Triterpenoid Saponins of Pulsatilla koreana Root Have Inhibition Effects of Tumor Necrosis Factor-α Secretion in Lipopolysaccharide-Induced RAW264.7 Cells

Abstract

In the present study, a new oleanane-type triterpenoid saponin, pulsatilloside F (1), along with 21 known compounds (2–22), were isolated from the root of Pulsatilla koreana. Their chemical structures were elucidated by mass, ¹H-, ¹³C-NMR, correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond connectivity (HMBC) spectroscopy. Anti-inflammatory effects of the compounds were evaluated in terms of inhibitory of tumor necrosis factor α (TNF-α) secretion in the lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cell line. Compounds 19 and 20 exhibited particularly inhibitory effects with respective IC₅₀ values of 0.32 and 0.65 µm. Compounds 1–4, 7 and 10–13 exhibited inhibitory effects with inhibition rates up to 41.55–73.76% at a concentration of 5 µm, respectively.

The Pulsatilla genus, Pulsatilla koreana Nakai (Ranuculaceae), from Korea, is an important herb in traditional medicine used to treat amoebic dysentery and malaria.¹⁾ Phytochemical studies on P. koreana root have demonstrated the presence of protoanemonin,²⁾ deoxypodophyllotoxin,³⁾ oleanane and lupane-type triterpenoid saponins.^4–6) Root extract of P. koreana has been reported to possess antitumor,⁷⁾ antibiotic,²⁾ and anti-inflammatory activities.⁸⁾ In this report, a new compound (1), and 21 known compounds (2–22), were isolated from a methanol extract of P. koreana. The structures were elucidated by one-dimensional (1D)-, and two-dimensional (2D)-nuclear magnetic resonance (NMR) and mass spectrometry.

Inflammatory cytokines are produced by innate immune cells, such as macrophages and dendritic cells, during infection. Lipopolysaccharide (LPS) is an endotoxin that induces septic shock syndrome and stimulates the production of inflammatory mediators such as tumor necrosis factor (TNF)-α.⁹⁾ TNF-α is a well-characterized, pro-inflammatory cytokine released primarily from monocytes and macrophages upon invasion of the host by a wide variety of pathogens. It plays a crucial role in host defense and the inflammatory response. Although it has numerous beneficial roles in immune regulation, it has also been implicated in the pathogenesis of both acute and chronic inflammatory disease.¹⁰⁾ A reduction in the secretion of TNF-α would greatly aid in the treatment or mitigation of chronic inflammatory diseases.¹¹⁾ Here, we report the effects of compounds isolated from P. koreana on the inhibitory activity of TNF-α secretion in the presence of LPS in a murine RAW264.7 macrophage cell line.

Results and Discussion

A MeOH extract (150 g) of the roots of P. koreana was suspended in H₂O and successively extracted with EtOAc and n-BuOH. Using combined chromatographic separations, a new oleanane-type triterpenoid saponin (1), along with 21 known compounds (2–22) were isolated.

Pulsatilloside F (1) was obtained as a white crystal. The molecular formula, C₇₇H₁₂₆O₄₁, was determined from the pseudomolecular ion peak [M−H]⁻ at m/z 1705.7692 (Calcd for C₇₇H₁₂₅O₄₁, 1705.7696) in the high-resolusion electrospray ionization quadrupole time-of-flight mass spectrometry (HR-ESI-QTOF-MS). Its Fourier transform infrared (FT-IR) spectrum showed absorption bands at 3367 and 1731 cm⁻¹, suggesting the presence of hydroxyl and carbonyl groups, respectively. Acid hydrolysis of compound 1 with 10% HCl resulted in hederagenin, l-arabinose, d-glucose, and l-rhamnose. The ¹³C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra exhibited 30 aglycone carbon signals, which were assignable to six methyl groups at δ_C 14.0 (C-24), 16.0 (C-25), 17.3 (C-26), 23.4 (C-30), 25.8 (C-27), and 32.9 (C-29); eleven methylene groups at δ_C 17.9 (C-6), 23.1 (C-16), 23.6 (C-11), 26.2 (C-2), 28.1 (C-15), 32.3 (C-22), 32.5 (C-7), 33.7 (C-21), 38.8 (C-1), 45.9 (C-19), and 63.7 (C-23); four methine groups at δ_C 41.4 (C-18), 47.3 (C-5), 47.9 (C-9), and 80.6 (C-3); six quaternary carbons at δ_C 30.5 (C-20), 36.6 (C-10), 39.6 (C-8), 41.9 (C-14), 43.3 (C-4), and 46.8 (C-17); two olefinic carbons at δ_C 122.7 (C-12) and 143.9 (C-13); and one carbonyl group at δ_C 176.3 (C-28). Another 47 carbon signals were assigned to eight sugar moieties. The ¹H-NMR spectrum revealed six quaternary methyl groups at δ_H 0.87 (Me-29), 0.88 (Me-30), 0.98 (Me-25), 1.12 (Me-26), 1.16 (Me-24), and 1.18 (Me-27), and a broad singlet for an olefinic proton at δ_H 5.31 (br s) characteristic of the Δ¹² proton in pentacyclic triterpenes.¹²⁾ Oxymethylene groups at δ_H 3.80 (d, J=11.2 Hz, H-23a) and 4.23 (d, J=11.2 Hz, H-23b) were assigned to H-23 by heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond correlation (HMBC) spectra (Table 1). All of the above signals showed a characteristic hederagenin skeleton.¹³⁾ In contrast, eight anomeric proton signals at δ_H 4.80 (d, J=6.9 Hz, ara-H1), 4.91 (d, J=7.0 Hz, glc⁗-H1), 5.01 (d, J=7.7 Hz, glc-H1), 5.07 (d, J=7.0 Hz, glc″-H1), 5.37 (d, J=7.7 Hz, glc′-H1), 5.77 (br s, rha′-H1), 6.16 (d, J=8.0 Hz, glc‴-H1), and 6.17 (br s, rha-H1) showed HMQC correlations with anomeric carbon signals at δ_C 104.6 (ara-C1), 104.7 (glc⁗-C1), 106.6 (glc-C1), 104.8 (glc″-C1), 106.4 (glc′-C1), 102.5 (rha′-C1), 95.4 (glc‴-C1) and 101.1 (rha-C1), indicating that compound 1 possessed eight sugar units (Table 2). The downfield chemical shift of C-3 (δ_C 80.6) and the upfield chemical shift of C-28 (δ_C 176.3) indicated that compound 1 was a bisdesmosidic saponin. The sequence of sugar residues of compound 1 was assigned unambiguously on the basis of HMBC spectra. The location of the sugar chain at C-3 was identified as O-β-d-glucopyranosyl-(1→4)-[O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→3)-O-α-l-rhamnopyranosyl-(1→2)]-O-α-l-arabinopyranosyl based on the HMBC correlations: H-1 of glucose″ (δ_H 5.07) with C-4 of glucose′ (δ_C 80.9), H-1 of glucose′ (δ_H 5.37) with C-3 of rhamnose (δ_C 82.9), H-1 of rhamnose (δ_H 6.17) with C-2 of arabinose (δ_C 75.5), H-1 of glucose (δ_H 5.01) with C-4 of arabinose (δ_C 80.5), and a cross-peak between H-1 of arabinose (δ_H 4.80) and C-3 of the aglycone (δ_C 80.6). The sugar chain at C-28 was established as O-α-l-rhamnopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl from the following HMBC correlations: H-1 of rhamnose′ (δ_H 5.77) with C-4 of glucose⁗ (δ_C 77.9), H-1 of glucose⁗ (δ_H 4.91) with C-6 of glucose‴ (δ_C 68.9), and H-1 of glucose‴ (δ_H 6.16) with C-28 of the aglycone (δ_C 176.3) (Fig. 1). On the basis of the above analyses, the structure of compound 1 was established as 3-O-β-d-glucopyranosyl-(1→4)-[O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→3)-α-O-l-rhamnopyranosyl(1→2)]-O-α-l-arabinopyranosyl hederagenin 28-O-α-l-rhamnopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl ester, and was termed pulsatilloside F.

Fig. 1. Key HMBC Correlations of Compound 1

Table 1. NMR Spectral Data for Aglycone of Compound 1

Position	δCa,b)	δHa,c) [mult. (J in Hz)]	Position	δCa,b)	δHa,c) [mult. (J in Hz)]
1	38.8	1.07 (m)	16	23.1	1.91 (m)
		1.54 (m)			2.05 (m)
2	26.2	2.00 (m)	17	46.8
		2.25 (m)	18	41.4	3.17 (d, 11.7)
3	80.6	4.30 (dd, 3.5, 10.5)	19	45.9	1.20 (m)
4	43.3				1.69 (m)
5	47.3	1.75 (d, 11.8)	20	30.5
6	17.9	1.55 (m)	21	33.7	1.08 (m)
7	32.5	1.56 (m)	22	32.3	1.73 (m)
		1.56 (m)			1.87 (m)
8	39.6		23	63.7	3.80 (d, 11.2)
9	47.9	1.77 (m)			4.23 (d, 11.2)
10	36.6		24	14.0	1.16 (s)
11	23.6	1.96 (m)	25	16.0	0.98 (s)
12	122.7	5.31 (br s)	26	17.3	1.12 (s)
13	143.9		27	25.8	1.18 (s)
14	41.9		28	176.3
15	28.1	1.09 (m)	29	32.9	0.87 (s)
		2.25 (m)	30	23.4	0.88 (s)

Assignments were done by HMQC, HMBC and ¹H–¹H COSY experiments; a) measured in pyridine-d₅; b) 150 MHz; c) 600 MHz.

Table 2. NMR Spectral Data for Suger Moieties of Compound 1

Position	δCa,b)	δHa,c) [mult. (J in Hz)]	Position	δCa,b)	δHa,c) [mult. (J in Hz)]
C-3			glc″-1	104.8	5.07 (d, 7.0)
ara-1	104.6	4.80 (d, 6.9)	2	75.1	3.92 (m)
2	75.5	4.13 (m)	3	77.9	4.15 (m)
3	73.8	4.35 (m)	4	71.2	4.87 (m)
4	80.5	4.76 (m)	5	78.2	4.10 (m)
5	65.7	3.70 (d, 10.8)	6	62.2	4.24 (m)
		4.40 (m)			4.50 (m)
glc-1	106.6	5.01 (d, 7.7)	C-28
2	75.3	4.13 (m)	glc⁗-1	95.4	6.16 (d, 8.0)
3	78.2	4.18 (m)	2	74.5	4.10 (m)
4	71.0	4.88 (m)	3	78.5	4.33 (m)
5	78.6	3.92 (m)	4	70.5	4.15 (m)
6	62.3	4.37 (m)	5	77.8	3.87 (m)
		4.54 (m)	6	68.9	4.30 (m)
rha-1	101.1	6.17 (br s)			4.59 (m)
2	71.4	4.92 (m)	glc⁗-1	104.7	4.91 (d, 7.0)
3	82.9	4.75 (dd, 2.5, 9.3)	2	75.0	3.93 (m)
4	72.7	4.50 (m)	3	76.5	4.12 (m)
5	69.5	4.98 (m)	4	77.9	4.07 (m)
6	18.3	1.47 (d, 6.0)	5	77.0	3.60 (d, 8.9)
glc′-1	106.4	5.37 (d, 7.7)	6	61.0	4.05 (m)
2	75.2	3.92 (m)			4.15 (m)
3	76.5	4.28 (m)	rha′-1	102.5	5.77 (br s)
4	80.9	4.31 (m)	2	72.3	4.65 (m)
5	76.3	4.08 (m)	3	72.5	4.53 (m)
6	61.5	3.83 (m)	4	73.6	4.33 (m)
		4.44 (m)	5	70.1	4.29 (m)
			6	18.3	1.60 (d, 6.0)

Assignments were done by HMQC, HMBC and ¹H–¹H COSY experiments; glc: d-glucopyranosyl; rha: l-rhamnopyranosyl; ara: l-arabinopyranosyl; a) Measured in pyridine-d₅; b) 150 MHz; c) 600 MHz.

Comparison of the 1D- and 2D-NMR and mass spectral data with reported values led to the identification of structures of the 21 known compounds (2–22) as hederacolchiside F (2),¹⁴⁾ pulsatilla saponin A (3),¹⁴⁾ cernuoside A (4),¹⁵⁾ hederacholchiside E (5),¹⁶⁾ beesioside Q (6),¹⁷⁾ oleanolic acid 3-O-β-d-glucopyranosyl(1→4)-β-d-glucopyranosyl(1→3)-α-l-rhamnopyranosyl(1→2)[β-d-glucopyranosyl(1→4)]-α-l-arabinopyranosyl-28-O-α-l-rhamnopyranosyl(1→4)-β-d-glucopyranosyl(1→6)-β-d-glucopyranosyl ester (7),¹⁸⁾ hederacoside B (8),¹⁹⁾ 3-O-β-d-glucopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→2)-[O-β-d-glucopyranosyl-(1→4)]-O-α-l-arabinopyranosyl oleanolic acid (9),²⁰⁾ raddeanoside R17 (10),²¹⁾ 3-O-β-d-glucopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopyranosyl oleanolic acid (11),⁶⁾ raddeanoside R13 (12),⁶⁾ 3-O-β-d-glucopyranosyl(1→4)-β-d-glucopyranosyl(1→3)-α-l-rhamnopyranosyl(1→2)[β-d-glucopyranosyl(1→4)]-α-l-arabinopyranosyl oleanolic acid (13),⁶⁾ 3-O-β-d-glucopyranosyl(1→4)-β-d-glucopyranosyl(1→3)-α-l-rhamnopyranosyl(1→2)-α-l-arabinopyranosyl oleanolic acid (14),⁶⁾ 23-hydroxy-3β-[(O-α-l-rhamnopyranosyl-(1→2)-O-[O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl(1→4)]-O-α-l-arabinopyranosyl)oxy]lup-20(29)-en-28-oic acid 28-O-α-l-rhamnopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl ester (15),²²⁾ 3β-[O-β-d-glucopyranosyl-(1→3)-O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopyranosyl)oxy]lup-20(29)-en-28-oic acid (16),⁶⁾ pulsatilloside E (17),⁸⁾ 3β-[(O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopyranosyl)oxy]lup-20(29)-en-28-oic acid 28-O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl ester (18),⁸⁾ pulchinenoside C (19),²²⁾ cussosaponin C (20),⁸⁾ hederagenin 3-O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopyranoside (21),²³⁾ and 23-hydroxy-3β-[O-α-l-rhamnopyranosyl-(1→2)-[O-β-d-glucopyranosyl-(1→4)]-O-α-l-arabinopyranosyl)oxy]lup-20(29)-en-28-oic acid (22)⁸⁾ (Fig. 2).

When RAW264.7 cells were cultured with the compounds isolated from P. koreana, the secretion of TNF-α was severely decreased by treatment with compounds 1–4, 7, and 10–13, and exhibited inhibitory effects with inhibition rates up to 41.55–73.76% at a concentration of 5 µm, respectively. Compounds 19 and 20 showed the strongest inhibitory effect with IC₅₀ values of 0.32±0.1 µm and 0.65±0.1 µm, respectively (Table 3). Other compounds had negligible effects at the same concentration. Therefore, compounds 1–4, 7, 10–13, 19, and 20 may be excellent therapeutics for the treatment of abnormally-increased-TNF-α levels, such as rheumatoid arthritis,²⁴⁾ atherosclerosis,²⁵⁾ and graft rejection.²⁶⁾

Fig. 2. Structures of Compounds 1–22 from the Roots of P. koreana

Table 3. IC₅₀ Values of Compounds 1–22 for TNF-α Production in LPS-Stimulated RAW264.7 Cells

Compounds	IC50 (µm)
1	>5
2	2.38±0.4
3	2.01±0.01
4	2.89±0.2
5	>5
6	>5
7	3.78±0.4
8	>5
9	>5
10	2.76±0.2
11	3.89±0.4
12	3.64±0.3
13	2.81±0.1
14	>5
15	>2
16	>2
17	>2
18	>2
19	0.32±0.1
20	0.65±0.1
21	>2
22	>2
Dexsamethasonea)	0.01±0.0001

a) Dexsamethasone was used as positive control. Data are presented as the mean±S.D. and all experiments measured in triplicate.

Upon examination of the structure–activity relationship of the lupane-type triterpenoid saponins (15–22), compounds 19 and 20 showed the strongest inhibitory effects on TNF-α secretion, and their structures were similar. When the sugar unit at C-3 of the aglycone was linked to a rhamnose-arabinose chain, the anti-inflammatory activity increased significantly when compared to a rhamnose-(glucose-glucose-)arabinose chain (15), and a rhamnose-(glucose-)arabinose chain (17). Additionally, the sugar moieties of compounds 19 and 20 at C-28 linked to a rhamnose-glucose-glucose chain of the aglycones compared to a carboxyl group (21) and a glucose-glucose chain (18) indicate that the anti-inflammatory activity could be increased significantly. Compounds 15 and 17 were also linked to a rhamnose-glucose-glucose chain; however, they did not display significant anti-inflammatory effects. As previous reported, compounds 19 and 20 showed significant inhibitory activities against nitric oxide (NO) production on LPS-stimulated RAW264.7 cells, with relative NO production values of 12.7±3.1 and 13.0±2.5% and at 100 µm, respectively.⁸⁾ Moreover, bisdesmosidic lupane-type saponin also showed significant effect against carrageenan, tetradecanoylphorbol acetate, arachidonic acid and ethyl phenylpropiolate acute edemas.²⁷⁾ Therefore, the rhamnose-arabinose chain at C-3 and the rhamnose-glucose-glucose chain at C-28 of the bisdesmosidic lupane-type saponin appear to be the key functional elements. These data may be useful in evaluating the structure–activity relationships of other lupane-type triterpenoid saponins and in developing anti-inflammatory agents for medical uses.

Experimental

General Experimental Procedures

Melting points were determined using an Electrothermal IA-9200 system. Optical rotations were determined using a Jasco DIP-370 automatic polarimeter. The FT-IR spectra were measured using a Nicolet 380 FT-IR spectrometer. GC was carried out on a Shimadzu-2010 spectrometer: detector, flame ionization detector (FID); detection temperature, 300°C; column, SPB-1 (0.25 mm i.d.×30 m); column temperature, 230°C; carrier gas, He (2 mL/min) injection temperature, 250°C; injection volume, 0.5 µL; The NMR spectra were recorded using a JEOL ECA 600 spectrometer (¹H, 600 MHz; ¹³C, 150 MHz), and high-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained using an Agilent 6530 Accurate-Mass Q-TOF LC/MS system. Column chromatography was performed using a silica gel (Kieselgel 60, 70–230, and 230–400 mesh, Merck, Darmstadt, Germany), YMC RP-18 resins, and thin layer chromatography (TLC) was performed using pre-coated silica-gel 60 F₂₅₄ and RP-18 F₂₅₄S plates (both 0.25 mm, Merck, Darmstadt, Germany).

Plant Material

Dried roots of P. koreana were purchased from herbal market, Kumsan, Chungnam, Korea in March 2009 and identified by one of the authors (Prof. Young Ho Kim). A voucher specimen (CNU 09106) was deposited at the Herbarium of College of Pharmacy, Chungnam National University, Korea.

Extraction and Isolation

Dried roots of P. koreana (2.0 kg) were extracted with MeOH under reflux for 10 h (7 L×3 times) to yield 500.0 g of extract. This extract was suspended in water and partitioned with ethyl acetate to yield 37.0 g ethyl acetate extract and 463.0 g water extract. The water extract was partitioned with n-BuOH to yield 130.0 g BuOH extract. The ethyl acetate extract was subjected to silica gel (5×30 cm) column chromatography with a gradient of CHCl₃–MeOH (1 : 0, 50 : 1, 20 : 1,10: 1 and 1 : 1; 2 L for each step) to give 3 fractions (Frs. 1A–C). Fraction 1B was separated using an YMC column (3×80 cm) with a MeOH–acetone–H₂O (1.3 : 1.3 : 1, 1.2 L) elution solvent to give compound 21 (62.0 mg). Fraction 1C was separated using a YMC column (1×60 cm) with a MeOH–H₂O (2.7 : 1–3.5 : 1, 800 mL) elution solvent to give compounds 22 (28.0 mg) and 14 (19.0 mg). The BuOH extract was subjected to silica gel (5×30 cm) column chromatography with a gradient of CHCl₃–MeOH–H₂O (7 : 1 : 0.1, 5 : 1 : 0.1, 2 : 1 : 0.1 and 0 : 1 : 0; 3 L for each step) to give 4 fractions (Frs. 2A–D). Fraction 2B was separated using a silica gel column (3×80 cm) with CHCl₃–MeOH–H₂O (5 : 1 : 0.1, 4 : 1 : 0.1 and 3 : 1 : 0.1, 1 L for each step) to give 4 sub-fractions (Frs. 2B1–B4). Fraction 2B1 was separated using an YMC column (1×80 cm) with a MeOH–H₂O (4.5 : 1, 1.1 L) elution solvent to give compound 3 (37.0 mg). Fraction 2B2 was separated using an YMC column (1.5×80 cm) with a MeOH–acetone–H₂O (2.5 : 0.7 : 1, 650 mL) elution solvent to give compound 16 (12.0 mg). Fraction 2B4 was separated using an YMC column (1×80 cm) with a MeOH–acetone–H₂O (2 : 0.5 : 1, 700 mL) elution solvent to give compound 17 (12.0 mg). Fraction 2C was separated using a silica gel column (3×80 cm) with CHCl₃–MeOH–H₂O (3.5 : 1 : 0.1, 2 : 1 : 0.1 and 1 : 1 : 0.2, 1 L for each step) to give 3 sub-fractions (Frs. 2C1–C3). Fraction 2C1 was further chromatographed on RP chromatography column with acetone–MeOH–H₂O (0.7 : 2.3 : 1–0.85 : 2.3 : 1) to yield compounds 11 (78.0 mg), 12 (110.0 mg), and 13 (25.0 mg). Fraction 2C2 was separated using a silica gel column (1.5×80 cm) with CHCl₃–MeOH–H₂O (2.9 : 1 : 0.1, 1.5 L) to give compound 18 (130.0 mg). Fraction 2C3 was separated using an YMC column (1.5×80 cm) with a MeOH–acetone–H₂O (1 : 0.5 : 1, 1.5 : 0.5 : 1, each 550 mL) elution solvent to give compounds 19 (7.0 mg) and 20 (20.0 mg). Water extract was chromatographed on a column of highly porous polymer (Diaion HP-20) and eluted with H₂O and MeOH, successively, to give three fractions (Frs. 3A–C). Fraction 3B was subjected to silica gel (8×30 cm, 70–230 µm) column chromatography with a gradient of CHCl₃–MeOH–H₂O (6 : 1 : 0.1, 4 : 1 : 0.1, 2 : 1 : 0.1 and 0 : 1 : 0; 4 L for each step) to give 5 fractions (Frs. 3B1–B5). Fraction 3B2 using an YMC column (2×80 cm) with a MeOH–acetone–H₂O (1 : 0.3 : 1, 650 mL) elution solvent to give compound 2 (12.0 mg). Fraction 3B4 was separated using a silica gel column (1.5×80 cm) with CHCl₃–MeOH–H₂O (1.2 : 1 : 0.1, 1.5 L) to give compound 15 (4.0 mg) and 1 (130.0 mg). Fraction 3B4 was further chromatographed on RP chromatography column with acetone–MeOH–H₂O (0.3 : 1 : 1–0.4 : 1 : 1–0.45 : 1 : 1.4) to yield compounds 5 (50.0 mg), 6 (44.0 mg). Compound 4 (44.0 mg) was isolated from 3B6 using RP chromatography column with MeOH–H₂O (2.5 : 1). Fraction 3B5 was further chromatographed on RP chromatography column with acetone–MeOH–H₂O (0.3 : 1 : 1–0.6 : 1 : 1–0.7 : 1 : 1.5) to yield compounds compounds 7 (78.0 mg), 8 (300.0 mg), 9 (12.0 mg), and 10 (36.0 mg).

Pulsatilloside F (1): White crystals (MeOH); mp 270–275°C; [α]_D²⁸−68 (c=0.25, MeOH); FT-IR (CH₃CN) ν_max 3373, 2933, 1731, 1621, 1377, 1257, 1057, 1031 cm⁻¹; HR-ESI-QTOF-MS: m/z 1705.7692 [M−H]⁻, (Calcd for C₇₇H₁₂₅O₄₁: 1705.7696); ¹H (pyridine-d₅, 600 MHz) and ¹³C-NMR (pyridine-d₅, 150 MHz) data: see Tables 1 and 2.

Acid Hydrolysis

Compound 1 (5 mg) was heated in 3 mL 10% HCl (dioxane–H₂O, 1 : 1) at 90°C for 3 h. The residue was partitioned between EtOAc and H₂O to give the aglycone and sugar, respectively. The aqueous layer was evaporated until dry to yield a residue; this was dissolved in anhydrous pyridine (200 µL) and then mixed with a pyridine solution of 0.1 m l-cysteine methyl ester hydrochloride (200 µL). After warming to 60°C for 1 h, trimethylsilylimidazole solution was added, and the reaction solution was warmed at 60°C for 1 h. The mixture was evaporated in vacuo to yield a dried product, which was partitioned between n-hexane and H₂O. The n-hexane layer was filtered and analyzed by gas chromatography. Retention times of the persilylated monosaccharide derivatives were as follows: l-arabinose (t_R, 4.72 min), l-rhamnose (t_R, 5.31 min) and d-glucose (t_R, 14.11 min) were confirmed by comparison with those of authentic standards.

Cell Culture

RAW264.7 cells were obtained from American Type Culture collection (ATCC, Manassas, VA, U.S.A.) and maintained in Dulbecco’s modified Eagle’s medium (DMEM), DMEM containing 2 mm l-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a 5% CO₂ humidified incubator.

Inflammatory Cytokine Assay

RAW264.7 cells were cultured at 2×10⁵ cells/mL in DMEM containing 10% fetal bovine serum in 24-well tissue culture plates. The cells were pretreated with 0.5, 1 and 2 µm concentrations of compound 1 h before LPS stimulation. Twenty four hours after LPS stimulation (1 µg/mL), TNF-α level in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA) according to the commercial instruction (BD Biosciences, San Diego, CA, U.S.A.). For the cytokine assay using the sandwich method, a capture antibody (1 : 250 dilution) solution was incubated overnight at 4°C in 96-well plates. The plates were washed three times with washing buffer (0.05% Tween-20 in phosphate buffered saline (PBS)) and blocked with assay diluent (10% fetal bovine serum in PBS) for 1 h at room temperature, and then 100 µL of the culture supernatants was added to the wells and was incubated at room temperature. After 2 h, the plates were washed and incubated for 1 h with a detection antibody solution (1 : 250 dilution), biotinylated anti-mouse TNF-α antibody and a Strepavidin–Linkde peroxidase conjugate (each at 0.5 µg/mL, respectively). After incubation 1h, the plates were washed and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution to each well. Incubate plate for 30 min at room temperature in the dark. A stop solution was then added to the plates, and the optical density of 450 nm was read. The concentrations of the cytokines were calculated using the cytokines’ standard calibration curve.

Statistical Analysis

All data represent the mean±S.D. of at least three independent experiments performed in triplicates. Statistical significance is indicated as determined by one-way ANOVA followed by Dunnett’s multiple comparison test.

Supporting Information

FT-IR, ¹H- and ¹³C-NMR, HMQC, HMBC, COSY and HR-ESI-QTOF-MS spectrum of compound 1 are available as Supporting Information.

Acknowledgment

This study was supported by the Priority Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0093815), and by the Ministry of Knowledge Economy (MKE), Korea Institute for Advancement of Technology (KIAT) through the Inter-ER Cooperation Projects (R0002019), Republic of Korea.

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