Screening and Characterization of an α-Amylase Inhibitor from Carya cathayensis Sarg. Peel

Inhibiting α-amylase can lower postprandial blood glucose levels and delay glucose absorption, offering an effective approach for the development of antidiabetic diets. In this study, an active constituent with inhibitory activity against α-amylase was isolated and purified by bioassay-guided fractionation from Carya cathayensis Sarg. peel (CCSP). The active constituent was identified by NMR and Q-Exactive Orbitrap Mass Spectrometry as 5-O-p-coumaroylquinic acid (5-CQA). 5-CQA possessed strong inhibitory activity against α-amylase, with an IC50 value of 69.39 µM. In addition, the results of the kinetic study indicated that 5-CQA was a potent, reversible, noncompetitive inhibitor against α-amylase. The findings indicate that 5-CQA derived from CCSP has potential as a novel inhibitor against α-amylase, which can help mitigate postprandial blood sugar spikes, making it suitable for inclusion in antidiabetic diets.


Introduction
Starch is the primary carbohydrate storage molecule in grains and is a significant source of energy for the human body.Starch digestive enzymes, α-amylase and αglucosidase, play pivotal roles in the digestive breakdown and subsequent absorption of dietary starches [1].Specifically, α-amylase catalyzes the hydrolysis of the α-D-1,4glucosidic bonds in starch, yielding dextrins and oligosaccharides [2].Subsequently, αglucosidase facilitates the conversion of these dextrins and oligosaccharides into glucose [3].Glucose undergoes cellular respiration to produce ATP, which cells then utilize to perform various vital functions.Nonetheless, excessive consumption of starch, especially from refined sources, may heighten the risk of chronic conditions, including type 2 diabetes, cardiovascular disease, and specific cancers, attributable to the elevated glycemic index associated with these foods [4,5].Targeting the digestive mechanisms of starch, specifically through the inhibition of starch hydrolases, offers a promising strategy for promoting foods with a reduced glycemic index [6].Inhibitors against α-amylase are used as antidiabetic drugs to control postprandial hyperglycemia.However, these pharmaceuticals have been reported to cause side effects, such as diarrhea, flatulence, and stomachache [7].Acarbose, a notable α-amylase inhibitor, impedes the breakdown of starch and sucrose, thereby moderating the absorption of starch from food [8].However, its use is somewhat limited due to side effects like flatulence and other symptoms stemming from carbohydrate malabsorption [9].In contrast, natural food sources such as vegetables, fruits, and nuts are abundant in phytochemicals, including potent secondary metabolites like flavonoids, phenolic acids, and saponins, which exhibit α-amylase inhibitory activity [10].The extraction of α-amylase inhibitors from plant sources is increasingly regarded as sustainable, given the lower environmental impact of plant cultivation and harvesting compared to chemical study were purchased from Aladdin Reagent Co., Ltd.(Shanghai, China).Ultra-pure water was used throughout the study.

Preparation of Extracts from CCSP
The extraction of extracts from Carya cathayensis Sarg.peel (CCSP) was conducted based on a previously reported method, with certain modifications [17].Initially, 5 kg of CCSP was thoroughly washed using tap water and subsequently dried in an oven set at 45 • C for a duration of 24 h.Post drying, the peel was ground into a fine powder using an ultrafine grinder (model DFY-500, Wenling Linda Instrument Co., Ltd., Taizhou, China), ensuring the particle size passed through a 40-mesh sieve.This powdered CCSP was then soaked in a 75% (v/v) ethanol aqueous solution, maintaining a ratio of 1:15 (w/v).The mixture underwent intermittent stirring for a period of 24 h, followed by centrifugation at 7500× g for 15 min.The resulting supernatant was concentrated under vacuum using a rotary evaporator (model RE-2000A, Shanghai Yarong Co., Ltd., Shanghai, China).Finally, the concentrated extract was freeze-dried, preparing for subsequent analytical procedures.

α-Amylase Inhibition Activity Assay
The inhibition assay was performed as described before, with some modifications [24].Briefly, 100 µL of α-amylase (1.25 U/mL) was mixed with 100 µL of different concentrations of test samples.Then, 200 µL of starch (0.5%, w/v) dissolved in phosphate buffer was added to the mixture and incubated at 37 • C for 8 min.To terminate the enzymatic reaction, 100 µL of DNS color reagent was added.After incubation at 100 • C for 10 min, the mixture was cooled to room temperature.Finally, the mixture was diluted with 1.5 mL of water, and the absorbance at 540 nm was recorded by an UV spectrophotometer (UV-1100, Mapada Instruments Co., Ltd., Shanghai, China).The assays of the control (the inhibitor solution was replaced by the carrier solvent), the sample background (the enzyme solution was replaced by PBS buffer), and the control background (the inhibitor solution and the enzyme solution were replaced by their carrier solvents) were performed by the same method as mentioned above.Each experiment was performed in triplicate.Acarbose is a specific inhibitor of pancreatic α-amylase enzyme and acted as the positive control.The inhibition activity was calculated using the following formula: where A S , A SB , A C, and A CB are the absorbances of the sample, sample background, control and control background, respectively.

Isolation of α-Amylase Inhibitor
The bioassay-guidance isolation was performed using a method previously reported with some modifications [14,25].The lyophilized crude extract powder (25 g) was dissolved in 350 mL of water and applied to an AB-8 macroporous adsorption resin column (600 mm × 40 mm) pre-equilibrated with water.Following this, the column was purged with 2 bed volumes (BV) of water to eliminate the unadhered compounds prior to successive elution.Subsequently, the column underwent stepwise desorption using 2 BV of aqueous ethanol solution, with ethanol concentrations of 15%, 30%, 45%, 60%, and 80% (v/v), respectively.All five collected fractions (F1-F5) were vacuum-concentrated to dryness and lyophilized for the bioactive assay.Then, the fraction (F2) was applied onto a Sephadex LH-20 column (300 mm × 35 mm) pre-equilibrated with water using a gradient elution of 2 BV of 5, 10, 15, 20, and 25% ethanol aqueous solution (v/v) to give five subfractions (F2-1-F2-5).Subfraction F2-2, guided by HPLC profiles, was further purified using a C 18reversed-phase silica gel column (175 mm × 18 mm).Initial elution was performed with 2% methanol and 0.5% formic acid for the first 5 min, followed by a gradient increase to 15% methanol from 5 to 20 min.After four rounds of elution purification, four eluates were obtained.

HPLC Analysis
HPLC analysis was conducted using an Agilent 1260 system equipped with a UV detector (Agilent Technologies Co., Ltd., Palo Alto, CA, USA).For preparation, the lyophilized sample powder was dissolved in water and then filtered through a 0.22 µm syringe filter.The filtered sample solution was introduced into a Welch Ultimate LP-C 18 column (250 mm × 4.6 mm, 5 µm), which was maintained at a constant temperature of 25 • C. The flow rate was set at 1 mL/min, and the absorbance of the eluent was monitored at 280 nm.The mobile phases used for the separation included water/formic acid (99:1, v/v, as solvent A) and acetonitrile (as solvent B).Initially, the column was equilibrated with 5% solvent B. The elution of sample compounds was conducted in a stepwise manner, with the following gradient profile: from 0 to 20 min, the concentration of solvent B was increased from 5% to 35%; from 20 to 25 min, it was ramped up from 35% to 100%; and from 25 to 30 min, the system was held at 100% solvent B.

Nuclear Magnetic Resonance (NMR) and Q-Exactive Orbitrap Mass Spectrometry Assays
The Nuclear Magnetic Resonance (NMR) and Q-Exactive Orbitrap Mass Spectrometry assays were employed to analyze the purified compound, adhering to established protocols with modifications for enhanced accuracy and sensitivity [11,26].NMR spectra ( 1 H and 13 C) were recorded on a Bruker Avance III 400 MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany).The compound was dissolved with CD 3 OD for measurement.Mass spectrometry was determined on a Thermo Scientific Q Exactive™ quadrupole-Orbitrap mass spectrometer (San Jose, CA, USA).The MS analysis conditions were as follows: scan type was Full-ddMS2; polarity was positive/negative modes; sheath gas flow rate was 45 arbitrary units; aux gas flow rate was 10 arbitrary units; spray voltage was set at 3.5 kV; capillary temperature was 275 • C; and gas heater temperature was set at 400 • C.

Inhibitory Kinetics Analysis
The inhibitory kinetic characteristics of 5-CQA against α-amylase were assessed based on a modified method from a previous study [15].Calculations were performed using the Michaelis−Menten equation to determine the Michaelis constant (K m ) and maximum reaction velocity (V max ) at various concentrations of 5-CQA solution.To calculate the dissociation constants for competitive inhibition (K ic ) and noncompetitive inhibition (K in ), the Michaelis−Menten equations were presented as follows [27]: Noncompetitive inhibition type : where V is the initial velocity, V m is the maximum velocity, K m is the Michaelis constant for the substrate (starch), [I] is the concentration of the inhibitor, [S] is the concentration of substrate (starch), K ic is the constant for competitive inhibition, and K in is the constant for noncompetitive inhibition.
To generate the Lineweaver-Burk plot, 1/V was plotted against 1/[S], with varying inhibitor concentrations.Additionally, plots of 1/[V] and [S]/V were constructed to conduct the Dixon plot and Eisenthal−Cornish−Bowden plot, correspondingly, at various substance concentrations.

Statistical Analysis
The results were presented as mean ± standard deviation (SD) for triplicate.Data were analyzed using SPSS version 26.0 software (Chicago, IL, USA), employing one-way ANOVA followed by Duncan's test to evaluate significant differences, with a signifi-cance level set at p < 0.05.Graphical representations of the data were generated using Origin 2021.

Purification of α-Amylase Inhibitor from CCSP
The purification procedure to obtain the component with α-amylase inhibitory activity is shown in Figure 1.Macroporous adsorption resin has been extensively used to separate bioactive components from plant [11].The crude extract of CCSP was loaded on an AB-8 macroporous adsorption resin column, and the eluent was partitioned into five fractions (F1-F5).Compared with other four fractions, F1 showed the highest activity (38.81%) at 1000 µg/mL, as shown in Figure 2A.In chromatography utilizing macroporous adsorption resin, an inverse relationship was observed between the polarity of the eluted component and the concentration of the alcohol solution when alcohol-water mixtures are employed as eluents [25,28,29].In this study, the active fraction (F1) was eluted using a 15% alcohol solution, suggesting that strongly hydrophilic compounds contributed to the α-amylase inhibitory activity.Hence, F1 was subjected to further separation.
duct the Dixon plot and Eisenthal−Cornish−Bowden plot, correspondingly, at various substance concentrations.

Statistical Analysis
The results were presented as mean ± standard deviation (SD) for triplicate.Data were analyzed using SPSS version 26.0 software (Chicago, IL, USA), employing one-way ANOVA followed by Duncan's test to evaluate significant differences, with a significance level set at p < 0.05.Graphical representations of the data were generated using Origin 2021.

Identification of Active Compound
The active compound obtained was a red amorphous powder.Mass spectra are shown in Figure S1.The molecular formula was determined to be C 16  ).The NMR data were closely consistent with those previously reported [30,31].Thus, the compound was identified as 5-O-p-coumaroylquinic acid (5-CQA), and the chemical structure is shown in Figure 4. To our knowledge, this compound was first reported in Carya cathayensis Sarg.

Identification of Active Compound
The active compound obtained was a red amorphous powder.Mass spectra are shown in Figure S1 ).The NMR data were closely consistent with those previously reported [30,31].Thus, the compound was identified as 5-O-p-coumaroylquinic acid (5-CQA), and the chemical structure is shown in Figure 4. To our knowledge, this compound was first reported in Carya cathayensis Sarg.

Identification of Active Compound
The active compound obtained was a red amorphous powder.Mass spectra are shown in Figure S1.t, C-6).The NMR data were closely consistent with those previously reported [30,31].Thus, the compound was identified as 5-O-p-coumaroylquinic acid (5-CQA), and the chemical structure is shown in Figure 4. To our knowledge, this compound was first reported in Carya cathayensis Sarg.
Though 5-CQA was less potent than acarbose, it demonstrated a stronger inhibitory effect compared to certain previously studied phenolic acids, such as gallic acid (IC50 of 1.25 mM), vanillic acid (IC50 of 27.89 mM), and syringic acid (IC50 of 44.81 mM) [32,33].A significant advantage of 5-CQA, similar to other natural plant-derived inhibitors, lied in its enhanced biocompatibility with human physiological systems.This compatibility generally translates into a lower probability of adverse side effects, a notable benefit when contrasted with many synthetic inhibitors, which may present higher toxicity [34].Furthermore, natural inhibitors like 5-CQA extend health benefits beyond mere enzyme inhibition.The utilization of plant-derived inhibitors such as 5-CQA offered not only the primary function of α-amylase inhibition but also additional advantages, including antioxidant properties, often absent in synthetic hypoglycemic drugs [35].Another aspect to consider is the costeffectiveness of natural inhibitors like 5-CQA.Depending on the source and extraction methods, these inhibitors might be more economical, particularly in regions with abundant source plants [36].Together, these characteristics emphasize the potential of 5-CQA from natural sources as a valuable option in both therapeutic and economic terms.
tion results in a consequent reduction in the glycemic index of the consumed carbo drates, contributing to a moderated rise in blood glucose levels post-meal [38].Inhibi of α-amylase indicates that 5-CQA has the ability to retard dietary starch liberation reduce its absorption, resulting in controlled postprandial hyperglycemia.Similar res were also described in other phenolic acid compounds, such as chlorogenic acid [39] ferulic acid [40], which effectively inhibited α-amylase.Key amino acid residues within the α-amylase enzyme, namely Glu233, Asp197, Asp300, play a crucial role in starch hydrolysis.These residues also form the binding for enzyme inhibitors at the active site [41].The interaction of inhibitors with α-amy either directly or indirectly, induces conformational changes in the enzyme, potent In the process of dietary carbohydrate digestion, α-amylase plays a pivotal role by catalyzing the breakdown of starch into oligosaccharides.These oligosaccharides are then further hydrolyzed into glucose by α-glucosidase, facilitating rapid absorption by the body [37].The interaction of 5-CQA with α-amylase leads to an inhibition of the enzyme's activity, thereby slowing down the rate of starch hydrolysis in ingested food.This inhibition results in a consequent reduction in the glycemic index of the consumed carbohydrates, contributing to a moderated rise in blood glucose levels post-meal [38].Inhibition of αamylase indicates that 5-CQA has the ability to retard dietary starch liberation and reduce its absorption, resulting in controlled postprandial hyperglycemia.Similar results were also described in other phenolic acid compounds, such as chlorogenic acid [39] and ferulic acid [40], which effectively inhibited α-amylase.
Key amino acid residues within the α-amylase enzyme, namely Glu233, Asp197, and Asp300, play a crucial role in starch hydrolysis.These residues also form the binding sites for enzyme inhibitors at the active site [41].The interaction of inhibitors with α-amylase, either directly or indirectly, induces conformational changes in the enzyme, potentially leading to the deactivation of the active sites.These interactions encompass a spectrum of molecular forces, including hydrogen bonding, van der Waals forces, electrostatic interactions, and hydrophobic contacts [42].Plant-derived phenolic acids, such as gallic acid, vanillic acid, and cinnamic acid, are well documented for their pronounced inhibitory effects on α-amylase [41,43].The inhibitory activity of phenolic acids is largely dependent on their specific chemical structures.It has been demonstrated that the hydrogen bonding interaction between phenolic acids and α-amylase plays a crucial role in this inhibitory mechanism [44,45].Recently, an investigation employing saturation transfer difference NMR revealed the hydrophobic interaction between the aromatic ring of phenolic acid and α-amylase, thereby inhibiting the enzyme [46].It is hypothesized that the hydrogen bonding and hydrophobic interactions are key contributors to the inhibitory activity of phenolic acids.These interactions with α-amylase potentially induce conformational alterations in the enzyme's protein structure.

Inhibitory Kinetics of 5-CQA against α-Amylase
Figure 6A displays the plot of the initial velocity (V) against the concentration of α-amylase, while considering various concentrations of 5-CQA.As the inhibitor concentration increased, all the linear trends intersected at the origin of coordinate, and the slope gradually diminished.The evidence suggests that 5-CQA could act as a reversible inhibitor, diminishing the catalytic activity of α-amylase.The Lineweaver-Burk plots of 5-CQA against α-amylase are presented in Figure 6B.These plots converged at a single point on the X-axis.Using Equation ( 2), the values of K m and V max were calculated through linear fitting, as detailed in Table 1.As the concentration of 5-CQA increased, the K m values remained consistent at approximately 0.64 µM, while the V max values decreased from 144.30 to 58.51 µM•min −1 .These findings suggest that 5-CQA behaves as a noncompetitive inhibitor against α-amylase.Dixon and Eisenthal-Cornish-Bowden plots are shown in Figure 6C,D, respectively.All lines intersected at a single point on the X-axis in both plots, further supporting that 5-CQA was a noncompetitive inhibitor.The results indicate that 5-CQA acted as a noncompetitive inhibitor of α-amylase by binding to the inactive sites on α-amylase and the starch-α-amylase complex.Values of K ic and K in were calculated according to Equations ( 2) and (3) and shown in Table 1.The K ic value and K in value were approximately the same (0.38 µM), indicating 5-CQA bound to the α-amylase with the same affinity as the starch-α-amylase complex [47].Similar findings were corroborated by previous studies [37,48], affirming that phenolic acids functioned as noncompetitive inhibitors of α-amylase from hog pancreas.
The commercially available α-amylase inhibitor acarbose is a competitive inhibitor.This means it competes with starch, the natural substrate of α-amylase, for binding to the enzyme.By doing so, it effectively reduces the rate at which starch is broken down into glucose, leading to a slower and reduced absorption of glucose in the intestines [49].Since acarbose works in the gastrointestinal tract and inhibits carbohydrate digestion, it can lead to gastrointestinal side effects like flatulence, bloating, and diarrhea.These effects are due to the fermentation of undigested carbohydrates in the large intestine [50].5-CQA, in contrast, is a noncompetitive inhibitor of α-amylase.It binds to a site on the enzyme different from the active site (allosteric site), altering the enzyme's shape or function.This alteration reduces the enzyme's activity without direct competition with the substrate (starch) [51].Although acarbose and 5-CQA both effectively reduce postprandial blood glucose levels, the efficacy of acarbose, being a competitive inhibitor, diminishes in environments with elevated substrate concentrations [52,53].In contrast, the effectiveness of 5-CQA, functioning as a noncompetitive inhibitor, remains unaffected by the concentration of carbohydrate substrates, thereby indicating a potential risk for hypoglycemia [52,54].Acarbose is primarily used as a therapeutic agent for diabetes management, whereas 5-CQA, found in dietary sources, might be viewed more as a preventive or complementary intervention with additional health benefits.

Conclusions
In summary, this study successfully isolated and identified 5-O-p-coumaroylquinic acid (5-CQA) as an α-amylase inhibitor from CCSP, demonstrating its inhibitory activity in a reversible and noncompetitive manner.These findings indicate that 5-CQA is a novel and promising candidate for α-amylase inhibition and has potential as a functional food ingredient for managing postprandial hyperglycemia.To further explore the efficacy of 5-CQA within a complex physiological environment, future in vivo studies, utilizing diabetic mouse models, are essential.

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Figure6Adisplays the plot of the initial velocity (V) against the concentration of αamylase, while considering various concentrations of 5-CQA.As the inhibitor concentration increased, all the linear trends intersected at the origin of coordinate, and the slope gradually diminished.The evidence suggests that 5-CQA could act as a reversible inhibitor, diminishing the catalytic activity of α-amylase.