Food Chemistry 170 (2015) 110–117

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Food Chemistry

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Changes of major tea polyphenols and production of four new B-ringfission metabolites of catechins from post-fermented Jing-WeiFu brick tea

http://dx.doi.org/10.1016/j.foodchem.2014.08.0750308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +86 551 5786401; fax: +86 551 5786765 (G.-H. Bao). Tel.: +86 551 5786002; fax: +86 551 5786765 (X.-C. Wan).E-mail addresses: baoguanhu@ahau.edu.cn (G.-H. Bao), xcwan@ahau.edu.cn (X.-C. Wan).

Yun-Fei Zhu a, Jing-Jing Chen a, Xiao-Ming Ji b, Xin Hu b, Tie-Jun Ling a, Zheng-Zhu Zhang a, Guan-Hu Bao a,⇑,Xiao-Chun Wan a,⇑a Key Laboratory of Tea Biochemistry and Biotechnology, Anhui Agricultural University, 130 West Changjiang Road, Hefei City, Anhui Province 230036, Chinab Shaanxi Zhong Fu Tea Research Institute, Xianyang City, Shaanxi Province 712044, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 18 April 2014Received in revised form 3 August 2014Accepted 14 August 2014Available online 23 August 2014

Chemical compounds studied in this article:Gallic acid (GA, PubChem CID: 370)(+)-Catechin (C, PubChem CID: 9064)(�)-Epicatechin (EC, PubChem CID: 72276)(�)-Epigallocatechin gallate (EGCG,PubChem CID: 65064)Epicatechin-3-gallate (ECG, PubChem CID:65056)Epigallocatechin (EGC, PubChem CID: 72277)Xanthocerin (PubChem CID: 5315332)Gallicin (PubChem CID: 7428)(�)-Epiafzelechin (PubChem CID: 443639)Phloroglucinol (PubChem CID: 359)Pyrogallol (PubChem CID: 1057)2,5-Dihydroxy benzoic acid (PubChem CID:3469)Quercetin (PubChem CID: 5280343)Kaempferol (PubChem CID: 5280863)Myricetin (PubChem CID: 5281672)Canophyllol (PubChem CID: 623591)a-Spinasterol (PubChem CID: 5281331)Theobromine (PubChem CID: 2519)Epicatechin-3-O-(40-O-methyl) gallate(PubChem CID: 21146794)Astragalin (PubChem CID: 5282102)Nicotiflorin (PubChem CID: 5318767)Rutin (PubChem CID: 5280805)

Keywords:Fuzhuan brick tea (FBT)B-ring fission metabolites of catechins (BRFCs)Fuzhuanin C–FCamellia sinensisEurotium spp.Aspergillus spp.

HPLC analysis of samples from four major fermentation procedures of Jing-Wei Fu brick tea showed thatthe level of major tea catechins epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) droppedincreasingly to about 1/3 in the final product. Phytochemical study of the final product led to the discov-ery of four new B-ring fission metabolites of catechins (BRFCs) Fuzhuanin C–F (1–4) together with threeknown BRFCs (5–7), six known catechins (8–13), five simple phenols (14–18), seven flavones and flavoneglycosides (19–25), two alkaloids (26, 27), three triterpenoids (28–30) and one steroid (31). The struc-tures were elucidated by spectroscopic methods including 1D and 2D NMR, LC–HR-ESI-MS, IR, and CDspectra. Five compounds (16–18, 28, 29) were reported for the first time in tea. Possible pathways forthe degradation of major tea catechins and the generation of BRFCs were also provided.

� 2014 Elsevier Ltd. All rights reserved.

Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117 111

1. Introduction

The manufacturing process substantially determines the typesof tea and also relevant chemical constituents of tea, such as thecontent and type of tea polyphenols (Harbowy, Balentine, Davies,& Cai, 1997; Wang et al., 2014). Accordingly, tea could be catego-rised into four major types: unfermented (green tea belongs to thistype), semi-fermented (such as Oolong tea), fully fermented (blacktea), and post-fermented (dark tea, such as Fu brick tea, FBT) basedon the increasing degree of fermentation of tea (Ho, Lin, & Shahidi,2008; Jiang et al., 2011; Kim, Goodner, Park, Choi, & Talcott, 2011).

Dark tea is of great interest due to the upsurge in popularity ofPuer tea. Brick dark tea is a kind of unique post-fermented tea withbrick form compressed from the older, coarse and rough leaves,and small branches of Camellia sinensis var. sinensis and C. sinensisvar. assamica, mainly in Hunan, Sichuan, Shaanxi, and Yunnanprovinces in China. It has been reported that brick dark tea has spe-cial health benefits, such as antihyperlipidaemic (Fu et al., 2011),anti-obesity (Li, Liu, Huang, Luo, et al., 2012), antibacterial (Amy,Tiffany, Corey, & Elizabeth, 2013), antioxidant (Cheng et al.,2013), inhibiting fat deposition (Peng et al., 2014) and so on. Itstypical fungal aroma has also been studied (Xu, Mo, Yan, & Zhu,2007). According to the material and manufacturing process, brickdark tea products can be categorised into several types: Heizhuanbrick tea, Huazhuan brick tea, Fu brick tea (FBT), Qingzhuan bricktea, Kangzhuan brick tea, and Pu-erh brick tea (Wan, 2010).

The changes of major tea polyphenols, especially EGCG, werereported in partially fermented Oolong tea, fully fermented blacktea, as well as post fermented dark tea. Tea catechins containedin green tea are higher than other types of tea because polyphenoloxidase and native microflora are inactivated in freshly plucked tealeaves after being immediately steamed or pan-fired (Toschi et al.,2000). However, during fermentation of black tea, polyphenol oxi-dase in the tea leaves catalyses the oxidation of the major cate-chins into theaflavin, hence reducing the catechins content(Friedman, Levin, Choi, Lee, & Kozukue, 2009). The characteristicreddish-black colour, reduced bitterness and astringency, andremoval of leafy and grassy flavour are derived from this oxidationprocess, giving black tea a marked distinction from green tea(Wang & Helliwell, 2000). As for puer tea, it has also been sug-gested that the levels of major catechins are reduced by oxidationand polymerisation through thermal and enzymatic reactions (Xieet al., 2009; Zuo, Chen, & Deng, 2002). For FBT, during the floweringprocedure, the level of tea catechins especially EGCG and ECGgreatly decreased, mainly due to enzymatic oxidation when thelarge population of microorganisms appear and release extracellu-lar enzymes. It was reported that the major reaction in the process-ing of FBT is oxidative polymerisation which lead to reduction ofcoarse astringency and increase of alcoholic taste and thusimproves the quality and taste of FBT (Fu et al., 2008). Several Bring fission metabolites of catechins (BRFCs) were also reported(Jiang et al., 2011; Kanegae et al., 2013; Luo et al., 2013;Wulandari et al., 2011), which are obviously distinct from the poly-merised products and may also contribute to the unique flavourand quality of FBT.

A number of studies have reported on FBT, including chemicalanalysis (Amy et al., 2013) and components purification (Linget al., 2010; Luo et al., 2012, 2013). However, to date, we are notaware of any HPLC analysis of the major tea polyphenolic changesduring the processing of FBT. Additionally, there is no systematicchemical purification study on Jing-Wei FBT, a kind of FBT tradi-tionally consumed in the northwestern area of China. In this paper,we studied the chemical constituents of Jing-Wei Fu brick tea.Through extensive liquid chromatography, 31 compounds wereisolated. Four new BRFCs, Fuzhuanin C–F, together with 27 known

compounds, were identified by IR, NMR, HR-ESI-MS, and CD spec-troscopy. A dynamic HPLC analysis of the degree of fermentationon the levels of major tea catechins during processing was con-ducted and a possible pathway for the generation of BRFCs wasalso provided.

2. Materials and methods

2.1. Instrumentation

IR was measured on a Thermo Nicolet 8700 FT-IR spectropho-tometer. 1H NMR and 13C NMR, HSQC and HMBC spectra wererecorded in dimethyl-d6 sulfoxide (DMSO-d6) with Bruker AM-400 spectrometers operating at 400 MHz for 1H NMR and100 MHz for 13C NMR, respectively. The Agilent 6210 HPLC/time-of-flight MS system with a binary high-pressure mixing pump, anauto sampler, a column oven, a photodiode array detector (PAD),a high resolution (HR) time-of-flight (TOF) MS, an ESI source (neg-ative mode) and an Agilent workstation was from Agilent Technol-ogies (Santa Clara, CA). HPLC was performed on a Waters 2695separation module combined with a Waters 2489 UV detector. Cir-cular dichroism (CD) spectra were detected with a JASCO J-815spectropolarimeter (JASCO, Tokyo, Japan).

2.2. Chemicals, tea materials, and extraction for HPLC analysis

Gallic acid (GA), epicatechin (EC), epigallocatechin (EGC), epi-catechin gallate (ECG), epigallocatechin gallate (EGCG) standardswere purchased from Chengdu Ultrapure Technology Co. Ltd.(Chengdu, China) and identified in our laboratory for analysis. Allof these standards were of a purity higher than 98%.

Jing-Wei FBT for phytochemical research (produced in 2011)was provided by Cangshan tea Company (Xianyang, China). Theprocessing samples of FBT were also supplied by the same com-pany. Briefly, the manufacture of FBT is summarised as steaming,rolling, microbial fermentation and drying (Wan, 2010). Thedetailed processing procedure has been described in several arti-cles (Jin, Chen, & Ji, 2003; Mo, Zhu, & Chen, 2008; Xu et al.,2011). The fungal growth stage (known as flowering stage) is thekey procedure for FBT, during which the chemical constituentschange a lot. HPLC analysis of the major tea catechins was there-fore carried out at the pre-growing stage of the fungi (pre-flower-ing, PF), the sixth day of fungal growth (six days of flowering, SDF),the ninth day of fungal growth (nine days of flowering, NDF), andthe final FBT products (FBTP).

Extracts of different degree of fermentation during processingof Jing-Wei FBT were prepared by ultrasonic extracting 2.5 g ofground tea powder in 100 mL of 70% aqueous methanol twice in12 h (15 min each time). A 2-mL aliquot of the liquid extractswas centrifuged at 10,000 rpm for 10 min and then the superna-tant was passed through a 0.22-lm filter. This filtrate was usedfor HPLC analysis.

2.3. HPLC analysis of major tea polyphenols of Jing-Wei Fu brick tea

HPLC analysis was carried out using a XP ODS-A C18 column(250 mm � 4.6 mm i.d., 5 lm, H&E Co. Ltd., PR China). Columntemperature was set at 30 �C. The eluant was composed of mobilephase A (water containing 0.17% acetic acid) and mobile phase B(acetonitrile). The gradient of mobile phase B was as follows:0–4 min, 6%; 4–16 min, from 6% to 14%; 16–22 min. from 14% to15%; 22–32 min, from 15% to 18%; 32–37 min, from 18% to 29%;37–45 min, from 29% to 45%; 45–50 min, 45%; 50–51 min, from45% to 6%; then kept at 6% for 10 min. Solvent flow rate was

112 Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117

1.0 mL/min and injection volume was 5 lL. The UV detectionwavelength was 280 nm.

The linear calibration curves contained five different concentra-tions of each reference compound diluted with methanol. Eachconcentration was measured three times. All calibration curveswere constructed by plotting the peak areas of the standard sub-stances versus the corresponding concentration of the injectedstandard solutions for quantitative analysis: EGCG (r2 = 0.9994,37.5–1000 lg/mL, RT = 29.15 min), EGC (r2 = 0.9995, 37.5–1000 lg/mL, RT = 20.80 min), ECG (r2 = 0.9997, 12.5–625 lg/mL,RT = 40.56 min), EC (r2 = 0.9998, 12.5–630 lg/mL, RT = 27.92 min);GA (r2 = 0.9996, 11.25–300 lg/mL, RT = 7.06 min).

2.4. Isolation of phytochemicals from Jing-Wei Fu brick tea

Jing-Wei FBT (15 kg) was ground and extracted with petroleumether, ethyl acetate, and methanol three times (3 � 20 L) at roomtemperature. The extracts were concentrated under reduced pres-sure to afford three residues: fraction A (the petroleum ether frac-tion, 165 g), fraction B (the ethyl acetate fraction, 200 g), andfraction C (the methanol fraction, 2 kg). Fraction C was extractedwith dichloromethane and n-butyl alcohol, respectively. The n-butyl alcohol fraction (fraction D) was concentrated under reducedpressure to afford residue (450 g).

Fraction A was separated by silica gel column chromatography(CC), eluting with a mixture of petroleum ether:ethyl acetate withincreasing polarity (1:0 to 0:1), yielding thirteen fractions (A1 toA13). Fractions A10 (6 g) and A12 (5 g) were subjected to ODS CCeluted with methanol to yield 28 (122 mg, 2-hydroxydiplopterol),29 (32 mg, canophyllol), 30 (12 mg, 3b, 6a, 13b-trihydroxyolean-7-one), 31 (1.2 g, a-spinasterol). Fraction B was applied to apolyamide CC, eluting with a mixture of dichloromethane:ethylacetate:methanol with increasing polarity (2:1:0–0:1:2), yieldingeight fractions (B1–B8). Fraction B5 (11 g) was subjected to a silicagel CC using dichloromethane:methanol:formic acid (20:1:0.5) asthe eluant, yielding 15 (260 mg, 2,5-dihydroxybenzoic acid), 19(136 mg, quercetin), 20 (154 mg, kaempferol). Fraction B7 (4.2 g)was subjected to a Sephadex LH-20 CC, yielding 21 (112 mg,myricetin). Fraction B8 (1.1 g) was subjected to a Sephadex LH-20 CC, yielding 8 (110 mg, epicatechin), 9 (396 mg, epigallocate-chin), 14 (80 mg, gallic acid). Fraction D was applied to a silicagel CC using dichloromethane:methanol:water (6:1:0.1) as the

Table 11H (d ppm, J Hz, s: single peak; d: double peaks; br s: broad single peak; m: multipeaks)

Pos. 1 2

C + DEPT H C + DEPT H

2 82.4d 4.37(1H, br d, J = 10.6 Hz) 77.3d 43 73.2d 4.33(1H, m) 71.6d 44b 21.2t 2.53(1H, dd, J = 15.2, 6.0 Hz) 20.1t 24a 2.64(1H, dd, J = 15.2, 3.6 Hz)5 155.7s 156.1s6 96.6d 5.94(1H, d, J = 2.0 Hz) 95.9d 57 156.3s 156.4s8 96.2d 5.71(1H, d, J = 2.0 Hz) 94.3d 59 154.8s 153.1s10 100.9s 97.0s10 51.3d 2.30(1H, dd, J = 11.6, 5.2 Hz) 79.2d 420b 32.1t 2.47(1H, m) 36.1t 220a 2.53(1H, m) 330 172.1s 175.4s40 105.4s 93.1s50 19.8q 1.20(3H, s, CH3) 35.5t 360 48.0q 3.10(3H, s, OCH3) 169.9s70 51.3q 3.62(3H, s, OCH3) 51.6q 35-OH 9.19(1H, s) 97-OH 8.99(1H, s) 9

eluant, yielding four fractions (D1–D4). Fraction D1 (26 g) was sub-jected to a silica gel CC eluted with dichloromethane:methanol(30:1), yielding four subfractions (D1-1 to D1-4). Compound 26(1.0 g, caffeine) was obtained from subfraction D1-1 by recrystalli-sation. Subfraction D1-2 (2.0 g) was subjected to Sephadex LH-20,polyamide CC, and HPLC to yield 2 (3.5 mg, Fuzhuanin D), 3(1.2 mg, Fuzhuanin E), 4 (1.0 mg, Fuzhuanin F). Subfraction D1-3(4.2 g) was subjected to Sephadex LH-20, polyamide, and silicagel CC to yield 1 (4.2 mg, Fuzhuanin C), 5 (22 mg, planchol A), 6(3.0 mg, xanthocerin), 18 (32 mg, gallicin). Subfraction D1-4(3.3 g) was subjected to Sephadex LH-20 and MCI gel CC to yield13 (23 mg, epiafzelechin), 16 (56 mg, phloroglucinol), 17 (62 mg,pyrogallol). Fraction D2 (16 g) was subjected to Sephadex LH-20and polyamide CC to yield 7 (38 mg, teadenol A), 25 (12 mg, taxif-olin), 27 (250 mg, theobromine). Fraction D3 (23 g) was subjectedto Sephadex LH-20, polyamide and MCI gel CC to yield 10 (119 mg,epicatechin gallate), 11 (31 mg, epigallocatechin gallate), 12(12 mg, epicatechin-3-O-(40-O-methyl)gallate). Also fraction D4(33 g) was subjected to Sephadex LH-20, polyamide and MCI gelCC to yield 22 (22 mg, astragalin), 23 (21 mg, nicotiflorin) and 24(52 mg, rutin).

HPLC separation of compounds 3 and 4 was carried out using anXP ODS-A C18 column (250 mm � 4.6 mm i.d., 5 lm). Columntemperature was set at 30 �C. The eluant was composed of mobilephase A (methanol) and mobile phase B (water). The gradient ofmobile phase A was as follows: 0–15 min, 50%; 15–17 min, from50% to 100%; 17–20 min, 100%; 20–22 min, from 100% to 50%; thenkept at 50% for 10 min. Eluant was performed at a solvent flow rateof 1.0 mL/min. The injection volume was 20 lL. The UV detectionwavelength was monitored at 280 nm and 210 nm.

The detailed purification procedure can be found in Supplemen-tary Fig. 1 and the structures of all these compounds are given inSupplementary Fig. 2.

Fuzhuanin C (1): white powder (CH3OH). IR (KBr) mmaz 3181,1731, 1605, 1518, 1467, 1380, 1139, 996, 822 cm�1. CD De (nm):+10.8 (240), �3.6 (280) (c 0.10, CH3OH). 1H and 13C NMR data(DMSO-d6) d see Table 1. HR-ESI-MS�: m/z 323.12384 [M�H]�,(Calc. 323.11363 for C16H19O7). IR, 1D and 2D NMR, HRMS, UVspectra are arranged in the Supplementary material.

Fuzhuanin D (2): white powder (CH3OH). IR (KBr) mmaz 3157,1785, 1732, 1607, 1521, 1469, 1373, 1150, 995, 823 cm�1. CD De(nm): +12.7 (240), �3.4 (280) (c 0.10, CH3OH). 1H and 13C NMR

and 13C NMR (d ppm, d: CH; t: CH2; q: CH3) data for compounds 1, 2 and 5.

5 (planchol A)

C + DEPT H

.47 (1H, br s) 79.1d 4.27 (1H, d, J = 2.0 Hz)

.43 (1H, br s) 73.0d 4.53 (1H, dd, J = 5.0, 2.0 Hz)

.67 (2H, m) 19.9t 2.72 (1H, m)2.65 (1H, m)

156.2s.92 (1H, d, J = 1.6 Hz) 95.6d 5.91 (1H, d, J = 2.0 Hz)

156.4s.71 (1H, d, J = 1.6 Hz) 94.0d 5.70 (1H, d, J = 2.0 Hz)

153.9s96.7s

.68 (1H, d, J = 7.0 Hz) 50.5d 3.02 (1H, s)

.56 (1H, d, J = 18.0 Hz) 31.2t 3.00 (1H, m)

.13 (1H, dd, J = 18.0, 7.0 Hz) 2.68 (1H, m)174.7s115.6s

.16 (2H, s) 24.7q 1.51 (3H, s)

.65 (3H, s, OCH3)

.28 (1H, s) 9.29 (1H, s)

.00 (1H, s) 8.98 (1H, s)

Table 21H (d ppm, J Hz, s: single peak; d: double peaks; brs: broad single peak; m: multipeaks) and 13C NMR (d ppm, d: CH; t: CH2; q: CH3) data for compounds 3, 4 and 6.

Pos. 3 4 6 (xanthocerin)

C + DEPT H C + DEPT H C + DEPT H

2 71.9d 4.58 (1H, br d, J = 10.8 Hz) 138.4s 68.3d 4.43 (1H, br s)3 73.6d 4.52 (1H, ddd, J = 10.8, 10.4, 6.0 Hz) 71.0d 5.18 (1H, br s) 70.8d 4.87 (1H, m)4b 26.0t 2.58 (1H, dd, J = 15.2, 10.4 Hz) 25.9t 2.37 (1H, dd, J = 16.0, 10.0 Hz) 23.4t 2.78 (2H, dd, J = 6 Hz)4a 2.95 (1H, dd, J = 15.2, 6.0 Hz) 3.20 (1H, dd, J = 16.0, 6.5 Hz)5 157.1s 156.5s 156.5s6 96.4d 5.99 (1H, d, J = 2.4 Hz) 96.6d 6.00 (1H, d, J = 2.4 Hz) 95.9d 5.95 (1H, d, J = 2.4 Hz)7 157.4s 157.9s 156.6s8 94.2d 5.79 (1H, d, J = 2.4 Hz) 93.5d 5.85 (1H, d, J = 2.4 Hz) 94.0d 5.70 (1H, d, J = 2.4 Hz)9 154.2s 152.6s 154.8s10 98.0s 96.4s 96.3s10 162.4s 167.2s 163.7s20 117.2d 5.90 (1H, q, J = 1.6 Hz) 33.8t 3.05 (1H, m) 118.6d 5.99 (1H, q, J = 1.6 Hz)

3.24 (1H, m)30 158.4s 103.2s 154.9s40 17.0q 2.07 (3H, brs) 13.2q 1.69 (3H, s) 20.4q 2.10 (3H, d, J = 1.6 Hz)5-OH 9.52 (1H, s) 9.58 (1H, s) 9.41 (1H, s)7-OH 9.17 (1H, s) 9.27 (1H, s) 9.07 (1H, s)

Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117 113

data (DMSO-d6) d see Table 1. HR-ESI-MS�: m/z 335.07678 [M�H]�

(Calc. 335.07724 for C16H15O8). IR, 1D and 2D NMR, HRMS, UVspectra are arranged in the Supplementary material.

Fuzhuanin E (3): white powder (CH3OH). IR (KBr) mmaz 3334,1685, 1622, 1524, 1478, 1394, 1183, 1066, 809 cm�1. CD De(nm): +12.5 (210), �2.3 (250) (c 0.10, CH3OH). 1H and 13C NMRdata (DMSO-d6) d see Table 2. HR-ESI-MS�: m/z 247.06156 [M�H]�

(Calc. 247.0612 for C13H11O5). IR, 1D and 2D NMR, HRMS, UV spec-tra are arranged in the Supplementary material.

Fuzhuanin F (4): white powder (CH3OH). IR (KBr) mmaz 3209,1705, 1605, 1518, 1444, 1392, 1164, 1046, 824 cm�1. CD De(nm): +7.1 (192.5), �9.3 (250) (c 0.10, CH3OH). 1H and 13C NMRdata (DMSO-d6) d see Table 2. HR-ESI-MS�: m/z 247.06007 [M�H]�

(Calc. 247.0612 for C13H11O5). IR, 1D and 2D NMR, HRMS, UV spec-tra are arranged in the Supplementary material.

3. Results and discussion

3.1. HPLC analysis of changes in major tea polyphenols during thepost-fermentation

The results are given in Fig. 1 and the HPLC chromatograms ofthe four extracts during fermentation are given in SupplementaryFig. 3. During the period of the fungal post-fermentation, the levelsof galloyl catechins dropped, especially those of EGCG and ECG,which dropped from the beginning of the fermentation to the final

Fig. 1. HPLC analysis of major tea catechins of Jing-Wei FBT during post-fermentation; samples were PF (preflowering), SDF (sixth day of flowering), NDF(ninth day of flowering), and FBTP (final Fuzhuan brick tea product); the data werepresented as means ± SD in triplicate.

product. Both levels of EGCG and ECG of the FBT products droppedto about one-third level of PF while the level of EGC increased. Thelevel of GA increased till the sixth day of fermentation because ofproduction of GA from degradation of galloyl catechins and/or gal-lotannins by microbial tannase (Bhat, Singh, & Sharma, 1998).However, the level of GA and EC in the final product decreased,suggesting that GA and EC continued to degrade from the ninthday of flowering to form more simple phenols (Tanaka et al.,2011). Interestingly, simple phenols such as 2,5-dihydroxybenzoicacid (15), phloroglucinol (16), and pyrogallol (17) were purifiedfrom the FBT product. In addition, EC and EGC also encounteredchanges, especially on the B-ring, to form B-ring fission metabo-lites of catechin derivatives (BRFCs, 1-7). EGCG is the most abun-dant and active catechin and it is often used as a qualityindicator (Lakenbrink, Lapczynski, Maiwald, & Engelhardt, 2000;Wang & Helliwell, 2000; Wang, Zhou, & Jiang, 2008). As there areobvious changes in the levels of EGCG, ECG, and GA during process-ing, these three chemicals could be used as indices for quality con-trol and real-time study of the processing of Jing-Wei Fu Brick teaafter further analysis of more samples.

To better understand the above changes of major tea polyphe-nols in final Jing-Wei FBT and what the catechins were trans-formed into, a system purification of the chemical constituentswas conducted. Thirty-one compounds were isolated and identi-fied including simple phenols and seven BRFCs. Detailed structuralelucidation of the new BRFCs (Fuzhuanin C–F, 1–4) is given in thefollowing section.

3.2. Fuzhuanin C and D

Compound 1 was isolated as a colourless powder. Its molecularweight was decided as C16H20O7 by HR-ESI-MS, with 7 degrees ofunsaturation. The IR spectrum (cm�1) indicated the presence ofhydroxyl group (broad peak around 3181), ester carbonyl group(1731), and aromatic ring (1605, 1518). The 1H-NMR spectrum issimilar to that of epicatechin (8) and exhibits the signals attribut-able to the A-ring (d 5.71 and 5.94, d, J = 2.0, H-8 and H-6; d 8.99and 9.19, each s, OH-7 and OH-5) and the C-ring (d 4.37 and4.33, H-2 and H-3; 2.53 and 2.64, H2-4). However, it has no aro-matic B ring signals compared with epicatechin, which indicatesthat it may be a B-ring fission metabolite of catechin (Jiang et al.,2011; Luo et al., 2013). The TLC showed very weak colourationwith the FeCl3 reagent, which also confirmed it as a BRFC (Jianget al., 2011). Compared with known BRFC planchol A (5), com-pound 1 has two methoxyl groups (dH 3.62 and 3.10; dC 51.3q,

114 Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117

48.0q). The 13C-NMR and DEPT spectra of compound 1 also indi-cated the presence of A- and C-ring moieties of flavan-3-ol: onephloroglucinol type aromatic A-ring (dC 155.7s, 96.6d, 156.3s,96.2d, 154.8s, 100.9s), two oxygenated methine (dC 82.4d, 73.2d),and one methylene (dC 21.2t). The remaining signals observed in1H-NMR, 13C-NMR, and DEPT spectra were assigned to onemethine (dC 51.3d, dH 2.30), one methylene (dC 32.1t, dH 2.47 and2.53), one ester carbonyl (dC 172.1s), one oxygenated quaternarycarbon in very low field (dC 105.4s), and two methoxyl groups (dC

51.3q, 48.0q and dH 3.62s, 3.10s).The HMBC correlations [dC 172.1 (C-30)/dH 3.62 (OCH3); C-30/dH

2.47 and 2.53 (H2-20); H-20/dC 105.4 (C-40), 82.4 (C-2) and 51.3(C-10); dH 1.20 (H3-50)/C-10 and C-40; C-40/dH 3.10 (OCH3)] can easilydecide the fragment CH3OCO-CH2-CH-C(-O-) (OCH3)-CH3 which isattached to positions C-2 and C-3, to form a saturated furan ring(THF). Thus, the proposed structure is shown in Fig. 2. The NOESYcorrelation dH 3.10 for (OCH3)/H-3 suggest that they have the same

Fig. 2. The structures o

Fig. 3. The key HMBC (solid single arrowhead line), NOESY (dashed double arro

orientation while the H-50/H-10 correlation suggests these two pro-tons also have the same orientation (Fig. 3). Thus, the stereochem-istry of the compound can be decided. The similar CD spectrum tothat of planchol A (5) further confirmed the structure of compound1 as shown in Fig. 2. Compound 1 was named as Fuzhuanin C.

Compound 2 was isolated as a colourless powder. Its molecularweight was decided as C16H16O8 by HR-ESI-MS, with 9 degrees ofunsaturation. The IR spectrum (cm�1) suggested the presence ofhydroxyl group (broad peak around 3157), ester carbonyl group(1732), five-membered lactone (1785), and aromatic ring (1607,1521). Just like Fuzhuanin C (1), the 1H NMR spectrum showed thatcompound 2 has no aromatic B-ring signals either. An ABX systemat d 4.68 (d, J = 7.0 Hz, H-10), 3.13 (dd, J = 18.0, 7.0 Hz, H-20a), 2.56(d, J = 18.0 Hz, H-20b) indicated the presence of an O-CHCH2-frag-ment. These units (fragments) can also be confirmed by the1H-1H COSY spectrum. In addition, it has a unique singlet methy-lene unit at d 3.16 (s, H-50) and a methoxyl group (3.65, s, OCH3).

f compounds 1–8.

whead line) correlations of 1–4 (up), and CD spectra of 1–6 and 8 (down).

Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117 115

The 13C NMR and DEPT spectrum indicated the presence of twocarbonyl groups (d 175.4 (s, C-30) and 169.9 (s, C-60), one quater-nary carbon at d 93.1 (s, C-40). The HMBC correlations at dH 3.65(OCH3)/C-60, C-60/H-50, H-50/C-40 and 79.2(C-10), dH 4.68 (H-10)/C-30 and 36.1 (C-20) indicated the presence of a CH3OCO-CH2-C-CH-CH2-CO-unit. The HMBC signals at H-10/71.6 (C-3) and H-50/dC

77.3 (C-2) deduced that the above CH3OCO-CH2-C-CH-CH2-CO-unitwas connected with C-2 and C-3. Therefore, the structure can bededuced as shown in Fig. 2. The stereochemistry can be deducedby coupling constants (J-values) and NOESY spectrum. The smallcoupling constants between H-2 and H-3 (both broad singlet) indi-cated a cis-orientation for these two protons. The NOESY correla-tions H-10/H-50 indicated that these two protons were at the a-orientation (Fig. 3). The similar CD spectrum to that of plancholA (5) further confirmed the structure of compound 2, as shownin Fig. 2. Compound 2 was named as Fuzhuanin D.

Biosynthetically, Fuzhuanin C (1), Fuzhuanin D (2), and plancholA (5) may derive from epicatechin (8) through enzyme-catalysedoxidative cleavage of the B-ring and then a sequential or simulta-neous cycloaddition procedure (Luo et al., 2013). These proposedbiosynthetic pathways and the similar CD spectra of the four com-pounds further confirmed the stereochemistry of the two newcompounds (Fig. 3).

Fig. 4. Possible pathways for degradation of tea cate

3.3. Fuzhuanin E and F

Compound 3 was isolated as a colourless powder. Its molecularweight was decided as C13H12O5 by HR-ESI-MS, with 8 degrees ofunsaturation. The IR spectrum (cm�1) suggested the presence ofa hydroxyl group (broad peak around 3334), a conjugated carbonylgroup (1685), and aromatic ring (1622, 1524). The 1H-NMR spec-trum suggested it could be a derivative of (+)-catechin, by analysisof the characteristic signals at d 5.79, 5.99 (both d, J = 2.4 Hz, H-8and H-6) at the A-ring and d 4.58 (brd, J = 10.8 Hz, H-2), 4.52(ddd, J = 10.8, 6.0, 10.4 Hz, H-3), 2.58 (dd, J = 15.2, 10.4 Hz, H-4b),2.95 (dd, J = 10.8, 6.0 Hz, H-4a). Again the signals of the B-ringwere not observed. In addition, TLC showed very weak colourationwith the FeCl3 reagent. Thus, compound 3 is another BRFC. Its NMRdata are very similar to those of xanthocerin (6) (Table 2) exceptfor signals at positions 2–4. The bigger coupling constant of H-2and H-3 (J = 10.8) suggested a trans-orientation for the two pro-tons, which is different from those of xanthocerin, which has acis-orientation for the corresponding two protons. In addition,the NOESY correlation H-2/H-4b and the methyl group (d 2.07, s)indicated that they had the same orientation while the NOESY cor-relation H-3/H-4a indicated that these two protons also had thesame orientation (Fig. 3). Its CD spectrum is very similar to that

chins in brick dark tea and generation of BRFCs.

116 Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117

of xanthocerin, which suggested that they have the same stereo-chemistry at position C-2 (2R) (Korver & Wilkins, 1971). Basedon the coupling constant of H-2/H-3, the NOESY correlations andCD spectrum, the stereochemistry of compound 3 was determinedas 2R, 3S. Based on the above evidence, the structure was estab-lished as shown in Fig. 2. Compound 3 was named as Fuzhuanin E.

Compound 4 was isolated as a colourless powder. Its molecularweight was decided as C13H12O5 by HR-ESI-MS, with 8 degrees ofunsaturation. The IR spectrum (cm�1) suggested the presence ofa hydroxyl group (broad peak around 3209), carbonyl group(1705), and aromatic ring (1605, 1518). Compound 4 is very similarto compound 3. Interestingly, compounds 3 and 4 were initiallypurified as one compound. All the 2D-NMR was thus conductedon the mixture of the two compounds in DMSO-d6 (Supplementarymaterial). The two compounds were finally separated by HPLC. TheIR, CD, HPLC-PDA–HR-ESI-MS, and 1H NMR spectra of compounds3 and 4 were then measured again (Supplementary material).Compounds 3 and 4 have the same molecular weight, confirmingthey are a pair of isomers. NMR data of 4 indicated the presenceof a methyl group (dH 1.69 s, dC 13.2q), a lactone (dC 167.2s), onetotally substituted double bond (dC 103.2s, 138.4s), and one oxy-genated methine (dH 5.18 brs, dC 71.0d). In addition, it has onemore methylene signal (dC 33.8t, C-20; dH 3.05 and 3.24, H2-20).The above data are different from those of compound 3, suggestedthat a double bond had moved from one side to the other side atthe position of C-30. Thus, the structure of compound 4 was decidedas shown in Fig. 2 and it was named as Fuzhuanin F.

Fuzhuanin E (3) and Fuzhuanin F (4) are two isomers of xan-thocerin (6). Their CD spectra are similar except that the positiveCotton effect is at shorter and shorter wavelength (3 is at210 nm, 4 at 192.5 nm) compared to that of xanthocerin(220 nm) (Fig. 3). Biosynthetically, the small amount of compounds3 and 4 may derive from (+) catechin, which is also present in smallamounts in tea infusion. It was reported that (+)-catechin can betransformed into BRFCs by many microorganisms (Das, Lamm, &Rosazza, 2011); in this paper two new B-ring fission lactone prod-ucts of (+)-catechin were produced. It was proposed that the diaci-dic compound from the cleavage of the B-ring could be theintermediate of the new BRFCs (Das et al., 2011; Luo et al., 2013).The linear 6/6/6 tricyclic BRFCs 3 and 4 could be formed throughrecyclisation from a diacidic intermediate (Luo et al., 2013; Fig. 4).

A Japanese fermented tea, which was selectively fermentedwith Aspergillus spp., was reported to produce teadenol A (7) andteadenol B (Wulandari et al., 2011), both of which are linear 6/6/6 tricyclic BRFCs which share the same skeleton with compounds3 and 4. In addition, Aspergillus spp. was also reported to exist inthe final products of FBT (Luo et al., 2013) and teadenol A (7)was also found in this study, which suggested that compounds 3and 4 may be transformed by Aspergillus spp.

Since very few BRFCs have been found, very little bioassay workhas been reported on them, such as their antiproliferative (Luoet al., 2013) and cytotoxic effects (Chang & Case, 2005; Luo et al.,2013). More thorough bioactivity research on BRFCs is needed forcomprehensive evaluation of the health benefits of brick dark tea.

3.4. Possible pathway for degradation of tea catechins and generationof BRFCs in brick dark tea

The post-fermented tea is characterised by the microbial fer-mentation procedure. During the post-fermentation procedure,complex biochemical reactions such as microbial enzyme andautoxidation lead to the polymerisation or decomposition of teacatechins, which produce catechin polymers or simple phenoliccompounds, respectively. The polymerisation of catechins occursthrough hydrogen-peroxide-dependent peroxidase oxidation, pol-yphenol oxidase oxidation and autoxidation (Das et al., 2011;

Tanaka, Matsuo, & Kouno, 2010); such reactions occur mainly onthe A-ring of catechins. Interestingly, since the first BRFC wasfound from FBT, seven BRFCs had been found only in brick darktea up to date (Jiang et al., 2011; Kanegae et al., 2013; Luo et al.,2013; Wulandari et al., 2011), in which all reactions happenedon the B-ring. It was suggested that BRFCs originated from tea cat-echins through oxidation and subsequent recyclisation of theB-ring by the micro-organisms in FBT (Jiang et al., 2011; Luoet al., 2013). The biosynthetic pathway of BRFCs was firstly studiedby Das et al. (2011), based on 18O2 labelling. Possible pathways forlactone formation of the two new BRFCs transformed from (+)-cat-echin involved initial dioxygenase-mediated meta-B-ring cleavagefollowed either by aldehyde oxidation to a dicarboxylic acid thatlactonises, or by hemiacetal (lactal) formation followed by alcoholoxidation. Possible pathways for the production of Fuzhuanin Aand B, planchol A (5), and xanthocerin (6) were proposed in arecent paper (Luo et al., 2013). Both of the pathways suggestedthe formation of BRFCs through a diacidic or dicarboxylic acidintermediate. As shown in Fig. 4, galloyl catechins (EGCG, ECG,GCG, CG) endured a gradual decomposition during microbial fer-mentation. At the beginning, they were degalloylated throughhydrolysis by the effect of heat treatment during the first stageof processing of FBT, and related degalloyl catechins (EGC, EC, C,GC) together with GA were produced. This step was confirmedby the HPLC analysis of changes in the levels of major tea polyphe-nols during post-fermentation procedure (Fig. 2).

In conclusion, besides being degraded to form simple phenols,the degalloyl catechins were also degraded to three major classesof BRFCs (the B-ring lactone type, the linear 6/6/6 tricyclic type,and the angular 6/6/5/5 tetracyclic type) through three differentrecyclisation pathways of the oxidised dicarboxylic acid intermedi-ate (Fig. 4).

4. Conclusion

In the present study, HPLC-based analysis of major tea polyphe-nolic profiles can easily provide fermentation behaviour of tea cat-echins and GA during processing of Jing-Wei FBT, and thus providea better understanding of unique changes in tea metabolites duringtea fermentation. A systematic study on the chemical componentsof FBT was performed and four new BRFCs together with 27 othercompounds were isolated and identified from Jing-Wei FBT for thefirst time. The possible mechanism of the changes of major teapolyphenols and the possible pathway of the formation of BRFCswere also provided. The galloyl catechins firstly decomposed intonon-galloyl catechins and GA. On the one hand, the subsequentnon-galloyl catechins and GA continued the decompositiontendency to firm simple phenols. On the other hand, they weretransformed into three types of BRFCs through initial dioxygen-ase-mediated meta-B-ring cleavage followed by oxidation to adicarboxylic intermediate that was subsequently recyclisedthrough three different pathways.

Funding

The authors declare no competing financial interest.

Acknowledgements

We thank Ming-Jie Chu, Department of Chemistry, AnhuiAgricultural University, for IR recording. Financial assistance wasreceived with appreciation from Anhui Agricultural UniversityTalents Foundation (YJ2011-06), the Earmarked Fund for ModernAgro-industry Technology Research System in Tea Industry ofChinese Ministry of Agriculture (nycytx-26), and Program for Chang-jiang Scholars and Innovative Research Team in University IRT1101.

Y.-F. Zhu et al. / Food Chemistry 170 (2015) 110–117 117

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2014.08.075.

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