2',4'-二羟基-3'-甲基-3-甲氧基查耳酮上调p21抑制人肝癌HepG2细胞的生长

目的 探讨2'',4''-二羟基-3''-甲基-3-甲氧基查耳酮(C20)对人肝癌HepG2细胞的体外抗肿瘤作用及其潜在的作用机制。方法 通过CCK-8法、集落形成实验、5-乙炔基-2''-脱氧尿苷(EdU)染色法检测C20对人肝癌HepG2细胞增殖的影响;通过彗星实验检测C20(10 μmol·L^-1)对HepG2细胞DNA损伤的影响;通过流式细胞术检测C20(5、10 μmol·L^-1)对HepG2细胞周期阻滞的影响;通过Hoechst染色和流式细胞术检测C20(5、10 μmol·L^-1)对HepG2细胞凋亡的影响。借助Western blotting法检测C20(5、10 μmol·L^-1)处理对HepG2细胞中与凋亡、DNA损伤、细胞周期阻滞相关蛋白表达水平的调控作用。结果 与对照组比较,C20显著抑制HepG2细胞的活力(P<0.001),给药48 h的半数抑制浓度(IC₅₀)为7.937 μmol·L^-1;5 μmol·L^-1 C20能够显著抑制HepG2细胞的集落形成能力(P<0.01);EdU染色结果显示5、10 μmol·L^-1的C20能够抑制人肝癌HepG2细胞的增殖能力;5、10 μmol·L^-1的C20显著诱导HepG2细胞G₂/M期阻滞(P<0.001);5、10 μmol·L^-1的C20显著促进HepG2细胞凋亡(P<0.001),并显著上调Caspas-3、Caspase-9以及PARP的剪切水平(P<0.01);10 μmol·L^-1的C20能够诱导HepG2细胞发生DNA损伤,并且5、10 μmol·L^-1的C20显著上调γH2AX、p21的蛋白水平(P<0.01)。结论 C20能够造成HepG2细胞发生DNA损伤,上调p21蛋白水平,导致细胞G₂/M期阻滞,并进一步诱发凋亡,发挥体外抗肝癌作用。     

HPLC波长切换联合梯度洗脱法同时测定参苏宣肺丸中的6种成分

目的 建立HPLC波长切换联合梯度洗脱法同时测定参苏宣肺丸中咖啡酸、迷迭香酸、3’-羟基葛根素、葛根素、3’-甲氧基葛根素、大豆苷的含量。方法 采用ZOBAX SB-C₁₈色谱柱（4.6 mm×250 mm,5 μm）;流动相A为甲醇-乙腈（1：1）,流动相B为0.1%冰醋酸溶液,进行梯度洗脱;流速为1.0 mL·min^-1;咖啡酸和迷迭香酸的检测波长为320 nm,3’-羟基葛根素、葛根素、3’-甲氧基葛根素和大豆苷的检测波长为250 nm;进样量为20 μL。结果 咖啡酸、迷迭香酸、3’-羟基葛根素、葛根素、3’-甲氧基葛根素、大豆苷的浓度与峰面积分别在2.32~46.40 μg·mL^-1（r=0.999 8）、3.06~61.20 μg·mL^-1（r=0.999 5）、4.45~89.00 μg·mL^-1（r=0.999 9）、14.48~289.60 μg·mL^-1（r=0.999 9）、4.86~97.20 μg·mL^-1（r=0.999 7）、3.69~73.80 μg·mL^-1（r=0.999 6）内具有较好的线性关系,平均加样回收率和相应的RSD分别为99.0%（1.6%）,97.5%（0.6%）,99.3%（0.9%）,98.6%（1.3%）,97.6%（1.3%）,97.0%（0.9%）。结论 方法操作准确、简便,可用于参苏宣肺丸的质量控制。     

新型苯并噁唑酮衍生物W3D的含量测定及体内抗炎活性研究

目的 建立全新结构化合物4-（5''-二甲氨基）-萘磺酰氧基苯并噁唑酮（W3D）的含量测定方法,并对其进行体内抗炎活性评价。方法 采用Diamonsil C₁₈（250 mm×4.6 mm,5 μm）色谱柱,以甲醇-水（75∶25）为流动相,体积流量1.0 mL·min^-1,检测波长257 nm,柱温28℃,测定W3D的峰面积,建立其含量测定方法;采用二甲苯致小鼠耳肿胀模型评价化合物W3D（25.000、12.500、6.250、3.125 mg·kg^-1）体内抗炎活性。结果 供试品主峰专属性良好,在1～100 μg·mL^-1线性关系良好（R²＝0.999 9）,精密度、重复性、稳定性的RSD分别为0.61%、0.19%、0.84%,回收率为99.55%（RSD＝0.81%）;与对照组比较,W3D各剂量组耳肿胀率均显著下降（P＜0.05、0.01）,12.5 mg·kg^-1组抑制率可达53.88%,优于阳性药塞来昔布。结论 建立的HPLC法简便、准确,且专属性好,可用于W3D的含量检测;W3D具有较好的体内抗炎活性,作为新结构抗炎小分子化合物具有深入研究的价值。     

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。     

高灵敏度HPLC测定大鼠血浆中益母草碱浓度及其药动学研究

目的 建立一种高灵敏度HPLC测定大鼠血浆中益母草碱浓度,并研究益母草碱在大鼠体内的药动学特征。方法 大鼠口服益母草碱混悬溶液（50 mg·kg^-1）后,不同时间点尾静脉采血,以苯甲酰精氨酸乙酯为内标,血浆样品经酸化后乙酸乙酯萃取,采用HPLC进行测定。色谱条件：采用Diamonsil C₁₈（250 mm×4.6 mm,5 μm）为色谱柱,以乙腈-0.02 mol·L^-1磷酸二氢钾缓冲溶液（pH 3.0）（22：78）为流动相,流速1.0 mL·min^-1,柱温35℃,检测波长277 nm。并利用PKS 1.0软件计算药动学参数。结果 益母草碱血浆浓度在0.05~1.5 μg·mL^-1内线性关系良好（r=0.999 1）。方法的定量下限（LLOQ）为0.05 μg·mL^-1（RSD=12.8%,n=5）;提取回收率为76.5%~82.5%;批内、批间准确度为96.9%~104.9%;日内、日间精密度均<10%;质控样品经反复冻融3次及-20℃放置1个月后均较稳定。大鼠口服益母草碱后,药-时曲线符合二室开放模型,主要药动学参数为t_max=0.95 h,C_max=0.51 μg·mL^-1,t_1/2=3.64 h,AUC_0-t=1.56 μg·mL^-1·h^-1,AUC_0-∞=1.78 μg·mL^-1·h^-1。结论 该方法准确度、灵敏度高,重复性好,可用于生物样品中益母草碱浓度的测定。     

HPLC测定沅江产芦笋中3种酚酸和6种黄酮类成分含量

目的 建立同时测定沅江产芦笋中原儿茶酸、绿原酸、香草酸、柚皮苷、芦丁、槲皮素、木犀草素、山奈酚、芹菜素含量的HPLC。方法 采用Diamonsil C₁₈色谱柱（250 mm×4.6 mm,5 μm）,以甲醇-0.1%甲酸水溶液为流动相,梯度洗脱,双波长检测（酚酸：283,327 nm;黄酮：283,335 nm）,流速1 mL·min^-1,柱温35℃,进样量为10 μL。结果 3种酚酸及6种黄酮分别在25 min和50 min内分离,线性范围为14.8~500 μg·mL^-1（r=0.999 0~0.999 7）,检测限0.006 3~0.053 9 μg·mL^-1,定量限0.021 1~0.179 7 μg·mL^-1,加样回收率为98.73%~102.44%,RSD为1.27%~2.20%（n=6）。结论 该方法准确灵敏、重复性好,可用于检测芦笋中9种成分含量。     

平消胶囊联合左甲状腺素钠治疗结节性甲状腺肿的临床研究

目的 对狭叶薰衣草Lavandula angustifolia中的木脂素类化合物进行研究。方法 运用RP-HPLC、TLC、硅胶、凝胶、MCI-gel树脂等方法进行分离纯化,并根据理化性质和波谱数据鉴定化合物的结构。结果 从狭叶薰衣草中分离得到11个木脂素类化合物,分别鉴定为松脂醇（1）、丁香树脂醇（2）、fraxiresinol-4''-O-β-D-glucopyranoside（3）、syringaresinol-4''-O-β-D-glucopyranoside（4）、8-hydroxypinoresinol-4-O-β-D-glucopyranoside（5）、rel-（2α,3β）-7-O-methylcedrusin（6）、落叶松脂醇-4''-O-β-D-葡萄糖苷（7）、（2S,3R）-2,3-dihydro-2-（4-hydroxy-3-methoxyphenyl）-3-hydroxymethyl-7-methoxybenzofuran-5-（trans） propen-1-ol-3-O-β-glucoside（8）、（7S,8R）-dihydrodehydrodiconiferyl alcohol-9-β-D-glucopyranoside（9）、（7R,8R）-7,8-dihydro-9''-hydroxyl-3''-methoxyl-8-hydroxymethyl-7-（4-hydroxy-3-methoxyphenyl）-1''-benzofuranpropanol-9''-O-β-D-glucopyranoside（10）、（E）-3-（（2S,3S）-2-（4-hydroxy-3-methoxyphenyl）-3-hydroxymethyl-7-methoxy-2,3-dihydrobenzofuran-5-yl） allyl-2-hydroxyacetate（11）。结论 11个化合物均首次从狭叶薰衣草中分离得到。     

邻苯二甲醛柱前衍生化-HPLC测定复方氨基酸注射液中蛋氨酸亚砜的含量

目的 建立测定复方氨基酸注射液中蛋氨酸亚砜含量的方法。方法 采用邻苯二甲醛（O-phthaladehyde,OPA）柱前衍生HPLC。色谱柱为Agilent ZORBAX Eclipse AAA C₁₈柱（150 mm×4.6 mm,5µm）,检测波长为338 nm,流动相A为0.04 mol·L^-1磷酸二氢钠溶液（调节pH至7.8）,流动相B为乙腈-甲醇-水（45：45：10）,梯度洗脱,流速为2.0 ml·min^-1,柱温为40℃,供试品溶液与OPA衍生剂及硼酸盐缓冲液按设定程序自动混匀后进样。结果 蛋氨酸亚砜的质量浓度线性范围为0.000 5~0.050 0 mg·mL^-1（R=0.999 9）;定量限为0.250 5 μg·mL^-1,检测限为0.083 5 μg·mL^-1;精密度、稳定性、重复性试验的RSD<3.0%;平均回收率为99.1%（RSD=1.33%,n=9）。结论 该方法操作简便,可用于复方氨基酸注射液中蛋氨酸亚砜的含量测定。     

柱前衍生-HPLC测定白消安在家兔体内的药动学参数

目的 建立测定家兔血清中白消安浓度和家兔体内药动学特征的柱前衍生化HPLC。方法 以1,5-戊二醇二甲磺酸酯为内标,二乙基二硫代氨基甲酸钠为衍生化试剂。流动相：甲醇-水（54∶46）,流速：0~20 min（1.0 mL·min^-1）,20~27 min（1.3 mL·min^-1）。柱温：30℃,检测波长：280 nm,进样量：25 μL。家兔分别以灌胃、静注的方式给予白消安,按本法测定血药浓度,DAS 3.0计算药动学参数。结果 白消安的血药浓度在0.1~3.4 mg·-L¹ 内线性关系良好（r=0.999 7）,日内、日间精密度以及样品稳定性符合中国药典2015年版的规定。低、中、高浓度的萃取回收率分别为90.0%,89.0%,91.5%。不同给药途径获得的药动学参数：单剂量口服t_1/2=（2.26±0.66）h,k=（0.33±0.12）·h^-1,k_a=（2.54±1.3）·h^-1,AUC_0–t=（1.95±0.18）h·mg·mL^-1;单剂量静脉注射t_1/2=（1.53±0.09）h,k=（0.45±0.03）·h^-1,AUC_0–∞=（4.38±0.26）h·mg·mL^-1。多剂量口服后C_ss=（0.48±0.03）mg·mL^-1,AUC_0–τ=（3.87±0.26）h·mg·mL^-1。结论 建立的柱前衍生-HPLC法适用于白消安血药浓度测定及药动学研究,不同给药途径的药动学参数为临床药动学研究提供了依据。     

RP-HPLC测定4，5，2’-三吗啉酰氧基-2，5'-二氯二苯甲酮原料药的含量及有关物质

目的 建立测定原料药4,5,2''-三吗啉酰氧基-2,5''-二氯二苯甲酮（LF1）的含量及有关物质的RP-HPLC方法。方法 采用Diamonsil C₁₈（250 mm×4.6 mm,5 μm）色谱柱,乙腈-磷酸水（60：40,pH3.0）为流动相,检测波长230 nm,体积流量1 mL/min,柱温25℃。结果 主峰与杂质峰分离良好,LF1和杂质A分别在质量浓度1.0~100（r=0.999 8）和0.2~2.4 mg/L （r=0.999 6）线性关系良好,最低检测限分别为1和2 ng/mL,平均回收率分别为100.7%和102.0%。结论 本法简便、快速、准确,可用于LF1原料药的含量及有关物质的测定。     

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.     

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.     

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.     

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。     

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.