Steroidal Sapogenins and Glycosides from the Fibrous Roots of Ophiopogon japonicus and Liriope spicata var. prolifera with Anti-inflammatory Activity

Abstract

Two new steroidal glycosides (1 and 2), together with 15 known compounds (3–17) were isolated from the fibrous roots of Ophiopogon japonicus, and three new steroidal glycosides (18–20), together with 14 known compounds (21–34) were isolated from the fibrous roots of Liriope spicata var. prolifera. The structures of the new compounds were elucidated on the basis of extensive one-dimensional (1D)- and 2D-NMR spectroscopic analyses and mass spectrometry. The isolated compounds were evaluated for their anti-inflammatory activity in vitro. Most of these steroidal glycosides showed significant inhibitory activity against neutrophil respiratory burst stimulated by phorbol myristate acetate.

Ophiopogon japonicus (L. f.) KER-GAWL (Liliaceae), widely distributed in South-east Asia, especially in most areas of China, has been used as a famous traditional medicine (locally known as maidong) in China to cure acute and chronic inflammation and cardiovascular diseases including thrombotic diseases for thousands of years.¹⁾ Liriope spicata (THUNB.) LOUR. var. prolifera Y. T. MA (Liliaceae), commonly known as Hubei-maidong, is another medicinal herb mainly cultivated in Hubei Province of China. Its roots have been used by native people to treat cough, heart diseases and diabetes and to be as the succedaneum of the official crude drug O. japonicus for centuries.^2,3) Previous phytochemical investigation revealed the abundant presence of steroidal saponins both in fibrous and tuber roots of the two medical plants.^4–10) Unfortunately, our preliminary analytic study also indicated that there were still some unknown steroidal constituents in fibrous roots of these two medicinal herbs by high-performance liquid chromatography (HPLC)-diode array detection (DAD)-tandem mass spectrometry (MS). As part of our systematic study on steroidal constituents with biological activities from the fibrous roots of O. japonica and L. spicata var. prolifera, a total phytochemical fractionation led to the isolation of a series of steroidal constituents including two new steroidal saponins (1, 2), eleven known steroidal saponins along with five known steroidal sapogenins from O. japonica, and three new steroidal saponins (18–20), together with twelve known steroidal saponins and two known steroidal sapogenins from L. spicata var. prolifera. Their structures were elucidated on the basis of spectroscopic and physicochemical analysis. In addition, their in vitro anti-inflammation activities were evaluated to go through neutrophil respiratory burst inhibition by chemiluminescence assay, and some subtle structural features are critical to the activity were analyzed.

Results and Discussion

The saponin-enriched fraction prepared from 60% EtOH extracts of the fibrous roots of O. japonica and L. spicata var. prolifera were subjected to column chromatography over D101 macroporous resins and then respectively extracted with EtOAc and n-BuOH successively. The respective EtOAc and n-BuOH parts were loaded on silica gel, Sephadex LH-20, and octadecyl silica (ODS) silica gel to afford compounds 1–34, including five new steroidal saponins (1, 2, 18–20). The twenty nine known compounds were identified as ruscogenin (3),¹¹⁾ ophiopogonin D (4),¹²⁾ ophiopogonin B (5),¹²⁾ ruscogenin 1-O-β-D-fucopyranoside (6),¹³⁾ 1-O-sulfate-ruscogenin (7),¹²⁾ ruscogenin 1-O-α-L-rhamnopyranosyl-(1→2)-4-O-sulfate-α-L-arabinopyranoside (8),¹⁴⁾ ruscogenin 1-O-α-L-rhamnopyranosyl-(1→2)-4-O-sulfate-α-L-fucopyranosido-3-O-β-D-glucopyranoside (9),¹⁴⁾ diosgenin 3-O-[2′-O-acetyl-α-L-rhamnopyranosyl-(1→2)]-β-D-xylopyranosyl-(1→3)-β-D-glucopyranoside (10),¹⁵⁾ ophiopogonin D′ (11),¹⁶⁾ ophiogenin (12),¹⁷⁾ ophiogenin 3-O-α-L-rhamnopyanosyl-(1→2)-β-D-glucopyranoside (13),¹⁷⁾ pennogenin 3-O-α-L-hamnopyranosyl-(1→2)-β-D-xylopyranosyl-(1→4)-β-D-glucopyranoside (14, 34),¹⁸⁾ prazerigenin A (15),¹⁹⁾ prazerigenin A 3-O-β-D-glucopyranoside (16),²⁰⁾ prazerigenin A 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (17),²¹⁾ 25(R,S)ruscogenin (21),¹¹⁾ 25(R,S)ruscogenin 1-O-β-D-fucopyranoside (22),¹³⁾ 25(R,S)ruscogenin 1-O-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranoside (23),¹¹⁾ 25(R,S)ruscogenin 1-O-[3-O-acetyl-α-L-rhamnopyranosyl-(1→2)]-β-D-fucopyranoside (24),⁸⁾ 25(R,S)ruscogenin 1-O-[β-D-glucopyranosyl-(1→2)] [β-D-xylopyranosyl-(1→3)]-β-D-fucopyranoside (25),⁸⁾ 25(S)ruscogenin 3-O-α-L-rhamnopyranoside (26),¹¹⁾ Glycoside B (27),¹¹⁾ 25(S)ruscogenin 1-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside (28),⁸⁾ 25(S)ruscogenin 1-O-[3-O-acetyl-α-L-rhamnopyranosyl-(1→2)][β-D-xylopyranosyl-(1→3)]-β-D-fucopyranoide (29),⁸⁾ 25(S)ruscogenin 1-O-[α-L-rhamnopyranosyl-(1→2)]-β-D-xylopyranosyl-(1→3)]-β-D-fucopyranoside (30),⁹⁾ neoruscogenin (31),²²⁾ neoruscogenin 1-O-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranoside (32),²²⁾ yamogenin1-O-[α-L-rhamnopyanosyl-(1→2)][β-D-xylopyranosyl-(1→3)]-β-D-glucopyranoside (33),⁸⁾ respectively, by comparison of spectrometric data with the literature (Fig. 1).

Fig. 1. Structure of Compounds 1–34

Compound 1 was obtained as white amorphous powder. It showed positive reaction in the Liebermann–Burchard test. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) of 1 showed a quasimolecular ion [M+Na]⁺ at m/z 875.4399 (calcd 853.4409) consistent with a molecular formula of C₄₄H₆₈O₁₆. Its IR spectrum displayed strong absorption bands for hydroxyl groups at 3425 cm⁻¹, and typical absorption bands of isospirostanol saponin at 982, 922, 900, and 866 cm⁻¹. The intensity of the bands (922>900 cm⁻¹) indicated that the absolute configuration of C-25 was R.²³⁾ The ¹H-NMR spectrum of 1 showed four methyl proton signals of a typical steroidal skeleton. Two appeared as singlets at δ_H 1.09 (3H, s, Me-18) and 0.95 (3H, s, Me-19), and the other two as doublets at δ_H 1.13 (3H, d, J=6.5 Hz, Me-21), and 0.70 (3H, d, J=5.5 Hz, Me-27). Proton signal of one hydroxylated methylene group noted at δ_H 3.60 (2H, m, CH₂-26) further supported the absolute configuration of C-25 was R,²⁴⁾ as well as signal at δ_H 5.20 (1H, br s, H-6) for an olefinic proton. The ¹³C-NMR spectrum showed two more olefinic carbon signals at δ_C 141.8 and 124.5 than diosgenin. The presence of the other double bond between C-8 and C-14 was further supported by the heteronuclear multiple bond connectivity (HMBC) correlation between methyl H-18 (δ_H 1.09) and C-14 (δ_C 141.8). Hence, the aglycone of compound 1 was likely a (25R)-spirost-5,8(14)-diene-3β-ol.

In the ESI-MS spectrum, molecular ion peak at m/z 851 [M−H]⁻ and fragment ion peaks at m/z 719 [M−H−132]⁻, 573 [M−H−132−146]⁻ showed that there were at least two sugar units in 1. Three anomeric proton signals at δ_H 4.94 (d, J=7.2 Hz), 5.02 (d, J=7.3 Hz), and 6.25 (br s) were observed in the low-field region of the ¹H-NMR spectrum with their corresponding anomeric carbon signals at δ_C 105.8, 100.1and 101.9, respectively in the ¹³C-NMR spectrum. Acid hydrolysis of 1 yielded D-glucose, D-xylose and L-rhamnose, which were confirmed by co-TLC comparison with the standard sugars and GC analysis of their corresponding trimethylsilylthiazolidine derivatives. This procedure was also applied to the new compounds (2, 18, 19+20). The coupling constants (³J_{1, 2}>7 Hz) of the anomeric protons suggested that the anomeric carbon orientations were β for the D-glucopyranose and D-xylopyranose moieties. An α-orientation for L-rhamnopyranose was deduced from the C-5 signal of rhamnopyranose at δ_C 69.5 in the ¹³C-NMR spectrum.²⁵⁾ The spectroscopic and chemical evidences therefore supported the presence of one D-glucopyranose, one D-xylopyranose and one L-rhamnopyranose in 1. Long-range correlations between the following proton and carbon signals: H-1′ (δ_H 5.02) of the glucopyranose and C-3 (δ_C 77.3) of the aglycone of 1, H-1″ (δ_H 6.25) of the rhamnopyranose and C-2′ (δ_C 77.5) of the glucopyranose, and H-1‴ (δ_H 4.94) of the xylopyranose and C-4′ (δ_C 81.7) of the glucopyranose in the HMBC spectrum (Fig. 2), indicated that a rhamnopyranose was linked at C-2′ and a xylopyranose at C-4′ of the inner glucopyranose that was attached to C-3 of the aglycone. All proton and carbon signals were fully assigned by total correlation spectroscopy (TOCSY), distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC) and HMBC experiments (see Tables 1, 2). The structure of 1 was thus elucidated as 25(R)-spirost-5,8(14)-diene-3β-ol-3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→4)]-β-D-glucopyranoside.

Fig. 2. Key HMBC Correlations of 1

Table 1. ¹H- and ¹³C-NMR Data of the Aglycone Moieties of 1, 2, 18, 19 and 20 in Pyridine-d₅ (δ in ppm, J in Hz)

Position	1		2		18		19		20
	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC
1	1.66	36.4	3.64	82.8	3.76, m	84.7	3.78, m	83.7	3.78, m	83.6
2	1.60, 1.85	30.2	2.22, 2.66	34.7	2.45, 2.73, m	38.1	2.41, 2.64, m	38.0	2.41, 2.64, m	37.9
3	3.86	77.3	3.93	74.7	3.85, m	68.1	3.86, m	68.3	3.86, m	68.2
4	2.70, 2.80	38.5	2.48, 2.66	39.7	2.56, 2.68, m	43.7	2.59, 2.64, m	43.7	2.59, 2.64, m	43.8
5		140.8		138.4		139.5		139.5		139.5
6	5.20, br s	120.8	5.42, d (15)	125.5	5.58, d (5.6)	124.8	5.61, d (6.0)	124.7	5.61, d (6.0)	124.7
7	2.43, 2.82	33.8	1.62	31.9	1.55, 2.02, m	32.3	1.53, 1.93, m	31.9	1.53, 1.93, m	31.9
8		124.5	1.45	33.0	1.55, m	33.0	1.53, m	33.1	1.53, m	33.1
9	2.00	46.6	1.40	50.0	1.56, m	50.5	1.59, m	50.4	1.59, m	50.5
10		37.8		42.9		42.8		42.6		42.8
11	1.05,1.55	19.6	1.50, 2.75	23.9	2.96, d (10.5)	23.9	2.98, br s	24.3	2.98, br s	24.3
12	1.22, 1.70	38.3	1.10, 1.37	39.6	1.31, 1.62, m	40.4	1.43, 1.81, m	40.4	1.43, 1.81, m	40.7
13	—	39.8		40.0		40.2		40.2		40.2
14	—	141.8	1.05	56.5	1.18, m	57.2	1.27, m	57.5	1.27, m	57.5
15	2.10, 2.60	29.2	1.80	32.0	1.48, 1.88, m	32.0	1.48, 2.05, m	32.3	1.48, 2.05, m	32.3
16	4.18	79.5	4.48	81.0	4.80, m	81.2	4.54, m	81.5	4.54, m	81.5
17	2.08	63.1	1.70	62.7	1.73	62.9	1.88, d (6.0)	63.3	1.88, d (6.0)	63.1
18	1.09, s	18.6	0.80, s	16.5	0.86, s	16.8	0.96, s	16.9	0.96, s	16.8
19	0.95, s	19.2	1.25, s	14.7	1.41, s	14.7	1.43, s	15.0	1.43, s	15.0
20	2.05	43.8	1.86	42.0	1.85, m	42.7	2.06, m	41.9	2.06, m	41.9
21	1.13, d (6.5)	14.6	1.04, d (7.0)	14.9	1.06, d (7.0)	14.9	1.08, d (5.0)	14.9	1.08, d (5.0)	14.9
22		108.9		109.2		109.7		109.5		109.5
23	1.62	31.9	1.52, 1.97	32.4	1.41, 1.88, m	26.3	1.56, 1.79, m	33.2	1.56, 1.79, m	33.2
24	1.60	29.4	1.54	29.2	1.32, 2.11, m	26.1	2.25, 2.71, m	29.0	2.25, 2.71, m	29.0
25	1.60	30.6	1.55	30.5	1.55	27.5		144.5		144.5
26	3.60	67.1	3.46, 3.53	66.8	4.02,3.33	65.0	4.03, 4.46, d (11.0)	65.0	4.03, 4.46, d (11.0)	65.0
27	0.70, d (5.5)	17.3	0.66, d (5.4)	17.2	1.05, d (7.0)	16.2	4.81, 4.78, br s	108.6	4.81, 4.78, br s	108.6

Table 2. ¹H- and ¹³C-NMR Data of the Sugar Moieties of 1, 2, 18, 19 and 20 (in Pyridine-d₅, δ in ppm, J in Hz)

Position	1		2		18		19		20
	δH	δC	δH	δC	δH	δC	δH	δC	δH	δC
1′	5.02, d (7.3)	100.1	4.41	99.9	4.64, d (7.5)	100.2	4.69, d (6.0)	99.8	4.69, d (6.0)	99.6
2′	4.10	77.5	4.40	75.3	4.58	72.7	4.39	74.8	4.30	74.3
3′	4.20	77.7	4.20	74.8	4.02	85.2	4.25	74.2	4.25	73.9
4′	4.18	81.7	5.24	76.4	4.19	72.6	4.60	72.7	4.66	72.3
5′	3.84	76.2	3.53, 3.64	65.6	3.73	70.8	4.74	69.8	4.75	69.7
6′	4.42, 4.50	61.8			1.48, d (6.0)	17.0	1.27, d (6.0)	17.0	1.27, d (6.0)	17.0
1″	6.25, br s	101.9	6.19, br s	101.2	6.45, br s	98.2	6.29, d (1.0)	101.9	6.17, br s	98.9
2″	4.78	72.5	4.75	72.4	6.14	70.8	4.72	70.0	5.47	74.9
3″	4.58	72.8	4.55	72.4	5.9	73.7	5.47	75.0	4.74	70.4
4″	4.35	74.2	4.28	73.9	4.24	70.8	4.69	72.5	4.24	74.1
5″	4.92	69.5	4.70	69.4	5.01	69.2	4.89	69.5	3.78	69.1
6″	1.75,d (6.5)	18.6	1.66,d (6.0)	18.9	1.73, d (6.0)	18.8	1.78, d (7.0)	19.1	1.76, d (7.0)	19.0
1‴	4.94, d (7.2)	105.8	5.05, d (7.9)	102.6	4.85, d (7.5)	106.5
2‴	3.95	74.9	3.98	75.2	3.76	74.6
3‴	4.10	78.3	4.27	78.4	4.04	78.2
4‴	4.15	70.8	4.25	71.7	4.03	71.0
5‴	3.68, 4.26	67.4	3.95	78.3	4.22, 3.64	67.1
6‴			4.38, 4.50	62.7
CH3CO
2-Rha					1.95, s	170.4			1.93, s	170.6
						21.0				21.0
3-Rha					1.94, s	170.7	2.02, s	171.1
						20.8		20.7

Compound 2 was obtained as white amorphous powder and showed positive reaction in the Liebermann–Burchard test. The molecular formula of 2 was defined as C₄₄H₇₂SO₂₀ by HR-ESI-time-of-flight (TOF)-MS data (m/z 949.4105 [M−H]⁻, calcd 949.4108). Compound 2 showed strong hydroxyl group absorption, the characteristic bands (919<899 cm⁻¹) of an isospirostanol moiety and an S–O stretching absorption band at 1210 cm⁻¹ indicative of a sulfate group in its IR spectrum. The ¹H-NMR spectrum of 2 showed the aglycone signals for four typical steroid angular methyl groups at δ_H 0.80 (3H, s, Me-18), 1.25 (3H, s, Me-19), 0.66 (3H, d, J=5.4 Hz, Me-27) and 1.04 (3H, d, J=7.0 Hz, Me-21). The ¹³C-NMR spectral data (Table 1) of the aglycone moiety of 2 were similar to those of the known (25R)-ruscogenin, suggesting that the aglycone of 1 was (25R)-ruscogenin, whose structure is (25R)-spirost-5-ene-1β,3β-diol.²⁶⁾ The ¹H- and ¹³C-NMR spectra of 2 showed three anomeric protons at δ_H 4.41 (1H, br s), 5.05 (1H, d, J=7.9 Hz), 6.19 (1H, br s), and their corresponding anomeric carbons at δ_C 99.9, 102.6 and 101.2, suggesting that 2 was a spirostanol triglycoside with the aglycone of (25R)-ruscogenin. Acid hydrolysis of 2 gave L-rhamnose, L-arabinose and D-glucose, which was in good agreement with the ESI-MS spectral fragmentation pattern of 2 observed as follows: m/z (negative-ion) 949 [M−H]⁻, 803 [M−146−H]⁻, 787 [M−162−H]⁻, indicating that rhamnose and glucose were both at the terminal positions in the sugar chains. Acid hydrolysis of 2, followed by treatment with BaCl₂, demonstrated the presence of a sulfate residue and the 4-sulfo-α-arabinopyranose was indicated by the downfield shifts of the arabinosyl H-4 (δ_H 5.24) and arabinosyl C-4 (δ_C 76.4) signals, as well as the upfield shifts of the arabinosyl C-3 and C-5 at δ_C 74.8 and 65.6, respectively.²⁷⁾ In the HMBC spectrum H-1″ (δ_H 6.19) of the rhamnose showed a ³J_C,H correlation with C-2′ (δ_C 75.3) of the arabinose, H-1′ (δ_H 4.41) of the arabinose with C-1 (δ_C 82.8) of the aglycone, and H-1‴ (δ_H 5.05) of the glucose with C-3 (δ_C 74.7) of the aglycone, indicating that the rhamnosyl-(1→2)-arabinosyl-unit was attached to C-1 of the aglycone and a glucosyl unit to C-3 of the aglycone. All the proton and carbon signals were fully assigned by DEPT, HSQC, HMBC and TOCSY experiments (see Tables 1, 2). In conclusion, the structure of 2 could be deduced to be ruscogenin 1-O-α-L-rhamnopyranosyl-(1→2)-4-O-sulfo-α-L-arabinopyranoside-3-O-β-D-glucopyranoside.

Compound 18 was a white amorphous powder with a molecular formula of C₄₈H₇₄O₁₈ on the basis of HR-ESI-TOF-MS data (m/z 983.4869 [M+HCOOH−H]⁻, calcd 983.4857). The IR spectrum of 18 displayed a (25S)-spirostanol moiety characteristic bands [984, 921, 903 and 840 cm⁻¹ (intensity 919<899)]. Comparison of ¹H- and ¹³C-NMR spectroscopic data of the aglycone moieties of 18 with those of 2 suggested that they contain the similar aglycone, except for the typical F ring carbon resonances of C-23, C-24, C-25 and C-27 at δ_C 26.3, 26.1, 27.5 and 16.2, respectively, instead of those carbons in 2 at δ_C 32.4, 29.2, 30.5 and 17.2, respectively, strongly supported a (25S)-spirostanol skeleton for 18.²⁷⁾ Thus, the aglycone of 18 was determined as (25S)-ruscogenin, whose structure is (25S)-spirost-5-ene-1β,3β-diol. The NMR spectroscopic data also showed two acetate groups [δ_H 1.94, 1.95 (3H×2, s); δ_C 170.4, 170.7 (C=O×2), 20.8, 21.0 (CH₃×2)]. Acid hydrolysis of 18 gave L-rhamnose, D-fucose and D-xylose, which were identified by TLC and GC analysis. The HMBC spectrum of 18 showed correlations between H-1′ (δ_H 4.64) of the fucose and C-1 (δ_C 84.7) of the aglycone, between H-1″ (δ_H 6.45) of the rhamnose and C-2′ (δ_C 72.7) of the fucose, and between H-1‴ (δ_H 4.85) of the xylose and C-3′ (δ_C 85.2) of the fucose, which suggested the linkage of fucose to C-1 of the aglycone, rhamnose to C-2′ of the fucose, and xylose to C-3′ of the fucose, respectively. Moreover, long-range correlations between H-2″ (δ_H 6.14) of rhamnose and C=O (δ_C 170.4), and between H-3″ (δ_H 5.90) of rhamnose and C=O (δ_C 170.7) permitted the assignment of two acetate groups at C-2″ and C-3″ of rhamnose, respectively (Fig. 3). All the proton and carbon signals were fully assigned by DEPT, HSQC, HMBC and TOCSY experiments (see Tables 1, 2). The structure of 18 was unambiguously elucidated as (25S)-ruscogenin1-O-2,3-O-diacetyl-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-fucopyranoside.

Fig. 3. Key HMBC Correlations of 2

Compounds 19 and 20 were obtained as an inseparable mixture of two isomers, which could hardly be separated by various solvent systems and reverse phased materials. The molecular formula was defined as C₄₁H₆₂O₁₃ by HR-ESI-TOF-MS data (m/z 807.4180 [M+HCOOH−H]⁻, calcd 807.4172). The MS spectrum from LC-ESI-MS equipment system showed two near peaks with the same [M−H]⁻ ion, thus further indicating a pair of isomers. This was clarified by the proton and carbon signals in pairs observed in the ¹H- and ¹³C-NMR spectra of the mixture (the ratio of 19 and 20 was estimated as ca. 9 : 8 based on the intensity of ¹³C-NMR signals). Compound 19 showed another exo-olefinic group (δ_C 144.5 and 108.6) compared with 18, and the aglycone of 19 was deduced as spirost-5,25(27)-diene-1β,3β-diol (neoruscogenin), by comparison of ¹H- and ¹³C-NMR spectroscopic data of those in ref. 28. The NMR spectroscopic data also showed an acetate groups [δ_H 2.02 (3H, s); δ_C 171.1, (C=O), 20.7 (CH₃)]. Acid hydrolysis of 19 and 20 gave L-rhamnose and D-fucose, which were identified by TLC and GC analysis. The HMBC spectrum of 19 showed correlations between H-1′ (δ_H 4.69) of the fucose and C-1 (δ_C 83.7) of the aglycone, between H-1″ (δ_H 6.29) of the rhamnose and C-2′ (δ_C 74.8) of the fucose, which suggested the linkage of fucose to C-1 of the aglycone, rhamnose to C-2′ of the fucose, respectively. Moreover, long-range correlation between H-3″ (δ_H 5.47) of rhamnose and C=O (δ_C 171.7) permitted the assignment of the acetate group at C-3″ of rhamnose (Fig. 4). All the proton and carbon signals were fully assigned by DEPT, HSQC, HMBC and TOCSY experiments (see Tables 1, 2). Thus, the structure of 19 was elucidated as neoruscogenin 1-O-3-O-acetyl-α-L-rhamnopyranosyl-(1→2)]-β-D-fucopyranoside. 20 had the same aglycone and the similar sugar chain of 19. The only difference was the location of acetate group at C-2″ of rhamnose, confirmed by the long-range correlation between H-2″ (δ_H 5.47) of rhamnose and C=O (δ_C 170.6) (Fig. 5). The structure of 20 was elucidated as neoruscogenin 1-O-2-O-acetyl-α-L-rhamnopyranosyl-(1→2)]-β-D-fucopyranoside. Considering a potential aceyl migration of 2″- and 3″-acetyl of these two compounds, compounds 19 and 20 are a pair of isomers which might interconvert in solution.²⁹⁾

Fig. 4. Key HMBC Correlations of 18

Compounds were evaluated for their anti-inflammation activities in vitro and the results were summarized in Table 3. Compounds 2–6, 8–11, 14, 16, 18, 19+20, 22–30 and 33 have displayed very significant inhibitory activity against neutrophil respiratory burst stimulated by phorbol myristate acetate (PMA). Owing to the limited number and structure types of compounds, it is not feasible to delineate a structure–activity relationship (SAR) to account for the different activity potency among these compounds with very similar structures. Nevertheless, it seems that some subtle structural features are critical to the activity. Compound 33 showed the most strong inhibitory activity neutrophil respiratory burst stimulated by PMA whereas compounds 12, 13 and 21 showed only marginal inhibitory activity, indicating the key roles of the types of steroidal sapogenins. The longer length of the sugar chain led to the better activities (4 vs. 5, 6; 22 vs. 23; 28 vs. 30). The substitute groups of steroidal saponins also have important influence on the activities. The sulfate substitute resulted in the lower activity for example 2, 8 and 9. In conclusion, the steroidal saponins from O. japonica and L. spicata var. prolifera showed excellent anti-inflammation activities in vitro, meanwhile the types of steroidal sapogenins, length of the sugar chain and substitute groups played important roles in their activities.

Table 3. Inhibition of PMN-CL by Compounds 1–33 (n=3)

Compound	IC50 (µM)	Compound	IC50 (µM)	Compound	IC50 (µM)
1	—	12	>100	24	0.99±0.06
2	35.90±3.00	13	>100	25	1.63±0.16
3	15.90±0.29	14	5.61±0.03	26	1.13±0.06
4	2.01±0.09	15	—	27	1.01±0.10
5	2.22±0.05	16	53.50±9.21	28	1.51±0.08
6	3.22±0.05	17	—	29	0.65±0.02
7	—	18	0.98±0.03	30	0.59±0.02
8	12.97±0.24	19+20	1.01±0.08	31	—
9	16.36±0.24	21	>100	32	—
10	1.68±0.04	22	5.45±0.26	33	0.34±0.01
11	1.35±0.03	23	1.08±0.03	Rutin	71.84±3.86

Fig. 5. Key HMBC Correlations of 19 and 20

Experimental

Plant Material

Dried fibrous roots of O. japonicus and L. spicata var. prolifera were collected Sichuan Province, China in October 2005, and from Hubei Province, China in April 2006, respectively. They were identified by Prof. Bo-Yang Yu. Voucher specimens O. japonicas (NO. MD2051010) and L. spicata var. prolifera (No. 20060428) are on deposited at the herbarium of the Department of Complex Prescription of TCM, China Pharmaceutical University.

Apparatus

Optical rotations were measured using a JASCO P-1020 digital polarimeter. IR spectra were measured on a Shimadzu FTIR-8400s spectrophotometer. NMR spectra were recorded on a Brucker AV-500 spectrometers with tetramethylsilane (TMS) as internal standard. ESI-MS and HR-ESI-TOF-MS experiments were performed on an Agilent 1100 HPLC system with an Agilent 1100 Series MSD Trap mass spectrometer and an Agilent 1200 HPLC system with an Agilent 6210 ESI-TOF spectrometer, respectively. TLC was performed on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., China). Detection was done by spraying the plates with 10% sulfuric acid, followed by heating. Column chromatography was carried out with silica gel H (Qingdao Haiyang Chemical Co., Ltd., China), D101 macroporous resin (Tianjin Pesticide Co., China), ODS-A (50 µm, YMC, Kyoto, Japan), and Sephadex LH-20 (Pharmacia, Sweden). GC analysis was run on an Agilent 6890 gas chromatograph. L-Cysteine methyl ester hydrochloride, trimethylchlorosilane, hexamethyldisilazane, standard D-glucose, D-fucose, and D-xylose, L-arabinose and L-rhamnose were purchased from Sigma (St. Louis, MO, U.S.A.). The chemiluminescence value was recorded by BPCL-1-G-C Ultra-weak Luminescence Analyzer (Beijing Institutes for Biophysics, Chinese Academy of Science). Luminol and phorbol 12-myristate 13-acetate were obtained from Sigma.

Extraction and Isolation

Air-dried fibrous roots of O. japonicus (40 kg) were pulverized and extracted with 60% EtOH under reflux. The ethanol extract was filtered and dried using a rotary evaporator under reduced pressure. The concentrated extract was subjected to D-101 macroporous resin column chromatography eluted gradiently with EtOH–H₂O (0 : 100, 30 : 70, 90 : 10) to give three fractions (I–III). The concentrated residue of fraction III (330 g) further suspended in water, and extracted with EtOAc and n-BuOH successively. The EtOAc extract (107 g) was subjected to column chromatography (CC) on silica gel 200–300 mesh eluted successively in a gradient of CHCl₃–CH₃OH (100 : 1, 40 : 1, 20 : 1, 10 : 1, 5 : 1, 2 : 1, 0 : 100, v/v) with increasing amounts of MeOH to give a total of eighteen fractions (A–R). Fraction F (2.3 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH (40 : 1, v/v) repeatedly, and then purified on ODS CC eluted with (CH₃)₂CO–H₂O (9 : 1, v/v) to yield compounds 3 (21 mg) and 15 (15 mg). Fraction G (1.8 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH (20 : 1, v/v) repeatedly to give a residue (0.5 g), which was further successively chromatographed on Sephadex LH-20 eluted with CHCl₃–CH₃OH (1 : 1) to give 12 (10 mg). Fraction L (5.3 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH (8.5 : 1.5, v/v) repeatedly to give a brown residue (0.8 g), which was loaded on a ODS column eluted with CH₃OH–H₂O (8 : 2, v/v) to afford 6 (120 mg), 7 (3 mg) and 16 (20 mg). The n-BuOH extract (200 g) was subjected to CC on silica gel 200–300 mesh eluted successively in a gradient of CHCl₃–CH₃OH–H₂O (100 : 0 : 0, 95 : 5 : 0, 90 : 10 : 0, 85 : 15 : 1, 80 : 20 : 2, 70 : 30 : 3, 50 : 50 : 5, 0 : 0 : 100, v/v/v) to give a total of seventeen fractions (a–q). Fraction d (4.7 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH–H₂O (8 : 2 : 0.2, v/v/v) twice to give two brown residues (1.3 g and 0.4 g, respectively). The former residue (1.3 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH–H₂O (8 : 2 : 0.2, v/v/v) and then was loaded on an ODS column eluted with CH₃OH–H₂O (8 : 2, v/v) to afford 13 (260 mg). The later residue (0.4 g) was purified on ODS CC eluted with CH₃OH–H₂O (8 : 2, v/v) to yield 17 (9 mg). Fraction e (8.8 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH–H₂O (7.5 : 2.5 : 0.2, v/v/v) repeatedly, and then purified on ODS CC eluted successively in a gradient of CH₃OH–H₂O (7 : 3, 8 : 2, v/v) to yield compounds 4 (500 mg) and 11 (250 mg). Fraction h (3.3 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH–H₂O (7 : 3 : 0.3, v/v/v) thrice and then purified on Sephadex LH-20 CC eluted with CHCl₃–CH₃OH (1 : 1, v/v). The obtained residue (0.3 g) was further purified on ODS CC eluted successively in a gradient of CH₃OH–H₂O (6 : 4, 7 : 3, 8 : 2, v/v) to yield compounds 5 (20 mg), 1 (7 mg) and 8 (7 mg). Fraction i (4.5 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH–H₂O (7 : 3 : 0.2, v/v/v) twice and then was purified on ODS CC eluted with CH₃OH–H₂O (6 : 4, v/v) to yield compounds 10 (45 mg) and 14 (55 mg). Fraction k (3.1 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH–H₂O (6 : 4 : 0.2, v/v/v) twice and then was purified on ODS CC eluted with CH₃OH–H₂O (6 : 4, v/v) to yield compounds 9 (10 mg) and 2 (6 mg).

Air-dried fibrous roots of L. spicata var. prolifera (31 kg) were pulverized and extracted with 60% EtOH under reflux. The ethanol extract was filtered and dried using a rotary evaporator under reduced pressure. The concentrated extract was suspended in water and then extracted with Et₂O and EtOAc successively. The concentrated water layer was subjected to D-101 macroporous resin column chromatography eluted gradiently with EtOH–H₂O (0 : 100, 30 : 70, 70 : 30, 95 : 5, v/v) to give four fractions (I–IV). The EtOAc extract (250 g) was subjected to CC on silica gel 200–300 mesh eluted successively in a gradient of CHCl₃–CH₃OH (100 : 0, 90 : 10, 80 : 20, 70 : 30, 60 : 40, 1 : 1, v/v) with increasing amounts of CH₃OH and yielded seven fractions (LA–LG). Fraction LB (15.0 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH (50 : 1, v/v) repeatedly, and then purified on ODS CC eluted with (CH₃)₂CO–H₂O (9 : 1, v/v) to yield compounds 21 (300 mg) and 31 (5 mg). Fraction LC (28.0 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH (8.5 : 1.5, v/v) repeatedly, and then purified on Sephadex LH-20 eluted with CHCl₃–CH₃OH (1 : 1, v/v). The obtained residue (1.1 g) was further purified on ODS CC eluted successively in a gradient of CH₃OH–H₂O (9 : 1, 8 : 2, v/v) to give compounds 19+20 (5 mg), 23 (3.5 mg), 24 (8 mg), 28 (50 mg) and 32 (3.0 mg). Fraction LD (37.0 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH (8 : 2, v/v) repeatedly, and then was purified on ODS CC eluted with CH₃OH–H₂O (7 : 3, v/v) to afford compounds 18 (40 mg) and 27 (2 g). Fraction LE (12.0 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH (7.5 : 2.5, v/v) repeatedly, and then purified on Sephadex LH-20 eluted with CHCl₃–CH₃OH (1 : 1, v/v) to obtain compound 28 (80 mg). The concentrated residue of fraction III (300 g) was subjected to CC on silica gel 200–300 mesh eluted successively in a gradient of CHCl₃–CH₃OH–H₂O (100 : 0 : 0, 90 : 10 : 0, 85 : 15 : 1, 80 : 20 : 2, 70 : 30 : 3, 60 : 40 : 4, 50 : 50 : 5, v/v/v) to give a total of nine fractions (La–Li). Fraction Lb (8.0 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH (9 : 1, v/v) repeatedly, and then purified on ODS CC eluted with CH₃OH–H₂O (9 : 1, v/v) to yield compounds 22 (50 mg) and 31 (50 mg). Fraction Ld (25.4 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH–H₂O (8 : 2 : 0.2, v/v/v) repeatedly, and then purified on Sephadex LH-20 eluted with CHCl₃–CH₃OH–H₂O (1 : 1 : 0.1, v/v) to yield compound 33 (20 mg). Fraction Lf (25.4 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH–H₂O (7.5 : 2.5 : 0.25, v/v/v) repeatedly, and then purified on ODS CC eluted with CH₃OH–H₂O (6 : 4, v/v) to give compounds 25 (30 mg) and 30 (120 mg). Fraction Lg (18.9 g) was subjected to CC on silica gel eluted with CHCl₃–CH₃OH–H₂O (7 : 3 : 0.3, v/v/v) repeatedly, and then purified on ODS CC eluted with CH₃OH–H₂O (6 : 4, v/v) to afford compound 34 (10 mg).

Compound Characterization

Compound 1: White amorphous powder; [α]_D²⁵ −86.4 (c=0.10, MeOH); IR (KBr) ν_max 3425 (OH), 982, 922, 900, and 866 (spirostane) cm⁻¹; ESI-MS m/z 851 [M−H]⁻; HR-ESI-MS m/z 875.4399 (Calcd for [C₄₄H₆₈O₁₆+Na]⁺, 875.4409); ¹H- and ¹³C-NMR: data are shown in Tables 1 and 2.

Compound 2: White amorphous powder; [α]_D²⁵ −54.3 (c=0.05, MeOH); IR (KBr) ν_max 3425 (OH), 983、919、899, 864 (spirostane), 1210 (S–O) cm⁻¹; ESI-MS m/z 949 [M−H]⁻; HR-ESI-MS m/z 949.4105 (Calcd for [C₄₄H₇₂SO₂₀−H]⁻, 949.4108); ¹H- and ¹³C-NMR: data are shown in Tables 1 and 2.

Compound 18: White amorphous powder; [α]_D²⁵ −95.3 (c=0.10, pyridine); IR (KBr) ν_max 3443 (OH), 984, 921, 903, and 840 (spirostane) cm⁻¹; ESI-MS m/z 937 [M−H]⁻; HR-ESI-MS m/z 983.4869 (Calcd for [C₄₈H₇₄O₁₈+HCOOH−H]⁻, 983.4857); ¹H- and ¹³C-NMR: data are shown in Tables 1 and 2.

Compounds 19 and 20: White amorphous powder; [α]_D²⁵ −47.0 (c=0.07, pyridine); IR (KBr) ν_max 3438 (OH), 2927 and 2852 (CH), 1070 cm⁻¹; ESI-MS m/z 761 [M−H]⁻; HR-ESI-MS m/z 807.4180 (Calcd for [C₄₁H₆₂O₁₃+HCOOH−H]⁻, 807.4172); ¹H- and ¹³C-NMR: data are shown in Tables 1 and 2.

Acid Hydrolysis of 1–2, 18 and 19+20

Each compound (2 mg of 1, 2, 18, respectively, 4 mg of 19+20) was refluxed with 2 mL of 2 M HCl (dioxane–H₂O, 1 : 1) at 100°C for 4 h. After the dioxane was removed, the solution was diluted with H₂O and extracted with EtOAc (1 mL×3). The aqueous layer was evaporated under vacuum, and the residue was diluted with H₂O. This procedure was repeated until a neutral residue was obtained. The residue was dissolved in pyridine (300 µL), and 4 mg of L-cysteine methyl ester hydrochloride was added to it. The mixture was kept at 60°C in oil bath for 1.5 h. Then 300 µL of HMDS–TMCS (hexamethyldisilazane–trimethylchlorosilane, 2 : 1) was added and the mixture was heated at 60°C in oil bath for another 30 min.³⁰⁾ After centrifugation, the supernatant was analyzed by GC under the following conditions: capillary column, DB1701 (0.53 mm×30 m×1.0 µm); detection, flame ionization detector (FID); detector temperature, 280°C; injection temperature, 250°C; initial temperature 140°C, then raised to 250°C at 11°C/min; carrier, N₂; split ratio, 1/5. The standard monosaccharides were subjected to the same reaction and GC-MS analysis. Under these conditions, the derivatives of rhamnose, fucose, arabinose, xylose and glucose were detected at 14.578, 14.780, 13.689, 13.706 and 16.542 min, respectively.

Biological Tests

Neutrophils were obtained using the methods in the literature.³¹⁾ A chemiluminescence assay was used to test neutrophil respiratory burst inhibitory activity with a method similar to the previous report.³²⁾ Rutin was chosen as the positive control, which was isolated by us before, and its purity was >98% as determined by HPLC.

Acknowledgments

This research was financially supported by the Major National Science and Technology Project of China for Significant New Drugs Development (No. 2012ZX09102201-015), the National Natural Science Foundations of China (No. 81274004, 81473317), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 2011’ Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, and the Postgraduate Innovation Project of Jiangsu Province, China (No. CX09B_285Z).

Conflict of Interest

The authors declare no conflict of interest.

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