Syzygium samarangense fruit extract attenuates hyperglycemia in type 2 diabetic rats through modulation of oxidative stress and inflammation

Introduction

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and dysfunction of pancreatic β cells (1). The inability of pancreatic β cells to produce sufficient insulin to counteract insulin resistance leads to hyperglycemia (2). Elevated blood sugar levels result in excessive production of reactive oxygen species (ROS), which can damage pancreatic β cells, thereby decreasing their number and function (3,4). Hyperglycemia not only increases ROS generation but also compromises the antioxidant defense system in pancreatic β cells. Research on antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase and thioredoxin, has shown that prolonged exposure to high glucose levels diminishes the antioxidant capacity of pancreatic β cells (5). Additionally, hepatic insulin resistance disrupts normal glucose metabolism by impairing the ability to suppress gluconeogenesis and glycogenolysis. This impairment results from decreased inhibition of key enzymes, including glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK) via the PI3K/Akt pathway (6,7). Consequently, hepatic glucose output increases while glycogen storage decreases (8). Simultaneously, insulin resistance in adipose tissue enhances lipolysis, increasing circulating fatty acids that fuel hepatic gluconeogenesis through β-oxidation, further elevating PEPCK activity (9). These combined effects contribute to hyperglycemia and the progression of T2DM.

Syzygium samarangense [SS; (Blume)] Merr. & L. M. Perry, commonly known as rose or wax apple, belongs to the Myrtaceae family and is widely cultivated across Southeast Asia. Rose apple is rich in polyphenolic compounds, including phenols, flavonoids, anthocyanins, ellagitannins, carotenoids and triterpenoids (10,11). In type 1 diabetic rats induced by streptozotocin (STZ), unprocessed rose apple powder decreases hyperglycemia while preserving pancreatic β cell mass through antioxidant and anti-inflammatory mechanisms (12). Our recent study employed microwave-assisted extraction combined with response surface technology to extract rose apple fruits, and ultrahigh-performance liquid chromatography (UHPLC)-microtime of flight (micrOTOF) Q II-tandem mass spectrometry (MS/MS) analysis was used to identify the phytochemical profiles of the extract (13). High concentrations of 12 bioactive compounds, including matairesinol and phenolic substances, were discovered (13). Furthermore, antioxidant properties of rose apple extracts can prevent glucotoxicity in a rat insulinoma cell line (INS-1 cells) (13). However, the effects of rose apple extracts on type 2 diabetic rats remain unexplored.

Materials and methods

Plant material and preparation of SS extract (SSE)

SSE was prepared and the major bioactive compounds were identified as previously described (13).

Animals

A total of 20 adult male Wistar rats (age, 6 weeks; weight, 180-220 g) were obtained from Nomura Siam International in Bangkok, Thailand. All rats were housed in a temperature- and humidity-controlled environment (23±3˚C; humidity, 60±10%) with a 12/12-h light/dark cycle at the Animal Center of Ubon Ratchathani University, Ubon Ratchathani, Thailand. They had unrestricted access to rodent diet and water and were allowed 1 week for acclimatization. The Animal Ethics Committee of Ubon Ratchathani University approved the protocol for animal experimentation (approval no. 37/2564 IACUC).

Induction of T2DM using high-fat diet (HFD) and low-dose STZ

The rats were divided into two dietary groups: Normal pellet diet (NPD; 12% calories from fat) and HFD; 58% fat, 25% protein and 17% carbohydrate, as percentages of total kcal). The HFD consisted of powdered NPD, 365 g/kg (National Laboratory Animal Center, Mahidol University, Nakhon Pathom, Thailand); casein, 250 g/kg (Difco, Becton Dickinson); cholesterol, 10 g/kg (Loba Chemie PVT Ltd.); vitamin and mineral mixture, 60 g/kg (Sigma-Aldrich; Merck KGaA); 2-amino-4-(methylthio) butanoic acid-methionine, 3 g/kg (Sigma-Aldrich; Merck KGaA); yeast powder, 1 g/kg and sodium chloride, 1 g/kg (14). Following a 2 week dietary modification period, the rats on HFD received an intraperitoneal injection of STZ at a dosage of 45 mg/kg body weight, dissolved in 0.1 M sodium citrate buffer (pH 4.5). At 3 days post-STZ injection, glucose levels were assessed in all rats using blood samples obtained from the tail tip using a glucometer (Accu-Chek Performa; Roche Diagnostics). Only rats with fasting blood glucose (FBG) levels ≥200 mg/dl were included in the diabetic group (DM) (15). The control rats were administered an injection of 0.1 M sodium citrate buffer at pH 4.5.

Experimental design

Following the establishment of diabetes, diabetic rats were orally administered glibenclamide or SSE at a volume of 1 ml/kg vehicle once daily. The rats were randomly assigned into four groups (n=5/group) as follows: Control and DM rats received only the vehicle; DM + SSE comprised DM rats treated with SSE at a dosage of 400 mg/kg body weight (; chosen based on preliminary findings showing effective antihyperglycemic activity without detectable toxicity in diabetic rats) and glibenclamide group (DM + GB) included DM rats receiving glibenclamide [Innova CapTab, Solan (H.P.)] at a dosage of 5 mg/kg body weight, serving as the positive control group (16). The treatment period lasted for 4 weeks, based on a previous study (12), and was considered sufficient for assessing sub-chronic exposure. The experimental protocol is illustrated in Fig. 1.

Figure 1 Experimental protocol. STZ, streptozotocin; DM, diabetes mellitus; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; H&E, hematoxylin and eosin; BW, body weight; SSE, Syzygium samarangense extract; FBG, fasting blood glucose; i.p., intraperitoneal.

Collection of blood samples

At the end of the study period, the rats underwent an overnight fast and were euthanized via intraperitoneal administration of thiopental sodium (120 mg/kg body weight; Scott-Edil Pharmacia Limited). Death was confirmed by the absence of heartbeat, respiration, corneal reflex and response to toe pinch, in accordance with the American Veterinary Medical Association guidelines for the euthanasia of animals (2020) (17). Blood samples were obtained through heart puncture. The blood collection tubes were immediately placed on ice, and serum was separated by centrifugation at 3,500 x g for 15 min at 4˚C. The serum samples were then stored at -80˚C until further analysis. Fresh anticoagulated blood was transported to the Pathological Laboratory at Ubon Ratchathani University Hospital for the measurement of liver enzyme levels, including alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST), using kinetic ultraviolet spectrophotometry with a Beckman Coulter AU680 Analyzer (Beckman Coulter).

Determination of pancreatic and serum insulin levels

Pancreatic tissue (80 mg) was homogenized in a Teflon homogenizer (Glas-Col homogenizer system) using RIPA lysis buffer with protease inhibitor (Thermo Fisher Scientific, Inc.). Following sonication (20 kHz; three cycles of 10 sec with 30-sec intervals), the homogenate was centrifuged at 2,000 x g for 10 min at 4˚C. The pancreatic protein concentration was measured using a micro-BCA kit (cat. no. SK3061; Bio Basic). Serum insulin levels and pancreatic proteins were determined using a Rat Insulin ELISA kit (cat. no. RAB0904; Sigma-Aldrich; Merck KGaA) according to the manufacturer's guidelines.

Assay for lipid peroxidation

Malondialdehyde (MDA), a product of lipid peroxidation, interacts with thiobarbituric acid (TBA) to generate TBA reactive substances (TBARS), which are used to assess lipid peroxidation. Lipid peroxidation was evaluated according to the manufacturer's instructions (cat. no. MAK085, Sigma-Aldrich; Merck KGaA). To produce TBARS, 600 µl TBA solution was added to each Eppendorf tube containing either a pancreatic protein sample or an MDA standard (0-20 mM). The tubes were incubated at 95˚C for 1 h. Samples and MDA standards were transferred into a 96-well plate at a volume of 200 µl/well. The concentration of TBARS in pancreatic protein samples and MDA standards was quantified using a spectrophotometer at 532 nm (Fluostar Omega, BMG Labtech).

Determination of antioxidant markers

CAT activity in pancreatic tissue was measured using a CAT assay kit (cat. no. MAK381; Sigma-Aldrich; Merck KGaA) following the manufacturer's instructions. CAT activity was quantified as the amount of enzyme required to degrade 1 µmol hydrogen peroxide/min. The activity of SOD was evaluated with a SOD Assay kit (cat. no. 574601; Merck Millipore) according to the manufacturer's guidelines. SOD activity was quantified as the amount of enzyme necessary to achieve 50% dismutation of the superoxide radical.

Determination of inflammatory markers

Quantitative measurement of TNF-α levels in the pancreas was conducted using a Rat TNF-α ELISA kit (cat. no. RAB0479; Sigma-Aldrich, Merck KGaA) in accordance with the manufacturer's guidelines.

Western blotting

Liver tissue (80 µg) was lysed in 200 µl RIPA solution containing a protease inhibitor cocktail (Wuhan Servicebio Technology Co., Ltd.). The tissue homogenates were centrifuged at 14,000 x g for 15 min at 4˚C and the supernatant was collected. The total protein concentration was measured using a Micro BCA protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of total protein (150 µg/lane) were resolved using 12% (w/v) SDS-PAGE and subsequently transferred to a PVDF membrane (Bio-Rad Laboratories, Inc.). Non-specific binding was blocked using 5% (w/v) skimmed milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 60 min at room temperature. The membranes were incubated overnight at 4˚C with rat monoclonal anti-PEPCK (cat. no. 12940S; Cell Signaling Technology, Inc.) and mouse monoclonal anti-β-actin (both 1:1,000; cat. no. STCSC-47778; Santa Cruz Biotechnology, Inc.). After washing with TBST buffer, the membranes were incubated with HRP-conjugated secondary antibodies (PEPCK, 1:3,000; β-actin, 1:5,000; both Cell Signaling Technology, Inc. cat. nos. 7074S and 7076S, respectively) for 1 h at room temperature. Protein bands were detected using DAB substrate (cat.no. E-IR-R101; Elabscience, Wuhan, China), and band intensities were assessed with ImageJ software (version 1.43; National Institutes of Health).

Histopathological examination

The pancreatic tissue was excised and weighed. Half of each tissue specimen was preserved in 10% neutral buffered formalin at room temperature for 24 h and processed for embedding in paraffin blocks. Thin slices (5 µm) of paraffin-embedded tissue were prepared, deparaffinized and stained with hematoxylin for 8 min and eosin for 1 min at room temperature. Images (x200) were captured using Nikon ECLIPSE Ni-U light microscope and analyzed using NIS-Elements software (Nikon Corporation).

Statistical analysis

All data were analyzed using SPSS software (version 23; IBM Corp.). All data are presented as the mean ± SEM. Normality of the data distribution was assessed using the Shapiro-Wilk test. Differences were analyzed using one-way ANOVA followed by Tukey-Kramer's post hoc test. P#x003C;0.05 was considered to indicate a statistically significant difference.

Results

Bioactive compounds derived from SSE

The total phenolic compound content was 10.21±0.22 mg GAE/g, as reported in our previous work (13).

Effect of SSE on FBG, body weight and food intake

FBG levels were elevated in diabetic (DM) compared with control rats (Table I). Diabetic rats treated with SSE at 400 mg/kg (DM + SSE) exhibited decreased FBG levels, however, this reduction was not statistically significant. By contrast, diabetic rats treated with glibenclamide at 5 mg/kg (DM + GB) demonstrated a significant decrease in FBG compared with DM rats. Despite increased food intake, DM rats showed a marked decrease in body weight compared with control rats. Following 28 days of SSE treatment, the DM + SSE rats exhibited an increase in body weight, however, this was not significant compared with untreated DM rats.

Table I Effects of SSE on FBG, body weight and food intake in diabetic rats.

Table I

Effects of SSE on FBG, body weight and food intake in diabetic rats.

	Mean FBG, mg/dl		Mean body weight, g		Mean food intake, g
Group	Week 1	Week 4	Week 1	Week 4	Week 1	Week 4
Control	100.33±22.06	106.50±24.37	255.23±6.54	508.12±16.56	22.98±3.78	17.73±2.01
DM	389.33±31.20a	425.00±34.47a	250.15±8.01	334.33±20.29a	29.87±4.62	35.83±2.46a
DM + SSE	401.33±31.20a	314.67±35.79a	263.60±7.16	372.14±18.14a	28.20±4.62	35.95±2.46a
DM + GB	311.40±24.17a	278.80±21.55a,b	249.08±7.16	370.62±18.14a	23.36±4.14	36.52±2.20a

[i] n=5/group.

[ii] ^aP#x003C;0.001 vs. control;

[iii] ^bP#x003C;0.05 vs. DM. FBG, fasting blood glucose; SSE, Syzygium samarangense extract; DM, diabetes mellitus; GB, glibenclamide.

Effect of SSE on serum and pancreatic insulin levels

Serum (Fig. 2A) and pancreatic insulin levels (Fig. 2B) were significantly decreased in DM compared with control rats. Notably, DM + SSE or DM + GB rats exhibited elevated serum and pancreatic insulin levels compared with untreated DM rats. These findings underscore the potential of SSE to alleviate hyperglycemia in diabetic rats by preserving pancreatic β cells, as indicated by the maintenance of islet area and enhancing insulin release.

Figure 2 Insulin levels. (A) Serum and (B) pancreatic insulin levels following 28 days of treatment with SSE or GB. n=5. *P#x003C;0.05, **P#x003C;0.01. SSE, Syzygium samarangense extract; GB, glibenclamide; DM, diabetes mellitus.

Effect of SSE on lipid peroxidation in pancreatic tissue

TBARS levels indicate MDA as a byproduct of lipid peroxidation. Compared with control rats, MDA levels in DM rats were significantly elevated (Fig. 3). However, after 28 days of treatment with SSE and glibenclamide, MDA levels in the treated diabetic rats significantly decreased.

Figure 3 MDA levels after 28 days of treatment with SSE or GB. n=5. *P#x003C;0.05, **P#x003C;0.01. MDA, malondialdehyde; SSE, Syzygium samarangense extract; GB, glibenclamide; DM, diabetes mellitus.

Effect of SSE on CAT and SOD activities

DM rats exhibited increased CAT activity compared with control rats, indicating heightened oxidative stress (Fig. 4A). SSE significantly decreased CAT activity compared with untreated DM rats. The amount of SOD required to eliminate superoxide radicals was elevated in DM rats, indicating increased SOD activity. SSE treatment led to a substantial reduction in SOD activity compared with untreated DM rats (Fig. 4B). These data suggest that the decrease in antioxidant enzyme activity in SSE-treated diabetic rats resulted from a reduction of free radicals in pancreatic tissue.

Figure 4 Antioxidant enzyme activity. (A) CAT and (B) SOD activity after 28 days of treatment with SSE or GB. n=5. *P#x003C;0.05, **P#x003C;0.01. CAT, catalase; SOD, superoxide dismutase; SSE, Syzygium samarangense extract; GB, glibenclamide; DM, diabetes mellitus.

Effect of SSE on proinflammatory cytokine markers

The apoptosis of pancreatic β cells in diabetes is associated with proinflammatory cytokines (18). Therefore, TNF-α, a key proinflammatory cytokine, was measured using ELISA to determine if SSE preserves pancreatic β cells by reducing the synthesis of proinflammatory cytokines. DM rats had significantly increased TNF-α levels in pancreatic tissue compared with control rats. SSE or glibenclamide resulted in a significant decrease in TNF-α levels in DM rats (Fig. 5).

Figure 5 Pancreatic TNF-a levels after 28 days of treatment with SSE or GB. *P#x003C;0.05, ***P#x003C;0.001. SSE, Syzygium samarangense extract; GB, glibenclamide; DM, diabetes mellitus.

Effect of SSE on hepatic gluconeogenic enzymes

The elevation of hepatic glucose synthesis, driven by the activation of gluconeogenic enzymes, contributes to hyperglycemia in diabetic conditions (19). The present study evaluated the expression of PEPCK, a key gluconeogenic enzyme in the liver, via western blot analysis. The results demonstrated that hepatic PEPCK expression was significantly increased in DM rats compared with controls. However, compared with no treatment, SSE treatment significantly reduced hepatic PEPCK expression in DM rats (Fig. 6).

Figure 6 Protein expression of PEPCK was determined via western blot analysis. (A) Intensity of the PEPCK protein band was compared to that of the β-actin protein band, which served as a loading control. (B) Mean protein expression levels of PEPCK (n=3). *P#x003C;0.05. PEPCK, phosphoenolpyruvate carboxykinase; SSE, Syzygium samarangense extract; DM, diabetes mellitus.

Histopathological changes in pancreatic islets

Histo-pathological alterations in the pancreatic islets were observed using H&E staining (Fig. 7). In control rats, the histological examination revealed the typical structure of islet cells. Conversely, DM rats exhibited abnormalities, as evidenced by a notable decrease in islet size and a deviation from the typical spherical morphology observed in control rats. DM rats administered SSE, along with those receiving glibenclamide, showed an increase in pancreatic islet size compared with DM rats; however, this difference was not statistically significant.

Figure 7 Histopathological changes in the pancreatic islets. (A) Pancreatic tissue was stained with hematoxylin and eosin and examined under a light microscope (magnification, x200; scale bar, 100 µm). (a) Normal control; (b) diabetic rats; (c) diabetic rats treated with SSE at a dose of 400 mg/kg and (d) diabetic rats treated with GB. Arrow indicates a pancreatic islet. (B) Quantitative analysis of pancreatic islet area. n=3. *P#x003C;0.05. NS, not significant; SSE, Syzygium samarangense extract; GB, glibenclamide; DM, diabetes mellitus.

Liver function enzyme levels

Hyperglycemia is recognized as a contributor to oxidative stress and hepatic damage (20). The present study assessed serum liver enzyme levels, which serve as indicators of hepatic injury. DM rats exhibited signs of hepatic injury, as evidenced by substantial elevations in serum concentrations of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) (Fig. 8). SSE or glibenclamide markedly reduced the increased levels of ALT, ALP, and AST, suggesting a protective effect of SSE against diabetes-induced hepatic damage.

Figure 8 Serum liver enzyme levels. (A) ALT, (B) ALP and (C) AST levels. n=5. *P#x003C;0.05, **P#x003C;0.01, ***P#x003C;0.001. ALT, alanine aminotransferase; ALP, alkaline phosphatase; AST, aspartate aminotransferase; DM, diabetes mellitus; SSE, Syzygium samarangense extract; GB, glibenclamide.

Discussion

Rats with T2DM induced by a combination of HFD and low-dose STZ were used to examine the potential antidiabetic effects of SSE, as well as its impact on pancreatic oxidative stress and inflammation. Consistent with findings in the literature (21), the present results demonstrated a notable increase in glucose levels and food intake, accompanied by a decrease in body weight in rats subjected to HFD and low-dose STZ treatment. Additionally, diabetic rats presented decreased serum and pancreatic insulin levels. The weight loss in diabetic rats can be attributed to increased protein breakdown and fat mobilization due to diminished insulin production, which impairs glucose absorption and utilization (12). Moreover, impaired insulin secretion contributes to increased appetite, leading to polyphagia in diabetic rats (22). Treatment with SSE over a 28-day period resulted in decreased hyperglycemia in diabetic rats, alongside significantly elevated serum and pancreatic insulin levels, indicating the potential of SSE to preserve pancreatic β cell mass and enhance insulin production. This aligns with a prior study involving STZ-induced type 1 diabetic rats, where administration of SSE fruit powder (100 mg/kg) attenuated hyperglycemia, increased serum insulin concentrations and enhanced pancreatic β cell activity, as evidenced by increased Homeostatic Model Assessment of β cell Function (12). SSE fruit powder contains notable quantities of phenolics, flavonoids and anthocyanins, which exhibit considerable antidiabetic properties by improving pancreatic β cell function and promoting insulin production in individuals with diabetes (23,24). Furthermore, our previous investigation revealed that SSE comprises at least 12 compounds, including cinnamic and glucaric acid, maloyl-hexose, citric, mono-caffeoylquinic, gallic, 2-methylcitric and ellagic acid, naringin-O-glucoside, kaempferol deoxyhexose, benzyl-diglycoside and matairesinol (13). Among these compounds, naringin has been extensively studied for its diverse antidiabetic effects (23-26). Naringin has demonstrated a dose-dependent decrease in blood glucose levels and an elevation in insulin concentrations through its antioxidative effects in STZ-induced diabetic rats (25). Additionally, naringin effectively decreases inflammation, oxidative stress and mitochondrial apoptosis caused by T2DM-related steatohepatitis via the RAGE/NF-κB pathway (26). Kaempferol, another flavonoid, also exhibits antidiabetic properties. The administration of kaempferol to STZ-induced diabetic rats is associated with a return to near-normal levels of plasma glucose, insulin, lipid peroxidation products and both enzyme- and non-enzyme-dependent antioxidants (27). In diabetic mice, kaempferol restores hexokinase activity while inhibiting hepatic pyruvate carboxylase activity and gluconeogenesis, indicating its ability to enhance skeletal muscle glucose metabolism and diminish liver gluconeogenesis (28). To the best of our knowledge, matairesinol, a notable secondary metabolite found in SSE, has been less extensively studied for its antidiabetic effects. However, in a rat model of brain sepsis, matairesinol increases antioxidant enzyme levels in brain tissue and inhibits neuronal death (29).

Hyperglycemia induces the generation of ROS while simultaneously impairing the antioxidant defense mechanisms. This imbalance can lead to oxidative stress, promoting pancreatic β cell death and a subsequent decline in insulin production over time (30). The present study investigated the antioxidant properties of SSE in pancreatic tissue. SSE in diabetic rats not only enhanced the activity of antioxidant enzymes but also decreased levels of oxidative stress markers. In the pancreatic tissue of rats with STZ-induced diabetes, SSE fruit powder demonstrates antioxidant activity, associated with an increase in insulin-expressing pancreatic β cells and pancreatic insulin protein levels (12). Additionally, SSE exerts notable antioxidant effects in liver damage induced by carbon tetrachloride (31). These beneficial effects may be due to the presence of potent antioxidant compounds in SSE, including phenolics, gallic acid, kaempferol, naringin and matairesinol.

Oxidative stress and inflammatory responses are associated with hyperglycemia. Hyperglycemia triggers oxidative stress and inflammatory signaling pathways, resulting in the synthesis of inflammatory cytokines within pancreatic β cells, ultimately leading to cellular damage and dysfunction (32). Consistent with prior studies (33,34), the present study observed elevated levels of TNF-α in the pancreatic tissue of diabetic rats, while treatment with SSE resulted in a decrease in TNF-α levels. Furthermore, SSE alleviates insulin resistance and mitigates inflammation in the liver following TNF-α exposure (35).

Dysregulation of glucose metabolism is a common feature of diabetes and contributes to increased hepatic glucose production through gluconeogenesis. This upregulation is primarily driven by the increased expression of key gluconeogenic enzymes such as PEPCK and glucose-6-phosphatase, which exacerbates hyperglycemia under diabetic conditions (20). Here, SSE significantly downregulated the expression of hepatic PEPCK, indicating its potential role in suppressing gluconeogenesis. This effect may be due to kaempferol, a major bioactive compound found in SSE. Additionally, other phytochemicals present in SSE, such as matairesinol and glucaric acid, suppress PEPCK expression by inhibiting the signaling pathways of PGC-1α and FOXO1, key transcription factors that regulate gluconeogenic gene expression (36,37).

SSE exhibited hepatoprotective effects, as evidenced by a significant decrease in elevated liver enzyme levels, including AST, ALT and ALP, in diabetic rats. These beneficial effects may be associated with the antioxidant and anti-inflammatory properties of the bioactive constituents within SSE.

To the best of our knowledge, the present study is the first investigation of the antidiabetic properties of SSE in rats with diabetes induced by HFD and low-dose STZ. The effects are attributed to the enhancement of antioxidant defense mechanisms, reduction in oxidative stress levels and suppression of the proinflammatory cytokine TNF-α. These findings suggest that SSE may serve as a supplementary treatment to help preserve β cell function in individuals with T2DM. However, the present study had limitations, including the use of a single SSE dose (400 mg/kg), a relatively small sample size and a short treatment duration (4 weeks). Future investigations should include multiple doses, extended treatment periods and larger sample sizes to optimize therapeutic efficacy and assess potential long-term effects and safety of SSE.

Acknowledgements

The authors would like to thank Associate Professor Songsak Chumpawadee (Department of Agricultural Technology, Mahasarakham University, Maha Sarakham, Thailand) for producing the high-fat diet used to induce T2DM in the rats. The authors would also like to thank Mr Tanapat Wisapon (Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, Thailand), who contributed to the design of equipment for pellet production and drying, facilitating the preparation of high-fat diet for the experimental rats.

Funding

Funding: The present study was supported by the National Science, Research, and Innovation Fund (Fundamental Fund 2565-2566) and the Faculty of Pharmaceutical Sciences, Ubon Ratchathani University (grant no. phar.ubu.0604-11-3/2567).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

NM and KS conceived the study and wrote the manuscript. BY designed and performed experiments, constructed figures and wrote the manuscript. NM, KS, PP, SP, SS, JK, PK, JO, CP and YB designed and performed experiments and analyzed data. NC performed experiments and analyzed data. NM and KS confirm the authenticity of all the raw data. NM edited the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The animal experiments were approved by the Animal Ethics Committee of Ubon Ratchathani University (approval no. 37/2564 IACUC).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References