Flavonoid constituents of Euonymus fortunei

Six flavonoid glycosides (1–6), together with three known compounds (7–9), were isolated from the 70% ethanol extract of Euonymus fortunei and identified on the basis of nuclear magnetic resonance (NMR) data, mass spectroscopy (MS) data, and comparison with literature data. Compound 1 was obtained as a new compound. Compounds 1–5, 7, and 9 were isolated from this plant for the first time.

Previous studies showed that the genus Euonymus possessed antifeedant, narcotic, and insecticidal properties as well as antitumor properties, mainly due to β-dihydroagarofuran sesquiterpene polyol esters and pyridine alkaloids [1–3]. The current taxonomic classification of Euonymus according to chemical constituents is based on the skeleton of β-dihydroagarofuran. However, recent research has reported that the extracts of Euonymus contain several flavonoids with antidiabetic and radical scavenging properties [4, 5]. These suggest flavonoids may be one of the most important components in Euonymus plants. Euonymus fortunei, belonging to the genus Euonymus, is a folk medicine and famous ornamental plant in China. Several sesquiterpenoid, triterpenoids, alkaloids, organic acids, and β-sitosterol have been isolated from this plant [2, 6–8]. To the best of our knowledge, flavonoid constituents of E. fortunei are rare.

In this paper, six flavonoid glycosides (1–6) and three other compounds (7–9) were isolated from the 70% ethanol extract of E. fortunei. Identification of compounds 2–9 was performed by comparison of the physical constants and spectral data with those of known compounds.

Compound 1, obtained as a yellow amorphous powder, was assigned the molecular formula C₃₉H₅₀O₂₄ by analyses of EI-MS at m/z 903.38 [M + H]⁺ and from ¹³C NMR spectrum. The UV spectrum at 265 and 343 nm (MeOH) showed characteristic bands of a flavonoid. The ¹H NMR spectrum exhibited an AB system at δ 7.80 and 6.94 (J = 8.4 Hz) typical of the four protons H-2′, H-6′ and H-3′, H-5′ of the p-substituted B ring of the flavonoid, and a 2H singlet at δ 6.79 and 6.46 attributed to H-8 and H-6 of the A ring, indicating the presence of a kaempferol skeleton. This spectrum also exhibited four anomeric protons (δ 5.51, 5.15, 4.39, and 4.26) and two 3H singlets at δ 1.18 (3H, d, J = 6.0 Hz) and 0.88 (3H, d, J = 6.0 Hz) relative to two methyl groups of rhamnose, suggesting this is a flavonol with four glycosides. The ¹³C NMR data further supported the presence of a kaempferol skeleton and four sugar moieties, which are indicated by two rhamnose (δ 18.8 and 18.4) and two glucose (δ 62.3 and 62.1) moieties. In comparison with kaempferol [9] the ¹³C NMR spectrum of 1 exhibited an upfield shift for C-3 and C-7 by 0.8 and 1.0 ppm, respectively, indicating glycosylation at both positions. In addition, in the HMBC spectrum the anomeric proton of one rhamnopyranosyl unit (H-1″, δ 5.51) showed a correlation with C-7 (δ 162.7), and the second rhamnopyranosyl unit (H-1″″, δ 5.15) showed a correlation with C-3 (δ 135.8). The anomeric proton of one glucopyranosyl unit (δ 4.39) showed a correlation with C-4″″ (δ 83.0), and the second glucopyranosyl unit (δ 4.26) also showed a correlation with C-4″″ (δ 82.4). The rhamnose and glucose units were found to be linked (1→4), as the signal at δ 83.0/82.4 is characteristic for C-4″/4″″ in a 4-substituted rhamnose unit. Other two-dimensional NMR techniques (¹H–¹H COSY, ROSEY, NOESY, and HSQC) verified the orientation of the complex conjugate as a rhamnosyl-glucosyl-O ether as opposed to a glucosyl-rhamnosyl-O ether. Furthermore, acid hydrolysis also confirmed that compound 1 contains kaempferol, D-glucose, and L-rhamnose. From the spectroscopic data and also from comparison with [9], compound 1 is identified as kaempferol-3-O-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-7-O-β-D-glucopyranosyl-(1→4)-α-Lrhamnopyranoside, a new flavonoid glycoside not reported before.

Experimental

General Procedures. The NMR spectra were run on a DRX-500 MHz spectrometer using TMS as an internal standard. The EI-MS spectra were measured with a Bruker Esquire 3000 spectrometer. The melting point was measured using an X-4 micromelting point apparatus (Beijing Second Optical Instrument Factory, China) and was uncorrected.

Fungus Material. The aerial parts of E. fortunei were collected from Yongfu County of Guangxi on October 2008. A voucher specimen was deposited at the Key Laboratory of Medicinal Chemical Resources and Molecular Engineering, Guangxi Normal University, Guilin, Guangxi.

Extraction and Isolation. The powdered aerial parts of E. fortunei (1.2 kg) were repeatedly extracted with aqueous ethanol (70%, v/v) by heating under reflux. The extract was then concentrated under reduced pressure to give a residue (194.5 g), which was suspended in deionized water and then successively partitioned with petroleum ether, EtOAc, and finally with n-BuOH, affording a known weight of each respective fraction. The n-BuOH fraction (49.3 g) was passed through an ODS-C₁₈ column and successively eluted with 0% MeOH, 30% MeOH, 60% MeOH, and 95% MeOH. The 60% MeOH eluate portion was subjected to silica gel column chromatography, eluting with CHCl₃–MeOH (10:1–1:1), to give compound 7. A portion of the 30% MeOH eluate was subjected to Sephadex LH-20 column chromatography and eluted with MeOH to obtain compound 1. The EtOAc fraction (25.5 g) was applied to a silica gel column (200–300 mesh) and eluted with a stepwise gradient mixture of CH₂Cl₂–MeOH (40:1→1:1) to obtain 11 fractions. Fraction 4 was applied to a silica gel column and eluted with CH₂Cl₂–MeOH (20:1, v/v) to obtain 6. Fraction 5 was repeatedly chromatographed on Sephadex LH-20 (60% MeOH, v/v) to obtain 3. Fraction 6 was applied to a silica gel column (300–400 mesh) and eluted with CH₂Cl₂–MeOH (8:1, v/v) to obtain 4 and 9. Fraction 8 was recrystallized twice from MeOH–H₂O to give the pure compounds 2 and 5. The petroleum ether fraction was subjected to a series of chromatographic techniques using a silica gel column (200–300 mesh) to give compound 8.

Compound 1. Yellow amorphous powder, mp 231–232°C. ¹H NMR (500 MHz, DMSO-d₆, δ, ppm, J/Hz): 7.80 (2H, d, J = 8.4, H-2′, 6′), 6.94 (2H, d, J = 8.4, H-3′, 5′), 6.79 (1H, s, H-8), 6.46 (1H, s, H-6), 5.51 (1H, br.s, H-1″), 5.15 (1H, br.s, H-1″′), 4.39 (1H, d, J = 1.2, H-1″″), 4.26 (1H, d, J = 1.2, H-1″″′), 1.18 (3H, d, J = 6.0, H-6″), 0.88 (3H, d, J = 6.0, H-6″″). ¹³C NMR (125 MHz, DMSO-d₆, δ, ppm): 158.9 (s, C-2), 135.8 (s, C-3), 179.0 (s, C-4), 162.0 (s, C-5), 100.6 (d, C-6), 162.7 (s, C-7), 95.7 (d, C-8), 157.2 (s, C-9), 107.0 (s, C-10), 121.4 (s, C-1′), 131.8 (d, C-2′, 6′), 116.6 (d, C-3′, 5′), 161.3 (s, C-4′), Rha1: 103.1 (d, C-1″), 71.0 (d, C-2″), 70.8 (d, C-3″), 83.0 (d, C-4″), 71.3 (d, C-5″), 18.4 (q, C-6″), Glc1: 105.8 (d, C-1″′), 75.6 (d, C-2″′), 78.1 (d, C-3″′), 71.4 (d, C-4″′), 77.7 (d, C-5″′), 62.3 (t, C-6″′), Rha2: 99.2 (d, C-1″″), 70.5 (d, C-2″″), 70.5 (d, C-3″″), 82.4 (d, C-4″″), 70.1 (d, C-5″″), 18.8 (q, C-6″″), Glc2: 105.6 (d, C-1″″′), 75.6 (d, C-2″″′), 78.1 (d, C-3″″′), 71.2 (d, C-4″″′), 77.7 (d, C-5″″′), 62.1 (t, C-6″″′).

Kaempferol-3,7- O -α-dirhamnopyranoside (2). Pale-yellow needles (MeOH), mp 185–187°C. EI-MS m/z 589 [M + H]⁺. ¹H NMR (500 MHz, DMSO-d₆, δ, ppm, J/Hz): 12.5 (1H, s, 5-OH), 10.12 (1H, s, 4′-OH), 7.80 (2H, d, J = 8.7, H-2′, 6′), 6.93 (2H, d, J = 8.7, H-3′, 5′), 6.80 (1H, d, J = 1.7, H-8), 6.46 (1H, d, J = 1.7, H-6), 5.56 (1H, br.s, H-1″), 5.02 (1H, s, H-1″′), 1.10 (3H, d, J = 6.4, H-6″), 0.81 (3H, d, J = 6.4, H-6″′). ¹³C NMR (125 MHz, DMSO-d₆, δ, ppm): 158.3 (s, C-2), 135.0 (s, C-3), 178.4 (s, C-4), 161.4 (s, C-5), 99.9 (d, C-6), 162.2 (s, C-7), 95.1 (d, C-8), 156.6 (s, C-9), 106.3 (s, C-10), 120.8 (s, C-1′), 131.2 (d, C-2′, 6′), 115.9 (d, C-3′, C-5′), 160.6 (s, C-4′), 102.3 (d, C-1″), 70.6 (d, C-2″), 71.2 (d, C-3″), 72.1 (d, C-4″), 70.7 (d, C-5″), 18.4 (q, C-6″), 98.9 (d, C-1″′), 70.6 (d, C-2″′), 70.8 (d, C-3″′), 71.6 (d, C-4″′), 70.3 (d, C-5″′), 17.9 (q, C-6″′) [10].

Kaempferol-3-(4″- O -acetyl)- O -α-L-rhamnopyranoside-7- O -α-L-rhamnopyranoside (3). Yellow solid, mp 206–208°C. EI-MS m/z 619.26 [M –H]^–. ¹H NMR (500 MHz, DMSO-d₆, δ, ppm, J/Hz): 8.08 (2H, d, J = 8.8, H-2′, 6′), 6.90 (2H, d, J = 8.8, H-3′, 5′), 6.81 (1H, s, H-8), 6.44 (1H, s, H-6), 5.54 (1H, br.s, H-1″), 5.47 (1H, d, J = 7.2, H-1″′), 2.01 (3H, s, H-6″), 1.10 (3H, d, J = 6.4, H-6″′). ¹³C NMR (125 MHz, DMSO-d₆, δ, ppm): 158.8 (C-2), 135.5 (C-3), 179.1 (C-4), 162.5 (C-5), 100.1 (C-6), 162.9 (C-7), 95.1 (C-8), 157.3 (C-9), 106.9 (C-10), 122.0 (C-1′), 131.5 (C-2′, 6′), 116.1 (C-3′, 5′), 160.9 (C-4′), 102.1 (C-1″), 70.5 (C-2″), 71.8 (C-3″), 74.2 (C-4″), 69.5 (C-5″), 17.9 (C-6″), 99.2 (C-1″′), 71.1 (C-2″′), 71.2 (C-3″′), 73.1 (C-4″′), 68.8 (C-5″′), 17.3 (C-6″′), 170.5 (-C=O), 20.7 (CH₃) [11].

Apigenin-7- O -β-D-glucopyranoside (4). Pale-yellow solid. ¹H NMR (500 MHz, DMSO-d₆, δ, ppm, J/Hz): 12.95 (1H, s, 5-OH), 10.39 (1H, s, 4′-OH), 7.95 (2H, d, J = 8.8, H-2′, 6′), 6.93 (2H, d, J = 8.8, H-3′, 5′), 6.85 (1H, s, H-3), 6.81 (1H, d, J = 2.0, H-8), 6.42 (1H, d, J = 2.0, H-6), 5.06 (1H, d, J = 7.3, H-1″), 3.6–3.7 (1H, m), 3.4–3.5 (2H, m), 3.1–3.2 (1H, m). ¹³C NMR (125 MHz, DMSO-d₆, δ, ppm): 164.7 (C-2), 103.4 (C-3), 182.2 (C-4), 161.7 (C-5), 99.9 (C-6), 163.0 (C-7), 94.9 (C-8), 157.2 (C-9), 106.0 (C-10), 121.5 (C-1′), 128.1 (C-2′), 116.2 (C-3′), 161.4 (C-4′), 116.2 (C-5′), 128.1 (C-6′), 100.5 (C-1″), 73.2 (C-2″), 76.8 (C-3″), 76.9 (C-4″), 70.0 (C-5″), 61.5 (C-6″) [12].

Kaempferol-3- O -β-D-glucopyranosyl-7- O -α-L-rhamnopyranoside (5). Pale-yellow solid. EI-MS m/z 593 [M – H]^–. ¹H NMR (500 MHz, DMSO-d₆, δ, ppm, J/Hz): 8.08 (2H, d, J = 8.0, H-3′, 5′), 6.87 (2H, d, J = 8.0, H-2′, 6′), 6.82 (1H, s, H-8), 6.44 (1H, s, H-6), 5.54 (1H, d, J = 2.0, H-1″), 5.46 (1H, d, J = 6.4, H-1″′), 1.10 (3H, d, J = 6.4, H-6″′). ¹³C NMR (125 MHz, DMSO-d₆, δ, ppm): 156.7 (C-2), 133.5 (C-3), 177.6 (C-4), 160.9 (C-5), 98.4 (C-6), 161.6 (C-7), 94.5 (C-8), 156.0 (C-9), 105.7 (C-10), 120.8 (C-1′), 130.0 (C-2′, C-6′), 115.1 (C-3′, C-5′), 160.1 (C-4′), 100.8 (C-1″), 74.2 (C-2″), 76.4 (C-3″), 70.2 (C-4″), 77.6 (C-5″), 60.8 (C-6″), 99.4 (C-1″′), 70.1 (C-2″′), 70.2 (C-3″′), 71.6 (C-4″′), 69.8 (C-5″′), 17.8 (C-6″′) [13].

Kaempferol-7- O -α-L-rhamnopyranoside (6). Yellow needles (MeOH). EI-MS m/z 431 [M – H]^–. ¹H NMR (500 MHz, DMSO-d₆, δ, ppm, J/Hz): 8.13 (2H, d, J = 9.0, H-3′, 5′), 6.92 (2H, d, J = 9.0, H-2″, 6″), 6.76 (1H, d, J = 2.0, H-8), 6.44 (1H, d, J = 2.0, H-6), 5.58 (1H, d, J = 2.0, H-1″), 1.28 (3H, d, J = 6.5, H-6″). ¹³C NMR (125 MHz, DMSO-d₆, δ, ppm): 149.7 (C-2), 138.4 (C-3), 178.4 (C-4), 163.2 (C-5), 100.9 (C-6), 164.2 (C-7), 96.3 (C-8), 158.7 (C-9), 107.1 (C-10), 124.5 (C-1′), 131.7 (C-2′), 117.2 (C-3′), 161.6 (C-4′), 117.2 (C-5′), 131.7 (C-6′), 100.8 (C-1″), 74.6 (C-2″), 73.0 (C-3″), 72.7 (C-4″), 72.1 (C-5″), 19.0 (C-6″) [14].

Syringin (7). White powder, mp 188–190°C. EI-MS m/z 395.07 [M + Na]⁺. ¹H NMR (500 MHz, DMSO-d₆, δ, ppm, J/Hz): 6.74 (2H, s, H-3, 5), 6.49 (1H, d, J = 15.8, H-7), 6.37 (1H, dt, J = 15.8, 5.5, H-8), 4.95 (2H, d, J = 2.1, H-9), 4.90 (1H, d, J = 5.5, H-1′), 3.79 (6H, s, 2 × OCH₃). ¹³C NMR (125 MHz, DMSO-d₆, δ, ppm): 134.0 (C-1), 152.6 (C-2, 6), 104.5 (C-3, 5), 132.6 (C-4), 130.1 (C-7), 128.3 (C-8), 77.1 (C-9), 102.6 (C-1′), 69.9 (C-2′), 76.5 (C-3′), 61.4 (C-4′), 74.1 (C-5′), 60.9 (C-6′), 56.3 (2 × OCH₃) [15].

Friedelin (8). White powder, mp 260–261°C. ¹H NMR (500 MHz, CDCl₃, δ, ppm, J/Hz): 0.72 (3H, s, H-24), 0.87 (3H, d, J = 7.0, H-23), 0.95 (3H, s, H-29), 0.98 (3H, s, H-30), 1.00 (3H, s, H-26), 1.01 (3H, s, H-27), 1.05 (3H, s, H-25), 1.18 (3H, s, H-28), 2.38–2.39 (1H, m, H-4), 2.23–2.29 (2H, m, H-2). ¹³C NMR (125 MHz, CDCl₃, δ, ppm): 22.3 (C-1), 41.4 (C-2), 213.2 (C-3), 58.1 (C-4), 42.1 (C-5), 41.2 (C-6), 18.1 (C-7), 53.0 (C-8), 37.4 (C-9), 59.4 (C-10), 35.5 (C-11), 30.5 (C-12), 39.6 (C-13), 38.2 (C-14), 32.3 (C-15), 36.0 (C-16), 30.0 (C-17), 42.7 (C-18), 35.2 (C-19), 28.1 (C-20), 32.7 (C-21), 39.2 (C-22), 6.7 (C-23), 14.6 (C-24), 17.8 (C-25), 20.3 (C-26), 18.6 (C-27), 32.0 (C-28), 34.9 (C-29), 31.7 (C-30) [16].

Acetamide (9). Colorless needles, mp 81–82°C. ¹H NMR (500 MHz, CDCl₃, δ, ppm, J/Hz): 6.3 (2H, br.s, NH₂), 1.9 (3H, s, H-2). ¹³C NMR (125 MHz, CDCl₃, δ, ppm): 173.8 (C-1), 22.6 (C-2).