Source: MICHIGAN STATE UNIV submitted to
ANTI-OXIDANT, ANTI-INFLAMMATORY AND ANTI-CANCER COMPOUNDS FROM TART CHERRIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0183131
Grant No.
99-35503-8147
Project No.
MICL08228
Proposal No.
1999-01676
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Dec 1, 1999
Project End Date
Nov 30, 2003
Grant Year
2000
Project Director
NAIR, M.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
NATIONAL FOOD SAFETY AND TOXICOLOGY CENTER
Non Technical Summary
Anecdotal reports indicate that consumption of tart cherries alleviates arthritic and gout related pain. Also, consumption of cherries are reported to reduce the incidence of heart diseases in human. This project will evaluate the antioxidant, anti-inflammatory and anticancer efficacies of tart cherry compounds. Also, the coloring compound in cherries will be evaluated for their activity against colon cancer in mouse models.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5021112200060%
5021112118040%
Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
1112 - Cherry;

Field Of Science
1180 - Pharmacology; 2000 - Chemistry;
Goals / Objectives
Tart cherries as a commodity have suffered in recent years from over production and consequent low economic returns for producers. Our preliminary experiments have shown that whole cherries and cherry compounds are inexpensive and valuable food ingredients in processed meat products. The thrust of this project is to identify and study compounds from tart cherries which will be used to improve quality and safety of existing value-added foods which potentially will benefit human health. This will improve cherry utilization in the USA and provide tart cherry growers with increased returns for their product. The specific objectives of our proposal are as follows: (1) isolate and identify tart cherry chemical components and study their potential, in model systems, to act as antioxidants, to prevent inflammation, and to protect against cancer and (2) to determine if tart cherry anthocyanins, phenolic compounds rich in tart cherries, can reduce the incidence and burden of intestinal tumors in Min mice, an animal model of human colon cancer. The proposed research is expected to demonstrate long-term health effects derived from the consumption of tart cherry products. Overall, this line of research is expected to lead to a greater consumption of cherry-containing foods by humans and increase the demand for cherries and value-added cherry products to be used in human foods.
Project Methods
We will analyze fresh and lyophilized tart cherry fruits. The lyophilized fruits will be sequentially extracted with hexane, ethyl acetate, methanol and water. Each extract will be concentrated in vacuo to afford the respective fractions. Organic extracts will be evaporated to dryness under reduced pressure. The crude extracts from each fruit will be screened for antioxidant activity using the liposome oxidation bioassay (Arora and Strasburg, 1997; Arora et al., 1997), anticancer activity using the topoisomerase inhibitory bioassay, and anti-inflammatory bioassay using the cyclooxygenase assay (Laneuville et al, 1994).The active fractions will be purified using the above bioassay guided fractionation to obtain pure bioactive components. The crude extract will be separated initially into various fractions by silica or C18 VLC, MPLC or by HPLC. The pure and active compound will be subjected to spectral analyses to obtain structural identity. The effects of feeding cherry anthocyanins and cyanidin on small intestinal and colonic tumor development in Min mice will be determined. Diets will be based on modified AIN93G rodent diets(Reeves et al.,1993). Experiment 1 will be to determine the doses of cherry anthocyanins and cyanidin to reduce tumor development in Min mice. Treatments will consist of control diet, 200 ppm cyanidin, 650 ppm mixed anthocyanins, and 248 ppm sulindac. Sulindac will be used as a control (Bourquin et al., unpublished data). These concentrations were selected based on the concentrations of NSAIDS previously demonstrated to reduce tumorigenesis in Min mice (Boolbol et al., 1996, Jacoby et al., 1996). Provided that cyanidin, anthocyanins, and sulindac are efficacious in reducing tumor multiplicity in Min mice, the next series of experiments will be conducted to determine the dose-response of tumor development to these compounds. Min positive mice (25 per treatment) will be fed the experimental diets from weaning (4 weeks of age) for a period of ten weeks. Mouse weights will be measured weekly. After ten weeks on treatments, mice will be sacrificed and the colon, cecum, and small intestine removed. Tissues will be oriented and fixed in neutral buffered formalin for 24 hours and then stained with 0.2 percent methylene blue in phosphate buffered saline for five minutes to facilitate tumor identification. Tumor number and size (2 or 3-dimensional measurements in 0.5 mm increments) in each intestinal segment will be determined with the aid of a stereo microscope. Essentially all adenomas in the small intestine of Min mice have flat, 2-dimensional morphology. Hence, total tumor burden in the small intestine of each mouse will be determined by summing the average diameter of each adenoma. Colonic and cecal tumors in Min mice will be summed for each mouse to estimate solid tumor incidence and solid tumor multiplicity.

Progress 12/01/99 to 11/30/03

Outputs
The Montmorency (Prunus cerasus) variety constitutes more than 95 percent of tart cherry cultivations in Michigan and USA. However, Balaton tart cherry, a new tart cherry cultivar, is being planted to replace Montmorency in several Michigan orchards. This cherry has higher anthocyanin contents. We have determined the total anthocyanin contents of Montmorency and Balaton tart cherries. In addition, a detailed investigation of other phenolic compounds in Balaton and Montmorency tart cherries was carried out. The bioactive anthocyanins present in tart cherries are cyanidin 3-glucosylrutinoside, cyanidin 3-rutinoside and cyanidin 3-glucoside. Cyanidin is the major anthocyanidin in tart cherries. Several biofalvonoids present in tart cherries were characterized and determined their antioxidant and anti-inflammatory activities. In our evaluation of the in vivo and in vitro efficacy of cherry anthocyanins to prevent inflammation and colon cancer, we found that the anthocyanins in tart cherries are the most abundant and active compound. Cherry bioflavonoids and anthocyanins are excellent antioxidants and inhibit enzymes that cause inflammation. Based on our results that tart cherry anthocyanins and their respective aglycone, cyanidin, can inhibit cyclooxygenase enzymes, we conducted experiments to test the potential of anthocyanins to inhibit intestinal tumor development in Apc Min mice and growth of human colon cancer cell lines. Anthocyanins, cyanidin, and tart cherries reduced the numbers and size of tumors in the cecum of Apc Min mice. Anthocyanins and cyanidin also reduced the proliferation of human colon cancer cell lines HT 29 and HCT 116. These results suggest that tart cherry anthocyanins and cyanidin may reduce the risk of colon cancer. We have examined the in vivo effects of tart cherry anthocyanins (400 mg/kg) on the nociceptive behaviors and edema associated with inflammation induced by intraplantar injection of 1 percent carrageenan in rats. These studies also included tests of motor coordination. The second set of experiments determined if tart cherry anthocyanins (15, 85, and 400 mg/kg) dose-dependently affected the inflammation. We founded that tart cherry extracts reduce inflammation-induced thermal hyperalgesia, mechanical hyperalgesia, and paw edema. The suppression of thermal hyperalgesia was dose-dependent and the efficacy of highest dose (400 mg/kg) was similar to indomethacin (5 mg/kg). The highest dose anthocyanin (400 mg/kg) had no effects on motor function. These data suggest that tart cherry anthocyanins may have a beneficial role in the treatment of inflammatory pain. The antihyperalgesic effects may be related to the anti-inflammatory and antioxidant properties of anthocyanins. A better understanding of the modulatory role of dietary constituents and phytonutrients on pain will offer further therapeutic options for treating patients with persistent and chronic pain conditions. Our research on tart cherries has generated tremendous public interest in addition to numerous presentations, publications and patents.

Impacts
Our research confirming tart cherries contained antioxidant and anti-inflammatory compounds enhanced value to tart cherry and tart cherry products. It also increased demand for cherry products and helped cherry growers to earn higher income due to its health benefits. and promoted the planting of a new variety of tart cherry cultivar high in anthocyanin content.

Publications

  • US Patent/Invention NO: 6,576,271. DATE: June 10,2003.
  • US Patent/Invention NO: 6,623,743. DATE: September 23, 2003.
  • US Patent/Invention NO: 6,656,914. DATE: December 3, 2003.
  • Seeram, N.P., Zhang, Y., Nair, M.G. 2003. Inhibition of proliferation of human cancer cell lines and cyclooxygenase enzymes by anthocyanidins and catechins. Nutrition and Cancer. 46:101-106.
  • Tall,J.M., Seeram,N.P., Zhao,C., Nair,M.G., Meyer, R.A., Raja, S.N. 2003. Tart cherry anthocyanins suppress inflammation-induced pain behavior in rat. Behavioural Brain Research. In press.
  • Kang, S-Y., Seeram, N.P., Bourquin, L.D., Nair, M.G. 2003. Tart cherry anthocyanins inhibit tumor development in ApcMin mice and reduce proliferation of human colon cancer cells. Cancer Letters. 194, 13-19, 2003.


Progress 01/01/02 to 12/31/02

Outputs
The antioxidant activities of a series of commonly consumed and biogenetically related plant phenolics, namely anthocyanidins, anthocyanins and catechins, in a liposomal model system have been investigated. The antioxidant efficacies of the compounds were evaluated on their abilities to inhibit the fluorescence intensity decay of an extrinsic probe, 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenyl propionic acid (DPH-PA), caused by free radicals generated during metal-ion-induced peroxidation. Distinct SAR were revealed for the antioxidant abilities of these structurally related compounds. Whereas antioxidant activity increased with an increasing number of hydroxyl substituents present on the B-ring for anthocyanidins, the converse was observed for catechins. However, substitution by methoxyl groups diminished antioxidant activity of the anthocyanidins. Substitution at position 3 of ring C played a major role in determining antioxidant activity of these classes of compounds studied. The anthocyanidins, which possess a hydroxyl group at position 3, demonstrated potent antioxidant activities. For the cyanidins, an increasing number of glycosyl units at position 3 resulted in decreased antioxidant activity. Similarly, the substitution of a galloyl group at position 3 of the flavonoid moiety resulted in significantly decreased antioxidant activity for the catechins. Among catechins, cis-trans isomerism, epimerization and racemization did not play a role in overall antioxidant activity. The antioxidant activities of test compounds (at 40 ppm) were compared to the commercial antioxidants TBHQ, BHT and BHA and vitamin E (all at 10 ppm). We have reported that anthocyanins, plant pigments present in tart cherries, exhibit antioxidant and cyclooxygenase inhibitory activities in vitro comparable to NSAIDs. We investigated the effects of tart cherry extract (TCE) on the paw edema and hyperalgesia induced by intraplantar carrageenan administration. Male Sprague-Dawley rats received oral gavage for 4 consecutive days with TCE (250 mg in 1 ml), indomethacin (5 mg/kg) or vehicle. On day 4, gavage was followed by an intraplantar injection of 1% carrageenan (0.1 ml). Paw thickness and paw withdrawal latency (PWL) to a radiant heat stimulus were determined before and 0.5,1.5, 2.5, 4.5 and 24 hours after the administration of carrageenan. A researcher blinded to the treatment groups collected all data. Gavage pretreatment did not affect pre-carrageenan paw thickness or PWL measurements in any treatment group. At 2 and 5 hours following intraplantar carrageenan, edema was significantly less in TCE-treated rats as compared to the other treatment groups (p<0.04). Rats treated with vehicle and indomethacin developed significant thermal hyperalgesia by 2.5 and 4.5 hours post-carrageenan, respectively (p<0.01). At all time points tested, PWL after carrageenan was not significantly different from baseline values in TCE-treated rats. Our observations suggest that the gastric administration of an extract from tart cherries significantly decreases edema and prevents thermal hyperalgesia following carrageenan-induced inflammation in rats.

Impacts
Colon cancer is the third leading cause of cancer mortality in the US, resulting in over 50,000 deaths each year. Based on the results of a number of epidemiological studies, there is strong evidence that 66-75% of colon cancer cases may be preventable through a proper diet. It is well known that increased consumption of fruits and vegetables correlates with reduced colon cancer incidences in human. Fruits and vegetables contain a diverse array of phytochemicals, of these flavonoids have shown particular promise as cancer-preventive compounds of dietary origin. Our studies have indicated that anthocyanins are one such family of flavonoids with potential ability to prevent cyclooxygenase mediated inflammatory pain and possibly cancers due to its remarkable antioxidant activities.

Publications

  • Seeram, N.P., Nair, M.G. 2002. Inhibition of lipid oxidation and Structure-Activity-Related studies of dietary constituents anthocyanins, anthocyanidins and catechins. J. Agric. Food Chem. 50:5308-5312.
  • Kang, S-Y., Bourquin, L.D., Nair, M.G., Seeram, N.P. 2002. Tart cherry anthocyanins and sulindac reduce adenomas in ApcMin mice. Experimental Biology: Meeting Abstracts 565.5 (Abstract).
  • Tall, J.M., Seeram, N.P., Nair, M.G., Meyer, R.A., Raja, S.N. 2002. Carrageenan-induced edema and hyperalgesia is suppressed by oral administration of tart cherry extract in the rat. Annual Society Meeting of Neuroscience, Nov. 2-7, Orlando, Florida (Abstract).
  • US Patent/ Invention NO: 6,423,365. DATE: July 23, 2002.


Progress 01/01/01 to 12/31/01

Outputs
Anthocyanins from tart cherries,sweet cherries,bilberries, blackberries, blueberries, cranberries, elderberries, raspberries, and strawberries, were investigated for cyclooxygenase inhibitory and antioxidant activities. The presence and levels of anthocyanins were determined in the fruits by HPLC. The antioxidant activity of anthocyanins from cherries was comparable to the commercial antioxidants, tert-butylhydroquinone, butylated hydroxytoluene and butylated hydroxyanisole and superior to vitamin E at a test concentration of 125 g/ml. Anthocyanins from raspberries and sweet cherries demonstrated 45% and 47% cyclooxygenase-I and cyclooxygenase-II inhibitory activities, respectively, when assayed at 125 g/ml. The cyclooxygenase inhibitory activities of anthocyanins from these fruits were comparable to ibuprofen and naproxen at 10 M concentrations. Anthocyanins 1 and 2 are present in both cherries and raspberry. The yields of pure anthocyanins 1 and 2 in 100 g fresh Balaton and Montmorency tart cherries, sweet cherries and raspberries were 21, 16.5; 11, 5; 4.95, 21; and 4.65, 13.5 mg, respectively. Fresh blackberries and strawberries contained only anthocyanin 2 in yields of 24 and 22.5 mg/100g, respectively. Anthocyanins in cherries were not found in bilberries, blueberries, cranberries or elderberries. We have added these compounds physiological medium in an effort to identify their degradation products during in vitro cell culture studies. This resulted in the isolation and characterization of protocatechuic acid,2,4-dihydroxybenzoic acid and 2,4,6-trihydroxybenzoic acid as degradation products of anthocyanins. In cyclooxygenase (COX)-I and -II enzyme inhibitory assays, compounds 5-9 did not show significant activities when compared to the NSAIDs, naproxen, celebrex and vioxx and ibuprofen at 50 micromolar concentrations . However, at a test concentration of 50 micromoles, the antioxidant activities of both protocatechuic acid (5) and benzyl glucoside (8) were comparable to the commercial antioxidants, tert-butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) and superior to vitamin E at 10 micro molar concentrations.

Impacts
Colon cancer is the third leading cause of cancer mortality in the US, resulting in over 50,000 deaths each year. Based on the results of a number of epidemiological studies, there is strong evidence that 66-75% of colon cancer cases may be preventable through a proper diet. It is well known that increased consumption of fruits and vegetables correlates with reduced colon cancer incidences in human. Fruits and vegetables contain a diverse array of phytochemicals, of these flavonoids have shown particular promise as cancer-preventive compounds of dietary origin. Our studies have indicated that anthocyanins are one such family of flavonoids with potential ability to prevent cyclooxygenase mediated inflammatory pain and possibly cancers due to its remarkable antioxidant activities.

Publications

  • N.P. Seeram, L.D. Bourquin and M.G. Nair. Degradation Products of Cyanidin Glycosides from Tart Cherries and their Bioactivities. J. Agric. Food Chem. 49,4924-4929, 2001.
  • N. P. Seeram, R.A. Momin, L.D. Bourquin and M.G. Nair. Cyclooxygenase and antioxidant cyanidin glycosides in cherries and berries. Phytomedicine. 8, 362-369,2001.
  • S-Y. Kang, L.D. Bourquin, M.G. Nair and N.P. Seeram. Tart cherry anthocyanins suppress growth of human colon cancer cell lines HT 29 and HCT 116. Annual Meeting of Experimental Biology, Orlando, Florida, April 2001.
  • A patent application has been filed with the US PTO (Application # 09/776,527, filing date 02/02/2001. It was published on 08/23/2001(publication number US-2001-0016573-A1).


Progress 01/01/00 to 12/31/00

Outputs
Anthocyanins from tart cherries, Prunus cerasus L. (Rosaceae) cv. Balaton and Montmorency, sweet cherries, Prunus avium L. (Rosaceae), bilberries, Vaccinum myrtillus L. (Ericaceae), blackberries, Rubus sp. (Rosaceae), blueberries var. Jersey, Vaccinium corymbosum L. (Ericaceae), cranberries var. Early Black, Vaccinium macrocarpon Ait. (Ericaceae), elderberries, Sambucus canadensis (Caprifoliaceae), raspberries, Rubus idaeus (Rosaceae) and strawberries var. Honeoye, Fragaria x ananassa Duch. (Rosaceae), were investigated for cyclooxygenase inhibitory and antioxidant activities. The presence and levels of cyanidin-3-glucosylrutinoside 1 and cyanidin-3-rutinoside 2 were determined in the fruits by HPLC. The antioxidant activity of anthocyanins from cherries was comparable to the commercial antioxidants, tert-butylhydroquinone, butylated hydroxytoluene and butylated hydroxyanisole and superior to vitamin E at a test concentration of 125 mg/ml. Anthocyanins from raspberries and sweet cherries demonstrated 45% and 47% cyclooxygenase-I and cyclooxygenase-II inhibitory activities, respectively, when assayed at 125 mg/ml. The cyclooxygenase inhibitory activities of anthocyanins from these fruits were comparable to ibuprofen and naproxen at 10 mM concentrations. Anthocyanins 1 and 2 are present in both cherries and raspberry. The yields of pure anthocyanins 1 and 2 in 100 g fresh Balaton and Montmorency tart cherries, sweet cherries and raspberries were 21, 16.5; 11, 5; 4.95, 21; and 4.65, 13.5 mg, respectively. Fresh blackberries and strawberries contained only anthocyanin 2 in yields of 24 and 22.5 mg/100 g, respectively. Anthocyanins 1 and 2 were not found in bilberries, blueberries, cranberries or elderberries. We have now processed more than 3 tons of cherries and produced 1.5 kg of pure anthocyanins for in vivo studies.

Impacts
(N/A)

Publications

  • No publications reported this period