Globally, diabetes mellitus (DM) or hyperglycemia is one of the most common metabolic disorders in both developed and developing nations. According to the International Diabetes Federation (IDF), diabetes affected 415 million people in 2015, and this number is expected to increase to 552 million by 2040 (Zimmet et al., 2016). A high blood glucose level leads to the accumulation of reducing sugars in the blood and the development of advanced glycation end products (AGEs) through non-enzymatic glycation of plasma proteins such as hemoglobin, serum albumin, and transferrin. In addition to altering and modifying other proteins, AGEs contribute to oxidative stress in the body by generating reactive oxygen species (Dariya and Nagaraju, 2020; Khalid et al., 2022), ultimately leading to several long-term complications of diabetes (Brownlee, 2001; Ahmed, 2005; Xu et al., 2018). These complications are considered a major concern after the onset of diabetes. Therefore, inhibition of AGEs may be one of the important prerequisites for preventing the post-translational modification of proteins (Gill et al., 2019). The most prevalent form of diabetes, type 2, is associated with a major pathophysiological condition termed insulin resistance, in which insulin-sensitive tissues (skeletal muscle, adipose tissue, and liver tissue) fail to respond adequately to the physiological concentration of insulin, altering glucose homeostasis in the process (Goldstein, 2002; Garneau and Aguer, 2019). Skeletal muscle has a major role in postprandial glucose disposal and accounts for more than 80% of insulin-dependent glucose disposal in humans (Vincent et al., 2004; Chadt and Al-Hasani, 2020).

In addition to AGE levels, GLUT4 surface translocation is also an important measure of antidiabetic activity. Insulin promotes a major signaling pathway—namely, the phosphatidyl inositol-3-kinase (PI-3-K)/protein kinase B (AKT) pathway—which stimulates the translocation and distribution of GLUT4 at the cell surface (Mackenzie and Elliott, 2014). AKT is a serine/threonine kinase that mediates most of the PI-3-K-mediated metabolic actions of insulin through the phosphorylation of several substrates, including other kinases, signaling proteins, and transcription factors. In skeletal muscle, the rate-limiting step for glucose utilization is the uptake of glucose by the cells. Glucose uptake is facilitated inside the cells by the translocation and redistribution of glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane (Vincent et al., 2004). Upon insulin stimulation, the insulin receptor is phosphorylated, leading to the activation of the insulin signal transduction pathway that upregulates glucose uptake in the cells by stimulation of the translocation of GLUT4 transporters to the plasma membrane. This peripheral glucose utilization is reduced in diabetic individuals due to a reduction in GLUT4 translocation, which leads to secondary complications such as increased AGE production (Figure 1) (Klip et al., 1990; Scheepers et al., 2004). Reports also suggest that AGEs interfere with insulin signaling and prevent GLUT4 membrane translocation, promoting the accumulation of more glucose in the blood (Diamanti-Kandarakis et al., 2016) (Figure 2). Research has revealed that aminoguanidine (AG) is an effective antiglycation agent at higher doses, but cannot be recommended due to its side effects (Sadowska-Bartosz et al., 2014). Thus, there is an unmet need for new herbal antiglycation and anti-diabetic agents that not only prevent the formation of AGEs and development of insulin resistance but are also safer to use and relatively cost-effective. Estimates indicate that approximately 80% of the world’s population relies on the use of herbal medicines, which thus have better prospects on the global market (Shakya, 2016).

Today, it is widely acknowledged that medicinal plants play a significant role in conventional healthcare operations, in directing research toward new areas, and in safeguarding biodiversity (Thakur et al., 2022; Singh et al., 2022). The plant Lyonia ovalifolia, locally known as “Angyar”, belongs to the Ericaceae family and is distributed in the mountain zones of India, Nepal, China, Japan, Cambodia, Myanmar, Thailand, Vietnam, and Malaysia. The leaves and stems of this plant have traditionally been used to treat pimples, boils, cuts, and wounds (Shah and Joshi, 1971; Bhandari et al., 2020), and have been investigated for their natural insecticide, antioxidant, antimicrobial, and immunomodulatory properties (Acharya, 2015; Sahu and Arya, 2017). Various compounds, such as grayanane diterpenoids (lyonin A-C, lyoniol D, grayanane, lyoniol-A, secorhodomollolides A and D), triterpenoids and triterpenoid glycosides (Hebecarposides A−K), iridoids (lyonofolins A-C, gelsemiol), flavonoids (quercetin 3-galactoside, apigenin, luteolin, quercetin, epicatechin, eriodictyol), lignans (lyoniside, lyoniresinol, ovafolinins A-E), and benzodioxane lignans (cupressoside A), have been isolated from the leaves and stem of this plant (Lv et al., 2016; Sahu and Arya, 2017; Yang et al., 2017; Teng et al., 2018; Zhao et al., 2018; Zhang et al., 2020). These compounds exhibit diverse biological activities. However, the bioactive compounds of L. ovalifolia with antidiabetic potential have been little studied, although the very small number of studies that have been conducted have revealed the presence of compounds with potential hyperglycemic properties, such as iridoids (lyonofolins A-C, gelsemiol) and flavonoids (eriodictyol), in the aerial parts of L. ovalifolia (Hameed et al., 2018; Hussain et al., 2018). However, the plant has yet not been explored for its targeted antiglycation and GLUT4 translocation properties. Therefore, there is a need for extensive research on this plant in terms of antidiabetic and antiglycation activity. Hence, this study evaluated the antiglycation activity of the ethanolic extract and solvent fractions of L. ovalifolia in a stepwise manner. In addition, extracts and bioactive fractions were examined for their role in anti-diabetic mechanisms, such as the rate of surface GLUT4 translocation in skeletal muscles. The LC-ESI-QTOF-MS/MS technique was used to identify bioactive phytochemical constituents. Furthermore, to elucidate the binding interaction of bioactive compounds, docking against the diabetes receptors aldose reductase (AR) (PDB ID: 1USO), tyrosine kinase (TK) (PDB ID: 1IR3), and SIRT6 (PDB ID: 3K35) was carried out.

The production of AGEs in serum albumin samples is characterized by the presence of a series of fluorescent compounds associated with strong fluorescent emission. In the present experiments, incubation of BSA (bovine serum albumin) with fructose (BF) for 28 days showed enhanced fluorescent emission, indicating increased formation of AGEs. Moreover, incubation of BF with L-01 (100 μg/mL) for 15, 21, and 28 days showed a significant (p < 0.01) reduction in AGE-associated fluorescent emission of 19.56%, 27.63%, and 36.95%, respectively, compared to positive control AG (59.01%) (Figure 5 and Table 1). Among the solvent fractions, L-03 at 100 μg/mL concentration (L-03b) was found to be the most effective (p < 0.01), with 23.45% and 38.40% AGE inhibition at 15 and 21 days, respectively, as compared to the other fractions (Figure 6 and Table 1). Remarkably, after 28 days of incubation, L-03 showed a significant increase in percent AGE inhibition (51.12%), compared to the positive control of 59.01% inhibition at 5 mM concentration (BFG5) (p < 0.01). The inhibitory effects of L-03 on fluorescent emission of AGEs prove its potential in inhibiting the biosynthesis of AGEs (Table 1).

GLUT4 translocation assay showed that L-01 significantly stimulated the abundance of GLUT4 molecules on the surface by up to 1.5- and 1.8-fold at 10 and 25 μg/mL concentrations, respectively, as compared to unstimulated samples (Figure 7). Moreover, the ethyl acetate fraction (L-03) also showed 1–1.2-fold GLUT4 translocation compared to unstimulated samples (Figure 7). This result meant that L-01 at 25 μg/mL was found to be most effective in GLUT4 translocation (Figure 8). The translocation rate further increased when insulin-stimulated cells were treated with L-01 (25 μg/mL), revealing the highest-fold GLUT4 translocation in a dose-dependent manner (Figure 8). This observed biological response of L-01 was comparable to the positive control rosiglitazone.

Moreover, LC-ESI-QTOF-MS/MS determination of L-01 and L-03 showed the presence of 12 major compounds belonging to the classes of lignans, flavonoids, flavonoid glycosides, and terpenoids (Table 2 and Figures 9A, B). Of these 12 major compounds, nine had been previously reported for this plant: (1) 5,5’dimethoxysecoisolariciresinol, (2) lyoniresinol, (3) syringaresinol, (4) astilbin or dihydroquercetin-3-O-rhamnoside, (5) quercetin 3-galactoside, (6) eriodictyol, (7) quercetin-3-O-rhamnoside or quercitrin, (8) luteolin, and (9) lyoniol C (Kashima et al., 2010; Acharya, 2015; Sahu & Arya, 2017). The remaining three compounds—(10) leucothol B, (11) rhodoterpenoids, and (12) leucothol A—are reported for the first time in this species. Of these 12 compounds, the three lignans (5,5’dimethoxysecoisolariciresinol, syringaresinol, and lyoniresinol) were identified and characterized by MS spectra and (RT) retention time compared with their corresponding standards analyzed under identical conditions (Table 2 and Figures 10A, B). The remaining compounds (4–12) were tentatively identified on the basis of their positive ESI mass spectra. Moreover, phytochemical analysis of the ethyl acetate fraction (L-03) also indicated the presence of the same 12 compounds identified in the ethanolic extract (L-01), with the exception of lyoniresinol. This indicates that most of the bioactive compounds of L-01 possess ethyl acetate solubility.

The receptors used for docking with bioactive compounds were aldose reductase (AR) (PDB ID: 1USO), tyrosine kinase (TK) (PDB ID: 1IR3), and SIRT6 (PDB ID: 3K35) (Figures 10A–C). After docking, it was observed that luteolin achieved the best docking score (12.3 kcal/mol) against the target receptor aldose reductase (PDB ID: 1USO). On the basis of the highest docking score and lowest binding energy, the top ligand was identified and hypothesized to be the most effective ligand against target receptor aldose reductase (PDB ID: 1USO). Among the ten ligands that were screened against the three selected receptor targets, the compounds with the best docking pose scores were predicted to be effective; their results are shown in Table 3 which presents the binding energy of ligand–receptor interactions. In our in silico analyses, we found that syringaresinol had a binding energy of −11.4, −7.9, and −7.4 kcal/mol against aldose reductase (AR) (PDB ID: 1USO), tyrosine kinase (TK) (PDB ID: 1IR3), and SIRT6 (PDB ID: 3K35), respectively. Astilbin had a binding energy of −11.5, −9.0, and −8.2 kcal/mol against aldose reductase (AR), SIRT6, and tyrosine kinase (TK), respectively. Lyoniol-A had a binding energy of −9.2, −6.8, and −6.7 kcal/mol against aldose reductase (AR), tyrosine kinase (TK), and SIRT6, respectively. Eriodictyol had a binding energy of −12.1, −9.1, and −8.0 kcal/mol against aldose reductase (AR), SIRT6 and tyrosine kinase (TK), respectively. Lyoniresinol had a binding energy of −9.0, −6.5, and −6.0 kcal/mol against aldose reductase (AR), SIRT6 and tyrosine kinase (TK), respectively. Luteolin had a binding energy of −12.3, −8.9, and −8.6 kcal/mol against aldose reductase (AR), SIRT6, and tyrosine kinase (TK), respectively. Quercitrin had a binding energy of −10.4, −8.9, and −8.6 kcal/mol against aldose reductase (AR), tyrosine kinase (TK), and SIRT6, respectively. Rutin had a binding energy of −10.7, −8.7, and −8.3 kcal/mol against aldose reductase (AR), tyrosine kinase (TK), and SIRT6, respectively. Leucothol A had a binding energy of −10.7, −9.1, and −7.5 kcal/mol against aldose reductase (AR), SIRT6, and tyrosine kinase (TK), respectively. Finally, leucothol B had a binding energy of −9.1, −8.3, and −7.7 kcal/mol against aldose reductase (AR), SIRT6, and tyrosine kinase (TK), respectively. As aldose reductase is reported to be involved in the rate-limiting enzyme of the polyol pathway, converting excess D-glucose to D-sorbitol with NADPH as a co-factor, it is crucial in the treatment of diabetic microvascular problems. Aldose reductase is also involved in lipid metabolism. One of the best known sirtuins in mammals, SIRT6 is involved in a number of cellular functions, including DNA repair, maintenance of glucose balance, and longevity. SIRT6 is a NAD+-dependent deacetylase, enabling it to control the cellular activity of numerous acetylated proteins in mammals (Singh et al., 2019).

Our study revealed that luteolin (−12.3 kcal/mol) was most effective against the target receptor aldose reductase (PDB ID: 1USO), followed by eriodictyol (−12.1 kcal/mol), astilbin (−11.5 kcal/mol), and syringaresinol (11.4 kcal/mol). The molecular interaction of the binding pocket suggests that the bound ligand luteolin can potentially inhibit aldose reductase (PDB ID: 1USO); three key amino acid residues of AR (ASP134, THR135, and ASN136) were strongly forming hydrogen bonds, indicating strong binding to the active pocket (Figure 11A). Ligand eriodictyol can potentially inhibit aldose reductase (PDB ID: 1USO); four key amino acid residues of AR (SER133, ASP134, THR135, and ASN136) were strongly forming hydrogen bonds, suggesting strong binding to the active pocket (Figure 11B). Similarly, seven residues of AR (PDB ID: 1USO) protein (GLU193, LYS194, LEU195, ARG296, PRO310, PHE311, and HIS312) were strongly forming hydrogen bonds with astilbin, suggesting strong binding to the active pocket (Figure 11C). In the same way, ligand syringaresinol was involved in inhibiting the AR protein by binding with the 11 residues of AR protein (ASN162, GLN192, GLU193, LYS194, LEU195, ASN292,ASN294, ARG296, PRO310, PHE311, and HIS312) that were strongly forming hydrogen bonds, suggesting strong binding to the active pocket (Figure 11D). As a result, it may be concluded that luteolin is the most effective inhibitor of the AR protein and may act to improve glucose absorption as well as lowering hyperglycemia.

Flavonoids are the largest group of polyphenols and are well known as AGE inhibitors of BSA glycation (Yeh et al., 2017). It is also evident that flavonoids have antioxidant and antiglycation properties which might be associated with their anti-oxidation function (Anusiri et al., 2014). Lyonia ovalifolia has previously been reported to exhibit antioxidant, antimicrobial, and immuno-modulatory effects (Acharya, 2015), and to show cytotoxic and antidiabetic activity (Yang et al., 2017). In this study, the effects of the ethanolic extract and solvent fractions of L. ovalifolia on AGE production were found to indicate that ethanolic extract has considerable potential to inhibit AGE formation at both early and advanced stages of glycation. Moreover, at 100 μg/mL, the L-03 bioactive fraction showed better AGE inhibition in the early phases of glycation compared to AG (positive control).

Similarly, L-01 was found to stimulate the translocation of GLUT4 molecules at the cell surface, dose dependently, and alone as well as in combination with insulin. This suggests that L-01 increases GLUT4 translocation in L6 GLUT4 myc skeletal muscle cells and has potential implications for the management of Type 2 diabetes. These findings further validate our discovery that L-01 follows a similar pathway to insulin for the stimulation of GLUT4 translocation to the cell surface. A similar finding regarding the antidiabetic potential of L. ovalifolia has been reported recently, in which eriodictyol isolated from ethanolic extract was found to stimulate insulin secretion though cAMP/PKA signaling (Hameed et al., 2018). Moreover, three new iridoid compounds have been isolated that also showed antihyperglycemic effects (Hussain et al., 2018). Cytochrome (CYP450) is well known for drug metabolism; here, we observed that isomers of CYP450 are not affected by any of the four compounds identified, meaning that compounds do not suppress the metabolic functions of involved enzymes. Regarding log Kp values, the more negative the value, the lower the availability of a compound to pass through the skin (Hussain et al., 2021). The bioavailability scores of luteolin, eriodictyol, and syringaresinol suggested that they follow the Lipinski, Veber, Ghose, Egan, and Muegge rule.

The antiglycation and GLUT4 translocation potential of L. ovalifolia is supported by the presence of flavonoids in the LCMS determinants of the L-03 fraction, namely, eriodictyol, quercitrin (quercetin-3-O-rhamnoside), quercetin-3-O-galactoside, luteolin, and astilbin (dihydroquercetin-3-O-rhamnoside). The docking studies also supported the hypothesis of GLUT4 translocation potential. Various reports support the flavonoids and their antidiabetic activity (Yasue and Kato, 1959; Yasue et al., 1961; Rahman et al., 2007; Dong et al., 2017; Hameed et al., 2018; Ma et al., 2021). There are several pieces of evidence linking the intake of flavonoids with favorable outcomes among patients with Type 2 diabetes (Wu and Yen, 2005; Wedick et al., 2012). The literature also reveals that flavonoids from the leaf extract of Thuja orientalis; their isolated flavonoids (i.e., quercitrin and amentoflavone) inhibit aldose reductase as well as the formation of AGEs and exhibit the strongest potential for AGE inhibition (Lee et al., 2009). Three flavonoids isolated from Cephalotaxus sinensis leaves have also been found to show an association with elevated GLUT4 protein levels in diabetic rats (Li et al., 2007). The experimental evidence reported in this article suggests that L. ovalifolia is a rich source of naturally occurring flavonoids. These compounds showed strong inhibitory potential against AGE formation and, hence, antiglycation activity, along with enhanced GLUT4 activity; this finding was also supported by docking studies.

This study shows that crude extract of L. ovalifolia has the potential to perform dual activities: antiglycation of advanced glycated endproducts (AGEs) as well as GLUT4 translocation. The strong antiglycation activity of the ethyl acetate fraction might be due to the involvement of flavonoids. The reported evidence strongly supports the anti-diabetic properties of L. ovalifolia, which may be considered to be a new and renewable herbal source for the management of insulin resistance and long-term diabetic complications. The promising potential of all four compounds, as evaluated in docking studies, MD simulations, and ADMET analysis, recommends them for in vitro investigation as well as in vivo experimentation to confirm their value in drug development.