NO Release Inhibitory Activity of Flavonoids from Aesculus Wilsonii Seeds through MAPK (P38), NF-κB, and STAT3 Cross-Talk Signaling Pathways

Abstract

The flavonoid constituents of Aesculus wilsonii, a source of the Chinese medicinal drug Suo Luo Zi, and their in vitro anti-inflammatory effects were investigated. Fifteen flavonoids, including aeswilflavonosides IA–IC (1–3) and aeswilflavonosides IIA–IIE (4–8), along with seven known derivatives were isolated from a seed extract. Their structures were elucidated by extensive spectroscopic methods, acid and alkaline hydrolysis, and calculated electronic circular dichroism (ECD) spectra. Among them, compounds 3 and 7 possess a 5-[2-(carboxymethyl)-5-oxocyclopent-yl]pent-3-enylate or oleuropeoylate substituent, respectively, which are rarely reported in flavonoids. Compounds 2, 3, 7, and 1215 were found to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cell lines. In a mechanistic assay, the flavonoid glycosides 2, 3, and 7 reduced the expressions of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) induced by LPS. Further investigations suggest that 2 and 3 down-regulate the protein expression of TNF-α and IL-6 by inhibiting the phosphorylation of p38. Compound 7 was found to reduce the production of inducible nitric oxide synthase (iNOS), and the secrection of TNF-α and IL-6 through inhibiting nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) signaling pathway. Compounds 2, 3 and 7 possessed moderate inhibitory activity on the expression of signal transducer and activator of transcription 3 (STAT3). Taken together, the data indicate that the flavonoid glycosides of A. wilsonii seeds exhibit NO release inhibitory activity through mitogen-activated protein kinase (p38) [MAPK (p38)], NF-κB and STAT3 cross-talk signaling pathways.

Potential Anti-Inflammatory Constituents from Aesculus wilsonii Seeds

“…The compound underwent hydrolysis using 1 M HCl, and the resulting hydrolysate was subjected to HPLC analysis with a polarimetric detector. As a result, l -rhamnose and d -glucose were identified in it [ 11 ]. Combing with the anomeric proton signals at δ H 4.71 (1H, d, J = 1.2 Hz, H-1″) and 5.65 (1H, d, J = 8.4 Hz, H-1′), as well as the cross peaks shown in the 1 H 1 H COSY ( Figure 2 ), the existence of α- l -rhamnopyranosyl and β- d -glucopyranosyl was clarified.…”

“…439.18100 for C 18 H 31 O 12 ). Using a similar method to compound 1 , l -rhamnose and d -glucose were detected from its acid hydrolysate [ 11 ]. Compared with aeswilosides I ( 1 ) and II ( 2 ), the 1 H and 13 C NMR ( Table 3 ) spectra suggested the existence of α- l -rhamnopyranosyl(1→6)-β- d -glucopyranosyl.…”

“…After hydrolyzing compound 4 with 1 M HCl, the product was analyzed by HPLC which was combining with an optical rotation detector [ 11 ]. As a result, d -glucose was detected [ 11 ]. Its 1 H NMR (CD 3 OD, 500 MHz) ( Table 4 ) spectrum suggested the presence of one β- d -glucopyranosyl signal at δ H 4.45 (1H, d, J = 8.0 Hz, H-1′)], four methyl signals at δ H 1.12, 1.16, 1.27, and 1.60 (3H each, all s, H 3 -12, 13, 14, and 15)], three methylene signals at δ H [2.16 (1H, ddd, J = 7.5, 7.5, 15.0 Hz), 2.35 (1H, ddd, J = 3.5, 7.5, 15.0 Hz), H 2 -5], [2.05 (1H, ddd, J = 8.5, 8.5, 14.0 Hz), 2.24 (1H, ddd, J = 4.0, 8.5, 14.0 Hz), H 2 -8], and 1.36, 1.75 (1H each, both m, H 2 -9), two oxygenated methine signals at δ H 3.53 (1H, dd, J = 3.5, 9.0, H-4) and 3.25 (1H, m, overlapped, H-10)], one trisubstituted olefinic proton [δ H 5.53 (1H, dd, J = 7.5, 7.5 Hz, H-6)], and one pair of terminal olefinic protons [δ H 5.11 (1H, br.…”

“…However, the literature research results indicated that research on the anti-inflammatory activity of A. wilsonii is mostly limited to escin saponins A–D, while related studies on other components are rarely reported. In our previous study, multiple saponins [ 10 ], flavonoids [ 11 ] and nitrogenous compounds [ 9 ] were proved to be its potential anti-inflammatory active components. In this article, the study of phenolic acid glycosides, terpenes, terpenoids, and organic acids was supplemented.…”

“…( Aesculus genus, Hippocastanaceae family) is mainly used in TCM to cure liver and stomach qi stagnation, chest and abdominal distension, and stomach pain, and has been found to show various activities such as anti-inflammatory [ 5 ], anti-edema [ 6 ], anti-viral [ 7 ], and anti-tumor [ 8 ]. In our previous studies, its nitrogenous compounds [ 9 ], saponins [ 10 ], and flavonoids [ 11 ] were found to exert anti-inflammatory activity through the suppression of MAPK (P38), nuclear factor kappa-B (NF-κB), and the signal transducer and activator of transcription 3 (STAT3) cross-talk signaling pathways or the NF-κB and NLR family pyrin domain containing 3 (NLRP3) pathway. These findings suggest that A. wilsonii seeds are a good source for discovering anti-inflammatory constituents.…”