenicol Gallium Nitrate Escherichia coli Pseudomonas aeroginosa Staphylococcus aureus Enterococcus aerogenes Proteus vulgaris CONCLUSION These results suggest that pentacyclic triterpenoid compound Lupeol and steroidal compound Stigmasterol isolated from the stem of Costus igneus had good antibacterial activity against five selected urinary tract infection causing organisms as compared with crude ethanolic Costus igneus extracts. Lupeol from Costus igneus represent a significant activity against selected urinary tract infection pathogens as compared with Stigmasterol compound. 144

8 Figure 8.1 The Antibacterial activity of crude ethanolic extract of stem of Costus igneus on the test organisms 145

9 Figure 8.2 The Antibacterial activity of Lupeol from crude ethanolic extract of stem of Costus igneus on the test organisms 146

10 Figure 8.3 The Antibacterial activity of Stigmasterol from crude ethanolic extract of stem of Costus igneus on the test organisms 147

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