Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 Namadina et al.,2019 Pharmacognostic and Acute Toxicity Studies of the Albizia chevalieri Hams (Mimosaceae) Namadina, M. M1., Bako, A.T1., Aliko, A.A1., Galalain, A.M1., Abbas, R.L1.,Hamisu, H4., Umar, A.M9., Kamal, R.M2., Sunusi, U3., Idris, A.M5., Nafisa, M.A6., Sanusi, H7., Hafiz, S.S7., Muttaka, A8., Umar, M.K4., Sale, A.I10., Adamu, M.M11 1 Department of Plant Biology, Bayero University, Kano, Nigeria. 2 Department of Pharmacology, Federal University, Dutse, Jigawa State, Nigeria 3 Department of Biochemistry, Faculty of Basic Medical Science, Bayero University, Kano 4 Department of Microbiology, Federal University, Dustinma, Katsina State, Nigeria 5 Department of Medical Microbiology, Bayero University, Kano, Nigeria. 6 Department of Biology, Saadatu Rimi Collage of Education, Kumbotso, Kano 7 Department of Biological Sciences, Federal University Dutsinma 8 Department of Biochemistry, Federal University Gusau 9 Department of Remedial and General Studies, Audu Bako Collage of Agriculture, Dambatta, Kano State, Nigeria 10 Department of Biology, Kano University of Science and Technology, Wudil 11 Department of Integrated Science, Saadatu Rimi Collage of Education, Kumbotso, Kano Hajiyaiyalle@gmail.com ABSTRACT Albizia chevalieri Hams (Mimosaceae), mostly found in the Northern Sahel Savannah region of Nigeria as well as in Nigér and Senegal is a tree of the acacia type with a long list of folklore therapeutic claims which include its use as purgative, taenicidal, cough remedy, dysentery, cancer, diabetes mellitus, tuberculosis and snake bite remedy. The stem bark was collected, dried and powdered. Physicochemical constants vis; moisture content, total ash, acid insoluble ash, water soluble ash, alcohol-soluble extractive value, water-soluble extractive value and elemental analysis vis ; acid digestion of the powdered stem was carried out using standard methods. Findings from this study revealed the presence of some diagnostic microscopical features such as calcium oxalate, starch, gum/mucilage, lignin, 186 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 Namadina et al.,2019 Aleurone grain, suberized/Cuticular cell wall and insulin but calcium carbonate was absent. Quantitative physical constants include moisture contents (6.50%), ash value (8.50%), acid insoluble ash value (2.70%), water and ethanol extractive indices 20.50% and 18.20% respectively. Trace metals such as Fe, Mn and Ni detected in A. chevalieri were below the permissible limit for edible plants. Phytochemical screening of aqueous and methanol extracts revealed the presence of alkaloid, flavonoids, saponins, tannins while anthraquinones was absent in all the extracts. The acute toxicity studies on the methanol extract showed that it is safe at 5000mg/kg. The aim of this research is to established pharmacognostic parameters of A. chevalieri. The results obtained also provided scientific basis for the use of A. chevalieri in folklore medicine. Keywords: Albizia chevalieri, quantitative, qualitative, phytochemical INTRODUCTION Natural products from medicinal plants, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug, because of the unmatched availability of chemical diversity they contain (Cos et al., 2006). Botanicals and herbal preparations for medicinal usage contain various types of bioactive compounds (Chen et al., 2011). Reported literature revealed scientific work on A. chevalieri, recording great medicinal values and thereby validating some therapeutic folklore claims. The leaves of A. chevalieri are widely used for the management of diabetes mellitus by traditional medicinal practitioners in some part of Nigeria. Consequently, hypoglycaemic effects of the leaves (Saidu et al., 2007a) and root (Saidu et al., 2010) have been reported for which the aqueous leaf extract showed some significant hypoglycemic and hypolipidaemic effect in alloxan induced diabetic rats. A. chevalieri Harms (Mimosaceae) is a tree of acacia type native to tropical and subtropical regions including Nigeria and Niger Republic, with loose balls of whitish fragrant flowers and flat brown pods. A. chevalieri Shrub or small tree, up to 6 m, occasionally 12 m, with an open and rounded or umbrella shaped canopy, bark pale-greyish, corky and deeply creviced, dark brown slash. Twigs pubescent with white lenticels, leaves with 8-12 pairs of pinnae, with 20-40 pairs of leaflets each. Leaflets 1 cm long x 2-3 mm wide, sometimes slightly curved, greyish-pubescent on both sides and apiculate (Geerling, 1988). A. chevalieri is used in Borno, NorthEastern Nigeria as purgative, taenicide and also as a remedy for coughs (Aliyu et al., 2009). A decoction of the leaves is used in Northern Nigeria as remedy for 187 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 dysentery (Burkill, 1995 as cited in Aliyu et al., 2009). There are also reports on the local use of the leaves extract for cancer treatment in Zaria, Kaduna State (Abubakar et al., 2007), while in Northern Sahel Savannah of SokotoNigeria and Niger Republic, the leaf extract is used either as cold water decoction or the dried ground sieved leaf powder is mixed with pap and consume for the management of diabetes mellitus (Saidu et al., 2007a). The unmatched availability of chemical diversity present in plants brings to bare the so many active constituents still unknown in most species, especially in the tropical world where facilities for modern scientific research is still a challenge. Consequently, literature search has shown that empirical research leading to the investigations of active compounds of A. chevalieri was not available, despite its ethnobotanical exploits. In conclusion, the established pharmacognostic standards for the powder of A. chevalieri stem could be used as a diagnostic tool for the standardization and identification of this medicinal plant for its purity and quality in the future and hence, inclusion into the pharmacopoeia for official use. Namadina et al.,2019 MATERIALS AND METHODS Collection and identification of plant material A.chevalieri stem were collected in the month of July 2018 in Madobi Local Government Area of Kano State NorthWest Nigeria, latitude and longitude 11o 46´38”N and 8o 17´ 18” E, 11.77722o N 8.28833oE respectively, and was identified and authenticated by Mal. Bahawuddeen Said of Herbarium, Department of Plant Biology, Bayero University, Kano-Nigeria Drying and preservation of plant material The stem of the plant was shed dried in a well-ventilated room until a constant weight was obtained. The plant material was grounded to coarse powder using pestle and mortar. The powder was stored in 1Liter beaker until required for use. Extraction of Plant Materials Dried plant materials (50 g) were extracted using cold maceration with 500 ml of both distilled water and methanol. The contents were then be filtered using a filter paper (Whatman no.1), the filtrate was concentrated to dryness using water bath which was kept in desiccator 188 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 Namadina et al.,2019 Figure 1: A. chevalieri stem and leaves Chemo-microscopic Studies on the Stem of A. chevalieri Powdered sample (stem) of A. chevalieri was used for this study to detect the presence of cell wall materials and cell inclusions. Finely ground sample of plant was cleared in a test tube containing 70% chloral hydrate solution. It was then boiled on a water bath for about thirty minutes to remove obscuring materials. The cleared sample was mounted with dilute glycerol onto a microscope slide. Using various detecting reagents the presence of cell wall materials and cell inclusions was detected in accordance to WHO (2011) guidelines. Cell wall Materials Test for Cellulose A drop or two of iodinated zinc chloride was added to the powdered sample and allowed to stand for a few minutes and 189 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 observed under a microscope. It stained cellulose cell wall blue to blue- violet. Test for Lignin The powdered plant material was moistened on a slide with a small volume of phloroglucinol and allowed to stand for about two minutes or until almost dry. A drop of hydrochloric acid was added and view under a microscope. Pink stained or cherry red was observed, for the presence of lignin. Test for Suberized or Cuticular cell walls A drop or two of Sudan red was added to the cleared powdered sample and allowed to stand for few minutes and observed under a microscope. Orange red or red colour was observed presence of suberin or cutin on the cell. Test for Gum and Mucilage To a small portion of the cleared powdered sample of the plant, a drop of ruthenium red was added. Appearance of pink coloration was considered positive for gums and mucilage. Cell Inclusions/ Cell Contents Test for Starch grains To a small portion of the cleared powder sample of the plant, N/50 iodine was added. Appearance of blue-black or reddish-blue coloration on some grains would be considered positive for starch. Namadina et al.,2019 Test for Calcium oxalates and Calcium Carbonates To a small portion of the cleared powdered sample of the plant, HCl was added, dissolution of crystals in the powdered drug without effervescence was considered positive for calcium oxalate while slow dissolution with effervescence was considered positive for calcium carbonate. Inulin A drop of 1-naphthol and that of sulphuric acid was added to the powdered sample and viewed under the microscope. Spherical aggregations of crystals of inulin turned brownish red and dissolve. Test for Tannins To a small portion of the cleared powdered sample of the plant, 5% ferric chloride solution was added. Appearance of greenish black colour was considered as positive for tannins. Extraction of Plant Materials Stem powder (50g) of Albizia chevalieri was extracted successively in 500 ml of distilled water and methanol for 72 hrs. using successive cold maceration and the concentrate was evaporated to dryness on a water bath and stored in desiccator for further use. Determination of Physicochemical Constants of the Powdered Stem Some physicochemical parameters of the powdered sample of the plant such as 190 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 moisture content, total ash, acid-insoluble ash, water-soluble ash, alcohol and water extractive values was determined as described in the updated edition of quality control methods for medicinal plant materials (WHO, 2011). Namadina et al.,2019 accurately weighed and placed in some clean, dried evaporating dishes of known weights. They were placed in an oven and o heated at a temperature of 105 C for 1 hour, then cooled in a dessicator and reweighed. Heating and weighing were repeated until a constant weight was obtained. The weight loss on drying was computed following the formula below: Moisture Content 3 g each of the powdered sample was % Moisture content = Initial Weight of Powder - Final Weight of Powder X 100 Initial Weight of Powder Total Ash Value 2 g of powdered plant materials was accurately weighed and placed separately in a crucible of known weight. It was heated gently and the heat gradually increased until it is white indicating the % Ash Value = absence of carbon. It was allowed to cool in a desiccator and weighed; this was repeated until a constant weight was obtained. The total ash value was determined as a percentage with the formula below Weight of Residual Ash X 100 Original Weight of Powder Acid-insoluble ash This was determined for the powdered plant material. 25 ml of dilute hydrochloric acid was added to the crucible containing ash. It was covered with a watch glass and gently boiled for 5mins. The watch glass was rinsed with 5 ml of hot water and the liquid added to the crucible. The insoluble matter was collected on an ash less filter-paper and washed with hot water until the filtrate is neutral. The filter-paper containing the insoluble matter was transferred to the original crucible, dried in an oven and ignited to a constant weight. The residue was allowed to cool in a suitable desiccator for 30 minutes and then weighed without delay (Evans, 2002). The acid-insoluble ash will then be calculated as a percentage for each of the two plants with the formula 191 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 % Acid insoluble Ash = Namadina et al.,2019 Weight of Residual Ash X 100 Original Weight of Powder Water soluble ash To the crucible containing the total ash, 25 ml of water was added and boiled for 5 minutes. The insoluble matter was collected in a sintered glass crucible. It was then be washed with hot water and ignited in a crucible for 15 minutes at 105oC. The weight of the residue was subtracted from the weight of the total ash. The content of water soluble ash per air dried powdered sample was calculated and recorded (WHO, 2011). % Water Soluble Ash = Weight of Total Ash - Weight of Residual Ash X 100 Original Weight of Powder Alcohol-Soluble Extractive Value 4 g of each of the plant material was separately weighed in a conical flask. 100 ml of ethanol was added and macerated for 24 hours, during which the mixture was frequently shaken within the first 6 hours using a mechanical shaker. It was % Alcohol Extractive Value = filtered and 25 ml of the filtrate transferred into an evaporating dish of known weight and evaporated to dryness on a water bath. It was dried to a constant weight, the percentage of alcohol-soluble extractive value was then determined for the plant as Weight of Extract in 25ml X 4 X 100 Original Weight of Powder sample, but solvent for extraction here was water. Water-Soluble Extractive Value Same procedure as in alcohol-soluble extractive value was repeated here for the Weight of Extract in 25ml X 4 X 100 Original Weight of Powder % Water Extractive Value = 192 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 Elemental analysis of Powdered stem of A. chevalieri Acid Digestion of the samples 0.5 grams of the powdered plant material was weighed into 10 different beakers each of 50 ml, to which 2.5 ml of hydrochloric acid (HCl) and 7.5 ml Nitric Acid (HNO3) were added to each beaker. The 10 beakers used were placed in an open space for 2 hours and mixture of hydrochloric acid (HCl) and nitric acid (HNO3) in 1:1 ratio was added to each o beaker. It was kept on a hot plate at 100 Co 170 C for 1- 4 hours. After the contents in beakers is about to dried; 5 ml of Hydrochloric acid (HCl) was added to each beaker and be kept on the hot plate until the entire liquid content in the beakers got evaporated. Then, 5 ml of deionized water was added to each beaker and the solutions were poured in sterile bottles and tested for the quantification of the metals. The concentration of Fe, Mg, Zn, Cu was read using the flame atomic absorption spectrophotometer (FAAS), AA 500 model, Atomic Emission Spectrophotometer, HACH Spectrophotometry (DR/4200) and Atomic Absorption Spectrophotometer were used for other elements detected The elemental analyses of the plant materials were carried out in Ahmadu Bello University Zaria, Multi-user Research Laboratory,. The mineral elements estimations indicated the amount of macro, trace elements and heavy metals present in the Plant Namadina et al.,2019 samples. The mineral elements detected include; Zinc (Zn), Magnesium (Mg), Lead (Pb), Manganese (Mn), Selenium (Se), Copper (Cu), Iron (Fe), Cadmium (Cd), Arsenic, Nickel and these were done by Spectrophotometric methods. Before determining the concentration of any element in the sample, calibration curve of the element in the sample was prepared using prepared standard stock solutions for the elements as reported by AOAC, 2000; 2005; Akpabio and Ikpe (2013). Phytochemical screening of aqueous and methanolic extract of Albizia chevalieri stem: The aqueous and methanol extracts were subjected to phytochemical screening using standard method as described below. Test for Saponins Frothing Test: About 0.5 g of the extracts was shaken with water in a test tube followed by warming on a water bath. Frothing which persists on warming was taken as an evidence for the presence of saponins (Sofowora, 1993). Test for steroids/ terpenes Lieberman-Buchard test: A small portion of the extracts was dissolved in chloroform. Equal volume of acetic anhydride was then added, followed by concentrated sulphuric acid down the side of the test tube. The solution was 193 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 observed for the presence of a brown ring at the interphase which indicates the presence of steroids/triterpenes (Evans, 2002). Test for flavonoids Shinoda Test: 0.5 g of the extracts was dissolved in 5 ml 95 % ethanol, warmed and filtered. Three (3) pieces of magnesium chips were added followed by five drops of concentrated hydrochloric acid. The appearance of a pink, orange or red to purple color was used as an indication for the presence of flavonoids (Evans, 2002). Namadina et al.,2019 aqueous layer was divided into three. To the first portion, few drops of freshly prepared Dragendorff?s reagent was added and observed for formation of orange to brownish precipitates. To the second portion, one drop of Mayer?s reagent was added and observed for formation of white to yellowish or cream color precipitates. To the third portion, 1ml of Wagner's reagent was added to give a brown or reddish or reddish-brown precipitates, in the presence of alkaloids (Evans, 2002). Test for anthraquinones Borntrager's Test: A small portion of the sample (extracts) was shaken with 10 ml of benzene, the content was filtered and 5 ml of 10% ammonia solution was added to the filtrate, then the mixture was shaken. Formation of a pink, red or violet colors in the lower part of the aqueous layer indicates the presence of free anthraquinones (Evans, 2002). Test for Tannins Ferric chloride Test: 0.5g of the sample (extracts) was stirred with 10 ml distilled water and filtered. Two drops of 1% ferric chloride solution was added to 2 ml of the filtrate. Formation of a blue-black (Hydrolysable/Gallitannins) or green or blue-green (Condensed/Cathehic tannins) precipitate was used as an indication for the presence of phenolic compounds (Evans, 2002). Test for cardiac glycosides Keller-Killiani test: About 0.5 g of the sample (Extracts) was dissolved in 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was then under layered with 1ml of concentrated sulphuric acid. A brown ring obtained at the interphase indicates the presence of a deoxy sugar characteristic of cardenolides (Evans, 2002). Test for Carbohydrates Molisch Test: To small portion of the Test for Alkaloids About 0.5 g of the extracts was stirred with 5 ml of 1 % aqueous hydrochloric acid on a water bath and filtered. Ammonia solution was added to the filtrate until it was basic then chloroform was added and shaken gently to allow separation. The chloroform layer was collected then diluted HCl added and shaken gently for separation. The 194 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 extract in a test tube, few drops of Molisch reagent was added and concentrated sulphuric acid was then applied down the side of the test tube to form a lower layer, a reddish colour ring at the interphase indicates presence of carbohydrate (Evans, 2002). Namadina et al.,2019 Acute Toxicity Study Lorke method (1983) was adopted for the acute toxicity test in rats. Thirteen (13) of each animal species were used. Nine (9) of rats in 3groups each of 3 animal per group for the three graded doses of 10,100 and 1000mg/kg were treated orally per body weight and observed for 24hrs for signs of changes in the behavioral pattern and/or death was observed. In a 2 n d phase of the experiment, the remaining 4rats in each of the 4groups of one animal per group respectively were given lower or higher doses of Albizia chevalieri depending on the occurrence of death or no death in the first phase and observed again for 24hrs. The oral median lethal doses were then calculated as geometric mean of the highest non-lethal and the lower lethal doses as follows: LD50 = Maximum non lethal dose * Minimum lethal dose for both animal species (Lorke, 1983). Thin Layer Chromatography Profile of Crude Extracts TLC aluminum sheet of 20 x 20cm silica gel pre-coated plate using the one way ascending techniques was employed for this analysis. The plates were cut into sizes of 5 x 10 cm. The extract was dissolved in the initial extraction solvents and spots were applied manually on the cut plate using capillary tubes, after which the plates were dried and developed in different solvents ratios of: Dichloromethane : Methanol (3:2), Dichloromethane : Methanol (2:3) in the chromatographic tank. Developed plates were sprayed using general detecting reagents (panisaldehyde/H 2 SO 4, 10% H 2 SO 4 in methanol) and specific detecting reagents: Borntragers, Dragendoff, ferric chloride, Libermann-buchards and alminium chloride (it was viewed under UV 365nm) and heated at 110o C for 2 minutes where applicable. Number of spots and retardation factors (Rf values) for each of the spots were determine and recorded while the chromatograms was scanned accordingly (Gennaro, 2000; Stahl, 2005). RESULTS Chemo-microscopical examination of powdered stem of A. chevalieri revealed the presence of cellulose, tannins, starch, lignin, calcium oxalate, inulin, suberin, aleurone grain and mucilage but calcium carbonate was absent (Table 1). 195 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 Namadina et al.,2019 Table 1 Chemomicroscopical studies of A. chevalieri stem Constituents Starch Gum and Mucilage Cellulose cell walls Lignin Aleurone grain Calcium oxalate crystals Calcium carbonate Suberized/Cuticular cell wall Inulin Tannins Inference + + + + + + + + + The average moisture contents in the powdered plant material using loss on drying method was calculated to be 6.5%, the total ash was 8.1% while acid insoluble and water soluble were 2.7% and 4.2% respectively. The alcohol and water extractives values were obtained to be 18.0% and 20.5% respectively (Table 2). Table 2 Physicochemical Constants of A. chevalieri powdered stem Parameters Values (%w/w) ± SEM* Albizia Chevalieri Moisture content Ash content Acid insoluble ash Water soluble ash Water extractive value Ethanol 6.5 8.1 2.7 4.2 20.5 18.2 EP (2011) 10-12% 6-19% ?1 *Average values of three determinations. 196 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 Namadina et al.,2019 Table 3: Elemental analysis of Albizia chevalieri powdered stem Element Concentration (ppm) Iron(Fe) Copper (Cu) Lead (Pb) Zinc (Zn) Nickel (Ni) Manganese (Mn) Aluminum (Al) Cadmium (Cd) Selenium (Se) Chromium (Cr) Arsenic (As) 2.294 0.067 0.513 0.210 0.893 0.242 2.915 0.000 1.007 0.023 -0.230 FAO/WHO (1984) limit* (ppm) 20.00 3.00 0.43 27.40 1.63 2.00 0.21 - Table 4: Some Physical Properties of A. chevalieri Extracts Properties Weight of plant material (g) Weight of extract (g) Percentage (%) yield Colour of extract Odour of extract Texture of extract Aqueous extract 50 7.2 14.4 Brown Fruity solid and Dry Phytochemical screening of aqueous and methanol extracts revealed the presence of alkaloid, flavonoids, saponins, tannins Methanolic extract 50 9.1 18.2 Red Fruity Solid and Dry while anthraquinones was absent in all the extracts. 197 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 Namadina et al.,2019 Table 5 Qualitative Phytochemical screening of aqueous and methanolic stem extract of A. chevalieri Metabolite Inference Aqueous + + + + + + + + + + - Alkaloid Flavonoid Saponins Cardiac glycoside Tannins Steroid Triterpenes Phenol Amino acid Carbohydrate Anthraquinones Methanolic extract + + + + + + + + + - Key;+presence,-absence Chromatographic analysis of methanol extract of A. chevalieri stem were carried out on TLC plate (silica gel) using suitable solvent systems and sprayed with panisaldehyde reagent. TLC Profile of A. chevalieri extract using Solvent system Dichloromethane: Methanol (3:2). Four spots were detected with p-Anisaldehyde spray and the Rf values were shown alongside the spots. Table 6: Chromatograms analysis of methanolic stem extracts of A. chevalieri Plant species Solvent system Distance travel by spot No of spot A. Chevalieri DCM:ME(3:2) 4.0 4 0.23,0.47,0.67,0.95 DCM:ME(2:3) 4.1 5 0.12,0.29,0.53,0.61,0.97 Key: DCM- Dichloromethane ME- Methane 198 Rf-value Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 No death was recorded in the first phase of the study in rats. In the second phase, doses of 1500, 2250, 3250 and 5000mg/kg were used and no death was also recorded. The oral median lethal Namadina et al.,2019 dose (LD50) for the methanol stem-extract of A. chevalieri was therefore estimated to be greater than 5000mg/kg and no sign of behavioural changes were also observed. Table 7 Acute toxicity studies of methanolic stem extract of Plant species Phase I Phase II Group Number of Animals Dose (mg/kg) Mortality recorded after 24hrs I 3 10 0/3 II 3 100 0/3 III 3 1000 0/3 I 1 1500 0/1 II 1 2250 0/1 III 1 3250 0/1 IV 1 5000 0/1 identification of powdered drug as their identification is largely based on the form, the presence or absence of certain cell types and cell inclusions. These are very important diagnostic pharmacognostic parameters for the identification and authentication of crude drugs especially in powdered plants (Chanda, 2011). The physicochemical constants of A. chevalieri stem determined include the moisture content, total ash value, acid insoluble ash, water soluble ash, alcohol DISCUSSION The studies carried out on the stem of A. chevalieri have established some pharmacognostic standards that will guide its utilization as crude drug in pharmacy and other fields. Chemomicroscopical examination of the powdered stem of A. chevalieri revealed the presence of cellulose, tannins, starch, lignin, calcium oxalate, suberin, aleurone grain and mucilage but calcium carbonate was absent. The chemo-microscopic features are most valuable in the 199 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 (ethanol) extractives value and water extractives value. These values are useful as criteria to evaluate the identity and purity of crude drugs (Evans, 2009; WHO, 1996). It also indicates the presence of various inorganic materials like carbonate, oxalate and silicate in plant materials. The average moisture content of the powdered plant material using loss on drying method was found to be 6.5%, and this value is within the permissible limits of British Herbal Pharmacopoeia B.H.P (1990) and WHO, (2011). Low or permissible moisture in crude drugs may discourage the growth of bacteria, yeast, mould and fungi and will stand for long period of time during storage without spoilage for better stability against degradation of product (WHO, 1996). Ash values obtained include total ash as 8.1%, acid insoluble ash 2.7% and water soluble 4.2%. These Ash values indicate the presence of various impurities such as carbonate, oxalate, sand and silicate in plant materials (Kaneria and Chanda, 2011). The alcohol and water extractive values were 18.0% and 20.5% respectively. The result showed that water have higher extractive value (20.5%) than ethanol. This is because water is a universal solvent that has high polarity and is able to extract more phytochemical constituents than alcohol that has less polarity. This verified why water is mostly used as solvent by traditional medical practitioner and individuals in Namadina et al.,2019 preparation of dosage forms (Ajazuddin and Shailendra, 2010). Despites alcohol's low extraction capacity, it is sometimes more preferred than water especially in researches that deals with natural products because it serves as preservative against microbial growth and easy to evaporate and handle. The powdered stem of A. chevalieri was extracted using successive cool maceration in aqueous and methanol as extracting solvents. These solvents were used in order to separate the phytochemical constituents of the powdered stem of A. chevalieri on the basis of their polarity (increasing polarity). The percentage yield of different extracts from 50g of powdered stem of A. chevalieri includes: methanol (18.20%), aqueous (14.40%). Methanol extract has the highest yield (18.2%). However, Kokate et al. (2003) reported the ability of highly polar solvents to extract most of the phytochemical constituents present in plant materials. The elemental analysis revealed some of the elements that are present in the twig of A. chevalieri. The elements are rich sources of macro and minor elements that aid in the growth of plants, and as well in human body functions such as muscle contraction, bone formations, growth, metabolism, osmotic balance, regulatory processes activation and other organic bimolecular activities (Rabia et al., 2012). The concentrations of elements gotten from this study were within 200 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 FAO/WHO (2004) permissible limits for edible plants. Zinc (Zn) is an essential component of a number of enzymes present in animal tissue including alcohol dehydrogenase, carbonic anhydrase, procarboxypeptidase and aids in normal growth, reproduction, tissue repair and wound healing. Zinc deficiency causes growth retardation and skin lesions (Chatterjee and Shinde, 1995). Concentration of elements in plants varies from region to region due to factors such as environmental, atmospheric, pollution, season of collection of sample, age and soil conditions in which plant grows (Faizul and Rahat, 2011). Preliminary phytochemical screening gives a brief idea about the qualitative nature of active phytochemical constituents present in plant extracts, which will helps the future investigators regarding the selection of the particular extract for further investigation or isolating the active principle (Mishra et al., 2010). Phytochemical analysis of the stem-bark extracts had revealed the presence of some secondary metabolites namely carbohydrate, alkaloids, tannins, flavonoids, cardiac glycosides, saponins, steroid/triterpenes and absences of anthraquinones; this result is in agreement with the finding of Mann et al. (2010) and Aliyu and Sani (2011). The information on the presence or absence and the type of phytochemical constituents especially the secondary metabolites are useful taxonomic keys in Namadina et al.,2019 identifying a particular species and distinguishing it from a related species, thus helping in the delimitation of taxa (Jonathan and Tom, 2008). These phytochemicals are known to exhibit medicinal activity as well as pharmacological activity. Alkaloids have a wide range of pharmacological activities including antimalarial (e.g., quinine), anticancer (e.g., homoharringtonine) (Kittakoop et al., 2014), antibacterial (e.g., chelerythrine) (Cushnie et al., 2014), and antihyperglycemic activities (e.g., piperine) (Qiu et al., 2014). Tannin is one of the major active ingredients found in plant based medicines (Haslam, 1996); they are used in the dyestuff industry as caustics for cationic dyes. Chromatographic analysis of methanol extracts of A. chevalieri stem were carried out on TLC plate (silica gel) using suitable solvent systems and sprayed with p- anisaldehyde reagent. The Thin Layer Chromatography chemical screening is usually done to target isolation of new or very important constituent present in the plant extracts which has marked pharmacological activities and also serves as an important tool in recognizing how metabolite for isolation behaved and can be purified; hence, channeling scientific efforts towards the desired compound(s) and prevent waste of resources and time (Patra et al., 2012). The success of separation of bio-molecules by 201 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 chromatographic technique is markedly influence by the suitability of the separating solvent systems largely influences the successful and also rely upon an ideal range of partition c o e ff i c i e n t ( k ) f o r e a c h t a rg e t compound(s) (Ito, 2005). Acute toxicity studies of Albizia chevalieri revealed that there were no deaths and any sign of toxicity such as loss or increase in weight, tiredness, abdominal constrict convulsion, hyperactive, weakness, diarrhea or increased diuresis within the short and long term effect in rats dosed with 5000 mg/kg body weight of the Albizia chevalieri extract (methanol). The outcome of the study of Alhassan et al., (2014) gave an LD50 of 2000 mg/kg and this guided our choice of dose used (5000 mg/kg). The LD50 was found to be greater than 5000 mg/kg body weight orally, and this suggested that the extract has low acute toxicity when administered orally. This may be attributed to the incomplete absorption brought about by inherent factors limiting absorption in the gastro intestinal tract (Dennis, 1984). The present study agrees with the work done by Prasanth et al., (2015); Ugbogu et al., (2016); Kofi et al., (2014) and Adesegun et al., (2016); . Bruce, (2006) reported that any substance with LD50 estimated to be greater than 2000-5000 mg/kg body weight given orally could be considered to be of low toxicity and safe. The very Namadina et al.,2019 high LD50 observed is not a conclusive finding about the safety of the extracts of A. chevalieri, higher doses could be tested for better understanding of its effects if use for a long period of time and for proper recommendation on its future utilization (Olson et al., 2000; Rang et al., 2001; Maikai et al., 2008; Ogbonnia et al., 2011). CONCLUSION From the results obtained, A. chevalieri possess secondary metabolites which include alkaloids, tannins, flavonoids and saponins. The values of Fe, Mn and Ni in the plant were below the FAO/WHO (1984) permissible limit for edible plants. However, Pb, Zn, Cd and Cu were found to be within the safety limit. The Acute toxicity (LD50) of the methanolic stem extract of A. chevalieri was found to be greater than 5000 mg /kg and is considered safe for use. Nonetheless, further studies are encouraged to evaluate toxicity at much higher doses. Acknowledgement Special thanks to the Director Academic Planning, Bayero University, Kano ,Prof. B.S.Aliyu, the entire academic and technical staffs of Department of Plant Biology, especially Dr. A.A Aliko, Dr. Muhammad Hayatu, Dr. Lawan Sani Abdu, Aisha Muhammad Galalain for their contribution towards the achievement of this work. 202 Faculty of Agriculture, Ibrahim BadamasiBabangida University, Lapai 2019 Lapai International Journal of Agriculture Vol. 1(2) December 2019 pp 186- 207 ISSN:2705-2869 Namadina et al.,2019 Extract of Albizia chevalieri Harms. Bajopas, 2 (1):149-53 Aliyu, B.S., and Sani, H. D. (2011). Invitro Antibacterial Activity of Anogeissus leiocarpus (stem bark) Extracts against Escherichia coli and Staphylococcus aureus. 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