Antimicrobial effect of isolated compound of Anadelphia afzeliana (Rendle) Stapf on selected wood fungi and bacteria in Makurdi, Nigeria

Anadelphia afzeliana was assessed for antimicrobial activity as an alternative to synthetic chemicals. A. afzeliana was collected from Orkar village, Gboko, Benue State. The specimen was pulverized. Methanol, ethyl acetate, and n-hexane solvents were used for extraction. Each solvent measured 1800 ml was added to 300 g of A. afzeliana. The mixture was left to soak for 48 hours. Extract was filtered and allowed to dry by evaporation. Dried extract was mixed with silica gel to run column chromatography. Compounds were analysed by Nuclear magnetic resonance (NMR). Concentrations of 200, 100, 50 and 12.5 μg/m were constituted for antimicrobial sensitive test on ten wood bacteria and nine fungi. NMR showed the presence of stigmasterol (C H O). Antifungal 29 48 test revealed A. fumigatus, C. puteana, G. sepiarium, P. schweinitzii, Rhizopus spp. and S. rolfsii as sensitive to stigmasterol at zones of inhibition (ZOI) of 19, 21, 22, 23, 24 and 20 mm, respectively. Antibacterial showed A. proteobacteria, B. subtilis, B. proteobacteria, E. faecium, E. coli, P. aeruginosa and P. mirabilis sensitive at ZOI of 30, 28, 29, 26, 27, 23, and 23 mm, respectively. Minimum inhibition concentration (MIC) of 25 μg/ml completely inhibited Alpha proteobacteria, Bacillus subtilis and Beta proteobacteria while Minimum Bactericidal Concentration (MBC) of 50 μg/ml completely killed A. proteobacteria, B. subtilis, and B. proteobacteria. MIC of stigmasterol at 50 μg/ml completely inhibited Coniophora puteana, Gloeophyllum sepiarium, Phaeolus schweinitzii, Rhizopus spp. and Sclerotium rolfsii while at 100 μg/ml of Minimum fungicidal concentration (MFC), Phaeolus schweinitzii and Rhizopus spp. were completely killed. A. afzeliana proved potent in the control of wood fungi and bacteria. MFC and MBC for tested pathogens were observed to be most effective at 50 μg/ml and is therefore recommended as concentration for A. afzeliana compound in the control of fungi and bacteria infections. Keyword: Anadelphia afzeliana, bacteria, fungi, stigmasterol, sensitive, resistant. *Corresponding author: davidekhuemelo@gmail.com Department of Forest Production and Products, Federal University of Agriculture Makurdi, Nigeria. Department of Chemistry, Federal University of Agriculture Makurdi, Nigeria. Introduction According to Jerrold (2005), wood is recognized Wood is described a permeable and as a multipurpose material with varied of tough basic tissue located in the roots and stem mechanical and physical properties. It is a of trees as well as woody plants. Wood is organic natural resource that is renewable with an in nature which is composed of complex cellulose excellent strength that can be used for a very and hemicellulose entrenched in an atmosphere long time. Although wood is proven to be very of lignin that prevents its compression. u s e f u l n e s s , i t s s e r v i c e l i f e 108 Nig. J. Biotech. Vol. 35 No. 2 (2018): 108-120 ISSN: 0189 1731 Available online at


Introduction
According to Jerrold (2005), wood is recognized Wood is described a permeable and as a multipurpose material with varied of tough basic tissue located in the roots and stem mechanical and physical properties.It is a of trees as well as woody plants.Wood is organic natural resource that is renewable with an in nature which is composed of complex cellulose excellent strength that can be used for a very and hemicellulose entrenched in an atmosphere long time.Although wood is proven to be very of lignin that prevents its compression.
u s e f u l n e s s , i t s s e r v i c e l i f e can be degraded by various bio-deteriorating et al., 2007).agents such as bacterial and fungal infection, Researchers are now seeking for termites, insects, marine borer, fire attack and alternative non-hazardous and biological mechanical failure that contribute to its limited methods of control of fungi and bacteria related selectivity (Areo, 2002).
diseases (Arldo et al.,2009;Femi-Ola et al., Fungi are eukaryote, they digest food 2008).Over a thousand plants species have outwardly and injects the nutrient straight across been stated to have chemicals in leaves, stems, its cell walls.Majority of fungi replicate by spores flowers, seeds, and roots which have insecticidal and have a body (thallus) that if comprised of properties.However, only a few of them have microscopic tubular cells referred to as hyphae.
been explored for practical disease control on a Fungi are heterotrophic in nature and they commercial scale.Biological control agents are derived the energy and carbon from other potent, biodegradable, environment friendly, organisms like animals (Carris et al., 2012).
economically worthwhile, and socially tolerable are method of pest management and disease pathogens or parasites of other organisms and control.Biological control method is encouraged decomposers.Fungi are a vital collection of plant by humid and warm micro condition favoured by pathogens which cause many plant diseases bacteria and fungi (Verma et al., 2009).(Knogge, 1996).Some types of fungi degrade Anadelphia afzeliana commonly called moist wood thereby causing wood rot.Brown rot thatch grass belongs to the family Poaceae.It fungi degrade dead while Armillaria commonly occurs mainly in the Sudanian zone and from known as honey fungus is a parasite of living Senegal eastward, southward to DR Congo trees.Excess moisture in wood that is above the (Bague, 2011).It is locally dominant near fibre saturation point increases the activities of waterholes and in low-lying regularly flooded wood fungal (Harris, 2001).The fungi which and poorly drained wetland savanna.In Senegal grow and penetrate wood fibrous structure it is dominant in savannas receiving 900-1100 causing decay are termed lignicolous fungi.
mm annual rainfall in a wet season of 5-7 Bacteria are prokaryotic and known as months; in southern Cote d'Ivoire it occurs in the the most abundant organisms present coastal zone in drier savannas (Bague, 2011).A. everywhere.They can attack wood either in afzeliana is a poor grazing grass but grazed anaerobic or aerobic conditions.Although fungi when it is young.It is used as a thatching grass are the major causes of wood decomposition, used for roofing and has a history of pesticidal however, it was also discovered that bacteria activities.Therefore, this study was aimed at attack dead wood.It is now known that there is assessing the fungicidal and bactericidal boundless diversity of bacteria within wood.
properties of A. afzeliana solvent extracts.Nevertheless, in the same environment, fungi are more understood compared to bacteria Materials and Methods (Johnston et al., 2016).

Study area Fungi and bacteria are largely controlled
The research was carried out at the by synthetic chemical.It is reported that there Federal University of Agriculture Makurdi, Benue are currently over 113 active ingredients listed as State, Nigeria.It is located in the Guinea fungicides globally (Knight et al., 1997).
Savannah zone between latitudes 8 35" E and 8 However, synthetic chemical lead to serious and 0 0 41" E longitudes 7 45" N and 7 52" N (Jimoh et continuing environmental pollution.They are al., 2009).Makurdi is located in the middle belt of extremely and intensely toxic (Daoubi et al., Nigeria and situated sideways of River Benue.As 2005).These chemicals are also known to be of 2007, Makurdi human population was carcinogenic to wild animals and humans.estimated to be 500,797.The main ethnic groups Besides, pathogens developed resistance to of Makurdi are in decreasing order of Tiv, Idoma, several of these chemicals.Thus, there is an Igede and Etulo.Land area of Makurdi is 34,059 apparent demand to explore alternate 2 km (13,150 sq.mi).Makurdi vegetation is compounds that are harmless to animals and characteristically of guinea savannah and this in environmentally friendly, biodegradable to a d d i t i o n t o i t s c l i m a t e o f control bacterial and fungal diseases (Makovitzki Fungi has hundreds of species of which some favours the production and harvest of Orkar village near Gando, Mbayion, Gboko LGA, agricultural crops (cassava, yam, banana, sweet Benue State. A. afzeliana was air dried for three potatoes, rice, maize) and forest products which weeks and pounded using mortar and pestle as mainly timber produce (Mage and Agber 2017).
shown in plate 1.The pounded material was ground using a grinding machine to a fine Plant collection and preparation powder.Fresh A. afzeliana was collected from Plate 1: A: Dried A. afzeliana B: Pounded A. afzeliana was run with solvent mixtures of increasing Method of extraction polarity of hexane and ethyl acetate in ratio of Three solvents were used in the 95%:5% -0%:100%.The fractions were extraction.They include methanol, n-hexane collected in labeled vials and allowed to dry as and ethyl acetate.The pounded plant (300 g) shown in Table 2. Dried fractions were on the was extracted using the various solvents (1800 basis of Tin Layer Chromatography (TLC) ml) respectively, for 48 hours with intermittent similarities.Fraction (Aa26) with clean coolening four hourly shaking.The extract was filtered into crystals was sent to Strathclyde Institute of clean glass jars using a filter paper (Whatman Pharmacy and Biomedical Sciences, Glasgow, no. 1) as shown in plate 2, 3 and 4. The extracts Scotland for NMR analysis and Aa25 was sent for were evaporated to dryness in a stream of air as anti-microbial screening.shown in plate 2, 3 and 4. Dried extracts were dissolved using chloroform and pre-absorbed N u c l e a r M a g n e t i c R e s o n a n c e ( N M R ) into silica gel (3 g) prior to column chromatography.
spectroscopic analysis Nuclear Magnetic Resonance (NMR) Column chromatography analysis was carried out on Aa26.NMR A ball of cotton wool was gently dropped spectrometer (400mH ) spectra acquired were z into chromatography column and tucked into further processed using mestrenora 12 software.place using a metal wire.A bigger ball of wool It was composed of white needle-shaped 1 about the diameter of the column (3 cm) was crystals.The characterization was done using H also tucked into the bottom of the column to NMR.NMR was carried out in Glasgow, Scotland.provide an even base for the silica gel bed.A gel was prepared using (hexane: ethyl acetate 95:5) Antimicrobial Screening and silica gel (60 g) and introduced into the The anti microbial screening was carried column.The column was tapped lightly on the out at Nigerian Institute for Leather Science and side with a thick rubber nose to release trapped Technology (NILEST), Zaria.The antimicrobial air bubbles.The solvent level was reduced to 2 activities of Aa25 compound was determined cm above the column bed before introducing using selected wood pathogens.pre-absorbed crude solvent extract.The column o medium.Incubation was made at 30 C for 1-7 Screening of bacteria days for the fungi after which the plate of the Screening of compound was done by media was observed for the zone of inhibition of diffusion method.Antibacterial activities were growth.The zone was measured with a evaluated by using initial concentration of transparent meter rule and results recorded in compounds determined by dissolving 0.002 mg of millimeters.each compound in 10 ml of DMSO to obtain a concentration of 200 µg/ml.Mueller Hinton agar Procedure for Minimum Inhibition Concentration and Sabouraud Dextrose agar were the media used as the growth media bacteria.Media were (MIC) prepared in accordance to manufacturer's MIC of compound was determined using o guidelines.It was purified at 121 C for 15 mins the broth dilution method.Muller Hinton broth and introduced into germ-free Petri dishes and and Sabouraud dextrose broth were prepared.was left to cool and congeal.
Ten millilitre was dispensed into test tubes and o Muller Hinton agar was seeded with 0.1 ml the broth was sterilized at 121 C for 15mins.The standard inoculums of the test bacteria.The broth was allowed to cool.Mc-farland turbidity inoculum was expanse equally on the surface of scale number 0.5 was made ready to produce the medium with aid of a disinfected swab.A well turbid solution.Normal saline was prepared while at the middle of each injected medium was cut 10 ml was poured into sterilized test tube and the with a typical cork borer of 6 mm in diameter.test microbe was inoculated and incubated at the o Solution of 0.1ml of compound of 200 µg/ml temperature of 37 C for a period of 6 hours for the o concentration was then introduced into the well bacteria and at a temperature of 30 C for a period on the inoculated medium.At a temperature of of 6 hours for the fungi.had a concentration of about 1.5x10 cfu/ml.Twofold serial dilution of the compound was carried Screening of fungi out in the sterilized broth to attain a concentration Screening of compound was done by levels of 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 diffusion method.Initial concentration of µg/ml and 12.5 µg/ml.compounds was determined by dissolving 0.002 Initial concentration was obtained by mg of the compound in 10 mls of DMSO to obtain dissolving 0.00 2mg of the compound in 10 ml of a concentration of 200 µg/ml.This was used to the sterile broth and 0.1 ml of the Microbe in the assessed its antifungal activities.Mueller Hinton normal saline was then introduced into the agar and sabouraud dextrose agar were the different concentrations.The bacteria were media used as the growth media for fungi.Media introduced into the Mueller Hinton broth while the were prepared in accordance to manufacturer's fungi were introduced into the Sabouraud o o manual and purified at temperature of 121 C for a Dextrose broth.Incubation was made at 37 C for o period of 15mins.Thereafter, it was emptied into 24hrs for the bacteria and at 30 C for 1-7 days for germ-free Petri dishes and was left to cool and the fungi after which the test tubes of the broth harden.
were observed for turbidity (growth).The least Sabouraud Dextrose agar was seeded concentration of the compound in the sterilized with a 0.1 ml standard inoculum of the test fungi.broth that indicate no turbidity was documented The inoculum was uniformly spread on the as the MIC.surface of the medium with aid of a disinfected swab.A typical cork borer measuring 6 mm in Procedure for Minimum Bacterial Concentration diameter was utilized to make cut a well at the (MBC) and Minimum Fungicidal Concentration middle of each injected medium.Solution of 0.1 (MFC) ml of compound of 200 µg/ml of concentration MBC and MFC was done to assess if the test was introduced into the well on the inoculated microbes were killed or only their growth was  Table 2 shows Minimum inhibition rolfsii.While MIC for Aspergillus fumigatus was concentration (MIC) of A. afzeliana compound 100 µg/ml.However, Fibroporia vaillantii, against test fungi.The MIC was found to be 50 Fomitopsis pinicoca, and Serpula lacrymans were µg/ml for Coniophora puteana, Gloeophyllum resistant to all concentration levels of A. sepiarium, Phaeolus schweinitzii, and Sclerotium afzeliana compound.    . afzeliana compound (Aa25).
bacteria was observed to be resistant to ciproflaxin, cefuroxime, and Aa25 but, sensitive The results indicate that Acido bacteria was to sparfloxacin at ZOI of 30 mm.Alpha resistant to ciproflaxin, cefuroxime, and Aa25, proteobacteria was sensitive (32 mm and 30mm) respectively, but resistant to sparfloxacin.
observed to be sensitive to ciprofloxacin, sparfloxacin, cefuroxime and Aa25 at ZOI of 32 Bacillus subtilis was sensitive (28 mm, 35 mm mm, 29 mm, 39 mm and 27 mm respectively.and 28 mm to ciprofloxacin, sparfloxacin and Gama proteobacteria was sensitive (31 mm) to Aa25 respectively, but resistant to cefuroxime.

Enterococcus faecium was sensitive to
Proteus mirabilis was sensitive (32 mm, 30 mm, ciprofloxacin cefuroxime and Aa25 at ZOI of 26 31 mm and 23 mm) to ciprofloxacin sparfoxacin mm, 30 mm, and 26 mm respectively, but cefuroxime and Aa25 respectively.resistant to sparfloxacin.Escherichia coli was Table 5 shows MIC of Aa25 compound µg/ml for Enterococcus faecium, Pseudomonas against test pathogens.The results showed that aeruginosa, and Proteus mirabilis.Acido bacteria, the MIC of A. afzeliana compound was 25 µg/ml Actino bacteria and Gama proteobacteria for Alpha proteobacteria, Bacillus subtilis, Beta pathogens were all resistant.proteobacteria and Escherichia coli while is it 50  rolfsii were found to be completely inhibited at Goins (2017) also described Minimum 50 µg/ml MIC of A. afzeliana compound while Aspergillus fumigatus was inhibited at 100 Bactericidal Concentration (MBC) as the lowest µg/ml.Compounds with MICs less than 100 concentration of an antibacterial agent desirable µg/ml are regarded as having strong to kill a bacterium over a fixed, slightly extended antimicrobial property (Tang et al., 2003).This period, for example 18 hours or 24 hours, below a precise set of conditions.The MBC of A.
implies that A. afzeliana compound was very afzeliana compound at 50 µg/ml completely a c t i v e a g a i n s compound had different antifungi activities and properties and controlled some wood fungi zone of inhibition against the tested pathogens.
(Aspergillus fumigatus, , Comparing the standard antibiotics and A. Fomitopsis pinicoca, Gloeophyllum sepiarium, afzeliana compound, the highest ZOI was , Rhizopus sp.Sclerotium observed with fulcin against Coniophora puteana rolfsii) and wood bacteria (Alpha proteobacteria, and .Anti-fungi activities of A. Ba c i l l u s s u b t i l i s , B e ta p ro t e o b a c t e r i a , afzeliana compound was observed to be Enterococcus faecium, Pseudomonas aeruginosa sensitive against Aspergillus fumigatus, Escherichia coli and Proteus mirabilis).Its ZOI Coniophora puteana, Gloeophyllum sepiarium, on test microbes was >19 mm which makes A. , Rhizopus sp., afzeliana very active and compete favourably and Sclerotium rolfsii but was with standard antibiotics.Stigmasterol in A. resistence against Fibroporia vaillantii and afzeliana was more potent in the control of wood Fomitopsis pinicoca.A. afzeliana compound had bacteria than fungi.MBC/MFC for all tested the highest zone of inhibition on Rhizopus sp.
pathogens were observed to be most effective at Guevara et al., (2005) stated that ZOI >19 mm is 50µg/ml level of concentration.
o 37 C and within 24hrs, incubation was made for Dilution of the test microbes carried out in bacteria after which the plate of the media was the standard saline until the turbidity viewed for the zone of inhibition of growth.The synchronised with the scale of Mc-farland by zone was measured with a translucent meter rule visual assessment.At this time, the test microbe 8 and results documented in millimeters.
t C o n i o p h o r a p u t e a n a , killed Alpha proteobacteria, Bacillus subtilis, and Gloeophyllum sepiarium, Phaeolus schweinitzii, Beta proteobacteria while at 100 µg/ml and Sclerotium rolfsii microbes.Enterococcus faecium and Escherichia coli were Aspergillus fumigatus, Coniophora completely killed.However, Proteus mirabilis was puteana and Sclerotium rolfsii microbes were completely killed at 200 µg/ml.Shan et al., killed at MFC of 100 µg/ml, whereas Phaeolus (2014) noted that MBC indicates the bacterial schweinitzii and Rhizopus sp.microbes were killing capability of the test substance.The MBC observed to be completely killed at MFC 200 result of this study indicates that A. afzeliana µg/ml.The result indicates that A. afzeliana extract might represents a potential source of compound was more active against bacteria plant antibiotics for the treatment of wood than fungi.This implies that wood bacteria will bacterial diseases.This agrees with Tamokou et readily be controlled than wood fungi.Tamokou al., (2011) who stated that such finding supports et al., (2011) reported that antimicrobial the traditional use of plant in the treatment of activities vary with the bacterial and fungal infectious diseases.species and claimed the variations could be due t o g e n e t i c d i f fe r e n c e s b e t w e e n t h e microorganisms.Anti-fungi characteristics of A. afzeliana compound Conclusion S t a n d a r d a n t i b i o t i c s ( f u l c i n , This study showed that A. afzeliana ketecanazole and fluonazole) and A. afzeliana contains stigmasterol (C H O) with antibiotic 29 48

Table shows
antibacterial activities, ZOI sensitive to sparfloxacin at ZOI of 32 mm.Actino of antibiotics and A Therefore, all ZOI of A. afzeliana study, MIC of A. afzeliana compound completely compound were very active against the test inhibited Alpha proteobacteria, Bacillus subtilis, pathogens except for , Beta proteobacteria and Escherichia coli at 25Fomitopsis pinicoca and Serpula lacrymans.µg/ml.Enterococcus faecium, Pseudomonas Coniophora puteana, Gloeophyllum aeruginosa, and Proteus mirabilis were sepiarium, Phaeolus schweinitzii, and Sclerotium completely inhibited at 50 µg/ml.