1. INTRODUCTION
After cancer and cardiovascular/cerebrovascular disease, diabetes mellitus (DM) is the third leading cause of death [1]. Currently, DM accounts for about 5% of all deaths worldwide, but that number is predicted to rise to 50% in the next 10 years [2]. It is an endocrine disease attributed to abnormalities in insulin secretion (deficiency or insufficient insulin synthesis from the pancreas), action (insulin resistance and hyperinsulinemia), or both [3–5], resulting in hyperglycemia and severe irreversible micro-and macrovascular complications [6]. These complications include diabetic-(neuropathy, retinopathy, nephropathy, and foot), atherosclerosis, and cardiovascular disease [7]. A previous study reported that in DM patients, approximately 70% of the total mass of host pancreatic β cells was destroyed due to immune-mediated processes detected during clinical diagnosis [8]. Therefore, management of hyperglycemia is very important to reduce or prevent severe complications [6]. Conventional DM treatment comprises insulin injections and some antidiabetic drugs, including biguanides [9], sulfonylureas [10], glinides [11], thiazolidinediones [12], alpha-glucosidase inhibitors [13], dipeptidyl peptidase-4 inhibitors [14], amylin analogues [15], and glucagon-like peptide 1 [16]. Although these drugs are successful in reducing and controlling blood glucose level (BGL), the majority hold unfavorable side effects, such as digestive disorders, hypoglycemia, anemia, weight gain, and kidney failure. This shows the need for new natural medicines with safer and effective properties that are capable of protecting and regenerating pancreatic β cells as a focus for the finding of new antidiabetic drugs [17–19].
Medicinal plants are already utilized for the treatment and prevention of diverse diseases, such as DM [19], malaria [20,21], hepatoprotective [22], antipyretic [23], skin protection from UV rays [24], and wound healing [25]. Furthermore, more than 800 species of medicinal plants were reported to have antihyperglycemic effects [26]. The content of active compounds is known to affect blood glucose through several different mechanisms, such as increasing insulin secretion [27] and β-cells in the pancreas by activating regeneration [28,29]. Some compounds also function as antioxidants by lessening oxidative stress caused by free radicals in the pancreas [30,31]. Castanopsis costata known as Cep-cepan is among the medicinal plants widely distributed in Thailand, Peninsular Malaysia, and Indonesia (Kalimantan and Sumatra). It is a woody plant thriving at an altitude of 1,200–1,421 masl to a height of 12–15 m. Castanopsis costata has a round, hard, and strong stem with a diameter of approximately 80 cm and branches. The leaf stalks are in the proper shape, featuring pinnate veins, reddish-white flowers, and green fruit measuring 3 cm, with 2 cm cone-shaped brown seeds [32]. Previous studies have reported that ethanol extract from C. costata (EECC) possesses various pharmacological activities, including antimalarial [33], antioxidant [34], antipyretic [34], antihyperlipidemic [35], antidiarrheal [36], and anti-inflammatory [37]. Other studies have also confirmed the anti-diabetic effects of leaf extracts and fractions in alloxan-induced mice [38]. The antidiabetic effects reported are due to the high content of flavonoids and phenolic compounds found in C. costata [35], which can affect glucose metabolism in the body [39].
Therefore, this study aimed to test the chemical compounds contained in EECC using liquid chromatography tandem mass spectrometry (LC-MS/MS). The hypoglycemic effects were also determined, including normoglycemic mice and oral glucose tolerance test (OGTT), antihyperglycemic effects in vitro (α-glucosidase and α-amylase enzyme inhibition models) and in vivo (alloxan-induced hyperglycemic mice model), as well as histopathological examination of pancreatic tissue.
2. MATERIALS AND METHODS
2.1. Chemicals and drugs
The chemicals used included glibenclamide (PT. Kimia Farma Tbk, Indonesia), alloxan monohydrate (Sigma Chemical Company, USA), diethyl ether (PT. Brataco, Indonesia), 0.9% NaCl (PT. Widatra Bhakti, Indonesia), paraffin, 10% formalin solution, xylene, acarbose, α-amylase solution, α-glucosidase solution, p-nitrophenyl-α-D-glucoside, and 3,5-dinitrosalicylic acid (DNSA). Others included potassium sodium tartrate tetrahydrate, starch solution, glucose (Sigma-Aldrich, Merck, Germany), hematoxylin-eosin stains (HiMedia, USA), 70% ethanol, acetonitrile, aquadest, sodium hydroxide, disodium hydrogen phosphate anhydrous, sodium bicarbonate, and methanol (EMSURE® ACS Merck, Darmstadt, Germany) with analytical grade.
2.2. Plant materials and determination
Fresh leaf samples used in this study were obtained in February 2022 from Pancur Batu Traditional Market, North Sumatra, Indonesia. The plant was determined at Herbarium Medanense, Universitas Sumatera Utara (No. 183/MEDA/2022) by Dr. Etti Sartina Siregar, and identified as Castanopsis costata species of the Fagaceae family.
2.3. Preparation of C. costata extract
Castanopsis costata leaf was collected, cleaned with water, cut into small pieces, dried, and blended to obtain powder form. Furthermore, 2 kg of the powder was macerated for 3 × 24 hours with 70% ethanol. A rotary evaporator (Eyela OSB-2100) was employed to collect and concentrate the liquid extract at 50°C [40].
2.4. Analysis of EECC using LC-MS/MS
As much as 50 mg of EECC was dissolved in acetonitrile and filtered utilizing a Millipore 0.54 μ filter unit. Subsequently, a Quadrupole Time-of-Flight (QTOF) LC-MS/MS apparatus system was filled with 5 μl of sample filtrate. LC-MS/MS analysis was provided with a binary pump and QTOF mass spectrometer linked to liquid chromatography (LC) with an electrospray ionization (ESI) source. The QTOF system with negative and positive ionization modes was employed in the mass spectrometry process. ESI parameters utilized were atomic gas of 500 L/hour, a voltage source of 3 kV, and a capillary temperature of 120oC. The source temperature was set to 110oC for the whole scan mode from m/z 100–5,000. Furthermore, LC column used was C18 (2.1 × 150 mm, 1.8 μm) (Acquity UPLC® HSS), with eluents comprising H2O (A) and acetonitrile p.a (B) at a total flow rate of 0.3 ml/minute. The isocratic elution system was performed at 0-1 minute with a 95:5 ratio, 0 minute linear gradient elution of solvent A from 95% to 5%, and 6–7 minute isocratic elution with a 0:100 ratio. Other conditions included 6–7 minute linear gradient elution of solvent A from 0% to 100%, 7.5–9 minute isocratic elution with a 95:5 ratio, 7.5–9 minute linear gradient elution of solvent A from 95% to 5%. LC-MS/MS data interpretation referred to the personal compound database and library [41].
2.5. α-Amylase inhibition assay
In this assay, EECC was initially diluted in phosphate buffer (pH 6.9) to get diverse test concentrations of 250, 500, 750, and 1,000 μg/ml. Subsequently, a test concentration of 200 μl was intermixed with α-amylase solution of 200 μl (2 units/ml) into each tube and subjected to incubation at 30oC for 10 minutes. Each tube acquired around 200 μl of starch solution (1% in water w/v), which was then subjected to incubation for 3 minutes. The reaction was halted by providing 200 μl of DNSA reagent (12 g of KNaC4H4O6·4H2O in 8 ml of 2 M NaOH and 20 ml of 96 mM 3,5-DNSA solution) and simmered in a water bath for 10 minutes at 85 oC–90oC. After cooling to room temperature, the liquid was diluted with distilled water of 5 ml, followed by measuring the absorbance at 540 nm with a UV-visible spectrophotometer. Blanks with 100% enzyme activity were formulated by substituting EECC with 200 μl of buffer. Moreover, EECC at any concentration without the use of an enzyme solution was employed to create blank reactions. Positive control samples were prepared using acarbose (250, 500, 750, and 1,000 μg/ml), and the reactions were carried out similarly to EECC [42]. The α-amylase inhibition was represented as % inhibition and computed using the equation below:
where AzC and AzS indicate the absorbance of the control and sample.
The percentage of α-amylase inhibition was plotted against EECC concentration and IC50 value was acquired from the graph.
2.6. α-Glucosidase inhibition assay
EECC was diluted in phosphate buffer (pH 6.9) to acquire diverse test concentrations of 250, 500, 750, and 1,000 μg/ml. This was followed by mixing EECC of 20 μl from each test concentration with α-glucosidase solution of 10 μl (1 unit/ml) into each tube and incubating at 37oC for 5 minutes. After adding around 20 μl of a 1 mM p-nitrophenyl-α-D-glucoside solution, the mixture was subjected to incubation at 37°C for 30 minutes. The reaction was halted by providing 50 μl of 0.1 M Na2CO3 and absorbance was gauged at 405 nm employing a UV-Visible spectrophotometer. Blank with 100% enzyme activity was formulated by substituting EECC with buffer of 50 μl. Moreover, blank reactions were formulated utilizing EECC at any concentration without the use of enzyme solution. Positive control samples were prepared using acarbose (250, 500, 750, and 1,000 μg/ml), and the reaction was carried out similarly to EECC [43]. The α-glucosidase inhibition was represented as % inhibition and computed using the equation:
where AzC and AzS indicate the absorbance of the control and sample.
The percentage inhibition of α-glucosidase was plotted against EECC concentration and IC50 value was acquired from the graph.
2.7. Experimental animals
As many as 80 male Swiss albino mice with a weight of 25–30 g and 8–12 weeks old were acquired from the animal house of CV. Mitra Putra Animal, Cicalengka, Indonesia. The animals obtained were reared properly in the Pharmacology Laboratory, Universitas Buana Perjuangan Karawang. Ad libitum access to water and standard pellets was provided to mice housed in plastic cages with softwood shavings on a 12-hour light/dark cycle.
2.8. Hypoglycemic test on normal mice
Despite a 6-hour fast, all male Swiss albino mice were allowed unlimited access to water ad libitum. Subsequently, mice were diverged randomly into five groups (n = 5 in each group). Group I (normal control) obtained 10 ml/kg distilled water, group II was provided glibenclamide 5 mg/kg, while groups III, IV, and V were administered EECC at 50, 100, and 200 mg/kg, respectively. Blood samples were aseptically taken from the mice’s tail vein and BGL was measured at 0, 1, 2, 3, and 4 hours after treatment utilizing a digital glucometer (Accu-chek® Advantage, Roche Diagnostic, Mannheim, Germany) [44].
2.9. Oral glucose tolerance test in mice
Mice were randomly diverged to five groups (n = 5 mice per group) and allowed unlimited access to water ad libitum despite fasting for 6 hours prior to treatment. Mice in group I (normal control) obtained distilled water (10 ml/kg), group II was provided 5 mg/kg standard drug glibenclamide, while groups III, IV, and V received 50, 100, and 200 mg/kg EECC, respectively. This was followed by administering glucose solution of 2 g/kg 30 minutes after treatment. Blood samples were taken from the mice’s tail vein and glucose levels were defined at 0 minutes (before treatment), 30, 60, 120, and 180 minutes after, using a digital glucometer [45].
2.10. Induction of hyperglycemia in mice
After the acclimatization period was completed, all mice were prepared for hyperglycemia induction with alloxan monohydrate after fasting for 18 hours. The body weight and BGL were recorded before and after induction. In this experiment, mice were allocated randomly into six groups (n = 5 mice in each group). Group I served as a normal control (NC), while II to VI were injected intraperitoneally with alloxan in saline solution (0.9% NaCl) at 150 mg/kg. After 1 hour of alloxan administration, all mice were fed ad libitum [7].
2.11. Antihyperglycemic activity
After 72 hours of alloxan induction, mice with BGL >200 mg/dl were deemed hyperglycemic and incorporated in this study (day 0). Treatment was given for 14 days followed by BGL measurement on days 0, 5, 10, and 15, which was acquired from the tail vein utilizing a digital glucometer [46]. Subsequently, treatment was given once a day in the morning.
Group I: noninduced (normal control) obtained distilled water (10 ml/kg) orally.
Group II: alloxan-induced nontreated mice (hyperglycemic control) obtained distilled water (10 ml/kg) orally.
Group III: alloxan-induced and treated with 5 mg/kg of glibenclamide orally.
Group IV: alloxan-induced and treated with 50 mg/kg of EECC orally.
Group V: alloxan-induced and treated with 100 mg/kg of EECC orally.
Group VI: alloxan-induced and treated with 200 mg/kg of EECC orally.
The percentage DBGL (decline in blood glucose level) was computed using the formula:
2.12. Pancreatic tissue sampling
Diethyl ether was used to anesthetize the mice in each treatment group on the 15th day of the trial. All mice were sacrificed by cervical dislocation, where an incision was made in the midline of the abdomen approximately ≈ 1.5 cm long to remove the pancreas, and wrapped immediately in filter paper. Subsequently, the pancreas was rinsed with normal saline, dried, as well as placed into a sample bottle for histological examination [19].
2.13. Histological examination
For histological examination, pancreatic tissue samples taken during the autopsy were directly processed at the Histology Laboratory, Universitas Jenderal Achmad Yani, Indonesia. Pancreatic tissue samples were processed via alcohol dehydration, heeded by xylene cleaning and paraffin infiltration. The preparations were deparaffinized in xylene and stained with H&E (hematoxylin and eosin) after paraffin blocks were sliced to a thickness of 7 μm [47]. These preparations were viewed at 100x magnification employing a light microscope provided with a camera linked to a computer for histopathological examination. Histopathological changes were recorded using a standard nonlinear semiquantitative scoring system and a scale from 0 to 3 adapted from Dyson et al. [48]. Significant findings were scored 0 (normal architecture), 1 for mild changes (0%–25% of cell), 2 for moderate changes (25%–50% of cell), and 3 for severe changes (>50% of cell) that could be observed by light microscopy.
2.14. Statistical analysis
All of the experiment’s data are displayed as the mean ± SEM of five replicates for each group. Differences between means of the parameters measured were compared utilizing one-way analysis of variance continued with Tukey’s post hoc test employing GraphPad Prism version 8. A p < 0.05 value was deemed significant.
3. RESULTS AND DISCUSSION
3.1. LC-MS/MS analysis of EECC
Based on the chemical compound identification results using LC-MS/MS, a total of 14 compounds were found in EECC (Table 1). These compounds were described through the chromatogram results showing peaks with various molecular weights, as presented in the supplementary data.
Table 1. Chemical compounds identified from EECC using LC-MS/MS.
| No | Category | RT (minutes) | Molecular mass (m/z) | Ionization (ESI−/ESI+) | Mass error (ppm) | Molecular formula | Identified compounds |
|---|---|---|---|---|---|---|---|
| 1 | Flavonoids | 9.78 | 317.06487 | [M + H]+ | −2.2 | C16H12O7 | 3- Methoxyherbacetin |
| 2 | 17.4 | 553.28059 | [M + H]+ | 1.8 | C32H40O8 | 6,17-Epoxylathyrol-5,15-diacetate-3-phenylacetate | |
| 3 | 8.76 | 303.04948 | [M + H]+ | −1.5 | C15H10O7 | Isoetin | |
| 4 | 16.58 | 403.13775 | [M + H]+ | −2.1 | C21H22O8 | Nobiletin | |
| 5 | 10.02 | 317.06498 | [M + H]+ | −1.9 | C16H12O7 | Rhamnetin | |
| 6 | 9.67 | 303.04944 | [M + H]+ | −1.6 | C15H10O7 | Robinetin | |
| 7 | 12.91 | 593.12941 | [M-H]− | −1.1 | C30H26O13 | Buddlenoid A | |
| 8 | 9.99 | 623.16327 | [M-H]− | 2.4 | C28H32O16 | Isorhamnetin-3-O-β-D-robinobioside | |
| 9 | 11.16 | 431.09917 | [M-H]− | 1.9 | C21H20O10 | Kaempferol-3-O-rhamnoside | |
| 10 | 10.29 | 417.08346 | [M-H]− | 1.8 | C20H18O10 | Kaempferol-3-O-α-L-arabinoside | |
| 11 | 9.78 | 593.15211 | [M-H]− | 1.5 | C27H30O15 | Kaempferol-3-O-β-rutinoside | |
| 12 | 11.13 | 563.14296 | [M-H]− | 4.1 | C26H28O14 | Patuletin-7-O-[6′′- (2-methylbutyryl)]- glucoside | |
| 13 | Triterpenoids | 17.99 | 441.37161 | [M + H]+ | −2.5 | C30H48O2 | Kansenol |
| 14 | 16.95 | 617.38401 | [M-H]− | −1.2 | C39H54O6 | 3β-O-trans-p-Coumaroyl alphitolic acid |
Table 2. IC50 of EECC on α-amylase inhibitory activity.
| α-Amylase inhibitory | |
|---|---|
| Samples | IC50 (µg/ml) |
| EECC | 454.72 ± 0.86 |
| Acarbose | 115.45 ± 0.75 |
The data are presented as the mean ± SEM of five replicates for each group.
3.2. In vitro α-amylase inhibition activity of EECC
In this study, α-amylase enzyme inhibitory activity test was carried out in vitro. EECC and acarbose indicated concentration-dependent inhibitory activity against the α-amylase enzyme (Fig. 1) with respective IC50 values of 454.72 ± 0.86 μg/ml and 115.45 ± 0.75 μg/ml (Table 2).
Table 3. IC50 of EECC on α-glucosidase inhibitory activity.
| α-Glucosidase inhibitory | |
|---|---|
| Samples | IC50 (µg/ml) |
| EECC | 575.08 ± 0.93 |
| Acarbose | 438.01 ± 0.78 |
The data are presented as the mean ± SEM of five replicates for each group.
 | Figure 1. α-Amylase inhibitory activity of EECC. [Click here to view] |
3.3. In vitro α-glucosidase inhibition activity of EECC
Activity study on α-glucosidase inhibition in vitro showed that EECC had a concentration-dependent α-glucosidase inhibitory effect, as presented in Figure 2. EECC and acarbose showed α-glucosidase inhibitory activity with respective IC50 of 575.08 ± 0.93 μg/ml and 438.01 ± 0.78 μg/ml (Table 3).
Table 4. Effect of administration of EECC on DBGL of
alloxan-induced hyperglycemic mice.
| Groups | DBGL (%) |
|---|---|
| HC | 18.88 ± 1.04 |
| GL 5 mg/kg | 51.48 ± 2.39*** |
| EECC 50 mg/kg | 50.19 ± 4.11*** |
| EECC 100 mg/kg | 59.23 ± 2.56**** |
| EECC 200 mg/kg | 64.95 ± 1.52**** |
The data are shown as the mean ± SEM of five replicates for each group. HC: hyperglycemic control group; GL 5 mg/kg: Glibenclamide dose 5 mg/kg. ***p < 0.001, ****p < 0.0001 versus HC group.
 | Figure 2. α-Glucosidase inhibitory activity of EECC. [Click here to view] |
3.4. Hypoglycemic activity of EECC in normoglycemic mice
This study evaluated the hypoglycemic effect after EECC administration in normoglycemic mice. The results indicated that the group given EECC at 200 mg/kg experienced a substantial decline in BGL after 2 (p < 0.05), 3 (p < 0.01), and 4 (p < 0.0001) hours of treatment. EECC group at 100 mg/kg indicated a substantial decline after 3 (p < 0.05) and 4 (p < 0.001) hours. Meanwhile, 50 mg/kg groups had a substantial decrease after 4 (p < 0.01) hours of treatment in comparison with mice in the NC. The glibenclamide group at 5 mg/kg indicated a substantial decline in BGL after 1 (p < 0.05), 2, 3, and 4 (p < 0.0001) hours of treatment. The effect of EECC administration on BGL of normoglycemic mice is outlined in Figure 3.
 | Figure 3. Effect of EECC administration on BGL of normoglycemic mice. The data are displayed as the mean ± SEM of five replicates for each group. NC: Normal control group; GL 5 mg/kg: Glibenclamide dose 5 mg/kg. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus NC group. [Click here to view] |
3.5. Effect of EECC on OGTT in normal mice
The results showed no substantial difference in BGL across all groups before glucose administration (p > 0.05). However, all groups indicated a significant increase in BGL 30 minutes after glucose administration, verifying the hyperglycemia induction. Further analysis indicated a substantial decline in BGL in the EECC group at 50 mg/kg (p < 0.05), 100 mg/kg (p < 0.05), and 200 mg/kg (p < 0.01) only after 60 minutes of glucose administration in comparison with mice in the NC group. Meanwhile, the glibenclamide group at 5 mg/kg indicated a substantial decline in BGL after 30 (p < 0.001), 60 (p < 0.0001), 120 (p < 0.001), and 180 (p < 0.0001) minutes. The effect of EECC administration on BGL of mice in the OGTT is presented in Figure 4.
 | Figure 4. Effect of EECC administration on BGL of mice loaded with oral glucose. The data are displayed as the mean ± SEM of five replicates for each group. NC: Normal control group; GL 5 mg/kg: Glibenclamide dose 5 mg/kg. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus NC group. [Click here to view] |
3.6.Effect of EECC on BGL of alloxan-induced hyperglycemic mice
Based on the results, all EECC and the glibenclamide groups showed significant BGL-lowering effects in comparison with the hyperglycemia control (HC) group. EECC administration at 50, 100, and 200 mg/kg significantly (p < 0.001 – p < 0.0001) lowered BGL from day 5 to 15. Meanwhile, a substantial decline in BGL (p < 0.0001) was found in the glibenclamide group at 5 mg/kg from day 5 to 15. The effect of EECC administration on the BGL of alloxan-induced hyperglycemic mice is summarized in Figure 5. After 15 days of treatment with EECC at 50, 100, and 200 mg/kg, the percentage DBGL was 50.19%, 59.23%, and 64.95%, respectively, and 51.48% for glibenclamide at 5 mg/kg (Table 4).
Table 5. Semiquantitative analysis of pancreatic histopathology.
| Histopathological parameters | ||
|---|---|---|
| Groups | Islet cell damage | Acinar cells vacuolization |
| NC | 0.00 ± 0.00 | 0.00 ± 0.00 |
| HC | 3.00 ± 0.00 | 3.00 ± 0.00 |
| GL 5 mg/kg | 1.00 ± 0.00 | 1.00 ± 0.00 |
| EECC 50 mg/kg | 1.40 ± 0.24 | 1.60 ± 0.24 |
| EECC 100 mg/kg | 1.20 ± 0.20 | 1.00 ± 0.00 |
| EECC 200 mg/kg | 1.00 ± 0.00 | 1.00 ± 0.00 |
The data are presented as the mean ± SEM of five replicates for each group. NC: Normal control group; HC: hyperglycemic control group; GL 5 mg/kg: Glibenclamide dose 5 mg/kg.
 | Figure 5. Effect of EECC administration on BGL of alloxan-induced hyperglycemic mice. The data are displayed as the mean ± SEM of five replicates for each group. NC: Normal control group; HC: hyperglycemic control group; GL 5 mg/kg: Glibenclamide dose 5 mg/kg. ***p < 0.001, ****p < 0.0001 versus HC group. [Click here to view] |
3.7. Effect of EECC administration on the histological appearance of pancreatic tissues
The histological examination of hyperglycemic mice induced by alloxan is depicted in Table 5 and Figure 6. Alloxan-induced hyperglycemia in mice causes significant histopathological damage to the pancreas, specifically targeting the islet cells and leading to acinar cell vacuolization. This damage is a consequence of alloxan’s toxic effects on the pancreatic beta cells [49]. The administration of EECC significantly reduced the pathology caused by alloxan induction compared to the hyperglycemic mice group, but was not significantly different from glibenclamide (Table 5).
 | Figure 6. Effect of EECC administration on the pancreatic histopathological of alloxan-induced hyperglycemic mice. (A) Pancreatic tissue in normal control mice showed the normal histological structure of parenchyma, lobules encircled by connective tissue capsules, excretory ducts, islets, and acini that appeared normal. (B) Pancreatic tissue in hyperglycemic control mice showed histopathological changes, including irregular pancreatic acini tissue, severe shrinkage and necrosis of islets with fat infiltration in pancreatic parenchyma, severe blockage of blood vessels, and vacuolation of pancreatic acini, as well as disruption of islet outline, and dilation of intralobular ducts. (C) Pancreatic tissue in hyperglycemic mice treated with glibenclamide at 5 mg/kg indicated mild histopathological changes. (D-F) Pancreatic tissue in hyperglycemic mice treated with EECC at 50, 100, and 200 mg/kg, everyday for 14 days, showed significant recovery from histopathological changes, where normal pancreatic acini and islets were observed, similar to the normal control group. Haematoxylin-eosin stain: 100x magnification. [Click here to view] |
The results for the normal group showed a normal pancreatic structure, where pancreatic lobules were encircled by connective tissue capsules comprising nerves, blood vessels, excretory ducts, and lymphatics, along with regular islets and clustered β-cells located in the center. The secretory acini has masses of spherical and tubular cells, while islets comprise anastomotic cords of polygonal endocrine cells (Fig. 6A). On the other hand, pancreatic tissues from the hyperglycemic mice group showed histological damage, such as fatty changes in the parenchyma. There was also an indication of extreme necrosis and shrinkage of the pancreatic islets, with severe vascular obstruction and vacuolization of the acini, as shown in Figure 6B. The histological results of the EECC groups at 50 mg/kg (Fig. 6D), 100 mg/kg (Fig. 6E), and 200 mg/kg (Fig. 6F) showed significant recovery in the histopathological changes of the pancreas. Based on observation, normal acini and pancreatic islets were found similar to the normal control. The standard drug glibenclamide at 5 mg/kg also caused restoration of histopathological changes in pancreatic tissue, as observed in intact islets (Fig. 6C).
In fasting conditions, low insulin levels can increase glucose production by stimulating gluconeogenesis and glycogenolysis reactions in the liver [50]. Glucagon is also known to stimulate these reactions in the liver and renal medulla [51]. After eating, the glucose produced triggers an increase in insulin secretion and a decline in glucagon. Most of this postprandial glucose will be used by skeletal muscle, which is the effect of insulin-stimulated glucose absorption [52]. However, increased glucose production in the liver and impaired uptake in peripheral tissues, as well as decreased insulin secretion and excess counter-regulatory hormones (glucagon, cortisol, catecholamines, and growth hormone) that can facilitate lipolysis and protein breakdown (proteolysis), may cause hyperglycemia [53]. A good antihyperglycemia drug does not reduce BGL below normal limits in normoglycemic patients, so it does not cause hypoglycemia and has a good BGL-reducing effect in hyperglycemic patients [54].
In this study, EECC administration at 50, 100, and 200 mg/kg in normoglycemic mice showed DBGL effect after 1–4 hours, but the value was still in the normal range. Based on the results, there was a progressive decrease from the first to the fourth hour in glibenclamide group at 5 mg/kg. DBGL effect of EECC in normoglycemic mice did not cause severe hypoglycemia as shown by the glibenclamide group. Meanwhile, oral administration of EECC at 50, 100, and 200 mg/kg caused a significant decline in BGL (p < 0.05—p < 0.01) at 60 minutes after glucose administration in comparison with normal group given glucose without treatment. This suggested that EECC could increase glucose tolerance in mice, showing the potential to reduce DM complications related to postprandial hyperglycemia (PPHG). Controlling PPHG is an essential strategy for managing type 2 DM. The body’s ability to use glucose is measured by OGTT, which can be used to control the amount of sugar that DM patients can consume [44].
To investigate antihyperglycemic effect of EECC, in vitro and in vivo antihyperglycemic experiments were conducted. For in vitro, antihyperglycemic experiments were conducted using α-amylase and α-glucosidase enzyme inhibition models. These enzymes regulate postprandial blood glucose, which can hydrolyze carbohydrates into glucose [55]. Specifically, α-amylase enzyme initiates the carbohydrate digestion process by hydrolyzing the 1,4-glycosidic bond of polysaccharides (starch) into disaccharides (maltose). α-Glucosidase catalyzes disaccharides into monosaccharides (glucose), causing PPHG [56]. The administration of agents that have an inhibitory effect on the activity of α-amylase and α-glucosidase enzymes is effective in controlling hyperglycemia to postpone carbohydrate digestion, thereby reducing postprandial plasma glucose levels [57]. The results indicated that EECC administration at 250, 500, 750, and 1,000 μg/ml had an inhibitory effect on the α-amylase and α-glucosidase enzymes in vitro with respective IC50 of 454.72 and 575.08 μg/ml. The inhibitory potency of the EECC on α-amylase and α-glucosidase (IC50 > 450 μg/ml) is moderate compared to acarbose, a known α-amylase and α-glucosidase inhibitor. This indicates that a higher concentration of EECC is needed to achieve the same level of inhibition that a concentration of acarbose would produce. In addition, further investigation into the specific mechanisms of action of the EECC bioactive compounds is needed to understand why it is less potent and to potentially identify ways to improve its efficacy.
Regarding in vivo, antihyperglycemia experiments were conducted using an alloxan-induced hyperglycemic mice model. Alloxan can induce glucose accumulation in pancreatic β-cells through GLUT2 and selectively inhibit insulin secretion by specifically hindering glucokinase and glucose sensors in pancreatic β-cells [58]. This was confirmed by an increase in fasting BGL in mice measured 72 hours after alloxan injection. For 14 days, EECC was administered orally to mice that had experienced hyperglycemia. EECC administration at 50, 100, and 200 mg/kg significantly (p < 0.001—p < 0.0001) lowered BGL of alloxan-induced hyperglycemic mice from day 5 to day 15. It was also observed that the higher the dose of EECC given, the greater the decrease in BGL of mice. The results indicated that EECC administration is able to reduce BGL of alloxan-induced hyperglycemic mice in a time and dose-dependent manner. According to previous studies, the administration of alloxan caused ROS formation leading to selective necrosis of pancreatic islets which were important target organs for insulin. Generally, pancreatic islets can produce insulin to maintain glucose homeostasis and damage to the organ potentially affects β-cell function [55]. Based on the results, administration of EECC with various doses can repair pancreatic islet damage due to alloxan induction. This was confirmed by the histological observation results of pancreatic tissue in hyperglycemic mice. Significant improvements were observed in the pancreatic islets of EECC group compared to the hyperglycemic control group. Additionally, the histological appearance of EECC group was similar to that found in the normal control. The results of this study are consistent with previous studies, which stated that other members of the Fagaceae family, namely Quercus coccifera extract, have antihyperglycemic activity in vitro and in vivo. Quercus coccifera extract can inhibit α-amylase and α-glucosidase activity with IC50 values > 100,000 μg/ml, respectively. Meanwhile, with the same dose as EECC (200 mg/kg), Q. coccifera extract was able to significantly reduce BGL in diabetic mice, but not as strongly as EECC [59].
The hypoglycemic and antihyperglycemic activities (in vitro and in vivo), as well as the repair effects on pancreatic tissue of mice by the EECC cannot be separated from the active compounds contained. The chemical compound identification results using LC-MS/MS showed that EECC contains 14 compounds, including three kaempferol derivatives, namely kaempferol-3-O-rhamnoside, kaempferol-3-O-α-L-arabinoside, and kaempferol-3-O-β-rutinoside. Kaempferol is known to inhibit intestinal glucose absorption by inhibiting the enzyme α-glucosidase [60], increase insulin secretion and sensitivity by activating mitochondrial calcium uptake [61], facilitate glucose utilization in peripheral tissues [62], and up-regulate blood glucose absorption into muscle cells by increasing GLUT4, AMPK, LKB1, JAK2, STAT3 translocation, as well as PI3K phosphorylation [63,64]. Additionally, kaempferol can lower BGL by increasing glucokinase levels and glycogen synthesis [65], reducing liver glucose production [66], protecting against pancreatic islet damage, and helping β-cell regeneration [67]. The isorhamnetin-3-O-β-D-robinobioside compound contained in EECC has also been reported to raise glucose absorption and reduce insulin resistance by activating PGK1 enzyme that plays an important role in the glycolysis [68]. This compound reportedly increases blood glucose absorption into muscle cells by enhancing the regulation of GLUT4 and p-AMPK-α expression [69]. It also has a protective effect on β cell integrity and regenerates islets in pancreatic cells [70]. Meanwhile, the nobiletin compound is known to increase glucose absorption in skeletal muscle [71], improve glucose tolerance by protecting pancreatic β cells from oxidative stress [72], raise glucose-induced insulin secretion, and prevent apoptosis of pancreatic β cells [73]. A previous study showed that EECC has strong antioxidant activity [34], with a significant potential to repair pancreatic β cells damage caused by exposure to ROS formed due to alloxan induction. Antioxidant compounds were also reported to have effects similar to insulin in peripheral tissues and stimulate the regeneration process or the release of insulin in pancreatic β cells. This can increase the rate of glucose removal from the circulation and enhance metabolism [74].
Based on the cited literature and the results of LC-MS/MS analysis, we can hypothesize that the main bioactive compound contained in C. costata extract responsible for the antihyperglycemic effect is Kaempferol. This is because the kaempferol compound dominates the results of the LC-MS/MS analysis of C. costata extract (3 types of kaempferol: kaempferol-3-O-rhamnoside, kaempferol-3-O-α-L-arabinoside, and kaempferol-3-O-β-rutinoside). Meanwhile, the limitations in the present study were that the sample size was too small, a lack of toxicity studies, and limited duration of in vivo treatment.
4. CONCLUSION
In conclusion, this study showed that EECC has hypoglycemic activity, suppressed PPHG, and demonstrated antihyperglycemic effects in vitro (inhibition model of α-amylase and α-glucosidase enzymes) and in vivo (hyperglycemic mice model induced by alloxan). EECC also repaired pancreatic tissue damage confirmed by the recovery of histopathological changes. These results provided a pharmacological basis for EECC application in DM management, particularly for traditional practitioners in North Sumatra. However, further study needed to be carried out to characterize and isolate the bioactive compounds in charge of the antihyperglycemic activity, define possible mechanisms, and test safety.
5. ACKNOWLEDGMENT
This research was supported by the Institute for Research and Community Services, Universitas Buana Perjuangan Karawang (grant no. 847/LPPM/XI/2023).
6. LIST OF ABBREVIATIONS
AMPK, adenosine monophosphate-activated protein kinase; GLUT2, glucose transporter 2; GLUT4, glucose transporter 4; JAK2, janus kinase 2; LKB1, liver kinase B1; p-AMPK-α; phosphorylated adenosine monophosphate-activated protein kinase alpha; PI3K, phosphoinositide 3-kinases; ROS, reactive oxygen species; STAT3, signal transducer and activator of transcription 3.
7. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agreed to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
8. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
9. ETHICAL APPROVALS
This study protocol was approved by the Research Ethics Commission, Universitas Padjadjaran, Indonesia (approval number: 1212/UN6.KEP/EC/2023; dated: September 22, 2023). Universitas Buana Perjuangan Karawang has an MOU with Universitas Padjadjaran, and the Research Ethics Committee of Universitas Padjadjaran has full access to all the experiments conducted.
10. DATA AVAILABILITY
The data that supports the findings of this study are available in the supplementary material of this article.
11. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors, and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
12. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
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SUPPLEMENTARY MATERIALS
This supplementary material contains a presentation of chromatograms from the results of analysis using LC-MS/MS QTOF in positive and negative ion modes. Each chromatogram contains information on the observed mass and its intensity which shows the profile of a component. The results of identifying chemical compounds using LC-MS/MS revealed that there are 14 compounds contained in EECC, which are divided into 2 groups of compounds, namely flavonoids (12 components) and triterpenoids (2 components).
The supplementary material can be accessed at the link here: [https://japsonline.com/admin/php/uploadss/4659_pdf.pdf]