Int J Pharm Pharm Sci, Vol 8, Issue 1, 205-208Original Article


THERAPEUTIC POTENTIAL OF LICHEN PARMELIA PERLATA AGAINST DUAL DRUG-RESISTANT HELICOBACTER PYLORI ISOLATES

VALENTINA GEHLOT1, SHWETA MAHANT1, POOJA VIJAYRAGHWAN1, KUNAL DAS2,3, SHANU HODA1,
RAJASHREE DAS1*

1Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India, 2Max Superspeciality Hospital, Patparganj, New Delhi, India, 3Yasodha Superspeciality Hospital, Ghaziabad, Uttar Pradesh, India
Email: rajashreepatra79@yahoo.co.in

 Received: 13 Oct 2015 Revised and Accepted: 18 Nov 2015

ABSTRACT

Objective: H. pylori have acquired resistance to the commonly used antibiotics due to their use in many anaerobic and parasitic infections leading to treatment failure of various gastroduodenal diseases associated with H. pylori infection. Our aim was to test the efficacy of Parmelia perlata which is traditionally used for the treatment of dysentery, diarrhea, and dyspepsia against antibiotic resistant gastric pathogen H. pylori.

Methods: The antimicrobial activity of an ethanolic and methanolic extract of P. perlata against drug-resistant H. pylori isolates from North India in vitro was carried out by determining the Minimum inhibition concentration (MIC) using disk diffusion method and microdilution method.

Results: Two H. pylori isolates were included in this study. One was resistant to both metronidazole (MIC of 64 µg/ml) and clarithromycin (MIC of 4 µg/ml) and another one was resistant to metronidazole only having a MIC of 64 µg/ml. Methanolic and ethanolic extract of P. perlata showed its effectiveness in inhibiting drug resistant H. pylori isolates with maximum inhibition at 500 × 103 µg/ml concentration of P. perlata.

Conclusion: Prevalence of metronidazole resistant ranges between 50–90% in developing countries including India with the emergence of dual-drug resistance was reported in many studies. This study suggests that P. perlata used commonly as a spice in food has a potential for the treatment of drug-resistant H. pylori infection in a safe and effective manner.

Keywords: Helicobacter pylori, Parmelia perlata, Dual-drug resistance.

© 2016 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/)

INTRODUCTION

Helicobacter pylori are a gastric pathogen and are associated with several gastroduodenal diseases such as chronic gastritis, peptic ulcer, and gastric cancer [1-3]. More than one-half of world’s population carries the H. pylori infection and in India its prevalence exceeds 90% of the population. Eradication of H. pylori from infected individuals remains the best choice for an effective treatment of H. pylori-associated diseases. Triple therapy, consisting of the combination of two antibiotics and a proton pump inhibitor, gives a high eradication rate [4]. The common antibiotics used against H. pylori are Metronidazole, Clarithromycin, Furazolidone, Amoxicillin, and Tetracycline. Misuse of antibacterial agents in general population for various respiratory and anaerobic infections have resulted in the emergence of antibiotic-resistant strains which is the prime cause of treatment failure apart from potential side effects [5, 6]. Metronidazole and clarithromycin are the most commonly used antibiotics in H. pylori treatment. Prevalence of resistance against metronidazole has been found to be high in many studies [4, 7, 8].

 The emergence of dual and multidrug resistance has also increased significantly in many countries and has become a major obstacle in eradicating the H. pylori infection [8-11]. In view of the incomplete cure achieved with a conventional triple therapy comprising metronidazole and clarithromycin, there is an urgent need to develop new treatment strategies for H. pylori infection.

Therefore, in such a scenario, there is a crucial need to find new antimicrobial agents. Unlike synthetic drugs, bioactive natural products are beneficial for the humans without causing unwanted side-effects. In this search lichens are the new bioactive preparations of natural origin. Lichens produce secondary metabolites the “lichen substances”, which comprise depsides, depsidones, dibenzofurans, xanthones and terpene derivatives. Lichens and their metabolites have multiple biological activities: antiviral, antibiotic, antitumor, allergenic, plant growth inhibitory, anti-herbivore, ecological roles and enzyme inhibitory [12, 13]. Parmelia perlata, commonly called as the stone flower is generally used as a spice to enhance the taste and flavor of the foods. P. perlata is lichen that is widely distributed in the hilly areas of the Indian subcontinent. In ancient folklore it has been used in cosmetic and has been reported to have medicinal value. It was used for the treatment of dysentery, wound healing, diarrhea, and dyspepsia [14, 15].

Studies have been conducted using different extracts of P. perlata against various bacterial and fungal pathogens. It contains many compounds like tridecyl myristate, 3-ketooleanane, icosan-1ol etc. [16]. Recent studies have shown that methanolic extract of Lichen possesses anti-microbial potential [17]. Our aim is to study the antimicrobial activity of the methanolic and ethanolic extracts of the lichen P. perlata against dual-drug resistant H. pylori in safe and effective manner.

MATERIALS AND METHODS

Collection of sample

P. perlata was collected from the local Indian market and authenticated in the Botany Department of Amity University. The plant was properly washed under running tap water and then rinsed in distilled water. Then it was air dried in shade and grinded into powder using mortar and pestle.

Preparation of extracts

In order to obtain the extract, about 100 gms of P. perlata were crushed with mortar and pestle and sieved. The dried powder was then extracted with 400 ml n-Hexane, chloroform, Methanol and Ethanol consecutively for 72 h/solvent under constant stirring. The extract was then filtered and dried under pressure and resuspended in the solvent. Methanolic and ethanolic extract of P. perlata were used in the present study.

H. pylori strains and culture

Patients suffering from various gastroduodenal diseases were included in this study. H. pylori strains were isolated from antral biopsies collected from patients and were identified on the basis of colony appearance, gram staining, and positive reactions in biochemical tests (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% Isovitale X (Becton Dickinson, MD), trimethoprim (5µg/ml), vancomycin (8µg/ml), and polymixin B (10µg/ml). The plates were incubated at 37 oC in a microaerophilic atmosphere (5% O2, 10% CO2, 85% N2) (Double gas incubator, Hera cell 150i) for 3 to 6 d. Stock cultures were maintained until use at–70 oC in Brain heart infusion broth with 20% glycerol.

Suspension preparation

The bacterial suspension was prepared by the direct colony method [18]. 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 (3 µl of which contains about 1 ×108 colony forming units (CFU)/ml) [19].

Determination of antimicrobial susceptibility and resistance

H. pylori cells growing exponentially on antibiotic free BHI agar were suspended in Phosphate buffered saline (PBS) buffer, a series of 10-fold dilutions of these cell suspensions was prepared, and 10 µl of each dilution was spotted on freshly prepared BHI agar containing various concentration of different antibiotics (µg/ml) viz. Metronidazole (0.2, 0.5, 1.5, 3, 8, 16, 32, 64), Clarithromycin (0.125, 025, 1, 2), Furazolidone (0.2, 0.5, 1, 2), Amoxicillin (0.125, 0.25, 1, 2) and Tetracycline (1, 2, 3, 4).

Minimum inhibitory concentration (MIC)

After 72 h incubation under microaerophilic conditions, the minimal inhibitory concentration was recorded as the lowest concentration that inhibited visible growth of organisms. Minimal inhibitory concentration (MIC) for different antibiotics was defined as Metronidazole (>8 µg/ml) [20], Clarithromycin (>0.5 µg/ml) [20], Amoxicillin (>0.12 µg/ml) [20], Tetracycline (>1 µg/ml) [20] and Furazolidone (>2 µg/ml) [7].

Anti- H. pylori activity assay of P. perlata extracts

  1. Disk diffusion method

Sterile Whatman paper disks (6 mm in diameter) were loaded with different concentration of methanolic and ethanolic extracts of P. perlata and placed on the inoculated plates with 3 ×109 colony forming unit (CFU) of H. pylori. The plates were kept under observation for 2 d at 37o C under microaerophilic conditions (5% O2, 10% CO2 and 85% N2). All experiments were performed in triplicates. Pure methanol and ethanol were used as a negative control and amoxicillin were used as positive control.

  1. Micro-dilution method

Minimum inhibitory concentration (MIC) was also determined by microdilution method. The 96-well plate was prepared by dispensing 80 µl of BHI broth into first well. A 20 µl from the stock solution of lichen extract (5 × 104 µg/ml) was added to the first well. Then, five-fold serial dilution was performed till 8th well. The obtained concentration range was from 4000 µg/ml to 0.05 µg/ml. To each well 150 µl of the diluted bacterial cells were added to give final concentration of 3 × 109 CFU/ml. The inoculated plates were incubated at 37 ° C for 2 d at microaerophilic conditions. MIC90 was defined as the lowest concentration of extract sample that inhibited the 90 % of H. pylori cells when to compare to control i.e. H. pylori cells without extract sample. Reading was noted on Elisa reader (Erba Lisa Scan II).

Rapid urease test (RUT) was also done for visualization of microbial growth. 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 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 (fig. 2).

RESULTS

Two clinical H. pylori isolates were included in this study isolated from a patient suffering from Non-erosive reflux disease (NERD) and Gastroesophageal reflux disease (GERD). The MIC of H. pylori isolate for metronidazole, clarithromycin, furazolidone, amoxicillin and tetracycline were determined by agar dilution method. Out of the two strains one strain was found resistant to metronidazole only having MIC of 64 µg/ml but sensitive to all drugs namely clarithromycin, furazolidone, amoxicillin, and tetracycline having MIC of 0.125 µg/ml, 0.2 µg/ml, 0.125 µg/ml and 1 µg/ml respectively and another strain was resistant to both clarithromycin and metronidazole having MIC of 4 µg/ml and 64 µg/ml respectively but sensitive to furazolidone, amoxicillin, and tetracycline with MIC of 2 µg/ml, 0.125 µg/ml and 1 µg/ml respectively.

Antimicrobial activity

Disc diffusion method

Different concentration of methanolic and ethanolic extract of P. perlata was loaded onto the disc and was air dried. 100 µl of the suspended H. pylori strain in PBS having Mac Farland 2 (3× 109 CFU/ml) was spread plated onto BHI medium. The disc loaded with the extracts was placed upside down in the H. pylori plates (fig. 1). After 72 h of incubation, we found that the methanolic and ethanolic extract of P. perlata has anti-H. pylori activity. MIC of P. perlata was < 50 × 103 µg/ml (table 1). Among the different concentration tested for both the extracts 500 × 103 µg/ml displayed maximum inhibition against the drug resistant H. pylori strain. The diameter of the zone of inhibition was 15 mm for the methanolic extract of P. perlata. (table 1)

4

5

(a)

(b)

Fig. 1: Disk diffusion method: Antimicrobial activity of P. perlata extracts against metronidazole-resistant H. pylori strain (a) Methanolic extract of P. perlata. (b) Ethanolic extract of P. perlata.

Table 1: Antimicrobial activity of methanolic and ethanolic extract of Parmelia perlata against drug-resistant H. pylori strains

Strain No.

MIC for MTZ* and

CLR*

(Agar dilution

method)

Ethanolicextracts of P. perlata (Disk

diffusion method)

MIC90 for P. perlata
(micro-dilution  method)

Methanolicextracts of P. perlata (Disk

diffusion method)

MIC90 for P. perlata

(microdilution method)

Concentration (in

µg/ml)

Zone of inhibition (in

mm)a

  

Concentration (in

µg/ml)

Zone of inhibition (in

  mm)a

  

HP 1

MTZ=

64µg/ml

CLR=

4 µg/ml

50 × 103

8.0±0.1

800 µg/ml

50 × 103

10.0±0.0

32 µg/ml

250 × 103

10.0±0.5

250 × 103

12.0±0.1

500 × 103

14.0±0.2

50 × 103

15.0±0.0

HP 2

MTZ=

64µg/ml

CLR=

<0.125µg/ml

50 × 103

8.0±0.0

800 µg/ml

50 × 103

8.0±0.1

32 µg/ml

250 × 103

11.0±0.1

250 × 103

13.0±0.0

500 × 103

13.0±0.5

500× 103

15.0±0.5

a= mean±SD (n = 3), MTZ = Metronidazole, CLR = Clarithromycin, GERD = Gastro-esophageal reflux disease, NERD = Non-erosive reflux disease

Microdilution method

The antimicrobial activity of lichen extract was evaluated by microdilution method. The MIC was determined in two drugs resistant clinical H. pylori isolates. MIC values ranged between 4000 µg/ml to 0.05 µg/ml (fig. 2). MIC90 values of a methanolic and ethanolic extract of P. perlata were 0.1 µg/ml and 31.3 µg/ml respectively against H. pylori isolate resistant to both metronidazole and clarithromycin.

6

Fig. 2: Micro-dilution method: lane 1 to 8 shows the Five-fold serial dilution of lichen extract from lane 1 to lane 8 with a constant cell count of H. pylori. Lane 9 shows media control without cells. Lane 10 shows the cell control without lichen extract. Lane 11 shows extract control without cells. Lane 12 shows the control in which the lichen extract dissolved with cells. MIC90 was observed at lane 4 and Lane 2 for methanolic and ethanolic extract of P. perlata respectively

DISCUSSION

Due to increasing use of antibiotics over the last two decades, H. pylori have acquired antimicrobial resistance and are the main cause of treatment failure. Previous studies have shown that the prevalence of resistance to metronidazole in different parts of India were Delhi (37.5%) [21], Chandigarh (38.2%) [21], Lucknow (68%) [21], Gangetic belt of North India (100%)[22]Hyderabad (100%) [21] Chennai (88.2%) [21], Kolkata (85%) [7] and Gujarat (83.8%) [23]. High prevalence of metronidazole-resistant H. pylori has also been reported from other developing countries (China, 77.8%; Bangladesh, 77.8%; Mexico, 76.3%) [24, 25]. In India, the high prevalence of multidrug-resistant strain was observed in (Multicentric study in India, 43.2%; Gujarat, 85%)[21, 23]. Our recent studies have shown the prevalence of Dual and multidrug resistance in 26.5% (18/68) and 8.9% (6/68) of cases in India [8]. The high prevalence of dual drug resistant H. pylori isolates has prompted us to look into the natural products which can be effective against H. pylori.

Anti- H. pylori potential of many plant and plant products and their usefulness for treatment of H. pylori infection has been studied. De et al. has mentioned the therapeutic potential of curcumin against H. pylori-associated gastroduodenal diseases and have shown eradication of H. pylori from the infected mouse stomach [26]. Similarly, our previous study has shown that ethanolic extract of Embilica officinalis can effectively treat H. pylori-associated gastric ulcer [27]. We have also found that methanolic extract of Paedra foetida was effective in inhibiting the growth of metronidazole-resistant H. pylori strain in vitro [28]. In India, Parmelia is used in treating a number of diseases and as a food supplement which is mentioned in Indian Materia medica [29]. Previous studiesshowed its antimicrobial activity against different bacteria namely Clavibacter michiganensis, Pseudomonas solanacearum and fungi like Fusarium oxysporum and Rhizopus nigricans [29]. Antimicrobial properties of different lichens have also been tested against different pathogenic bacterial and fungal strains. Tippeswamy et al. used hot and cold extracts of the lichen in various solvents and tested them against the variety of bacterial and fungal pathogens [29]. Gulluce et al. found that methanolic extract of the lichen Parmelia saxatilis has stronger antibacterial property as compared to antifungal activity [30].

We have included dual drug-resistant H. pylori strain in this study. Both the methanolic and ethanolic extracts of P. perlata were studied. In the present study, we found that methanolic and ethanolic extract of P. perlata potentially inhibited the growth of the H. pylori isolates in vitro that were isolated from the infected patient suffering from NERD and GERD. It is noteworthy that one of the H. pylori strain (HP1) was resistant to both clarithromycin and metronidazole and another strain (HP2) was resistant to metronidazole only which is the important component used in the first line therapy for the H. pylori treatment. So, our results suggest that methanolic and ethanolic extract of P. perlata acts through mechanisms distinctly different from the mode of action of these antibiotics for inhibition of H. pylori growth.

Further research needs to be done to determine the compounds that are responsible for antibacterial activity against H. pylori. The findings also indicate that scientific studies carried out on medicinal plants having traditional claims of effectiveness [31, 32] can give fruitful results.

ACKNOWLEDGEMENT

We thank Amity University for providing the infrastructure and support to carry out the work.

CONFLICT OF INTERESTS

Declared None

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