Vitexin isolated from Acanthus ilicifolius L. leaf enhances GLUT-4 translocation in experimental diabetic rats

Acanthus ilicifolius L. leaf is extensively used in the Indian and Chinese medicine systems to treat diabetes mellitus. In this study, the antidiabetic effect of vitexin isolated from A. ilicifolius leaf extract and their effect on glucose transporter protein type-4 (GLUT-4) translocation and peroxisome proliferator-activated receptor gamma (PPAR-γ) expression was evaluated in high-fat diet-streptozotocin (HFD-STZ) induced rats. In vitro antidiabetic effect of vitexin was investigated through glucose uptake activity in L6 (rat skeletal muscle) cell lines. Vitexin (10 and 20 mg/kg BW) was administered orally to HFD-STZ-induced diabetic rats for 48 days. The effect of vitexin on body weight, fasting blood glucose, serum insulin, total protein, urea, creatinine, and liver enzymes was examined. GLUT-4 translocation and PPAR-γ expression were studied in the skeletal muscle and adipocytes of experimental rats. The interaction of vitexin with GLUT-4 and PPAR-γ was validated by molecular docking analysis. Vitexin significantly lowered the blood glucose and also normalized other biochemical parameters. Furthermore, the treatment with vitexin up-regulates the mRNA expression of GLUT-4 and PPAR-γ in diabetic rats. In silico analysis also supports the promising interactions between vitexin and target proteins. These results explained that vitexin up-regulates the mRNA expression of GLUT-4 and PPAR-γ and enhanced the translocation of GLUT-4 which maintains glucose homeostasis. Thus, vitexin can serve as a novel antidiabetic drug in future.


Introduction
Type II diabetes mellitus (DM) is a major metabolic disorder, affecting approximately 5-6% of the global population annually and becoming a serious health issue (Kifle and Enyew 2020).Type II DM is diagnosed by the elevated level of glucose in the blood for a prolonged time which might be due to reduced uptake of glucose and metabolism by peripheral tissues such as skeletal tissue and adipocytes.Insulin is a peptide hormone secreted from pancreatic β-islets, enhances blood glucose uptake into peripheral tissues, thereby regulating glucose homeostasis (Juvekar and Bandawane 2009;Ashok Kumar et al. 2012).The rate-limiting stage in carbohydrate metabolism is glucose absorption, which in mammals are aided by glucose transporters (GLUTs) (Maughana 2009;Scheper et al. 2023).GLUT-4 is the major glucose transporter mainly expressed in adipocytes and skeletal muscles.The binding of insulin to its receptors in the cell membrane stimulates various intra-cellular proteins for the transport of GLUT-4 from the storage vesicles to the plasma membrane for further metabolism (Zaid et al. 2008;Van Gerwen et al. 2023).Since type II diabetes is characterized by insulin resistance, a defect in GLUT-4 translocation affects glucose uptake and consequently results in a higher level of blood glucose (Sameer et al. 2006).Currently, available synthetic drugs for up regulating GLUT-4 transportation are reported for their side effects (Sangeetha et al. 2013;Ganjayi et al. 2023).Metformin, thiazolidinediones, and sulfonylureas are the drugs employed to improve hyperglycemia (Giglio et al. 2022).Metformin, a biguanide, reduces hepatic glucose production and improves insulin resistance.Metformin has side effects including gastrointestinal disturbances, and rarely hypoglycemia, anemia, lactic acidosis, and liver inflammation (Du et al. 2022).Thiazolidinediones or glitazone improve insulin sensitivity and it mainly includes pioglitazone and rosiglitazone.Rosiglitazone is banned in UK due to its serious side effects including increased risk of heart attack and stroke.Pioglitazone side effects include weight gain, chest infections, allergies and rarely edema, heart failure, liver problems, and anemia (Rashid and Fatima 2024).Both sulfonylureas and meglitinides stimulate the release of insulin from pancreatic β-cells and induce hypoglycemic effect and its side effects include hypoglycemia, weight gain, nausea, and rarely liver and heart problems (Goncalves and Farooki 2022).Alpha-glucosidase inhibitor including acarbose blocks, the absorption of carbohydrate and there by improve DM, but it causes gastrointestinal problems and rarely liver problems and edema (Banwari et al. 2023).Sodium-glucose co-transporter 2 (SGLT2) inhibitors reduce blood glucose levels, but it may cause the development of diabetic ketoacidosis, hypoglycaemia, gastrointestinal disturbances, and liver inflammation (Ravindran and Munusamy 2022).Dipeptidyl peptidase-4 (DPP-4) inhibitors or gliptins reduces blood glucose levels and induce weight loss, but it has side effects including gastrointestinal problems, allergies, and rarely liver and kidney diseases (Palikhey et al. 2022).To avoid side effects, we search for an Indian medicinal plant compound that can be used for the potential treatment of type II DM (Nazarian-Samani et al. 2018).Acanthus ilicifolius L. (Acanthaceae) is a medicinal plant from the mangrove region but grows in normal salinity.The plant is commonly known as sea holy or Holly mangrove which has been employed in traditional Indian and Chinese medicine to cure different ailments (Amritpal et al. 2009;Babu et al. 2001).The A. ilicofolius plant is referred to as Sahachara in Ayurveda and is used to cure rheumatism, asthma, and coughs in India.The entire plant is used to make tonic, expectorant, and stimulant medications (Saranya et al. 2015).Seaside residents frequently use boiled root in milk to treat weakness (Dey et al. 2012).The leaf is used to prepare formulations in 1 3 Ayurveda to treat diabetes and snake bite (Mastaller et al. 1997;Zohora et al. 2023).The leaf is frequently used in Malaysia to cure rheumatism, neuralgia, and wounds from poison arrows.People who live close to mangroves practice chewing plant leaves to ward off snake bites (Ganesh and Vennila 2010;Islam et al. 2022).The leaf juice is used to treat hepatic ailments and prevent alopecia (Chakraborty et al. 2007;Islam et al. 2022).Different parts of the plant have been proven for their pharmacological activities such as anti-inflammatory (Mani Senthil Kumar et al. 2008), antioxidant (Asha et al. 2012), antinociceptive and analgesic activity (Wang et al. 2013), anticancer activity (Poorna et al. 2011), anti-leishmanial activity (Kapil et al. 1994), hepatoprotective activity (Babu et al. 2001), antiasthmatic activity (Ahmed et al. 2005), antimicrobial activity (Ravikumar et al. 2012), osteoblastic activity (Van Kiem et al. 2008), antilipidemic activity and activity against Alzheimer's disease (Burg et al. 2013), antiadipogenic activity (Gire et al. 2021), and antiulcer activity (Nizamuddin et al. 2011).2-Benzoxazolinone (Murty and Solimabi Kamat 1984), isoxazoline (D 'Souza et al. 1997), coumaric acid derivatives acancifoliuside (Kanchanapoom et al. 2001), vitexin (Wu et al. 2003) andalpha-amyrin (Van Kiem et al. 2008) are some of the compounds reported from this plant.
In our preliminary studies, the methanolic leaf extract of A. ilicifolius significantly enhanced glucose uptake activity in L6 cell lines and experimental rats than other prepared extracts.Hence, the same extract was chosen for further isolation and identification of an antidiabetic compound that enhances glucose uptake through GLUT-4 translocation.The present study explored apigenin-8-C-beta-D-glucopyranoside, known as vitexin, the bioactive compound present in the methanolic extract by bioactive-guided separation.Vitexin was isolated from various medicinal plants and proven for their pharmacological activities such as anti-inflammatory, antioxidant, antinociceptive, and anti-depressant (He et al. 2016).However, no research has reported vitexin for its glucose uptake activity with its underlying mechanism of action.Hence, the current study was focused to evaluate the antidiabetic effect of vitexin isolated from Acanthus ilicifolius L. leaf and its effects on GLUT-4 translocation in type II diabetic rats.

Plant collection and extraction
Fresh greenish leaves of A. ilicifolius were collected from Parangipettai, near Pichavaram forest, Cuddalore district, Tamil Nadu, India in November 2016 and authenticated by Dr. Kathiresan, Annamalai University, Chidambaram.The leaf of A. ilicifolius was washed and shade dried.About 3 kg of the powdered leaf was macerated with methanol for 3 days.The extract was filtered and concentrated at 40-45 °C using a rotary vacuum evaporator.The percentage yield of extract was 9.8 g/100 g.

Isolation and identification of active compound
The methanol extract of A. ilicifolius was fractioned under silica gel chromatography (100-200 mesh, 800 gm, 70 cm × 4 cm).The column was eluted with solvents of increasing polarity hexane, chloroform, ethyl acetate, and their mixtures.In total, 84 minor fractions were collected in a conical flask and transferred with proper labeling.The solvents in the separated fractions were recovered by simple distillation.Then, all the separated and concentrated fractions were placed in thin layer chromatography (TLC) for isolation and identification of active principle.The concentrated fractions were placed in alumina sheets (0.2 mm) the different ratios of solvents such as petroleum ether, ethyl acetate, and methanol at different ratios were used as mobile phase.The details of the solvent system are petroleum ether (5%), ethyl acetate (4%), and methanol (1%); petroleum ether (4%), ethyl acetate (4%), and methanol (2%); petroleum ether (3%), ethyl acetate (1%), methanol (1 %); and petroleum ether (1%), ethyl acetate (6%), and methanol (3%).
The fractions with similar R f (retention factor) values were pooled as major fractions.The major fraction which was identified as an active fraction by glucose uptake activity in L6 cell lines was further purified for identification through spectroscopic analysis.The isolated purified fraction was tested for phenol (brown color obtained in ferric chloride test) and quinine (given deep pink color with alcoholic NaOH).The physical data like color, appearance, melting point, and spectroscopic analysis like gas chromatography (GC) (Acq Method AUTO GC MS), Fourier transform infrared (FTIR) spectroscopy (ABB Bomem Inc., Canada), liquid chromatography-mass spectrometry (LC-MS) (Mass Lynx 4.1), hydrogen-1 nuclear magnetic resonance ( 1 H NMR), and carbon-13 nuclear magnetic resonance ( 13 C NMR) data were compared with the literature.The active compound was identified as apigenin-8-C-beta-D-glucopyranoside (vitexin).

Cytotoxicity and glucose uptake activity of vitexin
Cytotoxicity and glucose uptake activity of isolated vitexin in L6 cell lines was examined by the standard protocols (Gupta et al. 2009).

In silico molecular docking of isolated compound vitexin
The structures of vitexin and pioglitazone were sketched using Chemsketch.The structures were energy minimized using Argus Lab.The structures of PPAR-γ (ID: 2XKW) were retrieved from Protein Data Bank (PDB) (Berman et al. 2000).The structure of GLUT-4 was modeled using the SWISS-MODEL server based on the sequence obtained from UniProt (Acc.no: P14672) (Schwede et al. 2003).The details of the template and sequence identity of the modeled structure are given below.The protein structure was energy minimized using the GROMOS96 force field using GROMACS 4.5.6 (Berendsen et al. 1995).Modeling of GLUT-4 was done by following specifications: Template: 17-483 (4ZWC: A); solute carrier family 2, facilitated glucose transport member 3 Resolution: 2.60 Å Sequence identity: 60.86 Molecular docking analysis was performed using the Patch Dock server (Schneidman-Duhovny et al. 2005).The lowest energy conformation was chosen as the best binding conformation of the ligand with the protein based on the score and atomic contact energy (ACE).The interaction pattern of the ligand with the proteins was investigated and visualized using PyMol (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC).

Experimental induction of diabetes
Except for normal group rats, all other animals were supplied with a high-fat diet (HFD) for 5 weeks (Srinivasan et al. 2005) to induce obesity and insulin resistance.After 5 weeks, except for normal and HFD control rats, others were administered with a single intraperitoneal injection of streptozotocin (STZ) (40 mg/kg BW) dissolved in fresh ice-cold citrate buffer (pH 4.5, 0.1 M).The normal and HFD control rats were administered with citrate buffer alone to equalize the hormonal changes and stress.After 72 h of STZ administration, the experimental rats were tested for fasting blood glucose (FBG) by measuring tail vein blood glucose level in a glucose meter (Accucheck-Johnson & Johnson Company).Rats that have shown FBG over 240 mg/dL were considered diabetic and included in further studies.

Experimental design
Wistar rats of both sexes were randomly separated into six groups consisting of six numbers each.Normal HFD control and diabetic control animals received the vehicle alone (Normal saline).Positive control rats received the standard drug pioglitazone (10 mg/kg BW).The isolated vitexin from A. ilicifolius was administrated by oral gavage at doses of 10 and 20 mg/kg BW not exceeding 1.5 mL per day.Briefly, group I (normal control): normal rats + normal saline (1.5 mL/kg BW), group II (HFD control): HFD rats + normal saline (1.5 mL/kg BW), group III (diabetic control): diabetic rats + normal saline (1.5 mL/ kg BW), group IV (experimental I): diabetic rats + isolated vitexin (10 mg/kg BW), group V (experimental II): diabetic rats + isolated vitexin (20 mg/kg BW), and group VI (positive control): diabetic rats + pioglitazone (10 mg/kg BW).After 48 days of treatment, all experimental animals were sacrificed by the cervical dislocation method under anesthesia.

Processing of blood samples
The blood samples were collected by cardiac puncture in sterile test tubes and incubated for 20 min at room temperature for coagulation.The blood samples were collected in serum tubes and centrifuged at 3000 rpm for 10 min to separate the serum and stored in sterile vials at −80 °C for further experiments.The plasma was collected in ethylene diamine tetraacetic acid (EDTA)-added tubes after centrifugation at 3000 rpm for 10 min and stored in sterile vials.The whole blood was utilized to calculate glycated hemoglobin.

Estimation of biochemical parameters
The blood collected in serum tubes was incubated for complete coagulation.The clotted blood was centrifuged at 3000 rpm for 12 min and serum samples were separated and stored at −80 °C until further experiments.The fasting serum insulin (FSI) concentration was estimated by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics, Germany).Homeostasis of model assessment insulin resistance (HOMA-IR) was determined by the following formula: Glycosylated hemoglobin (HbA1c %) was estimated in EDTA blood samples by the commercial test kit from Crest Biosystems, Goa, India.The levels of serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), urea, creatinine, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), chloride, and calcium were determined spectrophotometrically according to the instructions of commercially available ready to use kits from Span Diagnostics ltd, Surat, Gujarat, India.Very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) were determined using the Friedewald formula (Mohammed et al. 2016):

Gene expression by quantitative polymerase chain reaction
Total RNA extraction Ribonucleic acid (RNA) extraction was done by using RNA iso plus from TAKARA.The 100 mg of adipose and muscle tissue sample was taken from −80 °C.The tissues were homogenized by an appropriate volume of RNA iso plus using a micro pestle.The homogenized sample was incubated for 5 min at room temperature and centrifuged at 12,000 rpm at 4 °C for 5 min.Then, 0.2 mL of chloroform was mixed with the supernatant and a milky white layer was formed.The sample was incubated at room temperature for 5 min and then centrifuged at 12,000 rpm for 15 min at 4 °C.Then, an HOMA − IR = fasting serum insulin × fasting blood glucose∕22.5 equal volume of isopropanol was mixed with the clear aqueous phase to precipitate RNA and spinned at 12,000 × g for 10 min at 4 °C.An equal volume of 75% ethanol was mixed into the solution to washed it thoroughly and to remove salt precipitates.The solution was centrifuged at 7500 rpm at 4 °C for 5 min.Then, the supernatant was removed and the final pellet was air-dried.Finally, the pellet was dissolved in diethyl pyrocarbonate (DEPC)treated water or ribonuclease (RNAse)-free water.The RNA quantity was quantified using UV spectrometer and the ratio was 1:7-2:1.
cDNA synthesis using iScript real-time The first strand of complementary DNA (cDNA) was synthesized from 1000 ng of total RNA by the real-time method by utilizing iScript's first cDNA synthesis kit from BIO-RAD according to the manufacturer's instructions.

Real-time polymerase chain reaction
The expression of gene levels was quantified by real-time polymerase chain reaction (RT-PCR) by using SYBR green dye from applied biosystems life technologies and the method used to analyze the gene expression was the 2−ΔΔ Ct method.Beta-actin acts as a housekeeping gene.This real-time assay was done by using different polymerase chain reaction (PCR) conditions and protocols.For one reaction 10 μL of the reaction mix, 0.5 μL of 10 pico molar diluted forward and reverse primer, 3 μL of water, and 1μL of 200 ng cDNA.The target genes were quantified using 0.2 mL strips.The amplification of the target genes was Roche Light Cycler Nano.Negative temperature coefficient (NTC) acts as a negative control.The defined program of this real-time PCR was initial denaturation of 95 °C for 10 min, 40 cycles of 3-step amplification consisting of denaturation −95 °C for 15 s, annealing 60 °C for 1 min and extension of 72 °C for 30 s, and the melting curve was analyzed from 60 to 95 °C for 15 s.The expression of each target gene has its own (ct) amplification rate value at the threshold level where the final transcripted products of the PCR start to sense the fluorescence.The details of the primers used are given in Table 1.The annealing temperature for all the primers was −60 °C.

Statistical analysis
All the data are expressed as mean ± SEM and were determined by one way analysis of variance (ANOVA), followed by Student's t-test and Tukey's multiple comparison tests by using GraphPad Prism version 5.0, and the values of p < 0.05 were considered as significant statistically.Microsoft Excel 2007 was used to plot the graphs.

Separation and in vitro glucose uptake activity of fractions
The minor fractions are pooled as major fractions and the results of the percentage of glucose uptake activity of each fraction are listed in Table 2.Among all the 22 major fractions 14, 15, 16, 17, and 18 fractions exhibited higher glucose uptake activity in L6 cell lines 22.93 ± 0.67, 27.46 ± 0.62, 72.99 ± 0.62 *** , 34.91 ± 0.62, and 21.08 ± 0.62, respectively.Major fraction, 16 showed a higher percentage of glucose uptake (72.99 ± 0.62 *** ) in L6 cell lines when compared to other major fractions.Hence, this fraction is further purified in column chromatography and analyzed spectroscopically for the identification of antidiabetic molecules present in it.

Glucose uptake activity of isolated vitexin in L6 cell lines
Vitexin isolated from methanolic leaf extract of A. ilicifolius increases the glucose uptake in L6 cell lines even at the lower concentration, but vitexin was less efficient when compared with the standard drug pioglitazone.The results are given in Fig. 10.

In silico molecular docking of isolated compound vitexin
To evaluate the antidiabetic effect of vitexin, molecular docking analysis was performed against its molecular targets.The docking results are compared with that of the standard drug pioglitazone to check the efficiency of vitexin (Table 3).The compounds were found to interact with their respective targets by forming hydrogen bonds.The GLUT-4-vitexin complex (Fig. 11) and GLUT-4-pioglitazone complex (Fig. 12) had an ACE score of 147.60 and −296.08,respectively.The PPAR-γ-vitexin complex (Fig. 13) and PPAR-γ-pioglitazone complex (Fig. 14) had an ACE score of −212.35 and −140.49,respectively.

Acute toxicity of vitexin
The oral acute toxicity of vitexin was tested with several dose levels in experimental animals for 14 days.No signs and symptoms of acute toxicity were noticed in any one of the experimental groups of treated rats.None of the rats showed changes in normal behavior or death throughout the 14 days of the experimental period.No significant changes were observed in the weight of the liver, kidney, and heart when compared with the zero doses (administered with distilled water) of rats.Hence, the minimum lethal dose (LD 50 ) could not be calculated.Since the acute toxicity study of vitexin resulted in a high margin of safety and the absence of mortality at a single dose of up to 800 mg/kg BW, further in vivo studies were undertaken.No behavioral changes were recorded between the 1/10th and 1/20th doses, and were considered a safe dose that was followed for this experiment.Therefore, doses of 10 mg/kg BW and 20 mg/kg BW were fixed as test doses for further studies.

Effect of vitexin on body weight
The effects of vitexin on body weight in normal, HFD, and diabetic control animals are given in Table 4. HFD animals demonstrated a statistically significant (p < 0.05) increase in body weight (399.50 ± 5.731 g) at end of the treatment due to the accumulation of fat in adipose tissue.Besides, HFD-fed diabetic control rats have also explained a significant elevation in body weight (354.80 ± 2.626 g) compared to the normal control rats (241.20 ± 1.046 g) but slightly less than HFD control rats.HFD-fed STZ-induced diabetic control rats treated with vitexin at 10 and 20 mg/kg BW for 48 days demonstrated a statistically significant reduction in body weight (272.70 ± 4.410 g and 265.00 ± 3.697 g, respectively) to a normal level.

Effect of vitexin on fasting blood glucose
The elevation in the level of fasting blood glucose (FBG) was observed in diabetic control (308.80 ± 6.896 mg/dL), but treatment with vitexin (10 and 20 mg/kg BW) for 48 days significantly (p < 0.05) reduced FBG (116.20 ± 3.591 and 114.50 ± 2.432 mg/dL, respectively) when compared with diabetic control rats.The HFD-fed control group also showed increased FBG levels (174.20 ± 3.167 mg/dL) but not as much as diabetic rats (Table 5).Diabetic rats treated with vitexin (10 and 20 mg/kg BW) explained a significant decrease in the FBG level compared to diabetic control rats.

Effect of vitexin on insulin, HOMA-IR, and HbA1c
The result of the effect of vitexin on insulin, HOMA-IR, and glycated hemoglobin levels are listed in Table 6.The increased level of insulin, HOMA-IR, and HbA1c was noted in diabetic control rats when compared to normal control group rats.Oral administration of vitexin (10 and 20 mg/kg BW) for 48 days significantly reduced these levels to the normal range.The results were statistically significant p < 0.05 when compared to untreated diabetic rats.

Effect of vitexin on liver function markers
The liver function marker enzymes such as ALT, AST, and ALP levels in serum are depicted in Table 7.There was a significant increase (p ≤ 0.05) in the concentration of these hepatic enzymes in the untreated diabetic rats observed when compared to

Effect of vitexin on renal function markers
In diabetic control rats, significant elevation of urea and creatinine levels, and a decrease in total proteins, albumin, and globulin levels were observed (Table 8).Treatment by vitexin at 10 and 20 mg/kg BW for 48 days exhibited a significant (p ≤ 0.05) decrease in the levels of urea and creatinine as well as significant (p ≤ 0.005) elevation in total protein compared to diabetic control rats.The pioglitazone (10 mg/kg BW) treated group rats also significantly (p ≤ 0.005) decreased the urea and creatinine compared to untreated diabetic rats but not increased the total protein significantly compared to normal and the diabetic rats treated with vitexin 10 and 20 mg/kg BW.

Effect of vitexin on GLUT-4 expression
Figures 15 and 16 show the GLUT-4 mRNA expression level of normal control, diabetic control, and treated rats in adipose tissue and skeletal muscle, respectively.The GLUT-4 mRNA expression level was significantly (p ≤ 0.005) improved in vitexin (20 mg/kg BW) treated diabetic rats than in HFD and diabetic control rats.

Effect of vitexin on PPAR-γ expression
Figure 17 shows PPAR-γ mRNA expression level of normal control, diabetic control, and treated rats in adipose tissue.There was a significant (p ≤ 0.005) importance in PPAR-γ mRNA expression level of vitexin (20 mg/kg BW) treated diabetic rats than HFD and diabetic control rats.

Discussion
Diabetes mellitus (DM) is one of the most challenging multifactorial disorders affecting approximately 347 million people globally.DM is characterized by hyperglycemia emerging as a result of reduced glucose uptake, and the metabolism of dietary nutrients Fig. 15 GLUT-4 mRNA expression level of normal control, diabetic control, and treated rats in adipose tissue Fig. 16 GLUT-4 mRNA expression level of normal control, diabetic control, and treated rats in skeletal muscle such as carbohydrates, protein, and fat due to a defect in the secretion and action of insulin (Juvekar and Bandawane 2009;Gao et al. 2022).Following the advancement of macro and microvascular problems such as retinopathy, nephropathy, neuropathy, and coronary heart diseases for an extended period of time, these altered metabolic processes become chronic (Ashok Kumar et al. 2012;Li et al. 2023).Many modern drugs currently available are reported for their side effects and reduction in the quality of life.In contrast, phytomedicines are highly expected for their therapeutic efficacy without harmful effects.The current study was designed to explore the possible mode of action and margin of safety of one such compound present in A. ilicifolius in HFD-STZ diabetic rats.
Bioactive guided fractionation of methanolic extract of A. ilicifolius explored the antidiabetic molecule.Based on the one-dimensional and two-dimensional nuclear magnetic resonance, the obtained compound was characterized and identified as vitexin, an apigenin flavone molecule, whose occurrence has never been reported from this extract.The L6 cell line isolated from rat skeletal muscle cells was chosen to investigate the glucose uptake activity of vitexin because 70% of glucose uptake occurs through the peripheral tissues.Vitexin proves its non-toxic nature and significantly enhances the glucose uptake activity in L6 cell lines; however, vitexin was less efficient compared to the standard drug pioglitazone.Since the glucose uptake was highly regulated by transporter proteins,in silico and gene expression study was carried out with special reference to its GLUT-4 translocation.
GLUT-4 showed a better binding preference for pioglitazone; however, PPAR-γ showed a higher preference for vitexin at the same active site.By comparing the binding energy observed, vitexin shows a higher preference towards PPAR-γ.From the in silico studies, it was understood that vitexin could act as a ligand for the activation of GLUT-4.The binding of the vitexin may induce the function of these proteins which are deregulated in the diseased condition.Furthermore, to understand the beneficial effects of vitexin in diabetic conditions an extensive in vivo study was undertaken.Due to the intake of HFD, the body weight of the rats increased which results in obesity and insulin resistance which are the characteristic of type II DM.In the untreated group, HFD enhances the storage of excess fat in adipose tissue whereas treatment with vitexin (10 and 20 mg/kg BW) effectively reduced the weight by promoting glycogen storage in the liver.In contrast to this, the standard drug pioglitazone fails to reduce body weight which is one of the remarkable side effects of the drug.But vitexin maintains the body weight more or less to the normal level and prevents harmful effects on the body.
After 3 days of STZ induction, an increased level of blood glucose was noticed due to insulin deficiency or resistance in all STZ-treated groups.The HFD rats also showed elevation in FBG after 48 days which indicates the prediabetic stage is influenced by diet.The administration of vitexin enhanced the uptake of glucose and utilization by the peripheral tissue thereby fasting serum glucose level was reduced.It was concluded that vitexin possesses antihyperglycemic activity because of its capability to enhance peripheral uptake of glucose by glucose transporters.
Because of the HFD feeds, there was a compensatory insulin production in HFD and untreated diabetic control rats which results in hyperinsulinemia.Besides, the normal level of insulin in treated rats is due to enhanced sensitivity to insulin and good glycemic control.HOMA-IR is the calculated factor used to measure insulin resistance.The increased HOMA-IR in diabetic and HFD rats indicates increased insulin resistance.The treatment with vitexin effectively (20 mg/kg BW) reduced insulin resistance.Vitexin treatment significantly reduced glucose to the normal level and it may be due to possible extra-pancreatic mechanism(s) such as glucose uptake through glucose transporters in adipose tissues and skeletal muscles.
In contrast to single glucose measurement, which only offers a result that is accurate at the moment a blood sample is taken, glycosylated hemoglobin is a reliable index that indicates the average blood glucose across the lifetime of RBCs (Weng et al. 2014).The elevation in HbA1c explains the poor glycemic control in the diabetic control group.High risks for the advancement of comorbidities such as retinopathy, nephropathy, and neuropathy are predicted by elevated HbA1c values (Calisti 2005;Pan et al. 2023).Administration of vitexin significantly decreased the increased level of glycosylated hemoglobin in diabetic rats by which validating its efficiency in long-term glycemic control of diabetes mellitus and its associated complications.
The liver is the metabolic organ in the body, maintaining and controlling lipid balance (Thilakarathna et al. 2012;London and Stratakis 2022).The liver function test is assessed by measuring the enzymes AST, ALT, and ALP in serum.These enzymes are regarded as reliable hepatic biomarker enzymes, which are present in the cytoplasm of hepatocytes under normal physiological conditions.Elevation of serum ALT, AST, and ALP indicates the impaired liver function observed in STZ-induced diabetic rats.This is due to the possibility that diabetic rats may develop hepatocellular necrosis changes, which point to both the hepatotoxic effects of STZ and oxidative stress.Chafiaa et al. 2016 also reported that significantly enhanced levels of ALT, AST, and ALP activities in diabetic control rats are due to the hepatotoxic effect of STZ.In addition to this, some plant extracts used for the control of various diseases frequently lead to subsequent hepatic toxicity indicates as the management persists for a longer period.In the current study, liver function was estimated to ensure whether vitexin is safe to be employed for the management of diabetes and dyslipidemia for a longer period.The abnormal level of total protein, albumin, and globulin is common in hepatotoxicity due to intracellular damage and irregular metabolism of proteins.Treatment with vitexin caused a decrease in the ALT, AST, and ALP and increased levels of proteins to the normal level, which can improve the damage caused by oxidative stress or STZ and thereby show its hepatoprotective effect in diabetic rats.
The kidney is crucial in the removal of metabolic waste products from blood circulation, such as urea and creatinine, and it also regulates electrolyte levels, which help to maintain physiological homeostasis (Patel et al. 2014;Adkine et al. 2022).Elevations in urea, creatinine, and reduction in protein levels are remarkable biomarkers of kidney dysfunction.The elevation was due to altered protein metabolism under diabetic conditions.Urea, the excretory substance produced through the metabolism of protein and creatinine is formed endogenously which indicates the glomerular infiltration rate (Perrone et al. 1992;Thompson and Joy 2022).An increase in urea and creatinine, in addition, to a decrease in total proteins, demonstrated the dysfunction of kidneys in diabetic control rats.In the current study, it is proved that administration of vitexin in diabetic rats prevented kidney damage by decreasing the levels of serum urea and creatinine and restoring the total proteins.From these results, it is understood that vitexin exerted a protective role against kidney dysfunction in treated diabetic rats.
Maintaining a normal level of electrolytes is important for many biological functions.Under diabetic conditions, the levels of sodium, potassium, calcium, and chloride were reduced due to abnormal metabolism.The present results demonstrate that the treatment with vitexin restores electrolytes which helps to alleviate complications due to diabetes.
Glucose uptake is the rate-limiting step in glucose metabolism.Glucose transporters facilitate glucose uptake across the cell membrane.Since glucose is a polar molecule, the metabolism of glucose mainly depends on glucose transporters (Rhodes and White 2002;Williams and Wasserman 2022).GLUT-4 is the important glucose transporter present predominantly in adipocytes, skeletal muscle, and heart muscle cells that maintain the whole body's glucose homeostasis.In normal conditions, insulin binds with insulin receptors and phosphorylates are the cascade proteins which trigger GLUT-4 translocation from cytosol vesicles to the plasma membrane for glucose uptake and utilization (Rakhshandehroo et al. 2010;Song et al. 2022).In diabetic conditions, because of the absence of an insulin signaling mechanism due to insulin resistance, glucose absorbed from the diet does not enter the cells for further metabolism and hence there is an elevation in glucose level.Insulin resistance is the main characteristic of type II DM mainly promoted by a high-fat diet which disturbs the metabolism of glucose and lipids by down-regulating the transcription proteins such as GLUT-4 and PPAR-γ.
In the current study down regulation of GLUT-4 expression in skeletal muscle and adipose tissues in HFD-STZ-induced diabetic rats was observed.Hence, the activation of GLUT-4 plays a crucial role in insulin signaling and is considered one of the important targets for the management of type II DM.Several studies reported that the phytoconstituents isolated from medicinal plants can enhance the expression of GLUT-4 for glucose disposal.Treatment by vitexin at 20 mg/kg BW up regulated the expression of GLUT-4 and showed the increased utilization of glucose in skeletal muscle by promoting transportation of GLUT-4 from intracellular vesicles to the cellular membrane.
PPAR-γ predominantly found in adipocytes is a ligand-dependent transcription factor that governs the genes involved in lipid metabolism .The activation of PPAR-γ improves the fatty acid storage and oxidation of adipocytes (Kershaw and Flier 2004;Sharma and Patial 2022).Thus, PPAR-γ is crucial in maintaining energy homeostasis by binding with specific ligands and has been considered a target for the management of type II DM.The binding of ligands with PPAR-γ activates the transcription factors that promote adipogenesis in adipocytes and sensitizes the genes of insulin signaling.PPAR-γ activation also enhances the expression of genes that trigger the metabolism of glucose in the adipocytes including GLUT-4 (Gandhi et al. 2013;Ganjayi et al. 2023).
In this study, it is proved that during the 48 days of treatment with flavonoids ligand vitexin, the expression of PPAR-γ in adipocytes of diabetic rats was improved significantly.Therefore, these results explained that treatment by vitexin in HFD-STZ induced diabetic rats' up-regulated PPAR-γ expression and explained a significant effect on preventing the resistance to insulin.Furthermore, this study shows down-regulation of PPAR-γ expression in the adipose tissue of diabetic control rats although treatment with vitexin significantly up-regulated expression of PPAR-γ.Many phytoconstituents isolated from medicinal plants act as a natural ligand for PPAR-γ and enhance its expression (Liang et al. 2003).Vitexin is a flavonoid molecule that improved PPAR-γ expression in adipocytes as well as a decrease in weight in treated diabetic rats.Vitexin enhances the activation of PPAR-γ which in turn up-regulates the GLUT-4 expression and thereby enhances the glucose uptake in adipocytes.Thus, vitexin enhances the expression of PPAR-γ and GLUT-4 in type II diabetic rats.

Conclusion
The present study provides evidence for vitexin as an antidiabetic agent.In vitro glucose uptake assay explains that it enhances the uptake of glucose by skeletal cells, which is a major defect in type II diabetes.A long-term study of vitexin revealed its significant effect on blood glucose, insulin, lipid profiles, hepatic enzymes, and other body profiles in HFD-STZ-induced diabetic rats.In addition, treatment with vitexin up-regulates PPAR-γ expression in adipocytes and thereby facilitates the transportation of glucose by the activation of GLUT-4 in peripheral tissues.Hence, vitexin can become a promising new-generation antidiabetic drug for insulin resistance-related type II diabetic patients.

Fig. 17
Fig. 17 PPAR-γ mRNA expression level of normal control, diabetic control, and treated rats in adipose tissue

Table 2
Glucose uptake activity of fractions in L6 cell lines *** indicates highest percentage of glucose uptake

Table 4
Effect of vitexin on body weight Values are the mean ± SEM of six rats in each group D diabetic, HFD high-fat diet Values with superscript "*" statistically differ from normal control rats (p ≤ 0.05) and "#" statistically differ from diabetic control rats (p < 0.05) (one way ANOVA followed by Tukey's multiple comparison test)

Table 5
Effect of vitexin on fasting blood glucoseValues are the mean ± SEM of six rats in each group

Table 6
Effect of vitexin on insulin, HOMA-IR, and HbA1c Values are the mean ± SEM of six rats in each group D diabetic, HFD high-fat diet, HOMA-IR homeostatic model assessment-insulin resistance Values with superscript "*" statistically differ from normal control rats (p ≤ 0.05) and "#" statistically differ from diabetic control rats (p < 0.05) (one way ANOVA followed by Tukey's multiple comparison test)

Table 7
Effect of vitexin on liver enzymesValues are the mean ± SEM of six rats in each group D diabetic, HFD high-fat diet, ALT alanine transaminase, AST aspartate transaminase, ALP alkaline phosphatase Values with superscript "*" statistically differ from normal control rats (p ≤ 0.05) and '#' statistically differ from diabetic control rats (p < 0.05) (one way ANOVA followed by Tukey's multiple comparison test)

Table 8
Effect of vitexin on renal functionValues are expressed as the mean ± SEM for six rats in each group