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DOI: 10.35845/kmuj.2019.19041 ORIGINAL ARTICLE

ANTIMICROBIAL ACTIVITY OF THUJA 1 Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan ORIENTALIS IN COMBINATION WITH 2 Khyber Medical University Institute of Medical Sciences (KMU-IMS), Kohat, CIPROFLOXACIN AGAINST Pakistan Email : [email protected] AERUGINOSA 3 Rehman Medical College, Peshawar, 1,2 1 Pakistan Hajra Mateen , Shafiq Ahmad Tariq , 4 Northwest School of Medicine, Peshawar, Syed Hamid Habib1, Syeda Huma Khizar1,2, Pakistan Abdul Jalil Popalzai3, Abid Hussain4 Date Submitted: February 15, 2019 Date Last Revised: June 16, 2019 ABSTRACT Date Accepted: June 20, 2019 OBJECTIVE: To compare the combined antimicrobial effect of Thuja orientalis and ciprofloxacin against in vitro samples. health care, approximately 80% of the population depend on traditional METHODS: In-vitro antibacterial activity of plant-extracts was evaluated alone 4 treatments using herbal medicines. and in combination with ciprofloxacin against Pseudomonas aeruginosa using Poor sanitary conditions among the disc-diffusion susceptibility assay (Kirby Bauer method) and minimum inhibitory developing countries4 and synthetic concentration (96 well broth microdilution method) following CLSI guidelines. antimicrobial agents have provoked Pseudomonas aeruginosa clinical strains were collected from Rehman Medical the development of drug resistant Institute (RMI) and Northwest General Hospital Peshawar, Pakistan and ATCC 5 strains (no.9721) of this bacterium were collected from Agriculture University strains among the infectious agents. In Peshawar. The organism was tested six times with crude extract and r e c e n t y e a r s , p h a r m a c e u t i c a l fractionation with different solvents such as n-hexane, , ethyl companies have developed many acetate and butanol at concentrations of 1, 4, 8, 12, 16, 20, 24, 30 and 36 mg/ml. antimicrobial drugs, however, bacterial The mean MIC and FICI (fractional inhibitory concentration index) was resistance to these drugs is increasing and is now becoming one of the main obtained to report the synergism. The data were analysed using SPSS version- 5 21. global problems. Medicinal plants extracts are important sources for anti- RESULTS: In combination, methanolic crude extract, chloroform and butanol microbial and other sources of new fraction showed synergistic effect at all tested concentrations against agents that can help in reducing the Pseudomonas aeruginosa (ATCC 9721 and clinical) except with 1, 4, 16 mg/ml .1,6-8 The rationalization of concentrations. Ethyl acetate and aqueous fractions shows indifference and using medicinal plants combined with synergistic effect against Pseudomonas aeruginosa (ATCC 9721 and clinical) at the existing antimicrobial agents will different concentrations. The fractional inhibitory concentration index (FICI) help in overcoming the bacterial ranged from 1.24 to 3.24 against Pseudomonas aeruginosa alone and in resistance.9,10 Furthermore, recently it combination with ciprofloxacin. has been reported that drug synergism CONCLUSION: By disc diffusion method, this study shows synergistic effect c a n b e a c h i e v e d b y m i x i n g against Pseudomonas aeruginosa in combination with ciprofloxacin. However, antimicrobial agents with plant extracts through minimum inhibitory concentration method, it shows antagonism and to yield the desirable results. indifference but no synergistic effect against different fractions of plant. Thuja orientalis is a monoecious and KEY WORDS: Drug Resistance, Microbial (MeSH); Drug Resistance (MeSH); evergreen tree belonging to the family Thuja (MeSH); Thuja Orientalis (MeSH); Synergism (Non-MeSH); Fractional Cupresseaceae (Cypress). 1 1 In Inhibitory Concentration Index (Non-MeSH); Antagonism (Non-MeSH); traditional medicine, Thuja orientalis is Microbial Sensitivity Tests (MeSH); Pseudomonas (MeSH); Pseudomonas used for treatment of different diseases (MeSH); Pseudomonas aeruginosa (MeSH). like bronchial catarrh, enuresis, uterine carcinomas, and rheumatism.12 It is also THIS ARTICLE MAY BE CITED AS: Mateen H, Tariq SA, Habib SH, Khizar effective in the treatment of psoriasis, SH, Popalzia AJ, Hussain A. Antimicrobial activity of Thuja orientalis in amenorrhea, cystitis and worm combination with ciprofloxacin against Pseudomonas aeruginosa. Khyber Med infestation. 1 2 In addition, it has Univ J 2019;11(2):90-7. DOI: 10.35845/kmuj.2019.19041 molluscicidal and nematicidal activity.13 Leaves of Thuja orientalis produce the

1 essential oil that has its active developing countries. New herbal INTRODUCTION ingredient “Alpha-” which is medicines are developed from natural effective as anti-fungal and insecticidal nfectious diseases remain main cause products which make the basis of 13 2,3 agent. In traditional medicines it is also Iof morbidity and mortality in the traditional medicine. In primary

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used for the treatment of blood methanol was added and the process University of Agriculture, Peshawar. In diseases, gastrointestinal symptoms, was repeated three times. In rotatory laminar flow hood all microbial stock chronic cough, asthma and skin evaporator the filtered methanolic culture were streaked on nutrient agar diseases etc.12 Furthermore, the solution was dried under vacuum medium plates by using a sterile phytochemicals in Thuja orientalis have pressure below 45°C. The semi-solid inoculation loop and incubation at significant hepatoprotective activity as extract was collected and kept in dark 37ºC for 24 hours. reported by Dubey and Batra.14,15 The for drying in a glass vial at room different parts of the Thuja orientalis temperature. The crude extract is Disc Diffusion Susceptibility Assay: plant are used for different purposes divided into two parts. One part is kept Extracted samples of Thuja orientalis 18 for example leaves are used as anti- as methanolic crude extract and were tested for antibacterial activity by pyretic, anti-diuretic and as astringent. second part was used for fractionation disc diffusion assay following Clinical Seeds are used as aperients, laxative, with different solvents such as n- and Laboratory Standard Institute lenitive, sedative and different nervous hexane, chloroform, ethyl acetate and (CLSI) guidelines. Nutrient agar media disorder like insomnia. Scalds and butanol in the following way. plates were inoculated for 18-24 hours pustules are treated by bark part of the cultures of microbial inoculums. Each plant.16 Pseudomonas aeruginosa is 1. Methanol crude extract (M.C.E) (E1), disc will represent a separate amongst the most common causes of replication. In the first round of the localized suppurative lesions in human 2. N-hexane fraction (E2), experiment 1 mg plant extracts alone in beings. Especially in hospital 6 µl dimethyl sulfoxide (DMSO) and in environment, it is capable of 3. Chloroform fraction (E3), combination with ciprofloxacin (50 µg) d e v e l o p i n g r e s i s t a n c e t o 4. Ethyl acetate fraction (E4), will be applied on the discs. In separate fluoroquinolones (e.g. ciprofloxacin) petri plates as positive and other antibiotics enhance its 5. Butanol fraction (E5), control (ciprofloxacin 50 μg per 6 μl) importance as human . -1 6. Aqueous fraction (E6) and absolute DMSO (6 μl disc ) as negative control will be also applied on Thuja orientalis have different Bacterial Isolates Collection the discs incubated at 37ºC for 18-24 composition of chemicals among them Method: hours. The zones of inhibition around three substances are very important i.e the discs will be noted next day in mm alpha (α) thujaplicin, beta (β) thujaplicin Clinical bacterial isolates were 17 by using vernier calipers. and gamma (∞) thujaplicin. The randomly collected from Rehman bioactive constituents of Thuja Medical Institute (RMI) and Northwest Susceptibility Test Interpretative orientalis such as saponins, tannins, General Hospital Peshawar, Pakistan. Criteria for Ciprofloxacin against , , and alkaloids ATCC strain number 9721 of the Pseudomonas aeruginosa by have great antimicrobial activity. bacteria was obtained from The NCCLS (Kirby-Bauer) Essential oils of Thuja orientalis also have activity against different viruses like herpes simplex virus type-1 (HSV- Medicinal plant 1) and severe acute respiratory (13kg) syndrome coronavirus (SARS- Coronavirus). Looking at the multiple Shade Drying uses of Thuja orientalis we aimed to Coarse Grinding discover the synergistic effects of Thuja (3.7 kg) orientalis in combination with ciprofloxacin against Pseudomonas Maceration aeruginosa in vitro samples. Rotary Evaporation METHODS Methanolic extract of Plant Material. Fresh plant leaves Thuja orientalts were collected from University of (340 g) Peshawar, Pakistan and washed thoroughly with distilled water, dried Fractionation for seven days and grinded. The dried powdered leaves were soaked in methanol. The macerated material was N-hexance Chloroform Ethyl acetate Butanol Aqueous kept in dark for 2 weeks at room (40.6 g) (70.1 g) (15.52 g) (75.39 g) (160.61 g) temperature, shaken vigorously and filtered using Whatman filter paper Figure 1: Plant Extraction and Fractionation Process no.1. In the solid residue fresh

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Bacteria (Pseudomonas aeruginosa) was ciprofloxacin on each isolate was also antibacterial activity against the clinical interpreted as sensitive (zone of studied to assess the synergistic strain of Pseudomonas aeruginosa at inhibition ≥21), intermediate (zone of activity. Each organism were tested different concentrations but on inhibition 16-20) or resistant (zone of three times and the mean MIC and FICI combination with ciprofloxacin it inhibition ≤15) according to CLSI (fractional inhibitory concentration showed synergistic effect (Table 1). guidelines of National Committee for index) was obtained to report the However, again at concentrations of 1, Clinical Laboratory Standards synergism. 4, 16 mg/ml it did not show synergistic (NCCLS).18 effect. The N-hexane fraction (E2) of The minimum inhibitory concentration Thuja orientalis shows no significant Minimum inhibitory concentration results was interpreted as one of the antibacterial activity against clinical (MIC) following categories; ; isolates of Pseudomonas aeruginosa. indifference; additive effect or The N-hexane fraction (E2) of Thuja After establishing antimicrobial activity antagonism.20 through disc diffusion method assay, orientalis showed a significant minimum inhibitory concentration Formula to Determine Synergy synergistic activity in combination with (MIC) was determined by a “broth (MIC and FIC) c i p r o f l o x a c i n a t d i f f e r e n t microdilution method” following CLSI concentrations against Pseudomonas MIC in MIC in guidelines. A B a e r u g i n o s a ( ATC C 9 7 2 1 ) . A t c o n c e n t r a t i o n o f 8 m g / m l a n FIC*A=combination FICB=combination McFarland 0.5 barium sulphate indifference effect was observed with MIC MIC turbidity standard: A B zone of inhibition 28.66±2.51 for both samples. No synergistic activity was McFarland standard (0.5) was Where A and B are the two antimicrobial found at concentration of 1, 4 and 16 prepared by adding 0.05 ml of barium under investigation. m g / m l a g a i n s t a l l t e s t e d c h l o r i d e d i h y d r a t e ( 1 . 1 7 5 % FICI = FIC + FIC A B microorganisms. BaCl .2H 0) to 9.95 ml of sulphuric acid 2 2 Mean FICI** = Sum of FICI calculated (1%H2SO4) with constant mixing. The antibacterial activity of chloroform Bacterial colonies were transferred to Number of FICI calculated fraction of Thuja orientalis (E3) and its 3-4 ml of sterile normal saline solution. synergistic effect with ciprofloxacin This inoculum was then matched with Interpretation against Pseudomonas aeruginosa strain 0.5 McFarland standard. Turbidity of Synergy = mean FICI < 0.5, Partial (ATCC 9721 and clinical) is shown in inoculum was adjusted by adding more synergy/addition = mean FICI > 0.5 < Table 1, the Chloroform fraction of saline solution (to decrease the 1.0, Thuja orientalis showed a synergistic turbidity) or by adding bacterial growth effect with highest zone of inhibition (to increase turbidity).19 Indifference = mean FICI >1 - <2.0, b e t w e e n 3 5 . 3 3 ± 2 . 5 2 t o Antagonism = mean FICI > 2.0 39.33±5.51.Clinical strains shows 96-well Microdilution Tray: * F I C : F r a c t i o n a l i n h i b i t o r y synergistic effect at different 96-well plates were loaded with 100 µl concentration. concentration. There is no significant (0.1 ml) of two fold dilutions of extracts antibacterial activity at 1, 4, 16 mg/ml into each well. The reconstituted * * F I C I : Fr a c t i o n a l i n h i b i t o r y concentration of chloroform against all extracts were serially diluted two fold concentration inde tested bacteria. in nutrient broth in order to make RESULTS All tested concentrations of ethyl different concentrations i.e 24 mg/ml, acetate fraction (E4) shows resistance 12 mg/ml, 6 mg/ml, 3 mg/ml, 1.5 The antimicrobial activities of alone. But in combination it shows mg/ml, 0.75 mg/ml and were assayed methanolic crude extract (M.C.E) of synergistic effect at 8 and 12 mg/ml against the tested microorganism. Each Thuja orientalis with ciprofloxacin are (clinical strain) and indifference effect well was filled with 5 µl bacterial shown in Table 1 in which methanol at 20 and 30 mg/ml (ATCC 9721) inoculum (1.5×105 CFU/ml). Plates crude extract (E1) showed significant concentration. In combination butanol without any plant extracts served as antibacterial activity at different fraction (E5) shows synergistic effect at growth control. The trays were concentrations with mean differences 24 and 30 mg/ml concentration and incubated at 37o C for 18-24 hours. The as (33.66±0.57, 34.66±0.57, shows no antibacterial activity at 1, 4, highest dilution of extract that showed 35.33±1.15 and 36.66±1.15) mm 16 concentrations alone and in no visible bacterial growth and against the Pseudomonas aeruginosa combination. turbidity was considered as MIC.19 strain (ATCC 9721). Synergistic effect The aqueous fraction of Thuja orientalis Synergistic Antimicrobial Assays was observed in means of all tested (E6) exhibited both synergistic and concentrations with ciprofloxacin indifference effects at different Subsequently by doing a checkerboard against Pseudomonas aeruginosa (ATCC titration, the combination action of concentrations for both ATCC 9721 9721) except at 1, 4 and 16 mg/ml. and clinical strain. both Thuja orientalis extract and While M.C.E alone exhibited no

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TABLE II: FRACTIONAL INHIBITORY CONCENTRATION INDEX (FICI) resistance to fluoroquinolones makes FOR THUJA ORIENTALIS WITH CIPROFLOXACIN AGAINST their treatment a major problem.23 PSEUDOMONAS AERUGINOSA In this study, medicinal effects of Thuja MIC MIC Concentrations a c FIC FICI o r i e n t a l i s w e r e e v a l u a t e d M.C.E. (mg/ml) 3 6 2 M.C.E-Cip 2.33 independently and in combination with Cip (µg/ml) 0.25 0.75 0.33 ciprofloxacin. In order to discover a N-hexane (mg/ml) 6 3 0.5 N-Hexane-Cip 1.24 better solution for increasing the Cip (µg/ml) 0.25 0.187 0.74 effectiveness of concerned Chloroform (mg/ml) 6 3 0.5 Chloroform-Cip 2 under study, six extracts in various Cip (µg/ml) 0.25 0.375 1.5 organic and aqueous solvent were E.A (mg/ml) 3 3 1 E.A-Cip 1.74 o b t a i n e d a n d e v a l u a t e d f o r Cip (µg/ml) 0.25 0.187 0.74 c o m p a r i s o n . D i s k d i f f u s i o n Butanol (mg/ml) 6 1.5 0.25 Butanol-Cip 3.25 susceptibility assay method was Cip (µg/ml) 0.25 0.75 3 employed and different concentrations Auqeous (mg/ml) No MIC 1.5 1.5 Aqueous Cip 2.24 Cip (µg/ml) 0.25 0.187 0.74 were tested. MIC of Thuja orientalis ciprofloxacin for Pseudomonas Evaluation of Synergistic effect of along with Thuja orientalis has indicated aeruginosa were separately obtained as ciprofloxacin / extracts t h a t s y n e r g i s m a t c e r t a i n standard and checked against MIC of concentrations can be achieved and combination of Thuja orientalis and Interactions of antimicrobial agents and may be effective for resistant strains ciprofloxacin. The results of check Thuja orientalis plant in different such as Pseudomonas aeruginosa. Khadir board studies of the fractions of Thuja mediums of extract showed indifferent et al. observe similar findings that o r i e n t a l i s w i t h c i p r o f l o x a c i n or antagonistic effect as no significant Pseudomonas aeruginosa resisted the combination are indicated in Table I reduction in MIC of the tested action of the essential oil of Thymus and II. Mean FICI calculated for all antibiotics against Pseudomonas 21 lanceolatus. fractions of Thuja orientalis for aeruginosa was observed as evident Pseudomonas aeruginosa showed from Table II. Medicinal plants have long been an antagonism as mean FICI was around important source of medicinal agents. 2.13 (>2). Synergistic FICI values were DISCUSSION The rationalization of using medicinal not observed with any of the strains. plants combined with the existing Interactions of antimicrobial agents and The main aim of the study was to study antimicrobial agents is an effective Thuja orientalis plant in different the synergistic activity of plant extract method to overcome resistance. mediums/fractions of extract showed with the use of antibiotics in order to Simultaneous administration of two or indifferent or antagonistic effect, as enhance the effectiveness of antibiotics more drugs or an antibiotic with a there was no significant reduction in against multidrug resistance bacteria. medicinal plant has shown to provide MIC of the tested antibiotics against Antimicrobial agents are the important effective treatment for the resistant Pseudomonas aeruginosa. Although, at drugs to counter infectious diseases, . Drug synergism between varied level of concentrations in however, distinct characteristic of known antimicrobial agents and various fractions of methanolic, N- microbes and pathogens cause bioactive plant extracts is a novel hexane, chloroform, ethyl acetate, development of resistance with time. concept, recently reported to yield butanol and aqueous fraction, This is decreasing the effectiveness of some desirable results. Thuja orientalis synergism was observed. However, the antibiotics against certain commonly known as Pankh, is an the observations remained below the pathogen; thus, posing a serious threat evergreen, monoecious tree or shrub. 20 deduced synergistic effects; thereby to health of the individuals. Evaluation It is an important plant ever since validation of synergistic effect of Thuja to use medicinal plant extracts and medieval ages due to its multiple orientalis with ciprofloxacin remained their essential oils with synthetic reported medicinal value due to the doubtful. Similar findings were medicines and antibiotics have been presence of one or more of the active observed by Kim and Choi by using carried out in various parts of the world constituents. Many researches have Thuja orientalis extract with different with varied results. Study carried out shown that Thuja orientalis has antibiotics.13 by Moussaoui and Alaoui showed that significant antibacterial, anti-fungal, all tested bacteria are sensitive to five anti-viral, anti-inflammatory, anti- Although the results of observations of essential oils of medicinal plants except cancer, larvicidal, insecticidal, various fractions and calculated MIC 21 Pseudomonas aeruginosa. This is also n e m a t i c i d a l a n d a n t i - o x i d a n t did not support the synergistic observed from our results showing a activities.22 Pseudomonas aeruginosa is administration of extract of Thuja zone of inhibition of Thuja orientalis amongst the most common causes of o r i e n t a l i s w i t h c i p r o f l o x a c i n , alone and is also supplemented in localized suppurative lesions in nevertheless, it does not close the previous studies. However, the humans, especially in hospitals. window of opportunity here, as combined effect with ciprofloxacin Furthermore, their ability to develop combination with other antimicrobial

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TABLE I: ANTIMICROBIAL ACTIVITY OF DIFFERENT FRACTIONS OF THUJA ORIENTALIS AND ITS SYNERGISTIC EFFECT WITH CIPROFLOXACIN Fractions Microorganism Conc. (mg/ml) Zone of Inhibition (mm) Stnd. Cip. Disc T.O & Cip Zone of Inhibition (mm) 01 0.33±0.57 28.33±0.57 1+cip 0.00±0.00 04 0.33±0.57 28.33±0.57 4+cip 0.00±0.00 08 15.33±0.57 28.33±0.57 8+cip 33.66±0.57 12 16.33±0.57 28.33±0.57 12+cip 34.66±0.57 P. aeruginosa 16 0.33±0.57 28.66±0.57 16+cip 0.33±0.57 (ATCC 9721) 20 16.33±0.57 28.66±0.57 20+cip 34.66±1.15 24 17.33±0.57 28.66±0.57 24+cip 36.66±1.15 30 17.66±0.57 27.66±1.52 30+cip 34±1 36 19±1 27.66±1.52 36+cip 35.33±1.15 E1 01 0.66±0.01 28.33±1.52 1+cip 0.33±0.57 04 0.00±0.01 28.33±1.52 4+cip 0.00±0.01 08 0.33±0.57 28.33±1.52 8+cip 41±1 12 7.66±4.93 28.66±0.57 12+cip 41.33±1.15 P. aeruginosa 16 0.01±0.06 28.66±0.57 16+cip 0.33±0.57 (Clinical) 20 8±3.46 28.66±0.57 20+cip 34.66±1.15 24 11.33±4.72 28.66±0.57 24+cip 36.66±1.15 30 9.66±6.35 28±2 30+cip 34±1 36 9.33±5.77 28±2 36+cip 35.33±1.15 01 0.00±0.00 27.66±1.15 1+cip 0.00±0.00 04 0.00±0.00 27.66±1.15 4+cip 0.00±0.00 08 12.66±1.52 27.66±1.15 8+cip 28.66±2.51 12 14.66±1.15 27.66±1.15 12+cip 30±3.60 P. aeruginosa 16 0.66±0.57 27.66±0.57 16+cip 0.00±0.00 (ATCC 9721) 20 15.66±0.57 27.66±0.57 20+cip 32±1 24 16.66±0.57 27.66±0.57 24+cip 33±1 30 17.66±0.57 28±1 30+cip 36±1 36 18.66±0.57 28±1 36+cip 37±1 E2 01 0.00±0.06 27.66±0.57 1+cip 0.00±0.00 04 0.00±0.00 27.66±0.57 4+cip 0.00±0.00 08 2.66±1.52 27.66±0.57 8+cip 28.66±2.51 12 14.66±1.15 27.66±0.57 12+cip 30±3.60 P. aeruginosa 16 0.02±0.88 27.66±0.57 16+cip 0.00±0.00 (Clinical) 20 8±3.21 27.66±0.57 20+cip 32±1 24 8±3.21 27.66±0.57 24+cip 33±1 30 9±4.35 27.66±0.57 30+cip 36±1 36 9±6.35 27.66±0.57 36+cip 37±1 01 0.33±0.00 28.33±0.58 1+cip 0.00±0.00 04 0.30±0.00 28.33±0.58 4+cip 0.00±0.00 08 14.67±1.15 28.33±0.58 8+cip 35.33±2.52 12 14.67±1.15 28.33±0.58 12+cip 37.33±2.03 P. aeruginosa 16 0.00±1.00 28.33±0.58 16+cip 0.00±0.00 (ATCC 9721) 20 19.00±1.00 28.33±0.58 20+cip 37.00±4.58 24 21.00±1.00 28.33±0.58 24+cip 39.00±3.61 30 19.67±3.51 27.67±2.31 30+cip 37.67±5.13 36 20.33±3.06 27.67±2.31 36+cip 39.33±5.51 E3 01 0.00±1.00 28.67±0.58 1+cip 0.00±0.00 04 0.33±0.58 28.67±0.58 4+cip 0.00±0.00 08 7.33±2.31 28.67±0.58 8+cip 31.00±1.00 12 9.00±2.65 28.67±0.58 12+cip 31.33±2.52 P. aeruginosa 16 0.67±0.89 27.67±0.58 16+cip 0.00±0.00 (Clinical) 20 9.00±3.61 27.67±0.58 20+cip 30.00±1.00 24 7.00±2.00 27.67±0.58 24+cip 31.00±1.00 30 9.00±3.00 27.67±1.53 30+cip 31.00±1.00 36 8.33±4.04 27.67±1.53 36+cip 31.00±1.00

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01 0.00±0.00 28.00±0.00 1+cip 0.00±0.00 04 0.00±0.02 28.00±0.00 4+cip 0.00±0.00 08 9.67±6.35 28.00±0.00 8+cip 27.00±3.61 12 10±6.93 28.00±0.00 12+cip 28.33±4.04 P. aeruginosa 16 0±0.05 28.67±0.58 16+cip 0.00±0.00 (ATCC 9721) 20 7.67±4.93 28.67±0.58 20+cip 30.67±0.58 24 6.67±6.43 28.67±0.58 24+cip 31.67±0.58 30 6.33±5.51 27.67±0.58 30+cip 29.00±1.00 36 6.33±5.51 27.67±0.58 36+cip 30.00±1.00 E4 01 000±0.00 28.00±0.00 1+cip 0.00±0.00 04 0.33±0.01 28.00±0.00 4+cip 0.00±0.00 08 6.33±7.51 28.00±0.00 8+cip 31.33±4.16 12 6.67±8.08 28.00±0.00 12+cip 31.67±6.66 P. aeruginosa 16 0.33±0.58 28.33±0.58 16+cip 0.00±0.00 (Clinical) 20 11.67±0.58 28.33±0.58 20+cip 30.33±0.58 24 12.67±0.58 28.33±0.58 24+cip 31.67±0.58 30 12.33±0.58 28.00±1.00 30+cip 29.33±0.58 36 13.33±0.58 28.00±1.00 36+cip 31.33±0.58 01 0.00±0.00 28.00±1.00 1+cip 0.00±0.00 04 0.00±0.00 28.00±1.00 4+cip 0.00±0.00 08 6.00±5.20 28.00±1.00 8+cip 32.67±3.79 12 6.33±7.51 28.00±1.00 12+cip 32.33±6.66 P. aeruginosa 16 0.00±0.00 27.33±0.58 16+cip 0.00±0.00 (ATCC 9721) 20 11.00±1.00 27.33±0.58 20+cip 31.00±1.00 24 13.00±1.00 27.33±0.58 24+cip 33.00±1.00 30 13.67±0.58 27.33±0.58 30+cip 30.00±1.00 36 14.67±0.58 27.33±0.58 36+cip 31.67±1.53 E5 01 0.00±0.00 28.33±1.53 1+cip 0.00±0.00 04 0.33±0.04 28.33±1.53 4+cip 0.00±0.00 08 6.00±5.20 28.33±1.53 8+cip 25.67±5.13 12 6.33±7.51 28.33±1.53 12+cip 29.00±2.65 P. aeruginosa 16 0.01±0.05 27.33±0.58 16+cip 0.00±0.00 (Clinical) 20 6.67±4.51 27.33±0.58 20+cip 29.00±1.00 24 7.67±5.51 27.33±0.58 24+cip 30.00±1.00 30 6.33±5.77 27.33±0.58 30+cip 30.00±1.00 36 11±5.51 27.33±0.58 36+cip 29.67±1.15 01 0.00±0.00 27.67±0.58 1+cip 0.00±0.00 04 0.00±0.00 27.67±0.58 4+cip 0.00±0.00 08 0.00±0.00 27.67±0.58 8+cip 29.00±1.00 12 8±2.08 27.67±0.58 12+cip 30.00±1.00 P. aeruginosa 16 0.00±0.00 28.33±0.58 16+cip 0.00±0.00 (ATCC 9721) 20 0.00±0.00 28.33±0.58 20+cip 39.33±0.58 24 8±4.04 28.33±0.58 24+cip 29.33±1.15 30 8.67±1.53 27.33±2.08 30+cip 30.00±1.00 36 6.67±5.86 27.33±2.08 36+cip 30.33±1.15 E6 01 0.67±0.58 27.33±1.15 1+cip 0.00±0.00 04 0.67±0.58 27.33±1.15 4+cip 0.00±0.00 08 9±5.20 27.33±1.15 8+cip 35.00±2.65 12 9±5.73 27.33±1.15 12+cip 37.00±1.73 P. aeruginosa 16 0.00±0.00 28.00±1.00 16+cip 0.00±0.00 (Clinical) 20 11.00±1.00 28.00±1.00 20+cip 30.67±0.58 24 12.00±1.00 28.00±1.00 24+cip 31.67±0.58 30 12.67±0.58 28.00±1.00 30+cip 30.67±1.15 36 13.67±0.58 28.00±1.00 36+cip 32.33±1.15

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agents is a vast avenue, which can be 0960312031 000102750. Demonstrates Major Anti-Cancer explored in search for right grouping Potentials: Evidences from In Vitro 5. Rahman BA, Wasfy MO, Maksoud against resistance pathogens. Studies on A375 Cells. Evid Based MA, Hanna N, Dueger E, House B. Importance of identifying new and Complement Alternat Med Multi-drug resistance and reduced effective antimicrobial agents along 2011;2011:568148. DOI: 10.1093/ susceptibility to ciprofloxacin with the value of medicinal plants helps ecam/neq042. among Salmonella enterica serovar reduce the use of antibiotics against Typhi isolates from the Middle East 13. Kim CS, Choi SU, Lee KR. Three resistant strains in clinical practice. This and Central Asia. New Microbes new diterpenoids from the leaves particular study observed synergism of New Infect 2014 Jul;2(4):88-92. of Thuja orientalis. Planta Med Thuja orientalis with ciprofloxacin, DOI: 10.1002/nmi2.46. 2012;78(5):485-7. 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AUTHORS' CONTRIBUTIONS Following authors have made substantial contributions to the manuscript as under: HM, SAT: Concept, acquisition of data, drafting the manuscript, final approval of the version to be published SHH, SHK: Analysis and interpretation of data, drafting the manuscript, critical review, final approval of the version to be published AJP, AH: Concept and study design, critical review, final approval of the version to be published Authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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