Sains Malaysiana 52(6)(2023): 1635-1648

http://doi.org/10.17576/jsm-2023-5206-03

In Vitro and In Silico Study on the Interaction between Apigenin, Kaempferol and 4-Hydroxybenzoic Acid in Xanthine Oxidase Inhibition

(Kajian Secara In Vitro dan In Silico pada Interaksi antara Apigenin, Kaempferol dan Asid 4-Hidroksibenzoik dalam Perencatan Xantina Oksidase)

CHIN YONG SIN¹, LOH KHYE ER^1,*, WEE SZE PING¹ & ONG GHIM HOCK²

¹Department of Bioscience, Faculty of Applied Sciences, Tunku Abdul Rahman University of Management and Technology, Jalan Genting Kelang, Setapak. 53300 Kuala Lumpur, Malaysia

²Faculty of Health and Life Sciences, INTI International University, Persiaran Perdana BBN, Putra Nilai, 71800 Nilai, Negeri Sembilan, Malaysia

Diserahkan: 17 Ogos 2022/Diterima: 23 Mei 2023

Abstract

Xanthine oxidase (XO) is a biological enzyme that takes part in purine catabolism. It catalyses the conversion of hypoxanthine to xanthine and eventually xanthine to uric acid. The catabolism reaction increases the level of uric acid and subsequently leads to hyperuricemia. Allopurinol is a XO inhibitor that is used clinically to prevent purine catabolism. Although it is an effective XO inhibitor, it causes some side effects. Therefore, a more effective inhibitor with fewer side effects is in an urgent need. Phenolic compounds have been identified as effective XO inhibitors in many studies. In vitro and in silico study were conducted to investigate the interaction between apigenin, kaempferol and 4-hydroxybenzoic acid in XO inhibition. Apigenin was found to be the most effective XO inhibitor among the compounds tested with the best docking score of -8.2 kcal/mol as demonstrated in the molecular docking simulation which indicated its favourable interaction with XO enzyme. Additive interactions between compounds namely apigenin-kaempferol, apigenin-4-hydroxybenzoic acid and 4-hydroxybenzoic acid-kaempferol were demonstrated in both in vitro and in silico studies. The results showed that 4-hydroxybenzoic acid- apigenin (-7.4 kcal/mol) was the most stable ligands combination docked to XO. The multiple ligands docking simulation showed independent ligands bound to the XO active site at non-interfering regional location. In conclusion, the combination of these three compounds can be explored further for their additive interaction in XO inhibition, which could be beneficial in terms of the enhanced effectiveness and lower side effects when each is used at lower dose to give the same effect.

Keywords: Additive interaction; molecular docking; multiple ligands; phenolic compounds; xanthine oxidase inhibitor

Abstrak

Xantina oksidase (XO) ialah sejenis enzim biologi yang terlibat dalam metabolisme purin. Ia memangkinkan penukaran hipozantin kepadaxantina dan akhirnya daripadaxantina kepada asid urik. Tindak balas katabolisme meningkatkan tahap asid urik dan seterusnya membawa kepada hiperurisemia. Allopurinol adalah sejenis perencat XO yang digunakan secara klinikal untuk mencegah katabolisme purin. Walaupun ia adalah sejenis perencat XO yang berkesan, ia menyebabkan kesan sampingan. Oleh itu, perencat yang lebih berkesan serta kurang kesan sampingan adalah amat diperlukan. Sebatianfenolik telah dikenal pasti sebagai perencat XO yang berkesan dalam banyak kajian. Kajian in vitro dan in siliko telah dijalankan untuk mengkaji interaksi antara apigenin, kaempferol dan asid 4-hidrosibenzoik semasa perencatan XO. Apigenin didapati merupakan perencat XO yang paling berkesan dalam kalangan sebatian yang dikaji dengan skor dok yang terbaik sebanyak -8.2 kcal/mol sebagaimana yang ditunjukkan oleh simulasi dok molekul yang menunjukkan interaksi yang menggalakkan dengan enzim XO. Interaksi secara tambahan antara sebatian iaitu apigenin-kaempferol, apigenin-asid 4-hidroksibenzoik dan asid 4-hidroksibenzoik-kaempferol telah ditunjukkan dalam kajian in vitro dan in siliko. Hasil kajian menunjukkan asid 4-hidroksibenzoik-apigenin (-7.4 kcal/mol) adalah gabungan ligan yang paling stabil semasa didokkan pada XO. Simulasi dok berbilang ligan menunjukkan ligan bebas terikat pada tapak aktif XO di lokasi yang tidak mengganggu antara satu sama lain. Secara kesimpulannya, gabungan ketiga-tiga sebatian ini boleh diterokai dengan lebih lanjut dari segi interaksi tambahan mereka dalam perencatan XO, yang boleh dimanfaatkan dari segi peningkatan keberkesanan dan pengurangan kesan sampingannya dapat dipertingkatkan apabila setiap satu digunakan pada dos yang lebih rendah untuk memberikan kesan yang sama. 

Kata kunci: Dok molekul; interaksi tambahan; pelbagai ligan; perencat xantina oksidase; sebatian fenolik

RUJUKAN

Cao, H., Pauff, J.M. & Hille, R. 2014. X-ray crystal structure of a xanthine oxidase complex with the flavonoid inhibitor quercetin. Journal of Natural Products 77(7): 1693-1699.

Cao, H., Pauff, J.M. & Hille, R. 2010. Substrate orientation and catalytic specificity in the action of xanthine oxidase. Journal of Biological Chemistry 285(36): 28044-28053.

Cos, P., Ying, L., Calomme, M., Hu, J.P., Cimanga, K., Poel, B.V., Pieters, L., Vlietinck, A.J. & Berghe, D.V. 1998. Structure-activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers. Journal of Natural Products 61(1): 71-76.

Du, X., Li, Y., Xia, Y.L., Ai, S.H., Liang, J., Sang, P., Ji, X.L. & Liu, S.Q. 2016. Insights into protein-ligand interactions: Mechanisms, models, and methods. International Journal of Molecular Science 17(2): 144-178.

Enroth, C., Eger, B.T., Okamoto, K., Nishino, T., Nishino, T. & Pai, E.F. 2000. Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion. Proceedings of the National Academy of Sciences of the United States of America 97(20): 10723-10728.

Ichide, K., Matsuo, H., Takada, T., Nakayama, A., Murakami, K., Shimizu, T., Yamanashi, Y., Kasuga, H., Nakashima, H., Nakamura, T., Takada, Y., Kawamura, Y., Inoue, H., Okada, C., Utsumi, Y., Ikebuchi, Y., Ito, K., Nakamura, M., Shinohara, Y., Hosoyamada, M., Sakurai, Y., Shinomiya, N., Tatsuo, H. & Suzuki, H. 2012. Decreased extra-renal urate excretion is a common cause of hyperuricemia. Nature Communications 3(1): 764-777.

Li, Q.Q., Yang, Y.X., Qv, J.W., Hu, G., Hu, Y.J., Xia, Z.N. & Yang, F.Q. 2018. Investigation of interactions between thrombin and ten phenolic compounds by affinity capillary electrophoresis and molecular docking. Journal of Analytical Methods in Chemistry 2018: 4701609.

Lin, C.M., Chen, C.S., Chen, C.T., Liang, Y.C. & Lin, J.K. 2002. Molecular modeling of flavonoids that inhibits xanthine oxidase. Biochemical and Biophysical Research Communications 29(1): 167-172.

Lin, S., Zhang, G., Liao, Y., Pan, J. & Gong, D. 2015a. Dietary flavonoids as xanthine oxidase inhibitors: Structure-affinity and structure-activity relationships. Journal of Agricultural and Food Chemistry 63(35): 7784-7794.

Lin, S.Y., Zhang, G.W., Liao, Y.J. & Pan, J.H. 2015b. Inhibition of chrysin on xanthine oxidase activity and its inhibition mechanism. International Journal of Biological Macromolecules 81: 274-282.

Liu, L., Zhang, L., Ren, L. & Xie, Y. 2020. Advances in structures required of polyphenols for xanthine oxidase inhibition. Food Frontiers 1: 152-167.

Loh, K.E., Chin, Y.S., Ismail, I.S. & Tan, H.Y. 2021. Rapid characterisation of xanthine oxidase inhibitors from the flowers of Chrysanthemum morifolium Ramat. using metabolomics approach. Phytochemical Analysis 33(1): 12-22.

Malik, N., Dhiman, P. & Khatkar, A. 2019. In silico design and synthesis of hesperitin derivatives as new xanthine oxidase inhibitors. BMC Chemistry 13(1): 53.

Masuda, A., Takahashi, C., Inai, M., Miura, Y. & Masuda, T. 2013. Chemical evidence for potent xanthine oxidase inhibitory activity of Glechoma hederacea var. Grandis leaves (Kakidoushi-Cha). Journal of Nutritional Science and Vitaminology 59: 570-575.

Masuoka, N. & Kubo, I. 2018. Characterization of the xanthine oxidase inhibitory activity of alk(en)yl phenols and related compounds. Phytochemistry 155: 100-106.

Murata, K., Nakao, K., Hirata, N., Hirata, N., Namba, K., Nomi, T., Kitamura, Y., Moriyama, K., Shintani, T., Munekazu, I. & Matsuda, H. 2009. Hydroxychavicol: A potent xanthine oxidase inhibitor obtained from the leaves of betel, Piper betle. Journal of Natural Medicines 63(3): 355-359.

Narayanaswamy, R., Isha, A., Lam, K.W. & Ismail, I.S. 2016. Molecular docking analysis of selected Clinacanthus nutans constituents as xanthine oxidase, nitric oxide synthase, human neutrophil elastase, matrix metalloproteinase 2, matrix metalloproteinase 9 and squalene synthase inhibitors. Pharmacognosy Magazine 12(45): 21-26.

Ng, T.L., Loh, K.E., Tan, S.A., Tan, H.Y., Yue, C.S., Wee, S.P. & Tey, Z.T. 2022. Anti-hyperuricemic effect of ethyl acetate sub-fractions from Chrysanthemum morifolium Ramat. dried flowers on potassium oxonate-induced hyperuricemic rats. Applied Sciences 12: 3487.

Okamoto, K., Eger, B.T., Nishino, T., Pai, E.F. & Nishino, T. 2008. Mechanism of inhibition of xanthine oxidoreductase by allopurinol: Crystal structure of reduced bovine milk xanthine oxidoreductase bound with oxipurinol. Nucleosides, Nucleotides and Nucleic Acids 27(6-7): 888-893.

Reynolds, C.H., Bembenek, S.D. & Tounge, B.A. 2007. The role of molecular size in ligand efficiency. Bioorganic & Medicinal Chemistry Letters 17(15): 4258-4261.

Santoyo, A.H., Tenorio-Barajas, A.Y., Altuzar, V., Vivanco-Cid, H. & Mendoza-Barrera, C. 2013. Protein-protein and protein-ligand docking. In Protein Engineering - Technology and Application. InTech Open Science. pp. 63-84.

Shani, M., Vinker, S., Dinour, D., Leiba, M., Twig, G., Holtzman, E.J. & Leiba, A. 2016. High normal uric acid levels are associated with an increased risk of diabetes in lean, normoglycemic healthy women. Journal of Clinical Endocrinology and Metabolism 101(10): 3772-3778.

Tao, X., Huang, Y.K., Wang, C., Chen, F., Yang, L.L., Ling, L., Che, Z.M. & Chen, X.G. 2019. Recent developments in molecular docking technology applied in food science: A review. International Journal of Food Science and Technology 55(44): 33-45.

Tung, Y.T. & Chang, S.T. 2010. Inhibition of xanthine oxidase by Acacia confusa extracts and their phytochemicals. Journal of Agricultural and Food Chemistry 58(2): 781-786.

Umamaheswari, M., Madeswaran, A., Asokkumar, K., Sivashanmugam, T., Subhadradevi, V. & Jagannath, P. 2011. Study of potential xanthine oxidase inhibitors: in silico and in vitro biological activity. Bangladesh Journal of Pharmacology 6(2): 117-123.

Wang, Y.J., Zhang, G.W., Pan, J.H. & Gong, D.M. 2015. Novel insights into the inhibitory mechanism of kaempferol on xanthine oxidase. Journal of Agricultural and Food Chemistry 63: 526-534.

Xie, Y., Yang, W., Tang, F., Chen, X. & Ren, L. 2014. Antibacterial activities of flavonoids: Structure-activity relationship and mechanism. Current Medicinal Chemistry 22(1): 132-149.

Zhao, C., Yang, C.F., Liu, B., Lin, L., Sarker, S.D., Nahar, L., Yu, H., Cao, H. & Xiao, J. 2018. Bioactive compounds from marine macroalgae and their hypoglycemic benefits. Trends in Food Science & Technology 72: 1-12.

*Pengarang untuk surat-menyurat; email: lohke@tarc.edu.my