1.1.

THE WINE

Wine is a health drink resulting from complete or partial fermentation of grapes, either exclusively by natural micro-flora of grapes or the added wine yeast culture (Joshi and Attari, 1990). Wine is an alcoholic beverage made from the fermentation of fruit juice (grapes, apple, etc). The natural chemical balance of grapes is such that they can ferment without addition of sugars, acids, enzymes or other nutrient. Although other fruits such as apples and berriers can also be fermented, the resultant wines are normally named after the fruit from which they are produced. Wine is produced by fermenting crushed fruits using various type of yeast which consume the sugars found in the fruits and convert them into alcohol. Various varieties of fruits and strains of yeasts are used depending on the type of wine produced.

1.1.1.

HISTORY OF THE WINE

The latest evidence suggests that wine was being made during the Neolithic period (approximately 6000 B.C.) in the Near East. Many lines of evidence indicate that wine making and grape cultivation began in the Transcaucasian region encompassing the northern portion of Turkey, Iran and Iraq, the southern region of Armenmia and Azerbaijan. This zone corresponds to the closest overlap of natural distribution of the wild Vitis vinifera (spp.silvatica) with the Northern spread of agriculture out of its Near Eastern centre of origin.

Figure 1.1: A 19th century reproduction of the Rigveda Viticulture was believed to have been introduced to India by Persian traders sometime in the 4th millennia BC. Historians believe that these early plantings were used mostly for table

grapes or grape juice rather than the production of an alcoholic beverage. During the Vedic period of the 2nd and 1st millennia, the Aryans tribes of the region were known for their indulgence of intoxicating drink and it seems probable that wine was a pleasant beverage. The religious text of the Vedas mentions at least one alcoholic drink that may have been wine related-sura which seems to have been a type of rice wine that was fermented with honey. The first known mentioning of grape- based wines was in the late 4th century B.C. writings of Chanakya who was the chief minister of Emperor Chandragupta Maurya. In his writings, Chanakya condemns the use of alcohol while chronicling the emperor and his court's frequent indulgence of a style of grape wine known as Madhu. In the centuries that would follow, wine became the privileged drink of the Kshatriya or noble class while the lower caste typically drank alcohol made from wheat, barley and millet. Under the rule of Muslim Mughal Empire alcohol was prohibited in accordance to Islamic dietary laws. However, there are written reports about at least one Mughal ruler, Jahangir who was fond of brandy wine. In the 16th century, Portuguese colonists at Goa introduced port style wine and the production of fortified wines soon spread to other regions. Under British rule during the Victoria era, viticulture and wine making was strongly encouraged as a domestic source for the British colonists. Vineyards were planted extensively through the Baramati, Kasmir and Surat regions. In 1883, at the Calcutta International Exibition, Indian wines were showcased to a favorable reception. The Indian wine industry was reaching a peak by the time the phylloxera epidemic made its way to country and devastated its vineyards. It was a long road for the Indian wine industry to recover from the devastation at the end of the 19th century. Unfavorable religious and public opinion on alcohol developed and culminated in the 1950s when many of India's states prohibited alcohol. Vineyards were either uprooted or encouraged to convert to table grape and raisin production. Some areas, like Goa, continued to produce wine but the product was normally very sweet and highly alcoholic. The turning part of the modern Indian wine industry occurred in early 1980s with the founding of Chateu Indage in the state of Maharashtra With the assistance of French winemakers, Chateau Indage began to import Vits vinifera grape varieties like Cabernet Sauvignon, Chardonnay, Pinot blanc, Pinot noir and Ugni blanc and started making still

and sparkling wines. Other wineries soon followed as the emergence of India's growing middle class fueled the growth and development of the Indian wine industry. However, Indian wine yards were totally destroyed by unknown reasons in the 1890.

1.1.2. WINE GROWING REGIONS IN INDIA

Following are the major wine growing regions in the country:<1> Nasik Region (Maharashtra State): It is the biggest wine producing region in India. This region includes Pune, Nasik and Ahmed Nagar. It is above 800 meter sea level. Several top wineries are located in this area including Chateau Indage and Sula Wines. <2> Sangli Region (Maharashtra State): This region includes Solapur, Sangali, Satara and Latur. It is above 800 meter sea level. <3> Bangalore Region (Karnataka State): Nandi Hills located about around 45 kilometer north of Bangalore City. Grover Vineyards is located in Nandi Hills. It is above 800 meter sea level. <4> Himachal Region: It is located at Northern India. It is upcoming state for the wine production. Temperature varies from 20 degree C to 40 degree C. Unique climate of this region attracts the wine makers to produce delicate wine grapes.

Figure 1.2: Wine producing regions of India

1.1.3. WINE INDUSTRY: INDIAN PERSPECTIVE

Approximately 40 wineries are presently operating in the country with a total production of 6.2million litres annually. Maharashtra is leading among the states with 36 wineries and 5.4 million litres production. Eighty percent of wine consumption in the country is confined in major cities such as Mumbai (39%), Delhi (23%), Bangalore (9%) and Goa (9%). There is growing awareness about the wine as a product in the domestic market. Major wineries such as Champagne Indage, Grover Vineyards and Sula Vineyards make invaluable contribution for production of wines in the country. Chateau Indage at Narayangaon is a pioneer of French style wines in India, produces exquisite qualities in

both still and sparkling wines. The company has the capacity of producing over 3 million bottles annually. Sula vineyards at Nasik are new vineyards in India for making wine. Chenin Blanc is predominantly grown but emphasis should be given to red wine varieties. Sangali is another region for wine production. Reportedly the world wine market is growing at 14% annually whereas the Indian market at more than 20%. Central governments have announced number of financial incentives and administrative reforms. Quality wines can only be prepared from quality grapes. Thus cultivation of good quality of grapes is very much important. Several research workers in India have concentrated their efforts to produce quality wines with improved health benefits.

1.1.4. TYPES OF WINES

Various research workers specified different compositional requirements of grapes and must for different wines. Wine is classified as dry table wines, which contain alcohol less than 14% and little or no unfermented sugar and sweet dessert wines are those having 17 to 20% alcohol and some unfermented grape sugars.Dry table wines require grapes of fairly low pH (3.00 to 3.35), high acidity (0.6 to 0.9%) and moderate sugar content (19 to 23 Brix) (Bammi, 1968).For sweet desert wines, the must should be of relatively higher pH (3.30 to 3.65), medium acidity (0.5 to 0.6%) and higher sugar content (atleast 24 Brix). Some common types of wine are:1. Red Wines: Red wine is simply wine produced from red (or black) grapes. Everyone knows that almost all grapes have colourless juice. The way that the red wine gets its colour is by letting the skins soak in the juice until the red colour bleeds out. Soaking the skins give red wine its colour and also imparts a substance known as tannin. Tannin is what gives red wines a complexity that is beyond that of most white wines.

Tannin has a mouth drying quality that causes the wine to feel firm in your mouth. When a red wine is young, this firmness can be quite intense. Over time, the qualities of the tannin will mellow and blend harmoniously with the other characteristics of the wine. This is one of the main reasons that red wines usually age better than whites. Table: 1.1 Major Red Wine (Grape) Varieties

Varieties Cabernet Sauvignon Merlot

Properties Thick skinned grape with lots of tannin, and blackcurrant flavor. Higher in alcohol, taste of black cherries and sometimes mint.

Nebbiolo Pinot Noir

High in tannin and acid and need aging to mellow, grown in the Piedmont region of Italy. The most finicky of grapes producing the widest range of quality.

Syrah/Shiraz Zinfandel

Rich and spicy wine with lots of tannin and the sweetness of blackberries. Ranging from light and fruity to big and spicy depending on the quality. Examples are from California.

Table: 1.2 Other Red Wine (Grape) Varieties

Varieties Barbera Carmenere Gamay Grenache/Garnacha Malbec Sangiovese

Properties Low tannin with high acidity. A rich and spicy wine and is popular in Chile. They actually taste of grapes and are low in tannin. Found in France. High in alcohol and is usually sweet and peppery. A smooth and plummy variety from Argentina.

This wine has medium acidity and tannin.

2. White Wines Most white wine is produced from white grapes. Wine gets its colour from letting the skins soak in the juice. It is possible to make white wine out of black grapes by carefully extracting the juice and keeping the skins separated. Champagne is the most famous example. It is made from a blend of grapes which include Pinot Noir and Pinot Meunier (black grapes). Besides colour, not allowing the skins and stems to soak in the juice also reduces the amount of tannin in the wine. Sometimes though, a white wine will be allowed to ferment or age in oak barrels. The oak barrels will impart some tannin to the wine, but not as much as in a typical red wine. Believe it or not, a blush, which is just another term for ros, is considered a white wine. They are made by allowing the skins to soak for only a short period of time before extracting. A good ros should be delicate and refreshing, not cloyingly sweet. The best ross are made from the Grenache grape. Ross have been given a bad reputation from

some of the extremely sweet and cheap varieties on the market. Don't let that stop you from finding some that are truly delicious and worth savoring. Table: 1.3 Major White Wine (Grape) Varieties

Varieties Chardonnay Chenin Blanc Gewrztraminer Muscat Reisling Sauvignon Blanc

Properties It is usually oak aged and has a buttery flavor. It is a highly acidic wine that can range from very dry to very sweet. The most intensely aromatic of all wines. The aromas are of florals and spice. Produces the only wine to actually smell like grapes. A low alcoholic wine with striking acidity. These are the most tangy and pungent of the wine varieties.

Table: 1.4 Other White Wine (Grape) Varieties

Varieties Verdelho Colombard Garganega Mller Thurgau Pinot

Properties A rich, white wine with the taste of limes. Produces a crisp every day wine with tropical fruit aromas. Famous for making Italy's Soave. This wine is fresh and tangy like green apples. A faily unexciting grape popular in cooler climates for its early ripening qualities. Highly acidic and low sugar levels resulting in a dry, crisp

Blanc Viognier 3. Port Wine

wine. A low acid wine with floral aromas and an apricot quality.

Port wine (also known as Vinho do Porto, Porto, and often simply Port) is a portugese style of fortified wine originating from the douro valley in the northern provinces of Portugal. It is also grown in Australia, India, South Africa, Canada, US. Port is produced from grapes grown and processed in the demarcated Douro region. The wine produced is then fortified by the addition of a neutral grape spirit known as Aguardente in order to stop the fermentation, leaving residual sugar in the wine, and to boost the alcohol content. The wine received its name, "Port", in the latter half of the 17th century from the seaport city of Porto at the mouth of the Douro river where much of the product was brought to market or for export to other countries in Europe.

1.2.

ANTIOXIDANTS AND FREE RADICALS

Antioxidants are intimately involved in the prevention of cellular damage -- the common pathway for cancer, aging, and a variety of diseases. The scientific community has begun to unveil some of the mysteries surrounding this topic, and the media has begun whetting our thirst for knowledge. Athletes have a keen interest because of health concerns and the prospect of enhanced performance and/or recovery from exercise. The purpose of this article is to serve as a beginners guide to what antioxidants are and to briefly review their role in exercise and general health. What follows is only the tip of the iceberg in this dynamic and interesting subject. Free radicals are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain molecules. Once formed these highly reactive radicals can start a chain reaction, like dominoes. Their chief danger comes from the damage they can do when they react with important cellular components

such as DNA, or the cell membrane. Cells may function poorly or die, if this occurs. To prevent free radical damage, the body has a defense system of antioxidants. Antioxidants are molecules which can safely interact with free radicals and terminate the chain reaction before vital molecules are damaged. Although there are several enzyme systems within the body that scavenge free radicals, the principle antioxidants are vitamin E, beta-carotene, and vitamin C. Additionally, selenium, a trace metal that is required for proper function of one of the body's antioxidant enzyme systems, is sometimes included in this category. The body cannot manufacture these micronutrients so they must be supplied in the diet. Vitamin E: d-alpha tocopherol. A fat soluble vitamin present in nuts, seeds, vegetable and fish oils, whole grains (esp. wheat germ), fortified cereals, and apricots. Current recommended daily allowance (RDA) is 15 IU per day for men and 12 IU per day for women. Vitamin C: Ascorbic acid is a water soluble vitamin present in citrus fruits and juices, green peppers, cabbage, spinach, broccoli, kale, cantaloupe, kiwi, and strawberries. The RDA is 60 mg per day. Intake above 2000 mg may be associated with adverse side effects in some individuals. Beta-carotene is a precursor to vitamin A (retinol) and is present in liver, egg yolk, milk, butter, spinach, carrots, squash, broccoli, yams, tomato, cantaloupe, peaches, and grains. Because beta-carotene is converted to vitamin A by the body there is no set requirement. Instead, the RDA is expressed as retinol equivalents (RE), to clarify the relationship. (NOTE: Vitamin A has no antioxidant properties and can be quite toxic when taken in excess.)

1.2.1. Types of antioxidant

Table 1.5: Some important enzymatic and non-enzymatic physiological antioxidants

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Sr.no

Antioxidant

Location

Function/property

A. ENZYMATIC OXIDANT 1. Glutathione peroxidase (GSH) Mitochondria & Cytosol Removal of H2O2 &Organic hydroperoxide 2. Catalase (CAT) Mitochondria & Cytosol Removal of H2O2

B. NON ENZYMATIC OXIDANT

1.

Carotenoids

Lipid soluble antioxidants Removal in membrane tissue reactive species

of Oxygen

2.

Bilirubin

Product

of

heme Extracellular antioxidants

metabolism in blood

3.

Glutathione

Non-protein thiol in cell

Cellular defense Serves

oxidant

as for

4.

-lipoic acid

Endogenous thiol

substitute Glutathione, Recycling Vit-C

5.

Vitamin-C

Aqueous phase of cell

Free

scavenger,

recycle Vit-E Chain 6. Vitamin-E Cell breaking

antioxidants

11

7.

Uric Acid

Product metabolism

of

Purine Scavenging of OH radical

1.2.2. Antioxidants based on defense mechanism

Antioxidants based on defense mechanism are of four types: A. Preventive antioxidants - These suppress the free radical formation. Ex. enzymes such as peroxidase, catalase, lactoferrin, carotenoids, etc B. Radical scavenging antioxidants - These suppress the chain initiation reaction. Eg: Vitamin-C & Carotenoids. C. Repair and de novo antioxidant - It comprises of proteolytic enzymes and repair enzymes of DNA and genetic materials. D. Enzyme inhibitor antioxidants - These induce production and reaction of free radicals and the transport of appropriate antioxidants to appropriate active site.

2.1. THE YEAST

It is very important to use high quality yeast in all winemaking. Yeast is the workhorse that converts the initial sweet syrupy must into great-tasting wine. Using Lalvin wine yeast made by Lallemand is recommended. Lallemand is the world's largest producer of specialty yeast for wine making and produce over 10 different wine yeast strains for the professional winemaker in active dried form. It is not surprising that the finest wines are made using only the finest ingredients and also the finest winemakers use Lalvin dried wine yeast. The primary role of wine yeast is to catalyse the rapid, complete and efficient conversion of grape sugars to ethanol, carbon dioxide and other minor, but important metabolites without the development of off-flavours.

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A lot of research has been done that confirm that the yeast play a critical role in determining the flavour, aroma, body, viscosity and colour of wines.

2.1.1 TAXONOMY
Out of the 100 yeast genera representing over 700 species described in the latest edition of the monographic series. A taxonomic study, Kurtzmann suggested that 15 are associated with winemaking Brettanomyces; Candida; Cryptococcus; Debaromyces; Hanseniaspora and its asexual counterpart Kloeckera; Kluyveromyces; Metschnikowia; Pichia; Rhodotorula; saccharomyces and Zygosaccharomyces. Table 2.1. The scientific classification of Saccharomyces cerevisiae Kingdom Phylum Subphylum Class Order Family Genus Species Fungii Ascomycota Saccharomycotina Saccharomycetes Saccharomycetals Saccharomycetaceae Saccharomyces cerevisiae

2.1.2. STRAINS OF WINE YEAST

i) Red Star Active Dry Yeasts Table 2.2

STRAIN NAME

WINE

ALCOHOL CONTENT

TEMPERATURE OTHER RANGE INFORMATION

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Assmannshausen

Red wines

Adds is a

spicy its

aroma, drawback ineffectiveness in high solid content. slow fermenting anaerobic fermentations It tolerates sulfur dioxide well, but does well Pasteur Champagne Premier Curvee Red, White 18% 45-95F Sparkling wine 13-15%. 59-86F not with work high of

Epernay Flor Sherry Montrachet

White wines Port, Madeira Red, wine White

12-14% 18-20%. 13%

low temperatures 59-86F 59-86F

sugar levels tolerance medium-high

alcohol conditions qualified for barrel fermentations it is tolerant to heat and sulfur dioxide

Pasteur Red

Red wine

16%

64-86F

ii) Lalvin Active Dry Yeasts Many of Lalvin's yeasts are intended for commercial rather than home use. Some are as follows: - Table 2.3- Lalvin active dry yeast STRAIN NAME ALCOHOL CONTENT Lalvin 43
18%

TEMPERATURE RANGE 55-95F

OTHER INFORMATION Restart abilities, fast fermenter.

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Lalvin 71B-1122

14%

60-85F

Metabolizes more of the malic acid during fermentation.

Lalvin AC

14%

85 F

produce low levels of SO2 and H2S

Lalvin AMH

15%

68-86 F

enhances spicy and fruity flavors, aroma

Lalvin BA11

16%

68-86F

It requires a highnitrogen nutrient

Lalvin BDX

16%

64-86 F

It

is

highly

recommended for the production of quality dry red wines. a It requires high-

nitrogen nutrient. Lalvin BGY

15%

75-86F

It is used in reds, particularly Noir, for fermentations. Pinot slow

Lalvin CSM

14%

59-89 F

This strain requires high levels of nitrogen and nutrients but will promote malolactic fermentation. It works particularly well in low maturity white grapes from cool regions.

Lalvin MO5

14%

59-90 F

15

Lalvin M1

16%

down to 54 F

This strain is used to produce aromatic ros and white wines, especially wines with residual sugar.

Lalvin QA23

16%

50-90 F

Used for Sauvignon Blanc, Chenin Blanc, Colombard, Semillon fruity, clean wines. and for

production of fresh,

Lalvin R2

16%

42-86 F

It may be used for fruit wines whenever a Sauternes wine yeast is specified.

Lalvin W15

16%

50-81 F

It was developed to ferment dry whites and ross at moderate speeds where bright fruit and heavy mouth feel are desired.

iii) White Labs Yeasts White Labs wine yeasts are "pitchable" yeast cultures, meaning they contain sufficient living yeast to inoculate five gallons of must without rehydration or starter propagation. Because they are fresh, in theory they impart better flavours to the must. Refrigerated, they have a four-to-six-month shelf-life. They should be attemperated to within 100F of the must for several hours before "pitching."

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Table 2.4- white labs yeast STRAIN NAME ALCOHOL CONTENT WLP715 Champagne 17% TEMPERATURE RANGE 70-75 F OTHER INFORMATION Classic yeast, used to produce wines, champagne, or to fully barley cider, dry meads, dry attenuate

wines/ strong ales. WLP720 Sweet Wine 15% 70-75 F A good choice for sweet mead and cider, as well as Blush wines, Gewurztraminer, and Riesling WLP730 Chardonnay White Wine 14% 50-90 F This is a good choice for all white and blush wines, Chablis, including Chenin

Blanc, Semillon, and Sauvignon Blanc. WLP740 Merlot Red Wine 18% 60-90 F This is a vigorous fermenter well suited for Cabernet, Shiraz, Pinot Noir,

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Chardonnay, Sauvignon Blanc, and Semillon. WLP760 Red Wine Cabernet 16% 60-90 F High tolerance, fermentation Chardonnay, Blanc. temperature moderate speed, Chenin

suitable for Merlot, Blanc, and Sauvignon

2.2. WINE
In the last 90 years, the wine industry has seen a revolution in the wine production technology. This is largely due to the scientific development. Annually, about 26 billion litres of wine are produced from about 8 million hectares of vineyards across the world. Wine contains a naturally rich source of antioxidants, which may protect the body from oxidative stress, a determinant of age-related disease. The current study set out to determine the in vivo effects of moderate red wine consumption on antioxidant status and oxidative stress in the circulation. The role of nutrition in health has captured researcher's interest in antioxidants and their capacity to protect the body from damage induced by oxidative stress. Extensive research has demonstrated the protective properties of antioxidants, which scavenge reactive oxygen species (ROS) and their precursors, as well as up-regulate enzymes involved in the repair of cellular damage. Red wine contains a rich source of a large number of antioxidants, namely the phenolic acids and polyphenols, which provide it with its protective redox potential. Studies have shown that despite the high intake of saturated fatty acids within the diets of some populations, a reduced mortality rate from cardiovascular disease is attributed to the high consumption of red wine, independent of its alcohol content. Moderate

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consumption of red wine has also been shown to retard or slow the plasma clearance of high density lipoproteins (HDL), a negative risk factor for the development of cardio vascular disease (CVD). The incubation of low density lipoproteins LDL in varying concentrations of red and white wine showed a 50% decline in oxidation at concentrations of 0.04 and 0.7 mg/ethanol/mL respectively, up to a concentration of 1.0 mg/mL. These results indicate that red wine inhibits cell mediated LDL oxidation more efficiently than white wine and at much lower concentrations. A large body of evidence which indicates that free radical production can directly or indirectly play a major role in cellular processes implicated in atherosclerosis and CVD. Therefore, the aim of this study was firstly to understand how moderate red wine consumption (400 ml/day) for two weeks effected circulating lipids, antioxidant level and total antioxidant capacity in the circulation and secondly assess the differences in bioefficacy of red wine in young and older populations(Cui,J. et al,.2002) Recent study on wine against cardiovascular diseases have shown that red wine have high content of antioxidant especially polyphenols, whereas white wine lacks polyphenols, but it contains other compound such as hydroxycinnamic acids (caffeic acids) and monophenols (tyrosol), which are known to have antioxidant properties. These compounds possess antioxidant activity and are able to reduce the release of pro-inflammatory cytokines. The experimental rats were given white wine and it also showed protection against cardiovascular disease (Mahesh et al., 2007). It is also being observed that consumption of white wine in moderate quantity help in reducing weight and other problem (Lamuela-Raventos et al,.1999). A group of obese patients were taken and till 3 months they were given moderate quantity of wine. It is being observed that there is redution in weight upto 4.73 0.53kg. It is also observed that percent body fat, waist circumference, blood pressure, blood glucose, insulin, triglycerides, and cholesterol were reduced, other safety parameters were unchanged ( Flechtner Mors, 2004). The absorption in human body of phenolic acids from white wine, particularly hydroxycinnamic acids from white wine are absorbed from the gastrointestinal tract and being circulated in the blood after being largely metabolized to the form of glucornide and sulfate conjugates. Unmodified tartaric acid esters of hydroxycinnamic acids from wine are

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human plasma at low levels. Wine hydroxycinnamic acids although present in wine as conjugate form, are bioavailable to humans (Nardini, Mirella et al., 2009). A study was carried out on daily consumption of wine on oxidative stress. Volunteers were taken and each of them was supplied 375ml of white wine daily. Each participant provided their venous blood samples. After that levels of glutathione peroxidase, reduced glutathione, total antioxidant capacity, total cholesterol. HDL cholesterol, TBARS, superoxide dismutase is being measured and after a month there is significant increase in HDL- cholesterol, paraoxonase, glutathione peroxidase and reduced glutathione levels, a decrease in superoxide dismutase activities and TBARS concentrations. However, there was also a clear increase in homocysteine after a month of wine consumption. This is clear that daily consumption of white wine is not only associated with both oxidative and antiatherogenic effects but also with a potentially proatherogenic increase of homocysteine concentration (Rajdl,D. et al., 2007). Lipofuschin is an end product of lipid peroxidation which dramatically increases ethanol consumption (Assuncao, Marco et al,.2007). Crude methanol extracts of red and white wines were added to diethyl ether in order to divide them into the anthrocyanin fraction and fractions containing other flavonoids and their derivatives. Whereas white wine did not contain anthrocyanins, it is being seen that when HCT-15 cells, which is derived from human colon cancer or AGS cells, derived from human gastric cancer, were cultured with these fractions. The anthrocyanin fraction from the red wine and the non-anthrocyanin substances extracted from red and white wine suppressed the growth of the cells, and the suppression rate by the anthrocyanin fraction was significantly higher than that of other fractions (Hashimoto, Yoko 1998).

2.2.1. CHEMICAL COMPOSITION AND SENSORY PROPERTIES OF WINE

Wine is produced by fermentation of fruit juice and therefore, contains all the nutrients present in the fruit juice (Bhutani et al., 1989). In addition it contains alcohol, vitamins, and other compounds synthesized by fermenting yeasts. A typical wine contains ethyl

20

alcohol, sugars, acids, higher alcohols, tannins, aldehydes, esters, amino acids, anthocyanins, fatty acids and minor constituents like flavouring compounds (Joshi, 1998).

Figure 2.1: Wine composition Component Water Alcohol Everything Else Percentage 85 12 3

Principal components of a litre of wine (1000 ml) Figure 2.2:

Water

glycerol tartaric acid malic acid lactic acid

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potassium succinic acid a) Water reducing sugar phenolic compound azoted substances

It is the major constituent of wine. Wine may contain 85% to 88% and 12 to 15% dry matter (Jarczyk and Wzorek, 1977). b) Alcohol Wine contains mainly ethyl alcohol and traces of methyl and higher alcohols (Amerine, 1954). The percentage of alcohol in wine is approximately equal to the brix multiplied by the factor 0.57 (Cruess 1958). reported production of 50% alcohol from the total sugar content of the berries (Pandey and Pandey 1990). The alcohol content of the wine prepared from four cultivars of grape was in the range of 11.2 to 15.5% (Suresh et al., 1985). c) Sugars The sugars present in the wine include glucose, fructose, sucrose, arabinose, xylose, rhamnose. Sucrose is rapidly hydrolysed during fermentation and is present in wine in very small amounts (0.01 to 0.06%) (Amerine et al., 1980 and Joslyn, 1961). The dry wines are practically free of sugars and contain 0 to 1.0% sugars. While semidry, sweet and very sweet wines contain 2.0 to 3.0, 8 to 11 and 7 to 12% sugars, respectively (Jarczyk and Wzorek, 1977). d) Organic acid The titrable acidity in the wines prepared from four cultivars ranged from 3.6 to 4.2 pH (Suresh et al., 1985). It is reported that 0.248 0.586% tartaric acid and 0.058 0.144%

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mallic acid is present in Thompson Seedless wines (Chikkasubbana 1990). According to Indian standards for wine, a wine should contain total acids in the range of 4 to 15g/l (as tartaric acid) (Bhalerao, 2001) e) Proteins The nitrogenous compounds present in wine include protein, peptones, polypeptides, amides, amino acid and ammonia. About half of the wine proteins are bound to phenolics. Protein content is in the range of 0.050 to 0.063% in wine (Patil 1994). It is being analysed that the total nitrogen of 0.01 to 0.09% and proteins of 0.07 to 0.56% is present in 16 young wines (Somer and Ziemelis 1973). f) Minerals The mineral elements present in wine include magnesium, sodium, potassium, calcium, phosphorous, iron, zinc, copper, cobalt, bromine, iodine, silica, aluminium and lead. They are present in varying proportion in different type of wine. g) Phenolic compounds The phenolic compounds isolated from wines include catechin, 1-epictechin, 1epigallocatechin, gallic acid, caffeic acid and ellagic acid (Henning and Brukhardt, 1960). A wide range of total phenols in white wines ranging from 50 to 6500 mg/l and red wines from 1000 to 4000 mg/l were reported (Joseph et al., 1983). A wide range of tannin content in white wine (46 to 143mg/ 100ml) and red wines (28 to 328 mg/100ml) was reported (Suresh et al., 1985). New wines contain nearly 200mg/l anthocyanin, whereas old wines contain 20mg/l anthocyanin (Joshi 1998) and 273 mg/l anthocyanin content in red wines. (Ribereau Gayon et al., 1998)

2.2.2. THE HEALTH BENEFITS OF WINE

When word got out about the health benefits of wine, most wine drinkers stopped swirling for a long minute and took notice. It was the mid 90s when story of the "French Paradox" poured from all media sources, and wine drinkers across the world swirled and sipped and rejoiced.

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a) Overall Health Benefits: Anti-aging effects in red grape skins (Harvard Medical School in Boston, 2004). b) Lung Health Benefits: Improved lung function from antioxidants in white wine (American Thoracic Society, 2002). c) Heart Health Benefits: Coronary heart disease reduced (University of California, Davis, 1995). Healthier blood vessels in elderly (University of Ferrara in Italy, 2004). d) Ulcer Prevention: Ulcer-causing bacteria reduced (American Journal of Gastroenterology, 2003). e) Cancer Prevention: Cancer cells killed by protein in red grape skins (University of Virginia Health System, 2004). f) Stroke Prevention: Arteries kept clean by polyphenols in red grape skins (William Harvey Research Institute, 2002). g) Womens Health Benefits: Decreased ovarian cancer risk (The Queensland Institute of Medical Research in Australia, 2004). Stronger bones (Twin Research and Genetic Epidemiology Unit, St. Thomas Hospital in London, 2004).

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 Lower risk of stroke (Centers for Disease Control and Prevention, 2001). h) Mens Health Benefits: Lower risk of heart attack for men with high blood pressure (Worcester Medical Center in Massachusetts, 2004). In fact, too much wine per day or per week can lead to negative health effects in many cases and rather than prevent disease, one will actually up your odds of acquiring such diseases. But the best of all is Concentrated Resveratrol. So with all of the benefits of wine discussed, resveratrol is getting all the praise from the medical field. The problem with getting it naturally from wine is that the quantity is very low. To really benefit, one must get it in larger quantities. The way to do that is in pill form.

2.2.3. HARMFUL EFFECTS OF WINE

i) Alcohol Although, the nomenclature Alcohol denotes the entire family of organic compounds like methanol, ethanol, isopropanol, in more general terms we denote Ethanol when we discuss alcohol, the most commonly ingested of these chemicals. Alcohol, Ethanol (C2H5OH) in specific, is an organic compound made up of molecules of Carbon, Hydrogen, and Oxygen. It is highly volatile liquid with distinct odour having great affinity towards water. In fact, its high solubility in water makes it one of the most potent depressant of human central nervous system. It is a powerful hypnotic sedative with an array of side effects. The amount to which extent the central nervous system is affected depends upon the concentration of alcohol in blood, usually denoted by BAC (Blood Alcohol Content).

25

ii) Effects at Initial Stages When ingested, the effects of alcohol on human body changes gradually over the time. At initial stages, person feels more relaxed and cheerful which is followed by more stumbling movements and animated speech. They become more confident, shying off their inhibitions. This happens because of the increased metabolism in nigrostriatal pathway of brain which is associated with body movements. While, increased alpha waves from brain makes person more cheerful, relaxed; stimulation by alcohol to cortex, hippocampus of brain helps to shed off inhibitions. This stage is often termed as Euphoria where BAC is around 0.03 to 0.12%. iii) Absorption and Distribution Mechanism When consumed, alcohol first irritates the mucous lining of mouth and then esophagus causing an anesthetic effect. Then it goes to stomach where only 20% of the total quantity is absorbed by it. Rest 80% is then absorbed by small intestine from where it gets distributed throughout the human body. Alcohol travels through blood and come into the vicinity of cells of almost every organ. As mentioned earlier, due to its high affinity towards water, it can penetrate almost all cellular membranes resulting in absorption by all organs. iv) Metabolism and its effects Alcohol dehydrogenase, the enzyme secreted by hepatic cells converts alcohol to acetaldehyde. This acetaldehyde further gets converted into acetic acid and then acetate by acetaldehyde dehydrogenase. Acetate is a compound of fats which gets deposited locally. Because of the chronic and continuous consumption of alcohol, the increase in fatty acid levels results in forming of plaque in the hepatic capillaries. This situation leads to Liver Cirrhosis. As liver performs vital role in filtration mechanism of body, malfunctioning of liver often leads to jaundice (Hepatitis). As alcohol enhances antidiuretic hormone secretion, more urine is formed which results in dehydration. v) Carcinogenic Effects Alcohol comes under Group1 carcinogens as classified by WHO. Although, previous studies have failed to establish direct connection between alcohol and its effect on cancer, there is a strong indication to suggest that alcohol enhances the effects of other

26

carcinogenic chemicals like tobacco. Acetaldehyde, the byproduct of metabolism of alcohol, if gets concentrated in high amounts then it can damage the DNA of cells. Their reaction with polyamines can end up in forming mutagenic DNA. The excessive consumption of ethanol also makes mouth, larynx, pharynx, esophagus more prone to cancer. vi) General Effects on Human Body There are different effects of alcohol on human body subject to their concentrations in blood. They are generally classified as follows1. When BAC count ranges from 0.09 to 0.23%, the condition is known as Lethargy. In

this condition, people become sleepy, their movements loose coordination and start losing their body balance. It is also characterized by blurred vision.
2. When BAC count ranges from 0.17 to 0.28%, the condition is known as Confusion. It

is characterized by aggravated emotional state where people try to be sentimental, overly aggressive. They are not certain of what they are doing. Dizziness continues. Nausea is the common symptom of this phase.
3. When BAC count ranges from 0.25 to 0.39%, the condition is known as Stupor. In

this stage, body movements are severely affected and patients lose and regain consciousness intermittently. They have high risk of coma or even death.
4.

When BAC count ranges from 0.35 to 0.50%, the condition is known as Coma. It is characterized by unconsciousness when body reflexes are low, breathing rate declines resulting in dropping of heart beat rate.

5. When BAC crosses the mark of 0.5%, it results in failure of CNS (Central Nervous System) ultimately resulting in Death. Alcoholism is a major public health problem. While consumption of alcohol develops an array of diseases, its withdrawal also develops symptoms like delirium tremens which has high percentage of mortality rate (35%), if not treated. During Pregnancy, alcohol consumption can lead to fetal alcohol spectrum disorder.

27

2.3. RESVERATROL
Resveratrol is an antioxidant that is commonly found in many plants. Red wine, peanuts, grapes and blueberries are some of its rich and popular sources. Food supplements often comprise in Resveratrol in pills that contain grape seed extract, polygonum cuspidatum extracts and red wine extracts. Originally, it was isolated from white hellebore roots by Takaoka. Later, Japanese knotweed was known to comprise in Resveratrol. Though red wine had been known to have health benefits since long but in 1992, it was recommended that probably the secret of these benefits lies in increased Resveratrol content in red wine. Muscadine grapes are utilized in making red wine, whose skin consists of Resveratrol that serves as the protective shield against diseases. This substance is usually created in the outer layers of fruits and plants and defends us against diseases caused by pathogens along with other benefits. Recent research indicates that resveratrol, found in red grapes, extends the life of yeast cells by 70 per cent. Mans pursuit of long life, the so-called fountain of youth, edged closer to fruition with the recent announcement that a dietary component may increase the human lifespan to the point where it would be common to live 125 years. This redcoloured youth potion can be obtained from a bottle of vino, and perhaps as a dietary supplement. Lead researcher David Sinclair, PhD, assistant professor of pathology at Harvard University Medical School, says the lifespan of all life forms tested so faryeast cells, fruit flies, worms and micehas been dramatically lengthened by minute amounts of a red wine extract, resveratrol. The skin of red wine is the most abundant source of resveratrol, a unique antioxidant that red grapes produce in great amounts as a defence against fungi. The process of winemaking utilises alcohol to extract resveratrol (or 3,4,5trihydroxystilbene) and then preserves it in an airtight bottle; otherwise it would vanish in days. Data in the literature indicates that its quantity is very variable (Lamuela-Reventros et al., 2004, Jeandet et al., 1995) Resveratrol is synthesized in the skin but not in the fleshy part of the berries of Vitis vinifera and Vitis labrusca grapes. Black grape is

28

nature's best source of resveratrol. Resveratrol concentration increases during fermentation on the skins but the amount extracted depends on the variety and enological conditions. Both cis- and trans-isomers of resveratrol have been analyzed in skin, seeds and must of grape berries of Gama, starting from must to skin separation and in wine after malolactic fermentation. Trans-resveratrol concentration in red wines ranges from 1.33 mg/l to 7.17 mg/l. The addition of polyvinylpolypyrrolidone (PVPP) significantly lowers the resveratrol content. Resveratrol can reduce serum lipid levels and can prevent or inhibit cellular events associated with tumor initiation, promotion and progression, thus may help prevent cardiovascular disease and cancer (Joshi, V. K 2009). In recent studies, it is being seen that resveratrol is able to neutralize free radicals, which can damage DNA and may lead to cancer onset. In this study we have indagated resveratrol anticancer action, analyzing its effects on both cell cycle and growing of human lymphoma B (DHL-4) cells. MTT colorimetric test, trypan blue dye exclusion assay, and cell cycle analysis showed that resveratrol has a dose-dependent antiproliferative and antiapoptotic action on DHL-4 cells. These results confirm resveratrol's potential therapeutic role on tumors. (Bruno.P. et al., 2003). Resveratrol reduces viability and DNA synthesis capability of cultured human promyelocytic leukemia (HL-60) cells. The growth inhibitory and antiproliferative properties of resveratrol appear to be attributable to its induction of apoptotic cell death as determined by morphological and ultrastructural changes, internucleosomal DNA fragmentation, and increased proportion of the sub diploid cell population. Resveratrol treatment resulted in a gradual decrease in the expression of anti-apoptotic Bcl-2 (Young-Joon et al., 1999).

2.3.1. MECHANISM OF ACTION

Since 1935, researchers have recognised that severe restriction of calories can significantly broaden the lifespan of insects, animals and probably humans (Heilbronn LK, Ravussin E., 2003). The mechanism behind calorie restriction appears to be a survival factor that is turned on when living organisms are exposed to harsh conditions. When a living organism is deprived of calories, the sirtuin gene upregulates the activity of an enzyme (histone deacetylase) that prolongs the time cells have in which to repair

29

their damaged genetic material, their strands of DNA. The enzymatic activity also silences the genes responsible for protein production (ribosomal DNA). Therefore, resveratrol inhibits the over-production of proteins within cells that leads to accelerated ageing. Aged cells typically produce hundreds of thousands of extra copies of ribosomal DNA. The accumulation of these proteins in living cells has been likened to an ageing clock. Slowing down the rate at which proteins are produced slows the rate of ageing itself. Sinclair began to research the dynamics of this survival mechanism. A family of ironcontrolling antioxidant molecules was screened for its ability to increase the activity of the enzyme. From a library of thousands of molecules, 17 activated the human survival/longevity gene. Resveratrol, the extract obtained from red wine, did indeed turn on the survival switch and extend the life of yeast cells by 70 per cent. In human terms, that would be equivalent to 30 to 50 years of added life! Resveratrol was superior to the 16 other molecules tested (Howitz KT, Bitterman KJ et al., 2003). The uniqueness of resveratrol may be partly explained by the fact it is utilised by cells and orally absorbed by humans better than other antioxidants found in grapes (Soleas GJ, et al., 2002). Humans have a similar gene, SIRT1, responsible for activating the same enzyme. The enzyme itself cannot be bottled because to work, it has to be delivered to cells at the right place and time. Cells have machinery to increase enzyme activity on their own. What Sinclair discovered was the dietary switch to turn on this mechanism. What grapes use to turn on this survival mechanism, the calorie restriction mimic, can be transferred to humans in a glass of wine; cross-species transfer process scientists now call xenohormesis (Hall SS, 2003).

2.3.2. SUPPLEMENTS FALL SHORT

Dietary supplements providing resveratrol from red wine or knotweed are available. Surprisingly, tests conducted at Harvard University by Sinclair have failed to find any significant biological activity in resveratrol dietary supplements in tablets, capsules or as

30

liquid herbal extracts (David Sinclair 2003). Leroy Creasy, PhD, a professor of plant science at Cornell University in Ithaca, New York, reported that resveratrol supplements failed to exhibit much biological activityevidenced by the ability to activate an enzyme that promotes DNA repair and lengthen the life of yeast cellscompared to wine, but apparently his report went unnoticed by manufacturers. Creasy claims it would take thousands of capsules of resveratrol to provide the equivalent amount of resveratrol found in a glass of red pinot noir wine (Leroy Creasy 2003). Encapsulation fails to duplicate the airtight environment found in a wine bottle, which preserves the resveratrol. Although studies are lacking that show resveratrol in pills work, resveratrol appears to work in wine, and also under laboratory conditions as a pure 100 per cent molecule, produced under nitrogen and preserved in part by refrigeration. However, a relatively new technology called Licaps, developed by Capsugel specifically for liquid ingredients, fills gelatin capsules in nitrogen rather than an oxygen environment and seals dietary supplements ingredients in an airtight pill. A nitrogen bubble inside the capsule also retards any spoilage. Licaps technology is being utilised to produce the first stabilised red wine extract, which has demonstrated biological activity, showcasing the need for special measures that should be undertaken in the manufacture of resveratrol supplements.

2.3.3. REQUIRED AMOUNTS

A small amount of resveratrol was found to increase the survival of yeast cells by threefold even when the cells were exposed to ionizing radiation (Zoberi I et al., 2002). Mega doses of resveratrol do not produce greater longevity and in fact may work in an opposite manner and become problematic to genes. Three five-ounce glasses of red wine per day, which provide about 3mg resveratrol, would be sufficient for humans to achieve enzyme activity levels equivalent to those achieved in the laboratory. However, much higher amounts of resveratrol have been used successfully in animal tests for treating cancer. (Kimura Y, Okuda 2001)

31

2.3.4. BENEFITS OF RESVERATROL

Years of researches have shown several benefits of this natural compound:

It helps you to reverse diseases that come handy as you grow old, such as diabetes, cancer and heart diseases. This can be done with regular intake of Resveratrol while you are still quite young.

It increases your life span as high resveratrol levels counteract with cell death and damage in brain and heart. It is an effective artery protector, antioxidant and inflammation damper. Low Resveratrol doses provide the same effect as that of a calorie-reduced diet. This would also slow the ageing process if you start taking in small quantities in your middle age.

Resveratrol contain in one red wine glass can suppress carcinogenesis that otherwise cause breast cancer. Last but not least, it ensures sound cardiovascular system.

2.4. ANTIOXIDANTS
Antioxidants are a type of complex compounds found in our diet that act as a protective shield for our body against certain disastrous enemies (diseases) such as arterial and cardiac diseases, arthritis, cataracts and also premature ageing along with several chronic diseases Antioxidants are molecules which can safely interact with free radicals and terminate the chain reaction before vital molecules are damaged. Although there are several enzyme systems within the body that scavenge free radicals, the principle micronutrient (vitamin) antioxidants are vitamin E, beta-carotene, and vitamin C. Additionally, selenium, a trace metal that is required for proper function of one of the body's antioxidant enzyme systems, is sometimes included in this category. The body cannot manufacture these micronutrients so they must be supplied in the diet.

32

Antioxidant help in many ways:Epidemiologic observations show lower cancer rates in people whose diets are rich in fruits and vegetables. This has lead to the theory that these diets contain substances, possibly antioxidants, which protect against the development of cancer. There is currently intense scientific investigation into this topic. Thus far, none of the large, well designed studies have shown that dietary supplementation with extra antioxidants reduces the risk of developing cancer. In fact one study demonstrated an increased risk of lung cancer in male smokers who took antioxidants vs. male smoker who did not supplement. Whether this effect was from the antioxidants is unknown but it does raise the issue that antioxidants may be harmful under certain conditions. Antioxidants are also thought to have a role in slowing the aging process and preventing heart disease and strokes, but the data is still inconclusive. Therefore from a public health perspective it is premature to make recommendations regarding antioxidant supplements and disease prevention. New data from ongoing studies will be available in the next few years and will shed more light on this constantly evolving area. Perhaps the best advice, which comes from several authorities in cancer prevention, is to eat 5 servings of fruit or vegetables per day.

2.4.1. EXERCISE AND OXIDATIVE DAMAGE

Endurance exercise can increase oxygen utilization from 10 to 20 times over the resting state. This greatly increases the generation of free radicals, prompting concern about enhanced damage to muscles and other tissues. It is not possible to directly measure free radicals in the body, scientists have approached this question by measuring the byproducts that result from free radical reactions. If the generation of free radicals exceeds the antioxidant defenses then one would expect to see more of these by-products. These measurements have been performed in athletes under a variety of conditions. Several interesting concepts have emerged from these types of experimental studies. Regular physical exercise enhances the antioxidant defense system and protects against exercise induced free radical damage. This is an important finding because it shows how

33

smart the body is about adapting to the demands of exercise. These changes occur slowly over time and appear to parallel other adaptations to exercise. On the other hand, intense exercise in untrained individuals overwhelms defenses resulting in increased free radical damage. Thus, the "weekend warrior" who is predominantly sedentary during the week but engages in vigorous bouts of exercise during the weekend may be doing more harm than good. To this end there are many factors which may determine whether exercise induced free radical damage occurs, including degree of conditioning of the athlete, intensity of exercise, and diet. Antioxidant supplements prevent exercise induced damage or enhance recovery from exercise:Although it is well known that vitamin deficiencies can create difficulties in training and recovery, the role of antioxidant supplementation in a well nourished athlete is controversial. The experimental studies are often conflicting and conclusions are difficult to reach. Nevertheless, most of the data suggest that increased intake of vitamin E is protective against exercise induced oxidative damage. It is hypothesized that vitamin E is also involved in the recovery process following exercise. Currently, the amount of vitamin E needed to produce these effects is unknown. The diet may supply enough vitamin E in most athletes, but some may require supplementation. There is no firm data to support the use of increased amounts of the other antioxidants.

2.4.2. PERFORMANCE
In general, antioxidant supplements have not been shown to be useful as performance enhancers. The one exception to this is vitamin E which has been shown to be useful in athletes exercising at high altitudes. A placebo controlled study done on mountaineers demonstrated less free radical damage and decline in anaerobic threshold in those athletes supplemented with vitamin E. Although difficult to generalize, this finding suggests that supplementation with vitamin E might be beneficial in those triathletes who are adapting to higher elevations.

34

2.4.3. AMOUNT REQUIRED BY BODY

Although there is little doubt that antioxidants are a necessary component for good health, no one knows if supplements should be taken and, if so, how much. Antioxidants supplements were once thought to be harmless but increasingly we are becoming aware of interactions and potential toxicity. It is interesting to note that, in the normal concentrations found in the body, vitamin C and beta-carotene are antioxidants; but at higher concentrations they are pro-oxidants and, thus, harmful. Also, very little is known about the long term consequences of mega doses of antioxidants. The body's finely tuned mechanisms are carefully balanced to withstand a variety of insults. Taking chemicals without a complete understanding of all of their effects may disrupt this balance.

35

3.1. WINE SAMPLE COLLECTION:Different samples of wine are purchased from different wine shops and restuarent.12 wine samples were collected- 4 of red wine, 4 of port wine and 4 of white wine. All 12 samples are stored in air tight centrifuge tubes.

3.2. CHEMICAL AND REAGENTS:Most of the chemicals used in this investigation were of analytical grades. They were obtained from Qualigens, Mumbai; Sigma, Mumbai and Hi-media, Mumbai. They were DPPH, dichromate reagents, potassium iodide, BSA, ascorbic acid, TBA reagents, HPLC grade water etc according to different biochemical test.

3.3. OTHER IMPORTANT MATERIALS:Autoclaved flask and beaker, distilled water, pH meter, measuring cylinder, pipettes, momo pan balance, forceps, blade, gloves.

3.4. BIOCHEMICAL STUDIES ON WINE:3.4.1. pH ESTIMATION OF THE INDIAN WINES

pH of the wine samples was measured by using Toshniwal pH meter.

3.4.2. ALCOHOL ESTIMATIONA. REAGENTS AND STANDARDS:

1% starch, 1.96gm of potassium dichromate, 450ml of sulphuric acid, 0.1N sodium thiosulphate, ethanol were taken.
B. PROTOCOL :

The estimation of total alcohol content is being measured by dichromate method. For estimation of alcohol content we need 1% starch, sodium thiosulphate (0.1N NaOH),

36

25% KI in distilled water and K2Cr2O7. For making this we add 1.96gm of K2Cr2O7 add 550ml of water and add 450ml of conc. H2SO4. Ethanol is taken as standard alcohol. (A) Take 3.8 ml (3gm absolute alcohol) in flask make volume upto 100 ml with distilled water. (B) Mix thoroughly and remove 4 ml and add 96 ml water and make volume upto 100 ml. 1.2mg/ml stock solution is being taken. Six flasks were taken and in each flask different amount of std. alcohol is poured which range in between 1ml to 3.5ml and add distilled water to make the volume upto 3.5 ml. After that 10 ml of dichromate is added to each flask and kept at room temperature for 30 min. after that 100ml of distilled water is added then 4ml of KI is added. Then few drops of starch are added as an indicator. Titration is carried out and result is being noted.

3.4.3. ESTIMATION OF REDUCING SUGAR BY DNSA METHODA. REAGENTS:-

Crystalline phenol, 50mg sodium sulphite, 100ml 1%NaOH, 40% Rochelle salt solution (Potassium sodium tartarate).
B. PROTOCOL:-

DNSA is prepared. Maltose is taken as standard. 0.5 mg/ml of maltose is taken and stock solution of 100ml is prepared. From this 50mg/100 ml i.e. 0.05gm/100ml is taken. Distilled water is taken as blank. Different concentration of standard is taken which range between 0.2 ml to 1ml and after that distilled water is added to make the volume upto 2ml. whereas in case of wine sample 0.5 ml of sample is taken and 1.5ml of d/w is added to make the total volume upto 2ml. After that 2 ml of DNSA is added and kept for incubation for 5 min in water bath. After that 10ml of distilled water is added and then reading is taken at 520nm by spectrophotometer.

37

3.4.4. ANTIOXIDANT ACTIVITY BY DPPH ASSAY A. REAGENTS:DPPH, methanol, ascorbic acid

B. PROTOCOL:-

The principle for the reduction of 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical is that, the antioxidant reacts with stable free radical, DPPH and converts it to 1,1-diphenyl2-picryl hydrazine. DPPH has an absorption maximum at 517 nm, which disappears on reduction by an antioxidant compound. The calibration curve was plotted with % DPPH scavenged versus concentration of standard oxidants (Sanchez-Moreno et al, .1999). The 0.1 mmoles/liter solution of DPPH radical in methanol was prepared and 2 ml of this solution was added to 2 ml of water solution containing 100l of wine samples. After 30 minutes of preparation in dark, absorbance was measured at 517nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. Different concentrations were tested using ascorbic acid as standard for calibration and expressed as mg ascorbic acid equivalents per litre of sample.

3.4.5. DETERMINATION OF TOTAL PHENOLIC COMPOUNDSA. REAGENTS:-

Tannic acid, Folin Ciocalteu

B. PROTOCOL:-

1ml of wine samples in a volumetric flask was taken and 46 ml of distilled water is added to it. 1 ml of Folin Ciocalteu was added and the content in the flask was mixed thoroughly. After 3 minute of incubation 3ml of 2g/100ml Na2CO3 was added then the mixture was allowed to stand for 2hr with intermittent shaking. The absorbance was

38

measured at 760nm using spectrophotometer. The concentration of total phenolic compounds was determined with microgram of tannic acid.

3.4.6. LIPID PEROXIDATION ASSAY BY TBARSA. REAGENTS:-

500ml K2HPO4, 500ml KH2PO4, saline, 0.25 M sucrose, 1Mm EDTA, TBA reagent, ascorbic acid, Fe2+
B. OTHER MATERIAL:-

Wistar rats, homogenizer, sterile forceps and blade, gloves. C. PROTOCOL:i) Isolation of Mitochondrial fraction from Rat Liver Wistar rats (weighing about 24020 g) were used for the preparation of mitochondria. Rat liver were excised and homogenized in 0.25Msucrose containing 1mM EDTA.The homogenate was centrifuged at 3000 g for 10 mins, to remove cell debris and the nuclear fractions. The supernatant was centrifuged at 10,000 g for 10 mins to sediment mitochondria. The mitochondrial pellet was washed thrice with 50mM KPO 4 buffer, pH 7.4, to remove sucrose. All the experiments were carried out thrice at 4C. Protein was estimated and pellets were suspended in the above buffer at the concentration of 5 mg protein/ml. ii) Exposure of rat liver mitochondria to oxidative stress Oxidative damage was induced by ascorbate- Fe2+ - system as described by (Devasagayan, T.P.A., 1986). The incubations were carried out at 37C in a water bath. After the incubation, samples were boiled with TBA reagent for 30 mins. The pink colour of thiobarbituric acid reactive substances (TBARS) formed were estimated at 532nm spectrophotometrically as malondialdehyde equivalents after accounting for appropriate

39

blanks. Malondialdehyde standard was prepared by the acid hydrolysis of tetraetoxypropane (Vinayak V et al, .2007).

3.4.7. HPLC ANALYSIS OF WINESA. REAGENTS:-

Tannic acid, ferulic acid, caffeic acid, catechol and vanillin, 25% methanol and 75% HPLC water
B. OTHER MATERIAL:-

0.22 micron filter, HPLC unit

C. PROTOCOL:Different wine samples (red, white and port wine) were used for the determination of different polyphenolics and flavonoid compounds. The samples were filtered through 0.22 micron filter. The filtered extracts were used for HPLC analysis. The standard phenolic acids and flavonoids used for HPLC analysis were tannic acid, ferulic acid, caffeic acid, catechol and vanillin. The qualitative analysis of the polyphenol contents was performed using a HPLC system which consists of a model 515 pump, a model 2487 dual wavelength absorbance detector and a manual injector. The separation of the polyphenols was conducted in a column. Flow was set to 0.75 ml/min. the two solvents used to make the gradient were as followsa) 25% methanol in 1% acetic acid. b) 75% HPLC water in 1% acetic acid

40

4.1. pH ESTIMATION OF THE INDIAN WINES

Table 4.1: pH readings SAMPLE Red 1 Red 2 Red 3 Red 4 Port 1 Port 2 Port 3 Port 4 White 1 White 2 White 3 White 4 pH readings 3.81 3.68 3.68 3.39 3.56 3.78 3.72 3.77 3.22 3.36 3.4 3.05

41

4.2. ALCOHOL ESTIMATION

ALCOHOL TOLERANCE BY DICROMATE METHOD STANDARD

Figure 4.1.: Standard graph for alcohol tolerance by dichromate method. Table 4.2.: Standard readings for alcohol tolerance by dichromate method.
X axis conc. (mg/ml) 0 0.34 0.51 0.68 0.85 1.028 Y axis differences (B E) 0 1.1 1.4 1.9 2.4 2.9

42

1.2

ALCOHOL ESTIMATION BY DICHROMATE REAGENT

Blank: 4.3 Dilution: 1:700 Table 4.3.: Readings for alcohol estimation. Samples Readings (B-D) Concentration (mg/ml) Alcohol estimation of wine (%v/v) 13.20 13.20 13.20 11.48 16.64 16.64 16.64 16.64 13.20 11.48 13.20 13.20

Red 1 Red 2 Red 3 Red 4 Port 1 Port 2 Port 3 Port 4 White 1 White 2 White 3 White 4

0.5 0.5 0.5 0.45 0.6 0.6 0.6 0.6 0.5 0.45 0.5 0.5

104.22 104.22 104.22 90.65 131.36 131.36 104.22 131.36 104.22 90.65 104.22 104.22

43

4.3. ESTIMATION OF REDUCING SUGAR BY DNSA METHOD

REDUCING SUGAR ASSAY STANDARD

Figure 4.2.: Standard graph for reducing sugar assay. Table 4.4.: Standard readings for reducing sugar assay. X-axis(mg/ml) 0 0.1 0.2 0.3 0.4 (0.2ml) (0.4ml) (0.6ml) (0.8ml) Y-axis (O.D Reading in nm) 0 0.228 0.282 0.31 0.398

44

0.5

(1.0ml)

0.48

READINGS OF REDUCING SUGAR IN WINE SAMPLES

Table 4.5.: Readings for reducing sugar assay. Samples Red 1 Red 2 Red 3 Red 4 Port 1 Port 2 Port 3 Port 4 White 1 White 2 White 3 White 4 0.D Reading at 520 nm
0.281 0.248667 0.242 0.293333 1.343333 0.738 0.254333 0.586667 0.290333 0.212333 0.23 0.271333

Reducing sugar for wine (mg/ml)

3.469 0.001732 3.069 0.002887 2.987 0.001732 3.617 0.001528 16.58 0.001155 9.11 0.003464 3.135 0.001155 7.234 0.003512 3.580 0.000577 2.617 0.000577 2.839 0 3.345 0.001155

45

4.4. ANTIOXIDANT ACTIVITY BY DPPH ASSAY

Table 4.6.: Readings for DPPH assay.

Samples Red 1 Red 2 Red 3 Red 4 Port 1 Port 2 Port 3 Port 4 White 1 White 2 White 3 White 4

Readings at 517 nm
0.177 0.18 0.093 0.047 0.087 0.071 0.113 0.088 0.193 0.111 0.162 0.17

DPPH assay (% scavenging activity)

80.22 79.88 89.60 100 90.24 92.06 87.37 90.16 78.43 87.59 81.89 81.00

46

4.5. DETERMINATION OF TOTAL PHENOLIC COMPOUNDS

STANDARD FOR TOTAL PHENOLIC COMPOUNDS
Standard: Tannic acid Stock: 10 mg in 10 ml

Figure 4.3.: Standard graph for determination of total phenolic compounds. Table 4.7.: Standard readings for determination of total phenolic compounds.
Sr.no 1 2 3 4 5 6 7 8 X - axis 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 Y - axis 0.02 0.054 0.08 0.11 0.13 0.16 0.2 0.24

47

9 10

0.09 0.1

0.26 0.25

READINGS FOR TOTAL PHENOLIC CONTENT

Dilution = 100 microliters of the sample + 990 microliters of the distilled water Table 4.8.: Readings for determination of total phenolic compounds. SAMPLES Red 1 Red 2 Red 3 Red 4 Port 1 Port 2 Port 3 Port 4 White 1 White 2 White 3 White 4 Readings at 760 nm
0.152 0.18 0.144 0.086 0.177 0.164 0.137 0.167 0.02 0.029 0.026 0.025

Total phenolic content (mg/ml)

5.6 0.00057735 6.6 0 5.3 0 3.1 0 6.5 0.0057735 6.07 0 5.07 0.0057735 6.18 0.0057735 0.74 0 1.07 0 0.96 0 0.92 0.00057735

48

4.6. LIPIDS PEROXIDATION ASSAY BY TBARS

STANDARD ASSAY
Standard : Malondialdehyde (MDA)

Figure 4.4.: Standard graph for lipid peroxidation. Table 4.9.: Standard readings for lipid peroxidation.
X axis 100 50 25 12.5 Y - axis 0.55 0.26 0.145 0.07

49

READINGS FOR TBARS

CONTROL- 1.35nmol/ml DAMAGE- 23.89nmol/ml Table 4.10.: Readings for lipid peroxidation. SAMPLES
RED 1 RED 2 PORT 1 PORT 2 WHITE 1 WHITE 3

Readings at 532 nm
0.098 0.093 0.074 0.071 0.486 0.54

Lipid peroxidation of wine (nmole/ml)

13.34 0.18 12.80 17.70 16.920.2 8.730.58 9.820.28

% avg protection
44.23333333 46.49 26.01 29.27 63.47666667 58.91666667

4.6.1. Isolation of mitochondria fraction from rat liver and protein estimation by Bradford reagent

50

Figure 4.5.: Standard graph for protein estimation.

Table 4.11.: Readings for protein estimation. X - axis 0 10 20 40 60 80 100 Y - axis 0 0.176 0.333 0.674 0.966 1.119 1.802 Reading at 595nm 10 microliter of mitochondria sample = 13.06 micrograms/10 microlitres (correspondence to 1.306 mg/ml) 50 microliter of mitochondria sample = 23.43 micrograms/10 microlitres (correspondence to 2.343 mg/ml)

51

100 microliter of mitochondria sample = 32.87 micrograms/10 microlitres (correspondence to 3.287 mg/ml)

4.7. HPLC ANALYSIS OF WINES

1) Sample Name: std-25

52

2) Sample Name: std-50

53

3) Sample Name: std-100

54

4) Sample Name: Sample- Red wine R1

55

5) Sample Name: Sample- Red wine R2

56

6) Sample Name: Sample- Red wine R3

57

7) Sample Name: Sample- Red wine R4

58

59

8) Sample Name: sample-port wine 1000

60

9) Sample Name: sample-port wine 9

61

10) Sample Name: sample-port wine 99

62

11) Sample Name: sample-port wine 4

63

12) Sample Name: Sample- White wine W1

64

13) Sample Name: Sample- White wine W2

65

66

14) Sample Name: sample- White wine W3

67

15) Sample Name: sample- White wine W4

68

4.7.1. HPLC ANALYSIS

Table 4.12.: Concentration of tannic acid for the samples. SAMPLES Concentration of Tannic acid (ppm)
25.000 50.000 100.000 56.544 72.099 64.438 36.430 46.038 83.852 73.659 65.403 48.013 47.137 46.990 54.294

STANDARD 25 STANDARD 50 STANDARD 100 RED 1 RED 2 RED 3 RED 4 PORT 1 PORT 2 PORT 3 PORT 4 WHITE 1 WHITE 2 WHITE 3 WHITE 4

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Table 4.13. Combined data for wines

Types of wine pH of Wine Reducing sugar for wine (mg/ml) Alcohol estimation of wine (%v/v) Total phenolic content (mg/ml) DPPH assay (% scavenging activity) Lipid peroxidation of wine (nmole/ml) Lipid peroxidatio n of wine (% avg protection) Conc. of tannic acid(ppm)

Port 1

3.56

16.58 0.0011 9.11 0.0034 64 3.135 0.0011 7.234 0.0035 3.469 0.0017 3.069 0.0028 2.987 0.0017 3.617 0.0015 3.580 0.0005

16.64

6.5 0.0057735 6.07 0

90.24

17.70

26.01

46.038

Port 2

3.78

16.64

92.06

16.920. 2

29.27

83.852

Port 3

3.72

16.64

5.07 0.0057735 6.18 0.0057735 5.6 0.00057735 6.6 0

87.37

73.659

Port 4

3.77

16.64

90.16

65.403

Red 1

3.81

13.20

80.22

13.34 0.18 12.80

44.23333

56.544

Red 2

3.68

13.20

79.88

46.49

72.099

Red 3

3.68

13.20

5.3 0

89.60

64.438

Red 4

3.39

11.48

3.1 0

100

36.430

White 1

3.22

13.20

0.74 0

78.43

8.730.5 8

63.47666 67

48.013

70

White 2 White 3 White 4

3.36

2.617 0.0005 2.839 0 3.345 0.0011

11.48

1.07 0

87.59

47.137

3.4

13.20

0.96 0

81.89

9.820.2 8

58.91666 67

46.990

3.05

13.20

0.92 0.00057735

81.00

54.294

71

It is being reported that there is increase in prevalence of coronary heart disease (CHD) in India and other developing countries, with a gradual increase in the number of patients with acute myocardial infarction (Krishnaswami, S., 1998). It has been shown that people of Indo-origin may be more prone to CHD due to metabolic syndrome comprising of resistance to insulinmediated glucose uptake, serum triglycerides, low levels of HDL cholesterol etc. (Bhatnagar, D.1998). Various antioxidants may prevent and/or improve different diseased states. Natural product especially derived from dietary components such as fruits and vegetables yield rich divided in terms of potential benefits in controlling diseases. Polyphenolic antioxidants such as flavonoids occur naturally in vegetables, fruits and beverages such as tea and wine. Their intake was significantly and inversely associated with mortality from CHD and also showed an inversely associated with mortality from CHD and also showed an inverse correlation with incidence of myocardial infarction (Hertog, M.G.L et al., 1993). Consumption of wine also can help. Hence we have estimated antioxidant abilities of Indian wine varieties. Biochemical test of wine is successfully carried out by using different sample of red, white, and port wine. Wine sample were collected from different wineries shop. The pH of Wine sample is carried out and from the study it is being clear that white wine is more acidic in comparison of red and port wine. Its acidity lies below 3.4 to 3.05 where as average reading of red wine is 3.65 and average reading of port wine is 3.7. According to this result port wine is much better after that red wine followed by white wine. There is slight difference between red and port wine. After doing alcohol estimation, we know that port wine consist of more alcohol content in comparison of white and red wine. Red wine consists of less alcohol in comparison of other wines. According to the result red wine is good for drinking. Reducing sugar for wine is being carried out taking maltose as standard. Reducing sugar was found more in comparison of red and white wine. For measuring antioxidant activity, we have used different methods such as radical scavenging by using DPPH and inhibition of lipid peroxidation by measuring TBARS in rat liver mitochondria. The model of scavenging the stable DPPH radical is a widely used method to

72

evaluate the free radical scavenging ability of various sample (Lee et al., 2003). DPPH is a stable nitrogen-centered free radical, the colour of which changes from violet to yellow upon reduction by either the process of hydrogen- or electron- donation. Substances which are able to perform this reaction can be considered as antioxidants and therefore radical scavengers (Brand-Williams et al., 1995). It was found that the radical scavenging activity of the extracts increased with increasing concentration. DPPH activity was found maximum in red wine. The high total phenol and flavonoid contents of wine may lead to its good DPPH-scavenging activity. Inhibitory effects of the six samples of wine were carried against lipid peroxidation induced by ascorbate-Fe2+ in rat liver mitochondria. In this we found that white wine show greater percent of protein protection followed by red and port wine. The content of phenolic compounds was determined by the Folin-Ciocalteu for the different wine samples (V.Katalinic et al., 2003). The result showed that the increase of phenolic content is higher in port wine, than in red wine and very less in white wine (range between-6.5 to 5.07, 6.6 to 3.1 and 1.07 to 0.74 respectively). The Comparative studies were made considering tannic acid as standard. As expected, the red wines had significantly higher amounts of total phenolic content. All the 12 varieties of wines were analyzed for total phenol and total flavonoid contents and some select varieties by HPLC to identify different phenolics and flavonoids such as tannic acid, cathachol, vanillin, caffic and ferulic acid. It was found that port wine has greater amount of tannic acid in comparison of red and white wine. From all the above data it is clear that red wine is better, followed by port and white wine for health and also for antioxidant activity.

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In conclusion, our studies show that among the different varieties of wines examined red wine have the most potent antioxidant activities and after that port wine followed by white wine. Their antioxidant activities, assayed at different levels correlate with their chemical composition in terms of total phenolics and flavonoids. These varieties of wine, if consumed in adequate amounts, may confer health benefits, especially in population prone to CVD. Wines also help from cancer and many heart disease when it is taken in moderate amount. Plato may have been wiser than he knew when he said, "Nothing more excellent or valuable than wine was ever granted by the Gods to man." Those of us who have come to enjoy the variety and tastes that wine have to offer can now look to red wines for greater health benefits. Qualitatively there is little difference between port, red and white wine. In many cases it is being observed that red wine and port wine are similar. The difference between red and white wines is that red wines contain anthocyanins, the pigments molecules, large amounts of catechins and phenolic content. Red wine has a high concentration of resveratrol because the skins and seeds ferment in the grapes' juices during the red wine-making process. This prolonged contact during fermentation produces significant levels of resveratrol in the finished red wine.

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