Shikonin derivatives as potent xanthine oxidase inhibitors: in-vitro study

Abstract Induction of hypersensitivity reactions (may be fatal too) by specific XO inhibitors has led to development of new molecules that are efficacious and have safer ADME profile. Among natural compounds, biologically active Alkannin/Shikonin (A/S) derivatives have unexplored XO inhibition potential. Therefore, their iso-hexenylnaphthazarin nucleus was studied and found that the nucleus is similar to that of allopurinol, signifying the XO inhibitory potential of these derivatives. For confirmation of their potential, β,β-dimethylacrylshikonin and deoxyshikonin were successfully isolated and characterised from Arnebia euchroma (Royle.) Johnst. (Boraginaceae) and were evaluated for in vitro XO inhibitory potential. β,β-dimethylacrylshikonin and deoxyshikonin showed a good XO inhibition potential with IC50 values of 7.475 ± 1.46 µg/mL and 4.487 ± 0.88 µg/mL, respectively. Results also validated the pharmacophore hypothesis, and it was concluded that nucleus iso-hexenylnaphthazarin can be remodelled for optimising the efficacy. Graphical Abstract


Introduction
Xanthine Oxidase (EC 1.17.3.2) is the key enzyme involved in the catalytic conversion of purines, xanthine and other related compounds to uric acid (UA) (Baker et al. 2005). Within physiological limits i.e., serum UA levels < 6.5 mg/dL, UA acts as a potent antioxidant, immune system stimulant and helps in maintaining blood pressure in saltpoor conditions (Pasalic et al. 2012). But during hyperuricemic condition, UA crystallises in the form of monosodium urate monohydrate microcrystals and starts depositing in various parts of the body mainly in joints and impacts mobility of the patient (Gulati et al. 2020). In US, 32.5 million individuals (14.6%) were found to be affected by hyperuricemia which may reach a value of 78 million (26%) by 2040 (Singh et al. 2019). Hence, this surge makes it mandatory to control the hyperuricemic patient which can be done by halting the biosynthesis of UA via inhibition of XO enzyme.
Specific XO inhibitors (allopurinol and febuxostat) are available for treatment, but they possess excessive toxicity (Singh et al. 2020). Patients treated with allopurinol and febuxostat have developed serious hypersensitivity reactions such as Stevens-Johnson syndrome, which can be fatal too (Duzallo 2020; Febuxostat Mylan 2022). Additionally, febuxostat showed an increase in liver enzyme levels which may progress to acute liver dysfunction (Ito et al. 2014). Therefore, this builds up a need to search for new molecules that are efficacious and specifically inhibit the XO enzyme and have better ADMET profile.
Therefore, the main pharmacophore of A/S derivatives i.e., iso-hexenylnaphthazarin (yellow) nucleus was aligned on allopurinol (red) using DE Shaw Desmond to predict their XO inhibition potential. This hypothesis elucidated that the iso-hexenylnaphthazarin nucleus has all the desired pharmacophoric features i.e., four acceptor groups and at least one aromatic ring to act as a potent XO inhibitor ( Figure S1). Hence, this builds up curiosity to evaluate and confirm the XO inhibitory potential of A/S derivatives biologically.

Results and discussion
For this study, Arnebia euchroma (Royle.) Johnst. plant (Boraginaceae family) was chosen as it is a rich source for isolating A/S derivatives. After completion of extraction and isolation process (approx. time 6 months), multiple compounds were collected from which only two compounds were isolated in pure and good amounts (A1 ¼ 990 mg and A2 ¼ 860 mg) which were then sent for evaluation through spectroscopic method for structural elucidation.
After procuring mass, proton and carbon spectra, protons were assigned to carbons by studying the correlation of each hydrogen via homonuclear (COSY) and heteronuclear correlation (HMBC, HSQC) methods as shown in Figures S2-S4, and Table S2. NMR data of these compounds showed some similarities in their structure such as two carbonyl carbon peaks ranging between 183.2 and 177.5 ppm, two carbon peaks (162-166 ppm) which show the presence of hydroxyl group, and four carbon peaks in the aromatic region. Moreover, two identical protons between 12.66 and 12.38 ppm signifies the presence of hydroxyl or acidic proton, two aromatic protons between 7.23 and 7.12 ppm, and one proton within the range of 6.91-6.87 ppm. These similarities confirm that the compounds have a common nucleus in them, and on further evaluation, this data was found to be similar to spectral data of iso-hexenylnaphthazarin nucleus. Furthermore, this structural data was then compared with already discovered A/S derivatives and it was found that the structures were possessing characteristic peaks of protons of Shikonin's hydroxyl groups at 12 ppm. Moreover, spectral data matched with the A/S derivatives already being reported by Han et al. i.e., A1¼ b, b-dimethylacrylshikonin (BBSH) and A2 ¼ deoxyshikonin (DOSH) as shown in Figure S5 (Han et al. 2008). Unfortunately, the compounds were not novel and have already been reported to be present in A. euchroma (Zhu et al. 2019). But still, no study has been reported to confirm the hypouricemic effect of A/S derivatives. Therefore, after successful characterisation, compounds were subjected to in-vitro evaluation i.e., XO inhibitory potential and antioxidant activity. For XO inhibitory potential, a per cent decrease in the conversion of xanthine to UA was monitored and respective IC 50 values were calculated. On comparing the results with allopurinol (standard drug), compounds A1 and A2, and methanolic extract (ME), showed a dosedependent response as shown in Figure S6, indicating that the derivatives have XO inhibitory potential. Compounds A1, A2 and ME possess IC 50 values of 7.475 ± 1.46 mg/ mL, 4.487 ± 0.88 mg/mL and 40.03 ± 1.42 mg/mL, respectively, whereas it was 2.468 ± 0.91 mg/mL for allopurinol. On statistical evaluation via one-way ANOVA, the compounds A1, A2 and allopurinol were having no significant difference in their activity at a p-value < 0.05, hence concluding that the inhibitory potential of isolated compounds was the same as that of allopurinol. Moreover, compounds A1 and A2 were also more potent than ME as shown in Figure S6. Hence, these results confirm that A/ S derivatives possess XO inhibitory potential, and their pharmacophore can be remodelled for optimisation of their potency.
During conversion of xanthine to UA by XO, the Molybdopterin (involved in the catalytic activity of the enzyme) nucleus gets reduced from VI to IV oxidation state which leads to generation of reactive oxygen species (ROS) (Singh et al. 2020). Therefore, during overactivity of the XO enzyme, it's very likely to observe a surge in ROS generation and this surge increases the risk of development of other associated disorders such as Gout, diabetes mellitus, metabolic syndrome, renal and cardiovascular diseases (Many et al. 1996;Dawson and Walters 2006;Harzand et al. 2012). Hence, to combat this surge in ROS generation, XO inhibitors must possess an antioxidant nature. Therefore, we have evaluated the free radical scavenging potential of isolated compounds by using DPPH scavenging assay and the results were compared with potent antioxidant compound i.e., gallic acid. The results of these compounds and ME were statistically compared with the reference antioxidant compound i.e., gallic acid by using one-way ANOVA and concluded that at a p-value < 0.05, there was no significant difference in the free radical scavenging potential of ME and compound A1 and A2 w.r.t. gallic acid as shown in Figure S7. Compounds A1, A2 and ME possess IC 50 values of 28.7 ± 0.94 mg/mL, 34.2 ± 1.00 mg/mL and 44.6 ± 1.66 mg/mL, respectively, whereas it was 3.843 ± 0.64 mg/mL for standard gallic acid.
At last, correlation studies were performed between XO inhibition potential and antioxidant potential of A1 and A2 by using Pearson's coefficient of correlation and their r 2 values were compared. These results showed that the r 2 value of the datasets was more than 0.90 as shown in Figure S8, hence this signifies a strong correlation between them. Moreover, the datasets were following the same trend i.e., with an increase in the dose of the compound there is an increase in the activity of the compounds. From this we can conclude that dual nature of compounds A1 and A2 i.e., a potent XO inhibitor as well as a potent antioxidant. Hence, this dual property will synergistically lower the UA levels of the patient with significant reduction in oxidative stress.

Conclusion
In search of an alternative for the currently available treatment of hyperuricemia, the XO inhibitory potential of A/S derivatives was predicted by the pharmacophore hypothesis. Two derivatives i.e., BBSH and DOSH were isolated from the root bark of Arnebia euchroma and characterised. From the above in-vitro screening, it was concluded that A/S derivatives have XO inhibitory potential and the scaffold iso-hexenylnaphthazarin of these derivatives can be remodelled for optimisation of the efficacy. Additionally, derivatives possessed a good antioxidant profile making them an eligible candidate for the treatment of hyperuricemia.