## 4 Discussion  
Despite hyperuricemia being prevalent in young adults and its incidence increasing in the past ten years [19], the development of specific medicine with minor side effects is still rare. Sunflower calathide, which could be used as a byproduct of food and agriculture, showed its potential to lower the effects of high uric acid and perhaps less toxicity than clinical medicine, such as allopurinol, benzbromarone, and febuxostat [20]. To determine  
Figure 6: The docked complexes (**a**) scopoletin with XO and (**b**) chlorogenic acid with XO. Residues around inhibitor binding.  
the active ingredients, we identified two water-soluble active components of SCHE, SP and CA, although their extraction methods remain to be developed.  
Scopoletin (6-methoxy-7-hydroxycournarin), a naturally occurring phenolic coumarin isolated from medicinal herbs [21, 22], has been verified to lower UA levels in hyperuricemic mice induced by potassium oxyzinate [23] or yeast extract/potassium oxyzinate [24], even if the uricase acts in parallel on hyperuricemic mice; thus, it is essential to investigate the regulation of urate transporters and XO activities in vitro. The same is true for chlorogenic acid, one of the most abundant polyphenols in herbs, whose isolation and identification methods have been extensively explored and it could be extracted from _Eucommia ulmoides_ ([PERSON]) leaf [25], green coffee [26], and _Eupatorium adenophorum_ ([PERSON]) [27], etc. While chlorogenic acid also contributes to hyperuricemia, gout, and inflammation [28, 29, 30], the evidence on the potential molecular mechanism of the regulation of urate transporters is lacking. Given the bioactive ingredients in sunflower calathide, the mechanism of scopoletin and chlorogenic acid was investigated theoretically and experimentally in this paper.  
Consistent with a previous study, in this article we showed that SP and CA have excellent potential as antioxidants [31]. We also demonstrated that the SCHE has the ability to reduce UA-induced oxidase stress stimulated by UA. Additionally, the urate resorptive transporter URAT1 (solute carrier family 22 member 12) [32] and the glucose transporter GLUT9 (solute carrier family 2 member 9) play crucial roles in urate transport associated with hyperuricemia [33, 34]. Therefore, in this study, we used HEK293T cells to determine the regulation of urate transporters with our compounds of interests. Although the UA treatment alone in HEK293T cells did not affect the expression of urate transporters, we found that the SCHE downregulated the expression of URAT1 and GLUT9 in parallel with CA. On the contrary, the significant activation of the GLUT9 transporter occurs in SP treatment, which implies that SP cannot be applied to promote UA excretion, despite its inhibition of the formation of UA and the inhibitory activity of XO (IC\({}_{50}\) = 577.7 uM). Based on the docking results, both SP and CA displayed the same binding pockets as xanthine and closed to the Mo = S center, which just was the important functional site of XO [35]. Moreover, the affinity of XO-CA was much stronger than that of the SP below 260 uM, which was consistent with the in vitro results according to the XO inhibition curve. Accordingly, CA and SP probably interfered with the interaction of xanthine and XO, thus competitively inhibit the production of UA.  
Overall, the results showed that CA, rather than SP, not only achieved the downregulation of the expression of URAT1 and GLUT9, but also inhibited the formation of UA in vitro and the stable binding to the traditional active pocket of XO. It can be concluded that chlorogenic acid has an excellent affinity to XO, which could be used as a potential therapy for the treatment of hyperuricemia.  
Given the above, future studies are needed to determine other potential ingredients in sunflower calathide. Moreover, it might be of great significance to develop drugs with chlorogenic acid and scopoletin that are widespread in botany as alternative anti-hyperuricemia therapy.