Dietary silymarin improves performance by altering hepatic lipid metabolism and cecal microbiota function and its metabolites in late laying hens.

BACKGROUND: Liver lipid dysregulation is one of the major factors in the decline of production performance in late-stage laying hens. Silymarin (SIL), a natural flavonolignan extracted from milk thistle, is known for its hepatoprotective and lipid-lowering properties in humans. This study evaluates whether SIL can provide similar benefits to late-stage laying hens. A total of 480 68-week-old Lohmann Pink laying hens were randomly assigned into 5 groups, each group consisting of 6 replicates with 16 hens each. The birds received a basal diet either without silymarin (control) or supplemented with silymarin at concentrations of 250, 500, 750, or 1,000 mg/kg (SIL250, SIL500, SIL750, SIL1000) over a 12-week period. RESULTS: The CON group exhibited a significant decline in laying rates from weeks 9 to 12 compared to the initial 4 weeks (P = 0.042), while SIL supplementation maintained consistent laying rates throughout the study (P > 0.05). Notably, the SIL500 and SIL750 groups showed higher average egg weight than the CON group during weeks 5 to 8 (P = 0.049). The SIL750 group had a significantly higher average daily feed intake across the study period (P < 0.05), and the SIL500 group saw a marked decrease in the feed-to-egg ratio from weeks 5 to 8 (P = 0.003). Furthermore, the SIL500 group demonstrated significant reductions in serum ALT and AST levels (P < 0.05) and a significant decrease in serum triglycerides and total cholesterol at week 12 with increasing doses of SIL (P < 0.05). SIL also positively influenced liver enzyme expression (FASN, ACC, Apo-VLDL II, FXR, and CYP7A1; P < 0.05) and altered the cecal microbiota composition, enhancing species linked to secondary bile acid synthesis. Targeted metabolomics identified 9 metabolites predominantly involved in thiamin metabolism that were significantly different in the SIL groups (P < 0.05). CONCLUSIONS: Our study demonstrated that dietary SIL supplementation could ameliorate egg production rate in late stage laying hens, mechanistically, this effect was via improving hepatic lipid metabolism and cecal microbiota function to achieve. Revealed the potentially of SIL as a feed supplementation to regulate hepatic lipid metabolism dysregulation. Overall, dietary 500 mg/kg SIL had the best effects.

Construction of a water-soluble and photostable rubropunctatin/ß-cyclodextrin drug carrier.

The purpose of the current study was to construct a ß-cyclodextrin drug carrier for rubropunctatin to improve its water solubility and light stability for future cytotoxicity studies. The inclusion complexation behavior of rubropunctatin with ß-cyclodextrin was investigated using FESEM, FT-IR and XRD. A molecular docking study was performed to elucidate the most probable inclusion structure. The inclusion complex could be completely dispersed in water and had a small size of 121.87 ± 2.13 nm (n = 3), a good PDI (0.320 ± 0.017), and an acceptable potential value of -27.7 ± 0.32 mV (n = 3). Furthermore, the stability of the rubropunctatin in water under light irradiation was found to be greatly enhanced after being encapsulated in cyclodextrin, and it exhibited a retention rate of over 70% vs. 10.17%. In addition, the cytotoxicity of the inclusion complex was evaluated by MTT assay and Annexin V-FITC/PI detection using cervical adenocarcinoma HeLa cells. The results showed that the inclusion complex had comparable toxicity compared to rubropunctatin solubilized with 0.4% DMSO. More importantly, the formation of the inclusion complex contributed greatly to the intensification of the bioavailability of rubropunctatin because the use of organic solvent was avoided.

Nonionic surfactants and their effects on asymmetric reduction of 2-octanone with Saccharomyces cerevisiae.

In an aqueous buffer system, serious reverse and side reactions were found in the asymmetric reduction of 2-octanone with Saccharomyces cerevisiae. However, some nonionic surfactants added to the aqueous buffer system improved the bioreduction process by decreasing the reverse and side reaction rates in addition to effectively increasing the average positive reaction rate. Further, a shorter carbon chain length of hydrophilic or hydrophobic moieties in surfactants resulted in a higher yield of (S)-2-octanol. The alkylphenol ethoxylate surfactants had a less influence than polyoxyethylenesorbitan trialiphatic surfactants on the product e.e. It suggested that the product e.e. resulting from the change of carbon chain length of the hydrophobic moieties varied markedly compared with the change of carbon chain length of the hydrophilic moiety. Emulsifier OP-10 and Tween 20 markedly enhanced the yield and product e.e. at the concentration of 0.4 mmol L-1 with a yield of 73.3 and 93.2%, and the product e.e. of 99.2 and 99.3%, respectively, at the reaction time of 96 h.

Monascus pigment rubropunctatin derivative FZU-H reduces Aß(1-42)-induced neurotoxicity in Neuro-2A cells.

Alzheimer's disease (AD) is an extremely complex disease, characterized by several pathological features including oxidative stress and amyloid-ß (Aß) aggregation. Blockage of Aß-induced injury has emerged as a potential therapeutic approach for AD. Our previous efforts resulted in the discovery of Monascus pigment rubropunctatin derivative FZU-H with potential neuroprotective effects. This novel lead compound significantly diminishes toxicity induced by Aß(1-42) in Neuro-2A cells. Our further mechanism investigation revealed that FZU-H inhibited Aß(1-42)-induced caspase-3 protein activation and the loss of mitochondrial membrane potential. In addition, treatment of FZU-H was proven to attenuate Aß(1-42)-induced cell redox imbalance and Tau hyperphosphorylation which caused by okadaic acid in Neuro-2A cells. These results indicated that FZU-H shows promising neuroprotective effects for AD.

Maslinic acid inhibits impairment of endothelial functions induced by high glucose in HAEC cells through improving insulin signaling and oxidative stress.

Impaired endothelial functions are closely associated with many chronic vascular-related diseases. Maslinic acid (MA), a natural pentacyclic triterpene compound, has received more and more attentions in recent years due to a variety of bioactivities. In the present study, we investigated the effect of MA on impaired endothelial functions in human aortic endothelial cells (HAEC) induced by high glucose treatment and discussed its possible mechanism. We showed that MA decreased the ROS level, inhibited the inflammatory cytokines expression, facilitated insulin-mediated IRS-1/PI3K/Akt/eNOS signaling and suppressed the cellular apoptosis ratio induced by high glucose. The molecular docking results showed that MA may regulate multiple targets to improve the endothelial dysfunction in HAECs exposed to high glucose. These results revealed potential application of MA in improving endothelia dysfunction.

Acute and Subacute Oral Toxicity Evaluation of Eriobotrya japonica Leaf Triterpene Acids in ICR Mice.

The interest focusing on Eriobotrya japonica leaf triterpene acid (ELTA) has increased recently because of its beneficial effects on health. However, there has been a lack of experimental data on its toxicity. The present study therefore was conducted to evaluate its toxicity in ICR mice. The results showed that ELTA produced neither mortality nor toxicity of the main organs in ICR male and female mice in both acute (0.30, 0.65, 1.39, and 3.00 g·kg-1 body weight) and subacute (150, 300, and 600 mg·kg-1 BW) 28-day toxicity studies. Because of lacking apparently adverse effects found in the hematology, clinical biochemistry, and histopathology evaluation, results of the present study together with the beneficial effects make ELTA as a promising dietary supplement and indicated that ELTA administered orally might have a large safety margin for human.

Monascus Pigment Rubropunctatin: A Potential Dual Agent for Cancer Chemotherapy and Phototherapy.

The Monascus pigment, rubropunctatin, was extracted and purified from red mold rice (RMR), and its cytotoxic activities against human cervical carcinoma HeLa cells were studied under the conditions with or without light irradiation. The IC50 value of rubropunctatin against HeLa cells in the dark was 93.71 ± 1.96 µM (24 h), while the cytotoxic activity was enhanced more than 3 times (IC50 = 24.02 ± 2.17 µM) under light irradiation (halogen lamp, 500 W; wavelength, 597-622 nm; and fluence rate, 15 mW cm(-2), for 30 min). However, the IC50 value of rubropunctatin against the immortalized human cervical epithelial H8 cells was more than 300 µM, even under light irradiation, indicating that rubropunctatin has a favorable selectivity index (SI). Treatment of HeLa cells with rubropunctatin in the dark or under light irradiation resulted in a dose-dependent apoptosis, as validated by the increase in the percentage of cells in the sub-G1 phase and phosphatidylserine externalization, and the inductive effect on HeLa cell apoptosis was boosted by the light irradiation. In addition, treatment with rubropunctatin alone or under light irradiation was found to induce apoptosis in HeLa cells via the mitochondrial pathway, including loss of mitochondrial membrane potential, activation of caspase-3, caspase-8, and caspase-9, and increase of the level of intracellular reactive oxygen species (ROS). It was suggested that rubropunctatin could be a promising natural dual anticancer agent for photodynamic therapy and chemotherapy.

Development of a conjugate vaccine against invasive pneumococcal disease based on capsular polysaccharides coupled with PspA/family 1 protein of Streptococcus pneumoniae.

The efforts were focused on exploring alternative pneumococcal vaccine strategies, aimed at addressing the shortcomings of existing formulations, without compromising efficacy. Our strategy involved the use of the carrier protein, pneumococcal surface protein A (PspA), conjugated with capsular polysaccharides (CPS), to provide effective and non-serotype-dependent protection. In this study, we generated a stable Escherichia coli construct expressing functional PspA from a capsular serotype 6B strain and confirmed it belonging to family 1, which was conjugated with CPS. The distribution of anti-CPS antibody response was almost completely of IgG2a subclass followed by IgG3 and low level of IgG1 subclass, but that of anti-PspA IgG subclass antibodies was almost equal IgG1 and IgG2a subclasses. Though PspA was less conspicuous on the surface of pneumococci than the capsule, the antibodies induced with CPS-rPspA conjugate possessed more accessibility to the surface of Streptococcus pneumoniae serotype 6B and 19F (the same family 1 PspA). By survival experiment, the result suggested that the level of cross-protection after immunized with the conjugate was more measurable within the same family 1. The CPS-rPspA conjugate not only induced CPS-specific protection but also provided PspA specific cross-protection.

Synthesis and evaluation of ursolic acid derivatives as potent cytotoxic agents.

Structural modification was performed at the C-3 and C-28 positions of ursolic acid (UA). Ten UA derivatives with distinct electrical property were synthesized. They could be divided into two groups according to their charge under physiological conditions: (1) Group I negatively charged and (2) Group II positively charged. The anti-proliferative capability of the derivatives was evaluated against HepG2, AGS, HT-29 and PC-3 cells by the MTT assay. Flow cytometry and Annexin V/PI dual staining assay were carried out to explore the antitumor mechanism. The results showed the cytotoxic capacity of the compounds was: Group I

Microsphere-based flow cytometric immunoassay for the determination of citrinin in red yeast rice.

The determination of citrinin (CIT) by a microsphere-based flow cytometric immunoassay (MFCI) has been developed. In the method, the carboxyl-modified microspheres were conjugated with CIT-Ovalbumin (OVA) antigen. CIT competed with the CIT-OVA antigen on the surface of the microspheres for the anti-CIT McAb. Under the optimised conditions, IC(50) value was 1.0 ng/mL and the limit of detection reached 0.005 ng/mL. The cross-reactivity was less than 0.01% against each of the four mycotoxins such as aflatoxin B(1) (AFB(1)), ochratoxin A (OTA), zearalenone (ZEA), deoxynilvalenol (DON). In the work, the MFCI could accurately determine CIT in the real red yeast rice. The systematic error was low with the coefficient of variation (CV) from 5.24% to 8.16% by the MFCI. The mean recovery of CIT from artificially contaminated red yeast rice was from 89% to 94%, with CV from 7.2% to 8.7%. The experimental data showed that the precision, sensitivity and specificity of the developed MFCI method for the determination of CIT were satisfactory.

A sensitive and selective DNAzyme-based flow cytometric method for detecting Pb2+ ions.

A sensitive and selective Pb(2+) sensor based on GR-5 DNAzyme has been developed by a flow cytometric method.

A continuous and adsorptive bioprocess for efficient production of the natural aroma chemical 2-phenylethanol with yeast.

Natural 2-phenylethanol is a high value aroma chemical and can be produced from l-phenylalanine via Ehrlich pathway by yeasts. Due to serious product inhibition, the space-time yield is usually low. A continuous approach using macroporous resin as in situ adsorbent made it possible that the quantity and viability of the cells were improved simultaneously. With Saccharomyces cerevisiae sp. strain R-UV3, the highest space-time yield of 0.90 gL(-1)h(-1) reported so far was obtained.

The mechanism of the G0/G1 cell cycle phase arrest induced by activation of PXR in human cells.

CONTEXT: Pregnane X receptor (PXR) is an important transcriptional regulator that plays important roles in the cell metabolism and cell growth by regulating the transcriptional of a sort of metabolizing enzymes. OBJECTIVE: To investigate whether rifampicin effected HepG2 cells growth and the inhibition was due to the G0/G1 phase arrest. METHODS: PXR-knockdown experiments using RNAi showed that the cell cycle phase arrest mediated by rifampicin based on activation of PXR. The results also indicated that cell phase arrest by rifampicin could protect cells form UVB-induced DNA damage. Retinoid X receptor alpha (RXRα) expression level in cells is another key factor for cell cycle phase arrest mediated by rifampicin. Both over expression and lacking expression of RXRα in cell reduced the cell arrest efficiency mediated by rifampicin. In the study, we found that rifampicin inhibited HepG2 cells growth and demonstrated that the inhibition is due to the G0/G1 phase arrest through flow cytometry analysis. CONCLUSION: The results showed that RXRα promote cell cycle phase transition rate of HepG2. Competitive bind of rifampicin-activated PXR with RXRα is one main reason to arrest cell cycle phase through inhibiting combination of RXRα with other partners. Rifampicin could promote cell growth rate when RXRα expressed more excessively than PXR in cells.

Cloning and expression of hydroxynitrile lyase gene from Eriobotrya japonica in Pichia pastoris.

Hydroxynitrile lyase gene (hnl) from Eriobotrya japonica was successfully amplified using the method of SEFA PCR (Self-Formed Adaptor PCR). The complete sequence was 5.5 kbp in length, including 3100 bp of the upstream promoter region, 1659 bp of the coding sequence, three introns and 315 bp of the downstream transcription terminator. The phylogenetic analysis illustrated that the obtained hnl exhibited 66-70% identity to the reported isozymes from almond, black cherry and Japanese apricot. The EjHNL had 552 amino acids including a 25 amino acid-long signal peptide. The conserved characteristic structures of HNLs, such as FAD-binding motif, N-glycosylation sites and active sites were observed. The coding sequence of the hnl was inserted into pPIC9K vector for heterologous expression in Pichia pastoris. The HNL activity of the culture supernatant reached 15 U/ml after 96 h of induction by methanol. The specific activity of the recombinant HNL was about 197 U/mg. The enantiomeric excess value of the product R-mandelonitrile attained 98.6% and the value of K(m) of the recombinant HNL was determined to be 0.47 mM based on the kinetic data. The optimum temperature and pH of the recombinant HNL were 40°C and 6.0 respectively. The experimental data indicated that the obtained recombinant HNL showed similar catalytic characteristics with the natural EjHNL. The expression of the recombinant HNL in P. pastoris could present another available biocatalyst for the synthesis of R-selective cyanohydrins.

[Preparation and identification of OmpW monoclonal antibodies].

AIM: To prepare and characterize the mouse monoclonal antibodies against Vibrio parahaemolyticus OmpW. METHODS: The OmpW amino acid sequence from three diseased Vibrio was analyzed by Bioinformatics. Mice were immunized with r-OmpW which was highly expressed and purified in E.coli. Five Vibrio(Va, Vp, Vh, Vv, Van) were chosen as antigen for mAb selection.The characters of the anti-OmpW monoclonal antibodies were studied by Western blot, Flow Cytometry, indirect immunofluorescence. RESULTS: OmpW was testified a highly conservative membrane protein.Three clones of anti-OmpW mAb was obtained. The Ig subclass of the mAb secreted from fused cell S5C10 was IgG3, which of the titer was 4.6×10(4);. The mAb could specifically recognize Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, which could not react with Pseudomonas flurosecens, Aeromonas hydrophila, Aeromonas sobria, Aeromonas hydrophila, Escherichia coli. CONCLUSION: The mAb could specially recognize five diseased Vibrio, which is a useful tool for the further study of the diagnosis of Vibrio.

Synthesis of [3ß-acetoxy-urs-12-en-28-oyl]-1-monoglyceride and investigation on its anti tumor effects against BGC-823.

Ursolic acid (UA) as the leader compound was designed to prepare a series of derivatives (three novel compounds UA-1a, UA-1b and UA-2) by modification at the C3 and C28 positions. Their chemical structures were confirmed by IR, (1)H NMR and MS. The cytotoxic activity of the derivatives was evaluated against HepG2, BGC-823 and HT-29 by the MTT assay. The novel derivative UA-1a, [3ß-acetoxy-urs-12-en-28-oyl]-1-monoglyceride showed significant anti-growth ability against the assayed cancer cell lines, particularly against BGC-823, while low cytotoxicity to human normal gastric cell line GES-1. Further investigation revealed that UA-1a could induce apoptotic events of the treated BGC-823 cells, such as comet-like DNA bend, sub-G0/G1 phase accumulation and phosphatidylserine externalization. The activity of Caspase-3 was found to be up-regulated, while the expression of Bcl-2 and Survivin were down-regulated in UA-1a treated cells. UA-1a might trigger the death of BGC-823 cells by inducing apoptosis via the mitochondria pathway. UA-1a exerted stronger ability than Taxol to retard tumor growth in nude mice without leaving apparent toxicity to the hosts. The experimental data suggested that UA-1a would have a therapeutic potential in the treatment of gastric cancer.

Synergistic inhibition effect of 2-phenylethanol and ethanol on bioproduction of natural 2-phenylethanol by Saccharomyces cerevisiae and process enhancement.

Natural 2-phenylethanol (PEA) could be produced on a large scale by way of bioconversion with yeast from l-phenylalanine. In this work the synergistic inhibition effect of the target product PEA and the byproduct ethanol on the bioconversion rate by Saccharomyces cerevisiae R-UV3 was systematically studied and a new kinetic model with an item representing the synergistic effect was proposed. Optimization strategies to repress the inhibition effect of PEA and ethanol were carried out in the mode of fed-batch culture with ISPR. The glucose concentration was regulated at the level of 0.2±0.1g/L by controlling a suitable respiratory quotient on line, which could limit the accumulation of the ethanol lower than 10g/L. In the presence of resin FD0816 with a weight of 10% of the medium, PEA was removed from the broth and the overall PEA concentration and the space-time yield reached 13.7g/L and 0.39g L(-1) h(-1) respectively. The semi-continuous process with ISPR was performed, in which the replacement of the resin was operated repeatedly when the aqueous PEA was over 2.7g/L and bioconversion continued until the bioactivity of the yeast cells declined, consequently achieving a final overall PEA concentration of 32.5g/L and a space-time yield of 0.45g L(-1) h(-1).

In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives.

Twenty-three ursolic acid (1) derivatives 2-24 (ten novel compounds 8-10, 14-17 and 22-24) modified at the C-3 and the C-28 positions were synthesized, and their structures were confirmed by IR, (1)H NMR, MS, and elemental analysis. The single crystals of compounds 15 and 17 were obtained. The cytotoxic activity of the derivatives was evaluated against HepG2, BGC-823, SH-SY5Y, HeLa and HELF cells by the MTT assay. The induction of apoptosis and affects on the cell cycle distribution with compound 14 were assessed by fluorescence microscopy, flow cytometry and the activity of caspase-3 in HepG2 cells. Compounds 14-17 had more significant antiproliferative ability against the four cancer cell lines and low cytotoxicity to human embryonic lung fibroblast cells (HELF). Compounds 11, 14-16, 21 and 23 were particularly active against HepG2 cell growth. Compound 14 was selected to investigate cell apoptosis and cell cycle distribution. Flow cytometric analysis and morphologic changes of the cell exhibited that treatment of HepG2 cells with compound 14 led to cell apoptosis accompanied by cell cycle arrest at the S phase in a dose-dependent manner. Furthermore, the activity of the caspase-3 enzyme was increased in the treated cells. In vivo studies using H22 xenografts in Kunming mice were conducted with compound 14 at doses of 50, 100 and 150 mg/kg body weight. The results revealed that the medium dosage group (100 mg/kg) showed significant anticancer activity (45.6 ± 4.3%) compared to the control group.

Anti-cancer effect of rubropunctatin against human gastric carcinoma cells BGC-823.

The Monascus pigment, rubropunctatin, was extracted and purified from red mold rice (RMR) and its cytotoxic activities against human gastric adenocarcinoma BGC-823 cells were studied both in vitro and in vivo. Rubropunctatin inhibited the proliferation of BGC-823 cells with an inhibitory concentration (IC50) of 12.57 µM, while it exhibited no significant toxicity to normal gastric epithelial cell GES-1 at the same concentration. Treatment of BGC-823 cells with rubropunctatin resulted in a dose- and time-dependent apoptosis, as validated by the increase in the percentage of cells in sub-G1 phase and phosphotidylserine externalization. The in vivo experimental data demonstrated that rubropunctatin could offer similar therapeutic benefits in comparison with the same dose of taxol. After five times of intravenous injection, tumor weight in BGC-823-bearing nude mice reduced 23.5% at the dose of 8 mg/kg and 37.7% at the dose of 32 mg/kg, respectively. The expressions of 30 genes related to induction of apoptosis were found up-regulated significantly. The two most expressed genes were tumor necrosis factor (TNF) and DNA-damage inducible transcript 3. TNF was considered as a major mediator of apoptosis induced by rubropunctatin. This is the first report describing the anti-proliferative effect of rubropunctatin and its apoptosis mechanism on BGC-823 cells. Rubropunctatin has potential to be developed as a new natural anti-cancer agent.

Cytotoxicity of Monascus pigments and their derivatives to human cancer cells.

Six pigments were separated from Monascus product, and some derivatives were chemically synthesized. The cytotoxicity of different Monascus pigments to various human cancer cells (SH-SY5Y, HepG2, HT-29, BGC-823, AGS, and MKN45) was evaluated. Rubropunctatin showed the greatest anticancer effect within the tested compounds. The inhibition effect of rubropunctatin was higher than that of taxol on the growth of the human gastric cancer cell SH-SY5Y (P<0.05), BGC-823 (P<0.01), AGS (P<0.01), and MKN45 (P<0.05). On the other hand, its cytotoxicity to the normal human gastric epithelial cell GES-1 was less than that of taxol (P<0.01). The experimental data demonstrated that rubropunctatin was a valuable compound with high anticancer activity, which could offer better therapeutic benefits than taxol. Cell apoptosis stages were assayed by annexin V-EGFP/PI staining experiments using flow cytometry. The data showed that 87.63% of tested BGC-823 cells entered the early phase of apoptosis when treated with 5 microM rubropunctatin for 24 h. A drug concentration-dependent cell apoptosis was observed. The analysis of the relationship between pharmaceutical activity and the chemical structure of the tested compounds led to the conclusion that 6-internal ether, 4-carbonyl, and conjugated double bonds in the tricyclic structure of rubropunctatin were necessary to the anticancer effect, whereas the difference of C2H4 in the side chain showed little influence. Rubropunctatin could be supplied as a precursor compound in the development of a new natural anticancer reagent.