Non-alkaloid constituents of Vinca major

Chinese Journal of Natural Medicines 2016, 14(1): 00560060

Chinese Journal of Natural Medicines

doi: 10.3724/SP.J.1009.2016.00056

Non-alkaloid constituents of Vinca major CHENG Gui-Guang 1, 2, ZHAO Hai-Yun 1, LIU Lu 2, ZHAO Yun-Li 2, SONG Chang-Wei 2, GU Ji 2, SUN Wei-Bang 2, LIU Ya-Ping 2*, LUO Xiao-Dong 2* 1

Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming, 650500, China;

2

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of

Sciences, Kunming 650201, China Available online 20 Jan., 2016

[ABSTRACT] The present study was designed to investigate the non-alkaloid compounds from the leaves and stems of Vinca major cultivated in Yunnan Province, China. The compounds were isolated using chromatographic techniques. The structures were elucidated by 1D- and 2D-NMR spectroscopic methods in combination with UV, IR, and MS analyses. The 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging activity of Compounds 1–7 were evaluated. One new iridoid glycoside (compound 1), together with 11 known compounds, were isolated from Vinca major. Compounds 1, 5, and 6 showed moderate DPPH-scavenging activity, with IC50 values being 70.6, 32.8, and 62.2 μmol·L−1, respectively. In conclusion, compound 1 is a newly identified iridoid glycoside with moderate antioxidant activity. [KEY WORDS] Vinca major; Iridoid glycoside; Vinmaside A; Antioxidant

[CLC Number] R284

[Document code] A

[Article ID] 2095-6975(2016)01-0056-05

Introduction The genus Vinca, belonging to the Apocynaceae family, is mainly native to Europe, northwest Africa, and southwest Asia, which has attracted considerable attention as a source of indole alkaloids with diverse structures and biological properties [1-6]. Vinca major has been cultivated widely in China as a flowering evergreen ornamental plant. Previous phytochemical investigations on the leaves and stems of V. major are focused on the alkaloid fraction and have led to the discovery of a series of indole alkaloids [7-11]. However, the non-alkaloid constitutes of this plant have seldomly been reported. As a continued systematic research on the chemical constitutes, our investigation on the non-alkaloid part of V. major resulted in the isolation a new iridoid glycoside, together with 11 known compounds (2–12). The structure of compound 1 was elucidated by means of spectroscopic

[Received on] 15-Feb.-2015 [Research funding] This work was supported by National Natural Science Foundation of China (No. 81225024) and National Science and Technology Support Program of China (No. 2013 BAI11B02). [*Corresponding author] E-mail: [email protected] (LUO Xiao-Dong); [email protected] (LIU Ya-Ping). These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

methods and the known compounds were identified by comparison of their spectroscopic data with those reported in the literature. In addition, the antioxidant activities of compounds 1–7 were determined using the 1, 1-diphenyl2-picryldyhydrazyl (DPPH) radical-scavenging assay were determined.

Results and Discussion The molecular formula of compound 1 was assigned as C27H34O13 on the basis of the negative quasi-molecular ion peak at m/z 565 [M – H]– and positive-ion HR-EI-MS at m/z 566.1993 [M]+, in combination with its 13C NMR spectrum. Its UV spectrum exhibited maximum absorptions at 204 and 290 nm. The 1H NMR spectrum displayed the signals for an aromatic ABX spin system at δH 7.08 (1H, d, J = 8.2 Hz), 6.81 (1H, dd, J = 8.2, 1.9 Hz), and 7.21 (1H, d, J = 1.9 Hz), and two trans-substituted double bonds proton signals at δH 7.61 (1H, d, J = 15.9 Hz) and 6.39 (1H, d, J = 15.9 Hz), indicating the existence of a feruloyl moiety in combination with the methoxy group signal at δH 3.89 (3H, s) [12] (Table 1). Additionally, a methyl doublet signal at δH 1.12 (3H, d, J = 6.8 Hz, CH3-10), a methoxy group signal at H 3.69 (3H, s, COOCH3), a trisubstituted olefinic proton signal at H 7.44 (1H, s), an oxymethine doublet signal at δH 5.30 (1H, d, J = 5.0 Hz), and an anomeric proton signal at δH 4.66 (1H, d, J = 7.9 Hz)

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CHENG Gui-Guang, et al. / Chin J Nat Med, 2016, 14(1): 5660

were also observed, which were characteristic signals for an iridoid glycoside. Besides feruloyl and glucoside groups, compound 1 possessed 12 carbon signals in the 13C NMR spectrum, ascribable to one sp2 methine (δC 152.6), five sp3 methines (δC 97.6, 78.4, 47.0, 41.0, and 32.7), one sp3 methylene (δC 40.4), one methoxyl group (δC 51.7), one methyl group (δC 13.7), and two sp2 quaternary carbons (δC 169.3 and Table 1

113.1). All the above spectroscopic data were in a good agreement with 7-O-(p-coumaryl)-loganin [13], suggesting that compound 1 was an iridoid glycoside bearing a feruloyl moiety and a loganin moiety. In the HMBC spectrum, the methine proton signal at H 5.27 (m, H-7) showed correlation with feruloyl carbonyl signal at δC 168.8 (C-9''), which revealed that the feruloyl moiety was located at C-7 (Fig. 2).

1

H and 13C NMR spectral data of compound 1 in methnol-d4 at 400 MHz

entry

δH

δC

entry

δH

δC

1 3

5.30, d (5.0)

97.6, d

4'

3.28, m

71.5, d

7.44, s

152.6, d

5'

3.32, m

78.4, d

113.1, s

6'a

3.91, d (11.7)

62.7, t

3.67, m

4 5

3.15, m

32.7, d

6'b

40.4, t

1''

6a

2.33, m

6b

1.78, m

7

5.27, m

78.4, d

3''

8

2.18, m

41.0, d

4''

9

2.11, m

47.0, d

5''

7.08, d (8.2)

116.4, d

10

1.12, d (6.8)

13.7, q

6''

6.81, dd (8.2, 1.9)

124.1, d

11

2''

127.6, s 7.21, d (1.9)

111.6, d 149.3, d 150.6, s

169.3, s

7''

7.61, d (15.9)

115.6, d

COOCH3

3.69, s

51.7, q

8''

6.39, d (15.9)

146.8, d

1'

4.66, d (7.9)

100.1, d

9''

2'

3.21, d (8.0)

74.7, d

OCH3

3'

3.38, m

77.9, d

Fig. 1 Structures of compounds 1−12

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168.8, s 3.89, s

56.4, q

CHENG Gui-Guang, et al. / Chin J Nat Med, 2016, 14(1): 5660

Experimental

Fig. 2 Selected HMBC (

) correlations of compound 1

Acid hydrolysis of compound 1 afforded D-glucose as measured by thin layer chromatography (TLC) and gas chromatography (GC) analyses of its corresponding trimethylsilylated L-cysteine adduct [14]. The β-configuration of glucoside was determined on the basis of the coupling constant (J = 7.9 Hz) of its anomeric proton [15]. The location for the sugar moiety of compound 1 were confirmed by HMBC spectrum, which showed key cross-peaks from H-1′ (H 4.66) to C-1 (C 97.6), and from H-1 (H 5.30) to C-1′ (C 100.1), C-5 (C 32.7), and C-3 (C 152.6). In the ROESY spectrum, the observed correlations of Me-10/H-9 and H-9/H-5 indicated the β orientation of H-9 and H-5. The ROESY correlations of H-7/H-8 and H-1/H-8 suggested α oriented for H-7 and H-1 (Fig. 1). These ROESY correlations suggested the relative configuration was the same as 7-O-(p-coumaryl)-loganin. Therefore, the structure of compound 1 was elucidated as 7-O-E-feruloyl-loganin. By comparison of the NMR data with the literature, 11 known compounds were identified as 7-O-(p-coumaryl) -loganin (2) [13], dearabinosyl pneumonanthoside (3) [16], byzantionoside B (4) [17], syringaresinol-4-O-β-D-glucopyranoside (5) [18], syringaresinol (6) [19], lyoniresinol 9-O-rhamnoside (7) [20], methyl ferulate (8) [21], sinapic acid methylester (9) [22], vanillin (10) [23], syringaldehyde (11) [23], and acetovanillone (12) [24], while compounds 3−4, 6−7, and 12 were first obtained from Vinca major (Fig. 1). The DPPH scavenging activity of compounds 1−7 were measured as described previously [25]. It was observed that compounds 1, 5, and 6 showed good radical scavenging activity on DPPH radicals with IC50 values being 70.6, 32.8, and 62.2 μmol·L−1, respectively. Table 2 DPPH radical scavenging activities of compounds 1-7 Sample 7-O-E-feruloyl-loganin (1) 7-O-(p-coumaryl)-loganin (2)

DPPH IC50/(μmol·L−1) 70.6 201

dearabinosyl pneumonanthoside (3)

NA

byzantionoside B (4)

NA

syringaresinol-4-O-β-D-glucopyranoside (5)

32.8

syringaresinol (6)

62.2

lyoniresinol 9-O-rhamnoside (7) L-Ascorbic acid NA: Not active

125 56.8

Apparatus and reagents Optical rotation was measured with a Horiba SEPA-300 polarimeter (Horiba Scientific, Kyoto, Japan). UV spectrum was obtained using a Shimadzu UV-2401A spectrometer (Shimadzu Corp., Kyoto, Japan). IR spectrum was obtained by a Bruker FT-IR Tensor 27 spectrometer (Bruker BioSpin GmBH, Rheinstetten, Germany) using KBr pellets. 1D and 2D spectra were obtained on Bruker AVANCE III-600, DRX-500, and AV-400 MHz spectrometers (Bruker BioSpin GmBH, Rheinstetten, Germany) with TMS as an internal standard. Chemical shifts () were expressed in ppm with reference to solvent signals. HREIMS was recorded on a Waters AutoSpec Premier P776 spectrometer (Waters Corp., Milford, MA, USA). Column chromatography was performed on Silica gel (200−300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), RP-18 gel (20–45 µm, Fuji Silysia Chemical Ltd., Tokyo, Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Uppsala, Sweden). Fractions were monitored by TLC (GF254, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Medium pressure liquid chromatography was employed using a Büchi pump system coupled with C18-silica gel-packed glass column (15 mm × 230 mm, and 26 mm × 460 mm). High performance liquid chromatography (HPLC) was performed using an Agilent 1260 pump (Agilent Technologies Co. Ltd., Palo Alto, USA) coupled with Agilent analytical, semi-preparative, or preparative Zorbax SB-C18 columns (150 mm × 4.6 mm, 150 mm× 9.4 mm, and 250 mm × 21.2 mm, respectively). Plant Materials V. major was collected from Kunming Botanical Garden, Yunnan province, China, and identified by Prof. SUN Wei-Bang, Kunming institute of Botany. A voucher specimen (No. Sun20110820) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. Extraction and Isolation An air-dried and powdered sample (20 kg) was extracted with MeOH (3 × 50 L) at room temperature and the solvent removed in vacuo. The residue was partitioned with EtOAc (3 × 10 L) to give EtOAc and H2O extracts. The EtOAc extract was subjected to a silica gel column (CHCl3−MeOH, 1 : 0 to 0 : 1) to afford fractions (I–VII). Fraction I (4.6 g) was purified on a preparative C18 HPLC column with a gradient of MeOH−H2O (20 : 80−80 : 20) to yield five subfractions I-1–5. Subfraction I-2 (460 mg) was further applied to a silica gel column using a petroleum ether−acetone eluent (5 : 1) to yield compounds 10 (130 mg), 11 (111 mg), and 12 (24 mg). Subfraction I-3 (380 mg) was separated by silica gel chromatography column (petroleum ether−acetone, 6 : 1–2 : 1) to yield compounds 8 (21 mg) and 9 (310 mg). Fr. III (26 g) was separated on a preparative C18 column with a gradient

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MeOH−H2O (30 : 70−80 : 20), then purified on a silica gel CC (CHCl3–MeOH, 15 : 1–10 : 1) to yield compound 7 (3.8 g). Fr. IV (8.2 g) was purified by C18 medium pressure liquid chromatography with a MeOH–H2O gradient (40 : 60−80 : 20) to yield subfractions IV-1–3. Subfraction IV-1 (1.1 g) was chromatographed on a silica gel column (CH2Cl3–MeOH, 15 : 1–10 : 1), then purified on a semi-preparative C18 HPLC column with a gradient of MeOH–H2O (30 : 70−45 : 55) to yield compounds 3 (12 mg) and 4 (8 mg). Subfraction IV-2 (480 mg) was further separated on a preparative C18 HPLC column with a gradient CH3CN-H2O (30 : 60–40 : 60) to afford compounds 1 (6 mg), 2 (65 mg) and 5 (23 mg). Subfraction IV-3 (1.2 g) was separated on a preparative C18 column with a gradient MeOH–H2O (30 : 70−80 : 20), then separated on a semipreparative C18 HPLC column (CH3CN– H2O, 25 : 75) to produce compound 6 (21 mg). Acidic hydrolysis of 1 and GC analysis Compound 1 (2 mg) was hydrolyzed with 2 mol·L−1 of HCl/dioxane (1 : 1, 10 mL) under reflux for 3 h. The reaction mixture was partitioned between CHCl3 and H2O. The aqueous layer was neutralized with MeOH and then dried to give a neutral residue. The residue was dissolved in anhydrous pyridine (1 mL) and reacted with L-cysteine methyl ester hydrochloride (1.5 mg) stirred at 60 ºC for 1.5 h. Then trimethylsilylimidazole (1.0 mL) was added to the reaction sugar moiety, and they were kept at 60 ºC for 30 min. The mixture (4 μL) was subjected to GC analysis with an HP 5890 gas chromatograph (Agilent Technologies Co. Ltd., Palo Alto, USA) with a quartz capillary column (30 mm × 0.32 mm × 0.25 μm): H2 flame ionization detector, column temp 180–280 °C at 3 °C·min−1, carrier gas N2 (1 mL·min−1), injector and detector temp 250 °C, split ratio 1 : 50. Peaks of the hydrolysate were detected by comparison with retention times of authentic samples of D-glucose, after treatment with trimethyl-chlorsilan (TMCS) in pyridine. The absolute configurations of the sugar residues were determined to be D-glucose (tR = 19.01 min). Assay for DPPH scavenging activity The DPPH assay was determined the radical scavenging activity of the samples [25]. L-Ascorbic acid was used as a positive control. About 0.4 mL of methanolic samples at different concentrations were added to 2 mL of the DPPH methanolic solution (0.1 mol·L−1). Shake incubated at 37 °C for 30 min in the dark. Then the absorbance was measured at 517 nm. The IC50 values were obtained through extrapolation from linear required to scavenge 50% of the DPPH radicals. DPPH scavenging activity (%) = [A0 – (A2 – A1)]/A0 × 100, A0: The absorbance of the blank; A1: The absorbance of the contrast sample; A2: The absorbance of the sample at different concentrations. Identification of compound 1 Compound 1: white amorphous powder; []18D −35.4 º (c 0.04, MeOH); UV λmax (log ε) = 204 (4.45), 290 (4.87) nm; IR (KBr): νmax = 3 439, 2 928, 1 633, 1 515, 1 458, 1 412,

1 281, 1 167, 1 075, 1 010; 1H (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectral data (Tabel 1); negative ESIMS m/z 565 [M – H]–; positive-ion HR-EI-MS m/z 566.199 3 [M]+ (Cald. for C27H34O13, 566.199 9).

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Cite this article as: CHENG Gui-Guang, ZHAO Hai-Yun, LIU Lu, ZHAO Yun-Li, SONG Chang-Wei, GU Ji, SUN Wei-Bang, LIU Ya-Ping, LUO Xiao-Dong. Non-alkaloid constituents of Vinca major [J]. Chinese Journal of Natural Medicines, 2016, 14(1): 56-60.

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