You Wu, Zisheng Han, Minghun Wen, Chi-Tang Hob,, Zongde Jiang, Yijun Wang,Na Xu, Zhongwen Xie, Jinsong Zhang, Liang Zhang,*, Xiaohun Wan,*

a State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, China

b International Joint Laboratory on Tea Chemistry and Health Effects of Ministry of Education, Anhui Agricultural University, Hefei 230036, China

c Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA

Keywords:

Larger-leaf yellow tea

Anti-hyperglycemia

α-Glucosidase

Mass spectrometry

Separation

A B S T R A C T

Larger-leaf yellow tea (LYT) is a characteristic type of Chinese tea produced in Huoshan County, Anhui Province, which is made by mature leaves with stems.According to recent report, LYT showed competitive effects in anti-hyperglycemia in comparison to other teas such as green or black tea.However, the bioactive compounds of LYT are still undiscovered so far.For this purpose, 5 fractions of LYT were prepared by sequential extraction.The in vitro bioassay results indicated that the ethyl acetate fraction of LYT had the strongest inhibitory effects on α- glucosidase and α-amylase.Fluorescence-quenching analysis and proteinbinding test revealed that the compounds of ethyl acetate fraction could inhibit α-glucosidase and α-amylase activities through binding to enzymes or other mechanisms.All chromatographic peaks of high-performance liquid chromatography (HPLC) of ethyl acetate fraction were separated and collected.The purified compounds were identified by liquid chromatography-mass spectrometry (LC-MS), and subsequently screened by calculating their inhibition ratio on α-glucosidase at the real concentration in LYT infusion.The results showed that (-)-epigallocatechin gallate, (-)-gallocatechin gallate, caffeine, N-ethyl-2-pyrrolidone-substituted flavan-3-ols were effective inhibitors for α-glucosidase.

1.Introduction

Larger-leaf yellow tea (LYT) is made in Huoshan County,Anhui Province, which is a kind of yellow teas characterized by its processing technology [1,2].Traditionally, LYT is produced by m ature leaves with stems plucked in May and June through fixation,rolling, yellowing and high-temperature roasting processes [3].Specific material and processing procedure contribute to the specific flavor and bioactivity of LYT, of which the final roasting process exert remarkable effects on primary compounds such as theanine and tea polyphenols [2-6].In our previous articles, LYT showed the best anti-hyperglycemic efficacy among four tea types on diabetic mice [7].Furthermore, it also induced energy consumption, improved chronic inflammatory and inhibited adipocyte differentiation [8,9].However,the bioactive compounds of LYT responsible for regulating metabolic syndrome are still unknown.

The unique chemical characteristics of LYT was determined by high-temperature roasting during manufacture process [2,10].Zhou et al.[2]discovered thatL-theanine was reduced by 99% and phenotypic catechins were all reduced in different degrees, meanwhile, new compounds were identified asN-ethyl-2-pyrrolidinone-substituted flavan-3-ols [2,11].To search for the bioactive compounds of LYT, a more efficient strategy should be developed.

In the digestion of carbohydrates,α-glucosidase andα-amylase are two critical digestive enzymes in the intestinal tract [12].They could hydrolyze polysaccharides, oligosaccharides and starch into monosaccharides,which would be further absorbed into blood circulation [13,14].For healthy population, plasma levels of glucose absorbed from carbohydrates could be regulated by insulin secretion [15-17].For the metabolic syndrome, insulin resistance usually leads to loss of control on the blood glucose level, especially for people who consumed high dose of carbohydrates [18].Therefore, advanced intervention on carbohydrate hydrolysis is recognized as an effective tool on preventing the increase of blood glucose level [19,20].The roasted tea has shown better inhibitory effects onα-glucosidase andα-amylase than non-roasted teas, but the main contributors for inhibiting digestive enzymes need further exploration [3].

The aim of the present study was to find bioactive compounds of LYT.The main and minor compounds of yellow tea were separated,purified and identified by high-performance liquid chromatography(HPLC) and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS).Subsequently, the purified compounds were screened by of fline bioassay ofα-glucosidase andα-amylase.The inhibitory mechanism of bioactive compounds was studied by fluorescence detection, protein binding and molecular docking.Finally, purification of 31 main compounds by preparative liquid chromatography to detect their inhibit ratio onα-glucosidase were designed to show their respective contribution.

2.Material and methods

2.1 Materials

Larger-leaf yellow tea (2 000 g) was obtained from Hengda Tea Company, Huoshan County, China.Gallic acid (GA), gallocatechin(GC), epigallocatechin (EGC), catechin (C), theobromine (THB),epicatechin (EC), epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), epicatechin gallate (ECG) and caffeine (CAF),pNPG,α-glucosidase andα-amylase were obtained from Yuanye Biological Technology Co., Ltd.(Shanghai, China) and Sigma-Aldrich Trading Company Ltd.(Shanghai, China).

2.2 Separation and quantitative analysis of LYT by HPLC

Two thousand grams LYT was boiled with 90 °C pure water.40 L of LYT extract was concentrated into 1.3 L (1.53 g/mL), then tea polysaccharides were removed by 75% alcohol precipitation.25 mL of LYT extract was continuously extracted by petroleum ether,dichloromethane, ethyl acetate andn-butanol (3 times, 25 mL for each solvent).Five fractions including petroleum ether, dichloromethane,ethyl acetate,n-butanol and water fractions (A, B, C, D and E) were obtained.All fractions were freeze dried.

The contents of main compounds in each fraction were determined by HPLC according to established method.The dry extract of each fraction was totally dissolved in 10 mL of respective solvent.The solution was filtered through a 0.22 μm Millipore filter before determining the contents of GA, GC, EGC, C, THB, EC, EGCG,GCG, ECG and CAF by HPLC [21].Detection was performed on an Agilent HPLC system (Agilent Technologies, Palo Alto, CA,USA) consisted of in finity binary pump, integrated vacuum degasser,autosampler, thermostated column compartment and diode array detector (DAD) with an Agilent ZORBAX shield SB-aq C18column(250 mm × 4.6 mm, 5 μm).The chromatographic conditions were performed similarly as described by Guoet al.[21].Each sample was measured for 3 times as parallel control.The contents of the chemical compounds were estimated using calibration curves (R2> 0.99) and proportions of each compound were calculated.

2.3 α-Glucosidase and α-amylase inhibition test

The inhibition ratio was measured by a SpectraMax M2 microplate reader (Molecular Devices, San Jose, CA, USA).Calculated as the equivalent dose of dry tea leaves, 5 fractions were diluted into various concentrations of 3.846, 1.538, 1.231, 0.769, 0.615, 0.308 and 0.015 mg/mL.50 μL of sample solution with different concentrations and 100 μL ofα-glucosidase (pH 6.8, 1 U/mL) were added into 96-well plates and incubated at 37 °C for 10 min, then 50 μLpNPG(pH 6.8, 5 mmol/L) were added and mixed equably.The absorbance was recorded under 405 nm after 5 min to calculate the inhibition ratio and IC50value.Each sample was measured for 3 times as parallel control group.

Similarly, to detect the inhibition effects onα-amylase, 5 fractions were diluted into various concentrations of 384.62, 256.67, 128.33,64.17, 12.83 and 1.28 mg/mL.300 μL sample solution and 300 μLα-amylase (pH 6.8, 13 U/mg) were added into 10 mL centrifuge tube and incubated at 37 °C for 5 min, then 300 μL of 1% soluble starch solution (m/V) were added and incubated at 37 °C for 25 min.After that, 500 μL DNS reagent were added, and the mixed solution was heated in water bath at 100 °C for 5 min, followed by dilution to 5 mL with water.The absorbance was recorded under 520 nm and used to calculate the inhibition ratio and IC50value, and each sample was measured in triplicate.IC50value was calculated by GraphPad Prism 7 software.Inhibition rate of enzyme activity was calculated by using the following equation:

2.4 Chemical analysis by LC-Q-TOF-MSn

The ethyl acetate fraction was diluted to approximately 100 μg/mL and filtered through a 0.22 μm Millipore filter before LC-MS analysis.The analysis was performed in negative ion mode on a UHPLC-ESIMS system (Agilent Technologies, Palo Alto, CA, USA) consisting of an Agilent 6545 tandem Q-TOF-MS coupled to an Agilent 1290 series HPLC system, which is equipped with an auto injector and a binary solvent delivery system.Separation was achieved using an Agilent ZORBAX shield SB-aq C18column (250 mm × 4.6 mm,5 μm) at a flow rate of 1 mL/min (with equipped t-branch pipe).The chromatographic condition was the same as mentioned in section 2.2 and the instrument parameters were similar as described by Zhou et al.[2].

2.5 Fluorescence-quenching detection

Fluorescence-quenching detection was performed by a Cary Eclipse fluorescence spectrometer (Agilent Technologies, Palo Alto, CA, USA).The ethyl acetate fraction was diluted to various concentrations of 1 040, 208, 8.32, 1.664, 0.332 8, 0.066 56 and 0 μg/mL to obtain fluorescence intensities.1 mL sample solution and 3 mLα-glucosidase (pH 6.8, 10 U/mL) were mixed equably and incubated at 37 °C for 10 min, then transformed into a 10 mm path length quartz cells.Fluorescence spectra ofα-glucosidase mixed with sample solutions of different concentrations were measured, the excitation wavelength was set at 280 nm and emission wavelength was set from 300 nm to 400 nm, with 5 nm excitation and emission slits.

Since theα-amylase group shares similar fluorescence pattern with theα-glucosidase group, fluorescence spectra ofα-amylase mixed with sample solutions of different concentrations were measured under the same excitation wavelength and emission wavelength.1 mL sample solution and 3 mLα-amylase (pH 6.8, 5 mg/mL) were mixed equably and incubated at 37 °C for 30 min before detecting.

2.6 Enzyme binding

The ethyl acetate fraction was diluted to the concentration of 1.3 mg/mL.250 μL sample solution and 250 μLα-glucosidase(pH 6.8, 10 U/mL) were mixed equably into ultra filtration centrifuge tube (10 kDa, 4 mL) before centrifuging (5 000 ×g, 30 min).Then 250 μL pure water was added for second centrifugation, followed by repetition (3 times of centrifugation in total).The control group was designed to contain 250 μL sample solution and 250 μL pure water.The percolates (< 10 kDa) of the test group and control group were extracted and filtered through a 0.22 μm Millipore filter prior to HPLC analysis.

The ethyl acetate fraction was diluted to the concentration of 32.5 μg/mL.1 mL sample solution and 1 mLα-glucosidase(pH 6.8, 1 U/mL) were mixed equably into the ultra filtration centrifuge tube (10 kDa, 4 mL) and incubated at 37 °C for 10 min, followed by centrifugation at room temperature (5 000 ×g, 30 min).After that,1 mL pure water was added for second centrifugation, followed by repetition (3 times of centrifugation in total).The control group was designed to contain 1 mLα-glucosidase and 1 mL pure water.The upper filter layer (＞ 10 kDa) was added with 500 μL pure water and washed repeatedly to be sample A (the control group was labeled as a),while the percolate (< 10 kDa) was labeled as sample B (the control group was labeled as b).Their inhibitors were detected respectively.150 μL sample A and a were added into 96-well plates.Additionally,50 μL sample B and b were mixed with 100 μLα-glucosidase(pH 6.8, 1 U/mL) respectively, incubated at 37 °C for 10 min.Then 50 μLpNPG (pH 6.8, 5 mmol/L) were added uniformly, the absorbance values were recorded under 405 nm after 5 min.Meanwhile, the inhibitor of ethyl acetate fraction at the concentration of 32.5 μg/mL was detected.

2.7 Molecular docking

The molecular docking was operated by MOE software (versions 2015.10, CCG Company, Canada).Chemical structural formula was optimized by ChemBio3D 14.0.The protein structural formula model ofα-glucosidase (Protein ID: 3WEO) was downloaded at Protein Data Bank (RSCB PDB: http://www.rcsb.org/).

2.8 Separation and inhibiting-detection of each chromatographic peak

Thirty-one confirmed compounds were separated by preparative liquid chromatography, and the injection volume was 60 μL for 100 times.Each chromatographic peak was separated, and dried by rotary vacuum evaporator, then re-dissolved with 10 mL pure water to detect their inhibitors.Inhibiting-detection ofα-glucosidase was similar as mentioned in section 2.3, 50 μL sample solution and 100 μLα-glucosidase (pH 6.8, 0.5 U/mL) were added into 96-well plates and incubated at 37 °C for 10 min, then 50 μLpNPG (pH 6.8, 5 mmol/L)was added and mixed equably.The absorbance was recorded under 405 nm after 5 min to calculate the inhibition ratio, each sample was detected for 3 times as parallel control group.A blank-control group(without sample) and a self-control group (withoutα-glucosidase)were also designed.

3.Results and discussion

3.1 Proportion and main compounds of 5 fractions

After freeze-drying, the dry weights of 5 fractions were 90.09,449.78, 1 304.75, 1 215.24 and 1 747.21 mg in sequence.As shown in Fig.1, the ethyl acetate fraction and water fraction occupied larger proportions of the LYT extracts, which suggested they contained more medium polar and polar compounds.

Fig.1 Proportion of 5 fractions to the LYT extract (m/m).

The results of quantitative analysis were listed in Table 1.The ethyl acetate fraction contained the higher contents of catechins, and the dichloromethane fraction mainly contained two purine alkaloids.Petroleum ether fraction,n-butanol fraction and water fraction contained lower levels of catechins and alkaloids.

Table 1Proportion of 10 compounds in 5 fractions (%, m/m).

3.2 Inhibitory effects of 5 fractions on α-glucosidase and α-amylase

As shown in Fig.2a, ethyl acetate fraction andn-butanol fraction had higher inhibition ratios onα-glucosidase at 3.846 mg/mL, but at lower concentration ethyl acetate fraction was better.In Fig.2b, ethyl acetate fraction had highest inhibitory effects onα-amylase, followed byn-butanol fraction.Petroleum ether fraction, dichloromethane fraction and water fraction were obviously less efficient in inhibitingα-glucosidase andα-amylase.To sum up, the ethyl acetate fraction was the most effective part for further experiment.Besides,n-butanol fraction also had higher bioactivity and lower IC50value, which was deserved to study (Table 2).

Fig.2 The inhibitor of 5 fractions on (a) α-glucosidase and (b) α-amylase.

Table 2The IC50 of 5 fractions on α-glucosidase and α-amylase’s inhibitory effects.

3.3 Fluorescence spectra of α-amylase and α-glucosidase

In Fig.3, the results showed that the binding ability of the compounds in LYT withα-amylase andα-glucosidase increased with the concentrations of these compounds [22].

Fig.3 The fluorescence spectra of (a) α-glucosidase and (b) α-amylase at different concentrations of the extract.Measured in phosphate buffer, pH 6,kex = 280 nm.

3.4 Identification and inhibition ratio on α-glucosidase of main compounds of LYT

In total, 31 main compounds were separated and identified by LC-Q-TOF-MS/MS (Table 3) and were labelled in the HPLC (Fig.4),which were some phenolic acids, procyanidin dimer, flavonoids andN-ethyl-2-pyrrolidone-substituted flavan-3-ols (EPSFs) except catechins and alkaloids.These compounds were detected under 278 nm(red line) and 360 nm (blue line), as the maximum absorbance wavelength of flavan-3-ols is about 280 nm, while that of flavanones and their glycosides is 360 nm, usually.

Fig.4 The inhibition ratio of each compound of ethyl acetate fraction on α-glucosidase (%).

Table 3The identification of main and minor compounds in each fraction of LYT by LC-DAD-Q-TOF-MSn.

Table 3 gave the main mass fragment ions for each compound in Fig.4, most of these compounds have been identified by standards,however, there were still some compounds need to be identified or deduced by LC-Q-TOF-MS/MS through their product ions under different levels of collision energy.In our previous report, the EPSFs,which are a category of conjugates ofL-theanine and catechins, are the newly-formed compounds during roasting [2].They exert several bioactivities, such as protecting human blood vessels, significant antioxidative activity [23], scavenging free radicals and inhibitory effects on acetylcholinesterase [24].Peak 17, 18, 24–26 were identified as EPSFs and the product ions and fragmentation regularity of EPSFs were shown in Fig.5.Them/zvalues of main product ions of EPS-EGCG/GCG (m/z568.143 8) were 169.013 8, 236.092 6,248.092 8 and 416.134 7; them/zvalues of main product ions of EPS-EGC/GC (m/z416.133 7) were 125.023 5, 177.019 1 and 248.092 8; them/zvalues of main product ions of EPS-ECG/CG(m/z552.148 3) were 125.023 4, 169.014 4, 236.092 8, 248.092 1,400.140 7 and 416.134 7; them/zvalue of main product ion of EPS-EC/C (m/z400.138 3) was 145.029 1.Besides the fragment ion withm/zvalues of 169, 125, 400 and 416 which were the fragments of GA group and the remaining moiety part after losing GA group, there were two typical mass fragment ions withm/zvalues of 248 and 236 which wereN-ethyl-2-pyrrolidone-substituted A ring of flavan-3-ols.For EPS-EC/C, there was a special product ion (response values of them are not high):m/z165.054 5, which might form due to the loss ofN-ethyl group and its adjacent carbonyl group.Furthermore, some flavonoid glycosides (or aglycones) were also identified as peak 20,27-31.The flavonoid glycosides were studied to contribute to the color formation of tea infusion [25].

Fig.5 The MS/MS fragmentation of EPSFs.(a) Proposed fragmentation, and LC-Q-TOF-MS/MS spectrum of (b) EPS-EGCG/GCG, (c) EPS-ECG/CG, (d) EPSEGC/GC, and (e) EPS-EC/C.

To explore the specific inhibitory effect of each compound in ethyl acetate fraction, every chromatographic peak was collected by preparative HPLC.31 compounds were collected, dried and then dissolved with the same concentration equivalent to their concentrations in the dry extract to measure the inhibitory effects onα-glucosidase.Therefore, the inhibition ratio of purified compound can directly represent their contribution toα-glucosidase inhibition.In Fig.4, the histogram under each peak indicates the corresponding inhibitory effect toα-glucosidase.We can see the potent inhibitors were mainly distributed in the 25-30 min range of HPLC.Among these compounds, catechins such as (-)-epigallocatechin gallate(peak 16) was the dominating compound in LYT, followed by(-)-gallocatechin gallate and (-)-epicatechin gallate, which had similar inhibition ratios.Furthermore, although EPSFs were not the main compounds and have lower content compared with tea catechins in LYT, they (peak 17, 18, 24–26) also showed great inhibition ratios onα-glucosidase.Besides, quercetin-3-O-glucoside has a low content in LYT and great inhibitory ratios onα-glucosidase.

3.5 Binding effects of ethyl acetate fraction on α-glucosidase

To study the inhibitory mechanism of bioactive compounds onα-glucosidase activities, the binding ability of10 main compounds of tea in ethyl acetate fraction were analyzed.After the ethyl acetate fraction andα-glucosidase were added into the 10 kDa ultrafiltration centrifuge tube to centrifuge, the bound part and unbound part were separated.As shown in Fig.6, GC, EGC,EGCG and GCG had great binding ability withα-glucosidase,other compounds were just partly bond, which means that GC,EGC, EGCG and GCG could bind with some groups preferentially and easily while EC, C, GA, ECG, caffeine and theobromine would have certain degrees of difficulties in binding withα-glucosidase, probably due to the same form of GA binding at C-2 of EGC/GC and EGCG/GCG.Then the unbound compounds were separated and eluted to detect the inhibitory effect onα-glucosidase, and enzymatic inhibitory effect of the bound part were detected directly to find out their own contribution to the inhibition of enzymatic activities.As shown in Table 4, the overall inhibition ratio of the extract was about 71%, however, the unbound part had a higher inhibition ration (37.59%) than the bonded part(28.10%).Therefore, it was suggested that the main polyphenols including GC, EGC, EGCG and GCG inhibited the bioactivity by binding with it, but others including EC, C, GA, ECG, caffeine and theobromine inhibited the enzymatic bioactivity through other mechanisms which might be a more basic mechanism than binding.

Fig.6 The comparison of LYT extract (a) before and (b) after binding with α-glucosidase protein by HPLC.

Table 4Inhibitor of bound part and unbound part.

3.6 Molecular docking of identified compounds on α-glucosidase

The results of molecular docking are shown in the Table 5, the binding affinity refers to the energy released during binding, and when the absolute value of binding affinity is larger, binding would occur more easily.As we can see, the absolute values of binding affinity of GC, EGC, EGCG and GCG were larger than EC, C, GA,ECG, caffeine and theobromine, which is corresponding with the result in section 3.5.At the same time, peak 26, C-8/6N-ethyl-2-pyrrolidone-substituted EGCG/GCG showed the strongest binding affinity among all tested compounds.The strong binding may be due to the formation of hydrogen bonds and also the hydrophobic interactions in theN-ethyl-2-pyrrolidone moiety, which was a Strecker degradation product of theanine.Other EPSFs also showed strong binding effects withα-glucosidase.Furthermore,the main galloylated catechins indicated by peaks 16, 19, 21 and 23 can bindα-glucosidase to form specific and tight hydrogen bonds, as well as hydrophobic interactions [26].

Table 5Binding energy of each compound.

Acknowledgements

This work was supported by Natural Science Foundation of China (32072633, 32072634, 31201335), earmarked fund for China Agriculture Research System (CARS-19), Anhui Key research and development plan (1804b06020367, 202004b11020004) and Young Elite Scientist Sponsorship Program by National CAST(2016QNRC001).