Two new phenolic acids from the fruits of Forsythia suspense

Article abstract of DOI:10.1080/10286020.2016.1205589

Two new phenolic acids forsythiayanoside C (1) and forsythiayanoside D (2), were isolated from the fruits of Forsythia suspense (Thunb.) Vahl. Their structures were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses,

Full text of DOI:10.1080/10286020.2016.1205589

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Two new phenolic acids from the fruits of
Forsythia suspense
Xin-Jia Yan, Jing Wen, Zheng Xiang, Dong Cai, Chong-Ning Lv, Ying Zhao,
Zhong-Yuan Qu, Yu-Jia Liu & Jia-Lin Qu
To cite this article: Xin-Jia Yan, Jing Wen, Zheng Xiang, Dong Cai, Chong-Ning Lv, Ying
Zhao, Zhong-Yuan Qu, Yu-Jia Liu & Jia-Lin Qu (2016): Two new phenolic acids from
the fruits of Forsythia suspense, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2016.1205589
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Published online: 20 Jul 2016.
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Date: 02 August 2016, At: 00:03

Journal of asian natural Products research, 2016
http://dx.doi.org/10.1080/10286020.2016.1205589
Two new phenolic acids from the fruits of Forsythia suspense
Xin-Jia Yana,b,c, Jing Wena,b,c, Zheng Xianga, Dong Caic, Chong-Ning Lvb,c, Ying Zhaoa,
Zhong-Yuan Qua, Yu-Jia Liua and Jia-Lin Qub,c
b
acollege of Pharmacy, harbin university of commerce, harbin 150076, china; school of traditional chinese
Materia Medica, shenyang Pharmaceutical university, shenyang 110016, china; cKey laboratory of structure-
Based drug design & discovery (Ministry of education), shenyang Pharmaceutical university, shenyang
110016, china
ABSTRACT
ARTICLE HISTORY
received 17 March 2016
accepted 21 June 2016
Two new phenolic acids forsythiayanoside C (1) and forsythiayanoside
D (2), were isolated from the fruits of Forsythia suspense (Thunb.)
Vahl. Their structures were elucidated on the basis of chemical and
spectral analysis, including 1D, 2D NMR analyses, HRESIMS and CD
spectrometry. The cytotoxic and antioxidant activities testing showed
that compound 2 exhibited free radical scavenging activity.
KEYWORDS
oleaceae; Forsythia suspense;
phenolic acid; cytotoxic
activity; antioxidant activity
1. Introduction
Forsythia suspense (unb.) Vahl, a member of the family Oleaceae, is widely distributed
in China including Henan, Shanxi, and Shandong Provinces. Its fruits are from one of the
most important original plants of Traditional Chinese Medicine which have been used
for antibacterial, antiviral, anti-inflammation, diuretic and antidotal purposes in oriental
medicine [1]. e literature survey revealed that many kinds of compounds have been
isolated from this plant, such as caffeoyl glycosides, phenylethanoid glycosides, lignans,
terpenoids, alkaloids and cyclohexylethanes [2–4]. In this paper, we describe the isolation
and the structural elucidation of two new phenolic acids obtained from 50% EtOH extract
of F. suspensa. eir structures (Figure 1) were established by extensive spectroscopic data
analysis and comparison with literature values. Furthermore, the cytotoxic and antioxidant
activities of the isolated compounds were evaluated.
2. Results and discussion
Compound 1 was obtained as a sticky oil (MeOH), with the molecular formula C15H22O8
as determined by the HRESIMS at m/z 353.1226 [M + Na]+, indicating the presence of five
degrees of unsaturation. e 1H-NMR spectrum of compound 1 (Table 1) displayed three
aromatic proton signals at δH 6.74 (1H, d, J = 3.0 Hz, H-6), 6.60 (1H, dd, J = 8.7, 3.0 Hz,
H-4), and 6.68 (1H, d, J = 8.7 Hz, H-3), which were assigned to one 1,3,4-trisubstituted
CONTACT Xin-Jia Yan
yanxinjia@hrbcu.edu.cn
© 2016 informa uK limited, trading as taylor & francis Group

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X.-J. YaN ET aL.
Figure 1. structures of compounds 1 and 2.
Table 1. 1h-nMr (600 Mhz) and 13c-nMr (150 Mhz) spectral data of compounds 1 and 2.
1a
2b
No.
δH
δC
δH
δC
1
127.5
3.98 (d, 8.0)
62.9
3.47–3.49 (m)
4.35–4.37 (m)
4.57 (d, 5.1)
2
3
4
5
6
7
8
150.7
116.3
113.6
154.4
117.4
32.1
75.6
84.3
6.68 (d, 8.7)
6.60 (dd, 8.7, 3.0)
6.74 (d, 3.0)
2.89–2.91 (m)
3.75–3.79 (m)
4.03–4.07 (m)
4.32 (d, 7.7)
3.50–3.52 (m)
3.41–3.42 (m)
3.30–3.32 (m)
3.36–3.37 (m)
3.70–3.72 (m)
3.85–3.87 (m)
3.70 (s)
70.7
1′
2′
3′
4′
5′
6′
104.4
75.2
78.1
71.6
78.0
62.7
130.8
111.8
147.8
146.4
115.5
120.4
6.81 (brs)
6.65 (d, 8.1)
6.73 (d, 8.1)
–och3
–och3
56.5
3.11 (s)
3.74 (s)
56.6
56.0
note: coupling constants (J) in hz are given in parentheses; chemical shift values are expressed in ppm.
aMeasured in methanol-d4.
bMeasured in dMso-d6.
benzene ring. In addition, the 1H-NMR spectrum also revealed two methylene protons at
δH 3.75–3.79, 4.03–4.07 (each 1H, m) and δH 2.89–2.91 (2H, m), one methoxyl group at δH
3.70 (3H, s), and a glucopyranosyl anomeric proton at δH 4.32 (1H, d, J = 7.7 Hz). e 13C-
NMR spectrum of 1 (Table 1) showed 15 carbon signals, including six aromatic (δC 127.5,
150.7, 116.3, 113.6, 154.4, and 117.4), a glucose unit (δC 104.4, 75.2, 78.1, 71.6, 78.0, and
62.7), one methoxy carbon at δC 56.5 and two methylenes (δC 32.1 and 70.7).
e HMBC correlation (Figure 2) between CH3O group protons at δH 3.70 (–OCH3)
and C-5 (δC 154.4) indicated that the methoxyl group was located at C-5. e correlations
from H-7 (δH 2.89–2.91) to C-8 (δC 70.7), C-1 (δC 127.5), C-2 (δC 150.7), and C-6 (δC 117.4)
suggested that C-7 was connected with C-1 of the benzene ring. e anomeric proton of
glucose at δH 4.32 (1H, d, J = 7.7 Hz) showed HMBC correlation with the aromatic C-8
(δC 70.7) indicated that C-1 should be connected with C-8. Accordingly, compound 1
was established as 2-(2-hydroxy-5-methoxyphenyl)-ethyl-β-D-glucopyranoside, named
forsythiayanoside C.

JouRNaL of aSIaN NaTuRaL PRoDuCTS RESEaRCH
3
Figure 2. Key hMBc correlations of compound 1.
Compound 2 was isolated as colorless needles (MeOH). HRESIMS gave a quasimolecular
ion peak at m/z 251.0897 [M + Na]+, corresponding to the molecular formula C11H16O5.
1
e H-NMR spectrum (Table 1) showed one ABX system of aromatic protons [δH 6.65
(1H, d, J = 8.1 Hz, H-5′), 6.73 (1H, d, J = 8.1 Hz, H-6′), and 6.81 (1H, brs, H-2′)], indicating
1
the presence of one trisubstituted benzene ring. In addition, the H-NMR spectrum also
revealed one oxymethylene at δH 3.98 (1H, d, J = 8.0 Hz), 3.47–3.49 (1H, m), two methine
protons at δH 4.35–4.37 (1H, m), 4.57 (1H, d, J = 5.1 Hz), two methoxyl groups at δH 3.11
(3H, s, 3-OCH3), 3.74 (3H, s, 3′-OCH3). In the 13C-NMR spectrum (Table 1), apart from six
aromatic signals [δC 111.8, 115.5, 120.4, 130.8, 146.4 and 147.8], two methoxyl signals [δC
56.0 and 56.6], two oxymethines [δC 84.3 and 75.6] and one oxymethylene [δC 62.9] were
observed. Comparison showed that the above spectroscopic data were similar to those of
3-(4-hydroxy-3-methoxyphenyl)-3-methoxypropane-1,2-diol [5]. erefore, the structure
of 2 was determined as shown in Figure 1. According to the literature, a smaller coupling
constant (J2,3 = 5.1 Hz) between H-2 and H-3 confirmed the erythro configuration of 2,
combining the negative Cotton effect at 280 nm [6,7], the absolute configurations C-2 and
C-3 of 2 were determined as 2S,3S. us, the structure of 2 was characterized as (2S,3S)-3-
(4-hydroxy-3-methoxyphenyl)-3-methoxypropane-1,2-diol, named forsythiayanoside D.
Compounds 1 and 2 were evaluated for their cytotoxic activities against breast cancer
(Bcap-37), hepatoma (Hep-3B) and breast cancer (MCF-7) cell lines by MTT Method [8],
using 5-fluorouracil as positive control. However, none of the isolated compounds exhibited
cytotoxicity.
Phenolic acids have been claimed to possess antioxidant activity and could serve as lead
compounds for the development of antioxidant agents [9]. e antioxidant effects of the
two isolates were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Compound
1 (IC50 = 98.61 μg/ml) and compound 2（IC50 = 45.12 μg/ml) were found to be less active
than the positive control trolox (IC50 8.29 μg/ml) in the DPPH assay.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a JASCOP-1020 Polarimeter (Jasco Co., Tokyo, Japan).
UV spectra were carried out on a Shimadzu UV-1700 Spectrophotometer (Shimadzu
Corporation, Kyoto, Japan). IR spectra were obtained on a Shimadzu FTIR-8400s spectro-
photometer (Shimadzu Corporation, Kyoto, Japan). NMR spectra were recorded on Bruker
ARX-600 instruments (Bruker Co., Billerica, MA, USA). HR-ESI-TOF-MS experiments were
performed on a Micro TOF spectrometer (Bruker Co., Karlsruhe, Germany). CD spectra

4
X.-J. YaN ET aL.
were measured by MOS 450 detector (Bio-Logic Co., Claix, France). High performance
liquid chromatography (HPLC) preparation was performed on a Hitachi preparative HPLC
system (Hitachi Ltd, Tokyo, Japan) equipped with Refractive Index Detector (L-2490) and
prep-ODS column (10 × 250 mm). GC was done on an Agilent 7890A Gas Chromatograph
(Agilent technologies, Inc., Santa Clara, CA, USA) equipped with HP-5 capillary column
(30 m × 320 mm × 0.25 μm). Sephadex LH-20 (20–100 μm, Pharmacia Fine Chemical
Co., Ltd, NJ, USA), silica gel (200–300 mesh, Qingdao Marine Chemistry Ltd, Qingdao,
China), macroporous resin (D101, Cangzhou Bon Adsorber Technology Co., Ltd, Cangzhou,
China) and Cosmosil octadecyl silane (ODS) (40–80 μm, Nacalai Tosoh, Inc., Uetikon,
Switzerland) were used for column chromatography (CC). TLC was conducted on silica
gel GF254 (Qingdao Marine Chemistry Ltd).
3.2. Plant material
e fruits (8.3 kg) of F. Suspense (unb.) Vahl were collected from Lushi County, Henan
province of China in June 2009, and authenticated by Prof Jin-Cai Lu, Department of
Pharmacognosy, Shenyang Pharmaceutical University.A voucher specimen (No. 20091011)
is kept in the Nature Products Laboratory of Shenyang Pharmaceutical University, Shenyang,
China.
3.3. Extraction and isolation
e fruits of F. suspense (8.3 kg) were crushed to pieces and refluxed with 50% EtOH for
three times. e extract was evaporated under reduced pressure to afford a residue (350 g).
e residue was suspended in H2O and then successively extracted with CHCl3, EtOAc and
n-butanol. e n-butanol extract was evaporated in vacuo to give a residue (100 g), which was
chromatographed over D101 macroporous resin with a gradient of aqueous EtOH (water,
30, 60 and 95%, v/v) to yield four major fractions (A-D). Fraction A (25.0 g) was subjected to
silica gel column chromatography (12 × 60 cm) with a gradient mixture of CH2Cl2-MeOH
(100:0–40:60) to afford five fractions (1–5). Fraction 1 (12.4 g) was further purified over an
ODS column chromatography (4 × 45 cm) using MeOH and H2O as the mobile phase with
a gradient from 10 to 40% to afford fractions F2-1–F2-8 based on HPLC analysis. F2-1 (9.5 g)
was subjected to another silica gel column chromatography (2 × 30 cm) and eluted with
CH2Cl2:MeOH:water (8:2:0.25) to afford fractions F2-1-1–F2-1-5 based on TLC analysis. F2-1-2
(200 mg) was subjected to preparative HPLC eluted with CH3OH-H2O (10:90) at 3 ml/min
(tR 15 and 17 min), to yield compounds 1 (5 mg) and 2 (36 mg).
3.3.1. Forsythiayanoside C (1)
Sticky oil (MeOH),[ꢀ]2D5 − 10.1(c 0.05, MeOH). UV (MeOH) λmax: 278.6, 227.4 and 207.0 nm;
IR (KBr) vmax (cm−1): 3416, 1574, 1414, 1384, 1270, 1127 and 1074; 1H and 13C-NMR spectral
data (MeOD-d4), see Table 1. HRESIMS: m/z 353.1206 [M + Na]+ (calcd for C15H22O8Na,
353.1207).
3.3.2. Forsythiayanoside D (2)
Colorless needles (MeOH),[ꢀ]2D5 + 19.2(c 0.10, MeOH). UV (MeOH) λmax: 280.2, 229.0 and
208.4 nm; IR (KBr) vmax (cm−1): 3439, 1631, 1466, 1384, 1271, 1123, and 1032; CD (MeOH) :

JouRNaL of aSIaN NaTuRaL PRoDuCTS RESEaRCH
5
Δε212.4 nm −4.12, Δε236.0 nm −5.53, Δε278.6 nm −8.12; 1H and 13C-NMR spectral data (DMSO-d6),
see Table 1; HRESIMS: m/z 251.0897 [M + Na]+ (calcd for C11H16O5Na, 251.0890).
3.4. Acid hydrolysis of compound 1
Compound 1 (3.0 mg) was hydrolyzed with 2 M HCl (5.0 ml), heated for 4 h at 95 °C and
extracted with CHCl3 (3 × 5.0 ml). en the aqueous layer was concentrated in vacuo to
appropriate volume, and the solution was examined by TLC (EtOAc-BuOH-H2O-HOAc,
4:4:1:1), and compared with the authentic sample, glucose was detected. Each remaining
aqueous layer was concentrated to dryness to give a residue, which was dissolved in pyridine
(1.0 ml), and then L-cysteine methyl ester hydrochloride (2.0 mg) was added to the solution.
e mixture was heated at 60 °C for 2 h, and 0.5 ml TMSI (N-Trimethylsilyimidazole) was
added, followed by heating at 60 °C for 2 h. e reaction product was subjected to GC analy-
sis on Agilent 7890A (HP-5, 30 m × 320 mm, 0.25 μm) with flame ionization detector (FID)
detection. Column temperature was kept at 120–280 °C with the rate of 8 °C/min, and the
carrier gas was N2 (1.4 ml/min), injection temperature: 250 °C; injection volume: 1 μl. e
absolute configuration of the monosaccharide was confirmed to be D-Glu by comparison of
the retention time of its Me3Si ethers with standard sample [tR (D-glucose) = 25.873 min].
3.5. Cytotoxicity assay
Breast cancer (Bcap-37), hepatoma (Hep-3B) and breast cancer (MCF-7) cells were obtained
from the National Center for Medical Culture Collection (Shanghai, CHN). ey were
routinely cultured in Roswell Park Memorial Institute (RPMI) 1640 or DMEM (Dulbecco’s
Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and maintained
at 37 °C in a humidified incubator with 5% CO2. e in vitro cell viability effects of com-
pounds were determined by MTT assay. e cells (1 × 105 cells/ml) were seeded into 96-well
culture plates.Afer overnight incubation, the cells were treated with various concentrations
of agents for 72 h. en 10 μl MTT solution (2.5 mg/ml in PBS) was added to each well, and
the plates were incubated for an additional 4 h at 37 °C.Afer centrifugation (200 g, 10 min),
the medium with MTT was aspirated, followed by the addition of 100 μl DMSO. e optical
density of each well was measured at 492 nm with a Biotek Synergy TM HT Reader.
3.6. Antioxidant assay
e DPPH radical scavenging activity of the samples was measured using a published
method with some modifications. e samples in different concentrations of EtOH (100 μl)
were added to 0.2 mM DPPH in EtOH (150 μl). Each mixture was shaken vigorously and
then immediately incubated in darkness for 1 h. e absorbance of the reaction solution was
determined in a varioskan flash instrument (ermo scientific, USA) at 515 nm. Trolox, a
stable antioxidant, was used as a positive reference. e DPPH radical scavenging activity
in terms of the percentage of sample was calculated as follows: DPPH scavenging activity
(%) = [1 − (S − SB)/(C − CB)] × 100%.Where S, SB, C and CB is the absorption of the sample,
the blank sample, the control, and the blank control, respectively. Tests were performed
in triplicate.

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X.-J. YaN ET aL.
Acknowledgments
Authors thank Mrs. Ying Peng, Mrs. Wen Li and Mr. Yi Sha of Shenyang Pharmaceutical University
for their support in recording of HRESIMS and NMR spectra.
Disclosure statement
No potential conflict of interest was reported by the authors.
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