Identification,Characterization and Anti-inflammatory Activity of a New Flavonoid From Hibiscus mutabilis L.

Abstract

Hibiscus mutabilis L (Malvaceae) was a traditional Chinese medicine with significant anti-inflammatory activity. We isolated 3 compounds from the flowers of H mutabilis L , including a new flavonoid, (1″R)-8-(1-(3,4-dihydroxyphenyl)ethyl)-3,3',5,7-tetrahydroxy-4′-methoxyl flavone (1), and 2 known flavonoids (2-3). The structures of these compounds were elucidated by various spectroscopic methods. In addition, we evaluated the anti-inflammatory effect of compound 1. The bioactivity assay showed that 1 significantly inhibit the excessive production of IL-6, TNF-α and NO in LPS-induced RAW264.7 macrophages. Western blot analysis demonstrated that 1 inhibit the expressions of COX-2 and iNOS protein in the macrophages induced by LPS in a concentration-dependent manner. In addition, 1 also significantly suppress the expression of the key proteins P65 and p-P65 in the NF-κB signaling pathway. Our results revealed that 1 exert potential anti-inflammatory activity through inhibiting the activation of NF-κB signaling pathway.

Graphical Abstract

Introduction

Medicinal plants plays a significant role in health care because of their special active ingredients. It was reported that around 80 % of the global population still relies on botanical drugs. ¹ Lots of medicinal plants extracts show therapeutic effects to some specific diseases. For example, Astragalus membranaceus (Fisch). Bunge extract is beneficial to prevent asthma, ² broad-spectrum cannabis oil can ameliorate fibromyalgia, ³ and so on. Medicinal plants contribute significantly to human livelihood and development.

H mutabilis L (Malvaceae) is a medicinal deciduous shrub, originally came from China and widely planted throughout the world. ⁴ The leaves, flowers and roots of the plant can be used as medicine. H mutabilis L reportedly contains a range of active constituents, including flavonoids, organic acids, amino acids, and other compounds. ⁵ And flavonoids are considered as the most important active components of H mutabilis L. ⁶ The separated flavonoids from H mutabilis L display identified bioactivities such as anti-influenza ⁷ and anti-oxidative activity, ⁸ among others. This paper shows the isolation and structure elucidation of a new flavonoid, named (1″R)-8-(1-(3,4-dihydroxyphenyl)ethyl)-3,3',5,7-tetrahydroxy-4′-methoxyl flavone (1), and 2 known flavonoids (2-3) from H mutabilis L (Figure 1). Furthermore, anti-inflammatory property and its mechanism of compound 1 in LPS-induced RAW264.7 macrophages are investigated.

Figure 1.

Structures of compounds 1-3.

Materials and Methods

General

Column chromatography: silica gel (200-300 mesh, Qingdao hailang silica gel desiccant Inc., China), and MCI gel (8A801, Mitsubishi chemical Inc., Japan). Semi-prep HPLC: 7725i-049, Chengdu laipu technology Inc., DAD detector at 210 and 280 nm, AQ-C18 column (10 × 210 mm, Welch). ¹H NMR and ¹³C NMR spectra: Bruker AVIII HD-600 spectrometer (600 and 151 MHz, resp.). HR-ESI-MS: UPLC-Q-Orbitrap-HRMS spectrometer (Thermo Scientific, USA). Multi label Plate Reader (Spectra MAX M5, Molecular Devices).

Plant Material

We collected the flowers of Hibiscus mutabilis from the H mutabilis L. planting base, Sanhe Town, Chengdu, Sichuan. The flowers were identified by Dr Fei Long (Chengdu University of TCM, Chengdu, China).

Extraction and Isolation

We extracted the air-dried flowers of H mutabilis L. (20 kg) with 90% EtOH (2 × 160 L, 2 h) under reflux. A brown residue (3.34 kg) was obtained after concentrating the EtOH extract under reduced pressure, which was suspended in H₂O (3.5 L) and then partitioned sequentially with EtOAc (3 × 3.5 L). Seven fractions (Fr.1∼Fr.7) were obtained from the EtOAc extract (80.0 g) with a gradient elution of petroleum ether-EtOAc (100:1-0:1, v/v). Ten subfractions (Fr.4-1-Fr.4-10) were obtained from Fr.4 (12.64 g). Compound 1 (6.0 mg) and 3 (1.5 mg) were obtained from Fr.4-5 via reversed-phase semipreparative HPLC (35% acetonitrile in H₂O). Purification of Fr.4-3 with reversed-phase semipreparative HPLC (45% MeOH in H₂O) yield 2 (5.3 mg).

$\left(1^{″}\mathrm{R}\right)-8-\left(1-\left(3,4-\text{dihydroxyphenyl}\right)\text{ethyl}\right)-3,3^{\prime },5,7-\text{tetrahydroxy}-4^{\prime }-\text{methoxyl}\hspace{0.17em}\text{flavone}$

(1)

Brown powder; [α]25 D-60 (c 0.05, MeOH); UV (λ_max, MeOH, nm): 260, 380; IR (KBr) V_max 3432, 1601, 1555, 1516 cm⁻¹; HRESIMS: m/z 451.10 [M-H]^- (calcd. for C₂₄H₁₉O₉ 451.11). ¹H-NMR (600 MHz, CD₃OD) δ: 7.69 (1H, d, J = 2.2 Hz, H-2′), 7.18 (1H, dd, J = 8.2, 2.2 Hz, H-6′), 6.93 (1H, d, J = 2.0 Hz, H-2‴), 6.80 (1H, d, J = 8.2 Hz, H-5′), 6.79 (1H, dd, J = 8.2, 2.0 Hz, H-6‴), 6.69 (1H, d, J = 8.2 Hz, H-5‴), 6.29 (1H, s, H-6), 4.88 (1H, q, J = 7.2 Hz, H-1″), 3.69 (3H, s, 4′-OCH₃), 1.76 (3H, d, J = 7.3 Hz, H-2″). ¹³C-NMR (151 MHz, CD₃OD) δ: 148.52 (C-2), 136.97 (C-3), 177.67 (C-4), 160.45 (C-5), 99.17 (C-6), 163.10 (C-7), 112.46 (C-8), 155.67 (C-9), 104.97 (C-10), 124.24 (C-1′), 116.57 (C-2′), 146.13 (C-3'), 148.70 (C-4′), 56.26 (4′-OCH₃), 116.09 (C-5′), 121.81 (C-6′), 33.95 (C-1''), 18.85 (C-2''), 138.33 (C-1'''), 112.02 (C-2'''), 145.34 (C-3'''), 148.62 (C-4'''), 115.87 (C-5'''), 120.73 (C-6''').

Cell Viability

Cell viability was evaluated by CCK-8 assay ⁹ in RAW264.7 macrophages. The macrophages were plated at a density of 3 × 10⁴ per well into 96-well plates and incubated for 24 h in a humidified atmospheres with 5% CO₂ at 37°C. After the addition of 1 (0-50 μM), the plates were incubated in a 37°C, 5% CO₂ incubator for 48 h. After removing the supernatant, 10 μL CCK-8, and 150 μL serum-free DMEM medium was added into the plates. The absorbance of each hole was measured by the microplate reader at 450 nm after the macrophages were cultured for 3 h.

Griess Assay

The effect of 1 on inhibiting NO production in the LPS-induced RAW264.7 cell model was investigated using the Griess reagent Kits. ¹⁰ Briefly, we inoculated macrophages into 96-well plates (3 × 10⁴ per well) and cultivated them for 24 h. In the model group, macrophages were cultured with LPS (1 μg/mL), while in the drug group, macrophages were incubated with 1 (10, 20, 30 μM) and LPS (1 μg/mL). Furthermore, dexamethasone (DXMS, 5 μM) as a positive drug in the positive group. After incubation for 24 h, the NO concentration was measured by microplate reader at 540 nm following the manufacturer’s instructions.

Elisa Assay

Compound 1 were evaluated its effect on the production of IL-6 and TNF-α proinflammatory cytokine in LPS-stimulated macrophages using the Elisa Kits. ¹¹ In short, macrophages were plated in 96-well plates and cultured for 24 h. In the model group, macrophages were incubated with LPS (1 μg/mL), while in the drug group, macrophages were incubated with LPS (1 μg/mL) and 1 (10, 20, 30 μM). In the positive drug group, DXMS (5 μM) as a positive drug. After cultivating for 24 h, the NO concentration was determined by measuring the optical density at 540 nm following the manufacturer’s instructions.

Western Blot Analysis

RAW264.7 macrophages (5 × 10⁵ per well) were seeded in 6-well plates and incubated for 24 h, and then pre-treated with 1 (10, 20, 30 μM) or DXMS (5 μM) for 2 h. After that, the macrophages were further stimulated with LPS (1 μg/mL) for 30 min (24 h for iNOS). Then the macrophages were collected and washed twice with cold PBS (Gibco, USA), and lysed with RIPA buffer containing proteinase inhibitors for 30 min on ice. After that, the lysates were clarified by centrifugation at 12,000×g for 20 min at 4°C. After centrifugation, the protein concentration was determined using BCA protein assay kit (Beyotime, Shanghai, China). Protein (40 μg/lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Then the membranes were probed with specific antibody and visualized by ECL (Millipore, USA). GAPDH was used as internal control.

Results

Structural Elucidation of Compound 1

Compound 1 was obtained as a brown powder. The UV spectrum showed maximum absorption at 260 and 380 nm. The HR-ESI-MS [ m/z [M - H]^- 451.10 (calcd. for C₂₄H₂₀O₉, 451.11)] was corresponding to the molecular formula of C₂₄H₂₀O₉ with fifteen degrees of unsaturation. Characteristic signals of 3′, 4′-dihydroxyflavonol was revealed by proton signals at δ 7.69 (1H, d, J = 2.2 Hz, H-2′), 6.80 (1H, d, J = 8.2 Hz, H-5′) and 7.18 (1H, dd, J = 8.2, 2.2 Hz, H-6′) in the ¹H NMR and ¹H-¹H COSY spectra data. Comparing the ¹³C NMR spectra data of compound 1 with those of quercetin, we speculated that the flavone moiety in the molecule was quercetin. In the ¹H NMR spectra data, the singlet signal of H-6 indicated C-8 was substituted. Furthermore, the HMBC spectra data (Figure 2) showed that the proton signal of the methyl carbon at δ 56.26 was correlated to carbon at δ 148.70 (C-4′), indicating the hydroxyl proton of C-4' in the quercetin moiety was substituted by methyl. In the ¹H NMR and ¹H-¹H COSY spectra, a methine and a methyl were revealed by proton signals at δ 4.88 (1H, q, J = 7.2 Hz) and δ 1.76 (3H, d, J = 7.3 Hz) respectively. The HSQC spectra data demonstrated the proton signal at δ 4.88 and 1.76 was correlated to carbon at δ 33.95 and δ 18.85 respectively, suggesting the existence of an ethyl group in the molecule. Except for the aromatic carbons related to quercetin, there were 2 oxygenated aromatic carbons at δ 145.34 and 148.62 and 4 non-oxygenated aromatic carbons at δ 138.33, 115.87, 112.02 and 120.73, indicating the presence of another phenyl ring in the molecules. In addition, the ABX-pattern were revealed by proton signals at δ 6.93 (1H, d, J = 2.0 Hz), 6.69 (1H, d, J = 8.2 Hz) and 6.79 (1H, dd, J = 8.2, 2.0 Hz), directly connected with the carbons at δ 112.02, 115.87 and 120.73 respectively. The HMBC spectra data (Figure 2) demonstrated that the proton signal of the carbon at δ 33.95 was correlated to carbons at δ 18.85, 112.02, 120.73, 155.67, 112.46 and 163.10, revealing the position of methyl, benzene ring, to the flavone moiety. Furthermore, the unsaturation demonstrated no other cyclic structure in the molecule. The absolute configuration at C-1″ position of 1 was assigned to be R on the basis of its negative optical rotation ([α]25 D-60 (c 0.05, MeOH)) and by the comparison of NMR characteristics and optical rotation values with those of similar flavonoids. ¹² Therefore, we finally confirmed compound 1 was (1″R)-8-(1-(3,4-dihydroxyphenyl)ethyl)-3,3',5,7-tetrahydroxy-4′-methoxyl flavone.

Figure 2.

¹H-¹H COSY and HMBC correlations of compound 1.

Two known compounds were identified by comparison of spectra data with those reported in the literatures as (1″R)-8-(1-(3,4-dihydroxyphenyl)ethyl)quercetin (2), ¹³ 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-8-[1-(4-hydroxy-3,5dimethoxyphenyl)ethyl]-4H-1-benzopyran-4-one (3). ¹⁴

Effect of Compound 1 on Cell Viability of RAW264.7 Macrophages

The potential cytotoxicity of 1 was evaluated by CCK-8 assay. As shown in Figure 3, 1 had no cytotoxicity on RAW264.7 macrophages at 10 and 20 μM, and exert less cytotoxicity at 30 μM. Therefore, 1 at 10-30 μM were used in subsequent anti-inflammatory experiments.

Figure 3.

Cell viability of compound 1 against RAW264.7 cells. Note: Compared with the control group^***P < 0.001; ns means no significance.

Effect of Compound 1 on NO Production in RAW264.7 Macrophages

The levels of NO production in macrophages were measured using the Griess reagent Kits. Treatment with LPS markedly increased NO production in macrophages. Administration of 1 significantly inhibited NO production in a dose-dependent manner (Figure 4).

Figure 4.

The inhibitory effect of compound 1 on NO production in RAW264.7 cells. Note: Compared with the control group ^###P < 0.001; compared with the model group (treatment with LPS) ^***P < 0.001, ^**P < 0.01. Abbreviations: DXMS, dexamethasone; LPS, Lipopolysaccharide (Table 1).

Effect of Compound 1 on IL-6 and TNF-α Production in RAW264.7 Macrophages

The levels of IL-6 and TNF-α in RAW264.7 cells were investigated using the Elisa Kits. In comparison with the control group, LPS treatment increased IL-6 and TNF-α concentrations (Figure 5). Treatment with 1 reduced the concentrations of IL-6 (Figure 5A) and TNF-α (Figure 5B) in macrophages activated by LPS. In particular, low concentration of 1 was more beneficial to reduce IL-6 and TNF-α concentrations.

Figure 5.

The inhibitory effect of compound 1 on IL-6 and TNF-α production in RAW264.7 cells. Note: Compared with the control group ^###P < 0.001; compared with the LPS group ^**P < 0.01, ^***P < 0.001; ns means no significance; a and b indicated that concentration is below the minimum detection limit of Kits. Abbreviations: DXMS, dexamethasone; LPS, Lipopolysaccharide; IL-6, Interleukin-6; TNF-α, tumor necrosis factor α (Table 1).

Table 1.

List of Abbreviations.

Abbreviation	Full Name
LPS	Lipopolysaccharide
NMR	Nuclear magnetic resonance
HSQC	1H detected heteronuclear single quantum coherence
HMBC	1H detected heteronuclear multiple bond correlation
1H-1H COSY	1H-1H correlated spectroscopy
DMEM	Dulbecco’s modification of Eagle’s medium
TNF-α	Tumor necrosis factor α
IL-6	Interleukin-6
PBS	Phosphate buffered saline
iNOS	Inducible nitric oxide synthase
COX	Cyclooxygenase
PMSF	Phenyl methane sulfonyl fluoride
NF-κB	Nuclear transcription factor-κB
DXMS	Dexamethasone

Effect of Compound 1 on iNOS, COX-2, P65 and p-P65 Protein Expression in RAW264.7 Macrophages

We carried out Western blot analysis on RAW264.7 macrophages to explore the potential anti-inflammatory mechanism of 1. Briefly, we investigated expression levels of critical enzymes (COX-2 and iNOS) related to inflammation and key protein (P65 and p-P65) in the NF-κB signaling pathway. As shown in Figure 6, LPS treatment (1 μg/mL) increased iNOS and COX-2 protein expressions, while 1 significantly inhibited the expressions of iNOS and COX-2 in macrophages activated by LPS in a concentration-dependent manner. In addition, 1 also suppressed the excessive phosphorylation of P65 induced by LPS in a concentration-dependent manner.

Figure 6.

Effects of compound 1 on LPS-induced iNOS and COX-2 expressions and activation of NF-κB signaling pathway in RAW264.7 cells. Note: Compared with the control group ^###P < 0.001; compared with the LPS group ^**P < 0.01, ^***P < 0.001 Abbreviations: DXMS, dexamethasone; LPS, Lipopolysaccharide; COX-2, Cyclooxygenase 2; iNOS, inducible nitric oxide synthase (Table 1).

Discussion

Hibiscus mutabilis L was a traditional Chinese medicine with significant anti-inflammatory activity, normally applied to the clinical treatment of various inflammation-related diseases such as epidemic parotitis. ¹⁵ At present, there are few reports on the anti-inflammatory effect of H mutabilis L, and it’s basic research is weak. The present study aimed to investigate the potential anti-inflammatory effect of a new flavonoid extracted from H mutabilis L. The results revealed that the flavonoid inhibits LPS-induced IL-6 and TNF-α release and iNOS and COX-2 expressions in RAW264.7 cells, suggesting its potential as a therapeutic agent for inflammation treatment. Furthermore, our results realed that the critical mechanism by the flavonoid may exert it’s anti-inflammatory effect is through the down-regulation of NF-κB signaling pathway.

The study findings demonstrate that the lowest dose of the flavonoid exert a greater inhibitory effect to IL-6 and TNF-α vs the other 2 higher concentrations. This result indicate that the anti-inflammatory effect of the compound may be not dose-dependent. The sesult might be related to the complex mechanism by which flavonoids act on cells. First, flavonoids can affect cellular immune responses through multiple signaling pathways and contribute to the expression of downstream key immune proteins. Meanwhile, downstream key proteins may also act on cells through certain positive and negative regulatory mechanisms. Our results only demonstrate that the flavonoid can act on immunity through NF-κB signaling pathway. However, it is reported that flavonoids also can affect immunity via TLR, MAPK and other signaling pathway. ¹⁶ Second, the flavonoid may also act on other targets to affect related signaling pathway through crosstalk, thereby regulating the secretion of IL-6 and TNF-α. In addition, different types of cell lines also have different responses to the same drug. All of the above reasons may lead to a non dose-dependent response of the flavonoid to IL-6 and TNF-α in RAW264.7 cells.

Furthermore, except for IL-6, other inflammation related immune factors are significantly inhibited. And IL-6 can also be significantly inhibited at lowest dose of the flavonoid. The results suggests the flavonoid’s potential as a targeted therapeutic agent against inflammation. Due to various reasons, this article only detected key targets on limited signaling pathways, and further exploration is needed to fully elucidate the mechanism of the flavonoid’s anti-inflammatory effect.

In a word, our study revealed the potential of the flavonoid as an inflammatory therapeutic agent. Despite the interesting finding, our study has a few limitations. On the 1 hand, the study was conducted in vitro using RAW264.7 macrophages. Therefore, our study finding may not reflect anti-inflammatory effect of the flavonoid in vivo or in other cell lines. So it is necessary to further evaluate the anti-inflammatory effect using animal models or other cell lines. On the other hand, our study only analyze the effect of the flavonoid on downstream proteins in the NF-κB signaling pathway. Further mechanistic studies are needed to fully elucidate the specific targets and signaling pathways invoved.

Finally, we hope that our finding can provide reference for future studies.

Conclusion

In this study, 1 new flavonoid, named (1″R)-8-(1-(3,4-dihydroxyphenyl)ethyl)-3,3',5,7-tetrahy-droxy-4′-methoxyl flavone (1), was separated from the flowers of H mutabilis L. In addition, this article provided the in vitro anti-inflammatory activities of 1. In RAW264.7 macrophages activated by LPS (1 μg/mL), 1 treatment significantly inhibited NO production and reduced TNF-α and IL-6 release. In Elisa assay, interestingly, low concentration of 1 was more beneficial to reduce IL-6 and TNF-α concentrations. Western blot analysis demonstrated that 1 significantly inhibited iNOS and COX-2 expressions in macrophages activated by LPS in a concentration-dependent manner. Additionally, 1 suppressed the excessive phosphorylation of P65, which was key protein in the NF-κB signaling pathway, induced by LPS in a concentration-dependent manner. Our results revealed that the anti-inflammatory effect of 1 was closely related with down-regulating NF-κB signaling pathway.