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This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.
Subject(s)
Mice , Animals , Reactive Oxygen Species/metabolism , Molecular Docking Simulation , Apoptosis , Oxygen , Adenosine Triphosphate/pharmacology , Mitogen-Activated Protein Kinase Kinases/pharmacology , Glucose/metabolismABSTRACT
This study aimed to investigate the enhancing effect of muscone on the transdermal penetration of traditional Chinese medicine ingredients and explore its possible mechanism of action. The Franz diffusion cells were employed to investigate the effect of muscone on the transdermal permeation of a series of model drugs with a wide range of log P values. The solubilities at saturation and the stratum corneum(SC)/vehicle partition coefficients of model drugs were measured to evaluate the effect of muscone on drug thermodynamic activities and partition of drugs into SC. Attenuated total reflectance-Fourier transform infrared spectroscopy(ATR-FTIR) was employed to explore the effect of muscone on the molecular structure of SC. The results showed that muscone significantly promoted the transdermal penetration of hydrophilic and lipophilic drugs, and the enhancement ratio(ER) increased with the decrease in the log P. Muscone could interact with the SC lipids to increase the disorder and fluidity of lipid bilayer packing, which improved skin permeability and promoted transdermal absorption of drugs. This study provides a scientific basis for the application of muscone in traditional Chinese medicine topical preparations.
Subject(s)
Animals , Rats , Administration, Cutaneous , Cycloparaffins , Medicine, Chinese Traditional , Permeability , Rats, Sprague-Dawley , Skin/metabolism , Skin AbsorptionABSTRACT
Objective To establish a quality control method for bomeol and artificial musk in Xinfufang-Zhenzhusan and Xinfufang-Zhenzhugao.Methods We used petroleum ether-toluene-ethyl acetate (9:3:2)as developer for TLC to identify isoborneol and borneol and petroleum ether-dichloromethane (2:3) as developer for TLC to identificate musk ketone.Agilent 7890 B gas chromatograph,FDI detector;Column:Thermo-TG-WaxMS GC (0.25 mm × 30 m,0.25 mm) was employed;the carrier gas was high purity nitrogen and flow rate for 1 mg/ml,the injection port temperature is 200 C and detector temperature is 250 ℃;the split ratio is 10:1 and injection volume was 1 μl,using temperature programmed.Results The isoborneol,borneol and musk ketone in the range of 0.001-10 mg/ml showed good linearity.The recovery of the method is in the range of 95 ％ to 105 ％.The TLC for isobomeol,bomeol,musk ketone can be identified easily.Conclusions The method was simple and reasonable,which can be used for the quality control of borneol and artificial musk in the Xinfufang-Zhenzhusan and Xinfufang-Zhenzhugao.
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Objective: To develop a GC-MS method for the simultaneous determination of six active constituents ( borneol, isoborneol, cinnamaldehyde, cinnamic acid, muscone and benzyl benzoate) in Shexiang Baoxin pills. Methods: A gas chromatogra-phy-mass spectrography (GC-MS) method was adopted using a DB-5 capillary column (30 m × 0. 25 mm,0. 1 μm). The column tem-perature was raised as the following program:the initial temperature was 70℃, maintained for 2 min, raised the temperature (5℃· min-1) to 150℃, maintained for 4 min and then raised the temperature (20℃·min-1) to 260℃, and maintained for 3 min. The range of mass-to-electric charge ratio was 10 to 425. Results:The calibration curves of borneol, isoborneol, cinnamaldehyde, cinnam-ic acid, muscone and benzyl benzoate were linear within the range of 0.022-22.000 μg·ml-1(r=0.999 6),0.024-24.000 μg· ml-1(r=0.999 6),0.028-28.000μg·ml-1(r=0.999 8),0.034-34.000μg·ml-1(r=0.999 6),0.040-40.000μg·ml-1(r=0.999 7) and 0.050-50. 000 μg·ml-1 (r =0.999 9), respectively. The average recovery was 97.20%, 97.40%, 97.53%, 99. 60%, 98. 78% and 98. 27% and RSD was 0. 89%, 1. 18%, 1. 52%, 1. 49%, 0. 79% and 1. 74%(n=6), respectively. Con-clusion:The method is accurate, suitable, convenient and reproducible, which can be used for the quality control of multi components in Shexiang Baoxin pills.
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OBJECTIVE:To establish a method for the content determination of muscone in Aiweixin oral liquid. METHODS:After extracting sample by butanol,GC-MS was performed on the column of BD-17 MS with the volume temperature of 270 ℃;the carrier gas was helium with the sample size of 1 μl at the flow rate of 10 ml/min;the split ratio was 20∶1. EI was ion source with the electron energy of 70 eV,ion source temperature of 230 ℃ and quadrupole temperature of 150 ℃;the tuning mode was automatic tuning by quality full scanning with the threshold value of 30 in the range of 30-600 aum. RESULTS:The linear range of muscone was 12.216-61.08 ng(r=0.999 6);RSD of precision test was lower than 2% and the RSDs of reproducibility and stability tests were lower than 3%;the average recovery was 100.24%(RSD=1.58%,n=6). CONCLUSIONS:The method is sensitive, rapid and simple,and can be used for the content determination of muscone in Aiweixin oral lipid.
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OBJECTIVE: To develop a UPLC-MS method for determination of seven components (muscone, hypaconitine, aconitine, protocatechuic acid, ferulic acid, ligustilide, 11-keto-β-acetylboswellic acid) in Xiaojin pills. METHODS: The analysis was performed on a Waters Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with gradient elution of the mixture of acetonitrile-wa-ter-formic acid, and the flow rate was 0.45 mL · min-1; positive MS scanning mode was used; the injection volume was 2.0 μL. RESULTS: Linear relationship was obtained between the concentrations and peak areas of the seven compounds (r > 0.999 6). The average recoveries (n=6) were between 99%- 101%. CONCLUSION: This method is simple, accurate, and reliable to determine the contents of the seven components in Xiaojin pills for the quality control.
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OBJECTIVE To investigate the develop mental toxicity of muscone to embryos. METHODS With zebrafish embryos as a model,The 3 h post fertilization (hpf)embryos were exposed to muscone at 5,10,20,40,80 and 160 μmol·L -1 culture solutions for 96 h and inspected daily with mi-croscopy for larval morphology.The drug solution was replaced every 24 h.Spontaneous move ments were checked at 24 hpf.Heart rate at 48 hpf,hatching rate,e mbryo deformity rate and mortality rate were evaluated.The expression of sepn1 was determined with real-ti me quantitative PCR technique at 96 hpf.RESULTS The 24 hpf spontaneous move ments showed no significant difference.At 48 hpf, spine curvature,pericardial ede ma,yolk sac ede ma,and abnormal swi mming were observed.In addition, the 48 hpf heart beats(10 s)was decreased fro m 26.5 ±1 .0 to 18.0 ±1 .9(P <0.01 ).At 48 hpf , hatching rate of 5 ~40 μmol·L -1 decreased(P <0.05),while of 160 μmol·L -1 increased (P <0.05) co mpared with muscone 0 μmol·L -1 .Muscone had little effect on hatching rate at other ti me points;Mal-formation rate and mortality rate at higher concentrations were up to 100%.The sepn1 gene expression at 96 hpf in the exposure groups decreased co mpared with that of control group(P <0.01 ).CONCLU-SION Muscone had toxic effects on the develop ment of zebrafish embryos,including spine curvature, abnormal swi mming,and pericardial ede ma.These effects may be related to the inhibition of sepn1 gene expression by muscone.
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Objective: To compare the absorption characteristics of Luodian Borneolum and muscone after nasal unidirectional perfusion with different Xingnaojing preparations in rats. Methods: The nasal unidirectional perfusion model was used, the contents of Luodian Borneolum and muscone were determined by GC method, and the absorption of Luodian Borneolum and muscone in Xingnaojing Nasal Drop (XN) and Xingnaojing Microemulsion (XM) were compared. Results: The absorption rate constants (Ka) of Luodian Borneolum in XN and XM were (1.00 ± 0.02)and (0.76 ± 0.03) min-1, respectively, and the Ka values of muscone in XN and XM were (0.76 ± 0.02) and (0.58 ± 0.10) min-1, respectively. Conclusion: The absorption of Luodian Borneolum and muscone in XN are both faster than that in XM.
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AIM:To develop a method for determining cholic acid by HPLC-ELSD and GC was applied to determing muscone;in Jawei Xihuang Soft Capsule(Calculus Bovis,Moschus,Venenum Bufonis,Olibanum,Myrrha).METHODS:AC_(18) column(Kromasil C_(18),5 μm,4.6 mm×250 mm)was used as stationary phase,the mobile phase was methanol-0.01% glacial acetic acid(73:21)at a flow rate of 1.0 mL/min.The parameters of ELSD were set as follows:evaporation temperature was 40℃,carrier gas(N_2)pressure was 200 kPa.The GC system consisted of DB-1 capillary column(30 m×0.32 mm×0.25 μm)and FID as the detector.The programmed temperature-GC and internal standard method were employed to determine the content of muscone.RESULTS:The linear ranges of cholic acid and muscone were in the range of 45.2 ng-904 ng and 0.05 mg/mL-0.5 mg/mL respectively.The average recoveries were 99.06% and 99.40% with RSD of 1.56% and 0.95% respectively.CONCLUSION:The method is convenient and accurate,and it can be used for the quality evaluation of Jawei Xihuang Soft Capsule.
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Gas chromatography combined with mass spectrography (GC - MS) was used to analyze the content of Muscone, a form of natural Moschus in high purity, with N2 as the carrier gas and isopsoralen as the internal standard. Results: The linear range of Muscone was from 0.056 to 0.451 g/L, regression equation: y=0. 3454 x-0. 1076,r=0.9999,the recovery was 97.99%and its RSD was 5.0%. RSD of within - day precision was 3.85% and RSD of day - to - day was 2.20% . The contents of Muscone from three batches of natural Moschus were 1.82% (RSD= 2.50%) ,3.33%(RSD = 1.36%) and 4.40%(RSD3.85%) respectively. The method is simple, rapid, reliable, and can be used as quality control for Muscone of Moschus and its preparation.
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Object To determine the content of muscone in preparation Methods Standard addition, DNP derivatization and HPLC with electrospray ionization tandem mass spectrometry (LC/ESI MS/MS) were utilized Results Identification of muscone hydrazone in the sample was based on the unique mass spectra of the standard Under an optimum condition peak corresponding to + ion and characteristic fragments for muscone hydrazone were observed ESI and APCI full scan m/z 419 MS/MS had the same relative molecular mass and fragmentation pattern The recovery was 102 5% with RSD ≤ 2 76% (n=5) Conclusion The qualitative and quantitative determination on muscone could be fulfilled simultaneously
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Objective To observe the effect of muscone on the blood-brain barrier and neuropathological changes after complete cerebral ischemia/reperfusion of rats.Methods The global cerebral ischemia/reperfusion injury model was established by the four-vessel occlusion(4VO) method,and then the model animals were given high-,middle-and low-dose muscone.Meanwhile,sham-operation group,model group,and positive control group were also set up.The rats neuroethology behavior,and the contents of superoxide dismutase(SOD),malondialdehyde(MDA) and excitatory amino acid(EAA) in ischemic brain tissue were examined.Results Muscone increased SOD content,lowered MDA content,reduced the increase of EAA content caused by ischemia and hypoxia,and inhibited the excitatory neurotoxicity caused by EAA.Conclusion Muscone has obvious protective effect on rats with complete cerebral ischemia/reperfusion injury.
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Objective To establish the quality standard for Zaizao Nongsuo Wan. Methods Artificial Calculus Bovis, Radix Paeoniae Rubra, Rhizoma Coptidis and Herba Ephedrae were identified by TLC. The content of Muscone was determined by GC. Results Under the selected conditions, the clear and distinguished spots were obtained. For content determination, the linear range of Muscone was 0.101 2 ? g～ 1.012 ? g, r=0.999 9, the average recovery was 99.11 % ( n=5) and RSD was 3.59 % . Conclusion The methods are proved to be simple, specialization with a good reproducibility, and can be used to control the quality of Zaizao Nongsuo Wan.
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AIM: To study the quality standard for Xingnaojing Injection (Moschus, Borneolum Syntheticum, Fructus Gardeniae, Radix Curcumae). METHODS: Two main components, muscone and bornel, were determined by HPLC and GC, respectively. RESULTS: The average recoveries were 98.3%( RSD was 0.41% , n=3 ) for muscone and 97.7%( RSD =0.54%, n=3 ) for borneol, respectively. CONCLUSION: The method is available with a good reproducibility and can control the quality of this preparation effectively.
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AIM: To establish the quality standard for Shexiangtongbi Babu plaster (Radix Astragali, Radix Angelicae sinensis, moschus, Commiphora myrrha, Resina Boswelliae Carterii, Radix Angelicae Pubescentis, Sanguis Draxonis, etc.) METHODS: Borneolum Syntheticum, Commiphora Myrrha, Radix Angelicae Pubescentis, Sanguis Draxonis, Radix Angelicae Sinensis were identified by TLC, and the content of Muscone was determined by GC. RESULTS: Borneolum Syntheticum, Commiphora Myrrha, Radix Angelicae Pubscentis, Radix Angelicae Sinensis could be identified by TLC. Muscone showed a good linear relationship at range of 0.026?g～0.078?g, r =0.9992. The average recovery was 97.57%, and RSD 1.50% respectively. CONCLUSION: The method is accurate and can be used to control the quality of Shexiangtongbi Babu plaster.
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Objective: To investigate the effect of muscone on infarction volume and neuronal glutamate transporter EAAC1 mRNA expression in rats. Methods: Rats were divided randomly, focal ischemic models of middle cerebral artery occlusion (MCAO) were achieved by inserting nylon surgical thread through the internal carotid artery(ICA) into the anterior cerebral artery(ACA). The rats were sacrificed, the brain was stained by TTC and the area of infarcton was measured. The expression of EAACl mRNA in the hippocampal regions was assessed by RT PCR. Results: Muscone could remarkably reduce tbe infarction volume and expression of EAAC1 mRNA. Conclusion: Muscone plays a significant role in against local cerebral ischemia, the mechanism may be related to reducing EEACl mRNA expression.
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AIM:To develop a method for determining cholic acid by HPLC-ELSD and GC was applied to determing muscone;in Jawei Xihuang Soft Capsule(Calculus Bovis,Moschus,Venenum Bufonis,Olibanum,Myrrha).METHODS:A C18 column(Kromasil C18,5 ?m,4.6 mm?250 mm)was used as stationary phase,the mobile phase was methanol-0.01% glacial acetic acid(73:27) at a flow rate of 1.0 mL/min.The parameters of ELSD were set as follows:evaporation temperature was 40 ℃,carrier gas(N2) pressure was 200 kPa.The GC system consisted of DB-1 capillary column(30 m?0.32 mm?0.25 ?m) and FID as the detector.The programmed temperature-GC and internal standard method were employed to determine the content of muscone.RESULTS:The linear ranges of cholic acid and muscone were in the range of 45.2 ng-904 ng and 0.05 mg/mL—0.5 mg/mL respectively.The average recoveries were 99.06% and 99.40% with RSD of 1.56% and 0.95% respectively.CONCLUSION:The method is convenient and accurate,and it can be used for the quality evaluation of Jawei Xihuang Soft Capsule.
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AIM:To establish the simultaneous determination of menthol, borneol, patchoulic alcohol and muscone in Compound Shexiang Injection (Moschus, Herba Pogostemonis, Borneolum Syntheticum, Mentholum, etc) by GC. METHODS: The GC system consisted of capillary column, Rtx-Wax-30 M; FID as the detector. The programmed temperature-GC and internal standard method were employed to determine four kinds of components in Compound Shexiang Injection. RESULTS: Menthol, borneol, patchoulic alcohol and muscone in Compound Shexiang Injection and n-octadecane have been separated well under the same chromatographic condition. The average recoveries of menthol, borneol, patchoulic alcohol and muscone were 99.76% (RSD=1.48%), 99.36%(RSD=1.99%), 99.15%(RSD=1.67%), 98.87%(RSD=1.57%), respectively. CONCLUSION: The method is sensitive, accurate, separable and reproducible, and can be used to control the quality of Compound Shexiang Injection.