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Research Article
 

Activity of Terminalia chebula and Erythrina variegata Extracts Against Pathogen Causing Respiratory Tract Infection



Chandrika Das, Dil Umme Salma Chowdhury, Abdul Mannan and Nazneen Naher Islam
 
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ABSTRACT

Background and Objective: Respiratory Tract Infections (RTIs) are one of the leading causes of morbidity and mortality worldwide. As the bacterial resistance towards commercially available antibiotics indicated for microorganisms associated with RTIs is increasing, the need of finding an appropriate alternative antimicrobial agent is also on the rise. Therefore, the present study was executed to explore the antibacterial activity of the acetone extracts of two medicinal plants, Terminalia chebula and Erythrina variegata against antibiotic-resistant Staphylococcus aureus. Materials and Methods: For this study, the presence of S. aureus in the human sputum samples was ensured by growing culture on Mannitol Salt Agar (MSA) and performing a battery of biochemical tests. Culture sensitivity tests were done to determine the antibiotic susceptibility pattern of S. aureus. MAR index of S. aureus isolates was also calculated to determine if they are resistant to more than one antibiotic. The antimicrobial activity of the plant extracts was determined using a disc diffusion assay. Results: The isolates were found highly resistant to Erythromycin (68.97%) and least resistant towards Chloramphenicol (13.79%). The high MAR index (>0.2) has signified the exposure of S. aureus to multiple antibiotics. Acetone extracts collected from the two plants showed the highest activity at 5 mg mL–1 among the three different concentrations used (1.25, 2.5 and 5 mg mL–1) in the study. It was also observed that the extracts from T. chebula exhibited better antibacterial activity than that of E. variegata against S. aureus isolates. Conclusion: T. chebula and E. variegata extracts could be further investigated as potential antibacterial agents to treat respiratory tract infection.

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Chandrika Das, Dil Umme Salma Chowdhury, Abdul Mannan and Nazneen Naher Islam, 2021. Activity of Terminalia chebula and Erythrina variegata Extracts Against Pathogen Causing Respiratory Tract Infection. Research Journal of Phytochemistry, 15: 51-57.

URL: https://scialert.net/abstract/?doi=rjphyto.2021.51.57
 

INTRODUCTION

Respiratory Tract Infections (RTIs), are one of the leading causes of mortality, morbidity, malnutrition, impaired growth and cognitive development in children as well as in older age groups1. They are classified into two broad categories based upon which part of the respiratory region is affected: Upper Respiratory Tract Infections (URTIs) and Lower respiratory tract infections (LRTIs). Pharyngitis, nasopharyngitis, otitis media, tonsillitis and sinusitis are the major infections of the upper respiratory tract whereas pneumonia is the formidable one among the lower RTIs since it can easily lead to death2. URTIs are generally caused by viruses (adenovirus, rhinovirus, parainfluenza virus and influenza virus). Following the viral invasion, secondary infection ensues by various types of bacteria which results in chronic obstructive lung disease and high fever3.

Several factors can influence the incidence and associated mortality owing to RTI notably, demographic risk profile, irrational antibiotic prescribing, the prevalence of causative agents and antibiotic resistance4. The different types of bacteria which are involved in RTIs are Staphylococcus aureus, Haemophilus influenzae, Streptococcus pyogenes, Moraxella catarrhalis, Streptococcus pneumonia, Klebsiella pneumonia and Mycobacterium tuberculosis. All of these bacteria liable for respiratory infections have been reported to become resistant towards different antibiotics including Amoxicillin, Cephalosporin (Ceftriaxone), Trimethoprim, Sulfamethoxazole, Erythromycin, Chloramphenicol, Ciprofloxacin, Penicillin, Medemycin, Aztreonam, Streptomycin, Ampicillin and Tetracycline5-8. Increased fatality in patients with serious infections has been linked with excessive and improper initial antibiotic therapy which is a potentially modifiable factor9.

The main reason for increased resistance to antibiotics in bacteria is due to the mobile genes present on the plasmids which spread and transfer through bacterial populations10. Another reason for antibiotic resistance of bacteria has been considered to be the production of biofilms during the quorum-sensing-regulated mechanism which releases beta-lactamase responsible for the degradation of various antibiotics5. Nowadays, antibiotic resistance developed by microorganisms is recognized as a far-reaching health concern by the global research community11. Researchers around the world have been focusing on obtaining knowledge of bacterial etiology and drug-resistance pattern of evolving microorganisms and employ necessary strategies to decrease the development of resistance and produce a better therapeutic outcome. So, there is an urgent need for alternative antibacterial agents which can target these drug- resistant pathogenic microbes. Many attempts have been made to investigate the potential role of plant extracts containing active compounds to combat the problem of antibiotic resistance in bacteria. Various essential oils (EOs) have been shown to exhibit noteworthy antimicrobial actions against a wide range of both gram-positive and gram-negative bacteria12. The Essential Oils (EOs) and their components cause permeabilization of the cell wall and cytoplasmic membrane of bacteria which results in ion leakage and reduction of the membrane potential and ATP pool13. EOs have been traditionally used for treating respiratory tract infections and are currently used as alternative medicines for the treatment of colds14. Moreover, to treat acute and chronic bronchitis, acute sinusitis and inhalation therapy using essential oils have previously been used. It has been reported that inhalation therapy using volatile essential oil vapours is effective in reducing asthma-associated respiratory distress and inflammation of the trachea as well as increasing the respiratory tract fluid output1,15.

Some plants have been used in folk medicines for a long period by traditional medicine practitioners in the South Asian region to treat cold and chronic asthma among which Terminalia chebula (seed) and Erythrina variegata (leaf) are included16,17. The claim of therapeutic efficacy of these plants has not yet been validated adequately through in vitro or in vivo methods which is crucial to give them a secure place in modern health care. Hence, this study has been conducted to explore primarily the effectiveness of the crude extracts of these medicinal plants against drug-resistant bacteria isolated from a human sputum sample.

MATERIALS AND METHODS

Study area, population and sample collection: Sputum samples of patients (N = 30) with respiratory tract infections were collected from different diagnostic centres of Chattogram Metropolitan area. Samples were collected in sterile sealed plastic vials and stored in the refrigerator at 4°C for further analysis. This study was carried out from February, 2019 to February, 2020. Ethical clearance was obtained from the ethical review committee of Chattogram Maa-O-Shishu Hospital, Chattogram. The patient consents were also taken as forms of the questionnaire.

Isolation of Staphylococcus aureus: Luria Bertani (LB) broth media was prepared in the sterile conical flask and sterilized by autoclaving. After that, the media was cooled to 45°C and poured into sterile test tubes. Then, a loopful of freshly collected sputum sample was inoculated in LB broth. The same step was followed for the rest of the samples. All of the culture tubes were then kept in an incubator at 37°C for 24 hrs. The growth of bacterial culture was determined by measuring optical density in a spectrophotometer at a wavelength of 600 nm. Then, Mannitol Salt Agar (MSA) media was prepared, cooled down and poured on sterile Petri plates and kept to solidify. One loop of liquid culture was streaked on the MSA plate and the same procedure was followed for each plate. The plates were then incubated for 24 hrs at 37°C in an incubator in the inverted position. Yellow coloured colonies that grew on MSA plates were identified as S. aureus18,19.

Biochemical identification of Staphylococcus aureus: Isolated S. aureus colonies were identified by performing the Coagulase test, Catalase test and Gram staining test18. Indole test, Methyl-Red (MR) test, Voges-Proskauer (VP) test, Citrate utilization test was also performed20.

Antibiotic susceptibility test: Kirby-Bauer disk diffusion assay was followed to perform antibiotic susceptibility test21. All S. aureus positive samples were subjected to disc diffusion assay to determine their antibiotic susceptibility pattern against five antibiotics. The selected antibiotics for assay were Erythromycin (15 μg), Chloramphenicol (30 μg), Amoxicillin (30 μg), Cefepime (30 μg) and Ciprofloxacin (5 μg). About 200 μL of overnight grown bacterial culture in LB media was spread on Mueller Hinton agar plate with a sterile glass spreader. Then after 5-10 min, commercially available standard antibiotic discs were placed on Mueller-Hinton agar (MHA) plates with sterile forceps. The plates were then incubated and then the organisms were categorized as “resistant”, “sensitive” or “intermediate” based on their production of the zone of inhibition which was compared with the obtained standard data from Clinical Laboratory Standards Institute.

Multiple antibiotic resistance (MAR) indexes: The MAR index of an isolate is calculated as a/b. Where, a = no. of antibiotics to which the isolates were resistant and b = no. of antibiotics to which the isolates were subjected22.

Collection and processing of plant sample: Plant samples were collected from two different locations of Chattogram regions namely, Anwara upazila and Botanical garden of the University of Chittagong. Healthy and matured leaf samples of Erythrina variegata and seeds of Terminalia chebula were collected and transported to the lab on the day of collection. Then the samples were properly washed with sterile distilled water and dried at 45°C in an oven followed by grinding and packing into thimbles of 8-9 cm which were made of clean dry clothes.

Preparation of plant extract: The Soxhlet extraction method was employed using acetone as extraction solvent23. The extract was filtered through Whatman no. 1 filter paper. Acetone was evaporated using a vacuum rotary evaporator at 50°C and then dried at room temperature to remove the residual solvent. The collected extracts were again filtered and finally stored in a sterile sealed glass bottle at 4°C before use for antimicrobial assay. Plant extracts obtained from T. chebula and E. variegata were diluted into 1.25, 2.5 and 5 mg mL–1.

Determination of the antimicrobial activity of plant extract: The antimicrobial activity of the acetone extracts of both plants was determined against the test organisms using disc-diffusion assay24. Sterilized and dried filter paper discs (6 mm in diameter) were soaked with plant extracts of T. chebula and E. variegata. About 200 μL of overnight grown LB media culture of S. aureus was spread on MHA media plates with a sterile spreader. After that, a disc containing test extracts were placed on MHA plates. The plates were kept in incubation overnight at 37°C. Standard discs of erythromycin (15 μg) were used as the positive control. Clear and distinct zone of inhibitions were visualized around the plant extract impregnated discs.

Statistical analysis: The experiment regarding the determination of the antimicrobial activity of the plant extracts was carried out in triplicate and zones of inhibition were calculated as mean±SD (mm).

RESULTS

Prevalence of S. aureus: The results of the study revealed that out of thirty (30) collected samples, the prevalence of S. aureus was 96.6% (29) as shown in Fig. 1.

Bacteriological analysis: Among the 29 positive S. aureus isolates of this study, 24 (82.76%) isolates were Coagulase-positive and 5 (17.24%) isolates were coagulase-negative. All of the 29 isolates (100%) were found positive for the catalase production test, Methyl Red, Voges Proskauer, Citrate utilization test and Gram staining but negative for Indole production test (Table 1).

Image for - Activity of Terminalia chebula and Erythrina variegata Extracts Against Pathogen Causing Respiratory Tract Infection
Fig. 1:
Percentage of sputum samples infected with Staphylococcus aureus
Co P: Coagulase positive and Co N: Coagulase-negative


Image for - Activity of Terminalia chebula and Erythrina variegata Extracts Against Pathogen Causing Respiratory Tract Infection
Fig. 2:
Antibiotic resistance profile of S. aureus isolates


Table 1: Different biochemical tests of S. aureus isolated from a sputum sample
Total positive samples Catalase test Coagulase test MR test VP test Indole test Citrate utilization test Gram staining
29 29 24 29 29 - 29 29

Antimicrobial susceptibility pattern: The antimicrobial susceptibility pattern was examined for 29 S. aureus isolates against 5 commercially available standard antibiotics by using disc diffusion assay. The whole analysis was done by following the standard data available from National Committee for Clinical Laboratory Standards. According to the result of culture sensitivity, the isolates were found to show the highest resistance towards erythromycin (68.97%) which was followed by ampicillin and Cefepime (65.52%). The resistance was moderate towards ciprofloxacin (51.72%) and least for chloramphenicol (13.79%) which meant that the highest susceptibility of S. aureus was found against chloramphenicol (75.86%). The results of the antibiotic resistance profile of S. aureus are displayed in Fig. 2.

Table 2: Multiple antibiotic resistance (MAR) indexes
No. of isolates
No. of antibiotics they are subjected to (b)
No. of antibiotics to which they showed resistance (a)
MAR Index (a/b)
4
5
1
0.2
6
5
2
0.4
11
5
3
0.6
7
5
4
0.8
0
5
0
0


Table 3: Antibacterial activity of plant extract collected from Erythrina variegata against S. aureus isolates
Diameter of inhibition zone (mm) of Erythrina variegata extract
Drug resistance pattern 5 mg mL1
2.5 mg mL1
1.25 mg mL1
E, AML, CPM, CIP 7.5±0.408
7.0±0.707
5.83±0.408
E, AML, CPM, CIP 11.5±1.225
11.0±0.408
10.83±0.471
E, C, AML, CIP 16.0±0.408
13.0±0.408
13.00±0.408
E, AML, CPM, CIP 10.0±0.00
7.5±0.408
6.50±0.408
C, AML, CPM, CIP 9.5±0.408
8.0±1.225
6.00±0.408
E, C, CPM, CIP 13.5±0.408
11.0±0.00
9.50±0.408
E, C, CPM, CIP 8.0±0.408
0.0
0
E: Erythromycin, AML: Amoxicillin, CPM: Cefepime, CIP: Ciprofloxacin and C: Chloramphenicol


Table 4: Antibacterial activity of plant extract collected from Terminalia chebula against S. aureus isolates
Diameter of inhibition zone (mm) o f Terminalia chebula extract
Drug resistance pattern 5 mg mL1 2.5 mg mL1 1.25 mg mL1
E, AML, CPM, CIP 19±1.080 16.0±1.080 14.0±0.408
E, AML, CPM, CIP 11±0.408 10.5±0.707 9.0±1.225
E, C, AML, CIP 19±1.225 19.0±1.225 18.0±1.225
E, AML, CPM, CIP 14±1.225 13.0±1.225 12.0±0.408
C, AML, CPM, CIP 13±0.816 13.0±0.408 10.0±1.225
E, C, CPM, CIP 17±0.408 14.0±0.408 10.5±0.408
E, C, CPM, CIP 13±0.408 11.0±1.225 8.5±0.408
E: Erythromycin, AML: Amoxicillin, CPM: Cefepime, CIP: Ciprofloxacin and C: Chloramphenicol

Multiple antibiotic resistance (MAR) indexes: In this study, among the 29 isolates, 4 isolates had a MAR index of 0.2 while 24 isolates showed a MAR index value of greater than 0.2 in Table 2.

Antimicrobial activity of plant extracts: It was revealed from the disc diffusion assay that seven S. aureus isolates exhibited resistance towards four different antibiotics (out of five antibiotics) used in this study. Then, the antimicrobial activity of extracts obtained from both of the plants was investigated against these seven S. aureus antibiotic-resistant isolates. Erythromycin (15 μg) was used as a positive control because the isolates showed maximum resistance (68.97%) against it. In the case of extracts from E. variegata, the highest zones of inhibition found for 7 different S. aureus isolates in 5 mg mL–1 were 7.5±0.408, 11.5±1.225, 16±0.408, 10±0, 9.5±0.408, 13.5±0.408 and 8±0.408, respectively while the highest zones of inhibition for T. chebula were 19±1.080, 11±0.408, 19±1.225, 14±1.225, 13±0.816, 17±0.408 and 13±0.408, respectively (Table 3 and 4).

DISCUSSION

In developing countries, RTIs are considered to be the second most common cause of death in children due to pneumonia. In Bangladesh, pneumonia is responsible for around 15% of the deaths of children under five years of age25. Bacteria responsible for respiratory infections have been reported to become resistant to different antibiotics. Though pharmaceutical companies are producing several new antimicrobial drugs, the resistance gain rate is pretty high. So, the need of producing alternate pharmacological products cannot be overlooked. Medicinal plants having antimicrobial potential can exert a better effect in this regard12.

The prevalence rate of S. aureus (96.6%) found in the current study aligned with the finding of Ullah et al.26 in which the highest occurrence rate was observed for S. aureus (57.81%) in sputum samples collected from patients with lower respiratory tract infections. The highest frequency of resistance exhibited by the S. aureus was against Erythromycin (68.97%) whereas the least resistance was observed against Chloramphenicol (13.79%). The reason for developing increased resistance of S. aureus isolates against these antibiotics could be their frequent administration. In a different study conducted by Ullah et al.26, it was shown that S. aureus showed a high rate of resistance against Amoxicillin (89.2%) which corroborates with the findings of our study (Amoxicillin: 65.52%). Moreover, it was observed that Chloramphenicol was the most effective antibiotic used in this study which is in agreement with the findings (53.3% susceptibility) of Olugbue et al.27. The MAR index indicates whether the isolates are from high or low antibiotic using region making it an effective tool for health risk assessment. MAR index values higher than 0.2 are considered to have originated from high-risk sources where antibiotics are often used. And MAR index values of less than or equal to 0.2 indicates a strain originated from sources where antibiotics are seldom or never used22,28. In this study, it was observed that out of 29 isolates, 24 isolates had a MAR index of greater than 0.2 which suggested that the S. aureus isolates have been identified from a high-risk source (human sputum sample). It was seen that extracts of both plants showed maximum activities at the highest concentration used in the study (5 mg mL–1). The antibacterial activities of the extracts were found better compared to the standard antibiotic (Erythromycin). Moreover, between the two acetone extracts used in this study, increased diameter for inhibition zone (mm) was observed for T. chebula extract in all three different concentrations which suggested that it has better antibacterial activity against S. aureus isolates. It is stated in a report that fruiting bodies of T. chebula exhibited antibacterial activity against methicillin-resistant S. aureus and the main components responsible for this effect are gallic acid and its ethyl ester. Another study observed that ethanedioic acid and ellagic acid from the fruits of T. chebula exhibited moderate inhibitory activity against intestinal E. coli, respectively29.

On the other hand, a study on E. variegata and its antibacterial effects against cariogenic oral bacteria revealed that isoflavonoids of E. variegata had bactericidal activity against methicillin-resistant S. aureus and various other strains among which the most active compounds are erycristagallin and orientanol B30. Another study revealed that isoflavonoids extracted from E. Variegata had shown substantial anti-MRSA activity31. So, it could be said that these two plant extracts could be used in future as alternatives to antibiotics to which S. aureus has grown resistant.

CONCLUSION

This study has found the potential effect of Terminalia chebula and Erythrina variegata plant extracts against antibiotic-resistant bacterial isolates. Moreover, T. chebula and E. variegata have shown better inhibitory activity than the standard antibiotic (Erythromycin) used in this study to which most of the bacterial isolates were found resistant.

SIGNIFICANCE STATEMENTS

In brief, T. chebula and E. variegata plant extracts exhibited good antibacterial activity towards S. aureus indicating the possibility of further exploration of their potential anti-microbial activity through in vitro and in vivo study. This study will help the researchers to embark on the further explorative study on the active compounds isolated from these plants which are required to understand their pharmacological effect as well as employ them as an antimicrobial agent in future.

ACKNOWLEDGMENT

This study was fully funded by the research and publication cell, University of Chittagong, Chattogram-4331, Bangladesh.

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