Antidiabetic and Antioxidant Potential of Detarium microcarpum and Zanthoxylum zanthoxyloides and Combined Extract Efficacy in Rats

Background and Objective: Detarium microcarpum and Zanthoxylum zanthoxyloides are both tropical medicinal plants used for the treatment and management of numerous ailments including diabetes. This study sought to evaluate the antidiabetic potential and efficacy, antioxidant and safety profile of the plants and combined extracts of the Detarium microcarpum and Zanthoxylum zanthoxyloides . Materials and Methods: The combined ( Detarium microcarpum and Zanthoxylum zanthoxyloides ) and separate plant parts were extracted with different solvents. Phytochemical analysis, radical scavenging activity and heavy metal content assessments were conducted. The antidiabetic potential of the extracts was further evaluated in vivo by employing the Oral Glucose Tolerance Test (OGTT) in 21 Wistar rats. The safety profile of the extracts was evaluated by assessing the effect of the extracts on the percentage change in body weights, hematological parameters and biochemical parameters as well. One-way and two-way analysis of variance followed by Dunnett’s multiple comparison test was used at p<0.05. Results: Alkaloids, terpenoids, saponins and tannins were present in aqueous extracts. The combined aqueous extract showed better radical scavenging activity. In a glucose tolerance test on rats, the combined extract demonstrated the most effective glucose reduction (from 6.83-4.60 mmol L G 1 ). Combined extract also had better antioxidant and antihyperglycemic effects compared to individual extracts. Conclusion: All extracts were safe concerning hematological and biochemical parameters. Combined extracts showed better therapeutic potential compared to the individual extracts.


INTRODUCTION
Diabetes is a chronic metabolic condition characterized by the inadequate secretion of insulin or the development of insulin resistance.Hyperglycaemia, polyuria, polydipsia, polyphagia, altered metabolism of carbohydrates, proteins and lipids and increased risk of vascular complications are some of the conditions associated with diabetes.Diabetes can be classified into three types namely, type 1 diabetes, type 2 diabetes and gestational diabetes with most diagnosed cases of diabetes being cases of type 2 diabetes [1][2][3][4] .
In Sub-Saharan Africa, approximately 19.4 million individuals have diabetes.Projections indicate that by 2030, this number will rise to 28.6 million, reaching 34.2 million by 2040.In Ghana, around 6.46% of adults are with diabetes mellitus 5 .
Although the control of diabetes is by synthetic oral hypoglycaemic drugs alongside insulin, these fail to reverse the course of the complication these drugs come along with and further exacerbate it by the expression of prominent side effects 6 .Aside from the prominent side effects of therapeutics, these drugs have high costs, with poor tolerability in patients and sometimes the development of therapeutic resistance 7 .
Diabetes management varies by type, typically involving insulin injections for type 1 and antidiabetic drugs for type 2. Lifelong treatment and regular follow-ups are essential.This complex approach can lead to economic strain on individuals, particularly in low and middle income countries.In such contexts, individuals often turn to cost-effective traditional plant remedies 8 .These medicinal plants are valued due to their accessibility, affordability and availability, offering a dependable treatment option.They are also considered safe mainly due to their natural origin and traditional usage, though scientific systematic studies on their usage remain limited 9 .
Zanthoxylum zanthoxyloides and Detarium microcarpum are both plants believed to have some medicinal properties.Zanthoxylum zanthoxyloides have been used in West African traditional medicine to manage and treat malaria, fever, sickle cell anaemia, tuberculosis, paralysis, elephantiasis, toothache, venereal diseases, dysmenorrhea, abdominal pain, cough, tuberculosis, urinary disorders, cancers and arthritis 10 .Detarium microcarpum has also been used traditionally in the treatment and management of numerous ailments such as stomach disorders, venereal diseases and gastrointestinal ailments.Investigations of the biological activities of D. microcarpum plants have shown the plant possesses some antimicrobial, antimalarial and cytotoxic effects 11 .
To fully have the desired therapeutic effect and benefits of medicinal plants, their quality must be assured in terms of their safety which includes contamination by heavy metals as well as safety and toxicological profile and assessment 12 .
Ensuring the therapeutic benefits of medicinal plants requires guaranteeing their quality in terms of safety, including heavy metal contamination, alongside overall safety and toxicity assessment 12 .Existing research on the anti-diabetic effects of medicinal plants primarily focuses on individual plants or specific plant parts.There's a lack of scientific data on the combined effects of multiple anti-diabetic plants.Consequently, the reliance on affordable, safe and effective natural remedies has gained importance.Thus, this study sought to evaluate the antidiabetic potential, efficacy, effect and safety profile of combined extracts of Z. zanthoxyloides and D. microcarpum on rats.
Processing of plant samples: The leaves, stem and roots of D. microcarpum and the leaves and stem bark of Z. zanthoxyloides were washed and air-dried at room temperature under shade for 28 days.After drying, the samples were pulverized and stored in a zip lock bag and kept at room temperature until needed for use.

Extraction of plant samples:
The extraction of the leaves, stems and roots of D. microcarpum and the leaves and stem bark of Z. zanthoxyloides was done by decoction for 48 hrs at room temperature.The aqueous, 50% ethanolic, methanolic, ethyl acetate and chloroform extraction of the various parts of D. microcarpum and Z. zanthoxyloides was carried out by suspending 60 g of the powder of the D. microcarpum leaves, stem and roots and Z. zanthoxyloides leaves and stem bark in 60 mL of the respective solvents namely distilled water, 50% ethanol (prepared in distilled water at 50:50 v/v), methanol, ethyl acetate and chloroform respectively.The extracts were then filtered through cotton wool and concentrated over a water bath at 60EC to obtain the various extracts.
The extraction of the best two performing extracts and their combination was done by decoction for 48 hrs at room temperature in the dark.

Phytochemical analysis:
The extracts were screened for the presence of alkaloids, tannins, terpenoids, saponins, phenols, flavonoids and cardiac glycosides according to the methods described by Thilagavathi et al. 13 with slight modifications.

Glucose uptake assay:
The assay was performed as employed by Jayatilake and Munasinghe 14 with some modifications.One gram of commercial baker's yeast was placed in a falcon tube and 10 mL of distilled water was added.Washing was through repeated centrifugation at 2800 g for 8 min, until the supernatant was clear.A 10% v/v solution of the extract was made with distilled water, discarding the pellets.Stock solutions of D. microcarpum and Z. zanthoxyloides extracts were formed by dissolving 20 mg in 2 mL of distilled water in Eppendorf tubes (2 mL).Concentrations of 1-5 mg mLG 1 for each extract were then prepared.Metformin (500 mg), the standard antidiabetic drug, was dissolved in 50 mL of distilled water with concentrations of 1-5 mg mLG 1 .Glucose solutions of 5, 10 and 25 mM were prepared by dissolving 4.5, 9 and 22.5 mg of glucose in 50 mL of distilled water.
In the assay, 400 µL of extract and metformin were added to 400 µL of glucose solution in 2 mL Eppendorf tubes.Incubation followed at 37EC for 10 min.The reaction was initiated by adding 40 µL of yeast solution to each tube, which was vortexed and then incubated at 37EC for an hour.After incubation, tubes were centrifuged at 2500 g for 5 min.Then, 200 µL of each reaction mixture were transferred into 96-well plate wells in triplicate.Absorbance at 540 nm was measured using a microplate spectrophotometer (Synergy H1, USA).The percentage increase in glucose uptake by yeast cells was calculated using the equation: Absorbance of control-Absorbance of sample Increase in glucose uptake (%) = ×100 Absorbance of control EC 50 values were determined by nonlinear regression analysis.

Determination of total phenolic content:
The total phenolic content (TPC) was assessed using the Folin-Ciocalteu method as described by Singleton et al. 15 and procedures as employed by Torkornoo et al. 16 .This method relies on the reduction of the Folin-Ciocalteu reagent by phenolics, resulting in the production of molybdenum-tungsten blue 17 .The concentration of phenolic compounds in each sample https://doi.org/10.3923/ajbs.2024.69.84 | Page 71 was determined using the gallic acid calibration curve and the gallic acid equivalence for each extract was calculated using the formula as mg of GAE/g of extract: V TPC = C× M where, 'C' is the concentration of gallic acid mg mLG 1 , 'V' is the volume of plant extract in mL and 'M' is the weight of the extract in grams.

DPPH radical scavenging activity:
The stock solutions of the aqueous extracts, hydro-ethanolic and methanolic extracts were used to determine the antioxidant activity as described by Brand-Williams et al. 18 with slight modifications.The percentage scavenging activity was determined by: Absorbance of blank -Absorbance of test Radical scavenging activity (%) = ×100 Absorbance of blank The mean percentage antioxidant activity for triplicates was graphically represented for both the standard and the extracts.The effective concentration at 50% (EC 50 ) values, indicating the amount of antioxidant required to reduce the initial DPPH concentration by 50%, were determined by nonlinear regression analysis.
Heavy metal analysis: Each powdered sample (raw powder and extract) of about 1g was weighed into a 50 mL digestion tube.Subsequently, 1 mL of H 2 O, 2 mL HCl, 5 mL of 1:1 HNO 3 :HClO 4 and 2 mL H 2 SO 4 were added.Allowing the samples to stand at room temperature for about 20 minutes to facilitate the foam formed to settle.The samples underwent heating on a digestion block on a hot for about 2 hrs at a temperature of 150 until a cleared solution was attained.The resultant clear solution was then filtered into a 100 mL volumetric flask using a 0.45 pm pore size membrane filter paper (Whatman filter paper No. 41).The filtrate was adjusted to the mark by topping up with distilled water and digested samples were transferred into the plastic bottles and stored at 4EC prior to analysis with blanks prepared alongside.Heavy metals including chromium, copper, iron, nickel and lead was analysed using the atomic absorption spectrophotometer (Nov 400P, Analytik Jena GmbH, Jena, Germany).Animals: Thirty Wistar rats were procured from School of Medicine, University of Ghana's animal holding facility.The rats were relocated to the Department of Biochemistry and Biotechnology at Kwame Nkrumah University of Science and Technology (KNUST), where they were housed in plastic cages with wood shavings.A 14-day acclimatization period to laboratory conditions was observed for the rats.The maintenance of optimal conditions of temperature between 24 and 26EC and 40-70% relative humidity was observed throughout the study.The Wistar rats were provided with unrestricted access to commercial feed and water.The identification of the rats involved in the study was by marking their tails with permanent makers.The conduct of the animal study followed the guidelines as set by the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA, New Delhi, India) and the Guide for Care and Use of Laboratory Animals 19 .
Grouping of animals: Twenty-one Wistar rats (of average weight 117 g) were randomly divided into seven groups each containing three rats as shown in Table 1.The rats were allowed access to feed and water ad libitum for 28 days.Control group received distilled water only and the treatment groups received 100 and 250 mg kgG 1 b.wt., using oral gavage.Treatment was done daily for 28 days.
Oral Glucose Tolerance Test (OGTT) in normoglycemic rats: Prior to the OGTT to determine the glucose lowering activity of the DMLAQ, ZZSAQ and their combined extracts, rats were fasted for 16  with access to water.Distilled water (normal control), a reference drug glibenclamide (5 mg kgG 1 b.wt.) or each of the two varying doses of DMLAQ, ZZSAQ and their combined extract (100 and 250 mg kgG 1 b.wt.) were administered to the rats by oral gavage to the various groups of rats each.After 30 min of the administration of the extracts and reference drugs to the various groups, glucose (10 g/kg) was administered to each rat.Blood samples from the twenty one rats were taken from the tail vein using a sterile lancet and blood glucose levels checked using Eco-Check Strips and Eco-Check Glucometer.The tail vein of all rats was cleaned and disinfected before and after use of the lancet.The blood glucose levels were taken before the administration of glucose, 1, 2 and 4 hrs after the administration of glucose as described by Larbie et al. 20 .

Extract treatment effect on body weight:
The body weight of each animal was measured before the start of extract treatment (day 0) and subsequently measured every other fourth day till the last day of extract treatment (day 28) using a mass balance (Zhejiang Haoyu, Jinhua, Zhejiang, China).The percentage change in body weights of the rats were computed using the following formula: where, Weight n is the body weight on day 4, 8-28 and Weight initial is the body weight on D0.

Termination of treatment:
The rats were sacrificed on the 28th day after an overnight fast.Animals were then exposed to cervical dislocation.With a sterile blade, incisions were made in the cervical region of the sacrificed rats and blood samples collected for haematological and biochemical analysis.

Evaluation of relative organ weight:
The procured lungs, kidney, pancreas, liver and heart were washed with normal saline, blotted dried with tissue paper and weighed.The relative weight of the excised organs with respect to the body weight of the rat at sacrifice was calculated from the formula: Absolute organ weight (g) Relative organ weight (%) = ×100 Body weight on day of sacrifice (g) Evaluation of haematological parameters: About 2 mL of blood samples were collected into EDTA K3 tubes for various haematological parameters using the Sysmex haematology system.The blood samples were evenly mixed to prevent blood clotting.The parameters assessed included white blood cell count (WBC), red blood cell (RBC) count, haemoglobin (Hgb), haematocrit, platelet count, lymphocytes, neutrophils, Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), Red Cell Distribution Width (RDW), plateletcrit, Platelet Distribution Width (PDW) and Platelet Larger Cell Ratio (P-LCR).
Statistical analysis: Data were analysed by GraphPad Prism 8 for Windows and the results were expressed as Mean±Standard Error (SEM).Data was then assessed by one-way and two-way analysis of variance followed by Dunnett's multiple comparison test.Values for which p<0.05 was considered significant.The EC 50 values were determined by nonlinear regression analysis.

Phytochemical constituent:
The aqueous extract of DML and ZSS had all phytochemicals assayed present except flavonoids.However, the combined extract of these two extracts had all phytochemicals present apart from phenols just as the aqueous extract of ZZS.The phytochemical constituents present in the various extracts were presented in Table 2.
The glucose uptake activity of D. microcarpum extracts remained relatively consistent at 5 and 10 mM glucose concentrations, with minor variations across concentrations, unlike the extracts of Z. zanthoxyloides as seen in Table 3 and 4. The maximum increase in glucose uptake by yeast cells was observed at 1 mg mLG 1 for most of the analyzed extracts.

Total phenolic content:
The ZZL plant part exhibited the highest total phenolic content among all samples.The hydroethanolic extract of ZZL had the greatest content (81.47±1.65 mg mLG 1 ), followed by ZZS with its highest content in the hydroethanolic extract (60.16±0.59mg mLG 1 ).While the combined aqueous extract showed higher phenolic content than DML aqueous extract, it was lower than ZZS aqueous extract.Hydroethanolic extracts had the highest phenolic content, followed by methanolic extracts, except for ZZL and ZZS extracts which had the highest content.This suggests higher phenolic content in Z. zanthoxyloides compared to D. microcarpum as seen in Table 7.

DPPH radical scavenging activity:
All the extracts reduced DPPH (2,2-diphenyl-1-picrylhydrazyl) to diphenylpicrylhydrazine at 517 nm.From the Table 8, the methanol extract of ZZL had the best DPPH radical scavenging activity followed closely by the hydroethanolic extract of ZZS and ZZL.However, the effective concentration (EC 50 ) at which half (50%) of free radicals were scavenged was low in all the aqueous extracts compared to the hydroethanolic and methanolic extracts.Of the aqueous extract, ZZL and the combined extract performed better with all the aqueous extract being statistically different from the standard ascorbic acid.All aqueous extracts of both plants and the combined extracts were statistically different from the standard ascorbic acid.
The glibenclamide group showed a further reduction, while most extract groups maintained elevated levels.The natural decline in blood glucose occurred in the normal group, resulting in no significant differences between normal and positive control (glibenclamide) groups, except for a significant decrease at 240 min (p<0.05-0.001).
At the 240 min mark, the 250 mg kgG 1 combined extract effectively suppressed postprandial hyperglycemia, with final blood glucose levels nearly matching the normal group.Notably, at 2 hrs, the 250 mg kgG 1 ZZS extract achieved better glucose reduction (6.20±1.11mmol LG 1 ) than the normal control (6.33±0.69mmol LG 1 ) and other extracts (excluding glibenclamide).
Haematology: There was no significance difference between the control group and both COMBO extracts in all haematological parameters.Furthermore, there were no significance difference between DML and ZZS extract for all haematological parameters except significant difference in platelet count for both extracts.There were significant increases (p<0.05-0.001) in platelet count for the DML 100 extracts and both ZZS extracts (100 and 250 mg) as seen in Table 13.

DISCUSSION
The mechanism of glucose transport across yeast cell membranes has garnered attention as an in vitro method for assessing plant extracts' hypoglycaemic potential.Interestingly, this study noted a general decline in glucose uptake by yeast cells as sample and glucose concentrations increased.Glucose uptake by cells is indicated by residual glucose in the medium after a specific time.Most extracts enhanced yeast cell glucose uptake, though some ethyl acetate extracts did not at 5 and 10 mM glucose concentrations 21 .This glucose uptake enhancement holds promise for better blood glucose level management and hyperglycaemia control 22 .
Yeast cell glucose uptake may differ from that of human cells.Yeast cell glucose transport might involve facilitated diffusion, unlike phosphotransferase enzyme systems seen in human cells.Muscle cell glucose uptake is driven by insulin-triggered glucose transport molecules accumulation in cell membranes.In yeast cells, glucose uptake is influenced by variables such as intracellular glucose concentration and metabolism.Increased glucose metabolism favours higher glucose uptake.diffusion and high levels of glucose metabolism.Investigating in vivo activity of these extracts could reveal enhanced glucose uptake in muscle and adipose tissues 23 .
The FBS, OGTT and HbA1c are clinically used to diagnose and manage prediabetes and diabetes.The OGTT is a clinically sensitive criterion that helps in the detection of early irregularities in the disposal of glucose 24 .In a diabetic state, the ability of insulin to stimulate the uptake of glucose from the bloodstream into cells is compromised.As a result, after the intake of carbohydrate-rich foods, maintaining the optimum blood glucose levels becomes challenging which reduces the body's ability to response to insulin which is released.This leads to the rise in blood glucose levels resulting in postprandial hyperglycaemia (PPHG) 25 .
Studies in relation to the effects of drugs on the reduction of PPHG have been one of the numerous key aspects in management of diabetes mellitus that has been a well-focused therapeutic approach up to date 23 .Most extracts known to have antidiabetic properties exert its abilities by suppressing postprandial hyperglycaemia.To manage PPHG, the digestion of carbohydrates into monosaccharides by the carbohydrate digestive enzymes and the absorption of these monosaccharides by the intestinal glucose transporters can be targeted 25 .From the results of OGTT, all extracts helped in the clearance of glucose after four hours to levels compared to normal and the standard drug glibenclamide, without any negative effect such as hypoglycaemia as seen with the standard drug glibenclamide.It could mean that the extracts especially the combined extract (COMBO) work in a similar mechanism like the standard drug and as well increase the permeability of glucose into the cells by increasing the activity of glucose transporters to take up more glucose into the cells 25,26 .
Drug toxicity can lead to body weight abnormalities, with decreased body weight and altered organ weight as indicators of toxicity with subtle changes in weight significant.During the study, all extracts increased rat body weight compared to control.Substantial body weight loss can be fatal 9 .Herbal products might impact organs like the liver, kidneys, spleen, brain and heart.Varied body weights and mostly unchanged relative organ weight, except lungs for ZZL 100 mg kgG 1 , suggest non-toxicity 27 .
All extracts were safe from heavy metals such as lead (Pb) and chromium (Cr), toxic metals absent in detectable amounts.The Pb poses risks like reproductive and renal system issues 28 .The absence of Pb suggests no harm to the renal system from extracts.With respect to all heavy metals analysed, the various plant extract and raw powder will not cause any negative effect resulting from excess concentration, when taken in as medicine.
Serum transaminase enzymes, ALT and AST, indicate hepatocyte toxicity.Elevated ALT and AST levels signal liver damage.The ALT is mainly found in cytoplasm of hepatocyte.The AST is found in heart, kidney, liver, muscle, pancreas and red blood cells 26 .The observed significant decline in levels of ALT in ZZS and DML extracts, as well as the non-significant decline of the combined extracts suggest possible hepatoprotective effects.All extracts at 100 mg kgG 1 showed non-significant triglyceride reduction and 250 mg kgG 1 extracts showed non-significant total cholesterol reduction, indicating potential lipidlowering effects, dose-dependent for cholesterol 29 .
The methanolic extracts of both plants exhibited better radical scavenging activity in the DPPH antioxidant assay, having the most effective EC 50 value.This was higher than the aqueous extract and combined aqueous extract, with the methanolic DMR extract having a better activity compared to ascorbic acid.EC 50 of the COMBO extract compared to individual components underscores the synergistic and/or antagonistic interactions of antioxidant-capable phytochemicals 30,31 .Future recommendations studies can be carried out to separate the active components exclusive to the combined extract of Zanthoxylum zanthoxyloides leaf extract and Detarium microcarpum stem bark and isolate the components to study the molecular interactions that contribute to the synergy and efficacy of the combine extract.Researches on the antidiabetic potential can be carried in vitro by glucose uptake activity with HEPG2 from rat diaphragm and L6 myoblast and in vivo in the STZ/NA rat model as the antidiabetic potential of the combined extract has been evaluated.

CONCLUSION
The combined aqueous extract (COMBO) had the best effect of suppressing postprandial hyperglycaemia than the individual aqueous extract of DML and ZZS and the best in vitro antioxidant activity due to the presence of phenols, alkaloids, terpenoids and flavonoids.The combined extracts also had lipid lowering and hepatoprotective potential.This indicates that the combined extract (COMBO) has a better therapeutic potential as an antidiabetic agent.

SIGNIFICANCE STATEMENT
Prior researches predominantly have delved into the antidiabetic attributes of individual plants or their specific plant parts.However, critical gaps exist in understanding the combined effects and efficacy of multiple antidiabetic plants.Recognizing the growing reliance of the individuals on a cost effective and safe natural remedies, this study sought to assess the collective antidiabetic potential, efficacy and safety profile of combined extracts from Zanthoxylum zanthoxyloides and Detarium microcarpum which seeks to provide basis for future development in the use of plant combinations for enhanced therapeutic benefits.

Table 3 :
Effect of various plant extracts on the percent increase in glucose uptake by yeast cell at 5 mM glucose concentration Concentration of extracts (mg mLG 1 ) Percent glucose uptake by yeast cells at different extract concentrations -

Table 4 :
Effect of various plant extracts the percent increase in glucose uptake by yeast cell due to the at 10 mM glucose concentration Concentration of extracts (mg mLG 1 ) Percent glucose uptake by yeast cells at different extract concentrations -

Table 13 :
Effect of DML, ZZS and COMBO (100 and 250 mg kgG 1 , p.o) on haematological indices in Wistar rats after 28 days of administration compared to control The combined aqueous extract (COMBO) performed better than the individual components (aqueous DML and ZZS extracts).The extracts having the most potent value of EC 50 values of extracts can be attributed to extracts rich in bioactive phytochemicals like phenols, alkaloids, terpenoids and flavonoids.The lower https://doi.org/10.3923/ajbs.2024.69.84 | Page 81