Three new caffeoyl glycosides from the roots of Picrorhiza scrophulariiflora.

From the underground parts of Picrorhiza scrophulariiflora, three new caffeoyl glycosides, scrocaffeside A-C (1-3), together with two caffeic acid derivates, 4-O-beta-D-glucopyranosyl caffeic acid (4) and 4-methoxy caffeic acid (5) and a phenylethanoid glycoside, scroside D (6), were isolated. Their structures were elucidated on the basis of chemical and spectroscopic evidence and comparisons with literature data of related compounds.

Introduction

The plant Picrorhiza scrophulariiflora (Scrophulariaceae) grows in the high altitude regions (over 4400 m) in the southeast of Tibet and the northwest of Yunnan in China. The roots of this plant are used in traditional Chinese medicine for the treatment of damp-heat dysentery, jaundice and steaming bone disorder [1]. Previous phytochemical investigations of this plant led to the isolation of terpenoids [2,3,4], iridoid glycosides [5,6,7], phenolic glycosides and phenylethanoid glycosides [8,9,10,11,12,13,14]. Here we report the isolation and characterization of three new caffeoyl glycosides 1-3, as well as three known compounds.

Results and Discussion

The three known compounds were identified as 4-O-β-D-glucopyranosyl cafeic acid (4) [15], n class="Chemical">4-methoxycaffeic acid (5) [16] and scroside D (6) [17] by comparing their physical and spectroscopic data with literature values.

Compounds Scrocaffeside A (1), n class="Chemical">Scrocaffeside B (2) and Scrocaffeside C (3)

Scrocaffeside A (1) was obtained as a white amorphous powder, with [α]D-33.7° (c 0.18, MeOH) and its molecular formula was determined as C30H34O17 by the [M+H]+ quasi-ion peak at m/z 667.1824 (calc. for C30H35O17: 667.1810) in the HR-ESIMS. In the IR spectrum, 1 exhibited bands at 3293 (hydroxy group), 1708 (C=O in conjugated esters), 1632 (C=C in α,β-unsaturated acid derivatives), 1599 and 1515 cm-1 (aromatic ring), and its UV spectrum showed absorption maxima at 249 and 379 nm.

The 1H-NMR spectrum of 1 (Table 1) exhibited the characteristic signals of two (trans)-caffeoyl units and two β-glucose unites [anomeric protons at δH 5.50 (1H, d, J = 8.0 Hz) and 4.78 (1H, d, J = 6.8 Hz)]. Twenty nine signals were exhibited in the 13C-NMR spectrum. The carbon resonances at δC 115.8 represent two methines, based on the HMQC experiment. Comparison of the 13C-NMR data with those of 4 also suggested the presence of two glucopyranosyl and two (trans)-caffeoyl moieties [15].

Table 1

13C- (100 MHz) and 1H- (400 MHz) NMR Data of 1, 2 and 3 (DMSO-d6)a

Moiety	position	1		2		3
		C	H (J, Hz)	C	H (J, Hz)	C	H (J, Hz)
Caffeoyl-1	1	125.3		128.9		128.4
	2	114.9	7.06, br.s	115.1	7.12﹡	115.1	7.18, br.s
	3	145.6		146.9		146.8
	4	148.7		146.9		147.7
	5	115.8	6.77, d, (8.4)	115.9	7.08, d, (8.8)	116.1	7.12, m
	6	121.7	7.02, br.d (8.4)	120.3	6.96, d, (8.4)	121.2	7.12, m
	7	146.6	7.57, d, (16.0)	143.5	7.38, d, (16.0)	146.0	7.62, d, (16.0)
	8	113.2	6.26, d, (16.0)	117.5	6.24, d, (16.0)	115.3	6.45, d, (16.0)
	9	165.2		167.7		165.1
Caffeoyl-2	1′	128.6		128.5		128.6
	2′	115.1	7.18, br.s	114.9	7.19, s	115.1	7.18, br.s
	3′	146.8		146.8		146.8
	4′	147.5		147.5		147.5
	5′	116.1	7.12, m	116.0	7.12﹡	116.1	7.12, m
	6′	120.9	7.12, m	120.8	7.12﹡	120.9	7.12, m
	7′	144.8	7.52, d, (16.0)	144.7	7.53, d, (16.0)	144.8	7.52, d, (16.0)
	8′	115.8	6.45, d, (16.0)	115.8	6.45, d, (16.0)	115.8	6.44, d, (16.0)
	9′	166.3		166.2		166.3
Glc-1	1′′	94.1	5.50, d, (8.0)	101.2	4.86, d, (6.8)	94.2	5.52, d, (8.0)
	2′′	72.4	3.20-3.38, m﹡	75.5	3.30﹡	72.4	3.17-3.39, m﹡
	3′′	76.1	3.20-3.38, m﹡	73.9	3.71﹡	76.1	3.17-3.39, m﹡
	4′′	69.6	3.20-3.38, m﹡	69.7	3.20, m	69.6	3.17-3.39, m﹡
	5′′	74.6	3.58, m	73.2	3.30﹡	74.6	3.51, m
	6′′	63.4	4.41, br.d, (11.2), 4.16, dd, (6.0, 12.0)	63.3	4.23, dd, (6.8, 11.6) 4.46, d, (11.2)	63.4	4.41, br.d, (11.2)4.16, d d, (6.0, 12.0)
Glc-2	1′′′	101.6	4.78, d, (6.8)	101.5	4.80, d, (6.8)	101.4	4.78, d, (6.0)
	2′′′	75.8	3.20-3.38, m﹡	75.7	3.32﹡	75.8	3.17-3.39, m﹡
	3′′′	73.2	3.20-3.38, m﹡	73.3	3.32﹡	73.2	3.17-3.39, m﹡
	4′′′	69.8	3.20-3.38, m﹡	69.9	3.30﹡	69.8	3.17-3.39, m﹡
	5′′′	77.2	3.20-3.38, m﹡	77.1	3.32﹡	77.2	3.17-3.39, m﹡
	6′′′	60.7	3.71, br.d, (8.4)3.47, m	60.7	3.48, dd, (5.6, 11.6) 3.71, d, (10.4)	60.7	3.70, br.d, (11.2)3.47, m
Glc-3	1′′′′					101.5	4.80, d, (6.0)
	2′′′′					75.8	3.17-3.39, m﹡
	3′′′′					73.2	3.17-3.39, m﹡
	4′′′′					69.8	3.17-3.39, m﹡
	5′′′′					77.2	3.17-3.39, m﹡
	6′′′′					60.7	3.73, br.d, (11.2)3.47, m

a Chemical shifts (δ) given in ppm; * signal pattern unclear due to overlapping signals.

The presence of the glucopyranosyl and (trans)-caffeoyl moieties were further confirmed by the acid hydrolysis of 1, which resulted in a release of glucose and caffeic acid, identified by TLC comparison with the authentic samples. The configuration of the glucopyranosyl was assigned to be β-D- according to the procedure of Oshima, Yamauchi and Kumanotani [18] and the coupling constant of the anomeric proton [19].

The detailed analysis of 1H-NMR, TCOSY, 13C-NMR, HSQC and HMBC spectra of 1 fixed the connections between the moieties. The deshielded shifts of H-1" (δH 5.50) and H-6" (δH 4.39, 4.14) indicated that the caffeoyl moieties were attached to C-1" and C-6", respectively. This was confirmed by HMBC correlations between H-1′′ (δH 5.50) and C-9 (δC 165.3), and between H-6′′ (δH 4.39 and 4.14) and C-9′ (δC 166.5). A HMBC correlation between another anomeric proton H-1′′′ (δH 4.78) and C-4′ (δC 147.5) demonstrated that the remaining glucopyranosyl was connected to C-4′. Therefore, the structure of scrocaffeside A (1) was concluded to be E-caffeoyl-6-O-[4-O-(β-D-glucopyranosyl) E-caffeoyl]-β-D-glucopyranoside, for which the trivial name scrocaffeside A is proposed.

Important HMBC correlations (H→C) for Scrocaffeside A (1)

Scrocaffeside B (2), a white amorphous powder, showed [α]D-23.5° (c 0.20, MeOH). Its molecular formula, C30H34O17, was established by the [M+H]+ quasi-ion peak at m/z 667.1830 (calc. for C30H35O17 667.1809) in the HR-ESIMS. The presence of β-D-glucopyranosyl and (trans)-caffeoyl moieties were confirmed using the same methods as for the structure elucidation of 1. Close structural similarity of 2 and 1 followed from the general congruence of 1H- and 13C-NMR data (Table 1). Notable differences were signals from a (trans)-caffeoyl moiety in 2. Comparison of the NMR spectra of 2 and 1 also showed a conspicuous deshielding of H-1′′ (ΔδH 0.64) and C-1′′ (ΔδC 7.1) of the central glucose core (Glc 1), indicating the diferent glycosidic linkage between the glucopyranosyl moiety and caffeoyl moiety 1. In the HMBC spectrum correlations between H-1′′ (Glc 1-1) and C-4 demonstrated that the glucose 1 attached at C-4 of caffeoyl moiety 1. Thus, the structure of compound 2 was established as 4-O-[6-O-[4-O-(β-D-glucopyranosyl) E-caffeoyl]-β-D-glucopyranosyl] E-caffeic acid, and it was named scrocaffeside B.

Scrocaffeside C (3) was obtained as a white amorphous powder with [α]D-50.5° (c 0.17, MeOH). The molecular formula of C36H44O22 was assigned for 3 on the basis of HR-ESIMS at m/z 829.2329 [M+H]+ (calc. for C36H45O22 829.2338). The UV, 1H-NMR and the 13C-NMR spectroscopic data of 3 together with the chemical test results were very similar to those of 1, suggesting a close relationship. Structural assessment of 3 was accomplished using a combination of NMR techniques, along with comparisons to the assignments of analogues 1 and 2.

The 13C-NMR spectra experiments gave a total of 29 resonance lines, of which 12 signals (δC 102~60) could be assigned to three glucosy moieties by the aid of DEPT and HMQC experimrnts. Analysis of 1H-NMR, TCOSY, 13C-NMR, HSQC and HMBC spectra of 3 indicated again the glycosidic linkages between the glucopyranosyl and caffeoyl moieties. The connections between glucose 1, glucose 2 and the two caffeoyl moieties of 3 shared the same pattern with those of 1. The glucose 3 should be linked to C-4, as indicated by the HMBC crosspeak between this carbon (δC 147.7) and the anomeric proton (δH 4.80).

On the basis those data the structure of 3 was determind to be 1, 6-di-O-[4-O-(β-D-glucopyranosyl) E-n class="Chemical">caffeoyl]-β-D-glucopyranoside, and the compound was designated as scrocaffeside C.

13C- (100 MHz) and n class="Chemical">1H- (400 MHz) NMR Data of 1, 2 and 3 (DMSO-d6)a

Experimental

General

UV spectra were recorded on a Milton Roy Spectronic 1201 spectrophotometer, and FTIR spectra were measured on a Perkin-Elmer 157G infrared spectrophotometer. Optical rotations were obtained using a Perkin-Elmer 241-MC polarimeter. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were obtained on a Bruker AV400 spectrometer with DMSO-d6 as solvent and TMS as an internal standard. HR-ESIMS data were measured with a Bruker AOEXIII 7.0 TESLA FTMS. HPLC were carried out on the reverse phase columns (mighty sil RP-18 and 8, Kantho Chemical Co. Ltd) with the MeOH-H2O solvent system. Colum chromatography was carried out on silica gel (Qingdao Marine Chemical Company China, 200-300 mesh), Sephadex LH-20 (Amersham Pharmacia Biotech AB). Silica GF254 for TLC was produced by Qingdao Marine Chemical Company China and Merck Company. All chemicals used were of biochemical reagent grade.

Plant Material

Roots of Picrorhiza scrophulariiflora were collected in October 2006 in Sichuan Province of China and identified by Prof. Qi-shi Sun (Shenyang Pharmaceutical University). The voucher specimen has been deposited in the Herbarium of School of Traditional Chinese Medicines of Shenyang Pharmaceutical University, China.

Extraction and Isolation

The dried and ground roots (underground parts) of Picrorhiza scrophulariiflora (3.0 kg) were successively extracted three times with 90% EtOH under reflux. After removal of the solvent in vacuo, the residue (1.6 kg) was suspended in H2O and then extracted successively with petroleum ether (b.p. 60-90°C), EtOAc and n-BuOH. The n-BuOH layer was concentrated in vacuo to give a viscous residue (500 g), which was then dissolved in water (2 L) and subjected to a macro-porous resin D-101 column chromatography eluting successively with water and ethanol (water, 30%, 50% and 100% ethanol). The 50% EtOH eluted fraction was evaporated in vacuo to yield a residue (140 g), which was subjected to silica gel column chromatography eluting with mixtures of CHCl3-MeOH of increasing polarity to give 8 fractions (frs.1-8).

Fraction 2 (43.0 g) was further separated by silica gel column chromatography, using an EtOAc-MeOH gradient as eluent, to afford six fractions (frs. 2A-2F). Fractions 2B (5.3 g) and 2C (4.6 g) were further purified separately by Sephadex LH-20 to yield compounds 4 (42.3 mg) and 5 (11.3 mg). Fraction 2D was purified by Sephadex LH-20 and further separated by reversed-phase HPLC using MeOH-H2O (57:43 and 44:56) as a mobile phase to afford compounds 1 (38.7mg) and 2 (15.9 mg). Fraction 3 (28.5 g) was chromatographed over silica gel using a EtOAc-MeOH gradient system as eluent to afford five fractions (frs. 3A-2E). Fraction 3B (6.7 g) was purified by Sephadex LH-20 chromatography and further separated by reversed-phase HPLC using MeOH-H2O (43:57) as a mobile phase to afford compounds 3 (17.6 mg) and 6 (18.4 mg).

Scrocaffeside A (1): White amorphous powder; [α]D-33.7°; UV (n class="Chemical">MeOH) λmax nm (log ε): 379 (2.75), 249 (5.51); IR (KBr) νmax cm-1: 3293, 2922, 2359, 1708, 1632, 1599, 1515; for 1H- and 13C-NMR data see Table 1; HR-ESIMS m/z: 667.1824 (calc. for C30H35O17: 667.1810).

Scrocaffeside B (2): White amorphous powder; [α]D-23.5°; UV (n class="Chemical">MeOH) λmax nm (log ε): 332 (2.35), 245 (5.50); IR (KBr) νmax cm-1: 3324-2260, 2905, 2257, 1700, 1635, 1510; for 1H- and 13C-NMR data see Table 1; HR-ESIMS m/z: 667.1830 (calc. for C30H35O17: 667.1809).

Scrocaffeside C (3): White amorphous powder; [α]D-50.5°; UV (n class="Chemical">MeOH) λmax nm (log ε): 361 (2.51), 249 (5.50); IR (KBr) νmax cm-1: 3309, 2918, 2255, 1711, 1634, 1510; for 1H- and 13C-NMR data see Table 1; HR-ESIMS m/z: 829.2329 (calc. for C36H45O22: 829.2338).

Acid hydrolysis of , and

A solution of the compound (8 mg) in 2 N TFA (3 mL) was refluxed at 100°C for 3 h. The reaction mixture was extracted with EtOAc. The EtOAc extract was proven to contain caffeic acid by direct TLC comparison with authentic samples. D-Glucose was found as the only sugar present in the water part following the procedure of Oshima, Yamauchi and Kumanotani [18].