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

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][5][6][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][9][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 antiinflammatory activities were also evaluated.

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 antiinflammatory 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 IC 50 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 IC 50 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.

Conclusion
In this study, a new flavonoid named 4-hydroxy-7-O-(4hydroxybenzyl)-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 IC 50 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.

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

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 × 10 5 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.