Abstract

Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia, affecting millions globally. Traditional medicinal plants, like Chrysophyllum albidum (African star apple), have been used for their purported health benefits, including anti-diabetic properties. This study investigated the antidiabetic effect of Chrysophyllum albidum pulp extract on streptozotocin-induced diabetic rats, providing scientific validation for its traditional use. This experimental study used a randomized control trial design involving streptozotocin-induced diabetic rats. The study was conducted in accordance with the guidelines for the care and use of laboratory animals. The study was carried out after ethical approval was obtained from University of Ilorin ethical review committee. Twenty-five (25) male Wister rats were grouped into five groups. Each group consisted of five animals. The rats were subjected to the following treatment:

Group 1: Normal control (non-diabetic, received Water only)

Group 2: Diabetic control group (streptozotocin-induced, received Water only)

Group 3: Diabetic + Standard drug (metformin)

Group 4: Diabetic + 100mg/kg Chrysophyllum albidum pulp extract

Group 5: Diabetic + 200mg/kg Chrysophyllum albidum pulp extract.

Fresh pulps of Chrysophyllum albidum were collected, authenticated, and sun-dried. The dried pulps were grounded into powdered form. To 250g of the powdered crude sample,500Ml of 100% ethanol was added. The mixture was sealed with foil paper and allowed to stand at room temperature for7 days with frequent. The mixture was sieved, strained and the marc (damp solid material) was pressed. The solvent extract was filtered using Whatman No 1 filter paper as fine solvent extract was obtained. After the induction of diabetes using streptozotocin, the rats were treated daily for two (2) weeks with Chrysophyllum albidum pulp extract and the standard drug. Blood glucose level was monitored at baseline, 2 days intervals (e.g., Day 1, Day 3, Day 5, up till day 13) using a glucometer. The set of data obtained in this study was analyzed using SPSS statistical software version 23.0. ANOVA was the statistical analysis method used for this project work. Results of the phytochemical screening of Chrysophyllum albidum pulp extract revealed the presence of alkaloids, tannins, flavonoids, terpenoids and phenols. However, saponins were found to absent. Phenols were observed to have the highest concentration (20.1 mg/g), followed by alkaloids (15.2 mg/g), then flavonoids (10.8 mg/g). Terpenoids were found to have the least concentration (2.3 mg/g) compared to all other phytochemicals present. The highest amount of insulin was secreted by experimental animals in the normal control group (15.2 µU/mL) throughout the period of treatment. Experimental animals in group 2 showed the lowest amount of insulin (5.4 µU/mL) produced during the entire period of treatment. The extract of Chrysophyllum albidum pulp demonstrated significant antidiabetic effects, improved insulin levels translating to preserved pancreatic health in the diabetic rats in this study. Comparatively, Chrysophyllum albidum pulp extract showed efficacy similar to metformin, highlighting its potential as a natural anti-diabetic treatment

Keywords: Diabetes; Chrysophyllum albidum; Streptozotocin; Phytochemicals; Insulin; Antidiabetic; Rats

Introduction

Traditional medicine has been a cornerstone in the management of various diseases, including diabetes, particularly in developing countries where access to modern healthcare may be limited [1,2]. Medicinal plants, used in folk remedies, offer a reservoir of bioactive compounds that could be developed into modern pharmaceuticals [3]. One of such diseases that has been managed using traditional medicine is diabetes mellitus. Diabetes mellitus describes a cluster of metabolic disorders defined by elevated glucose levels [4,5]. It results from either insulin deficiency or insulin resistance, leading to serious complications if not managed properly [6,7]. Globally, the prevalence of diabetes has been rising, posing a significant public health challenge.

The International Diabetes Federation (IDF) reported that approximately 463 million adults were living with diabetes in 2019, and this number is projected to reach 700 million by 2045 if current trends continue [8]. This increase underscores the urgent need for effective and accessible therapeutic strategies. The diabetic population worldwide is predicted to rise to 693 million worldwide from its current figure of 451 million in the next 25 years with the majority of this increase coming from Sub-Saharan Africa [9,10]. Type 2 diabetes dominates the disease burden in Africa, with less than 10% of diabetic cases presenting as type 1. This has been attributed to rapid urbanization and economic development with its attendant behavioral changes such as sedentary lifestyles and unhealthy eating habits [11]. The costly nature of available synthetic hypoglycemic drugs in addition to their adverse effects limits their use in low- and middle-income countries like Nigeria. One of the experimental models to study diabetes and its complications involves the use of streptozotocin-induced diabetic rats [12]. Streptozotocin is known to selectively destroys insulin-producing beta cells in the pancreas, mimicking the pathophysiology of diabetes [13].

Chrysophyllum albidum (C. albidum) commonly known as the African star apple, is a tree well distributed in the coastal regions of West Africa [8]. Various parts of this tropical plant have been traditionally used in folk medicine for their purported health benefits. The anti-plasmodial, antihistaminic, anti-inflammatory and antimicrobial properties of the plant have been reported [14]. The ethanol extract of the seed cotyledons and root bark demonstrated moderate anti-hyperglycemic and hypolipidemic effects in alloxan-induced diabetic rats [15]. Phenolic compounds namely epicatechin, epigallocatechin and procyanidin B5 were isolated from the stem bark whereas the alkaloids skatole, eleagnine and tetrahydro-2-methylharman have been reported from the seed testa [15-17]. Traditionally, the stem bark is used in treating sleeping sickness, yellow fever, diabetes and malaria. Preliminary studies suggest that Chrysophyllum albidum possesses antioxidant and hypoglycemic properties, making it a potential candidate for managing diabetes [18,19]. However, its antidiabetic properties have not been fully explored.

Previous studies on the anti-diabetic potential of Chrysophyllum albidum have yielded promising results. For instance [20]. demonstrated that the leaf extract of Chrysophyllum albidum significantly lowered blood glucose levels in diabetic rats. However, comprehensive studies investigating the mechanisms by which Chrysophyllum albidum exerts its hypoglycemic effects are still needed. Understanding these mechanisms could facilitate the development of new, plant-based therapeutic agents for diabetes management. Given the increasing burden of diabetes and the limitations of current treatments [21,22], there is a pressing need to explore alternative therapies. Natural products like Chrysophyllum albidum offer a promising avenue for the discovery of new anti-diabetic agents that are both effective and affordable. This study aims to build on the existing body of knowledge by systematically investigating the antidiabetic effects of Chrysophyllum albidum in streptozotocin-induced diabetic rat model.

Materials and Method

Study Design

This experimental study used a randomized control trial design involving streptozotocin-induced diabetic rats. The study was conducted in accordance with the guidelines for the care and use of laboratory animals.

Ethical Approval

The study was carried out after ethical approval was obtained from University of Ilorin ethical review committee. We received an ethical approval number UERC/ASN/2017/907 of 12th Jun, 2017.

Experimental Animals

Male Wistar rats weighing between 150-200 grams were purchased and used in for this study. The animals were kept in an animal house under standard laboratory conditions The conditions included 12 hrs of light and dark cycles at a temperature of 25±2EC, as well as unlimited access to standard pellet food and water.

Sample Collection and Preparation of Chrysophyllum Albidum Pulp Extract

Fresh pulps of Chrysophyllum albidum were collected, authenticated, and sun-dried. The dried pulps were grounded into powdered form. To 250g of the powdered crude sample,500Ml of 100% methanol was added. The mixture was sealed with foil paper and allowed to stand at room temperature for7 days with frequent. The mixture was sieved, strained and the marc (damp solid material) was pressed. The solvent extract was filtered using Whatman No 1 filter paper as fine solvent extract was obtained. The ethanolic extract was concentrated by evaporating the solvent (100% ethanol) at50 ̊C using a rotary evaporator and vacuum oven to obtain the oily-sticky extract. It was dissolved in 80% for an aqueous extract of Chrysophyllum albidum pulp. The extract was then concentrated under reduced pressure and stored at 25°C until it was required for use.

Experimental Design

Twenty-five (25) male Wister rats were grouped into five groups. Each group consisted of five animals. The rats were subjected to the following treatment:

Group 1: Normal control (non-diabetic, received vehicle only)

Group 2: Diabetic control group (streptozotocin-induced, received vehicle only)

Group 3: Diabetic + Standard drug (metformin)

Group 4: Diabetic + 100mg/kg Chrysophyllum albidum pulp extract

Group 5: Diabetic + 200mg/kg Chrysophyllum albidum pulp extract

Induction of Diabetes in Experimental Animals

Induction of diabetes in experimental animals was done after 8-10 hrs of fasting by intraperitoneal injection of STZ dissolved in 0.1 M cold citrate buffer, pH 4.5, at a single dose of 35 mg/kg body wt. After STZ injection, the animals were allowed to drink a 10.0% glucose solution overnight to overcome the initial drug-induced hypoglycaemia. After 14 days, rats with glycaemia>250 mg dLG1 were selected for further experimentation.

Treatment of Animals and Blood Glucose Measurement

After the induction of diabetes, the rats were treated daily for two (2) weeks with Chrysophyllum albidum pulp extract and the standard drug. Blood glucose level was monitored at baseline, 2 days intervals (e.g., Day 1, Day 3, Day 5, up till day 13) using a glucometer; with the common routine of diabetic studies all the rats’ body weights and fasting blood glucose levels were measured on weekly basis and monitored for any behavioural changes during the study period.

Biochemical and Histological Analysis

Evaluation of Antidiabetic Effect of Chrysophyllum Albidum Pulp Extract on Experimental Animals

Blood glucose test was performed to investigate the anti-hyperglycemic effect of Chrysophyllum albidum pulp extract on experimental the experimental animals. Blood glucose levels were estimated by glucose oxidase/peroxidase reactive strips. Blood glucose levels were measured on weekly basis and monitored for any behavioral changes during the study period.

Evaluation of Insulin Level in Experimental Animals

Plasma insulin was measured using enzyme-linked immunosorbent testing kits (ELISA) on day 1 and day 13 [23].

Phytochemical Analysis

Determination of Total Flavonoid Content (TFC)

The flavonoid content in the sample was determined using the method described by [24]. About l0g of the plant sample was extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. The whole solution was filtered through whatman filter paper No 42 (125 mm). The filtrate was later transferred into a crucible and evaporated into dryness over a water bath and weighed to a constant weight.

Determination of Total Tannins

Tannin determination was carried out using the method described by [25]. About 500mg of the sample was weighed into a 50 ml plastic bottle. 50 ml of distilled water was added and shaken for 1 h in a mechanical shaker. This was filtered into a 50 ml volumetric flask and made up to the mark. Then 5 ml of the filtered was pipetted out into a test tube and mixed with 2 ml of 0.1 M FeCh in 0.1NHC1 and 0.008 M potassium Ferro-cyanide. The absorbance was measured at 720 nm within 10 min.

Determination of Total Phenols

Total phenols were determined using a spectrophotometric technique as described by [26]. For 15 minutes, the sample was boiled in 50 ml of ether to extract the phenolic component. 5 mL of the extract was pipetted into a50mL flask, followed by 10 mL of distilled water. There was also 2 mL of ammonium hydroxide solution and 5mL of strong amylalcohol added. The samples were prepared to the specification and left to react for 30 minutes to allow for colour development. The wavelength was measured at 505 nm.

Determination of Alkaloid Content

[27] approach was used to determine this. 5g of the sample was weighed into a 250 ml beaker, and 200 ml of 10% acetic acid in ethanol was poured in and sealed for 4 hours. This was filtered, and the extract was concentrated to one-quarter of its original volume in a water bath. Drop by drop, concentrated ammonium hydroxide was added to the extract until the precipitation was finished. Allowing the situation to settle, the precipitate was collected, washed with weak ammonium hydroxide, and then filtered. The residual was the dried and weighed alkaloid.

Determination of Saponin Content

[28] approach was employed in determining the saponin content of the samples. The samples were pulverised, and 20g of each was placed in a conical flask with 100 cm3 of 20% aqueous ethanol. The samples were heated in a hot water bath approximately 90°C. The concentrate was placed in a 250 ml separatory funnel, then 20 ml of diethyl ether was poured into it and vigorously agitated. The aqueous layer was saved, but the ether layer was not. The purifying procedure was carried out once again. N - butanol (60 mL) was added. The n-butanol extracts were rinsed twice with 10 ml of 5% aqueous sodium chloride. In a water bath, the residual solution was heated. Following evaporation, the samples were oven dried to a consistent weight, and the saponin concentration was measured as percentage. The statistical difference was judged significant at P < 0.05

Determination of Terpenoid Content

The method described by [24] was used to estimate terpenoid content in the samples. 100 mg (initial weight-wi) dried pulp extract was steeped in 9 ml ethanol for 24 hours and then filtered using Whatmann filter paper. Using a separating funnel, the filtrate was extracted with 10 ml of petroleum ether. The ether extract was separated and dried completely in pre-weighed glass vials (final weight-wf). Ether was evaporated and the yield (%) of total terpenoids was calculated by using the formula. (Wi-wf/wi x 100).

Statistcal Analysis

The set of data obtained in this study was analyzed using SPSS statistical software version 23.0. ANOVA was the statistical analysis method used for this project work. Values of all experimental variables were given as Mean ± Standard deviation

Results

Phytochemical Screening of Chrysophyllum albidum Pulp Extract

The present study investigated the phytochemical content of Chrysophyllum albidum pulp extract. Phytochemical screening of Chrysophyllum albidum pulp extract revealed the presence of alkaloids, tannins, flavonoids, terpenoids and phenols. However, saponins were found to absent as presented in Table 1 and Figure 1. Phenols were observed to have the highest concentration (20.1 mg/g), followed by alkaloids (15.2 mg/g), then flavonoids (10.8 mg/g). Terpenoids were found to have the least concentration (2.3 mg/g) compared to all other phytochemicals present (Table 1) (Figure1).

*Key: - = absent; + = present

Evaluation of Antidiabetic Effect of Chrysophyllum albidum Pulp Extract on Experimental Animals

Table 2 and Figure 2 present the result of average blood glucose level of experimental animals treated within a period of two (2) weeks. The lowest level of blood glucose was noticed on day when the treatment began across all groups of experimental animals. Group 3 experimental animals showed the highest blood glucose level (86 mg/dl) whereas the normal control showed the lowest blood glucose level (82 mg/dl).On day of treatment, blood glucose level of experimental animals experienced a shoot with group 3 and group 5 presenting the highest blood glucose level (240 mg/dl) whereas group 4 showed the lowest blood glucose level (83 mg/dl). A decrease in blood glucose level was observed in the experimental animals after 3 up till day 13. This trend was consistent for group 3, group 4 and group 5 experimental animals. Across all groups except group 4, day 2 treatment produced the highest blood glucose level amongst the experimental animals (Table 2) (Figure 2).

Evaluation of Insulin Level of Experimental Animals

The present study investigated the level of insulin in the experimental animals of various groups. The highest amount of insulin was secreted by experimental animals in the normal control group (15.2 µU/mL) throughout the period of treatment. Experimental animals in group 2 showed the lowest amount of insulin (5.4 µU/mL) produced during the entire period of treatment. Group 3 experimental animals produced the highest amount of insulin (12.7 µU/mL) compared to Group 4 (10.1 µU/mL) and Group 5 experimental animals (11.8 µU/mL) (Table 3).

Discussion

Globally, the prevalence of diabetes has been rising, posing a significant public health challenge [29]. The International Diabetes Federation (IDF) reported that approximately 463 million adults were living with diabetes in 2019, and this number is projected to reach 700 million by 2045 if current trends continue [30]. This increase underscores the urgent need for effective and accessible therapeutic strategies. The present study investigated the antidiabetic effect of Chrysophyllum albidum on streptozotocin-induced diabetic rats. The phytochemical composition of Chrysophyllum albidum pulp extract provides insight into the bioactive compounds present in the plant, which may contribute to its medicinal properties in managing diabetes. The phytochemical screening carried out revealed the presence of alkaloids, tannins, flavonoids, terpenoids and phenols. These compounds are known for their antioxidant, anti-inflammatory, and anti-diabetic properties. For instance, flavonoids and phenols are potent antioxidants that can neutralize free radicals, reducing oxidative stress, which is a key factor in the development of diabetes complications [31]. The identification of these compounds supports the traditional use of Chrysophyllum albidum pulp extract in managing diabetes and other ailments.

The antidiabetic effect of Chrysophyllum albidum pulp extract was evaluated by measuring the average blood glucose levels of streptozotocin-induced diabetic rats over a period of two (2) weeks. The study involved various groups, including normal control, diabetic control, diabetic rats treated with a standard drug (metformin), and diabetic rats treated with 100mg/kg and 200mg/kg of Chrysophyllum albidum pulp extract. The results indicated a significant reduction in blood glucose levels in the Chrysophyllum albidum pulp extract-treated groups compared to the diabetic control group. 200mg/kg of Chrysophyllum albidum pulp extract was particularly effective, showing antidiabetic effect comparable to that of metformin and other groups induced with streptozotocin. This suggests that Chrysophyllum albidum pulp extract is potentially an anti-diabetic agent, which is most likely due to its phytochemical constituents [32]. To further understand the anti-diabetic potential of Chrysophyllum albidum pulp extract, its effect on insulin levels was assessed. Diabetic rats treated with Chrysophyllum albidum pulp extract showed an increase in serum insulin levels compared to untreated diabetic control. This indicates that the extract may enhance insulin secretion or improve insulin sensitivity [33]. These findings suggest that Chrysophyllum albidum pulp extract not only lowers blood glucose but also helps preserve pancreatic function and integrity. STZ partially damages pancreatic islet cells, reducing the number of cells and insulin globules [34,35]. This damage leads to inflammation in the pancreas, which is called insulitis. In the untreated diabetic rats, the significantly low level of insulin suggests that the number and size of islets decreased greatly, and the degree of insulitis was also very high compared to the non-treated non-diabetic rats. However, administering does of Chrysophyllum albidum pulp extract significantly improved the level of serum insulin suggesting that significantly, the number and size of islets were increased, thus decreasing degree of insulitis.

Comparing the antidiabetic effect of Chrysophyllum albidum pulp extract and standard anti-diabetic drug (metformin), valuable insight was provided into the efficacy of the plant extract. Both treatments effectively reduced blood glucose levels and improved insulin levels in diabetic rats. Additionally, that both treatments preserved pancreatic tissue structure and function. While metformin is a well-established anti-diabetic drug, Chrysophyllum albidum pulp extract demonstrated comparable efficacy, particularly at higher doses. This suggests that Chrysophyllum albidum pulp extract could be considered as a complementary or alternative treatment for diabetes. However, further studies, including clinical trials, are necessary to fully establish its safety and efficacy in humans.

Conclusion

In conclusion, findings from this study confirms that the treatment groups responded differently over time, with varying doses of Chrysophyllum albidum pulp extract. Group 5 experimental animals that received the highest dose (200mg/kg) of Chrysophyllum albidum pulp extract showed the most promising results compared to the group that was treated with metformin and the group that received 100mg/kg of Chrysophyllum albidum pulp extract. The phytochemical analysis of Chrysophyllum albidum pulp extract supports its traditional use in managing diabetes due to its rich composition of bioactive compounds. The extract of Chrysophyllum albidum pulp demonstrated significant antidiabetic effects, improved insulin levels translating to preserved pancreatic health in the diabetic rats in this study. Comparatively, Chrysophyllum albidum pulp extract showed efficacy similar to metformin, highlighting its potential as a natural anti-diabetic treatment.

Acknowledgments

We want to thank all the researchers who contributed to the success of this research work.

Conflict of interest

Conflict of interest

The authors declared that there are no conflicts of interest.

Funding

No funding was received for this research work.

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