34种吴茱萸中吴茱萸碱和吴茱萸次碱的含量测定

建立了吴茱萸中吴茱萸碱和吴茱萸次碱的反相高效液相色谱法 ,并用该法测定了 34种不同产地及不同炮制方法吴茱萸中吴茱萸碱和吴茱萸次碱的含量 .采用峰面积内标法定量 .吴茱萸碱和吴茱萸次碱在 0 5～ 2 0g/mL范围内均呈线性 ,相关系数分别为 0 9999、0 9998,平均回收率分别为 96 6 %、96 0 %     

Contents of constituents in mature and immature fruits of evodia species

A total of 33 commercial samples of Evodiae fructus originated from the rutaceous plants Evodia rutaecarpa (Juss.) Benth. and E. rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang (= E. officinalis Dode), respectively, were collected from Taiwan's herbal market. The contents of eighteen constituents (including 15 alkaloids, 2 flavonoids and 1 diterpenoid) in these samples were determined by high-performance liquid chromatography within 60 minutes. Analysis results showed that the constituent contents were almost unrelated to the species, but rather closely related to the degrees of maturity of the fruit. Those fruits with open mouths and unsplit ovaries had the highest total contents, fruits with open mouths and split ovaries had lower contents, and fruits with closed mouths had the lowest contents. In terms of the individual constituents, the open-mouth fruits contained higher contents of dehydroevodiamine, evodiamine, evocarpine and rutaecarpine, whereas the closed-mouth fruits had higher contents of dehydroevodiamine, epimedoside C, evocarpine and evodiamine.     

Anti-inflammatory principles from the fruits ofEvodia rutaecarpa and their cellular action mechanisms

The fruits of Evodia rutaecarpa Benth (Rutaceae) has long been used for inflammatory disorders and some anti-inflammatory actions of its constituents such as dehydroevodiamine, evodiamine and rutaecarpine were previously reported. Since the pharmacological data is not sufficient to clearly establish the scientific rationale of anti-inflammatory medicinal use of this plant material and the search for its active principles is limited so far, three major constituents (evodiamine, rutaecarpine, goshuyuamide II) were evaluated for their anti-inflammatory cellular action mechanisms in the present study. From the results, evodiamine and rutaecarpine were found to strongly inhibit prostaglandin E2 synthesis from lipopolysaccharide-treated RAW 264.7 cells at 1-10 microM. Evodiamine inhibited cyclooxygenase-2 induction and NF-kappaB activation, while rutaecarpine did not. On the other hand, goshuyuamide II inhibited 5-lipoxygenase from RBL-1 cells (IC50 = 6.6 microM), resulting in the reduced synthesis of leukotrienes. However, these three compounds were not inhibitory against inducible nitric oxide synthase-mediated nitric oxide production from RAW cells up to 50 micorM. These pharmacological properties may provide the additional scientific rationale for anti-inflammatory use of the fruits of E. rutaecarpa.     

In vitro and in vivo antiallergic effect of the fructus of Evodia rutaecarpa and its constituents

The unripe fruit of Evodia rutaecarpa (JUSS) BENTH (ER, Family Rutaceae) has been used frequently as a traditional medicine against inflammatory diseases in Korea, China and Japan. To evaluate antiallergic effect of ER, we isolated its main constituents, evodiamine and rutaecarpine, and evaluated in vivo their inhibitory effects against passive cutaneous anaphylaxis (PCA) reaction induced by IgE-antigen complex and scratching behaviors by compound 48/80. ER and its constituents, evodiamine and rutaecarpine, potently inhibited PCA reaction and scratching behaviors in mice, although ER weakly inhibited scratching behaviors. Evodiamine and rutaecarpine inhibited TNF-alpha and IL-4 protein expression in RBL-2H3 cells induced by IgE-antigen complex, although these did not inhibit degranulation of RBL-2H3 cells induced by IgE-antigen complex and rat peritoneal mast cells induced by compound 48/80. These findings suggest that ER and its constituents, evodiamine and rutaecarpine, may be effective for IgE-induced allergic diseases such as atopic dermatitis and rhinitis.     

吴茱萸不同炮制品中吴茱萸碱、吴茱萸次碱、柠檬苦素含量测定

目的:考察不同炮制方法及炮制前后吴茱萸中吴茱萸碱、吴茱萸次碱和柠檬苦素的含量变化情况。方法:采用高效液相色谱法,Hypersil ODS C₁₈（250 mm×4.6 mm,5μm）;流动相:乙腈-水-四氢呋喃-乙酸（41:59:1:0.2）;流速:1.0 ml·min^-1;检测波长:225 nm;柱温:25℃。结果:吴茱萸碱、吴茱萸次碱、柠檬苦素进样量分别在0.156～3.120μg（r=0.999 6,n=6）、0.116～2.320μg（r=1.000 0,n=6）、0.620～12.400μg（r=0.999 1,n=6）范围内与峰面积呈良好线性关系,平均回收率分别为97.2%（RSD=1.33%）、97.1%（RSD=1.56%）、97.3%（RSD=1.52%）。结论:不同炮制方法吴茱萸碱、吴茱萸次碱、柠檬苦素含量不同,砂烫法优于其他炮制方法。     

薄层荧光扫描法测定吴茱萸药材及其制剂中5种生物碱的含量

目的：用薄层荧光扫描法测定吴茱萸及加味左金丸中5种生物碱:羟基吴茱萸碱(hydroxyevodiamine, I)、N-甲酰二氢吴茱萸次碱(N-formyldihydrorutaecarpine, II)、吴茱萸酰胺甲(goshuyuamide-I, III)、吴茱萸碱（evodiamine, IV)及吴茱萸次碱(rutaecarpine, V)。方法：在高效硅胶薄层板上点样后,二次展开。展开剂:（1）氯仿—石油醚—甲醇—环己烷（5∶3∶0.4∶0.4）;(2)氯仿—甲醇(4.1∶0.3)。展开后用浓盐酸衍生化,荧光扫描测定。结果：各生物碱的线性范围在20.5～245 ng,回收率为96.8%～104.5%,并用此法测定了不同产地吴茱萸及不同批号的加味左金丸中生物碱的含量。结论：方法灵敏度高、样品用量少,可为寻找和利用植物资源,控制生药及方剂质量提供科学依据。     

吴茱萸化学成分研究

目的研究吴茱萸Evodia rutaecarpa ［Juss.］ Benth.的化学成分。方法采用溶剂法、硅胶柱色谱和重结晶等方法进行分离纯化，根据化合物的理化性质和光谱数据鉴定结构。结果从吴茱萸中分离得到14个化合物。鉴定出其中的10个化合物，分别为吴茱萸宁碱(evodianinine, 1)、吴茱萸次碱(rutaecarpine, 2)、 吴茱萸碱(evodiamine, 3)、吴茱萸酰胺I(wuchuyuamide I, 4)、羟基吴茱萸碱(hydroxyevodiamine, 5)、柠檬苦味素(limonin, 6)、胡萝卜苷(daucosterol, 7)、三十碳酸(triacontanoic acid, 8)、二十九烷(nonacosane, 9)和β-谷甾醇(β-sitosterol, 10)。结论通过UV，IR，ESI-MS，HREIMS，¹HNMR，¹³CNMR，HMQC，HMBC和NOESY分析，鉴定化合物1是新化合物，命名为吴茱萸宁碱。     

一株产吴茱萸碱结构类似物的内生真菌的筛选和初步鉴定

目的筛选产吴茱萸碱结构类似物的内生真菌。方法采用平板分离法对湖南新晃、湘乡、娄底,贵州铜仁,江西万载和浙江温州等地的吴茱萸内生真菌进行分离纯化。采用TLC、LC-MS等分析技术对产吴茱萸碱、吴茱萸次碱及其结构类似物等成分的内生菌进行分析。结果从6个吴茱萸产区共分离到31种内生真菌。分别从吴茱萸的叶、茎和果实中分离到不同的内生真菌,表现为组织特异性,且在不同产地的内生真菌的种类和优势种群存在明显的差异,即具有地域特异性;通过筛选找到了5株产吴茱萸碱和吴茱萸次碱类似成分的内生真菌,该成分可能为去氢吴茱萸碱。并对其中的1株内生真菌进行了形态学鉴定和ITS序列分析,初步判断该菌属子囊菌纲。结论从吴茱萸内生真菌中分离得到1株产去氢吴茱萸碱的子囊菌,为去氢吴茱萸碱的资源开发开辟了新途径。     

The positive inotropic and chronotropic effects of evodiamine and rutaecarpine, indoloquinazoline alkaloids isolated from the fruits of Evodia rutaecarpa, on the guinea-pig isolated right atria: possible involvement of vanilloid receptors

Cardiotonic effects of evodiamine and rutaecarpine, constituents of the fruits of Evodia rutaecarpa Bentham Rutaceae, were evaluated on guinea pig isolated atria. Comparison with capsaicin, a vanilloid receptor agonist, revealed similar positive inotropic and chronotropic activity, as judged from antagonistic effects of the competitive vanilloid receptor (capsaicin receptor) antagonist capsazepine, the non-competitive vanilloid receptor antagonist ruthenium red, the calcitonin gene related peptide antagonist CGRP(8-37), the P2X purinoceptor antagonist PPADS, and various desensitization studies. Evodiamine and rutaecarpine produced transient positive inotropic and chronotropic effects on the guinea-pig isolated atria, followed by a desensitizing effect to additional administration. Dose-response relationships for evodiamine, rutaecarpine and capsaicin were obtained. All the compounds evoked positive inotropic and chronotropic effects in a concentration-dependent manner. Maximal contractions for evodiamine, rutaecarpine and capsaicin were observed at concentrations of 1 microM, 3 microM and 0.3 microM, respectively. The cardiotonic responses evoked by both evodiamine and rutaecarpine were shifted to the right by capsazepine, an established antagonist of vanilloid receptor (capsaicin-receptor). The effects of both evodiamine (1 microM) and rutaecarpine (3 microM) were abolished by pretreatment with a desensitizing dosage of capsaicin (1 microM), developing cross-tachyphylaxis between these compounds. The effects of evodiamine (1 microM), rutaecarpine (3 microM) and capsaicin (0.3 microM) were also significantly reduced by pretreatment with ruthenium red (10 microM) and CGRP (8-37) (10 microM). The effects of evodiamine, rutaecarpine and capsaicin were not affected by pretreatment with PPADS (100 microM), a highly selective P2X purinoceptor antagonist, and the possibility of the involvement of the P2X purinoceptor was excluded. These results suggest that the positive inotropic and chronotropic effects on the guinea-pig isolated right atria induced by both evodiamine and rutaecarpine could be attributed to their interaction with vanilloid receptors and the resultant release of CGRP, a cardiotonic neurotransmitter, from capsaicin-sensitive nerves as with capsaicin.     

Synthesis, physicochemical and pharmacological properties of pentacyclic alkaloid-analogues

Quinazolinocarboline rutaecarpine and evodiamine (Evodia rutaecarpa) are main alkaloid components of traditional Chinese folk-remedies. Evodiamine exhibited selective antitumor and antimetastatic effects on several cancer cell lines and became lead structure of anticancer agents. During our synthetic research we achieved to gain alkaloid hybrid derivatives by combining the structural elements of quinazolinocarbolines with analogous alkaloids or drug molecules having similar effects by bioisosteric replacements. 8-norrutaecarpine, a hybrid molecule of rutaecarpine and luotonin A containing the indolo-pyrroloquinazolinone ring system has been synthesized. The hybrids of rutaecarpine and piroxicam bearing the indolo-pyridobenzothiazine and the 12-azaindolo-pyridobenzothiazine structures were prepared on two alternative routes. Two new heterocondensed pentacyclic compounds, 5-sulfarutaecarpine and 5-sulfa-8-norrutaecarpine were reached via bioisosteric replacement on the structure of rutaecarpine and 8-norrutaecarpine. Two new tricyclic ring systems, pyrido-benzothiadiazine and pyrrolo-benzothiadiazine were produced as intermediaries of these pentacyclic molecules. Series of substituted derivatives were prepared for pharmacological studies by modification of the structures with various substituents and solubilizing groups. During our work alternative way for synthesis of nauclefine (Nauclea latifolia) was laboured, and we published the synthesis of indolylquinazolinone derivative bouchardatine (Bouchardata neurococca) for the first time. Some of the physicochemical attributes of the synthesized intermediaries were defined, such as the pKa constants of 2,3-poly-methylene-benzothiadiazines. Proton/deuteron exchange kinetic constants of active methylene-groups of five tricyclic compounds were measured by 1H NMR technique. Solvent-dependent ratio of the Z/E isomers of phenyhydrazone-derivatives in polar and apolar solvents were determined. In the case of 18 produced compounds our work was completed by in vitro pharmacological studies performed within co-operation with the Institute of Pharmacology. The viability of HeLa cells was inhibited by five of our compounds to similar extent as the effect of evodiamine. Eight of our compounds induced apoptosis on HeLa cells to similar extent as evodiamine.     

HPLC法同时测定吴茱萸中吴茱萸碱、吴茱萸次碱与吴茱萸内酯的含量

目的:建立同时测定吴茱萸中吴茱萸碱、吴茱萸次碱与吴茱萸内酯含量的方法。方法:采用高效液相色谱法。色谱柱为迪马C1(8200mm×4.6mm,5μm),流动相为乙腈-0.04%庚烷磺酸钠溶液(48:52,V/V),流速为1.0mL·min-1,检测波长为225nm,柱温为35℃。结果:吴茱萸碱、吴茱萸次碱、吴茱萸内酯的进样浓度分别在5.38～53.80、5.02～50.20、10.30～103.00μg·mL-(1r均为0.9999)范围内与各自峰面积积分值呈良好线性关系;三者平均加样回收率分别为99.75%、97.66%、85.68%,RSD分别为0.67%、1.16%、1.54%(n=6)。结论:本方法简便、可行、重复性好,可用于吴茱萸中吴茱萸碱和吴茱萸次碱的含量测定;但吴茱萸内酯的回收率较低,需进一步改进其测定方法。     

Antinociceptive Activity of Aqueous and Alcohol Extract of Evodia Rutaecarpa

Water, methanol and ethanol extracts of Evodia rutaecarpa were tested for antinociceptive activity, which were correlated with the contents of evodiamine, rutaecarpine and evodine. Determination of contents was achieved by chromatographic techniques. Extracts were evaluated for antinociceptive activities using hot-plate test; acetic acid-induced writhing test and formalin test. All three extracts of Evodia rutaecarpa showed antinociceptive activities but the ethanol extract exhibited better effect. The better antinociceptive activity appeared to be related to higher contents of evodiamine, rutaecarpine and evodine in ethanol extract of Evodia rutaecarpa.     

DAD-HPLC法同时测定戊己丸中4种成分的含量

目的:建立戊己丸中芍药苷、盐酸小檗碱、吴茱萸碱与吴茱萸次碱4种成分的含量测定方法。方法:采用高效液相色谱法,应用XSelect CSH-C₁₈(4.6 mm×150 mm,5μm)色谱柱,以乙腈-0.1%磷酸溶液(含0.05 mol·L^-1磷酸二氢钾)为流动相,梯度洗脱,流速为1.0 mL·min^-1,柱温30℃,检测波长:芍药苷232 nm,盐酸小檗碱230 nm,吴茱萸碱226 nm,吴茱萸次碱342 nm。结果:芍药苷、盐酸小檗碱、吴茱萸碱、吴茱萸次碱分别在3.856×10²~3.856×10³ ng、4.239×10²~4.239×10³ ng、16.46~1.646×10² ng、8.340~83.40 ng范围内与峰面积呈良好的线性关系,加样回收率分别为98.7%、103%、95.3%、94.3%。结论:本方法专属性强,前处理操作简单,检测结果准确,可为戊己丸全面质量控制提供参考。     

New glycosidic alkaloid from the nearly ripe fruits of Euodia rutaecarpa

A new natural product, 10-hydroxyrutaecarpine (1), and a rarely new glycosidic alkaloid, rutaecarpine-10-O-rutinoside (2), along with the known compounds rutaecarpine (3), evodiamine, wuzhuyuamide-I, and dehydroevodiamine were isolated from the butanol fraction of 70% ethanol aqueous extract of the dried and nearly ripe fruits of Euodia rutaecarpa (Juss.) Benth. Their structures were elucidated on the basis of their spectroscopic data.     

市售制吴茱萸中甘草汁成分质量标准研究

目的探讨市售制吴茱萸中甘草汁成分的质量标准。方法用甘草药材与吴茱萸药材自行炮制制吴茱萸,采用高效液相色谱梯度洗脱法检测并比较自行炮制吴茱萸与市售吴茱萸中甘草汁成分含量。结果自行购买吴茱萸与甘草中特征性成分吴茱萸碱、吴茱萸次碱、柠檬苦素及甘草苷、甘草酸符合2015年版《中国药典(一部)》要求,且其中甘草汁成分相近,均符合2015年版《中国药典(一部)》要求。结论通过试验可掌握市售吴茱萸甘草汁炮制品中甘草苷、甘草酸含量情况。应以2015年版《中国药典(一部)》对制吴茱萸的各项规定为基础,明确规定制吴茱萸的制法、检查、浸出物、含量测定,为制订甘草汁制吴茱萸更完善的质量标准提供参考。     

吴茱萸中4种单体成分致肾细胞毒性的初步研究

目的探讨吴茱萸中成分吴茱萸碱、吴茱萸次碱、吴茱萸内酯和辛弗林在体外对人胚肾细胞(HEK-293)的影响。方法采用MTT法检测吴茱萸碱、吴茱萸次碱、吴茱萸内酯和辛弗林对肾细胞活力的影响;给药后检测肾细胞培养上清液中的功能性指标乳酸脱氢酶(LDH)含量;采用倒置相差显微镜对给药后的细胞形态进行观察。结果 MTT法显示,8.3～33.2 g.mL-1的吴茱萸碱、5～40 g.mL-1的吴茱萸次碱和50～200 g.mL-1的吴茱萸内酯对HEK-293细胞活力有明显的抑制作用(P<0.01 or 0.05),辛弗林对肾细胞活力无明显影响。4.15～33.2 g.mL-1的吴茱萸碱、5～20 g.mL-1的吴茱萸次碱和50～200 g.mL-1的吴茱萸内酯能显著升高肾细胞上清液中的LDH(P<0.01),给予辛弗林后LDH无变化。给予4.15～33.2 g.mL-1的吴茱萸碱和100～200 g.mL-1的吴茱萸内酯后,肾细胞均不同程度的皱缩、减少、甚至死亡,吴茱萸次碱和辛弗林对肾细胞形态无明显影响。结论吴茱萸碱、吴茱萸次碱和吴茱萸内酯对肾细胞均有毒性作用,而辛弗林对肾细胞无毒性作用。     

不同比例黄连-吴茱萸配方颗粒/单/合煎对比分析

目的： 通过考察不同比例的黄连与吴茱萸药对,其配方颗粒与单煎液、合煎液的关键成分含量差异,探索药对配方颗粒与传统煎法的等效性。方法： 采用高效液相色谱法测定不同制法下6种生物碱含量,并利用SPSS18.0进行统计检验。结果： 与单煎相比,配方颗粒中4种黄连生物碱和吴茱萸次碱含量均与合煎中没有明显差异（小檗碱P=0.881;表小檗碱P=0.810;黄连碱P=0.961;巴马汀P=0.220;吴茱萸次碱P=0.137）,单煎中黄连生物碱含量平均值约为配方颗粒或合煎的2倍（P<0.01）,吴茱萸次碱单煎含量明显降低（配方颗粒/合煎平均值为单煎的3倍以上,P<0.01）,而吴茱萸碱在配方颗粒与单煎中的差异小（P=0.314）,平均值均在合煎液含量的40%以下（P<0.01）。另外,随着吴茱萸占比提高,合煎液中4种黄连生物碱、吴茱萸碱和吴茱萸次碱含量明显降低,但比例对配方颗粒和单煎影响不大。结论： 在黄连和吴茱萸药对中,黄连生物碱配方颗粒与合煎差异不显著,吴茱萸生物碱在配方颗粒中可能发生转变,导致与合煎有差异,而且配方颗粒并不能重现合煎中药对比例对成分含量的影响趋势。     

反相高效液相色谱法测定吴茱萸中吴茱萸碱和吴茱萸次碱含量

目的建立测定吴茱萸中吴茱萸碱及吴茱萸次碱含量的反相高效液相色谱法。方法采用Boston Symmetrix ODS-R C18色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-0.04%辛烷磺酸钠溶液(50∶50),流速为1 mL/min,检测波长为225 nm。结果该法准确可靠,重现性好。结论反相高效液相色谱法可以同时测定吴茱萸中吴茱萸碱及吴茱萸次碱的含量。     

Simultaneous determination of five main active constituents of Erigeron multiradiatus by HPLC-DAD-MS

An HPLC-DAD-MS method was developed for simultaneous determination of the five major active constituents in Erigeron multiradiatus (Wall.) Benth, namely 6'-O-cafferylerigeroside (1), scutellarin (2), apigenin-7-O-beta-d-glucuronide (3), apigenin (4) and kaempferol (5), respectively. They were identified by ESI-MS and comparisons with literature. A comprehensive validation of the method included tests of sensitivity, linearity, precision and accuracy. The linear regressions were acquired with r>0.999. The precision was evaluated by intra- and inter-day assays, and relative standard deviation (R.S.D.) values were reported within 2.7%. The recovery studies for the quantified compounds were observed in the range of 95.3-102.4% with R.S.D. values less than 2.3%. The overall procedure may be suitable for the qualitative and quantitative analyses of a large number of E. multiradiatus samples. Hierarchical clustering analysis based on the characteristics of the 5 investigated compound peaks in HPLC profiles showed that 18 samples were divided into 2 main clusters. The clusters corresponded to their content. The five constituents in E. multiradiatus are generally regarded as an index for the quality assessment of this herb.     

大鼠灌胃不同纯度吴茱萸提取物后吴茱萸碱及吴茱萸次碱的药动学研究

目的:研究灌胃给予不同纯度吴茱萸提取物后大鼠体内吴茱萸次碱(Rut)和吴茱萸碱(Evo)的药动学特点。方法:以Rut、Evo分别为40、31mg·kg-1的剂量,灌胃给予雌性大鼠高、中、低不同纯度(Rut:45%、28%、9%,Evo:35%、21%、7%)的吴茱萸提取物混悬液(n=6),分别于灌胃后0·25、0·5、0·75、1·0、1·5、2·0、2·5、3·0、4·0h眼底静脉丛采血0·5mL,高效液相色谱法测定血样中Rut和Evo含量,并计算药动学参数cmax、tmax和AUC0～4h。结果:高、中、低纯度组中,cmax:Rut:(215·3±80·4)、(94·5±28·8)、(22·8±4·4)ng·mL-1,Evo:(164·8±65·1)、(78·8±23·5)、(43·4±17·2)ng·mL-1(两两比较P<0·01);tmax:Rut:(0·5±0·0)、(0·6±0·1)、(0·5±0·0)h,Evo:(0·5±0·2)、(0·7±0·2)、(0·5±0·0)h;AUC0～4h:Rut:(117·1±26·5)、(117·4±42·2)、(36·8±5·6)ng·h·mL-1,Evo:(148·9±39·1)、(122·2±23·3)、(80·4±14·3)ng·h·mL-1。3种纯度组中Rut和Evo的cmax具有统计学差异,AUC0～4h随纯度的升高而增加(P<0·01)。结论:吴茱萸提取物的纯度与Rut和Evo的吸收明显有关,在纯度为16%～80%(以二者总含量计)范围内,随着纯度的提高,二者生物利用度均明显增加。