Tetracycline-induced gut community dysbiosis and Israeli Acute Paralysis Virus infection synergistically negatively affect honeybees.

Antibiotics are frequently employed to control bacterial diseases in honeybees, but their broad-spectrum action can disrupt the delicate balance of the gut microbiome, leading to dysbiosis. This imbalance in the gut microbiota of honeybees adversely affects their physiological health and weakens their resistance to pathogens, including viruses that significantly threaten honeybee health. In this study, we investigated whether tetracycline-induced gut microbiome dysbiosis promotes the replication of Israeli acute paralysis virus (IAPV), a key virus associated with colony losses and whether IAPV infection exacerbates gut microbiome dysbiosis. Our results demonstrated that tetracycline-induced gut microbiome dysbiosis increases the susceptibility of honeybees to IAPV infection. The viral titer in worker bees with antibiotic-induced gut microbiome dysbiosis prior to IAPV inoculation was significantly higher than in those merely inoculated with IAPV. Furthermore, we observed a synergistic effect between tetracycline and IAPV on the disruption of the honeybee gut microbiome balance. The progression of IAPV replication could, in turn, exacerbate antibiotic-induced gut microbiome dysbiosis in honeybees. Our research provides novel insights into the role of the gut microbiota in host-virus interactions, emphasizing the complex interplay between antibiotic use, gut microbiome health, and viral susceptibility in honeybees. We highlight the crucial role of a balanced gut microbiota in honey bees for their immune response against pathogens and emphasize the importance of careful, safe antibiotic use in beekeeping to protect these beneficial microbes.

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.

Chinese Propolis Suppressed Pancreatic Cancer Panc-1 Cells Proliferation and Migration via Hippo-YAP Pathway.

Pancreatic cancer is one of the most malignant cancers with high mortality. Therefore, it is of great urgency to develop new agents that could improve the prognosis of Pancreatic cancer patients. Chinese propolis (CP), a flavonoid-rich beehive product, has been reported to have an anticancer effect. In this study, we applied CP to the human Pancreatic cancer cell line Panc-1 to verify its impact on tumor development. CP induced apoptosis in Panc-1 cells from 12.5 µg/mL in a time- and dose-dependent manner with an IC50 value of approximately 50 µg/mL. Apoptosis rate induced by CP was examined by Annexing FITC/PI assay. We found that 48 h treatment with 50 µg/mL CP resulted in 34.25 ± 3.81% apoptotic cells, as compared to 9.13 ± 1.76% in the control group. We further discovered that the Panc-1 cells tended to be arrested at G2/M phase after CP treatment, which is considered to contribute to the anti-proliferation effect of CP. Furthermore, our results demonstrated that CP suppressed Panc-1 cell migration by regulating epithelial-mesenchymal transition (EMT). Interestingly, the Hippo pathway was activated in Panc-1 cells after CP treatment, serving as a mechanism for the anti-pancreatic cancer effect of CP. These findings provide a possibility of beehive products as an alternative treatment for pancreatic cancer.

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.

Trans-10-hydroxy-2-decenoic acid protects against LPS-induced neuroinflammation through FOXO1-mediated activation of autophagy.

PURPOSE: Neuroinflammation is thought to be associated with the pathogenesis of a series of neurodegenerative diseases. We have previously reported that royal jelly (RJ) has an anti-inflammatory effect on microglial BV-2 cells. However, components contributing to the effect of RJ were largely unexplored. The aim of this study was to assess whether trans-10-hydroxy-2-decenoic acid (10-HDA), the exclusive fatty acid in RJ, can alleviate neuroinflammation and to further explore the underlying mechanisms. METHODS: Immunohistochemistry staining, ELISA, qRT-PCR and Western blot were used to assess the effect of 10-HDA on LPS-induced neuroinflammation both in vivo and in vitro. To determine the extent of inflammatory changes after 10-HDA treatment, RNAseq transcriptomic analysis was conducted. RESULTS: 10-HDA pretreatment significantly reduced the production of pro-inflammatory mediators in LPS-treated C57BL/6J mice and microglial BV-2 cells. 10-HDA inhibited the activation of the TNF-α/NF-κB axis and NLRP3 inflammasome-IL-1ß pathway, which may be the anti-neuroinflammatory mechanism of 10-HDA. We also demonstrated that 10-HDA triggered cell autophagy, as evidenced by elevated levels of microtubule-associated protein 1 light chain 3-II (LC3-II) and decreased expression of SQSTM1. More importantly, 10-HDA increased the transcriptional activity of FOXO1 by increasing FOXO1 nuclear localization. Inhibition of FOXO1 and autophagy using chemical inhibitors markedly blunted the effect of 10-HDA on the TNF-α pathway and NLRP3 inflammasome-IL-1ß pathway, indicating that 10-HDA alleviates neuroinflammation in BV-2 cells by modulating FOXO1-mediated autophagy. CONCLUSIONS: 10-HDA may be a promising agent for various neuroinflammation-associated diseases.

Plant microRNAs in larval food regulate honeybee caste development.

The major environmental determinants of honeybee caste development come from larval nutrients: royal jelly stimulates the differentiation of larvae into queens, whereas beebread leads to worker bee fate. However, these determinants are not fully characterized. Here we report that plant RNAs, particularly miRNAs, which are more enriched in beebread than in royal jelly, delay development and decrease body and ovary size in honeybees, thereby preventing larval differentiation into queens and inducing development into worker bees. Mechanistic studies reveal that amTOR, a stimulatory gene in caste differentiation, is the direct target of miR162a. Interestingly, the same effect also exists in non-social Drosophila. When such plant RNAs and miRNAs are fed to Drosophila larvae, they cause extended developmental times and reductions in body weight and length, ovary size and fecundity. This study identifies an uncharacterized function of plant miRNAs that fine-tunes honeybee caste development, offering hints for understanding cross-kingdom interaction and co-evolution.

Grouping, Spectrum-Effect Relationship and Antioxidant Compounds of Chinese Propolis from Different Regions Using Multivariate Analyses and Off-Line Anti-DPPH Assay.

49 samples of propolis from different regions in China were collected and analyzed for their chemical compositions, contents of total flavonoids (TFC), total phenolic acid (TPC) and antioxidant activity. High-performance liquid chromatography (HPLC) analysis identified 15 common components, including key marker compounds pinocembrin, 3-O-acetylpinobanksin, galangin, chrysin, benzyl p-coumarate, pinobanksin and caffeic acid phenethyl ester (CAPE). Cluster analysis (CA) and correlation coefficients (CC) analysis showed that these propolis could be divided into three distinct groups. Principal component analysis (PCA) and multiple linear regression analysis (MLRA) revealed that the contents of isoferulic acid, caffeic acid, CAPE, 3,4-dimethoxycinnamic acid, chrysin and apigenin are closely related to the antioxidant properties of propolis. In addition, eight peak areas decreased after reacting with 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals, indicating that these compounds have antioxidant activity. The results indicate that the grouping and spectrum-effect relationship of Chinese propolis are related to their chemical compositions, and several compounds may serve as a better marker for the antioxidant activity of Chinese propolis than TFC and TPC. The findings may help to develop better methods to evaluate the quality of propolis from different geographic origins.

Occurrence of multiple honeybee viruses in the ectoparasitic mites Varroa spp. in Apis cerana colonies.

In this study, we investigated the prevalence of honeybee viruses in Varroa destructor and Varroa underwoodi infesting Apis cerana colonies in China. Deformed wing virus (DWV) was the most prevalent virus in these two mite species, followed by Israeli acute paralysis virus (IAPV), Black queen cell virus (BQCV), Kashmir bee virus (KBV), Chronic bee paralysis virus (CBPV), Apis mellifera filamentous virus (AmFV) and Sacbrood virus (SBV) in V. destructor, while in V. underwoodi, it was followed by CBPV, AmFV, BQCV, IAPV and KBV. In addition, multiple viruses were commonly detectable in both mite species.

Anti-inflammatory effects of Chinese propolis in lipopolysaccharide-stimulated human umbilical vein endothelial cells by suppressing autophagy and MAPK/NF-κB signaling pathway.

This study aimed to investigate the possible benefits of Chinese poplar propolis (CP) in inhibiting inflammation using vascular endothelial cells (VECs) cultured in a nutrient-rich condition exposed to lipopolysaccharide (LPS). Cell proliferation was detected by sulforhodamine B assay and EdU kit. The production of reactive oxygen species (ROS) and level of mitochondrial membrane potential were determined with fluorescent probe DCHF and JC-1, respectively. Protein expression was examined by immunofluorescence staining and western blotting. The results showed that CP (6.25, 12.5, and 25 µg/mL) significantly reduced LPS-induced cytotoxicity, and when challenged with CP substantially suppressed ROS overproduction and protected mitochondrial membrane potential. CP treatment significantly inhibited autophagy by inhibiting LC3B distribution and accumulation, and elevating the p62 level in an mTOR-independent manner but mainly by suppressing the translocation of p53 from the cytoplasm to the nucleus. Furthermore, CP treatment markedly reduced protein levels of TLR4 at 12 and 24 h and significantly suppressed nuclear translocation of NF-κB p65 from cytoplasm to nucleus. In addition, CP treatment significantly reduced the phosphorylation of JNK, ERK1/2, and p38 MAPK. Our findings demonstrated that CP protects VECs from LPS-induced oxidative stress and inflammation, which might be associated with depressing autophagy and MAPK/NF-κB signaling pathway. The results provided novel insights for the potential use of nutrient-rich propolis against inflammation.

A New Propolis Type from Changbai Mountains in North-east China: Chemical Composition, Botanical Origin and Biological Activity.

Propolis is a bee product with a wide range of biological activities and its chemical compounds depend highly on the type of plant accessible to the bees. The Changbai Mountains are a major mountain range in Northeast China and are one of the major bee product-producing areas in China. In this study, we evaluated the total phenolic acids and flavonoid contents as well as the antioxidant activity of propolis sampled from the Changbai Mountains area (CBM). We identified the major compounds and qualified their contents by HPLC-ESI/MS and HPLC-UV, and found that the content of p-coumaric acid and an unknown peak (CBE) in CBM propolis was higher than in propolis from other parts of China. The unknown compound CBE was isolated, purified, and identified as benzyl p-coumarate by MS and NMR. Possible plant sources of CBM propolis are Populus davidiana dode and Populus simonii Carr, which widely distributed in the Changbai Mountains area. CBM propolis is a new propolis type, that could be an excellent raw material for health foods and pharmaceuticals.

Royal Jelly Ameliorates Behavioral Deficits, Cholinergic System Deficiency, and Autonomic Nervous Dysfunction in Ovariectomized Cholesterol-Fed Rabbits.

Estrogen deficiency after menopause is associated with autonomic nervous changes, leading to memory impairment and increased susceptibility to Alzheimer's disease (AD). Royal jelly (RJ) from honeybees (Apis mellifera) has estrogenic activity. Here, we investigated whether RJ can improve behavior, cholinergic and autonomic nervous function in ovariectomized (OVX) cholesterol-fed rabbits. OVX rabbits on high-cholesterol diet were administered with RJ for 12 weeks. The results showed that RJ could significantly improve the behavioral deficits of OVX cholesterol-fed rabbits and image structure of the brain. RJ reduced body weight, blood lipid, as well as the levels of amyloid-beta (Aß), acetylcholinesterase (AchE), and malonaldehyde (MDA) in the brain. Moreover, RJ also increased the activities of choline acetyltransferase (ChAT) and superoxide dismutase (SOD) in the brain, and enhanced heart rate variability (HRV) and Baroreflex sensitivity (BRS) in OVX cholesterol-fed rabbits. Furthermore, RJ was also shown to reduce the content of Evans blue and the expression levels of Aß, beta-site APP cleaving enzyme 1(BACE1), and receptor for advanced glycation end products (RAGE), and increase the expression level of LDL(low density lipoprotein) receptor-related protein 1 (LRP-1) in the brain. Our findings suggested that RJ has beneficial effects in neurological disorders of postmenopausal women, which were associated with reducing cholesterol and Aß deposition, enhancing the estrogen levels and the activities of cholinergic and antioxidant systems, and ameliorating the blood⁻brain barrier (BBB) permeability and restoring autonomic nervous system.

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.

Sublethal doses of neonicotinoid imidacloprid can interact with honey bee chemosensory protein 1 (CSP1) and inhibit its function.

As a frequently used neonicotinoid insecticide, imidacloprid can impair the chemoreceptive behavior of honey bees even at sublethal doses, while the physiochemical mechanism has not been further revealed. Here, multiple fluorescence spectra, thermodynamic method, and molecular docking were used to study the interaction and the functional inhibition of imidacloprid to the recombinant CSP1 protein in Asian honey bee, Apis cerana. The results showed that the fluorescence intensity (λem = 332 nm) of CSP1 could be significantly quenched by imidacloprid in a dynamic mode. During the quenching process, ΔH > 0, ΔS > 0, indicating that the acting forces of imidacloprid with CSP1 are mainly hydrophobic interactions. Synchronous fluorescence showed that the fluorescence of CSP1 was mainly derived from tryptophan, and the hydrophobicity of tryptophan decreased with the increase of imidacloprid concentration. Molecular docking predicted the optimal pose and the amino acid composition of the binding process. Circular dichroism (CD) spectra showed that imidacloprid reduced the α-helix of CSP1 and caused the extension of the CSP1 peptide chain. In addition, the binding of CSP1 to floral scent ß-ionone was inhibited by nearly 50% of the apparent association constant (KA) in the presence of 0.28-2.53 ng/bee of imidacloprid, and the inhibition rate of nearly 95% at 3.75 ng/bee of imidacloprid at sublethal dose level. This study initially revealed the molecular physiochemical mechanism that sublethal doses of neonicotinoid still interact and inhibit the physiological function of the honey bees' chemoreceptive system.

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.

Antioxidant activities and molecular mechanisms of the ethanol extracts of Baccharis propolis and Eucalyptus propolis in RAW64.7 cells.

Context Numerous studies have reported that propolis possesses strong antioxidant activities. However, their antioxidant molecular mechanisms are unclear. Objective We utilize ethanol extracts of Chinese propolis (EECP) as a reference to compare ethanol extracts of Eucalyptus propolis (EEEP) with ethanol extracts of Baccharis propolis (EEBGP) based on their antioxidant capacities and underlying molecular mechanisms. Materials and methods HPLC and chemical analysis are utilized to evaluate compositions and antioxidant activities. ROS-eliminating effects of EEBGP (20-75 µg/mL), EEEP (1.25-3.75 µg/mL) and EECP (1.25-5 µg/mL) are also determined by flow cytometry analysis. Moreover, we compared antioxidant capacities by determining their effects on expressions of antioxidant genes in RAW264.7 cells with qRT-PCR, western blot and confocal microscopy analysis. Results EEBGP mainly contains chlorogenic acid (8.98 ± 0.86 mg/g), kaempferide (11.18 ± 8.31 mg/g) and artepillin C (107.70 ± 10.86 mg/g), but EEEP contains 10 compositions, whereas EECP contains 17 compositions. Meantime, although EEEP shows DPPH (IC50 19.55 ± 1.28), ABTS (IC50 20.0 ± 0.31) and reducing power (2.70 ± 0.08 mmol TE/g) better than EEBGP's DPPH (IC50 43.85 ± 0.54), ABTS (IC50 38.2 ± 0.33) and reducing power (1.53 ± 0.05 mmol TE/g), EEBGP exerts much higher ROS inhibition rate (40%) than EEEP (under 20%). Moreover, EEBGP strengthen antioxidant system by activating p38/p-p38 and Erk/p-Erk kinase via accelerating nucleus translocation of Nrf2. EEEP and EECP improve antioxidant gene expression only via Erk/p-Erk kinase-Nrf2 signalling pathway. Discussion and conclusion EEBGP and EEEP exert antioxidant activities via different molecular mechanisms, which may depend on chemical compositions.