INTRODUCTION:

Diabetes mellitus (DM) is a widespread health issue with a growing prevalence, posing significant challenges to individual well-being and quality of life.¹ Diabetes management essentials remains difficult, largely due to the demand for medications that are more effective, safe, and affordable². As a result, there is increasing interest in exploring natural sources, particularly ethnopharmacological plants, for the development of innovative antidiabetic drugs.³

Research has highlighted several ethnopharmacological plants from Sulawesi, such as Cordia myxa L., Syzygium cumini, S. malaccense, and Antidesma bunius, as promising candidates for antidiabetic drug development.^4,5 Studies have investigated their biological activities and chemical constituents, revealing significant findings. For instance, an ELISA analysis of C. myxa L. leaf extracts demonstrated α-glucosidase inhibitory activity,⁵ while in silico methods identified potential α-glucosidase inhibitors among its compounds.^6,7 Furthermore, active compound mapping in the n-hexane fraction of C. myxa L. leaves, along with postprandial bioassay and radical scavenging activity tests, confirmed the antidiabetic and antioxidant potential of these plants.⁸

While previous research has offered important findings, additional studies are essential to fully investigate healing possibilities of Sulawesi's ethnopharmacological plants as antidiabetic agents. The primary goal is to identify bioactive compounds from these plants with α-glucosidase inhibitory activities and to elucidate their mechanisms of action and pharmacological effects⁹. Furthermore, combining traditional knowledge with modern scientific approaches is crucial for developing effective and sustainable antidiabetic treatments¹⁰.

MATERIALS AND METHODS:

Samples Preparation:

The leaves of C. myxa L. were sourced from Rante Padang-Rante Mario village, Malua district, Enrekang regency, South Celebes, Indonesia (3°22'26"S; 119°53'15"E). Meanwhile, the leaves of A. bunius, S. cumini, and S. malaccense were collected from Kalebarembeng village, Bontonompo district, Gowa regency, South Sulawesi, Indonesia (5°18'21"S; 119°23'48"E). The taxonomic identification of these plants was verified by the botanical division, department of Biology Faculty of Math and Science, Makassar State University. After collection, the fresh leaves were cleaned and air-dried at normal air temperature for a week. Subsequently, 300g of the dried samples were ground and subjected to three rounds of extraction with 2 liters of 95% ethanol.

Phytochemical Screening:

Alkaloid Test:

A 10mg sample is dissolved with 2 drops of 2N sulfuric acid (H₂SO₄) and then added with 3 drops of Dragendorff's reagent. The presence of alkaloids is indicated by the formation of red to orange precipitate. A further 10mg sample is dissolved with 2 drops of 2N solution of sulfuric acid (H₂SO₄) and then added with 3 drops of Mayer's reagent. Positive alkaloids are indicated by the formation of a yellowish-white precipitate.¹¹

Flavonoid Test:

A 10mg extract is dissolved with 96% ethanol, then a small amount of magnesium powder and a few drops of concentrated HCl are added. The presence of flavonoids is indicated by the formation of an orange-red color.¹²

Steroid/Terpenoid Test:

Steroid and triterpenoid testing is performed using the Lieberman-Burchard reaction. A concentrated extract is dissolved with 96% ethanol, then 2mL of Lieberman-Burchard reagent is added. Positive steroid content is indicated by the formation of a blue-green color, and positive triterpenoid content is indicated by the formation of a red or purple color.¹³

Saponin Test:

A 10mg extract is dissolved with 96% ethanol and then added with 10mL of hot water. After shaking for 10 seconds and then letting it sit for 10 seconds, saponins are indicated by the formation of stable foam 1-10cm for not less than 10 minutes, and the foam does not disappear upon the addition of 1 drop of concentrated HCl.¹⁴

Tannin Test:

An approximately 10 mg extract is boiled with 100 mL of water for 15 minutes, then filtered while cold using filter paper. The addition of 1% FeCl₃ will result in a dark blue or greenish-black color.¹⁵

Microplate Reader Test on Samples (In Vitro Bioassay):

The microplate reader test was conducted on fractions based on a scale derived from previous studies, using a reaction mixture consisting of 25µL of 2mM p-nitrophenyl α-D-glucopyranoside (Sigma Chemical Co.), 49.5µL of phosphate buffer (pH 7.0) added to a flask containing 0.5µL of sample dissolved in DMSO at various concentrations (0.31-2.5µg/mL). The reaction mixture was pre-incubated for 5 minutes at 37°C, the reaction was initiated by adding 25µL of α-glucosidase (Sigma) and incubation continued for 30minutes. The reaction was stopped by adding 1mL of 0.01M Na₂CO₃. α-glucosidase activity was determined by measuring the release of p-nitrophenol at 400nm. Acarbose (Glucobay^®) was used as the positive control for α-glucosidase.⁵

Inhibition Kinetics Against α-glucosidase:

The sample's inhibition against α-glucosidase activity was measured by increasing the concentration of p-nitrophenyl α-D-glucopyranoside as the substrate in the absence or presence of the sample at different concentrations. The type of inhibition was determined through analysis of the Lineweaver-Burk plot from the data, calculated according to Michaelis-Menten kinetics.⁵

RESULT:

Samples Extraction:

The extraction results for each sample yielded extract percentages as follows: A. bunius 2.28%, C. myxa 1.93%, S. cumini 1.87%, and S. malacense 2.97%. The complete results are shown in table 1.

Table 1. Results of Phytochemical Screening

Sample

Sample Weight (gram)

Extract Weight (gram)

% yield

A. bunius

C. myxa

S. cumini

S. malaccense

300

6.84

5.79

5.62

8.91

2.28

1.92

1.87

2.97

Phytochemical Screening:

The phytochemical screening vealed diverse bioactive compounds across the tested plant extracts. A. bunius tested positive for flavonoids, terpenoids, saponins, and tannins, but alkaloids were absent. Similarly, C. myxa L. indicated flavonoid presence, terpenoids, saponins, and tannins, while alkaloids were not detected. In S. cumini, alkaloids, flavonoids, and tannins were identified, though steroids were absent. Meanwhile, S. malaccense demonstrated the presence of terpenoids/steroids, flavonoids, and tannins, but lacked alkaloids and saponins. These findings highlight the unique phytochemical profiles of each plant species, underscoring their potential pharmacological applications. The detailed results as shown in Table 2.

Table 2. Results of Phytochemical Screening

Sample	Alkaloid	Flavonoid	Steroid/Terpenoid	Saponin	Tanin
A. bunius	-	+	+	+	+
C. myxa	-	+	+	-	+
S. cumini	+	+	-	+	+
S. malaccense	-	+	+	-	+

Microplate Reader Test on Samples (In Vitro Bioassay):

Each extract and fraction tested demonstrated α-glucosidase inhibitory activity that surpassed Acarbose, with the n-hexane and methanol fractions being especially noteworthy. Notably, among the samples, A. bunius consistently had the lowest IC50 values, suggesting it is the most promising candidate for antidiabetic applications. The detailed results as shown in Table 3.

Table 3. Results of IC50 Value (μg/ml) on Samples

Sample

EtOH extract

n-hexane fraction

EtOAc Fraction

MeOH Fraction

Acarbose (Standart)

A. bunius

5.66±

0.33*

5.59±

0.30

5.91±

0.35

5.50±

0.33

6.85±

0.01^a

C. myxa

6.03±

0.33*

5.73±

0.21

5.79±

0.34

5.95±

0.26

S. cumini

6.17±

0.19*

5.48±

0.19

6.09±

0.29

5.44±

0.12

S. malaccense

6.13±

0.20*

5.90±

0.29

5.89±

0.19

6.10±

0.41

^aAnalyzed using Tukey’s HSD

*Statistically significant at p=0.05

The kinetic reaction mechanism based on the Lineweaver-Burk plot analysis for the sample has been determined and is illustrated in Figure 1 below.

Figure 1. The Lineweaver-Burk plot depicts the reaction of α-glucosidase in the presence of the sample.

DISCUSSION:

The phytochemical screening results are highly relevant to antidiabetic research, as they reveal the presence of bioactive compounds such as flavonoids, terpenoids, saponins, and tannins in the plant extracts¹⁶. Flavonoids renowned for their antioxidant qualities, which help reduce oxidative stress a significant factor in diabetes while also enhancing insulin sensitivity and glucose uptake, thereby lowering blood sugar levels.^17,18 Terpenoids exhibit hypoglycemic effects and anti-inflammatory properties¹⁹, which are crucial for reducing blood sugar and preventing complications associated with diabetes.²⁰ Saponins play a role in stimulating insulin secretion from the pancreas and improving glucose metabolism, in addition to their cholesterol-lowering effects, which benefit cardiovascular health in diabetic individuals.^21,22 Tannins, with their enzyme-inhibiting properties, slow down carbohydrate digestion by targeting α-glucosidase and α-amylase, preventing rapid post-meal spikes in blood sugar levels.^23,24 Together, these bioactive compounds suggest that the extracts from A. bunius, C. myxa L., S. cumini, and S. malaccense hold significant potential as natural antidiabetic agents, offering promising applications for managing blood sugar levels and reducing diabetes-related complications²⁵.

The Microplate Reader Test was crucial in revealing the α-glucosidase inhibitory activity of extracts and fractions from A. bunius, C. myxa, S. cumini, and S. malaccense, highlighting their potential as natural antidiabetic agents. The test results showed that all plant samples had superior inhibitory activity compared to Acarbose, a standard antidiabetic drug. This significant finding emphasizes the potential of these plants in addressing key metabolic processes involved in diabetes, such as carbohydrate digestion and glucose absorption. Among the samples, the n-hexane and methanol fractions consistently exhibited the most potent inhibitory effects, underscoring the importance of solvent selection in extracting bioactive compounds²⁶.

A. bunius stood out as the leading contender, displaying the lowest IC₅₀ values across all fractions. Its strong activity is likely due to the presence of bioactive compounds like flavonoids and tannins, which are known for their enzyme inhibition and antioxidant properties two critical factors in diabetes management. These compounds not only slow down carbohydrate breakdown but also mitigate the harmful effects of free radicals, which are often elevated in diabetic conditions. Similarly, C. myxa showed robust α-glucosidase inhibition, particularly in its n-hexane and ethyl acetate fractions. This activity may be attributed to its flavonoid and saponin content, which contribute to glucose regulation by enhancing insulin secretion and improving carbohydrate metabolism.²⁷

S. cumini also demonstrated notable inhibitory activity, especially in its n-hexane and methanol fractions. Its effectiveness is likely due to the synergistic effects of flavonoids and alkaloids, both of which have been documented to interfere with α-glucosidase activity²⁸. On the other hand, S. malaccense showed slightly reduced activity relative to the other samples but still demonstrated significant potential as an antidiabetic agent²⁹. Its terpenoid and flavonoid content likely play a key role in its inhibitory effects, making it a promising candidate for further exploration.^30,31

To better understand the mechanism of enzyme inhibition, the study used the Lineweaver-Burk plot to analyze the kinetic reaction mechanism of α-glucosidase in the presence of these plant extracts.³² This method provided a clear representation of the interaction between the extracts and the enzyme, shedding light on how these natural compounds influence enzyme activity. The analysis revealed the mode of inhibition exhibited by the extracts, offering deeper insights into their therapeutic potential. For example, the consistently low IC₅₀values of A. bunius across different solvent fractions suggest that its active compounds may function as noncompetitive inhibitors, directly targeting the enzyme's active site.

These findings underscore the immense potential of these plant extracts in the development of natural antidiabetic therapies. The consistently strong inhibitory activities observed, particularly in A. bunius, pave the way for further studies to isolate and characterize the specific bioactive compounds responsible for these effects. Additionally, understanding the synergistic interactions between compounds in each extract could lead to the formulation of more effective therapeutic agents. The research highlights the importance of exploring traditional ethnopharmacological plants as a sustainable source for developing cost-effective, natural antidiabetic treatments that can address the growing global burden of diabetes.

CONCLUSION:

The results from the Microplate Reader Test demonstrate the potential of A. bunius, C. myxa, S. cumini, and S. malaccense as natural antidiabetic agents. Their significant α-glucosidase inhibitory activities, especially in the n-hexane and methanol fractions, highlight their effectiveness in diabetes management. Among the samples, A. bunius consistently exhibited the lowest IC50 values, making it the most promising candidate for further research and development. These findings provide a strong foundation for future studies aimed at isolating and identifying the active compounds responsible for the observed inhibitory effects, supporting the advancement of natural antidiabetic therapies.

CONFLICT OF INTEREST:

All the authors declare that they have no known conflict of interest concerning the publication of this work.

ACKNOWLEDGMENTS:

We extend our gratitude to the Directorate of Research, Technology, and Higher Education, Ministry of Research, Technology, and Higher Education of the Republic of Indonesia, for providing funding support for this research through the KATALIS Grant (DRTPM-DIKTI) 2024.

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Received on 01.02.2025 Revised on 16.06.2025

Accepted on 17.09.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):56-60.

DOI: 10.52711/0974-360X.2026.00009

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.