Novel C-17 spirost protostane-type triterpenoids from Alisma plantago-aquatica with anti-inflammatory activity in Caco-2 cells

Abstract

Twenty-one protostane-type triterpenoids with diverse structures, including nine new compounds (1–9), were isolated from the of Alisma plantago-aquatica Linn. Structurally, alisolides A‒F (1–6), composed of an oxole group coupled to a five-membered ring, represent unusual C-17 spirost protostane-type triterpenoids. Alisolide H (8) is a novel triterpenoid with an unreported endoperoxide bridge. Alisolide I (9) represents the first example of 23,24-acetal triterpenoid. Their structures were elucidated based on spectroscopic analysis, wherein the absolute configurations of 4‒6, 8 were further confirmed by the Mo₂(OAc)₄-induced ECD method. Furthermore, all isolates were evaluated for their inhibitory effects on LPS-induced NO production in Caco-2 cells, and all the compounds showed remarkable inhibitory activities, with IC₅₀ values in the range of 0.76–38.20 μmol/L.

Graphical abstract

Nine novel compounds (1–9), were isolated from the of Alisma plantago-aquatica Linn. The isolated compounds proved to possess a very regular structure–activity relationship on inhibitory effects on LPS-induced NO production in Caco-2 cells.

1. Introduction

Protostane-type triterpenoids (PTs), mainly reported in plants of the genus Alisma Linn., are a class of natural products with fascinating and diverse carbon skeletons and intriguing biological activities. The characteristic groups of β-CH₃ in position C-10 and C-14, α-CH₃ groups in position C-8 and 21R configurations have made PTs hot molecules in the novel natural products discovery fields. According to our database, there are 115 bioactive PTs, such as alisols A‒V¹, alismanols A‒Q² and Alismanins A‒C³, that have been isolated from Alisma Linn. genus, and some of them displayed remarkable pharmacological effects, including anti-hyperlipidemic, antidiabetes, anti-tumor, anti-complement, anti-inflammatory, anti-HBV4, 5, 6, 7, 8, 9, etc. Of these, however, the majority just belongs to carbonylation derivatives of alisols A‒F type PTs. Some other novel and diverse PTs have rarely been reported, such as spirost protostane-type PTs, which not only have incredibly complicated structures but also show significant biological activities in our preliminary study.

To investigate unique and biologically active natural spirost protostane-type triterpenoids from plants of the genus Alisma Linn., we described the investigation on systematic phytochemical profiles of 17-spirost PTs in the rhizome of Alisma plantago-aquatica, called “Chuan Zexie”, which is widely distributed in the marshes in Sichuan province of China. However, the low concentrations of these compounds prevented us from isolating and identifying them. In this work, UPLC-Orbitrap-QDa guided isolation as well as rapid separation were performed and resulted in the isolation of nine undescribed PTs, alisolides A‒I (1–9), with an spirost functionality at C-17, together with twelve known PTs, neoalisol (10)¹⁰, alisol B (11)¹¹, alisol B 23 acetate (12)¹¹, alisol C 23 acetate (13)¹¹, alisol A (14)¹², alisol A 24 acetate (15)¹¹, 16-oxo-alisol A(16)¹³, alisol E 23 acetate(17)¹⁴, alisol F (18)¹⁴, alisol F 24 acetate (19)¹⁵, 11-anhydroalisol F (20)¹⁶, alisol O (21)¹⁷, which can be divided into nor-protostane-type PTs, carbonylation derivatives of alisol A and alisol B-type PTs, and dehydroxylation derivatives of alisol A and alisol B-type PTs (see Fig. 1). A. plantago-aquatica has been widely used in traditional Chinese medicine for excreting dampness and eliminating edema¹ and for the treatment of diarrhea¹⁸. As we all know, sometimes diarrhea is related to intestinal inflammations, hence all isolates were evaluated for their inhibitory effects on LPS-induced NO production in Caco-2 cells.

Figure 1.

The structures of compounds 1–21.

2. Results and discussion

Alisolide A (1), was purified as a white, amorphous powder, and its molecular formula was found to be C₂₆H₃₆O₅, established from HR-ESI-MS data at m/z 429.2617 ([M + H]⁺, Calcd. for 429.2641), indicative of nine degrees of unsaturation. The ¹H NMR spectrum (Table 1) of 1 displayed characteristic signals attributed to six methyl protons at δ_H 1.08 (3H, s, CH₃-29), 1.11 (3H, s, CH₃-28), 1.12 (3H, s, CH₃-19), 1.17 (3H, s, CH₃-18), 1.19 (3H, d, J = 7.0 Hz, CH₃-21), 1.53 (3H, s, CH₃-30), one oxygenated methane or oxymethine at δ_H 4.11 (1H, m, H-16), and one olefinic proton at δ_H 5.87 (1H, s, H-12). The ¹³C NMR spectrum (Table 1) of 1 showed 26 carbon signals, indicating the presence of six methyls, six methylenes, five methines (one oxygenated and one olefinic), nine quaternaries (three carbonyls, one olefinic), and chemical structure of 1 was similar to that of alisolide (23-nor-protostane)¹⁹. A detailed comparison of its ¹H and ¹³C NMR data (Table 1) with those of alisolide, the C-16 carbon signal was shifted to δ_C 73.8 toward low field, suggesting that a free hydroxy group was attached to C-16, as confirmed by the 2D HMBC correlations (Fig. 2), including key correlations from H-16 to C-15, C-17, and C-20, as well as ¹H–¹H COSY correlations of H-16/H-15. Moreover, the relative configuration of 1 was determined by NOESY data (Fig. 3). The key NOESY cross-peaks of H-20/H-12, and CH₃-21/H-16 indicated that C-20 and C-17 were R* stereochemistry.¹⁹ The NOESY spectrum correlations were also observed between H-15α to H-16, CH₃-18; CH₃-18 to H-5, indicating that H-5, H-16, and CH₃-18 were α-orientation, and H-9β to CH₃-19, CH₃-30 indicated that CH₃-19, CH₃-30 were β-configuration (Fig. 3). Therefore, the structure of 1, which belongs to PT with an unusual C₁₇ spirost, was asserted as a 23-nor-protostane and named alisolide A.

Table 1.

¹H and ¹³C NMR data of compounds 1‒3 (CDCl₃).

No.	1a		2a		3b
	δH	δC	δH	δC	δH	δC
1	1.89, m	32.3	2.45, m; 1.88, m	32.5	2.11, m; 1.98, m	32.5
2	2.71, m (α); 2.33, m (β)	33.6	2.69, m; 2.31, m	33.9	2.66, m; 2.43, m	33.9
3		219.2		219.3		219.2
4		47.0		47.1		47.0
5	2.19, m	48.5	2.16, d, 12.0	48.8	1.98, m	49.4
6	1.32, m	20.1	1.57, m; 1.30, m	20.4	1.53, m; 1.31, m	20.4
7	2.18, m; 1.39, m	33.2	2.32, m; 1.39, m	33.3	1.99, m; 1.27, m	34.3
8		44.6		45.0		42.4
9	2.73, s	56.0	2.63, s	56.1	1.95, d, 9.0	49.0
10		37.3		37.5		36.9
11		199.4		198.6	4.32, dd, 9.0, 4.0	69.0
12	5.87, s	124.1	5.89, s	124.6	5.66, d, 4.0	124.6
13		162.4		163.7		144.1
14		49.3		47.0		46.2
15	2.37, m (α); 1.60, m (β)	40.6	2.27, m (α); 1.55, m (β)	39.0	1.35, m (α); 2.15, m (β)	38.7
16	4.11, m	73.8	4.35, m	77.9	4.14, t, 8.0	78.8
17		91.3		94.9		95.5
18	1.17, s	24.5	1.21, s	24.9	1.02, s	23.9
19	1.12, s	24.9	1.10, s	25.2	1.11, s	25.4
20	2.84, m	32.5	2.91, m	32.5	2.78, t, 7.0	32.5
21	1.19, d, 7.0	14.3	1.02, d, 7.0	18.6	1.04, d, 7.0	18.7
22	2.62, dd, 17.0, 12.0	36.2	3.34, dd, 17.5, 8.0	38.8	3.28, dd, 19.0, 8.0	39.1
	2.69, dd, 17.0, 9.0		2.21, d, 17.5		2.16, d, 19.0
23		175.3		177.0		177.6
28	1.11, s	29.5	1.10, s	29.6	1.08, s	29.4
29	1.08, s	19.4	1.07, s	19.5	1.07, s	20.3
30	1.53, s	24.8	1.44, s	25.5	1.33, s	28.0

^aIn ¹H (600 MHz) and ¹³C NMR (150 MHz).

^bIn ¹H (500 MHz) and ¹³C NMR (125 MHz).

Figure 2.

Selected HMBC (→) and COSY (—) correlations of compounds 1–9.

Figure 3.

Selected NOESY () correlations of compounds 1–9.

Alisolide B (2), originally obtained as a white, amorphous powder, was designated with the molecular formula C₂₆H₃₆O₆ (nine degrees of unsaturation), as determined by HR-ESI-MS data at m/z 445.2572 ([M + H]⁺, Calcd. for 445.2590). Based on the detailed analysis of its ¹H and ¹³C NMR spectra (Table 1), the chemical structure of 2 was found to be similar as that of 1. The difference was that substitution at C-16 location was hydroxy unit (‒OH) in 1, whereas hydroxyl peroxide unit (‒OOH) in 2. Compared with 1, the H-16 NMR signal was shifted to low field with Δδ_H +0.24 in ¹H NMR data, and Δδ_C +3.6 in ¹³C NMR data, suggesting that 2 possessed a free hydroxyl peroxide group at C-16²⁰, as confirmed by the tandem mass spectrometric fragmentation behaviors (ESI-MSⁿ) of 2. The structures of 2 and 1 were similar, so the tandem mass spectrometric fragmentation behaviors (ESI-MSⁿ) of 1 and 2 were investigated by UHPLC/LTQ-Orbitrap performed in the positive mode as shown in Supporting Information Figs. S11–12 and S23–24. In the ESI-MS³ spectra of compound 2, we can see the cleavage of the peroxide-bridge generating an abundant ion at m/z 427.2743 which represents the loss of a H₂O (18 Da). And the fragmentation behaviors of compound 2 are similar to compound 1, the difference is that there are 16 (O) units more than that of compound 1 for all corresponding fragments, which means that an carbonyl group was generated after the cleavage of the peroxide-bridge21, 22.

The planar structure of 2 was supported based on its HSQC, HMBC, and ¹H–¹H COSY data. The NOESY correlations of H-20/H-15β, and CH₃-21/CH₃-30, H-12 indicated that C-20 and C-17 were R* and S* configurations, respectively¹⁹. In the NOESY spectrum (Fig. 3), the correlations between H-15α and H-16, CH₃-18; CH₃-18 and H-5 indicated that H-5, H-16, and CH₃-18 were all α-orientation, while interactions of H-9β and CH₃-19, CH₃-30 showed that CH₃-19, CH₃-30 were β-configuration. Accordingly, the structure of 2, which represents a previously undescribed C₁₇ spirost PT, was defined and named alisolide B.

Alisolide C (3) was originally obtained as a white, amorphous powder and had the molecular formula C₂₆H₃₈O_5, according to the ¹³C NMR and HR-ESI-MS analysis (m/z 431.2789, [M + H]⁺ (Calcd. for 431.2797), which was 2 mass units more than that of compound 1. The 1D NMR data (Table 1) of 3 showed close similarities to those of 1, and the main difference was that chemical shift at C-11 (δ_C 69.0, δ_H 4.32, dd, 9.0, 4.0) in 3 was reduced, compared to that carbon (δ_C 199.4) in 1. Moreover, the C-12 (δ_H 5.66, d, 4.0) in 3 was deshielded. The data indicated that a hydroxyl group was substituted in C-11. The planar structure of 3 was supported by its HSQC, HMBC, and COSY data. The same relative configuration was inferred for compounds 3 and 2 on the NOESY spectra (Fig. 3). Besides, 11-OH and 16-OH was assigned to a β position because the NOESY correlation was observed between H-11, H-16 and CH₃-18. Therefore the structure of 3 was elucidated and given a trivial name alisolide C.

Alisolide D (4), isolated as a white, amorphous powder, had a molecular formula of C₃₀H₄₆O_6, indicative of eight degrees of unsaturation, as assigned by a protonated molecular ion at m/z 503.3368 ([M + H]⁺ Calcd. for 503.3373) from its HR-ESI-MS data. The ¹H NMR spectrum showed eight methyl signals at δ_H 0.96 (3H, d, 7.0 Hz, CH₃-21), 1.06 (3H, s, CH₃-28), 1.08 (3H, s, CH₃-19), 1.11 (3H, s, CH₃-29), 1.22 (3H, s, CH₃-26), 1.24 (3H, s, CH₃-27), 1.27 (3H, s, CH₃-18), and 1.36 (3H, s, CH₃-30). Analysis of the ¹³C NMR and DEPT-135 NMR data of 4 revealed 30 carbon signals, which were attributed to eight methyls, seven methylenes, five methines, and ten quaternary carbons. The presence of a carbonyl carbon at δ_C 195.8 (C-11), and two olefinic carbons δ_C 140.9 (C-12), and δ_C 138.6 (C-13) accounted for two of eight degrees of unsaturation, illustrating that 4 had a α,β-unsaturated ketones in C ring. In the HMBC spectrum (Fig. 2), the key correlations of H-9 (2.84, s) with C-11, C-12, and C-14, of CH₃-30 with C-13, and C-14 further verified our conclusion. A side-by-side comparison of ¹H and ¹³C NMR data of 4 with those of neoalisol (10)¹⁰ indicated that 4 had a similar side chain structure with neoalisol, which was supported based on the key HMBC correlations of CH₃-21 to C-17 (δ_C 92.4), C-20 (δ_C 38.1), and C-22 (δ_C 37.8), of CH₃-26 and CH₃-27 to C-24 (δ_C 78.4), and C-23 (δ_C 77.6); and key ¹H–¹H COSY correlations of H-20 (δ_H 3.20, m) to CH₃-21, and H-22 (δ_H 2.19, m; 1.83, m), of H-23 (δ_H 4.43, br d, 9.5 Hz) to H-22, and H-24 (δ_H 3.15, br d, 10.0 Hz) (Fig. 2). Therefore, the planar structure of 4 was determined. Furthermore, in the NOESY experiment (Fig. 3), the interaction of CH₃-21 with H-16α indicated that C-17 was R^* configuration. The absolute configuration of C-24 was determined via Mo₂(OAc)₄-induced ECD method developed by Snatzke23, 24, 25, 26. The positive Cotton effect at 316 nm indicated a 24 S configuration²⁷. Thus the structure of 4 was defined and named alisolide D.

Alisolide E (5) was isolated as a white, amorphous powder with a molecular formula of C₃₀H₄₈O₆, as deduced from the HR-ESI-MS data at m/z 487.3416 ([M-H₂O + H]⁺, Calcd. for 487.3423). The ¹H and ¹³C NMR spectra showed characteristic signals attributed to eight methyl characteristic signals of protostane triterpenoid, and then an enol unit, three oxygenated methine groups. The structure of 5 was deduced based on the HMBC correlations (Fig. 2) from H-11 to C-9, C-10, C-12, and C-13; H-12 to C-9, C-14, and C-17; H-16 to C-13, C-14, C-15, and C-17; CH₃-21 to C-17, C-20, and C-21; H-23 to C-17, C-22, C-24; CH₃-26 and 27 to C-24, and C-25, coupled with the NOESY experiment (Fig. 3). The NOESY experiment showed correlation between CH₃-21 to H-12 indicated that C-17 spirost ring group was S^* configuration. The interaction of H-16 to CH₃-18 suggested that 16-OH was β orientation. The Mo₂(OAc)₄-induced ECD spectrum of 5 (Fig. 4) showed a positive Cotton effect at 313 nm, indicating a C-24 S configuration. The structure of 5 was therefore elucidated and given a trivial name alisolide E.

Figure 4.

Mo₂(OAc)₄-induced ECD spectra of compounds 4–6.

Alisolide F (6) was obtained as a white, amorphous powder. Its molecular formula was established as C₃₀H₄₆O₅ according to the HR-ESI-MS peak at m/z 487.3412 [M + H]⁺ (Calcd. for 487.3423), which was 18 (H₂O) mass units less than that of compound 5. The similarities of ¹H and ¹³C NMR data of 6 and 5 suggested that these two compounds belong to 17-spirost PTs, and their structures were found to be exactly similar. The difference was that the hydroxyl group (11-OH) was dehydrated with the proton (H-9) in 5 to form a double bond (Δ_9,11) in 6. The plane structure of 6 was supported by its HSQC, HMBC, and COSY data. The same relative configuration was inferred for compounds 6 and 5 on the NOESY spectra (Fig. 3). The Mo₂(OAc)₄-induced ECD spectrum of 6 (Fig. 4) showed a positive Cotton effect at 296 nm, indicating a C-24 S configuration. Hence, the structure of 6 was defined and named alisolide F.

Alisolide G (7), isolated as a white, amorphous powder, showed a protonated molecular ion at m/z 529.3519 ([M + H]⁺, Calcd. for 529.3529) in the HR-ESI-MS analysis, in accordance with the molecular formula C₃₂H₄₈O₆. Inspection of the 1D and 2D NMR spectra of 7 and alisol Q 23 acetate²⁸ suggested that these two compounds shared the same planar structure. An α,β-unsaturated ketones unit was also located in 7, which was confirmed by the HMBC correlations of C-ring (H-9 to C-11, C-12, and C-14; H-12 to C-9, and C-14; CH₃-30 to C-13). A hydroxy group was present in 7, and the position of hydroxyl group was assigned to be at C-17 based on the HMBC correlations (Fig. 2) from both CH₃-21 to C-17, and H-12 to C-17. The structure of 7 was supported by its HSQC, HMBC, and COSY data, together with the NOESY correlations. The NOESY correlations of CH₃-30 (δ_H 1.34, s) and H-20 (δ_H 2.10, m), H-20 and H-12 suggested the β configuration of C-17, and the NOSEY correlation of H-20 (δ_H 2.10, m) and H-12 (δ_H 6.16, s) revealed that C-20 was R^∗ configuration. In addition, the NOSEY correlations from H-24 to CH₃-27, and H-23 to CH₃-26 in combination with the characteristic NMR data of δ_H 5.25 (1H, td, J = 9.0, 4.5 Hz, H-23) and δ_H 3.03 (1H, d, J = 9.0 Hz, H-24) suggested that 7 shared the same side chain with 16β-acetoxy alisol B24, 29. Thus, the structure of 7 was determined and named as alisolide G.

Alisolide H (8), isolated as a white, amorphous powder, and showed a protonated molecular ion at m/z 501.3208 ([M + H]⁺, Calcd. for 501.3216) in the HR-ESI-MS analysis, corresponding to the molecular formula C₃₀H₄₄O₆. The ¹H NMR and ¹³C NMR data of 8 showed close resemblances to the known compound 16-oxo-11-anhydroalisol A³⁰ with same structure of A, B, C and D rings, differing only in that of their side chains. A detailed comparison of its ¹H and ¹³C NMR data with those of toonaciliatavarin C³¹ and garcimultiflorone Q³² indicated the presence of the same hydroperoxy ring substructure in 8 according to its degree of unsaturation, in which the peroxide-bearing quaternary carbon was substantially shifted to low filed compared to oxygen-bearing quaternary carbon, as confirmed by the HMBC correlations (Fig. 2) from CH₃-21 (δ_H 1.31, s) to C-17 (δ_C 139.2), C-20 (δ_C 82.4), and C-22 (δ_C 42.5); CH₃-26 (δ_H 1.37, s) and 27 (δ_H 1.19, s) to C-24 (δ_C 82.8), and 25 (δ_C 83.3). In order to confirm the peroxide-bearing substitutation, the tandem mass spectrometric fragmentation behaviors (ESI-MSⁿ) of 8 was investigated (Supporting Information Figs. S85 and 86). In the ESI-MS² spectra, we can see the cleavage of the peroxide-bridge generating an ion at m/z 467.3172, which represents the loss of two hydroxyl groups (34 Da)21, 22. This hydroperoxy ring structure was rarely seen in triterpenoids. The relative configuration of 8 was deduced from the NOESY correlations and Mo₂(OAc)₄-induced ECD method. The key correlations between H-12 to CH₃-21; H-23 to CH₃-21, and CH₃-26; H-24 to CH₃-27, and a positive Cotton effect at 303 nm in Mo₂(OAc)₄-induced ECD spectrum of 8 (Fig. 5) indicated that C-23 and C-24 were S and R configuration, respectively. Thus, the structure of 8 was determined and named as alisolide H.

Figure 5.

Mo₂(OAc)₄-induced ECD spectra of compound 8.

Alisolide I (9), obtained as a white, amorphous powder, and its HR-MS showed a protonated molecular ion at m/z 569.3840 ([M + H]⁺, Calcd. 569.3842), corresponding to the molecular formula C₃₅H₅₂O₆. The NMR data were analyzed and the structures of 9 and 8 were found to be very similar, differing only in C-17 side chain. This conclusion was further supported by the HMBC spectrum (Fig. 2) of CH₃-21 (δ_H 1.20, d, 7.0) to C-17 (δ_C 138.3), C-20 (δ_C 27.6), and C-22 (δ_C 38.8); CH₃-26 (δ_H 1.17, s) and 27 (δ_H 1.04, s) to C-24 (δ_C 88.3), and 25 (δ_C 70.1); H-24 (δ_H 3.40, d, 6.0) to C-22; H-23 (δ_H 3.69, m) to C-20, C-25, and C-1′ (δ_C 101.5); H-1′ (δ_H 5.02, t, 4.5) to C-2′ (δ_C 27.8); CH₃-5′ (δ_H 2.15, s) to C-3′ (δ_C 37.8), and C-4′ (δ_C 208.6). The relative configuration of 9 was deduced from the NOESY correlations (Fig. 3). The doublet of H-24 (J = 6.0 Hz) observed in the ¹H NMR spectrum indicated the threo 23, 24 configuration. On the basis of the 20R configuration in protostane triterpenoids isolated from Alisma. orientale and the correlations of H-20β (δ_H 2.91, m) to H-23 and H-12 (δ_H 6.65, dd, 10.0, 3.5 Hz), H-24 to H-1′ observed in its NOESY spectrum, the 23R* and 24S* configurations of compound 9 were established24, 31. Thus, the structure of 9 was defined and named alisolide I.

Chronic and acute diarrhea is strongly correlated with intestinal inflammations³³. Considering the treatment for diarrhea of the A. plantago-aquatica, the aim of this study is therefore to further investigate the anti-inflammatory activities of the identified PTs of A. plantago-aquatica, including the six unique 17-spirost PTs, using an in vitro model of inflammation in Caco-2 epithelial cell lines.

The anti-inflammatory activities of the novel 17 spirost PTs (1–7) and alisolides H–I (8–9), and other PTs (10–21) were evaluated on the NO production in LPS-stimulated Caco-2 cells, and the inhibition results are shown in Table 4. All the PTs showed remarkable inhibitory activities against the NO production with IC₅₀ ranging from 0.76 to 38.20 μmol/L, compared to dexamethasone, a drug currently used for the treatment of anti-inflammation. Of these, 17R-spirost PTs showed significant inhibitory activities, displaying an IC₅₀ of 1.98–12.00 μmol/L, and, in contrast, 17-spirost nor-PTs existed moderate inhibitory with IC₅₀ from 14.60 to 36.15 μmol/L. Alisol B 23 acetate (12), the major PT in Alisma, showed an IC₅₀ value of 0.76 μmol/L.

Table 4.

Effects of compounds 1–21 on NO production in LPS-stimulated Caco-2 cells.

Compd.	IC50 (μmol/L)	Compd.	IC50 (μmol/L)	Compd.	IC50 (μmol/L)
1	36.15	8	11.48	15	4.64
2	14.60	9	12.00	16	34.69
3	26.93	10	4.43	17	23.49
4	1.98	11	0.88	18	27.15
5	38.20	12	0.76	19	21.86
6	20.56	13	12.91	20	1.17
7	11.20	14	2.19	21	1.67

Positive control: dexamethasone (0.5 μmol/L, inhibition rate 75.26%).

The structure‒activity relationships of the isolated compounds were illustrated in Fig. 6, in which 17R-spirost protostane-type PTs showed dramatic improvement in anti-inflammatory activities than 17S close analogues, suggesting that spatial configuration is essential to the anti-inflammatory activity. For nor-PTs (1–3), in contract, a decrease in activity was observed when the 17R absolute configuration changed to 17S, which verified the configuration issue of crucial importance. Moreover, the hydroxy group in position 11 (3) displayed moderate reduced anti-inflammatory activity compared to ketone replacement (2). In carbonylation derivatives of alisol F type PTs (18–21), there was no significant difference between alisol F (18) and alisol F 24 acetate (19) suggesting that C-24 acetate has no effect on anti-inflammatory activity, and the analogues (20–21) showed similar potent activities. Additionally, compound 20 achieved a significantly improved anti-inflammatory activity versus the 11-hydrogen analogue 18, identifying that the double bond in position 11 provides a significant improvement in potency. Similarly, alisol A 24 acetate (15) appeared in close anti-inflammatory activity (IC₅₀ = 4.64 μmol/L) versus alisol A (14, IC₅₀ = 2.19 μmol/L). Replacement of the 16-hydrogen with ketone (16) remained to achieve reduced activity for alisol A-type PTs. For alisol B-type PTs, and replacement of the 16-hydrogen with either ketone (13) resulted in a modest loss of anti-inflammatory activities and, in contrast, the 23-acetyl group (compound 12) proved to be not changed compared to 11.

Figure 6.

Structure‒activity relationships of isolated compounds.

3. Conclusions

To sum up, twenty-one protostane-type triterpenoids with diverse structures, including nine new ones (1–9), were isolated and identified from the rhizome of A. plantago-aquatica Linn. distributed in the marshes in Sichuan province of China. Structurally, alisolides A‒F (1–6) represent an unprecedented C₁₇ spirost protostanes-type triterpenoids with an oxole group coupled to a five-membered ring; Alisolide G (8) is a novel triterpenoid with a specific endoperoxide bridge; Alisolide I (9) represents the first example of 23,24-acetal triterpenoid. All the PTs showed remarkable inhibitory activities against the NO production with IC₅₀ ranging from 0.76 to 38.20 μmol/L, and 17R-spirost PTs showed significant inhibitory activities, displaying an IC₅₀ of 1.98–12.00 μmol/L. Compounds 11, 12, 20 and 21 exhibited significant inhibitory effects against NO production in Caco-2, with IC₅₀ values of 0.88, 0.76, 1.17 and 1.67 μmol/L, respectively.

4. Experimental section

4.1. General experimental procedures

Optical rotation data were acquired on an Autopol VI polarimeter from Rudolph Research Analytical (Hackettstown, NJ, USA). IR data were determined on a Thermo Scientific Nicolet iS5 FT-IR spectrometer and processed using the Thermo Scientific OMNIC v9.1 software (San Jose, CA, USA). The Shimadzu UV-2450 spectrophotometer (Shimadzu, Japan) was used to measure the UV data. HR-ESI-MS data were measured on an LTQ-Orbitrap Velos Pro hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). NMR spectra were acquired on a Bruker Avance III HD Ascend 500 MHz spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) or Bruker Avance III HD 600 MHz spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) using tetramethylsilane as an internal standard, with chemical shifts (δ) expressed in ppm. All of the NMR data were reported in the paper were derived from ¹H NMR, ¹³C NMR, DEPT-135 NMR, ¹H–¹H COSY, HSQC, HMBC, NOESY experiments. CD spectra were recorded in DMSO determined on a JASCO J-815 spectropolarimeter. Preparative HPLC was carried out on Agilent 1200 HPLC system with a PRC-ODS column (50 cm × 34 mm). Semi-preparative high-performance liquid chromatography (HPLC) separations were carried out on an Agilent 1100 liquid chromatograph with a DAD or VWD detector using a ZORBAX SB-C18 (25 cm × 9.4 mm, 5 μm; Agilent Technologies, Palo Alto, CA, USA) and Eclipse XDB-C18 column (25 cm × 9.4 mm, 5 μm; Agilent Technologies, Palo Alto, CA, USA). Column chromatography (CC) was carried out on silica gel (200–300 mesh; Qingdao Marine Chemical, Inc., Qingdao, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). The UPLC–PDA-QDA analysis was performed on Waters UPLC H-Class system (Waters Corporation, Milford, MA, USA) with PDA and QDa detectors conducting on a Waters BEH C18 column (100 mm × 2.1 mm, 1.8 μm).

4.2. Plant material

The rhizome of A. plantago-aquatica Linn. was collected from Sichuan province, China, in December 2016 and identified by Prof. De-an Guo. A voucher specimen (Y141029) was deposited at the National Engineering Laboratory for Traditional Chinese Medicine Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

4.3. Extraction and isolation

The powdered rhizome of A. plantago-aquatica (5.0 kg) was extracted by infusion with 100% MeOH at room temperature (24 h, 3 times). Subsequently, the residue was filtered and the solvent was evaporated under reduced pressure in a rotary evaporation equipment (Buchi, Switzerland) at a temperature of 40 °C. And then the obtained extract (573.2 g) was dissolved in distilled H₂O and successively partitioned with n-hexane, CH₂Cl₂, EtOAc, and BuOH. The CH₂Cl₂ portion (142.5 g) was purified on a silica gel column (1.5 kg) to give thirty-five fractions, Fr1‒Fr35, through gradient elution with changing ratios of n-hexane:EtOAc from 100:0 to 0:100.

Fraction 9 (3.5 g) was loaded onto RP-C18 column on MHPLC system with a gradient system of CH₃CN–H₂O (15:85–40:60, v/v) to obtain 10 fractions (F9a‒F9i) and three compounds, 14 (15.0 mg) , 11 (20.2 mg) and 13 (10.5 mg). Fraction 9i (55.0 mg) afforded 7 (2.5 mg) after semipreparative HPLC eluted with a gradient of MeOH‒acetonitrile‒H₂O (75:10:15, v/v) on an Eclipse XDB-C18 column. Fraction 10 (4.1 g) was applied to RP-C18 column with CH₃CN–H₂O (stepwise 20:80–40:60, v/v) as mobile phase to obtain four fractions (F10a‒F10d), and F10c further purified by semi-preparative HPLC (MeOH–H₂O, 85:15, 3.0 mL/min) to give compounds 1 (1.9 mg) and F10d was separated over a ZORBAX SB-C18 column (MeOH–H₂O, 80:20, 3.0 mL/min) to yield 20 (3.5 mg) and 21 (4.0 mg). Fraction 12 (4.5 g), collected in the radio of n-hexane:EtOAc at 1:1 of the gradient elution, was purified by preparative HPLC with a gradient elution (CH₃CN–H₂O, 15:85–80:20, 25.0 mL/min) to yield compound 18 (15.0 mg) and eight fractions (F12a‒F12h). The fraction 12c (80.0 mg) was separated on a semipreparative HPLC using an Eclipse XDB-C18 column (20% CH₃CN:80% H₂O to 100% CH₃CN:0% H₂O), giving 2 (1.8 mg) and in the same condition, compound 3 (1.1 mg) was obtained from F12d. Semipreparative HPLC of Fr12e (55.0 mg) on an Eclipse XDB-C18 column yielded 19 (8.0 mg). Further purification of Fr12f and Fr12g by semipreparative HPLC separately on an Eclipse XDB-C18 column (CH₃CN–H₂O, 70:30) and an SB-C18 column (CH₃CN–H₂O, 85:15) afforded 8 (2.3 mg) and 9 (2.6 mg). Fr12h (400 mg) was separated on an Eclipse XDB-C18 column (CH₃CN–H₂O, 70:30–100:0), giving 6 (2.3 mg). Fraction F13 was repeatedly purified by silica gel CC (Hexane‒EA, from 20:1 to 50:50), preparative HPLC (CH₃CN–H₂O, 10:90–100:0, 10.0 mL/min) and semi-preparative HPLC (CH₃CN–H₂O, 70:30–100:0, 3.0 mL/min) to give compounds 4 (2.0 mg), 10 (3.5 mg) and 12 (200 mg). The fraction F14 was separated on Sephadex LH-20 (500 mm × 30 mm, 50 g) eluted with MeOH–Cl₂Cl₂ (1:1) to afford three main fractions F14a–c and F14a was further fractionated via RP-C18 column chromatography eluted with a step gradient of CH₃CN–H₂O (from 10:90–30:70, 25.0 mL/min) to obtain three fractions (F14a1‒3). Fraction F14a2 (70.2 mg) was separated over a ZORBAX SB-C18 column (CH₃CN–H₂O, 50:50–80:20, 3.0 mL/min) to yield compound 15 (5.1 mg) and 17 (3.5 mg). Fraction F14a3 (82.2 mg) was purified by semipreparative HPLC on an Eclipse XDB-C18 column, developed with acetonitrile‒H₂O (30:70–100:0, v/v) to give compound 5 (2.8 mg) and 16 (3.2 mg).

4.3.1. Alisolide A (1)

White, amorphous powder; UV (MeOH) λ_max (logε): 203.8 (4.0), 242.6 (4.2); IR (KBr) ν_max: 3431, 2973, 2947, 2872, 1780, 1694, 1664, 1456, 1372, 1176 cm⁻¹; HR-ESI-MS (positive) at m/z 429.2617 [M + H]⁺ (Calcd. for C₂₆H₃₆O₅ 429.2641); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data see Table 1.

4.3.2. Alisolide B (2)

White, amorphous powder; UV (MeOH) λ_max (logε): 203.6 (3.9), 241.6 (4.0); IR (KBr) ν_max: 3437, 2973, 2925, 2878, 1771, 1700, 1670, 1376 cm⁻¹; HR-ESI-MS (positive) at m/z 445.2572 [M + H]⁺ (Calcd. for C₂₆H₃₇O₆ 445.2590); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data see Table 1.

4.3.3. Alisolide C (3)

White, amorphous powder; UV (MeOH) λ_max (logε): 202.0 (4.1), 248.0 (3.5); IR (KBr) ν_max: 3432, 2962, 2941, 2878, 1742, 1691, 1465, 1382, 1260 cm⁻¹; HR-ESI-MS (positive) at m/z 431.2789 [M + H]⁺ (Calcd. for C₂₆H₃₉O₅ 431.2797); ¹H (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data see Table 1.

4.3.4. Alisolide D (4)

White, amorphous powder; ${\left[\alpha \right]}_{\text{D}}^{20}$+ 5.8 (c 0.1, MeOH); UV (MeOH) λ_max (logε): 201.6 (4.0), 284.4 (4.0); IR (KBr) ν_max: 3452, 2970, 2929, 2869, 1700, 1646, 1462, 1376 cm⁻¹; HR-ESI-MS (positive) at m/z 503.3368 [M + H]⁺ (Calcd. for C₃₀H₄₇O₆ 503.3373); Mo₂(OAc)₄-induced ECD (DMSO) 316 (Δε 7.48) nm; ¹H (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data see Table 2.

Table 2.

¹H (500 MHz) and ¹³C (125 MHz) NMR data of compounds 4‒7.

No.	4a		5a		6a		7b
	δH	δC	δH	δC	δH	δC	δH	δC
1	1.95, m	32.5	2.15, m	32.4	2.22, m; 1.68, m	39.6	2.66, m; 2.04, m	33.4
2	2.71, m;	33.8	2.67, m	33.9	2.80, td, 14.5, 6.0	34.8	2.81, m	34.4
	2.32, m		2.45, m		2.30, ddd, 14.5, 4.5, 3.0		2.32, m
3		219.3		219.7		216.5		218.8
4		47.1		47.0		48.5		47.5
5	2.19, m	48.3	2.00, m	49.3	1.86, dd, 10.5, 8.5	44.9	2.30, m	48.3
6	1.48, m; 1.30, m	20.3	1.51, m; 1.32, m	20.4	1.73, m	17.5	1.48, m; 1.34, m	20.7
7	2.17, m	33.6	1.97, m	34.2	2.01, m, 1.33, m	22.8	2.27, m; 2.11, m	33.9
8		50.6		42.3		39.5		44.9
9	2.84, s	54.8	1.92, d, 9.0	49.3		153.5	2.82, s	56.2
10		37.4		36.8		38.3		38.0
11		195.8	4.30, dd, 9.0, 4.0	69.3	5.62, d, 5.0	114.4		200.6
12	6.58, s (OH)	140.9	5.58, d, 4.0	123.2	5.77, d, 5.0	118.2	6.16, s	121.8
13		138.6		147.7		144.9		177.4
14		44.8		45.3		42.1		52.7
15	1.72, m	30.9	2.06, m	37.8	2.14, dd, 14.0, 8.5	37.2	2.24, m	30.4
	1.40, m		1.19, m		1.24, dd, 14.0, 11.0		1.38, m
16	2.08, m; 1.86, m	32.3	3.76, dd, 10.5, 9.0	80.6	4.06, dd, 11.0, 8.5	81.2	1.90, m	33.5
17		92.4		95.2		95.3		84.3
18	1.27, s	25.1	0.97, s	23.8	1.029, s	21.3	1.40, s	24.8
19	1.08, s	24.4	1.10, s	25.4	1.36, s	23.3	1.28, s	25.7
20	3.20, m	38.1	2.46, m	34.1	2.45, t, 7.0	34.4	2.10, m	35.9
21	0.96, d, 7.0	13.7	0.92, d, 7.0	18.3	0.89, d, 7.0	18.2	1.40, d, 7.0	16.1
22	2.19, m	37.8	2.66, m	36.7	2.66, m	36.5	2.05, m	36.4
	1.83, m		1.69, m		1.70, m		1.82, m
23	4.43, br d, 9.5	77.6	4.51, dd, 10.0, 7.0	76.5	4.52, dd, 10.0, 7.0	76.5	5.25, td, 9.0, 4.5	73.5
24	3.15, br d, 10.0	78.4	3.27, br s	76.0	3.27, s	76.1	3.03, d, 9.0	65.4
25		73.4		73.4		73.4		59.5
26	1.22, s	27.0	1.25, s	27.5	1.27, s	26.9	1.30, s	25.0
27	1.24, s	26.7	1.37, s	26.8	1.38, s	27.4	1.35, s	20.3
28	1.06, s	19.4	1.07, s	20.2	1.01, s	24.5	1.12, s	29.5
29	1.11, s	29.5	1.06, s	29.3	1.15, s	21.3	1.18, s	20.1
30	1.36, s	21.8	1.26, s	27.3	1.034, s	19.6	1.34, s	22.4
24-OH	3.66, d, 10.0
-OAc							2.07, s	21.5,
								170.6

^aIn CDCl₃.

^bIn pyridine-d₅.

4.3.5. Alisolide E (5)

White, amorphous powder; ${\left[\alpha \right]}_{\text{D}}^{20}$+ 53.8 (c 0.1, MeOH); UV (MeOH) λ_max (logε): 204.0 (4.0), 244.4 (3.8); IR (KBr) ν_max: 3426, 2967, 2938, 2869, 1697, 1465, 1378 cm⁻¹; HR-ESI-MS (positive) at m/z 487.3416 [M-H₂O + H]⁺ (Calcd. for C₃₀H₄₇O₅ 487.3423); Mo₂(OAc)₄-induced ECD (DMSO) 313 (Δε 4.09) nm; ¹H (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data see Table 2.

4.3.6. Alisolide F (6)

White, amorphous powder; ${\left[\alpha \right]}_{\text{D}}^{20}$+ 8.0 (c 0.05, MeOH); UV (MeOH) λ_max (logε): 202.0 (4.0), 274.0 (3.9); IR (KBr) ν_max: 3435, 2962, 2925, 2866, 1738, 1447, 1372 cm⁻¹; HR-ESI-MS (positive) at m/z 487.3412 [M + H]⁺ (Calcd. for C₃₀H₄₇O₅ 487.3423); Mo₂(OAc)₄-induced ECD (DMSO) 296 (Δε 3.90) nm; ¹H (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data see Table 2.

4.3.7. Alisolide G (7)

White, amorphous powder; ${\left[\alpha \right]}_{\text{D}}^{20}$+ 130.0 (c 0.05, MeOH); UV (MeOH) λ_max (logε): 202.0 (4.2), 248.6 (4.4); IR (KBr) ν_max: 3461, 2965, 2934, 2875, 1733, 1697, 1649, 1453, 1370, 1235 cm⁻¹; HR-ESI-MS (positive) at m/z 529.3519 [M + H]⁺ (Calcd. for C₃₂H₄₉O₆ 529.3529); ¹H (500 MHz, pyridine-d₅) and ¹³C NMR (125 MHz, pyridine-d₅) data see Table 2.

4.3.8. Alisolide H (8)

White, amorphous powder; ${\left[\alpha \right]}_{\text{D}}^{20}$+ 148.2 (c 0.1, MeOH); UV (MeOH) λ_max (logε): 201.8 (3.9), 286.2 (4.3); IR (KBr) ν_max: 3444, 2970, 2866, 1697, 1602, 1453, 1376, 1233 cm⁻¹; HR-ESI-MS (positive) at m/z 501.3208 [M + H]⁺ (Calcd. for C₃₀H₄₅O₆ 501.3216); Mo₂(OAc)₄-induced ECD (DMSO) 303 (Δε 2.13) nm; ¹H (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data see Table 3.

Table 3.

¹H (500 MHz) and ¹³C (125 MHz) NMR data of compounds 8 and 9 (CDCl₃).

No.	8		9
	δH	δC	δH	δC
1	2.12, m	32.4	2.10, m	32.4
2	2.70, m	33.4	2.74, m	33.4
	2.10, m		2.32, m
3		219.4		219.3
4		47.3		47.3
5	2.30, m	46.2	2.33, m	46.2
6	1.60, m; 1.21, m	19.5	1.56, m; 1.17, m	19.5
7	2.15, m	31.4	2.11, m	31.4
8		48.5		48.2
9	2.37, m	47.6	2.39, m	47.9
10		36.1		36.1
11	7.30, dd, 10.5, 3.5	124.9	6.16, dd, 10.0, 2.0	122.3
12	6.15, dd, 10.5, 2.0	138.1	6.65, dd, 10.0, 3.5	138.2
13		170.5		171.7
14		40.2		39.2
15	2.36, d, 18.5	44.7	2.36, m	44.8
	1.94, d, 18.5		1.91, d, 18.5
16		206.5		207.8
17		139.2		138.3
18	0.94, s	21.8	0.97, s	22.1
19	0.95, s	24.8	0.95, s	24.9
20		82.4	2.91, m	27.6
21	1.31, s	23.4	1.20, d, 7.0	19.6
22	2.70, m	42.5	2.02, m	38.8
	2.17, m		1.67, m
23	3.88, t, 9.5	68.7	3.69, m	75.7
24	3.19, d, 9.0	82.8	3.40, d, 6.0	88.3
25		83.3		70.1
26	1.37, s	26.1	1.17, s	27.1
27	1.19, s	23.9	1.04, s	24.9
28	1.09, s	19.3	1.09, s	19.3
29	1.06, s	29.4	1.06, s	29.4
30	1.18, s	23.9	1.13, s	24.2
1ʹ			5.02, t, 4.5	101.5
2ʹ			1.97, m	27.8
3ʹ			2.52, m	37.8
4ʹ				208.6
5ʹ			2.15, s	29.9

4.3.9. Alisolide I (9)

White, amorphous powder; ${\left[\alpha \right]}_{\text{D}}^{20}$+ 32.0 (c 0.1, MeOH); UV (MeOH) λ_max (logε): 201.8 (4.0), 285.8 (4.2); IR (KBr) ν_max: 3449, 2956, 2932, 2869, 1706, 1682, 1616, 1450, 1369, 1239 cm⁻¹; HR-ESI-MS (positive) at m/z 569.3840 [M + H]⁺ (Calcd. for C₃₅H₅₃O₆ 569.3842); ¹H (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃) data see Table 3.

4.4. Measurement of LPS-induced NO production on Caco-2 cells

Caco-2 Cells, purchased from cell bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in Dulbecco's modified eagle's medium (DMEM) with 20% fetal bovine serum (FBS, ThermoFisher, Cat. No. 10099-141) and 1% streptomycin/penicillin (ThermoFisher, Cat. No. 15140148). The Caco-2 cells were seeded into a 96-well microstate plate at a density of 2 × 10⁶ cells per well and allowed 24 h to adhere before introducing Lipopolysaccharides (1 μg/mL), and then co-incubated with or without increasing the concentrations of the isolates for 24 h at 37 °C. After treatment, the Nitric Oxide concentration in the medium was measured with Nitric Oxide Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) based on the nitrate reductase method. Absorbance was measured at 550 nm against a calibration curve with sodium nitrite standards. The nitric oxide concentration of the wells with cells was subtracted from the nitric oxide concentration of the blank wells without cells to calculate the nitric oxide production. Cell viability of the remaining cells was determined by an MTT (Sigma Chemical Co., St. Louis, MO, USA)-based colorimetric assay34, 35.

Appendix A. Supplementary data

The following is the Supplementary data to this article: