Novel Acylated Flavonol Tetraglycoside with Inhibitory Effect on Lipid

Mar 24, 2017 - Tea Natural Product Laboratory of International Joint Laboratory of Tea Chemistry and Health Effects, State Key Laboratory of Tea Plant...
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A Novel Acylated Flavonol Tetraglycoside with Inhibitory Effect on Lipid Accumulation in 3T3-L1 Cells from Lu'an GuaPian Tea and Quantification of Flavonoid Glycosides in Six Major Processing Types of Tea Running title: A Novel Acylated Flavonol Tetraglycoside Against Lipid Accumulation from Lu'an GuaPian Tea Wu-Xia Bai, Chao Wang, Yijun Wang, Wen-Jun Zheng, Wei Wang, Xiaochun Wan, and Guan-Hu Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00239 • Publication Date (Web): 24 Mar 2017 Downloaded from http://pubs.acs.org on March 26, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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A Novel Acylated Flavonol Tetraglycoside with Inhibitory Effect on Lipid

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Accumulation in 3T3-L1 Cells from Lu'an GuaPian Tea and Quantification of

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Flavonoid Glycosides in Six Major Processing Types of Tea

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Wu-Xia Bai⊥, Chao Wang⊥, Yi-Jun Wang, Wen-Jun Zheng, Wei Wang, Xiao-Chun

5

Wan, Guan-Hu Bao *

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Tea natural product laboratory of International Joint Lab of Tea Chemistry and Health

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effects, State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural

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University, Hefei, P.R. China

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These two authors contribute equally to the paper

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* Corresponding Author

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Professor Guan-Hu Bao

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Tea natural product laboratory of International Joint Lab of Tea Chemistry and Health

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effects, State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural

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University

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Office Phone: 0551-65786401

16

Office Fax: 0551-65786765

17

[email protected]

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ABSTRACT: A novel acylated flavonol tetraglycoside, kaempferol 3-O-[(E)-p-

19

coumaroyl-(1→2)][α-L-arabinopyranosyl-(1→3)][β-D-glucopyranosyl

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rhamnopyranosyl (1→6)]-β-D-glucopyranoside (camellikaempferoside C, 1), together

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with two flavonol and eighteen flavone and flavonol glycosides (FGs) (2-21) was

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isolated from the green tea Lu'an GuaPian (Camellia sinensis L.O. Kuntze). Their

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structures were identified by spectroscopic and chemical methods. Four acylated FGs

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(1, 7, 8, 9) were found to inhibit the proliferation and differentiation of 3T3-L1

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preadipocytes at the concentrations 25, 50, 100 µM (P < 0.05). Furthermore, we

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established a rapid UPLC method to quantify nine FGs in six major processing types

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of tea. The results showed that dark tea had the highest amount of 20 (0.70 ± 0.017

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mg/g) and black tea had the highest amount of 8 (0.09 ± 0.012 mg/g) while the

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amounts of 10 and 16 basically decreased with the increasing degree of fermentation

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and could contribute to the discrimination of different processing types of tea.

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KEYWORDS: Flavone and flavonol glycosides (FGs), camellikaempferoside C,

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Lu'an GuaPian, 3T3-L1 cells, Camellia sinensis

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(1→3)-α-L-

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INTRODUCTION

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Tea (Camellia sinensis), a traditional beverage originated in China, has a wide range

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of consumers among different ages, places, cultures, societies, etc. due to its unique

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flavor and healthy benefits. According to the different manufacturing processes, tea is

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generally divided into six major types as green, yellow, white, oolong, black and dark

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tea.1,2 The manufacturing processes basically determine the flavor, type and content of

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components of tea.3 Lu'an GuaPian tea, a type of baked green tea produced in Lu'an,

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Anhui province, is one of the top ten tea consumed in China. It is well-known for its

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unique quality and processing methods. Only fresh leaves without buds or stems of

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Camellia sinensis (C. sinensis) are used as the material and high baking temperature is

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needed during its drying step. However, few studies have been conducted on

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systematic purification and structural identification of chemical constituents from this

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unique processed green tea.

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Recently, flavone and flavonol glycosides (FGs) have drawn increasing attention due

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to their contribution to tea taste and bioactivities. Tea FGs was firstly studied over 50

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years ago.4 Major types of flavonoids presented in tea are kaempferol, quercetin,

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myricetin and apigenin, conjugated with glycosides through hydroxyl groups in the

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case of flavonoid O-glycosides or bound directly to the carbon atoms as flavonoid

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C-glycosides.5 The glycosides usually are glucoside, galactoside, rhamnoside and

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arabinoside and the number of the glycosides substituted on the aglycone varies from

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one to four. Researches showed that FGs affected the astringent taste of tea at a very

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low threshold concentration in tea, and also FGs can enhance bitterness by

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augmenting the bitterness of caffeine in tea infusions.6,7 Furthermore, the aqueous

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solution of FGs has a yellow-green color, which is a key factor to the appearance of

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green tea soup.8 Besides the main bitterness contributing components of tea, FGs also

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have various biological activities including anti-oxidant, anti-obesity, anti-cancer and

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hypoglycemic effects.9-12

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In this study, we purified and identified the structures of FGs in Lu’an GuaPian green

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tea. The cytotoxicity and inhibitory effect of four acylated FGs (1, 7, 8, 9) against lipid

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differentiation and accumulation in 3T3-L1 cells were tested as well. Furthermore, a

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rapid UPLC method was also established to quantify nine FGs conjugated with sugar

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units from one to four including four acylated FGs in six major processing types of

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tea prepared from the same tea leaves.

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MATERIALS AND METHODS

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Chemicals. HPLC grade acetonitrile, methanol, formic acid and acetic acid were

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purchased from Duksan (Ansansi, Korea). The standard sugar of L-rhamnose (≥ 99%)

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was purchased from Shanghai Hushi Laboratorial Equipment Co.Ltd. (Shanghai,

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China). D-glucose (≥ 99.5%), L-arabinose (98%), and trimethylsilylimidazole (98%)

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were purchased from Shanghai Energy chemical (Shanghai, China). 3T3-L1 cells

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were provided by Jiangsu KeyGEN BioTECH Corp., Ltd. Dulbecco’s modified

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Eagle’s medium (DMEM) were purchased from GIBCO (Invitrogen Co. Ltd., USA).

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Standard FGs (compounds 1, 7, 8, 9, 10, 16, 17, 20, 21) were isolated from Lu’an

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GuaPian tea in our tea natural product laboratory and the purity of these compounds

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was ≥ 98% confirmed by UPLC analysis.

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HPLC purification was carried out with a Waters e2695 separation module

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accompanied with a Waters 2998 photodiode detector array (PDA) detector (Waters,

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USA). The column used for separation was a semipreparative X Bridge Prep C18

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Column (10 × 250 mm, 5 µm, Waters, Ireland) at 30 °C. Samples for LC-MS analysis

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were performed on a Agilent 1100 HPLC with a PDA combined with a 6210

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time-of-flight (TOF) mass spectrometer with electrospray ionization (ESI) source by

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negative mode (Agilent Technologies, USA). A SunFire™ C18 column (4.6 × 150

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mm i.d., 3.5 µm, Waters, USA) was applied for analyzing at 30 °C. GC-MS were

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performed on a HP-5MS column (length = 30 m, i.d. = 0.25 µm, Agilent Technologies,

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USA) with GCMS-QP2010S (Shimadzu Corp., Japan). 1H NMR,

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COSY, HMQC and HMBC spectrum were performed in dimethyl-d6 sulfoxide

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(DMSO-d6) with a Agilent DD2 spectrometer (600 MHz, Agilent Technologies, USA).

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IR spectrum was obtained with an iS50 FI-IR spectrometer (Thermo, USA). MTT and

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oil-red O assays were applied on a Tunable Microplate Reader (EL-x800, BioTek

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Instruments, USA). UPLC analysis for quantification of FGs was performed on

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Waters ACQUITY UPLCTM HClass UPLC system equipped with a 2489 UV detector

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on a Waters C18 column (2.1 × 150 mm, 1.7 µm, Waters, Ireland) at 350 nm (Waters,

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USA).

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Tea Material and Extraction for UPLC Analysis. Lu’an GuaPian tea used for

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isolation (produced in 2014) was purchased from Anhui Lu’an GuaPian Tea Company

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(Anhui, China). Six processing types of tea samples (green, yellow, white, Oolong,

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C NMR, 1H-1H

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black and dark tea) used for UPLC analysis were processed using same material

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plucked from the cultivars Longjingchangye (C. sinensis var. sinensis) with the

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corresponding tea manufacturing processes (Figure 1)

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plucked in May 2015 from the tea base of Anhui Agricultural University (Hefei,

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Anhui, China). 0.25 g of ground tea powder were stored in 10 mL 70% aqueous

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methanol which we habitually used in analysis of chemical constituents of tea samples,

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and extracted twice within 12 h (15 min each time) by ultrasonic extraction. Before

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UPLC analysis, the tea infusion extracts were centrifuged at 10,000 rpm for 10 min.

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Then the supernatant was saved after running through a 0.22 µm filter.

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Extraction and Isolation. Thinking of the large volume of solvent and the low

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boiling point of acetone easily being dry as well as without apparent difference of the

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FGs profile between 70% methanol and 80% acetone extraction (Figure S1), Lu’an

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GuaPian tea (9 kg) was ground and extracted with 80% aqueous acetone for three

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times at room temperature and concentrated to a water-soluble extract. The water

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extract

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dichloromethane-soluble fraction (400 g) and a water-soluble fraction. The

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water-soluble fraction was further extracted by ethyl acetate (1:1, v/v) to provide ethyl

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acetate-soluble fraction (760 g) and residue water-soluble fraction (630 g). The

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water-soluble phase of Lu’an GuaPian tea (400 g) was subjected to MCI-Gel CHP20P

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gel column chromatography (CC) with methanol:water (1:0 to 0:1, v/v) in a gradient

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elution, giving five fractions A1-A5. Fraction A2 (40 g) was applied to Sephadex

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LH-20 CC to get fractions B1-B13. Fraction B13 was eluted with methanol:water (3:7,

was

then

mixed

with

dichloromethane

1-3

. The tea leaves were

(1:1,

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to

provide

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v/v) on ODS CC to give compound 2 (10 mg). Fraction B12 (107 mg) was eluted with

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30% aqueous methanol on ODS CC and Sephadex LH-20 CC with methanol to get

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fraction C. Fraction C was separated by HPLC (water:acetonitrile = 8:2) to give

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compound 3 (3.7 mg) and compound 4 (3.5 mg). Fraction A5 (1 g) was eluted with

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dichloromethane methanol solution with increasing polarity (50:1 to 0:1, v/v) applied

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on silica gel CC to get compound 5 (43 mg). Fraction A4 (1 g) was applied to silica

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gel CC with dichloromethane:methanol mixture with increasing polarity (40:1 to 0:1,

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v/v) and with methanol on Sephadex LH-20 CC to get compound 6 (22 mg). Silica gel

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CC was applied on fraction A3 (1.6 g) eluted with dichloromethane methanol solution

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from 20:1 to 0:1, (v/v), and Sephadex LH-20 CC with methanol to get fractions

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D1-D2. Fraction D1 was subjected to toyopearl CC for purifying to give compound 7

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(9 mg) and compound 8 (5 mg). Fraction D2 was performed on toyopearl CC to give

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compound 1 (18.7 mg) and compound 9 (5.6 mg,). Fraction B2 to B7 (35 g) were

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merged and subjected to silica gel CC eluting with ethyl acetate methanol solution

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from 20:1 to 0:1 (v/v) to obtain ten fraction F1 to F10. Applying Sephadex LH-20 CC

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on Fraction F9 with methanol and toyopearl CC with methanol gave fraction G1 to

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G3. Fraction G2 was compound 10 (20 mg). Fraction G3 was subjected to polyamide

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CC (ethyl acetate:methanol:water = 3:1 to 0:1, v/v ) to get fraction H1 (compound 11,

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10 mg) and fraction H2. Fraction H2 was subjected to toyopearl CC eluting with

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methanol to give three fractions, fraction I1 to I3. Fraction I3 was compound 12 (2.7

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mg). Fraction I1 was separated by HPLC (water:acetonitrile = 85:15, v/v) to get

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compound 15 (3.2 mg) and compound 16 (37.4 mg). Fraction I2 was separated by

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HPLC (water:methanol = 6:4, v/v) to get compound 13 (9.6 mg) and compound 14

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(3.2 mg). Fraction F4 and F5 were merged, and then subjected to a silica gel CC with

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dichloromethane methanol solution from 100:1 to 0:1 (v/v), which step produced

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eight fractions, fraction J1 to J8. Fraction J5 was subjected to toyopearl CC with

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methanol to give three fractions, fraction K1 to K3. Fraction K1 was applied to ODS

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CC with methanol:water (3:7, v/v) to give fraction L1 and fraction L2. Fraction L1

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was applied to toyopearl CC with methanol to give compound 17 (43.5 mg). Fraction

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L2 was applied to toyopearl CC with methanol to give ten fractions, fraction M1 to

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M10. Fraction M10 was applied to toyopearl CC with methanol:water (3:7 to 7:3, 1:0,

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v/v) and 50% aqueous methanol to give compound 19 (13.8 mg). Fraction K2 was

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applied to toyopearl CC eluting with methanol:water (3:7 to 7:3, 1:0, v/v) and

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methanol:water (1:1, v/v) to give compound 18 (17.0 mg). Fraction J6 was applied to

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toyopearl CC eluting with 50% aqueous methanol to give compound 20 (10.5 mg).

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Fraction J3 was performed on toyopearl CC eluting with methanol:water (3:7 to 7:3,

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1:0, v/v) and 50% aqueous methanol to give compound 21 (6.0 mg).

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Acid Hydrolysis and Sugar Analysis of Compound 1. The method was modified

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according to reference.13 Briefly, compound 1 (0.8 mg) was melted in 2 M HCl (0.8

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mL), and heated in water bath for 4 h at 80 °C. The reactants were extracted using

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chloroform to get the supernatant and vacuum freeze-dried. The sample was melted in

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0.4 mL of pyridine mixed with 10 mg/mL L-cysteine methyl ester hydrochloride at

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60 °C for 2 h. The solution was then dried by vacuum freeze-drying, following with

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the addition of 0.2 mL trimethylsilylimidazole to the solid. The mixture was then

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heated for 1.5 h at 70 °C, and then partitioned between n-hexane and water. GC-MS

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was carried out to analyze the n-hexane fraction with injector temperature at 280 °C.

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The oven temperature firstly started at 160 °C for 1 min, and then rose up to 200 °C at

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a speed of 6 °C/min , and then kept warming-up until a temperature of 280 °C at a

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speed of 3 °C/min and held for 5 min. The sugar standards were analyzed under the

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same condition. A comparison between the sugar standards and the sugar units of

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compound 1 was made with peak and retention time (RT). As a result, D-glucose (RT

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22.16 min), L-rhamnose (RT 18.55 min) and L-arabinose (RT 16.97 min) were

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confirmed in a ratio of 2:1:1 for compound 1.

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Cell Culture. 3T3-L1 cells were cultured in DMEM supplemented with 10% fetal

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bovine serum (FBS) together with 1% penicillin/streptomycin antibiotics, stored in a

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moisturized atmosphere with 5% CO2 at 37 °C .14

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Cell

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2H-tetrazolium bromide (MTT) assay, cell toxicity of the four acylated FGs (1, 7, 8, 9)

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was determined.15 3T3-L1 cells were seeded on 96-well plates at a density of 3 × 104

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cells/well. After 24 h, the media for each compound at three concentrations (25, 50,

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and 100 µM) was removed and 100 µL of the media was added into the respective

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wells containing the cells. The negative control was wells with untreated cells, and the

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positive control was wells treated with taxol (10 µM). After cultured, the 20 µL 5

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mg/mL MTT was added into every well. The cells culture medium was discarded after

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incubation for 4 h and the purple precipitate attached to the bottom of the plates were

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melted in DMSO for 10 min in the dark. The optical density (OD) of each well was

Viability

Assay.

By

using

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-

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measured at 490 nm.

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3T3-L1 Cell Adipocyte Differentiation and Oil-red O staining. To stimulate the

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differentiation, the confluent 3T3-L1 preadipocytes (day 0) were treated with 10

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µg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine and 1 µM dexamethasone,

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respectively.15 After 2 days, the medium was changed to DMEM supplemented with

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10% FBS. 3T3-L1 preadipocytes were administrated with compound (0, 25, 50 and

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100 µM) from day 0 to day 8, and the positive control resveratrol (88 µM). After then,

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mature adipocytes needed leaching with phosphate buffered saline (PBS), and fixed

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with 10% formalin, and then leached with 60% isopropanol. After air drying, 0.5%

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oil-red O in isopropanol and water (3:2, w/v) was added to every well at room

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temperature for 3-4 h. Then the solution was withdrawn and distilled water was used

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for washing cells. Pictures of stained cells were saved. The oil-red O in triglyceride

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droplets was extracted with 100% isopropanol and determined at OD510

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quantification.

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UPLC Analysis for Quantification of the FGs. The gradient elution of mobile phase

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B (acetonitrile) in UPLC method was set as follows: 0-2 min, 15%; 2-8 min, from

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15% to 25%; 8-12 min, from 25% to 30%; 12-16 min, kept at 30%; 16-18 min, from

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30% to 43%; 18-20 min, kept at 30%; 20-22 min, from 43% to 15%; then returned to

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15% for 2 min. Mobile phase A was 0.17% aqueous acetic acid with a flow rate of

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0.22 mL/min under the wavelength of 350 nm. The injection volume was 1 µL. Three

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replicates were analyzed for each sample. To validate the method described above, we

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examined the standard curve, linear range, precision, repeatability, stability, limit of

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for

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detection (LOD), limit of quantification (LOQ) and the recovery ratio (supplementary

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data).

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Statistical Analysis. All assay experiments were done in triplicate (n = 3) and the

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values were presented as mean ± SD, unless otherwise specified. One-way ANOVA

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with Tukey tests was applied to determine significant differences. GraphPad Prism

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(version 6.0) software was applied for statistical analysis.

215 216

RESULTS AND DISCUSSION

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Identification of Compound 1. The extract of Lu'an GuaPian tea was suspended in

218

aqueous solution and partitioned successively with dichloromethane and ethyl acetate

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to provide dichloromethane-soluble fraction, ethyl acetate-soluble fraction, and the

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residual water-soluble fraction. The water-soluble fraction was subjected to multiple

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chromatographic purification steps to produce a novel acylated flavonol

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tetraglycoside (1) and 20 known compounds (2-21) (Figure 2).

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Compound 1 was observed as yellow amorphous powder with molecular formula of

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C47H54O26 based on its HR-ESI-MS- (m/z 1033.28429 [M-H]-, calcd 1033.28251). The

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IR spectrum of 1 indicated the absorption bands of hydroxyl (3394 cm-1), carbonyl

226

(1700 cm-1), double bonds (1604, 1511 cm-1) and O-glycosidic group (1078 cm-1).

227

The 1H NMR and 13C NMR data of compound 1 were shown in Table 1, which were

228

similar to those of compound 9, except for the differences in signals of the B-ring

229

moiety.16 The 1H NMR and 13C NMR spectrum of 1 presented two doublet signals at

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δH 7.94 (2H, d, J = 9.0 Hz) and δH 6.87 (2H, d, J = 9.0 Hz), and δC 131.3 and δC 115.6,

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respectively, which suggested presence of a kaempferol aglycone. Furthermore, the

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signals appeared at δH 7.55 (1H, d, J = 15.6 Hz), δH 7.50 (2H, d, J = 9.0 Hz), δH 6.77

233

(2H, d, J = 9.0 Hz), and δH 6.34 (1H, d, J =15.6 Hz) indicated the presence of

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trans-4-hydroxycinnamic [(E)-p-coumaric] acid fragment. The presence of four

235

anomeric 1H signals and 13C signals in the NMR indicated compound 1 is conjugated

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with four sugars. The absolute configurations of L-rhamnose, L-arabinose, and

237

D-glucose on compound 1 were confirmed by chemical and GC-MS analysis. The

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HMBC spectrum (Figure 3) determined the linkages between kaempferol,

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(E)-p-coumaroyl, arabinopyranosyl, rhamnopyranosyl and glucopyranosyl, which

240

showed long-range correlations as follows: δH 5.54 (H-1, Glc1) to δC 133.2 (C-3 of

241

kaempferol unit), δH 4.39 (H-1, Rha) to δC 67.9 (C-6, Glc1), δH 4.27 (H-1’, Glc2) to δC

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82.2 (C-3, Rha), δH 4.33 (H-1, Ara) to δC 80.7 (C-3, Glc1), and δH 4.98 (H-2, Glc1) to

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δC 166.0 (carbonyl carbon of E-p-coumaroyl). Thus, the structure of 1 was identified

244

as kaempferol 3-O-[(E)-p-coumaroyl-(1→2)] [α-L-arabinopyranosyl-(1→3)][β-D-

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glucopyranosyl-(1→3)-α-L-rhamnopyranosyl(1→6)]-β-D-glucopyranoside,

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was named as camellikaempferoside C.

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Compounds 2-21 were confirmed by comparison of their spectral data with those

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reported literatures and Tea Metabolome Database (TMDB)17 as myricetin

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3-O-galactoside (2),7 quercetin 3-O-galactoside (3),18 myricetin 3-O-glucoside (4),7

250

kaempferol

251

[α-L-arabinopyranosyl-(1→3)][α-L-rhamno-pyranosyl (1→6)]-β-D-glucopyranoside

252

(7),20 quercetin 3-O-[(E)-p-coumaroyl-(1→2)] [α-L-arabinopyranosyl-(1→3)][α-L-

(5),19

quercetin

(6),19

which

kaempferol-3-O-[(E)-p-coumaroyl-(1→2)]

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(8),16

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rhamnopyranosyl(1→6)]-β-D-glucopyranoside

254

coumaroyl-(1→2)] [α-L-arabinopyranosyl-(1→3)] [β-D-glucopyranosyl-(1→3)-α-L-

255

rhamnopyranosyl(1→6)]-β-D-glucopyranoside

256

rutinoside (10),7 kaempferol 3-O-galactosylrutinoside (11),7 apigenin 6, 8-C-di

257

glucoside

258

arabinosyl-8-C-glucoside (14),21 quercetin 3-O-galactosylrutinoside (15),7 quercetin

259

3-O-glucosylrutinoside

260

3-O-rhamnosylgalactoside (18),23

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Vitexin-4’’-O-glucoside (20),24 and kaempferol 3-O-glucoside (21)18.

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These 21 compounds include the main four kinds of flavonoid aglycone glycosides in

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tea: kaempferol, quercetin, myricetin and apigenin glycosides. The glycosides

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substituted on the aglycone contain monosaccharides, disaccharides, trisaccharides

265

and tetrasaccharides. Among these FGs, four are acylated ones: 1, 7, 8, 9. Twelve

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compounds are groups of isomers: 2 and 4, 10 and 11, 13 and 14, 15 and 16, 12, 17,

267

19 and 20.

268

Effect of Four Acylated FGs (1, 7, 8, 9) on 3T3-L1 Cells Viability. The four

269

acylated FGs (1, 7, 8, 9) were investigated on their cell viability in 3T3-L1 cells using

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MTT cell assay (Table 2). The 3T3-L1 cells inhibition activities of 1, 7, 8, 9 with 100

271

µM were 6.72±0.79%, 7.88 ±1.26%, 8.57 ±1.33%, 9.10 ± 0.78%, respectively

272

(inhibition ratio of the positive control taxol was 90.86 ± 3.04% at 10 µM). It

273

indicated that the IC50 values of these four compounds for 3T3-L1 cells were all higher

274

than 100 µM. So we can conclude that 1, 7, 8, 9 are non-cytotoxic against 3T3-L1 cells.

(9),16

quercetin

kaempferol

(12),21 apigenin-6-C-glucosyl-8-C-arabinoside

(16),7

kaempferol

3-O-[(E)-p-

3-O-glucosyl

(13),21 apigenin-6-C-

3-O-rutinoside

(17),22

quercetin

kaempferol 3-O-rhamnosylgalactoside (19),23

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Inhibitory Effects of Four Acylated FGs against Lipid Accumulation in 3T3-L1

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Cells. In order to assess the effect of the four acylated FGs (1, 7, 8, 9) on the

277

proliferation and differentiation of 3T3-L1 preadipocyte, 3T3-L1 preadipocyte cells

278

were incubated with each compound (25, 50 and 100 µM) for 8 days after stimulation

279

of differentiation, and then Oil-red O staining was carried out (Figure 4). In Figure 4,

280

AI-II were normal 3T3-L1 adipocytes (negative control, lipid accumulation as 100%)

281

and adipocytes treated with resveratrol (positive control, 88 µM), which indicated that

282

there was lipid accumulation in adipocytes and resveratrol can successfully reduce the

283

lipid accumulation to 40.35 ± 2.13%. In Figure 4, BI-V microscopic observation

284

showed that the number of oil droplets in the adipocytes treated with 1, 7, 8, 9 (50 µM)

285

was reduced compared with that in the negative control. Figure 5 showed that the

286

four acylated FGs (1, 7, 8, 9) can significantly reduce the lipid accumulation in

287

3T3-L1 adipocytes compared with normal 3T3-L1 adipocytes (negative control) at

288

different concentrations (25, 50, 100 µM) (P < 0.05). Therefore, we can conclude that

289

1, 7, 8 and 9 show good suppression against lipid accumulation in 3T3-L1 adipocytes.

290

UPLC Method Validation. As the main component of tea, the content of FGs in tea

291

is between 1% and 3%. In this study, we established a UPLC method to detect FGs

292

and to quantify nine FGs including flavonol mono to tetraglycosides in 24 minutes in

293

the six major types of tea processed from the same material (Figure 6). Calibration

294

curves of nine FGs showed good linearity over the concentration range with

295

correlation coefficients (R2) ranged from 0.9970 to 0.9998 in a certain concentration

296

range (Supporting Table 2). Method validation details were shown in Supporting

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Table 3. Relative standard deviation (RSD) of the precision, repeatability and stability

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were all less than 5.36%. The value of LOD and LOQ tested in UPLC were less than

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0.61 and 2.04 ng, respectively. And the value of recovery varied from 94.71% to

300

107.57% with RSD values less than 5.5% for the investigated FGs.

301

UPLC Analysis of FGs in Six Processing Types of Tea. Six different types of tea

302

samples were processed through the corresponding manufacturing processes (Figure

303

1). 1-3 Previous quantification of FGs in tea has tended to focus on FGs in different

304

varieties of tea. little is know on the concentration of FGs in the six major processing

305

types of tea prepared from the same material. In our study, the content of nine FGs

306

varied among the six types of tea. Kaempferol 3-O-glucosyl rutinoside (10) is the

307

highest one in all six types of tea and quercetin 3-O-glucosylrutinoside (16) is the

308

second one (Table 3). The amounts of both 10 and 16 basically decrease with the

309

degree of fermentation of processing tea. And the amounts of both 10 and 16 are high

310

enough to be easily detected, which could contribute as factors to discriminate the

311

different processing types of tea17. Dark tea was also found to have the highest

312

amount of the flavone C-diglycosides, Vitexin-4’’-O-glucoside (20) (0.70 ± 0.017

313

mg/g), and black tea was found to have the highest amount of the acylated flavonol

314

tetraglycoside, quercetin 3-O-[(E)-p-coumaroyl-(1→2)][α-L-arabinopyranosyl-(1→3)]

315

[α-L-rhamnopyranosyl (1→6)]-β-D-glucopyranoside (8) (0.09 ± 0.012 mg/g). The

316

level of acylated flavonol triglycoside was lower than that of acylated flavonol

317

tetraglycosides and even lower than that of non-acylated flavonol triglycosides. The

318

amount of the kaempferol acylated triglycosides (7) in some tea is even

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non-detectable.

320

Obesity is a serious metabolic syndrome that has association with many diseases, such

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as type II diabetes mellitus, coronary heart disease, respiratory complications,

322

hypertension, hyperlipidemia and cancer.25 A surfeit of calories and dearth of energy

323

expenditure is the basic factor of obesity. Tea has been used as an anti-obesity therapy

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to prevent lipid accumulation in Traditional Chinese Medicine and tea polyphenols

325

have been regarded as the main contributors.26 FGs are widely distributed in plants

326

with potential therapeutic effects. Recent studies have demonstrated that FGs have

327

anti-obesity effects, which suggests that FGs-enriched plants can be considered as

328

supplements or functional foods to prevent lipid accumulation.27,28 In our research, we

329

systematically investigated FGs in the Chinese famous green tea, Lu'an GuaPian.

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From Lu'an GuaPian tea, we obtained 21 flavonol and FGs with a novel acylated

331

flavonol tetraglycoside named as camellikaempferoside C. Here, the four acylated

332

FGs we isolated displayed non-cytotoxic effect against 3T3-L1 cells and compounds 1,

333

7, 8, 9 exhibited good inhibition against lipid accumulation in 3T3-L1 adipocytes at

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25, 50, and 100 µM. The average amount of daily tea intake can be around 10 g.29 The

335

amount of FGs could be around 1-3 mg/g dry tea (Table 3). 17-52% FGs can be

336

absorbed through dietary intake.30 Therefore, daily intake of FGs in tea (around

337

0.20-3.0 µM) is far below the amount we used here, which means extra addition of

338

FGs is needed for meeting anti-obesity effect besides the daily dietary intake. In

339

addition, a UPLC method to quantified FGs including acylated FGs and flavonol

340

triglycosides in six major processing types of tea prepared from the same material has

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been developed for the first time. The relation between some of the FGs and tea types

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were also observed. The result showed that acylated FGs can be found and quantified

343

in tea. The content of two main FGs (10 and 16) in all types of tea decreases basically

344

with the increasing degree of fermentation, which could be developed as contributors

345

to efficiently discriminate the different processing types of tea combined with other

346

factors. 31 Moreover, the UPLC method we established could serve as a reference

347

method for the determination of different flavonol glycosides with one to four sugar

348

units at the same time in six processing types of tea.

349 350

ACCOCIATED CONTENTS

351

Supplementary Information

352

Supplementary data can be found in the online version. These data include

353

spectroscopic and physical data of compound 1, cell toxicity and inhibitory effects on

354

lipid accumulation in 3T3-L1 cells of four acylated FGs (1, 7, 8, 9), and quantification

355

method validation of nine FGs in tea for UPLC analysis.

356 357

ACKNOWLEDGEMENT

358

Financial assistances were received with appreciation from National Natural Science

359

Foundation of China 81170654/H0507, Anhui Agricultural University Talents

360

Foundation (YJ2011-06), Nutrition and Quality & Safety of Agricultural Products,

361

National Modern Agriculture Technology System (CARS-23).

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REFERENCES

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(1) Engelhardt, U. H. 3.23 Chemistry of Tea, Comprehensive Natural Products II

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Chemistry and Biology, Volume 3: Development & Modification of Bioactivity.

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Elsevier Ltd, Oxford, United Kingdom. 2010, 999-1032.

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(2) Han, M.; Zhao, G.; Wang, Y.; Wang, D.; Sun, F.; Ning, J.; Wan, X.; Zhang, J.

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Safety and anti-hyperglycemic efficacy of various tea types in mice. Sci. Rep., 2016, 6,

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(3) Wang, Y.; Yang, Y.; Wei, C.; Wan, X.; Thompson, H. J. Principles of biomedical

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agriculture applied to the plant family theaceae to identify novel interventions for

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cancer prevention and control. J. Agric. Food. Chem. 2016, 64, 2809-2814.

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(4) Roberts, E. A. H.; Cartwright, R. A.; Wood, D. J. The flavonols of tea. J. Sci. Food.

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(5) Balentine, D. A.; Wiseman, S. A.; Bouwens, L. C. The

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flavonoids. Crit. Rev. Food Sci. Nutr. 1997, 37, 693-704.

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(6) Scharbert, S.; Hofmann, T. Molecular definition of black tea taste by means of

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quantitative studies, taste reconstitution, and omission experiments. J. Agric. Food.

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Chem. 2005, 53, 5377-5384.

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(7) Scharbert, S.; Holzmann, N.; Hofmann, T. Identification of the astringent taste

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compounds in black tea Infusions by combining instrumental analysis and human

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bioresponse. J. Agric. Food. Chem. 2004, 52, 3498-3508.

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(8) Tsanova-Savova, S.; Ribarova, F. Free and conjugated myricetin, quercetin, and

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kaempferol in bulgarian red wines. J. Food Compos Anal. 2003, 15, 639-645.

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(9) Takuya, K.; Naoto, I.; Yasuhiro, K.; Yasuhiro, K.; Yoshimitsu, Y.; Kuninori,

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S.; Yosuke, Y. Antioxidant flavonol glycosides in mulberry (Morus alba L.) leaves

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isolated based on LDL antioxidant activity. Food Chem. 2006, 97, 25-36.

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(10) Morikawa, T.; Ninomiya, K.; Miyake, S.; Miki, Y.; Okamoto, M.; Yoshikawa,

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M.; Muraoka, O. Flavonol glycosides with lipid accumulation inhibitory activity and

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simultaneous quantitative analysis of 15 polyphenols and caffeine in the flower buds

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of Camellia sinensis from different regions by LCMS. Food Chem. 2013, 140,

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353-360.

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(11) Ahn, E. M.; Han, J. T.; Kwon, B. M.; Kim, S. H.; Baek, N. I. Anti-cancer activity

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of flavonoids from Aceriphyllum rossii. J. Korean Soc. Appl. Biol. Chem. 2008, 51,

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(12) Yoshikawa, M; Wang, T; Morikawa, T.; Xie, H. H.; Matsuda, H. Bioactive

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constituents from chinese natural medicines. XXIV.1) hypoglycemic effects of

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Sinocrassula indica in sugar-loaded rats and genetically diabetic KK-Ay mice and

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structures of new acylated flavonol glycosides, Sinocrassosides A1, A2, B1, and B2.

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Chem. Pharm. Bull. 2007, 55, 1308-1315.

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(13) Zong, J. F.; Wang, R. L.; Bao, G. H.; Ling, T. J.; Zhang, L.; Zhang, X. F.; Hou, R.

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Y. Novel triterpenoid saponins from residual seed cake of Camellia oleifera Abel.

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show anti-proliferative activity against tumor cells. Fitoterapia 2015, 104, 7-13.

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(14) Li, K. K.; Wong, H. L.; Hu, T. Y.; Zhang, C.; Han, X. Q.; Ye, C. X.; Leung, P. C.;

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Cheng, B. H.; Ko, C. H. Impacts of Camellia kucha and its main chemical

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components on the lipid accumulation in 3T3-L1 adipocytes. Int. J. Food Sci. Tech.

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2016, 51, 2546-2555

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(15) Yang, Y.; Qiao, L. L.; Zhang, X.; Wu, Z.; Weng, P. Effect of methylated tea

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catechins from Chinese oolong tea on the proliferation and differentiation of 3T3-L1

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preadipocyte. Fitoterapia 2015, 104, 45-49.

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(16) Manir, M. M.; Kim, J. K.; Lee, B. G.; Moon, S. S. Tea catechins and flavonoids

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from the leaves of Camellia sinensis inhibit yeast alcohol dehydrogenase. Bioorg.

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Med. Chem. 2012, 20, 2376-2381.

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(17) Yue, Y.; Chu, G.X.; Liu, X. S.; Tang, X.; Wang, W.; Liu, G. J.; Yang, T.; Ling, T.

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J.; Wang, X. G.; Zhang, Z. Z.; Xia, T.; Wan, X. C.; Bao, G. H.

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TMDB: A literature-curated database for small molecular compounds found from tea.

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BMC Plant Biol. 2014, 14, 1-8.

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(18) Deng, S. G.; Deng, Z. Y.; Fan, Y. W.; Peng, Y.; Li, J.; Xiong, D. M.; Liu, R.

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Isolation and purification of three flavonoid glycosides from the leaves of Nelumbo

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nucifera (Lotus) by high-speed counter-current chromatography. J. Chromatogr. B

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2009, 877, 2487-2492.

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(19) Li, Y. L.; Li, J.; Wang, N. L.; Yao, X. S. Flavonoids and a new polyacetylene

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from Bidens parviflora Willd. Molecules 2008, 13, 1931-41.

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(20) Tian, Y. Z.; Liu, X.; Liu, W.; Wang, W. Y.; Long, Y. H.; Zhang, L.; Xu, Y.; Bao, G.

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H.; Wan, X. C.; Ling, T. J. A new anti-proliferative acylated flavonol glycoside from

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Fuzhuan brick-tea. Nat. Prod. Res. 2016, 30, 2637-2645.

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(21) Engelhardt, U. H.; Finger, A.; Kuhr, S. Determination of flavone C-glycosides in

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tea. Eur. Food. Res. Technol. 1993, 197, 239-244.

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(22) Cheng, N. A. L.; Tako, M.; Hanashiro, I.; Tamaki, H. Antioxidant flavonoid

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glycosides from the leaves of Ficus pumila L. Food Chem. 2008, 109, 415-420.

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(23) Brasseur, T.; Angenot, L. Flavonol glycosides from leaves of Strychnos variabilis.

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Photochem. 1986, 25, 563-564.

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(24) Kwon, Y. S.; Kim, E. Y.; Kim, W. J.; Kim, K. W.; Kim, C. M. Antioxidant

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constituents from Setaria viridis. Arch Pharm Res. 2002, 2, 300-305.

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(25) Kopelman, P. G. Obesity as a medical problem. Nature 2000, 404, 635-643.

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(26) Wolfram, S.; Wang, Y.; Thielecke, F. Anti-obesity effects of green tea: from

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bedside to bench. Mol. Nutr. Food Res. 2006, 50, 176-187.

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(27) Tadahiro, Y.; Akihiro, D.; Susumu, K. Flavonol acylglycosides from flower of

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Albizia julibrissin and their inhibitory effects on lipid accumulation in 3T3-L1 cells.

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Chem. Pharm. Bull. 2012, 60, 129-136.

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(28) Takuya, K.; Masayuki, Y.; Kuninori, S.; Tomoko, I.; Ichiro, M.; Keiko,

442

A.; Yukikazu, Y. Effect of flavonol glycoside in mulberry (Morus alba L.) leaf on

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glucose metabolism and oxidative stress in liver in diet-induced obese mice. J. Sci.

444

Food Agric. 2010, 90, 2386-2392.

445

(29) Peng, C. Y.; Cai, H. M.; Zhu, X. H.; Li, D. X.; Yang, Y. Q.; Hou, R. Y.; Wan, X.

446

C. Analysis of naturally occurring fluoride in commercial teas and estimation of its

447

daily intake through tea consumption. J. Food Sci. 2016, 81(1), H235-H239.

448

(30) Hollman, P. C.; deVries, J. H.; vanLeeuwen S. D.; Mengelers, M. J.; Katan, M. B.

449

Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy

450

volunteers. Am. J. Clin. Nutr. 1995, 62(6), 1276-1282.

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(31) Ning, J.; Li, D.; Luo, X.; Ding, D.; Song, Y.; Zhang, Z.; Wan, X. Food Anal.

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Methods, 2016, 9(11), 3242-3250.

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Figure captions

454

Figure 1. The six major manufacturing processes of tea

455

Figure 2. The structures of compounds 1-21

456

Figure 3. The Key HMBC correlations of compound 1

457

Figure 4. Effects of four acylated FGs (1, 7, 8, 9) on lipid accumulation in 3T3-L1

458

adipocytes. A: (I) 3T3-L1 adipocytes in negative control group; (II) 3T3-L1

459

adipocytes were treated with resveratrol (88 µM); B: (I-V) 3T3-L1 adipocytes were

460

treated with 1, 7, 8, 9 (50 µM), respectively. The oil droplets were stained with

461

Oil-Red O.

462

Figure 5. Effect of the resveratrol (positive control, 88 µM), four acylated FGs (1, 7, 8,

463

9) on lipid accumulation in 3T3-L1 adipocytes at different concentrations (25, 50 and

464

100 µM, results are expressed as mean ± SD of triplicate tests). The lipid

465

accumulation without being added any compound in cells (negative control) was

466

supposed to be 100%. Data with different letters are significantly different at P < 0.05

467

among different treatments.

468

Figure 6. The UPLC analysis of nine FGs in Lu'an GuaPian green tea at 350 nm. (The

469

above curve was Lu'an GuaPian green tea, the below was FG standards. Peak

470

numbers corresponded to Table 3, from left to right are compound 20, 16, 10, 17, 21,

471

9, 1, 8 and 7, respectively.)

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Table 1. NMR spectroscopic data of compound 1a Position

δH

(J in Hz)

δC

HMBC (1H to 13C)

Position

δH

(J in Hz)

δC

HMBC (1H to 13C)

2

157.4

5'

3.07 br s

76.9

C-Glc-(4', 6')

3

133.2

6'a

3.55 dd (1.8, 10.8)

61.1

C-Glc-(4', 5')

4

177.4

6'b

3.41 dd (3.6, 11.4)

5

161.6

Rha 1

4.39 br s

101.5

C-Glc-6, Rha-3

2

3.70 br s

69.7

C-Rha-1

3

3.29 m

b

82.2

C-Rha-4

4

3.25 t (9.0)

71.1

C-Rha-(3, 6)

68.2

C-Rha-1

6

6.14 d (1.8)

7

99.3

C-5, 7, 8, 10

164.7

8

6.34 d (1.8)

94.3

C-6, 7, 9, 10

b

C-Glc-(4', 5')

9

156.9

5

3.30 m

10

104.4

6

0.93 d (6.0)

18.0

C-Rha-(4, 5)

1'

121.1

Ara 1

4.33 d (5.4)

2'

7.94 d (9.0)

3'

6.87 d (9.0)

4'

131.3 115.6

C-2, 4', 6' C-1', 4', 5'

160.4

103.1

C-Glc-3, Ara-(3, 5)

3.31 m

b

70.6

C-Ara-(1, 3)

3

3.31 m

b

72.6

C-Ara-2

4

3.61 br s

67.2

C-Ara-5

65.1

C-Ara-(1, 4, 3)

2

5'

6.87 d (9.0)

115.6

C-1', 3', 4'

5a

3.74 dd (5.4, 12.0)

6'

7.94 d (9.0)

131.3

C-2, 4', 2'

5b

3.40 dd (3.0, 10.2)

Glc 1

5.54 d (7.8)

99.1

C-3

p-Cou

2

4.98 dd (9.0, 8.4)

73.2

C-Glc-(1, 3), Cou-1

1



166.0

3

3.82 t (9.6)

80.7

C-Glc-(2, 4), Ara-1

2

6.34 d (15.6)

114.7

C-Cou-1, 1'

4

3.23 m

69.3

C-Glc-(3, 5)

3

7.55 d (15.6)

145.3

C-Cou-1, 2, 2'

5

3.50 t (9.0)

75.6

C-Glc-(1, 4)

1'

6a

3.69 d (10.8)

67.9

C-Rha-1

2'

7.50 d (9.0)

130.7

C-Cou-3, 4', 6'

C-Rha-1

3'

6.77 d (9.0)

116.2

C-Cou-1', 4', 5'

b

C-Ara-(1, 4, 3)

125.6

6b

3.31 m

Glc 1'

4.27 d (7.8)

104.8

C-Rha-3, Glc-3'

4'

2'

3.00 dd (8.0, 9.0)

74.5

C-Glc-(1', 3', 4')

5'

6.77 d (9.0)

116.2

C-Cou-1', 3', 4'

3'

3.17 m

76.4

C-Glc-(2', 4')

6'

7.50 d (9.0)

130.7

C-Cou-3, 4, 2'

4'

3.09 d (9.6)

70.0

C-Glc-(2', 3')

a

1

b

signals were overlapped.

160.2

H at 600 MHz and 13 C NMR at 150 MHz in DMSO-d6.

Glc: glucopyranosyl, Ara: arabinopyranosyl, Rha: rhamnopyranosyl, p-Cou: E-p-hydroxycoumaroyl

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Table 2. Cytotoxicity of four acylated FGs (1, 7, 8, 9) on 3T3-L1 cell compounds 100 µM IC50 (µM)

1 6.72 ± 0.79% > 100 µM

7

8

9

7.88 ± 1.26% 8.57 ± 1.33% 9.10 ± 0.78% > 100 µM

> 100 µM

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Table 3. The content of nine FGs in different tea (mg/g, n = 3, mean ± SD) FGs

Green tea

White tea b

20

0.57±0.015

0.37±0.036

16

2.10±0.008a

10

Yellow tea c

b

Oolong tea c

Black tea

Dark tea d

0.27±0.027

0.70±0.017a

0.60±0.025

0.40±0.008

1.86±0.161b

1.84±0.079b

1.23±0.003c

1.09±0.011c

1.17±0.025c

3.44±0.045a

2.75±0.177bc

3.02±0.115b

2.67±0.071c

2.50±0.232c

1.95±0.315d

17

0.30±0.014a

0.27±0.034a

0.28±0.026a

0.28±0.005a

0.26±0.032a

0.17±0.003b

21

0.08±0.001a

0.06±0.005c

0.07±0.003b

0.06±0.001c

0.06±0.003c

0.07±0b

9

0.26±0.008a

0.25±0.019a

0.22±0.01bc

0.24±0.013ab

0.19±0.019bc

0.20±0.003c

1

0.07±0c

0.08±0.001c

0.08±0.009ab

0.08±0.004bc

0.07±0.007a

0.09±0.013a

8

0.02±0.004d

0.04±0.003c

0.02±0d

0.05±0.003b

0.09±0.012a

0.02±0.002d

7

0.01±0a

0.01±0a

0.01±0.001a

ND

ND

ND

20: Vitexin 4''-O-Glc, 16: Q 3-O-Glc-Rha-Glc, 10: K 3-O-Glc-Rha-Glc, 17: K 3-O-Glc-Rha, 21: K 3-O -Glc, 9: Q 3-O-p-Cou-Glc-Ara-Rha-Glc, 1: K 3-O-p-Cou-Glc-Ara-Rha-Glc, 8: Q 3-O-p-Cou-Glc-RhaGlc, 7: K 3-O-p-Cou-Glc-Rha-Glc. ND: not detected. One-way ANOVA with Tukey tests was applied to determine significant differences. Different superscripts show significant differences (P < 0.05) for different processing types of tea sample.

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