Properties of ACE inhibitory peptide prepared from protein in green tea residue and evaluation of its anti-hypertensive activity

Journal Pre-proof Properties of ACE inhibitory peptide prepared from protein in green tea residue and evaluation of its anti-hypertensive activity Xingfei Lai (Data curation) (Writing - original draft) (Formal analysis), Shunshun Pan (Data curation) (Investigation), Wenji Zhang (Resources), Lingli Sun (Resources), Qiuhua Li (Resources), Ruohong Chen (Resources), Shili Sun (Conceptualization) (Methodology) (Writing - review and editing) (Supervision)

PII:

S1359-5113(19)31318-2

DOI:

https://doi.org/10.1016/j.procbio.2020.01.021

Reference:

PRBI 11906

To appear in:

Process Biochemistry

Received Date:

2 September 2019

Revised Date:

16 January 2020

Accepted Date:

20 January 2020

Please cite this article as: Xingfei L, Shunshun P, Wenji Z, Lingli S, Qiuhua L, Ruohong C, Shili S, Properties of ACE inhibitory peptide prepared from protein in green tea residue and evaluation of its anti-hypertensive activity, Process Biochemistry (2020), doi: https://doi.org/10.1016/j.procbio.2020.01.021

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Properties of ACE inhibitory peptide prepared from protein in green tea residue and evaluation of its anti-hypertensive activity

Xingfei Lai, Shunshun Pan, Wenji Zhang, Lingli Sun, Qiuhua Li, Ruohong Chen,

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Shili Sun*

Tea Research Institute, Guangdong Academy of Agricultural Sciences / Guangdong Provincial Key Laboratory of Tea Plant Resources Innovation & Utilization, China;

[email protected]

(X.L.);

[email protected]

(S.P.);

[email protected]

(W.Z.);

[email protected]

(L.S.);

[email protected]

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[email protected] (R.C.)

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510640,

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Guangzhou

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*Correspondence: [email protected] (S.S.)

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Graphical abstract

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(Q.L.);

Highlights: 

The extraction rate of green tea protein from green tea residue under the optimised enzyme-alkali complex method was 74.27%.

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The ACE inhibitory rate of the green tea proteolytic product under the optimised hydrolysis condition was determined to be 77.00%.

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The molecular weight distribution of the polypeptide in the green tea proteolytic product was widely between 45.0–1.2 kDa. The green tea proteolytic product had good blood pressure lowering activity in

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vivo.

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Abstract

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Green tea contains active ingredients which are beneficial for health. While numerous studies have been conducted on the components extracted from green tea, few studies have investigated

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the active ingredients in tea residue. In this study, proteins were extracted from green tea residue

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via an optimised alkaline extraction combined with enzymatic hydrolysis, of which, an acidic protease was selected to prepare an enzymatic hydrolysate because of its high angiotensin

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converting enzyme (ACE) inhibitory activity. The composition characteristics of extracted green tea proteolysis products were elucidated, including amino acid composition, molecular weight

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distribution and possible amino acid sequences. In addition, the protein hydrolysate had anti-digestive properties, maintained its activity of inhibiting ACE enzyme at different temperatures, pH and metal ions, and exhibited antihypertensive activity in animals. In conclusion, the optimised alkaline extraction and enzymatic hydrolysis conditions of a ACE inhibitory peptide from green tea residue is an optimal extraction method to maintain its antihypertensive activity,

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providing the basis for the clinical application of green tea for blood pressure reduction.

Keywords: green tea; residue; protein hydrolysates; ACE inhibitory peptide; hypertensive

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1. Introduction

China is the largest tea producer in the world, with a net gross tea output of approximately

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2.62 billion kg in 2018. With the advancement of tea deep processing technology, the output of tea

deep processing products has increased rapidly, accompanied by the production of a large amount

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of waste tea residues. According to incomplete statistics, the annual output of wet tea residue in

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China's deep processing industry is 200-300 million kg. Internationally, the utilisation of waste tea residue is mostly used to produce feed, soil fertiliser and other low-value products, which fails to

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make full use of tea residue resources. Tea polyphenols, caffeine, tea polysaccharides, amino acids and vitamins are the main components of tea, accounting for only about 30% of the proportion of

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tea [1-4]. The waste tea residue contains many active ingredients, with a crude protein content of 18-20%, which has the potential for development and utilisation. It has been reported that tea

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protein is composed of a variety of amino acids, more than that of soybean protein, therefore is of high nutritional value and a high quality plant protein source. Studies have also shown that tea protein and its enzymatic hydrolysates have antioxidant, hypolipidemic, hypoglycaemic, anti-atherosclerotic, anti-mutation and other biological activities. As one of the most common chronic cardiovascular and cerebrovascular diseases,

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hypertension seriously threatens human life and health. The number of patients with hypertension is increasing worldwide, exceeding 1.1 billion worldwide [5]. The prevalence of adult hypertension in Europe and America accounts for one third of the total population. At present, angiotensin converting enzyme (ACE) inhibitors are the main therapeutic drugs used in the treatment of hypertension, but some studies have found that patients taking synthetic ACE inhibitors have different degrees of side effects, such as cough, oedema, rash and other

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uncomfortable reactions [6, 7]. Therefore, natural antihypertensive peptides have gradually been favoured by researchers. Antihypertensive peptide, also known as ACE inhibitory peptide, is a

polypeptide that can reduce blood pressure by inhibiting ACE. At present, there are three methods

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to obtain natural antihypertensive peptides: I. direct extraction of natural active peptides, i.e. direct

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purification of various natural antihypertensive peptides from organisms without hydrolysis; II.

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hydrolysis of proteins by chemical (heating or acid treatment) or enzymatic methods (direct enzymatic hydrolysis and indirect enzymatic hydrolysis by microorganisms), and III. recombinant

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DNA technology and chemical synthesis of active peptide [8]. Although ACE inhibitors have been recognised for the treatment of hypertension, due to their

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toxicity and side effects on kidney for example, more safe and efficient ACE inhibitors are required, such as the preparation of ACE inhibitory peptides by enzymatic hydrolysis of food

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proteins. ACE inhibitory peptides derived from food proteins are usually obtained by protease hydrolysis under mild conditions, which are safe with no toxic side effects [9]. The preparation of ACE inhibitory peptides from plant proteins has become a research hotspot due to its low cost, high safety and non-toxic side effects. Food or plant proteins are rich in valine, leucine, phenylalanine, proline, glutamine, histidine, threonine, arginine, methionine and lysine, which are

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appropriate for the preparation of ACE inhibitory peptide. There are a few reports regarding the preparation of ACE inhibitory peptide from green tea residue by hydrolysis of protein. Therefore, the aim of this study was to optimise the extraction process of proteins from green tea residue with ACE inhibitory activity and evaluate the antihypertensive activity of the extracted proteins to provide a theoretical basis for the utilisation of tea residue resources and the development of

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natural blood pressure lowering peptides.

2. Material and Methods

2.1. Materials and reagents

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Green tea processed to produce green tea residue was provided by the Tea Research Institute,

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Guangdong Academy of Agricultural Sciences. ACE (0.25 U), N-hippuryl-his-leu hydrate (HHL), and hippuric acid (HA) standards were purchased from Sigma-Aldrich. Captopril was purchased

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from Shanghai Yuanye Biotechnology Co., Ltd. Viscozyme L, acid protease, alcalase, neutrase,

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pepsin, and trypsin were purchased from Novozymes, Denmark. Vivaflow 50 Flip-flow Ultra-filtration (100 kDa) was purchased from Vivascience. Dialysis bag (500 Da, MD77MM, 49

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mm) were purchased from Viskase (USA).

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2.2. Extraction of green tea proteolytic products

Green tea was added to boiling water at a ratio of 1:30, then placed in a water bath at 90°C

for 0.5 h. The tea soup was discarded and the leaching was repeated three times to obtain the green tea residue. The green tea residue was dried in an oven at 90°C, pulverised, and passed through a 0.425 mm standard sieve. The green tea residue was dissociated by Viscozyme L and extracted with a solution of sodium hydroxide; the supernatant was collected by centrifugation and 5

precipitated with acid for 30 min. The precipitate was collected for dialysis and desalting by centrifugation, decoloured with 75% acetone, then freeze-dried to obtain green tea residue proteins, which were further dissociated using different bioproteolytic enzymes to obtain green tea proteolytic products as shown in Fig. 1.

2.3. Optimisation of green tea residue protein extraction process

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2.3.1. Single factor test

The effects of different NaOH concentration, temperature, extraction time and solid-liquid

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ratio on the extraction rate of protein from green tea residue (GTPE rate) were studied. The fixed conditions were: 0.08 mol/L NaOH, extraction temperature 70°C, extraction time 60 min,

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solid-liquid ratio (w/v) 1/40 and the variable conditions were: NaOH concentration (0.04, 0.06,

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0.08, 0.10, 0.12 mol/L), extraction temperature (50, 60, 70, 80, 90°C), extraction time (20, 40, 60, 80, 100 min), solid-liquid ratio (1/10, 1/20, 1/30, 1/40, 1/50, 1/60). The GTPE rate was calculated

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according to the following formula: GTPE rate = (total content of protein in the green tea protein/total content of protein in green tea residues) × 100. The protein content of green tea and

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green tea residues was determined by the Kjeldahl method.

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2.3.2. Orthogonal test to determine the optimal alkaline extraction process

According to the results of single factor test, the alkali extraction temperature, time, alkali

concentration and solid-liquid ratio were investigated. The GTPE rate was taken as the index, and the optimal alkaline extraction process was determined by the L9 (34) orthogonal test (Table S1).

2.3.3. Total factor test to determine the optimal concentration and time of hydrolysis by hydrolase

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According to the protein extraction process in Fig. 1, a complex hydrolase pre-treatment step was added before the alkaline extraction step. The enzyme concentration range was set to 1.0%, 1.5%, 2.0%, 2.5%, 3.0% and 3.5%. The extraction time range was set to 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h and 3.5 h. The full factor hydrolysis test was conducted at pH 3.4, a temperature of 45°C, and a solid-liquid ratio of 1:20. Finally, the GTPE rate was determined, and the results were

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analysed by full factor analysis of variance.

2.4. ACE enzyme activity assay

The protein from the green tea residue was hydrolysed under the same conditions

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(solid-liquid ratio (w/v) 1/50, reactant ratio (w/w) 100/2, 2 h) using acid protease, neutral protease,

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alkaline protease, trypsin and pepsin, respectively. The effect of green tea proteolytic products on ACE enzyme inhibitory activity in vitro was examined. The ACE enzyme inhibitory activity was

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determined by the modified liquid chromatography method of Liu et al [10]. Specifically, the column was an Agilent Eclipe Plus C18 column (4.6 × 150 mm, 5 μm), and the mobile phase was

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acetonitrile-water (30:70, V/V, 0.05% formic acid) at a flow rate of 0.8 mL/min, wavelength was 228 nm, followed by equal gradient elution, auto injection and 10 μL injection volume. The green

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tea proteolysis product was dissolved in a sodium borate buffer solution and filtered to prepare a

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1.0 mg/mL solution. A 1.0 mg/mL captopril solution was prepared as a control sample. Then, 30 μL of sample solution, 30 μL of sodium borate buffer solution were taken, and 20 μL of ACE solution was mixed. The solution was preheated in a constant temperature water bath at 37°C for 10 min, and then thoroughly mixed with 100 μL of HHL solution. After the solution was incubated at 37°C for 60 min, the reaction was stopped with 200 μL of HCL solution and passed through a 0.45 μm filter for use. An equal amount of buffer solution was used instead of the sample as a 7

blank control.

2.5. Optimisation of hydrolysis conditions of green tea proteolytic products

2.5.1. Single factor test

The effects of hydrolysis temperature, time, enzyme dosage and pH on the activity of green tea proteolytic products were examined. The fixed conditions were as follows: the amount of acid

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protease was 2.0% of the amount of protein, the pH value was 3.0, the hydrolysis temperature was 40°C, and the time was 2.0 h. Variable conditions were: hydrolysis temperature (30°C, 35°C, 40°C,

45°C, 50°C), hydrolysis time (1.0 h, 2.0 h, 3.0 h, 4.0 h, 5.0 h), enzyme dosage (protein 1.0%, 2.0%,

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3.0%, 4.0%, 5.0%), and pH (2.0, 3.0, 4.0, 5.0, 6.0). The effect of various factors on the ACE

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determined by neutral formaldehyde titration.

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inhibition rate of green tea proteolytic products was examined. The degree of hydrolysis (DH) was

2.5.2. Orthogonal test to determine the optimal hydrolysis conditions

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On the basis of the single factor test, the hydrolysis temperature, hydrolysis time, enzyme amount and reaction pH were investigated, and the ACE inhibition rate was used as an indicator.

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The hydrolysis conditions were optimised by L9 (34) orthogonal test (Table S2). Under the optimal

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hydrolysis conditions, the obtained green tea proteolytic products were used for further composition analysis and antihypertensive activity detection in vivo.

2.6. Composition analysis of green tea proteolytic products

The amino acid content and distribution in the green tea proteolytic products were determined by the method for determination of amino acids in the National Food Safety National

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Standard (GB 5009.124-2016). The molecular weight distribution of the hydrolysed product was conducted according to the Tricine-SDS-PAGE electrophoresis method of Haider et al [11].

2.7. LC-MS/MS determination

The appropriate amount of the enzymatic hydrolysed product was dissolved in distilled water, and the ultrafiltrate having a molecular weight of less than 100 kDa was cut off after ultrafiltration

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at 100 kDa. The analysis was performed according to the following mass spectrometry conditions. The LC-MS/MS analysis was conducted in a Triple TOF 5600 system (AB SCIEX) combined

with a nanospray III ion source (AB SCIEX, USA). The hydrolysate fractions were resuspended in

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mobile phase A (98.0% (v/v) water, 2.0% (v/v) acetonitrile, and 0.1% (v/v) formic acid), and 10

μL sample was loaded on a C18NanoLC trap column (100 μm × 3 cm, C 18, 3 μm, 150 Å) and

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washed by mobile phase A at 4 μL/min for 10 min. After 10 min of pre-concentration, the trap

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column was automatically switched in-line to a ChromXPC18 column (75 μm × 15 cm, C18, 3 μm, 120 Å). Mobile phase A contained 98% (v/v) water, 2.0% (v/v) acetonitrile, and 0.1% (v/v) formic

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acid. Mobile phase B contained 2.0% (v/v) water, 98.0% (v/v) acetonitrile, and 0.1% (v/v) formic acid. Peptides were eluted with a linear gradient from 5% to 35% of solvent B over 90 min at a

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flow rate of 0.3 μL/min and a running temperature of 25°C. The following conditions were used to

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characterise the peptides: spray voltage, 2.5 kV; air curtain pressure, 30 PSI; and atomisation pressure, 5 PSI. The data were processed using Mascot 2.3 (Matrix Science) mass spectrometry software, and homologous protein analysis was performed with a protein database (15566 sequences; 4692812 residues).

2.8. Determination of factors affecting the stability of green tea proteolytic products

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2.8.1. Temperature The green tea proteolysis product (200 μL of 1.0 mg/mL) was placed in a water bath at 25°C, 37°C, 50°C, 60°C and 80°C for 2 h, then 40 μL of each solution was taken for assessment of the ACE inhibition rate by liquid chromatography.

2.8.2. pH

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The green tea proteolysis product (10.0 mg/mL) was incubated at room temperature for 2 h at pH 2.0, 4.0, 6.0, 8.0, and 10.0, then diluted 10 times and the pH was adjusted to 8.3 before the

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ACE inhibition rate was measured by liquid chromatography.

2.8.3. Simulation of gastrointestinal tract environment in vitro

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The stability of green tea proteolytic products in simulated gastrointestinal tract environment

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in vitro was investigated by the method of the Chinese Pharmacopoeia (2015 edition). The green tea proteolysis product (10.0 mg/mL) was added to artificial gastric juice or artificial intestinal

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juice at 1:9 and placed in a 37°C water bath. After incubation for 0, 30, 60, 90 and 120 min at room temperature, the solutions was separately diluted 10 times and the pH was adjusted to 8.3,

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before the ACE inhibition rate was measured.

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2.8.4. Metal ions

The green tea proteolysis product (10.0 mg/mL) was dissolved in Mg2+, Zn2+, K+, Ca2+, Cu2+

solutions at concentrations of 0, 1, 3, 5, 7 mmol/L, respectively. After incubation for 2 h at room temperature, the solution was diluted 10 times and the pH was adjusted to 8.3 for measurement of the ACE inhibition rate.

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2.9. Antihypertensive activity detectionin vivo

SPF grade 12-week-old male SHR rats (Spontaneously hypertensive rats) and male WKY rats (Wistar-Kyoto rats) were purchased from Beijing Vital River Laboratory Animal Technology. All experimental procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals. The protocols were approved by the Ethical Committee of Tea Research Institute at Guangdong Academy of Agricultural Sciences. Nine WKY rats served as a

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normal control group (control group). Thirty-six SHR rats were randomly divided into four groups: model group (distilled water gavage), positive control group (captopril group, 5.0 mg/kg·BW

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captopril), low-dose group (400 mg/kg·BW green tea proteolytic product) and high-dose group (800 mg/kg·BW green tea proteolysis product). Systolic blood pressure (SBP) and diastolic blood

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pressure (DBP) were measured before the start of the experiment, then at 0.25 h, 0.5 h, 1.0 h, 2.0 h,

2.10. Statistical analysis

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4.0 h and 6.0 h after intragastric administration. The measurements were repeated three times.

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The results are presented as mean ± SD. Analyses were performed with SPSS 19.0 or

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GraphPad Prism 7.0 software for Windows (GraphPad Software Inc., San Diego, CA, USA). The level of confidence required for significance was set at p<0.05.

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3. Results and Discussion

3.1. Optimisation of the alkaline extraction processof proteinfrom green tea residue

3.1.1. Single factor test

The results showed the extraction rate of protein from green tea residue gradually increased

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with the increase of NaOH concentration (Fig. 2A) until 0.08mol/L, after which it gradually slowed down, with no further change at 0.12 mol/L. Therefore, the most appropriate NaOH concentration for extraction of protein from green tea residue is 0.08-0.12 mol/L. The extraction rate of protein from green tea residue increased significantly with the increase of liquid-solid ratio (Fig. 2B) until 50:1-60:1, after which it increased slowly. However, the higher liquid-solid ratio caused lye waste, which affected subsequent processing, so the 50:1 liquid-solid

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ratio was selected as the optimum liquid-solid ratio for subsequent tests.

The effect of alkali extraction time on the extraction rate of protein from green tea residues was shown in Fig. 2C. With the increase of alkali extraction time, the extraction rate of protein

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increased significantly. As the alkali extraction time exceeded 100 min, the extraction rate of

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protein decreased, therefore 100 min was selected as the best alkali extraction time.

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As shown in Fig. 2D, the extraction rate of protein increased slowly with increasing temperature from 50°C to 80°C, increasing significantly at 90°C. However, the extraction rate

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remained unchanged at 100°C, so 90°C was selected as the optimum alkali extraction temperature.

3.1.2. Orthogonal test to determine the best alkaline extraction process

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According to the results of the single factor test, the orthogonal test was performed using the

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L9(34) orthogonal table, and the effects of each factor is shown in Table S1. Through the visual analysis method comparing the extreme R values of the four factors in the table, the NaOH concentration in the alkali extraction step of protein from green tea residue was the most important factor affecting the protein extraction rate (Table 1). The factors affecting the extraction rate of tea protein were as follows: A (NaOH concentration) > D (temperature) > B (liquid-solid ratio) > C (time). 12

Analysis of variance was performed to investigate the error and fine effect of the experiment, showing that the four factors significantly effecting the extraction rate of tea protein (Table S3) were as follows: A > D > B > C. In summary, the optimal conditions for the combined alkali extraction process were A3B2C3D3: 0.12 mol/L NaOH solution, 50:1 liquid-solid ratio at 90°C, and 100 min extraction

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time, yielding a GTPE of 52.35%.

3.2. Optimisation of the enzyme-alkali complex method of protein extration from green tea residue

Compared with single-alkali extraction, Viscozyme L enzymatic hydrolysis followed by

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alkali extraction increased the protein solubility in green tea residues, thereby increasing the

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protein extraction rate [12]. Therefore, we explored the hydrolysis conditions of hydrolase before alkaline extraction, finding that the factors affecting the extraction rate of protein from green tea

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residue in the hydrolysis method of Viscozyme L included hydrolysis time, enzyme concentration, pH value and hydrolysis temperature [13, 14]. Since the optimum pH of Viscozyme L is 3.3-3.4,

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the optimum temperature is 40-45°C, the pH of the hydrolysis was set to 3.4, and the hydrolysis temperature was set to 45°C. The hydrolysis time and enzyme concentration were investigated

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using a full factor test to determine the optimal hydrolysis conditions as shown in Table S4.

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According to the results of the analysis of variance (Table S5), two factors, hydrolysis time and enzyme concentration, significantly affected the extraction rate of protein from green tea residue. There were significant differences in protein extraction rates at almost all hydrolysis times (Table S6), except for 3 h and 3.5 h. so a hydrolysis time of 3 h was chosen as the optimal hydrolysis time. Similarly, there were significant differences in protein extraction rates at almost all enzyme concentrations (Table S7), except for enzyme concentrations of 2.50% and 3.00%, so considering 13

the extraction rate of protein and cost of saving reagent, the enzyme concentration of 2.50% was selected as the optimal hydrolysis condition. In summary, the optimal extraction conditions for protein from green tea residue were Viscozyme L hydrolysis for 2 h at an enzyme concentration of 2.50%, and NaOH solution with an alkali extraction condition of 0.12 mol/L, extraction temperature of 90°C, liquid-solid ratio of 50:1, and extraction time of 120 min. The extraction rate of protein from green tea residue under these

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conditions was 74.27%.

3.3. Comparison of in vitro inhibition of ACE activity by green tea proteolytic products

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Five green tea proteolysis products were obtained respectively by acid protease, neutral

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protease, alkaline protease, trypsin and pepsin, and the inhibitory ACE activity was detected in vitro. The in vitro inhibition of ACE enzymatic activity of the green tea proteolytic product

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obtained by acid protease digestion was the highest (70.01%), which was only lower than that of captopril (80.67%, Fig. 3). Therefore, acid protease was used to prepare green tea proteolysis

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products in hydrolysis conditions.

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3.4. Optimisation of hydrolysis conditions of green tea proteolytic products

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3.4.1. Single factor test

At the hydrolysis temperature of 45°C, the acid hydrolysis of green tea proteolytic products

yielded the highest ACE inhibitory rate and hydrolysis (Fig. 4A). As the hydrolysis time increased, the ACE inhibitory rate and hydrolysis degree of green tea proteolysis products gradually increased. At 3 h of hydrolysis, ACE inhibitory activity and DH reached the highest at 3 h and 4 h, respectively (Fig. 4B). Therefore, we determined the optimal temperature at 45°C and the optimal 14

hydrolysis time of 3 h for subsequent orthogonal experiments. The acid protease concentration affected the ACE inhibitory rate and DH. When the concentration of acid protease was 1.0-4.0%, the ACE inhibitory rate and DH of green tea proteolysis products increased significantly with increasing concentration (Fig. 4C). The ACE inhibitory rate and DH of the green tea proteolytic product reached a maximum at pH 3.0 (Fig. 4D). Taken together, we determined the optimum hydrolysis concentration of 3.0% and pH 3.0 for

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the next orthogonal test.

3.4.2. Orthogonal test to determine the optimal hydrolysis conditions

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The orthogonal test was performed using the L9 (34) orthogonal table, and the range of the

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three levels of each factor is shown in Table S2. Comparing the extreme R values of the four factors by visual analysis (Table 2), the factors affecting the ACE inhibitory rate of green tea

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proteolytic products were: D (pH) > A (hydrolysis temperature) > B (hydrolysis time) > C (enzyme concentration). The results of variance analysis showed that pH (D factor) had a

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significant effect on the ACE inhibitory rate of green tea proteolytic products, with the effects of A (hydrolysis temperature), B (hydrolysis time), and C (enzyme concentration) on the ACE

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inhibitory rate not significant (Table S8). Therefore, in terms of energy saving and reagent cost,

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the hydrolysis conditions of A1 (40°C), B1 (2 h), and C1 (2.0%) were selected. In summary, the optimal conditions (A1, B1, C1, D3) were: hydrolysis temperature 40°C,

hydrolysis time 2 h, enzyme concentration 2.0% and pH 4.0. The ACE inhibitory rate of the green tea proteolytic product obtained under these hydrolysis conditions was determined to be 77.00%.

3.5. Composition analysis of green tea proteolytic products

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3.5.1. Amino acid composition of green tea proteolytic products

The green tea proteolytic product extracted by the above optimised process contained 16 amino acids, including seven essential amino acids and nine non-essential amino acids. The ratio of essential amino acids to non-essential amino acids was 0.67, which was higher than the 0.6 specified by the FAO/WHO standard. The essential amino acid content accounted for 40% of the total amino acids and met the FAO/WHO standard (Table 3), indicating that the green tea

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proteolytic product had a high nutritional value. In addition, its hydrophobic amino acid content

was 15.74 g/100 g, accounting for 46.69% of the total amino acid, therefore an ideal raw material

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for the preparation of ACE inhibiting peptides.

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3.5.2. Molecular weight distribution of green tea proteolytic products

The Tricine-SDS-PAGE electrophoresis map (Fig. 5) showed that the molecular weight

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distribution of the polypeptide in the green tea proteolytic product was widely distributed between

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45.0-1.2 kDa.

3.5.3. Structure analysis of green tea proteolytic products by LC-MS/MS

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Mass spectrometry results of green tea proteolytic products were analysed by sequence

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information using the Mascot database and NCBI. The four amino acid matching sequences were: .MAEIPTSDR.S, R.LATGEPLR.V, K.GMMLEDSSR.S, and R.LENMCWR.I (Fig. 6-9). These polypeptides were searched by PROTEIN BLAST and aligned with the amino acid sequence of the tea-related proteins reported in PubMed [15, 16]. In addition, the molecular weight range of the four polypeptides was 855.44814-1056.4216 Da, with R.LENMCWR.I having the highest hydrophobic amino acid content of 57.14% (Table 4). 16

3.6. Stability analysis of green tea proteolytic products

The ACE inhibitory activity of the green tea proteolytic product did not change significantly in the range of 25-80°C (Fig. 10A), indicating that temperature did not influence the hypotensive activity. As the pH increased, the ACE inhibitory activity of the green tea proteolytic product decreased significantly (Fig. 10B), indicating that the green tea proteolytic product was more active under acidic conditions, which was advantageous for its activity in acidic gastric juice.

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We simulated the in vitro digestive environment of the stomach and intestines to study the

anti-digestibility of green tea proteolytic products (Fig. 10C). The ACE inhibitory activity of green

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tea proteolysis products did not change significantly within 0-60 min of pepsin action. However,

the activity of the green tea proteolytic product decreased significantly after 90 min, whereas

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under the action of trypsin, the ACE inhibitory activity increased with time. These results indicate

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that the green tea proteolytic product had good anti-digestibility and maintained high ACE inhibitory activity within 60 min.

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Metal ions affected the ACE inhibitory activity of green tea proteolytic products (Fig. 10D). At low Mg2+ concentration (1 mmol/L), the ACE inhibitory rate of green tea proteolytic products

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was only 26.21%, decreasing with increasing Mg2+ concentration. Similarly, the ACE inhibitory activity decreased with increasing Ca2+ concentration, and the degree of inhibition was higher than

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that of Mg2+. In the food industry, Ca2+ is abundant in dairy products, meat products, and legumes [17], therefore, green tea proteolytic products should not be consumed with these foods, avoiding their antagonism with Ca2+ and affecting their activity. Lower concentrations of Cu2+ reduced ACE inhibitory activity, but high concentrations of Cu2+ increased ACE inhibitory activity. The concentration of Zn2+ at 3 mmol/L promoted an increase in ACE inhibitory activity, reaching its

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maximum, whereas K+ had little effect on the activity of green tea proteolytic products. Potassium is a major component of daily food and important for maintaining body fluid osmotic pressure and cardiac function [18, 19], so green tea proteolytic products can be consumed with such foods without affecting their activity.

3.7. Antihypertensive activity of green tea proteolysis products in vivo

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In Fig. 11, SHR rats were intragastrically administered low-dose and high-dose green tea proteolytic products for 1 h, and the SBP and DBP decreased. After 2 h, the SBP and DBP of the

rats in the treatment group decreased to the lowest level, which was significantly (p<0.05) or

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extremely significant (p<0.01) lower in comparison with the model control group. The blood

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pressure lowering effect in the high-dose group was close to the positive control group. Subsequently, SBP and DBP gradually increased, and returned to the pre-administration level at 6

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h, indicating that the green tea proteolytic product possessed effective blood pressure lowering

4. Conclusions

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activity in vivo.

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The ACE inhibitory peptides in green tea residue have potential antihypertensive activity. This study optimised the extraction of tea residue protein using an alkaline extraction method

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combined with enzymatic hydrolysis. The extracted green tea residue proteolytic products possessed significant ACE inhibitory activity and demonstrated effective antihypertensive activity in vivo. This study detailed an optimal extraction method for green tea ACE inhibitory peptides and laid the foundation for clinical application of green tea residue for blood pressure reduction.

Credit Author Statement 18

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Xingfei Lai: Data curation, Writing-Original draft preparation, Formal analysis.

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Shunshun Pan: Data curation, Investigation.

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Wenji zhang: Resources.

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Lingli Sun: Resources.

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Qiuhua Li: Resources.



Ruohong Chen: Resources.



Shili Sun: Conceptualization, Methodology, Writing-Review & Editing,

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Supervision.

Author Contributions

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All authors’ contributions as follows-Shili Sun conceived and designed the experiments. Xingfei

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Lai and Shunshun Pan performed the experiments and analyzed the data. Wenji zhang, Lingli Sun, Qiuhua Li and Ruohong Chen contributed reagents. Xingfei Lai wrote the paper, and Shili Sun

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revised the manuscript. All authors approved the final version of the manuscript.

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Declarations of interest: none.

Acknowledgements

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The work was supported by the National Natural Science Foundation of China (31800295,

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81803236, 81903319), the Guangdong Science and Technology Program (2017A070702004, 2016B090918118, 2017A020224015, 2018KJYZ002), the Natural Science Foundation of Guangdong Province (2017A030310504), the Guangdong Provincial Agriculture Department Program (2017LM2151), the President Foundation of Guangdong Academy of Agricultural Sciences (201720), special fund for scientific innovation strategy-construction of high level

19

Academy of Agriculture Science (R2019PY-JX004, R2018YJ-YB3002, R2016YJ-YB3003, R2018PY-QF005, R2018QD-101).

References [1] Kellogg J, Graf TN, Paine MF, Mccune JS, Kvalheim OM, Oberlies NH, Cech NB. Comparison of Metabolomics Approaches for Evaluatingthe Variability of Complex Botanical Preparations: Green Tea (Camellia sinensis) as a Case Study. Journal of Natural

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Products vol. 80; 2017. p. 1457-1466. [2] Skrzydlewska E, Ostrowska J, Stankiewicz A, Farbiszewski R. Green tea as a potent antioxidant in alcohol intoxication. Addiction Biology 2010;7:307-314.

[3] Rameshrad M, Razavi BM, Hosseinzadeh H. Protective effects of green tea and its main

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constituents against natural and chemical toxins: A comprehensive review. Food & Chemical Toxicology 2016;100:115.

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[4] Bedrood Z, Rameshrad M, Hosseinzadeh H. Toxicological effects of Camellia sinensis (green tea): A review. Phytotherapy Research 2018;32.

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[5] Subhija P, Fatima J, Enisa R, Ibrahim G, Vesna F, Budimka N, Emir H. Obesity as a Risk Factor for Artherial Hypertension. Materia Socio-Medica 2012;24:87-90.

na

[6] Sun WP, Zhang HB, Guo JC, Zhang XK, Zhang LX, Li CL, Zhang L. Comparison of the Efficacy and Safety of Different ACE Inhibitors in Patients With Chronic Heart Failure: A PRISMA-Compliant Network Meta-Analysis. Medicine 2016;95:e2554.

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[7] Shahani L. Case Report: ACE inhibitor-induced intestinal angio-oedema: rare adverse effect of a common drug. Bmj Case Rep 2013;2013:bcr2013200171.

Jo

[8] Chen Y, Wei Y, Donghai SU, Chen T. Optimization for the Fermentation Process With Bacillus natto to Obtain ACE Inhibitory Peptides From Ruditapes philippinarum. Journal of Nuclear Agricultural Sciences 2016.

[9] Wang YL, Huang Q, Kong D, Xu P. Production and Functionality of Food-derived Bioactive Peptides: A Review. Mini Reviews in Medicinal Chemistry 2018;18. [10] Liu J, Luo Y, Su Q, Fang C. Rapid determination of the angiotensin I-converting enzyme inhibitory activity of peptide by HPLC method: A simulated gastrointestinal digestion study. 20

Journal of Liquid Chromatography 2016;39:830-836. [11] Haider SR, Reid HJ, Sharp BL. Tricine-SDS-PAGE. Methods in Molecular Biology 2012;869:81-91. [12] Zhang C, Sanders JPM, Xiao TT, Bruins ME. How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves. Plos One 2015;10:e0133046. [13] Hong YH, Jung EY, Park Y, Shin KS, Kim TY, Kwang-Won YU, Chang UJ, Suh HJ. Enzymatic Improvement in the Polyphenol Extractability and Antioxidant Activity of Green

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Tea Extracts. Journal of the Agricultural Chemical Society of Japan 2013;77:22-29. [14] Jang JH, Park YD, Ahn HK, Kim SJ, Lee JY, Kim EC, Chang YS, Song YJ, Kwon HJ. Analysis of Green Tea Compounds and Their Stability in Dentifrices of Different pH Levels. Chemical & Pharmaceutical Bulletin 2014;62:328-335.

-p

[15] Alyousef AA, Mateen A, Al-Akeel R, Alqasim A, Al-Sheikh Y, Alqahtani MS, Syed R. Screening & analysis of anionic peptides from Foeniculum vulgare Mill by mass

re

spectroscopy. Saudi journal of biological sciences 2019;26:660-664.

[16] Fu J, Liu G, Yang M, Wang X, Chen X, Chen F, Yang Y. Isolation and functional analysis of

PPB 2019;142:53-58.

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squalene synthase gene in tea plant Camellia sinensis. Plant physiology and biochemistry :

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[17] Rohrmann S, Hemelrijck MV. The Association of Milk and Dairy Consumption and Calcium Intake With the Risk and Severity of Prostate Cancer. Current Nutrition Reports 2014;4:1-6. [18] Liu Z, Du L, Li M. Update on the slow delayed rectifier potassium current (I(Ks)): role in

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modulating cardiac function. Current medicinal chemistry 2012;19:1405-1420. [19] Li Y, Zhang L, Zhang L, Zhang H, Zhang N, Yang Z, Gao M, Yang X, Cui L. High-dose

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glucose–insulin–potassium has hemodynamic benefits and can improve cardiac remodeling in acute myocardial infarction treated with primary percutaneous coronary intervention: From a randomized controlled study. Journal of Cardiovascular Disease Research 2010;1:104-109.

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Figure legends

Fig. 1. The extraction process of green tea proteolytic product.

Fig. 2. Single factor test for the alkaline extraction process of green tea protein extraction. A is NaOH concentration, B is liquid-solid ratio, C is extraction time and D is extraction temperature.

Fig. 3. Comparison of in vitro inhibition of ACE activity of green tea proteolytic products hydrolysed by different proteases. Data are presented as mean ± SD (n=3). Values with different

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letters (a−f) are significantly different from each other (p<0.05).

Fig. 4. Single factor test for hydrolysis conditions of green tea proteolytic products. A is the ACE

inhibitory rate and DH of green tea proteolytic products under different hydrolysis temperatures, B

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is different hydrolysis time, C under different concentrations of acid protease and D under

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different pH.

Fig. 5. Tricine-SDS-PAGE for green tea proteolytic products hydrolysed by different proteases.

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The Tricine-SDS-PAGE mass range is between 1.2–45.0 kDa. a is tea protein from green tea residue, b is green tea proteolytic products hydrolysed by acid protease, c is acid protease, d is green tea proteolytic products hydrolysed by alkaline protease, e is alkaline protease, f is green tea

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proteolytic products hydrolysed by neutral protease, g is neutral protease, h is green tea proteolytic products hydrolysed by pepsin, i is pepsin, j is green tea proteolytic products hydrolysed by

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trypsin and k is trypsin.

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Fig. 6. Sequence MAEIPTSDR mass spectrum (protein: gi|570709911|gb|AHE93348.1|)

Fig. 7. Sequence LATGEPLR mass spectrum (protein: gi|594021565|gb|AHL69788.1|)

Fig. 8. Sequence GMMLEDSSR mass spectrum (protein: gi|555947674|gb|AGZ20100.1|)

Fig. 9. Sequence LENMCWR mass spectrum (protein: gi|430802664|gb|AGA82513.1|)

Fig. 10. Stability analysis of green tea proteolytic products. A is the ACE inhibitory rate of green 22

tea proteolytic products under different temperature, B is different pH, C is the ACE inhibitory rate of green tea proteolytic products under in vitro digestive environment of the stomach and intestines in 120 min and D is the ACE inhibitory rate of green tea proteolytic products under different concentrations of Mg2+, Zn2+, K+, Ca2+, and Cu2+ solutions.

Fig. 11. Antihypertensive activity of green tea proteolysis products in vivo. A is the SBP and B is the DBP of the rats in the different treatment groups. SBP and DBP were measured by ZH-HX-Z non-invasive blood pressure meter and measurements were repeated three times. Data are

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presented as mean ± SD (n=9). The level of significance relative to the model group is represented

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ur

na

lP

re

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by *p<0.05 or **p<0.01 (One-way ANOVA).

23

ro of

-p

re

lP

na

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Jo Fig. 1.

24

ro of

-p

re

lP

na

ur

Jo Fig. 2.

25

ro of

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re

lP

na

ur

Jo Fig. 3.

26

ro of

-p

re

lP

na

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Jo Fig. 4.

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ro of

-p

re

lP

na

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Jo Fig. 5.

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Fig. 6. L1 #4475 RT: 18.53 AV: 1 NL: 1.06E3 T: ITMS + c NSI r d w Full ms2 [email protected] [130.00-1035.00] 524.3701

100 95 90 85 80 75 70

Relative Abundance

65 60 55 50 45 40

514.4816

30 25 20

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35

521.3903

510.3731

15 10

525.3436

518.3548

5 0 509

510

511

512

513

514

515

516

517

518 m/z

519

520

521

522

523

524

525

526

527

528

529

-p

508

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Fig. 7. L1 #11498 RT: 38.10 AV: 1 NL: 3.67E2 T: ITMS + c NSI r d w Full ms2 [email protected] [105.00-870.00] 100

437.3462

428.2617

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95 90 85 80 75

na

70

60 55 50

40 35 30 25

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45

429.4139

427.2617

436.5699

424.6483

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Relative Abundance

65

435.8961

20 15

430.7021

10

5 0

421

422

423

424

425

426

427

428

429

29

430

431 m/z

432

433

434

435

436

437

438

439

440

441

Fig. 8. L1 #17663 RT: 56.14 AV: 1 NL: 2.36E2 T: ITMS + c NSI r d w Full ms2 [email protected] [125.00-995.00] 492.3918

100 95 90 491.2769

85 80 75 70

Relative Abundance

65 60 55 50 45 40 35

25 20

493.4141

15 10 5 0 490.5

491.0

491.5

492.0 m/z

492.5

493.0

493.5

494.0

-p

490.0

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Fig. 9. L1 #8751 RT: 30.24 AV: 1 NL: 2.17E2 T: ITMS + c NSI r d w Full ms2 [email protected] [130.00-1040.00] 100 95

513.4875

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90 85 80 75 70

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65 60 55 50 45

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40 35 30 25 20

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Relative Abundance

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30

512.3211

511.6989

15 10

5 0

510.8

511.0

511.2

511.4

511.6

511.8

512.0

512.2

30

512.4 m/z

512.6

512.8

513.0

513.2

513.4

513.6

513.8

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re

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na

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Jo Fig. 10.

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Fig. 11.

1 2 3 4 5 6 7 8 9 K1 K2 K3 k1 k2 k3 R

0.08 0.08 0.08 0.10 0.10 0.10 0.12 0.12 0.12 91.53 112.80 128.04 30.51 37.60 42.68 12.17

concentration

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Liquid-solid ratio B

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NaOH (mol/L) A

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Item

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Table 1 L9 (34) orthogonal test and results of green tea protein extraction (GTPE)

1:40 1:50 1:60 1:40 1:50 1:60 1:40 1:50 1:60 101.40 116.70 114.30 33.80 38.90 38.10 5.10

Time (min) C

Temperature (°C) D

GTPE (%)

80 100 120 100 120 80 120 80 100 109.92 106.77 115.68 36.64 35.59 38.56 2.97

70 80 90 90 70 80 80 90 70 100.65 109.56 122.16 33.55 36.52 40.72 7.17

23.70 30.73 37.10 36.92 37.82 38.07 40.77 48.15 39.13

32

Note: Ki is the experiment value of i value of this factor. ki is the experiment average value of i value of this factor.

Table 2 L9 (34) orthogonal test and results of GTPH Temperature (°C)

Time (min)

Acid protease concentration (%)

pH

GTPH (%)

1 2 3 4 5 6 7 8 9 K1 K2 K3 k1 k2 k3 R

40 40 40 45 45 45 50 50 50 207.16 150.90 142.70 69.53 50.30 47.23 22.30

2 3 4 2 3 4 2 3 4 184.51 156.75 159.50 61.50 52.25 53.17 9.25

2 3 4 3 4 2 4 2 3 175.51 169.85 155.40 58.50 56.62 51.80. 6.70

2 3 4 4 2 3 3 4 2 116.61 180.15 204.00 38.87 60.05 68.00 29.13

61.01 71.05 75.10 69.50 26.30 55.10 54.00 59.40 29.30

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Item

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Note: Ki is the experiment value of i value of this factor. ki is the experiment average value of i value of this factor. R is the maximum difference between the three ki values.

Table 3 Amino acid composition of GTPH

Amino acid (AA)

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Item

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Content (g/100g)

Non-essential amino acid (NEAA)

1.69 2.37 0.87 1.69 1.71 3.24

Lys

1.91

His Asp Glu Ser Gly Arg Ala Tyr Pro

1.35 3.97 5.28 1.65 1.84 0.28 2.63 1.31 1.92

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Essential amino acid (EAA)

Thr Val Met Phe Ile Leu

13.48

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Total EAA (TEAA)

20.23

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Total NEAA (TNEAA)

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TAA

TEAA/TAA

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TEAA/TNEAA

0.67

0.40

15.74

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Hydrophobic amino acid

33.71

Note: The concentration of hydrophobic aminoacid is the sum of Ala, Val, Ile, Leu, Phe, Pro, Met

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and Tyr.

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Table 4 The structure characterisation of GTPH

Matching protein name

1018.4622

1018. 4753

Fructokinase

R.LATGEP LR.V

855.5176

855.4 814

Hypothetical protein

1056.4034

1056. 4216

Serine/threonin e-protein kinase

1023. 4266

Sucrose phosphate synthase, partial

K.GMMLE DSSR.S

R.LENMC WR.I

1023.4061

Cam ellia sinen sis Cam ellia sinen sis Cam ellia sinen sis Cam ellia sinen sis

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.MAEIPTS DR.S

Speci es

Isoelectri c point

Hydrop hobic amino acid (%)a

5.92

33.33%

6.51

37.5%

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Match Calcul peptide Theoretical value ated amino acid of MW (Da) MW sequence (Da)

6.35

33.33%

6.03

57.14%

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a: Calculated from the percentage of hydrophobic radidues (I, V, L, F, C, M, A, W) in the peptides

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sequence.

35