Antimicrobial potencies of selected native African herbs against water microbes

2.1 Plant sampling and microbial culture collections

Zanthoxylum zanthoxyloides and Gongronema latifolium samples were collected from plant material market (8.1294^oN, 4.2005^oE) Ogbomoso, Oyo State, Nigeria. Plants were washed, air dried and grinded to powder, then sieved to get a fine powder. Dry running through published literatures was used in identification of emerging microbial contaminants in water and identified bacteria species (P. aeruginosa, Klebsiella sp., E. coli, S. pneumonia, B. cereus) and fungal isolates (A. flavus, A. niger, Trichoderma sp and Candida sp.) were sourced from local strains collection from Culture Bank, Microbiology Unit, Pure and Applied Biology Department, LAUTECH, Ogbomoso, Nigeria.

2.2 Preparation of samples

50 g of dried and grinded samples of Zanthoxylum zanthoxyloides and Gongronema latifolium were extracted separately by maceration using 150 ml of ethyl acetate and chloroform as extraction solvent for 48 hrs. The solvent fractions were separated using vacuum filtration aided by a sterile Whatmann No 1 filter paper and filtering crucible. The filtered extracts were then dried with the help of a freeze drier and stored in sterile polypropylene airtight container at 4 °C or dispensed in appropriate sterile dissolving solution when required.

2.3 Antimicrobial activity, preparation of antimicrobial disc and sensitivity test

Crude extracts of G. latifolium and Z. zanthoxyloides eluted by ethyl acetate and chloroform and freeze dried were prepared into solutions of different concentrations ranging between 25 and 500 mg/ml by dissolving appropriate amount of crystallised extracts in sterile deionised water. About 20 µl of extract solution corresponding to a range of (0.5 mg – 10 mg) was then transferred on sterile 6 mm diameter sterile paper discs prior to antimicrobial sensitivity testing by disc diffusion method as described by (Adebayo et al., 2014; Lateef and Adeeyo, 2015). Briefly, 1 to 2 colonies of each bacteria culture were introduced into nutrient broth, and incubated at 37 °C for 24 hrs. A 1 to 50 dilution of the stock culture was then adjusted to tune of 0.5 McFarland standard (1.0 × 10⁶ cfu/ml). About 100 µl of the test microbes were then swabbed over the agar plate surface using surface spread method. Treated paper discs were afterward arranged serially and radially but firmly unto seeded agar plates. The plates were afterward incubated at 37 °C for 24 hrs. Fungal plates were incubated at room temperature (27 ± 2 °C) for 48 hrs. The degree of microbial susceptibility was determined by measuring the visible diameter of inhibition on microbial growth after incubation. Test concentrations of crude extract ranged from 500 and 25 mg/ml. Standard reference commercial antibiotic disc for Gram-negative (streptomycin), Gram-positive (gentamycin) and fungi (nystatin) were used for a comparative study.

2.4 Phytochemical analyses

The screening for plant extract composition was assayed according to standard methods and as modified by Adeeyo et al., 2018. Screening for tannins, saponnin, reducing sugars, alkaloids, terpenoids, flavonoids, steroids, anthraquinones and phlobatannins were carried out on extracted crude products.

2.5 Data analysis

For Data analysis, SPSS, version 19.0 was used in analysing the different means of data obtained. ANOVA test was used to determine the level of significance of the result at various concentrations. Also, comparison of the different solvent extracts (chloroform and ethyl acetate) were made statistically. Antimicrobial efficacy of the extracts were examined against that of standard antibiotics. Zones of inhibition was compared with reference standard to assign the level of activities of extracts.

3.1 Dose dependent activities

The degree of antimicrobial activity by measuring the visible diameter of inhibition on microbial growth has been described as strong (23–38 mm), moderate (19–22 mm) and low (12–18 mm) (Ahmad et al., 1999). The dose response to plant extracts can be observed in Figs. 1–5 and Table 2. At the lower doses (25–50 mg/ml), the extracts show low to moderate antimicrobial activities across the various plant extracts irrespective of the extraction solvent. The range of inhibition zone is between 7.5 and 18.50 mm. Noteworthy is the activity of ethyl acetate extract of Z. zythoxyliodes at this dose (18.50 mm; Fig. 1C) against E. coli. At the higher dosage, extracts showed moderate to high antibiosis with a range of 10.5 to 27.0 mm. Noteworthy is the activities of Z. zythoxyloide’s ethyl acetate extract against E. coli (Fig. 1C; 27.0 mm), as well as its chloroform extract against S. pneumoniae (21.50 mm) (Fig. 1D) and Trichoderma sp (Table 2; 20.50). G. latifolium chloroform extract also exhibited appreciable activity against all tested microbes and its activity against S. pneumonia (Fig. 1D; 20.00 mm) is remarkable. These effects are qualitatively alike to that of Shaheed et al. (2009) and Jones and Bridgeman (2017) who reported increase in performances of herbal extracts at higher dosages against microbial pollutants of water.

3.2 Comparative phytobiotic activities (with commercial antibiotics)

The abilities of phytoactive extracts of plants materials investigated as compared with commercial antibiotics were investigated. Plant extracts investigated performed beyond the activities of commercial streptomycin, gentamycin and nystatin within selected concentration range investigated. Statistical analysis revealed notable significant differences from 25 mg/ml and above, but mostly at higher concentrations. The comparative antibiosis of commercial antibiotics, as well as minimum and maximum extract concentrations against tested microbes were presented in Tables 3 and 4. The results revealed that potent phytoactive formulations of the plant materials tested can be developed within the range researched in this work, and with better performance than available commercial antibiotics. The advent of such formulations as envisaged in this work may be the dawn of novel technology in achievement of the World Health Organisation (WHO) recommendation of zero faecal count/100 ml water sample (WHO, 1998) especially when developed for water disinfection. Potent antimicrobial activities of plant materials have been reported for different formulations such as antipsoriatic cream with varied combinations of neem, bakuchi, sarsaparilla and daruhaldi which resulted in good anti-inflammatory and antimicrobial results (Mundada et al., 2009); herbal gel comprising Curcuma- longa, Aloe-vera and A. indica resulted in a good antimicrobial activity against the tested microbes in comparison to commercially available antimicrobials (Pandey et al., 2010). In a study where polyherbal ointment was made using methanolic extracts of M. pudica, S. indica A. indica, C. odorata, and assessed for bioactivities, the results showed better antibacterial properties (Rajasree et al., 2012). These reported research findings supported phytoactivities of plant materials as indicated in this work. Herbal extracts of Ocimum samctum and Eucalyptus globules as anti-microbial agents were found to exhibit major activity against P. aeuroginosa, B. subtilis, Escherichia. coli, S. aureus, S. cerevisiae as well as C. albicans and were safer for human usage when compared to reference standards Amphoterecin and Ampicillin respectively (Wani et al., 2013).

3.3 Antibacterial activities

The antibacterial activity of the extracts differs with respect to extraction solvents and susceptibility of microbial isolates increases with concentration of extract in solution (Figs. 1–5). Noteworthy is the activities of the extracts against E. coli which is a conventional indicator of water pollution. The activities of extracts tested were all notable against E. coli. Antibiosis of extracts on E. coli showed moderate to high performance at concentrations of 50 to 500 mg/ml. It is, however, notable that significant susceptibility was recorded at a concentration of 25 mg/ml of ethyl acetate extract of Z. zanthoxyloides which was the least concentration tested and may confirm possible potent antimicrobial activities of the investigated plants against groups of coliforms and microbial pathogens in water. The effects of the plant extracts against E. coli, Kelsiella sp and P. aeruginosa indicated the activities of these plant materials against a range of Gram-negative bacteria. For P. aeruginosa, susceptibility to plant extracts increases with concentration of the extracts in solution from 25 to 500 mg/ml. At dosages of 25 and 50 mg/ml, maximum values of 12.0 and 14.50 mm zones of inhibition respectively were observed when compared across all plant materials and extraction solvent use, which are low levels of susceptibility. The susceptibility of P. aeruginosa at this dose is, however, comparable to that of streptomycin. At doses of 100, 250 and 500 mg/ml maximum zones of inhibition recorded with respect to plant material and extraction solvents were 17.00, 17.50 and 19.00 mm respectively. Chloroform extracts of both plants seemed to have effective antimicrobial action against P. aeruginosa. Considering Klebsiella sp., a low level susceptibility to concentrations of extracts at 25 and 50 mg/ml was observed. The minimum inhibition zones recorded at 25 and 50 mg/ml were 8.50 mm and 10.50 mm while the maximum zones were 10 and 13 mm respectively. These were low level antibiosis. However, the activity of chloroform extract of the two plants were significant at 50 mg/ml against Klebsiella sp. All other concentrations of extracts irrespective of solvent material possessed significant antimicrobial activity against Klebsiela sp. Maximum zone was observed at 18.00 mm with chloroform extract of G. latifolium at 500 mg/ml.

S. pneumonia and B. cereus are Gram-positive bacteria, the least antibiosis recorded for S. pneumoniae was 9.50 mm at concentration of 25 mg/ml of G. latifolium ethyl acetate extract while the optimum recorded was 20.00 mm (G. latifolium) and 21.50 mm (Z. zanthozyloides) from chloroform extract. Highest activity of plant extract against B. cereus was found to be 17.00 mm from chloroform extract of Z. zanthoxyloide at concentration of 500 mg/ml. These results also indicates the ability of extract to inhibit the growth of Gram-positive bacteria. The susceptibility of all tested microbial isolates to the plant extracts is therefore a clear indication that plants studied would be good candidates for disinfection product development.

3.4 Antifungal activities

Susceptibility of the fungal isolates to tested plant extracts were observed. The maximum antifungal activity of the extracts recorded were in the order of 20.20 mm (Zanthoxylum chloroform extract against Trichoderma at 500 mg/ml) > 17.50 mm (Zanthoxylum chloroform extract against A. flavus at 500 mg/ml) > 17.00 mm (Gongronema chloroform extract against A. niger at 500 mg/ml) > 16.00 mm (Zanthoxylum chloroform extract against Candida sp at 500 mg/ml). Activities of extracts were significant from a concentration of 50 mg/ml to 500 mg/ml and show low to moderate antimicrobial activities (Table 2).

The overall results revealed that antimicrobial activities of the plant extracts are dose dependent with comparatively greater efficacy than commercial antibiotics at the concentration of extracts tested. E. coli was the most susceptible microbial isolate tested representing the potentials of the extracts against a group of coliform which are important indicators of microbial pollution in water. Other microbial isolates also recorded susceptibility to extracts tested to varying degrees. It was observed in this finding that microbes tested were most susceptible to chloroform extract of Z. zanthoxyloides and G. latifolium except for the activity of ethyl acetate extract of Z. zanthoxyloide against E. coli. Results of phytochemical screening revealed the varied presence of tannins, flavonoids, terpenoids, anthraquinones, alkaloids and saponins, (Table 5). The observed trends of phytochemicals of Z. zanthoxyloides in this work is similar with the earlier reports on same plant (Adesina, 1986; Chaaib, 2004; Adesina, 2005). Plant phytochemicals have been established to exhibit antimicrobial properties (Iwu et al., 1999; Banso and Adeyemo, 2006). Antimicrobial potency as reported in this work with a broad range of activity against Gram-positive and Gram-negative organisms is similar to that reported in the work of Adegbolagun and Olukemi (2010) when reporting the activity of Z. zanthxyloides against selected microbial isolates. Optimum antimicrobial inhibition reported in this work is, however, greater than that reported by Adegbolagun and Olukemi (2010) for Bacillus, P. aeruginosa and E. coli which may be attributed to the concentrations of extract used. Adesina (2005) has described the broad spectrum antibacterial activity of many species of Zanthoxylum against different microbes, although aqueous extracts was reported to be less effective against microbial sample of P. aeruginosa and E. coli (Ndukwe et al., 2005; Adebiyi et al., 2009). Extracts from Z. zanthoxyloides have previously also been documented for notable activity against bacteria associated with periodontal diseases (Taiwo et al., 1999). In the work of Olila et al. (2001), there was no antimicrobial activity reported from Zanthoxylum chalybeum against E. coli and Candida albicans tested. This was attributed to low dosage and solubility of extract in extraction solvent used (Nair and Chanda, 2006).

The doses of antimicrobial samples used in this work were found to be effective in the treatment of the microbial isolates investigated with effectiveness against all bacterial and fungal isolates. Phytochemical products with minimum inhibitory concentrations (MIC) ranging from 100 to 1000 mg mL⁻¹ are referred to as antimicrobials (Abreu et al., 2012).

The activity of G. latifolium as reported in this work is also similar to other reports on the activity of G. latifolium’s extracts to inhibit different microbes (Morebise et al., 2002; Ugochukwu and Babady, 2002; Farombi, 2003). The presence of saponins and flavonoids in G. latifolium have been attributed to its antimicrobial efficacy (Morebise and Fafunso, 1998). In a similar manner to the findings of this study, Staphylococcus sp., Pseudomonas sp., Shigella sp., K. Pneumonia, Salmonella sp., E. coli and O. anthropi were all inhibited at 100 mg/ml in Adeleye et al. (2011). The presence of essential oil was attributed to high antimicrobial efficacy against tested microbes including C. albicans. Inhibition ranged from 7.5 mm for P. aeruginosa to 11.25 mm for Shigella flexneri, respectively. However, activity of extracts were comparatively lesser than that of commercial antibiotics at the concentration tested. This suggests the inefficiency of active ingredient present at the concentration of extract tested which was not the case in this work. The extracts tested over the range of concentrations considered showed improved antimicrobial activities over all antibiotics used, mostly, with significant differences

Inhibition activities observed against bacterial and fungal cell as exerted by phytochemicals in this study may be due to the collapsing of cell walls and membranes, resulting in leakage of cell component, interruption of proton motive force, dysfunctioning of efflux pump and enzyme, leading to cytosis (Burt, 2004). Phytochemical activities of plant extract against microbes from disruption of nucleic acid synthesis, interfering with intermediate metabolism, initiation of coagulation of cytoplasmic components and disruption of normal cell communication in quorum sensing have also been reported (Sanchez et al., 2010; Chitemerere and Mukanganyama, 2014; Anandhi et al., 2014; Nazzaro et al., 2013; Radulovic et al., 2013; Mogosanu et al., 2015). A common phenomenon is that several compounds in crude plant extract act at different target sites in pathogens and contribute to optimum efficacy of plant extracts (Gupta et al., 2014). Phytochemicals may exhibit antimicrobial effect in microbes not only through direct lethal activity but also by altering key events in pathogenesis (Brijesh et al., 2009). The activity of inhibition as observed in this study can therefore be bactericidal in which the zone of inhibition experienced results from complete lethal action against the microbes or bacteriostatic in which the inhibition zones results in a state of viable but dormant situation referred to as viable but non culturable (VBNC) state. Conventional chemically synthesised antibiotics and phytobiotics significantly differ with respect to frequency in radicals and spatial structure (Koehn and Carter, 2005). The latter is with a fewer nitrogen, sulphur, halogens, phosphorus and exhibit enhanced scaffold variety, stereo chemical abundance, diversity in ring system, molecular complexity and carbohydrate contents (Schmidt et al., 2008). Also, plant products can alter or prevent protein-protein interactions, thus acting as effective modulators of immune response, apoptosis, mitosis and signal transduction (Koehn and Carter, 2005) making them suitable alternatives to synthetic antibiotics.