Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1.

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

Revealing binding mechanism of ß-casein to chrysin, apigenin, and luteolin and locating its binding pockets by molecular docking and molecular dynamics.

Revealing the interaction mechanism of proteins with bioactive molecules and the location of their binding pockets is crucial for predicting the structure-function relationship of proteins in drug discovery and design. Despite some published papers on the interaction of ß-casein with small bioactive molecules, the ambiguity of the location and constituent amino acids of ß-casein binding pockets prompted us to identify them by in silico simulation of its interaction with three polyphenols, chrysin, apigenin, and luteolin. Molecular docking revealed that the primary ß-casein binding pocket for chrysin consists of five nonpolar amino acids (Leu73, Phe77, Pro80, Ile89, and Pro196), three polar neutral amino acids (Ser137, Gln138, and Gln197), and two polar charged amino acids (Glu136, and Arg198). For ß-casein/apigenin and ß-casein/luteolin complexes, Asn83 also contributes to forming the pocket. Molecular dynamics provided more details, such as the relative contribution of determinative amino acids and the role of various forces. For example, we found that Glu210, Glu132, and Glu35 are the most destructive residues in the binding of chrysin, apigenin, and luteolin to ß-casein, respectively. Also, we observed that hydrophobic forces mainly stabilize ß-casein/chrysin and ß-casein/apigenin, and polar solvation (including hydrogen bonds) stabilizes ß-casein/luteolin, all by spontaneous processes.

Bioflavonoid luteolin prevents sFlt-1 release via HIF-1α inhibition in cultured human placenta.

Preeclampsia (PE) is a serious hypertensive complication of pregnancy and is a leading cause of maternal death and major contributor to maternal and perinatal morbidity, including establishment of long-term complications. The continued prevalence of PE stresses the need for identification of novel treatments which can target prohypertensive factors implicated in the disease pathophysiology, such as soluble fms-like tyrosine kinase 1 (sFlt-1). We set out to identify novel compounds to reduce placental sFlt-1 and determine whether this occurs via hypoxia-inducible factor (HIF)-1α inhibition. We utilized a commercially available library of natural compounds to assess their ability to reduce sFlt-1 release from primary human placental cytotrophoblast cells (CTBs). Human placental explants from normotensive (NT) and preeclamptic (PE) pregnancies were treated with varying concentrations of luteolin. Protein and mRNA expression of sFlt-1 and upstream mediators were evaluated using ELISA, western blot, and real-time PCR. Of the natural compounds examined, luteolin showed the most potent inhibition of sFlt-1 release, with >95% reduction compared to vehicle-treated. Luteolin significantly inhibited sFlt-1 in cultured placental explants compared to vehicle-treated in a dose- and time-dependent manner. Additionally, significant decreases in HIF-1α expression were observed in luteolin-treated explants, suggesting a mechanism for sFlt-1 downregulation. The ability of luteolin to inhibit HIF-1α may be mediated through the Akt pathway, as inhibitors to Akt and its upstream regulator phosphatidylinositol-3 kinase (PI3K) resulted in significant HIF-1α reduction. Luteolin reduces anti-angiogenic sFlt-1 through inhibition of HIF-1α, making it a novel candidate for the treatment of PE.

The establishment of transient expression systems and their application for gene function analysis of flavonoid biosynthesis in Carthamus tinctorius L.

BACKGROUND: Safflower (Carthamus tinctorius L.) is an important economic crop and a traditional medicinal material rich in flavonoids, which can alleviate cardiovascular and cerebrovascular pathologies. Thus, many candidate genes involved in safflower flavonoid biosynthesis have been cloned. However, owing to the lack of a homologous gene expression system, research on gene function is limited to model plants. Therefore, a gene function identification protocol for safflower must be established. RESULTS: In the present study, using safflower callus as the experimental material, Agrobacterium and biolistic transient expression systems were established. In the Agrobacterium transient expression system, the highest transformation rate was obtained at the original Agrobacterium concentration of OD600 0.4, infiltration concentration of OD600 0.6, infection for 20 min, co-culture for 3 days, and acetosyringone concentration of 100 µmol·L-1. In the biolistic transient expression system, the highest transformation efficiency was observed at helium pressure of 1,350 psi, vacuum degree of -0.8 bar, flight distance of 6.5 cm, one round of bombardment, plasmid concentration of 3 µg·shot-1, and gold particle concentration of 100 µg·shot-1. Further, these two transient expression systems were used for the functional analysis of CtCHS1 as an example. After overexpression, relative CtCHS1 expression increased, particularly in Agrobacterium-transformed calli. Additionally, the contents of some flavonoids were altered; for instance, naringenin and genistein levels were significantly increased in Agrobacterium-transformed calli, whereas luteolin, luteolin-7-O-rutinoside, and apigenin derivative levels were significantly decreased in biolistic-transformed calli. CONCLUSION: Using safflower callus as the experimental material, highly efficient Agrobacterium and biolistic transient expression systems were successfully established, and the utility of both systems for investigating gene function was demonstrated. The proposed safflower callus transient expression systems will be useful for further functional analyses of flavonoid biosynthetic genes in safflower.

Engineering of flavonoid 3'-O-methyltransferase for improved biosynthesis of chrysoeriol in Escherichia coli.

O-Methylation catalyzed by O-methyltransferases (OMTs) is an important modification of flavonoids for improving the transport efficiency across membranes and metabolic stability in mammalian cells. Chrysoeriol, also known as 3'-O-methylated luteolin, is a methylated flavonoid compound with health-promoting activities. The generation of chrysoeriol from luteolin can be catalyzed by a rice-derived 3'-OMT named ROMT-9, which has a high regiospecificity and activity toward flavonoids in vitro. Herein, we explored the potential of ROMT-9 for in vivo biosynthesis of chrysoeriol in Escherichia coli and adopted semi-rational enzyme engineering guided by homology modeling and molecular docking to improve the bio-production. Two positive variants including L34Q and W284A were obtained which promoted chrysoeriol formation to more than 85 mg/L from 200 mg/L of luteolin in 24 h compared with a titer of 55 mg/L for the strain expressing the native enzyme. Further biochemical analysis confirmed that such improvement in production stemmed from a higher enzyme expression level for the L34Q variant and higher efficiency in substrate binding and catalysis for the W284A variant. This study provides some insights into the engineering of other flavonoid OMTs and will facilitate high-level biosynthesis of methylated flavonoids in engineered microorganisms. KEY POINTS: • Biosynthesis of chrysoeriol from luteolin in E. coli using ROMT-9 • Engineering of ROMT-9 for better bio-production • ROMT-9 variants promote production via better expression or better catalysis.

The C-glucosyl flavone isoorientin pretreatment attenuates the methylglyoxal-induced mitochondrial dysfunction in the human neuroblastoma SH-SY5Y cells: role for the AMPK-PI3K/Akt/Nrf2/γ-GCL/GSH axis.

The reactive dicarbonyl methylglyoxal (MG) behaves as a pro-oxidant agent, causing redox dysfunction and cell death by different mechanisms in mammalian cells. MG is also a mitochondrial toxicant, impairing the oxidative phosphorylation (OXPHOS) system and leading to bioenergetics and redox collapses. MG induces glycation and exerts an important role in neurodegenerative and cardiovascular diseases. Isoorientin (ISO), a C-glucosyl flavone found in Aspalathus linearis, Fagopyrum esculentum, and Passiflora edulis, among others, is an antioxidant and anti-inflammatory molecule. ISO is a potent inducer of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), the master modulator of the redox environment in mammals. We investigated here whether ISO would prevent the mitochondria-related redox and bioenergetics impairments induced by MG in the human neuroblastoma SH-SY5Y cells. The cells were administrated with ISO at 20 µM for 18 h prior to the exposure to MG at 500 µM for further 24 h. It was observed that ISO efficiently prevented the mitochondrial impairments caused by MG. ISO upregulated the activity of the enzyme γ-glutamate-cysteine ligase (γ-GCL), consequently stimulating the synthesis of glutathione (GSH). The inhibition of γ-GCL, adenosine monophosphate-activated protein kinase (AMPK), and phosphoinositide 3-kinase/Akt (PI3K/Akt) suppressed the beneficial effects induced by ISO on the MG-challenged cells. Moreover, silencing of Nrf2 blocked the ISO-dependent γ-GCL and GSH upregulation and the effects on the mitochondria of the MG-challenged cells. Then, ISO caused mitochondrial protection by an AMPK-PI3K/Akt/Nrf2/γ-GCL/GSH-dependent manner in MG-administrated SH-SY5Y cells.

Adding genistein or luteolin decreased the yield of citrinin and without reducing pigments in yam solid-fermentation by Monascus.

BACKGROUND: Chinese yam fermented by Monascus, namely red mold dioscorea (RMD), has the potential of treating diseases. However, the production of citrinin limits the application of RMD. In the present study, the fermentation process of Monascus was optimized by adding genistein or luteolin to reduce citrinin yield. RESULTS: The results showed that citrinin in 25 g of Huai Shan yam was reduced by 48% and 72% without affecting the pigment yield by adding 0.2 g of luteolin or genistein, respectively, to a 250-mL conical flask after fermentation for 18 days at 28 °C, whereas the addition of luteolin increased the content of yellow pigment by 1.3-fold. Under optimal conditions, citrinin in 20 g of iron bar yam decreased by 55% and 74% after adding 0.2 g of luteolin or genistein. Luteolin also increased yellow pigment content by 1.2-fold. Ultra HPLC coupled to quadrupole time-of-flight mass spectrometry was used for the preliminary analysis of Monascus fermentation products. It was found that the amino acid types in RMD are similar to those in yams, but there are fewer polysaccharides and fatty acids. CONCLUSION: The results obtained in the present study showed that the addition of genistein or luteolin could reduce citrinin on the premise of increasing pigment yield, which laid a foundation for the better use of yams in Monascus fermentation. © 2023 Society of Chemical Industry.

Targeting protein tyrosine phosphatase 1B in obesity-associated colon cancer: Possible role of sweet potato (Ipomoea batatas).

Protein tyrosine phosphatase 1B (PTP1B) has emerged as one of the links between obesity and colon cancer (CC). Anti-obesity and anti-CC attributes of sweet potato (Ipomoea batatas) reported sparsely. Here, we aimed to study the potential of PTP1B as a target in CC, particularly in obese population. Expression and genomic alteration frequency of PTPN1 (PTP1B) were checked in CC. Interacting partners of PTP1B through STRING and hub genes through Cytoscape (MCODE) were identified. Hub genes were subjected to functional enrichment analyses (via Metascape), differential gene expression, copy number variation, and single nucleotide variation analyses (GSCA database). Cancer-related pathways and associated immune infiltrates of the hub genes were checked too. Eleven sweet potato-derived compounds selected through drug likeness (DL) and toxicity filters were explored via molecular docking (AutoDock Vina) to reveal the interactions with PTP1B. Genomic alteration frequency of the PTPN1 was highest in CC compared to all the other TCGA cancers, and a high expression (RNA and protein) is also observed in CC that correlated well to a poor overall survival (OS). Furthermore, PTP1B and related proteins were enriched in different biological processes and signaling pathways related to carcinogenesis including epithelial-mesenchymal transition. Overall, PTP1B identified as a potential target in obesity-linked CC and sweet potato might exert its protective action by targeting the PTP1B. Sweet potato compounds (e.g., pelargonidin and luteolin) interacted with the catalytic P loop and the WPD loop of the PTP1B. Furthermore, MD simulation study ascertained that luteolin has the highest affinity against the PTP1B, whereas pelargonidin and quercetin showed good binding affinity too, thus can be explored further.

Genomics and transcriptomics insights into luteolin effects on the beta-rhizobial strain Cupriavidus necator UYPR2.512.

Cupriavidus necator UYPR2.512 is a rhizobial strain that belongs to the Beta-subclass of proteobacteria, able to establish successful symbiosis with Mimosoid legumes. The initial steps of rhizobium-legumes symbioses involve the reciprocal recognition by chemical signals, being luteolin one of the molecules involved. However, there is a lack of information on the effect of luteolin in beta-rhizobia. In this work, we used long-read sequencing to complete the genome of UYPR2.512 providing evidence for the existence of four closed circular replicons. We used an RNA-Seq approach to analyse the response of UYPR2.512 to luteolin. One hundred and forty-five genes were differentially expressed, with similar numbers of downregulated and upregulated genes. Most repressed genes were mapped to the main chromosome, while the upregulated genes were overrepresented among pCne512e, containing the symbiotic genes. Induced genes included the nod operon and genes implicated in exopolysaccharides and flagellar biosynthesis. We identified many genes involved in iron, copper and other heavy metals metabolism. Among repressed genes, we identified genes involved in basal carbon and nitrogen metabolism. Our results suggest that in response to luteolin, C. necator strain UYPR2.512 reshapes its metabolism in order to be prepared for the forthcoming symbiotic interaction.

Modulatory influences of antiviral bioactive compounds on cell viability, mRNA and protein expression of cytochrome P450 3A4 and P-glycoprotein in HepG2 and HEK293 cells.

The induction of cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (ABCB1) influence drug plasma, and eventually decreases the drugs' therapeutic effects. The effects of Plant-derived compounds (PCs) on drug-metabolising proteins are largely unknown. This study investigated the cytotoxicity, cell viability profiles and regulatory influences of four PCs (epigallocatechin gallate (EGCG), kaempferol-7-glucoside (K7G), luteolin (LUT) and ellagic acid (EGA)) on the mRNA and protein expressions of CYP3A4 and ABCB1 in HepG2 and HEK293 cells. After treatment with the PCs (0-400 µM) for 24 h, 80% (IC20) and 50% (IC50) cell viability were determined. The PCs were not toxic to HepG2 (ATP levels increased at IC20, insignificant change in LDH (lactate dehydrogenase) with the exception of LUT, and ABCB1 protein expressions decreased. The PCs decreased CYP3A4 at IC20 (except LUT), EGCG and K7G at IC20 decreased mRNA expression. For HEK293 cells, no significant change in ATP, except for EGCG IC20 and K7G IC50 which decreased and increased, respectively. LDH decreased at IC20, but LUT IC50 significant increase LDH. ABCB1 protein expression increased at both IC20 and IC50, but LUT and EGA at IC50 decreased mRNA expression. The PCs at IC20, and IC50 of LUT, K7G and of EGCG may enhance drug bioavailability.

Hepatoprotective Effects of Bioflavonoid Luteolin Using Self-Nanoemulsifying Drug Delivery System.

Luteolin (LUT) is a natural pharmaceutical compound that is weakly water soluble and has low bioavailability when taken orally. As a result, the goal of this research was to create self-nanoemulsifying drug delivery systems (SNEDDS) for LUT in an attempt to improve its in vitro dissolution and hepatoprotective effects, resulting in increased oral bioavailability. Using the aqueous phase titration approach and the creation of pseudo-ternary phase diagrams with Capryol-PGMC (oil phase), Tween-80 (surfactant), and Transcutol-HP (co-emulsifier), various SNEDDS of LUT were generated. SNEDDS were assessed for droplet size, polydispersity index (PDI), zeta potential (ZP), refractive index (RI), and percent of transmittance (percent T) after undergoing several thermodynamic stability and self-nanoemulsification experiments. When compared to LUT suspension, the developed SNEDDS revealed considerable LUT release from all SNEDDS. Droplet size was 40 nm, PDI was <0.3, ZP was -30.58 mV, RI was 1.40, percent T was >98 percent, and drug release profile was >96 percent in optimized SNEDDS of LUT. For in vivo hepatoprotective testing in rats, optimized SNEDDS was chosen. When compared to LUT suspension, hepatoprotective tests showed that optimized LUT SNEDDS had a substantial hepatoprotective impact. The findings of this investigation suggested that SNEDDS could improve bioflavonoid LUT dissolution rate and therapeutic efficacy.

Suppressing Cdk5 Activity by Luteolin Inhibits MPP+-Induced Apoptotic of Neuroblastoma through Erk/Drp1 and Fak/Akt/GSK3ß Pathways.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons. The cause of PD is still unclear. Oxidative stress and mitochondrial dysfunction have been linked to the development of PD. Luteolin, a non-toxic flavonoid, has become interested in an alternative medicine, according to its effects on anti-oxidative stress and anti-apoptosis, although the underlying mechanism of luteolin on PD has not been fully elucidated. This study aims to investigate whether luteolin prevents neurotoxicity induction by 1-methyl-4-phenylpyridinium iodide (MPP+), a neurotoxin in neuroblastoma SH-SY5Y cells. The results reveal that luteolin significantly improved cell viability and reduced apoptosis in MPP+-treated cells. Increasing lipid peroxidation and superoxide anion (O2-), including mitochondrial membrane potential (Δψm) disruption, is ameliorated by luteolin treatment. In addition, luteolin attenuated MPP+-induced neurite damage via GAP43 and synapsin-1. Furthermore, Cdk5 is found to be overactivated and correlated with elevation of cleaved caspase-3 activity in MPP+-exposed cells, while phosphorylation of Erk1/2, Drp1, Fak, Akt and GSK3ß are inhibited. In contrast, luteolin attenuated Cdk5 overactivation and supported phosphorylated level of Erk1/2, Drp1, Fak, Akt and GSK3ß with reducing in cleaved caspase-3 activity. Results indicate that luteolin exerts neuroprotective effects via Cdk5-mediated Erk1/2/Drp1 and Fak/Akt/GSK3ß pathways, possibly representing a potential preventive agent for neuronal disorder.

Comparative biotransformation of luteolin and apigenin from the flower extract and the stem-and-leaf extract of Dendranthema morifolium Ramat. Tzvel. in rats.

BACKGROUND: The flower of Dendranthema morifolium Ramat Tzvel has been widely used as a nutritional health supplement worldwide. However, most of the studies have focused on the flower and the rest of the plant was neglected. Our hypothesis is that similar flavonoids may be present at different parts of D. morifolium, and the flavonoids may undergo a similar biotransformation pathway within the body. To investigate this hypothesis, an in vivo pharmacokinetic experimental model was developed to explore the comparative biotransformation of luteolin and apigenin after administration of D. morifolium extracts (10 g kg-1 , p.o.) in freely moving rats. Because luteolin and apigenin mainly underwent phase II metabolism, the metabolic enzymes of ß-glucuronidase/sulfatase or ß-glucuronidase were used to hydrolyze the plasma sample, depending on the biotransformation pathway involved. RESULTS: The results revealed that luteolin and apigenin mainly went through glucuronide and sulfate conjugations, respectively, in both the extract of flowers and the stem-and-leaf group. In addition, the area under the concentration curve (AUClast ) of luteolin glucuronides and sulfates in the group administered the stem-and-leaf extract was approximately 4.6 times higher than that of the flower extract group. The dominant products of biotransformation for apigenin were sulfates. CONCLUSION: These findings support our hypothesis that not only the flower parts of D. morifolium, but also the stem-and-leaf parts contain rich flavones, including glycosides and aglycone, and they undergo similar biotransformation pathways. © 2021 Society of Chemical Industry.

Physiological and biochemical responses of green and red perilla to LED-based light.

BACKGROUND: Light-emitting diodes (LEDs) are widely used in closed-type plant production systems to improve biomass and accumulate bioactive compounds in plants. Perilla has been commonly used as herbal medicine because of its health-promoting effects. This study aimed to investigate the physiological and biochemical responses of green and red perilla under various visible-light spectra. RESULTS: Results showed that red (R) LEDs improved fresh weights of shoots and roots, plant height, internode length, node number and leaf area, as well as photosynthetic rate of green and red perilla plants compared to blue (B) LEDs and RB combined LEDs. Meanwhile, B resulted in higher stomatal conductance, transpiration rate and Fv/Fm compared to R. Supplementation of green (G) and far-red (FR) did not enhance perilla growth. Reduction or absence of B decreased leaf thickness, adaxial and abaxial epidermis, and palisade and spongy mesophyll. Total phenolic content, antioxidant capacity, rosmarinic acid content and caffeic acid content of green perilla were higher under R, R8B2 and RGB + FR, while greater values were obtained in red perilla under R. Accumulation of perillaldehyde, luteolin and apigenin presented different trends from those of rosmarinic and caffeic acids in both cultivars. CONCLUSIONS: Growth and accumulation of bioactive compounds in green perilla were greater than in red perilla under similar light quality, and R LEDs or a higher R ratio in combination treatments were suitable for cultivating high-quality green and red perilla plants in closed-type plant factories. © 2020 Society of Chemical Industry.

Luteolin suppresses bladder cancer growth via regulation of mechanistic target of rapamycin pathway.

Luteolin is a natural flavonoid with strong anti-oxidative properties that is reported to have an anti-cancer effect in several malignancies other than bladder cancer. In this study, we describe the effect of luteolin on a human bladder cancer cell line, T24, in the context of the regulation of p21, thioredoxin-1 (TRX1) and the mechanistic target of rapamycin (mTOR) pathway. Luteolin inhibited cell survival and induced G2/M cell-cycle arrest, p21 upregulation and downregulation of phospho(p)-S6, which is downstream of mTOR signaling. Luteolin also upregulated TRX1 and reduced intracellular reactive oxygen species production. In a subcutaneous xenograft mouse model using the rat bladder cancer cell line, BC31, tumor volumes were significantly decreased in mice orally administered luteolin compared to control. Immunohistochemical analysis revealed that increased p21 and decreased p-S6 expression were induced in the luteolin treatment group. Moreover, in another in vivo N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced rat bladder cancer model, the oral administration of luteolin led to a trend of decreased bladder tumor dimension and significantly decreased the Ki67-labeling index and p-S6 expression. Furthermore, the major findings on the metabolism of luteolin suggest that both plasma and urine luteolin-3'-O-glucuronide concentrations are strongly associated with the inhibition of cell proliferation and mTOR signaling. Moreover, a significant decrease in the squamous differentiation of bladder cancer is attributed to plasma luteolin-3'-glucuronide concentration. In conclusion, luteolin, and in particular its metabolized product, may represent another natural product-derived therapeutic agent that acts against bladder cancer by upregulating p21 and inhibiting mTOR signaling.

Bioinspired oxidation in cytochrome P450 of isomers orientin and isoorientin using Salen complexes.

RATIONALE: Orientin and isoorientin are C-glycosidic flavonoids, considered as markers of some plant species such as Passiflora edulis var. flavicarpa Degener, and reported in the literature to have pharmacological properties. In order to evaluate and characterize the in vitro metabolism of these flavonoids, phase I biotransformation reactions were simulated using Salen complexes. METHODS: These flavonoids were oxidized separately in biomimetic reactions in different proportions, using one oxidant, m-chloroperbenzoic acid or iodosylbenzene, and one catalyst, the Jacobsen catalyst or [Mn(3-MeOSalen)Cl]. The [Mn(3-MeOSalen)Cl] catalyst was synthesized and characterized using spectrometric techniques. The oxidation potentials of the catalysts were compared. All reactions were monitored and analyzed using ultrahigh-performance liquid chromatography diode-array detection (UHPLC-DAD) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). RESULTS: The analysis by UHPLC-DAD and HPLC/MS/MS showed that isoorientin produces more products than orientin and that [Mn(3-MeOSalen)Cl] produces more products than the Jacobsen catalyst. In addition, [Mn(3-MeOSalen)Cl], which has a higher oxidation potential, formed products with the addition of one or two atoms of oxygen, while the Jacobsen catalyst formed compounds with only one added oxygen atom. The products with the addition of one oxygen atom were mainly epoxides, while those with two added oxygens formed an epoxide in the C-ring and incorporated the other oxygen into the glycosidic moiety. CONCLUSIONS: The formation of epoxides is common in biomimetic reactions and they may represent a safety risk in medicinal products due to their high reactivity. This study may serve as a basis for subsequent pharmacological and toxicological studies that investigate the presence of these compounds as phase I metabolites, and ensure the safe use of plant products containing orientin as a chemical marker.

Luteolin modulates gene expression related to steroidogenesis, apoptosis, and stress response in rat LC540 tumor Leydig cells.

In males, androgens are mainly produced by Leydig cells from the testis. A critical and highly regulated step of steroidogenesis involves the importation of cholesterol within the mitochondria by the steroidogenic acute regulatory (STAR) protein. During aging, STAR protein levels in Leydig cells gradually decrease, leading to a reduced entry of cholesterol into mitochondria and lower testosterone production. In addition to preserving its steroidogenic capacity, tumor Leydig cells can also be excellent models for evaluating the mechanisms of action of anticancer agents. In this study, we examined whether polyphenolics having structural similarities to luteolin could promote steroidogenic and cancer-related gene expressions within rat L540 tumor Leydig cells. In this cell model, luteolin activated Star expression and increased progesterone as well as testosterone productions. Interestingly, luteolin decreased gene expression related to cholesterol biosynthesis, possibly inhibiting membrane synthesis and cell proliferation. In addition, increased expression of genes such as Fas, Cdkn1a, Atp7b, and Tp53, as well as increased accumulation of cleaved caspase 3 and PARP, in response to luteolin treatment indicates that apoptosis is being activated. Luteolin also modulated the expression of genes involved in stress response, such as glutathione-S transferases Gsta1 and Gstt2, and the unfolded protein response. Thus, dietary luteolin may be effective in Leydig cell tumor chemoprevention and in maintaining steroidogenesis in aging males.

Luteolin protects against lead acetate-induced nephrotoxicity through antioxidant, anti-inflammatory, anti-apoptotic, and Nrf2/HO-1 signaling pathways.

Lead (Pb) is one of the most common heavy metal pollutants affecting living organisms. It induces nephrotoxicity with significant alterations in renal structure and function. Luteolin (LUT) a flavonoid present in various plant products is well known for exhibiting numerous pharmacological properties. We evaluated the protective efficacy of LUT against Pb-induced renal injury in male Wistar rats. Four experimental groups: control, LUT (50 mg/kg, orally), PbAc (20 mg/kg, i.p.), LUT + PbAc (at the aforementioned doses) were maintained for 7 days. PbAc administration significantly increased renal Pb accumulation, urea, and creatinine levels in serum, and induced renal histological alterations. Additionally, compared to the control rats, PbAc-treated rats exhibited significantly low levels of antioxidant enzyme activity and expression (SOD, CAT, GPx and GR), as well as high MDA levels. Moreover, PbAc exposure downregulated Nfe212 and Homx1 mRNA expression and significantly increased inflammatory marker (TNF-α, IL-1ß and NO) levels in renal tissue. PbAc significantly upregulated the synthesis of apoptotic related proteins and downregulated antiapoptotic protein expression. Notably, LUT pretreatment of PbAc-treated rats provided significant nephroprotection and reversed the alterations in the abovementioned parameters. In conclusion, LUT provided significant protection against PbAc intoxication via antioxidant, anti-inflammatory, and anti-apoptotic activities by activating the Nrf2/ARE signaling pathway.

SP and IL-33 together markedly enhance TNF synthesis and secretion from human mast cells mediated by the interaction of their receptors.

The peptide substance P (SP) and the cytokine tumor necrosis factor (TNF) have been implicated in inflammatory processes. Mast cells are recognized as important in inflammatory responses. Here, we report that IL-33 (30 ng/mL), a member of the IL-1 family of cytokines, administered in combination with SP (1 µM), markedly increase (by 1,000-fold) TNF gene expression in cultured human LAD2 and primary mast cells derived from umbilical cord blood. SP (0.01-1 µM) and IL-33 (1-100 ng/mL) in combination also greatly stimulate TNF secretion (by 4,500-fold). Pretreatment of LAD2 cells with two different neurokinin-1 (NK-1) receptor antagonists and siRNA inhibits TNF secretion by 50% (P < 0.001) when stimulated by SP and IL-33. Pretreatment of LAD2 cells with a neutralizing antibody for IL-33 receptor, ST2, inhibits TNF secretion by 50% (P < 0.001), and ST2 siRNA decreases TNF secretion by 30% (P < 0.05), when stimulated by SP and IL-33. Surprisingly, NK-1 antagonists also inhibit 50% of TNF secretion (P < 0.001) when stimulated only by IL-33, and ST2 receptor reduction also decreases SP-stimulated TNF secretion by 30% (P < 0.05), suggesting an interaction between NK-1 and ST2 receptors. Moreover, IL-33 increases NK-1 gene and surface protein expression, as well as IKß-α phosphorylation. Pretreatment of LAD2 cells with 5,7,3',4'-tetramethoxyflavone (methoxyluteolin) (1-100 µM) inhibits (P < 0.001) TNF gene expression (98%) and secretion (64%) at 50 µM and phosphorylation of p-IKB-α at 1 µM when stimulated by SP and IL-33. These findings identify a unique amplification process of TNF synthesis and secretion via the interaction of NK-1 and ST2 receptors inhibitable by methoxyluteolin.

Transcriptional regulation of Lonicera japonica Thunb. during flower development as revealed by comprehensive analysis of transcription factors.

BACKGROUND: Lonicera japonica Thunb. flower has been used for the treatment of various diseases for a long time and attracted many studies on its potential effects. Transcription factors (TFs) regulate extensive biological processes during plant development. As the restricted reports of L. japonica on TFs, our work was carried out to better understand the TFs' regulatory roles under different developmental stages in L. japonica. RESULTS: In this study, 1316 TFs belonging to 52 families were identified from the transcriptomic data, and corresponding expression profiles during the L. japonica flower development were comprehensively analyzed. 917 (69.68%) TFs were differentially expressed. TFs in bHLH, ERF, MYB, bZIP, and NAC families exhibited obviously altered expression during flower growth. Based on the analysis of differentially expressed TFs (DETFs), TFs in MYB, WRKY, NAC and LSD families that involved in phenylpropanoids biosynthesis, senescence processes and antioxidant activity were detected. The expression of MYB114 exhibited a positive correlation with the contents of luteoloside; Positive correlation was observed among the expression of MYC12, chalcone synthase (CHS) and flavonol synthase (FLS), while negative correlation was observed between the expression of MYB44 and the synthases; The expression of LSD1 was highly correlated with the expression of SOD and the total antioxidant capacity, while the expression of LOL1 and LOL2 exhibited a negative correlation with them; Many TFs in NAC and WRKY families may be potentially involved in the senescence process regulated by hormones and reactive oxygen species (ROS). The expression of NAC19, NAC29, and NAC53 exhibited a positive correlation with the contents of ABA and H2O2, while the expression of WRKY53, WRKY54, and WRKY70 exhibited a negative correlation with the contents of JA, SA and ABA. CONCLUSIONS: Our study provided a comprehensive characterization of the expression profiles of TFs during the developmental stages of L. japonica. In addition, we detected the key TFs that may play significant roles in controlling active components biosynthesis, antioxidant activity and flower senescence in L. japonica, thereby providing valuable insights into the molecular networks underlying L. japonica flower development.