Investigation of Black and Brown Rice as Inhibitors of Tyrosinase and Xanthine Oxidase Enzymes: An in vitro and in silico Investigation

Xanthine oxidase (EC 1.17.3.2) is an enzyme that catalyses the conversion hypoxanthine to xanthine and then finally to uric acid. In this process, xanthine oxidase produces reactive oxygen species. High activity of xanthine oxidase leads to deposition of uric acid leading to hyperuricemia.¹ High levels of xanthine oxidase has also been reported in parkinson’s disease.² Xanthine oxidase is one of the major enzymes that promote ROS production, leading to death of dopamine producing cells which is a prime cause of parkinson’s disease.³ A high level of xanthine oxidase can lead to several other diseases. As such, the inhibition of xanthine oxidase becomes necessary in different disease situations and the current challenge is to obtain a xanthine oxidase inhibitor that is safe for consumption.⁴

Tyrosinase (EC 1.14.18.1.) is an enzyme involved majorly in the synthesis of melanin. It catalyses the conversion of L-tyrosine to 3,4-Dihydroxyphenylalanine (DOPA) followed by its conversion to o-dopaquinone which ultimately leads to melanin production.⁵ Based on the tissue of occurrence, the higher activity of tyrosinase can lead to different issues. In skin tissues, the higher activity of tyrosinase can lead to diseases like hyperpigmentation, melanoma etc.⁶ Tyrosinase overexpression in the brain can lead to increased production of neuromelanin. Accumulation of neuromelanin has been regarded as a major cause of the onset of Parkinson’s disease.⁷ There are reports of the correlations between the occurrence of melanoma and parkinson’s disease and tyrosinase overexpression has been the connecting link between the two pathologies.⁸ The current crop of tyrosinase inhibitors suffer issues of severe toxicity along with low efficacy.⁷ Thus it necessitates the search for new inhibitors from plant sources.

Black rice and brown rice are popular rice varieties in the South East Asia along with the North-Eastern states of India. The bran of both the rice varieties is rich in various polyphenols like anthocyanins, tocopherols, etc., and have various health benefits.⁹ In our current study, we analyse the ability of the methanolic extracts of the brown and black rice varieties to inhibit the enzymes tyrosinase and xanthine oxidase.

The methanolic and aqueous extracts of the black and brown rice varieties were prepared and the extracts were checked for the ability to inhibit the activity of the xanthine oxidase and tyrosinase enzymes. The methanolic and aqueous extracts of black rice showed the best inhibition of xanthine oxidase with the percent enzyme activity dropping to 30.65±2.39 and 31.56±1.48 at a dose of 1.5 mg/mL with respect to the untreated control (p<0.01 in both the cases). In both the extracts, a dose dependent decrease in enzyme activity was observed (Figure 1, Table 1).

Table 1:
Table showing the percent enzyme activity of xanthine oxidase under different treatments.

In case of tyrosinase, the methanolic and aqueous extracts of black rice showed the best inhibition with the percent enzyme activity dropping to 43.42±1.80 and 53.77±1.97 respectively at a dose of 1.5 mg/mL with respect to the untreated control (p<0.01) (Figure 2, Table 2).

Table 2:
Table showing the percent enzyme activity of tyrosinase under different treatments.

To identify the phytochemicals responsible for the lowering of the enzymatic acivities, we carried out a literature survey to obtain the major phytochemicals present in the rice varieties.^19,20 For us in silico analysis, we docked the reported compounds with the pdb structures of the enzymes tyrosinase and xanthine oxidase. With respect to xanthine oxidase, the percentage identity of the template and target protein is 100% as per BLAST analysis. From our dockings, we observed that cathechin hydrate from black rice showed the best docking with xanthine oxidase and β-sitosterol from brown rice showed the best docking with tyrosinase (Tables 3 and 4).

Table 3:
Binding energy of the black rice phytochemicals with xanthine oxidase and tyrosinase.

Table 4:
Binding energy of the brown rice phytochemicals with xanthine oxidase and tyrosinase.

Catechin hydrate formed hydrogen bonds with conserved residues in the xanthine oxidase active site. β-sitosterol docked with several conserved residues of the tyrosinase active site (Figure 3). We carried out molecular simulation dynamics studies to look into the stabilities of catechin hydrate-xanthine oxidase and β-sitosterol-tyrosinase complexes.

In the present work, it is seen that the target protein Xanthine Oxidase shows higher variations in Root Mean Square Deviation (RMSD) in the presence of ligand catechin hydrate. Similarly, the complex of tyrosinase and β-sitosterol also show higher RMSD value as compared to the tyrosinase protein alone. The shift in RMSD value is more prominent in case of the complex of xanthine oxidase-catechin hydrate with a 0.2 nm deviation (Figure 4a, 4b).

Figure 1:
Percent enzyme activity of xanthine oxidase in the presence of (a) methanolic extract of black rice, (b) aqueous extract of black rice, (c) methanolic extract of brown rice, (d) aqueous extract of brown rice. All values expressed as mean± standard deviation. *p>0.01 vs Control.

Figure 2:
Percent enzyme activity of tyrosinase in the presence of (a) methanolic extract of black rice, (b) aqueous extract of black rice, (c) methanolic extract of brown rice, (d) aqueous extract of brown rice. All values expressed as mean± standard deviation. *p>0.01 vs Control.

The Root Mean Square Fluctuation (RMSF) values of the amino acids in the presence and absence of catechin hydrate show a difference in the amino acids in the range of residue number 400 to 500 of the xanthine oxidase. In case of tyrosinase, there is a deviation in the in the RMSF values in the amino acid residues in the range of 50 to 90 in the presence of β-sitosterol (Figure 4c, 4d).

Radius of gyration (Rg) is a measure of the compactness or spatial extent of a molecular structure. Figure 5a and 5b shows the fluctuations in Rg for both the bound and unbound forms of Xanthine Oxidase with the selected ligand. It is clearly evident that the protein in bound form shows increased Rg with respect of the simulation time. For tyrosinase, there is an increase in the Rg values at 120 ns. We observed an increase in the Solvent Accessible Surface Area (SASA) values of the proteins when bound to their respective ligands (Figure 5c, 5d).

Figure 3:
2D images of the binding of catechin hydrate with (a) xanthine oxidase (PDB- 3B9J), (b) xanthine oxidase (model structure) and (c) β-sitosterol with tyrosinase (PDB- 4OUA).

In the current analysis, Principal Component Analysis (PCA) is calculated on the backbone Cα atoms in the proteins both in presence and absence of their respective ligands; catechin hydrate and β-sitosterol. Projection of two-dimensional principal components PC1 and PC2 for the two largest Eigen values is shown in Figure 6a and 6b, which represents the relevant conformational space. It is evident that the free proteins in absence of ligand show a reduced space as compared to the more dispersed region in the bound state with ligand.

The significant inhibition of xanthine oxidase by the methanolic and aqueous extracts of black rice point the potential of the black rice bran constituents like anthocyanins to act as potential inhibitors of xanthine oxidase. Although brown rice extracts also inhibited the xanthine oxidase activity, but the inhibition of black rice extract was more prominent. As such black rice could be further studied as a potential therapy against gout.²¹ Both black rice and brown rice extracts were able to lower the activity of tyrosinase enzyme significantly as compared to the untreated control. However, brown rice extract was more prominent in its inhibiting effect. Thus, both of the rice extracts can be further researched for their potential to act as therapeutic against diseases like melanoma, hyperpigmentation and neurodegradative issues like parkinson’s disease.²²

The major phytochemicals in black and brown rice which are reported in the methanolic and aqueous extracts showed favourable binding with xanthine oxidase and tyrosinase proteins. There was bond formation between the xanthine oxidase amino acid residues and black rice polyphenols like cyaniding-3 glucoside, cyaniding-3 galactoside, catechin hydrate (Figure S1 to S4) and brown rice polyphenols like peonidin-3-O-glucoside, epicatechin, gallic acid and luteolin (Figure S5 to S8). These polyphenols formed hydrogen bonds with the conserved active site amino acids like ASP233, ARG311. Binding of small compounds to these amino acids has led to the inhibition of the xanthine oxidase.²³ The black rice phytotchemicals like hydroxybenzoic acid, vallinic acid, syringic acid bound with critical amino acids like HIS82, CYS80 and PHE454 of tyrosinase (Figure S9 to S10). The brown rice polyphenols like peonidin-3-O-glucoside, β-sitosterol, vanillic acid, cinnamic acid and ellagic acid formed bonds with conserved HIS residues present in positions 82, 251 and 255 of tyrosinase (Figure S11 to S12). Compounds that bind to these amino acids have caused inhibition of tyrosinase activities.²⁴ We further proceeded with the molecular dynamic’s simulation of the catechin hydrate-xanthine oxidase complex and β-sitosterol-tyrosinase complex.

Figure 4:
RMSD values of (a) xanthine oxidase alone and in complex with catethin hydrate, (b) tyrosinase alone and in complex with β-sitosterol. RMSF values of (c) xanthine oxidase alone and in complex with catethin hydrate, (d) tyrosinase alone and in complex with β-sitosterol.

Figure 5:
Rg values of (a) xanthine oxidase alone and in complex with catethin hydrate, (b) tyrosinase alone and in complex with β-sitosterol. SASA values of (c) xanthine oxidase alone and in complex with catethin hydrate, (d) tyrosinase alone and in complex with β-sitosterol.

Figure 6:
Range of Conformational space related to (a) xanthine oxidase alone and xanthine oxidase-catechin hydrate complex and, (b) tyrosinase alone and tyrosinase-β-sitosterol complex as calculated from the first two principal components PC1 and PC2.

The increase in RMSD values of xanthine oxidase and tyrosinase observed in the presence of their respective ligands indicates the change in the structure of the proteins caused by the binding of the ligand.²⁵ The increased RMSF values of amino acids residues the increased flexibility of the amino acids in that region that allows them to interact with the ligand.²⁶ The increase in the Rg and SASA values in the presence of ligand correlates with the increase in RMSD values all of which indicates a distinct change in the shape of the protein in the presence of the ligand to accommodate better interactions with the ligand. The PCA analysis reveals that the protein in absent of ligand was limited to a lower number of conformations, depicting stability in structural dynamics. In contrast to this, a larger space in the protein conformations in presence of ligand reflects the dynamic nature of the protein visiting more conformations in the course of simulation, speculative of higher interactions of the protein and ligand. Thus, the presence of catechin hydrate has a distinct consequence on the protein motions with greater movement which will thus change the functional attributes of the target protein.^27,28

From our study, we have observed that both the black rice extracts and the brown rice extracts have shown inhibitory activity towards xanthine oxidase and tyrosinase enzymes. Molecular docking and molecular simulation dynamics studies reveal that catechin hydrate from black rice and β-sitosterol from brown rice are the potential phytochemicals which could bind to xanthine oxidase and tyrosinase enzymes respectively to give the observed inhibitory effects.

Authors would like to thank financial support in the form of ‘Student Research Project’ provided by the Assam Royal Global University for carrying out the work. Authors would also like to thank Guwahati Biotechnology Park for providing the facility to carry out the molecular dynamic’s simulation studies.

The authors declare that there is no conflict of interest.