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Abstract

Background

Diabetes mellitus, a metabolic disorder, leads to complications via oxidative stress and AGEs formation. Antioxidants are promising therapeutic agents for reducing these complications. Eremochloa ophiuroides (Munro) Hack. (centipedegrass), known for its diverse bioactivities, remains understudied for its anti-glycative properties.

Objective

This study aimed to isolate and characterize bioactive compounds from the aerial part of centipedegrass and evaluate their inhibitory effects on AGEs formation and oxidative stress to identify potential therapeutic candidates for diabetic complications.

Methods

The aerial parts of centipedegrass were extracted with 70% ethanol, followed by bioactivity-guided fractionation using Diaion HP-20 column chromatography. The most active fraction (CGE03) was analyzed for total flavonoid content and further purified using Toyopearl HW-40, Sephadex LH-20, and YMC gel ODS columns. The structures of the isolated compounds were elucidated using NMR and UV spectroscopy in combination with mass spectrometry. Anti-glycation activity was assessed via fluorescence-based AGEs inhibition assays, and hydroxyl radical scavenging activity was evaluated using a deoxyribose degradation assay.

Results

Bioactivity-guided fractionation of centipedegrass extract led to the isolation of nine C-glycosylated flavones (1‒9). Structural elucidation using 1D and 2D NMR spectroscopy revealed a rotational isomer of maysin (1), allowing for a more refined understanding of its structure. Among the isolated compounds, maysin exhibited the most potent anti-glycation (IC₅₀ = 5.0 ± 0.1 μM) and hydroxyl radical scavenging activities (IC₅₀ = 1.5 ± 0.2 μM), significantly outperforming the positive controls. The glycosylation pattern, particularly the rare ketosugar moiety at the C-6 position, was found to play a crucial role in bioactivity.

Conclusion

This study presents a detailed structural analysis of the rotational isomer of maysin, revealing its potent therapeutic potential for managing oxidative stress-related diabetic complications and offering novel insights into C-glycosylated flavonoids from Eremochloa ophiuroides (Munro) Hack.

Keywords

maysin advanced glycation end products natural antioxidants

Introduction

Diabetes mellitus is a metabolic disorder marked by chronic hyperglycemia. It is one of the most prevalent diseases worldwide, contributing to rising mortality rates and complications such as coronary heart disease, nephropathy, and neurodegeneration.¹ Diabetes-associated metabolic syndrome is commonly characterized by hyperglycemia, elevated triacylglycerol (TG) levels, and HDL dyslipidemia. Among these factors, hyperglycemia plays an essential role in the pathogenesis of diabetic complications by triggering multiple pathways, including the increased formation of advanced glycation end products (AGEs), overexpression of AGE receptor, activation of protein kinase C isoform, excessive oxidative stress, and increased flux through the aldose reductase (AR)-related polyol pathway.^2,3 Several recent reports have also shown that synthetic and natural antioxidants may help mitigate diabetic complications in humans.⁴ Therefore, inhibiting AGE formation and oxidative stress is considered a promising therapeutic approach for managing diabetic-related complications.

Eremochloa ophiuroides (Munro) Hack. (centipedegrass) a widely cultivated warm-season lawn belonging to the Poaceae family, is native to China, Southeast Asia, and South America. It is known for its thick sod formation, low-growing stoloniferous dispersion, and a medium to light green color.⁵ Previous biological and phytochemical investigations have identified C-glycosidic flavones and phenolic constituents as its secondary metabolites, which have been reported to inhibit osteoclast differentiation.^6,7 In an ongoing study on the bioactive constituents of South Korea native E. ophiuroides, the isolation and characterization of C-glycosylated flavones and their pancreatic lipase inhibitory activity were reported.⁸ Further screening of naturally occurring bioactive compounds in E. ophiuroides led to the identification of an ethanolic extract from the aerial part, which significantly inhibited AGE formation (IC₅₀ values of 42.1 ± 1.0 μg/mL). Based on this promising activity, bioactivity-guided fractionation was conducted to isolate and characterize its active constituents. The isolation and structural characterization of nine C-glycosidic flavones are presented herein, and their inhibitory effects against oxidative stress-related diabetic complications were evaluated using in vitro AGEs formation and hydroxyl radical scavenging assays.

Materials and Methods

General Experimental Procedures

Nuclear magnetic resonance (NMR) spectra for ¹H and ¹³C were measured on an Avance NEO-600 instrument (Bruker, Karlsruhe, Germany) operated at 600 and 150 MHz, respectively. Chemical shifts are reported in δ (ppm) using tetramethylsilane (TMS) as an internal standard. For calibration, DMSO-d₆ (δ_H 2.50; δ_C 39.5) and CD₃OD (δ_H 3.35; δ_C 49.8) were used as reference solvents. The electrospray ionization (ESI) mass spectra were recorded using a Vanquish UPLS System (Thermo Fisher Scientific, MA, USA). The ultraviolet (UV) spectrum was measured on a T-60 spectrophotometer (PG Instrument, Leicestershire, UK), the circular dichroism (CD) spectrum and optical rotation were recorded on a JASCO J-1500 and P-2000 spectrometer (JASCO, Tokyo, Japan). Column chromatography was performed using Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan), Toyopearl HW-40 (coarse grade; Tosoh Co., Tokyo, Japan), Sephadex LH-20 (particle size 25-100 μm; GE Healthcare Biosciences AB, Uppsala, Sweden), and YMC gel ODS AQ 120-50S (particle size 50 μm; YMC Co., Kyoto, Japan) gel columns. A microplate reader (Infinite F200, Tecan Austria GmBH, Grodig, Austria) was used for absorbance measurements. Semi-preparative high-performance liquid chromatography (HPLC) was performed using an Agilent HPLC 1200 system (Agilent Technologies, Palo Alto, CA, USA) equipped with a photodiode array detector (PDA, 1200 Infinity series, Agilent Technologies). YMC-Pack ODS A-302 column (4.6 mm i.d. × 150 mm, particle size 5 μm; YMC Co., Kyoto, Japan) was used for compound purification. All other reagents and chemicals purchased and used in this study were of analytical grade.

Plant Material

Dried aerial parts of Eremochloa ophiuroides (Munro) Hack. were collected at the Advanced Radiation Technology Institute (ARTI), Jeongeup, Korea, in May 2024. The sampling site was located at 29 Geumgu-gil, Jeongeup-si, Jeollabuk-do, Republic of Korea (Latitude: 35.6056° N, Longitude: 126.8706° E, Altitude: approximately 33 m). The collection complied with institutional and national guidelines for the ethical use of plant materials. A voucher specimen (Lot no. 202410A) representing this collection was identified by Dr. Seung Sik Lee and deposited at the Natural Product Chemistry Laboratory of Daegu University.

Extraction and Isolation

The plant material (4 kg) was extracted three times with 70% EtOH (80 L per extraction) at room temperature, each for 24 h, over a total period of 3 d. The extracts were filtered, and the solvent was evaporated under reduced pressure. The combined crude EtOH extract (500 g) was suspended in a mixture of MeOH–H₂O (9:1) solution (1 L), and subjected directly to a Diaion HP-20 column (10 cm i.d. × 45 cm) with H₂O containing increasing amounts of MeOH in stepwise gradient mode, and then fractionated into six subfractions CGE01–CGE06, respectively. Among these subfractions, CGE03 exhibited the most potent inhibitory activity against AGEs formation, with an IC₅₀ value of 9.2 ± 0.5 μg/mL. Moreover, CGE03 exhibited the highest flavonoid content (310 ± 2.7 mg LE/g), outperforming other extracts or subfractions. Bioactive fraction CGE03 (2.1 g) was further fractionated using a Toyopearl HW-40 (2.5 cm i.d. × 45 cm) column, yielding five subfractions (CGE0301‒CGE0305). Subfraction CGE0304 (305.1 mg) was subjected to column chromatography over a YMC gel ODS AQ 120-50S (1.5 cm i.d. × 38 cm) column with aqueous MeOH to yield pure maysin (1) (135.9 mg, t_R 31.6 min), ax-4″-hydroxymaysin (2) (9.0 mg, t_R 32.7 min), and derhamnosylmaysin (4) (7.6 mg, t_R 33.8 min). Isoorienetin (8) (89.4 mg, t_R 23.3 min), orientin (9) (38.0 mg, t_R 23.7 min), and isoorientin 2″-O-α-L-rhamnoside (3) (12.0 mg, t_R 28.7 min) were then isolated from CGE0302 (506.0 mg) using YMC gel ODS AQ 120-50S (1.0 cm i.d. × 45 cm) column with aqueous MeOH. In a similar fractionation, CGE0303 (350.2 mg) was chromatographed on a Sephadex LH-20 (1.0 cm i.d. × 35 cm) column with EtOH to yield luteolin 6-C-α-L-rhamnoside (7) (4.6 mg, t_R 26.7 min), luteolin 6-C-α-L-arabinoside (6) (10.5 mg, t_R 28.5 min), and luteolin 6-C-β-D-boivinopyranoside (5) (4.2 mg, t_R 37.4 min).

Maysin (1): yellow amorphous powder, [α]²⁰_D −68.1 (c 1.0, MeOH); UV λ_max MeOH nm (log ε): 212 (3.09), 269 (1.61), 352 (1.65) nm; ¹H and ¹³C NMR, see Table 1; ESIMS m/z 577 [M + H]⁺, HRESIMS m/z 577.1550 [M + H]⁺ (calculated for C₂₇H₂₉O₁₄, 577.1551) (Figure S1–S9).

Table 1.

¹H and ¹³C NMR Data of Compound 1.^a

Position	1 (Ma, 65%)^b		1 (Mi, 35%)^b
Position	δ_H (J in Hz)^c	δ_C, type^d	δ_H (J in Hz)^c	δ_C, type^d
2	—	163.6, C	—	163.7, C
3	6.70 (s)	102.7, CH	6.68 (s)	102.9, CH
4	—	182.1, C	—	181.7, C
5	—	161.2, C	—	160.0, C
6	—	107.5, C	—	107.3, C
7	—	162.3, C	—	163.5, C
8	6.49 (s)	93.0, CH	6.48 (s)	93.8, CH
9	—	156.5, C	—	156.7, C
10	—	103.6, C	—	103.0, C
1′	—	121.2, C	—	121.3, C
2′	7.41 (d, 2.4)	113.3, CH	7.41 (d, 2.4)	113.3, CH
3′	—	145.8, C	—	145.8, C
4′	—	149.8, C	—	149.8, C
5′	6.89 (d, 8.4)	116.1, C	6.89 (d, 8.4)	116.1, CH
6′	7.43 (dd, 8.4, 2.4)	119.0, C	7.43 (dd, 8.4, 2.4)	119.0, CH
1″	4.85 (d, 10.2)	73.3, CH	4.83 (d, 10.2)	72.8, CH
2″	5.30 (br d, 10.2)	75.4, CH	5.24 (br d, 10.2)	75.9, CH
3″	3.92 (d, 9.6)	78.0, CH	3.91 (d, 9.6)	78.0, CH
4″	—	206.1, C	—	206.1, C
5″	3.40 (overlap)	78.1, CH	3.40 (overlap)	78.1, CH
6″	1.30 (d, 6.0)	19.0, CH₃	1.30 (d, 6.0)	19.0, CH₃
1‴	4.64 (d, 1.8)	99.1, CH	4.63 (d, 1.8)	99.2, CH
2‴	3.69 (dd, 4.2, 1.8)	70.1, CH	3.69 (dd, 4.2, 1.8)	70.1, CH
3‴	3.04 (dd, 9.6, 4.2)	70.2, CH	3.05 (dd, 9.6, 4.2)	70.2, CH
4‴	2.97 (t, 9.6)	71.2, CH	2.97 (t, 9.6)	71.1, CH
5‴	2.35 (dq, 9.6, 6.0)	68.9, CH	2.41 (dq, 9.6, 6.0)	68.8, CH
6‴	0.67 (d, 6.0)	17.3, CH₃	0.82 (d, 6.0)	17.6, CH₃
5-OH	13.8 (s)	—	13.6 (s)	—

Measured in DMSO-d₆, and assignments of chemical shifts were based on the analysis of 1D and 2D NMR spectra. Overlapping signals were assigned from the HSQC, HMBC, and ¹H-¹H COSY spectra without designating the multiplicity.

Ma and Mi denote the major and minor rotamer of each flavone, respectively.

Data (δ) measured at 600 MHz.

Data (δ) measured at 150 MHz.

ax-4″-Hydroxymaysin (2): yellow amorphous powder, ¹H NMR (600 MHz, CD₃OD) δ 7.38 (1H, d, J = 1.8 Hz, H-2′), 7.37 (1H, dd, J = 1.8, 7.8 Hz, H-6′), 6.89 (1H, d, J = 7.8 Hz, H-5′), 6.54 (1H, s, H-3), 6.51 (1H, s, H-8), 5.17 (1H, s-like, H-1‴), 4.91 (1H, d, J = 9.6 Hz, H-1″), 4.26 (1H, t, J = 9.6 Hz, H-2″), 3.84 (1H, dd, J = 9.6, 1.8 Hz, H-3″), 3.77 (1H, m, H-5″), 3.73 (1H, dd, J = 2.4 Hz, H-4″), 3.68 (1H, s-like, H-2‴), 3.45 (1H, dd, J = 2.4, 9.6 Hz, H-3‴), 3.09 (1H, t, J = 9.3 Hz, H-4‴), 2.55 (1H, q, J = 6.6, 9.6 Hz, H-5‴), 1.28 (3H, d, J = 6.6 Hz, H-6″), 0.70 (3H, d, J = 6.6 Hz, H-6‴) (Figure S10–S14); ¹³C NMR (150 MHz, CD₃OD) δ 184.9 (C-4), 167.1 (C-2), 165.4 (C-7), 161.4 (C-5), 159.6 (C-9), 151.8 (C-4′), 147.8 (C-3′), 124.4 (C-1′), 121.1 (C-2′), 117.6 (C-5′), 114.9 (C-6′), 110.7 (C-6), 106.2 (C-10), 104.8 (C-3), 103.1 (C-1‴), 96.9 (C-8), 78.4 (C-4″), 77.0 (C-5″), 76.5 (C-2″), 74.7 (C-1″), 74.4 (C-2‴), 74.2 (C-4‴), 73.0 (C-3″), 72.9 (C-3‴), 70.6 (C-5‴), 18.8 (C-6″), 18.0 (C-‴).

Isoorientin 2″-O-α-L-rhamnoside (3): yellow amorphous powder, ¹H NMR (600 MHz, CD₃OD) δ 7.37 (1H, br d, J = 8.4 Hz, H-6′), 7.36 (1H, br s, H-2′), 6.89 (1H, d, J = 8.4, Hz, H-5′), 6.54 (1H, s, H-3), 6.49 (1H, s, H-8), 5.18 (1H, d, J = 1.2 Hz, H-1‴), 4.79 (1H, d, J = 10.2 Hz, H-1″), 4.24 (1H, m, H-5‴), 4.00 (1H, m, H-2‴), 3.98 (1H, m, H-3‴), 3.95 (1H, m, H-6″), 3.85 (1H, m, H-2″), 3.82 (1H, m, H-6″), 3.72 (1H, m, H-3″), 3.70 (1H, m, H-4″), 3.60 (1H, m, H-4‴), 3.41 (1H, m, H-5″), 1.12 (3H, d, J = 6.6 Hz, H-6‴) (Figure S15); ¹³C NMR (150 MHz, CD₃OD) δ 186.9 (C-4), 167.2 (C-2), 165.6 (C-7), 162.2 (C-5), 159.6 (C-9), 151.9 (C-4′), 147.9 (C-3′), 124.5 (C-1′), 121.2 (C-6′), 117.7 (C-5′), 115.0 (C-2′), 110.2 (C-6), 106.0 (C-10), 104.8 (C-3), 103.3 (C-1‴), 96.5 (C-8), 77.1 (C-5″), 76.7 (C-3″), 73.2 (C-1″), 72.9 (C-3‴), 72.8 (C-2″), 72.6 (C-4‴), 71.7 (C-2‴), 71.6 (C-5‴), 70.7 (C-6″), 18.8 (C-6‴).

Derhamnosylmaysin (4): yellow amorphous powder, ¹H NMR (600 MHz, CD₃OD) δ 7.37 (1H, d, J = 1.8 Hz, H-6′), 7.36 (1H, dd, J = 7.8, 1.8 Hz, H-2′), 6.89 (1H, d, J = 7.8, Hz, H-5′), 6.55 (1H, s, H-3), 6.52 (1H, s, H-8), 4.80 (1H, d, J = 9.0 Hz, H-1″), 4.04 (1H, m, H-2″), 3.96 (1H, m, H-3″), 3.16 (1H, m, H-5″), 1.43 (3H, d, J = 6.0 Hz, H-6″); ¹³C NMR (150 MHz, CD₃OD) δ 207.6 (C-4″), 184.2 (C-4), 166.3 (C-2), 164.3 (C-7), 162.6 (C-5), 158.9 (C-9), 151.2 (C-4′), 147.2 (C-3′), 123.7 (C-1′), 120.5 (C-2′), 116.9 (C-5′), 114.3 (C-6′), 109.1 (C-6), 105.7 (C-10), 104.1 (C-3), 95.6 (C-8), 82.5 (C-3″), 78.2 (C-5″), 73.5 (C-1″), 72.7 (C-2″), 17.4 (C-6″) (Figure S16 and S17).

Luteolin 6-C-β-D-boivinopyranoside (5): yellow amorphous powder, ¹H NMR (600 MHz, CD₃OD) δ 7.44 (1H, d, J = 1.8 Hz, H-2′), 7.42 (1H, dd, J = 7.8, 1.8 Hz, H-6′), 6.94 (1H, d, J = 7.8 Hz, H-5′), 6.60 (1H, s, H-3), 6.52 (1H, s, H-8), 5.53 (1H, dd, J = 12.6, 2.4 Hz, H-1″), 4.18 (1H, q, J = 6.6 Hz, H-5″), 4.04 (1H, d, J = 3.0 Hz, H-3″), 3.43 (1H, d, J = 3.0 Hz, H-4″), 2.31 (1H, m, H-2″), 1.77 (1H, dd, J = 14.4, 3.0 Hz, H-2″), 1.33 (3H, d, J = 6.6 Hz, H-6″); ¹³C NMR (150 MHz, CD₃OD) δ 184.0 (C-4), 166.5 (C-2), 164.6 (C-7), 158.7 (C-5), 158.3 (C-9), 151.1 (C-4′), 147.1 (C-3′), 123.6 (C-1′), 120.4 (C-2′), 116.8 (C-5′), 114.2 (C-6′), 111.6 (C-6), 104.9 (C-10), 103.8 (C-3), 96.0 (C-8), 72.7 (C-5″), 70.6 (C-4″), 70.0 (C-1″), 68.8 (C-3″), 17.4 (C-6″) (Figure S18 and S19).

Luteolin 6-C-α-L-arabinoside (6): yellow amorphous powder, ¹H NMR (600 MHz, CD₃OD) δ 7.39 (1H, d, J = 2.4 Hz, H-2′), 7.37 (1H, dd, J = 8.4, 2.4 Hz, H-6′), 6.90 (1H, d, J = 8.4 Hz, H-5′), 6.56 (1H, s, H-3), 6.50 (1H, s, H-8), 4.79 (1H, d, J = 9.6 Hz, H-1″), 4.24 (1H, t, J = 9.6 Hz, H-2″), 4.00 (1H, m, H-5″), 3.95 (1H, br s, H-4″), 3.73 (1H, m, H-5″), 3.60 (1H, dd, J = 9.0, 3.0 Hz, H-3″); ¹³C NMR (150 MHz, CD₃OD) δ 184.0 (C-4), 166.4 (C-2), 161.4 (C-5), 159.1 (C-9), 158.8 (C-7), 151.1 (C-4′), 147.1 (C-3′), 123.6 (C-1′), 120.3 (C-2′), 116.8 (C-5′), 114.1 (C-6′), 109.3 (C-6), 105.1 (C-10), 103.9 (C-3), 95.6 (C-8), 76.3 (C-1″), 75.9 (C-3″), 71.9 (C-5″), 70.8 (C-4″), 70.7 (C-2″) (Figure S20 and S21).

Luteolin 6-C-α-L-rhamnoside (7): yellow amorphous powder, ¹H NMR (600 MHz, CD₃OD) δ 7.36 (1H, d, J = 2.4 Hz, H-2′), 7.35 (1H, dd, J = 7.8, 2.4 Hz, H-6′), 6.89 (1H, d, J = 7.8 Hz, H-5′), 6.54 (1H, s, H-3), 6.51 (1H, s, H-8), 5.18 (1H, brs, H-1″), 3.85 (1H, m, H-2″), 3.43 (1H, dd, J = 10.2, 3.0 Hz, H-3″), 3.10 (1H, m, H-4″), 2.57 (1H, m, H-5″), 0.71 (3H, d, J = 6.0 Hz, H-6″) (Figure S22); ¹³C NMR (150 MHz, CD₃OD) δ 184.9 (C-4), 167.1 (C-2), 165.4 (C-7), 165.3 (C-5), 159.6 (C-9), 151.8 (C-4′), 147.8 (C-3′), 124.4 (C-1′), 121.1 (C-6′), 117.5 (C-5′), 115.0 (C-2′), 106.3 (C-6), 104.9 (C-10), 104.8 (C-3), 103.2 (C-8), 75.1 (C-1″), 74.3 (C-4″), 73.1 (C-2″), 72.9 (C-3″), 70.7 (C-5″), 18.8 (C-6″).

Isoorienetin (8): Yellow amorphous powder, ¹H NMR (CD₃OD, 600 MHz): δ 7.31 (1H, d, J = 2.4 Hz, H-2′), 7.30 (1H, dd, J = 8.4, 2.4 Hz, H-6′), 6.86 (1H, d, J = 8.4 Hz, H-5′), 6.47 (1H, s, H-8), 6.42 (1H, brs, H-3), 4.89 (1H, d, J = 10.2 Hz, H-1″), 3.30∼3.80 (6H, m, H-2″∼6″) (Figure S23).

Orientin (9): Yellow amorphous powder, ¹H NMR (CD₃OD, 600 MHz): δ 7.42 (1H, d, J = 7.8 Hz, H-6′), 7.41 (1H, s, H-2′), 6.94 (1H, d, J = 7.8 Hz, H-5′), 6.59 (1H, s, H-6), 6.54 (1H, s, H-3), 4.83 (1H, d, J = 9.6, H-1″), 4.16 (1H, br t, J = 10.2 Hz, H-2″), 4.05 (1H, dd, J = 12.0, 1.8, H-6″), 3.75 (1H, dd, J = 12.0, 5.4 Hz, H-6″), 3.66 (1H, m, H-3″), 3.64 (1H, m, H-5″), 3.36 (1H, m, H-4″) (Figure S24).

Total Flavonoid Content

The total flavonoid content was determined using the aluminum chloride colorimetric method, as previously reported.⁹ Luteolin (0.05 g) was dissolved in 50 mL of 95% MeOH (v/v) and diluted to obtain calibration standards (31.25 to 500 μg/mL). Then, 0.5 mL of the standards, blank, or centipedegrass extract and subfractions (1 mg/mL) were mixed with 0.1 mL of aluminum chloride (10%), 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water in a test tube and incubated for 30 min at room temperature. The absorbance of the reaction mixture was measured at 432 nm. Finally, the total flavonoid content was calculated from the luteolin calibration equation (y = 0.0002x + 0.0377, R² = 0.9998) and expressed as milligrams of luteolin equivalent per gram of dry extract (mg LE/g).

AGEs Formation Inhibitory Activity Assay

The ability of the compounds to inhibit AGE formation was evaluated using previously reported methods with a minor modification.¹⁰ Briefly, an AGE reaction solution was prepared by the addition of 10 mg/mL bovine serum albumin in 50 mM sodium phosphate buffer (pH 7.4), along with 0.02% sodium azide to prevent bacterial growth, was added to 0.2 M fructose and 0.2 M glucose. The reaction mixture (950 μL) was then combined with various concentrations of the samples (50 μL, final concentration: 200 μg/mL for the extracts, fractions, and compounds) dissolved in 10% DMSO. Following incubation at 37 °C for 7 days, the fluorescence intensity of the reaction products was determined using a spectrofluorometric detector (Infinite F200; Tecan Austria GmBH, Grödig, Austria), with excitation and emission wavelengths at 350 and 450 nm, respectively. The nucleophilic hydrazine compound aminoguanidine was used as a positive control substance in the AGE assay.

Hydroxyl Radical Scavenging Activity Assay

The hydroxyl radical scavenging activity of the C-flavones was determined using the original deoxyribose degradation assay.¹¹ In brief, the isolated flavones were first dissolved in EtOH to prepare the sample solution (at 10-300 μM). An aliquot of the sample solution (400 μL) was added to phosphate buffer (10 mM, pH 7.4). Then, 50 μL of deoxyribose (50 mM), 50 μL of ethylenediaminetetraacetic acid (1 mM), 50 μL of FeCl₃ (3.2 mM) and 50 μL of H₂O₂ (50 mM) were added. The reaction was initiated by mixing 50 μL of _L-ascorbic acid (1.8 mM) and the total volume of the reaction mixture was adjusted to 400 μL with buffer. After incubation at 37 °C for 4 h, the reaction was terminated by 250 μL of trichloroacetic acid (2.8%, w/w). The color was then developed by addition of 150 μL of thiobarbituric acid (0.5%, w/w) and heating in a water-bath at 105 °C for 15 min. The mixture was cooled and absorbance was measured at 530 nm (ELISA reader) against the buffer (as blank). The IC₅₀ value (50% inhibition) was evaluated using linear regression analysis of scavenging activities under the assay conditions. The natural radical scavenger (+)-catechin was used as the positive control substance in this assay.

Acid Hydrolysis of Maysin (1)

Compound 1 (5.0 mg) was dissolved in 1 mL of 1 N HCl and heated at 90 °C for 1 h, following the method described. After cooling, the reaction mixture was extracted three times with EtOAc (1.0 mL × 3). The combined EtOAc layers were concentrated under reduced pressure to yield a hydrolysis product (1.8 mg). The product was subsequently analyzed by LC–MS, which confirmed its identity as derhamnosylmaysin (4) (Figure S25).

Statistical Analysis

All data were evaluated by one-way ANOVA, followed by Duncan's multiple range test. Results were considered statistically significant when P < .05. Each experiment was conducted at least three times independently.

Results

Identification of Maysin (1) and Characterization of C-flavones

Eremochloa ophiuroides is recognized for its diverse biological activities, such as anti-inflammatory,¹² anticancer,^13,14 anti-adipogenesis,¹⁵ and anti-osteoporotic effects.⁶ These activities are primarily attributed to its phenolic and flavonoid constituents.⁸ Despite the known pharmacological benefits, its potential role in diabetic complications remains unexplored. Studies characterizing its major components or evaluating the bioactivity of individual compounds are still limited. To identify bioactive compounds derived from natural resources, bioactivity-guided fractionation was performed on an ethanolic extract using Diaion HP-20 column chromatography. The 40% MeOH in H₂O eluate (fraction CGE03) exhibited potent AGEs inhibition activity with an IC₅₀ value of 9.2 ± 0.5 μg/mL (Table 2). Additionally, total flavonoid content analysis revealed that CGE03 contained a significantly higher flavonoid content (310.0 ± 2.7 mg LE/g) compared to the 70% EtOH extract (Table 2). Reverse-phase HPLC analysis revealed a significant increase in the area values of major components in fraction (CGE03), compared to the 70% EtOH extract (Figure 1). Following this, a bioactivity-guided isolation of 40% MeOH (CGE03) eluates of aqueous EtOH extract of E. ophiuroides was performed to isolate and characterize nine C-glycosidic flavones (Figure 2).

Figure 1.

HPLC Chromatograms of 70% EtOH Extract and 40% MeOH Fraction (CGE03) of Eremochloa ophiuroides. Analysis Concentration: 2 mg/mL, Injection Volume: 5 µL. 1: Maysin, 2: ax-4″-Hydroxymaysin, 3: Isoorientin 2″-O-α-_L-rhamnoside, 4: Derhamnosylmaysin, 5: Luteolin 6-C-β-_D-boivinopyranoside, 6: Luteolin 6-C-α-_L-arabinoside, 7: Luteolin 6-C-α-_L-rhamnoside, 8: Isoorienetin, 9: Orientin.

Figure 2.

Structures of Isolated C-glycosidic Flavones 1‒9 from Eremochloa Ophiuroides.

Table 2.

Total Flavonoid Content and Inhibition of AGEs Formation of the Extract and Subfractions of Eremochloa Ophiuroides.

Extract and fractions	IC₅₀ values (μg/mL) of AGEs formation^a	Total flavonoid content (mg LE/g)^b
70% EtOH extract	42.1 ± 1.0	57.6 ± 1.5
CGE01 (H₂O elution)	>500	3.8 ± 0.3
CGE02 (20% MeOH elution)	82.9 ± 1.9	42.3 ± 0.9
CGE03 (40% MeOH elution)	9.2 ± 0.5	310.0 ± 2.7
CGE04 (60% MeOH elution)	32.9 ± 1.1	127.1 ± 1.9
CGE05 (MeOH elution)	>500	2.4 ± 0.2
CGE06 (70% Acetone elution)	>500	6.7 ± 0.3

The values (μM) are defined as the concentration that results in a 50% decrease of the bioassay data are mean ± Standard deviation (SD) from triplicate experiments. Different letters (w–z) within the same column indicate significant differences (p < .05).

LE: luteolin equivalents.

Compound 1 was obtained as a yellow amorphous powder with a molecular formula C₂₇H₂₈O₁₄ confirmed by positive HRESIMS at m/z 577.1550 [M + H]⁺ (calculated for C₂₇H₂₉O₁₄, 577.1551). UV absorption spectra of 1 exhibited characteristic absorption of flavones at maxima 212 (3.09), 269 (1.61), and 352 (1.65) nm.¹⁵ The ¹H NMR spectrum (Table 1) of 1 displayed resonances characteristic for ABX-type aromatic ring system at δ_H 7.43 (dd, J = 8.4, 2.4 Hz, H-6′), 7.41 (d, J = 2.4 Hz, H-2′), and 6.89 (d, J = 8.4 Hz, H-5′). Additionally, a pair of isolated aromatic signals was observed for H-3 at δ_H 6.70 and 6.68, alongside a pair of singlet proton signals for H-8 at δ_H 6.49 and 6.48, as well as a pair of aromatic hydroxyl protons for 5-OH at δ_H 13.8 and 13.6. This indicates the presence of a luteolin moiety in 1.¹⁶ The presence of anomeric protons at δ_H 4.85 (or 4.83, d, J = 10.2 Hz, H-1²¢) and 4.64 (or 4.63, d, J = 1.8 Hz, H-1″), suggested the presence of the disaccharide moiety in 1. The chemical shifts of the anomeric carbon signals at δ_C 99.2 (or 99.1, C-1²¢), and 73.3 (or 72.8, C-1″) are in accordance with O- and C-glucoside, respectively. The small coupling constant (J = 1.8 Hz) of H-1²¢ and doublet methyl signals at δ_H 0.82 (or 0.67, d, J = 6.0 Hz, H-6²¢) in the ¹H NMR suggested the presence of the α-rhamnopyranosyl residue.¹⁷ The other sugar residue was identified as β-fucopyranosyl, based on the large coupling constant (J = 10.2 Hz) of H-1″ and the doublet methyl signal at δ_H 1.30 (d, J = 6.0 Hz, H-6²¢), which indicates the presence of β-fucopyranose.¹⁸ Consistent with these ¹H NMR observations, the ¹³C and HSQC NMR spectra also revealed an aliphatic ketone signal at δ_C 206.1 (C-4″), which, through HMBC correlations with H-6″, confirmed the presence of 4″-ketofucopyranoside (Figure 2). The linkage position of the luteolin residue on the sugar moieties in compound 1 was unambiguously determined from the key HMBC spectrum, which showed correlations of H-1²¢/C-2″, H-1″/C-5, C-6, and C-7, as well as 5-OH/C-5 (Figure 3). To further verify the structure, compound 1 was subjected to acid hydrolysis using 1 N HCl, followed by LC–MS analysis. The resulting product exhibited molecular ion peaks at m/z 431 [M + H]⁺ and 429 [M – H]^–, consistent with derhamnosylmaysin (4) (Figure S25). These correlations confirmed that compound 1 is identical to the previously reported maysin.¹⁹

Figure 3.

Key HMBC and COSY Correlations of 1.

Interestingly, NMR spectra of compound 1 showed signal duplication, indicative of rotameric isomerism, likely due to rotational hindrance at the C-C glycosyl-flavone linkage in flavone C-glycoside.²⁰ Based on the integration of proton signals in the ¹H NMR data, the relative proportions of the major and minor rotamers of 1 were calculated as 65% and 35%, respectively. To verify the persistence of rotamerism, the ¹H NMR spectrum of compound 1 was additionally measured in CD₃OD at 37 °C, as suggested in prior studies (Figure S9).²¹ Despite the elevated temperature, the duplicated proton signals remained clearly distinguishable, indicating that restricted rotation persisted and that the interconversion rate between rotamers was still slow on the NMR time scale.²² While previous reports suggest that signal coalescence may occur with increasing temperature for some C-glycosylated flavonoids, our results demonstrate that this behavior is not universal. While compound 1 has previously been identified as a constituent of Zea mays L. silk,²³ its detailed structure had not been characterized using 1D and 2D NMR spectral techniques. This study provides a comprehensive structural elucidation of the rotational isomer of maysin (1), using advanced NMR techniques, revealing previously unreported signal duplication attributed to rotamerism, and advancing our understanding of C-glycosidic flavonoid dynamics, which may influence biological activity and stability.

Based on the spectroscopic analysis and comparisons with previously reported data, compounds 2‒9 were identified as known C-glycosyl flavones: ax-4″-hydroxymaysin (2),²⁴ isoorientin 2″-O-α-L-rhamnoside (3),²⁵ derhamnosylmaysin (4),²¹ luteolin 6-C-β-D-boivinopyranoside (5),⁸ luteolin 6-C-α-L-arabinoside (6),²⁶ luteolin 6-C-α-L-rhamnoside (7),²⁷ isoorienetin (8),²⁸ and orientin (9) (Figure 2).²⁸

Suppression of AGEs Formation and Hydroxyl Radical Scavenging Activities of C-glycosidic Flavones

All pure isolates in the present investigation were evaluated for their anti-glycation properties against AGEs formation using aminoguanidine as a positive control (Table 3). The C-glycosyl flavone 1, which was isolated in the highest quantity and contained an unusual ketosugar moiety at the C-6 position, exhibited potent inhibitory activity in this bioassay system with an IC₅₀ value of 5.0 ± 0.1 μM compared to the positive control aminoguanidine (IC₅₀ value: 513.4 ± 6.9 μM), a well-known AGE inhibitor, highlighting its superior inhibitory effect. Compounds 2 and 3, both of which contain disaccharides at the C-6 position in the luteolin skeleton, also demonstrated potent anti-glycation activity, with IC₅₀ values of 10.5 ± 0.7 and 14.0 ± 0.9 μM, respectively. On the other hand, derhamnosylmaysin (4), derived from the hydrolysis of rhamnose in maysin (1), exhibited slightly reduced activity compared to the most potent compound 1 (Table 3). Among the 6-C-monoglycoside flavones (4-8), compounds with ketofucosyl (4), glucosyl (8), boivinosyl (5), rhamnosyl (7), and arabinosyl (6) moieties exhibited anti-glycation activity in descending order. Based on the structure-activity relationship analysis in the evaluated active compounds in the AGEs formation assay, it can be suggested that glycosylation at the C-6 position significantly influences activity.

Table 3.

Effects on the Inhibition of AGEs Formation and Hydroxyl Radical Scavenging Activities of the Isolated Compounds 1–9.

Compounds	IC₅₀ values (μM)^a
Compounds	AGEs formation assay	Hydroxyl radical assay
1	5.0 ± 0.1	1.5 ± 0.2
2	10.5 ± 0.7	5.5 ± 0.5
3	14.0 ± 0.9	2.9 ± 0.2
4	9.9 ± 0.8	19.3 ± 1.0
5	17.4 ± 1.1	81.5 ± 2.1
6	50.7 ± 1.7	134.7 ± 3.6
7	25.0 ± 1.0	88.0 ± 2.5
8	11.6 ± 0.4	18.6 ± 1.7
9	20.2 ± 0.8	27.3 ± 1.8
Aminoguanidine^b	513.4 ± 6.9	—
(+)-Catechin^b	—	69.0 ± 2.2

Used as a positive control.

In addition, the radical scavenging activity of the isolated compounds was evaluated based on the previously reported procedure using hydroxyl radical.¹¹ As summarized in Table 3, the C-glycosylated flavone 1, which contains a rare ketosugar with a rhamnose in the A-ring of luteolin, exhibited the highest hydroxyl radical scavenging activity, with an IC₅₀ value of 1.5 ± 0.2 μM, outperforming the positive control ((+)-catechin). In contrast, the structurally similar C-diglycosylated derivatives 2 and 3 exhibited slightly weaker radical scavenging activity in this antioxidant assay than the most potent 1. These results suggest that the type of sugar moiety at C-6 in the luteolin nucleus may influence antioxidant activity. Although various natural products, including flavonoids and polyphenolic constituents, have been reported as antioxidant and AGEs formation inhibitors, this is the first report demonstrating that C-glycosylated flavones 1–9 exhibit potent inhibitory effects against AGEs formation and hydroxyl radical scavenging activity.

Discussion

Eremochloa ophiuroides (Munro) Hack. (centipedegrass) extract has been reported to exhibit various bioactivities, including antioxidant and anti-obesity effects, due to its rich composition of polyphenolic compounds.^6–8 In particular, flavone derivatives are abundant in centipedegrass and are considered key contributors to its biological activities.^6–8 In this study, nine C-glycosylated flavones were successfully isolated from the aerial parts of Eremochloa ophiuroides (Munro) Hack. Among them, maysin (1) has been previously reported as a constituent of corn silk (Zea mays L)²³; however, its structural characterization using advanced spectroscopic techniques, such as 2D NMR and HRMS, had not been performed. This study offers a comprehensive structural characterization of maysin (1) using modern analytical techniques. Notably, unique NMR spectral features indicative of rotational isomerism were observed in C-glycosylated flavones, a phenomenon that had not been previously documented for maysin (1). Although maysin was previously isolated and characterized by UV, IR, and ¹H NMR techniques in early studies no duplicated NMR signals or discussion of rotational isomerism were reported.²¹ In contrast, our ¹H NMR spectra revealed clear signal duplication, particularly for H-3, H-8, 5-OH, and H-1″, which strongly suggests the presence of rotamers due to restricted rotation around the C6–C1″ bond. The duplicated NMR signals observed for compound 1 are attributed to restricted rotation around the C6–C1″ bond, leading to the formation of rotamers. This phenomenon has been reported for other C-glycosylated flavonoids, particularly when bulky or di-substituted sugar moieties are present.²² Although compounds 2–9 share similar 6 or 8-C-glycosylated flavone backbones, no spectroscopic evidence (e.g., signal duplication or broadening) indicating the presence of stereoisomers was observed under our experimental conditions.^8,24–29 Their structures were confirmed by comparison with both literature data and commercially available standards. Furthermore, although two rotameric forms of maysin (1) were clearly distinguishable by NMR, they were not resolved as separate peaks in the HPLC analysis, likely due to rapid interconversion and insufficient chromatographic resolution. This observation aligns with previous reports suggesting that rotamers of C-glycosylated flavonoids generally cannot be chromatographically separated due to their low rotational energy barriers.²² This finding not only provides novel insights into the structural dynamics of C-glycosidic flavones but also underscores the importance of detailed spectroscopic analysis in natural product research. Also, among the isolates, ax-4″-hydroxymaysin (2), luteolin 6-C-α-L-arabinoside (6), and luteolin 6-C-α-L-rhamnoside (7) were identified in the centipedegrass for the first time. These results not only clarify the structural behavior of maysin but also contribute to a better understanding of conformational dynamics in C-glycosylated flavonoids, which may have implications for their biological function and stability.

Diabetes and hyperglycemia lead to increased oxidative stress, which presumably has an important role in the onset of diabetic complications such as neuropathy, cataracts, and retinopathy.^29,30 Among the proposed mechanisms for the pathogenesis of diabetic complications, AGE formation is considered a major contributor to oxidative stress in ocular and neural tissues.³¹ Recently, a more effective therapeutic strategy for diabetic complications has focused on preventing AGE formation and AGE-mediated damage by scavenging free radicals, trapping reactive dicarbonyl species, chelating transition metal ions, blocking AGE receptors, and inhibition of aldose reductase.³² Thus, oxidative stress is closely linked to AGE formation, and antioxidants that inhibit AGE formation are valuable therapeutic agents for diabetic complications in humans. Naturally occurring flavonoids are ubiquitous phytochemicals in the plant kingdom, especially in vegetables and fruits. To date, more than 6000 flavonoids have been identified with their potential antioxidant properties varying significantly based on the linkage position and number of hydroxyl groups on flavonoid aglycones.³³ Previous studies have demonstrated that the antioxidant activity of flavonoids contributes to their protective effects against diabetic complication.³¹ In present investigation, C-glycosylated flavones 1–9 were isolated from this biomass. Their structures were established through spectroscopic data interpretation. Among the tested compounds, maysin (1) exhibited the most potent radical scavenging and anti-glycation effects, with the lowest IC₅₀ values. The results of this investigation indicate that compound 1 might be a potential therapeutic agent for diabetic complication and related diseases. Compared to aminoguanidine, a well-known synthetic AGE inhibitor (IC₅₀= 513.4 ± 6.9 μM), maysin (1) demonstrated significantly stronger anti-glycation activity (IC₅₀= 5.0 ± 0.1 μM) and also exhibited potent hydroxyl radical scavenging activity (IC₅₀= 1.5 ± 0.2 μM). These results suggest that maysin exerts multi-targeted effects, inhibiting both AGE formation and oxidative stress—two key mechanisms involved in diabetic complications. Moreover, as a naturally occurring flavonoid, maysin may offer a more favorable safety profile than synthetic agents.

Limitation of the Study

Although anti-glycative C-glucosylated flavones, including maysin (1), were successfully isolated from the aerial part of Eremochloa ophiuroides (Munro) Hack., their biological efficacy has only been demonstrated in vitro. The oral bioavailability, metabolic stability, and pharmacokinetic properties of maysin have yet to be fully elucidated, and in vivo studies using cellular and animal models are essential to confirm its therapeutic potential. Nevertheless, the potent dual anti-glycation and antioxidant activities observed in vitro support maysin (1) as a promising candidate for future development as a multi-functional therapeutic agent against oxidative stress-related diabetic complications.

Conclusion

This study demonstrated a pair of rotational isomers (rotamers) of maysin (1) from the aerial part of Eremochloa ophiuroides (Munro) Hack., highlighting its structural novelty and bioactive significance. Among the isolated compounds, maysin (1) demonstrated remarkable anti-glycation (IC₅₀ = 5.0 ± 0.1 μM) and hydroxyl radical scavenging activities (IC₅₀ = 1.5 ± 0.2 μM), significantly outperforming positive controls. Structural elucidation using 1D and 2D NMR techniques revealed duplicated signals due to restricted rotation, supporting the presence of a rotameric mixture and providing detailed structural information regarding this behavior in maysin. Furthermore, the rare ketosugar moiety at the C-6 position was found to play a key role in bioactivity. These findings position maysin (1) as a promising therapeutic candidate for oxidative stress-related diabetic complications and offer new insights into the structure–activity relationships of C-glycosylated flavonoids from E. ophiuroides.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251348565 - Supplemental material for Anti-glycative C-glycosidic Flavones from the Aerial Part of Eremochloa Ophiuroides (Munro) Hack

Supplemental material, sj-docx-1-npx-10.1177_1934578X251348565 for Anti-glycative C-glycosidic Flavones from the Aerial Part of Eremochloa Ophiuroides (Munro) Hack by Gyeong Han Jeong, Seong Hee Kang, Hanui Lee, Tae Hoon Kim, Hyoung-Woo Bai, Seung Sik Lee and Byung Yeoup Chung in Natural Product Communications

Footnotes

ORCID iD

Gyeong Han Jeong

Author Contributions

Gyeong Han Jeong: Conceptualization, Formal Analysis, Investigation, Methodology, Resource, Validation, Writing - Original Draft Preparation, Data Curation.

Seong Hee Kang: Methodology, Resource, Validation, Writing - Original Draft Preparation.

Hanui Lee: Conceptualization, Methodology, Writing - Original Draft Preparation.

Tae Hoon Kim: Formal Analysis, Investigation, Resource.

Hyoung-Woo Bai: Methodology, Resource, Validation.

Seung Sik Lee: Conceptualization, Visualization, Writing - Original Draft Preparation, Writing - Review & Editing.

Byung Yeoup Chung: Conceptualization, Methodology, Supervision, Writing - Original Draft Preparation, Writing - Review & Editing.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the KAERI Institutional Program (Project No. 523310-25) funded by the Nuclear R&D Program of the Ministry of Science and ICT, and by the Innopolis Foundation through the Technology Commercialization Project funded by the Ministry of Science and ICT (Grant No. RS-2024-00416870).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

Statement of Informed Consent

There are no human subjects in this article, and informed consent is not applicable.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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Anti-glycative C -glycosidic Flavones from the Aerial Part of Eremochloa Ophiuroides (Munro) Hack

Abstract

Background

Objective

Methods

Results

Conclusion

Keywords

Introduction

Materials and Methods

General Experimental Procedures

Plant Material

Extraction and Isolation

Total Flavonoid Content

AGEs Formation Inhibitory Activity Assay

Hydroxyl Radical Scavenging Activity Assay

Acid Hydrolysis of Maysin (1)

Statistical Analysis

Results

Identification of Maysin (1) and Characterization of C-flavones

Suppression of AGEs Formation and Hydroxyl Radical Scavenging Activities of C-glycosidic Flavones

Discussion

Limitation of the Study

Conclusion

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251348565 - Supplemental material for Anti-glycative C-glycosidic Flavones from the Aerial Part of Eremochloa Ophiuroides (Munro) Hack

Footnotes

ORCID iD

Author Contributions

Funding

Declaration of Conflicting Interests

Data Availability Statement

Statement of Informed Consent

Statement of Human and Animal Rights

Supplemental material

References

Supplementary Material