ANTI-INFLAMMATORY ACTIVITY

Perhaps the most important effect of curcumin is its anti-inflammatory properties and this is the major focus of this review. Only a few clinical studies have been reported on the effect of administration of curcumin on inflammatory diseases ^6-9. However, curcumin has been known to possess anti-inflammatory activity in experimental animals¹⁰. In this regard, we have recently shown that curcumin has beneficial effects in sepsis. Male Sprague Dawley rats were treated with a bolus intravenous injection of 0.2 μmol of curcumin followed by a continuous infusion of 0.24 μmol/day for 3 days via a 2-mL alzet pump. Then the rats were subjected to sepsis by cecal ligation and puncture (CLP), a widely used animal model of sepsis. Twenty hours following CLP (i.e., the late stage of sepsis), the rats were killed and the blood and tissue samples were collected.The blood samples were analyzed for tissue injury parameters, alanine aminotransferase, aspartate aminotransferase, lactate, and TNF-α. As expected, sepsis induced a two-tothree-fold increase in the circulating levels of these injury markers compared to sham controls. Pretreatment with curcumin significantly reduced these levels to that of sham. Similar results were observed when curcumin was administered 5 hours after the onset of sepsis. In an additional group of animals, a 10-day survival study was conducted after CLP in animals pretreated with curcumin for 3 days. Sepsis caused a 56–69% mortality rate while pretreatment with curcumin improved the survival rate to 100% throughout the 10-day observation period. Thus, we have demonstrated the anti-inflammatory effect of curcumin in an in vivo experimental model of sepsis. We have also shown that pretreatment with 50 μM curcumin in a macrophage cell line, RAW 264.7 cells, produced 23% and 71% reduction in LPS-induced increases in TNF-α gene expression and protein levels, respectively. At 100 μM curcumin, a reduction by 60% and 99% in the LPS-stimulated increases in TNF-α gene expression and protein levels were observed, respectively. These data prompted us to explore the potential mechanisms associated with curcumin-induced anti-inflammatory effects.

POTENTIALMECHANISMS

The mechanism by which curcumin induces its anti-inflammatory effects is yet to be elucidated. Studies have shown that peroxisome proliferator-activated receptor gamma (PPAR-γ) has been associated with anti-inflammatory effects. PPARs belong to the superfamily of nuclear receptors consisting of three genes that give rise to three different subtypes, PPAR-α, PPAR-δ, and PPAR-γ. Among them, PPAR-γ is the most widely studied form. Upon ligand binding, PPAR-γ forms heterodimers with the retinoid X receptor and binds to a peroxisome proliferation response element (PPRE) in a gene promoter leading to regulation of gene transcription ¹¹. In that regard, we have recently shown that gene and protein levels of PPAR-γ in the liver decreased by approximately 50% at 20 hours after the onset of sepsis. Pretreatment with curcumin for 3 days at 0.24 μmol/kg body weight in these septic rats produced 45% and 65% increase in PPAR-γ mRNA and protein levels, respectively.The mRNA and protein levels of PPAR-γ in the treatment group were similar to sham controls. To confirm that the beneficial effect of curcumin in sepsis is mediated through PPAR-γ pathway, a separate group of animals were treated for 3 days with PPAR-γ antagonist, GW9662, at 1.5mg/kg along with curcumin at 0.24 μmol/kg body weight.Then, rats were subjected to sepsis by CLP and 20 hours after surgery, blood and tissue samples were collected. Concurrent administration of curcumin and GW9662 in the septic rats completely abolished the effects of curcumin on serum levels of the liver enzymes, ALT and AST, lactate, and TNF-α. Furthermore, in vitro using RAW 264.7 cells, pretreatment with 50 and 100 μM curcumin increased PPAR-γ mRNA levels by 86% and 125%, respectively, compared to LPS treatment alone. Consistent with this, immunohistochemical staining of RAW 264.7 cells with PPAR-γ antibody showed increased nuclear PPAR-γ staining in cells pretreated with 100 μMcurcumin compared to LPS alone.This suggests that the beneficial effect of curcumin appears to be mediated by the upregulation of PPAR-γ.Both in vivo and in vitro studies have shown that activation of PPAR-γ by thiazolidinediones (TZDs), the class of insulin-sensitizing drugs, or 15d-PG-J2 has anti-inflammatory effects ^12-14. TZDs are the synthetic agonists of PPAR-γ and PGJ2 series have been identified as the natural ligand of PPAR-γ. In that regard, Zingarelli et al. showed that PPAR-γ expression was markedly reduced in lung and thoracic aorta after CLP sepsis. Furthermore,in vivo treatment with 15d-PGJ2 or ciglitazone, one of the TZDs, following CLP ameliorated hypotension and survival,blunted cytokine production and reduced neutrophil infiltration in lung, colon, and liver. These beneficial effects of PPAR-γ ligands were associated with the reduction of IκB kinase complex, JNK activation, and reduction of NF-κB and AP-1 pathways ¹².

Curcumin interferes with inflammatory pathways by blocking the transcription factor NFκB. The numbers 1, 2, and 3 represent the pathways that are described to be affected by curcumin as detailed in Brennan et al., 1998,Jobin et al., 1999 and Plummer et al., 1999, respectively. NFκB: Nuclear transcription factor required for transcription of genes involved in the inflammatory responses; IκB: Cytosolic inhibitor of NFκB; NIK: NFκBinducing kinase; IKK:IκB kinases.

Schematic representation of the molecular mechanisms for the anti-inflammatory activity of curcumin. Curcumin is known to exert anti-inflammatory effects significantly by interrupting NF-B signaling at multiple levels. For example, ROS mediate inflammation through the activation of stress kinases and redox-sensitive transcription factors such as NF-B, however, curcumin is a ROS scavenger and thus prevents the inflammatory signaling. In addition, curcumin can interfere with the functions of Akt and MAPKs, and in turn down-regulate the downstream molecule, NF-B

Recent evidence suggests that PPAR-γ ligands exert their effects in HT-29 colon cancer cells by phosphorylation of the PPAR-γ by the extracellular signalregulated kinase 1/2, thereby causing a physical interaction with the p65 subunit of the NF-κB preventing the activation of the NF-κB pathway ¹⁵. The inhibition of cell signaling pathways, Akt, NF-κB, AP-1, or JNK, has been implicated as the mechanism responsible for apoptosis induction by curcumin.A recent study reported that curcumin potentiates the antitumor effect of gemcitabine in pancreatic cancer by suppressing proliferation, angiogenesis, and downregulating NF-κB andNF-κB-regulated gene products ¹⁶. However, it is plausible that curcumin induced anti-inflammatory effect caused by the upregulation of PPAR-γ is associated with the NF-κB pathway. Numerous studies have shown the importance of curcumin as a potent immunomodulatory agent in T cells, B cells, neutrophils, natural killer cells, dendritic cells, and macrophages ¹⁷. In that regard, we have shown that curcumin induces apoptosis in human neutrophils ¹⁸. Neutrophils are the first line of host immune defense against foreign substances and their biological activities are tightly regulated by apoptosis. Delayed neutrophil apoptosis has been associated with acute lung injury and sepsis ^19-21.We first examined the effect of curcumin on both spontaneous neutrophil apoptosis and apoptosis of neutrophils following transmigration across a human lung endotheliumepithelium bilayer. The results showed that curcumin increased constitutive neutrophil apoptosis and abrogated the transbilayer migration-induced delay in neutrophil apoptosis.To determine the impact of curcumin on neutrophil function, we performedmyeloperoxidase activity andmigration assays. Curcumin treatment decreased neutrophil migration and myeloperoxidase release indicating a reduction in neutrophil activation. To elucidate the potential mechanism,we have examined the effect of curcumin on p38 mitogen-activated protein kinase and caspase-3 activity. A marked increase in p38 phosphorylation and caspase-3 activity was observed in the presence of curcumin. Treatment of p38-specific inhibitor, SB203580, suppressed both curcumin-induced apoptosis and caspase-3 activation. From 4 PPAR Research this study, we concluded that curcumin induces apoptosis in human neutrophil and its effect is mediated by the activation of p38 and caspase-3 activity^22-25. Curcumin was found to be a very potent antioxidant^26-29Curcumin was found to generate hydroxyl radicals through the Fenton reaction by reducing Fe3+ to Fe2+³⁰ .Effect of curcumin as superoxide scavenger was studied and curcumin was found to be a potent scavenger of superoxide³¹. They also reported a better correlation between antiinflammatory activity and superoxide scavenging property. Balasubramanyam et al demonstrated that curcumin abolished both PMA and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggest that curcumin mechanistically interferes with PKC and calcium regulation³².Priyadarsini et al tested the antioxidant activity of curcumin and dimethoxy curcumin by radiation-induced lipid peroxidation in rat liver microsomes³³. They found that at equal concentration, the efficiency to inhibit lipid peroxidation is changed from 82% with curcumin to 24% with dimethoxycurcumin. These results suggested that, although the energetic to remove hydrogen from both phenolic OH and the CH (2)group of the beta-diketo structure were very close, the phenolic OH was essential for both antioxidant activity and free radical kinetics. This was further confirmed by density functional theory (DFT) calculations where it was shown that the –OH hydrogen was more labile for abstraction compared to the –CH (2) hydrogen in curcumin suggesting that phenolic OH plays a major role in the activity of curcumin.Inhibition of monocyte chemoattractant protein-1 (mcp-1) by curcumin Nakayama et al described a novel effect of proteosome inhibitors on the expression of the monocyte chemoattractant protein 1 (MCP-1) in mesangial cells. They found that proteosome inhibitors MG 132 dose-dependently induced the expression of MCP-1 at the transcriptional level. The 5’-flanking region of the MCP-1 gene contains multiple AP-1 sites. A reporter assay showed that AP-1 activity was up-regulated after treatment with MG 132 and kinase assay revealed that c-jun-N-terminal kinase (JNK) was rapidly activated by MG132.Curcumin, a pharmacological inhibitor of the JNK-AP-1 pathway, abrogated the induction of MCP-1 by MG132. These data revealed that proteosome inhibition triggered the expression of MCP-1 and other genes via the multistep induction of the JNK-c-Jun/AP-1 pathway³⁴.Kohli K, et al.tyInhibition of acidic glycoprotein (gp a 72) by curcumin.Joe et al. observed an increased level of acidic glycoprotein Gp A 72 in the sera of arthritic rats³⁶ .The appearance of Gp A 72 in the serum preceded the onset of the paw inflammation in the arthritic rats and persisted in the chronic phase.They found that oral administration of antiinflammatory spices like capsaicin and curcumin lowered the levels of Gp A72 by 88% and 73%, respectively, with concomitant lowering of paw volume in the arthritic rats.Zsila et al demonstrated binding of curcumin molecule to human alpha1-acid glycoprotein (AGP), an acute phase protein in blood³⁴.Oppositely signed induced circular dichroism (CD) bands measured in the visible spectral region in pH 7.4 phosphate buffer indicated that the protein bounded curcumin molecule in a left-handed chiral conformation. Curcumininduced changes in the tertiary structure of AGP, which lead to the decreased binding affinity.Curcumin, an antioxidant present in the spice turmeric (Curcuma longa), has been shown to inhibit chemical carcinogenesis in animal models and has been shown to be an anti-inflammatory agent. While mechanisms of its biological activities are not understood, previous studies have shown that it modulates glutathione (GSH)-linked detoxification mechanisms in rats. In the present studies, we have examined the effects of curcumin on GSH-linked enzymes in K562 human leukemia cells. One micromolar curcumin in medium (16 h) did not cause any noticeable change in glutathione peroxidase (GPx), glutathione reductase, and glucose-6-phosphate dehydrogenase activities. Gamma-glutamyl-cysteinyl synthetase activity was induced 1.6-fold accompanied by a 1.2-fold increase in GSH levels. GSH S-transferase (GST) activities towards 1-chloro-2,4-dinitrobenzene, and 4-hydroxynonenal (4HNE) were increased in curcumin-treated cells 1.3- and 1.6-fold, respectively (P = 0.05). The GST isozyme composition of K562 cells was determined as follows:
66% of GST Pl-1, 31% of Mu class GST(s), and 3% of an anionic Alpha-class isozyme hGST 5.8, which was immunologically similar to mouse GSTA4-4 and displayed substrate preference for 4HNE. The isozyme hGST 5.8 appeared to be preferentially induced by curcumin, as indicated by a relatively greater increase in activity toward 4HNE. Immunoprecipitation showed that GPx activity expressed by GST 5.8 contributed significantly (approximately 50%) to the total cytosolic GPx activity of K562 cells to lipid hydroperoxides. Taken together, these results suggest that GSTs play a major role in detoxification of lipid peroxidation products in K562 cells, and that these enzymes are modulated by curcumin.

ANTICANCER ACTIVITY

Schematic representation of chemopreventive targets of curcumin in curtailing tumor proliferation and progression.

COX indicates cyclooxygenase; MMP, matrix metalloproteinase;OPN, osteopontin; VEGF, vascular endothelial growth factor;VCAM, vascular cell adhesion molecules; NF к B, nuclear factor kappa B; TCF/LEF, T-cell factor/lymphoid enhancer factor; IL,interleukin.

INVOLVEMENT OF CURCUMIN IN THE DOWNREGULATION OF ANTI-APOPTOTIC PROTEINS

Curcumin down-regulates the expression of genes involved in cell growth by inhibiting the activation of transcription factors involved in cell proliferation and survival. It has been shown that the activation of nuclear factor-kappa B (NF-êB),an inducible transcription factor, is critical to the establishment of cancer. Inactive NF-êB in the cytoplasm is a heterotrimer composed of three subunits p50 (NF-êB1), p65 (RelA) and inhibitor êB (IêBá). Upon stimulation, IêBá is phosphorylated by IêB kinase complex (IKK),followed by ubiquitination-dependent degradation of IêBá, leading to nuclear translocation, and binding of NF-êB to a specific DNA sequence. This results in transcription of multiple êB-dependent genes, including TNF-á,IL-6, IL-8 and other chemokines, MHC class II,ICAM-1, inducible nitric oxide synthase (iNOS),Cox-2, as well as, apoptosis suppressing proteins such as Bcl-2 and Bcl-xL which inturn induce cellular transformation, proliferation,differentiation, growth and inflammation.The inhibitory activity of curcumin is not only restricted to the NF-êB pathway, but also, it inhibits the pathway of activator protein-1(AP-1), another important transcription factor involved in cell proliferation and survival. AP-1 consists of a homodimer of c-Jun, or a heterodimer of c-Jun/c-Fos family members. Like NF-kB, AP-1 regulates the expression of several genes that are involved in cell differentiation and proliferation. Phosphorylation of c-Jun by c-Jun N-terminal kinases (JNKs; also named stress activated protein kinases, SAPKs) is important for c-Jun transcriptional activity. These kinases (JNK1, JNK2, and JNK3) are members of the mitogen activated protein kinase (MAPK) family that is involved in cellular responses to mitogen stimulation, environmental stress,proinflammatory cytokines, and apoptotic stimuli. Besides c-Jun, the JNK pathway also activates the transcription factors ATF-2 ³⁷, Elk-1 ³⁸, and Sap-1a ³⁹, and interacts with the NF-êB pathway ⁴⁰. Curcumin also has been shown to inhibit JNK activation. Several studies have shown that curcumin inhibits the activation of NF-êB and Ap-1, and down-regulates the expression of their target gene products, finally leading to cell cycle arrest,suppression of proliferation, and induction of apoptosis.

INHIBITION OF NF-êB ACTIVATION UPON CURCUMIN TREATMENT

The three NF-êB stimuli; TNF-á, Phorbolester, and Hydrogen peroxide, could not activate NF-êB when a human myelomonoblastic leukemia cell, ML-1a, was pre-treated with curcumin. Curcumin treatment totally suppressed TNF-á induced NF-êB activation, even after treating the cells with reducing agents like dithiothreitol (DTT) or 2,3-dimercaptopropanol (DMP). These reducing agents have been shown to reverse the inhibitory effect of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) and phenylarsineoxide on NF-êB activation ⁴¹. The effect of curcumin regulation of the IêB/NF-êB pathway in nontransformed intestinal epithelial cell line IEC-6, human HT-29 colonic epithelial cells and Caco-2 epithelial cells, have been examined by inducing cells with IL-1â. In these studies, it was shown that curcumin downregulates IL-1â-mediated ICAM-1 and IL-8 gene expression by inhibiting NF-êB activation,through blocking an upstream signal leading to NIK (NF-êB inducing kinase) activity, that phosphorylates and activates IêB kinase complex ⁴². The critical anti-apoptotic role of NF-êB in curcumin induced cell regulation is strongly supported by a study conducted with a relA gene,encoding the p65 (RelA) subunit of NF-êB, in transfected L-929 (mouse fibrosarcoma) cells.The transfected cells showed significant resistivity to curcumin induced apoptosis when compared to the parent cell line. On the other hand, resistivity of the transfected cells was totally demolished by co-transfection with a super-repressor form of IêB-á, which is known to inhibit NF-êB ⁴³.Mantle cell lymphoma (MCL) cell line JeKo-1, Mino, SP-53, and Granta 519 have been used in studying the effect of curcumin in downregulation of cyclin D1 expression, as these MCL cells are characterized by overexpression of cyclin D1. Upon curcumin treatment, NF-êB is inactivated through the inactivation of IêB kinase (IKK) by inhibiting Akt activation. As a consequence, curcumin treatment attenuated the expression of NF-êB regulated genes such as IêBá, cyclin D1, Bcl-2, Bcl-xL and Cox-2.Curcumin treatment also down-regulated the expression of NF-êB targeted tumor cell survival genes cIAP1, xIAP, TRAF1 and survivin leading to G1/S arrest, suppression of proliferation, and finally apoptosis ⁴⁴. A study conducted on the effect of curcumin on immature B cell lymphoma cell line (BKS-2) demonstrated curcumin induced apoptosis through repression of NF-êB binding activity, and down-regulation of the survival genes egr-1, which has been shown to be essential for the growth of B lymphoma cells ⁴⁵, c-myc, Bcl-xL as well as tumor suppressor gene p53 (2). Ku70, a subunit of Ku protein complex, plays a major role in keeping Bax protein in an inactive conformation during apoptosis ⁴⁶. Overexpression of Ku70 and BclxL proteins in human colon cancer cell line (SW480) inhibited curcumin induced apoptosis.This inhibition is achieved through blocking the release of cytochrome c, apoptosis inducing factor (AIF), and second mitochondria derived factor of caspase (Smac) from mitochondria,therefore, inactivating caspase cascade. This study supported the role that Ku70 plays in the retention of Bax in the cytosol ⁴⁷

INHIBITION OF AP-1 AND JNK ACTIVATION UPON CURCUMIN TREATMENT

Activator protein-1 has a central role in controlling the eukaryotic gene expression.Activation of c-un/AP-1 plays an important role in signal transduction of phorbol 12-myristate 13-acetate (PMA) induced tumor promotion. It has been reported that curcumin can suppress the PMA induced activation of c-Jun/AP-1 in mouse fibroblast cells NIH 3T3 ⁴⁸. The transcriptional activity of c-Jun is dependent on JNK activation, and is essential for its gene expression ⁴⁹. Thus,inhibition of JNK by curcumin would result in inhibition of c-Jun activation, and transcription of the c-Jun gene. In support of the inhibitory action of curcumin on c-Jun/AP-1 activation, curcumin completely blocks JNK activation by various agents such as PMA, ionomycin, ãradiation,UV-C, TNF-á, and sodium orthovanadate in Jurkat cells ⁵⁰. Suppression of NF-êB and AP-1 activation upon curcumin treatment is well demonstrated in human promyelotic leukemia (HL-60) cells ⁵¹. Phorbolester induced activation of NF-êB, AP-1 and its DNA binding to its response elements was completely interrupted by curcumin pretreatment. Sustained JNK activity, is found to be proapoptotic, whereas, rapid transient JNK activation could be anti-apoptotic ^52-54. Unlike the inhibitory effect of curcumin on JNK activation, curcumin induced apoptosis in the human colon cancer cell line HCT166 is accompanied by sustained phosphorylation and activation of JNK and p38 MAPK. Curcumin treatment inhibited NF-êB transcriptional activity, but it showed a significant increase in AP-1 transcriptional activity ⁵⁵.

INVOLVEMENT OF CURCUMIN IN UP-REGULATION OF APOPTOTIC AGENTS Curcumin is shown to induce apoptosis in several cancer cell lines through the downregulation of the expression of anti-apoptotic proteins. However, curcumin treatment upregulates the expression of proteins involved in apoptosis and exhibits common apoptotic features like altered expression of Bcl-2 family of proteins, imbalanced mitochondrial transmembrane permeability (ÄØm), release of cytochrome c, Smac, and AIF from mitochondria which in turn induce apoptosis via caspasedependent and independent pathways .Curcumin also facilitates the rapid generation of ROS that leads to cell death in several cancer cell lines.

INVOLVEMENT OF p53 AND CASPASE CASCADE IN CURCUMIN INDUCED APOPTOSIS

The inhibitory action of curcumin on colon adenocarcinoma was has been proven by treating the human HT-29 colon adenocarcinoma cell line with curcumin. These undergo apoptosis by activating p53 through phosphorylation at Ser15 residue, and by decreased expression of the antiapoptotic protein Bcl-2, increased expression of pro-apoptotic protein Bax, and increased caspase-3 and caspase-9 activation ⁵⁶.

Similarly, curcumin induced apoptosis in the human breast cancer cell line MCF-7 by potentiating p53 DNA binding activity which induces Bax expression ⁵⁷. Another interesting result was obtained when MCF-7 cells and normal mammary epithelial cells (NME) were treated with curcumin. In this case curcumin induced apoptosis at G2 phase of MCF-7 cells,while it blocked NME cell cycle progression without apoptosis. Curcumin induces apoptosis in carcinoma cells through increased expression of p21Waf-1, a cell cycle inhibitory protein, p53,and cytochrome c release ⁵⁸. Curcumin has been shown to arrest cell cycle progression, and induce apoptosis in vascular smooth muscle cell line A7r5 through reduced expression of c-myc,and Bcl-2, without altering p53 expression level ⁵⁹. Curcumin posses chemopreventive potentials against a panel of acute lymphoblastic leukemia cells (T-ALL) including CEM, HSB2,Jurkat and Molt-4 cells by inducing apoptosis. It has been demonstrated that curcumin suppresses targets of PI3'-kinase i.e. Akt, FOXO, and GSK3â, and it induces caspase-dependent apoptosis through cytochrome c release,activation of caspase-3 and PARP cleavage. At the same time, curcumin down regulates the expression of survival proteins such as cIAP,xIAP and survivin ⁶⁰. Curcumin treatment effectively suppressed the AK-5 (a rat histiocytic tumor) development in an in vivo study. In vitro study with single AK-5 tumor cell BC-8 exhibited apoptosis through ROS generation,caspase-3 activation, but not caspase-1, PARP cleavage, and DNA fragmentation (8). It has been reported that p53 is not necessary for intercellular induction of apoptosis ⁶¹, and the relationship between p53 and c-myc in the process of apoptosis is still controversial ^62,63. Curcumin has been reported to induce apoptosis in p53 proficient A549 and p53 deficient H1299 human lung cancer cell lines. In both cell lines curcumin induces apoptosis by up-regulation of c-myc, and down regulation of anti-apoptotic genes BclXL and Bcl-2. This result suggests a multiple p53 independent pathways in lung cancer cells where c-myc is possibly playing a major role ⁶⁴.

INVOLVEMENT OF REACTIVE OXYGEN SPECIES IN CURCUMIN INDUCED APOPTOSIS

Several studies have shown that curcumin acts as an anti-oxidant, and as a potent scavenger of free radicals, thereby, usually being considered as protecting cells from oxidative stress.Curcumin acts as a potent scavenger of a variety of reactive oxygen species (ROS) including superoxide anion ⁶⁵, hydroxyl radical, singlet oxygen ⁶⁶, and nitric oxide radicals ⁶⁷. On the other hand, curcumin has been shown to induce free radical generation and significant cell death through apoptosis under certain experimental conditions. There are conflicting reports as to the role of curcumin in redox balance. An investigation aimed at finding out the role of curcumin as an antioxidant against the oxidative stress damage induced by H2O2 on the neuronal cell NG108-15 leads to a contradictory result. Co-treatment of curcumin with H2O2 increased cell viability of NG108-15 cells, but when curcumin was pretreated, not only curcumin was unable to inhibit H2O2 induced cell death, it actually significantly decreased cell viability ⁶⁸. The role of ROS generation in curcumin induced apoptosis or necrosis is well studied using the human osteoblast cell line HFOb1.19. Low concentration of curcumin treatment leads to increased ROS generation,JNK activation, loss of ÄØm, caspase-3 activation, PARP cleavage and finally to apoptosis. Whereas, high dose treatment led to less ROS generation, loss of ÄØm and necrosis,while, JNK and caspase-3 had no effect.Moreover, intracellular ATP levels, important mediators capable of switching the mode of cell death from apoptosis to necrosis ⁶⁹, play a major role in switching the mechanism of cell death from apoptosis to necrosis ⁷⁰. Apart from these, several studies have reported that curcumin induces apoptosis through ROS generation ^71,72.Lipid peroxidation induced by free radicals is believed to be one of the major causes of cell membrane damage leading to lysis of cell⁷³. Curcumin inhibits iron catalysed lipid peroxidation in rat brain tissue homogenates by chelation of iron⁷⁴. The effect of curcumin on lipid peroxidation has also been studied in various models by several authors⁷⁵. Curcumin is a good antioxidant and inhibits lipid peroxidation in rat liver microsomes, erythrocyte membrane and brain homogenated⁷⁶.Several studies established the ability of curcumin to mainly eliminate hydroxy radical⁷⁷,singlet oxygen⁷⁸, nitrogen dioxide⁷⁹, and nitric oxide⁸⁰. It has also been demonstrated that curcumin inhibits the generation of the superoxide radicals^81,82.

INHIBITION OF ENZYMATIC ACTIVITY BY CURCUMIN

Being an antioxidant, anti-inflammatory and anti-carcinogenic agent, curcumin has been shown to inhibit some enzyme activities that have important roles in cell regulation. Some of the in vitro experimental studies have shown that curcumin directly acts as a potent inhibitor of certain enzymes. It has been reported that curcumin inhibits the activity of different protein kinases such as protein kinase A (PKA), protein kinase C (PKC), cytosolic protamine kinase (cPK), phosphorylase kinase (PhK), autophosphorylation-activated protein kinase (AK), and pp60c-src tyrosine kinase. Among these kinases, curcumin acted as a selective and potent inhibitor of PhK⁸³ The effect of curcumin has been tested on the enzyme activities of phospholipases such as ARF/GTPãS-dependent phospholipase D (PLD), phosphatidylinositol specific phospholipase C, phosphatidyl cholinephospholipase C, phospholipase A2 and sphingomyelinase in a cell free system. Curcumin inhibited enzyme activities of all phospholipases except sphingomyelinase. Amongst these, PLD was effectively inhibited at lower concentration of curcumin, which was further confirmed in intact mouse macrophage J774.1 cell model by inducing the cells with TPA ⁸⁴Apart from its in vitro inhibitory action,curcumin has been shown to inhibit some of the enzyme activities in vivo by interfering with their gene expression level. Curcumin inhibited P185neu autophosphorylation and transphosphorylation by inhibiting P185neu tyrosine kinase, a potent oncoprotein that is overexpressed in breast cancers, under in vitro condition and completely depleted the protein invivo and suppressed the growth of breast cancer cell line AU-565 ⁸⁵. An inhibitory effect of curcumin on nitric oxide synthase (NOS) was observed when murine macrophage RAW 264.7 cells were treated with curcumin and its hydrogenated metabolites tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin. Lipopolysaccharide (LPS) has been shown to induce the expression of iNOS by the activation NF-êB ⁸⁶. Only curcumin completely suppressed the expression of iNOS mRNA level when macrophages were induced with LPS ⁸⁷.Apart from these, curcumin has been shown to act as a novel inhibitor of class I histone deacetylase (class I HDACs) such as HDAC1,HDAC3, and HDAC8. Evidence suggests that a family of histone deacetylases may exist in order to regulate diverse cellular functions, including chromatin structure, gene expression, cell cycle progression, and oncogenesis ⁸⁸. A curcumin induction study conducted on the Burkkit lymphoma cell line Raji, has shown that curcumin induced apoptosis by inhibiting the histone deacetylase enzymes HDAC1, HDAC3 and HDAC8 and up-regulating the expression of Achistone H4 ⁸⁹. Telomerase, a reverse transcriptase enzyme highly expressed in tumor cells, activity was suppressed by curcumin and induced apoptosis in human chronic myelogenic leukemia (K-562) cells. The mode of curcumin inhibition of the enzyme activity was due to the suppression of translocation of telomerase reverse transcriptase (TERT) from cytosol to nucleus ⁹⁰.

The intraperitoneal administration of MPTP increases the SOD and CAT activities in the striatal and mid brain regions. The consequent augmented oxidative stress is considered a cardinal feature of MPTP neurotoxicity. Increases in SOD and CAT enzymatic activities were observed in MPTP treated animals⁹¹.

The repeated administration of curcumin causes positive influence on CAT and SOD activities.Both curcumin and MPTP caused a parallel change in the antioxidant enzymatic activities suggesting a repair mechanism in the mice brain.The chronic treatment of curcumin improved the levels of two key antioxidant enzymes SOD and CAT⁹².We observed that intraperitoneal administration of MPTP increases oxidative stress estimated by SOD, and CAT activities. The significantly decreased levels of GSH may impair H2O2 clearance.The increase of GSH suggests it’s role in neurotoxicity by MPTP, since GSH depleted animals have shown more vulnerability to MPTP insult⁹³.GSH, a major non protein thiol in living organisms plays a crucial role in co-ordinating the body’s antioxidant defence process. The results of the present study indicate that the MPTP administration drastically lowered the levels of GSH in the brain of mice. Our observation confirms the earlier data that the administration of curcumin increases the levels of glutathione reductase in ischemic brains of rats as well as alveola and human leukemia cells^94,95

REFERENCES

1. B. B. Aggarwal,A.Kumar, andA. C. Bharti, “Anticancer potential of curcumin: preclinical and clinical studies,” Anticancer Research, vol. 23, no. 1 A, pp. 363–398, 2003.

2. R. A. Sharma, A. J. Gescher, and W. P. Steward, “Curcumin:the story so far,” European Journal of Cancer, vol. 41, no. 13,pp. 1955–1968, 2005.

3. S. V. Jovanovic, C. W. Boone, S. Steenken, M. Trinoga, and R. B. Kaskey, “How curcumin works preferentially with water soluble antioxidants,” Journal of the American Chemical Society,vol. 123, no. 13, pp. 3064–3068, 2001.

4. Y.-J. Wang, M.-H. Pan, A.-L. Cheng, et al., “Stability of curcumin in buffer solutions and characterization of its degradation products,” Journal of Pharmaceutical and BiomedicalAnalysis, vol. 15, no. 12, pp. 1867–1876, 1997.

5. M.-T. Huang, N. Ma, Y.-P. Lu, et al., “Effects of curcumin,demethoxycurcumin, bisdemethoxycurcumin and tetrahydrocurcumin on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion,” Carcinogenesis, vol. 16,no. 10, pp. 2493–2497, 1995.

6. S. D. Deodhar, R. Sethi, and R. C. Srimal, “Preliminary study on antirheumatic activity of curcumin (diferuloyl methane),”The Indian Journal of Medical Research, vol. 71, pp. 632–634,1980.

7. B. Lal, A. K. Kapoor, P. K. Agrawal, O. P. Asthana, and R.C. Srimal, “Role of curcumin in idiopathic inflammatory orbital pseudotumours,” Phytotherapy Research, vol. 14, no. 6,pp. 443–447, 2000.

8. B. Lal,A.K.Kapoor, O. P. Asthana, et al., “Efficacy of curcumin in the management of chronic anterior uveitis,” Phytotherapy Research, vol. 13, no. 4, pp. 318–322, 1999.

9. R. R. Satoskar, S. J. Shah, and S. G. Shenoy, “Evaluation of anti-inflammatory property of curcumin (diferuloyl methane) in patients with postoperative inflammation,” International Journal of Clinical Pharmacology, Therapy, and Toxicology, vol. 24,no. 12, pp. 651–654, 1986.

10. R. C. Srimal and B. N. Dhawan, “Pharmacology of diferuloyl methane (curcumin), a non-steroidal anti-inflammatory agent,” The Journal of Pharmacy and Pharmacology, vol. 25,no. 6, pp. 447–452, 1973

11. B. M. Forman, J. Chen, and R. M. Evans, “The peroxisome proliferator-activated receptors: ligands and activators,” Annals of the New York Academy of Sciences, vol. 804, no. 1, pp.266–275, 1996.

12. B. Zingarelli, M. Sheehan, P. W. Hake, M. O’Connor,A. Denenberg, and J. A. Cook, “Peroxisome proliferator activator receptor-gamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J2 and ciglitazone, reduce systemic inflammation in polymicrobial sepsisby modulation of signal transduction pathways,” Journal of Immunology, vol. 171, no. 12, pp.6827–6837, 2003.

13. M. Ricote, A. C. Li, T. M. Willson, C. J. Kelly, and C. K. Glass,“The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation,” Nature, vol. 391,no. 6662, pp. 79–82, 1998.

14. C. Jiang, A. T. Ting, and B. Seed, “PPAR-γ agonists inhibit production of monocyte inflammatory cytokines,” Nature,vol. 391, no. 6662, pp. 82–86, 1998.

15. F. Chen,M.Wang, J. P. O’Connor, M. He, T. Tripathi, and L. E.Harrison, “Phosphorylation of PPARγ via active ERK1/2 leads to its physical association with p65 and inhibition of NF-κβ,” Journal of Cellular Biochemistry, vol. 90, no. 4, pp. 732–744,2003.

16. A. B. Kunnumakkara, S. Guha, S. Krishnan, P. Diagaradjane, J.Gelovani, and B. B. Aggarwal, “Curcumin potentiates antitumor activity of gemcitabine in an orthotopic model of pancreatic cancer through suppression of proliferation, angiogenesis,and inhibition of nuclear factor-κβ-regulated gene products,”Cancer Research, vol. 67, no. 8, pp. 3853–3861, 2007.

17. G. C. Jagetia and B. B. Aggarwal, ““Spicing up” of the immune system by curcumin,” Journal of Clinical Immunology, vol. 27,no. 1, pp. 19–35, 2007.

18. M. Hu, Q. Du, I. Vancurova, et al., “Proapoptotic effect of curcumin on human neutrophils: activation of the p38 mitogenactivated protein kinase pathway,” Critical Care Medicine,vol. 33, no. 11, pp. 2571–2578, 2005.

19. D. C. Angus, W. T. Linde-Zwirble, J. Lidicker, G. Clermont, J.Carcillo, and M. R. Pinsky, “Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care,” Critical Care Medicine, vol. 29, no. 7, pp.1303–1310, 2001.

20. A. Ayala, C.-S. Chung, J. L. Lomas, et al., “Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency,” American Journal of Pathology, vol. 161, no. 6, pp. 2283–2294, 2002.

21. R. Taneja, J. Parodo, S. H. Jia, A. Kapus, O. D. Rotstein, and J.C. Marshall, “Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity,” Critical Care Medicine,vol. 32, no. 7, pp. 1460–1469, 2004.

22. M. Collin, N. S. Patel, L. Dugo, and C. Thiemermann, “Role of peroxisome proliferator-activated receptor-γ in the protection afforded by 15-deoxyΔ12,14 prostaglandin J2 against the multiple organ failure caused by endotoxin,” Critical CareMedicine,vol. 32, no. 3, pp. 826–831, 2004.

23. M. Abdelrahman, A. Sivarajah, and C. Thiemermann, “Beneficial effects of PPAR-γ ligands in ischemia-reperfusion injury, inflammation and shock,” Cardiovascular Research, vol. 65, no. 4, pp. 772–781, 2005.

24. M. Abdelrahman, M. Collin, and C. Thiemermann, “The peroxisome proliferator-activated receptor-γ ligand 15-deoxyΔ12,14 prostaglandin J2 reduces the organ injury in hemorrhagic shock,” Shock, vol. 22, no. 6, pp. 555–561, 2004

25. Asha Jacob,1, 2 RongqianWu,1, 2 Mian Zhou,1, 2 and PingWang1, 2 Mechanism of the Anti-inflammatory Effect of Curcumin: PPAR-γ Activation; PPAR Research Volume 2007,PP1-5

26. Sharma OP. Antioxidant activity of curcumin and related compounds. Biochem Pharmacol 1976;25:1811-5.

27. Unnikrishnan MK, Rao MN. Inhibition of nitrite induced oxidation of hemoglobin by curcuminoids. Pharmazie 1995;50:490-2.

28. Osawa T, Sugiyama Y, Inayoshi M, Kawakishi S. Antioxidative activity of tetrahydrocurcuminoids. Biosci Biotechnol Biochem 1995;59:1609-12.

29. Iqbal M, Sharma SD, Okazaki Y, Fujisawa M, Okada S. Dietary supplementation of curcumin enhances antioxidant and phase II metabolizing enzymes in ddY male mice: Possible role in protection against chemical carcinogenesis and toxicity. Pharmacol Toxicol 2003;92:33-8.

30. Elizabeth K, Rao MNA. Effect of curcumin on hydroxyl radical generation through Fenton reaction. Int J Pharm 1989;57:173-6.

31. Elizabeth K, Rao MNA. Oxygen radical scavenging activity of curcumin. Int J Pharm 1990;58:237-40.

32. Balasubramanyam M, Koteswari AA, Kumar RS, Monickaraj SF, Maheswari JU, Mohan V. Curcumin-induced inhibition of cellular reactive oxygen species generation: Novel therapeutic implications. J Biosc 2003;28:715-21.

33. Priyadarsini KI, Maity DK, Naik GH, Kumar MS, Unnikrishnan MK, Satav JG,et al. Role of phenolic O-H and methylene hydrogen on the free radical reactions and antioxidant activity of curcumin. Free Radic Biol Med 2003;35:475-84.

34. Nakayama K, Furusu A, Xu Q, Konta T, Kitamura M. Unexpected transcriptional induction of monocyte chemoattractant protein 1 by proteasome inhibition:involvement of the c-Jun N-terminal kinase-activator protein 1 pathway. J Immunol 2001;167:1145-50.

35. Joe B, Rao UJ, Lokesh BR. Presence of an acidic glycoprotein in the serum of arthritic rats: modulation by capsaicin and curcumin. Mol Cell Biochem 1997;169:125-34.

36. Zsila F, Bikadi Z, Simonyi M. Induced circular dichroism spectra reveal binding of the anti-inflammatory curcumin to human alpha1-acid glycoprotein. Bioorg Med Chem 2004;12:3239-4

37. Gupta, S., Campbell, D., Derijard,B. and Davis, R.J. (1995). Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science, 267: 389-393.

38. Whitmarsh, A.J., Shore, P., Sharrocks, A.D.and Davis, R.J. (1995). Integration of MAP kinase signal transduction pathways at the serum response element. Science, 269:403-407.

39. Janknecht, R. and Hunter, T. (1997).Activation of the Sap-1a transcription factor by the c-Jun N-terminal kinase (JNK) Mitogen-activated Protein Kinase.Journal of Biological Chemistry, 272:4219-4224.

40. Meyer, C.F., Wang, X., Chang, C. and Tan,T.H. (1996). Interaction between c-Rel and the Mitogen-activated Protein Kinase Kinase Kinase 1 signaling cascade in mediating êB enhancer activation. Journal of Biological Chemistry, 271: 8971-8976.

41. Singh, S. and Aggarwal, B.B. (1995).Activation of transcription factor NF-êB is suppressed by curcumin (Diferuloylmethane). The Journal of Biological Chemistry, 270: 24995-25000.

42. Jobin, C., Bradham, C.A., Russo, M.P.,Juma, B., Narula, A.S., Brenner, D.A. and Sartor, R.B. (1999). Curcumin blocks cytokine-mediated NF-êB activation and proinflammatory gene expression by inhibiting inhibitory factor I-êB kinase activity. The Journal of Immunology, 163:3474-3483.

43. Anto, R.J., Maliekal, T.T. and Karunagaran, D. (2000). L-929 cells harboring ectopically expressed RelA resist curcumin-induced apoptosis. The Journal of Biological Chemistry, 275: 15601-15604.

44. Shishodia, S., Amin, H.M., Lai, R. and Aggarwal, B.B. (2005). Curcumin (diferuloylmethane) inhibits constitutive NF-êB activation, induces G1/S arrest,suppresses proliferation, and induces apoptosis in mantle cell lymphoma.Biochemical Pharmacology, 70: 700-713.

45. Muthukkumar, S., Han, S.S., Rangnekar,V.M and Bondada, S. (1997). Role of Egr-1 gene expression in B cell receptorinduced apoptosis in an immature B cell lymphoma. Journal of Biological Chemistry, 272: 27987-27993.

46. Sawada, M., Sun, W., Hayes, P., Leskov,K., Boothman, D.A. and Matsuyama, S.(2003). Ku70 supresses the apoptotic translocation of Bax to mitochondria.Nature Cell Biology, 5: 320-329.

47. Rashmi, R., Kumar, S. and Karunagaran,D. (2004). Ectopic expression of Bcl-xL or Ku70 protects human colon cancer cells (SW480) against curcumin-induced apoptosis while their down-regulation potentiates it. Carcinogensis, 25: 1867-1877.

48. Huang, T.S., Lee, S.C. and Lin, J.K.(1991). Suppression of c-Jun/AP-1 activation by an inhibitor of tumor promotion in mouse fibroblast cells. Proceedings of the National Acadamy of Sciences of the United States of America,88: 5292-5296.

49. Karin, M. (1995). The regulation of AP-1 activity by mitogen-activated protein kinases. Journal of Biological Chemistry,270: 16483-16486.

50. Chen, Y.R. and Tan, T.H. (1998). Inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway by curcumin. Oncogene, 17: 173-178.

51. Han, S.S., Keum, Y.S., Seo, H.J. and Surh,Y.J. (2002). Curcumin suppresses activation of NF-êB and AP-1 induced by phorbol ester in cultured Human Promyelocytic Leukemia cells. Journal of Biochemistry and Molecular Biology, 35:337-342.

52. Liu, B., Fang, M., Lu, Y., Mills, G.B. and Fan, Z. (2001). Involvement of JNK mediated pathway in EGF-mediated protection against paclitaxel-induced apoptosis in SiHa human cervical cancer cells. British Journal of Cancer, 85: 303-311.

53. De Smaele, E., Zazzeroi, F., Papa, S.,Nguyen, D.U., Jin, R., Jones, J., Cong, R.and Franzoso, G. (2001). Induction of gadd45â by NF-êB down-regulates proapoptotic JNK signaling. Nature, 414: 308-313.

54. Tang, G., Minemoto, Y., Dibling, B.,Purcell, N., Li, Z., Karin, M. and Lin, A.(2001). Inhibition of JNK activation through NF-êB target genes. Nature, 414:313-317.

55. Collet, G.P. and Campbell, F.C. (2004).Curcumin induces c-Jun N-terminal kinase-dependent apoptosis in HCT116 human colon cancer cells. Carcinogenesis,25: 2183-2189

56. Song, G., Mao, Y.B., Cai, Q.F., Yao, L.M.,Ouyang, G.L. and Bao, S.D. (2005).Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosisrelated protein expression. Brazilian Journal of Medical and Biological Research, 38: 1791-1798.

57. Choudhuri, T., Pal, S., Agwarwal, M.L.,Das, T. and Sa, G. (2002). Curcumin induces apoptosis in human breast cancer cell through p53-dependent Bax induction.FEBS Letters, 512: 334-340.

58. Choudhuri, T., Pal, S., Das, T. and Sa, G.(2005). Curcumin selectively induces apoptosis in deregulated cyclin D1-expressed cells at G2 phase of cell cycle in a p53-dependent manner. The Journal of Biological Chemistry, 280: 20059-20068.

59. Chen, H.W. and Huang, H.C. (1998).Effect of curcumin on cell cycle progression and apoptosis in vascular smooth muscle cells. British Journal of Pharmacology, 124: 1029-1040.

60. Hussain, A.R., Al-Rasheed, M., Manogaran, P.S., Al-Hussein, K.A., Platanias, L.C., Al Kuraya,K., and Uddin, S. (2006). Curcumin induces apoptosis via inhibition of PI3'-kinase/AKT pathway in acute T cell leukemias. Apoptosis, 11: 245-254.

61. Hipp, M.L. and Bauer, G. (1997).Intercellular induction of apoptosis in transformed cells does not depend on p53.Oncogene, 15: 791-797.

62. Hermeking, H. and Eick, D. (1994).Mediation of c-myc induced apoptosis by p53. Science, 265: 2091-2093.

63. Sakamuro, D., Eviner, V., Elliott, K.J.,Showe, L., White, E. and Prendegast, G.C.(1995). c-Myc induces apoptosis in epithelial cells by both p53-dependend and p53-independent mechanism. Oncogene, 11: 2411-2418.

64. Radhakrishna pillai, G., Srivastava, A.S.,Hassanein, T.I., Chauhan, D.P. and Carrier,E. (2004). Induction of apoptosis in human lung cancer cells by curcumin. Cancer Letters, 208: 163-170.

65. Kunchandy, E. and Rao, M.N.A. (1990).Oxygen radical scavenging activity of curcumin. International Journal of Pharmaceutics 58:237-240

66. Subramanian, M., Sreejayan., Rao, M.N., Devasagayam, T.P.A. and Singh, B.B.236 Chathoth et al (1994). Diminution of singlet oxygenindused DNA damage by curcumin and related antioxidants. Mutation. Research,311: 249-255.

67. Sreejayan., and Rao, M.N. (1997). Nitric oxide scavenging by curcuminoids. Journal of Pharmacy and Pharmacology, 49: 105-107.

68. Mahakunakorn, P., Tohda, M., Murakami,Y., Matsumoto, K., Watanabe, H. and Vajaragupta, O. (2003). Cytoprotective and cytotoxic effects of curcumin: Dual action on H2O2-induced oxidative cell damage in NG108-15 cells. Biological Pharmaceutical Bulletin, 26: 725-728.

69. Leist, M., Single, B., Castoldi, A.F.,Kuhnle, S. and Nicotera, P. (1997).Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. Journal of Experimental Medicine, 185: 1481-1486.

70. Chan, W.H., Wu, H.Y. and Chang, W.H.(2006). Dosage effects of curcumin on cell death types in a human osteoblast cell line. Food and Chemical Toxicology, 44: 1362-1371.

71. Woo, J.H., Kim, Y.H., Choi, Y.J., Kim,D.G., Lee, K.S., Bae, J.H., Min, D.S.,Chang, J.S., Jeong, Y.J., Lee, Y.H., Park,J.W. and Kwon, T.K. (2003). Molecular mechanism of curcumin- induces cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-xL and IAP, the release of cytochrome c and inhibition of Akt. Carcinogenesis, 24:1199-1208.

72. Bhaumik, S., Anjum, R., Rangaraj, N.,Pardhasaradhi, B.V. and Khar, A. (1999).Curcumin mediated apoptosis in AK-5 tumor cells involves the production of reactive oxygen intermediates. FEBS Letters, 456: 311-314.

73. Halliwell B. Reactive oxygen species and the central nervous system. J Neurochem 1992; 59:1609-1623.

74. Sreejayan N, Rao MN. Curcuminoids as potent inhibitors of lipid peroxidation. J Pharm Pharmacol 1994; 46: 1013-1016.

75. Reddy AC, Lokesh BR. Studies on spice principles as antioxidants in the inhibition of lipid peroxidation of rat liver microsomes. Mol Cell Biochem 1992; 111: 117-124.

76. Reddy AC, Lokesh BR. Effect of dietary turmeric (Curcuma longa) on iron-induced lipid peroxidation in the rat liver. Food Chem Toxicol 1994; 32:279-283.

77. Reddy AC, Lokesh BR. Studies on the inhibitory effects of curcumin and eugenol on the formation of reactive oxygen species and the oxidation of ferrous iron. Mol Cell Biochem 1994; 137: 1-8.

78. Rao CV, Rivenson A, Simi B, Reddy BS. Chemoprevention of colon carcinogenesis by dietary curcumin,a naturally occurring plant phenolic compound.Cancer Res 1995; 55: 259-266.

79. Unnikrishnan MK, Rao MN. Curcumin inhibits nitrogen dioxide induced oxidation of hemoglobin.Mol Cell Biochem 1995; 146: 35-37.

80. Sreejayan N, Rao MN. Nitric oxide scavenging by curcuminoids. J Pharm Pharmacol 1997; 49: 105-107.

81. Ruby AJ, Kuttan G, Babu KD, Rajasekharan KN, Kuttan R. Anti-tumour and antioxidant activity of natural curcuminoids. Cancer Lett 1995; 94: 79-83.

82. Sreejayan N, Rao MN. Free radical scavenging activity 26) Chen J, Wanming D, Zhang D, Liu Q, Kang J. Water-soluble antioxidants improve the antioxidant and anticancer activity of low concentrations of curcumin in human leukemia cells. Pharmazie 2005; 60: 57-61.

83. Reddy, S. and Aggarwal, B.B. (1994).Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase. FEBS Letters, 341: 19-22.

84. Yamamoto, H., Hanada, K., Kawasaki, K.and Nishijima, M. (1997). Inhibitory effect of curcumin on mammalian phospholipase D activity. FEBS Letters, 417: 196-198.

85. Hong, R.L., Spohn, W.H. and Hung, M.C.(1999). Curcumin inhibits Tyrosine kinase activity of p185neu and also depletes p185neu1. Clinical Cancer Research, 5:1884-1891.

86. Griscavage, J.M., Wilk, S. and Ignarro, L.J.(1996). Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophage by blocking activation of transcription factor NF-êB.Proceedings of the National Acadamy of Sciences of the United States of America,93: 3308-3312.

87. Pan, M.H., Lin Shiau, S.Y. and Lin, J.K.(2000). Comparative studies on the suppression of Nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IêB kinase and NF-êB activation in macrophages.Biochemical Pharmacology, 60: 1665-1676.

88. Graessle, S., Loidl, P. and Brosch, G.(2001). Histone acetylation: plants and fungi as model system for the investigation of histone deacetylases. Cellular and Molecular Life Sciences, 58: 704-720.

89. Liu, H., Chen Y., Cui, G.H. and Zhou, J.F.(2005). Curcumin, a potent anti-tumor reagent, is a novel histone deacetylase inhibitor regulating B-NHL cell line Raji proliferation. Acta Pharmacologica Sinica, 26: 603-609.

90. Chakraborty, S., Ghosh, U., Bhattacharyya,N.P., Bhattacharya, R.K. and Roy, M.(2006). Inhibition of telomerase activity and induction of apoptsis by curcumin in K-562 cells. Mutation Research, 596: 81-90

91. Muralikrishnan D, Mohanakumar KP. Neuroprotection by bromocriptine against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- induced neurotoxicity in mice. FASEB J 1998; 12: 905-912.

92. Tirkey N, Kaur G, Vij G, Chopra K. Curcumin, a diferuloylmethane, attenuates cyclosporine-induced renal dysfunction and oxidative stress in rat kidneys. BMC Pharmacology 2005; 5: 15.

93. Thomas B, Muralikrishnan D, Mohanakumar KP. In vivo hydroxyl radical generation in the striatum following systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. Brain Res 2000: 852: 221-224.

94. Chen J, Wanming D, Zhang D, Liu Q, Kang J. Water-soluble antioxidants improve the antioxidant and anticancer activity of low concentrations of curcumin in human leukemia cells. Pharmazie 2005; 60: 57-61.

95. Thiyagarajan M, Sharma SS. Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats. Life Sci 2004; 74: 969-985.

Received on 07.07.2009

Accepted on 10.08.2009

Research Journal of Pharmacognosy and Phytochemistry. 1(3): Nov. – Dec 2009, 153-161