STUDY OF THE STABILITY OF NATURAL EXTRACT OF GREEN TEA IN AQUEOUS SOLUTIONS AND THEIR KINETICS

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STUDY OF THE STABILITY OF NATURAL EXTRACT

OF GREEN TEA IN AQUEOUS SOLUTIONS AND

THEIR KINETICS

By

Rajae Hani Saadeh

Supervisor

Dr. Abeer Al-Bawab

Co-Supervisor

Dr. Ahmad Abdoh

Submitted in Partial fulfillment of the Requirements for the Degree of

Master of Science in Chemistry

Faculty of Graduated Studies

University of Jordan

March 2007

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Study of the stability of natural extract of green tea in aqueous solutions and their kinetics was Successfully Defended and Approved on 11/03/2007.

Examination Committee

Signature

Dr. Abeer Al-Bawab, Advisor

Associat. Prof. of Physical chemistry ---

Dr. Ahmad Abdoh, Co-Advisor

Dr. of Physical Chemistry ---

Dr. Rebhi Al-Zaru, Member

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DEDICATION

To My Family,

My Wife,

My Friends,

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ACKNOWLEDGMENT

I would like to express my sincere thanks and gratitude to my advisors Dr. Abeer Al-Bawab and Dr. Ahmad Abdoh for their assistance in guiding the whole work, and for their continuous encouragement and support.

Special thanks are also due to the examination committee members, Dr. Rebhi Al-Zaru, Dr. Manar Fayyad and Dr. Musa El-Bargothi for their valuable comments.

Special thanks are also due to the Advanced Pharmaceuticals team especially Dr. Nadia Ghazal and Eng. Lina Rezeq for the unlimited support and cooperation.

Special thanks are also due to my father, mother, wife, sisters and my brother for the unlimited support and cooperation.

Special thanks are also due to my friends Ala` Al-hares, Ashraf Kahlawe, Baha` Hammad, Ibrahim Nshash, M. Mansour, M. Tamimi, Rami Abusadah and Samer Baha`i for their support.

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Table of Contents

Subject Page

Committee Decision II Dedication III Acknowledgement IV List of contents V

List of Tables VII

List of Figures X

List of Equations XI

List of Abbreviation XII

Abstract XIII

INTRODUCTION 1

1. Preface 2

2. Green Tea Healthy Benefits 3

2.1 Antioxidant Effect 3

2.2 Anti-Endothelial Dysfunction Effect 3

2.3 Anti-inflammatory effect 4

2.4 Hypertension 4

2.5 Diabetes 5

3.0 Tea Catechins 5

4.0 Aim of This Study 7

LITERATURE REVIEW 9

1. Factors Affect Tea Color and Taste 10

2. Method of Evaluation of Antioxidant Activity of Poly Phenols 11

3. Method of Extraction and Separation of Poly Phenols 14

3.1 HPLC Methods 14

3.2 High Performance Capillary Electrophoresis (HPCE) Methods 17

3.3 Gas Chromatography Methods 18

4.0 Stability Studies for Tea Catechins 21

4.1 Stability of Green Tea Catechins in Solutions 21

4.2 Stability of Green Tea Catechins in Bread Making Process 24

5.0 Kinetic Studies for The Tea Catechins 25

5.1 Autoxidation of Catechins 25

5.2 Kinetic of Catechin Oxidation by Polyphenols Oxidase 26

5.3 Oxidation of Tea Catechins by Molecular Oxygen 28

EXPERIMENTAL 30

1. Experimental Plan 31

2. Material and Methods 33

2.1 Chemicals 33

2.2 Tea Material 33

2.3 Instrumentations 34

3. High Performance liquid chromatographic analysis 34

4. Experimental Procedure 35 4.1 Method Validation 35 4.1.1 Linearity 35 4.1.2 Precision 35 4.1.3 Repeatability 35 4.1.4 Selectivity 35 4.1.5 Limit of Quantitation 36

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4.1.6 Limit of Detection 36

5. Investigation of the Factors Affecting the Stability of Green Tea

Catechins in Aqueous Solutions 36

5.1 The Effect of Atmospheric Air on the Stability 36

5.2 The Effect of Temperature on the Stability 36

5.3 The Effect of pH on the Stability 36

5.4 The Effect of Buffer Species on the Stability 37

5.5 The Effect of Co-solvency on the Stability 38

5.6 Kinetics for the Reaction 39

RESULTS 40

1. Identification of the examined Tea Catechins 41

2. Validation of Analytical Method 42

2.1 Linearity 42 2.2 Precision 45 2.3 Reproducibility 46 2.4 Selectivity 46 2.5 Limit of Quantification 48 2.6 Limit of Detection 49

3.0 Investigation of the Factors Affecting the Stability of Tea Catechins in

Aqueous Solutions. 51

3.1 The Effect of Atmospheric Air on the Stability: 51

3.2 The Effect of Temperature on the Stability: 53

3.3 The Effect of pH on the Stability: 56

3.4 The Effect of Co-Solvency on the Stability 60

4.0 Kinetics for the Reaction 63

CONCLUTIONS 69

REFERANCES 71

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VII

List of Tables

Page Table 1 Description of the performed experiments to study different factors

affecting the stability of Tea Catechins in this study 32

Table 2 Specification of the chemicals used in the present study 33 Table 3 The added amount of NaCl to 100 ml of 0.05 phosphate buffer

solution at each pH value 37

Table 4 The added amount of NaCl to 100 ml of 0.05 Citrate buffer

solutions at each pH value 38

Table 5 Calibration Data for EC 43

Table 6 Calibration Data for EGCG 43 Table 7 Calibration Data for ECG 44 Table 8 Precision for EC,RGC,EGCG 0.5mg/ml 45 Table 9 Intermediate precision for EGCG, NC, and ECG in 0.5mg/ml

extract solution in water 46

Table 10 The limits of quantitation and detection for EC, EGCG and ECG 51 Table 11

The assay of EGCG after incubation of 0.5 mg/ml of tea extract solutions with different filling levels after incubation at 40oC for 15 days

52 Table 12

The assay of NC after incubation of 0.5 mg/ml of tea extract solutions with different filling levels after incubation at 40oC for 15 days

52 Table 13 The assay of ECG after incubation of 0.5 mg/ml of tea extract with

different filling levels after incubation at 40oC for 15 days. 53

Table 14 Thermal stability of 0.5 mg/ml tea extract water solution for 15

Days stored in 95% filled vials at different temperature 54

Table 15

The assay of tea catechins after storage of 0.5 mg/ml tea extract in 0.05 M citrate buffer with 0.2 M ionic strength at 40oC for five days

56 Table 16

The assay of tea catechins after storage of 0.5 mg/ml tea extract in 0.05 M phosphate buffer with 0.2 M ionic strength at 40oC for five days.

56 Table 17

The assay of EGCG in 0.05M citric at pH 3.0 and 0.2M ionic strength in the presence of different types and amounts of co solvents after 15 days of storage at 40oC

60 Table 18

The assay of NC in 0.05M citric at pH 3.0 and 0.2M ionic strength in the presence of different types and amounts of co solvents after 15 days of storage at 40oC

61 Table 19

The assay of ECG in 0.05M citric at pH 3.0 and 0.2M ionic strength in the presence of different types and amounts of co solvents after 15 days of storage at 40oC

61 Table 20 Dielectric constant ( ) for the different concentration of aqueous

solutions of co-solvent at 20oC 62

Table 21 The HLB values for the examined co-solvents 63 Table 22 Catechins concentration at different times at 25oC 64 Table 23 Catechins concentration at different times at 30oC 65

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Table 24 Catechins concentration at different times at 35oC 66 Table 25 Catechins concentration at different times at 40oC 66 Table 26 Catechins oxidation reaction rates at different catechins

concentrations 67

Table 27 The rate constants for catechins oxidation reaction at different

temperatures 68

Table 28 Activation energies for tea catechins oxidation reactions 68

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List of Figures Page

Figure 1 Tea catechins main group (Flavan-3-ol) 6

Figure 2 Major tea catechins 7

Figure 3 HPLC chromatogram for 0.5 mg/ml green tea extract in

water 41

Figure 4 HPLC chromatogram for 0.5 mg/ml EC in water 41 Figure 5 HPLC chromatogram for 0.5 mg/ml EGCG in water 42 Figure 6 HPLC chromatogram for 0.5 mg/ml ECG in water 42 Figure 7 Calibration curve of the area of the EC peak against its

concentration 43

Figure 8 Calibration curve of the area of the EGCG peak against

its concentration 44

Figure 9 Calibration curve of the area of the ECG peak against its

concentration 45

Figure 10 Peak purity for EC peak in 0.5 mg/ml tea extract water

solution 47

Figure 11 Peak purity for EGCG peak in 0.5 mg/ml tea extract

water solution 47

Figure 12 Peak purity for NC peak in 0.5 mg/ml tea extract water

solution 47

Figure 13 Peak purity for ECG peak in 0.5 mg/ml tea extract water

solution 48

Figure 14 HPLC chromatogram for 0.5 mg/ml tea extract water

solution after incubation for 15 days at 40oC 48

Figure 15 HPLC chromatogram for 1.50 x 10-3 mg/ml EC in water 49 Figure 16 HPLC chromatogram for 1.62 x 10

-3

mg/ml EGCG in

water 49

Figure 17 HPLC chromatogram for 1.49 x 10-3 mg/ml ECG in water 49 Figure 18 HPLC chromatogram for 0.50 x 10-3 mg/ml EC in water 50 Figure 19 HPLC chromatogram for 0.56 x 10

-3

mg/ml EGCG in

water 50

Figure 20 HPLC chromatogram for 0.51 x 10-3 mg/ml ECG in water 50 Figure 21 The effect of atmospheric air on the stability of EGCG in

0.5 mg/ml tea extract solution at 40oC 52

Figure 22 The effect of atmospheric air on the stability of NC in 0.5

mg/ml tea extract solution at 40oC 53

Figure 23 The effect of atmospheric air on the stability of ECG in

0.5 mg/ml tea extract solution at 40oC 53

Figure 24

The plot of assay of 0.5 mg/ml aqueous solution of EGCG against time at different temperature in 95% filled vial.

54 Figure 25

The plot of assay of 0.5 mg/ml aqueous solution of NC against time at different temperature in 95% filled vial.

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Figure 26

The plot of assay of 0.5 mg/ml aqueous solution of ECG against time at different temperature in 95% filled vial.

55 Figure 27 Comparison between the thermal stability of EGCG,

NC and ECG at 40oC in 95% filled vials 55

Figure 28 The structure of EGCG and ECG 59 Figure 29

pH stability profile of 0.5 mg/ml Catechins solutions in 0.05 M phosphate buffer and 0.2 M ionic strength at 40oC

59 Figure 30

pH stability profile of 0.5 mg/ml Catechins solutions in 0.05 M citrate buffer and 0.2 M ionic strength at 40oC

60 Figure 31

The stability of EGCG in 0.05M citric at pH 3.0 and 0.2M ionic strength in the presence of different types and amounts of co solvents after 15 days of storage at 40oC

61 Figure 32

The stability of NC in 0.05M citric at pH 3.0 and 0.2M ionic strength in the presence of different types and amounts of co solvents after 15 days of storage at 40oC

62

Figure 33

The stability of ECG in 0.05M citric at pH 3.0 and 0.2M ionic strength in the presence of different types and amounts of co solvents after 15 days of storage at 40oC

62 Figure 34 The plot of Ln (ao/(ao-x)) catechins versus time for

0.5 mg/ml tea extract aqueous solution at 25oC 64

Figure 35 The plot of Ln( ao/(ao-x)) catechins versus time for

0.5 mg/ml tea extract aqueous solution at 30oC 65

Figure 36 The plot of Ln( ao/(ao-x)) catechins versus time for 65

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List of equations

Page Equation 1 The reaction between catechins and molecular oxygen 57 Equation 2 The reaction between catechins and oxygen radical 57 Equation 3 The reaction between oxygen radical and hydrogen ion 57 Equation 4 The reaction between semiquinone radical intermediate and

oxygen 57

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Abreviation Name

LDL Low density lipoprotein

UV Ultra violet

EGCG (-)-epigallocatechin 3-gallate

ECG (-)-epigallocatechin

EC (-)-epicatechin

GC (-)-gallocatechin

C (+)-catechin

GTE Green tea extract

CuTAAB-SG Tetrabenzo-[b,f,j,n][1,5,9,13]-tetraaza-cyclohexadecine copper(II) complex immobilized on silica gel

TEAC Trolox equivalent antioxidant capacity

ABTS + 2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid

HPLC The high-performance liquid chromatography

RP Reverse phase

BHA Butylated hydroxyanisole

PDA Photo diode array detector

8-OHdG 8-Hydroxy-2`-deoxyguanosine

HPCE High performance capillary electrophoresis

GC Gas chromatography

HMDS Hexamethyldisilazane

TMCS Trimethylchlorosilane

PPO Polyphenol oxidase

SOD Superoxide dismutase

PP Polyphenols

HLB Hydrophilic-Lipophilic Balance

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XIII

STUDY OF THE STABILITY OF NATURAL EXTRACT

OF GREEN TEA IN AQUEOUS SOLUTIONS AND

THEIR KINETICS

By

Rajae Hani Saadeh

Supervisor

Dr. Abeer Al-Bawab

Co-Supervisor

Dr. Ahmad Abdoh

Abstract

A stability study of tea catechins extract aqueous solutions was carried out using a validated HPLC method. The HPLC was used to monitor the changes in assay of tea catechins with time. The effect of different factors on the tea catechins stability were investigated in this work including the effect of atmospheric air, temperature, pH value, buffer species and the effect of adding different material to the tea solutions.

Determination of the reaction order, rate constants, and activation energies for the oxidation reaction of the tea catechins was done also in this work. It has been found that the tea catechins were sensitive to atmospheric air and temperature. On the other hand it was found that the catechins were stable at low pH (2-3) but very unstable at high pH (6-8) with more stability in citrate buffers than in phosphate buffers. Enhancement of the tea catechins stability was achieved by adding ethanol, propylene glycol or sorbitol to the aqueous solutions of tea catechins.

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1. Preface

Tea (Camellia Sinensis) is one of the most widely consumed beverages in the world, next only to water and coffee, and carbonated soft drinks. It can be categorized into three types, depending on the level of fermentation, i.e., green (unfermented), oolong (partially fermented) and black (fermented) tea. Although this process is often assumed, incorrectly, to be fermentation which usually implies additives, the more correct term should be oxidation which means exposure to air while drying (Cheng T.O., 2005).

In general, green tea has been found to be superior to black tea in terms of antioxidant activity owing to the higher content of chatechins. The processes used in the manufacture of black tea are known to decrease levels of the monometric catechins to a much greater extent than the less severe conditions applied to other teas. The production and consumption of the partially fermented oolong tea are confined to China. In the Far East the drink is normally prepared from green tea by infusing it in hot (but not boiling) water. Generally the first infusion is discarded and it is the second and subsequent infusion that are consumed. Recently, in the Western countries, green teas (often contained in teabags) have gained its popularity. The effects of these wide product and preparation differences on the in-cup chemical composition of tea infusions are of interest. this is because the quality and health properties of the consumed drink are associated with the chemical components (in particular the polyphenols and caffeine) extracted from the leaf. The primary components under study in this respect are the flavonoid polyphenols, which have been demonstrated to have strong antioxidant effect

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