2. MATERIALS AND METHODS

Plant Material Collection and Authentication

A. digitata leaves were obtained at Ayedade farm in Ogbomosho, Oyo State, southwest of Nigeria. The Herbarium Unit of the Department of Plant Biology at the University of Ilorin conducted the identification and authentication, assigning a voucher specimen number (UILH/0011/951/2024). After that, the leaves were washed and allowed to dry at room temperature until they reached a consistent weight before being ground.

Feed Components

Soybeans, sucrose, yellow corn, and maize husk were obtained from the Oja Oba market in Ilorin, Kwara State. Sunola produced the soyabean oil, and Kewalram Nigeria Limited, Nigeria, refined the soybeans. Vitamin and mineral mix, cellulose, and methionine were purchased at the Aromokeye store in Ilorin, Kwara State, Nigeria.

Experimental Animals

Fifty (50) female Wistar rats were obtained from the Animal Housing Unit at the Department of Biochemistry, University of Ilorin, each with an average of 100.15 ± 14.24. Before the experiment started, they were housed in a well-ventilated section of the animal house.

Nutritional /Chemical Analysis of A. Digitata Leaves.

Proximate Analysis of A. Digitata Leaves.

To identify the leaves' proximate elements, methods detailed by the Association of Official Analytical Chemists (A.O.A.C.) (2010) were em ployed.

Determination of Carbohydrate Content in A. Digitata Leaves

The method of estimation by difference was used to calculate carbohydrate content of A. digitata leaves and the formulated diet.

Carbohydrate = 100 – (% moisture + % ash + % protein + % lipids + % fiber).

Assessment of A. Digitata Leaves' Mineral Content

The mineral content of A. digitata leaves was ascertained by wet oxidation or perchloric acid digestion (AOAC, 1990).

Determination of Vitamin Content of A.Digitata Leaves

The Association of Official Analytical Chemists' official procedures were used to identify the vitamins present in A. digitata leaves (AOAC, 1990).

Determination of Secondary Metabolites Constituents of A. Digitata Leaves

The secondary metabolites in A. digitata leaves were determined using standard analytical tests for both qualitative and quantitative analyses.

Qualitative Secondary Metabolites: A. digitata leaves weighing 10 g were extracted in 200 ml of distilled water and left at room temperature for 48 hours. Following this, the determination of alkaloids (Harborne, 1998), saponins (Wall et al., 1954), anthraquinones, steroids, and phlobatannins (Trease and Evans, 1989), cardiac glycosides, cardenolides, and dienolides (Sofowora, 1993), triterpenes and tannins (Odebiyi and Sofowora, 1978), flavonoids and phenolics (Milutinović et al., 2018) were all conducted on the aqueous extract.

Quantitative Secondary Metabolites: The determination of saponins (Obadoni and Ochuko, 2001), flavonoids (Boham and Kocipai, 1974), terpenoids and glycosides (Sofowora, 1993), and steroids (Wall et al., 1954) were carried out on the A. digitata leaves.

Animal Grouping

The rats (50) were acclimatized for one week before being divided into five groups as outlined below:

Group A – Rats feed on basal control diet

Group B - Rats feed on 25% inclusion of A. digitata leaves

Group C - Rats feed on 50% inclusion of A. digitata leaves

Group D - Rats feed on 75% inclusion of A. digitata leaves

Group E - Rats feed on 100% inclusion of A. digitata leaves

The animals were fed their designated diets for six weeks prior to being sacrificed.

Composition f Diets (G)

The procedure described by the American Institute of Nutrition (1977) was adopted for the feed formulation.

Feed Analysis

Determ ination of Feed Intake (Fi):

To obtain the quantity of feed consumed by each group per day feeds given to different animals per group were measured, and the remains the next day were measured to determine how much was consumed according to the formula.

FI (g) = Total feed given– Feed remaining.

Determination of Body Weight Gain (BWG): The body Weight of each animal in various groups was taken weekly, and the average was calculated and recorded.

BWG (g)= Final Weight- Initial Weight.

Determination of Feed Conversion Ratio (FCR), expressed as grams of feed per gram of weight gain, was determined by dividing the FI by the BWG (Mwale et al., 2008). Mathematically as;

FCR = FI (G) / BWG (Go

Anthropometric Analysis

Determination of Body Mass Index (BMI) was calculated as the ratio of the animal's weight (g) to the square of its length (cm²) (Bernardis and Patterson, 1968).

BMI = Rats Weight (g) / Length (cm²).

Determination Abdominal Circumference (AC): This was measured using a tape rule around the rat's abdomen in centimeters (Novelli et al., 2007).

Determination of Arm Circumference (ArC): This was measured using a tape rule to measure around the rat's arm (Novelli et al., 2007).

Determination of Length of Animal (Naso-anal length): The length of the body was recorded from the snout to the pelvic-caudal junction at the base of the tail (Novelli et al., 2007). Every measurement was taken with a non-elastic tape measure.

Statistical Analysis

All results were reported as the average of five replicates ± (SEM). One-way analysis of variance (ANOVA) was used to analyze the data statistically, and Duncan's multiple range test was used for post hoc comparisons, utilizing SPSS version 20. Results were deemed significant at p < 0.05.

3. RESULTS AND DISCUSSION

Nutritional/ Chemical Constituents of A. Digitata Leaves

Proximate Composition of A. Digitata Leaves

Table 2 presents the proximate composition of A. digitata leaves. The leaves contain high fiber and carbohydrate content and a moderate amount of moisture, ash, protein, and lipid. According to Ashikkumar et al. (2007), proximate analysis of food provides estimates of its carbohydrate, lipid, crude protein, and fat content. The fiber content was far higher than those obtained in the same species grown in northern Nigeria, as documented by previous research (Belewu et al., 2008; Wudil et al., 2020), although it compares favorably with Yahauri spices reported by Oyegoke et al. (2024). This high fiber content is beneficial because fiber plays an essential role in regulating intestinal transit by absorbing trace elements (Abolaji et al., 2007) and increases dietary bulk and therefore prevents constipation (Abolaji et al., 2007; Olowokudejo et al., 2008). Fiber, therefore, helps to bring down the levels of cholesterol and the risk of cardiovascular disease and diabetes (Mgbemena et al., 2022). The high carbohydrate content means A. digitata serves as a credible source of energy as a carbohydrate, especially glucose, which is an energy source for muscle cells, brain cells, and blood cells (Ejelonu et al., 2011), which are important for the maintenance of cellular function and biochemical reactions. While the protein content of A. digitata leaves is moderately available, it remains greater than that of certain leafy vegetables, such as Ficus capensis leaves cultivated in southeastern Nigeria (Achi, 2017). Mgbemena and Amako (2020) state that proteins are necessary for hormones, enzymes, and blood plasma production in the body. According to past studies, fresh green leafy vegetables contain low levels of protein, located as enzymes rather than storage pools, as in grains and nuts (Ifon and Bashir, 1989; Wills et al., 1998; Oboh and Masodje, 2009). The lipid content of this leaf is lesser than that of some leafy vegetables commonly consumed in Nigeria, as recorded in the literature (Sena et al., 1998). It is well-documented that most leafy vegetables are generally poor sources of lipids (Ifon and Bassir, 1980; Ejoh et al., 1996). This is very advantageous as its low-fat intake lowers the risk of aging, cancer, and cardiovascular diseases (Kris-Etherton et al., 2002). The moderate ash level of this leaf compared favorably with that of those grown in the south-savanna region of Nigeria (Wudil et al., 2020). The leaves ash content indicates the proportion of mineral elements they contain. This implies that a high ash content will mean a high mineral content and, therefore, higher nutritional quality (Fagbohun, 2012). The moderate moisture content of the leaves can give it a two-way definition. According to research, the moisture contents of most leaves, especially vegetables, range between 6 -15% (Rishi et al., 2012), and our leaves fall within this range in a moderately available range. This signifies that the growth and development of microorganisms will be inhibited at this percentage, thereby extending their shelf life (Dike, 2010). In the same way, the moderate moisture content of these leaves may promote a moderate activation of co-enzymes and water-soluble enzymes essential to their metabolic activities (Iheanacho and Udebuani, 2009).

Iron is essential for the production of hemoglobin and myoglobin, which transport oxygen in the muscles and blood. Additionally, it supports the activity of antioxidant enzymes like peroxidase and catalase, which safeguard cells against oxidative damage. The moderate iron content in A. digitata leaves contributes to their role in preventing oxidative stress and maintaining overall cellular health (Makena et al., 2021).

The magnesium content of the leaves is higher than that of some other leafy vegetables. Therefore, its roles cannot be underemphasized. It is involved in over 300 enzymatic reactions in the body, including those that regulate blood glucose levels. Proper magnesium intake can improve insulin sensitivity, which is necessary for maintaining a healthy body weight and preventing BWG (de Sousa Melo et al., 2022).

Copper is an essential trace mineral that contributes to the antioxidant defense system by acting as a cofactor for superoxide dismutase (SOD), an enzyme that safeguards cells against reactive oxygen species (ROS)-induced damage. Even in small amounts, the copper in A. digitata leaves enhances their antioxidant potential, contributing to overall health and disease prevention (Du et al., 2010; Olayinka et al., 2024).

Zinc is a crucial component of numerous enzymes and proteins that protect against oxidative stress and support the immune system. Therefore, the zinc content in these leaves boosts their antioxidant properties, helping to mitigate oxidative damage and maintain immune function (Prasad, 2020).

From the above, deductions can be made that the mineral composition of A. digitata leaves underscores their potential benefits.

Vitamin Content of A. Digitata Leaves

Table 4 presents the results of the vitamin content determination of A. digitata leaves. Six vitamins were detected, with vitamins C and E being the most abundant.

These are compounds that are vital for various physiological functions in the body (Habbib et al., 2014). Vitamin A and B complexes assayed in this research (B₁, B_2,and B₃) are found in minute quantities in A. digitata leaves. However, the quantity is enough to elicit their physiological activities since they are only needed in small amounts. Vitamin A is involved in maintaining immune function, good vision, and healthy skin. (Harrison, 2012). Vitamin B₁ plays a crucial role in carbohydrate metabolism, helping convert carbohydrates into energy. Efficient metabolism of carbohydrates is essential for energy balance and weight management (Gropper et al., 2018). The vitamins B₂ and B₃ are crucial for oxidative phosphorylation and the creation of coenzymes (Adesina, 2006). Vitamin C is an antioxidant that helps neutralize free radicals, protecting cells from oxidative damage (Carr and Maggini, 2017). It also regenerates other antioxidants within the body, such as vitamin E. The high vitamin C content in A. digitata leaves enhances their antioxidant capacity, contributing to overall well-being and helping prevent chronic diseases (Carr and Maggini, 2017).As a lipid-soluble antioxidant, vitamin E guards against oxidative damage to cell membranes (Burton and Traber, 1990). It performs a crucial role in preventing the oxidation of body lipids. The significant amount of vitamin E in the leaves supports their antioxidant potential, helping to reduce oxidative stress and inflammation (Burton and Traber, 1990).

Vitamin K is essential for blood clotting and maintaining bone health (Shea and Booth, 2016). It also has antioxidant properties that help protect cells from oxidative damage. The presence of vitamin K in A. digitata leaves adds to their overall antioxidant potential (Shea and Booth, 2016).

Secondary Metabolites Constituents of A. Digitata Leaves

Table 5 displays the plant secondary metabolite constituents of the leaves. Steroids, cardiac glycosides, triterpenes, flavonoids, and saponins were detected in the leaves, while alkaloids, tannins, and phenolics were not detected. Saponins and flavonoids were the most abundant in the leaves. Plants' medicinal value is attributed to their bioactive phytochemical compounds, which produce specific physiological effects (Akinmoladun et al., 2007). A. digitata leaves contain high concentrations of saponins. This antioxidant acts by counteracting free radicals, lowering oxidative stress, and exhibiting antimicrobial properties, allowing it to be effective against bacteria, fungi, and viruses. It also possesses the potential to lower cholesterol levels due to its hypercholesteric effect (Nnam, 2011); glycosides have antibacterial and antiviral properties and anti-inflammatory effects, as well as useful in treating various diseases (Harborne, 1986). Flavonoid has anti-inflammatory and antioxidant characteristics, enhance endothelial function, and reduce blood pressure (Beecher, 2003). This suggests that the plant can act as a radical scavenger and exhibit a variety of metabolic and signaling enzymes, thus preventing chronic disease. Triterpenes have anti-cancer and anti-inflammatory characteristics and are useful in protecting the liver from damage (Yadav et al., 2010). Steroids act by regulating various physiological functions and reducing inflammation (Kaur et al., 2018), all of which are important for the proper maintenance of health.

Table 6: Growth Performance Characteristics of Female Wistar Rats Fed on A. digitata Leaves-based Diet

*A. digitata* Leaves-based Diet
Parameters	Control	25%	50%	75%	100%
IW(g)	123.26 ± 1.03^a	112.11 ± 0.91^b	98.74 ±0.68^c	90.21 ± 0.79^d	76.45 ± 1.02^e
FW(g)	142.18 ± 1.39^a	130.34 ± 0.91^b	118. .27 ± 1.05^c	97.16 ± 0.90 ^d	81.44 ± 0.80^e
BWG (g)	19.68 ± 0.36^a	18.17 ± 0.01^ab	20.14 ± 0.38^a	7.05 ± 0.11^c	5.01 ± 0.23^d
FI (g)	49.12 ± 0.78^a	43.41 ± 0.78^ab	46.64 ± 0.71^a	35.94 ± 0.72^b	38.35 ± 0.66^c
FCR	2.49 ± 0.81^a	2.39 ± 0.92^b	2.32 ± 0.53^b	5.10 ± 1.11^c	7.65 ± 0.23^d

IW (g); Initial weight, FW; Final weight, BWG (g); body weight gain, FI (g); Feed intake, FCR; Feed conversion ratio.

Anthropometric Indices of Rats Fed on A. Digitata Leave–Based Diet

Table 7 shows the anthropometric index of female Wistar rats fed an A. digitata leaf-based diet.

Table 7: Anthropometric Index of Female Wistar Rats Fed with A. digitata Leave-based Diet (Before and After feeding Period)

Parameters	Groups (A. digitata leaves)
	Control	25%	50%	75%	100%
BMI day 0 (g/cm²)	0.33 ± 0.22^a	0.31 ± 0.03^a	0.30 ± 0.01^a	0.27 ± 0.02^a	0.25 ± 0.02^a
BMI day 42 (g/cm²)	0.37 ± 0.03^b	0.34 ± 0.02^b	0.32 ±0.03^b	0.25 ±0.04^b	0.25 ± 0.02^a
AC day 0 (cm)	11.22 ± 0.98^a	10.78 ± 0.87^a	10.21 ± 0.78^a	10.56 ± 0.78^a	8.98 ± 1.01^a
AC day 42 (cm)	13.69 ± 1.01^b	12.23 ± 1.21^b	11.89 ± 1.11^b	10.92 ± 0.77^a	9.34 ± 0.78^a
ArC day 0 (cm)	1.65 ± 0.11^a	1.45 ± 0.41^a	1.32 ± 0.12^a	1.11 ± 0.12^a	1.11 ± 0.13^a
ArC day 42 (cm)	1.82 ± 0.12^b	1.57 ± 0.12^b	1.48 ± 0.16^b	1.19 ± 0.08^a	1.18 ± 0.09^a
NA length day 0 (cm)	19.23 ± 0.94^a	19.02 ± 0.78^a	18.23 ± 0.86^a	18.34 ± 0.54^a	17.35 ± 0.33^a
NA length day 42 (cm)	19.92 ± 0.34^b	19.89 ± 0.88^b	19.14 ± 0.78^b	19.11 ± 0.24^b	18.22 ± 0.45^b

Values are means of 5 replicates ± SEM. Values with superscripts along the same row different from the control for each parameter are significantly different (p<0.05). BMI; Body Mass Index, AC; Abdominal Circumference, ArC; Arm Circumference, NA Length; Naso-anal Length

CONCLUSION

This study concludes that A. digitata leaves possess essential nutrients needed by the body for proper functioning. These nutrients were well absorbed and digested, making them bioavailable to the body system. This was achieved effectively at 25% and 50% inclusion in the diet.

FUNDING TO THE STUDY

This research did not receive any specific funding from public, commercial, or non-profit organizations.

CONFLICT OF INTEREST

None declared.

AUTHORS CONTRIBUTION DETAILS

A- Oyegoke Rukayat Abiodun

B- Abdulwahab Amina Abebi

C- Musa, Fasilat Jumoke

D- Osifade, Jahswill Toluwanimi

E- Nafiu, Mikhail Olugbemiro

F- Moronfolu, Ibrahim Adeola

A.E. conceived and planned the experiments. A.B.C. carried out the experiments. A.B. D, F. took the lead in writing the manuscripts. All authors provided critical feedback and helped shape the research, analysis, and manuscript.

DECLARATION STATEMENT

The authors state that all information in this article is accurate to the best of our knowledge, and we take full responsibility for its correctness.

ACKNOWLEDGEMENT

The research team hereby acknowledges the University of Ilorin, Ilorin, Nigeria, for the facilities provided in the department's Biochemistry Laboratory, which were used during this study.

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Parameters	Moisture	Ash	Crude Fiber	Lipid	Protein	Carbohydrate
Composition (100%)	8.63 ± 0.15	11.41 ± 0.54	38.24 ± 0.42	5.54 ± 0.34	7.27 ± 0.20	29.06 ± 0.09

Vitamins	A	B₁	B₂	B₃	C	E	K
Composition (g/100g)	0.01 ± 0.01	2.60±0.06	0.001±0.00	0.43±0.02	23.03 ± 1.55	19.93 ± 0.00	0.38 ± 0.05

Secondary plant metabolites	Alkaloids	Tannins	Saponins	Glycosides	Phenolics	Flavonoids	Triterpenes	Steroids
Concentration (%)	Nd	Nd	137.17 ± 0.76	34.62 ± 0.18	Nd	62.51 ± 1.01	33.30 ± 0.32	36.15 ± 0.28

Ingredients	Treatments (A. digitata leave-based diet)
	Group A (Control / Basal diet)	Group B (25%)	Group C (50%)	Group D (75%)	Group E (100%)
Corn starch	506	379.50	253	126.50	-
A. digitata leaves	-	126.50	253	126.50	506
Cellulose	40	40	40	40	40
Sucrose	100	100	100	100	100
Soybean	250	250	250	250	250
Soybean oil	50	50	50	50	50
Vitamin/Mineral mix	50	50	50	50	50
D-methione	4	4	4	4	4
Total	1000	1000	1000	1000	1000

Mineral	Cu	Zn	Ca	Fe	Mg
Composition (mg/100g)	0.22 ± 0.00	1.08 ±0.04	20.08 ± 0.00	6.11 ± 0.04	7.19 ± 0.00