HPLC波长切换法同时测定心神安胶囊中9种成分的含量

目的 建立HPLC波长切换法同时测定心神安胶囊中9种成分的含量。方法 采用Agilent Eclipse XDB-C₁₈色谱柱,流动相乙腈（A）-0.1%甲酸溶液（B）,梯度洗脱;流速0.9 mL·min^-1;检测波长分别为320 nm[检测远志（口山）酮Ⅲ、3,6''-二芥子酰基蔗糖]、203 nm （检测人参皂苷Rb₁、绞股蓝皂苷XLIX、绞股蓝皂苷XVⅡ）和254 nm （检测毛蕊异黄酮葡萄糖苷、芒柄花苷、毛蕊异黄酮、芒柄花素）;柱温25℃。结果 远志（口山）酮Ⅲ、3,6''-二芥子酰基蔗糖、人参皂苷Rb₁、绞股蓝皂苷XLIX、绞股蓝皂苷XVⅡ、毛蕊异黄酮葡萄糖苷、芒柄花苷、毛蕊异黄酮、芒柄花素分别在2.070~41.40 μg·mL^-1（r=0.999 2）、3.860~77.20 μg·mL^-1（r=0.999 6）、11.29~225.8 μg·mL^-1（r=0.999 8）、5.070~101.4 μg·mL^-1（r=0.999 9）、19.86~397.2 μg·mL^-1（r=0.999 5）、1.280~25.60 μg·mL^-1（r=0.999 1）、0.960 0~19.20 μg·mL^-1（r=0.999 3）、0.670 0~13.40 μg·mL^-1（r=0.999 7）、2.580~51.60 μg·mL^-1（r=0.999 1）内线性关系良好,平均回收率分别为98.04%,99.26%,99.05%,97.42%,100.0%,98.27%,97.81%,96.84%和99.86%,RSD分别为1.28%,0.82%,1.43%,1.43%,0.86%,1.26%,1.38%,1.16%和0.69%。结论 本方法操作简便、准确、重复性好,能够对心神安胶囊中9种成分进行同时含量测定,为提高和完善心神安胶囊的质量标准提供了有效方法。     

HPLC测定异维A酸有关物质

目的 建立HPLC测定异维A酸有关物质的方法。方法 色谱柱为NUCLEOSIL 100-3 C₁₈（4.6 mm×150 mm,3 μm）,以甲醇-水-冰醋酸（770：225：5）为流动相,流速为1.0 mL·min^-1;柱温为25℃,检测波长为355 nm。结果 异维A酸峰与杂质H、I、维A酸、强制降解杂质峰分离良好;异维A酸、杂质H、I和维A酸的线性范围分别为0.000 545 5~21.82 μg·mL^-1（r=0.999 9）,0.002 856~7.14 μg·mL^-1（r=0.999 0）,0.002 789~6.97 μg·mL^-1（r=0.999 1）、0.017 07~22.76 μg·mL^-1（r=0.999 6）;检测限分别为0.27,0.60,0.65,5.50 ng·mL^-1,定量限分别为0.55,2.85,2.80,17.00 ng·mL^-1;杂质H、I和维A酸的平均回收率分别为101.57%,102.02%,101.03%,RSD分别为0.5%,0.8%,1.5%。结论 建立的HPLC方法准确、专属性强,可用于异维A酸有关物质的测定。     

高效液相色谱法测定盐酸赛庚啶的有关物质

目的 建立测定盐酸赛庚啶有关物质的高效液相色谱法。方法 选用Agilent Eclipse XDB-C₁₈柱（4.6 mm×250 mm,5 μm）;流动相：乙腈-缓冲液[称取辛烷磺酸钠2.16 g,加水约500 ml,溶解,加入冰醋酸10.0 ml和三乙胺5.0 ml,加水至1 000 ml,摇匀,用三乙胺调节pH至7.0]（85：15,V/V）,过滤并脱气;流速：1.0 ml/min;检测波长：286 nm;进样量：10 μl;柱温：25℃。结果 盐酸赛庚啶和杂质A、B、C的线性范围分别为0.056 2~5.620 μg/ml（r=0.999 8）,0.052 4~5.240 μg/ml（r=1.000 0）,0.050 3~5.032 μg/ml（r=0.999 9）,0.053 2~5.316 μg/ml（r=0.999 7）;盐酸赛庚啶和杂质A、B、C的定量限在0.049~0.054 μg/m1之间,检测限在0.019~0.022 μg/ml之间;回收率在98%~100%之间;重复性RSD为5.5%（n=6）。结论 该方法简便灵敏,结果准确可靠,重复性好,可用于盐酸赛庚啶有关物质的质量控制。     

HPLC测定奥美沙坦酯中的基因毒性杂质

目的 建立HPLC测定奥美沙坦酯中潜在的基因毒性杂质[杂质1 ：N-（三苯基甲基）-5-（4''-溴甲基联苯-2-基）四氮唑,杂质2 ：N-三苯甲基-5-（4'',4''-二溴甲基联苯-2-基）四氮唑]的含量和限度。方法 采用Phenomenex C₁₈柱（250 mm×4.6 mm,5 μm）;流动相：0.1%冰乙酸水溶液-0.1%冰乙酸乙腈溶液（15：85）;检测波长：254 nm;流速：1.5 mL·min^-1;柱温：25℃。结果 杂质1 和杂质2 均在0.030 97~0.247 7 μg·mL^-1内线性良好（r分别为0.999 6和0.998 7）,平均回收率分别为94.37%和94.43%,RSD分别为2.38%和2.72%（n=9）。结论 该方法专属性强,准确、灵敏,可以作为奥美沙坦酯中基因毒性杂质1 和杂质2 的液相分析方法。     

UPLC法同时测定市售菟丝子中5种化学成分的含量

目的 采用UPLC法同时测定市售菟丝子中5种化学成分的含量。方法 采用Agilent SB-C₁₈（100 mm×2.1 mm,1.8 μm）;流动相为乙腈-0.2%甲酸水溶液进行梯度洗脱;流速：0.4 mL·min^-1;柱温：35℃;分段检测波长：1~5 min,325 nm;5~12 min,260 nm;12~30 min,360 nm。结果 5种成分的检测范围分别为绿原酸：0.50~200.09 mg·L^-1（r =0.999 9）、金丝桃苷：0.56~226.32 mg·L^-1（r =0.999 9）、异槲皮苷：0.45~180.60 mg·L^-1（r =0.999 9）、紫云英苷：0.43~173.07 mg·L^-1（r =0.999 9）、山柰酚：0.43~173.28 mg·L^-1（r =0.999 9）;平均回收率（n = 9）分别为 102.0%（RSD=1.56%）,99.0%（RSD=2.90%）,100.1%（RSD=3.19%）,100.2%（RSD=3.00%）,99.3%（RSD=4.25%）。结论 该方法简便、可靠,可用于菟丝子的质量评价,为进一步提高和完善菟丝子质量标准提供参考。     

RP-HPLC法测定田七痛经胶囊中阿魏酸、藁本内酯、欧当归内酯A

目的 建立高效液相色谱法测定田七痛经胶囊中阿魏酸、藁本内酯、欧当归内酯A的方法。方法 选用Thermo Acclaim C₁₈色谱柱,Thermo SCIENTIFICSyncronis C₁₈色谱柱;检测波长为321、280 nm;体积流量1.0 mL/min;柱温35℃结果 阿魏酸、藁本内酯、欧当归内酯A的质量浓度与峰面积分别在1.12～112.30、1.33～132.60、0.96～95.50 μg/mL呈良好的线性关系;回归方程分别为Y=46 335 540 X+44 186.71（r=0.998）,Y=14 514 060 X+8 715.27（r=0.999）,Y= 22 031 250 X+16 472.10（r=0.999）。结论 该方法准确,简便,灵敏,可作为田七痛经胶囊质量控制的方法。     

高效分子排阻色谱法测定抗毒素/抗血清类制品纯度

目的 建立高效分子排阻色谱法（high performance size exclusion chromatography，HPSEC）测定抗毒素/抗血清类制品纯度，并应用该方法对52批样品进行测定。方法 采用TOSOH TSKgelG3000SWxl色谱柱，以含1%异丙醇，0.2 mol·L^-1磷酸盐缓冲液（pH 7.0）为流动相，流速0.6 mL·min^-1，检测波长280 nm，进样体积20 μL，柱温室温，按面积归一化法计算各组分相对百分含量。结果 选取抗毒素类和抗血清类制品各1批，在蛋白质浓度为1.2~24 mg·mL^-1时，主峰F（ab''）₂、大分子杂质峰和小分子杂质峰峰面积均与相应蛋白质浓度呈良好线性关系（n=5，r>0.999 0），重复性RSD均在0.4%~0.9%（n=6），主成分F（ab''）₂定量限分别为0.1 μg和0.2 μg，检出限分别为0.02 μg和0.1 μg，制备的供试品溶液在室温条件下12 h内各组分稳定性良好。结论 建立的HPSEC可用于抗毒素/抗血清类制品纯度的测定。     

UHPLC测定芎菊上清丸中9种成分含量及化学模式分析

目的 建立UHPLC波长切换法同时测定芎菊上清丸中9种成分的含量方法。方法 采用Agilent Ecilipse C₁₈（2.1 mm×100 mm,1.6 μm）色谱柱,流动相：甲醇-0.05%磷酸水溶液,梯度洗脱;流速为0.3 mL·min^-1;检测波长：327,237,320,345,278,254 nm;柱温30℃;进样量2 μL;并采用SPSS 22.0统计软件对含量测定结果进行主成分分析与聚类分析。结果 绿原酸、3,5-二咖啡酰奎宁酸、栀子苷、甘草苷、阿魏酸、盐酸小檗碱、黄芩苷、升麻素苷、5-O-甲基维斯阿米醇苷线性范围分别为4.30~68.80 μg·mL^-1（r=0.999 0）、6.66~106.56 μg·mL^-1（r=0.999 2）、7.67~122.72 μg·mL^-1（r=0.999 4）、4.88~78.08 μg·mL^-1（r=0.999 1）、2.37~37.92 μg·mL^-1（r=0.999 1）、6.50~103.92 μg·mL^-1（r=0.999 2）、8.85~141.60 μg·mL^-1（r=0.999 4）、0.88~14.08 μg·mL^-1（r=0.999 7）、0.74~11.92 μg·mL^-1（r=0.999 3）;平均加样回收率（n=9）均在99.42%~103.10%,RSD均<2.0%。主成分分析与聚类分析均可将不同生产厂家的芎菊上清丸很好地分类,且分类结果一致。结论 所建立的多成分方法快捷、准确、重复性好,可用于芎菊上清丸的质量控制。     

HPLC同时测定藤珠胃康颗粒中4个皂苷类成分

目的 建立HPLC同时测定藤珠胃康颗粒中4个皂苷类成分（人参皂苷Re、人参皂苷Rb₁、人参皂苷Ro和竹节参皂苷Ⅳa）的方法。方法 采用Hypersil GOLD C₁₈色谱柱（250 mm×4.6 mm,5 μm）,流动相为乙腈-0.2 %磷酸水溶液,梯度洗脱（-5~0 min,19%乙腈;0~14 min,19%→26%乙腈;14~22 min,26%→29%乙腈;22~30 min,29%乙腈;30~40 min,29%→35%乙腈;40~55 min,35%乙腈）,体积流量1.0 mL·min^-1,检测波长203 nm,柱温30 ℃。结果 人参皂苷Re在0.080 4~1.608 μg（r=1）,人参皂苷Rb₁在0.108 8~2.176 μg（r=0.999 9）,人参皂苷Ro在0.288 8~5.776 μg（r=0.999 9）,竹节参皂苷Ⅳa在0.176 0~3.520 μg（r=0.999 9）内线性关系良好;平均回收率（n=6）分别为99.41%,101.92%,99.76%,100.31%,RSD值分别为2.22%,2.07%,0.33%,0.64%。结论 本实验所建立的方法简单,专属性强,重复性好,可用于藤珠胃康颗粒中人参皂苷Re、人参皂苷Rb₁、人参皂苷Ro和竹节参皂苷Ⅳa的测定。     

百贝益肺胶囊含量测定的方法改进

目的 增加评价指标,建立科学、合理的百贝益肺胶囊含量测定方法。方法 C₁₈小柱纯化样品,优化色谱条件为：Vp-ODS色谱柱（150 mm×4.6 mm,5 μm）;柱温：20℃;变动流速;流动相：乙腈（A）-水（B）,梯度洗脱;进样量：10 μL;检测波长：203 nm。结果 所测成分三七皂苷R₁、人参皂苷Rg₁、人参皂苷Rb₁分别在40.24~241.44（r=0.999 6）,91.50~549.00（r=0.999 9）,35.72~241.32（r=0.999 8）μg·mL^-1内线性关系良好;加样回收率为94.15%~99.81%,RSD为1.12%~1.94%。结论 该方法准确,重复性良好,可为修订标准提供依据。     

Effects of local anesthetics on calmodulin-dependent guanylate cyclase in the plasma membrane of Tetrahymena pyriformis

A highly purified preparation of Tetrahymena calmodulin activated a membrane-bound guanylate cyclase by more than 40-fold. This activation of guanylate cyclase by calmodulin was inhibited completely by local anesthetics such as dibucaine, tetracaine, lidocaine and procaine at concentrations that had no appreciable effect on the activities of basal guanylate cyclase (without calmodulin) and adenylate cyclase. The inhibition by dibucaine of calmodulin-mediated activation of the enzyme activity was not reversed by calcium but was partially overcome by increasing the concentration of calmodulin. Kinetic analysis of local anesthetic-induced inhibition of activation of guanylate cyclase demonstrated a mixed type of antagonism. These results suggest the possibility that the inhibition of calmodulin-dependent guanylate cyclase resulted, in part, from interaction of the drugs with calmodulin.     

Age related changes in cardiac and aortic phosphodiesterase activities in normotensive and hypertensive rats.

The activities of cAMP¹ and cGMP phosphodiesterase were studied in the aorta (freed of adventitia layer) and in the heart (ventricles) of normotensive and mincralocorticoid hypertensive rats of 8 or 16 weeks of age. The enzyme activities were determined at low (1 μM) and high (100 μM) substrate concentrations. The changes in activity were compared to the changes in organ weight, protein and DNA content. The increase in organ weight that occurred with both age and hypertensive treatment corresponded mostly to a marked elevation in protein content in the aorta, but not in the heart, where the DNA content increased without any significant variation in protein content. In both tissues. eGMP phosphodiesterase activity measured at low substrate concentration was sensitive to endogenous Ca²⁺-dependent activation and markedly increased with age. This increase was proportionally larger than the variations in DNA content of the tissues, but lower than those of total protein in the aorta. It could not be ascribed to an increase in the activator content of the tissues, which was in excess. By contrast. cGMP phosphodiesterase activity measured at high substrate concentration and cAMP phosphodiesterase activity, measured at either substrate concentration, were not sensitive to the Ca²⁺-dependent activation and did not undergo large changes with age except for a significant decrease in cAMP phosphodiesterase activity at high substrate concentration per mg heart cytosol protein. No relationship could be found between the elevation of blood pressure, due to age or to the influence of the mineralocorticoid treatment, and phosphodiesterase activities, which varied in a similar manner in control and hypertensive rats. The results are consistent with the view that a cGMP phosphodiesterase. which is sensitive to Ca²⁺-dependent endogenous activation, increases in aorta and heart cells with the age of the rat.     

Increased thymidylate synthetase in 5-fluorodeoxyuridine resistant cultured hepatoma cells

A FdUrd resistant line of cultured mouse hepatoma cells has been obtained. The resistant cell line had 6- to 10-fold higher levels of thymidylate synthetase, but dihydrofolate reductase and thymidine kinase were unchanged. No impairment of FdUrd incorporation by the resistant cell line could be detected. The increased thymidylate synthetase in resistant cells had the same turnover number and I₅₀ for FdUMP as the enzyme found in sensitive cells, making it unlikely that a new gene product had been obtained. Sensitive cells could be completely rescued by the addition of thymidine, suggesting that the primary mode of drug action is to diminish thymidine metabolites. Resistant cells, removed from FdUrd for several generations, did not proliferate immediately upon reintroduction of the drug; however, loss of sensitivity was much more rapid than upon initial exposure. These results are interpreted in terms of a mechanism for resistance.     

Structural analogs of 5'-methylthioadenosine as substrates and inhibitors of 5'-methylthioadenosine phosphorylase and as inhibitors of human lymphocyte transformation

5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase was purified 13.4-fold from human peripheral lymphocytes. The enzyme demonstrated normal Michaelis-Menten kinetics with Km values of 26 microM and 7.5 mM for the two substrates, MTA and phosphate, respectively. The rate of MTA degradation was temperature dependent, 47 degrees being the optimum temperature. Five structural analogs served as alternative substrates with Km values ranging from 31 to 53 microM while two compounds, 5'-deoxy-5'-methylthiotubercidin (MTT) (Ki = 31 microM) and adenine (Ki = 172 microM), were inhibitory. These same analogs were examined as inhibitors of mitogen-induced human lymphocyte blastogenesis. MTT was found to be the most effective inhibitor of lymphocyte transformation with an I50 of 80 microM.     

Stimulatory effect of ionophores on adenosine 3',5'-monophosphate content in human mononuclear leukocytes

Ionophores A23187 and bromo-lasalocid ethanolate enhanced the cyclic AMP content in human mononuclear leukocytes. The maximum effect of A23187 with a 10-min incubation was found with 0.3–1.0μM concentrations with or without l-isoproterenol (1 μM) or prostaglandin E ₁ (pge ₁) (0.3 μM). The maximum effect after 5 min of incubation at 37° was observed with 0.05, 0.2 and 1 μm A23187. The effect of ionophore A23187 was enhanced by both aminophylline (1 mM) and isobutyl-methylxanthine (1 mM). Calcium (1 mM). aspirin (1 mM) and indomethacin (100 μM) decreased the stimulatory action of A23187. Bromo-lasalocid ethanolate increased cyclic AMP content in cells maximally at a 3 μM concentration with or without 0.3 μM pge ₁.     

UPLC-TOF-MS法鉴定胀果甘草药渣中黄酮类成分

目的建立胀果甘草药渣中黄酮类成分的超高效液相色谱-高分辨飞行时间质谱(UPLC-TOF-MS)定性分析方法。方法 AgilentSB-C18柱(100mm×4.6mm,1.8μm);流动相乙腈-0.1%甲酸水溶液,梯度洗脱;体积流量0.4mL/min;柱温25℃;检测波长254nm。ESI离子源,飞行时间质谱检测器。对比自制对照品进行鉴别。结果共鉴定出8个黄酮类成分,分别为2’,4,4’-三羟基查耳酮、甘草查耳酮D、甘草查耳酮甲、4’-羟基-2’’,2’’-二甲基吡喃[5’’,6’’,6,7]黄酮、甘草黄酮C、光甘草酮、甘草黄酮B和kanzonolE。结论建立了一种简单、可靠的UPLC-TOF-MS方法对胀果甘草药渣中黄酮类成分进行了鉴定,对胀果甘草药渣综合利用有一定参考价值。     

HPLC测定当归中1-(3'',4''-dihydroxycinnamoyl)-cyclopentane-2,3-diol的含量

目的 建立HPLC测定当归中1-（3'',4''-dihydroxycinnamoyl）-cyclopentane-2,3-diol含量的方法。方法 采用Inertsil ODS-3 C₁₈柱（4.6 mm×250 mm,5 μm）,流动相为乙腈-0.05%三氟乙酸水溶液梯度洗脱,流速为1.0 mL·min^-1,检测波长为325 nm,柱温为30℃。结果 1-（3'',4''-dihydroxycinnamoyl）-cyclopentane-2,3-diol进样量在0.023 36~1.168 0 μg（r=1.000 0）内与峰面积呈良好的线性关系,平均回收率为95.33%,RSD为1.88%。结论 该方法简便、准确、重复性好,可为当归药材的质量控制提供参考。     

Mitochondrial Biology and Neurological Diseases

Mitochondria are extremely active organelles that perform a variety of roles in the cell including energy production, regulation of calcium homeostasis, apoptosis, and population maintenance through fission and fusion. Mitochondrial dysfunction in the form of oxidative stress and mutations can contribute to the pathogenesis of various neurodegenerative diseases such as Parkinson’s (PD), Alzheimer’s (AD), and Huntington’s diseases (HD). Abnormalities of Complex I function in the electron transport chain have been implicated in some neurodegenerative diseases, inhibiting ATP production and generating reactive oxygen species that can cause major damage to mitochondriaMutations in both nuclear and mitochondrial DNA can contribute to neurodegenerative disease, although the pathogenesis of these conditions tends to focus on nuclear mutations. In PD, nuclear genome mutations in the PINK1 and parkin genes have been implicated in neurodegeneration [1], while mutations in APP, PSEN1 and PSEN2 have been implicated in a variety of clinical symptoms of AD [5]. Mutant htt protein is known to cause HD [2]. Much progress has been made to determine some causes of these neurodegenerative diseases, though permanent treatments have yet to be developed. In this review, we discuss the roles of mitochondrial dysfunction in the pathogenesis of these diseases.     

Effects of phenothiazines on the membrane-bound guanylate and adenylate cyclase in tetrahymena pyriformis

The particulate-bound guanylate cyclase activity of Tetrahymena pyriformis was shown previously to be Ca²⁺-dependent and to be activated by an endogenous calmodulin-like protein (Tetrahymena Ca²⁺-binding protein, TCBP) [S. Nagao, Y. Suzuki, Y. Watanabe and Y. Nozawa, Biochem. biophys. Res. Commum.90, 261 (1979)]. Phenothiazine derivatives, such as chlorpromazine and trifluoperazine, that interact with calmodulin were found to inhibit the Ca²⁺-dependent guanylate cyclase activity and the TCBP-induced activation of the guanylate cyclase activity. Ethylene glycol-bis (β-aminoethyl ether)-N, N'-tetraacetic acid (EGTA), a Ca²⁺ chelator, also inhibited the activation of guanylate cyclase. However, the mechanisms by which EGTA and trifluoperazine act were different. The EGTA-induced inhibition could not be overcome by increasing the concentration of TCBP, whereas the trifluoperazine-induced inhibition could be overcome by increasing the concentration of TCBP, but not by increasing the concentration of Ca²⁺. These findings suggest that the mechanism by which trifluoperazine inhibits the activation of guanylate cyclase involves competition with TCBP.