Simin Hu, Xiolei Li, Chungchung Go, Xinyu Meng, Mingcho Li,Yuqin Li, Tinrui Xu,*, Qin Ho,*

a Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China

b Research Center for Analysis and Measurement, Kunming University of Science and Technology, Kunming 650093, China

Keywords:

Pu-erh tea

Polymer

Theabrownin

Thiol degradation

A B S T R A C T

Habitual tea consumption can reduce the risk of ASCVD morbidity and mortality and all-cause mortality in China, and long-term adherence to this habit can provide greater protection.Pu-erh tea is one of the representatives of post-fermented teas, and the polymers in Pu-erh tea have anti-cancer and anti-hyperlipidemic activities, but their chemical composition is still unknown.In this study, the chemical composition of Pu-erh tea was investigated in detail and eight (1–8) known compounds were isolated.The polymers in Pu-erh tea were also degraded by thiol degradation, and the fragments of quercetin and (-)-epicatechin-3-O-gallate were found in the thiol degradation products of Pu-erh tea polymers by UPLC observation and separation and purification.This is the first time that this fragment was obtained in Pu-erh tea polymer.

1.Introduction

According to a study on the project of Prediction for ASCVD Risk in China (China-PAR), habitual tea consumption could reduce the risk for both ASCVD morbidity and mortality, as well as all-cause mortality in China, and long-term adherence to the habit can provide greater protection [1].Pu-erh tea, as one of the representatives of postfermented tea, is produced in Puer City, Yunnan Province, China,and was once popular overseas for its unique flavor and its good anti-diabetic and anti-hyperlipidemic effects [2,3].Post-fermented tea is unique to China, and the unique process of Pu-erh tea makes its material composition different from other teas, such as green tea,black tea and oolong tea [4].During the pile-fermentation process,the tea polyphenols in Pu-erh tea will be oxidized into thearubigins(TRs) and thea flavins (TFs) under the action of microorganisms and moisture and heat, and TRs and TFs will be further oxidized and polymerized and combined with polysaccharides, proteins, lipids and other compounds to form a large number of polymers, also known as theabrownins (TBs) [5,6].These polymers have active functions such as anti-cancer [7], anti-fatigue [8], anti-hyperlipidemia [9], and regulation of intestinal flora [10].In particular, the hypoglycemic and hypolipidemic activity, thea flavin inhibits the intestinal FXR-FGF15 signaling pathway through interaction with intestinal microbes,leading to increased hepatic production and fecal excretion of BAs,lower hepatic cholesterol as well as reduced lipogenesis [11].Its chemical composition and structure are still unknown due to its large molecular weight, high degree of polymerization, complex structure and difficulty in isolation [12].In this study, 8 known compounds (1-8)were isolated from Pu-erh tea, and the thiol degradation method was used to investigate the polymers in Pu-erh tea.

2.Materials and methods

2.1 General experimental procedures

The Pu-erh tea sample was determined using Agilent 1290 Infinity ultra-high performance liquid chromatography system (Agilent Technologies, USA) with an EclipsePlus C183.0 mm × 150 mm(1.8 μm) column (Agilent, USA).The mobile phase was composed of acetonitrile (solvent A) and water (solvent B) with a flow rate of 0.15 mL/min, 4%-30% (18 min), 30%-75% (9 min), 75%-95%(3 min), injection volume was 1 μL, detection wavelength was 278 nm,column temperature was 35 °C.NMR spectra was measured on an Avance III 600 MHz (Bruker BioSpin, Switzerland) with tetramethylsilane (TMS) as the internal reference, and chemical shifts are expressed inδ(ppm).Column chromatography was performed by using ODS (YMC, Japan), Sephadex LH-20 (Pharmacia, USA),Diaion HP 20SS (Mitsubishi, Japan), Toyopearl HW40F (Tosoh,Japan), MCI GEL CHP20/P120 (Mitsubishi, Japan).Fractions were monitored by TLC (silica gel GF25410-40 μm, Marine Chemical Factory, Qingdao, China).UP Water Purification Sestem (Nanjing QuanKun biotechnology Co., Ltd.China).Ultrasonic Cleaner(Ningbo Xinzhi Biotechnology Co., Ltd.China).Deuterium reagent(Cambridge Isotope Laboratories, Inc, USA).Pure methanol and acetonitrile by chromatography (BCR, USA).Other reagents are of analytical grade (Xilong Chemical Co., Ltd.China).

2.2 Preparation of sample and standard solution

Samples ofL-theanine, (–)-epigallocatechin gallate (EGCG),(–)-gallocatechin gallate (GCG), (–)-epigallocatechin (EGC),(–)-gallocatechin (GC), (–)-epicatechin (EC), (–)-epicatechin gallate(ECG), caffeine and gallic acid were accurately weighed for 1 mg each, dissolved in 1 mL of ethanol and filtered through a 0.45 μm microporous membrane to obtain standard solutions for UPLC analyses.1.0 g of tea samples (raw Pu-erh tea and ripe Pu-erh tea,Menghai, 2015) were immersed in 50 mL of 70% acidic ethanol for ultrasonic extraction, and the supernatant was taken as the UPLC sample after filtering through a microporous membrane.

2.3 Plant materials

The Pu-erh tea used Ripe Tea produced in September 2017 at Zhong Yi Tea Factory, Menghai County, Yunnan Province, with ingredients selected from Yunnan big leaf kind of drying green Maocha.A sample (PRT201709) has been deposited in laboratory 403, Fundamental chemical experiment center of Kunming University of Science and Technology.

2.4 Extraction and separation

Pu-erh tea (160.00 g) was ultrasound extracted three times with 1 500 mL 70% acetone at 25 °C for 30 min each time.Then the extract of Pu-erh tea was extracted and separated with ethyl acetate to obtain ethyl acetate phase (19.07 g) and aqueous phase (15.20 g).

The ethyl acetate extract (10.05 g) was fractionated by column chromatography on Diaion HP 20SS and gradient eluted with Methanol-water (0%–100%) to give two fractions (Fr.).Fr.1 (2.90 g)was separated by gradient elution of methanol-water (20%-100%)by Toyopearl HW-40C column chromatography and methanol-water(20%-100%) by MCI gel column chromatography to give gallic acid (1) (97.2 mg) [13].Fr.2 (5.90 g) was eluted by gradient of methanol-water (20%-100%) by ODS column chromatography to give Fr.2-1-Fr.2-6.Fr.2-3 (280.5 mg) was eluted by gradient chromatography ethanol-acetone-water (1:0:0, 8:1:1, 6:2:2, 0:5:5,0:7:3,V/V) on Toyopearl HW 40F column to give Fr.2-3-1-Fr.2-3-6.Fr.2-3-4 (50.0 mg) was eluted by Sephadex LH-20 column chromatography chloroform-methanol (1:1,V/V) to give caffeine (2)(5.6 mg) and EC (3) (13.8 mg) [14].Fr.2-3-5 (17.2 mg) was eluted by ODS column chromatography 70% methanol-water to give EGC (4)(11.2 mg) [14].Fr.2-4 (2.10 g) were eluted by Toyopearl HW 40F column chromatography ethanol-acetone-water (1:0:0, 8:1:1, 6:2:2,0:5:5, 0:8:2,V/V), ODS column chromatography methanol-water(30%) and Sephadex LH-20 column chromatography chloroformmethanol (1:1,V/V), respectively, to give (-)-epicatechin-3-O-gallate (5) (6.9 mg) [14].Fr.2-5 (1.30 g) were eluted by Toyopearl HW-40C column chromatography ethanol-acetone-water (1:0:0,8:1:1, 6:2:2, 0:5:5, 0:8:2,V/V), ODS column chromatography methanol-water (20%-100%) and Toyopearl HW-40C column chromatography methanol-water (20%-100%) for gradient elution to give rutin (6) (2.0 mg) [14], quercetin-3-O-α-L-rhamnopyranoside (7) (4.0 mg) [15], and kaempferol-3-O-β-D-glucopyranoside (8) (1.0 mg) [14].

The aqueous phase was concentrated under reduced pressure,then alcoholic precipitation with ethanol ( filtrate-anhydrous ethanol 1:4,V/V) and centrifuged at 4 000 r/min for 20 min after standing for 6 h.The supernatant was poured off and the precipitate was obtained as polymer sample [16].

2.5 Thiol degradation

Pu-erh tea polymer (5 g) was added to 600 mL of ME reagent(2-mercaptoethanol-ethanol-hydrochloric acid, 10:55:6,V/V) in an oil bath at 80 °C for 12 h.

2.6 Separation of thiol degradation products

The thiol degradation product (5.0 g) was eluted by gradient chromatography ethanol-acetone-water (1:0:0, 0:5:5, 0:7:3,V/V) on Toyopearl HW 40C column to give Fr.w1–Fr.w5.Fr.w2 (840.0 mg)was eluted on Sephadex LH-20 column using chloroform-methanol(20:1, 1:1, 0:1,V/V) gradient chromatography to give Fr.w2-1–Fr.w2-4.Fr.w2-4 (25.6 mg) was isocratic eluted by ODS column chromatography acetonitrile-water (35%) to give quercetin (a) (10.2 mg)(Fig.1) [14]: yellow powder, HR-ESI-MSm/z: 303.050 5 [M+H]+(Calcd for C15H11O7: 303.049 9);1H-NMR (600 MHz, Acetone-d6)δ: 7.75 (1H, d,J= 2.2 Hz, H-2’), 7.62 (1H, dd, J = 8.5, 2.2 Hz, H-6’), 6.91 (1H, d,J= 8.5 Hz, H-5’), 6.45 (1-H, d,J= 2.1 Hz, H-8), 6.18 (1H, d,J= 2.0 Hz,H-6).13C-NMR (150 MHz, Acetone-d6)δ: 176.65 (C-4), 165.15(C-7), 162.42 (C-9), 157.85 (C-5), 148.54 (C-2), 147.05 (C-4’), 145.99(C-3’), 136.85 (C-1’), 123.79 (C-3), 121.52 (C-6’), 116.41 (C-2’),115.89 (C-5’), 104.20 (C-10), 99.25 (C-8), 94.56 (C-6).

Fig.1 Structures of compounds 1–8 and a.

3.Results and discussion

As we all know, raw Pu-erh tea and ripe Pu-erh tea are classified according to their processing and quality characteristics.Raw Pu-erh tea is made from sun-dried tea, which is made from fresh leaves of Yunnan large leaf species by killing, kneading, and sun-drying,and then steam-formed into tightly pressed tea, whose chemical composition and quality are very similar to those of green tea [17].Ripe Pu-erh tea is a kind of post-fermented tea made from sundried tea through microbial solid-state fermentation technology.Post-fermentation has a significant impact on the final composition of raw tea, especially in terms of polyphenols [18]and amino acids [19].A large number of previous studies have shown that ripe Pu-erh tea contains less catechins compared with raw Pu-erh tea.This is due to that the catechins in raw Pu-erh tea are oxidized into TRs and TFs under the action of microorganisms and humid heat during the process of ripe Pu-erh tea fermentation, and TRs, TFs are further oxidized,polymerized and combined with polysaccharides, proteins, lipids, and other compounds to form a large number of polymer (theabrownins).The ripe Pu-erh tea contains more gallic acid, which is the consequence of catechin-gallate degradation by the microorganisms involved in the fermentation process [5,6,20-22].

In addition, we have conducted a comparative analysis of the chemical composition of raw Pu-erh tea and ripe Pu-erh tea(Table 1).It is showed that the content of catechins in ripe Pu-erh tea was significantly lower than raw Pu-erh tea, while the content of gallic acid was significantly higher.This result is consistent with the results of previous studies.

Table 1Comparison of the chemical composition (mg/g) of raw tea and ripe tea.

Therefore, we studied on the chemical constituents of the ripe Pu-erh tea.The chromatographic separation of the ethyl acetate phase fraction of puerh tea was performed and 8 known compounds(1-8) were isolated, gallic acid (1), caffeine (2), EC (3), EGC (4),(-)-epicatechin-3-O-gallate (5), rutin (6), quercetin-3-O-α-L-rhamnopyranoside (7), kaempferol-3-O-β-D-glucopyranoside (8).These compounds were identified by comparing the spectral data of the known compounds with the reported values (Fig.1).

The aqueous phase of Pu-erh tea was obtained as a polymer by water extraction-ethanol precipitation technology.UPLC analysis showed that the polymer was detected as a large broad hump at the baseline (Fig.2), and the chromatographic properties were similar to those of Chinese Dragon’s Blood [23], the black tea thearubigins [24,25]and polymeric proanthocyanidins [26].Therefore, degradation of the polymer fraction with mercaptoethanol under acidic conditions was used, and compound (a) was obtained by isolation and purification.Compound (a) was inferred from1H NMR and13C NMR data as quercetin.UPLC shows (Fig.2) that we obtained partial fragments of the polymer quercetin, (-)-epicatechin-3-O-gallate.Previous studies have been conducted to elucidate the structure of Pu-erh teapolymers by atomic force microscop (AFM), ross polarization-magic angle spinning NMR (CP-MAS NMR) and curie-point pyrolysis gas chromatography-massspectrosco-PY (CP-GC/MS) to elucidate the structure of Pu-erh tea polymers, it was only found that Pu-erh tea polymers are a benzene ring as the main structure, complexed with polysaccharides and proteins, rich in carboxyl, hydroxyl and methyl groups, etc.[16,27,28].Unlike previous studies, we obtained quercetin (a) fragments from Pu-erh tea for the first time as well as(-)-epicatechin-3-O-gallate.Furthermore, it can be found that the large broad hump of the Pu-erh tea polymer is still remains after thiol degradation, which indicates that the degradation method is not sufficient for too mild degradation of the Pu-erh tea polymer.Since the degradation method is very important, it generally needs to be as mild as possible to keep the structural fragments of the original sample.However, if it is too mild, the desired product will not be obtained, and if it is too drastic, the sample may be decomposed into small meaningless molecules such as carbon dioxide and water, and the method needs to be improved.Based on these data,the chemical composition of Pu-erh tea differs from other teas,because the components of polyphenols, proteins, polysaccharides and polyphenol conjugates form a class of structurally complex polymers during the post-fermentation process [7,8].

Fig.2 UPLC profile of Pu-erh tea polymer degradation.UPLC profile of (A) before degradation, (B) after thiol degradation, (C) blank 1 (ME reagent), and(D) blank 2 (77% ethanol).Detection at 278 nm UV absorbance.

4.Conclusion

In the present study, we isolated 8 known compounds and polymer from Pu-erh tea.In addition, the polymer was investigated by spectroscopic and chemical methods, it is the main functional component also known as theabrownins in Pu-erh tea.The NMR,UPLC spectroscopy and thiol degradation experiments showed that the polymer structure contains fragments of quercetin and(-)-epicatechin-3-O-gallate.This is the first time that the fragment structure of this polymer has been obtained, and further studies on the chemical mechanism of Pu-erh tea polymers and their suitable degradation methods are currently underway.

Conflicts of interest

There are no conflicts to declare.

Acknowledgment

This study was supported by the National Natural Science Foundation of China (31660099).