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.

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.

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.

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.

Discrimination of the entomological origin of honey according to the secretions of the bee (Apis cerana or Apis mellifera).

The eastern honeybee Apis cerana and the western honeybee Apis mellifera are the two most economically valuable honeybee species used in apiculture. In market, the price of Apis cerana honey (ACH) is usually several times higher than that of Apis mellifera honey (AMH) due to the production limit, resulting in wide adulteration and counterfeiting of ACH by AMH. In the present study, we compared honeybee secretions in these two kinds of honey, and found significant differences in protein profiles and hydrocarbon components. The SDS-PAGE pattern showed three species-specific bands with molecular weights between 15.0 and 29.4 KDa in ACH, and six species-specific bands in AMH with molecular weights between 13.8 and 33.1 KDa. The GC-MS-MS detection of the petroleum ether extracts of the two kinds of honey showed that 17-Pentatriacontene and Hentriacontane were the characteristic constituents of ACH and AMH, respectively. These two methods constitute a system to satisfy different needs for entomological authentication of honey samples.

Ethanol Extract of Chinese Propolis Attenuates Early Diabetic Retinopathy by Protecting the Blood-Retinal Barrier in Streptozotocin-Induced Diabetic Rats.

Propolis has been shown to reduce the level of blood glucose and suppress the histopathological changes in diabetics. However, it still remains unknown if propolis has a similar effect on diabetic retinopathy (DR). Our study aimed to evaluate the effect of the ethanol extract of Chinese propolis (EECP) on early DR in streptozotocin (STZ)-induced diabetic rats. EECP was given to diabetic rats by oral intubation for 12 weeks. The concentrations of fasting blood glucose (FBG), glycated hemoglobin (HbA1c), malondialdehyde (MDA), reactive oxygen species (ROS), and reactive nitrogen species (RNS) were measured. Pathological examinations, including hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and immunofluorescence, were also conducted to provide further evidence of EECP's effect on early DR. EECP was able to attenuate diabetes via directly decreasing the levels of FBG and HbA1c, which also resulted in the reduction of MDA, ROS, and RNS. Furthermore, EECP could protect against the damages of photoreceptor cells, as well as retinal thickening. And the inhibition of blood-retinal barrier (BRB) leakage was also observed in EECP-treated diabetic rats, along with the inhibition the loss of tight junction proteins (occludin, ZO-1). These results suggest that EECP has an ameliorating effect on early DR by inhibition of blood-retinal barrier breakdown. PRACTICAL APPLICATION: This study sheds light on the protective effect of the ethanol extract of Chinese propolis on early diabetic retinopathy and the molecular actions underlying the inhibition of blood-retinal barrier breakdown. Our study suggests that ethanol extract of Chinese propolis can be considered as a potential therapeutic agent in the treatment of early diabetic retinopathy.

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.

Mechanisms underlying the wound healing potential of propolis based on its in vitro antioxidant activity.

BACKGROUND: Propolis is a resinous substance collected by honeybees, Apis mellifera, from various plant sources. Having various pharmacological and biological activities, it has been used in folk medicine and complementary therapies since ancient times. PURPOSE: To evaluate the effects and underlying mechanism of the protective effects of the ethanol extract of Chinese propolis (EECP) on L929 cells injured by hydrogen peroxide (H2O2). STUDY DESIGN: The wound healing activities of EECP in L929 cells with H2O2-induced damage were investigated. METHODS: The main components of EECP were analyzed by RP-HPLC, and the free radical scavenging capacity and reducing power were also measured. The effects of EECP on the expression of antioxidant-related genes in fibroblast L929 cells were determined using qRT-PCR and western blotting. RESULTS: EECP had significant protective effects against cell death induced by H2O2 and significantly inhibited the decline of collagen mRNA expression caused by H2O2 in L929 cells. CONCLUSION: EECP induced the expression of antioxidant-related genes, such as HO-1, GCLM, and GCLC, which has great implications for the potential of propolis to alleviate oxidative stress in wound tissues. The protective effects of propolis have great implications for using propolis as a wound healing regent.

The effects of clove oil on the enzyme activity of Varroa destructor Anderson and Trueman (Arachnida: Acari: Varroidae).

Varroa destructor, a key biotic threat to the Western honey bee, has played a major role in colony losses over the past few years worldwide. Overuse of traditional acaricides, such as tau-fluvalinate and flumethrin, on V. destructor has only increased its tolerance to them. Therefore, the application of essential oils in place of traditional pesticides is an attractive alternative, as demonstrated by its high efficiency, lack of residue and tolerance resistance. To study the acaricidal activity of essential oils, we used clove oil (Syzygium aromaticum L.), a typical essential oil with a wide range of field applications, and examined its effects on the enzyme activities of Ca2+-Mg2+-ATPase, glutathione-S-transferase (GST) and superoxide dismutase (SOD) and its effects on the water-soluble protein content of V. destructor body extracts after exposure to 0.1 µl and 1.0 µl of clove oil for 30 min. Our results showed that the water-soluble protein content significantly decreased after the treatments, indicating that the metabolism of the mites was adversely affected. The bioactivity of GSTs increased significantly after a low dosage (0.1 µl) exposure but decreased at a higher dosage (1.0 µl), while the activities of SOD and Ca2+-Mg2+-ATPase were significantly elevated after treatments. These results suggest that the protective enzyme SOD and detoxifying enzymes Ca2+-Mg2+-ATPase and GST contributed to the stress reaction of V. destructor to the essential oils and that the detoxification ability of V. destructor via GST was inhibited at higher dosages. Our findings are conducive to understanding the physiological reactions of V. destructor to treatment with essential oils and the underlying mechanisms behind the acaricidal activities of these natural products.

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.

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.

Chemosensory proteins of the eastern honeybee, Apis cerana: Identification, tissue distribution and olfactory related functional characterization.

Chemosensory proteins (CSPs), a class of small soluble proteins, are thought to be involved in insect chemoreceptive behavior. Here, six CSP genes, AcerCSP1-6 from Apis cerana, were cloned and characterized from worker bees' antennae. Results revealed that the AcerCSPs' amino acid sequences shared high similarity with the homologous genes of Apis mellifera, but low similarity with other insect species. Compared with corresponding CSPs of A. mellifera, AcerCSPs (1, 3, 4, and 6) exhibit quite similar gene expression profiling. On the contrary, AcerCSP2 showed a higher expression level in the forager antennae and legs than CSP2 of A. mellifera. Furthermore, AcerCSP5 was not specifically expressed in larvae, unlike CSP5 of A. mellifera. In a ligand-binding assay, AcerCSP1 and AcerCSP2, which exhibited the highest expression in antennae of A. cerana, had a stronger affinity with candidate floral chemicals and pheromones than AcerCSP4, the results of which was supported by docking analysis, suggesting that the relevance of them with A. cerana olfactory functions. Taken together, these results suggest that despite the quasi-similarity of protein sequences between A. cerana and A. mellifera, differences in tissue expression and functional characteristics between the two species still exist, indicating that homologous proteins potentially perform different tasks even in related species.

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.