Triterpenoid Saponins From the Fruit of Acanthopanax senticosus (Rupr. & Maxim.) Harms

Five new oleanane-type triterpenoid saponins (1–5), together with 24 known saponins (6–29) were isolated from the fruit of Acanthopanax senticosus. Their structures were determined by extensive spectroscopic analysis, including 1D, 2D nuclear magnetic resonance (NMR), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), in combination with chemical methods (acid hydrolysis). The neuroinflammation model was established by lipopolysaccharide (LPS)-induced BV2 microglia, and the neuroprotective effects of all compounds (1–29) were evaluated.

Introduction

Acanthopanax senticosus (Rupr. & Maxim.) Harms, commonly known as Ci Wu Jia or Siberian Ginseng, is a well-known traditional Chinese medicine widely distributed in the northeast of China. With high medicinal value, A. senticosus is popularly used as an “adaptogen” like Panax ginseng. Modern pharmacology study shows that this plant was used for antifatigue, anti-depression, anxiolytic, anti-irradiation, anticancer, anti-inflammatory, hypolipidemic, etc. (Huang et al., 2011; Li et al., 2016a), and these activities may be attributed to triterpenoid saponins. Insuperably, modern pharmacological studies have confirmed that A. senticosus fruits possess significant activities of antifatigue (Cong et al., 2010), antioxidant (Kim et al., 2015; Zhao et al., 2013), hypolipidemic (Yan et al., 2009), anti-obesity (Li et al., 2007; Saito et al., 2016), anti-inflammatory (Li et al., 2013), and so on. However, for the past few years, most of the phytochemical studies have been mainly focused on the root, stem, and leaves of A. senticosus, and limited researches have been investigated on its fruits (Ge et al., 2016; Huang et al., 2011; Li et al., 2016b; Li et al., 2017; Wang et al., 2012; Zhang et al., 2017).

In the present paper, we continue to further explore the active component triterpenoid saponins from the fruits of A. senticosus. The results found five previously undescribed triterpenoid saponins (1–5) (Figure 1), together with known 24 triterpenoid saponins (6–29). Their structures were elucidated mainly by spectroscopic methods including 1D and 2D nuclear magnetic resonance (NMR) experiments in combination with high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and by comparison of their physical and spectral data with literature. Meanwhile, their neuroprotective effects were evaluated by lipopolysaccharide (LPS)-induced BV2 microglia.

FIGURE 1

FIGURE 1. Chemical structures of compounds 1–5.

Experimental Section

General Experimental Procedures

The HR-ESI-MS data of the new triterpenoid saponins were obtained on a Thermo Orbitrap Fusion Lumos Tribrid Mass Spectrometer. The 1D and 2D NMR spectra were acquired on a Bruker DPX-600 spectrometer in Pyridine-d5 using TMS as internal standard. Preparative high-performance liquid chromatography (HPLC) (LC-20AR, Shimadzu) was performed on Waters Atlantis^® Prep T3 (5 μm, 10 × 250 mm column) with a RID-20 A detector, with flow rates of 3 ml/min. Optical rotation measurements were conducted on a JASCO P-2000 instrument. Gas chromatography-mass spectrometry (GC-MS) analysis was performed on an Agilent 7890A system with a DB-5 capillary column. Absorbance (OD) value was detected on a BioTek Epoch™2 Microplate Reader. The FT-IR data of the new triterpenoid saponins was performed on Thermo Scientific Nicolet iS10. Silica gel column chromatography (CC) and octadecyl silica (ODS) chromatography were used in the separation of extracts.

Plant Material

The fruit of A. senticosus was collected in October 2018 from the Yichun, Heilongjiang Province. The plant was identified by the Professor Rui-Feng Fan of the Heilongjiang University of Chinese Medicine, and its voucher specimen (NO. 20190330) has been deposited at Heilongjiang University of Chinese Medicine.

Extraction and Isolation

The dry fruits (20 kg) of A. senticosus were extracted with 70% EtOH three times, under reflux for 2 h each time to afford a crude extract (2,216 g). The crude extract was extracted with petroleum ether, EtOAc, and n-BuOH successively, and the corresponding extract was obtained after removing the solvent, namely, PE fraction (320.0 g), EtOAc fraction (470.0 g), and n-BuOH fraction (510.0 g). The ethyl acetate layer (360.0 g) was chromatographed on a silica gel column (200–300 mesh) eluted successively with CH₂Cl₂/MeOH (100:1–0:1) to obtain nine fractions. Fr. VI was separated on a silica gel column (200–300 mesh) eluted successively with CH₂Cl₂/MeOH (50:1–0:1) to obtain five fractions (Fr. VI 1–5). Fr. VI 4 was purified by ODS chromatography to afford 48 fractions. Fr. VI 4–(46) were purified by semi-preparative HPLC (MeOH/H₂O 84%) to afford compounds 14 (5.3 mg), 7 (4.9 mg), 15 (5.0 mg), 8 (37.5 mg), 18 (9.7 mg), 16 (45.9 mg), 10 (7.1 mg), and 11 (5.4 mg). Fr. VII was separated on a silica gel column (200–300 mesh), using solvent system CH₂Cl₂/MeOH (30:1 to 0:1) to give five fractions (Fr. VII 1–7) based on TLC analysis. Fr. VII 5 was purified by ODS chromatography to afford sixty-three fractions. Fr. VII 5–(54) were purified by semi-preparative HPLC (MeOH/H₂O 78%) to afford compounds 5 (5.3 mg) and 6 (3.6 mg). Fr. VIII was separated on a silica gel column (200–300 mesh, 1 kg), using solvent system CH₂Cl₂/MeOH (30:1 to 0:1) to give nine fractions (Fr. VIII 1–9) based on TLC analysis. Fr. VIII 5 was purified by ODS chromatography to afford fifty fractions. Fr. VIII 5–(47) were purified by semi-preparative HPLC (MeOH/H₂O 84%) to afford compounds 13 (66.4 mg) and 9 (52.8 mg). Fr. VIII 6 was purified by ODS chromatography to afford forty-two fractions. Fr. VIII 6-(24) was purified by semi-preparative HPLC (MeOH/H₂O 73%) to afford compound 3 (8.8 mg). Fr. VIII 6–(25) was purified by semi-preparative HPLC (MeOH/H₂O 73%) to afford compound 12 (5.3 mg). Fr. VIII 6–(27) were purified by semi-preparative HPLC (MeOH/H₂O 68%) to afford compounds 1 (26.0 mg), 4 (4.2 mg), 17 (147.1 mg), and 22 (26.4 mg). Fr. VIII 6–(29) was purified by semi-preparative HPLC (MeOH/H₂O 73%) to afford compound 25 (42.0 mg). Fr. VIII 6–(29D) were purified by semi-preparative HPLC (MeOH/H₂O 78%) to afford compound 19 (3.6 mg). Fr. VIII 6–(30) were purified by semi-preparative HPLC (MeOH/H₂O 70%) to afford compound 23 (28.1 mg). Fr. VIII 6–(30C) were purified by semi-preparative HPLC (MeOH/H₂O 80%) to afford compound 2 (8.7 mg). Fr. VIII 6–(32) were purified by semi-preparative HPLC (MeOH/H₂O 80%) to afford compound 26 (5.4 mg). Fr. VIII 6–(33) were purified by semi-preparative HPLC (MeOH/H₂O 81%) to afford compound 24 (9.1 mg). Fr. VIII 6–(34) were purified by semi-preparative HPLC (MeOH/H₂O 82%) to afford compounds 28 (8.2 mg) and 27 (7.6 mg). Fr. VIII 6–(35) were purified by semi-preparative HPLC (MeOH/H₂O 84%) to afford compounds 29 (2.8 mg), 20 (7.7 mg), and 21 (23.5 mg).

Spectroscopic Data

Acasentrioid A (1): amorphous powder; ${\left[\text{α}\right]}_{\text{D}}^{24}=+14.7$, (c = 0.15, MeOH); HR-ESI-MS m/z: 784.4845 [M + NH₄]⁺ (calculated to be 784.4842 for C₄₁H₇₀NO₁₃). The ¹H (pyridine-d₅, 600 MHz) and ¹³C NMR (pyridine-d₅, 150 MHz) data are shown in Tables 1, 2.

TABLE 1

TABLE 1. ¹³C NMR data (δ) for compounds 1–5 (150, MHz in pyridine-d₅).

TABLE 2

TABLE 2. ¹H NMR data (δ) for compounds 1–5 (600, MHz in pyridine-d₅).

Acasentrioid B (2): amorphous powder; ${\left[\text{α}\right]}_{\text{D}}^{24}=+7.5$, (c = 0.32, MeOH); HR-ESI-MS m/z: 856.5043 [M + NH₄]⁺ (calculated to be 856.5053 for C₄₄H₇₄NO₁₅). The ¹H (pyridine-d₅, 600 MHz) and ¹³C NMR (pyridine-d₅, 150 MHz) data are shown in Tables 1, 2.

Acasentrioid C (3): amorphous powder; ${\left[\text{α}\right]}_{\text{D}}^{24}=+2.1$, (c = 0.28, MeOH); HR-ESI-MS m/z: 737.4490 [M + H]⁺ (calculated to be 737.4471 for C₄₀H₆₅O₁₂). The ¹H (pyridine-d₅, 600 MHz) and ¹³C NMR (pyridine-d₅, 150 MHz) data are shown in Tables 1, 2.

Acasentrioid D (4): amorphous powder; ${\left[\text{α}\right]}_{\text{D}}^{24}=+19.1$, (c = 0.22, MeOH); HR-ESI-MS m/z: 798.4648 [M + NH₄]⁺ (calculated to be 798.4634 for C₄₁H₆₈NO₁₄). The ¹H (pyridine-d₅, 600 MHz) and ¹³C NMR (pyridine-d₅, 150 MHz) data are shown in Tables 1, 2.

Acasentrioid E (5): amorphous powder; ${\left[\text{α}\right]}_{\text{D}}^{24}=-3.5$, (c = 0.23, MeOH); HR-ESI-MS m/z: 663.4121 [M + H]⁺ (calculated to be 663.4103 for C₃₇H₅₉O₁₀). The ¹H (pyridine-d₅, 600 MHz) and ¹³C NMR (pyridine-d₅, 150 MHz) data are shown in Tables 1, 2.

Hydrolysis of Compounds 1–5

Monosaccharide was determined by GC (Teng et al., 2018). Compounds 1–5 (each 1.0 mg) were dissolved in 2 ml of 2 M HCl (dioxane/H₂O, 1:1, v/v), and hydrolyzed at 90°C for 3 h. After removing dioxane in a vacuum, the solution was diluted with H₂O and extracted with EtOAc (3 × 1 ml). The aqueous layer was evaporated to dryness. The dried residue was dissolved in pyridine (200 μl) and treated with L-cysteine methyl ester hydrochloride (2.0 mg). After stirring the mixture for 1 h at 60°C, 100 μl of N-trimethylsilylimidazole was added, and they were kept at 60°C for 1 h. The reaction mixture was suspended in 1.0 ml H₂O and extracted with n-hexane (3 × 1.0 ml). The layer of n-hexane was directly analyzed by GC with a DM-5 column (30 m × 0.25 mm, 0.25 μm) with the elution of N₂ as carrier gas. Other GC conditions are as follows: column temperature: 220–270°C with the rate of 3°C/min; injector and detector temperature: 250°C; split ratio: 10:1; and injection volume:1 μl. The configurations of D-glucose, L-arabinose, D-glucuronic acid, and L-rhamnose in compounds 1–5 were determined by comparison of their retention times with those of standard samples.

Bioassay for Cytotoxicity Activities

In each well of a 96-well plate, 100 μl of logarithmic growth phase cells (density 1.5 × 105/ml) was inoculated and cultured at 37°C and 5% CO₂ until the cells attached to the wall. A drug-containing medium (0, 50, 100, 200, 400, and 600 μM) was added to each well of the administration group, and an equal volume of medium was also added to the blank group, and they were incubate at 37°C and 5% CO₂ for 24 h. After the culture, 10 μl of CCK-8 was added to each well and incubated for 1–4 h at 37°C and 5% CO₂. The absorbance (OD) value of each well at 450 nm was detected with a microplate reader, repeating three times, and its IC₅₀ value was calculated.

Bioassay for NO Production Inhibitory Activities

The anti-neuroinflammatory effect of compounds 1–29 was evaluated by LPS-induced BV2 microglia reported previously (Luo et al., 2020). The BV2 microglia cells were plated into a 96-well plate. After adding LPS (1 μg/ml) to each well for 12 h, it was treated with or without compounds of various concentrations (0, 100, 200, 300, 400, and 600 μM) for 12 h. The NO production in the supernatant was measured by the Griess reaction. The absorbance at 570 nm was measured using a microplate reader. The NO concentration and the inhibitory rate were calculated through a calibration curve. Quercetin was used as the positive control. Experiments were repeated three times.

Results and Discussion

Structure Elucidation of Compounds

Compound 1 was obtained as an amorphous powder. The negative HR-ESI-MS showed a deprotonated molecular ion peak at m/z 784.4845 [M + NH₄]⁺ (calculated for C₄₁H₇₀NO₁₃, 784.4842), indicating its molecular formula of C₄₁H₆₆O₁₃. The ¹H NMR spectrum displayed characteristic resonances of an olean-12-ene skeleton, namely, six methyls [δ_H 0.83, 0.96, 1.00, 1.22, 1.29, and 1.31 (3H each, all s, H-25, 24, 26, 30, 23, and 27)], one oxygenated methylene [δ_H 3.61 (2H, s, H-29)], one oxygenated methine [δ_H 3.33 (1H, dd, J = 11.5, 3.8 Hz, H-3)], one olefin [δ_H 5.51 (1H, br. s, H-12)], and two anomeric proton signals [δ_H 4.74 (1H, d, J = 7.3 Hz, H-1′) and 5.39 (1H, d, J = 7.7 Hz, H-1″)] (see Tables 1, 2). Coupled with DEPT spectrum, the ¹³C NMR spectrum showed the presence of 41 signals, of which 30 signals were assigned to a triterpene of oleanane skeleton, containing one carboxyl group (δ_C 180.2), two olefinic carbons (δ_C 122.5 and 144.9), two anomeric carbons (δ_C 107.3 and 106.3), one downfield glycosylation-shifted oxygenated methine (δ_C 88.5), a hydroxymethyl carbon (δ_C 73.8), and six methyls (δ_C 15.4, 16.9, 17.3, 19.7, 26.1, and 28.0) (see Tables 1, 2). These observations implied that compound 1 might be an oleanane-type triterpenoid saponin.

The HMBC cross-peaks of the anomeric proton H-1′ (δ_H 4.74)/C-3 showed the by ether bond location of the sugar chain at C-3. The HMBC connections of H₂-22 (δ_H 2.16)/C-17, C-28 indicated a carboxy fragment attached at C-17 (Figure 2). Based on the above analysis, the structure of compound 1 was similar to compound 21, and the major difference was the substituent C-29 is changed from methyl to oxymethylene in compound 1, which was supported by the HMBC correlation of the anomeric protons of H₂-29 (δ_H 3.61) with C-19, C-20, C-21, and C-30 (Figure 2). The β orientations of both pyranose sugars were deduced according to the large coupling constants of the anomeric protons (J = 7.3 Hz, H-1′; J = 7.7 Hz H-1″). To determine the absolute configuration of the arabinopyranose and glucopyranose, compound 1 was hydrolyzed by 2 mM HCl to obtain the sugar, and then, the trimethylsilyl thiazolidine derivatives of the sugar and standards, L-arabinose, and D-glucose were prepared. By comparing the retention times of these three trimethylsilyl thiazolidine derivatives obtained from GC, the absolute configuration of the arabinopyranose and glucopyranose in 1 was determined to be L and D, respectively.

FIGURE 2

FIGURE 2. ¹H–¹H COSY and key HMBC correlations of compounds 1–5.

In the NOESY spectrum (Figure 3), the correlation peaks of H₃-24/H₃-25/H₃-26/H-18/H₃-30 suggested the β orientations of H₃-24, H₃-25, H₃-26, H-18, and H₃-30. Conversely, the correlation peaks of H-3/H₃-23/H-5/H-9/H₃-27/H-22α (δ_H 2.16)/H₂-29 indicated that H-3, H-5, H-9, H₃-23, H₃-27, and H₂-29 were α-oriented. Therefore, the structure of compound 1 was elucidated to be 3-O-β-glucopyranosyl-(1→3)-β-arabinopyranosyl-29-hydroxy-olean-12-en-28-oic acid, named acasentrioid A.

FIGURE 3

FIGURE 3. Key NOESY correlations of compounds 1–5.

Compound 2 was isolated as an amorphous powder. Its molecular formula, C₄₄H₇₀O₁₅, was determined by the negative HR-ESI-MS at m/z 856.5043 [M + NH₄]⁺ (calculated for C₄₄H₇₄NO₁₅, 856.5053). The ¹H NMR spectrum displayed a skeleton characteristic similar to compound 1, namely, six methyls [δ_H 0.87, 0.88, 0.92, 0.93, 1.11, and 1.19 (3H each, all s, H-30, 29, 25, 24, 26, and 27)], together with one methyl triplet at δ_H 1.14 (3H, t, J = 7.1 Hz, H-8′), one hydroxymethyl [δ_H 3.70 (1H, d, J = 10.9 Hz, H-23a), δ_H 4.34 (1H, d, J = 10.9 Hz, H-23b)], one oxygenated methine [δ_H 4.30 (1H, dd, J = 4.3, 12.3 Hz, H-3)], one olefin [δ_H 5.41 (1H, t, J = 3.1 Hz, H-12)], and two anomeric proton signals [δ_H 5.20 (1H, d, J = 7.7 Hz, GlcA-H-1′) and 6.33 (1H, d, J = 8.1 Hz, Glc-H-1″)] (see Tables 1, 2). There are 44 signals displayed in the ¹³C NMR spectrum, of which 30 signals corresponded to the triterpene of the oleanane skeleton. Combined with the DEPT spectrum, the ¹³C NMR spectrum showed resonances for one carboxyl group (δ_C 176.4), two olefinic carbons (δ_C 122.9 and 144.1), two anomeric carbons (δ_C 106.4 and 95.6), one downfield glycosylation-shifted oxygenated methine (δ_C 82.2), oxygenated methylene (δ_C 64.3), and seven methyls (δ_C 13.5, 14.1, 16.0, 17.5, 23.6, 26.0, and 33.0) (see Tables 1, 2). These observations implied that compound 2 might be as well an oleanane-type triterpenoid saponin.

The heteronuclear multiple bond coherence (HMBC) cross-peaks of the anomeric protons H-1′ (δ_H 5.20)/C-3 and H-1″ (δ_H 6.33)/C-28 show that sugar is attached to the ether bond of C-3 and C-28, respectively. The HMBC connections of H₂-22 (δ_H 1.74, 1.81)/C-17, C-28 indicated a glycosylated carboxyl fragment attached at C-17 (Figure 2). Based on the above analysis, the structure of 2 was similar to ilexoside XLVIII (Amimoto et al., 1993), except for the ethyl group (δ_C 14.1 and 61.1) linked to C-6′ (δ_C 170.2) by ether bond in compound 2, which was supported by the HMBC correlation of the ethyl protons H₃-8′ and H-7′ with C-7′ and C-6′, respectively (Figure 2). The β orientations of both pyranose sugars were deduced according to the large coupling constants of the anomeric protons (J = 7.7 Hz, H-1′; J = 8.1 Hz, H-1″). The absolute configurations of both glucuronopyranosyl and glucopyranosyl were determined to be D by the same chemical methods and GC analysis as 1.

In the NOESY spectrum (Figure 3), the correlation peaks of H₃-24/H₃-25/H₃-26/H-18/H₃-30 suggested the β orientations of H₃-24, H₃-25, H₃-26, H-18, and H₃-30. Conversely, the correlation peaks of H-3/H₂-23/H-5/H-9/H-27/H-21β (δ_H 1.07)/H₃-29 indicated that H-3, H-5, H-9, H₂-23, H₃-27, and H₃-29 were α-oriented. Thus, the structure of compound 2 was defined as 3-O-β-D-6′-ethyl-glucuronopyranosyl-23-hydroxy-olean-12-en-28-β-D-glucopyranosyl, named acasentrioid B.

Compound 3 was also obtained as an amorphous powder with the molecular formula of C₄₀H₆₄O₁₂ as indicated by the molecular ion peak m/z 737.4490 [M + H]⁺ (calculated 737.4471 for C₄₀H₆₅O₁₂) in the HR-ESI-MS spectra. The ¹H NMR spectra gave seven angular methyl signals at δ_H 0.82 (3H, s, H-25), 0.99 (3H, s, H-26), 1.06 (3H, s, H-24), 1.17 (3H, s, H-23), 1.26 (3H, s, H-27), 1.58 (3H, s, H-30), and 1.63 (3H, d, J = 5.9 Hz, Ara-H-6″); one oxygenated methine at δ_H 3.25 (1H, dd, J = 4.1, 11.5 Hz, H-3); one olefin at δ_H 5.53 (1H, br.s, H-12); and two anomeric proton signals at δ_H 4.91 (1H, d, J = 5.1 Hz, Ara-H-1′) and 6.16 (1H, s, Rha-H-1″) (see Tables 1, 2). Accordingly, the ¹³C NMR spectra also revealed seven methyl signals at δ_C 15.5 (C-25), 17.0 (C-24), 17.3 (C-26), 18.5 (C-6″), 26.0 (C-27), 28.0 (C-23), and 25.7 (C-30); an oxygen-substituted methine signal at δ_C 88.7 (C-3); oxygen-substituted quaternary carbon signal at δ_C 69.8 (C-20); two olefinic carbon signals at δ_C 122.6 (C-12) and 144.3 (C-13); and one carboxyl carbon signal at δ_C 180.0 (C-28), along with two anomeric carbon signals at δ_C 101.7 (C-1″) and 104.9 (C-1′) as determined by the HSQC and DEPT spectra. All these NMR data were characteristic resonances of olean-12-ene skeleton triterpenes. The NMR data of 3 resembled those of 17, and the major difference was the substituent C-29 is changed from methyl to hydroxyl in compound 3, which was supported by the chemical shift δ_C 69.8 (C-20) and the HMBC correlation of the anomeric protons of H₃-30 (δ_H 1.58) with C-19, C-20, and C-21 (Figure 2). The HMBC connections of H₂-22 (δ_H 2.07) and H-18 (δ_H 3.35)/C-28 indicated a carboxy fragment attached at C-17 (Figure 2). The absolute configurations of both arabinopyranose and rhamnopyranose were determined to be L by the same chemical methods and GC analysis as 1. The coupling constant of the anomeric hydrogen J = 5.1 Hz (δ_H 4.91, d, H-1′) established the α-arabinopyranosyl linkage in 3. In the NOESY spectrum (Figure 3), the correlation peaks of H₃-24/H₃-25/H₃-26/H-15β/H-18/H₃-30 indicated that the H₃-24, H₃-25, H₃-26, H-18, and H₃-30 were β-oriented. Conversely, the correlation peaks of H-3/H₃-23/H-5/H-9/H₃-27 suggested the α orientations of H-3, H-5, H-9, H₃-23, H₃-27, and OH-20. Thus, compound 3 was defined as 3β-[(α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl)oxy]-20α,30-dihydroxy-norolean-12-en-28-oic acid, named acasentrioid C.

Compound 4 was obtained as an amorphous powder. Its molecular formula C₄₁H₆₄O₁₄ was established by the HR-ESI-MS spectrum m/z 798.4648 [M + NH₄]⁺ (calculated for C₄₁H₆₈NO₁₄, 798.4634) and was supported by the ¹³C NMR spectroscopic data. The ¹³C NMR spectrum of 4 displayed 41 carbons, of which 30 were assigned to the aglycone part and the remaining 11 were assigned to two sugar units comprising one pentose and one hexose. The NMR spectra showed signals for six angular methyl at δ_H 0.80, 0.96, 1.00, 1.18, 1.25, and 1.55 (3H each, all s, H-25, 26, 24, 23, 27, and 30), and their corresponding carbons at δ_C 15.3 (C-25), 17.2 (C-26), 16.6 (C-24), 28.0 (C-23), 25.9 (C-27), and 19.9 (C-30); an olefinic group at δ_H 5.51 (1H, t, J = 2.9 Hz, H-12) and δ_C 123.0 (C-12) and 144.1 (C-13); one oxygenated methine at δ_H 3.17 (1H, dd, J = 4.2 and 11.8 Hz, H-3) and δ_C 88.7 (C-3); two anomeric proton signals at δ_H 4.93 (1H, d, J = 5.7 Hz, Ara-H-1′) and 5.17 (1H, d, J = 7.8 Hz, Glc-H-1″) and their corresponding carbons at δ_C 104.6 (C-1′) and 105.7 (C-1″); and two carboxy groups at δ_C 180.0 (C-28) and 181.1 (C-29), which were characteristic for the triterpenoid saponin with oleanane skeleton. Two-dimensional NMR data of 4 resembled those of 20, and the major difference was the substituent C-29 is changed from methyl to carboxyl in compound 3, which was supported by the chemical shift δ_C 181.1 (C-29) and the HMBC correlation of H-19 (δ_H 2.57)/C-29 and H₃-30 (δ_H 1.55)/C-20, C-21, and C-29 (Figure 2). The absolute configurations of the arabinopyranose and glucopyranose were determined to be L and D, respectively, by the same chemical methods and GC analysis as 1, and the coupling constant of anomeric protons J = 5.7 Hz (δ_H 4.93, d, H-1′) and J = 7.8 Hz (δ_H 5.17, d, H-1″) established the α-arabinopyranosyl and β-glucopyranosyl linkage in 4. In the NOESY spectrum (Figure 3), the correlation peaks of H₃-24/H₃-25/H₃-26/H-15β/H-18/H₃-30 indicated that the H₃-24, H₃-25, H₃-26, H-18, and H₃-30 were β-oriented. Conversely, the correlation peaks of H-3/H₃-23/H-5/H-9/H₃-27 suggested the α orientations of H-3, H-5, H-9, H₃-23, H₃-27, and COOH-20. Thus, compound 4 was defined as 3β-[(O-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl)oxy]olean12-ene-28,29-dioic acid, named acasentrioid D.

Compound 5 was obtained as an amorphous powder. The HR-ESI-MS indicated a precise [M + H]⁺ ion at m/z 663.4121 (calculated for C₃₇H₅₉O₁₀, 663.4103), indicating an empirical molecular formula of C₄₁H₆₄O₁₄. In the ¹H NMR spectrum, six quaternary methyl group protons at δ_H 0.83, 0.89, 0.92, 1.02, 1.04, and 1.11 (3H each, all s, H-25, 27, 23, 26, 30, and 29); a methoxy group proton at δ_H 3.69 (3H, s, H-7′); an olefinic proton at δ_H 5.27 (1H, s, H-19); an oxygenated methine proton at δ_H 4.32 (1H, dd, J = 4.3, 12.1 Hz, H-3), hydroxymethyl protons at δ_H 3.70 (1H, d, J = 11.0 Hz, H-24a) and δ_H 4.34 (1H, d, J = 11.0 Hz, H-24b); and an anomeric proton at δ_H 5.22 (1H, d, J = 7.7 Hz, GlcA-H-1′) along with six quaternary methyl group carbons at δ_C 17.3 (C-25), 15.2 (C-27), 13.3 (C-23), 16.2 (C-26), 29.2 (C-30), and 30.7 (C-29); methoxy group carbons at δ_C 51.9 (C-7′); an oxygen-bearing methine carbon at δ_C 82.0 (C-3); a set of olefinic carbons at δ_C 138.9 (C-18) and 131.9 (C-19); a hydroxymethyl carbon at δ_C 64.1 (C-24); a carboxyl carbon at δ_C 179.4 (C-28); an ester group carbon at δ_C 170.8 (C-6′); and an anomeric carbon at δ_C 106.4 (C-1′) in its 13C NMR suggested the aglycone belongs to oleanane-type triterpene (see Tables 1, 2). A detailed analysis of HSQC, HMBC, COSY, and NOESY spectra of 5 assisted the complete assignment of its ¹H and ¹³C NMR data, which were similar to those of 3β,23-dihydroxyolean-18-en-28-oic acid (Cai and Geng, 2016). The only difference is the presence of glucuronopyranoside-6′-O-methyl ester at C-3 in 5, while there is no glycoside at C-3 in 3β,23-dihydroxyolean-18-en-28-oic acid, which was further confirmed by the HMBC correlations of H-1′ with C-3 and of H₃-7′ with C-6′ (Figure 2). The absolute configurations of the glucuronopyranosyl were determined to be D by the same chemical methods and GC analysis as 1, and the coupling constant of anomeric proton J = 7.7 Hz (δ_H 5.22, d, H-1′) established the β-glucuronopyranoside in 5. In the NOESY spectrum (Figure 3), the correlation peaks of H₂-24/H₃-25/H₃-26/H-13/H₃-30 indicated that the H₂-24, H₃-25, H₃-26, H-13, and H₃-30 were β-oriented. Conversely, the correlation peaks of H-3/H₃-23/H-5/H-9/H₃-27 suggested the α orientations of H-3, H-5, H-9, H₃-23, and H₃-27. Thus, compound 5 was defined as 3β,23-dihydroxyolean-18-en-28-oic acid 3-O-β-D-glucuronopyranoside-6′-O-methyl ester, named acasentrioid E.

The structures of known compounds 6–29 were determined as HN-saponin D1 (6) (Kizu et al., 1985), hederagenin glycosides 3-O-α-L-arabinopyranoside (7) (Grishkovets et al., 2005), oleanolic acid 3-O-β-D-glucuronopyranoside (8) (Li et al., 2012), HN-saponin K (9) (Kizu et al., 1985), 3-O-β-D-glucuronopyranosyl-3β,16α-dihydroxyolean-12-en-28-oic acid (10) (Ushijima et al., 2008), gypsogenin 3-O-glucuronide (11) (Bouguet-Bonnet et al., 2002), elatoside G (12) (Yoshikawa et al., 1995), hederagenin-3-O-β-D- glucuronopyranoside 6′-O-methyl ester (13) (Cao et al., 2011), tragopogonsaponin A methyl ester (14) (Warashina et al., 1991), 3-O-6′-O-methyl-β-D-glucuronopy-ranoside of gypsogenin (15) (Iwamoto et al., 1985), 3-O-β-D-(6′-O-methyl-glucuronopyranosyl) oleanolic acid (16) (Melek et al., 1996), 3-O-α-rhamnopyranose- (1→2)-α-arabinopyranosyl-29-hydroxy-olean-12-en-28-oic acid (17) (Shao et al., 1989), 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] oleanolic acid (18) (Nakanishi et al., 1993), HN-saponin F (19) (Mizui et al., 1988), saponin PE (20) (Zhong et al., 2001), oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-α-L-arabinopyranoside (21) (Satoh et al., 1994), lucyoside F (22), lucyoside H (23) (Takemoto et al., 1984), 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl oleanolic acid (24) (Reginatto et al., 2001), 3-O-β-D-glucuronopyranosyl methyl ester-28-O-β-D-glucopyranoside (25) (Li et al., 2007), oleanolic acid 3-O-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-28-O-β-D-glucopyranosyl ester (26) (Fan et al., 2013), paritriside E (27) (Wu et al., 2012), 3-O-α-arabinopyranosyl-(1→2)-β-glucopyranoside-30-norolean-12,20(29)-dien-28-oic acid (28) (Shao et al., 1989), and 3-[(O-β-D-glucopyranosyl-(1→3)-α-L-arabinopyranosyl)oxy]-30- noroleana-12,20(29)-dien-28-oic acid (29) (Jitsuno and Mimaki, 2010) by comparison with literature data (Supplementary Table S1). Compounds 7 and 20 were isolated from A. senticosus for the first time. Compounds 6, 9, 12, 16, 19, 21, 23, and 24, were isolated from Acanthoganax Miq. species for the first time. Compounds 10, 11, 14, 15, 22, 26, 27, and 29 were isolated from the family Araliaceae for the first time.

Bioactive Activity

The cytotoxicity of compounds 1–29 on BV2 microglia was determined by the CCK-8 assay, and the results are listed in Table 3. The neuroinflammation model was established by LPS-induced BV2 microglia, and the neuroprotective effect of compounds (1–29) in vitro was evaluated. Unfortunately, the results of the evaluation were not ideal. Compounds 5, 10, 12, 13, and 16 had moderate inhibitory effects on neuroinflammation, as indicated in Table 4, and other compounds had no anti-neuroinflammatory activity. Based on the existing results and analyzing its structure–activity relationship, we speculate in the structure of oleanane-type triterpene saponins; when the C-16 hydroxyl group is substituted or the structure contains only one methyl glucuronate, the compound has moderate anti-neuroinflammatory effects.

TABLE 3

TABLE 3. Cytotoxic activities of compounds 1–29 on BV2 Cells (IC₅₀, μM).

TABLE 4

TABLE 4. Inhibitory effects of compounds 1–29 on NO in LPS-induced BV-2 Cells (n = 3, x ± s).

Conclusion

In summary, five previously undescribed oleanane-type triterpenoid saponins (1–5), together with twenty-four known saponins (6–29), were isolated from the fruit of A. senticosus. The structures of all compounds were elucidated by extensive spectroscopic analysis, including 1D, 2D NMR, and HR-ESI-MS, in combination with chemical methods (acid hydrolysis). The neuroinflammation model was established by LPS-induced BV2 microglia, and the neuroprotective effects of all compounds (1–29) were evaluated. Unfortunately, the results of the evaluation were not ideal. Compounds 5, 10, 12, 13, and 16 had moderate inhibitory effects on neuroinflammation, while other compounds have no anti-neuroinflammatory activity.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.

Author Contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Funding

This work was financially supported by Heilongjiang Touyan Innovation Team Program, Heilongjiang University of Chinese Medicine Funds (2018pt01 and 2018bs03).

Conflict of Interest

Author M-LZ is employed by China Resources Double-Crane Pharmaceutical Co., Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2022.825763/full#supplementary-material

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