Acorn (Quercus variabilis BL.) shells extract alleviating postprandial blood glucose via α-amylase and α-glucosidase inhibition and starch digestion resistance

Acorn (Quercus variabilis BL.) shells extract alleviating postprandial blood glucose via α-amylase and α-glucosidase inhibition and starch digestion resistance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Acorn (Quercus variabilis BL.) shells extract alleviating postprandial blood glucose via α-amylase and α-glucosidase inhibition and starch digestion resistance Zixuan Han, Nan Lin, Zongyao Liang, Xuchang Duan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9496475/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Acorn ( Quercus variabilis BL.) shells as by-product were identified to rich in phenolic acids, flavonoids and triterpene, exhibiting the excellent inhibition of α-amylase and α-glucosidase. Subsequently, this study was performed to evaluate the inhibitory mechanism of acorn shells extract against the enzymes and the regulatory of postprandial blood glucose. Enzymatic kinetics assays and spectroscopy revealed the mixed inhibition of α-amylase and α-glucosidase were attributed to the hydrophobic interaction between the extract and enzymes, which promoted the tryptophan energy transfer and α-helix unfolding in the enzymes. Moreover, in vitro starch digestion test, 25% extract-starch significantly reduced the starch digestibility (60.48%), while 5% extract-starch also delayed the digestion (60min). Furthermore, starch and glucose tolerance tests showed that the extract down-regulated glycemia via delaying the starch hydrolysis into glucose and improving glucose metabolism to the blood. Thus, acorn shells extract had comprehensive exploration values in food formulation against hyperglycemia. Acorn shells extract α-amylase α-glucosidase Starch digestion Postprandial blood glucose Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Diabetes is a metabolic disease characterized by chronic hyperglycemia caused by multiple factors, which has been spreading worldwide (Pappachan, Fernandez, & Chacko, 2019 ). Balancing optimal glycemic control with the most appropriate diabesity management regime should be the priority for clinicians in choosing the antidiabetic agent (Deol, Lekkakou, Viswanath, & Pappachan, 2017 ; Pappachan et al., 2019 ; Pappachan & Viswanath, 2017 ), including lifestyle changes, medication therapies, and surgery (Laiteerapong & Cifu, 2016 ). Despite the widening choice of oral and injectable antihyperglycemic agents to treat type 2 diabetes (T2DM) and the availability of long- and fast-acting insulin analogues, many people struggled to achieve recommended glycemic targets and were at risk of developing long-term micro- and macrovascular complications. (Daly & Hovorka, 2021 ; Khunti, Wolden, Thorsted, Andersen, & Davies, 2013 ). Natural products with low toxicity have been explored as drug sources for a long history to treat infectives, cancer, hypertension and diabetes (Newman, 2022 ). The potential effects of experimental research and clinical applications have been verified. (Hu, Jiang, Yan, Zeng, Ma, & Zhao, 2023 ). Compared with the synthetic drugs, the treatment benefits of natural products were attributed to multi-components and multi-targets to produce combined or synergistic effects. Nowadays, multi-components and multi-targets therapies have been proved to be more effective and less toxicity than traditional single-target drugs (Espinoza-Fonseca, 2006 ). There were various extracts used for the T2DM treatment, such as Annona muricata aqueous extract was daily administrated to diabetic rats for 28 days, the blood glucose levels and serum creatinine were reduce (Florence et al., 2014 ); the extract of apple trees bark (Moradi-Marjaneh, Paseban, & Sahebkar, 2019 ) was used to decrease blood glucose by the inhibition of SGLT2; and Abroma augusta L. (Malvaceae) leaf extract could be considered as a kind of prophylactic agent against T2DM (Khanra et al., 2015 ). Phytochemical investigations revealed the presence of tannins, flavonoids, terpenoids and phenolic compounds from all of these extracts. Polyphenols widely distributed in natural products tissues as secondary metabolites, which had been proved effectively impede the activity of the starch digestion enzymes to diminish the amylolysis. α-amylase and α-glucosidase, which were the key enzymes located in the cell membrane of small intestine for digestion and degradation of starch compounds, played crucial roles in the metabolism of carbohydrate and the maintenance of normal physiological functions (Li et al., 2018 ). Therefore, inhibition of α-amylase and α-glucosidase had become an important target for the research and treatment of diabetes based on the factor that hydrolysis of carbohydrate after meal was the main pathway of blood glucose increase. It is of great significance to find a digestive enzyme inhibitor with high activity and derived from natural plants for the treatment of diabetes. Acorn shells were rich in polyphenols and the extract exhibited in vitro α-amylase and α-glucosidase inhibitory activities (Güvenalp, Yuca, Gözcü, Dursunoğlu, Tosun, & Demirezer, 2016 ), but it was still unclear that if the binding of the extract with the enzymes and starch affect their hypoglycemic activity and starch digestion, which was crucial important for the development of more powerful antidiabetic drugs and efficacious utilization of acorn shells. Thus, the inhibition of the extract on α-amylase and α-glucosidase activity were investigated and proposed a reasonable inhibition kinetics in this study. Following, the inhibition mechanism of the extract was analyzed during the interaction with the enzymes using fluorescence quenching and circular dichroism spectra. Furthermore, whether acorn shells extract could interact with starch were examined by in vitro starch digestibility. Finally, the effect mechanism of the extract on the hypoglycemic was discussed based on the starch tolerance and glucose tolerance were verified by animal experiments. Our work might provide a perspective regarding the application of acorn shells extract in functional starch products. 2. Materials and methods 2.1 Preparation of acorn shells extract Quercus variabilis acorn shells were plucked from an experimental field at Northwest Agriculture and Forestry University, dried at 50°C for 24 h and crushed into powders. The acorns shells powders were used to extract polyphenols as the protocol previously described by (Oroian, Ursachi, & Dranca, 2020 ; Sun, Guo, Fu, Li, & Li, 2013 ) with slight modifications. Firstly, the powders were extracted with 95% ethyl alcohol in a proportion of 1:5 (w/v) under ultrasound at 40°C, 300 W for 30 min, followed by filtration to gather the remainder to repeatedly extracted twice. Subsequently, the ethanol-extract were concentrated by a vacuum rotary evaporator at 45°C,0.09 Mpa (LGJ-100, Beijing Sihuan Instrument Co., Ltd., China), and dried with Freeze Dryer (LGJ-10) at -80°C for 8 h to obtain the acorn shells extract. The chemical composition of the extract was analyzed by HRLC-TOF-MS (Appendix Table S1 ). 2.2 Inhibition assays of α-amylase and α-glucosidase The inhibition assay of α-amylase was adapted from Shen et al. (2012). Briefly, 30 µL the extract (0.1 ~ 2.0 mg/mL, diluted with the sodium phosphate buffer, PBS, pH 6.8) were thoroughly mixed with 30 µL α-amylase suspension (0.148 U/mL, diluted in PBS). After the mixture was incubated at 37°C for 10 min, 60 µL wheat starch solution (2%, w/v, gelatinized at 98°C for 20 min) was added and reacted at 37°C for 10 min. The reaction was stopped by the addition of 120 µL 3,5-dinitrosalicylic acid reagent (DNS) in a boiling water bath for 5 min, followed by cooling to 25°C. Subsequently, the reaction products were transferred into 96-well microplates for absorbance measurements at 540 nm using an enzyme-labeled instrument (Victor X3, PerkinElmer Enterprise Management Co., Ltd., Shanghai, China). Acarbose was used as a positive control. α-glucosidase activity assay was performed according to (Zhu, Zhang, Wang, Li, Fu, & Huang, 2019 ). Briefly, 20 µL α-glucosidase (1.5 U/mL) and 20 µL the extract (0.01 ~ 1.5 mg/mL) were mixed in 20 µL PBS and incubated at 37°C for 30 min. And then 20 µL PNPG (5 mmol/L) was added to react for 10 min prior to terminating the reaction with 150 µL Na 2 CO 3 (1mol/L). The absorbance of the mixture was recorded at 405nm. Acarbose was used as a positive control. The inhibition rates of α-amylase and α-glucosidase were calculated using Eq. ( 1 ). $$\:\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\text{r}\text{a}\text{t}\text{e}\:\left(\text{%}\right)=\left[1-\frac{\left({\text{A}}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}-{\text{A}}_{\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{b}\text{l}\text{a}\text{n}\text{k}}\right)}{\left({\text{A}}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}-{\text{A}}_{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{b}\text{l}\text{a}\text{n}\text{k}}\right)}\right]\times\:100$$ 1 where A control , A control blank , A sample , A sample blank represent the absorbance values of the sample with enzymes and PBS, PBS, extract with enzymes and PBS, extract with PBS, respectively. 2.3 Inhibition Kinetic of α-amylase and α-glucosidase The inhibitory type of the acorn shells extract (0.5 mg/mL) against α-amylase and α-glucosidase were determined by varying the concentrations of the enzymes (0, 10, 20, 40, 50 µg/mL). The α-amylase and α-glucosidase inhibition test were carried out as the methods described above (Section 2.2 ). The kinetic of α-amylase and α-glucosidase was carried out according to the previous method (Huang, Wu, Ying, Dong, & Chen, 2021 ) with a slight modification. Michaelis-Menten constants were determined via varying the concentrations of substrates (wheat starch: 0.5%, 0.75%, 1.0%, 1.25%, 1.5%; PNPG: 1, 2, 4, 5, 10 mmol/L) and extract (0, 0.2, 0.5 mg/mL for α-amylase; 0, 0.1, 0.2 mg/mL for α-glucosidase). Subsequently, Lineweaver-Burk double inverse curves were plotted based on the enzymatic reaction rates of α-amylase and α-glucosidase. Finally, the kinetic constants of inhibition were calculated using the following equation: The kinetic equation: $$\:V=\frac{{V}_{m}·\left[S\right]}{{K}_{m}\left(1+\frac{I}{{K}_{i}}\right)+\left[S\right]\left(1+\frac{I}{{K}_{is}}\right)}$$ 2 Plotted as 1/V against 1/[S], a double inverse equation: $$\:\frac{1}{V}=\frac{{K}_{m}}{{V}_{m}}\left(1+\frac{I}{{K}_{i}}\right)\frac{1}{\left[S\right]}+\frac{1}{{V}_{m}}\left(1+\frac{I}{{K}_{is}}\right)$$ 3 $$\:Slope=\frac{{K}_{m}}{{V}_{m}}\left(1+\frac{\left[I\right]}{{K}_{i}}\right)=\frac{{K}_{m}}{{V}_{m}}+\frac{{K}_{m}\left[I\right]}{{V}_{m}{K}_{i}}$$ 4 $$\:Intercept=\frac{1}{{V}_{m}}\left(1+\frac{\left[I\right]}{{K}_{is}}\right)=\frac{1}{{V}_{m}}+\frac{\left[I\right]}{{V}_{m}{K}_{is}}$$ 5 where V is the initial reaction speed, V max is the maximum initial reaction speed, S is the substrate concentration, I is the inhibitor concentration, K m is the Michaelis constant, K i is the competitive inhibition constant, and K is is the noncompetitive inhibition constant. 2.4 Fluorescence quenching assay Fluorescence spectra were recorded using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) according to the previously reported method (X. Li, Cai, Yu, Wang, Copeland, & Wang, 2021 ; Tian et al., 2023 ). The α-amylase (0.148 U/mL, 3.0 mL) or α-glucosidase (1.5 U/mL, 3.0 mL) were mixed with 3 mL the extract solution (0, 0.001, 0.05, 0.1,0.2, 0.3, 0.4 and 0.5 mg/mL) and incubated for 5 min at 298.15 K and 310.15 K. Equivalent PBS was used as the blank control. The fluorescence of the enzymes was measured upon excitation at 280 nm and the emission wavelengths ranging from 300 to 500 nm, with the 10.0 nm slit width and the scanning speed at 240 nm/min. The quenching constant K sv was calculated using the Stern-Volmer equation: $$\:\frac{{F}_{0}}{F}=1+{K}_{sv}\left[Q\right]=1+{K}_{q}{\tau\:}_{0}\left[Q\right]$$ 6 where F 0 and F are the fluorescence intensities of enzyme with and without the extract, respectively. τ 0 is the lifetime of fluorophore. K sv is the Stern-Volmer quenching constant (K sv = K q τ 0 ). [Q] is the concentration of the quencher (extract). K q is the quenching rate constant of the biomolecule. τ 0 is the average lifetime of enzyme fluorophore in the absence of quencher and was taken as 1 × 10 − 8 s. Static quencher equation: $$\:\text{log}\left(\frac{{F}_{0}-F}{F}\right)=\text{log}{K}_{a}+n\text{log}\left[\:Q\right]$$ 7 Where n is the number of binding sites and K a is the static binding constant. The slope of the straight line is n and the vertical axis intercept is logK a when log[(F 0 -F)/F] is plotted against log[Q]. The intermolecular forces between the extract and the enzyme can be determined by the van't-Hoff equation: $$\:\varDelta\:{\rm\:H}=\frac{\text{ln}\left(\frac{{K}_{a2}}{{K}_{a1}}\right)\times\:R}{\frac{1}{{T}_{1}}-\frac{1}{{T}_{2}}}$$ 8 $$\:\varDelta\:G=\varDelta\:{\rm\:H}-{\rm\:T}\varDelta\:S$$ 9 Calculate the Gibbs free energy change (ΔG) from Eq. ( 11 ) to determine the spontaneity of the reaction. $$\:\varDelta\:G=-R{\rm\:T}ln{K}_{a}$$ 10 Where K a1 and K a2 are the binding constants at different reaction temperatures T 1 and T 2 . R is the atmospheric constant (8.314 K/(J·mol)). 2.5 Circular dichroism Circular dichroism was measured according to the previously reported method (Wu, Hu, Hu, Ding, Gong, & Zhang, 2019 ). Equal volume of α-amylase (0.148 U/mL, 400 µL) or α-glucosidase (1.5 U/mL, 400 µL) mixed with the different concentrations of the acorn shells extract (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 mg/mL). After incubation at 37°C for 15 min, the circular dichroic spectrum of the mixture was measured using a spectrometer (Chirascan V100, Applied Optical Physics, UK) with a 1.0 mm path length cuvette and a scan speed of 120 nm/min in the range of 190 ~ 260 nm. PBS served as the blank control. 2.6 In Vitro Starch Digestion The starch digestion in vitro was measured according to a modified version of Englyst’s method. The wheat starch (1 g, dw) with different proportion of acorn shells extract (0%, 5%, 10% and 15%, w/w) were dispersed in 50 mL of 0.2 mol/L sodium acetate buffer (pH 5.2) and cooked in a boiling water bath for 30 min, then cooled to 37°C. After that, 6 mL starch-extract mixture were enzymolized by 1 mL preincubated enzymes solution (α-amylase and α-glucosidase mixed in a proportion of 4:1 µg/mL) at 37°C for 120 min. During enzymatic hydrolysis, 1 mL enzymatic hydrolysate were collected at 0, 40, 60, 90, and 120 min, respectively, and put in an ice water bath. The cooling hydrolysate were centrifuged at 3500 g for 10 min. The supernatant was used to analyze the glucose content using DNS colorimetry. $$\:\text{D}\text{i}\text{g}\text{e}\text{s}\text{t}\text{i}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\:\left(\text{%}\right)=\frac{\text{m}}{M}\times\:100$$ 11 Where m is the glucose content, and M is the starch content. 2.7 Postprandial glycemic response measurement Animal experiments were executed according to the guidelines of the institutional animal ethics committee and were supported by the institutional animal committee of Northwest A&F University (XN2023-0605). The male mice (C57BL/6J, 7 weeks old, n = 24; SPF (Beijing) Biotechnology Co., Ltd, China) of similar weight were randomly selected for measuring postprandial glycemic response measurement. The temperature and the relative humidity were set at 24 ± 1°C and 50 ± 5%, respectively. Food and water were freely available. After a week of adjustable feeding, mice were randomly assigned to 3 groups (eight mice per group): positive control group (100 mg/kg acarbose + 2 g/kg starch or glucose), acorn shells extract group (100 mg/kg extract + 2 g/kg starch or glucose), negative control group (same volume saline + 2 g/kg starch or glucose). The interval between the two tolerance tests was one week, fasting overnight before the test, as well as the interval between administration and starch (or glucose) was 15 min. Blood samples from the lateral tail vein were collected at 0, 30, 45, 60, 90, and 120 min and measured using a glucose analyzer (Sano Biosensing Co., Ltd., China) to obtain the glycemic value and the area under the glycemic curve were calculated. 2.8 Statistical analysis All the values were expressed as means±standard deviation (means ± SD) of three replications. Statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS (version 16.0). p < 0.05 was considered to indicate statistically significant difference. 3. Results and discussion 3.1 Inhibition effect of acorn shells extract against enzymes Inhibition of digestion enzymes was one of the most effective approaches for postprandial glycemic control, which was also believed to be the most important ways of polyphenols to exert their glycemic index (GI) reducing effect in vivo (K. Li et al., 2018 ). α-amylase and α-glucosidase were the main digestive enzymes involved in the hydrolysis of dietary starch (Barrett et al., 2013 ). Therefore, we plotted the inhibition curve of the acorn shells extract against α-amylase and α-glucosidase (Fig. 1 B and C), showing that the inhibition rates of both α-amylase and α-glucosidase increased dramatically with the increase of the acorn shells extract concentration. The IC 50 values of the acorn shells extract for α-amylase and α-glucosidase were 0.352 mg/mL and 0.0158 mg/mL, respectively. Surprisingly, the extract had more excellent inhibition on α-glucosidase than acarbose (IC 50 =0.024 mg/mL). According to the previous reports, the more powerful inhibition of the acorn shells extract against α-glucosidase was attributed to its own galloyl substituent on triterpene derivative (Jing Xu, Cao, Yue, Zhang, & Zhao, 2018 ). Additionally, inhibitory strength was also related to the formation of hydrogen bonds between hydroxyl groups of polyphenols and amino acid residues in the active sites of the enzyme (Lo Piparo, Scheib, Frei, Williamson, Grigorov, & Chou, 2008 ). Therefore, the influencing mechanism of the extract binding to the enzymes were further discussed as follow. 3.2 Inhibition kinetics of acorn shells extract against enzymes The reversibility was determined by plotting the change curves of absorbance of reaction product against the concentrations of α-amylase or α-glucosidase (Fig. 2 ), because all the curves passed through the origin (Feng et al., 2022 ). Moreover, the reversible inhibition revealed that the acorn shells extract binds to α-amylase or α-glucosidase through a non-covalent bond, and the enzyme activity could return to normal after the extract was removed (Zhang et al., 2023 ). The types of reversible inhibition, including competitive, uncompetitive, anti-competitive and mixed inhibition, were required to further discriminate by obtaining the Lineweaver-Burk plot to calculate the corresponding inhibition constants (Fig. 2 and Table S2). All lines intersected at the third quadrant after linear fitting, while not on the x or y axis; and the slope of these lines could gradually rose with increasing concentrations of the extract (Fig. 2 A and D), implying that the inhibition type didn’t belong to competitive or uncompetitive. Both K m and V m decreased in the reaction system of α-amylase or α-glucosidase with the increasing concentration of the extract, indicating that the extract bound to both the enzyme and to the enzyme-substrate complex, that was, mixed inhibition. The extract from different sources had different inhibition types on α-amylase or α-glucosidase: the grape pomace extract bound to both free α-amylase and α-amylase -substrate complex, which was consistent with the acorn shells extract above (Kato-Schwartz et al., 2020 ); the extract from finger millet (Eleusine coracana L.) seed coat inhibition reaction type was non-competitive inhibition (Shobana, Sreerama, & Malleshi, 2009 ), which bound to the enzyme molecule outside the substrate binding site and does not affect the binding of the enzyme to the substrate; the inhibition of rhein on α-amylase was proved a competitive inhibition, which competing with the substrate for the active site of the enzyme (Liu at al., 2015). The reason for the different inhibitory effect were possibly related to differences in molecular structure and composition of phytochemical identities (Feng et al., 2022 ; Lo Piparo et al., 2008 ). By further calculating and comparing the inhibition constants (appendix Table S2), it was found that K is values of both α-amylase and α-glucosidase were less than K i values, suggesting that the inhibition capacity on enzyme-substrate complex was stronger than enzyme. Namely, the extract bound to the enzyme-substrate complex because of uncompetitive inhibition prior to the enzymes due to the competitive inhibition, ultimately to form a more stable extract-enzyme-substrate ternary complex, which impeded the intermediate ESI (enzyme-substrate-inhibitor) not be decomposed into products, but also occupied the active center of the enzymes reversibly, thereby inhibiting the activity of the enzymes. 3.3 Fluorescence quenching of acorn shells extract against enzymes In order to reveal the interaction details of α-amylase or α-glucosidase with the acorn shells extract, fluorescence intensities (appendix Fig. S1 ) were obtained to assess the binding affinity and the conformational changes of the enzymes. Tryptophan (Try) residues displayed an inherent fluorescence excitation peak in α-amylase and α-glucosidase (λ ex = 280nm), which was the high sensitivity to its local environment, thus could be utilized as an indicator of binding interactions (Abdollahi, Ince, Condict, Hung, & Kasapis, 2020 ). The intensity of the intrinsic fluorescence of the enzymes was gradually quenched with the increasing addition of the extract (0 ~ 0.5 mg/mL), until disappeared completely for α-glucosidase at 0.5 mg/mL extract. The fluorescence quenching of the Try residues was probably attributed to the interaction with compounds in the acorn shells extract through hydrogen bonds or hydrophobic forces (Fan et al., 2020 ). Moreover, the maximum emission (λ em ) of α-amylase and α-glucosidase shifted to lower wavelength (blue shift) with increasing addition of the extract (appendix Fig. S1 ). The blue shift indicated alterations to secondary structure of the enzymes with the extract addition since the enhancement of hydrophobicity around Try fluorescent carriers, and Try residues were moving to a less polar environment closer to the edge of the protein molecule. Further analysis of the quenching mechanism of the extract with two enzymes by Stern Volmer kinetic equation. The fluorescence quenching Stern-Volmer equation curves of acorn shells extract on α-amylase (Fig. 3 A) or α-glucosidase (Fig. 3 D) were not linear, but quadratic curves concaved toward the y-axis, which indicated that both static and dynamic quenching existed between the extract and the enzymes. By investigating the impact of temperature on the fluorescence quenching, both the fluorescence quenching constant (K sv ) and the fluorescence quenching rate constant (K q ) were growing with the increasing temperature from 298 K to 310 K. However, K q were lower than the maximum biomolecular collision quenching constant (2×10 10 L·(mol·s)). These results implied that the fluorescence quenching process of α-amylase or α-glucosidase by acorn shells extract was dominated by dynamic quenching on account of intermolecular collisions. (Roy, Nandi, Ganai, Majumdar, & Das, 2017 ). As a hypothetical dynamic quenching process, the data of fluorescence intensities were further transferred to draft the double logarithmic curve and natural logarithmic (Fig. 3 B, C, E and F) to obtain the values of n and K a . With the rise of temperature, the binding site (n) of α-amylase was exceed 1 but of α-glucosidase was getting closer to 2 and the static binding constant (K a ) of α-glucosidase also increased more obviously, indicating the larger values of n and K a correspond to tighter and stronger binding. Furthermore, the interaction between the extract and the enzymes were no distance effect due to the binding constant (K) above K a . To sum up, the temperature had an enhancing effect on the affinity between the extract and the enzymes, the extract was more inclined to combined with α-glucosidase resulting in less value of IC 50 for α-glucosidase than α-amylase. 3.4 Thermodynamic parameters and the nature of the binding forces Thermodynamic parameters were calculated using the van't-Hoff Eq. ( 8 ~ 10) to characterize the interaction forces between the extract and α-amylase or α-glucosidase (Table 3). Based on the calculated (ΔG 0 and ΔS > 0), the proceeds of forming enzyme-extract complexes were spontaneously endothermic at the test temperatures (Ross & Subramanian, 1981 ). Moreover, the positive and negative values of thermodynamic parameters were associated with various individual interactions, including hydrogen bonding, van der Waals forces, electrostatic forces, and hydrophobic interactions, which may occur during the process of enzymes bind to inhibitors (Chen et al., 2014 ). From the thermodynamic standpoint (ΔH > 0 and ΔS > 0), the hydrophobic force as the main driving force led to collisions and energy transfer between molecules, resulting in the enzyme molecule returning to the ground state and losing the luminescence effect. Combined with the discussion of fluorescence quenching, the secondary structure of the enzymes changed, which were further analyzed as follows. 3.5 Circle dichroism spectroscopy of acorn shells extract against enzymes Circle dichroism (CD) spectroscopy of proteins in 178 ~ 250 nm (far-UV region) was generally analyzed the secondary structure of proteins (Andrew J. Miles, Drew, & Wallace, 2023 ; A. J. Miles, Janes, & Wallace, 2021 ). CD spectra used the circular dichroism of peptide bonds at 178 ~ 250 nm to obtain the secondary structure information of the enzymes with or without the effect of acorn shells extract (Fig. 4 ). Due to the n→π* and π→π* electron transfer of the peptide bond, the free α-amylase had two distinctive negative peaks near 208 and 228 nm, which characterized α-helix and β-sheet structure, respectively (Xue et al., 2022 ). Similarly, the free α-glucosidase had two obvious negative peaks at 209 and 222 nm. The binding of the extract to the enzymes changed both these bands and also shifting in the negative minima and the results suggested that interaction of the extract with the enzymes may cause some conformational change of the enzymes. The decrease of peak intensity indicated that the addition of acorn shells extract led to the decrease of α-helical structure content and the change of secondary structure, which resulted in the inhibition of the enzymes. These secondary structure changes brought from acorn shells extract were similar to the case of epigallocatechin gallate and luteolin (Wu et al., 2019 ; Yan, Zhang, Pan, & Wang, 2014 ). Overall, the decrease of α-amylase or α-glucosidase catalytic activity could be attributed to the unfolding of enzymes structure, which involved with unwound α-helix and he destruction of hydrogen bonds and some functional units. 3.6 Effect of acorn shells extract on starch digestibility in vitro Based on the starch digestion related to the rise of postprandial blood glucose, the in vitro digestibility of wheat starch-acorn shells extract system was evaluated within 120 min. The highest in vitro starch digestion rate was observed in wheat starch without the extract at all digestion times up to 120 min (Fig. 1 A). Rapidly digestible starch, referred to the starch that can be digested and absorbed within 20 min in the small intestine, decreased significantly with the addition of the extract (5%~25%) and the digestibility of starch-extract decreased from 60.48% to 14.96% as a significant dose-effect positive correlation. Starch that could be completely digested and absorbed within 20 ~ 120 min in the small intestine was defined as slowly digestible starch, which reduced by 28.34% at addition of 25% extract obviously. In general, the starch digestibility presented visibly decreasing at the high enough addition of the extract (25%), signifying that the extract had a certain resistance to the digestion of starch. Combined with the analysis of inhibition kinetics, this phenomenon could be explained that the extract prevented the adhesion of α-amylase and α-glucosidase to starch by occupying its hydrophobic helical region to bind to starch. Even if the addition was at low level, the retardation of digestion remained noticeable, which might be the destruction of rheological properties, gelatinization, retrogradation and gelling as well as the establishment of an ordered structure or crystallinity in the starch-extract mixture system (Guzar, Ragaee, & Seetharaman, 2012 ; Maibam, Chakraborty, Nickhil, & Deka, 2023 ; Sun & Miao, 2020 ; Jingwen Xu, Wang, & Li, 2019 ). Besides, the polyphenols and terpenoids from the extract that could still retained enzyme inhibition after being bound onto starch, even if they participated in the high-temperature (90 ~ 100°C) gelatinization of starch. (Barros, Awika, & Rooney, 2012 ; Chi, Li, Zhang, Chen, Li, & Wang, 2017 ; Mrázková, Sumczynski, & Orsavová, 2023 ). Therefore, the extract directly or indirectly inhibited the digestion of starch and absorption of glucose in vitro. 3.7 Postprandial glycemic response on mice With the intention of further exploration of the extract resistance against starch and glucose in vivo, the postprandial glucose responses of the extract-starch and extract-glucose mixture were investigated (Fig. 5 ). The blood glucose of all three groups rose firstly, then declined and finally stabilized within 120 min. In contrast to the control group, the elevated level of blood glucose of the extract group was significantly lower (p < 0.05) than that of, indicating that the extract had inhibitory effect on starch digestion and glucose metabolism. To be specific, the acorn shells extract reduced the peak value of blood glucose by 29.15% and 30.77% after starch and glucose loading (Fig. 5 A and C). Whereas the peak delay (15 min) was only observed after glucose loading (Fig. 5 C). Subsequently, the areas under the blood glucose curves were further calculated and found no significant differences (p < 0.05) in the relief on postprandial blood glucose between the group of the extract (decreased by 14.39% or 9.64% after starch or glucose loading, respectively) and acarbose (by 15.46% or 14.20%, respectively). Dietary polyphenols were reported to be able to alleviate elevation of blood glucose level after meal, which resulted from interactions between polyphenols and starch, as well as from inhibition of key digestive enzymes, including α-amylase and α-glucosidase (Sun et al., 2020). However, acorn shells extract also repressed in the level of blood glucose in glucose tolerance test, which can be reasonably speculated that it prevented the generation of blood glucose and improved the rapid increase of postprandial blood glucose. Based on the analysis of in vitro and in vivo experimental results, the reasons for the extract to reduce postprandial blood glucose were summarized for three aspects as follow. Acorn shells extract, as a kind of mixed enzymes inhibitor, especially α-glucosidase inhibitor, not only reversibly competed with the active site of the substrate, but also formed a substrate-extract complex to inhibit the formation of the product. Next, the extract interacted with starch directly or indirectly to format resistant starch or inhibiting the hydrolysis of starch by the enzymes. Lastly, the extract regulated the transport of glucose in the blood, promoting glucose metabolism. According to the above results, food-borne acorn shells extracts might be added to food such as bread and noodles to develop food for special medical purpose. Conclusion Acorn shells extracts suppressed α-amylase and α-glucosidase activity in mixed-type manners with IC 50 values of 0.352 and 0.0158 mg/mL, respectively. Through hydrophobic force, the extract destroyed the luminescent groups of the enzymes mainly along with dynamic quenching and ruined the secondary structure of the enzymes with the α-helix content reduce. Besides, the extract directly interacted with starch to affect digestion. From the starch and glucose tolerance tests, the dropping blood sugar level showed that significantly delay the digestion of starch and the glucose transfer. Therefore, acorn shells extract offered prophylactic role against hyperglycemia and assisted its associated intervention studies in vivo, which could be used as a potentially important source in the management of diabetes and functional food additives. Declarations Author Contribution Zixuan Han: Conceptualization, Investigation, Formal analysis, Writing-original draft.Nan Lin: Investigation.Zongyao Liang: Investigation.Xuchang Duan: Project administration, Conceptualization, Writing-review & editing. Acknowledgement This work was supported by Key Research and Development Program of Shaanxi Province (2021NY-158), and Science and Technology Innovation and Achievement Transformation Project of Northwest A&F University Experiment and Demonstration Station (TGZX2020-29). References K. Abdollahi, C. Ince, L. Condict, A. Hung, S. Kasapis, Combined spectroscopic and molecular docking study on the pH dependence of molecular interactions between β-lactoglobulin and ferulic acid. Food Hydrocoll. 101 , 105461 (2020). https://doi.org/https://doi.org/10.1016/j.foodhyd.2019.105461 A. Barrett, T. Ndou, C.A. Hughey, C. Straut, A. Howell, Z. Dai, G. Kaletunc, Inhibition of α-Amylase and Glucoamylase by Tannins Extracted from Cocoa, Pomegranates, Cranberries, and Grapes. J. Agric. Food Chem. 61 (7), 1477–1486 (2013). https://doi.org/10.1021/jf304876g F. Barros, J.M. Awika, L.W. Rooney, Interaction of Tannins and Other Sorghum Phenolic Compounds with Starch and Effects on in Vitro Starch Digestibility. J. Agric. Food Chem. 60 (46), 11609–11617 (2012). https://doi.org/10.1021/jf3034539 H. Chen, Y. Jin, X. Ding, F. Wu, M. Bashari, F. Chen, X. Xu, Improved the emulsion stability of phosvitin from hen egg yolk against different pH by the covalent attachment with dextran. Food Hydrocoll. 39 , 104–112 (2014). https://doi.org/https://doi.org/10.1016/j.foodhyd.2013.12.031 C. Chi, X. Li, Y. Zhang, L. Chen, L. Li, Z. Wang, Digestibility and supramolecular structural changes of maize starch by non-covalent interactions with gallic acid. Food Funct. 8 (2), 720–730 (2017). https://doi.org/10.1039/C6FO01468B A. Daly, R. Hovorka, Technology in the management of type 2 diabetes: Present status and future prospects. Diabetes Obes. Metabolism. 23 (8), 1722–1732 (2021). https://doi.org/https://doi.org/10.1111/dom.14418 H. Deol, L. Lekkakou, A.K. Viswanath, J.M. Pappachan, Combination therapy with GLP-1 analogues and SGLT-2 inhibitors in the management of diabesity: the real world experience. Endocrine. 55 (1), 173–178 (2017). https://doi.org/10.1007/s12020-016-1125-0 L.M. Espinoza-Fonseca, The benefits of the multi-target approach in drug design and discovery. Bioorg. Med. Chem. 14 (4), 896–897 (2006). https://doi.org/https://doi.org/10.1016/j.bmc.2005.09.011 M. Fan, W. Lian, T. Li, Z. Rao, Y. Li, H. Qian, L. Wang, Characterization of promising natural blue pigment from Vaccinium bracteatum thunb. leaves: Insights of the stability and the inhibition of α-amylase. Food Chem. 326 , 126962 (2020). https://doi.org/https://doi.org/10.1016/j.foodchem.2020.126962 J. Feng, F. He, Y. Huang, M. Zhou, X. Liu, X. Ye, H. Chen, Inhibitory effects of phenolic glycosides from Trollius chinensis Bunge on α-glucosidase: inhibition kinetics and mechanisms. Food Funct. 13 (5), 2857–2864 (2022). https://doi.org/10.1039/D1FO03347F N.T. Florence, M.Z. Benoit, K. Jonas, T. Alexandra, D.D.P. Désiré, K. Pierre, D. Théophile, Antidiabetic and antioxidant effects of Annona muricata (Annonaceae), aqueous extract on streptozotocin-induced diabetic rats. J. Ethnopharmacol. 151 (2), 784–790 (2014). https://doi.org/https://doi.org/10.1016/j.jep.2013.09.021 Z. Güvenalp, H. Yuca, S. Gözcü, B. Dursunoğlu, F. Tosun, L. Demirezer, In vitro α-glucosidase and α-amylase inhibitory activities of acorn, acorn shell and cupule extracts of Quercus robur L. Planta Med. 82 , 01 (2016). P180. https://doi.org/10.1055/s-0036-1596337 I. Guzar, S. Ragaee, K. Seetharaman, Mechanism of Hydrolysis of Native and Cooked Starches from Different Botanical Sources in the Presence of Tea Extracts. J. Food Sci. 77 (11), C1192–C1196 (2012). https://doi.org/https://doi.org/10.1111/j.1750-3841.2012.02929.x Q. Hu, L. Jiang, Q. Yan, J. Zeng, X. Ma, Y. Zhao, A natural products solution to diabetic nephropathy therapy. Pharmacol. Ther. 241 , 108314 (2023). https://doi.org/https://doi.org/10.1016/j.pharmthera.2022.108314 Y. Huang, P. Wu, J. Ying, Z. Dong, X.D. Chen, Mechanistic study on inhibition of porcine pancreatic α-amylase using the flavonoids from dandelion. Food Chem. 344 , 128610 (2021). https://doi.org/https://doi.org/10.1016/j.foodchem.2020.128610 C.G. Kato-Schwartz, R.C.G. Corrêa, de D. Souza Lima, de A.B. Sá-Nakanishi, de G. Almeida Gonçalves, F.A.V. Seixas, R.M. Peralta, Potential anti-diabetic properties of Merlot grape pomace extract: An in vitro, in silico and in vivo study of α-amylase and α-glucosidase inhibition. Food Res. Int. 137 , 109462 (2020). https://doi.org/https://doi.org/10.1016/j.foodres.2020.109462 R. Khanra, S. Dewanjee, K. Dua, T. Sahu, R. Gangopadhyay, M. De Feo, V., M. Zia-Ul-Haq, Abroma augusta L. (Malvaceae) leaf extract attenuates diabetes induced nephropathy and cardiomyopathy via inhibition of oxidative stress and inflammatory response. J. Translational Med. 13 (1), 6 (2015). https://doi.org/10.1186/s12967-014-0364-1 K. Khunti, M.L. Wolden, B.L. Thorsted, M. Andersen, M.J. Davies, Clinical Inertia in People With Type 2 Diabetes: A retrospective cohort study of more than 80,000 people. Diabetes Care. 36 (11), 3411–3417 (2013). https://doi.org/10.2337/dc13-0331 N. Laiteerapong, A.S. Cifu, Screening for Prediabetes and Type 2 Diabetes Mellitus. JAMA 315 (7), 697–698 (2016). https://doi.org/10.1001/jama.2015.17545 K. Li, F. Yao, J. Du, X. Deng, C. Li, Persimmon Tannin Decreased the Glycemic Response through Decreasing the Digestibility of Starch and Inhibiting α-Amylase, α-Glucosidase, and Intestinal Glucose Uptake. J. Agric. Food Chem. 66 (7), 1629–1637 (2018). https://doi.org/10.1021/acs.jafc.7b05833 X. Li, J. Cai, J. Yu, S. Wang, L. Copeland, S. Wang, Inhibition of in vitro enzymatic starch digestion by coffee extract. Food Chem. 358 , 129837 (2021). https://doi.org/https://doi.org/10.1016/j.foodchem.2021.129837 H. Liu, S. Li, Y. Zhong, D. Wu, Inhibition mechanism analysis and molecular simulation of rhein on α-amylase. Mod. Food Sci. Technol. 31 (02), 47–51 (2015). (in Chinese). https://doi.org/10.13982/j.mfst.1673-9078.2015.2.009 E. Lo Piparo, H. Scheib, N. Frei, G. Williamson, M. Grigorov, C.J. Chou, Flavonoids for Controlling Starch Digestion: Structural Requirements for Inhibiting Human α-Amylase. J. Med. Chem. 51 (12), 3555–3561 (2008). https://doi.org/10.1021/jm800115x B.D. Maibam, S. Chakraborty, C. Nickhil, S.C. Deka, Effect of Euryale ferox seed shell extract addition on the in vitro starch digestibility and predicted glycemic index of wheat-based bread. Int. J. Biol. Macromol. 226 , 1066–1078 (2023). https://doi.org/https://doi.org/10.1016/j.ijbiomac.2022.11.223 A.J. Miles, E.D. Drew, B.A. Wallace, DichroIDP: a method for analyses of intrinsically disordered proteins using circular dichroism spectroscopy. Commun. Biology. 6 (1), 823 (2023). https://doi.org/10.1038/s42003-023-05178-2 A.J. Miles, R.W. Janes, B.A. Wallace, Tools and methods for circular dichroism spectroscopy of proteins: a tutorial review. Chem. Soc. Rev. 50 (15), 8400–8413 (2021). https://doi.org/10.1039/D0CS00558D R. Moradi-Marjaneh, M. Paseban, A. Sahebkar, Natural products with SGLT2 inhibitory activity: Possibilities of application for the treatment of diabetes. Phytother. Res. 33 (10), 2518–2530 (2019). https://doi.org/https://doi.org/10.1002/ptr.6421 M. Mrázková, D. Sumczynski, J. Orsavová, (2023). Influence of Storage Conditions on Stability of Phenolic Compounds and Antioxidant Activity Values in Nutraceutical Mixtures with Edible Flowers as New Dietary Supplements. Antioxidants, 12 (4), 962. https://www.mdpi.com/2076-3921/12/4/962 D.J. Newman, Natural products and drug discovery. Natl. Sci. Rev. 9 (11) (2022). https://doi.org/10.1093/nsr/nwac206 M. Oroian, F. Ursachi, F. Dranca, Ultrasound-Assisted Extraction of Polyphenols from Crude Pollen. Antioxidants. 9 (4), 322 (2020). https://www.mdpi.com/2076-3921/9/4/322 J.M. Pappachan, C.J. Fernandez, E.C. Chacko, Diabesity and antidiabetic drugs. Mol. Aspects Med. 66 , 3–12 (2019). https://doi.org/https://doi.org/10.1016/j.mam.2018.10.004 J.M. Pappachan, A.K. Viswanath, Medical Management of Diabesity: Do We Have Realistic Targets? Curr. Diab. Rep. 17 (1), 4 (2017). https://doi.org/10.1007/s11892-017-0828-9 P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry. 20 (11), 3096–3102 (1981). https://doi.org/10.1021/bi00514a017 S. Roy, R.K. Nandi, S. Ganai, K.C. Majumdar, T.K. Das, Binding interaction of phosphorus heterocycles with bovine serum albumin: A biochemical study. J. Pharm. Anal. 7 (1), 19–26 (2017). https://doi.org/https://doi.org/10.1016/j.jpha.2016.05.009 S. Shobana, Y.N. Sreerama, N.G. Malleshi, Composition and enzyme inhibitory properties of finger millet (Eleusine coracana L.) seed coat phenolics: Mode of inhibition of α-glucosidase and pancreatic amylase. Food Chem. 115 (4), 1268–1273 (2009). https://doi.org/https://doi.org/10.1016/j.foodchem.2009.01.042 L. Sun, Y. Guo, C. Fu, J. Li, Z. Li, Simultaneous separation and purification of total polyphenols, chlorogenic acid and phlorizin from thinned young apples. Food Chem. 136 (2), 1022–1029 (2013). https://doi.org/https://doi.org/10.1016/j.foodchem.2012.09.036 L. Sun, M. Miao, Dietary polyphenols modulate starch digestion and glycaemic level: a review. Crit. Rev. Food Sci. Nutr. 60 (4), 541–555 (2020). https://doi.org/10.1080/10408398.2018.1544883 J.-L. Tian, M. Zhao, J.-Y. Xu, T.-M. Lv, X.-C. Liu, S. Sun, P. Hu, Inhibitory Mechanism of Prenylated Flavonoids Isolated from Mulberry Leaves on α-Glucosidase by Multi-Spectroscopy and Molecular Dynamics Simulation. J. Agric. Food Chem. 71 (23), 9135–9147 (2023). https://doi.org/10.1021/acs.jafc.3c00776 X. Wu, M. Hu, X. Hu, H. Ding, D. Gong, G. Zhang, Inhibitory mechanism of epicatechin gallate on α-amylase and α-glucosidase and its combinational effect with acarbose or epigallocatechin gallate. J. Mol. Liq. 290 , 111202 (2019). https://doi.org/https://doi.org/10.1016/j.molliq.2019.111202 J. Xu, J. Cao, J. Yue, X. Zhang, Y. Zhao, New triterpenoids from acorns of Quercus liaotungensis and their inhibitory activity against α-glucosidase, α-amylase and protein-tyrosine phosphatase 1B. J. Funct. Foods. 41 , 232–239 (2018). https://doi.org/https://doi.org/10.1016/j.jff.2017.12.054 J. Xu, W. Wang, Y. Li, Dough properties, bread quality, and associated interactions with added phenolic compounds: A review. J. Funct. Foods. 52 , 629–639 (2019). https://doi.org/https://doi.org/10.1016/j.jff.2018.11.052 B. Xue, J. Tian, Y. Wang, B. Jin, H. Deng, N. Gao, B. Li, Mechanism underlying the interaction of malvidin-3-O-galactoside with protein tyrosine phosphatase-1B and α-glucosidase. J. Mol. Struct. 1253 , 132249 (2022). https://doi.org/https://doi.org/10.1016/j.molstruc.2021.132249 J. Yan, G. Zhang, J. Pan, Y. Wang, α-Glucosidase inhibition by luteolin: Kinetics, interaction and molecular docking. Int. J. Biol. Macromol. 64 , 213–223 (2014). https://doi.org/https://doi.org/10.1016/j.ijbiomac.2013.12.007 S. Zhang, Y. Zhang, E. Karrar, Q. Jin, H. Zhang, G. Wu, X. Wang, Mechanisms of sesamol and sesamin inhibiting α-glucosidase activity by spectroscopy and molecular docking. Food Bioscience. 53 , 102680 (2023). https://doi.org/https://doi.org/10.1016/j.fbio.2023.102680 J. Zhu, B. Zhang, B. Wang, C. Li, X. Fu, Q. Huang, In-vitro inhibitory effects of flavonoids in Rosa roxburghii and R. sterilis fruits on α-glucosidase: Effect of stomach digestion on flavonoids alone and in combination with acarbose. J. Funct. Foods. 54 , 13–21 (2019). https://doi.org/https://doi.org/10.1016/j.jff.2019.01.009 Tables Table 1 Reaction kinetic parameters, changes in enthalpy, entropy and free energy of extract on α-amylase and α-glucosidase at different temperatures T/K K sv (L/g) K q (L/(g·s)) n K a K ΔH (KJ/mol) ΔS (J/(mol·K)) ΔG (KJ/mol) α-amylase 298 5.03 5.03×10 8 1.1094 4.27 2.4499 8.153 39.43 -3.597 310 5.50 5.50×10 8 1.122 4.850 2.6044 -4.070 α-glucosidase 298 13.531 1.35×10 9 1.619 18.69 3.2929 16.41 79.40 -7.26 310 17.462 1.75×10 9 1.637 24.15 3.5795 -8.21 Additional Declarations No competing interests reported. Supplementary Files AppendixA.Supplementarydata.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 14 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers invited by journal 24 Apr, 2026 Editor assigned by journal 23 Apr, 2026 Submission checks completed at journal 23 Apr, 2026 First submitted to journal 22 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9496475","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634315513,"identity":"052cda72-1e39-441f-a61d-ffe9b76c891d","order_by":0,"name":"Zixuan 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1","display":"","copyAsset":false,"role":"figure","size":116902,"visible":true,"origin":"","legend":"\u003cp\u003eThe digestion rate of wheat starch in vitro (A), and the inhibitory effect of acorn shells extract on the α-amylase (B) and α-glucosidase (C).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9496475/v1/44f102f6963124d21a1974d5.png"},{"id":108489008,"identity":"449efe83-9fd5-46ab-ad9c-842c5d577441","added_by":"auto","created_at":"2026-05-05 09:26:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155965,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory type of extract on α-amylase (inset of A) and α-glucosidase (inset of D) , Lineweaver-Burk plot for inhibition of extract on α-amylase (A~C) and α-glucosidase (D~F).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9496475/v1/92fb7cf8373fb51b0537e8d6.png"},{"id":108488992,"identity":"6fc2113f-64bc-4b3a-8f41-ab1e1babc8a8","added_by":"auto","created_at":"2026-05-05 09:26:42","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":174228,"visible":true,"origin":"","legend":"\u003cp\u003eStern-Volmer plots, Logarithmic curve and Natural logarithm curve for the fluorescence quenching of α-amylase (A~C) and α-glucosidase (D~F).\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9496475/v1/412f37844fca0c179eb2a055.jpeg"},{"id":108804540,"identity":"d3b5de4d-a12f-4117-aaff-b20eb104fc67","added_by":"auto","created_at":"2026-05-08 15:21:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":104818,"visible":true,"origin":"","legend":"\u003cp\u003eCircular dichroism of different concentrations of extract on α-amylase (A) and α-glucosidase (B).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9496475/v1/133045766fa8a741b707035e.png"},{"id":108488987,"identity":"d323f325-4685-446b-9ee1-7b044639c19e","added_by":"auto","created_at":"2026-05-05 09:26:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":125249,"visible":true,"origin":"","legend":"\u003cp\u003eThe postprandial blood glucose level in normal mice after oral administrated starch (A) or glucose (C), area under curve after orally administration test with starch (B) or glucose (D) for 2 h.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9496475/v1/e65389ed4f1842a7e6297d96.png"},{"id":108809111,"identity":"9aec666c-8531-46ba-b82d-7e62333d234f","added_by":"auto","created_at":"2026-05-08 15:50:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":915494,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9496475/v1/58f9cafd-1ce9-4161-a855-0037b113335d.pdf"},{"id":108488997,"identity":"d19203a7-4bd2-41c7-83eb-a49ffc4bff7a","added_by":"auto","created_at":"2026-05-05 09:26:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":250977,"visible":true,"origin":"","legend":"","description":"","filename":"AppendixA.Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-9496475/v1/ee1347d57a14301061705705.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Acorn (Quercus variabilis BL.) shells extract alleviating postprandial blood glucose via α-amylase and α-glucosidase inhibition and starch digestion resistance","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDiabetes is a metabolic disease characterized by chronic hyperglycemia caused by multiple factors, which has been spreading worldwide (Pappachan, Fernandez, \u0026amp; Chacko, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Balancing optimal glycemic control with the most appropriate diabesity management regime should be the priority for clinicians in choosing the antidiabetic agent (Deol, Lekkakou, Viswanath, \u0026amp; Pappachan, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pappachan et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pappachan \u0026amp; Viswanath, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), including lifestyle changes, medication therapies, and surgery (Laiteerapong \u0026amp; Cifu, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Despite the widening choice of oral and injectable antihyperglycemic agents to treat type 2 diabetes (T2DM) and the availability of long- and fast-acting insulin analogues, many people struggled to achieve recommended glycemic targets and were at risk of developing long-term micro- and macrovascular complications. (Daly \u0026amp; Hovorka, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Khunti, Wolden, Thorsted, Andersen, \u0026amp; Davies, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNatural products with low toxicity have been explored as drug sources for a long history to treat infectives, cancer, hypertension and diabetes (Newman, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The potential effects of experimental research and clinical applications have been verified. (Hu, Jiang, Yan, Zeng, Ma, \u0026amp; Zhao, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Compared with the synthetic drugs, the treatment benefits of natural products were attributed to multi-components and multi-targets to produce combined or synergistic effects. Nowadays, multi-components and multi-targets therapies have been proved to be more effective and less toxicity than traditional single-target drugs (Espinoza-Fonseca, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). There were various extracts used for the T2DM treatment, such as \u003cem\u003eAnnona muricata\u003c/em\u003e aqueous extract was daily administrated to diabetic rats for 28 days, the blood glucose levels and serum creatinine were reduce (Florence et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e); the extract of apple trees bark (Moradi-Marjaneh, Paseban, \u0026amp; Sahebkar, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) was used to decrease blood glucose by the inhibition of SGLT2; and \u003cem\u003eAbroma augusta\u003c/em\u003e L. (Malvaceae) leaf extract could be considered as a kind of prophylactic agent against T2DM (Khanra et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Phytochemical investigations revealed the presence of tannins, flavonoids, terpenoids and phenolic compounds from all of these extracts.\u003c/p\u003e \u003cp\u003ePolyphenols widely distributed in natural products tissues as secondary metabolites, which had been proved effectively impede the activity of the starch digestion enzymes to diminish the amylolysis. α-amylase and α-glucosidase, which were the key enzymes located in the cell membrane of small intestine for digestion and degradation of starch compounds, played crucial roles in the metabolism of carbohydrate and the maintenance of normal physiological functions (Li et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, inhibition of α-amylase and α-glucosidase had become an important target for the research and treatment of diabetes based on the factor that hydrolysis of carbohydrate after meal was the main pathway of blood glucose increase. It is of great significance to find a digestive enzyme inhibitor with high activity and derived from natural plants for the treatment of diabetes.\u003c/p\u003e \u003cp\u003eAcorn shells were rich in polyphenols and the extract exhibited in vitro α-amylase and α-glucosidase inhibitory activities (G\u0026uuml;venalp, Yuca, G\u0026ouml;zc\u0026uuml;, Dursunoğlu, Tosun, \u0026amp; Demirezer, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), but it was still unclear that if the binding of the extract with the enzymes and starch affect their hypoglycemic activity and starch digestion, which was crucial important for the development of more powerful antidiabetic drugs and efficacious utilization of acorn shells. Thus, the inhibition of the extract on α-amylase and α-glucosidase activity were investigated and proposed a reasonable inhibition kinetics in this study. Following, the inhibition mechanism of the extract was analyzed during the interaction with the enzymes using fluorescence quenching and circular dichroism spectra. Furthermore, whether acorn shells extract could interact with starch were examined by in vitro starch digestibility. Finally, the effect mechanism of the extract on the hypoglycemic was discussed based on the starch tolerance and glucose tolerance were verified by animal experiments. Our work might provide a perspective regarding the application of acorn shells extract in functional starch products.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of acorn shells extract\u003c/h2\u003e \u003cp\u003e \u003cem\u003eQuercus variabilis\u003c/em\u003e acorn shells were plucked from an experimental field at Northwest Agriculture and Forestry University, dried at 50\u0026deg;C for 24 h and crushed into powders. The acorns shells powders were used to extract polyphenols as the protocol previously described by (Oroian, Ursachi, \u0026amp; Dranca, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sun, Guo, Fu, Li, \u0026amp; Li, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with slight modifications. Firstly, the powders were extracted with 95% ethyl alcohol in a proportion of 1:5 (w/v) under ultrasound at 40\u0026deg;C, 300 W for 30 min, followed by filtration to gather the remainder to repeatedly extracted twice. Subsequently, the ethanol-extract were concentrated by a vacuum rotary evaporator at 45\u0026deg;C,0.09 Mpa (LGJ-100, Beijing Sihuan Instrument Co., Ltd., China), and dried with Freeze Dryer (LGJ-10) at -80\u0026deg;C for 8 h to obtain the acorn shells extract. The chemical composition of the extract was analyzed by HRLC-TOF-MS (Appendix Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Inhibition assays of α-amylase and α-glucosidase\u003c/h2\u003e \u003cp\u003eThe inhibition assay of α-amylase was adapted from Shen et al. (2012). Briefly, 30 \u0026micro;L the extract (0.1\u0026thinsp;~\u0026thinsp;2.0 mg/mL, diluted with the sodium phosphate buffer, PBS, pH 6.8) were thoroughly mixed with 30 \u0026micro;L α-amylase suspension (0.148 U/mL, diluted in PBS). After the mixture was incubated at 37\u0026deg;C for 10 min, 60 \u0026micro;L wheat starch solution (2%, w/v, gelatinized at 98\u0026deg;C for 20 min) was added and reacted at 37\u0026deg;C for 10 min. The reaction was stopped by the addition of 120 \u0026micro;L 3,5-dinitrosalicylic acid reagent (DNS) in a boiling water bath for 5 min, followed by cooling to 25\u0026deg;C. Subsequently, the reaction products were transferred into 96-well microplates for absorbance measurements at 540 nm using an enzyme-labeled instrument (Victor X3, PerkinElmer Enterprise Management Co., Ltd., Shanghai, China). Acarbose was used as a positive control.\u003c/p\u003e \u003cp\u003eα-glucosidase activity assay was performed according to (Zhu, Zhang, Wang, Li, Fu, \u0026amp; Huang, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Briefly, 20 \u0026micro;L α-glucosidase (1.5 U/mL) and 20 \u0026micro;L the extract (0.01\u0026thinsp;~\u0026thinsp;1.5 mg/mL) were mixed in 20 \u0026micro;L PBS and incubated at 37\u0026deg;C for 30 min. And then 20 \u0026micro;L PNPG (5 mmol/L) was added to react for 10 min prior to terminating the reaction with 150 \u0026micro;L Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (1mol/L). The absorbance of the mixture was recorded at 405nm. Acarbose was used as a positive control. The inhibition rates of α-amylase and α-glucosidase were calculated using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{I}\\text{n}\\text{h}\\text{i}\\text{b}\\text{i}\\text{t}\\text{i}\\text{o}\\text{n}\\text{r}\\text{a}\\text{t}\\text{e}\\:\\left(\\text{%}\\right)=\\left[1-\\frac{\\left({\\text{A}}_{\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}-{\\text{A}}_{\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\:\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}}\\right)}{\\left({\\text{A}}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}-{\\text{A}}_{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}}\\right)}\\right]\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere A\u003csub\u003econtrol\u003c/sub\u003e, A\u003csub\u003econtrol blank\u003c/sub\u003e, A\u003csub\u003esample\u003c/sub\u003e, A\u003csub\u003esample blank\u003c/sub\u003e represent the absorbance values of the sample with enzymes and PBS, PBS, extract with enzymes and PBS, extract with PBS, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Inhibition Kinetic of α-amylase and α-glucosidase\u003c/h2\u003e \u003cp\u003eThe inhibitory type of the acorn shells extract (0.5 mg/mL) against α-amylase and α-glucosidase were determined by varying the concentrations of the enzymes (0, 10, 20, 40, 50 \u0026micro;g/mL). The α-amylase and α-glucosidase inhibition test were carried out as the methods described above (Section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e). The kinetic of α-amylase and α-glucosidase was carried out according to the previous method (Huang, Wu, Ying, Dong, \u0026amp; Chen, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) with a slight modification. Michaelis-Menten constants were determined via varying the concentrations of substrates (wheat starch: 0.5%, 0.75%, 1.0%, 1.25%, 1.5%; PNPG: 1, 2, 4, 5, 10 mmol/L) and extract (0, 0.2, 0.5 mg/mL for α-amylase; 0, 0.1, 0.2 mg/mL for α-glucosidase). Subsequently, Lineweaver-Burk double inverse curves were plotted based on the enzymatic reaction rates of α-amylase and α-glucosidase. Finally, the kinetic constants of inhibition were calculated using the following equation:\u003c/p\u003e \u003cp\u003eThe kinetic equation:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:V=\\frac{{V}_{m}\u0026middot;\\left[S\\right]}{{K}_{m}\\left(1+\\frac{I}{{K}_{i}}\\right)+\\left[S\\right]\\left(1+\\frac{I}{{K}_{is}}\\right)}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ePlotted as 1/V against 1/[S], a double inverse equation:\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\frac{1}{V}=\\frac{{K}_{m}}{{V}_{m}}\\left(1+\\frac{I}{{K}_{i}}\\right)\\frac{1}{\\left[S\\right]}+\\frac{1}{{V}_{m}}\\left(1+\\frac{I}{{K}_{is}}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:Slope=\\frac{{K}_{m}}{{V}_{m}}\\left(1+\\frac{\\left[I\\right]}{{K}_{i}}\\right)=\\frac{{K}_{m}}{{V}_{m}}+\\frac{{K}_{m}\\left[I\\right]}{{V}_{m}{K}_{i}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ5\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ5\" name=\"EquationSource\"\u003e\n$$\\:Intercept=\\frac{1}{{V}_{m}}\\left(1+\\frac{\\left[I\\right]}{{K}_{is}}\\right)=\\frac{1}{{V}_{m}}+\\frac{\\left[I\\right]}{{V}_{m}{K}_{is}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere V is the initial reaction speed, V\u003csub\u003emax\u003c/sub\u003e is the maximum initial reaction speed, S is the substrate concentration, I is the inhibitor concentration, K\u003csub\u003em\u003c/sub\u003e is the Michaelis constant, K\u003csub\u003ei\u003c/sub\u003e is the competitive inhibition constant, and K\u003csub\u003eis\u003c/sub\u003e is the noncompetitive inhibition constant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Fluorescence quenching assay\u003c/h2\u003e \u003cp\u003eFluorescence spectra were recorded using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) according to the previously reported method (X. Li, Cai, Yu, Wang, Copeland, \u0026amp; Wang, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The α-amylase (0.148 U/mL, 3.0 mL) or α-glucosidase (1.5 U/mL, 3.0 mL) were mixed with 3 mL the extract solution (0, 0.001, 0.05, 0.1,0.2, 0.3, 0.4 and 0.5 mg/mL) and incubated for 5 min at 298.15 K and 310.15 K. Equivalent PBS was used as the blank control. The fluorescence of the enzymes was measured upon excitation at 280 nm and the emission wavelengths ranging from 300 to 500 nm, with the 10.0 nm slit width and the scanning speed at 240 nm/min. The quenching constant K\u003csub\u003esv\u003c/sub\u003e was calculated using the Stern-Volmer equation:\u003cdiv id=\"Equ6\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ6\" name=\"EquationSource\"\u003e\n$$\\:\\frac{{F}_{0}}{F}=1+{K}_{sv}\\left[Q\\right]=1+{K}_{q}{\\tau\\:}_{0}\\left[Q\\right]$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere F\u003csub\u003e0\u003c/sub\u003e and F are the fluorescence intensities of enzyme with and without the extract, respectively. τ\u003csub\u003e0\u003c/sub\u003e is the lifetime of fluorophore. K\u003csub\u003esv\u003c/sub\u003e is the Stern-Volmer quenching constant (K\u003csub\u003esv\u003c/sub\u003e = K\u003csub\u003eq\u003c/sub\u003eτ\u003csub\u003e0\u003c/sub\u003e). [Q] is the concentration of the quencher (extract). K\u003csub\u003eq\u003c/sub\u003e is the quenching rate constant of the biomolecule. τ\u003csub\u003e0\u003c/sub\u003e is the average lifetime of enzyme fluorophore in the absence of quencher and was taken as 1 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e s.\u003c/p\u003e \u003cp\u003eStatic quencher equation:\u003cdiv id=\"Equ7\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ7\" name=\"EquationSource\"\u003e\n$$\\:\\text{log}\\left(\\frac{{F}_{0}-F}{F}\\right)=\\text{log}{K}_{a}+n\\text{log}\\left[\\:Q\\right]$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e7\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere n is the number of binding sites and K\u003csub\u003ea\u003c/sub\u003e is the static binding constant. The slope of the straight line is n and the vertical axis intercept is logK\u003csub\u003ea\u003c/sub\u003e when log[(F\u003csub\u003e0\u003c/sub\u003e-F)/F] is plotted against log[Q].\u003c/p\u003e \u003cp\u003eThe intermolecular forces between the extract and the enzyme can be determined by the van't-Hoff equation:\u003cdiv id=\"Equ8\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ8\" name=\"EquationSource\"\u003e\n$$\\:\\varDelta\\:{\\rm\\:H}=\\frac{\\text{ln}\\left(\\frac{{K}_{a2}}{{K}_{a1}}\\right)\\times\\:R}{\\frac{1}{{T}_{1}}-\\frac{1}{{T}_{2}}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e8\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ9\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ9\" name=\"EquationSource\"\u003e\n$$\\:\\varDelta\\:G=\\varDelta\\:{\\rm\\:H}-{\\rm\\:T}\\varDelta\\:S$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e9\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eCalculate the Gibbs free energy change (ΔG) from Eq.\u0026nbsp;(\u003cspan refid=\"Equ11\" class=\"InternalRef\"\u003e11\u003c/span\u003e) to determine the spontaneity of the reaction.\u003cdiv id=\"Equ10\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ10\" name=\"EquationSource\"\u003e\n$$\\:\\varDelta\\:G=-R{\\rm\\:T}ln{K}_{a}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e10\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere K\u003csub\u003ea1\u003c/sub\u003e and K\u003csub\u003ea2\u003c/sub\u003e are the binding constants at different reaction temperatures T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e. R is the atmospheric constant (8.314 K/(J\u0026middot;mol)).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Circular dichroism\u003c/h2\u003e \u003cp\u003eCircular dichroism was measured according to the previously reported method (Wu, Hu, Hu, Ding, Gong, \u0026amp; Zhang, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Equal volume of α-amylase (0.148 U/mL, 400 \u0026micro;L) or α-glucosidase (1.5 U/mL, 400 \u0026micro;L) mixed with the different concentrations of the acorn shells extract (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 mg/mL). After incubation at 37\u0026deg;C for 15 min, the circular dichroic spectrum of the mixture was measured using a spectrometer (Chirascan V100, Applied Optical Physics, UK) with a 1.0 mm path length cuvette and a scan speed of 120 nm/min in the range of 190\u0026thinsp;~\u0026thinsp;260 nm. PBS served as the blank control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 In Vitro Starch Digestion\u003c/h2\u003e \u003cp\u003e The starch digestion in vitro was measured according to a modified version of Englyst\u0026rsquo;s method. The wheat starch (1 g, dw) with different proportion of acorn shells extract (0%, 5%, 10% and 15%, w/w) were dispersed in 50 mL of 0.2 mol/L sodium acetate buffer (pH 5.2) and cooked in a boiling water bath for 30 min, then cooled to 37\u0026deg;C. After that, 6 mL starch-extract mixture were enzymolized by 1 mL preincubated enzymes solution (α-amylase and α-glucosidase mixed in a proportion of 4:1 \u0026micro;g/mL) at 37\u0026deg;C for 120 min. During enzymatic hydrolysis, 1 mL enzymatic hydrolysate were collected at 0, 40, 60, 90, and 120 min, respectively, and put in an ice water bath. The cooling hydrolysate were centrifuged at 3500 \u003cem\u003eg\u003c/em\u003e for 10 min. The supernatant was used to analyze the glucose content using DNS colorimetry.\u003cdiv id=\"Equ11\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ11\" name=\"EquationSource\"\u003e\n$$\\:\\text{D}\\text{i}\\text{g}\\text{e}\\text{s}\\text{t}\\text{i}\\text{b}\\text{i}\\text{l}\\text{i}\\text{t}\\text{y}\\:\\left(\\text{%}\\right)=\\frac{\\text{m}}{M}\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e11\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere m is the glucose content, and M is the starch content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Postprandial glycemic response measurement\u003c/h2\u003e \u003cp\u003eAnimal experiments were executed according to the guidelines of the institutional animal ethics committee and were supported by the institutional animal committee of Northwest A\u0026amp;F University (XN2023-0605). The male mice (C57BL/6J, 7 weeks old, n\u0026thinsp;=\u0026thinsp;24; SPF (Beijing) Biotechnology Co., Ltd, China) of similar weight were randomly selected for measuring postprandial glycemic response measurement. The temperature and the relative humidity were set at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, respectively. Food and water were freely available. After a week of adjustable feeding, mice were randomly assigned to 3 groups (eight mice per group): positive control group (100 mg/kg acarbose\u0026thinsp;+\u0026thinsp;2 g/kg starch or glucose), acorn shells extract group (100 mg/kg extract\u0026thinsp;+\u0026thinsp;2 g/kg starch or glucose), negative control group (same volume saline\u0026thinsp;+\u0026thinsp;2 g/kg starch or glucose). The interval between the two tolerance tests was one week, fasting overnight before the test, as well as the interval between administration and starch (or glucose) was 15 min. Blood samples from the lateral tail vein were collected at 0, 30, 45, 60, 90, and 120 min and measured using a glucose analyzer (Sano Biosensing Co., Ltd., China) to obtain the glycemic value and the area under the glycemic curve were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the values were expressed as means\u0026plusmn;standard deviation (means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of three replications. Statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS (version 16.0). p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistically significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Inhibition effect of acorn shells extract against enzymes\u003c/h2\u003e \u003cp\u003eInhibition of digestion enzymes was one of the most effective approaches for postprandial glycemic control, which was also believed to be the most important ways of polyphenols to exert their glycemic index (GI) reducing effect in vivo (K. Li et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). α-amylase and α-glucosidase were the main digestive enzymes involved in the hydrolysis of dietary starch (Barrett et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, we plotted the inhibition curve of the acorn shells extract against α-amylase and α-glucosidase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C), showing that the inhibition rates of both α-amylase and α-glucosidase increased dramatically with the increase of the acorn shells extract concentration. The IC\u003csub\u003e50\u003c/sub\u003e values of the acorn shells extract for α-amylase and α-glucosidase were 0.352 mg/mL and 0.0158 mg/mL, respectively. Surprisingly, the extract had more excellent inhibition on α-glucosidase than acarbose (IC\u003csub\u003e50\u003c/sub\u003e=0.024 mg/mL). According to the previous reports, the more powerful inhibition of the acorn shells extract against α-glucosidase was attributed to its own galloyl substituent on triterpene derivative (Jing Xu, Cao, Yue, Zhang, \u0026amp; Zhao, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, inhibitory strength was also related to the formation of hydrogen bonds between hydroxyl groups of polyphenols and amino acid residues in the active sites of the enzyme (Lo Piparo, Scheib, Frei, Williamson, Grigorov, \u0026amp; Chou, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, the influencing mechanism of the extract binding to the enzymes were further discussed as follow.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Inhibition kinetics of acorn shells extract against enzymes\u003c/h2\u003e \u003cp\u003eThe reversibility was determined by plotting the change curves of absorbance of reaction product against the concentrations of α-amylase or α-glucosidase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), because all the curves passed through the origin (Feng et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, the reversible inhibition revealed that the acorn shells extract binds to α-amylase or α-glucosidase through a non-covalent bond, and the enzyme activity could return to normal after the extract was removed (Zhang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe types of reversible inhibition, including competitive, uncompetitive, anti-competitive and mixed inhibition, were required to further discriminate by obtaining the Lineweaver-Burk plot to calculate the corresponding inhibition constants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table S2). All lines intersected at the third quadrant after linear fitting, while not on the x or y axis; and the slope of these lines could gradually rose with increasing concentrations of the extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and D), implying that the inhibition type didn\u0026rsquo;t belong to competitive or uncompetitive. Both K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003em\u003c/sub\u003e decreased in the reaction system of α-amylase or α-glucosidase with the increasing concentration of the extract, indicating that the extract bound to both the enzyme and to the enzyme-substrate complex, that was, mixed inhibition. The extract from different sources had different inhibition types on α-amylase or α-glucosidase: the grape pomace extract bound to both free α-amylase and α-amylase -substrate complex, which was consistent with the acorn shells extract above (Kato-Schwartz et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); the extract from finger millet (Eleusine coracana L.) seed coat inhibition reaction type was non-competitive inhibition (Shobana, Sreerama, \u0026amp; Malleshi, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), which bound to the enzyme molecule outside the substrate binding site and does not affect the binding of the enzyme to the substrate; the inhibition of rhein on α-amylase was proved a competitive inhibition, which competing with the substrate for the active site of the enzyme (Liu at al., 2015). The reason for the different inhibitory effect were possibly related to differences in molecular structure and composition of phytochemical identities (Feng et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lo Piparo et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). By further calculating and comparing the inhibition constants (appendix Table S2), it was found that K\u003csub\u003eis\u003c/sub\u003e values of both α-amylase and α-glucosidase were less than K\u003csub\u003ei\u003c/sub\u003e values, suggesting that the inhibition capacity on enzyme-substrate complex was stronger than enzyme. Namely, the extract bound to the enzyme-substrate complex because of uncompetitive inhibition prior to the enzymes due to the competitive inhibition, ultimately to form a more stable extract-enzyme-substrate ternary complex, which impeded the intermediate ESI (enzyme-substrate-inhibitor) not be decomposed into products, but also occupied the active center of the enzymes reversibly, thereby inhibiting the activity of the enzymes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Fluorescence quenching of acorn shells extract against enzymes\u003c/h2\u003e \u003cp\u003eIn order to reveal the interaction details of α-amylase or α-glucosidase with the acorn shells extract, fluorescence intensities (appendix Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were obtained to assess the binding affinity and the conformational changes of the enzymes. Tryptophan (Try) residues displayed an inherent fluorescence excitation peak in α-amylase and α-glucosidase (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;280nm), which was the high sensitivity to its local environment, thus could be utilized as an indicator of binding interactions (Abdollahi, Ince, Condict, Hung, \u0026amp; Kasapis, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The intensity of the intrinsic fluorescence of the enzymes was gradually quenched with the increasing addition of the extract (0\u0026thinsp;~\u0026thinsp;0.5 mg/mL), until disappeared completely for α-glucosidase at 0.5 mg/mL extract. The fluorescence quenching of the Try residues was probably attributed to the interaction with compounds in the acorn shells extract through hydrogen bonds or hydrophobic forces (Fan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, the maximum emission (λ\u003csub\u003eem\u003c/sub\u003e) of α-amylase and α-glucosidase shifted to lower wavelength (blue shift) with increasing addition of the extract (appendix Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The blue shift indicated alterations to secondary structure of the enzymes with the extract addition since the enhancement of hydrophobicity around Try fluorescent carriers, and Try residues were moving to a less polar environment closer to the edge of the protein molecule.\u003c/p\u003e \u003cp\u003eFurther analysis of the quenching mechanism of the extract with two enzymes by Stern Volmer kinetic equation. The fluorescence quenching Stern-Volmer equation curves of acorn shells extract on α-amylase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) or α-glucosidase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) were not linear, but quadratic curves concaved toward the y-axis, which indicated that both static and dynamic quenching existed between the extract and the enzymes. By investigating the impact of temperature on the fluorescence quenching, both the fluorescence quenching constant (K\u003csub\u003esv\u003c/sub\u003e) and the fluorescence quenching rate constant (K\u003csub\u003eq\u003c/sub\u003e) were growing with the increasing temperature from 298 K to 310 K. However, K\u003csub\u003eq\u003c/sub\u003e were lower than the maximum biomolecular collision quenching constant (2\u0026times;10\u003csup\u003e10\u003c/sup\u003e L\u0026middot;(mol\u0026middot;s)). These results implied that the fluorescence quenching process of α-amylase or α-glucosidase by acorn shells extract was dominated by dynamic quenching on account of intermolecular collisions. (Roy, Nandi, Ganai, Majumdar, \u0026amp; Das, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As a hypothetical dynamic quenching process, the data of fluorescence intensities were further transferred to draft the double logarithmic curve and natural logarithmic (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C, E and F) to obtain the values of n and K\u003csub\u003ea\u003c/sub\u003e. With the rise of temperature, the binding site (n) of α-amylase was exceed 1 but of α-glucosidase was getting closer to 2 and the static binding constant (K\u003csub\u003ea\u003c/sub\u003e) of α-glucosidase also increased more obviously, indicating the larger values of n and K\u003csub\u003ea\u003c/sub\u003e correspond to tighter and stronger binding. Furthermore, the interaction between the extract and the enzymes were no distance effect due to the binding constant (K) above K\u003csub\u003ea\u003c/sub\u003e. To sum up, the temperature had an enhancing effect on the affinity between the extract and the enzymes, the extract was more inclined to combined with α-glucosidase resulting in less value of IC\u003csub\u003e50\u003c/sub\u003e for α-glucosidase than α-amylase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Thermodynamic parameters and the nature of the binding forces\u003c/h2\u003e \u003cp\u003eThermodynamic parameters were calculated using the van't-Hoff Eq.\u0026nbsp;(\u003cspan refid=\"Equ8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u0026thinsp;~\u0026thinsp;10) to characterize the interaction forces between the extract and α-amylase or α-glucosidase (Table\u0026nbsp;3). Based on the calculated (ΔG\u0026thinsp;\u0026lt;\u0026thinsp;0, ΔH\u0026thinsp;\u0026gt;\u0026thinsp;0 and ΔS\u0026thinsp;\u0026gt;\u0026thinsp;0), the proceeds of forming enzyme-extract complexes were spontaneously endothermic at the test temperatures (Ross \u0026amp; Subramanian, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). Moreover, the positive and negative values of thermodynamic parameters were associated with various individual interactions, including hydrogen bonding, van der Waals forces, electrostatic forces, and hydrophobic interactions, which may occur during the process of enzymes bind to inhibitors (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). From the thermodynamic standpoint (ΔH\u0026thinsp;\u0026gt;\u0026thinsp;0 and ΔS\u0026thinsp;\u0026gt;\u0026thinsp;0), the hydrophobic force as the main driving force led to collisions and energy transfer between molecules, resulting in the enzyme molecule returning to the ground state and losing the luminescence effect. Combined with the discussion of fluorescence quenching, the secondary structure of the enzymes changed, which were further analyzed as follows.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Circle dichroism spectroscopy of acorn shells extract against enzymes\u003c/h2\u003e \u003cp\u003eCircle dichroism (CD) spectroscopy of proteins in 178\u0026thinsp;~\u0026thinsp;250 nm (far-UV region) was generally analyzed the secondary structure of proteins (Andrew J. Miles, Drew, \u0026amp; Wallace, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; A. J. Miles, Janes, \u0026amp; Wallace, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). CD spectra used the circular dichroism of peptide bonds at 178\u0026thinsp;~\u0026thinsp;250 nm to obtain the secondary structure information of the enzymes with or without the effect of acorn shells extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Due to the n\u0026rarr;π* and π\u0026rarr;π* electron transfer of the peptide bond, the free α-amylase had two distinctive negative peaks near 208 and 228 nm, which characterized α-helix and β-sheet structure, respectively (Xue et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, the free α-glucosidase had two obvious negative peaks at 209 and 222 nm. The binding of the extract to the enzymes changed both these bands and also shifting in the negative minima and the results suggested that interaction of the extract with the enzymes may cause some conformational change of the enzymes. The decrease of peak intensity indicated that the addition of acorn shells extract led to the decrease of α-helical structure content and the change of secondary structure, which resulted in the inhibition of the enzymes. These secondary structure changes brought from acorn shells extract were similar to the case of epigallocatechin gallate and luteolin (Wu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yan, Zhang, Pan, \u0026amp; Wang, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Overall, the decrease of α-amylase or α-glucosidase catalytic activity could be attributed to the unfolding of enzymes structure, which involved with unwound α-helix and he destruction of hydrogen bonds and some functional units.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effect of acorn shells extract on starch digestibility in vitro\u003c/h2\u003e \u003cp\u003eBased on the starch digestion related to the rise of postprandial blood glucose, the in vitro digestibility of wheat starch-acorn shells extract system was evaluated within 120 min. The highest in vitro starch digestion rate was observed in wheat starch without the extract at all digestion times up to 120 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Rapidly digestible starch, referred to the starch that can be digested and absorbed within 20 min in the small intestine, decreased significantly with the addition of the extract (5%~25%) and the digestibility of starch-extract decreased from 60.48% to 14.96% as a significant dose-effect positive correlation. Starch that could be completely digested and absorbed within 20\u0026thinsp;~\u0026thinsp;120 min in the small intestine was defined as slowly digestible starch, which reduced by 28.34% at addition of 25% extract obviously. In general, the starch digestibility presented visibly decreasing at the high enough addition of the extract (25%), signifying that the extract had a certain resistance to the digestion of starch. Combined with the analysis of inhibition kinetics, this phenomenon could be explained that the extract prevented the adhesion of α-amylase and α-glucosidase to starch by occupying its hydrophobic helical region to bind to starch. Even if the addition was at low level, the retardation of digestion remained noticeable, which might be the destruction of rheological properties, gelatinization, retrogradation and gelling as well as the establishment of an ordered structure or crystallinity in the starch-extract mixture system (Guzar, Ragaee, \u0026amp; Seetharaman, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Maibam, Chakraborty, Nickhil, \u0026amp; Deka, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sun \u0026amp; Miao, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jingwen Xu, Wang, \u0026amp; Li, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Besides, the polyphenols and terpenoids from the extract that could still retained enzyme inhibition after being bound onto starch, even if they participated in the high-temperature (90\u0026thinsp;~\u0026thinsp;100\u0026deg;C) gelatinization of starch. (Barros, Awika, \u0026amp; Rooney, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Chi, Li, Zhang, Chen, Li, \u0026amp; Wang, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mr\u0026aacute;zkov\u0026aacute;, Sumczynski, \u0026amp; Orsavov\u0026aacute;, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, the extract directly or indirectly inhibited the digestion of starch and absorption of glucose in vitro.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Postprandial glycemic response on mice\u003c/h2\u003e \u003cp\u003eWith the intention of further exploration of the extract resistance against starch and glucose in vivo, the postprandial glucose responses of the extract-starch and extract-glucose mixture were investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The blood glucose of all three groups rose firstly, then declined and finally stabilized within 120 min. In contrast to the control group, the elevated level of blood glucose of the extract group was significantly lower (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that of, indicating that the extract had inhibitory effect on starch digestion and glucose metabolism. To be specific, the acorn shells extract reduced the peak value of blood glucose by 29.15% and 30.77% after starch and glucose loading (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and C). Whereas the peak delay (15 min) was only observed after glucose loading (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Subsequently, the areas under the blood glucose curves were further calculated and found no significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the relief on postprandial blood glucose between the group of the extract (decreased by 14.39% or 9.64% after starch or glucose loading, respectively) and acarbose (by 15.46% or 14.20%, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDietary polyphenols were reported to be able to alleviate elevation of blood glucose level after meal, which resulted from interactions between polyphenols and starch, as well as from inhibition of key digestive enzymes, including α-amylase and α-glucosidase (Sun et al., 2020). However, acorn shells extract also repressed in the level of blood glucose in glucose tolerance test, which can be reasonably speculated that it prevented the generation of blood glucose and improved the rapid increase of postprandial blood glucose. Based on the analysis of in vitro and in vivo experimental results, the reasons for the extract to reduce postprandial blood glucose were summarized for three aspects as follow. Acorn shells extract, as a kind of mixed enzymes inhibitor, especially α-glucosidase inhibitor, not only reversibly competed with the active site of the substrate, but also formed a substrate-extract complex to inhibit the formation of the product. Next, the extract interacted with starch directly or indirectly to format resistant starch or inhibiting the hydrolysis of starch by the enzymes. Lastly, the extract regulated the transport of glucose in the blood, promoting glucose metabolism. According to the above results, food-borne acorn shells extracts might be added to food such as bread and noodles to develop food for special medical purpose.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAcorn shells extracts suppressed α-amylase and α-glucosidase activity in mixed-type manners with IC\u003csub\u003e50\u003c/sub\u003e values of 0.352 and 0.0158 mg/mL, respectively. Through hydrophobic force, the extract destroyed the luminescent groups of the enzymes mainly along with dynamic quenching and ruined the secondary structure of the enzymes with the α-helix content reduce. Besides, the extract directly interacted with starch to affect digestion. From the starch and glucose tolerance tests, the dropping blood sugar level showed that significantly delay the digestion of starch and the glucose transfer. Therefore, acorn shells extract offered prophylactic role against hyperglycemia and assisted its associated intervention studies in vivo, which could be used as a potentially important source in the management of diabetes and functional food additives.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZixuan Han: Conceptualization, Investigation, Formal analysis, Writing-original draft.Nan Lin: Investigation.Zongyao Liang: Investigation.Xuchang Duan: Project administration, Conceptualization, Writing-review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was supported by Key Research and Development Program of Shaanxi Province (2021NY-158), and Science and Technology Innovation and Achievement Transformation Project of Northwest A\u0026amp;F University Experiment and Demonstration Station (TGZX2020-29).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eK. Abdollahi, C. Ince, L. Condict, A. Hung, S. Kasapis, Combined spectroscopic and molecular docking study on the pH dependence of molecular interactions between β-lactoglobulin and ferulic acid. Food Hydrocoll. \u003cb\u003e101\u003c/b\u003e, 105461 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.foodhyd.2019.105461\u003c/span\u003e\u003cspan address=\"10.1016/j.foodhyd.2019.105461\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Barrett, T. Ndou, C.A. Hughey, C. Straut, A. Howell, Z. Dai, G. Kaletunc, Inhibition of α-Amylase and Glucoamylase by Tannins Extracted from Cocoa, Pomegranates, Cranberries, and Grapes. J. Agric. Food Chem. \u003cb\u003e61\u003c/b\u003e(7), 1477\u0026ndash;1486 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf304876g\u003c/span\u003e\u003cspan address=\"10.1021/jf304876g\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF. Barros, J.M. Awika, L.W. Rooney, Interaction of Tannins and Other Sorghum Phenolic Compounds with Starch and Effects on in Vitro Starch Digestibility. J. Agric. Food Chem. \u003cb\u003e60\u003c/b\u003e(46), 11609\u0026ndash;11617 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf3034539\u003c/span\u003e\u003cspan address=\"10.1021/jf3034539\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Chen, Y. Jin, X. Ding, F. Wu, M. Bashari, F. Chen, X. Xu, Improved the emulsion stability of phosvitin from hen egg yolk against different pH by the covalent attachment with dextran. Food Hydrocoll. \u003cb\u003e39\u003c/b\u003e, 104\u0026ndash;112 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.foodhyd.2013.12.031\u003c/span\u003e\u003cspan address=\"10.1016/j.foodhyd.2013.12.031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC. Chi, X. Li, Y. Zhang, L. Chen, L. Li, Z. Wang, Digestibility and supramolecular structural changes of maize starch by non-covalent interactions with gallic acid. Food Funct. \u003cb\u003e8\u003c/b\u003e(2), 720\u0026ndash;730 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C6FO01468B\u003c/span\u003e\u003cspan address=\"10.1039/C6FO01468B\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Daly, R. Hovorka, Technology in the management of type 2 diabetes: Present status and future prospects. Diabetes Obes. Metabolism. \u003cb\u003e23\u003c/b\u003e(8), 1722\u0026ndash;1732 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1111/dom.14418\u003c/span\u003e\u003cspan address=\"10.1111/dom.14418\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Deol, L. Lekkakou, A.K. Viswanath, J.M. Pappachan, Combination therapy with GLP-1 analogues and SGLT-2 inhibitors in the management of diabesity: the real world experience. Endocrine. \u003cb\u003e55\u003c/b\u003e(1), 173\u0026ndash;178 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12020-016-1125-0\u003c/span\u003e\u003cspan address=\"10.1007/s12020-016-1125-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL.M. Espinoza-Fonseca, The benefits of the multi-target approach in drug design and discovery. Bioorg. Med. Chem. \u003cb\u003e14\u003c/b\u003e(4), 896\u0026ndash;897 (2006). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.bmc.2005.09.011\u003c/span\u003e\u003cspan address=\"10.1016/j.bmc.2005.09.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Fan, W. Lian, T. Li, Z. Rao, Y. Li, H. Qian, L. Wang, Characterization of promising natural blue pigment from Vaccinium bracteatum thunb. leaves: Insights of the stability and the inhibition of α-amylase. Food Chem. \u003cb\u003e326\u003c/b\u003e, 126962 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.foodchem.2020.126962\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2020.126962\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Feng, F. He, Y. Huang, M. Zhou, X. Liu, X. Ye, H. Chen, Inhibitory effects of phenolic glycosides from Trollius chinensis Bunge on α-glucosidase: inhibition kinetics and mechanisms. Food Funct. \u003cb\u003e13\u003c/b\u003e(5), 2857\u0026ndash;2864 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D1FO03347F\u003c/span\u003e\u003cspan address=\"10.1039/D1FO03347F\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN.T. Florence, M.Z. Benoit, K. Jonas, T. Alexandra, D.D.P. D\u0026eacute;sir\u0026eacute;, K. Pierre, D. Th\u0026eacute;ophile, Antidiabetic and antioxidant effects of Annona muricata (Annonaceae), aqueous extract on streptozotocin-induced diabetic rats. J. Ethnopharmacol. \u003cb\u003e151\u003c/b\u003e(2), 784\u0026ndash;790 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.jep.2013.09.021\u003c/span\u003e\u003cspan address=\"10.1016/j.jep.2013.09.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZ. G\u0026uuml;venalp, H. Yuca, S. G\u0026ouml;zc\u0026uuml;, B. Dursunoğlu, F. Tosun, L. Demirezer, In vitro α-glucosidase and α-amylase inhibitory activities of acorn, acorn shell and cupule extracts of Quercus robur L. Planta Med. \u003cb\u003e82\u003c/b\u003e, 01 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eP180. https://doi.org/10.1055/s-0036-1596337\u003c/span\u003e\u003cspan address=\"P180. 10.1055/s-0036-1596337\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI. Guzar, S. Ragaee, K. Seetharaman, Mechanism of Hydrolysis of Native and Cooked Starches from Different Botanical Sources in the Presence of Tea Extracts. J. Food Sci. \u003cb\u003e77\u003c/b\u003e(11), C1192\u0026ndash;C1196 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1111/j.1750-3841.2012.02929.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1750-3841.2012.02929.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQ. Hu, L. Jiang, Q. Yan, J. Zeng, X. Ma, Y. Zhao, A natural products solution to diabetic nephropathy therapy. Pharmacol. Ther. \u003cb\u003e241\u003c/b\u003e, 108314 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.pharmthera.2022.108314\u003c/span\u003e\u003cspan address=\"10.1016/j.pharmthera.2022.108314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY. Huang, P. Wu, J. Ying, Z. Dong, X.D. Chen, Mechanistic study on inhibition of porcine pancreatic α-amylase using the flavonoids from dandelion. Food Chem. \u003cb\u003e344\u003c/b\u003e, 128610 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.foodchem.2020.128610\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2020.128610\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC.G. Kato-Schwartz, R.C.G. Corr\u0026ecirc;a, de D. Souza Lima, de A.B. S\u0026aacute;-Nakanishi, de G. Almeida Gon\u0026ccedil;alves, F.A.V. Seixas, R.M. Peralta, Potential anti-diabetic properties of Merlot grape pomace extract: An in vitro, in silico and in vivo study of α-amylase and α-glucosidase inhibition. Food Res. Int. \u003cb\u003e137\u003c/b\u003e, 109462 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.foodres.2020.109462\u003c/span\u003e\u003cspan address=\"10.1016/j.foodres.2020.109462\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR. Khanra, S. Dewanjee, K. Dua, T. Sahu, R. Gangopadhyay, M. De Feo, V., M. Zia-Ul-Haq, Abroma augusta L. (Malvaceae) leaf extract attenuates diabetes induced nephropathy and cardiomyopathy via inhibition of oxidative stress and inflammatory response. J. Translational Med. \u003cb\u003e13\u003c/b\u003e(1), 6 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12967-014-0364-1\u003c/span\u003e\u003cspan address=\"10.1186/s12967-014-0364-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Khunti, M.L. Wolden, B.L. Thorsted, M. Andersen, M.J. Davies, Clinical Inertia in People With Type 2 Diabetes: A retrospective cohort study of more than 80,000 people. Diabetes Care. \u003cb\u003e36\u003c/b\u003e(11), 3411\u0026ndash;3417 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2337/dc13-0331\u003c/span\u003e\u003cspan address=\"10.2337/dc13-0331\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN. Laiteerapong, A.S. Cifu, Screening for Prediabetes and Type 2 Diabetes Mellitus. JAMA \u003cb\u003e315\u003c/b\u003e(7), 697\u0026ndash;698 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1001/jama.2015.17545\u003c/span\u003e\u003cspan address=\"10.1001/jama.2015.17545\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Li, F. Yao, J. Du, X. Deng, C. Li, Persimmon Tannin Decreased the Glycemic Response through Decreasing the Digestibility of Starch and Inhibiting α-Amylase, α-Glucosidase, and Intestinal Glucose Uptake. J. Agric. Food Chem. \u003cb\u003e66\u003c/b\u003e(7), 1629\u0026ndash;1637 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.7b05833\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.7b05833\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eX. Li, J. Cai, J. Yu, S. Wang, L. Copeland, S. Wang, Inhibition of in vitro enzymatic starch digestion by coffee extract. Food Chem. \u003cb\u003e358\u003c/b\u003e, 129837 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.foodchem.2021.129837\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2021.129837\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Liu, S. Li, Y. Zhong, D. Wu, Inhibition mechanism analysis and molecular simulation of rhein on α-amylase. Mod. Food Sci. Technol. \u003cb\u003e31\u003c/b\u003e(02), 47\u0026ndash;51 (2015). (in Chinese). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13982/j.mfst.1673-9078.2015.2.009\u003c/span\u003e\u003cspan address=\"10.13982/j.mfst.1673-9078.2015.2.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eE. Lo Piparo, H. Scheib, N. Frei, G. Williamson, M. Grigorov, C.J. Chou, Flavonoids for Controlling Starch Digestion: Structural Requirements for Inhibiting Human α-Amylase. J. Med. Chem. \u003cb\u003e51\u003c/b\u003e(12), 3555\u0026ndash;3561 (2008). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jm800115x\u003c/span\u003e\u003cspan address=\"10.1021/jm800115x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB.D. Maibam, S. Chakraborty, C. Nickhil, S.C. Deka, Effect of Euryale ferox seed shell extract addition on the in vitro starch digestibility and predicted glycemic index of wheat-based bread. Int. J. Biol. Macromol. \u003cb\u003e226\u003c/b\u003e, 1066\u0026ndash;1078 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.ijbiomac.2022.11.223\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2022.11.223\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA.J. Miles, E.D. Drew, B.A. Wallace, DichroIDP: a method for analyses of intrinsically disordered proteins using circular dichroism spectroscopy. Commun. Biology. \u003cb\u003e6\u003c/b\u003e(1), 823 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42003-023-05178-2\u003c/span\u003e\u003cspan address=\"10.1038/s42003-023-05178-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA.J. Miles, R.W. Janes, B.A. Wallace, Tools and methods for circular dichroism spectroscopy of proteins: a tutorial review. Chem. Soc. Rev. \u003cb\u003e50\u003c/b\u003e(15), 8400\u0026ndash;8413 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D0CS00558D\u003c/span\u003e\u003cspan address=\"10.1039/D0CS00558D\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR. Moradi-Marjaneh, M. Paseban, A. Sahebkar, Natural products with SGLT2 inhibitory activity: Possibilities of application for the treatment of diabetes. Phytother. Res. \u003cb\u003e33\u003c/b\u003e(10), 2518\u0026ndash;2530 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1002/ptr.6421\u003c/span\u003e\u003cspan address=\"10.1002/ptr.6421\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Mr\u0026aacute;zkov\u0026aacute;, D. Sumczynski, J. Orsavov\u0026aacute;, (2023). Influence of Storage Conditions on Stability of Phenolic Compounds and Antioxidant Activity Values in Nutraceutical Mixtures with Edible Flowers as New Dietary Supplements. \u003cem\u003eAntioxidants, 12\u003c/em\u003e(4), 962. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mdpi.com/2076-3921/12/4/962\u003c/span\u003e\u003cspan address=\"https://www.mdpi.com/2076-3921/12/4/962\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD.J. Newman, Natural products and drug discovery. Natl. Sci. Rev. \u003cb\u003e9\u003c/b\u003e(11) (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nsr/nwac206\u003c/span\u003e\u003cspan address=\"10.1093/nsr/nwac206\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Oroian, F. Ursachi, F. Dranca, Ultrasound-Assisted Extraction of Polyphenols from Crude Pollen. Antioxidants. \u003cb\u003e9\u003c/b\u003e(4), 322 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mdpi.com/2076-3921/9/4/322\u003c/span\u003e\u003cspan address=\"https://www.mdpi.com/2076-3921/9/4/322\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.M. Pappachan, C.J. Fernandez, E.C. Chacko, Diabesity and antidiabetic drugs. Mol. Aspects Med. \u003cb\u003e66\u003c/b\u003e, 3\u0026ndash;12 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.mam.2018.10.004\u003c/span\u003e\u003cspan address=\"10.1016/j.mam.2018.10.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.M. Pappachan, A.K. Viswanath, Medical Management of Diabesity: Do We Have Realistic Targets? Curr. Diab. Rep. \u003cb\u003e17\u003c/b\u003e(1), 4 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11892-017-0828-9\u003c/span\u003e\u003cspan address=\"10.1007/s11892-017-0828-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry. \u003cb\u003e20\u003c/b\u003e(11), 3096\u0026ndash;3102 (1981). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/bi00514a017\u003c/span\u003e\u003cspan address=\"10.1021/bi00514a017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Roy, R.K. Nandi, S. Ganai, K.C. Majumdar, T.K. Das, Binding interaction of phosphorus heterocycles with bovine serum albumin: A biochemical study. J. Pharm. Anal. \u003cb\u003e7\u003c/b\u003e(1), 19\u0026ndash;26 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.jpha.2016.05.009\u003c/span\u003e\u003cspan address=\"10.1016/j.jpha.2016.05.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Shobana, Y.N. Sreerama, N.G. Malleshi, Composition and enzyme inhibitory properties of finger millet (Eleusine coracana L.) seed coat phenolics: Mode of inhibition of α-glucosidase and pancreatic amylase. Food Chem. \u003cb\u003e115\u003c/b\u003e(4), 1268\u0026ndash;1273 (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.foodchem.2009.01.042\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2009.01.042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL. Sun, Y. Guo, C. Fu, J. Li, Z. Li, Simultaneous separation and purification of total polyphenols, chlorogenic acid and phlorizin from thinned young apples. Food Chem. \u003cb\u003e136\u003c/b\u003e(2), 1022\u0026ndash;1029 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.foodchem.2012.09.036\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2012.09.036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL. Sun, M. Miao, Dietary polyphenols modulate starch digestion and glycaemic level: a review. Crit. Rev. Food Sci. Nutr. \u003cb\u003e60\u003c/b\u003e(4), 541\u0026ndash;555 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/10408398.2018.1544883\u003c/span\u003e\u003cspan address=\"10.1080/10408398.2018.1544883\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.-L. Tian, M. Zhao, J.-Y. Xu, T.-M. Lv, X.-C. Liu, S. Sun, P. Hu, Inhibitory Mechanism of Prenylated Flavonoids Isolated from Mulberry Leaves on α-Glucosidase by Multi-Spectroscopy and Molecular Dynamics Simulation. J. Agric. Food Chem. \u003cb\u003e71\u003c/b\u003e(23), 9135\u0026ndash;9147 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.3c00776\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.3c00776\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eX. Wu, M. Hu, X. Hu, H. Ding, D. Gong, G. Zhang, Inhibitory mechanism of epicatechin gallate on α-amylase and α-glucosidase and its combinational effect with acarbose or epigallocatechin gallate. J. Mol. Liq. \u003cb\u003e290\u003c/b\u003e, 111202 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.molliq.2019.111202\u003c/span\u003e\u003cspan address=\"10.1016/j.molliq.2019.111202\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Xu, J. Cao, J. Yue, X. Zhang, Y. Zhao, New triterpenoids from acorns of Quercus liaotungensis and their inhibitory activity against α-glucosidase, α-amylase and protein-tyrosine phosphatase 1B. J. Funct. Foods. \u003cb\u003e41\u003c/b\u003e, 232\u0026ndash;239 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.jff.2017.12.054\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2017.12.054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Xu, W. Wang, Y. Li, Dough properties, bread quality, and associated interactions with added phenolic compounds: A review. J. Funct. Foods. \u003cb\u003e52\u003c/b\u003e, 629\u0026ndash;639 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.jff.2018.11.052\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2018.11.052\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB. Xue, J. Tian, Y. Wang, B. Jin, H. Deng, N. Gao, B. Li, Mechanism underlying the interaction of malvidin-3-O-galactoside with protein tyrosine phosphatase-1B and α-glucosidase. J. Mol. Struct. \u003cb\u003e1253\u003c/b\u003e, 132249 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.molstruc.2021.132249\u003c/span\u003e\u003cspan address=\"10.1016/j.molstruc.2021.132249\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Yan, G. Zhang, J. Pan, Y. Wang, α-Glucosidase inhibition by luteolin: Kinetics, interaction and molecular docking. Int. J. Biol. Macromol. \u003cb\u003e64\u003c/b\u003e, 213\u0026ndash;223 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.ijbiomac.2013.12.007\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2013.12.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Zhang, Y. Zhang, E. Karrar, Q. Jin, H. Zhang, G. Wu, X. Wang, Mechanisms of sesamol and sesamin inhibiting α-glucosidase activity by spectroscopy and molecular docking. Food Bioscience. \u003cb\u003e53\u003c/b\u003e, 102680 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.fbio.2023.102680\u003c/span\u003e\u003cspan address=\"10.1016/j.fbio.2023.102680\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Zhu, B. Zhang, B. Wang, C. Li, X. Fu, Q. Huang, In-vitro inhibitory effects of flavonoids in Rosa roxburghii and R. sterilis fruits on α-glucosidase: Effect of stomach digestion on flavonoids alone and in combination with acarbose. J. Funct. Foods. \u003cb\u003e54\u003c/b\u003e, 13\u0026ndash;21 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1016/j.jff.2019.01.009\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2019.01.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eReaction kinetic parameters, changes in enthalpy, entropy and free energy of extract on \u0026alpha;-amylase and \u0026alpha;-glucosidase at different temperatures\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eT/K\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eK\u003csub\u003esv\u003c/sub\u003e\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(L/g)\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eK\u003csub\u003eq\u003c/sub\u003e\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(L/(g\u0026middot;s))\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003en\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eK\u003csub\u003ea\u003c/sub\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eK\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u0026Delta;H\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(KJ/mol)\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u0026Delta;S\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(J/(mol\u0026middot;K))\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u0026Delta;G\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(KJ/mol)\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u0026alpha;-amylase\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e298\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e5.03\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e5.03\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.1094\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e4.27\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2.4499\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e8.153\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e39.43\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-3.597\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e310\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e5.50\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e5.50\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.122\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e4.850\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2.6044\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-4.070\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u0026alpha;-glucosidase\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e298\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e13.531\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.35\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.619\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e18.69\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e3.2929\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e16.41\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e79.40\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-7.26\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e310\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e17.462\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.75\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1.637\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e24.15\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e3.5795\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e-8.21\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Acorn shells extract, α-amylase, α-glucosidase, Starch digestion, Postprandial blood glucose","lastPublishedDoi":"10.21203/rs.3.rs-9496475/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9496475/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcorn (\u003cem\u003eQuercus variabilis\u003c/em\u003e BL.) shells as by-product were identified to rich in phenolic acids, flavonoids and triterpene, exhibiting the excellent inhibition of α-amylase and α-glucosidase. Subsequently, this study was performed to evaluate the inhibitory mechanism of acorn shells extract against the enzymes and the regulatory of postprandial blood glucose. Enzymatic kinetics assays and spectroscopy revealed the mixed inhibition of α-amylase and α-glucosidase were attributed to the hydrophobic interaction between the extract and enzymes, which promoted the tryptophan energy transfer and α-helix unfolding in the enzymes. Moreover, in vitro starch digestion test, 25% extract-starch significantly reduced the starch digestibility (60.48%), while 5% extract-starch also delayed the digestion (60min). Furthermore, starch and glucose tolerance tests showed that the extract down-regulated glycemia via delaying the starch hydrolysis into glucose and improving glucose metabolism to the blood. Thus, acorn shells extract had comprehensive exploration values in food formulation against hyperglycemia.\u003c/p\u003e","manuscriptTitle":"Acorn (Quercus variabilis BL.) shells extract alleviating postprandial blood glucose via α-amylase and α-glucosidase inhibition and starch digestion resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-05 09:26:13","doi":"10.21203/rs.3.rs-9496475/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-14T22:09:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2453619239179153444899803174405376623","date":"2026-05-07T05:57:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216662391751236598180697944506972526490","date":"2026-05-04T17:18:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-24T14:34:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-23T05:24:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-23T05:23:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Biophysics","date":"2026-04-22T12:34:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Biophysics](https://www.springer.com/journal/11483)","snPcode":"11483","submissionUrl":"https://submission.nature.com/new-submission/11483/3","title":"Food Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d6dec82f-296d-4a38-baa6-7b08fe20cd38","owner":[],"postedDate":"May 5th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-14T22:09:05+00:00","index":24,"fulltext":""},{"type":"reviewerAgreed","content":"2453619239179153444899803174405376623","date":"2026-05-07T05:57:55+00:00","index":23,"fulltext":""},{"type":"reviewerAgreed","content":"216662391751236598180697944506972526490","date":"2026-05-04T17:18:05+00:00","index":22,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T09:26:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-05 09:26:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9496475","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9496475","identity":"rs-9496475","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}