Network pharmacology and in vivo experimental studies reveal the protective effects of 6-hydroxygenistein against hypobaric hypoxia-induced brain injury.

Hypobaric hypoxia-induced brain injury (HHBI) is a progressive neurodegenerative disease that has still not been effectively treated. There are several different mechanisms involved in HHBI. Among them, oxidative stress and inflammation response predominate. 6-hydroxygenistein (4',5,6,7-tetrahydroxyisoflavone, 6-OHG) is a hydroxylated derivative of genistein with excellent antioxidant activity, however, the protective effects and underlying mechanisms against HHBI have not been clarified. In the present study, we aimed to explore the mechanisms of action of 6-OHG on HHBI using network pharmacology and experimental validation. Network pharmacology analysis revealed 186 candidate targets through the intersection of the targets of 6-OHG and related genes in HHBI, which were mainly enriched in oxidative stress and inflammation response. Moreover, key targets of 6-OHG against HHBI, namely Nrf2 and NF-κB, were screened and found to be closely related to oxidative stress and inflammation response. Subsequent in vivo experiments revealed that 6-OHG treatment attenuated oxidative stress and inflammation response, prevented energy disorder and apoptosis as well as maintained the BBB integrity in HHBI mice. In addition, 6-OHG administration up-regulated the expressions of Nrf2 and HO-1 and down-regulated the expressions of NF-κB and NLRP3, thereby inhibiting oxidative stress and inflammation response. Hence, the present study demonstrates that 6-OHG protects against HHBI by stimulating the Nrf2/HO-1 signaling pathway and suppressing the NF-κB/NLRP3 signaling pathway.

Synthesis, antioxidant and anti-hypoxia activities of 6-hydroxygenistein and its methylated derivatives. / 6-ç¾ŸåŸºæŸ“æ–™æœ¨ç´ åŠå ¶ç”²åŸºåŒ–è¡ç”Ÿç‰©çš„åˆæˆå’ŒæŠ—æ°§åŒ–ä¸ŽæŠ—ç¼ºæ°§æ´»æ€§.

OBJECTIVES: Hypoxia is a common pathological phenomenon, usually caused by insufficient oxygen supply or inability to use oxygen effectively. Hydroxylated and methoxylated flavonoids have significant anti-hypoxia activity. This study aims to explore the synthesis, antioxidant and anti-hypoxia activities of 6-hydroxygenistein (6-OHG) and its methoxylated derivatives. METHODS: The 6-OHG and its methoxylated derivatives, including 4',6,7-trimethoxy-5-hydroxyisoflavone (compound 3), 4',5,6,7-tetramethoxyisoflavone (compound 4), 4',6-imethoxy-5,7-dihydroxyisoflavone (compound 6), and 4'-methoxy-5,6,7-trihydroxyisoflavone (compound 7), were synthesized by methylation, bromination, methoxylation, and demethylation using biochanin A as raw material. The structure of these products were characterized by 1hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR) and mass spectrometry (MS). The purity of these compounds was detected by high pressure chromatography (HPLC). The antioxidant activity in vitro was investigated by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) free radical scavenging assay. PC12 cells were divided into a normal group, a hypoxia model group, rutin (1×10-9-1×10-5 mol/L) groups, and target compounds (1×10-9-1×10-5 mol/L) groups under normal and hypoxic conditions. Cell viability was detected by cell counting kit-8 (CCK-8) assay, the target compounds with excellent anti-hypoxia activity and the drug concentration at the maximum anti-hypoxia activity were screened. PC12 cells were treated with the optimal concentration of the target compound or rutin with excellent anti-hypoxia activity, and the cell morphology was observed under light microscope. The apoptotic rate was determined by flow cytometry, and the expressions of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) were detected by Western blotting. RESULTS: The structure of 6-OHG and its 4 methylated derivatives were correct, and the purity was all more than 97%. When the concentration was 4 mmol/L, the DPPH free radical removal rates of chemical compounds 7 and 6-OHG were 81.16% and 86.94%, respectively, which were higher than those of rutin, the positive control. The removal rates of chemical compounds 3, 4, and 6 were all lower than 20%. Compared with the normal group, the cell viability of the hypoxia model group was significantly decreased (P<0.01). Compared with the hypoxia model group, compounds 3, 4, and 6 had no significant effect on cell viability under hypoxic conditions. At all experimental concentrations, the cell viability of the 6-OHG group was significantly higher than that of the hypoxia model group (all P<0.05). The cell viability of compound 7 group at 1×10-7 and 1×10-6 mol/L was significantly higher than that of the hypoxia model group (both P<0.05). The anti-hypoxia activity of 6-OHG and compound 7 was excellent, and the optimal drug concentration was 1×10-6 and 1×10-7 mol/L. After PC12 cells was treated with 6-OHG (1×10-6 mol/L) and compound 7 (1×10-7 mol/L), the cell damage was reduced, the apoptotic rate was significantly decreased (P<0.01), and the protein expression levels of HIF-1α and VEGF were significantly decreased in comparison with the hypoxia model group (both P<0.01). CONCLUSIONS: The optimized synthesis route can increase the yield of 6-OHG and obtain 4 derivatives by methylation and selective demethylation. 6-OHG and compound 7 have excellent antioxidant and anti-hypoxia activities, which are related to the structure of the A-ring ortho-triphenol hydroxyl group in the molecule.

Moslosooflavone protects against brain injury induced by hypobaric hypoxic via suppressing oxidative stress, neuroinflammation, energy metabolism disorder, and apoptosis.

OBJECTIVES: To investigate the protect effect of moslosooflavone against brain injury induced by hypobaric hypoxia (HH) in mice. METHODS: Protective effects of moslosooflavone in oxidative stress, neuroinflammation, energy metabolism disorder, and apoptosis were studied in HH-induced brain damage mice. The pathological morphology in the cortex of mice was determined by hematoxylin and eosin staining. The related protein expressions were detected by western blot. KEY FINDINGS: Moslosooflavone improved HH-induced brain histopathological changes, reduced the contents of ROS and MDA, and elevated the levels of antioxidant enzymes and GSH in HH-exposed brains of mice. Moslosooflavone also markedly enhanced the ATPase activities and PK, ATP contents, while reducing LDH activity and the LD, TNF-α, IL-1ß, and IL-6 contents HH-exposed brains of mice. In addition, moslosooflavone notably decreased the expression of HIF-1α, VEGF, Bax, and cleaved caspase-3 dramatically increasing the expression of Bcl-2, Nrf2, and HO­1 in HH-exposed brains of mice. CONCLUSIONS: Our current studies indicate that moslosooflavone protects HH-induced brain injury possibly through alleviating oxidative stress and neuroinflammation, maintaining the balance of energy metabolism, and inhibiting cell apoptosis.

Research progress on the preparation and application of flavonoid nanocrystals. / é»„é ®ç±»åŒ–åˆç‰©çº³ç±³æ™¶ä½“çš„åˆ¶å¤‡åŠåº”ç”¨ç ”ç©¶è¿›å±•.

Flavonoids have been reported to possess significant pharmacological activities,such as antioxidant, anti-inflammatory and anticancer effects. However, the low solubility and low bioavailability limits their clinical application. Nanocrystal technology can solve the delivery problems of flavonoids by reducing particle size, increasing the solubility of insoluble drugs and improving their bioavailability. This article summaries nanosuspension preparation methods and the stabilizers for flavonoid nanocrystals, and reviews the drug delivery routes including oral, Injection and transdermal of flavonoid nanocrystals, to provide information for further research on nanocrystal delivery system of flavonoids.

Design, synthesis and anti-hypoxia activity of HPN derivatives containing lipophilic long chains.

OBJECTIVE: To design and synthesize long-chain substituted 2-[(4'-hydroxyethoxy) phenyl]-4,4,5,5-tetramethyl-2-imidazoline-1-oxyl 3-oxide (HPN) derivatives with enhanced anti-hypoxic activity. METHODS: HPN derivatives 1, 3, 5 containing lipophilic long chains were synthesized via the alkylation of HPN with 6-bromohexan-1-ol, ethyl 6-bromohexanoate or 6-bromohexane, respectively using acetonitrile as the solvent and K 2CO 3 as the acid-binding agent at 60 ℃. Derivative 2 was synthesized via hydrolysis reactions of derivative 1 in the NaOH/CH 3OH/H 2O system. Using dichloromethane as the solvent and N, N'-diisopropylcarbodiimide as the dehydrating agent, HPN underwent esterification with hexanoic acid to obtain derivative 4. The structures of derivatives 1-5 were characterized by infrared spectroscopy, electron paramagnetic resonance and high resolution mass spectrometry. The purities of derivatives were detected by high performance liquid chromatography, and the lipid solubilities of derivatives were evaluated by calculating the oil-water partition coefficients (log P). Anti-hypoxia activities of HPN and its long-chain lipophilic derivatives 1-5 were evaluated using normobaric hypoxia test and acute decompression hypoxia test. RESULTS: The structures of the derivatives were confirmed by infrared spectroscopy, electron paramagnetic resonance and high resolution mass spectroscopy. The yields of target derivatives were all above 92%, and the purities were all above 96%. The log P values of derivatives 1-5 were 2.78, 2.00, 2.04, 2.88 and 3.10, which were higher than that of HPN (0.97). Derivatives 1-5 significantly prolonged the survival time of mice at the dose of 0.3 mmol/kg in normobaric hypoxic test and reduced the mortality rate of acute decompression hypoxic mice to 60%, 70%, 60%, 70% and 40%, respectively. CONCLUSIONS: The synthesis of derivatives 1-5 is convenient, and the yield is high. The synthesized derivatives especially derivative 5 show anti-hypoxic activity similar to or better than HPN at lower doses.

Protective effects of active compounds from on heart and brain of mice at simulated high altitude.

To investigate the active compounds from on the heart and brain of mice at simulated high altitude.Fifty healthy male adult BALB/c mice were randomly divided into normal control group, hypoxic model group, acetazolamide group, petroleum ether extract of (PESI) group and octacosan group with 10 mice in each group. Acetazolamide group, PESI group and octacosan group were treated with acetazolamide PESI (200 mg/kg) or octacosan by single tail vein injection, respectively. Except normal control group, the mice were exposed to a simulated high altitude of for in an animal decompression chamber. After the mice were sacrificed by cervical dislocation, the heart and brain were histologically observed by HE staining; superoxide dismutase (SOD) activity, total anti-oxidant capacity (T-AOC) and the content of malondialdehyde (MDA) in plasma, heart and brain tissues were detected by WST-1 method, ABTS method and TBA method, respectively; lactic acid and lactate dehydrogenase (LDH) activity in plasma, heart and brain tissues were detected by colorimetric method and microwell plate method, respectively; ATP content and ATPase activity in heart and brain tissues were detected by colorimetric method. PESI and octacosane significantly attenuated the pathological damages of heart and brain tissue at simulated high altitude; increased SOD activity, T-AOC and LDH activity, and decreased the contents of MDA and lactic acid in plasma, heart and brain tissues; increased the content of ATP in heart and brain tissues; increased the activities of Na-K ATPase, Mg ATPase, Ca ATPase and Ca-Mg ATPase in myocardial tissue; and increased the activities of Mg ATPase, Ca-Mg ATPase in brain tissue. PESI and octacosan exert anti-hypoxic activity by improving the antioxidant capacity, reducing the free radical levels, promoting the anaerobic fermentation, and alleviating the energy deficiency and metabolic disorders caused by hypoxia in mice.