Qualitative phytochemical analysis of methanol crude extract of Detarium microcarpum leaves

Introduction

In recent years, as infectious diseases have spread and harmful organisms have developed resistance to therapy, the quest for new treatments has increased substantially (Abdulrahman et al., 2018) [1] . Humans have always drawn inspiration from plants due to their unique medical characteristics. Traditional African medicine relies heavily on the use of medicinal plants for the treatment of a wide variety of illnesses (Kankara et al., 2015; Abubakar et al., 2022) [2] . Herbal medicine (also called botanical medicine or phytomedicine) refers to using plant's seeds, berries, roots, leaves, bark, or flowers for medicinal purposes. Traditional medicine refers to health practices, approaches, knowledge and beliefs incorporating plants, animals and mineral based medicines, spiritual therapies, manual techniques and exercises, applied singly or in combination to treat or to diagnose and prevent illnesses or maintain well-being (WHO, 2003) [24] . A renewed global interest in the use of herbal medicines even among developed nations has necessitated the need for the standardization of herbal products. The World Health Organization has emphasized the need to ensure the quality of medicinal plant products by using modern control techniques and applying suitable standards. Standardization is necessary to mitigate the incidence of lack of efficacy, poor quality, adverse effects and toxicity (WHO, 1998; WHO, 2007) [26,23] . The tallow tree, Detarium microcarpum, is widely distributed in tropical West Africa's arid Savannah ecosystem. Because every part of the D. microcarpum plant has therapeutic applications, the plant itself is referred to as a miracle plant by traditional herbalists (Idris et al., 2021) [11] . Folk medicine heavily relies on its leaves and fruits. The need for new drugs derived from numerous species of medicinal plants is continuously growing today (Ashraf et al., 2020) [4] . Plants consist of various kinds of chemical constituents known as phytoconstituents (phytochemical constituents) (Mercy et al., 2017) [15] .

The secondary roles that phytoconstituents perform for plants include promoting plant development, protecting them by triggering defense mechanisms, and giving them color, flavor, and odor (Molyneux et al., 2007) [16] . In comparison to their synthetic counterparts, natural chemicals and their derivatives have less adverse effects and greater efficacy. These plant-derived components, such as flavonoids, quinine, and terpenoids, perform certain biological functions that enhance therapeutic activities, such as anti-carcinogenic, anti-mutagenic, anti-inflammatory, and antioxidant properties (Batiha and Beshbishy, 2020) [5] . D. microcarpum leaves have traditionally been used to treat diabetes mellitus in most regions of Africa (Manukumar et al., 2017) [13] . Total phenolic content and radical-scavenging activities have been assessed in D. microcarpum methanol extracts (Meda et al., 2017) [14] . Silver nanoparticles (AgNPs) have strong antimicrobial and antioxidant activities and have many uses, including catalysis, synergistic oxidation of carbon monoxide, biological research, dye reduction, selective oxidation of benzene, and chemotherapy (Priya et al., 2016) [19] . D. microcarpum has been used in the synthesis of AgNPs, with an aqueous extract of the plant being applied as a stabilizing and reducing agent. The antioxidant activity of AgNPs stabilized by D. microcarpum is between medium and good activity (Labulo et al., 2016) [12] . The anti-inflammatory properties of D. microcarpum aqueous extracts, which contain polyphenolic compounds such as vanillic acid, gallic acid, and protocatechuic acid, imply that it is a promising herbal remedy for pain and inflammation management (Windmi et al., 2021) [25] . Lupeol, betulinic acid, β-sitoterol glucoside, methyl gallate (Feudjou et al., 2020) [9] , humulene-1,2-epoxide, caryophyllene oxide,eryl acetone-caryophyllene, salvia-4(14)-ene-1-one, and linalool are some of the main components of the essential oil of D. microcarpum leaves, which have antimicrobial properties (Semde et al., 2018) [20] . Detarium microcarpum flavanes had potent inhibitory effects on HIV-1 and HIV-2 infection in a methanol extract. In order to identify different classes of bioactive constituents present in various parts of the plant for the discovery of drugs, scientists use a process called phytochemical screening. The active components identified from this process can be used for further investigation and research. Depending on the plant species, soil type, and their relationship to microbes, medicinal plants' physiologically active chemical composition varies greatly (Morsy, 2014) [17] . These bioactive secondary metabolites produced by medicinal plants have a significant impact on the physiological processes of the microbial communities that are connected with plants as well (Phillipot et al., 2013) [18] . The research is significant because:  It provides empirical evidence on the bioactive constituent present in D. microcarpum  It validates traditional claims on therapeutic activities of the plant.  It establishes a foundation for future quantitative analyses and pharmacological evaluations that could lead to the development of novel therapeutic formulations.

Detarium microcarpum is widely recognized in West African ethnomedicine, but there is paucity of detailed about the phytochemical makeup of its leaves, which have been hypothesized to contain diverse bioactive phytoconstituents that traditional medicinal efficacy.

Detarium microcarpum leaf extracts are rich in nutrients and medicinal properties; therefore, performing phytochemical screening will not only help to identify the components of the leaf extracts and which one predominates over the others, but also be helpful in investigating bioactive agents that can be used in the synthesis of useful drugs, that could be used for treating a variety of diseases and maintaining health.

The aim of the study is determine the phytoconstituents that are present in the methanol crude extract of the leaf (MEL) of Detarium microcarpum.

The primary objectives of the study are:  To extract the bioactive compounds from D.

microcarpum leaves using the maceration method.  To conduct a qualitative phytochemical screening of the methanol crude extract of the leaf (MEL) of D. microcarpum to determine the presence of key secondary metabolites.

Materials and Methods

Materials Equipment/apparatus used

Methods

Sample Collection

Fresh leaves of Detarium microcarpum were harvested in September, 2023 from Saki, Nigeria. The plant was identified by Mr. Waliu Olamide Azeez. The leaves were collected using a cleaned plastic container.

Macroscopical Evaluation

Macroscopical (organoleptic) Analysis: The leaf's size, shape, margin, apex, flavor, color, surface texture, petiole's presence or absence, base, lamina, and venation were all seen and noted (Evans, 2009) [7,8] .

Sample preparation

The leaves were cleaned, and air dried at room temperature. The dried leaves were crushed into a fine powder and stored in an airtight container after being pulverized using a pestle and mortar.

Sample Extraction

A total of 5 g of the powdered leaves were macerated in 100 cm 3 of 90% v/v methanol in a conical flask. It was allowed to stand at room temperature for 24 hours while stirring occasionally. The mixture was filtered using Whatman filter paper (No. 1), and the filtrate was allowed to evaporate in a water bath at 30 °C to obtain the methanol extract of the leaf (MEL).

Percentage Yield

The mass of the dried extract obtained after evaporation = 1.875 g Mass of the dried powdered leaves used for extraction = 5.000 g To calculate the percentage yield of the methanol extract, the expression below was used:

percentage yield = mass of extract mass of sample used × 100 percentage yield = 1.875 g 5.000 g × 100 percentage yield = 0.375 × 100 percentage yield = 37.5%

Thus, the percentage yield of the extract was determined to be 37.5%

Preparation of reagents

All the reagents were prepared using analytical reagentgrade chemicals.

Wagner's reagent

In a beaker, 2 g of sublimed iodine and 6g of potassium iodide were dissolved in 100 cm 3 of distilled water.

In a 100 cm 3 volumetric flask, 5g of ferric chloride was weighed and dissolved in 50 cm 3 of distilled water, and made up to the mark with distilled water.

The reagent was prepared by dissolving 10g of sodium hydroxide pellet in 40 cm 3 of distilled water in a beaker; the solution was then poured into a 100 cm 3 volumetric flask and made up to the mark with distilled water.

Exactly 40 cm 3 of distilled was measured and then transfer into 100 cm 3 volumetric flask. 10 cm 3 of concentrated HCl was added and then made up to the mark with distilled water.

The reagent was prepared by measuring 10 cm 3 of ammonia, and then transferred it into a 100 cm 3 volumetric flask. 90 cm 3 of distilled water was added, diluting it to the mark indicated on the flask.

Fehling's solution

Fehling's solution is prepared by combining two separate solutions: Fehling's A, which is a deep blue aqueous solution of copper (II) sulfate, and Fehling's B, which is a colorless solution of aqueous potassium sodium tartrate (also known as Rochelle salt) made strongly alkaline with sodium hydroxide in a 1:1 ratio.

Mayer's reagent

Two solutions were made, one containing 1.36g of mercury chloride dissolved in 60 cm 3 of distilled water and the other containing 5g of potassium iodide dissolved in 20 cm 3 of distilled water. The two solutions were blended, poured into a 100 cm 3 beaker, and filled up to the mark with distilled water.

Qualitative phytochemical screening of the extract

The methanol extract was then subjected to qualitative phytochemical screening. Using the methodology described by Evans and Trease (1999) [7,8] ; Sofowora (1993) [21] ; El-Oleye et al. (1994) [6] , phytochemical screening was done to determine the presence of the following secondary metabolites: alkaloids, tannins, saponins, anthraquinones, glycosides, steroids, saponin glycosides, cardiac glycosides, volatile oils, balsams, and flavonoids.

Test for Alkaloids

To prepare for the next test, 2 cm 3 of the extract was stirred with 2 cm 3 of 10% aqueous hydrochloric acid. 1 cm 3 of the mixture was treated with a few drops of Wagner's reagent, and the second portion (1 cm 3 ) was treated similarly with Mayer's reagent. Turbidity or precipitation with either of these reagents is taken as preliminary evidence for the presence of alkaloids (Harbone, 1998) [10] .

Test for Tannins

To 3 cm 3 of the extract in a test tube, a few drops of 5% FeCl 3 solution were added, and then the test tube was shaken. A dirty green color observed indicates the presence of tannins (Evans and Trease, 1999) [7,8] .

Test for Flavonoids

The extract (3 cm 3 ) was treated with 1 cm 3 of 10% NaOH.

According to Evans and Trease (1999) [7,8] , the yellow color observed indicates the presence of flavonoids.

Test for Saponins

A test tube containing a mixture of 5 cm 3 of the extract and 5 cm 3 of distilled water was forcefully shaken. The development of foams or stable frothing observed indicates the presence of saponins.

Test for Steroids

A volume of 0.5g of the extract was dissolved in 2 cm 3 of chloroform, and 2 cm 3 of H 2 SO 4 is carefully added to form the lower layer. A reddish-brown color observed at the interface indicates the presence of a steroidal ring.

Test for Anthraquinones

About 0.5g of the extract is shaken with 10 cm 3 of benzene, and 5 cm 3 of 10% ammonia is added. The mixture was shaken, and a pink, red, or violet color was observed, which indicates the presence of anthraquinones.

Test for Glycosides

In a test tube, 5 cm 3 of the extract was combined with precisely 2.5 cm 3 of 50% sulfuric acid. After cooling and being neutralized with 10% NaOH, the liquid was boiled in boiling water for 15 minutes. The liquid was then heated while 5 cm 3 of Fehling's solution was added. The brick-red precipitate formed indicates the presence of glycosides.

Test for Cardiac glycosides (Keller-Killiani's Test) 2 cm 3 of a 3.5% ferric chloride solution were added to 1 cm 3 of the extract and left to stand for a minute. A lower layer was created by gently pouring 1 cm 3 of concentrated H 2 SO 4 down the tube's wall. A reddish brown ring formed at the contact indicates the presence of cardiac glycosides.

Test for Saponin glycosides

Fehling's solution (2.5 cm 3 ) was added to 2.5 cm 3 of the extract. The bluish-green precipitate formed indicates the presence of saponin glycosides (El-Oley et al., 1994) [6] .

Balsam's Test

The extract (0.2 g) was mixed with an equal volume of 90% ethanol. 2 drops of alcoholic ferric chloride solution were added to the mixture. The dark green color observed indicates the presence of balsams.

Test for Volatile oils

The extract (1 cm 3 ) was mixed with 1 cm 3 of dilute HCl. The white precipitate formed indicates the presence of volatile oils.

Results and Discussion Results

Results of the Phytochemical Screening

The phytochemical screening performed on the methanol extract of Detarium microcarpum leaves revealed the presence of all targeted phytoconstituents.

Discussion

The macroscopical evaluation of Detarium microcarpum leaves revealed significant morphological characteristics, including an ovate shape, glossy green surface, entire margin, and a rounded base. These observations are consistent with the findings of Idris et al. (2021) [11] , who described D. microcarpum leaves as green, odorless, tasteless, with a thickly glabrescent surface and ovate to elliptical shape. The phytochemical screening of the methanol crude extract confirmed the presence of various secondary metabolites, including alkaloids, flavonoids, tannins, saponins, steroids, cardiac glycosides, saponin glycosides, anthraquinones, balsams, volatile oils, and glycosides (Table 3). These bioactive compounds contribute to the plant's diverse pharmacological properties. Previous studies by Ugwoke et al. (2017) [22] and Semde et al. (2018) [20] similarly reported the presence of flavonoids, tannins, saponins, and steroids in the methanol extract of D. microcarpum leaves. Flavonoids and tannins are well known for their strong antioxidant and anti-inflammatory properties, while saponins and alkaloids exhibit antimicrobial and immunomodulatory effects (Evans & Trease, 1999; Idris et al., 2021) [7,8,11] . The percentage yield of the methanol crude extract was determined to be 37.5% (Table 2). This is comparable to the yield obtained in previous studies on methanol extracts of medicinal plants, suggesting that methanol is an efficient solvent for extracting the bioactive compounds present in D. microcarpum (Meda et al., 2017) [14] . The high presence of flavonoids and glycosides may contribute to the plant's potential as a source of therapeutic agents, particularly in antioxidant and cardiovascular treatments (Morsy, 2014; Batiha & Beshbishy, 2020) [5] . The presence of volatile oils and balsams suggests antimicrobial and anti-inflammatory properties, aligning with the findings of Semde et al. (2018) [20] , who characterized the essential oils of D. microcarpum and found antimicrobial activity against several pathogens. The detection of cardiac glycosides indicates potential cardiovascular benefits, supporting the traditional use of the plant in managing heart-related ailments (Feudjou et al., 2020) [9] . The study's results align with previous phytochemical studies, further validating the medicinal properties of D. microcarpum. The combination of alkaloids, flavonoids, saponins, tannins, and steroids suggests that the methanol extract could serve as a promising candidate for future drug development. However, while this qualitative analysis provides valuable insights, further quantitative phytochemical analysis, pharmacological evaluations, and toxicity studies are essential to confirm the efficacy and safety of D. microcarpum extracts (WHO, 2007; Labulo et al., 2016) [23,12] . To fully explore the potential of D. microcarpum, advanced analytical techniques such as High-Performance Liquid Chromatography (HPLC), Gas Chromatography-Mass Spectrometry (GC-MS), and Nuclear Magnetic Resonance (NMR) spectroscopy should be employed to isolate and characterize individual bioactive compounds (Priya et al., 2016) [19] . Additionally, in-vitro and in-vivo studies will be required to establish bioavailability, pharmacodynamics, and toxicity levels before developing standardized herbal formulations (Windmi et al., 2021) [25] .

Conclusion

This study has demonstrated that the methanol crude extract of Detarium microcarpum leaves contains a diverse range of bioactive phytochemicals, including alkaloids, flavonoids, tannins, saponins, steroids, cardiac glycosides, saponin glycosides, anthraquinones, balsams, volatile oils, and glycosides. These findings validate the traditional medicinal use of D. microcarpum and suggest its potential as a source of therapeutic compounds. The presence of these bioactive constituents indicates possible pharmacological applications, particularly in antimicrobial, antiinflammatory, and antioxidant therapies. While this study provides qualitative insights into the phytochemical composition of the plant, further research is necessary to conduct quantitative analysis, isolate and characterize individual compounds, and evaluate their pharmacological efficacy and safety. This work lays the groundwork for future studies aimed at developing standardized herbal formulations and exploring the potential of D. microcarpum in drug discovery.

Recommendation

Based on the findings of this study, several recommendations for further research can be proposed to deepen the understanding of Detarium microcarpum leaves and their potential applications:

Quantitative phytochemical Analysis: To determine the concentration of each bioactive compound and correlate this with biological activities.

Pharmacological Evaluation:

In-vitro and in-vivo studies to assess antimicrobial, anti-inflammatory, anticancer and anti-diabetic potentials. This will be essential to confirm the therapeutic efficacy and safety of the extract.

Acknowledgement

AcknowledgementsThe authors are grateful to the management and staff of Usman DanFodio University department of chemistry, for providing facilities and support for this research. We also appreciate the technical assistance from the colleagues and peers, and then our families and friends for their encouragement

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