Demethoxycurcumin Research Articles

Comparison of inhibitory potency of three different curcuminoid pigments on nitric oxide and tumor necrosis factor production of rat primary microglia induced by lipopolysaccharide

Microglia are the resident innate immune cells in the central nervous system. Evidence supports that the unregulated activation of microglia results in the production of pro-inflammatory cytokines and chemokines that propagate neuronal injury and finally cause neurodegenerative diseases. Curcuminn (Cur), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are curcuminoid pigments extracted from turmeric ( Curcuma longa L.). Cur has been reported to suppress the activation of microglia by reducing toxic factors production, but little is known about whether the two natural demethoxy derivatives of Cur, DMC and BDMC, have the similar effects as Cur. In the present study, we found that all of the three curcuminoid pigments significantly suppressed nitric oxide (NO) production by LPS-activated microglia and the relative potency was DMC > BDMC > Cur. This result was verified by RT-PCR analysis of iNOS mRNA. The NO-scavenging abilities of three curcuminoid pigments are very weak, which suggested that the indirect effect may not be critical in inhibiting NO production by LPS-activated microglia. Moreover, these three curcuminoid pigments attenuated the expression of mRNA and proteins of tumor necrosis factor-alpha (TNF-α) in a concentration-dependent manner and the relative potency was also DMC > BDMC > Cur. In conclusion, Cur, DMC and BDMC were found as potent microglia-activation inhibitors, and DMC exhibited the strongest inhibitory activity on NO and TNF-α production. These results provided an interesting clue for designing new compounds which could have better potential therapeutic implications for various neurodegenerative diseases.

The Biosynthetic Pathway of Curcuminoid in Turmeric (Curcuma longa) as Revealed by13C-Labeled Precursors

In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding (13)C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by (13)C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid >> ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.

Curcumin, demethoxycurcumin and bisdemethoxycurcumin differentially inhibit cancer cell invasion through the down-regulation of MMPs and uPA

Curcumin (Cur), a component of turmeric ( Curcuma longa), has been reported to exhibit antimetastatic activities, but the mechanisms remain unclear. Other curcuminoids present in turmeric, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) have not been investigated whether they exhibit antimetastatic activity to the same extent as curcumin. The regulation of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) play important role in cancer cell invasion by cleavage of extracellular matrix (ECM). In this line, we comparatively examined the influence of Cur, DMC and BDMC on the expressions of uPA, MMP-2, MMP-9, membrane Type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinases (TIMP-2), and in vitro invasiveness of human fibrosarcoma cells. The results indicate that the differential potency for inhibition of cancer cell invasion was BDMC≥DMC>Cur, whereas the cell migration was not affected. Zymography analysis exhibited that curcumin, DMC and BDMC significantly decreased uPA, active-MMP-2 and MMP-9 but not pro-MMP-2 secretion from the cells in a dose-dependent manner, in which BDMC and DMC show higher potency than curcumin. The suppression of active MMP-2 level correlated with inhibition of MT1-MMP and TIMP-2 protein levels involved in pro-MMP-2 activation. Importantly, BDMC and DMC at 10 μM reduced MT1-MMP and TIMP-2 protein expression, but curcumin slightly reduced only MT1-MMP but not TIMP-2. In addition, three forms of curcuminoids significantly inhibited collagenase, MMP-2, and MMP-9 but not uPA activity. In summary, these data demonstrated that DMC and BDMC show higher antimetastasis potency than curcumin by the differentially down-regulation of ECM degradation enzymes.

HPLC Analysis of Curcuminoids in Dietary Supplements and in vitro Digestion Fractions

Curcumin (Cur), demethoxycurcumin (DMC), and bis‐demethoxycurcumin (BDMC) are the main curcuminoids in Curcuma longa used to prepare the spice turmeric. Cur has been shown to reduce the risk of cancer, inflammation and Alzheimers. As such, it has become increasingly popular in foods and dietary supplements. To examine content and bioaccessibility of commercial Cur products, we developed and validated an HPLC method to measure Cur and metabolites. Dietary supplements were processed via an in vitro digestion (simulated gastric and small intestinal phases) and Caco‐2 cellular uptake (Chitchumroonchokchai et al., J. Nutr. 2004). Cur, DMC, BDMC were monitored at 425nm for sensitive detection and 280nm was monitored to detect the internal standard, Flavone, and cellular metabolites of curcuminoids. Three products were tested containing from 10% to 95% curcuminoids. The micellarized Cur was stable in cell culture medium for at least 4 hrs. Transfer of Cur from the supplements to micelles during the small intestinal phase of digestion varied markedly between the two products. Micellarization was more efficient as polarity increased, i.e., BDMC> DMC>Cur. Efficiency (%) of incorporation into micelles was higher for Curcu‐Gel™ than Turm’Brite 10% sd Cur, 29.1% vs 8.7%. Cur in synthetic micelles was taken up by the cells in a time and concentration dependent manner. (Supported in part by Tishcon Corp., Westbury, NY)

Regulation of heme oxygenase-1 expression by demethoxy curcuminoids through Nrf2 by a PI3-kinase/Akt-mediated pathway in mouse β-cells

Curcumin (diferuloylmethane), a component of turmeric, has been shown to have therapeutic properties. Induction of phase 2 detoxifying enzymes is a potential mechanism through which some of the actions of curcumin could proceed. Heme oxygenase-1 (HO-1), an antioxidant phase 2 enzyme, has been reported to have cytoprotective effects in pancreatic beta-cells. Curcumin on further purification yields demethoxy curcumin (DMC) and bisdemethoxy curcumin (BDMC). The objective of the present study was to determine the mechanism by which these purified curcuminoids induce HO-1 in MIN6 cells, a mouse beta-cell line. Demethoxy curcuminoids induced HO-1 promoter linked to the luciferase reporter gene more effectively than curcumin. The induction was dependent on the presence of antioxidant response element (ARE) sites containing enhancer regions (E1 and E2) in HO-1 promoter and nuclear translocation of nuclear factor-E2-related factor (Nrf2), the transcription factor that binds to ARE. Curcuminoids stimulated multiple signaling pathways that are known to induce HO-1. Inhibition of specific signaling pathways with pharmacological inhibitors and cotransfection experiments suggested the involvement of phosphotidylinositol 3-kinase and Akt. Real-time quantitative RT-PCR analysis showed significant elevation in the mRNA levels of HO-1 and two other phase 2 enzymes, the regulatory subunit of glutamyl cysteine ligase, which is needed for the synthesis of glutathione, and NAD(P)H:quinone oxidoreductase, which detoxifies quinones. DMC and BDMC induced the expression of HO-1 and translocated Nrf2 to nucleus in beta-cells of mouse islets. Our observations suggest that demethoxy curcuminoids could be used to induce a cellular defense mechanism in beta-cells under conditions of stress as seen in diabetes.

Curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and turmerones differentially regulate anti-inflammatory and anti-proliferative responses through a ROS-independent mechanism

Curcumin, a component of turmeric (Curcuma longa), has been shown to exhibit chemopreventive activity. Whether analogs of curcumin (Cur), such as demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), tetrahydrocurcumin (THC) and turmerones, modulate inflammatory signaling and cell proliferation signaling to same extent as curcumin was investigated. The results indicate that the relative potency for suppression of tumor necrosis factor (TNF)-induced nuclear factor-kappaB (NF-kappaB) activation was Cur > DMC > BDMC; thus suggesting the critical role of methoxy groups on the phenyl ring. THC, which lacks the conjugated bonds in the central seven-carbon chain, was completely inactive for suppression of the transcription factor. Turmerones also failed to inhibit TNF-induced NF-kappaB activation. The suppression of NF-kappaB activity correlated with inhibition of NF-kappaB reporter activity and with down-regulation of cyclooxygenase-2, cyclin D1 and vascular endothelial growth factor, all regulated by NF-kappaB. In contrast to NF-kappaB activity, the suppression of proliferation of various tumor cell lines by Cur, DMC and BDMC was found to be comparable; indicating the methoxy groups play minimum role in the growth-modulatory effects of curcumin. THC and turmerones were also found to be active in suppression of cell growth but to a much lesser extent than curcumin, DMC and BDMC. Whether suppression of NF-kappaB or cell proliferation, no relationship of any of the curcuminoid was found with reactive oxygen species (ROS) production. Overall, our results demonstrated that different analogs of curcumin present in turmeric exhibit variable anti-inflammatory and anti-proliferative activities, which do not correlate with their ability to modulate the ROS status.

Development and Validation of Improved Reversed Phase-HPLC Method for Simultaneous Determination of Curcumin, Demethoxycurcumin and Bis-Demethoxycurcumin

Isocratic reversed phase high performance liquid chromatographic (HPLC) method using RP C18 column was developed for simultaneous determination of the curcuminoids. Mobile phase consisted of acetonitrile:0.1% trifluro-acetic acid (50:50) and flow rate was 1.5 mL min−1 and elution was monitored at 420 nm. Validation in selected conditions showed that the chosen method is sensitive, selective, precise and reproducible with linear response of detector for the simultaneous determination of curcumin (C), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC). The limits of detection were 27.99, 31.91 and 21.81 ng mL−1 for C, DMC and BDMC, respectively. Limits of quantitation for C, DMC and BDMC, were 84.84, 96.72 and 66.10 ng mL−1, respectively. Linear range was form 100 to 600 ng mL−1. The mean ± SD percent recoveries of curcuminoids were 99.87 ± 0.34, 100.09 ± 0.48 and 100.10 ± 0.60% of C, DMC and BDMC, respectively. Further, the method was used for quantitation of curcuminoids from turmeric rhizome.

Curcuminoids, Curcumin, and Demethoxycurcumin Reduce Lead-Induced Memory Deficits in Male Wistar Rats

This study investigated the neuroprotective effects of the curcuminoids against lead-induced neurotoxicity. The results show that lead significantly increases lipid peroxidation and reduces the viability of primary hippocampal neurons in culture. This lead-induced toxicity was significantly curtailed by the co-incubation of the neurons with the curcuminoids. In a whole animal experiment, rats were trained in a water maze and thereafter dosed with lead and/or curcumin (CURC), demethoxycurcumin (DMC), or bisdemethoxycurcumin (BDMC) for 5 days. Animals treated with curcumin and demethoxycurcumin but not bisdemethoxycurcumin had more glutathione and less oxidized proteins in the hippocampus than those treated with lead alone. These animals also had faster escape latencies when compared to the Pb-treated animals indicating that CURC- and DMC-treated animals retain the spatial reference memory. The findings of this study indicate that curcumin, a well-established dietary antioxidant, is capable of playing a major role against heavy metal-induced neurotoxicity and has neuroprotective properties.

Comparative effects of curcuminoids on endothelial heme oxygenase-1 expression: ortho-methoxy groups are essential to enhance heme oxygenase activity and protection

Recently, it has been reported that curcumin, which is known as a potent antioxidant, acts as a non- stressful and non-cytotoxic inducer of the cytoprotective heme oxygenase (HO)-1. In this study, naturally occurring curcuminoids, such as pure curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), were compared for their potential ability to modulate HO-1 expression and cytoprotective activity in human endothelial cells. All three curcuminoids could induce HO-1 expression and HO activity with differential levels. The rank order of HO activity was curcumin, DMC and BDMC. In comparison with endothelial protection against H2O2-induced cellular injury, cytoprotective capacity was found to be highest with curcumin, followed by DMC and BDMC. Interestingly, cytoprotective effects afforded by curcuminoids were considerably associated with their abilities to enhance HO activity. Considering that the main difference among the three curcuminoids is the number of methoxy groups (none for BDMC, one for DMC, and two for curcumin), the presence of methoxy groups in the ortho position on the aromatic ring was suggested to be essential to enhance HO-1 expression and cytoprotection in human endothelial cells. Our results may be useful in designing more efficacious HO-1 inducers which could be considered as promising pharmacological agents in the development of therapeutic approaches for the prevention or treatment of endothelial diseases caused by oxidative damages.

Microarray-based analysis of anti-angiogenic activity of demethoxycurcumin on human umbilical vein endothelial cells: crucial involvement of the down-regulation of matrix metalloproteinase.

cDNA microarray‐based gene expression analysis has been successfully employed to explore the action mechanism and to validate the targets of several drugs. In the present study, we evaluated anti‐angiogenic activity of demethoxycurcumin (DC), a structural analog of curcumin, isolated from Curcuma aromatica, and investigated the effect of DC on genetic reprogramming in cultured human umbilical vein endothelial cells (HUVECs) using cDNA microarray analysis. Of 1024 human cancer‐focused genes arrayed, 187 genes were up‐regulated and 72 genes were down‐regulated at least 2‐fold by DC. Interestingly, 9 angiogenesis‐related genes were down‐regulated over 5‐fold in response to DC, suggesting that the genetic reprogramming was crucially involved in anti‐angiogenesis by the compound. To verify the results obtained from cDNA microarray analysis, matrix metalloproteinase‐9 (MMP‐9), the product of one of the angiogenesis‐related genes down‐regulated over 5‐fold by DC, was investigated using gelatin zymography. DC potently inhibited the expression of MMP‐9, yet showed no direct effect on its activity. These data show that gene expressional change of MMP‐9 is a major mediator for angiogenesis inhibition by DC. All genes identified and microarray data are available on the web at http://dasan.sejong.ac.kr/bioprobe/.

SIMULTANEOUS DETERMINATION OF CURCUMINOIDS IN CURCUMA SAMPLES USING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

A rapid and simple high performance thin layer chromatographic method has been developed for the simultaneous quantitation of pharmacologically important diaryl heptanoids curcumin, demethoxy curcumin, and bis-demethoxy curcumin in Curcuma longa and C. amada. The assay combines the isolation and separation of curcuminoids on silica gel 60F254 high performance thin layer chromatographic plates, followed by scanning of the spots at 366 nm using a UV detection mode.

Kinetics of Alkaline Degradation of the Food Pigments Curcumin and Curcuminoids

ABSTRACTAlkaline degradation of the pigments curcumin (CC), demethoxycurcumin (DMC) and bis‐demethoxycurcumin (BDMC) in aqueous solutions of purified and commercial oleoresin sources was investigated. Alkaline degradation of the compounds corresponded to pseudo‐first‐order kinetics. Responses were similar for pigments combined or alone in solutions. Profiles of rate constants were closely associated to polynominal curves. Degradation rate constants rapidly increased from pH 7.45 to a maximum at about 10.2 and then tended to decline at higher pH. Rates of alkaline degradation were lower for BDMC than for CC and DMC. Halflives of CC, DMC and BDMC at pH 7.45 were 900, 1700 and 2200 hr, respectively. In contrast, half‐lives of CC, DMC and BDMC at pH 10.2 were 0.4, 1.0 and 5.0 hr, respectively. Use of BDMC instead of CC in alkaline foods would be expected to improve color stability.