**1. Introduction**

Advanced glycation end products (AGEs) are a heterogeneous group of reactive, crosslinking compounds produced from nonenzymatic glycation, also known as the Maillard reaction, which occurs between reducing sugars and the amino groups of proteins, nucleic acids, and phospholipids [1]. Protein glycation is formed from Schiff bases, followed by Amadori rearrangements. Amadori products are the initial products of AGEs and can undergo additional glycoxidative modifications [2]. In addition to the above reaction, glucose and Schiff bases can undergo auto-oxidation to form reactive 1,2-dicarbonyl

**Citation:** Chung, C.-P.; Hsia, S.-M.; Chang, W.-S.; Huang, D.-W.; Chiang, W.-C.; Ali, M.; Lee, M.-Y.; Wu, C.-H. Antiglycation Effects of Adlay Seed and Its Active Polyphenol Compounds: An In Vitro Study. *Molecules* **2022**, *27*, 6729. https:// doi.org/10.3390/molecules27196729

Academic Editor: Nour Eddine Es-Safi

Received: 29 August 2022 Accepted: 5 October 2022 Published: 9 October 2022

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compounds, such as methylglyoxal (MGO) and 3-deoxyglucosone [1,3]. Amadori products also undergo glycoxidation to yield glyoxal and glucosone [2]. The formation of AGEs is relatively slow under physiological status, but significantly accelerated in hyperglycemia conditions. Thus, AGEs may play an important role in the pathogenesis of diabetic complications and aging-related diseases [3,4], and may even have an undefined relationship with COVID-19 morbidity and mortality [5].

AGEs adversely affect our body through several mechanisms [2,3]. The first is the modification of intracellular proteins, including the protein regulation of gene transcription. The second mechanism is through these AGE precursors that diffuse out of the cell and modify the extracellular matrix. These modifications dampen the signaling transduction between the matrix and cells, leading to cellular dysfunction. Finally, the third mechanism is these AGE precursors that modify circulating proteins in the blood, such as albumin. The glycated proteins then bind to the receptor for AGEs (RAGE) and activate AGE–RAGE signaling pathways, evoking oxidative stress and an inflammatory reaction [6].

There are several known mechanisms of AGE inhibition [6]. First, AGE formation can be reduced by tight glycemic control. Carbonyl-trapping agents can block AGE formation, reducing the harmful effects of reactive carbonyl species (RCS). Metal ion chelators can also reduce glycoxidative stress due to suppressing the redox reaction, and finally, AGE levels in vivo can be decreased by crosslink breakers. Aminoguanidine (AG), an AGE inhibitor, is often used as a positive control in antiglycation studies. AG is a nucleophilic agent that traps RCS, such as MGO, by forming non-toxic stable adducts [7]. On the other hand, phenolic antioxidants have demonstrated antiglycative properties primarily by scavenging free radicals and chelating metal ions [6,8]. Theoretically, if an inhibitor possesses more than one of the mechanisms mentioned above, it would make an ideal AGE inhibitor for mitigating diabetic complications and other chronic diseases [9].

Adlay (*Coix lachryma-jobi* L. var. *ma-yuen Stapf*), commonly known as Job's tears, is widely cultivated in Asia and is utilized as a Chinese folk medicine, as well as a nutritious food [10]. Structurally, adlay seeds consist of the following four parts from the outside to the inside: the hull (AH), testa (AT), bran (AB), and polished adlay (PA). Recent studies have indicated that adlay and its solvent extracts consist of more than 30 ingredients with 20 biofunctional effects, based on clinical and experimental studies [10–17]. In addition, adlay has been shown to regulate blood sugar [14], blood pressure [18], immunity [19], uterine contractions [20], anti-influenza viruses [21], and osteoporosis preventive activities [12]. However, there are still limited studies that explore the effect of adlay on protein glycation and AGE formation. Thus, this study aimed to evaluate the antiglycation potential of adlay using in vitro glycation assays and to investigate the active fractions and compounds of adlay.

#### **2. Results**

#### *2.1. Effects of the Individual Parts of Adlay on Protein Glycation According to BSA-Glucose Assay*

In the BSA-glucose assay, glucose was used as the glycating agent, and BSA served as the amine group donor, as the glycated target of glucose. This assay aimed to determine whether adlay could inhibit post-Amadori glycation based on the development of AGErelated fluorescence [8]. Firstly, the adlay seeds were divided into the following four parts: hull (AH), testa (AT), bran (AB), and polished adlay (PA). A solvent extraction technique with column chromatography was used to investigate the active fractions and components of adlay (Figure 1). Ethanol extracts of the hull, testa, bran, and polished adlay were referred to as AHE, ATE, ABE, and PAE, respectively. The results showed that among the individual parts of the adlay, ATE and ABE demonstrated greater inhibitory capacities against protein glycation (Figure 2).

**Figure 1.** Partitioning scheme for preparing and isolating active fractions and components from adlay ethanolic extracts. ATE-Ea was dissolved in Ea and subjected to column chromatography on a silica gel with a Hex/Ea gradient from 0 to 100% EA (every 10%) to yield a–h. ATE-BuOH was dissolved in MeOH and subjected to column chromatography on a Diaion HP-20 resin with an H2O/MeOH gradient from 0 to 100% MeOH (every 25%)to yield a–f.

**Figure 2.** Effects of ethanolic extracts of adlay hull (AHE), adlay testa (ATE), adlay bran (ABE), and polished adlay (PAE) on glucose-mediated development of fluorescence of AGEs (BSA-glucose assay). Results are means ± SD for *n* = 3.
