Isolation of two flavonol glycosides from Rosa rugosa pollen and their anti-inflammatory effect in lipopolysaccharide-stimulated macrophages

Abstract

Rosa rugosa is a rosaceous plant used in traditional Chinese medicine to treat various diseases, such as rheumatism. Although the chemical constituents of R. rugosa petals have been reported, those of the pollen and their physiological functions remain unclear. In this study, two major compounds were isolated from R. rugosa pollen extracts for the first time, and their structures were identified using instrumental analyses as 8-methoxykaempferol 3-O-sophoroside and 8-methoxykaempferol 3-O-(2″-O-α-L-rhamnosyl)-β-D-glucoside. The anti-inflammatory activity of flavonol glycosides isolated from R. rugosa pollen was evaluated in lipopolysaccharide-stimulated RAW 264 macrophages in vitro. They significantly inhibited nitric oxide release and induced nitric oxide synthase protein expression. They also significantly suppressed the release of inflammatory cytokines, such as interleukin (IL)-1β and IL-6. These results suggest that the two flavonol glycosides in R. rugosa pollen have anti-inflammatory properties.

Introduction

Rosa rugosa Thunb. is a deciduous shrub belonging to the Rosaceae family and is native to East Asia. R. rugosa is distributed along the coasts of Hokkaido, Okhotsk, and Kamchatka and its petals are used in herbal teas and cosmetics. The flower buds of R. rugosa have been used in traditional Chinese medicine to treat abdominal pain, hepatodynia, anorexia, vomiting, menstrual disorders, and rheumatic arthralgia (Lu and Wang, 2018). R. rugosa anthers, which contain pollen in the buds, are believed to improve diabetes in Uyghur folk medicine. Previous phytochemical studies have reported that the petals of R. rugosa contain bioactive compounds consisting mainly of hydrolyzable tannins and small amounts of flavonoids (Hashidoko, 1996; Sarangowa et al., 2010a; Sarangowa et al., 2010b). However, reports on the bioactive compounds in plant pollen are scarce due to the challenge of collecting sufficient amounts for analysis (Ferreres et al., 1989). Although bee pollen contains flavonoids such as kaempferol, quercetin, and isorhamnetin (Campos et al., 2008; Komosinska-Vassev et al., 2015), the chemical constituents of R. rugosa pollen remain unclear.

Inflammation is a protective response that involves immune cells responding to noxious stimuli, such as toxic chemicals and pathogen infection by parasites, viruses, and bacteria, resulting in swelling, redness, fever, pain, and tissue dysfunction. Inflammation plays an important role in the removal of foreign substances; however, excessive inflammation can lead to chronic diseases. Macrophages are important immune effector cells involved in inflammation (Watanabe et al., 2019). Stimulation of macrophages with lipopolysaccharide (LPS), a component of the bacterial cell wall (Maldonado et al., 2016), induces the production of nitric oxide (NO) by inducible NO synthase (iNOS) (Sharma et al., 2007) and inflammatory cytokines, a type of signaling molecule, such as interleukin (IL)-1β and IL-6 via intracellular signal transduction (Shapouri-Moghaddam et al., 2018). Excessive NO production causes tissue injury, and inflammatory cytokines secreted by macrophages play an important role in the upregulation of inflammatory responses (Yoon et al., 2009).

In recent years, natural substances have been used to reduce inflammation instead of drugs which can have side effects. Flavonoids in edible medicinal herbs have been suggested to suppress inflammation both in vitro and in vivo (Al-Khayri et al., 2022). It has been reported that the whole-flower extract of R. rugosa has anti-inflammatory activity in LPS-induced macrophages in vitro (Tursun et al., 2016). In this study, major flavonoids were isolated from R. rugosa pollen, and their structures were identified. Additionally, their anti-inflammatory activities were evaluated in a macrophage cell line.

Materials and Methods

Materials R. rugosa flowers were obtained from a farm in Kitami, Hokkaido, Japan. The stamens were separated from the flowers and air dried. The pollen was separated from the anthers and filaments by filtration through a 90 µm sieve. All chemicals used were of reagent grade.

Extraction and isolation Dried powder of R. rugosa pollen (2.84 g) was mixed with 1 L of 70 % ethanol and sonicated three times for 30 min. The extract was centrifuged, and the supernatant was lyophilized. The extract (928.6 mg) was dissolved in water and applied to an open column (Wakosil 40C18, 4 i.d. × 40 cm, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). The column was eluted with an H₂O/ethanol (EtOH) solvent system to obtain 10 fractions. Fraction 5 (58.4 mg), containing the flavonol glycoside, was further purified using a Sephadex LH-20 column (3 i.d. × 16 cm, GE Healthcare, Uppsala, Sweden). Compound 1 (16.6 mg) was isolated from the fraction eluted with EtOH/H₂O (30:70, v/v). Compound 2 (3.2 mg) was isolated from the fraction eluted with EtOH/H₂O (35:65, v/v). The structures of these compounds were characterized by 1D and 2D NMR spectra and HR-ESI-MS spectra, and compared with previously published data.

Instrumental analyses Optical rotation was determined using a Jasco P-2200 polarimeter. The UV spectra were recorded using a Shimadzu UV-1601 UV-VIS spectrophotometer. HR-ESI-MS data were obtained using a Waters Xevo G2-XS Q-TOF mass spectrometer. 1D and 2D NMR spectra were recorded on a Bruker Avance Neo 600 MHz digital NMR spectrometer (Bruker, Fällanden, Switzerland) in methanol (MeOH)-d₄, which was used as an internal reference (δ_h: 3.3 ppm, δ_C: 49.0 ppm).

8-Methoxykaempferol 3-O-sophoroside (Compound 1): Light yellow amorphous powder. [α]²⁶d −14.7 (c 0.10, MeOH); UV λmax (MeOH) nm (log ε): 282 (3.37), 355 (3.16); HR-ESI-MS m/z: 663.1502 [M+Na]⁺ (calcd for C₂₈H₃₂O₁₇Na: 663.1532); ¹H NMR (MeOH-d₄, 600 MHz) δ: 3.21 (1H, dd, J = 2.4, 9.7, H-5″), 3.28 (1H, m, H-5‴), 3.35 (1H, m, H-2‴), 3.38 (1H, t, J = 7.5, H-4‴), 3.48 (1H, dd, J = 12.2, 5.3, H-6″), 3.49 (1H, dd, J = 9.0, 2.4, H-4″), 3.61 (1H, t, J = 9.0, H-3″), 3.67 (1H, d, J = 11.9, H-6‴), 3.69 (1H, dd, J = 7.5, 9.8, H-3‴), 3.74 (1H, dd, J = 7.5, 9.0, H-2″), 3.78 (1H, dd, J = 2.4, 12.2, H-6″, 6‴), 3.88 (3H, s, 8-OCH₃), 4.75 (1H, d, J = 7.5, H-1‴), 5.47 (1H, d, J = 7.5, H-1″), 6.25 (1H, s, H-6), 6.92 (2H, d, J = 8.9, H-3′,5′), 8.10 (2H, d, J = 8.9, H-2′,6′). ¹³C NMR (MeOH-d₄, 150 MHz) δ: 61.9 (8-OCH₃), 62.5 (C-6‴), 62.6 (C-6″), 71.1 (C-4″), 71.3 (C-4‴), 75.6 (C-2‴), 78.2 (C-5″,5‴), 78.3 (C-3″, 3‴), 82.6 (C-2″), 100.0 (C-6), 100.9 (C-1″), 104.7 (C-1‴), 105.8 (C-10), 116.3 (C-3′,5′), 122.9 (C-1′), 129.1 (C-8), 132.3 (C-2′,6′), 134.9 (C-3), 150.4 (C-2), 158.1 (C-9), 158.4 (C-5), 158.7 (C-7), 161.6 (C-4′), 179.9 (C-4).

8-Methoxykaempferol 3-O-(2′-O-α-L-rhamnosyl)-β-D-glucoside (Compound 2): Light yellow amorphous powder. [α] ²⁶d -34.1 (c 0.05, MeOH); UV λmax (MeOH) nm (log ε): 283 (3.05), 355 (2.89); HR-ESI-MS m/z: 647.1551 [M+Na]⁺ (calcd for C₂₈H₃₂O₁₆Na: 647.1583); ¹H NMR (MeOH-d₄, 600 MHz) δ: 0.96 (1H, d, J = 6.4, H-6‴), 3.23 (1H, dd, J = 2.4, 5.8, H-5″), 3.28 (1H, dd, J = 2.4, 9.8, H-4″), 3.34 (1H, t, J = 9.8, H-4‴), 3.50 (1H, dd, J = 5.8, 12.2, H-6″), 3.55 (1H, t, J = 9.0, H-3″), 3.62 (1H, dd, J = 7.6, 9.0, H-2″), 3.74 (1H, dd, J = 2.4, 12.2, H-6″), 3.76 (1H, dd, J = 3.4, 9.8, H-3‴), 3.88 (3H, s, 8-OCH₃), 3.99 (1H, dd, J = 1.5, 3.3, H-2‴), 4.02 (1H, dq, J = 6.4, 9.8, H-5‴), 5.23 (1H, d, J = 1.5, H-1‴), 5.74 (1H, d, J = 7.6, H-1″), 6.23 (1H, s, H-6), 6.90 (2H, d, J = 8.9, H-3′,5′), 8.11(2H, d, J = 9.2, H-2′,6′). ¹³C NMR (MeOH-d₄, 150 MHz) δ: 17.6 (C-6‴), 61.9 (C-OCH₃), 62.7 (C-6″), 69.9 (C-5‴), 71.9 (C-4″), 72.3 (C-3‴), 72.4 (C-2‴), 74.1 (C-4‴), 78.4 (C-5″), 78.9 (C-3″), 80.1 (C-2″), 100.2 (C-6), 100.3 (C-1″), 102.6 (C-1‴), 105.6 (C-10), 116.2 (C-3′,5′), 123.2 (C-1′), 129.2 (C-8), 132.1 (C-2′,6′), 134.4 (C-3), 150.6 (C-9), 158.1 (C-2), 158.2 (C-7), 159.0 (C-5), 161.4 (C-4′), 179.5 (C-4).

Ultra-performance liquid chromatography (UPLC) analysis The extracts of R. rugosa pollen and petals were dissolved in EtOH/H₂O (50:50, v/v) at a concentration of 50 mg/mL and passed through a 0.45 µm filter, and 1 µL of the sample was subjected to UPLC analysis as follows. UPLC was performed using a Waters Acquity system (Milford, MA, USA) consisting of a binary solvent manager, a sample manager, a column heater set at 40°C, a PDA detector, and an Acquity UPLC BEH C₁₈ column (2.1 i.d. × 100 mm, 1.7 µm). The initial mobile phase (A) was acetonitrile/water (2.5:97.5, v/v) with 0.1 % formic acid, and the final mobile phase (B) was acetonitrile with 0.1 % formic acid. Gradient conditions were as follows: 0–1 min, 0 % B; 2 min, 10 % B; 4 min, 15 % B; 15 min, 20 % B; 25 min, 40 % B; 28 min, 60 % B; 30 min, 100 % B. The flow rate was 0.2 mL/min. The detector wavelength range was set from 200 to 500 nm, and comparisons between pollen and petals were made using their chromatograms at 350 nm.

Cell culture The murine macrophage-like cell line RAW 264 (cell number: RCB0535) was obtained from the RIKEN BRC Cell Bank (Tsukuba, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wako, Japan) containing 1 mM sodium pyruvate, 4 mM L-glutamine, 10 % fetal bovine serum (HyClone Laboratories, Logan, UT, USA), 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C under 5 % CO₂ and 95 % air.

NO production and iNOS analysis Cells were cultured in 24-well plates at a density of 4 × 10⁵ cells/well for 2 h. After pretreatment with different concentrations of the samples for 30 min, the cells were incubated with LPS (10 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. NO released into the culture supernatant was determined using a NO₂/NO₃ assay kit with 2,3-diaminonaphthalene (Dojindo Laboratories, Kumamoto, Japan). LPS-stimulated cells were solubilized under the same conditions described above using an EzRIPA lysis kit (ATTO Corporation, Tokyo, Japan) according to the supplier’s protocol. The protein expression level of iNOS in the cell lysate (30 µL) was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an acrylamide slab gel system (AnykD TGX gel, Bio-Rad, Hercules, CA, USA) (Zorig et al., 2021), followed by Western blotting using rabbit anti-iNOS polyclonal antibody (GeneTex, Inc., Irvine, CA, USA) and anti-β-actin monoclonal antibody (8H10D10, Cell Signaling Technology, Danvers, MA, USA) as primary antibodies. Immunoreactive substances were visualized by near-infrared fluorescence (800 and 680 nm) using an Odyssey CLx (LI-COR, Lincoln, NE, USA) with goat anti-rabbit IgG conjugated to IRDye 800CW and goat anti-mouse IgG conjugated to IRDye 680RD as secondary antibodies (LI-COR).

Determination of inflammatory cytokines Cells were cultured in 24-well plates at a density of 4 × 10⁵ cells/well for 2 h. After pretreatment with the samples for 30 min, the cells were stimulated with LPS (10 µg/mL). IL-1β and IL-6 released into the supernatant after the incubation for 24 h and 6 h, respectively, were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s protocol (Mabtech, Nacka Strand, Sweden).

Cytotoxicity assay Cells were cultured in 96-well plates for 2 h and incubated for 24.5 h in the presence of the samples. Cell viability was evaluated using a Cell Counting Kit-8 (Dojindo) (Gan et al., 2017).

Statistical analysis All experiments were performed in triplicate and the data are presented as the mean ± S.D. The statistical significance of the differences was analyzed using the Tukey–Kramer multiple comparison test. Differences were considered statistically significant at p < 0.05.

Results and Discussion

Phytochemical composition of R. rugosa pollen Compounds 1 and 2 were isolated from 70 % ethanol extracts of R. rugosa pollen using column chromatography eluted with EtOH/H₂O. The structures of Compounds 1 and 2 were characterized by UV, HR-ESI-MS, and NMR analyses. The ¹H NMR spectra of Compounds 1 and 2 indicated the presence of kaempferol as an aglycone (Zhu et al., 2017), and a methoxy group appeared as a single peak at δ_h 3.88, correlated with δ_c 61.9 in the HSQC spectra, and correlated with δ_c 129.2 (C₈ position of kaempferol) in the HMBC spectra. The ¹³C NMR spectrum of Compound 1 indicated the presence of two hexoses, whereas that of Compound 2 indicated the presence of both a hexose and a deoxy-hexose. Analysis of the HMBC spectra confirmed the position of the sugar moieties. The C₁-H of the sugar moiety correlated with the C₃ position of kaempferol, and the C₂-H of the sugar moiety correlated with the C2 position of the other sugar moiety. The ¹H, ¹³C NMR, and MS data were compared with values reported in the literature (Ferreres et al., 1989; Rychlinska and Gudej, 2002), and the structures of Compounds 1 and 2 were characterized as 8-methoxykaempferol 3-O-sophoroside and 8-methoxykaempferol 3-O-(2″-O-α-L-rhamnosyl)-β-D-glucoside, respectively, as shown in Fig. 1. These compounds have also been identified in the flowers of Pyrus communis L. (Rychlinska and Gudej 2002). Compound 1 has been isolated from almond pollen (Ferreres et al., 1989), Sophora japonica L. flowers (Shi et al., 2017), and Potentilla anserina flowers (Kombal and Glasl, 2017). In this study, we isolated these compounds from R. rugosa pollen for the first time. In addition, compounds with a methoxy group at the C₈ position of the kaempferol benzopyran ring, such as 8-methoxykaempferol 3-O-neohesperidoside, 8-methoxykaempferol 3-O-glucoside, and 8-methoxykaempferol, have been found in bee pollen from Crataegus monogyna (Dauguet et al., 1993), a species of the Rosaceae family. Therefore, these kaempferol derivatives may be specific to pollen of the Rosaceae family. To investigate whether Compounds 1 and 2 are also present in R. rugosa petals, 70 % ethanol extracts of R. rugosa pollen and petals were analyzed by UPLC at 350 nm. Compounds 1 and 2 were the major flavonoids in the pollen extract (Fig. 2A) but were not detected in the petal extract (Fig. 2B). Additionally, the chromatogram patterns of the pollen and petal extracts were completely different, indicating that the flavonoids in the pollen and petals of R. rugosa are different.

Fig. 1

Chemical structures of Compounds 1 and 2 isolated from Rosa rugosa pollen.

1: 8-Methoxykaempferol 3-O-sophoroside; 2: 8-Methoxykaempferol 3-O-(2″-O-α-L-rhamnosyl)-β-D-glucoside.

Fig. 2

Ultra-performance liquid chromatograms of the extract of R. rugosa. (A): pollen extract; (B): petal extract. 1: 8-Methoxykaempferol 3-O-sophoroside; 2: 8-Methoxykaempferol 3-O-(2″-O-α-L-rhamnosyl)-β-D-glucoside.

Anti-inflammatory effect of Compounds 1 and 2 on LPS-induced RAW 264 cells Previous studies have shown that methoxykaempferol 3-sophoroside from Flos Sophorae has inhibitory effects on triglyceride accumulation in HepG2 cells (Shi et al., 1993); however, other physiological activities are unclear. In this study, the anti-inflammatory activities of Compounds 1 and 2 were evaluated in RAW 264 macrophages.

The cytotoxicity of Compounds 1 and 2 against RAW 264 cells was not observed at up to 0.4 mg/mL for 24 h (data not shown). LPS stimulation significantly increased the NO concentration in the culture supernatant of RAW 264 cells (Fig. 3A). As shown in Fig. 3B and C, NO release from LPS-stimulated RAW 264 cells was significantly inhibited in a dose-dependent manner by Compounds 1 and 2 at 0.1, 0.2, and 0.4 mg/mL, respectively. The inhibitory activities of Compounds 1 and 2 were similar. To elucidate the mechanism of NO production inhibition in LPS-stimulated RAW 264 cells, the effects of Compounds 1 and 2 on iNOS protein expression were analyzed using Western blotting. Figure 4A shows bands of iNOS with a molecular weight of 131 kDa in the RAW 264 cell lysate. Compared to unstimulated cells (lane 1), LPS stimulation for 24 h (control) significantly increased the iNOS level (lane 2). In contrast, the increase in iNOS expression was suppressed by treatment with Compound 1 (lane 3) and Compound 2 (lane 4) at 0.4 mg/mL. There was no difference in the levels of β-actin at 42 kDa, a loading control of total cell protein, between the unstimulated (lane 1), control (lane 2), Compound 1 (lane 3), and Compound 2 (lane 4), as shown in Fig. 4B. The fluorescence intensities of the iNOS bands (n = 3) were quantified using the imaging analyzer, and the values were normalized to the corresponding fluorescence intensities of the β-actin bands. The suppression of iNOS by Compounds 1 and 2 was significant (p < 0.05) compared to that of the control, whereas the inhibitory activities of Compounds 1 and 2 were not significantly different (Fig. 4C). These results indicate that the suppression of NO release from LPS-stimulated RAW 264 cells by Compounds 1 and 2 may be caused by the inhibition of iNOS overexpression. Figure 5 shows the effect of Compounds 1 and 2 on IL-1β (A) and IL-6 (B), inflammatory cytokines, released from LPS-stimulated RAW 264 cells. Compounds 1 and 2 significantly suppressed the release of cytokines. In a comparison of the inhibitory effects of Compounds 1 and 2 at a concentration of 0.4 mg/mL, the inhibitory activity of Compound 2 was much stronger than that of Compound 1, whereas their inhibitory activities against NO and iNOS were comparable. The transcription factor Nrf2 plays an important role in regulating the expression of antioxidant genes such as heme oxygenase-1, which exerts anti-inflammatory functions (Saha et al., 2020). Caspase activation through inflammasomes is suggested to be involved in the production of IL-1β (Rathinam and Fitzgerald, 2013). The effect of Compounds 1 and 2 on these mechanisms should be investigated in future studies. Some flavonoid glycosides with the same aglycone have different physiological functions depending on the number and type of sugars. It has been suggested that solubility, hydrolysis, and metabolism may be involved, but the mechanism responsible for the difference is controversial (Xiao, 2017; Johnson et al., 2021). The contribution of the glycoside moiety of Compounds 1 and 2 requires further investigation. This is the first report of the inhibitory effects of 8-methoxykaempferol glycosides on LPS-stimulated inflammatory responses in RAW 264 cells, while the effects of kaempferol and its glycosides on inflammation have been previously reported (Devi et al., 2015; Lee et al., 2018; Fang et al., 2022). Furthermore, comparison of the anti-inflammatory activity with the chemical substances in R. rugosa petals, such as hydrolyzable tannins, should be clarified in future studies.

Fig. 3

Inhibitory effect of Compounds 1 and 2 on nitric oxide (NO) production in RAW 264 cells. RAW 264 cells were stimulated with lipopolysaccharide (LPS) for 24 h after 30 min pretreatment with the samples. The culture supernatant was analyzed for NO released from unstimulated and LPS-stimulated RAW 264 cells (A). The dose-dependent suppressive effect of Compounds 1 and 2 on NO production was determined (B and C). Means without a common letter are significantly different (p < 0.05).

Fig. 4

Effect of Compounds 1 and 2 on induce nitric oxide synthase (iNOS) expression in RAW 264 cells. LPS-stimulated cells were solubilized and the cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting using anti-iNOS (A) and anti-β-actin antibodies (B). Lane 1, unstimulated; lane 2, control; lane 3, Compound 1; lane 4, Compound 2. The fluorescence intensities of iNOS bands were normalized by each β-actin band (C). Means without a common letter are significantly different (p < 0.05).

Fig. 5

Inhibitory effect of Compounds 1 and 2 on inflammatory cytokine production, IL-1β (A) and IL-6 (B), in RAW 264 cells. enzyme-linked immunosorbent assay was performed on the culture supernatant after the LPS stimulation. Means without a common letter are significantly different (p < 0.05).

In conclusion, we found 8-methoxykaempferol 3-O-sophoroside and 8-methoxykaempferol 3-O-(2″-O-α-L-rhamnosyl)-β-D-glucoside in R. rugosa pollen. Furthermore, these two flavonol glycosides inhibited the inflammatory response in a macrophage cell line, suggesting that R. rugosa pollen could be used as a natural medicine to alleviate inflammation-related diseases.

Acknowledgements This work was supported by grants from the Foreign Researchers’ Project under the “Fiscal 2019 JASSO Follow-up Research Fellowship” of Japan and the International Cooperation Fund of the Inner Mongolia Cooperative Innovation Center for Mongolian Medicine of China (No. MYYXTGJ202301).

Conflict of interest There are no conflicts of interest to declare.

Abbreviations

LPS

lipopolysaccharide

NO

nitric oxide

iNOS

inducible nitric oxide synthase

IL

interleukin

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Ig

immunoglobulin

ELISA

enzyme-linked immunosorbent assay

HRMS

high-resolution mass spectroscopy

UPLC

ultra-performance liquid chromatography

NMR

nuclear magnetic resonance

HMBC

heteronuclear multiple bond correlation

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