A propolis-derived small molecule ameliorates metabolic syndrome in obese mice by targeting the CREB/CRTC2 transcriptional complex

The molecular targets and mechanisms of propolis ameliorating metabolic syndrome are not fully understood. Here, we report that Brazilian green propolis reduces fasting blood glucose levels in obese mice by disrupting the formation of CREB/CRTC2 transcriptional complex, a key regulator of hepatic gluconeogenesis. Using a mammalian two-hybrid system based on CREB-CRTC2, we identify artepillin C (APC) from propolis as an inhibitor of CREB-CRTC2 interaction. Without apparent toxicity, APC protects mice from high fat diet-induced obesity, decreases fasting glucose levels, enhances insulin sensitivity and reduces lipid levels in the serum and liver by suppressing CREB/CRTC2-mediated both gluconeogenic and SREBP transcriptions. To develop more potential drugs from APC, we designed and found a novel compound, A57 that exhibits higher inhibitory activity on CREB-CRTC2 association and better capability of improving insulin sensitivity in obese animals, as compared with APC. In this work, our results indicate that CREB/CRTC2 is a suitable target for developing anti-metabolic syndrome drugs.

Molecular Insights into the Antistress Potentials of Brazilian Green Propolis Extract and Its Constituent Artepillin C

Propolis, also known as bee-glue, is a resinous substance produced by honeybees from materials collected from plants they visit. It contains mixtures of wax and bee enzymes and is used by bees as a building material in their hives and by humans for different purposes in traditional healthcare practices. Although the composition of propolis has been shown to depend on its geographic location, climatic zone, and local flora; two largely studied types of propolis: (i) New Zealand and (ii) Brazilian green propolis have been shown to possess Caffeic Acid Phenethyl Ester (CAPE) and Artepillin C (ARC) as the main bioactive constituents, respectively. We have earlier reported that CAPE and ARC possess anticancer activities, mediated by abrogation of mortalin-p53 complex and reactivation of p53 tumor suppressor function. Like CAPE, Artepillin C (ARC) and the supercritical extract of green propolis (GPSE) showed potent anticancer activity. In this study, we recruited low doses of GPSE and ARC (that did not affect either cancer cell proliferation or migration) to investigate their antistress potential using in vitro cell based assays. We report that both GPSE and ARC have the capability to disaggregate metal- and heat-induced aggregated proteins. Metal-induced aggregation of GFP was reduced by fourfold in GPSE- as well as ARC-treated cells. Similarly, whereas heat-induced misfolding of luciferase protein showed 80% loss of activity, the cells treated with either GPSE or ARC showed 60–80% recovery. Furthermore, we demonstrate their pro-hypoxia (marked by the upregulation of HIF-1α) and neuro-differentiation (marked by differentiation morphology and upregulation of expression of GFAP, β-tubulin III, and MAP2). Both GPSE and ARC also offered significant protection against oxidative stress and, hence, may be useful in the treatment of old age-related brain pathologies.

Artepillin C Reduces Allergic Airway Inflammation by Induction of Monocytic Myeloid-Derived Suppressor Cells

Propolis is a natural product produced by bees that is primarily used in complementary and alternative medicine and has anti-inflammatory, antibacterial, antiviral, and antitumoral biological properties. Some studies have reported the beneficial effects of propolis in models of allergic asthma. In a previous study, our group showed that green propolis treatment reduced airway inflammation and mucus secretion in an ovalbumin (OVA)-induced asthma model and resulted in increased regulatory T cells (Treg) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) frequencies in the lungs, two leukocyte populations that have immunosuppressive functions. In this study, we evaluated the anti-inflammatory effects of artepillin C (ArtC), the major compound of green propolis, in the context of allergic airway inflammation. Our results show that ArtC induces in vitro differentiation of Treg cells and monocytic MDSC (M-MDSC). Furthermore, in an OVA-induced asthma model, ArtC treatment reduced pulmonary inflammation, eosinophil influx to the airways, mucus and IL-5 secretion along with increased frequency of M-MDSC, but not Treg cells, in the lungs. Using an adoptive transfer model, we confirmed that the effect of ArtC in the reduction in airway inflammation was dependent on M-MDSC. Altogether, our data show that ArtC exhibits an anti-inflammatory effect and might be an adjuvant therapy for allergic asthma.

Baccharis dracunculifolia extract-loaded chitosan nanoparticles: development, physicochemical characterization and cytotoxicity evaluation/ Extrato de Baccharis dracunculifolia encapsulado em nanopartículas de quitosana: desenvolvimento, caracterização físico-química e avaliação da citotoxicidade

Nesse estudo investigamos a adequabilidade da encapsulação do extrato em acetato de etila do ápice foliar de Baccharis dracunculifolia(BdAA) (Asteraceae), uma planta medicinal brasileira com promissoras aplicações farmacêuticas, em nanopartículas de quitosana (BdAA-Qui). As nanopartículas foram desenvolvidas pelo método de gelificação iônica e caracterizadas em relação ao diâmetro hidrodinâmico médio (DHm), índice de polidispersividade (IP), potencial zeta (PZ), eficiência de encapsulação (EE), com quantificação do Artepillin C, marcador químico do extrato, por CLAE-DAD, morfologia (por microscopia eletrônica de transmissão – MET) e citotoxicidade em linhagem celular de fibroblastos (L929). As BdAA-Qui apresentaram DHm superior às nanopartículas controle (sem o extrato). Esse pode ser considerado o primeiro indício da ocorrência da encapsulação dos constituintes do extrato na matriz polimérica das nanopartículas. Além disso, o IP obtido apresentou valor próximo a de sistemas monodispersos. Essa característica foi comprovada pelas imagens de MET. O valor de PZ positivo (20,4±0,85 mV) é característico da protonação da molécula de quitosana e pode implicar em estabilidade satisfatória das nanopartículas desenvolvidas. Ainda, esse valor foi semelhante àquele obtido para as nanopartículas controle, o que pode sugerir que os constituintes do extrato estejam no interior da nanopartícula e não adsorvidos em sua superfície. Em relação à EE, encontramos um valor elevado (cerca de 85%). Esse dado pode ser devido à interação eletrostática entre a quitosana e o Artepillin C (pka 4.65), o qual encontra-se prioritariamente sob a forma ionizada no pH padronizado para o preparo das nanopartículas (4.7). Por fim, a formulação desenvolvida não alterou a viabilidade das células durante o período de exposição (24 ou 48h). Esse dado preliminar é particularmente importante para garantir a segurança de uso do extrato encapsulado em nanopartículas de quitosana. Pelo exposto, o carreador escolhido mostrou-se adequado para encapsular o extrato BdAA visando futuras aplicações farmacêuticas.

Two Types of PPARγ Ligands Identified in the Extract of Artemisia campestris

The 70% ethanol extract of Artemisia campestris was screened to find PPARγ ligands using the PPARγ ligand-responsive chimera luciferase reporter system. Capillartemisin B was identified as a PPARγ ligand that stimulated lipid accumulation in 3T3-L1 cells. By further purification of PPARγ ligands from a large-scale preparation of the methanol extract of Artemisia campestris, we isolated and identified eupatilin and santaflavone as PPARγ ligands. Weak PPARγ ligand activity of eupatilin or santaflavone in reporter assay was enhanced by a PPARγ antagonist, GW9662, suggesting that santaflavone or eupatilin and GW9662 bound simultaneously to the multiple sub-pockets of the PPARγ ligand-binding domain (LBD) and cooperatively activated PPARγ. Docking simulation suggested that eupatilin binds to the Ω-pocket but not to the AF-2 pocket of Y-shaped PPARγ LBD where artepillin C that differs from capillartemisin B at the C-5′ position without hydroxy group binds. Eupatilin or santaflavone with or without GW9662 did not stimulate lipid accumulation in differentiated 3T3-L1 cells, suggesting that binding of each compound alone or with GW9662 to the Ω-pocket which stimulated the PPARγ-responsive reporter expression was not enough to stimulate lipid accumulation. The PPARγ ligands found in this study have a potential to design the fragment-based drug design of a novel PPARγ ligand that cover the Y-shaped PPARγ LBD.

Artepillin C Inhibits Cell Proliferation by Cell Cycle Arrest at G0/G1 Phase Accompanied by Up-Regulation of p27Kip1 in Human Hepatoma HepG2 Cells

Artepillin C, 3, 5-diprenyl-4-hydroxycinnamic acid, is one of the bioactive constituents in Brazilian propolis. In the present study, the anticarcinogenic activity of this compound was investigated in human hepatoma HepG2 cells. Artepillin C inhibited the cell proliferation in a dose- and time-dependent manner accompanied by G0/G1 phase arrest in the cell cycle. This compound caused a decrease in the phosphorylation levels of the retinoblastoma protein at Ser780 and Ser807/811 and a decrease in the kinase activity of the cyclinD and CDK4 complex without any change in these protein levels. Artepillin C increased the protein level of p27Kip1, known as a CDK inhibitor. This up-regulation was regulated by both the transcriptional and post-transcriptional levels, i.e., the treatment increased the mRNA of p27Kip1 and decreased the proteosome activity. Thus, artepillin C induces cell cycle arrest at G0/G1 phase accompanied by up-regulation of p27Kip1, resulting in the inhibition of cell proliferation in HepG2 cells. This study suggested that artepillin C will be a promising anti-cancer agent against hepatoma cancer.

Przeciwnowotworowe działanie składników propolisu. Cz. 3. Związki o różnej strukturze chemicznej

The paper reviews the research on the antitumor activity of propolis components belonging to different, previously (Part 1 and 2), not discussed chemical groups. The in vitro (human and animal cancer cell lines) and in vivo tests performed showed differentiated cytotoxic activity of the tested compounds (IC50 from 2 to > 100.0 g/ml), including weak, moderate or strong (IC50 = 4.0 μg/ml). The strongest cytotoxic activity was shown by prenylated aromatic acids (artepillin C), isoflavones (mucronulatol) and prenylated benzophenones (nemorosan and plukenethione). The studies also showed a reduction in the degree of neoplastic metastases to various organs under the influence of the test substances (83.0%), as well as inhibition of neoplastic cells (90%) compared to control cultures.