STUDY OF THE STABILITY OF NATURAL EXTRACT
OF GREEN TEA IN AQUEOUS SOLUTIONS AND
Rajae Hani Saadeh
Dr. Abeer Al-Bawab
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
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.
Dr. Abeer Al-Bawab, Advisor
Associat. Prof. of Physical chemistry ---
Dr. Ahmad Abdoh, Co-Advisor
Dr. of Physical Chemistry ---
Dr. Rebhi Al-Zaru, Member
To My Family,
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.
Table of Contents
Subject PageCommittee 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
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
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
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
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
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
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
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
Table 27 The rate constants for catechins oxidation reaction at different
Table 28 Activation energies for tea catechins oxidation reactions 68
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
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
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
Figure 10 Peak purity for EC peak in 0.5 mg/ml tea extract water
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
Figure 13 Peak purity for ECG peak in 0.5 mg/ml tea extract water
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
mg/ml EGCG in
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
mg/ml EGCG in
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
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.
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
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
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
LDL Low density lipoprotein
UV Ultra violet
EGCG (-)-epigallocatechin 3-gallate
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
HPCE High performance capillary electrophoresis
GC Gas chromatography
PPO Polyphenol oxidase
SOD Superoxide dismutase
HLB Hydrophilic-Lipophilic Balance
STUDY OF THE STABILITY OF NATURAL EXTRACT
OF GREEN TEA IN AQUEOUS SOLUTIONS AND
Rajae Hani Saadeh
Dr. Abeer Al-Bawab
Dr. Ahmad Abdoh
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.
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
Akhadov, Y.Y. (1981). Dielectric properties of binary solutions; A data handbook. Pergamon Press.
Aneja R., Hake PW., Burroughs TJ., Denenberg AG., Wong HR. and Zingarelli B. (2004). Epigallocatechin, a green tea polyphenol, attenuates myocardial ischemia reperfusion injury in rats. Mol Med, 10, 55-62.
Anderson RA. and Polansky MM.(2002). Tea enhances insulin activity. J Agric
Food Chem, 50, 7182-7186.
Arts I., Putte B. and Hollman P.(2000). Catechin contents of foods commonly consumed in the netherlands. 2. Tea, Wine, Fruit Juices, and Chocolate Milk. J. Agric.
Food Chem., 48, 1752-1757.
Attwod, D. and Florence, A.(1983). Surfactant systems; their chemistry, pharmacy and biology. London. New York Chapman and Hall.
Bonoli M., Pelillo M., Toschi T.G., and Lercker G. (2003). Analysis of green tea catechins:comparative study between HPLC and HPCE. Food Chemistry, 81, 631-638.
Boylan, J. (1976). The theory and practice of industrial pharmacy. (2nd edition), London: Kimpton Publisher.
Brown, A.G., Falsaw, C.P., Haslam, E., Hohnes, A.and Ollis,W.D. (1966). The constitution of theaflavins. Tetrahedron Letters, 11, 1193-1204.
Cheng TO. (2004) Effect of green tea on smokers. Heart. http://heart.bmjjournals. com/ cgi/eletters/90/12/1485.
Cheng T.O. (2005). All teas are not created equal; The Chinese green tea and cardiovascular health. International Journal of Cardiology, (Journal in press).
Henning S.M., Fajardo-Lira C., Lee H.W., Youssefian A.A, Vay L. W. Go, and Heber D. (2003). Catechin Content of 18 Teas and a Green Tea Extract Supplement Correlates With the Antioxidant Capacity. Nutrition and Cancer, 45(2), 226 235
Hodgson JM. and Puddey IB. (2004). Acute effects of tea on fasting and post meal blood pressure. Asia Pac J Clin Nutr, 71 (13) [Suppl].
Gramza A., and Korczak J. (2005). Tea constituents(Camellia sinensis L.) as antioxidants inlipid systems. Trends in Food Science & Technology 16, 351 358.
Jimenez Atienzar M., J. Cabanes, Gand aherrero F. ,and Garc acarmona F. (2004) Kinetic analysis of catechin oxidation by polyphenols oxidase at neutral pH.
Biochemical and Biophysical Research Communications, 319, 902-910.
Kim DW., Park YS. and Kim YG. (2004). Local delivery of green tea catechins inhibits neointimal formation in the rat carotid artery injury model. Heart Vessels, 19, 242-247.
Mochizuki M., Yamazaki S., Kano K. and Ikeda T.(2002). Kinetic analysis and mechanistic aspects of autoxidation of catechins. Biochimica et Biophysica Acta, 1569, 35-44.
Nederkassel A.M., Daszykowski M., Massart D.L., Heyden Y. (2005). Prediction of total green tea antioxidant capacity from chromatograms by multivariate modeling.
Journal of Chromatography A, (in press article)
Nishitani E. and Sagesaka M.Y. (2004). Simultaneous determination of catechins, caffeine and other phenoliccompounds in tea using new HPLC method. Journal of
Food Composition and Analysis, 17, 675-685.
Owuor, P.O., and Obanda,M.(1998).The changes in black tea quality due to variations of plucking standards and fermentation time. Food Chemistry, 61 (4), 435-441.
Pierce A. R., H. N. Graham, Glassner S., Madlin H. and Gonzalez J.G.(1969). Analysis of Tea Flavanols by Gas Chromatography of Their Trimethylsilyl Derivatives. Analytical chemistry, 41(2), 298-302.
Roginsky V. and Alegria A. E. (2005). Oxidation of Tea Extracts and Tea Catechins by Molecular Oxygen. J. Agric. Food Chem., 53, 4529-4535
Sueoka N., Suganuma M., Sueoka E., Okabe S., Matsuyama S. and Imai K. (2001). A new function of green tea: prevention of lifestyle-relateddiseases. Ann N Y Acad
Sci., 928, 274-280.
Toschi T.G., Bordoni A., Hrelia S., Bendini A., Lercker G. and Pier L. (2000). The protective role of different green tea extracts after oxidative damage is related to their catechin composition. J. Agric. Food Chem., 48, 3973-3978
Wang R. and Zhou W. (2004). Stability of tea catechins in the bread making process. J. Agric. Food Chem., 52, 8224-8229.
Waltner-Law ME., Wang XL., Law BK., Hall RK, Nawano M., and Granner DK. (2002). Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production. J Biol Chem, 277, 34933-34940.
Yang Y.C., Lu F.H., Wu J.S., Wu C.H., Chang C.J. (2004). The protective effect of habitual tea consumption on hypertension. Arch Intern Med.,164, 1534-1540.
Yao L.H., and Jiang Y.M. (2005). Phenolic compounds in tea from Australian supermarkets. Food Chemistry. (Journal in press).
Zaporozhets O. A., Krushynska O.A., Lipkovska N. A., and Barvinchenko. V. (2004) A new test method for the evaluation of total antioxidant activity of herbal products. J. Agric. Food Chem., 52, 21-25.
Zhu Q.Y., Hackman R. M., Ensunsa J.L.,R.R. Holt and C. L. Keen. (2002). Antioxidative activities of oolong Tea. J. Agric. Food Chem, 50, 6929-6934.
Zhu Q. Y., Zhang A., Tsang D., Huang Y. and Chen Z. (1997). Stability of green tea catechins. J. Agric. Food Chem, 45, 4624-4628.
Zuo Y. Chen H. and Deng Y. (2002). Simultaneous determination of catechins, caffeine and gallic acids in green, Oolong, black and pu-erh teas using HPLC with a