Anti-gout potential of selected edible flowers

Gout is a form of inflammatory arthritis triggered by the interaction between monosodium urate crystals and tissues during the purine metabolism by xanthine oxidase. This study aimed to determine the xanthine oxidase inhibitory activity, total phenolic content, total flavonoid content and total anthocyanin content of 10 selected edible flowers, namely Rosa sp., Malus sp., Lavandula sp., Lilium sp., Hibiscus sabdariffa L., Chrysanthemum sp., Matricaria sp., Gomphrena sp., Myosotis sp. and Jasminum sp. extracted using hot water infusion method. Phytochemical contents and the anti-gout activity of the flower extracts using the xanthine oxidase inhibition assay were determined spectrophotometrically. The results revealed that three aqueous flower extracts (Rosa sp., Hibiscus sabdariffa L. and Malus sp.) exhibited potent xanthine oxidase inhibitory activity (IC50 values, 0.10±0.15 μg/mL, 0.12±0.11 μg/mL and 2.59±3.8 μg/mL, respectively), which were comparable to the positive control, allopurinol (IC50 value, 4.9±0.00 μg/mL). The highest phenolic and flavonoid contents were found in Lavandula sp. (4.39±0.13 mg GAE/g and 63.46±1.07 mg RE/g) while Rosa sp. showed the highest content of anthocyanin (70.14±4.82 mg c-3-gE/g). Positive correlations were observed between the phytochemicals and xanthine oxidase inhibitory activity of the flower extracts. Hence, this study suggests that Rosa sp., Hibiscus sabdariffa L. and Malus sp. possess anti-gout potential, which is associated with the presence of possible anti-gout phytochemicals. The isolation of the bioactive compounds that exhibit significant anti-gout activity among the selected flowers is recommended for future research.


Introduction
Gout is a type of arthritis associated with joint pain and swelling which occurs due to the increase of uric acid in the blood. Xanthine oxidase is an enzyme that controls the uric acid level by catalyzing the oxidative hydroxylation of hypoxanthine to xanthine, then to uric acid (Abu Bakar et al., 2018). The inflammation response is initiated with the formation of monosodium urate crystals at the joints and tissues under abnormally high uric acid levels as a consequence of gout development (Abu Bakar et al., 2018). Allopurinol is the most common drug used for gout treatment and acts as a xanthine oxidase inhibitor, but it causes several side effects including acute renal and hepatic failure, gastrointestinal distress, hypersensitivity reactions and skin rash (Chen et al., 2005;Pacher et al., 2006). Hence, searching for novel xanthine oxidase inhibitors with greater therapeutic potential and fewer side effects is highly needed.
There is a vast array of natural products potentially to be developed as xanthine oxidase inhibitors, at present, the research related to this discovery is still largely unexplored . Flower and herbal tea infusions have been gaining popularity among researchers due to their nutritional value and therapeutic importance. In addition, refreshing tea infusions made from the pure or powdered forms of flowers are popular due to their fragrance, antioxidant properties and therapeutic applications (Aoshima et al., 2007). Its taste, aroma and health benefits also contribute to increased attention and interest for flower teas consumption (Hussain et al., 2019).
Phenolics and flavonoids, including anthocyanins, are the most common phytochemicals found in edible flowers (Kumari et al., 2021). Phytochemical screening eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER of plant extracts has revealed that these compounds may act as anti-gout agents by inhibiting the biosynthesis of uric acid and it is worthy of further research on other samples (Unno et al., 2004). Thus, this study aimed to evaluate the anti-gout activity and phytochemical contents such as phenolics, flavonoids and anthocyanins among the selected edible flowers.

Chemicals and reagents
Gallic acid, rutin, allopurinol and xanthine substrate were purchased from Acros Organics (Fair Lawn, New Jersey, United States). Folin Ciocalteu reagent, aluminium chloride hexahydrate, sodium hydroxide, disodium hydrogen phosphate heptahydrate and potassium chloride were obtained from Merck (Darmstadt, Germany). Sodium carbonate and sodium nitrite were acquired from Fisher Scientific (Hampton, New Hampshire, United States). Xanthine oxidase from bovine milk, hydrochloric acid (0.5 M) and sodium phosphate monobasic was bought from Sigma-Aldrich (St. Louis, Missouri, United States).

Flowers selection and identification
A total of ten dried flowers, namely Rosa sp., Malus sp., Lavandula sp., Lilium sp., Hibiscus sabdariffa L., Chrysanthemum sp., Matricaria sp., Gomphrena sp., Myosotis sp. and Jasminum sp. were purchased in the marketplace at Jonker Walk, Malacca, Malaysia. Two main criteria were established for the sample selection; (1) plant parts used such as a flower in this case, and (2) anti-inflammatory properties of flowers based on the literature. All the flower samples were identified through the guidance of information such as plant morphological description, photographs and illustrations from the botanical databases. Next, the dried flowers were pulverized using a grinder (Panasonic, MX-GM1011H, Japan) and kept in the zip lock bags.

Extraction of flower
Each powdered flower (1 g) was mixed with 100 mL of boiling water for 3 mins (Abu Bakar et al., 2006). The solution was then filtered through the tea filter bag and left to cool down. The aqueous flower extract was used for further phytochemical content determination and anti -gout activity.

Phytochemical testing 2.4.1 Determination of total phenolic content
Total phenolic content was determined by Folin-Ciocalteu assay (Mohd Noor et al., 2020) using gallic acid as the standard. An amount of 0.9 mL of distilled water was mixed with 2 mL of ten-fold diluted Folin Ciocalteu reagent and 0.1 mL of aqueous flower extract. After 5 mins, a 2 mL of 7% (w/v) sodium carbonate solution was added to the mixture and incubated for 30 mins at room temperature. The absorbance values of the reaction mixtures were measured at 760 nm using T60 UV-Vis spectrophotometer (PG Instruments Limited, United Kingdom) and the results were expressed as mg gallic acid equivalent (GAE) per gram of flower sample.

Determination of total flavonoid content
Total flavonoid content was determined using the aluminium chloride colourimetric method (Ali Hassan and Abu Bakar, 2013) and rutin was used as the standard. An amount of 1 mL of aqueous flower extract was diluted with 4 mL of distilled water and then mixed with 0.3 mL of 5% sodium nitrite solution and 0.3 mL of 10% aluminium chloride hexahydrate. The mixture was kept for 5 mins. About 2 mL of 1 M sodium hydroxide was added to the mixture and mixed using a vortex mixer (Labmart, LM-3000, Malaysia). The absorbance values of the reaction mixtures were measured at 510 nm using a T60 UV-Vis spectrophotometer (PG Instruments Limited, United Kingdom). The results were expressed as mg rutin equivalent (RE) per gram of flower sample.

Determination of total anthocyanin content
Total anthocyanin content was determined using spectrophotometric pH differential protocol with some modification (Giusti and Wrolstad, 2001). A 3.5 mL of potassium chloride buffer (0.025 M; pH 1.0) was added into 0.5 mL of aqueous flower extract. The mixture was mixed using a vortex mixer (Labmart, LM-3000, Malaysia) and allowed to stand for 15 mins. The absorbance values of the reaction mixtures were measured at 515 nm and 700 nm against blank as distilled water using a T60 UV-Vis spectrophotometer (PG Instruments Limited, United Kingdom). The results were expressed as mg cyanidin-3glucoside equivalent (c-3-gE) per gram of flower sample.

Anti-gout activity
Xanthine oxidase inhibition assay is an enzyme assay used to determine the anti-gout activity of the plant extracts (Abu  and it was conducted based on the study of Unno et al. (2004) with some modifications. Allopurinol (100 μg/mL) was used as the positive control in this study. The inhibitory effect on xanthine oxidase was measured spectrophotometrically at 295 nm (Ahmad et al., 2006). The reaction mixture consisted of 0.3 mL of 50 mM sodium phosphate buffer (pH 7.5), 0.1 mL of flower extract or standard solution (10-100 μg/mL) dissolved in distilled water, 0.1 mL of freshly prepared enzyme solution (0.2 units/mL of XO in phosphate buffer) and 0.1 mL of distilled water. The eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER assay mixture was pre-incubated at 37°C for 15 mins. Then, a 0.2 mL of substrate solution (0.15 mM of xanthine) was added to the mixture. The reaction mixture was incubated at 37°C for 30 mins. Next, the reaction was stopped with the addition of 0.2 mL of 0.5 M hydrochloric acid. The absorbance values of the reaction mixtures were measured using a UV-VIS spectrophotometer (PG Instruments Limited, United Kingdom) against a blank prepared in the same way but the enzyme solution was replaced with the phosphate buffer. Another reaction mixture was prepared (control) having 0.1 mL of distilled water instead of test compounds in order to have maximum uric acid formation. The degree of xanthine oxidase inhibitory activity was evaluated according to equation (1) (Nessa et al., 2010): Where A is the absorbance of the enzyme without extract and B is the absorbance of the enzyme with the extract. The half-maximal inhibitory concentration (IC 50 ) is defined as the concentration of the test samples required to inhibit 50% of the xanthine oxidase enzyme, which concomitantly decreases the uric acid production by 50% (Nessa et al., 2010). IC 50 values were obtained through the slope of the plot of the percentage inhibition against various concentrations of samples.

Statistical analysis
All experiments were carried out in triplicate. The correlation analysis was done to evaluate the association between the phytochemicals and xanthine oxidase inhibitory activity using the Pearson correlation test.

Phytochemical contents of the selected flowers
The total phenolic, flavonoid and anthocyanin contents in the selected flowers were determined in this study (Table 1). Overall, the total phenolic content of flower extracts ranged from 0.45±0.03 to 4.39±0.13 mg GAE/g. The highest phenolic content was observed in Lavandula sp. extract with the value of 4.39±0.13 mg GAE/g. The second and third highest phenolic contents were recorded in Rosa sp.  FULL PAPER also known to possess antioxidant, anti-inflammatory, anti-cancer and other pharmacological activities (Tungmunnithum et al., 2018). As shown in the result of the current study, the presence of phenolics, flavonoids and anthocyanins was detected in all the flower extracts. Phenolic compounds including phenolic acid and polyphenols are a major class of secondary metabolites existing in most plants and have proven their ability able to delay the ageing process as well as reduce the health risk (Minatel et al., 2017). Among 10 selected flower extracts, their total phenolic content (0.45-4.39 mg GAE/ g) was found to be much lower than the result shown (8.23-284.8 mg GAE/g) in the study of Zheng et al. (2018). The result of the top 3 samples (Lavandula sp., Rosa sp. and Malus sp.) with the highest phenolic content in this study was lower as compared to the extracts (50.8, 39.47-284.8, 8.23-60.58 mg GAE/g, respectively) studied by Zheng et al. (2018). Besides, the overall phenolic content in the current study was lesser than the study of Jin et al. (2016), which ranged from 1.26 to 24.55 g GAE/100 g dw, but the present result of Lavandula sp. (4.39 mg GAE/g) is comparable to the content of L. angustifolia Mill. (3.50 g GAE/100 g dw) and Rosa spp. were also reported to have a high content of phenolics in the study of Jin et al. (2016).
Flavonoids are an important class of secondary metabolites consisting of a large group of polyphenolic compounds and exhibit various health-promoting effects (Kumar and Pandey, 2013). In total, 10 selected flower extracts were found to have rich flavonoid content (2.56-63.46 mg RE/g) and this was higher than the overall content observed (1.18-25.39 mg CAE/g) among the similar samples in the previous study (Zheng et al., 2018). Most of the flower extracts possessed higher flavonoid content as compared to the study of Zheng et al. (2018), except the Myosotis sp. extract. The difference between sample origins, the influence of environmental conditions and extraction methods could lead to the variation in the phytochemical composition of phenolics and flavonoids (Abu Bakar et al., 2016;Md Akhir et al., 2017).
Anthocyanins are a subclass of flavonoids that are responsible for pigmentation in plants and fruits and have been widely studied for various medicinal purposes (Khoo et al., 2017). Most red, blue and purple coloured flowers contain anthocyanins and this could relate to the high content of anthocyanins (2.23-70.14 mg c-3-gE/g) observed among the selected flowers, especially Rosa sp., Myosotis sp. and Lilium sp. extracts with their stronger colouration. The comparison between current and previous studies showed that the anthocyanin content of Rosa sp. (70.14 mg c-3-gE/g) was found to be slightly higher than the anthocyanin content of Rosa indica (64.52 mg c-3-gE/g) (Vankar and Srivastava, 2010). The environmental factors such as temperature and light as well as the presence of complex compounds including phenols and other constituents could affect the anthocyanin content present in every plant species, resulting in a variety of flower colours (Bolling et al., 2010;Mohd Noor et al., 2020). Table 2 displays the inhibition percentages of the standard, allopurinol as well as the flower extracts and their IC 50 values. As a result, all flower extracts showed potent xanthine oxidase inhibitory activity, but the inhibition percentages of flower extracts were not in a concentration-dependent manner. The trend is also observed in the study of Wahyuningsih et al. (2016) and this could be due to the increasing metabolites along with the sample concentrations that disturb the inhibition of the xanthine oxidase enzyme. In the present study, Rosa sp. exhibited the strongest xanthine oxidase inhibitory activity with the IC 50 value of 0.10 µg/mL, as compared to the other 9 flower extracts including Hibiscus sabdariffa L. Xanthine oxidase inhibitors are proven to be effectively used for the treatment of hepatic disease and gout, which is caused by the increased uric acid level and the generation of excessive amounts of superoxide anion radical (Lin et al., 2000). Rosa sp. could be developed as a xanthine oxidase inhibitor because it showed the most significant xanthine oxidase inhibitory activity with the lowest IC 50 value in this study. Previous studies stated that rosehip, the fruit of rose plants within the genus Rosa, in particular, Rosa canina L. exhibited potent biological activity including the anti-inflammatory effect (Jäger et al., 2007;Orhan et al., 2007). As reported in the literature, the presence of flavonoid and phenolic compounds (catechin and syringic acid), as well as anthocyanins (cyanidin and pelargonidin), was identified in the Rosa sp. extracts and these compounds have shown their capabilities to inhibit xanthine oxidase (Kovatcheva-Apostolova et al., 2008;Yang and Shin, 2017;Abu Bakar et al., 2018;Malik et al., 2019).  Kong et al. (2000), whereas Bustanji et al. (2011) reported that the calyx of H. sabdariffa L. inhibited 19.4% of xanthine oxidase enzyme activity, which was lower as compared with this study. The previous and current results are varied in terms of the sample localities and the plant part used for analysis. Furthermore, the flavonoid compounds such as luteolin and apigenin isolated from the flower of Chrysanthemum indicum were shown to exhibit potent xanthine oxidase inhibitory activity (Cos et al., 1998). The major flavonoid, ferulic acid that can be found in Lavandula angustifolia was reported to significantly inhibit more than 50 of xanthine oxidase enzyme activity (Spiridon et al., 2011;Nile et al., 2016).

Xanthine oxidase inhibitory activity of the selected flowers
Hence, the results suggest that all flower extracts are considered as a promising anti-gout agent and the aqueous extracts of Rosa sp., H. sabdariffa L. and Malus sp. with their remarkable xanthine oxidase inhibitory activities should be given priority for further analysis. In addition to the anti-inflammatory and antioxidant activities, the presence of active phytochemicals could act as the natural xanthine oxidase inhibitor and contribute to the anti-gout potential in the flower extracts.

Correlation between phytochemicals and xanthine oxidase inhibitory activity
From the correlation analysis in the study, the total phenolic content was strongly correlated with xanthine oxidase inhibitory activity, which was about 70%. Meanwhile, positive relationships were observed between the total flavonoid content and total anthocyanin content with the xanthine oxidase inhibitory activity, which were 30% and 40%, respectively. As the result shown in the present study, phytochemical compositions such as phenolics, flavonoids and anthocyanins were positively correlated with the xanthine oxidase inhibitory activity, and the phenolic compounds contributed primarily to the anti-gout activity among the flower extracts. High content of phenolics in Rosa sp. extract resulted in its highest xanthine oxidase inhibitory activity and the strong positive correlation was supported by Kılıçgün and Altıner (2010). A similar observation was also found in Malus sp. due to its high content of phenolics, contributing to its stronger xanthine oxidase inhibition. Other than phenolics, flavonoids and anthocyanins, the compounds including alkaloids, cardiac glycosides, steroids, tannins and terpenoids are responsible for the xanthine oxidase inhibitory activity (Apaya and Chichioco-Hern, 2011) and this could explain the strong anti-gout potency in H. sabdariffa L. extract.

Conclusion
This study clearly demonstrated the potential of Rosa sp., H. sabdariffa L. and Malus sp. as an anti-gout agent due to their stronger xanthine oxidase inhibition abilities recorded with lower IC 50 values and rich content of phytochemicals. The preliminary results of this study could be further investigated through the isolation and identification of active constituents among the flower extracts, toxicity test as well as the in vivo experiments for the verification of their abilities in inhibiting the xanthine oxidase enzyme.

Conflict of interest
The authors do not have any conflicts of interest regarding the content of the present work.