A New Sexangularetin Derivative From Camellia hakodae

Abstract

A new flavonoid, sexangularetin 3-O-(2″-O-(E)-p-coumaroyl-β-d-glucopyranoside) (1), together with 9 known flavonoid compounds (2-10), was isolated from flowers of Camellia hakodae Ninh. Their structures were established based on physicochemical and spectroscopic analyses. The new compound displayed moderate to weak cytotoxic activities against HepG2 (IC₅₀ 192.0 μg/mL), Lu (IC₅₀ 90.2 μg/mL), and KB (IC₅₀ 72.7 μg/mL) cell lines, and inactivity against MCF7 (IC₅₀ 256.0 μg/mL).

Camellia L. is the most important genus in the family Theaceae. A total of 310 Camellia species are widely distributed throughout East and South-East Asia. Camellia plants are well-known for their high economic values. ¹ The leaves of some species are used to produce tea and the seeds of some species are used to produce cooking or cosmetic oil. Several species are also used as ornamental plants. Camellia species are especially important in South-East Asian culture as an ingredient for traditional medicine. ² Camellia hakodae Ninh, commonly called Tra hoa vang, is an endemic ornamental plant in Tam Dao province, Vietnam. It is a shrub that grows in the valleys of evergreen forests at 400 to 500 m altitude. Its flowers are yellow, with diameters ranging from 6 to 8 cm. The flowering period is from December to February. ^{3 -5}

Phytochemical and pharmacological studies on Camellia species have demonstrated the presence of different chemical constituents such as flavonoids, triterpenes, tannins, and saponins from their seeds, leaves, and flowers ⁶ with antioxidant, ⁷ anti-inflammatory, antifungal, ⁸ hepatoprotective, ⁹ and cytotoxic activities. ¹⁰ However, to the best of our knowledge, our report is the first phytochemical report on C. hakodae. In this paper, we describe the isolation from its flowers and structural determination of a new sexangularetin glycoside, and 9 known compounds; we also analyzed the new compound for cytotoxic activity.

The ethanol extract of C. hakodae flowers was fractionated and purified by column chromatography (CC) using silica gel, and Sephadex LH-20 to afford a new sexangularetin glycoside (sexangularetin 3-O-(2″-O-(E)-p-coumaroyl-β-d-glucopyranoside) (1)) (Figure 1), and 9 known flavonoids (2-10). The known compounds were established as naringenin (2), ¹¹ kaempferol (3), ¹² quercetin (4), ¹³ taxifolin (5), ¹⁴ (−) epicatechin (6), ¹⁵ epigallocatechin (7), ¹⁶ epigallocatechin gallate (8), ¹⁷ quercetin 3-O-β-d-glucopyranoside (9), ¹⁸ and quercetin 7-O-β-d-glucopyranoside (10) ¹⁹ based on nuclear magnetic resonance spectroscopy (NMR) data and comparison with the reported literature.

Figure 1

Isolated flavonoids 1 to 10 from the flowers of Camellia hakodae.

Compound 1 was isolated as a yellow powder. Its molecular formula was established by high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) to be C₃₁H₂₈O₁₄ from the ion at m/z 625.1548 [M+H]⁺.

From the ¹H NMR and ¹³C NMR spectra, compound 1 was confirmed to be a flavone (E)-p-coumaroyl-β-d-glucopyranosyl derivative. The ¹H NMR spectrum showed the signals of 1 methoxy group (δ_H 3.84, s, 3 H, 8-OCH₃) and 1 aromatic proton (δ_H 6.21, 1 H, s, H-6) attributed to a 1,2,3,4,5-pentasubstituted benzene ring A. In addition, 2 aromatic signals of 4 protons at δ_H 6.92 (2H, d, J = 9.0 Hz, H-3′, 5′) and 8.08 (2H, d, J = 9.0 Hz, H-2′, 6′) belong to a p-disubstituted benzene ring B. The ¹³C NMR and distortionless enhancement by polarization transfer (DEPT) spectra demonstrated signals of the flavonol moiety with a conjugated ketone carbon signal at δ_C 179.2 (C-4), 2 olefinic quaternary carbon signals at δ_C 105.2 (C-10) and 122.9 (C-1′); 7 oxygenated quaternary carbon signals at δ_C 157.9 (C-2), 134.8 (C-3), 158.1 (C-5), 160.1 (C-7), 129.4 (C-8), 150.3 (C-9), and 161.6 (C-4′); and 5 olefinic methine carbon signals at δ_C 100.6 (C-6), 132.1 (C-2′, 6′), and 116.8 (C-3′, 5′). In particular, the heteronuclear multiple bond correlation (HMBC) correlations between the signal at δ_H 3.84 (3H, s, -OCH₃) and a carbon at δ_C 129.4 (C-8) proved the position of the methoxy group at C-8. From these data and comparison with the literature data, ²⁰ the aglycone part of 1 was suggested to be sexangularetin, a flavonol with 3 hydroxyl and 1 methoxy groups.

The sugar part was confirmed to have a β-configuration based on the large coupling constant (J = 8.0 Hz) of the anomeric proton at δ_H 5.70 in the ¹H NMR spectrum. In addition, heteronuclear single quantum coherence (HSQC) interactions from proton signals at δ_H 5.70, 5.05, 3.62, 3.44, 3.35, 3.81/3.62 (2H) to carbon signals at δ_C 100.6, 75.8, 76.3, 71.6, 78.8, and 62.5, respectively, further assigned for the glucopyranosyl unit. Moreover, 4 aromatic proton signals at δ_H 7.45 (2H, d, J = 8.0 Hz, H-2′′′, 6′′′) and 6.82 (2H, d, J = 8.0 Hz, H-3′′′, 5′′′) and 2 trans-olefinic proton signals at δ_H 6.34 (1H, d, J = 16.0 Hz, H-8′′′) and 7.66 (1H, d, J = 16.0 Hz, H-7′′′) were observed as signals of the (E)-p-coumaroyl moiety, which were confirmed based on HSQC correlations with δ_C 131.2 (C-2′′′, 6′′′), 116.3 (C-3′′′, 5′′′), 115.2 (C-8′′′), and 146.9 (C-7′′′), respectively. Furthermore, the HMBC correlations between H-8′′′ with C-7, C-9 (168.4), H-7′′′ with C-2′′′, 6′′′, C-1′′′ (127.3), H-1′′ and C-3, and between H-2′′ and C-9′′′ indicated that the glucopyranoside moiety was attached at the C-3 position and the (E)-p-coumaroyl moiety to C-2′′. The key HMBC and correlated spectroscopy (COSY) correlations of 1 are shown in Figure 2.

Figure 2

Key HMBC (arrows) and H-H COSY (bold lines) correlations of compound 1.

Based on the above evidence, the structure of 1 was determined as sexangularetin 3-O-(2″-O-(E)-p-coumaroyl-β-d-glucopyranoside) (Figure 2); this is a new flavonoid-type glycoside from C. hakodae.

The cytotoxicity of compound 1 against 4 human cancer cell lines consisting of lung (Lu), breast (MCF7), epidermoid (KB), and hepatoma (HepG2) were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Supplemental Table S1). The effect of the sample was tested at a series of concentration in the range of 1.0 to 0.256 µg/mL. Ellipticine was used as a positive control. Compound 1 showed weak or moderate activity against KB (IC₅₀ 72.7 µg/mL), HepG2 (IC₅₀ 192.0 µg/mL), and Lu (IC₅₀ 90.2 µg/mL) cell lines, but was inactive against MCF7 (IC₅₀ 256.0 µg/mL) (Supplemental Table S1).

In conclusion, phytochemical study of the ethanol extract of C. hakodae flowers led to the isolation of a new flavonoid, sexangularetin 3-O-(2″-O-(E)-p-coumaroyl-β-d-glucopyranoside), ¹ together with 9 known flavonoid compounds naringenin, ² kaempferol, ³ quercetin, ⁴ taxifolin, ⁵ (−) epicatechin, ⁶ epigallocatechin, ⁷ epigallocatechin gallate, ⁸ quercetin 3-O-β-d-glucopyranoside, ⁹ and quercetin 7-O-β-d-glucopyranoside. ¹⁰ The new compound 1 showed moderate antioxidant activity and either moderate to weak or no cytotoxic activity against different cancer cell lines. Further phytochemical and biological activity study of this plant is ongoing.

Experimental

General Procedure

The ¹H NMR and ¹³C NMR spectra were performed on a Bruker AM500 FT-NMR spectrometer (Bruker Spin, Germany) using tetramethylsilane (TMS) as internal standard and resonating at 500 and 125 MHz, respectively. The HR-MS was recorded in the ESI mode on an Agilent 6530 Accurate-Mass Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) spectrometer (Agilent Technologies, United States). Column chromatography was carried out with silica gel 60 Å grade (particle sizes 40-63 µm), which was purchased from Merck (Darmstadt, Germany). Sephadex LH-20 beads (size 25-100 µm) were purchased from Sigma-Aldrich (St Louis, MO, United States). For thin-layer chromatography, precoated silica gel 60 F₂₅₄ plates were used, thickness 0.2 mm (Merck, Germany) and visualized by spraying with 10% H₂SO₄ in methanol. All solvents were distilled and purified before use.

Plant Materials

The flowers of C. hakodae were collected in the Cooperative of Conservation and Development of Medicinal Plant in Bac Son garden, Soc Son district, Hanoi, and identified by Professor Tran Ninh, Hanoi University of Science. A voucher specimen (CH.01) has been deposited in the Soc Son Cooperative of Conservation and Development of Medicinal Herbs.

Extraction and Isolation

Dried and powdered flowers of C. hakodae (5.0 kg) were extracted with EtOH 96% at 60°C (3× 1 hour). The extracts were combined and concentrated under vacuum at 50°C. The obtained residue (200.0 g) was fractionated on a silica gel column [400 g silica gel 60 Å (63-200 µm)] eluting with n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), acetone (3 L each), and methanol (MeOH) to afford 20.8 g n-hexane, 35.1 g DCM, 40.1 g EtOAc, 14.2 g acetone, and 50.5 g MeOH fraction, respectively.

The EtOAc fraction was chromatographed on a silica gel column [400 g silica gel 60 Å (40, 63 µm)] using a gradient of DCM:MeOH (1:0-0:1, v/v) to give 6 fractions (F1-F6). Fraction F2 (10 g) was subjected to silica gel CC [200 g silica gel 60 Å (40-63 µm)] and eluted with a gradient of DCM:EtOH (1:0-0:1, v/v) to give 6 fractions (F2.1-F2.6). Fraction F2.3 (1.5 g) was purified by silica gel CC [150 g silica gel 60 Å (40-63 µm)] with a gradient of DCM:acetone (1:0-0:1, v/v) to yield compounds 2 (10 mg) and 3 (11 mg). Fraction F2.5 (2.0 g) was repeatedly chromatographed on a silica gel column [200 g silica gel 60 Å (40-63 µm)] using mixtures of DCM-acetone to yield 4 fractions (F2.5.1-F2.5.4). Fraction F2.5.2 was purified by Sephadex LH-20 CC with EtOH to furnish compound 4 (7 mg). Fraction F2.6 (1.3 g) was repeatedly chromatographed on a silica gel column [150 g silica gel 60 Å (40-63 µm)] using mixtures of DCM-acetone to yield compound 5 (8 mg).

Fraction F3 (19 g) was subjected to silica gel CC [300 g silica gel 60 Å (40-63 µm)] with a gradient of DCM:EtOH (1:0-0:1, v/v) to give 5 fractions (F3.1-F3.5). Fraction F3.3 (2.0 g) was purified by Sephadex LH-20 CC with MeOH to give compound 1 (15 mg). Fraction F3.2 (2.6 g) was repeatedly chromatographed on a silica gel column [260 g silica gel 60 Å (40-63 µm)] using mixtures of DCM-MeOH to yield compound 6 (200 mg). Fraction F3.4 (5 g) was subjected to CC [300 g silica gel 60 Å (40-63 µm)] with a gradient of DCM:MeOH (1:0-0:1, v/v) to yield compound 7 (6.7 mg) and compound 8 (11 mg).

Fraction F5 (1.0 g) was subjected to silica gel CC [100 g silica gel 60 Å (40-63 µm)] with a gradient of DCM:EtOH (1:0-0:1, v/v) to give 5 fractions (F5.1-F5.5). Fraction F5.1 (2.0 g) was purified by Sephadex LH-20 CC with MeOH to give compound 9 (20 mg).

Fraction F6 (1.0 g) was repeatedly chromatographed on a silica gel column [100 g silica gel 60 Å (40-63 µm)] using mixtures of EtOAc-EtOH to yield 7 fractions (F6.1-F6.7). Fraction F6.7 (430 mg) was purified by Sephadex LH-20 CC with MeOH to yield compound 10 (16 mg).

Sexangularetin 3-O-(2″-O-(E)-p-Coumaroyl-β-d-Glucopyranoside) (1)

Yellow amorphous powder.

¹H NMR (CD₃OD, 500 MHz): δ 6.21 (1H, s, H-6), 8.08 (2H, d, J = 9.0 Hz, H-2′, 6′), 6.92 (2H, d, J = 9.0 Hz, H-3′, 5′), 3.84 (3H, s, 8-OCH₃), 5.70 (1H, d, J = 8.0 Hz, H-1′′), 5.05 (1H, dd, J = 8.0, 9.0 Hz, H-2′′), 3.62 (2H, m, H-3′′, 6β′′), 3.44 (1H, dd, J = 9.0, 9.0 Hz, H-4′′), 3.35 (1H, m, H-5′′), 3.81 (1H, dd, J = 2.0, 13.5 Hz, H-6α′′), 7.45 (2H, d, J = 8.0 Hz, H-2′′′, 6′′′), 6.82 (2H, d, J = 8.0 Hz, H-3′′′, 5′′′), 7.66 (1H, d, J = 16.0 Hz, H-7′′′), 6.34 (1H, d, J = 16.0 Hz, H-8′′′). ¹³C NMR (CD₃OD, 125 MHz): 157.9 (C-2), 134.8 (C-3), 179.2 (C-4), 158.1 (C-5), 100.6 (C-6), 160.1 (C-7), 129.4 (C-8), 150.3 (C-9), 105.2 (C-10), 61.8 (8-OCH₃), 122.9, (C-1′), 132.1 (C-2′, 6′), 116.8 (C-3′, 5′), 161.6 (C-4′), 100.6 (C-1′′), 75.8 (C-2′′), 76.3 (C-3′′), 71.6 (C-4′′), 78.8 (C-5′′), 62.5 (C-6′′), 127.3 (C-1′′′), 131.2 (C-2′′′, 6′′′), 116.3 (C-3′′′, 5′′′), 161.3 (C-4′′′), 146.9 (C-7′′′), 115.2 (C-8′′′), 168.4 (C-9′′′).

HR-ESI-MS m/z: 625.1548 [M+H]⁺

Cytotoxicity Assay

For cytotoxicity assay, see supporting information.

Supplemental Material

Supplemental material - Supplemental material for A New Sexangularetin Derivative From Camellia hakodae

Supplemental material, Supplemental material, for A New Sexangularetin Derivative From Camellia hakodae by Nguyen T. Tuyen, Tran Van Hieu, Pham G. Dien, Tran Ninh, Nguyen T. Hung and Vu D. Hoang in Natural Product Communications