Pharmacognostic profiles and toxicity studies of Melastomastrum capitatum R. & A. Fern. (Melastomataceae) leaf methanol extract

Cletus A. U. et al., Pharmacognostic profiles and ……. 127 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.2 August, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 Pharmacognostic profiles and toxicity studies of Melastomastrum capitatum R. & A. Fern. (Melastomataceae) leaf methanol extract Cletus A. Ukwubile 1*, Matthew O. Agu 2, Hassan B. Yesufu 3, Hananiya H. Milagawanda 3, Bashir K. Bello 3, Simon Paul 4 1. Department of Pharmacognosy, Faculty of Pharmacy, University of Maiduguri, Nigeria. 2. Department of Pure and Industrial Chemistry, Nnamdi Azikiwe University Awka, Nigeria. 3. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Maiduguri, Nigeria. 4. Department of Biological Sciences, National Open University, Abuja, Nigeria. *Email: [email protected]; caukwub[email protected] DOI No.: http://doi.org.10.55639/607.phar.101201.0016 Abstract Melastomastrum capitatum is a shrubby herb whose leaf has been used for treating diseases like cancer, diabetes, inflammation, pains, and hypertension in Mambila plateau, Taraba State, Nigeria. This study was aimed evaluating the pharmacognostic profiles of leaf and toxicity profiles of methanol leaf extract. Pharmacognostic studies of the leaves were carried out by determining various parameters such as chemomicroscopy, qualitative microscopy, physicochemical, and phytochemical contents, and acute and sub-chronic toxicities following standard procedures. Pharmacognostic studies showed that the leaf contains anomocytic type of stomata which are more on the upper surface (12-13.4-16 mm2) than the lower surface (8-9.5-12 mm2), many covering trichomes with acute apex and swollen base as well as simple starch with centric hilum while cutin, suberin gums and mucilage were absent. Phytochemical screening of the leaf methanol extract revealed the presence of metabolites like carbohydrates, alkaloids, saponins and flavonoids. All the physicochemical parameters were within the acceptable ranges. Acute oral toxicity of the leaf extract administered to rats at a dose of 5,000 mg/kg body weight (b.w.) does not show any sign of toxicity after 14 days. Sub-chronic toxicity evaluation of rats for 28 days administration of extract at doses of 150, 300, 600 and 1200 mg/kg b.w. orally resulted in a slight increase in body weights, relative organ weights, and hematological parameters like packed cell volume (PCV), white blood cells (WBC) and platelet counts in a dose-dependent fashion in all groups. There was no rise in the values of liver function parameters like aspartate amino transferase (AST) and bilirubin at the dose of 1,200 mg/kg b.w. Photomicrographs from histological examinations of some vital organs of rats showed no abnormalities in the liver, lungs, ovary, testis, heart, and pancreas. Necrosis was, however, observed in the kidney at the dose of 1,200 mg/kg b.w. The study showed that M. capitatum leaf contain important pharmacognostic features which will help in preventing adulteration by ensuring quality standards. It further showed that the leaf extract is safe for use in ethnomedicine for treatment of diseases. Keywords: Melastomastrum capitatum, Pharmacognostic, Necrosis, Toxicity studies, Wistar rats. Introduction Cletus A. U. et al., Pharmacognostic profiles and ……. 128 Plants have been used for medicinal purposes long before orthodox medicines came into existence. Ancient men observed and appreciated the great diversity of plants within their arsenal for use in the treatment of various diseases (Iwu, 2014). Plants provide the following to man: food, clothing, shelter, and medicine. Most of the medicinal uses of plants seem to have evolved through observations of wild animals, and by trial and error (Sofowora et al., 2013; Iwu, 2014). As time proceeds, each geographical entity added the medicinal power of herbs in their area to its knowledge base. Ancient people used observable features and collected information on the herbs and developed welldefined herbal pharmacopoeias which are still in use today. Many drugs listed as conventional medications were originally derived from plants. For example, salicylic acid, which is a precursor of aspirin was originally derived from white willow bark and the meadow sweet plant. Cinchona bark is the source of antimalarial drug quinine. The opium poppy gave rise to morphine, codeine, and paregoric, which is also a remedy for diarrhoea. Melastomastrum capitatum is a shrubby herb that grows up to 1∙25 m high, and it is found in dry situations and streambanks in Nigeria especially in Mambila Plateau, Taraba State (Ukwubile and Agabila, 2015), Guinea, Mali, Uganda, and Angola. In Nigeria, it is locally called ‘’Belkon’’ by the Fulani tribe (in Mambila Plateau, Nigeria) who use the leaf to treat cancers traditionally (Ukwubile, 2017). A large part of the plant has a sweetto-sour taste. The leaf-sap diluted into a little water is used in Ivory Coast as a sedative. Its leaf can reduce cholesterol, act as an analgesic, and clean the blood vessels in traditional medicine. In South-south Nigeria, the leaves have been used to heal many wounds (Hutchings, 1996). The methanol leaf extract has been reported to be cytotoxic on ovarian (OV7), breast (MCF-7) and vaginal melanoma (HMVII) cancer cells (Ukwubile et al., 2023). The leaves are somewhat distinctive, being opposite decussate and usually contain sweet and sour tastes, with 3-7 longitudinal veins arising either from the base of the bladder (inner veins diverging above the base of the blade) veins diverging or pinnately nerved with three or pairs of primary veins diverging from the mid-vein at succession point above the base. Flowers are perfect and borne either singly or in terminal or axillary; paniculate cymes (Burkill, 1984; Hutchings, 1996). Several Melastomastrum capitatum are regarded as invasive species once naturalized in tropical and subtropical environments outside of their normal range. The leaf extract contains mainly glycosides, alkaloids, and carbohydrates. The leaf extract possesses analgesic and anti-inflammatory properties as well as anti-hypercholesterolemic activity (Ukwubile et al., 2016). The leaf methanol extract has been shown to possess analgesic and antiinflammatory activity in Swiss albino mice. The leaves are used as an antirheumatic agent; cure stomach-aches, purification of blood vessels and blood as well as for alleviating diuresis, as sedatives, and correcting pulmonary problems. The plant contains alkaloids, glycosides, carbohydrates, sterols, and tannin (Ukwubile and Agabila, 2015). Cletus A. U. et al., Pharmacognostic profiles and ……. 129 The present study was aimed at evaluating the pharmacognostic profiles of leaf and toxicity of methanol leaf extract of M. capitatum for taxonomic and quality standards. Materials and materials Chemicals and reagents Methanol, ethanol, formalin, sulphuric acid, hydrochloric acid, H & E stains, chloral hydrate, safranin, and other necessary reagents were obtained from Sigma-Aldrich (St Louis Mo, USA) unless mentioned otherwise. Every chemical and reagent used in the experiment were of analytical grades. Equipment and apparatus Compound microscope (Olympus, UK), camera lucida (ThermoFisher Scientific Ltd., UK), oven, rotary evaporator (ThermoFisher Scientific Ltd., UK), auto hematology analyzer (BC2800, China), RefltronPlus (Roche, USA), flasks, beakers, etc. Collection, identification, and preparation of plant materials The fresh leaves of M. capitatum were collected into a clean sack in the evening hour in December 2017 at Mambila Plateau, Taraba State, Nigeria. The plant was identified and authenticated by a taxonomist Mr Namadi Sanusi in the Department of Botany, Ahmadu Bello University, Zaria. A voucher specimen number of 2761 was the reference number of the plant at the herbarium. The leaves were then air-dried under shade for two weeks until constant weight was obtained and were pulverized into fine powder using an electronic blender. The powder was then sieved using sieve number 20 mesh to obtain a fine powder and remove any debris and was weighed on a scale balance to know the initial weight of the powder. The powdered plant material was then kept in a clean and dried container for further use. Plate I. Picture of M. capitatum in its habitat in Mambila plateau Taraba State, Nigeria (Source: Ukwubile and Agabila, 2015) Cletus A. U. et al., Pharmacognostic profiles and ……. 130 Chemomicroscopy of powdered leaf Chemomicroscopical analysis of the powdered leaves was carried out to determine cell wall components such as cellulose, hemicelluloses, suberin, cutin, gums and mucilage and lignin, as well as cell inclusions like starch grains, calcium oxalate crystals, calcium carbonate, fats, and oils. The following were determined following the procedures described by Evans (2009): i) Cellulose and Hemicelluloses: 0.5 g of powdered leaf was stained in N/50 iodine solution and observed under the microscope using 4x objective. The appearance of blue colour indicated the presence of hemicelluloses. When the powder was treated with 80 % (w/w) H2SO4, a blue colour indicated cellulose. When the powdered leaf was treated with ammoniacal solution of copper II oxide and dilute H2SO4, cellulose was dissolved. ii) Suberin and Cutin: 0.5 g of powdered leaf was treated with a solution of chlor-zinciodine and observed under the microscope using 4x objective. The formation of a yellow brown colour showed the presence of suberin or cutin. When the solution was stained with potash, suberin and cutin were stained yellow, but on warming with 20 % (v/v) solution of potash, yellowish droplets exuded indicating the presence of suberin. Cutin is more resistant to colour change on warming with potash (Kokate, 1986). iii) Gums and Mucilage: 0.5 g of powdered leaf of M. capitatum was stained with Ruthenium red solution and observed under the microscope using 4x objective, A pink colour indicated the presence of mucilage, while bright red colour on the addition of two drops of lead acetate solution indicated the presence of gums (Kokate, 1986). iv) Lignin: 0.5 g of powdered leaf of M. capitatum was mounted in 1 % phloroglucinol solution in 90 % (v/v) ethanol with Conc. HCl and observed under the microscope using 4x objective. The presence of pink colouration indicated lignin (Evans, 2009). v) Starch: 0.5 g of powdered leaf of M. capitatum was stained with the solution of N/50 iodine and observed under the microscope using 4x objective. The formation of a blue-black colour indicated the presence of starch (Evans, 2009). vi) Calcium oxalate crystals: 0.5 g of powdered leaf of M. capitatum was mounted in glycerin solution and viewed under polarized light. The presence of shining crystals showed the calcium oxalate. vii) Calcium carbonate (CaCO3): 0.5 g of powdered leaf of M. capitatum was mounted in HCl solution. The appearance of effervescence revealed the presence of CaCO3. viii) Fats and oils: 0.5 g of powdered leaf of M. capitatum was mounted in Sudan IV solution and heated gently on a Bunsen burner. The presence of red colouration was used as an indication of fats and oils. Examination of M. capitatum leaf epidermal characters The fresh leaf of M. capitatum was nicked with a razor blade. The epidermis was then peeled with forceps on both upper and lower Cletus A. U. et al., Pharmacognostic profiles and ……. 131 epidermal surfaces and mounted in a drop of alcohol and 1 drop of dilute glycerol solution. It was then observed under the microscope using the 10x objective. Images of epidermal features were taken using a dual camera lens (Infinix S5 smartphone) with high-resolution cameras (Kokate, 1986). Quantitative microscopic features of M. capitatum fresh leaf To examine leaf surface data of the plant, the methods described by Kokate (1986) and Evans (2009) were used for the following fresh leaf features: stomatal number, stomatal index, vein-islet and veinlet termination numbers as well as palisade ratio. Twenty consecutive readings (under 4x objective) were taken and represented as means of original values. The following were examined: i) Stomatal number and stomatal index: Briefly, a fragment of leaf from the middle lamina was cleared in chloral hydrate solution. The upper and lower epidermis of the leaf were separately mounted in a solution of glycerin water. A square 2 x 4 mm was drawn using a stage micro-meter and a digital device on drawing paper, and the stage micro-meter was replaced by the cleared leaf preparation and focused using 4x objective. The number of the stomata and epidermal cells were counted (two guard cells and ostiole being counted as one) within the square. The stomatal index (S.I) was calculated using the formula below: S.I = (S/E+S) 100 Where, E = epidermal cells, and S = number of stomata. ii) Vein-islet and veinlet termination numbers: In determining this, the leaf sample was boiled in a solution of chloral hydrate in a test tube until it was cleared. After clearing, it was mounted in a solution of glycerin. Camera Lucida was set up to divide the paper into two square mm using the stage micrometer. The stage micrometer was then replaced by the cleared leaf preparation, and the veins were traced in a square of 2 mm x 4 mm, following the superimposition of the image of the leaf portion on paper. The number of vein-islets and veinlets termination present within the four contiguous squares was counted and the total number of each was divided by four to get the value in square mm. A total of three sets of counts were made. iii) Palisade Ratio: Powdered leaves from each of the leaf surfaces were cleared by boiling in chloral hydrate solution mounted in glycerin water and then focused under 4x magnification. A camera Lucida was attached and the outlines of four contiguous epidermal cells were traced and marked using pencil. Palisade cells within each group were counted including those that were more than half covered by the epidermal cells. The values obtained from the counts were divided by four to obtain the palisade ratio (Kokate, 1986). Physicochemical evaluation of M. capitatum leaf powder The methods described by Brain and Turner (1975) were used to determine the following physical constants: i) Moisture Content: Three grams of the powdered leaves were accurately weighed in Cletus A. U. et al., Pharmacognostic profiles and ……. 132 a tarred silica dish. It was then heated for one hour at 105 0C in an oven, cooled in desiccators and then reweighed. This procedure was repeated until a constant weight was obtained. The percentage moisture content was then calculated concerning the initial weight of the powdered leaves. Values obtained were also compared with those in the official book. ii) Total ash: Three grams (3 g) of the powdered drugs were weighed accurately into nickel crucibles for each part. The contents were heated gently until it was moisture free and then completely charred at a temperature not greater than 450 0C. The heating was continued to remove all the carbon contents and allowed to turn to ash. Finally, the content was cooled and weighed to obtain a constant weight. The ash values were then calculated concerning the initial weight of the powder. iii) Acid insoluble ash: The total ash produced from the above (ii) was transferred to a beaker containing 25 mL of dilute HCl solution, and boiled for 5 min. The insoluble matter was collected on an ashless filter paper. The content in the beaker was washed with hot water. The washing was allowed to pass through the filter paper. The washing was continued until the beaker was free from acid. The residue and the filter paper were dried gently and ignited in tarred crucibles. The crucibles were cooled and weighed. Acid insoluble ash values were then calculated regarding the initial weight of the powdered leaves (British Pharmacopeia, 1968). iv) Water-soluble Ash Value: The total ash produced was boiled in 25 mL of water for 5 minutes. The insoluble matters were collected as in (iii) above. The same procedure above was repeated. The difference between the insoluble matter and the weight of the ash was taken as the watersoluble ash value (British Pharmacopeia, 1968). v) Determination of alcohol extractive value: One gram of the powdered drug was shaken with 95 % ethanol 50cm3 in 250 mL stopperd flasks for 6 h using a mechanical shaker and allowed to stand for 18 h. and was filtered immediately. 20 mL of each filtrate was transferred into a tarred evaporating dish whose weight and that of the flat bottom flask were already known. Using a hot plate, they were evaporated to dryness and dried to constant weight at 105 0C in an oven and the final weight was noted. The alcohol extractive values were calculated with reference to the initial weight of powdered drugs (Ukwubile and Nuhu, 2010). vi) Determination of water extractive value: The above procedure in (v) was repeated using chloroform – water (0.25% V/V chloroform in distilled water), in place of ethanol as the extracting solvent. Values were calculated with reference to the initial weight of drugs. Extraction of plant material Three and a half kilograms (3.5 kg) of powdered leaves of M. capitatum were defatted with petroleum ether to remove fat, latex, and non-polar compounds of high molecular weights (Altemimi et al., 2017). The defatted plant residue was extracted with 10 litres of absolute methanol using the Soxhlet apparatus for 12 h at a temperature of 64.7 °C, to obtain the methanol extract Cletus A. U. et al., Pharmacognostic profiles and ……. 133 (MCE). The solvent was regularly changed until no colouration was observed. The collected extract was filtered through a sieve cloth of small diameter. Finally, the filtrate was concentrated in a vacuum using a rotary evaporator. The final weight of the extract was then noted, and the percentage yield was calculated with reference to the initial weight of the powder as given below: % 𝑌𝑖𝑒𝑙𝑑 = 𝐹𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑜𝑤𝑑𝑒𝑟𝑒𝑑 𝑑𝑟𝑢𝑔 ×100 The extract was then stored in a clean dried sample bottle and kept in a desiccator for further use. Phytochemical screening of M. capitatum leaf methanol extracts The methods described by Odebiyi and Sofowora (1978), Evans (2009), and Koparde et al. (2021) were used to test for the presence of secondary metabolites below: i) Tests for saponins To test for the presence of saponins in M. capitatum leaf methanol extract, the following tests were carried out: Frothing test: 0.5 g of the extract was dissolved in 5 mL chloroform, shaken vigorously for 30 sec, and then allowed to stand for 30 min. The formation of foam which persisted for 5 min or more indicated the presence of saponins. Hemolysis test: 2 mL of extract solution was put in a test tube containing 5 mL 1.8 % aqueous NaCl solution. After this, a few drops of blood from rats were added to the test tube. It was allowed to stand for 30 min and observed for occurrence of hemolysis of the RBC which indicated the presence of saponins. ii) Tests for flavonoids To test for the presence of flavonoids in the leaf extract, the following tests were carried out: Sodium hydroxide test: 0.5 g of the extract dissolved in 5 mL distilled water, and two drops of aqueous NaOH solution were added. The appearance of yellow colour was an indication of flavonoids. Shinoda’s test: This was carried out by heating the extract in 2 mL of 50 % methanol followed by the addition of metallic magnesium. Four drops of conc. HCl solution was then added. The formation of an orange colour indicated the presence of flavonoids. iii) Tests for tannins To test for the presence of tannins in the leaf methanol extract, the following tests were carried out: Ferric chloride test: 0.5 g of the extract was dissolved in 5 mL of distilled water and filtered. Then, two drops of 5 % FeCl3 solution were added to the solution. The formation of a dark blue colour showed the presence of condensed tannins. Lead acetate test: To 0.5g of extract dissolved in 5 mL of distilled water, two drops of lead acetate solution. The formation of brown colour was taken for the presence of tannins. Goldbeater’s skin test: A small piece of Goldbeater’s skin was soaked in 2 % HCl and rinsed with 10 mL distilled water. It was then placed in the extract solutions for 5 min and Cletus A. U. et al., Pharmacognostic profiles and ……. 134 washed with distilled water. The skin was then transferred to a 1 % (v/v) solution of ferrous sulphate. The formation of a black colour on the skin was taken for the presence of tannins. iv) Tests for alkaloids To test for the presence of alkaloids in the methanol leaf extract of M. capitatum, the following tests were carried out following the methods described by Evans (2009): Dragendorff test: A few drops of Dragendorff reagent were added to 0.5 g of the extracts dissolved in 5 mL distilled water. The formation of orange precipitate indicated the presence of alkaloids. Mayer’s test: Two drops of the reagent were added to extract dissolved in distilled water in a test tube. The formation of a creamy precipitate showed the presence of alkaloids. Wagner’s test: Two drops of the reagent were added to extract the solution in a test tube. The formation of a brown precipitate showed the presence of alkaloids. v) Tests for cardiac glycosides To detect the presence of cardiac glycosides in the leaf methanol extract, the following tests were carried out: Baljet’s test: 0.5 g of extract was dissolved in a test tube containing 5 mL of distilled water. Two drops of sodium picrate were added. The formation of yellow to orange colour was an indication of cardiac glycosides. Kedde’s test: 0.5 g of the extract was dissolved in 5 mL distilled water. Then, 2 mL of pyridine and sodium nitroprusside were each added. The formation of pink to red colour indicates the presence of cardenolides Keller-Kiliani test: 0.5 g of the extract was dissolved in 5 mL 0.5 mL glacial acetic acid followed by a drop of 5 % FeCl3, and Conc. H2SO4 by the wall of the test tube. The formation of a reddish-brown colour at the interphase of the two liquids confirmed the presence of deoxysugar characteristics of cardenolides. Liebermann’s test: 0.5 g of the extract was dissolved in 5 mL distilled water. It was followed by the addition of 1 mL acetic anhydride, gently heated for 5 min, and cooled. Subsequently, a drop of Conc. H2SO4 were added to the wall of the test tube. The formation of a reddish-brown colour at the interface and a blue colour at the upper level indicated the presence of a steroidal nucleus. vi) Tests for anthraquinones To test for the presence of free or combined anthracene in the extract, the following tests were carried out: Borntrager’s test: 0.5 g of the extract was dissolved in 5 mL distilled water followed by the addition of 2 mL dil. H2SO4 solution. The mixture was boiled for 5 min in a water bath filtered using filter paper and allowed to cool. The cold filtrate was then added to an equal volume of chloroform, shaken vigorously for 2 min and filtered. To one portion of the filtrate, 2 mL of 10 % NH3 solution was added. The appearance of pink colour at the ammoniacal layer confirmed the presence of anthracenes. Modified Borntrager’s test: To a fresh portion from the above, 5 mL 5 % FeCl3 and Cletus A. U. et al., Pharmacognostic profiles and ……. 135 2 mL dil. HCl solutions were added and then heated for 5 min, filtered while hot followed by the addition of 2 mL benzene and shaken very well for 3 min. The benzene layer was filtered, and an equal volume of 10 % dil. ammonia solution was added to it. The formation of a pink red colour at the ammoniacal layer indicates the presence of free anthracene. vii) Tests for steroids and triterpenoids To test for the presence of steroids and triterpenoids in the extract, the following were carried out: Salkowski reaction: 0.5 g of extract was added to 2 mL chloroform followed by 3 mL Conc. H2SO4 solution. The formation of a reddish-brown colour at the interface indicated the presence of steroids and terpenoids. Liebermann-Burchard reaction: 0.5 g of the extract was dissolved in 2 mL chloroform and 1 mL acetic anhydride was added. Finally, 2 mL of conc. H2SO4 solution was added to the side of the test tube. The formation of a reddish-brown ring at the interface showed the presence of steroids and triterpenoids. Experimental animals Adult Wistar rats of either sex weighing between 160-200 g were purchased from the Animal House of the Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Ahmadu Bello University (ABU), Zaria, Nigeria. Ethical approval for the use of the animals were obtained from the Ahmadu Bello University Zaria Committee on Animal Use and Care with the approval number ABUCAUC/2021/073. Ethical considerations guiding the conduct of research with life animals of the University were strictly followed and the guidelines as outlined by ABU research policy (NIH, 1985; NRC, 1996). Acute (LD50) and s-chronic toxicity study on extract Acute oral toxicity study on M. capitatum leaf extract (limit test) The acute oral toxicity study was carried out following OECD guideline 452, which stipulates the use of only five animals (OECD, 2009; Sankhari et al., 2010; Johnson et al., 2013). Five rats of either sex (3 males plus 2 females) was weighed and fasted overnight. Test doses of M. capitatum MCE were calculated in terms of their body weight and administered via oral gavage at 5,000 mg/kg body weight (b.w) maximum dose at once. The animals were regularly and individually observed for behavioral changes and general toxicity signs after dosing for the first 24 h, with special attention being given during the first 4 h. After these, observations continued daily for a total of 14 days (Nana et al., 2011; Bello et al., 2016). Oral subchronic toxicity study (28 days) A total of thirty (30) Wistar rats of either sex was divided into five groups of six each following the methods described by the OECD guidelines paragraph 425 (2009). Animals in Groups II, III, IV, and V were orally administered 24 % of the maximum dose from acute toxicity study i.e., 150, 300, 600 and 1200 mg/kg b.w. M. capitatum crude leaf extract respectively, once daily for 28 Cletus A. U. et al., Pharmacognostic profiles and ……. 142 Physicochemical contents of powdered M. capitatum leaf Table 5 shows the results of the physiochemical parameters of M. capitatum leaf powder. The data obtained showed the percentage total ash value of 12.39 ± 1.02 % w/w. The extractive value of water was 6.82 ± 0.10 % w/w while the moisture content was 6.16 ± 0.01 %w/w. Acid insoluble ash value was 0.56±0.01 % w/w. Table 5. Physicochemical parameters of M. capitatum leaf Parameter Mean ± SD (%w/w) Moisture content 6.16±0.01 Total ash 12.39 ± 1.02 Acid insoluble ash 0.56±0.01 Water soluble ash Water extractive Alcohol extractive 3.83±0.02 6.82 ± 0.10 4.14 ± 0.20 Results are mean ± SD, n= 3. Phytochemical contents of M. capitatum leaf ethyl acetate extract The results of phytochemical screening of ethyl acetate leaf extract of M. capitatum revealed the presence of carbohydrates, saponins, alkaloids and flavonoids while other metabolites were absent as summarized in Table 6 below. Cletus A. U. et al., Pharmacognostic profiles and ……. 143 Table 6. Phytochemical content of M. capitatum leaf ethyl acetate extract Constituent Tests Observation Inference Carbohydrate Molisch Barfoed’s Blue-black Blue Present Present Saponin Frothing Haemolysis 30 min Bursting RBC Present Present Anthraquinones Borntrager Mod. Borntrager Pink-red Bright-pink Absent Absent Cardiac glycosides KellerKiliani Kedde’s No brown ring No colour Absent Absent Flavonoids Shinoda NaOH Red colour Yellow colour Present Present Tannins Ferric chloride Lead subacetate. Goldbeater’s No colour No ppt No colour Absent Absent Absent Alkaloids Dragendorff Mayer’s Wagner’s Test Orange ppt Creamy ppt Cloudy ppt Present Present Present Steroids/Triterpenes Salkowski Lieberman-Burchard Reddish-brown Reddish-brown Present Present Acute and subchronic toxicities of M. capitatum leaf extract in Wistar rats In the acute toxicity study, there were no deaths, or any signs of toxicity observed in the rats administered with a limited dose of 5,000 mg/kg of MCE after 14 days. Clinically, the rats did not show any sign of toxicity within the observed period. They were active and their eyes were clear with no body itching. However, increased water intake which lasted for about 10 min was witnessed in the animals at 5, 000 mg/kg b.w. extract (Table 7) Cletus A. U. et al., Pharmacognostic profiles and ……. 144 Table 7. Median lethal dose of M. capitatum leaf extract in Wistar rats. __________________________________________________________________ Dose (mg/kg) Animal died/Animal survived 5000 0/5_________________________________ n = 5; hyperactivity was witnessed at 5,000 mg/kg b.w. in the animals only for 10 secs; LD50 > 5,000 mg/kg Similarly, in subchronic toxicity study, the body weights of rats slightly increased in Groups I-V at doses 150, 300, 600 and 1200 mg/kg of MCE orally from day one to day 28 of the study. The highest dose of 1200 mg/kg body weight (b.w.) extract administration did not reduce the animal’s weight implying that the extract was well tolerated by the animals orally. However, these increases were significantly different (p < 0.05) from the normal control group that received distilled water within the same period (Table 8). According to the results of the analysis, it is seen that the time frame (four weeks) at which the extract was administered based on the dosage and normal control has significant effect on the weight of the rats (𝐹(5,24)= 8.46, 𝑝 < 0.05). Considering the data in Table 8, it can be concluded that the effect of the application of the different dosage over a four-week period was significant 𝐹(5,24)=13.45, 𝑝 < 0.05. In addition, SPANOVA test results comparing the interaction of these two factors (group * time) are significant (𝐹(5,24)=13.45, 𝑝 < 0.05). The results further showed that there were no toxicities in liver function enzymes, hematological parameters, lipid profiles and histological profiles of the rats after 28 days of subchronic study as shown in the tables below. All values were within the acceptable ranges (Tables 8 to 14; Plate V). Table 8. Effects of M. capitatum leaf methanol extract on body weight of Wistar albino rats following 28 days of oral administration Weeks of oral administration of M. capitatum leaf extract Group WK 1 WK 2 WK 3 WK 4 I NC 161.18±0.42 163.85±0.91 163.50±1.73 164.44±0.28* II 150 mg/kg 160.08±0.12 167.95±0.32 168.25±0.36* 168.40±0.01* III 200 mg/kg 179.90±0.22 182.25±0.08 182.59±0.02* 182.69±0.02* IV 600 mg/kg 199.87±0.20* 200.17±0.05* 200.19±0.02 200.07±0.05* V 1200 mg/kg 200.10±0.02* 200.30±0.04 200.31±0.05* 200.13±0.05* Results are mean ± SD of six rats in each group, WK1-4 (weeks 1-4), NC (normal control with normal saline). *Statistically significant at p < 0.05 (p = 0.000) using split plot ANOVA followed by DMRT’s post hoc test. Cletus A. U. et al., Pharmacognostic profiles and ……. 145 Table 9. Effect of M. capitatum leaf methanol extract on organ weight of rats after 28 days of oral administration. __________________________________________________________________ Relative organ weight after extract administration (mg/kg) Organ (g) NC 150 300 600 1200___ Heart 0.63±0.01 0.78±0.01* 0.82±0.01 0.83±0.02 0.90±0.02* Liver 6.50±0.01 6.30±0.01* 7.49±0.01 7.60±0.02 0.81±0.01* Kidney 0.83±0.01 1.85±0.01* 1.90±0.02 1.93±0.01 1.98±0.02* Lung 1.80±0.01 1.20±0.01 1.28±0.01 1.32±0.01 1.36±0.01* Testis 1.40±0.01 1.49±0.01 1.53±0.01 1.58±0.01* 1.61±0.01 Ovary 1.31±0.01 1.32±0.01 1.38±0.01* 1.42±0.01 1.43±0.01__ Results are mean ± SD of six rats in each group, * significant difference at p ≤ 0.05 (one-way ANOVA) followed by DMRT post hoc test, NC is the normal control group. Table 10. Effect of M. capitatum leaf Methanol Extract on Haematological Parameters of Wistar rats after 28 days of oral administration Dose (mg/kg) Parameter Control 150 300 600 1200 PCV (%) 45.07 ± 0.06 48.17 ± 0.06* 49.31 ± 0.01 49.50 ± 0.01 50.07 ± 0.06* MCV (fL) 52.60 ± 0.01 53.57 ± 0.06 53.80 ± 0.01 56.60 ± 0.01 59.07 ± 0.06* MCH (pg) 48.30 ± 0.01 56.45 ± 0.01 57.90 ± 0.01 67.21 ± 0.01 68.72 ± 0.01* MCHC (g/L) 330.33 ± 0.58 320.40 ± 0.52 323.97 ± 0.41 326.07 ± 0.06 328.98 ± 0.59 RBC (x1012/L) 6.80 ± 0.01 6.78 ± 0.01* 6.81 ± 0.02 6.88 ± 0.01* 8.24 ± 0.01* RDW (%) 19.47 ± 0.06 19.60 ± 0.01 22.27 ± 0.06 24.05 ± 0.05 28.32 ± 0.01* WBC (x109/L) 12.60 ± 0.01 6.90 ± 0.01* 7.20 ± 0.01 7.32 ± 0.01 10.14 ± 0.01* Hb (g/dL) 11.50 ± 0.04 11.80 ± 0.01* 12.20 ± 0.01 12.40 ± 0.01 14.80 ± 0.01* PLT (x109/L) 150.27 ± 0.06 151.07 ± 0.06 250.27 ± 0.06 300.25 ± 0.01 350.10 ± 0.01 Results are mean ± SD of six rats in each group, * Statistically significant at p≤ 0.05 (one-way ANOVA followed by DMRT post hoc test) versus normal control. PLT (platelets), WBC (white blood cells), RBC (red blood cells), PCV (packed cell volume), RDW (red blood cell distribution width), MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), Hb (Haemoglobin count). Cletus A. U. et al., Pharmacognostic profiles and ……. 146 Table 11. Effects of 28 days of oral administration of M. capitatum extract on WBC differentials in Wistar rats __________________________________________________________________ WBC Dose (mg/kg) ____________________________________ Control 150 300 600 1200_ NTR 1.78±0.01 1.79±0.01* 1.88±0.01 2.50±0.01 2.58±0.01* LYM 4.78±0.01 4.88±0.01* 4.95±0.01 8.44±0.01 10.15±0.01* MON 0.01±0.01 0.02±0.01* 0.03±0.01 0.04±0.01 0.04±0.01* EOS 0.03±0.01 0.04±0.00* 0.05±0.01 0.06±0.00 0.07±0.01* BAS 0.01±0.01 0.02±0.01* 0.02±0.01 0.03±0.01 0.03±0.01* Results are mean ± SD of six rats in each group, *p≤ 0.05; significant (one-way ANOVA); NTR (Neutrophils), LYM (Lymphocytes), MON (Monocytes), EOS (Eosinophils), BAS (Basophiles). Table 12. Effects of M. capitatum leaf extract on the liver of Wistar rats after 28 days of subchronic toxicity studies Dose (mg/kg) Parameter Control 150 300 600 1200 ALP (U/L) 56.50 ± 0.01 58.30 ± 0.01* 68.50 ± 0.01 74.29 ± 0.01 80.59 ± 0.01* AST (U/L) 45.30 ± 0.01 45.59 ± 0.01* 48.30 ± 0.01 48.09 ± 0.01 46.44 ± 0.01* ALT (U/L) 20.09 ± 0.52 20.89 ± 0.01* 23.10 ± 0.00 25.49 ± 0.01 25.79 ± 0.01* ALB (g/L) 3.79 ± 0.01 3.81 ± 0.01 3.86 ± 0.01 3.92 ± 0.01* 4.51 ± 0.01* BIL (mg/dL) 0.31 ± 0.01 0.36 ± 0.01* 0.34 ± 0.01 0.32 ± 0.01 0.31 ± 0.01 GLO (g/dL) 1.50 ± 0.01 1.52 ± 0.00 1.88 ± 0.01 1.96 ± 0.01* 2.56 ± 0.01* TPRN (g/dL) 5.61 ± 0.01 5.61 ± 0.00 5.88 ± 0.01* 5.93 ± 0.01 6.90 ± 0.10 Results are mean ± SD of six rats in each group, ALP (alkaline phosphatase), AST (aspartate transaminase), ALT (alanine transaminase), ALB (albumin), BIL (bilirubin), GLO (globulin), TPRN (total protein), *p≤ 0.05 statistically significant (one-way ANOVA). (x109/L) Cletus A. U. et al., Pharmacognostic profiles and ……. 147 Table 13. Effects of M. capitatum extract on serum electrolyte levels after 28 days of subchronic toxicity study Dose (mg/kg) Serum Electrolyte (mmol/L) Control 150 300 600 1200 Na+ 120.60±0.01 118.17±0.06* 124.90±0.10 126.17±0.06 128.17±0.10* Cl76.30±0.01 82.40±0.01* 84.10±0.01 86.30±0.01 88.90±0.02* Ca2+ 1.60±0.01 1.83 ± 0.01* 1.89±0.01 1.90±0.01 2.44±0.01* K+ 4.80±0.01 5.61±0.01* 5.68±0.01 5.88±0.00* 5.91±0.01* PO-4 1.50±0.01 1.44±0.01* 1.42±0.01 1.20±0.01 1.11±0.01* Urea 15.10±0.01 15.30±0.01* 15.55±0.01 16.10±0.01 18.00±0.01* UA 0.05±0.00 0.05±0.01* 0.09±0.01 0.12±0.00* 0.05±0.01* CRE 0.20±0.01 0.20±0.01* 0.25±0.01 0.28±0.00 0.45±0.05* Results are mean ± SD of six rats in each group (n = 6), *p≤ 0.05 statistically significant versus control (one-way ANOVA followed by DMRT post hoc), Ser (serum), PO-4(Phosphate), UA (Uric acid), CRE (Creatinine in mg/dL). Table 14. Effects of M. capitatum Leaf Extract on serum lipid profiles and plasma glucose parameters of Wistar rats after 28 days of subchronic toxicity studies Profiles Dose (mg/kg) ______________________________________ Control 150 300 600 1200________ HDL 2.13±0.01 0.60±0.00* 0.84±0.01 1.27±0.01 2.50±0.00* LDL 0.40±0.00 0.19±0.01* 0.14±0.00 0.11±0.01 0.03±0.01* VLDL 1.20±0.01 1.44±0.00* 1.45±0.01 1.48±0.01* 1.88±0.02* TCH 7.74±0.01 2.34±0.01* 2.44±0.00 2.95±0.01* 4.42±0.00* TRG 0.82±0.01 0.99±0.01* 1.12±0.01 1.63±0.01* 1.67±0.01* GLU 65.10±0.00 68.46±0.42* 80.49±0.01 84.90±0.01 92.09±0.01* Results are mean ± SD of six rats in each group, *p < 0.05 statistically significant (one-way ANOVA followed by DMRT post hoc), HDL (high-density lipoproteins), LDL (low-density lipoproteins), VLDL (very low-density lipoproteins), TCH (total cholesterol), TRG (triglycerides), GLU (plasma glucose). Note: All profiles are expressed in mg/dL. In the photomicrograph sections of the organs shown overleaf (Plate V), there were no major pathological changes in the organs after 28 days of subchronic toxicity study with 1200 mg/kg b.w. dose of M. capitatum leaf extract in rats. However, necrosis was Cletus A. U. et al., Pharmacognostic profiles and ……. 148 observed in the kidney, vascular congestion in the liver, lung, and spleen as well as increased spermatogenesis in the testis at the highest dose (1200 mg/kg b.w.) . Kidney (control) Kidney 1200 mg/kg Liver (control) Liver 1200 mg/kg dose Testis (control) Testis 1200 mg/kg dose Lung (control) Lung 1200 mg/kg dose Cletus A. U. et al., Pharmacognostic profiles and ……. 149 Spleen (control) Spleen 1200 mg/kg dose Plate V: Photomicrographic section of vital organs following a 28-day subchronic administration of M. capitatum in rats. One arrow (normal epithelial cell), two arrows (necrosis), three arrows (vascular congestion); 40x. Discussion and Conclusion Before the era of orthodox medicines, medicinal plants played major roles in the treatment of diseases mostly in developing and underdeveloped nations (Sofowora, 2006; Iwu, 2014). This is owing to their numerous advantages which include accessibility, affordable, mode of preparation and traditional beliefs. Despite the growing use of medicinal plants, some of them have not been evaluated to determine the effects of long-term use (Sofowora, 2006; Mbaka et al., 2010). For instance, in Nigeria, it is estimated that about 20 % of deaths that occur annually result from the long-term use as well as abuse of traditional medicinal plants in the treatment of diseases (WHO, 2018). Evaluation of Pharmacognostic parameters of medicinal plants helps to ensure quality standards, discover lead compounds in drug development, prevent adoration of crude drugs, reveal whether the plant is toxic or not, and develop conservation strategies for medicinal plants (Iwu, 2014). The use of medicinal plants in the treatment of diseases has not yielded immediate therapeutic results due to the uneven distribution of drugs to almost every body’s tissue (Koparde et al., 2021). In this present study, macroscopic features of M. capitatum revealed that mature leaves were pink in colour due to the presence of anthraquinones while young leaves are green. This is because anthraquinones are known to be responsible for colours like yellow, orange, pink and red in plants like apples, strawberries, etc. (Simpson and Amos, 2017; Yadav et al., 2019). The role of this secondary metabolite in the leaf of M. capitatum was similar as seen from the macroscopic studies on the leaves. The leaf also possessed a mixture of sweet and sour tastes when chewed. This characteristic was discovered to be unique to M. capitatum leaf as there were no previous reports on any members of the family Melastomataceae. Although, the berry of Synsepalum dulcificum (Sapotaceae) has been reported to possess sweet and sour tastes due to the presence of glycosides (Wiersema and Leon, 1999). In the current study, the sweet and sour tastes of leaves were also due to the presence of glycosides as revealed from the phytochemical screening. This observation Cletus A. U. et al., Pharmacognostic profiles and ……. 150 was unique to the plant probably due to its habitat within a mash land with a very low temperature of -20 oC. Similarly, the presence of anomocytic (or ranunculaceous) stomata which are more on the upper surface than on the lower surface of M. capitatum leaf is of taxonomic significance in that plants tend to increase the rate of their transpiration with numerous stomata on the upper surface of their leaves (Liu et al., 2020). It is not surprising, therefore, that the presence of more stomata on the upper surface of the leaves was due to its habitat being a wetland with a regular supply of water. This structural adaptation was further supported by the possession of numerous unicellular covering trichomes with acute apex, swollen base, and extended appendages. The extended appendages which are slightly emergent of epidermal cells (Wang et al., 2021), might serve the purpose of anchorage to withstand the force of wind since the plant is in streams and along riverbanks as well as very cold temperate regions. Trichomes have been used in taxonomic identification and a diagnostic feature of a species and may vary among species. This type of trichome observed in the leaf of M. capitatum will serve as a diagnostic feature for its identification from closely related species (Wang et al., 2021). Chemomicroscopic evaluation of the powdered leaves in this current study indicated the absence of cutin, suberin, gums and mucilage were not detected (or absent) whereas cellulose and hemicellulose as well as lignin were present. The study further revealed that the powdered leaves possessed oval-shaped simple starch grains with centric hilum and striations. The absence of some inclusions such as cutin, suberin, gums and mucilage are strategies to facilitate water loss. This is because, cutin and suberin have been reported to serve as water-repellent in plants to prevent excessive water loss in plants (Bernards, 2002). Since the plant was domiciled in a water-rich environment, the results from this study support the absence of these features in the plant. Similarly, the TS of the leaves also revealed that the plant’s leaf possessed thin cuticles which are almost obliterated, collenchyma, compact spongy parenchyma, and collateral vascular bundles (i.e., phloem surrounds the xylem tissue) in concentric (amphicribal or hydrocentric) arrangement. Both the spongy and palisade parenchyma did not contain calcium oxalate crystals. The presence of collateral vascular bundles has also been previously reported in many monocots’ families like Graminea, Solanaceae, and Apocynaceae as well as dicots families like Burseraceae, Fabaceae, etc. (Zhong et al., 1999). These features observed in the leaf were aimed at adapting the plant to its environment (Iwu, 2014). For example, the compact nature of the spongy cells was an adaption to minimize the intake of atmospheric oxygen and facilitate the absorption of dissolved oxygen (Iwu, 2014; Malviya et al., 2023). The moisture content of leaves obtained in the study was relatively low with a value of 6.16± 0.01 %(w/w), while fresh weight and acid insoluble ash values are 3.88 ± 0.01% (w/w) and 0.56 ±0.01 % (w/w) respectively. These values are within the acceptable range because a high moisture content will lead to microbial infestation and deterioration of the leaf (Brain and Turner, 1975). These values Cletus A. U. et al., Pharmacognostic profiles and ……. 151 will further help to check possible adulteration of the leaf powder by closely related species. In another development, extracting the powdered leaves in methanol yielded 16.73 % (w/v). Because it is generally known that polar solvents extract more compounds than non-polar solvents like n-hexane (Abubakar and Haque, 2020). Phytochemicals screening of the methanol leaf extract revealed that it contains alkaloids, saponins, flavonoids fats/oils, steroids and triterpenes. Other metabolites tannins, and cardiac glycosides were not detected. These secondary metabolites detected were responsible for the biological activity of the leaf. For example, flavonoids and alkaloids have played crucial roles as antioxidant and anticancer agents (Zhao et al., 2018), thus, these metabolites played similar roles in this present study as seen from the biological activity of the extract observed in this study. From the current study, acute toxicity evaluation of the leaf extract at a dose of 5,000 mg/kg body weight (b.w.) showed behavioural changes such as body itching, reddish eyes and weight loss which are salient indices for determining the effects of short-term use of herbal drugs on animals (Lorke, 1983). The acute (LD50) study further showed that there were no observable signs of toxicity throughout the experimental periods. This implies that the extract was well tolerated by the animals at the highest dose of 5,000 mg/kg b.w. In the subchronic toxicity test, the animals showed a slight increase in weight from week one to week four among the treatment groups in a dose-dependent manner. These increases were significantly different from the normal control group, which might suggest that the extract has supplied the animals with other body supplements such as proteins, fats, and vitamins (Simpson and Amos, 2017). The animals did not show much increase in the relative weights of organs at the doses administered, but the weights of lungs and testis decreased at the dose of 1200 mg/kg b.w. in group V. These results further suggest that the extract did not induce any form of infection in the rats at these doses. However, the increased activity like a high rate of food consumption witnessed in all the groups during the acute study further revealed that the extract was tolerated in the animals at these doses. This is because, oral administration of plant extract doses has resulted in increased food intake in rats, dogs, and rabbits (Tiwari, 2008). In haematology and clinical pathology, damage to the cardiovascular systems is usually indicated by the values of the haematological parameters. For instance, a damaged heart does not pump blood properly thereby resulting in high pressure in the heart (Ozer et al., 2008). From this study, all haematological parameters increased in a dose-dependent fashion. Monocytes and basophil did not show a further increase between 600 and 1200 mg/kg b.w. doses in the rats which suggest that high doses of the extract do not have a significant effect on these WBC differentials. However, these values were within the acceptable range, and any significant decrease in them could suggest a sign of anomalies in the body, hence the 28 days of subchronic administration of this extract further suggests that M. capitatum methanol leaf extract did