Two New Phenolic Glycosides from Gnaphalium affine D. Don and Their Anti-Complementary Activity

Two new phenolic glycosides, named gnaphaffine A and B (compounds 1 and 2), were isolated from Gnaphalium affine. together with six known compounds, including caffeic acid (3), everlastoside L (4), isorhamnetin-7-O-β-d-glucopyranoside (5), quercetin-3-O-β-d-glucopyranoside (6), scutellarein-7-O-β-d-glucoside (7) and api-genin-7-O-β-d-glucopyranoside (8). Their structures were elucidated by spectroscopic methods, including ESI-MS and 2D NMR spectroscopy (HMQC and HMBC). All compounds were evaluated for their anti-complementary activity on the classical pathway of the complement system in vitro.


Results and Discussion
An 80% ethanolic extract of dried G. affine whole plant was suspended in distilled water and extracted with EtOAc. The EtOAc soluble fraction was concentrated under reduced pressure to produce a residue that was subjected multiple chromatography, two new compounds 1 and 2 and six known compounds 3-8 were isolated and identified.
Gnaphaffine A (1) was isolated as a yellow, amorphous powder. On the basis of a HR-ESI-MS peak at m/z 501.1393 [M+H] + and 13 C-NMR data the molecular formula of 1 was determined to be C 25 H 24 O 11 , indicating 14 degrees of unsaturation. The IR spectrum showed the presence of hydroxyl (3535 cm −1 ), carbonyl (1668 cm −1 ) and benzene ring (1606, 1506 cm −1 ) groups. The structure of the compound was established from detailed analysis of its 1 H-and 13 C-NMR spectra, including 2D NMR. The 13 C-NMR spectrum of 1 (Table 1), exhibited 25 signals that together with the information from a DEPT spectrum, indicated two methylene, fourteen methine, and nine quaternary carbons. Among these were one carbonyl group at δ 196.1 (C-9) and one ester function at δ 166.1 (C-19); as well as one anomeric methine at δ 100.7 (C-1') and four methines at δ 72.7 (C-2'), 75.  Figure 2). Since the above-mentioned groups accounted for 13 degrees of unsaturation, the remaining degree suggested the presence of an additional ring system in the structure of 1. The HMBC experiment ( Figure 2) showed clear correlations of ABX protons H-2 and H-6 with C-7, H-7 with C-2 and C-6 and of H-8 with C-1, which confirmed the presence of the carbon-carbon double bond attached at C-1 of the ABX system, while correlations of H-7 with C-9 and of H-8 with C-9 and C-10 confirmed the presence of the carbonyl group C-9 attached at C-8.
Correlations of H-10a and H-10b with C-9, C-12 (δ 123.4) and C-16 (δ 117.9), and of H-16 with C-10 confirmed the attachment of C-10 at carbonyl group (C-9) and C-11 of the tetrasubstituted aromatic ring at C-10.  Another carbon-carbon double bond attached at C-12 was deduced from correlations of H-18 with C-12, H-17 with C-11 (δ 127.5) and C-13 (δ 113.2) and of H-13 (δ 7.00) with C-17 (δ 140.9). In addition, correlations of H-17 and H-18 with C-19 suggested the ester function was attached to C-18. The glucopyranose moiety was found to be attached at C-4 as evidenced by the HMBC correlation from H-1' to C-4 (δ 149.4). Besides, the glucopyranose group was linked to C-19 through C-6' as determined by the HMBC correlations from H-6' (δ 4.38 and 4.29) to C-19. Based on this combined evidence, structure of compound 1 was confirmed as that of a novel phenolic glycoside that was trivially named gnaphaffine A. A plausible biogenetic pathway has been discussed for compound 1 herein (Scheme 1). Everlastoside L (4) was proposed as the precursor of compound 1 via Friedel-Crafts reaction. The  Acid hydrolysis of 2 yielded a free sugar that was identified as D-glucopyranose by measurement of the corresponding optical rotation  (Figure 2) correlation of H-1'' (5.05) to C-7 (165.0). The glucopyranose group was linked to C-9''' through C-6''', as confirmed by the HMBC correlation from H-6'' (δ 4.42 and 4.11) to C-9'''. These suggested the glucopyranose group was linked to C-9''' through C-6''. All proton and carbon signals were assigned via HMQC, HMBC ( Figure 2) and 1 H-1 H COSY spectra. Therefore, compound 2 was identified as naringenin-7-O-β-D-(6''-E-caffeoyl)-glucopyranoside, and named gnaphaffine B.
Previous studies have already reported on the anti-complementary activity of components from G. affine [2,16]. Compounds 1-8 were also evaluated in vitro for anti-complementary activity on the classical pathway of the complement. Heparin, with an IC 50 value of 0.016 mg/mL, was used as positive control in this study. Compounds 2, 3, 4 and 7 caused moderate inhibtion, showing IC 50 values of 0.471, 0.221, 0.577 and 1.041 mg/mL, respectively. IC 50 values for the remaing compounds 1, 5, 6 and 8 were 69.63, 81.50, 23.01, and 13.36 mg/mL, respectively.

General
Optical rotations were measured with Perkin-Elmer 341 polarimeter. UV and IR spectra were recorded on Shimadzu UV-2550 and Perkin-Elmer 577 (using KBr disks) spectrophotometers, respectively. NMR spectra were acquired on a Bruker Avance III spectrometer (600 MHz for 1 H-NMR, 150 MHz for 13 C-NMR, data in ppm relative to TMS). ESI-MS spectra were recorded on an Agilent 1200 series HPLC interfaced to an Agilent 6410 triple-quadrupole mass spectrometer equipped with an electrospray ionization source, and HR-ESI-MS spectra were recorded on an Agilent 1290 series HPLC interfaced to an Agilent 6538 UHD Accurate-Mass Q-TOF LC/MS (Agilent Corporation, Santa Clara, MA, USA). Semi-preparative RP-HPLC isolation was performed with an Agilent 1200 instrument with a refractive index detector (RID) using a YMC 5 μm C8 column (250 mm × 10 mm

Extraction and Isolation
The dried, whole plant materials of G. affine (2.7 kg) were ground and extracted with 80% EtOH three times (25 L, each for 2 h) under reflux at 70-80 °C. Evaporation of the solvent at 60 °C yielded a crude extract (338 g), which was suspended in distilled water and successively partitioned with petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate extract (60 g) was fractionated by silica gel column chromatography (100-200 mesh, 720 g), using a gradient of CH 2 Cl 2 : MeOH (50:1→1:1; each 5 L, v/v) to yield seven fractions (A→G).

Acid Hydrolysis of Compounds 1 and 2
A solution of compound 1 (10 mg) or compound 2 (10 mg) in 2 N aqueous CF 3 COOH (10 mL) was refluxed at 80 °C for 2 h. The mixture was then diluted in water (10 mL) and extracted with EtOAc (3 × 3 mL). The combined EtOAc layers were washed with H 2 O and evaporated to dryness to afford the glycoside. The residue was purified over an ODS column to afford D-glucopyranose (1.2 mg), which was identified on the basis of its specific rotation: