RESEARCH AND EDUCATION ON PHYTOCHEMICALS AND NUTRACEUTICAL FOODS

Many components in food have been identified as therapeutic substances that prevent certain cancers. Initial studies of ellagic acid in rodents by Dr. Gary Stoner have shown significant prevention and reduction of certain cancers. Preliminary studies by Funt, Bash and Schwartz have shown the highest level of ellagic acid in unripe raspberries, blackberries and strawberries. Different cultivars of these small fruits contain different levels of ellagic acid. We propose to do research and education on ellagic acid and determine the benefits of ellagic acid to human health.

We will grow berry cultivars and cultural methods to achieve the highest level of ellagic acid. We will investigate methods to extract ellagic acid. We will test processed products for cancer prevention in rodents and, ultimately, in humans at the Arthur James Cancer Hospital and Research Institute. We will disseminate results by way of newspapers, proceedings, Web site and seminars.

This proposal will insure a safe, competitive, nutritional and accessible food and will identify phytochemicals with cancer prevention potential. This will impact the health of millions of people, maintain profitability to small fruit growers, and provide jobs in the food industry.

Participants:

The Ohio State University

Ellagic acid is a naturally occurring phenolic constituent of many species from a diversity of flowering plant families. It is present in plants in the form of hydrolyzable tannins called ellagitannins. Ellagitannins are esters of glucose with hexahydroxydiphenic acid; when hydrolyzed, they yield ellagic acid, the dilactone of hexahydroxydiphenic acid. The content of ellagic acid in a series of fruits and nuts has been determined, and the highest amounts were found in blackberries, raspberries, strawberries, cranberries, walnuts and pecans. Ellagic acid, a very stable compound, is moderately soluble in dimethysulfoxide, slightly soluble in other organic solvents, and relatively insoluble in water. It is pharmacologically active and has been found to control hemorrhage in animals and in humans, presumably as a result of its ability to activate Hageman factor.

Initial studies of the antitumor activity of ellagic acid were conducted in mice using lung and skin tumors as endpoints. Lesca⁽¹⁾ investigated the effect of ellagic acid on benzo(a) pyrene-induced lung tumors in A/J mice. When administered ip or as a dietary admixture, ellagic acid decreased the multiplicity of B(a)P-induced tumors; however, significant toxicity was observed following up administration of ellagic acid. Chang et al. ⁽²⁾ showed that treatment of preweanling mice with a total dose of 300 nmol of ellagic acid shortly before an injection of B(a)P diol-epoxide caused a 44-75% inhibition in the number of diol-epoxide-induced lung tumors.

Other tissues in which ellagic acid has been shown to exhibit anticarcinogenic effects include the esophagus and liver. Mandal and Stoner ⁽³⁾ reported inhibitory effects of ellagic acid on N-nitrosomethylbenzylamine (NMBA) tumorigenesis in the esophagus of F-344 rats. The ellagic acid inhibited the development of both preneoplastic and neoplastic lesions by 25-50%. These results were confirmed in a subsequent experiment by Daniel and Stoner ⁽⁴⁾ . In a recent experiment in our laboratory, ellagic acid was found to be an effective inhibitor of NMBA-tumorigenesis in the rat esophagus only when administered before, during and after the carcinogen; there was no significant inhibition of esophageal tumorigenesis when the inhibitor was administered in the post-initiation phase only ⁽⁵⁾. Tanaka et al. ⁽⁶⁾ investigated the effect of ellagic acid on hepatocarcinogenesis induced by N-2-fluorenylacetamide (FAA) in male ACE/N rats. Rats were fed a diet containing 400 ppm of ellagic acid before, during and after administration of FAA in the diet. The ellagic acid reduced the number of altered foci and the incidence of hepatocellular neoplasms in carcinogen-treated rats. A summary of the cancer preventative properties of ellagic acid was presented by Stoner & Mukhtar ⁽⁸⁾.

It is recognized that basic and applied research needs to be expanded to a much higher level so that our knowledge of phytochemicals and nutraceuticals can be applied to better human diet and nutrition. In order to accomplish this end, the research information must be disseminated to our constituents. The reduction in cancer that might be expected from improved nutrition could have many impacts including a reduction in human suffering, in health care costs, increased opportunities for Ohio agriculture to produce nutraceutical containing crops such as raspberries, blackberries, strawberries and others and provide a new industry in the processing of these products into acceptable forms to meet human dietary needs.

The total research requirement dealing with these nutraceutical compounds is so broadly based that many research programs with individual objectives will need to be formulated. Research already conducted by the principal investigators of this project have determined that ellagic acid in strawberries prevents esophagus cancer in rats when they are given high doses of carcinogens and then treated by feeding lyophilized strawberries or other small fruits. Work has been initiated to determine the ellagic acid content of various small fruit cultivars and the impact of varying cultural practices on the ellagic acid potency within these berries. The one year's data indicates a wide variability among blackberries, red raspberries, black raspberries and strawberries in ellagic acid content by cultivar, harvest date and portion of the fruit tested ⁽⁷⁾. Work has also been undertaken to evaluate processing techniques of freezing, drying and in-plant unit operations to produce a product suitable for higher-dose level consumption by humans.

Additional research is necessary to investigate both chemical and physical techniques to enhance extraction yields of ellagic acid from fruit as well as to investigate novel processing technology such as membrane separations and concentration, high pressure processing, pulsed electronic fields and testing for improvement of extraction efficiency and recovery.

Of considerable importance is the establishment of a berry production system and grower network to produce sufficient quantities of berries. These berries have the potential of generating a high degree of value-added product. New harvesting and production practices will allow mechanical harvesting of fruit such that sorting will produce a high quality fresh market component to be sold to consumers as fresh berries. Fruit not handled in this vain can be further processed and the fruit segments separated so that seeds may be used for ellagic acid production and juice and pulp can be used for single-strength or concentrated food production. Thus, one crop can produce high value-added products for fresh market, process and nutraceutical use. Additional work needs to be done in the area of processing to determine for sure what segments of the berries contain the higher levels of ellagic acid and how and what unit operations must be performed to attain the most economical and efficient processing methods.

A significant part of this project request is the work necessary to disseminate the research information to consumers, the medical field and food processors.

The Ohio State University Extension Service will provide a vehicle by which information can be readily distributed to growers, food processors and consumers. The Ohio Extension Service already has direct communication networks established with all three of these groups through trade association interaction such as the Ohio Fruit & Vegetable Growers Association, the Ohio Fruit Growers Society, the Mid-America Food Processors Association, the many home, yard and garden clubs and county homemaker groups. These communications extend all the way from the group activities down to individual grower, processor and consumer contacts. Similar communication systems exist through the medical profession by way of OSU Seminars routinely presented for physician updates and with several Ohio and national medical associations and journals that are the usual pathway of disseminating information to the medical community.

1) Determine the different levels of ellagic acid from small fruit cultivars;

2) Determine the cultural practices that produce high levels of ellagic acid;

3) Investigate physical and chemical methods to enhance extraction yields of ellagic acid;

4) Test new processing technologies for improvement of extraction efficiency;

5) Evaluate lyophilized fruits containing various amounts of ellagic acid for their ability to prevent cancer;

6) Disseminate information and educate growers, processors and the general public.

Eastern strawberries and California strawberry cultivars will be grown in replicated plots under Ohio soil and climatic conditions. Soil and leaf analysis will be conducted with the fruit elemental content. Harvested fruit will be tested for high and low levels of ellagic acid.

Berries will be harvested as immature (large white berry), mature (full red berry) and over mature (soft dark red berry) fruit. The amount of ellagic acid in each maturity will be evaluated and recorded. Strawberries and raspberries will be tested from three different levels of compost.

Soil moisture, rainfall and soil temperature will be recorded from replicated plots to understand the conditions that will produce the highest level of ellagic acid. This information is vital to present educational programs to growers who ultimately benefit from the demand for the end product. An economic analysis of different production, harvesting and marketing methods will be completed to aid growers in their decisions to produce berries for processing.

The same type of data will be recorded for black raspberries. Non-replicated survey work on different Eastern thornless blackberries, high bush and fall red raspberry cultivars will be completed before additional cultural systems will be planted for future evaluations of ellagic acid.

Reports showing progress and conclusive data will be presented annually to the Ohio Fruit Growers Society in educational programs, proceedings and on the Midwest Web site. This will impact more than 1,500 growers.

The results of this work will provide a base for further research to optimize the levels of ellagic acid, the use of mechanical harvesting and sorting for efficient and profitable production. It will encourage growers to plant specific cultivars under prescribed cultural practices for a nutraceutical and other processed products and provide income to rural communities. Animal tests will be done to assess the efficacy of various cultivars to prevent carcinogen induced cancer and the most effective cultivar will ultimately be evaluated for cancer-inhibiting effects in humans. These tests will determine whether ellagic acid has real potential for cancer prevention and treatment in humans.

Our activities can be listed in three distinct areas:

The concept of being able to utilize berries in multi value added products should make this an attractive and economically feasible undertaking for Ohio growers. Producing the multiple products of whole, fresh market berries, ellagic acid from the berry seed component, and use either single strength or concentrated in the remainder of the berry makes for a desirable product utilization scheme.

The limitations of this work are:

1) Test several cultivars for ellagic acid;

2) Test berry maturity levels in berries;

3) Retest and expand sample preparation for ellagic acid analysis;

4) Expand number of ellagic acid samples to verify existing data;

5) Test new sample preparation unit operations in a pilot plant;

6) Develop use for berry parts not used for ellagic acid production.

7) Report to berry growers and processors at meetings and on Web site.

1) Test different soil nutrient levels for ellagic acid;

2) Continue to expand data base on ellagic acid content in berries and the specific area of the berry;

3) Develop lyophilization procedure for sample preparation;

4) Produce sufficient dried sample for animal and clinical (human) testing;

5) Continue product development of product from berry parts not used for ellagic acid.

6) Prepare news articles for general public; add to Web site.

1) Make recommendations to growers;

2) Produce sufficient product for clinical testing;

3) Develop and disseminate educational program for food processors, growers, etc.

4) Conduct one-day seminar for university personnel

The accomplishments will be assessed and evaluated by the principal investigator who will request an annual progress report. In addition, the number of people who attend specific seminars, the number of requests for reports and the number of news articles will be recorded. Presentations of scientific talks, seminars, lectures to students, peer reviewed papers, popular articles and proceedings will be documented. Reports will be put onto the Midwest Small Fruit Web site and the number of inquiries will be monitored.

Dr. Richard Funt, Coordinator of the Ohio Small Fruit Team, will coordinate, inform and advise the team members concerning the project. He will establish collaborator planning meetings and coordinate reports and other information channels. The information for the Web site will be under his leadership. Additional information will be written for general public newspapers and magazines through the Communications & Technology Section at Ohio State. This will reach over 25 million people.

• Dr. Winston Bash, Food Technologist, will prepare the berries for testing. He will conduct other studies on quality standards for berries for a nutraceutical. He will coordinate collaborators at the farm and processing plant.

   

• Dr. Steve Schwartz and his graduate students will coordinate the in-depth analytical studies on the berries and collaborate with Dr. Gary Stoner. Dr. Schwartz will be looking at the various chemical components of berries and investigating new techniques for extraction and concentration.

   

• Dr. Gary Stoner, Chair, Division of Environmental Health Sciences, James Cancer Research Institute, will conduct animal bioassays in rodents and clinical trials in humans on different levels of strawberries and raspberries to test the efficacy of ellagic acid for cancer-inhibitory effects. Specifically selected cultivars will be evaluated for their relative ability to inhibit cancers of the esophagus, colon and lungs.