Because hepatic XOD inhibition involvement was crucial to the intended anti-hyperuricemic activity investigation, the induced hyperuricemia would require at least some allusions to hepatic UA biosynthesis contribution. Hence, the adoption of the ethanol induction method depends primarily on the accumulation of purines from excess ATP consumption accompanying ethanol metabolism and its consequent enhanced hepatic XOD bioconversion of purines to UA . This precluded the use of potassium oxonate (PO), commonly employed for hyperuricemia induction in rodents, as its action mechanism, predicated on the rodent-encoded uricase inhibition, hinges on UA urinary elimination impairment with no modicum of hepatic XOD biosynthetic activity contribution . All the same, the suitability of the adopted ethanol induction method was manifest in the significant increases in SUA (p < 0.0001) and LUA (p < 0.05) levels of the negative control group compared to those of the normal control group (Figs. 3 and 4).
SUA- and LUA-lowering effects of FATT
Each of the three doses of FATT investigated showed a significant SUA-lowering effect compared to the negative control (p < 0.0001). While the activity of each dose was comparable to that of allopurinol, no significant dose dependence was observable, probably because the dose range over which such observation could be made had been exceeded. And though this anti-hyperuricemic activity only partly, by virtue of the induction mechanism, alluded to XOD inhibition, it sufficed greatly in establishing the anti-hyperuricemic activity of FATT, thereby providing a strong scientific basis for the application of the plant in its earlier-mentioned treatment claims of kidney stone and other hyperuricemia-related disorders in (“Background” section).
A comparison of SUA and LUA data (Figs. 3 and 4) showed that only the highest dose of FATT (200 mg/Kg) produced a significant LUA lowering (p < 0.05). A comparison of this p value with that of SUA lowering (p < 0.0001) even at the lowest (50 mg/Kg) dose showed FATT demonstrating much greater potency for lowering SUA than for LUA. Notwithstanding, given the fact that LUA accumulation could only have occurred by overwhelming hepatic UA biosynthesis, unlike SUA accumulation, which could occur either as a result of excessive UA biosynthesis and/or its impaired urinary elimination, LUA-lowering data appear more associable with hepatic XOD inhibition than do the corresponding SUA data.
However, the huge variation in the intensities of these two responses over the same dose range was indicative of variation in action mechanisms. For instance, the fact that FATT did not show LUA lowering effect at 50 mg/Kg dose, at which its SUA-lowering activity was not only highly significant (p < 0.0001) but also above which there was no dose dependency (Fig. 3), showed that the XOD inhibitory SUA-lowering effect of FATT must have been greatly augmented by some additional SUA-lowering means. And recalling that the only known alternative LUA-lowering means is UA urinary elimination enhancement, anti-hyperuricemic effects of flavonoid aglycones could be conjectured as jointly uricostatic and uricosuric.
Xanthine oxidase (XOD) model and chain selection
The unavailability of an inhibitor conformation of the human XOD in the Protein Databank (PDB) at the time of this investigation warranted using the 3NVY-coded XOD model as substitute. It is the X-ray crystal model of bovine milk XOD co-crystallized with a flavonoid inhibitor, quercetin . It should be noted that the factor of primary consideration in this selection was the existence of the protein in its inhibitor conformation, the presence of flavonoid as co-crystallized ligand being merely fortuitous. Additional key considerations in this bovine XOD choice, however, were its mammalian origin and the superimposability of its active site on that of the human enzyme [41, 42]. Moreover, the 3NVY homodimer model was particularly suitable considering its good resolution (2.00 Å) and distinct demarcation of the three domains in each monomer into chains, A B C in one and J K L, respectively, in the other . This enabled the use of only chain C, one of the two active site-containing chains (chains C and L) of the homodimer, for the docking and other in silico studies, making time-involving in silico processes (e.g. protein preparation) achievable within practicable computational time.
Docking validation and binding affinities of the aglycones with XOD
Figure 5, a stick model of the overlay of the coordinates of the co-crystallized quercetin conformation and those of its docked best pose, visually shows excellent correspondence calculated as 1.00 Å RMSD by the Discovery Studio software. The docking protocol was thereby validated.
The binding energies of the three flavonoid aglycones (− 7.8, − 8.1, − 8.2 kcal/mol for kaempferol, quercetin and isorhamnetin, respectively) were significantly lower than that of allopurinol (− 5.2 kcal/mol). This portends a higher binding affinity of each of the aglycones for XOD compared to that of allopurinol, projecting flavonoid aglycones as more-XOD specific than allopurinol and, invariably, indicating that more potent and safer uricostatics than allopurinol could emanate from the flavonoid aglycone chemical space .
Molecular basis for the facility of flavonoid aglycone–XOD binding
The comparable in vivo activity of allopurinol and those of the flavonoid aglycones notwithstanding, molecular docking experiment results indicated that the flavonoid aglycones (FAs) showed more affinity to XOD than allopurinol did. The higher affinity of the FAs compared to allopurinol’s could be speculated based on active site amino acid residues involved in their interactions as follows: Three active site amino acid residues (GLU1261, GLU802 and ARG880) have been recognized to be catalytically crucial to XOD biotransformation of xanthine to uric acid [45,46,47]. In addition, the active site bears other residues which, though non-catalytic, play significant roles in the purine substrate and cofactors binding. They include GLN767, PHE789, ARG912, ALA1079, PHE914 and THR1010 . A careful analysis of the 2D and 3D active site interaction simulations of the aglycones (Figs. 7, 8 and 9) showed that each aglycone showed a pi-alkyl interaction with the substrate-binding ARG912, while isorhamnetin, in addition, showed a carbon–hydrogen interaction with the rather catalytic GLU802. On the other hand, allopurinol’s hydrogen bonding with THR1093 and pi-sigma bonding with GLN1040 (Fig. 6) were neither of substrate-binding nor of catalytic consequence, thereby explaining the higher binding affinities of the aglycones than that of allopurinol for XOD (Table 2). This, in a way, provides a strong molecular support for the possible discovery of potent anti-hyperuricemic agents from the flavonoid aglycones of TT and, by extension, the flavonoid aglycone chemical space in general.
Molecular dynamics of flavonoid aglycone–XOD complexes
Every atom in a physiological system is in a constant state of motion, being found in the field of forces exerted by surrounding atoms within and without the molecule of which it is a part . Binding affinity outcomes of molecular docking would therefore often need to be validated by the stability of ligand–protein complexes in such dynamic environments simulated over a suitable period of time. Arguably, the two most useful stability-assessing parameters deductible from a typical ligand–protein complex dynamics simulation trajectory are radius of gyration (Rg) and root-mean-square deviation (RMSD) .
Rg is the time-based displacement of the coordinates of parts of the complex structure from a fixed point, usually its main axis. It specifically evaluates the compactness of the macromolecular structure or the proneness of its secondary structure to changes that could lead to folding perturbations and, hence, new tertiary structures, over a given period of time . Rg values depend on the secondary structure of the protein involved. Predominantly, α-helix proteins tend to have higher Rg values than those of their β-sheet counterparts while those with mixtures of both have Rg values in between the two extremes . Other critical factors affecting Rg values are the number of units the protein is made up of and the number of domains (or chains) making up each unit . Analysis of the Rg-time plots of the aglycone–XOD complexes (Fig. 10) showed that each complex was largely maintained at an Rg below 28 Å, notably modest for an α-helix dominated huge (85 Kda) XOD C-terminal domain under consideration .
RMSD, in molecular dynamics parlance, is the deviation between the coordinates of atoms of a biomolecule at an instant and those of its initial structure. It is calculated by spatial rotation of an instantaneous structure to superimpose with the initial (or reference) structure with the maximum overlap possible. In other words, RMSD is a measure of deviation in the overlay of two compared structures, i.e. instantaneous and initial structures. It is used to assess the degree of conformational changes of the macromolecular structure in the course of the dynamics simulation . RMSD-time plot of each aglycone–XOD complex (Fig. 11) showed the compared structures converging at around 2 ns, broadly plateauing for the rest of the 20 ns simulation time, with a deviation of instantaneous from initial structures averagely maintained around 3 Å during the period. This observation implies the general stability of the three aglycone–XOD complexes [51,52,53]. However, kaempferol–XOD complex demonstrated the least fine fluctuations associated with the RMSD plots particularly over the plateaued region, revealing it as the most stable of the three complexes. Molecular dynamics simulations therefore, in general, revealed a stable aglycone–XOD complex for each of the three flavonoid aglycones of TT and suggests kaempferol as a better inhibitor despite its least docking score (− 7.8 kcal/mol) compared to quercetin (− 8.1 kcal/mol) and isorhamnetin (− 8.2 kcal/mol).
In vivo XOD inhibition assay
The IC50 values of kaempferol (8.2 ± 0.9 μg/ml), quercetin (20.4 ± 1.3 μg/ml) and isorhamnetin (22.2 ± 2.1 μg/ml) were conspicuously lower than that of allopurinol (30.2 ± 3.0 μg/ml), showing that the three flavonoids are better XOD inhibitors than allopurinol, a standard XOD inhibiting antihyperuricemic drug. Further analysis of the IC50 values, however, showed that the three flavonoids do not have equal potencies, kaempferol (IC50: 8.2 ± 0.9 μg/ml) being more potent than isorhamnetin and quercetin which have comparable potencies (IC50 22.2 ± 2.1 μg/ml and 20.4 ± 1.3 μg/ml, respectively). This result is in tandem with the molecular dynamics simulation RMSD plots (Fig. 11) which showed kaempferol–XOD complex as more stable than the isorhamnetin–XOD and quercetin–XOD complexes. At this juncture, it is intellectually gratifying to speculate on the possible correlation of kaempferol’s better-stabilized XOD complex and its associated stronger XOD inhibition potency to its distinct structure compared to those of isorhamnetin and quercetin: The three flavonoid aglycones are flavonols, essentially comprising a flavone skeleton with multiple oxygenated substituents, mostly hydroxyls (Fig. 2) . Kaempferol, however, appears different from the other two in its lack of substitution at the 3ʹ position which is methoxylated and hydroxylated in isorhamnetin and quercetin, respectively. Recalling that each of isorhamnetin and quercetin showed unfavourable donor–donor interactions in the analyses of active site interaction simulations (Figs. 6, 7, 8 and 9), it is safe to link the lack of 3′ oxygenation in kaempferol to its lack of unfavourable active site interaction and hence the edge over its counterparts in XOD inhibition.