Phytochemical Analysis Of Indian Tea Biology Essay

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The aim of the research is to perform photochemical analysis of Indian tea and to evaluate their antioxidant properties ad different time points after brewing

Tea is a drink made from the leaves and buds and is the most important non-alcoholic beverage in the world. Tea infusions, consumed by two thirds of the world's population, are obtained from the leaves of one kind of a plant named Camellia sinensis. Tea is for the most part simply considered a tasteful drink, but the scientific community has recently re-discovered the therapeutic potential of this beverage.

Critical consideration of the physiological and pharmacological effects of tea requires background information concerning production, leaf composition, availability of the various types of tea, and most importantly the chemical changes that take place during the manufacture of the commercial product. Consumption patterns of the different types of the beverages should also be noted. Tea is manufactured in three basic forms. Green tea is prepared by dehydration of Camellia sinensis leaves which does not lead to the oxidation of constituent polyphenols, therefore green tea, contains high concentrations of monomeric polyphenols from the catechins group. However black tea, obtained by tea leaves fermentation, is oxidized and contains mainly multimeric polyphenols, whose biological activity is not well documented. The third form of tea is oolong tea, which is a partially oxidized product.  Botanical evidence indicates that India and China were among the first countries to cultivate tea.

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Medicinally tea has been most used as a stimulant, or as an astringent lotion which may be used as a gargle or injection. An infusion of tea leaves was once used as a remedy for insect blights. An infusion of tea has been used for some digestive problems and to reduce sweating in fevers. In Tamil Nadu, tea leaves have been used homoeopathically for mania, paralysis, nervousness, neuralgia and sleeplessness.

COMPONENTS OF TEA:

The components of tea include Polyphenols, flavonoids, catechins and tannins. Tannins are astringent, bitter-tasting plant polyphenols that bind and precipitate proteins. The term tannin refers to the source of tannins used in tanning animal hides into leather; however, the term is applied to any large polyphenolic compound containing sufficient hydroxyls and other suitable groups (such as carboxyls) to form strong complexes with proteins and other macromolecules.

Catechins belong to the flavan-3-ol class of flavonoids. They are all tannins which are polyphenolic substances. The four main catechins found in tea are: Gallocatechin (GC), Epigallocatechin (EGC), Epicatechin (EC), and Epigallocatechin Gallate (ECGC). They have an antioxidant activity and have also been associated with tea's anticarcinogenic, anti-inflammatory, anti-atherogenic, thermogenic and antimicrobial properties.

The major tea purine alkaloids include caffeine, theobromine, theophylline and theacrine. Theanine is considered as the main amino acid found in tea.

Caffeine:

Caffeine is an odourless, slightly bitter-tasting alkaloid found in tea. When used in moderation, caffeine acts as a mild stimulant to the nervous system, blocking the neurotransmitter adenosine and resulting in a feeling of well-being and alertness. It increases the heart rate, blood pressure, and urination and stimulates secretion of stomach acids.

It is a stimulant that has been shown to speed reaction time, increase alertness, and improve concentration. The physical effects include stimulation of digestive juice, the kidneys, and the metabolism in ways that possibly help eliminate toxins. An increasing of mental alertness, shortening of reaction time, and improving efficiency of muscle action is brought about by caffeine's stimulation of the heart and respiratory system, bringing more oxygen to the brain. Caffeine from coffee or other beverages is absorbed by the stomach and small intestine within 45 minutes of ingestion and then distributed throughout all tissues of the body. Caffeine is metabolized in the liver by the cytochrome P450 oxidase enzyme system into three metabolic dimethylxanthines.

Caffeine increases energy metabolism throughout the brain but decreases at the same time cerebral blood flow, inducing a relative brain hypoperfusion. Caffeine activates noradrenaline neurons and seems to affect the local release of dopamine. Many of the alerting effects of caffeine may be related to the action of the methylxanthine on serotonin neurons. The methylxanthine induces dose-response increases in locomotor activity in animals. The effects of caffeine on learning, memory, performance and coordination are rather related to the methylxanthine action on arousal, vigilance and fatigue. Caffeine exerts obvious effects on anxiety and sleep which vary according to individual sensitivity to the methylxanthine. The central nervous system does not seem to develop a great tolerance to the effects of caffeine. Caffeine molecule is structurally similar to adenosine, and binds to adenosine receptors on the surface of cells without activating them. Interaction between adenosine A2a receptors and dopamine D2 receptors in the striatum might underlie some of the behavioral effects of methylxanthines. By antagonizing the negative modulatory effects of adenosine receptors on dopamine receptors, caffeine leads to inhibition and blockade of adenosine A2 receptors, causing potentiation of dopaminergic neurotransmission. The latter interaction might explain the adenosine receptor antagonists-induced increase in behaviors related to dopamine (eg, caffeine-induced rotational behavior).

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Caffeine induces hepatic CYP IA2 in rats, acts similar to polyphenol but the induction is weaker. It stimulates central nercous system, acts on the kidney to produce dieresis, relax smooth muscle and stimulate cardiac muscle.

Extraction of caffeine from coffee, to produce decaffeinated coffee and caffeine, is an important industrial process and can be performed using a number of different solvents. Benzene, chloroform, trichloroethylene and dichloromethane have all been used over the years but for reasons of safety, environmental impact, cost and flavor, they have been superseded.

Figure 1.Chemical structure of caffeine.

Theanine:

Theanine is a glutamic acid analog or amino acid derivative commonly found in tea (infusions of Camellia sinensis),In 1950 the Tea laboratory of Kyoto successfully separated theanine from Gyokuro leaf, which has the highest theanine content among all teas. Theanine is an analog to glutamine and glutamate, and can cross the blood-brain barrier.

Theanine has psychoactive properties which reduces the mental and physical stress, and improves cognition and mood in a synergistic manner with caffeine.

l-theanine is helpful in improving learning performance, heightening mental acuity, and promoting concentration, acting antagonistically against high doses of caffeine, calming nervous agitation, lowering blood pressure, diminishing symptoms of PMS. Theanine produces these effects by increasing the level of GABA (gamma-amino-butyric acid), an important inhibitory neurotransmitter in the brain. GABA serves a sedative function that brings balance to excitability that can lead to restlessness, insomnia, and other disruptive conditions. Theanine also appears to increase levels of dopamine, another brain chemical with mood-enhancing effects, which can reduce blood pressure.

Figure 2. Chemical structure of theanine.

Epicatechin:

Epicatechin is a flavonol belonging to the group of flavonoids. It is an odourless white powder found in many plants like tea and cocoa. Epicatechin is a strong antioxidant which improves heart health with its insulin mimic action. Epicatechin reduces lipid peroxidation and inhibits platelet aggregation. It causes blood vessel dilation by regulating nitric oxide, a molecule secreted by the blood vessel endothelium to signal surrounding muscle to relax.

The mechanism of action of epicatechin is different to that of insulin and remains speculative. In diabetic red blood cells epicatechin causes an increase in acetylcholinesterase activity. This activity is significantly lower in type 2 diabetic patients.

Figure 3. Chemical structure of epicatechin.

Epigallocatechin gallate is the most abundant catechin in tea. epicatechin can reduce the risk of four of the major health problems: stroke, heart failure, cancer and diabetes. Several studies have shown that tea polyphenols prevent tumour irritation. Tumour irritation is usually the result of permanent covalent DNA modification. This causes alterations of the genetic codes. This may lead to tumorigenesis. Polyphenols have been shown to prevent or reduce tumour promotion. Tea polyphenols are modulators of various cell growth regulators.

ORIGIN OF INDIAN TEA:

The cultivation and brewing of tea in India has a long history of applications in traditional systems of medicine and for consumption. The production of tea started in India in 1830 in the jungles of northeast Assam. Joseph banks, the British botanist in 1788 reported to British east india company that some parts of northeast india were ideal for tea growing.

In 1823 and 1831 Robert Bruce and Charles Bruce sent some tea plant and specimens to the botanical garden of Calcutta. Charles Bruce then collected seeds from China and planted them in the botanical gardens of Calcutta. In the south western tip of the country, experimental plantings had been made in 1835, while the first nurseries were being established in Assam, and by the mid 1850s tea was growing successfully alongside coffee. The climate of the Nilgiri Hills, or Blue Mountains, seemed to suit the plant, and the area under tea steadily expanded.

In 1853, India exported 183.4 tons of tea. By 1870, this has been increased to 6,700 tons and by 1885, 35,274 tons. Today, India is one of the world's largest producers of tea with 13,000 gardens and a workforce of more than 2 million people.

DIFFERENT TYPES OF INDIAN TEA:

There are two main kinds of tea produced in India

Assam tea

Darjeeling black tea

Assam tea:

Assam tea plants are indigenous tea plants of India. A full flavor, very satisfying tea. Assam teas are strong, well rounded and malty with rich aroma and flavor. Usually served with milk and sugar. Assam tea comes from the North Eastern section of the country. This heavily forested region is home to much wildlife. Tea from here is rich and full-bodied. It was in Assam that the first tea estate was established, in 1837. Assam is the world's largest tea-growing region, lying on either side of the Brahmaputra River, and bordering Bangladesh and Burma (Myanmar). This part of India experiences high precipitation. The leaves of the Assam tea bush are dark green and glossy and fairly wide compared to those of the Chinese tea plant. The bush produces delicate white blossoms.

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Discovery of the Assam tea bush is attributed to Robert Bruce, a Scottish adventurer, in 1823. Bruce reportedly found the plant growing wild in Assam while trading in the region. He noticed local tribesman brewing tea from the leaves of the bush and arranged with the tribal chiefs to provide him with samples of the leaves and seeds, which he planned to have scientifically examined. Robert Bruce died shortly thereafter, without having seen the plant properly classified.

Robert's brother Charles has arranged some leaves from the assam tea bush and sent to botanical gardens in Calcutta for examination. There, the plant was finally identified as variety of tea or camellia sinensis.

Darjeeling tea:

It was launched in 1983. It is a symbol that verifies that the packet / caddy etc contains 100 % pure Darjeeling, unblended with teas from any other growth. Darjeeling Tea is the world's most expensive and exotically flavoured tea. Darjeeling is located on the Northern part of West Bengal, India. There are only 86 Tea Estates, which produce Darjeeling Tea. All of these are in Darjeeling District and only teas coming from these estates can be called Darjeeling Tea. It cannot be produced anywhere else in the World. The Annual production of Darjeeling Tea is approximately 10 Million Kg.

Its flavour is so unique that it cannot be replicated anywhere else in the world. Grown in the romantic and mystical mountainous region of Darjeeling at an elevation of 750 - 2000 metre, the tea is imbued with an incomparable charisma and quality. Grown in century old Tea Gardens, these tea bushes are nurtured by intermittent rainfall, sunshine and moisture laden mellow mists. The soil is rich and the hilly terrain provides natural drainage for the generous rainfall the district receives.Even the tea pluckers, well aware of the status their produce enjoys throughout the world, pick only the finest two leaves and the bud to enhance the unique flavour which has been described as "Muscatel".

Adherence to this high quality profile, results in extremely low yields. The Darjeeling Planter has never succumbed to the temptation of increasing yields at the expense of quality and makes every effort to ensure the highest quality standards, inspite of the high costs involved. It would not be an overstatement to say that Darjeeling Tea is by far the finest and most sought after Tea in the world.

Black tea is process goes through the most stages. Once the leaves are picked, they are left to wither for several hours. After the leaves are rolled, oils from the leaves are brought to the surface. These aromatic oils aid in the oxidation process, which last for several hours. The last step consists of placing the leaves in an oven with temperatures reaching up to 200 degrees Fahrenheit. When the leaves are 80 percent dry, the leaves complete their drying over wood fires. The resulting product is brownish (sometimes black) in color and is sorted accordingly to size, the larger grade is considered "leaf grade," and smaller "broken grade" are usually used for tea bags.

OBJECTIVES:

The objectives are to quantify the contents of caffeine, theanine and epicatechin by HPLC, to determine their antioxidant activities by ABTS assay, to determine their total polyphenol content by Folin-Ciocalteu phenol reagent, and to correlate TPC and antioxidant activity.

MATERIALS AND METHODS:

Five types of teas were used in the analysis:

Chai spice tea

Lychee tea

Mango tea

Darjeeling tea

Three roses

Chai spice tea:

Chai from India is a spiced milk tea that has become increasingly popular throughout the world. It is generally made up of, rich black tea, heavy milk, a combination of various spices, a sweetener. The most common spices are cardamom, cinnamon, ginger, cloves, and pepper. Indian chai produces a warming, soothing effect, acts as a natural digestive aid and gives one a wonderful sense of well being. It's difficult to resist a second cup.

Lychee tea:

Lychee Black Tea is made from Black Tea which has been scented with lychee peels. Lychee or lichee is a sweet tropical fruit that is popular in Asia. When preparing and drinking this tea, one is instantly amazed with its strong pleasantly sweet fruity aroma. When brewed the tea has a reddish brown hue and a light sweet honey-like taste. Lychee Black Tea makes an excellent ice tea and can be served with sugar and/or cream.

Mango tea:

Mango tea contains papin, a known digestive aid. It is also useful in breaking down protein. Mango tea is believed to cleanse the intestines and boost metabolism and thus help with weight control and in reducing appetite. Mango tea contains many of mango's powerful antioxidant benefits. It claims to guard against cancer, regulate blood sugar levels and to be advantageous for those with cardiovascular diseases. Mango tea is said to be helpful for those who are under stress, suffer from muscle cramps and who have a high metabolic acidosis count seen in alcoholics, diabetics, and the malnourished.

Darjeeling tea:

Darjeeling tea is normally made from the small-leaved Chinese variety of Camellia sinensis var. sinensis, rather than the large-leaved Assam plant (C. sinensis var. assamica). Traditionally Darjeeling tea is made as black tea; however, Darjeeling oolong and green teas are becoming more commonly produced and is easier to find.

Many Darjeeling teas also appear to be a blend of teas oxidized to levels of green, oolong, and black.

1st Flush is harvested in mid-March following spring rains, and has a gentle, very light colour, aroma, and mild astringency. In Between is harvested between the two "flush" periods. 2nd Flush is harvested in June and produces an amber, full bodied, muscatel-flavoured cup. Monsoon or Rains tea is harvested in the monsoon (or rainy season) between 2nd Flush and Autumnal, is less withered, consequently more oxidized, and usually sold at lower prices. It is rarely exported, and often used in Masala chai. Autumnal Flush is harvested in the autumn after the rainy season, and has somewhat less delicate flavour and less spicy tones, but fuller body and darker colour.

Three roses:

Brook Bond Three Roses Tea is one of the best product from Hindustan Lever Private Limited, Mumbai. The three roses meant for perfect colour, perfect strength, and perfect aroma. That's why they are using the logo of three red roses in their pocket. It is one of the high graded tea product in India. It is really rich in taste. This is one of the nice tea brands in India. This product is also available with some other flavours too.

Materials:

Chai spice tea, lychee tea, mango tea, Darjeeling tea, and three roses tea powders. The standards of caffeine, theanine and epicatechin.

Gallic acid (3,4,5-trihydrobenzoic acid), Trolox standard (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Folin-Ciocalteu's phenol reagent, ABTS-2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and potassium persulfate, sodium carbonate.

Preparation of tea samples:

In each tea two different weights were used for all the analysis throughout the project and their weights are presented in table no 1. 0.5g of all tea samples were accurately weighed into beakers.100ml of water was added to each of the tea leaves at 70°C controlled by thermometer. Wait for five minutes and stir the tea leaves. After stirring collect the samples from each of the tea leaves. This sample was collected immediately and was used for analysis. In the same way sample was collected after 60 minutes and then after 24 hours respectively and were used for analysis.

Table 1. Weight of tea samples

Teas

Weights

Chai spice 1

0.5092g

Chai spice 2

0.5136g

Lychee 1

0.5091g

Lychee 2

0.5098g

Mango tea 1

0.5088g

Mango tea 2

0.5060g

Darjeeling 1

0.5063g

Darjeeling 2

0.5021g

Three roses 1

0.5081g

Three roses 2

0.5061g

Preparation of stock solution:

28.5mg of caffeine was dissolved in 25mL of volumetric flask with water to produce a concentration of 1.14mg/mL

26.8mg of theanine was dissolved in 25mL of vlolumetric flask with water to produce a concentration of 1.072mg/mL

27.5mg of epicatechin was dissolved in 25ml of volumetric flask with water to produce concentration of 1.104mg/mL

Table 2. Concentrations of caffeine, theanine and epicatechin in each mixture standards.

Caffeine conc. (mg/ml)

Theanine conc. (mg/ml)

Epicatechin conc.(mg/ml

Standard1 1.14mg/ml

Standard1 1.072mg/ml

Standard1 1.104mg/ml

Standard2 0.912mg/ml

Standard2 0.8576mg/ml

Standard2 0.88mg/ml

Standard3 0.684mg/ml

Standard3 0.6432mg/ml

Standard3 0.66mg/ml

Standard4 0.456mg/ml

Standard4 0.4288mg/ml

Standard4 0.44mg/ml

Standard5 0.228mg/ml

Standard5 0.2144mg/ml

Standard5 0.22mg/ml

Method for high performance liquid chromatography:

The analysis of the standard of caffeine, theanine and epicatechin was performed using high performance liquid chromatography (HPLC). HPLC analysis of standards was performed on the Dionex machine (Dionex HPLC)

ABTS radical cation decolourisation assay:

ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) radical cation was produced by reaction between ABTS stock solution and potassium persulfate. ABTS stock solution is prepared by weighing 0.36gm of ABTS and added to water. The potassium persulfate solution is prepared by weighing 0.06gm of potassium persulfate and added to water. Now both ABTS solution and potassium persulfate solution is mixed together and made up to 100ml. The testtube containing the mixture solution was kept at room temperature for 12-16hours before use. For the study of antioxidant activity of citrus juices, the ABTS stock solution was diluted with water to an absorbance of 0.70 (+0.02) at 734 nm and equilibrated at 30 °C. This was done by taking 1ml of ABTS stock solution and made up to 75ml with water. Then the absorbance was noted which was 0.705. 1mL diluted ABTS cation was added into 10µL of sample and incubated in a water bath at 30°C exactly 1 minute before reading at 734 nm in a spectrophotometer with water as a blank solvent. Trolox was used as a standard with final concentration of 10, 5, 2.5, 1.25, 0.63 µg/mL.

Total polyphenol concentration (TPC)

50µL of tea sample was added to a test tube containing 450µL of filter water and 250µL of Folin-Ciocalteu reagent. The mixture was allowed to stand for 5min before the addition of 1.5mL of 20% Na2CO3. Then the solution was kept for 20 min at room temperature. The absorbance was measured at 735nm versus water blank. Gallic acid was used as the analytical standard with the final concentration of 10, 5, 2.5, 1.25µg/mL

RESULTS AND DISCUSSION:

Analysis of standards and tea samples:

HPLC is the technique used for the analysis of caffeine, theanine, and epicatechin contents in five different tea samples. HPLC utilizes different types of stationary phase (typically, hydrophobic saturated carbon chains), a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic information. Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase. Common solvents used include any miscible combination of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as trifluoroacetic acid which acts as an ion pairing agent.

The mobile phase used for analytical separation was acetonitrile and orthophosphoric acid in deionized water. Acetonitrile is a colourless liquid used as a polar solvent with a wide solubility range. Acetonitrile can dissolve many non-polar and ionic compounds. 0.1% ortho-phosphoric acid in deionised water was used so as not to exihibit ion exchange that might affect the results of the analysis. In this method two-gradient elution system was used,mobile phase A contained acetonitrile (4%) and mobile phase B was ortho-phosphoric acid (96%).

Mixed standards were made which show caffeine, theanine, and epicatechin. The retention time of caffeine, theanine, and epicatechin are in the same range in all standards. The retention time is in the range of 3.5-3.6 for theanine, 24.3-24.5 for caffeine and 25.6-25.8 for epicatechin. These were used to plot the standard calibration curve of caffeine, theanine and epicatechin.

Standard concentrations of caffeine, theanine, and epicatechin:

Caffeine mg/mL

Area

1.14

750.12

0.912

609.12

0.684

489.802

0.456

328.633

0.228

158.616

Standard calibration curve of caffeine

Theanine mg/mL

Area

1.1

31.162

0.88

25.323

0.66

19.137

0.44

12.937

0.22

6.544

Standard calibration curve of theanine

Epicatechin mg/mL

Area

1.072

901.444

0.8576

755.325

0.6432

596.901

0.4288

400.430

0.2144

206.173

Standard calibration curve of epicatechin

Mixed standards were analyzed using HPLC. The graphs clearly illustrate a direct relationship between the concentrations and the HPLC areas of the three antioxidants. Concentrations of standards were plotted against the HPLc area to obtain the calibration curve of caffeine, theanine and epicatechin respectively. The standard caffeine has a linear equation of y=641.88x + 28.21 while y= 28.01x + 0.534 is for standard calibration curve of theanine and the standard calibration curve of epicatechin has an equation of y= 814.1x + 48.424

Concentrations of the antioxidants of the five different tea samples were identified at different time frames and were performed in two trials in average values of mg/mL. The antioxidants of the five tea samples were analysed at three different points. First sample is collected after 5mins, then after one hour and then for 24 hours. All the tea samples showed the readings of the presence of caffeine, theanine, epicatechin.

Average concentrations in mg/mL of caffeine, theanine, and epicatechin in five types of teas done in two trials:

Type of tea average values, mg/mL

Theanine caffeine epicatechin

Standard1 0.610 1.09 1.07

Standard2 0.471 0.9002 0.877

Standard3 0.752 0.670 0.682

Standard4 0.482 0.441 0.426

Standard5 0.214 0.211 0.183

Type of Tea

Duration

Average values, mg/ml

Caffeine

Theanine

Chai spice tea

5mins

cs51

0.27

0.02

cs52

0.255

0.03

1 hour

cs601

0.298

0.027

cs602

0.306

0.037

24hours

cs241

0.301

0.027

cs242

0.328

0.03

Lychee tea

5mins

lt51

0.303

0.088

Lt52

0.369

0.007

1 hour

Lt601

0.363

0.015

Lt602

0.414

0.013

24hours

Lt241

0.411

0.015

Lt242

0.43

0.014

Mango tea

5mins

mt51

0.35

0.012

mt52

0.371

0.014

1hr

mt601

0.455

0.019

mt602

0.408

0.017

24hrs

mt241

0.405

0.018

mt242

0.403

0.017

Darjeeling tea

5mins

dt51

416

0.112

dt52

0.426

0.13

1hr

dt601

0.533

0.224

dt602

0.546

0.206

24hrs

dt241

0.614

0.237

dt242

0.5966

0.288

Three roses tea

5mins

3r51

0.514

0.073

3r52

0.55

0.0703

1hr

3r601

0.574

0.0701

3r602

0.585

0.0702

24hrs

3r241

0.55

0.0704

3r242

0.576

0.068

Total polyphenol concentration:

Absorbance used in the polyphenol concentration assay

Gallic acid mg/mL

Absorbance

10.00

3.926

5.00

1.864

2.5

0.786

1.25

0.278

Folin-Ciocalteu reagent is used for the calorimetric assay of phenolic antioxidants and functions by measuring the amount of substance being tested in inhibiting the reagent to undergo oxidation.

Gallic acid (3,4,5-trihydroxybenzoic acid) was used as an analytical standard for total polyphenol concentration. Na2CO3 is an electrolyte which was used to stabilize the pH