CHAPTER 6 EVALUATION OF SELECTED PLANT EXTRACTS FOR EVALUATION OF SELECTED PLANT EXTRACTS FOR ANTI-ACNE ACTIVITY

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1 CHAPTER 6 EVALUATION OF SELECTED PLANT EXTRACTS FOR School of Science, SVKM s NMIMS University Page 119

2 6. EVALUATION OF SELECTED PLANT EXTRACTS FOR 6.1 MATERIALS AND METHODS Antimicrobial assays Enzyme assay 6.2 RESULTS AND DISCUSSION 6.3 CONCLUSION School of Science, SVKM s NMIMS University Page 120

3 6.1 MATERIALS AND METHODS a) List of Reagents and Chemicals used in the experiments Sr. No Name Make 1 Sodium chloride Loba Chemie Pvt Ltd 2 Mueller-Hinton agar (MHA) Hi-media 3 Brain Heart Infusion agar (BHI) Hi-media 4 Tetracycline SRL Pvt Ltd 5 Anaerobic gas pack Hi-Media 6 Sodium chloride Loba Chemie Pvt 7 Mueller-Hinton broth Hi-media 8 Mueller-Hinton agar (MHA) Hi-media 9 Brain Heart Infusion broth Hi-media 10 Brain Heart Infusion agar (BHI) Hi-media 11 Tetracycline SRL Pvt Ltd 12 Polysorbate 80 (Tween 80) Fisher Scientific 13 Lecithin Hi-Media 14 Anaerobic gas pack Hi-Media 15 p-nitrophenol SRL Pvt Ltd 16 Tris (hydroxymethyl) aminomethane SRL Pvt Ltd 17 p-nitrophenyl laurate SRL Pvt Ltd 18 Dimethyl Sulfoxide (DMSO) Qualigens b) List of Instruments used in the experiments Sr. No Instrument Make 1 UV Visible Spectrophotometer LAMBDA 25 Perkin Elmer 2 Anaerobic Gas Jar Equitron 3 Vortex Mixer REMI 4 5 Water Bath Tabletop Centriguge Metalab REMI School of Science, SVKM s NMIMS University Page 121

4 6.1.1 Antimicrobial assays A. Determination of MIC and MBC by agar dilution method Principle: The agar dilution method for determining antimicrobial susceptibility is a quantitative method for determination of MIC of antimicrobial agent against the test organisms. The antimicrobial agent i.e. plant extract is incorporated into the agar medium, with each plate containing a different concentration of the extract. Then the inoculum is applied on dried surface of the fresh agar plates and incubated at required conditions. Plates are examined for presence or absence of growth of test organisms. The lowest concentration at which the bacterial growth is completely inhibited is considered as the MIC of the plant extract. Further, samples were taken from all the plates and subcultured on freshly prepared agar medium and incubated. The MBC is considered as the lowest concentration of the extract that did not allow any bacterial growth on surface of the agar plates (Wiegand et al. 2008). Test microorganisms: S. aureus (MTCC 737) S. epidermidis (MTCC 435) P. acnes (MTCC 1951) These lyophilised cultures were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. Inoculum preparation: Test organisms were inoculated into sterile saline (0.85% NaCl) and O.D. was adjusted to 0.2 at 620 nm to obtain the cell suspension at 10 7 CFU/ ml. Growth conditions: S. aureus and S. epidermidis were grown aerobically at 37 ºC for hrs. P. acnes was incubated anaerobically at 37 ºC for hrs. Plant extract preparation: 25 mg of dried plant extract was weighed, dissolved in 100 µl of methanol by sonication and volume was then made up to 1 ml. School of Science, SVKM s NMIMS University Page 122

5 Standard solution preparation: Tetracycline stock solution was prepared by considering the given potency of antibiotic. 10 µg/ ml solution was prepared in distilled water and kept in amber colored volumetric flask. Procedure: The MIC for all extracts was determined by the agar dilution method (Russell & Furr 1977). Different concentrations of the extracts ( mg/ ml) and ( mg/ ml) were prepared. 0.5 ml of the extract from each dilution was mixed with 4.5 ml of molten agar (MHA for S. aureus and BHI for P. acnes) and poured into sterile petri dishes. The surface of the agar was streaked with the test organisms (10 7 CFU/ ml). The plates were incubated at 37 ºC for hrs for S. aureus and S. epidermidis and at 37 ºC for hrs for P. acne. The MIC was observed as the lowest inhibitory concentration of the extract, inhibiting the visible growth of test organism. Further, the MBC of the extracts was determined (Olorundare et al. 1992). MBC was determined by sub-culturing the samples on to a fresh agar plates and incubated at above mentioned conditions. The MBC was taken as the lowest concentration of the extract that did not allow any bacterial growth on the surface of the agar plates. Tetracycline was used as positive control. Statistical analysis: The MIC and MBC values were expressed as mean ± SD. Statistical analysis was carried out by ANOVA followed by Bonferroni s Test (P < 0.05). All calculations were performed using Graph Pad Prism (version 5.0). B. Time kill efficacy study Principle: Time-kill studies are used to determine the rate of bactericidal activity. The time kill efficacy test evaluates the microbial reduction by an antimicrobial agent. The test sample is brought in contact with known population of microorganisms for a specified period of time at specified temperature. At selected time points, aliquots are removed and placed in neutralizer, which is further diluted and plated onto agar and colonies School of Science, SVKM s NMIMS University Page 123

6 are enumerated. A 3-fold reduction in bacterial counts is compared to initial count, indicates an adequate bactericidal response. The reliability of the neutralizer used should be checked. The neutralizer should not prevent the growth of organism by itself. It should be efficient enough to neutralize the active component of the sample. Hence, neutralizer validity and neutralizer control are carried out (CLSI 1999). Test microorganisms: S. aureus (MTCC 737) S. epidermidis (MTCC 435) P. acnes (MTCC 1951) These lyophilised cultures were obtained from the Microbial Type Culture Collection and Gene Bank, Chandigarh, India. Inoculum preparation: Test organisms were inoculated into sterile saline (0.85% NaCl) and O.D. was adjusted to 0.2 at 620 nm to obtain the cell suspension at 10 7 CFU/ ml. Growth conditions: S. aureus and S. epidermidis were grown aerobically at 37 ºC for hrs. P. acnes was incubated anaerobically at 37 ºC for hrs. Plant extract preparation: 25 mg of dried plant extract was weighed, dissolved in 100 µl of methanol by sonication and volume was then made up to 1 ml. Standard solution preparation: Tetracycline stock solution was prepared by considering the given potency of antibiotic. 10 µg/ ml solution was prepared in distilled water and kept in amber colored volumetric flask. School of Science, SVKM s NMIMS University Page 124

7 Procedure: In Time kill efficacy study, four plant extracts were tested against three test organisms i.e. S. aureus, S. epidermidis and P. acnes for three time points (5 min, 10 min and 15 min) (CLSI 1999). The test involves four major steps:- Initial Count: - For input counts, 10-fold serial dilution of the culture suspension was carried out from 10-1 to 10-7 and dilutions from 10-5 to10-7 were plated out on sterile agar plates using sterile non absorbent cotton swabs and the plates were incubated at above mentioned growth conditions. Neutralizer Control: - 9 ml Neutralizer, 1 ml Test culture was added. Hold for 15 min and viable count was carried out up to 10-7 dilution. Neutralizer Validity: - In, 8.9 ml Neutralizer, 1 ml extract was added. Hold for 5 min and 0.1 ml culture was added. After 15 min., viable count was done up to 10-7 dilution. Actual Time Points: - This is the step where the actual time kill efficacy of the sample is determined for different time points against the given organism. In 0.9 ml extract, 0.1 ml culture was added and hold for the required time period (5 min, 10 min, 15 min) and 9 ml Neutralizer was added. Further, viable count was carried out up to 10-7 dilution. At the end, log reduction and % kill efficacy of test extracts were calculated. Calculations: Log = Log (Initial Count) Log (Recovery Count) = [(Recovery Count / Initial Count) X 100] Statistical analysis: The reduction values and percentage kill efficacy values were expressed as mean ± SD. Statistical analysis was carried out by ANOVA followed by Bonferroni s Test (P < 0.05). All calculations were performed using Graph Pad Prism (version 5.0). School of Science, SVKM s NMIMS University Page 125

8 6.1.2 Enzyme assay A. Anti-lipase assay Principle: In lipase enzyme assay, p-nitrophenyl laurate substrate hydrolyses in presence of lipase enzyme and p-nitrophenol was released hence standard curve of p-nitrophenol was prepared to calculate the activity of P. acnes lipase enzyme. Further, Anti-lipase activity of test extracts was obtained by pre-incubating test extract with lipase and p- nitrophenyl laurate substrate was added to measure the released p-nitrophenol in presence of test extracts. For each concentration, suitable blanks were kept and percentage of anti-lipase activity was calculated (Patil et al. 2012). Test microorganisms: P. acnes (MTCC 1951). Growth conditions: Test organism was inoculated into BHI broth and incubated anaerobically at 37 ºC for hrs. Plant extract preparation: 10 mg of dried plant extract was weighed, dissolved in 100 µl of methanol by sonication and volume was then made up to 1 ml. Further, dilutions were prepared ranging from mg/ ml. Standard solution preparation: Tetracycline stock solution was prepared by considering the given potency of antibiotic. 10 mg/ ml solution was prepared in distilled water in amber colored volumetric flask. Further, dilutions were prepared ranging from mg/ ml. Procedure In this assay, plant extracts were tested for anti-lipase activity of crude lipase enzyme from P. acnes (Pinsirodom & Parkin 2001). Three major steps are involved: - School of Science, SVKM s NMIMS University Page 126

9 Lipase extraction: - Lipase extraction was carried out by growing P. acnes anaerobically in BHI broth at 37 ºC for hrs. This solution was subjected to centrifugation at 10,000 r.p.m. for 20 min. After centrifugation, supernatant was collected in sterile container and stored at 4 ºC until further use. p-nitrophenol standard curve: to 0.5 ml of 0.5 mm p-nitrophenol standard solution was put into ten individual test tubes and dilute each to 5 ml with 0.1 M Tris HCl buffer (ph 8.2) and absorbance was measured at 410 nm. This obtained a standard curve of to 0.05 µmol p-nitrophenol/ ml. Anti-lipase Assay: - Anti-lipase activity of test extracts was obtained as follows, 0.5 ml plant extract was mixed with 1.25 ml of 0.1 M Tris HCl buffer (ph 8.2). This mixture was preincubated for 1 hr at 37 o C and reaction was started by addition of 1.25 ml of 420 μm p-nitrophenyl laurate substrate. Absorbance was measured at 410 nm. Calculations: p-nitrophenol standard curve was used to convert absorbences to mm substrate hydrolyzed as follows: µmol p-nitrophenol/ ml reaction mixture = [A 410 y intercept)/ [slope 6 ml reaction mixture] (Pinsirodom & Parkin 2001). Percentage lipase inhibition activity = 100 [(B b) / (A a) X 100] Where, A = Activity without inhibitor a = Negative control without inhibitor B = Activity with inhibitor b = Negative control with inhibitor (Roh & Jung 2012). Statistical analysis: The EC 50 values were expressed as mean ± SD (n=3). Statistical analysis was carried ANOVA followed by Bonferroni's Multiple Comparison Test (**P < 0.01, *** P < 0.001). All calculations were performed using Graph Pad Prism (version 5.0). School of Science, SVKM s NMIMS University Page 127

10 6.2 RESULTS AND DISCUSSION A. Determination of MIC and MBC by agar dilution method MICs are mainly used to determine in vitro activity of antimicrobial compounds (Andrews 2001). MIC values indicate the minimum concentration required for inhibition of visible growth of test organisms whereas MBC values report the bactericidal concentration (Parija 2009). Antimicrobial agents with low activity against a particular organism usually exhibit high MIC and MBC values, while a highly potent agents show low MIC and MBC values. The MIC and MBC techniques are used to evaluate the efficacies of antimicrobial agent (Abubakar 2009). In the present study, MIC values for all extracts and standard (Tetracycline) were determined by agar dilution method against acne inducing bacteria i.e. S. aureus, S. epidermidis and P. acnes. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into an agar medium followed by the application of inoculums with calibrated loop to the surface of the agar plate (Wiegand et al. 2008). Agar dilutions are most often prepared in petri dishes and have advantage that it is possible to test several organisms on each plate (Nad & Agents 2000). In this study, we have used glass petri plates of smaller diameter to reduce the amount of plant extract used for preparation of various concentrations. Fig. 6.1 represents the MIC determination of CR HAE against S. aureus and S. epidermidis by agar dilution method. The concentration range prepared for CR HAE was from 0.1 mg/ ml to 15 mg/ ml. Positive control showed the growth of both the test organisms. Solvent control was kept to check the intervention of solvent on the growth of organisms however both the test organisms showed growth in solvent control plate. Negative control was kept without any test culture and to confirm the absence of any contamination. Further, MBC was determined by streaking sample from each concentration onto a fresh agar medium and after incubation, the concentration at which no growth was observed was considered as bactericidal concentration. MBC determination of CR HAE against S. aureus and S. epidermidis is represented in Fig Similarly, for all plant extracts and Tetracycline; MIC and MBC values were determined against three test organisms by agar dilution method. School of Science, SVKM s NMIMS University Page 128

11 0.1 mg 0.5 mg 1 mg 5 mg 10 mg 15 mg Positive control Solvent control Negative control Fig. 6.1: MIC Determination of CR HAE against S. aureus and S. epidermidis by agar dilution method Positive growth No growth Fig. 6.2: MBC Determination of CR HAE against S. aureus and S. epidermidis School of Science, SVKM s NMIMS University Page 129

12 OT HAE OT CAE CR HAE CR CAE OT HAE OT CAE CR HAE CR CAE Values (mg/ ml) EVALUATION OF SELECTED PLANT EXTRACTS FOR MIC and MBC values of all selected plant extracts against S. aureus are presented in Table 6.1. In MIC and MBC determination against S. aureus, OT HAE exhibited 2.0 ± 0.5 mg/ ml and 6.17 ± 0.29 mg/ ml of MIC and MBC values respectively. OT CAE showed lower values (MIC: 1.3 ± 0.3, MBC: 3.50 ± 0.50) than OT HAE. CR CAE demonstrated less MIC and MBC values than CR HAE. The MIC and MBC values of CR CAE were significant (*P < 0.05) in comparison with MIC and MBC values of CR HAE against S. aureus (Fig. 6.3). Results indicate that cold alcoholic extract of Ocimum tenuiflorum Linn (OT CAE) has strong antibacterial activity against S. aureus. Table 6.1: MIC and MBC values of plant extracts against S. aureus. Plant Extract MIC (mg/ ml) S. aureus MBC (mg/ ml) OT HAE 2.0 ± ± 0.29 OT CAE 1.3 ± ± 0.50 CR HAE 6.7 ± ± 1.89 CR CAE 3.3 ± ± S.aureus MIC MBC * * 2 0 Fig. 6.3: MIC and MBC values of plant extracts against S. aureus. School of Science, SVKM s NMIMS University Page 130

13 OT HAE OT CAE CR HAE CR CAE OT HAE OT CAE CR HAE CR CAE Values (mg/ ml) EVALUATION OF SELECTED PLANT EXTRACTS FOR MIC and MBC values of all plant extracts against S. epidermidis are represented in Table 6.2. OT HAE showed higher MIC and MBC values than OT CAE indicating that more concentration is required for inhibition of S. epidermidis. MIC and MBC values of CR HAE were found to be more (MIC: 14.8 ± 0.2 mg/ ml, MBC: ± 0.29) than CR CAE (MIC: 10.5 ± 0.5 mg/ ml, MBC: ± 2.89 mg/ ml). The MIC values of OT CAE and CR CAE were significant (***P < 0.001) than MIC and values of their hot alcoholic extracts i.e. OT HAE and CR HAE respectively (Fig 6.4). Table 6.2: MIC and MBC values of plant extracts against S. epidermidis Plant S. epidermidis Extract MIC (mg/ ml) MBC (mg/ ml) OT HAE 6.5 ± ± 1.52 OT CAE 3.83 ± ± 0.29 CR HAE 14.8 ± ± 0.29 CR CAE 10.5 ± ± S. epidermidis *** MIC *** MBC Fig. 6.4: MIC and MBC values of plant extracts against S. epidermidis School of Science, SVKM s NMIMS University Page 131

14 OT HAE OT CAE CR HAE CR CAE OT HAE OT CAE CR HAE CR CAE Values (mg/ ml) EVALUATION OF SELECTED PLANT EXTRACTS FOR MIC and MBC values of test extracts against P. acnes are shown in Table 6.3. MIC and MBC values of OT CAE were 0.67 ± 0.10 mg/ ml and 1.50 ± 0.87 mg/ ml correspondingly. OT HAE exhibited slightly higher values (MIC: 1 ± 0.2 mg/ ml and 2.83 ± 0.76 mg/ ml) than OT CAE. In the case of Citrus peel extracts, cold alcoholic extract (CR CAE) was found to be more potent than hot alcoholic extract (CR HAE). MIC and MBC values of CR CAE against P. acnes were significant (***P < 0.001) compared to the values of CR HAE (Fig. 6.5). Table 6.3: MIC and MBC values of plant extracts against P. acnes Plant Extract MIC (mg/ ml) P. acnes MBC (mg/ ml) OT HAE 1.00 ± ± 0.76 OT CAE 0.67 ± ± 0.87 CR HAE 6.76 ± ± 0.29 CR CAE 3.83 ± ± P. acnes MIC *** MBC *** Fig. 6.5: MIC and MBC values of plant extracts against P. acnes School of Science, SVKM s NMIMS University Page 132

15 S. aureus S. epidermidis P. acnes S. aureus S. epidermidis P. acnes Values ( g/ ml) EVALUATION OF SELECTED PLANT EXTRACTS FOR Tetracycline was used as a positive control. MIC and MBC values of Tetracycline were determined against S. aureus, S. epidermidis and P. acnes and presented in Table 6.4. MIC and MBC values of Tetracycline against S. aureus were found to be 6.33 ± 0.57 µg/ ml and 7.33 ± 1.15 µg/ ml respectively and for S epidermidis these values were found to be 8.67 ± 1.15 to 9.33 ± 1.15 µg/ ml. Results showed that, P. acnes was more susceptible microorganism than other two test organisms. MIC required for P. acnes was 2.67 ± 1.15 µg/ ml and MBC was 4.00 ± 2.00 µg/ ml. MIC and MBC values of Tetracycline against P. acnes were statistically significant (*P < 0.05) than other two test organisms (Fig. 6.6). Table 6.4: MIC and MBC values of Tetracycline against S. aureus, S. epidermidis and P. acnes Test organisms MIC (µg/ ml) Tetracycline MBC (µg/ ml) S. aureus 6.33 ± ± 1.15 S. epidermidis 8.67 ± ± 1.15 P. acnes 2.67 ± ± MIC Tetracycline * MBC * Fig. 6.6: MIC & MBC values of Tetracycline against S. aureus, S. epidermidis and P.acnes School of Science, SVKM s NMIMS University Page 133

16 Present investigation focus on the antibacterial activity of test extracts in order to find out the new antibacterial bioactive compounds against acne inducing bacteria. MIC and MBC values were determined for all test extracts to understand their nature as bacteriostatic or bactericidal agents. Antimicrobial substances are considered as bactericidal agents when the ratio MBC/MIC 4 and bacteriostatic agent when the ratio MBC/MIC > 4 (Gatsing et al. 2007) For most of the test extracts, the ratio MBC/ MIC was 4 against the acne inducing strains, indicating that these extracts may be classified as bactericidal in nature. The MBC/MIC ratio of positive control i.e. Tetracycline also indicates its bactericidal effect against all test bacteria Time kill efficacy study Time kill efficacy study measures the time dependant antibacterial activity. Time-kill methods have been commonly used for testing bactericidal activity of antimicrobial agents (Mukherjee et al. 2005). Time kill curves demonstrate better correlations with the in vivo efficacy than other microbiological methods (Zeitlinger et al. 2004). This study was performed by measuring the bacterial count (CFU/ ml) of surviving bacteria and expressed as log reduction and % time kill efficacy of the antimicrobial agent. In the present study, plant extracts were tested at their MBC concentrations against acne inducing bacteria at three time points. Input count is the viable count of the microbial cell suspension used in the test. This provides information about the original or total number of organisms initially present in the suspension. In this study, input counts (CFU/ ml) of S. aureus, S. epidermidis and P. acnes were calculated and presented in Table 6.5. Table 6.5: Input, neutralizer control and neutralizer validity counts (CFU/ ml) Test Input Neutralizer control Neutralizer validity organisms S. aureus 3.26 x 10 7 ± x 10 7 ± x 10 7 ± 0.22 S. epidermidis 2.32 x 10 7 ± x 10 7 ± x 10 7 ± 0.24 P. acnes 1.91 x 10 7 ± x 10 7 ± x 10 7 ± 0.27 Neutralizer control and neutralizer validity counts were expressed as CFU/ ml. Neutralizer control was kept to check if the neutralizer has any antimicrobial activity against test organisms. Results indicate that, the neutralizer has no effect on all three School of Science, SVKM s NMIMS University Page 134

17 test organisms. To check the neutralizer validity, the plant extract was kept in contact with neutralizer for specified period of time, after which culture is added to it and kept in contact for specified time period. Results obtained showed that, neutralizer has no effect on plant extracts and on microorganisms as well. Hence, neutralizer play important role in neutralising the reaction at specified period of time without any inhibitory effect. This study allows the determination of the rate of bactericidal activity of the plant extracts (Aiyegoro et al. 2009). The number of surviving microorganisms in the extract was determined by plate count method at sampling time and enumerated. The log reduction and percentage kill efficacy for each time point was calculated (Oladosu et al. 2013). Table 6.6 represents the time kill efficacy of OT HAE against S. aureus, S. epidermidis and P. acnes. Average reduction against S. aureus ranged from 0.64 ± 0.03 to 0.91 ± 0.08 CFU/ ml. Percentage kill efficacy is ranged from ± 1.47 to ± 2.06% between 5 to 15 min of interaction. For S. epidermidis highest reduction was observed at 15 min as 1.05 ± 0.12 CFU/ ml. P. acnes was found to be most susceptible organism as OT HAE exhibited highest percent kill efficacy i.e ± 0.78% after 15 min of interaction. Table 6.6: Time kill efficacy of OT HAE against S. aureus, S. epidermidis, P.acnes S. aureus S. epidermidis P. acnes Time (min) 5 min 0.64 ± ± ± ± ± ± min 0.71 ± ± ± ± ± ± min 0.91 ± ± ± ± ± ± 0.78 School of Science, SVKM s NMIMS University Page 135

18 Time kill efficacy of OT CAE is shown in Table 6.7. For S. aureus, reduction was obtained as 2.37 ± 0.06 CFU/ ml and percentage kill efficacy was ± 0.06 %. reduction was increased up to 2.63 ± 0.01 CFU/ ml after 10 min of interaction of extract and at 15 minutes it reached to 4.02 ± 0.29 CFU/ ml and percentage kill efficacy obtained was ± 0.01%. A bactericidal effect is defined as a 3 log decrease in the CFU/ ml or a 99.9% kill over a specified time (CLSI 1999) Thus, OT CAE exhibited stong bactericidal effect against S. aureus after 15 min of interaction. For S. epidermidis, reduction obtained at 15 min was 2.36 ± 0.11 CFU/ ml and corresponding percentage kill efficacy was ± These results indicate that more time of interaction between extract and test organism is required to achieve the bactericidal effect against S. epidermidis. For P. acnes, reduction ranged from 1.17 ± 0.12 to 3.69 ± 0.39 CFU/ ml. Percentage reduction ranged from ± 1.83 to ± 0.56%, between 5 to 15 min of interaction. Table 6.7: Time kill efficacy of OT CAE against S. aureus, S. epidermidis, P. acnes Time (min) 5 min 10 min 15 min 2.37 ± ± ±0.29 S. aureus S. epidermidis P. acnes ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.57 School of Science, SVKM s NMIMS University Page 136

19 Time kill efficacy of CR HAE is presented in Table 6.8. The reduction obtained for S. aureus was ranged from 1.38 ± 0.04 to 1.74 ± 0.10 CFU/ ml and percentage kill efficacy was up to ± 0.43% after 15 min of contact time. CR HAE showed highest reduction i.e ± 0.02 CFU/ ml against S. epidermidis after 15 min of interaction. The percentage reduction obtained was up to ± 0.01%. The least activity was observed against P. acnes as only 0.83 ± 0.11 CFU/ ml reduction was obtained after 5 min of interaction and corresponding percentage kill efficacy was ± 3.43%. Further, it increased up to ± 1.49%. Table 6.8: Time kill efficacy of CR HAE against S. aureus, S. epidermidis, P. acnes Time (min) 5 min 1.38 ± 0.04 S. aureus S. epidermidis P. acnes ± ± ± ± ± min 1.55 ± ± ± ± ± ± min 1.74 ± ± ± ± ± ± 1.49 Time kill efficacy of CR CAE is presented in Table 6.9. CR CAE extract was found to be more effective against P. acnes as reduction was ranged between 1.64 ± 0.07 to 3.38 ± 0.39 CFU/ ml. Percent reduction obtained was from ± 0.38 to ± 0.25% for specified time points. For S. aureus and S. epidermidis, percentage kill efficacy was found to be ± 0.84 and ± 2.44 after 15 min of interaction. Results indicate that, S. epidermidis was found to be more resistant than S. aureus. School of Science, SVKM s NMIMS University Page 137

20 Time (min) Table 6.9: Time kill efficacy of CR CAE against S. aureus, S. epidermidis, P. acnes S. aureus S. epidermidis P. acnes 5 min 1.24 ± ± ± ± ± ± min 1.51 ± ± ± ± ± ± min 1.74 ± ± ± ± ± ± 0.25 Time kill efficacy of standard antibiotic, Tetracycline is presented in Table 6.10, against all three test organisms. For S. aureus, reduction obtained was 0.70 ± 0.06 CFU/ ml after 5 min and it was 1.32 ± 0.06 CFU/ ml after 15 min. For S. epidermidis, reduction was ranged between 1.29 ± 0.47 to 1.44 ± 0.52 CFU/ ml. Tetracycline showed, up to 1.12 ± 0.14 CFU/ ml reduction after 15 min against P. acnes. Percentage kill efficacy of Tetracycline against all acne inducing bacteria was found in between ± 2.64 to ± 1.04%. Table 6.10: Time kill efficacy of Tetracycline against S. aureus, S. epidermidis, P. acnes S. aureus S. epidermidis P. acnes Time (min) 5 min 10 min 15 min 0.70 ± ± ± ± ± ± 0.67 Reducti on 1.29 ± ± ± ± ± ± ± ± ± ± ± ± 2.69 School of Science, SVKM s NMIMS University Page 138

21 Results of time kill efficacy study demonstrated that, the percentage kill efficacy obtained for test extracts against acne inducing bacteria are varied among the strains and are concentration and time dependent. MBC values obtained against S. epidermidis were comparatively higher than other two organisms. The variation in susceptibility may be due to the differences in cell wall composition (Karaman et al. 2003). Among all tested extract, time kill profile of OT CAE was found to be distinct against all test bacteria and especially against S. aureus. This could relate to the synergistic action of active constituents of crude extract to generate the good antimicrobial effects (Eloff 1998). Fig. 6.7 represents the time kill efficacy pattern of OT CAE against S. aureus, S. epidermidis and P. acnes. Initial counts of all test bacteria were indicated as input counts. The final output counts, after specified time interval were named as 5 min, 10 min and 15 min counts. It can be clearly seen that, there was a reduction in viable cell counts after reaction with the test extract. Similarly, for other test extracts and Tetracycline, decrease in viable cell density was observed in comparison with initial cell density. It was observed that, cold alcoholic extracts of both the plants exhibited potent antimicrobial activity than the extracts obtained by Soxhlation. OT CAE and CR CAE exhibited strong time kill activity than OT HAE and CR HAE respectively. The difference in activity of standard Tetracycline and test extracts is may be due to the fact that, the crude extracts are mixtures of bioactive compounds where as standard antibiotic is a pure compound (Gatsing et al. 2010). Evaluation of selected plant extracts for anti-acne activity was performed by determination of MIC and MBC values and by time kill efficacy study. Results suggest that, the biocidal effect of crude extracts could be expected on most of the tested organisms. This antimicrobial effect could necessitate the identification of phytoconstituents responsible for the activity. The results of time kill study provide the strong support for the further study of isolation and identification of new antimicrobial agents against acne vulgaris. School of Science, SVKM s NMIMS University Page 139

22 P. acnes S. epidermidis S. aureus EVALUATION OF SELECTED PLANT EXTRACTS FOR Input 5 min 10 min 15 min Input 5 min 10 min 15 min Input 5 min 10 min 15 min Fig. 6.7: Time kill efficacy study of OT CAE against S. aureus, S. epidermidis and P. acnes School of Science, SVKM s NMIMS University Page 140

23 6.2.3 Anti-lipase assay P. acnes organism is a gram- positive anaerobe, resides under the skin and considered as one of the major causative agent of acne vulgaris (Strauss et al. 2007). P. acnes is responsible for inflammation occurs in acne. Several factors released by P. acnes are accountable for inflammation. These factors are lipases, proteases, hyaluronidase, and chemotactic products (Heymann 2006). Lipase enzyme produced by P. acnes hydrolyses the sebum triglycerides and release inflammatory fatty acids in pilosebaceous follicles. Various species of P. acnes showed higher production of lipase enzyme, triggering inflammation. Hence, a novel approach of targeting this enzyme for treatment of acne vulgaris has been developed (Higaki 2003). Batubara et al screened the anti-acne potency of Indonesian medicinal plants by using lipase inhibition as one of the parameter (Batubara et al. 2009). In the present study, anti-lipase activity of selected plant extracts was investigated. Initially, standard curve of p-nitrophenol was obtained to calculate the activity of crude lipase enzyme, extracted from P. acnes. Table 6.11 represents the absorbances of p-nitrophenol, obtained at 410 nm. Standard curve of p-nitrophenol is shown in Fig It was observed that the, absorbance pattern was concentration dependant and the linear equation is y = x , R 2 = Table 6.11: Standard curve of p-nitrophenol Concentration Absorbance ± SD (µmol) at 410 nm ± ± ± ± ± ± ± ± ± School of Science, SVKM s NMIMS University Page 141

24 Lipase Inhibition (%) Absorbance at 410 nm EVALUATION OF SELECTED PLANT EXTRACTS FOR y = x R² = Concentration (µ mol) Fig. 6.8: Standard curve of p-nitrophenol Lipase inhibitory effects of test extracts and Tetracycline were expressed as percentage lipase inhibition activity. Tetracycline was used as a positive standard and its activity is shown in Fig The percentage lipase inhibition activity obtained for Tetracycline was up to ± 0.46 and was concentration dependant. Several studies showed that the, Tetracycline inhibited the lipase enzyme produced by P. acnes (Uncles & Gemmell 1982) (Webster et al 1981) Tetracycline Conc (mg/ ml) Fig. 6.9: Lipase inhibition activity of Tetracycline School of Science, SVKM s NMIMS University Page 142

25 Lipase Inhibition (%) Lipase Inhibition (%) EVALUATION OF SELECTED PLANT EXTRACTS FOR Lipase inhibition activity of OT HAE and OT CAE is shown in Fig For OT HAE, percentage lipase inhibition activity ranged from ± 0.07 to ± 0.65%. Whereas, OT CAE exhibited higher activity ranging from ± 0.62 to ± 0.47%. This could probably relate to our earlier findings, where cold alcoholic extract of Ocimum tenuiflorum Linn (OT CAE) showed significant antimicrobial effect against P. acnes. 100 OT HAE OT CAE Conc (mg/ ml) Fig. 6.10: Lipase inhibition activity of OT HAE and OT CAE Percentage lipase inhibition activity of CR HAE and CR CAE extracts was not very significant. Fig shows that only up to ± 0.92% activity was obtained for CR HAE and CR CAE exhibited anti-lipase activity from ± 0.95 to ± 0.68% CR HAE CR CAE Conc (mg/ ml) Fig. 6.11: Lipase inhibition activity of CR HAE and CR CAE School of Science, SVKM s NMIMS University Page 143

26 EC 50 VALUE (mg/ ml) EVALUATION OF SELECTED PLANT EXTRACTS FOR Fig represents the EC 50 values (mg/ ml ± SD) of all test extracts and Tetracycline. The EC 50 value of OT CAE was lesser and statistically significant (**P < 0.01) than EC 50 value of OT HAE. Similarly, CR CAE showed significant EC 50 value than CR HAE (*** P < 0.001). EC 50 value of Tetracycline was found to be ± 0.01%, which was very low than tested extracts ** *** Tetracycline OT HAE OT CAE CR HAE CR CAE Tetracycline OT HAE OT CAE CR HAE CR CAE Fig. 6.12: EC 50 values of Lipase inhibition assay Results of anti-lipase assay indicate that, all the tested plant extracts have potential of inhibition of lipase enzyme. However, extracts of Ocimum tenuiflorum Linn (OT HAE and OT CAE) were found to be more potent lipase inhibitors than CR HAE and CR CAE. School of Science, SVKM s NMIMS University Page 144

27 6.3 CONCLUSION The results of the present study provided scientific justification for the use of selected plant extracts against acne inducing bacteria. The degree of the antibacterial activity indicated that, the crude extracts are potential source of bioactive compounds that could be useful for the development of new antimicrobial agents for acne vulgaris. MBC values indicate that, most of the test extracts possesses bactericidal activity. Time kill efficacy results demonstrated the time dependant antibacterial activity of extracts and provide the path for the further study of isolation and identification of new antimicrobial agents against acne vulgaris. Selected plant extracts have been investigated for their effect on inhibition of lipase activity of P. acnes. The results clearly suggest that extracts chosen here are significant inhibitors of lipase activity and OT CAE was found to be the most potent inhibitor of lipase enzyme. School of Science, SVKM s NMIMS University Page 145