Online - 2455-3891 Print - 0974-2441 Vol 11, Issue 12, 2018 Research Article MORINDA COREIA HAS AN ANTI-HELICOBACTER PYLORI EFFECT AGAINST THE MULTIDRUG-RESISTANT CLINICAL ISOLATE OF NORTH-EAST INDIA SHWETA MAHANT1, VALENTINA GEHLOT1, SHANU HODA1, POOJA VIJAYARAGHAVAN1, SELVAMURTHY W1, THIRUMURTHY N3, RAJASHREE DAS1* 1 Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India. 2Amity Science, Technology & Innovation Foundation, Amity University, Noida, Uttar Pradesh, India. 3BT Herbal, Sivakasi, Tamil Nadu, India. Email: rajashreepatra79@yahoo.co.in Received: 23 June 2018, Revised and Accepted: 21 August 2018 ABSTRACT Objective: Antimicrobial resistance is a growing problem in Helicobacter pylori eradication which is a microaerophilic bacterium causing various gastroduodenal diseases. The present study has been designed to test the efficacy of Morinda coreia against the metronidazole clarithromycin and levofloxacin-resistant H. pylori strains isolated from the biopsy taken from the patient suffering from gastric erosion in Guwahati, Assam. Method: The antimicrobial activity of n-hexane and chloroform extract of M. coreia was tested against multidrug-resistant H. pylori isolate of Guwahati, Assam, by agar well method and microdilution method. Result: In the present study, the H. pylori strain resistant for metronidazole (minimal inhibitory concentration [MIC] >64 µg/mL), clarithromycin at (MIC >0.5 µg/mL), and levofloxacin at (MIC >1 µg/mL) was tested against the n-hexane and chloroform extract of M. coreia. Both the extracts of M. coreia showed good efficacy against the multidrug-resistant strain of H. pylori shown inhibition at 1.2 µg/mL with n-hexane extract and 2 µg/mL with chloroform extract of M. coreia. Conclusion: The prevalence of metronidazole-resistant ranges between 50% and 90% in the developing countries, including India, clarithromycin ranges from 0% to 15% in India, and levofloxacin ranges between 50% and 70% in India, so there is a need of alternative therapy for the eradication of this bacterium from the stomach. Hence, this study suggests that M. coreia, which has been used traditionally as a folk medicine for the treatment of many gastric diseases, has also shown good efficacy against the multidrug-resistant H. pylori strain of North-east India. Keywords: Helicobacter pylori, Morinda coreia, Resistance, Antibiogram. © 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2018.v11i12.28064 INTRODUCTION The Gram-negative, microaerophilic gastric bacteria Helicobacter pylori is recognized as an important human pathogen and classified as a Group I carcinogen by the International Agency for Research on Cancer [1]. H. pylori has infected over half of the world’s population, and its infection usually occurs in early childhood and can persist throughout a lifespan if it is untreated with a specific antibiotic regimen [2]. H. pylori infections are largely asymptomatic, but the studies have also shown that, if its association lasts for long term with the host, it can cause more serious disease such as gastritis, peptic ulcer, gastric cancer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma [3,4]. H. pylori eradication from the infected individuals is the best and only choice for an effective treatment of its associated diseases. Eradication by triple therapy regimen consists of a proton-pump inhibitor in combination with two antibiotics, such as clarithromycin and amoxicillin or metronidazole, which has been recommended as the first-line therapy and results in a high eradication rate [5]. The other antibiotics which are also used for the eradication of H. pylori as a second-line therapy or third-line therapy are levofloxacin, furazolidone, and tetracycline. Due to the use of these antibiotics for the treatment of other diseases like respiratory, anaerobic infection, dental infection, which have resulted in the emergence of antibiotic resistance which may be the major cause of treatment failure [6,7]. Metronidazole and clarithromycin are the most commonly used antibiotics in the treatment of H. pylori infection as compared to other antibiotics, but many studies from India and abroad have reported that the resistance for both the antibiotics has been increasing nowadays which may be the reason for the treatment failure [8-10]. The prevalence of dual drug resistance and multidrug resistance has been observed in many countries which has also become the major obstacle in the treatment for H. pylori infection. In the geographical areas where the resistance rate ranges between 15% and 90%, there is an urgent need to develop new treatment strategies for H. pylori infection. Unlike synthetic drugs, bioactive natural products which are benefical without any side effects as they are part of our day to day life and other culinary items [11]. In this search, Morinda coreia which is generally found in dry forests of the greater part of India and Sri Lanka belongs to the family Rubiaceae, which is one of the largest and the most widely distributed plants in approximately 400 genera in this family. M. coreia is also known by its Tamil name as “Nuna.” The plant is small- to medium-sized tree with a straight cylindrical stem 3.6–4.2 m in length and 90 cm in width [12,13]. However, it has been shown that the beverage produced by fermenting the fruits was able to inhibit enteropathogenic bacteria and also had a high amount of potassium. It is also extensively used in Thai traditional medicine to cure stomach infection and blood stasis [14]. A dye obtained from the root bark is used for coloring linen and woolen goods [14]. Studies have conducted using extracts of M. coreia against various Gram-negative and Gram-positive pathogenic bacteria but not with H. pylori [15]. Therefore, in the present study, we tested the antimicrobial activity of M. coreia against the multidrug-resistant strain of H. pylori isolated from the patient suffering from gastric erosion in North-East India. METHODS Source of plant material Healthy leaves of M. coreia were collected from Sivakasi, Tamil Nadu, India. The leaves of M. coreia were authenticated at the Botany Mahant et al. Asian J Pharm Clin Res, Vol 11, Issue 12, 2018, 143-148 Department of Amity University. The leaves were washed properly under running tap water and then rinsed in distilled water. Then, it was air dried in the shade and grinded into powder using mortar and pestle. Extract preparation of M. coreia To obtain the extract of M. corea, 100 g of leaves was crushed with mortar and pestle and sieved. The dried powder was then extracted with 400 mL of n-hexane, chloroform, methanol, water, and ethanol consecutively for 72 h solvent under constant stirring. The extract was then filtered and dried under pressure and resuspended in the solvents. The concentrated extract was then kept in the dark bottles at 4°C until used. The n-hexane and chloroform extracts of M. coreia were used in the present study to test the antimicrobial activity against multidrugresistant H. pylori isolates from North-East India. Isolation of H. pylori strains and culture The patient suffering from gastric erosion was included in the study. The H. pylori strains were isolated from the antral biopsies of the patient who underwent endoscopy procedure in the Gastroenterology Department of Gauhati Medical College, Guwahati, Assam. The identification of the H. pylori was done on the basis of colony morphology, gram staining, and positive reaction in the biochemical test (catalase, urease, and oxidase). H. pylori strain was isolated and cultured on brain heart infusion (BHI) agar (Difco Laboratories, Detroit, MI) supplemented with 5% horse serum (Invitrogen, NY), 0.4% IsovitaleX (Becton Dickinson, MD), trimethoprim (5 μg/mL), vancomycin (8 μg/mL), and polymixin B (10 μg/mL). The plates were incubated at 37°C under microaerophilic condition (5% O2, 10% CO2, and 85% N2) (Double gas incubator, Hera cell 150i) for 3–6 days. Stock cultures were maintained until use at –80°C in BHI broth with 20% glycerol. Suspension preparation of H. pylori strain The bacterial suspension of H. pylori was prepared by direct colony method [16]. The colonies were taken directly from the plate and were suspended in 5 mL of sterile 0.85% phosphate buffer saline (PBS). The turbidity of the initial suspension was adjusted by comparing with McFarland’s standard number 2 (which contains about 1×108 colonyforming units (CFU)/mL) [17]. Antimicrobial susceptibility and resistance determination Exponentially grown H. pylori cells on antibiotic-free BHI agar were further suspended in PBS buffer, 10-fold serial dilution of these cell suspensions was prepared, and 10 μL of each dilution was spotted on freshly prepared BHI agar containing various concentrations of different antibiotics in μg/mL, namely metronidazole (0.2, 0.5, 1.5, 3, 8, 16, 32, and 64), clarithromycin (0.125, 025, 1, and 2), furazolidone (0.2, 0.5, 1, and 2), amoxicillin (0.125, 0.25, 1, and 2), levofloxacin (0.2, 0.4, 1, and 2), and tetracycline (1, 2, 3, and 4). Determination of minimum inhibitory concentration (MIC) by agar dilution method The antibiotics plates with H. pylori cultures were incubated for 72 h under microaerophilic conditions; the MIC was defined as the lowest concentration that inhibited visible growth of organisms. The H. pylori isolates are considered to be resistance if the MIC for different antibiotics is metronidazole (>8 μg/mL) [18], clarithromycin (>0.5 μg/mL) [18], amoxicillin (>0.12 μg/mL) [18], tetracycline (>1 μg/mL) [18], levofloxacin (>1 μg/mL) [18], and furazolidone (>2 μg/mL) [17]. Conformation of antibiotic resistance by sequence analysis of 23S rRNA, gyraseA, rdx, and frx genes of H. pylori isolate Antibiotic-resistant strain by agar dilution method was further confirmed by polymerase chain reaction (PCR) followed by sequencing for the amplification of 23S rRNA, gyraseA, rdx, and frx gene mutations responsible for the resistance of clarithromycin, levofloxacin, and metronidazole, respectively. Genomic DNA was isolated from the H. pylori strain by C-TAB method [19], and the total DNA concentration was determined by NanoDrop 1510 (IS10-03571C, Multiskan GO, Thermo Fisher Scientific). The PCR for 23S rRNA, gyraseA, rdx, and frx gene was performed to detect the resistance for all the antibiotics. All the genes were amplified using the primer sets as listed in Table 1. The PCR was carried out in 20 µL reaction containing 10 pmol dNTP (Bangalore Genei, Bengaluru, India), 10 pmole primer sets (Sigma-Aldrich), 1.5U Taq DNA polymerase (Bangalore Genei), and 10 ng of H. pylori-positive genomic DNA Eppendorf Thermocycler (vapo.protectTM) for 35 cycles under the following cycling conditions 94°C for 1 min, annealing for 1 min, and 72° for 1min. The product was finally analyzed in 2% agarose gel stained with ethidium bromide under standard procedure. All the amplified PCR products were further purified by QIAquick PCR Purification kit (QIAGEN, Hilden, Germany). The purified products were further sequenced by ABI sequencer 3100XL consisting of Big DyeR Terminator (PerkinElmer) with Ampli Taq FS. The sequence was edited after aligning with SeqMan program (DNASTAR Inc., Madison, WI). For the detection of point mutation in various genes of H. pylori, multiple sequence alignment was performed by CLUSTAL OMEGA taking 26695 strains of H. pylori as a reference strain. Anti-H. pylori activity of M. coreia against multidrug-resistant strain MIC determination by the agar well method The different concentration of n-hexane and chloroform extract of M. coreia was loaded into the well of 6 mm in diameter in inoculated plates with 3 × 109 CFU of multidrug-resistant strain of H. pylori isolated from the patient of North-East India. The plates were kept under microaerophilic conditions (5% O2, 10% CO2, and 85% N2) for 2 days at 37°C. All experiments were performed in triplicates. Pure n-hexane and chloroform were used as a negative control, and amoxicillin was used as positive control. MIC determination by microdilution method The 96-well microtiter plate was prepared by dispensing 80 μL of BHI broth into first well. A 20 μL from the stock solution of M. coreia extract (120 μg/mL) was added to the first well. Then, five-fold serial dilution was performed until 8th well. The obtained concentration range was from 12 μg/mL to 12 × 10−7 μg/mL. To each well, 150 μL of the diluted bacterial cells was added to give a final concentration of 3 × 109 CFU/mL. The inoculated plates were incubated at 37°C for 72 h under microaerophilic conditions. MIC90 was defined as the lowest concentration of extract sample that inhibited the 90% of H. pylori cells when compared to control, i.e., H. pylori cells without extract sample. Reading was noted on the Elisa reader (Erba Lisa Scan II). Table 1: PCR primers of 23S rRNA, gyraseA, rdx, and frx genes of H. pylori Gene Primer sequence Size of the product References 23S rRNA F-5ˈGGCTCTTTGAGTCCTTTAGGACAA-3ˈ forward sense positions 2020-2044 of U27270) R-3ˈCTCCATAAGAGCCAAAGCCCTTACT-5ˈ reverse antisense position 2612-2636 of U27270) F-5ˈ-TTTRGCTTATTCMATGAGCGT-3ˈ R-3ˈ -GCAGACGGCTTGGTARAATA-5ˈ F-5ˈ-GCAGGAGCATCAGATAGTTCT-3ˈ F-3ˈ-GGGATTTTATTGTATGCTACAA-5ˈ F-5ˈ-GGATATGGCAGCCGTTTATCATT -3ˈ F-3ˈ-GAATAGGCATCATTTTAAGAGATT -5ˈ 613 [20] 428 [21] 886 [22] 780 [22] gyraseA rdx frx F: Forward primer; R: Reverse primer; bp: Base pairs, PCR: Polymerase chain reaction 144 Mahant et al. Asian J Pharm Clin Res, Vol 11, Issue 12, 2018, 143-148 Rapid urease test (RUT) For the microbial growth visualization, RUT was done. The basis of the test is the ability of H. pylori to secrete urease enzyme which hydrolyzes the urea to ammonia and carbon dioxide and raises the pH of the medium from yellow (H. pylori negative) to a pink color (H. pylori positive). MIC was defined as the lowest concentration of extract sample that prevented RUT medium color change from yellow to pink. RESULT One clinical isolate was included in the study from the patient suffering from gastric erosion. The MIC for various antibiotics, namely metronidazole, clarithromycin, amoxicillin, tetracycline, levofloxacin, and furazolidone, was determined by agar dilution method against H. pylori isolate of North-East India. The strain which was selected to test the efficacy of M. corea was multidrug resistant showing resistance against metronidazole at the MIC of >64 µg/mL, clarithromycin at MIC >0.5 µg/mL, and levofloxacin at MIC >1 µg/mL but sensitive to other drugs, namely furazolidone, amoxicillin, and tetracycline. The genomic DNA was further PCR amplified for 23S rRNA, gyraseA, rdx, and frx gene of H. pylori for sequencing and sequence analysis (Fig. 1a-c). Sequence analysis of various genes of H. pylori for the conformation of resistance pattern Mutation analysis of 23S rRNA gene of H. pylori for clarithromycin resistance The sequence of 23S rRNA gene was aligned with the reference sequence U27270 using CLUSTAL OMEGA for the presence of mutation at A to G at 2142, A to G at 2143, and T to C at 2182 positions in the V domain of 23S rRNA gene of H. pylori (Fig. 2). The strong association was observed in the agar dilution method, and sequence analysis method showed that the H. pylori strain resistant for clarithromycin was having a mutation at T to C at the 2182 position and MIC of >0.5 μg/mL. Mutation analysis of gyraseA gene of H. pylori for Levofloxacin resistance: The resistance for levofloxacin was observed due to a mutation in gyraseA gene of H. pylori at various amino acid positions, namely asparagine (N) to lysine (K) at 87 position, alanine (A) to valine (V) at 88 position, and aspartic acid (D) to glycine (G)/tyrosine (Y)/ asparagine (N) at 91 position. The H. pylori nucleotide sequence was converted to protein sequence by EXPASY TRANSLATE TOOL and was aligned with the reference sequence HP0701 using CLUSTAL OMEGA for mutation analysis. Point mutation was observed from aspartic acid (D) to glycine(G) at 91 position in gyraseA gene of H. pylori (Fig. 3). The strong association was observed in the sequence analysis method and agar dilution method showing MIC at >1 ug/mL for levofloxacin. Mutation analysis of rdx and frx gene of H. pylori for metronidazole resistance The resistance for metronidazole was observed by the mutation in rdx and frx gene of H. pylori at various amino acid positions as these gene are highly conserved genes of H. pylori [22]. The H. pylori nucleotide sequence was converted to protein sequence by EXPASY TRANSLATE TOOL and was aligned with the reference sequence HP0954 for rdx and HP0642 for frx using Clustal Omega. Point mutation was observed as various amino acid positions of rdx and frx gene of H. Pylori (Fig. 4a and b). Hence, the strong association was observed in the sequence analysis method and agar dilution method which has shown MIC at >64 ug/mL for metronidazole. Agar well method to test the efficacy of M. coreia Different concentration of n-hexane and chloroform extract of M. coreia was loaded onto the well and was air dried. 100 μL of the suspended H. pylori strain in PBS having McFarland 2 (1×108 CFU/mL) was spreaded and plated onto BHI medium. The well was made in the plate using well puncher and the wells were loaded with different concentrations of the extract. After 72 h of incubation, we found that the n-hexane and chloroform extract of M. coreia has anti-H. pylori activity showing the zone of inhibition of 7 mm at 0.4 mg/mL for n-hexane extract (Fig. 5a and b) and 7 mm for 2.4 mg/mL chloroform extract (Fig. 6a and b) by agar well method, and the MIC of M. coreia was <12 μg/mL for n-hexane extract and <2 μg/mL for chloroform extract against multidrugresistance H. pylori strain of North-East India by microdilution method (Fig. 7 and Table 2). DISCUSSION Fig. 1: DNA amplification of Helicobacter pylori gene: (a) 23S rRNA gene of 613bp. Lane 1 - positive control, 26695 Lane 2 H. pylori clinical isolate,and Lane M - Marker (100bp). (b) gyraseA gene of 428 bp. Lane 1 - positive control, 26695 Lane 2 - H. pylori clinical isolate, and Lane M - Marker (100bp). (c) rdx and frx gene of 886bp and 780bp. Lane1 - positive control of rdx gene, 26695 Lane 2 - H. pylori clinical isolate, Lane M - Marker (100bp), Lane 3 - positive control of frx gene, 26695 and Lane 4 - H. pylori clinical isolate H. pylori have acquired antibiotic resistance for several antibiotics in the past two decades, which is the major cause of treatment failure. The antibiotic resistance varies geographically due to its varied use in other diseases for the treatment of the local population. The geographical resistance scenario of antibiotic resistance pattern provides a guideline to all the clinicians for the treatment regimen. Previous studies have shown that resistance toward metronidazole varies geographically in India such as Chandigarh (38.2%) [23], Delhi NCR (48.5%) [24], Lucknow (68%) [23], Hyderabad (100%) [23], Chennai (82.2%), [23], Kolkata (85%) [8], Gangetic belt of North India (100%) [25], and Gujarat (83.2%) [26]. High prevalence of metronidazole was also found in other developing countries such as Pakistan (97.8%) [27], China (78.8%), Nepal (88.1%), Bangladesh (77.8%), Bhutan (83%), Vietnam (72%), and Mexico (76.3%) [28,29]. The resistance to metronidazole is Fig. 2: Multiple sequence alignment of domain V of 23S rRNA gene of clarithromycin of Helicobacter pylori strains. The strain number is indicated on the left-hand side of each sequence. U27270 is a representative sequence showing T to C point mutations marked in yellow at positions 2182 of the 23S rRNA gene. Numbering of nucleotide position followed the proposed system by Taylor et al. (position [2515 373] + 1 = position 2143) 145 Mahant et al. Asian J Pharm Clin Res, Vol 11, Issue 12, 2018, 143-148 Fig. 3: Multiple sequence alignment of gyraseA gene of Helicobacter pylori for levofloxacin resistance pattern. The strain number is indicated on the left-hand side of each sequence. HP0701 is a representative sequence showing D to G point mutations marked in yellow at amino acid positions 91 of the gyraseA gene Fig. 4: (a) Multiple sequence alignment of rdx gene of Helicobacter pylori for metronidazole resistance pattern. The strain number is indicated on the left-hand side of each sequence. HP0954 is a representative sequence showing multiple point mutations marked in yellow at amino acid positions, (b) multiple sequence alignment of rdx gene of H. pylori for metronidazole resistance pattern. The strain number is indicated on the left-hand side of each sequence. HP0642 is a representative sequence showing multiple point mutations marked in yellow at amino acid positions resistance for levofloxacin is due to the mutation in the amino acid chain of gyraseA gene of H. pylori at various positions [21]. The resistance to clarithromycin also observed and it is considered as one of the main drugs in the treatment regime in combination with protonpump inhibitor and amoxicillin or metronidazole. The resistance to clarithromycin also varies geographically such as Kolkata (0%) [8], Gujarat (58.8%) [26], Delhi NCR (11.8%) [31], Lucknow (4%) [23], Pakistan (5.4%) [27], China (12.8–23.8%), Spain (35.6), and USA (10–15%) [32]. Hence, the emergence of resistance to various key antibiotics, which are mainly used as a first-line or a second-line therapy in the eradication of H. pylori, has prompted us to look into the non-antibiotic compound that inhibits H. pylori growth. a b Fig. 5: (a and b) Agar well method: Antimicrobial activity of n-hexane extract of Morinda coreia against the multidrugresistant strains of Helicobacter pylori due to the mutation in oxygen-insensitive NADPH nitroreductase (rdx) and NADPH flavin oxidoreductase (frx) gene of H. pylori [22]. The resistance for levofloxacin was also reported high in many countries such as Delhi NCR (73.2%) [30], Gujarat (13.8%) [26], Bangladesh (66%), Bhutan (3%), and Vietnam (25%) [28,29]. The There are many plant and plant products which are having anti-H. pylori effect and showed good efficacy against the drug-resistant H. pylori strain. De et al. showed the good efficacy of curcumin in H. pyloriinfected mouse [33]. Our other studies have shown that the methanolic extract of Paederia foetida [34], methanolic and ethanolic extract Parmelia perlata [35], Embilica officinalis [36], and methanolic and n-hexane extract of Brassica capitata is effective against dual and multidrug resistance H. pylori stain [37]. Our various studies prompted us to explore the efficacy of M. coreia, which is being extensively cultivated in India for the dye which is obtained from the root bark and it is a popular medicinal plant in Thailand. It has been used to 146 Mahant et al. 2 μg/mL 7 Zone of inhibition (in mm) b Fig. 6: (a and b) Agar well method: Antimicrobial activity of chloroform extract of Morinda coreia against the multidrugresistant strains of Helicobacter pylori Fig. 7: Microdilution method: Lanes 1–8 show the 5-fold serial dilution of Morinda coreia extract from Lanes 1–8 with a constant cell count of Helicobacter pylori. Lane 9 shows the cell control without the extract. Lane 10 shows media control without cells. Lane 11 shows extract control without cells. Lane 12 shows the H. pylori cell control. Minimal inhibitory concentration 90 was observed at Lane 2 and Lane 1 for n-hexane and chloroform extract of M. coreia, respectively 7 treat menstrual disorders, as a tonic for stomach infection and blood stasis, and has shown antibacterial properties against Gram-negative and Gram-positive bacteria [15,38]. In our study, we found that the n-hexane and chloroform extract of M. coreia has shown good efficacy against the multidrug-resistant H. pylori stain of North-East India by agar well method and microdilution method. H. pylori: Helicobacter pylori, M. coreia: Morinda coreia, MIC: Minimal inhibitory concentration Zone of inhibition (in mm) 0.4 MTZ=64 µg/mL CLR=0.5 µg/mL LEV=1 µg/mL Amount (in mg) 1.2 μg/mL 2.4 Amount (in mg) a Strain 1 Chloroform extract of M. coreia (Agar well method) MIC90 for M. coreia (Microdilution method) n-Hexane extract of M. coreia (Agar well method) MIC for MTZ, CLR, LEV (agar dilution method) Strain No. Table 2: Antimicrobial activity of n-hexane and chloroform extract of M. coreia against multidrug-resistant H. pylori strain MIC90 for M. coreia (Microdilution method) Asian J Pharm Clin Res, Vol 11, Issue 12, 2018, 143-148 However, further research needs to be done to determine the compounds that are responsible for antibacterial activity against multidrug-resistant H. pylori strain of North-East India. CONCLUSION In our present study, we have primarily shown that M. coreia, which is a traditional plant used for the treatment of various diseases, have potentially inhibited the growth of multidrug-resistant H. pylori in vitro that were isolated from the patient suffering from gastric erosion. It is noteworthy that the strain was resistant for metronidazole at MIC > 64 µg/mL, clarithromycin at MIC >0.5 µg/mL, and levofloxacin >1 µg/mL. Overall, this study provides novel insights in the therapeutic potential of n- hexane and chloroform extract of M. coreia against multidrug-resistant H. pylori strain, although further studies are required to determine the pathway of its mechanism and its active compounds. ACKNOWLEDGMENT We thank Amity University for providing the infrastructure and support to carry out the research work. AUTHORS’ CONTRIBUTION RD has given the concept of antibiogram profiling of H. pylori strains. RD and PV have finalized the manuscript. SM has performed the experiments, analyzed the data, and drafted the manuscript. WS and NT have provided the M. coreia sample. VG and SH have performed some of the experiments. 147 Mahant et al. Asian J Pharm Clin Res, Vol 11, Issue 12, 2018, 143-148 CONFLICTS OF INTEREST The authors declared they have no conflicts of interest. FUNDING This study was supported by the Department of Biotechnology (DBT) grant no. (BT/240/NE/TBP/2011) REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Dunn BE, Cohen H, Blaser MJ. Helicobacter pylori. Clin Microbiol 1997;10:720-41. Blaser MJ. Epidemiology and pathophysiology of Campylobacter pylori infections. Infect Dis 1990;12 Suppl 1:S99-106. Matthews GM, Butler RN. Cellular mucosal defense during Helicobacter pylori infection: A review of the role of glutathione and oxidative pentose pathway. Helicobacter 2005;10:298-306. Montecucco C, Rappuoli R. Living dangerously: How Helicobacter pylori survive in the human stomach. 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