Kaempferol; Quinoa seed extract; Quercetin; Pancreatic and pancreatic protection; High-fructose diet

Metabolic syndrome, a medical condition worldwide, is generally associated with diabetes mellitus (DM), insulin resistance, dyslipidemia, and increased triglyceride and cholesterol levels, which are often accompanied by liver and pancreas dysfunction [1]. Excessive fructose intake is one of the contributing factors to type 2 diabetes mellitus (T2DM). High fructose intake causes oxidative stress and inflammation in pancreatic beta cells, leading to impaired insulin production and secretion, thus causing hyperglycemia [2]. Excess fructose intake contributes to visceral adipose deposition and hepatic steatosis to promote the production of inflammatory mediators, increasing the risk of chronic diseases such as T2DM and nonalcoholic fatty liver disease (NAFLD) [3]. NAFLD, a disease characterized by fat deposition on hepatocytes, is associated with an abnormal lipid profile, including low-density lipoprotein (LDL) and high cholesterol levels [4]. Biomarkers such as aspartate aminotransferase (AST) and alkaline phosphatase (ALP) increase due to hepatocellular injury. Chronic fructose consumption significantly contributes to cirrhosis, hypertriglyceridemia and NAFLD [5].
Pakistan ranks third in the world in terms of diabetes prevalence, followed by China and India. The national prevalence of diabetes has consistently increased over recent years, increasing from 11.77% in 2016 to 16.98% in 2018 and 17.1% in 2019 [6]. According to the latest International Diabetes Foundation (IDF) atlas in 2023, approximately 33 million adults are living with T2DM in Pakistan, making it the third-largest population with diabetes in the world. In addition, approximately 11 million adults have impaired glucose tolerance, placing them at high risk of progression to diabetes, whereas approximately 8.9 million people with diabetes are still undiagnosed [7]. The variability in the prevalence of gestational diabetes mellitus (GDM) in Pakistani people is also reflected by its wide range, ranging from 4.41% to 57.90%, across different studies [8].
The therapeutic potential of medicinal plants has gained increased scientific attention. Bioactive plant constituents such as kaempferol have shown beneficial effects on hypertension, pancreatic disorders, and DM [9]. Flavonoids are naturally occurring polyphenolic compounds that are widely distributed among vegetables and fruits, including radish, parsley, grains, oregano, leafy greens, and citrus fruits such as oligomers, aglycones and glucosides [10]. Glucosides include quercetin, naringin, kaempferol, and hesperidin [11].
Among these, the most powerful hepatoprotective and antidiabetic properties are exhibited by kaempferol and quercetin. Quercetin is involved in reducing pancreatic inflammation, which moderates Carnitine palmitoyltransferase I (CPT-1) expression to increase fatty acid oxidation, thereby reducing NAFLD progression [12]. Kaempferol stabilizes normal liver function by inhibiting inflammatory mediators and reducing hepatic triglyceride accumulation through the downregulation of peroxisome proliferator-activated receptors (PPAR) and sterol regulatory element binding transcription factor 1 (SREBP-1) [13]. Quercetin offers further protective benefits against metabolic syndrome, liver cancer, and cardiovascular disease by suppressing NF-κB activation and reducing the secretion of proinflammatory cytokines such as IL-6, TNF-α and IL-1α [14,15]. Flavonoids have been demonstrated to be effective antidiabetic therapeutic agents because they protect pancreatic beta-cell integrity and support glucose homeostasis. In type 1 diabetes mellitus (T1DM), autoimmune beta-cell destruction results in high blood sugar levels, whereas long-standing insulin resistance contributes to T2DM [16]. Kaempferol and quercetin protect beta cells from apoptosis. Kaempferol has been shown to specifically inhibit the activity of caspase-3 [17].
Chenopodium quinoa Willd. (Chenopodiaceae) consists of approximately 250 species that are spread all over the world. Owing to its gluten-free nature, quinoa is consumed by patients suffering from celiac disease and is considered highly nutritious. Quinoa seed extract (QSE) includes various flavanols, such as kaempferol glycosides, apigenin and quercetin [18]. QSE improves the lipid profile, mineral balance, protein metabolism, and blood glucose regulation [19]. It has been shown to lower total cholesterol, triglyceride, LDL, blood sugar, and circulating protein levels [20]. Therefore, this study aimed to compare the effects of standard therapy (metformin) and natural compounds (quercetin, kaempferol, and QSE) on metabolic syndrome and to evaluate the protective effects of these compounds against high-fructose diet (HFrD)-induced hepatic and pancreatic alterations.

2.1. Study design and approval

This experimental study was approved by the Directorate of Graduate Studies, University of Agriculture, Faisalabad (No. 5738-27/DGS). The study was based on an animal model; young albino Wistar rats were obtained from Government College University, Faisalabad, and the study was conducted at the Institute of Physiology and Pharmacology, University of Agriculture Faisalabad, Pakistan.

2.2. Selection of drugs

Kaempferol and quercetin were selected as representative pure flavonoids because of their well-documented antidiabetic, antioxidant, and hepatoprotective activities, including the modulation of insulin signaling, lipid metabolism, and pancreatic β-cell protection [21,22]. QSE was included to assess the synergistic effects of multiple phytochemicals naturally present in quinoa, particularly kaempferol and quercetin glycosides, which may act additively or synergistically to enhance therapeutic outcomes [23]. The comparative evaluation of these pure compounds and a whole-plant extract allow a broader assessment of both isolated bioactive molecules and complex phytochemical mixtures in mitigating high-fructose diet (HFrD)-induced hepatic and pancreatic alterations.

2.3. Collection of drugs and chemicals

All the compounds were prepared in vehicle dimethyl sulfoxide (DMSO, 250 µL) diluted with 60% distilled water for administration. Metformin (chemical formula C₄H₁₁N₅, molar mass ~129.1 g/mol) is a first-line medication for T2DM and was obtained commercially under the name Glucophage. Quercetin and kaempferol were obtained from commercial sources [24]. Quinoa seeds were sourced and extracted via standard solvent extraction methods [25]. D-fructose (Sigma–Aldrich) was purchased to induce HFrD.

2.4. Experimental grouping

This study involved 30 albino Wistar rats weighing 150–200 grams. Thirty rats were divided into 6 groups (n = 5 per group). Group 1 served as the control group and was given a standard diet. Group 2 served as the disease model (HFrD 61% only). Group 3 received HFrD + metformin (M) (HFrD + M, 61% fructose + 100 mg/kg body weight, orally). Group 4 received HFrD + kaempferol (K) (HFrD + K, 61% fructose + 100 mg/kg). Group 5 received HFrD + quercetin (Q) (HFrD + Q, 61% fructose + 100 mg/kg). Group 6 received HFrD + QSE (HFrD + QSE, 61% fructose + 200 mg/kg). All the treatments were given orally.

2.5. Administration of drugs

In the first week of the study, a HFrD was given to all the groups except the control group (which was fed a normal diet and received no treatment). Drug administration was initiated in the second week. Kaempferol, quercetin, and QSE were administered to the respective treatment groups (HFrD + K, HFrD + Q, and HFrD + QSE) via oral gavage for 3 weeks. All the compounds were delivered in 250 µL of DMSO diluted with 60% distilled water. The treatments were given daily, and the experiment lasted a total of 28 days. The body weights of all the rats were measured daily throughout the 28 days. On day 29 of the trial, the animals were euthanized for sample collection; blood samples and liver and pancreas organs were collected from all 6 groups in accordance with ethical guidelines.

2.6. Data collection procedure

The rats were anesthetized via the inhalation of chloroform via cotton swabs. Under deep anesthesia, an incision was made in the left jugular vein to collect a blood sample (~2 mL) in an EDTA vial for complete blood count (CBC) analysis. The remaining blood was collected into clot activator (red-top) tubes to obtain serum for biochemical analyses [alanine aminotransferase (ALT) and AST levels]. After dissection, the liver and pancreas from each rat were harvested and placed in 10% neutral buffered formalin for preservation.

2.7. Histopathological analysis

Liver and pancreas tissue samples were fixed in 10% neutral buffered formalin and then processed through graded ethanol for dehydration. Xylene was used to clear the tissues, which were then embedded in paraffin wax. Five-micrometer-thick sections were cut via a microtome. Hematoxylin and eosin (H&E) staining was performed following standard protocols. The stained sections were mounted and examined via light microscopy for histopathological changes.

2.8. Statistical analysis

Descriptive statistics [mean ± standard error (SE)] were calculated for body weight, white blood cell (WBC) count, and hemoglobin (Hb), ALT, and AST levels for each group via SPSS version 24.00.