In-Vitro Antibacterial Properties of Crude Aqueous and n-Hexane Extracts of the Husk of Cocos nucifera

The increasing numbers of cases of antibiotic resistance among pathogenic bacteria such as Vibrio species poses a major problem to the food and aquaculture industries, as most antibiotics are no longer effective in controlling pathogenic bacteria affecting these industries. Therefore, this study was carried out to assess the antibacterial potentials of crude aqueous and n-hexane extracts of the husk of Cocos nucifera against some selected Vibrio species and other bacterial pathogens including those normally implicated in food and wound infections. The crude extracts were screened against forty-five strains of Vibrio pathogens and twenty-five other bacteria isolates made up of ten Gram positive and fifteen Gram negative bacteria. The aqueous extract was active against 17 of the tested bacterial and 37 of the Vibrio isolates; while the n-hexane extract showed antimicrobial activity against 21 of the test bacteria and 38 of the test Vibrio species. The minimum inhibitory concentrations (MICs) of the aqueous and n-hexane extracts against the susceptible bacteria ranged between 0.6–5.0 mg/mL and 0.3–5.0 mg/mL respectively, while the time kill study result for the aqueous extract ranged between 0.12 Log10 and 4.2 Log10 cfu/mL after 8 hours interaction in 1 × MIC and 2 × MIC. For the n-hexane extract, the log reduction ranged between 0.56 Log10 and 6.4 Log10 cfu/mL after 8 hours interaction in 1 × MIC and 2 × MIC. This study revealed the huge potential of C. nucifera extracts as alternative therapies against microbial infections.

Nowadays, coconut oil, obtained from the fruit of coconut palm, has been relegated mainly to non food uses in the developed countries but retains its importance for traditional uses in producing countries [10]. Coconut oil has been confirmed to possess antimicrobial, antiviral and antiprotozoal activities [12,13]. Though the antibacterial potential of the husk of C. nucifera has been reported before [11,14], these studies have not been elaborate enough and have covered very few bacterial strains. For example, Esquenazi et al. [11] used only one bacterial species (Staphylococcus aureus), which though pathogenic is not a known diarrheic pathogen. Also, Srinivas et al. [14] relied only on five bacterial species. There is need for a more detailed assessment of the antibacterial potential of the husk of C. nucifera against a wide panel of bacterial species including referenced, environmental and clinical strains which is the focus of this current report. In this paper, we report on the antibacterial properties of the aqueous and n-Hexane extracts of the husk of C. nucifera against some Vibrio pathogens and other bacteria as part of our exploration for new and novel bioactive compounds.

Results and Discussion
The antibacterial activity of Cocos nucifera husk extract was investigated. Sixteen of the test bacteria were susceptible to the aqueous extract with a zone of inhibition value ranging between 11 and 20 mm, while twenty bacteria were susceptible to the n-hexane extract with inhibition zones ranging between 12 and 18 mm at the test concentration of 25 mg/mL (Table 1). Thirty-seven of the Vibrio isolates were susceptible, with inhibition zone diameters ranging between 10 and 18 mm for the aqueous extracts; while thirty-eight were susceptible to the n-hexane extract with inhibition zone diameters ranging between 12 and 21 mm ( Table 2). The minimum inhibitory concentrations (MICs) of the extract against the susceptible bacteria generally ranged between 0.6-5.0 mg/mL for both extracts. Specifically, MICs for the aqueous and n-hexane extracts ranged between 0.6-5.0 mg/mL and 0.3-5.0 mg/mL respectively (Table 1).  Key: -represents no antibacterial activity; MIC represents minimum inhibitory concentration; ND represents not determined, AMP -ampicillin, TET -tetracycline.
The results of time-kill studies are presented in Table 3. Data are presented in terms of the Log 10 cfu/mL reduction in viable cell count and interpretations are based on the conventional bactericidal activity standard, which is, a 3 Log 10 cfu/mL or greater reduction in the viable colony count [15]. For the aqueous extract, average log reduction in viable cell count in time kill assay ranged between 0.12 Log 10 and 4.2 Log 10 cfu/mL after 8 hours interaction at 1 × MIC and 2 × MIC. For the n-hexane extract, the log reduction ranged between 0.56 Log 10 and 6.4 Log 10 cfu/mL after 8 hours interaction in 1 × MIC and 2 × MIC. The greatest reductions in cell density achieved with the aqueous extract were on Vibrio vulnificus EL039, with the average value of 4.2 Log 10 cfu/mL, S. aureus OKOH2B (clinical strain) with the average reduction in viable cell count of 3.46 log 10 cfu/mL, while the greatest reduction in viable cell volume achieved by the n-hexane extract were on the environmental strain Bacillus substilis, with the average value of 6.40 log 10 cfu/mL and the reference strain Escherichia coli ATCC 8739, with an average reduction in viable cell count of 5.6 log 10 cfu/mL.  The use of plant extracts with medicinal potential represents a valid alternative for the treatment of different ailments and diseases. The antivibrio and antibacterial properties of the husk of C. nucifera were investigated against a number of Vibrio pathogens and other bacteria pursuant to contributing to our body of knowledge on the potentials o the plant in the management of Vibrio and other bacterial infections in support of previous report [16]. The aqueous and n-hexane extracts of the husk of our study plant exhibited potent antivibrio and antibacterial activity against about 90% of the bacteria strains tested. The result from this study confirms that both the aqueous and n-hexane fraction of the husk possess antimicrobial properties against Vibrio species and other bacteria thus supporting the traditional use of this plant in the treatment of wound, respiratory and gastro intestinal tract infections.
The diameters of the zones of inhibition exhibited by the extracts against the text bacteria are similar to those reported elsewhere, viz. by Ravikumar et al. [17] and Chandrasekaran et al. [18], who reported on the chloroform extracts of Exoecaria agallocha leaves; as well as methanol and aqueous extracts of mangrove respectively. The limited activity of the aqueous extract in comparison to the n-Hexane extract corroborate previous reports [19,20] where they reported lower activity in the aqueous extracts compared to other solvent extracts.
The MICs values observed in this study varied depending on the strain and ranged from 0.6 to 5.0 mg/mL for the Vibrio bacteria and from 0.3 to 5.0 mg/mL for the other bacterial isolates. The observation that some of the Vibrio and bacteria strains were susceptible to the plant extract at a concentration as low as 0.3125 mg/mL strongly suggest that of C. nucifera plant can be effective in the treatment of infections caused by these pathogens. A similar result was reported by Sharma et al. [21] in their studies on the vibriocidal activities of 16 Indian medicinal plants, wherein 70% of the Vibrio pathogens tested were susceptible to the plant extract at a concentration ranging between 2.5 and 20 mg/mL.
The bactericidal activities of the aqueous extracts of this plant at 2 × MIC after 8 h exposure against S. aureus OKOH2B (a clinical isolate from wound sepsis); V. vulnificus and V. fluvialis is worth noting and further supports its use as a folklore remedy. At 1 × MIC, the n-hexane extract showed bactericidal activity against three of the six bacterial species tested as well as the entire cohort of Vibrio isolates. At 2 × MIC the entire population of the Vibrio and the other bacteria species (except S. faecalis) tested had been wiped out after 8 hr exposure. It would appear that the observed bactericidal or bacteriostatic activity of this plant is both time and concentration dependent.
Considering the crude nature and low toxicities of the solvent extracts used in this study, our results allow us to conclude that the crude extract from C. nucifera exhibited significant antibacterial activity and properties that support its folkloric use in the treatment of some food borne diseases as well as its potential wound healing activities. Plants that have tannins as their components are astringent in nature and are used for treating intestinal disorder such as dysentery and diarrhea [22] thus exhibiting antimicrobial activity. Esquenazi et al. [11] reported that C. nucifera aqueous extract is rich in catechin and epicatechin together with condensed tannin. Edeoga et al. [23] had reported that the curative potentials of plants are locked-up and embedded in some chemical components that effect physiological responses in man. Some of these ingredients act synergistically to confer bioactivity on a plant an active material.
To further buttress the phytochemical importance of C. nucifera, Zakaria et al. [24] administered the coconut juice extract as part of a dietary supplement at low concentrations and also the coconut cream and oil. The application of C. nucifera extract as food supplement is both an economical and an eco-friendly alternative in antimicrobial chemotherapy. Although coconut fruit is meant for human consumptions, this present study suggests the need for characterizing the antibacterial active principle(s) of Cocos nucifera. Understanding the chemical nature of the active principle(s), it will provide an opportunity to synthesize new and effective antibacterial (including antivibrio) drugs.

Plant material
The plant specimens were collected from the vicinity of the Research Farm of the Obafemi Awolowo University, Ile Ife, Nigeria and identified by the curator of the Herbarium at the Department of Botany, Obafemi Awolowo University, and a voucher specimen kept there.

Preparation of extracts
The coconut husk was sun-dried, milled and sieved manually to obtain fine powdered particles. About 50 g dried of powdered husk of the plant was extracted at room temperature and for 48 h with 95% n-hexane (200 mL) using a Soxhlet extraction method. The mixture was then filtered using Whatman No. 1 filter paper. The filtrates of each extraction were pooled together and concentrated to dryness in vacuo using a rotary evaporator to remove the n-hexane. The concentrated extract was then allowed to dry at room temperature to a constant weight. For the aqueous extract, about 50 g of the powdered extract was dissolved in sterile distilled water (500 mL) for 24 h with shaking. The resulting extracts were centrifuged at 3,000 rpm for 5 min at 4 °C. The supernatant was filtered through a Whatman No. 1 filter paper and the filtrate was lyophilized.

Test bacterial strains
The bacterial isolates used in this study included forty-five Vibrio strains and twenty-five bacteria pathogens as part of the culture collection of the Applied and Environmental Microbiology Research Group (AEMREG), Department of Biochemistry and Microbiology, University of Fort Hare, Alice, South Africa. The Vibrio species were isolated from waste-water effluent in the Eastern Cape Province, South Africa. The bacterial isolates include reference strains (9) obtained from the South African Bureau of Standard (SABS), environmental strains (12) and clinical isolates (4).

Antibacterial susceptibility test
The susceptibility screening of the test bacteria to both crude extracts and standard antibiotics were done in accordance with the methods described elsewhere [25,26]. The inoculum size of each test strain was standardized at 5 × 10 5 cfu/mL using McFarland Nephelometer standard. Sterile Mueller-Hinton agar plates were seeded with test bacterial strains and allowed to stand at 37 °C for 3 h. Wells were then bored into the agar media using a sterile 6 mm cork borer and the wells filled with the solution of the extracts and antibiotics taking care not to allow spillage of the solution onto the surface of the agar. The plates were allowed to stand on the laboratory bench for 1 h to allow proper diffusion of the extract and antibiotics into the media and thereafter incubated at 37 °C for 24 h, after which they were observed for zones of inhibition. The effects of the extracts on the test bacterial isolates were compared with those of the standard antibiotics tetracycline and ampicillin which served as a negative and positive control at a concentration of 1 mg/mL and 10 μg/mL, respectively.

Determination of minimum inhibitory concentration (MIC)
The MIC of the crude aqueous and n-hexane extract was carried out using the method of Akinpelu and Kolawole [27]. Two-fold dilutions of the extracts were prepared and 2 mL aliquot of different concentrations of the solution were added to 18 mL of pre-sterilized molten Mueller-Hinton agar at 40 °C to give final concentration regimes of 5.0 to 0.156 mg/mL. The medium was then poured into sterile Petri dishes and allowed to set. The surfaces of the media were allowed to dry under a laminar flow before streaking with 18 h old bacterial cultures. The plates were later incubated at 37 °C for up to 72 h after which they were examined for the presence or absence of growth. The MIC was taken as the least concentration of extracts that will prevent the visible growth of the test bacteria.

Time-kill assay
Determination of the kill rate of the crude extracts was done following the procedure as described by Okoli and Iroegbu [28]. Inocula were prepared following the described guidelines of EUCAST [29]. The resultant suspension were diluted 1:100 with fresh sterile broth and used to inoculate 50 mL volumes of Mueller Hinton broth incorporated with extracts at MIC and 2 × MIC to a final cell density of approximately 5 × 10 5 cfu/mL. The flasks were incubated at 37 °C on an orbital shaker at 120 rpm. A 500 μL sample was removed from cultures at 0, 4 and 8 h, diluted serially and 100 μL of the diluted samples were plated on Mueller Hinton agar plates and incubated at 37 °C for 24 h. Controls included extract-free Mueller Hinton broth seeded with the test inoculum.

Conclusion
This study has demonstrated the antibacterial activities of C. nucifera especially against Vibrio bacteria and suggests that the plant has immense potentials as an alternative to synthetic antibiotics in the management of Vibrio and other bacterial infections. Further studies are needed to elucidate the active components and their modes of action as well as their potentials in combination chemotherapy with synthetic drugs which is the subject of ongoing research in our group.