Hirundigenin type C₂₁ steroidal glycosides from Cynanchum stauntonii and their anti-inflammatory activity

Abstract

Six new hirundigenin-type C₂₁ steroidal glycosides, namely hirundigosides E–J (1–6), were obtained from the dried roots of Cynanchum stauntonii. Their chemical structures were elucidated by analyses of HR ESI-TOF MS, 1D, 2D-NMR, and X-ray crystallographic methods together with acidic hydrolysis. Interestingly, they exist in nature as epimers owing to the presence of 14-hemiketal hydroxyl group. Moreover, it is striking that only one set of resonance signals (14β-OH epimer) appeared in the ¹H and ¹³C NMR spectra of compounds 1–6 when measured in CDCl₃, while two sets of resonance signals (14α- and 14β-OH epimers) were observed when measured in pyridine-d₅. It may ascribe to the favorable formation of intramolecular hydrogen bond of aglycone due to the proximate distance of hydroxyl group and oxygen atoms (1.86 nm) in 14β-OH epimer in CHCl₃. The difference of potential energy (ΔG) of 14α-OH epimer to 14β-OH epimer is about 0.73 kcal mol⁻¹ for compound 1 in CHCl₃, while it is unfavorable for the formation of intramolecular hydrogen bond in pyridine and the G values of them are almost equal in 1. Also, CDCl₃ is neither protic solvent nor Lewis acid and it can't induce the isomerization of hemiketal hydroxyl group in hirundigosides, while pyridine is Lewis base and it promotes the isomerization of hirundigosides. In addition, secondary anhydrohirundigenin-type glycosides and anhydrohirundigenin were obtained by mild acidic hydrolysis and their anti-inflammatory activities were also discussed.

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The dried roots of Cynanchum stauntonii (Decne.) Schltr.ex Lévl., together with the other species of the same genus, C. glaucescens (Decne.) Hand.-Mazz., are recorded in the Chinese Pharmacopoeia (ChP) as “Bai-Qian”, and have been used as antitussive and expectorants in China for a long time.¹ Chemical investigation revealed that they contain characteristic C₂₁ steroidal glycosides, especially with the aberrant 13,14:14,15-disecopregnane-type skeleton or 14,15-secopregnane-type skeleton, which exhibited antitumor activity and inhibitory effects on Na⁺/K⁺-ATPase and alpha virus-like RNA viruses.^2–4

In our search for anti-inflammatory constituents from the roots of C. stauntonii,⁵ six new hirundigenin-type C₂₁ steroidal glycosides, namely hirundigosides E–J (1–6), were obtained from the dried roots of C. stauntonii. Herein, we described the isolation and structural elucidation of the new compounds. Meanwhile, we provided the evidence that they existed in nature as epimers owing to the presence of 14-hemiketal hydroxyl group by UPLC-MS/MS and chemical derivatization. Also, the abnormal behavior of their NMR spectrum in different deuterated solvents was discussed by computational calculation. In addition, secondary anhydrohirundigenin-type glycosides and anhydrohirundigenin were obtained by mild acidic hydrolysis of compounds 2 and 6 and their anti-inflammatory activities were also discussed.

Results and discussion

The 95% (v/v) aqueous EtOH extract of the roots of C. stauntonii was chromatographed over an open macroporous resin Diaion HP 20 column eluted with gradient EtOH–H₂O to obtained 4 fractions (A–D). The fraction D (93.0 g, 95% EtOH) was repeatedly subjected to silica gel, Sephadex LH-20, and RP-C₁₈ column chromatography (CC) and semipreparative RP-HPLC to afford 6 new 14,15-secopregnane steroidal glycosides (1–6) (Fig. 1).

Fig. 1 The structures of compounds 1–6 from Cynanchum stauntonii.

Compounds 1–6 showed positive reactions to Libermann–Burchard and Keller–Kiliani reagents, suggesting that they were likely steroidal glycosides with 2-deoxysugar units in the structures.

Hirundigoside E (1), [α]²⁷_D −15.2 (MeOH, c = 0.5), was obtained as colorless needle crystals (CHCl₃/MeOH). Its molecular formula, C₂₈H₄₂O₉, was deduced from the positive-ion HR ESI-TOF MS (m/z 545.2732 [M + Na]⁺, calcd for C₂₈H₄₂O₉Na, 545.2727), indicating eight degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3441 cm⁻¹) and olefinic (1455, 1165 cm⁻¹) functionalities. The ¹H NMR spectrum of aglycone portion exhibited two tertiary methyl protons at δ_H 0.91 (3H, s, H-19) and 1.44 (3H, s, H-21), one olefinic proton signal at δ_H 5.40 (1H, d, J = 4.9 Hz, H-6), two oxygen-substituted methine protons at δ_H 3.49 (1H, m, H-3) and 4.69 (1H, q, J = 3.5 Hz, H-16), two methylene protons at δ_H 3.72 (1H, m, H-15α)/4.16 (1H, d, J = 10.9, H-15β) and 3.73 (1H, m, H-18α)/4.48 (1H, d, J = 9.0, H-18β) (Table 1). The ¹³C NMR spectrum showed two oxygenated tertiary carbon signals at δ_C 108.3 (C-14) and 118.6 (C-20), two oxygenated methylene carbon signals at δ_C 75.3 (C-15) and 77.9 (C-18), and two olefinic carbons at δ_C 139.1 (C-5) and 122.7 (C-6) (Table 3). Comparison of ¹H and ¹³C NMR data with that of hirundigoside D,⁶ a known steroidal glycoside isolated from the roots of Vincetoxicum hirundinaria, revealed that compound 1 contained the aglycone of hirundigenin. An anomeric proton and carbon signals at δ_H 4.29 (1H, d, J = 7.7 Hz)/δ_C 101.1 in the ¹H and ¹³C NMR spectra, along with the presence of one secondary methyl proton signals at δ_H 1.26 (3H, d, J = 6.1 Hz), suggested that 1 contained one 6-deoxysugar with β glycosidic linkage. The sugar moiety was defined as thevetose by its ¹³C resonance data, which matched well with that of anhydrohirundigenin monothevetoside.⁷ The HMBC correlation from δ_H 4.29 (H-1′) to δ_C 79.1 (C-3) attached the sugar moiety at C-3 position of the aglycone. The relative configuration of 1 was established by ROESY experiment (Fig. 2). The ROESY correlation from CH₃-19 to H-8 indicated that H-8 was β-oriented; while the correlation from 14-OH to 19-CH₃, 15β-H, and 18β-H suggested the relative orientation of 14-OH was β. The relative configuration of compound 1 was confirmed by X-ray diffraction analysis (Fig. 3). Mild acid hydrolysis of 1 followed by column chromatography yielded thevetose. The specific rotation value of thevetose ([α]²⁷_D + 19.9) was obtained from an aqueous solution after 24 h equilibration. The D-configuration of thevetose was defined by comparison of the experimental and reported specific rotation values.^5,8 Thus, the structure of 1 was defined as hirundigenin 3-O-β-D-thevetopyranoside named hirundigoside E.

Table 1 Assignment of ¹H NMR data for the aglycone part of compounds 1–6 in CDCl₃ (δ in ppm, J values in Hz)

Position	1^b	2^a	3^b	4^b	5^b	6^a
a Spectra in 600 MHz.b Spectra in 400 MHz.
1	1.79, m/1.00, td(13.5, 3.5)	1.80, m/0.98, td(13.5, 3.5)	1.79, m/1.00, td(13.9, 3.6)	1.82, m/1.00, td(13.7, 3.5)	1.79, m/1.00, td(13.9, 3.6)	1.80, m/0.98, td(13.7, 3.3)
2	1.88, m/1.57, m	1.87, m/1.54, m	1.88, m/1.55, m	1.89, m/1.58, m	1.88, m/1.55, m	1.88, m/1.55, m
3	3.49, m	3.48, m	3.49, m	3.49, m	3.49, m	3.48, m
4	2.35, m/2.13, m	2.30, m/2.14, m	2.30, m/2.15, m	2.35, m/2.16, m	2.34, m/2.17, m	2.34, m/2.17, m
5
6	5.40, d(4.9)	5.38, d(4.4)	5.39, d(4.4)	5.41, d(5.2)	5.40, d(5.3)	5.39, d(5.3)
7	2.29, m/2.13, m	2.30, m/2.13, m	2.30, m/2.13, m	2.31, m/2.13, m	2.30, m/2.13, m	2.30, m/2.13, m
8	1.80, m	1.79, m	1.80, m	2.09, m	2.10, m	2.07, m
9	1.29, m	1.30, m	1.30, m	1.30, m	1.30, m	1.28, m
10
11	1.46, m/1.22, m	1.43, m/1.25, m	1.46, m/1.24, m	1.47, m/1.23, m	1.46, m/1.24, m	1.46, m/1.24, m
12	1.66, m/1.48, m	1.67, m/1.45, m	1.68, m/1.47, m	1.68, m/1.47, m	1.68, m/1.47, m	1.66, m/1.47, m
13
14-OH	5.15, s	5.15, s	5.15, s	5.15, s	5.15, s	5.15, s
15	4.16, d(10.9)/3.72, m	4.15, d(10.9)/3.72, m	4.16, d(10.9)/3.71, m	4.17, d(11.0)/3.73, dd(11.0, 3.8)	4.16, d(10.8)/3.71, m	4.16, d(10.8)/3.71, m
16	4.69, q(3.5)	4.69, q(3.6)	4.70, q(3.6)	4.70, m	4.70, m	4.70, m
17	2.84, d(7.2)	2.83, d(7.2)	2.84, d(7.1)	2.84, d(7.2)	2.84, d(7.1)	2.84, d(7.1)
18	4.48, d(9.0)/3.73, m	4.47, d(9.0)/3.74, m	4.48, d(9.2)/3.74, m	4.49, d(9.0)/3.75, m	4.48, d(9.0)/3.74, m	4.47, d(9.0)/3.73, m
19	0.91, s	0.90, s	0.91, s	0.92, s	0.91, s	0.91, s
20
21	1.44, s	1.43, s	1.44, s	1.45, s	1.44, s	1.43, s

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Table 2 Assignment of ¹H NMR data for the sugar chains of compounds 1–6 in CDCl₃ (δ in ppm, J values in Hz)

Position	1^b	2^a	3^b	4^b	5^b	6^a
a Spectra in 600 MHz.b Spectra in 400 MHz.
	The	Can	Ole	The	The	The
1′	4.29, d(7.7)	4.55, d(9.6)	4.50, dd(9.8, 1.8)	4.29, d(7.8)	4.28, d(7.8)	4.27, d(7.9)
2′	3.36, m	2.30, m/1.54, m	2.21, m/1.49, m	3.37, m	3.33, m	3.33, m
3′	3.07, m	3.54, m	3.32, m	3.19, m	3.19, m	3.17, m
4′	3.14, m	2.92, t(8.8)	3.15, t(8.8)	3.25, m	3.25, m	3.16, m
5′	3.32, m	3.26, m	3.25, m	3.30, m	3.30, m	3.30, m
6′	1.26, d(6.1)	1.26, d(6.6)	1.26, d(6.1)	1.22, d(6.2)	1.23, d(5.9)	1.24, d(6.6)
3′-OCH3	3.60, s			3.58, s	3.57, s	3.56, s
		Digt	Digt	Digt	Cym	Digt
1′′		4.77, d(9.6)	4.96, dd(9.7, 1.9)	4.92, dd(9.7, 1.6)	4.69, d(8.5)	4.92, d(9.5)
2′′		2.14, m/1.70, m	2.09, m/1.66, m	2.09, m/1.65, m	2.20, m/1.59, m	2.07, m/1.63, m
3′′		4.03, d(2.5)	4.02, d(3.2)	4.19, d(2.8)	3.74, m	4.18, d(2.9)
4′′		3.21, m	3.20, m	3.17, m	3.20, m	3.17, m
5′′		3.84, m	3.73, m	3.78, m	3.81, m	3.74, m
6′′		1.22, d(5.6)	1.21, d(6.1)	1.24, d(6.1)	1.19, d(6.0)	1.25, d(6.6)
3′′-OCH3					3.39, s
		Cym	Cym	Dign	Cym	Cym
1′′′		4.85, d(3.6)	4.87, d(3.9)	4.70, d(7.9)	4.85, dd(9.2, 1.3)	4.75, d(9.3)
2′′′		2.26, m/1.67, m	2.33, m/1.71, m	2.21, m/1.58	2.20, m/1.51, m	2.18, m/1.57, m
3′′′		3.59, m	3.59, m	3.60, m	3.34, m	3.71, m
4′′′		3.21, m	3.21, m	3.15, m	3.18, m	3.16, m
5′′′		3.82, m	3.81, m	3.57, m	3.76, m	3.71, m
6′′′		1.21, d(6.0)	1.22, d(6.2)	1.23, d(6.3)	1.18, d(6.0)	1.18, d(6.6)
3′′′-OCH3		3.36, s	3.36, s	3.41, s	3.37, s	3.37, s
					Dign	Dign
1′′′					4.95, d(3.3)	4.94, d(2.8)
2′′′					1.92, m/1.80, m	1.86, m/1.55, m
3′′′					3.62, m	3.33, m
4′′′					3.81, m	3.74, m
5′′′					3.95, m	3.94, m
6′′′					1.26, d(6.6)	1.22, d(6.2)
3′′′-OCH3					3.35, s	3.34, s

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Table 3 Assignment of ¹³C NMR data for compounds 1–6 in CDCl₃ (δ in ppm)

Position	1^b	2^a	3^b	4^b	5^b	6^a
a Spectra in 150 MHz.b Spectra in 100 MHz.
1	36.9	36.9	36.9	36.9	36.9	36.9
2	29.4	29.4	29.4	29.5	29.4	29.4
3	79.1	78.1	78.1	79.0	78.9	78.9
4	38.6	38.5	38.6	38.6	38.6	38.6
5	139.1	139.5	139.5	139.3	139.2	139.2
6	122.7	122.3	122.4	122.6	122.6	122.6
7	26.2	26.2	26.2	26.3	26.2	26.2
8	37.5	37.5	37.5	37.5	37.5	37.5
9	44.1	44.1	44.2	44.2	44.1	44.1
10	38.1	38.1	38.1	38.1	38.1	38.1
11	21.0	20.9	21.0	21.0	21.0	20.9
12	32.5	32.5	32.6	32.6	32.5	32.5
13	59.7	59.7	59.7	59.7	59.7	59.7
14	108.3	108.3	108.3	108.3	108.3	108.2
15	75.3	75.3	75.3	75.4	75.3	75.3
16	80.7	80.7	80.7	80.8	80.7	80.7
17	65.8	65.8	65.8	65.8	65.8	65.8
18	77.9	77.9	78.0	78.0	77.9	77.9
19	18.3	18.3	18.5	18.3	18.3	18.3
20	118.6	118.6	118.6	118.7	118.6	118.6
21	22.5	22.5	22.5	22.5	22.5	22.5
	The	Can	Ole	The	The	The
1′	101.1	97.9	97.8	100.9	100.9	100.9
2′	74.4	38.9	36.8	73.7	73.8	73.7
3′	85.6	69.9	79.3	84.3	84.3	84.4
4′	74.8	88.5	82.8	82.2	82.6	82.5
5′	71.7	70.5	71.3	71.5	71.5	71.4
6′	17.9	17.9	18.1	18.4	18.5	18.4
3′-OCH3	60.7		56.5	59.9	60.0	59.9
		Digt	Digt	Digt	Cym	Digt
1′′		99.4	98.8	98.9	99.6	98.3
2′′		36.8	37.2	37.2	35.5	37.2
3′′		67.5	67.8	66.7	77.1	66.7
4′′		79.7	79.7	82.6	81.9	82.1
5′′		69.0	68.8	68.4	68.9	69.1
6′′		17.9	18.3	18.4	18.2	18.2
3′′-OCH3					58.1
		Cym	Cym	Dign	Cym	Cym
1′′′		97.8	97.6	98.3	98.7	98.7
2′′′		31.1	31.1	33.9	34.0	34.2
3′′′		75.3	75.2	77.5	77.2	77.0
4′′′		72.1	72.2	72.4	81.8	81.7
5′′′		66.0	65.9	71.2	67.7	67.7
6′′′		18.0	18.3	18.4	18.3	18.3
3′′′-OCH3		56.6	56.7	57.6	57.0	57.2
					Dign	Dign
1′′′					100.7	100.7
2′′′					30.0	29.9
3′′′					74.5	74.5
4′′′					68.9	68.4
5′′′					66.2	66.3
6′′′					17.2	17.1
3′′′-OCH3					55.7	55.6

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Fig. 2 Key ROESY correlations of the aglycone of hirundigenin type C₂₁ steroidal glycosides in CDCl₃.

Fig. 3 Plot of the X-ray crystallographic data of 1.

Hirundigoside F (2) was obtained as colorless amorphous powder. Its molecular formula was determined as C₄₀H₆₂O₁₄ based on the positive HR ESI-TOF MS (m/z 789.4027 [M + Na]⁺). The IR spectrum showed the absorption bands for hydroxyl (3441 cm⁻¹) and olefinic (1454 cm⁻¹) groups. The ¹H and ¹³C NMR data were assigned by analyses of ¹H–¹H COSY, HSQC and HMBC spectra (Tables 1 and 3). By comparing its NMR data with that of 1, compound 2 had the same aglycone as that of 1. Three anomeric carbon signals at δ_C 99.4, 97.9, and 97.8 in the ¹³C NMR spectrum were observed, corresponding to the anomeric proton resonances at δ_H 4.55 (1H, d, J = 9.6 Hz), 4.77 (1H, d, J = 9.6 Hz), and 4.85 (1H, d, J = 3.6 Hz) in the ¹H NMR spectrum, respectively, suggesting that it contained three sugar units in the structure. The presence of three secondary methyl proton signals at δ_H 1.21 (3H, d, J = 6.0 Hz), 1.22 (3H, d, J = 5.6 Hz), and 1.26 (3H, d, J = 6.6 Hz) revealed that all of them were 6-deoxysugars. Analyses of ¹H–¹H COSY, HSQC and HMBC spectra enabled the identification of the three sugar units as cannarose, digitoxose, and cymarose (Fig. 4). The HMBC correlations from δ_H 4.85 (H-1′′′) to δ_C 79.7 (C-4′′) and from δ_H 4.77 (H-1′′) to δ_C 88.5 (C-4′) suggested that they were 1 → 4 linkages. The HMBC correlation from δ_H 4.55 (H-1′) to δ_C 78.1 (C-3) linked the sugar chain to C-3 position of the aglycone. The ¹³C NMR spectroscopic data of the sugar chain for 2 was quite similar with that of glaucogenin C 3-O-α-L-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-β-D-cannaropyranoside,⁹ which also supported above deduction. The monosugars were obtained by mild acidic hydrolysis of 2 and silica gel column chromatography purification. Their absolute configurations were identified to be L-cymarose, D-digitose, and D-cannose according to their specific rotation values.^5,10–12 The splitting patterns of anomeric proton signals indicated that the L-cymaropyranose was α-linkage and the other two sugars were β-linkages. Therefore, 2 was identified to be hirundigenin 3-O-α-L-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-β-D-canaropyranoside named hirundigoside F.

Fig. 4 Key ¹H–¹H COSY and HMBC correlations of compounds 2 and 6.

Hirundigoside G (3) was obtained as colorless amorphous powder. It had the molecular formula of C₄₁H₆₄O₁₄ by its pseudomolecular ion at m/z 803.4194 [M + Na]⁺ in the positive HR ESI-TOF MS. By comparing its NMR data with that of 1 (Tables 1 and 3), compound 3 was deduced to have the same aglycone as 1 and the difference between them lay in the sugar chains. The ¹H NMR spectrum of 3 showed the presence of three anomeric proton signals at δ_H 4.50 (1H, dd, J = 9.8, 1.8 Hz), 4.87 (1H, d, J = 3.9 Hz), and 4.96 (1H, dd, J = 9.7, 1.9 Hz) and three secondary methyl proton signals at δ_H 1.21 (3H, d, J = 6.1 Hz), 1.22 (3H, d, J = 6.2 Hz), and 1.26 (3H, d, J = 6.1 Hz), which revealed that it was a triglycoside with 6-deoxysugars. The sugar moieties were determined to be cymarose, digitoxose and oleandrose by analyzing their ¹³C resonance data (Table 3). Furthermore, comparison of its NMR data with that of stauntoside K¹³ suggested that compound 3 had the same sugar chain in the structures, which supported the above deduction. The 1 → 4 connections of the three sugar units were determined by HMBC correlations from δ_H 4.87 (H-1′′′) to δ_C 79.7 (C-4′′) and from δ_H 4.96 (H-1′′) to δ_C 82.8 (C-4′). The HMBC correlation from δ_H 4.50 (H-1′) to δ_C 78.1 (C-3) enabled the location of the sugar chain at C-3 of the aglycone. The splitting patterns of anomeric proton signals indicated that the cymaropyranose was α-linkage and the other two sugars were β-linkages. The L configuration of cymarose and D configurations of digitoxose and oleandrose were defined according to their specific rotation values after mild acidic hydrolysis of 3 and subsequent purification.^5,8,11,12 Thus, compound 3 was identified as hirundigenin 3-O-α-L-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-β-D-oleandropyranoside named hirundigoside G.

Hirundigoside H (4) was obtained as colorless amorphous powder. It had the molecular formula of C₄₁H₆₄O₁₅ based on the HR ESI-TOF MS (m/z 803.4194 [M + Na]⁺). By comparing its NMR data with that of 1 (Tables 1 and 3), compound 4 was deduced to share the same aglycone as 1. Three glycosyl units should be present in the structure of 4 according to three anomeric proton signals at δ_H 4.29 (1H, d, J = 7.8 Hz), 4.70 (1H, d, J = 7.9 Hz), and 4.92 (1H, dd, J = 9.7, 1.6 Hz) in its ¹H NMR spectrum. The presence of three secondary methyl proton signals at δ_H 1.22 (3H, d, J = 6.2 Hz), 1.23 (3H, d, J = 6.3 Hz), and 1.24 (3H, d, J = 6.1 Hz) revealed that all of them were 6-deoxysugars. Comparison of the ¹H and ¹³C NMR data with that of stauntoside I¹³ suggested that they had the same sugar chain in the structure, which was confirmed by analyses of ¹H–¹H COSY, HSQC and HMBC spectra. The HMBC correlation from δ_H 4.29 (H-1′) to δ_C 79.0 (C-3) enabled the location of sugar chain at C-3 of the aglycone. The L-configuration of diginose and D-configurations of digitoxose and oleandrose were defined according to their specific rotation values after mild acidic hydrolysis of 4 and subsequent purification.^5,8,11,12 The splitting patterns of anomeric proton signals indicated that the three sugars were β-linkages. Thus, 4 was identified to be hirundigenin 3-O-β-L-diginopyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-β-D-oleandropyranoside named hirundigoside H.

Hirundigoside I (5) was obtained as colorless amorphous powder. The positive mode HR ESI-TOF MS showed a pseudomolecular ion at m/z 977.5084 [M + Na]⁺, revealing its molecular formula to be C₄₉H₇₈O₁₈. By comparing its NMR data with that of 1 (Tables 1 and 3), compound 5 was deduced to have the same aglycone as 1. The ¹H NMR spectrum of 5 showed the presence of four anomeric proton signals at δ_H 4.28 (1H, d, J = 7.8 Hz), 4.69 (1H, d, J = 8.5 Hz), 4.85 (1H, dd, J = 9.2, 1.3 Hz), and 4.95 (1H, d, J = 3.3 Hz) and four secondary methyl proton signals at δ_H 1.18 (3H, d, J = 6.0 Hz), 1.19 (3H, d, J = 6.0 Hz), 1.23 (3H, d, J = 5.9 Hz), and 1.26 (3H, d, J = 6.6 Hz), which revealed that it was a tetraglycoside with 6-deoxysugars. Assignments of ¹H and ¹³C NMR data for each sugar unit were achieved by analyses of ¹H–¹H COSY, HSQC, HMBC and HSQC-TOCSY spectra. The sugar moieties in 5 were determined to be cymarose (×2), diginose, and thevetose by comparing its ¹³C NMR data with those of stauntoside E and stauntoside H.¹³ The 1 → 4 connections of the four sugar units were determined by the HMBC correlations from δ_H 4.95 (H-1′′′) to δ_C 81.8 (C-4′′′), from δ_H 4.85 (H-1′′′) to δ_C 82.8 (C-4′′), and from δ_H 4.69 (H-1′′) to δ_C 81.9 (C-4′). The HMBC correlation from δ_H 4.28 (H-1′) to δ_C 78.9 (C-3) enabled the location of the sugar chain at C-3 of the aglycone. The splitting patterns of anomeric proton signals indicated that the diginopyranose was α-linkage and the other three sugars were β-linkages. The D-cymarose, D-thevetose, and L-diginose were determined by their specific rotation values after mild acidic hydrolysis of 5 and subsequent purification.^5,8,11 Thus, compound 5 was identified to be hirundigenin 3-O-α-L-diginopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-thevetopyranoside named hirundigoside I.

Hirundigoside J (6) was obtained as colorless amorphous powder. The molecular formula was deduced as C₄₈H₇₆O₁₈ by the pseudomolecular ion at m/z 963.4912 [M + Na]⁺ in the positive mode HR ESI-TOF MS. Compounds 6 and 1 possessed the identical aglycones according to their ¹H and ¹³C NMR data (Tables 1 and 3). The ¹H and ¹³C NMR spectra of 6 showed four anomeric proton and carbon signals at δ_H 4.27 (d, J = 7.9 Hz)/δ_C 100.9, 4.94 (d, J = 2.8 Hz)/100.7, 4.75 (d, J = 9.3 Hz)/98.7, and 4.92 (d, J = 9.5 Hz)/98.3, indicating that it was a tetraglycoside. The presence of four secondary methyl proton signals at δ_H 1.18 (3H, d, J = 6.6 Hz), 1.22 (3H, d, J = 6.2 Hz), 1.24 (3H, d, J = 6.6 Hz), and 1.25 (3H, d, J = 6.6 Hz) revealed that all of them were 6-deoxysugars. Analyses of the ¹H–¹H COSY, HSQC, HMBC and HSQC-TOCSY spectra enabled the identification of these four sugar units as thevetose, digitoxose, cymarose, and diginose (Fig. 4). The ¹³C NMR spectroscopic data of the sugar chain in 6 was quite similar with that of stauntoside A,¹⁴ which also supported the above deduction. The 1 → 4 connections of the four sugar units were determined by HMBC correlations from δ_H 4.94 (H-1′′′) to δ_C 81.7 (C-4′′′), from δ_H 4.75 (H-1′′′) to δ_C 82.1 (C-4′′), and from δ_H 4.92 (H-1′′) to δ_C 82.5 (C-4′). The HMBC correlation from δ_H 4.27 (H-1′) to δ_C 78.9 (C-3) enabled the location of the sugar chain at C-3 of the aglycone. The absolute configurations of D-cymarose, D-thevetose, D-digitoxose, and L-diginose were determined by their specific rotation values after mild acidic hydrolysis of 6 and subsequent purification.^5,8,11 The splitting patterns of anomeric proton signals indicated that the L-diginopyranose was α-linkage and the other three sugars were β-linkages. Therefore, compound 6 was identified to be hirundigenin 3-O-α-L-diginopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-β-D-thevetopyranoside named hirundigoside J.

During the purification of compounds 1–6 by preparative HPLC, we found that they were unstable. Two discrete peaks always appeared in the HPLC by repeated preparative HPLC purification. Previous papers reported that hirundigenin was easy to transfer into anhydrohirundigenin by thermal dehydration or under acidic condition due to the presence of a free hemiketal hydroxyl group in its structure.^15,16 Firstly, UPLC-MS/MS was applied to analyze their molecular formulas of the two discrete peaks. The result showed that they had the same molecular formulas, which excluded the presence of dehydration product (data not shown). Then, methylation of compounds 2 and 6 were performed under the condition of CH₃I and NaH in anhydrous DMF. Four permethylated epimers, 14β-(2a and 6a) and 14α-OCH₃ derivatives (2b and 6b), were obtained by semipreparative HPLC. Their chemical structures were identified by HR ESI-TOF MS, 1D and 2D-NMR experiments (Fig. S36–S67†). The relative configurations were determined by sel-NOE experiments (Fig. 5). These results revealed that hirundigosides E–J (1–6) exist in nature as epimers due to isomerization of the 14-hemiketal hydroxyl group, which also appeared in some other types of natural products containing this functionality.^17,18

Fig. 5 Selected NOE spectra of compounds 2a and 2b.

It is striking that only one set of resonance signals appeared in the ¹H and ¹³C NMR spectra of compounds 1–6 when measured in CDCl₃, while two sets of resonance signals were observed when measured in pyridine-d₅. This may ascribe to lower molecular potential energy (G) of 14β-OH epimers than that of 14α-OH epimers. Since it is favorable for the formation of intramolecular hydrogen bond in 14β-OH epimer due to the proximate distance of hydroxyl group and oxygen atoms (1.86 nm) in 14β-OH epimer in CHCl₃ and the difference of potential energy (ΔG) of 14α-OH epimer to 14β-OH epimer is about 0.73 kcal mol⁻¹ for compound 1 (Fig. 6). Meanwhile, it is unfavorable for the formation of intramolecular hydrogen bond in pyridine and the ΔG of 14α-OH epimer to 14β-OH epimer is about −0.10 kcal mol⁻¹. Also, CDCl₃ is neither protic solvent nor Lewis acid and it can't induce the isomerization of hemiketal hydroxyl group in hirundigosides, while pyridine is Lewis base and it promotes the isomerization of hemiketal hydroxyl group.

Fig. 6 The difference of potential energy (ΔG) of 14α-OH (S-1) and 14β-OH epimer (1-R) in chloroform and pyridine. All calculations were performed using Gaussian09 suite of program.

In addition, secondary anhydrohirundigenin-type glycosides (2d–2f and 6c–6f) and anhydrohirundigenin (2c) were obtained by mild acidic hydrolysis of compounds 2 and 6 followed by semipreparative HPLC purification in order to discuss the structure–activity relationship (SAR) of their anti-inflammatory activity. Among them, compounds 2d–2f and 6d–6f were new compounds and their chemical structures were determined by HR ESI-TOF MS, 1D- and 2D-NMR experiments (Fig. S36–S67, Tables S1–S3†). The known structures of 2c and 6c were identified through comparison of their physical and spectroscopic data with those reported previously.^15,7

We evaluated the anti-inflammatory activity of hirundigosides E–J (1–6) and their derivatives (2a–2f and 6a–6f) by using a LPS-stimulated macrophage RAW264.7 cell model. As shown in Fig. 7(A and B), compounds 1–5, 2b–2f, 6a, and 6c–6f obviously inhibited LPS-induced iNOS protein expression compared to single LPS stimulation (blank control) in RAW264.7 cells. Meanwhile, results showed that all tested compounds couldn't remarkably suppress COX-2 protein expression. In contrast, compound 6e obviously increased COX-2 protein expression compared to single LPS stimulation. Structure–activity relationship (SAR) analysis revealed that anhydrohirundigenin-type C₂₁ glycosides exhibited stronger inhibitory effects on the LPS-induced iNOS protein expression compared to that of hirundigenin-type C₂₁ glycosides.

Fig. 7 Effect of hirundigosides E–J (1–6) and their derivatives (2a–2f and 6a–6f) on iNOS and COX-2 protein expression in LPS-stimulated RAW264.7 cells. Cells were pretreated with different compounds or DEX for 1 hour at 100 μM (DEX at 0.5 μM), and then stimulated with 100 ng mL⁻¹ LPS for another 18 hours. Cell lysates were prepared and analyzed for iNOS, COX-2 and β-actin (loading control) by western blotting. The protein level was quantitated using Odyssey v3.0 software (Li-COR, USA), and to count the density ratio of iNOS/COX-2 to β-actin. The density ratio of LPS group (blank control) was set to 100%.

Mature macrophages are generally located tissues throughout the body, including spleen, lung, interstitial connective tissue, and liver, where they ingest and process foreign materials, dead cells and debris, and play a critical role in the initiation, maintenance and resolution of local inflammatory response.¹⁹ Upon soluble stimuli stimulation such as LPS, TNF-α and IL-1β, macrophages produce pro-inflammatory mediators, including nitric oxide (NO). NO is synthesized from the amino acid L-arginine using NADPH and molecular oxygen, and is one of the most versatile players in the immune system.²⁰ However, overexpression of NO which is catalyzed by the enzymatic activity of nitric oxide synthases (NOS), is associated with a variety of pathological states. Therefore, NOS inhibitors could be as therapeutic agents for inflammatory diseases.²¹ Our studies revealed that most of the compounds could obviously inhibit LPS-induced iNOS protein expression in LPS-stimulated RAW264.7 cells. Results implied that these compounds probably possessed the anti-inflammatory properties and could, to a certain extent, suppress the NO release in the acute and chronic inflammatory response and had the potential to medicate anti-inflammatory action.

Experimental section

General experimental procedures

Optical rotations were obtained on a P-1020 digital polarimeter (Jasco Corporation). IR spectra were recorded on a JASCO FTIR-480 plus spectrometer. NMR spectra were measured on Bruker AV 300 and 600. The chemical shifts were given in ppm relative to chemical shifts of solvent resonances (C₅D₅N: δ_H 8.74/δ_C 150.3, CDCl₃: δ_H 7.24/δ_C 77.2). HR-ESI-MS spectra were obtained on a Micromass Q-TOF mass spectrometer. A single-crystal X-ray diffraction was measured on Agilent Super-Nova. Analytical HPLC was performed on a Shimadzu LC-20AT Liquid Chromatograph with SPD-M20A Detector and an Alltech ELSD 2000 detector using a Phenomex C₁₈ column (4.6 × 250 mm, 5 μm). Preparative HPLC was performed on a Shimadzu LC-6AD Liquid Chromatograph with SPD-20A Detector using an ODS column [YMC-Pack ODS-A (10.0 × 250 mm, 5 μm, 250 and 210 nm)]. Open column chromatography (CC) was performed using silica gel (200–300 mesh, Qingdao Haiyang Chemical Group Corp., Qingdao), ODS (50 μm, YMC, Japan), and Sephadex LH-20 (Pharmacia). TLC analysis was performed on pre-coated silica gel GF254 plates (Qingdao Haiyang Chemical Group Corp., Qingdao). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY, USA). Lipopolysaccharide (LPS, Escherichia coli 055: B5) and dexamethason (DEX) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). iNOS and COX-2 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA), and β-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IRDye 800CW goat anti-mouse IgG (H + L) and IRDye 800CW goat anti-rabbit IgG (H + L) secondary antibodies were purchased from Li-COR Biotechnology (Lincoln, NE, USA).

Plant material

The Chinese Medicine “Bai-Qian”, the dried roots of Cynanchum stauntonii, was obtained from Guangzhou Xiangxue pharmaceutical Co., Ltd. in November, 2012. It was identified by associated Prof. Zhang Ying, College of Pharmacy, Jinan University. A voucher specimen (CS201211-1) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University.

Extraction and isolation

The air-dried and chopped roots of C. stauntonii (9.5 kg) were refluxed with 95% (v/v) aqueous EtOH (90 L, ×3). The extract was evaporated under reduced pressure to yield a dark-brown residue (ca. 1100 g). The residue was directly chromatographed over an open macroporous resin Diaion HP-20 column (ϕ 12.0 × 63.0 cm), eluted with gradient EtOH–H₂O (0%, 30%, 50%, and 95%) to yield 4 fractions (A–D). The fraction D (93.0 g, 95%) was applied to silica gel column chromatography (ϕ 9.5 × 68.0 cm) using a gradient of mixtures of petroleum ether–EtOAc (100 : 0, 98 : 2, 95 : 5, 9 : 1, 7 : 3, 0 : 100) and EtOAc–CH₃OH (95 : 5, 9 : 1, 7 : 3, 0 : 100) to yield 13 fractions (D1–D13) according to their TLC profiles and HPLC behavior. Fraction D10 (20.8 g) was subjected to open ODS CC (ϕ 4.0 × 30.0 cm) using a CH₃OH–H₂O gradient to afford 13 subfractions (D10-1–D10-13). Subfraction D10-4 (1.8 g) was separated by ODS CC using gradient solvents of CH₃OH–H₂O to yield 9 subfractions (D10-4-1–D10-4-9). Subfraction D10-4-5 was further purified by semipreparative RP-C₁₈ HPLC with MeOH–H₂O (50 : 50) to afford compound 1 (10.0 mg). Compound 4 (50.0 mg) was obtained from D10-4-9 by semipreparative RP-C₁₈ HPLC using MeOH–H₂O (60 : 40). Subfraction D10-5 (2.4 g) was subjected to ODS CC using a stepwise gradient of MeOH in H₂O from 60% to 80% to give 4 subfractions (D10-5-1–D10-5-4). Subfractions D10-5-1 was chromatographed over Sephadex LH-20 CC eluting with MeOH–H₂O (70 : 30) to afford 5 fractions (D10-5-1-1–D10-5-1-5). Fraction D10-5-1-3 (77 mg) was purified by semipreparative RP-C₁₈ HPLC with MeOH–H₂O (70 : 30) to afford compound 3 (38.0 mg). Compound 5 (48.0 mg) was obtained from D10-5-1-4 (237 mg) by semipreparative RP-C₁₈ HPLC using MeCN–H₂O (40 : 60). Fraction D12 (24.0 g) was subjected to open ODS CC (ϕ 4.0 × 30.0 cm) using a CH₃OH–H₂O gradient to give 9 subfractions (D12-1–D12-9). Subfraction D12-2 (16.0 g) was chromatographed over Sephadex LH-20 CC eluting with MeOH–H₂O (70 : 30) to afford 9 subfractions (D12-2-1–D12-2-9). Compound 6 (500 mg) was obtained from D12-2-2 (1.57 g) by preparative RP-C₁₈ HPLC using MeOH–H₂O (65 : 35). Subfraction D12-3 (6.0 g) was chromatographed over silica gel CC (ϕ 2.5 × 25.0 cm) eluting with gradient CHCl₃–MeOH (95 : 5, 9 : 1, 85 : 15, 8 : 2, 0 : 100) to yield 7 subfractions (D12-3-1–D12-3-7). Subfraction D12-3-2 (1.57 g) was subjected to Sephadex LH-20 CC and then purified by preparative RP-C₁₈ HPLC with MeOH–H₂O (70 : 30) to yield compound 2 (480 mg).

Permethylation of hirundigoside F (2) and J (6)

To a solution of compound 2 (50 mg) in anhydrous DMF (3 mL) was added NaH. The reaction mixture was stirred for half an hour in ice bath and was added CH₃I (1 mL). After 1 h, 5 mL of H₂O was added to the reaction mixture to stop the reaction. The reaction mixture was extracted with CHCl₃ (5 mL × 3). The organic layer was concentrated under reduced pressure to provide crude product, which was purified by semipreparative RP-HPLC using MeOH–H₂O (80 : 20) to afford compounds 2a and 2b. Permethyl derivatives of hirundigoside J (6a and 6b) were prepared in the same way as described above.

Mild acid hydrolysis of hirundigoside F (2) and J (6) and preparation of their secondary glycosides

To a solution of compound 2 (80 mg) in MeOH (2 mL) was added 0.05 N HCl (3 mL). The reaction mixture was stirred for 15 min at 70 °C and evaporated to remove MeOH under reduced pressure. The aqueous solution was extracted with CHCl₃ (3 mL × 3). The recovered organic layers were concentrated to provide crude product, which was purified by semipreparative RP-HPLC using MeOH–H₂O (70 : 30) to afford anhydrohirundigenin (2c) and secondary anhydrohirundigenin-type glycosides (2d–2f). The secondary anhydrohirundigenin-type glycosides (6c–6f) derived from hirundigoside J (6) were prepared in the same way as described above.

Hirundigoside E (1)

Colorless needle crystals (CHCl₃/MeOH), [α]²⁷_D −15.2 (c = 0.5, CH₃OH); IR (KBr) ν_max 3441, 2939, 1706, 1455, 1379, and 1069 cm⁻¹; ESI-MS (positive mode) m/z 545.9 [M + Na]⁺, HR ESI-MS (positive mode) m/z 545.2732 [M + Na]⁺ (calcd for C₂₈H₄₂O₉Na, 545.2727); ¹H (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃) data, see Tables 1–3.

Hirundigoside F (2)

Colorless amorphous powder; [α]²⁷_D −20.0 (c = 0.5, CH₃OH); IR (KBr) ν_max 3436, 2934, 1708, 1454, 1378, and 1063 cm⁻¹; ESI-MS (positive mode) m/z 789.7 [M + Na]⁺, HR ESI-MS (positive mode) m/z 789.4027 [M + Na]⁺ (calcd for C₄₀H₆₂O₁₄Na, 789.4037); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Tables 1–3.

Hirundigoside G (3)

Colorless amorphous powder; [α]²⁷_D −35.6 (c = 0.5, CH₃OH); IR (KBr) ν_max 3445, 2976, 1692, 1514, 1389, and 1061 cm⁻¹; ESI-MS (positive mode) m/z 803.9 [M + Na]⁺, HR ESI-MS (positive mode) m/z 803.4194 [M + Na]⁺ (calcd for C₄₁H₆₄O₁₄Na, 803.4194); ¹H (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃) data, see Tables 1–3.

Hirundigoside H (4)

Colorless amorphous powder; [α]²⁷_D −6.4 (c = 0.5, CH₃OH); IR (KBr) ν_max 3445, 2934, 1644, 1456, 1377, and 1073 cm⁻¹; ESI-MS (positive mode) m/z 819.7 [M + Na]⁺, HR ESI-MS (positive mode) m/z 819.4133 [M + Na]⁺ (calcd for C₄₁H₆₄O₁₅Na, 819.4143); ¹H (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃) data, see Tables 1–3.

Hirundigoside I (5)

Colorless amorphous powder; [α]²⁷_D −22.4 (c = 0.5, CH₃OH); IR (KBr) ν_max 3445, 2936, 1455, 1375, and 1060 cm⁻¹; ESI-MS (positive mode) m/z 977.8 [M + Na]⁺, HR ESI-MS (positive mode) m/z 977.5084 [M + Na]⁺ (calcd for C₄₉H₇₈O₁₈Na, 778.5164); ¹H (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃) data, see Tables 1–3.

Hirundigoside J (6)

Colorless amorphous powder; [α]²⁷_D −34.4 (c = 0.5, CH₃OH); IR (KBr) ν_max 3474, 2934, 1644, and 1080 cm⁻¹; ESI-MS (positive mode) m/z 963.9 [M + Na]⁺, HR ESI-MS (positive mode) m/z 963.4912 [M + Na]⁺ (calcd for C₄₈H₇₆O₁₈Na, 963.4929); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Tables 1–3.

14β-OCH₃-permethyl hirundigoside F (2a)

Colorless amorphous powder; [α]²⁷_D −67.9 (c = 0.5, CH₃OH); IR (KBr) ν_max 2934, 1455, 1380, and 1002 cm⁻¹; ESI-MS (positive mode) m/z 845.9 [M + Na]⁺, HR ESI-MS (positive mode) m/z 845.4666 [M + Na]⁺ (calcd for C₄₄H₇₀O₁₄Na, 845.4663); ¹H (600 MHz, C₅D₅N) and ¹³C NMR (150 MHz, C₅D₅N) data, see Tables S1–S3.†

14α-OCH₃-permethyl hirundigoside F (2b)

Colorless amorphous powder; [α]²⁷_D −50.0 (c = 0.5, CH₃OH); IR (KBr) ν_max 2934, 1455, 1380, and 1002 cm⁻¹; ESI-MS (positive mode) m/z 845.9 [M + Na]⁺, HR ESI-MS (positive mode) m/z 845.4647 [M + Na]⁺ (calcd for C₄₄H₇₀O₁₄Na, 845.4663); ¹H (600 MHz, C₅D₅N) and ¹³C NMR (150 MHz, C₅D₅N) data, see Tables S1–S3.†

Anhydrohirundigenin β-D-canaropyranoside (2d)

Colorless amorphous powder; [α]²⁷_D +2.6 (c = 0.5, CH₃OH); IR (KBr) ν_max 3441, 2933, 1645, 1375, and 1068 cm⁻¹; ESI-MS (positive mode) m/z 497.7 [M + Na]⁺, HR ESI-MS (positive mode) m/z 475.2697 [M + H]⁺ (calcd for C₂₇H₃₉O₇, 475.2696); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Tables S1–S3.†

Anhydrohirundigenin β-D-digitoxopyranosyl-(1 → 4)-β-D-canaropyranoside (2e)

Colorless amorphous powder; [α]²⁷_D −43.2 (c = 0.5, CH₃OH); IR (KBr) ν_max 3441, 2934, 1645, 1375, and 1068 cm⁻¹; ESI-MS (positive mode) m/z 627.8 [M + Na]⁺, HR ESI-MS (positive mode) m/z 605.3336 [M + H]⁺ (calcd for C₃₃H₄₉O₁₀, 605.3326); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Tables S1–S3.†

Anhydrohirundigenin 3-O-α-L-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-β-D-canaropyranoside (2f)

Colorless amorphous powder; [α]²⁷_D −71.4 (c = 0.5, CH₃OH); IR (KBr) ν_max 3441, 2934, 1645, 1375, and 1068 cm⁻¹; ESI-MS (positive mode) m/z 771.9 [M + Na]⁺, HR ESI-MS (positive mode) m/z 771.3906 [M + Na]⁺ (calcd for C₄₀H₆₀O₁₃Na, 771.3932); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Tables S1–S3.†

14β-OCH₃-permethyl hirundigoside J (6a)

Colorless amorphous powder; [α]²⁷_D −41.0 (c = 0.5, CH₃OH); IR (KBr) ν_max 2933, 1455, 1379, and 1061 cm⁻¹; ESI-MS (positive mode) m/z 1020.1 [M + Na]⁺; ¹H (600 MHz, C₅D₅N) and ¹³C NMR (150 MHz, C₅D₅N) data, see Tables S1–S3.†

14α-OCH₃-permethyl hirundigoside J (6b)

Colorless amorphous powder; [α]²⁷_D −37.7 (c = 0.5, CH₃OH); IR (KBr) ν_max 2934, 1455, 1380, and 1002 cm⁻¹; ESI-MS (positive mode) m/z 1020.2 [M + Na]⁺; ¹H (600 MHz, C₅D₅N) and ¹³C NMR (150 MHz, C₅D₅N) data, see Tables S1–S3.†

Anhydrohirundigenin β-D-digitoxopyranosyl-(1 → 4)-β-D-thevetopyranoside (6d)

Colorless amorphous powder; [α]²⁷_D −8.3 (c = 0.5, CH₃OH); IR (KBr) ν_max 3441, 2934, 1645, 1375, and 1068 cm⁻¹; ESI-MS (positive mode) m/z 657.9 [M + Na]⁺, HR ESI-MS (positive mode) m/z 657.3245 [M + Na]⁺ (calcd for C₃₄H₅₀O₁₁Na, 657.3251); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Tables S1–S3.†

Anhydrohirundigenin β-D-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-β-D-thevetopyranoside (6e)

Colorless amorphous powder; [α]²⁷_D +4.7 (c = 0.5, CH₃OH); IR (KBr) ν_max 3441, 2934, 1645, 1375, and 1068 cm⁻¹; ESI-MS (positive mode) m/z 802.1 [M + Na]⁺, HR ESI-MS (positive mode) m/z 801.4035 [M + Na]⁺ (calcd for C₄₁H₆₂O₁₄Na, 801.4037); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Tables S1–S3.†

Anhydrohirundigenin 3-O-α-L-diginopyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-β-D-thevetopyranoside (6f)

Colorless amorphous powder; [α]²⁷_D − 33.5 (c = 0.5, CH₃OH); IR (KBr) ν_max 3441, 2933, 1645, 1375, and 1068 cm⁻¹; ESI-MS (positive mode) m/z 946.0 [M + Na]⁺, HR ESI-MS (positive mode) m/z 945.4819 [M + Na]⁺ (calcd for C₄₁H₆₂O₁₄Na, 945.4824); ¹H (600 MHz, CDCl₃) and ¹³C NMR (150 MHz, CDCl₃) data, see Tables S1–S3.†

X-ray crystallographic analysis of 1

Crystals of 1 were obtained in the mixture solvent of CHCl₃ and CH₃OH. Colorless blocks, C₂₉H₄₅O₁₀, M = 553.65, monoclinic, space group P1211, a = 8.6095(14) Å, b = 7.8910(8) Å, c = 21.093(3) Å, V = 1412.5(3) Å, Z = 2, T = 293(2) K, μ(Cu Kα) = 0.804 mm⁻¹, D_calcd = 1.302 mg m⁻³, 3444 reflections collected (4.25 ≤ θ ≤ 62.66), 2669 unique (R_int = 0.0284), which were used in all calculations. The final R₁ was 0.0552[I > 2σ(I)] and wR₂ was 0.1432[I > 2 sigma(I)]. Data collection was performed in a SMART CCD by using graphite monochromated radiation (l = 1.54184). The structure of compound 1 was solved by the direct methods (SHELXTL version 6.1) and refined by full-matrix least-squares on F². Crystallographic data (excluding structure factors) for compound 1 (Cu Kα radiation) reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1433862.

Computational calculations

All calculations were performed using Gaussian09 suite of program.²² Density functional theory method was used, employing uB3LYP hybrid functional.^23,24 Geometry optimization was done using a combined basis set 6-31G(d). Frequency calculation was done at the same level of theory as geometry optimization, to confirm the stationary points to be minima. Single point energy calculations were done using B3LYP method at a larger basis set 6-31++G(d,p). Solvent effect was accounted for using self-consistent reaction field (SCRF) method using SMD model and UAKS radii.^25,26 Chloroform and pyridine were used as solvents.

Cells and treatments

The murine macrophage cell line RAW264.7 (American Type Culture Collection, Manassas, VA, USA) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) FBS (Gibco BRL Co, Grand Island, NY, USA), penicillin G (100 units per ml), streptomycin (100 mg mL⁻¹), and L-glutamine (2 mM) (Gibco BRL Co, Grand Island, NY, USA) in a 5% CO₂-humidified incubator at 37 °C. All of tested compounds were dissolved in DMSO in 100 mM and work concentration was 100 μM (DMSO concentration was less than 0.1% in assay). To analysis the anti-inflammatory activity of these compounds derived from the dried roots of Cynanchum stauntonii. RAW264.7 cells were plated at a density of 8 × 10⁴ cells per well in 24-well plates for 24 h. Cells were pretreated for 1 h with the test compounds or DEX before treatment with 100 ng mL⁻¹ LPS. DEX was chosen as positive control in this study. Cells stimulated by LPS without any intervention were observed as blank control. Cells incubated by DMEM medium were as normal control.

Western blot analysis

Total proteins were prepared from RAW264.7 cells through the use of RIPA lysis buffer (Cell Signaling technology, Boston, MA, USA) and protease inhibitors (Roche Applied Science, Germany). Lysate protein (30 μg per lane) was separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Buckinghamshire, UK). The membrane was blocked for 1 h with 5% skimmed milk, and then incubated with the primary antibody (iNOS and COX-2) and mouse antibodies specific for β-actin at 4 °C for overnight. The antigen–antibody complexes were visualized using IRDye 800CW goat anti-mouse IgG (H + L) or IRDye 800CW goat anti-rabbit IgG (H + L) secondary antibodies (Li-COR, Lincoln, NE, USA). The protein expression level was quantitated using Odyssey v3.0 software (Li-COR, USA).

Statistical evaluation

All data are given as the mean ± standard deviation for the number of experiments. Statistical significance was compared each treated with compounds group with the blank control and determined by one-way analysis of variance (ANOVA) using SPSS statistical program. P values less than 0.05 was accepted as statistically significant.

Acknowledgements

We are grateful to the State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology for the anti-inflammation activity testing. We are also grateful to Mr T. Shi, Mr J. Geng, and Dr Y. Yu for the HR ESI-MS and NMR measurements. Part of the chemical work was accomplished in Guangzhou Xiangxue Pharmaceutical Ltd. Co.

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Footnotes

† Electronic supplementary information (ESI) available: The 1D and 2D NMR spectra of compounds 1–6, 2d–2f and 6d–6f (Fig. S1–S67). Assignments of the ¹H and ¹³C NMR data for compounds 2d–2f and 6d–6f (Tables S1–S3). CCDC 1433862. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra11957c

‡ Contributed equally to this work.

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