基于HPLC指纹图谱、多指标成分含量测定结合化学计量学的怀牛膝质量评价＊

目的 本实验采用HPLC方法建立来自3个产地15批怀牛膝药材的指纹图谱，并对β-蜕皮甾酮，25R-牛膝甾酮，25S-牛膝甾酮进行含量测定，同时通过化学计量学的方法对15批怀牛膝进行质量评价。方法 选用Waters SunFire C₁₈（150 mm×4.6 mm，5 μm）色谱柱，检测波长280 nm，以乙腈-0.1%甲酸水梯度洗脱，流速为1.0 mL·min^-1，柱温25℃，进样量10 μL。以β-蜕皮甾酮峰为参照峰，运用软件《中药色谱指纹图谱相似度评价系统（2012版）》对15批怀牛膝药材进行相似度分析，使用Metabo Analyst 5.0网站进行聚类热图分析，使用SIMCA 14.1软件进行PCA和PLS-DA分析。结果 标定了31个共有指纹峰，指认了3个已知成分，并对其进行含量测定，化学计量学分析结果将15批怀牛膝药材样品分为3类，并且筛选了不同产地间潜在的差异性成分。结论 该方法具有良好的精密度、重复性以及稳定性，可为怀牛膝的质量评价与控制提供科学依据及参考。     

A fully automated sex estimation for proximal femur X-ray images through deep learning detection and classification

PurposeTo develop a fully automated deep learning pipeline using digital radiographs to detect the proximal femur region for accurate automated sex estimation.MethodRadiograph predictive features from 2122 Chinese Han clinical pelvic with ages ranging from 18 to 26 years were collected retrospectively to train and test the sex prediction model using deep machine learning’s convolutional neural networks (CNN). Model performance was assessed using a Chinese Han population with 361 samples and a white population with 50 samples. The average accuracy of the sex estimation of the two test datasets was determined.ResultsFor the Chinese Han population test dataset, the sex estimation accuracy was 94.6% (males: 93.9% and females: 94.7%). For the white population samples, the accuracy of sex estimation was 82.9% (males: 80.9% and females: 88.6%). The accuracy of CNN tested in the Chinese population was significantly higher than that tested in the White population (p < 0.001)ConclusionsThe model based on convolutional neural networks has an accuracy similar to that of current state-of-the-art mathematical functions using manually extracted features for the Chinese Han population samples, proving to be a reliable choice for the human sex estimation.     

藏药细叶亚菊的质量标准研究＊

目的 建立细叶亚菊的质量标准，为其质量控制和合理开发利用提供科学参考。方法 采用性状鉴别、显微鉴别、薄层色谱法（TLC）对细叶亚菊进行定性分析；参照《中国药典》（2020年版）通则方法对细叶亚菊水分、总灰分、酸不溶性灰分、水溶性浸出物进行含量检查；利用高效液相色谱法（HPLC）测定细叶亚菊中绿原酸、异绿原酸A的含量。结果 确定了细叶亚菊的药材性状及显微特征。TLC鉴别显示，供试品（细叶亚菊药材）与对照品（绿原酸、异绿原酸A）在相应位置上均显示相同颜色的荧光斑点。13批细叶亚菊水分、总灰分、酸不溶性灰分、浸出物的含量分别为8.55%-13.07%、6.81%-12.68%、1.11%-3.53%、8.41%-11.64%；绿原酸、异绿原酸A的含量范围分别为0.072%-0.440%、0.283%-1.324%（n=3）。结论 本研究建立的方法准确稳定，可为细叶亚菊的质量控制提供参考。暂规定细叶亚菊水分不得过12.0%，总灰分不得过10.0%，酸不溶性灰分不得过2.5%，水溶性浸出物不得少于8.0%，绿原酸不得少于0.2%，异绿原酸A不得少于0.6%。     

慢性高原病脑部多体素1H-MRS研究

目的利用多体素氢质子磁共振波谱成像(hydrogen proton magnetic resonance spectroscopy,¹H-MRS)探讨慢性高原病(chronic mountain sickness,CMS)长期缺氧状态下脑部代谢物的特点,并比较各代谢物与血液指标之间的相关性。材料与方法前瞻性纳入青海大学附属医院经临床确诊为CMS的17例男性患者作为实验组,年龄(53.29±9.03)岁,居住海拔(3989.12±937.45)m,血红蛋白(hemoglobin,HGB)(224.35±11.81)g/mL,并招募与实验组年龄及居住海拔相匹配的18名健康男性志愿者作为对照组,年龄(48.61±8.76)岁,居住海拔(3674.94±634.27)m,HGB(156.67±9.46)g/mL。采用Siemens Prisma 3.0 T MR扫描仪20通道头颅线圈对所有受试者行常规头颅MRI及多体素;H-MRS检查,通过Syngo.via后处理软件获得;H-MRS图,ROI选取双侧额叶及海马区,并获得相应脑区N-乙酰天门冬氨酸/肌酸(NAA/Cr)、胆碱/肌酸CHo/Cr、乙酰天门冬氨酸/胆碱(NAA/CHo)、乳酸/肌酸(Lac/Cr)的比值。结合独立样本t检验及非参数曼-惠特尼U检验以比较两组间代谢物相对浓度差异,然后将CMS组双侧额叶及海马区各代谢物比值与血液生化指标作相关性分析。结果(1)两组受试者年龄、长期居住海拔差异均无统计学意义(P>0.05),而与对照组相比,CMS组HGB、红细胞计数(red blood cell count,RBC)、血细胞比容(hematocrit,HCT)增高,血小板(blood platelet,简称PLT)减低,差异均有统计学意义(P<0.01);(2)与对照组相比,CMS组双侧额叶及海马区NAA/Cr及NAA/CHo均减低(P<0.05),Lac/Cr增高(P<0.05),差异均具有统计学意义;(3)与对照组相比,CMS组双侧额叶及双侧海马区CHo/Cr均增高(P>0.05),差异无统计学意义;(4)CMS组右侧额叶及左侧海马CHo/Cr与RBC明显正相关,左侧额叶CHo/Cr与HCT低度正相关,右侧额叶及左侧海马Lac/Cr与HCT低度正相关。结论CMS长期缺氧状态下脑组织局部代谢物改变,神经元受损,无氧代谢增加,且这些代谢物的改变与血液指标呈一定的相关性,可为临床后续进一步预防或干预CMS患者脑损害提供影像学证据及监测指标。     

Highly sensitive sex determination method using the exon 1 region of the amelogenin gene

Sex determination is a crucial factor in the identification of unidentified human remains. Sex determination by DNA analysis is particularly useful because it can be applied to samples for which morphological characteristics are unavailable. Because samples handled in forensic DNA typing are easily degraded by environmental factors and microorganisms, there is a need for a method that can accurately determine sex even in highly decayed samples. Previous studies mainly used sex differences in an intron of the amelogenin gene. However, this region is highly polymorphic, and there are cases where accurate sexing cannot be performed because of genetic mutations in the target region. Thus, for reliable sex determination, it is desirable to select loci with as few non-sexual polymorphisms as possible. In this study, we focused on the exon 1 region of the amelogenin gene, which has very little polymorphism other than sex differences. We developed a primer set for sex determination and compared it with the GlobalFiler™ PCR Amplification Kit (GF), which is widely used for forensic DNA typing. The results showed that the amount of DNA required for accurate sex determination was 25 pg for both methods, achieving equivalent sensitivity. Next, we compared the two methods using ancient human skeletons and found that the present method with its shorter amplicon was considerably superior to GF. The present method is simple, rapid, inexpensive, and suitable for analyzing highly degraded samples. Therefore, this method is expected to contribute to forensic sciences and physical anthropology.     

Cyanide and lactate levels in patients during chronic oral amygdalin intake followed by intravenous amygdalin administration

The natural compound amygdalin has gained high popularity among tumor patients as a complementary or alternative treatment option. However, due to metabolization of amygdalin to cyanide (HCN) following oral consumption, there could be a high risk of lactic acidosis caused by cyanide intoxication. The present retrospective study was undertaken to evaluate cyanide blood and lactate plasma levels of tumor patients (n = 55) before and after intravenous (i.v.) amygdalin infusion. All patients had also continuously ingested amygdalin tablets (3 x 500 mg/day), excepting on the days of i.v. administration. Each patient received one to five intravenous amygdalin treatments. The time period between each i.v. application ranged between 4–6 days. The initial i.v. dose was 6 mg (n = 28), 9 mg (n = 1), 15 mg (n = 1) or 18 mg (n = 25). The mean cyanide blood level before i.v. amygdalin administration was 34.74 μg/L, which increased significantly to a mean value of 66.20 μg/L after i. v. amygdalin application. In contrast, lactate decreased significantly from 1266 μmol/L pre-infusion to 868 μmol/L post-infusion. Increasing i.v. amygdalin by 1 mg was also associated with a significant increase in the cyanide level, while the lactate blood level significantly decreased. This is the first study evaluating cyanide levels under conditions employed by amygdalin administrators, i.e. after chronic oral amygdalin intake and then again after a closely subsequent intravenous amygdalin administration. Since lactate decreased, whilst cyanide increased, it is concluded that elevation of cyanide does not induce metabolic acidosis in terms of an increased lactate level.     

灰毡毛忍冬LmC3H1基因的克隆及表达模式与绿原酸含量相关性分析

目的:以灰毡毛忍冬为材料,克隆对-香豆酸3-羟化酶(LmC3H1)基因,进行生物信息学和表达模式分析,结合绿原酸含量,研究推测灰毡毛忍冬LmC3H1基因的功能。方法:通过逆转录聚合酶链式反应(RT-PCR)和RACE技术克隆LmC3H1基因的全长c DNA序列,对该序列进行生物信息学分析,并利用实时荧光定量PCR(Real-time PCR)和HPLC分别测定灰毡毛忍冬茎、叶及不同花期花中LmC3H1的相对表达量及绿原酸含量。结果:克隆得LmC3H1(Gen Bank:MN177695)基因,开放阅读框(ORF)长度为1 533 bp,编码510个氨基酸,推测其分子式为C_(2618)H_(4134)N_(718)O_(727)S_(22),相对分子质量为58 005.32,等电点8.92,为亲水性蛋白,定位于叶绿体中,具有跨膜区域LLLIPAVLFLISLVYPLI,含有细胞色素P450的保守结构域CYTOCHROME_P450(422-433 aa);Real-time PCR结果显示,LmC3H1在灰毡毛忍冬茎、叶及不同花期花有不同程度的表达,其中在花发育阶段,白色花蕾期相对表达量最高,花蕾初期及白色开花期次之;白色花蕾期花与茎、叶比,花的相对表达量最高,叶的最低;HPLC结果显示,从绿白色花蕾期到金黄色开花期绿原酸含量呈上升趋势,金黄色开花期含量最高,不同器官中,花中绿原酸最高,茎最低。结论:克隆得到灰毡毛忍冬LmC3H1基因,推测LmC3H1可能参与灰毡毛忍冬花绿原酸的生物合成。该研究为进一步研究该基因的功能及探究灰毡毛忍冬绿原酸生物合成和调节机制提供了依据,同时为遗传改良灰毡毛忍冬品质奠定了基础。     

HPLC同时测定归芍疏肝散中芍药苷阿魏酸和甘草酸铵的含量

目的:建立同时测定归芍疏肝散中芍药苷、阿魏酸和甘草酸铵含量的高效液相色谱法。方法:采用Amethyst C18色谱柱(200 mm×4.6 mm,5μm),乙腈-0.1%磷酸为流动相,梯度洗脱,流速为1.0 mL·min-1,检测波长为237 nm,柱温为30℃。结果:在线性范围内3种成分线性良好(r≥0.9998),精密度RSD均<2%;重复性RSD均<3%;平均回收率98.81%~100.25%,RSD均<2%。结论:该方法快捷,操作简便,结果可靠,可用于归芍疏肝散的质量控制。     

生物样品中天麻素测定方法及药代动力学研究进展＊

天麻素是从兰科植物天麻的块茎中提取分离而得，现已人工合成。天麻素药理作用广泛，如镇痛、镇静安眠、促智、保护神经元、降血压、抗惊厥、抗癫痫、抗氧化、抗衰老、改善微循环等，且安全、无明显毒副作用，其相关制剂在临床上亦被广泛用于心脑血管及神经精神系统疾病，如头痛、冠心病、高血压、后循环缺血性眩晕、突发性耳聋、癫痫、神经衰弱及脑卒中等。考虑到天麻素临床治疗上述疾病常与其他药物或治疗方法联用，故其体内过程及其药代动力学性质不容忽视。目前，有关天麻素综述多集中于药理作用和临床应用方面，而其药代动力学综述鲜见报道。随着天麻素药代动力学研究日渐深入，本研究将从其样品前处理、测定方法、药物ADME过程及影响体内过程的因素等方面较系统全面地对天麻素近年来的药代动力学研究作一综述，以期对天麻素制剂工艺及临床联合配伍应用有所裨益。