Antimicrobial Efficacy of Vitellaria paradoxa fractions and compounds on some wood Fungi and Bacteria

This study examined antifungal efficacy of Vitellaria paradoxa fractions and compounds in the control of some wood degrading fungi. Stem bark and heartwood parts of Vitellaria paradoxa were collected, dried, pulverised and macerated sequentially in nhexane, methanol and ethyl acetate solvents. The mixtures were filtered, evaporated and the dried samples were mixed and run over silica gel in column chromatography with a mixture of n -hexane and ethyl acetate solvents to obtain fractions. The fractions collected were evaporated and those with white needles were subjected to Magnetic Resonance (NMR) spectroscopic analysis. Spinasterol was isolated and characterised from the heartwood fraction while the stem bark fractions were fatty. Vitellaria paradoxa fractions were active against Serpula lacrymans, Sclerotium rolfsii, Aspergillus fumigatus, Fomitopsis pinicoca, Phaeolus schweinitzii, Rhizopus sp., Coniophora puteana, Gloeophyllum sepiarium, and Fibroporia vaillantii at zones of inhibition (ZOI) of 18 mm 24 mm. Although the antibiotics were active (25 – 31 mm), they were found inactive against the Fomitopsis pinicoca fungus which was sensitive to all the V. paradoxa fractions at zones of inhibition of 18 24 mm. The minimum inhibitory concentrations (MIC) of the V. paradoxa fractions were active at 50 μg/mL against all test fungi. At minimum fungicidal concentration (MFC) of between 50 200 μg/mL, all the test fungi were killed. Based on the ZOI, MIC and MFC, the V. paradoxa stem bark heartwood fractions have been proven to be very efficient in inhibiting the growth of test wood rot fungi; hence the species could be explored as a potential source of bioactive fungicides.

The species originated in African and is found in areas with 400 -1800 mm rainfall per year (IPGRI, INIA. 2006). It either grows naturally or is farmed as a tree crop in the arid Savanna region of West Africa countries (Audu & Awulu, 2017). It is believed to have spread from Senegal in West Africa to Uganda in East Africa till Adamaoua Province in Cameroon which is in North-South Africa (IPGRI, INIA. 2006). Vitellaria paradoxa grows in the wild area of dry savannah region of West Africa from Senegal in the west to Sudan in the east to the foothills of Ethiopian highlands. This species also grows in nineteen countries of Africa. Namely Burkina Faso, Ghana, Chad, Cameroon, Central African Republic, Ethiopia, Niger, Ivory Coast, Mali, Nigeria, Sierra Leone, Guinea Bissau, Togo Uganda, Zaire, Senegal, Guinea, Benin and Sudan (Warra, 2011). Vitellaria paradoxa is multipurpose agroforestry indigenous tree species that contribute immensely to the livelihood of rural communities for income generation and as source of raw material for many industries. The ripe fruits are eaten as a snack and as famine food. Shea butter, or Shea oil, is a raw material used in factories to manufacture margarine, baking fat, cocoa butter substitutes as well as different pharmaceutical and moisturising beauty products (IPGRI, INIA. 2006). Shea butter leaves are good forage for animal feeding, soil improvement (Ziblim et al., 2015). The species has the capacity to improve nutrition, increase healthcare, decrease rural poverty and aid sustainable land care (Moore, 2008).The wooden stem is resistant to termites and a good timber used for various constructing purposes.
Several authors have reported the use of Shea butter in medicine (Prescott et al., 2002;El-Mahmoud et al., 2008;Ahmed and Sani, 2013;Ahmed et al., 2012;Fodouop et al., 2015). It is used in the treatment of rheumatic and joint pains and healing of wounds, managing swellings and bruises, dermatitis and other skin (Fodouop et al., 2015). Some people have been reported to consume extract of V. paradoxa for treatment of various bacterial and fungal infections (Kalgo et al., 2019). The stem back extract possesses immunomodulatory properties which have effect on human neutrophils viability and function (Kalgo et al., 2019) and contain antioxidant constituents with antimicrobial activities (Olasunkanmi et al., 2017). Shea tree bark, if purified, could be used to produce an antiseptic agent capable of treating skin infections caused by groups of fungi (Ahmed et al., 2009). Vitellaria paradoxa has been researched as an effective medicinal plant (Prescott et al., 2002).
It has proved to be active against bacterial and fungal diseases (El-Mahmoud et al., 2008;Ahmed and Sani, 2013). The barks, leaves and roots extracts of V. paradoxa contain phytochemical constituents inhibits growth of some dermatophytes (Boyejo et al., 2019) and could be used as a potential source of antibiotic substance for a drug development (Ajijolakewu and Awarun, 2015). Vitellaria paradoxa may be used as potent sources of bioactive substances in the production of drugs against diseases caused by superficial and enteric organisms (Ajijolakewu and Awarun, 2015). The leaf, stem bark, and seed of shea tree contain a host of bioactive compounds that can be utilized in the control of infections caused by T. mentagrophyte, A. fumigatus, E. flocossum, T. rubrum, and M. Audouinii (Ahmed et al., 2012).
Wood is versatile and can be used for a very long time but it is subject to biodeterioration. Wood deterioration is a very essential process in the environment that recycles complex organic materials and it is a fundamental component of life (Blanchette, 2000). Primarily, fungi, inserts, ants and bacteria are some of the agents of deterioration. Wood decay fungi are classified into two major groups: white-and brown-rot fungi (Blanchette, 2000). White rot fungi can degrade all cell wall components, including lignin. They cause bleaching of common wood colouration, metabolize large amounts of lignin in wood is unique among microorganisms, degrade cells and reduce the strength properties of wood in the late stages of decay. On other hand, brown-rot fungi depolymerise cellulose quickly during initial stages of wood colonization. Substantial losses in wood strength start very early in the decay process, frequently before decay evidence is visually shown (Blanchette, 2000). Bacteria are known to have the ability to decompose wood cellulose although their influence on wood decay is restricted. Different bacteria from woodland soil contain enzymes used in the breakdown of cellulose and cellulose products (Llado´ et al., 2015).
Fungi and bacteria play a major role in the well being, diversity, and productivity of forest ecosystems. Fungi feed on woody products and serve to recycle nutrients. As a result, they physically and chemically break down wood products thereby causing lots of economic hazards (Marcot, 2007). Despite the many studies that have been done on the medicinal and antimicrobial properties of V. paradoxa, not much has been done on the effect of V. paradoxa activity on wood degrading fungi and bacteria. Therefore, this study was undertaken to evaluate the compounds present in the stem bark and heartwood fractions of V. paradoxa and assess their effect on selected wood decay fungi and bacteria.

Plant parts Collection and preparation
Stem bark of V. paradoxa was collected from the wild at the Federal University of Agriculture, Makurdi campus, located between longitudes 8° 21ˈ and 9° E and latitudes 7° 21ˈ and 8° N in Benue State, Nigeria, within the southern guinea savannah ecological zone (Seibert, 2007). The stem bark was air-dried during harmattan season and pulverised. The heartwood was sawn at Makurdi Head Bridge Timber Shed to collect its sawdust which was also dried to avoid being damp.
Plate 1: Stem and canopy of Vitellaria paradoxa tree at College of Animal Science, FUAM Extraction of plant materials Extraction of V. paradoxa stem bark and heartwood was done successively by dissolving 1,000 g and 600 g of stem bark and heartwood, respectively, into 1,000 mL (w/v) of n-hexane for 24 hours, followed by filtration of the n' hexane extract. The residue left from the hexane extraction was again macerated with ethyl acetate and methanol, respectively, for 24 hours each. The mixtures were filtered with Whatman No.1 filter papers into well labelled glass bottles as n -hexane, ethyl acetate and methanol extracts, respectively. The filtrates were evaporated to obtain dried extracts (Plate 1). Column Chromatography and Nuclear Magnetic Resonance (NMR) spectroscopic analysis Dried crude mixture of n-hexane and ethyl acetate extracts was run over silica gel with solvent mixtures of successive increasing polarity of hexane and ethyl acetate in the ratio of 95:5 -0:100, respectively, in column to produce pure compounds or fractions according to Ekhuemelo et al. (2018). The fractions of the pure compounds were collected in well-labelled vials and allowed to dry (Plate 2). Nuclear Magnetic Resonance (NMR) spectroscopic analysis was done on selected fractions which contained white needle-shaped crystals. NMR data was acquired on a Bruker-Avance 500MHz spectrometer. The NMR data was processed using MestreNova® 12 software. The characterization was done using the 1 H NMR data. Determination of antimicrobial activity of Vitellaria paradoxa fractions The activity of V. paradoxa fractions on test and bacteria and fungi samples was investigated using agar diffusion method. Concentrations of 200 μg/mL and 100 μg/mL were prepared from each fraction and antibiotics respectively. The antibiotics used as control were Ketoconazole, fluconazole and Fulcin for fungi; and ciprofloxacin, sparfloxacin and cefuroxime for bacteria according to Ekhuemelo et al. (2018).
The Muller-Hinton and Sabouraud Dextrose agar were used in the culture of bacteria and fungi, respectively. The incubation of test fungi was made at 30 o C for 7 days and at 37 o C for 24 hours for bacteria. During the period plates of the media were observed for inhibition of growth. Zones of inhibition were measured and results recorded in millimetres (mm).

Minimum inhibitory concentration (MIC)/ Minimum Fungicidal concentration (MFC)
The MIC of the antifungal fractions and compounds was determined by broth dilution methods. Different concentrations (200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL and 12.5 μg/mL) of fractions were added to media in the Petri dishes and inoculated with the test fungi. The mixture was incubated and examined for growth according to Ekhuemelo et al. (2018). The lowest concentration of the fraction that inhibits visible growth of the test fungi was recorded as the MIC. The MFC of the fraction was determined by subculturing the contents of the Petri dishes that showed inhibition on Muller-Hinton and Sabouraud Dextrose agar plates for bacteria and fungi, respectively, while the absence of growth on incubation was recorded as MFC.

Characterization of VPH22 as Spinasterol
Vitellaria paradoxa heartwood fraction (VPH22) was obtained as white needles. It's proton nuclear magnetic resonance spectrum ( 1 H-NMR) gave the following data (  (Cheung, and Williamson, 1969). The data acquired was reminiscent of that for sterols and triterpenes. A literature look up revealed the data to be unambiguously identical to that of spinasterol (Villaseñor and Domingo, 2008).
The zones of inhibition of V. paradoxa fractions (18 -24 mm) were at the same range of 25 mm -31 mm recorded for the three antibiotics.
Fomitopsis pinicoca fungus was resistant to all antibiotics but was sensitive to all fractions of V. paradoxa at zones of inhibition of 18 -24 mm.
These values are within the very active (>19 mm) range described by Guevara, (2005) Although ZOI recorded for V. paradoxa fractions were slightly lower than the ones for the three antibiotics, they both were very active according to Guevara, (2005) who reported ZOI greater than 19 mm to be very active. Garba and Salihu, (2011) observed that ketone (2), 1-phenyl-1,4pentanedione and ester (1) Freire et al. (2005) reported the presence of spinasterol as natural sources of bioactive sterols from the wood and bark of the Acacia species (A. longifolia. A. dealbata, A. melanoxylon and A. retinodes).
Spinasterol and a-Spinasterol have been reported by Ravikumar et al. (2010) to show antiproliferative effects that inhibit cell growth. Spinasterol has also been demonstrated to be a powerful inhibitor of glomerular mesangial cell multiplication (Ravikumar et al., 2010). Villasenor and Domingo (2000) reported result of in vivo studies that proved the efficacy of antitumorigenic activity of spinasterol to skin tumours without co-tumour or co-carcinogen promoter activities as well as possessing active antiproliferative effect on gynaecological cancer cells.
The MIC of V. paradoxa on all the test bacteria was between 25 -50 µg/mL while the MBC was between 50 -200 µg/mL. Least MIC (25 µg/mL) and MBC (50 µg/mL) were recorded for the stem bark. This implies that fractions from the stem bark were more potent on the test bacteria than on the fractions from the heartwood. Kuete et al. (2009)  This study revealed that V. paradoxa heartwood fractions possess spinasterol. The stem bark was more active on wood fungi compared to the heartwood fraction. This agrees with Boyejo et al. (2019) who reported that stem bark extract of V. paradoxa was most active compared to the leaves and roots extracts. The presence of spinasterol in the heartwood could be due to the deposition and accumulation of extractives in the heartwood of the tree over a period of time.

Conclusion
Spinasterol compound was isolated from the heartwood of Vitellaria paradoxa. All fractions exhibited high antifungal and antibacterial activity as it controlled all test wood fungi and bacteria.
Although Fomitopsis pinicoca fungus and Pseudomonas aeruginosa bacteria were resistant to all antibiotics, they were sensitive to fractions of V. paradoxa probably because of its spinasterol compound which is a natural source of bioactive sterols. The stem bark was more active on wood fungi compared to the heartwood fraction. The results of ZOI, MIC and MFC showed that V. paradoxa stem bark heartwood fractions were very efficient in inhibiting the growth of test wood rot fungi and wood colonising bacteria; hence the species should be explored as a potential source of bioactive fungicides.