PHYTOCHEMICAL SCREENING, ELEMENTAL ANALYSIS AND ACUTE TOXICITY OF METHANOL EXTRACT OF MONOON LONGIFOLIUM (SONN.) IN WISTAR RATS

310 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.3 December, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 PHYTOCHEMICAL SCREENING, ELEMENTAL ANALYSIS AND ACUTE TOXICITY OF METHANOL EXTRACT OF MONOON LONGIFOLIUM (SONN.) IN WISTAR RATS Yabalu Zakari Abacha1*, Hannah Alim Madziga2, Elijah Tonde Gargah3, Ahidiyu Anaryu Mamza,4 Ijuptil Chiroma4 and Mbursa Chiroma2. 1Department of Pharmacognosy, Faculty of Pharmacy, University of Maiduguri 2Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, University of Maiduguri 3Department of Internal Medicine, Modibbo Adama University Yola 4Department of Internal Medicine, University of Maiduguri Teaching Hospital Corresponding author: [email protected] http://doi.org/10.55639/607.phar.101001.0010 Abstract The plant Monoon longifolium (Sonn.) Thwaites, (PL) is a tall evergreen tree and it is cultivated all over the world especially in Asian Countries. The plant has been commonly used in traditional system of medicine for the treatment of various ailments such as diabetes and fever. This study investigated the phytochemical and elemental contents of M longifolium using well established methods. The study also investigated acute toxicity of the plant in Wistar rats using Up and Down acute toxicity testing according to CIOMS guidelines. Phytochemical analysis of methanol extract of Monoon longifolium leaves indicated the presence of Carbohydrates, flavonoids, tannins, terpenoids, saponins and cardiac glycosides while elemental constituents of the plant included Ca, K, Na and Mg as the most abundant ones. The acute toxicity testing revealed that the plant extract was safe at a dose of 5000mg/kg but histopathology revealed Mesangial cell proliferation, glomerular destruction and tubular sloughing in the kidney at same dose. This study shows the importance of phytochemical, elemental analysis of medicinal plants and their effect on the kidney. Key Words: Phytochemistry, Elemental analysis, Acute toxicity, Histopathology and Monoon longifolium Introduction Many plants are used in traditional medicine practices around the world to treat diseases and these plants are used either in their crude forms or developed into standardised products (Jamshidi-kia et al, 2017). The study of these plants has led to discovery of many new therapeutic remedies to fill the ever-growing need for more efficient and cost-effective medicines. Medicines from renewable sources such as plants and minerals contribute to the reduction in the cost of health care services (Fabricant and Farnsworth, 2001). The plant, Monoon longifolium (Sonn.) also known as Polyathia longifolia (Sonn.) belongs to the family Annonaceae and is a 311 native to India and Sri Lanka (POWO, 2024). It grows throughout the tropical and subtropical parts of these countries and is commonly called Ashoka in India (Dattatray et al.,2021). According to Faizi et al. (2003), it is also known as Buddha tree, mast tree or cemetery tree. It has now been introduced and cultivated in Southeast Asia, Australia, New Zealand and Africa (Katkar et al., 2010). The genus Monoon now includes about 77 accepted species occurring mainly in South and South-Eastern Asia, Australia, New Zealand and some parts of sub-Saharan Africa (POWO, 2024). The plant as shown in Figure 1 is tall, straight, undivided, evergreen, pyramid-like, columnar in nature and growing up to 12 m or more, (Katkar et al., 2010). In herbal systems of medicine, M longifolium has been used in the treatment of various ailments including diabetes, fever, helminthiasis and various cardiac problems (Vishala,2021). Figure 1 Picture of M longifolium tree (left) near a 2 storey building and the cross section of the leaves (right). Pharmacological investigations have shown that the plant possesses significant biological and pharmacological activity which may include antidiabetic activity. A study of the hypoglycaemic activity of the plant showed that it had similar effect as glipizide in reducing blood glucose levels (Lakshmi et al 2011). With regards to antiinflammation activity, Sharma et al., 2011, investigated the activity using a sub-acute inflammation model where the study found that a diterpenoid isolated from the plant 312 was responsible for the activity. A cell cycle analysis on the effect of the plant methanol extract found that it can decrease cell growth by blocking the growth of Prostate cancer cells in the G1/S phase leading induction of apoptosis. This activity may explain why the plant is used traditionally in the treatment of cancer (Afolabi et al, 2019). Other studies have shown that the plant has antibacterial, antifungal, anti-ulcer and antioxidant properties which makes it a versatile medicinal plant (Faizi et al.,2003, Chang et al., 2006, Sashidhara et al.,2011, Vishala 2021). With regards to toxicity, administration of the methanol extract of M longifolium in Wistar Albino rats was not toxic and safe at a high dose of 3240 mg/kg (Chanda et al., 2012). The aim of this study is to investigate the elemental and phytochemical constituents of M longifolium tree found in North-Eastern Nigeria and to further investigate the histopathology of vital organs after exposure to high dosages. Material and Methods The leaves of M longifolium were collected from the environs of the University of Maiduguri, Borno State, latitude 11.80240 N and longitude 13.19310 E, Nigeria in the month of Sept 2023. It was then submitted to the department of biological sciences and identified by Prof S S Sanusi, a botanist in the department. A voucher specimen was deposited in the herbarium of Veterinary Physiology and Biochemistry with herbarium number Vet. Species 301 A. The fresh leaves were air-dried under shade and ground into fine powder using a milling machine. Approximately 750 g of the pulverized leaves was subjected to Soxhlet extraction using methanol as a solvent and then filtered using Whatman filter paper (18 cm). The filtrate was thereafter transferred to an evaporating dish to concentrate in a hot air oven at 40-50oC. The dry extract was then kept in a clean container and stored at 4oC for future use. Phytochemical Analyses The phytochemical analyses were conducted using standard procedures by as described (Evans 2009). Elemental Analysis This was performed using Atomic Absorption Spectrophotometer (AAS) and the method used is briefly explained below. Samples were prepared via microwave digestion method Sample Pre-Treatment/Digestion 313 The samples were allowed to dry using hot oven (Model 30GC lab oven) and then ground into fine powder by using a porcelain mortar and pestle. The sample was then pre-treated via wet digestion using alcohol and microwave heating as describe below. Microwave Digestion Method 1.0 g of each sample was weighed into thoroughly clean digestion vessel (microwave tube) and then 6 mL of Concentrated HNO3 (65%) were added followed by 2 mL of H2O2 (30% v/v). The mixture was allowed to stand for a while. The digestion vessel was then covered and placed in to microwave digester (Master 40 serial No: 40G106M) to undergo the digestion process. The digestion was carried out at 950 ℃ temperature for 40 minutes. As thereafter followed by a cooling to room temperature in the microwave and the sample was diluted with de-ionized water. Potential presence of heavy metals/metalloids in chemicals used in digestion were then determined. Blanks were used in between each batch of the analysis to authenticate the analytical quality of the samples. Preparation of 1000 mg/Litre stock AAS standard solution The determination of a given metal concentration in the experimental solution was based on its respective calibration curve. In plotting the calibration curve for each element; a stock solution of the element (1000 ppm) supplied by manufacturers company was used, from which a standard working solution of 100ppm was prepared. Standard working solution: 100 ppm was prepared as working solution from the 1000 ppm already prepared. A simple dilution formula (C1V1 = C2V2) was used to determine the volume of the stock solution needed to get a desired concentration. 1 mL of concentrated HNO3 was added to each working standard and finally diluted to the desired volume with deionised water. To prepare 100 ppm, 10 ml of the standard stock solutions were pipetted and added in to 100 ml calibrated flasks finally diluted with deionized water and the solution was mixed thoroughly. The other standard working solutions were prepared from100 ppm by pipetting out appropriate volume in to calibrated flasks and made up to volume with deionized water. In each step of the measurement, the solution and the 314 containers were weighted to ensure accurate measurement (the concentration is reported in w/w instead of v/v). Determination of metal content by AAS Preparation of calibration curve The instrument was calibrated using series of working standards. Calibration curves were prepared to determine the concentration of the elements in the sample solution by measuring the absorbance of each standard and then plotting the absorbance as a function of standard concentration. Determination of metal contents of each sample The metals were determined by absorption/concentration mode and the instrument readout was recorded for each solution manually. The same analytical procedure was employed for the determination of elements in digested blank solutions and for the spiked samples. At least three replicate determinations were carried out on each sample. Concentration of the metal ions present in the sample was determined by reading their absorbance using AAS (Buck Scientific model 210GP) and the concentrations were extrapolated from the respective standard calibration curve. Data Analysis: Data was analysed using Microsoft Office Excel. The data were presented with Mean values as (Mean ± SD). Acute Toxicity study All experiments on animals were carried out according to the biomedical principles involving animals (CIOMS and ICLAS, 2012) and ethical clearance was obtained from the university’s Faculty of Pharmacy Research and Ethics Committee and a reference number FP/052024/UGP01 was assigned to the project. The Up and Down method of acute toxicity testing was used during the study. The rats were kept for acclimatization and observation in the animal house of the Department of Veterinary Physiology and Biochemistry, University of Maiduguri for two weeks. Three (3) Wistar rats were used in this method as briefly described. The first rat was used as control; therefore, it did not receive any extract while the second received 3240 mg/kg and kept under observation for 4 hrs and subsequently up to 72 hrs for clinical signs and mortality. The third was given 5000 mg/kg and observed 315 for the same period as the second rat. At the end of 72 hrs the rats were humanely sacrificed, vital organs were harvested and submitted for histopathology. Results and Discussion In this work, the phytochemical screening of methanol extract of M longifolium leaves indicated the presence of useful compounds such as flavonoids, terpenoids, tannins, saponin and cardiac glycosides. Some of these chemical constituents have many known therapeutic values for instance, flavonoids have antioxidant activity and many health promoting effects. Some of the activities attributed to flavonoids include anti-platelet, antiallergic anti-cancer, antioxidant, anti-inflammatory and antiviral (Ullah et al.,2020). Terpenes have been shown to reduce stress and have antidepressant properties (Bhattacharyya et al. 2007; Subhan et al. 2010), while exhibiting antidiabetic and inflammation properties at the same time (Nabavi et al. 2015). Tannins are diverse organic substances with various compositions that have produced physiological astringent properties that hasten wound healing and ameliorate inflamed mucus membrane (Shenefelt, 2011). Tannins also has haemostatic properties as they are known to maintain balance in biological systems (Awosika 1991). Glycosides are known to produce physiological action. Cardiac glycosides are still the choice drugs for the treatment of congestive heart failure. Additionally, glycosides with laxative, diuretic and antiseptic properties are used for different therapeutic purposes (Fasuyi, 2006). The presence of flavonoids, saponins and tannins in this plant may account for the health promoting activities of Monoon longifolium. Saponins which were identified to be present in the extract have been described as active immune boosters; hence they may be acting in synergy with zinc to promote the immune boosting activity of M longifolium (Jiang et al 2021). The result of the phytochemical analysis of the methanol leaf extract of M longifolium is shown in Table 1 which reveals the presence of carbohydrates, flavonoids, tannins, terpenoids, cardenolides, cardiac glycosides and saponins. The results of this study differ from a recent study by Celestine et al. 2024, where the presence of alkaloids was detected in an ethanol extract of the plant whereas, it was not detected in this study. It is interesting to note that another study by Ankwai et al. 2023 also showed the presence of alkaloids but absence of terpenes and cardiac glycosides in the 316 aqueous leave extract of the plant. These variations may be due to the geographical regions where the plants grow or the type of extraction methods or solvents used in processing the plant extracts (Uddin et al, 2019). Table.1 Qualitative Phytochemical Constituents of Methanol Leaf Extract of Monoon longifolium. Phytochemical Test Inference Carbohydrates Flavonoids Tannins Terpenoids Cardenolides Alkaloid Anthraquinones Cardiac glycosides Saponin glycoside Molisch’s test Monosaccharide Combined reducing sugar Ketones Free reducing sugar Shinoda’s Ferric chloride Sodium hydrochloride Lead acetate Lead acetate Salkowski’s Kellerkiliani Dragendorf’s Mayer’s Free anthraquinones Combined anthraquinones Liebermann-Burchard’s Frothing + - + + + + + + - + + + - - - - + + Extract was subjected to qualitative phytochemical analyses and presence or absence is indicated as + = Positive or - = Negative The concentration of heavy metals in plant materials is of utmost importance as levels above the safe limits can lead to toxicity and illness or they may contribute to the activity of the plant (Elliud & Peter, 2012). It is therefore important to evaluate the levels of heavy metals in herbal plant materials (Luo et al, 2021). The elemental constituents of M longifolium leaf extract included, Cl, K, S, Fe, Mn and Zn, some of which have been known to exert pharmacological effects. Calcium and potassium were the most abundant elements present in our sample as shown in Table 2. Sodium and magnesium were also present in high concentrations while cadmium, chromium, nickel and selenium were present in low concentrations compared to the others. Elements such as Zn, Mn and Se have been postulated to have hypoglycaemic effect (Al-Awadi et al., 317 2004). The presence of Zinc in M longifolium may prove useful in enhancing body immunity (Ngugi et al., 2012). Impairment of chromium and zinc status has been reported as aggravating factors in the progression of diabetes. Chromium potentiates the action of insulin, acting as a co-factor. Also, the elements Zinc and Manganese, have been postulated to possess hypoglycaemic activity (Al-Awadi et al., 2004), therefore, the presence of these phytochemicals and elements in M longifolium might be accountable for the hypoglycaemic effect observed when used traditionally. The plant has been found to contain essential microelements such as sodium, sulphur, manganese, iron, chlorine and zinc which are useful for diverse biochemical and physiological activities of the body. The M longifolium extract also contains chemically active compounds of Pharmacological importance such as tannins, flavonoids, saponin, terpenoids and cardiac glycosides (Sok Yen et al., 2021). Table 2. Elemental Analyses of M longifolium Element Mean ± SD Cadmium 0.013 ± 0.04 Calcium 941.7 ± 10.13 Chromium 0.049 ± 0.004 Iron 1,75 ± 0.03 Magnesium 234.6 ± 13.6 Manganese 0.611± 0.010 Nickel 0.061±0.002 Phosphorus 106.8 ± 3.1 Potassium 928.4 ± 25.5 Selenium 0.039 ± 002 Sodium 758.1 ± 28.7 Zinc 1.88 ± 0.01 The elemental analyses show the quantity of elements present in the extract of M longifolium. Results are presented as SD of 3 independent experiments. The oral administration of M longifolium leaves extract to rats at 3200 and 5000 mg/kg did not produce death in the rats; this indicates a very low toxicity level of the extract. Clarke and Clarke (1977) opined that any substance with LD50 above 1000 mg/kg in rats possesses low toxicity. Acute toxicity of M longifolium leaves at different dosage is shown in Table 3. First phase was dosed at 3240 mg/kg and the 318 second phase at 5000 mg/kg. The rats were observed for signs of toxicity for 2 hours after extract administration in the second phase. The clinical signs to watch out for include abdominal stretching, restriction movement, piloerection, lethargy, and droopy eyes. No death of rats was recorded at the highest dose of 5,000 mg/ kg in the second phase. Therefore, the crude M longifolium methanol extract was observed to have high safety margin with 5000 mg/kg oral dose which is not considered clinically lethal to rats although the kidney showed mesangial cell proliferation, glomerular destruction and tubular sloughing which implies that the plant is not safe for human consumption at very high doses. The results are presented in Table 3. Table 3. Acute Toxicity of methanol extract of leaves of M. longifolium Animals (mg/kg) Duration (Hrs) Result First Rat (0) 72 No death Second Rat (3240) 1st Phase 2nd Phase 4 72 No death No death Third Rat (5000) 1st Phase 2nd Phase 4 72 No death No death The histopathology results are consistent with pathological characteristics in the photomicrograph of liver tissue is shown in Figure 2, where there were no signs of toxicity or lesions. Normal features of a kidney are shown in the photomicrograph in Figure 3. However, Mesangial cell proliferation, glomerular destruction and tubular sloughing are seen in the kidney at 5000 mg/kg as shown in Figure 4.