IN SILICO STUDY OF α-AMYLASE AND α-GLUCOSIDASE INHIBITORY COMPOUNDS IN Aloe vera AS ANTIDIABETIC AGENT

Type 2 diabetes mellitus (T2DM) is a chronic disease that poses a significant global health issue. The most commonly given drug to treat diabetes is acarbose, which works as an inhibitor of polysaccharide-degrading enzymes, such as α-amylase dan α-glucosidase so that the blood glucose level in T2DM patients could be reduced. This study aims to analyse five bioactive aloe vera compounds as inhibitors of α-amylase and α-glucosidase enzymes. This research was conducted through in silico studies based on molecular docking using PyRx 0.8 and Biovia Discovery Studio software, cytotoxicity testing through pkCSM and feasibility of medicinal materials through ADMET analysis. The results showed that hesperidin has a binding affinity of -9.1 kcal/mol against the α-amylase enzyme. Meanwhile, acarbose, as a control, showed a binding affinity of -8.5 kcal/mol. In addition, osajin, pomiferin, cosmosiin, and hesperidin had a binding affinity of -7.8, -8.1, -7.7, and - 8.8 kcal/mol, respectively. These values were also higher than acarbose (-7.3 kcal/mol). Three compounds, namely osajin, pomiferin, and aloesin, met the drug-likeness criteria and had absorption rates in the digestive tract between 31.48-96.59%. The toxicity analysis using the AMES test revealed that all compounds, except aloesin, did not exhibit properties that could potentially cause hepatotoxicity or promote mutagenesis. These results indicate that the bioactive compounds in Aloe vera have the potential to be used as antidiabetic agents through the inhibition of polysaccharide-degrading enzymes. Further research is needed to analyze the antidiabetic properties of the compounds in Aloe vera through in vivo studies with more optimal drug delivery innovations.


INTRODUCTION
Diabetes mellitus (DM) is a chronic, nontransmittable disease that represents a crucial issue in the world's health sector today.There are two types of diabetes mellitus, namely type 1 (T1DM) and type 2 (T2DM), with T2DM constituting 90-95% of cases.This rate of incidence is drastically increasing year by year [1].According to BPS data from 2019, the number of people with DM in Indonesia reached 10.6 million, while the International Diabetes Federation (IDF) reported 463 million cases of diabetes worldwide [2].WHO has predicted an increase of at least 21.3 million people in DM cases by 2030 [3].Therefore, the rise in diabetes mellitus cases will result in a burden in managing its treatment.
Diabetes is a metabolic disorder that causes increased blood glucose levels, also known as hyperglycemia, including type 2 diabetes (T2DM).The root cause of this disease is the decreased insulin levels from the pancreas, which leads to insufficient insulin secretion and impaired insulin activity or both [4].The key difference between both types is that T2DM often results in weight gain and decreased physical activity due to the body's inability to utilize insulin, and most patients are not dependent on insulin [5].This puts patients at risk for serious health problems, particularly cardiovascular disorders.Without appropriate treatment, chronic diabetes can cause organ failure and often leads to complications that can be fatal in the long term [6].
The primary approach to treat T2DM patients involves inhibiting polysaccharide-degrading enzymes, such as α-amylase and α-glucosidase.Blocking the activity of α-amylase could halt the degradation of amylase into shorter amylose chains, whereas inhibiting α-glucosidase could impede the conversion of disaccharides into simpler sugars like glucose [7].This inhibitory mechanism could also hinder the activity of αamylase to hydrolyse polysaccharides in the intestinal tract.The purpose of inhibiting these two enzymes is to reduce the digestion and absorption of carbohydrates into glucose in the digestive tract, resulting in decreased blood sugar levels in patients with T2DM by delaying glucose absorption.These mechanisms are the main targets with significant potential for the development of drug therapy for T2DM [8,9].
However, polysaccharides-degrading drugs such as acarbose, voglibose, and miglitol have strong side effects on the patient's body, such as gastrointestinal disorders, impaired liver and kidney function, and dizziness, nausea, and vomiting.According to doctors, these side effects can worsen patients' compliance with medication, leading to further risks to their health.Moreover, improper management of T2DM treatment, such as irregular treatment and incomplete drug combinations, can increase the risk of treatment failure [10,11].
The increasing prevalence of T2DM and the resistance to conventional drugs have led to the need for innovative solutions in diabetes management, such as using natural ingredients as alternative treatments.One such natural product is the bioactive compounds derived from the gel of the Aloe vera plant, which has been shown to possess anti-diabetic properties by reducing blood glucose levels and inhibiting the breakdown of polysaccharides into glucose, thus increasing insulin levels [12].The use of plant bioactive compounds as the main component of T2DM drugs is an essential prototype molecule for developing new conventional medicines [13].
Considering this potential, it is important to conduct a study to optimize the potential of bioactive compounds in A. vera based on in silico screening studies with molecular docking.This study will select bioactive compounds with the highest activity carried out through the prediction of candidate drug compound molecules and whether the compound molecules exhibit activity before being tested by aligning the ligand and the selected receptor by considering the nature of both.The goal is to mimic the interaction of a ligand molecule with the target protein in the in vitro test [14].This study aims to screen several bioactive compounds in A. vera that have the ability as antidiabetic agents through the mechanism of polysaccharide degrading enzyme inhibitors.Therefore, the in silico test in this research is expected to be an initial study for the development of new drugs for T2DM based on natural products with high effectiveness and no side effects.

3D structure of target protein preparation.
The 3D crystal structures of α-amylase enzyme (PDB ID: 1HNY) and α-glucosidase (PDB ID: 8D43) were retrieved from the Protein Data Bank database (https://www.rcsb.org/)and downloaded in pdb format.The 3D structures were opened and prepared using Biovia Discovery Studio 2021 software by removing water molecules, native ligands, and other residue molecules, then saved in pdb format.

Ligand preparation.
The bioactive compounds in A. vera were used as ligands against α-amylase and α-glucosidase enzymes with the mechanism of inhibiting the active site of the enzyme with polysaccharides.Acarbose was used as a control compound because it has the same mechanism.The ligand compounds were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) in sdf format.There were five ligand compounds tested, namely cosmosiin (CID 5280704), osajin (CID 95168), pomiferin (CID 4871), hesperidin (CID 10621), and aloesin (CID 160190), with acarbose (CID 41774) as the control.The identity of each compound can be seen in Table 1.The ligands were opened and prepared using PyRx 0.8 software by minimizing the molecule and then saved in pdb format.
Molecular docking simulation and analysis.Molecular docking simulations were performed using PyRx 0.8 software to determine the binding affinity between ligands and target proteins.The Vina Wizard tab was selected in the PyRx software and loaded the prepared proteins and ligands.Acarbose, which is the α-amylase and αglucosidase inhibitor drug, was used as a control.The binding site's dimensions and amino acid residues were controlled by the initial imposing of acarbose with the α-amylase and α-glucosidase enzymes.The binding dimensions for the αamylase enzyme were found to be X: 25.9457 Å; Y: 28.2985 Å; Z: 20.1066 Å, and the dimensions for the α-glucosidase enzyme were X: 23.4080 Å; Y: 18.0569 Å; Z: 27.3834 Å.Five A. vera compounds were used as ligands and docked to both enzymes using the same dimensions as acarbose.The docking results for all ligands to both enzymes are presented in a table, showing the binding affinity and amino acid residue profiles.The binding affinity between the ligand and protein is measured in units of Gibbs free energy (ΔG), which has a negative value.The more negative the ΔG value, the stronger the bond formed.Potential protein-ligand complexes characterized by higher binding strengths than the control will then be visualized [14,24,29,32].
Protein-ligand complex visualization.The potential protein-ligand complexes were visualized for their binding morphology using Biovia Discovery Studio 2021 software for 2D visualization and UCSF Chimera for 3D visualization.Two-dimensional visualization can facilitate the analysis of the structure, distance, and types of bonds between the ligand and receptor molecules.
Pharmacological characteristics analysis.Pharmacological characteristic predictions were performed to determine the safety and bioavailability level of the compounds as drug candidates.The feasibility was predicted on the the canonical SMILES index obtained from PubChem.Feasibility predictions were performed using Lipinski's Rule of Five parameters, which included molecular weight, hydrophobicity, hydrogen bond donors, and hydrogen bond acceptors.The analysis results were then displayed in the form of a table.
Toxicity prediction.Toxicity predictions for the compounds were performed using the pkCSM webserver (https://biosig.lab.uq.edu.au/pkcsm/), and the prediction results were displayed in the form of a table.

RESULTS AND DISCUSSION
Prior research on antidiabetic properties of Aloe vera. A. vera has various bioactive compounds that have the potential to be used as natural-based diabetes medication.In vivo studies using male albino Wistar rats induced with type 1 diabetes via streptozotocin injection at a dose of 50 mg/kg body weight showed that A. vera gel extract could decrease blood glucose concentration and increase insulin levels compared to the control group [15].A. vera extract was also able to significantly reduce malondialdehyde (MDA), superoxide dismutase (SOD) levels and increase blood glutathione (GSH) levels, with a better effect compared to the current commercial drug for type 2 diabetes, glimepiride [15].MDA is a toxic free radical produced from lipid oxidation by products, and its increase in the blood indicates the development of diabetes mellitus [16].GSH, on the other hand, is an antioxidant commonly found in mammalian cells, and abnormalities in GSH concentration indicate the development of diseases such as Alzheimer's, Parkinson's, cancer, and diabetes [17].An in vitro study using female albino rat pancreatic Langerhans cell cultures also showed that A. vera extract could increase insulin secretion in cell cultures compared to the control group [15].
The methanol extract of A. vera has been proven to inhibit the glycation reaction of Bovine Serum Albumin (BSA) protein through the oxidative degradation of fructosamine [18].Fructosamine is an intermediate compound for the formation of advanced glycation end products (AGEs), which are the end products of proteins or lipids that have been glycated due to exposure to sugar [19].A. vera methanol extract has also been shown to inhibit the activation of several polysaccharidedegrading enzymes, such as α-amylase and αglucosidase, thus reducing blood glucose levels [18].
Pharmacological characteristics analysis.The potential of bioactive compounds in A. vera as inhibitors of polysaccharide-degrading enzymes needs to be further explored through in silico molecular docking approaches.These compounds were first analyzed for their physicochemical aspects to validate their safety as drug candidates using Lipinski's rule of five.According to Lipinski's rule, a compound can be considered a drug candidate if it meets the following criteria: molecular weight (MW) ≤ 500 daltons; number of hydrogen acceptors (HA) ≤ 10; number of hydrogen donors (HD) ≤ 5 daltons, and lipophilicity (LogP) ≤ 5.An orally active drug should comply with a maximum of one violation of these criteria [20].Screening results for five compounds in A. vera showed that three compounds, osajin, pomiferin, and aloesin, met Lipinski's rule of five, which enabled these three compounds to be promising drug candidates for oral delivery and adequate capacity to be transported along tissues within human's body.Whereas there were violations for cosmosiin and hesperidin, LogP scores indicated the likeliness of nature for solubility in water, and it was interesting to figure out more about their activity in molecular scope [21] (Table 1 & 2  Acarbose is a commercial drug used for T2DM.It works by inhibiting the enzymes α-amylase and α-glucosidase.Both enzymes play a role in breaking down polysaccharides into monosaccharides in the digestive tract, allowing them to be absorbed by the small intestine.By inhibiting these enzymes, acarbose can slow down the process of glucose breakdown and absorption in the small intestine, preventing a significant increase in blood glucose levels [22].However, the use of acarbose in patients with diabetes mellitus often causes serious side effects such as diarrhoea, stomach distension, bloating, dyspepsia, and prolonged disturbance of sugar metabolism [23]. Molecular docking results.Molecular docking tests were conducted to analyze the strength and stability of the bonds formed by five bioactive compounds in A. vera towards the enzymes αamylase (PDB ID: 1HNY) and α-glucosidase (PDB ID: 8D43), with acarbose as the control.The test results showed that one compound had a higher binding affinity than acarbose in binding to the αamylase enzyme, as well as four compounds that could bind more strongly to the α-glucosidase enzyme (Figure 1 & 2).
The potential compound that can act as an inhibitor of the α-amylase enzyme is hesperidin, with a binding affinity of -9.1 kcal/mol, higher than acarbose which is -8.5 kcal/mol.Meanwhile, four potential compounds as inhibitors of the αglucosidase enzyme are osajin, pomiferin, cosmosiin, and hesperidin, with binding affinity values of -7.8 kcal/mol, -8.1 kcal/mol, -7.7 kcal/mol, and -8.8 kcal/mol respectively, also higher than acarbose which is -7.3 kcal/mol.The binding interactions formed between these A. vera compounds and the α-amylase and α-glucosidase enzymes are relatively stable, as evidenced by the similarity of amino acid residues formed with acarbose as a control (Table 3 & 4).
The amino acid residues marked in bold indicate the presence of common interactions formed between A. vera compounds and acarbose.Among these amino acid residues, glycine (GLY) is the most essential type for forming bonds between the ligand and α-amylase receptor, with four bonds in the control compound.On the other hand, for the α-glucosidase receptor, isoleucine (ILE) is the most essential amino acid, with three bonds present in the control compound.

Pharmacokinetics and toxicity prediction.
The pharmacokinetic characteristics of the bioactive compounds of A. vera have an advantage in higher absorption rates compared to acarbose as a control.The absorption rates of the bioactive compounds of A. vera via the gastrointestinal tract range from 31.48 -96.59% (Table 5).All compounds -including acarbose -are unable to diffuse freely through the blood-brain barrier.In terms of toxicity, all candidate bioactive compounds are safe as they do not show toxic properties based on AMES testing.The AMES test shows the potential of a compound to induce mutagenesis and is categorized as a carcinogenic substance [24].In addition, the bioactive compounds of A. vera are predicted to have no potential as hepatotoxic compounds, except for aloesin.The solubility of the compounds in water needs to be considered when determining the administration route to improve the effectiveness of compound absorption at the target.

The effectiveness of bioactive compound in
Aloe vera as an antidiabetic agent.The screening results of the five A. vera compounds showed that hesperidin has the highest binding strength to the α-amylase and α-glucosidase enzymes compared to acarbose.The utilization of hesperidin as an antidiabetic agent has been studied by scientists.Previous research on male albino rats induced with a high-fat diet and streptozotocin showed that administration of hesperidin at a dose of 50 mg/kgBW/day for 30 days could increase insulin secretion by pancreatic β cells and lower blood glucose levels.Hesperidin acts as an antioxidant that can prevent oxidative stress, thus increasing insulin receptor sensitivity in peripheral tissue cells, making them more effective in absorbing glucose [25].In addition, the compound profile data from the web server showed that hesperidin was not toxic to liver cells (hepatocytes) and did not cause skin sensitization when given at a maximum dose of 0.525 log mg/kgBW/day.Knowing its promising potential as an antidiabetic Table 5.The pharmacokinetic profile and toxicity prediction of the A. vera's bioactive compounds and acarbose agent, isolating hesperidin from A. vera extract at the appropriate dose can certainly become the latest solution in the management of T2DM.
Osajin is one of the isoflavones from A. vera, which acts as an antidiabetic agent [26].Osajin administration was able to improve blood glucose levels, insulin levels, and C-peptide numbers towards normal values in diabetic model mice [27].The structure of osajin allows this compound to have a key role in improving diabetes conditions.Evidently, the structure and molecular formula of osajin are quite similar to another antidiabetic isoflavone compound, scandenone (C25H24O5), so it has similar efficacy [28].Thus, many studies have examined the role of osajin as a superior agent in PDE5 inhibition in diabetes [29].
Pomiferin showed less efficient interactions than hesperidin.However, several studies have shown the efficacy of pomiferin on diabetic symptoms.One of the symptoms of diabetes can be observed through significant weight loss due to decreased sugar metabolism and increased protein metabolism from body tissues [30].Earlier studies showed that when diabetic model rats were administered with pomiferin at a dosage of 300 mg/kg bw, they were able to maintain their weight loss, unlike the control group, which had a significant decrease in their weight [31].Also, in the analysis of blood glucose levels, pomiferin can be referred to as one of the anti-diabetic agents through the interaction of PDE5 enzyme inhibition, thus increasing insulin synthesis [28].
In vitro tests on T3-L1 adipocyte cell culture showed that cosmosiin compounds have antidiabetic effects through increased adiponectin secretion, insulin receptor-β (IR-β) phosphorylation, and GLUT4 translocation.This indicates that cosmosiin activity has insulin-like properties in stimulating glucose uptake into muscle cells and adipocytes, so it proves useful in managing T2DM [31].In vivo and in vitro tests, cosmosiin bioactive compounds are also able to produce better hypoglycemic effects compared to antidiabetic drugs such as metformin and glibenclamide, as evidenced by a significant improvement in plasma lipid profiles [32].In addition, cosmosiin, which is a flavonoid group, also has an antidiabetic mechanism by strongly inhibiting the digestive enzymes -glucosidase of the intestine and -amylase of the pancreas [33].
Aloesin is one of the components found in A. vera leaf pulp.A previous study found the modulating effect of adiponectin by aloesin as an active component in Aloe ferox extract during their extensive assays and screening [34].Adiponectin is a hormone released from adipose tissue (fat) that helps with insulin sensitivity and inflammation.Adiponectin deficiency has been linked to a number of diseases, including obesity, type 2 diabetes, and atherosclerosis [35].In addition, aloesin has antioxidant properties, especially its radical scavenging activity [36].In accordance with the finding that adiponectin hormone was found in conditions of high glucose-induced ROS suppression based on in vitro models [37].Therefore, aloesin can work synergistically as an antidiabetic compound with its ability as an antioxidant.

CONCLUSION
The bioactive compound found in A. vera has the potential to be used as an antidiabetic agent in the treatment of T2DM by inhibiting the degradation of polysaccharides into glucose by the enzymes α-amylase and α-glucosidase.The inhibition of these enzymes prevents the breakdown of polysaccharides, thus hindering the elevation of blood glucose levels.Further studies are indispensable to assess the efficacy of A. vera bioactive compounds as drug candidates for T2DM, mainly through in vivo studies.

Figure 1 .
Figure 1.Molecular docking visualization of α-amylase enzyme with ligand (A) acarbose, and (B) hesperidin.Cyan and red colours represent the enzyme and A. vera's bioactive compound, respectively.

Table 1 .
). 3D and 2D structure of A. vera bioactive compounds and acarbose

Table 3 .
Molecular docking results of A. vera's bioactive compounds with α-amylase enzyme

Table 4 .
Molecular docking results of A. vera's bioactive compounds with α-glucosidase enzyme