Abstract
Seeds of Eugenia jambolana Lam. (Myrtaceae) are used by many tribes in India to treat diarrhea and dysentery. The crude extracts of seeds of this plant demonstrated zones of inhibition in the range of 14– 21 mm against the isolated β-lactamase-producing drug-resistant bacteria. The methanol extract showed promising antibacterial activity which was subjected to fractionation. The effective fraction (F2) showed a minimum inhibitory concentration (MIC) ranging from 31.75 to 62.5 μg/mL. Phytochemical analysis and thin layer chromatography of the most promising fraction showed the presence of saponin as the active phytoconstituent. The active fraction was further tested for its in vitro hemolytic activity in sheep and human erythrocytes and no hemolysis was seen. Thus, the use of this plant by tribals to treat bacterial infections has some scientific basis.
Introduction
Eugenia jambolana Lam. (Myrtaceae) is widely used in India to treat several ailments in the traditional system of medicine. The seeds are reported to possess antidiabetic activity (CitationSridhar et al., 2005) and are also used to treat chronic diarrhea and other enteric disorders (CitationVeigas et al., 2007). CitationChandrasekaran and Venkatesalu (2004) reported that methanol crude extracts of E. jambolana seeds showed promising activity against Gram-positive bacteria. Traditional healers of Madhya Pradesh use the seeds of this plant to treat pimples (CitationRoy & Chaturvedi, 1986). Bhoxa tribals of Uttar Predesh use the leaf of this plant to treat blisters in the mouth (CitationMaheswari & Singh, 1984). Tribals of Rajasthan use the leaf to treat stomach ache (CitationSebastian & Bhandari, 1984).
The potential of this plant against the commonly encountered multidrug-resistant bacteria has not been evaluated. Among several drug-resistant bacteria, β-lactamase production is the most important mechanism of resistance to penicillin and cephalosporins (CitationAyyagari & Bhargava, 2001). Plasmids responsible for extended spectrum β-lactamase (ESβL) production tend to be large and carry resistance to several agents, which is an important limitation in the design of treatment alternatives (CitationJacoby & Medeiros, 1991). The most frequent co-resistances found among ESβL-producing organisms are aminoglycosides, fluoroquinolones, tetracyclines, chloramphenicol, and sulfamethoxazole-trimethoprim (CitationNathisuwan et al., 2001). Major outbreaks involving ESβL strains have been reported from all over the world, hence making them emerging pathogens (CitationAnanthakrishnan et al., 2000). Thus, the number of effective exogenous antibiotics is decreasing; therefore, concerted efforts are to be made to identify antimicrobial materials from natural products and traditional medicines. Antibacterial activity of medicinal plants against drug-resistant bacteria including ESβL-producing bacteria has been reported (CitationLiu et al., 2000; CitationShimizu et al., 2001; CitationPalombo & Semple, 2002). Very few reports of antibacterial activity against drug-resistant bacteria are available for Indian medicinal plants (CitationAhmad & Aqil, 2006). No reports are available for E. jambolana for antibacterial activity against ESβL-producing bacteria. The present study was carried out to assess the efficacy of Eugenia jambolana against β-lactamase-producing drug-resistant bacteria.
Materials and methods
Plant material
Seeds of E. jambolana were collected during May 2004 from a tree growing at Holy Cross College, Tiruchirapalli, Tamil Nadu and authenticated by Dr. Rosalie, a taxonomist from the Department of Botany, Holy Cross College, Tiruchirappalli. A voucher (HCH:171) specimen was deposited at the herbarium of the above department.
The seeds were air-dried and powdered. The powder (1 kg) was extracted with n-hexane, acetone, and methanol sequentially in a Soxhlet apparatus and evaporated to dryness under reduced pressure in a rotary evaporator (CitationDuraipandiyan & Ignacimuthu, 2007). The yields of the n-hexane, acetone, and methanol extracts were 10.9, 12.1, and 14.3 g, respectively. The dry residues of the crude extracts were stored at 4°C.
Fractionation of the crude extract
The most active crude extract (methanol) was chromatographed on a silica gel column. The methanol crude extract was dissolved in n-hexane and then loaded on the silica gel column. Initial elution was done with a discontinuous gradient of 50% ethyl acetate and 50% n-hexane, 75% ethyl acetate and 25% n-hexane, and 100% ethyl acetate; then a continuous gradient from 90% ethyl acetate and 10% methanol untill 100% methanol was used. This yielded 13 fractions (F1–13) which were combined into five fractions using TLC according to their Rf (retention factor) values.
Test organisms
Bacterial isolates were obtained from symptomatic urinary tract-infected patients admitted to the CSI Mission General Hospital in Tiruchirappalli from June 2004 to June 2005 (). The bacterial strains were grown and maintained on nutrient agar slants. The isolated bacterial strains were identified by conventional biochemical methods (CitationKoneman et al., 1997).
Table 1. Antibiogram pattern of the screened extended spectrum β-lactamase (ESβL)-producers showing the number and percentage of resistance of the organisms to the listed antibiotics.
Screening for drug resistance
The antibiogram obtained for the isolated bacteria revealed them to be multidrug-resistant clinical isolates. The tested isolates were screened for ESβL production following a double-disk synergy test (DDST) (CitationMiles & Amyes, 1996). ESβL presence was assayed using the following antibiotic disks: cefotaxime (30 μg), cefotaxime clavulanic acid (30/10 μg), ceftazimide (30 μg), and ceftazidime clavulanic acid (30/10 μg). In the DDST, synergy was determined between a disk of augmentin (20 μg amoxycillin and 10 µg clavulanic acid) and a 30 μg disk of each third-generation cephalosporin test antibiotic, placed at a distance of 30 mm on a lawn culture of the resistant isolate on Mueller–Hinton agar medium (MHA; Himedia Pvt. Ltd., Mumbai, India). The test organism was considered to produce ESβL if the zone size around the test antibiotic disk increased toward the augmentin disk.
Antibacterial activity
The antibacterial activity of the extract was evaluated by the disk diffusion method (CitationBauer et al., 1966). The test organisms, whose concentration was adjusted using a 0.5 std McFarland’s opacity tube (CitationMcFarland, 1907), were inoculated on the surface of MHA medium. About 10 μL of the test extracts (1g crude extract dissolved in 10 mL dimethylsulfoxide (DMSO)) were impregnated on sterile disks (Himedia, Mumbai, India), allowed to dry for 5 min, and placed on MHA medium. After incubation for 24 h at 37°C the presence or absence of clear zones of inhibition was recorded. DMSO- and solvent-only disks were used as controls. The assessment of the antibacterial activity was based on measurement of the diameter of the zone of inhibition formed around the standard antibiotic disks (CitationNCCLS, 2008).
Determination of minimal inhibitory concentration of the plant extracts
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined for the plant extracts and the fractions by the broth dilution method, as described by CitationAyafor et al. (1994), and the microbroth dilution method (CitationEloff, 1998), respectively. The concentration at which there was no visually detectable bacterial growth was taken as the MIC, and the concentration at which there was no bacterial growth after inoculation in Mueller–Hinton agar was taken as the MBC.
Phytochemical analysis of plant extracts
The methanol extract of E. jambolana and the most active fraction (F2) were selected for preliminary phytochemical screening. Tests for alkaloids, steroids, flavonoids, terpenoids, and proteins were carried out using standard methods (CitationHarborne, 1973).
Determination of cellular toxicity using sheep erythrocytes
The method described by CitationXian-guo and Ursula (1994) was employed to study cellular toxicity. Briefly, 10-fold serial dilutions of the extract were made in phosphate buffered saline. A total volume of 0.8 mL for each dilution was placed in an Eppendorf tube. A negative control tube (containing saline only) and a positive control tube (containing saponin, 5mg/mL) were also included in the analysis. Fresh sheep erythrocytes were added to each tube to give a final volume of 1 mL. Solutions were incubated at 37°C for 30 min, and all tubes were centrifuged for 5 min and then observed for hemolysis. Complete hemolysis was indicated by a clear red solution without any deposit of erythrocytes. Hemolysis was also checked microscopically for the presence or absence of intact red blood cells (RBCs).
Results
Antibiotic sensitivity test for ESβL-producing strains of gram-negative bacteria showed multidrug resistance to β-lactam antibiotics. The antibiogram pattern of the isolates selected for the study is shown in . The MIC values of penicillin, ampicillin, cefuroxime, and cefotaxime ranged from 128 to 1024 µg/mL. Only a few species such as Aeromonas hydrophila and Citrobacter freundii showed low resistance to ampicillin (MIC 32 µg/mL) but produced β-lactamase, hydrolyzing ampicillin (). All the test strains could hydrolyze the common substrate drugs, ampicillin, cefotaxime, ceftazidime, and cefuroxime, confirming their ESβL production as detected by double-disk synergy test. The fractions of the methanol extract of E. jambolana exhibited broad-spectrum activity against all the ESβL-producing multidrug-resistant strains. The zones of inhibition for all three plants extracts are presented in . The methanol extract produced larger zones of inhibition, and no zones of inhibition were observed for control disks (methanol, acetone, n-hexane, and DMSO). Varying levels of activity for these extracts were observed against different bacteria. The MIC values for all three crude extracts are presented in . MIC values for the methanol crude extract ranged from 0.2 mg/mL in A. hydrophila to 12.5 mg/mL in E. aerogenes and K. pneumoniae. Results for zones of inhibition and MICs of the fractions of the methanol extract are listed in and , respectively. The disk diffusion assay revealed that fractions F1, F2, F3, and F4 inhibited the growth of all the tested bacteria strongly; F5 inhibited only three of the tested bacteria moderately (). With regard to the MIC, F2 exhibited good antibacterial activity against all the tested bacteria, with values ranging from 31.75 to 62.5 µg/mL, followed by F3 and F4 with MIC values ranging from 7.9 to 125 µg/mL. Moderate activity was seen in F5. Poor activity was seen in F1 (). The results of phytochemical screening of F2 of the methanol extract showed the presence of saponin (). The absence of hemolysis of the erythrocytes by the plant extracts was observed at all the dilutions used.
Table 2. Antibiotic sensitivity behavior and β-lactamase production by the test isolates.
Table 3. Disk diffusion assay of three extracts of Eugenia jambolana against ESβL-producing clinical isolates.
Table 4. Minimal inhibitory concentrations (mg/mL) of the different extracts of E. jambolana on ESβL-producing clinical isolates.
Table 5. Disk diffusion assay of fractions of the methanol extract of E. jambolana against ESβL-producing clinical isolates.
Table 6. MIC values of fractions of E. jambolana on ESβL producers (μg/mL).
Table 7. Phytochemicals present in E. jambolana test materials.
Discussion
β-Lactam resistance among clinical isolates is a growing problem (CitationLivermore et al., 2001). Many Gram-negative bacilli produce ESβL, enzymes that mediate resistance to all β-lactams except cephamycins and carbapenems (CitationPhilippon et al., 1989). Organisms producing ESβL are typically multidrug resistant (CitationTankhiwale et al., 2004). Compared with ESβL-negative isolates, ESβL-positive isolates are more often resistant to aminoglycosides, ciprofloxacin, and cotrimoxazole. In our study, most ESβL-positive strains resistant to several antibiotics were susceptible to plant extracts.
The inhibition halos of different extracts indicated that the methanol extract was the most effective. Comparison of the sizes of inhibition halos of different extracts alone cannot be considered for determination of the relative antimicrobial potency, since a more diffusible but less active extract could give a bigger diameter than a non-diffusible but more active extract. Hence, MIC studies were also done, which indicated that the methanol extract showed the lowest MIC values. Among the different fractions of the methanol extract, fraction 2 showed effective zones of inhibition. Though fractions F2, F3, and F4 were active against β- lactam-resistant clinical isolates, consistent effective results were obtained only for fraction 2. This was also true for the MIC values.
CitationChandrasekaran and Venkatesalu (2004) reported that the methanol extract of Syzygium jambolanum seeds exhibited good MIC values against many microorganisms. Similarly, Citationda Silva et al. (2008) have reported significant MIC values against many gram-positive and gram-negative bacteria. Novel antibacterial actions of plant extracts or phytocompounds have been demonstrated, which include inhibition of the MDR-efflux pump (CitationDixon et al., 1983), β-lactamase activity (CitationTsuchiya et al., 1994), anti-antibiotic resistance properties (CitationBatista et al., 1994), and R-plasmid elimination (CitationBorris, 1996). In our study, most ESβL-positive strains resistant to several antibiotics were found to be sensitive to the plant extracts. Their activity is probably due to their ability to complex with bacterial cell walls, disrupting microbial membranes. Fraction 2 of the methanol extract revealed the presence of saponins. All ESβL-positive isolates were susceptible to fraction 2. Though a few reports are available on the role of saponin against bacteria (CitationWang et al., 2000), there is not much literature available for the inhibitory action of these compounds on multidrug-resistant ESβL-producing human pathogens. Thus, our study adds credence to the use of this plant by tribal people to treat diarrhea and dysentery.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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