INTRODUCTION:

Aldose reductase (AR) and NADPH oxidase-1 (NOX-1) are a key enzyme of polyol pathway, catalyse NADPH-dependent reduction of glucose to sorbitol (Sorbitol pathway)^1,2.

The excessive accumulation of intracellular sorbitol found in various tissues of diabetic animals and in cells cultured under high glucose conditions has been proposed to be an important factor for the pathogenesis of diabetic complications. The condition is mediated by the production of Reactive Oxygen Species (ROS) from sorbitol pathway². The only strategy shown to be consistently beneficial in the treatment of diabetic complications is meticulous control of blood glucose³. However, both AR and NOX-1 inhibition are becoming one of the therapeutic strategies that have been proposed to prevent long-term diabetic complications^2,3. Therefore, AR inhibitors (ARIs) hold promise for reducing metabolic nerve injury, although the further study is required^2,4.

On the other hand, there is strong evidence to show that diabetes is associated with the increase of oxidative stress. The studies suggest that hydroxyl radical is indirectly inhibited by ARIs resulting from decreasing hydroxyl radical formation and polyol levels. It is related to the early stages of diabetic complications, possibly via the Fenton reaction involving H₂O₂ produced from the activated polyol pathway³. Therefore, it is proposed that hydroxyl radical may accelerate damage to the cell membranes resulting from polyol accumulation^2,3. The search for specific inhibitors of AR enzyme has still become a major pharmaceutic challenge, though a number of AR inhibitors must be assessed for diabetic complications.

Glycine max (G. max) or soybean and Zingiber officinale (Z. officinale) or ginger are functional food are known to cure some diseases empirically.Animal study showed G. max have a hypoglycemic activity⁵. Isoflavon compound like daidzein (0.85% w/w) and genistein (1.2% w/w) were predicted as active compound in G. Max^5,6. Meanwhile, Z. officinale rhizome has anti-hyperlipidemic and antioxidant potential by gingerol (20% w/w) and shogaol (7% w/w) compounds. Other studies have reported that ginger has anti-diabetes and antioxidant effects, therefore it can help reduce blood glucose levels and also its complication^7,8. Several studies have proven the potential of soybean seeds and ginger rhizome as anti-diabetes^6,8,9, however the active substances of soybean seed extract and ginger rhizome to inhibit complication of diabetic through oxidative pathway are still unknown clearly.

The purpose of this study was to evaluate phytochemical substances of soybean seed extract and ginger rhizome on NOX-1 and AR that play a role in diabetic complication using in-silico study.

MATERIAL AND METHODS:

Preparation of Glycine max and Zingiber officinale Extract:

Soybean seeds were obtained from Argo Mulyo variety with a letter of determination number 074/241/102.7/2017. Meanwhile, the ginger rhizome was obtained from UPT Laboratorium Herbal Materia Medika Batu with a letter of determination 074/211/102.7/2017. Extraction of the two herbs was conducted using the maceration method with slightly modification. Both of soybean seed and ginger root were extracted by ethanol (E.Merck, pro analysis) solvent at 50℃ for 48 hours and shacked using a waterbath-shacker. The extract was obtained after evaporation process.

Identification of phytochemical substances

Ethanolic extract of G. max seed and Z. officinale rhizome was performed a qualitative analysis using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) TSQ Quantum Access Max Thermo Scientific. The mobile phase of solution A contain 0.1% formic acid (E.Merck, pro analysis) in H₂O and solution B contain 0.1% formic acid in acetonitrile (E.Merck, pro analysis) with a flow rate of 300µL/min. Hypersilgold as a stationary phase¹⁰.

Molecular docking study:

The chemical structure of ligands (phytosterol compounds of soybean seeds and terpenoids from ginger rhizome) and reference drugs are downloaded on the PubChem website (https://pubchem.ncbi.nlm.nih.gov) with a 3D SDF file extension. Therefore, the file type was changed to a PDB extension file with the Open Babel software version 2.4.1. The FASTA format of the target protein (AR with UniProt ID: P27487 and NOX-1 with UniProt ID: O43451) is downloaded on the Uniprot website (http://www.uniprot.org/). Moreover, protein homology on the Swiss Model (http://www.swissmodel.expasy.org/interactive), the modeling results can be downloaded in a PDB file¹¹. The ligand-protein docking was performed using web-based software (dockingserver.com). Inhibition constant (Ki), free energy of binding, and surface interactions were observed by this method to examine their activity on AR and NOX-1 ¹².

RESULTS AND DISCUSSION:

Identification of phytochemical substances in Glycine max and Zingiber officinale Extract:

The phytochemical compounds from ethanolic extract of soybean seeds and ginger rhizomes using the LC-MS / MS method can be seen in Figure 1, Figure 2, and Table 1.

Figure 1. Chromatogram of phytochemical substances in ethanolic extract of Glycine max seed. (A) Stigmasterol, (B) Campesterol, (C) β-Sitosterol, (D) Lanosterol

Figure 2. Chromatogram of phytochemical substances in ethanolic extract of Zingiber officinale rhizome. (A) 6-Gingerdiol, (B) 10-Gingerol, (C) 12-Shogaol

The qualitative analysis using LC-MS/MS showed that phytochemical substances contained in the ethanol extract of soybean seeds were Stigmasterol, Campesterol β-Sitosterol, and Lanosterol. The highest level is β-Sitosterol, whereas the lowest level is Lanosterol. Meanwhile, the active compounds contained in the ethanol extract of ginger rhizoma are 6-Gingerdiol, 10-Gingerol, and 12-Shogaol. The highest levels is 12-Shogaol, meanwhile the lowest level is 6-Gingerdiol.

Four phytochemical compounds were identified in G. max seed extract and three phytochemical substances in Z. officinale rhizome extract in ethanol. The active compound can be determined by evaluating the value of the Selected Reaction Monitoring (SRM) on the chromatogram. SRM is a measurement parameter on LC-MS/MS to measure protein and active substances accurately and consistently. SRM is also used as a validation method to confirm the list of target proteins and active compounds obtained in global research or previous findings^13,14. The active substances of soybean seeds and ginger rhizome are secondary metabolites and have biological activity, moreover they can be used as candidates phytopharmaca^15,16.

The main components of phytosterols in soybean seeds are β-sitosterol, campesterol, and stigmasterol^16,17. All of them are classified into secondary metabolite groups and have biological activity that can be used to cure diseases. Stigmasterol is one of the phytosterol groups in plants to maintain the balance of phospholipid membranes from plant cells which are chemically similar to cholesterol in animal cell membranes. Stigmasterol can inhibit cholesterol biosynthesis through the inhibition of sterol reductase in cells ^18,19. On the other hand, stigmasterol has the potential to be anti-inflammatory, anti-tumor, anti-osteoarthritis, hypoglycemic and antioxidant effects^18,20. Meanwhile, β-Sitosterol acts as anti-cholesterol, anti-inflammatory, immunomodulatory, and antioxidant^18,21. Campesterol plays a role in lowering blood cholesterol and has anti-carcinogenic effects²². However, these compounds also have anti-angiogenic effects by inhibiting endothelial cell proliferation and capillary differentiation²³. Some studies both of in vivo and in vitro showed that lanosterol has activity as anti-cataract²⁴.

Ginger contains many active phenolic components such as gingerol and shogaol that have antioxidant and anti-cancer effects^25,26. Phenolic compounds have activity as antioxidants due to their ability to stabilize free radicals by providing hydrogen atoms to free radicals. Meanwhile, radicals derived from antioxidants of phenolic compounds are more stable than free radicals^25,27. The results of pre-clinical study showed that gingerol and shogaol compounds in the ginger extract can increase insulin secretion through protection activity from free radical on β-cells pancreas²⁶. Other research indicated that administration of ginger extract can reduce cholesterol, glucose, and triglyceride levels in experimental animals induced by Diabetes Mellitus^21,28,29.

Prediction of physicochemical properties

The result of the physicochemical properties of Glycine max and Zingiber officinale phytochemical compound can be seen at Table 2.

Table 2. Prediction of physicochemical properties of phytochemical substances G. max and Z. officinale

Herbs	Active Compounds	MW	Log P	Fr. Csp3	Torsion	HBA	HBD	PSA (A²)	Water Solubility
G. max	Stigmasterol	412.69	7.80	0.86	5	1	1	20.23	-5.47
	Kampesterol	400.68	7.63	0.93	5	1	1	20.23	-5.79
	Β-Sitosterol	414.71	8.02	0.93	6	1	1	20.23	-6.19
	Lanosterol	426.72	8.48	0.87	4	1	1	20.23	-7.20
Z. officinale	6-Gingerdiol	296.40	3.03	0.65	10	4	3	69.92	-4.11
	10-Gingerol	350.49	4.79	0.67	14	4	2	66.76	-6.17
	12-Shogaol	360.53	6.38	0.61	15	3	1	46.53	-7.19

MW = Molecular weight; Log P = logarithm of octanol/water partition coefficient; Torsion = bond between rotating atoms; HBA = H-bond acceptors; HBD = H-bond donors; PSA = polar surface activity

It can be seen that the molecular weight values of the active compound ranged from 296 to 426 (less than 500), the value of log of the octanol/water partition coefficient (log P) ranged from 3.03 to 8.48 (> 5), the amount of HBD ranged from 1 to 3 (less than 5), and the amount of HBA ranged from 1 to 4 (less than 10). 6-gingerdiol and 10-gingerol meet Lipinski Rules of Five completely, meanwhile, others compound did not meet in log P value only.

Chemical databases contain many of molecules that could be suitable ligands for an enzyme. However, no matter how good the fit with the protein target, the candidate molecule is of no use if the absorption is poor or if the drug is eliminated too slowly from the body. The World Drugs Index database were analysed and it was concluded that a compound is more likely to have poor absorption or permeability if the molecular weight exceeds 500; the calculated octanol/water partition coefficient (log P) exceeds +5; there are more than 5 H-bond donors (HBD) expressed as the sum of O–H and N–H groups; and there are more than 10 H-bond acceptors (HBA) expressed as the sum of N and O atoms. The above analysis is called the Lipinski Rules of Five because all values are multiples of five³⁰.

Based on Table 2, this means that 6-gingerdiol and 10-gingerol meet the Lipinski Rules of Five completely, meanwhile five others did not fulfill these rules³⁰. Hence, it can be predicted that 6-gingerdiol and 10-gingerol will be easily absorbed and have high permeability.

Molecular docking of Glycine max and Zingiber officinale on AR:

Activity of soybean seed and ginger rhizome extracts on AR were evaluated by in-silico approach and the results can be seen at Table 3.

Molecular docking study showed that the lowest inhibition constant (Ki) and free energy binding in each herb were β-sitosterol and 12-shogaol, although the surface interaction was high. Meanwhile, 10-Gingerol have a higher free energy binding score compared to β-sitosterol and epalrestat (a reference standard). On the other hand, epalrestat indicated binding free energy higher than other substance of herbs. The difference in the score of each parameter causes differences in ARIs. Free energy of binding and surface interaction between protein target and ligand influences the inhibitory activity of AR³¹. However, the lowest score of binding free energy results in a strong binding molecule, it causes an increase in their biological activity of soybean seed and ginger rhizome to inhibit the AR^31,32.

Phytosterol indicated ARIs activity stronger than terpenoid, however phytosterol and terpenoid from the herbs showed ARIs activity more potent than epalrestat as AR inhibitor. Phytochemical substances that are identified in both of herbs also indicated anti-diabetic through inhibitory activity of DPP-4 and alpha-glucosidase³³. research. Many studies have shown anti-hiperglycemic activity of herbs and have been applied in therapy type 2 diabetes mellitus^21,34 therefore, it supports also the prevention of diabetic complication. Study indicated soybean and ginger prevent nephropathy diabetic as a diabetic complication by decrease of plasma protein profile of diabetic rat³⁵. Furthermore, it is required an in vitro and in vivo study for the herb in order to confirm the results from molecular docking.

Table 3. Molecular docking of phytochemical substances in Glycine max and Zingiber officinale extracts with AR

Herbs	Ligand	Binding Free Energy (Kcal/mol)	Inhibition Constant (µM)	Surface Interaction (Å)
Glycine max	Stigmasterol	-8.47	0.62	923.96
	β-Sitosterol	-8.60	0.49	918.40
	Kampesterol	-8.30	0.82	906.31
	Lanosterol Epalrestat*	-8.17 -7.29	1.03 4.50	920.43 762.57
Zingiber officinale	6-Gingerdiol	-7.32	4.28	797.06
	10-Gingerol	-7.21	5.18	896.75
	12-Shogaol	-7.44	3.54	952.55
	Epalrestat*	-7.29	4.50	762.57

* reference standard

Molecular docking of Glycine max and Zingiber officinale on NOX-1:

Activity of soybean seed and ginger rhizome extracts on NOX-1 were evaluated by in-silico approach and the results can be seen at Table 4. In-silico studies indicates that lanosterol and 6-gingerdiol have a lower inhibition constant (Ki) and free energy binding compared to others active compound in both of herbs, although the surface interaction was high. Meanwhile, stigmasterol and 10-gingerol was found to have a higher free energy binding compared to β-sitosterol and 6-gingerdiol. Acetovanilone (a reference standard) indicated binding free energy and Ki score higher than other substance in this herb. The difference in the score of each parameter causes differences in inhibitory activity on NOX-1^{31, 32}. Phytosterol from soybean more potential to inhibit NOX-1 compared to terpenoid of ginger, however, they indicated NOX-1 inhibitor activity stronger than acetovanilone as NOX-1 inhibitor.

Docking molecular research is widely used to predict potential drug candidates in the pharmaceutical field. The orientation of the binding of this active substance to the molecular target indicates activity and affinity as a possible drug candidate. In addition, bioinformatics could also examine the bioactivity of a compound by predicting its inhibition constant (Ki). The lowest Ki show the most potentially active substances³¹however, it is accompanied by an increase of toxicity also³³. Therefore, the toxicity of compound must be tested, and the research also encouraged the in vivo and in vitro evaluation for the proposed designed compounds to validate the computational findings¹⁹.

Table 4. Molecular docking of phytochemical substances in Glycine max and Zingiber officinale extracts with NOX-1

Herbs	Ligand	Binding Free Energy (Kcal/mol)	Inhibition Constant (µM)	Surface Interaction (Å)
Glycine max	Stigmasterol	-8.69	0.42	849.86
	β-Sitosterol	-9.65	0.84	871.95
	Kampesterol	-9.14	0.20	860.80
	Lanosterol Acetovanilone*	-9.16 -4.78	0.19 312.86	866.23 485.89
Zingiber officinale	6-Gingerdiol	-6.11	33.45	757.09
	10-Gingerol	-5.28	135.37	897.03
	12-Shogaol	-5.52	89.59	1005.10
	Acetovanilone*	-4.78	312.86	485.89

* reference standard

CONCLUSION:

G. max seeds were found to contain phytosterols, mainly beta sitosterol, campesterol, stigmasterol, and lanosterols, while Z.officinale contained 6-gingerdiol, 10-gingerol and 12-shogaol. Based on in silico evaluation, β-Sitosterol and 12-Shogaol strongly inhibit AR enzyme, meanwhile NOX-1 activity was strongly inhibited by Lanosterol and 6-Gingerdiol.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

ACKNOWLEDGEMENT:

This research is supported financially by Ministry Education and Culture Indonesia. Therefore, we are grateful for the funding and support of this research.

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Received on 15.04.2024 Revised on 20.09.2024

Accepted on 10.12.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2095-2100.

DOI: 10.52711/0974-360X.2025.00300

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Herbs	Active Compound	SRM *Transition (m/z)*	Identified SRM Score	Surface Area (AA)	Ethanol Extract of Soybean Seeds
Glycine max	Stigmasterol	395-297	395	1015	++
	Campesterol	383-161	383	5795	++
	β-Sitosterol	397-161	397	14547	+++
	Lanosterol	409-109	409	282	+
Zingiber officinale	6-Gingerdiol	295-280	295	443	+
	10-Gingerol	399-193	399	446	++
	12-Shogaol	359-223	359	1561	+++