INTRODUCTION:

Diabetes mellitus (DM) has recognized as a global health problem with increasing cases in forthcoming years. A total of 6.28% of the global populations were affected by DM, contributing to the ninth cause of mortality worldwide¹. Further, it is estimated that around 642 million people will suffer from DM in 2040². With those conditions, health management and prevention play a vital role in delaying DM development day by day.

Health management has been applied to halt DM's progression, including lifestyle changes and dietary intervention^3-5. A few nutritional compositions, particularly natural products^6–8, have been suggested for people with DM, including legumes consumption⁹. One of the high potential legumes for dietary intake in DM conditions is Dolichos lablab (DL). With the high content of fibres and other nutritional compositions, DL has a good potential as a dietary supplement for DM¹⁰. Previous studies have reported DL's efficacy in regulating glycaemic levels, despite the precise mechanism still unresolved^11,12.

Since metabolic disease like DM involved many proteins for its progression, targeting specific proteins becomes the promising way to develop anti-diabetic drugs¹³. Altering glucose metabolism and insulin signalling can turn into an effective way of controlling DM^13,14. As previously described, ɑ-amylase plays a role in starch metabolism and contributes to plasma glucose enhancement¹⁵. On the other hand, DPP4 and PTP1B orchestrate insulin signalling, mainly involved in insulin sensitization, secretion, and post-prandial blood glucose levels^16,17. Some studies have been employed to inhibit those proteins for achieving average blood glucose concentration and improving insulin performance^18–20. Thus, targeting ɑ-amylase, DPP4, and PTP1B have favourable results in preventing DM advancement^21–25. Nevertheless, there were no studies for understanding the role of bioactive compounds in DL to inhibit those proteins and regulate DM conditions. Therefore, this study will discover the potential bioactivity of DL as dietary intervention for DM based on nutritional and phytochemical contents using computational approach.

MATERIALS AND METHODS:

Plant Samples and Extraction:

Sample was obtained from Madura Island, Indonesia. Detailed characteristics of the sample as explained in previous work²⁶. Bean was ground prior to the extraction process. Extraction was carried out by soaking in 96% ethanol in a 1:3 ratio (weight/volume) for 24 hours. After submerging, the solvent was evaporated and freeze-dried to obtain DL extract.

Total Protein, Crude Fat, Crude Fiber, and Gross Energy Determination:

Determination of total protein, crude fat, and crude fiber was performed according to a previously described method²⁷. Gross energy was measured using IKA C2000 Calorimeter System (IKA Works, Germany) following the manufacturer's protocol.

Bioactive Metabolites Identification:

Thermo Scientific Dionex Ultimate 3,000 RSLCnano Liquid Chromatography (LC) linked with Thermo Scientific Q Exactive High Resolution Mass Spectrometry (HRMS) was employed to identify the presence of bioactive compound in DL extract. Detailed protocols for chromatography as mentioned in earlier work ²⁸. Total ionic chromatograms then analyzed using Compound Discoverer and matched with mzCloud in the MS/MS Library. Compound with match score higher or equal with 80 then selected for molecular docking simulations as the ligands.

Data Mining of Protein and Ligand Structures:

Three-dimensional (3D) structures of protein were retrieved from Protein Data Bank (PDB; https://www.rcsb.org/), while 3D ligand structures were obtained from PubChem database (https://pubchem.ncbi.nlm.nih.gov/). In detail, the structures of protein used in this study are ɑ-amylase (PDB ID: 1HNY), DPP4 (PDB ID: 5Y7K), and PTP1B (PDB ID: 1BZC). The list of phytochemicals and their identity (PubChem CID) as shown in table 2²⁸.

Binding Energy Calculations:

AutoDock Vina integrated into PyRx software was employed for molecular docking simulations^29,30. Water molecules and the previous-attached ligand in each 3D protein structure were removed prior to the docking process. Protein structures were set as a rigid molecule, while the phytochemicals as the ligands were set as a flexible molecule. Blind docking was applied with a maximum grid setting for searching binding sites.

Data Analysis:

Complex with binding energy lower or equal with -7 kcal/mol was directed into further investigation for amino acid-ligand interaction. Interacted residues in each complex and visualization were analyzed using Discovery Studio 2019 to determine the chemistry of formed interaction.

Drug-Likeness Evaluation and Structure-Activity Relationship (SAR):

The drug-likeness characteristics was determined by Swiss-ADME webserver³¹, while Molinspiration (https://www.molinspiration.com/cgi-bin/properties) was used to determine SAR of selected compounds based on the molecular docking result.

RESULT:

Nutritional Values of DL:

The protein was the higher constituent from the analyzed nutritional contents, followed by crude fibers, while fat content has the smallest portion. Protein constitutes 24.91±0.08% of the total contents, while crude fiber and fat have 7.03±0.02% and 0.36±0.01%, respectively. Also, gross energy measurement showed that DL has a high energy source for daily energy uptake (table 1).

Table 1. Nutritional value of DL.

Protein (%)	Fat (%)	Crude Fiber (%)	Gross Energy (kcal/g)
24.91±0.08	0.36±0.01	7.03±0.02	3.86±0.007

Bioactive Compounds in DL:

Phenolic acid and flavonoid were the most abundance compound in DL (table 2). Pipecolic acid, trans-3-Indoleacrylic acid, caffeine, choline, and trigonelline were major constituent according to peak area. Some amino acids like arginine and histidine also found since DL is a legumes species. In addition, a common isoflavones in legumes, daidzein, also identified in DL extract. From the screening revealed that DL has diverse phytochemical compounds with possible potential to contribute in biological mechanism, particularly for health purposes. The identified compounds from this step then continued for the screening of anti-diabetic

Properties.

Table 2: Identified bioactive compounds from ethanolic extract of DL using LC-HRMS

Name	Formula	Molecular Weight	Retention Time (min.)	Area (max.)	PubChem CID
Octyl decyl phthalate	C₂₆ H₄₂ O₄	418.308	0.54	936,248.27	8380
L-Histidine	C₆H₉ N₃ O₂	155.06944	0.778	2,000,058.30	6274
DL-Arginine	C₆ H₁₄ N₄ O₂	174.11162	0.784	10,687,018.08	232
Trigonelline	C₇ H₇ N O₂	137.04752	0.853	114,012,627.64	5570
Betaine	C₅H₁₁ N O₂	117.07902	0.854	5,520,343.02	247
N3,N4-Dimethyl-L-arginine	C₈ H₁₈ N₄ O₂	202.14284	1.258	7,730,350.73	169148
N6-Methyladenine	C₆ H₇ N₅	149.06996	1.277	3,306,074.91	67955
Pipecolic acid	C₆ H₁₁ N O₂	129.07887	1.289	541,352,086.71	849
Adenine	C₅ H₅N₅	135.05433	1.314	36,842,152.69	190
Nicotinic acid	C₆ H₅ N O₂	123.0321	1.328	6,123,811.35	938
2-Hydroxyphenylalanine	C₉ H₁₁ N O₃	164.04732	1.348	7,003,574.34	91482
δ-Valerolactam	C₅ H₉ N O	99.06864	1.459	5,682,585.39	12665
4-Piperidone	C₅ H₉ N O	99.06864	1.61	10,433,494.32	33721
L-(+)-Arginine	C₆ H₁₄ N₄ O₂	174.11162	1.714	1,196,106.39	6322
Senkyunolide H	C₁₂ H₁₆ O₄	206.09422	1.738	11,410,856.53	13965088
N-Acetyldopamine	C₁₀ H₁₃ N O₃	195.08943	1.996	11,282,218.02	100526
trans-3-Indoleacrylic acid	C₁₁ H₉ N O₂	187.06311	2.019	360,015,558.16	5375048
4-Indolecarbaldehyde	C₉ H₇ N O	145.05263	2.023	7,753,170.99	333703
Ferulic acid	C₁₀ H₁₀O₄	194.05786	2.194	18,955,718.90	445858
8-Hydroxyquinoline	C₉ H₇ N O	145.05263	2.445	42,324,020.88	1923
4-Hydroxybenzaldehyde	C₇ H₆O₂	122.03679	2.49	22,922,938.63	126
Sinapinic acid	C₁₁ H₁₂ O₅	224.06836	2.578	16,788,350.39	637775
Pyrogallol	C₆ H₆ O₃	126.03161	2.686	7,197,375.67	1057
Caffeine	C₈ H₁₀ N₄O₂	194.08026	2.765	130,944,470.60	2519
4-Coumaric acid	C₉ H₈ O₃	164.04722	3.255	12,099,306.61	637542
Isovanillic acid	C₈H₈ O₄	168.04215	3.264	2,838,264.12	12575
Icariside B	C₁₉ H₃₀ O₈	386.19365	3.398	4,745,053.93	45783010
Jasmonic acid	C₁₂ H₁₈ O₃	210.12553	3.823	14,410,134.05	5281166
7-Methyl-3-methylene-6-(3-oxobutyl)-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-2-one	C₁₅ H₂₀ O₃	230.13053	4.611	7,148,969.45	540288
Psilostachyin B	C₁₅ H₁₈ O₄	262.12035	4.615	2,093,036.74	5320768
Maltol	C₆ H₆ O₃	126.03165	4.69	2,094,936.46	8369
Butyl benzoate	C₁₁ H₁₄ O₂	178.09931	4.766	2,374,368.41	8698
Scopoletin	C₁₀ H₈ O₄	192.04213	4.91	38,999,675.85	5280460
Rutin	C₂₇ H₃₀ O₁₆	610.15325	4.919	461,606.60	5280805
D-(+)-Camphor	C₁₀ H₁₆ O	152.12008	4.926	5,934,546.27	159055
Isoquercetin	C₂₁ H₂₀ O₁₂	464.09508	5.093	773,077.83	5280804
Citral	C₁₀ H₁₆ O	152.12007	5.191	23,207,422.31	638011
(3aR,8R,8aR,9aR)-8-Hydroxy-8a-methyl-3,5-bis(methylene)decahydronaphtho[2,3-b]furan-2(3H)-one	C₁₅ H₂₀ O₃	248.14108	5.673	2,132,451.00	23928145
Ilicic Acid	C₁₅ H₂₄ O₃	274.15422	6.365	332,753.11	496073
Ageratriol	C₁₅H₂₄ O₃	234.1619	6.369	3,667,818.31	181557
Daidzein	C₁₅ H₁₀ O₄	254.05779	6.37	2,651,879.82	5281708
9S,13R-12-Oxophytodienoic acid	C₁₈H₂₈ O₃	292.20354	7.329	3,829,088.87	14037063
Oleanolic acid	C₃₀ H₄₈ O₃	456.35965	7.585	8,995,648.05	10494
9-Oxo-10(E),12(E)-octadecadienoic acid	C₁₈H₃₀ O₃	294.21916	7.723	3,519,971.30	5283011
19-Nortestosterone	C₁₈ H₂₆ O₂	274.19298	7.934	153,999.92	9904
Ursolic acid	C₃₀ H₄₈ O₃	456.35964	8.122	44,150,341.55	64945
OPEO	C₁₆H₂₆ O₂	250.19298	8.456	307,176.99	201055
Dimethomorph	C₂₁ H₂₂ClNO₄	387.12352	9.059	186,440.71	5889665
19-Norandrostenedione	C₁₈ H₂₄ O₂	272.1772	9.812	105,991.24	92834
α-Eleostearic acid	C₁₈ H₃₀ O₂	278.22432	10.443	3,022,006.29	5282820
(+/-)12(13)-DiHOME	C₁₈ H₃₄ O₄	296.23476	10.465	6,551,886.55	5282961
Benzoic Acid	C₁₅ H₂₂ O₃	250.15666	10.575	866,671.61	15007
1-Tetradecylamine	C₁₄ H₃₁ N	213.24547	11.03	2,455,695.81	16217
Methyl palmitate	C₁₇ H₃₄ O₂	287.28197	11.037	8,756,300.57	8181
Diazinon	C₁₂H₂₁N₂O₃P S	304.10073	11.727	262,096.73	3017
Tributyl phosphate	C₁₂ H₂₇ O₄ P	266.16435	11.908	597,823.49	31357
Nootkatone	C₁₅ H₂₂ O	218.16689	12.628	253,913.08	1268142
Galaxolidone	C₁₈ H₂₄ O₂	272.1772	12.967	500,319.44	69131857
Dibutyl phthalate	C₁₆ H₂₂ O₄	278.15139	13.031	76,116,574.22	3026
Bis(2-ethylhexyl) amine	C₁₆ H₃₅ N	241.27667	13.62	436,429.49	7791
Mesterolone	C₂₀ H₃₂ O₂	304.23998	13.835	1,250,518.08	15020
Citroflex A-4	C₂₀ H₃₄ O₈	402.22486	14.317	552,076.62	10222764
1-Linoleoyl glycerol	C₂₁ H₃₈ O₄	354.27628	15.062	859,472.95	5283469
Oleoyl ethanolamide	C₂₀ H₃₉ N O₂	325.29772	15.646	2,237,329.66	5283454
Palmitoyl ethanolamide	C₁₈ H₃₇ N O₂	299.28206	15.739	4,281,993.75	4671
Monoolein	C₂₁ H₄₀ O₄	356.29213	16.57	491,056.13	5283468
Oleamide	C₁₈ H₃₅ N O	281.27153	17.112	3,894,904.96	5283387
Hexadecanamide	C₁₆ H₃₃ N O	255.25594	17.826	1,591,948.23	69421
Eicosapentaenoic acid ethyl ester	C₂₂ H₃₄ O₂	330.25536	18.249	3,519,978.76	9831415
(9cis)-Retinal	C₂₀ H₂₈O	284.2134	18.25	23,205,473.84	6436082
Bis(2-ethylhexyl)adipate	C₂₂ H₄₂ O₄	370.30776	19.249	648,409.29	7641
Phthalic acid	C₈ H₆ O₄	166.02602	19.249	468,285.38	1017
Bis(2-ethylhexyl) phthalate	C₂₄ H₃₈ O₄	390.27613	19.257	66,697,492.22	8343
Stearamide	C₁₈ H₃₇ N O	283.28713	20.144	1,184,511.61	31292
Choline	C₅ H₁₃ N O	103.09976	25.18	128,907,165.10	305

Potential Mechanism of Phytochemicals from DL in Diabetic Pathway:

Eighteen compounds could interact with a minimum of one of the target proteins at low binding energy. Ursolic acid, rutin, and 19-Nortestosterone are the compounds with the lowest binding energy for ɑ-Amylase, DPP4, and PTP1B, respectively (table 3). Unfortunately, not all of the screened compounds have good potential for protein target inhibitors. Protein-ligand structure analysis revealed that only 14 compounds could interact directly with several essential residues in each targeted protein (figure 1-3).

Table 3. Selected compounds based on binding affinity lower than or equal to –7 kcal/mol.

Compound	Binding Energy (kcal/mol)
Compound	ɑ-Amylase	DPP4	PTP1B
(3aR,8R,8aR,9aR)-8-Hydroxy-8a-methyl-3,5-bis(methylene)decahydronaphtho [2,3-b]furan-2(3H)-one	-7.9	-8.4	-6.6
(9cis)-Retinal	-7.0	-8.0	-6.4
19-Norandrostenedione	-8.4	-8.6	-7.1
19-Nortestosterone	-8.0	-8.9	-9.3
Icariside B1	-7.4	-7.8	-6.8
Coumaric acid	-6.0	-6.2	-7.2
Daidzein	-8.1	-7.7	-7.8
Galaxolidone	-8.2	-8.2	-6.4
Ilicic Acid	-7.7	-8.3	-7.1
Isoquercetin	-8.2	-8.1	-7.2
Mesterolone	-8.6	-8.6	-6.8
Nootkatone	-7.5	-7.6	-6.2
Oleanolic acid	-9.5	-8.9	-8.5
Psilostachyin B	-7.8	-8.8	-7.1
Rutin	-8.8	-9.1	-7.6
Scopoletin	-5.8	-6.7	-7.2
Trans-3-Indoleacrylic Acid	-6.5	-7.0	-7.4
Ursolic acid	-10.1	-8.9	-7.9

Ursolic Acid, Oleanolic Acid, Isoquercetin, Psilostachyin B, Rutin, 9-cis-Retinal, and Icariside B1 were the compounds that been able to bind directly to the active sites of ɑ-Amylase. Those compounds could interact with the ɑ-Amylase mostly at HIS305 by hydrophobic or hydrogen bond interaction. Some compounds also bind with other key residues in the active sites, including ASP197, GLU233, and ASP300. Rutin and oleanolic acid are the compounds with the most binding sites in the active sites of ɑ-Amylase with three different interaction at the key residues (figure 1).

Figure 1. Structural orientation and residues interaction of ɑ-amylase along with ursolic acid (A, H), oleanolic acid (B, I), isoquercetin (C, J), psilotachyin B (D, K), rutin (E, L), 9cis-retinal (F, M), and icariside B1 (G, N).

Different from the ɑ-Amylase, DPP4 has higher selectivity to bind with the analyzed compounds. There were three compounds bound to DPP4 at the active sites, i.e., Isoquercetin, Rutin, and Icariside B1. GLU205, GLU206, TYR547, SER630, HIS740 were the active sites of DPP4, which interacted with all of those three compounds. Interestingly, Isoquercetin and Rutin have similar binding sites with one additional interaction of catalytic residues at ARG125 (figure 2).

Figure 2. The visualization of structural orientation and residues interaction of DPP4 along with isoquercetin (A, D), rutin (B, E), and icariside B (C, F).

Seven compounds could bind with the PTP1B at its catalytic sites. 19-Nortestosterone, Ilicic Acid, 19-Norandrostenedione, Scopoletin, Coumaric Acid, Trans-3-Indoleacrylic Acid, and Daidzein were the compounds that have interaction with the catalytic sites of PTP1B. Remarkably, Scopoletin and Trans-3-Indoleacrylic Acid were the compounds that could interact with more catalytic residues. In contrast, Daidzein was the compound that has less interaction with catalytic residues. In general, PHE182, ALA217, and ARG221 are the most preferred residues of those compounds (figure 3).

Figure 3. Structural orientation and residues interaction of PTP1B along with 19-Nortestosterone (A, H), ilicic acid (B, I), 19-Norandrostenedione (C, J), scopoletin (D, K), coumaric acid (E, L), trans-3-indoleacrylic acid (F, M), and daidzein (G, N).

Drug Likeness Characteristics of Screened Phytochemicals:

Drug-likeness properties and SAR were predicted using Swiss-ADME webserver and molinspiration, respectively. Six criteria, including lipophilicity, molecular size, polarity, insolubility, unsaturation, and flexibility, were employed to predict the drug-likeness properties of each screened compound. The pink areas represent the most favorable criterias with high similarity as the drug. Accordingly, 19-Norandrostenedione, 19-Nortestosterone, Icariside B1, Ilicic Acid, and Psilostachyin B were the compounds with the most resemblance with drug (figure 4A). Further, SAR prediction discovered that nine out of fourteen compounds have potential as both enzyme inhibitors and nuclear receptor ligands (figure 4B).

Figure 4. Drug-likeness properties of each screened compound according to bioavailability radar from Swiss-ADME (A) and SAR prediction using Molinspiration (B).

DISCUSSION:

Natively grown in Africa and Indian subcontinent, DL has been labeled as underutilized crops due to its limited global market potential and unpopular nutritional sources ¹⁰. Nevertheless, DL has been used in different regions of the world as human food and animal feed³². Consisting of adequate main macronutrients needed for daily food intake, DL has promising potential as nutritional therapy for several metabolic diseases including DM¹⁰. Diet management has been suggested for diabetic patients to maintain plasma glycemic levels^33,34. Consuming high fiber and protein content can increase insulin response and prevent plasma glycemic augmentation^33,35–37. Also, low-fat nutritional sources help fulfill energy requirement and prevent cardiovascular risk³³. With the high protein, fibers, and low-fat composition, DL has worthy potential for dietary intervention in diabetes management.

Phenolic acid is major secondary metabolite founded in DL, particularly in raw beans³⁸. Some phenolic acids including ferulic acid and coumaric acid make several major phenolic acid in DL, and those compounds were identified and confirmed at present study³⁹. Other dominant polyphenol compound, rutin, also identified³⁹. Phenolic acid has been proved to exhibits an anti-diabetic nature, particularly by inhibiting ɑ-amylase ^{21,23,24,40–42}. Therefore, this result discover a wide potential of DL as anti-diabetic agent.

Regulating glucose metabolism and insulin performance are the key factors in diabetes management^21,24,43,44. An enzyme called ɑ-amylase plays a vital role in glucose metabolism from dietary intake ⁴⁵. Targeting its catalytic sites could lead to inhibition of the catalytic activity of ɑ-amylase then prevent uprising glycemic levels^15,19. In the present study, Ursolic Acid, Oleanolic Acid, Isoquercetin, Psilostachyin B, Rutin, 9-cis-Retinal, and Icariside B1 from DL extract could bind with some key residues of ɑ-amylase in the catalytic sites as mentioned in the earlier experiments^19,46. Therefore, the interaction of those compounds with ɑ-amylase implies plasma glucose regulation.

Insulin sensitization also the primary outcome in diabetes therapy⁴. As the proteins involved in the insulin signaling process, DPP4 and PTP1B frequently used as the target for increasing insulin sensitivity^17,25,47. ARG125, GLU205, TYR547, SER630, ASP708, ASN710, and HIS740 have been reported as catalytic residues in DPP4²⁰. Interaction in those residues could alter the biomechanism of DPP4, driving to the enhancement of glucose-dependent insulin secretion⁴⁸. Also, addressing DPP4 for diabetes therapy has gained more attention and gave promising recovery effects⁴⁹. Thus, blocking DPP4 by Isoquercetin, Rutin, and Icariside B1 from DL has immense opportunity to improve the health of diabetic patients.

Augmenting insulin sensitization can be reached by altering PTP1B activity^50,51. Recently, allosteric and catalytic sites blocking of PTP1B have been reported. Directing LEU192, ASN193, PHE196, GLU276, PHE280, and TRP291 generate allosteric inhibition⁵², while ARG47, ASP48, PHE182, SER216, ALA217, GLY218, ILE219, GLY220, ARG221, and GLN266 perform catalytic inhibition⁵³. With some compounds interacting at the catalytic sites, particularly PHE182, ALA217, and ARG221, DL may serve as a catalytic inhibitor for PTP1B and ameliorates insulin-signaling impairments.

The drug-likeness and drug promiscuity of a compound strongly associate with its physicochemical properties (PP)^54,55. With the suitable PP, a compound will achieves an adequate absorption, distribution, efficacy, metabolism, and excretion (ADME) and prevent adverse drug reactions^54,56. Lipophilicity, molecular size, polarity, solubility, saturation, and flexibility were determined based on XLOGP3, molecular weight, total polar surface area (TPSA) value, log S, fraction of carbons in the sp3 hybridization, and number of rotatable bond, respectively³¹. 19-Norandrostenedione, 19-Nortestosterone, Icariside B1, Ilicic Acid, and Psilostachyin B were the most compatible compound with those described properties. Thus, those compounds has high probability to have excellent bioavailability, flexibility, and affinity to the target proteins. In advance, 19-Nortestosterone, 19-Norandrostenedione, Icariside B1, and Psilostachyin B also have a reasonable probability of giving biological activity as an enzyme inhibitor and nuclear receptor ligands. Consequently, those compounds seem to have great potential for modulating glucose metabolism and insulin signaling fault in diabetes mellitus patients and good diet therapy for complementary medicine.

CONCLUSION:

DL may serves as suitable dietary interventions for diabetes therapy with good nutritional contents and numerous biologically active compounds. Several compounds, mainly 19-Norandrostenedione, 19-Nortestosterone, Icariside B1, Ilicic Acid, and Psilostachyin B highly probable to act as glucose metabolism modulator and insulin signalling repairmen agent through inhibiting ɑ-amylase, DPP4, and PTP1B, correspondingly.

CONFLICT OF INTEREST:

The authors declare no potential conflicts of interest concerning this research.

ACKNOWLEDGMENTS:

The authors thank to Ministry of Research, Technology, and Higher Education, the Republic of Indonesia for funding this research (Grant no. 229/SP2H/LT/DRPM/2019).

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Received on 07.08.2021 Modified on 23.11.2021

Research J. Pharm. and Tech. 2022;15(7):3233-3240.

DOI: 10.52711/0974-360X.2022.00542