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Antibacterial Activity of Mimusops elengi Leaf, Seed and Bark Extracts Alone and in Combination with Antibiotics against Human Pathogenic Bacteria

Aims: The current study determines the antibacterial activity of ‘Bakul’ (Mimusops elengi) leaf, seed and bark extracts against gram-negative clinical bacterial isolates as well as the standard bacterial strains. Methods: The disc diffusion method was followed to determine the antibacterial activity of M. elengi leaf, seed and bark extracts against the clinical isolates of Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa. The antibiotic susceptibility of the bacterial isolates was determined by disc diffusion. Results: The concentration dependent activity of the extracts against the bacteria was recorded with zone diameter of inhibition 7-21 mm. The extracts in combination with antibiotics (cefpodoxime, gentamycin and ciprofloxacin) had growth inhibitory indices (GIIs), 0.5-0.6, 0.5-0.89 and 0.73-0.82 against E. coli, K. pneumoniae and Pr. vulgaris clinical isolates, respectively, while for the standard strains (E. coli, K. pneumoniae and Ps. aeruginosa) the GIIs ranged 0.56-0.86. Most of the extracts were tested positive for the presence of flavonoids, glycosides, steroids, terpenoids, quinone and phenol. Conclusion: The M. elengi leaf, seed and bark extracts, in combination with antibiotics, had synergistic interactions against all the standard bacterial strains and Pr. vulgaris clinical isolate, while both synergistic and additive interactions were recorded against E. coli and K. pneumoniae clinical isolates.


Introduction
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][2][3][4]. This plant has also been reported for analgesic, diuretic, antiulcer, antipyretic, anti-inflammatory and antimicrobial activities [5][6][7][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 ethanol extracts 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 of silver nanoparticles is known [23], and the biogenic silver Translational Medicine Mandal  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 (leaf and seed) and 20 g (bark), 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 indices) 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.

Results
The antibacterial activity of the plant extracts is shown in Table 1 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.     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.

Discussion
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] [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  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.