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Chemical Structures of Curcumin Metabolites

Comparative effects of curcumin and an analog of curcumin on alcohol and PUFA induced oxidative stress



Abstract

PURPOSE: Alcoholic liver disease is a major medical complication of alcohol abuse and a common liver disease in western countries. Increasing evidence demonstrates that oxidative stress plays an important etiologic role in the development of alcoholic liver disease. Alcohol alone or in combination with high fat is known to cause oxidative injury. The present study therefore aims at evaluating the protective role of curcumin, an active principle of turmeric and a synthetic analog of curcumin (CA) on alcohol and thermally oxidised sunflower oil (DPUFA) induced oxidative stress. METHODS: Male albino Wistar rats were used for the experimental study. The liver marker enzymes: g-glutamyl transferase (GGT), alkaline phosphatase (ALP), the lipid peroxidative indices: thiobarbituric acid reactive substances (TBARS) and hydroperoxides (HP) and antioxidants such as vitamin C, vitamin E, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) were used as biomarkers for testing the antioxidant potential of the drugs.


RESULTS: The liver marker enzymes and lipid peroxidative indices were increased significantly in alcohol, DPUFA and alcohol + DPUFA groups. Administration of curcumin and CA abrograted this effect. The antioxidant status which was decreased in alcohol, DPUFA and alcohol + DPUFA groups was effectively modulated by both curcumin and CA treatment. However, the reduction in oxidative stress was more pronounced in CA treatment groups compared to curcumin.


CONCLUSION: In conclusion, these observations show that CA exerts its protective effect by decreasing the lipid peroxidation and improving antioxidant status, thus proving itself as an effective antioxidant.



Introduction

Oxidative stress plays an important role in the development of alcohol induced tissue injury . Oxidative stress is generally considered as an imbalance between pro oxidant/antioxidant. Intake of alcohol results in excessive generation of free radicals, which alter the bio membranes and cause severe damage. Alcohol alone or in combination with high fat is known to cause oxidative injury.

Fat is an important dietary component, which affects both growth and health. It is widely accepted that a high level of fat in the diet is detrimental to health. Replacing the traditional cooking fats, considered atherogenic with refined vegetable oils promoted as `heart friendly' because of their PUFA content, has resulted in increased prevalence of heart disease in India . Current data on dietary fats indicate that it is not just the presence of PUFA but the type of PUFA that is important. A high PUFA n-6 content and a high n-6/n-3 ratio in dietary fats are considered to be dangerous (4). The newer heart friendly oils like sunflower oil possess this undesirable PUFA content and thus excess intake of these vegetable oils is actually detrimental to health. Moreover heating of oil is known to alter its nutritional properties especially when it is rich in PUFA. During deep fat frying many volatile and non-volatile products are produced, some of which are toxic depending on the level of intake.

Alcoholics usually after a heavy binge of alcohol, take fried food items normally made up of PUFA. Our previous studies have shown that the intake of sunflower oil along with alcohol aggravates the toxicity, especially when it is heated.

Curcumin (C) and bisdemethoxy curcumin are natural phenolic curcuminoids present in turmeric, a spice used in Indian food. Curcumin, the active principle of turmeric has been extensively investigated for its antioxidant potential. In experimental animals, curcumin has been shown to prevent lipid peroxidation.



Figure 1: (a) Curcumin and (b) Curcumin Analog

A report has shown that an ortho hydroxyl group substituted analog of curcumin is very effective compared to all other existing curcuminoids in treating skin tumours. Previous studies from our lab have shown that this curcuminoid is effective against colon cancer and diabetes. Since little or no work has been done to evaluate the other therapeutic strategy of this curcuminoid, in the present study, we synthesized an o-hydroxy substituted analog of curcumin (Figure 1b) and compared its effects with curcumin over alcohol and PUFA induced oxidative stress.



Materials and Methods

Animals

Male Albino rats, Wistar strain of body weight ranging 140-160 g bred in Central Animal House, Rajah Muthiah Medical College, Tamil Nadu, India, fed on standard pellet diet (Agro Corporation Private Limited, Bangalore, India) were used for the study and water was given ad libitum . The standard pellet diet comprised 21% protein, 5% lipids, 4% crude fibre, 8% ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, 2% vitamin and 55% nitrogen free extract (carbohydrates). It provides metabolisable energy of 3600 K Cal.

The animals were housed in plastic cages under controlled conditions of 12 h light/12 h dark cycle, 50% humidity and at 30° ± 2°C. The animals used in the present study were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the Animal Ethical Committee, Annamalai University.

Materials used

Ethanol: Absolute ethanol (AR) was obtained from Hayman limited, England.

Thermally oxidised PUFA (D PUFA): Sunflower oil (Gold Winner) was subjected to heating at 180°C for 30 minutes, twice (Fatty acid composition given in Table 1) (6).

Table 1: Fatty Acid Composition of Sunflower Oil (Percentage of Fatty Acid/g Oil)



Values are mean ± S.D. of six values.

Curcumin: Curcumin was obtained from Central drug house private limited, Mumbai, India.

Curcumin analog: Curcumin analog was synthesized as per the method described by Dinesh Babu and Rajasekaran (13). Briefly, the procedure is as follow, Acetyl acetone was mixed with boric acid and dimethyl formamide (DMF), heated for 15 minutes. To that hot mixture, salicylaldeyde was added and heating was continued further for 5min. Few drops of catalyst (2:1 mixture of glacial acetic acid and diethanolamine) was added, refluxed for 5 to 6 hours and kept overnight. Few ml of DMF was added, warmed and the flowy paste was poured to 10% acetic acid slowly with stirring. The drug separates as a yellow solid mass. The product thus obtained was purified by column chromatography packed with silica gel using chloroform as solvent. Purity was checked by thin layer chromatography and the structure was further confirmed by FTIR and H1 NMR.

All other chemicals and reagents used in the present study were of analytical grade and were obtained from Sigma Chemical Company, Saint Louis, USA and Hi media laboratories, Mumbai, India.


Experimental design

The animals were divided into 12 groups of 6 rats each.

Group 1 (Control): Control rats were given glucose solution isocalorific to ethanol and high fat diet.

Group 2 (Alcohol): Rats given 20% ethanol (7.9 g/kg body weight) (14) orally, using an intragastric tube.

Group 3 ( D PUFA): Rats given high fat diet (15% thermally oxidised sunflower oil) mixed with the diet.

Group 4 (Alcohol + D PUFA): Rats given 20% ethanol + 15% thermally oxidised sunflower oil.

Group 5 (Alcohol + C): Rats given curcumin (80 mg/kg body weight) dissolved in 20% ethanol.

Group 6 (Alcohol + CA): Rats given curcumin analog (80 mg/kg body weight) dissolved in 20% ethanol.

Group 7 ( D PUFA + C): Rats given 15% thermally oxidised sunflower oil + curcumin (80 mg/kg body weight) dissolved in distilled water.

Group 8 ( D PUFA + CA): Rats given 15% thermally oxidised sunflower oil + curcumin analog (80 mg/kg body weight) dissolved in distilled water.

Group 9 (Alcohol + D PUFA + C): Rats given curcumin (80 mg/kg body weight) dissolved in 20% ethanol + 15% thermally oxidised sunflower oil.

Group 10 (Alcohol + D PUFA + CA): Rats given curcumin analog (80 mg/kg body weight) dissolved in 20% ethanol + 15% thermally oxidised sunflower oil.

Group 11 (Curcumin): Rats given curcumin (80 mg/kg body weight) dissolved in distilled water orally using an intragastric tube.

Group 12 (CA): Rats given Curcumin analog (80 mg/kg body weight) dissolved in distilled water orally using an intragastric tube.

Rats were maintained in isocalorific diet using glucose solution. (Total calories per day: 508 K Cal/kg body weight). At the end of the experimental period of 45 days, the rats were killed by cervical decapitation and the blood and tissues (liver, heart and kidney) were collected for various biochemical estimations.

Preparation of Plasma: Blood was collected in a heparinised tube and plasma was separated by centrifugation at 1000 g for 15 min for the estimation of GGT and ALP.

Preparation of Tissue Homogenate: Known amount of tissue was weighed and homogenised in appropriate buffer for the estimation of lipid peroxidative indices and enzymic and non-enzymic antioxidants.



Biochemical investigation

Estimation of liver marker enzymes

Hepatic damage was assessed by estimating the activities of ALP by King and Armstrong method and GGT by Orlowski and Meister method. Based on the method of King and Armstrong, alkaline phosphatase activity was assayed using disodium phenyl phosphate as substrate. After preincubation of buffer (0.1 M Bicarbonate buffer pH 10) with substrate for 10 minutes, 0.2 ml of serum was added and incubated for 15 min at 37°C. The liberated phenols from substrate, reacts with Folin - Phenol reagent (1 ml). The suspensions were centrifuged and supernatant was collected. 2 ml of 10% sodium bicarbonate was added to supernatant and the colour developed was read at 680 nm after 10 min.

GGT was analysed by adding 2ml buffer (Tris HCl 120 mm , MgCl2 12mM glycyl glycine 90 mM , pH 7.8) to 0.2ml substrate (L- g-glutamyl p-nitro anilide 48 mm in 150mM HCl ), warmed to 37°C. 0.1 ml serum was added, mixed and incubated at 37°C. The reaction was then stopped by adding 2ml of glacial acetic acid and the absorbance was read at 405nm.


Estimation of lipid peroxidative indices

Lipid peroxidation as evidenced by the formation of TBARS and HP were measured by the method of Niehaus and Samuelsson (17) and Jiang et al. (18) respectively. In brief, 0.1 ml of tissue homogenate (Tris-Hcl buffer, pH 7.5) was treated with 2 ml of (1:1:1 ratio) TBA-TCA-HCl reagent (thiobarbituric acid 0.37%, 0.25N HCl and 15% TCA) and placed in water bath for 15 min, cooled and centrifuged at room temperature for 10 min at 1,000 rpm. The absorbance of clear supernatant was measured against reference blank at 535 nm.

For hydroperoxides 0.1 ml of tissue homogenate was treated with 0.9 ml of Fox reagent (88 mg butylated hydroxytoluene (BHT), 7.6 mg xylenol orange and 9.8 mg ammonium ion sulphate were added to 90 ml of methanol and 10 ml 250 mM sulphuric acid) and incubated at 37°C for 30 min. The colour developed was read at 560 nm colorimetrically.


Determination of non-enzymic antioxidant status

Estimation of Reduced glutathione

Reduced glutathione (GSH) was determined by the method of Ellman (19). To the homogenate added 10% TCA, centrifuged. 1.0 ml of supernatant was treated with 0.5 ml of Ellmans reagent (19.8 mg of 5, 5'-dithiobisnitro benzoic acid (DTNB) in 100 ml of 0.1% sodium nitrate) and 3.0 ml of phosphate buffer (0.2M, pH 8.0). The absorbance was read at 412 nm.


Estimation of vitamin E

Vitamin E was estimated by Baker and Frank method (20). Lipid extract was prepared by the method of Folch et al . To 0.5ml of lipid extract, 1.5ml ethanol, 2.0ml of petroleum ether were added and centrifuged. The supernatant was evaporated to dryness at 80°C, to that added 0.2ml of 2-2' dipyridyl solution (0.2%) and ferric chloride (0.5%), kept in dark for 5 minutes and then 4ml of butanol was added. The colour developed was read at 520nm.


Estimation of ascorbic acid

Vitamin C was estimated by Roe and Kuether method (21). To 0.5 ml of tissue homogenate, 1.5ml 6% TCA was added, centrifuged. To the supernatant added, acid washed norit and filtered. To the filterate, added 0.5ml of DNPH and incubated at 37°C for 3 hours and then added 85% H2SO4 and incubated for30 min. The colour developed was read at 540nm.


Determination of superoxide dismutase, catalase and glutathione peroxidase

Superoxide dismutase (SOD) was assayed utilizing the technique of Kakkar et al . (22). A single unit of enzyme was expressed as 50% inhibition of NBT (Nitroblue tetrazolium) reduction/min/mg protein.

Catalase (CAT) was assayed colorimetrically at 620 nm and expressed as mmoles of H2O2 consumed/min/mg protein as described by Sinha (23). The reaction mixture (1.5ml) contained 1.0 ml of 0.01M pH 7.0 phosphate buffer, 0.1 ml of tissue homogenate and 0.4 ml of 2M H2O2 . The reaction was stopped by the addition of 2.0 ml of dichromate-acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in 1:3 ratio).

Glutathione peroxidase (GPx) activity was measured by the method described by Ellman (19). Briefly, reaction mixture contained 0.2 ml of 0.4M phosphate buffer pH 7.0, 0.1 ml of 10 mM sodium azide, 0.2 ml of tissue homogenate (homogenised in 0.4M, phosphate buffer pH 7.0), 0.2 ml glutathione, 0.1 ml of 0.2 mM hydrogen peroxide. The contents were incubated at 37°C for 10 min. The reaction was arrested by 0.4 ml of 10% TCA, and centrifuged. Supernatant was assayed for glutathione content by using Ellmans reagent.

The total protein was estimated by total protein and albumin Kit (No-72111) from Qualigens fine chemicals, Worli, Mumbai.


Statistical Analysis

Statistical analysis was done by analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT). Values were considered statistically significant when P ≤ 0.05.

Results

This study analyses the protective role of curcumin and CA on oxidative stress induced by alcohol and Δ PUFA. Our results showed that there was a significant reduction in oxidative stress in both curcumin and CA treatment.

Figure 2a and 2b show the changes in the activities of ALP and GGT in plasma.


Figure 2: (a) Activities of γ-glutamyltranseferase in Plasma. (values are mean ± S.D from 6 rats in each group). Figure 2: (b) Activities of Alkaline Phosphatase in Plasma. (values are mean ± S.D from 6 rats in each group).

Their activities were increased significantly in alcohol, ∆ PUFA and alcohol + ∆ PUFA groups, which were decreased on treatment with curcumin and CA. However, CA treatment decreased their activity more significantly compared to curcumin.

Figure 3 and 4 show the levels of lipid peroxidative indices in different tissues.


Figure 3: Levels of TBARS in Tissues. (values are mean ± S.D from 6 rats in each group)


Figure 4: Levels of Hydroperoxides in Tissues. (values are mean ± S.D from 6 rats in each group)

The levels of TBARS (Figure 3) and HP (Figure 4) were increased significantly in alcohol, ∆ PUFA and alcohol + ∆ PUFA groups, which were decreased significantly on treatment with curcumin and curcumin analog. The decrease was more significant in CA treated groups compared to curcumin. The levels of non-enzymic antioxidants: vitamin C, vitamin E (Figure 5) and GSH (Figure 6) and enzymic antioxidants: SOD (Figure 7), CAT (Figure 8) and GPX (Figure 9) were significantly depleted in alcohol, ∆ PUFA and alcohol + ∆ PUFA groups which were increased in both curcumin and CA treatment.


Figure 5: Levels of Vitamin C and Vitamin E in Tissues. (values are mean ± S.D from 6 rats in each group)


Figure 6: Levels of Reduced Glutathione in Tissues. (values are mean ± S.D from 6 rats in each group)


Figure 7: Activities of superoxide dismutase in Tissues. (values are mean ± S.D from 6 rats in each group)


Figure 8: Activities of Catalase in Tissues. (values are mean ± S.D from 6 rats in each group)


Figure 9: Activities of Glutathione Peroxidase in Tissues. (values are mean ± S.D from 6 rats in each group)

The CA treatment was found to be more effective compared to curcumin. Over all the reduction in oxidative stress was ~ 50% in curcumin, treated groups and ~60% in CA treated groups.


Discussion

The increase in plasma liver markers is a direct reflection of oxidative injury of liver. Various pathways play a role in ethanol induced tissue injury, including changes in cellular oxidized NAD+, NADH, production of acetaldehyde protein adducts, induction of CYP2E1, formation of 1-hydroxyethyl free radicals , ethanol mediated mitochondrial damage , endotoxin derived activation of Kupffer cells and subsequent production of tumour necrosis factor a . These changes perturb the biomembranes and cause severe damage and leakage of liver markers into the circulation. Moreover, increased intake of PUFA increases the degree of unsaturation of the biomembrane and makes them more susceptible to lipid peroxidation . Wide utilization of fats which are highly susceptible to oxidation during cooking and frying may alter physiological effects of their PUFA content and generate lipid peroxides that cause membrane damage and increase lipid infiltration and hence make the membrane leaky to liver markers. Thus, the increased activities of GGT and ALP in our study are suggestive of severe hepatic injury during alcohol and PUFA ingestion.


The enhanced lipid peroxidation is one of the toxic manifestations of acute ethanol ingestion. evidences have indicated that free radicals or Reactive Oxygen Species (ROS) such as hydroxy ethyl radical, superoxide radical (O2°-), hydroxy radical (OH•), peroxy radical and hydrogen peroxide are implicated in ethanol induced lipid peroxidation. Ethanol is extensively metabolised to cytotoxic acetaldehyde by alcohol dehydrogenase enzyme in the liver and acetaldehyde is oxidised to acetate by aldehyde dehydrogenase or xanthine oxidase giving rise to ROS . Moreover, ethanol metabolism is associated with increase in CYP2E1 activity. CYP2E1 catalyses the conversion of ethanol to acetaldehyde and at the same time reduces dioxygen to a variety of ROS, including O2°- . These enhanced O2°- and other ROS increases the degree of LPO during alcohol ingestion. The excess LPO in alcohol-ingested group as measured by the formation of TBARS and HP in our study corroborate these findings.


The changes in the composition of erythrocyte with increased erythrocyte deformability ex vivo have been reported with increased intake of PUFA. It has been demonstrated that fatty acid composition of membranes can be affected by dietary variation of saturated and unsaturated fat. The increase in dietary unsaturated fat increases the degree of unsaturation of the membranes and unsaturated bonds are more susceptible to lipid peroxidation. Moreover, heating of oil rich in PUFA produces various toxic metabolites, which may increase the lipid peroxidative changes. Thus, the observed increase in lipid peroxidative indices in our study during ∆ PUFA ingestion is in correlation with other findings.


Antioxidant defense system protects the aerobic organism from the deleterious effects of reactive oxygen metabolites. Vitamin E, a major lipophilic antioxidant and vitamin C, play a vital role in the defense against oxidative stress. In our study, the levels of vitamin E and C were decreased significantly during alcohol and PUFA ingestion. This is in agreement with the previous reports that chronic alcoholics are deficient in vitamin C and E. The increased oxidative stress due to alcohol and PUFA ingestion might have resulted in complete utilization of vitamin C and E thus depleting their levels.


Glutathione, an important cellular reductant is involved in protection against free radicals, peroxides and other toxic components. In addition to serving as a substrate for glutathione related enzymes, GSH acts as a free radical scavenger, a generator of a α-tocopherol and plays an important role in the maintenance of protein sulfhydryl groups. Previous studies have shown that acute ethanol ingestion depletes GSH levels. In the present study, the levels of GSH were decreased significantly in alcohol and Δ PUFA ingestion indicating the oxidative stress.


GPx has a well-established role in protecting cells against oxidative injury. GPx is non-specific for H2O2 and lack of this substrate specificity extends a range of substrates from H2O2 to organic hydroperoxides. Therefore, the excess H2O2 and lipid peroxides generated during alcohol and PUFA ingestion are efficiently scavenged by GPx activity. The depression of this enzyme activity reflects perturbations in normal oxidative mechanisms during alcohol and PUFA ingestion. Catalase, which acts as preventative antioxidant plays an important role in protection against the deleterious effects of lipid peroxidation). The inhibition of CAT activity is suggestive of enhanced synthesis of O2°- during the ingestion of alcohol and PUFA since O2°- is a powerful inhibitor of catalase.


SOD catalyses the dismutation of O2°- radical anions to H2O2 and O2. Numerous studies have shown the importance of SOD in protecting cells against oxidative stress . Our study has shown a decrease in SOD activity in tissues during alcohol and PUFA ingestion. This decrease could be due to a feed back inhibition or oxidative inactivation of enzyme protein due to excess ROS generation . The generation of the α-hydroxy ethyl radical may also lead to inactivation of the enzyme.


Administration of curcumin and curcumin analog (CA), decreased the LPO, improved the antioxidant status and thereby prevented the damage to the liver and leakage of enzymes GGT and ALP. This is mainly because of the antioxidant sparing action of curcumin and CA.


The antioxidant mechanism of curcumin may include one or more of the following interactions. Scavenging or neutralizing of free radicals, interacting with oxidative cascade and preventing its outcome, oxygen quenching and making it less available for oxidative reaction, inhibition of oxidative enzymes like cytochrome P450 and chelating and disarming oxidative properties of metal ions such as iron. Thus in this work curcumin effectively prevented tissue damage by decreasing the oxidative stress and restoring the antioxidant status.


However, the treatment with CA was found to be more effective compared to curcumin. Among many classes of compounds, phenolics have been recognized as a powerful counter measure against LPO. Normally phenolic compounds act by scavenging free radicals and quenching the lipid peroxidative side chain. Phenolic compounds can act as free radical scavengers by virtue of their hydrogen donating ability, forming aryloxyl radicals . It has been proposed that hydroxy and hydroperoxy radicals initiate H+ abstraction from a free phenolic substrate to form phenoxy radical that can rearrange to quinonemethide radical intermediate which is excreted via bile.


Moreover the introduction of a hydroxyl group in the `O' and `P' position is known to increase antioxidant activity in peroxidizing lipid system. Several investigators have shown that `O' substitution with an e- donor group increases the stability of the aryloxyl radical and thus antioxidant activit. The increased efficacy of this novel curcuminoid may be attributed to the presence of hydroxyl group at ortho position. The o -hydroxyl group, because of its resonance property, easily donates e− to free radicals and effectively neutralizes them. This property makes the CA, a novel compound for treating oxidative stress.


Conclusion

Thus, CA effectively quenches free radicals and LPO, decreases release of liver markers and positively modulates antioxidant status. Thus by the property of eliciting a significant effect on LPO, this synthetic curcuminoid may become a promising candidate for the treatment of oxidative stress. Further, more studies that are mechanistic are essential to elucidate the exact mechanism of its modulatory effects.


Metabolism and Bioavailability

Clinical trials in humans indicate that the systemic bioavailability of orally administered curcumin is relatively low . Curcumin is readily conjugated in the intestine and liver to form curcumin glucuronides and curcumin sulfates or reduced to hexahydrocurcumin . Curcumin metabolites may not have the same biological activity as the parent compound. In one study, conjugated or reduced metabolites of curcumin were less effective inhibitors of inflammatory enzyme expression in cultured human colon cells than curcumin itself. In a clinical trial conducted in Taiwan, serum curcumin concentrations peaked 1-2 hours after an oral dose, and peak serum concentrations were 0.5, 0.6 and 1.8 micromoles/liter at doses of 4, 6 and 8 g/day, respectively. Curcumin could not be detected in serum at lower doses than 4 g/day. More recently, a clinical trial conducted in the UK, found that plasma curcumin, curcumin sulfate and curcumin glucuronide concentrations were in the range of 10 nanomoles/liter (0.01 micromole/liter) one hour after a 3.6 g dose of oral curcumin . Curcumin and its metabolites could not be detected in plasma at lower doses than 3.6 g/day. Curcumin and its glucuronidated and sulfated metabolites were also measured in urine at a dose of 3.6 g/day. There is some evidence that orally administered curcumin accumulates in gastrointestinal tissues. When colorectal cancer patients took 3.6 g/d of curcumin orally for 7 days prior to surgery, curcumin was detected in malignant and normal colorectal tissue. In contrast, curcumin was not detected in the liver tissue of patients with liver metastases of colorectal cancer after the same dose of oral curcumin, suggesting that oral curcumin administration may not effectively deliver curcumin to tissues outside the gastrointestinal tract



Biological Activities

Antioxidant Activity

Curcumin is an effective scavenger of reactive oxygen species and reactive nitrogen species in the test tube However, it is not clear whether curcumin acts directly as an antioxidant in vivo. Due to its limited oral bioavailability in humans (see Metabolism and Bioavailability above), plasma and tissue curcumin concentrations are likely to be much lower than that of other fat-soluble antioxidants, such as alpha-tocopherol (vitamin E). However, the finding that 7 days of oral curcumin supplementation (3.6 g/day) decreased the number of oxidative DNA adducts in malignant colorectal tissue suggests that curcumin taken orally may reach sufficient concentrations in the gastrointestinal tract to inhibit oxidative DNA damage. In addition to direct antioxidant activity, curcumin may function indirectly as an antioxidant by inhibiting the activity of inflammatory enzymes or by enhancing the synthesis of glutathione, an important intracellular antioxidant (see below).


Anti-inflammatory Activity

The metabolism of arachidonic acid in cell membranes plays an important role in the inflammatory response by generating potent chemical messengers known as eicosanoids . Membrane phospholipids are hydrolyzed by phospholipase A2 (PLA2), releasing arachidonic acid, which may be metabolized by cyclooxygenases (COX) to form prostaglandins and thromboxanes, or lipoxygenases (LOX) to form leukotrienes. Curcumin has been found to inhibit PLA2, COX-2 and 5-LOX activity in cultured cells. Although curcumin inhibited the catalytic activity of 5-LOX directly, it inhibited PLA2 by preventing its phosphorylation and COX-2 mainly by inhibiting its transcription. Nuclear factor-kappa B (NF-kB) is a transcription factor that binds DNA and enhances the transcription of the COX-2 gene and other pro-inflammatory genes, such as inducible nitric oxide synthase (iNOS). In inflammatory cells, such as macrophages, iNOS catalyzes the synthesis of nitric oxide, which can react with superoxide to form peroxynitrite, a reactive nitrogen species that can damage proteins and DNA. Curcumin has been found to inhibit NF-kB-dependent gene transcription, and to inhibit the induction of COX-2 and iNOS in cell culture and animal studies.


Glutathione Synthesis

Glutathione is an important intracellular antioxidant that plays a critical role in cellular adaptation to stress . Stress-related increases in cellular glutathione levels result from increased expression of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione synthesis. Studies in cell culture suggest that curcumin can increase cellular glutathione levels by enhancing the transcription of the genes for GCL .


Effects on Biotransformation Enzymes Involved in Carcinogen Metabolism

Biotransformation enzymes play important roles in the metabolism and elimination of a variety of biologically active compounds, including drugs and carcinogens. In general, phase I biotransformation enzymes, including those of the cytochrome P450 (CYP) family, catalyze reactions that increase the reactivity of hydrophobic (fat-soluble) compounds, preparing them for reactions catalyzed by phase II biotransformation enzymes. Reactions catalyzed by phase II enzymes generally increase water solubility and promote the elimination of these compounds. Although increasing biotransformation enzyme activity may enhance the elimination of potential carcinogens, some carcinogen precursors (procarcinogens) are metabolized to active carcinogens by phase I enzymes . CYP1A1 is involved in the metabolic activation of several chemical carcinogens. Curcumin has been found to inhibit increases in CYP1A1 activity induced by procarcinogens in cell culture and animal studies. Increasing phase II biotransformation enzyme activity is generally thought to enhance the elimination of potential carcinogens. Several studies in animals have found that dietary curcumin increased the activity of phase II enzymes, such as glutathione S-transferases (GSTs). However, curcumin intakes ranging from 0.45-3.6 g/day for up to 4 months did not increase leukocyte GST activity in humans .


Induction of Cell Cycle Arrest and Apoptosis

After a cell divides, it passes through a sequence of stages collectively known as the cell cycle before it can divide again. Following DNA damage, the cell cycle can be transiently arrested to allow for DNA repair or activation of pathways leading to cell death (apoptosis) if the damage cannot be repaired Defective cell cycle regulation may result in the propagation of mutations that contribute to the development of cancer. Curcumin has been found to induce cell cycle arrest and apoptosis in a variety of cancer cell lines grown in culture. The mechanisms by which curcumin induces apoptosis are varied but may include inhibitory effects on several cell signaling pathways. However, not all studies have found that curcumin induces apoptosis in cancer cells. Curcumin inhibited apoptosis induced by the tumor suppressor protein p53 in cultured human colon cancer cells, and one study found that curcumin inhibited apoptosis induced by several chemotherapeutic agents in cultured breast cancer cells at concentrations of 1-10 micromoles/liter.


Inhibition of Tumor Invasion and Angiogenesis

Cancerous cells invade normal tissue aided by enzymes called matrix metalloproteinases. Curcumin has been found to inhibit the activity of several matrix metalloproteinases in cell culture studies. Invasive tumors must also develop new blood vessels to fuel their rapid growth by a process known as angiogenesis. Curcumin has been found to inhibit angiogenesis in cultured vascular endothelial cells and in an animal model.

Note: It is important to keep in mind that that many of the biological activities discussed above were observed in cells cultured in the presence of curcumin at higher concentrations than are likely to be achieved in humans consuming curcumin orally (see Metabolism and Bioavailability above).


Disease Prevention

Cancer

The ability of curcumin to induce apoptosis in cultured cancer cells by several different mechanisms has generated scientific interest in the potential for curcumin to prevent some types of cancer . Oral curcumin administration has been found to inhibit the development of chemically-induced cancer in animal models of oral stomach, , liver  and colon cancer . ApcMin/+ mice have a mutation in the Apc (adenomatous polyposis coli) gene similar to that in humans with familial adenomatous polyposis, a genetic condition that is characterized by the development of numerous colorectal adenomas (polyps) and a high risk for colorectal cancer. Oral curcumin administration has been found to inhibit the development of intestinal adenomas in ApcMin/+ mice. In contrast, oral curcumin administration has not consistently been found to inhibit the development of mammary (breast) cancer in animal models.

Although the results of animal studies are promising, particularly with respect to colorectal cancer, there is presently little evidence that high intakes of curcumin or turmeric are associated with decreased cancer risk in humans. A phase I clinical trial in Taiwan, examined the effects of oral curcumin supplementation up to 8 g/day for 3 months in patients with precancerous lesions of the mouth (oral leukoplakia), cervix (high grade cervical intraepithelial neoplasia), skin (squamous carcinoma in situ) or stomach (intestinal metaplasia). Histologic improvement on biopsy was observed in 2 out of 7 patients with oral leukoplakia, 1 out of 4 patients with cervical intraepithelial neoplasia, 2 out of 6 patients with squamous carcinoma in situ and 1 out of 6 patients with intestinal metaplasia. However, cancer developed in 1 out of 7 patients with oral leukoplakia and 1 out of 4 patients with cervical intraepithelial neoplasia by the end of the treatment period. This study was designed mainly to examine the bioavailability and safety of oral curcumin, and interpretation of its results is limited by the lack of a control group for comparison. As a result of the promising findings in animal studies, several controlled clinical trials in humans designed to evaluate the effect of oral curcumin supplementation on precancerous colorectal lesions, such as adenomas, are under way .


Alzheimer’s Disease

In Alzheimer’s disease, a peptide called amyloid beta forms aggregates (oligomers), which accumulate in the brain and form deposits known as amyloid plaques . Inflammation and oxidative damage are also associated with the progession of Alzheimer’s disease. Curcumin has been found to inhibit amyloid beta oligomer formation in vitro. When injected peripherally, curcumin was found to cross the blood brain barrier in an animal model of Alzheimer’s disease . Dietary curcumin has been found to decrease biomarkers of inflammation and oxidative damage and to decrease amyloid plaque burden in the brain and amyloid beta-induced memory deficits in animal models of Alzheimer’s disease. It is not known whether curcumin taken orally can cross the blood brain barrier or inhibit the progression of Alzheimer’s disease in humans. As a result of the promising findings in animal models, several clinical trials of oral curcumin supplementation in patients with early Alzheimer’s disease are under way .


Disease Treatment

Cancer

The ability of curcumin to induce apoptosis in a variety of cancer cell lines in culture and its low toxicity have led to scientific interest in its potential for cancer therapy as well as cancer prevention. To date, most of the controlled clinical trials of curcumin supplementation in cancer patients have been Phase I trials. Phase I trials are clinical trials in small groups of people, aimed at determining bioavailability, optimal dose, safety and early evidence of the efficacy of a new therapy . A phase I clinical trial in patients with advanced colorectal cancer found that doses up to 3.6 g/day for 4 months were well tolerated, although the systemic bioavailability of oral curcumin was low. When colorectal cancer patients with liver metastases took 3.6 g/day of curcumin orally for 7 days, trace levels of curcumin metabolites were measured in liver tissue, but curcumin itself was not detected. In contrast, curcumin was measured in normal and malignant colorectal tissue after patients with advanced colorectal cancer took 3.6 g/day of curcumin orally for 7 days.

These findings suggest that oral curcumin is more likely to be effective as a therapeutic agent in cancers of the gastrointestinal tract than other tissues. Phase II trials are clinical trials designed to investigate the effectiveness of a new therapy in larger numbers of people, and to further evaluate short-term side effects and safety of the new therapy. Phase II clinical trials of curcumin in patients with advanced pancreatic cancer are currently under way, and phase II trials of curcumin for colorectal cancer have been recommended.


Inflammatory Diseases

Although the anti-inflammatory activity of curcumin has been demonstrated in cell culture and animal studies, few controlled clinical trials have examined the efficacy of curcumin in the treatment of inflammatory conditions. A preliminary intervention trial that compared curcumin with a nonsteroidal anti-inflammatory drug (NSAID) in 18 rheumatoid arthritis patients found that improvements in morning stiffness, walking time and joint swelling after 2 weeks of curcumin supplementation (1200 mg/day) were comparable to those experienced after 2 weeks of phenylbutazone (NSAID) therapy (300 mg/day). A placebo-controlled trial in 40 men who had surgery to repair an inguinal hernia or hydrocele found that 5 days of oral curcumin supplementation (1200 mg/day) was more effective than placebo in reducing post surgical edema, tenderness and pain, and was comparable to phenylbutazone therapy (300 mg/day). Two uncontrolled studies found that oral curcumin (1125 mg/day) for 12 weeks or longer improved anterior uveitis and idiopathic inflammatory orbital pseudotumor, inflammatory conditions of the eye. However, without a control group, it is difficult to draw conclusions regarding the anti-inflammatory effects of curcumin in these conditions. Larger randomized controlled trials are needed to determine whether oral curcumin supple

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