RESVERATROL AND PROCYANIDIN CONTENT IN SELECT MISSOURI RED WINES

A Thesis presented to the Faculty of the Graduate School

at the University of Missouri

In Partial Fulfillment of the Requirements for the Degree

Master of Science

By LAURA ORTINAU

Dr. Ingolf Gruen, Thesis Supervisor

DECEMBER 2009

The undersigned, appointed by the dean of the Graduate School, have examined the thesis entitled

RESVERATROL AND PROCYANIDIN CONTENT IN MISSOURI RED WINES

presented by Laura Ortinau, a candidate for the degree of Master of Science, and hereby certify that in their opinion it is worthy of acceptance.

Ingolf Gruen, Ph.D., Department of Food Science

Keith Striegler, Ph.D., Department of Food Science

Mark Ellersieck, Ph.D., Experiment Station Statistics

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ACKNOWLEDGEMENTS

I would like to take this opportunity to thank my advisor Dr. Gruen for helping

me work through the complications of this project (the HPLC breaking twice), and also

my committee members Dr. Ellersieck and Dr. Striegler. Each added their expertise to

my project, which was greatly appreciated. Also I would like to give a shout out to

JoAnn for answering all my questions on editing and formatting. Well and I suppose I

could thank the parentals as well for creating me.

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TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………………

LIST OF FIGURES…………………………………………………………………

LIST OF TABLES ………………………………………………………………….

ABSTRACT ………………………………………………………………………..

Chapter

1. INTRODUCTION ………………………………………………………….

2. LITERATURE REVIEW …………………………………………………. 2.1 Wine …………………………………………………………………

2.1.1 Wine Production and Consumption ………………………... 2.1.2 Missouri Wine History …………………………………….. 2.1.3 Red Wine Production ………………………………………. 2.1.4 Norton Grape ………………………………………………. 2.1.5 Health Benefits of Alcohol …………………………………

2.1.5.1 Blood Pressure ……………………………………. 2.1.5.2 Platelet Aggregation ……………………………… 2.1.5.3 Quantity of Alcohol Consumed ………………….. 2.1.5.4 Type of Alcohol Consumed ……………………….

2.1.6 Health Benefits of Wine …………………………………... 2.1.6.1 Anti-Cancer ………………………………………. 2.1.6.2 Oxidative Stress …………………………………... 2.1.6.3 Nitric Oxide ……………………………………….

2.2 Resveratrol …………………………………………………………. 2.2.1 Structure and Formation …………………………………... 2.2.2 Chemopreventative Action ………………………………..

2.2.2.1 Hormone Signaling ………………………………. 2.2.2.2 Cell Surface Structure ……………………………. 2.2.2.3 Cell Cycle and Apoptosis ………………………… 2.2.2.4 Carcinogens ……………………………………….

2.2.3 Oxidative Stress …………………………………………… 2.2.4 Nitric Oxide ………………………………………………. 2.2.5 Platelet Aggregation ………………………………………. 2.2.6 Functional Dose …………………………………………… 2.2.7 Absorption and Bioavailability ……………………………. 2.2.8 Reported Levels ……………………………………………

2.3 Procyanidin …………………………………………………………. 2.3.1 Structure and Formation …………………………………... 2.3.2 Chemoprevention …………………………………………. 2.3.3 Reactive Oxygen Species …………………………………..

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3 3 3 4 5 7 8 9 9

10 11 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 19 20 24 24 25 26

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2.3.4 Heart Health ……………………………………………….. 2.3.5 Functional Dose …………………………………………… 2.3.6 Absorption and Bioavailability ……………………………. 2.3.7 Reported Levels ……………………………………………

3. MATERIALS AND METHODS ………………………………………….

3.1 Materials ……………………………………………………………. 3.1.1 Chemicals …………………………………………………. 3.1.2 Wine Samples ……………………………………………...

3.2 Resveratrol Method ………………………………………………… 3.2.1 Sample Preparation ……………………………………….. 3.2.2 HPLC Conditions …………………………………………. 3.2.3 Validation of Resveratrol Method …………………………

3.3 Flavon-3-ol Method ………………………………………………… 3.3.1 Sample Preparation ……………………………………….. 3.3.2 HPLC Conditions …………………………………………. 3.3.3. Validation of Method ………………………………………. 3.3.4 Statistical Analysis …………………………………………

4. RESULTS …………………………………………………………………. 4.1 Resveratrol Results …………………………………………………. 4.2 Procyanidin Results ………………………………………………… 4.3 Variation Between Varietals and Vintages …………………………. 4.4 Variation Between Location and Compound ………………………. 4.5 Relationship Between Compounds ………………………………….

5. DISCUSSION ……………………………………………………………..

APPENDIX …………………………………………………………………………

A-1 Resveratrol Standard Curve ………………………………………………. A-2 Procyanidin B1 Standard Curve and Points ………………………………. A-3 Catechin Standard Curve and Points …………………………………....... A-4 Procyanidin B2 Standard Curve and Points ………………………………. A-5 Epicatechin Standard Curve and Points ………………………………….. A-6 Coefficient of Variance of Resveratrol Method ………………………....... A-7 Coefficient of Variance of HPLC for Resveratrol Method ………………. A-8 Coefficient of Variance of HPLC for Procyanidin Method ………………. A-9 Coefficient of Variance of Procyanidin Method ………………………….. A-10 Catechin Results ……………………………………………………….... A-11 Epicatechin Results …………………………………………………….... A-12 Procyanidin B1 Results …………………………………………………. A-13 Procyanidin B2 Results …………………………………………………. A-14 Resveratrol Results …………………………………………………….... A-15 pH Results ……………………………………………………………….. A-16 SAS Results ……………………………………………………………...

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32 32 32 32 34 34 34 35 35 35 35 36 37

38 38 42 49 50 52

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55 56 57 58 59 60 61 61 62 62 63 65 67 69 71 73 74

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REFERENCES ………………………………………………………………… 111

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LIST OF FIGURES

Figure

Page

2.2.1 2.3.1 4.1 4.2

Chemical Structure of trans-resveratrol (left) and cis-resveratrol (right) …………………………………………………………………. Procyanidin A and B Structures ………………………………………… Resveratrol Chromatograph of Augusta Norton 2005 Bottle3 …………. Procyanidin Chromatograph of Mount Pleasant Norton 2005 Bottle 3…………………………………………………………………

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LIST OF TABLES

Table

Page

2.1.1 2.2.8.1 3.1.2 3.3.2 4.1 4.2.1 4.2.2 4.4.1 4.4.2 4.4.3 4.4.4 4.4.5

United States Top Ten Wine Producing States by Amount and Percentage of Production in 2006 ……………………………………… Reported Levels of Resveratrol After Strevbo and others 2007 ………. Winery Name, Wine, and Vintage of Wine Sample …………………… Gradient System for Procyanidin HPLC Solvents ……………………. Resveratrol and pH Results …………………………………………… Catechin and Epicatechin Results ……………………………………... Procyanidin B1 and B2 Results ……………………………………….. Epicatechin Results by Location ………………………………………. Catechin Results by Location …………………………………………. Procyanidin B1 Results by Location …………………………………. Procyanidin B2 Results by Location …………………………………. Resveratrol Results by Location ………………………………………..

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26-27

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RESVERATROL AND PROCYANIDIN CONTENT IN SELECT MISSOURI RED

WINES

Laura Ortinau

Dr. Ingolf Gruen, Thesis Supervisor

ABSTRACT

Many health benefits have been attributed to procyanidins and resveratrol,

including increased nitric oxide (NO) production, decreased platelet aggregation, and

chemopreventative actions. These polyphenolic molecules are predominantly found in

the skins of grapes and are present in higher quantities in red wines than white wines.

These polyphenols levels have been reported for wines around the world. However, the

Norton grape is one of the predominant grapes grown in Missouri, and there is limited

information on its polyphenolic makeup. This study analyzed wines produced in

Missouri, predominantly Norton, and determined the resveratrol and procyanidin

contents. Resveratrol ranged from 0.07 mg/L to 1.52 mg/L. Procyanidins were of

higher ranges consisting of 0.17 mg/L to 20.79 mg/L catechin, 0.22 mg/L to 29.48 mg/L

epicatechin, ND to 52.16 mg/L procyanidin B1, and ND to 23.95 mg/L procyanidin B2.

There was a significant correlation between the overall resveratrol content and

procyanidin B1, as well as between catechin and procyanidin B1 and B2 (p<0.0001).

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CHAPTER 1

INTRODUCTION

Heart Disease is the leading cause of death in America with 631,636 deaths in

2006, and cancer was a close second with 559,888 deaths in 2006 (Control, 2009). Since

the 1990’s alcohol, specifically wine, has become of great interest because of the “French

Paradox”. The French Paradox is the relationship seen in France of a high saturated fat

diet and a low incidence in coronary heart disease (CHD) (Renaud and De Lorgeril

1992). Resveratrol was originally thought to be the polyphenolic compound mainly

responsible for the cardioprotective effects of red wine consumption. However,

resveratrol concentrations are so low in wines that in order to obtain a functional level

within the body one would have to consume large quantities of red wine. In 2007 Corder

claimed that the health benefits associated with moderate red wine consumption were

from the procyanidin compounds in wine (Marian 2007). Resveratrol and procyanidin

levels have been reported for European countries, Australia, Canada, and the United

States of America. However, only limited reports have been published on American

wines, mainly for those from California.

The objectives of this study were to analyze the resveratrol and procyanidin

content of red wines in Missouri, specifically those of the Norton variety. Since there are

no reported values for this variety, we compared the quantities of resveratrol and

procyanidin to that of wines from other countries. Also, resveratrol and procyanidin

levels were related to the different geographic locations of the wineries in Missouri.

Lastly, each compound was correlated to each other to see if there was a significant

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relationship between the quantity of resveratrol and the procyanidins as well as a

relationship between the quantities of procyanidins themselves.

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CHAPTER 2

LITERATURE REVIEW

2.1 Wine

2.1.1 Wine Production and Consumption

In 2005, 2.37 billion liters of wine were consumed in the world. The USA wine

industry reached a retail value of $30 billion and produced 2.85 billion liters of wine in

2007. The United States wine market has increased in recent years. Table wines

consisted of 2.46 billion liters, dessert wines consisted of 234.7 million liters, and

sparkling wines consisted of 124.9 million liters. Imported table and sparkling wines

accounted for more than 25% of the United States consumption. The top five countries

that the United States imports wine from are France (31%), Italy (28%), Australia (17%),

Spain (5.8%), and Chile (4.5%). From 2002 to 2007 the United States exports grew 56%

in volume and 66% in value. The countries that imported the most wine from the United

States in 2007 include the United Kingdom, Canada, Japan, Italy, and Germany. Of all

the wines sold in the United States, the Department of Commerce estimates that 61% of

wine sold is from California and 26% is from imports. This leaves only 13% from all the

other US states. California wineries produced over 2.18 billion liters of wine in 2006,

which accounted for 89.25% of the total US production. In comparison, Missouri

produced 3.38 million liters in 2006, ranking it below the top 10 producer states

(Norton/Cynthiana 2005; Agriculture 2007; Hodgen 2008; Vitis 2009).

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Table 2.1.1 United States Top Ten Wine Producing States by Amount and

Percentage of Production in 2006 State Amount (L) Percent of U.S. Production California 2.18 billion 89.25 New York 106.8 million 4.37 Washington 75.9 million 3.11 Oregon 15.6 million 0.64 Florida 6.6 million 0.27 New Jersey 6.3 million 0.26 Kentucky 4.7 million 0.19 Ohio 4.2 million 0.18 Virginia 3.7 million 0.15 North Carolina 3.5 million 0.14

Table information from (Hodgen, 2008)

2.1.2 Missouri Wine History

In 1837 German settlers founded the town of Hermann, MO. Hermann was an

ideal place to cultivate grapes because the growing conditions were similar to those in

Germany. Before the Prohibition, Missouri produced over 2 million gallons of wine per

year. This ranked Missouri second in the nation for production of wine. However,

during the prohibition all of the Missouri wineries closed with the exception of the St.

Stanislaus Novitiate in St. Louis, which was allowed to continue limited production of

wine for sacramental purposes. Then in 1965, Stone Hill Winery reopened and started a

trend for other wineries to reopen as well. In 1980 Augusta, Missouri became the first

wine appellation in the United States. As of 2005 Missouri was ranked 11th in grape

production, producing approximately 2.65 million liters (702,000 gallons) wine, of which

the St. James Winery produced approximately a third of the state’s wine sales with Stone

Hill Winery coming in second. Missouri’s production increased in 2007 to 3.38 million

liters (894,391 gallons) of wine and Missouri is still currently growing as a wine

producing state. In 2002 there were 31 wineries in Missouri whereas in 2007 there were

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72. Currently the price per ton of grapes in Missouri is increasing in value, and the

tonnage per acres is decreasing, which normally indicates an increased quality of the

fruit. Some of the premier growing regions of Missouri include Augusta, Hermann,

Ozark Highlands, and the Ozark Mountains. These regions often contain optimal

growing conditions for vines, with south facing slopes, and protection from the cold

winters. Some of the varietals that have been successfully grown within these regions

include Norton (Cynthiana), Chardonel, Concord, Vignoles, Catawaba, Vidal Blanc, and

Chambourcin. The Norton grape accounts for 20% of the grapes grown in Missouri.

Many of the varietals grown in Missouri are native to America, or are hybrids of French

vines, because Missouri’s cold winters and relatively humid climate during the summer

will not support the growth of the Noble Grape varietals. Due to these climate

conditions, many of the vines face multiple challenges such as mold, fungi, mildew,

insects, and wildlife (Management 2000; Norton/Cynthiana 2005; Hodgen 2008; Vitis

2009).

2.1.3 Red Wine Production

Approximately 4,000 varieties of Vitis vinifera are used in wine production. There

are different styles of creating wine that can be specific to the type of grape; this is often

how the wine making process is selected. Steps to general red wine production include:

1. Harvesting – can either be done by hand (slow process, but harvesters can

determine if all the berries are ripe and it doesn’t damage the grapes) or by

machine (this is a much faster process, but the berries can be damaged, and all

grapes are harvested, the good and bad)

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2. Crushing – this is done normally within 24 hours of harvesting. The grapes are

usually destemmed because of unwanted flavor compounds (but stems can add

astringency back to the wine if desired because of their high tannin content). The

grapes are crushed between rollers to the desire of the wine maker.

3. Fermentation – the major choices start here by choosing natural vs. inoculated

yeasts, or open vs. closed tanks,

4. Maceration - punch down cap vs. pump-over cap vs. rotary fermenters

5. Pressing – this is done once the free wine has been removed from the pomace. 2

days to 3 weeks after fermentation.

6. Malo-lacto fermention - changes malic acid into lactic acid lowering the

harshness of the acid, which is done in oak barrels or vats.

7. Maturation – can occur from 3 months to 3 years depending on the wine. Can be

done in vats or barrels. Barrels allow for oxidative reactions and wood

extractives.

8. Clarification – removing any sediment from the wine prior to bottling (last

minute changes)

9. Aging – is done in the bottle, it allows for the formation of different flavors to

develop

These steps depend on the wine, winery, and winemaker’s preferences on how to make

the best product from their grapes (Soleas and others 1997; Hornsey 2007). Red wine

processing is uniquely different from that of white wine production. These differences

lead to more phenolic compounds in red wine due to the increased contact time of skins

with the juice.

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2.1.4 Norton Grape

The Norton grape is of the family and genus Vitis aestivalis Michx (Tarara 1991).

It was found in 1835 near Richmond, Virginia. In 1873, the Monticello Wine Company’s

(Charlottesville) Norton Claret won the gold medal in Vienna, and then in Paris the silver

medal in 1878, showing that the American wines could compete at the same level as the

European varieties (Adams 1973). Norton is the oldest American grape variety that is

commercially grown today. It is commercially produced in Arkansas, Illinois, Indiana,

Kansas, Louisiana, Maryland, Missouri, Oklahoma, New Jersey, Pennsylvania,

Tennessee, Texas, Virginia, and West Virginia. The vines are relatively cold hardy and

disease resistant. Because of Missouri’s warm and humid summers, vines are susceptible

to many diseases, insects and molds. Norton is extremely vigorous and needs a divided

canopy training system. The preferred growing conditions of this plant are well drained

soils in full sun, medium moisture, and preferably a south facing slope. Norton is the

latest ripening grape in Missouri. The vines produce medium clusters with blue-black

berries. These grapes produce a very dark colored dry wine that is medium bodied with

fruity overtones. These grapes should be used to produce young style wines, and not be

aged much longer than a year because of their high pH and high titratable acidity.

Norton’s high pH and titratable acidity is from the presence of weak acids (tartaric and

malic acid) that are present in their undissociated forms. Differences in pH can be

attributed to soil type, rootstock, vine vigor, leaf shading, cultivar crop level, and

seasonal variations. ( Tarara 1991; Walker and others 2002; Norton/Cynthiana 2005;

Viti, 2009)

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2.1.5 Health Benefits of Alcohol

Light to moderate drinkers have an inverse protective relationship between

alcohol consumption and coronary heart disease (CHD) (Suh and others 1992; Klatsky

and others 2005; Mukamal and others 2005). After taking confounding variables into

account, the multivariate relative risk of alcohol was inversely associated with CHD

(p=0.0005), and adding the individuals with medical conditions that were possible

precursors to coronary artery disease to the data still produced an inverse association

between alcohol consumption and coronary artery disease incidence (p=0.02) (Rimm and

others 1991). After successful coronary stenting, the C reactive protein (CRP) and

inflammation are indicators that identify groups of individuals with an increased risk of

ischemic complications later. Patients with a high plasma CRP ( ≥68 mg/dl) and who

consumed one to two alcoholic beverages a day (moderate drinking) had a significantly

lower incidence of non-fatal myocardial infarction (MI), cardiac death, and unstable

angina (p<0.001) (Zairis and others 2003). The first study to analyze a dose-response

model of drinking patterns in males and females in relation to myocardial infarctions was

just published in 2009. Taking confounding variables into account, as alcohol

consumption increased, the risk for MI also increased. Whereas, increasing the

frequency of alcohol consumption decreased the increased risk of CHD. According to

dosages, men were at a lower MI risk if consumption was less than 4.55 drinks per

drinking day and less than 3.08 drinks per drinking day for women (Russell and others

2009). Health effects that have been attributed to alcohol include reduction of

atherosclerosis, platelet aggregation, inflammation, and increased blood pressure, all of

which are symptoms associated with coronary heart disease (CHD).

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2.1.5.1 Blood Pressure

The normal blood pressure range of a healthy adult is 120/80 mmHg, and

hypertension is classified as 149/90 mmHg. An elevated blood pressure is a known risk

factor for CHD. The relative risk of death from CHD ranges from 1.09-1.25 for every 10

mmHg increase, and for every 5 mmHg diastolic blood pressure the relative risk range is

1.06-1.19. Hypertension was a significant risk factor for death in all of the populations

studied (Van Den Hoogen and others 2000). Another study included covariates such as

age, body mass index (BMI), mean corpuscular volume, γ-Glutamyl transferase, systolic

blood pressure, diastolic blood pressure, serum cholesterol, glucose, smoking, and

sedentary. Individuals at the highest risk of death were those with a systolic blood

pressure of 158 mmHg, and only moderate drinking (<60g alcohol/day and no beer)

lowered that risk of all-cause mortality (Renaud and others 2004).

2.1.5.2 Platelet Aggregation

Platelet aggregation can be induced by adenosine-diphosphate (ADP), collagen,

epinephrine, and thrombin, the aggregation of all has been reported to be inhibited by

resveratrol (Renaud and Ruf 1996). Inhibition of platelet aggregation is thought to be

through the thromboxane A2 pathway including some of its metabolites, such as

thromboxane B2 (Rubin 1999). Resveratrol inhibited platelet aggregation significantly in

vitro and in vivo in a dose dependent manner, and in some cases it reduced the particle

size of aggregates (Renaud and others 1992; Pace-Asciak and others 1996; de Lange and

others 2004). Further studies on quantity of consumption showed acute alcohol

consumption by binge drinkers caused an initial decrease in platelet aggregation, but after

hours of no consumption of alcohol these individuals were susceptible to the rebound

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effect. This happens after large quantities of alcohol have been consumed, which induces

an initial reduction in platelet aggregation, but then hours or even weeks later platelet

aggregation is increased after alcohol is no longer present and does not provide protective

action any longer (Renaud and Ruf 1996). Thus, acute alcohol consumption significantly

increased platelet aggregation over time, which could be the cause of the increased rate of

mortality associated with binge drinking (de Lange and others 2004). Chronic

consumption of red and white wine, however, decreased platelet aggregation, and

thromboxane B2 (Pace-Asciak and others 1996).

2.1.5.3 Quantity of Alcohol Consumed

Suh and others (1992) showed that after seven drinks each additional drink

consumed increased the relative risk of death from CHD. Other studies support this by

showing that heart failure not associated with CHD was associated with heavy alcohol

consumption, as well as an increased risk of CHD associated with individuals who have

high alcohol consumption (140 - <240 g/week) (Fushs and others 2004; Kauhanen and

others 1999). In a study by Kauhanen and others (1999) multivariate analysis showed

that not only quantity of alcohol consumed but also the time frame in which it was

consumed were associated with atherosclerosis. Foerster and others (2009) reported a J-

shape relationship of risk of coronary artery disease (CAD) and alcohol consumption,

because the protective effect of alcohol disappeared as alcohol consumption increased.

The protective effect of the increase in HDL is offset by the increase in blood pressure

(Foerster and others 2009). Many of the above studies have shown that there is an

inverse relationship between CHD, MI, CAD, and moderate consumption of alcohol, but

the type of alcohol consumed also has an important effect.

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2.1.5.4 Type of Alcohol

High progression of atherosclerosis has been shown in men who consumed >12

portions of spirits (p<0.05), and in men who consumed ≥ 6 bottles of beer (p<0.10)

(Kauhanen and others 1999). There was no relationship between the type of alcohol and

protective effect (Fushs and others 2004). Another study indicated that the type of

beverage consumed and consumption of alcohol with meals did not reveal a reduced risk

of myocardial infarction (Mukamal and others 2003). In a 1986 study, non-smokers who

consumed beer and wine had a reduced risk of coronary heart disease, over that of those

who consumed spirits (Friedman and Kimball 1986). These relationships are thought to

be from the effect of the ethanol, and possibly the antioxidant compounds within the type

of alcohol consumed.

2.1.6 Health Benefits of Wine

2.1.6.1 Anti-Cancer

Numerous studies have looked at the effect of wine on cancer. In one study, cells

were incubated for five days in varying concentrations of a cabernet sauvignon wine. A

dose-dependent effect on inhibition of prostate cancer cell proliferation was observed

(Kampa and others 2000). Another study showed that a 1:25 dilution of wine added to

oral squamous carcinoma cells was capable of significant (35.0±2.9%) inhibition of cell

growth and DNA synthesis (34.2±0.5%). The same dilution also showed a loss of tumor

nest structure and almost a complete destruction of the cells, displaying an inhibition on

cancerous cell growth (Elattar and Virji 1999).

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2.1.6.2 Oxidative Stress

The urinary excretion of isoprostanes can be used to determine the oxidative

stress on an individual. In a 2006 study, human subjects were given 300 mL/day of wine

for 15 days, specifically a red wine containing a phenolic content of 1.8 g/L and a white

wine with 0.25 g/L phenolics and another group as a control group. The oxidative stress

was decreased by 38.5±6% for the red wine group and 23.1±6% for the white wine group

as compared to the control group (Pignatelli and others 2006). Another study on

oxidative stress used doses of 40 mg/Kg red wine polyphenols for 5 weeks on rats. A

reduction in systolic blood pressure, urinary isoprotaglandins F2α, and aortic 02-

production was observed. This study showed that chronic treatment with red wine

polyphenols reduces the blood pressure and vascular dysfunction by reducing oxidative

stress in female rats (Lopez-Sepulveda and others 2008).

2.1.6.3 Nitric Oxide

Nitric oxide (NO) is a vascular relaxation promoter, which is stimulated by

guanosine 3’, 5’-cyclic monophosphate within the vascular endothelium. NO also

inhibits the platelet aggregation to the endothelium. Human umbilical vein endothelial

cells (HUVEC) were treated with an alcohol-free red wine polyphenol extract (RWPE).

This study showed that RWPE significantly increases the production of endothelial NO

synthase (eNOS) and in relation, NO production from endothelial cells in a concentration

dependent manner, demonstrating that the polyphenols in the extract could be responsible

for the cardio-protective effect (Leikert and others 2002). Different wines were

compared for their effect on NO. German wines showed a slight increase in eNOS

mRNA expression, whereas French wines had a significant dose-dependent increase in

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eNOS mRNA expression as well as NO production. The different effects of the various

types of wine can be attributed to particular compounds found within the type of

wine/grapes (Wallerath and others 2003). In a study by Freedman and others (1986)

purple grape juice used in venous blood showed that NO release from platelets increased

with the increasing amounts of purple grape juice. There was also a dose-dependent

decrease in superoxide release, a compound that is known to decrease the bioactivity of

NO. The effect of red wine polyphenol extracts and grape juice on increased NO

production implies that it is not only the alcohol in wine that has heart healthy benefits,

but also the compounds within the grapes and juice themselves that contribute to the

effect.

2.2 Resveratrol

2.2.1 Structure/Formation

Resveratrol is created using a Wittig reaction, which includes connecting 2

aromatic rings by a methylene bridge (styrene double bond) with varying hydroxyl

groups. These hydroxyl groups can be substituted with sugars, methyl, and methoxy

groups. Resveratrol is a phytoalexin because of its ability to inhibit the progression of

fungal infection (Moreno-Manas and Plexats, 1985). It is also produced in response to

infection or injury, and is produced in quantities of 50-400µg/g fresh weight of damaged

leaves (Langcake and Pryce, 1976). It is often found in the wood of the vines but is also

located in the leaves, and in small quantities in the skin of the grapes, but not the flesh

(Jeandet and others 1991). Resveratrol can be produced in grapevine leaf discs at

wavelengths of 260-270 nm. Resveratrol would not be produced in sunlight outside

because the wavelength range of sunlight starts at 300 to 310 nm. When leaf disc were

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exposed to UV-irradiation the peak concentration of resveratrol was 20 minutes after the

initial exposure (Langcake and Pryce 1977).

Figure 2.2.1 Chemical Structure of trans-resveratrol (left) and cis-resveratrol (right)

(Fremont, 2000)

2.2.2 Chemopreventative Action

Chemoprevention “refers to the administration of chemical agents to prevent the

initiation and promotional events associated with carcinogenesis” (Clement and others

1998). There are several different actions in which cancer cells can be inhibited from

growth and production. Some of these include hormone signaling, cell surface structure,

suppression of anti-apoptotic gene expression, and protection from aryl hydrocarbons.

2.2.2.1 Hormone Signaling

Cyclooxygenase (COX) produces prostaglandins, which are the hormones

responsible for signaling within cells. There are two forms of COX; these include COX-

1 which is responsible for the baseline levels of prostaglandins within the body, and

COX-2 which is activated by mitogenic and inflammatory stimulation. It has been shown

that COX-2 is up-regulated in transformed cells, and it also inhibits apoptosis, which

increases the effects of the malignant cells (Subbaramaiah and others 1998). Resveratrol

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inhibited COX-2 enzyme activity and COX-2 induction by phorbol 12-myristat 13-acetat

(PMA) in a dose dependent manor (Subbaramaiah and others 1998; Lu and others 2001).

2.2.2.2 Cell Surface Structure

The filopodia and lamellipodia are actin structures involved in the protrusion of

cell surfaces during metastasis. Rho, Rac, and Cdc42 are intermediates involved in the

signaling of the actin cytoskeleton during migration. Cdc42 is implicated in filopodia

formation and Rac regulates lamellipodia (Azios and others 2001). In human

cholangiocarcinoma and glimoa cells, resveratrol had an effect on the cell membrane by

producing shrunken cells, irregular shapes, and membrane swelling (Zhang and others

2007; Roncoroni and others 2008). These cell defects contribute to the

chemopreventative action by making the cells more vulnerable to apoptosis.

2.2.2.3 Cell Cycle and Apoptosis

Cell stages include interphase (G1, S, and G2) and mitosis (M), and cells contain

genes that are pro-apoptotic and anti-apoptotic, which have an effect on cell death. Some

of the pro-apoptic genes consist of p53 and Bax, while the anti-apoptotic genes consist of

bcl-2, bcl-x, and mcl-1 genes. These genes can be either enhanced or suppressed by

resveratrol (Lu and others 2001). With the addition of resveratrol to Lewis Lung

Carcinoma cells there was a decrease in the S phase cells and an increase in proportion of

cells in the G2/M phase of the cell cycle (Kimura and Okuda 2001). Resveratrol coupled

with tumor necrosis factor-related apoptosis-inducing ligand (TRIAL) caused an

accumulation of cells in the G1 Phase, and an increase in p53 expression. Resveratrol

suppressed the anti-apoptotic gene (bcl-2, bcl-x, and mvl-1) expression of cancerous cells

and induced the expression of pro-apoptotic genes (p53 and Bax). In glioma, melanoma,

16

and cholangiocarcinoma cells, resveratrol had a concentration and time dependent effect

on apoptosis. Resveratrol had specific apoptosis effects on carcinoma cells, but has no

inhibitory effect on regular (non-transformed) cells (Tseng and others 2004; Hsieh and

others 2005; Zhang and others 2007; Roncoroni and others 2008).

2.2.2.4 Carcinogens

Benzo[a]pyrene (BaP) is present in a wide variety of environmental conditions

caused by the combustion of organic materials. BaP is one of many polycyclic aromatic

hydrocarbons (PAHs). Aryl hydrocarbon receptor (AhR) is responsible for the effects of

PAHs on human tissue. The AhR binds with a ligand, such as BaP, and forms a ligand-

AhR complex that moves to the nucleus of the cell were it binds to the DNA and

regulates gene transcription (phase 1 enzymes, cytochromes P-450 (CYP) 1A1, 1A2 and

1B1). The oxidative metabolism of ligand-AhR complex by CYP1A1(cytochrome

enzyme) produces reactive oxygen species, carcinogenic metabolites, and DNA adducts

(Revel and others 2003). Resveratrol prevented the expression of CYP1A1 in vivo and in

vitro (Casper and others 1999 Revel and others 2003) and thus may decrease the

carcinogenicity of compounds, such as BaP.

2.2.3 Oxidative Stress

Reactive oxygen species (ROS) have been implicated in the commencement of

several diseases, which include cancer, diabetes, and neurodegenerative disorders.

Hydroxyl radicals react with lipids, proteins, and DNA, which causes changes in the

structure/form of these molecules (Jang and Surh 2001). Changes in DNA can have

effects on cell proliferation, differentiation, adhesion, inflammation, immune function,

and neoplastic transformation. Resveratrol being an amphipathic molecule, can easily

17

move throughout the cell to provide protection from oxidative injury (Chanvitayapongs

and others 1997). Resveratrol was added to hydrogen peroxide induced cells and

decreased the number of ROS by 80.4% (Jang and Surh 2001). When coupled with

Vitamin E, resveratrol provided more protection than Vitamin E by itself, preventing cell

death by ROS (Chanvitayapongs and others 1997).

2.2.4 Nitric Oxide

Preconditioning rats with resveratrol induced the production of eNOS and NO,

which was responsible for the protective actions of reduced cardiac infarct size and

reduced rhythm disturbances (Hung and others 2004). In plasma, resveratrol

significantly increased NO production and vasodilator-stimulated phosphoprotein

(Gresele and others 2008). After an ischemic event, rats were given resveratrol for four

weeks, which provided cardio protective activities, such as reducing left ventricular

dilatation, left ventricular end-diastolic pressure, and infarct size (Lin and others 2008).

In a rabbit model, resveratrol protected the spinal cord from ischemic events by

decreasing oxidative stress and increasing production of NO (Kiziltepe and others 2004).

Resveratrol increased the production of eNOS and NO, and reduced the oxidative stress

in animal models before and after an ischemic event.

2.2.5 Platelet Aggregation

Platelet aggregation can be induced by thrombin, adenosine diphosphate, and

collagen (inflammation mediators). The arachidonic acid released from the platelet

plasma membrane is converted to thromboxane A2 (TXA2), which acts as a positive

feedback mediator. Surface P-selectin-positive platelets are produced by these same

stimuli. Resveratrol inhibited platelet aggregation through the TXA2 pathway as well as

18

surface P-selectin-positive platelets (Yang and others 2008a, 2008b). Resveratrol also

inhibited platelet aggregation in patients that were aspirin-resistant and were at high risk

for a cardiac event (Stef and others 2006).

2.2.6 Functional Dose

The amount of resveratrol needed to see some of the chemopreventative actions,

decreased oxidative stress, increased NO production, and platelet aggregation benefits

discussed above varied for each function. For chemopreventative actions, concentrations

from 15-50 µM of resveratrol have been used to inhibit hormone signaling throughout the

carcinoma cells (Subbaramaiah and others 1998; Lu and others 2001). Cell surface

studies indicate that 8-210 µM resveratrol disrupted the cell surface (Zhang and others

2007; Roncoroni and others 2008). Cell cycle inhibition and apoptosis have happened at

concentrations of 10-210 µM resveratrol in vivo (Elattar and Virji 1999; Roncoroni and

others 2008; Hsieh and others 2005), whereas 40mg/kg/day in vitro suppressed glioma

cell growth (Tseng and others 2004). For preventing induction of aryl hydrocarbons, 10-7

M in vivo or 50mg/kg/wk of resveratrol were needed in mice (Casper and others 1999;

Revel and others 2003). However, for resveratrol to reduce oxidative stress, resveratrol

levels needed to be 25-50 µM (Jang and Surh 2001). On a related note, in vitro

concentrations of 0.1-0.5 µmol/L of resveratrol were needed to increase NO production

and vasodilator-stimulated phosphoprotein, whereas the in vivo functional amount of

resveratrol to increase the production of NO and eNOS was 0.5-1.0 mg/kg/day

(Chanvitayapongs and others 1997; Gresele and others 2008). Resveratrol displayed a

dose-dependent inhibition on platelet aggregation in vitro using 25 – 100 µM (Yang and

others 2008a, 2008b).

19

2.2.7 Absorption and Bioavailability

Rat models have been used to determine the absorption of resveratrol into the

body and specific organs. Radio isotope labeled resveratrol was orally administered, and

50-75% of the resveratrol administered was absorbed (Soleas and others 2001).

Resveratrol is absorbed in the aglycone and glucuronide forms. Resveratrol glucuronide

consist of 95 ± 4.6% resveratrol and its glucuronide conjugate, leaving a small percentage

to be the aglycone form. It is thought that it may transform back to its original form in

the presence of β-glucuronidases, which are present in macrophages around inflammation

and cells under oxidative stress (Kuhnle and others 2000a; Marier and others 2002).

Soleas and others (2001) observed that rats given 0.5, 1.5. 2.5 mg resveratrol in 1 mL

20% ethanol had serum concentrations of 2.5, 3.6, and 5.7 µg/L, respectively, sixty

minutes after the dose. A chronic dosage of resveratrol (6.5mg/L) showed initial

concentrations of resveratrol in the plasma and liver within 30 minutes of administration,

whereas the kidney and heart contained measureable levels of resveratrol 60 minutes after

ingestion (Bertelli and others 1996a). Acute dosing of red wine with a total resveratrol

content of 28.24 µg showed that plasma and liver tissues had peak concentrations 1 hour

after ingestion and steadily declined after that. Heart and kidney tissues showed a delay

in peak concentration (Bertelli and others 1996b). In another study, bile, urine, and blood

radioactivity was taken 1.5, 3, and 6 hours after administration of 5 mg/kg radio labeled

resveratrol through gastric intubation. Initially, the blood had low concentrations, while

bile and urine had increased concentrations, with urine levels declining throughout the

experiment. Using autoradiography, the highest concentrations after 3 hours were in the

stomach, liver, kidneys, and intestines. The autoradiograph also showed that resveratrol

20

was able to penetrate the tissues of organs (Vitrac and others 2003). From the reported

information, resveratrol blood concentrations were detectable within 30 minutes after

dosing, but have a peak around 60 minutes. Liver tissues were also high in resveratrol

within 30 minutes because the liver acts as a filter after ingestion. This also correlates

with the increased plasma levels because plasma is needed in order to carry resveratrol to

other organs in the body explaining the delay in heart and kidney concentrations (Bertelli

and others 1996b) Resveratrol is entrohepatically recirculated by the small intestines

which explains the delay in measureable levels of resveratrol throughout the body

(Marier and others 2002). Urine and kidney resveratrol levels increased 60 minutes after

ingestion and then steadily decreased (Bertelli and others 1996a). Both Bertelli studies

indicate the kidney is the route of elimination of resveratrol for the body. In a human

study, resveratrol in doses of 25 mg/70 kg obtained a concentration of 10-40 nmol/L

blood. This was much lower (5-100µM/L) than in vitro reports for anti-cancer, anti-

inflamitory, and chemopreventative effects cited in the functional dose section of this

paper (Goldberg and others 2003).

2.2.8 Reported Levels of Resveratrol

A review by Stervbo (2007) reported the high, low, and average concentrations of

resveratrol in different types of wine by country as seen in Table 2.2.8.1. The average

levels of resveratrol reported for each variety were 3.6 ± 2.9 mg/L Pinot Noir, 3.2 ± 1.8

mg/L St. Laurent, 3.0 ± 2.1 mg/L Marzemino, 2.8 ± 2.6 mg/L Merlot, 2.6 ± 1.3 mg/L

Blaufränkisch, 1.9 ± 1.7 mg/L Protugieser, 1.9 ± 0.8 mg/L Grenache, 1.9 ± 1.2 mg/L

Zweigelt, 1.8 ± 0.2 mg/L Negroamaro, 1.8 ± 0.9 mg/L Shiraz, 1.7 ± 1.7 mg/L Cavernet

Sauvignon, 1.6 ± 1.1 mg/L Nero d’ Avola, 1.5 ± 0.3 mg/L Teroldego, 1.3 ± 0.7 mg/L

21

Tempranillo, 1.2 ± 0.5 mg/L Cabernet Franc, 1.0 ± 0.3 mg/L Liatiko, 1.0 ± 0.5 mg/L

Xinomauro, 0.8 ± 0.5 mg/L Muscat Bailey A, 0.7 ± 0.9 Zinfandel, and 0.6 ± 0.2 mg/L

Agiorgitiko. The standard deviations for these averages is quite high indicating that there

is a large variation between the amounts of resveratrol for each specific wine from

different regions. Some of the notably high levels of resveratrol include Pinot Noir from

Switzerland (11.9mg/L), Portugieser from Czech Republic (4.1 ± 3.0 mg/L), Merlot

from Brazil (4.0 ± 1.0 mg/L) and Spain (4.0 ± 2.9), and Cabernet Sauvignon from Italy

(4.0 ± 3.1 mg/L) (Stervbo 2007). Since the Stervbo review there has been some data

added to that information, including data from areas such as Croatia, Turkey, Italy,

Hungry, and Greece as seen in Table 2.2.8.1. Some of the notably high levels of

resveratrol were Dingač from Croatia (4.9 ± 0.3 mg/L), Merlot from Hungary (3.9 ± 4.0

mg/L), and Syrah from Greece (2.55 mg/L) (Nikfardjam and others 2006; Kallithraka and

others 2007; Rastija and others 2009). Resveratrol and other polyphenols could be used

to determine geographical regions for wines in Croatia and Sicily utilizing a cluster

analysis (Dugo and others 2006; Rastija and others 2009). Higher levels of resveratrol

were reported in cooler climates such as Canada, and areas of the warmest climates,

California, South America, and Australia had the lowest levels of resveratrol in Cabernet

Sauvignon wines. The opposite relationship was seen for Pinot Noir wines (Goldberg

and Ng 1996). Resveratrol levels for those wines produced in the United States have

comparable levels to those of the averages reported for other countries. Oregon,

California, Idaho, and Washington are the only states to have reported levels of

resveratrol at this time ( Goldberg and Ng, 1996; Gu and others 1999; Lee and Rennaker,

2007). So, with a range of 11.9 mg/L to “not detectible limits” in red wine, moderated

22

drinking at the highest average reported values with individuals consuming 2 glasses of

wine (5 fl. oz.), they would consume 3.52 mg of resveratrol. This level would only be

0.0154 µmoles, which, according to the absorption and bioavailability, would not be

sufficient to achieve high enough levels within the body to have a significant contribution

to the health benefits associated with moderate red wine consumption.

23

Table 2.2.8.1 Reported Levels of Resveratrol After the Stervbo 2007 Review

Location Wine Amount (mg/L) Reference Greece Merlot 0.86 (Kallithraka and others, 2007) Cabernet Sauvignon 0.21 Syrah 2.55 Agiorgitiko 0.4 Xinomavro 0.6 Mandilaria 1.49

Agiorgitico 1.213 (Gerogiannaki-Christopoulou and others, 2006)

Xinomvro 0.699 Kalambaki 0.488 Negosca 0.352 Mantilaria 0.1991 Kotsifali 1.331 Cabernet 0.741 Fokiano 0.985 Romeiko 1.158 Verzami 0.736 Mavrodafni 0.521 Mavro Mesenikola 0.966 Turkey Kalecikkarasi 0.0056 ±0.0014 (Gürbüz and others, 2007) Calkarasi 0.0007 ± 0.0001 Boğazkere 0.0246 ± 0.013 Öküzgözü 0.102 ± 0.033 Cabernet Sauvignon 0.0004 ± 0.0003 Cinsaut 0.001 ± 0.00003 Merlot 0.0008 ± 0.000006 Hungary Cabernet Franc 0.8 ± 0.5 (Nikfardjam and others 2006) Cabernet Sauvignon 2.8 ± 2.4 Cabernet Sau/Fr 1.2 ± 0.8 Cuvee 1.9 ± 0.4 Kadarka 0.9 ± 1.1 Kékfrankos 2.8 ± 1.7 Merlot 3.9 ± 4.0 Oportó 1.2 ± 0.7 Pinot Noir 3.2 ± 0.5 Portugieser 1.4 ± 0.9 Royal Cuvee 1.9 ± 0.9 Rubin Cuvee 3.1 ± 2.8 Shiraz 1.1 ± 0.2 Zweigelt 2.7 ± 1.7

24

Table 2.2.8.1 Continued

Location Wine Amount (mg/L) Reference Croatia Frakovka 1.9 ± 0.3 (Rastija and others 2009) Pinot crni 1.7 ± 0.3 Zweigelt 2.2 ± 0.3 Postup 0.8 ± 0.2 Plavac 3.2 ± 0.4 Dingač 4.9 ± 0.3 Merlot 1.2 ± 0.2 Borganja 0.4 ± 0.2

Cabernet Sauvignon 0.4 ± 0.1

2.3 Procyanidin

2.3.1 Structure and Formation

Procyanidins are part of a polyphenolic group called proanthocyanidins, which

are colorless compounds extracted from plants. When heat and acid is applied to

proanthocyanidins they form anthocyanidins. Flavan-3-ol molecules make up the

backbone of proanthocyanidins, which can be esterified with gallic acid.

Proanthocyanidins consisting of epiafzelechin, afzelechin, gallocatechin, and

epigallocatechin subunits are referred to as propelargonidin and prodelphinidin,

respectively. The term procyanidins refers to those compounds composed of catechin

and epicatechin. Procyanidin is the most common proanthocyanidin found in nature.

Procyanidins are linked by a C4C8 interflavon bond (B type), C4C6 interflavon

bond, or a C4C6 interflavon bond with an additional ether bond between C2 and O7 (A

type) as shown in Figure 2.3.1 (Haslam 1982).

25

Figure 2.3.1 Procyanidin A and B structure (Prior and others 2001)

Proanthocyanidins are localized in the skin of the grape and the grape seed

(Kennedy, 2008). Proanthocyanidin content is related to vine vigor. Vines with low-

vigor fruits had a significant increase in skin tannin extracted into wines, and pigment

polymers and proanthocyanidins were of higher degrees of polymerization (higher

molecular mass). However, flavan-3-ol monomer concentrations were lower and grape

seed proanthocyanidin remained the same (Cortell and others 2005). Procyanidins are

also affected by oxidative degradation within wines. While the size of procyanidins

changed very little due to oxidative deterioration, epigallocatechin subunits and seed

procyanidins showed significant degradation (Jorgensen and others 2004). Procyanidins

are responsible for color stabilization and astringency in red wines (Somers 1971; Noble

1990; Gawel 1998; Vidal and others 2004).

2.3.2 Chemoprevention

Procyanidins inhibit cancer through DNA regulation, enzymes, and cell apoptosis.

Topoisomerases are involved in the DNA processes of replication, transcription,

translation, and recombination. Topoisomerase II is an ATP-dependent enzyme that

separates both DNA strands at the same time, which allows for fragmentation or even

mutation of the DNA. Transcription factor NF-κB regulates the expression of COX-2,

which is responsible for regulation of inflammation and carcinogenesis in the promotion

26

stage. Procyanidin B2 caused a dose-dependent inhibition of NF- κB in cancerous cells

(Mackenzie and others 2008; Kang and others 2008). Grape cell culture extracts, which

included procyanidin B1, B2, and B3, significantly inhibited topoisomerase II catalytic

activity (Jo and others 2005). Another polyphenolic grape fraction containing catechins,

procyanidin dimers, and flavones inhibited catalytic topoisomerase II activity. The most

abundant polyphenols in the fraction included epicatechin gallate, myricetin, procyanidin

B2, and resveratrol (Jo and others 2006). Procyanidin B2 interaction with metal ions

and hydrogen peroxide exhibited both antioxidant and pro-oxidant properties on DNA in

human leukemia cells (Sakano and others 2005).

2.3.3 Reactive Oxygen Species

β-amyloid peptide induces oxidative stress and plays a role in cell death. An

oligomer polyphenol extract from grape seeds decreased β-amyloid peptide-induced

intracellular ROS accumulation, as well as apoptosis (Li and others 2004). A cocoa

procyanidin fraction, as well as Procyanidin B2, decreased ROS and the down-regulation

of anti-apoptotic proteins (Cho and others 2009). Cells treated with hydrogen peroxide

caused a down regulation of anti-apoptotic genes. Pretreatment with procyanidin B2

decreased cell death caused by hydrogen peroxide, as well as increased the expression of

anti-apoptotic genes (Cho and others 2008). Epicatechin, catechin, and procyanidins

inhibited oxidation of liposomes, and protected membranes from disruption by a

surfactant (Verstraeten and others 2003). Antioxidant activity of procyanidins is related

to linkage type (C4-C6 or C4-C8) as well as the increased degree of polymerization

(Verstraeten and others 2003; da Silva Porto and others 2003).

27

2.3.4 Heart Health

Thrombin induced matrix metalloproteinase (MMPs), which is responsible for

structural reorganization in vascular smooth muscle cells, plays an important role in

atherosclerosis, and may cause fissure or rupture within the vascular endothelium.

Procyanidin B2 inhibited matrix metalloproteinase (Lee and others 2008). Human

umbilical vein endothelial cells cultured with epicatechin protected vascular endothelial

cells by oxygen radical scavenging as well as by increasing the production of NO.

Epicatechin functions as an oxygen radical scavenger but does not inhibit NADPH

oxidase activity. Procyanidin and epicatechin gluconide, however, inhibited NADPH

oxidase as well as being oxygen radical scavengers (Steffen and others 2008).

Differentiated human monocytic cells treated with procyanidin B2 inhibited COX-2

expression and enzymes that regulate DNA transcription for COX-2. This significantly

reduced inflammation providing increased protection from atherosclerosis (Zhang and

others 2006).

2.3.5 Functional Dose

The amount of procyanidin needed to induce chemopreventative actions varies by

function. To inhibit Topoisomerase II activity, 5µg/mL of grape cell culture extract

containing 4.5µM procyanidin B2 was used (Jo and others 2005, 2006). To reduce the

expression of COX-2, 5µM cocoa polyphenol extract and 40 µM procyanidin B2 were

effective doses, and 25 µM procyanidin B2 reduced the induction of COX-2 by inhibiting

NF-κB (Kang and others 2008; Mackenzie and others 2008). Procyanidin B2 had

protective effects of oxidative DNA damage at 20µM but had the opposite effect at

200µM (Sakano and others 2005). However, to protect cells from ROS these amounts

28

are within a lower range than that of chemopreventative actions. Catechin, epicatechin

Procyanidin B1 through B8, and ester gallates at concentrations of 1.5 µM individually

had the capability of free radical trapping (da Silva Porto and others 2003). According to

Verstraeten and others (2008) 25 µM monomer equivalents of procyanidins protected

liposomes as well as cell membranes from oxidation. Again, procyanidin B2 at

concentrations of 1-5 µM reduced cell death and DNA damage by hydrogen peroxide

(Cho and others 2008). Procyanidin B2 and Cocao Polyphenol Fraction reduced ROS

and decreased the anti-apoptotic proteins with concentrations at 10-20 µM and 5-10 µM,

respectively (Cho and others 2009). Oligonol, a mixture of 30-50% oligomeric

compounds reduced ROS at concentrations of 1-10 µM (Li and others 2004). Lastly,

protective effects of procyanidins were seen in protection of heart tissue at concentrations

ranging from 1-50 µM procyanidins. Procyanidin B2 inhibited MMP at concentrations of

1, 3, 10, 30 µM, and it also reduced the expression of COX-2 with 0.1,1, and 10 µM.

However, 50 µM had the greatest effect on transcription factors for the COX-2 enzyme

(Zhang and others 2006; Lee and others 2008).

2.3.6 Absorption and Bioavailability

Procyanidin B2 was orally administered to rats in 50 mg/kg body weight doses.

Eighteen hours after administration, urine samples were analyzed. Procyanidin was

measureable and was 0.34% of oral dose. Epicatechin and 3’-O-methyl – epicatechin

were also present at concentrations of 0.025% and 0.027%, respectively, of the oral dose

present in the urine (Baba and others 2002). Procyanidin B1 was absorbed into the blood

of human subject 2 hours after a 2.0 g oral dose, resulting in a serum concentration of

10.6 ±2.5 nmol/L (Sano and others 2003). Procyanidins, even those with high degrees of

29

polymerization, were stable through the gastric transition of the stomach. So, all were in

their original forms when they went into the small intestine (Rios and others 2002). The

jejunum and ileum were used to study the absorption of catechin and epicatechin. In the

jejunum 13.1 % and 11.6% of catechin and epicatechin was absorbed, respectively. The

majority of the absorbed compounds was in the glucuronide and methylated forms.

Whereas absorption in the ileum was 66.2% and 55.9% for catechin and epicatechin

respectively, and the majority of the absorption was in the original unmetabolized form

(Kuhnle and others 2000b). Procyanidin A1, A2, and B2 were absorbed by rat small

intestines in very small quantities (5-10%), and epicatechin was absorbed in its

methylated form (Appeldoorn and others 2009). An in vitro study of the effect of human

colonic microflora, showed that proanthocyanidins were degraded into aromatic

compounds of a lower molecular weight after 48 hours (Deprez and others 2000).

Catechin, procyanidin B3, and C2 were individually administered in a rat diet. Urine was

collected for 24 hrs. Catechin excreted in the urine consisted of catechin and 3’-O-

methylated catechin. Procyanidin B3 and C2 however were not found in the urine, but

many of their metabolites, such as phenylvaleric, phenylpropionic, phenylacetic, and

benzoic acid derivatives, were present (Gonthier and others 2003).

2.3.7 Reported Values

The catechin, epicatechin, procyanidin B1, and procyanidin B2 content in wines

vary in concentration from different varietals of the same vintages and vice versa. One of

the most notable reports included 54 French wines from different varietals and vintages,

34 of which were red wines. The ranges for catechin, epicatechin, procyanidin B1, and

procyanidin B2 were 1.35 – 1689 mg/L, 1.0 – 100 mg/L, 0.8 – 50.8 mg/L, and 1.1 – 58.2

30

mg/L, respectively (Landrault and others 2001). Procyanidin levels have also been

reported for wines from Spain, Greece, and Turkey. Spanish wines had ranges of 17.24 –

29.53 mg/L catechin, 12.04 – 13.21 mg/L epicatechin, 0.88 – 5.75 mg/L procyanidin B1,

and 1.1 – 58.2 mg/L procyanidin B2 (Monagas and others 2003). Greek wines had

values ranging from not detectible to 144.64 mg/L for catechin, 6.15 – 150.77 mg/L for

epicatechin, not detectable to 38.07 mg/L for procyanidin B1, and not detectable to 33.88

mg/L for procyanidin B2 (Kallithraka and others 2007). Turkish red wines contained

14.505 – 104.29 mg/L of catechin, 11.33 – 55.93 mg/L of epicatechin, 16.88 – 61.3 mg/L

of procyanidin B1, and 4.66 – 37.65 mg/L of procyanidin B2 (Anli and others 2006).

More catechin and epicatechin levels have been reported from regions such as Croatia,

Hungary, Italy, Spain, Sicily, Turkey, Australia, California, France, and Oregon.

Catechin levels range from 2 – 179.29 mg/L and from 0.04 - 37.01 mg/L for epicatechin

(Goldberg and Ng 1996; Nikfardjam and others., 2006; Dugo and others 2006; Gomez-

Alonso and others 2007; Tarola and others 2007; Gürbüz and others 2007; Rastija and

others 2009). Catechin and epicatechin levels from Oregon and Californian wines range

from 33.5 – 119 mg/L and 21.4 – 41.6 mg/L, respectively with Oregon’s Pinot Noir

having the highest concentrations (Goldberg and Ng 1996). With these reported values,

if an individual consumed two 5oz glasses of wine, they would consume 42.77 mg

catechin, 44.58 mg epicatechin, 18.13 mg procyanidin B1, and 24.60 mg procyanidin B2.

The concentration needed to produce positive health benefits within the body are 0.1 – 50

µM. With the molar concentration of catechin being 0.8633 µM, epicatechin, 0.1535 µM,

procyanidin B1 0.0313 µM, and 0.0425 µM procyanidin B2, the lowest dose to obtain

health benefits would be possible with moderate consumption for catechin. However,

31

when adding the concentration of catechin, epicatechin, procyanidin B1, and procyanidin

B2 together the concentration would be high enough to produce increased NO

production, reduction in ROS, and chemopreventative actions with moderate drinking

suggesting a synergistic effect of the polyphenols found in wine.

32

CHAPTER 3

MATERIALS AND METHODS

3.1 Materials

3.1.1 Chemicals

Phosphoric acid, HPLC grade water, sodium chloride, methanol, acetonitrile,

formic acid, and ammonium phosphate were obtained from Fisher Scientific (St. Louis,

MO). The standards Catechin, Epicatechin, Procyanidin B1, Procyanidin B2, and

Resveratrol were obtained through Sigma-Aldrich (St. Louis, MO).

3.1.2 Wine Samples

Table 3.1.2 Winery Name, Wine, and Vintage of Wine Samples

Wine Samples Winery Wine 2000 2001 2002 2003 2004 2005 2006 2007 Winery 1 Norton x x x x Winery 2 Chambourcin x x Norton x x Winery 3 Chambourcin x x Norton x x Winery 4 Chambourcin x Norton x Premium Claret x x x Winery 5 Norton x x x x Winery 6 Blackberry x Chambourcin x x x Cherry x Norton x x x x Strawberry x School House Red x x Winery 7 Norton x x x x x

33

Winery 1 donated 2001 to 2004 vintages of Norton wines. All of these samples

were 100% Estate grown in the Winery 1 vineyard. The Winery 3 wine samples included

a Chambourcin 2003 and 2004 as well as a Cynthiana (another name for Norton) 2003

and 2004. The 2003 Cynthiana consisted of 90% Cynthiana and 10% Chambourcin. The

2004 Cynthiana, 2003 Chambourcin, and 2004 Chambourcin were all 100% varietal

specific. Winery 7 donated Norton vintages from 2003 to 2007. Each of the vintages

was 100% Norton with no other varieties added to them. Also, the 2006 and 2007 Norton

samples were from the barrel and had not been bottled. Winery 6 donated Norton

samples from 2000, 2002, 2003, and 2004, as well as Friendship School Red 2005 and

2006, Cherry, Strawberry, Blackberry, and Chambourcin 2003, 2004, and 2005.

According to the Winery 6, the 2002 and 2003 Norton vintages are of a 100% Norton

Grape blend, the 2004 vintage is a blend of Norton and Cabernet Sauvignon

(86.41%/13.39%), and the 2000 vintage is of an unknown blend. The Chambourcin

2003, 2004, and 2005 vintages are all made from 100% Chambourcin. The blackberry,

strawberry, and cherry wines were all from 2006, and the School House Red vintages

were 2005 and 2006. Between the 2005 and 2006 vintages, the winery switched from

using a cork to a screw cap. Winery 4’s Norton Premium Claret 2004 was made from

100 % Norton Grapes from Missouri river hills (2 different vineyards) and High Plains in

the Ozarks (2 different vineyards). The contribution of grapes from each vineyard was

21% Winery 4, 40% Native Stone, 30% McMurtry, and 9% Neobo. The 2005 Premium

Claret was made from a variety of grapes. Norton was 42% from Winery 4, a mixed

Norton from McMurtry Vineyards contributed to 38% of the final product, 11% was

Cabernet Sauvignon from Oak Grove Vineyards, 6% was Chambourcin from Drunken

34

Monkey Vineyards, and 3% was Syrah from Oak Grove Vineyards. As for the 2005

Norton, it was also 100% Norton and the contribution of grapes was from Winery 4,

McMurtry vineyards (Mountain Grove), and Gordon Vineyard (Neobo Cemetary

Columbia/Rocheport). Lastly, the 2005 Chambourcin was a 100% Chambourcin blend

from the Drunken Monkey and Sugar Branch Vineyards. Wines from Winery 5 and

Winery 2 are of unknown blends.

3.2 Resveratrol Method

3.2.1 Sample Preparation

A 1 mL sample of wine was taken from each bottle and placed in a 5 mL vial that

was covered with aluminum foil to protect the sample from UV light. The Samples were

then dried under nitrogen in a chemical fume hood at room temperature (between 25°C

and 28°C). Once dried, the solids were redissolved in the mobile phase consisting of 25%

acetonitrile in HPLC water + 0.1% H3PO4 + 5 mM NaCl. The samples were then placed

in a sonicator for 10-15 minutes. Samples were filtered using a 3 mL syringe and 0.45

µm filter (Kankakee, IL) and transferred into a 5 mL amber vial. Samples were either

analyzed that day or placed into the refrigerator until they were analyzed (less than a

week).

3.2.2 HPLC Conditions

The HPLC system consisted of a Perkin Elmer series 410 pump, a Perkin Elmer

LC 90 UV Spectrophotometric Detector, and a C18 Column 25cm x 4.6mm, 5µm

(Supelcosil 5-8298, COL 018341AP). The mobile phase was isocratic and consisted of,

as stated before: 25% acetonitrile in HPLC water + 0.1% H3PO4 + 5 mM NaCl. The flow

rate was 1 mL/min for 35 minutes with a 3 minute wash-out phase of 50%

35

methanol/water (Kolouchová-Hanzliková and others 2004). Detection was set at 306 nm,

and the data were analyzed using Star Chromatography Workstation Version 4.51 by

Varian Associates Inc.

3.2.3 Validation of Resveratrol Method

Standards to create a standard curve were at concentrations of 0.01, 0.1, 0.25, 0.5,

0.75, 1.0, 2.5, and 5.0 µL/mL. The resveratrol standard curve’s regression coefficient

was 0.999638. The coeffiecent of Variation of the HPLC system was determined by

injecting the same sample 10 times. This gave a coefficient of variation of 11.41% for

the HPLC system. As for sample preparation, ten samples were taken from the same

bottle of wine, and each were injected once into the HPLC system. The coefficient of

variation was 2.5% for the sample preparation.

3.3 Flavan-3-ol Method

3.3.1 Preparation of Sample

A 5mL sample of wine was pipetted into a glass vial. The sample was acidified

by the addition of formic acid 1.5% (v/v). The sample was then filtered into 1.5 mL

sample vials for HPLC analysis using a 3 mL syringe and a 0.45 µm filter (Gao and

others 1997; Gomez-Alonso and others 2007).

3.3.2 HPLC Conditions

The HPLC system consisted of a ProStar model 410 Auto Sampler Perkin Elmer

series 410 pump, an SSI 500 Detector variable UV/Vis, a Waters 474 scanning

fluorescence detector, and a C18 Column 25cm x 4.6mm, 5µm (Supelcosil 5-8298, COL

018341AP). The column was kept at ambient temperature throughout the analysis. The

solvent system was a gradient system consisting of three solvents based on the

36

procedures of Gao and others (1997) with slight modifications. Solvent A: 50mM

NH4H2PO4, pH=2.6; Solvent B: 20% solvent A and 80% Acetonitrile; Solvent C: 200mM

H3PO4, pH=1.5. The gradient was as shown in Table 3.3.2.

Table 3.3.2 Gradient System for Procyanidin HPLC Solvents

Flavon-3-ols Mobile Phase Gradient of the HPLC Method Step Time

(min) Flow

Rate(mL/min) % of

solvent A % of

Solvent B % of

solvent C Curve

0 5 1 100 0 0 0 1 3 1 100 0 0 0 2 12 1 92 8 0 1 3 5 1 0 14 86 1 4 7.5 1 0 18 82 1 5 25.5 1 0 21 79 1 6 15 1 0 33 97 1 7 5 1 0 50 50 1 8 3 1 0 50 50 0 9 3 1 20 80 0 1

Twenty micro liters were injected into the column. Standards were done linearly at

concentrations in the range of 1.0 mg/L to 500 mg/L. For more complete details refer to

appendix A-2 to A-5 pages 69-72. Data analysis was computed using a linear standard

curves with Galaxie version 1.9.302.530.

3.3.3 Validation of Method

The coefficient of variation of the HPLC instrument was 7.60% for catechin,

2.30% for epicatechin, 5.02% for procyanidin B1, and 1.50% for procyanidin B2. The

coefficient of variation of the overall method was determined by taking 10 samples from

the same bottle and analyzing them. The coefficent of variation was 1.57% for catechin,

4.35% for epicatechin, 15.04% for procyanidin B1, and 4.48% for procyandin B2. The

Standard Curves produced regression coefficients of 0.9893 for procyanidin B1, 0.9995

37

for catechin, 0.9911 for procyanidin B2, and 0.9994 for epicatechin. The recovery rate

was done by adding approximately ½ the known amount of either catechin, epicatechin,

procyanidin B1, or procyanidin B2 to a sample. The recovery rate, done in triplicate, of

each compound was as follows; catechin 123%, epicatechin 106%, procyanidin B1

108%, and procyanidin B2 137%. This comes to an average recovery rate of 118.5%.

The detection limit was determined by diluting the standards until no peaks were present

at a threshold of 10 and peak width of 0.4. The detection limit was 0.19 mg/L for

procyanidin B1, 0.078 mg/L for catechin, 0.16 mg/L for procyanidin B2, and 0.078 mg/L

for epicatechin.

3.4 Statistical Analysis

Three bottles were randomly selected from the Winery’s stores, and then one

sample was taken from each bottle. Statistical analysis was conducted using SAS (Cary,

NC). Tukey’s HSD, Fisher’s LSD, and Pearson’s Correlation Coefficients were used to

analyze the data. All individual statistical results can be seen in the Appendix A-16

pages 74-110.

38

CHAPTER 4

RESULTS

4.1 Resveratrol Results

Resveratrol eluted at 12.3 minutes (Figure 4.1). The results are well within the

range of other reported values for resveratrol. Some of the notably high amounts of

resveratrol were from the Winery 1 Norton 2004 (1.33 mg/L), Winery 2 Norton 2003

(1.49 mg/L), Winery 2 Norton 2004 (1.22 mg/L), Winery 5 Norton 2003 (1.12 mg/L),

and Winery 5 Norton 2004 (1.52 mg/L). Some of the considerably low levels of

resveratrol of the Norton wines were Winery 1 2002 (0.14 mg/L), Winery 4 Norton 2005

(0.14 mg/L), and Winery 6 2000 (0.12 mg/L). Of the other wine varieties, Winery 6’s

Blackberry, Cherry, Strawberry, and School House Red wines had the lowest levels of

resveratrol present. For individual results refer to Table 4.1 or Appendix A-14 pages 71-

72.

39

Figure 4.1 Resveratrol Chromatograph of Winery 2 Norton 2005 bottle 3

40

Table 4.1 Resveratrol and pH Results

Resveratrol (mg/L) and pH Results Winery Wine Vintage Resveratrol Standard

Deviation pH Standard

Deviation Winery 1 Norton 2001 0.21IJKLijk 0.17 3.68 0.02 Norton 2002 0.14KLMjk 0.12 3.50 0.03 Norton 2003 0.30HIJKLhijk 0.19 3.53 0.01 Norton 2004 1.33ABabjk 0.19 3.86 0.01 Winery 2 Chambourcin 2003 0.32HIJKhijk 0.06 3.62 0.00 Chambourcin 2004 0.80DEdef 0.01 3.60 0.01 Norton 2003 1.50Aab 0.07 3.68 0.01 Norton 2004 1.22BCabc 0.19 3.45 0.02 Winery 3 Chambourcin 2003 0.80DEdef 0.08 3.57 0.04 Chambourcin 2004 0.37HIJhijk 0.06 3.68 0.01 Norton 2003 1.14BCabcd 0.09 3.76 0.03 Norton 2004 0.81DEdef 0.05 3.63 0.06 Winery 4 Chambourcin 2005 0.17KLMjk 0.01 3.67 0.02 R. Norton 2005 0.14KLMjk 0.03 3.60 0.02 Premium Claret 2002 0.17KLMjk 0.06 3.67 0.00 Premium Claret 2003 0.32HIJKhijk 0.10 3.52 0.11 Premium Claret 2005 0.19JKLMijk 0.03 3.53 0.02 Winery 5 Norton 2003 1.12Cbcd 0.14 3.63 0.08 Norton 2004 1.52Aa 0.41 3.66 0.02 Norton 2005 0.38GHIJhijk 0.02 3.65 0.03 Norton 2006 0.65EFefgh 0.09 3.66 0.02 The t-test and Tukey’s studentized test results are compound specific ABCDEFGHIJKLM – t-test, each letter is significantly different from the other abcdefghijk –Tukey’s Studentized Range Test, each letter is significantly different

41

Table 4.1 continued

Resveratrol (mg/L) and pH Results Winery Wine Vintage Resveratrol Standard

Deviation pH Standard

Deviation Winery 6 Blackberry 2006 0.12LMjk 0.04 3.54 0.01 Chambourcin 2003 0.32HIJKhijk 0.06 3.42 0.01 Chambourcin 2004 0.78DEdefg 0.04 3.91 0.06 Chambourcin 2005 0.31HIJKLhijk 0.00 3.74 0.01 Cherry 2006 0.05Mk 0.00 3.72 0.01 Norton 2000 0.12LMJK 0.01 3.85 0.03 Norton 2002 0.40GHghijk 0.04 3.70 0.02 Norotn 2003 0.39GHIhijk 0.05 3.71 0.03 Norton 2004 0.44GHfghi 0.04 3.82 0.01 Strawberry 2005 0.08Mjk 0.00 3.43 0.04

School House Red 2005 0.17KLMjk 0.01 3.77 0.59

School House Red 2006 0.07Mjk 0.01 3.52 0.02

Winery 7 Norton 2003 0.89Dcde 0.08 3.75 0.13 Norton 2004 0.78DEdefg 0.02 3.68 0.04 Norton 2005 0.57FGefghi 0.33 3.64 0.01 Norton 2006 0.66EFefgh 0.03 3.66 0.02 Norton 2007 0.38GHIhijk 0.03 3.78 0.02 The t-test and Tukey’ studentized test results are compound specific ABCDEFGHIJKLM – t-test, each letter is significantly different from the other abcdefghijk –Tukey’s Studentized Range Test, each letter is significantly different

42

4.2 Procyanidin Results

All the levels for catechin, epicatechin, procyanidin B1, and procyanidin B2 were

within the ranges previously reported for other wine varietals. Norton Wines from

Winery 5 2006 (15.91 mg/L) and Winery 7 2007 (11.22 mg/L) had the highest levels of

catechin, whereas Norton wines from Winery 3 2003 (0.19 mg/L) and Winery 7 2005

(0.81 mg/L) had the lowest catechin levels. For individual results refer to Table 4.2.1.1

or Appendix A-10 pages 63-64

As for epicatechin, the reported levels again varied by vintage and varietal. The

highest levels were found in Winery 1 Norton 2003 (14.49 mg/L), Winery 2

Chambourcin 2004 (27.73 mg/L), Winery 6 Chambourcin 2003 (11.66 mg/L), and

Winery 6 Norton 2004 (16.23 mg/L). The lowest levels were found in Winery 1 Norton

2001 (0.31 mg/L), Winery 4 Norton 2005 (0.23 mg/L), Winery 4 Premium Claret 2002

(0.24 mg/L), Winery 6 Strawberry 2005 (0.22 mg/L), and Winery 7 Norton 2005 (0.40

mg/L). For individual results refer to Table 4.2.1 or Appendix A-11 pages 65-66.

43

Figure 4.2 Procyanidin Chromatogram of Winery 5 Norton 2005 bottle 3

44

Table 4.2.1 Catechin and Epicatechin Results

Catechin and Epicatechin Results(mg/L) Winery Wine Vintage Catechin Standard

Deviation Epicatechin Standard

Deviation

Winery 1

Norton 2001 1.57KLMNjklmn 0.13 0.31OPkl 0.02 Norton 2002 1.76JKLjklmn 0.39 6.41Ed 0.51 Norton 2003 0.63MNOlmn 0.27 15.91Bb 0.36 Norton 2004 0.59MNOlmn 0.28 5.28Fdefgh 1.20 Winery 2 Chambourcin 2003 14.22Cbc 0.66 2.84GHIefg 0.21 Chambourcin 2004 2.87HIJhijkl 0.04 29.48Aa 0.54 Norton 2003 3.64HIfghij 0.33 0.55NOPjkl 0.08 Norton 2004 12.04Dc 0.45 2.59HI 0.14 Winery 3 Chambourcin 2003 5.48Fefg 2.38 1.29KLMhijkl 0.30 Chambourcin 2004 3.00HIhijk 0.21 10.45Dc 0.02 Norton 2003 0.19Om 0.17 3.18GHef 0.76 Norton 2004 0.88LMNOklmn 0.12 2.18JIefghi 0.12 Winery 4 Chambourcin 2005 1.68KLMijklmn 1.30 0.82 MNOPijkl 0.35 R. Norton 2005 1.15LMNOklmn 0.33 0.23Pkl 0.10 Premium Claret 2002 0.17Omn 0.04 0.24OPkl 0.08 Premium Claret 2003 0.41Om 0.17 0.42OPkl 0.26 Premium Claret 2005 0.19Om 0.04 0.48NOPkl 0.12 Winery 5 Norton 2003 3.45HIghij 1.62 0.68MNOPjkl 0.50 Norton 2004 3.74HIfghij 0.80 0.87MNOijkl 0.31 Norton 2005 5.68Ed 4.79 1.88JKfghi 0.17 Norton 2006 15.91Bb 0.88 3.37Ge 0.23 The t-test and Tukey’s studentized test results are compound specific ABCDEFGHIJKLMNOP – t-test results, each letter is significantly different than the other abecdefghijklmn – Tukey’s Studentized Range (HSD) Test, each letter is significantly different than the other.

45

Table 4.2 continued

Catechin and Epicatechin Results(mg/L)

Winery Wine Vintage Catechin Standard Deviation Epicatechin Standard

Deviation

Winery 6

Blackberry 2006 5.70Fdefg 0.23 3.35Ge 0.20 Chambourcin 2003 0.88LMNOklmn 0.29 11.66Cc 1.11 Chambourcin 2004 0.24On 0.21 2.20IJefghi 0.17 Chambourcin 2005 5.78Fdef 0.41 1.34KLMhijkl 0.14 Cherry 2006 1.80JKLjklmn 0.55 0.42 OPkl 0.73 Norton 2000 7.70Ede 0.03 1.63JKLghijk 0.02 Norton 2002 1.54KLMNjklmn 0.22 2.58HIefgh 0.15 Norton 2003 0.88LMNOklmn 0.14 11.69Cc 0.59 Norton 2004 1.04LMNOklmn 0.43 16.23Bb 0.93 Strawberry 2005 3.91GHfghi 0.06 0.22Pl 0.02

School House Red 2005 4.91FGfgh 0.30 0.93MNOijkl 0.02

School House Red 2006 20.79Aa 0.58 5.63Fd 0.25

Winery 7 Norton 2003 2.64IJKhijklm 0.32 0.30OPkl 0.26 Norton 2004 0.50NOmn 0.09 1.13LMNijkl 0.03 Norton 2005 1.14LMNO 0.35 0.40OPkl 0.06 Norton 2006 7.46Ede 0.91 3.49Ge 0.22 Norton 2007 15.71Bb 1.29 3.19GHef 0.29 The t-test and Tukey’s studentized test results are compound specific ABCDEFGHIJKLMNOP – t-test results, each letter is significantly different than the other abecdefghijklmn – Tukey’s Studentized Range (HSD) Test, each letter is significantly different than the other.

46

Winery 5 Norton 2006 (52.39 mg/L), Winery 6 Norton 2002 (40.06 mg/L), and

Winery 7 Norton 2004 (138.68 mg/L) had the highest levels of procyanidin B1. The

lowest levels consisted of Winery 1 Norton 2001 (3.60 mg/L) and Winery 6 Blackberry

2006 (0.59 mg/L), Cherry 2006 (ND), Strawberry 2005 (1.14 mg/L), and School House

Red 2005 (0.30 mg/L). For individual results refer to Table 4.2.2 or Appendix A-12

pages 67-68.

As for Procyanidin B2 the highest concentrations came from Winery 3

Chambourcin 2004 (23.95 mg/L) and Winery 7 Norton 2006 (12.95 mg/L) and 2007

(15.69 mg/L), and the lowest observed levels were found in Winery 2 Chambourcin 2004

(1.27 mg/L), Winery 4 Norton 2005 (1.41 mg/L), Winery 4 Premium Claret 2005 (1.70

mg/L), Winery 5 Norton 2003 (1.83 mg/L), Winery 6 Cherry 2006 (ND) and Strawberry

2005(1.14 mg/L), and Winery 7 Norton 2003 (1.01 mg/L), 2004 (1.23 mg/L), and 2005

(1.23 mg/L). For individual results refer to Table 4.2.2 or Appendix A-13 pages 69-70.

47

Table 4.2.2 Procyanidin B1 & B2 Results

Procyanidin B1 & B2 Results(mg/L) Winery Wine Vintage Procyanidin

B1 Standard Deviation

Procyanidin B2

Standard Deviation

Winery 1

Norton 2001 0.81STr 0.15 4.85JKLfghijkl 0.40

Norton 2002 10.62KLMNlmno 1.68 3.40MNOjklmnop

q 0.64

Norton 2003 18.19Jijk 0.12 3.95LMNijklmno 0.28 Norton 2004 30.38EFdef 3.02 4.52KLMghijklm 0.06 Winery 2 Chambourcin 2003 13.50Kjkl 0.77 4.99JKLfghijk 1.02 Chambourcin 2004 6.08OPQmnopq 1.72 0.58STrs 0.18 Norton 2003 11.89KLMklmn 0.63 4.91JKLfghijkl 0.86 Norton 2004 8.42NOPlmnop 1.81 5.55HIJKfghij 1.27 Winery 3 Chambourcin 2003 5.36PQnopq 1.00 2.47OPlmnopqr 1.52 Chambourcin 2004 10.13LMNlmnop 1.38 23.95Aa 0.68 Norton 2003 22.33HIghi 0.35 3.20NOklmnopq 1.05 Norton 2004 27.95FGefg 0.64 6.02GHIJfghi 0.28 Winery 4 Chambourcin 2005 4.20QRopqr 0.22 4.89JKLfghijkl 1.69 R. Norton 2005 7.92NOPlmnopq 0.62 1.41PQRSpqrs 0.18

Premium Claret 2002 9.31MNlmnop 0.38 3.98LMNijklmn 0.34

Premium Claret 2003 12.52KLMklm 0.94 4.37KLMNhijklm

n 0.39

Premium Claret 2005 8.47NOPlmnop 0.33 1.70PQRSopqrs 0.08

Winery 5 Norton 2003 26.69Gefgh 0.46 2.39OPQmnopqrs 0.46 Norton 2004 32.56DEcde 0.13 6.74FGHIefgh 0.97 Norton 2005 32.97DEcde 2.35 5.54IJKfghij 0.35 Norton 2006 52.16Aa 1.83 6.76FGHefgh 0.11 The t-test and Tukey’ studentized test results are compound specific ABCDEFGHIJKLMNOPQRST - t test results, each letter is significantly different abcdefghijklmnopqrs – Tukey’s Studentized Range Test, each letter is significantly different ND – Not Detectable

48

Table 4.2.2 continued

Procyanidin B1 & B2 Results (mg/L) Winery Wine Vintage Procyanidin

B1 Standard Deviation

Procyanidin B2

Standard Deviation

Winery 6

Blackberry 2006 0.59STr 1.02 10.56Dcd 0.53 Chambourcin 2003 1.48RSTqr 0.37 5.25JKfghij 0.98 Chambourcin 2004 13.81K 0.03 7.23Fef 0.47 Chambourcin 2005 13.44KLjkl 0.55 2.55OPklmnopqr 0.18 Cherry 2006 NDTr 0.00 NDTs 0.00 Norton 2000 31.12EFdef 0.80 1.40PQRSpqrs 0.21 Norton 2002 40.06Bb 0.91 1.97PQRnoprs 0.39 Norton 2003 25.23GHfgh 1.25 6.84FGefg 0.99 Norton 2004 11.20LKMNlmn 3.95 1.57PQRSopqrs 0.69 Strawberry 2005 3.81QRSqr 6.59 1.14RSTqrs 0.99

School House Red 2005 0.30Tr 0.18 3.79LMNijklmnop 0.05

School House Red 2006 38.31BCbc 1.48 8.68Ede 0.46

Winery 7 Norton 2003 22.12HIghi 1.43 1.01RSTqrs 0.38 Norton 2004 35.06CDbc 0.51 1.23QRSqrs 0.07 Norton 2005 19.91IJhij 0.93 1.23QRSqrs 0.27 Norton 2006 25.11GHfgh 0.94 12.95Cc 0.29 Norton 2007 37.98BCbc

7.71 15.69Bb 2.01 The t-test and Tukey’ studentized test results are compound specific ABCDEFGHIJKLMNOPQRST - t test results, each letter is significantly different abcdefghijklmnopqrs – Tukey’s Studentized Range Test, each letter is significantly different ND – Not Detectable

49

4.3 Variations between Varietals and Vintages

Using a t-test and HSD test, each compound measured was analyzed comparing

the specific compound with that of other varietals and vintages. For epicatechin, Winery

2 Chambourcin 2004 had the highest mean and was significantly different from all of the

other wines sampled. Winery 6 Norton 2004 and Winery 1 Norton 2003, the next highest

means, were not significantly different from each other but were significantly different

from all the others. For catechin, Winery 6 School House Red 2006 had the highest

mean, and was significantly different from all of the other samples. The next highest

values were from Winery 5 Norton 2006 and Winery 7 Norton 2007. These two samples

were not significantly different from one another but were significantly different from the

rest of the samples when using the t-test, but for the HSD test Winery 7 Norton 2007 and

Winery 2 Chambourcin 2003 were also in the second grouping that was significantly

different for the rest of the samples, with the exception of Winery 2 Chambourcin 2003

not being significantly different than Winery 2 Norton 2004. Procyanidin B1 content in

Winery 5 Norton 2006 was significantly higher than that of other samples when using the

t-test and HSD. However many of the other samples had overlapping significant

differences from one another. The t-test of the procyanidin B2 indicated that the five

highest means were significantly different from not only one another but also from the

rest of the samples. These five samples included Winery 3 Chambourcin 2004, Winery 7

Norton 2007, Winery 7 Norton 2006, Winery 6 BlackBerry 2006, and Winery 6 School

House Red 2006. For the HSD test, however, only Winery 3 Chambourcin 2004 and

Winery 7 Norton 2007 are significantly different from each other as well as that of the

rest of the samples. The highest mean values of resveratrol were found in Winery 5

50

Norton 2004, Winery 2 Norton 2003, and Winery 1 Norton 2004. As a group these

samples were significantly different from the rest of the samples with the exception of

Winery 1 Norton 2004. Winery 1 2004 was not significantly different from Winery 2

Norton 2004 and Winery 3 Norton 2004 according to the t-test. However, with the HSD

test Winery 5 Norton 2004, Winery 2 Norton 2003, Winery 1 Norton 2004, Winery 2

Norton 2004, and Winery 3 Norton 2003 were considered as a group that was

significantly different from the rest of the samples, but not from one another. Winery 2

Norton 2003, Winery 1 Norton 2004, Winery 2 Norton 2004 and Winery 3 Norton 2003

were also grouped with Winery 5 Norton 2003 as not being significantly different from

one another but different from the rest of the samples.

4.4 Relationship between Winery Location and Compound Concentration

The t-test, as well as HSD, was also done to see if there is a relationship between

the quantity of each compound in the wines and the location in which the wineries are

located. Wineries in Winery 6 and Hermann had significantly different quantities of

epicatechin as determined by the t-test. Using HSD, St. James, Herman, and Waverly

had significantly higher quantities of epicatechin. Catechin results for Augusta wines had

the highest quantity by location based on the t-test, and with the HSD test Augusta,

Hermann, and St. James grouped together and had significantly higher quantities of

catechin than the other locations. However, this test also grouped Hermann, St. James,

and Waverly together as well. Procyanidin B1 showed no significant differences

between the locations and the quantity measure for either test. Hermann, Augusta, and

Waverly were grouped together for the t-test. Again however, Waverly and St. James

were also grouped together. Using the HSD test all locations were considered equal.

51

Lastly, the relationship between resveratrol and winery locations created quite different

results. Based on the t-test, Augusta and Waverly were grouped together, and Hermann

and St. James were grouped together, indicating that the two groups were different from

one another. With the HSD test, however, three groups were created with all three

overlapping.

Table 4.4.1 Epicatechin Concentration by Location

Table 4.4.2 Catechin Concentration by Location

Epicatechin Catechin Location Mean t-test HSD Location Mean t-test HSD St. James 6.81 ± 7.10 A A August 7.76 ± 5.22 A A Herman 4.05 ± 4.98 AB AB Herman 3.56 ± 5.04 B A Waverly 2.68 ± 0.71 B AB St. James 3.43 ± 3.28 B AB Augusta 1.66 ± 1.15 B B Waverly 0.53 ± 0.49 B B

*AB – each letter is significantly different from one another

*AB – each letter is significantly different from one another

Table 4.4.3 Procyanidin B1 Concentration by Location

Table 4.4.4 Procyanidin B2 Concentration by Location

Procyanidin B1 Procyanidin B2 Location Mean t-test HSD Location Mean t-test HSD St. James 27.45 ± 12.11 A A Herman 5.43 ± 5.29 A A Augusta 27.45 ± 15.95 A A Augusta 5.32 ±1.61 A A Waverly 25.14 ± 3.97 A A Waverly 4.61 ± 1.99 AB A Herman 22.24 ±11.74 A A St. James 1.65 ± 2.61 B A

*AB – each letter is significantly different from one another

*AB – each letter is significantly different from one another

52

Table 4.4.5 Resveratrol Concentration by Location

Resveratrol Location Mean t-test HSD Augusta 1.07 ± 0.54 A A Waverly 0.97 ± 0.24 A AB Herman 0.57 ± 0.38 B BC St. James 0.32 ± 0.14 B C

*ABC – each letter is significantly different from one another

4.6 Relationship between Compounds

Using the Pearson’s Correlation Coefficients, a significant correlation between the

quantities of catechin measured and the quantities of procyanidin B1 and B2 were

observed. There was also a significant correlation between the quantity of procyanidin

B1 and the resveratrol content in the samples (p<0.001). For full results refer to

Appendix A-16 page 111.

53

CHAPTER 5

DISCUSSION

There are many variables that will affect the quantity of resveratrol and

procyanidin content in wine. Some of them include climate conditions, soil type,

geographical location, cultivar practices, varietal blend, and the wine making processes.

Fining agents were shown to decrease the resveratrol content in red wines (Threlfall and

others 1999). Vine vigor has a significant influence on the polyphenolic content of the

grapes skins, with a lower vine vigor more proanthocyanidins were extracted from the

skins of the grapes into the wine (Cortell and others 2005). Cultivar practices, such as

leaf removal, can influence the wine composition as well (Main 2004). Thus, there are

many confounding variables that could neither be controlled nor are they known, and thus

are not reported for the data analyzed. One assumption we can make about the wine is

that the varietal name of the wine on the bottle must be at least 75% of that varietal.

For the variation between wines and procyanidin and resveratrol content, these

often are a reflection of what varietals were used and the percentage contained in the

wine. Winery 2 Chambourcin 2004 was ≥75% Chambourcin, and had the highest content

of epicatechin. Winery 6 School House Red 2006 had the highest content of catechin,

and was of an unknown blend of varietals. Winery 5 Norton 2006 was at least 75%

Norton and had the highest content of procyanidin B1. As for procyanidin B2, Winery 3

Chambourcin 2004 had the highest concentration and was 100% Chambourcin. Winery 5

Norton 2004 had the highest resveratrol content of the samples and was at least 75%

Norton.

54

Analysis of Norton Wines by location indicates that there may not be a location

that is significantly better than another. Again this is due to the many confounding

variables in the cultivation of the grapes and production of the wines.

It is hard to distinguish if the reported results were a contribution from the

Norton Grape or the varietal blended with it. However, within the top five highest values

for resveratrol, catechin, epicatechin, procyanidin B1, and procyanidin B2 there was

consistently at least one sample that was 100% Norton, and often at least two others that

were at least 75% Norton. More analysis of 100% Norton wines as well as other 100%

varietal specific wines will need to be conducted in the future to compare procyanidins

and resveratrol content, and to determine if there is still a correlation between resveratrol

content and procyanidin B1 as well as a correlation between catechin content and

procyanidin B1 and B2 when using 100% of the same grape to produce a wine.

55

APPENDIX A

A-1 Resveratrol Standard Curve

Calibration Curve ReportFile: steve113-b.mthDetector: Unknown Detector (5), Address: 16, Channel ID: B

t-ResveratrolExternal Standard Analysis Resp. Fact. RSD: 30.28%Curve Type: Linear Coeff. Det.(r_): 0.999638Origin: Includey = +2.010875e+005x -4.382202e+003

Peak Size

Amount (ug)1 2 3 4 5

0

250000

500000

750000

1000000

Replicates 1 1 1 1 1 11 1

56

A-2 Procyanidin B1 Standard Curve and Concentrations used

Procyanidin B1 Concentration

(mg/L) Area

(µV/min) 0.00 0.00 1.80 90.57 4.50 335.81

54.00 3698.29 108.00 7538.83 360.00 19508.35

y = 55.745xR² = 0.9893

0.00

5000.00

10000.00

15000.00

20000.00

25000.00

0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00

µV/m

in

mg/L

Procyanidin B1 Standard Curve

57

A-3 Catechin Standard Curve and Concentrations used

Catechin Concentration (mg/L)

Area (µV/min)

0.00 0.00 1.00 1669.52 1.49 0.87 2.00 607.13 3.73 30.92

10.00 3291.95 200.00 65156.19 250.00 82807.34

y = 329.06xR² = 0.9995

0.0010000.0020000.0030000.0040000.0050000.0060000.0070000.0080000.0090000.00

0.00 50.00 100.00 150.00 200.00 250.00 300.00

µV/m

in

mg/L

Catechin Standard Curve

58

A-4 Procyanidin B2 Standard Curve and Concentrations used

Procyanidin B2 Concentration

(mg/L) Area

(µV/min) 0.00 0.00 1.53 1063.04 3.83 49.06 9.65 199.40

19.13 387.35 72.00 24241.81

250.00 76479.48

y = 306.29xR² = 0.9911

0.0010000.0020000.0030000.0040000.0050000.0060000.0070000.0080000.0090000.00

0.00 50.00 100.00 150.00 200.00 250.00 300.00

µV/m

in

mg/L

Procyanidin B2 Standard Curve

59

A-5 Epicatechin Standard Curve and Concentrations used

Epicatechin Concentration

(mg/L) Area

(µV/min)

0.00 0.00 1.49 103.69 3.73 520.86 9.38 24.09

250.00 18345.15 500.00 37793.21

y = 75.128xR² = 0.9994

0

5000

10000

15000

20000

25000

30000

35000

40000

0.00 100.00 200.00 300.00 400.00 500.00 600.00

µV/m

in

mg/L

Epicatechin Standard Curve

60

A-6 Coefficient of Variation for the Resveratrol Method

COEFFICIENT OF VARIANCE RESVERATROL SAMPLE METHOD

Sample Resveratrol (mg/mL

1-1SH N07 0.42 1-2 SH N07 0.41 1-3 SH N07 0.41 1-4 SH N07 0.41 1-5 SH N07 0.38 1-6 SH N07 0.40 1-7 SH N07 0.40 1-8 SH N07 0.40 1-9SH N07 0.40 1-10 SH N07 0.40 AVERAGE 0.40

STANDARD DEVIATION 0.01

COEFFICIENT OF VARIANCE 2.50 A-7 Coefficient of Variation of the HPLC for the Resveratrol Method

COEFFICIENT OF VARIANCE OF HPLC SYSTEM FOR RESVERATROL METHOD

Sample Resveratrol (mg/mL) 1-1 AP N01 0.27 1-2 AP N01 0.19 1-3 AP N01 0.18 1-4 AP N01 0.21 1-5 AP N01 0.21 1-6 AP N01 0.22 1-7 AP N01 0.21 1-8 AP N01 0.21 1-9 AP N01 0.22

1-10 AP N01 0.20

AVERAGE 0.21

STANDARD DEVIATION 0.02

COEFFICIENT OF VARIANCE 11.41

61

A-8 Coefficient of Variation of the HPLC for the Procyanidin Method

COEFFICIENT OF VARIANCE OF THE HPLC SYSTEM (mg/L) FOR THE PROCYANIDIN METHOD

Sample Catechin Epicatechin Procyanidin B1 Procyanidin B2 2 BB C04_8 0.047 0.908 1.399 1.619 2BB C043_8 0.063 0.931 1.398 1.626 2 BB C044_1 0.082 0.922 1.395 1.605 2 BB C044_2 0.134 0.836 1.361 1.3 2 BB C045_8 0.159 0.966 1.266 1.545 2 BB C045_8 0.158 0.96 1.276 1.477

AVERAGE 0.10717 0.92050 1.34917 1.52867 STANDARD DEVIATION

0.04939 0.04699 0.06226 0.12545

COEFFICIENT OF VARIANCE

46.08326 5.10465 4.61438 8.20633

A-9 Coefficient of Variation of the Procyanidin Sample Method

COEFFICIENT OF VARIANCE OF THE PROCYANIDIN SAMPLE METHOD (mg/L) Sample Catechin Epicatechin Procyanidin B1 Procyanidin B2

1-1MP N067_1 17.38 4 17.14 4.66 1-3 MP N068 17.57 4.3 18.01 9.83 1-4 MP N069 18.22 4.34 20.89 10.31

1-5 MP N0610 17.48 4.16 17.36 9.99 1-6 MP N0611 17.98 4.2 18.36 9.76 1-7 MP N063 17.96 4.19 20.05 10.12 1-8 MP N064 17.36 3.9 11.53 9.83 1-2 MP N068 17.94 4 15.26 9.03 AVERAGE 17.73625 4.13625 17.32500 9.19125

STANDARD DEVIATION

0.32663 0.15528 2.91687 1.86891

COEFFICIENT OF VARIANCE

1.84157 3.75417 16.83620 20.33363

62

A-10 Catechin Results

Catechin Measured Per Bottle of Wine (mg/L)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 1 Norton 2001 1.44 1.69 1.57 1.57 0.13 Norton 2002 1.36 1.78 2.14 1.76 0.39 Norton 2003 0.46 0.48 0.94 0.63 0.27 Norton 2004 0.37 0.91 0.50 0.59 0.28 Winery 2 Chambourcin 2003 14.97 13.91 13.77 14.22 0.66 Chambourcin 2004 2.83 2.89 2.90 2.87 0.04 Norton 2003 3.40 3.51 4.01 3.64 0.33 Norton 2004 11.88 11.70 12.55 12.04 0.45 Winery 3 Chambourcin 2003 8.23 3.94 4.28 5.48 2.38 Chambourcin 2004 2.79 3.21 2.99 3.00 0.21 Norton 2003 0.24 0.32 0.00 0.19 0.17 Norton 2004 1.02 0.81 0.80 0.88 0.12 Winery 4 Chambourcin 2005 0.44 3.03 1.58 1.68 1.30 R. Norton 2005 0.81 1.16 1.47 1.15 0.33 Premium Claret 2002 0.16 0.14 0.21 0.17 0.04 Premium Claret 2003 0.61 0.31 0.32 0.41 0.17 Premium Claret 2005 0.17 0.24 0.16 0.19 0.04 Winery 5 Norton 2003 2.57 2.46 5.32 3.45 1.62 Norton 2004 2.86 3.95 4.41 3.74 0.80 Norton 2005 6.56 7.75 9.08 7.80 1.26 Norton 2006 16.15 16.65 14.93 15.91 0.88

63

A-10 continued Catechin Measured Per Bottle of Wine (mg/L)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 6 Blackberry 2006 5.45 5.89 5.76 5.70 0.23 Chambourcin 2003 0.84 1.19 0.61 0.88 0.29 Chambourcin 2004 0.39 0.00 0.32 0.24 0.21 Chambourcin 2005 5.32 6.11 5.91 5.78 0.41 Cherry 2006 2.35 1.79 1.26 1.80 0.55 Norton 2000 7.72 7.70 7.67 7.70 0.03 Norton 2002 1.40 1.43 1.80 1.54 0.22 Norton 2003 0.87 1.03 0.75 0.88 0.14 Norton 2004 1.20 1.36 0.55 1.04 0.43 Strawberry 2005 3.98 3.89 3.86 3.91 0.06 School House Red 2005 4.56 5.10 5.07 4.91 0.30 School House Red 2006 20.90 21.30 20.16 20.79 0.58 Winery 7 Norton 2003 2.86 2.80 2.27 2.64 0.32 Norton 2004 0.40 0.55 0.56 0.50 0.09 Norton 2005 0.75 1.22 1.44 1.14 0.35 Norton 2006 6.79 8.50 7.10 7.46 0.91 Norton 2007 14.25 16.18 16.69 15.71 1.29

64

A-11 Epicatechin Results

Epicatechin Measured Per Bottle of Wine (mg/L)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 1 Norton 2001 0.31 0.33 0.30 0.31 0.02 Norton 2002 6.88 6.49 5.87 6.41 0.51 Norton 2003 16.14 16.09 15.49 15.91 0.36 Norton 2004 6.65 4.81 4.39 5.28 1.20 Winery 2 Chambourcin 2003 3.08 2.76 2.68 2.84 0.21 Chambourcin 2004 29.90 28.87 29.66 29.48 0.54 Norton 2003 0.58 0.46 0.61 0.55 0.08 Norton 2004 2.52 2.50 2.76 2.59 0.14 Winery 3 Chambourcin 2003 0.95 1.48 1.45 1.29 0.30 Chambourcin 2004 10.48 10.44 10.44 10.45 0.02 Norton 2003 4.01 2.53 3.01 3.18 0.76 Norton 2004 2.09 2.13 2.31 2.18 0.12 Winery 4 Chambourcin 2005 0.56 1.22 0.69 0.82 0.35 R. Norton 2005 0.31 0.27 0.12 0.23 0.10 Premium Claret 2002 0.15 0.28 0.30 0.24 0.08 Premium Claret 2003 0.22 0.34 0.71 0.42 0.26 Premium Claret 2005 0.59 0.48 0.36 0.48 0.12 Winery 5 Norton 2003 0.40 0.38 1.25 0.68 0.50 Norton 2004 0.58 0.82 1.20 0.87 0.31 Norton 2005 2.06 1.73 1.85 1.88 0.17 Norton 2006 3.45 3.55 3.11 3.37 0.23

65

A-11 continued Epicatechin Measured Per Bottle of Wine (mg/L)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 6 Blackberry 2006 3.18 3.30 3.57 3.35 0.20 Chambourcin 2003 12.34 12.27 10.38 11.66 1.11 Chambourcin 2004 2.27 2.01 2.33 2.20 0.17 Chambourcin 2005 1.19 1.36 1.46 1.34 0.14 Cherry 2006 0.00 0.00 1.26 0.42 0.73 Norton 2000 1.64 1.61 1.64 1.63 0.02 Norton 2002 2.69 2.64 2.41 2.58 0.15 Norton 2003 11.95 12.10 11.01 11.69 0.59 Norton 2004 17.25 16.03 15.42 16.23 0.93 Strawberry 2005 0.25 0.21 0.21 0.22 0.02 School House Red 2005 0.93 0.95 0.92 0.93 0.02 School House Red 2006 5.76 5.78 5.34 5.63 0.25 Winery 7 Norton 2003 0.06 0.58 0.26 0.30 0.26 Norton 2004 1.16 1.12 1.10 1.13 0.03 Norton 2005 0.38 0.36 0.47 0.40 0.06 Norton 2006 3.56 3.67 3.25 3.49 0.22 Norton 2007 3.28 2.86 3.42 3.19 0.29

66

A-12 Procyanidin B1 Results

Procyanidin B1 Measured Per Bottle of Wine (mg/L)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 1 Norton 2001 0.85 0.64 0.93 0.81 0.15 Norton 2002 11.23 11.91 8.72 10.62 1.68 Norton 2003 18.32 18.10 18.15 18.19 0.12 Norton 2004 28.10 33.80 29.24 30.38 3.02 Winery 2 Chambourcin 2003 14.19 12.67 13.63 13.50 0.77 Chambourcin 2004 4.91 8.06 5.28 6.08 1.72 Norton 2003 12.29 12.21 11.16 11.89 0.63 Norton 2004 6.93 7.91 10.43 8.42 1.81 Winery 3 Chambourcin 2003 6.51 4.68 4.89 5.36 1.00 Chambourcin 2004 11.37 10.38 8.64 10.13 1.38 Norton 2003 22.64 21.95 22.40 22.33 0.35 Norton 2004 27.28 28.01 28.56 27.95 0.64 Winery 4 Chambourcin 2005 4.38 3.95 4.27 4.20 0.22 R. Norton 2005 7.25 8.48 8.04 7.92 0.62 Premium Claret 2002 9.31 9.69 8.93 9.31 0.38 Premium Claret 2003 12.05 11.90 13.60 12.52 0.94 Premium Claret 2005 8.81 8.44 8.15 8.47 0.33 Winery 5 Norton 2003 26.28 27.19 26.60 26.69 0.46 Norton 2004 32.46 32.70 32.51 32.56 0.13 Norton 2005 34.68 33.93 30.29 32.97 2.35 Norton 2006 50.14 53.69 52.65 52.16 1.83

67

A-12 continued Procyanidin B1 Measured Per Bottle of Wine (mg/L)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 6 Blackberry 2006 0.00 1.77 0.00 0.59 1.02 Chambourcin 2003 1.43 1.14 1.88 1.48 0.37 Chambourcin 2004 13.84 13.79 13.81 13.81 0.03 Chambourcin 2005 14.08 13.15 13.10 13.44 0.55 Cherry 2006 0.00 0.00 0.00 0.00 0.00 Norton 2000 31.96 30.37 31.02 31.12 0.80 Norton 2002 40.23 40.87 39.07 40.06 0.91 Norton 2003 26.27 25.57 23.84 25.23 1.25 Norton 2004 12.44 6.77 14.38 11.20 3.95 Strawberry 2005 0.00 11.42 0.00 3.81 6.59 School House Red 2005 0.50 0.19 0.20 0.30 0.18 School House Red 2006 38.96 39.35 36.62 38.31 1.48 Winery 7 Norton 2003 21.45 21.15 23.76 22.12 1.43 Norton 2004 35.56 35.08 34.54 35.06 0.51 Norton 2005 18.98 19.91 20.83 19.91 0.93 Norton 2006 24.77 24.39 26.17 25.11 0.94 Norton 2007 35.75 31.64 46.56 37.98 7.71

68

A-13 Procyanidin B2 Results

Procyanidin B2 Measured Per Bottle of Wine (mg/L)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average

Standard Deviation

Winery 1 Norton 2001 5.25 4.45 4.85 4.85 0.40 Norton 2002 4.13 2.93 3.15 3.40 0.64 Norton 2003 4.02 3.65 4.19 3.95 0.28 Norton 2004 4.51 4.47 4.58 4.52 0.06 Winery 2 Chambourcin 2003 5.59 5.57 3.81 4.99 1.02 Chambourcin 2004 0.78 0.49 0.46 0.58 0.18 Norton 2003 4.24 5.88 4.61 4.91 0.86 Norton 2004 4.28 6.82 5.55 5.55 1.27 Winery 3 Chambourcin 2003 4.18 1.26 1.98 2.47 1.52 Chambourcin 2004 23.71 24.72 23.42 23.95 0.68 Norton 2003 2.75 2.45 4.40 3.20 1.05 Norton 2004 6.33 5.94 5.78 6.02 0.28 Winery 4 Chambourcin 2005 6.83 4.01 3.82 4.89 1.69 R. Norton 2005 1.21 1.47 1.56 1.41 0.18 Premium Claret 2002 4.34 3.66 3.95 3.98 0.34 Premium Claret 2003 4.01 4.31 4.79 4.37 0.39 Premium Claret 2005 1.78 1.62 1.70 1.70 0.08 Winery 5 Norton 2003 2.92 2.09 2.16 2.39 0.46 Norton 2004 7.76 6.62 5.83 6.74 0.97 Norton 2005 5.65 5.15 5.83 5.54 0.35 Norton 2006 6.78 6.65 6.86 6.76 0.11

69

A-13 continued Procyanidin B2 Measured Per Bottle of Wine (mg/L)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 6 Blackberry 2006 9.94 10.89 10.84 10.56 0.53 Chambourcin 2003 6.24 5.22 4.28 5.25 0.98 Chambourcin 2004 6.76 7.70 7.24 7.23 0.47 Chambourcin 2005 2.36 2.58 2.72 2.55 0.18 Cherry 2006 0.00 0.00 0.00 0.00 0.00 Norton 2000 1.30 1.25 1.64 1.40 0.21 Norton 2002 1.84 1.66 2.41 1.97 0.39 Norton 2003 7.34 7.47 5.70 6.84 0.99 Norton 2004 1.32 1.05 2.35 1.57 0.69 Strawberry 2005 1.73 1.69 0.00 1.14 0.99 School House Red 2005 3.84 3.78 3.75 3.79 0.05 School House Red 2006 8.88 9.01 8.15 8.68 0.46 Winery 7 Norton 2003 0.93 0.67 1.42 1.01 0.38 Norton 2004 1.31 1.18 1.19 1.23 0.07 Norton 2005 1.01 1.14 1.53 1.23 0.27 Norton 2006 12.62 13.19 13.03 12.95 0.29 Norton 2007 16.51 17.17 13.40 15.69 2.01

70

A-14 Resveratrol Results

Resveratrol results (mg/mL)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 1 Norton 2001 0.33 0.02 0.27 0.21 0.17 Norton 2002 0.24 0.01 0.18 0.14 0.12 Norton 2003 0.09 0.47 0.34 0.30 0.19 Norton 2004 1.53 1.31 1.15 1.33 0.19 Winery 2 Chambourcin 2003 0.39 0.28 0.29 0.32 0.06 Chambourcin 2004 0.79 0.81 0.80 0.80 0.01 Norton 2003 1.42 1.56 1.51 1.50 0.07 Norton 2004 1.43 1.18 1.05 1.22 0.19 Winery 3 Chambourcin 2003 0.71 0.83 0.85 0.80 0.08 Chambourcin 2004 0.43 0.35 0.32 0.37 0.06 Norton 2003 1.13 1.06 1.24 1.14 0.09 Norton 2004 0.77 0.86 0.78 0.81 0.05 Winery 4 Chambourcin 2005 0.17 0.15 0.18 0.17 0.01 R. Norton 2005 0.11 0.17 0.15 0.14 0.03 Premium Claret 2002 0.24 0.13 0.13 0.17 0.06 Premium Claret 2003 0.41 0.21 0.33 0.32 0.10 Premium Claret 2005 0.22 0.16 0.19 0.19 0.03 Winery 5 Norton 2003 0.97 1.13 1.26 1.12 0.14 Norton 2004 1.05 1.74 1.77 1.52 0.41 Norton 2005 0.37 0.38 0.40 0.38 0.02 Norton 2006 0.55 0.72 0.68 0.65 0.09

71

A-14 continued Resveratrol results (mg/mL)

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 6 Blackberry 2006 0.13 0.15 0.08 0.12 0.04 Chambourcin 2003 0.33 0.37 0.26 0.32 0.06 Chambourcin 2004 0.80 0.74 0.81 0.78 0.04 Chambourcin 2005 0.31 0.31 0.31 0.31 0.00 Cherry 2006 0.05 0.05 0.04 0.05 0.00 Norton 2000 0.12 0.12 0.11 0.12 0.01 Norton 2002 0.36 0.40 0.44 0.40 0.04 Norton 2003 0.33 0.41 0.44 0.39 0.05 Norton 2004 0.41 0.44 0.48 0.44 0.04 Strawberry 2006 0.08 0.08 0.08 0.08 0.00 School House Red 2005 0.17 0.16 0.17 0.17 0.01 School House Red 2006 0.07 0.07 0.06 0.07 0.01 Winery 7 Norton 2003 0.91 0.97 0.80 0.89 0.08 Norton 2004 0.76 0.79 0.79 0.78 0.02 Norton 2005 0.18 0.79 0.74 0.57 0.33 Norton 2006 0.70 0.64 0.65 0.66 0.03 Norton 2007 0.38 0.41 0.35 0.38 0.03

72

A-15 pH Results

pH

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 1 Norton 2001 3.66 3.69 3.69 3.68 0.0173 Norton 2002 3.47 3.50 3.52 3.50 0.0252 Norton 2003 3.53 3.53 3.54 3.53 0.0058 Norton 2004 3.85 3.86 3.87 3.86 0.0100 Winery 2 Chambourcin 2003 3.62 3.62 3.62 3.62 0.0000 Chambourcin 2004 3.59 3.60 3.60 3.60 0.0058 Norton 2003 3.67 3.69 3.69 3.68 0.0115 Norton 2004 3.43 3.46 3.45 3.45 0.0153 Winery 3 Chambourcin 2003 3.52 3.59 3.59 3.57 0.0404 Chambourcin 2004 3.69 3.69 3.67 3.68 0.0115 Norton 2003 3.73 3.76 3.78 3.76 0.0252 Norton 2004 3.57 3.67 3.66 3.63 0.0551 Winery 4 Chambourcin 2005 3.64 3.68 3.68 3.67 0.0231 R. Norton 2005 3.58 3.61 3.62 3.60 0.0208 Premium Claret 2002 3.67 3.67 3.67 3.67 0.0000 Premium Claret 2003 3.45 3.65 3.46 3.52 0.1127 Premium Claret 2005 3.51 3.53 3.55 3.53 0.0200 Winery 5 Norton 2003 3.54 3.66 3.69 3.63 0.0794 Norton 2004 3.64 3.65 3.67 3.66 0.0212 Norton 2005 3.62 3.66 3.68 3.65 0.0306 Norton 2006 3.64 3.67 3.67 3.66 0.0173

73

A-15 continued pH

Winery Wine Vintage Bottle 1 Bottle 2 Bottle 3 Average Standard Deviation

Winery 6 Blackberry 2006 3.54 3.54 3.55 3.54 0.0058 Chambourcin 2003 3.41 3.43 3.42 3.42 0.0100 Chambourcin 2004 3.85 3.92 3.96 3.91 0.0557 Chambourcin 2005 3.73 3.75 3.75 3.74 0.0115 Cherry 2006 3.72 3.72 3.73 3.72 0.0058 Norton 2000 3.83 3.84 3.88 3.85 0.0265 Norton 2002 3.68 3.70 3.72 3.70 0.0200 Norton 2003 3.68 3.73 3.71 3.71 0.0252 Norton 2004 3.81 3.81 3.83 3.82 0.0115 Strawberry 2005 3.40 3.43 3.47 3.43 0.0351

School House Red 2005 3.43 3.43 4.45 3.77 0.5889

School House Red 2006 3.50 3.52 3.53 3.52 0.0153

Winery 7 Norton 2003 3.67 3.69 3.90 3.75 0.1274 Norton 2004 3.63 3.70 3.71 3.68 0.0436 Norton 2005 3.63 3.64 3.65 3.64 0.0100 Norton 2006 3.64 3.67 3.67 3.66 0.0173 Norton 2007 3.76 3.78 3.79 3.78 0.0153

74

A-15 1 The SAS System 11:38 Monday, October 19, 2009 NOTE: Copyright (c) 2002-2003 by SAS Institute Inc., Cary, NC, USA. NOTE: SAS (r) 9.1 (TS1M3) Licensed to THE CURATORS OF THE UNIV OF MISSOURI - T&R, Site 0001242001. NOTE: This session is executing on the XP_PRO platform. NOTE: SAS initialization used: real time 0.53 seconds cpu time 2.34 seconds NOTE: AUTOEXEC processing beginning; file is C:\Program Files\SAS\SAS 9.1\autoexec.sas. NOTE: AUTOEXEC processing completed. 1 options ls=100 ps=70; 2 data one; infile 'e:\documents\e.csv' dsd firstobs=3 missover; 3 input loc$ winery$ wine$ vin e1-e3; 4 length trt$ 30; 5 trt=compress(winery||wine||vin); 6 title 'epicatechin'; NOTE: Numeric values have been converted to character values at the places given by: (Line):(Column). 5:28 NOTE: The infile 'e:\documents\e.csv' is: File Name=e:\documents\e.csv, RECFM=V,LRECL=256 NOTE: 39 records were read from the infile 'e:\documents\e.csv'. The minimum record length was 6. The maximum record length was 57. NOTE: The data set WORK.ONE has 39 observations and 8 variables. NOTE: DATA statement used (Total process time): real time 0.23 seconds cpu time 0.23 seconds 7 proc print; 8 NOTE: There were 39 observations read from the data set WORK.ONE. NOTE: The PROCEDURE PRINT printed page 1. NOTE: PROCEDURE PRINT used (Total process time): real time 0.14 seconds cpu time 0.14 seconds 9 data two; set one; 10 e=e1; bot=1; output; 11 e=e2; bot=2; output; 12 e=e3; bot=3; output; 13 14 *proc print; NOTE: There were 39 observations read from the data set WORK.ONE. NOTE: The data set WORK.TWO has 117 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 15 proc glm; class trt; 16 model e=trt;

75

17 means trt/lsd tukey lines; 18

76

2 The SAS System 11:38 Monday, October 19, 2009 NOTE: The PROCEDURE GLM printed pages 2-7. NOTE: PROCEDURE GLM used (Total process time): real time 0.18 seconds cpu time 0.17 seconds 19 data three; set two; 20 if wine='Norton'; NOTE: There were 117 observations read from the data set WORK.TWO. NOTE: The data set WORK.THREE has 60 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 21 proc glm; class loc; 22 model e=loc; 23 means loc/lsd tukey lines; 24 NOTE: The PROCEDURE GLM printed pages 8-11. NOTE: PROCEDURE GLM used (Total process time): real time 0.06 seconds cpu time 0.04 seconds 25 data foure; set two; NOTE: There were 117 observations read from the data set WORK.TWO. NOTE: The data set WORK.FOURE has 117 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 26 proc sort; by trt bot; 27 28 run; NOTE: There were 117 observations read from the data set WORK.FOURE. NOTE: The data set WORK.FOURE has 117 observations and 10 variables. NOTE: PROCEDURE SORT used (Total process time): real time 0.06 seconds cpu time 0.06 seconds 29 30 31 data one; infile 'e:\documents\c.csv' dsd firstobs=3 missover; 32 input loc$ winery$ wine$ vin c1-c3; 33 length trt$ 30; 34 trt=compress(winery||wine||vin); 35 title 'catechin'; NOTE: Numeric values have been converted to character values at the places given by: (Line):(Column). 34:28 NOTE: The infile 'e:\documents\c.csv' is: File Name=e:\documents\c.csv, RECFM=V,LRECL=256 NOTE: 38 records were read from the infile 'e:\documents\c.csv'. The minimum record length was 42. The maximum record length was 60. NOTE: The data set WORK.ONE has 38 observations and 8 variables. NOTE: DATA statement used (Total process time):

77

3 The SAS System 11:38 Monday, October 19, 2009 real time 0.03 seconds cpu time 0.03 seconds 36 proc print; 37 NOTE: There were 38 observations read from the data set WORK.ONE. NOTE: The PROCEDURE PRINT printed page 12. NOTE: PROCEDURE PRINT used (Total process time): real time 0.00 seconds cpu time 0.00 seconds 38 data two; set one; 39 c=c1; bot=1; output; 40 c=c2; bot=2; output; 41 c=c3; bot=3; output; 42 43 *proc print; NOTE: There were 38 observations read from the data set WORK.ONE. NOTE: The data set WORK.TWO has 114 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 44 proc glm; class trt; 45 model c=trt; 46 means trt/lsd tukey lines; 47 NOTE: The PROCEDURE GLM printed pages 13-18. NOTE: PROCEDURE GLM used (Total process time): real time 0.11 seconds cpu time 0.10 seconds 48 data three; set two; 49 if wine='Norton'; NOTE: There were 114 observations read from the data set WORK.TWO. NOTE: The data set WORK.THREE has 60 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 50 proc glm; class loc; 51 model c=loc; 52 means loc/lsd tukey lines; 53 54 NOTE: The PROCEDURE GLM printed pages 19-22. NOTE: PROCEDURE GLM used (Total process time): real time 0.04 seconds cpu time 0.04 seconds 55 data fourc; set two; NOTE: There were 114 observations read from the data set WORK.TWO. NOTE: The data set WORK.FOURC has 114 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds

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4 The SAS System 11:38 Monday, October 19, 2009 56 proc sort; by trt bot; 57 58 run; NOTE: There were 114 observations read from the data set WORK.FOURC. NOTE: The data set WORK.FOURC has 114 observations and 10 variables. NOTE: PROCEDURE SORT used (Total process time): real time 0.03 seconds cpu time 0.03 seconds 59 60 61 data one; infile 'e:\documents\b1.csv' dsd firstobs=3 missover; 62 input loc$ winery$ wine$ vin b1-b3; 63 length trt$ 30; 64 trt=compress(winery||wine||vin); 65 title 'procyanidin B1'; NOTE: Numeric values have been converted to character values at the places given by: (Line):(Column). 64:28 NOTE: The infile 'e:\documents\b1.csv' is: File Name=e:\documents\b1.csv, RECFM=V,LRECL=256 NOTE: 40 records were read from the infile 'e:\documents\b1.csv'. The minimum record length was 6. The maximum record length was 60. NOTE: The data set WORK.ONE has 40 observations and 8 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 66 proc print; 67 NOTE: There were 40 observations read from the data set WORK.ONE. NOTE: The PROCEDURE PRINT printed page 23. NOTE: PROCEDURE PRINT used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 68 data two; set one; 69 ba=b1; bot=1; output; 70 ba=b2; bot=2; output; 71 ba=b3; bot=3; output; 72 73 *proc print; NOTE: There were 40 observations read from the data set WORK.ONE. NOTE: The data set WORK.TWO has 120 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 74 proc glm; class trt; 75 model ba=trt; 76 means trt/lsd tukey lines; 77 NOTE: The PROCEDURE GLM printed pages 24-29. NOTE: PROCEDURE GLM used (Total process time):

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5 The SAS System 11:38 Monday, October 19, 2009 real time 0.12 seconds cpu time 0.12 seconds 78 data three; set two; 79 if wine='Norton'; NOTE: There were 120 observations read from the data set WORK.TWO. NOTE: The data set WORK.THREE has 60 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 80 proc glm; class loc; 81 model ba=loc; 82 means loc/lsd tukey lines; 83 84 NOTE: The PROCEDURE GLM printed pages 30-33. NOTE: PROCEDURE GLM used (Total process time): real time 0.04 seconds cpu time 0.04 seconds 85 data fourb1; set two; NOTE: There were 120 observations read from the data set WORK.TWO. NOTE: The data set WORK.FOURB1 has 120 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 86 proc sort; by trt bot; 87 88 run; NOTE: There were 120 observations read from the data set WORK.FOURB1. NOTE: The data set WORK.FOURB1 has 120 observations and 10 variables. NOTE: PROCEDURE SORT used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 89 90 91 92 data one; infile 'e:\documents\b2.csv' dsd firstobs=3 missover; 93 input loc$ winery$ wine$ vin b1-b3; 94 length trt$ 30; 95 trt=compress(winery||wine||vin); 96 title 'procyanidin b2'; NOTE: Numeric values have been converted to character values at the places given by: (Line):(Column). 95:28 NOTE: The infile 'e:\documents\b2.csv' is: File Name=e:\documents\b2.csv, RECFM=V,LRECL=256 NOTE: 38 records were read from the infile 'e:\documents\b2.csv'. The minimum record length was 42. The maximum record length was 57. NOTE: The data set WORK.ONE has 38 observations and 8 variables. NOTE: DATA statement used (Total process time): real time 0.03 seconds

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6 The SAS System 11:38 Monday, October 19, 2009 cpu time 0.03 seconds 97 proc print; 98 NOTE: There were 38 observations read from the data set WORK.ONE. NOTE: The PROCEDURE PRINT printed page 34. NOTE: PROCEDURE PRINT used (Total process time): real time 0.00 seconds cpu time 0.00 seconds 99 data two; set one; 100 bb=b1; bot=1; output; 101 bb=b2; bot=2; output; 102 bb=b3; bot=3; output; 103 104 *proc print; NOTE: There were 38 observations read from the data set WORK.ONE. NOTE: The data set WORK.TWO has 114 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.03 seconds cpu time 0.03 seconds 105 proc glm; class trt; 106 model bb=trt; 107 means trt/lsd tukey lines; 108 NOTE: The PROCEDURE GLM printed pages 35-40. NOTE: PROCEDURE GLM used (Total process time): real time 0.10 seconds cpu time 0.09 seconds 109 data three; set two; 110 if wine='Norton'; NOTE: There were 114 observations read from the data set WORK.TWO. NOTE: The data set WORK.THREE has 60 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 111 proc glm; class loc; 112 model bb=loc; 113 means loc/lsd tukey lines; 114 115 NOTE: The PROCEDURE GLM printed pages 41-44. NOTE: PROCEDURE GLM used (Total process time): real time 0.04 seconds cpu time 0.04 seconds 116 data fourb2; set two; NOTE: There were 114 observations read from the data set WORK.TWO. NOTE: The data set WORK.FOURB2 has 114 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.00 seconds cpu time 0.00 seconds

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7 The SAS System 11:38 Monday, October 19, 2009 117 proc sort; by trt bot; 118 119 run; NOTE: There were 114 observations read from the data set WORK.FOURB2. NOTE: The data set WORK.FOURB2 has 114 observations and 10 variables. NOTE: PROCEDURE SORT used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 120 121 122 data one; infile 'e:\documents\r.csv' dsd firstobs=3 missover; 123 input loc$ winery$ wine$ vin r1-r3; 124 length trt$ 30; 125 trt=compress(winery||wine||vin); 126 title 'resveratrol'; NOTE: Numeric values have been converted to character values at the places given by: (Line):(Column). 125:28 NOTE: The infile 'e:\documents\r.csv' is: File Name=e:\documents\r.csv, RECFM=V,LRECL=256 NOTE: 38 records were read from the infile 'e:\documents\r.csv'. The minimum record length was 48. The maximum record length was 63. NOTE: The data set WORK.ONE has 38 observations and 8 variables. NOTE: DATA statement used (Total process time): real time 0.03 seconds cpu time 0.03 seconds 127 proc print; 128 NOTE: There were 38 observations read from the data set WORK.ONE. NOTE: The PROCEDURE PRINT printed page 45. NOTE: PROCEDURE PRINT used (Total process time): real time 0.00 seconds cpu time 0.00 seconds 129 data two; set one; 130 r=r1; bot=1; output; 131 r=r2; bot=2; output; 132 r=r3; bot=3; output; 133 134 *proc print; NOTE: There were 38 observations read from the data set WORK.ONE. NOTE: The data set WORK.TWO has 114 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 135 proc glm; class trt; 136 model r=trt; 137 means trt/lsd tukey lines; 138 NOTE: The PROCEDURE GLM printed pages 46-51. NOTE: PROCEDURE GLM used (Total process time): real time 0.12 seconds

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8 The SAS System 11:38 Monday, October 19, 2009 cpu time 0.09 seconds 139 data three; set two; 140 if wine='Norton'; NOTE: There were 114 observations read from the data set WORK.TWO. NOTE: The data set WORK.THREE has 60 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 141 proc glm; class loc; 142 model r=loc; 143 means loc/lsd tukey lines; 144 145 NOTE: The PROCEDURE GLM printed pages 52-55. NOTE: PROCEDURE GLM used (Total process time): real time 0.06 seconds cpu time 0.06 seconds 146 data fourr; set two; NOTE: There were 114 observations read from the data set WORK.TWO. NOTE: The data set WORK.FOURR has 114 observations and 10 variables. NOTE: DATA statement used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 147 proc sort; by trt bot; 148 149 NOTE: There were 114 observations read from the data set WORK.FOURR. NOTE: The data set WORK.FOURR has 114 observations and 10 variables. NOTE: PROCEDURE SORT used (Total process time): real time 0.01 seconds cpu time 0.01 seconds 150 data all; merge foure fourc fourb1 fourb2 fourr; by trt bot; 151 drop e1-e3 c1-c3 b1-b3 r1-r3; NOTE: There were 117 observations read from the data set WORK.FOURE. NOTE: There were 114 observations read from the data set WORK.FOURC. NOTE: There were 120 observations read from the data set WORK.FOURB1. NOTE: There were 114 observations read from the data set WORK.FOURB2. NOTE: There were 114 observations read from the data set WORK.FOURR. NOTE: The data set WORK.ALL has 120 observations and 11 variables. NOTE: DATA statement used (Total process time): real time 0.04 seconds cpu time 0.04 seconds 152 proc print; NOTE: There were 120 observations read from the data set WORK.ALL. NOTE: The PROCEDURE PRINT printed pages 56-57. NOTE: PROCEDURE PRINT used (Total process time): real time 0.00 seconds cpu time 0.00 seconds

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9 The SAS System 11:38 Monday, October 19, 2009 153 proc corr; 154 var e c ba bb r; 155 156 run; NOTE: The PROCEDURE CORR printed page 58. NOTE: PROCEDURE CORR used (Total process time): real time 0.03 seconds cpu time 0.03 seconds NOTE: SAS Institute Inc., SAS Campus Drive, Cary, NC USA 27513-2414 NOTE: The SAS System used: real time 3.31 seconds cpu time 4.24 seconds Epicatechin 11:38 Monday, October 19, 2009 4 epicatechin 11:38 Monday, October 19, 2009 3 The GLM Procedure Dependent Variable: e Sum of Source DF Squares Mean Square F Value Pr > F Model 37 3989.436042 107.822596 595.99 <.0001 Error 76 13.749533 0.180915 Corrected Total 113 4003.185575 R-Square Coeff Var Root MSE e Mean 0.996565 10.36929 0.425341 4.101930 Source DF Type I SS Mean Square F Value Pr > F trt 37 3989.436042 107.822596 595.99 <.0001 Source DF Type III SS Mean Square F Value Pr > F trt 37 3989.436042 107.822596 595.99 <.0001 The GLM Procedure t Tests (LSD) for e NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 0.180915 Critical Value of t 1.99167

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Least Significant Difference 0.6917 Means with the same letter are not significantly different. t Grouping Mean N trt A 29.4767 3 Winery 2Chambour2004 B 16.2333 3 St.JameNorton2004 B B 15.9067 3 AdamPucNorton2003 C 11.6867 3 St.JameNorotn2003 C C 11.6633 3 St.JameChambour2003 D 10.4533 3 BaltimorChambour2004 E 6.4133 3 AdamPucNorton2002 F 5.6267 3 St.JameSchoolH2006 F F 5.2833 3 AdamPucNorton2004 G 3.4933 3 StoneHiNorton2006 G G 3.3700 3 MountPlNorton2006 G G 3.3500 3 St.JameBlackber2006 G H G 3.1867 3 StoneHiNorton2007 H G H G 3.1833 3 BaltimorNorton2003 H G H G I 2.8400 3 Winery 2Chambour2003 H I H I 2.5933 3 Winery 2Norton2004 H I H I 2.5800 3 St.JameNorton2002 I J I 2.2033 3 St.JameChambour2004 J I J I 2.1767 3 BaltimorNorton2004 J K J 1.8800 3 MountPlNorton2005 K J K J L 1.6300 3 St.JameNorton2000 K L K M L 1.3367 3 St.JameChambour2005 K M L K M L 1.2933 3 BaltimorChambour2003 M L N M L 1.1267 3 StoneHiNorton2004 N M N M O 0.9333 3 St.JameSchoolH2005 N M O N P M O 0.8667 3 MountPlNorton2004 N P M O N P M O 0.8233 3 LesBourChambour2005 N P M O N P M O 0.6767 3 MountPlNorton2003 N P O N P O 0.5500 3 Winery 2Norton2003 N P O N P O 0.4767 3 LesBourPremium2005 P O P O 0.4233 3 LesBourPremium2003 P O P O 0.4200 3 St.JameCherry2006

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P O P O 0.4033 3 StoneHiNorton2005 P O P O 0.3133 3 AdamPucNorton2001 P O P O 0.3000 3 StoneHiNorton2003 P O P O 0.2433 3 LesBourPremium2002 P P 0.2333 3 LesBourR.Norto2005 P P 0.2233 3 St.JameStrawber2005 epicatechin 11:38 Monday, October 19, 2009 9 The GLM Procedure Dependent Variable: e Sum of Source DF Squares Mean Square F Value Pr > F Model 3 171.489465 57.163155 3.12 0.0331 Error 56 1025.880169 18.319289 Corrected Total 59 1197.369633 R-Square Coeff Var Root MSE e Mean 0.143222 118.6172 4.280104 3.608333 Source DF Type I SS Mean Square F Value Pr > F loc 3 171.4894648 57.1631549 3.12 0.0331 Source DF Type III SS Mean Square F Value Pr > F loc 3 171.4894648 57.1631549 3.12 0.0331

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epicatechin 11:38 Monday, October 19, 2009 6 The GLM Procedure Tukey's Studentized Range (HSD) Test for e NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 0.180915 Critical Value of Studentized Range 5.68949 Minimum Significant Difference 1.3972 Means with the same letter are not significantly different. Tukey Grouping Mean N trt A 29.4767 3 Winery 2Chambour2004 B 16.2333 3 St.JameNorton2004 B B 15.9067 3 AdamPucNorton2003 C 11.6867 3 St.JameNorotn2003 C C 11.6633 3 St.JameChambour2003 C C 10.4533 3 BaltimorChambour2004 D 6.4133 3 AdamPucNorton2002 D D 5.6267 3 St.JameSchoolH2006 D D 5.2833 3 AdamPucNorton2004 E 3.4933 3 StoneHiNorton2006 E E 3.3700 3 MountPlNorton2006 E E 3.3500 3 St.JameBlackber2006 E F E 3.1867 3 StoneHiNorton2007 F E F E 3.1833 3 BaltimorNorton2003 F E F E G 2.8400 3 Winery 2Chambour2003 F E G F H E G 2.5933 3 Winery 2Norton2004 F H E G F H E G 2.5800 3 St.JameNorton2002 F H E G I F H E G 2.2033 3 St.JameChambour2004 I F H E G I F H E G 2.1767 3 BaltimorNorton2004 I F H G I F H J G 1.8800 3 MountPlNorton2005 I H J G I K H J G 1.6300 3 St.JameNorton2000 I K H J I K H J L 1.3367 3 St.JameChambour2005 I K H J L I K H J L 1.2933 3 BaltimorChambour2003 I K J L I K J L 1.1267 3 StoneHiNorton2004

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I K J L I K J L 0.9333 3 St.JameSchoolH2005 I K J L I K J L 0.8667 3 MountPlNorton2004 I K J L I K J L 0.8233 3 LesBourChambour2005 K J L K J L 0.6767 3 MountPlNorton2003 K J L K J L 0.5500 3 Winery 2Norton2003 K L K L 0.4767 3 LesBourPremium2005 K L K L 0.4233 3 LesBourPremium2003 K L K L 0.4200 3 St.JameCherry2006 K L K L 0.4033 3 StoneHiNorton2005 K L K L 0.3133 3 AdamPucNorton2001 K L K L 0.3000 3 StoneHiNorton2003 K L K L 0.2433 3 LesBourPremium2002 K L K L 0.2333 3 LesBourR.Norto2005 L L 0.2233 3 St.JameStrawber2005

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epicatechin 11:38 Monday, October 19, 2009 10 The GLM Procedure t Tests (LSD) for e NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 18.31929 Critical Value of t 2.00324 Least Significant Difference 3.6897 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. t Grouping Mean N loc A 6.814 9 St. Jame A B A 4.047 27 Herman B B 2.680 6 Waverly B B 1.656 18 Winery 2 epicatechin 11:38 Monday, October 19, 2009 11 The GLM Procedure Tukey's Studentized Range (HSD) Test for e NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 18.31929 Critical Value of Studentized Range 3.74475 Minimum Significant Difference 4.8771 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. Tukey Grouping Mean N loc A 6.814 9 St. Jame A B A 4.047 27 Herman B A B A 2.680 6 Waverly B B 1.656 18 Winery 2

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catechin 11:38 Monday, October 19, 2009 14 The GLM Procedure Dependent Variable: c Sum of Source DF Squares Mean Square F Value Pr > F Model 37 2901.723775 78.424967 162.44 <.0001 Error 76 36.691467 0.482782 Corrected Total 113 2938.415242 R-Square Coeff Var Root MSE c Mean 0.987513 16.10158 0.694825 4.315263 Source DF Type I SS Mean Square F Value Pr > F trt 37 2901.723775 78.424967 162.44 <.0001 Source DF Type III SS Mean Square F Value Pr > F trt 37 2901.723775 78.424967 162.44 <.0001

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catechin 11:38 Monday, October 19, 2009 15 The GLM Procedure t Tests (LSD) for c NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 0.482782 Critical Value of t 1.99167 Least Significant Difference 1.1299 Means with the same letter are not significantly different. t Grouping Mean N trt A 20.7867 3 St.JameSchoolH2006 B 15.9100 3 MountPlNorton2006 B B 15.7067 3 StoneHiNorton2007 C 14.2167 3 Winery 2Chambour2003 D 12.0433 3 Winery 2Norton2004 E 7.7967 3 MountPlNorton2005 E E 7.6967 3 St.JameNorton2000 E E 7.4633 3 StoneHiNorton2006 F 5.7800 3 St.JameChambour2005 F F 5.7000 3 St.JameBlackber2006 F F 5.4833 3 BaltimorChambour2003 F G F 4.9100 3 St.JameSchoolH2005 G G H 3.9100 3 St.JameStrawber2005 H I H 3.7400 3 MountPlNorton2004 I H I H 3.6400 3 Winery 2Norton2003 I H I H 3.4500 3 MountPlNorton2003 I H I H 2.9967 3 BaltimorChambour2004 I H I H J 2.8733 3 Winery 2Chambour2004 I J I K J 2.6433 3 StoneHiNorton2003 K J L K J 1.8000 3 St.JameCherry2006 L K J L K J 1.7600 3 AdamPucNorton2002 L K L K M 1.6833 3 LesBourChambour2005 L K M L N K M 1.5667 3 AdamPucNorton2001 L N K M L N K M 1.5433 3 St.JameNorton2002 L N M

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L N O M 1.1467 3 LesBourR.Norto2005 catechin 11:38 Monday, October 19, 2009 16 The GLM Procedure t Tests (LSD) for c Means with the same letter are not significantly different. t Grouping Mean N trt L N O M L N O M 1.1367 3 StoneHiNorton2005 L N O M L N O M 1.0367 3 St.JameNorton2004 L N O M L N O M 0.8833 3 St.JameNorotn2003 L N O M L N O M 0.8800 3 St.JameChambour2003 L N O M L N O M 0.8767 3 BaltimorNorton2004 N O M N O M 0.6267 3 AdamPucNorton2003 N O M N O M 0.5933 3 AdamPucNorton2004 N O N O 0.5033 3 StoneHiNorton2004 O O 0.4133 3 LesBourPremium2003 O O 0.2367 3 St.JameChambour2004 O O 0.1900 3 LesBourPremium2005 O O 0.1867 3 BaltimorNorton2003 O O 0.1700 3 LesBourPremium2002 catechin 11:38 Monday, October 19, 2009 17 The GLM Procedure Tukey's Studentized Range (HSD) Test for c NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 0.482782 Critical Value of Studentized Range 5.68949 Minimum Significant Difference 2.2824 Means with the same letter are not significantly different. Tukey Grouping Mean N trt A 20.7867 3 St.JameSchoolH2006 B 15.9100 3 MountPlNorton2006 B

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B 15.7067 3 StoneHiNorton2007 B C B 14.2167 3 Winery 2Chambour2003 C C 12.0433 3 Winery 2Norton2004 D 7.7967 3 MountPlNorton2005 D E D 7.6967 3 St.JameNorton2000 E D E D 7.4633 3 StoneHiNorton2006 E D E D F 5.7800 3 St.JameChambour2005 E D F E G D F 5.7000 3 St.JameBlackber2006 E G F E G F 5.4833 3 BaltimorChambour2003 G F G H F 4.9100 3 St.JameSchoolH2005 G H F I G H F 3.9100 3 St.JameStrawber2005 I G H F J I G H F 3.7400 3 MountPlNorton2004 J I G H F J I G H F 3.6400 3 Winery 2Norton2003 J I G H J I G H 3.4500 3 MountPlNorton2003 J I H J I H K 2.9967 3 BaltimorChambour2004 J I H K J I L H K 2.8733 3 Winery 2Chambour2004 J I L H K J I L H K M 2.6433 3 StoneHiNorton2003 J I L K M J I L N K M 1.8000 3 St.JameCherry2006 J I L N K M J I L N K M 1.7600 3 AdamPucNorton2002 J I L N K M J I L N K M 1.6833 3 LesBourChambour2005 J L N K M J L N K M 1.5667 3 AdamPucNorton2001 J L N K M J L N K M 1.5433 3 St.JameNorton2002 L N K M L N K M 1.1467 3 LesBourR.Norto2005 L N K M L N K M 1.1367 3 StoneHiNorton2005 L N K M L N K M 1.0367 3 St.JameNorton2004 L N K M L N K M 0.8833 3 St.JameNorotn2003 L N K M L N K M 0.8800 3 St.JameChambour2003 L N K M L N K M 0.8767 3 BaltimorNorton2004 L N M L N M 0.6267 3 AdamPucNorton2003 L N M L N M 0.5933 3 AdamPucNorton2004 N M N M 0.5033 3 StoneHiNorton2004 N M N M 0.4133 3 LesBourPremium2003 N N 0.2367 3 St.JameChambour2004 N N 0.1900 3 LesBourPremium2005 N N 0.1867 3 BaltimorNorton2003 N N 0.1700 3 LesBourPremium2002

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catechin 11:38 Monday, October 19, 2009 20 The GLM Procedure Dependent Variable: c Sum of Source DF Squares Mean Square F Value Pr > F Model 3 320.646468 106.882156 5.34 0.0026 Error 56 1121.512972 20.027017 Corrected Total 59 1442.159440 R-Square Coeff Var Root MSE c Mean 0.222338 99.53638 4.475156 4.496000 Source DF Type I SS Mean Square F Value Pr > F loc 3 320.6464678 106.8821559 5.34 0.0026 Source DF Type III SS Mean Square F Value Pr > F loc 3 320.6464678 106.8821559 5.34 0.0026 catechin 11:38 Monday, October 19, 2009 21 The GLM Procedure t Tests (LSD) for c NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 20.02702 Critical Value of t 2.00324 Least Significant Difference 3.8578 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. t Grouping Mean N loc A 7.763 18 Winery 2 B 3.556 27 Herman B B 3.426 9 St. Jame B B 0.532 6 Waverly

94

catechin 11:38 Monday, October 19, 2009 22 The GLM Procedure Tukey's Studentized Range (HSD) Test for c NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 20.02702 Critical Value of Studentized Range 3.74475 Minimum Significant Difference 5.0994 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. Tukey Grouping Mean N loc A 7.763 18 Winery 2 A B A 3.556 27 Herman B A B A 3.426 9 St. Jame B B 0.532 6 Waverly procyanidin B1 11:38 Monday, October 19, 2009 25 The GLM Procedure Dependent Variable: ba Sum of Source DF Squares Mean Square F Value Pr > F Model 37 20043.92573 541.72772 126.65 <.0001 Error 76 325.08340 4.27741 Corrected Total 113 20369.00913 R-Square Coeff Var Root MSE ba Mean 0.984040 11.69571 2.068191 17.68333 Source DF Type I SS Mean Square F Value Pr > F trt 37 20043.92573 541.72772 126.65 <.0001 Source DF Type III SS Mean Square F Value Pr > F

95

trt 37 20043.92573 541.72772 126.65 <.0001 procyanidin B1 11:38 Monday, October 19, 2009 26 The GLM Procedure t Tests (LSD) for ba NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 4.277413 Critical Value of t 1.99167 Least Significant Difference 3.3633 Means with the same letter are not significantly different. t Grouping Mean N trt A 52.160 3 MountPlNorton2006 B 40.057 3 St.JameNorton2002 B C B 38.310 3 St.JameSchoolH2006 C B C B 37.983 3 StoneHiNorton2007 C C D 35.060 3 StoneHiNorton2004 D E D 32.967 3 MountPlNorton2005 E D E D 32.557 3 MountPlNorton2004 E E F 31.117 3 St.JameNorton2000 E F E F 30.380 3 AdamPucNorton2004 F G F 27.950 3 BaltimorNorton2004 G G 26.690 3 MountPlNorton2003 G G H 25.227 3 St.JameNorotn2003 G H G H 25.110 3 StoneHiNorton2006 H I H 22.330 3 BaltimorNorton2003 I H I H 22.120 3 StoneHiNorton2003 I I J 19.907 3 StoneHiNorton2005 J J 18.190 3 AdamPucNorton2003 K 13.813 3 St.JameChambour2004 K K 13.497 3 Winery 2Chambour2003 K L K 13.443 3 St.JameChambour2005 L K L K M 12.517 3 LesBourPremium2003 L K M L K M 11.887 3 Winery 2Norton2003 L K M

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L N K M 11.197 3 St.JameNorton2004 L N K M L N K M 10.620 3 AdamPucNorton2002 L N M L N M 10.130 3 BaltimorChambour2004 N M N O M 9.310 3 LesBourPremium2002 N O P N O 8.467 3 LesBourPremium2005 P N O P N O 8.423 3 Winery 2Norton2004 P N O P N O 7.923 3 LesBourR.Norto2005 P O P O Q 6.083 3 Winery 2Chambour2004 P Q P Q 5.360 3 BaltimorChambour2003 Q R Q 4.200 3 LesBourChambour2005 R Q S R Q 3.807 3 St.JameStrawber2005 S R S R T 1.483 3 St.JameChambour2003 S T S T 0.807 3 AdamPucNorton2001 S T S T 0.590 3 St.JameBlackber2006 T T 0.297 3 St.JameSchoolH2005 T T 0.000 3 St.JameCherry2006 The GLM Procedure Tukey's Studentized Range (HSD) Test for ba NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 4.277413 Critical Value of Studentized Range 5.68949 Minimum Significant Difference 6.7937 Means with the same letter are not significantly different. Tukey Grouping Mean N trt A 52.160 3 MountPlNorton2006 B 40.057 3 St.JameNorton2002 B C B 38.310 3 St.JameSchoolH2006 C B C B 37.983 3 StoneHiNorton2007 C B C B D 35.060 3 StoneHiNorton2004 C D C E D 32.967 3 MountPlNorton2005 C E D C E D 32.557 3 MountPlNorton2004 E D F E D 31.117 3 St.JameNorton2000 F E D

97

F E D 30.380 3 AdamPucNorton2004 F E F E G 27.950 3 BaltimorNorton2004 F E G F H E G 26.690 3 MountPlNorton2003 F H G F H G 25.227 3 St.JameNorotn2003 F H G F H G 25.110 3 StoneHiNorton2006 H G H I G 22.330 3 BaltimorNorton2003 H I G H I G 22.120 3 StoneHiNorton2003 H I J H I 19.907 3 StoneHiNorton2005 J I J I K 18.190 3 AdamPucNorton2003 J K J L K 13.813 3 St.JameChambour2004 J L K J L K 13.497 3 Winery 2Chambour2003 J L K J L K 13.443 3 St.JameChambour2005 L K M L K 12.517 3 LesBourPremium2003 M L K M N L K 11.887 3 Winery 2Norton2003 M N L M N L 11.197 3 St.JameNorton2004 M N L M N L O 10.620 3 AdamPucNorton2002 M N L O P M N L O 10.130 3 BaltimorChambour2004 P M N L O P M N L O 9.310 3 LesBourPremium2002 P M N L O P M N L O 8.467 3 LesBourPremium2005 P M N L O P M N L O 8.423 3 Winery 2Norton2004 P M N L O P M N L O Q 7.923 3 LesBourR.Norto2005 P M N O Q P M N R O Q 6.083 3 Winery 2Chambour2004 P N R O Q P N R O Q 5.360 3 BaltimorChambour2003 P R O Q P R O Q 4.200 3 LesBourChambour2005 P R Q P R Q 3.807 3 St.JameStrawber2005 R Q R Q 1.483 3 St.JameChambour2003 R R 0.807 3 AdamPucNorton2001 R R 0.590 3 St.JameBlackber2006 R R 0.297 3 St.JameSchoolH2005 R R 0.000 3 St.JameCherry2006 procyanidin B1 11:38 Monday, October 19, 2009 31 The GLM Procedure Dependent Variable: ba Sum of Source DF Squares Mean Square F Value Pr > F Model 3 366.703916 122.234639 0.79 0.5060

98

Error 56 8693.162369 155.235042 Corrected Total 59 9059.866285 R-Square Coeff Var Root MSE ba Mean 0.040476 50.08677 12.45934 24.87550 Source DF Type I SS Mean Square F Value Pr > F loc 3 366.7039165 122.2346388 0.79 0.5060 Source DF Type III SS Mean Square F Value Pr > F loc 3 366.7039165 122.2346388 0.79 0.5060 procyanidin B1 11:38 Monday, October 19, 2009 32 The GLM Procedure t Tests (LSD) for ba NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 155.235 Critical Value of t 2.00324 Least Significant Difference 10.741 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. t Grouping Mean N loc A 27.457 9 St. Jame A A 27.447 18 Winery 2 A A 25.140 6 Waverly A A 22.242 27 Herman procyanidin B1 11:38 Monday, October 19, 2009 33 The GLM Procedure Tukey's Studentized Range (HSD) Test for ba NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ.

99

Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 155.235 Critical Value of Studentized Range 3.74475 Minimum Significant Difference 14.197 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. Tukey Grouping Mean N loc A 27.457 9 St. Jame A A 27.447 18 Winery 2 A A 25.140 6 Waverly A A 22.242 27 Herman procyanidin b2 11:38 Monday, October 19, 2009 36 The GLM Procedure Dependent Variable: bb Sum of Source DF Squares Mean Square F Value Pr > F Model 37 2363.902745 63.889263 114.64 <.0001 Error 76 42.354733 0.557299 Corrected Total 113 2406.257478 R-Square Coeff Var Root MSE bb Mean 0.982398 14.98914 0.746525 4.980439 Source DF Type I SS Mean Square F Value Pr > F trt 37 2363.902745 63.889263 114.64 <.0001 Source DF Type III SS Mean Square F Value Pr > F trt 37 2363.902745 63.889263 114.64 <.0001 procyanidin b2 11:38 Monday, October 19, 2009 37 The GLM Procedure t Tests (LSD) for bb NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate.

100

Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 0.557299 Critical Value of t 1.99167 Least Significant Difference 1.214 Means with the same letter are not significantly different. t Grouping Mean N trt A 23.9500 3 BaltimorChambour2004 B 15.6933 3 StoneHiNorton2007 C 12.9467 3 StoneHiNorton2006 D 10.5567 3 St.JameBlackber2006 E 8.6800 3 St.JameSchoolH2006 F 7.2333 3 St.JameChambour2004 F G F 6.8367 3 St.JameNorotn2003 G F G F H 6.7633 3 MountPlNorton2006 G F H G I F H 6.7367 3 MountPlNorton2004 G I H G I J H 6.0167 3 BaltimorNorton2004 I J H K I J H 5.5500 3 Winery 2Norton2004 K I J K I J 5.5433 3 MountPlNorton2005 K J K J 5.2467 3 St.JameChambour2003 K J K J L 4.9900 3 Winery 2Chambour2003 K J L K J L 4.9100 3 Winery 2Norton2003 K J L K J L 4.8867 3 LesBourChambour2005 K J L K J L 4.8500 3 AdamPucNorton2001 K L K M L 4.5200 3 AdamPucNorton2004 K M L K N M L 4.3700 3 LesBourPremium2003 N M L N M L 3.9833 3 LesBourPremium2002 N M L N M L 3.9533 3 AdamPucNorton2003 N M L N M L 3.7900 3 St.JameSchoolH2005 N M O N M 3.4033 3 AdamPucNorton2002 O N O N 3.2000 3 BaltimorNorton2003 O O P 2.5533 3 St.JameChambour2005 O P O P 2.4733 3 BaltimorChambour2003 O P O P Q 2.3900 3 MountPlNorton2003 P Q R P Q 1.9700 3 St.JameNorton2002 R P Q R S P Q 1.7000 3 LesBourPremium2005 R S P Q

101

R S P Q 1.5733 3 St.JameNorton2004 R S P Q R S P Q 1.4133 3 LesBourR.Norto2005 R S P Q R S P Q 1.3967 3 St.JameNorton2000 R S Q R S Q 1.2267 3 StoneHiNorton2004 R S Q R S Q 1.2267 3 StoneHiNorton2005 R S R S T 1.1400 3 St.JameStrawber2005 R S T R S T 1.0067 3 StoneHiNorton2003 S T S T 0.5767 3 Winery 2Chambour2004 T T 0.0000 3 St.JameCherry2006 procyanidin b2 11:38 Monday, October 19, 2009 39 The GLM Procedure Tukey's Studentized Range (HSD) Test for bb NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 0.557299 Critical Value of Studentized Range 5.68949 Minimum Significant Difference 2.4522 Means with the same letter are not significantly different. Tukey Grouping Mean N trt A 23.9500 3 BaltimorChambour2004 B 15.6933 3 StoneHiNorton2007 C 12.9467 3 StoneHiNorton2006 C D C 10.5567 3 St.JameBlackber2006 D D E 8.6800 3 St.JameSchoolH2006 E F E 7.2333 3 St.JameChambour2004 F E F E G 6.8367 3 St.JameNorotn2003 F E G F H E G 6.7633 3 MountPlNorton2006 F H E G F H E G 6.7367 3 MountPlNorton2004 F H G F H I G 6.0167 3 BaltimorNorton2004 F H I G J F H I G 5.5500 3 Winery 2Norton2004 J F H I G J F H I G 5.5433 3 MountPlNorton2005 J F H I G J F H I G 5.2467 3 St.JameChambour2003 J F H I G J F H I G K 4.9900 3 Winery2Chambour2003 J F H I G K

102

J F H I L G K 4.9100 3 Winery 2Norton2003 J F H I L G K J F H I L G K 4.8867 3 LesBourChambour2005 J F H I L G K J F H I L G K 4.8500 3 AdamPucNorton2001 J H I L G K J M H I L G K 4.5200 3 AdamPucNorton2004 J M H I L K J M H I L N K 4.3700 3 LesBourPremium2003 J M I L N K J M O I L N K 3.9833 3 LesBourPremium2002 J M O I L N K J M O I L N K 3.9533 3 AdamPucNorton2003 J M O I L N K J M O P I L N K 3.7900 3 St.JameSchoolH2005 J M O P L N K J M O P Q L N K 3.4033 3 AdamPucNorton2002 J M O P Q L N K J M O P Q L N K 3.2000 3 BaltimorNorton2003 M O P Q L N K R M O P Q L N K 2.5533 3 St.JameChambour2005 R M O P Q L N R M O P Q L N 2.4733 3 BaltimorChambour2003 R M O P Q N R M O P Q N S 2.3900 3 MountPlNorton2003 R O P Q N S R O P Q N S 1.9700 3 St.JameNorton2002 R O P Q S R O P Q S 1.7000 3 LesBourPremium2005 R O P Q S R O P Q S 1.5733 3 St.JameNorton2004 R P Q S R P Q S 1.4133 3 LesBourR.Norto2005 R P Q S R P Q S 1.3967 3 St.JameNorton2000 R Q S R Q S 1.2267 3 StoneHiNorton2004 R Q S R Q S 1.2267 3 StoneHiNorton2005 R Q S R Q S 1.1400 3 St.JameStrawber2005 R Q S R Q S 1.0067 3 StoneHiNorton2003 R S R S 0.5767 3 Winery2Chambour2004 S S 0.0000 3 St.JameCherry2006

103

procyanidin b2 11:38 Monday, October 19, 2009 42 The GLM Procedure Dependent Variable: bb Sum of Source DF Squares Mean Square F Value Pr > F Model 3 104.8602165 34.9534055 2.63 0.0591 Error 56 744.8340019 13.3006072 Corrected Total 59 849.6942183 R-Square Coeff Var Root MSE bb Mean 0.123409 76.87875 3.647000 4.743833 Source DF Type I SS Mean Square F Value Pr > F loc 3 104.8602165 34.9534055 2.63 0.0591 Source DF Type III SS Mean Square F Value Pr > F loc 3 104.8602165 34.9534055 2.63 0.0591 procyanidin b2 11:38 Monday, October 19, 2009 43 The GLM Procedure t Tests (LSD) for bb NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 13.30061 Critical Value of t 2.00324 Least Significant Difference 3.1439 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. t Grouping Mean N loc A 5.425 27 Herman A A 5.316 18 Winery 2 A B A 4.608 6 Waverly B B 1.647 9 St. Jame

104

procyanidin b2 11:38 Monday, October 19, 2009 44 The GLM Procedure Tukey's Studentized Range (HSD) Test for bb NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 13.30061 Critical Value of Studentized Range 3.74475 Minimum Significant Difference 4.1557 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. Tukey Grouping Mean N loc A 5.425 27 Herman A A 5.316 18 Winery 2 A A 4.608 6 Waverly A A 1.647 9 St. Jame resveratrol 11:38 Monday, October 19, 2009 47 The GLM Procedure Dependent Variable: r Sum of Source DF Squares Mean Square F Value Pr > F Model 37 19.53164060 0.52788218 38.08 <.0001 Error 76 1.05354667 0.01386246 Corrected Total 113 20.58518727 R-Square Coeff Var Root MSE r Mean 0.948820 22.23031 0.117739 0.529632 Source DF Type I SS Mean Square F Value Pr > F trt 37 19.53164060 0.52788218 38.08 <.0001 Source DF Type III SS Mean Square F Value Pr > F

105

trt 37 19.53164060 0.52788218 38.08 <.000 resveratrol 11:38 Monday, October 19, 2009 48 The GLM Procedure t Tests (LSD) for r NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 0.013862 Critical Value of t 1.99167 Least Significant Difference 0.1915 Means with the same letter are not significantly different. t Grouping Mean N trt A 1.52187 3 MountPlNorton2004 A A 1.49617 3 Winery 2Norton2003 A B A 1.33053 3 AdamPucNorton2004 B B C 1.21950 3 Winery 2Norton2004 B C B C 1.14123 3 BaltimorNorton2003 C C 1.11993 3 MountPlNorton2003 D 0.89220 3 StoneHiNorton2003 D E D 0.80503 3 BaltimorNorton2004 E D E D 0.80160 3 Winery 2Chambour2004 E D E D 0.79557 3 BaltimorChambour2003 E D E D 0.78050 3 St.JameChambour2004 E D E D 0.78027 3 StoneHiNorton2004 E E F 0.66390 3 StoneHiNorton2006 E F E F 0.65063 3 MountPlNorton2006 F G F 0.56903 3 StoneHiNorton2005 G G H 0.44063 3 St.JameNorton2004 G H G H I 0.39810 3 St.JameNorton2002 G H I G H I 0.39283 3 St.JameNorotn2003 G H I G H I 0.38253 3 StoneHiNorton2007 G H I G J H I 0.38177 3 MountPlNorton2005 J H I J H I 0.36750 3 BaltimorChambour2004 J H I K J H I 0.32367 3 St.JameChambour2003 K J H I

106

K J H I 0.32113 3 Winery 2Chambour2003 K J H I K J H I 0.31627 3 LesBourPremium2003 K J H I L K J H I 0.30863 3 St.JameChambour2005 L K J H I 0.30293 3 AdamPucNorton2003 L K J I L K J M I 0.20740 3 AdamPucNorton2001 L K J M L K J M 0.19067 3 LesBourPremium2005 L K M L K M 0.16860 3 LesBourChambour2005 L K M L K M 0.16717 3 St.JameSchoolH2005 L K M L K M 0.16607 3 LesBourPremium2002 L K M L K M 0.14493 3 AdamPucNorton2002 L K M L K M 0.14440 3 LesBourR.Norto2005 L M L M 0.11893 3 St.JameNorton2000 L M L M 0.11883 3 St.JameBlackber2006 M M 0.07947 3 St.JameStrawber2005 M M 0.06910 3 St.JameSchoolH2006 M M 0.04650 3 St.JameCherry2006 resveratrol 11:38 Monday, October 19, 2009 50 The GLM Procedure Tukey's Studentized Range (HSD) Test for r NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 76 Error Mean Square 0.013862 Critical Value of Studentized Range 5.68949 Minimum Significant Difference 0.3868 Means with the same letter are not significantly different. Tukey Grouping Mean N trt A 1.52187 3 MountPlNorton2004 A B A 1.49617 3 Winery 2Norton2003 B A B A 1.33053 3 AdamPucNorton2004 B A B A C 1.21950 3 Winery 2Norton2004 B A C B D A C 1.14123 3 BaltimorNorton2003 B D C B D C 1.11993 3 MountPlNorton2003 D C D E C 0.89220 3 StoneHiNorton2003 D E

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F D E 0.80503 3 BaltimorNorton2004 F D E F D E 0.80160 3 Winery 2Chambour2004 F D E F D E 0.79557 3 BaltimorChambour2003 F D E F D E G 0.78050 3 St.JameChambour2004 F D E G F D E G 0.78027 3 StoneHiNorton2004 F E G F H E G 0.66390 3 StoneHiNorton2006 F H E G F H E G 0.65063 3 MountPlNorton2006 F H E G I F H E G 0.56903 3 StoneHiNorton2005 I F H G I F H J G 0.44063 3 St.JameNorton2004 I H J G I K H J G 0.39810 3 St.JameNorton2002 I K H J I K H J 0.39283 3 St.JameNorotn2003 I K H J I K H J 0.38253 3 StoneHiNorton2007 I K H J I K H J 0.38177 3 MountPlNorton2005 I K H J I K H J 0.36750 3 BaltimorChambour2004 I K H J I K H J 0.32367 3 St.JameChambour2003 I K H J I K H J 0.32113 3 Winery 2Chambour2003 I K H J I K H J 0.31627 3 LesBourPremium2003 I K H J I K H J 0.30863 3 St.JameChambour2005 I K H J I K H J 0.30293 3 AdamPucNorton2003 I K J I K J 0.20740 3 AdamPucNorton2001 I K J I K J 0.19067 3 LesBourPremium2005 K J K J 0.16860 3 LesBourChambour2005 K J K J 0.16717 3 St.JameSchoolH2005 K J K J 0.16607 3 LesBourPremium2002 K J K J 0.14493 3 AdamPucNorton2002 K J K J 0.14440 3 LesBourR.Norto2005 K J K J 0.11893 3 St.JameNorton2000 K J K J 0.11883 3 St.JameBlackber2006 K J K J 0.07947 3 St.JameStrawber2005 K J K J 0.06910 3 St.JameSchoolH2006 K K 0.04650 3 St.JameCherry2006 resveratrol 11:38 Monday, October 19, 2009 53 The GLM Procedure Dependent Variable: r Sum of

108

Source DF Squares Mean Square F Value Pr > F Model 3 4.45305467 1.48435156 10.44 <.0001 Error 56 7.96481780 0.14222889 Corrected Total 59 12.41787247 R-Square Coeff Var Root MSE r Mean 0.358600 51.77712 0.377132 0.728377 Source DF Type I SS Mean Square F Value Pr > F loc 3 4.45305467 1.48435156 10.44 <.0001 Source DF Type III SS Mean Square F Value Pr > F loc 3 4.45305467 1.48435156 10.44 <.0001

109

resveratrol 11:38 Monday, October 19, 2009 54 The GLM Procedure t Tests (LSD) for r NOTE: This test controls the Type I comparisonwise error rate, not the experimentwise error rate. Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 0.142229 Critical Value of t 2.00324 Least Significant Difference 0.3251 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. t Grouping Mean N loc A 1.0650 18 Winery 2 A A 0.9731 6 Waverly B 0.5860 27 Herman B B 0.3192 9 St. Jame resveratrol 11:38 Monday, October 19, 2009 55 The GLM Procedure Tukey's Studentized Range (HSD) Test for r NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 56 Error Mean Square 0.142229 Critical Value of Studentized Range 3.74475 Minimum Significant Difference 0.4297 Harmonic Mean of Cell Sizes 10.8 NOTE: Cell sizes are not equal. Means with the same letter are not significantly different. Tukey Grouping Mean N loc A 1.0650 18 Winery 2 A B A 0.9731 6 Waverly B B C 0.5860 27 Herman C C 0.3192 9 St. Jame

110

resveratrol 11:38 Monday, October 19, 2009 58 The CORR Procedure 5 Variables: e c ba bb r Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum e 114 4.10193 5.95201 467.62000 0 29.90000 c 114 4.31526 5.09938 491.94000 0 21.30000 ba 114 17.68333 13.42597 2016 0 53.69000 bb 114 4.98044 4.61458 567.77000 0 24.72000 r 114 0.52963 0.42681 60.37810 0.01160 1.77340 Pearson Correlation Coefficients, N = 114 Prob > |r| under H0: Rho=0 e c ba bb r e 1.00000 -0.07840 -0.08906 0.08330 -0.00507 0.4070 0.3460 0.3783 0.9573 c -0.07840 1.00000 0.40166 0.34016 -0.07112 0.4070 <.0001 0.0002 0.4521 ba -0.08906 0.40166 1.00000 0.14418 0.28842 0.3460 <.0001 0.1259 0.0019 bb 0.08330 0.34016 0.14418 1.00000 -0.01708 0.3783 0.0002 0.1259 0.8568 r -0.00507 -0.07112 0.28842 -0.01708 1.00000 0.9573 0.4521 0.0019 0.8568

111

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