A study on the anti-senescent effects of flavones derived from Prinsepia utilis Royle seed residue.

ETHNOPHARMACOLOGICAL RELEVANCE: Prinsepia utilis Royle, also known as the Anas fruit, is a unique perennial woody oil plant from Yunnan Province, China. In the ancient texts of Dongba sutras and Yunnan Southern Materia Medica, it has been documented that the local Naxi, Tibetan, and Mosuo communities extensively utilize the root and leaf fruits of green thorns for various purposes. These include treating mild-to-moderate specific dermatitis, moisturising the skin, providing protection against UV damage, aiding childbirth in pregnant women, safeguarding stomach health, reducing the risk of arteriosclerosis, and delaying aging. AIM OF THE STUDY: In this study, leftover residues from oil extraction were efficiently reused, and flavonoids were identified during subsequent extraction and separation processes. The anti-senescent effects of flavonoids in P. utilis Royle have not been systematically studied. Therefore, the objective of this study was to explore the anti-senescent properties of the flavonoids obtained from P. utilis Royle. METHODS: First, HPLC and other analytical techniques were used to identify the components of the P. utilis Royle flavonoid (PURF). Next, DPPH, hydroxyl radicals, superoxide anion O2-, collagenase, and elastase were initially detected using in vitro biochemical assays. To examine its antioxidant properties, a zebrafish model was used, and to confirm its anti-senescent effects, a d-galactose-induced mouse aging model was employed. The anti-senescent mechanism of PURF was examined using a natural senescence HFF model. Furthermore, the anti-senescent target was confirmed using a 3D full T-Skin™ model. RESULTS: In vitro biochemical assays demonstrated that flavones exhibited potent antioxidant activity and anti-senescent potential by inhibiting DPPH, hydroxyl radicals, superoxide anion O2-, collagenase, and elastase. It significantly enhanced the antioxidant effect on zebrafish while suppressing ROS and inflammatory injury, up-regulating COL1A1, COL3A1, AMPK, and mTOR gene expression and down-regulating MMP-9, TGF-ß, p21, and p16 gene expression suggesting its potential anti-senescent ability. Findings from the D-galactose-induced aging mouse model showed that PURF greatly increased SOD levels, while simultaneously decreasing HYP and MDA levels. In addition, when PURF was given to the HFF cell and 3D full T-Skin™ model, consistent trends were observed in gene and protein expression, with up-regulation of COL1A1, COL3A1, AMPK, and mTOR genes and down-regulation of TGF-ß, MMP-1, MMP-9, p21, and p16 genes. Therefore, these preliminary findings indicate that flavones can modulate AMPK/mTOR/TGF-ß signalling pathways to exert its influence. CONCLUSION: The kernel residue of natural P. utilis Royle oil extracted from Yunnan province was previously considered agricultural waste, but we successfully extracted and isolated its flavonoid components. Our preliminary studies demonstrated its potential as an environmentally friendly anti-senescent raw material.

Superoxide, nitric oxide and peroxynitrite production by macrophages under different physiological oxygen tensions.

Macrophages count on two O2-consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O2•-) and nitric oxide (•NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O2•- and •NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO2) normally ranges within 2-14 %, with a typical average pO2 value for most tissues ca. 5 %. Given that O2 is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O2•- and •NO production. Also, O2 is a modulator of the macrophage adaptative response and may influence iNOS expression in a hypoxia inducible factor 1-α (HIF1α-)-dependent manner. However, most of the reported experiments in cellula, analyzing the formation and effects of O2•- and •NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO2; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O2) which are far from the physiological pO2. Here, the role of O2 as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO2 and O2•- and •NO formation rates were measured, obtaining a KM of 26 and 42 µM O2 for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO2, reaching a maximum at ≥ 10 % O2, but even at levels as low as 2 % O2, a substantial formation rate of this oxidant was detected. Indeed, the cytotoxic capacity of immunostimulated macrophages against the intracellular parasite T. cruzi was significant, even at low pO2 values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions.

Antioxidant and Antiproliferative Activities of Several Garlic Forms.

It is hypothesized that garlic, Allium sativum, might protect against oxidative stress that causes damage to cells and tissues leading to the development of various health conditions including cancer. However, it is not known whether garlic's potential anticancer benefits differ by form of garlic consumed. This study aimed to quantify and compare the in vitro antioxidant and antiproliferative activity of several garlic forms in water and alcohol extracts including fresh garlic, fresh garlic set aside, heated garlic, heated garlic set aside, garlic powder, black garlic, two commercially available garlic supplements. Antioxidant activity of different garlic forms were measured using three assays: DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay, superoxide assay, and hydroxyl assay. In vitro effects of garlic extracts were investigated against the most common lung cancer subtypes: H520, H1975, and A549 cell lines using the sulforhodamine B (SRB) assay. Among free radical scavenging assays, Garlicin®, a commercially available supplement, displayed high antioxidant activity in water and alcohol extracts (DPPH assay: 2.02 mg AAE (mg ascorbic acid equivalent)/g garlic and 3.53 mg AAE/g garlic, respectively; superoxide assay: 6.73 mg AAE/g garlic and 7.13 mg AAE/g garlic, respectively). In the hydroxyl assay, water extract of fresh garlic crushed and set aside for 10 min showed the highest antioxidant activity. Garlicin® alcohol extract and fresh garlic water extracts strongly inhibited the proliferation of H1975, A549 and H520 cells. Other forms of garlic including garlic powder and black garlic exhibited low antioxidant and antiproliferative activity. Our results demonstrate that the preparation and processing methods of garlic may lead to different antioxidant benefits.

Lipid Peroxidation and Antioxidant Protection.

Lipid peroxidation (LP) is the most important type of oxidative-radical damage in biological systems, owing to its interplay with ferroptosis and to its role in secondary damage to other biomolecules, such as proteins. The chemistry of LP and its biological consequences are reviewed with focus on the kinetics of the various processes, which helps understand the mechanisms and efficacy of antioxidant strategies. The main types of antioxidants are discussed in terms of structure-activity rationalization, with focus on mechanism and kinetics, as well as on their potential role in modulating ferroptosis. Phenols, pyri(mi)dinols, antioxidants based on heavy chalcogens (Se and Te), diarylamines, ascorbate and others are addressed, along with the latest unconventional antioxidant strategies based on the double-sided role of the superoxide/hydroperoxyl radical system.

ZnO/black phosphorus/C3N4 composite: An effective photocatalyst for Cr (VI) reduction and degradation of rhodamine B.

The utilization of photocatalysts offers a promising approach for the removal of Cr (VI) and rhodamine dyes. Through the generation of reactive species and subsequent degradation reactions, photocatalysis provides an efficient and environmentally friendly method for the remediation of wastewater. In this study, we have synthesized an n-p-n heterojunction of carbon nitride (C3N4), zinc oxide (ZnO), and black phosphorus (BP) through the sonication-stirring method. The photocatalytic ability of this composite was examined for the decomposition rhodamine B (RhB) and detoxification of hexavalent chromium ion (up to 97% during 80 min) under Xenon irradiation. The results of trapper experiments indicated that the active species were hydroxyl radical (˙OH), electron (e-), and superoxide anion radical (˙O2-). Based on the obtained potential of the lowest unoccupied molecular orbitals (LUMO) and the highest occupied molecular orbital (HOMO) for the mentioned semiconductors, through Mutt-Schottky results, the double Z-scheme mechanism was proposed for the studied process. The electrochemical impedance spectroscopy data exhibited good charge transfer for the evaluated composite versus the pure compounds. The impressive separation of holes and electrons along with the low recombination were confirmed by the responses of photocurrent and quenching the photoluminescence (pl) intensity for the composite, respectively. The current density of the composite recorded 66.6%, 87.3%, and 92% higher than those of BP, C3N4, and ZnO, indicating an excellent electron-hole separation for the ternary composite compared to the pure semiconductors. Diffuse reflectance spectra (DRS) data revealed 2.9, 3.17, 1.15, and 2.63 eV as the band gap values for C3N4, ZnO, BP, and composite. The rate constant of the new composite to remove RhB and reduce hexavalent chromium were about 4.79 and 2.64 times higher than that of C3N4, respectively.

The Protective Effect of Lithium Against Rotenone may be Evolutionarily Conserved: Evidence from Eisenia fetida, a Primitive Animal with a Ganglionic Brain.

Chronic exposure to stress is a non-adaptive situation that is associated with mitochondrial dysfunction and the accumulation of reactive oxygen species (ROS), especially superoxide anion (SA). This accumulation of ROS produces damage-associated molecular patterns (DAMPs), which activate chronic inflammatory states and behavioral changes found in several mood disorders. In a previous study, we observed that an imbalance of SA triggered by rotenone (Ro) exposure caused evolutionarily conserved oxi-inflammatory disturbances and behavioral changes in Eisenia fetida earthworms. These results supported our hypothesis that SA imbalance triggered by Ro exposure could be attenuated by lithium carbonate (LC), which has anti-inflammatory properties. The initial protocol exposed earthworms to Ro (30 nM) and four different LC concentrations. LC at a concentration of 12.85 mg/L decreased SA and nitric oxide (NO) levels and was chosen to perform complementary assays: (1) neuromuscular damage evaluated by optical and scanning electron microscopy (SEM), (2) innate immune inefficiency by analysis of Eisenia spp. extracellular neutrophil traps (eNETs), and (3) behavioral changes. Gene expression was also evaluated involving mitochondrial (COII, ND1), inflammatory (EaTLR, AMP), and neuronal transmission (nAchR α5). LC attenuated the high melanized deposits in the circular musculature, fiber disarrangement, destruction of secretory glands, immune inefficiency, and impulsive behavior pattern triggered by Ro exposure. However, the effects of LC and Ro on gene expression were more heterogeneous. In summary, SA imbalance, potentially associated with mitochondrial dysfunction, appears to be an evolutionary component triggering oxidative, inflammatory, and behavioral changes observed in psychiatric disorders that are inhibited by LC exposure.

Slit dual-frequency ultrasound-assisted pulping of Lycium barbarum fresh fruit to improve the dissolution of polysaccharides and in situ real-time monitoring.

In this study, the slit dual-frequency ultrasound-assisted pulping of fresh Lycium barbarum fruit was optimized to improve the dissolution of polysaccharides. The microscopic mechanism of polysaccharide dissolution was explored through establishing polysaccharides dissolution kinetics model and visualizing the multi-physical fields during ultrasonic process, and an in situ real-time monitoring model was established by the relationship between the chemical value and spectral information collected by near-infrared spectroscopy. The results showed that, under optimal conditions, treatment with ultrasound (28-33 kHz, 250 W, 30 min) not only significantly promoted the dissolution rate of polysaccharides in Lycium barbarum pulp (LBPPs, increased by 43.64 %, p < 0.01), reduced its molecular weight, but also improved the arabinose molar ratio, the uniformity of polysaccharide particles, and the antioxidant activity of LBPPs. Correlation analysis indicated that ultrasonic treatment is closely related to LBPPs content, particle size and scavenging capacity against superoxide anion radicals (ptotal sugar content < 0.01, pparticle size < 0.05 and psuperoxide anion scavenging < 0.05). Moreover, the in situ real-time monitoring model for the pulping process could quantitatively predict LBPPs dissolution rate and its superoxide anion radical scavenging capacity with good calibration and prediction performance (Rc = 0.9841, RMSECV = 0.0873, Rp = 0.9772, RMSEP = 0.0530; Rc = 0.9874, RMSECV = 0.1246, Rp = 0.9868, RMSEP = 0.0665). These results indicated that slit dual-frequency ultrasound has great potential in improving the quality of Lycium barbarum pulp, which may provide theoretical support for the industrial development of intelligent systems for polysaccharides preparation.

Maytenus octogona Superoxide Scavenging and Anti-Inflammatory Caspase-1 Inhibition Study Using Cyclic Voltammetry and Computational Docking Techniques.

The relationship between oxidative stress and inflammation is well known, and exogenous antioxidants, primarily phytochemical natural products, may assist the body's endogenous defense systems in preventing diseases due to excessive inflammation. In this study, we evaluated the antioxidant properties of ethnomedicines from Peru that exhibit anti-inflammatory activity by measuring the superoxide scavenging activity of ethanol extracts of Maytenus octogona aerial parts using hydrodynamic voltammetry at a rotating ring-disk electrode (RRDE). The chemical compositions of these extracts are known and the interactions of three methide-quinone compounds found in Maytenus octogona with caspase-1 were analyzed using computational docking studies. Caspase-1 is a critical enzyme triggered during the activation of the inflammasome and its actions are associated with excessive release of cytokines. The most important amino acid involved in active site caspase-1 inhibition is Arg341 and, through docking calculations, we see that this amino acid is stabilized by interactions with the three potential methide-quinone Maytenus octogona inhibitors, hydroxytingenone, tingenone, and pristimerin. These findings were also confirmed after more rigorous molecular dynamics calculations. It is worth noting that, in these three compounds, the methide-quinone carbonyl oxygen is the preferred hydrogen bond acceptor site, although tingenone's other carbonyl group also shows a similar binding energy preference. The results of these calculations and cyclovoltammetry studies support the effectiveness and use of anti-inflammatory ethnopharmacological ethanol extract of Maytenus octogona (L'Héritier) DC.

Mn Single-Atom Nanozymes with Superior Loading Capability and Superb Superoxide Dismutase-like Activity for Bioassay.

Single-atom nanozymes (SANs) with highly exposed active sites and remarkable catalytic activity have shown noteworthy practicability in heterogeneous catalysis-based bioassay. Nevertheless, most of them were reported with peroxidase-like activity and ordinary loading capability. It is still a challenge to prepare high-loading SANs with desirable superoxide dismutase (SOD)-like activity. In this work, Mn SAN was successfully confined in the frameworks of Prussian blue analogues formed on Ti3C2 MXene sheets with the assistance of massive surfactants, which show a superior loading efficiency of 13.5 wt % (typically <2.0 wt %). The prepared Mn SAN exhibits desirable superoxide radical anion elimination capability because of its SOD-like activity. Moreover, due to the wide-spectrum absorption behavior of the carriers, Mn SAN shows a synergistically quenching efficiency up to 98.89% on the emission of the reactive oxygen species-mediated chemiluminescent (CL) system. Inspired by these features, a CL quenching method was developed on a lateral flow test strip platform by utilizing Mn SAN as a signal quencher and acetamiprid as a model analyte. The method for detecting acetamiprid shows a detection range of 1.0-10,000 pg mL-1 and a limit of detection of 0.3 pg mL-1. Its accuracy has been validated by detecting acetamiprid in medicinal herbs with acceptable recoveries. This work opens an avenue for preparing SANs with a surfactant-assisted protocol and pioneers the study of SANs with SOD-like activity in bioassay.

Vascular protection afforded by zinc supplementation in human coronary artery smooth muscle cells mediated by NRF2 signaling under hypoxia/reoxygenation.

Zinc (Zn) has antioxidant, anti-inflammatory and anti-proliferative actions, with Zn dysregulation associated with coronary ischemia/reperfusion injury and smooth muscle cell dysfunction. As the majority of studies concerning Zn have been conducted under non-physiological hyperoxic conditions, we compare the effects of Zn chelation or supplementation on total intracellular Zn content, antioxidant NRF2 targeted gene transcription and hypoxia/reoxygenation-induced reactive oxygen species generation in human coronary artery smooth muscle cells (HCASMC) pre-adapted to hyperoxia (18 kPa O2) or normoxia (5 kPa O2). Expression of the smooth muscle marker SM22-α was unaffected by lowering pericellular O2, whereas calponin-1 was significantly upregulated in cells under 5 kPa O2, indicating a more physiological contractile phenotype under 5 kPa O2. Inductively coupled plasma mass spectrometry established that Zn supplementation (10 µM ZnCl2 + 0.5 µM pyrithione) significantly increased total Zn content in HCASMC under 18 but not 5 kPa O2. Zn supplementation increased metallothionein mRNA expression and NRF2 nuclear accumulation in cells under 18 or 5 kPa O2. Notably, NRF2 regulated HO-1 and NQO1 mRNA expression in response to Zn supplementation was only upregulated in cells under 18 but not 5 kPa. Furthermore, whilst hypoxia increased intracellular glutathione (GSH) in cells pre-adapted to 18 but not 5 kPa O2, reoxygenation had negligible effects on GSH or total Zn content. Reoxygenation-induced superoxide generation in cells under 18 kPa O2 was abrogated by PEG-superoxide dismutase but not by PEG-catalase, and Zn supplementation, but not Zn chelation, attenuated reoxygenation-induced superoxide generation in cells under 18 but not 5kPaO2, consistent with a lower redox stress under physiological normoxia. Our findings highlight that culture of HCASMC under physiological normoxia recapitulates an in vivo contractile phenotype and that effects of Zn on NRF2 signaling are altered by oxygen tension.

Selenium-Containing Type-I Organic Photosensitizers with Dual Reactive Oxygen Species of Superoxide and Hydroxyl Radicals as Switch-Hitter for Photodynamic Therapy.

Organic type-I photosensitizers (PSs) which produce aggressive reactive oxygen species (ROS) with less oxygen-dependent exhibit attractive curative effect for photodynamic therapy (PDT), as they adapt better to hypoxia microenvironment in tumors. However, the reported type-I PSs are limited and its exacted mechanism of oxygen dependence is still unclear. Herein, new selenium-containing type-I PSs of Se6 and Se5 with benzoselenadiazole acceptor has been designed and possessed aggregation-induced emission characteristic. Benefited from double heavy-atom-effect of selenium and bromine, Se6 shows a smaller energy gap (ΔEST ) of 0.03 eV and improves ROS efficiency. Interestingly, type-I radicals of both long-lived superoxide anion (O2 •‾ ) and short-lived hydroxyl (• OH) are generated from them upon irradiation. This may provide a switch-hitter of dual-radical with complementary lifetimes for PDT. More importantly, simultaneous processes to produce • OH are revealed, including disproportionation of O2 •‾ and reaction between excited PS and water. Actually, Se6 displays superior in-vitro PDT performance to commercial chlorin e6 (Ce6), under normoxia or hypoxia. After intravenous injection, a significantly in-vivo PDT performance is demonstrated on Se6, where tumor growth inhibition rates of 99% is higher than Ce6. These findings offer new insights about both molecular design and mechanism study of type-I PSs.

Revisit the E2 Domain of Amyloid Precursor Protein: Ferroxidase, Superoxide and Peroxynitrite Scavenging Activities.

Amyloid precursor protein (APP) is the biological precursor of ß-amyloids, a known histopathological hallmark associated with Alzheimer's disease (AD). The function of APP is of great interest yet remains elusive. One of the extracellular domains of APP, the E2 domain, has been proposed to possess ferroxidase activity and affect neuronal iron homeostasis. However, contradicting evidence has been reported, and its precise role remains inconclusive. Here, we studied the Cu-binding site of the E2 domain using extended X-ray absorption fine structure (EXAFS), UV-vis, and electron paramagnetic resonance (EPR) and discovered that a new labile water ligand coordinates to the Cu(II) cofactor in addition to the four known histidines. We explored the proposed ferroxidase activity of the Cu(II)-E2 domain through reactions with ferrous iron and observed single-turnover ferrous oxidation activity with a rate up to 1.0 × 102 M-1 s-1. Cu(I)-E2 reacted with molecular oxygen at a rate of only 5.3 M-1 s-1, which would restrict any potential multiturnover ferroxidase activity to this slow rate and prevents observation of activity under multiturnover conditions. The positive electrostatic potential surface of the protein indicates possible reactivity with negatively charged small substrates such as superoxide radicals (O2•-) and peroxynitrite (ONOO-) that are major contributors to the oxidative stress prevalent in the extracellular environment. Our assays showed that Cu(I)-E2 can remove O2•- at a rate of 1.6 × 105 M-1 s-1, which is slower than the rates of native SODs. However, the reaction between Cu(I)-E2 and ONOO- achieved a rate of 1.1 × 105 M-1 s-1, comparable to native ONOO- scavenger peroxiredoxins (105-107 M-1 s-1). Therefore, the E2 domain of APP can serve as an enzymatic site that may function as a ferroxidase under substrate-limiting conditions, a supplemental O2•- scavenger, and an ONOO- remover in the vicinity of the cellular iron efflux channel and protect neuron cells from reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage.

Simple assay for quantifying xanthine oxidase activity.

This paper reports a sensitive method for assaying xanthine oxidase (XO) enzyme activity. XO produces hydrogen peroxide (H2O2) and superoxide anion radicals (O2•-), promoting the development of oxidative stress-related diseases, and is inhibited by various plant extracts. XO activity is quantified by incubating enzyme samples with an appropriate xanthine concentration as the substrate. The proposed method requires XO activity to be quantified based on H2O2 generation using a 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system catalyzed by cupric ions. After a 30-min incubation at 37 °C, sufficient cupric ion and TMB amounts are added. The assay produces optical signals that can be visually recognized or detected with a UV-visible spectrometer. A direct correlation was found between XO activity and the absorbance at 450 nm of the resulting di-imine (dication) yellow product. The proposed method uses sodium azide to prevent catalase enzyme interference. The new assay's function was confirmed using the TMB-XO assay and a Bland-Altman plot. The resulting correlation coefficient was 0.9976. The innovative assay was relatively precise and comparable to the comparison protocols. In conclusion, the presented method is very efficient at measuring XO activity.

Diclofenac exhibits cytotoxic activity associated with metabolic alterations and p53 induction in ESCC cell lines and decreases ESCC tumor burden in vivo.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human malignancy, often displaying limited therapeutic response. Here, we examine the non-steroidal anti-inflammatory drug diclofenac (DCF) as a novel therapeutic agent in ESCC using complementary in vitro and in vivo models. DCF selectively reduced viability of human ESCC cell lines TE11, KYSE150, and KYSE410 as compared with normal primary or immortalized esophageal keratinocytes. Apoptosis and altered cell cycle profiles were documented in DCF-treated TE11 and KYSE 150. In DCF-treated TE11, RNA-Sequencing identified differentially expressed genes and Ingenuity Pathway Analysis predicted alterations in pathways associated with cellular metabolism and p53 signaling. Downregulation of proteins associated with glycolysis was documented in DCF-treated TE11 and KYSE150. In response to DCF, TE11 cells further displayed reduced levels of ATP, pyruvate, and lactate. Evidence of mitochondrial depolarization and superoxide production was induced by DCF in TE11 and KYSE150. In DCF-treated TE11, the superoxide scavenger MitoTempo improved viability, supporting a role for mitochondrial reactive oxygen species in DCF-mediated toxicity. DCF treatment resulted in increased expression of p53 in TE11 and KYSE150. p53 was further identified as a mediator of DCF-mediated toxicity in TE11 as genetic depletion of p53 partially limited apoptosis in response to DCF. Consistent with the anticancer activity of DCF in vitro, the drug significantly decreased tumor burdene in syngeneic ESCC xenograft tumors and 4-nitroquinoline 1-oxide-mediated ESCC lesions in vivo. These preclinical findings identify DCF as an experimental therapeutic that should be explored further in ESCC.

[Blueberry attenuates liver injury in metabolic dysfunction-associated liver disease by promoting the expression of mitofilin/Mic60 in human hepatocytes and inhibiting the production of superoxide].

Objective To study the effect and mechanism of blueberry on regulating the mitochondrial inner membrane protein mitofilin/Mic60 in an in vitro model of metabolic dysfunction-associated liver disease (MAFLD). Methods L02 human hepatocytes were induced by free fatty acids (FFA) to establish MAFLD cell model. A normal group, a model group, an 80 µg/mL blueberry treatment group, a Mic60 short hairpin RNA (Mic60 shRNA) transfection group, and Mic60 knockdown combined with an 80 µg/mL blueberry treatment group were established. The intracellular lipid deposition was observed by oil red O staining, and the effect of different concentrations of blueberry pulp on the survival rate of L02 cells treated with FFA was measured by MTT assay. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) contents were measured by visible spectrophotometry. The expression of reactive oxygen species (ROS) in hepatocytes was observed by fluorescence microscopy, and the mRNA and protein expression of Mic60 were detected by real-time quantitative PCR and Western blot analysis, respectively. Results After 24 hours of FFA stimulation, a large number of red lipid droplets in the cytoplasm of L02 cells was observed, and the survival rate of L02 cells treated with 80 µg/mL blueberry was higher. The results of ALT, AST, TG, TC, MDA and the fluorescence intensity of ROS in blueberry treated group were lower than those in model group, while the levels of SOD, GSH, Mic60 mRNA and protein in blueberry treated group were higher than those in model group. Conclusion Blueberry promotes the expression of Mic60, increases the levels of SOD and GSH in hepatocytes, and reduces the production of ROS, thus alleviating the injury of MAFLD hepatocytes and regulating the disorder of lipid metabolism.

BP@Au undergoes rapid degradation and releases singlet oxygen under dark conditions: Doping effect and detrimental effects on superoxide-producing marine algae.

Black phosphorus (BP) shows encouraging utility in many fields, and metal doping has been suggested as an efficient way to improve stability. However, controversial results and inconsistent mechanisms have been reported for doping modulation and stability change. We observed the unforeseen evolution of singlet oxygen (1O2) from BP integrated with gold nanoparticles (BP@Au) under dark conditions, and this led to rapid BP deterioration, even though enhanced stability is commonly thought via surface doping. Briefly, the BP reacted with oxygen and water to yield superoxide (O2•-) and hydrogen peroxide. Au0 acted as an enzyme mimic and catalyzed the conversion of these derivatives, and Au0 was converted to a mixture of Au3+ and Au+. The O2•- was converted to 1O2 via direct donation of electrons to the Au3+/+. The Au-catalyzed redox reactions accelerated the degradation of the BP nanosheets. BP@Au showed significant toxicity toward marine alga that produce O2•- in the dark, as indicated by a more than 30% reduction in cell viability after 12 h of incubation with 7.56 mg/L BP@Au. The novelty of this work lies in the demonstration of a dopant-related degradation pathway of BP that shows unrevealed toxicity toward O2•--producing marine algae.

Phytochemicals, Antioxidant and Antimicrobial Potentials and LC-MS Analysis of Centaurea parviflora Desf. Extracts.

Centaurea parviflora (C. parviflora), belonging to the family Asteraceae, is an Algerian medicinal plant used in folk medicine to treat different diseases related to hyperglycemic and inflammatory disorders, as well as in food. The present study aimed to assess the total phenolic content, in vitro antioxidant and antimicrobial activity and phytochemical profile of the extracts of C. parviflora. The extraction of phenolic compounds from aerial parts was conducted using solvents of increasing polarity starting from methanol, resulting in crude extract (CE), to chloroform extract (CHE), ethyl acetate extract (EAE) and butanol extract (BUE). The total phenolic, flavonoid and flavonol contents of the extracts were determined using the Folin-Ciocalteu and AlCl3 methods, respectively. The antioxidant activity was measured with seven methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, galvinoxyl free-radical-scavenging test, 2,2'-Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) assay, cupric reducing antioxidant capacity (CUPRAC), reducing power, Fe+2-phenanthroline reduction assay and superoxide-scavenging test. The disc-diffusion method aimed at testing the sensitivity of bacterial strains toward our extracts. A qualitative analysis with thin-layer chromatography of the methanolic extract was performed. Moreover, HPLC-DAD-MS was used to establish the phytochemical profile of the BUE. The BUE was found to contain high amounts of total phenolics (175.27 ± 2.79 µg GAE/mg E), flavonoids (59.89 ± 0.91 µg QE/mg E) and flavonols (47.30 ± 0.51 µg RE/mg E). Using TLC, different components such as flavonoids and polyphenols were noted. The highest radical-scavenging ability was recorded for the BUE against DPPH (IC50 = 59.38 ± 0.72 µg/mL), galvinoxyl (IC50 = 36.25 ± 0.42 µg/mL), ABTS (IC50 = 49.52 ± 1.54 µg/mL) and superoxide (IC50 = 13.61 ± 0.38 µg/mL). The BUE had the best reducing power according to the CUPRAC (A0.5 = 71.80 ± 1.22 µg/mL), phenanthroline test (A0.5 = 20.29 ± 1.16 µg/mL) and FRAP (A0.5 = 119.17 ± 0.29 µg/mL). The LC-MS analysis of BUE allowed us to identify eight compounds including six phenolic acids and two flavonoids: quinic acid, five chlorogenic acid derivatives, rutin and quercetin 3-o-glucoside. This preliminary investigation revealed that the extracts of C. parviflora have a good biopharmaceutical activity. The BUE possesses an interesting potential for pharmaceutical/nutraceutical applications.

Effect of nanoclay supported nanosilver on the growth inhibition of aquatic pathogens and immunomodulatory effect in Penaeusvannamei.

Hybrid of nanosilver and nanoscale silicate platelet (AgNSP) is a safe, non-toxic nanomaterial which has been applied in medical use due to its strong antibacterial activity. The application of AgNSP in aquaculture was first proposed in the present study by evaluating the in vitro antibacterial activities against four aquatic pathogens, in vitro effects toward shrimp haemocytes as well as the immune responses and disease resistance in Penaeus vannamei fed with AgNSP for 7 days. For evaluating the antibacterial activities of AgNSP in culture medium, the minimum bactericidal concentration (MBC) values against Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus and Vibrio parahaemolyticus were 100, 15, 625 and 625 mg/L, respectively. Moreover, the inhibition of pathogen growth over a period of 48 h could be achieved by the appropriate treatment of AgNSP in culturing water. In freshwater containing bacterial size of 103 and 106 CFU/mL, the effective doses of AgNSP against A. hydrophila were 12.5 and 450 mg/L, respectively while the effective doses against E. tarda were 0.2 and 50 mg/L, respectively. In seawater with same bacterial size, the effective doses against V. alginolyticus were 150 and 2000 mg/L, respectively while the effective doses against V. parahaemolyticus were 40 and 1500 mg/L, respectively. For the in vitro immune tests, the superoxide anion production and phenoloxidase activity in haemocytes were elevated after in vitro incubation with 0.5-10 mg/L of AgNSP. In the assessment of dietary supplemental effects of AgNSP (2 g/kg), no negative effect on the survival was found at the end of 7 day feeding trail. In addition, the gene expression of superoxide dismutase, lysozyme and glutathione peroxidase were up-regulated in haemocytes taken from shrimps received AgNSP. The following challenge test against Vibrio alginolyticus showed that the survival of shrimp fed with AgNSP was higher than that of shrimp fed with control diet (p = 0.083). Dietary AgNSP improved the Vibrio resistance of shrimp by increasing 22.7% of survival rate. Therefore, AgNSP could potentially be used as a feed additive in shrimp culture.

α-Amylase inhibitory potential of Thunbergia mysorensis leaves extract and bioactive compounds by in vitro and computational approach.

In this study, we aim to evaluate the anti-diabetic potential of Thunbergia mysorensis leaves methanolic extract (MeL) using inhibitory assays for α-glucosidase (AG), α-amylase (AM) (carbohydrate digestive enzymes) and aldose reductase (AR) (an enzyme involved in the polyol pathway responsible for glycation). In addition to antidiabetic studies, antioxidant studies were also performed due to the fact that reactive oxygen species (ROS) are produced by various pathways under diabetic conditions. Hyperglycemia induces ROS by activating the glycation reaction and the electron transport chain in mitochondria. The MeL effectively inhibited the enzymes (AG IC50: 27.86 ± 1.0, AM IC50: 12.00 ± 0.0, AR IC50: 4.50 ± 0.09 µg/mL) and showed effective radical ion scavenging activity during the antioxidant assay (DPPH EC50: 30.10 ± 0.75, ABTS EC50: 27.25 ± 1.00, Superoxide EC50: 35.00 ± 1.50 µg/mL). Using activity-guided repeated fractionation on a silica gel column chromatography, two compounds including 3,4-dimethoxy benzoic acid (DMBA) (101 mg) and 3,4-dimethoxy cinnamic acid (DMCA) (87 mg) with potent anti-diabetic activity were extracted from the MeL of T. mysorensis leaves. Both DMBA (IC50 AG: 27.00 ± 1.05, IC50 AM: 12.15 ± 0.10, IC50 AR: 4.86 ± 0.30 µg/mL) and DMCA (IC50 AG: 27.25 ± 0.98, IC50 AM: 12.50 ± 0.20, IC50 AR: 5.00 ± 1.00 µg/mL) were subjected for enzyme inhibition. Since both compounds significantly inhibited AM, enzyme kinetics for AM inhibition was performed. The compounds also showed effective antioxidant potential (DPPH EC50: 30.50 ± 0.99, ABTS EC50: 27.86 ± 0.16, Superoxide EC50: 36.10 ± 0.24 µg/mL), and DMCA (DPPH EC50: 31.00 ± 1.00, ABTS EC50: 28.00 ± 0.25, Superoxide EC50: 36.25 ± 0.37 µg/mL). Further, to elucidate the role of DMBA and DMCA in enzyme inhibition and stability at the molecular level, both compounds were subjected for in silico enzyme inhibitory studies using molecular docking simulation, molecular dynamics (MD) simulation, and binding free energy calculations. Compared to AR and AG, AM was the most significantly inhibited enzyme (DMBA: -6.6 and DMCA: -7.8 kcal/mol), and compounds combined with AM were subjected to MD simulation. Both compounds were stable in the binding pocket of AM till 100 ns and chiefly use Van der Waal's energy to bind. Compared to the controls, both DMBA and DMCA had a higher efficiency in the inhibition of target enzymes in vitro and in silico. The presence of DMBA and DMCA is more likely to be associated with the potential of MeL in antihyperglycemic activity. This bio-computational study indicates DMBA and DMCA as potential lead inhibitors of AM and could be used as effective anti-diabetic drugs in the near future.

Potato Stu-miR398b-3p Negatively Regulates Cu/Zn-SOD Response to Drought Tolerance.

One of the main impacts of drought stress on plants is an excessive buildup of reactive oxygen species (ROS). A large number of ·OH, highly toxic to cells, will be produced if too much ROS is not quickly cleared. At the heart of antioxidant enzymes is superoxide dismutase (SOD), which is the first antioxidant enzyme to function in the active oxygen scavenging system. To shield cells from oxidative injury, SOD dismutation superoxide anion free radicals generate hydrogen peroxide and molecule oxygen. Cu/Zn SOD is a kind of SOD antioxidant enzyme that is mostly found in higher plants' cytoplasm and chloroplasts. Other studies have demonstrated the significance of the miR398s family of miRNAs in the response of plants to environmental stress. The cleavage location of potato stu-miR398b-3p on Cu/Zn SOD mRNA was verified using RLM-5'RACE. Using the potato variety 'Desiree', the stu-miR398b-3p overexpression mutant was created, and transgenic lines were raised. SOD activity in transgenic lines was discovered to be decreased during drought stress, although other antioxidant enzyme activities were mostly unaltered. Transgenic plants will wilt more quickly than wild-type plants without irrigation. Additionally, this demonstrates that the response of Cu/Zn SOD to drought stress is adversely regulated by potato stu-miR398b-3p.