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

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).

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.

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.

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).

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.

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).

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.

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.

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).

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).

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.

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

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.

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.

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.

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.

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).

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).

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.

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.