Introduction

Diabetes Mellitus (DM) is a metabolic illness that is defined by insufficient insulin production and/or impaired insulin use within the body. Diagnosis of DM involves the observation of a notable elevation in blood glucose levels (Azis et al., 2020). The global prevalence of diabetes mellitus (DM) has exhibited a consistent upward trend across diverse nations, encompassing both high-income, low-income, and middle-income countries. According to data collected by the World Health Organization (WHO) since 1980, there has been a fourfold increase in the global population of people diagnosed with diabetes mellitus (DM), reaching a staggering 422 million individuals. According to the World Health Organization (2016), diabetes was responsible for around 1.5 million deaths out of a total of 89 million mortalities in the year 2012. According to the findings of the Sample Registration Survey conducted in 2014, it is evident that diabetes mellitus (DM) ranks as the third leading cause of mortality in Indonesia, accounting for 6.7% of deaths. This places DM behind stroke, which accounts for 21.1% of deaths, and coronary heart disease, responsible for 12.9% of deaths. According to Riska (2016), the International Diabetes Federation reported an estimated population of 10 million individuals diagnosed with diabetes mellitus in Indonesia in 2015. If this pattern persists, it may have adverse consequences for the human population, such as reduced productivity, disability, and early mortality.

Diabetes mellitus (DM) has the potential to give rise to numerous problems, hence elevating the likelihood of macrovascular and microvascular abnormalities in multiple organs, including the heart, kidneys, eyes, and brain.

Disorders of the metabolism of carbohydrates may give rise to DM. a-glucosidase is the principal enzyme involved in the metabolism of carbohydrates. At the periphery of small intestinal cells, this enzyme facilitates the hydrolysis of complex oligosaccharides into monosaccharides or glucose, which are subsequently absorbed into the bloodstream (Chatsumpun, et al, 2017). Despite ongoing advancements in the scientific understanding of the biology of diabetes, current therapeutic interventions mostly focus on antihyperglycemic effects and do not effectively mitigate the development of diabetes-related comorbidities. A therapeutic strategy for managing diabetes mellitus involves the use of measures to impede the uptake of glucose into the bloodstream, namely by the inhibition of a-glucosidase enzyme activity (Garber, et al, 2013). Inhibition of this enzyme will have an impact on delaying glucose absorption (Khatri and Juvekar, 2014). Therefore, it is imperative to investigate potential innovative antidiabetic compounds to improve the overall well-being of individuals with diabetes in the future. Numerous individuals suffering from chronic illnesses, including diabetes patients, hold the belief that herbal medications might serve as a complementary form of therapy to enhance their overall well-being. This underscores the need for additional investigation into the potential benefits of this therapeutic approach for such patients (Ghorbani, 2017).

In a study conducted by Indra in 2019, it was found that the ethanol extract derived from the leaves of the Glochidion arborescens (Müll. Arg.) Boerl. plant shown promising qualities as a natural antioxidant. The extract demonstrated a total polyphenol content of 33.32 mg QE/g and a flavonoid content of 3.02 mg QE/g. The free radical reduction test results, specifically the DPPH assay, indicated an IC₅₀ value of 5.62 µg/mL. This value was found to be higher than that of vitamin C, which exhibited an IC5₀ value of 3.34 µg/mL. Furthermore, it has been demonstrated that the ethanol extract derived from the leaves of the Mareme plant exhibits significant antioxidant properties. Mareme fraction leaves also have activity that could inhibit an active a-amylase enzyme inhibitory activity with an IC₅₀ value of 28.262 ppm (Ambarsari and Haryoto, 2022).

According to Rostiani (2019), the concentration of 1.60 ppm aligns with the findings of antidiabetic experiments conducted on male mice. These experiments demonstrated a significant reduction in blood glucose levels by 72.2% following seven days of supplementation. According to Li et al. (2019), the potential antidiabetic benefits of flavonoid compounds can be attributed to their ability to enhance insulin production, modulate enzymes involved in glucose metabolism, improve insulin sensitivity, and facilitate glucose absorption by skeletal muscle and adipose tissue.

The plant often referred to as Sala, scientifically known as Cynometra ramiflora Linn, is frequently employed as a medicinal remedy for many ailments such as gout, diabetes, hypertension, and other medical conditions inside the Kraton Kasunanan Surakarta. The Sala plant is considered to be a plant species that is quite uncommon. Therefore, there is a limited body of research on this substance's pharmacological actions and chemical ingredients. The water extract (godogan) derived from the leaves and stems of the Sala plant is purported to possess curative properties for various ailments, based on empirical evidence.

 The medical conditions encompassed in this list consist of hypertension, diabetes, gout, and hypercholesterolemia. Prior research has indicated that Cynometra ramiflora Linn plants originating from several nations had promising qualities as antidiabetic agents, antimicrobial agents (Khan et al., 2006), and antioxidants (Bunyapraphatsara et al., 2003) cytotoxic and in vivo biological activity (Sabiha et al, 2022), also antiviral activity (Meutia and Dewi, 2017; Yusuf et al., 2018). Consequently, it is imperative to examine the chemical composition, pharmacological properties, potential toxicity, and appropriate formulations of this plant to establish standardized herbal medications, thereby establishing a robust scientific basis (Haryoto et al., 2013). This study aimed to investigate the potential antidiabetic properties of the ethanol extract derived from the leaves of the Mareme plant (Glochidion arborescens (Müll. Arg.) Boerl.). The inhibitory activity of the leaves of the Cynometra ramiflora Linn plant against the a-glucosidase enzyme was assessed in vitro. Additionally, chemical ingredients were identified by the utilization of phytochemical screening methods.

Materials And Methods

Materials

The foliage of the Mareme plant (Glochidion arborescens (Müll. Arg.) Boerl.) was procured from the region of Ciamis. In contrast, the leaves of the Sala plant (Cynometra ramiflora Linn.) were gathered in the vicinity of Kasunanan, the Palace Surakarta, located in Central Java, employs many chemical substances in its research and experiments. These substances include ethanol with a concentration of 96%, aquadest, pyrogen-free aquadest, dimethyl sulfoxide (DMSO), phosphate buffer with a pH value of 6.8, the enzyme a-glucosidase, p-nitrophenyl-D-glucopyranoxide (pNPG), acarbose, sodium carbonate (Na2CO3), concentrated sulfuric acid, and ferric chloride (FeCl3).

Methods

Extraction

200 grams of powdered leaves from both plants were individually subjected to extraction using 96% ethanol in a round bottom flask. The solvent was replaced at four-hour intervals, while the extraction process was repeated three times.

The filtrate underwent concentration through the utilization of a rotary evaporator and subsequent vaporization in a water bath maintained at a temperature of 60°C, as described by Indra et al. (2019).

Phytochemical tests

1. The identification and recognition of steroids

The sample was solubilized in 0.5 mL of chloroform and combined with 0.5 mL of acetic acid anhydride. Subsequently, a volume of 2 mL of concentrated sulfuric acid was carefully introduced into the mixture using the tube's walls. The bluish-green color indicates the presence of sterols, whereas the presence of triterpenoids is suggested by the brown ring or violet coloration (Jones & Kinghorn, 2006; Evans, 2009).

2. The identification of tannins

A quantity of 1 gram of extract was introduced into a test tube. Subsequently, a volume of 10 mL of heated water was introduced into the tube. The solution was heated to its boiling point for 5 minutes, followed by adding approximately 3 to 4 drops of FeCl₃. The presence of tannin catechol is indicated by the production of a green-blue (green-black) color, whereas the presence of tannin pyrogallol and triterpenoids is suggested when the color turns blue-black (Jones & Kinghorn, 2006; Evans, 2009).

3. The identification and quantification of polyphenols

A quantity of 0.5 grams of powdered samples was dissolved in 10 milliliters of distilled water, after that heated, and subsequently filtered while still hot. FeCl3 was added dropwise to the filtrate. According to Kachkoul et al. (2018), the filtrate will exhibit a blackish blue or dark green coloration due to polyphenols.

4. The identification and quantification of flavonoids

A test tube contained a quantity of leaf oil, namely 3-7 drops. Subsequently, a little quantity of concentrated sulfuric acid solution (H2SO4) was introduced into the test tube. The study documented variations in coloration, with the emergence of dark red or yellow pigmentation as an indicator of flavonoid chemicals' existence (Harbone, 1987).

5. The identification of saponins

3-7 drops of leaf oil were introduced into a test tube, followed by adding 5 mL of water (H2O). The combination was subjected to agitation for 30 seconds, during which the observation of foam development served as an indicator for the presence of saponins. The solution was after that allowed to equilibrate at ambient temperature for a brief duration. The confirmation of saponin compounds was established if the foam lasted within the range of 1-10 cm, as stated by Harbone (1987).

6. The assay for inhibiting the a-glucosidase enzyme.

Preparation of the Sample Solution. A stock solution with a concentration of 1000 ppm was prepared by measuring 5 mg of an ethanol extract containing 96% ethanol from the leaves of Glochidion arborescens (Müll. Arg.) Boerl. Additionally, 5 mg of leaves from the Cynometra ramiflora Linn. plant was dissolved in DMSO to a final volume of 10 mL. The concentration series comprises five different concentrations, namely 50, 100, 150, 200, and 250 ppm, which are obtained from the stock solution sequentially. The activity of the a-glucosidase enzyme to liberate pNPG, as the substrate, takes place at a temperature of 37°C under conditions of pH 6.8 (Nursalinda, et al., 2021). The a-glucosidase enzyme inhibition experiment was conducted in accordance with the instructions provided by the kit's manufacturer, adhering to the specific reaction protocol outlined in Table 1. The solution's absorbance measurement was thereafter conducted at a wavelength of 450 nm using an ELISA reader.

 The procedure for preparing an acarbose solution

Ten mg of acarbose powder was mixed into 10 mL DMSO. A 1000 ppm stock solution of acarbose was made. A viable resolution was achieved with serial dilution, wherein concentrations of 50, 100, 150, 200, and 250 ppm were utilized.

Data analysis

The data analysis determined the percentage of a-glucosidase inhibition based on absorbance measurements. This was achieved by the utilization of the following equation:

The IC₅₀ value was determined by employing a linear regression model, which involved plotting the sample concentration on the x-axis and the corresponding percentage of inhibition on the y-axis. The IC₅₀ value was then calculated using the formula IC₅₀ = (50 - a)/b  according to Sugiwati et al. (2009).

Results And Discussion

The initial step in this project involved conducting phytochemical screening. Phytochemical screening serves as a preliminary step in identifying compound groups present in certain plant species (Dewatisari et al., 2018). Table 2 displays the outcomes of the phytochemical screening conducted on the leaves of Mareme and Sala.

The tube test method validated the presence of steroids, tannins, polyphenols, flavonoids, and saponins group compounds, utilizing different reagents as outlined in Table 2. Notably, the test for flavonoids group exhibited the most pronounced alteration in color, suggesting a substantial concentration of flavonoid compounds in both plant specimens. The pharmacological activities attributed to flavonoid families encompass a range of effects, notably antioxidant, anti-inflammatory, and antidiabetic properties (Ullah et al., 2020).

The assay for inhibiting the a-glucosidase enzyme was conducted using combinations of Mareme and Sala leaf extracts at final concentrations of 50, 100, 150, 200, and 250 ppm. The positive control in this study was acarbose, a commonly prescribed antihyperglycemic medicine that functions as an a-glucosidase inhibitor. Acarbose is classified as an oral anti-diabetic medication belonging to the Alpha-glucosidase inhibitor category. Its mechanism of action involves the inhibition of the a-glucosidase enzyme, which is responsible for the breakdown of complex carbohydrates located on the surface of the small intestine membrane. Acarbose achieves this without inducing hypoglycemia (Tjay and Rahardja, 2007). The positive control was assessed by conducting experiments on two reaction systems, one of which involved the reaction without a specific variable.

The extraction process involves obtaining a substance (S₀) and subsequently subjecting it to a reaction using an extract (S₁). The IC₅₀ value was established in order to quantify the effectiveness of inhibition. This was achieved by employing a linear regression equation that relates the concentration of the extract (in parts per million) or acarbose (in parts per million) to the percentage of inhibition. The IC₅₀ value represents the level at which 50% of enzyme inhibitory activity is observed (Meila & Purwandarie, 2017). Mohan et al. (2015) observed a significant suppression of the a-glucosidase enzyme, as indicated by the reduced IC₅₀ value.

The findings of the enzyme inhibition experiment are presented in Table 3. In the present study, the IC₅₀ value of acarbose was determined to be 14.17 ppm, indicating a significant level of effectiveness in inhibiting a-glucosidase activity. In contrast, the co-administration of Mareme and Sala leaf extracts did not exhibit significant inhibitory effects compared to acarbose, as seen by the IC₅₀ value of 374.47 ppm. According to the categorization of IC₅₀ values, acarbose demonstrates activity as an antidiabetic agent, but the combination extract of Mareme and Sala leaves exhibits inactivity as an antidiabetic agent. According to the findings of Ariani et al. (2017), the results indicate that acarbose exhibits inhibitory potential against the enzyme a-glucosidase, as evidenced by its IC₅₀ value. On the other hand, the combination of 96% ethanol extract of Mareme and Sala leaves has a relatively modest inhibitory potential against the same enzyme.

In this investigation, acarbose was employed to test its efficacy against the conventional medication in managing diabetes mellitus. The mechanism by which acarbose exerts its effects involves the inhibition of the enzyme a-glucosidase, so preventing the hydrolysis of polysaccharides into glucose. This inhibition ultimately leads to a reduction in blood sugar levels.

According to the study conducted by Darmawi et al. (2015), The ethanol extract derived from the leaves of Mareme and Sala plants has been found to contain flavonoid chemicals, which have been previously identified as inhibitors of the alpha-glucosidase enzyme (Proenca et al., 2017). The inhibition of a-glucosidase enzymes leads to a subsequent delay in the absorption of carbohydrates, hence contributing to the reduction of blood glucose levels (Madduluri, 2013). According to Rostiani (2019), prior studies have indicated that Mareme leaves possess flavonoid compounds with a concentration of 3.02 mg QE/g. Similarly, Afrin et al. (2016) found that Sala leaves contain a higher concentration of flavonoid compounds, precisely 86.2 mg QE/g. Nevertheless, irrespective of the flavonoid composition, our research demonstrates that a combination of Mareme and Sala leaves extracted using 96% ethanol did not exhibit any inhibitory effects on a-glucosidase enzymes. It is noteworthy that previous research by Patel et al. (2012) has identified the leaves of Mareme and Sala as effective antidiabetic drugs, operating through an alternative mechanism of action by promoting insulin secretion. According to Nadilah et al. (2019), the lack of inhibition of the enzyme a-glucosidase suggests that the combination of Mareme Sala plant leaf extract's antidiabetic properties may not be mediated through the enzymatic pathway.

In addition, various additional parameters can contribute to variations in IC₅₀ values and a-glucosidase enzyme activity. These factors include temperature, enzyme reaction rate, substrate concentration, and pH levels (Champe et al., 2010).

The experiment for evaluating the inhibitory activity of the a-glucosidase enzyme involved using a positive control, acarbose, and 96% ethanol extract samples derived from a combination of Mareme and Sala leaves. This assay was designed to measure the hydrolysis reaction of the substrate, namely p-nitrophenyl-a-D-glucopyranoside (PNPG). The effectiveness of enzyme inhibition was assessed by measuring the absorbance value of p-nitrophenol, which served as a substrate. The intensity of the resulting yellow color was quantified using an ELISA reader set at a wavelength of 450 nm (Maryam et al., 2020). The enzyme a-glucosidase catalyzes the hydrolysis of p-nitrophenyl-a-D-glucopyranoside, forming p-nitrophenol and glucose, as depicted in Figure 1. The magnitude of the yellow hue generated is proportional to the quantity of glucose synthesized. According to Ariani et al. (2017), the presence of a vibrant yellow hue during the reaction signifies a substantial production of glucose, which suggests a diminished inhibition of the a-glucosidase enzyme.

Table 1.  a-glucosidase inhibition reaction system

Blanks = Reaction system without extracts and enzymes, C = Mixture without extract (Blank Control), S0 = Mixture without enzyme with extract (Sample), S1 = Mixture of enzymes and extracts (Sample Control)

Table 2. Phytochemical Screening of Mareme and Sala Plant Leaves

Table 3. The results of alpha-glucosidase enzyme inhibition assay

*Ave= average, **Average of % inhibition and IC₅₀ values were calculated from two replicates for acarbose and three replicates for combination extracts

Conclusion

The study's results indicate that when combined, the ethanolic extracts of Mareme and Sala leaves yielded an IC₅₀ value of 374.47 ppm. This value classifies the combination as an inactive inhibitor of a-glucosidase. However, the results of the phytochemical screening indicated that the ethanolic extracts of Mareme and Sala leaves exhibited the presence of steroids, tannins, polyphenols, flavonoids, and saponins. These findings have potential significance for future investigations into additional pharmacological properties.

Acknowledgment

The authors are thankful to Universitas Muhammadiyah Surakarta which has funded this research through the HIT (Tridharma Integration Grant) program.

Author Contributions

H. conceptualized, prepared the method, managed the project, and wrote. H., A. V. A. investigated, collected data, and prepared graphs. P. I., C. H. M. wrote, drafted, edited, prepared graphs and analyzed data. K. H. Y. wrote, reviewed, and edited the paper.

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