Brazilian Green Propolis Research Articles

Brazilian green propolis suppresses microglia-mediated neuroinflammation by inhibiting NF-kB activation

Brazilian green propolis suppresses microglia-mediated neuroinflammation by inhibiting NF-kB activation

Brazilian green propolis extracts obtained by conventional processes and by processes at high pressure with supercritical carbon dioxide, ethanol and water

Extracts of green propolis were obtained by conventional process at atmospheric pressure and at high pressure using supercritical carbon dioxide (scCO2) as solvent and, ethanol and water and its mixtures as solvents or as CO2 co-solvents. All extracts were evaluated with regards to the total phenol content, total flavonoids, Artepillin C, p-Coumaric Acid and Kaempferide and also was evaluated the antioxidant activity, and in vitro antimicrobial activity. The highest overall extraction yields were 53.5% in the three-step sequential extraction at high pressure and 44.7% at low pressure in a Soxhlet extractor. All extracts showed high antioxidant activity and presented antimicrobial activity. The highest levels of phenols of 222mg GAE/g extract, of flavonoids of 67.0mg CE/g extract, of artepillin C of 62.4mg Artepillin C/g extract and high antioxidant activity were obtained in the ethanolic extract (80%) of the second stage of the two-step sequential process.

Mechanisms underlying the cytotoxic effect of propolis on human laryngeal epidermoid carcinoma cells

Propolis has been used as a traditional remedy for centuries because of its beneficial effects, including anticancer properties. The aim of this study was to compare the cytotoxic mechanism of Cuban red propolis (CP) and Brazilian green propolis (BP) on human laryngeal carcinoma (HEp-2) cells. Cell viability, leakage of lactate dehydrogenase, fluorescence staining, mitochondrial membrane potential (ΔΨm) and the expression of pro/anti-apoptotic genes were assessed. Cell viability and cytotoxic assays suggested a dose-dependent effect of CP and BP extracts with a possible association of intracellular reactive oxygen species production and decreased ΔΨm. Both samples induced apoptosis via activation of TP53, CASP3, BAX, P21 signalling, and downregulation of BCL2 and BCL-XL. CP exerted a higher cytotoxic effect than BP extract. Our findings suggest further investigation of the main components of each propolis sample, what may lead to the development of strategies for the treatment of laryngeal cancer.

The effects of Brazilian green propolis that contains flavonols against mutant copper-zinc superoxide dismutase-mediated toxicity

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. The purpose of this study was to clarify effects of brazilian green propolis and the active ingredient against ALS-associated mutant copper-zinc superoxide dismutase (SOD1)-mediated toxicity. Ethanol extract of brazilian green propolis (EBGP) protected N2a cells against mutant SOD1-induced neurotoxicity and reduced aggregated mutant SOD1 by induction of autophagy. Kaempferide and kaempferol, the active ingredients of EBGP, also inhibited mutant SOD1-induced cell death and reduced the intracellular mutant SOD1 aggregates. Both kaempferide and kaempferol significantly suppressed mutant SOD1-induced superoxide in mitochondria. Western blot analysis showed that kaempferol potentially induced autophagy via the AMP-activated protein kinase (AMPK) - the mammalian target of rapamycin (mTOR) pathway. These results suggest that EBGP containing the active ingredient against mutant SOD1-mediated toxicity is a promising medicine or health food for prevention and treatment of ALS.

Identification of Free Radical Scavengers from Brazilian Green Propolis Using Off-Line HPLC-DPPH Assay and LC-MS.

Brazilian green propolis is known as an appreciable natural antioxidant with abundant polyphenolic compounds. For quality control, a fingerprint-efficacy study of Brazilian green propolis was carried out in this work. Chemical fingerprints of Brazilian green propolis from 22 different sources were determined by HPLC and investigated by similarity analysis. The fingerprint-efficacy relationships between chemical fingerprint and DPPH radical-scavenging activity were established. The results showed that 14 characteristic common peaks were identified, and 9 compounds were discovered with free radical-scavenging activities. Caffeoylquinic acids and artepillin C might be the major effective components for quality control of Brazilian green propolis due to their specificity and strong antioxidant activity. This study provides new markers for the quality assessment of Brazilian green propolis and its derived products.

Anti-Inflammatory Properties of Brazilian Green Propolis Encapsulated in a γ-Cyclodextrin Complex in Mice Fed a Western-Type Diet.

Ageing is often accompanied by chronic inflammation. A fat- and sugar-rich Western-type diet (WTD) may accelerate the ageing phenotype. Cell culture studies have indicated that artepillin C-containing Brazilian green propolis exhibits anti-inflammatory properties. However, little is known regarding its anti-inflammatory potential in mouse liver in vivo. In this study, female C57BL/6NRj wild-type mice were fed a WTD, a WTD supplemented with Brazilian green propolis supercritical extract (GPSE) encapsulated in γ-cyclodextrin (γCD) or a WTD plus γCD for 10 weeks. GPSE-γCD did not affect the food intake, body weight or body composition of the mice. However, mRNA levels of the tumour necrosis factor α were significantly downregulated (p < 0.05) in these mice compared to those in the WTD-fed controls. Furthermore, the gene expression levels of other pro-inflammatory markers, including serum amyloid P, were significantly (p < 0.001) decreased following GPSE-γCD treatment. GPSE-γCD significantly induced hepatic ferritin gene expression (p < 0.01), which may contribute to its anti-inflammatory properties. Conversely, GPSE-γCD did not affect the biomarkers of endogenous antioxidant defence, including catalase, glutathione peroxidase-4, paraoxonase-1, glutamate cysteine ligase and nuclear factor erythroid 2-related factor-2 (Nrf2). Overall, the present data suggest that dietary GPSE-γCD exhibits anti-inflammatory, but not antioxidant activity in mouse liver in vivo. Thus, GPSE-γCD has the potential to serve as a natural hepatoprotective bioactive compound for dietary-mediated strategies against chronic inflammation.

Brazilian Green Propolis Extract Synergizes with Protoporphyrin IX-mediated Photodynamic Therapy via Enhancement of Intracellular Accumulation of Protoporphyrin IX and Attenuation of NF-κB and COX-2.

Brazilian green propolis (BGP) is noted for its impressive antitumor effects and has been used as a folk medicine in various cultures for many years. It has been demonstrated that BGP could enhance the cytotoxic effect of cytostatic drugs on tumor cells. Photodynamic therapy (PDT) is a therapeutic approach used against malignant cells. To assess the synergistic effect of BGP extract on protoporphyrin IX (PpIX)-mediated photocytotoxicity, MTT assays were performed using A431 and HeLa cells. TUNEL assay and Annexin V-FITC/PI staining were performed to confirm the induction of apoptosis. Western blotting analysis was performed to examine the pro-apoptotic proteins, anti-apoptotic proteins and inflammation related proteins in A431 cells. Intracellular accumulation of PpIX was examined by flow cytometry. The synergistic effect of BGP extract in PpIX-PDT was also evaluated with a xenograft model. Our findings reveal that BGP extract increased PpIX-mediated photocytotoxicity in A431 and HeLa cells. PpIX-PDT with BGP extract treatment resulted in a decrease in Bcl-xL and an increase in NOXA, Bax and caspase-3 cleavage. The protein expression levels of p-IKKα/β, NF-κB and COX-2 were upregulated by PpIX-PDT but significantly attenuated when in combination with BGP extract. BGP extract was also found to significantly enhance the intracellular accumulation of PpIX in A431 cells. BGP extract increased PpIX-mediated photocytotoxicity in a xenograft model as well. Our findings provide evidence for a synergistic effect of BGP extract in PpIX-PDT both in vitro and in vivo.

Physicochemical Characteristics of Brazilian Green Propolis Evaluated During a Six-Year Period

Propolis has been used as a natural health product mainly due to the presence of polyphenols, flavonoids, phenolic aldehydes, amino acids, vitamins and others bioactive constituents. To this natural substance are attributed different biological and pharmacological properties which are influenced by its chemical composition and organoleptic properties. The aim of this work was to evaluate the physicochemical properties and parameters of green propolis collected during a period of six years (2008-2013) in the state of Minas Gerais, located at the southeastern region of Brazil. The methodology were in accordance with Brazilian legislation on the identity and quality standards of propolis. The evaluated parameters of hydroalcoholic from green propolis were total flavonoids, antioxidant activity - DPPH method, oxidation index, wax content, humidity and insoluble impurities. Propolis samples collected in different seasons during the years 2008 to 2013 presented mean values of total flavonoids (3.4 ± 0.11 mg/g), antioxidant activity DPPH (4.76 ± 0.16 μg/mL), oxidation index (3, 4 ± 0.33 seconds) and wax (15.14 ± 0.78% m/m), which are in accordance with Brazilian legislation. Green propolis did not show abrupt seasonal changes during the six years of investigation, and may be considered as an adequate functional ingredient.

Artepillin C, is it a good marker for quality control of Brazilian green propolis?

As commercialisation of Brazilian green propolis is going on, quality evaluation and authenticity are important. The result demonstrated that artepillin C found by far in Brazilian green propolis by HPLC-ESI-MS/MS analysis, while a small interferent may be mistaken as artepillin C in some propolis from China. A new HPLC quality control method as artepillin C for marker was developed, which is the primary assessment criteria for quality control of Brazilian green propolis.

Chemopreventive Action by Ethanol-extracted Brazilian Green Propolis on Post-initiation Phase of Inflammation-associated Rat Colon Tumorigenesis.

Propolis has since long been utilized in numerous folk medicines with a variety of medicinal properties. In this study, the effects of ethanol-extracted (EEP) and water-extracted (WEP) Brazilian green propolis on the post-initiation phase of inflammation-associated rat colon tumorigenesis were directly compared. Male F344 rats at 6 weeks of age were subcutaneously injected with 1,2-dimethylhydrazine (DMH) at 40 mg/kg body weight twice during the first week, followed by 1% dextran sodium sulfate (DSS) in drinking water for one week. After a 1-week no-treatment period, animals were administered either basal Oriental MF powdered diet, or 1% EEP or 1% WEP in the basal diet until week 32. Post-initiation treatment with EEP significantly reduced the multiplicity of colorectal carcinomas compared to the control (0.40±0.13/rat vs. 2.29±0.84/rat, respectively, p<0.05), and EEP also reduced the tumor volume. Immunohistochemically, expression of inflammation-associated proteins inducible nitric oxide synthase, tumor necrotic factor alpha, nuclear factor kappa B and glutathione peroxidase-2 were significantly diminished in colorectal tumors from EEP-treated rats. Suppression of inflammation and oxidative stress, which had been triggered by DMH and promoted by DSS, was a primary mechanism by which EEP suppressed carcinogenesis.

New propolis type from north-east Brazil: chemical composition, antioxidant activity and botanical origin.

Propolis is a bee product with wide diversity of biological activity. It has a complex composition, which is dependent on its botanical source. The present study aimed to determine the chemical profile, antioxidant activity and botanical origin of two samples of a propolis type from two locations of the state of Rio Grande do Norte (RN, north-east Brazil). The standard chemical characteristics of the RN propolis are similar or superior to the internationally marketed Brazilian green propolis. RN propolis from two locations have high antioxidant activity, corresponding to 10% (municipality of Afonso Bezerra) and 13% (municipality of Alto do Rodrigues) of quercetin activity by the 2,2-diphenyl-1-picrylhydrazyl method and to 15% (both locations) by the β-carotene discoloration method. High-performance liquid chromatography with diode array detection (HPLC-DAD)-electrospray ionization-tandem mass spectrometry analyses revealed that most constituents of the RN propolis are flavonoids, mainly flavonols and chalcones. HPLC-DAD analysis of ethanol extracts revealed a great similarity between the chemical profile of RN propolis and shoot apices of 'jurema-preta' (Mimosa tenuiflora, Leguminosae, Mimosoideae). 'Jurema-preta' shoot apices are likely resin sources of RN propolis. The chemical characteristics and antioxidant property of RN propolis provide promising prospects for the introduction of this type of propolis into the apicultural market. © 2017 Society of Chemical Industry.

In Vitro Metabolism of Artepillin C by Rat and Human Liver Microsomes.

Artepillin C, a natural product present in the Brazilian green propolis, has several biological properties. Among these properties, the antitumor action of this product is noteworthy and makes it a promising drug candidate for the treatment of several types of cancer. This paper describes the in vitro metabolism of Artepillin C in rat and human liver microsomes. The rat model suggested a sigmoidal profile for the metabolism, adapted to the Hill's kinetic model. The enzymatic kinetic parameters were as follows: maximal velocity = 0.757 ± 0.021 µmol/mg protein/min, Hill coefficient = 10.90 ± 2.80, and substrate concentration at which half-maximal velocity of a Hill enzyme is achieved = 33.35 ± 0.55 µM. Based on these results, the calculated in vitro intrinsic clearance for Artepillin C was 16.63 ± 1.52 µL/min/mg protein. The in vitro metabolism assay conducted on the human model did not fit any enzymatic kinetic model. Two novel metabolites were formed in both mammal microsomal models and their chemical structures were elucidated for the first time. The main human cytochrome P450 isoforms involved in Artepillin C metabolism had been identified, and the results suggest a majority contribution of CYP2E1 and CYP2C9 in the formation of the two metabolites.

The Neuroprotective Effects of Brazilian Green Propolis on Neurodegenerative Damage in Human Neuronal SH-SY5Y Cells.

Oxidative stress and synapse dysfunction are the major neurodegenerative damage correlated to cognitive impairment in Alzheimer's disease (AD). We have found that Brazilian green propolis (propolis) improves the cognitive functions of mild cognitive impairment patients living at high altitude; however, mechanism underlying the effects of propolis is unknown. In the present study, we investigated the effects of propolis on oxidative stress, expression of brain-derived neurotrophic factor (BDNF), and activity-regulated cytoskeleton-associated protein (Arc), the critical factors of synapse efficacy, using human neuroblastoma SH-SY5Y cells. Pretreatment with propolis significantly ameliorated the hydrogen peroxide- (H2O2-) induced cytotoxicity in SH-SY5Y cells. Furthermore, propolis significantly reduced the H2O2-generated reactive oxygen species (ROS) derived from mitochondria and 8-oxo-2′-deoxyguanosine (8-oxo-dG, the DNA oxidative damage marker) but significantly reversed the fibrillar β-amyloid and IL-1β-impaired BDNF-induced Arc expression in SH-SY5Y cells. Furthermore, propolis significantly upregulated BDNF mRNA expression in time- and dose-dependent manners. In addition, propolis induced Arc mRNA and protein expression via phosphoinositide-3 kinase (PI3K). These observations strongly suggest that propolis protects from the neurodegenerative damage in neurons through the properties of various antioxidants. The present study provides a potential molecular mechanism of Brazilian green propolis in prevention of cognitive impairment in AD as well as aging.

Chronic Intake of Green Propolis Negatively Affecting the Rat Testis.

Background:Human and animal evidence suggests that environmental toxicants may have an adverse impact on male reproductive health, reducing the population's reproductive output. Owing to the renewed attraction for natural products, some of them constitute effective alternatives to mitigate these effects. Propolis is a candidate for this use because of its intrinsic properties. In many situations, it improved the testicular damage and alleviated the toxic effects induced by environmental contaminant exposure.Objective:The aim of this study was to investigate possible alterations of testicular parameters and certify if its use is really advantageous to the testis, since this could affect rat reproductive function.Materials and Methods:Forty-eight adult male Wistar rats were divided into four groups (Co = control, T1 = 3 mg propolis/kg/day, T2 = 6 mg/kg/day, T3 = 10 mg/kg/day) and were exposed during 56 days. The testes were assessed with morphometrical, stereological, and ultrastructural analyses. Cell proliferation and death were diagnosed, respectively, by immunocytochemistry. Connexin 43 (Cx43) and N-cadherin transcript levels were determined by reverse transcription-polymerase chain reaction.Results:Increased cell proliferation and Leydig cell volume were observed in T2, and in contrast, Cx43 upregulation and cell death were observed in T3. Both T2 and T3 showed ultrastructural abnormalities in testicular parenchyma.Conclusion:We recommend a cautious intake of propolis to avoid deleterious effects.SUMMARY Chronic intake of Brazilian green propolis induced N.-cadherin downregulation and decreased on seminiferous tubule volumeIncrease on connexin 43 expression and cell death and decrease in Leydig cell.(LC) number/testis with the concentration of 10 mg/kg/day were observedIncrease on cell proliferation, cytoplasmic proportion, and volume of LC with the concentration of 6 mg/kg/day was detectedThe presence of empty spaces between spermatids and malformed spermatozoa in the lumen of seminiferous tubule was showedThis male reproductive disruption can be linked to phenolic compounds present in Brazilian green propolis. Abbreviation Used: AEC: 3-amino-9-ethylcarbazole; AJ: Adherens junction; AME: Aromadendrin-40-methyl ether; CAPE: Caffeic acid phenethyl ester; Co: Control group; C×43: Connexin 43; DAB: Diaminobenzidine; dNTP: Deoxyribonucleotide phosphate; DSP: Daily sperm production; FA: Ferulic acid; FSH: Follicle-stimulating hormone; GJ: Gap junction; GJIC: Gap junction intercellular communication; HPLC: High-performance liquid chromatography; LC: Leydig cell; LH: Luteinizing hormone; N-cad: N-cadherin; PCNA: Proliferating cell nuclear antigen; PCR: Polymerase chain reaction; RT-PCR: Reverse transcription-polymerase chain reaction; SDM: Standard deviation of mean; T1: Group exposed to 3 mg of propolis/kg/day; T2: Group exposed to 6 mg of propolis/kg/day; T3: Group exposed to 10 mg of propolis/kg/day; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; WB-ras 2 cells: Ras-transformed rat liver epithelial cell line.

Phenolic composition and antioxidant activity assessment of southeastern and south Brazilian propolis

Propolis is a resinous substance collected by honey bees Apis mellifera from several plant sources and used in the hive to seal the walls, to strengthen the ends of the honey comb or embalm dead invaders. The chemical specificity of propolis is directly determined by the variability of the plant origins and by geographical and climatic features of the collection site. The aim of this work was the quality assessment of 16 south and southeast Brazilian propolis samples through the identification and quantification of phenolic compounds using chromatographic and spectroscopic techniques such as HPLC and LC/DAD/ESI-MSn. Generally, the samples presented a phenolic profile related to Brazilian green propolis with origin in Baccharis spp. leaves, where the caffeoylquinic acid derivatives as well as dihydrokaempferide and artepillin C were the main compounds. Moreover, DPPH• free radical-scavenging activity, reducing power and differential pulse voltammetry were applied to evaluate the antioxidant activity. Differential pulse voltammetry proved to be a rapid and easy tool for the quantification of the total electroactive species present in the samples. The results revealed a richer phenolic composition and higher bioactivity in Minas Gerais samples rather than the southern ones.

Brazilian Green Propolis Promotes Weight Loss and Reduces Fat Accumulation in C57BL/6 Mice Fed A High-Fat Diet.

Propolis is a bee product with various biological properties. C57BL/6 mice were fed a high-fat diet and treated with propolis for 14 weeks. Body weight in mice treated with 2% propolis was less than that in control mice from 3 weeks after the start of treatment until 14 weeks except for the 7th week. Mice treated with propolis showed significantly lower epididymal fat weight and subcutaneous fat weight. Infiltration of epididymal fat by macrophages and T cells was reduced in the propolis group. Supplementation of propolis increased feces weight and fat content in feces, suggesting that mechanisms of weight reduction by propolis partly include a laxative effect and inhibition of fat absorption.

16 Discrimination of Brazilian green propolis and Chinese propolis based on high-performance liquid chromatographic fingerprints and multivariate statistical analysis

Objectives The determination of chemical components is usually used in the quality control of propolis. However, chemical components from different types of propolis are similar. The objective of this investigation was to establish a method based on a specific chemical fingerprint profile and a multivariate mixed model statistical analysis which could easily distinguish propolis of different origins and promote the quality control of propolis. Methods A novel approach using high performance liquid chromatography (HPLC) coupled with multivariate statistical analysis was established for profiling and distinguishing Chinese and Brazilian green propolis. A batch of 22 propolis samples was analyzed, and the datasets on retention time, peak area and sample codes were subjected to mixed multivariate statistical analysis consisting of principal component analysis (PCA) and a self-organization mapping net (SOM). Results The fingerprints were profiled. PCA score plots showed Chinese and Brazilian green propolis clearly classified into two groups. The visualized SOM results showed data from the two groups projected to the adjacent neurons clearly separated from each other. Artepillin C, which contributed greatly to the differentiation, was screened out and identified as the reference compound. Artepillin C is the characteristic component in Brazilian propolis which can be used as chemical marker to distinguish propolis of different origins. Conclusions In this study, fingerprints coupled with multivariate statistical analysis have been successfully applied to distinguish Chinese from Brazilian green propolis. The research identified a chemical marker, and thus helps to investigate and promote the quality control of propolis.

Fouling evaluation on nanofiltration for concentrating phenolic and flavonoid compounds in propolis extract

Nanofiltration is useful to concentrate propolis extract. During the selection of membrane, both compound rejection and permeate flux are important indicators of process economy. Brazilian green propolis extract was studied to evaluate the separation performance of Startmen 122 and NF270 membranes. Compared to Starmen 122, NF270 membrane showed better rejection of bioactive compounds. The flux decline patterns were further studied using Hermia's model. Cake formation is the major fouling mechanism on the hydrophobic surface of Starmen 122. While the fouling mechanism for NF270 is pore blocking. The fouled membranes were further characterized using SEM and FT-IR to confirm on the predicted fouling mechanisms.

Ethanol-Extracted Brazilian Propolis Exerts Protective Effects on Tumorigenesis in Wistar Hannover Rats.

The present study was conducted over a course of 104 weeks to estimate the carcinogenicity of ethanol-extracted Brazilian green propolis (EEP). Groups of 50 male and 50 female Wistar Hannover rats, 6-week-old at commencement were exposed to EEP at doses of 0, 0.5 or 2.5% in the diet. Survival rates of 0.5% and 2.5% EEP-treated male and female rats, respectively, were significantly higher than those of respective control groups. Overall histopathological evaluation of neoplasms in rat tissues after 2 years showed no significant increase of tumors or preneoplastic lesions in any organ of animals administered EEP. Significantly lower incidences of pituitary tumors in 0.5% EEP male and 2.5% EEP female groups, malignant lymphoma/leukemia in both 2.5% EEP-treated males and females and total thyroid tumors in 0.5% EEP male group were found. Administration of EEP caused significant decreases of lymphoid hyperplasia of the thymus and lymph nodes in 2.5% EEP-treated rats, tubular cell hyperplasia of kidneys in all EEP groups, and cortical hyperplasia of adrenals in EEP-treated females. In the blood, significant reduction of neutrophils in all EEP-treated males and band neutrophils in 2.5% EEP-treated females was found indicating lower levels of inflammation. Total cholesterol and triglicerides levels were significantly lower in the blood of 2.5% EEP-treated female rats. In conclusion, under the conditions of the 2-year feeding experiment, EEP was not carcinogenic, did not induce significant histopathological changes in any organ, and further exerted anti-inflammatory and antitumorigenic effects resulting in increase of survival of Wistar Hannover rats.

Brazilian Green Propolis Improves Antioxidant Function in Patients with Type 2 Diabetes Mellitus.

Propolis contains a variety of bioactive components and possesses many biological properties. This study was designed to evaluate potential effects of Brazilian green propolis on glucose metabolism and antioxidant function in patients with type 2 diabetes mellitus (T2DM). In the 18-week randomized controlled study, enrolled patients with T2DM were randomly assigned to Brazilian green propolis group (900 mg/day) (n = 32) and control group (n = 33). At the end of the study, no significant difference was found in serum glucose, glycosylated hemoglobin, insulin, aldose reductase or adiponectin between the two groups. However, serum GSH and total polyphenols were significantly increased, and serum carbonyls and lactate dehydrogenase activity were significantly reduced in the Brazilian green propolis group. Serum TNF-α was significantly decreased, whereas serum IL-1β and IL-6 were significantly increased in the Brazilian green propolis group. It is concluded that Brazilian green propolis is effective in improving antioxidant function in T2DM patients.