HPLC测定不同采收年限雷公藤根及不同部位雷公藤甲素含量

目的 用高效液相色谱法测定雷公藤中雷公藤甲素的含量,研究雷公藤植株不同部位及不同采收年限雷公藤根中雷公藤甲素含量的差异情况。方法 采用外标法,以甲醇-水(45∶55)为流动相;检测波长为218 nm;柱温为28 ℃;流速为1.0 mL·min^-1。结果 雷公藤甲素浓度在1.026~10.26 μg·mL^-1呈良好的线性关系(r=0.999 9),回收率为95.90%,RSD=3.49%(n=6)。结论 本法简便、准确、灵敏、重复性良好,适用于雷公藤甲素的含量测定。在不同采收年限中,3年生雷公藤全根中雷公藤甲素含量最高,1年生最低;在不同部位中,根皮中含量最高,其次是叶片,根木质部最低。     

HPLC测定不同产地雷公藤中雷公藤甲素含量

目的建立高效液相色谱法测定雷公藤中雷公藤甲素的含量,研究不同产地雷公藤药材中雷公藤甲素含量的差异情况。方法采用外标法,以乙腈-水(30∶70)为流动相;检测波长为220nm;流速为1.0mL·min-1。结果雷公藤甲素进样量在0.04392μg～3.5136μg范围内呈良好的线性关系(r=0.9998),回收率为98.70%,RSD=3.27%(n=9)。其中湖南产(批号:B)的雷公藤含量最高。结论本法简便、准确,适用于雷公藤中雷公藤甲素的含量测定。另外不同产地、来源的雷公藤药材中雷公藤甲素的含量差异较大,在临床用药时应引起重视。     

HPLC法测定粉背雷公藤中雷公藤甲素的含量

目的：建立高效液相色谱法测定广西产粉背雷公藤中不同部位雷公藤甲素的含量方法。方法：采用C18色谱柱（250mm×4．6mm,5μm）,流动相为甲醇-水（45：55）,检测波长217nm,流速1mL／min,柱温24℃,进样量为20μL。结果：雷公藤甲素在0．5～10μg／mL范围内呈良好的线性关系;平均加样回收率为99．61％,RSD为1．69％（n=5）;粉背雷公藤根部和茎枝中雷公藤甲素的平均含量分别为14．98和3．64μg/g。结论：本方法简便可行、定量准确、重现性好,可用于粉背雷公藤中雷公藤甲素的质量控制。     

HPLC法测定雷公藤粗制品中雷公藤甲素的含量

目的：建立高效液相色谱法测定雷公藤粗制品中雷公藤甲素的含量。方法：雷公藤粗制品经洗脱提取后，采用十八烷基键合硅胶柱，以乙腈-水（30：70）为流动相，以218nm为检测波长，检测雷公藤粗制品中雷公藤甲素的含量。结果：雷公藤甲素在1．92～11．52μg／ml范围内线性良好，r=0．9999，平均回收率为95．32％，RSD为1．16％。结论：本法操作简便、结果准确，可用于雷公藤粗制品中雷公藤甲素的含量测定。     

不同雷公藤制剂雷公藤甲素、雷公藤内酯甲及雷公藤总内酯含量的比较

目的比较雷公藤片、雷公藤多苷片、昆明山海棠片中雷公藤甲素、雷公藤内酯甲及雷公藤总内酯日用量的差异,为该类制剂的质量控制及临床应用评价提供参考。方法采用HPLC法测定雷公藤甲素、雷公藤内酯甲的含量,采用紫外分光光度法测定雷公藤总内酯的含量,比较不同厂家3种成分日用量的差异。雷公藤甲素以甲醇-水(45:55)为流动相,柱温为30℃,检测波长为218 nm;雷公藤内酯甲以乙腈-水(85:15)为流动相,柱温为30℃,检测波长为210 nm。结果雷公藤片中雷公藤甲素、雷公藤内酯甲及雷公藤总内酯3种成分的含量为最高,其次为雷公藤多苷片,昆明山海棠片最低,雷公藤甲素仅为雷公藤片的1/50、雷公藤内酯甲及总内酯为雷公藤片的1/8~1/10。临床上雷公藤甲素最高用量为53μg·d-1、最低用量仅为1.8μg·d-1,雷公藤内酯甲最高用量为1.2 mg·d-1、最低用量为0.1 mg·d-1,雷公藤总内酯最高用量为8.4mg·d-1、最低用量为1.3 mg·d-1。同一厂家不同批号的雷公藤片雷公藤甲素相差近5倍、雷公藤内酯甲相近、总内酯相差近1倍。结论不同雷公藤片、雷公藤多苷片、昆明山海棠片中雷公藤甲素、雷公藤内酯甲及雷公藤总内酯的日用量存在明显差异,以雷公藤片最高、其次为雷公藤多苷片、昆明山海棠片为最低。同一厂家不同批号的雷公藤片之间、雷公藤多苷片之间及不同厂家的雷公藤多苷片之间所测成分均有一定的差异,但不同厂家的昆明山海棠片相差不大。     

HPLC法测定复方雷公藤糖浆中雷公藤甲素的含量

目的：建立高效液相色谱法测定复方雷公藤糖浆中雷公藤甲素的方法。方法：采用DiamonsilC18（150mm×4．6mm,5μm）色谱柱分离,甲醇-水（46：54）为流动相,流速为1．0ml·min-1,检测波长为224nm,柱温为30℃,进样量为10μl。结果：雷公藤甲素在5．66～33．96μg·ml-1（r=0．9998）范围内质量浓度与峰面积呈良好的线性关系,其平均回收率为99．35％（RSD=1．02％,n=9）。结论：苓方法简便、灵敏、重复性好,可用于复方雷公藤糖浆中雷公藤甲素的质量控制。     

高效液相色谱法测定不同采收月份雷公藤中雷藤甲素

目的采用高效液相色谱法测定雷公藤中雷藤甲素含量,研究不同采收月雷公藤根中雷藤甲素含量差异。方法采用外标法,以水 乙腈 甲醇（70：10：20）为流动相;检测波长218 nm;流速1.0 mL•min－1。结果雷藤甲素在 2.015～20.150 μg•mL－1呈良好的线性关系,r＝0.999 9,回收率96.3%,RSD＝1.1%（n＝6）。结论该方法简便,准确,灵敏,重复性良好,适用于雷藤甲素的含量测定。5年生雷公藤全根中以当年11月到次年6月雷藤甲素含量最高。     

不同水浴回流时间对粉背雷公藤茎枝中雷公藤甲素含量测定的影响

［摘要］目的探讨不同水浴回流时间对粉背雷公藤茎枝中雷公藤甲素含量测定的影响。方法水浴回流时间分别为0.5,1.0,2.0,3.0,4.0 h,采用高效液相色谱（HPLC）法测定粉背雷公藤茎枝中有效活性成分雷公藤甲素的含量;色谱条件：色谱柱为C8柱（150 mm×4.6 mm,5 μm）;流动相为甲醇 水（45：55）;检测波长为217 nm;柱温29 ℃;流速1.0 mL• min 1;进样量20 μL。结果以水浴回流时间2.0 h、加水量与药物之比为6：1时雷公藤甲素含量最高。结论缩短水浴回流时间可提高粉背雷公藤茎枝水提液中雷公藤甲素含量。     

HPLC法测定昆明山海棠片中雷公藤甲素的含量

目的：应用高效液相色谱法测定昆明山海棠片中雷公藤甲素的含量。方法：采用Diamonsil^TM C18色谱柱（5μm，250mm×4．6mm），流动相为乙腈-水（25：75），流速1．0mL·min^-1，检测波长220nm。结果：雷公藤甲素进样量在0．143～0．858μg范围内线性关系良好（r=0．9999）；平均加样回收率（n=6）为97．56％，RSD=0．8％。结论：本法操作简便、准确，灵敏度高，重复性好，可用于昆明山海棠片中雷公藤甲素的含量测定。     

反相高效液相色谱法测定雷公藤口服液中雷公藤甲素含量

目的 建立反相高效液相色谱法测定雷公藤口服液中雷公藤甲素含量的方法.方法 以ODS-BP(4.6 mm×250 mm)为色谱柱,水:甲醇(57:43)为流动相,流速1 mL·min-1,检测波长218 nm,分析时间90 min,测定雷公藤口服液中雷公藤甲素含量;结果 雷公藤甲素进样量在0.0247～0.7904 μg范围内峰面积线性关系良好,r=0.9995,加样回收率为99.56%.结论 该方法简便、准确、稳定性好,线性范围宽,可作为雷公藤甲素含量测定方法.     

Quantitative determination of 1-deoxynojirimycin in mulberry leaves using liquid chromatography-tandem mass spectrometry

A novel HPLC-MS/MS method was developed for the quantitative determination of 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor present in mulberry leaves (Morus alba L.). DNJ was isolated from the mulberry leave extract on a TSKgel Amide-80 column using a mixture of 0.1% formic acid and acetonitrile as a mobile phase at a flow rate of 0.6 ml/min. A triple quadrupole mass spectrometry using electrospray ionization source in a positive ion mode under multiple reaction monitoring with the [M+H]+ ions, m/z 164.4/109.9 was used. The detection limit (S/N=3) was 75 pg and quantitation limit (S/N=10) was 100 pg. The comparison of mulberry leaves of different ages showed that the DNJ level was higher in mulberry shoots than young and mature leaves.     

Carboxymethylcellulose sodium improves the pharmacodynamics of 1-deoxynojirimycin by changing its absorption characteristics and pharmacokinetics in rats

1-Deoxynojirimycin (DNJ) has excellent inhibitory activity against alpha-glucosidase and can therefore decrease the postprandial blood glucose level in humans. However, a major limitation of DNJ is its fast absorption rate compared with other alpha-glucosidase inhibitors. In this study, we investigated the effect of adjuvants on the pharmacokinetics of DNJ, and found that carboxymethylcellulose sodium (CMCNa) can remarkably improve the activity of DNJ on glucose levels. When DNJ was used in combination with CMCNa, its absorption was suppressed and delayed by CMCNa. CMCNa can also change the pharmacokinetics of DNJ in rats. Pharmacodynamics were further studied using the oral glucose tolerance test, and the results confirmed that CMCNa can enhance DNJ's modulation of glucose level. All the results indicate that carboxymethylcellulose sodium can improve the pharmacodynamics of 1-deoxynojirimycin by changing the absorption characteristics and pharmacokinetics of DNJ in rats.     

1-Deoxynojirimycin isolated from Bacillus subtilis improves hepatic lipid metabolism and mitochondrial function in high-fat–fed mice

The aim of this study was to determine whether 1-deoxynojirimycin (DNJ) isolated from Bacillus subtilis MORI beneficially influences lipid metabolism and mitochondrial function in the liver of mice fed a high-fat diet in addition to the anti-obesity properties of DNJ. Male C57BL/6 mice (n = 29; 5 weeks old) were randomly assigned to three groups: normal control diet (CTL, n = 10), high-fat diet (HF, n = 10), and high-fat diet supplemented with DNJ (DNJ, n = 9). After 12 weeks, the HF group exhibited higher overall weight gain, of the liver, and of various fat pads than the CTL and DNJ groups did. The HF group also showed greater expression of C/EBPα and CD36 mRNA in the liver than that in the CTL and/or DNJ groups. In addition, mRNA expressions of AAC and FAS were lower, while mRNA expression of PGC-1β was higher in the liver of the DNJ group than that of the HF group. The hepatic expression of p-AMPK/AMPK was higher in the DNJ group than in the HF group. This study provides novel insight into the protective effect of DNJ supplementation against obesity-induced hepatic lipid abnormalities and mitochondrial dysfunction.     

脱氧野尻霉素衍生物抗乙型肝炎病毒的体外试验研究

目的:观察脱氧野尻霉素(DNJ)衍生物N—苄基—1—脱氧野尻霉素(P-DNJ)与N—壬基—1—脱氧野尻霉素(NN-DNJ)的体外抗乙型肝炎病毒(HBV)作用。方法:以HBVDNA转染的人肝癌细胞株HepG22,2,15细胞为靶细胞,以不同浓度的试验药物培养细胞,在第6天、第10天时应用时间分辨免疫荧光分析法和荧光实时定量聚合酶链反应法检测培养基上清液中HBsAg、HBeAg和HBVDNA,同时应用MTT法检测药物对细胞的毒性。结果:P-DNJ、NN-DNJ在试验浓度(5~125μg.mL-1)内未见细胞毒性作用,在浓度为125μg.mL-1时能显著减少细胞培养基上清液中HBsAg、HBeAg、HBVDNA的分泌。结论:P-DNJ、NN-DNJ在体外细胞培养试验中发现有抗HBV作用。     

The relationship between 1-deoxynojirimycin content and α-glucosidase inhibitory activity in leaves of 276 mulberry cultivars (<Emphasis Type="Italic">Morus</Emphasis> spp.) in Kyoto,Japan

The relationship between 1-deoxynojirimycin (DNJ) content and α-glucosidase inhibitory activity in mulberry (Morus) leaves is discussed. Mulberry leaves were collected from the Center for Bioresource Field Science, Kyoto Institute of Technology, Kyoto, Japan on 19 May, 9 July, and 9 August, 2003. Mulberry leaves were extracted with 75% ethanol. The inhibitory activity for rat intestinal crude enzyme was measured using maltose. The content of DNJ in the extracts was measured using HPLC. The mean DNJ content in the 0.04–0.06% range was high in collected samples. The inhibitory activities in July and August were higher (P < 0.01) than in May, and the activity in July was higher (P < 0.01) than in August. A strong correlation (r = 0.901, r ² = 0.811, n = 15) existed between DNJ content and α-glucosidase inhibition in leaves of Morus bombycis harvested in July. Similarly, correlation coefficients of the other mulberry varieties in July were higher than they were in May or August. The inhibitory activity and the DNJ content of Morus latifolia in August were lower than for any other mulberry variety. These results show that the high inhibitory cultivars harvested in July, except for M. latifolia, are more suited to products that contain high DNJ contents.     

氨苄西林含量测定方法的比较

对氨苄西林的三种含量测定方法即电位滴定法、硫醇汞盐－UV法和高效液相色谱法进行对比分析三种方法测得结果没有显著性差异（P＞0．05），硫醇汞盐－UV法和电位滴定法与高效液相色谱法测得结果具有可比性，高效液相法能与杂质分离，结果精密度较好，且更准确，可靠。     

两种方法测定比沙可啶纯度及不确定度评定

目的:采用两种不同原理的测定方法对比沙可啶纯度进行联合定值,并建立其不确定度评定方法。方法:采用高效液相色谱法(HPLC)与非水溶液电位滴定法测定比沙可啶纯度,并依据相关规范要求,对两种方法测定过程的不确定度进行了系统分析。结果:比沙可啶HPLC法、非水溶液电位滴定法纯度测定值分别为(99.81±0.29)%、(99.82±0.51)%,两种方法联合测定比沙可啶纯度的标准值及其不确定度分别为99.82%、(±)0.59%(k=2,P=0.95)。结论:采用HPLC与非水溶液电位滴定法联合测定比沙可啶纯度及不确定度评定结果准确可靠,避免了采用一种技术带来的分析方法缺陷,有利于提高比沙可啶的质量评价与控制水平,同时为比沙可啶纯度标准物质的研制提供了科学依据。     

不同提取方法对石斛多糖含量测定的影响

目的:研究不同提取方法对石斛多糖含量测定的影响。方法:分别采用回流提取、改进回流提取、超声波提取与超声辅助热回流提取法对石斛多糖进行提取,通过苯酚-硫酸法检测其多糖含量。结果:4种提取方法所测得的多糖含量分别为(13.473±0.634)%、(15.692±0.743)%、(11.87±0.536)%、(16.29±0.407)%。结论:不同提取方法对石斛多糖含量测定影响较大,采用超声辅助热回流提取法所得含量最高,而且提取时间短、提取较为彻底,适合于石斛多糖含量测定的样品前处理。     

Biological study of the Angiogenesis Inhibitor N-(8-(3-ethynylphenoxy)octyl-1-deoxynojirimycin

The α-glucosidase inhibitors N-methyl-1-deoxynojirimycin (MDNJ) and castanospermine have been shown to inhibit angiogenesis. A hybrid of 1-deoxynojirimycin (DNJ) and an aryl-1,2,3-triazole, which inhibits both an α-glucosidase and methionine aminopeptidase-2 (MetAP2), displayed properties associated with inhibition of angiogenesis (Bioorg. Med. Chem., 16, 2008 , 6333–7). The biological evaluation of a structural analogue N-(8-(3-ethynylphenoxy)octyl-1-deoxynojirimycin is described herein. Although this alkyne derivative did not inhibit MetAP2, it inhibited a bacterial α-glucosidase, altered bovine aortic endothelial cell (BAEC) surface oligosaccharide expression and inhibited BAEC proliferation by inducing G1 phase cell cycle arrest. Experiments showed G1 arrest was attributable to the α-glucosidase inhibitor inducing an increase in p27^Kip1 expression and high phosphorylation of ERK1/2 without a reduction in cyclin D1. The DNJ derivative (0.1 mm ) prevented capillary tube formation from bovine aortic endothelial cells, whereas DNJ or other analogues were unable to inhibit tube formation at the same concentration. Stress fiber assembly in bovine aortic endothelial cells was abolished, and BAEC migration was inhibited indicating the inhibition of tube formation by this derivative is partially a result of a reduction in cell motility. The agent also caused a reduction in secretion of MMP-2 from bovine aortic endothelial cells. Therefore, the new α-glucosidase inhibitor has a different mechanism by which it inhibits angiogenesis in vitro when compared with deoxynojirimycin, the deoxynojirimycin -triazole hybrid, N-methyl-1-deoxynojirimycin and castanospermine.     

Different methods for toxin analysis in the cyanobacterium Nodularia spumigena (Cyanophyceae).

The brackish water cyanobacterium Nodularia spumigena produce the hepatotoxic cyclic pentapeptide nodularin. Intoxications for both human as well as animal may arise when water reservoirs are contaminated with potentially toxic Nodularia species. Here, results of three independent methods for the determination of nodularin in different strains of N. spumigena are presented. The results obtained with a protein phosphatase assay and a HPLC/UV/MS method are compared with the results obtained with a bioluminescence assay, which is successfully introduced here for nodularin determination. Statistical evaluation of the three applied methods revealed a good comparability towards the detected toxin content. The methods were evaluated taking into consideration the parameters: handling, efficiency, sensitivity and selectivity. The detection limit in the protein phosphatase assay is highest (0.05ng nodularin) and lowest (250ng nodularin) in the bioluminescence assay- it was determined with 5ng (MS) and 25ng (UV) for the HPLC/UV/MS methods. The different selectivities and sensitivities are critically discussed and an analytical pathway for the determination of the biotoxin nodularin from Nodularia samples is proposed.