Study on Optimal Extraction and Hypoglycemic Effect of Quercetin

Quercetin was extracted from Portulaca oleracea L. through biphasic acid hydrolysis to investigate its potential as a suppressor of dipeptidyl peptidase IV (DPP-IV) and its hypoglycemic effect in type 2 diabetic mice. The extraction procedure was optimized utilizing the response surface method (RSM) in a single-factor experimental setting. An extraction efficiency of 0.675% was achieved using the following optimized parameters: 0.064 mol/L vitriol, 1 : 109.155 solid-liquid ratio, and 21.408 min ultrasonication. Overall, findings indicate the effectiveness of quercetin extraction. A mouse model for type 2 diabetes was established to receive oral treatment with various quercetin concentrations for 8 weeks. Fasting blood glucose (FBG) and the DPP-IV activity in the serum were significantly reduced. The weight and insulin levels of the mice in the quercetin group were raised compared to those in the model group (P < 0.01). Quercetin dose-dependently inhibited postprandial blood glucose excursions, as demonstrated by the oral glucose tolerance test. These results confirmed that quercetin has hypoglycemic effects and considerably improves insulin sensitivity via DPP-IV targeting.


Introduction
In 2021, according to the most recent data from the International Diabetes Federation, 537 million adults between the ages of 20 and 79 were living with diabetes, accounting for 10.5% of the world's population in this age group.Te total number is projected to reach 643 million (11.3% of the world's population) by 2030 and 783 million (12.2%) by 2045 [1].Te most common form of this disease is type 2 diabetes mellitus (T2DM) which amounts to approximately 90% of all cases worldwide.T2DM is recognized as a set of metabolic ailments characterized by hyperglycemia, dyslipidemia, and insulin resistance in target metabolic tissues [2,3].Most diabetes' clinical manifestations are characterized by a syndrome of substitution.Some patients have polyuria, polydipsia, increased food intake, and weight loss, as well as an itchy vulva and blurred vision.A prolonged hyperglycemic environment can also result in immune cell abnormalities.Tese diseases harm multiple organs and result in various common complications.Examples include diabetic cardiomyopathy, diabetic nephropathy, diabetic neuropathy, diabetic wounds, and diabetic retinopathy [4].Insulin, metformin, sodium-glucose cotransporter 2 (SGLT-2) inhibitors, incretin-based agent-like glucagon-like peptide-1 (GLP-1) receptor agonists (RAs), α-glucosidase inhibitors, sulfonylureas (SUF), dipeptidyl peptidase-4 (DPP-4) inhibitors, glinides, and thiazolidinediones (TZDs) are the currently available drugs for the treatment of diabetes [4].Although the drugs mentioned above are widely used in clinical practice, they can cause side efects.
Traditional herbal remedies have evolved into potentially benefcial biological products that efectively manage or control diabetes.P. oleracea.is a medicinal plant that is widely grown in China and is easy to pick, which is rich in favonoids.Studies have found that favonoids have obvious hypoglycemic efects [5].Quercetin is a frequently consumed favonoid, ubiquitous in beverages and plant-based foods, and abundant in P. oleracea.[4,6].Like most favonoids, this pigment typically exists in a range of glycosidic forms rather than as a free aglycone.Most glycosides cannot enter blood due to the obstruction of the small intestine wall; therefore, they must be hydrolyzed into glycosides by hydrolases secreted by probiotics in the intestine and reabsorbed into blood.Only favones in the form of glycosides can directly enter blood and play various physiological roles.Terefore, quercetin can be detected in plasma after acid hydrolysis.Te bioavailability of favonoid aglycones in vivo is higher than that of favonoid glycosides [7,8].Tis study aimed to optimize the acid hydrolysis of quercetin from P. oleracea.by applying response surface methodology.Te treatment was administered to the T2DM mouse model to explore how powerful the hypoglycemic efect is and to understand the underlying mechanisms controlling it.

Experimental Material.
Quercetin standards were purchased from Aladdin.Ethanol, hydrochloric acid, and vitriol were acquired from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China).HPLC-grade methanol was procured from Merck (Darmstadt, Germany).Te ICR mice were purchased from Mudanjiang Medical College, and the bioethics protocol number is 20211015-1.P. oleracea.was bought from Mudanjiang Pharmacy.High-fat and highsugar feed was purchased from Xiaoshu Youtai (Beijing) Biotechnology Co., LTD (Table 1).Mouse INS (Insulin) ELISA Kit was bought from Sangon Biotech.STZ was acquired from Beijing DingGuo Biotechnology Company.

HPLC Analysis.
Te samples were separated into four concentration gradients with concentrations of 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL, and 1.0 mg/mL, and the quercetin standard and samples were fltered.As mobile phases for HPLC analysis, methanol (solvent A, 45%) and water (solvent B, 55%) were used at a fow rate of 1 mL/min.Te injection volume was 10 μL, the column temperature was 30 °C, and the detection wavelength was 360 nm.For linear regression, the peak area of the standard was used as the ordinate, and the mass concentration of the reference product was 1.56, 6.25, 12.5, 25, 50, and 100 μg/mL as the abscissa.Te calibration curve is obtained by the formula: Y � 11309X + 36215 (R 2 � 0.9994).

Extraction of Free and Conjugated Quercetin.
Approximately, 900 g of dried and crushed P. oleracea.was placed in a 1 L round bottom fask, along with the addition of 80% volume fraction ethanol according to the material-toliquid ratio of 1 : 7 (m : m), refuxed in a 60 °C water bath for 2 h, and extracted for three times in total.All extracts were combined, and the fnal product was obtained by spinning and steaming under 60 °C.
To understand the interaction among optimized conditions, RSM was employed by utilizing the Box-Behnken data processor.Based on a single-factor analysis, the quercetin extraction rate was chosen as the dependent variable, whereas solid-liquid ratios, vitriol concentrations, and extraction duration were selected as independent variables.All other conditions were optimized after fxing the extraction temperature at 60 °C.

Establishment of the T2DM Mouse Model.
Eighty male ICR mice with a weight ranging between 18 and 22 g were exposed to an ambient temperature of 22 °C-25 °C and a 12 : 12 h light/dark cycle, following housing them in a cage (fve animals each).Ten mice in the normal group were given ordinary chow as feed.A high-fat diet was given to the other mice for 1 week.Tese mice were then made to fast for 8 h; however, they were allowed free access to water.Intraperitoneal injections of STZ (35 mg/kg in 0.1 mol/L citrate-bufered saline, pH 4.4; injection given daily for 3 days) were then given to these mice to induce T2DM.Free access to water and high-fat food were given to the STZtreated group.Te mouse's fasting blood glucose levels were consistently higher than 11.1 mmol/L and remained above 15 mmol/L two hours after gavage, indicating that the model was successfully established.

Treatment Protocol.
Five groups of the mice were made, including a quercetin high-dose group that was treated with 80 mg/kg quercetin, a quercetin low-dose group that was treated with 20 mg/kg quercetin, the isoquercitrin group that was treated with 20 mg/kg isoquercitrin, a diabetes model group was treated with 0.9% saline (vehicle), and the normal group was treated with 0.9% saline (vehicle).Among them, sixteen mice were in the high-dose group of quercetin, eighteen mice in the low-dose group of quercetin, eighteen mice in the isoquercetin group, eighteen mice in the diabetes model group, and ten mice in the normal group.Saline EDTA), 10 mL, and 50 ng DPP-IV and recombinant DPP-IV, respectively, along with or without diferent concentrations of isoquercetin diluted in dimethylsulfoxide (DMSO).After incubation for 10 min at 37 °C, the reactions were terminated with 0.1 mol•L −1 NaHCO 3 , and the amount of the product, pentose nucleic acid (PNA), was measured by UV absorbance at a wavelength of 405 nm.Te inhibitory potency of inhibitors was evaluated by IC 50 values [9].Based on the GPO-PAP method determined using the instructions in a commercial kit, total cholesterol (TC) and triglycerides (TGs) in serum were calculated.According to the ELISA method, insulin was measured using Mouse INS (Insulin) ELISA Kit.

OGTT Test.
After the administration, OGTT tests were performed on mice fasted for 8 hours.Each mouse was given a dose of glucose solution of 2 g/kg by intragastric administration, and glucose content in the caudal vein was measured by using a glucose meter at 0 h, 0.5 h, 1 h, and 2 h, respectively.

Statistical Analysis.
Data were presented as the mean ± SD.Using GraphPad Prism 5 software, statistical analysis was conducted using Student's t-test or one-way ANOVA.A probability value of P < 0.05 was considered statistically signifcant, and the LSD test was used for postmortem examination.

Single-Factor Experimental Design.
When diferent solvents were used for extraction, ethyl acetate had a signifcantly higher extraction efciency than n-butanol and dichloromethane (Figure 1(a)).A notable increase in the quercetin extraction rate was observed when the solid-liquid ratios were maintained between 1 : 50 and 1 : 150 (Figure 1(d)).Tis fnding suggests that the extract was highly viscous at ratios below 1 : 50, thereby resulting in inefcient extraction.An increment in the solid-liquid ratio can result in an enhancement in quercetin extraction.At a ratio of 1 : 100, peak extraction was attained.A 1 : 100 solid-liquid ratio was therefore employed for subsequent single-factor experiments.

RSM-Mediated Parameter Optimization.
After the extraction-related parameters were individually evaluated, their interactions were explored via RSM for optimization.Tese three parameters served as independent variables for this analysis, whereas the dependent variable was taken to be the quercetin extraction index.As detailed in Table 2, experimental randomization was carried out to maximize the infuence of inexplicable variability on the extraction rate.Seventeen tests with fve replicates (runs 2, 8, 10, 14, and 17, Table 1) were performed to calculate the pure error sum of squares.
Te projected R 2 value of 0.7346 corroborated quite well with the adjusted R 2 value of 0.9336.Te value of the precision ratio is equivalent to 12.639, suggesting an adequate precision (Table 3).Moreover, the established model had low P values (P < 0.00001) and extremely high F values for a pair of responses.Te F value of 25.98 indicates only a minimum chance (0.01%) of this value being a consequence of noise.All "Prob > F" values of <0.0500 were signifcant, while values >0.1 were insignifcant.Following these specifcations, the signifcant terms in the model are as follows: A, B, C, AB, A 2 , B 2 , and C 2 (Table 4).

Evidence-Based Complementary and Alternative Medicine
Te diference between the predicted R 2 value of 0.7346 and the adjusted R 2 value of 0.9336 was less than 0.2, indicating reasonable agreement.Te signal-to-noise ratio is estimated by "Adeq precision."Te desirable ratio is a value greater than 4. Te obtained ratio of 12.639 suggests an adequate signal.Hence, the design space can be navigated using this model.
Te analysis revealed that the three independent variables were related based on the second-order polynomial expression.Extraction efciency % � 0.67 + 0.04A + 0.044B + 0.057C + 0.00725AB − 0.00525AC + 0.075BC − 0.38A 2 − 0.21B 2 − 0.018C 2  Figure 2 shows the response surfaces complementing the efect of the independent variables on the mean quercetin extraction efciency.Te interactions between the acid concentration and time, between the acid concentration and solid-liquid ratio, and between the time and solid-liquid ratio are illustrated in Figures 3(a  Whereas a stable increase in the body weight was observed in the normal mice, a decline was observed in the mouse model (Figure 4(a)).During the early treatment, the value of body weight in the quercetin-and isoquercitrintreated mice decreased, then underwent a gradual increase, and hardly demonstrated any diference in comparison to that in the normal group.Te mice in the quercetin highdose group and isoquercitrin group showed higher weight than the members of the low-dose group, and the weight shift was more or less dose-dependent.A signifcant decline was observed in the body weight in the diabetic mouse model following a passage of 8 weeks; however, there was an increase in the other groups of mice.After quercetin and isoquercitrin treatments, the mice's body weight was higher than before treatment (P < 0.01).Compared with the normal mice and isoquercitrin-treated mice, the body mass of the quercetin-treated mice manifested no statistically signifcant diference after therapy.Te outcome suggested that   Evidence-Based Complementary and Alternative Medicine quercetin has the potential to ameliorate body weight disorder in T2DM mice.Compared with the model group, the quercetin administration group showed no signifcant difference in TG and TC contents.

Efect of Quercetin on Glucose Tolerance in Mice.
After 8 weeks of mice given quercetin and isoquercetin, the body weight values of mice in the high-dose quercetin group and the isoquercetin group were signifcantly diferent from those in the model group (P < 0.01), while the low-dose quercetin group was signifcantly diferent from the model group (P < 0.05) (Figure 4(a)).Te blood glucose values of mice in the high-dose quercetin group and the isoquercetin group were very signifcantly different from those in the model group (P < 0.01), while the low-dose quercetin group was signifcantly diferent from the model group (P < 0.05) (Figure 4(b)).Te glucose tolerance values of mice in the high-dose quercetin group and the isoquercetin group were very diferent from those in the model group (P < 0.01), while the low-dose quercetin group was signifcantly diferent from the model group (P < 0.05) (Figure 4(c)).Te increase in the FBG in the diabetic mouse model was relatively gradual and was considerably higher than that in the normal mice (Figure 4(b)).After 8 weeks, the blood glucose level manifested a signifcant increase in the diabetic mouse model.At the end of the 8-week regimen, the high-dose quercetin group and the isoquercitrin group exhibited a notable decrease in their blood glucose level compared to the diabetic mice (P < 0.01).However, there was a noticeable decline in blood glucose levels among the group receiving the low dose (P < 0.05).Te outcome suggested an efcient hypoglycemic activity of quercetin.Additional in vivo investigations, particularly diferent dosage variants, are key to accurately determining its therapeutic potential.OGTT demonstrated a signifcant decrease in the blood glucose concentration within the treatment group compared to the model group.Figure 4(c) shows that treatments of isoquercitrin and high-dose quercetin antagonized the blood glucose increase resulting from exogenous glucose and that lowdose quercetin also caused a reduction in the blood glucose level.

Efect of Quercetin Terapy on the Insulin Level and DPP-IV Activity in Serum.
In an attempt to address how quercetin exerts its ameliorating efect on hyperglycemia in T2DM mice, the activity in serum was evaluated.
Tere was a rapid decrease in the DPP-IV activity in serum in the diabetic mice that were given quercetin and isoquercitrin treatment.Various quercetin concentrations exhibited a dose-dependent inhibitory impact in the DPP-IV serum of the mice that were treated for 8 weeks (Figure 2(a)).Figure 2(b) shows that in comparison to those of the model group, the levels of serum insulin of mice in the quercetin group and the isoquercitrin group were considerably higher, in particular in the high-dose isoquercitrin group (P < 0.01).
Te IC 50 value is 29.6 mg/kg.

Discussion
Flavonoids are glycosylated derivatives in plant materials, frequently occurring as glycosides [10].In general, these pigments are absorbed as aglycones after the prior hydrolysis of glycosides along the aerodigestive tract.Hence, the oral favone glycosides' efect arises from their glycosides' activity [11].In recent studies, plants containing favonoids, such as procyanidin, epicatechin, and rhodomyrtone E, have also been indicated to have antiglycation properties [12].P. oleracea.is an annual herb of the family Portulaca oleracea, edible vegetables, and widely planted in the country, has high yield, is easy to pick, and has the efect of reducing swelling and can promote ulcer healing.Quercetin extracted from P. oleracea.isless expensive than other hypoglycemic drugs.Quercetin, chemical name 3,3′,4′,5,7-pentahydroxy favones, is found in many Chinese herbs, mostly in the form of binding glycosides.Acid hydrolysis is a chemical reaction in which an organic molecule is decomposed by water, and acids act as catalysts to hydrolyze glycosides into sugars and aglycones [13].
Quercetin and isoquercitrin are similar in structure and belong to the class favonoids.Isoquercitrin is hydrolyzed to produce quercetin.In this work, conjugated quercetin was dissociated via acid hydrolysis to increase its extraction rate.Te results showed that the extraction rate of quercetin could be improved by adding acids because hydrolysis cleaves the glycoside linkage.Many of the conjugated quercetin molecules are dissociated through acid hydrolysis.Since the dissociation efect of sulfuric acid is enhanced compared to hydrochloric acid, the hydrolysis of favonoid glycosides proceeded using sulfuric acid as a catalyst [10].Te degradation of favonoids at high acid concentrations is probably the underlying cause behind the reduction in extraction yields [14].Type 2 diabetes and obesity are intricately linked chronic disorders, so the body mass index is a valuable marker for manifesting insulin resistance and glucose tolerance [17].Quercetin is characterized by alleviation potency on body weight disorder.Oral gavage treatment with various quercetin concentrations led to a profound decrease in hyperglycemia (Figure 4).Glucose tolerance is the embodiment of the body's ability to regulate blood glucose concentration.Te high-dose quercetin treatment successfully antagonized the increase in blood glucose caused by exogenous glucose.
Studies have found in mouse models of type 2 diabetes that quercetin regulates hyperglycemia by increasing pancreatic antioxidant levels and enzyme activity associated with glucose metabolism [17].GLP-1 has been identifed as an incretin hormone produced by L cells in the intestine and neurons in the nucleus of the solitary tract (NTS) of the brainstem; together with GLP-1 receptor (GLP-1R), this hormone enhances glycemic control [18][19][20].DPP-IV activity is associated with the Glp-1 level in plasma [21,22] because native GLP-1 undergoes swift degradation by enzyme dipeptidyl peptidase-IV (DPP-IV) [23].Stable GLP-1 mimetics and DPP-IV inhibitor drugs have been introduced into clinical practice to treat obesity and T2DM [24].Te favonoid-rich fractions from Allophylus cominia Sw. inhibit the DPPIV enzyme [25].Tus, DPP-IV could be one of the frst possible targets of quercetin to improve insulin sensitivity.DPP-IV inhibition may stimulate insulin secretion, which substantially improves the characteristic T2DM symptoms.Zhang et al. [9] reported that quercetin impedes the activity of DPP-IV under in vitro and in vivo conditions, subsequently leading to enhanced secretion of GLP-1 and insulin and ameliorating hyperglycemia in STZ-induced T2DM and high-fat diet mice.Quercetin and isoquercitrin are similar in structure and belong to the class favonoids.Te serum of the mice treated with diferent quercetin doses showed a signifcant inhibitory efect on DPP-IV at 8 weeks after administration, and the inhibitory efect increased with the administration dose.Terefore, a reduction in the intensity of hyperglycemia in db/db mice was observed following oral treatment with quercetin.Tis hypoglycemic potency, in particular, was at least as good as that observed with isoquercitrin.Te mechanistic details may point towards improving DPP-IV activity with quercetin; however, this hypothesis requires further studies.In addition, the dose and side efects of quercetin on the human body need to be further studied.

Conclusion
In conclusion, this study demonstrates that quercetin and isoquercitrin extracted from P. oleracea.exert a hypoglycemic efect in a common mouse model of type 2 diabetes.Its function may be related to blood glucose and insulin levels, glucose tolerance, and DPP-IV activity.In addition, it can ameliorate the weight disorder caused by type 2 diabetes.Recent studies [26] and these data support the future use of P. oleracea.as a dietary supplement for type 2 diabetes, and it is anticipated that extracted quercetin and isoquercitrin will become new therapeutic agents.

Table 1 :
List of mouse feed ingredients.9%) was used to dilute all drug stock solutions, administered once daily through oral gavage for 8 weeks.Te blood glucose and body weight of mice fasted for 8 h were detected weekly during treatment.Glucose tolerance and serum DPP-IV activity were measured 8 weeks after administration.Measurement of Parameters in Serum.Mice were made to fast for 8 h and were tested weekly for DPP-IV during treatment.Te suppression efect of serum on DPP-IV was tested in vitro.Te tail vein of the mice was used to draw blood samples.Te DPP-IV activities were measured by the addition of 10 μL of 10 mmol•L −1 Gly-Pro-PNA (the substrate) to a bufer solution (pH 8.0) containing 0 Te concentration of 0.05 mol/L vitriol was utilized in downstream experiments because peak extraction was achieved at this concentration.It is evident from the observed dose-dependent relationship between extraction efciency and vitriol concentration that the utilization of high viscosity vitriol solutions may lead to poor penetration of plant raw materials (Figure1(b)).
Concentration on Quercetin Extraction.Te outcome implies that sulfuric acid is better in terms of its extraction efect than hydrochloric acid.Te quercetin extraction rates increased with vitriol concentrations of 0.025-0.1 mol/L but decreased signifcantly at higher concentrations.3.1.3.Efect of Solid-Liquid Ratios on Quercetin Extraction.A key determinant of extraction efciency is the ratio of the amount of raw plant material to the amount of solvent.Enhanced extraction efciency generally results from the high solvent volume; nevertheless, exceedingly large volumes can lead to relatively complex extractions and generate additional unneeded waste.

Table 3 :
Credibility analysis of the regression equations.
dose STZ therapy.Te outcome suggested the successful creation of the T2DM model: the mice showed polyuria and polydipsia, subsequently losing body weight.Te model group exhibited extremely dull hair, high water consumption, and listless spirit compared to the normal group.Te quercetin group had a notably better condition than the model group.

Table 4 :
Test of signifcance for the regression coefcient.