Antioxidative Flavan-3-ol Dimers from the Leaves of Camellia

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Anti-oxidative Flavan-3-ol Dimers from the Leaves of Camellia fangchengensis Xiu-Hua Meng, Chang Liu, Rong Fan, Li-Fang Zhu, Shi-Xiong Yang, Hong-Tao Zhu, Dong Wang, Chong-Ren Yang, and Ying-Jun Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04572 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Journal of Agricultural and Food Chemistry

Anti-oxidative Flavan-3-ol Dimers from the Leaves of Camellia fangchengensis

Xiu-Hua Meng,†,‡ Chang Liu,† Rong Fan,†,┴ Li-Fang Zhu,† Shi-Xiong Yang,§ Hong-Tao Zhu,† Dong Wang,† Chong-Ren Yang,† and Ying-Jun Zhang*,†

†

State Key Laboratory of Phytochemistry and Plant Resources of West China, Kunming Institute

of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China ‡ §

University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China Key Lab of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of

Science, Kunming 650201, People's Republic of China ┴

Kunming Medical University Haiyuan College, Kunming 650106, People's Republic of China

_______________________________ * Corresponding author. Tel: + 86 871 6522 3235; E-mail address: [email protected] 1

ACS Paragon Plus Environment


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ABSTRACT

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Camellia fangchengensis Liang et Zhong, belonging to the genus Camellia sect. Thea

3

(Theaceae), is an endemic tea species to the south and southwest areas of Guangxi

4

province, P. R. China. Known as a wild tea plant, the leaves have been used for

5

producing green tea or black tea by the local people of its growing area. HPLC and

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LC-MS analysis showed the leaves contain oligomeric catechins as major phenolic

7

components. Further detailed phytochemical study led to the identification of five

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flavan-3-ol dimers (1 − 5) including two new ones, fangchengbisflavans A (1) and B

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(2) from the leaves of C. fangchengensis, together with six known monomers (6 − 11)

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and one glucoside (12), in addition to gallic acid (13). Their structures were

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determined by extensive spectroscopic analysis. Most of the isolates displayed

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significant antioxidant activities on DPPH and ABTS+ radical scavenging assays. The

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results suggested that the leaves of C. fangchengensis, rich in flavan-3-ol oligomers

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and monomers as potent antioxidants, could be a valuable plant resource for the

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production of tea and natural beverage.

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KEYWORDS: Camellia fangchengensis; Theaceae, fangchengbisflavans A and B;

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flavan-3-ol dimers; antioxidant activity; HPLC; LC-MS

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INTRODUCTION

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Tea is one of the most popular and healthy beverages consumed in the world, due to

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its high content in polyphenols with a broad bioactivity spectrum, e.g., antimicrobial,

21

anti-mutagenesis, anti-oxidative, anti-tumor, and so on.1-3 It is generally manufactured

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from the fresh leaves of two widely cultivated tea plants, Camellia sinensis L. O.

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Kuntze and C. sinensis var. assamica (Masters) Kitamura (Theaceae), both belonging

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to the genus Camellia section Thea. Among which, another 11 wild tea species and

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five varieties are also included according to Min's taxonomic system.4 Most of them,

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known as wild tea plants, have been used for making tea and consumed by the local

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people of its growing areas.

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Camellia fangchengensis Liang et Zhong, a small tree about 3-5 m high with dark

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green, thinly leathery and large leaves, is endemic to the south and southwest areas of

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Guangxi province, China. From where, the type specimen was collected in Fangcheng

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county. Our previous study suggested that wild tea species from the genus Camellia

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section Thea contained flavan-3-ols, e.g., epicatechin (EC), catechin (C), epicatechin-

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3-O-gallate (ECG), gallocatechin (GC), and epigallocatechin-3-O-gallate (EGCG),5,6

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which are similar to those of the two widely cultivated tea plants, C. sinensis and C.

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sinensis var. assamica.7,8 A new flavan-3-ol dimer, talienbisflavan A, was also

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identified from C. taliensis, a wild tea plant from the east side of Ai-Lao mountain,

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Yunnan province, China.9 As a continuing study on species from the genus Camellia

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section Thea, the chemical constituents of the leaves of C. fangchengensis was

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studied. HPLC and LC-MS analyses showed the leaves contain oligomeric

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procyanidins as major phenolic components. Further detailed phytochemical study led

41

to the identification of 12 flavan-3-ol analogues. All the isolates were evaluated by

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1,1-diphenyl-2-picrylhydrazyl (DPPH) and ABTS+ radical scavenging assays for their

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antioxidant activities.

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MATERIALS AND METHODS

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General Procedure. HPLC analysis was operated on a Waters 2695 separation

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module combined with the accessory of the Waters 2996 photodiode array detector

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(Waters, USA) and Millennium ®32 software (Waters). HPLC-DAD-MS analysis was

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performed on a Agilent series 1200 (Agilent Technologies) liquid chromatography,

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equipped with a 1260 diode array detector (DAD)，and an ion-trap mass spectrometer

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(Bruker HCT, Germany) with electrospray interface (ESI), operating in full scan MS

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mode from 100 to 1500 amu. Optical rotations were measured with a Horiba

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SEPA-300 high-sensitive polarimeter. IR spectra were measured on a Bio-Rad

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FTS-135 series spectrometer with KBr pellets. UV spectra were recorded on a UV

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210A Shimadzu spectrometer. 1D and 2D NMR spectra were recorded in CD3OD

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with Bruker AM-400, DRX-500 and DRX-600 spectrometers operating at 400, 500

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and 600 MHz for 1H, and at 100, 125 and 150 MHz for 13C, respectively. Coupling

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constants are expressed in Hertz, and chemical shifts are given on a δ (ppm) scale

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with TMS as an internal standard. ESIMS were recorded on a VG Auto Spec-300

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spectrometer. HRESIMS were recorded on an API QSTAR Pular-1 spectrometer.

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DPPH and ABTS+ radical scavenging assays were performed on an Emax precision

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microplate reader. Column chromatography (CC) was performed on 25 - 100 µm

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Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden), 75 - 100 µm

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MCI-gel CHP20P (Mitsubishi Chemical Co. Ltd., Tokyo, Japan), and 37 - 70 µm

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Toyopearl HW-40F (Tosoh Co. Ltd., Tokyo, Japan). Thin-layer chromatography was

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performed on precoated 0.2 - 0.25 mm thick silica gel H plates (Qingdao Haiyang

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Chemical Co., Qingdao, China), with benzene / ethyl formate / formic acid (1:7:1,

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2:7:1, 3:6:1, v/v/v) or chloroform/methanol/water (7:3:0.5 or 8:2:0.2, v/v/v), and spots

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were detected by spraying with 2% ethanolic FeCl3 or anisaldehyde-H2SO4 reagent

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followed by heating.

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Chemicals and Reagents. Authentic samples including GC, EGC, C, EC, EGCG,

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GCG and ECG used for high-performance liquid chromatography (HPLC) analysis

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were purchased from Shunbo Biotech Limited Company (Shanghai, China).

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Acetonitrile and methanol (chromatographic grade) were purchased from Merck

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(Darmstadt, FR, Germany). Water was purified in a Milli-Q (Millipore, America).

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1,1-Diphenyl-2-picryl-hydrazyl (DPPH), trolox, ABTS, and potassium persulfate

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were purchased from Sigma-Aldrich Chemicals (Steinheim, Germany), and ascorbic

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acid was obtained from Xinxing Chemical Industrial Reagent Institute (Shanghai,

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China).

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Materials. The leaves of C. fangchengensis Liang et Zhong collected at the

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Botanical Garden, Guangxi Institute of Botany (GIB), Chinese Academy of Sciences

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(CAS), Guilin city, Guangxi province, P. R. China, in April of 2012. The materials

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were identified by Prof. Dian-Peng Li from GIB, CAS. A voucher specimen

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(KIB-ZL-20120406) was deposited in State Key Laboratory of Phytochemistry and

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Plant Resources of West China, Kunming Institute of Botany (KIB), CAS.

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HPLC and LC-MS analysis. The fine powder (1.0 g) of the air-dried leaves of C.

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fangchengensis was saturated in 70% aqueous methanol (100 mL) at room

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temperature for 12 hours, during which ultrasonic bathes were carried out three times,

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each time 15 min. The extract was filtered with a 0.45 µm nylon membrane filter

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before injected into HPLC and LC-DAD-MS analysis.

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HPLC analysis (Fig. 1A) was performed at 30 oC using an Agilent Zorbax SB-C18

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column (4.6 × 250 mm, 5 µm). The gradient of mobile phase was acetonitrile in 0.1%

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formic acid aqueous solution as follows: 4 to 10% in 10 min, remained 10% for 6 min,

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then 10 to 20% from 16 to 25 min, 20 to 30% from 25 to 44 min, 30 to 50% from 44

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to 50 min, and finally to reach 100% in 53 min, at a flow rate of 1.0 mL/min. The

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injection volume was 20 µL, and UV detection was carried out between 210 and 400

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nm.

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MS analysis (Fig. 1B) was performed with electrospray interface (ESI), operating

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in full scan MS mode from 100 to 1500 amu. Sample was analyzed by both negative

99

and positive ionization modes. ESI-MS parameters were as follows: potential of the

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ESI source, 4 kV; capillary temperature, 400 oC.

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Extraction and Isolation. The air-dried leaves (700 g) of C. fangchengensis were

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soaked with 70% aqueous methanol at room temperature for three times (2 L × 3,

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each time one week). The filtrates were combined and concentrated to a small volume

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(500 mL) under reduced pressure and then partitioned with petroleum ether, EtOAc

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and n-BuOH, consecutively, to give petroleum ether fraction (1 g), EtOAc fraction

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(26.5 g), n-BuOH fraction (30 g) and water fraction (50 g). TLC detection

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demonstrated that phenolic compounds were contained in the EtOAc and n-BuOH

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fractions, on which further investigation was focused, particularly the oligomers.

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The EtOAc fraction (26.5 g) was subjected to Sephadex LH-20 column

110

chromatography (CC), eluting with water-methanol (1:0-0:1), to give four fractions,

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Fr. E1 - Fr.E4. Fractions E2 (7.9 g) and E3 (4.5 g) were separately applied to

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repeated CC over MCI-gel CHP20P, Sephadex LH-20, and Toyopearl HW-40F,

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eluting with water-methanol (1:0−0:1), to give compounds 1 (10 mg), 2 (80 mg), 4

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(400 mg), and 5 (600 mg) from Fr. E2, and 3 (120 mg), 6 (100 mg), 7 (10 mg), 8

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(100 mg), 9 (100 mg), 10 (60 mg), 11 (10 mg) from Fr. E3.

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The n-BuOH fraction (30 g) were chromatographed over Sephadex LH-20, eluting

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with water-methanol (1:0-0:1), to afford five fractions (Fr. B1 - Fr. B5). Repeated 6


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CC over MCI-gel CHP20P, Sephadex LH-20, and Toyopearl HW-40F, eluted with

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water-methanol (1:0-0:1) resulted the isolation of compounds 4 (100 mg), 5 (100

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mg), 12 (5 mg) and 13 (8 mg) from Fr. B3 (10.0 g), and 9 (190 mg) from Fr. B4 (8.0

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g), respectively.

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Fangchengbisflavan A (1). Brownish yellow amorphous powder; [α]D25 −96.0 (c

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0.1, methanol). ESI-MS m/z 591 [M−H]−; HRESI-MS m/z 591.1494 [M−H]− (calcd

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for C31H27O12, 591.1503). IR (KBr): νmax 3451, 2922, 1630, 1452, 1284, 1115 cm-1.

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UV λmax (methanol) (log ε): 281 (3.20), 204 (4.28) nm. 1H (600 MHz, CD3OD): δH

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3.73 (2H, s, CH2), upper unit: 4.72 (1H, br s, H-2), 4.10 (1H, dd, J = 4.6, 3.4 Hz, H-3),

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2.78 (1H, dd, J = 16.8, 4.6 Hz, H-4a), 2.67 (1H, dd, J = 16.8, 3.4 Hz, H-4b), 6.03 (1H,

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s, H-8), 6.90 (1H, d, J = 2.0 Hz, H-2'), 6.70 (1H, d, J = 8.2 Hz, H-5'), 6.73 (1H, dd, J

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= 8.2, 2.0 Hz, H-6'), lower unit: 4.98 (1H, br s, H-2), 4.17 (1H, dd, J = 4.6, 3.2 Hz,

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H-3), 2.88 (1H, dd, J = 16.8, 4.6 Hz, H-4a), 2.77 (1H, dd, J = 16.8, 3.2 Hz, H-4b),

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6.04 (1H, s, H-6), 7.06 (1H, d, J = 2.1 Hz, H-2'), 6.79 (1H, d, J = 8.2 Hz, H-5'), 6.88

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(1H, dd, J = 8.2, 2.1 Hz, H-6'). 13C NMR (150 MHz, CD3OD): δC 17.4 (CH2), upper

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unit: 79.8 (C-2), 67.5 (C-3), 29.6 (C-4), 156.2 (C-5), 106.6 (C-6), 154.7 (C-7), 97.1

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(C-8), 155.3 (C-9), 101.3 (C-10), 132.2 (C-1'), 115.2 (C-2'), 146.1 (C-3'), 145.7 (C-4'),

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115.8 (C-5'), 119.4 (C-6'), lower unit: 81.4 (C-2), 67.0 (C-3), 29.3 (C-4), 155.3 (C-5),

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97.1 (C-6), 154.2 (C-7), 108.0 (C-8), 153.1 (C-9), 100.8 (C-10), 131.1 (C-1'), 115.6

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(C-2'), 146.3 (C-3'), 145.9 (C-4'), 116.0 (C-5'), 119.9 (C-6').

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Fangchengbisflavan B (2). Brownish yellow amorphous powder; [α]D20 −331.6 (c

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0.14, methanol). ESI-MS m/z 767 [M+Na]+; HRESI-MS m/z 767.1574 [M+Na]+,

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calcd for C38H32O16, 767.1588). IR (KBr): νmax 3444, 1687, 1619, 1522, 1453, 1368,

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1284, 1228, 1146, 1093, 1034 cm-1. UV λmax (methanol) (log ε): 294 (3.63), 213 (4.40)

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nm.

1

H (400 MHz, CD3OD): upper unit: 4.36 (1H, d, J = 9.2 Hz, H-2), 4.57 (1H, m, 7



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H-3), 4.52 (1H, br d, J = 8.3 Hz, H-4), 6.00 (1H, s, H-6), 5.93 (1H, s, H-8), 6.99 (1H,

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s, H-2'), 6.63 (1H, d, J = 8.4 Hz, H-5'), 6.45 (1H, d, J = 8.4 Hz, H-6'), lower unit: 5.03

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(1H, s, H-2), 5.37 (1H, m, H-3), 2.87 (2H, br d, J = 17.6 Hz, H-4), 6.12 (1H, s, H-6),

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6.68 (1H, s, H-2'), 6.63 (1H, d, J = 9.3 Hz, H-5'), 6.41 (1H, d, J = 9.3 Hz, H-6'),

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3-O-(3''-O-methyl)-gallate: 6.88 (1H, s, H-2''), 6.99 (1H, s, H-6''), 3.81 (3H, s,

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3''-OMe).

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(C-4), 157.3 (C-5), 97.6 (C-6), 157.1 (C-7), 97.1 (C-8), 158.4 (C-9), 107.2 (C-10),

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132.5 (C-1'), 116.3 (C-2'), 146.1 (C-3'), 145.5 (C-4'), 116.0 (C-5'), 120.5 (C-6'), lower

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unit: 78.4 (C-2), 70.5 (C-3), 26.9 (C-4), 156.2 (C-5), 96.4 (C-6), 156.2 (C-7), 107.9

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(C-8), 157.1 (C-9), 100.6 (C-10), 131.1 (C-1'), 114.6 (C-2'), 145.8 (C-3'), 145.6 (C-4'),

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116.0 (C-5'), 120.3 (C-6'), 3-O-(3''-O-methyl)-gallate: 121.4 (C-1''), 106.3 (C-2''),

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148.9 (C-3''), 140.5 (C-4''), 145.8 (C-5''), 112.1 (C-6''), 167.8 (C-7''), 57.1 (3''-OMe).

13

C NMR (100 MHz, CD3OD): upper unit: 83.9 (C-2), 73.8 (C-3), 38.7

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DPPH Radical Scavenging Activity. The DPPH assay was performed as

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previously described in our previous paper,5,6 and ascorbic acid was used as positive

157

control. Reaction mixtures containing an ethanolic solution of 200 µM DPPH (100 µL)

158

and 2-fold serial dilutions of sample (dissolved in 100 µL of ethanol, with amounts of

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sample ranging from 2 to 1000 µg/mL) were placed in a 96 well microplate and

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incubated at 37 °C for 30 min. After incubation, the absorbance was read at 517 nm

161

and the scavenging activity was determined by following equation: percentage of

162

DPPH reduction (%) = [Acontrol - Asample]/Acontrol × 100. The SC50 value was obtained

163

through extrapolation from linear regression analysis and denoted the concentration of

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sample required to scavenge 50% of DPPH radicals. The data presented are means ±

165

SD of three determinations.

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ABTS+ Radical Scavenging Activity. ABTS+ assay was performed as reported

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previously,9,10 and trolox was used as positive control. Briefly, the ABTS radical

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cation (ABTS+) solution was prepared by reaction of ABTS (7 mM) and potassium

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persulfate (2.45 mM) after incubation at room temperature in the dark for 16 h. The

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ABTS+ solution was then diluted with 80% ethanol to obtain an absorbance of 0.700

171

± 0.002 at 734 nm. The ABTS+ solution (200 µL) was added to a 96-well microplate

172

containing 10 µL of each sample and incubated at room temperature for 6−8 min, and

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the absorbance at 405 nm was immediately recorded. The scavenging activity was

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determined according to the following equation: percentage of ABTS+ reduction (%)

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= [Acontrol - Asample] / Acontrol × 100. The SC50 value was obtained through extrapolation

176

from linear regression analysis and denoted the concentration of sample required to

177

scavenge 50% of ABTS+ radicals. The data presented are means ± SD of three

178

determinations.

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RESULTS AND DISCUSSION

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HPLC and LC-MS analysis. The 70% aqueous methanol extract of the leaves of C.

181

fangchengensis was applied to HPLC (Figure 1A) and LC-DAD-MS (Figure 1B)

182

analyses. Twelve catechin derivatives, including five monomers, five dimers and two

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trimers were identified by LC-DAD-MS analysis, on the basis of their retention time

184

(tR), UV absorption, the quasi-molecular and fragment ions, and co-HPLC

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comparison with standards, as well as the previous reference data, and the result was

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shown in Table 1. Most of the identified compounds were obtained from the leaves

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of C. fangchengensis, during the further isolation.

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Peaks 1, 4, 7, 12 and 14 in Figure 1B were identified as catechin monomers. Peak

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1 was supposed to be GC (11) by the negatively charged quasi-molecular ions at m/z

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305 [M-H]- and 611 [2M-H]-, and confirmed by comparing the tR (9.0 min) and UV

191

absorptions (λmax 225, 271 nm) with the standard sample. In the same way, peak 4

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(tR16.5 min; λmax 227, 278 nm), peaks 7 (tR 22.8 min; λmax 227, 278 nm) and peak 12

9



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(tR 30.1 min; λmax 225, 277 nm) were identified as C (10), EC (6) and ECG (7),

194

respectively. Peak 14 (tR 34.5 min; m/z 455 [M-H]-, 911 [2M-H]-, 479 [M+Na]+, 935

195

[2M+Na]+) was identified as epicatechin 3-O-(3"-O-methyl)-gallate (9). These

196

monomers were obtained during the further isolation and purification.

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Peaks 2, 3, 5, 9, and 11 in Figure 1B were supposed to be procyanidin dimers. Due

198

to the quasi-molecular ions (m/z 577 [M-H]-, 579 [M+H]+, 601 [M+Na]+, 617

199

[M+K]+), their tR and MS/MS data (Table 1), peaks 2, 3, and 5 were identified to be

200

procyanidins B1, B3 (4) and B2 (5), resp., also based on the comparison with

201

standards and the published data.11,12,13 Peak 9 (tR 25.4 min; m/z 729 [M-H]-, 731

202

[M+H]+, 753 [M+Na]+, 769 [M+K]+) was identified to be a procyanidin dimer

203

monogallate (DP2G, DP: degree of polymerisation). Peak 11 (tR 29.3 min; m/z 743

204

[M-H]- , 745 [M+H]+, 763 [M+H+H2O]+, 767 [M+Na]+, 783 [M+K]+), whose

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molecular weight was 14 Da more than that of peak 9, was supposed to be a

206

methylate of DP2G, and confirmed to be fangchengbiflavan B (2) by comparing

207

directly with the standard.

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Similarly, peak 6 was supposed to be a procyanidin trimer, due to its molecular

209

weight and the main fragmentation of MS/MS, while peaks 8 was also a procyanidin

210

trimer, or could be a dimer composing of afzelechin and catechin with two galloyl

211

groups, e.g., afzelechin(4-8)catechin or afzelechin(4-6)catechin 3,3'-di-O-gallate,

212

due to the molecular weight (Table 1).

213

The structures of procyanidin oligomers (B-type) and their gallate with C4-C8 or

214

C4-C6 linkages varied depending on the C-2 and C-3 configurations of their

215

composing units, catechin or epicatechin. The MSn data were useful for their

216

structural determination14,15,16. A procyanidin dimer monogallate (DP2G) loses its

217

galloyl group firstly, to form the corresponding non-galloyl procyanidin dimers

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(DP2), or loses a catechin or epicatechin unit to form the lower procyanidin unit.

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Usually, it has two main fragmentation paths for procyanidins, dehydration and

220

Retro-Diels-Alder (RDA) reaction. Take DP2G (m/z 579) for an example, a fragment

221

ion at m/z 579 ([M-152]+) was firstly generated due to the loss of galloyl group, and

222

then the first path leads to an RDA fission providing an ion at m/z 427 ([579-152]+),

223

which was followed by a dehydration reaction, providing an ion at m/z 409

224

([427-18]+). The second path leads to an dehydration firstly, generating an ion at m/z

225

561 ([579-18]+), and then an RDA reaction product at m/z 409 [427-18]+. On basis of

226

these fragmentation paths, MS/MS can successful confirm the structure of these

227

procyanidins (Table 1).

228

Isolation and Characterization of Flavan-3-ol Dimers. The EtOAc and n-BuOH

229

fractions from the leaves of C. fangchengensis with flavan-3-ol oligomers were

230

subjected to repeated CC over Sephadex LH-20, MCI-gel CHP20P, and Toyopearl

231

HW-40F to yield five flavan-3-ol dimers (1 − 5), two of which are new compounds,

232

namely fangchengbisflavans A (1) and B (2). Meanwhile, seven known monomers (6

233

− 12) were also obtained, together with gallic acid (13)17. The known dimers and

234

monomers were determined to be bis(8-epicatechinyl)-methane (3),18 procyanidins B3

235

(4)19,20 and B2 (5),20 (-)-EC (6),21 (-)-ECG (7),22 (-)-EGCG (8),22 (-)-EC-3-O-(3-O-

236

methyl)-gallate (9),23 (+)-C (10),21 and (±)-GC (11),7 and catechin-7-O-β-D-gluco-

237

pyranoside (12),24 respectively, by comparison of their physical and spectroscopic

238

data with reference values and authentic samples (Figure 3).

239

Compound 1 was obtained as brownish amorphous powder. Its molecular formula

240

was deduced to be C31H28O12, on the basis of the negative HRESIMS (m/z 591.1494

241

[M − H]−, calcd for 591.1503). The IR spectrum showed the presence of hydroxy

242

group (3451 cm−1) and benzene rings (1630 and 1452 cm−1) in 1. The

11


13

C NMR


243

spectrum showed the occurrence of 31 carbon signals, classified as seven aliphatic

244

carbons including three methylenes (δC 17.4, 29.6, 29.3) and four oxymethines (δC

245

81.4, 79.8, 67.5, 67.0), and 24 aromatic carbons referring to eight methines (δC 97.1

246

(x 2), 115.2, 115.6, 115.8, 116.0, 119.9, 119.4) and 16 quaternary carbons, by further

247

HSQC and DEPT experiments. The 1H NMR spectrum displayed characteristic

248

signals for two flavan-3-ol C-rings [upper unit: δH 4.72 (1H, br s, H-2), 4.10 (1H, dd,

249

J = 4.6, 3.4 Hz, H-3), 2.78 (1H, dd, J = 16.8, 4.6 Hz, H-4a), 2.67 (1H, dd, J = 16.8,

250

3.4 Hz, H-4b), lower unit: δH 4.98 (1H, br s, H-2), 4.17 (1H, dd, J = 4.6, 3.2 Hz, H-3),

251

2.88 (1H, dd, J = 16.8, 4.6 Hz, H-4a), 2.77 (2H, dd, J = 16.8, 3.5 Hz, H-4b)], and two

252

1,3,4-tri- substituted B-rings [upper unit: δH 6.90 (1H, d, J = 2.0 Hz, H-2'), 6.70 (1H,

253

d, J = 8.2 Hz, H-5'), 6.73 (1H, dd, J = 8.2, 2.0 Hz, H-6'), lower unit: δH 7.06 (1H, d, J

254

= 2.1 Hz, H-2'), 6.79 (1H, d, J = 8.2 Hz, H-5'), 6.88 (1H, dd, J = 8.2, 2.1 Hz, H-6')].

255

The above data suggested that 1 should comprise of two catechin units. Due to the

256

coupling constants of H-3/H-4 (J3,4a = 4.1 Hz, J3,4b = 4.1 Hz for (-)-epicatechin, and

257

J3,4a = 5.5 Hz, J3,4b = 8.4 Hz for (+)-catechin),25 the two flavan-3-ol units in 1 (J3,4a =

258

4.6 Hz / J3,4b = 3.5 Hz) were concluded to be both as epicatechin. However, instead of

259

the two A-ring aromatic methines (CH-6, CH-8) in epicatechin, the NMR data of 1

260

display only two aromatic methines [δC 97.1 (x 2)] for two epicatechin A-rings and

261

two additional quaternary aromatic carbons at δC 108.0 and 106.6, as well as an

262

additional benzylic methylene [δH 3.73 (s), δC 17.4 (CH2)]. These data indicated that

263

the two epicatechin units in 1 are connected between their C-6 and C-8 positions

264

through a methylene bridge. The position of connection was further determined by 2D

265

NMR experiments.

266

In the HMBC spectrum (Figure 4), correlations from H-4 (δH 2.78, 2.67) to C-3 (δC

267

67.5), C-2 (δC 79.8), C-10 (δC 101.3), C-9 (δC 155.3) and C-5 (δC 156.2), and from

12


Page 12 of 29

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268

H-2 (δH 4.72) to C-9 (δC 155.3), C-1' (δC 132.2), C-2' (δC 115.2), C-6' (δC 119.4), and

269

C-4 (δC 29.6) confirmed that those signals belong to one (upper) of the epicatechin

270

units in 1. Signals from another epicatechin unit showing HMBC correlations from

271

H-4 (δH 2.88, 2.77) to C-3 (δC 67.0), C-2 (δC 81.4), C-10 (δC 100.8), C-9 (δC 153.1)

272

and C-5 (δC 155.3), from H-2 (δC 4.98) to C-9 (δC 153.1), C-1' (δC 131.1), C-2' (δC

273

115.6), C-6' (δC 119.9) and C-4 (δC 29.3), clearly assigned the signals of the lower

274

unit in 1. Moreover, HMBC correlations of the benzylic methylene at δH 3.73 (s) with

275

the upper C-5 (δC 156.2) and C-7 (δC 154.7), and the lower C-9 (δC 153.1) and C-7 (δC

276

154.2) revealed that the two epicatechin units in 1 were linked between the upper C-6

277

and the lower C-8 positions through a methylene bridge. Similar (−) signs of the

278

optical rotation of 1 ([α]ଶହ ୈ − 96.0 (c 0.1, methanol)) with that of oolonghomobis-

279

26 flavan A, comprising of two (-)-epigallocatechin ([α]ଶ଺ ୈ − 271.0 (c 1.0, acetone)),

280

further confirmed the two (-)-epicatechin units in 1. Therefore, compound 1 was

281

elucidated as bis-6,8-(-)-epicatechinylmethane, and named fangchengbisflavan A.

282

Compound 2, obtained as brownish amorphous powder, had a molecular formula of

283

C38H32O16, as deduced from the negative HRESIMS (m/z 767.1574 [M + Na]+). The

284

IR spectrum showed the presence of hydroxy group (3445 cm−1), ester carbonyl (1687

285

cm−1) and benzene rings (1619 and 1454 cm−1). The 13C NMR spectrum of 2 showed

286

the occurrence of 38 signals, classified as seven aliphatic including five methines with

287

four oxygen-bearing ones (δC 38.7, 83.9, 78.4, 73.8, 70.5), one methylene (δC 26.9),

288

and one methoxy (δC 57.1), one carbonyl (δC 167.8), and 30 aromatic carbons,

289

referring to 11 methines, and 19 quaternary carbons, by further HSQC and DEPT

290

experiments. Among which, six aromatic and a carboxyl signals at δC 121.4 (C-1''),

291

106.3 (C-2''), 148.9 (C-3''), 140.5 (C-4''), 145.8 (C-5''), 112.1 (C-6'') and 167.8 (C-7''),

292

together with the methoxy at δC 57.1, could be assigned undoubtedly as an 13



293

unsymmetric 3-O-methylgalloyl group. The left 30 signals referred to two flavan-3-ol

294

units. Of which, δC 83.9 (C-2), 73.8 (C-3) and 38.7 (C-4) were from the upper unit,

295

while δC 78.4 (C-2), 70.5 (C-3) and 26.9 (C-4) were from the lower unit. The 1H

296

NMR spectrum also displayed characteristic signals for the two flavan-3-ol C-rings

297

[upper unit: δH 4.36 (1H, d, J = 9.2 Hz, H-2), 4.57 (1H, m, H-3), 4.52 (1H, br d, J =

298

8.3 Hz, H-4), lower unit: δH 5.03 (1H, br s, H-2), 5.37 (1H, m, H-3), 2.87 (1H, br d, J

299

= 17.6 Hz, H-4)]. The aforementioned NMR data were closely related to those of

300

(+)-catechin and (-)-epicatechin-3-O-gallate, suggesting that 2 was comprised of these

301

two units.

302

However, instead of an aliphatic methylene at δC 28.7 (C-4) in catechin and an

303

aromatic methine at δC 96.4 (C-8) in epicatechin-3-O-gallate, the NMR data of 2

304

display an aliphatic methine at δC 38.7 and an aromatic quaternary carbon at δC 107.9,

305

indicating the C-4 of catechin and C-8 of epicatechin-3-O-gallate in 2 were

306

substituted. These NMR features were similar to those of procyanidin B4-3'-O-

307

gallate,23,27 except for the appearance of an additional methoxy group [δH 3.81 (s, 3H),

308

δC 57.1] in 2. The down field shift of the galloyl C-3'' at δC 148.9 in 2 from δC 145.6

309

in ECG and procyanidin B4-3'-O-gallate,22,27 together with the unsymmetric galloyl

310

signals in 2, indicated the methoxyl group was located on the galloyl C-3''. The

311

positions of substitutions were further confirmed by 2D NMR experiments.

312

In the HMBC spectrum of 2 (Figure 4), correlations from the methine proton at δH

313

4.52 (H-4) to C-3 (δC 73.8), C-2 (δC 83.9), C-10 (δC 107.2), C-9 (δC 158.4) and C-5

314

(δC 157.3), and H-2 (δH 4.36) to C-9 (δC 158.4), C-1' (δC 132.5), C-2' (δC 116.3), C-6'

315

(δC 120.5) and C-4 (δC 38.7) assigned these signals to the upper catechin unit in 2.

316

While, the HMBC correlations from the methylene protons at δH 2.87 (H-4) to C-3 (δC

317

70.5), C-2 (δC 78.4), C-10 (δC 100.6), C-9 (δC 157.1) and C-5 (δC 156.2), and from

14


Page 14 of 29

Page 15 of 29


318

H-2 (δH 5.03) to C-9 (δC 157.1), C-1' (δC 131.1), C-2' (δC 114.6), C-6' (δC 120.3), and

319

C-4 (δC 26.9), clearly assigned the signals of the lower unit as epicatechin in 2;

320

Moreover, according to the HMBC correlations from the upper H-2 (δH 4.36) to C-9

321

(δC 158.4), and H-6 (δH 6.00) to C-5 (δC 157.3) and C-7 (δC 157.0), and from the

322

lower H-2 (δH 5.03) to C-9 (δC 157.1), and H-6 (δH 6.12) to C-5 (δC 156.2) and C-7

323

(δC 156.2), the upper and lower C-5, C-7 and C-9 could be assigned, respectively. The

324

HMBC correlations of the lower H-3 (δH 5.37) and the methoxy proton at δH 3.81 with

325

the galloyl C-7'' (δC 167.8) and C-3'' (δC 148.9), respectively, revealed the

326

3"-O-methoxylgallate was located at C-3 of the lower unit. The optical rotation of 2

327

was [α]ଶହ ୈ − 331.3 (c 1.4, methanol). On the basis of the above evidences, compound

328

2 was deduced to be procyanidin B4-3'-O-(3''-O-methyl)gallate, and named as

329

fangchengbisflavan B.

330

In the 1H NMR spectrum of 2, the upper unit displayed two sets of H-2 (δH 4.36,

331

4.46), H-3 (δH 4.30, 4.57) and H-4 (δH 4.52, 4.69), which means it may exist

332

conformational isomerism caused by a steric interaction (restricted rotation ) between

333

the upper and lower units. This sort of isomerism is known to be observable only in

334

the cases of procyanidins B-3 and B-4, where a catechin unit (C2,C3: trans) is located

335

in the upper half and is bonded to the lower unit with α-configuration.27,28,29

336

Free Radical Scavenging Activities of Compounds 1-13. All the isolates (1 − 13)

337

were evaluated by DPPH and ABTS+ radical scavenging assays, and the results are

338

shown in Table 2. All of them, except 12, exhibited significant DPPH and ABTS+

339

radical scavenging activities, which were stronger than or comparable to those of the

340

positive controls (ascorbic acid and trolox).

341

All the flavan-3-ol dimers 1 – 5, including the new fangchengbisflavans A (1) and

342

B (2), monomers (7-9) with galloyl group at C-3, and gallic acid (13) showed stronger

15



343

DPPH radical scavenging activities than positive control (ascorbic acid) and the other

344

flavan-3-ols. Their activity order is 8 > 2 > 3 > 4 > 13 > 9 > 5 > 1 > ascorbic acid >

345

11 > 6 > 10 > 12. Though the 3-O-galloyl group was monomethylated, compound 9

346

displayed stronger activity than 6 without 3-O-galloyl group, suggesting that the

347

monomethylation of the galloyl group did not affect the DPPH radical scavenging

348

activity. On the other hand, compounds 6, 10 and 11 without 3-O-galloyl group, but

349

with a catechol- or pyrogallol-B-ring, showed less DPPH radical scavenging activities.

350

It is suggested that the 3-O-galloyl group attribute the most important role to the

351

DPPH radical scavenging activity.

352

On the ABTS+ radical scavenging assay, all the isolates, except 12, exhibited

353

stronger activities than positive control, ascorbic acid and trolox, with an activity

354

order of 4 > 3 > 2 > 8 > 5 > 7 > 13 > 11 > 9 > 1 > 6 >10 > trolox > ascorbic acid > 12.

355

The flavan-3-ol dimers 1-5 showed stronger activities than their monomer,

356

epicatechin (6). Moreover, catechins with 3-O-galloyl group (7 and 8) displayed

357

stronger activities than the ones (6 and 10) without galloyl unit. When the phenyl

358

hydroxy on galloyl group was monomethylated (9), the ABTS+ radical scavenging

359

activity was decreased. Same as that on DPPH radical scavenging assay, glycosylation

360

(12) on C-7 of catechin (10) led its (12) ABTS+ radical scavenging activity decreased.

361

The above result was consistent with the previously reported data5-7,30.

362

In conclusion, five flavan-3-ol dimers (1 - 5) including two new ones,

363

fangchengbisflavans A (1) and B (2), were identified from C. fangchengensis,

364

together with seven known monomers (6 - 11) and glucoside (12), in addition to a

365

simple phenolic compound, gallic acid (13). All of the isolates were reported from the

366

titled plant for the first time. Fangchengbisflavan A (1) and bis(8-epicatechinyl)-

367

methane (3) were epicatechin dimers connecting by C-8/C-6 (1) and C-8/C-8 (3) of

16


Page 16 of 29

Page 17 of 29


368

two units through a methylene group, fangchengbisflavan B (2) was a dimer of

369

catechin and epicatechin-3-O-gallate by C-4/C-8 linkage. The known dimer 3,

370

reported previously from the pericarps of Litchi chinensis was isolated from tea plant

371

for the first time. Same to the widely cultivated tea plant, C. sinensis var. assamica,

372

six flavan-3-ol monomers were obtained from C. fangchengensis. The flavan-3-ol

373

dimers 1-5, exhibited stronger antioxidant activities on both DPPH and ABTS+ assays,

374

were found to be the characteristic phenolic constituents in C. fangchengensis. They

375

may play important role for the health beneficial effects of tea produced from C.

376

fangchengensis, together with the flavan-3-ols monomers.30 The results suggested that

377

C. fangchengensis, as one member of the genus Camellia section Thea, with enriched

378

flavan-3-ol oligomers and monomers as potent antioxidants, could be a valuable plant

379

resource for the production of tea and natural beverage.

380

ASSOCIATED CONTENT

381

Supporting Information

382

1

383

available free of charge via the Internet at http://pubs.acs.org.

384

AUTHOR INFORMATION

385

Corresponding Author

386

* Tel: + 86 871 6522 3235. E-mail: [email protected].

387

ORCID

388

Ying-Jun Zhang: orcid.org/0000-0002-0295-337X

389

Funding

390

This work was supported by the National Natural Science Foundation of China (No.

391

31470429).

392

Notes

H, 13C, HSQC, and HMBC spectra of compounds 1 and 2 in CD3OD. This material is

17



393

The authors declare no competing financial interest.

394

ACKNOWLEDGMENTS

395

We are grateful to the staffs of the analytical group at State Key Laboratory of

396

Phytochemistry & Plant Resources in West China, Kunming Institute of Botany,

397

Chinese Academy of Sciences, for measuring the spectroscopic data.

398

REFERENCE

399

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400

Mitsunaga, T.; Hashimoto, F.; Kiso, Y., Inhibitory effects of oolong tea

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(10) Zhu, F.; Cai, Y. Z.; Sun, M.; Ke, J.; Lu, D.; Corke, H., Comparison of major

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(11) Rigaud, J.; Escribano-Bailon, M.T.; Prieur, C.; Souquet, J.M.; Cheynier, V.

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of

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apple musts and ciders. Journal of Chromatography A, 1996, 727 (2), 203-209.

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tannins of grape berries by liquid chromatography/electrospray ionization

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419-426.

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HPLC with electrospray ionization mass spectrometry. Eur Food Res Technol.

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444

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G. Isolation and structural elucidation of some proanthocyanidins from apple by

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3806~3813.

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of procyanidin dimers in water and hydro-alcoholic media as viewed by NMR

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(21) Hashimoto, F.; Nonaka, G.; Nishioka, I. Tannins and related compounds. CXIV.

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Structure of novel fermentation products, theogallinin, theaflavonin, and

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desgalloyl theaflavonin from black tea, and changes of tea leaf polyphenols

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during fermentation. Chem. Pharm. Bull. 1992, 40, 1383-1389.

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(22) Lee, M. W.; Morimoto, S.; Nonaka, G. I.; Nishioka, I. Flavan-3-ol gallates and

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proanthocyanidins from Pithecellobium lobatum. Phytochemistry 1992, 31,

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(23) Iijima, T.; Mohri, Y.; Hattori, Y.; Makabe, H. Synthesis of (-)-epicatechin 3-(3-

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O-methylgallate) and (+)-catechin 3-(3-O-methylgallate) and their anti-

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inflammatory activity. Chem. Biodivers. 2009, 6, 520-526.

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(24) Cui, E.-J.; Song, N.-Y.; Shrestha, S.; Chung, I.-S.; Kim, J.-Y.; Jeong, T.-S.; Baek,

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N.-I. Flavonoid glycosides from cowpea seeds (Vigna sinensis K.) inhibit LDL

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oxidation. Food Sci. Biotechnol. 2012, 21 (2), 619-624.

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(25) Seto, R.; Nakamura, H.; Nanjo, F.; Hara, Y., Preparation of epimers of tea catechins by heat treatment. Biosci. Biotechnol. Biochem. 1997, 61, 1434-1439.

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475

8-C-ascorbyl (-)-epigallocatechin 3-O-gallate and novel dimeric flavan-3-ols,

476

oolonghomobisflavans A and B, from oolong tea (3). Chem. Pharm. Bull. 1989,

477

37, 3255-3263.

478

(27) Nonaka, G. I.; Kawahara, O.; Nishioka, I. Tannins and related compounds. XV.

479

A new class of dimeric flavan-3-ol gallate, theasinensins A and B, and

480

proanthocyanidin gallates from green tea leaf. Chem. Pharm. Bull. 1983, 31(11),

481

3906-3914.

482

(28) Fletcher, A. C.; Poter, L. J.; Haslam, E.; Gupta, R. K. Plant proanthocyanidins.

483

Part III. Conformational and configurational studies of natural procyanidins. J.

484

Chem. Soc., Perkin Trans. 1, 1977, 1628-1637.

485

(29) Thompson, R. S.; Jacques, D.; Haslam, E.; Tanner, R. J. N. Plant

486

proanthocyanidins. Part I. Introduction, the isolation, structure, and distribution

487

in nature of plant proanthocyanidins. J. Chem. Soc., Perkin Trans. 1, 1972,

488

1387-1399.

489

(30) Nakajima, N.; Sakuda, H.; Saito, A.; Mizushina, Y.; Yoshida, H.; Tanaka, A.,

490

Synthesis of galloyl-substituted procyanidin B4 series, and their DPPH radical

491

scavenging activity and DNA polymerase inhibitory activity. Heterocycles 2006,

492

67, 175-189.

21



Figure captions 1.

Figure 1. HPLC (A) and LC-MS total ion (B) chromatograms of the 70% aqueous methanolic extract of the leaves of C. fangchengensis

2.

Figure 2. Two main fragmentation paths of procyanidins and their monogallates.

3.

Figure 3. Compounds 1-13 isolated from C. fangchengensis.

4.

Figure 4. Key HMBC correlations (H → C) of 1 and 2.

22


Page 22 of 29

Page 23 of 29


1.60

A

1.40

10

1.20

6 trimer*

procyanidin B1

AU

1.00 0.80

11

0.60

4

0.40

DP2G 7

trimer

9

2

5

8

20.00

25.00

3

1

0.20 0.00 0.00

5.00

10.00

15.00

30.00

35.00

40.00

45.00

Minutes

B

Figure 1. HPLC (A, numbers of compounds) and LC-MS total ion (B, numbers of peaks) chromatograms of the 70% aqueous methanolic extract of the leaves of C. fangchengensis (* or a dimer composing of afzelechin and catechin with two galloyls)

23



OH

H+

H+

OH HO

OH OH

HO

OH

OR1

HO

OH HO

O

OH

-H2O

OH OH HO

O

O

OH

OH

OH

OH

galloyl

DP2G [M+H]+ m/z 731

OH

H+

OH

RDA

OH

O

HO

OH -H2O

O

+

OH OH

OH HO

H H

OH m/z 409

O

OH

OH OH HO

O

H+

OH

OH HO

m/z 561 RDA

H+

OH

OH O

OH

OH DP2 [M+H]+ m/z 579

OH

R1 = galloyl, R2 = H R1 = H, R2 = galloyl

OH HO

O OH

OH

OH

- galloyl

O

OH

O

OH

OR2

HO

H+

OH

O

OH HO

Page 24 of 29

O

+

H

OH

OH m/z 427

OH

m/z 409

Figure 2. Two main fragmentation paths of procyanidins and their monogallates

24


H OH

Page 25 of 29


Figure 3. Compounds 1-13 isolated from C. fangchengensis. 25



OH O

HO

OH OH

OH O

HO

OH

OH OH

Page 26 of 29

OH HO

O

OH

upper unit OH OH HO

OH

O

OH OCH3

lower unit

O OH

OH

OH

O

1

2

Figure 4. Key HMBC correlations (H → C) of 1 and 2.

26


OH

Page 27 of 29


Table 1. Retention time (tR, min), ESI-MS (m/z), MS/MS (m/z) and the identified chemical compositions in the leaves of C. fangchengensis Peaks tR 1 9.0

Negative ESI-MS Positive ESI-MS 305 [M-H]NMS 611 [2M-H]

2

3

4

14.3

14.8

16.5

Compounds GC

-

577 [M-H]-

577 [M-H]

MS/MS

-

-

579 [M+H]+ 601 [M+Na]+

427, 409, 291 449, 431, 311

617 [M+K]+

465, 447, 327

+

579 [M+H] 601 [M+Na]+

427, 409, 291 449, 431, 311

617 [M+K]+

465, 447, 327

+

289 [M-H] 579 [2M-H]-

291 [M+H] 313 [M+Na]+

Procyanidin B1

Procyanidin B3

C

603 [2M+Na]+ 5

19.9

6

22.3

577 [M-H]-

579 [M+H]+ 601 [M+Na]+ 617 [M+K]+

427, 409, 291 449, 431, 311 465, 447, 327

Procyanidin B2

867 [M+H]+ 889 [M+Na]+

715, 427, 577, 409 737, 601

Procyanidin trimer

905 [M+K]+ 866 [M]7

22.8

695, 577, 543, 525, 407 -

+

289 [M-H] 579 [2M-H]-

291 [M+H] 603 [2M+Na]+

EC

Procyanidin trimer*

334 [M+HCOO-]8 9

23.2

865 [M-H]-

867 [M+H]+

25.4

-

+

729 [M-H]

731 [M+H] 753 [M+Na]+

579, 561, 427, 409 583, 465, 449, 431, 413

DP2G

769 [M+K]+

10 11

28.3 29.3

543 743 [M-H]

571 -

NI +

745 [M+H] 593, 575, 427, 409 763 [M+H+H2O]+ 427, 409 767 [M+Na]+ 783 [M+K]

12

30.1

441 [M-H]883 [2M-H]-

+

Fangchengbisflavan B

479, 449, 431, 413 768 ([M+K-CH3]+), 750

443 [M+H]+

ECG

13

30.8

543

571

NI

14

34.5

455 [M-H]911 [2M-H]-

479 [M+Na]+ 935 [2M+Na]+

EC-3-O-(3”-OMe)G

15

40.4

586 [M]871

587 [M+H]+ 1019

NI

1018 NI: not identified;

NMS: no mass signal;

*: or a dimer composing of afzelechin and catechin with two galloyls

27



Table 2. Antioxidant Activity of Compounds 1-13 from C. fangchengensis. SC50 (µM)a Sample Fangchengbiflavan A (1) Fangchengbiflavan B (2) bis(8-epicatechinyl)methane (3) Procyandin B3 (4) Procyandin B2 (5) (-)-Epicatechin (6) (-)-epicatechin gallate (7) (-)-epigallocatechin gallate (8) (-)-epicatechin3-( 3-O-methylgallate ) (9) (+)-Catechin (10) Gallocatechin (11) Catechin-7-O-β-glucopyransoide (12) Gallic acid (13) Ascorbic acidc Trolox c

ABTSb 109.3 ± 4.9 64.9 ± 5.9 59.3 ± 6.1 57.9 ± 5.5 98.4 ± 3.0 123.7 ± 4.8 102.9 ± 8.6 85.0 ± 1.4 107.4 ± 2.1 136.5 ± 3.7 105.7 ± 7.5 272.1 ± 14 104.1 ± 9.3 243.8 ± 8.1 197.9 ± 7.0

a

DPPHb 32.0 ± 0.5 18.9 ± 0.5 20.7 ± 0.9 24.6 ± 2.5 31.9 ± 2.0 46.6 ± 2.7 26.4 ± 2.0 15.0 ± 0.3 29.9 ± 1.7 55.7 ± 4.9 41.6 ± 1.4 188 ± 10 29.5 ± 5.3 36.0 ± 1.5

Values represent means ± SD (n = 3). bSC50 = concentration in µM required to scavenge 50% of DPPH and ABTS+ radical. cPositive control.

28


Page 28 of 29

Page 29 of 29


Graphic for table of contents Anti-oxidative Flavan-3-ol Dimers from the Leaves of Camellia fangchengensis Xiu-Hua Meng, Chang Liu, Rong Fan, Li-Fang Zhu, Shi-Xiong Yang, Hong-Tao Zhu, Dong Wang, Chong-Ren Yang, and Ying-Jun Zhang OH HO H 2C

HO

O

OH

O

O

OH OH

OH

HO

OH OH

O

2

0.60 0.40

O HO

1.00 0.80

O

OH

1

1.40 1.20

OH HO HO

OH OH

1.60

OH

AU

OH

OH

0.20 0.00

OH OCH3

0.00

5.00

10.00

15.00

20.00

25.00

Minutes

1


30.00

35.00

40.00

45.00

Antioxidative Flavan-3-ol Dimers from the Leaves of Camellia

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