Inhibitory Activities of Thai Culinary Vegetables against Key Enzymes Relevant to Diabetes Mellitus and the Kinetics of Enzyme Inhibitions

Diabetes mellitus (DM) is one of the most challenging noncommunicable diseases, as it causes signifcant costs for medical treatment as well as high morbidity and mortality rates. Dietary plants with antidiabetic properties have been explored as an alternative to synthetic medicines to treat DM because of their safety and nutrition. Hence, the objective of the present study was to determine the inhibitory activities of twenty commonly consumed Tai culinary vegetables against α -glucosidase and α -amylase. All vegetables were extracted using deionized water, ethanol, and hexane at 150rpm and 30 ° C for 24hours. Te enzyme inhibitory activities were performed using a colorimetric assay. Diverse results for α -glucosidase and α -amylase inhibitory activities were found for all vegetable extracts. Te most potent anti-α -glucosidase activity was obtained from the ethanolic extract of Leucaena leucocephala (Lamk.) de Wit with the half maximal inhibitory concentration (IC 50 ) of 13.39 ± 0.14 μ g/mL, followed by the aqueous and ethanolic extracts of Polygonum odoratum Lour with IC 50 of 25.60 ± 0.42 and 49.03 ± 0.72 μ g/mL, respectively. All the samples exhibited mixed, noncompetitive, and uncompetitive inhibition. It can be concluded that the α -glucosidase and α -amylose inhibitory efects of the investigated extracts may be an indicator of antidiabetic potency, and these extracts might potentially be benefcial as functional components for postprandial hyperglycemia treatment.


Introduction
Diabetes mellitus (DM), a modern lifestyle-related disease, has been classifed as one of the most challenging global public health problems.According to the World Health Organization (WHO), an estimated 463 million adults were living with diabetes as of 2021, and the number is projected to rise to 578 million by 2030 [1].In addition, diabetes is a leading cause of disability and mortality, accounting for an estimated 4.2 million deaths annually [2].According to the International Diabetes Federation (IDF), an estimated 6.0 million adults in Tailand were living with diabetes as of 2021, representing a prevalence of 10.6% [2].Furthermore, an estimated 2.4 million adults in Tailand were estimated to have undiagnosed diabetes.Te prevalence of diabetes in Tailand is projected to increase in the coming years, with an estimated 6.7 million adults living with diabetes by 2030.Diabetes is one of the leading causes of death in Tailand, and it is responsible for an estimated 62,000 deaths annually [2].Undoubtably, the rising number of DM patients every year generates tremendous expenses in medical care as well as high morbidity and death rates.
DM is a chronic health condition that occurs when the body does not produce enough insulin or does not use the insulin efectively.Insulin is a hormone produced by the pancreas that plays a key role in the regulation of glucose metabolism.It helps the body use glucose for energy and store excess glucose in the liver and muscles for future use.Insulin helps cells take up glucose from the blood, preventing it from reaching excessively high levels.It also stimulates the liver to take up and store glucose, helping to maintain stable blood glucose levels [3].Without insulin, too much glucose can build up in the bloodstream, resulting in several health complications.To control blood glucose levels, most DM patients take synthetic medicines, which have an inhibitory efect against the activity of the enzymes, especially α-amylase and α-glucosidase which break down carbohydrates in the gut, leading to a slowdown of glucose absorption into the bloodstream.Te digestion process is initiated by α-amylase by breaking down starches into smaller oligosaccharides and disaccharides, which are then further broken down by α-glucosidase to release glucose and other simple sugars.Tese monosaccharides are then absorbed into the bloodstream.By acting on the fnal step of carbohydrate digestion, α-glucosidase directly infuences the rate at which glucose enters the bloodstream [4].As a result, glucose levels can be maintained at a more consistent level, preventing spikes or drops in blood sugar.Nevertheless, these drugs often have unpleasant side efects such as fatulence, infation, diarrhea, nausea, and loss of appetite.Terefore, a long-term use of these medications would constitute a burden to DM patients and reduce their quality of life [5].To reduce the side efects caused by the synthetic antidiabetic medications and to lower the cost of medical treatments, many bioactive compounds from natural sources, especially plants, have been investigated.
Plants are rich in many secondary metabolites possessing potent biological properties to maintain blood glucose levels [6].Tus, many plants have recently been investigated for their inhibitory efects against α-amylase and α-glucosidase, including bitter gourd (Momordica charantia) [7], Java plum (Syzygium cumini) [8], turmeric (Curcuma longa) [9], and king of bitters (Andrographis paniculata) [10].Some have been studied and provided valid scientifc evidence, while others have not been scientifcally demonstrated.To pursue the fnding, in this study, twenty commonly consumed Tai culinary vegetables in the northeastern region of Tailand with herbal remedy backgrounds in diabetes management were screened and examined for their efect on α-amylase and α-glucosidase inhibition in vitro.Te knowledge gained from this study would be useful to identify potential culinary vegetables with α-amylase and α-glucosidase inhibitory activity as functional foods for postprandial hyperglycemia management as well as help establish the scientifc validity of folk medicine.

Plant Extraction.
Ten grams of freeze-dried vegetables were macerated using extraction solvents with diferent polarity including deionized water, ethanol, and hexane, in a ratio of 1 : 10 in a 30 °C water bath shaker for 24 hours at 150 rpm.Vegetable debris was removed by centrifugation, and the vegetable extract was obtained after the removal of the extraction solvent by rotary evaporation.Te vegetable extract was kept in darkness at 4 °C for further analysis.

α-Glucosidase Inhibitory Assay.
Te α-glucosidase inhibitory properties were analyzed using the method, explained by Kim et al. with minor modifcations [11].Briefy, 50 µL of 10 mg/mL vegetable extract was preincubated with 0.1 M phosphate bufer, pH 6.8 containing α-glucosidase for 10 min.After preincubation, 1 mM pnitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate bufer, pH 6.8 was added and further incubated at 37 °C for 10 min.Te reaction was stopped by adding 1 mL of 0.1 M sodium carbonate.Te α-glucosidase inhibitory activity was followed by the measurement of absorbance at 405 nm.Te α-glucosidase inhibitory property was expressed as the percentage of α-glucosidase inhibition and calculated according to the following equation: where A and B were the absorbance values for the control and sample, respectively.A control was prepared using the same procedure to replace the vegetable extract with distilled water.Te experiment was conducted in fve replicates.
After preincubation, 1% starch in phosphate bufer, pH 6.9 was added and further incubated for 10 min.Te reaction was stopped by adding 1 mL of the 3, 5-dinitrosalicylic acid reagent, then incubated in boiling water for 5 min and cooled to room temperature.Te α-amylase inhibitory activity was followed by the measurement of absorbance at 540 nm.Te α-amylase inhibitory property was expressed as the percentage of α-amylase inhibition and calculated according to the equation: where A and B were the absorbance values for the control and sample, respectively.A control was prepared using the same procedure, replacing the vegetable extract with distilled water.Te experiment was conducted in fve replicates.

Determination of the IC 50 .
Te vegetable extracts with more than 50% of α-glucosidase inhibitory or 50% of α-amylase inhibitory were selected for the evaluation of IC 50 value.Te IC 50 is defned as the concentration of vegetable extract that could reduce the α-glucosidase activity by 50% which was only determined for the vegetable extract with inhibition ≥50%.Te IC 50 was obtained graphically for the plot of percentage of inhibition versus concentration [11].
Te experiment was conducted in triplicate.

Kinetics of Enzyme Inhibition.
In the enzyme-kinetic measurement, an inhibition assay was performed according to the protocol described by Kim et al.Inhibition modes of selected vegetable extracts against α-glucosidase were determined by increasing concentration of p-nitrophenyl-α-Dglucopyranoside solution in the absence or presence of selected vegetable extracts.Te experiment was conducted in triplicate.Te type of inhibition of the vegetable extracts was determined by a Lineweaver-Burk plot [11].

Screening of In Vitro α-Glucosidase and α-Amylase
Inhibitory Activity.Te α-glucosidase inhibitory activities of the vegetable extracts, intentionally chosen for their Tai remedial background in diabetes management are presented in Table 2. Using diferent extraction solvents to extract antiα-glucosidase agents from each vegetable resulted in diferent anti-α-glucosidase activity levels.Nine out of twenty vegetables showed an inhibitory efect against α-glucosidase, whereas eleven showed no inhibition.Hexane did not appear to be a suitable solvent to extract antiα-glucosidase substances.Tese screening results indicated that L. leucocephala (Lamk.)de Wit, P. odoratum Lour, S. gratum (Wight) S.N.Mitra var.gratum, and C. grandis (L.) Voigt appeared to be good potential sources of anti-α-glucosidase agents.Terefore, these vegetable extracts were selected for further analysis on IC 50 determination and study for the kinetics of enzyme inhibition.
To determine the inhibition mechanism of selected vegetable extracts with high inhibitory activity against α-glucosidase, the inhibitory kinetics of the vegetable extracts were measured at various concentrations of substrate, and the data were exported using the method of Lineweaver-Burk plot.Table 4 shows the K m and V max values of the vegetable extracts towards α-glucosidase.Compared to the uninhibited reaction (reaction containing α-glucosidase without inhibitor), a decrease in V max was found for all vegetable extracts, but the efects of the vegetable extracts on K m values were diferent.Te K m values were reduced in the presence of an aqueous extract of S. gratum (Wight) S.N.Mitra var.gratum and the ethanolic extract of C. grandis (L.) Voigt.Te K m values were increased in the presence of the ethanolic extract of S. gratum (Wight) S.N.Mitra var.gratum and P. odoratum Lour.However, the aqueous extract of P. odoratum Lour and the ethanolic extract of L. leucocephala (Lamk.)de Wit did not change the K m values of the reactions.Tese results demonstrated the mode of inhibition for the vegetable extracts on α-glucosidase.Te aqueous extract of S. gratum (Wight) S.N.Mitra var.gratum and the ethanolic extract of C. grandis (L.) Voigt exhibited an uncompetitive inhibition mode.Te ethanolic extract of S. gratum (Wight) S.N.Mitra var.gratum and P. odoratum Lour were in a mixed inhibition mode, while the aqueous extract of P. odoratum Lour and the ethanolic extract of L. leucocephala (Lamk.)de Wit demonstrated a noncompetitive inhibitor mode.

. Discussion
Tere is growing interest in developing novel and potential antidiabetic properties with minimal adverse efects that can be derived from plants that have known, scientifcally  [6,13,14].
In the present study, the inhibition of carbohydratehydrolyzing enzymes of regularly consumed vegetables in Tailand was the subject of investigation.Te enzyme inhibitory property may be a practical method for regulating the control of blood glucose levels.Te target enzymes in this investigation were the hydrolyzing enzymes: α-amylase and α-glucosidase.Tese enzymes are primarily responsible for the decomposition of starch into glucose.Te inhibition of these enzymes results in a delay in glucose absorption into the blood vessels.
Te inhibition of carbohydrate-hydrolyzing enzymes of sixty diferent vegetable extracts from diferent twenty vegetables was tested.It was discovered that antiα-glucosidase and anti-α-amylase activities were afected by the type of vegetables and solvent utilized, which was in agreement with several previous studies [15][16][17][18].Plants have a wide range of phytochemicals that are structurally diferent, resulting in distinct polarity [16,17].In this study, solvents with a wide range of polarity from nonpolar to polar were used to ensure that plant materials that difered in their polarity were sequentially extracted based on their polarity, and all components were presented in the screening study.It was found that only four out of twenty vegetables evaluated in this study had high anti-α-glucosidase activity (>50% of inhibition).Tis circumstance could be explained by the phytochemical components present in plants varying contingent on plant origin, plant genotype, geography, climate, soil fertility, and stress level.Tese elements afect how plants create bioactive compounds, which might vary in quantity and form [19][20][21].In addition, the results of the current study suggested that the antiα-glucosidase agents from these vegetable extracts were also likely composed of functional groups that appear to be hydrophilic with polarity indices between 5.2 (absolute ethanol) and 10.2 (water) because the anti-α-glucosidase activities were found in the ethanolic and aqueous extracts.Five out of the twenty vegetables evaluated, however, had minimal inhibitory efects on α-glucosidase.Additionally, a few of the vegetable extracts exhibited minor inhibitory efects on α-amylase.Terefore, it is probable that these vegetables may not contain efective or sufcient antiα-glucosidase and anti-α-amylase agents.As mentioned previously, the type and concentration of bioactive chemicals found in various plants may vary.
Measuring enzyme inhibition at a fxed concentration provided rather limited information [22].Te IC 50 value was therefore used to compare the efciency of the vegetable extract to inhibit α-glucosidase in this study.Te IC 50 value means the concentration of vegetable extract that generates 50% inhibition under a particular assay condition, resulting in a diference in the IC 50 value found among the conditions used [23].
Te IC 50 results revealed that the aqueous and ethanolic extracts of S. gratum (Wight) S.N.Mitra var.gratum were approximately the same with values of 516.92 ± 5.08 and 542.50 ± 0.90 µg/mL, respectively.Tis result implied that  Journal of Food Quality the active compounds found in these two extracts were possibly hydrophilic-like compounds, which corresponded to the study from Syabana et al. [24].In their study, the leaf extract of S. gratum (Wight) S.N.Mitra var.gratum was found to be a potential source of α-glucosidase inhibitors, especially the fractions extracted by acetone-water 4 : 1 and 3 : 2. Te IC 50 of these two fractions were 24.8 and 31.8 μg/ mL, respectively [24].According to the study from Syabana et al. [24], the α-glucosidase inhibitory activity of S. gratum (Wight) S.N.Mitra var.gratum could be a result of myricetin-3-O-rhamnoside (myricitrin) and epigallocatechin-3gallate (EGCG) [24].Additionally, both aqueous extract and ethanolic extract from the leaves of S. gratum (Wight) S.N.Mitra var.gratum showed strong antioxidant and intercellular oxygen scavenging activity, according to the study from Senggunpri et al. [25].Te aqueous extract also showed a cytoprotective efect in vivo.Te activity of heme oxygenase (HO-1), a potent cytoprotective enzyme in the antioxidant defense system, was signifcantly increased in the high-dose-treated C57BL/6J mice, and the expression of HO-1 gene had a tendency to increase when treated with the aqueous extract.Te data extrapolated the beneft of S. gratum (Wight) S.N.Mitra var.gratum as a source of natural antidiabetic agents and antioxidants, and it could induce cytoprotective enzymes without toxicity being observed.
Te IC 50 values of the P. odoratum Lour extracts were 49.03 ± 0.72 μg/mL for the ethanolic extract and 25.60 ± 0.42 μg/mL for the aqueous extract, respectively.Tese results were supported by the study of Tongra-ar et al. [26], which specifed that the ethanolic extract of P. odoratum Lour strongly inhibited α-glucosidase with an IC 50 value of 9.82 ± 1.64 μg/mL [26].Moreover, Dedvisitsakul and Watla-Iad [27] further reported the inhibitory efect of the ethanolic extract from P. odoratum Lour towards α-glucosidase with the IC 50 of 0.66 ± 0.08 mg/mL.Teir study also discovered that the ethanolic P. odoratum Lour extract demonstrated signifcant inhibitory activity towards the formation of advanced glycation end product (AGEs) which derived from glucose using a BSA-glucose system with the IC 50 of 0.03 ± 0.01 mg/mL [27].Te phenolic compound (gallic acid and chlorogenic acid) and favonoid (isorhamnetin) were believed to respond to the inhibitory efect of α-glucosidase in accordance with their phytochemical study [26].Te in vivo study from Deng et al. [28] also indicated saponins found in this vegetable presented antidiabetic activity, whereas favonoids infuenced antioxidant activity.
Te ethanolic extract of C. grandis (L.) Voigt proved its IC 50 against α-glucosidase at 82.74 ± 1.39 μg/mL.Likewise, the study by Pulbutr et al. indicated the IC 50 value of the ethanolic extract against α-glucosidase at 77.66 ± 9.16 μg/mL [29].Te antidiabetic properties of the ethanolic extract were supported by the study from Astiti et al. [30], which identifed the compounds responsible for the antidiabetic efect from the extract of

-Oβ-D-apiofuranosyl-(1⟶2)-[α-L-rhamnopyranosyl-(1⟶6)]β-D-galactopyranoside.
Te most efcient anti-α-glucosidase activity in this study was obtained from the ethanolic extract from L. leucocephala (Lamk.)de Wit with the lowest IC 50 of 13.39 ± 0.14 μg/mL.In addition, the study by Renganathan et al. [31] revealed that L. leucocephala (Lam.)De Wit leaf extract inhibited enzyme activity in a dose-dependent manner.Te study from Wan-Nadilah et al. [32] also found the in vitro α-glucosidase inhibitory activity from the seed of L. leucocephala (Lam.)De Wit with the IC 50 of 30.80 ± 2.50 μg/mL.Parts of use in the study by Renganathan et al. [31] and the present study were not the same; their extracts were obtained from leaves, while the extracts in this study were from young shoots.Tis might have contributed to the diference in IC 50 values.However, these data indicated that leaves, seeds, and young shoots of L. leucocephala (Lam.)De Wit were a good source of antiα-glucosidase constituents.Moreover, in silico virtual screening was used to identify the phytochemicals involved in α-amylase enzyme inhibition, while hexadecenoic acid and oleic acid ((Z)-octadec-9-enoic acid) were identifed as α-amylase inhibitors.
Line-weaver-Burk plots revealed that the inhibition modes of all samples might be mixed-uncompetitive and noncompetitive inhibition.A mixed inhibitor can either bind to the free enzyme or the enzyme-substrate complex which results in an alteration of K m and V max values (an increase in K m and a decrease in V max ).For the uncompetitive inhibition and noncompetitive inhibition, the binding of these types of inhibitor can infuence the binding of the substrate by changing the conformation of the enzyme.Te uncompetitive inhibitor binds the enzymesubstrate complex, resulting in a decrease of K m and V max , while the noncompetitive inhibitor either binds to a free enzyme or the enzyme-substrate complex, which results in a decrease of V max value and no change in K m [11,18,23].It is likely that the capacity of these extracts as mixed, uncompetitive, and noncompetitive inhibitors to bind to extensive areas of the enzyme other than the active site allows them to show a broader specifcity of inhibition when compared to acarbose as a competitive inhibitor [11,18,23].Te explanation for various inhibition modes from the extracts could be as a result of the diferent bioactive constituents available in the extracts [18,22,23].Te various bioactive compounds presented in the extracts probably had diferent binding modes to α-glucosidase.Contrary to acarbose, these extracts may not be afected by increased quantities of the substrate, which is one advantage they have over acarbose.With increased carbohydrate meal consumption, higher dosages of acarbose as a competitive inhibitor would be necessary to have the same impact, but with the mixed, uncompetitive, and noncompetitive inhibition, the inhibitor would be efective at lower concentrations [18,23,33].Moreover, the stronger inhibition activity of the α-glucosidase than the α-amylase activity of these extracts revealed their medicinal potential to prevent 6 Journal of Food Quality some negative efects of utilizing synthetic α-glucosidase and α-amylase inhibitors.Te side efects of using synthetic enzyme-inhibitor drugs can include abnormal bacteria fermentation of undigested carbohydrates in the colon because these drugs strongly inhibit α-amylase over α-glucosidase.Terefore, more potent inhibitors for enzymes should have a strong inhibitory efect on α-glucosidase and a moderate inhibitory efect on α-amylase, which can improve the management of postprandial hyperglycemia with the fewest side efects [34,35].
Te results of the current study showed the potential of vegetable extracts toward enzyme-hydrolyzing carbohydrates.As a result, individuals should be encouraged to consume more of these vegetables as an alternative course for diabetic prevention and treatment.However, more information on in vivo bioactivity and the absorption of these bioactive components needs to be established before deciding on their applications.

Conclusion
Vegetables that are frequently consumed in the northeastern region of Tailand showed a varying range of α-glucosidase and α-amylase inhibitory efects.Promising α-glucosidase inhibitory activities were reported from L. leucocephala (Lamk.)de Wit, P. odoratum Lour, C. grandis (L.) Voigt, and S. gratum (Wight) S.N.Mitra var.gratum.Te results of this investigation suggest that these vegetables may be good dietary sources of extractable anti-α-glucosidase agents for preventing or managing postprandial hyperglycemiainduced complications.Nevertheless, this was an in vitro study with potential relevance concerning phytochemicals.
Most of the α-glucosidase inhibitory activities were discovered from the extracts using water and ethanol as extraction solvents.L. leucocephala (Lamk.)de Wit and P. odoratum Lour generated respectively.Moderate α-glucosidase inhibitory efects were also obtained from S. gratum (Wight) S.N.Mitra var.gratum, which gave lower anti-α-glucosidase activity when compared to the formers.Its inhibition rates against α-glucosidase were 55.12 ± 1.71% from the aqueous extract and 53.11 ± 1.44% from the ethanolic extract, respectively.Te ethanolic extract from C. grandis (L.) Voigt showed 67.30 ± 1.39% of inhibition, but no inhibition was found from the aqueous or hexane extract.Besides, other vegetable extracts produced either no or insufcient antiα-glucosidase activity; their inhibitory activities were less than 25% of inhibition against α-glucosidase activity.

Table 3 :
Te IC 50 and kinetics for enzyme inhibition of the vegetable extracts.
Remarks: All data was expressed as mean ± standard deviation (S.D.).

Table 4 :
Kinetic parameters for α-glucosidase inhibition of the vegetable extracts.
Remarks: All data was expressed as mean ± standard deviation (S.D.).