Conceptual functional-by-design optimisation of the antioxidant capacity of trans-resveratrol, quercetin, and chlorogenic acid: Application in a functional tea.

Designing functional foods as delivery systemsmay become a tailored strategy to decrease the risk of noncommunicable diseases. Therefore, this work aims to optimise a combination of t-resveratrol (RES), chlorogenic acid (CHA), and quercetin (QUE) based on antioxidant assays and develop a functional tea formulation enriched with the optimal polyphenol combination (OPM). Experimental results showed that the antioxidant capacity of these compounds is assay- and compound-dependent. A mixture containing 73% RES and 27% QUE maximised the hydroxyl radical scavenging activity and FRAP. OPM upregulated the gene expressions of heme oxygenase-1, superoxide dismutase, and catalase and decreased the reactive oxygen species generation in L929 fibroblasts. Adding OPM (100 mg/L)to a chamomile tea increased FRAP:39%, DPPH:59%; total phenolic content: 57%, iron reducing capacity: 41%, human plasma protection against oxidation: 67%. However, pasteurisation (63 °C/30 min) decreased onlythe DPPH. Combining technology, engineering, and cell biology was effective for functional tea design.

Australian propolis ethanol extract exerts antibacterial activity against methicillin-resistant Staphylococcus aureus by mechanisms of disrupting cell structure, reversing resistance, and resisting biofilm.

The antibacterial activity and mechanisms of Australian propolis ethanol extract (APEE) against methicillin-resistant Staphylococcus aureus (MRSA) were investigated herein. The diameter of inhibition zones (DIZ) of APEE was 19.7 mm, while the minimum inhibition concentration (MIC) and minimum bactericide concentration (MBC) of APEE were both 0.9 mg/mL against the tested strain of MRSA. Nucleic acid leakage and propidium iodide (PI) staining assays showed that APEE can stimulate the release of intracellular nucleic acids by disrupting the integrity of the cell wall and cytoplasmic membrane. Scanning electron microscopy (SEM) further confirmed that APEE could depress cellular activities via damaging the cell structure, including the cell wall and membrane. Western blot analysis and ß-lactamase activity assay showed that APEE could inhibit the expression of PBP2a and reduce the activity of ß-lactamase, suggesting that APEE is able to reverse the drug resistance of MRSA. XTT and crystal violet (CV) assays indicated that APEE had the capacity to prevent the formation of biofilms through decreasing cellular activities and biomass. Bacterial adhesion assay revealed that APEE could reduce the adhesive capacity of the strain, belonging to its antibiofilm mechanisms. Furthermore, nine main compounds of APEE were identified and quantified by HPLC-DAD/Q-TOF-MS. The results above all verified that the antibacterial activity of APEE against MRSA was mainly due to disrupting cell structure, reversing resistance, and resisting biofilm formation, which indicates that APEE is expected to be an efficient functional ingredient with great potential application in the field of medicine and food.

Brazilian Green Propolis Inhibits Ox-LDL-Stimulated Oxidative Stress in Human Umbilical Vein Endothelial Cells Partly through PI3K/Akt/mTOR-Mediated Nrf2/HO-1 Pathway.

Propolis has been widely used as a dietary supplement for its health benefits, including cardiovascular protective effects. The aim of this study was to investigate the cytoprotective effects of Brazilian green propolis (BP) against oxidized low-density lipoprotein (Ox-LDL) induced human umbilical vein endothelial cells (HUVECs) damage. Our results suggested that treatment with BP rescued Ox-LDL-stimulated HUVECs cell viability losses, which might be associated with its inhibitive effects on the cell apoptosis and autophagy. We also noticed that BP restored Ox-LDL-stimulated HUVECs oxidative stress, by induced antioxidant gene expressions, including Heme oxygenase-1 and its upstream mediator, Nrf2, which were mediated by the activation of the phosphorylation of PI3K/Akt/mTOR. Pretreatment with wortmannin, PI3K/AKT inhibitor, abolished BP induced Nrf2 nuclear translocation and HO-1 level. Our results demonstrated that BP protected HUVECs against oxidative damage partly via PI3K/Akt/mTOR-mediated Nrf/HO-1 pathway, which might be applied into preventing Ox-LDL mediated cardiovascular diseases.

Bioactive components and mechanisms of Chinese poplar propolis alleviates oxidized low-density lipoprotein-induced endothelial cells injury.

BACKGROUND: Propolis, a polyphenol-rich natural product, has been used as a functional food in anti-inflammation. However, its bioactive components and mechanisms have not been fully elucidated. To discover the bioactive components and anti-inflammatory mechanism, we prepared and separated 8 subfractions from ethyl acetate extract of Chinese propolis (EACP) and investigated the mechanism in oxidized low density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) damage. METHODS: Eight subfractions were prepared and separated from ethyl acetate extract of Chinese propolis (EACP) with different concentrations of methanol-water solution, and analysed its chemical constituents by HPLC-DAD/Q-TOF-MS. Then 80% confluent HUVECs were stimulated with 40 µg/mL ox-LDL. Cell viability and apoptosis were evaluated by Sulforhodamine B (SRB) assay and Hoechst 33,258 staining, respectively. Levels of caspase 3, PARP, LC3B, p62, p-mTOR, p-p70S6K, p-PI3K, p-Akt, LOX-1 and p-p38 MAPK were assessed by western blotting and immunofluorescence assay, respectively. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with fluorescent probes. RESULTS: Each subfraction exhibited similar protective effect although the contents of chemical constituents were different. EACP attenuated ox-LDL induced HUVECs apoptosis, depressed the ratio of LC3-II/LC3-I and enhanced the p62 level. In addition, treatment with EACP also activated the phosphorylation of PI3K/Akt/mTOR, and deactivated the level of LOX-1 and phosphorylation of p38 MAPK. The overproduction of ROS and the damage of MMP were also ameliorated after ECAP treatment. CONCLUSIONS: These findings indicated that the bioactive component of propolis on anti-inflammatory activity was not determined by a single constituent, but a complex interaction including flavonoids, esters and phenolic acids. EACP attenuated ox-LDL induced HUVECs injury by inhibiting LOX-1 level and depressed ROS production against oxidative stress in ox-LDL induced HUVECs, further to activate PI3K/Akt/mTOR pathway and deactivate p38 MAPK to inhibit apoptosis and autophagy, which provide novel insights into the potential application of propolis on modulating chronic inflammation.

Ethanol extract of propolis and its constituent caffeic acid phenethyl ester inhibit breast cancer cells proliferation in inflammatory microenvironment by inhibiting TLR4 signal pathway and inducing apoptosis and autophagy.

BACKGROUND: Propolis and its major constituent - caffeic acid phenethyl ester (CAPE) have good abilities on antitumor and anti-inflammation. However, little is known about the actions of propolis and CAPE on tumor in inflammatory microenvironment, and inflammatory responses play decisive roles at different stages of tumor development. To understand the effects and mechanisms of ethanol-extracted Chinese propolis (EECP) and its major constituent - CAPE in inflammation-stimulated tumor, we investigated their effects on Toll-like receptor 4 (TLR4) signaling pathway which plays a crucial role in breast cancer MDA-MB-231 cell line. METHODS: 80% confluent breast cancer MDA-MB-231 cells were stimulated with 1 µg/mL lipopolysaccaride (LPS). Then the cells were divided for treatment by CAPE (25 µg/mL) and EECP (25, 50 and 100 µg/mL), respectively. Cell viability, nitric oxide (NO) production and cell migration were measured by sulforhodamine B assay, chemical method and scratch assay. The levels of TLR4, MyD88, IRAK4, TRIF, caspase 3, PARP, LC3B and p62 were investigated through western blotting. The expression of TLR4, LC3B and nuclear factor-κB p65 (NF-κB p65) were tested by immunofluorescence microscopy assay. RESULTS: Treatment of different concentrations of EECP (25, 50 and 100 µg/mL) and CAPE (25 µg/mL) significantly inhibited LPS-stimulated MDA-MB-231 cell line proliferation, migration and NO production. Furthermore, EECP and CAPE activated caspase3 and PARP to induce cell apoptosis, and also upregulated LC3-II and decreased p62 level to induce autophagy during the process. TLR4 signaling pathway molecules such as TLR4, MyD88, IRAK4, TRIF and NF-κB p65 were all down-regulated after EECP and CAPE treatment in LPS-stimulated MDA-MB-231 cells. CONCLUSIONS: These findings indicated that EECP and its major constituent - CAPE inhibited breast cancer MDA-MB-231 cells proliferation in inflammatory microenvironment through activating apoptosis, autophagy and inhibiting TLR4 signaling pathway. EECP and CAPE may hold promising prospects in treating inflammation-induced tumor.

Bioactive Components of Chinese Propolis Water Extract on Antitumor Activity and Quality Control.

To understand the material basis of antitumor activity of Chinese propolis water extract (CPWE), we developed a simple and efficient method using macroporous absorptive resin coupled with preparative high performance liquid chromatography and separated and purified eleven chemical components (caffeic acid, ferulic acid, isoferulic acid, 3,4-dimethoxycinnamic acid, pinobanksin, caffeic acid benzyl ester, caffeic acid phenethyl ester, apigenin, pinocembrin, chrysin, and galangin) from CPWE; then we tested the antitumor activities of these eleven components using different human tumor cell lines (MCF-7, MDA-MB-231, HeLa, and A549). Furthermore, cell migration, procaspase 3 level, and reactive oxygen species (ROS) of effective components from CPWE were investigated. Our data showed that antitumor activities of the eleven components from CPWE were different from each other. CPWE and its effective components induced apoptosis by inhibiting tumor cell migration, activating caspase 3, and promoting ROS production. It can be deduced that the antitumor effects of propolis did not depend on a single component, and there must exist "bioactive components," which also provides a new idea for Chinese propolis quality control.

Anti-inflammatory effects of ethanol extracts of Chinese propolis and buds from poplar (Populus×canadensis).

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis is used widely in a number of cultures as a folk medicine and is gaining wider recognition for its potential therapeutic use, due to its wide range of biological properties and pharmacological activities, especially its anti-inflammatory effects. Despite an increasing number of studies focused on the biological activities of propolis together with its botanical sources, studies on Chinese propolis are insufficient. This study was designed to investigate the anti-inflammatory properties of ethanol extracts from Chinese propolis (EECP) and poplar buds (EEPB) from Populus×canadensis Moench (Salicaceae family). MATERIALS AND METHODS: Phytochemical analysis of EECP and EEPB was performed via total phenolic and flavonoid content measurements followed by high-performance liquid chromatography (HPLC) analysis. DPPH and ABTS free-radical scavenging methods were used to evaluate their anti-oxidant properties. The anti-inflammatory effects of EECP and EEPB were investigated in vitro by evaluating their modulating effects on the key inflammatory cytokines and mediators in LPS/IFN-γ co-stimulated RAW 264.7 cells and by measuring nuclear factor (NF)-κB activation in TNF-α or IL-1ß stimulation HEK 293 cells using reporter gene assays. Their effects on acute inflammatory symptoms (LPS-induced endotoxemia and acute pulmonary damage) were also examined in mice. RESULTS: EECP and EEPB exhibited strong free-radical scavenging activity and significant in vitro anti-inflammatory effects by modulating key inflammatory mediators of mRNA transcription, inhibiting the production of specific inflammatory cytokines, and blocking the activation of nuclear factor (NF)-κB. The administration of EECP and EEPB (25 and 100 mg/kg) provided significant protective effects by attenuating lung histopathological changes and suppressing the secretion of LPS-stimulated inflammatory cytokines, such as interleukin-6 (IL-6), IL-10, MCP-1, TNF-α and IL-12p70 production in endotoxemic mice. CONCLUSIONS: The results presented here reveal the potent anti-inflammatory properties of Chinese propolis and poplar buds, and provide biological information for developing suitable substitute(s) for propolis in the prevention and treatment of inflammatory diseases.

Antitumor Activity of Chinese Propolis in Human Breast Cancer MCF-7 and MDA-MB-231 Cells.

Chinese propolis has been reported to possess various biological activities such as antitumor. In present study, anticancer activity of ethanol extract of Chinese propolis (EECP) at 25, 50, 100, and 200 µ g/mL was explored by testing the cytotoxicity in MCF-7 (human breast cancer ER(+)) and MDA-MB-231 (human breast cancer ER(-)) cells. EECP revealed a dose- and time-dependent cytotoxic effect. Furthermore, annexin A7 (ANXA7), p53, nuclear factor- κ B p65 (NF- κ B p65), reactive oxygen species (ROS) levels, and mitochondrial membrane potential were investigated. Our data indicated that treatment of EECP for 24 and 48 h induced both cells apoptosis obviously. Exposure to EECP significantly increased ANXA7 expression and ROS level, and NF- κ B p65 level and mitochondrial membrane potential were depressed by EECP dramatically. The effects of EECP on p53 level were different in MCF-7 and MDA-MB-231 cells, which indicated that EECP exerted its antitumor effects in MCF-7 and MDA-MB-231 cells by inducing apoptosis, regulating the levels of ANXA7, p53, and NF- κ B p65, upregulating intracellular ROS, and decreasing mitochondrial membrane potential. Interestingly, EECP had little or small cytotoxicity on normal human umbilical vein endothelial cells (HUVECs). These results suggest that EECP is a potential alternative agent on breast cancer treatment.

Propolis Reduces Phosphatidylcholine-Specific Phospholipase C Activity and Increases Annexin a7 Level in Oxidized-LDL-Stimulated Human Umbilical Vein Endothelial Cells.

To understand the mechanisms underlying the regulating dyslipidemia action of Chinese propolis and Brazilian green propolis, we investigated their effects on phosphatidylcholine-specific phospholipase C (PC-PLC) activity and annexin a7 (ANXA7) level which play crucial roles in the control of the progress of atherosclerosis. Furthermore, active oxygen species (ROS) levels, nuclear factor-KappaB p65 (NF- κ B p65), and mitochondrial membrane potential (MMP) were also investigated in oxidized-LDL- (ox-LDL-) stimulated human umbilical vein endothelial cells (HUVECs). Our data indicated that the treatment of both types of propolis 12.5 µ g/mL significantly increased cell viability and attenuated apoptosis rate, increased ANXA7 level, and decreased PC-PLC activity. Both types of propolis also inhibited ROS generation as well as the subsequent MMP collapse, and NF- κ B p65 activation induced by ox-LDL in HUVECs. Our results also indicated that Chinese propolis and Brazilian green propolis had similar biological activities and prevented ox-LDL induced cellular dysfunction in HUVECs.

Molecular mechanisms underlying the in vitro anti-inflammatory effects of a flavonoid-rich ethanol extract from chinese propolis (poplar type).

China produces the greatest amount of propolis but there is still lack of basic studies on its pharmacological mechanisms. Our previous study found that ethanol extract from Chinese propolis (EECP) exerted excellent anti-inflammatory effects in vivo but mechanisms of action were elusive. To further clarify the possible mechanisms underlying the anti-inflammatory effects of Chinese propolis (poplar type), we utilized EECP to analyze its chemical composition and evaluated its potential anti-inflammatory effects in vitro. High-performance liquid chromatography (HPLC) profile indicated that EECP contained abundant flavonoids, including rutin, myricetin, quercetin, kaempferol, apigenin, pinocembrin, chrysin, and galangin. Next we found that EECP could significantly inhibit the production of NO, IL-1ß, and IL-6 in lipopolysaccharide- (LPS-) stimulated RAW 264.7 cells and suppress mRNA expression of iNOS, IL-1ß, and IL-6 in a time- and dose-dependent manner. Furthermore, we found that EECP could suppress the phosphorylation of IκBα and AP-1 but did not affect IκBα's degradation. In addition, using a reporter assay, we found that EECP could block the activation of NF-κB in TNF-α-stimulated HEK 293T cells. Our findings give new insights for understanding the mechanisms involved in the anti-inflammatory effects by Chinese propolis and provide additional references for using propolis in alternative and complementary therapies.

Effects of encapsulated propolis on blood glycemic control, lipid metabolism, and insulin resistance in type 2 diabetes mellitus rats.

The present study investigates the encapsulated propolis on blood glycemic control, lipid metabolism, and insulin resistance in type 2 diabetes mellitus (T2DM) rats. The animal characteristics and biological assays of body weight, fasting blood glucose (FBG), fasting serum insulin (FINS), insulin act index (IAI), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured and euglycemic hyperinsulinemic glucose clamp technique were used to determine these effects. Our findings show that oral administration of encapsulated propolis can significantly inhibit the increasing of FBG and TG in T2DM rats and can improve IAI and M value in euglycemic hyperinsulinemic clamp experiment. There was no significant effects on body weight, TC, HDL-C, and LDL-C in T2DM rats treated with encapsulated propolis. In conclusion, the results indicate that encapsulated propolis can control blood glucose, modulate lipid metabolism, and improve the insulin sensitivity in T2DM rats.

Inhibitory effect of chinese propolis on phosphatidylcholine-specific phospholipase C activity in vascular endothelial cells.

To understand the mechanisms underlying the anti-inflammatory action of Chinese propolis, we investigated its effect on the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) that plays critical roles in control of vascular endothelial cell (VEC) function and inflammatory responses. Furthermore, p53 and reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) were investigated. Our data indicated that treatment of Chinese propolis 6.25 and 12.5 µg/ml for 12 hours increased VEC viability obviously. Exposure to Chinese propolis 6.25, 12.5, and 25 µg/ml for 6 and 12 hours significantly decreased PC-PLC activity and p53 level, and ROS levels were depressed by Chinese propolis 12.5 µg/ml and 25 µg/ml dramatically. The Δψm of VECs was not affected by Chinese propolis at low concentration but disrupted by the propolis at 25 µg/ml significantly, which indicated that Chinese propolis depressed PC-PLC activity and the levels of p53 and ROS in VECs but disrupted Δψm at a high concentration.