Introduction

Trollius chinensis Bunge, also known as Jin Lian Hua, is a perennial herb which is widely distributed in the northeastern regions of China and Mongolia. As a well-known Chinese folk herb medicine, its flowers have been employed to treat respiratory infections, tonsillitis, pharyngitis and bronchitis owing to its actions of heat-clearing and detoxification [1]. To date, phytochemical investigations has revealed these flowers to contain mainly alkaloids, phenolic acids and flavonoids, while their effective components remain undetermined [2, 3]. As a follow-up study on the chemical constituents of Trollius chinensis, two new compounds (1, 2), together with one indole alkaloid and five known flavonoids, have been obtained. This paper deals with the isolation and structure determination of the constituents.

Results and discussion

Compound 1 was obtained as a yellow amorphous powder and exhibited a positive magnesium hydrochloric acid test, which suggested a flavonoid. Its molecular formula was assigned as C32H31O14 according to the HR-ESI–MS data (m/z 639.1707 [M+H]+ calcd. for C32H31O14, 639.1714). The UV spectrum of 1 showed absorption bands at 249, 266 and 336 nm, in accordance with a flavonoid skeleton. The 1H-NMR spectral data (Table 1) of 1 showed resonances at δ H 7.64 (1H, br.d, J = 8.0 Hz), 7.57 (1H, br.s) and 6.90 (1H, d, J = 8.0 Hz), suggesting the presence of an ABX system on the B-ring. Two singlets at δ H 6.73 and 6.42 were attributed to the aromatic protons at C-3 and C-6 in rings C and A of a flavone, respectively. Additionally, the anomeric proton at δ H 4.83 (1H, d, J = 9.9 Hz) and other signals between δ H 3.0 and 4.1 can be assumed to be the protons of sugar. The site of the sugar linkage in 1 was attached to the flavonoid skeleton at the C-8 position, which was further confirmed by the correlation of the anomeric proton H-1″ (δ H 4.83) with carbon signals at δ C 103.8 (C-8), 163.2 (C-7) and 156.0 (C-9) in the HMBC spectrum (Fig. 1). In addition, signals due to a trans double-bond proton at δ H 7.27 (1H, d, J = 15.8 Hz) and 6.17 (1H, d, J = 15.8 Hz), along with a methoxy proton signal at δ H 3.78 (3H, s) and an ABX system [δ H 7.22 (1H, br.s), 7.02 (1H, br.d, J = 7.8 Hz), 6.75 (1H, d, J = 7.8 Hz)] indicated the presence of a feruloyl moiety. In the NOESY spectrum (Fig. 2), the correlation between –OCH3 (δ H 3.78) and H-2′′′ (δ H 7.22) further corroborated the presence of a feruloyl group. Comparison of these data of 1 with those of isoswertiajaponin [4] led to the conclusion that 1 was a derivative of the latter, with an additional substituent at the glucosyl (Glc) moiety. This substituent was demonstrated to be a feruloyl group by analysis of 1D- and 2D-NMR spectra. The long-range correlations of H-2″ (δ H 5.42) and C-9′′′ (δ C 165.8) suggested that the glucopyranosyl moiety was connected to C-9′′′. Acid hydrolysis of 1 yielded d-glucose, which was confirmed by optical rotation using chiral detection in HPLC analysis after acid hydrolysis. On the basis of the above evidence, the structure of 1 was established as shown in Fig. 1, and named 2″-O-feruloylisoswertiajaponin.

Table 1 1H-NMR (400 MHz) and 13C-NMR (150 MHz) spectral data of compound 1 and 1H-NMR (600 MHz) and 13C-NMR (150 MHz) spectral data of compound 2 in DMSO-d 6
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Fig. 1
figure 1

Key HMBC correlations of compounds 1 and 2

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Fig. 2
figure 2

Key NOESY correlations of compounds 1 and 2

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Compound 2 was obtained as a colourless oil and its molecular formula as determined to be C19H26O10 by HR-ESI–MS at m/z 437.1420 [M+Na]+ (calcd. for C19H26O10Na 437.1424), requiring 7° of unsaturation. Combined analysis of the 1H- and 13C-NMR spectral data revealed that 2 contained a double bond [δ H 5.64 (1H, t, J = 6.1 Hz, H-3)], one methyl proton [δ H 1.70 (3H, s, H-5)] and two methylenes [δ H 4.67 (2H, s, H-1), 4.34 (1H, dd, J = 12.3, 6.1 Hz, H-4), 4.16 (1H, m, H-4)]. According to the above mentioned data, compound 2 was determined to have an moiety of 2-methyl-2-butene. Moreover, an anomeric proton signal of a glucosyl group at δ H 4.15 (1H, d, J = 7.8 Hz, H-1″) and the β-configuration was determined from the coupling constant (7.8 Hz) of the anomeric proton signal of the glucose. The HMBC spectral analysis (Fig. 1) revealed correlation peaks between the methylene proton H-4 (δ H 4.34, 4.16) with the carbon signals at δ C 102.3 (C-1″), 124.0 (C-3) and 134.1 (C-2), and therefore the connection of the 2-methyl-2-butene with C-1″ of the glucose was confirmed. The proton signal δ H 4.67 (2H, s, H-1) of the methylene group exhibited HMBC correlation with the carbon signal δ C 165.6 of C-7′, indicating that the 2-methyl-2-butene group is attached to the C-7′ position. In the 1H-NMR spectrum of compound 2, the occurrence of an ABX [δ H 7.50 (1H, dd, J = 8.2, 1.8 Hz), 7.44 (1H, d, J = 1.8 Hz), 6.88 (1H, d, J = 8.2 Hz)] and one methoxy proton [δ H 3.81 (3H, s)] could be assigned to a vanilloyl moiety. Furthermore, major HMBC correlations were also observed between a proton signal at δ H 3.81 (3′-OCH3) and δ C 147.8 (C-3′), between δ C 7.44 (H-2′), 7.50 (H-6′) and 165.6 (C-7′). Based upon the above observations, the presence of a vanilloyl group was also confirmed. The NOESY cross-peak between H-3 (δ H 5.64) and H-1 (δ H 4.67), H-4(δ H 4.34) and H-5 (δ H 1.70) indicated the olefinic bond at C-2 to be E (Fig. 2) On acid hydrolysis, 2 afforded one sugar moiety that was identified as d-glucose based on HPLC analysis by optical rotation using chiral detection. Thus, compound 2 was elucidated as (2E)-2-methyl-1-O-vanilloyl-4-β-d-glucopyranoside-2-butene.

In addition, by comparison with the NMR data reported in the literature [4–8], the known compounds 3–8 (Fig. 3) were identified as (R)-cyanomethyl-3-hydroxyoxindole (3), vitexin (4), isoswertisin (5), trollisin I (6), 2″-O-(2′′′-methylbutyryl)isoswertisin (7) and 6″-O-acetylorientin (8).

Fig. 3
figure 3

The structure of compounds 3–8

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Experimental

General experimental procedures

UV spectra were obtained on Shimadzu UV-2201 and UV-1700 spectrophotometers. HR-ESI–MS spectra were measured with an Agilent 6530 Accurate-Mass Q-TOF mass spectrometer. CD spectrum was obtained on a Bio-Logic Science MOS-450 spectrometer (Bio-Logic Science, France). NMR spectra were recorded on Bruker AV-400 and AV-600 MHz spectrometers with TMS as internal standard. Semipreparative HPLC was carried out on a Shimadzu LC-10ATvp series pumping system equipped with a Shimadzu SPD-10Avp spectrophotometric detector at 210 nm using a Phenomenex C18 reversed-phase column (5 μm, 250 mm × 10 mm, flow rate 2.0 mL/min). Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Group, Co., Qingdao, China), ODS (50–100 mesh, YMC, Co., Ltd., Japan), Sephadex LH-20 (Amersham Pharmacia Biotech AB Co.) and D101 macroporous adsorption resin (Shanghai Hualing Resin Group, Co., Shanghai, China). TLC was obtained on precoated silica gel GF254 glass plates (Qingdao Marine Chemical Group, Co., Qingdao, China).

Plant material

The flowers of Trollius chinensis were provided by Anhui Jiren Pharmacy Co., Ltd. (Anhui Province, China) and authenticated by Prof. Jincai Lu of Shenyang Pharmaceutical University. A voucher specimen (No. 20130121) was deposited in the Research Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University.

Extraction and isolation

Dried flowers of Trollius chinensis (7.5 kg) were extracted with 60 % EtOH (3 × 120 L) under reflux conditions for 1.5 h. The EtOH extract was condensed to 75 L by vacuum rotary evaporation. The extraction was carried out by D101 CC eluting in sequence with H2O, 50 % EtOH to yield 630 g of crude extract. A portion of the 50 % EtOH fraction (110 g) was subjected to silica gel column chromatography using a stepwise gradient elution of CH2Cl2–MeOH (100:0 to 0:100) to afford thirteen fractions (Fr.1–13). Fr.8 (3.3 g) underwent silica gel column eluting with CH2Cl2–MeOH (100:0 to 0:100) to afford two fractions (Fr.8.1–8.2). Compound 3 (10 mg) was purified by preparative HPLC eluted with MeOH–H2O (60:40) from Fr.8.1 (0.3 g). Fr.8.2 (2.6 g) was isolated with ODS column, employing gradient MeOH–H2O from 20:80 to 100:0 as the eluent, to give six fractions (Fr.8.2.1–8.2.6). Fr.8.2.6 (0.5 g) was further isolated with HPLC, employing MeOH–H2O (65:35) as eluent, to obtain 7 (26.5 mg) and 6 (10 mg). Fr.9 (5.2 g) was chromatographed by ODS column using a stepwise gradient elution of MeOH–H2O (20:80 to 100:0), to obtain six fractions (Fr.9.1–9.6). Further purification of Fr.9.3 (3.0 g) with silica gel column chromatography eluted with CH2Cl2–MeOH (95:5 to 0:100) provided five fractions (Fr.9.3.1–9.3.5). Fr.9.3.3 (1.8 g) was also separated by Sephadex LH-20 with MeOH as eluent to afford nine fractions (Fr.9.3.3.1–9.3.3.9). Fr.9.3.3.3 (0.3 g) was subsequently subjected to preparative HPLC, using a mobile phase of MeOH–H2O (70:30) to give compound 2 (11.8 mg). Fr.9.5 (0.8 g) was loaded onto a Sephadex LH-20 column chromatography eluted with MeOH and then Fr.9.5.8 (0.2 g) was further purified by RP-HPLC using MeOH–H2O (45:55) with solvent system to yield compound 1 (6.6 mg). Fr.9.3.4 (0.8 g) was purified by Sephadex LH-20 eluting with MeOH and then Fr.9.3.4.7 (0.15 g) was further purified by RP-HPLC using MeOH–H2O (50:50) as solvent system to yield compound 5 (21 mg). Using similar separation procedures, compounds 4 (1.5 mg) and 8 (16.5 mg) were also obtained from Fr.9.3.4.10 (0.18 g).

2″- O -Feruloylisoswertiajaponin (1): yellow amorphous powder; UV (MeOH) λ max: 249, 266, 336 nm; 1H-NMR (DMSO-d 6, 400 MHz) and 13C-NMR (DMSO-d 6, 150 MHz) spectroscopic data, see Table 1; HR-ESI–MS: (m/z 639.1707 [M+H]+ calcd. for C32H31O14, 639.1714).

(2 E )-4- O -vanilloyl-3-methyl-2-butenyl- β - d -glucopyranoside (2): colourless oil; UV (MeOH) λ max: 261, 291 nm; 1H-NMR (DMSO-d 6, 600 MHz) and 13C-NMR (DMSO-d 6, 150 MHz) spectroscopic data, see Table 1; HR-ESI–MS: (m/z 437.1420 [M+Na]+, calcd. for C19H26O10Na 437.1424).

Acid hydrolysis of compounds 1 and 2

Compounds 1 and 2 (3.0 mg each) were dissolved separately in 2 M HCl (3.0 mL) and heated at 85 °C on a water bath for 2 h. After cooling, the mixture was extracted with EtOAc (3 × 3.0 mL). The aqueous layer was then evaporated under vacuum to give a residue. The remaining sugar residue was dissolved in water (1.0 mL) and analyzed by HPLC with chiral detection under the following conditions: column (NH2P-50 4E, Shodex Asahipak); detector (JAsco OR-4090); column temperature (28 °C); mobile phase (acetonitrile and water 3:1); flow rate (0.8 mL/min). The peak shape (positive peak) and retention time (12.5 min) was consistent with d-glucose.