Changes in the volatile compounds and characteristic aroma during liquid-state fermentation of instant dark tea by Eurotium cristatum.

Instant dark tea (IDT) was prepared by liquid-state fermentation inoculating Eurotium cristatum. The changes in the volatile compounds and characteristic aroma of IDT during fermentation were analyzed using gas chromatography-mass spectrometry by collecting fermented samples after 0, 1, 3, 5, 7, and 9 days of fermentation. Components with high odor activity (log2FD ≥ 5) were verified by gas chromatography-olfactometry. A total of 107 compounds showed dynamic changes during fermentation over 9 days, including 17 alcohols, 7 acids, 10 ketones, 11 esters, 8 aldehydes, 37 hydrocarbons, 4 phenols, and 13 other compounds. The variety of flavor compounds increased gradually with time within the early stage and achieved a maximum of 79 compounds on day 7 of fermentation. ß-Damascenone showed the highest odor activity (log2FD = 9) in the day 7 sample, followed by linalool and geraniol. These results indicate that fungal fermentation is critical to the formation of these aromas of IDT.

Untargeted and targeted metabolomics reveal changes in the chemical constituents of instant dark tea during liquid-state fermentation by Eurotium cristatum.

Instant green tea powder was used as raw material to prepare an instant dark tea via liquid-state fermentation by Eurotium cristatum. To understand how the chemical constituents present in fermented green tea develop during fermentation, samples were collected on different days during fermentation for qualitative analyses by ultra-performance liquid chromatography-Q Exactive Orbitrap/Mass spectrometry. Untargeted metabolomics analyses revealed that the levels of original secondary metabolites in the instant green tea changed significantly from day 3 to day 5 during fermentation. Targeted metabolomics indicated that the levels of galloylated catechins (GCs) and free amino acids (FAAs) significantly decreased, but the nongalloylated catechins (NGCs), alkaloids, thearubigins and theabrownins increased dramatically after fermentation. The changes in the contents of catechins, gallic acid and free amino acids in the instant dark tea samples were positively related to the DPPH radical scavenging activities in vitro, and the phenolic acids and FAAs were positively related to the inhibitory effects towards α-glucosidase. These results showed that fermentation by Eurotium cristatum is critical to the formation of certain qualities of instant dark tea.

Homoharringtonine inhibits the progression of hepatocellular carcinoma by suppressing the PI3K/AKT/GSK3ß/Slug signaling pathway.

Homoharringtonine (HHT), was first isolated from the bark of Cephalotaxus harringtonia (Knight ex J. Forbes) K. Koch and Cephalotaxus fortunei Hook trees. The bark extract is used to treat leukemia and in recent years has also been used in traditional Chinese medicine (TCM) to treat solid tumors. However, the inhibitory mechanism of HHT in the progression of hepatocellular carcinoma (HCC) is rarely studied. We aimed to evaluate the antitumor efficacy of HHT on HCC in vitro and in vivo and elucidate the underlying molecular mechanism(s). HCC cell lines, including HCCLM3, HepG2, and Huh7, were used to evaluate the antitumor efficacy of HHT in vitro. Cytotoxicity and proliferative ability were evaluated by MTT and colony formation assays. Cell cycle progression and apoptosis in HHT-treated HCC cells were evaluated by flow cytometry. To determine the migration and invasion abilities of HCC cells, wound-healing and Transwell assays were used. Finally, western blot analysis was used to reveal the proteins involved. We also established a xenograft nude mouse model for in vivo assessments of the preclinical efficacy of HHT, mainly using hematoxylin and eosin staining, immunohistochemistry, ultrasound imaging (USI), and magnetic resonance imaging (MRI). HHT suppressed the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells, and induced cell cycle arrest at the G2 phase and apoptosis. In the HCC xenograft model, HHT showed an obvious tumor-suppressive effect. Surprisingly, Slug expression was also decreased by HHT via the PI3K/AKT/GSK3ß signaling pathway at least partially suppressed the growth of HCC via the PI3K/AKT/GSK3ß/Slug signaling pathway.

Influence of Eurotium cristatum and Aspergillus niger individual and collaborative inoculation on volatile profile in liquid-state fermentation of instant dark teas.

The three instant dark teas were produced from instant green tea (IGT) by liquid-state fermentations using the microorganisms Eurotium cristatum (EFT), Aspergillus niger (AFT), and sequential inoculation of E. cristatum/A. niger (EAFT), respectively. The volatile compounds of four tea samples were extracted by headspace-solid phase microextraction (HS-SPME) and analyzed using gas chromatography-mass spectrometry (GC-MS) coupled with chemometrics. A total of 97 volatile compounds were tentatively identified to distinguish three fermented instant dark from IGT. Alcohols, acids, esters, ketones, aldehydes, and heterocyclics could be clearly distinguished by principal component analysis (PCA), venn diagram, heatmap analysis and hierarchical cluster analysis (HCA). Descriptive sensory analysis revealed that AFT had a moldy, woody and herbal aroma; EFT showed woody and herbal aroma; and EAFT smelled an herbal, sweet, minty and floral aroma. This study indicates that fermentation using different microorganisms is critical in forming unique aroma characteristics of instant dark teas.

Feature-Based Molecular Networking Analysis of the Metabolites Produced by In Vitro Solid-State Fermentation Reveals Pathways for the Bioconversion of Epigallocatechin Gallate.

Dark teas are prepared by a microbial fermentation process. Flavan-3-ol B-ring fission analogues (FBRFAs) are some of the key bioactive constituents that characterize dark teas. The precursors and the synthetic mechanism involved in the formation of FBRFAs are not known. Using a unique solid-state fermentation system with ß-cyclodextrin inclusion complexation as well as targeted chromatographic isolation, spectroscopic identification, and Feature-based Molecular Networking on the Global Natural Products Social Molecular Networking web platform, we reveal that dihydromyricetin and the FBRFAs, including teadenol A and fuzhuanin A, are derived from epigallocatechin gallate upon exposure to fungal strains isolated from Fuzhuan brick tea. In particular, the strains from subphylum Pezizomycotina were key drivers for these B-/C-ring oxidation transformations. These are the same transformations seen during the fermentation process of dark teas. These discoveries set the stage to enrich dark teas and other food products for these health-promoting constituents.

Cyclovirobuxine D Exerts Anticancer Effects by Suppressing the EGFR-FAK-AKT/ERK1/2-Slug Signaling Pathway in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), the sixth most common malignancy worldwide, is characterized by a dismal prognosis due to high recurrence and metastasis rates. Thus, the need for the development of novel chemotherapeutic drugs is urgent. Cyclovirobuxine D (CVB-D), a steroidal alkaloid extracted from Buxus microphylla that has been extensively used to relieve the symptoms of cardiovascular diseases, has shown promising antineoplastic effects in recent studies. However, the therapeutic effects and underlying mechanisms of CVB-D on HCC remain largely unelucidated. This study experimentally indicated that CVB-D can repress HCC cell proliferation by arresting the cell cycle in G2 phase and can facilitate apoptosis. In addition, the migratory and invasive capabilities of HCC cells were noticeably attenuated by a nonlethal dose of CVB-D, and this attenuation was correlated with the inhibition of epithelial-mesenchymal transition (EMT). Moreover, in vivo, CVB-D displayed excellent anticancer effects in HCC tumor-bearing nude mice. Regarding the molecular mechanisms of CVB-D activity, decreased Slug expression was determined to be associated with the aforementioned anti-HCC functions of this extract, which might be regulated by epidermal growth factor receptor (EGFR) through the focal adhesion kinase (FAK)-associated PI3K/AKT and MEK/ERK1/2 signaling pathways. Collectively, our results revealed the suppressive effects of CVB-D on progressive behaviors of HCC, including proliferation, migration, invasion, and EMT, in addition to its outstanding proapoptotic effects, which were correlated with the inhibition of the EGFR-FAK-AKT/ERK1/2-Slug signaling pathway. These discoveries provide an experimental and theoretical foundation for the use of CVB-D as a promising candidate for HCC therapy.

Vitamin D3 Activates Phosphatidylinositol-3-Kinase/Protein Kinase B via Insulin-Like Growth Factor-1 to Improve Testicular Function in Diabetic Rats.

OBJECTIVE: In diabetes mellitus, vitamin D3 deficiency affects sex hormone levels and male fertility; however, the mechanism leading to the disorder is unclear. This research was designed to investigate the mechanism of vitamin D3 deficiency and hypogonadism in diabetic rats. Our aim was to assess serum vitamin D3 levels and the relationship among vitamin D3, insulin-like growth factor-1 (IGF-1), and testicular function. MATERIALS AND METHODS: Rats with streptozotocin-induced diabetes were randomly divided into four groups and treated with different doses of vitamin D3: no vitamin D3, low (0.025 µg/kg/day), high (0.1 µg/kg/day), and high (0.1 µg/kg/day) with JB-1 (the insulin-like growth factor-1 receptor inhibitor group, 100 µg/kg/day). The groups were compared with wild-type rats, which function as the control group. Various parameters such as vitamin D3 and IGF-1 were compared between the experimental and wild-type groups, and their correlations were determined. RESULTS: Twelve weeks of vitamin D3 supplementation improved the testosterone levels, as shown by the increase in the level of serum IGF-1 in diabetic rats. Phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT), which was a downstream of the signaling pathway of IGF-1, was significantly increased after vitamin D3 treatment. CONCLUSIONS: The study shows that vitamin D3 may promote the expression of testosterone and improve testicular function in diabetic rats by activating PI3K/AKT via IGF-1.

Oncogenic MSH6-CXCR4-TGFB1 Feedback Loop: A Novel Therapeutic Target of Photothermal Therapy in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) has been considered the most aggressive glioma type. Temozolomide (TMZ) is the main first-line chemotherapeutic agent for GBM. Decreased mutS homolog 6 (MSH6) expression is clinically recognized as one of the principal reasons for GBM resistance to TMZ. However, the specific functions of MSH6 in GBM, in addition to its role in mismatch repair, remain unknown. Methods: Bioinformatics were employed to analyze MSH6 mRNA and protein levels in GBM clinical samples and to predict the potential cancer-promoting functions and mechanisms of MSH6. MSH6 levels were silenced or overexpressed in GBM cells to assess its functional effects in vitro and in vivo. Western blot, qRT-PCR, and immunofluorescence assays were used to explore the relevant molecular mechanisms. Cu2(OH)PO4@PAA nanoparticles were fabricated through a hydrothermal method. Their MRI and photothermal effects as well as their effect on restraining the MSH6-CXCR4-TGFB1 feedback loop were investigated in vitro and in vivo. Results: We demonstrated that MSH6 is an overexpressed oncogene in human GBM tissues. MSH6, CXCR4 and TGFB1 formed a triangular MSH6-CXCR4-TGFB1 feedback loop that accelerated gliomagenesis, proliferation (G1 phase), migration and invasion (epithelial-to-mesenchymal transition; EMT), stemness, angiogenesis and antiapoptotic effects by regulating the p-STAT3/Slug and p-Smad2/3/ZEB2 signaling pathways in GBM. In addition, the MSH6-CXCR4-TGFB1 feedback loop was a vital marker of GBM, making it a promising therapeutic target. Notably, photothermal therapy (PTT) mediated by Cu2(OH)PO4@PAA + near infrared (NIR) irradiation showed outstanding therapeutic effects, which might be associated with a repressed MSH6-CXCR4-TGFB1 feedback loop and its downstream factors in GBM. Simultaneously, the prominent MR imaging (T1WI) ability of Cu2(OH)PO4@PAA could provide visual guidance for PTT. Conclusions: Our findings indicate that the oncogenic MSH6-CXCR4-TGFB1 feedback loop is a novel therapeutic target for GBM and that PTT is associated with the inhibition of the MSH6-CXCR4-TGFB1 loop.

Vitamin D3 supplementation improves testicular function in diabetic rats through peroxisome proliferator-activated receptor-γ/transforming growth factor-beta 1/nuclear factor-kappa B.

AIMS/INTRODUCTION: Vitamin D3 deficiency can lead to male hypogonadism in diabetes mellitus, but the target organs and the mechanism driving the disorder are unclear. This experiment was designed to study the relationship between vitamin D3 deficiency and hypogonadism in diabetes mellitus. MATERIALS AND METHODS: Rats with streptozotocin-induced diabetes were randomly divided into four groups and treated with different doses of vitamin D3 : blank (no vitamin D3 ), low (0.025 µg/kg/day), high (0.1 µg/kg/day), high (0.1 µg/kg/day) and with bisphenol A diglycidyl ether (peroxisome proliferator-activated receptor gamma inhibitor 30 mg/kg/day). They were compared with wild-type rats. RESULTS: After 12 weeks, the vitamin D3 supplements had partially restored testicular pathological changes, as shown by reduced testicular fibrosis related to downregulation transforming growth factor beta 1 and apoptosis related to downregulation of nuclear factor kappa B, but not the pituitary gland. The expression of peroxisome proliferator-activated receptor gamma, which can inhibit transforming growth factor beta 1 and nuclear factor kappa B, was significantly increased after treatment with vitamin D3 . CONCLUSIONS: These results suggest that treatment with vitamin D3 can improve testicular function in diabetic rats through the peroxisome proliferator-activated receptor gamma/transforming growth factor beta 1/nuclear factor kappa B signaling pathway.

Bioassay-guided isolation of a Mycobacterium tuberculosis bioflim inhibitor from Arisaema sinii Krause.

Mycobacterium tuberculosis biofilms harbour drug-tolerant bacteria. Identification of drugs that inhibit biofilm formation could enable the dramatic shortening of tuberculosis treatments using standard antibiotics. Arisaema sinii Krause is used to treat pulmonary and lymphatic tuberculosis by Dong People of China. Current study was aimed to purify the active components against M. tuberculosis biofilms from Arisaema sinii extract by using bioassay-guided isolation. (E)-2-(methyl (phenyl) amino) ethyl 2-(2-hydroxyundecanamido)-7, 11-dimethyl-3-oxotetradec-4-enoate, compound 1, was identified as the active component. It could inhibit mycobacterial biofilm formation, disperse the preformed biofilms, and disrupt the mature biofilms at concentration of 4, 8, and 32 µg/ml, respectively. At the dose of 32 µg/ml, it could potentiate the bactericidal activity of isoniazid against M. tuberculosis in mature biofilms. The results of this study indicate that compound 1 might be a novel lead compound against mycobacterial biofilm formation.

Paramagnetic CuS hollow nanoflowers for T2-FLAIR magnetic resonance imaging-guided thermochemotherapy of cancer.

The development of nanoplatforms with integrated therapeutic and imaging functions is necessary for highly efficient cancer therapy. Herein, 3D CuS hollow nanoflowers (HNs) consisting of 2D nanoplates are successfully fabricated through the technique of laser ablation in liquids followed by ion-exchange reactions and applied for the first time as a theranostic nanoagent for magnetic resonance imaging (MRI), photothermal therapy (PTT), and chemotherapy simultaneously. Due to the sufficient and immediate contact between the exposed cupric centers of nanoplates and protons from water molecules, CuS HNs are demonstrated to be capable of being a T1 positive contrast agent for efficient MRI of tumors on the T2-weighted fluid-attenuated inversion recovery imaging (T2-FLAIR) sequence. Besides, the hollow structure makes CuS HNs an efficient nanoplatform for drug loading with a laser-triggered drug release. Moreover, CuS HNs exhibit high photothermal conversion efficiency (30%) and good biocompatibility. The combination of PTT and chemotherapy with CuS HNs provides a significant synergistic therapeutic effect, resulting in a higher tumor inhibition ratio than PTT or chemotherapy alone. This study demonstrates a single-component multifunctional theranostic nanoagent for T2-FLAIR MRI guided thermochemotherapy, which has great potential application in theranostics of cancer.

Hyperthermia with different temperatures inhibits proliferation and promotes apoptosis through the EGFR/STAT3 pathway in C6 rat glioma cells.

Malignant gliomas are a group of aggressive neoplasms among human cancers. The curative effects of current treatments are finite for improving the prognosis of patients. Hyperthermia (HT) is an effective treatment for cancers; however, the effects of HT with different temperatures in treatment of MG and relevant mechanisms remain unclear. MTT assay and Annexin V­fluorescein isothiocyanate/propidium iodide staining were used for investigating the proliferation and apoptosis of C6 cells, respectively. Western blotting was applied to detect the expression of proteins. Ultrasonography was employed to evaluate the tumor formation rate, growth rate, angiogenesis rate and degree of hardness of tumors in vivo. The authors certified that HT with 42­46˚C x 1 h, 1 t could inhibit proliferation, promote apoptosis, reduce tumor formation rate, growth rate, angiogenesis rate, degree of hardness of tumors, ischemic tolerance and anoxic tolerance, and have synergy with temozolomide in C6 cells. Long­term HT (43˚C x 1 h, 1 t/5 d, 90 d) did not cut down the sensitivity of C6 cells to HT, and sustainably inhibited the proliferation of C6 cells. Furthermore, the authors proved HT produced these effects primarily through inhibition of the EGFR/STAT3/HIF­1A/VEGF­A pathway.