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 se nticosus (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

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; , (c = 0.15, MeOH); HR-ESI-MS m/z: 784.4845 [M + NH4]+ (calculated to be 784.4842 for C41H70NO13). The 1H (pyridine-d 5, 600 MHz) and 13C NMR (pyridine-d 5, 150 MHz) data are shown in Tables 1, 2.

TABLE 1

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

No	1	2	3	4	5
1	38.6	38.6	38.8	38.5	39.0
2	26.6	26.0	26.5	26.3	26.2
3	88.5	82.2	88.7	88.7	82.0
4	39.7	43.4	39.4	39.3	43.5
5	55.7	47.5	55.8	55.6	47.7
6	18.4	18.1	18.4	18.3	18.0
7	33.1	32.7	33.1	33.0	34.6
8	39.5	39.9	39.7	39.6	40.9
9	48.0	48.1	47.9	47.8	51.4
10	37.0	36.8	36.9	36.8	36.9
11	23.7	23.8	23.7	23.6	21.2
12	122.5	122.9	122.6	123.0	26.4
13	144.9	144.1	144.3	144.1	41.6
14	42.0	42.1	42.1	42.4	42.9
15	28.3	28.2	28.2	28.1	29.9
16	23.7	23.3	23.8	23.6	34.3
17	47.0	46.9	46.7	46.5	48.5
18	41.3	41.7	44.3	40.9	138.9
19	41.1	46.1	48.0	40.9	131.9
20	36.5	30.7	69.8	42.0	32.3
21	29.0	33.9	36.2	29.1	34.1
22	32.6	32.5	35.1	32.2	34.1
23	28.0	64.3	28.0	28.0	13.3
24	16.9	13.5	17.0	16.6	64.1
25	15.4	16.0	15.5	15.3	17.3
26	17.3	17.5	17.3	17.2	16.2
27	26.1	26.0	26.0	25.9	15.2
28	180.2	176.4	180.0	180.0	179.4
29	73.8	33.0	–	181.1	30.7
30	19.7	23.6	25.7	19.9	29.2
1′	107.3	106.4	104.9	104.6	106.4
2′	71.8	75.4	75.9	80.6	75.4
3′	84.1	77.9	73.9	73.3	77.8
4′	69.2	73.1	68.7	68.2	73.1
5′	67.0	77.4	64.8	64.8	77.2
6′	–	170.2	–	–	170.8
7′	–	61.1	–	–	51.9
8′	–	14.1	–	–	–
1″	106.3	95.6	101.7	105.7	–
2″	75.6	74.0	72.4	76.2	–
3″	78.3	78.9	72.6	78.0	–
4″	71.4	71.1	74.0	71.4	–
5″	78.6	79.2	69.8	78.0	–
6″	62.6	62.1	18.5	62.4	–

TABLE 2

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

No	1	2	3	4	5
1	0.94 o	0.95 o	0.90 t (13.7)	0.86 t (13.2)	0.93 o
	1.50 o	1.47 dt (3.5, 13.1)	1.47 br s	1.46 o	1.60 d (11.8)
2	1.88 o	1.98 dd (3.5, 13.1)	1.82 o	1.80 t (13.9)	1.99 t (12.9)
	2.14 o	2.22 m	2.07 o	2.05 m	2.24 m
3	3.33 dd (3.8, 11.5)	4.30 dd (4.3, 12.3)	3.25 dd (4.1, 11.5)	3.17 dd (4.2, 11.8)	4.32 dd (4.3, 12.1)
4	–	–	–	–	–
5	0.80 d (11.8)	1.64 o	0.75 d (11.3)	0.67 d (11.8)	1.60 d (11.8)
6	1.30 o	1.31 br s	1.28 o	1.22 o	1.30 m
	1.48 o	1.66 o	1.44 br s	1.42 o	1.67 d (12.1)
7	1.31 o	1.30 br s	1.28 o	1.22 o	1.41 br s
	1.47 o	1.58 t (11.9)	1.42 br d (14.2)	1.36 m	1.49 o
8	–	–	–	–	–
9	1.65 t (8.8)	1.72 o	1.60 o	1.57 t (8.6)	1.39 br s
10	–	–	–	–	–
11	1.91 o	1.90 m	1.87 o	1.87 m	1.17 d (11.1)
	–	–	–	–	1.46 o
12	5.51 br s	5.41 t (3.1)	5.53 br s	5.51 t (2.9)	1.28 o
	–	–	–	–	1.67 d (12.1)
13	–	–	–	–	2.69 d (11.9)
14	–	–	–	–	–
15	1.20 o	1.10 o	1.21 br s	1.18 o	1.24 o
	2.20 o	2.32 td (4.3, 13.6)	2.17 t (13.1)	2.15 m	1.99 t (12.9)
16	2.00 br d (10.7)	1.92 br s	2.03 o	2.00 br d (11.7)	1.41 br s
	2.23 o	2.03 td (3.7, 13.6)	2.26 t (13.1)	2.22 t (12.8)	2.51 br d (13.2)
17	–	–	–	–	–
18	3.42 dd (3.3, 13.7)	3.18 dd (4.0, 13.6)	3.35 br d (13.7)	3.41 dd (3.7, 13.7)	–
19	1.52 o	1.22 m	1.91 o	1.91 o	5.27 s
	2.18 o	1.70 o	2.44 t (13.7)	2.57 t (13.7)	–
20	–	–	–	–	–
21	1.40 br d (12.2)	1.07 m	1.82 o	1.80 t (13.9)	1.51 o
	1.85 m	1.33 m	2.03 o	2.30 td (4.3, 13.9)	1.74 t (9.3)
22	1.94 br d (13.6)	1.74 o	2.07 o	1.94 o	1.74 t (9.3)
	2.16 o	1.81 td (4.1, 13.8)	–	2.10 m	2.30 m
23	1.29 s	3.70 d (10.9)	1.17 s	1.18 s	0.92 s
	–	4.34 d (10.9)	–	–	–
24	0.96 s	0.93 s	1.06 s	1.00 s	3.70 d (11.0)
	–	–	–	–	4.34 d (11.0)
25	0.83 s	0.92 s	0.82 s	0.80 s	0.83 s
26	1.00 s	1.11 s	0.99 s	0.96 s	1.02 s
27	1.31 s	1.19 s	1.26 s	1.25 s	0.89 s
28	–	–	–	–	–
29	3.61 s	0.88 s	–	–	1.11 s
30	1.22 s	0.87 s	1.58 s	1.55 s	1.04 s
1′	4.74 d (7.3)	5.20 d (7.7)	4.91 d (5.1)	4.93 d (5.7)	5.22 d (7.7)
2′	4.58 t (7.3)	4.09 t (7.7)	4.57 o	4.58 t (5.7)	4.10 t (8.2)
3′	4.22 o	4.16 m	4.29 o	4.35 o	4.16 t (8.2)
4′	4.43 br s	4.46 br s	4.28 o	4.36 br s	4.45 t (9.6)
5′	3.75 d (11.9)	4.47 o	3.83 d (11.5)	3.78 o	4.47 t (9.6)
	4.20 o	–	4.32 o	4.28 o	–
6′	–	–	–	–	–
7′	–	4.23 d (7.1)	–	–	3.69 s
8′	–	1.14 t (7.1)	–	–	–
1″	5.39 d (7.7)	6.33 d (8.1)	6.16 s	5.17 d (7.8)	–
2″	4.03 t (7.7)	4.20 d (7.0)	4.75 br s	4.08 t (7.8)	–
3″	4.25 t (8.8)	4.27 d (8.8)	4.63 dt (3.1, 9.4)	4.18 t (8.9)	–
4″	4.24 o	4.37 t (9.2)	4.29 o	4.28 o	–
5″	3.98 br s	4.03 m	4.59 o	3.80 o	–
6″	4.39 dd (5.0, 11.7)	4.41 dd (4.3, 12.0)	1.63 d (5.9)	4.41 dd (4.2, 7.1)	–
	4.54 br d (10.8)	4.46 dd (2.2, 12.0)	–	–	–

Acasentrioid B (2): amorphous powder; , (c = 0.32, MeOH); HR-ESI-MS m/z: 856.5043 [M + NH4]+ (calculated to be 856.5053 for C44H74NO15). The 1H (pyridine-d 5, 600 MHz) and 13C NMR (pyridine-d 5, 150 MHz) data are shown in Tables 1, 2.

Acasentrioid C (3): amorphous powder; , (c = 0.28, MeOH); HR-ESI-MS m/z: 737.4490 [M + H]+ (calculated to be 737.4471 for C40H65O12). The 1H (pyridine-d 5, 600 MHz) and 13C NMR (pyridine-d 5, 150 MHz) data are shown in Tables 1, 2.

Acasentrioid D (4): amorphous powder; , (c = 0.22, MeOH); HR-ESI-MS m/z: 798.4648 [M + NH4]+ (calculated to be 798.4634 for C41H68NO14). The 1H (pyridine-d 5, 600 MHz) and 13C NMR (pyridine-d 5, 150 MHz) data are shown in Tables 1, 2.

Acasentrioid E (5): amorphous powder; , (c = 0.23, MeOH); HR-ESI-MS m/z: 663.4121 [M + H]+ (calculated to be 663.4103 for C37H59O10). The 1H (pyridine-d 5, 600 MHz) and 13C NMR (pyridine-d 5, 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

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

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 [δH 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 (δH 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

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

Compound	IC50 (μM)	Compound	IC50 (μM)
1	143.04 ± 10.89	16	102.31 ± 9.77
2	156.71 ± 12.67	17	234.75 ± 21.13
3	149.89 ± 13.55	18	203.62 ± 19.78
4	246.03 ± 21.16	19	465.70 ± 35.05
5	236.33 ± 20.21	20	136.24 ± 10.55
6	108.17 ± 8.24	21	306.45 ± 28.00
7	123.75 ± 10.56	22	145.61 ± 12.67
8	107.37 ± 9.59	23	131.47 ± 10.04
9	240.90 ± 18.55	24	188.55 ± 15.22
10	215.13 ± 20.24	25	276.32 ± 25.46
11	402.42 ± 39.54	26	160.31 ± 13.53
12	264.49 ± 22.89	27	132.56 ± 11.76
13	41.42 ± 3.93	28	565.45 ± 47.98
14	584.67 ± 45.59	29	126.57 ± 10.56
15	156.85 ± 11.00

TABLE 4

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

Compound	IC50 (μM)	Compound	IC50 (μM)
1	>100	16	92.55 ± 7.92
2	>100	17	>100
3	>100	18	>100
4	>100	19	>100
5	45.00 ± 3.89	20	>100
6	>100	21	>100
7	>100	22	>100
8	>100	23	>100
9	>100	24	>100
10	50.18 ± 4.72	25	>100
11	>100	26	>100
12	50.96 ± 5.05	27	>100
13	41.42 ± 3.93	28	>100
14	>100	29	>100
15	>100	Quercetin	10.50 ± 1.07

The IC50 > 100 μM was deemed inactive or meant ineffective.

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.