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Article

Potential Anti-Inflammatory Constituents from Aesculus wilsonii Seeds

by
Ping Zhang
1,
Lequan Yu
1,
Huina Cao
1,
Jingya Ruan
2,
Fei Li
1,
Lijie Wu
1,
Yi Zhang
1,2,* and
Tao Wang
1,2,*
1
Tianjin Key Laboratory of TCM Chemistry and Analysis, Tianjin University of Traditional Chinese Medicine, 10 Poyanghu Road, West Area, Tuanbo New Town, Jinghai District, Tianjin 301617, China
2
Institute of TCM, Tianjin University of Traditional Chinese Medicine, 10 Poyanghu Road, West Area, Tuanbo New Town, Jinghai District, Tianjin 301617, China
*
Authors to whom correspondence should be addressed.
Molecules 2024, 29(5), 1136; https://doi.org/10.3390/molecules29051136
Submission received: 9 January 2024 / Revised: 21 February 2024 / Accepted: 28 February 2024 / Published: 3 March 2024
(This article belongs to the Special Issue Anticancer, Antioxidant, and Anti-inflammatory Activities of Natural Products)
Browse Figures
Versions Notes

Abstract

:
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.

1. 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.
In the ongoing search for these kinds of bioactive components, many chromatographic materials, such as D101 resin, silica gel, ODS, MCI gel CHP 20P, and Sephadex LH-20 column chromatographies (CC), as well as preparative high-performance liquid chromatography (pHPLC), were used to fractionate and purify the compounds from the 70% ethanol extraction of A. wilsonii seeds. Various spectroscopic techniques, including UV, IR, NMR, [α]D, and MS spectra, were applied to identify the structure of the isolates. As results, eighteen compounds were identified from it. Retrieving results using SciFinder showed that there were four unreported compounds, named as aeswilosides I–IV (1–4), and fourteen known ones, including phenyl-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (5), benzyl-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (6) [15], 2-phenethyl-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (7) [15], asechipuroside A (8) [16], 1-[(2-methylpropanoyl)phloroglucinyl]-β-d-glucopyranoside (9) [17], 1-(2-methylbutyryl)phloroglucinyl-glucopyranoside (10) [18], (–)-pinoresinol 4-O-β-d-glucoside (11) [19,20], 3-O-[α-l-arabinopyranosyl(1→6)-β-d-glucopyranosyl]oct-1-ene-3-ol (12) [21], myrseguinoside A (13) [22], lippianoside E (14) [23], pisuminic acid (15) [24], fulgidic acid (16) [25], (9S,10E,12S,13S)-9,12,13-trihydroxy-10-octadecenoic acid (17) [26], and methyl (9S,10E,12S,13S)-9,12,13-trihydroxy-10-octadecenoate (18) (Figure 1).
Additionally, an LPS-induced RAW264.7 macrophage model was established to assess the anti-inflammatory efficacy of the identified compounds through a quantification of NO and interleukin (IL)-1β levels in RAW264.7 cells.

2. Results

Aeswiloside I (1) was obtained as a white powder. The molecular formula of it was assigned as C20H28O13 from the HRESIMS at m/z 475.14618 [M − H]− (calcd. for C20H27O13, 475.14461). Its IR spectrum presented the characteristic absorptions for hydroxyl (3362 cm−1), unsaturated ester carbonyl (1703 cm−1), aromatic ring (1599, 1519, 1457 cm−1), and ether bond (1067 cm−1). 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 δH 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 1H 1H COSY (Figure 2), the existence of α-l-rhamnopyranosyl and β-d-glucopyranosyl was clarified. In addition, the signals for methoxyl at δH 3.91 (3H, s, 3-OCH3), three aromatic protons with ABX spin coupling systems at δH 6.86 (1H, d, J = 8.4 Hz, H-5), 7.62 (1H, d, J = 1.8 Hz, H-2), and 7.65 (1H, dd, J = 1.8, 8.4 Hz, H-6)] were detected by its 1H NMR spectrum. And the 13C NMR (Table 1) spectrum presented one unsaturated ester carbonyl at δC 166.7 (C-7).
According to the HMBC correlations from δH 7.62 (H-2), 7.65 (H-6) to δC 166.7 (C-7); δH 3.91 (3-OCH3) to δC 148.8 (C-3); δH 7.62 (H-2) to δC 125.8 (C-6), 153.4 (C-4); δH 6.86 (H-5) to δC 121.8 (C-1), 148.8 (C-3); and δH 7.65 (H-6) to δC 114.0 (C-2) 153.4 (C-4) (Figure 2), the aglycon of aeswiloside I (1) was elucidated to be 3-methoxy-4-hydroxybenzoic acid. Finally, the linkage location between glycosyl and aglycon, as well as between glycosyls, were determined by the long-range correlations from δH 5.65 (H-1′) to δC 166.7 (C-7) and δH 4.71 (H-1″) to δC 67.8 (C-6′) (Figure 2).
Aeswiloside II (2) was obtained as a white powder with the molecular formula, C20H30O12, gained from the HR-ESI-MS at m/z 461.16696 [M − H]− (calcd. 461.16535 for C20H29O12). The 1H and 13C NMR (Table 2) spectra indicated that it had the same groups including one methoxyl, one benzene ring with an ABX spin coupling system, one α-l-rhamnopyranosyl, and one β-d-glucopyranosyl as compound 1. Compared with 1, the signal for ester carbonyl disappeared, while the signals at δH 4.54, 4.77 (1H each, both d, J = 11.5 Hz, H2-7] for methtylene appeared. Then, the structure of aeswiloside II (2) was elucidated according to the HMBC correlations from δH 6.82 (H-6), 7.03 (H-2) to δC 71.2 (C-7); δH 4.29 (H-1′) to δC 71.2 (C-7); and δH 4.79 (H-1″) to δC 68.2 (C-6′) (Figure 2).
Aeswiloside III (3) was a white powder. HR-ESI-MS analysis proved its molecular formula to be C17H30O10 through the presence of a peak at m/z 439.18240 [M + COOH]− (calcd. 439.18100 for C18H31O12). 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 1H and 13C NMR (Table 3) spectra suggested the existence of α-l-rhamnopyranosyl(1→6)-β-d-glucopyranosyl. Moreover, 1H, 13C NMR and HSQC spectra indicated the existences of two methyls [δH 1.76, 1.70 (3H each, both s, H3-4, 5)], one oxygenated methylene [δH 4.21, 4.25 (1H each, both dd, J = 7.8, 11.4 Hz, H2-1)], and one trisubstituted olefin group [δH 5.36 (1H, m, H-2)].
In its HMBC spectrum, the long-range correlations from δH 4.21, 4.25 (H2-1) to δC 139.0 (C-3); δH 5.36 (H-2) to δC 18.2 (C-5), 26.0 (C-4); δH 1.76 (H3-4) to δC 18.2 (C-5), 121.5 (C-2), 139.0 (C-3); and δH 1.70 (H3-5) to δC 26.0 (C-4), 121.5 (C-2), 139.0 (C-3) were displayed (Figure 2), which indicated that its aglycon was 3-methyl-2-butene-1-ol. Finally, the linkage positions between the above-mentioned moieties were elucidated according to the correlations from δH 4.26 (H-1′) to δC 66.1 (C-1) and δH 4.75 (H-1″) to δC 68.1 (C-6′).
Aeswiloside IV (4) was a white powder with a negative optical rotation ([α]D25 −14.1, MeOH). The molecular formula, C21H38O9 (m/z 479.25037 [M + COOH]−; calcd. for C22H39O11, 479.24869) of it was established on negative-ion HRESIMS analysis. 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 1H NMR (CD3OD, 500 MHz) (Table 4) spectrum suggested the presence of one β-d-glucopyranosyl signal at δH 4.45 (1H, d, J = 8.0 Hz, H-1′)], four methyl signals at δH 1.12, 1.16, 1.27, and 1.60 (3H each, all s, H3-12, 13, 14, and 15)], three methylene signals at δH [2.16 (1H, ddd, J = 7.5, 7.5, 15.0 Hz), 2.35 (1H, ddd, J = 3.5, 7.5, 15.0 Hz), H2-5], [2.05 (1H, ddd, J = 8.5, 8.5, 14.0 Hz), 2.24 (1H, ddd, J = 4.0, 8.5, 14.0 Hz), H2-8], and 1.36, 1.75 (1H each, both m, H2-9), two oxygenated methine signals at δH 3.53 (1H, dd, J = 3.5, 9.0, H-4) and 3.25 (1H, m, overlapped, H-10)], one trisubstituted olefinic proton [δH 5.53 (1H, dd, J = 7.5, 7.5 Hz, H-6)], and one pair of terminal olefinic protons [δH 5.11 (1H, br. d, ca. J = 11 Hz), 5.26 (1H, br. d, ca. J = 17 Hz), H2-1]. Then, the four moieties shown in bold lines in Figure 2 were determined by the cross peaks observed in its 1H 1H COSY spectrum. The planar structure of aeswiloside IV (4) was clarified by the HMBC correlations from δH 6.05 (H-2) to δC 24.5 (C-14), 77.2 (C-3), 89.9 (C-4); δH 3.53 (H-4) to δC 24.5 (C-14), 142.8 (C-2); δH 2.16, 2.35 (H2-5) to δC 77.2 (C-3), 136.3 (C-7); δH 5.53 (H-6) to δC 16.5 (C-15), 37.9 (C-8), 136.3 (C-7); δH 2.05, 2.24 (H2-8) to δC 16.5 (C-15), 123.7 (C-6), 136.3 (C-7); δH 1.36, 1.75 (H2-9) to δC 73.8 (C-11), 136.3 (C-7); δH 3.25 (H-10) to δC 24.9 (C-12), 25.9 (C-13), 73.8 (C-11); δH 1.12 (H3-12) to δC 25.9 (C-13), 73.8 (C-11), 78.9 (C-10); δH 1.16 (H3-13) to δC 24.9 (C-12), 73.8 (C-11), 78.9 (C-10); δH 1.27 (H3-14) to δC 77.2 (C-3), 89.9 (C-4), 142.8 (C-2); δH 1.60 (H3-15) to δC 37.9 (C-8), 123.7 (C-6), 136.3 (C-7); and δH 4.45 (H-1′) to δC 89.9 (C-4) (Figure 2), which was a sesquiterpene glycoside. Finally, Δ6 was elucidated to be an E configuration according to the NOE correlations between δH 2.16, 2.35 (H2-5) and δH 1.60 (H3-15) and δH 5.53 (H-6) and δH 2.05, 2.24 (H2-8) (Figure 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-d-glucopyranoside (5) were firstly assigned. Meanwhile, comparing the 1H, 13C NMR spectra and the [α]D25 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).

3. 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.

4. 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.

5. Experimental Section

5.1. 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).
Macroporous resin D101 (Haiguang Chemical Co., Ltd.), silica gel (48–75 μm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, CN), YMC × Gel ODS-A-HG (50 μm, AAG12S50, YMC Co., Ltd., Kyoto, JPN), and Sephadex LH-20 (Ge Healthcare Bio-Sciences, DE, USA) were used for column chromatography (CC). Analytical HPLC was performed on Cosmosil 5C18-MS-II (4.6 mm i.d. × 250 mm, 5 μm, Nakalai Tesque, Inc., Kyoto, JPN) and Cosmosil PBr (4.6 mm i.d. × 250 mm, 5 μm, Nakalai Tesque, Inc.) columns. Cosmosil 5C18-MS-II (20 mm i.d. × 250 mm, 5 μm, Nakalai Tesque, Inc.) and Cosmosil PBr (20 mm i.d. × 250 mm, 5 μm, Nakalai Tesque, Inc.) columns were used for preparative isolation. All reagents used for phytochemical investigation were analytical grade (Concord Technology Co. Ltd., Tianjin, CN).
The RAW264.7 cell lines (Chinese Academy of Medical Science, Beijing, CN), Dulbecco’s modified eagle medium (DMEM) (Biological Industries, Kibbutz Beit Haemek, Israel) and 10% (v/v) fetal bovine serum (FBS) (Biological Industries, Israel), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), LPS (Sigma-Aldrich, St. Louis, MO, USA), thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA), Mouse TNF-α ELISA kits (Biotechnology Co., Ltd., Shanghai, China), and IL-1β ELISA kits (Biotechnology Co., Ltd., Shanghai, China) were used for bioassay.

5.2. Plant Material

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

5.3. 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 H2O and 95% EtOH to yield the H2O (717.8 g) and 95% EtOH (SA, 847.5 g) eluates.
SA (400.0 g) was fractionated by silica gel CC [CH2Cl2-MeOH (100:0 → 100:1 → 100:3 → 100:7 → 10:1 → 8:1 → 7:1 → 5:1 → 3:1 → 1:1 → 0:100, v/v)] to produce SA1–SA14. SA8 (3.2 g) was further subjected to silica gel CC [CH2Cl2-MeOH (100:1 → 100:5 → 100:7 → 10:1 → 0:100, v/v)], and SA8-1–SA8-7 were given. SA8-3 (450.0 mg) was separated by pHPLC [MeOH-1% HAc (78:22, v/v), Cosmosil 5C18-MS-II column] to yield methyl (9S,10E,12S,13S)-9,12,13-trihydroxy-10-octadecenoate (18, 97.2 mg, tR 16.7 min). SA8-5 (450.0 mg) was purified by pHPLC [MeOH-1% HAc (68:32, v/v), Cosmosil 5C18-MS-II column] to gain fulgidic acid (16, 22.0 mg, tR 14.1 min) and (9S,10E,12S,13S)-9,12,13-trihydroxy-10-octadecenoic acid (17, 180.2 mg, tR 16.0 min). SA9 (5.0 g) was loaded onto ODS CC [MeOH-H2O (20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v)] to give SA9-1–SA 9-14. SA9-6 (120.0 mg) was purified by pHPLC [MeOH-1% HAc (60:40, v/v), Cosmosil PBr column] to produce 1-[(2-methylpropanoyl)phloroglucinyl]-β-d-glucopyranoside (9, 8.8 mg, tR 17.0 min). SA9-7 (350.0 mg) was isolated by pHPLC [MeOH-1% HAc (40:60, v/v), Cosmosil 5C18-MS-II column] to gain SA9-7-1–SA9-7-5. Among them, SA9-7-5 (25.0 mg) was elucidated to be myrseguinoside A (13, 25.0 mg, tR 31.3 min). SA9-7-2 (69.8 mg) was separated by pHPLC [CH3CN-1% HAc (20:80, v/v), Cosmosil 5C18-MS-II column], and (–)-pinoresinol 4-O-β-d-glucoside) (11, 30.3 mg, tR 26.6 min) was yielded. SA9-9 (1000.0 mg) was purified by pHPLC with MeOH-1% HAc (65:35, v/v, Cosmosil PBr column) and CH3CN-1% HAc (31:69, v/v, Cosmosil 5C18-MS-II column) to obtain 1-(2-methylbutyryl)phloroglucinyl-glucopyranoside (10, 13.5 mg, tR 17.6 min). SA10 (4.8 g) was fractionated by ODS CC [MeOH-H2O (20:80 → 40:60 → 45:55 → 50:50 → 55:45 → 60:40 → 100:0, v/v)] to yield SA10-1–SA10-13. SA10-3 (1000.0 mg) was subjected to pHPLC [MeOH-1% HAc (30:70, v/v), Cosmosil 5C18-MS-II column] to obtain SA10-3-1–SA10-3-11. SA10-3-3 (18.5 mg) was further purified by pHPLC [CH3CN-1% HAc (14:86, v/v), Cosmosil 5C18-MS-II column] to give lippianoside E (14, 9.6 mg, tR 23.8 min). SA10-3-10 (35.3 mg) was separated by pHPLC [CH3CN-1% HAc (16:84, v/v), Cosmosil 5C18-MS-II column], to produce 2-phenethyl-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (7, 13.7 mg, tR 30.7 min). SA10-5 (500.0 mg) was loaded onto pHPLC [CH3CN-1% HAc (20:80, v/v), Cosmosil 5C18-MS-II column] to obtain SA10-5-1–SA10-5-10. SA10-5-4 (11.5 mg) and SA10-5-6 (34.7 mg) were purified by pHPLC [MeOH-1% HAc (43:57, v/v), Cosmosil 5C18-MS-II column] to gain aeswiloside IV (4, 3.0 mg, tR 41.6 min) and pisuminic acid (15, 12.7 mg, tR 47.1 min), respectively. SA 10-6 (100.0 mg) was isolated by pHPLC [CH3CN-1% HAc (23:77, v/v), Cosmosil 5C18-MS-II column] to produce 3-O-[α-l-arabinopyranosyl(1→6)-β-d-glucopyranosyl]oct-1-ene-3-ol (12, 9.2 mg, tR 31.3 min). SA11 (30.0 g) was subjected to MCI gel CHP 20P CC [MeOH-H2O (20:80 → 30:70 → 40:60 → 50:50 → 60:40 → 70:30 → 80:20 → 100:0, v/v)] to obtain SA11-1–SA11-19. SA11-4 (200.0 mg) was separated by pHPLC [MeOH-1% HAc (31:69, v/v), Cosmosil 5C18-MS-II column] to produce aeswiloside I (1, 16.0 mg, tR 11.4 min), asechipuroside A (8, 12.8 mg, tR 14.5 min), phenyl-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (5, 12.9 mg, tR 16.8 min), and aeswiloside III (3, 9.1 mg, tR 25.6 min). SA11-5 (150.0 mg) was purified by pHPLC [CH3CN-1% HAc (11:89, v/v), Cosmosil 5C18-MS-II column] to obtain aeswiloside II (2, 10.1 mg, tR 23.2 min). SA11-8 (300.0 mg) was purified by pHPLC [CH3CN-1% HAc (13:87, v/v), Cosmosil 5C18-MS-II column] to give benzyl-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (6, 22.7 mg, tR 31.7 min).

5.4. Spectral Data of 1–18

5.4.1. Aeswiloside I (1)

White powder; [α]D25 −36.4 (conc. 0.044, MeOH); UV λmax (MeOH) nm (log ε): 221 (4.29), 264 (4.02), 296 (3.77); IR νmax (KBr) cm−1: 3362, 2926, 1703, 1599, 1519, 1457, 1285, 1217, 1127, 1067; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data: see Table 1; ESI-Q-Orbitrap MS: m/z 475.14618 [M − H]− (calcd. for C20H27O13, 475.14461).

5.4.2. Aeswiloside II (2)

White powder; [α]D25 −57.1 (conc. 0.035, MeOH); UV λmax (MeOH) nm (log ε): 231 (3.67), 282 (3.23); IR νmax (KBr) cm−1: 3368, 2927, 1703, 1606, 1560, 1518, 1455, 1277, 1161, 1126, 1067, 1046; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data: see Table 2; ESI-Q-Orbitrap MS: m/z 461.16696 [M − H]− (calcd. for C20H29O12, 461.16535).

5.4.3. Aeswiloside III (3)

White powder; [α]D25 −50.0 (conc. 0.032, MeOH); UV λmax (MeOH) nm (log ε): 284 (2.96); IR νmax (KBr) cm−1: 3363, 2924, 1658, 1067, 1048; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) data: see Table 3; ESI-Q-Orbitrap MS: m/z 439.18240 [M + COOH]− (calcd for C18H31O12, 439.18100).

5.4.4. Aeswiloside IV (4)

White powder; [α]D25 −14.1 (conc. 0.043, MeOH); IR νmax (KBr) cm−1: 3352, 2973, 2926, 2849, 1450, 1164, 1077, 1017, 923; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data: see Table 4; ESI-Q-Orbitrap MS: m/z 479.25037 [M + COOH]− (calcd. for C22H39O11, 479.24869).

5.4.5. Phenyl-O-α-l-Rhamnopyranosyl(1→6)-β-d-Glucopyranoside (5)

White powder; 1H NMR (CD3OD, 600 MHz) δH: 7.08 (2H, dd, J = 1.2, 7.2 Hz, H-2,6), 7.29 (2H, dt, J = 1.2, 7.2 Hz, H-3,5), 7.00 (1H, tt, J = 1.2, 7.2 Hz, H-4), 4.85 (1H, d, J = 8.4 Hz, H-1′), 3.46 (1H, dd, J = 8.4, 9.0 Hz, H-2′), 3.44 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 3.37 (1H, dd, J = 9.6, 9.6 Hz, H-4′), 3.55 (1H, m, H-5′), [3.60 (1H, dd, J = 6.6, 11.4 Hz), 4.02 (1H, dd, J = 1.8, 11.4 Hz), H2-6′], 4.70 (1H, d, J = 1.8 Hz, H-1″), 3.83 (1H, dd, J = 1.8, 3.0 Hz, H-2″), 3.70 (1H, dd, J = 3.0, 9.6 Hz, H-3″), 3.36 (1H, dd, J = 9.6, 9.6 Hz, H-4″), 3.65 (1H, m, H-5″), 1.20 (3H, d, J = 6.0 Hz, H3-6″); 13C NMR (CD3OD, 150 MHz) δC: 159.1 (C-1), 117.9 (C-2,6), 130.5 (C-3,5), 123.5 (C-4), 102.4 (C-1′), 75.0 (C-2′), 78.1 (C-3′), 71.6 (C-4′), 77.0 (C-5′), 67.9 (C-6′), 102.2 (C-1″), 72.2 (C-2″), 72.5 (C-3″), 74.1 (C-4″), 69.9 (C-5″), 18.0 (C-6″); ESI-Q-Orbitrap MS: m/z 447.15134 [M + COOH]− (calcd. for C19H27O12, 447.14970).

5.4.6. Benzyl-O-α-l-Rhamnopyranosyl(1→6)-β-d-Glucopyranoside (6)

White powder; 1H NMR (CD3OD, 600 MHz) δH: 7.41 (2H, br. d, ca. J = 7 Hz, H-2,6), 7.33 (2H, t like, ca. J = 7 Hz, H-3,5), 7.27 (1H, t like, J = 7 Hz, H-4), [4.64 (1H, d, J = 11.4 Hz), 4.88 (1H, d, J = 11.4 Hz), H2-7], 4.32 (1H, d, J = 7.8 Hz, H-1′), 3.24 (1H, dd, J = 7.8, 9.0 Hz, H-2′), 3.34 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 3.29 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.38 (1H, m, H-5′), [3.64 (1H, dd, J = 6.0, 11.4 Hz), 4.00 (1H, dd, J = 1.8, 10.8 Hz), H2-6′], 4.78 (1H, d, J = 1.2 Hz, H-1″), 3.87 (1H, dd, J = 1.2, 3.6 Hz, H-2″), 3.70 (1H, dd, J = 3.6, 9.6 Hz, H-3″), 3.38 (1H, J = 9.6, 9.6 Hz, H-4″), 3.69 (1H, m, H-5″), 1.27 (3H, d, J = 6.0 Hz, H3-6″); 13C NMR (CD3OD, 150 MHz) δC: 138.9 (C-1), 129.4 (C-2,6, 3,5), 128.8 (C-4), 71.8 (C-7), 103.2 (C-1′), 75.1 (C-2′), 78.1 (C-3′), 71.8 (C-4′), 77.0 (C-5′), 68.2 (C-6′), 102.3 (C-1″), 72.3 (C-2″), 72.4 (C-3″), 74.1 (C-4″), 69.9 (C-5″), 18.1 (C-6″); ESI-Q-Orbitrap MS: m/z 461.16733 [M + COOH]− (calcd. For C20H29O12, 461.16535).

5.4.7. 2-Phenethyl-O-α-l-Rhamnopyranosyl(1→6)-β-d-Glucopyranoside (7)

White powder; 1H NMR (DMSO-d6, 500 MHz) δH: 7.27 (2H, m, H-2,6), 7.28 (2H, m, H-3,5), 7.27 (1H, t like, ca. J = 8 Hz, H-4), 2.86 (2H, t, J = 7.0 Hz, H-7), [3.68 (1H, q like, ca. J = 8 Hz), 3.89 (1H, q like, ca. J = 8 Hz), H2-8], 4.19 (1H, d, J = 7.5 Hz, H-1′), 2.96 (1H, dd, J = 7.5, 8.5 Hz, H-2′), 3.14 (1H, dd, J = 8.5, 9.0 Hz, H-3′), 3.00 (1H, dd, J = 9.0, 9.0 Hz, H-4′), 3.27 (1H, m, H-5′), [3.43 (1H, dd, J = 6.5, 11.0 Hz), 3.82 (1H, br. D, ca. J = 11 Hz), H2-6′], 4.60 (1H, br. S, H-1″), 3.61 (1H, br. D, ca. J = 4 Hz, H-2″), 3.42 (1H, dd, J = 3.5, 9.5 Hz, H-3″), 3.17 (1H, dd, J = 9.5, 9.5 Hz, H-4″), 3.45 (1H, m, H-5″), 1.13 (3H, d, J = 6.5 Hz, H3-6″); 13C NMR (DMSO-d6, 125 MHz) δC: 138.6 (C-1), 128.8 (C-2,6), 128.1 (C-3,5), 125.9 (C-4), 35.6 (C-7), 69.4 (C-8), 102.8 (C-1′), 73.2 (C-2′), 76.5 (C-3′), 70.1 (C-4′), 75.3 (C-5′), 66.9 (C-6′), 100.7 (C-1″), 70.4 (C-2″), 70.6 (C-3″), 71.8 (C-4″), 68.2 (C-5″), 17.8 (C-6″); ESI-Q-Orbitrap MS: m/z 429.17685 [M − H]− (calcd. for C20H29O10, 429.17552).

5.4.8. Asechipuroside A (8)

White powder; 1H NMR (CD3OD, 600 MHz) δH: 7.06 (2H, d, J = 8.0 Hz, H-2,6), 6.69 (2H, d, J = 8.0 Hz, H-3,5), 2.83 (2H, m, H2-7), [3.68 (1H, m), 3.96 (1H, m), H2-8], 4.28 (1H, d, J = 7.8 Hz, H-1′), 3.17 (1H, dd, J = 7.8, 9.0 Hz, H-2′), 3.33 (1H, dd, J = 9.0, 9.0 Hz, H-3′), 3.27 (1H, dd, J = 9.0, 9.6 Hz, H-4′), 3.37 (1H, m, H-5′), [3.61 (1H, dd, J = 6.0, 11.4 Hz), 3.97 (1H, m), H2-6′], 4.74 (1H, d, J = 1.8 Hz, H-1″), 3.83 (1H, dd, J = 1.8, 3.6 Hz, H-2″), 3.67 (1H, dd, J = 3.6, 9.0 Hz, H-3″), 3.36 (1H, dd, J = 9.0, 9.0 Hz, H-4″), 3.66 (1H, m, H-5″), 1.25 (3H, d, J = 6.0 Hz, H3-6″); 13C NMR (CD3OD, 150 MHz) δC: 130.7 (C-1), 131.0 (C-2,6), 116.2 (C-3,5), 156.8 (C-4), 36.5 (C-7), 72.3 (C-8), 104.5 (C-1′), 75.1 (C-2′), 78.1 (C-3′), 71.7 (C-4′), 76.9 (C-5′), 68.1 (C-6′), 102.3 (C-1″), 72.2 (C-2″), 72.4 (C-3″), 74.1 (C-4″), 69.8 (C-5″), 18.1 (C-6″); ESI-Q-Orbitrap MS: m/z 445.17215 [M − H]− (calcd. For C20H29O11, 445.17044).

5.4.9. 1-[(2-Methylpropanoyl)Phloroglucinyl]-β-d-Glucopyranoside (9)

White powder; 1H NMR (CD3OD, 600 MHz) δH: 5.95 (1H, d, J = 2.4 Hz, H-4), 6.18 (1H, d, J = 2.4 Hz, H-6), 3.99 (1H, sept, J = 6.6 Hz, H-2′), 1.15 (3H, d, J = 6.6 Hz, H3-3′), 1.13 (3H, d, J = 6.6 Hz, H3-4′), 5.04 (1H, d, J = 7.8 Hz, H-1″), 3.50 (1H, dd, J = 7.8, 9.0 Hz, H-2″), 3.47 (1H, dd, J = 9.0, 9.0 Hz, H-3″), 3.39 (1H, dd, J = 9.0, 9.6 Hz, H-4″), 3.46 (1H, m, H-5″), [3.72 (1H, dd, J = 6.0, 12.0 Hz), 3.91 (1H, dd, J = 1.8, 12.0 Hz), H2-6″]; 13C NMR (CD3OD, 150 MHz) δC: 161.7 (C-1), 106.3 (C-2), 167.5 (C-3), 98.3 (C-4), 165.6 (C-5), 95.4 (C-6), 212.0 (C-1′), 40.5 (C-2′), 20.3 (C-3′), 19.5 (C-4′), 101.6 (C-1″), 74.9 (C-2″), 78.7 (C-3″), 71.2 (C-4″), 78.4 (C-5″), 62.5 (C-6″); ESI-Q-Orbitrap MS: m/z 357.11917 [M − H]− (calcd. for C16H21O9 357.11801).

5.4.10. 1-(2-Methylbutyryl)Phloroglucinyl-Glucopyranoside (10)

White powder; CD (conc. 0.0027 M, MeOH) mdeg (λnm): +0.98 (271), −4.20 (304); 1H NMR (CD3OD, 600 MHz) δH: 5.96 (1H, d, J = 2.4 Hz, H-4), 6.19 (1H, d, J = 2.4 Hz, H-6), 3.90 (1H, sex, J = 6.6 Hz, H-2′), 1.38, 1.80 (1H each, both m, H2-3′), 0.89 (3H, t, J = 7.8 Hz, H3-4′), 1.13 (3H, d, J = 6.6 Hz, H3-5′), 5.03 (1H, d, J = 7.2 Hz, H-1″), 3.52 (1H, dd, J = 7.2, 9.0 Hz, H-2″), 3.46 (1H, dd, J = 8.4, 9.0 Hz, H-3″), 3.39 (1H, dd, J = 8.4, 9.0 Hz, H-4″), 3.46 (1H, m, H-5″), [3.71 (1H, dd, J = 5.4, 12.0 Hz), 3.92 (1H, dd, J = 1.8, 12.0 Hz), H2-6″]; 13C NMR (CD3OD, 150 MHz) δC: 161.8 (C-1), 106.9 (C-2), 167.5 (C-3), 98.4 (C-4), 165.6 (C-5), 95.4 (C-6), 211.8 (C-1′), 47.0 (C-2′), 28.3 (C-3′), 12.1 (C-4′), 16.9 (C-5′), 101.7 (C-1″), 74.8 (C-2″), 78.8 (C-3″), 71.3 (C-4″), 78.5 (C-5″), 62.5 (C-6″); ESI-Q-Orbitrap MS: m/z 371.13489 [M − H]− (calcd. For C17H23O9, 371.13365).

5.4.11. (−)-Pinoresinol 4-O-β-d-Glucoside (11)

White powder; CD (conc. 0.0001 M, MeOH) mdeg (λnm): −11.92 (200), −0.94 (229); 1H NMR (CD3OD, 500 MHz) δH: 7.01 (1H, d, J = 1.5 Hz, H-2), 7.13 (1H, d, J = 8.5 Hz, H-5), 6.89 (1H, dd, J = 1.5, 8.5 Hz, H-6), 4.73 (1H, d, J = 3.5 Hz, H-7), 3.09 (1H, m, H-8), 3.84, 4.21 (1H each, both m, H2-9), 6.93 (1H, d, J = 1.5 Hz, H-2′), 6.76 (1H, d, J = 8.5 Hz, H-5′), 6.78 (1H, dd, J = 1.5, 8.5 Hz, H-6′), 4.68 (1H, d, J = 4.5 Hz, H-7′), 3.09 (1H, m, H-8′), 3.84, 4.21 (1H each, both m, H2-9′), 4.87 (1H, d, J = 7.0 Hz, H-1″), 3.49 (1H, dd, J = 7.0, 9.5 Hz, H-2″), 3.47 (1H, dd, J = 9.5, 9.5 Hz, H-3″), 3.39 (1H, m, overlapped, H-4″), 3.39 (1H, m, overlapped, H-5″), [3.68 (1H, dd, J = 4.5, 11.0 Hz), 3.86 (1H, br. D, ca. J = 11 Hz), H2-6″], 3.85, 3.84 (3H each, both s, 3, 3′-OCH3); 13C NMR (CD3OD, 125 MHz) δC: 137.5 (C-1), 111.7 (C-2), 151.0 (C-3), 147.5 (C-4), 118.0 (C-5), 119.8 (C-6), 87.1 (C-7), 55.5 (C-8), 72.7 (C-9), 133.8 (C-1′), 111.0 (C-2′), 149.1 (C-3′), 147.3 (C-4′), 116.1 (C-5′), 120.1 (C-6′), 87.5 (C-7′), 55.3 (C-8′), 72.7 (C-9′), 102.8 (C-1″), 74.9 (C-2″), 77.8 (C-3″), 71.4 (C-4″), 78.2 (C-5″), 62.5 (C-6″), 56.8 (3-OCH3), 56.5 (3′-OCH3); ESI-Q-Orbitrap MS: m/z 519.18768 [M − H]− (calcd. For C26H31O11, 520.19391).

5.4.12. 3-O-[α-l-Arabinopyranosyl(1→6)-β-d-Glucopyranosyl]oct-1-ene-3-ol (12)

Colorless oil; [α]D25 −23.5 (conc. 0.026, MeOH); 1H NMR (C5D5N, 600 MHz) δH: [5.20 (1H, d, J = 10.8 Hz), 5.44 (1H, d, J = 16.8 Hz), H2-1], 6.13 (1H, m, H-2), 4.48 (1H, m, H-3), 1.64, 1.77 (1H each, both m, H2-4), 1.46 (2H, m, H2-5), 1.18 (4H, m, H2-6 and 7), 4.90 (1H, d, J = 7.8 Hz, H-1′), 4.00 (1H, dd, J = 7.8, 8.4 Hz, H-2′), 4.20 (1H, dd, J = 8.4, 9.0 Hz, H-3′), 4.14 (1H, dd, J = 9.0, 9.6 Hz, H-4′), 4.05 (1H, m, H-5′), [4.30 (1H, dd, J = 6.0, 12.0 Hz), 4.77 (1H, br. d, ca. J = 12 Hz), H2-6′], 4.99 (1H, d, J = 6.0 Hz, H-1″), 4.46 (1H, dd, J = 6.0, 7.8 Hz, H-2″), 4.19 (1H, dd, J = 7.8, 9.0 Hz, H-3″), 4.34 (1H, m, H-4″), [3.76 (1H, br. d, ca. J = 12.0 Hz), 4.31 (1H, dd, J = 5.4, 12.0 Hz), H2-5″]; 13C NMR (C5D5N, 150 MHz) δC: 115.3 (C-1), 140.8 (C-2), 80.9 (C-3), 35.1 (C-4), 24.8 (C-5), 32.2 (C-6), 22.8 (C-7), 14.2 (C-8), 103.6 (C-1′), 75.3 (C-2′), 78.6 (C-3′), 71.9 (C-4′), 77.1 (C-5′), 69.5 (C-6′), 105.3 (C-1″), 72.4 (C-2″), 74.4 (C-3″), 69.1 (C-4″), 66.4 (C-5″); ESI-Q-Orbitrap MS: m/z 467.21439 [M + COOH]− (calcd. for C20H35O12, 467.21230).

5.4.13. Myrseguinoside A (13)

White powder; 1H NMR (CD3OD, 500 MHz) δH: 5.63 (1H, br. d, ca. J = 5 Hz, H-2), 1.77, 2.13 (1H each, both m, H2-3), 1.88 (1H, m, H-4), [1.25 (1H, dt like, ca. J = 4, 14 Hz), 2.22 (1H, m), H2-5], 1.79 (3H, br. s, H3-7), 1.20 (3H, s, H3-9), 1.16 (3H, s, H3-10), 4.37 (1H, d, J = 8.0 Hz, H-1′), 3.19 (1H, dd, J = 8.0, 8.5 Hz, H-2′), 3.39 (1H, dd, J = 8.5, 8.5 Hz, H-3′), 3.32 (1H, dd, J = 8.5, 8.5 Hz, H-4′), 3.30 (1H, m, H-5′), [3.70 (1H, dd, J = 5.0, 11.5 Hz), 3.90 (1H, dd, J = 1.5, 11.5 Hz), H2-6′]; 13C NMR (CD3OD, 125 MHz) δC: 134.1 (C-1), 127.4 (C-2), 28.3 (C-3), 39.7 (C-4), 29.9 (C-5), 76.0 (C-6), 21.1 (C-7), 73.0 (C-8), 27.8 (C-9), 26.1 (C-10), 102.2 (C-1′), 75.1 (C-2′), 78.2 (C-3′), 71.9 (C-4′), 78.0 (C-5′), 63.0 (C-6′); ESI-Q-Orbitrap MS: m/z 377.18222 [M + COOH]− (calcd. for C17H29O9, 377.18061).

5.4.14. Lippianoside E (14)

White powder; 1H NMR (DMSO-d6, 500 MHz) δH: [1.20 (1H, dd, J = 11.5, 11.5 Hz), 1.80 (1H, ddd, J = 3.0, 4.0, 11.5 Hz), H2-2], 4.15 (1H, m, H-3), [1.18 (1H, dd, J = 11.5, 11.5 Hz), 2.38 (1H, ddd, J = 3.0, 4.0, 11.5 Hz), H2-4], 5.86 (1H, s, H-7), 2.12 (3H, s, H3-10), 1.28 (3H, s, H3-11), 1.05 (3H, s, H3-12), 1.33 (3H, s, H3-13), 4.36 (1H, d, J = 7.8 Hz, H-1′), 2.91 (1H, dd, J = 7.5, 8.5 Hz, H-2′), 3.15 (1H, dd, J = 8.5, 8.5 Hz, H-3′), 3.00 (1H, dd, J = 8.5, 9.0 Hz, H-4′), 3.06 (1H, m, H-5′), [3.37 (1H, dd, J = 6.0, 11.5 Hz), 3.62 (1H, br. d, ca. J = 11 Hz), H2-6′]; 13C NMR (DMSO-d6, 125 MHz) δC: 35.4 (C-1), 49.1 (C-2), 61.0 (C-3), 45.9 (C-4), 76.9 (C-5), 117.4 (C-6), 99.8 (C-7), 210.6 (C-8), 197.5 (C-9), 26.2 (C-10), 29.0 (C-11), 31.7 (C-12), 96.8 (C-1′), 73.6 (C-2′), 77.2 (C-3′), 70.1 (C-4′), 76.6 (C-5′), 61.1 (C-6′); ESI-Q-Orbitrap MS: m/z 425.17201 [M + Na]+ (calcd. for C19H30O9Na, 425.17820).

5.4.15. Pisuminic Acid (15)

White powder; [α]D25 −37.7 (conc. 0.027, MeOH); 1H NMR (C5D5N, 600 MHz) δH: [1.01 (1H, dt, J = 3.0, 13.2 Hz), 1.87 (1H, m, overlapped), H2-1], [1.50 (1H, br. D, ca. J = 14 Hz), 2.23 (1H, m, overlapped), H2-2], [1.24 (1H, dt, J = 4.2, 13.8 Hz), 2.51 (1H, br. D, ca. J = 13 Hz), H2-3], 2.41 (1H, d, J = 10.8 Hz, H-5), 4.97 (1H, br. D, ca. J = 11 Hz, H-6), 4.18 (1H, m, H-7), 1.83 (1H, br. D, ca. J = 8 Hz, H-9), 1.60, 1.68 (1H each, both m, H2-11), 1.55, 1.92 (1H each, both m, H2-12), [2.12 (1H, d, J = 14.4 Hz), 2.59 (1H, d, J = 14.4 Hz), H2-14], [1.99 (1H, d, J = 11.4 Hz), 2.23 (1H, m, overlapped), H2-15], 2.48 (1H, m, H-16), [4.10 (1H, d, J = 10.8 Hz), 4.17 (1H, d, J = 10.8 Hz), H2-17], 1.87 (3H, s, H3-18), 1.28 (3H, s, H3-20); 13C NMR (C5D5N, 150 MHz) δC: 41.1 (C-1), 19.8 (C-2), 40.6 (C-3), 44.6 (C-4), 52.0 (C-5), 72.0 (C-6), 82.6 (C-7), 49.4 (C-8), 50.0 (C-9), 41.1 (C-10), 18.7 (C-11), 27.4 (C-12), 81.3 (C-13), 51.0 (C-14), 36.9 (C-15), 45.8 (C-16), 66.5 (C-17), 33.3 (C-18), 181.3 (C-19), 17.2 (C-20). ESI-Q-Orbitrap MS: m/z 367.21310 [M − H]− (calcd. For C20H31O6, 367.21152).

5.4.16. Fulgidic Acid (16)

White powder; [α]D25 −14.8 (conc. 0.054, MeOH); 1H NMR (CD3OD, 500 MHz) δH: 2.27 (2H, t, J = 7.5 Hz, H2-2), 1.59 (2H, m, H2-3), 1.27–1.45 (8H, H2-4–7), 1.52 (2H, H2-8), 4.05 (1H, q like, ca. J = 6 Hz, H-9), 5.71 (1H, dd, J = 5.0, 15.5 Hz, H-10), 5.69 (1H, dd, J = 5.0, 15.5 Hz, H-11), 3.95 (1H, t like, ca. J = 5 Hz, H-12), 3.45 (1H, m, ca. J = 5 Hz, H-13), [2.11 (1H, m), 2.34 (1H, ddd, J = 5.5, 5.5, 14.5 Hz), H2-14], 5.43 (1H, dd, J = 6.0, 11.0 Hz, H-15), 5.47 (1H, dd, J = 6.5, 11.0 Hz), H-16], 2.06 (2H, m, H2-17), 0.96 (3H, t, J = 7.5 Hz, H3-18); 13C NMR (CD3OD, 125 MHz) δC: 177.7 (C-1), 35.0 (C-2), 26.1 (C-3), 30.2 (C-4), 30.4 (C-5), 30.6 (C-6), 26.5 (C-7), 38.4 (C-8), 73.1 (C-9), 136.6 (C-10), 131.1 (C-11), 75.8 (C-12), 75.9 (C-13), 31.6 (C-14), 126.4 (C-15), 134.4 (C-16), 21.7 (C-17), 14.6 (C-18); ESI-Q-Orbitrap MS: m/z 327.21808 [M − H]− (calcd. for C18H31O5, 327.21660).

5.4.17. (9S,10E,12S,13S)-9,12,13-Trihydroxy-10-Octadecenoic Acid (17)

White powder; [α]D25 −19.5 (conc. 0.021, MeOH); 1H NMR (CD3OD, 500 MHz) δH: 2.27 (2H, t, J = 7.5 Hz, H2-2), 1.59 (2H, m, H2-3), 1.32–1.52 (18H, H2-4–8 and 14–17), 4.05 (1H, q like, ca. J = 6 Hz, H-9), 5.72 (1H, dd, J = 5.5, 15.5 Hz, H-10), 5.67 (1H, dd, J = 5.5, 15.5 Hz, H-11), 3.90 (1H, t like, ca. J = 6 Hz, H-12), 3.42 (1H, m, H-13), 0.90 (3H, t, J = 7.0 Hz, H3-18); 13C NMR (CD3OD, 125 MHz) δC: 177.6 (C-1), 34.9 (C-2), 26.0 (C-3), 30.1 (C-4), 30.3 (C-5), 30.5 (C-6), 26.5 (C-7), 38.3 (C-8), 73.0 (C-9), 136.5 (C-10), 131.0 (C-11), 76.5 (C-12), 75.7 (C-13), 33.5 (C-14), 26.4 (C-15), 33.1 (C-16), 23.7 (C-17), 14.4 (C-18); ESI-Q-Orbitrap MS: m/z 329.23392 [M − H]− (calcd. For C18H33O5, 329.23225).

5.4.18. Methyl (9S,10E,12S,13S)-9,12,13-Trihydroxy-10-Octadecenoate (18)

White powder; [α]D25 −13.5 (conc. 0.103, MeOH); 1H NMR (CD3OD, 500 MHz) δH: 2.31 (2H, t, J = 7.5 Hz, H2-2), 1.59 (2H, m, H2-3), 1.32–1.52 (18H, H2-4–8 and 14–17), 4.05 (1H, q like, ca. J = 6 Hz, H-9), 5.72 (1H, dd, J = 5.0, 15.5 Hz, H-10), 5.67 (1H, dd, J = 5.5, 15.5 Hz, H-11), 3.90 (1H, t like, ca. J = 6 Hz, H-12), 3.41 (1H, m, H-13), 0.90 (3H, t, J = 7.0 Hz, H3-18), 3.64 (3H, s, 1-COOCH3); 13C NMR (CD3OD, 125 MHz) δC: 175.9 (C-1), 34.8 (C-2), 26.0 (C-3), 30.1 (C-4), 30.4 (C-5), 30.6 (C-6), 26.5 (C-7), 38.3 (C-8), 73.0 (C-9), 136.5 (C-10), 131.1 (C-11), 76.5 (C-12), 75.8 (C-13), 33.5 (C-14), 26.6 (C-15), 33.1 (C-16), 23.7 (C-17), 14.5 (C-18), 52.0 (1-COOCH3); ESI-Q-Orbitrap MS: m/z 367.24493 [M + Na]+ (calcd. for C19H36O5Na, 367.24550).

5.5. 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 NH2-60-5 column (4.6 mm i.d. × 250 mm, Tokyo Kasei Co., Ltd., Tokyo, Japan). The mobile phase consisted of CH3CN-H2O (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 (tR: 10.2 min, positive optical rotation) and l-rhamnose (tR: 7.4 min, negative optical rotation) were clarified.

5.6. 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 I (1): O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO[C@H]2[C@H](O)[C@H](O)[C@@H](O)[C@H](C)O2)O[C@H]1OC(C3=CC(OC)=C(O)C=C3)=O
Aeswiloside II (2): O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO[C@H]2[C@H](O)[C@H](O)[C@@H](O)[C@H](C)O2)O[C@H]1OCC3=CC(OC)=C(O)C=C3
Aeswiloside III (3): C/C(C)=C\CO[C@@H]1O[C@H](CO[C@H]2[C@H](O)[C@H](O)[C@@H](O)[C@H](C)O2)[C@@H](O)[C@H](O)[C@H]1O
Aeswiloside IV (4): O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(C)(O)C=C)C/C=C(C)/CCC(C(C)(O)C)O

5.7. 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.

5.8. Supplementary Data

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

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29051136/s1, Supplementary data including the NMR, HRESIMS spectra of compounds 1–4, and cell viability assay associated with this article can be found in the online version.

Author Contributions

P.Z., L.Y. and H.C. conducted research, collected data, performed analysis and interpretation, and prepared graphic elements and illustrative materials. J.R., F.L. and L.W. collected data and revised the article for significant intellectual content. Y.Z. and T.W. conceptualized and designed the study, drafted the article text, and critically revised the article for important intellectual content. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82074118, 82104369) and the Tianjin Committee of Science and Technology of China (No. 23ZYJDSS00030).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The author does not guarantee the provision of original data.

Conflicts of Interest

The authors declare no conflict of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Molecules 29 01136 g001
Figure 1. The structures of compounds 1–18 obtained from A. wilsonii seeds.
Figure 1. The structures of compounds 1–18 obtained from A. wilsonii seeds.
Molecules 29 01136 g001
Molecules 29 01136 g002
Figure 2. The main 1H 1H COSY and HMBC correlations of compounds 1–4.
Figure 2. The main 1H 1H COSY and HMBC correlations of compounds 1–4.
Molecules 29 01136 g002
Molecules 29 01136 g003
Figure 3. Inhibitory effects of compounds 1, 4, 10, and 15 at concentrations of 10, 25, and 50 μM on NO production in RAW264.7 cells, respectively. Nitrite relative concentration (NRC): percentage of control group (set as 100%). Values represent the mean ± SD of six determinations. * p < 0.05; ** p < 0.01; *** p < 0.001 (Differences between compound-treated group and control group). ### p < 0.001 (differences between control group and normal group).
Figure 3. Inhibitory effects of compounds 1, 4, 10, and 15 at concentrations of 10, 25, and 50 μM on NO production in RAW264.7 cells, respectively. Nitrite relative concentration (NRC): percentage of control group (set as 100%). Values represent the mean ± SD of six determinations. * p < 0.05; ** p < 0.01; *** p < 0.001 (Differences between compound-treated group and control group). ### p < 0.001 (differences between control group and normal group).
Molecules 29 01136 g003
Molecules 29 01136 g004
Figure 4. Effect of compounds 1, 4, 10, and 15 on the concentration of TNF-α and IL-1β in LPS-induced RAW264.7 cells. Cells were incubated with LPS (0.5 μg/mL) with or without compounds 1, 4, 10, and 15 for 18 h. The results are presented as the mean ± standard error (n = 3), and a p-value less than 0.05 was expressed as statistically significant (### p < 0.001 vs. normal group; ** p < 0.01, *** p < 0.001 vs. LPS-treatment group).
Figure 4. Effect of compounds 1, 4, 10, and 15 on the concentration of TNF-α and IL-1β in LPS-induced RAW264.7 cells. Cells were incubated with LPS (0.5 μg/mL) with or without compounds 1, 4, 10, and 15 for 18 h. The results are presented as the mean ± standard error (n = 3), and a p-value less than 0.05 was expressed as statistically significant (### p < 0.001 vs. normal group; ** p < 0.01, *** p < 0.001 vs. LPS-treatment group).
Molecules 29 01136 g004
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data for compound 1 in CD3OD.
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data for compound 1 in CD3OD.
No.δCδH (J in Hz)No.δCδH (J in Hz)
1121.8—5′77.83.56 (m)
2114.07.62 (d, 1.8)6′67.83.66 (dd, 3.0, 12.0)
3148.8— 3.97 (dd, 1.8, 12.0)
4153.4—1″102.34.71 (d, 1.2)
5116.06.86 (d, 8.4)2″72.13.83 (dd, 1.2, 9.6)
6125.87.65 (dd, 1.8, 8.4)3″72.4
7166.7—4″74.03.33 (dd, 9.6, 9.6)
1′96.15.65 (d, 8.4)5″69.93.65 (m)
2′74.13.48 (dd, 8.4, 9.6)6″18.01.19 (d, 6.6)
3′78.13.49 (dd, 9.6, 9.6)3-OCH356.53.91 (s)
4′71.2
There is no corresponding hydrogen data for quaternary carbons or carbonyl groups, they are represented as “—”.
Table 2. 1H (500 MHz) and 13C (125 MHz) NMR data for 2 in CD3OD.
Table 2. 1H (500 MHz) and 13C (125 MHz) NMR data for 2 in CD3OD.
No.δCδH (J in Hz)No.δCδH (J in Hz)
1130.2—4′71.83.27 (dd, 9.0, 9.0)
2113.37.03 (br. s)5′77.03.37 (m)
3149.0—6′68.23.64 (dd, 6.0, 11.0)
4147.4— 4.00 (br. d, ca. 11)
5115.86.75 (d, 8.0)1″102.44.79 (br. s)
6122.66.82 (br. d, ca. 8)2″72.33.87 (br. d, ca. 3)
771.24.54 (d, 11.5)3″72.43.69 (m, overlapped)
4.77 (d, 11.5)4″74.13.38 (dd, 9.0, 9.5)
1′102.74.29 (d, 8.0)5″69.93.69 (m, overlapped)
2′75.13.22 (dd, 8.0, 8.5)6″18.11.27 (d, 6.5)
3′78.13.31 (dd, 8.5, 9.0)3-OCH356.43.86 (s)
Table 3. 1H and (600 MHz) 13C (150 MHz) NMR data for 3 in CD3OD.
Table 3. 1H and (600 MHz) 13C (150 MHz) NMR data for 3 in CD3OD.
No.δCδH (J in Hz)No.δCδH (J in Hz)
166.14.21 (dd, 7.8, 11.4)5′76.93.35 (m)
4.25 (dd, 7.8, 11.4)6′68.13.59 (dd, 6.0, 10.8)
2121.55.36 (m) 3.98 (dd, 1.8, 10.8)
3139.0—1″102.34.75 (d, 1.2)
426.01.76 (s)2″72.23.83 (dd, 1.2, 3.6)
518.21.70 (s)3″72.43.65 (dd, 3.6, 9.6)
1′102.54.26 (d, 7.8)4″74.13.36 (dd, 9.6, 9.6)
2′75.13.16 (dd, 7.8, 9.0)5″69.83.66 (m)
3′78.23.33 (dd, 9.0, 9.0)6″18.11.26 (d, 6.0)
4′71.83.26 (dd, 9.0, 9.6)
Table 4. 1H (500 MHz) and 13C (125 MHz) NMR data for 4 in CD3OD.
Table 4. 1H (500 MHz) and 13C (125 MHz) NMR data for 4 in CD3OD.
No.δCδH (J in Hz)No.δCδH (J in Hz)
1113.95.11 (br. d, ca. 11)1078.93.25 (m, overlapped)
5.26 (br. d, ca. 17)1173.8—
2142.86.05 (dd, 11.0, 17.0)1224.91.12 (s)
377.2—1325.91.16 (s)
489.93.53 (dd, 3.5, 9.0)1424.51.27 (s)
531.22.16 (ddd, 7.5, 7.5, 15.0)1516.51.60 (s)
2.35 (ddd, 3.5, 7.5, 15.0)1′106.24.45 (d, 8.0)
6123.75.53 (dd, 7.5, 7.5)2′75.93.23 (dd, 8.0, 9.0)
7136.33.33 (dd, 9.0, 9.0)3′78.33.35 (dd, 9.0, 9.0)
837.92.05 (ddd, 8.5, 8.5, 14.0)4′71.63.33 (dd, 9.0, 9.5)
2.24 (ddd, 4.0, 8.5, 14.0)5′78.03.25 (m, overlapped)
930.51.36 (m)6′62.83.71 (dd, 5.0, 12.0)
1.75 (m) 3.86 (dd, 2.0, 12.0)
Table 5. Inhibitory effects of compounds 1–18 on NO production in RAW264.7 cells.
Table 5. Inhibitory effects of compounds 1–18 on NO production in RAW264.7 cells.
No.NRC (%)No.NRC (%)No.NRC (%)
Normal2.2 ± 0.2599.8 ± 4.91295.0 ± 2.3
Control100 ± 3.0693.0 ± 3.91397.3 ± 3.8
DEX76.8 ± 3.8 ***795.0 ± 4.31498.5 ± 2.2
191.7 ± 3.5 *895.6 ± 3.11591.2 ± 4.9 *
294.1 ± 3.7993.9 ± 4.71694.5 ± 3.4
396.7 ± 4.21088.1 ± 3.2 ***1798.1 ± 3.4
490.2 ± 3.5 **1193.6 ± 2.81894.2 ± 2.9
Positive control: Dexamethasone (Dex). Nitrite relative concentration (NRC): percentage of control group (set as 100%). Values represent the mean ± SD of six determinations. * p < 0.05; ** p < 0.01; *** p < 0.001 (Differences between compound-treated group and control group). Final concentration was 50 μM for compounds 1–18 and 1.0 μg/mL for Dex, respectively.
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Zhang, P.; Yu, L.; Cao, H.; Ruan, J.; Li, F.; Wu, L.; Zhang, Y.; Wang, T. Potential Anti-Inflammatory Constituents from Aesculus wilsonii Seeds. Molecules 2024, 29, 1136. https://doi.org/10.3390/molecules29051136

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Zhang P, Yu L, Cao H, Ruan J, Li F, Wu L, Zhang Y, Wang T. Potential Anti-Inflammatory Constituents from Aesculus wilsonii Seeds. Molecules. 2024; 29(5):1136. https://doi.org/10.3390/molecules29051136

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Zhang, Ping, Lequan Yu, Huina Cao, Jingya Ruan, Fei Li, Lijie Wu, Yi Zhang, and Tao Wang. 2024. "Potential Anti-Inflammatory Constituents from Aesculus wilsonii Seeds" Molecules 29, no. 5: 1136. https://doi.org/10.3390/molecules29051136

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