Food Chemistry 152 (2014) 539–545

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8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols as the marker compounds of Chinese dark teas formed in the post-fermentation process provide significant antioxidative activity Weinan Wang 1, Liang Zhang 1, Shu Wang, Shepo Shi, Yong Jiang, Ning Li, Pengfei Tu ⇑ State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

a r t i c l e

i n f o

Article history: Received 25 July 2013 Received in revised form 6 September 2013 Accepted 21 October 2013 Available online 1 November 2013 Keywords: Antioxidant Aspergillus niger Chinese dark tea Flavan-3-ols Post-fermentation

a b s t r a c t Phytochemical investigation of the aqueous extract of pu-erh tea afforded eight novel 8-C N-ethyl-2pyrrolidinone substituted flavan-3-ols (puerins I–VIII) by 1H, 13C, two-dimensional nuclear magnetic resonance (NMR) and high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD–ESI/MS) analysis. Comparative chemical analysis of green tea, black tea and Chinese dark teas confirmed that these compounds were the marker compounds of Chinese dark teas. Furthermore, fungal fermentation was indispensable for the biosynthesis of these novel compounds. Through single fungal fermentation, it was proved that catechins and theanine were the precursors of puerins I–VIII. HPLC-DAD-ESI/MS analysis elucidated the biosynthetic pathway for puerins I–VIII. Puerins I–IV have potential protective effects for the human micro-vascular endothelial cells (HMEC) injury induced by hydrogen dioxide compared to other tea polyphenols. 8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols could be used in the quality control and authentication of Chinese dark teas. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Chinese dark tea (CDT) is the special post-fermented tea product based on mature tea leaves treated by microbial fermentation. In China and southeast Asia, CDTs are popular for their special flavour, being quite different from green tea. Post-fermentation process has been trusted to be critical for the formation of CDTs’ characteristics. The production places of CDTs are mainly located in southwestern China, such as Yunnan, Hunan and Shaanxi province. As a famous CDT, the so called pu-erh tea has been widely reported with many healthcare functions and chemical constituents (Hwang, Lin, Liuchang, & Shiao, 2002; Wu et al., 2007; Xie et al., 2009). Flavan-3-ols are the major compounds of green tea. They basically inherit the main secondary metabolites of tea plants (Camellia sinensis and C. assamica). However, these tea polyphenols have varied considerably by different manufacture processes. Fermentation highly changes the contents of tea polyphenols. For example, fullfermentation promoted the oxidation of catechins, and formed theaflavins of black tea (Aneja, Odoms, Denenberg, & Wong, 2004), while semi-fermented oolong tea possessed a series of oolonghomobisflavans (Hashimoto, Nonaka, & Nishioka, 1989). In CDTs, post-fermentation decreased the contents of catechin ⇑ Corresponding author. Fax: +86 10 8280 2750. 1

E-mail address: [email protected] (P. Tu). These authors contribute equally to this work.

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.10.117

-gallates and increased the content of gallic acid (Zhang, Li, Ma, & Tu, 2011). It also structurally produced some catechin derivatives. It was reported that two 8-C substituted flavan-3-ols had already been identified in pu-erh tea, but the marker compounds of CDTs formed within the post-fermentation process remains unknown (Zhou, Zhang, Xu, & Yang, 2005). In this study, we aim to find the compounds that exclusively exist in ripened pu-erh tea, by the HPLC-DAD-ESI/MS. Through microbial isolation and identification, the interaction of fungi and tea polyphenols were also studied.

2. Materials and methods 2.1. Instrumentation Optical rotations were measured on a P-1020 Polarimeter (Jasco, Tokyo, Japan). Infrared spectra were measured on Thermo Nicolet Nexus 470 fourier transform infrared spectroscopy spectrophotometer. Proton nuclear magnetic resonance (1H NMR) and carbon-13 nuclear magnetic resonance (13C NMR), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) spectra were recorded in dimethyl-d6 sulfoxide (DMSO-d6) with Varian 500 spectrometers operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR, respectively. Mass spectra were performed at a Bruker Daltonics Apex IV 70e spectrometer. High-performance liquid chromatography (HPLC) was performed

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on an Agilent 1100 instrument. Fungal fermentation was carried out with biological safety cabinet (Thermo scientific 1300 series A2) and Thermostat incubator (Thermo scientific Heratherm IGS 60). Circular dichroism (CD) spectrum was detected with JASCO J815 spectropolarimeter. 2.2. Chemicals and plant materials Fresh tea leaves (C. assamica) were collected from the tea garden in Menghai, Yunnan province. Ripened pu-erh tea fermentation materials were provided by the Yunnan Agricultural University and Dayi Pu-erh Tea Company. Green tea and black tea were the famous Longjing green tea and Dianhong black tea, which were bought from Zhejiang and Yunnan province, respectively. Chinese dark teas were purchased from Yunnan, Shaanxi and Hunan province. Pu-erh tea was purchased from Dayi pu-erh Tea Company, Xishuangbanna, Yunnan province. Fu brick tea was purchased from Yiyang Tea Company, Yiyang, Hunan province. Jing-wei Fu tea was provided by Cangshan Tea Company, Xianyang, Shaanxi province. Gallic acid (GA), caffeine (CA), catechin (C), gallocatechin (GC), epigallocatechin gallate (EGCG), epicatechin (EC), epigallocatechin (EGC), and gallocatechin gallate (GCG) standards were purchased from Shanghai Tongtian Biotechnology Co. and identified in our laboratory for fermentation and analysis. All of these standards were with the purity of more than 98%. 2.3. Extraction and isolation Pu-erh tea (1 kg) was extracted with ten times of water at 95 °C for 30 min, continuously extracted for three times. After removal of the water under wave membrane concentration, the aqueous solution afforded precipitates, which were removed by filtration. The filtrate was extracted with CHCl3, ethyl acetate and n-BuOH in sequence. The ethyl acetate extract was separated into 10 fractions by silica gel column chromatography (6.5  35.0 cm) with gradient elution of chloroform and methanol from (1:0 to 0:1). The fraction 7 was subjected to Sephadex LH-20 column chromatography (4.0  50.0 cm) with H2O containing increasing proportions of acetone. The eluent which was obtained by elution of 30–40% acetone revealed the presence of special flavonal-3-ols derivatives by HPLC-DAD-ESI/MS analysis. This fraction was successively subjected to reversed phase C18 column by elution of 30% methanol to get the fraction containing compound 1–6. This fraction was subjected to preparative HPLC to yield compound 1 (5), 2 (10), 3 (5) 4 (10), 5 (3), and 6 (8 mg), respectively. These compounds were used as chemical standards (purity of more than 95%) for identification of individual puerins in tea samples. Compound 1–6 were dissolved in methanol at the concentration of 0.1 mg/ml for HPLC-DAD-ESI/MS analysis. 2.4. HPLC-DAD-ESI/MS analysis Agilent G6300 series HPLC-DAD-ESI/MS system (Santa Clara, CA) consisted of a Surveyor MS pump, an auto sampler, a diode array detector, and an LC/MSD ion trap mass spectrometer with Xcalibur software for data acquisition and analysis. Separations were carried out using an Agilent SB-Aq C18 reverse phase column (250  4.6 mm i.d., 5 lm) protected with a security guard cartridge (Gemini C18, 4  2.0 mm i.d., Phenomenex). The elution used a linear gradient program from 5% to 30% acetonitrile in 0.6% formic acid aqueous solution over 60 min, and then changed to 100% acetonitrile for 10 min. The flow rate was 0.8 ml/min. One gram of each tea sample was extracted with 25 ml of 50% ethanol by ultrasound-assisted extraction for 30 min. The extract was filtered with a 0.22 lm micropore filter before analysis. The injection volume of

each sample was set to be 10 ll. A 15 min re-equilibration time was used between each HPLC run. After passing through the flow cell of the DAD, the column eluate was split to 0.2 ml/min, which was directed to a trap mass spectrometer with an electrospray interface (ESI) operating in full scan MS mode from m/z 100 to 2000. Mass spectra were acquired in both negative and positive modes with an ion spray voltage of 3.5 kV, a capillary temperature of 350 °C, a capillary voltage of 35 V, a sheath gas pressure of 241.3 kPa, and an auxiliary gas pressure of 82.7 kPa. The parameters to build a multiple reaction monitoring (MRM) method were as follow: (1) (2) (3) (4) (5)

Puerins I–IV: m/z 402 ? 250 [M + H]+. Puerins V–VIII: m/z 418 ? 250 [M + H]+. C/EC:m/z 291 ? 123 [M + H]+. GC/EGC:m/z 307 ? 139 [M+H]+. Theanine:m/z 175 ? 158 [M+H]+.

2.5. Isolation, screening and identification of the fungi Six batches of fermentation materials from different stages of pu-erh tea fermentation were selected and screened by the solidphase fermentation. One gram of the fermentation material was washed with sterile saline (10 ml), and then diluted to 101, 102, 103 and 104 of levels. These solutions were respectively incubated on potato dextrose agar (PDA) to allow growth and isolation of microorganisms. As a result, sixteen molds, one yeast and four bacteria were obtained. Through a standard solid-state fermentation protocol, five molds were verified to have potential ability of producing eight marker compounds. The extracted DNA of these five molds was subjected to the amplification of the ITS region fragment for gene sequencing by the China Center of Industrial Culture Collection and they were identified to be: (1) (2) (3) (4) (5)

Aspergillus fumigatus CBS 133.61T (AY685150). Absidia corymbifera NRRL2982 (AF157227). Aspergillus tubingensis CBS 134.48T (FJ629305). Aspergillus niger CBS 554.65T (FJ629288). Aspergillus flavus CBS 100927T (AY819992).

They were preserved in our laboratory at 80 °C. The culture broth used for fungal incubation was Martin Broth, Modified (MBM) agar composing of 5 g/l peptone, 2 g/l yeast extract powder, 20 g/l D-(+)-glucose, 1 g/l K2HPO4, 0.5 g/l MgSO4, and 15 g/l potatodextrose agar. The producing strain was prepared on MBM and stored at 4 °C. 2.6. Culture medium and fermentation conditions A. niger CBS 554.65T (FJ629288) was the predominant fungus of fermentation material. It was used to conduct the solid-state fermentation. The medium used for solid-state fermentation was MBM agar composing of MBM and 15 g/l agar. The substrates used for fermentation experiments were C, EC, GC, EGC, theanine and sterile leaves of C. assamica. Sterilization of the medium and substrates was performed at 121 °C for 20 min. The solid-phase fermentation (SSF) process was as follows: One milliliter of fungus inoculums was inoculated on MBM agar with 3  105 CFU/ml for pure cultures at 37 °C and humidity at 75%, and then the fungus was maintained under this condition on a MBM Petri dish for 2 days. During SSF, 0.1 g of sample was removed after 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 h, subsequently extracted with 2 ml of methanol at 37 °C by sonic extraction for one hour. The extracts were filtered through a 0.22 lm filter for HPLCMS analysis.

W. Wang et al. / Food Chemistry 152 (2014) 539–545

2.7. Cell culture and treatments Human micro-vascular endothelial cells (HMECs) were cultured in 100-mm tissue culture plates in high glucose Dulbecco’s modification of Eagles’s minimal essential medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and penicillin solution (10,000 U/ml; Invitrogen) and streptomycin (1 lg/ml; Invitrogen). Cells were maintained at 37 °C in a humidified incubator containing 5% CO2 and grown to 80–90% confluency before being passed or used in an experiment. For cell viability experiments, cells were seeded at a density of 105 cells/ml in 96-well culture plates. Following seeding, cells were allowed to adhere to plates for 16 h prior to beginning experiments. Once the cells were adhered, appropriate volumes of fresh compounds stock were added to plates to achieve the final concentration of 1 lM. Cells were incubated with compounds for 2 h prior to induction of stress by 500 lM H2O2. Cells were subjected to stress for 48 h prior to experimental analysis.

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The detailed data of 1H NMR (500 MHz, DMSO-d6) and 13C NMR 125 MHz, DMSO-d6) of puerins I, II, III and IV see Table 1. On the basis of 2D-NMR data and related articles (Ren et al., 2008; Tanaka, Watarumi, Fujieda, & Kouno, 2005), the N-ethyl-2-pyrrolidinone was identified to be substituted in the C-8 of A-ring of catechin and epicatechin. With reference to the published method (Ren et al., 2008), the configuration of compounds 1–4 were identified as shown in Fig. 1. The CD contribution of C-500 can be clarified by subtracting one CD spectrum from that of the other stereoisomer with the same configuration at C-2/3. In this study, compounds 2 and 4 had the moiety of (–)-epicatechin. So, to determine the configuration of 2 and 4, the CD spectrums of 2 and 4 were compared after subtracting the CD spectrum of each other with Origin 7.5 software (Origin Lab Corp., North Hampton, MA, USA). As shown in Fig. 1, a positive CE at 210 nm (De + 37.1) confirmed an S-configuration at C-500 of compound 2, while a negative CE at 210 nm (De -37.1) confirmed an R-configuration at C-500 of compound 4. With the same method, the configurations of compound 1 and 3 were also determined.

3. Results 3.1. Puerins I (1), II (2), III (3) and IV (4) They are all white amorphous powders, puerins I[a]21 + 74.0(MeOH), puerins II [a]21 D + 17.0(MeOH), puerins III [a]21 D D 83.0(MeOH), puerins IV [a]21 D 84.0(MeOH). Puerins I, II, III and IV UV kmax (MeOH): 230, 282 nm. Puerins I, II, III and IV IR (KBr) kmax (cm1): 3747, 3200, 2927, 1728, 1610, 1524, 1453, 1377, 1110, 1023, 996, 822, 768. All of these four compounds show nearly the same high resolution fast atom bombardment mass spectrometry (HRFABMS) m/z [M+H]+, which are 402.15204, 402.15232, 402.15445 and 402.15432, calculated for C21H23O7N1. But during the separation of these isomers, we clearly detected four peaks on the HPLC spectrum. Through this HPLC preparation, these four isomers can be purified. The 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) spectras of the isomers clearly showed some differences, as shown in Table 1. Firstly, the occurrence of a flavan-3ol skeleton in the molecule could be easily deduced from the 1H NMR and 13C NMR spectrum. More specifically, the chemical shift variation resulted from different relative configurations of C2/C3 of catechin and epicatechin, and this contributed to structurally distinguish the puerins I–IV. For example, compared to the 1H NMR of catechin and epicatechin, puerins II and IV showed the chemical shifts at dH 4.79/4.73 (s), 3.92/3.97 (m), and 2.47, 2.67/ 2.50, 2.72 (2H, m), which were ascribable to C2, C3, and C4 protons on a C-ring of epicatechin, respectively. Moreover, puerins I and III had signals at dH 4.53/4.42 (s), 3.75/3.71 (m), and 2.36, 2.70/2.38, 2.74 (2H, m) ascribable to C2, C3, and C4 protons on C-ring of catechin. From the 13C NMR spectrum, puerins II and IV have the signals at dC 78.9/78.4 and 66.2/66.1 ascribable to the C2 and C3 on the C-ring of epicatechin. The signals at dC 78.9/78.6 and 64.8/64.9 of puerins I and III were ascribable to the C2 and C3 on the C-ring of catechin. In the high field of 1H NMR and 13C NMR of puerins I (1), a group of signals of high field [dC 30.4, 23.6, 34.1, 12.4] and CH part [dC 50.6, dH5.10] are confirmed by the 1H–1H two-dimensional correlated spectroscopy (COSY) and HMBC spectra. These signals were also observed on the 1H NMR and 13C NMR of puerins II (2), III (3) and IV (4). Furthermore, a signal at dC 173.0 showed the presence of a carboxyl function. This was supported by the IR spectrum showing a strong band at 1728 cm1. In the HMBC of puerin II, the CH group at dH 5.21 correlated with the carbon signal at dC 156.0, which showed the CH [dC 50.6, dH5.10] group was directly linked to C-8 of A-ring of catechin.

3.2. Puerins V (5), VI (6), VII (7) and VIII (8) In the chemical constituents of tea materials, catechins are paired with the gallocatechins, the C-20 of which is substituted by a hydroxyl group. The identification of compounds 1–4 led us to believe that the other four corresponding compounds composing of the gallocatechins existed in pu-erh tea. Aiming at these targeted ions traces, the ions m/z at 417 (puerinsV–VIII) were positioned by HPLC-DAD-ESI/MS. Then, the mass fragments of puerins I–VIII were studied with collision-induced dissociation (CID) of HPLC-DAD-ESI/MS. From the fragmentation of puerins I–VIII, the dissociation regulation of this type of compounds was established as shown in Fig. 2. Puerins V–VIII with the molecular weight of 417 was speculated to be other four N-ethyl-2-pyrrolidinone substituted 8-C flavan-3-ols. After isolation of targeted compound 5 and 6 with HPLC, these were identified using NMR. The data of 1H and 13C NMR of compound 5 and 6 are listed in Table 1. As shown in Table 1, the chemical shifts of compound 5 and 6 were similar to those of compounds 2 and 4. At the C-20 of B-ring of gallocatechins, the chemical shifts of dc 147 corresponded to the signal of ()-epigallocatechins. Using the same method to determine the configuration of 2 and 4, the configurations of C-500 of compound 5 and 6 were confirmed as the S and R-configuration, respectively. The structure of compound 5 and 6 are shown in Fig. 2. Furthermore, other pairs of enantiomers were derived from HPLC-DAD-ESI/MS. As shown in Fig. 2, compound 7 and 8 showed the same mass fragmentation of 5 and 6. With reference to the retention time and mass fragmentations of compound 1–4, the structures of compounds 5–8 could be clarified. 3.3. The detection of puerins I–VIII in various CDTs Puerins I–VIII were used as standards in the HPLC-DAD-ESI/MS analysis of Chinese dark teas from Shaanxi, Hunan, and Yunnan province. To obtain high resolution and accuracy, the precursor MS ions m/z at 402 and 418 of puerins I–VIII were selected as target ions in HPLC-DAD-ESI/MS. The 50% ethanol extracts of these three kinds of famous CDTs were analyzed as shown in Fig. 3. Furthermore, the fresh dry leaves of two main plants for making CDTs, C. sinensis and C. assamica were also comparatively analyzed to explore the source of puerins I–VIII. Black tea and green tea were simultaneously analyzed.

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Table 1 H NMR and

1

1

13

C NMR spectroscopic data of compounds 1, 2, 3, 4, 5 and 6 (d Value, in DMSO-d6).

1 2 4.53(1H, br s) 3 3.75(1H, dd, J = 7.5, 13.0) 4 2.36(1H, dd, J = 3.5, 1.5 Hz), 2.70(1H, dd, J = 3.5, 16.3 Hz) 5 6 6.01(1H, s) 7 8 9 10 10 20 6.70(1H, d, J = 2.0) 30 40 50 60 200 300 400

13

H NMR

C-NMR

2

3

4

5

6

4.79(1H, s) 3.92 (1H, m) 2.47, 2.69(each 1H, br d)

4.42(1H,d, J = 7.5) 3.71(1H, br d) 2.38(1H, dd, J = 8.5, 16.0 Hz), 2.74(1H,br d)

4.73(1H, s) 3.97(1H, m) 2.72, 2.50(each 1H, br d)

4.66(1H, br s) 3.99 (1H, m) 2.53, 2.73(each 1H, dd, J = 4.4, 16.6)

4.68(1H, br s) 4.06(1H, m) 2.53, 2.75(each 1H, dd, J = 4.4, 16.6)

6.02(1H, s)

6.02(1H, s)

6.01(1H, s)

6.02(1H, s)

6.02(1H, s)

6.87(1H, br d)

6.67(1H, s)

6.70(1H, br d)

6.36(1H, s)

6.39(1H, s)

500 5.10 (1H, s) 600 2.44 (2H, m) 700 0.84(3H, t, J = 6.9)

5.10 (1H, s) 3.33 (2H, m) 0.89(3H, t, J = 7.2)

From the total ion chromatograms of targeted ion m/z at 402 and 418, all of CDTs were detected with puerins I–VIII, while the leaves of tea plants and other teas did not contain these eight marker compounds. These results indicate that these puerins I–VIII were produced during the post-fermentation process of CDTs, rather than originating from tea plants. 3.4. Study on the biosynthetic pathway for puerins I–VIII by fungal fermentation The substituted part of the N-ethyl-2-pyrrolidinone moiety on puerins was supposed to be derived from the structure of theanine in tea. In order to investigate the biosynthetic pathway of puerins I-–VIII, the changes of C, EC, GC, EGC and theanine were quantitatively analyzed by MRM mode during the solid-state fermentation

80.3 66.5 30.9

155.1 94.7 155.1 114.4 155.1 103.4 130.7 114.4

154.8 95.9 156.0 115.2 156.0 104.0 130.8 115.2

155.2 94.7 155.2 114.8 155.2 104.0 130.1 114.8

155.7 96.0 156.1 114.9 156.1 104.3 131.0 114.9

155.5 99.7 155.5 103.1 155.5 103.9 132.1 107.5

157.2 100.4 157.5 105.9 157.5 105.9 131.7 107.7

144.9 144.9 115.3 118.3 173.3 31.3 22.9

144.8 144.8 115.1 117.8 172.8 30.6 23.0

144.8 144.8 115.4 117.9 173.4 31.3 23.5

146.7 132.2 146.7 107.3 172.9 34.2 29.0

147.4 134.0 147.4 107.7 175.1 33.0 25.1

51.3 34.6 12.9

50.5 34.1 12.3

51.3 34.4 12.8

50.9 39.0 12.5

50.6 36.0 14.3

50.6 34.1 12.4

2.08 (2H, m) 2.31, 2.06(2H, m) 5.26 (1H, s) 3.36 (2H, m) 0.86(3H, t, J = 7.2)

6

78.5 64.4 34.2

5.27(1H, t, J = 6.7) 3.38, 2.57(2H, m) 0.85(1H, t, J = 7.0)

2.13 (2H, m) 2.38, 1.99(2H, m) 5.21 (1H, s) 2.44 (2H, m) 0.92(3H, t, J = 7.2)

5

78.6 64.9 29.3

5.22(1H, t, J = 6.7) 3.38, 2.57(2H, m) 0.86(1H, t, J = 7.0)

2.12 (2H, m) 2.32 (2H, m)

6.39(1H, s)

4

81.4 66.1 28.7

2.09(2H, m) 2.20, 2.08(2H, m)

6.61(1H, s) 6.69(1H, br s) 6.36(1H, s)

3

78.9 64.8 28.6

2.09(2H, m) 2.21, 2.09(2H, m)

6.59(1H, br s) 6.66(1H, s) 6.65(1H, br s) 6.59(1H, br s)

2

81.4 66.2 27.8

144.8 144.8 115.7 118.1 172.7 30.4 23.6

6.67(1H, d, J = 8.0) 6.57(1H, dd, J = 2.0, 8.0)

2.09 (2H, m) 2.35 (2H, m)

1

on fresh tea leaves of C. assamica. Firstly, the content of theanine decreased sharply at the beginning stage (0–12 h), and was hardly detected after 24 h of fermentation. Secondly, the contents of C and GC showed the same tendency of reduction. Thirdly, all of eight puerins had emerged after 24 h fermentation and reached the peak of their content at around 60–72 h. Based on the results above, the correlation between formation of puerins with theanine and catechins can be accepted. To confirm the biosynthetic pathway of puerins I–VIII, A. niger was used as a single strain to ferment C, EC and theanine mixture or GC, EGC and theanine mixture. HPLC-DAD-ESI/MS analysis of the fermentation products showed that either the C/EC/theanine group could produce puerins I–IV, or the GC/EGC/theanine group could produce puerins V–VIII. A biosynthetic pathway of puerins was profiled as shown in Fig. 4.

Fig. 1. The configuration of compounds 1–4 by arithmetically CD curves subtracted each other for two couples of stereoisomer (1/3 and 2/4).

W. Wang et al. / Food Chemistry 152 (2014) 539–545

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Fig. 2. (A) The chemical structures of compounds 5–8; (B) the arithmetically CD curves subtracted each other for compound 5/6; (C) the extract ions and fragmentation of compounds 5–8; (D) the dissociation regulation of compounds 5–8 under positive CID.

3.5. Protective effects of puerins I–IV on HMEC injured by H2O2

4. Discussions

Puerins I–IV were tested for their protective effects on HMEC injured by H2O2. Compared with the H2O2 group, the result showed that it had cytoprotective activity. Some major compounds of green tea such as GA, CA, EGCG, EC, EGC and GCG were also comparatively tested. The cell viabilities of HMEC injured with H2O2 after treatment with each compound at 1.0 lM is shown in Fig. 5. Puerins I–IV showed potential protective effects on the HMEC from injury induced by H2O2.

We structurally represented the chemical characteristics of Chinese dark teas using eight catechin derivatives, puerins I–VIII. Study on the biosynthesis of these compounds showed that the fungal fermentation process was critical for the formation of puerins I–VIII. Different from the oxidation of B-ring on catechins under polyphenol oxidase, the A-ring on catechins of CDTs was prone to be affected by fungi.

Fig. 3. The confirmation of puerins I–VIII in different kinds of teas with HPLC-MRM-MS.

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Fig. 4. (A) The variations of C, EC, GC and EGC during solid fermentation with Aspergillus niger; (B) the variations of puerins I–VIII during solid fermentation with A. niger; (C) the pathway for the biosynthesis of puerins; (D) MRM analysis of fermented samples and controls.

Fig. 5. The advantages of protective effects of puerins I–IV on HMEC injury induced by H2O2 compared to other tea polyphenols.

Although some authors described that the chemical constituents of pu-erh tea is different from other teas, the catechin derivatives found in pu-erh tea were still composed of carbon, oxygen and hydrogen (Zhou et al., 2005). Puerins I–VIII are special because they contain the moiety deriving from theanine, which has been proved to decrease sharply after post-fermentation (Syu, Lin, Huang, & Lin, 2008). At present, the identification of CDT origin and quality have relied on the subjective evaluation rather than objective chemical analysis. The results indicated that these compounds exclusively existed in Chinese dark teas. A more reasonable quality control is expected to be achieved by puerins I–VIII. From this study, a more distinctive standard for the chemical description of CDTs with these marker compounds can be used. Chinese dark teas have been consumed by many people of Southeast Asia, Europe and America. Most of the preparation of CDTs in animal experiments and clinical trials were crude extracts, the quality of which was still represented by total tea polyphenols (Cao et al., 2011; Duh, Yen, Yen, Wang, & Chang, 2004). From this study, it can be accepted that microbial metabolites of tea polyphenols are stronger antioxidant agents than common tea polyphenols. The studies on disease models, such as hyperlipemic and hyperglycemic animals with puerins I-VIII, will contribute to explain the biological activities of CDTs. Acknowledgment This work was supported by the Grant (81172944) from the National Science Foundation of China and ‘Study on the Active

⁄⁄

P < 0.01 compared with control.

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8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols as the marker compounds of Chinese dark teas formed in the post-fermentation process provide significant antioxidative activity.

Phytochemical investigation of the aqueous extract of pu-erh tea afforded eight novel 8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols (puerins I-...
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