In Vitro Antimicrobial Activity Studies

Transcription

1 Chapter - 1 In Vitro Antimicrobial Activity Studies Water is H 2 O, hydrogen two parts, oxygen one, but there is also a third thing, that makes it water and nobody knows what that is. - D.H. Lawrence

2 Introduction Introduction The use of plants and plant products as medicine could be traced far back as the beginning of human civilization. From ancient literature it is evident that various parts of the plants were used in Ayurveda, Unani and Siddha medicines for the treatment of disease of human beings (Das et al., 2009). Medicinal plants constitute the main source of new pharmaceuticals and health care products (Ivanova et al., 2005). Extraction and characterization of several active phytocompounds from these green factories have given birth to some of the high activity profile drugs (Mandal et al., 2007). It is believed that crude extracts from medicinal plants are more biologically active than isolated compounds due to their synergistic effects (Jana and Shekhawat, 2010). Phytochemical screening of plants has revealed the presence of numerous chemicals including phenols, flavonoids, alkaloids, steroids, saponins and glycosides. These secondary metabolites of plants serve as medicine in the treatment of various infectious diseases (Cowan, 1999). Herbal medicines have become more popular in the treatment of many diseases due to the belief that green medicine is safe, easily available and with fewer side effects. Given the alarming incidence of antibiotic resistance in bacteria of medical importance, there is a constant need for new and effective therapeutic agents which are natural, stable, non toxic and multifunctional. A systematic review of literature on research work of A. paniculata, T. cordifolia and T. foenum-graecum have revealed that there are very few studies showing the antibacterial activity of these plants, especially the leaves. Surprisingly, there are no reports on the antifungal activities of these plants on dermatophytes. Hence, in this study we have screened for the antibacterial activity in different solvent leaf extracts of these plants using clinical isolates causing urinary tract infections. Antifungal activity studies were performed on fungi causing dermatomycosis. Phytochemical screening of the leaf extracts were also carried out to know the active compounds responsible for the antimicrobial activity. The details of the work carried out are presented in this chapter. 28

3 Material and Methods Material and Methods Plant material Fresh and healthy leaves of A. paniculata and T. cordifolia were obtained from local growers of Mysore. T. foenum-graecum leaves were obtained from the local market, Mysore. The sample specimen was identified based on the taxonomical characteristics. The leaves were washed thoroughly in distilled water and the surface water was removed by air drying under shade. The leaves were subsequently dried in a hot air oven at 40 C for 48 h, powdered to mesh in an apex grinder [Apex Constructions, London] and used for extraction. Chemicals Solvents viz., chloroform, hexane, methanol and ethanol were of AR grade from Merck (Mumbai, India). Nutrient agar, nutrient broth, saborauds dextrose broth and saborauds dextrose agar were from Himedia Laboratories (Mumbai, India). Ferric chloride, phenol reagent, α-napthol, sodium carbonate, sodium hydroxide, copper sulphate, acetic anyhydride, glacial acetic acid, iodine and potassium iodide were from SD Fine Chemicals (Mumbai, India). Test microorganisms The test organisms used were clinical isolates viz., Escherichia coli, Proteus vulgaris, Klebsiella sp., Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogens, Trichophyton rubrum and Epidermophyton floccosum which were obtained from Department of Microbiology, JSS Medical College, Mysore. The bacterial and the fungal cultures were maintained on nutrient agar medium and saborauds dextrose agar (SDA) medium respectively. Preparation of aqueous extract Fifty grams each of powdered leaves of A. paniculata, T. cordifolia and T. foenum-graecum were macerated with 100 ml sterile distilled water in a blender for 10 min. The macerate was first filtered through double layered muslin cloth and centrifuged at 4000 rpm for 30 min. The supernatant was filtered through Whatman No.1 filter paper and heat sterilized at 120 C for 30 min. The extracts were preserved aseptically in brown bottles at 4 C until further use. 29

4 Material and Methods Preparation of solvent extract Extraction was carried out according to the method of Okigbo et al. (2005). Fifty grams each of the powdered material was extracted initially with 300 ml of chloroform, hexane, methanol and ethanol separately for 24 h at 23 ± 2 C. The extract was filtered with Whatman No. 1 filter paper into a clean conical flask. Second extraction was carried out with same amount of solvent for another 24 h at 23 ± 2 C and filtered. The extracts were later pooled and transferred into the sample holder of the rotary flash evaporator [Buchi Rotavapor R-124, Switzerland] for the evaporation of the solvents. The evaporated extracts so obtained were preserved at 4 C in airtight bottles until further use. Antibacterial screening The antibacterial activity was carried out by disc diffusion method (Bauer et al., 1966). Bacterial cultures (adjusted to CFU/ml using spectrophotometer) were used to lawn nutrient agar plates evenly using a sterile swab. The plates were dried for 15 min and sterile discs (5 mm in diameter, Whatman No.1) impregnated with 10 µl (1 mg/ml) of the plant extracts were placed on the nutrient agar surface. 10 µl of the respective solvent served as the negative control. Streptomycin standard antibiotic disc served as the positive control (10 µg/disc). The plates were then incubated at 37 C for h. After overnight incubation the plates were examined for the zone of inhibition. Determination of Minimum Inhibitory Concentration The minimum inhibitory concentration (MIC) was carried out by broth dilution method (Brantner and Grein, 1994). The test organisms were grown in nutrient broth medium to a concentration of CFU/ml. Extract of about 0.5 ml ( mg/ml) was mixed with 4 ml of nutrient broth inoculated with 0.5 ml of bacterial suspension. The tubes containing 4.5 ml of broth and 0.5 ml of bacterial suspension served as bacterial control and 5 ml of un-inoculated broth served as blank. The tubes were incubated at 37 C for 18 h. Inhibition of bacterial growth was determined by measuring the absorbance at 600 nm in a colorimeter. The lowest concentration of the compound that inhibits the growth of the organism was 30

5 Material and Methods determined as the MIC. The percentage of growth inhibition was calculated according to the formula: Per cent growth inhibition = [(A control A test ) / A control ] 100 Antifungal activity assay Mycelial dry weight method was carried out to determine the antifungal activity of the ethanol extracts at concentrations ranging from 1 to 10 mg/ml according to the method of Rasooli and Abyaneh (2004). The dermatophytes grown on SDA medium for a week were flooded with 0.85% saline. After settling of the larger particles, conidia were counted with a haemocytometer and diluted in saborauds dextrose broth to a final spore concentration of spores/ml. For antidermatophytic assay in broth, 5 ml of sterile saborauds dextrose broth medium taken in screw capped tubes were inoculated with 20 µl of fungal suspension and 1-10 mg/ml concentration of the extract. The tubes were incubated for a week at 30 C. The visible mycelial growth in the tubes expressed the degree of activity of the extract. Fungal mycelia from the above tubes were separated by passing through Whatman No. 1 filter paper. The filter paper was allowed to dry at 60 C to reach a constant weight. Fungal growth inhibition was calculated by considering the control and sample mycelial dry weights. Ketaconozole was used as a standard antifungal agent. The percentage of growth inhibition was calculated according to the formula: Per cent growth inhibition = [(A control A test ) / A control ] 100 Phytochemical screening Phytochemical analysis of ethanol leaf extracts of A. paniculata, T. cordifolia and T. foenum-graecum was carried out qualitatively to test for the presence of carbohydrates, proteins, tannins, phenols, flavonoids, cardiac glycosides, phytosterols, saponins and alkaloids (Harborne, 1998). Test for carbohydrates Molisch s test: Two drops of Molisch s reagent was added to 2-5 ml of the extract in a test tube to which 1 ml of concentrated sulphuric acid was allowed to flow down the side of the tube. Appearance of a violet ring at the junction indicated the presence of carbohydrates. 31

6 Material and Methods Test for proteins Biuret test: To 2 ml of the extract, 2 ml of 10% sodium hydroxide was added and mixed well. To this, 2 drops of 0.1% copper sulphate solution was added. Formation of violet or pink colour indicated the presence of proteins. Test for phenols FC reagent method: A known volume of the extract was made up to 3 ml with distilled water to which 0.5 ml of freshly prepared FC reagent was added. After 3 5 min, 2 ml of 20% sodium carbonate was added. Appearance of blue colour indicated the presence of phenols. Test for flavonoids Ferric chloride test: To a small amount of extract, neutral ferric chloride solution was added. Appearance of blackish red colour indicated the presence of flavonoids. Test for tannins Ferric chloride test: Extract was treated with alcoholic ferric chloride solution. Formation of blue colour indicated the presence of tannins. Test for terpenoids Libermann Burchard test: Ten milligram of the extract was dissolved in 1 ml of chloroform and 1 ml of acetic anhydride. Concentrated sulphuric acid (2 ml) was added along the sides of the test tube. Formation of reddish violet colour ring at the junction indicated the presence of terpenoids. Test for steroids Salkowski test: Extract was dissolved in chloroform and few drops of concentrated sulphuric acid were added to it. Formation of red colour in chloroform layer suggested the presence of steroids. Test for cardiac glycosides Keller Kiliani test: The extract was treated with 1 ml of ferric chloride reagent (mixture of 1 volume of 5% ferric chloride and 99 volumes of glacial acetic acid). To this solution a few drops of concentrated sulphuric acid was added. Appearance of greenish blue colour indicated the presence of cardiac glycosides. 32

7 Material and Methods Test for alkaloids Mayers test: To a small amount of the extract, potassium mercuric iodide solution was added. Formation of cream precipitate indicated the presence of alkaloid. Wagners test: To a small amount of extract, iodine-potassium solution was added. Appearance of reddish brown precipitate indicated the presence of alkaloid. Test for saponins Frothing test: The dry extract was vigorously shaken with distilled water and was allowed to stand for 10 min. Stable froth indicated the presence of saponins. Statistical analysis The experimental results are expressed as mean ± standard deviation (SD) of triplicate measurements. The data was subjected to One Way Analysis of Variance (ANOVA) and the significance of differences between the sample means was calculated by Turkeys test. Data was considered statistically significant at P value Statistical analysis was performed using Graph Pad statistical software. 33

8 Results and discussion Antibacterial screening of solvent extracts The antibacterial activity of different solvent extracts of A. paniculata leaf against the pathogenic bacteria showed varied levels of inhibition. As shown in Table 1.1, among the solvent extracts tested, ethanol extract had a broad spectrum of activity against all the bacteria tested and showed the highest zones of inhibition against P. aeruginosa (15.0 mm) and S. aureus (13.0 mm). The least zone of inhibition was observed with the chloroform extract which showed an inhibition of 1.0 mm against E. coli, 2.2 mm against S. aureus and 3.0 mm against P. aeruginosa and did not show any inhibition against E. aerogenes, P. vulgaris and Klebsiella sp. Table 1.1 : Zone of inhibitory activity (in millimeter) of different solvent extracts of Andrographis paniculata leaves against the test bacteria Solvent extract E. coli E. aerogenes Klebsiella sp. P. vulgaris S. aureus P. aeruginosa Aqueous Ethanol Methanol Hexane Chloroform Streptomycin 2.0 ± 2.0 a 11.0 ± 1.0 bc 8.0 ± 2.64 b 3.0 ± 1.0 a 1.0 ± 0.0 a 15.0 ± 2.0 c 0.0 ± 0.0 a 10.0 ± 1.73 c 6.0 ± 1.73 b 1.0 ± 1.0 a 0.0 ± 0.0 a 14.0 ± 1.73 d 0.0 ± 0.0 a 8.0 ± 3.0 bc 5.0 ± 1.0 b 0.0 ± 0.0 a 0.0 ± 0.0 a 11.0 ± 1.72 c 1.0 ± 1.0 a 9.0 ± 1.70 bc 7.0 ± 2.0 b 2.0 ± 1.0 a 0.0 ± 0.0 a 12.0 ± 1.73 c 3.0 ± 1.73 a 13.0 ± 2.64 b 9.0 ± 1.0 b 4.0 ± 1.73 a 2.2 ± 0.26 a 11.0 ± 2.0 b 4.0 ± 1.72 a 15.0 ± 1.74 c 10.5 ± 1.80 b 4.5 ± 1.32 a 3.0 ± 1.0 a 14.0 ± 1.70 bc (10µg/disc) 1 mg/ml concentration of the extract used Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) Table 1.2 represents the antibacterial activity of T. cordifolia leaf extracts in different solvent extracts against the pathogenic bacteria. The highest antibacterial activity as indicated by the zone of inhibition was achieved with ethanol extract which showed inhibition zones of 9.0 mm, 6.6 mm, 6.1 mm, 6.0 mm, 12.0 mm and 9.6 mm against E. coli, E. aerogenes, Klebsiella sp., P. vulgaris, S. aureus and P. aeruginosa respectively. Among the bacteria tested, S. aureus showed maximum susceptibility to T. cordifolia ethanol extract. Chloroform and aqueous extracts showed the least antibacterial activity. 34

9 Table 1.2 : Zone of inhibitory activity (in millimeter) of different solvent extracts of Tinospora cordifolia leaves against the test bacteria Solvent extract E. coli E. aerogenes Klebsiella sp. P. vulgaris S. aureus P. aeruginosa Aqueous 1.0 ± 2.0 ab 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 3.0 ± 1.0 a 2.0 ± 0.0 a Ethanol 9.0 ± 1.0 d 6.6 ± 0.57 d 6.1 ± 0.9 c 6.0 ± 1.0 c 12.0 ± 2.0 d 9.6 ± 0.5 c Methanol 5.6 ± 0.57 c 4.4 ± 0.50 c 4.3 ± 0.4 bc 4.0 ± 1.0 bc 10.0 ± 0.0 cd 8.3 ± 0.5 bc Hexane 3.1 ± 0.17 bc 2.2 ± 0.20 b 2.6 ± 0.2 b 2.3 ± 0.1 ab 7.2 ± 1.3 bc 6.8 ± 0.7 b Chloroform 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 4.1 ± 0.3 ab 3.5 ± 0.9 a Streptomycin (10µg/disc) 15.0 ± 2.0 e 14.0 ± 1.73 e 11.0 ± 1.72 d 12.0 ± 1.73 d 11.0 ± 2.0 d 14.0 ± 1.70 d 1 mg/ml concentration of the extract used Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) The efficacy of various solvent extracts of T. foenum-graecum leaf against the pathogenic bacteria showed varied levels of inhibition (Table 1.3). Among the various extracts, maximum in vitro inhibition of the tested bacteria E. coli, E. aerogenes, Klebsiella sp., P. vulgaris., S. aureus and P. aeruginosa was achieved in ethanol extract with zones of inhibition of 12 mm, 11 mm, 10 mm, 12 mm, 14 mm and 13 mm respectively. Methanol extract showed an inhibition of 12 mm against S. aureus and 10 mm against P. aeruginosa. As with other solvent extracts tested, a zone of inhibition greater than 10 mm could not be achieved with any of the bacteria. The least inhibition was observed with the aqueous extract. Table 1.3 : Zone of inhibitory activity (in millimeter) of different solvent extracts of Trigonella foenum-graecum leaves against the test bacteria Solvent extract E. coli E. aerogenes Klebsiella sp. P. vulgaris S. aureus P. aeruginosa Aqueous 2.0 ± 0.2 cd 1.0 ± 0.4 c 1.0 ± 0.5 d 2.0 ± 0.7 d 3.0 ± 0.5 ef 2.0 ± 0.0 de Ethanol 12.0 ± 0.8 ab 11.0 ± 0.2 a 10.0 ± 0.3 a 12.0 ± 0.5 a 14.0 ± 0.5 a 13.0 ± 0.0 a Methanol 9.0 ± 0.4 b 8.0 ± 0.4 b 8.0 ± 0.2 b 10.0 ± 0.4 bc 12.0 ± 0.7 b 10.0 ± 0.5 b Hexane 5.0 ± 0.7 c 5.0 ± 0.4 c 4.0 ± 0.4 c 5.0 ± 0.5 c 7.0 ± 0.2 d 5.0 ± 0.3 c Chloroform 3.0 ± 0.4 c 2.0 ± 0.7 c 1.0 ± 0.2 d 2.0 ± 0.0 d 4.0 ± 0.7 e 3.0 ± 0.5 d Streptomycin (10µg/disc) 15.0 ± 2.0 e 14.0 ± 1.73 e 11.0 ± 1.72 d 12.0 ± 1.73 d 11.0 ± 2.0 d 14.0 ± 1.70 d 1 mg/ml concentration of the extract used Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) 35

10 Fig. 1.1 : Antibacterial activity of ethanol leaf extract of Andrographis paniculata against the test bacteria (a) Escherichia coli (b) Enterobacter aerogenes (c) Klebsiella sp. (d) Proteus vulgaris (e) Staphylococcus aureus and (f) Pseudomonas aeruginosa 36

11 Fig. 1.2 : Antibacterial activity of ethanol leaf extract of Tinospora cordifolia against the test bacteria (a) Escherichia coli (b) Enterobacter aerogenes (c) Klebsiella sp. (d) Proteus vulgaris (e) Staphylococcus aureus and (f) Pseudomonas aeruginosa 37

12 Fig. 1.3 : Antibacterial activity of ethanol leaf extract of Trigonella foenumgraecum against the test bacteria (a) Escherichia coli (b) Enterobacter aerogenes (c) Klebsiella sp. (d) Proteus vulgaris (e) Staphylococcus aureus and (f) Pseudomonas aeruginosa 38

13 Fig. 1.4 : Antibacterial activity of the standard antibiotic streptomycin (10 µg/disc) against the test bacteria (a) Escherichia coli (b) Enterobacter aerogenes (c) Klebsiella sp. (d) Proteus vulgaris (e) Staphylococcus aureus and (f) Pseudomonas aeruginosa 39

14 The traditional medicinal methods, especially the use of medicinal plants, still play a vital role to cover the basic health needs in the developing countries and moreover, the use of herbal remedies has risen in the developed countries in the last decade. Plants have provided a source of inspiration of novel drug compounds, as plant derived medicines have made large contributions to human health and well being. Their role is twofold namely; they provide key chemical structure for the development of new antimicrobial drugs and also as a phytomedicine to be used for the treatment of disease (Abukakar et al., 2008). It is anticipated that phytochemicals with adequate antibacterial efficacy will be used for the treatment of bacterial infections (Balandrin et al., 1985). The first step towards this goal is the in vitro antibacterial activity assay (Tona et al., 1998). Diffusion method is extensively used to investigate the antimicrobial activity of natural substances and plant extracts. These assays are based on the use of discs or holes as reservoirs containing the solutions of substances to be examined (Rauha et al., 2000). In the present study, bacteria commonly causing urinary tract infection (UTI) have been selected. Urinary tract infection represents one of the most common diseases occurring from the neonates to the geriatric age groups (Raju and Tiwari, 2004). The increasing drug resistance among the bacteria causing UTI has made therapy difficult and has led to greater use of expensive broad spectrum drugs. This resistance problem needs a renewed effort resulting in searching of effective antibacterial agents against pathogenic microorganisms resistant to current antibiotics (Soulsby, 2005). The chloroform, hexane, methanol, ethanol and water extracts of the leaves of A. paniculata, T. cordifolia and T. foenum-graecum were subjected to a preliminary screening for antimicrobial activity against six human pathogenic bacteria commonly associated with UTI infections. It was clear from the present results that ethanol leaf extract of all the three plants had a broad spectrum of activity against all the tested six bacteria. This tends to show that the active ingredients of the leaves are better extracted with ethanol than with any other solvent. Since, nearly all of the identified compounds from plants active against microorganisms are aromatic or saturated organic compounds, they are most often obtained through initial ethanol or methanol extraction (Cowan, 1999). Earlier studies have also reported the greater activity of 40

15 ethanol extracts compared to other solvent extracts (Aqil and Ahmad 2003; Allero and Afolayan, 2006; Parekh and Chanda, 2007b; Kannan et al., 2009). Least antibacterial activity was observed in the aqueous extracts of A. paniculata, T. cordifolia and T. foenum-graecum. Investigators in the past have also clearly shown that ethanol extracts are more effective than water extracts (Koduru et al., 2006; Aiyegoro et al., 2008; Ashrafa et al., 2008). Two possibilities that may account for the higher antibacterial activity of alcoholic extracts are the nature of bioactive components (polyphenols, flavonoids, alkaloids, essential oil and terpenoids) which may be enhanced in the presence of ethanol; and the stronger extraction capacity of ethanol that may have yielded a greater number of active constituents responsible for antibacterial activity (Ming et al., 2005; Vaghasiya and Chanda, 2007; Ghosh et al., 2008). Prajjal et al. (2003) have shown the antibacterial activity of aqueous extract of A. paniculata against E. coli, S. aureus and P. aeruginosa at a concentration of 10 mg/disc. But studies by Leelarasamee et al. (1990), Xu et al. (2006) and Anjana et al. (2009) have shown no antibacterial activity of A. paniculata aqueous extract against these organisms. The result of the antibacterial activity of A. paniculata aqueous extract in the present study is in agreement with the earlier done studies. Antibacterial activity was not observed with the ethanol extract of A. paniculata against E. coli, S. aureus and P. aeruginosa in a study by Xu et al. (2006). But, the present study showed significant antibacterial activity of A. paniculata ethanol extract against these organisms which supports the earlier studies by Zaidan et al. (2005) and Anjana et al. (2009). Srinivasan et al. (2001) have shown the broad spectrum activity of T. cordifolia root and stem extracts. Similarly in the present study, T. cordifolia leaf extracts exhibited antibacterial activity against both Gram positive and Gram negative bacteria. The antibacterial activity of aqueous, ethanol and chloroform stem extracts of T. cordifolia (100 mg/ml) against E. coli, P. vulgaris and S. aureus were carried out by Jeyachandran et al. (2003). They found that the ethanol extract displayed significant antibacterial activity while the chloroform and aqueous extracts displayed least antibacterial activity. Mahesh and Satish (2008) have shown the antibacterial activity of methanol leaf extract of T. cordifolia against E. coli, P. fluorescens and 41

16 S. aureus at a concentration of 0.1 mg/ml. The findings from the present study also indicate significant antibacterial activity in a polar solvent like ethanol. Aqueous extract of T. cordifolia has shown the least antibacterial activity in the present study which is contrary to the study by Upadhyay et al. (2011) which has shown higher percentage of growth inhibition of K. pneumoniae, E. coli and S. aureus in T. cordifolia stem aqueous extracts in comparison to other solvent extracts. Ethanol leaf extract of T. foenum-graecum exhibited broad spectrum activity against both Gram positive and Gram negative bacteria. A study by Aqil and Ahmad (2003) has also shown the broad spectrum activity of fenugreek leaves against S. aureus, E. coli and P. aeruginosa in the ethanol extract. El-Kamali and El-Karim (2009) in their study on T. foenum-graecum seeds indicated pronounced antibacterial activity of ethanol seed extract. Bonjar (2004) has shown the antibacterial activity of fenugreek seed extract in a polar solvent like methanol at a concentration of 20 mg/ml against E. coli. Similar to the earlier findings in fenugreek seed, the findings of the present study also show significant antibacterial activity in a polar solvent like ethanol. The variation in the antimicrobial activity of different solvents can be rationalized in terms of the polarity of the solvents used, polarity of the compounds being extracted by each solvent and, in addition to their extrinsic bioactivity and by their ability to dissolve or diffuse in the media used in the assay (Anjana et al., 2009). The other possibility may be due to loss of some active compounds during extraction process of the sample, lack of solubility of active constituents in the solvent (Kumar et al., 2008) or the active compounds may be present in insufficient quantities in the crude extracts to show their activity with the dose levels employed (Taylor et al., 2001). Alternatively, if the active principle is present in high enough quantities there could be other constituents exerting antagonistic effects (Jager et al., 1996). Extracts which have shown least antibacterial activity may be active against other bacterial species which were not tested (Shale et al., 1999). As shown in Table 1.8, leaves of A. paniculata, T. cordifolia and T. foenumgraecum contain phenols, flavonoids, terpenoids and tannins which have biological significance as antimicrobials. The antibacterial activity as seen in this study might be due to the presence of these compounds. 42

17 Determination of MIC Broth dilution method which was used to determine the MIC values of A. paniculata ethanol leaf extract was carried out with concentrations ranging from mg/ml (Table 1.4). Minimum inhibitory concentration values for P. aeruginosa and S. aureus were found to be 0.75 mg/ml and 1.0 mg/ml respectively. At 600 nm, P. aeruginosa and S. aureus showed an absorbance of 0.02 (83.3% growth inhibition) and 0.03 (84.8% growth inhibition) as against a control absorbance of 0.14 and 0.22 respectively. Minimum inhibitory concentration values were found to exceed 2.0 mg/ml for other bacteria tested. The per cent growth inhibition of bacteria is shown in Figure 1.5. Table 1.4 : Minimum inhibitory concentration (MIC) of Andrographis paniculata leaf ethanol extract using broth dilution method Bacteria Control Concentration (mg/ml) E. coli 0.27 ± 0.05 c 0.27 ± 0.01 c 0.17 ± 0.02 b 0.14 ± 0.03 ab 0.13 ± 0.00 ab 0.11 ± 0.00 a E. aerogenes 0.30 ± 0.02 d 0.29 ± 0.01 d 0.24 ± 0.01 c 0.19 ± 0.02 bc 0.15 ± 0.01 ab 0.14 ± 0.01 a Klebsiella sp ± 0.00 c 0.24 ± 0.01 bc 0.20 ± 0.02 ab 0.20 ± 0.02 ab 0.18 ± 0.02 a 0.16 ± 0.01 a P. vulgaris 0.25 ± 0.01 c 0.24 ± 0.00 c 0.20 ± 0.00 b 0.19 ± 0.01 b 0.16 ± 0.00 a 0.15 ± 0.01 a S. aureus 0.22 ± 0.04 e 0.21 ± 0.01 de 0.15 ± 0.00 cd 0.09 ± 0.02 bc 0.03 ± 0.01 ab 0.02 ± 0.00 a P. aeruginosa 0.14 ± 0.03 b 0.13 ± 0.02 b 0.06 ± 0.01 a 0.02 ± 0.01 a 0.02 ± 0.00 a 0.01 ± 0.01 a Values are absorbance at 600 nm Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) Fig. 1.5 : Per cent growth inhibition of bacteria at different concentrations ( mg/ml) of Andrographis paniculata ethanol leaf extract Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) 43

18 The MIC values of ethanol leaf extract of T. cordifolia at different concentrations ( mg/ml) against the tested six bacteria are summarized in Table 1.5. The ethanol extract was more effective in inhibiting S. aureus. The MIC value of S. aureus was found to be 1.75 mg/ml. Staphylococcus aureus showed an absorbance of 0.07 at 1.75 mg/ml as against a control absorbance of 0.22 at 600 nm. For other bacteria tested the MIC values exceeded 2 mg/ml. There was 68.18% growth inhibition of S. aureus at 1.75 mg/ml (Fig. 1.6). Pseudomonas aeruginosa recorded 61.90% growth inhibition at 2 mg/ml. Table 1.5 : Minimum inhibitory concentration (MIC) of Tinospora cordifolia leaf ethanol extract using broth dilution method Bacteria Control Concentration (mg/ml) E. coli 0.27 ± 0.05 d 0.28 ± 0.00 d 0.23 ± 0.00 c 0.20 ± 0.05 b 0.19 ± 0.00 b 0.17 ± 0.01 a 0.16 ± 0.00 a E. aerogenes 0.30 ± 0.02 b 0.30 ± 0.00 b 0.30 ± 0.01 b 0.28 ± 0.01 ab 0.26 ± 0.01 a 0.25 ± 0.00 a 0.25 ± 0.01 a Klebsiella sp ± 0.00 cd 0.26 ± 0.00 d 0.25 ± 0.00 cd 0.24 ± 0.00 bc 0.23 ± 0.01 ab 0.21 ± 0.00 ab 0.21 ± 0.01 a P. vulgaris 0.25 ± 0.01 b 0.25 ± 0.00 b 0.25 ± 0.01 ab 0.24 ± 0.01 ab 0.24 ± 0.00 ab 0.22 ± 0.00 a 0.22 ± 0.01 a S. aureus 0.22 ± 0.04 c 0.23 ± 0.01 c 0.18 ± 0.01 c 0.12 ± 0.00 b 0.08 ± 0.00 ab 0.07 ± 0.00 a 0.07 ± 0.01 a P. aeruginosa 0.14 ± 0.03 cd 0.14 ± 0.01 d 0.13 ± 0.00 cd 0.10 ± 0.01 bc 0.09 ± 0.00 ab 0.06 ± 0.01 ab 0.05 ± 0.00 a Values are absorbance at 600 nm Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) Fig. 1.6 : Per cent growth inhibition of bacteria at different concentrations ( mg/ml) of Tinospora cordifolia ethanol leaf extract Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) 44

19 % growth Inhibition of Bacteria Chapter 1 : In vitro antimicrobial activity studies The MIC values of ethanol extract of T. foenum-graecum against the tested six bacteria are represented in Table 1.6. Maximum antibacterial activity was observed against S. aureus followed by P. aeruginosa with a MIC value of 1 mg/ml. Staphylococcus aureus and P. aeruginosa recorded an absorbance of 0.03 and 0.05 as against a control absorbance of 0.22 and 0.14 respectively at 600 nm. In this study, S. aureus recorded a growth inhibition of around 86% and P. aeruginosa showed a growth inhibition of nearly 65% at a concentration of 1 mg/ml (Fig. 1.7). The MIC values exceeded 2 mg/ml as seen by the absorbance values in case of other bacteria. Table 1.6 : Minimum inhibitory concentration (MIC) of Trigonella foenumgraecum leaf ethanol extract using broth dilution method Bacteria Control Concentration (mg/ml) E. coli 0.27 ± 0.0 a 0.26 ± 0.0 a 0.17 ± 0.0 b 0.19 ± 0.0 b 0.25 ± 0.0 a E. aerogenes 0.30 ± 0.0 a 0.3 ± 0.01 a 0.28 ± 0.0 a 0.27 ± 0.0 a 0.3 ± 0.0 a Klebsiella sp ± 0.0 a 0.23 ± 0.01 ab 0.19 ± 0.0 b 0.19 ± 0.0 b 0.23 ± 0.0 ab P. vulgaris 0.25 ± 0.0 a 0.23 ± 0.01 ab 0.23 ± 0.0 ab 0.2 ± 0.0 b 0.22 ± 0.0 ab S. aureus 0.22 ± 0.0 ab 0.21 ± 0.01 ab 0.17 ± 0.01 b 0.03 ± 0.0 c 0.09 ± 0.0 c P. aeruginosa 0.14 ± 0.01 b 0.14 ± 0.01 b 0.06 ± 0.01 c 0.05 ± 0.0 c 0.07 ± 0.01 c Values are absorbance at 600 nm Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) Fig. 1.7 : Per cent growth inhibition of bacteria at different concentrations ( mg/ml) of Trigonella foenum-graecum ethanol leaf extract c E.coli E.aerogenes Klebsiella sp. sp. P.vulgaris S.aureus P.aeruginosa P. aeruginosa a ab a b c a c b b c c d c ab a c c d d bc e d d Concentrations (mg/ml) (mg/ml) Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) 45

20 As ethanol extracts of A. paniculata, T. cordifolia and T. foenum-graecum showed potent antibacterial activity against both Gram positive and Gram negative organisms, the determination of MIC values was carried out only with the ethanol extract. Broth dilution method which was used for the determination of MIC values of ethanol extract of all the three plants indicated that S. aureus and P. aeruginosa were more susceptible than any other bacteria at lower concentrations. Earlier findings have reported that most medicinal plants attack Gram positive bacteria while few are active against Gram negative bacteria (Srinivasan et al., 2001; Priscilla et al., 2007; Pavithra et al., 2010). The reason for the different sensitivity between Gram positive and Gram negative bacteria could be ascribed to the morphological differences between these microorganisms. The Gram negative bacteria have an outer phospholipidic membrane carrying the structural lipopolysaccharidic components which makes the cell wall impermeable to lipophilic solutes, while porins constitute a selective barrier to the hydrophilic solutes with an exclusion limit of about 600 Da (Nikaido and Vaara, 1985). The Gram positive bacteria are more susceptible having only an outer peptidoglycon layer which is not an effective permeability barrier (Scherrer and Gerhardt, 1971). The results of the present study are contrary with the earlier reports which have shown greater activity of medicinal plants against Gram positive bacteria. In spite of this permeability difference between Gram positive and Gram negative bacteria, the ethanol extract of all the three plants had a broader spectrum of inhibitory activity. This tends to show the involvement of more than one active principle of biological significance (Ming et al., 2005). The possible mechanism for their broad spectrum activity against both Gram positive and Gram negative bacteria may be due to their ability to complex with the cell wall (Cowan, 1999). The findings of the present study are in fair correlation with the study carried out by Calabrese et al. (2000) who reported the antibacterial activity of A. paniculata against both Gram positive and Gram negative organisms. Upadhyay et al. (2011) have shown the MIC value of methanol extract of T. cordifolia stem against S. aureus to be 0.06 mg/ml. But, the MIC value of T. cordifolia leaf ethanol extract in the present study was found to be 1.75 mg/ml. The reason for this variation may be attributed to the presence of different biological substances in the stem and leaf (Shan et al., 2005). Earlier studies by Aqil and Ahmad (2003) and Bonjar (2004) have shown the broad spectrum activity of fenugreek seeds against both Gram positive and Gram negative bacteria. Similar to the earlier findings in fenugreek seeds, we have also shown the wide spectrum activity of fenugreek leaves. 46

21 Antifungal activity assay Mycelial dry weight method was used to determine the MIC values of A. paniculata, T. cordifolia and T. foenum-graecum leaf ethanol extracts against two dermatophytes, T. rubrum and E. floccosum. Table 1.7 represents the MIC values and the per cent inhibition of ethanol leaf extracts of A. paniculata, T. cordifolia and T. foenum-graecum against the dermatophytes as compared to the antifungal drug, ketaconozole. Among the two dermatophytes, E. floccosum was found to be more susceptible to the ethanol leaf extract of all the three plants. Andrographis paniculata extract had the MIC values of 1.75 mg/ml (74.6% growth inhibition) and 3.0 mg/ml (70.9% growth inhibition) against E. floccosum and T. rubrum respectively which was lower when compared to other plant extracts. The MIC value of T. cordifolia extract exceeded more than the used concentration range of 10 mg/ml against T. rubrum. The MIC values obtained against T. rubrum and E. floccosum with all the three plant extracts were higher than the standard antifungal drug ketaconazole. Table 1.7 : Minimum Inhibitory Concentration (MIC) of Andrographis paniculata, Tinospora cordifolia and Trigonella foenum-graecum leaf ethanol extracts against dermatophytes using mycelial dry weight method T. rubrum E. floccosum MIC ( mg/ ml) % growth inhibition MIC ( mg/ ml) % growth inhibition Ketaconazole 0.4 ± 0.01 a ± 0.02 a 93.0 A. paniculata 3.0 ± 0.04 b ± 0.05 b 74.6 T. cordifolia > 10 ± 0.06 d ± 0.03 d 56.3 T. foenum-graecum 6.5 ± 0.02 c ± 0.04 c 65.0 Values are means of three independent replicates Mean values with different superscripts are significantly different from each other as indicated by Turkey s HSD (P 0.05) Dermatomycoses is the disease caused by a group of fungi known as dermatophytes. Skin disease such as tinea is the most frequent mycoses which involves superficial infections of keratinized tissue of the skin which occur in most classes of patients, especially in immunosuppressed patients (Chuang et al., 2007). Clinical surveys carried out in India have shown ringworm as one of the most common dermatomycoses caused by species of Trichophyton, Epidermophyton and Microsporum. The disease is predominant in tropical and subtropical countries due to 47

22 their prevailing moisture and temperature regimes, and poses a therapeutic problem despite several antimycotic drugs available in the market. These drugs are mostly fungistatic. Besides, these drugs have been found to possess various side effects (Shahi et al., 1998). Recently, products of higher plant origin have been shown as effective sources of chemotherapeutic agents without undesirable side effects and with strong fungicidal activity (Igbinosa et al., 2009; Mathur et al., 2011). Although advances have been made in pharmacology and synthetic organic chemistry, the reliance on natural products, particularly on plants, remains largely unchanged. It is well established that some plants contain compounds that inhibits microbial growth (Evarando et al., 2005). These plant compounds have different structures and different actions when compared with antimicrobials conventionally used to control microbial growth (Nascimento et al., 2000). These findings prompted us to explore other plant products which could be exploited as effective antifungal agents. A study by Wanchaitanawong et al. (2005) has shown the in vitro antifungal activity of ethanol extract of A. paniculata plant against Aspergillus niger, Aspergillus oryzae and Penicillium. Antifungal activity of hydroalcoholic extract of A. paniculata against A. niger and Candida albicans has been shown by Mathur et al. (2011) with a MIC value of 0.5 mg/ml. Similar to the earlier reports on antifungal activity of A. paniculata ethanol extract, the result of the present study also indicates its antifungal activity against dermatophytes. Based on the review of current literature, there are no previous reports showing the antifungal activity of A. paniculata leaf ethanol extract on dermatophytes. Satish et al. (2007) have shown the antifungal activity of T. cordifolia stem extract against eight species of Aspergillus. Methanol extract of T. cordifolia root and stem exhibited antifungal activity against Helminthosporium sp. at a concentration of 5 mg/ml (Singh et al., 2010). Antifungal activity of T. cordifolia methanol leaf extract against Aspergillus flavus, Dreschlera turcica and Fusarium verticilloides has been shown by Mahesh and Satish (2008) at a concentration of 0.1 mg/ml. Hydroalcoholic extract of T. cordifolia stem was found to have maximum antifungal activity against A. niger and Candida albicans with a MIC value of 0.5 mg/ml (Mathur et al., 2011). Eventhough, T. cordifolia stem and root extracts have shown antifungal activity against the field fungi with lower MIC values, the MIC values in the present study for 48

23 T. cordifolia ethanol leaf extract against E. flocossum was quite high and with T. rubrum it could not be achieved even at a higher concentration of 10 mg/ml. This could be due to the presence of lower concentration of antifungal substances in the extract. In the present study, T. foenum-graecum ethanol leaf extract showed antifungal activity against both fungi with MIC values ranging between 4.0 and 7.0 mg/ml. There are no previous reports showing the antifungal activity of T. foenum-graecum leaf extract on dermatophytes. But, a study by Aqil and Ahmad (2003) has shown less antifungal activity of T. foenum-graecum leaf extract against the field fungi viz., A. niger, Alternaria alternata, Fusarium chlamydosporum and Rhizoctonia bataticola. It is well established that the potential antimicrobial properties of plants are related to their ability to synthesize by secondary metabolism several chemical compounds of relatively complex structures including tannins, alkaloids, cardiac glycosides, saponins, terpenes, phenolics, flavonoids and coumarins (Evarando et al., 2005; Matasyoh et al., 2009). The antifungal activity of plant extracts in the present study may be due to the presence of any one or more of these active compounds which might be acting synergistically. Phytochemical screening Ethanol extract which had shown good antibacterial and antifungal activities was used for screening the phytochemical compounds. Investigations on the phytochemical screening of A. paniculata, T. cordifolia and T. foenum-graecum leaf ethanol extracts are summarized in Table 1.8. Results from the current study indicate that ethanol leaf extracts of A. paniculata, T. cordifolia and T. foenum-graecum contain varied types of pharmacologically active compounds. The commonly identified components of pharmacological importance in the three plant extracts included phenols, flavonoids and cardiac glycosides. In addition, tannins and terpenoids were present in A. paniculata and T. cordifolia, polysterols were present in T. cordifolia and T. foenum-graecum, saponins were present in T. foenum-graecum and carbohydrates and proteins were present in all the three plant extracts. Alkaloids were found to be absent in the plant extracts. 49

24 Table 1.8 : Phytochemical constituents of ethanol leaf extracts of Andrographis paniculata, Tinospora cordifolia and Trigonella foenum-graecum Phytochemical constituents Ethanol leaf extracts A. paniculata T. cordifolia T. foenum-graecum Phenols Flavonoids Tannins + + _ Terpenoids + + _ Alkaloids _ Polysterols _ + + Cardiac glycosides Saponins + Carbohydrates Protiens Represents presence of the phytoconstituent; - represents absence of the phytoconstituent Bioactive secondary metabolites have been utilized as natural medicines and plants containing these compounds have been used as medicinal plants and prescribed in many recipes as forms of crude drugs (Zuin and Vilegas, 2002; Rios and Recio, 2005). The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive compounds are flavonoids, tannins, alkaloids and phenolic compounds (Weimann and Heinrich, 1997; Atindehou et al., 2002; Edeoga et al., 2005). Phytochemical compounds are heterogeneous mixtures of substances acting in a synergistic or antagonistic manner (Musyimi et al., 2008). Mixtures of active constituents show a broad spectrum of biological and pharmacological activity (Coelho-de-Souza et al., 2002). The need of the hour is to screen plants that are traditionally used and also to evaluate their probable phytoconstituents (Parekh and Chanda, 2007a; Parekh and Chanda, 2007c). Knowledge of the chemical constituents of plants is desirable because such information will be of great value for the synthesis of complex chemical substances. Many studies have been undertaken with the aim of determining the different antimicrobial and phytochemical constituents of medicinal 50

25 plants and using them for the treatment of both topical and systemic microbial infections as possible alternatives to chemical synthetic drugs to which many infectious microorganisms have become resistant (Akinpelu and Onakoya, 2006). Phytochemical analysis of the ethanol leaf extracts of A. paniculata, T. cordifolia and T. foenum-graecum has revealed the presence of biologically active compounds such as phenols, flavonoids, saponins, tannins, terpenoids, cardiac glycosides and polysterols. The antimicrobial (antibacterial and antifungal) activity of the plant extracts is due to the presence of these active compounds which may be acting independently or synergistically with other compounds. The phenolic compounds are one of the largest and most ubiquitous groups of plant metabolites (Singh et al., 2007). Baravalia et al. (2009) have shown the importance of polyphenols as antimicrobial compounds. Polyphenols and flavonoids exhibit wide range of biological effects and antibacterial activity is one of them (Hartung, 1990; Edenharder and Grunhage, 2003; Wang et al., 2003; Kathad et al., 2010). According to Fatope (1995) polyphenols are important fungitoxic phytochemical compounds. Flavonoids have shown a wide range of biological activities like antimicrobial, antiinflammmatory, antiallergic and antioxidant properties (Hodek et al., 2002). They are found to be effective antimicrobial substances against a wide array of infectious agents (Mendosa et al., 1997; Erdogrul, 2002; Jamine et al., 2007; Mostahar et al., 2007). Flavonoids have been shown to be effective antimicrobial agents by complexing with extracellular and soluble proteins and also with bacterial cells (Tsuchiya et al., 1996). They also act by inhibiting enzymes that regulate cell proliferation (Fatope, 1995). Tannins are known for their antimicrobial activity (Stern et al., 1996; Rios et al., 2000). Prasad et al. (2008) have reported the growth inhibition of many fungi, bacteria and viruses by tannins. Tannins have been reported to prevent the development of microorganisms by precipitating microbial proteins. They have been found to form irreversible complexes with proline rich proteins resulting in the inhibition of cell protein synthesis (Shimada, 2006). 51

26 Quinlan et al. (2000) have shown the presence of antibacterial activities of steroidal extracts from medicinal plants. Terpenoids have been demonstrated to be active against bacteria, fungi, viruses and protozoa (Aurelli et al. 1992; Tassou et al., 1995). The mechanism of action of terpenes is by lipophilic membrane disruption (Cowan, 1999). Saponins are known for their medicinal properties as a natural blood cleanser, expectorant and antibiotic (Kalanithi and Lester, 2001). Earlier studies on aerial parts of A. paniculata have shown the presence of diterpenoids, flavonoids and polyphenols (Reddy et al., 2003; Rao et al., 2004; Chao and Lin, 2010). Sule et al. (2010) in their study have shown the presence of flavonoids, glycosides and saponins in methanol extract of A. paniculata. Similar to the earlier findings, ethanol extract of A. paniculata leaves in the present study has shown the presence of phenols, flavonoids, terpenoids and cardiac glycosides. Reports on T. cordifolia stem extracts have shown the presence of phenols, tannins, cardiac glycosides and steroids (Sivakumar et al., 2010; Vaghasiya et al., 2011). Rose et al. (2010) have reported the presence of alkaloids, glycosides, steroids, saponins, tannins, phenols and flavonoids in root extract of T. cordifolia. The result of the present study is in agreement with an earlier study by Murthy et al. (2010) which has shown the presence of tannins, flavonoids, phytosterols, glycosides in ethanol leaf extract of T. cordifolia. Trigonella foenum-graecum seeds have been shown to contain alkaloids, saponins and flavonoids by Dheeraj et al. (2010). But in the present study, T. foenumgraecum leaf extracts showed absence of alkaloids which may be due to the early harvesting of the leaves (Srinivasan, 2006). 52