A New Flavone C-Glycoside from Clematis rehderiana

Structure Elucidation

The extract of C. rehderiana obtained with 90% EtOH was then successively partitioned between H₂O and EtOAc, followed by H₂O and n-butanol. The n-butanol soluble fraction was separated by different chromatographic procedures to afford one new C-flavone glycoside 1 and two known flavonoid glycosides 2 and 3. Their structures were determined by examination of their spectral data and comparison of the data with reported literature values.

Compound 1 was obtained as a yellow powder. The molecular formula C₃₀H₂₆O₁₂ was determined based on the high resolution FABMS data (m/z 577.1352 [M−H]‾, calcd: 577.1346). It gave a positive green coloration with 1% FeCl₃ reagent suggesting it was a flavonoid. The ¹H-NMR spectrum of 1 (Table 1) analyzed with the aid of HSQC and HMBC, showed two hydroxyl proton signals at δ_H 13.64 and δ_H 9.88; two one-proton singles at δ_H 6.54 and δ_H 6.63, attributed to H-8 and H-3, respectively; and two doublets at δ_H 7.91 (2H, d, J = 8.8 Hz, H-2′, 6′) and δ_H 6.98 (2H, d, J = 8.8 Hz, H-3′, 5′), suggesting that 1 was a 5,7,4′-trihydroxyflavone derivative. The ¹H-NMR also showed sugar proton signals at δ_H 3.54 (2H, m), 3.70 (1H, m), 4.12 (1H, m), 4.34 (1H, dd, J = 6.0, 12 Hz), 4.56 (1H, dd, 1.52, 12.0 Hz), 4.92 (1H, d, J = 9.8 Hz) attributed to H-4″ and 5″; 3″; 2″; 6″; 6″ and the anomeric proton H-1″. The position of attachment of the sugar moiety to the flavonol skeleton was determined by HMBC experiments which showed long range coupling between the anomeric H-1″ (δ 4.92) and the C-5 at δ 161.7 and C-7 at δ 164.1. The ¹H- and ¹³C-NMR spectroscopic data of the aglycone and sugar moieties of 1 were similar to those of isovitexin [8], except for the presence of nine additional carbon signals. These nine carbon signals are compatible with an E-p-coumaroyl moiety [9]. Two doublets at δ_H 6.85 (J = 8.6 Hz) and δ_H 7.53 (J = 8.6 Hz) were attributed to H-3'''/H-5''' and H-2'''/H-6'''of the E-p-coumaroyl moiety, respectively. Two other doublets (J = 15.9 Hz) at δ_H 6.37 and δ_H 7.62 were assigned to H-8''' and H-7''' of the p-coumaroyl moiety with E-configuration, respectively. The acylation of C-6″ with a p-coumaroyl unit was deduced from the downfield shift of CH₂-6″ diastereomeric protons (~ + 1 ppm) and its ¹³C-resonance at 64.7 (+ 4 ppm). This evidence was confirmed by the HMBC correlation between δ_H 4.34, 4.56 (H-6″) and C-9''' at δ 167.5 Figure 1). Based on the spectral data, the structure of 1 was characterized as the new natural product isovitexin 6″-O-E-p-coumarate (Figure 2).

Compound 2 was isolated as a yellow powder and had a molecular formula of C₂₁H₁₈O₁₃, as determined by the high resolution FABMS (m/z 477.0669 [M−H]‾). The ¹H-NMR showed two doublets at δ_H 6.83 (1H, d, J = 8.3 Hz, H-2′) and δ_H 7.42 (1H, br d, J = 8.2 Hz, H-6′) and one singlet at δ_H 8.05 (H-5′), suggesting (ortho)-hydroxylation of the B-ring, whereas 5,7-dihydroxylation of A-ring was duduced from two meta-coupled doublets that appeared as br-singlets at δ_H 6.39 (H-8) and δ_H 6.19 (H-6). The presence of a glucuronide moiety was suggested by the signals at δ_H 3.11–3.25 (3H, m, H-2″, 3″, 4″), δ_H 5.32 (H-1″) and a set of carbon signals at δ_C 102.4 (C-1″), 74.1 (C-2″), 76.4 (C-3″), 71.7 (C-4″), 74.7 (C-5″) and 171.7 (C-6″). The assignment of ¹H- and ¹³C-NMR resonances for the glucuronide moiety were decided by HMBC and H–H COSY correlations, as well as comparison with the reported literature data [10]. The position of attachment of glucuronide moiety to the flavonol skeleton was determined by HMBC experiments which showed long range correlation (³J-coupling) between the anomeric proton H-1″ (δ 5.32) and the C-3 (δ 133.8). It was further confirmed by the absence of the characteristic H-3 proton singlet at around δ_H 6.7 associated with the C-3 in the C-ring [10]. Therefore, compound 2 was identified as quercetin 3-O-β-D-glucuronopyranoside (miquelianin; Figure 2). Finally, compound 3 was identified as isoorientin (Figure 2) by its spectral data (Table 1) and comparison of this data with the reported literature values [8].

Figure 1

Key HMBC interactions of compound 1.

Figure 2

Structures of compounds 1–3.

Biological Activity

Since phenolics are characterized by antioxidant activities [11,12,13,14], two isolates were subjected to antioxidant activity experiments using both the MTT and DPPH assays (Table 2 and Table 3). Compound 1 showed potent activity against H₂O₂-induced impairment in PC12 cells within the concentration range tested (0.4 to 50 μM), whereas isoorientin (3) scavenged DPPH radical strongly, with an IC₅₀ value of 13.5 μM. This indicated that compound 1 might be an indirectly acting antioxidant, while 3 might be a directly acting antioxidant.

General

Optical rotations were measured with a JASCO DIP-370 digital polarimeter in MeOH solutions. IR spectra were measured on a Bio-Rad FTS-135 infrared spectrometer with KBr pellets. UV spectra were obtained with a Shimadzu UV-2401PC spectrometer. Mass spectra were measured on a VG Auto Spec-3000 spectrometer. NMR spectra were recorded on a DRX-500 NMR spectrometer with TMS as internal standard. Silica gel (200-300 mesh) for column chromatography and precoated TLC plates (Si gel G) were purchased from the Qingdao Marine Chemical Factory (Qingdao, P. R. China). Reversed-phase C₁₈ silica gel for column chromatography were obtained from Merck. Sephadex LH-20 for column chromatography was purchased from Amersham Biosciences.

Plant material

Aerial parts of Clematis rehderiana was collected from Lijian in Yunnan Province, China, in August 2005. A voucher specimen (KUN0816301) is stored at the herbarium of Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and isolation

The air-dried aerial parts of C. rehderiana (3 kg) were ground and extracted four times with 90% EtOH (8 L each time) at room temperature. After removal of the solvent, the residue was suspended in water (2 L) and then extracted successively with petroleum ether (PE), EtOAc and n-butanol (4 × 2L each). The n-butanol extract was subjected to column chromatography (CC) over macroporous resin D101 eluting with EtOH-H₂O (0%–95%) and afforded five fractions Fr. I–V. Fr. III (18.1 g) was subjected to CC over silica gel (100 g) and eluted with CHCl₃/MeOH 20:1→ 9:1→ 4:1 → 7:3 and EtOH to give 10 subfractions (Fr. III1 – Fr. III10). Fr. III5 was further isolated by repeated vacuum liquid chromatography (VLC) over RP-18 and eluted with MeOH-H₂O (0% to 50%) to afford compounds 2 (4 mg) and 3 (50 mg). Fr. IV (3.9 g) was subjected to CC over silica gel (60 g) and eluted with CHCl₃/MeOH 20:1→ 9:1→ 7:3 to give 14 subfractions (Fr. IV1 – Fr. III14). Fr. IV8 was further fractionated by repeated CC over SephadexLH-20 and eluted with MeOH-CHCl₃ and MeOH to afford compound 1 (12 mg).

Compound 1: = +32.9 (c 0.76, MeOH); IR (KBr): 3,420, 2,920, 1,720 cm^-1; ¹H- and ¹³C-NMR see Table 1. HRFABMS (neg): m/z 577.1352 [M-H]⁻, calcd: 577.1346.

Compound 2: = −8.6 (c 0.58, MeOH); UV (MeOH) λ_max (log ε) 404, 270, 210 nm; IR (KBr): 3,408, 2,922, 1,653, 1,604; HRFABMS (neg): m/z 477.0669 [M-H]⁻, calcd: 477.0669; ¹H-NMR (DMSO-d₆) δ: 12.35 (1H, br s 5-OH), 8.05 (1H, s, H-5′), 7.42 (1H, br d, J = 8.2 Hz, H-6′), 6.83 (1H, d, J = 8.3 Hz, H-2′), 6.39 (1H, br s, H-8), 6.19 (1H, br s, H-6), 5.32 (1H, d, J = 7.0 Hz, H-1″), 3.39 1H, m, H-5″), 3.11-3.25 (3H, m, H-2″, 3″, 4″); ¹³C-NMR (DMSO-d₆) δ: 157.2 (C-2), 133.8 (C-3), 177.4 (C-4), 161.0 (C-5), 98.9 (C-6), 164.5 (C-7), 93.7 (C-8), 156.4 (C-9), 103.7 (C-10), 120.6 (C-1′), 115.3 (C-2′), 144.8 (C-3′), 148.5 (C-4′), 117.5 (C-5′), 120.9 (C-6′), 102.4 (C-1″), 74.1 (C-2″), 76.4 (C-3″), 71.7 (C-4″), 74.7 (C-5″), 171.7 (C-6″).

Antioxidant assays

The antioxidant assay against H₂O₂-induced impairment in PC12 cells was conducted according to the reported protocol [14]. Briefly, PC12 cells were seeded into 96-well plates in RPMI 1640 medium with 10% characterized Newborn Bovine Serum. Twenty-four hours later, different concentrations of compounds 1 and 3 together with freshly prepared H₂O₂ were added and incubation continued for 1 hour. Then MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added and the incubation continued for 4 hours. Finally, solution (100 μL) containing 5% iso-butanol, 10% SDS (Sigma) and 0.004% HCl was added. The mixtures were kept overnight and the index of cell viability (% of control) was calculated by measuring the optical density of the color produced by MTT dye reduction with a microplate reader at 570 nm.

DPPH radical-scavenging activity assays were performed according to our previously reported procedures [11]. Each compound (100 μL) at five different concentrations was added to DPPH solution (100 μL). The absorbance was measured with a microplate reader at 517 nm after 30 min of reaction at 37 °C. IC₅₀ values denote the concentration of sample required to scavenge 50% DPPH free radicals.