超声提取-分光光度法测定远志总皂苷的含量

目的:超声提取远志总皂苷,并用分光光度计进行总皂苷含量的测定.方法:超声提取远志总皂苷,以远志皂苷元为对照品,香草醛-冰醋酸-高氯酸为显色剂,采用分光光度法在585 nm处测定远志总皂甙含量.结果:对照品在28～63 μg范围内线性良好(r2=0.9984),平均回收率为100.45%,RSD=1.59%(n=5).超声提取和热回流提取的远志总皂苷含量分别为1.63%,1.50%.结论:超声提取法用时短,提取率高,能达到比传统回流法更理想的效果.分光光度法测定含量简便、快速、准确、可靠.     

黑灵芝中三萜及其皂苷类化合物总量的光度测定

采用正交试验法优化了香草醛高氯酸法测定黑灵芝三萜及其皂苷类化合物总量的显色条件,建立了测定黑灵芝中三萜及其皂苷类化合物总量的分光光度法,检测波长为550 nm,回归方程为:A=0.0093C-0.0032(μg),R2=0.9986(n=6),齐墩果酸对照品在0～128.4μg范围内呈良好线性关系,RSD为4.09%,对样品进行加标实验,平均回收率为103.9%.用于黑灵芝中三萜及其皂苷类成分的溶剂回流提取、超声提取的测定,结果证明这种方法是可靠的,可供灵芝质量控制.     

测定山楂及提取物总三萜酸的含量

目的:测定山楂及其提取物的总三萜酸的含量.方法:选用熊果酸为对照品,采用5%香草醛冰醋酸溶液、高氯酸显色后,进行山楂药材及提取物总三萜酸的含量测定.考察了显色剂香草醛、高氯酸用量,水浴温度等.结果:5%香草醛-冰醋酸溶液0.4 mL,高氯酸1.0 mL,60℃水浴15 min,冰浴冷却,加冰醋酸5.0 mL摇匀,在548 nm处测定,其效果最佳.标准曲线方程y=0.006 178x 0.005 600(r=0.999 8),药材的平均回收率为97.22%,RSD为1.32%;总三萜酸提取物的平均回收率为100.6%,RSD为0.82%.结论:该比色法操作简便,吸收值稳定,并且干扰小.     

龙牙楤木总皂苷含量测定及其抗炎镇痛活性评价

建立了一种龙牙楤木中总皂苷的含量测定方法,比较了龙牙楤木根皮和茎皮中总皂苷含量,并研究了龙牙愡木的抗炎镇痛作用。采用超声辅助提取,用紫外-可见分光光度法,以齐墩果酸为对照品建立标准曲线;以5%香草醛-冰醋酸为显色剂,检测波长550nm,用比色法测定总皂苷含量。结果显示所建立的方法稳定可靠,龙牙楤木不同部位皂苷含量差异较大,根皮总皂苷含量为43.50mg/g,而茎皮为24.38mg/g。总皂苷在0.02~0.10mg/mL(R~2=0.999 6)质量浓度范围内呈良好线性关系,根皮和茎皮的平均回收率分别为100.12%(RSD=1.10%)和99.84%(RSD=1.77%)。采用醋酸致小鼠扭体试验和耳廓肿胀试验研究龙牙楤木总皂苷抗炎镇痛活性,结果表明龙牙楤木总皂苷能显著减轻醋酸对试验小鼠内脏所致的疼痛(P<0.05)和降低由二甲苯所致实验小鼠耳廓的肿胀程度(P<0.05),显示出较强的抗炎镇痛作用,且根皮的作用效果比茎皮好。龙牙愡木抗炎镇痛活性可能与其总皂苷含量呈正相关。建立的总皂苷含量测定方法简便、准确、重现性好,可作为龙牙楤木根皮与茎皮中总皂苷的含量测定方法。     

分光光度法测定降血脂片中总皂苷含量的方法研究

建立一种简便、可靠的降血脂片中总皂苷含量的测定方法。以香草醛-高氯酸为显色体系,以人参皂苷为对照品,采用分光光度法测定降血脂片中总皂苷含量。比较分析了索氏提取法和超声波提取法对总皂苷提取分离效果的影响,并对大孔树脂填充高度进行了优化,最后对所建立的方法进行了重复性和加标回收率实验验证。结果表明,所测标准曲线的相关系数为09994,确立超声波提取法和3 cm大孔树脂填充高度为最佳实验条件,样品中总皂苷重复性相对标准偏差为18%,加标回收率为964%～990%,总皂苷测定重复性和回收率均良好。     

五叶木通营养器官解剖结构与三萜皂苷的积累关系

采用显微制片技术、组织化学定位和三萜皂苷定量分析方法,对五叶木通的营养器官分别进行了三萜皂苷的组织化学定位、含量和解剖结构特征研究。结果表明：三萜皂苷在五叶木通不同营养器官中的含量不同,含量测定与组织化学定位推测的结果吻合,叶＞根＞茎;三萜皂苷在同一营养器官的不同发育时期含量不同,总的规律是：老叶＞幼茎＞幼根＞幼叶﹥老根﹥老茎。三萜皂苷在不同器官中具有明确的积累位置：根中主要是次生韧皮部和周皮;皮层和次生韧皮部是茎中三萜皂苷积累部位;叶内则以栅栏组织为主。五叶木通根中初生木质部二原型,次生木质部内导管多为单管孔,少数为管孔链,纤维含量丰富。茎中韧皮纤维丰富,在幼茎中纤维成束状罩在初生韧皮部的外方,在次生韧皮部中纤维呈带状环绕在外方。叶为异面叶。     

酸性染料比色法测定花椒总生物碱的含量

建立了酸性染料比色法测定花椒中总生物碱的含量的方法.以白屈菜红碱作为对照品,加入pH值=7.6缓冲溶液,溴甲酚绿指示液、氯仿混合振摇,静置分层,于 425 nm 波长处测定氯仿层吸收度.得到标准曲线:y= 4.2566 x-0.006 6,R2 = 0.999 4,生物碱检测浓度在0 ～ 0.2 mg/mL 范围内与吸收度线性关系良好,此方法具有较好的稳定性和精密度.用此方法测定花椒中总生物碱含量为0.95%.本方法操作简便、准确,可用于花椒的质量控制.     

以沙苑子苷为对照品测定沙苑子总黄酮含量

目的 :以沙苑子苷为对照品测定沙苑子总黄酮含量。方法 :80 %乙醇提取液 ,2 6 6nm测定紫外吸收度。结果 :总黄酮含量为 2 1.5mg/g ,RSD =1.0 0 % ;回收率 95 .4 % ,RSD =2 .2 7% ,线性范围 4～ 2 4ug/ml。结论 :本法测定沙苑子总黄酮含量 ,操作简便 ,稳定性好 ,可用于沙苑子的质量控制。     

用微孔板分光光度法测定薯蓣皂苷元的含量

为了对植物样品中薯蓣皂苷元的含量进行高通量快速测定,本研究采用高压酸解制备薯蓣皂苷元,以高氯酸为显色剂,用微孔板分光光度法测定样品中薯蓣皂苷元的含量。合适的分析条件为:反应温度为30℃、高氯酸用量为200μL、振荡时间2 min后静置10 min,在410 nm处测定光吸收值。该方法的线性范围为每孔薯蓣皂苷元2～10μg(R=0.9988),平均回收率为99.9%,精密度的RSD为1.65%。该方法操作简单、准确稳定,可实现大批量样品中薯蓣皂苷元的快速检测。     

溶石胶囊中威灵仙总皂苷的含量测定研究

本文报道了溶石胶囊中威灵仙总皂苷的含量测定方法。用铁线莲皂苷Ⅰ为对照品,采用香草醛一硫酸为显色剂,475nm波长处检测。浓度在22．40～201．60μg／mL范围内呈现良好的线性关系,其回归方程为Y=213．2X-2．311,相关系数R=0．9993,加样回收率为100．31％RSD=1．02％。本方法简便,快速,灵敏,适用于该制剂的质量控制。     

HPLC法测定灵芝孢子粉中三萜含量

为准确测定灵芝孢子粉中三萜的含量,运用高效液相建立适合孢子粉的分析测定方法。通过对前处理条件的优化,确定40%乙醇为孢子粉中等极性三萜酸类的最佳提取溶剂,浓缩倍数是子实体提取条件的50倍。通过色谱柱和洗脱条件的优化,建立了包括灵芝酸I、灵芝烯酸C、灵芝酸C2等13种标准品测定方法,方法学考察显示该分析方法精密度、重复性、稳定性的RSD值均小于5%,可以用于灵芝孢子粉中三萜类成分的定量检测。通过5组样品的分析发现,灵芝酸C6、灵芝酸G、灵芝酸A、灵芝酸D、灵芝酸F是灵芝孢子粉中的主要三萜类成分,其中灵芝酸A含量最高,平均占样品三萜总量的比例达19.71%;三萜类成分的溶出量与是否破壁没有相关性。三萜类成分在灵芝孢子粉和灵芝孢子油产品中的含量非常低,孢子粉的三萜含量为14.24-99.70μg/g,仅为子实体的1/100,灵芝孢子油中三萜含量也均低于50μg/g,因此三萜类成分不适合作为灵芝孢子粉及其相关产品的定量检测指标。     

Conformation of abortifacient proteins: trichosanthin, alpha-momorcharin and beta-momorcharin

The conformations of three abortifacient proteins, trichosanthin from the Chinese herb Tianhuafen and alpha- and beta-momorcharin from the Chinese herb Kuguazi, were studied by circular dichroism. Their spectra in the ultraviolet region (188-250 nm) were similar to each other and also pH-independent (between pH 5 and 9). All three proteins contained about 30% helix, as compared with 39% for trichosanthin by X-ray diffraction study, and 40-60% beta-sheets, but had no beta-turns, suggesting a structural homology among the proteins. The addition of 20 mM sodium dodecyl sulfate nearly doubled the helicity of beta-momorcharin at the expense of beta-sheets but had a small effect on the conformation of alpha-momorcharin, whereas the corresponding change in conformation for trichosanthin was in between the two momorcharins. This implies a marked difference in stability of the three proteins against the surfactant.     

Effect of naturally occurring triterpenoids glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin on the immune system.

The effect of naturally occurring triterpenoid compounds such as glycyrrhizic acid, ursolic acid, oleanolic acid, and nomilin on the immune system was studied using Balb/c mice. Intraperitoneal treatments with 5 doses of these terpenoid compounds were found to enhance the total white blood cells (WBC) count. In ursolic acid, oleanolic acid and nomilin treated animals the maximum total WBC count was observed on the 6th day, while in glycyrrhizic acid treated animals it was observed only on the 9th day after the drug treatment. In ursolic acid, oleanolic acid and nomilin treated animals the percentage of increase in the total WBC count was to 91.48 +/- 4.6%, 135.75 +/- 6.4% and 117.33 +/- 17.9% respectively. In the glycyrrhizic acid treated animals the total WBC count was increased to 114.9 +/- 18%. Bone marrow cellularity and alpha-esterase positive cells were also enhanced by the treatment with these terpenoids. Treatment with various triterpenoids along with antigen produced an enhancement in the specific antibody titre and the number of plaque forming cells (PFC) in the spleen. Triterpenoids remarkably inhibited delayed type hypersensitivity reaction (DTH). These results indicate the immunomodulatory activity of naturally occurring triterpenoids such as glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin.     

The mechanism of action of trichosanthin on eukaryotic ribosomes--RNA N-glycosidase activity of the cytotoxin.

Trichosanthin is a ribosome-inactivating protein from root tubers of Trichosanthes kirilowii Maxim. In this paper, the mechanism of action of trichosanthin on eukaryotic ribosomes was studied. A fragment of about 450 nucleotides was released from 28S ribosomal RNA after treatment of rat liver ribosome with trichosanthin and its isolated ribosomal RNAs were treated with aniline. Analysis of nucleotide sequence of 5' terminus of this fragment revealed that the aniline-sensitive site of the phosphodiester bond was between positions A4324 and G4325 in the 28S rRNA. Adenine was recovered by ion-exchange column chromatography from the 50% ethanol soluble fraction of the reaction mixture in which rat liver ribosomes were treated with trichosanthin. Thin-layer chromatographic analysis indicated that 1 mol of adenine was released from 1 mol of ribosomes. When the ribosomes were incubated with trichosanthin in the presence of inorganic [32P]phosphate, little incorporation of radioactivity into 28S rRNA was observed, indicating that the release of adenine was not mediated by phosphorolysis. These results demonstrate that trichosanthin inactivates the ribosomes by cleaving the N-C glycosidic bond of adenylic acid at 4324 of 28S rRNA in a hydrolytic fashion.     

HPLC法测定3种老鹳草药材中没食子酸和柯里拉京的含量

目的：建立老鹳草、野老鹳草、牻牛儿苗中没食子酸、柯里拉京含量测定方法.方法：采用HPLC法,色谱条件：色谱柱为Diamonsil C18 （2）色谱柱（150 mm×4.6 mm,5μm）;流动相为乙腈-0.1％磷酸梯度洗脱;流速为1.0 mL/min;检测波长为274 nm;柱温为25℃.结果：没食子酸和柯里拉京的线性范围分别是：0.2024μg～2.024 0 μg（r =0.999 6）、0.126 0 ～2.520 0μg（r =0.999 2）,平均回收率分别是97.4％（RSD=1.4％）、96.7％（RSD=0.9％）.结论：本法简便、准确、重复性好,可用于老鹳草药材的质量评价.     

Purification and characterization of trichosanthin. Homology to the ricin A chain and implications as to mechanism of abortifacient activity

Trichosanthin, a protein from the Chinese medicinal herb Trichosanthes kirilowii, was purified in two essentially quantitative steps involving CM-Sephadex chromatography and reverse-phase high performance liquid chromatography. The protein was found to have a molecular mass of 25-26 kDa, to contain no cysteine, and to contain no glycosidic linkages. Pure trichosanthin was found to have potent abortifacient activity in pregnant mice. In order to understand the molecular basis of this unique biological activity, we have examined the amino acid sequence of the protein. As purified, trichosanthin was found to contain two amino-terminal sequences which differed only in the absence or presence of a tyrosine at residue 1. Sequence analysis of trichosanthin has allowed for determination of the NH2-terminal 38-amino acid residues. Comparison of this sequence to those present in a data base revealed homology with the ricin A-chain. Consistent with this structural homology, we have found that trichosanthin is a potent inhibitor of protein synthesis in a reticulocyte lysate system.     

黄芪和酸应激对中华鳖幼鳖血清补体C3和C4含量的影响

首次探讨了黄芪和酸应激对中华鳖（Pelodiscus sinensis)幼鳖血清补体C3和C4含量的影响。实验设对照组和实验组,实验组在饵料中按5％添加黄芪原料粉。持续饲喂中华鳖幼鳖4周后取一半样,其余作酸处理24h后再取样,测定补体C3和C4的含量。结果表明,黄芪对补体C3和C4的合成有明显的促进作用;酸应激可导致血清补体C3和C4的含量下降,而黄芪能抵抗酸应激所致的下降。提示黄芪具有抗酸应激的作用。     

HPLC法同时测定鸡矢藤中熊果酸和齐墩果酸的含量

目的：建立高效液相色谱法同时测定鸡矢藤中熊果酸和齐墩果酸的含量。方法：色谱柱为DiamonsilTM C18（250×4.6 mm,5滋m）,流动相为甲醇-0.05mol.L-1磷酸溶液（93∶7）为流动相,检测波长210 nm,进样量10滋L。结果：熊果酸在21.28滋g.mL-1～212.8滋g.mL-1范围内线性关系良好（r=0.9997）,平均回收率为97.79%（RSD=1.30%）;齐墩果酸在24.02滋g.mL-1～240.2滋g.mL-1范围内线性关系良好（r=0.9996）,平均回收率为98.36%（RSD=1.11%）。结论：该方法简便、灵敏、准确,可用于鸡矢藤药材的质量评价。     

桦褐孔菌三萜化合物提取工艺的优选

为了提高桦褐孔菌三萜化合物的产量,对其三萜化合物的提取工艺进行了初步优选。试验以桦褐孔菌发酵菌丝体为材料,齐墩果酸为标准品,用香草醛比色法测桦褐孔菌三萜化合物含量。通过单因素实验考察了不同提取剂、提取剂用量、提取温度、提取时间对桦褐孔菌三萜化合物提取的影响。结果表明,提取的最佳工艺为：以异丙醇为提取溶剂,用量为7倍,最佳提取温度为80℃,提取时间2h,在此条件下三萜化合物占菌丝体干质量的9．03％。该工艺的三萜桦褐物的提取率高,可用于桦褐孔菌中三萜化合物的提取。     

Properties of bromodextran-trichosanthin: a comparison with trichosanthin, an anti-AIDS protein.

Trichosanthin was coupled with bromodextran and the reaction mixture chromatographed on Sephadex G-75 fine to yield two peaks. The first peak was judged to be bromodextran-trichosanthin by SDS-polyacrylamide gel electrophoresis. The yield was optimal when 4% trichosanthin and 8% bromodextran T20 were reacted for 210 hours. Bromodextran-trichosanthin exhibited an ultraviolet absorption spectrum similar to that of free trichosanthin and the complex reacted positively with the trichosanthin antiserum. It had lower abortifacient and protein synthesis inhibiting activities but it also possessed lower allergenicity, suggesting that it may be useful as a substitute of trichosanthin, especially when the problem of hypersensitivity caused by prolonged administration of trichosanthin is serious.