Four New Compounds Isolated from the Aerial Part of Belamcanda chinensis (L.) and Their Effect on Vascular Smooth Muscle Cell (VSMC) Proliferation

Abstract

Bio-guided fractionation of the 70% ethanol extract of Belamcanda chinensis (L.) DC. revealed four new compounds, including 6″-O-acetylembinin (5), 3″-O-acetylembinin (6), irigenin 3′-O-β-glucopyranoside (8), and 2′-acetyl-1,3-O-diferuloylsucrose (9), along with five known compounds (1–4, 7). Their chemical structures were determined using extensive NMR data, mass spectroscopy, and comparison with published literature. Among the isolates, compounds 1 and 4–7 achieved good regulation of the growth and proliferation of vascular smooth muscle cells.

Introduction

Belamcanda chinensis (L.) DC. (BC) is a popular ornamental and medicinal plant in the Iridaceae family. The dried rhizomes of BC are used in Vietnamese traditional medicine for treatment of inflammation and respiratory disorders, such as asthma, coughing, tonsillitis, and pharyngitis.¹⁾ Previous phytochemical studies have identified isoflavones and isoflavone aglycones as the main components of this plant. Other chemical groups found in this plant include benzoquinones, phenolics, iridal-type triterpenoids, and steroids.²⁾ The pharmacological effects of the BC rhizomes have been demonstrated by modern biological assays. BC rhizome properties include anti-inflammatory,³⁾ anti-tumor,^4,5) antioxidant, hepatoprotection,^6,7) estrogen receptor modulation,^8,9) and aldose reductase inhibition.¹⁰⁾ Recent studies have reported the hypoglycemic effect of BC leaf extract in normal and streptozotocin (STZ)-induced diabetic rats,^11,12) due to the presence of isoflavones.¹³⁾ In this study, we focused on the phytochemical extraction, isolation, and identification of new compounds from the aerial part of BC and evaluated effects on vascular smooth muscle cell (VSMC) proliferation.

Results and Discussion

The 70% ethanol extract from the aerial parts of BC was suspended in distilled water and then successively partitioned with n-hexane and EtOAc. The total extract and all the fractions, including n-hexane and EtOAc and the remaining water layer, were screened for their effects on the proliferation of VSMCs. Since the EtOAc-soluble fraction showed the most potent inhibitory activity on VSMC proliferation at the concentration of 20 µg/mL, this fraction was used to isolate compounds using repeated silica gel, reversed phase column chromatography and preparative (p)-HPLC purifications. The isolation process revealed four new compounds (5, 6, 8, and 9) and five known compounds (1–4, 7).

Compound 5 was obtained as a yellow amorphous solid. The maximum UV absorptions at 216, 273, and 329 nm suggested that this compound was a flavone.¹⁴⁾ The IR spectrum showed absorption bands for hydroxyl (3360 cm⁻¹), conjugated ketone (1649 cm⁻¹), conjugated olefin (1606 cm⁻¹), and aromatic moiety (1489 cm⁻¹). The molecular formula was determined by high-resolution electrospray ionization (HR-ESI)-MS at m/z 647.1995 [M−H]⁻ (Calcd for C₃₁H₃₅O₁₅, 647.1976). The ¹H-NMR data (Table 1) of 5 showed the signals of two AA′BB′ systems of 4′-substituted B-rings at δ_H 8.10 (or 8.09) (2H, d, J = 9.0 Hz, H-2′ and H-6′) and 7.13 (2H, d, J = 9.0 Hz, H-3′ and H-5′); a pair of singlet signals for H-3 at δ_H 6.97 and 6.95; a pair of singlet signals for H-8 at δ_H 6.88 and 6.89; a singlet signal for chelated 5-OH at δ_H 13.43; a pair of singlet signals for 7-OCH₃ at δ_H 3.91 and 3.90; and a singlet signal for 4′-OCH₃ at δ_H 3.87. The signals of anomeric protons at δ_H 4.69 (or 4.70) (1H, d, J = 10.0 Hz, H-1″) and 5.07 (or 4.98) (1H, d, J = 1.0 Hz, H-1‴) together with heteronuclear multiple bond correlations (HMBCs) of the proton at δ_H 5.07 (4.98) with the carbon signal at δ_C 74.3 (75.8) indicated the existence of the disaccharide moiety in 5. The chemical shifts of the anomeric carbons at δ_C 71.1 (or 70.8) and 100.3 (or 100.8) indicated C- and O-glycones, respectively. The doublet methyl signal at δ_H 0.47 (or 0.59) (3H, d, J = 6.0 Hz, H-6‴) in the ¹H-NMR revealed a rhamnopyranosyl unit. The α-rhamnopyranosyl unit was assigned by the small coupling constant (1.0 Hz) of H-1‴. The other sugar was determined to be a β-glucopyranosyl unit based on the J value (10.0 Hz) of H-1″. In the HMBC spectra, the correlations of H-1″ with C-5, C-6, and C-7 indicated that the β-glucopyranosyl moiety was attached at the C-6 position via a C–C bond, while the correlation of H-1‴ with C-2″ indicated that the connection between α-rhamnopyranosyl and β-glucopyranosyl was a C(1‴)-O-C(2″) linkage. The spectroscopic data of 5 were similar to those of embinin (4),¹⁵⁾ except for an additional proton chemical shift of an acetoxy group at δ_H 1.99 (3H, s) and ¹³C chemical shifts at δ_C 20.7 and 170.4. The obvious downfield shift of H-6″ (δ_H 4.39 and 3.86) in 5 compared with that of 4 (δ_H 3.73 and 3.36), as well as the HMBC correlation of H-6″ with the carboxy carbon of an acetoxy group, confirmed the position of the additional acetoxy group at C-6″. Based on the above analysis, the structure of 5 was determined to be 5-hydroxyl-7,4′-dimethoxy-6-[2-O-(α-rhamnopyranosyl)-6-O-acetyl-glucopyranosyl]flavone and was named 6″-O-acetylembinin (Fig. 1).

Table 1. NMR Data of Compounds 5 and 6 (DMSO-d₆)

No.	5								6
	Major				Minor				Major				Minor
	δCa)	δHb) (J in Hz)			δCa)	δHb) (J in Hz)			δCa)	δHb) (J in Hz)			δCa)	δHb) (J in Hz)
2	163.3				163.3				163.6				163.6
3	103.6	6.97	s		103.8	6.95	s		103.7	6.87	s		103.9	6.87	s
4	182.3				181.9				182.5				182.0
5	160.3				159.2				160.6				159.6
6	109.6				109.4				108.9				108.8
7	164.8				163.3				165.1				163.6
8	90.5	6.88	s		91.5	6.89	s		90.6	6.92	s		91.6	6.90	s
9	156.9				157.1				157.2				157.4
10	104.9				104.2				105.0				104.3
5-OH		13.43	s			13.43	s			13.50	s			13.48	s
7-OMe	56.6	3.91	s		56.3	3.90	s		56.8	3.91	s		56.5	3.90	s
1′	122.5				122.6				122.6				122.6
2′, 6′	128.4	8.10	d	(9.0)	128.4	8.09	d	(9.0)	125.5	8.06	d	(9.0)	125.8	8.04	d	(9.0)
3′, 5′	114.7	7.13	d	(9.0)	114.7	7.13	d	(9.0)	114.8	7.10	d	(9.0)	114.8	7.10	d	(9.0)
4′	162.5				162.5				162.7				162.6
4′-OMe	55.6	3.87	s		55.6	3.87	s		55.7	3.85	s		55.7	3.85	s
6-C-Glc
1″	71.1	4.69	d	(10.0)	70.8	4.70	d	(10.0)	71.3	4.79	d	(10.0)	71.1	4.80	d	(10.0)
2″	74.3	4.32	t	(10.0)	75.8	4.16	t	(10.0)	74.1	4.50	t	(9.5)	75.9	4.33	t	(9.5)
3″	77.8	3.37	m		77.8	3.37	m		80.3	4.97	t	(9.0)	80.1	4.97	t	(9.0)
4″	70.5	3.59	m		70.5	3.61	m		68.4	3.31	m		68.5	3.31	m
5″	79.5	3.37	m		79.3	3.37	m		81.5	3.31	m		81.5	3.31	m
6″	64.4	4.39	m		64.3	4.39	m		61.4	3.73	m		61.4	3.73	m
		3.86	m			3.86	m			3.43	m			3.43	m
3″-OAc									21.2	2.07	s		21.2	2.08	s
									170.3				170.2
6″-OAc	20.7	1.99	s		20.7	1.99	s
	170.4				170.4
1‴	100.3	5.07	d	(1.0)	100.8	4.98	d	(1.0)	101.2	4.58	br s		101.9	4.53	br s
2‴	70.4	3.06	ddd	(9.0, 6.0, 3.0)	70.4	3.12	ddd	(9.0, 6.0, 3.0)	70.9	3.38	m		71.0	3.38	m
3‴	70.3	3.17	m		70.2	3.17	m		70.3	3.06	dd	(9.5, 3.0)	70.1	3.11	dd	(9.5, 3.0)
4‴	71.5	2.90	td	(9.0, 4.5)	71.6	2.90	td	(9.0, 4.5)	71.6	2.91	m		71.7	2.91
5‴	68.2	2.12	dp	(12.0, 6.0)	68.2	2.24	dp	(12.0, 6.0)	68.9	2.23	m		69.0	2.37	dp	(12.0, 6.0)
6‴	17.5	0.47	d	(6.0)	18.0	0.59	d	(6.0)	17.7	0.46	d	(6.0)	18.0	0.55	d	(6.0)

a) Recorded at 125 MHz. b) Recorded at 500 MHz.

Fig. 1. Chemical Structures of Isolates (1–9) from the Aerial Part of B. chinensis

The ¹H- and ¹³C-NMR signals of 6 (Table 1) shared similarities with those of 4, except for the presence of an additional acetoxy group (δ_H 2.07 [or 2.08], δ_C 21.2 and 170.3 [or 170.2]). The position of this acetoxy group at C-3″ was determined based on the HMBC correlation between the signals of δ_H 4.97 (H-3″) and δ_C 170.3/170.2 and the correlation spectroscopy (COSY) correlations of H-3″ (δ_H 7.97) with both H-2″ (δ_H 4.50/4.33) and H-4″ (δ_H 3.31). Moreover, the chemical shift of H-3″ (δ_H 4.97) in 6 differed from that of 4 (δ_H 3.36), indicating that acetoxy group was connected to C-3″ position. This result was supported by HMBC analysis (Fig. 2). Thus, the chemical structure of 6 was shown to be 3″-O-acetylembinin (Fig. 1).

Fig. 2. Selected COSY (—) and HMBC (→) Correlations of 5, 6, 8, and 9

Duplicate signals shown in the NMR spectra of 5 and 6 were caused by rotational hindrance at the C(6)–C(1″) linkage, which is very common for 6-C-glycosylflavones.¹⁶⁾

Compound 8 was purified as a yellow amorphous solid. The UV spectrum was characteristic of isoflavones showing absorption maxima at 244 and 270 nm.¹⁴⁾ The molecular formula of 8 was determined to be C₂₄H₂₆O₁₃ using HR-ESI-MS at m/z 521.1328 [M−H]⁻ (Calcd for C₂₄H₂₅O₁₃, 521.1295). The ¹H-NMR spectrum of 8 displayed one C-8 proton at δ_H 6.45 (1H, s) in an A-ring, one C-2 proton at δ_H 8.20 (1H, s), three methoxy groups at δ_H 3.89 (3H, s, 5′-OCH₃), 3.87 (3H, s, 4′-OCH₃), 3.88 (3H, s, 6-OCH₃), and an anomeric proton at δ_H 4.98 (1H, d, J = 7.5 Hz, H-1″). Two protons in ring B appeared as two doublets at δ_H 7.03 (1H, d, J = 1.5 Hz, H-2′) and 6.99 (1H, d, J = 1.5 Hz, H-6′). The position of methoxy groups at C-6, C-4′, and C-5′ was identified by the HMBC correlations between the proton signals of methoxy groups at δ_H 3.89, 3.87, 3.88 (3H each, s) and C-5′ (δ_C 154.0), C-4′ (δ_C 140.0), and C-6 (δ_C 133.2), respectively. The existence of a β-glucopyranosyl moiety was suggested by the coupling constant of the anomeric proton and ¹³C-NMR data. The ¹H- and ¹³C-NMR spectra of 8 resembled those of irigenin,¹⁷⁾ except for the presence of a β-glucopyranosyl moiety. The HMBC correlations of the anomeric proton at δ_H 4.98 and H-2′ at δ_H 7.03 with a carbon signal at δ_C 152.1 suggested that the β-glucopyranosyl moiety was placed on C-3′ (Fig. 2). From the results described above, the structure of 8 was determined to be irigenin 3′-O-β-glucopyranoside (Fig. 1).

Compound 9 was isolated as a pale amorphous solid. The UV spectrum exhibited absorption maxima at 264 and 328 nm. The IR spectrum showed absorption bands at 3331 cm⁻¹ (OH), 1651 cm⁻¹ (conjugated CO), and 1604 cm⁻¹ (aromatic C=C). The molecular formula was determined to be C₃₄H₄₀O₁₈ using HR-ESI-MS at m/z 735.2176 [M−H]⁻ (Calcd for C₃₄H₃₉O₁₈, 735.2136). The ¹H-NMR spectrum of 9 showed two sets of signals for the trans olefinic protons at δ_H 7.65 (1H, d, J = 16.0 Hz, H-7″), 6.37 (1H, d, J = 16.0 Hz, H-8″), 7.71 (1H, d, J = 16.0 Hz, H-7‴) and 6.49 (1H, d, J = 16.0 Hz, H-8‴); two sets of 1,3,4-trisubstituted aromatic ring protons at δ_H 7.18 (1H, d, J = 2.0 Hz, H-2″), 6.77 (1H, d, J = 8.0 Hz, H-5″), 7.06 (1H, dd, J = 8.0, 2.0 Hz, H-6″), 7.23 (1H, d, J = 2.0 Hz, H-2‴), 6.82 (1H, d, J = 8.0 Hz, H-5‴), and 7.12 (1H, dd, J = 8.0, 2.0 Hz, H-6‴); and two methoxy groups at δ_H 3.90 (3H, s, 3″-OCH₃) and 3.84 (3H, s, 3‴-OCH₃), which suggested the existence of two feruloyl units in 9. A doublet signal with a small coupling constant at δ_H 5.65 (1H, d, J = 3.5 Hz, H-1′), as well as signals at δ_C 91.2, 74.5 (2C), 72.3, 71.3 and 62.2 indicated the presence of a α-glucopyranosyl moiety in 9.¹⁸⁾ Furthermore, signals at δ_C 66.4, 103.4, 79.8, 73.4, 84.0, and 63.1 suggested that 9 contained a fructofuranosyl moiety. The existence of a sucrose moiety in 9 was revealed due to the HMBC correlation of H-1′ (δ_H 5.65) with C-2 (δ_C 103.4). The positions of two feruloyl groups at C-1 and C-3 of the fructose moiety of sucrose were illustrated by the HMBC correlations of H-1 (δ_H 4.29 and 4.20) with C-9‴ (δ_C 168.3), and H-3 (δ_H 5.51) with C-9″ (δ_H 168.4) (Fig. 2). The spectroscopic data of 9 was similar to that of 1,3-O-diferuloylsucrose,¹⁹⁾ except for the presence of an acetoxy group at δ_H 2.11 (3H, s) and δ_C 172.8 and 21.1, which linked to C-2′ (δ_C 74.5) based on the HMBC cross-peak from H-2′ (δ_H 4.64) to the carboxy carbon of the acetoxy group (δ_C 172.8), in conjunction with the COSY correlations of H-2′ (δ_H 4.64) with both H-1′ (δ_H 5.65) and H-3′ (δ_H 3.86). Based on the above spectroscopic data, 9 was determined to be 2′-acetyl-1,3-O-diferuloylsucrose (Fig. 1).

Based on spectroscopic analysis and comparison with the literature, the structures of known compounds were determined to be isoswertisin (1),²⁰⁾ 2″-O-α-L-rhamnosyl-4′-O-methylisovitexin (2),²¹⁾ 2″-O-rhamnosylswertisin (3),²²⁾ embinin (4),¹⁵⁾ and iridin (7)²³⁾ (Fig. 1).

Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall.²⁴⁾ VSMC proliferation plays an important role in vessel-wall remodeling and is a crucial event in the formation of atherosclerosis. Thus, inhibition of VSMC proliferation can potentially decrease the incidence of vascular occlusive diseases.^25,26)

In a preliminary cell proliferation assay, 70% ethanol extract (BCL 70EtOH), n-hexane (BCL HF), ethyl acetate (BCL EF), and water (BCL WF) fractions were screened for their effects on VSMC proliferation in human VSMC cells. The results showed that the BCL EF significantly inhibited VSMC proliferation at a concentration of 20 µg/mL (Fig. 3A). Eight compounds (1–7 and 9) were obtained from the most active BCL EF fraction and were screened for their effect on VSMC proliferation. Compounds 1 and 4–7 showed considerable inhibitory effects, whereas compounds 2, 3, and 9 demonstrated weak inhibitory effects on VSMC proliferation at a concentration of 30 µM (Fig. 3B). These results provide initial evidence and incentive to further study the aerial part of BC and associated compounds on VSMC proliferation as a treatment for atherosclerosis.

Fig. 3. Effect of Extracted, Fractionated, and Isolated Compounds on VSMC Proliferation

(A) VSMC proliferation with the BC aerial part of 20 µg/mL of 70% ethanol extract (BCL 70EtOH), and its n-hexane (BCL HF), ethyl acetate (BCL EF), and water fractions (BCL WF). (B) VSMC proliferation with 30 µM of the isolated compounds from an ethyl acetate fraction of the aerial part of BC (compounds 1–7, and 9).

Experimental

General Experimental Procedures

UV spectra were recorded in MeOH on a JASCO V-550 UV/Vis spectrometer with a 0.5 nm resolution, and IR spectra were measured in MeOH on Shimadzu FT-IRAffinity-1S and presented in cm⁻¹. NMR spectra were recorded on a Bruker Avance Digital 500 MHz NMR spectrometer (Karlsruhe, Germany) with tetramethylsilane (TMS) as the internal standard, J in Hz at 294 K. HR-ESI-MS data were analyzed via an Agilent 6530 Q-TOF (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.) spectrometer. Silica gel (Merck, 63–200 mm particle size), RP-18 (Merck, 40–63 mm particle size), and MCI gel (75–150 µm) were used for column chromatography. TLC was carried out with silica gel 60F₂₅₄ and RP-18F₂₅₄ plates. Preparative HPLC was performed with a HPLC Water system (Waters, Middleton, U.S.A.): 1525 Binary pump; Water 2998 photodiode array detector; YMC Park ODS column (20 × 250 mm, 4 µm); t_R in min.

Cell Culture

Human vascular smooth muscle cells were obtained from ATT C and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium (Biochrom, FG 4815) supplemented with 10% fetal bovine serum (FBS, Biochrom, S 0115) and 50 U/50 µg/mL penicillin/streptomycin (Biochrom, A 2212) at 37°C and 5% CO₂.

Cell Proliferation

Initially, the cells were seeded into 96-well culture plates at 5 × 10³ cells/well and cultured in DMEM containing 10% FBS at 37°C for 24 h. When cells reached up to 70% confluence, the medium was replaced with serum-free DMEM containing 20 µg/mL of the aerial extract and fractions of BC or 30 µM of the isolated compounds. After 24 h incubation, they were treated with 25 ng/mL PDGF-BB (Upstate Biotechnology, NY, U.S.A.) and further incubated for another 24 h. After the incubation period, add 10 µL of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) labeling reagent and follow protocol from cell proliferation kit (11465007001, Sigma).

Statistical Analysis

Quantitative data are presented as mean ± standard error of the mean (S.E.M.). Data were analyzed using GraphPad Prism Software 5. Statistical analysis was performed by Student’s t-test or one-way ANOVA for multiple comparisons. Differences were considered significant when p < 0.05.

Plant Material

The aerial parts of Belamcanda chinensis were collected in Thanh Hoa province (Vietnam), in July 2013 and botanically identified by Tran The Bach, Ph.D., Department of Herbarium, Institute of Ecology and Biological Resources, Vietnam. A voucher specimen (BUITHIBINH-1) has been deposited at the Herbarium of NIMM, Hanoi, Vietnam.

Extraction and Isolation

The dried leaves of BC (2.0 kg) were extracted and refluxed with 70% ethanol (10 L × 3 h × 3 times) at 70°C. The combined extracts were filtered and evaporated under reduced pressure to yield a residue (500.0 g, BCL 70EtOH), which was suspended in H₂O and successively partitioned with organic solvents, then concentrated to yield 3 fractions of n-hexane (78.0 g, BCL HF), EtOAc (54.0 g, BCL EF), and a water-soluble layer (335.0 g, BCL WF). The EtOAc extract (54.0 g) was fractionated via silica gel column chromatography and eluted with MC–MeOH (from 100 : 0 to 0 : 100, v/v) to yield 12 fractions (1A–1M). Fraction 1G (4.75 g) was separated using MCI gel CC with a solvent mixture of MeOH–H₂O (50–100%, v/v) to yield 7 fractions (2A–2G). Fractions 2D (226.2 mg), 2E (226.2 mg), and 2G (1.12 g) were recrystallized to yield compounds 1 (BCLE-2D1, 161.6 mg), 7 (BCLE-2E1, 45.2 mg), and 5 (BCLE-2G1, 115.3 mg), respectively. Fraction 2F (1.50 g) was chromatographed on MCI gel CC with an elution mixture of MeOH–H₂O (50–100%, v/v) to yield 6 fractions (3A–3F). Fraction 3C (401.6 mg) was further isolated by preparative HPLC with an isocratic mixture of MeOH–H₂O (55%, 5 mL/min, UV 210, 254, and 275 nm) to yield compounds 9 (BCLE-3C2, 8.9 mg, t_R = 21.0 min), 6 (BCLE-3C4, 136.5 mg, t_R = 33.0 min), and a mixture of 3C3 (22.6 mg, t_R = 26.5 min). The mixture of 3C3 was purified via p-HPLC with an isocratic elution of 45% MeOH in 0.5% acetic acid–H₂O (5 mL/min, UV 210, 254, and 275 nm) to isolate compounds 8 (BCLE-3C33, 1.5 mg, t_R = 40 min) and 2 (BCLE-3C34, 10.0 mg, t_R = 48.0 min). Compounds 3 (BCLE-4B, 216.2 mg) and 4 (BCLE-4D, 984.3 mg) were obtained from a fraction of 1K (2.83 g) using MCI gel CC with a gradient elution of MeOH–H₂O (60–100%, v/v).

6″-O-Acetylembinin (5)

Yellow amorphous solid. UV λ_max (MeOH) 329, 273, 216 nm. IR (ν_max): 3360 (OH), 1649 (conjugated CO), 1606 (aromatic C=C), 1489 (aromatic moiety) cm⁻¹. ¹H- and ¹³C-NMR (DMSO-d₆) data, see Table 1. HR-ESI-MS m/z 647.1995 [M−H]⁻ (Calcd for C₃₁H₃₅O₁₅, 647.1976).

3″-O-Acetylembinin (6)

Yellow amorphous solid. UV λ_max (MeOH) 331, 273, 215 nm. IR (ν_max): 3381 (OH), 1649 (conjugated CO), 1602 (aromatic C=C), 1489, 1442 (aromatic moiety) cm⁻¹. ¹H- and ¹³C-NMR (DMSO-d₆) data, see Table 1. HR-ESI-MS m/z 647.1999 [M−H]⁻ (Calcd for C₃₁H₃₅O₁₅, 647.1976).

Irigenin 3′-O-β-D-Glucopyranoside (8)

Yellow amorphous solid. UV λ_max (MeOH) 270, 244 nm. ¹H- and ¹³C-NMR (CD₃OD) data, see Table 2. HR-ESI-MS m/z 521.1328 [M−H]⁻ (Calcd for C₂₄H₂₅O₁₃, 521.1295).

Table 2. NMR Data of Compound 8 (CD₃OD)

No.	δCa)	δHb) (J in Hz)
2	155.9	8.20 (1H, s)
3	123.8
4	182.1
5	155.1
6	133.2
7	159.7
8	95.3	6.45 (1H, s)
9	155.1
10	106.5
1′	128.4
2′	111.7	7.03 (1H, d, 1.5)
3′	152.1
4′	140.0
5′	154.0
6′	109.2	6.99 (1H, d, 1.5)
1″	102.8	4.98 (1H, d, 7.5)
2″	75.0	3.51 (2H, m)
3″	78.1
4″	71.5	3.37 (1H, m)
5″	78.3	3.44 (1H, m)
6″	62.7	3.91 (1H, overlap), 3.67 (1H, dd, 12.0, 6.0)
5′-OCH3	56.7	3.89 (3H, s)
4′-OCH3	61.6	3.87 (3H, s)
6-OCH3	60.9	3.88 (3H, s)

a) Recorded at 125 MHz. b) Recorded at 500 MHz.

2′-O-Acetyl-1,3-O-diferuloylsucrose (9)

Pale amorphous solid. UV λ_max (MeOH) 328, 264 nm. IR (ν_max): 3331 (OH), 1651 (conjugated CO), 1604 (aromatic C=C) cm⁻¹. ¹H- and ¹³C-NMR (CD₃OD) data, see Table 3. HR-ESI-MS m/z 735.2176 [M−H]⁻ (Calcd for C₃₄H₃₉O₁₈, 735.2136).

Table 3. NMR Data of Compound 9 (CD₃OD)

No.	δCa)	δHb) (J in Hz)
1	66.4	4.29 (1H, d, 11.5)
		4.20 (1H, d, 11.5)
2	103.4
3	79.8	5.51 (1H, d, 8.0)
4	73.4	4.40 (1H, t, 8.0)
5	84.0	3.95 (1H, m)
6	63.1	3.83 (2H, m)
1′	91.2	5.65 (1H, d, 3.5)
2′	74.5	4.64 (1H, d, 10.0, 3.5)
3′	72.3	3.86 (1H, m)
4′	71.3	3.49 (1H, m)
5′	74.5	3.95 (1H, m)
6′	62.2	3.89 (1H, m)
		3.77 (1H, dd, 12.0, 5.0)
2′-COCH3	172.8
	21.1	2.11 (3H, s)
1″	127.6
2″	111.6	7.18 (1H, d, 2.0)
3″	149.4
4″	150.9
5″	116.5	6.77 (1H, d, 8.0)
6″	124.4	7.06 (1H, dd, 8.0, 2.0)
7″	147.5	7.65 (1H, d, 16.0)
8″	114.9	6.37 (1H, d, 16.0)
9″	168.4
1‴	127.6
2‴	112.0	7.23 (1H, d, 2.0)
3‴	149.4
4‴	150.8
5‴	116.5	6.82 (1H, d, 8.0)
6‴	124.4	7.12 (1H, dd, 8.0, 2.0)
7‴	148.1	7.71 (1H, d, 16.0)
8‴	114.7	6.49 (1H, d, 16.0)
9‴	168.3
3″-OCH3	56.5	3.90 (3H, s)
3‴-OCH3	56.4	3.84 (3H, s)

a) Recorded at 125 MHz. b) Recorded at 500 MHz.

Acknowledgments

The authors are grateful to the Center of Applied Spectroscopic Methods of Institute of Chemistry—Vietnam Academy of Science and Technology for NMR and MS spectroscopic measurements and Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Korea for HR-MS measurements.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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