Shea nut sample was obtained from various identified locations within the natural ecological regions of Shea tree within Ilorin metropolis, Kwara state Nigeria.

Fungal isolates, C. albican ATCC 14503, T. rubrum ATCC 28188 and T. mentagrophytes ATCC 9533 were obtained from American Typed Cultured Center (ATCC) United State of America. S. aureus ATCC 25925 was obtained from Microbiology Department, University of Ilorin, Nigeria.

Extract from nut of Shea plant was obtained from solvent extraction procedure as described by Ajala et al. 14 using a Soxhlet apparatus with n-Hexane, Methanol and Petroleum ether as the solvents and rotary evaporator was used to concentrate the filtrate.

Proximate analysis was carried out using a previously reported method 15. The moisture content was determined by drying five grams of ground samples of were weighed into a sterile aluminum dish, weight of the dish and weight of un-dried sample (in duplicate) were taken. This was transferred into an oven at 100°C for 3 hours respectively. Ash content determination was carried out by placing two grams of the sample placed in a muffle furnace which is maintained at 550 °C for 5 hours, while crude fibre content was estimated by consecutive acid and alkali digestion followed by washing, drying, ashing at 550 °C. Crude fat was obtained by exhaustively extracting about 30 grams of the sample in a Soxhlet apparatus using n-hexane solvent. Crude protein (% total Nitrogen 6.25) was determined by Kjeldahl method. The carbohydrate content was determined by difference.

For phytochemical analysis of shea nut, the different phytoconstituents present in the methanolic extracts of nut was screened qualitatively 14.

Agar well technique was employed in the assay of the extract against S. aureus and C. albican. Standardized isolates were spread on sterile Mueller Hinton and Sabouraud dextrose agar plates for growth of bacterial and fungal isolates respectively. Wells were made into the agar plates using sterile corkborer of 8.0 mm in diameter. The methanolic extract of 200 mg/ml concentration was introduced into the wells for bacterial isolates, plates were left for about one hour for diffusion to occur before incubating at 37^oC for 24 hours. For T. rubrum and T. mentagrophyte, agar plugs of uniform size (diameter, 8 mm) were picked using a sterilized corkborer from the Petri dish having full growth isolates and inoculated at the middle of every Petri dish containing 200 mg/ml kept at room temperature for 14 days. After incubation, radial growth of fungal mycelium was observed was measured and recorded accordingly. Control used includes standard antibiotics disk and ketoconazole as positive control, while the negative control was 4% dimethyl sulfur oxide (DMSO) used as diluent. The diameter of zones of clearance around the wells and disks were measured to show the degree to which the test organisms are susceptible to the sample 15.

The minimum inhibitory concentration (MIC) of the methanolic extract against test organisms was determined using the modified method of Ahmed et al. 15. Extract (1ml) at concentrations of 350, 300, 250, 200, 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 mg/ml was added each to 1 ml of sterile nutrient broth in different test tubes respectively. Eighteen hours culture (50 µl) was adjusted to 0.5 MacFarland standard with approximate cell number of 1.0 x 10⁸cfu/ml and inoculated in each test tube. The tubes were incubated at 37°C for 24 hours in the case of S. aureus ATCC 25925. For dermatophytes, spore suspension of each of the isolate was collected into sterile saline water as described by Ahmed et al. 15 and 1 ml of extract at concentrations of 350, 300, 250, 200, 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 mg/ml was added separately to 1 ml of sterile Sabouraud dextrose broth in different test tubes respectively. Spore suspension (50 μl) was inoculated into each of the tubes and incubated at room temperature for 14 days. Control tubes included reference antibiotics, growth medium and test isolates while a blank was set containing only sterile broth. The tube with the lowest concentration of the extracts (highest dilution) which had no detectable bacterial and fungal growth when compared with the control tube was considered the MIC.

Gas Chromatography-Mass Spectrometry was done as described by Khan and Bhadauria 16 using SHIMADZU QP2014 ULTRA apparatus operated in EI mode at 70ev. A Restek-5MS column (30 m × 0.25 mm × 0.25 μm) was employed in the determination of bioactive compounds present in the extract. The temperature of oven was projected at 60 °C raised to 280 °C at 5 °C min-1 and held for 2 min, then 250 °C to 280 °C and held for 14 min. The injector temperature was 290 °C with normal injection mode. The flow rate of carrier gas, helium was 1.00 ml min-1. Total running time for GC was 30 min. The compounds were identified by comparing the mass spectra data with data stored in GC-MS library.

The result of proximate analysis of shea nut subjected to varying treatment (dry and roasted, dried and freshly crushed) is presented in Table 1. Moisture content was seen to be highest in fresh nuts (19.56 ± 0.03) and lowest in the dried and roasted nuts (6.8 ± 0.01). This is in line with report by Ukpanukpong et al. (2016) that moisture content of shea nut was within the range of 6.33 ± 3.21 to 6.33 ± 3.21. It was obvious from the result that drying as a form of treatment reduced the moisture content of the nuts s compared to the fresh nuts. This also suggests that drying with roasting is an efficient treatment capable of preserving the nuts from microbial invasion for a longer time without spoilage. This is because higher moisture content results in increased microbial activity, since microorganism require moisture as a growth condition. Ash content was highest in dried and roasted nuts (4.93 ± 0.01) and lowest in freshly crushed nuts (3.72 ± 0.01). From literature, ash content connotes availability of minerals in a sample 17, 18. This information suggests that the dried and roasted nuts may be higher in mineral content than the other treatment method which exhibited lower ash contents. The ash content values in this study is in line with Abdul-Mumeen et al. 19 who reported ash content ranging from 4.16 to 4.44 in the analysis of shea nut cakes. The fact that higher mineral composition of sample is a function of ash content was further confirmed by Abdul-Mumeen et al. 19 where the shea nut sample with higher ash content contained more number of minerals than there were in the ones with lower ash content. From literature, shea butter comprises of a vast array of fatty acids and according to Maanikuu and Peker 20 the presence of relatively high amount of saturated fatty acids in shea butter gives it anti-inflammatory property which in turn confers on it great healing potential. The oil-soluble components of the fats in shea butter do not undergo saponification on contact with alkali. From this explanation, it is imperative to state that the exposure of extract from the shea nuts may be inhibitory against pathogens.

In this present study, five classes of phytochemicals were observed as follows; Saponins, tannins, alkaloids, terpenoids and reducing sugars (Table 2 ). From literatures, the medicinal effect of plants is as a result of certain chemical substances which possess bioactive uses. Saponins, known to possess detergent-like properties which may disrupt microbial cell membrane thus rendering them permeable to harmful substances. The antimicrobial activity of tannins is explained by their ability to penetrate bacterial cell wall thereby interfering with metabolism 21. Terpenes has been reported to be able to inhibit crucial processes in microbial survivability 22. Generally, the mechanism of action of most terpenes remains largely unknown. However, a study by Griffin et al. 23 reported that terpenes able to disrupt oxygen uptake as well as oxidative phosphorylation in microbial cells. The presence of reducing sugars can cause bacterial cells to lose water by osmosis which could result in cell lysis. From literature, alkaloids exert their inhibitory effects by various mechanisms, some of which are: disruption of virulent genes, inhibition of microbial destructive enzymes, inhibition of biofilm formation etc. 24.

The antimicrobial assay carried out of different extracts obtained from the solvent extraction processed revealed that only methanolic fraction showed activities against the test pathogens and is presented in Table 3. Methanolic extract inhibited both S. aureus (25.0mm) and C. albican (29.0mm) with 24 hours of exposure with the highest susceptibility by C. albican. The closeness in susceptibility demonstrated by S. aureus and C. albican may be due to the fact that C. albican are yeast and hence act like vegetative form of bacteria such as S. aureus. From the antifungal assay, no radial growth was observed at all for the first 10 days, owing to the growth pattern of T. rubrum and T. mentagrophyte. However, only T. rubrum exhibited total susceptibility towards the extract with no radial growth observed after 14 days indicating 100% growth inhibition of the isolate. T. mentagrophyte resisted the extract by demonstrating increased radial growth as the time incubation time increased. The resistance exhibited by T. mentagrophyte in this present study is in accordance with findings by Taghipour et al. 25 indicating that T. mentagrophyte and T. interdigitile demonstrated emergent antifungal drug resistance and global spread of such resistance mechanisms.

Minimum inhibitory concentration of methanolic extract against S. aureus was observed at 150g/ml (Table 4 ) and compared favourably with standard antibiotics, as S. aureus resisted all conventional antibiotics used except meropenem (Table 5 ). The effectiveness of meropenem on this isolate further confirm that meropenem is one of the strongest antibiotics in the treatment of infections caused by S. aureus. Several researchers have also reported the sensitivity of multidrug resistant S. aureus to meropenem 26. The MIC assay with C. albican revealed that the extract exhibited the lowest MIC value (100mg/ml) in comparison to all other test antifungals with lowest MIC of 150mg/ml (Table 6 ). The MIC assay against T. rubrum and T. mentagrophyte in comparison to conventional antifungals revealed that Itraconazole among other antifungals exhibited the lowest MIC (25mg/ml) value against both fungi (Table 7 ). However, MIC for the extract was recorded at 150mg/ml against T. rubrum only. T. mentagrophyte did not indicate any MIC effect, confirming its resistant nature as suggested by Taghipour et al. 25.

The chemical compounds detected in the methanolic extract are presented in Table 8. Some of the compound reported to possess antimicrobial activities include E- 15 Heptadecenal possess antifungi activity 27, Methyl palmitate possess anti-inflammatory 28, Palmitic acid possesses both antifungal and anti-inflammatory activities 29, Oleic acid possess anti-inflammatory activity 30. 10- Nonadecenoic acid 31, cis-13-Eicosenoic acid 32, 2-Monoolein 33.

Table 4

Concentration (mg/ml)	S. aureus 25923
Control (0)	+
1.56	+
3.125	+
6.25	+
12.5	+
25	+
50	+
75	+
100	+
150	–
200	–

Key: + = Turbid growth; – = No growth

Table 5

Antibiotic / Extract	Conc	ZOI (mm)	CLSI breakpoints
			R	I	S
Amoxicillin	10µg	0.00	≤25	28-36	≥37
Tetracyclin	30µg	0.00	≤24	24-30	≥30
Cotrimoxazole	30µg	10.50	≤12	13–14	≥15
Gentamacin	10µg	9.50	≤12	13-14	≥15
Cefuroxine	30µg	0.00	≤27	27-35	≥35
Cefazolin	30µg	0.00	≤29	29-35	≥35
Ceftriaxone	30µg	0.00	≤27	27-35	≥35
Cefotaxine	30µg	13.50	≤25	25-31	≥31
Ciprofloxacin	5µg	0.00	≤15	16–20	≥ 21
Meropenem	10µg	25.00	≤29	29-37	≥37
Amikacin	30µg	0.00	≤17	15-16	≤14
Levofloxacin	5µg	0.00	≤15	16-18	≥19
Vancomycin	30µg	15.00	-	-	-
Methanolic extract	75 mg/ml	12.0 ± 0.71
	100 mg/ml	13.5 ± 0.35
	150 mg/ml	18.0 ± 2.83
	200 mg/ml	25.0 ± 0.71

Conc- Concentration; ZOI- Zone of Inhibition

Table 7

Concentration (mg/ml)	T. rubrum				T. mentagrophyte
	M	G	I	F	M	G	I	F
Control (0)	+	+	+	+	+	+	+	+
1.56	+	+	+	+	+	+	+	+
3.125	+	+	+	+	+	+	+	+
6.25	+	+	+	+	+	+	+	+
12.5	+	+	+	+	+	+	+	+
25	+	+	–	+	+	+	–	+
50	+	+	–	+	+	+	–	+
75	+	+	–	+	+	+	–	+
100	+	+	–	–	+	+	–	+
150	–	–	–	–	+	+	–	+
200	–	–	–	–	+	+	–	–
250	–	–	–	–	+	+	–	–
300	–	–	–	–	+	+	–	–
350	–	–	–	–	+	–	–	–

Key: M = Methanolic extract; G = Griseofluvin; I = Itraconazole; F = Fluconazole; + = Aerial growth; – = No aerial growth

Table 8

RT	Compound name	Methanolic extract
30.98	E- 15 Heptadecenal	1.08
38.39	Methyl palmitate	10.02
39.02	Palmitic acid	10.78
39.71	cis-Oleic Acid	19.96
41.23	10-Nonadecenoic acid, methyl ester	0.64
41.32	cis-13-Eicosenoic acid	9.42
41.44	Eicosanoic acid	16.29
41.79	Dasycarpidan-1-methanol, acetate 2 (Octadecyloxy) ethyldocosanoic	3.51
41.91	acid ester	0.86
42.39	Docosanoic acid	2.68
43.04	N-Butylcinnamamide	2.20
43.49	2-Monoolein	22.55

The authors will like to acknowledge the contributions of Dr. Atolani from the Department of Chemistry and the laboratory technologists from the Faculty of Pharmaceutical Sciences, University of Ilorin, Nigeria.