Comparative Studies of the Chemical Constituents of Green Tea A Thesis Submitted in Partial Fulfillment of the Requirements for the Master Degree of Science in Chemistry (Analytical Chemistry) By Tharwat Nabil Abdul-Jabbar King Abdul Aziz University - Jeddah ١٤٢٩ A.H ٢٠٠٨ A.D
١. Introduction ١.١. Historical overview: Tea is one of the most widely consumed beverages in the world (Cheng, ٢٠٠٤ ; Vinson, ٢٠٠٠). All tea types come from the leaves of the Camellia sinensis plant (Fig.١.١), which is a member of theaceae family. The ancient use of tea as a beverage has been practised by mankind between ٥٠٠-٥٠٠٠ years ago (Gutman and Ryu, ١٩٩٦). Camellia sinensis tree is an evergreen shrub or tree (Alschuler, ١٩٩٨ ; Graham, ١٩٩٢). It can grow up to ١٦ m tall. Tea leaves alternate, exstipulate and lanceolate to obovate up to ٣٠ cm long, ٢ ٥ cm broad and pubescent. The tree sometimes becoming glabrous, serrate, acuminate, flowers ١ ٣ in axillary or subterminal cymes, deflexed, ٢ ٥ cm broad, white or pinkish, fragrant, actinomorphic, sepals and petals ٥ ٧, pedicels ٥ ١٥ mm long; approximately ٥٠٠ seeds/ kg (Ferrara et.al., ٢٠٠١; Pister et.al., ٢٠٠١). Three major types of tea are well known and are namely: black tea, green tea and oolong tea come from the same Camellia sinensis plant (Fig.١.١) as reported by Chow and Kramers, ١٩٩٠ and Duke and Atchley, ١٩٨٤. The main difference between the three tea types depends on the fermentation step during the processing. Traditional Chinese medicine has recommended the use of
green tea for headaches, pains, enhancement of immune defenses, detoxification and as an energizer (Duke and Waine, ١٩٨١ ; Leung, ١٩٨٠). The worldwide production of tea in ٢٠٠١ was over ٣ million tons as reported by Shehata et.al., ٢٠٠٤. The greatest production (about half of the total world production) comes from China and India. Indonesia, Sri Lanka and Kenya together account for another quarter of the total production. The other production countries of tea at lower scale are: Japan, Bangladesh, Argentina, Malawi and Tanzania (Chang and Yabuki, ٢٠٠٣). The quality of tea differs according to the climate condition and soil (Khaleel, ٢٠٠٤). The methods of harvesting and processing of the tea leaves determines the tea type and the content of the flavonoids (polyphenolic compounds) responsible for its chracteristics. The production process plays an important role on differentiating between the three types of tea (Alschuler, ١٩٩٨). ١.٢. Tea types: ١.٢.١. Black tea: Over seventy eight percent of the black tea are consumed in Europe, India, North America and North Africa (Chow and Kramer, ١٩٩٠; Chung et.al., ٢٠٠٣; Mackay and Blumberg, ٢٠٠٠). According to the Beilenson, ١٩٩٥, the processing of black tea consists of four steps namely: i-withering, ii-rolling,
المستخلص إن الھدف الري یسي لدراسة أنواع الشاي الا خضر المتوفرة داخل أسواق المملكة العربیة السعودیة ھو تقدیر تركیزات المكونات الكیمیاي یة العضویة و غیر العضویة في الشاي الا خضر باستخدام أجھزة التحلیل الكروماتوجرافي. تم تحلیل ١٢ عینة من الشاي الا خضر بواسطة جھاز IC و أوضحت الدراسة أن تركیزات الا نیونات المقاسة تتبع الترتیب F - ٣-٢- > PO ٤ > SO ٤ > Cl - - التالي: >NO ٣ تم تحلیل ٢٩ عینة من الشاي الا خضر بواسطة جھازي, AASو ICP-OES أوضحت النتاي ج تفاوتا كبیرا في تركیز ھذه العناصر كما أوضحت الدراسة أن متوسط تركیزات العناصر في أنواع الشاي المختلفة تتبع الترتیب التالي: K >P > Mg > Mn > Ca > Al > Si > Zn > Ni > Cu > Ba > Fe > Sr > Na > Mo > V وتم أیضا تحدید التصنیف الكیمیاي ي speciation) (Chemical لبعض العناصر قید الدراسة المتواجدة في عینة مختارة من الشاي الا خضر وذلك بتحدید تركیزات العناصر الموجودة بصورة حرة ions) (Labile والموجودة علي شكل معقدات ) species (Complexes مع بعض المكونات العضویة الا كثر تواجدا في الشاي الا خضر مثل عدید الفینولات. تضمنت الدراسة أیضا تا ثیر زمن النقع على مدى تحرر بعض الا نیونات والعناصر الفلزیة وغیر الفلزیة في عینات مختارة وكذلك على بعض المكونات العضویة قید الدراسة. أمكن تحدید تركیزات بعض الا یونات المختلفة مثل أیون الفلورید وبعض الا یونات الفلزیة التي یحصل علیھا الشخص البالغ من تناول كوب شاي ومقارنتھا بالكمیة التى یحتاجھا. تم تحلیل ٢٩ عینة من الشاي الا خضر بواسطة جھاز HPLC وتم التعرف على تركیز الكافیین و العدید من الكاتشینات و أوضحت النتاي ج أن متوسط توزیع ھذه المركبات یتبع الترتیب التالي: EGCG > EGC > ECG > EC > C > Caffeine لقد أوضحت نتاي ج الدراسة عدم وجود ارتباط بین أسعار الشاى الا خضر المتوفرة فى السوق المحلى وتركیزات المكونات البیولوجیة العضویة وغیر العضویة. فمضادات الا كسدة (الكاتشینات) ذات الفاي دة الكبیرة فى الحد من نمو الخلایا السراطانیة وجد أنھا موجودة في كل أنواع الشاي حتي الرخیصة منھا. وكذلك الحال بالنسبة لا یون الفلورید الذي یعتبر أساسیا في منع تسوس الا سنان وكذلك البوتاسیوم. أمكن حساب بعض الدوال المعبرة عن كفاءة العمود المستخدم في تقنیة الكروماتوجرافیا ذو الكفاءة العالیة (HPLC) وذلك بحساب عدد الطبقات (N ( أمكن وسمك الطبقات (HETP) ومدى درجة التماثل والفصل للعمود.(As, R s ) أیضا حساب بعض االدوال LOD) (LOQ, المعبرة عن مدى دقة وكفاءة الطریقة المستخدمة في تقدیر المركبات عدیدة الفینولات قید الدراسة.
Abstract The overall work presented in this thesis can be summarized as follow: - The anions fluoride, chloride, nitrate, phosphate and sulfate in the twelve selected green tea samples are present at acceptable concentration. The average concentration of the investigated anions at five min contact time in boiled water followed the sequence: F - ٣-٢- > PO ٤ > SO ٤ > Cl - - > NO ٣ - The mean distribution of the tested free ions in the twenty nine green tea leaves followed the order: K >P > Mg > Mn > Ca > Al > Si > Zn > Ni > Cu > Ba > Fe > Sr > Na > Mo > V - The total content and the chemical speciation (labile free ion and complex species of the element) of some selected metal ions (Al, Ca, Mg, Mn, Fe) and P in the tea sample was also performed. The results revealed that, the amount of complexed metal ions in particular Al is much higher than that present labile on boiled water. - The average concentration of caffeine, catechin, epicatechin, epigallocatechin, epicatechin ٣- gallate and epigallocatechin ٣- gallate in the infusion tea leaves in boiled water for five min employing HPLC at ٢٠٥ nm were found in the range ٠.٠٨٦-٢.٢٣, ٠.١١٣-٢.٩٤, ٠.٥٨-١٠.٢٢, ٠.١٩-٢٤.٩١, ٠.٢٢-١٣.٨٨ and ١.٠١-٤٣.٣٤ mg g -١, respectively. The content of caffeine and catechins in the tested tea samples followed the sequence: EGCG > EGC > ECG > EC > C > Caffeine - The performance of the employed HPLC method was determined from the calculated values of the number (N) and the height equivalent to the theoretical plates (HETP) and the separation factor (R s ). The average values of N, HETP from the chromatograms at ١٠ µg ml -١ for the four catechins: EC, EGC, EGCG and ECG were found in the range ٢.٦ ١٠⁴ ± ١.٢ ١٠ ٣ and١.٧ ١٠-٣ ± ٤.٧ ١٠-٤ cm, respectively. The value of R s calculated from the chromatogram for EGCG and EC was found equal ١.٧ ± ٥.٥٣ ١٠-٢. The asymmetry (As) and capacity (k`) factors for the four catechins were found equal ١.٠ and ٢.٩٧, respectively. The lower limit of detection (LOD) of the employed HPLC procedure for some catechins were found in the range ٠.٠٠٤ ٠.٠٥ µg ml-١ and the values of lower limit of quantification (LOQ) were found in the range ٠.٠١ ٠.١٧ g ml -١. The method was applied successfully for the determination of (+)-catechin even at trace and ultra trace concentration. The method was found rapid, ideal for routine analysis, reproducible, precise and accurate.
Table of Contents Subject Page Abstract Arabic Abstract Acknowledgment Preliminary Postgraduate Courses List of Abbreviations List of Figures List of Tables i ii iii iv v vii xii Chapter ١:General Introduction ١. Introduction ١.١. Historical overview ١.٢. Tea types ١.٢.١. Black tea ١.٢.٢. Oolong tea ١.٢.٣. Green tea ١.٣. Chemical composition ١.٣.١. Flavonoids ١.٣.١.١. Flavan-٣-ols ١.٣.١.٢. Flavonols ١ ١ ٢ ٢ ٥ ٧ ٨ ٨ ١٠ ١٤ ١٥
١.٣.١.٣. Flavones ١.٣.٢. Organic acids ١.٣.٣. Amino acids ١.٣.٤. Alkaloids ١.٣.٥. Vitamins ١.٣.٦. Minerals ١.٣.٧. Other compounds ١.٤. Health benefits of green tea ١.٤.١. Antioxidant and anti carcinogenic properties ١٧ ١٨ ١٨ ٢٠ ٢١ ٢٢ ٢٢ ٢٣ ٢٤ ١.٤.٢. Cholesterol, hypertension, diabetes, weight control and Alzheimer's disease ١.٤.٣. Miscellaneous uses of green tea ١.٥. Dosage and toxicity ١.٦. Mechanism of action and pharmacology of green tea ١.٧. Techniques used on the analysis of organic and inorganic species in tea ٢٥ ٢٧ ٢٩ ٣٢ ٣٦ ١.٨. Aim of the current study Chapter ٢: Experimental ٢. Experimental ٢.١. Reagents and materials ٢.٢. Apparatus ٢.٣. Recommended sample preparation ٢.٣.١. Preparation of the standard mixtures of the catechins and caffeine ٢.٣.٢. Digestion of tea samples ٣٨ ٣٨ ٤٢ ٤٥ ٤٥ ٤٦
٢.٣.٣. Analysis of some selected inorganic anions by ion chromatography ٢.٣.٤. Analysis of the essential and non essential elements and toxic metal ٤٩ ٥٠ ions by ICP-OES and AAS ٢.٣.٥. Analysis of caffeine and catechins by HPLC ٥١ Chapter ٣: Results and discussion ٣. Results and discussion ٣.١. Distribution behavior of some selected anions in the investigated green ٥٣ ٥٣ tea samples ٣.٢. Distribution behavior of some selected elements in the investigated ٦١ green tea samples ٣.٢.١. The influence of digestion time and microwave digestion on the ٦٧ release of labile essential and non essential elements in green tea ٣.٢.٢. Dietary elements intake from green tea consumption ٣.٣. Determination of catechins and caffeine in tea samples ٣.٤. Analytical performance ٤. Conclusion and recommendation References ٧٢ ٧٥ ١٠٨ ١١١ ١١٤