Flavonoids from the leaves of Apocynum venetum and their anti-inflammatory activity

Abstract

Chemical investigation of the EtOAc-soluble extract of the leaves of Apocynum venetum allowed the isolation of seven flavonoids including a new compound named 4′-hydroxy-7-O-(4-hydroxybenzyl)-3-methoxy-6-prenylflavone (1) and six known compounds (2–7). The structures of these compounds are elucidated by spectroscopic and physico-chemical analyses. All the isolates are evaluated for in vitro anti-inflammatory activity by measuring their inhibitory activities on nitric oxide and tumor necrosis factor-α production in lipopolysaccharide-induced mouse peritoneal macrophages (RAW 264.7). Among them, 5 exhibits significant inhibitory activity toward nitric oxide and tumor necrosis factor-α production with IC₅₀ values of 9.0 ± 0.7 and 42.1 ± 0.8 μM, respectively. In addition, 1 also shows moderate inhibitory activity toward nitric oxide production with an IC₅₀ value of 17.2 ± 0.9 μM.

Introduction

Apocynum venetum L. (Apocynaceae) is a small perennial shrub that is widely distributed in the temperate regions of Europe, North America, and Asia. ¹ In China, A. venetum commonly grows in the salt marshes of the Yellow River delta, and its flowers and leaves are used as a medicine and as tea. ² As a traditional Chinese medicine, the leaves of A. venetum are used to treat neurasthenia, hypertension, nephritis, and heart disease. ³ Pharmacological studies showed that the extract of A. venetum possesses significant antioxidant, anti-hyperlipidemic, anti-hyperglycemic effect and reverses the effects of depressive-like behaviors.^4–7 Phytochemical investigations on A. venetum revealed that flavonoids were the major active constituents which exhibited various pharmacological activities including anti-hypertensive, antioxidant, anti-depressant, anti-anxiety, hepatoprotective, and cardiotonic effects.^1,2,8–10 During our studies on finding novel natural products with potent anti-inflammatory activity, we found that the EtOAc-soluble extract of the leaves of A. venetum showed moderate inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced mouse peritoneal macrophages (RAW264.7). Thus, a series of studies focused on the leaves of A. venetum were carried out. As a result, a new flavonoid named 4′-hydroxy-7-O-(4-hydroxybenzyl)-3-methoxy-6-prenylflavone (1), together with six known compounds (2–7) were isolated. Herein, we report the isolation and structural elucidation of these seven compounds. In addition, their in vitro anti-inflammatory activities were also evaluated.

Results and discussion

Structural elucidation

Compound 1 was obtained as a yellow amorphous powder, and the molecular formula was established as C₂₈H₂₆O₆ based on the [M + H]⁺ HRESIMS mass ion at m/z 459.1652 (Supplemental Figure S4). The IR spectrum showed typical absorption bands due to hydroxy and carbonyl groups at 3408 and 1732 cm⁻¹, respectively. The ¹ H NMR spectrum (Table 1, Supplemental Figure S1) revealed the presence of two sets of AA′XX′-type aromatic protons at δ_H 7.60 (2H, d, J = 8.5 Hz, H-2′/6′), 7.26 (2H, d, J = 8.5 Hz, H-3′/5′), 7.51 (2H, d, J = 8.5 Hz, H-3‴/7‴), and 7.22 (2H, d, J = 8.5 Hz, H-4‴/6‴), a pair of singlet aromatic protons at δ_H 8.04 (1H, s, H-5) and 6.63 (1H, s, H-8), a prenyl group at δ_H 3.39 (1H, d, J = 7.5 Hz, H-1″), 5.44 (1H, t, J = 7.5 Hz, H-2″), 1.67 (3H, s, H-4″), and 1.68 (3H, s, H-5″), a methoxy group at δ_H 3.78 (3H, s) and an oxygenated methylene at δ_H 5.14 (2H, s, H-1‴). The ¹³ C NMR spectrum of 1 (Supplemental Figure S2) revealed 28 carbons including a carbonyl carbon at δ_C 178.3; 18 aromatic carbons at δ_C 164.5-100.0; a pair of typical oxygenated olefinic carbons at δ_C 156.5 and 138.1; a set of prenyl carbons at δ_C 133.2, 127.5, 26.1, 28.5, and 18.0; a methoxy carbon at δ_C 56.2; and an oxygenated methylene carbon at δ_C 70.5. The key HMBC correlations of δ_H 5.14 (H-1‴) with δ_C 164.5 (C-7), 125.6 (C-2‴), and 129.5 (C-3‴/C-7‴); δ_H 7.51 (H-3‴/7‴) with δ_C 70.5 (C-1‴), 125.6 (C-2‴), 116.1 (C-4‴/6‴), and 160.3 (C-5‴); δ_H 7.22 (H-4‴/6‴) with δ_C 125.6 (C-2‴), 129.5 (C-3‴/7‴), and 160.3 (C-5‴) revealed the presence of an oxygenated 4-hydroxybenzyl moiety which linked with C-7 (Figure 1, Supplemental Figure S3). Furthermore, the locations of the prenyl and methoxy groups could be deduced by the key HMBC correlations of δ_H 3.39 (H-1″) with δ_C 123.0 (C-5) and 124.9 (C-6), and δ_H 3.78 (OCH₃) with δ_C 138.1 (C-3). Thus, the structure of compound 1 was assigned as 4′-hydroxy-7-O-(4-hydroxybenzyl)-3-methoxy-6-prenylflavone.

Table 1.

¹ H and ¹³ C NMR spectral data of compound 1 in pyridine-d₅ ( ¹ H: 500 MHz, ¹³ C: 125 MHz).

Carbon no.	1
	δH (mult, J in Hz)	δC (mult)
2	‒	156.5
3	‒	138.1
4	‒	178.3
5	8.04, s	123.0
6	‒	124.9
7	‒	164.5
8	6.63, s	100.0
9	‒	163.2
10	‒	115.1
1′	‒	122.1
2′	7.60, d (8.5)	129.1
3′	7.26, d (8.5)	116.8
4′	‒	159.8
5′	7.26, d (8.5)	116.8
6′	7.60, d (8.5)	129.1
1″	3.39, d (7.5)	28.5
2″	5.44, t (7.5)	127.5
3″	‒	133.2
4″	1.67, s	26.1
5″	1.68, s	18.0
1‴	5.14, s	70.5
2‴	‒	125.6
3‴	7.51, d (8.5)	129.5
4‴	7.22, d (8.5)	116.1
5‴	‒	160.3
6‴	7.22, d (8.5)	116.1
7‴	7.51, d (8.5)	129.5
OCH3	3.78, s	56.2

Figure 1.

Key HMBC correlations of compound 1.

Moreover, six known compounds 2–7 were also obtained from the leaves of A. venetum and identified as bavachin (2), ¹¹ chrysoeriol (3), ¹¹ 6,7-dimethoxy-4′-hydroxy-8-formylflavone (4), ¹² 4′,7-dihydroxy-8-formyl-6-methoxyflavone (5), ¹² quercetin (6), ¹³ and quercetin-3-O-β-ᴅ-glycosides (7) ¹² based on the NMR data and by comparison with literature data (Figure 2).

Figure 2.

Structures of compounds 1–7.

Analysis of the biological activity results

Compounds 1–7 were evaluated for their anti-inflammatory in vitro. Based on NO and tumor necrosis factor-α (TNF-α) are two key mediators for the pathogenesis of inflammatory diseases such as psoriasis, ulcerative colitis, and osteoarthritis, thus, the anti-inflammatory activities of 1-7 were evaluated by measuring the production of NO and TNF-α in LPS-induced RAW264.7 cells. As shown in Table 2, compounds 1 and 3–7 showed different levels of anti-inflammatory activities with IC50 values ranging from 9.0 ± 0.7 to 81.2 ± 1.2 μM. Among these compounds, 4′,7-dihydroxy-8-formyl-6-methoxyflavone (5) exhibited significant inhibitory activity toward NO and TNF-α production with IC50 values of 9.0 ± 0.7 and 42.1 ± 0.8 μM, respectively. Compound 1 also exhibited moderate inhibitory activity toward NO production with an IC₅₀ value of 17.2 ± 0.9 μM. In addition, natural product 5 showed higher anti-inflammatory activity compared with compound 4, which suggested that the hydroxy group linked at C-7 in 5 might be responsible for the increased inhibitory activity toward NO and TNF-α production. Furthermore, a comparison of the IC₅₀ data between compounds 6 and 7 indicated that the sugar moiety located at C-3 of 7 might play a negative role on the inhibitory activity toward NO production. Based on the significant inhibitory activity of compound 5, in-depth pharmacological studies on 5 should be further explored.

Table 2.

Anti-inflammatory activities of compounds 1–7.

Compound	IC50 (μM) a
	NO	TNF-α
1	17.2 ± 0.9	71.4 ± 1.7
2	>100	>100
3	81.2 ± 1.2	>100
4	23.6 ± 0.9	57.4 ± 1.2
5	9.0 ± 0.7	42.1 ± 0.8
6	31.7 ± 1.0	>100
7	52.4 ± 1.3	>100
AH b	8.3 ± 0.4	-
Silybin b	-	65.2 ± 0.6

AH: aminoguanidine hydrochloride.

a

IC₅₀ values represent the means ± SEM of three parallel measurements.

b

Positive control.

Conclusion

In this study, a new flavonoid named 4-hydroxy-7-O-(4-hydroxybenzyl)-3-methoxy-6-prenylflavone (1), together with six known compounds (2–7), was isolated from the EtOAc-soluble extract of the leaves of A. venetum. All the compounds were evaluated for their in vitro inhibitory activities toward NO and TNF-α production in mouse macrophage RAW264.7 cells. It is worth noting that compound 5 exhibited significant inhibitory activities on NO and TNF-α production with IC50 values of 9.0 ± 0.7 and 42.1 ± 0.8 μM, respectively. In addition, flavone 1 also showed moderate inhibitory activity toward NO production with an IC₅₀ value of 17.2 ± 0.9 μM. Based on the potent anti-inflammatory activity and the few phytochemical investigations of A. venetum, efforts directed toward finding novel compounds with potential anti-inflammatory activity should be intensified.

Experimental

General

UV spectra were obtained on a Hitachi U-3310 UV/vis spectrometer (Hitachi, Tokyo, Japan). IR spectra were recorded with a Nicolet Avatar 370 FTIR spectrophotometer (Nicolet, Wisconsin, USA). Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker AV-500 MHz spectrometer with tetramethylsilane (TMS) as an internal standard (Bruker, Karlsruhe, Germany). Mass spectra were obtained on a QTOF2 high-resolution mass spectrometer (Micromass, Wythenshawe, UK). Column chromatography was conducted using silica gel 60 (100 and 200 μm particle size, Yantai Xinde Chemical Co., Ltd, Yantai, China) and RP-18 (150-63 μm particle size, Merck, Darmstadt, Germany). Thin-layer chromatography (TLC) was performed with precoated silica gel GF₂₅₄ glass plates (Qingdao Marine Chemical Co., Ltd, Qingdao, China). High-performance liquid chromatography (HPLC) was carried out using a Shimadzu System LC-20AT pump equipped with a SPD-10Avp UV detector (Shimadzu, Tokyo, Japan), and a YMC ODS-A column (250 mm × 4.6 mm, 5 μm).

Plant material

The leaves of A. venetum were collected in Tengchong, Yunnan Province, P.R. China, and authenticated by Professor Qiaofeng Wu (College of Pharmacy, Zhejiang Chinese Medical University). A voucher specimen of the plant (no. 20200724) was deposited at the College of Pharmacy, Zhejiang Chinese Medical University, Zhejiang, P.R. China.

Extraction and isolation

The dried leaves of A. venetum (10.0 kg) were extracted three times with 75% MeOH under reflux, and the solution was concentrated in vacuo to yield the extract (1.8 kg). This extract was suspended in H₂O, partitioned successively with petroleum ether (PE), CH₂Cl₂, EtOAc, and n-BuOH. The EtOAc extract of A. venetum showed moderate inhibitory activity toward NO production with an IC₅₀ value of 64.3 ± 0.2 μg/mL. Thus, the EtOAc fraction (161.5 g) was subjected to silica gel column chromatography eluting with a gradient of CH₂Cl2-MeOH (from 100:0 to 0:1) to give 12 fractions (Fr.1-Fr.12). Fr.6 (13.9 g) was applied to silica gel column using a gradient of PE-EtOAc (from 100:0 to 0:1) and was separated into 12 fractions (Fr.6.1-Fr.6.12). Furthermore, Fr.6.7 (1.8 g) was subjected to the RP-18 column and eluted using MeOH-H₂O (2:8 to 1:0) to afford 15 fractions (Fr.6.7.1-Fr.6.7.15). Fr.6.7.5 (121.2 mg) was further purified by HPLC and eluted with a gradient of 55%–65% MeOH in H₂O at a flow rate of 3.0 mL/min over 70 min. This resulted in the isolation of compounds 3 (6.7 mg, t_R = 37.2 min), 6 (3.7 mg, t_R = 41.0 min), and 1 (3.8 mg, t_R = 52.7 min). Fr.6.7.7 (141.7 mg) was separated via preparative HPLC using an isocratic solvent system of 50% MeCN in H₂O to yield compounds 2 (4.8 mg, t_R = 24.8 min), 7 (5.4 mg, t_R = 35.1 min), 4 (5.7 mg, t_R = 42.8 min), and 5 (4.2 mg, t_R = 53.6 min).

4′-Hydroxy-7-O-(4-hydroxybenzyl)-3-methoxy-6-prenylflavone ( 1 ). Yellow amorphous powder; m.p.: 152–155 °C. UV (MeOH): λ_max (log ε): 224 (3.72), 270 (3.94), 305 (3.87), 361 (3.40), 366 (4.08) nm. IR (KBr disk): 3408, 3171, 2928, 1732, 1654, 1600, 1537 cm^-. ¹H (500 MHz) and ¹³ C NMR (125 MHz) spectral data in pyridine-d₅, see Table 1. HRESIMS: m/z [M + H]⁺ calcd for C₂₈H₂₆O₆: 458.1729; found: 459.1652.

Anti-inflammatory assay

The anti-inflammatory activities were tested by measuring the inhibitory activity toward NO and TNF-α production in mouse macrophage RAW264.7 cells. ¹⁴ Briefly, RAW264.7 cells were cultured to the log phase, and seeded into 24-well plates at 1 × 10⁵ cells/well and the MTT method was used to evaluate the effect of different concentrations (100, 50, 25, 12.5, 6.25, 3.125 μM) of tested samples on cell proliferation of cells in response to LPS. A Griess kit was used to measure the effect of these sample concentrations on NO secretion of RAW 264.7 cells in response to LPS and aminoguanidine hydrochloride was used as the positive control. The levels of TNF-α were determined using an ELISA (enzyme-linked immunosorbent assay) kit (Solarbio, Beijing, P.R. China) according to the manufacturer’s instructions. TNF-α was determined from a standard curve and silybin was used as the positive control. Experiments were performed at least three times.

Supplemental Material

sj-doc-1-chl-10.1177_17475198211073871 – Supplemental material for Flavonoids from the leaves of Apocynum venetum and their anti-inflammatory activity

Supplemental material, sj-doc-1-chl-10.1177_17475198211073871 for Flavonoids from the leaves of Apocynum venetum and their anti-inflammatory activity by Hong-Min Fu, Chun-Ling Yin, Zhi-Yong Shen and Ming-Hua Yang in Journal of Chemical Research