Isolation and Characterization of a New Glycosylated Depside and Other Compounds with Antioxidant Activity from the Flower Buds of Rosa rugosa

Abstract

Objectives

This study focused on the isolation, structural elucidation, and free radical scavenging activities of compounds from the flower buds of Rosa rugosa.

Methods

The 30% ethanolic extract of R. rugosa was partitioned with organic solvents (n-hexane, chloroform, ethyl acetate, n-butanol). The ethyl acetate soluble layer was subjected to medium-pressure column chromatography, Sephadex LH-20 column chromatography, and preparative-high pressure liquid chromatography to yield five compounds (1-5). Structural elucidation of these compounds was conducted through nuclear magnetic resonance spectroscopy and high–resolution electrospray ionization mass spectrometry. These compounds were evaluated for their anti-oxidant activity using 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals.

Results

Rosarugoside F (1), a new compound was isolated and characterized as a depside glucoside. The remaining four known compounds were identified as sinensin (2), 3,4-dihydroxybenzoic acid (3), vanillic acid (4), and 4-hydrobenzoic acid (5). Compounds 1 and 3 exhibited moderate ABTS and DPPH radical scavenging activities.

Conclusions

A new depside glucoside and four known compounds were isolated from the flower buds of R. rugosa. In particular, a new compound 1 showed moderate anti-oxidant activities against ABTS and DPPH radicals, with IC₅₀ values of 70.1 ± 5.0 μM and 187.5 ± 14.2 μM, respectively.

Introduction

Rosa rugosa, a member of the Rosaceae family, is native to the temperate regions of eastern Asia, including Korea, China, and northern Japan. ¹ This species is renowned for its fragrant flowers and is a significant source of natural products that are widely used in cosmetics, aromatherapy, floral scent, and nutrition.^2-4 Moreover, R. rugosa has applications in the culinary fields such as production of teas, wines, and jams.^5,6 Notably, the flower buds of R. rugosa have been extensively used in traditional medicine for their potential therapeutic effects, including the management of diabetes mellitus, alleviation of pain, treatment of chronic inflammatory conditions, promotion of vasodilation, and enhancement of microcirculation.^7,8 Previous studies have confirmed that the flower buds of R. rugosa produce various secondary metabolites such as anthocyanins, flavonoids, ellagitannins, phenolic acids, and terpenoids.^9-11 Among its phytochemicals, previous research has reported on new glycosylated depsides that exhibited antioxidant, anti-inflammatory, anti-aging, whitening, and moisturizing effects.^12,13 During our continuous search for novel bioactive secondary metabolites, a new glycosylated depside (1) and four known compounds, including one flavonoid and three benzoic acid derivatives (2‒5) were isolated from the flower buds of R. rugosa (Figure 1). Herein, the structures of these compounds were elucidated, and their scavenging activity against 2,2′-azinobis-(3-ethylben-zothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2- picrylhydrazyl (DPPH) radicals were analyzed.

Figure 1.

Structures of 1-5.

Materials and Methods

General

The Nuclear Magnetic Resonance (NMR) spectra were recorded in CD₃OD using a Varian-VNMRS 500 MHz FT-NMR spectrometer (Varians, Palo Alto, CA, USA). The optical rotation was measured using a JASCO P-2000 Digital Polarimeter (Jasco, Tokyo, Japan). The UV spectrum was measured on a Thermo Scientific Multiskan GO spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) and the IR spectrum were recorded on a JASCO FT-IR 4100 spectrometer (Jasco, Tokyo, Japan). Chromatograms were obtained by using a medium-pressure liquid chromatography (MPLC) system (Biotage, Sweden, Uppsala) equipped with C₁₈ column (40 g, Biotage, Uppsala, Sweden) and were measured with high performance liquid chromatography (HPLC) (Waters Acquity Arc, Waters, Milford, US) equipped with a C₁₈ column (4.6 mm × 250 mm and 10.0 mm × 250 mm, Cosmosil, Kyoto, Japan). HR-ESI-MS was performed with a SCIEX X500R Q-TOF LC-MS/MS spectrometer (SCIEX, Framingham, MA, USA).

Plant Material

The flower buds of R. rugosa were collected in May, 2023 from Janghang-eup, Seocheon-gun, Chungchengnam-do, Korea (36°01′08″ N 126°39′58″ E). The species identification was carried out by one of the authors (Ji-Yul Kim). A voucher specimen (RR-01-2023) was kept in National Biodiversity institute of Korea.

Extraction and Isolation

The flower buds of R. rugosa (400 g) were dried in dried oven at 40 °C and finely ground using a blender. Powdered the flower buds of R. rugosa were extracted at three times with 30% ethanol at 50 °C for 1day each. The extract was evaporated and partitioned using organic solvents (n-hexane, chloroform, ethyl acetate, and n-butanol). The ethyl acetate fraction (18.8 g) was subjected to reversed-phase MPLC using a gradient with an increasing amount of methanol in water (0 → 50% aq. methanol) to obtain nine fractions (Fr. 1–Fr. 9). Fr. 5 and Fr. 6 (4.0 g) were combined and subjected to Sephadex LH-20 column chromatography and eluted with methanol to yield five subfractions (Fr. 5-1–Fr. 5-5). Fr. 5-3 (47.4 mg) was further purified via preparative HPLC using an acetonitrile (ACN)-water solvent system with increasing amounts of ACN (5% aq. ACN → 50% aq. ACN) to obtain 1 (2.9 mg), 4 (2.4 mg), and 5 (4.2 mg). Fr. 5-4 (32.7 mg) was subjected to preparative HPLC using an ACN-water solvent system (5% aq. ACN → 45% aq. ACN) to yield 2 (2.5 mg) and 3 (2.9 mg).

Rosarugoside F (1): yellow oil; [α]_D²⁰ + 76.4 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 220 (4.5), 267 (4.2), and 293 (4.1) nm; HR-ESI-MS m/z 495.1133 [M−H]⁻ (calcd. for C₂₂H₂₃O₁₃, 495.1139); for the ¹H and ¹³C NMR (500 and 125 MHz) spectral data, see Table 1.

Table 1.

¹H (500 MHz) and ¹³C NMR (125 MHz) Spectral Data for 1 in CD₃OD.

No.	δ C	δH (J in Hz) a
1	111.4
2	158.6
3	102.4	6.64 (d, J = 2.3)
4	158.7
5	105.3	6.39 (d, J = 2.3)
6	152.2
7	31.4	3.53 (m)
8	177.2
1´	121.7
2´	114.1	7.69 (d, J = 1.9)
3´	149.0
3′-OCH3	56.6	3.94 (s)
4´	153.7
5´	116.2	6.90 (d, J = 8.3)
6´	126.0	7.71 (dd, J = 8.3, 1.9)
7´	166.2
1´´	103.6	4.86 (d, J = 7.5)
2´´	75.0	3.48 (m)
3´´	78.0	3.47 (m)
4´´	78.4	3.41 (m)
5´´	71.3	3.40 (m)
6´´	62.7	3.91 (J = 12.0, 1.8)/3.72 (dd, J = 12.0, 5.0)

a

Proton multiplicity and coupling constants (J = Hz) in parentheses.

Acid Hydrolysis of 1

Compound 1 (1.0 mg) was hydrolyzed using 2 N CF₃COOH (2 mL) at 90 °C for 3 h. After cooling to room temperature, residue was partitioned with ethyl acetate to remove the aglycone. The aqueous layer, including the sugar unit, was evaporated to dryness under reduced pressure. The residue was dissolved in pyridine (100 μL) containing L-cysteine methyl ester hydrochloride (2.0 mg) at 60 °C for 1 h. Then, o-tolylisothiocyanate (10 μL) was added and heated at 60 °C for another 1 h. The reaction mixture was immediately analyzed using HPLC. HPLC was performed on a reversed-phase C₁₈ column (5 μm, i.d. 4.6 mm × 150 mm) under isocratic conditions of 22% ACN containing 0.1% formic acid for 30 min at a flow rate of 1.0 mL/min, with UV detection at 250 nm.^14,15

ABTS Radical Cation Decolorization Assay

ABTS radical scavenging activity was analyzed by using an ABTS radical cation decolorization assay with minor modifications.^16,17 ABTS was dissolved in water to a concentration of 7.0 mM, and ABTS cation radicals were produced by reacting the ABTS stock solution with 2.45 mM potassium persulfate in the dark for 12 h. After adding 190 μL of the ABTS radical cation solution to the samples (10 μL) dissolved in methanol, the absorbance was measured using a microplate reader at 734 nm after mixing for 7 min. The assays were performed in triplicate. Trolox and butylated hydroxyanisole (BHA) were used as positive controls.

DPPH Radical Scavenging Activity Assay

The ability of the samples to scavenge DPPH radicals was determined using previously reported method. ¹⁸ The samples (10 μL) dissolved in MeOH were treated in wells and added to a 320 μM DPPH (90 μL) ethanol solution. The mixture was incubated for 10 min at room temperature, and the absorbance was measured at 517 nm using a microplate reader. The assays were performed in triplicate. Trolox and BHA were used as positive controls.

Statistical Analysis

All bioassays were expressed as mean ± standard deviation. Statistical analysis was performed using Excel software. P value less than 0.05 (P < .05) was considered as statistical significance.

Results

Compound 1 was obtained as a yellow oil with a specific optical rotation value of +76.4° (c 0.1, MeOH). The molecular formula of 1 was established as C₂₂H₂₄O₁₃ on the basis of a quasi-molecular ion peak at m/z 495.1133 [M−H]⁻ (calcd. for C₂₂H₂₃O₁₃, 495.1139) observed in the high-resolution electrospray ionization (ESI)-mass spectrum. The UV spectrum of 1 showed maximum absorption (λ_max) at 220 (4.5), 267 (4.2), and 293 (4.1) nm. The IR spectrum showed absorption bands at 3334 (-OH), 2924 (C-H), and 1702 (C = O) cm⁻¹. The ¹H NMR spectrum of 1 (Table 1) featured the signals of five aromatic methines at δ_H 7.71 (dd, J = 8.3, 1.9 Hz, H-6′), 7.69 (d, J = 1.9 Hz, H-2′), and 6.90 (d, J = 8.3 Hz, H-5′), 6.64 (d, J = 2.3 Hz, H-3) and 6.39 (d, J = 2.3 Hz, H-5), one methylene at δ_H 3.53 (m, H-7), one methoxy at δ_H 3.94 (s, 3′-OCH₃), and a glucose unit at δ_H 4.86 (d, J = 7.5 Hz, H-1′′), 3.91 (dd, J = 12.0, 1.8 Hz, H-6′′), 3.72 (dd, J = 12.0, 5.0 Hz, H-6′′), 3.48 (m, H-2′′), 3.47 (m, H-3′′), 3.41 (m, H-4′′), and 3.40 (m, H-5′′). The ¹³C NMR spectrum of 1 (Table 1) indicated two carbonyl carbons at δ_C 177.2 (C-8) and 166.2 (C-7′), twelve sp² carbons including five sp² methine carbons at δ_C 126.0 (C-6′), 116.2 (C-5′), 114.1 (C-2′), 105.3 (C-5), and 102.4 (C-3), seven sp² quaternary carbons at δ_C 158.7 (C-4), 158.6 (C-2), 153.7 (C-4′), 152.2 (C-6), 149.0 (C-3′), 121.7 (C-1′), and δ_C 111.4 (C-1), one anomeric carbon at δ_C 103.6 (C-1′′), four oxygenated methine carbons at δ_C 78.4 (C-4′′), 78.0 (C-3′′), 75.0 (C-2′′), and 71.3 (C-5′′), one oxygenated methylene carbon at δ_C 62.7 (C-6′′), one methoxy carbon δ_C 56.6 (3-OCH₃), and one methylene carbon δ_C 31.4 (C-7). The NMR spectral data revealed that 1 structurally resembled rosarugoside C, a known depside. ¹³ Heteronuclear Multiple Bond Correlation (HMBC) correlations were observed from H-3 to C-1, C-4, and C-5; from H-5 to C-1, C-3, C-4, and C-6; and from H-7 to C-1, C-2, C-6, and C-8. These data confirmed the presence of the 24,6-trihydroxyphenylacetic acid moiety. Additionally, the HMBC correlations from H-2′ to C-4′, C-6′, and C-7′; from H-5′ to C-1′, C-3′, and C-4′; from H-6′ to C-1′, C-2′, and C-4′; and from 3′-OCH₃ to C-3′ were observed. These data, along with the carbon chemical shifts at δ_C 149.0 (C-3′) and 153.7 (C-4′) indicated the presence of a 4-hydroxy-3-methoxybenzoic acid moiety (Figure 2). Furthermore, we compared to the chemical shift of the free carboxyl carbon of vanillic acid (4), the upfield shift of about 4.2 ppm observed for C-7′ in 1 provided indirect evidence that C-7′ is esterified within the vanillic acid moiety^19-22 The attachment of the glucose unit at C-2 position was confirmed through the HMBC correlation from H-1′′ to C-2. The anomeric proton at H-1′′ (δ_H 4.87) exhibited a β-configuration, as determined by its ¹H NMR coupling constant (J = 7.5 Hz).^23,24 Moreover, the stereochemistry of the sugar moiety in 1 was elucidated through acid hydrolysis followed by HPLC analysis, which confirmed a retention time consistent with that of D-glucose (t_R : 14.5 min) rather than L-glucose (t_R of : 12.7 min). Therefore, the structure of 1 was elucidated as a new glycosylated depside, namely Rosarugoside F.

Figure 2.

HMBC and ¹H–¹H Correlated Spectroscopy (¹H–¹H COSY) Correlations for 1.

The antioxidant activity of the isolated compounds (1-5) was analyzed using ABTS and DPPH radical scavenging assays. Compound 1 exhibited moderate scavenging activity with IC₅₀ values of 70.1 µM for the ABTS radical and 187.5 µM for the DPPH radical. Compound 3 also exhibited moderate scavenging activity, with IC₅₀ values of 78.2 and 164.7 µM for the ABTS and DPPH radicals, respectively. Compound 2 demonstrated weak scavenging activity, with IC₅₀ values of 128.5 µM for the ABTS radical, and was inactive toward DPPH radical at concentrations up to 250 µM. Compounds 4 and 5 did not exhibit antioxidant activity in ABTS and DPPH radical scavenging assays at concentrations up to 250 µM (Table 2).

Table 2.

ABTS and DPPH Radical Scavenging Activities of 1-5.

Compounds	IC50 (μM)
	ABTS	DPPH
1	70.1 ± 5.0	187.5 ± 14.2
2	128.5 ± 1.3	- a
3	78.2 ± 2.2	164.7 ± 14.9
4	- a	- a
5	- a	- a
Trolox	32.4 ± 1.1	70.6 ± 3.0

a

No activity up to 250 μM.

Discussion

In this study, a new glycosylated depside, Rosarugoside E (1), along with four known compounds, sinensin (2), 3,4-hydroxybenzoic acid (3), vanillic acid (4), 4-hydroxybenzoic acid (5), was isolated from the flower buds of Rosa rugosa, a plant traditionally used in herbal medicine and widely employed in cosmetic applications. The structure of compound 1 was elucidated based on comprehensive spectroscopic analysis including NMR (1D and 2D), HR-ESI-MS, and acid hydrolysis. The antioxidant activities of compounds 1-5 were evaluated through ABTS and DPPH radical scavenging assays. Among them, compounds 1 and 3 exhibited moderate scavenging activity, while compounds 4 and 5 were inactive under the tested conditions. While this study employed ABTS and DPPH assays to evaluate the free radical scavenging activity of the isolated compounds, it is important to note that both methods mainly assess hydrogen atom transfer (HAT) mechanisms. Given that antioxidant activity can also involve electron transfer (ET) processes, the current study does not capture the full mechanistic profile of antioxidant behavior. Future investigations should consider integrating ET-based assays to more comprehensively assess the antioxidant potential of compound 1.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251349341 - Supplemental material for Isolation and Characterization of a New Glycosylated Depside and Other Compounds with Antioxidant Activity from the Flower Buds of Rosa rugosa

Supplemental material, sj-docx-1-npx-10.1177_1934578X251349341 for Isolation and Characterization of a New Glycosylated Depside and Other Compounds with Antioxidant Activity from the Flower Buds of Rosa rugosa by Dae-Cheol Choi, Kyung Lee, Gun-Woo Oh, Dae-Won Ki, Seok-Chun Ko, Kyung Woo Kim, Dongwoo Yang, Du-Min Jo, Mi-Jin Yim, Jeong Min Lee, Grace Choi, Dae-Sung Lee and Ji-Yul Kim in Natural Product Communications