Alpha-Glucosidase Inhibition, Antioxidant Activities, and Molecular Docking Study of Krom Luang Chumphon Khet Udomsak, a Thai Traditional Remedy

Krom Luang Chumphon Khet Udomsak remedy (KKR) has traditionally been used as an alternative treatment, particularly for hyperglycemia; however, its therapeutic efficacy has not been scientifically validated. Thus, this study aims to investigate the potential inhibitory and antioxidant effects of α-glucosidase enzyme and characterize the chemical profile of KKR extracts using gas chromatography-mass spectrometry (GC-MS). The investigation highlights both KKR extracts as potent inhibitors of α-glucosidase, with the ethanolic extract of KKR (KKRE) displaying an IC50 value of 46.80 µg/mL and a noncompetitive mode of action. The combination of ethanolic and aqueous extracts of KKR (KKRE and KKRA, respectively) with acarbose exhibited a synergistic effect against the α-glucosidase. The KKRE extract displayed strong scavenging effects in the DPPH assay (IC50 156.3 µg/mL) and contained significant total phenolic (172.82 mg GAE/g extract) and flavonoid (77.41 mg QE/g extract) contents. The major component of KKRE is palmitic acid (15.67%). Molecular docking revealed that the major compounds interacted with key amino acid residues (ASP215, GLU277, HIS351, ASP352, and ARG442), which are crucial for inhibiting α-glucosidase. Notably, campesterin had a more significant influence on α-glucosidase than acarbose, with low binding energy. These findings underscore the significance of KKR in traditional medicine and suggest that it is promising treatment for diabetes mellitus. Further studies using animal model will provide valuable insights for advancing this research.


Introduction
Diabetes mellitus (DM) is a global noncommunicable disease (NCD), afecting 422 million people in 2014, primarily in low-and middle-income nations, with a 13% increase in diabetes-related deaths between 2000 and 2019, causing 1.5 million deaths directly [1].Projections estimate a 25% increase in DM cases by 2030 and a 51% increase by 2045 [2].Diabetes mellitus, the most prevalent metabolic disorder, is characterized elevated blood glucose levels due to insulin imbalances [3].Type 2 diabetes involves insulin resistance and insufciency, leading to complications such as blindness, kidney failure, heart attack, stroke, and lower limb amputation.Prolonged hyperglycemia contributes to signifcant morbidity and mortality [4].Chemical and synthetic medications have been developed to manage metabolic disorders [5].
α-Glucosidase inhibitors mitigate postmeal hyperglycemia in individuals with DM by impeding the inhibitory efect of α-glucosidase in the intestinal tract.Tis mechanism extends carbohydrate digestion, curtails glucose absorption, and delays glucose release into the bloodstream [6].Notably, α-glucosidase inhibitors such as acarbose efectively lower postprandial blood glucose levels and are used in clinical practice [7].Acarbose functions as a reversible inhibitor of intestinal α-glucosidases, which are enzymes pivotal for breaking down complex carbohydrates into absorbable monosaccharides.Tis mechanism leads to a reduced and delayed surge in blood glucose levels after meals, consequently lowering postprandial hyperglycemia.In addition, when combined with other antidiabetic therapies, such as sulfonylureas and insulin, acarbose has demonstrated additional efcacy in glycemic control.Commonly reported adverse reactions include abdominal pain, diarrhea, and fatulence, which typically decrease over time [8].Despite the efectiveness of pharmaceutical drugs for blood glucose management, their extended use can lead to adverse efects [9].Consequently, many people have turned to traditional herbal remedies because of their perceived safety and reduced side efects.
Krom Luang Chumphon Khet Udomsak remedy (KKR) is a traditional Tai herbal medicine used for centuries to manage various health conditions, including DM [10].Tai herbs are rich in alkaloids and favonoids and are valued for therapeutic properties [11].Derived from herbal medicine scripture and Doctor Porn Krom Luang Chumphon Khet Udomsak's recipe, KKR aims to lower blood glucose levels.It comprised Phyllanthus amarus Schumach.and Tonn (PA), Smilax corbularia Kunth (SC), and Smilax glabra Roxb (SG), the evidence supporting KKR as a novel antidiabetic drug remains inconclusive.PA is known for its antidiabetic, antioxidant, anticancer, anti-infammatory, and hepatoprotective properties [12].Both SG and SC are rich in phenolic and favonoid compounds, including catechin, astilbin, isoastilbin, taxifolin, and smiglasides, which are known for their antioxidant, anti-infammatory, antibacterial, and anticancer properties [13].While there is no scientifc evidence supporting this remedy as an antidiabetic medicine, its potential in this area has not been fully explored.An initial study on its α-glucosidase inhibitory activity revealed promising efects, leading to a more detailed examination.Further analysis of the remedial extracts confrmed the strong α-glucosidase inhibitory efect of the KKR extract.
Tus, the primary objectives of this study are to evaluate the α-glucosidase enzyme inhibitory efect of KKR and its plant ingredient, elucidate the mechanisms of its principal botanical constituents, combination, and examine the interaction between these bioactive compounds and diabetic enzymes using molecular docking methodologies.Te anticipated outcomes of this investigative study have the potential to facilitate the integration of this botanical resource into the development of food supplements and herbal medicines specifcally designed to prevent hyperglycemia.

Plant Material and Management.
Te botanical components within KKR were purchased from a legally registered Tai traditional herbal pharmacy situated in the Hatyai District, Songkhla Province, Tailand.Tese botanical constituents encompass Phyllanthus amarus Schumach.and Tonn, Smilax corbularia Kunth., and Smilax glabra Roxb.Te authenticity of the plant materials was verifed by a qualifed and licensed traditional Tai pharmacist.As part of the reference collection process, samples of the remedies and each individual ingredient were gathered and subsequently preserved in the authors' herbarium, which was housed in the Department of Applied Tai Traditional Medicine, Walailak University, Nakhon Si Tammarat, Tailand (Table 1).

Preparation of Extraction.
Te extraction process was performed according to a meticulous protocol [14].Plant specimens were washed and desiccated at 55 °C for 24 h.After desiccation, the material was fnely pulverized to ensure uniformity.KKR, which is a blend of plant components, weighs approximately 60 g.Te preparation involved combining these components in equal proportions at a 1 : 1 : 1 ratio (20 g per plant component).One composite underwent ethanolic maceration, and the other was boiled in distilled water at 80 °C for 6 hours.Te extracts were rigorously fltered to remove impurities.Te ethanolic extracts were subjected to rotary evaporation, and the aqueous extracts were freeze-dried.All extracts were stored at 4 °C to preserve their bioactive constituents for subsequent bioassays.

α-Glucosidase Inhibitory Assay.
Te α-glucosidase inhibitory efect of KKR and its plant ingredient extracts was assessed using a colorimetric method [15].All samples, including acarbose (a standard drug), were dissolved in a 20% DMSO solution.Tese solutions were dispensed into a 96-well plate, each containing 200 µL, composed of 50 µL PBS (pH 7.0), 50 µL of 8 mg/mL sample solution (resulting in a fnal 2 mg/mL concentration), and 50 µL α-glucosidase enzyme solution (at 1 U/mL).Te plate was incubated at 37 °C for 2 minutes, and then 50 µL of 4 mM p-nitrophenylα-glucopyranoside solution was added.A microplate reader was used to measure the paranitrophenol content at 405 nm every 30 seconds for 10 minutes.Te percent inhibition was calculated using the following equation: where is the absorbance at 405 nm in the control sample without the extract, and A Control 405 is the absorbance at 405 nm after treatment with the extract.

Enzyme Kinetic Determination.
Te method for determining the mode of action and inhibition constant (Ki) against α-glucosidase inhibitory efect followed a precedent reference [16].Both the Lineweaver-Burk equation and Dixon plot were employed, with a slight modifcation in the enzymatic reaction.Tree concentrations of active crude extracts (25 to 500 µg/mL) were chosen based on their observed activity.Six diferent substrate concentrations (0.15625-5 mM) were used.Constant amounts of α-glucosidase were incubated with increasing substrate concentrations (PNPG) at 37 °C for 15 minutes, with or without samples (at concentrations equivalent to IC 50 ).Te necessary equations (2) and (3) were then applied to the analysis.
Line Weaver-Burk equation  [17,18].Te primary objective of this study was to provide insights into efective and safe strategies for managing diabetes, including the development of a novel Tai herbal remedy.

Antioxidant Activity (DPPH Radical Scavenging Assay).
Te free radical scavenging capacities of the test samples were determined following an established protocol [19].Each extract (100 µL) and a positive control solution (0.1-100 µg/mL) were combined in a 96-well plate.Ten, 100 µL of DPPH in methanol (6 × 10 −5 M) was added and mixed thoroughly.Te mixtures were kept in the dark for 30 minutes, and the absorbance was measured at 520 nm against a blank.Butylated hydroxytoluene (BHT) served as the standard.DPPH radical scavenging (RS) activity was determined using the following equation: 2.8.Total Phenolic and Total Flavonoid Contents.Total phenolics and favonoids in the crude extracts were evaluated Folin-Ciocalteu and aluminum chloride colorimetric methods [20,21].To estimate the total phenolic content, 100 µL of samples were mixed with 500 µL of 10% v/v Folin-Ciocalteu's reagent and 400 µL of 1 mM sodium bicarbonate.After incubation for 30 minutes, color intensity was measured at 765 nm.Gallic acid was used to generate a standard calibration curve.Te total favonoid content was quantifed using the aluminum chloride colorimetric method.A mixture of 100 µL 10% w/v aluminum chloride, 100 µL 1 M potassium acetate, 1,500 µL ethanol, and 500 µL samples was incubated for 30 minutes, and color intensity was measured at 415 nm.Quercetin served as the reference standard for calibration.

Gas Chromatography-Mass Spectrometric (GC-MS)
Analysis.Te analysis was conducted at the Scientifc and Technological Research Equipment Centre of Chulalongkorn University in Bangkok, Tailand [22].GC-MS analysis involved an Agilent Technologies 19091S-433 gas chromatograph (GC) coupled with an Agilent 5973 mass selective detector (MSD), controlled by Agilent Chemstation software.An HP-5MS capillary column (30 m length, 250 µm inner diameter, 0.25 µm flm thickness) was used, and ultrapure helium served as the carrier gas at 0.7 mL/ minute.Te injector temperature was maintained at 300 °C.Te initial oven temperature was set at 50 °C, increasing at 10 °C/minute until reaching 310 °C, and then held for 10 minutes.Injections of 1 µL were in splitless mode (manual Advances in Pharmacological and Pharmaceutical Sciences split ratio 10 : 1).Te mass spectrometer was operated in the electron ionization mode at 70 eV, with an electron multiplier voltage set at 1859 V. Other parameters included an ion source temperature of 230 °C, quadrupole temperature of 150 °C, solvent delay of 4 minutes, and a scan range from 50 to 700 amu.Compounds in the test samples were identifed by comparing their retention times (RT) and mass spectral data with those of standard compounds in the NIST library.

Statistical Analysis.
In this study, we averaged the values from three separate experiments and expressed them as the mean ± standard deviation (SD).To analyze the data, we used a statistical method called two-way analysis of variance (ANOVA) along with Tukey's post hoc test specifcally for total phenolic and favonoid contents.For the remaining results, we applied a diferent statistical approach called one-way analysis of variance (ANOVA) using the GraphPad Prism software.Te results were considered statistically signifcant if the p value was less than 0.05, which corresponds to a 95% confdence level.

Percentage Yield. KKR and its plant ingredient extracts
were subjected to a two-step process involving maceration in ethanol and boiling in distilled water.Te dried weights of each extract and their respective percentage yields are listed in Table 2. Te results reveal that, in each plant extract, aqueous extracts yielded higher percentages compared to the ethanolic extracts.Notably, the highest yield was obtained from the aqueous extract of P. amarus (9.91% w/w), whereas its ethanolic counterpart yielded only 8.20% w/w.For the KKR remedy itself, the aqueous extract exhibited a percentage yield of 6.11% w/w, while the ethanolic extract produced a percentage yield of 5.50% w/w.2).

Assessment of the Enzyme Kinetic Study of KKR.
Te enzyme kinetics study aimed to discern the type of inhibition exerted by the KKR extract, considering its traditional use as a remedy.Established methodologies, including the Lineweaver-Burk and Dixon equations, were used to elucidate the mode of action of the extracts.Analysis based on the Lineweaver-Burk equation revealed that KKRE exhibited noncompetitive inhibition with a Ki value of 0.672 mM.In contrast, the aqueous extract exhibited competitive inhibition, with a Ki value of 0.507 mM.Similarly, acarbose, a well-known inhibitor, demonstrated competitive inhibition, with a Ki value of 0.235 mM (Table 3 and Figure 3).

Combination Index Test of the KKR.
Acarbose was utilized in the combination index (CI) analysis of the two extracts, KKRA and KKRE, in support of the traditional use of KKR.A fxed IC 50 the amount of acarbose (170 µg/mL) was combined with varying amounts of each sample (which varied from IC 50 0.5-2).Tables 4 and 5 provide data on the CI test fndings.As shown in Figure 4 (y-axis) and Figure 5 (x-axis), the combination of KKRE and acarbose resulted in a fraction afected (Fa) greater than 0.5.Similarly, as shown in Figure 6 (y-axis) and Figure 7 (x-axis), the combination of   Advances in Pharmacological and Pharmaceutical Sciences  6 Advances in Pharmacological and Pharmaceutical Sciences 3.6.Total Phenolic and Total Flavonoid Contents.Te quantifcation of total phenolic and favonoid contents in the extracts of KKR and its individual components is presented in Figure 9.For the total phenolic content, the observed values varied within a range of 30.72 mg GAE/g to 319.50 mg GAE/g.Te highest total phenolic content was recorded for the ethanolic extract of S. corbularia, measuring 319.50 mg GAE/g, followed by KKRE (172.82 mg GAE/g).In contrast, the lowest value, (30.72 mg GAE/g) was observed in the ethanolic extract of S. glaba.Tis fgure also shows the favonoid content.Te ethanolic extract of S. corbularia exhibited the highest value (128.40 mg QE/g), followed by the aqueous extract of P. amarus at 85.49 mg QE/g.Te total favonoid content of KKRE was 77.41 mg QE/g.

Gas Chromatography-Mass Spectrometric (GC-MS)
Analysis.Te extract of KKRE demonstrated the most signifcant impact on α-glucosidase activity.Subsequently, gas chromatography-mass spectrometry (GC-MS) was conducted to determine its chemical composition.Te analysis revealed 86 compounds, of which 32 exhibited a matching score of >80%. Figure 10 shows the chromatogram of the compounds identifed in the KKRE extract.Te analysis identifed 32 compounds, which are listed in Table 6.Te most abundant compound detected was nhexadecanoic acid, also known as palmitic acid, with a retention time of 30.576 minute, constituting 15.67% of the total compound.Linolenic acid (14.06%), linoleic acid (6.75%), oleamide (3.71%), stearic acid (3.62%), oleic acid, and ethyl ester (1.75%) were identifed as the signifcant constituents.

Molecular Docking.
Eleven compounds present in KKRE that exhibited peak areas greater than 1% were selected for molecular docking.Table 7 provides information on the binding energy and amino acid residues of the α-glucosidase enzyme that interacts with each compound, including details of the hydrogen bonds and hydrophobic interactions.To identify the interacting amino acid residues and predict the binding modes of the compounds with      Advances in Pharmacological and Pharmaceutical Sciences  Advances in Pharmacological and Pharmaceutical Sciences with ARG213, HIS351, ASP352, and ARG442, and possessing a binding energy of −5.9 kcal/mol (Figure 12(a)).In addition, it engages in two hydrophobic interactions with residues PHE178 and VAL216.However, it is worth noting that these compounds interacted with fewer amino acids compared to acarbose, which formed thirteen hydrogen bonds with LYS156, TYR158, SER241, ASP242, GLU277, GLN279, HIS280, ARG315, HIS351, ASP352, and ARG442, resulting in a binding energy of −8.4 kcal/mol (Figure 11(b)).

Discussion
Delaying carbohydrate digestion and reducing glucose absorption through the inhibition of α-glucosidase represents a therapeutic avenue for managing type 2 diabetes (T2DM).Numerous studies have documented the efect of herbal medicine on the suppression of α-glucosidases [33,34].Krom Luang Chumphon Khet Udomsak remedy (KKR) has been used in Tai traditional medicine for centuries to treat DM [10].Te present study was undertaken to evaluate the potential anti-α-glucosidase and antioxidant activities of KKR and its plant ingredients, with a series of experiments conducted to substantiate their biological efects.From our results, both KKR remedies demonstrated a remarkably α-glucosidase inhibitory activity.Tese results suggest that the KKRE extract exhibited the highest inhibition of α-glucosidase activity through noncompetitive mechanisms, surpassing other extracts and demonstrating an IC 50 lower than that of the standard drug acarbose.Te presence of palmitic acid, identifed as a major component of KKRE through GC-MS analysis, suggests its potential to inhibit α-glucosidase, as previously reported [35].Importantly, the Ki value of KKRE associated with this mechanism was signifcantly higher than that of acarbose, suggesting a relatively low binding afnity of this extract for the enzyme [36].
Moreover, our fndings underscore the efcacy of ethanol as the optimal solvent for extracting secondary metabolites from KKR and its plant constituents, particularly evident in our observations of anti-α-glucosidase and antioxidant activities.Tis preference for ethanol arises from its intrinsic polarity, which allows the selective isolation of low molecular weight phenolic and favonoid compounds [37].Tese compounds are prevalent in plants and are associated with antidiabetic [38] and antioxidant [39] properties in biological systems.
Furthermore, our assessment of α-glucosidase inhibition across all extracts revealed that KKRA exhibited potent inhibition of the α-glucosidase enzyme, while KKRE demonstrated signifcantly higher α-glucosidase inhibitory activity compared to other extracts.Similarly, in terms of antioxidant activity, the ethanolic extract of S. corbularia displayed the highest efcacy.Palmitic acid, a fatty acid soluble in organic solvents [40], has been identifed as a signifcant contributor to α-glucosidase inhibition [35].Tis explains the superior results observed with the ethanolic extract of KKR and other plant components compared with the aqueous extract.In a previous study investigating herbal components, it was observed that the powdered ash obtained from P. amarus displayed inhibitory activity against α-glucosidase, with an IC 50 value of 982.13 ± 162.69 µg/mL [41].In addition, the ethyl acetate rhizome extract of S. glabra demonstrated inhibitory activity against α-glucosidase, with an IC 50 value of 5.5 µg/mL [42].Furthermore, the ethanolic rhizome extract of S. corbularia, at a concentration of 25 µg/mL, also displayed inhibitory activity against α-glucosidase at approximately 50% [43].Consequently, based on the results of the α-glucosidase inhibitory assay, it was determined that the KKR had potent inhibitory efects on the α-glucosidase enzyme.Tis observation aligns with previous research suggesting that phenolic and favonoid compounds can act as inhibitors of α-glucosidase, contributing to the regulation of hyperglycemia [44] and also can act as inhibitors of antioxidants activity [45].
Since, oxidative stress has been demonstrated to participate in the progression of diabetes which plays an important role during diabetes, including impairment of insulin action and elevation of the complication incidence [46].Oxidative stress involves the transfer of hydrogen or electrons from stable molecules to free radicals, which then convert them into stable molecules [47].Terefore, we assessed the DPPH free radical scavenging ability of KKR and its plant ingredients.According to our fndings, the ethanolic extract of S. corbularia demonstrated the highest inhibition of DPPH activity compared to the other extracts, and also exhibited the lowest IC 50 compared to the positive control, BHT.Te observed DPPH radical scavenging capacity of the ethanolic extract derived from S. corbularia rhizome can be attributed to the presence of polyphenolic compounds within the plant material as reported previously [48].As per a prior investigation, the ethanolic extract derived from the rhizome of S. corbularia demonstrated signifcant potency in inhibiting DPPH, with an IC 50 value of 9.24 ± 1.71 at concentrations ranging from 0.15 to 75 µg/mL [43].In our study, we observed that the ethanolic extract from S. corbularia, tested at concentrations ranging from 0.1 to 100 µg/mL, also exhibited notable potency in inhibiting DPPH, with an IC 50 value of 53.52 ± 0.59.Plants are abundant in polyphenols and favonoids, exhibit strong antioxidant activity, and have diverse defense and diseasefghting properties [49].Phenolics and favonoids present in medicinal plants and foods are essential components that contribute to a range of antidiabetic activities [50].Elevated levels of total phenolic content (TPC) and total favonoid content (TFC) serve as indicators of the potential therapeutic activities inherent in plant extracts [51].In a previous investigation on the TPC and TFC of the herbal components, it was determined that the ethanolic extract of S. corbularia contained quantifable amounts of phenolic and favonoid compounds, with Advances in Pharmacological and Pharmaceutical Sciences concentrations measuring 388.22 ± 1.92 mg GAE/g extract and 33.72 ± 1.18 mg QE/g extract, respectively.Similarly, the ethanolic extract of S. glabra was found to contain detectable levels of phenolic and favonoid compounds, with concentrations of 30.83 ± 1.18 mg GAE/g extract and 21.12 ± 0.54 mg QE/g extract, respectively [43].Tese fndings corroborate our observations that the ethanolic extract of S. corbularia exhibited higher TPC and TFC levels than those of S. glabra.GC-MS of the KKRE extract, which had the highest potential to inhibit α-glucosidase enzyme, revealed 86 compounds, among which 32 exhibited a matching score of more than 80%.Eleven compounds were selected for a better understanding of the compounds in KKRE, and molecular docking was performed.Tese compounds included pyranone, malic acid, pyrogallic acid, palmitic acid, ethyl palmitate, linoleic acid, linolenic acid, stearic acid, ethyl oleate, oleamide, and campesterin.Consequently, it can be inferred that KKRE induces a reduction in glucose levels in the presence of these compounds.Te main chemical constituents identifed in KKRE comprised palmitic acid, as reported in prior studies investigating the ethanolic leaf extract of P. amarus [52] and the methanolic extract of the Smilax China plant within the Smilax genus [53].Furthermore, margarinic acid was documented in the methanolic extract of Smilax zeylanica [54], while malic acid and oleic acid ethyl ester were also detected in the rhizome of the ethanolic Smilax domingensis extract [55].Te chemical constituents of KKRE exhibited α-glucosidase inhibitory activity; for instance, palmitic acid demonstrated potential inhibitory efects on the α-glucosidase enzyme [35], while malic acid also showed similar inhibitory potential [56].To explore the interactions of the identifed compounds with the α-glucosidase enzyme, we employed molecular docking, an integral part of in silico drug development to predict small molecule-protein interactions at the atomic level [57].Docking results revealed that campesterin exhibited the strongest binding afnity to the α-glucosidase enzyme, with a low binding energy of −8.9 kcal/mol.It forms a hydrogen bond with GLU277 and engages in hydrophobic interactions with residues TYR158, PHE159, PHE303, and ARG315.Tis fnding aligns with previous reports on the antidiabetic, cholesterol-lowering, anticarcinogenic [58], antioxidant, antibacterial, immunomodulatory, and anti-infammatory 14 Advances in Pharmacological and Pharmaceutical Sciences activities [59].In addition, linoleic acid has been identifed as a multi-target inhibitor associated with insulin resistance [60].
Tere has been a hypothesis that two inhibitors, each employing diferent modes of inhibition, might synergistically contribute to the inhibition of α-glucosidase [61,62].To test this hypothesis, we performed a combined assay.Te addition of KKR to the enzymatic reactions with acarbose revealed a synergistic efect, as evidenced by the CI value <1.Our fndings revealed, for the frst time, the potential of KKR extracts as a herbal medicinal therapy for diabetes.In addition, this information could ofer healthcare professionals valuable insights into the potential use of KKR in combination with the antidiabetic agent acarbose to enhance the control of blood glucose levels in diabetic patients.Tis may further contribute to our understanding of the potential synergies between KKR and other bioactive substances and medications.

Conclusions
Tis study represents the frst investigation of the biological activities of KKR, a Traditional Tai herbal remedy with a century-old history of managing various health conditions.Our fndings indicate that KKRA exerts potent inhibitory efects on the α-glucosidase via a competitive mechanism.In addition, KKRE exhibited the highest potency in inhibiting the α-glucosidase enzyme through a noncompetitive mechanism compared with the standard drug acarbose.Notably, the combination of KKR extract and acarbose had a remarkable synergistic efect on α-glucosidase inhibition.KKR and its plant ingredients exhibit potent antioxidant activities.Furthermore, a molecular docking study identifes campesterin, a constituent compound from KKRE, as having the most signifcant impact on α-glucosidase inhibition.Tese outcomes provide substantial evidence of the importance of KKR in traditional medicine and underscore its potential signifcance in contemporary therapeutic contexts.

10 Advances in Pharmacological and Pharmaceutical Sciences Table 7 :a
Te binding energy and amino acid residues of the α-glucosidase enzyme.Two interaction with amino acid residues.b Tree interaction with amino acid residues.

Figure 11 :
Figure 11: Predicted binding modes, H-bond and hydrophobic interactions of α-D-glucopyranose (a), acarbose (b), pyranone (c), and malic acid (d) with α-glucosidase.Te backbone of the α-glucosidase enzyme is presented in a blue-ribbon model, the yellow line dot represents the hydrogen bond, and the gray dot represents the hydrophobic interaction.

Figure 12 :
Figure 12: Predicted binding modes, H-bond and hydrophobic interactions of pyrogallic acid (a), palmitic acid (b), ethyl palmitate (c), and ethyl oleate (d) with α-glucosidase.Te backbone of the α-glucosidase enzyme is presented in a blue-ribbon model, the yellow line dot represents the hydrogen bond, and the gray dot represents the hydrophobic interaction.

Figure 13 :
Figure 13: Predicted binding modes, H-bond and hydrophobic interactions of linoleic acid (a), linolenic acid (b), stearic acid (c), oleamide (d), and campesterin (e) with α-glucosidase.Te backbone of the α-glucosidase enzyme is presented in a blue-ribbon model, the yellow line dot represents the hydrogen bond, and the gray dot represents the hydrophobic interaction.

Table 1 :
List of plant materials used in the study.

Table 2 :
Extraction yields of ethanolic and aqueous extracts of the medicinal plants in KKR.

Table 3 :
Te presented data showcases the kinetic parameters of α-glucosidase when exposed to the extract of KKRE and acarbose (standard drug).
* CI � Combination index.Advances in Pharmacological and Pharmaceutical Sciences α-glucosidase, 2D interaction diagrams were generated, as shown in Figures11-13.Among the eleven compounds, campesterin demonstrated the strongest binding afnity to the α-glucosidase enzyme, with a low binding energy of −8.9 kcal/mol, representing the highest observed afnity to an enzyme.Its afnity surpasses that of acarbose, the standard drug, which exhibits a binding energy of −8.4 kcal/ mol.Campesterin formed a hydrogen bond with GLU277 and exhibited hydrophobic interactions with TYR158, a binding afnity to the α-glucosidase enzyme with a binding energy of −6.8 kcal/mol.It strongly interacts with ARG213, ASP352, and ARG446 (Figure13(b)).Tis compound engages in eight hydrophobic interactions with residues TYR72, TYR158, PHE159, VAL216, PHE303, ASP352, and ARG442.Pyrogallic acid exhibited notable binding afnity to the α-glucosidase enzyme, forming seven hydrogen bonds

Table 6 :
Compounds identifed in the KKRE by GC-MS.