## 4 Discussion
### Putative Identification of XO Inhibitors Using HPLC-Q-TOF-MS/MS  
Based on HPLC-Q-TOF-MS/MS analysis, only five compounds, namely 3,4,5-tricaffeoylquinic acid, apigenin 7-O-6\({}^{\prime\prime}\)-acetylglucoside, 6-C-glucosyl-8-C-arabinosylapigenin, apigenin 7-O-glucose, and equatilin have been reported previously in _C. morifolium_, while other known compounds were reported for the first time in _C. morifolium_. Several phenolic compounds, including caffeoylquinic acid and flavonoids, were identified from EtOAc F10-4 (**6**, **12**), F10-8 (**17**, **18**, **19**, **21**), and F10-9 (**29**). The flavonoid compounds identified in this study were speculated to be responsible for the high XO inhibitory activity of F10. Flavonoid compounds such as apigenin isolated from _Chrysanthemum spp_. have been shown to possess good XO inhibitory activity [31]. Apigenin 7-O-glucose has been shown to have multiple significant biological activities, such as anti-inflammatory, antioxidant, anticancer, antiviral, antibacterial, and pro-apoptotic effects [32]. Apigenin, on the other hand, has been reported to be the most potent inhibitor for XO, attributed to its stable interaction with the enzyme active site in a molecular modelling study [33]. Prunetin and Prunetin 4\({}^{\prime}\)-O-galactoside were identified for the first time from _C. morifolium_ in the present study. It has been isolated from the leaves and stem bark of _Dalbergai spinose_ and has been related to anti-inflammatory activity [34]. Since the current treatment for gout include the use of anti-inflammatory agent, apigenin and prunetin could be investigated further as a lead compound for gout therapy.  
Other than the phenolic compounds, five terpenoid compounds were identified from F10-4 (**3**, **7**, **8**, **9**) and F10-10 (**33**). Medicagenic acid is a bioactive triterpenoid and has been demonstrated to exhibit only low XO inhibitory activity [35]. So far, there is no report about the XO inhibitory activity of schizonepetoside E, zerumbone, vulgarone A and B. Besides, sporalens identified from F10-8 (**23**) and F10-9 (**28**) have also not been reported to inhibit XO enzyme. The primary metabolites identified from different F10 sub-fractions were regarded as possessing important functions in the primary metabolism of plants; it was postulated that they might not possess any direct inhibitory potential on the XO enzyme. To date, there are a limited number of reports about the bioactivities of these compounds.  
The majority of the compounds in the highly bioactive F10 might be alkaloids, but only two known compounds were identified in this study, namely acetylimetermedine (**20**) and prosopinine (**32**) from F10-8 and F10-9, respectively. Acetylimetermedine is a naturally occurring pyrrolizidine alkaloid reported to be hepatotoxic. Pyrrolizidine alkaloids are common secondary metabolites in plants, which possess significant hepato-toxicity, genotoxicity, tumorigenicity, as well as neurotoxicity, on human and animal models [36]. Although alkaloid compounds are often toxic to human beings, they are widely used in modern medicines. For instance, colchicine is used as an anti-inflammatory agent for acute gout attack treatment and prevention [37], and is currently used as a clinically effective anti-gout medicine. However, its risk to benefit ratio is considered high, as gastrointestinal effects such as nausea, diarrhoea, vomiting, and stomach upset occur in about 80% of patients [38].  
Metabolite profiling of the EtOAc F10 suggested that flavonoid compounds might be responsible for the XO inhibitory activity and anti-hyperuricemic activity of _C. morifolium_ flowers. Since an abundance of alkaloids are also present in _Chrysanthemum_ flowers [39] and alkaloid has been shown to exhibit XO inhibitory activity [40], it is highly recommended to isolate and elucidate the structure of the unknown compounds in the bioactive sub-fractions by other spectroscopic methods, including 1 H and 13 C NMR.