Phytochemical and Acute Toxicity studies of the Stem bark of Irvingia gabonensis (Aubry-Lecomte ex O'Rorke) Baill. (IRVINGIACEAE)

Troy S. M., Cletus A. U. & Musa Y. D. Phytochemical and Acute Toxicity ….. 157 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.2 August, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 Phytochemical and Acute Toxicity studies of the Stem bark of Irvingia gabonensis (Aubry-Lecomte ex O’Rorke) Baill. (IRVINGIACEAE) T. S. Malgwi 1, K. Y. Musa2, I. M. Maje3, H. M. Mshelia4, Cletus A. Ukwubile 1, M. Y. Dibal 1 1Department of Pharmacognosy, Faculty of Pharmacy, University of Maiduguri, Maiduguri, Nigeria 2Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria. 3Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria 4Department of Pharmacognosy and Ethno Pharmacy,, Faculty of Pharmaceutical Sciences, Usman Dan Fodiyo University, Sokoto Corresponding Author Phone: +234 (80) 63738523 E-mail: [email protected] DOI No.: http://doi.org.10.55639/607.phar.101201.0017 Abstract Phytochemical analysis of plant extracts plays a crucial role in discovering bioactive compounds with potential pharmacological activities. Understanding the phytochemical profile of Irvingia gabonensis could provide insights into its medicinal properties and facilitate the development of novel therapeutics. The conducted research aimed to assess the phytochemical composition and acute toxicity profile of Irvingia gabonensis stem bark with the goal of establishing pharmacognostic standards for the plant. Samples of leaves, fruits, stem bark, and roots were collected from the Iragbiji community, Osun State, Nigeria, and a voucher number was obtained after identification. The stem bark was powdered and sequentially extracted using n-hexane, ethyl acetate, and methanol through maceration. Results revealed the presence of saponins, glycosides, flavonoids, tannins, and steroids in the stem bark extracts. The total phenolic content was found to be 122 mg/mL and 79.47 mg/mL (±SEM) in the ethyl acetate and methanol extracts, respectively. Similarly, the total flavonoid content was measured as 73.42 mg/mL and 36.61 mg/mL (±SEM) in the ethyl acetate and methanol extracts, respectively. The acute oral toxicity studies conducted on rats revealed no signs of toxicity at a dosage of 5000 mg/kg for all experimental animals. These findings suggest that Irvingia gabonensis stem bark possesses phytochemical compounds with potential medicinal benefits and a favorable safety profile. Keywords: Irvingia gabonensis, Phytochemical, Test, Acute, and Toxicity Introduction Traditional medicine remains a cornerstone in global healthcare, serving as the primary source of medical treatment for approximately 80% of the world's population (Owolabi et al., 2007). This reliance on traditional medicine underscores the enduring significance of natural remedies, particularly those derived from plants, in managing various ailments. In African Troy S. M., Cletus A. U. & Musa Y. D. Phytochemical and Acute Toxicity ….. 158 traditional medicine, the utilization of higher plants and their extracts has been welldocumented for treating numerous diseases, contributing to the development of new drugs, and enriching the drug discovery process (Farnsworth and Fabricant, 2001; Cragg et al., 1997). The resurgence of interest in natural remedies stems from their widespread availability, accessibility, and perceived minimal side effects (Chanda, 2014). Additionally, there is a growing inclination towards exploring the correlation between the phytochemical composition of medicinal plants and their pharmacological properties (Sharma et al., 2017). Amidst this backdrop, the Irvingiaceae family emerges as a significant botanical entity within traditional African medicine. The Irvingiaceae family comprises ten species of tropical trees primarily found across Africa, Southeast Asia, and western Malaysia. These trees thrive in regions characterized by specific environmental conditions, including altitudes ranging from 200 to 500 meters, annual rainfall between 1200 to 1500 mm, and temperatures spanning from 20 to 38 °C (Raj et al., 2022). Morphologically, members of the Irvingiaceae family exhibit distinct features such as glabrousness, extremely hard wood, and unique leaf structures with mucilagecontaining secretory canals. Among the genera within the Irvingiaceae family, Irvingia stands out as a particularly noteworthy one. Irvingia species, commonly referred to as wild African mango or bush mango, are valued for their edible fruits and fat/protein-rich nuts. The genus comprises several species, including Irvingia gabonensis, Irvingia smithii, Irvingia grandifolia, and others (Harris, 1996; Leakey, 1999). Irvingia gabonensis, also known as African Mango, is a prominent member of the Irvingiaceae family. Indigenous to West Africa, including countries like Nigeria, Cameroon, and Ghana, Irvingia gabonensis bears edible fruits and seeds that hold significant cultural and medicinal importance. The tree is referred to by various local names such as Apon, Ogbono, and Goron biri in different Nigerian dialects (Unaeze et al., 2017). Culturally, Irvingia gabonensis is cultivated across West African regions for its fruit, which is used in traditional cuisines. The seeds of Irvingia gabonensis yield dika fat, rich in C12 and C14 fatty acids, alongside essential vitamins, minerals, and flavonoids (Sun and Jen, 2012). Phytochemical analyses have revealed the presence of various bioactive compounds in Irvingia gabonensis seeds, including tannins, alkaloids, flavonoids, and terpenoids (Mahunu et al., 2019; Olorundare et al., 2020). Furthermore, Irvingia gabonensis seed extract has demonstrated notable antioxidant properties attributed to compounds like lupeol (Ekpe et al., 2017). The medicinal potential of Irvingia gabonensis extends beyond its nutritional value, with ongoing research exploring its therapeutic applications in diverse health contexts. Material and Methods: Ethanol (Analytical grade Sigma-Aldrich, USA), Methanol (Analytical grade SigmaAldrich, USA), Ethyl acetate (Analytical grade Sigma-Aldrich, USA), N-Hexane Troy S. M., Cletus A. U. & Musa Y. D. Phytochemical and Acute Toxicity ….. 159 (Analytical grade Sigma-Aldrich, USA), Aluminum Chloride powder (500g) (Reagent Grade), Quercetin (Reagent grade), FolinCiocalteu reagent (FCR), Sodium Carbonate Powder (Reagent Grade), Dragendorff Reagent, N/50 iodine solution, Sudan Red Solution, Picric acid reagent, Lead subacetate, Mayer’s reagent, Wagner’s reagent, Libermann-Buchard reagent, Ferric chloride reagent, Nitric acid, Gallic Acid, Distilled water, Hydrogen tetraoxosulphate IV acid, Hydrochloric acid, Chloral hydrate (BDH Laboratory Chemicals Division, POOLE, England), Rotary Evaporator (RE-210D-1L5L, 220V Rotavapor), UVSpectrophotometer (Model UV-2505 ~ultraviolet and visible range of 195-1050 nm, Labomeds) ,UV Lamp (Short/LongWave UV lamp; 4 watts, 254/365nm wavelength, 220V/50Hz, COLEPARMER®), and Oven. The leaves, fruits, stem-bark, and roots of the plant were collected in October 2018 at Iragbiji community, Boripe Local Government Area of Osun State, Nigeria. The plant was identified, and a voucher number was obtained. The plant materials, after collection, were air-dried, powdered, and 3 kilograms of the powdered plant material were extracted sequentially at room temperature with n-hexane, ethyl acetate, and methanol by maceration for a period of 48 hours, 72 hours, and then 72 hours, respectively (Chart 1). Troy S. M., Cletus A. U. & Musa Y. D. Phytochemical and Acute Toxicity ….. 160 All the extracts were filtered, and the filtrates were concentrated and stored properly in a desiccator for further analysis. The percentage yield was also calculated as well with reference to the weight of the powdered plant material (Table 1). Percentage yield of extracts = Weight of total 𝑒𝑒𝑒𝑒𝑒𝑒𝑒 / Weight of powdered material X 100 Qualitative Phytochemical Screening The stem bark of Irvingia gabonensis was subjected to qualitative phytochemical screening to identify various chemical constituents present in the plant material. The screening involved tests for carbohydrates, anthracenes, cardiac glycosides, flavonoids, tannins, alkaloids, and steroids/triterpenes. Tests for Carbohydrates: Three tests were conducted to detect carbohydrates in the stem bark extracts. The Molisch test, Fehling test, and Frothing test were performed on nHexane, ethyl acetate, and methanol extracts to identify the presence of carbohydrates based on specific chemical reactions. Tests for Anthracenes: The presence of anthracenes was tested using the Bontrager test and Modified Bontrager's test. These Troy S. M., Cletus A. U. & Musa Y. D. Phytochemical and Acute Toxicity ….. 161 tests involved the addition of specific reagents to the extracts and observing color changes indicative of the presence of anthracenes. Tests for Cardiac Glycosides: Various tests such as Keller-Killiani test, Kedde test, and Baljet test were conducted to detect cardiac glycosides in the extracts. These tests involved the addition of specific reagents and observing characteristic color changes. Tests for Flavonoids: Flavonoids were detected using the Shinoda test and Sodium hydroxide test. These tests involved the addition of specific reagents and observing color changes indicative of the presence of flavonoids. Tests for Tannins: Two tests, the Ferric chloride test and the Lead sub-acetate test, were conducted to detect tannins in the extracts. These tests involved the addition of specific reagents and observing precipitate formation. Tests for Alkaloids: Several tests, including Dragendorff’s reagent test, Mayer’s reagent test, and Wagner’s reagent test, were conducted to detect alkaloids. These tests involved specific chemical reactions leading to the formation of characteristic precipitates. Tests for Steroids/Triterpenes: The Lieberman-Burchard test and the Salkowski test were conducted to detect steroids and triterpenes. These tests involved the addition of specific reagents and observing color changes indicative of the presence of these compounds. Quantitative Phytochemical Determination: Total Phenolic Content (TPC): The TPC of the extracts was determined using the FolinCiocalteu method. Absorbance readings were taken at 765 nm, and the concentration of phenolic compounds was calculated using a calibration curve prepared with gallic acid solutions. Total Flavonoid Content (TFC): TFC was determined using the aluminum chloride colorimetric assay. Absorbance readings were taken at 533 nm, and the concentration of flavonoids was calculated using a calibration curve prepared with quercetin solutions. Total Tannin Content (TTC): The tannin content was determined using the FolinCiocalteu method. Absorbance readings were taken at 725 nm, and the concentration of tannins was calculated using a calibration curve prepared with gallic acid solutions. Acute Toxicity Studies: Acute oral toxicity studies were conducted according to OECD guidelines in rats (OECD Test Guideline 425, 2001). Rats were orally dosed with different concentrations of extracts and observed for signs of toxicity and mortality over a 14-day period. Results The yield obtained from n-Hexane was 110.16g, with a calculated percentage yield was calculated as 3.67%. Ethyl acetate gave a total yield of 160.86g, which was 5.36%. The methanol extract had a weight of 280.22 with a percentage yield of 9.34%, as presented in Table 1. Troy S. M., Cletus A. U. & Musa Y. D. Phytochemical and Acute Toxicity ….. 162 The preliminary phytochemical screening of Irvingia gabonensis stem bark was conducted using crude extracts in n-Hexane, ethyl acetate, and methanol. Various standard tests were employed to identify the presence of different phytochemical compounds in the extracts, as represented in Table 2 . Troy S. M., Cletus A. U. & Musa Y. D. Phytochemical and Acute Toxicity ….. 163 Molisch’s test showed a reddish-colored interfacial ring, indicating the presence of carbohydrates. Fehling’s test resulted in a brick-red precipitate, confirming the presence of reducing sugars. The frothing test displayed a persistent honeycomb froth for more than 15 minutes in all extracts, suggesting the presence of saponins. Anthracenes: Borntrager's test and modified Borntrager's test showed no formation of pinkish-red coloration in any of the extracts, indicating the absence of free and combined anthraquinones. The Keller-Killiani test revealed a brown ring at the liquid junction and a pale green upper acetic acid layer, indicating the presence of deoxy sugar. The Baljet test resulted in an Troy S. M., Cletus A. U. & Musa Y. D. Phytochemical and Acute Toxicity ….. 164 immediate red coloration, confirming the presence of cardiac glycosides. Shinoda test and sodium hydroxide test showed red and yellow solutions respectively, indicating the presence of flavonoids in all extracts. Ferric chloride test and lead sub-acetate test produced greenish-black and brownish-white precipitates respectively in all extracts, confirming the presence of condensed tannins. Mayer's test, Dragendorff test, and Wagner’s test all yielded precipitates (cream-colored, red, and reddish-brown respectively), indicating the presence of alkaloids in all extracts. The Lieberman-Burchard test resulted in a brownish-red ring at the interface of the organic and acid layers, along with a pale green upper organic layer, suggesting the presence of steroids and/or triterpenoids. The Salkowski test produced a reddish-brown ring at the interphase of the two liquids, confirming the presence of steroids. Quantitative phytochemical determination of Irvingia gabonensis stem bark using the spectrophotometric method: Table 3 shows the results for the quantitative phytochemical content of n-hexane, ethyl acetate, and methanol extracts of I. gabonensis stem bark. The table shows a total phenolic, flavonoid, and tannin content of the n-hexane extract as 41.76 mg/g, 43.19 mg/g, and 67.23 mg/g respectively, that of ethyl acetate as 122.18 mg/g, 73.42 mg/g, and 164.81 mg/g, and for the methanol extract as 79.47 mg/mL, 36.61 mg/g, and 159.37 mg/g respectively. Toxicological Profile of Irvingia. gabonensis stem bark. Acute oral median lethal dose (LD50) in Rats: Troy S. M., Cletus A. U. & Musa Y. D. Phytochemical and Acute Toxicity ….. 165 The limit test results revealed that a single oral dose of n-hexane ethyl-acetate, and methanol extracts of I. gabonensis stem bark did not cause mortality or any signs of toxicity in rats within 24 hours or during the 14-day observation period. It was observed that the LD50 was greater than 2000 mg/kg body weight in rats. When the dose was increased to 5000 mg/kg, no mortality or signs of toxicity were observed with the test within the first 24 hours or over the 14-day observation period Discussion The research conducted on Irvingia gabonensis stem bark revealed significant findings regarding the extraction process, chemical composition, and potential biological activities of the plant extracts. The extraction process involved using n-hexane, ethyl acetate, and methanol, resulting in extracts with varying yields and compositions. The n-hexane extraction yielded 3.67% (w/v) of extract, while the subsequent extractions with ethyl acetate and methanol produced higher yields of 5.30% (w/v) and 9.34% (w/v), respectively. This indicates that Irvingia gabonensis stem bark contains more polar constituents than nonpolar ones, consistent with previous research. The total tannin content of the stem bark extracts was determined, with the ethyl acetate and methanol extracts showing tannin content of 164.81 and 159.37 mg GAE/GAE g, respectively. Other studies reported varying tannin content, with values ranging from 85.5 mg to 773.3 mg GAE/g, indicating a high concentration of tannins in polar solvents. Similarly, the total phenolic content of the extracts was analyzed, and higher concentrations were found in polar extracts compared to non-polar ones. The phenolic content ranged from 79.47 to 253.39 mg/GAE g, indicating the presence of potent antioxidants. Flavonoid content analysis showed higher levels in ethyl acetate extracts compared to methanol extracts, with values of 73.42 and 36.61 mg/QE g, respectively. This discrepancy may be attributed to differences in extraction techniques and environmental factors. Phytochemical screening of the extracts revealed the presence of carbohydrates, alkaloids, saponins, and flavonoids, while tannins, glycoside proteins, fats/oils, steroids, terpenes, and cardiac glycosides were absent. These secondary metabolites contribute to the biological activity of the plant, including antioxidant and anticancer properties. Previous research has demonstrated the potential health benefits of Irvingia gabonensis extracts, including prophylactic effects against cadmium-induced toxicity, anti-diabetic, antihyperlipidemic, and antioxidant effects. Phytochemical analysis consistently identified alkaloids, glycosides, flavonoids, and tannins as predominant compounds in Irvingia gabonensis extracts. Acute toxicity studies indicate that Irvingia gabonensis extracts are well-tolerated by experimental animals, with no signs of morbidity or mortality observed at high doses. This suggests the safety of the extracts for potential therapeutic use. Conclusion Research on Irvingia gabonensis stem bark has revealed promising findings regarding its