Antimicrobial activity of the crude extracts of Parkia biglobosa ( Jacq ) seeds on selected clinical isolates

Parkia biglobosa (Jacq) is a wild leguminous plant found in North-Central zone of Nigeria with high calorific value, essential proteins, fatty acids, and vitamins. The study investigated the antimicrobial activity of crude extracts of fermented and unfermented P. biglobosa seeds on selected clinical microbial isolates namely, Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. P. biglobosa seeds were obtained from Oja-Oba market in Ilorin, Kwara State, Nigeria. The samples were pre-treated and pulverized into powder. The extraction was achieved with acetone and water and qualitative phytochemical analysis was performed following standard procedures. The antimicrobial activity of the extracts against the isolates was determined by agar well diffusion method. Qualitative phytochemical screening of the crude extracts showed the presence of tannins, alkaloid, flavonoid, saponin and glycosides. P. aeruginosa was sensitive to the aqueous extract of fermented seeds having a zone of inhibition of 14.00±1.00mm while for unfermented seeds it was 10.00±2.00 mm at 100 mg/ml. The acetone extracts of both fermented and unfermented seeds revealed antibacterial activity against P. aeruginosa with zone of inhibition of 17.00±3.00 mm and 18.00±0.00 mm respectively. In conclusion, the crude extracts of the fermented and unfermented P. biglobosa seeds at a concentration of 75 and 100 mg/ml respectively have antimicrobial effect on the clinical isolates.


INTRODUCTION
Parkia biglobosa also known as African locust beans is a dicotyledonous plant in the family Fabaceae -Mimosoideae. It is classified as a vascular plant (Thiombiano et al., 2012). African locust bean is a leguminous crop commonly grown in the tropics. It is very prominent in West Africa and can be found in the Northern and South -Western regions of Nigeria (Tee et al., 2009). Its height ranges between 7 and 20 metres (Fern, 2019). P. biglobosa is a leguminous plant commonly known for their readily available protein content, high calorific value, essential amino acids and fatty acids content, vitamin, and fiber. Fermented seeds are highly nutritional with several health benefits (Oloyede and Akintunde, 2019). However, the presence of anti-nutrients in the seeds has limited their use (Bhat and Karim, 2009). P. biglobosa plants are rich sources of phytochemicals, wood, fuel and gum and the seeds have been investigated for their protein and amino acid contents as reported by Ajaiyeoba (2002). The tree has a thick dark brown bark and it is fire-resistant while the pods which house the seeds are usually 30-45 centimeters long on average and are dark brown when mature (Janick, 2008;Abioye et al., 2013). The African locust bean is prepared locally by subjecting the seeds to natural fermentation of the boiled and de-hulled cotyledon. The different duration of fermentation of the seeds makes them edible by increasing their digestibility (Simonya, 2012). Fermentation involved in the preparation of the seeds promotes the desired nutritional value accentuates the organoleptic properties such as taste, flavour and texture (Akin-Osanaiye and Musa, 2017). The seeds are widely used in Africa and usually the fermented forms are processed to condiments such as, iru, dawadawa, afitin, soumbala, netetu and sonru with high nutritive value (Zannou et al., 2018). The fermented seeds of P. biglobosa are used as condiments in preparing local soups in Nigeria and it is known as 'iru', 'origili' and 'dawadawa' among the Yorubas, Ibos and Hausas respectively (Dosumu et al., 2012;Ojewunmi et al., 2016). In traditional medicine, seeds of P. biglobosa are used for the management and treatment of some infectious diseases; they are used as antimalarial and anti-bacterial agents (Balogun et al., 2018). However, there is limited scientific investigation on the antibacterial activity, although there are reports that the main microorganisms involved in the fermentation process also help to inhibit harmful bacteria such as Bacillus cereus, Staphylococcus aureus and Escherichia coli (Ouoba et al., 2005). Microorganisms such as S. aureus, E. coli, Pseudomonas aeruginosa amongst others are becoming increasingly resistant to antimicrobial agents. Antibiotic resistance has been reported to occur when a drug loses its ability to inhibit bacterial growth effectively and this is increasingly becoming a global concern (O' Neil, 2016). Bacteria which have become 'resistant' continue to multiply in the presence of therapeutic levels of the antibiotics. Antibiotics which are usually effective against these microbes become less effective or the microbes become resistant as such, requiring a higher than the normal concentration of the same drug to have an effect. The emergence of antimicrobial resistance was observed shortly after the introduction of new antimicrobial compounds (Levy, 2017).
Also, since antimicrobials are not fully degraded in human and animal body; antimicrobial compounds, their metabolites and transformation products are abundant in the environment (Segura et al., 2009;Michael et al., 2013). Consequently, this leads to the selection of antimicrobial resistant bacteria or the acquisition of resistance genes by horizontal gene transfer (Martinez, 2009). Emergence of less effective antibiotics lead to the risks of many treatments failures (O'Neill, 2016), resulting in the need for an alternative therapy. Therefore, the aim of this study is to determine phytochemical constituents and the antimicrobial activity of crude extracts of fermented and unfermented P. biglobosa seeds on selected clinical isolates.

Sample Collection and Preparation
Fresh samples of locust bean seeds (fermented and unfermented) were purchased from Oja-Oba market in Ilorin, Kwara State and placed in a clean plastic container during transportation to the laboratory. The fermented locust bean seeds were cleaned by hand picking of the dirt particles and other physical contaminants and was dried in an oven (DHG 9202) at 40 0 C for 3 days. However, the unfermented seeds were first soaked in water and thoroughly rinsed, afterwards boiled in order to enhance the removal of hull from the cotyledons. The cotyledons obtained were then dried in an oven at 40 0 C for 3 days. Both samples were then grounded with Flourish blender (FL1039) and kept in an airtight container prior to analysis.

Sample Extraction
Sterile warm water (40 0 C) and acetone were used to obtain extracts from the fermented and unfermented locust bean seeds. Seed extracts were obtained by maceration; 125 grams of the sample was dissolved in 1000 ml of water and was left to stand for 24 hours. The extracts were then filtered using muslin cloth and the aqueous filtrates was evaporated to dryness using a water bath at a temperature of 40 0 C for 3 hours while the filtrates obtained using acetone was dried in a fume cupboard. The crude warm water and acetone extracts of the seeds were then subjected to phytochemical and antimicrobial analysis (Oluwaniyi and Bazambo, 2014).

Test Microorganisms
The test microorganisms were clinical isolates obtained from University of Ilorin Teaching Hospital, Oke-Oyi, Ilorin, Kwara State. Bacteria obtained were Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa and fungus obtained was Candida albicans. The organisms were reconfirmed by sub-culturing and identification according to the standard methods of Cowan and Steel (l993).

Standardization of Inocula
McFarland standard was prepared by introducing 0.05 ml of 1% BaCl in 9.95 ml of 1% H2SO4, the solution was then shaken well (CLSI, 2009). The inocula were standardized by introducing 10 ml of nutrient broth into sterile test tubes which were then inoculated with the test organisms. Afterwards, the turbidity of the media containing the isolates were then adjusted by adding sterile water to match that of the McFarland standard which is equivalent to 1.5 X 10 8 CFU/ml.

Qualitative Phytochemical Screening of Crude Extracts
Alkaloids: Two grams of each extracts were heated on a boiling water bath with 2% Hydrochloric acid (50 ml), cooled, filtered, and treated with Mayer's reagent (5 drops). The samples were then observed for the presence of yellow precipitation or turbidity (Tyler, 1994). Flavonoids: Two ml of 50% methanol was added to 4 ml of the crude extracts at a concentration of 100 mg/ml. After warming, magnesium fillings followed by few drops of concentrated hydrochloric acid were then added. A pink/red colour indicates the presence of flavonoid (Tyler, 1994). Tannins: A portion of each extract was diluted with distilled water in a ratio of 1:4 and few drops of 10% ferric chloride solution were added. A blue/green color indicates the presence of tannins (Ahmed et al., 2013). Saponins: A small quantity (0.5g) of each extract was boiled with sterile distilled water (3 ml). The mixture was filtered, and 2.5 ml of the filtrate was added to 10 ml of the distilled water in a test tube, shake well for about 30 seconds and observed for frothing (Sofowora, 2008). Glycosides: to 2 ml of each crude extract (at a concentration of 100mg/ml in methanolic solution), Fehlings reagent was added and boiled for two minutes. A brick red coloration indicates the presence of glycosides (Ahmed et al., 2013).

Antibacterial Activity of P. biglobosa Seed Extract against Selected Clinical Microbial isolates
The agar well diffusion method as described by Irobi et al. (1996) and Okeke et al. (2001) was used for the antibacterial test. A pure culture of each test organism was grown in nutrient broth for 18 hours at 37 0 C. The broth culture was then standardized to match McFarland turbidity standard which was approximately 1.5 x l0 8 cfu/ml. One ml of inoculum was then used to seed 20 ml of cooled molten Mueller Hinton agar (MHA) medium in Petri dishes. A sterile cork borer (6mm in diameter) was used to dig wells equidistant from each other on the surface of the solidified MHA medium and 0.1ml of each extract of fermented and unfermented seeds at a concentration of 25, 50, 75 and 100 mg/ml was delivered into the wells. A control, 0.1ml of sterile distilled water and acetone were used. Antibiotics (streptomycin, ciprofloxacin and rocephin) and antifungal (sporanox) were used as positive controls. Duplicates of each plate were made and the plates were then incubated at 37 0 C for 24 hours after which zones of inhibition detected were measured using a meter rule, the efficiency of the extracts is directly related to the level of clearance (Dairo and Adanlowo, 2010)

Antifungal Activity of P. biglobosa Seed Extract against Selected clinical microbial isolates
The methods of Perez et al. (1990); Murugesan et al. (2011) with a little modification was employed in the test. Sterile Mueller Hinton agar was aseptically poured into sterile Petri-dishes and allowed to solidify properly. The fungal broth culture was then standardized to match McFarland turbidity standard which was approximately 1.5 x l0 8 cfu/ml. Approximately 0.1 ml was spread on the surface of the agar using sterile spreader. Wells, 6 mm in diameter, were made with a sterilized cork borer and different concentrations (25, 50, 75 and 100mg/ml) of the extracts, sterile distilled water and acetone as controls were dispensed, incubation was performed at 27 0 C for 48 hours. Antifungal activity was determined by measuring the zones of inhibition produced by the extracts and sporanox was used as the positive control. Tests were conducted in duplicate to ascertain the results obtained.

Antifungal Sensitivity Test
Antifungal susceptibility test was performed on the fungal isolate using sporanox ® (100 mg), which was adjusted to a concentration of 25 mg/ml. Sterile cork borer was used to bore two holes equidistance from each other on sterile solidified MHA plate containing the fungal isolate. Two drops of the adjusted concentration were then introduced into each well and the zone of inhibition obtained afterwards was measured.

Determination of Minimum Inhibitory Concentration (MIC) of the clinical isolates
The MIC of each extracts was determined as follows; the plant extracts used were aqueous and acetone extracts of the fermented and unfermented seed, 1 ml of each of the different concentrations-100 mg/ml, 75 mg/ml, 50 mg/ml, and 25 mg/ml was introduced into 1 ml of sterile nutrient broth tubes containing the test organisms, the tubes were then incubated at 37 0 C for bacterial isolates and 28 0 C for fungal isolate for 24 and 48 hours respectively. Tubes without plant extracts were used as positive control (Akintobi et al., 2016a).

Determination of Minimum Bactericidal and Fungicidal Concentration of the clinical isolates
Inoculum from the tubes showing no turbidity was introduced on a sterile nutrient agar and Sabourand dextrose agar for bacteria and fungi, respectively. The least concentration that produced no growth on the medium was taken as the minimum bactericidal concentration for the bacterial isolates and minimum fungicidal concentration for the fungal isolate (Ibekwe and Ezeji, 2011).

Data Analysis
Data were expressed as mean ± standard error of mean (SEM). Statistical analysis was performed using IBM SPSS (V21). The data were analyzed using one-way analysis of variance followed by LSD post-hoc test and independent samples T-test for comparison of means. P < 0.05 was considered significant.

Phytochemical Analysis of Extracts
Phytochemical analysis of the crude extracts shows the presence of tannins, alkaloid, flavonoid, saponin and glycosides in the acetone and aqueous extract of the fermented P. biglobosa seeds; alkaloid and tannin were present in the acetone extract of unfermented seeds and alkaloid, tannin, saponin and glycosides in aqueous extract of unfermented seeds. The result obtained from the qualitative phytochemical analysis of the extracts of both fermented and unfermented seeds of P. biglobosa is shown in Table 1. Key: + , present; -, absent; AFS, acetone extract of fermented seeds; AUFS, acetone extract of unfermented seeds; AQFS, aqueous extract of fermented seeds; AQUFS, aqueous extract of unfermented seeds

Antimicrobial Activity of Aqueous Extract of P. biglobosa Seeds against Selected Clinical Isolates
As shown in Table 2, the mean zone of inhibition obtained for the aqueous extract of fermented P. biglobosa seeds against C. albicans was highest at 100 mg/ml with a value of 11.50±1.50 mm and lowest at 25 mg/ml with a value of 0.00 mm; for P. aeruginosa, the highest zone observed at 100mg/ml was 14.00±1.00 mm and least zone at 25 mg/ml was 6.00±1.00 mm. Similar result was observed with E. coli; highest zone of 13.00±1.00 mm at 100 mg/ml and 6.50 ± 050 mm for S. aureus, 11.00±1.00 mm at 100 mg/ml and 0.00 mm at 25 mg/ml. The mean zone of inhibition obtained for the aqueous extract of unfermented P. biglobosa seeds against C. albicans was highest at 100 mg/ml with a value of 11.00±1.00 mm Similar result was observed with E. coli and S. aureus with the highest zone of 11.00±1.00 mm at 100mg/ml as shown in Table 3.  The result obtained for the fermented crude acetone extract of P. biglobosa seeds revealed that the mean zone of inhibition obtained with C. albicans was highest at 100 mg/ml with a value of 13.50±1.50 mm. For P. aeruginosa, the highest zone of inhibition was 17.00±3.00 at 100 mg/ml and for E. coli, the highest zone of inhibition was 15.00±1.00 mm at 100 mg/ml as shown in Table 4. For the unfermented crude acetone extract, the mean zone of inhibition obtained with C. albicans was highest at 100 mg/ml with a value of 13.00±1.00 mm for P. aeruginosa, the highest zone of inhibition was 18.00±0.00 mm at 100 mg/ml, for S. aureus, the highest zone of inhibition was 10.50±0.50 mm at 100 mg/ml as shown in Table 5. Values are expressed as mean ± SEM of duplicate readings of the zone of inhibition obtained for each isolate. Values with the same superscript in a row are statistically significant at P < 0.05 The Minimum Inhibitory Concentration (MIC) obtained for the fermented aqueous extract for S. aureus and C. albicans was 75 mg/ml while the MIC for P. aeruginosa and E. coli was 50 mg/ml. The same result was obtained for the MIC of the unfermented aqueous extract as shown in Table 6. The Minimum Inhibitory Concentration (MIC) obtained for the fermented acetone extract with S. aureus was 100 mg/ml and 75 mg/ml for C. albicans, E. coli and P. aeruginosa. However, the MIC obtained for the unfermented acetone extract of P. biglobosa seeds with S. aureus was 75mg/ml and 50mg/ml for C. albicans, E. coli and P. aeruginosa as shown in Table 6.

Minimum Bactericidal Concentration and Minimum Fungicidal Concentration of Crude Extracts of P. biglobosa Seeds against Selected Clinical Microbial Isolates
The minimum bactericidal concentration (MBC) of the aqueous and acetone extracts of fermented and unfermented P. biglobosa seeds against S. aureus was 100 mg/ml, 75 mg/ml and 50 mg/ml respectively while for E. coli, the MBC obtained using aqueous extract of fermented and unfermented seeds was 50mg/ml and 75mg/ml respectively while that of the acetone extract of fermented and unfermented seeds was 50mg/ml for both extracts. The Minimum Fungicidal Concentration (MFC) obtained with C. albicans using aqueous extract of fermented seeds, aqueous extract of unfermented seed and acetone extract of unfermented seeds was 75mg/ml while for the acetone extract of fermented seeds, the MFC was 50mg/ml, as shown in Table 7. Key: AQFS, aqueous extract of fermented seeds; AQUFS, aqueous extract of unfermented seeds; AFS, acetone extract of fermented seeds; AUFS, acetone extract of unfermented seeds.

Antibiotic and Antifungal Sensitivity Testing against Selected Clinical Isolates
The result obtained from antibiotic sensitivity test performed using commercial antibiotic disc is shown in the  Antimicrobial activity of the acetone extract of the fermented P. biglobosa seed showed no significant difference in the mean zone of inhibition obtained with S. aureus at all concentrations; for P. aeruginosa, a significant difference was only observed with the least concentration and the highest concentration. However, increasing concentration tend to produce a slight increase in the mean zone of inhibition obtained with C. albicans and E. coli. This tally with the work of Dosumu et al. (2012) who reported that the methanolic extract of P. biglobosa seeds had inhibitory effect on the test organisms. The unfermented seeds also showed inhibitory effect on the test organisms but with significant effect on P. aeruginosa, C. albicans and S. aureus. However, for E. coli, there was only a slight significant difference in the mean zone of inhibition with increasing concentration. This shows that the extract contains some active phytochemicals which when purified could serve as a potential drug to combat these highly resistant microorganisms. This agrees with the work of Ajaiyeoba (2002), who reported that the aqueous extract of the leaves of the same plant had inhibitory effect on the test organisms.
Analysis of the bactericidal and fungicidal activity of the extracts revealed that the acetone extract of fermented P. biglobosa seeds was bactericidal on all the organisms except S. aureus and P. aeruginosa. Also, the aqueous extract of the unfermented seeds was not bactericidal on P. aeruginosa. This may be due to the highly resistant nature of these organisms or probably because the concentration was not high enough to kill them. The aqueous extract of fermented seeds and the acetone extract of unfermented P. biglobosa seeds were bactericidal on all the test organisms. Candida albicans was susceptible to the commercial antifungal while the antibiotics were effective against both gram positive and Gram-negative bacteria as observed also by Akintobi et al. (2016b).

CONCLUSION
The crude extracts of both the fermented and unfermented seeds of P. biglobosa showed antimicrobial activity on the selected clinical isolates. The aqueous extract of fermented seeds had more inhibitory effect on E. coli and C. albicans while the acetone extract had pronounced inhibitory effect on P. aeruginosa and S. aureus. The result also shows that water was a better extracting solvent than acetone as it had a significant inhibitory effect on all the organisms than acetone extracts. The study therefore confirms the folklore use of the seeds of P. biglobosa for the treatment of infections that could arise from these microorganisms.