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Evaluation of Synergistic Effect Of Open Access Original Article Synergistic Effect of Thujaorientalis Pak Armed Forces Med J 2020; 70 (4): 975-82 EVALUATION OF SYNERGISTIC EFFECT OF THUJAORIENTALIS WITH CIPROFLOXACIN AGAINST ESCHERICHIA COLI Hajra Mateen, Rizwan Faisal*, Syeda Huma Khizar, Syed Hamid Habib** KMU Institute of Medical Sciences, Kohat Pakistan, *Rehman Medical College, Peshawar Pakistan, **Khyber Medical University, Peshawar Pakistan ABSTRACT Objectives: To determine the antimicrobial effect of Thujaorientalis (TO) and ciprofloxacin alone against Escherichia Coli (E.Coli). To compare the combined antimicrobial effect of TO and ciprofloxacin against E. Coli. Study Design: Laboratory experimental study. Place and Duration of Study: This in vitro study was performed at Khyber Girls Medical College, Peshawar, from Jan to Jul 2018. Methodology: Antibacterial activity of TO extracts alone and in combination with ciprofloxacin against E.Coli was evaluated by using disc-diffusion and minimum inhibitory concentration method. E. coli clinical strains were collected from Rehman Medical Institute and Northwest General Hospital Peshawar, and American Type Culture Collection [ATCC] strains (number 23922) of these bacteria were collected from Agriculture University Peshawar. The organisms were tested six times with crude extract and fractionation with different solvents such as n-hexane, chloroform, ethyl acetate and butanol at concentrations of 1, 4, 8, 12, 16, 20, 24, 30 and 36 mg/ml. The mean minimum inhibitory concentration and fractional inhibitory concentration index were obtained to report the synergism. The data was analysed using SPSS version 21. Results: Antibacterial activity of different fractions of plant was interpreted as synergy, indifference and antagonism against E.Coli. Fractional inhibitory concentration index for E.Coli ranged from 3 to 1.05, which showed antagonism and indifferent effect of combination of plant extract with ciprofloxacin. Conclusion: Synergism between TO and ciprofloxacin was shown against E.Coli by disc diffusion method while no synergistic effects were found through minimum inhibitory concentration. Keywords: Ciprofloxacin, Drug antagonism, Drug synergism, Escherichia Coli, Fractional inhibitory concentration index, Minimum inhibitory concentration, Thuja orientalis. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. INTRODUCTION with prolong use of antimicrobial agents is very One of the major causes of death in under alarming situation for both underdeveloped and developed countries is infectious diseases. Allo- developed countries. Therefore, alternative strate- pathic medicines basically play an important role gies are thought to combat this problem. This in reducing burden of infectious diseases by their situation makes the alternate way of therapeutic ability to treat diseases efficiently. However, resi- use of plants extracts as an ancient remedy5. Plant stance is produced with prolong usage of these extracts being new may not have the problem of drugs due to which their effectiveness is dec- microbial resistance. The usage of plant extract as reased1,2. The bacterial resistance is a great threat medicine for different diseases has become wide to the public health; and different types of anti- spread because it is realized that the effectiveness biotics, including the major last resort drugs are of traditional medicine has more efficacy com- 6 now becoming ineffective3. Antimicrobial drug pared to synthetic drugs . The rationalization of resistance in bacteria is frequently reported from using medicinal plants combined with the exis- all over the world4. Drug resistance associated ting antimicrobial agents may help in overcoming the bacterial resistance. Furthermore, recently Correspondence: Dr Hajra Mateen, Pharmacology Dept, KMU in year 2014 it has been reported by Brighenti Institute of Medical Sciences, Kohat Pakistan and Lairungruang in their separate studies that Received: 24 Jun 2019; revised received: 01 May 2020; accepted: 08 May drug synergism can be achieved by mixing 2020 975 Synergistic Effect of Thujaorientalis Pak Armed Forces Med J 2020; 70 (4): 975-82 antimicrobial agents with plant extracts to yield Bacterial Collection the desirable results6,7. Clinical strains were collected from Rehman Prehistorically medicinal plants were used as Medical Institute (RMI) and Northwest General traditional medicine that have been playing signi- Hospital Peshawar and ATCC strain number ficant role to heal human diseases and disorders. 23922 were obtained from Agriculture University Thujaorientalis (More Pankh) is one such plants Peshawar. which is being used since long because of its Disc Diffusion Susceptibility Assay medicinal value. In traditional medicine Thujao- This was a method which indicated the rientalis (TO) is used for treatment of different susceptibility of the organism to the tested anti- diseases like bronchial catarrh, enuresis, uterine biotic by clear zone of inhibited growth around carcinomas, and rheumatism8. It is also effective the filter paper discs. The nutrient agar medium in the treatment of psoriasis, amenorrhea, cystitis plates was allowed to solidify for about 24-48 and worm infestation. In addition, it has mollus- hrs at 37°C to check sterility. The bacteria were cicidal and nematicidal activity9. streaked on plates in Laminar flow hood. For Escherichia coli (E.coli) is a gram negative making the discs, Whatman filter paper No.1 (6 organism which is responsible for various bacte- mm) was used which was sterilized in autoclave. rial infections. They are developing resistance Crude extract solutions and solutions of fractions against flouroquinolones and other antibiotics at different concentrations i.e. 1, 4, 8, 12, 16, 20, which is becoming a problem. Research studies 24, 30 mg/ml were prepared in 5% DMSO. Sterile show that TO has great activity against Staphylo- coccus aureus, E.Coli, Bacillus subtilis and Agrobac- terium tumefaiens10. Therefore, looking at the mul- tiple uses of TO and the importance of synergism we aimed to evaluate the synergistic effects of this compound in combination with ciprofloxacin against in vitro samples of E. coli. METHODOLOGY Plant Material Fresh plant leaves of Thujaoriental is weigh- ting 13kg were collected from Bagh-e-Naran, Hayatabad, Peshawar. The plant specimen was identified by taxonomist in Botany department, University of Peshawar and a specimen was Figure: Plant extraction and fractionation process. placed there in the herbarium with voucher number Bot-224/2016. The sample was dried 6 mm discs were impregnated with 50 µl of the for 7 days and soaked in methanol. The filtered extract and placed on the surface of agar plates methanolic solution was dried under vacuum inoculated with microbial culture. Ciprofloxacin pressure below 45°C in rotatory evaporator. The (5 µg/disc) served as positive control and DMSO semi-solid extract was collected in a glass vial 6 µl served as negative control. Inoculated plates and divided into two parts. One part was the were then incubated at 37°C for 18-24 hrs. methanolic extract (E1) andthe second part was The next day zones of inhibition were measured used for refractionation with different solvents in mm around the discs. Reference table used to such as N-hexane (E2), chloroform (E3), ethyl- interpret bacteria as sensitive (S), intermediate or acetate (E4), butanol (E5) and aqueous extract resistant (R) according to clinical and laboratory (E6). standard institute (CLSI) guidelines of National 976 Synergistic Effect of Thujaorientalis Pak Armed Forces Med J 2020; 70 (4): 975-82 Committee for Clinical Laboratory Standards ned to report the synergism. The antibacterial (NCCLS)11. activity was interpreted as one of the following Minimum Inhibitory Concentration (MIC) categories; synergy; indifference; additive effect or antagonism12. It was the lowest concentration of an anti- microbial agent that prevents visible growth of Formula to Determine Synergy (MIC and FIC) a microorganism in an agar or broth dilution MICA in susceptibility test. Combination FIC*A = MIC Table: Interpretation and Zone of Diameter (mm) MICB in Interpretation and Zone of Diameter Combination FICB = (mm) MIC Bacteria B Sensitive Resistant Intermediate Where A and B are the two antimicrobial (S) (R) under investigation. Escherichia ≥21 16-20 ≤15 coli FICI = FICA + FICB Sum of FICI Calculated McFarland 0.5 Barium Sulphate Turbidity Mean FICI = Standard Number of FICI Calculated Interpretation 0.5 McFarland standard was prepared by adding 0.05 ml of barium chloride dihydrate Synergy = mean FICI <0.5, (BaCl2) to 9.95 ml of sulphuric acid (1%H2SO4) Partial Synergy/Addition = mean FICI >0.5 <1.0, with constant mixing. Bacterial colonies were Indifference = mean FICI >1 - <2.0, transferred to 3-4 ml of sterile normal saline Antagonism = mean FICI >2.0. solution. This inoculum was then matched with 0.5 McFarland standard11. RESULTS 96-Well Microdilution Tray Antimicrobial activities of TO fractions were observed in this study alone and in combination 96-well plates were loaded with 100 µl (0.1 with ciprofloxacin against E.coli. The results of ml) of two fold dilutions of extracts into each M.C.E (E1) fraction against E.coli (clinical and well. The extracts were serially diluted two fold ATTC 25922 strains) alone and with
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