Anti-adipogenic Effects of Sulforaphane-rich Ingredient with Broccoli Sprout and Mustard Seed in 3T3-L1 Preadipocytes.

Glucoraphanin (GRA) is a precursor of sulforaphane (SFN), which can be synthesized by the enzyme myrosinase. In this study, we developed and validated HPLC analytical methods for the determination of GRA and SFN in mustard seed powder (MSP), broccoli sprout powder (BSP), and the MSP-BSP mixture powder (MBP), and evaluated their anti-adipogenic effects in 3T3-L1 adipocytes. We found that the analysis methods were suitable for the determination of GRA and SFN in MSP, BSP, and MBP. The content of GRA in BSP was 131.11 ± 1.84 µmol/g, and the content of SFN in MBP was 162.29 ± 1.24 µmol/g. In addition, BSP and MBP effectively decreased lipid accumulation content without any cytotoxicity. Both BSP and MBP significantly inhibited the expression of adipogenic proteins and increased the expression of proteins related to lipolysis and lipid metabolism. BSP and MBP inhibited the expression of adipocyte protein 2 (aP2), CCAAT/enhancer-binding protein-α (C/EBP-α), and peroxisome proliferator-activated receptor-γ (PPAR-γ) in 3T3-L1 adipocytes, and inhibited the expression of fatty acid synthase (FAS) through AMP-activated protein kinase (AMPK). Meanwhile, BSP and MBP also increased the expression of the lipolysis-related proteins, uncoupling protein-1 (UCP-1) and carnitine palmitoyltransferase-1 (CPT-1). Moreover, MBP exerted anti-adipogenic to a greater extent than BSP in 3T3-L1 preadipocytes.

Effects of gamma aminobutyric acid on performance, blood cell of broiler subjected to multi-stress environments.

OBJECTIVE: Stress factors such as high temperatures, overcrowding, and diurnal temperature range exert profound negative effects on weight gain and productivity of broiler chickens. The potential of gamma aminobutyric acid (GABA) as an excitatory neurotransmitter was evaluated under various stress conditions in this study. METHODS: The experiment was conducted under four different environmental conditions: normal, high temperature, overcrowded, and in an overcrowded-diurnal temperature range. The experimental groups were divided into (-) control group without stress, (+) control group with stress, and G50 group (GABA 50 mg/kg) with stress. Weight gain, feed intake, and feed conversion ratio were measured, and stress reduction was evaluated through hematologic analysis. RESULTS: The effects of GABA on broilers in four experimental treatments were evaluated. GABA treated responded to environmental stress and improved productivity in all the experimental treatments. The magnitude of stress observed was highest at high temperature, followed by the overcrowded environment, and was least for the overcrowded-diurnal temperature range. CONCLUSION: Various stress factors in livestock rearing environment can reduce productivity and increase disease incidence and mortality rate. To address these challenges, GABA, an inhibitory neurotransmitter, was shown to reduce stress caused due to various environmental conditions and improve productivity.

Anti-Obesogenic Effects of Sulforaphane-Rich Broccoli (Brassica oleracea var. italica) Sprouts and Myrosinase-Rich Mustard (Sinapis alba L.) Seeds In Vitro and In Vivo.

Glucoraphanin (GRA), a glucosinolate particularly abundant in broccoli (Brassica oleracea var. italica) sprouts, can be converted to sulforaphane (SFN) by the enzyme myrosinase. Herein, we investigated the anti-obesogenic effects of broccoli sprout powder (BSP), mustard (Sinapis alba L.) seed powder (MSP), and sulforaphane-rich MSP-BSP mixture powder (MBP) in bisphenol A (BPA)-induced 3T3-L1 cells and obese C57BL/6J mice. In vitro experiments showed that MBP, BSP, and MSP have no cytotoxic effects. Moreover, MBP and BSP inhibited the lipid accumulation in BPA-induced 3T3-L1 cells. In BPA-induced obese mice, BSP and MBP treatment inhibited body weight gain and ameliorated dyslipidemia. Furthermore, our results showed that BSP and MBP could activate AMPK, which increases ACC phosphorylation, accompanied by the upregulation of lipolysis-associated proteins (UCP-1 and CPT-1) and downregulation of adipogenesis-related proteins (C/EBP-α, FAS, aP2, PPAR-γ, and SREBP-1c), both in vitro and in vivo. Interestingly, MBP exerted a greater anti-obesogenic effect than BSP. Taken together, these findings indicate that BSP and MBP could inhibit BPA-induced adipocyte differentiation and adipogenesis by increasing the expression of the proteins related to lipid metabolism and lipolysis, effectively treating BPA-induced obesity. Thus, BSP and MBP can be developed as effective anti-obesogenic drugs.

A formulation development strategy for dual-release bilayer tablets: An integrated approach of quality by design and a placebo layer.

Although dual-release mechanism bilayer tablets containing one drug in both immediate- and sustained-release layers are widely used to improve therapeutic efficiency, studies quantitatively analyzing the drug amount released from each layer and the mutual effect of each layer's mechanical properties on drug product quality are limited. Here, the formulation of a dual-release bilayer tablet containing sarpogrelate HCl was optimized with a placebo layer and quality by design (QbD) approach. The placebo layer was developed to replace the active pharmaceutical ingredient and its mechanical properties were evaluated. The formulation was developed using the placebo layer to quantitatively analyze the drug released from each layer. The mixture design and Monte Carlo simulation enabled robust design space identification. The mutual effect of each layer's mechanical properties on drug product quality was confirmed by multivariate analysis using the optimal settings in the design space. The optimized formulation was characterized by comparison with a reference drug for various quality attributes and in vivo pharmacokinetic parameters, which ensured the bioequivalence of the optimized bilayer tablet with the reference drug. This study shows that the integration of QbD and a placebo layer is an effective optimization strategy for dual-release bilayer tablets containing one drug in different layers.

Gamma-Aminobutyric Acid Increases Erythropoietin by Activation of Citrate Cycle and Stimulation of Hypoxia-Inducible Factors Expression in Rats.

Erythropoietin (EPO) is the primary regulator of erythropoiesis in the mammalian fetus and adult. Deficiency of EPO induces anemia. In this study, we investigated the effect of gamma-aminobutyric acid (GABA) on serum EPO levels and erythropoiesis in rats. Expression levels of Epo-related genes were measured by quantitative real-time PCR (qPCR) and expression of Epo and Epo receptor (Epor) proteins were measured by immunohistochemistry. The gene and protein expression profiles of kidney tissue in GABA-treated rats were evaluated by ribonucleic acid (RNA) sequencing and two-dimensional electrophoresis (2-DE), respectively. GABA significantly increased serum EPO levels and expression levels of Epo and Epor. GABA increased expression levels of hypoxia-inducible factor (Hif)-1 and Hif-2. Seven proteins with expression levels showing >2-fold change were identified by 2-DE followed by MALDI-TOF MS in GABA-treated rat kidney. The top KEGG pathway from the identified proteins was the tricarboxylic acid cycle, and nicotinamide adenine dinucleotide (NADH) dehydrogenase, succinate dehydrogenase, and isocitrate dehydrogenase were identified as key proteins. GABA treatment significantly increased ATP levels and NADH dehydrogenase activity in a dose-dependent manner. In conclusion, GABA shows a new physiological role in EPO production, and it can thus can contribute to the prevention of anemia when used alone or in combination with other anemia treating drugs.

Comprehensive Study of Intermediate and Critical Quality Attributes for Process Control of High-Shear Wet Granulation Using Multivariate Analysis and The Quality by Design Approach.

A robust manufacturing process and the relationship between intermediate quality attributes (IQAs), critical quality attributes (CQAs), and critical process parameters (CPPs) for high-shear wet granulation was determined in this study. Based on quality by the design (QbD) approach, IQAs, CQAs, and CPPs of a telmisartan tablet prepared by high-shear wet granulation were determined and then analyzed with multivariate analysis (MVA) to evaluate mutual interactions between IQAs, CQAs, and CPPs. The effects of the CPPs on the IQAs and CQAs were quantitatively predicted with empirical models of best fit. The models were used to define operating space, and an evaluation of the risk of uncertainty in model prediction was performed using Monte Carlo simulation. MVA showed that granule size and granule hardness were significantly related to % dissolution. In addition, granule FE (Flow Energy) and Carr's index had effects on tablet tensile strength. Using the manufacture of a clinical batch and robustness testing, a scale-up from lab to pilot scale was performed using geometric similarity, agitator torque profile, and agitator tip speed. The absolute biases and relative bias percentages of the IQAs and CQAs generated by the lab and pilot scale process exhibited small differences. Therefore, the results suggest that a risk reduction in the manufacturing process can be obtained with integrated process parameters as a result of the QbD approach, and the relationship between IQAs, CQAs, and CPPs can be used to predict CQAs for a control strategy and SUPAC (Scale-Up and Post-Approval Guidance).

Induction of functional erythropoietin and erythropoietin receptor gene expression by gamma-aminobutyric acid and piperine in kidney epithelial cells.

AIMS: The aim of this study was to evaluate gamma-aminobutyric acid (GABA)- and piperine-induced erythropoietin (EPO) and EPO-receptor expression. MATERIALS AND METHODS: The effect of GABA and piperine on cell viability was examined using kidney epithelial cells. Expression levels of EPO and EPO-R mRNA and protein were evaluated in response to GABA and piperine treatments. GABA- and piperine-mediated activation of the mitogen-activated protein kinase (MAPK) signaling pathway was investigated. Additionally, EPO function was evaluated using conditioned media containing EPO. The GABA receptor type involved in this process was identified. KEY FINDINGS: Messenger RNA and protein expression levels of EPO and EPO-R significantly increased in response to treatment with GABA, piperine, or the combination of both, compared with control. GABA plus piperine synergistically enhanced EPO and EPO-R expression through p38 and c-Jun N-terminal kinase (JNK) MAPK signaling pathways, but not through the extracellular signal-regulated kinase (ERK) MAPK pathway. SB203580 and SP600125 (p38 and JNK pathway inhibitors, respectively) attenuated GABA plus piperine-induced EPO and EPO-R expression. Treatment of macrophages with EPO-containing conditioned media induced mRNA expression of interleukin (IL)-10 and nuclear factor (NF)-κB due to the interaction between EPO and EPO-R. Interestingly, GABA-induced EPO and EPO-R expression was mediated through GABAA, not GABAB, receptor activation. SIGNIFICANCE: These findings demonstrate that GABA plus piperine-mediated p38 and JNK MAPK activation increases EPO and EPO-R expression, resulting in up-regulation of IL-10 and NF-κB.

Effects of herbal medicine on human uterine tumor-bearing nude mice.

AIM: Uterine leiomyomas are the most common benign uterine neoplasms associated with significant morbidity. Herbal formulas capable of restoring yin-yang balance by dispersing blood stasis may be useful for managing fibroid symptoms. MATERIALS AND METHODS: In this study, the antitumor properties of three herbs viz., Trogopterus xanthipes Milen-Edwards, Paeonia lactiflora Pallas, and Ulmus davidiana Planch were evaluated in nude mice injected intravenously with human malignant myomas. Tumor fragments were xenografted subcutaneously through a flank incision in female mice. The mice entered the study for 8 weeks when their tumors reached the threshold volume (260 mm3). The mice were randomly allocated to receive subcutaneous injections of normal saline (Group 1; negative control), P. lactiflora Pallas (Group 2), U. davidiana Planch (Group 3), T. xanthipes Milen-Edwards (Group 4), and intravenous injections of paclitaxel (Group 5; positive control). The weight and tumor volume were measured, followed by histopathology. RESULTS: A few cases of abdominal distention and death were observed in the negative control group. Furthermore, a considerable enlargement of the liver and spleen was observed in the negative control group at autopsy with a gradual increase in body weight during the experiment. The mean tumor volume which increased in negative control mice reduced in mice treated with herbal remedies or paclitaxel from day 14 onwards (P < 0.05). The degree of necrosis and apoptosis induction from herbal treatments was similar to that of paclitaxel. CONCLUSION: Collectively, three herbs viz., T. xanthipes Milen-Edwards, P. lactiflora Pallas, and U. davidiana Planch were able to induce necrosis and apoptosis of uterine leiomyoma cells, proving antitumor properties against uterine fibroids.

Effect of Coating Method on the Survival Rate of L. plantarum for Chicken Feed.

This study was designed to find the most suitable method and wall material for microencapsulation of the Lactobacillus plantarum to maintain cell viability in different environmental conditions. To improve the stability of L. plantarum, we developed an encapsulation system of L. plantarum, using water-in-oil emulsion system. For the encapsulation of L. plantarum, corn starch and glyceryl monostearate were selected to form gel beads. Then 10% (w/v) of starch was gelatinized by autoclaving to transit gel state, and cooled down at 60ºC and mixed with L. plantarum to encapsulate it. The encapsulated L. plantarum was tested for the tolerance of acidic conditions at different temperatures to investigate the encapsulation ability. The study indicated that the survival rate of the microencapsulated cells in starch matrix was significantly higher than that of free cells in low pH conditions with relatively higher temperature. The results showed that corn starch as a wall material and glycerol monostearate as a gelling agent in encapsulation could play a role in the viability of lactic acid bacteria in extreme conditions. Using the current study, it would be possible to formulate a new water-in-oil system as applied in the protection of L. plantarum from the gastric conditions for the encapsulation system used in chicken feed industry.

Tumoral acidic extracellular pH targeting of pH-responsive MPEG-poly(beta-amino ester) block copolymer micelles for cancer therapy.

The main objective of this study was to develop and characterize a pH-responsive and biodegradable polymeric micelle as a tumor-targeting drug delivery system. The pH-responsive block copolymer was synthesized by a Michael-type step polymerization of hydrophilic methyl ether poly(ethylene glycol) (MPEG) and pH-responsive and biodegradable poly(beta-amino ester), resulting in an amphiphilic MPEG-poly(beta-amino ester) block copolymer. This copolymer, which formed nano-sized self-assembled micelles under aqueous conditions, could be efficiently (74.5%) loaded with doxorubicin (DOX) using a solvent evaporation method. In an in vitro drug release study, these DOX-loaded polymeric micelles showed noticeable pH-dependent micellization-demicellization behavior, with rapid release of DOX from the micelles in weakly acidic environments (pH 6.4) but very slow release under physiological conditions (pH 7.4). Moreover, due to demicellization, the tumor cell uptake of DOX released from polymeric micelles was much higher at pH 6.4 than at pH 7.4. When in vivo anti-tumor activity of pH-responsive polymeric micelles was evaluated by injecting the DOX-loaded polymeric micelles into B16F10 tumor-bearing mice, these micelles notably suppressed tumor growth and also prolonged survival of the tumor-bearing mice, compared with mice treated with free DOX.