Ohio State
Evaluation of Ellagic Acid Content of Ohio Berries - Final ReportOhio State University Extension InformationIntroductionBerries contain many compounds that have been shown to inhibit carcinogen induced cancers in rats. Research since 1968 has shown that ellagic acid exhibits anticarcinogenic and antimutagenic activity (Daniel et al., 1990; Maas et al., 1991b). Ellagic acid (C14H608) is a natural occurring phenolic compound in many plants, particularly strawberries (Fragaria x ananassa Duch.), raspberries (Rubus species), and blackberries [Rubus L. subgenus Rubus (Eubatus)]. Medical research programs are studying ellagic acid and have demonstrated its ability to inhibit the number and size of chemically induced tumors of the esophagus of rats. (Stoner and Mukhtar, 1995). In a study in rats, freeze dried black raspberries reduced colon cancer tumors (Harris, et al., 2000 ). Ellagic acid has been shown to function as an antioxidant and to act as an exogenous antioxidant (Maas, et al., 1991b).
Early reports indicate that cultivars have different amounts of ellagic acid .The strawberry cultivar Earliglow, which is widely grown in Ohio, was tested for ellagic acid content in Maryland and the content was low in red-fruit pulp and highest among five genotypes for red-fruit achene values (Maas et al., 1991a). Also, the ellagic acid concentration in pulp and achenes from fruit harvested at green or red ripe stages of maturity and in leaves of different strawberry cultivars was found to vary.
In Ohio, growers have expressed interest in various components of berries that may develop into a phytochemical or nutraceutical product either as a processed product or as a specific fresh market health food. Our objective was to test different cultivars of Ohio strawberries, raspberries, and blackberries for ellagic acid content. As an interdisciplinary team our group worked closely with individuals in the Ohio State University (OSU) Arthur James Cancer Hospital and Richard Solove Research Institute, which is a nationally designated comprehensive cancer center.
MethodsIn 1996 and 1997, unripe (white) and ripe (red) berries from three strawberry cultivars were harvested at the OSU Waterman Farm in Columbus. In 1997 strawberry leaves were harvested after renovation in mid to late August and early September. Red (Rubus idaetus L.) and black raspberry (Rubus occidentalis L.) samples were harvested the same as strawberry samples. Blackberry (eastern thornless) samples from OSU Piketon Research and Education Center were frozen at Piketon and delivered to Columbus several days after harvest. In 1998, strawberry fruit and leaves, red, black raspberry and blackberry fruit similar to those in 1997 were tested for year-to-year variation in ellagic acid content.
In another experiment, black raspberry fruit was tested for ellagic acid content where plants were subjected to different composted yard waste treatments. Fruit samples from the same experiment were subjected to fruit elemental content to investigate the impact of elemental content on ellagic acid content. Also a study of old and new black raspberry leaves from the same compost study was conducted in 1998. Leaves were taken from near the soil (old) and near the top (new) of the plant. Leaves were dried and sent to Brunswick Laboratories (Wareham, MA) for ellagic acid content analysis.
Modified extraction methods were developed based on earlier work of Daniel et al., 1989. In 1996, Dr. Schwartz and his team were initially unable to obtain repeatable results on the ellagic acid content of samples using the procedure explained in the literature. It wasn't until several months passed and many, many tests were conducted that it was found that the procedure printed in the literature was incomplete. Even after this error was corrected (it was accurate as far as it went), it is believed that the procedure was not as accurate as desired, and it was further improved upon throughout the remainder of the study. Therefore, only selected data from 1996 is reported.
For the fruit extraction of ellagic acid, all samples were prepared the same for all years. Pulp and seed were separated. Dried leaves were placed in a food processor and the same steps were used as with fruit.
Ten gram individual samples were homogenized with a Brinkman polytron. This puree was mixed with approximately 50 ml methanol which extracts the ellagic acid and ellagitannins. The extract was collected by filtering and the process was repeated. Extracted ellagic acid and ellagitannins were then hydrolyzed with trifluorocetic acid in order to convert all ellagitannins to ellagic acid. The extracts were injected into a liquid chromatograph and peak areas were compared to a calibration curve generated with pure ellagic acid samples.
The ellagic acid content in berry seeds was examined separately from the pulp. Seeds were removed by hand to avoid rupturing any other fruit structure. Extracts were filtered into a 2 ml High Performance Liquid Chromatography (HPLC) vial. All HPLC analysis were reported in micrograms/gram dry weight of sample. In order to compare results between samples and to express ellagic acid content on a dry weight basis, the moisture content of all samples was determined.
In 1998, some samples were taken from replicated field plots. A statistical analysis was used to determine significant differences in ellagic acid content among cultivars and variables (SAS, Institute: 1990). Mean separation was determined by least significant difference (LSD) statistics at a probability at the 0.05 level of significance.
ResultsStrawberry - PulpIn a comparison of three strawberry cultivars in 1996 and 1997, ripe berries were equal or lower in ellagic acid content than unripe (white) berries (Table 1). 'Earliglow' and Kent had higher levels of ellagic acid in the pulp in 1997 than in 1996.
Table 1. Year to year comparison of ellagic acid content in strawberry pulp and seeds from
three cultivars, Waterman Farm, Columbus. Ripez % Differences Unripey % DifferenceCultivar 19961997 1996 to 1997 19961997 1996 to 1997Ug.g-1 (dry weight)Strawberry Pulpx Earliglow 4164.2 a +80% 6880.8 a +18%Kent 4874.8 a +50 4875.5 a +56Jewel 5651.0 a -11 7145.2 a -35 Strawberry Seeds Earliglow -1164 a - -657 ab -Kent -1003 a - -757 a -Jewel -617 b - -468 b -z Average micrograms per gram of product from harvests over entire season - Waterman Farm,Columbus.y Unripe berries are mature white or white with red color at tip.x There were a different number of samples tested among cultivars and years.(-) data not available.In replicate studies in 1998, the ellagic acid content in the pulp of ripe fruit from Earliglow appeared to be about one-half that observed in 1996 (Table 2). 'Mohawk' was significantly lower in pulp and seed ellagic acid contents than Earliglow.
Table 2. Ellagic acid content of strawberry fruit pulp, seeds and leaves, Piketon, 1998. Fruit Ratio Cultivarz PulpSeed Pulp to Seed LeavesUg.g-1 (dry weight)Earliglow 21.6 a182.1 a 8.6 a 2191.8 aMohawk 13.0 b68.9 b 5.4 a 3183.2 aStartyme 17.0 ab152.3 a 9.4 a 2381.5 az Mean separation determined by LSD statistic (P 0.05), 4 replications
Source: Piketon Research and Extension CenterStrawberry - Seeds'Jewel' had a significantly lower amount of ellagic acid in the seeds of ripe and unripe fruit than Kent in 1997 (Table 1). Earliglow and Kent had similar levels of ellagic acid in both ripe and unripe fruit.
The ellagic acid content of all seeds (achenes) tested was lower in 1998 than in 1997 (Table 3). Seneca and Northeaster cultivars tended to be the lowest among Ohio cultivars for ellagic acid content in the seed in 1998. Ohio cultivars ranged from 51 to 252 while non Ohio cultivars ranged from 298 to 405 micrograms/gram of ellagic acid in 1998. Thus non Ohio strawberry cultivars had 1.6 to 5.8 more ellagic acid in their seed in 1998 than Ohio cultivars.
Strawberry - LeavesThere were no significant differences among three strawberry cultivars for ellagic acid content in leaves (Table 2). However, Mohawk had the highest amount and Earliglow had the lowest. In another test higher amounts were found in leaves on August 12 as compared to leaves on August 25 for the same cultivars (Table 4). The difference between the two harvest dates ranged from a 38 to 53 percent reduction in ellagic acid content. In another test on frozen (fresh) leaves (September 5), Kent and Jewel leaves were 54 to 63 percent higher in ellagic acid content than freeze-dried leaves harvested on August 25.
Table 3. Ellagic Acid Content of Strawberry Seeds
1997 and 1998.Ohio Cultivarsx 1997y 1998zUg.g-1 (dry weight)Earligloww 1164 a 182Jewelw 1003 a 252Kentw 617 b ---Delmarvel --- 188 to 214Allstar --- 110 to 191Mohawk --- 120 to 144Northeaster --- 68Seneca --- 51 to 58 Non Ohio Cultivarsx Oso Grande 1208 ---Camarosa 1077 350Sweet Charlie 549 354Chandler 516 298 to 405z One or two samples per cultivar.y Mean of 4 replications for Earliglow, Jewel and Kent.xSource: Piketon Research/Extension Center, Piketon, OH.wWaterman Farm - Kent, Jewel, Earliglow in 1997.Table 4. Ellagic acid content of strawberry leaves taken at different times and tested as freeze
dried or fresh (frozen), Waterman Farm, 1997. Sample Date - Freeze Dried Sample Date - Fresh (Frozen)Cultivar Aug. 12Aug. 25%Difference1 Sept. 5% Difference2Ug.g-1 (dry weight)Earliglow 52053174-39 3028-5Kent 43662045-53 3154+54Jewel 45522822-38 4605+631Percent change in ellagic acid content from 8/12 to 8/25.2Percent change in ellagic acid from 8/25 freeze dried sample.Black Raspberry - pulp, seed, leafEllagic acid content of Jewel black raspberry pulp was markedly lower in 1996 than in 1997 (Table 5). There was 83 and 89 percent decrease for ripe and unripe berry pulp for 1996 versus 1997. However, seeds from ripe and unripe fruit ranged from a 10% increase to a 7% decrease, respectively. There was no significant difference in ellagic content among ripe or unripe pulp or seed 1997.
Table 5. Year to year comparison of ellagic acid content in 'Jewel' black raspberry pulp and
seeds, Ohio. Ripez % Differences Unripey % DifferencesCultivar 19961997 1996 to 1997 19961997 1996 to 1997Ug.g-1 (dry weight)Pulpx 9817 -83% 20723 -89%Seeds 218240 +10 251234 -7z Average microgram per gram of product from harvests over entire season, Waterman
Farm, Columbusy Unripe berries are mature white to full red color. Ripe fruit was glossy and black.x There were a different number of samples between years.
There were no significant differences in 1997 for ripe or unripe pulp or seeds with 4
replicationsIn a study using composted yard waste applied either to the soil surface, subsurface or surface and subsurface, there were no significant differences in ellagic acid content for 'Jewel' black raspberry fruit pulp or seeds in the treatment groups (Table 6). However, potassium and manganese in the fruit elemental content and leaf elemental content of phosphorus, potassium, and magnesium, respectively, were affected by treatment (data not shown).
Table 6. Ellagic acid content of 'Jewel' black raspberry fruit grown in composted yard waste
treatments, 1997 Waterman Farm, Columbus. Ripe Fruit RatioTreatmentz PulpSeedsPulp to SeedsyUg.g-1 (dry weight)Control 18.2 a261.2 a1:15.5Compost subsurface 20.5 a248.2 a1:12.6Compost surface 17.6 a250.3 a1:12.9Compost surface & subsurface 20.8 a288.4 a1:15.1z Mean separation determined by LSD statistic (P 0.05). 5 replications.y No statistical analysis.In the same 'Jewel' black raspberry study, old leaves had less ellagic acid content than new leaves (Table 7). In new leaves, surface applied compost had significantly lower ellagic acid content than surface plus subsurface treatments. When new and old leaf means were combined, the control and surface applied were significantly lower than the surface plus subsurface treatment.
Table 7. Ellagic acid content of 'Jewel' black raspberry leaves grown in composted yard waste
treatments, 1997 Waterman Farm, Columbus. Leaves Combined MeanTreatmentz OldNewOld and NewUg.g-1 (dry weight)Control 200 a480 ab340 bCompost subsurface 243 a577 ab410 abCompost surface 180 a237 b208 bCompost surface & subsurface 273 a1047 a660 az Mean separation determined by LSD statistic ( 0.05). 3 replications.Red Raspberry (Fall bearing) - pulp, seedIn 1997 Heritage red raspberry had the highest amount of ellagic acid in pulp among several raspberry cultivars (Table 8). Anne, a yellow raspberry (Rubus sp.), had only 25 percent of ellagic acid in pulp as Heritage. However, Heritage was lower in ellagic acid content in the seed as compared to Caroline, Ruby and Anne.
Table 8. Comparison of ellagic acid content for red raspberry cultivars
(ripe fruit), 1997 and 1998, Piketon. 1997z 1998yCultivar PulpSeed PulpSeedUg.g-1 (dry weight)Caroline 36.0173.4 52.5 a799.2 aA. Bliss 22.398.9 42.0 b263.6 cHeritage 40.5105.8 39.2 b467.2 bAnnex 11.1177.7 7.860.5Ruby 39.7176.4 10.085.6z From one sample - Piketon Research and Extension Centery Mean of 4 replications - Caroline, A. Bliss and Heritage- mean separation
determined by LSD statistic ( 0.05)x Anne is a yellow colored fruit which ripens in the fall and is grown as a red cultivarIn 1998, Caroline was significantly higher in ellagic acid content in both pulp and seed than Autumn Bliss and Heritage. The ellagic acid content in the seeds of Autumn Bliss was significantly lower than in Heritage. While Anne and Ruby samples were not replicated, their values were much lower than the other cultivars.
Blackberry - Eastern Thornless - pulp, seedThree eastern thornless blackberry cultivars, Navaho, Hull and Chester were tested for ellagic acid content in ripe and unripe fruit in 1997 (Table 9). Similar levels of ellagic acid were found for the three cultivars within pulp or seeds. Unripe berry pulp had 35%, 51% and 36% higher levels of ellagic acid than ripe berries, respectively. However, ripe seeds had 40%, 11% and 8% more ellagic acid than unripe seeds, respectively.
Table 9. Ellagic acid content of blackberry fruit, 1997 Piketon. Ripe1,2 Unripe Rate Pulp to SeedCultivar PulpSeed PulpSeed RipeUnripeUg.g-1 (dry weight)Navaho 24.1352.0 36.7214.2 1 to 14.61 to 5.8Hull 16.6346.7 33.5309.4 1 to 20.81 to 9.2Chester 22.7324.5 35.1299.3 1 to 14.31 to 8.51 From one sample2 Source: Piketon Research & Extension Center, Piketon, OH.Berry pulp had higher levels of ellagic acid in 1998 than in 1997 (Table 10). This year to year variation ranged from 71 to 84% and is similar to that reported in strawberry and black raspberry pulp for 1996 and 1997 (Tables 1 and 4). However, for seeds, the increase was 22% which is nearly the same year to year variation as in black raspberry seed content for 1996 and 1997. Chester thornless was significantly higher in ellagic acid content of pulp than any other cultivar tested in 1998.
All Berries TestedA list of berries and a range of ellagic acid content of ripe fruit and leaves is shown in Table 11. This data represents multiple samples which were taken from different locations and soil types in Ohio during 1996 to 1998. Generally, the seeds from ripe fruit had higher levels of ellagic acid than the pulp. Although a wide range and variation existed, leaves had higher levels of ellagic acid than the fruit.
Table 10. Comparison of ellagic acid content for eastern thornless
blackberry cultivars (ripe fruit), 1997 and 1998, Piketon. 1997a 1998Cultivar PulpSeed PulpSeedbUg.g-1 (dry weight)Navaho 24.1352.0 83.2b463.4aHull 16.6346.7 70.1b441.4aChester 22.7324.5 141.9a413.6aa From one sampleb Source: Piketon Research & Extension Center, Piketon, OH.
- Mean separation determined by LSD statistic ( 0.05) of 4 replicationsTable 11. Range of ellagic acid content from Ohio berries for pulp seed or leaves from tests in
Ohio 1996 to 1998.Berry Cultivar PulpSeed LeavesUg.g-1 (dry weight)Strawberry Earliglow 22 to 41184 to 1164 1743 to 2930Kent 481003 --Jewel 56252 to 617 --Mohawk 11 to 1467 to 144 --Startyme 14 to 21100 to 201 1302 to 3863Delmarvel --14 to 214 --Allstar --110 to 191 --Northeaster --68 --Seneca 50 to 57-- --Oso Grande --1208 --Camarosa --350 to 1077 --Sweet Charlie --354 to 549 --Chandler --298 to 516 -- Raspberry Black Jewel 17 to 98218 to 288 180 to 1047Red Autumn Bliss 22 to 4299 to 263 --Caroline 36 to 53173 to 799 --Heritage 39 to 40105 to 467 --Ruby 10 to 4085 to 176 --Yellow Anne 8 to 1160 to 178 --
Blackberry - Eastern Thornless Navaho 16 to 8740 to 439 --Hull 42 to 8874 to 687 --Chester 6 to 225268 to 1862 --C-65 31171 --zRanges are from multiple samples taken from different sites during 1996 to 1998.DiscussionSeveral strawberry, black raspberry, red raspberry (fall bearing) and blackberry (eastern thornless) cultivars were tested for ellagic acid content. Fruit and leaf samples were collected from Ohio State University research plots at Waterman Farm, Columbus and Piketon Research and Extension Center, Piketon, from 1996 to 1998. The reported extraction method was modified to provide more consistent results. However, the authors recognize that ellagic acid is a most difficult compound to extract and assess. While careful preparation and diligence was performed by all team members under current knowledge, it is hoped that other untried methods may prove to be more accurate and comparable than those in this report. Therefore, this initial work is reported for others to expand upon and to be used as a foundation for future research.
CultivarsCultivars within species contain different amounts of ellagic acid. Many of the cultivars that we tested had different levels of ellagic acid. However, ellagic acid content in the pulp of Earliglow is nearly the same as other cultivars tested (Bash et al., 1996). It is possible that non Ohio strawberry cultivars, grown in other states (California and Florida), may contain higher levels of ellagic acid in the seeds than those in Ohio. Earliglow does not appear to be different from other cultivars tested for ellagic acid content in the leaf. This needs to be verified with future research
In most cases unripe strawberry, raspberry or blackberry pulp had higher levels of ellagic acid than ripe fruit pulp. In all cultivars, berry seeds were always higher in ellagic acid than in pulp regardless of ripeness. In black raspberry, seeds were 12 to 15 times higher in ellagic acid content than in pulp. Ripe fruit seeds appear to be higher in ellagic acid than unripe fruit seeds by as much as 8 to 40 percent.
Maas and Galletta, 1991, indicated that genetic manipulation (breeding) could be used to increase ellagic acid, but the manner of ellagic acid inheritance was unknown because no correlation was found among pulp, seeds and leaf tissue among different strawberry cultivars. Thus selection for ellagic acid content would be highly specific for tissue type. Our data agree with that of Maas and Galletta.
The Jewel black raspberry cultivar has shown excellent fruit size and yield for Ohio growers. Jewel demonstrated no difference in ellagic acid among compost treatments in fruit (Funt et al., 1997). However, there were significant differences in leaf ellagic acid content. The difference of ellagic acid content of younger leaves of black raspberries treated with compost could be important to raspberry growers. Ellagic acid can be an endogenous inhibitor of insect feeding on certain plants (Klocke et al., 1986). Inhibitor mechanisms of ellagic acid to chewing insects and aphids may be different. Sensitivity among aphid species to several ellagitannins was shown in feeding-deterrent studies (Jones and Klocke, 1987). This needs to be verified with future research.
The Heritage fall red raspberry cultivar has been a standard in many mid-western states. Caroline, a new cultivar not widely grown in Ohio, had higher levels of ellagic acid in pulp and seed than Heritage in 1998. Autumn Bliss had lower levels of ellagic acid in the seeds than Caroline in both 1997 and 1998. Other cultivars that were tested appeared to have lower levels than Caroline or Heritage. Replicated trials for several years are needed to verify these results.
All three blackberry (eastern thornless) cultivars were similar in ellagic acid in the pulp and seed. However, Chester thornless had significantly greater ellagic acid in the pulp in 1998 than other cultivars (Funt et al., 1998). Chester thornless is a high yielding cultivar.
SeedsFor seeds, the Jewel strawberry cultivar was lower in ellagic acid than Earliglow in 1997. The data are mixed as to whether strawberry (Earliglow) seeds have greater ellagic acid levels than raspberry or blackberry seeds. Clearly in 1997 Earliglow and Kent ripe strawberry seeds had higher amounts than raspberry or blackberry cultivars studied. But in 1998 the Earliglow seeds were among the lowest levels of all berry types. In 1998 blackberry seeds were higher than either raspberry or strawberry seeds except for Caroline and Heritage fall red raspberry.
Year to year variation in ellagic acid content of pulp appears to occur in all berries tested. Less year to year variation appears to occur in black raspberry and blackberry seeds. Strawberries and red raspberry seeds had more year to year variation of ellagic acid than other berries. Leaves were not tested for year to year variation. However in one observation, strawberry leaves harvested after renovation were higher in ellagic acid than if harvested two weeks later. In all berries leaves were higher in ellagic acid content than seeds. Generally, strawberry leaves contained more ellagic acid than raspberry leaves. However, ellagic acid content varies within the growing season and may change dramatically within weeks. In raspberries treated with compost, older leaves had less ellagic acid than younger leaves. Maas et al in 1991c. indicated that ellagic acid content of red (ripe) fruit tissue varied little between two growing sites that were about 2 kilometers apart. In this study, the Earliglow strawberry was grown at two locations about 40 kilometers apart. We can not verify if location can influence ellagic acid content of strawberry pulp.
ConclusionEllagic acid exhibits anticarcinogenic and antiimutagenic activity in humans. It is a natural occurring phenolic compound found in many plants and berries. Medical research has shown reductions in the size and number of cancerous tumors when mice are fed a diet of freeze dried strawberries or raspberries.
In Ohio, ellagic acid content was found to be highest in the leaves, intermediate in seeds from ripe fruit and lowest in the pulp in strawberry and black raspberry fruit. Leaves can be much higher in ellagic acid content depending upon the time of year or physiological growth stage. The ellagic acid content in certain black raspberry seeds is 12 to 15 times higher than in the pulp.
Ellagic acid content in ripe fruit seeds of strawberry was highest in Earliglow, Startyme and Jewel cultivars. Strawberry cultivars specifically grown in Florida or California appeared to be higher in ripe seed ellagic acid content than those grown in Ohio (Earliglow or Jewel). Caroline and Heritage were highest in ellagic acid among other fall red or yellow raspberry cultivars. Generally, the seeds of Navaho, Hull and Chester eastern thornless blackberry contain about the same amounts of ellagic acid. Raspberry and blackberry seeds appear to have less year to year variation in ellagic acid content than strawberry seeds.
Variation in ellagic acid content is cultivar dependent. It does not appear that soil, site location, or management practices as fertilizer applications influence ellagic acid content of plant tissue. However, the incorporation of compost prior to planting of black raspberry may increase ellagic acid content of young leaves.
Ellagic acid is a most difficult compound to gather consistent and repeatable assays. Other methods of sample collection, preparation, extraction and analysis may prove to be better than those used in this work. Additional studies by public and private laboratories is required to validate our results to optimize the potential amount of ellagic acid per acre.
Future research in berries needs to verify year to year variation of ellagic acid in various plant tissues. Identifying ellagic acid content in tissues which show the least variation, would improve product quality and consistency for nutraceuticals. Secondly, growers could possibly benefit from insect deterrence. If certain soil amendments at planting such as compost can influence ellagic acid content in leaves, certain insects as aphids could be reduced on raspberry plants.
We have no published data as to the absorption and distribution of ellagic acid in humans. Human clinical studies utilizing berries containing ellagic acid are necessary to verify absorption and distribution into various human tissues. From these studies, recommendations for daily intake for humans can be made for fresh berries or a process product as a nutraceutical.
ReferencesBash, W.D., S.J. Schwartz and R.C. Funt. 1996. Small fruit evaluation for ellagic acid content. Proc. Ohio Fruit and Veg. Growers Congress and Roadside Marketing Conference. p. 126-129.
Daniel, E.M., A.S. Krupnick, Y. Hew, J.A. Blinzler, R.M. Nems and G.D. Stoner. 1990. Extraction, stability, and quanlitation of ellagic acid in various fruits and nuts. J. of Food Composition and Analysis 2, p. 338-349.
Funt, R.C., W.D. Bash, S.J. Schwartz, J. Collier and G. Wenneker. 1997. Small fruit cultivar characteristics for ellagic acid and neutracuetical development. Proc. Ohio Fruit and Veg. Growers Congress and Roadside Marketing Conference. p. 110-113.
Funt, R.C., W.D. Bash, S.J. Schwartz, C. Moxley and T.E. Wall. 1998. Evaluation of year to year variation in berries for ellagic acid content and neutracuetical development. Proc. Fruit, Vegetable, Roadside Marketing and Ohio Wine Producers Conf. p. 14-18.
Harris, G.K., G.D. Stoner and S.J. Schwartz. 2000. Effects of freeze dried black raspberries on ayoxymethane induced colon tumors in the F344 rat. OARDC poster session (unpublished).
Jones, K.C. and J.A. Klocke. 1987. Aphid feeding deterrence of ellagitannins, their phenolic hydrolysis products and related phenolic derivatives. Entomol. Expt. Appl. 44:229-234.
Klocke, J.A., B. VanWagenam, and M.F. Bolundrin. 1986. The ellagitannin geranium and its hydrolysis products isolated as insect growth inhibitors from semi-arid land plants. Phytochemistry 25:85-91.
Maas, J.L., S.Y. Wang., G.J. Galletta. 1991a. Evaluation of strawberry genotypes for ellagic acid, an antimutagenic and anticarcinogenic plant phenol. In: The Strawberry Into the 21st Century, A. Dale and J.J. Luby, editors. Timber Press. p. 115-117.
Maas, J.L., G.J. Galletta and G.D. Stoner. 1991b. Ellagic acid, an anticarcinogen in fruits, especially in strawberries. A review. HortScience 26(1)10-14.
Maas, J.L., S.Y. Wang, and G.J. Galletta. 1991c. Evaluation of strawberry cultivars for ellagic acid content. HortScience 26(1)66-68.
SAS Institute. 1990. SAS/STAT User's Guide. 4th Ed. Vol. 2. SAS Institute, Inc. Cary, NC.
Stoner, G.D. and H. Mukhtar. 1995. Polyphenols as cancer chemo-preventive agents. J. of Cellular Biochemistry, Supplement 22:169-180.
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Evaluation of Ellagic Acid Content of Ohio Berries - Final ReportOhio State University Extension InformationIntroductionBerries contain many compounds that have been shown to inhibit carcinogen induced cancers in rats. Research since 1968 has shown that ellagic acid exhibits anticarcinogenic and antimutagenic activity (Daniel et al., 1990; Maas et al., 1991b). Ellagic acid (C14H608) is a natural occurring phenolic compound in many plants, particularly strawberries (Fragaria x ananassa Duch.), raspberries (Rubus species), and blackberries [Rubus L. subgenus Rubus (Eubatus)]. Medical research programs are studying ellagic acid and have demonstrated its ability to inhibit the number and size of chemically induced tumors of the esophagus of rats. (Stoner and Mukhtar, 1995). In a study in rats, freeze dried black raspberries reduced colon cancer tumors (Harris, et al., 2000 ). Ellagic acid has been shown to function as an antioxidant and to act as an exogenous antioxidant (Maas, et al., 1991b).
Early reports indicate that cultivars have different amounts of ellagic acid .The strawberry cultivar Earliglow, which is widely grown in Ohio, was tested for ellagic acid content in Maryland and the content was low in red-fruit pulp and highest among five genotypes for red-fruit achene values (Maas et al., 1991a). Also, the ellagic acid concentration in pulp and achenes from fruit harvested at green or red ripe stages of maturity and in leaves of different strawberry cultivars was found to vary.
In Ohio, growers have expressed interest in various components of berries that may develop into a phytochemical or nutraceutical product either as a processed product or as a specific fresh market health food. Our objective was to test different cultivars of Ohio strawberries, raspberries, and blackberries for ellagic acid content. As an interdisciplinary team our group worked closely with individuals in the Ohio State University (OSU) Arthur James Cancer Hospital and Richard Solove Research Institute, which is a nationally designated comprehensive cancer center.
MethodsIn 1996 and 1997, unripe (white) and ripe (red) berries from three strawberry cultivars were harvested at the OSU Waterman Farm in Columbus. In 1997 strawberry leaves were harvested after renovation in mid to late August and early September. Red (Rubus idaetus L.) and black raspberry (Rubus occidentalis L.) samples were harvested the same as strawberry samples. Blackberry (eastern thornless) samples from OSU Piketon Research and Education Center were frozen at Piketon and delivered to Columbus several days after harvest. In 1998, strawberry fruit and leaves, red, black raspberry and blackberry fruit similar to those in 1997 were tested for year-to-year variation in ellagic acid content.
In another experiment, black raspberry fruit was tested for ellagic acid content where plants were subjected to different composted yard waste treatments. Fruit samples from the same experiment were subjected to fruit elemental content to investigate the impact of elemental content on ellagic acid content. Also a study of old and new black raspberry leaves from the same compost study was conducted in 1998. Leaves were taken from near the soil (old) and near the top (new) of the plant. Leaves were dried and sent to Brunswick Laboratories (Wareham, MA) for ellagic acid content analysis.
Modified extraction methods were developed based on earlier work of Daniel et al., 1989. In 1996, Dr. Schwartz and his team were initially unable to obtain repeatable results on the ellagic acid content of samples using the procedure explained in the literature. It wasn't until several months passed and many, many tests were conducted that it was found that the procedure printed in the literature was incomplete. Even after this error was corrected (it was accurate as far as it went), it is believed that the procedure was not as accurate as desired, and it was further improved upon throughout the remainder of the study. Therefore, only selected data from 1996 is reported.
For the fruit extraction of ellagic acid, all samples were prepared the same for all years. Pulp and seed were separated. Dried leaves were placed in a food processor and the same steps were used as with fruit.
Ten gram individual samples were homogenized with a Brinkman polytron. This puree was mixed with approximately 50 ml methanol which extracts the ellagic acid and ellagitannins. The extract was collected by filtering and the process was repeated. Extracted ellagic acid and ellagitannins were then hydrolyzed with trifluorocetic acid in order to convert all ellagitannins to ellagic acid. The extracts were injected into a liquid chromatograph and peak areas were compared to a calibration curve generated with pure ellagic acid samples.
The ellagic acid content in berry seeds was examined separately from the pulp. Seeds were removed by hand to avoid rupturing any other fruit structure. Extracts were filtered into a 2 ml High Performance Liquid Chromatography (HPLC) vial. All HPLC analysis were reported in micrograms/gram dry weight of sample. In order to compare results between samples and to express ellagic acid content on a dry weight basis, the moisture content of all samples was determined.
In 1998, some samples were taken from replicated field plots. A statistical analysis was used to determine significant differences in ellagic acid content among cultivars and variables (SAS, Institute: 1990). Mean separation was determined by least significant difference (LSD) statistics at a probability at the 0.05 level of significance.
ResultsStrawberry - PulpIn a comparison of three strawberry cultivars in 1996 and 1997, ripe berries were equal or lower in ellagic acid content than unripe (white) berries (Table 1). 'Earliglow' and Kent had higher levels of ellagic acid in the pulp in 1997 than in 1996.
Table 1. Year to year comparison of ellagic acid content in strawberry pulp and seeds from
three cultivars, Waterman Farm, Columbus. Ripez % Differences Unripey % DifferenceCultivar 19961997 1996 to 1997 19961997 1996 to 1997Ug.g-1 (dry weight)Strawberry Pulpx Earliglow 4164.2 a +80% 6880.8 a +18%Kent 4874.8 a +50 4875.5 a +56Jewel 5651.0 a -11 7145.2 a -35 Strawberry Seeds Earliglow -1164 a - -657 ab -Kent -1003 a - -757 a -Jewel -617 b - -468 b -z Average micrograms per gram of product from harvests over entire season - Waterman Farm,Columbus.y Unripe berries are mature white or white with red color at tip.x There were a different number of samples tested among cultivars and years.(-) data not available.In replicate studies in 1998, the ellagic acid content in the pulp of ripe fruit from Earliglow appeared to be about one-half that observed in 1996 (Table 2). 'Mohawk' was significantly lower in pulp and seed ellagic acid contents than Earliglow.
Table 2. Ellagic acid content of strawberry fruit pulp, seeds and leaves, Piketon, 1998. Fruit Ratio Cultivarz PulpSeed Pulp to Seed LeavesUg.g-1 (dry weight)Earliglow 21.6 a182.1 a 8.6 a 2191.8 aMohawk 13.0 b68.9 b 5.4 a 3183.2 aStartyme 17.0 ab152.3 a 9.4 a 2381.5 az Mean separation determined by LSD statistic (P 0.05), 4 replications
Source: Piketon Research and Extension CenterStrawberry - Seeds'Jewel' had a significantly lower amount of ellagic acid in the seeds of ripe and unripe fruit than Kent in 1997 (Table 1). Earliglow and Kent had similar levels of ellagic acid in both ripe and unripe fruit.
The ellagic acid content of all seeds (achenes) tested was lower in 1998 than in 1997 (Table 3). Seneca and Northeaster cultivars tended to be the lowest among Ohio cultivars for ellagic acid content in the seed in 1998. Ohio cultivars ranged from 51 to 252 while non Ohio cultivars ranged from 298 to 405 micrograms/gram of ellagic acid in 1998. Thus non Ohio strawberry cultivars had 1.6 to 5.8 more ellagic acid in their seed in 1998 than Ohio cultivars.
Strawberry - LeavesThere were no significant differences among three strawberry cultivars for ellagic acid content in leaves (Table 2). However, Mohawk had the highest amount and Earliglow had the lowest. In another test higher amounts were found in leaves on August 12 as compared to leaves on August 25 for the same cultivars (Table 4). The difference between the two harvest dates ranged from a 38 to 53 percent reduction in ellagic acid content. In another test on frozen (fresh) leaves (September 5), Kent and Jewel leaves were 54 to 63 percent higher in ellagic acid content than freeze-dried leaves harvested on August 25.
Table 3. Ellagic Acid Content of Strawberry Seeds
1997 and 1998.Ohio Cultivarsx 1997y 1998zUg.g-1 (dry weight)Earligloww 1164 a 182Jewelw 1003 a 252Kentw 617 b ---Delmarvel --- 188 to 214Allstar --- 110 to 191Mohawk --- 120 to 144Northeaster --- 68Seneca --- 51 to 58 Non Ohio Cultivarsx Oso Grande 1208 ---Camarosa 1077 350Sweet Charlie 549 354Chandler 516 298 to 405z One or two samples per cultivar.y Mean of 4 replications for Earliglow, Jewel and Kent.xSource: Piketon Research/Extension Center, Piketon, OH.wWaterman Farm - Kent, Jewel, Earliglow in 1997.Table 4. Ellagic acid content of strawberry leaves taken at different times and tested as freeze
dried or fresh (frozen), Waterman Farm, 1997. Sample Date - Freeze Dried Sample Date - Fresh (Frozen)Cultivar Aug. 12Aug. 25%Difference1 Sept. 5% Difference2Ug.g-1 (dry weight)Earliglow 52053174-39 3028-5Kent 43662045-53 3154+54Jewel 45522822-38 4605+631Percent change in ellagic acid content from 8/12 to 8/25.2Percent change in ellagic acid from 8/25 freeze dried sample.Black Raspberry - pulp, seed, leafEllagic acid content of Jewel black raspberry pulp was markedly lower in 1996 than in 1997 (Table 5). There was 83 and 89 percent decrease for ripe and unripe berry pulp for 1996 versus 1997. However, seeds from ripe and unripe fruit ranged from a 10% increase to a 7% decrease, respectively. There was no significant difference in ellagic content among ripe or unripe pulp or seed 1997.
Table 5. Year to year comparison of ellagic acid content in 'Jewel' black raspberry pulp and
seeds, Ohio. Ripez % Differences Unripey % DifferencesCultivar 19961997 1996 to 1997 19961997 1996 to 1997Ug.g-1 (dry weight)Pulpx 9817 -83% 20723 -89%Seeds 218240 +10 251234 -7z Average microgram per gram of product from harvests over entire season, Waterman
Farm, Columbusy Unripe berries are mature white to full red color. Ripe fruit was glossy and black.x There were a different number of samples between years.
There were no significant differences in 1997 for ripe or unripe pulp or seeds with 4
replicationsIn a study using composted yard waste applied either to the soil surface, subsurface or surface and subsurface, there were no significant differences in ellagic acid content for 'Jewel' black raspberry fruit pulp or seeds in the treatment groups (Table 6). However, potassium and manganese in the fruit elemental content and leaf elemental content of phosphorus, potassium, and magnesium, respectively, were affected by treatment (data not shown).
Table 6. Ellagic acid content of 'Jewel' black raspberry fruit grown in composted yard waste
treatments, 1997 Waterman Farm, Columbus. Ripe Fruit RatioTreatmentz PulpSeedsPulp to SeedsyUg.g-1 (dry weight)Control 18.2 a261.2 a1:15.5Compost subsurface 20.5 a248.2 a1:12.6Compost surface 17.6 a250.3 a1:12.9Compost surface & subsurface 20.8 a288.4 a1:15.1z Mean separation determined by LSD statistic (P 0.05). 5 replications.y No statistical analysis.In the same 'Jewel' black raspberry study, old leaves had less ellagic acid content than new leaves (Table 7). In new leaves, surface applied compost had significantly lower ellagic acid content than surface plus subsurface treatments. When new and old leaf means were combined, the control and surface applied were significantly lower than the surface plus subsurface treatment.
Table 7. Ellagic acid content of 'Jewel' black raspberry leaves grown in composted yard waste
treatments, 1997 Waterman Farm, Columbus. Leaves Combined MeanTreatmentz OldNewOld and NewUg.g-1 (dry weight)Control 200 a480 ab340 bCompost subsurface 243 a577 ab410 abCompost surface 180 a237 b208 bCompost surface & subsurface 273 a1047 a660 az Mean separation determined by LSD statistic ( 0.05). 3 replications.Red Raspberry (Fall bearing) - pulp, seedIn 1997 Heritage red raspberry had the highest amount of ellagic acid in pulp among several raspberry cultivars (Table 8). Anne, a yellow raspberry (Rubus sp.), had only 25 percent of ellagic acid in pulp as Heritage. However, Heritage was lower in ellagic acid content in the seed as compared to Caroline, Ruby and Anne.
Table 8. Comparison of ellagic acid content for red raspberry cultivars
(ripe fruit), 1997 and 1998, Piketon. 1997z 1998yCultivar PulpSeed PulpSeedUg.g-1 (dry weight)Caroline 36.0173.4 52.5 a799.2 aA. Bliss 22.398.9 42.0 b263.6 cHeritage 40.5105.8 39.2 b467.2 bAnnex 11.1177.7 7.860.5Ruby 39.7176.4 10.085.6z From one sample - Piketon Research and Extension Centery Mean of 4 replications - Caroline, A. Bliss and Heritage- mean separation
determined by LSD statistic ( 0.05)x Anne is a yellow colored fruit which ripens in the fall and is grown as a red cultivarIn 1998, Caroline was significantly higher in ellagic acid content in both pulp and seed than Autumn Bliss and Heritage. The ellagic acid content in the seeds of Autumn Bliss was significantly lower than in Heritage. While Anne and Ruby samples were not replicated, their values were much lower than the other cultivars.
Blackberry - Eastern Thornless - pulp, seedThree eastern thornless blackberry cultivars, Navaho, Hull and Chester were tested for ellagic acid content in ripe and unripe fruit in 1997 (Table 9). Similar levels of ellagic acid were found for the three cultivars within pulp or seeds. Unripe berry pulp had 35%, 51% and 36% higher levels of ellagic acid than ripe berries, respectively. However, ripe seeds had 40%, 11% and 8% more ellagic acid than unripe seeds, respectively.
Table 9. Ellagic acid content of blackberry fruit, 1997 Piketon. Ripe1,2 Unripe Rate Pulp to SeedCultivar PulpSeed PulpSeed RipeUnripeUg.g-1 (dry weight)Navaho 24.1352.0 36.7214.2 1 to 14.61 to 5.8Hull 16.6346.7 33.5309.4 1 to 20.81 to 9.2Chester 22.7324.5 35.1299.3 1 to 14.31 to 8.51 From one sample2 Source: Piketon Research & Extension Center, Piketon, OH.Berry pulp had higher levels of ellagic acid in 1998 than in 1997 (Table 10). This year to year variation ranged from 71 to 84% and is similar to that reported in strawberry and black raspberry pulp for 1996 and 1997 (Tables 1 and 4). However, for seeds, the increase was 22% which is nearly the same year to year variation as in black raspberry seed content for 1996 and 1997. Chester thornless was significantly higher in ellagic acid content of pulp than any other cultivar tested in 1998.
All Berries TestedA list of berries and a range of ellagic acid content of ripe fruit and leaves is shown in Table 11. This data represents multiple samples which were taken from different locations and soil types in Ohio during 1996 to 1998. Generally, the seeds from ripe fruit had higher levels of ellagic acid than the pulp. Although a wide range and variation existed, leaves had higher levels of ellagic acid than the fruit.
Table 10. Comparison of ellagic acid content for eastern thornless
blackberry cultivars (ripe fruit), 1997 and 1998, Piketon. 1997a 1998Cultivar PulpSeed PulpSeedbUg.g-1 (dry weight)Navaho 24.1352.0 83.2b463.4aHull 16.6346.7 70.1b441.4aChester 22.7324.5 141.9a413.6aa From one sampleb Source: Piketon Research & Extension Center, Piketon, OH.
- Mean separation determined by LSD statistic ( 0.05) of 4 replicationsTable 11. Range of ellagic acid content from Ohio berries for pulp seed or leaves from tests in
Ohio 1996 to 1998.Berry Cultivar PulpSeed LeavesUg.g-1 (dry weight)Strawberry Earliglow 22 to 41184 to 1164 1743 to 2930Kent 481003 --Jewel 56252 to 617 --Mohawk 11 to 1467 to 144 --Startyme 14 to 21100 to 201 1302 to 3863Delmarvel --14 to 214 --Allstar --110 to 191 --Northeaster --68 --Seneca 50 to 57-- --Oso Grande --1208 --Camarosa --350 to 1077 --Sweet Charlie --354 to 549 --Chandler --298 to 516 -- Raspberry Black Jewel 17 to 98218 to 288 180 to 1047Red Autumn Bliss 22 to 4299 to 263 --Caroline 36 to 53173 to 799 --Heritage 39 to 40105 to 467 --Ruby 10 to 4085 to 176 --Yellow Anne 8 to 1160 to 178 --
Blackberry - Eastern Thornless Navaho 16 to 8740 to 439 --Hull 42 to 8874 to 687 --Chester 6 to 225268 to 1862 --C-65 31171 --zRanges are from multiple samples taken from different sites during 1996 to 1998.DiscussionSeveral strawberry, black raspberry, red raspberry (fall bearing) and blackberry (eastern thornless) cultivars were tested for ellagic acid content. Fruit and leaf samples were collected from Ohio State University research plots at Waterman Farm, Columbus and Piketon Research and Extension Center, Piketon, from 1996 to 1998. The reported extraction method was modified to provide more consistent results. However, the authors recognize that ellagic acid is a most difficult compound to extract and assess. While careful preparation and diligence was performed by all team members under current knowledge, it is hoped that other untried methods may prove to be more accurate and comparable than those in this report. Therefore, this initial work is reported for others to expand upon and to be used as a foundation for future research.
CultivarsCultivars within species contain different amounts of ellagic acid. Many of the cultivars that we tested had different levels of ellagic acid. However, ellagic acid content in the pulp of Earliglow is nearly the same as other cultivars tested (Bash et al., 1996). It is possible that non Ohio strawberry cultivars, grown in other states (California and Florida), may contain higher levels of ellagic acid in the seeds than those in Ohio. Earliglow does not appear to be different from other cultivars tested for ellagic acid content in the leaf. This needs to be verified with future research
In most cases unripe strawberry, raspberry or blackberry pulp had higher levels of ellagic acid than ripe fruit pulp. In all cultivars, berry seeds were always higher in ellagic acid than in pulp regardless of ripeness. In black raspberry, seeds were 12 to 15 times higher in ellagic acid content than in pulp. Ripe fruit seeds appear to be higher in ellagic acid than unripe fruit seeds by as much as 8 to 40 percent.
Maas and Galletta, 1991, indicated that genetic manipulation (breeding) could be used to increase ellagic acid, but the manner of ellagic acid inheritance was unknown because no correlation was found among pulp, seeds and leaf tissue among different strawberry cultivars. Thus selection for ellagic acid content would be highly specific for tissue type. Our data agree with that of Maas and Galletta.
The Jewel black raspberry cultivar has shown excellent fruit size and yield for Ohio growers. Jewel demonstrated no difference in ellagic acid among compost treatments in fruit (Funt et al., 1997). However, there were significant differences in leaf ellagic acid content. The difference of ellagic acid content of younger leaves of black raspberries treated with compost could be important to raspberry growers. Ellagic acid can be an endogenous inhibitor of insect feeding on certain plants (Klocke et al., 1986). Inhibitor mechanisms of ellagic acid to chewing insects and aphids may be different. Sensitivity among aphid species to several ellagitannins was shown in feeding-deterrent studies (Jones and Klocke, 1987). This needs to be verified with future research.
The Heritage fall red raspberry cultivar has been a standard in many mid-western states. Caroline, a new cultivar not widely grown in Ohio, had higher levels of ellagic acid in pulp and seed than Heritage in 1998. Autumn Bliss had lower levels of ellagic acid in the seeds than Caroline in both 1997 and 1998. Other cultivars that were tested appeared to have lower levels than Caroline or Heritage. Replicated trials for several years are needed to verify these results.
All three blackberry (eastern thornless) cultivars were similar in ellagic acid in the pulp and seed. However, Chester thornless had significantly greater ellagic acid in the pulp in 1998 than other cultivars (Funt et al., 1998). Chester thornless is a high yielding cultivar.
SeedsFor seeds, the Jewel strawberry cultivar was lower in ellagic acid than Earliglow in 1997. The data are mixed as to whether strawberry (Earliglow) seeds have greater ellagic acid levels than raspberry or blackberry seeds. Clearly in 1997 Earliglow and Kent ripe strawberry seeds had higher amounts than raspberry or blackberry cultivars studied. But in 1998 the Earliglow seeds were among the lowest levels of all berry types. In 1998 blackberry seeds were higher than either raspberry or strawberry seeds except for Caroline and Heritage fall red raspberry.
Year to year variation in ellagic acid content of pulp appears to occur in all berries tested. Less year to year variation appears to occur in black raspberry and blackberry seeds. Strawberries and red raspberry seeds had more year to year variation of ellagic acid than other berries. Leaves were not tested for year to year variation. However in one observation, strawberry leaves harvested after renovation were higher in ellagic acid than if harvested two weeks later. In all berries leaves were higher in ellagic acid content than seeds. Generally, strawberry leaves contained more ellagic acid than raspberry leaves. However, ellagic acid content varies within the growing season and may change dramatically within weeks. In raspberries treated with compost, older leaves had less ellagic acid than younger leaves. Maas et al in 1991c. indicated that ellagic acid content of red (ripe) fruit tissue varied little between two growing sites that were about 2 kilometers apart. In this study, the Earliglow strawberry was grown at two locations about 40 kilometers apart. We can not verify if location can influence ellagic acid content of strawberry pulp.
ConclusionEllagic acid exhibits anticarcinogenic and antiimutagenic activity in humans. It is a natural occurring phenolic compound found in many plants and berries. Medical research has shown reductions in the size and number of cancerous tumors when mice are fed a diet of freeze dried strawberries or raspberries.
In Ohio, ellagic acid content was found to be highest in the leaves, intermediate in seeds from ripe fruit and lowest in the pulp in strawberry and black raspberry fruit. Leaves can be much higher in ellagic acid content depending upon the time of year or physiological growth stage. The ellagic acid content in certain black raspberry seeds is 12 to 15 times higher than in the pulp.
Ellagic acid content in ripe fruit seeds of strawberry was highest in Earliglow, Startyme and Jewel cultivars. Strawberry cultivars specifically grown in Florida or California appeared to be higher in ripe seed ellagic acid content than those grown in Ohio (Earliglow or Jewel). Caroline and Heritage were highest in ellagic acid among other fall red or yellow raspberry cultivars. Generally, the seeds of Navaho, Hull and Chester eastern thornless blackberry contain about the same amounts of ellagic acid. Raspberry and blackberry seeds appear to have less year to year variation in ellagic acid content than strawberry seeds.
Variation in ellagic acid content is cultivar dependent. It does not appear that soil, site location, or management practices as fertilizer applications influence ellagic acid content of plant tissue. However, the incorporation of compost prior to planting of black raspberry may increase ellagic acid content of young leaves.
Ellagic acid is a most difficult compound to gather consistent and repeatable assays. Other methods of sample collection, preparation, extraction and analysis may prove to be better than those used in this work. Additional studies by public and private laboratories is required to validate our results to optimize the potential amount of ellagic acid per acre.
Future research in berries needs to verify year to year variation of ellagic acid in various plant tissues. Identifying ellagic acid content in tissues which show the least variation, would improve product quality and consistency for nutraceuticals. Secondly, growers could possibly benefit from insect deterrence. If certain soil amendments at planting such as compost can influence ellagic acid content in leaves, certain insects as aphids could be reduced on raspberry plants.
We have no published data as to the absorption and distribution of ellagic acid in humans. Human clinical studies utilizing berries containing ellagic acid are necessary to verify absorption and distribution into various human tissues. From these studies, recommendations for daily intake for humans can be made for fresh berries or a process product as a nutraceutical.
ReferencesBash, W.D., S.J. Schwartz and R.C. Funt. 1996. Small fruit evaluation for ellagic acid content. Proc. Ohio Fruit and Veg. Growers Congress and Roadside Marketing Conference. p. 126-129.
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Funt, R.C., W.D. Bash, S.J. Schwartz, J. Collier and G. Wenneker. 1997. Small fruit cultivar characteristics for ellagic acid and neutracuetical development. Proc. Ohio Fruit and Veg. Growers Congress and Roadside Marketing Conference. p. 110-113.
Funt, R.C., W.D. Bash, S.J. Schwartz, C. Moxley and T.E. Wall. 1998. Evaluation of year to year variation in berries for ellagic acid content and neutracuetical development. Proc. Fruit, Vegetable, Roadside Marketing and Ohio Wine Producers Conf. p. 14-18.
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Maas, J.L., G.J. Galletta and G.D. Stoner. 1991b. Ellagic acid, an anticarcinogen in fruits, especially in strawberries. A review. HortScience 26(1)10-14.
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