Dimethylchalcone Research Articles

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone Promoted Glucose Uptake and Imposed a Paradoxical Effect on Adipocyte Differentiation in 3T3-L1 Cells

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), one of the flavonoids isolated and purified from the dried flower buds of Cleistocalyx operculatus, was explored for its function in glucose uptake/glycogen synthesis in insulin-sensitive tissue cells and its effect and mechanism on 3T3-L1 preadipocyte differentiation. DMC (10 μM) treatment remarkably promoted glucose uptake in differentiated 3T3-L1 adipocytes (P < 0.05 vs control group), whereas the glucose uptake in L6 myoblasts and glycogen synthesis in HepG2 hepatocytes were not affected by the treatment. DMC had paradoxical effects on lipid accumulation in 3T3-L1 cells compared with differentiation control. High concentrations of DMC (10 and 20 μM) markedly diminished lipid accumulation; however, a low concentration of DMC (2.5 μM) enhanced lipid storage in 3T3-L1 cells (P < 0.01 vs differentiation control group), and 5 μM DMC did not impose a significant effect. It was demonstrated that the effect of DMC in lipid accumulation was controlled by the expression of PPAR-γ.

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone Protects the Impaired Insulin Secretion Induced by Glucotoxicity in Pancreatic β-Cells

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), which is isolated and purified from the dried flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae), was investigated for its insulinotropic benefits against glucotoxicity using in vitro methods. When exposed to high glucose at the cytotoxicity level for 48 h, RIN-5F β-cells experienced a significant viability loss and impaired insulin secretion function, whereas cotreating with DMC could protect β-cells against glucotoxicity-induced decrease in glucose-stimulated insulin secretion in a dose-dependent manner without affecting basal insulin secretion. It was demonstrated that DMC increased insulin secretion against glucotoxicity by simulating the effect of GLP-1 and enhancing the expression of GLP-1R, followed by activating the signal pathway of PDX-1, PRE-INS, and GLUT2-GCK. Another mechanism was that DMC avoided the pancreatic islet dysfunction resulting from cellular damage by suppressing the production of nitric oxide (NO) by iNOS, and the expression of MCP-1. The results indicated the potential application of DMC in the intervention against glucotoxicity-induced hyperglycemia.

Protective effects of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone against hydrogen peroxide-induced oxidative stress in hepatic L02 cell

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) is a chalcone isolated from the buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry, and the hepatoprotective effects of DMC on Kunming mice have been studied in previous study. However, the effects of DMC on hepatocyte toxicity and corresponding mechanism remain unclear. The aim of this study was to evaluate the hepatoprotective mechanism of DMC in human hepatocytes (L02) treated with H₂O₂. The results demonstrated that pretreatment with DMC effectively protected H₂O₂-induced cell viability loss, cell membrane damage (lactate dehydrogenase, nitric oxide production and caspase-3 accumulation. Besides, DMC pretreatment increased the amount of glutathione, decreased malondialdehyde and the percentage of apoptotic L02 cells compared with only H₂O₂ treated group. Taken together, these results indicated that DMC had hepatoprotective effects against H₂O₂-induced liver injury by alleviating oxidative stress and apoptosis process in L02 cells, and DMC might be a potential candidate for the intervention of liver diseases.

Isolation, Characterization, Crystal Structure Elucidation, and Anticancer Study of Dimethyl Cardamonin, Isolated from Syzygium campanulatum Korth.

Syzygium campanulatum Korth is an equatorial, evergreen, aboriginal shrub of Malaysia. Conventionally it has been used as a stomachic. However, in the currently conducted study dimethyl cardamonin or 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) was isolated from S. campanulatum Korth, leaf extract. The structural characterization of DMC was carried out by making use of various techniques including UV, IR, NMR spectral followed by LC-MS, and X-ray crystallographic techniques. For determining the purity of compound, highly effective techniques including TLC, HPLC, and melting point were used. The cytotoxicity of DMC and three different extracts of S. campanulatum was evaluated against human colon cancer cell line (HT-29) by three different assays. DMC and ethanolic extract revealed potent and dose-dependent cytotoxic activity on the cancer cell line with IC50 12.6 and 90.1 µg/mL, respectively. Quite astonishingly to our knowledge, this is the very first report on S. campanulatum as being a rich source (3.5%) of DMC, X-ray crystallography, and anticancer activity on human colon cancer cells.

Induction of apoptosis in human hepatoma SMMC-7721 cells using 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone, a chalcone from buds of Cleistocalyx operculatus

The molecular mechanism to induce apoptosis in human hepatoma SMMC-7721 cells was investigated using 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) isolated from the buds of Cleistocalyx operculatus. Typical morphological changes in apoptotic body formation after DMC treatment were observed using acridine orange (AO)/ethidium bromide (EB) staining. After 48 h of incubation with 0, 6.25, 12.5, and 25 μM of DMC, the percentages of annexin V-FITC-positive/PI negative cells were 1.51, 9.33, 33.84, and 61.32%, respectively. Apoptosis induced by DMC was blocked by z-VAD-fmk, a caspase inhibitor. Analysis of fluorescence intensities of rhodamine 123 in cells showed that DMC induced a loss of mitochondrial membrane potential (ΔΨm). Western blot results showed that DMC decreased the Bcl-2/Bax ratio, suggesting that the Bcl-2 family is involved in control of apoptosis. All of these signal transduction pathways are involved in initiating apoptosis. DMC is a potential anticancer agent that deserves further exploration.

Ceriporia lacerata DMC1106, a new endophytic fungus: Isolation, identification, and optimal medium for 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone production

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), a bioactive flavonoid isolated from Cleistocalyx operculatus (Roxb.) Merr and Perry (Myrtaceae) exhibits significant anti-cancer effects and has received great attention recently. In this work, a new endophytic DMCproducing fungus, Ceriporia lacerata DMC1106, was isolated from the bud of C. operculatus. Its identity was confirmed based on rDNA ITS sequences (ITS1 and ITS2 regions and the intervening 5.8S rDNA region) and phylogenetic analysis. Furthermore, statistical screening designs were applied to identify significant medium variables for DMC production and to find their optimal levels. The difference between the optimized medium and the original medium suggested that L-phenylalanine, magnesium ion and oxalic acid might be attributed to the enhancement of DMC production. Compared with the initial medium, the production of DMC was increased 6-fold in optimum medium. These results demonstrated that the endophytic fungus Ceriporia lacerata DMC1106 has potential applications for DMC production.

Antioxidant activity and protection of human umbilical vein endothelial cells from hydrogen peroxide-induced injury by DMC, a chalcone from buds of Cleistocalyx operculatus

The aim of this work was to study the antioxidant activity and the protective effect of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), the main compound from the buds of Cleistocalyx operculatus, on human umbilical vein endothelial cells against cytotoxicity induced by H2O2. The antioxidant activities of DMC were measured by ABTS assay, ferric reducing antioxidant power (FRAP) and hydroxyl radical scavenging activity, and protective effects of DMC on human umbilical vein endothelial cells against cytotoxicity induced by H2O2 were tested. DMC was found to have high ABTS radical scavenging activity (176.5±5.2μmoltroloxequivalents/500μmol DMC) and strong ferric reducing antioxidant power (213.3±5.8μmoltroloxequivalents/500μmol DMC). In addition, DMC scavenged the hydroxyl radicals, with IC50 values of 243.7±6.3μM, slightly lower than the reference antioxidant ascorbic acid (ASC). Moreover, DMC could protect the human umbilical vein endothelial cells against H2O2-induced cytotoxicity by decrease intracellular and extracellular ROS levels, reduction in catalase (CAT) activity and increment in malondialdehyde (MDA) level. These results suggested that DMC has the potential to be used in the therapy of oxidative damage.

In Vitro Investigation of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone for Glycemic Control

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a compound isolated and purified from the dried flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae), was investigated for its glucose control benefits using in vitro methods. DMC showed strong noncompetitive (IC(50) of 43 μM) inhibition of pancreatic α-amylase; it was, however, ineffective against intestinal α-glucosidase. In addition, DMC exhibited remarkable glucose transport inhibition effects in both simulated fasting and fed states in Caco-2 cell monolayers (P < 0.05). Besides, exposure of MIN6 cells to 250 μM H(2)O(2) for 1 h caused a significant viability loss and insulin secretion reduction. Pretreatment of MIN6 cells with DMC for 2 h protected against the H(2)O(2)-induced decrease in glucose-stimulated insulin secretion in a dose-dependent manner and also enhanced the impaired basal insulin secretion. Such effects highlight the therapeutic potential of DMC in the management of hyperglycemia.

Inhibitory activities of extracts from Cleistocalyx operculatus flower buds on pancreatic lipase and α-amylase

In this work, we investigated the inhibitory activities of aqueous extract (Eaq), 30, 60, and 95 % ethanol extracts (E3et, E6et, and E9et), petroleum ether extract (Epe), ethyl acetate extract (Eae), and 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) from Cleistocalyx operculatus flower buds on pancreatic lipase and α-amylase, which were closely related to body weight and obesity. The inhibition effect of various extracts was determined by the Lineweaver–Burk plots. The results showed that the IC50 values of DMC, Epe, Eae, and E9et against pancreatic lipase were 101.5 μM, 38.65, 76.62, and 109.76 μg/ml, respectively, which were much lower than that of E3et and E6et. Eaq, E3et, E6et, and DMC strongly inhibiting the pancreatic α-amylase, with IC50 values of 73.10, 165.93, 231.23 μg/ml, and 69.35 μM, respectively. Furthermore, the kinetic analysis suggested that the inhibition mode of crude extract against pancreatic lipase was noncompetitive, while that against pancreatic α-amylase was competitive. The content of DMC and total polyphenols in each extract were determined by HPLC and spectrophotometric method, respectively. It was interestingly found that the DMC content had profound influence on pancreatic lipase activity, and the total polyphenols content significantly affected the pancreatic α-amylase activity. The findings in this work suggested that extracts from C. operculatus flower buds possess potent inhibitory activity against pancreatic lipase and α-amylase, and provided some new ideas for the prevention and treatment for obesity.

Hepatoprotective Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on CCl4-Induced Acute Liver Injury in Mice

In this paper, the hepatoprotective effects of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) on CCl(4)-induced acute liver injury in Kunming mice were investigated. DMC was administered intraperitoneally (ip) (5, 10, or 20 mg/kg of body weight) for 7 days prior to the administration of CCl(4) (0.1%, ip). Pretreatment with DMC significantly decreased activities of serum hepatic enzymes, namely alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, γ-glutamyl transferase, and total bilirubin, and decreased the elevation of lipid peroxidation, malondialdehyde, reactive oxygen species, and protein carbonyl content. Pretreatment with DMC markedly increased activities of enzymatic antioxidants such as superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase and increased levels of nonenzymatic antioxidant markers such as reduced glutathione, total sulfhydryl groups, vitamin C, and vitamin E in liver. These results combined with liver histopathology demonstrate that DMC has potential hepatoprotective effects, which may be related to the attenuation of oxidative stress, accelerating the antioxidant cascade and inhibition of lipid peroxidation.

Multidrug resistance reversal effect of DMC derived from buds of Cleistocalyx operculatus in human hepatocellular tumor xenograft model

Multidrug resistance (MDR) is a major obstacle in the chemotherapeutic treatment of many human cancers. 2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) isolated from the buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae), was investigated for its reversal effects on cancer cell MDR. A human hepatocellular tumor xenograft model was established with the BEL-7402/5-FU cell line. Combined 5-fluorouracil (5-FU) and DMC (40 mg kg(-1) ) treatment significantly elevated tumor inhibition rate to 72.2%. DMC could also increase 5-FU concentrations in tumor tissues and increase caspase-3 activity. Also, combined therapy resulted in enhanced tumor apoptotic and reduced proliferative activities relative to 5-FU alone. Examining body weight and other signs of unwanted toxicity of the different treatment groups revealed no significant signs of adverse effects. All results suggested that DMC reverses 5-FU resistance, with a benign side effects profile.

Reversal effect of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on multi-drug resistance in resistant human hepatocellular carcinoma cell line BEL-7402/5-FU

Multi drug resistance (MDR) is a major obstacle in the chemotherapeutic treatment of many human cancers. 2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a chalcone, isolated from the buds of Cleistocalyx operculatus, has been shown to have antitumor effects on human carcinoma SMMC-7721 cells in vitro and in vivo. In this paper, we studied the reversal effect and the mechanism of DMC on human hepatocellular carcinoma drug-resistant cells BEL-7402/5-FU in vitro. Administration of DMC reversed the multi-drug resistance of human hepatocellular carcinoma BEL-7402/5-FU cells significantly. DMC enhanced the sensitivity of BEL-7402/5-FU cells to 5-fluorouracil (5-FU) and doxorubicin (DOX). Staining with Hoechst 33258 and flow cytometric analysis showed that DMC has apoptosis-inducing effect on BEL-7402/5-FU cells. It could also increase the concentration of 5-FU in the resistant multi-drug-resistant cells. We also observed that over-expression of the multi-drug resistance-associated protein (MRP1) and of the glutathione S-transferase π (GST-π) contributed to MDR in BEL-7402/5-FU cells. The mRNA expressions of MRP1 and GST-π and the protein expression of MRP1 were decreased by DMC. These data demonstrated that DMC could effectively reverse MDR in BEL-7402/5-FU cells.

Protective Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone to PC12 Cells against Cytotoxicity Induced by Hydrogen Peroxide

Oxidative stress has been considered as a major cause of cellular injuries in various clinical abnormalities. One of the possible ways to prevent reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical therapies to augment the endogenous antioxidant defense capacity. The present study found that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a chalcone isolated from the buds of Cleistocalyx operculatus, possessed cytoprotective activity in PC12 cells treated with H(2)O(2). The results showed that DMC could effectively increase cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction], decrease the cell apoptotic percentage [annexin V/propidium iodide (AV/PI) assay], prevent the membrane from damage [lactate dehydrogenase (LDH) release], scavenge ROS formation, reduce caspase-3 activity, and attenuate the decrease of mitochondrial membrane potential (MMP) in PC12 cells treated with H(2)O(2). Meanwhile, DMC increased the catalytic activity of superoxide dismutase (SOD) and the cellular amount of glutathione (GSH), decreased the cellular amount of malondialdehyde (MDA), and decreased the production of lipid peroxidation in PC12 cells treated with H(2)O(2).

In Vitro and In Vivo Reversal of Cancer Cell Multidrug Resistance by 2',4'-Dihydroxy-6'-methoxy-3',5'- dimethylchalcone

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) isolated from the buds of Cleistocalyx operculatus, was investigated for its reversal effects on cancer cell multidrug resistance. DMC potentiated the cytotoxicity of the chemotherapeutic agent doxorubicin to drug-resistant KB-A1 cells. When 5 μM DMC was present simultaneously with doxorubicin, the IC50 of DOX on KB-A1 cells decreased from 13.9 ± 0.7 μg/ml to 3.6 ± 0.7 μg/ml. A human carcinoma xenograft model was established with the KB-A1 cell line. DMC could sensitize the tumors to doxorubicin as indicated by a considerable reduction in tumor weight. DMC increased the intracellular accumulation of doxorubicin in KB-A1 cells. When KB-A1 cells were exposed to 10 μg/ml doxorubicin combined with 5, 10, 20 μM DMC for 4 hours, the intracellular concentrations of doxorubicin were increased 1.4-, 1.8-, 3.1-fold, respectively, in comparison with doxorubicin alone treatment. All results indicated that DMC had reversal effects on the multidrug resistance phenotype.

In vivo antitumor activity by 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone in a solid human carcinoma xenograft model

Previously we have shown that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), which is isolated from the buds of Cleistocalyx operculatus, significantly inhibits the growth of human liver cancer SMMC-7721 cells and is able to induce apoptosis of SMMC-7721 cells in vitro. Here we report the antitumor effects of DMC in vivo, using a solid human tumor xenograft mouse model using human liver cancer SMMC-7721 cells. The average tumor weights in the control group and in mice injected with 150 mg/kg DMC were 1.42+/-0.11 g and 0.59+/-0.12 g, respectively. Flow cytometric analysis of the tumor cell population demonstrated an aneuploid peak (representing 33.60+/-0.80% of the total in mice injected with 150 mg/kg DMC). To our knowledge, this is the first time that chalcone compounds have been applied to a human tumor xenograft model.

Induction of apoptosis in K562 human leukemia cells by 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, was investigated in its cytotoxicity and anti-proliferation on K562 cell line. Our results revealed that the IC 50 was equal to 14.2 ± 0.45 μM and the EC 50 was 3.3 ± 0.14 μM. Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cells treated with 8 μM DMC for 48 h. Flow cytometric analysis was performed to determine hypodiploid cells. The results of flow cytometry assay indicated that the percentage of hypodiploid K562 cells was 76.15 ± 3.22% after 48 h treatment with 16.0 μM DMC. The treatment resulted in the appearance of a hypodiploid peak (A 0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2 n. Western blot results illustrated that in the same dosage and incubation time, DMC could down-regulate the level of Bcl-2 protein and did not influence the expression of Bax protein. The resulting net effect could thus lead to a lower ratio of Bcl-2/Bax, which might be responsible for the DMC-induced apoptosis in K562 cells.

In vivo antitumor activity by 2?,4?-dihydroxy-6?-methoxy-3?,5?-dimethylchalcone in a solid human carcinoma xenograft model

Previously, we showed that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, significantly inhibited the growth of human liver cancer SMMC-7721 cells and could induce SMMC-7721 cells apoptosis in vitro. Here, we report the antitumor effects of DMC in vivo, using a solid human tumor xenograft model with a human liver cancer SMMC-7721 cell line. Our results revealed that the average tumor weight in a control group and a 150-mg/kg DMC injection group was 1.42+/-0.11 g and 0.59+/-0.12 g, respectively. Flow cytometric analysis of the tumor cell population demonstrated the existence of an aneuploid peak (representing 33.60+/-0.80% of the total in the 150-mg/kg DMC injection group). To our knowledge, this is the first time that chalcone compounds were applied to a human tumor xenograft model.

In vitro anti-tumor activity of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone against six established human cancer cell lines

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, was investigated in its cytotoxicity and its influence on six human cancer cell lines. Among SMMC-7721, 8898, HeLa, SPC-A-1, 95-D and GBC-SD cell lines, SMMC-7721 cells was the most sensitive one in these tested cell lines, with IC 50 equal to 32.3 ± 1.13 μM, EC 50 equal to 9.00 ± 0.36 μM and the therapeutic index equal to 3.59. Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cells treated with 9 μM DMC for 48 h. Flow cytometric analysis was performed to determine hypodiploid cells. The results of flow cytometry assay indicated that the percentage of hypodiploid SMMC-7721 cells were 49.44 ± 1.06% after 48 h treatment with 18.0 μM DMC. The treatment resulted in the appearance of a hypodiploid peak (A 0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2 n. To our knowledge, this is the first report on anti-tumor activity by DMC.

Triterpenoids and chalcone from Syzygium samarangense

A new triterpene, methyl 3-epi-betulinate in its native form and 4′,6′-dihydroxy-2′-methoxy-3′,5′-dimethyl chalcone along with ursolic acid, jacoumaric acid and arjunolic acid have been isolated from the aerial parts of Syzygium samarangense.

Absolute configuration determination by anomalous scattering of light atoms. Have the limits been reached?

The absolute conformation determination of the achiral olefin 4,4'-dimethylchalcone (DMC), C17H16O, crystallizing in a chiral space group (P212121), was accomplished from the anomalous scattering of oxygen (Cu Kα) despite the small O:C ratio (0.059). The symmetry-equivalent intensities of seven enantiomer-sensitive reflections of two crystal specimens were measured with graphite-monochromated Cu Kα radiation on a Picker diffractometer. In addition, an extensive data set of one of the specimens was collected on a CAD4 diffractometer with Ni-filtered Cu Kα radiation. Bijvoet-difference analysis established that the two specimens were enantiomers. The probability that the absolute conformation has been wrongly determined is <0.1% in all three measurements. The results of this study established, for the first time, the correlation between the absolute conformation, in the crystal, of an achiral optically inactive molecule and the absolute configuration and optical rotation of one of the two possible enantiomers of its dibromide adduct obtained by gas/solid bromination reaction. Statistical analysis of the CAD4 data shows that the use of Friedel pairs tends to minimize the effects of systematic errors and yields a higher statistical confidence in the results.