不同产区木瓜药材质量对比分析

目的 考察市售木瓜药材主流商品质量,为木瓜药材商品规格等级标准的建立奠定基础.方法 收集三大主流产区的木瓜样品38批,采用HPLC特征图谱表征其化学成分,运用SPSS软件整体分析特征图谱相似度数据,采用偏最小二乘-判别分析(PLS-DA)特征图谱统计分析,并用正交偏最小二乘-判别分析(OPLS-DA)对宣木瓜和资丘木瓜...     

A combined HPLC-PDA and HPLC-MS method for quantitative and qualitative analysis of 10 major constituents in the traditional Chinese medicine Zuo Gui Wan

A method using a high-performance liquid chromatograph coupled with a photodiode array UV detector (HPLC-DAD) and an electrospray ionization mass spectrometer (HPLC-ESI/MS) was developed for the quality assessment (fingerprinting and simultaneous quantification of 10 major constituents) of Zuo Gui Wan (ZGW), a Traditional Chinese Medicine (TCM). The compounds were identified on the basis of comparison of their mass spectra with literature data and standard samples and quantified by the HPLC-DAD method. Baseline separation was achieved on an ODS-100V C₁₈ column (3.0 μm, 150 mm × 4.6 mm I.D.) with linear gradient elution of acetonitrile–0.1% formic acid. The method was validated for linearity (r² > 0.9996), repeatability (R.S.D. < 3.5%), intra- and inter-day precision (R.S.D. < 2.0%) with accuracy (91.8–99.7%), recovery (99.9–105.6%), limits of detection (1.4–22.0 ng), and limits of quantification (3.4–73.4 ng). The 10 compounds were selected for quality assessment of ZGW by using partial least squares-discrimination analysis (PLS-DA). The similarities of 20 batches of ZGW and their classification according to their manufacturers were based on the retention times and peak areas of the characteristic compounds.     

UPLC-Q-TOF-MS结合代谢组学分析冬虫夏草不同部位的差异性

目的:利用高分辨质谱结合代谢组学方法研究冬虫夏草不同部位化学成分差异及其与品质间的关联。方法:建立虫草类中药材化学成分数据库,采用UPLC-Q-TOF-MS对虫草子座、头及虫体3个部位及全虫样品进行数据采集,将结果导入Peak View软件,结合软件的Formula Finder,Mass Calculators,Fragment Matching功能及二级碎片裂解规律进行定性分析;将定性结果建立已知成分筛查表,并逐个提取分段样品离子强度,代入SIMCA-P软件中,进行可视化处理,构建PCA及PLS-DA数学模型。结果:冬虫夏草醇提样品中共找到了六类23个化合物,从子座、头及虫体中鉴定出11个差异性化合物,以脂肪酸及其衍生物居多,其中8个化合物为首次在冬虫夏草中发现;运用SIMCA-P11.5软件构建PCA及PLS-DA数学模型,分析结果显示子座所含成分与头及虫体有明显差异,按照对分类起作用强弱,依次为氨基酸、脂肪酸、维生素、生物碱、核苷及糖苷类。结论:该方法的建立为冬虫夏草药效学物质基础阐明及冬虫夏草品质形成机制提供了科学参考,并为冬虫夏草药材的快速鉴定、质量控制与深入地开发利用奠定了基础。     

Quality evaluation of Angelica acutiloba Kitagawa roots by 1H NMR-based metabolic fingerprinting

The prices of Angelica acutiloba Kitagawa (yamato-toki) and A. acutiloba Kitagawa var. sukiyamae Hikino (hokkai-toki) are now mainly determined according to the sensory quality determined by experts in addition to the physical properties. This method provides a low reliability result for differentiating and qualifying their qualities. In addition, the quality in terms of pharmacological efficiency is not taken into account for consideration in the ordinary sensory method. A combination of a (1)H NMR technique and a multivariate analysis was preliminarily applied for the quality evaluation of both toki roots with regard to their geographical and variety differences. A broad range of metabolites was detected by a single-run (1)H NMR spectrometry. Partial least-squares discrimination analysis (PLS-DA), a pattern recognition method, was applied to the (1)H NMR spectra of aqueous extracts of toki samples having different sensory qualities. The PLS-DA result showed a clear clustering corresponding to the cultivation area between toki samples cultivated in Hokkaido (Japan) and those cultivated in the southern part of China and the Nara prefecture (Japan), while there was no separation corresponding to the toki's variety and sensory qualities, indicating the inconsistency of the sensory evaluation result. The chemical metabolites contributing to the discrimination of toki samples in relation to pharmacological and sensory properties were reported for the first time. A reliable multivariate calibration model used to predict the sensory quality was successfully carried out by PLS regression.     

附子理中丸大蜜丸、水蜜丸和浓缩丸3种剂型的化学特征关键质量属性辨识研究

目的 建立附子理中丸（Fuzi Lizhong Pills,FLP）处方的大蜜丸（Dami Pills,FLP-D）、水蜜丸（Shuimi Pills,FLP-S）及浓缩丸（Nongsuo Pills,FLP-N）的HPLC指纹图谱,同时测定7种药效成分含量,寻找FLP处方与剂型关联的化学特征关键质量属性。方法 采用分层抽样法结合研究实际,随机抽取市售FLP-D 30批,FLP-S 12批,FLP-N 12批,建立以上54批FLP的HPLC指纹图谱,并同时测定甘草苷、苯甲酰新乌头原碱、党参炔苷、6-姜酚、白术内酯III、白术内酯I、10-姜酚7种有效成分含量;利用主成分分析（principal component analysis,PCA）和偏最小二乘法-判别分析（partial least squares-discriminant analysis,PLS-DA）方法对FLP-D、FLP-S和FLP-N各自的HPLC指纹图谱特征峰及7种有效成分进行分析。结果 FLP-D、FLP-S和FLP-N的HPLC指纹图谱分别标定出20、25、20个特征峰,通过PCA和PLS-DA,筛选出14个差异性标志物,其中8、9、12、17、18、25号峰来自甘草,29、32号峰来自干姜,22号峰来自白术。含量测定结果表明,甘草苷、苯甲酰新乌头原碱、6-姜酚、白术内酯III、白术内酯I在FLP-D、FLP-S和FLP-N中的含量差异显著,其中6-姜酚为变量重要性投影（variable importance in projection,VIP）值＞1.0的差异性标志物。结论 建立的HPLC指纹图谱与多成分同时测定方法能有效评价FLP-D、FLP-S和FLP-N的化学成分差异;识别出的14个差异性标志物可作为区分FLP-D、FLP-S和FLP-N的潜在化学特征关键质量属性;甘草苷、苯甲酰新乌头原碱、白术内酯III、白术内酯I可作为区分FLP-D与FLP-N或区分FLP-S与FLP-N的化学特征关键质量属性,6-姜酚可作为同时区分FLP-D、FLP-S和FLP-N 3种不同剂型的化学特征关键质量属性。为进一步完善FLP系列制剂的质量标准提供了参考,同时为解决同方异剂中药的质控指标同质化问题提供了新思路。     

不同采收及加工方式对穿心莲药材质量的影响

通过考察穿心莲的生长期、产地加工、营养器官和全草分段等因素对药材质量的影响,为穿心莲优质药材的生产提供科学依据。采用"一测多评"法,同步测定78份样品中穿心莲内酯(andrographolide,AD)、新穿心莲内酯(neoandrographolide,NAD)、14-去氧穿心莲内酯(14-deoxyandrographolide,14-DAD)和脱水穿心莲内酯(dehydroandrographolide,DHAD)的含量,并分别采用直观分析、聚类分析和偏最小二乘法判别分析(PLS-DA)等统计学方法进行数据分析和挖掘。结果表明:穿心莲按照快速生长期(Ⅰ期)、枝叶茂盛期(Ⅱ期)、花蕾期(Ⅲ期)、花盛期(Ⅳ期)、盛果期(Ⅴ期)和果期后期(Ⅵ期)6个生长期来划分,4种二萜内酯总量(TC)在Ⅰ~Ⅲ期内较为稳定,Ⅳ期开始下降,Ⅴ期下降最为迅速,Ⅵ期降至最低,因此穿心莲药材的采收期以花蕾期为最佳。产地加工时,尽管晒干和阴干对TC的影响较小,但与晒干比较,阴干样品中AD含量降低,DHAD和NAD含量升高,即阴干过程可以导致AD的分解和转化。因此,在天气条件允许的情况下,产地加工尽量采用晒干法进行干燥。AD,DHAD和TC在穿心莲不同全草分段和营养器官的分布分别为:全草上部>全草中部≈全草>全草下部,叶>全草>茎,且4种二萜内酯在叶和全草中的含量远高于茎,建议药材采收时以全草的上部和中部或含叶以上部位为佳,以保证其叶的占比要求。     

Comparison of toxic reaction of Tripterygium wilfordii multiglycoside in normal and adjuvant arthritic rats

Aim of the study

Tripterygium wilfordii multiglycoside (GTW), an authorized Chinese patent drug, is used for treatment of rheumatoid arthritis and other immune disease. This study was to determine whether GTW induced different toxic reactions in adjuvant arthritis rats (AA rats) compared to those in normal rats.

Materials and methods

To prepare arthritic rat model, male Sprague-Dawley (SD) rats were immunized by injecting complete Freund's Adjuvant into right hind footpad. And then, GTW was given to rats intragastrically at dosage of 7 or 105 mg kg⁻¹ day⁻¹ from day 15 to day 28 after immunization. Routine clinical parameters and histopathologic changes of liver, kidney and testis were examined. Metabolic profiling in serum of groups was analyzed by LC-MS. A principal component analysis (PCA) and partial-least-squares discriminate analysis (PLS-DA) were carried out combined with mass spectrometry (MS) data set. All the quantitative data were performed by two-way ANOVA analysis following Student's t-test.

Results and conclusions

Treatment with GTW at both doses could diminish the right and left hind paws swelling. There was slight lipoid degeneration in hepatic tissue of normal rats treated by high dose of GTW, but there were not distinctly pathological changes in hepatic tissue of AA rats treated by GTW. Compared normal rats administered with GTW, no statistically significant difference in the serum alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN) levels were observed. However, the serum aspartate aminotransferase (AST) level was significant decreased in AA rats under exposure GTW compared with normal rats in the same conditions (p < 0.05), which indicated that GTW could offer a different liver toxic reaction in normal and AA rats. The metabolic analysis showed that a clear separation of PCA and PLS-DA score spot in normal rats, but not separation was seen in AA rats perturbed with low dosage GTW. The result indicated low dosage GTW might arouse a general toxic in normal rats but not in AA rats. The biomarker analysis showed that the level of lysophosphatidylcholines (LPCs) was down-regulated, but the level of ursodeoxycholic acid (UDCA) and chenodexycholic acid (CDCA) was up-regulated in AA rats compared with normal rats under exposure GTW. According to pathway analysis of metabolic markers, we conceived that LPC, UDCA and CDCA were the critical intermediates of choline and fatty acid metabolism. And the lipid metabolism was a correlative outcome of GTW induced toxicity in the liver in physiological condition animals. Taken together, GTW could induce different toxic reactions between normal and AA rats, and the lipid metabolism might be part of the mechanism for the hepatic lipidosis or the other liver injury.     

牡丹皮炭给药后血清代谢物的~1H-NMR代谢组学研究

目的运用1H-NMR代谢组学方法研究正常人服用牡丹皮炭前后的血清代谢组变化及探讨牡丹皮炭的临床作用机制。方法采用磁共振技术对人体的血清进行测试分析,应用偏最小二乘判别分析法(PLS-DA)研究比较正常人服用牡丹皮炭前后血清之间的代谢谱差异。结果服用牡丹皮炭后,血清中变化最明显的为葡萄糖含量下降,乳酸和糖蛋白含量升高。结论正常人服用牡丹皮炭前后的血清在1H-NMR中出现的特征峰有明显差异,为医学临床检验和临床应用前期研究提供了一个参考。     

Better understanding of dissolution behaviour of amorphous drugs by in situ solid-state analysis using Raman spectroscopy

Amorphous drugs have a higher kinetic solubility and dissolution rate than their crystalline counterparts. However, this advantage is lost if the amorphous form converts to the stable crystalline form during the dissolution as the dissolution rate will gradually change to that of the crystalline form. The purpose of this study was to use in situ Raman spectroscopy in combination with either partial least squares discriminant analysis (PLS-DA) or partial least squares (PLS) regression analysis to monitor as well as quantify the solid-phase transitions that take place during the dissolution of two amorphous drugs, indomethacin (IMC) and carbamazepine (CBZ). The dissolution rate was higher from amorphous IMC compared to the crystalline α- and γ-forms. However, the dissolution rate started to slow down during the experiment. In situ Raman analysis verified that at that time point the sample started to crystallize to the α-form. Amorphous CBZ instantly started to crystallize upon contact with the dissolution medium. The transition from the amorphous form to CBZ dihydrate appears to go through the anhydrate form I. Based on the PLS analysis the amount of form I formed in the sample during the dissolution affected the dissolution rate. Raman spectroscopy combined with PLS-DA was also more sensitive to the solid-state changes than X-ray powder diffraction (XRPD) and was able to detect changes in the solid-state that could not be detected with XRPD.     

Dunaliella salina extract effect on diabetic rats: Metabolic fingerprinting and target metabolite analysis

Antioxidant therapy has been proposed to improve the oxidative stress status of diabetic patients. Natural products are a source of substances such as carotenoids, with known antioxidant properties with possible benefits on diabetes. Among them, Dunaliella salina is a microalga with high content in carotenoids that can be extracted via an environmentally clean process such as supercritical fluid extraction with CO₂.