Constituents and Anti-Hyperuricemia Mechanism of Traditional Chinese Herbal Formulae Erding Granule

Erding granule (EDG) is a traditional Chinese medicine that has recently been identified as having anti-hypouricemic effects. However, the active components and underlying mechanism for this new indication have not been elucidated. Therefore, we compared the effects of different EDG extracts (water, 50% ethanol and 95% ethanol) on serum uric acid concentrations in the hyperuricemia model mouse. We also analyzed the constituents of different extracts by ultra-high performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) to observe the variation between the active and inactive products. Extract activity and target site were evaluated by assessing uric acid- and inflammation-suppressing effects along with evaluating ability to regulate the uric acid transporter. The results showed that the 50% ethanol extract (EDG-50) had an obvious serum uric acid concentration lowering effect compared with water (EDG-S) and the 95% ethanol extract (EDG-95). UHPLC-Q-TOF-MS/MS analysis showed that EDG-50 was compositionally different to EDG-S and EDG-95. EDG-50 showed dose-dependent effects on reducing uric acid, suppressing inflammation and regulating uric acid transporters. Moreover, western blot analysis showed that EDG-50 down-regulated GLUT9 and URAT1 expression, and up-regulated OAT1 expression. Therefore, our findings enable the preliminarily conclusion that EDG-50 lowers serum uric acid concentrations, mainly by down-regulating the expression of GLUT9 and URAT1 proteins and up-regulating the expression of OAT1 proteins. This provides a research basis for clinical use of EDG as an anti-hyperuricemic agent.


Introduction
Hyperuricemia is human metabolic disorder resulting from increased uric acid formation and/or reduced uric acid elimination. Elevated serum uric acid concentrations may precipitate gout [1,2] and contribute to cardiovascular disease [3,4], type 2 diabetes mellitus [5], nonalcoholic fatty liver disease [6], and chronic kidney disease [7,8], which can seriously compromise human health. Reducing uric acid production with xanthine oxidase inhibitors and increasing uric acid excretion with diuretics are still the main treatments for lowering serum uric acid concentrations. However, with only allopurinol and febuxostat as the key drugs in clinical use, the availability of effective hypouricemic agents is very limited. The discovery of new uric acid-lowering drugs remains of considerable research interest [9][10][11][12]. Note: "*" was compared with the blank group, * P < 0.05, **.P < 0.01; "#" was compared with the model group, # P < 0.05, ## P < 0.01. CG-control group; MG-model group; APG-AP group; SG-EDG-S group; 50G-EDG-50 group; 95G-EDG-95 group.

UHPLC-Q-TOF-MS/MS Analysis of EDG Extracts
Comparison of the EDG-S, EDG-50 and EDG-95 preparations by UHPLC-Q-TOF-MS/MS analysis, revealed 74 components under positive and negative ion modes (positive ion mode: 23; negative ion mode: 51), with 72 being identified preliminarily ( Figure 1 and Table 2). Of the 72 compounds, there were  21 organic acids, 9 coumarins, 28 flavone, 12 alkaloids, and 2 lignins. The structures of these compounds were tentatively assigned by matching the MS/MS data with a reference or public database such as PubChem (https://pubchem.ncbi.nlm.nih.gov/) or MassBank (http://www.massbank.jp/). By comparing the MS/MS intensity of the 74 components from the three extracts, we found that the response of the 20 components in EDG-50 was higher than that of EDG-S and EDG-95 (expressed as "Y" in Table 2).

UHPLC-Q-TOF-MS/MS Analysis of EDG Extracts.
Comparison of the EDG-S, EDG-50 and EDG-95 preparations by UHPLC-Q-TOF-MS/MS analysis, revealed 74 components under positive and negative ion modes (positive ion mode: 23; negative ion mode: 51), with 72 being identified preliminarily ( Figure 1 and Table 2). Of the 72 compounds, there were 21 organic acids, 9 coumarins, 28 flavone, 12 alkaloids, and 2 lignins. The structures of these compounds were tentatively assigned by matching the MS/MS data with a reference or public database such as PubChem (https://pubchem.ncbi.nlm.nih.gov/) or MassBank (http://www.massbank.jp/). By comparing the MS/MS intensity of the 74 components from the three extracts, we found that the response of the 20 components in EDG-50 was higher than that of EDG-S and EDG-95 (expressed as "Y" in Table 2)

Effects of EDG-50 on Serum Uric Acid, Creatinine, and Blood Urea Nitrogen in the Hyperuricemia Rat Model
We investigated the effects of EDG-50 on serum uric acid, creatinine and blood urea nitrogen concentrations in the hyperuricemia rat model. Table 3 shows that there was no significant change in rat body weight in the groups after 7 days of drug administration. The MG had significant differences in serum uric acid, creatinine, and blood urea nitrogen concentrations compared with the CG (P < 0.01). Compared with the MG, serum uric acid concentrations in the APG significantly decreased (P < 0.01), but there was no significant difference in creatinine or blood urea nitrogen concentrations. Compared with the MG, the different EDG-50 dose groups (EDG-50H, -50M and -50L) reduced serum uric acid and creatinine to varying degrees, which was most pronounced in the 50HG (P < 0.01). None of the EDG-50 groups had a significant effect on blood urea nitrogen.    Note: "*": The components were unambiguously identified by comparison with the reference standards. "N": Not identified. "Y": The response of these compounds in EDG-50 is higher than that in EDG-95 and EDG-S. Note: "*" was compared with the blank group, * P < 0.05, ** P < 0.01; "#" was compared with the model group, # P < 0.05, ## P < 0.01. Figure 2 shows that glomerular atrophy and renal tubular dilation were present in the MG, compared with the CG. Compared with MG, renal tubules in the APG were slightly dilated and the glomerular structure was clear; whereas the renal tubules and glomeruli of the 50HG and 50MG showed some improvement, but there was no significant improvement in the EDG-50LG.

Pathological Slices of EDG-50 on the Kidney of Hyperuricemic Rats
Molecules 2019, 24, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules differences in serum uric acid, creatinine, and blood urea nitrogen concentrations compared with the CG (P < 0.01). Compared with the MG, serum uric acid concentrations in the APG significantly decreased (P < 0.01), but there was no significant difference in creatinine or blood urea nitrogen concentrations. Compared with the MG, the different EDG-50 dose groups (EDG-50H, -50M and -50L) reduced serum uric acid and creatinine to varying degrees, which was most pronounced in the 50HG (P < 0.01). None of the EDG-50 groups had a significant effect on blood urea nitrogen Note: "*" was compared with the blank group, * P < 0.05, ** P < 0.01; "#" was compared with the model group, # P < 0.05, ## P < 0.01. Figure 2 shows that glomerular atrophy and renal tubular dilation were present in the MG, compared with the CG. Compared with MG, renal tubules in the APG were slightly dilated and the glomerular structure was clear; whereas the renal tubules and glomeruli of the 50HG and 50MG showed some improvement, but there was no significant improvement in the EDG-50LG.

Effect of EDG-50 on the Expression of OAT1, GLUT9, and URAT1 in Kidney Tissue from Hyperuricemic Rats
Western blotting was used to detect the effects of different doses of EDG-50 (50H, 50M, and 50L) on the expression of uric acid excretion-related proteins (OAT1, GLUT9, and URAT1) in the kidney tissues of hyperuricemic rats. Figure 3 shows that the expression of GLUT9 and URAT1 was significantly up-regulated while the expression of OAT1 was significantly down-regulated in the C group after combined PO and HY administration. Compared with the MG, the expression of OAT1, GLUT9 and URAT1 protein in the AP group was down-regulated; whereas the expression of GLUT9 and URAT1 protein in the EDG-50 group was down-regulated, and the expression of OAT1 protein up-regulated in a dose-dependent manner; for the 50HG, the difference in down-regulated expression of GLUT9 and URAT1 protein was significant.

Discussion
Uric acid is the final product of human purine metabolism. Most uric acid in the human body is produced by the liver. Purine synthesis eventually leads to hypoxanthine, which is then

Discussion
Uric acid is the final product of human purine metabolism. Most uric acid in the human body is produced by the liver. Purine synthesis eventually leads to hypoxanthine, which is then metabolized by xanthine oxidase to uric acid. Therefore, Xanthine oxidase is the key factor to inhibit the production of uric acid [16,17]. In addition, uric acid excretion is mainly regulated by related uric acid transporters in the kidney, including fructose transporter (GLUT9), uric acid anion transporter 1 (URAT1), and organic anion transporters (OAT1, OAT3). URAT1 mediates the exchange of organic anions such as uric acid, lactic acid, chlorine, and other inorganic anions. Uric acid is reabsorbed into renal tubular epithelial cells from the tubular lumen. GLUT-9 mediates uric acid reabsorption across the basement membrane of tubular epithelial cells for return to the blood to help maintain equilibrium. OAT1 and OAT3 are expressed in the basolateral membrane of renal tubular epithelial cells and transport uric acid from peritubular capillaries to renal tubular epithelial cells to complete the first step of uric acid secretion [18][19][20][21]. These are the key ion channels that maintain uric acid concentrations in humans.
After we found that EDG has anti-hyperuricemic effects, we speculated that EDG might reduce serum uric acid concentration mainly via regulation of the uric acid transporter, and increase uric acid excretion. We confirmed that regulation of the uric acid transporter is in accordance with the findings for similar traditional Chinese medicines [22][23][24]. In addition, the previous experimental results were obtained with traditionally prepared EDG prescription drugs as a water extract. Therefore, in order to maximize the anti-hyperuricemic effect of EDG, the active fractions of EDG were screened. The experimental results showed that the anti-hyperuricemic effect of a 50% ethanol extract of EDG was better than that of traditional water and 95% ethanol extracts. Further, the anti-inflammatory effect of EDG-50 and the experimental results regarding regulation of the expression of uric acid transporter proteins showed a dose-response relationship. These results further revealed that a principle EDG effect is to lower serum uric acid concentrations. We concentrated on the 50% ethanol extract which down-regulated GLUT9 and URAT1 protein expression and up-regulated OAT1 protein expression. The results of UHPLC-Q-TOF-MS/MS analysis showed that the components of EDG-50 extracts were different from those of EDG-S and EDG-95 extracts. We inferred that the anti-hyperuricemic effect of EDG-50 was better than that of the EDG-S and EDG-95 extracts, mainly as a result of the different constituents, which preliminary results suggested amounted to 20 differences. These 20 different components include organic acids, alkaloids and flavonoids, which have been reported to have significant anti-hyperuricemia effects [25][26][27][28][29]. Therefore, it can be concluded that EDG produces anti-hyperuricemic effects by promoting uric acid excretion and improving renal function.
The above results only apply to the 50% ethanol extract of EDG, and the specific substances responsible for the anti-hyperuricemic actions are still unclear. Therefore, we will further separate and purify the 50% ethanol extract for future studies of EDG's serum uric acid lowering and anti-inflammatory effects, and its regulation of uric acid transporter proteins. This will enable refined research on the pharmacodynamics of EDG prescription drugs.

Establishment of the Hyperuricemic Mouse Model and Experimental Protocol
Mice (n = 60) were divided randomly into six groups (n = 10 each): control group (CG), model group (MG), AP group (APG; 10 mg/kg), EDG-S group (SG, 5.8 g/kg/day, equivalent to 18 g raw materials/kg/day), EDG-50 group (50G, 4.4 g/kg/day, equivalent to 18 g raw materials/kg/day), and EDG-95 group (95G, 1.8 g/kg/day, equivalent to 18 g raw materials/kg/day). For the treatment groups, AP and EDG treatments were dispersed in 0.5% (w/v) sodium carboxymethyl cellulose (CMC-Na) and administered orally once daily at 8:00 AM from days 1 to 7 whereas the CG and MG received CMC-Na solution alone. After an overnight fast, hyperuricemia was established in all mouse groups except the CG using an intraperitoneal injection of the uricase inhibitor, PO (450 mg/kg), on day 7; the CG was treated with same volume of CMC-Na solution. After 1 h, all groups received their assigned treatments. Blood was collected 1 h after drug administration, and centrifuged at 4000 rpm at 4 • C for 10 min to separate the serum. Serum uric acid concentration was determined using the Hitachi 7080 automatic biochemical analyzer (Kyoto, Japan).

Establishment of the Hyperuricemic Rat Model and Experimental Protocol
After acclimatizing 60 rats for one week, they were divided randomly into the following six groups (n = 10 each): control group (CG), model group (MG), AP group (APG; 10 mg/kg), EDG-50 high dose group (50HG, 2.9 g/kg/day, equivalent to 12 g raw materials/kg/day), EDG-50 medium dose group (50MG, 1.5 g/kg/day, equivalent to 6 g raw materials/kg/day), and EDG-50 low dose group (50LG, 0.8 g/kg/day, equivalent to 3 g raw materials/kg/day). During each of the 7 experimental days, HY 300 mg/kg was administered to all except the CG rats by oral gavage followed by intraperitoneal injection of OP 200 mg/kg at 0800 h each day to induce hyperuricemia. One hour later, the group-specific solvent/drug was given by oral gavage. Blood was collected from the rats 1 h after drug administration, and centrifuged at 4000 rpm at 4 • C for 10 min to separate the serum. After sacrificing the animals, the kidneys were collected and flash frozen in liquid nitrogen. All samples were stored at -80 • C until analysis. Serum uric acid concentrations were measured by the Hitachi 7080 automatic biochemical analyzer (Kyoto, Japan).
The UHPLC-Q-TOF-MS/MS detection was conducted on a Triple TOFTM 5600+ system with a Duo Spray source in the negative electrospray ion mode (AB SCIEX, Foster, CA, USA). The electrospray ionization was applied in the negative mode with the following parameters: ion spray voltage, −4500 V; ion source temperature, 500 • C; curtain gas, 25 psi; nebulizer gas (GS 1), 50 psi; heater gas (GS 2), 50 psi; and declustering potential (DP), −100 V. The mass ranges were set at m/z 50-1250 Da for the TOF-MS scan and 50-1250 Da for the TOF MS/MS experiments. In the IDA-MS/MS experiment, the collision energy (CE) was set at 35 eV, and the collision energy spread (CES) was (±) 15 eV for the UHPLC-Q-TOF-MS/MS detection. Accurate mass and composition for the precursor and fragment ions were analyzed using Peak View ® 1.2 software (AB SCIEX, Foster City, CA, USA)

Pathological Section Analysis
The collected right rat kidney was fixed in 4% paraformaldehyde solution for 24 h, and then cut into appropriate thicknesses, rinsed with running water for 2 h, dehydrated, embedded and sectioned. HE staining was used to observe the pathological damage under microscope.

Kidney Cytokine Analysis
Concentrations of three inflammatory factors (TNF-α, IL-1β and IL-6) were measured in tissues using ELISA kits, as per the manufacturer's instructions, at 450 nm absorbance. Inflammatory factor concentrations were calculated according to the formula of the drawn standard curves.

Western Blot Analysis of Kidney Tissues
Renal tissue was homogenized with RIPA buffer and centrifuged at 14,000 rpm and 4 • C for 30 min to obtain total protein. Protein concentration was then established by BCA Protein assay kit (Beyotime, Shanghai, China). Equal quantities of tissue lysate (30 µg protein) were separated by 8% SDS-PAGE, followed by western blot analysis after transfer of the separated proteins to PVDF membranes. After incubation in a 5% nonfat milk blocking suspension for 1 h, the membranes were incubated with antibodies against β-actin, URAT1, GLUT9, and OAT1 at 4 • C overnight. HRP-conjugated secondary antibodies were incubated at a 1:5000 dilution for 2 h with the membranes. The detection was done using a Molecular Imager ChemiDoc XRS + System (Bio-Rad, Hercules, CA, USA).

Statistical Analysis
Data are presented as the mean ± standard error (S.E.) and were statistically evaluated using one-way analysis of variance (ANOVA) with the GraphPad Prism (GraphPad Inc., San Diego, CA, USA) software program. The results were considered statistically significant at P < 0.05.

Conclusions
Our results showed that EDG-50 was more effective at lowering serum uric acid concentrations than EDG-S or EDG-95. Therefore, our pharmacodynamic evaluation focused mainly on the 50% ethanol extract. We found that EDG-50 had a dose-dependent relationship with anti-hyperuricemia and anti-inflammatory effects, as well as with the regulation of uric acid transporter protein expression.