Prenylflavonoids isolated from Epimedii Herba show inhibition activity against advanced glycation end-products

Introduction: As inhibitors of advanced glycation end products (AGEs), such as pyridoxamine, significantly inhibit the development of retinopathy and neuropathy in rats with streptozotocin-induced diabetes, treatment with AGE inhibitors is believed to be a potential strategy for the prevention of aging, age-related diseases, and lifestyle-related diseases, including diabetic complications. In the present study, the MeOH extract of Epimedii Herba (EH; aerial parts of Epimedium spp.) was found to inhibit the formation of N ε -(carboxymethyl)lysine (CML) and N ω -(carboxymethyl) arginine (CMA) during the incubation of collagen-derived gelatin with ribose. Materials and methods: EH was purchased from Uchida Wakan-yaku Co., and a MeOH extract was prepared. Several steps of column chromatography purified the extract. Each fraction was tested for inhibitory activity by ELISA using monoclonal antibodies for CML and CMA. Results: After activity-guided fractionation and purification by column chromatography, three new prenylflavonoids [named Koreanoside L (1), Koreanoside E1 (2), and Koreanoside E2 (3)] and 40 known compounds (4–43) were isolated from EH, and their inhibitory effects against CML and CMA formation were tested. Among these, epimedokoreanin B (8), epimedonin E (21), epicornunin B (22), and epicornunin F (24) inhibited the formation of both CML and CMA, with epimedokoreanin B (8) having the most potent inhibitory effect among the isolated compounds. To obtain the structure–activity relationships of 8, the phenolic hydroxy groups of 8 were methylated by trimethylsilyl-diazomethane to afford the partially and completely methylated compounds of 8. Prenyl derivatives of propolis (artepillin C, baccharin, and drupanin) were used in the assay. Discussion: As only 8 showed significant activity among these compounds, the catechol group of the B ring and the two prenyl groups attached to the flavanone skeleton were essential for activity. These data suggest that 8 could prevent the clinical complications of diabetes and age-related diseases by inhibiting AGEs.


Introduction
In recent years, preventive medicine has begun to play an important role in the aging population globally.Inhibiting the formation of advanced glycation end products (AGEs), which are involved in the progression of lifestyle-related diseases such as diabetic complications (Lin et al., 2011) and atherosclerosis (Torres et al., 2015), and aging-related diseases such as osteoporosis (Brandt et al., 2022) and Alzheimer's disease (Barnard et al., 2014), is an effective method for the prevention of these diseases using natural products ( Al-Musayeib et al., 2011;Harris et al., 2011;Aljohi et al., 2018;Tominaga et al., 2020).N ε -(Carboxymethyl) lysine (CML), a major antigenic AGE structure, accumulates in several human and animal tissues during aging (Araki et al., 1992;Schleicher et al., 1997), and in patients with various diseases, including diabetic nephropathy (Vlassara et al., 1994;Rabbani and Thornalley, 2018) and encephalopathy.N ω -(Carboxymethyl) arginine (CMA) is an acidlabile AGE structure discovered in the enzymatic hydrolysate of glycated collagen (Iijima et al., 2000).Collagen is an important protein that constitutes body tissues; however, it has been reported that when collagen becomes an AGE, it decreases both in strength and flexibility (Kitamura et al., 2021).CMA accumulation in tissue proteins may contribute to the pathophysiology of aging and agerelated diseases (Mera et al., 2008;Kinoshita et al., 2019).
Epimedii Herba (EH) has been used in traditional Chinese Medicine to treat erectile dysfunction, dysuria, waist and knee pain, infertility, and angina pectoris (Wu et al., 2003;Li C. et al., 2015;Chen et al., 2015;Qian et al., 2024).In Japan, the crude drugs listed in the Japanese Pharmacopeia and EH extracts are usually included in energy drinks for tonicity.Its main ingredients are prenylated flavonoids (Ma et al., 2011;Li et al., 2021), especially icariin (Figure 1), which suppresses nerve degeneration, improves cognitive function in neurological disorders (Guo et al., 2010), and has neuroprotective effects (Li et al., 2022).It has also been suggested that icariin inhibits AGE-derived neuropathy in PC12 cells (Zhao et al., 2019); RAGE might be a potential target for Epimedium's antineuroinflammatory role in vascular dementia, which is an insight from network pharmacology and molecular simulation (Yuan et al., 2023), and extracts including icariin may reduce the risk of atherosclerosis by inhibiting the formation of AGEs on HDL (Kim and Shim, 2019).However, these studies were comparisons using readily available icariin, and the anti-glycation activity of the main body of EH was not considered due to the limited number of samples used in these evaluations.We have previously shown that some compounds isolated from EH have significant inhibitory effects on AGE formation (Nakashima et al., 2016).In this study, the isolation and purification of EH's prenylflavonoid compounds was continued; 40 known compounds and three new compounds were isolated, and their chemical structures were determined.Based on the results of CML and CMA production inhibitory activity tests on 35 prenylflavonoids, of which quantities were available, the chemical structure characteristics necessary for the production inhibitory activity were summarized by synthesis of derivatives and comparison with related natural products and are reported.

Acid hydrolysis of compound 1
Compound 1 (1.0 mg) was hydrolyzed with 2 M HCl: dioxane = 1:1 solvent (1 mL) at 95 °C in a pear-shaped flask for 1.5 h and H 2 O (2 mL) was added to the mixture, and it was evaporated to dryness under vacuum to obtain a residue.The residue was loaded onto Amberlite   Effect of compounds from Epimedii Herba on CML formation.Gelatin (2.0 mg/mL) and ribose (30 mM) were incubated with the samples [(A): 100 and 10 μM, (B) 1.0 μM] in 10 mM phosphate buffer at 37 °C for 7 days.The CML content was determined using noncompetitive ELISA.Effect of compounds from Epimedii Herba on CMA formation.Gelatin (2.0 mg/mL) and ribose (30 mM) were incubated with samples [(C): 100 and 10 μM, (D) 1.0 μM] in 100 mM sodium phosphate buffer at 37 °C for 7 days.The CMA content was determined using noncompetitive ELISA (mean ± SD, n = 3).polarity of the standard [L-rhamnose: t R = 4.5 min (−)], the constituent sugars of compound 1 was identified as L-rhamnose [t R = 4.5 min (−)].

Determination of the inhibitory effects of compounds on CML and CMA formations
Gelatin (2 mg/mL) and ribose (30 mM) were incubated with the tested compounds in PBS for CML and in 100 mM sodium phosphate buffer for CMA at 37 °C for 7 days, followed by the determination of CML and CMA formation using a noncompetitive enzyme-linked immunosorbent assay (ELISA).

ELISA
ELISA was performed as previously described (Sugawa et al., 2016).Briefly, each well of a 96-well microtiter plate was coated with 100 μL of the sample in PBS, blocked with 0.5% gelatin, and washed three times with PBS containing 0.05% .The wells were incubated with 100 μL of anti-CML antibody 6D12 (0.1 μg/mL) or anti-CMA antibody 3F5 (1.0 μg/mL) dissolved in washing buffer for 1 h.The wells were then washed three times with washing buffer and incubated with horseradish peroxidaseconjugated anti-mouse IgG antibodies, followed by incubation with 1,2-phenylenediamine dihydrochloride.The reaction was terminated by the addition of 100 μL of 1 M sulfuric acid and the absorbance at 492 nm was read by a micro-ELISA plate reader.

Statistics
All data are representative of two or three independent experiments.Data are expressed as mean (SD).The Mann-Whitney U test was used for two-group comparisons.Statistical significance was set at p < 0.05.

Isolation and determination of new compounds (1-3) from EH
Following activity-guided fractionation of the three fractions prepared in a previous report (Nakashima et al., 2016) (frs. 1, 2, and 3), fr.Three was further separated by column chromatography combined with MCI gel CHP20P, Sephadex LH-20, μ-Bondapak C 18 , and preparative HPLC.Then, 43 candidate prenylated flavonoid derivatives, including new compounds (1-3), were isolated to test the inhibition of CML and CMA accumulation.
Compound 3 was similar to compound 2 in the 1 H-NMR spectrum, but its rotation value was different ([α] D −48.5 °(c = 0.36)).The 13 C-NMR date of three and two were identical but C-3 (δ C 130.5-133.7),C-4 (δ C 178.8-179.4), and C-15 (δ C 18.1-19.9)data were shifted lower in the field.Because of the possibility of stereoisomerism at the C-12 position, compound 3 was hydrolyzed by naringinase to get an aglycone 3a in 75% yield.Compound 3a was obtained as a yellow amorphous powder and the rotation value was [α] D −14.0 (c = 0.038).The 1 H-NMR of 3a was identical to that of 2a.The supernatant of reactant was also analyzed by HPLC connected to an optical rotatory detector, and the peak of L-(−)-rhamnose was observed.To adapt the modified Mosher method, the phenolic hydroxyl groups of 3a were methylated and purified to yield 3b. Subsequently, 3b was reacted with (R)-(−)-MTPA-Cl to give MTPA ester 3b-S in 47% yield.The 1 H-NMR data of 3b-S was superimposable to that of 2b-R, so that it was applied to the advanced Mosher method because the 1 H-NMR signals of 2b-S were the same as those of 3b-R.As a result, the distribution of chemical shifts, as shown in Figure 3B, was observed, and the absolute conformation at C-12 of compound 3 was determined as R. Compound 3 was given the trivial name koreanoside E2.This indicated that compound 3 was a diastereomer of compound 2 because of the rhamnose bond to C-3, which could be separated by HPLC.

The inhibitory effects of compounds on CML and CMA formation
Of the 43 compounds isolated and determined from EH, 1 mM DMSO solutions were prepared for 35 compounds, which were evaluated for their inhibitory activity against CML and CMA formation (Table 2).The results showed that samples 7,8,9,10,16,17,21,22, and 24 had vigorous CML production inhibitory activity of more than 80% at 10 μM (Figure 4A).All the nine prenylflavonoids that exhibited significant inhibitory activity were aglycones.For further comparison, when the sample concentration was examined at 1.0 μM, samples 8, 21, 22, and 24 showed more potent inhibition than pyridoxamine (PM) and luteolin as the positive controls (Figure 4B).Inhibitory formation of CMA also showed significant activity in the same compounds at 10 μM (Figure 4C).In contrast, weak inhibitory activity was observed for samples 8, 21, and 22, and the other compounds displayed no activity at 1.0 μM (Figure 4D).
When the compounds evaluated for activity were divided into glycosides and aglycones, the CML and CMA production inhibitory activities of the aglycons tended to be more pronounced.Compounds with vigorous CML inhibitory activity also showed intense CMA inhibitory activity, especially aglycones of the flavanone skeleton without an oxygen functional group at the three-position.However, among the glycosides, icariside I (2) and epimedoside C (5), in which glucose is attached only at the seven-position of the flavonol backbone, were active (Figures 4A, C).In contrast, no activity was observed for limonianin (6) or epimedokoreanin C (EK-C, 23), even as aglycones.The γ,γ-dimethylallyl group was on the dimethylpyrane ring of 6. Comparing 6 with epimedonin C (16), 16 showed activity even when a dimethylpyran ring was present on the B ring.In contrast, 16 and 23, which share the same A-and C-ring moieties, showed a marked decrease in activity when the B ring became a cyclopentane ring.Furthermore, when comparing the activity at lower concentrations, epimedokoreanin B (8), epimedonin E (21), epicornunin B ( 22), and F (24) (Figure 4B) showed greater muscular CML inhibitory activity at 1 μM than PM and luteolin, which were used as control drugs.These four compounds also showed vigorous CMA production inhibitory activity of more than 80% at 10 μM (Figure 4C).Based on these results, the standard chemical structure of prenylflavonoids with both CML and CMA formation inhibitory activity is an aglycon of the luteolin-type flavanone skeleton, with prenyl groups at the eight-position of the A ring and the 5′position of the B ring and a catechol group on the B ring.
Instead of gelatin, type I collagen, which is present in the dermis, has been used to inhibit CML and CMA production.Four compounds, , epimedonin E (21), epicornunin B ( 22), and F (24) that showed significant inhibitory activity in the gelatin evaluation system, were used in this experiment.Prenylflavonoids (8,21,22,and 24) in Figure 5A were added to a mixture of ribose (30 mM) and type I collagen (1.5 mg/mL), incubated at 37 °C for 7 days, and then measured by ELISA using monoclonal anti-CML and anti-CMA antibodies.The amounts of CML and CMA generated in the type I collagen were determined by ELISA using monoclonal anti-CML and anti-CMA antibodies, respectively (Figure 5B; C).The results showed that the four prenylflavonoid compounds in EH had significant inhibitory activity against both CML and CMA.In particular, , epimedonin E (21), and epicornunin B ( 22) almost wholly inhibited CML formation even at concentrations of 1 μM (Figure 5B).These compounds also inhibit CMA entirely at a concentration of 10 μM (Figure 5C).In contrast, epicornunin F (24) showed inhibitory activity against CML and CMA formation, comparable to that of luteolin, which was used as a control drug.
Gelatin is a hydrolyzed and solubilized form of collagen that has the same amino acid sequence as collagen but a different steric structure.The CML and CMA inhibitory activities of the four prenylflavonoids (Figure 5A) were also observed in collagen, which has a different conformation.In other words, gelatin used as a mediocre protein reproduced the same inhibitory activity as expensive collagen.Thus, the usefulness of gelatin in screening prenylflavonoids for inhibition of CML and CMA production was confirmed.

Structure-activity relationships on CML and CMA formations
All compounds with significant inhibitory activity against CML and CMA production (Figure 5A) contained a catechol group.To investigate the importance of the catechol group, we methylated the hydroxyl group of 8, which had the most potent inhibitory activity and the highest yield.We then measured the inhibitory effects on CML and CMA formation.First, the four phenol hydroxyl groups of 8 were partially methylated using TMS-diazomethane.The reaction was monitored using silica gel TLC and stopped when the fully methylated form (C) was formed (Figure 6A).The resulting mixture containing the partially methylated product was separated and purified using silica gel to isolate the three products, along with raw material recovery.The chemical structures of the partially methylated forms A, B, and the fully methylated form C were determined from various NMR data.The yield of each compound was 11% for A, 25% for B, and 19% for C. The derivatives obtained (A-C) were tested for their CML and CMA formation inhibitory activities.All the methylated compounds showed lower CML and CMA formation inhibitory activities than 8 (Figures 6B, C).Compared to compounds A, B, and C, the CML formation inhibitory activity (Figure 6B, at 10 μM) was significantly reduced when the hydroxyl group attached to the C-5 in ring A was methylated as shown in compound C. Silica gel TLC (Hexane: acetone = 2:1 (v/v)) analysis of compound B, in which the hydroxyl group at position five of the B ring remains, and the fully methylated compound C showed that the Rf value of compound B was 0.75.In contrast, that of compound C was as low as 0.20.Furthermore, in HPLC analysis [column: YMC C 18 (ϕ10 × 250 mm); flow rate: 2.0 mL/min; temperature: 40 °C; detection: RI], the retention time of compound C was 14.5 min, which was shorter than that of compound B (19.9 min).Thus, methylation of the hydroxyl group at the five-position of the A ring causes a marked change in the chemical and physical properties owing to the loss of the hydrogen bond with the carbonyl oxygen at the four-position of the C ring.Comparison of the CML and CMA inhibitory activities of eight reaffirmed the importance of the catechol group of the flavonoid B ring.They suggested that the hydrogen bond between the hydroxyl group at the five-position of the A ring and the carbonyl group at the four-position of the C ring is essential for the inhibitory activity against CML and CMA formation.Next, we focused on the prenyl group, a substructure other than the catechol group of 8, which exhibits inhibitory activity.Therefore, we examined its contribution to the inhibitory activities of CML and CMA formation.In this study, three prenylated cinnamic acid derivatives, artepillin C, baccharin, and drupanin (Figure 7A), which are the main components of propolis (Rodrigues et al., 2020), were used as prenyl-related compounds for the inhibitory activity test.As shown in Figures 7B, C, the prenylated cinnamic acid derivatives used in this study did not inhibit CML or CMA formation.This suggests that neither prenylated cinnamic acid derivatives (two prenyl groups for artepillin C and one for drupanin) nor their aromatic esters (baccharin) were active; rather, the binding of prenyl groups to the flavonoid backbone, which contains phenolic hydroxyl groups, was responsible for the inhibition of CML and CMA formation.This suggests that the binding of a prenyl group to the flavonoid skeleton may enhance the inhibitory activity against CML and CMA formation.
In summary, the structure-activity relationship of the prenylrelated compounds from EH revealed that a catechol group in the B ring and prenyl groups at the eight and 5′positions are essential for the inhibitory activity against CML and CMA formation, that the hydroxyl group at the five-position is hydrogen bonded, and that the three-position does not have an oxygen functional group.In other words, compound 8, which had the highest yield (0.084%) and the most potent inhibitory activity against CML and CMA formation, was found to be the active compound in the extract.Our findings are still experimental results at the test-tube level.However, we were able to clarify the partial structure of the prenylflavonoids required for anti-glycation activity.Although further studies should be conducted in animal models of diseases related to diabetes, atherosclerosis, and osteoporosis, these results suggest that compound 8 could be used as a therapeutic compound because it inhibits AGE formation and prevents the development of diabetic complications, such as diabetic nephropathy, retinopathy, and neuropathy, and age-related diseases, such as Alzheimer's disease.
FIGURE 5(A) The structures have strong inhibition activity of CML and CML formation.Effect of compounds from Epimedii Herba on CML (B) and CMA (C) formation.Type I collagen (1.5 mg/mL) and ribose (30 mM) were incubated with the compounds (100, 10, and 1 μM) in (B) 10 mM phosphate buffer; in (C) 100 mM sodium phosphate buffer at 37 °C for 7 days.The CML and CMA content was determined using noncompetitive ELISA (mean ± SD, n = 3).

FIGURE 7
FIGURE 7Chemical structures of prenylated cinnamic acid derivatives (PCAs) (A) artepillin C, baccharin, and drupanin.Comparative study of the effect of and PCAs on (B) CML and (C) CMA formation.Gelatin (2.0 mg/mL) and ribose (30 mM) were incubated with the compounds (100, 10, and 1 μM) in (B) 10 mM phosphate buffer; in (C) 100 mM sodium phosphate buffer at 37 °C for 7 days.The CML and CMA content was determined using noncompetitive ELISA.Data are presented as the mean ± SD.
°C.For the analysis of sugar moieties, HPLC was performed on the Shodex RS-Pak ϕ6.0 × 150 mm, Resonac Corp., Tokyo, Japan)column at a flow rate of 1.0 mL/min and a column temperature of 80 °C.TLC was performed using pre-coated silica gel 60 F 254 plates (Merck Ltd., Frankfurt, Germany).Detection was achieved by spraying the plates with 10% H 2 SO 4 followed by heating.Column chromatography was

TABLE 2
Prenylflavonoids from Epimedii Herba used for inhibiting activity test against CML and CMA formation.