Brazilian propolis (AF-08) inhibits collagen-induced platelet aggregation without affecting blood coagulation.

Brazilian propolis (AF-08) is a dietary supplement containing a variety of flavonoids. It is used worldwide as a folk medicine. Flavonoids and a diet of fruits and vegetables containing them have been shown to reduce the risk of cardiovascular diseases (CVDs). Most of CVDs are caused by arterial thrombus formation. A thrombus is formed by the interaction between adhesion and aggregation of platelets to damaged blood vessels and blood coagulation consisting of extrisic and intrinsic pathways. Platelet aggregation and blood coagulation are closely linked to thrombosis. Therefore, we evaluated the effectiveness of AF-08 or its component flavonoids against thrombosis by examining their inhibition of platelet aggregation and blood coagulation. Human platelet-rich plasma was incubated with serial dilutions of AF-08 for 10 min to assess its inhibitory effect on platelet aggregation caused by collagen. The inhibitory effect of AF-08 on blood coagulation was evaluated by the prothrombin time (PT) and activated partial thromboplastin time (APTT), which reflect the coagulation function of extrinsic and intrinsic pathways, respectively. AF-08 significantly inhibited collagen-induced platelet aggregation but not PT and APTT, indicating that AF-08 inhibited platelet aggregation but not blood coagulation. Among three flavonoids contained in AF-08, apigenin and chrysin obviously inhibited platelet aggregation but the inhibitory effect of kaempferol was less effective. The three flavonoids did not affect PT and APTT. The inhibitory activity of AF-08 on human platelet aggregation without affecting blood coagulation was suggested to be partially due to apigenin and chrysin. AF-08 may be effective in suppressing platelet-based arterial thrombus formation and reducing the risk of CVDs.

Inhibitory effects of compounds isolated from the dried branches and leaves of murta (Myrceugenia euosma) on lipid accumulation in 3T3-L1 cells.

As obesity is a global health concern the demand for anti-obesity drugs is high. In this study, we investigated the anti-obesity effect of the dried branches and leaves of murta (Myrceugenia euosma Legrand, Myrtaceae). A methanol extract of the dried branches and leaves of murta inhibited adipogenesis in 3T3-L1 cells. Three known flavanones-cryptostrobin (1), pinocembrin (4), and 5,7-dihydroxy-6,8-dimethylflavanone (6), and three chalcones-2',6'-dihydroxy-3'-methyl-4'-methoxychalcone (2), pinostrobin chalcone (3), and 2',6'-dihydroxy-4'-methoxy-3',5'-dimethylchalcone (5) were isolated from the active fraction. Structures of these compounds were identified using various spectral data. Each of these compounds also inhibited adipogenesis in 3T3-L1 cells. In particular, compound 3 was a more potent inhibitor of triglyceride accumulation than the positive control berberine. Gene expression studies revealed that treatment of 3T3-L1 cells with 3 lowers the expression levels of CCAAT/enhancer-binding protein α and peroxisome proliferator activator γ2 during adipogenesis without affecting cell viability. Treatment of 3T3-L1 cells with 3 reduced the expression levels of mRNAs encoding sterol regulatory element-binding protein 1c and several lipogenic enzymes, including fatty acid synthase and stearoyl CoA desaturase-1. These results indicate that the methanol extract and compounds isolated from the dried branches and leaves of murta exert their anti-obesity effects through the inhibition of adipogenesis.

Effect of Brazilian propolis (AF-08) on genotoxicity, cytotoxicity and clonogenic death of Chinese hamster ovary (CHO-K1) cells irradiated with (60)Co gamma-radiation.

The present study was conducted in order to evaluate the effect of Brazilian propolis (AF-08; 5, 10, 15, 30, 50, 100, and 200µg/mL) in protecting CHO-K1 cells against genotoxic and cytotoxic damage and clonogenic death induced by (60)Co gamma-radiation (1.0, 2.0, 4.0, and 6.0Gy). For this purpose, three interlinked endpoints were analyzed: induction of DNA damage by use of the micronucleus (MN) test (genotoxic damage), cell viability by means of the MTS assay, and differential staining (cytotoxic damage) and clonogenic death via the colony-formation test (cytotoxic damage). The MN test revealed that propolis alone (5-100µg/mL) was not genotoxic up to 100µg/mL and that 30µg/mL of propolis reduced the radiation-induced DNA damage (∼56% reduction, p<0.05), exhibiting a radio-protective effect on irradiated CHO-K1 cells. On the other hand, analysis of cytotoxicity showed that a concentration of 50µg/mL presented a significant proliferative effect (p<0.001) when associated with radiation, decreasing the percentage of necrotic cells (p<0.01). No mediated cytotoxic effect was found, but the concentration of 200µg/mL was toxic when analyzed at 24 and 48h via the differential staining technique, but not at 72h after irradiation, analyzed with the MTS assay. Differential staining also showed that necrosis was the main death modality in irradiated cells and that apoptosis was induced only at the toxic concentration of propolis (200µg/mL). Concerning the clonogenic capacity, a concentration of 50µg/mL also exhibited a significant stimulating effect on cell proliferation (p<0.001), in agreement with the data from differential staining. Taken together, these data suggest that the use of propolis AF-08 for the prevention of the adverse effects of ionizing radiation is promising. Nevertheless, additional investigations are necessary for a better understanding of potential applications of propolis to improve human health.

Effect of brazilian propolis on exacerbation of respiratory syncytial virus infection in mice exposed to tetrabromobisphenol a, a brominated flame retardant.

Tetrabromobisphenol A (TBBPA), a brominated flame retardant, has been found to exacerbate pneumonia in respiratory syncytial virus- (RSV-) infected mice. We examined the effect of Brazilian propolis (AF-08) on the exacerbation of RSV infection by TBBPA exposure in mice. Mice were fed a powdered diet mixed with 1% TBBPA alone, 0.02% AF-08 alone, or 1% TBBPA and 0.02% AF-08 for four weeks and then intranasally infected with RSV. TBBPA exposure increased the pulmonary virus titer and level of IFN- γ , a representative marker of pneumonia due to RSV infection, in the lungs of infected mice without toxicity. AF-08 was significantly effective in reducing the virus titers and IFN- γ level increased by TBBPA exposure. Also, AF-08 significantly reduced proinflammatory cytokine (TNF- α and IL-6) levels in the lungs of RSV-infected mice with TBBPA exposure, but Th2 cytokine (IL-4 and IL-10) levels were not evidently increased. Neither TBBPA exposure nor AF-08 treatment affected the anti-RSV antibody production in RSV-infected mice. In flow cytometry analysis, AF-08 seemed to be effective in reducing the ratio of pulmonary CD8a(+) cells in RSV-infected mice with TBBPA exposure. TBBPA and AF-08 did not exhibit anti-RSV activity in vitro. Thus, AF-08 probably ameliorated pneumonia exacerbated by TBBPA exposure in RSV-infected mice by limiting excess cellular immune responses.