Research Round-Up

New insights on the relationship between diet, body weight, inflammation and chronic disease will be presented by nutritional experts in the field at a symposium, “Taming the Fires of Inflammation Through Diet,” sponsored by the California Strawberry Commission at the American Dietetic Association Food and Nutrition Conference and Expo in Honolulu, Hawaii on September 16, 2006. Zhaoping Li, MD, PhD, Associate Professor of Medicine, Center for Human Nutrition, David Geffen School of Medicine at University of California, Los Angeles will share a detailed look at the process of inflammation, discuss the effects of inflammation in normal weight adults, and emerging research from clinical trials looking at strawberry consumption’s impact on inflammation in normal weight adults. Britt Burton- Freeman, PhD, Assistant Research Nutritionist, Department of Nutrition, University of California, Davis will discuss the effects of inflammation in overweight and obese adults and emerging research from clinical trials looking at strawberry consumption’s impact on inflammation in overweight and obese adults. Below is a summary of their presentation.

Zhaoping Li, MD, PhD – “The Secret Killer,” a Time magazine cover story in February 2004 brought inflammation center stage, discussing the role of inflammation in cancer, diabetes, and cardiovascular, pulmonary, neurological, and autoimmune diseases. Inflammation is the first response of the immune system to infection or irritation, causing the production of cytokines. A cascade occurs as various leukocytes cause the initiation and maintenance of inflammation, further stimulated by lymphocytes such as T cells, B cells and antibodies. C-reactive protein (CRP) is produced by the liver in response to cytokine production. High-sensitivity CRP (hs-CRP) has been suggested to predict future cardiovascular events in healthy men and women. In fact, a meta-analysis published in the New England Journal of Medicine indicated that hs-CRP is a stronger predictor of future cardiovascular events than the total cholesterol/HDL cholesterol ratio, LDL cholesterol, total cholesterol, and homocysteine.

A correlation has been established between inflammation and diet. There is an inverse relationship between phytonutrients, non-nutritive compounds found in fruits and vegetables, and CRP. In a study published in the American Journal of Clinical Nutrition, participants who consumed eight servings of fruits and vegetables per day had significantly lower CRP levels than those who consumed two and five per day. Researchers at UCLA are identifying phytonutrients in strawberries, developing methods to detect strawberry phytonutrients and their biological activities in human blood and urine. They have found that strawberry phytonutrients include flavanoids such as anthocyanins and flavonols, hydrolyzable tannins such as ellagic acid and glycosides, and phenolic acids such as hydroxycinnamic acids and esters. Their research has suggested that bioactives of compounds in human plasma and urine have desirable effects on hs-CRP and inflammation.

Britt Burton-Freeman, PhD – Obesity is a major risk factor for many chronic diseases and increasing adiposity is associated with increased CRP. In fact, multiple studies showed that CRP was significantly higher in overweight and obese individuals than normal weight individuals. Adipose tissue is actually a dynamic tissue with endocrine function. Obesity represents a pro-inflammatory state and weight loss decreases the inflammatory state. Obesity can be a model for studying inflammatory response and researchers are asking the following questions: Can changing the dietary pattern, independent of weight loss, decrease the pro/anti-inflammatory response profile, and reduce disease risk? Can changing the dietary pattern in addition to weight loss augment/enhance the anti- inflammatory benefits of weight loss? Dietary intervention that increases phytonutrient intake does make a difference according to research published in the American Journal of Clinical Nutrition. These bioactive compounds quiet the endothelium, increase anti-oxidant activity, and decrease cytokine synthesis.

Research at UC Davis is focusing on human studies in overweight and obese subjects. Individuals are being fed a Strawberry Intervention diet (4 servings per day) or a Standard Diet. Endpoints being investigated include changes in lipid profile, the inflammatory profile, endothelial function, and oxidative stress.

View from the Lab

Navindra Seeram PhD, assistant director of the UCLA Center for Human Nutrition and adjunct assistant professor at the David Geffen School of Medicine at UCLA, and a member of the Commission’s Scientific Advisory Committee, discusses the potential chemopreventative benefits of berries in a chapter of the recently-published Nutritional Oncology. In the chapter, Seeram discusses the research supporting the role of berry consumption on cancer prevention and the disparate nutrients that may be responsible for berries’ positive benefits. A wide number of laboratory and animal studies have shown that berries may have anticancer properties which can be attributed to bioactive phytochemicals including flavanoids (anthocyanins, flavonols, and flavanols); tannins, such as proanthocyanidins (PAs), ellagitannins (ETs), and gallotannins (GTs); stilbenoids, such as resveratrol; phenolic acid (hydroxybenzoic and hydroxycinnamic acids); and lignans. Below is a summary of the research in the chapter relating to strawberries.

While flavonols are typically present in the leaves of plants and skins of fruits, they are found in the flesh of strawberries. Two of the most common flavonols, quercetin and kaempferol are present in substantial quantities in strawberries. Lignans, which are commonly found in flaxseed and linseed, have also been found in strawberries. The main phenolics in strawberries after anthocyanins are ellagitannins (ETs). Studies have suggested that berries high in ETs, including ellagic acid, show greater effects against oral cavity and esophageal cancers than berries abundant in tannins.

In a study investigating effects of purified berry bioactives as well as whole strawberry fruit extract on the viability and apoptosis of human hepatoma HepG2 cells, only quercetin and whole strawberry fruit extract inhibited viability and induced apoptosis. Another study demonstrated the inhibitory effects of strawberries on well-known tumor promoters, tetradecanoylphorbol-13-acetate (TPA) and ultraviolet B (UVB), which may be due to the antioxidant properties of strawberries.

Seeram, NP; Berries. Nutritional Oncology, second edition. Eds. Heber, D, Blackburn, G, Go, VLW, Milner, J; Academic Press, London, UK.

Increased Fruit Consumption Associated with Lower HbA1C

NHANES III data on more than 14,000 adults were analyzed for HbA1C (glycosylated hemoglobin) levels by tertiles of fruit and vegetable intake. Fruit and vegetable intake was measured by food frequency questionnaire. Data was examined within groups stratified by BMI (normal, overweight, obese) and sex/age (males aged 19 to 65 and pre- and post- menopausal females). Individuals with diabetes were excluded, as were subjects with extreme weight or height, and women who were pregnant, lactating, or missing menopausal status. Results indicated that increased frequency of fruit consumption was associated with lower HbA1C among overweight premenopausal and normal and overweight men . Increased frequency of vegetable intake was associated with lower HbA1C levels among normal premenopausal women. There appears to be a relationship between increased frequency of fruit or vegetable consumption and this long term marker of glycemic regulation.

Kimmons, J.E., et. al. HbA1C levels by frequency of fruit and vegetable consumption and BMI from NHANES III. Center for Disease Control and Prevention.

Anthocyanin-rich Diet and Type 2 Diabetes Risk

Noting that low intake of fruits is a risk factor for the development of diseases such as Type 2 diabetes, researchers from The University of Maine suggest that this may due to the ability of flavonoid pigments in red and blue fruits rich in anthocyanins to modulate alpha-glucosidase and aldose reductase activity and insulin production in vitro. When fruits are lacking from the diet, anthocyanins would also be absent. The researchers investigated the potential benefits of anthocyanin consumption in individuals at risk for developing diabetes. Subjects were paired by gender, age, and fasting blood glucose levels, and then each member of the pair were randomly assigned to the control or treatment group. The control group was instructed to make no changes in their diet or exercise habits and to limit consumption of anthocyanin-containing foods to no more than 3 servings per week. The treatment group was provided with a list of serving sizes for common fruits that provided 150 mg anthocyanins and instructed to consume at least two servings daily for three months. Fasting blood glucose, insulin, glycosylated hemoglobin, cholesterol, triglycerides, antioxidant capacity and urinary microalbumin were measured at baseline and at 6 and 12 weeks of the study. Results from the study are anticipated this spring.

Henderson, A., et. al. Can an anthocyanin-rich diet reduce risks for developing Type 2 diabetes? The University of Maine.

Metabolites of Strawberry Anthocyanins

Among berry bioactives, the metabolism and bioavailability of anthocyanins in both human and animal models have been well studied. Researchers from the United States Department of Agriculture (USDA) are currently working to identify four urinary metabolites

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of strawberry anthocyanins. Results on the identity of these metabolites are expected in the spring. What they have determined thus far in their investigation is the stability of the metabolites. The results suggest that three metabolites degrade over time while one peak area increased over time, indicating it may be a degradation product of the other metabolites.

Carkeet, C., et. al. Stability of Urinary Metabolites of Strawberry Anthocyanins. Beltsville Human Nutrition Research Center, USDA.

Antioxidant Compounds in Strawberries Investigated

Fruits are known to contain antioxidant and anti- carcinogenic compounds, but what these compounds are and in what quantities they are present is still under investigation. Researchers at Cal Poly State University quantified the concentrations of different phenolics in six types of fruit (cranberries, blueberries, strawberries, blackberries, pomegranate and guava). The phenolic compounds gallic acid, catechin, epicatechin, and ellagic acid were detected at 280 nanomoles (nm). Caffeic acid, p-couaric acid, and trans-resveratrol were detected at 313 nm. Rutin and quercetin were detected at 365 nm. Preliminary results presented demonstrated that the concentrations of different phenolic compounds vary widely in different fruits. The sum of all the absorbance at either 280 nm (phenolics) or 520 nm (anthocyanins) was also presented. Only absorbance values have been calculated at this time. The researchers are calculating the absorptivity constants for these

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compounds to relate absorbance to concentrations and results are expected this spring.

Hitt, L., et. al. Concentrations of Anti-Oxidant Compounds in Different Fruits. Cal Poly State University

Strawberries to Enhance “Portfolio Diet”…?

The ability of strawberries to contribute to a cholesterol lowering diet will be examined by a research team at the University of Toronto headed up by David Jenkins, M.D. Armed with a body of research indicating that various foods and/or food components can help reduce serum cholesterol, including soy protein, viscous fibers found in oats and psyllium, plant sterols and stanols found in cholesterol-lowering margarines, and nuts, Dr. Jenkins’ team has studied the impact of all of these cholesterol-lowering components, which were combined into a single diet dubbed the

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“Portfolio Diet.” The eating regimen is comparable to the extremely high-fiber, vegetarian diet of prehistoric ancestors of humans, containing some 63 servings per day of fruits, vegetables and nuts.

When tested in humans, the Portfolio Diet lowered cholesterol two to three times better than the low- fat therapeutic diet recommended by the National Cholesterol Education Panel (NCEP Step 2 diet). The diet’s effects were also comparable to statin drugs’ ability to lower serum cholesterol as well as C- reactive protein, another marker of heart disease risk. Blood pressure was reduced in subjects who achieved and sustained weight loss of 1.5 kilograms (about three pounds) or more. The researchers concluded that approximately 30 percent of serious dieters can achieve a reduction in LDL cholesterol of 20 percent or more over a six-month period when following the Portfolio Diet, and that other risk factors for heart disease may also be reduced, including blood pressure and C-reactive protein.

This team has been commissioned by CSC to embark upon a new study that adds two pounds of strawberries into the daily mix of portfolio foods for one month to determine any additive effects on blood lipids and blood pressure.

Jenkins et al, University of Toronto and St. Michael’s Hospital

Phase II of Women’s Health Study Examines Diabetes and Hypertension

Researchers at Brigham & Women’s Hospital, Harvard Medical School recently completed their first project for CSC, assessing strawberry intake and cardiovascular disease (CVD) in middle-aged women enrolled in the Women’s Health Study. Although they found no clear association between higher levels of strawberry intake and the risk of total CVD, they did find a nonsignificant trend suggesting that those consuming more strawberries had a somewhat reduced likelihood of having high blood levels of C- reactive protein, a marker of inflammation and heart disease risk. The researchers are now beginning to analyze the same data set in search of any relationship between eating strawberries and the risks of developing either diabetes or hypertension.

The tremendous public health impact of diabetes and hypertension in the U.S. has drawn attention to these diseases, both of which greatly increase the risk of developing cardiovascular disease. A recent article in the New York Times called diabetes a modern epidemic and cited statistics showing that it afflicts one in eight New Yorkers. Fruit and vegetable intake is known to be an important component of primary prevention of diabetes and hypertension. In fact, the “DASH” diet (Dietary Approaches to Stop Hypertension), which includes liberal amounts of fruits and vegetables, has been clinically proven to lower blood pressure.

Therefore, this project will answer the following questions:

  1. Is eating strawberries associated with the risk of developing diabetes?
  2. Is there an association between strawberry intake and biomarkers of glycemic control such as hemoglobin A1c?
  3. Can strawberries reduce the risk of developing high blood pressure?
  4. Is there an association between strawberry intake and changes in blood pressure over a four-year period?

These analyses will take approximately one year to complete.

Sesso et al. Brigham & Women’s Hospital, Harvard Medical School

Animal Study Targets Oral Cancer

A second study conducted by researchers from the James Cancer Hospital and Ohio State University will evaluate whether strawberries can prevent or retard the development of oral cancers. Although prevalence of oral cancer is relatively low in the United States (2.5 percent of all cancers), there are high-risk areas in the Appalachian regions where rates are much higher. Like esophageal cancer, the prognosis for sufferers is not favorable: only half survive more than five years after diagnosis. In addition, current

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treatments are relatively ineffective and potentially harmful. Thus, identifying preventive measures could greatly benefit segments of the population at elevated risk, such as former smokers.

This study will use an animal model of oral carcinogenesis, the hamster cheek pouch (HCP), which has been shown to be an excellent model because the cell and tissue changes that occur during early cancer are similar between hamsters and humans. Two different protocols will be used. In the first, the hamsters will be given lyophilized strawberries in the diet beginning one week before induction of oral cancer and continuously for 13 weeks thereafter. This will reveal whether strawberries can prevent the formation of cancer.

In the second protocol, the animals’ HCPs will first be exposed to a chemical carcinogen for six weeks and then treated topically with a lyophilized strawberry solution. This will reveal whether strawberries can intervene in the cancer process in cells that have already been initiated. If successful, the study will pave the way for a clinical trial in humans.

Weghorst et al, James Cancer Hospital, Ohio State University

Strawberries to Be Tested in Prevention of Esophageal Cancer

A clinical study sponsored by the California Strawberry Commission (CSC) will evaluate whether strawberries can inhibit the development of cancer of the esophagus. The study will be carried out in China, which has one of the highest rates of this cancer in the world. The age-standardized incidence of esophageal cancer in China is 27.4 per 100,000 population, compared to an average rate of 13.8 in less developed regions and 6.8 in more developed regions worldwide.*

Overall, esophageal cancer is the 7th most common type of cancer in the world, but it is the second most deadly. It has a survival rate of 14 percent, second only to pancreatic cancer at 4 percent (lung cancer has the third lowest survival rate at 15 percent). Because of the lack of effective treatment and poor prognosis for its victims, esophageal cancer is a prime candidate for chemoprevention.

The exact causes are still unknown, but risk factors include vitamin and mineral inadequacies, excessive tobacco use, combined alcohol and tobacco use, viral infections or contaminants in foods and water, and high intakes of hot and spicy foods. One leading hypothesis is that esophageal cancer is triggered by chemical carcinogens called nitrosamines, which are found in smoked and pickled foods. Several of these compounds, notably N-nitrosodemethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-(1- methylacetonyl-N-3-methybutyl) (NMBA) have been shown to produce esophageal lesions in laboratory animals. These compounds have also been identified in pickled vegetables and other foods that are eaten in high amounts in the regions most affected by esophageal cancer.

Ninety to 95 percent of esophageal cancers are of a type called squamous cell carcinoma. The actual progression to cancer is preceded by a non- cancerous stage called dysplasia. Thus, people with dysplasia are at high risk, but may potentially be spared progression to cancer if preventive strategies are successful.

Studies conducted by researchers at the College of Medicine and Public

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Health at Ohio State University previously showed that freeze-dried black raspberry extract inhibited esophageal tumors induced by nitrosamines by up to 65 percent. Four biochemical markers of the cancer initiation process were identified and measured. These studies provided a biochemical rationale for attempting chemoprevention in humans using strawberry extracts, which contain protective compounds similar to those found in black raspberries.

In the new trial, subjects with mild to severe dysplasia will be given either 50 or 100 grams of freeze-dried strawberries mixed in a breakfast drink daily for six months. Subsequent biopsies will measure markers of anti-proliferation of cancer cells, anti-inflammatory effects, and the effects of strawberries on gene transcription.

Ohio State expects the trial to be completed over the course of the next three years.

*Data source: International Agency for Research on Cancer (IARC), a division of the World Health Organization. www-dep.iarc.fr/

Stoner et al, James Cancer Hospital, Ohio State University

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