Antioxidant Ellagic Acid Studies

Multiple studies have discovered that phytonutrients found in raspberries can protect us from cancer and can even shrink some types of cancer tumors. These substances can also act as an antibacterial and as an antiviral agent. Does this sound too good to be true? One particular substance found in this natural “medicine chest”, is a series of compounds called ellagitannins. The highest levels are found in raspberries, but the ellagitannins are also in certain types of grapes, strawberries, blackberries, blueberries and some nuts too. Recent work (2001), published by Dr. Gary Stoner at Ohio State University, showed that components in the seeds and berry, but particularly ellagitannins, inhibited the initiation and promotion/progression stages of esophageal cancer. This is an extremely important finding, considering the potential benefits.

We do not as yet know all of the functions of the ellagitannins in terms of cancer. A study at Hollings Cancer Center, Medical University of South Carolina has shown one of the ways they work is to “turn on” a normal cellular process called apoptosis. Apoptosis is “science speak” for something called programmed cell death. This natural cell death is just one of several ways our body protects us from cancer. As we age, cellular replication mistakes can occur. Cancer cells somehow become immune to the signals that cause cells to self-destruct, so they become virtually immortal and reproduce indefinitely. Another way ellagitannins work is to inhibit the growth of tumors. Because of the need for an independent blood supply inhibition of angiogenesis limits the size of the tumor to less then 2 cm.

The disease with a thousand faces

Cancer is not just one disease but is the general name for more than 200 different types of malignancies. Cancers are classified by the tissue type from which they arise, for example:

• osteosarcoma – bone cancer
• melanoma – skin cancer
• lymphoma – cancer of lymph nodes
• leukemia – blood cancer

Every cellular type has its own form of cancer. The one thing all cancers share in common is uncontrolled growth. Cancer occurs when cells lose control over critical checkpoints during the process of one cell splitting and becoming two cells. This control over cellular replication is in the hands of several specific types of genes.

Two classes of genes are suspected of being associated with the occurrence of cancer. A mutation in a tumor suppressor gene is like having faulty brakes in your car. Just as their name implies, tumor suppressor genes function by making sure there are no mistakes in the genes that are replicated prior to one cell becoming two. In this “quality control” process, if errors are detected, the cell is instructed not to divide. Thus, tumor suppressor genes put the brakes on cellular division. The genes of the other class thought to be involved with preventing cancer are called proto-oncogenes.

Researchers have found that these genes “code” for proteins involved in mechanisms that regulate the social behavior of cells. Signals from those cells in the immediate environment induce their neighbors to divide, differentiate and even undergo apoptosis. So, this type of gene is involved in promoting the normal growth and division of cells and could be likened to your car’s accelerator. A change in the genetic message – a mutation, can turn the proto-oncogene into an oncogene and cause your accelerator to become stuck, thus initiating “runaway” cellular replication. Nevertheless, there seem to be no pattern to these mutations. What is so frustrating for both researchers and clinicians alike is that different combinations of mutations are found in different types of cancer and even in cancers of supposedly the same type in different patients. What is most important to remember is that cancer begins as a single abnormal cell that begins to multiply out of control.

So, what causes most mutations?

We live in a polluted environment. For instance, the outgassing from asphalt on a hot summer day produces the deadly carcinogen benzo{a}pyrene, the same chemical found on meat that has been charcoal broiled. This is just but one example. Exposure to such chemicals in the environment can cause the mutations in our genetic material that lead to cancer. Even normal metabolic processes like breathing and exercise produce free radicals that can wreak havoc on our cellular DNA. We can protect ourselves from mutations caused by environmental toxins and free radicals by taking antioxidants. Guess what? Ellagitannins are also very good antioxidants and chemoprotective agents. Researchers at Wayne State University have a theory about how ellagitannins might work. The liver produces enzymes that rid the body of toxins. These enzymes break down or chemically change toxic substances we ingest or inhale so that they can be excreted. During this detox process, the breakdown products, called metabolites, are frequently more damaging then the original substance. It appears that ellagitannins are able to safeguard the liver from damage caused by these breakdown products. Another theory held by some investigators is that ellagitannins are able to protect our genetic material from certain types of chemical reactions that lead to misreading of damaged DNA.

Why does chemotherapy and radiation eventually stop working?

It is becoming clear that normal therapeutic cancer treatment works by turning on apoptosis. We used to think that chemotherapy and radiation killed rapidly dividing cells, which is why these procedures were able to shrink tumors. However, at some point these treatments begin to lose their effectiveness. Why is that? Scott Lowe, a research scientist at Cold Spring Harbor Laboratory may have found the answer. Instead of killing these cells, chemotherapy and radiation damage their cellular DNA. This alerts the cell watchdogs that control the cell cycle that something is wrong and tells the cell to stop dividing or to commit suicide. Therefore, chemotherapy and radiation act somewhat like a “vaccination” that works by helping the body help itself. The evidence for Dr. Lowe’s theory is pretty convincing, because when these treatments start to fail, researchers have found that the genes that control apoptosis are no longer functioning.

Why don’t ellagitannins induce normal cells to commit suicide?

As we know, cancer cells become immortal, this means that they are able to replicate themselves after something called the Hayflick limit has been reached. The Hayflick limit is the number of “allowed” cellular replications. Each cell type has its own limit. Human cancer studies show that mutations in the tumor suppressor gene called p53 account for many of the tumors found. One of the functions of this gene is that it normally prevents cells with damaged DNA from proceeding through the cell cycle. The presence of the protein product encoded by p53 turns on the waf-1 gene. The waf-1 gene produces a protein that normally inhibits the activity of several similar cellular proteins called kinases. These proteins are involved in stopping cell cycle progression. A mutation in either the p53 or waf-1 gene can cause the loss of that “emergency brake” function and allow uncontrolled growth. However, only “damaged” cells are induced to commit suicide and so normal cells are not effected.

An Antibacterial and an Antiviral agent

Ellagitannins can act as antibacterial agents and as antiviral agents too, and now we know how. Think of the genetic material of bacteria as a rubber band that is all twisted up. In order to replicate, the DNA must untwist itself through a process requiring the enzyme gyrase. Ellagitannins inhibits gyrase activity so replication of the bacterial DNA is restricted. Importantly, bacteria cannot easily become resistant to this type of antibacterial action. Resistance to antibiotics has become a real concern to the international medical community. A federal government task force noted that antibiotic resistance was “a growing menace to all people” but children, the elderly and those with weakened immune systems are especially at risk. Besides its antibacterial action, ellagitannins have antiviral activity also. Viruses do not have the ability to replicate themselves. Instead they must “hijack” the host cell and insert their own DNA into the host cell genome. This requires an enzyme called integrase and the ellagitannins inhibit this enzyme also.

Citizen: Heal Thyself. (With apologies to Mr. Hippocrates)

People are turning to alternative forms of medical treatment and prevention. Not only is the medical delivery system failing but, our costs for health services are rising at an astronomical rate. What this means for the medical consumer is that we need to be more responsible for our own health. We need to look at prevention instead of always looking to health care providers to “fix” what exposure to a toxic environment and/or years of unhealthy lifestyle practices have wrought. The quality of medical care is uneven at best. Too often, our insurance providers do not cover necessary tests and procedures, especially those of a preventative nature. However, we can become involved in our own health care. A diet rich in fresh fruits and vegetables is a good start towards preventing disease.

Unfortunately, current tests show that our soil is severely lacking in many minerals or electrolytes and other components that are essential for proper nutrition. It is necessary to sometimes take supplements as it may not be physically or economically possible to eat enough food to get the proper nutrition. In addition, the cost of fresh fruits and vegetables can be prohibitive. For instance, unless you grow your own raspberries, the cost of the American Cancer Institute’s recommended daily bowl of the whole berries could run as high as $300 a month. Not only that, but research has shown that the ellagitannin content is much higher in the seeds then in the fruit. So nutraceutical supplements may be the answer. Raspberry seeds contain many more times the ellagic acid than the fruit at one-tenth the cost. It’s your choice, whatever form you may decide to use- the take home message is: “Eat your ellagitannins!”

References

Study Abstract #1

Effect of chemopreventive agents on DNA adduction induced by the potent mammary carcinogen dibenzo[a,l]pyrene in the human breast cells MCF-7.

Taken from: Mutat Res 2001 Sep 1;480-481:97-108
Smith WA, Freeman JW, Gupta RC.
Graduate Center for Toxicology, 354 Health Sciences Research Building, University of Kentucky Medical Center, Lexington, KY 40536-0305, USA.

Over 1500 structurally diverse chemicals have been identified which have potential cancer chemopreventive properties. The efficacy and mechanisms of this growing list of chemoprotective agents may be studied using short-term bioassays that employ relevant end-points of the carcinogenic process. In this study, we have examined the effects of eight potential chemopreventive agents, N-acetylcysteine (NAC), benzylisocyanate (BIC), chlorophyllin, curcumin, 1,2-dithiole-3-thione (D3T), ellagic acid, genistein, and oltipraz, on DNA adduction of the potent mammary carcinogen dibenzo[a,l]pyrene (DBP) using the human breast cell line MCF-7. Bioactivation of DBP by MCF-7 cells resulted in the formation of one predominant (55%) dA-derived and several other dA- or dG-derived DNA adducts. Three test agents, oltipraz, D3T, and chlorophyllin substantially (>65%) inhibited DBP-DNA adduction at the highest dose tested (30 microM). These agents also significantly inhibited DBP adduct levels at a lower dose of 15 microM, while oltipraz was effective even at the lowest dose of 5 microM. Two other agents, genistein and ellagic acid were moderate (45%) DBP-DNA adduct inhibitors at the highest dose tested, while NAC, curcumin, and BIC were ineffective.

These studies indicate that the MCF-7 cell line is an applicable model to study the efficacy of cancer chemopreventive agents in a human setting. Moreover, this model may also provide information regarding the effect of the test agents on carcinogen bioactivation and detoxification enzymes.

Study Abstract #2

Tannins, xenobiotic metabolism and cancer chemoprevention in experimental animals.

Taken from: Eur J Drug Metab Pharmacokinet 1999 Apr-Jun;24(2):183-9
Nepka C, Asprodini E, Kouretas D.
Cytopathology Laboratory, Serres, Greece.

Tannins are plant polyphenolic compounds that are contained in large quantities in food and beverages (tea, red wine, nuts, etc.) consumed by humans daily. It has been shown that various tannins exert broad cancer chemoprotective activity in a number of animal models. This review summarizes the recent literature regarding both the mechanisms involved, and the specific organ cancer models used in laboratory animals. An increasing body of evidence demonstrates that tannins act as both anti-initiating and antipromoting agents. In view of the fact that tannins may be of valid medicinal efficacy in human clinical trials, the present review attempts to integrate results from animal studies, and considers their possible application in humans.

Study Abstract #3

The effects of dietary ellagic acid on rat hepatic and esophageal mucosal cytochromes P450 and phase II enzymes.

Taken from: Carcinogenesis 1996 Apr;17(4):821-8
Ahn D, Putt D, Kresty L, Stoner GD, Fromm D, Hollenberg PF.
Department of Surgery, Wayne State University, Detroit, MI 48201, USA.

Ellagic acid (EA), a naturally occurring plant polyphenol possesses broad chemoprotective properties. Dietary EA has been shown to reduce the incidence of N-2-fluorenylacetamide-induced hepatocarcinogenesis in rats and N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumors. In this study changes in the expression and activities of specific rat hepatic and esophageal mucosal cytochromes P450 (P450) and phase II enzymes following dietary EA treatment were investigated. Liver and esophageal mucosal microsomes and cytosol were prepared from three groups of Fisher 344 rats which were fed an AIN-76 diet containing no EA or 0.4 or 4.0 g/kg EA for 23 days.

In the liver total P450 content decreased by up to 25% and P450 2E1-catalyzed p-nitrophenol hydroxylation decreased by 15%. No changes were observed in P450 1A1, 2B1 or 3A1/2 expression or activities or cytochrome b5 activity. P450 reductase activity decreased by up to 28%. Microsomal epoxide hydrolase (mEH) expression decreased by up to 85% after EA treatment, but mEH activities did not change. The hepatic phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone reductase ?NAD-(P)H:QR? and UDP glucuronosyltransferase (UDPGT) activities increased by up to 26, 17 and 75% respectively.

Assays for specific forms of GST indicated marked increases in the activities of isozymes 2-2 (190%), 4-4 (150%) and 5-5 (82%). In the rat esophageal mucosa only P450 1A1 could be detected by Western blot analysis and androstendione was the only P450 metabolite of testosterone detectable. However, there were no differences in the expression of P450 1A1, the formation of androstendione or NAD(P)H:QR activities between control and EA-fed rats in the esophagus.

Although there was no significant decrease in overall GST activity, as measured with 1-chloro-2,4-dinitrobenzene (CDNB), there was a significant decrease in the activity of the 2-2 isozyme (66% of control). In vitro incubations showed that EA at a concentration of 100 microM inhibited P450 2E1, 1A1 and 2B1 activities by 87, 55 and 18% respectively, but did not affect 3A1/2 activity. Using standard steady-state kinetic analyses, EA was shown to be a potent non-competitive inhibitor of both liver microsomal ethoxyresorufin O-deethylase and p-nitrophenol hydroxylase activities, with apparent Ki values of approximately 55 and 14 microM respectively.

In conclusion, these results demonstrate that EA causes a decrease in total hepatic P450 with a significant effect on hepatic P450 2E1, increases some hepatic phase II enzyme activities ?GST, NAD-(P)H:QR and UDPGT? and decreases hepatic mEH expression. It also inhibits the catalytic activity of some P450 isozymes in vitro. Thus the chemoprotective effect of EA against various chemically induced cancers may involve decreases in the rates of metabolism of these carcinogens by phase I enzymes, due to both direct inhibition of catalytic activity and modulation of gene expression, in addition to effects on the expression of phase II enzymes, thereby enhancing the ability of the target tissues to detoxify the reactive intermediates.

Study Abstract #4

p53/p21(WAF1/CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells.

Taken from:Cancer Lett 1999 Mar 1;136(2):215-21
Narayanan BA, Geoffroy O, Willingham MC, Re GG, Nixon DW.
Cancer Prevention Program, Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, USA. [email protected]

Ellagic acid is a phenolic compound present in fruits and nuts including raspberries, strawberries and walnuts. It is known to inhibit certain carcinogen-induced cancers and may have other chemopreventive properties. The effects of ellagic acid on cell cycle events and apoptosis were studied in cervical carcinoma (CaSki) cells. We found that ellagic acid at a concentration of 10(-5) M induced G arrest within 48 h, inhibited overall cell growth and induced apoptosis in CaSki cells after 72 h of treatment. Activation of the cdk inhibitory protein p21 by ellagic acid suggests a role for ellagic acid in cell cycle regulation of cancer cells.

Study Abstract #5

Chemoprevention of esophageal tumorigenesis by dietary administration of lyophilized black raspberries.

Taken from: Cancer Res 2001 Aug 15;61(16):6112-9
Kresty LA, Morse MA, Morgan C, Carlton PS, Lu J, Gupta A, Blackwood M, Stoner GD.
Division of Environmental Health Sciences, School of Public Health, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA.

Fruit and vegetable consumption has consistently been associated with decreased risk of a number of aerodigestive tract cancers, including esophageal cancer. We have taken a “food-based” chemopreventive approach to evaluate the inhibitory potential of lyophilized black raspberries (LBRs) against N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in the F344 rat, during initiation and postinitiation phases of carcinogenesis. Anti-initiation studies included a 30-week tumorigenicity bioassay, quantification of DNA adducts, and NMBA metabolism study. Feeding 5 and 10% LBRs, for 2 weeks prior to NMBA treatment (0.25 mg/kg, weekly for 15 weeks) and throughout a 30-week bioassay, significantly reduced tumor multiplicity (39 and 49%, respectively). In a short-term bioassay, 5 and 10% LBRs inhibited formation of the promutagenic adduct O(6)-methylguanine (O(6)-meGua) by 73 and 80%, respectively, after a single dose of NMBA at 0.25 mg/kg. Feeding 5% LBRs also significantly inhibited adduct formation (64%) after NMBA administration at 0.50 mg/kg. The postinitiation inhibitory potential of berries was evaluated in a second bioassay with sacrifices at 15, 25, and 35 weeks. Administration of LBRs began after NMBA treatment (0.25 mg/kg, three times per week for 5 weeks). LBRs inhibited tumor progression as evidenced by significant reductions in the formation of preneoplastic esophageal lesions, decreased tumor incidence and multiplicity, and reduced cellular proliferation. At 25 weeks, both 5 and 10% LBRs significantly reduced tumor incidence (54 and 46%, respectively), tumor multiplicity (62 and 43%, respectively), proliferation rates, and preneoplastic lesion development. Yet, at 35 weeks, only 5% LBRs significantly reduced tumor incidence and multiplicity, proliferation indices and preneoplastic lesion formation. In conclusion, dietary administration of LBRs inhibited events associated with both the initiation and promotion/progression stages of carcinogenesis, which is promising considering the limited number of chemopreventives with this potential.

Study Abstract #6

DNA gyrase inhibitory activity of ellagic acid derivatives.

Taken from: Cancer Res 2001 Aug 15;61(16):6112-9
Bioorg Med Chem Lett 1998 Jan 6;8(1):97-100

Weinder-Wells MA, Altom J, Fernandez J, Fraga-Spano SA, Hilliard J, Ohemeng K, Barrett JF.
R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ 08869, USA.

Ellagic acid was found to inhibit E. coli DNA gyrase supercoiling with approximately the same potency as nalidixic acid. Tricyclic analogs of ellagic acid, which vary in the number and position of the hydroxy groups as well as their replacement with halogens, have been synthesized. The biological activity of these analogs is discussed.

Study Abstract #7

Antioxidant properties of novel preparations-bioflavonoid derivatives and tannins. (This is translation. Article written in Russian)

Taken from: Eksp Klin Farmakol 2001 Mar-Apr;64(2):55-9

Iakovleva LV, Gerasimova OA, Karbusheva IV, Ivakhnenko AK, Buniatian ND, Sakharova TS.
Central Research Laboratory, Ukrainian Pharmaceutical Academy, ul. Pushkinskaya 53, Kharkov, 310002 Ukraine.

New medicinal plant preparations of polyphenol nature, representing the derivatives of bioflavonoids (piflamin) and ellagotannins (altan and ellagic acid) were experimentally studied. The drugs exhibited antioxidant properties, which were manifested by inhibition of a pathological lipid peroxidation, restoration of the functional activity of the antioxidant system components, and stabilization of the hepatocyte membranes.

Study Abstract #8

Human immunodeficiency virus type 1 cDNA integration: new aromatic hydroxylated inhibitors and studies of the inhibition mechanism.

Taken from: Antimicrob Agents Chemother 1998 Sep;42(9):2245-53

Farnet CM, Wang B, Hansen M, Lipford JR, Zalkow L,
Robinson WE Jr, Siegel J, Bushman F.
Salk Institute for Biological Studies, La Jolla, California, USA.

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA is a required step for viral replication. Integrase, the virus-encoded enzyme important for integration, has not yet been exploited as a target for clinically useful inhibitors. Here we report on the identification of new polyhydroxylated aromatic inhibitors of integrase including ellagic acid, purpurogallin, 4,8, 12-trioxatricornan, and hypericin, the last of which is known to inhibit viral replication. These compounds and others were characterized in assays with subviral preintegration complexes (PICs) isolated from HIV-1-infected cells. Hypericin was found to inhibit PIC assays, while the other compounds tested were inactive. Counterscreening of these and other integrase inhibitors against additional DNA-modifying enzymes revealed that none of the polyhydroxylated aromatic compounds are active against enzymes that do not require metals (methylases, a pox virus topoisomerase). However, all were cross-reactive with metal-requiring enzymes (restriction enzymes, a reverse transcriptase), implicating metal atoms in the inhibitory mechanism. In mechanistic studies, we localized binding of some inhibitors to the catalytic domain of integrase by assaying competition of binding by labeled nucleotides. These findings help elucidate the mechanism of action of the polyhydroxylated aromatic inhibitors and provide practical guidance for further inhibitor development.

Study Abstract #9

Inhibition of liver fibrosis by ellagic acid.

Taken from: Indian J Physiol Pharmacol 1996 Oct;40(4):363-6

Thresiamma KC, Kuttan R.
Amala Cancer Research Centre, Amala Nagar, Trichur, Kerala.

Chronic administration of carbon tetrachloride in liquid paraffin (1.7) ip; 0.15 ml, (20 doses) has been found to produce severe hepatotoxicity, as seen from the elevated levels of serum and liver glutamate-pyruvate transaminase, alkaline phosphatase and lipid peroxides. The chronic administration of carbon tetrachloride was also found to produce liver fibrosis as seen from pathological analysis as well as elevated liver-hydroxy proline. Oral administration of ellagic acid was found to significantly reduce the elevated levels of enzymes, lipid peroxide and liver hydroxy proline in these animals and rectified liver pathology. These results indicate that ellagic acid administration orally can circumvent the carbon tetrachloride toxicity and subsequent fibrosis.

Study Abstract #10

The protective action of ellagic acid in experimental myocarditis [This is translation. Article written in Russian]

Taken from: Eksp Klin Farmakol 1998 May-Jun;61(3):32-4

Iakovleva LV, Ivakhnenko AK, Buniatian ND.
Central Research Laboratory, Ukranian Pharmaceutical Academy, Kharkov, Ukraine.

The article presents the material on the study of the cardioprotective effect of ellagic acid on a model of neoepinephrine myocarditis in rats. In doses of 0.5-1 mg/kg ellagic acid causes a marked antioxidant effect. Restores the disturbed myocardial functions. The reference-agent vitamin E (50 mg/kg) yields to ellagic acid as a cardioprotector. The effect of 0.5 mg/kg of ellagic acid was more stable than that of a 1 mg/kg dose. The cardioprotective activity of the drugs under study was determined according to the POL parameters in a myocardial homogenate and blood serum and according to the EEG parameters and the degree of cardiomyocyte cytolysis.