60 e- ISSN 0976-1047 Print ISSN 2229-7499 International Journal of Biopharmaceutics Journal homepage: www.ijbonline.com IJB EVALUATION OF SYNERGISTIC EFFECT OF GINGER, GARLIC, TURMERIC EXTRACTS ON THE ANTIMICROBIAL ACTIVITY OF DRUGS AGAINST BACTERIAL PATHOGENS Seema Rawat* Department of Botany and Microbiology, H.N.B Garhwal (Central) University, Srinagar, Uttarakhand, India. ABSTRACT The present work was carried out to determine the antimicrobial potential of spices and to study whether the crude extracts of spices exhibit any synergistic effect when used in combination with antibiotics. All extracts were most effective against E. coli. The ethanolic extract of turmeric exhibited maximum antimicrobial potential against E. coli (29.3±0.17 mm) followed by garlic (25±0.17 mm) and ginger(21.0±0.34 mm). Amongst methanolic extracts garlic exhibited maximum potential (15.8±0.19 mm) followed by that of ginger (15.0±0.12 mm) and turmeric (11.6±0.17 mm). The aqueous extract of turmeric was most effective against E. coli (14.3±0.21 mm) followed by ginger (13.0±0.16 mm) while aqueous extract of garlic showed maximum antimicrobial potential against Staphylococcus (12.6±0.13 mm). Maximum isolates were observed to be resistant to clindamycin, oxacillin and ampicillin. The crude extracts showed synergistic effect with antibiotics in exhibiting antimicrobial potential against bacterial pathogens. This synergistic effect can be used to design good therapeutic approach to combat with bacterial pathogens. Key words: Antimicrobial potential, Garlic, Ginger, Turmeric, E. coli, Staphylococcus. INTRODUCTION Desolate warnings issued by several health authorities are clear indication of us standing at the doorstep of a post-antibiotic era (CDC, 2013; WHO, 2014). Overuse of drugs has led to the evolution of drug resistance mechanism amongst the pathogens (Kiffer et al., 2007; AlJohani et al., 2010). The pathogens have evolved various mechanisms to counteract the effect of drugs. The resistance to antibiotics can be natural, acquired, genetic, phenotypic or biological (Thomas and Singh, 2013). The resistance may develop due to spontaneous mutation in gene, acquisition of plasmid or transposon, change in the physiological state of bacterial cell or reduced permeability of cell. Major mechanisms Corresponding Author Seema Rawat E-mail: seemamillenium@gmail.com of bacterial resistance to antimicrobial agents include the following: (a) enzymatic drug inactivation; (b) drug target modification; (c) drug permeability reduction; and (d) active efflux of drugs (Davies, 1994, Webber and Piddock, 2003; Fabrega et al., 2009; Drapeau et al., 2010). The bacteria harbouring these drugs resist, survive, or even grow, inthe presence of a given antimicrobial agent. Moreover, certain bacterial variants have evolved mechanisms to resist multiple drugs, making such variants obstinate to chemotherapy against such bacterial strains that are the causative agents of infection in patients. The various drug inactivation mechanisms involve enzymatic hydrolysis of antibiotics, group transfer, ribosome protection and biofilm formation (Wright, 2005; Roberts, 2005; Hoiby, 2010). There are several non-antibiotic approaches with specific regard to antimicrobials to the treatment and prevention of infection including probiotics, bacteriophages and
61 phytomedicines (Aliskyet al., 1998; Caelliet al., 2000; Fillho-Lima et al., 2000). The present study was carried out to investigate the synergistic effect of ginger, garlic and turmeric on the antimicrobial activity of antibiotics against the bacterial pathogens. MATERIALS AND METHODS Bacterial culture The pathogenic bacteria viz., E. coli, Pseudomonas, Proteus, Serratia, Staphylococcus and Klebsiella were taken from departmental culture collection. Acquisitions of spices& preparation of extracts Garlic, ginger and turmericwere procured from the local market. The spices were sorted for separation of dirt and unwanted materials and grounded into fine powder. Three extractantsi.e, water, ethanol and methanol were used. The extracts were prepared by dissolving spices in solvents in a concentration of 1:4 and keeping at room temperature for 24hrs in a sterile beaker covered with aluminium foil to avoid evaporation and then subjected to filtration through sterilized Whatman no. 1 filter paper. The solvent was dried and concentrated using orbital shaker at 40 C. The stock solutions of the extracts thus obtained were prepared by diluting the dried extracts with 50% of respective solvents. Evaluation of antimicrobial activity of extracts The antimicrobial activity of crude extracts against pathogenic bacteria was evaluated by using agar well diffusion method. The isolates were inoculated into 10ml of sterile nutrient broth, and incubated at 37±1 0 C overnight. The turbidity of culture was compared with Mac Farland standard number II. The cultures were swabbed on the surface of sterile Mueller-Hinton agar plates using a sterile cotton swab and allowed to dry for 3-5 minutes. Agar wells were prepared with the help sterilized borer with 10mm diameter. The extract of spices was diluted to give the final concentration 1000ppm, 2000ppm, 3000ppm and 4000ppm. 100 µl of different dilutions of the extracts was added to the wells of the inoculated plates. 50% ethanol and 50% methanol was used as control which was introduced into the well instead of the extract. The plates were incubated in an upright position at 37±1 0 C for 24hrs. The zone of inhibition was measured and expressed in millimetres. Antibiotic sensitivity assay All isolates were tested for antibiotic sensitivity by Kirby-Bauer disc diffusion method (Bauer et al., 1996) on Mueller-Hinton agar (MHA). The cultures were enriched in sterile nutrient Broth overnight at 37 C. Using a sterile cotton swabs, the cultures were aseptically swabbed on the surface of surface MHA plates and allowed to dry for 3-5 minutes before applying the antibiotic discs. Using a sterile forcep, 4 antibiotic discs were aseptically placed over the inoculated plates sufficiently separated from each other to avoid overlapping of inhibition zones. The plates were incubated in an upright position for 24hrs at 37 C and diameter of zone of inhibition was measured in mm. Evaluation of synergistic effect of crude extracts on the antimicrobial activity of drugs The antibiotic and dilution of the crude extracts exhibiting maximum antimicrobial potential against the bacterial pathogens was chosen for further study. This test was carried out in the similar as described under antibiotic sensitivity assay with an addition that 100 µl of the dilutions of the extracts exhibiting maximum antimicrobial potential was added to the antibiotic discs. The plates were incubated in an upright position at 37±1 0 C for 24hrs. The zone of inhibition was measured and expressed in millimetres. RESULTS Antimicrobial activity of crude extracts against bacterial pathogens All extracts exhibited good antimicrobial potential towards uropathogens (Table 1 to 3). The ethanolic extract of turmeric exhibited maximum antimicrobial potential against E. coli (29.3±0.17 mm) and least activity towards Serratia (7.3±0.14 mm). The methanolic extract exhibited maximum activity againste. coli (11.6±0.17 mm) and least against Serratia (6.5±0.14 mm). The aqueous extract exhibited maximum activity against E. coli (14.3±0.21 mm) and minimum against Serratia (6.9±0.12 mm). The ethanolic extract of garlic showed maximum antimicrobial potential towards E. coli (25±0.17 mm) and least towards Klebsiella (7.3±0.18 mm). The methanolic extract was most effective against E. coli (15.8±0.19 mm) and least towardsklebsiella (7.0±0.14 mm) while aqueous extract was most effective against Staphyloccus(12.6±0.13 mm) and least towards Klebsiella (5.6±0.14 mm). The ethanolic extract of ginger showed maximum activity against E.coli (21.0±0.34 mm) and least activity against Serratia (9.3±0.32 mm). The methanolic extract was most effective against E. coli (15.0±0.12 mm) and least against Serratia (6.3±0.12 mm). The aqueous extract was most effective against E. coli (13.0±0.16 mm) and least against Serratia (6.0±0.12 mm). Antimicrobial activity of antibiotics against bacterial pathogens Maximum isolates were observed to be resistant to clindamycin, oxacillin and ampicillin (Fig. 1). E. coli was found to be resistant to ciprofloxacin. Staphylococcus was observed to be resistant
62 to clindamycin, erthyromycin, oxacillin, vancomycin, ampicillin and ciprofloxacin. Pseudomonas was resistant to erythromycin while Klebsiella was resistant to clindamycin, chloramphenicol, oxacillin, vancomycin, ampicillin, ciprofloxacin and cephalothin. Proteus was resistant to clindamycin, eryhtromycin, oxacillin, ampicillin and cephalothin. Serratia was resistant to clindamycin, eryhtromycin, oxacillin, vancomycin, ampicillin, ciprofloxacin and cephalothin. Synergistic effect of crude extracts on the antimicrobial activity of drugs The extracts of different spices showed synergistic effect with antibiotics in exhibiting antimicrobial potential against bacterial pathogens (Table 4 to 6). The zone diameters were found to increase when extracts were used in combination with antibiotics. The combination was found to be more potent than either of the two. Fig 1. Effect of antibiotics against bacterial pathogens CLI- Clindamycin; TPM- Trimethoprim; CHL- Chloramphenicol; ERY- Erythromycin; TOB- Tobramycin; OX- Oxacillin; VAN- Vancomycin; AMP- Ampicillin; AMK- Amikacin; CIP- Ciprofloxacin, CEPH- Cephalothin. Table 1a. Antimicrobial activity of ethanolic extract of turmeric against bacterial pathogens Zone of inhibition E. coli 11.6±0.15 16.3±0.25 21.3±0.14 29.3±0.17 Staphylococcus 8.6±0.14 9.3±0.15 10.7±0.20 13.6±0.13 Pseudomonas 3.6±0.10 5.3±0.12 7.4±0.20 8.6±0.20 Klebsiella 8.6±0.10 10.3±0.21 12.6±0.14 14.3±0.18 Proteus 9.3±0.12 11.6±0.12 12.6±0.22 13.3±0.21 Serratia 3.3±0.15 4.3±0.21 5.3±0.12 7.3±0.14 Table 1b. Antimicrobial activity of methanolic extract of turmeric against bacterial pathogens Zone of inhibition E. coli 5.6±0.15 7.3±0.14 9.4±0.21 11.6±0.17 Staphylococcus 7.3±0.22 8.3±0.25 9.6±0.20 10.6±0.12 Pseudomonas 3.0±0.12 4.5±0.14 6.2±0.12 7.6±0.20 Klebsiella 7.4±0.10 8.6±0.12 9.0±0.12 10.6±0.24 Proteus 3.3±0.24 5.6±0.15 6.6±0.25 8.3±0.15 Serratia 2.3±0.15 3.8±0.15 4.9±0.22 6.5±0.14 Table 1c. Antimicrobial activity of aqueous extract of turmeric against bacterial pathogens Zone of inhibition E. coli 6.3±0.20 8.6±0.23 10.3±0.21 14.3±0.21 Staphylococcus 7.6±0.26 8.7±0.14 9.8±0.16 11.3±0.23 Pseudomonas 3.4±0.20 4.9±0.21 6.8±0.15 8.0±0.14 Klebsiella 7.8±0.20 9.3±0.15 10.6±0.30 12.8±0.14 Proteus 4.3±0.10 6.5±0.12 8.6±0.12 10.3±0.12 Serratia 3.0±0.12 4.0±0.12 5.1±0.24 6.9±0.12
63 Table 2a. Antimicrobial activity of ethanolic extract of garlic against bacterial pathogens Zone of inhibition E. coli 19±0.15 21±0.10 23±0.13 25±0.17 Staphylococcus 7.0±0.14 10±0.15 12±0.11 15±0.12 Pseudomonas 6.7±0.21 7.8±0.17 8.6±0.13 9.3±0.14 Klebsiella 4.0±0.12 5.4±0.16 6.7±0.12 7.3±0.18 Proteus 6.3±0.14 8.2±0.12 9.4±0.22 10.3±0.15 Serratia 3.3±0.12 5.7±0.20 7.5±0.14 9.3±0.12 Table 2b. Antimicrobial activity of methanolic extract of garlic against bacterial pathogens Zone of inhibition E. coli 9.3±0.17 11.6±0.13 12.8±0.23 15.8±0.19 Staphylococcus 6.6±0.14 7.3±0.20 9.3±0.16 11.5±0.13 Pseudomonas 6.0±0.18 7.3±0.11 8.2±0.17 9.0±0.17 Klebsiella 3.3±0.17 4.8±0.20 6.3±0.12 7.0±0.14 Proteus 6.0±0.12 7.5±0.16 9.0±0.10 10.0±0.12 Serratia 3.0±0.14 4.8±0.17 6.1±0.10 8.2±0.10 Table 2c. Antimicrobial activity of aqueous extract of garlic against bacterial pathogens Zone of inhibition E. coli 3.4±0.15 4.3±0.10 5.4±0.13 10.2±0.17 Staphylococcus 4.5±0.12 6.7±0.15 10.5±0.12 12.6±0.13 Pseudomonas 5.0±0.10 6.5±0.12 7.5±0.13 8.7±0.27 Klebsiella 2.2±0.17 3.4±0.14 4.1±0.12 5.6±0.14 Proteus 5.0±0.12 6.2±0.12 7.5±0.12 9.2±0.15 Serratia 2.5±0.10 3.7±0.17 4.9±0.13 6.0±0.10 Table 3a. Antimicrobial activity of ethanolic extract of ginger against bacterial pathogens Zone of inhibition E. coli 17.0±0.15 18.0±0.15 20.0±0.18 21.0±0.14 Staphylococcus 9.2±0.14 10.0±0.15 12.5±0.21 14.0±0.12 Pseudomonas 10.0±0.20 14.0±0.17 16.0±0.13 18.0±0.18 Klebsiella 6.0±0.12 8.4±0.16 10.7±0.12 12.3±0.18 Proteus 10.5±0.14 11.2±0.12 13.6±0.12 15.3±0.15 Serratia 4.7±0.12 6.7±0.21 7.4±0.14 9.3±0.12 Table 3b. Antimicrobial activity of methanolic extract of ginger against bacterial pathogens Zone of inhibition E. coli 11.0±0.25 12.0±0.15 14.0±0.13 15.0±0.12 Staphylococcus 8.0±0.14 9.2±0.15 10.7±0.21 11.7±0.12 Pseudomonas 8.5±0.21 10.0±0.17 11.6±0.10 12.5±0.24 Klebsiella 4.0±0.12 6.0±0.16 7.0±0.12 8.5±0.18 Proteus 6.2±0.24 7.2±0.12 8.4±0.20 9.7±0.25 Serratia 3.2±0.12 4.7±0.21 5.4±0.14 6.3±0.12.
64 Table 3c. Antimicrobial activity of aqueous extract of ginger against bacterial pathogens Zone of inhibition E. coli 9.0±0.15 10.6±0.17 11.4±0.10 13.0±0.16 Staphylococcus 7.3±0.14 8.7±0.24 10±0.21 12±0.21 Pseudomonas 4.57±0.17 5.8±0.22 6.4±0.20 8.3±0.12 Klebsiella 3.4±0.12 5.4±0.19 6.7±0.12 7.9±0.10 Proteus 2.3±0.14 3.6±0.12 4.75±0.22 6.5±0.15 Serratia 2.7±0.12 4.3±0.14 5.1±0.20 6.0±0.12. Table 4. Antimicrobial activity of turmeric extracts in combination with antibiotics against bacterial pathogens Antibiotic Zone of methanolic inhibition ethanolic extract aqueous extact extract E. coli Amp 29.0±0.67 45.5±0.20 36.0±0.16 38.1±0.30 Staphylococcus Chl 24.0 ±0.35 34.4±0.50 30.2±0.32 32.2±0.30 Pseudomonas Cip 19.0 ±0.48 25.0±0.34 22.0±0.36 23.0±0.22 Klebsiella Amk 11.0±0.62 20.0 ±0.24 14.0±0.30 17.0 ±0.24 Proteus Chl 23.0±0.56 40.5±0.36 34.0±0.32 37.2±0.35 Serratia Chl 13±0.45 18.0±0.30 13.7±0.25 16.4±0.35 Amp- Ampicillin; Chl- Chlorafloxacin; Cip- Ciprofloxacin; Amk- Amikacin Conc. of extracts used: 4000 ppm. Table 5. Antimicrobial activity of garlic extracts in combination with antibiotics against bacterial pathogens Zone of methanolic Antibiotic ethanolic extract inhibition extract aqueous extact E. coli Amp 29.0±0.67 46.0±0.39 36.3±0.34 31.0±0.35 Staphylococcus Chl 24.0 ±0.35 32.4±0.56 30.1±0.24 27.2±0.32 Pseudomonas Cip 19.0 ±0.48 24.2±0.40 23.0±0.30 22.4±0.24 Klebsiella Amk 11.0±0.62 18.2 ±0.34 16.4±0.24 13.2 ±0.27 Proteus Chl 23.0±0.56 30.4±0.33 27.1±0.32 20.4±0.21 Serratia Chl 13.0±0.45 19.4±0.37 16.2±0.25 15.1±0.26 Amp- Ampicillin; Chl- Chlorafloxacin; Cip- Ciprofloxacin; Amk- Amikacin; Conc. of extracts used: 4000 ppm. Table 6. Antimicrobial activity of ginger extracts in combination with antibiotics against bacterial pathogens Name of organism Antibiotic Zone of inhibition ethanolic extract methanolic extract aqueous extact E. coli Amp 29.0±0.67 41.0±0.24 36.3±0.16 30.0±0.35 Staphylococcus Chl 24.0 ±0.35 32.1±0.56 27.5±0.34 25.0±0.30 Pseudomonas Cip 19.0 ±0.48 30.4±0.34 26.3±0.30 20.4±0.24 Klebsiella Amk 11.0±0.62 20.5 ±0.24 17.4±0.26 15.2±0.27 Proteus Chl 23.0±0.56 33.1±0.33 30.4±0.32 26.5±0.21 Serratia Chl 13.0±0.45 19.3±0.35 17.0±0.25 14.5±0.30 Amp- Ampicillin; Chl- Chlorafloxacin; Cip- Ciprofloxacin; Amk- Amikacin Conc. of extracts used: 4000 ppm. DISCUSSION AND CONCLUSION The spices used in cooking are well known since ages for adding flavor, colour and aroma to the food. They are part of our daily diet. These spices also hold medicinal values and therefore widely used in traditional medical practices. The spices have started gaining attention of scientists as an alternative approach due to increasing resistance amongst pathogens towards antibiotics (Uraih, 2004; Souza, 2005; Pundir and Jain, 2010). They possess a number of pharmacological effects to treat different human ailments (Arora and Kaur, 1999; Gur et al., 2006). Many workers are now-a-days working on the antimicrobial potential of extracts of medicinal plants,
65 spices and herbs however there is a need to evaluate the synergism between these extracts and antibiotics usually prescribed to treat infections. This may prove to be beneficial and probably will not allow bacterial pathogens to easily develop resistance. The present work was focused on synergistic aspect only. All extracts were found to be most effective against E. coli. The ethanolic extract of turmeric exhibited maximum antimicrobial potential as compared to aqueous and aqueous was more potent than methanolic extract. However in case of ginger and garlic aqueous extract was found to be less effective as compared to methanolic extract. The solubility of phytochemicals in different solvents decides which will be most effective and therefore in this study ethanolic, aqueous and methanolic extracts were used. The active constituent of spices may exhibit their antimicrobial effect either by degradation of cell wall, disruption of cytoplasmic membrane, leakage of cellular components, damage protein, interfere with the enzymatic activities inside cell, affect synthesis of DNA and RNA, affect electron transport and nutrient uptake, leakage of cellular components, impair the energy production inside cell, change fatty acid and phospholipid constituents (Shan et al., 2007). The extracts showed synergistic effect with antibiotics in exhibiting antimicrobial potential against bacterial pathogens.the combination was found to be more potent than either of the two. Thus it may be concluded that the combination of antibiotics alongwith spices can be effectively used to combat various infections. REFERENCES Al Johani SM, Akhter J, Balkhy H, El-Saed A, Younan M and Memish Z. Prevalence of antimicrobial resistance among gram-negative isolates in an adult intensive care unit at a tertiary care center in Saudi Arabia. Annals Saudi Med. 2009; 30: 364-369. Alisky J, Iczkowski K, Rapoport A and Troitsky N. Bacteriophages show promise as antimicrobial agents. J. Hosp. Infect. 1998; 36: 5 15. Arora D and Kaur J. Antimicrobial activity of spices. Intl. J. Antimicrobial agents. 1999, 12: 257-262. Caelli M, Porteous J, Carson CF, Heller R and Riley TV. Tea tree oil as an alternative topical decolonization agent for methicillin-resistant Staphylococcus aureus. J. Hosp. Infect. 2000; 46: 236 237. Center for Disease Control and Prevention (CDC). Antibiotic resistance threats in the United States, Atlanta, 2013. Davies J. Inactivation of antibiotics and the dissemination of resistance genes. Science. 1994; 264: 375-82. Drapeau CM, Grilli E and Petrosillo N. Rifampicin combined regimens for gram-negative infections: data from the literature. Int. J. Antimicrobiol. Agents. 2010; 35: 39-44. Fabrega A, Madurga S, Giralt E and Vila J. Mechanism of action of and resistance to quinolones. Microbial biotechnol. 2009; 2: 40-61. Filho-Lima JVM, Viera EC and Nicoli JR. Antagonistic effect of Lactobacillus acidophilus, Saccharomyces boulardii and Escherichia coli combination against experimental infections with Shigellaflexneri and Salmonella enteritidis subsp. typhimurium in gnotobiotic mice. J. Appl. Microbiol. 2000; 88: 365 370. Gur S, Balik DT and Gur N. Antimicrobial activity and some fatty acids of turmeric, ginger root, and linseed used in the treatment of infectious disease. World J. Agri. Sci. 2006; 2: 439-442. Hoiby N, Bjarnsholt T, Givskov M, Molin S and Ciofu O. Antibiotic resistance of bacterial biofilms. Int. J. Antimicrobiol Agents. 2010; 35: 322-32. Kiffer CR, Mendes C, Oplustil CP and Sampaio JL. Antibiotic resistance and trend of urinary pathogens in general outpatients from a major urban city. Int. Braz. J. Urol. 2009; 33: 42-48. Pundir RK and Jain P. Comparative studies of antimicrobial activity of black pepper and turmeric extracts. Int. J. Biol. Pharm. Res. 2010; 1: 491-501. Roberts MC. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 2004; 245: 195-203. Shan B, Cai YZ, Sun M, Corke H. The in vitro antibacterial activity of dietary spice and medicinal herb extracts. Int. J. Food Microbiol. 2007; 117: 112-119. Souza EL, Stamford TLM, Lima EO, Trajano VN and Filho JB. Antimicrobial effectiveness ofspices: an approach for use in food conservation systems. Braz. Arch. Biol. Technol. 2005; 48: 549-558. Thomas MB and Singh S. Review article on Antimicrobial Resistance. Ind. J. Res. Pharmacy &Biotechnol. 2013; 62: 2320 3471. Uraih N. Food Microbiology. Bobpeco Publishers, Benin City, Nigeria, 2004; 92-130. Webber MA and Piddock LJ. The importance of efflux pumps in bacterial antibiotic resistance. J. Antimicrobial Chemo. 2003; 51: 9-11. WHO. Antimicrobial resistance: global report on surveillance, Geneva, 2014. Wright GD. Bacterial resistance to antibiotics: enzymatic degradation and modification. Adv. Drug Deliv. Rev. 2005; 57: 1451-70.