Ligustroflavone exerts neuroprotective activity through suppression of NLRP1 inflammasome in ischaemic stroke mice.

Inflammation is thought to play an important role in the pathophysiology of ischaemic stroke, which is a main cause of disability and morbidity worldwide. Inhibition of the NOD-like receptor protein 1 (NLRP1) inflammasome has been reported to alleviate the inflammatory response in cell and animal models. Ligustroflavone (LIG) is a compound derived from Ligustrum lucidum, which shows anti-inflammatory activity and may play a beneficial role in a number of neurological diseases. To date, the potential for LIG to act through NLRP1 as a treatment for ischemic stroke has not been studied. The present study established an ischaemic stroke model by middle cerebral artery occlusion (MCAO). Modified neurological severity scoring, open-field and the Rotarod test were used to assess neurological deficits. Staining with Hoechst 33258 and western blotting were used to evaluate neuronal damage. Expression levels of NLRP1 inflammasome complexes and inflammatory cytokines were determined using western blotting, enzyme-linked immunosorbent assay and reverse transcription-quantitative PCR. Treatment with LIG minimized the impairment of neurological function and blocked neuronal damage in MCAO mice. In addition, treatment with LIG attenuated the upregulation of expression levels of the NLRP1 inflammasome complexes and the inflammatory cytokines TNF-α, IL-18, IL-6 and IL-1ß. Overall, LIG played an important role in anti-inflammatory and neuroprotective activity in MCAO models of ischaemic stroke.

Ligustroflavone mediates the resistance of non-small cell lung cancer to osimertinib by arresting G1/S phase / 药学学报

Natural products are an important source for the development of antitumor lead compounds, but the pharmacological effects and regulatory mechanisms of natural products in osimertinib resistance in non-small cell lung cancer (NSCLC) are not well understood. The natural product ligustroflavone was used as the research object to analyze its efficacy in osimertinib-resistant NSCLC cells by cell proliferation assay and cell cycle detection. The potential targets of ligustroflavone in osimertinib-resistant NSCLC cells were screened by public databases and bioinformatics, molecular docking and microscale thermophoresis were used to identify the interaction between privet and target molecules. Western blot was used to detect the effect of privet on the target molecules and their downstream pathways. Ligustroflavone reduced the proliferation of osimertinib-resistant NSCLC cells, and could arrest the cell cycle. Cyclin-dependent kinase 6 (CDK6) was the potential target of ligustroflavone in osimertinib-resistant NSCLC cells. Ligustroflavone inhibited the activation of CDK6-Rb axis. Together, ligustroflavone could regulate osimertinib resistance in NSCLC cells by binding cell cyclin-related molecules. This study provides a theoretical basis for the targeted drug resistance of NSCLC with natural products, and also provides a new idea for the development of clinical drug combination.

Ligustroflavone ameliorates CCl4-induced liver fibrosis through down-regulating the TGF-ß/Smad signaling pathway.

Liver fibrosis is a pathological process characterized by excess deposition of extracellular matrix (ECM) that are mainly derived from activated hepatic stellate cells. Previous studies suggested that ligustroflavone (LF) was an ingredient of Ligustrum lucidum Ait. with activities of anti-inflammation and anti-oxidation. In this study, we investigated whether LF had any effect on liver fibrosis. In our study, we established a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis and used TGF-ß1-stimulated human hepatic stellate cell line (LX-2) to explore the effect of LF and associated underlying mechanism. LF was used in vivo with low dose (L-LF, 5 mg·kg-1, i.p., 3 times each week) and high dose (H-LF, 20 mg·kg-1, i.p., 3 times each week) and in vitro (25 µmol·L-1). Histopathological and biochemical assays investigations showed that LF delayed the formation of liver fibrosis; decreased AST, ALT activities and increased Alb activity in serum; decreased MDA level, Hyp content and increased GSH-Px concentration, SOD activity in liver tissues. Moreover, immunohistochemical, immunofluorescent and Western blot results showed that LF reduced the expressions of hepatic stellate cells specific marker proteins, including collagen I and α-SMA in vivo and in vitro. In addition, LF markedly suppressed TGF-ß1-upregulated protein expressions of TßR I, TßR II, P-Smad2, P-Smad3 and Smad4 in LX-2 cells. Taken together, these findings demonstrated LF could decrease histopathological lesions, ameliorate oxidative injury, attenuate CCl4-induced liver fibrosis, which may be associated with down-regulating the TGF-ß/Smad signaling pathway.

Ligustroflavone ameliorates CCl / 中国天然药物

Liver fibrosis is a pathological process characterized by excess deposition of extracellular matrix (ECM) that are mainly derived from activated hepatic stellate cells. Previous studies suggested that ligustroflavone (LF) was an ingredient of Ligustrum lucidum Ait. with activities of anti-inflammation and anti-oxidation. In this study, we investigated whether LF had any effect on liver fibrosis. In our study, we established a mouse model of carbon tetrachloride (CCl

Screening of type II 5α-reductase inhibitors from traditional Chinese medicine based on molecular docking and molecular dynamics simulation technology / 中草药

Objective: To screen the potential type II 5α-reductase inhibitors from active ingredients of traditional Chinese medicine (TCM) based on molecular docking and molecular dynamics (MD) simulation technology. Methods: The molecular docking was used to screen 26 monomer compositions from TCM. Based on the docking results, MD was performed to evaluate the binding strength of compounds with protein. The binding free energy of the system was calculated using the MM/PBSA method. The in vitro micro-reaction system was used to investigate biological activity. Results: The binding energies of 26 monomer compositions from TCM to type II 5-alpha Reductase were different. Among them, ligustroflavone, safflower yellow and hinokiflavone have low binding energies to type II 5-alpha reductase, and their binding abilities were strong. The molecular dynamics simulation results are consistent with the docking results (binding capacity: ligustroflavone-protein > safflower yellow-protein > hinokiflavone-protein). The three components ligustroflavone, safflower yellow and hinokiflavone have a certain inhibitory activity on type II 5α-reductase with the IC50 value of (42.12 ± 3.83), (69.06 ± 6.35), and (191.28 ± 5.90) μmol/L, respectively. Conclusion: Among the screened 26 monomer compositions, ligustroflavone, safflower yellow and hinokiflavone have the potential to be used in the study of treatment and prevention of androgen-dependent diseases, which provides a reference for further exploration and discovery of type II 5α-reductase inhibitors.

Ligustroflavone reduces necroptosis in rat brain after ischemic stroke through targeting RIPK1/RIPK3/MLKL pathway.

Receptor-interacting protein kinase 1/3 (RIPK1/3) and mixed lineage kinase domain-like (MLKL)-mediated necroptosis contributes to brain injury after ischemic stroke. Ligustroflavone is an ingredient of common privet with activities of anti-inflammation and complement inhibition. This study aims to explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the underlying mechanisms. A rat model of ischemic stroke was established by middle cerebral artery occlusion (MCAO), which showed ischemic injury (increase in neurological deficit score and infarct volume) and upregulation of necroptosis-associated proteins (RIPK1, RIPK3 and MLKL/p-MLKL). Administration of ligustroflavone (30 mg/kg, i.g.) 15 min before ischemia evidently improved neurological function, reduced infarct volume, and decreased the levels of necroptosis-associated proteins except the RIPK1. Consistently, hypoxia-cultured PC12 cells (O2/N2/CO2, 1:94:5, 8 h) caused cellular injury (LDH release and necroposis) concomitant with up-regulation of necroptosis-associated proteins, and these phenomena were blocked in the presence of ligustroflavone (25 µM) except the elevated RIPK1 levels. Using the Molecular Operating Environment (MOE) program, we identified RIPK1, RIPK3, and MLKL as potential targets of ligustroflavone. Further studies showed that the interaction between RIPK3 and RIPK1 or MLKL was significantly enhanced, which was blocked in the presence of ligustroflavone. Based on these observations, we conclude that ligustroflavone protects rat brain from ischemic injury, and its beneficial effect is related to the prevention of necroptosis through a mechanism involving targeting RIPK1, RIPK3, and/or MLKL.

Protective Effects of Ligustroflavone, an Active Compound from Ligustrum lucidum, on Diabetes-Induced Osteoporosis in Mice: A Potential Candidate as Calcium-Sensing Receptor Antagonist.

Ligustroflavone is one major compound contained in active fraction from Fructus Ligustri Lucidi (the fruit of Ligustrum lucidum), which could regulate parathyroid hormone (PTH) levels and improve calcium balance by acting on calcium-sensing receptors (CaSR). This study aimed to explore the potency of ligustroflavone as a CaSR antagonist and its protective effects against diabetic osteoporosis in mice. LF interacted well with the allosteric site of CaSR shown by molecular docking analysis, increased PTH release of primary parathyroid gland cells and suppressed extracellular calcium influx in HEK-293 cells. The serum level of PTH attained peak value at 2 h and maintained high during the period of 1 h and 3 h than that before treatment in mice after a single dose of LF. Treatment of diabetic mice with LF inhibited the decrease in calcium level of serum and bone and the enhancement in urinary calcium excretion as well as elevated circulating PTH levels. Trabecular bone mineral density and micro-architecture were markedly improved in diabetic mice upon to LF treatment for 8 weeks. LF reduced CaSR mRNA and protein expression in the kidneys of diabetic mice. Taken together, ligustroflavone could transiently increase PTH level and regulate calcium metabolism as well as prevent osteoporosis in diabetic mice, suggesting that ligustroflavone might be an effective antagonist on CaSR.

Dynamic changes of five constituents in Ligustri lucidi Fructus at five picking time / 中成药

AIM To analyze the dynamic changes of five constituents in Ligustri lucidi Fructus at five picking time (August,September,October,November,December).METHODS The HPLC analysis of Ligustri lucidi Fructus ethanol extract was performed on a 25 ℃ thermostatic Aglient Zorbax SB-C1s column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1％ phosphoric acid flowing at 1 mL/min in a gradient elution manner,and the detection wavelength was set at 224 nm.RESULTS Salidroside,tyrosol,luteolin-7-O-glucoside,ligustroflavone and specnuezhenide showed good linear relationships within their own ranges (r ＞0.999 0),whose average recoveries were 99.56％-100.30％ with the RSDs of 0.89％-1.23％.The contents of various constituents (except for tyrosol) were the highest in samples picked up in September,followed by those picked up in October.CONCLUSION The suitable picking time of Ligustri lucidi Fructus is September and October.

Simultaneous Determination of the Contents of 4 Ingredients in Zibu Ganshen Pill by Dual-wavelength HPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of the contents of ligustroflavone,specnuezhe-nide,demethylwedelolactone and wedelolactone in Zibu ganshen pill. METHODS:Dual-wavelength HPLC was performed on the column of Elite C18 with mobile phase of acetonitrile-0.5%acetic acid(gradient elution)at flow rate of 0.9 ml/min,column temper-ature was 25 ℃,detection wavelengths were 224 nm(0-30 min) and 351 nm (30-50 min),and the injection volume was 20 μl. RESULTS:The linear range was 6.75-135.00μg/ml(r=0.999 5)for ligustroflavone,6.54-130.80μg/ml(r=0.999 8)for specnuezhe-nide,4.90-98.00μg/ml(r=0.999 4)for demethylwedelolactone and 6.42-128.40μg/ml(r=0.999 6)for wedelolactone;RSDs of pre-cision,stability and reproducibility tests were no more than 1.25%;average recoveries were 96.15%-99.96%(RSD<2%,n=6). CONCLUSIONS:The method is rapid,sensitive and accurate,and can be used for the contents determination of ligustroflavone, specnuezhenide,demethylwedelolactone and wedelolactone in Zibu ganshen pill.

Simultaneous determination of six index components in ligustri lucidi fructus by MEKC-DAD / 中国药学杂志

OBJECTIVE: To establish a micellar electrokinetic chromatography(MEKC) method for simultaneous determination of salidroside, tyrosol, ligustroflavone, specnuezhenide, oleanolic acid, and ursolic acid in Ligustri Lucidi Fructus. METHODS: 60 mmol·L-1 borax-10 mmol·L-1 SDS-30 mmol·L-1 hydroxypropyl-beta-cyclodextrin-10% methyl alcohol was used as the buffer solution (pH 9.03). Uncoated fused silica capillary (75 μm×64.5 cm, 56 cm of effective length) was used with separation voltage of 20 kV. The detection wavelength was set at 210 nm. The column temperature was maintained at 25°C, and the sample was injected at 5 kPa×6 s. RESULTS: The calibration curves of the six index components showed good linearity (r>0.95) in the range of the tested concentrations, and the average recoveries of the method were between 94.57% and 102.07% (RSD<5%). CONCLUSION: The method is simple, accurate and reproducible, and can be used for the quality control of Ligustri Lucidi Fructus.