INTRODUCTION:

Zingiber officinale (Ginger) belong to the family Zingiberaceae. Since ancient times it’s been used in Ayurvedic Medicine with numerous biological applications¹. It has a history of medicinal use in India. Anti-inflammatory property of ginger has been known for centuries. Discovery of ginger inhibitory effect on prostaglandin has been reported in 20^th century itself. This property identified in Zingiber officinale as a medicinal product which as biochemical effects with non-steroidal anti-inflammatory drugs. Hyperuricemia can occur due to overproduction or under excretion of uric acid. Gout is an inflammatory arthritis lead to decrease in quality of life².

The disease occurs when body fluids are saturated due to high level of uric acid that eventually settles in the joints. Gout could be overcome by lowering the levels of uric acid in the blood to normal limits. Uricosuric drugs work by inhibiting there absorption of uric acid in the kidney tubules in a competitive fashion, so the removal of uric acid through the kidneys can be treated. Uricostatic drug decreases the enzyme xanthine oxidase that converts hypoxanthine into xanthine and in turn into uric acid, by lowering the formation of uric acid. The metabolized end product of purine in human is uric acid, which is excreted through urine. This has very low solubility and tends to form crystals. Uric acid accumulated in joints is commonly found in the form of monosodium uric crystals, that induces inflammatory reactions^3and4. Allopurinol drug is widely used in treatment of gout and hyperuricemia. Allopurinol inhibits the xanthine oxidase which prevents the formations of uric acid, is mainly useful in treating gout characterized by deposit of formed uric acid crystals in the joints, cartilage and skin. This eventually impairs renal function⁵. Apart from its therapeutic efficacy, allopurinol causes side effects such as nephropathy, toxicity, and allergic reactions⁶. Further, the drug can also induce hepatitis and allergic reactions⁷. This represents the need for new alternatives to decrease uric acid deposits with minimal side effects. Recent medications sources are turned towards plant-derived substances. The aim was to evaluate the hypouricemic property of Zingiber officinale and confirm the same with in-silico docking analysis.

MATERIALS AND METHODS:

Plant materials and extraction:

Zingiber officinale (Fresh Rhizome) was authenticated and obtained from The Department of Botany in Sri Venkateshwara University, Tirupati-517 502, Andhra Pradesh, India. The collected fresh rhizome was shade dried and powdered using mechanical grinder. Extraction process was carried out by cold maceration method using 95% ethanol. 150gm powdered rhizome was extracted with 1000ml of 95% ethanol for 3 days. The macerate was collected on 4^th day by filtering with Whatman No.1 filter paper and stored in desiccator throughout the period study to prevent spoilage or deterioration.

Chemicals and reagents:

DPPH, Butylhydroxyltoluene, Allopurinol, Ascorbic acid was obtained from Sisco research laboratories pvt. Ltd.

Phytochemical analysis:

Preliminary phytoconstituents were carried out according to the modern methods of phytochemical analysis⁸. GC-MS analysis of the ethanolic extract of Zingiber officinale was carried out using a Perkin-Elmer GC Clarus 500 system with an AOC-20i auto-sampler and a gas chromatograph. Compounds were compared and identified by using the mass spectra with the known compounds in NIST Library.

In-vitro anti-oxidants assay:

DPPH free radical scavenging activity:

The evaluation of the radical scavenging potential, carried out according to the method reported by Hwang et al and Mensoret al ^9and10. 0.3 mM alcohol DPPH solution (1 mL) was mixed to 2.5ml of extract. The samples was placed in a dark space at room temperature and the absorbance was measured at 518 nm after 30 min. Final sample mixture which contains 1 mL ethanol and 2.5 mL from various concentrations of extract; Control containing 1 mL of 0.3 mM DPPH and 2.5 mL ethanol. BHT (butylhydroxytoluene) was used as positive controls.

The antioxidant activity was evaluated using the formula:

% DPPH Inhibition=100-[(sample absorbance-control absorbance)X 100]/control absorbance

Nitric oxide scavenging activity:

Nitric oxide scavenging activity was carried by^11and12. Generating the nitric oxide from sodium nitroprusside solution in phosphate buffer saline (PBS) at physiological pH 7.4.Sodium nitroprusside solution (0.2 mL), with1 mL of test or standard antioxidant solution and 1.8 mL of phosphate buffer saline was added in various concentrations are incubated at 25°C for 180 minutes. 1 mL of incubated solution was added with 1 mL of Griess reagent. Using UV visible spectrophotometer absorbance was measured at 540 nm using ascorbic acid as positive control.

The antioxidant activity was calculated using the formula.

% NO Inhibition=100-[(sample absorbance-control absorbance)X 100]/control absorbance

Xanthine oxidase inhibitory assay:

An in-vitro method was carried out by measuring the activity of the enzyme xanthine oxidase using UV spectrophotometry^13-18. Xanthine oxidase enzyme extracted from cow milk by centrifuging at 3000rpm and supernatant layer was separated and washed with ethanol. Solubility of xanthine substrate was increased by adding 5 drops of 1.0 M NaOH¹⁹. Ginger extract was dissolved in ethanol and prepared the sample concentration at 50, 100, 200, 400 and 800 mg/mL. Allopurinol is used as a positive control. Final 3.2 mL mixture consisting of 1 mL extract determined at various concentrations, 1mL of 0.15 M PBS (Phosphate buffer saline) of pH (7.8), 100 µL xanthine oxidase enzyme solutions. Post preincubation of sample solution for 15 min at 37 °C, and by adding of 100 µL of xanthine oxidase enzyme solution reaction was initiated and again it was incubated for 30 min at 37 °C. Reaction was terminated by adding 1 mL of 1N HCl. Absorbance measured at 295 nm using spectrophotometer, illustrates the uric acid formation. Inhibition of xanthine oxidase activity of the test was estimated by comparing the absorbance of uric acid from the mixture with positive control with the absorbance of test samples.

% inhibition=[1-(Absorbance of Sample /Absorbance of Control)] × 100

Molecular docking studies:

Ligand Preparation:

The major phytoconstituents are identified from the ethanol extract of Zingiber officinale rhizome namely are Alpha-curcumene, Nerolidol, Dihydrocapsicin, Capsaicin, Gingerol, Shogaol, Alpha-terpineol with the standard drug Allopurinol^20-25. The structures of the phytoconstituents are downloaded from PubChem chemical databases²⁶ and saved in .mol format. Preparation for docking study done by importing the phytoconstituents to the workspace which are saved in mol formats.

Enzyme Preparation:

Xanthine oxidase is selected as a target for docking studies. Docking analysis is done by selecting the target for the disease and followed by obtaining the 3D structure of Xanthine oxidase (1FIQ)²⁷ from protein data bank²⁸ in .pdb format. Search space performed in the docking studies was studied as subset region of 25.0 Angstroms around active side cleft. The water molecules are also taken in to cogitation and the replaceable water molecules were given a score of 0.50.

Molegro Virtual Docker’s algorithms and scoring:

Molegro Virtual Docker (MVD) software is used to perform the ligand docking analysis, which has been widely used and popular among research scientist. It’s a rapid and adaptable docking simulation which fetches the appropriate conformation of binding ligand to macromolecule. MVD program is based on new interrogative search algorithm which predicts the cavity algorithm with a differential evolution²⁹. It’s a bilateral optimization method based up on Darwinian Evolution Theory, were the population of each is exposed to competing selection that intrudes to unpredictable solutions. To generate new solutions recombination and mutations are used. The scoring function of MolDock method is based on Piecewise Linear Potential, were the interpreted potential parameters were fit to protein-ligand structures and binding scoring function³⁰ which is more elaborated in Generic Evolutionary Method for molecular DOCK³¹ with charge schemes and new hydrogen bonding.

MolDock Optimizer:

In MolDock, selected criterions are utilized for instructed differential algorithm, number of runs is 5, maximum interactions are 2000, scaling factor is 0.5, population size 50 and cross over rate 0.9 Annova based termination scheme were selected. The most appropriate binding mode in binding cavity, Pose clustering were deployed, that provides various binding modes.

DruLi To Software:

DruLiTo software is an open source of National Institute of Pharmaceutical Education and Research. It’s used to calculate the various molecular properties and screen the molecules based up on the drug likeness rules like, ‘The Lipinski rule of five’.

RESULTS AND DISCUSSIONS:

Extraction was done on rhizome of Zingiber officinale rhizome with cold maceration followed by shadow drying to preserve active substances contained and the tentative yield value was found to be 5.925gms from 150gms.

Phytochemical screening results (Table 1), suggested that the drug contained alkaloid, saponins were in abundant, tannins, glycosides and flavonoids are moderately present. Flavonoid reduces blood uric acid through inhibiting the xanthine oxidase. The flavonoid content is a potential reactive free radical scavenging activity³³.

Table 1: Phytochemical analysis of Zingiber officinale rhizome:

Phytochemicals	Extract
Alkaloids	***
Tannins	**
Glycosides	**
Saponins	***
Steroids	#
Flavonoids	**
Terpenoids	*

Key: *** abundantly present, ** moderately present, *fairly present, # absent

GC-MS Analysis:

Interpretations of GC-MS were carried out by comparing the database of National Institute Standard and Technology (NIST). The spectrums of analyzed components were matched with spectrum of identified data stored in the NIST library. The retention time, molecular weight of the components of test materials was determined. GC-MS analysis of Zingiber officinale the major percentage area of phytochemical (Table 2).

Table 2: GC-MS Analysis of Ethanolic extract of Zingiber officinale rhizome:

S.No.	RT	Area%	Name	Molecular Weight
1	14.274	20.57	α Gingiberene	204
2	14.872	12.71	β Seiquphellandrene	204
3	14.042	11.27	α Curcumen	202
4	14.531	10.61	Cyclo Hexane	204
5	14.352	9.77	α Fernesene	204
6	23.693	7.45	Shagole	276
7	24.207	4.46	Gingerol	294
8	14.65	3.41	ϒ Cadinene	204
9	15.509	3.09	Nerolidol	222
10	7.434	3.06	Cineole	154
11	16.932	2.74	Guaiol	222
12	9.408	2.68	Camphol	154
13	25.64	2.62	Capsaicin	293

In-vitro anti-oxidants assay:

DPPH free radical scavenging activity:

The results DPPH inhibition activity (Fig:1) of ethanolic extract of Zingiber officinale IC₅₀ value was 42.09μg/mL and the positive control butylhydroxytolueneic IC₅₀ value was 66.67μg/mL.

Fig 1: DPPH inhibition activity of Zingiber officinale rhizome

Nitric oxide scavenging activity:

The results of nitric oxide inhibition activity (Fig: 2) of ethanolic extract of Zingiber officinale IC₅₀ value was 28.36μg/mL and the positive control ascorbic acidIC₅₀ value was 42.87 μg/mL.

Fig 2: In-vitro Nitrous oxide inhibitory assay of Zingiber officinale rhizome

Xanthine oxidase inhibitory assay:

The results of xanthine oxidase inhibition activity (Fig: 3) of ethanolic extract of Zingiber officinale IC₅₀value was 188.5 μg/mL and the positive control allopurinol IC₅₀ value was 499.2 μg/mL.

Fig 3: Xanthine oxidase inhibition assay of Zingiber officinale rhizome

Investigations of the anti-oxidant property of Zingiber officinale rhizome on DPPH, nitric oxide and xanthine oxidase inhibition assay suggests that it can be an effective alternative for the management of hyperuricemia. The uric acid synthesis in the oxidative pathway carried out through the activation of hypoxanthine into xanthine, which is catalyzed by xanthine oxidase enzyme and the oxidation of xanthine into uric acid. This is an essential biochemical intervention for gout and hyperuricemia. In addition to that results showed that the ethanolic extract gave similar inhibition of xanthine oxidase compared to the standard drug allopurinol.

Molecular docking studies:

The competency of the phytoconstituents towards binding with the targets is given in terms of MolDockScore. The MolDock Score and re-rank scoring are used as the criterion for analysing the docking results. The phytoconstituents are arranged according to their MolDock Score. The ligand possessing the highest mol dock and re rank score shows a highcompatibility towards its target.

In-silico docking analysis of Zingiber officinale phytoconstituents on Xanthine oxidase (PDB ID: 1FIQ) ranking based on MolDock Score is described in table 3 and H Bond is characterized in table 4. The binding arrangement of Gingerol, Dihydrocapsaicin, Capsaicin, Shogaol, Nerolidol, Alpha Curcumene, Alpha Terpineolare analysed using the ligand energy inspector tool built-in in the Molegro virtual docker. It was found that the binding patterns are stronger and it poses maximum MolDock Score as well as the rerank score compared to the standard drug Allopurinol. The structure 1FIQ has in total 3 chains (A, B and C chains). Each chain of Xanthine Oxidase possesses 219 residues. It was analysed that the chain B plays a major role in the binding to phytoconstituents including the standard drug.

Table 3: In-silico docking analysis of phytoconstituents from ethanolic extract of Zingiber officinale rhizome and crystal structure of xanthine oxidase from bovine milk (PDB ID: 1FIQ) ranking based on MolDock Score.

Name	Ligand	MolDock Score	Rerank Score	H Bond
[00]Gingerol	Gingerol	-95.2981	-78.7588	-2.39824
[00]DihydroCapsician	DihydroCapsician	-94.6751	-83.4946	1.08594
[00]Capsaicin	Capsaicin	-89.6908	-73.9623	-4.88201
[00]Shogaol	Shogaol	-84.042	-66.7936	-2.49955
[00]Nerolidol	Nerolidol	-80.9654	-68.9856	-0.844542
[00]Alpha Curcumene	Alpha Curcumene	-66.1783	-45.378	0
[00]Allopurinol	Allopurinol	-51.9283	-47.3437	-0.796146
[00]Alpha Terpineol	Alpha Terpineol	-48.5939	-43.4719	-2.5

Table 4: In-silico docking analysis of phytoconstituents from ethanolic extract of Zingiber officinale rhizome and crystal structure of xanthine oxidase from bovine milk (PDB ID: 1FIQ) ranking based on H Bond interaction.

Name	Ligand	MolDock Score	Rerank Score	H Bond
[00]DihydroCapsician	DihydroCapsician	-94.6751	-83.4946	1.08594
[00]Gingerol	Gingerol	-95.2981	-78.7588	-2.39824
[00]Capsaicin	Capsaicin	-89.6908	-73.9623	-4.88201
[00]Nerolidol	Nerolidol	-80.9654	-68.9856	-0.844542
[00]Shogaol	Shogaol	-84.042	-66.7936	-2.49955
[00]Allopurinol	Allopurinol	-51.9283	-47.3437	-0.796146
[00]Alpha Curcumene	Alpha Curcumene	-66.1783	-45.378	0
[00]Alpha Terpineol	Alpha Terpineol	-48.5939	-43.4719	-2.5

The residues in the 1FIQ chain B which are involved in the binding to the standard drug Allopurinol and Gingerol, Dihydrocapsaicin, Shogaol, Nerolidol are given in Table 5. The binding pattern for Allopurinol, Gingerol,Nerolidol, Shogaol and Dihydrocapsicinare represented in the figure 4, 5, 6, 7 and 8respectively.

‘The Lipinski rule of five’ was used to determine the drug likeness properties of the phytoconstituents and to develop them as competitive lead compound for hypouricemic activity. The analyzed phytoconstituents results are represented in table 6. All the phytoconstituents passes the drug likeness properties.

Table 5: Residues in the 1FIQ chain B which are involved in the binding

Compounds	Residues involved
Allopurinol	Ala 338, Gly 339, Val 342, Gly 339, Phe 337, Gly 145, Asn 351, Leu 348
Gingerol	Phe 337, Gln 144, Gly 339, Gln 341, Val 342, Ala 338, Thr 262, Gly 145, Arg 426
Dihydrocapsicin	Gly 145, Ser 1225, Thr 1226, Gly 46, Gln 341, Thr 262, Ala 338, Arg 426
Shogaol	Arg 426, Gly 145, Pro 1224, Gly 49, Lys 1228, Ser 1225, Leu 147, Thr 1226
Nerolidol	Lys 1228, Thr 1226, Ala 338, Glu 45, Gly 47, Cys 48, Pro 1224, Lys 1228

Table 6: The Lipinski rule of five for evaluation of ‘drug likeness properties’ using DruLiTo Software.

S.No	Title	Molecular Weight	Logp	HBA	HBD
1	Shogaol	276.17	3.775	3	1
2	Dihydrocapsicin	307.21	4.503	4	2
3	Alpha Curcumene	202.17	5.761	0	0
4	Gingerol	294.18	2.437	4	2
5	Capsaicin	305.2	3.983	4	2
6	Nerolidol	222.2	4.29	1	1
7	Allopurinol	136.04	-0.64	5	2
8	Alpha Terpineol	154.14	2.369	1	1

Fig 4: Docked view of Allopurinol with Xanthine Oxidase (1FIQ)

Fig 5: Docked view of Gingerol with Xanthine Oxidase (1FIQ)

Fig 6: Docked view of Nerolidol with Xanthine Oxidase (1FIQ)

Fig 7: Docked view of Shogaol with Xanthine Oxidase (1FIQ)

Fig 8: Docked view of Dihydrocapsicin with Xanthine Oxidase (1FIQ)

CONCLUSION:

Several methods were employed for the evaluation of antioxidant potential of Zingiber officinale. Investigations exhibit hypouricemic activity. Results of study suggests that the ethanolic extract of Zingiber officinale rhizome has the effective inhibition of xanthine oxidase activity and DPPH, nitric oxide assays to confirm the free radical scavenging activity indicating it’s potential as supplementary agent for treating hyperuricemia and gout. Further in-silico docking studies also suggest that most of the phytoconstituents of Zingiber officinale rhizome passes the drug likeness properties as such standard drug allopurinol. Further studies can be performed to estimate in-vivo hypouricemic activity of the Zingiber officinale to discover pharmacokinetic properties of the phytoconstituents.

ACKNOWLEDGEMENTS:

The authors thank the management of Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), Chennai, India for providing us with all the facilities for the successful completion of the project.

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Received on 28.08.2018 Modified on 03.10.2018

Research J. Pharm. and Tech 2019; 12(1): 314-320.

DOI: 10.5958/0974-360X.2019.00057.X