Kinetics Of Epimerization And Degradation Of Green Tea Biology Essay

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Around 2327 B.C. tea leaves utilization and consumption may have started for medicinal purposes. At the present time, tea has become the most widely consumed beverage in the world after water. Tea attracts the society not only from its taste and aroma but they also believe that consuming tea may contribute to many positive health benefits such as prevention of cancer, treating cardiovascular disease, and dental health. A lot of studies have also found that tea catechins have beneficial effects on overall good health such as anti-inflammatory, anti-diabetic, anti-bacterial, anti-hypertensive, anti-arteriosclerotic and anti-ageing functionality (Wang et al., 2008, Chen et al., 2000). Also, in these past decades a wide varieties of tea leaves are processed and utilized not only serving as a drinking beverage but also in food as a colourant and preservative, kitchen deodorizer and a lot of cosmetic products. In addition, a lot of varieties of bottled or canned tea drinks are now available in the market (Hara, 2001).

Tea is made from the leaves of Camellia sinensis that grows naturally in many tropical to temperate countries around the world. Following harvesting, the leaves are processed to undergo fermentation to some extents to produce different tea products: black tea, green tea or oolong tea. In particular, green tea which accounts for 22% of the world's tea consumption (Blumberg, 2002), made by steaming or drying fresh tea leaves at elevated temperatures to prevent polyphenol oxidation. Green tea is also believed to be the most beneficial among any other teas simply because its production only involves little processing and fermentation and therefore, green tea brews are richer in catechins (Boon, 2008). The amount of total catechins in a cup of tea is highly variable depending on the type of tea, the ratio of tea leaves/water, as well as the infusion time. Generally, an average serving of 250 ml of a cup of brewed green tea contains 620-880 mg of water-extractable materials of which one-thirds are catechins and 3-6% are caffeine (Yang et al., 2009)

All kinds of tea including green tea all contain polyphenols, flavandiols, flavonoids and phenolic acids. Green tea polyphenols or commonly known as green tea catechins are found to be the most abundant constituents. Green tea catechins are present mainly as catechin (C), catechin gallate (CG), gallocatechin (GC), gallocatechin gallate (GCG), epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), and epigallocatechin galate (EGCG). In addition, the most significant catechin that presents in green tea is EGCG (Hara, 2001, Yamamoto, 1997)

The many benefits of green tea catechins make them to be the most important constituents in green tea. Although catechins alone already make up 20% of the dry weight of green tea, which are mainly EGCG (Hara, 2001), they are very prone to epimerization and degradation reactions during production or storage of tea beverages. These reactions are undesirable in many food products, especially in bottled or canned tea drinks where only little amount of catechins was found (David Labbé, 2008). As a result, several studies have been done to investigate the kinetics of the stability of tea catechins in aqueous solutions (Wang and Helliwell, 2000, Wang et al., 2008b, Wang et al., 2006). In order to improve the quality of tea drinks in the food industry, it is important to fully understand the properties of green tea catechins not only at a higher temperature range, where most thermal processes occur, but also at a lower temperature range which is also commonly used in some food processes and storage. Some studies on the kinetics of the stability of green tea catechins in green tea solutions have been done both at a higher temperature (100-165°C) and a lower temperature range (25-100°C). Both studies have succeeded in developing a mathematical model regarding the stability of the catechins EGCG and GCG (Wang et al., 2006, Wang et al., 2008b). However, further studies still need to be done on the kinetics of the stability of tea catechins in a green tea infusion, in particular to study the effects of different values of pH at lower temperature range.

Green Tea Polyphenols

2.1 Chemistry of Green Tea

The largest components of green tea are carbohydrates and proteins but apparently these components are almost insoluble in a green tea infusion. Only relatively small molecular weight components can be soluble in hot water and they are mainly polyphenols (Yamamoto, 1997). Apart from polyphenols, a few other minor components are also present such as caffeine (0.15-0.2%), theophylline (0.02-0.04%) and other methylxanthines, lignin (6.5%), organic acids (1.5%), chlorophyll (0.5%), free amino acids (1-5.5%), and other minor flavour compounds (Hamilton-Miller et al., 2005)

The slight astringent and bitter taste of a green tea infusion is attributed to the catechins (Yamamoto, 1997). Almost all of the characteristics of manufactured tea, including its taste, colour, aroma are associated directly or indirectly with the modifications of catechins (Wang et al., 2000). Catechins or polyphenols belong to the group known as flavonoids which have a C6-C3-C6 carbon structure with two aromatic rings. The four major types of catechins in green tea are EGCG, EGC, ECG and EC. These catechins are present not only in tea leaves but also in many parts of a plant (Hara, 2001). In the case of green tea polyphenols, it is a class of flavanols which are C15 compounds, and their derivatives are composed of two phenolic nuclei (A ring and B ring) connected by three carbon units (C-2, C-3, and C-4) (Yamamoto, 1997). As can be seen from Figure 2.1, the structure of tea catechins is characterized by dihydroxyl or trihydroxyl substitutions on the B ring and the m-5,7-dihydroxyl substitutions on the A ring. Catechins and its derivatives also have nucleophilic centers at C-6 and C-8 which are reactive with electrophilic specimens. They are chemically reactive and able to have the properties of metal chelator, oxidative radicals scavenger, nitrosation inhibitor and etc (Susanne Valcic, 2000, Yang et al., 2009, Yamamoto, 1997)

Figure 2.1 Structure of basic polyphenols, EGCG, Epicatechin, EGC and ECG.

Adapted from (Yang et al., 2009)

Catechins synthesized inside the buds and tea leaves undergo malonic acid- and shikimic acid-metabolic pathways. Gallic acid is derived from an intermediary product produced in the shikimic acid-metabolic pathways (Yamamoto, 1997).

2.2 Biological Activities of Green Tea Polyphenols

2.2.1 Antioxidative Activity of Tea Polyphenols

It is believed that the presence of free radicals in our body can cause oxidation reactions that may harm human's DNA or cell membranes which will subsequently result in many diseases such as cancer, heart disease, and autoimmune diseases (Wang et al., 2000). Antioxidants are substances that significantly decrease the adverse effects of both reactive oxygen and nitrogen species. A lot of research have been done and proved that the flavonoids compounds in tea are even more effective as an antioxidant than Vitamin C and E (Wang et al., 2000, Hara, 2001) . Furthermore, both in vitro and in vivo experiments have given positive results, broadening the possibilities for practical applications of green tea catechins as an antioxidant (Hara, 2001, Yamamoto, 1997).

Furthermore, the antioxidative activities of green tea can also be applied in food industries, for example in preventing oxidation of fats and oils and discoloration of reddish food. In an investigation of antioxidative activity of tea extracts, green tea proved to be more effective antioxidants to canola oil compared to butylated hydroxytolune (BHT). In addition, in the case of oolong tea (semi fermented product) only a moderate activity of antioxidant was found and in black tea (fully fermented product) very little antioxidative activity was found (Yamamoto, 1997). These results are reasonable because that same amount of tea catechins have been destroyed during fermentation of oolong and black tea or in other words, oxidation reactions already proceed before heat treatment and drying, whereas green tea is produced by heat-treatment after harvest, therefore oxidation reaction can be prevented (Yamamoto, 1997, Boon, 2008).

In another study where antioxidative activity of individual catechins are examined, it was found that EGCG, which is the major component of tea polyphenols, is the strongest antioxidant (Yamamoto, 1997). The polyphenolic structure of EGCG allows electron delocalization and quenching of free radicals (Sang et al., 2006). This is supported in another study, which showed that catechins that have a galloyl moiety in the 3-position have the highest antioxidant activity and also the most effective inhibitors of lipid peroxidation (Valcic et al., 2000, Yang et al., 2008). As can be seen from Figure 2.1, EGCG and ECG have this kind of structure.

2.2.2 Antimicrobial Activity of Tea Polyphenols

A lot of studies have shown that polyphenols in tea can kill a wide range of pathogenic bacteria or at least reduce their concentration; such bacteria namely are Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Vibrio fluvialis and a few others (Hara, 2001). According to Hara (2001), green tea catechins EGC, EGCG, ECG inhibit the growth of methicilin-resistant Staphylococcus aureus in vitro by an equivalent of 1:10 dilution of tea leaves to water. This particular pathogenic bacteria, being resistant to the most common antibiotic used, is essential to be eliminated because it can cause some serious problems in hospitals where the patients have a lower immune system (Hara, 2001).

Another common example of the antimicrobial activity of tea polyphenols can be observed in the inhibition of the growth of Streptococcus mutans and Streptococcus sobrinus that are the major causes of teeth decaying (Yamamoto, 1997, Wang et al., 2000). It was proposed that the three major causes of dental carries are host (tooth), bacteria and substrate (sucrose) for the bacterial metabolism (Yamamoto, 1997).It was believed that under normal conditions tea would reduce acid on the tooth enamel where starch is trapped in the teeth, and this is how it exerts its anti-carries effect (Wang et al., 2000).

2.2.3. Cancer Prevention of Tea Polyphenols

It takes a long process for a cancer cell to develop (carcinogenesis), and it must goes through the stages of initiation, promotion and progression. The concept of cancer prevention is by using a synthetic or natural compound to slow, block, or reverses the development of cancer cell. It has been widely accepted that tea polyphenols, in particular EGCG catechins, has a cancer preventive effect. The evidence showed that the inhibitory effects of EGCG on carcinogensis is by the regulation of cell signalling pathways. EGCG activates cell death signals and induce apoptosis in the cancer cells, resulting the inhibition of cancer development (Khan et al., 2006). Also, there is a possibility that tea polyphenols will inactivate some viruses that cause cancer (Hara, 2001).

Numerous studies have been done on the anticarcinogenic effects of tea polyphenols but the actual relationship between tea consumption and occurrence of cancers is not conclusive because of the contradictory of the results of different studies (Yamamoto, 1997, Boon, 2008). This could be because of the diversity of cancer in different site of organs (Yamamoto, 1997), the difference of quality and quantity of tea consumption, and lifestyle factors such as cigarette smoking and alcohol consumption (Yang et al., 2009). Apart from that, the authors admited that green tea was the most potential natural cancer chemo preventive agent with low toxicity (Yamamoto, 1997, Wang et al., 2000).

2.2.4. Lipid Lowering Effects of Tea Polyphenols

Clustering of some diseases such as obesity, hypertension, hypercholesterolemia, diabetes and usually leading to cardiovascular diseases, can be termed as metabolic syndrome. The major determinant for this metabolic syndrome is the accumulation of fat or lipid (Alexander et al., 2003, Isomaa et al., 2001). A decrease of body weight, body mass index, body fat, waist circumference, systolic blood pressure, low density lipoprotein cholesterol and subcutaneous fat area, were found to be greater in a group of panel that consumed green tea extracts that were high in catechins compared to the control (Nagao et al., 2007, Boon, 2008, Blumberg, 2002). It is also important to note that waist circumference and systolic blood pressure are two important indicators of metabolic syndrome (Nagao et al., 2007). Therefore it is reasonable to suggest that the consumption of green tea may prevent obesity and decrease the risk of cardiovascular disease.

A lot of in vitro studies in animals as well as humans found that green tea catechins, especially EGCG, can inhibit the increase of mass in adipose tissue and also reduce carbohydrate and fat absorption by inhibition of various digestive enzymes (Kao et al., 2006, Moon et al., 2007, Juhel et al., 2000).

2.3 Bioavailability and Metabolism of Tea Polyphenols

The major health benefits of tea polyphenols have been studied in vitro extensively and some of them have been discussed in this report. Nevertheless, it is essential to discuss about how effective is the absorption of the catechins, especially EGCG, inside human's body. A proper metabolic pathway of green tea polyphenols in the human body is still under investigation (Yamamoto, 1997). Nonetheless, to give an idea, the metabolic pathway of EGCG in rats is shown in Figure 2.2. Kohri et al (2001) proposed that for tea catechins in rats after oral intake, firstly it would enter the small intestine and a part of them would undergo conjugation and be absorbed to the portal vein. At this stage the EGCG in the liver undergo both conjugation and methylations, and a part of them will be excreted into the bile and the rest of them (including intact EGCG) will enter the blood circulation. After 2 hours, the amount of EGCG in the blood will reach its maximum. However, it has also been reported that the bioavailability of EGCG in the blood circulation was very low, estimated to be 0.26% (Kohri et al., 2001). The author believed this was because a great proportion of EGCG that was absorbed undergone hepatic elimination reactions. On the other hand, the unabsorbed EGCG moves into the intestine and undergoes further degradation by intestinal bacteria to EGC then to 5-(3',5'-dihydroxyphenyl)-γ-valerolactone (M-1). Further, a large proportion of M-1 undergoes glucoronidation in the intestinal mucosa and/or liver, to form 5-(5'-hydroxyphenyl)-γ-valerolactone 3'-O-β-glucuronide (M-2), which enters the blood circulation, distributed to various tissues, and is finally excreted in the urine.

Figure 2.2. Possible metabolic route of EGCG orally administered to rats. GA, gallic acid; M-1, 5-(3',5'-dihydroxyphenyl)-γ-valerolactone; M-2, 5-(5'-hydroxyphenyl)-γ-valerolactone 3'-O-β-glucuronide.

Adapted from (Kohri et al., 2001)

According to Lipinski (2001), there are some compounds that will be poorly absorbed after administration, they are compounds that have; five or more hydrogen bond donors (OH and NH groups), ten or more hydrogen bond acceptors (notably N and O), molecular weight greater than 500 g/mol. In regards to the previous statement, tea catechins EGCG, which has a molecular weight of 458g/mol and have 8 hydroxyl groups (Yang et al., 2008), will be poorly absorbed. However, other catechins that have a smaller molecular weight and fewer hydrogen bonds such as (-)epicatechin and (+)-catechin (Yang et al., 2008), will be absorbed better than EGCG. This is proven in another investigation by Wang et al (1994), where 200 mg of catechins/kg body weight of rats was orally administered, radioactivity results showed about 30-40% of (+)-catechin was observed in the rat's small intestine after one hour. Thus, it can be suggested that more research is need to be done to understand the absorption of tea polyphenols especially EGCG in vivo. Nevertheless, the molecular weight of green tea catechins are still lower than theaflavins, which is the polyphenols in black tea (Yang et al., 2009), therefore it can be proposed that green tea catechins will have greater bioavailability compared to black tea's theaflavins.

3. Toxicology

Heavy tea drinkers generally consume more than 10 cups (150 cc) a day, which is also estimated to have consumed almost 1 g of tea catechins a day. As a result, the heavy tea drinkers have a decreased serum concentration of total cholesterol, LDL cholesterol, triglycerides, and an increased proportion of HDL cholesterol as compared with the moderate tea drinkers group (Hara, 2001). Besides, there are harmful effects due to over consumption according to Cabrera et al (2006). Notably there are three possible causes: tea's caffeine content, presence of aluminium, and the effects of tea polyphenols on the iron availability.

Different types of tea have different compositions, including its caffeine content. The order of caffeine content in different types of tea is black tea > oolong tea > green tea > fresh tea leaf (Lin et al., 2003). The caffeine content in green tea infusion is approximately to be 5% at the maximum (Yamamoto, 1997). There are harmful effects due to over consumption of caffeine, such as nervousness, hard to sleep, sleeping disorders, vomiting, headache, epigastric pain, and tachycardia (Cabrera et al., 2006).

It was found that some minerals especially aluminium is present in black and green tea. It will cause problems especially for patients with renal failure because Al can be accumulated in the body and cause many diseases, for example neurodegenerative disorders or Alzheimer's disease (Costa et al., 2002). In addition, it was suggested that Al dietary intake should be no more than 6mg/day (MINOIA, 1990). In many studies, it was found that the concentrations of Al in green tea is lower than black tea, and the authors proposed that the difference of Al content in tea is due to different soil conditions, harvesting periods and influence of water quality (Costa et al., 2002)

Apparently some studies have found that tea catechins may inhibit the bioavailability of non-heme iron by 79-94% whenever consumed altogether; however this depends on the iron status and intake of the individual (Tuntawiroon et al., 1991). It can be suggested to anaemia patients to avoid tea consumption (Costa et al., 2002).

It can be proposed that the main causes of harmful effects are from the other components of tea apart from tea polyphenols, such as caffeine and aluminium. Moreover, the content of those two compounds are lower in green tea than any other tea. Green tea catechins are also a natural product that is proven to have no acute toxicity when consumed orally and even if a huge amount of catechins are consumed over a lifetime will not harm human beings (Hara, 2001).

4. Separation of Individual Green Tea Catechins from Green Tea

Figure 4 presents a flow chart of preparation of green tea catehins from green tea. Hot water and organic solvents are commonly used in the extraction method. However, it is suspected by Masuzawa (2007) that hot water may destroy some of the catechins as well as the overall green tea quality. Extraction at low temperatures is more desirable even though the efficiency of the extraction will also be low.

Green tea

Extracted with hot water by spray drying

Water-soluble green tea powder

Dissolved in hot water

Washed with chloroform

Aqueous layer Chloroform layer

Extracted with ethyl acetate

Ethyl acetate layer Aqueous layer

Evaporated

Concentrated solution

Freeze dried

Green tea catechins

Figure 4.1 The preparation of green tea catechins

Adapted from (Hara, 2001)

In a lot of studies, reversed phase HPLC is frequently used for the analysis and quantification of tea catechins (Wang et al., 2008b, Wang et al., 2006, Chen et al., 2000, Wang and Helliwell, 2000). In the HPLC analysis, acidic mobile phases such as methanol or acetonitrile modifiers are commonly used because catechins are more stable at lower pH (Nanjo et al., 1996, Lun Su et al., 2003, Wang and Helliwell, 2000). Thus, it can be suggested to control the pH to be acidic in order to get a stable reaction and accurate results. Also, it was proved that the addition of small quantities of ascorbic acid in the mobile phase could be improving the peak shapes (Donovan et al., 1999).

Referring to Figure 4.2, it can be observed that a successful separation of green tea catechins was obtained by the HPLC procedures. Catechins standards were also used in the analysis, which enables calibration plots for the determination of catechin concentrations in tea infusion. Also, the effect of galloyl group on retention time can be found according to Table 4.1. It was found that the ratio of retention time of ECG/EC is nearly equal to the EGCG/EGC for both standards and tea extracts, suggesting that the presence of gallate residue in catechin molecule might have a consistent effect on retention time (He et al., 2010).

Figure 4.2 HPLC chromatograms of standards (A) and tea extracts (B). (mobile phase: M1=aqueous solution of 0.2% CH3CN, M2=CH3OH; Column: Hypersil ODS C18, 150x4.0 mm, 5 mm; Flow rate: 1.2mL=min; Detector: UV-DAD, 280 nm; Injection volume: 20 μl; Gradient: 0-12 min with 0-50% M2, 13-20 min with 50-100% M2)

Adapted from (He et al., 2010).

Table 4.1 Retention times (min) for Catechins and Caffeine (n=3)

Component

EGC (C)

EGCG

EC

GCG

ECG

Caffeine

Standards

9.186 (0.38a)

11.346 (0.19)

11.932 (0.19)

12.812 (0.66)

14.032 (0.45)

10.679 (0.71)

Extracts

9.219 (1.41)

11.352 (1.27)

11.949 (1.53)

12.851 (0.48)

14.109 (1.13)

10.709 (1.10)

aRSD values (%) are given in parantheses

Adapted from (He et al., 2010).

Many HPLC detection methods have been applied to detection of different types of catechins, such as UV or photodiode array (Wang et al., 2008a, Wang et al., 2006) fluorescence (Ho et al., 1995), electrochemical (Donovan et al., 1999), chemiluminescence, and mass spectrometry detection (Robb et al., 2002). Furthermore, HPLC separation with UV detector is the most common method used for the analysis of polyphenols in fruits and beverages (Donovan et al., 1999). UV-Vis absorbance spectra (210-400 nm) can be collected continuously for each chromatogram (Bronner and Beecher, 1998). In many studies, HPLC analysis with UV detection method is adequate for tea catechins quantification (Wang et al., 2008b, Wang et al., 2006). HPLC-UV detection ensures a correct identification of a single catechin and is also able to distinguish between the similarity of the UV spectra of the catechins (Pelillo et al., 2002). However, it was also proposed that UV detection was not suitable for the analysis of flavanols and their metabolites in complex samples such as human plasma due to its lack of selectivity, sensitivity and qualitative information available (Donovan et al., 1999). Therefore, different detections methods may be used depending on the suitability and complexity of the sample.

The content of catechins found in green tea may be varied between different species of green tea, climate, and cultural practices (Pelillo et al., 2002). More importantly, the content of green tea catechins also varied in different tea infusions (Wang et al., 2000). Based on Table 4.2 below, the content of catechins was found highest in the first infusion and decreased substantially in the later infusions. The amount of EGCG was also found to be the highest amongst the other catechins, this result is also consistent with other investigations (Wang et al., 2008a, Bronner and Beecher, 1998, Wang et al., 2008b, He et al., 2010)

Table 4.2 Comparison of catechins and flavonols in different tea infusionsa (mg L-1)

Infusions

1st

2nd

3rd

Total

Catechins

(+)-GC

30.0

18.7

3.80

52.5

(-)-EGC

228

131

37.2

396.2

(+)-C

10.4

5.90

0.90

17.2

(-)-EGCG

306

223

2.30

531.3

(-)-EC

52.8

28.1

6.30

87.2

(-)-GCG

10.0

9.60

Ndb

19.6

(-)-ECG

76.8

52.0

0.50

129.3

(-)-CG

1.40

1.40

Nd

2.8

Flavonols

Myricetin

6.40

4.20

2.70

13.3

Quercetin

23.9

15.9

10.0

49.8

Kaempferol

9.00

6.50

3.50

19.0

a According to the conventional brewing method, 1 g of gunpowder tea leaves was infused with 100 mL of boiling distilled water for 5 min, 70 mL liquid were filtered off and cooled to room temperature under running water; a 2nd infusion was added by adding a further 70 mL boiling distilled water to the tea leaves for 5 min, filtering off 70 mL which was cooled to room temperature under running water; a 3rd infusion was using the same procedure. b Nd= not detectable

Adapted from (Wang et al., 2000)

Stability of Green Tea Polyphenols.

5.1 Epimerization and Degradation Reactions

It has been reported the four major green tea catechins, namely EC, ECG, EGC, and EGCG, are very unstable when they are exposed to high temperatures, alkaline solutions, oxygen levels, metal ions and concentrations of other ingredients in the infusion (Wang et al., 2006, Sang et al., 2005, Wang and Helliwell, 2000). The four main green tea catechins mentioned previously are cis compounds and also be termed as epicatechins. In green tea infusions, the predominant change appears from epicatechins that convert to their epimers that are non-epicatechins, i.e. C, CG, GC and GCG respectively (Wang et al., 2008b). This reversible conversion of tea catechins to their corresponding isomers is called epimerization and is pictured on Figure 5.1 (Wang and Helliwell, 2000).

Figure 5.1 Epimerization between EGCG and GCG; k1: reate constant of the epimerization

from EGCG to GCG; k2: rate constant of the epimerization from GCG to EGCG

Adapted from (Wang et al., 2008a).

Green tea catechins are also prone to degradation and decomposition which lead to oxidation, dimerization and polymerization (Lun Su et al., 2003, Wang et al., 2008b). It was speculated by Chen et al. (2000) that the low concentrations of catechins found in many bottled or canned tea drinks was due to thermal degradation of catechins during the production, storage and transport of tea drinks and beverages. In addition, degradation and epimerization of catechins can occur simultaneously in a tea infusion both in higher (100-165°C) or lower temperature range (25-100°C) (Wang et al., 2008b, Lun Su et al., 2003). The conversion of green tea epicatechins to their corresponding epimers would also reduce the concentration of green tea catechins (Xu et al., 2003).Therefore, it is important to consider both reactions in studying the kinetic stability of green tea catechins.

In comparing the stability of epimer pairs in the solution, it was found that ECG-CG are more stable than EGCG-GCG in thermal processing since the value of A in EGCG-GCG was greater than the other pair. This statement is supported based on the fact that EGCG-GCG having three hydroxyl group (OH) at 3', 4', and 5' in the B-ring are more vulnerable to destructions than the others with only two adjacent - OH groups at 3' and 4' such as ECG and CG (Lun Su et al., 2003). In addition, the value of A remained unchanged in the same solution of a selected catechins pair in the temperature range of 25-165°C (Wang et al., 2008b).

5.2. Effect of Heating

Tea drinks must have undergone heat treatment either during the process of manufacturing, in canned or bottled tea drinks, or brewing tea leaves or bags before consumption. Therefore it is essential to know the effects of heating on the catechins content of tea. Despite the fact that epimerization and degradation reactions are temperature dependent, green tea catechins in general are more stable than theaflavins (Lun Su et al., 2003). According to Chen et al (2000), as the temperature increased the rate of epimerization also increased. In an investigation of the kinetic stability of green tea in higher temperature (100-165°C), it was found that the decrease in concentrations of epi-structured catechins was counteracted by the exponential increase of nonepi-structured catechins. Furthermore, the rate of increase in nonepi-structured catechins and the rate of decrease of epicatechins also gave a similar trend (Wang et al., 2006). This also proves that catechins and their epimers have similar kinetic characteristics.

It is also essential to study the stability of catechins under a lower temperature range where most tea leaves and beverages are stored and transported. Epimerization and degradation of catechins also occur in low temperature range, but takes a long period of time (Chen et al., 2000). The epimerization of catechins at 25-100°C showed a decrease of epi-structured catechins and an increase of the nonepi-structured catechins to a maximum then decreased with an increasing time (Wang et al., 2006). Further, it was reported that there was a reduction in catechins concentrations when tea infusions were kept at 40°C for 6 months (Wang and Helliwell, 2000).

5.3. Effect of pH

The degradation of catechins is pH dependent. Various studies have shown that in an acidic environment (pH ≤ 4) tea catechins are the most stable. As the pH increases the rate of degradation increases as well (Lun Su et al., 2003, Wang et al., 2006, Chen et al., 2000). In a kinetic study of green tea catechins done by Wang et al. (2006), it was found that as the rate constant increased with an increase of pH, the frequency factor was postulated to increase but Ea remained the same. It was found in another study that pH 7 tea catechins were very unstable and would degrade almost completely in a few minutes (Chen et al., 2000). At neutral or alkaline pH tea catechins are more susceptible to degradation, they are able to form their corresponding free radicals easier due to their increase of proton donating potential (Wang et al., 2006).

A study on the pH stability of individual catechin confirmed that among the four major green tea catechins, EGCG and EGC were most unstable. EGC was almost completely degraded when incubated for 3 hours, while EGCG was completely destroyed after 6 hours of incubation, both at pH 7.4 (Lun Su et al., 2003). Similarly, another study concluded that EGCG and EGC were extremely unstable in alkaline solutions while EC and ECG were relatively stable (Zhu et al., 1997). Again, this also confirmed that EGCG and EGC are more vulnerable to destructions (Lun Su et al., 2003).

The effect of pH on the epimerization of EGCG at 120°C for 30 min was studied by Seto et al (1997) which showed that at pH ≥ 5 conversion of EGCG proceeded readily. However, at pH 7 there was no significant concentration of EGCG and its epimer detected. The authors postulated that this outcome was because the catechins might have been degraded, polymerized or undergone oxidation reaction (Komatsu et al., 1992, Seto et al., 1997). It was also found that at lower pH values, the epimerization reaction would decrease until no epimerization reaction of EGCG will be detected anymore at pH 2. Additionally, another study suggested that the difference of pH was the main factor for the difference of the rate of epimerisation (Wang and Helliwell, 2000). To sum up, the lower the pH the greater the stability of the green tea catechins.

5.4. Effect of Ascorbic Acid, Metal Ions, and Oxygen Concentrations

According to Chen et al (1998), the addition of organic acids such as ascorbic acid, may increase the stability of green tea catechins although not directly lowering the pH value of the solution. In their experiment, ascorbic acid only dropped the pH value of the mixture from 7.42 to 7.38 but drastically improved the stability of the four major green tea catechins in the mixture, particularly EGC and EGCG. It is believed that ascorbic acid reacts as a reductant that can protect the catechins by recycling their free radical form.

It is generally agreed that ascorbic acid may improve the stability of catechins (Chen et al., 1998, Chen et al., 2000) but not necessarily improve the stability at longer period of time. Ascorbic acid may protect the solution of green tea catechins for the first month, but after that it may promote the degradation process because ascorbic acid is shown to have both antioxidant and prooxidant characteristics (Chen et al., 2000).

In a study where factors that influence the stability of catechins were looked at, it was shown that the stability of catechins was more affected by the effect of metal ions in the water than the pH of the water itself. However, in the same ionic environment, the catechins were still more stable in the lower pH (Wang and Helliwell, 2000). Also, the antioxidative activities of catechins were affected by the metal ions. Metal ions such as copper and manganese are able to activate oxygen in water to form complexes that may catalyse the oxidation of catechins, whereas iron inhibited the oxidation reaction (Kumamoto et al., 2001). Metal ions are able to disrupt the stability of tea catechins during reactions, therefore it can be suggested to replace normal water with deionized water for future experiments regarding the stability of catechins.

Zimeri and Tong (1999) have found that the rate constant of degradation kinetics of EGCG increased log-linearly with respect to pH and also dissolved oxygen concentration. This is in agreement with another study proposing the reduce in concentration of dissolved oxygen in the mixture may inhibit the oxidation of green tea catechins (Chen et al., 2000). This is reasonable because, oxygen is the most important acceptor of electron. Molecular oxygen O2 has two unpaired electrons occupying separate outer orbitals and each of these outer orbitals can accommodate an additional electron (Zhao et al., 1989). This transfer of electrons species may promote other reactions including oxidation.

6. Kinetic Study

In many studies, the degradation of tea catechins was found to follow first-order kinetics (Wang et al., 2008b, Wang et al., 2006, Komatsu et al., 1992), i.e. it was assumed that the concentrations decrease exponentially with time.

Or

where x is the concentration of catechins at the time t, is the initial concentration of the catechins, and is the rate constant of the degradation or epimerisation of the catechins (Wang et al., 2006).

However, this degradation can be disturbed by other competing reactions, mainly epimerization. This can be observed at 82°C where different modes of reaction kinetics can be observed (Wang and Helliwell, 2000). Among other reactions besides epimerization that also occurred is superoxide mediated autoxidation in the presence of oxygen (Wang et al., 2008b, Wang and Helliwell, 2000). As a result, there are limited studies on the kinetics especially mathematical modelling that consider all of the interfering reactions when measuring the stability of green tea catechins.

Another assumption was made in modelling the degradation of total epi- and nonepicatechins. The assumption was that the rate constants of degradation between epi- and nonepicatechins were similar. This was because of their similar structure, 2R, 3R (2,3-cis,epi form) and 2S,3R (2,3-trans, nonepi-form). Thus, the degradation rate constant of total epi- and non-epicatechins would follow the Arrhenius equation:

where A is the frequency factor, Ea is the activation energy, R is the ideal gas constant and T is the temperature in Kelvin. In the case of EGCG and GCG, the rate constant ky could be obtained from the gradient of the line of best fit of ln ([EGCG] + [GCG]) / ([EGCG]0 + [GCG]0) against time, t. It was later confirmed that ky was similar between pairs of catechins but shown to increase with an increase of temperature, regardless if the experiment was carried in higher or lower temperature conditions (Wang et al., 2006, Wang et al., 2008a).

Activation energy (Ea) refers to the change of potential energy of a chemical system that is required to convert reactants into products by a reaction. The activation energy was also found to be unchanged in a selected catechin solution regardless of the varying concentrations in the temperature range of 25-165°C. Table 7.1 shows the activation energy and frequency factor obtained from Purified EGCG Powder (PEP) solution at this temperature range (Wang et al., 2008b).

Table 6.1. Activation Energy (Ea) and Frequency Factor (A) of Tea Catechins in the PEP solutions

Adapted from (Wang et al., 2008b).

In another investigation of kinetic stability of catechins but at higher temperature, it was found that the A or frequency factor of the epimerization from epi-structured to nonepi-structured catechins was 104 greater than the epimerization from nonepi-structured to epi-structured catechins. Frequency factor refers to the number of collisions between the reactants to have the correct orientation leading to the final products. This indicates lower rate of collision in the epimerization of nonepi-structured to epi-structured catechins (Wang et al., 2006). This result is also in agreement with another study which proposed that GCG/CG in the 2,3-trans form had a smaller steric hindrance and a possibility to have a lower rate of collision than that of EGCG/ECG in the 2,3-cis form in a thermal reaction (Wang and Helliwell, 2000).

7. Conclusion

It is believed that the health benefits found in green tea are associated with the high content of catechins present in green tea. With the increasing market of tea drinks and products that use tea extracts, it is necessary to understand the stability of those catechins to be able to improve the quality of products and prevent its undesirable epimerization and degradation reactions. Thus, the main objective of this study is to analyse the kinetics of degradation and the epimerisation of green tea catechins in aqueous solutions under different conditions particularly at different pH and low temperature range.

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