Antidiabetic Activity, Phytochemical Analysis, and Acute Oral Toxicity Test of Combined Ethanolic Extract of Syzygium polyanthum and Muntingia calabura Leaves

Syzygium polyanthum is known for its capacity to regulate blood glucose levels in individuals with diabetes, while Muntingia calabura leaves have a traditional history as an alternative therapy due to their antidiabetic compounds. The combination of these two plants is expected to yield more optimized antidiabetic agents. This study aims to assess the antidiabetic activity of the combined ethanolic extract of S. polyanthum and M. calabura leaves by measuring the in vitro inhibition of the α-glucosidase enzyme and the blood glucose level in streptozotocin-induced rats and to determine the phytochemical contents of total phenolics, total flavonoids, and quercetine as marker compounds. Acute oral toxicity test was also evaluated. Both plants were extracted by maceration using 96% ethanol. Various combinations of S. polyanthum and M. calabura leaves extracts (1 : 1, 2 : 1, 3 : 1, 1 : 3, and 1 : 2) were prepared. The in vitro test, along with the total phenolic and total flavonoid content, were measured by using UV-Vis spectrophotometry, while quercetine levels were quantified through high-performance liquid chromatography (HPLC). The in vivo and acute toxicity tests were performed on rats as an animal model. The findings demonstrated that the 1 : 1 combination of S. polyanthum and M. calabura leaves ethanolic extract displayed the highest enzyme inhibitory activity with IC50 value of 36.43 µg/mL. Moreover, the combination index (CI) was found <1 that indicates the synergism effect. This combination also decreases the blood glucose level in rats after 28 days of treatments without significant difference with positive control glibenclamide (p > 0.005), and it had medium lethal doses (LD50) higher than 2000 mg/kg BW. Phytochemical analysis showed that the levels of total phenolics, total flavonoids, and quercetine were 30.81% w/w, 1.37% w/w, and 3.25 mg/g, respectively. These findings suggest the potential of combined ethanolic extracts of S. polyanthum and M. calabura leaves (1 : 1) as raw materials for herbal antidiabetic medication.


Introduction
Diabetes mellitus is a serious health issue that afects globally.According to the International Diabetes Federation, the global prevalence of diabetes was estimated to be 9.3% in 2019 and projected to rise by 2045 [1].Te chronic condition's diabetes elevates the incidence has spurred interest in alternative treatments beyond conventional synthetic drugs, which often have side efects including gastritis, diarrhea, hypoglycemia, abdominal pain, nausea, fatulence, headache, fatigue, vomiting, and pharyngitis.As a result, there has been a surge in exploring natural products as potential solutions for managing diabetes [2][3][4].
Medicinal plants ofer natural antidiabetic properties with fewer side efects [5].For instance, Gymnema sylvestre, an herb native to India and commonly used in Ayurvedic medicine, contains gymnemic acids that inhibit alphaglucosidase activity, reducing blood sugar levels [6].Similarly, mulberry leaves and bitter melon (Momordica charantia) extracts have shown promising results in glycemic control [4,7].Medicinal plants with analogous activity may form synergistic interactions that signifcantly enhance the therapeutic efcacy of treatments.Tis principle has been employed in traditional medicines for centuries, where combinations of herbs are used to efectively address various diseases.Moreover, recent studies have demonstrated that polyherbal extracts have also demonstrated enhanced therapeutic efcacy with minimal side efects [8,9].Studies have evaluated the α-glucosidase enzyme inhibitory activity of combination of extracts of Oryza sativa L. var glutinosa and Orthosiphon aristatus.Combinations of these extracts in a 1 : 1 ratio exhibited IC 50 values of 67.82 µg/mL [10].Furthermore, the ethanol extracts of turmeric, ginger, and black tea water extract were tested for their α-glucosidase inhibition.Te IC 50 values obtained were 9.48 ± 0.05 g/mL for turmeric, 66.64 ± 0.44 g/mL for ginger, and 9.52 ± 0.25 g/ mL for black tea.Among these extracts, the combination of turmeric, ginger, and black tea exhibited the highest α-glucosidase inhibition at 67.86 ± 0.93% [11].
Exploration of medicinal plants from Indonesia presents a promising source for alternative treatments including diabetes.Particularly, S. polyanthum and M. calabura have gained attention for their culinary uses as well as their reported therapeutic properties.S. polyanthum not only adds favor to Indonesian cuisine but also shows promise as a natural remedy for managing diabetes.S. polyanthum leaves have long enjoyed a reputation for their benefcial efects in diabetes management [12].Extensive research has revealed that S. polyanthum leaves possess compelling antidiabetic properties, including glucose-lowering efects and insulin-enhancing activity [13].Tis rich history of traditional use, combined with scientifc evidence, highlights S. polyanthum as a captivating natural resource ripe for exploration as an alternative for diabetes management.
Recent research has revealed its potential therapeutic efects in combating this prevalent metabolic disorder.S. polyanthum leaves are rich in bioactive compounds such as favonoids and polyphenols, which have demonstrated remarkable antidiabetic properties.Tese compounds have the potential to enhance glucose metabolism and improve insulin sensitivity, making S. polyanthum an intriguing natural product worth exploring for diabetes management [12,14].
Another captivating plant species, M. calabura, has garnered attention for its potential therapeutic value in diabetes management.Apart from bearing delicious fruits, M. calabura also holds the potential to assist in the fght against diabetes.Studies have identifed bioactive compounds like phenolics, favonoids, and triterpenoids in the leaves and fruits of M. calabura, which exhibit signifcant antidiabetic properties.Tese compounds play a vital role in regulating blood glucose levels, enhancing insulin secretion, and protecting pancreatic cells from damage, positioning M. calabura as a promising natural product for diabetes management [15].Similarly, M. calabura leaves have long been used as an alternative therapy due to their compounds with antidiabetic properties.Similarly, M. calabura leaves have been celebrated for their potential antidiabetic and antioxidant properties [16].Scientifc investigations have demonstrated that M. calabura leaf extracts exhibit remarkable glucose-lowering efects and ofer protection against the oxidative stress commonly associated with diabetes.Tese remarkable fndings further emphasize the therapeutic potential of M. calabura in the management of diabetes [17].Combining these two plants is expected to provide a synergistic efect, potentially enhancing their efcacy in diabetes management.Our previous in silico experiment found that alphaglucosidase was the target for metabolite compounds of both S. polyanthum and M. calabura [18].
Te primary objective of this study was to evaluate the in vitro and in vivo antidiabetic potential of the combined ethanolic extract of S. polyanthum and M. calabura leaves.Te assessment will be alpha-glucosidase activity inhibition assay and blood glucose decreasing level on streptozotocininduced diabetic rats.Additionally, the investigation will further determine the total phenol, total favonoid, and quercetine contents and also the acute oral toxicity test.Te outcomes of this research are anticipated to enrich our comprehension of the antidiabetic attributes intrinsic to the combined plant extracts of S. polyanthum and M. calabura leaves.Moreover, the study seeks to illuminate the viability of these common Indonesian plants as valuable natural sources of antidiabetic agents.In doing so, it aims to lay the groundwork for deeper exploration and the development of natural interventions for the management of diabetes.

Materials.
In this study, ethanol analytical grade was used as an organic solvent, along with phosphate bufer, 4nitrophenyl α-D-glucopyranoside, hexamethylenetetramine (HMT), HCl, glacial acetic acid, AlCl 3 , Folin-Ciocalteu reagent, NaOH, and gallic acid as part of our experimental setup.M. calabura and S. polyanthum leaves samples were collected from Lembasada Village, South Banawa District, Donggala Regency, Central Sulawesi Province, in March 2023.Taxonomists from the Department of Biology, Faculty of Mathematics and Natural Sciences, Tadulako University, verifed and authenticated the herbarium specimens of the plant samples under the identifcation number 218/UN28.1.28/BIO/2022.

Extraction.
All samples were thoroughly rinsed under a continuous stream of tap water to remove debris and then gently dried.Te cleaned M. calabura and S. polyanthum leaves were subsequently cut into smaller pieces, approximately 0.5 cm in length.Once cut, the samples were dried to complete dryness in a dehydrator set at 40 °C.Once fully dried, the samples were coarsely ground into powder using an electrical grinder.Te extraction process was carried out using 96% ethanol through the maceration technique for 3 × 24 hours per cycle at room temperature.After each maceration cycle, the mixture was fltered and the fltrates were evaporated by using a rotary evaporator to obtain a viscous extract.

Experimental Animals.
Male Wistar rats 3-4 months old weighing 200-230 g were selected for this study.Tey were fed with standard rats' pellet, and water was given ad libitum.Te animals were acclimatized for 1 week before the induction of experimental diabetes.Te experimental protocols were conducted in accordance with ethical guidelines as approved by the Ethics Committee for Medical and Health Research, Faculty of Medicine, Tadulako University (932/UN.28.1.30/KL/2023).

Induction of Diabetes Mellitus.
Experimental diabetes was induced in overnight fasted experimental rats by a single intraperitoneal injection of streptozotocin (30 mg/kg body weight) dissolved in 0.1 M freshly prepared cold citrate bufer pH 4.5.After 36 h for development of diabetes, blood glucose was measured and rats with fasting serum glucose levels more than 200 mg/dL were considered diabetic and selected for further study.

Experimental Design.
After the successful induction of experimental diabetes, the rats were divided into six groups each containing a minimum of 5 rats.Group 1 was given a suspension of M. calabura leaf extract at a dose of 200 mg/ kg BW.Group 2 was given a suspension of S. polyanthum leaf extract at a dose of 200 mg/kg BW, and Group 3 was given a suspension of combined extracts of M. calabura and S. polyanthum leaves at a dose of 1 : 1 (200 : 200 mg/kg BW).Group 4 was given a suspension of glibenclamide 0.45 mg/kg BW, Group 5 (negative control) was given 0.5% Na-CMC, and Group 6 was a normal control without any treatment.Body weight and plasma glucose level measurements were conducted weekly during the experiment.Te plasma glucose level was measured using a one-touch glucometer.Te dosage of the extracts was adjusted every week to accommodate changes in body weight to maintain the same dosage throughout the experiment.Tey were administered orally for 28 days.After 28 days, the rats were fasted overnight and euthanized under anesthesia (ketamine) following blood sample collection and the organs of heart and kidney were dissected and stored at −20 °C.

Acute Oral Toxicity
Test.Acute oral toxicity study was performed for the combined extracts of S. polyanthum and M. calabura (1 : 1) according to the guidelines of the Organisation for Economic Cooperation and Development (OECD) [22].Te rats were kept on fasting overnight, being provided only water prior to oral dosing.Ten, the extract was administered orally at diferent dose levels, that is, 175, 550, and 2000 mg/kg of body weight.Te rats were closely observed for specifc toxicities and behavioral alterations such as restlessness, tremors, diarrhea, sluggishness, weight loss, and paralysis at consistent intervals during the initial four hours following the administration of the extract for a total of 24 hours.Subsequently, daily observations were conducted over a two-week period to detect any changes in overall behavior and physical activities.Food access was provided four hours after administering the extracts [23].

Determination of Total Flavonoid.
Te quantifcation of favonoid content involved the utilization of a reagent comprising multiple components: HMT (0.5% w/v hexamethylenetetramine solution), HCl (25% w/v hydrochloric acid solution), glacial acetic acid (5% v/v glacial acetic acid solution in methanol), and AlCl 3 (2% w/v AlCl 3 solution in glacial acetic acid solution).In the preparation of the stock solution, an extract equivalent to 200 mg of the crude simplicia was combined with 1 mL of HMT solution, 20 mL of acetone, and 2 mL of HCl solution.Tis mixture was subjected to refux for 30 minutes, fltered, and the resulting fltrate was transferred into a 100 mL volumetric fask.Subsequently, the residue underwent another refux with 20 mL of acetone for 30 minutes, followed by fltration and combination with the volumetric fask, which was then augmented with acetone to achieve a total volume of 100 mL.A further step involved transferring 20 mL of the fltrate to a separating funnel, where it was combined with 20 mL of water.Tis mixture underwent thrice-extraction with 15 mL portions of ethyl acetate each time.Te collected ethyl acetate fraction was then amalgamated with additional ethyl acetate in the volumetric fask to attain a total volume of 50 mL.Blank solution was meticulously concocted by drawing 10 mL of the stock solution and adding glacial acetic acid solution until the total volume reached 25 mL in the volumetric fask.For the sample solution, 10 mL of the stock Te Scientifc World Journal solution was combined with 1 mL of AlCl 3 solution and glacial acetic acid solution, leading to a total volume of 25 mL within the volumetric fask.After the introduction of AlCl 3 , measurements were conducted utilizing a spectrophotometer set at a wavelength of 425 nm, employing quercetine as the reference.Tese measurements were carried out after a 30-minute interval [24].
2.9.Determination of Total Phenolics.Te procedure for determining the total phenol content was carried out as follows: for the standard (gallic acid), a gallic acid stock solution was prepared at a concentration of 5000 ppm.Tis was achieved by weighing 12.5 mg of gallic acid and dissolving it in analytical grade methanol within a 25 mL volumetric fask.Subsequently, a series of standard concentrations spanning 0, 10, 30, 50, 70, and 100 ppm were established within separate 25 mL fasks.From each concentration, 1 mL was pipetted into a test tube, followed by the addition of 1 mL of the gallic acid solution and 5 mL of the 7.5% Folin-Ciocalteu reagent.Te mixture was meticulously mixed and left to incubate in a dark environment for approximately 8 minutes.Afterwards, 4 mL of the 1% NaOH reagent was introduced, mixed once more, and left to incubate for 1 hour in the dark.Te solution was eventually assessed using a spectrophotometer, measuring at a wavelength of 730 nm.For the sample analysis, 10 mg of extract was precisely weighed and dissolved in a 25 mL volumetric fask using analytical grade methanol.Following this, 1 mL of the sample solution was drawn into a test tube, and 5 mL of the 7.5% Folin-Ciocalteu reagent was added, mixed, and allowed to incubate in the dark for around 8 minutes.Subsequently, 4 mL of the 1% NaOH reagent was introduced, mixed again, and incubated for 1 hour in a dark environment.Te resulting solution was then evaluated using a spectrophotometer at a wavelength of 730 nm [25,26].

Determination of Quercetine by HPLC.
Quercetine quantifcation was conducted employing high-performance liquid chromatography (HPLC) to ascertain the quercetine content within the combined extracts.Te analysis was executed utilizing a C18-250 × 4.60 mm, 5 μm, 100 Å column, with a mobile phase composed of HPLC-grade potassium hydrogen phosphate (pH 2.4) and acetonitrile (75 : 25) in an isocratic fashion over a 30-minute period.Te fow rate was consistently maintained at 1 ml/min, and the UVvisible detector was set to 340 nm [27,28].

Statistical Analysis.
IC 50 values were ascertained from dose-inhibition (curve ft) analysis using nonlinear regression within GraphPad Prism Software, version 9 (Graph Pad Software, San Diego, CA, USA).

In Vitro and In Vivo Antidiabetic Activity.
In this study, we investigated the potential synergistic efects of individual and combined extracts of S. polyanthum and M. calabura, both of which are recognized for their antidiabetic properties.Te primary objective was to evaluate their inhibitory activity against the α-glucosidase enzyme, thereby assessing the potential to enhance inhibition capacity through synergistic interactions.Te results, as depicted in Figure 1, exhibited a dosedependent profle of inhibition.Notably, increasing concentrations of the combined extracts correlated with higher percentages of inhibition.Remarkably, the inhibition rate observed in S. polyanthum extract demonstrated a lower IC 50 value of 24.93 µg/mL compared to that of M. calabura extract (81.05 µg/mL).Tis result aligns with the fndings observed when the extracts were combined in a 1 : 1 ratio, showcasing a signifcant inhibition rate of over 50% at a relatively low dose (62.5 µg/mL).Notably, this combination displayed the lowest IC 50 value of 36.42 µg/mL, with signifcant diferences (p < 0.05) compared among other combinations.Although the IC 50 value of this combination (1 : 1) is not yet stronger than that of acarbose (standard drug), nevertheless it underscores the potent inhibitory efect of this mixture, as demonstrated by the IC 50 value falling below 50 µg/mL.In contrast, other combinations of extracts exhibited IC 50 values ranging from 73.94 to 89.67 µg/mL, indicating a moderate inhibitory efect (above 50 µg/mL), as detailed in Table 1.
Table 2 presents the results of assessing the efcacy of a combination of a 96% ethanol extract of S. polyanthum and M. calabura in inhibiting the α-glucosidase enzyme, utilizing the Combination Index (CI) as the analytical parameter.Initiating with a low ratio of extracts in combinations serves as a straightforward method to ascertain the impact of each extract on both efcacy and toxicity.Tese preliminary data are crucial for identifying the infuence of individual extracts when combined, enabling the estimation of the optimal combination ratio.Both the S. polyanthum and M. calabura extracts individually exhibit commendable antidiabetic activity through α-glucosidase enzyme inhibition.Te synergistic efect of combining the 96% ethanol extract of S. polyanthum and M. calabura in a 1 : 1 ratio is evident, as indicated by a lower IC 50 value and a Combination Index (CI) of less than 1, compared to other combinations that exhibit antagonism, which is shown by a CI > 1. Tis fnding suggests a synergistic efect, consistent with prior research indicating that combining plant extracts enhances α-glucosidase enzyme inhibition through synergism, a positive interaction exceeding the sum of individual substances.However, beyond synergism, other combinations exhibit antagonism efects.Antagonism occurs when the combined activity is lower than that of each individual extract.
Te synergistic efect of a combined 1 : 1 ratio of S. polyanthum and M. calabura leaf extracts was investigated for its potential to ameliorate hyperglycemia in a streptozotocin-induced diabetic rat model.As depicted in Figure 2, the longitudinal assessment of blood glucose levels over a 28-day span revealed a consistent downward trend in the treatment group, in contrast to the negative control and normal control groups.Notably, glibenclamide, serving as a positive control, signifcantly attenuated blood glucose concentrations to 83 mg/dL throughout the duration of the treatment period.

4
Te Scientifc World Journal Te synergistic potential of a 1 : 1 combination of S. polyanthum and M. calabura leaves extract was further investigated for its efcacy in reducing blood sugar levels in streptozotocin-induced diabetic rats.According to Figure 2, the treatment group exhibited a consistent decrease in blood glucose levels over a 28-day period, in contrast to both the negative control and normal control groups.Tis outcome underscores the efcacy of the combined extracts in managing blood glucose levels.Notably, glibenclamide, employed as a positive control, signifcantly decreased blood glucose levels to 83 mg/dL, demonstrating a marked difference from the normal control (p < 0.005).Similarly, M. calabura extract exhibited superior efcacy in lowering blood glucose levels compared to S. polyanthum extract during the 28-day treatments.Surprisingly, when the two extracts were combined, their collective activity became more potent, resulting in a blood glucose level of 94 mg/dL, which is proved by a signifcant diference than the normal control (p < 0.005).Intriguingly, no signifcant diferences were observed between the 1 : 1 extract combination and positive control glibenclamide, indicating the efectiveness of the combined dose as an antidiabetic agent.Furthermore, the extract combination demonstrated the highest average percentage decrease in blood glucose levels (72.35%), reinforcing the synergistic efect observed (Table 3).

Acute Oral Toxicity.
Te acute toxicity study revealed that the combined administration of ethanolic extracts from S. polyanthum and M. calabura (1 : 1) exhibited no discernible signs of toxicity at doses up to 2000 mg/kg.Tis lack of toxicity was substantiated by the absence of signifcant alterations in various behavioral indicators, including alertness, motor activity, weight, sluggishness, paralysis, breathing, restlessness, diarrhea, convulsions, and coma.Furthermore, there were no fatalities observed over a twoweek period, and the subjects remained physically active.Te fndings indicate that the tested doses of the plant extracts did not result in any observable adverse efects, suggesting that the median lethal dose (LD 50 ) exceeds 2000 mg/kg body weight in rats.Consequently, the combined extract can be considered nontoxic, as its actual LD 50 surpasses the 2000 mg/kg threshold.Additionally, a subsequent organ histopathology test at a dose of 2000 mg/kg body weight revealed mild changes and damage to the organs (Figure 3).

Total Phenolic, Total Flavonoid, and Quercetine Content.
Table 4 presents the results for total phenolic and total phenolics.Te determination of total phenolic content relied on the linear regression of the standard quercetine compound, following the equation y � 0.0513x − 0.0461 (r 2 � 0.9979).Te combined extract exhibited varying total phenolic content within the range of 24.51 to 32.40% w/w.Te Scientifc World Journal Similarly, the quantifcation of total favonoids was based on the linear regression of the gallic acid standard compound, with the equation y � 0.0027x − 0.0441 (r 2 � 0.9997), as depicted in Figure 4. Te total favonoid content fell within the range of 0.67 to 1.37% w/w.Meanwhile, quercetine quantifcation showed that the combined extract (1 : 1) contained detectable levels of quercetine with a sharp peak that can be comparable with the quercetine standard at retention time of 14.947 minutes.Te concentration of quercetine in the combined extract was found to be 3.25 mg/g (Figure 5).

Discussion
Patients with diabetes require meticulous management of their blood glucose levels, particularly during fasting and postprandial states.An enzyme known as α-glucosidase, predominantly present in the small intestine, plays a pivotal role in carbohydrate metabolism.Targeting this enzyme has emerged as a promising strategy for antidiabetic treatment.6 Te Scientifc World Journal     Te Scientifc World Journal individuals with diabetes [29].Several studies found that drugs capable of inhibiting the action of digestive enzymes like α-glucosidase can efectively regulate postprandial blood glucose levels in diabetic patients [30].Moreover, studies have highlighted the inhibitory potential of various plant extracts on α-glucosidase, indicating their role in managing diabetes and postprandial hyperglycemia [31][32][33].Additionally, natural compounds from plants or even the extracts and fractions have been investigated for their inhibitory efects on α-glucosidase, ofering potential as alternative treatments for controlling high blood sugar levels [34,35].
Te exploration of natural products for antidiabetic properties has led to the identifcation of S. polyanthum and M. calabura as potential sources of antidiabetic agents.S. polyanthum and M. calabura are two medicinal plants that have been reported to possess antidiabetic properties based on an alpha-glucosidase inhibitory mechanism.Te 50% ethanol extract of M. calabura and the 70% ethanol extract of S. polyanthum inhibited the enzyme with low IC 50 values of 0.46 and 19.08 µg/mL, respectively [16,36].In this work, the 96% ethanolic extract of S. polyanthum and M. calabura showed IC 50 values of 24.39 and 81.05 µg/mL, respectively.Interestingly, when the extracts were combined in a 1 : 1 ratio, the IC 50 became 36.43 µg/mL.Te compounds with IC 50 values below 50 µg/mL are considered to have superior activity which made the combination of extracts from S. polyanthum and M. calabura show promising results in inhibiting key enzymes like α-glucosidase, which is essential in managing blood sugar levels in diabetes [37].Te low IC 50 values observed in this combination suggest a higher potency in inhibiting these enzymes, potentially leading to better control of blood glucose levels.Tis phenomenon suggests a nuanced interaction between the compounds present in the two extracts when combined, leading to a modifed bioactivity profle.Tis is substantiated by the fact in this study that reveal the combination manifested a synergistic efect, as evidenced by a Combination Index (CI) value of 0.94, which is less than 1.Te concept of combining extracts to achieve a modifed or enhanced biological efect or mitigate adverse efects.For instance, the combination of extracts has been shown to potentially ofer synergistic efects, where the combined activity of the extracts is greater than or equal to the sum of their individual activities.Synergistic interactions can lead to enhanced bioactivity, which might result in a more potent therapeutic efect than would be expected from the individual activities of the single compounds alone [38][39][40].Combining plant extracts has been shown to enhance the pharmacological activity potential due to the presence of multiple compounds, leading to synergistic or additive efects.Tis approach not only diversifes the sources of active ingredients but also reduces the pressure on specifc plant species, supporting sustainability [41].
Further in vivo analysis showed that the combination of S. polyanthum and M. calabura extracts in a 1 : 1 ratio at a dose of 200 mg/kg BW demonstrated signifcant synergistic efects in reducing blood glucose levels.Tis synergism was evidenced by a remarkable 72.35% reduction in blood glucose levels after 28 days of treatment, surpassing the efects of the individual extracts of S. polyanthum and M. calabura alone.Another study demonstrated that the methanolic extract of S. polyanthum, at doses of 250, 500, and 1000 mg/kg BW, could reduce blood glucose levels in streptozotocin-induced rats.Additionally, the water extract of M. calabura, at a dose of 400 mg/kg BW, also reduced blood glucose levels and increased insulin selectivity [15,42].
Te 1 : 1 extract combination exhibited a notably low IC 50 value in the inhibition of alpha-glucosidase and in vivo antidiabetic activity, a result that could be attributed to its elevated total favonoid content.Tis higher favonoid content likely played a pivotal role in conferring a strong category of alpha-glucosidase inhibitory and in vivo antidiabetic activity.Several reports have supported our results, showing that M. calabura contains several antidiabetic compounds derived from favonoids and favonoid glycosides such as geniposide, daidzein, quercitrin, 6hydroxyfavanone, kaempferol, and formononetin.Meanwhile, S. polyanthum also contains antidiabetic compounds with favonoids' type, including myricetin-3-O-rhamnoside (myricitrin) and epigallocatechin-3-gallate (EGCG) [16,43].Terefore, in this study, we propose quercetine, a favonoid, as a bioactive and marker compound.Its presence has been identifed in the extract combination (1 : 1) and exhibited a direct correlation with total favonoid content, in vitro alpha-glucosidase inhibitory activity, and in vivo antidiabetic activity.Quercetine itself has been reported as a potent α-glucosidase inhibitor, demonstrating a competitive mechanism [44,45].Tese fndings highlighted the potential for developing an extract combination of these two plants as a raw material for antidiabetic herbal medicine, particularly noteworthy for its demonstrated lack of toxicity in acute oral toxicity tests.

Conclusion
Te synergistic potential of the combined ethanolic extracts from M. calabura and S. polyanthum has been proven to yield a signifcant α-glucosidase inhibitory and blood glucose level decreasing efect.Particularly, the 1 : 1 combination displayed the most enzymatic inhibitory activity, with an IC 50 value of 36.43 mg/L and 73.25% of DGBL on streptozotocin-induced rats animal.Tis efcacy was found to correlate with the elevated concentrations of total phenolics, total favonoids, and quercetine, which measured at 30.81% w/w, 1.37% w/w, and 3.25 mg/g, respectively.Tese fndings highlighted the promising role of these combined extracts in the observed efects.

Figure 1 :
Figure 1: In vitro antidiabetic activity assay through inhibition of alpha-glucosidase enzyme activity.
Inhibitors of α-glucosidase have demonstrated signifcant potential in managing postprandial hyperglycemia and treating diabetes by impeding the rapid conversion of complex carbohydrates into glucose.Tis inhibition leads to a controlled release of glucose into the bloodstream, thereby averting sudden spikes in blood glucose levels after meals, which is crucial for maintaining glycemic control in * *

Figure 3 :
Figure 3: Histopathology of rat organs of 1 : 1 extract combination at a dose of 2000 mg/kg body weight (right) compared to normal control (left).

Figure 4 :
Figure 4: Linear regression curves of total favonoids by using quercetine standard (a) and total phenolic by using gallic acid standard (b).

Table 1 :
IC 50 value of alpha-glucosidase inhibition of a combined ethanolic extract of S. polyanthum and M. calabura.

Table 2 :
Te combination index of alpha-glucosidase inhibition of the combined ethanolic extract of S. polyanthum and M. calabura.
cTe diferent subscript letters in the table indicate a signifcant diference between the treatment groups with a 95% confdence level (p < 0.05) using post hoc Tukey's HSD test.

Table 4 :
Total phenolic, total favonoids, and quercetine levels of the combined extract of S. polyanthum and M. calabura leaves.