Identification of the free phenolic profile of Adlay bran by UPLC-QTOF-MS/MS and inhibitory mechanisms of phenolic acids against xanthine oxidase

Highlights

• A total of 20 phenolics were identified from adlay bran by UPLC-QTOF-MS/MS.

• Sinapic acid was identified from adlay bran for the first time.

• Sinapic acid had strong XOD inhibitory activity in a mixed noncompetitive manner.

• Sinapic acid had synergistic effects with adlay phenolic acids on XOD inhibition.

Abstract

Adlay bran free phenolic extract has been previously demonstrated to possess potent xanthine oxidase (XOD) inhibitory activity. The aims of this study were to characterize the free phenolic profile of adlay bran and investigate the structure-activity relationship, underlying mechanism and interaction of phenolic acids as XOD inhibitors. A total of twenty phenolics including ten phenolic acids, two coumarins, two phenolic aldedhyes and six flavonoids were identified in a phenolic compound-guided separation by UPLC-QTOF-MS/MS. Adlay bran free phenolic extract possessed strong XOD inhibitory activity related to hydroxycinnamic acids with methoxyl groups. The hydrogen bonding and hydrophobic interactions were the main forces in the binding of adlay phenolics to XOD. Sinapic acid, identified in adlay bran for the first time, possessed strong XOD inhibitory activity in a mixed non-competitive manner, and synergistic effects with other adlay phenolic acids at low concentrations, and would be a promising agent for preventing and treating hyperuricemia.

Introduction

Hyperuricemia is mostly due to regular intake of high purine-containing foods and invariably accompanied with gout and chronic kidney disease (Becker et al., 2005). Xanthine oxidase (XOD), an important enzyme associated with hyperuricemia, catalyzes the oxidation of xanthine to uric acid, causing the generation of reactive oxygen species (Chu, Chen, Wu, & Hsieh, 2014). Allopurinol, an effective XOD inhibitor, has been clinically used for the treatment of hyperuricemia and gout (Wang, Zhang, Pan, & Gong, 2015). However, allopurinol causes hepatitis, nephropathy, hypersensitivity and skin rash, raising safety concerns (Li, Zhang, Wang, Xie, & Kong, 2011). The alternative XOD inhibitors with minimal side effects for preventing and treating hyperuricemia are highly desirable.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf), a nourishing soft-shelled seed crop widely distributed in China, Japan and India, is used as a dietary supplement for humans and traditional Chinese medicine due to the bioactive phytochemicals including phenolic acids, phenolic aldehydes, phenolic alcohols, flavonoids, lignans, steroids and lactams (Chung et al., 2011, Wu et al., 2007). The previous studies indicated that adlay bran exhibited more effective anti-haemolysis (Zhao, Yang, Lin, Sun, & Wang, 2017), XOD inhibitory (Zhao et al., 2014) and anti-proliferative activities (Chung et al., 2011) than endosperm. Phenolics are the major phytochemicals in adlay bran and contributed largely to antioxidant (Yang, Zhao, & Lin, 2016), anti-haemolysis (Zhao et al., 2017), XOD inhibitory (Zhao et al., 2014), anti-allergic (Chen, Lo, & Chiang, 2012), anti-inflammatory (Chen, Chung, Chiang, & Lin, 2011) and gastroprotective activities (Chung et al., 2011).

Previous work from our group has shown that brown adlay extract could effectively decrease the serum uric acid levels of oxonate-induced hyperuricemic rats without any side effects (Zhao et al., 2014). The adlay bran free phenolic extract exhibited stronger XOD inhibitory, antioxidant and anti-haemolysis activities than the adlay bran bound phenolic extract (Zhao et al., 2017, Zhao et al., 2014). The most common phenolic compounds found in whole grains are phenolic acids and flavonoids. However, the content and type of phenolics varied depending on the type of cereal and variety (Liu, 2007). Identification of the free phenolic profile of adlay bran is needed to illustrate the relationship between the phenolics and pharmaceutical activity of adlay bran.

Flavonoids widely distributed in fruits and vegetables have attracted growing attention. The structure-activity relationship of dietary flavonoids as XOD inhibitors is clear. Chrysin, apigenin, luteolin, kaempferol and quercetin were considered as the effective XOD inhibitors (Cos et al., 1998, Dong et al., 2016, Nagao et al., 1999). Nevertheless, the structure-XOD inhibitory activity relationship of phenolic acids is not well understood due to different test methods (Masuda et al., 2014, Nile and Park, 2014, Ozyurek et al., 2009, Valentao et al., 2001). It is of high interest to evaluate the relationship between the chemical structures of phenolic acids and their XOD inhibitory activities and verify the potential use of phenolic acids as XOD inhibitors. In addition, the combination of phenolic acids is potentially useful for preventing and treating hyperuricemia.

In the current study, the free phenolic profile of adlay bran was characterized by UPLC-QTOF-MS/MS. The structure-activity relationship of phenolic acids found in adlay bran as XOD inhibitors was illustrated. The underlying mechanisms of their XOD inhibitory effects were also investigated. The interactions of adlay phenolics on XOD inhibition activity were conducted. This study is expected to benefit further application of adlay phenolic acids in functional foods for preventing and treating hyperuricemia.