Potential Anti-Inflammatory Constituents from Aesculus wilsonii Seeds

A chemical study of Aesculus wilsonii Rehd. (also called Suo Luo Zi) and the in vitro anti-inflammatory effects of the obtained compounds was conducted. Retrieving results through SciFinder showed that there were four unreported compounds, aeswilosides I–IV (1–4), along with fourteen known isolates (5–18). Their structures were elucidated by extensive spectroscopic methods such as UV, IR, NMR, [α]D, and MS spectra, as well as acid hydrolysis. Among the known ones, compounds 5, 6, 8–10, and 12–16 were obtained from the Aesculus genus for the first time; compounds 7, 11, 17, and 18 were first identified from this plant. The NMR data of 5 and 18 were reported first. The effects of 1–18 on the release of nitric oxide (NO) from lipopolysaccharide (LPS)-induced RAW264.7 cells were determined. The results showed that at concentrations of 10, 25, and 50 μM, the novel compounds, aeswilosides I (1) and IV (4), along with the known ones, 1-(2-methylbutyryl)phloroglucinyl-glucopyranoside (10) and pisuminic acid (15), displayed significant inhibitory effects on NO production in a concentration-dependent manner. It is worth mentioning that compound 10 showed the best NO inhibitory effect with a relative NO production of 88.1%, which was close to that of the positive drug dexamethasone. The Elisa experiment suggested that compounds 1, 4, 10, and 15 suppressed the release of TNF-α and IL-1β as well. In conclusion, this study enriches the spectra of compounds with potential anti-inflammatory effects in A. wilsonii and provides new references for the discovery of anti-inflammatory lead compounds, but further mechanistic research is still needed.


Introduction
Inflammation is often associated with the development and progression of cancer, immune system disorder, Alzheimer disease, depression, etc. [1].Therefore, easing inflammation plays a crucial role in the prevention and treatment of a variety of diseases.Current anti-inflammatory drugs primarily consist of steroidal and non-steroidal agents [2].Clinical studies have demonstrated that prolonged use of steroidal anti-inflammatory drugs can result in osteoporosis in 50% of patients [3].Non-steroidal anti-inflammatory drugs also exhibit notable adverse reactions, including peptic ulcers, renal failure, and cardiovascular diseases [4].Traditional Chinese medicine (TCM) has garnered significant attention from researchers due to its remarkable efficacy and minimal side effects.
Aesculus wilsonii Rehd.(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.This study focuses on the rarely reported potential of active phenolic acids and organic acids and is a supplement to previous research.
RAW 264.7 cells are a type of mouse macrophage commonly used for studying in vitro inflammation, immunity, etc. [12].Lipopolysaccharides (LPS) can induce acute inflammatory responses, releasing inflammatory factors such as nitric oxide (NO) and interleukin [13].In recent years, the macrophage inflammation model, established by inducing RAW264.7 cells with LPS, has been widely used for the preliminary screening of anti-inflammatory activities and their pathways of action [14], with simple operation and strong universality.
Aeswiloside IV (4) was a white powder with a negative optical rotation ([α] D 25 −14.Then, the four moieties shown in bold lines in Figure 2 were determined by the cross peaks observed in its 1   2) as shown in its NOESY spectrum.The structures of the known compounds 6-17 were identified by comparing the spectroscopic data with those reported in the literature.According to the 1D and 2D NMR spectra identification, the NMR data of phenyl-O-α-L-rhamnopyranosyl(1→6)-β-Dglucopyranoside (5) were firstly assigned.Meanwhile, comparing the 1 H, 13 C NMR spectra and the [α] D 25 data with those of compound 17, the structure of 18 was elucidated to be methyl (9S,10E,12S,13S)-9,12,13-trihydroxy-10-octadecenoate; the NMR data of 18 are also first reported here.
The RAW264.7 cells, known for their robust adhesion and phagocytic capacity towards antigens, are widely employed as an in vitro research model for screening bioactive compounds with anti-inflammatory activity.After stimulation by inducers, such as LPS, it will release or cause dissonance of inflammatory factors such as NO, IL-1β, TNF-α, etc.The expression levels of these factors can serve as indicators for in vitro anti-inflammatory activity screening, reflecting the severity of inflammation and commonly used in this context.
The inhibitory effects of compounds 1-18 on NO release were investigated by using LPS-stimulated RAW264.7 cells as an in vitro screening model.Based on the results of the MTT assay (Figure S32), the bioactivity of 1-18 at the concentration of 50 µM on the release of NO from RAW264.7 cells were determined.As a result, compounds 1, 4, 10, and 15 could inhibit NO release from RAW264.7 cells in a dose-dependent manner at 10, 25, and 50 µM (Table 5; Figure 3).Furthermore, an Elisa assay was applied to investigate the inhibition of active compounds 1, 4, 10, and 15 on the IL-1β and TNF-α produced by LPS-induced RAW264.7 cells to verify their anti-inflammatory activities.It was found that all of them could significantly down-regulate the levels of IL-1β and TNF-α stimulated by LPS in RAW264.7 cells at 50 µM (Figure 4).

Discussion
In the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the "Pharmacopoeia"), Suo Luo Zi is recorded as the dried and mature seeds of A. chinensis, A. chinensis var.chekiangensis, or A. wilsonii.Among them, A. chinensis and A. chinensis var.chekiangensis have been researched more extensively.They were found to be rich in various chemical components such as escin saponins, flavonoids, organic acids, and sterols.Modern pharmacological research has shown that saponins from them showed a

Discussion
In the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the "Pharmacopoeia"), Suo Luo Zi is recorded as the dried and mature seeds of A. chinensis, A. chinensis var.chekiangensis, or A. wilsonii.Among them, A. chinensis and A. chinensis var.chekiangensis have been researched more extensively.They were found to be rich in various chemical components such as escin saponins, flavonoids, organic acids, and sterols.Modern pharmacological research has shown that saponins from them showed a variety of activities such as anti-inflammatory [6], anti-edema [7], anti-viral [8], and anti-tumor [9], which have attracted the attention of many domestic and foreign experts and scholars.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.It was found that when comparing with the other two plants, phenolic acid glycosides from the seeds of A. wilsonii owned a wide variety of sugar groups, including rhamnose and glucose [27,28], while phenolic acid glycosides A. chinensis and A. chinensis var.chekiangensis were only substituted by glucuronic acid and glucose.
The variety of compounds found in A. wilsonii indicate that multiple biosynthetic routes exist in this ancient plant, such as the acetate-malonate pathway, the mevalonic acid pathway, the cinnamic acid pathway, and the shikimic acid pathway [29][30][31].
While there is too little research on these compounds, among the active compounds found in this study, only 1-(2-methylbutyryl)phloroglucinyl-glucopyranoside (10) has been reported to exhibit potential anti-inflammatory effects, which can significantly reduce the production of inflammatory cytokine COX-1 [17].Thus, it can be seen that more in-depth mechanism research still needs to be carried out.

Conclusions
In conclusion, four undescribed isolates, aeswilosides I-IV (1-4), and fourteen known ones were obtained from the 70% EtOH extraction of A. wilsonii seeds.Among the known isolates, compounds 5, 6, 8-10, and 12-16 were isolated from the Aesculus genus for the first time; compounds 7, 11, 17, and 18 were first isolated from this plant.The NMR data of 5 and 18 were reported first.
Furthermore, the NO release inhibitory effects of all the obtained compounds 1-18 were determined, and 1, 4, 10, and 15 were found to show significant bioactivity compared with the LPS-stimulated group in RAW264.7 cells.Then, their anti-inflammatory activities were confirmed by an Elisa assay of inflammatory factors IL-1β and TNF-α.
Combining our previous research on A. wilsonii seeds, we can conclude that it contains a variety of chemical components, including saponins [10], flavonoids [11], nitrogenous compounds [9], and phenolic acids, which contribute to the plant's chemical diversity.Additionally, we have discovered that all these compounds exhibit anti-inflammatory activity, indicating that A. wilsonii seeds possess significant potential as an inflammation inhibitor for the clinical treatment of inflammatory diseases.Furthermore, the identification of bioactive compounds from A. wilsonii seeds serves as a valuable reference for the development of novel anti-inflammatory agents.

General Experimental Procedures
Optical rotations were determined using a Rudolph Autopol V automatic polarimeter.UV spectra were acquired using a Varian Cary 50 UV-Vis spectrophotometer (Varian, Inc., DE, USA).IR spectra were recorded on a Varian 640-IR FT-IR spectrophotometer (Varian, Inc.).NMR spectra were collected on Bruker Ascend 500 MHz or 600 MHz NMR spectrometers (Bruker BioSpin AG, Mass, USA).Mass spectra were obtained in the negative ion mode using a Thermo ESI-Q-Orbitrap MS spectrometer connected to an UltiMate 3000 UHPLC instrument via ESI interface (Thermo Fisher Scientific, Mass, USA).MTT and nitrite levels were determined using a BioTek Cytation 5 cell imaging multi-mode reader (BioTek, VT, USA).

Plant Material
The seeds of Aesculus wilsonii Rehd were purchased, identified, and kept, as has been reported previously [11].

Extraction and Isolation
The dried seeds of A. wilsonii (15.0 kg) were extracted under reflux for three times (3, 2, and 2 h) using 75 L, 60 L, and 60 L of a solution containing 70% EtOH, respectively.After removal of the solvent under reduced pressure, a residue weighing 2.6 kg was obtained.The residue (2.2 kg) was loaded onto a D101 resin column, and sequentially eluted with H 2 O and 95% EtOH to yield the H 2 O (717.8 g) and 95% EtOH (SA, 847.5 g) eluates.

Acid Hydrolysis of Compounds 1-4
Compounds 1-4 (1.5 mg each) dissolved in 2.0 mL 1 M HCl were heated under reflux for 3 h, respectively.The reaction products were partitioned by EtOAc (3 × 2.0 mL).The aqueous layer extracts were analyzed using HPLC with a Kaseisorb LC NH 2 -60-5 column (4.6 mm i.d.× 250 mm, Tokyo Kasei Co., Ltd., Tokyo, Japan).The mobile phase consisted of CH 3 CN-H 2 O (80:20, v/v), and the flow rate was set at 0.7 mL/min.By comparing the retention time and optical rotation with those of D-glucose and L-rhamnose standards, the presence of D-glucose (t R : 10.2 min, positive optical rotation) and L-rhamnose (t R : 7.4 min, negative optical rotation) were clarified.

Scifinder Searching
The novelty of compounds 1-4 was determined by searching their SMILES codes in SciFinder in an absolute retrieval mode.Their SMILES codes are listed as follows. Aeswiloside

Bioassays
The NO production inhibitory assay was performed as we previously reported [5].The concentrations of TNF-α and IL-1β in RAW264.7 cell culture supernatants were quantified using Elisa kits according to the manufacturer's instructions.

Supplementary Data
The Supplementary data including the NMR, HRESIMS spectra of compounds 1-4, and cell viability assay was provided in Supplementary Materials.