Biotransformation and metabolite activity analysis of flavonoids from propolis in vivo.

Propolis is a natural resinous compound produced by bees, mixed with their saliva and wax, and has a range of biological benefits, including antioxidant and anti-inflammatory effects. This article reviews the in vivo transformation of propolis flavonoids and their potential influence on drug efficacy. Despite propolis is widely used, there is little research on how the active ingredients of propolis change in the body and how they interact with drugs. Future research will focus on these interactions and the metabolic fate of propolis in vivo.

Design, Synthesis and Molecular Docking of 1,3,4-Oxadiazole-2(3H)-thione Derivatives Containing 1,4-Benzodioxane Skeleton as Potential FabH Inhibitors.

Fatty acid biosynthesis is essential for bacterial survival. Of these promising targets, ß-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) is the most attractive target. A series of novel 1,3,4-oxadiazole-2(3H)-thione derivatives containing 1,4-benzodioxane skeleton targeting FabH were designed and synthesized. These compounds were determined by 1 H-NMR, 13 C-NMR, MS and further confirmed by crystallographic diffraction study for compound 7m and 7n. Most of the compounds exhibited good inhibitory activity against bacteria by computer-assisted screening, antibacterial activity test and E. coli FabH inhibitory activity test, wherein compounds 7e and 7q exhibited the most significant inhibitory activities. Besides, compound 7q showed the best E. coli FabH inhibitory activity (IC50 =2.45 µΜ). Computational docking studies also showed that compound 7q interacts with FabH critical residues in the active site.

Chemical Analyses and Antimicrobial Activity of Nine Kinds of Unifloral Chinese Honeys Compared to Manuka Honey (12+ and 20+).

Honey has good antimicrobial properties and can be used for medical treatment. The antimicrobial properties of unifloral honey varieties are different. In this study, we evaluated the antimicrobial and antioxidant activities of nine kinds of Chinese monofloral honeys. In addition, headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) technology was used to detect their volatile components. The relevant results are as follows: 1. The agar diffusion test showed that the diameter of inhibition zone against Staphylococcus aureus of Fennel honey (21.50 ± 0.41 mm), Agastache honey (20.74 ± 0.37 mm), and Pomegranate honey (18.16 ± 0.11 mm) was larger than that of Manuka 12+ honey (14.27 ± 0.10 mm) and Manuka 20+ honey (16.52 ± 0.12 mm). The antimicrobial activity of Chinese honey depends on hydrogen peroxide. 2. The total antioxidant capacity of Fennel honey, Agastache honey, and Pomegranate honey was higher than that of other Chinese honeys. There was a significant positive correlation between the total antioxidant capacity and the total phenol content of Chinese honey (r = 0.958). The correlation coefficient between the chroma value of Chinese honey and the total antioxidant and the diameter of inhibition zone was 0.940 and 0.746, respectively. The analyzed dark honeys had better antimicrobial and antioxidant activities. 3. There were significant differences in volatile components among Fennel honey, Agastache honey, Pomegranate honey, and Manuka honey. Hexanal-D and Heptanol were the characteristic components of Fennel honey and Pomegranate honey, respectively. Ethyl 2-methylbutyrate and 3-methylpentanoic acids were the unique compounds of Agastache honey. The flavor fingerprints of the honey samples from different plants can be successfully built using HS-GC-IMS and principal component analysis (PCA) based on their volatile compounds. Fennel honey, Agastache honey, and Pomegranate honey are Chinese honey varieties with excellent antimicrobial properties, and have the potential to be developed into medical grade honey.

Authentication of Apis cerana Honey and Apis mellifera Honey Based on Major Royal Jelly Protein 2 Gene.

In Asia, honey is mainly produced by Apis mellifera and Apis cerana. However, the price of A. cerana honey is usually much higher than A. mellifera honey. Seeing considerable profits, some dishonest companies and beekeepers mislabel A. mellifera honey as A. cerana honey or incorporate A. mellifera honey into A. cerana honey. In the present study, we developed methods to discriminate A. cerana honey from A. mellifera honey based on the MRJP2 (major royal jelly protein 2) gene. Two pairs of species-specific primers were designed. The amplification products of A. cerana and A. mellifera were 212 and 560 bp, respectively. As little as one percent incorporation of A. mellifera honey in the mixture can be detected by duplex PCR. Additionally, another method based on the melt curve analysis using the same primers was also developed, allowing a rapid discrimination of real-time PCR product of different species. Our study shows that the entomological authentication of honey samples can be identified by nuclear genes other than mitochondrial genes and this extends the possibility of gene selection in identification. The authentication system we proposed could be a useful tool for discriminating A. cerana honey from A. mellifera honey.

Royal Jelly Attenuates LPS-Induced Inflammation in BV-2 Microglial Cells through Modulating NF-κB and p38/JNK Signaling Pathways.

Royal jelly (RJ), a hive product with versatile pharmacological activities, has been used as a traditional functional food to prevent or treat inflammatory diseases. However, little is known about the anti-inflammatory effect of RJ in microglial cells. The aim of this study is to assess the anti-inflammatory effects of RJ in lipopolysaccharide- (LPS-) induced murine immortalized BV-2 cells and to explore the underlying molecular mechanisms. Our results showed that in LPS-stimulated BV-2 cells, RJ significantly inhibited iNOS and COX-2 expression at mRNA and protein levels. The mRNA expression of IL-6, IL-1ß, and TNF-α was also downregulated by RJ in a concentration-dependent manner. Additionally, RJ protected BV-2 cells against oxidative stress by upregulating heme oxygenase-1 (HO-1) expression and by reducing reactive oxygen species (ROS) and nitric oxide (NO) production. Mechanistically, we found that RJ could alleviate inflammatory response in microglia by suppressing the phosphorylation of IκBα, p38, and JNK and by inhibiting the nucleus translocation of NF-κB p65. These findings suggest that RJ might be a promising functional food to delay inflammatory progress by influencing the microglia function.

Evidence of Apis cerana Sacbrood virus Infection in Apis mellifera.

Sacbrood virus(SBV) is one of the most destructive viruses in the Asian honeybee Apis cerana but is much less destructive in Apis mellifera In previous studies, SBV isolates infecting A. cerana(AcSBV) and SBV isolates infecting A. mellifera(AmSBV) were identified as different serotypes, suggesting a species barrier in SBV infection. In order to investigate this species isolation, we examined the presence of SBV infection in 318A. mellifera colonies and 64A. cerana colonies, and we identified the genotypes of SBV isolates. We also performed artificial infection experiments under both laboratory and field conditions. The results showed that 38A. mellifera colonies and 37A. cerana colonies were positive for SBV infection. Phylogenetic analysis based on RNA-dependent RNA polymerase (RdRp) gene sequences indicated that A. cerana isolates and most A. mellifera isolates formed two distinct clades but two strains isolated fromA. mellifera were clustered with theA. cerana isolates. In the artificial-infection experiments, AcSBV negative-strand RNA could be detected in both adult bees and larvae ofA. mellifera, although there were no obvious signs of the disease, demonstrating the replication of AcSBV inA. mellifera Our results suggest that AcSBV is able to infectA. melliferacolonies with low prevalence (0.63% in this study) and pathogenicity. This work will help explain the different susceptibilities ofA. cerana and A. melliferato sacbrood disease and is potentially useful for guiding beekeeping practices.

In Vitro Anti-Inflammatory Effects of Three Fatty Acids from Royal Jelly.

Trans-10-hydroxy-2-decenoic acid (10-H2DA), 10-hydroxydecanoic acid (10-HDAA), and sebacic acid (SEA) are the three major fatty acids in royal jelly (RJ). Previous studies have revealed several pharmacological activities of 10-H2DA and 10-HDAA, although the anti-inflammatory effects and underlying mechanisms by which SEA acts are poorly understood. In the present study, we evaluated and compared the in vitro anti-inflammatory effects of these RJ fatty acids in lipopolysaccharide-stimulated RAW 264.7 macrophages. The results showed that 10-H2DA, 10-HDAA, and SEA had potent, dose-dependent inhibitory effects on the release of the major inflammatory-mediators, nitric oxide, and interleukin-10, and only SEA decreased TNF-α production. Several key inflammatory genes have also been modulated by these RJ fatty acids, with 10-H2DA showing distinct modulating effects as compared to the other two FAs. Furthermore, we found that these three FAs regulated several proteins involved in MAPK and NF-κB signaling pathways. Taken together, these findings provide additional references for using RJ against inflammatory diseases.

Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage.

Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis.

High Royal Jelly-Producing Honeybees (Apis mellifera ligustica) (Hymenoptera: Apidae) in China.

China is the largest producer and exporter of royal jelly (RJ) in the world, supplying >90% of the global market. The high production of RJ in China is principally owing to the high RJ-producing lineage of honeybees (Apis mellifera ligustica Spinola, 1806) established by beekeepers in the 1980s. We describe the development of high royal jelly-producing honeybees and the management of this lineage today. Previous research and recent advances in the genetic characterization of this lineage, and the molecular markers and mechanisms associated with high RJ production are summarized. The gaps in our knowledge and prospects for future research are also highlighted.

Identification of catechol as a new marker for detecting propolis adulteration.

Adulteration of propolis with poplar extract is a serious issue in the bee products market. The aim of this study was to identify marker compounds in adulterated propolis, and examine the transformation of chemical components from poplar buds to propolis. The chemical profiles of poplar extracts and propolis were compared, and a new marker compound, catechol, was isolated and identified from the extracts of poplar buds. The polyphenol oxidase, catechol oxidase, responsible for catalyzing oxidation of catechol was detected in poplar buds and propolis. The results indicate catechol can be used as a marker to detect propolis adulterated with poplar extract.

Recent advances in the chemical composition of propolis.

Propolis is a honeybee product with broad clinical applications. Current literature describes that propolis is collected from plant resins. From a systematic database search, 241 compounds were identified in propolis for the first time between 2000 and 2012; and they belong to such diverse chemical classes as flavonoids, phenylpropanoids, terpenenes, stilbenes, lignans, coumarins, and their prenylated derivatives, showing a pattern consistent with around 300 previously reported compounds. The chemical characteristics of propolis are linked to the diversity of geographical location, plant sources and bee species.

Geographical influences on content of 10-hydroxy-trans-2-decenoic acid in royal jelly in China.

The content of 10-hydroxy-trans-2-decenoic acid (10-HDA), a marker compound in royal jelly (RJ), is the most important criterion in grading RJ for commercial trade and varies with its origin. To identify the effect of geographical origin on 10-HDA content in RJ, 138 samples were collected from 19 provinces of China (divided into three groups) produced by either Apis mellifera ligustica Spinola, 1806 or a hybrid of A. m. ligustica and Apis mellifera carnica Pollman, 1879 and analyzed for moisture, sugar, crude protein, ash, acid, and 10-HDA concentration. The results show that RJ from western China has a significantly higher 10-HDA level (2.01 +/- 0.05%) than those from northeastern (1.87 +/- 0.05%) and eastern (1.75 +/- 0.03%) China. RJ secreted by hybrid bees contained more 10-HDA (1.89 +/- 0.03%) than that secreted by A. m. ligustica (1.78 +/- 0.03%). The 10-HDA content of RJ produced during flowering of rape (Brassica campestris L.), lime (Tilia amurensis Ruprecht), and vitex (Vitex negundo L. variety heterophylla (Franch.) Rehder) was 1.92, 1.80, and 1.68%, respectively. The results would be helpful during the process of price determination of RJ by providing some basis of geographical, bee strain, and botanical information for commercial trade.

[Advance in studies on antioxidant activity of propolis and its molecular mechanism].

Propolis is an adhesive substance mixed with plant resins collected by honeybees (Apis mellifera) and secretions from their mandibular gland and wax gland, with wide pharmacological activity and healthcare functions. Its antioxidant activity has long been regarded as one of the most important biological activities of propolis. This article summarizes studies on the antioxidant activity of propolis extracts from different geographic origins and with different extraction methods, as well as several important monomer active ingredients in propolis, and concludes the potential molecular mechanism of antioxidant activity of propolis and its monomer ingredients, with the aim of providing ideas for further studies on pharmacological activity of propolis, as well as reference for in-depth development of propolis products.

[Phylogenetic relationships of honey bees (Hymenoptera: Apinae) inferred from mitochondrial and nuclear DNA sequences].

The sequences of mitochondrial ND2, CO2, and 16S rRNA genes and nuclear ITPR gene were obtained from 22 samples of 5 Apis species from China. The characteristics of the sequences and the pairwise distances among species were analyzed. Phylogenetic trees were reconstructed for Apis species using maximum parsimony, neighbor-joining and maximum likelihood methods together with the sequences of the other 4 Apis species downloaded from GenBank. Results supported that Apis species were divided into three major clusters: dwarf bees (A. florea and A. andreniformis), giant bees (A. dorsata and A. laboriosa), and cavity-nesting bees (A. mellifera, A. cerana, A. koschevnikovi, A. nigrocinta, and A. nuluensis). The dwarf honey bees were confirmed as basal lineage. Our study also revealed a high level of genetic divergence between A. dorsata from Hainan Island and China mainland.

Investigation of the Maturity Evaluation Indicator of Honey in Natural Ripening Process: The Case of Rape Honey.

Honey maturity, a critical factor for quality evaluation, is difficult to detect in the current industry research. The objective of this study was to explore the changes in the composition and find potential maturity indicators of rape honey at different maturity stages through evaluating physicochemical parameters (moisture, sugars, pH, electrical conductivity, total protein, total phenols, total flavonoids, proline, and enzyme activity), the antioxidant capacity, and volatile components. The relevant results are as follows: 1. As the maturity increased, the moisture, sucrose, and maltose content of rape honey gradually decreased, while the glucose, fructose, and total protein content gradually increased. The activities of diastase, invertase, and ß-glucosidase showed a significant increase with the elevation of ripening days, and the activity of glucose oxidase reached the highest before completely capping. 2. The antioxidant capacity of honey increased with the increase in honey maturity. There is a significant and strong correlation between the bioactive components of rape honey and antioxidant capacity (p < 0.01, |r| > 0.857). 3. Thirty-five volatile components have been identified. Nonanal, benzaldehyde monomer, and benzaldehyde dimer can be used as potential indicators for the identification of honey maturity stages. Principal component analysis (PCA) based on antioxidant parameters and volatile components can identify the maturity of honey.

[Study on the identification of six kinds of bee pollens by three-step infrared macro-fingerprint method].

Six kinds of bee pollens, including apricot pollen, lotus pollen, rape pollen, camellia pollen, watermelon pollen and corn poppy pollen, were identified non-destructively by Fourier transform infrared spectroscopy (FTIR) combined with derivative spectra and two-dimensional correlation spectroscopy (2D) in the present article. Compared with conventional IR spectra of samples, some certain differences were found in the characteristic peaks of proteins, lipids and carbohydrates. Obvious differences of the six kinds of bee pollens were found in the second derivative spectra. And in the 2D-IR correlation spectra, the samples presented the differences in the position and intensity of the autopeaks and correlation peak clusters. Therefore, the three-step IR macro-fingerprint provides a more rapid and effective method for the identification of different kinds of bee pollens.

Unique dynamic mode between Artepillin C and human serum albumin implies the characteristics of Brazilian green propolis representative bioactive component.

As a representative bioactive component in Brazil green propolis, Artepillin C (ArtC; 3, 5-diprenyl-4-hydroxycinnamic acid) has been reported a wide variety of physiological activities including anti-tumor, anti-inflammatory, and antimicrobial activity etc. However, it seems incompatible that ArtC in vivo was characterized as low absorption efficiency and low bioavailability. In order to obtain the elucidation, we further investigated the physicochemical basis of ArtC interacting with human serum albumin (HSA) in vitro. We found a unique dynamic mode interaction between ArtC and HSA, which is completely different from other reported propolis bioactive components. Thermodynamic analysis showed that hydrophobic interactions and electrostatic forces are the main driving force. The competitive assay indicates that the binding site of ArtC with HSA is close to the Sudlow's site I. The findings of this study reveal the unique physicochemical transport mechanism of ArtC in the human body, which helps to further understand the uniqueness of the representative functional components of Brazilian green propolis in the human body.

Chinese Propolis Inhibits the Proliferation of Human Gastric Cancer Cells by Inducing Apoptosis and Cell Cycle Arrest.

Special Chinese propolis sourced from the Changbai Mountains (CBMP) in Northeast China is rich in specific flavonoids and phenolic acids and its bioactivity has not been reported. This study aimed to investigate the antiproliferative effect of CBMP on cancer cells and its molecular mechanisms. Different cancer cell lines were treated with the ethanol extracts of CBMP for 24 hours before the cell viability and mechanism measurements. The results showed CBMP had weak activities against human pancreatic cancer cell PANC1, human lung cancer cell A549, human colon cancer cell HCT116, human liver cancer cell HepG2, human bladder cancer cell T24, and human breast cancer cell MDA-MB-231, but it significantly inhibited the growth of human gastric cancer SGC-7901 cells, caused cell apoptosis and cell cycle arrest in S phase, with increased production of reactive oxygen species (ROS) and reduced mitochondrial membrane potential (MMP). The results indicate that Chinese propolis sourced from the Changbai Mountains selectively inhibits the proliferation of human gastric cancer SGC-7901 cells by inducing both death receptor-induced apoptosis and mitochondria-mediated apoptosis, and cell cycle arrest in S phase. These activities and mechanisms help understand the anticancer action of propolis and its active compounds.

Royal jelly attenuates nonalcoholic fatty liver disease by inhibiting oxidative stress and regulating the expression of circadian genes in ovariectomized rats.

Nonalcoholic fatty liver disease (NAFLD) has a high incidence in postmenopausal women and is accompanied by insulin resistance, obesity, and dyslipidemia. Royal jelly (RJ), a natural substance derived from hive, possesses numerous health-beneficial properties. Here, we evaluated the effects of RJ (150, 300, and 450 mg kg-1  day-1 , 8 weeks) on NAFLD in ovariectomized (OVX) rats. Based on the results, RJ ameliorated the degree of anxiety, improved serum lipid profile, and attenuated the hepatic steatosis and liver injury in OVX rats. Furthermore, the protective effects of RJ could be attributed to its antioxidant properties, which enhance the levels of hepatic antioxidant enzymes. The qRT-PCR results also suggest that RJ improves the disturbances of circadian genes by downregulating their expression, including that of Per1 and Per 2, in the liver of OVX rats. Altogether, our findings suggest that RJ may be a promising agent for the treatment of NAFLD. PRACTICAL APPLICATIONS: Postmenopausal women are at an increased risk of NAFLD. Currently, there are no licensed therapies for NAFLD. Although hormone replacement therapy (HRT) is reported to inhibit the development of NAFLD, it causes unexpected adverse effects. As HRT is controversial, the use of natural supplements to counteract the detrimental effects of menopause has recently attracted more attention. RJ is a natural product secreted from the hypopharyngeal and mandibular glands of worker bees. The present study illustrates the protective effect of the natural product, RJ, and its underlying mechanisms on NAFLD. This is the first study to assess the effect of RJ on NAFLD under estrogen deficiency. Such findings contribute to the further utilization of RJ, which might serve as a promising therapeutic option and natural food for the treatment of NAFLD.

[Research on overall assessment of royal jelly freshness by FTIR spectroscopy].

Fourier transform infrared spectra (FTIR) of royal jelly (RJ) stored at different temperature and after different storage periods were measured, a series of correlation analysis among the spectra were carried out by using the spectra of new-harvested RJ as a standard. The results showed that the correlation coefficient of amide band I and the relative intensity ratios of I 1 647 /I1 541, I1 647/I1 409, I1 647/I1 247 and I1 647/I1 054 of RJ samples' spectra decreased with extension of storage time and temperature, and existed good linear correlations with the storage time, with the order of their change extent being 28 degrees C > 16 degrees C > 4 degrees C > -18 degrees C. According to the spectra change laws and practical experiences of RJ storage, the correlation coefficient of amide band I and four relative intensity ratios I1 647/I1 541, I1 647/I1 409, I1 647/I1 247 and I1 647/I1 054 were selected as assessment indexes of RJ freshness. The threshold value of correlation coefficient was set to be 0.910 0, and the threshold values of the four relative intensity ratios were set to be 1.744, 2.430, 3.345 and 1.412 respectively. Once one or more indexes are lower than the corresponding threshold values, the RJ sample can be considered as a stale sample. So, FTIR spectroscopy combined with several data-processing methods would be an effective method for overall assessing the freshness of IRJ.