Anti-inflammatory alkaloid glycoside and quinoline alkaloid derivates from the stems of Clausena lansium

Abstract

Six new alkaloid glycosides, Clausenasides A–F (1–6), along with two new quinoline alkaloids, Clausenasides G–H (7–8), and ten known compounds (9–18) were obtained from the stems of C. lansium. The structures of the new compounds were elucidated on the basis of their spectroscopic analysis, and the absolute configurations of 1, 2, 3 and 7 were confirmed by Mosher's method, CD and ECD spectra, respectively. Compounds 4, 6, 9, 17, and 18 showed moderate inhibitory effects on LPS-induced NO production in murine microglial BV2 cells (IC₅₀ values < 10 μM).

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Introduction

Clausena lansium (Lour.) skeels (Rutaceae), a fruit tree, was widely distributed in southern China. In traditional Chinese medicine, the leaves and roots of C. lansium were used for coughs, asthma, dermatological disease, viral hepatitis, and gastro-intestinal diseases; the seeds are used to treat acute and chronic gastro-intestinal inflammation, ulcers, etc.¹ Various bioactive constituents including coumarins, carbazole alkaloids and amide alkaloids have been isolated and identified from this plant.^2–4 Previously, twenty new natural products including thirteen new carbazole alkaloids,^5,6 four new coumarins,⁷ a new amide and a new megastigmane glucoside⁸ from the leaves and skeels of C. lansium were reported by our research group, and some of these alkaloids showed selective neuroprotective effects. In order to continue research for microscale bioactive metabolites from C. lansium, 200 kg stems of C. lansium were extracted by EtOAc and n-BuOH. This paper reported further investigation of n-BuOH extract from the stems of C. lansium which led to the isolation and characterization of six new alkaloid glycosides (1–6), two new quinoline alkaloids (7–8) along with ten known compounds (9–18) from the stems of C. lansium (Fig. 1). It was the first time to obtain these alkaloid glycosides from C. lansium. The inhibitory effects on LPS-induced NO production in a murine microglial cell lines of 1–18 were also evaluated. We present herein the isolation and structural characterization of Clausenasides A–H, as well as their bioactivities.

Fig. 1 Alkaloid derivates (1–18) obtained from the stems of C. lansium.

Results and discussion

Clausenaside A (1) was obtained as a white powder. Its molecular formula was assigned as C₂₂H₂₇NO₈ based on the ¹³C NMR spectroscopic data and HRESIMS (m/z 456.1634 [M + Na]⁺, calcd for C₂₂H₂₇NO₈Na, 456.1629), implying ten indices of hydrogen deficiency. The IR spectrum displayed absorptions characteristic of amino (3315 cm⁻¹), amide (1640 cm⁻¹) and aromatic ring (1618, 1579 and 1505 cm⁻¹) groups, and the UV spectrum showed absorptions at λ_max 202 and 226 nm. A 1,4-disubstituted benzene ring at δ_H 7.32 (2H, d, J = 8.8 Hz), 6.87 (2H, d, J = 8.8 Hz) and a 1-substituted benzene ring at δ_H 7.79 (2H, d, J = 7.2 Hz), 7.51 (1H, m), 7.44 (2H, t, J = 7.6 Hz) were clearly observed in the ¹H NMR spectrum and were assigned with the aid of COSY correlations (Fig. 2). In addition, an amide group δ_H 8.33 (1H, t, J = 4.0 Hz, NH), a methoxy group [δ_H 3.73; δ_C 55.0], a methylene group [δ_H 3.69 (1H, m), 3.55 (1H, m); δ_C 44.5], an oxygenated methine group [δ_H 4.83 (1H, t, J = 4.0 Hz); δ_C 78.8], and a β-glucopyranosyl moiety [4.42 (1H, d, J = 7.6 Hz), δ_H 3.06–3.56 (5H, m); δ_C 102.9, 73.8, 76.5, 69.9, 76.8, 60.9] were clearly observed in the ¹H and ¹³C NMR spectra along with HSQC correlations. On the basis of the NMR techniques, compound 1 was elucidated as an amide alkaloid glycoside. In the HMBC spectrum (Fig. 2), the cross-peaks between H-3, H-5/C-1 (δ_C 134.4), H-2, H-6/C-7 (δ_C 166.2) demonstrated the 1-substituted benzene ring attached to the carbonyl group. H-8/C-7 (δ_C 166.2), N-H/C-7 (δ_C 166.2), C-9 (δ_C 78.8) indicated the methylene group attached to the N atom. H-9/C-10 (δ_C 132.5), C-11 (δ_C 127.9), H-1′/C-9 (δ_C 78.8) implying that 1,4-disubstituted benzene ring and β-glucopyranosyl unit were linked to C-9. The methoxy group resonated at C-13 (δ_C 158.5). Hydrolysis of 1 with snailase produced the aglycone (1a) and glucose. 1a was identified as tembamide⁹ by comparison of its ¹H and ¹³C NMR, and HRESIMS. The absolute configuration o of 1a was determined as 9S by comparison of the optical rotation of 1a {[α]²⁵_D +54.8 (c 0.20 CHCl₃)} and (S)-(+)-tembamide¹⁰ {[α]²⁵_D +56.9 (c 0.54 CHCl₃)}. Compound 1a was treated with (R)- and (S)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) in anhydrous CH₂Cl₂ (ref. 11) to afforded the 1a-(S)-MTPA ester (1aa) and 1a-(R)-MTPA ester (1ab), respectively. The Δδ^SR_H values were calculated as shown in Fig. 3. Application of Mosher's rule¹² revealed that 1a had the 9S configuration. The CD spectrum of 1a exhibited negative Cotton effect at 235 nm (Fig. 4), and the CD spectrum of 1 also showed negative Cotton effect at 238 nm (Fig. 4), which indicated that the structure of 1a had not changed in the process of enzymatic hydrolysis and the absolute configuration of 1 was elucidated as 9S. The D-glucose was identified by comparison of TLC and optical rotation data of D-glucose and the authentic sample.¹³ Thus, the structure of 1 was defined as (S)-(+)-tembamide-9-O-β-D-glucopyranoside.

Fig. 2 Key ¹H, ¹H-COSY and HMBC correlations of compounds 1, 2 and 3.

Fig. 3 The Mosher's method of 1a.

Fig. 4 The experimental CD spectrum of 1, 2, 3 and 1a.

Clausenaside B (2) was obtained as a white powder. Its molecular formula was established as C₂₂H₂₇NO₈ from the ¹³C NMR and positive-ion HRESIMS (m/z 456.1635 [M + Na]⁺, calcd for C₂₂H₂₇NO₈Na, 456.1629), indicating ten indices of hydrogen deficiency. Comparison of spectroscopic data of 2 with those of 1 revealed that the planar structure of 2 was established the same as that of 1. Therefore, compound 2 was defined to be a stereo-isomer of 1. The CD spectra of 2 which showed positive Cotton effect at 237 nm (Fig. 4) was contrary to 1. Thus, the structure of 2 was established as (R)-(−)-tembamide-9-O-β-D-glucopyranoside.

Clausenaside C (3) was obtained as a white powder. Its molecular formula was deduced as C₂₁H₂₅NO₇ on the basis of its ¹³C NMR and HRESIMS at m/z 426.1524 [M + Na]⁺, calcd for C₂₁H₂₅NO₇Na, 426.1523, implying ten indices of hydrogen deficiency. Comparison of the spectroscopic data of 3 with those of 1 revealed that compound 3 also was an amide alkaloid glycoside, with the primary difference being the absence of a methoxy group at C-13 (δ_C 127.8). Compound 3 showed similar CD Cotton effects to those of 1 (Fig. 4), and the absolute configuration of 3 was 9S. Thus, the structure of 3 was defined as (S)-(+)-demethoxy-tembamide-9-O-β-D-glucopyranoside.

Clausenaside D (4) was obtained as a white powder. Its molecular formula was determined as C₁₆H₁₉NO₇ based on its ¹³C NMR spectroscopic and HRESIMS at m/z 338.1232 [M + H]⁺ (calcd for C₁₆H₂₀NO₇, 338.1234), corresponding with eight indices of hydrogen deficiency. The UV spectrum showed absorptions at 226, 270 and 322 nm. The ¹H NMR spectrum (Table 2) showed o-substituted benzene ring [δ_H 8.04 (1H, dd, J = 7.8, 1.8 Hz, H-5); 7.28 (1H, dt, J = 7.8, 0.6 Hz, H-6); 7.66 (1H, dt, J = 8.4, 1.8 Hz, H-7); 7.52 (1H, d, J = 8.4 Hz, H-8)], a bond group δ_H 6.19 (1H, s, H-3), a methyl group δ_H 3.57, and a β-glucopyranosyl moiety δ_H 5.09 (1H, d, J = 7.8 Hz, H-1′). The ¹³C NMR spectrum (Table 2) exhibited 16 carbon signals including six in glucopyranosyl unit, a methyl group, and the remaining nine in the quinoline moiety. A comparison of the ¹H and ¹³C NMR of 4 with those of integriquinolone¹⁴ suggested that their structures are closely related, except for the presence of the β-glucopyranose unit signals in 4. The glucose was confirmed to be located at C-4 from the HMBC correlation between H-1′ (δ_H 5.09) and C-4 (δ_C 159.4) (Fig. 5). D-Glucose were isolated from the enzymatic hydrolysate of 4 and identified by TLC and specific rotation. Therefore, the structure of 4 was elucidated as integriquinolone-4-O-β-D-glucopyranoside.

Table 1 ¹H and ¹³C NMR spectroscopic data of compounds 1–3 (δ in ppm, J in Hz)

Position	1		2		3
	δH^a	δC^b	δH^a	δC^b	δH^a	δC^b
a In DMSO-d6 400 MHz for 1, 2, 3.b In DMSO-d6 100 MHz for 1, 2, 3. Coupling constants (J) in Hz are given in parentheses. The assignments were based on HSQC and HMBC experiments.c Signal overlapped.
1		134.4 s		134.6 s		134.8 s
2, 6	7.79, br d (7.2)	127.1 d	7.77, br d (7.2)	127.1 d	7.79, br d (7.6)	127.6 d
3, 5	7.44, t (7.6)	128.3 d	7.43, t (7.6)	128.4 d	7.46, t (7.6)	128.8 d
4	7.51, m	131.1 d	7.50, m	131.0 d	7.52, m	131.6 d
7		166.2 s		166.4 s		166.8 s
8	3.69, m; 3.55, m	44.5 t	3.50, m	45.6 t	3.73, m; 3.57, m	45.2 t
9	4.83, t (4.0)	78.8 d	4.99, t (6.6)	76.1 d	4.90, m	79.6 d
10		132.5 s		131.2 s		141.1 s
11, 15	7.32, br d (8.8)	127.9 d	7.36, br d (8.8)	128.2 d	7.41, br d (7.6)	127.1 d
12, 14	6.87, br d (8.8)	113.3 d	6.90, br d (8.8)	113.5 d	7.33, br d (7.4)	128.4 d
13		158.5 s		158.7 s	7.26, t (7.2)	127.8 d
Glc-1′	4.42, d (7.6)	102.9 d	3.98, d (7.6)	100.0 d	4.46, d (7.6)	103.5 d
Glc-2′	3.07,^c m	73.8 d	3.04,^c m	73.5 d	3.10, m	74.3 d
Glc-3′	3.16, m	76.5 d	3.04,^c m	76.6 d	3.18, m	77.0 d
Glc-4′	3.07,^c m	69.9 d	3.04,^c m	70.2 d	3.07,^c m	70.4 d
Glc-5′	3.07,^c m	76.8 d	2.96, m	76.9 d	3.08,^c m	77.4 d
Glc-6′a	3.56, m	60.9 t	3.39, m	61.2 d	3.37, m	61.4 t
Glc-6′b	3.37, m		3.65, m		3.56, m
NH	8.33, t (4.0)		8.23, t (5.5)		8.35, t (5.4)
OCH3	3.73, s	55.0 q	3.74, s	55.0 q

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Table 2 ¹H and ¹³C NMR spectroscopic data of compounds 4–6 (δ in ppm, J in Hz)

Position	4		5		6
	δH^a	δC^b	δH^a	δC^b	δH^c	δC^d
a In DMSO-d6 600 MHz for 4 and 5.b In DMSO-d6 150 MHz for 4 and 5.c In MeOH-d4 600 MHz for 6.d In MeOH-d4 150 MHz for 6. Coupling constants (J) in Hz are given in parentheses. The assignments were based on HSQC and HMBC experiments.e Signal overlapped.
2		162.3 s		163.3 s		164.8 s
3	6.19, s	99.4 d	6.02, s	96.7 d		105.1 s
4		159.7 s		163.5 s		159.2 s
4a		115.4 s		116.4 s		120.9 s
5	8.04, dd (7.8, 1.8)	123.2 d	7.87, dd (7.8, 1.2)	123.2 d	7.95, dd (8.4, 1.2)	117.0 d
6	7.28, m	121.4 d	7.28, m	122.9 d	7.39, dd (8.4, 7.8)	125.0 d
7	7.66, m	131.5 d	7.64,^e m	132.2 d	7.48, dd (7.8, 1.2)	114.4 d
8	7.52, br d (8.4)	114.6 d	7.62,^e m	116.3 d		153.0 s
8a		139.5 s		139.5 s		137.7 s
9	3.57, 3H, s	28.7 q	5.87, d (9.6); 5.75, d (9.6)	69.9 t
Glc-1′	5.09, d (7.8)	99.5 d	4.42, d (7.8)	101.9 d	5.04, d (7.8)	103.0 d
Glc-2′	3.90, m	73.0 d	2.95, m	73.9 d	3.74, m	74.7 d
Glc-3′	3.34, m	76.1 d	3.12,^e m	77.5 d	3.59, m	77.3 d
Glc-4′	3.19, m	69.5 d	3.07, m	70.6 d	3.47, m	71.6 d
Glc-5′	3.46, m	77.2 d	3.10,^e m	77.9 d	3.57, m	78.5 d
Glc-6′	3.48, m; 3.70, m	60.6 t	3.47, m; 3.66, m	61.7 t	3.98, m; 3.75, m	62.6 t
OCH3			3.96, s	57.1 q	4.52, s	60.2 q
α					7.86, d (2.4)	145.4 d
β					7.40, d (2.4)	106.5 d

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Fig. 5 Key ¹H, ¹H-COSY and HMBC correlations of compounds 4, 5 and 6.

Clausenaside E (5) was obtained as a white powder. Its HRESIMS displayed m/z [M + H]⁺ 368.1346 (calcd for C₁₇H₂₂NO₈, 368.1340), which was consistent with a molecular formula of C₁₇H₂₁NO₈ with eight indices of hydrogen deficiency. Compound 5 showed similar UV absorptions to those of 4, which suggested that their structure were closely related except that the 4-methoxy group and 9-hydroxymethyl-O-β-D-glucopyranoside of 5 replaced 4-O-β-D-glucopyranoside group and the 9-methyl group of 4 respectively. The linkage of the β-D-glucose moiety to C-9 was supported by the HMBC correlation observed between H-1′ (δ_H 4.42) and C-9 (δ_C 69.9) (Fig. 5). D-Glucose were isolated from the enzymatic hydrolysate of 5 and identified by TLC and specific rotation. Therefore, the structure of 5 was defined as 4-methoxy-1-hydroxymethyl-2(1H)-quinolinone-9-O-β-D-glucopyranoside.

Clausenaside F (6) was obtained as a white powder. Its molecular formula was assigned as C₁₈H₁₉NO₈ on the basis of its ¹³C NMR and HRESIMS (m/z 378.1180 [M + H]⁺, calcd for C₁₈H₂₀NO₈ 378.1183; 400.0999 [M + Na]⁺, calcd for C₁₈H₁₉NO₈Na, 400.1003), implying ten indices of hydrogen deficiency. The ¹H and ¹³C NMR of 6 displayed signals characteristic of quinoline alkaloid glycoside, which were similar to these reported for robustine.¹⁵ The only difference was that the β-D-glucopyranose unit in 6 substituted the hydroxyl group in robustine. The D-glucose was localized at C-8, which was supported by the HMBC correlations (Fig. 5) from H-1′ (δ_H 5.04) to C-8 (δ_C 153.0). D-Glucose were isolated from the enzymatic hydrolysate of 6 and identified by TLC and specific rotation. Therefore, the structure of 6 was elucidated as robustine-8-O-β-D-glucopyranoside.

Clausenaside G (7) was obtained as a yellow powder. Its molecular formula was established as C₁₅H₁₇NO₃ by the HRESIMS at m/z 282.1102 [M + Na]⁺, (calcd for C₁₅H₁₇NO₃Na, 282.1101), indicating eight indices of hydrogen deficiency. The ¹H and ¹³C NMR of 7 displayed signals characteristic of quinoline alkaloid, which was an analog of 3-hydroxyquinoline-2,4(1H,3H)-diones.¹⁶ In the HMBC spectrum (Fig. 6) of 7, correlations of H-1′ (δ_H 2.42)/C-2 (δ_C 171.9), C-3 (δ_C 82.0), C-4 (δ_C 195.1), C-2′ (δ_C 116.3), C-3′ (δ_C 135.2) and of H-4′ (δ_H 1.49) and H-5′ (δ_H 1.29)/C-2′ (δ_C 116.3), C-3′ (δ_C 135.2) suggested that the isopentene group was attached to C-2. The absolute configuration of 7 was defined as 3S by comparison of the experimental ECD spectra and calculated ECD data using the time-dependent density functional theory (TDDFT) method at the B₃LYP/6-31G (d) level.¹⁷ The calculated ECD spectrum of 3S (7a) (Fig. 7) matched the experimental spectrum very well. Thus, the structure of 7 was elucidated.

Fig. 6 Key ¹H, ¹H-COSY and HMBC correlations of compounds 7 and 8.

Fig. 7 Calculated ECD spectra of 7a (3S)- and 7b (3R)-isomers and the experimental ECD spectrum of 7.

Clausenaside H (8) was obtained as a white powder. Its molecular formula C₁₅H₁₇NO₃ was deduced from the HRESIMS (m/z 260.1280 [M + H]⁺, calcd for C₁₅H₁₈NO₃, 260.1281; 282.1102 [M + Na]⁺, calcd for C₁₅H₁₇NO₃Na 282.1101) and ¹³C NMR spectroscopic data, corresponding with eight indices of hydrogen deficiency. The ¹H and ¹³C NMR of 8 displayed signals characteristic of quinoline alkaloid, which were similar to these reported for atanine.¹⁸ The only difference between 8 and atanine was that a methyl group in atanine is replaced by a hydroxymethyl group [δ_H 3.76 (2H, s, H-4′), δ_C 66.2] in 8. This was supported further by the HMBC correlations (Fig. 6) between H-4′ (δ_H 3.76) and C-2′ (δ_C 120.7)/C-3′ (δ_C 135.8)/C-5′ (δ_C 13.7). The NOE difference experiment displayed that a strong enhancement of H-2′ was observed when H-4′ was irradiated, while H-2′ was no enhanced on irradiation of H-5′, indicating an E configuration of the double bond. Thus, the structure of 8 was elucidated.

The ten known alkaloids were identified as 4-methoxy-8-O-β-D-glucopyranosyloxy-2(1H)-quinolinone (9),¹⁹ integriquinolone (10),²⁰ araliopsine (11),²¹ clausenamide (12),²² 4-methoxy-2(1H)-quinolone (13),²³ 4-methoxy-1-methyl-2-quinolone (14),²⁴ neoclausenamide (15),²² demethylsecoclausenamide (16),²⁵ ribalinine (17),²¹ claulansine D (18)⁵ based on the analysis and comparison with their spectroscopic data (NMR, UV and MS) with literature data.

Compounds 1–18 were tested for their inhibitory effects on LPS-induced NO production in murine microglial BV2 cells. In the anti-inflammatory assay, compounds 4, 6, 9, 17, 18 exhibited moderate inhibitory effect on LPS-stimulated NO production in murine microglial BV2 cells with curcumin as a positive control as shown in Table 4, whereas the other compounds were inactive in this assay (IC₅₀ value > 10 μM).

Table 3 ¹H and ¹³C NMR spectroscopic data of compounds 7 and 8 (δ in ppm, J in Hz)

Position	7		8
	δH^a	δC^b	δH^a	δC^b
a In DMSO-d6 (600 MHz for 7, 400 MHz for 8).b In DMSO-d6 (150 MHz for 7, 100 MHz for 8). Coupling constants (J) in Hz are given in parentheses. The assignments were based on HSQC and HMBC experiments.
2		171.9 s		163.3 s
3		82.0 s		121.9 s
4		195.1 s		160.7 s
4a		120.6 s		116.0 s
5	7.76, dd (7.6, 1.6)	126.7 d	7.69, dd (8.0, 1.0)	122.5 d
6	7.20, t (7.6)	122.8 d	7.20, td (8.0, 0.8)	121.7 d
7	7.70, td (7.0, 1.6)	136.0 d	7.49, td (8.0, 1.4)	130.0 d
8	7.33, d (8.3)	115.5 d	7.33, dd (8.0, 1.4)	115.3 d
8a		142.6 s		137.7 s
1′	2.42, 2H, m	39.4 t	3.27, 2H, d (6.8)	22.4 t
2′	4.91, t (7.7)	116.3 d	5.40, td (6.8, 1.2)	120.7 d
3′		135.2 s		135.8 s
4′	1.49, s	25.8 q	3.76, 2H, s	66.2 t
5′	1.29, s	17.4 q	1.71, s	13.7 q
6′		171.9 s
NH			11.72, br s
N-CH3	3.36, 3H, s	29.7 q
OCH3			3.87, s	61.5 q

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Table 4 Inhibitory effects of compounds 1–18 against LPS-induced NO production in microglia BV2 Cells

Compound	IC50 (μM)	Compound	IC50 (μM)
a Positive control.
1	>10	10	>10
2	>10	11	>10
3	>10	12	>10
4	7.58	13	>10
5	>10	14	>10
6	6.28	15	>10
7	>10	16	>10
8	>10	17	5.70
9	5.03	18	5.93
Curcumin^a	5.59

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Experimental

Optical rotations were measured on a JASCO P2000 automatic digital polarimeter. UV spectra were recorded on a JASCO V-650 spectrophotometer, CD spectra were measured on a JASCO J-815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 spectrometer using an FT-IR microscope transmission method. NMR spectra were acquired with Bruker AVIIIHD 600, VNS-600, and Mercury-400 spectrometers in DMSO-d₆ and MeOH-d₄. HRESIMS spectra were collected on an Agilent 1100 series LC/MSD ion trap mass spectrometer. MPLC system was composed of two C-605 pumps (Büchi), a C-635 UV detector (Büchi), a C-660 fraction collector (Büchi), and an ODS column (450 mm × 60 mm, 50 μm, 400 g; YMC). Semi-preparative HPLC was conducted using a Shimadzu LC-6AD instrument with an SPD-20A detector and a Daicel Chiralpak AD-H column (250 × 10 mm, 5 μm). Preparative HPLC was also performed on a Shimadzu LC-6AD instrument with a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Column chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China), SF-PRP 512A (100–200 mesh, Beijing Sunflower and Technology Development Co., Beijing, People's Republic of China), ODS (50 μm, YMC, Japan), and Sephadex LH-20 (GE, Sweden). TLC was carried out on glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating. Curcumin (99%, Sinopharm Chemical Reagent Company Limited).

Plant materials

The stems of C. lansium were collected in Liuzhou, Guangxi, China, in March 2013 and identified by Engineer Guangri Long, Forestry of Liuzhou. A voucher specimen has been deposited at the Herbarium of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (ID-S-2320).

Extraction and isolation

Air-dried, powdered stems of C. lansium (200 kg) were extracted with 95% ethanol (1000 L × 2 h × 3). The residue was suspended in water and then partitioned with EtOAc (3 × 40 L), and n-BuOH (3 × 40 L), successively. After removing the solvent, the n-BuOH-soluble portion (850 g) was fractionated via macroporous adsorbent resin (HPD-100) column with H₂O, 30% EtOH, 60% EtOH, and 95% EtOH to yield four corresponding fractions A–D.

Fraction B (180 g) was fractionated via silica gel column chromatography, eluting with CHCl₃–MeOH–H₂O (10 : 1 : 0.05, 9 : 1 : 0.1, 8 : 2 : 0.2, 7 : 3 : 0.3, 6 : 4 : 0.4) to afford twelve fractions B₁-B₁₂ on the basis of TLC analysis. Fraction B₄ (5.9 g) was further separated by reversed-phase silica MPLC with 20%–50% MeOH (50 mL min⁻¹, 6 h) to afford B_4-1-B_4-47 fractions. Fractions B_4-38-B_4-42 were successively separated using preparative HPLC (detection at 210 nm, 18% CH₃CN, 8 mL min⁻¹) to yield 1 (29 mg, t_R 83.45 min), 2 (5.0 mg, t_R 73.87 min), 3 (19 mg, t_R 67.02 min) and 6 (9 mg, t_R 78.25 min). Fraction B₆ (8.3 g) was further separated by reversed-phase silica MPLC with 20%–45% MeOH (50 mL min⁻¹, 6 h) to afford fractions B_6-1-B_6-39. Fraction B_6-31-B_6-32 was successively separated using preparative HPLC (detection at 210 nm, 15% CH₃CN, 8 mL min⁻¹) to yield 5 (3 mg, t_R 51.14 min). Fraction B₇ (36.8 g) was fractionated via silica gel column chromatography, eluting with CHCl₃–MeOH–H₂O (10 : 1 : 0.5–8 : 2 : 0.3) to afford fractions B_7-1-B_7-18 on the basis of TLC analysis. Fraction B_7-6 (11.7 g) was further separated by reversed-phase silica MPLC with 20%–50% MeOH (50 mL min⁻¹, 8 h) to afford B_7-6-1-B_7-6-11 fractions. Fraction B_7-6-5 was successively separated using preparative HPLC (detection at 210 nm, 12% CH₃CN, 8 mL min⁻¹) to yield 4 (16 mg, t_R 40.59 min). Fraction B_7-6-8 was successively separated using preparative HPLC (detection at 210 nm, 11% CH₃CN, 8 mL min⁻¹) to yield 9 (16 mg, t_R 40.59 min).

Fraction C (220 g) was partitioned via kieselguhr eluting with CHCl₃, EtOAc, n-BuOH, acetone, and MeOH to afford five fractions C₁-C₅. After the solvent was removed, the C₁ fraction (43 g) was subjected to SF-PRP 512A resin eluting with 35%–70% MeOH to afford fractions C_1-1-C_1-6. C_1-2 (7.1 g) was further separated by reversed-phase silica MPLC with 30%–50% MeOH (50 mL min⁻¹, 6 h) to afford fractions C_1-2-1-C_1-2-11. Fraction C_1-2-4 was subjected to Sephadex LH-20 and preparative HPLC (detection at 210 nm, 19% CH₃CN, 8 mL min⁻¹) to yield 10 (8 mg, t_R 39.04 min). Fraction C_1-2-5 was subjected to Sephadex LH-20 and preparative HPLC (detection at 210 nm, 19% CH₃CN, 8 mL min⁻¹) to yield 11 (7 mg, t_R 32.04 min). Fraction C_1-2-8 was subjected to Sephadex LH-20 and preparative HPLC (detection at 210 nm, 25% CH₃CN, 8 mL min⁻¹) to yield 12 (153 mg, t_R 21.99 min) and 13 (8 mg, t_R 30.68 min). Fraction C₂ (14.6 g) was further separated by reversed-phase silica MPLC with 35%–55% MeOH (50 mL min⁻¹, 10 h) to afford fractions C_2-1-C_2-15. Fractions C_2-11 was subjected to Sephadex LH-20 and preparative HPLC (detection at 210 nm, 29% CH₃CN, 8 mL min⁻¹) to yield 7 (2 mg, t_R 46.59 min), 14 (80 mg, t_R 38.00 min) and 15 (54 mg, t_R 38.20 min). Fractions C_2-12 was subjected to Sephadex LH-20 and preparative HPLC (detection at 210 nm, 30% CH₃CN, 8 mL min⁻¹) to yield 8 (10 mg, t_R 28.20 min), 16 (84 mg, t_R 45.00 min), 17 (40 mg, t_R 41.81 min) and 18 (4 mg, t_R 42.87 min).

Structure characterization

Clausenaside A (1). White amorphous powder; [α]²⁵_D +18.5 (c 0.1 MeOH); UV (MeOH) λ_max (log ε) 201.6 (4.31), 225.6 (4.14) nm; IR (microscope) ν_max 3314, 2919, 1640, 1617, 1540, 1515, 1246, 1103, 1075, 1036, 828, 697 cm⁻¹; CD (MeOH) λ_max (Δε) 217.5 (+2.28), 238 (−2.86) nm; ¹H NMR (DMSO-d₆, 400 MHz) and ¹³C NMR (DMSO-d₆, 100 MHz), see Table 1; HRESIMS m/z 456.1634 [M + Na]⁺ (calcd for C₂₂H₂₇NO₈Na, 456.1629).

Clausenaside B (2). White amorphous powder; [α]²⁵_D −8.5 (c 0.1 MeOH); UV (MeOH) λ_max (log ε) 201.8 (4.61), 226.2 (4.38) nm; IR (microscope) ν_max 3361, 2924, 1641, 1613, 1577, 1543, 1513, 1306, 1248, 1077, 1029, 833, 713 cm⁻¹; CD (MeOH) λ_max (Δε) 218 (−1.48), 237 (+2.71) nm; ¹H NMR (DMSO-d₆, 400 MHz) and ¹³C NMR (DMSO-d₆, 100 MHz), see Table 1; HRESIMS m/z 456.1635 [M + Na]⁺ (calcd for C₂₂H₂₇NO₈Na, 456.1629).

Clausenaside C (3). White amorphous powder; [α]²⁵_D +8.2 (c 0.1 MeOH); UV (MeOH) λ_max (log ε) 201.6 (4.14) nm; IR (microscope) ν_max 3357, 2917, 1641, 1577, 1541, 1490, 1313, 1159, 1076, 701 cm⁻¹; CD (MeOH) λ_max (Δε) 203.5 (+2.94), 228 (−1.90) nm; ¹H NMR (DMSO-d₆, 400 MHz) and ¹³C NMR (DMSO-d₆, 100 MHz), see Table 1; HRESIMS at m/z 426.1524 [M + Na]⁺ (calcd for C₂₁H₂₅NO₇Na, 426.1523).

Clausenaside D (4). White amorphous powder; [α]²⁵_D −58.4 (c 0.1 MeOH); UV (MeOH) λ_max (log ε) 228.2 (4.39), 269.6 (3.48), 320.4 (3.34) nm; IR (microscope) ν_max 3501, 3362, 3273, 2930, 2882, 1640, 1615, 1574, 1109, 1080, 841, 755 cm⁻¹; ¹H NMR (DMSO-d₆, 600 MHz) and ¹³C NMR (DMSO-d₆, 150 MHz), see Table 2; HRESIMS m/z 338.1232 [M + H]⁺ (calcd for C₁₆H₂₀NO₇, 338.1234).

Clausenaside E (5). White amorphous powder; [α]²⁵_D −28.7 (c 0.1 MeOH); UV (MeOH) λ_max (log ε) 226.6 (4.38), 268 (3.50), 314.6 (3.22) nm; IR (microscope) ν_max 3391, 2923, 1647, 1581, 1463, 1400, 1326, 1245, 1113, 1071, 828, 775, 759 cm⁻¹; ¹H NMR (DMSO-d₆, 600 MHz) and ¹³C NMR (DMSO-d₆, 150 MHz), see Table 2; HRESIMS m/z 368.1346 [M + H]⁺ (calcd for C₁₇H₂₂NO₈, 368.1340), 390.1161 [M + Na]⁺ (calcd for C₁₇H₂₁NO₈Na, 390.1159).

Clausenaside F (6). White amorphous powder; [α]²⁵_D −57.8 (c 0.1 MeOH); UV (MeOH) λ_max (log ε) 202.4 (4.24), 243.2 (4.53) nm; IR (microscope) ν_max 3342, 2935, 1589, 1516, 1460, 1397, 1373, 1267, 1094, 1034, 748 cm⁻¹; ¹H NMR (MeOH-d₄, 600 MHz) and ¹³C NMR (MeOH-d₄, 150 MHz), see Table 2; HRESIMS m/z 378.1180 [M + H]⁺ (calcd for C₁₈H₂₀NO₈, 378.1183), 400.0999 [M + Na]⁺ (calcd for C₁₈H₁₉NO₈Na, 400.1003).

Clausenaside G (7). White amorphous powder; [α]²⁵_D −38.3 (c 0.1 MeOH); UV (MeOH) λ_max (log ε) 201.8 (4.13), 234.8 (4.27) nm; IR (microscope) ν_max 3394, 2920, 2849, 1709, 1663, 1603, 1472, 1368, 1302, 1123, 1102, 759 cm⁻¹; ¹H NMR (DMSO-d₆, 600 MHz) and ¹³C NMR (DMSO-d₆, 150 MHz), see Table 3; HRESIMS m/z 282.1102 [M + Na]⁺ (calcd for C₁₅H₁₇NO₃Na, 282.1101).

Clausenaside H (8). White amorphous powder; [α]²⁵_D +8.6 (c 0.1 MeOH); UV (MeOH) λ_max (log ε) 226.6 (4.41), 270.6 (3.74), 322.8 (3.71) nm; IR (microscope) ν_max 3295, 3174, 2997, 2945, 2898, 2853, 1659, 1612, 1571, 1501, 1434, 1379, 1267, 1094, 1001, 751 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) and ¹³C NMR (DMSO-d₆, 100 MHz), see Table 3; HRESIMS m/z 260.1280 [M + H]⁺ (calcd for C₁₅H₁₈NO₃, 260.1281), 282.1102 [M + Na]⁺ (calcd for C₁₅H₁₇NO₃Na, 282.1101).

Enzymatic hydrolysis of 1–6

Compounds 1–6 (1.0–8.0 mg) were hydrolyzed in H₂O (1 mL) with snailase at 37 °C for 4 h, then extracted by EtOAC (3 × 3 mL). The aqueous phase was dried by a stream of N₂, then chromatographed over silica gel eluting with CH₃CN–H₂O (8 : 1), to yield glucose. The glucose (0.3–1.2 mg) from the hydrolysates of 1–6 gave retention factor (R_f ≈ 0.33; CH₃CN–H₂O, 6 : 1) on TLC, [α]²⁵_D values +43.1 to +68.6 (c = 0.02–0.1, H₂O)], and ¹H NMR spectral data (D₂O) consistent with those of an authentic D-glucose. The EtOAC phase was concentrated under reduced pressure, and purified by RP-HPLC to obtain 1a (3.14 mg) from 1. (S)-(+)-tembamide (1a): white amorphous powder; [α]²⁵_D +54.8 (c 0.2 CHCl₃); CD (MeOH) λ_max (Δε) 216 (+0.21), 235 (−2.14) nm; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 8.46 (NH, t, J = 6.0 Hz), 7.83 (2H, d, J = 7.6 Hz), 7.50 (1H, m), 7.44 (2H, m), 7.28 (2H, d, J = 8.0 Hz), 6.89 (2H, d, J = 8.0 Hz), 5.41 (1H, d, J = 4.0 Hz), 4.72 (1H, m), 3.72 (3H, s), 3.43 (1H, m), 3.28 (1H, m); ¹³C NMR (DMSO-d₆, 150 MHz) δ_C 166.3, 158.3, 135.8, 134.5, 131.0, 128.2, 128.2, 127.2, 127.2, 127.1, 127.1, 113.4, 113.4, 70.7, 55.0, 47.7; HRESIMS at m/z 294.1109 [M + Na]⁺ (calcd for C₁₆H₁₇NO₃Na, 294.1101).

Preparation of (R)- and (S)-MTPA esters of 1a

A solution of 1a (1.09 mg) in dehydrated CH₂Cl₂ (2 mL) was treated with (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride [(S)-MTPA-Cl (10 mg)] in the presence of anhydrous pyridine, and the mixture was stirred at room temperature for 13 h. After cooling, the reaction mixture was poured into ice-water and extracted with EtOAC. The EtOAC extract was successively washed with 5% aqueous HCl, saturated aqueous NaHCO₃, and brine, then dried over Na₂SO₄ and filtered. The solvent was removed from the filtrate under reduced pressure to afford a residue.²⁶ The residue was purified by semi-preparative HPLC (C18 column, 3.0 mL min⁻¹, UV 210 nm, 58% CH₃CN–H₂O) to yield (S)-MTPA ester derivative of 1a (compound 1aa 1.12 mg). Using a similar procedure, (R)-MTPA ester derivative of 1a (compound 1ab 0.93 mg) was obtained from 1a (1.04 mg).

Compound 1aa. ¹H NMR (600 MHz, DMSO) δ_H 8.61 (1H, dd, J = 6.4, 4.5 Hz), 7.72 (2H, m), 7.53 (1H, m), 7.46 (2H, m), 7.39 (2H, d, J = 8.7 Hz), 7.36 (1H, m), 7.31 (2H, m), 7.23 (2H, dd, J = 8.4, 7.3 Hz), 7.00 (2H, d, J = 8.7 Hz), 6.25 (1H, dd, J = 9.3, 4.5 Hz), 3.80 (1H, m), 3.77 (3H, s), 3.52 (1H, dt, J = 13.9, 4.5 Hz), 3.35 (3H, s); ¹³C NMR (DMSO-d₆, 150 MHz) δ_C 166.4, 165.4, 159.6, 134.0, 131.3, 131.2, 129.8, 128.7, 128.7, 128.5, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 127.1, 127.1, 127.1, 127.1, 114.1, 114.1, 75.9, 55.2, 55.2, 43.6.

Compound 1ab. ¹H NMR (600 MHz, DMSO) δ_H 8.82 (1H, dd, J = 6.5, 4.3 Hz), 7.84 (2H, m), 7.55 (1H, m), 7.49 (2H, dd, J = 8.2, 6.8 Hz), 7.40 (1H, m), 7.25 (4H, m), 7.22 (2H, d, J = 8.7 Hz), 6.94 (2H, d, J = 8.7 Hz), 6.20 (1H, dd, J = 9.6, 4.1 Hz), 3.83 (1H, ddd, J = 14.0, 9.6, 6.6 Hz), 3.77 (3H, s), 3.53 (1H, dt, J = 14.0, 4.2 Hz), 3.40 (3H, s); ¹³C NMR (DMSO-d₆, 150 MHz) δ_C 166.4, 165.1, 159.5, 133.9, 131.4, 129.8, 129.6, 128.7, 128.5, 128.4, 128.4, 128.4, 128.4, 128.2, 128.2, 127.1, 127.1, 127.0, 126.9, 126.9, 113.9, 113.9, 76.3, 55.3, 55.2, 43.9.

Inhibition of nitric production assay

The murine microglial BV2 cells, purchased from the Cell Culture Centre at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), at 37 °C atmosphere, 5% CO₂, and 100% relative humidity. The microglia cells were placed in 96-well cell culture plates (2 × 104 cell per mL) and preincubated for 24 h. Then the cells were treated with various concentrations of isolated compounds in triplicate for 1 h and continuously incubated with LPS (Sigma-Aldrich) (0.3 μg mL⁻¹) for 24 h. Curcumin was used as the positive control. After incubation, the supernatants (100 μL) were added to a solution of 100 μL of Griess reagent (a 1 : 1 mixture of 0.1% naphthylethylenediamine and 1% sulfanilamide in 5% H₃PO₄) at room temperature for 20 min. NO concentration was quantified by a microplate reader at 540 nm for the amount of stable nitrite produced in the cell culture supernatants using the Griess assay.²⁷

Acknowledgements

We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, for the measurement of the UV, IR, CD, NMR, and HRESIMS spectra. This research program is financially supported by the National Natural Science Foundation of China (No. 21272278) and the National Megaproject for Innovative Drugs (No. 2012ZX09301002-002).

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Footnote

† Electronic supplementary information (ESI) available: The spectra including 1D-, 2D-NMR, HRESIMS of compounds 1–8 as well as related original CD spectrum for Clausenasides A (1), B (2) and C (3), and ECD calculation data for Clausenaside G (7). See DOI: 10.1039/c5ra14173g

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