The huge numbers of infectious diseases caused by the gramnegative bacteria that are resistant to many commonly used antibiotics are the causes of great concern to the clinicians as well as the microbiologists. Phytomedicines, prepared from various plant materials, such as Ayurvedic traditional medicine, are comparatively safe, inexpensive and have less antagonistic effects. The leaf, bark, fruit and seeds of Mimusops elengi possess several medicinal properties, viz., astringent and tonic in dental diseases and uterine disorders [1-4]. This plant has also been reported for analgesic, diuretic, antiulcer, antipyretic, anti-inflammatory and antimicrobial activities [5-8].

In rural areas of developing countries, like India, herbal materials are in use as the primary source of medicines [9]. Nearly 80% of the people in developing countries use traditional drugs for the purpose of primary health maintenance [10]. Among the plant species occurring worldwide [11], only a very less percentage has been investigated phytochemically. The medicines of plant origin used by the medical practitioners are in the form of extract of the whole plants or part of the plants. Some of the effects elaborated by the plant extracts used in the traditional medicine include antiviral, antitumor, antimicrobial, and having central nervous system effect [12]. The plants possess bioactive components of therapeutic value to cure several health disorders of humans [13]. The research interest on the antimicrobial activity of plant extracts is a raising one because of the current problems with bacterial antibiotic resistance, and the use of phytochemicals as natural antimicrobials is gaining popularity [14].

One such important traditional medicinal plant is M. elengi belonging to the Sapotaceae family, called as ‘Bakula’ in Bengali and it is well known in Ayurvedic medicine. All the parts of M. elengi have medicinal properties, and the leaves are reported to be used in the treatment of bacterial diseases by tradition [15]. The pharmacognostic and phytochemical screening reports on M. elengi stem bark has been documented [16]. Recently, estimation of triterpene acids using from M. elengi stem bark has been published [17,18]. Antimicrobial, antiviral and hepatoprotective and cytotoxic activities of M. elengi are well accepted because of the wealth of scientific literature supporting these effects [19]. The aqueous and ethanolextracts M. elengi leaves have been tested against Proteus mirabilis , Pseudomonas aeruginosa , Staphyloccus aureus , Salmonella enterica serovar Typhimurium and Bacillus cereus . The ethanol extracts had greater activity than the aqueous extracts of all the remedial plants [20]. The M. elengi leaf extracts showed great antioxidant activity in different solvent like nhexane, dichloromethane and methanol compared to different standered antioxidants [21]. Both enzymatic and non-enzymatic antioxidant activities were conducted by Kalaiselvi et al. [22] in assessing the antioxidant properties of M. elengi . The antibacterial activity silver nanoparticles is known [23], and the biogenic silver nanoparticles produced by M. elengi fruit (pericarp) and flower extracts have been reported as excellent antimicrobials against grampositive as well as gram-negative bacterial strains [24,25]. Therefore, in the present study, the ethanolic extracts of leaf, seed and bark of M. elengi were evaluated phytochemically and tested against six different bacterial strains to identify the potentiality of antibacterial activity.

Bacterial strains

The clinical isolates of Escherichia coli , Klebsiella pneumonia and Proteus vulgaris were used in the current study, and the standard strains included were E. coli ATCC 25922, K. Pneumonia MTCC 7407, Pseudomonas aeruginosa ATCC 27853.

Plant parts and preparation of extracts

Various parts of Bakul plant, M. elengi : leaf, seed (from mature fruits) and bark of were collected from naturally grown wild plants at Raiganj of Uttar Dinajpur district, West Bengal (India). The plant materials were washed repetitively with distilled water and shed-dried, and grinded thereafter using a grinding machine.

The granules prepared from different parts of the plant, 25 g each, were soaked separately in 100 ml of ethanol for a period of 48 h with manual shaking [26]. The extracts thus obtained were sieved through sterilized Whatman No.1 paper filter after straining through an autoclaved cheese cloth, and stored at 4°C for further use. The concentrations of the ethanolic Bakul (M. elengi ) leaf extract (BLE) and seed extract (BSE) in the stock solutions were 250 μg/μl, and bark extract (BBE) was 200 μg/μl.

Phytochemical analysis

The plant extracts were screened qualitatively for different types of bioactive components (phenol, quinone, flavonoids, steroids, terpenoids and glycosides) using the protocol of Radhakrishnan et al. [27].

Antibiotic susceptibility

The test bacterial strains were screened for susceptibility to antibiotics (Hi-Media, India): cefpodoxime(CPD; 10-μg/disc), gentamycin(GEN; 10-μg/disc) and ciprofloxacin(CIP; 5-μg/disc) following disk diffusion method [28], as per the recommendation of CLSI guidelines [29]. The inoculated nutrient agar plates with impregnated antibiotic discs were incubated at 35°C for 24 h, and the zone diameter of inhibition (ZDI) values obtained around each of the antibiotic discs were measured.

Antibacterial activity of plant extract

The Antibacterial activities of BLE, BSE and BBE were determined by disc diffusion method, as mentioned earlier [30]. In this process, on sterile nutrient agar plate young broth culture of the test bacteria was swabbed and dried, and 4 sterilized discs, each of 6 mm diameter (prepared in the laboratory from Whatman’s No. 1 filter paper), were placed on 4 different sectors and marked. Thereafter, the discs were soaked with four different concentrations: 20, 35, 50 and 65 μl/disc, equivalent to 5, 8.75, 12.5 and 16.25 mg/disc for BLE and BSE, while 4, 7, 10 and 13 mg/disc for BBE. The ZDIs were recorded and interpreted according to the CLSI criteria [31], for resistance and sensitivity of the isolates.

Combined antibacterial activity

The combined antibacterial activity of antibiotic and plant extract was determined following the protocol mentioned earlier [32]. Briefly, on nutrient agar plates, swabbed inoculated with test bacteria, three sectors were prepared and marked with BLE, BSE and BBE, and the antibiotic discs: CPD (10-μg/disc), GEN (10-μg/disc) and CIP (5-μg/ disc) were placed. On each of the antibiotic disc, 20 μl (i.e., 5 mg/disc for BLE and BSE, and 4 mg/disc for BBE was dropped, soaked and dried properly for about 30 min at room temperature. The ZDIs were measured for each combined action against the bacteria tested.

Growth inhibitory indices

The GII (growth inhibitory index) values were calculated following the formula stated earlier [33], in order to interpret the various effects of antibiotic-plant extract interaction. The synergistic, additive or antagonistic activities, if any, in between the two of the antimicrobial agents (plant extract and antibiotic) were defined with GIIs > 0.5, 0.5 and < 0.5, respectively [32].

Statistical analysis

The t-test was used in order to compare the antibacterial activity (in terms of ZDIs) of the antibiotics (CPD, GEN and CIP) alone and in combination with plant extracts (BLE, BSE and BBE), against the test bacterial isolates. The p-value of < 0.10 was considered significant.

The antibacterial activity of the plant extracts is shown in Table 1. The BLE, BSE and BBE, tested at various concentrations, against E. coli (ATCC 25922), E. coli (clinical), K. Pneumoniae (MTCC 7407), K. Pneumonia (clinical), Ps. aeruginosa (ATCC 27853) and Pr. vulgaris (clinical) had ZDIs 7-21mm. The BBE displayed ZDIs 12, 14, 20 and 21mm at 4, 7, 10 and 13 mg/disc, respectively, against K. Pneumonia (MTCC 7407), and BSE had antibacterial activity against K. Pneumonia (clinical) with ZDIs 7, 8, 9 and 10 mm at 5, 8.75, 12.5 and 16.25 mg/disc, respectively. The BLE, BSE and BBE had ZDIs 10-14 mm for E. coli (clinical) and 8-14 mm for E. coli ATCC 25922 strain. In case of Ps. aeruginosa , BSE exhibited ZDIs 10-17 mm at 5-16.25 mg/ disc, respectively; against Ps. aeruginosa BBE showed ZDIs 8-18 mm at 4-13 mg/disc.

Table 1: Antibacterial activity of ethanolic leaf, seed and bark extract of M. elengi.

The antibiotic susceptibility test results are presented in Table 2. The E. coli (clinical), K. Pneumonia (clinical) and Ps. aeruginosa were resistant to CPD, while E. coli (ATCC 25922), K. Pneumonia (MTCC 7407) and Pr. vulgaris (clinical) were sensitive to CPD having ZDIs 10, 18 and 17 mm, respectively. The CIP showed activity, with ZDIs 20-30 mm, for the test bacteria except the E. coli clinical isolate (6 mm); the ZDIs ranged 10-28 mm and 6-30 mm, due to GEN and CIP, respectively.

Table 2: Antibiotic susceptibility test results for clinical and standard bacterial strain; CPD: cefpodoxime, GEN: gentamycin; CIP: ciprofloxacin.

The combined antibacterial activity of the plant extracts and antibiotics against the test bacterial isolates is presented in Figure 1. The growth inhibitory indices (GIIs) of combined action are represented in Table 3.

Table 3: Growth inhibitory indices (GII) from the combined action of antibiotics and plant extracts against the test bacterial isolates. CPD: cefpodoxime; GEN: gentamicin; CIP: ciprofloxacin; BLE: bBakul leaf extract; BSE: bakul seed extract; BBE: bakul bark extract.

Figure 1: Combined susceptibility test results A: leaf extract; B: seed extract; C: bark extract; ZDI (zone diameter of inhibition), BI-1: E. coli (clinical); BI-2: K. Pneumonia (clinical); BI-3: Proteus vulgaris (clinical); BI-4: E. coli (ATCC 25922); BI-5: K. Pneumonia (MTCC 7407); BI-6: Ps. aeruginosa (ATCC 27853); CPD: cefpodoxime, GEN: gentamicin, CIP: ciprofloxacin, BLE: bakul leaf extract; BSE: bakul seed extract; BBE: bakul bark extracts. The p-values ranged 0.22-0.42 when the difference between antibacterial activity of antibiotic alone and in combination with plant extracts, were considered.

Glycosides, steroids, terpenoids and quinone were presented in the BLE, BSE and BBE whereas along with the above phytoconstituents, presence of flavonoids was seen in the BSE.

The therapeutic role of plants in the treatment of human health disorders of several kinds has been recorded worldwide [9]. Among 122 compounds, 80%, used in the ethnomedical purposes, have been derived from 94 plant species [11]. The ethyl acetate, hexane, methanol, and ethanol M. elengi extracts had antibacterial activity against the dental caries causing bacteria Streptococcus mutans isolated from the patients [34]. The acetone, petroleum ether, methanol and water extracts of M. elengi bark have been tested for their antibacterial activity against dental infection causing bacteria: Staph. aureus , Str. mutans, Str. salivarius , Str. sanguinis , and fungus (Candida albicans ) by well diffusion method [35,36]. M. elengi leaf extracts had antibacterial activity against Bacillus subtilis and Trichoderma viride [37]. In the current study, three different extratcs (BLE, BSE and BBE) showed antibacterial activity against E. coli , K. Pneumoniae , Ps. aeruginosa , Pr. vulgaris . The concentration dependant antibacterial activities, as indicated by the ZDI due to the action of BLE (250 μg/μl), BSE (250 μg/μl) and BBE (200 μg/μl) alone, and in combination with antibiotics (CPD, GEN and CIP), have been recorded. The ZDIs were increased from 8 mm to 14 mm, when the leaf extract concentration was increased from 5 mg/disc to 16.25 mg/disc; for seed extracts ZDI was 7 mm at 5 mg/disc, which was increased to 17 mm at the highest concentration (16.25 mg/disc), while the bark extracts had highest ZDI (21 mm) at 16.25 mg/disc and lowest (8mm) at 5mg/disc against the test bacterial isolates.

Lalitha et al. [38] reported that the aqueous extract of M. elengi (leaf) had a strong antibacterial activity having ZDIs 27 mm and 24 mm against E. coli and Str. pneumoniae ; the extract showed moderate growth inhibitory activity against Ps. aeruginosa , Vibrio cholerae and Salmonella typhi . In the present investigation, BLE showed antibacterial activity with ZDIs 14, 13, 13, 14, 12 and 14 mm, against E. coli (clinical), E. coli ATCC 25922, K. Pneumonia (clinical), K. Pneumonia MTCC 7407, Ps. aeruginosa ATCC 27853 and Pr. vulgaris (clinical), respectively in presence of 65 μl of the extracts (16.25 mg) per disc. The aqueous extract of M. elengi (leaf) was, thus, found more effective than the ethanolic extract. Kannadhasan et al. demonstrated that, the antibacterial activity of two different extracts of M. elengi leaf having ZDIs 8 – 10 mm and 11 mm against K. Pneumoniae and E. coli , respectively [39].

Lalitha et al. reported the top ZDI of GEN (36 mm) against E. coli [38]. The susceptibility to chloramphenicol and trimethoprim for S. enterica serovar Typhi isolates was determined with resistivity to both chloramphenicol and trimethoprim [40]. The study conducted by Reddy and Joss, utilized the gram-positive bacteria: Bacillus cereus (MTCC-1305), and Staph. aureus (MTCC 96), and gram-negative bacteria: Enterobacter faecalis (MTCC 5112), Salmonella paratyphi (MTCC 735), E. coli (MTCC 729), K. Pneumoniae (MTCC 109), Ps. aeruginosa (MTCC 647), Pr. vulgaris (MTCC 426) and Serratia marcescens (MTCC 86), and showed top ZDI (36 mm) against E. coli and K. Pneumonia [41]. Padhi and Mahapatra utilized E. coli (MTCC 40), Staph. aureus (MTCC 87), Staph. epidermidis (MTCC 2639), Ps. aeruginosa (MTCC 424), Str. pneumoniae (MTCC 237) and Pr. vulgaris (MTCC 426), and found highest susceptibility of E. coli to tetracycline (ZDI: 32 mm) [15]. The standard strains of E. coli DSM 1103 and Staph. aureus ATCC 25923 were sensitive to ampicillin and CIP [42]. In the present investigation, CPD, GEN and CIP were used to determine antibiotic susceptibility tests, where CIP showed greater ZDI (30 mm) against Pr. vulgaris , compared to the other antibiotics. The action of antibiotics can be stimulated by the plant extracts (BLE, BSE and BBE), as has been reflected in this study. In the present study calculated GIIs ranged 0.5-0.89, and thus, all test result showed synergistic and additive activities, but not antagonistic. However, no significant difference between the action of antibiotic alone and in combination was seen (p= 0.22-0.42).

The presence of alkaloids, carbohydrates, glycosides, tannins and phenolic compounds, steroids, saponins and flavonoids in the aqueous M. elengi leaf extract has been reported by Padhi and Mahapatra [15]. Lalitha et al. observed cardiac glycosides, tannins, alkaloids, flavonoids, saponins, steroids and reducing sugar present in BLE [38]. Dichloromethane, The M. elengi seed extracts prepared with petroleum ether, ethyl acetate and ethanol had antibacterial activity against E. coli , B. subtilis and S. enterica serovar Typhi, as has been reported by Hazra et al. [43]. The phytocomponents detected in BLE included β-carotene and glucose, quercitol and hentriacontane, β- sitosterol, β-sitosterol-β-D-glucoside, D-mannitol and quercetin [44-46]. M. elengi bark has been shown to contain alkaloids, saponins, tannin and ash forming inorganic salts [47,48]. In this study, the presence of glycosides, steroids, terpenoids and quinone was noted in BLE and BBE, and flavonoids, glycosides, steroids, terpinoids and quinone in BSE. The findings of the current study, along with the reports made by the others, the M. elengi might be useful in the preparation of cost effective new antimicrobials, alone and in combination with antibiotics, in combating bacterial antibiotic resistance.