VPS37C facilitates erythroid differentiation by promoting EKLF stability.

The process of erythroid differentiation is orchestrated at the molecular level by a complex network of transcription factors. Erythroid Krüppel-like factor (EKLF/KLF1) is a master erythroid gene regulator that directly regulates most aspects of terminal erythroid differentiation. However, the underlying regulatory mechanisms of EKLF protein stability are still largely unknown. In this study, we identified Vacuolar protein sorting 37 C (VPS37C), a core subunit of the Endosomal sorting complex required for transport-I (ESCRT-I) complex, as an essential regulator of EKLF stability. Our study showed that VPS37C interacts with EKLF and prevents K48-linked polyubiquitination of EKLF and proteasome-mediated EKLF degradation, thus enhancing EKLF protein stability and transcriptional activity. VPS37C overexpression in murine erythroleukemia (MEL) cells promotes hexamethylene bisacetamide (HMBA)-induced erythroid differentiation manifested by up-regulating erythroid-specific EKLF target genes and increasing benzidine-positive cells. In contrast, VPS37C knockdown inhibits HMBA-induced MEL cell erythroid differentiation. Particularly, the restoration of EKLF expression in VPS37C-knockdown MEL cells reverses erythroid-specific gene expression and hemoglobin production. Collectively, our study demonstrated VPS37C is a novel regulator of EKLF ubiquitination and degradation, which plays a positive role in erythroid differentiation of MEL cells by enhancing EKLF protein stability.

Effects of processing methods on quality, antioxidant capacity, and cytotoxicity of Ginkgo biloba leaf tea product.

BACKGROUND: Ginkgo biloba leaves contain beneficial flavonoids, bilobalide (BB), and ginkgolides. However, the toxic ginkgolic acid (GA) limit its application. In this study, various traditional processing methods were used to prepare G. biloba leaf tea (GBLT), including white tea, black tea, dark tea, green tea, and freeze-dried as control, followed by investigations of their effects on quality, antioxidant capacity, bioactive components, and cytotoxicity of the tea products. RESULTS: Results showed that different processing methods significantly impact the tea products' quality indexes and the principal component analysis (PCA) and hierarchical cluster analysis (HCA) corroborated it. White tea had the highest total sugar (TS) and GA content and the most potent cytotoxicity on HepG2 cells. However, TS and GA content and the cytotoxicity of GBLT markedly decreased during fermentation and fixation. Moreover, white tea possessed higher total phenolic content (TPC), total flavonoid content (TFC), and more vigorous antioxidant activities than green tea, black tea, and dark tea. Terpene trilactones value was stable, but different catechins contents fluctuated according to the manufacturing process of different GBLTs. Among the four GBLTs, dark tea combining fixation and fermentation had the lowest GA content and cytotoxicity, less bioactive components reduction, appropriate quality, and stronger flavor. CONCLUSION: These findings demonstrate that fixation and fermentation help reduce GAs during the manufacturing of GBLT. However, their ability to retain bioactive substances needs further optimization in future studies. © 2023 Society of Chemical Industry.

GPS2 promotes erythroid differentiation by control of the stability of EKLF protein.

Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2-/- embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.

EDAG mediates Hsp70 nuclear localization in erythroblasts and rescues dyserythropoiesis in myelodysplastic syndrome.

During human erythroid maturation, Hsp70 translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. Failure of Hsp70 to localize to the nucleus was found in Myelodysplastic syndrome (MDS) erythroblasts and can induce dyserythropoiesis, with arrest of maturation and death of erythroblasts. However, the mechanism of the nuclear trafficking of Hsp70 in erythroblasts remains unknown. Here, we found the hematopoietic transcriptional regulator, EDAG, to be a novel binding partner of Hsp70 that forms a protein complex with Hsp70 and GATA-1 during human normal erythroid differentiation. EDAG overexpression blocked the cytoplasmic translocation of Hsp70 induced by EPO deprivation, inhibited GATA-1 degradation, thereby promoting erythroid maturation in an Hsp70-dependent manner. Furthermore, in myelodysplastic syndrome (MDS) patients with dyserythropoiesis, EDAG is dramatically down-regulated, and forced expression of EDAG has been found to restore the localization of Hsp70 in the nucleus and elevate the protein level of GATA-1 to a significant extent. In addition, EDAG rescued the dyserythropoiesis of MDS patients by increasing erythroid differentiation and decreasing cell apoptosis. This study demonstrates the molecular mechanism of Hsp70 nuclear sustaining during erythroid maturation and establishes that EDAG might be a suitable therapeutic target for dyserythropoiesis in MDS patients.

Effect of different drying methods on product quality, bioactive and toxic components of Ginkgo biloba L. seed.

BACKGROUND: Ginkgo biloba seeds are used as a functional food across Asia. However, the presence of toxic compounds has limited their application. In this study, freeze drying, infrared drying, hot-air drying and pulsed-vacuum drying were used to dry G. biloba seeds. A comprehensive analysis was performed on their product quality, antioxidant activities, bioactive and toxic components. RESULTS: Results showed that the drying methods had a significant influence on product quality with freeze drying being superior due to the minimal microstructural damage, followed by infrared drying and pulsed-vacuum drying. Infrared-dried product possessed the strongest antioxidant activities and higher bioactive compound content than hot-air-dried and pulsed-vacuum-dried product. Toxic compounds in fresh G. biloba seeds (ginkgotoxin, ginkgolic acid and cyanide) were reduced markedly by drying. Ginkgotoxin was reduced fourfold, and the contents of acrylamide, ginkgolic acid and cyanide in dried G. biloba seeds were reduced to the scope of safety. Amongst the four drying methods, infrared drying had the shortest drying time, and its product showed higher quality and bioactive compound content, and stronger antioxidant activities. CONCLUSIONS: These findings will offer salient information for selecting a drying method during the processing of ginkgo seeds. Infrared drying could be considered as a multiple-effect drying method in the processing of ginkgo seeds. © 2020 Society of Chemical Industry.

NLRP3 is dispensable for d-galactosamine/lipopolysaccharide-induced acute liver failure.

The nucleotide-binding domain and leucine-rich repeat-containing family pyrin domain containing 3 (NLRP3) inflammasome is involved in various acute and chronic liver diseases, however, it is not clear whether NLRP3 contributes to d-Galactosamine (D-GalN) plus lipopolysaccharide (LPS)-induced acute liver failure (ALF). This study aims to investigate the role of NLRP3 inflammasome in D-GalN/LPS-induced fatal hepatitis. We found that Nlrp3-/- and WT mice showed similar mortality against a lethal dose of D-GalN/LPS treatment. Serum ALT and AST levels, as well as liver necrosis area and hepatocyte apoptosis, were not significantly different between Nlrp3-/- and WT mice at 6 h after D-GalN/LPS injection. Moreover, the numbers of intrahepatic F4/80+ cells and Ly6G+ cells were comparable in two genotype mice following D-GalN/LPS treatment. Besides, Nlrp3-/- mice had reduced IL-1ß levels but similar TNF-α, IL-6, and MCP-1 levels compared with WT mice upon D-GalN/LPS administration. Our findings revealed that NLRP3 ablation does not protect mice from D-GalN/LPS-induced fatal hepatitis and has a marginal effect on intrahepatic inflammatory response upon D-GalN/LPS treatment. This suggests that NLRP3 inflammasome does not appear to be a major contributor to D-GalN/LPS-induced ALF.

Toll-like receptor 5 signaling restrains T-cell/natural killer T-cell activation and protects against concanavalin A-induced hepatic injury.

Toll-like receptor-5 (TLR5) signaling regulates the immune privileged status of the liver and is involved in hepatic immune disorders. However, the role of TLR5 has not yet been investigated in experimental models of concanavalin A (Con A)-mediated liver injury. Here, we show that TLR5 is highly up-regulated in the hepatic mononuclear cells of mice during Con A-induced hepatitis. Increased mortality and liver histopathology of TLR5-deficient mice correlated with excessive production of proinflammatory cytokines, suggesting that TLR5 knockout mice were more susceptible to Con A-induced hepatitis. We also report that administration of CBLB502, an exogenous TLR5 agonist, substantially alleviated Con A-mediated hepatitis in wild-type mice as shown by increased survival rates, reduced aminotransferase and proinflammatory cytokine production, impaired lymphocyte infiltration, and ameliorated hepatocyte necrosis and/or apoptosis. Mechanistic studies revealed that CBLB502 acts as a negative regulator in limiting T-cell/natural killer T-cell activity and cytokine production in the Con A-hepatitis model. Bone marrow transplantation experiments showed that TLR5 in bone marrow-derived cells contributed to the hepatoprotective efficacy of CBLB502 against Con A-induced liver injury. Moreover, interleukin-6 elevation induced by CBLB502 is an important protective factor against Con A-induced liver injury. In addition, we demonstrate that CBLB502 suppresses α-galactosylceramide-induced natural killer T cell-dependent inflammatory liver injury. CONCLUSION: The TLR5 signaling pathway plays an important role in T cell-mediated hepatic injury and may be exploited for therapeutic treatment of inflammatory liver diseases. (Hepatology 2017;65:2059-2073).

Alkaloids from Oxytropis ochrocephala and antiproliferative activity of sophoridine derivatives against cancer cell lines.

Ten alkaloids (1-10), with sophoridine (1) as the most abundant component, were obtained from the whole plants of Oxytropis ochrocephala Bunge. Furthermore, eight new sophoridine derivatives (11-16, 20, 21), with modification on the C-14 position of 1 were synthesized. All compounds (1-16, 20, 21) were evaluated for antiproliferative activity against five human tumor cell lines. Among them, the newly synthesized derivative 20 exhibited the best inhibitory activity against the tested cell lines. Its activity was increased by more than fourfold as compared with parent compound 1.

Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1α transactivation.

Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1α and ATM. Moreover, ATM enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1α, a mutation in Ser249 resulted in a pronounced decrease in HNF1α transactivation, whereas no dominant-negative effect was observed. The HNF1αSer249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1αSer249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1α by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis.

Hepassocin is required for hepatic outgrowth during zebrafish hepatogenesis.

BACKGROUND & AIMS: Hepassocin (HPS) is a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage. In this paper, zebrafish were used to investigate the role of HPS in liver development. METHODS AND RESULTS: During zebrafish development, HPS expression is enriched in liver throughout hepatogenesis. Knockdown of HPS using its specific morpholino leads to a smaller liver phenotype. Further results showed that the HPS knockdown has no effect on the expression of the early endoderm marker gata6 and early hepatic marker hhex. In addition, results showed that the smaller-liver phenotype in HPS morphants was caused by suppression of cell proliferation, not induction of cell apoptosis. CONCLUSIONS: Current findings indicated that HPS is essential to the later stages of development in vertebrate liver organogenesis.

EDAG positively regulates erythroid differentiation and modifies GATA1 acetylation through recruiting p300.

Erythroid differentiation-associated gene (EDAG) has been considered to be a transcriptional regulator that controls hematopoietic cell differentiation, proliferation, and apoptosis. The role of EDAG in erythroid differentiation of primary erythroid progenitor cells and in vivo remains unknown. In this study, we found that EDAG is highly expressed in CMPs and MEPs and upregulated during the erythroid differentiation of CD34(+) cells following erythropoietin (EPO) treatment. Overexpression of EDAG induced erythroid differentiation of CD34(+) cells in vitro and in vivo using immunodeficient mice. Conversely, EDAG knockdown reduced erythroid differentiation in EPO-treated CD34(+) cells. Detailed mechanistic analysis suggested that EDAG forms complex with GATA1 and p300 and increases GATA1 acetylation and transcriptional activity by facilitating the interaction between GATA1 and p300. EDAG deletion mutants lacking the binding domain with GATA1 or p300 failed to enhance erythroid differentiation, suggesting that EDAG regulates erythroid differentiation partly through forming EDAG/GATA1/p300 complex. In the presence of the specific inhibitor of p300 acetyltransferase activity, C646, EDAG was unable to accelerate erythroid differentiation, indicating an involvement of p300 acetyltransferase activity in EDAG-induced erythroid differentiation. ChIP-PCR experiments confirmed that GATA1 and EDAG co-occupy GATA1-targeted genes in primary erythroid cells and in vivo. ChIP-seq was further performed to examine the global occupancy of EDAG during erythroid differentiation and a total of 7,133 enrichment peaks corresponding to 3,847 genes were identified. Merging EDAG ChIP-Seq and GATA1 ChIP-Seq datasets revealed that 782 genes overlapped. Microarray analysis suggested that EDAG knockdown selectively inhibits GATA1-activated target genes. These data provide novel insights into EDAG in regulation of erythroid differentiation.

Mutagenic and Estrogenic Effects of Organic Compounds in Water Treated by Different Processes: A Pilot Study.

OBJECTIVE: In this study, a pilot-scale investigation was conducted to examine and compare the biotoxicity of the organic compounds in effluents from five treatment processes (P1-P5) where each process was combination of preoxidation (O3), coagulation, sedimentation, sand filtration, ozonation, granular activated carbon, biological activated carbon and chlorination (NaClO). METHODS: Organic compounds were extracted by XAD-2 resins and eluted with acetone and dichlormethane (DCM). The eluents were evaporated and redissolved with DMSO or DCM. The mutagenicity and estrogenicity of the extracts were assayed with the Ames test and yeast estrogen screen (YES assay), respectively. The organic compounds were detected by GC-MS. RESULTS: The results indicated that the mutation ratio (MR) of organic compounds in source water was higher than that for treated water. GC-MS showed that more than 48 organic compounds were identified in all samples and that treated water had significantly fewer types and concentrations of organic compounds than source water. CONCLUSION: To different extents, all water treatment processes could reduce both the mutagenicity and estrogenicity, relative to source water. P2, P3, and P5 reduced mutagenicity more effectively, while P1 reduced estrogenicity, most effectively. Water treatment processes in this pilot plant had weak abilities to remove Di-n-butyl phthalate or 1, 2-Benzene dicarboxylic acid.

14-3-3ζ interacts with hepatocyte nuclear factor 1α and enhances its DNA binding and transcriptional activation.

14-3-3 proteins regulate numerous cellular processes through interaction with a variety of proteins, and have been identified as HNF1α binding partner by mass spectrometry analysis in our previous study. In the present study, the interaction between 14-3-3ζ and HNF1α has been further validated by in vivo and in vitro assays. Moreover, we have found that overexpression of 14-3-3ζ potentiated the transcriptional activity of HNF1α in cultured cells, and silencing of 14-3-3ζ by RNA interference in HepG2 cells specifically affected the HNF1α-dependent gene expression. Furthermore, we have demonstrated that 14-3-3ζ is recruited to endogenous HNF1α responsive promoters and enhances HNF1α binding to its cognate DNA sequences. In addition, we have also provided evidence that the association between HNF1α and 14-3-3ζ is phosphorylation-dependent. Taken together, these results suggest that 14-3-3ζ may be an endogenous physiologic regulator of HNF1α.

Expansion of the polyQ repeats in THAP11 forms intranuclear aggregation and causes cell G0/G1 arrest.

Polyglutamine diseases are a group of neurodegenerative disorders caused by expansion of a CAG repeat that encodes polyglutamine in each respective disease gene. The transcription factor THAP11, a member of THAP family, is involved in cell growth, ES cell pluripotency and embryogenesis. Previous studies suggest that THAP11 protein contains a 29-residue repeat polyglutamine motif and the number of polyglutamine ranges from 20 to 41 in Indian population. We have investigated the CAG numbers at the THAP11 locus in normal individuals and neurodegenerative disease patients of Chinese Han population and a 38Q expansion (THAP11(38Q)) was found in patients. Using fluorescence confocal-based cell imaging, THAP11(38Q) protein formed intranuclear inclusions easier than THAP11(29Q) in PC12 cells. Enhanced toxicity was investigated in THAP11(38Q)-expressing cells by growth inhibition and G0/G1 arrest. CREB-mediated transcription activity was inhibited by THAP11(38Q). The transcription factor, TBP, coactivator CBP, and chaperon protein, HSP70, could be recruited to THAP11(38Q). These results indicate that expansion of the polyglutamine in THAP11 forms intracellular aggregation and is toxic in PC12 cells, suggesting a putative role of THAP11 in polyglutamine disease.

Total synthesis of plagiochin G and derivatives as potential cancer chemopreventive agents.

A new and efficient total synthesis has been developed to obtain plagiochin G (22), a macrocyclic bisbibenzyl, and four derivatives. The key 16-membered ring containing biphenyl ether and biaryl units was closed via an intramolecular SNAr reaction. All synthesized macrocyclic bisbibenzyls inhibited Epstein-Barr virus early antigen (EBVEA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells and, thus, are potential cancer chemopreventive agents.

Intraductal radiofrequency ablation for refractory benign biliary stricture: pilot feasibility study.

BACKGROUND AND AIM: Endoscopic management of benign biliary stricture (BBS) remains challenging. There is no reported method for the amelioration of biliary fibroplasia endoscopically. We report our initial experience of radiofrequency ablation (RFA) for the management of BBS. METHODS: Nine patients with BBS (postoperation stricture four, liver transplant three, and chronic inflammation two), seven of whom had previously unsuccessful endoscopic or percutaneous interventions, were enrolled. Intraductal bipolar RFA was delivered at power of 10 W for 90 s per stricture segment, followed by balloon dilatation with/without stent placement. RESULTS: All patients had immediate stricture improvements after RFA. No severe adverse event occurred except for one patient with mild post-endoscopic retrograde cholangiopancreatography pancreatitis. During median (SD) follow-up duration of 12.6 (3.9) months, BBS resolution without the need for further stenting was achieved in four patients whereas two patients had stent(s) in situ waiting scheduled removal. However, one patient had stricture relapse after initial resolution, one underwent surgery, and another patient died of other cause. CONCLUSIONS: Endobiliary RFA appears to be safe and effective for the treatment of BBS, especially for refractory cases. Further studies are warranted.

Synthesis of novel spin-labeled derivatives of 5-FU as potential antineoplastic agents.

Chemotherapy is a general treatment option for various cancers, including lung cancer. In order to find compounds with superior bioactivity and less toxicity against lung cancer, novel spin-labeled 5-fluorouracil (5-FU) derivatives (3a-f) were synthesized and evaluated against four human tumor cell lines (A-549, DU-145, KB, and KBvin). Two promising compounds 3d and 3f exhibited IC50 values of 2.76 and 2.38 µM, respectively, against non-small cell lung carcinoma cell line A-549. These compounds were twofold more cytotoxic than 5-FU and less toxic against other tested cell lines. Compound 3f exhibited seven times more selective cytotoxicity against A-549 than 5-FU. Our results suggest that compounds 3d and 3f merit further investigation for development into clinical trial candidates for non-small cell lung cancer.

Development, Validation, and Application of High-Performance Liquid Chromatography with Diode-Array Detection Method for Simultaneous Determination of Ginkgolic Acids and Ginkgols in Ginkgo biloba.

Ginkgo biloba leaves (GBLs), which comprise many phytoconstituents, also contain a toxic substance named ginkgolic acid (GA). Our previous research showed that heating could decarboxylate and degrade GA into ginkgols with high levels of bioactivity. Several methods are available to measure GA in GBLs, but no analytical method has been developed to measure ginkgols and GA simultaneously. Hence, for the first time, an HPLC-DAD method was established to simultaneously determine GA and ginkgols using acetonitrile (0.01% trifluoroacetic acid, v/v) as mobile phase A and water (0.01% trifluoroacetic acid, v/v) as mobile phase B. The gradient elution conditions were: 0-30 min, 75-90% phase A; 30-35 min, 90-90% phase A; 35-36 min, 90-75% phase A; 36-46 min, 75-75% phase A. The detection wavelength of GA and ginkgol were 210 and 270 nm, respectively. The flow rate and injection volume were 1.0 mL/min and 50 µL, respectively. The linearity was excellent (R2 > 0.999), and the RSD of the precision, stability, and repeatability of the total ginkgols was 0.20%, 2.21%, and 2.45%, respectively, in six parallel determinations. The recoveries for the low, medium, and high groups were 96.58%, 97.67%, and 101.52%, respectively. The limit of detection of ginkgol C13:0, C15:1, and C17:1 was 0.61 ppm, 0.50 ppm, and 0.06 ppm, respectively. The limit of quantification of ginkgol C13:0, C15:1, and C17:1 was 2.01 ppm, 1.65 ppm, and 0.20 ppm, respectively. Finally, this method accurately measured the GA and ginkgol content in ginkgo leaves and ginkgo tea products (ginkgo black tea, ginkgo dark tea, ginkgo white tea, and ginkgo green tea), whereas principal component analysis (PCA) was performed to help visualize the association between GA and ginkgols and five different processing methods for GBLs. Thus, this research provides an efficient and accurate quantitative method for the subsequent detection of GA and ginkgols in ginkgo tea.

Combinative effect of pulsed-light irradiation and solid-state fermentation on ginkgolic acids, ginkgols, ginkgolides, bilobalide, flavonoids, product quality and sensory assessment of Ginkgo biloba dark tea.

Pulsed light (PL) is a prospective non-thermal technology that can improve the degradation of ginkgolic acid (GA) and retain the main bioactive compounds in Ginkgo biloba leaves (GBL). However, only using PL hasn't yet achieved the ideal effect of reducing GA. Fermentation of GBL to make ginkgo dark tea (GDT) could decrease GA. Because different microbial strains are used for fermentation, their metabolites and product quality might differ. However, there is no research on the combinative effect of PL irradiation fixation and microbial strain fermentation on main bioactive compounds and sensory assessment of GDT. In this research, first, Bacillus subtilis and Saccharomyces cerevisiae were selected as fermentation strains that can reduce GA from the five microbial strains. Next, the fresh GBL was irradiated by PL for 200 s (fluences of 0.52 J/cm2), followed by B. subtilis, S. cerevisiae, or natural fermentation to make GDT. The results showed that compared with the control (unirradiated and unfermented GBL) and the only PL irradiated GBL, the GA in GDT using PL + B. subtilis fermentation was the lowest, decreasing by 29.74%; PL + natural fermentation reduced by 24.53%. The total flavonoid content increased by 14.64% in GDT using PL + B. subtilis fermentation, whose phenolic and antioxidant levels also increased significantly. Sensory evaluation showed that the color, aroma, and taste of the tea infusion of PL + B. subtilis fermentation had the highest scores. In conclusion, the combined PL irradiation and solid-state fermentation using B. subtilis can effectively reduce GA and increase the main bioactive compounds, thus providing a new technological approach for GDT with lower GA.

Thermal fixation technologies affect phenolic profile, ginkgolides, bilobalide, product quality, and ginkgolic acids in Ginkgo biloba leaf tea.

Ginkgo biloba leaves (GBLs) contain high phytoconstituents, but ginkgolic acids (GAs, the main toxic compound in GBLs) have limited its applications. Processing Ginkgo biloba dark tea (GBDT) using fixation technology could decrease the toxic compounds; retain flavonoids, ginkgolides, and bilobalide; and improve the product quality. For the first time, various thermal fixations (hot air fixation [HAF], iron pot fixation [IPF], and boiled water fixation [BWF]) followed by rolling, fermentation, and drying were applied to produce GBDT. A comprehensive analysis of the toxicants (GAs), main bioactive compounds (ginkgolides and bilobalide, flavonoids, antioxidants, and phenolic profiles), and product qualities (moisture content, reducing sugar [RS], free amino acids [FAAs], enzyme activity, color properties, antioxidant capacity, etc.) were evaluated. The results revealed that thermal fixations BWF and HAF significantly reduced the GA contents (41.1%-34.6%). Most terpene lactones showed significant differences in control, IPF, and HAF. The HAF had lower total flavonoid content (TFC) than BWF and IPF. The control group (unfixated) had the highest toxic components (GA), terpene trilactones, and TFC compared with various fixations. Adding different fixations to rolling, fermentation, and drying had various impacts on GBDT, and principal component analysis supported the results. Among four thermal fixations, HAF yielded the best results in RS, FAA, total phenolic content, and antioxidant activities, while IPF had the highest TFC. BWF had the lowest content for GA. In conclusion, HAF (6) was chosen as the best technique for producing GBDT since it preserved GBDT's bioactive components while lowering its toxic components.