黄芩提取物中有效成分黄芩苷和汉黄芩苷在糖尿病大鼠中的药动学研究

目的比较黄芩提取物中主要成分黄芩苷和汉黄芩苷在糖尿病大鼠和正常大鼠体内的药动学.方法腹腔注射链脲佐菌素建立糖尿病大鼠模型,灌胃黄芩提取物后取血分离血浆,采用HPLC-Uv法检测血浆中黄芩苷和汉黄芩苷浓度,以矩量法计算药动学参数,AUC(0-τ)的计算采用梯形法.采用黄芩提取物与大鼠粪便温孵,从肠道代谢研究黄芩苷和汉黄芩苷动力学改变的初步机制.结果与正常大鼠比较,糖尿病大鼠给予黄芩苷和汉黄芩苷后体内Cmax1明显增加[黄芩苷(6.07±0.95)vs.(17.01±3.60)μg·mL-1,P＜0.01;汉黄芩苷(3.50±0.72)vs.(9.34±2.04)μg·mL-1,P＜0.01],Cmax2明显增加[黄芩苷(1.61±0.18)vs.(7.39±3.04)μg·mL-1,P＜0.01;汉黄芩苷(1.95±0.52)vs.(6.72±2.60)μg·mL-1,P＜0.01],AUC(0-τ)也明显增加[黄芩苷(38.72±7.25)vs.(86.70±20.91)μg·mL-1·h,P＜0.01;汉黄芩苷(39.20±12.10) vs.(69.40±24.20)μg·mL-1·h,P＜0.01],粪便悬浮液中黄芩苷降解也加快(Ke0.087 vs.0.173 min-1).结论黄芩苷和汉黄芩苷在糖尿病大鼠与正常大鼠之间存在明显的药动学差异,在糖尿病大鼠肠道内代谢加快.     

大鼠尿中黄芩汤多成分及其代谢物的分析比较

目的：通过大鼠尿中黄芩汤多成分及其代谢物的分析，比较复方和单味药中相应成分的代谢差异。方法：建立高效液相梯度洗脱的多成分分析方法，对口服黄芩汤及其各单味药后的大鼠尿中多种成分的排泄进行比较研究。结果：从尿中排泄物达峰时间（Tmax）比较，黄芩汤中的多种成分可以分为三种，一是快速排泄的成分，如芍药苷（PF）、甘草苷（LG）（Tmax为4h左右）；二是中等速度排泄的成分，如黄芩苷（BG）、汉黄芩苷（WG）、千层纸素A苷（OG）、黄芩素（B）和甘草酸（GL）（Tmax为8h-12h）；三是延迟排泄的成分，如汉黄芩素（W）、千层纸素A（O）、芍药苷代谢素I（PM-I）和甘草次酸（GA）（Tmax〉12h）。延迟排泄的成分都是代谢产物型化合物，经肠道内细菌作用，代谢转化后再吸收进入体内。在单味药中WG、OG、W、O、PM-I、LG和甘草素（L）的排泄总量和总排泄率高于在黄芩汤复方中。其中W、O、LG和L具有显著性差异，尤其是LG和L，其总排泄量是复方中的3倍左右。排泄速率在复方和单味药中也有差别，其中BG、WG、OG、B和GL的Tmax在复方中为8h，在单味药中缩短为4h，结论：黄芩汤大多数成分及代谢产物经尿的排泄在复方和在单味药中具有明显的差别，复方成分的代谢排泄较缓慢。     

黄芩苷PC12细胞跨膜转运的实验研究

目的研究黄芩苷神经元跨膜转运的特点,部分阐释黄芩苷发挥神经系统保护作用的物质性基础。方法以PC12细胞为研究对象,使用高效液相色谱(HPLC)、质谱(MS)法检测黄芩苷孵育细胞内的物质含量及相关代谢产物。结果 100 mg.L-1黄芩苷给药30 min后,胞内黄芩苷浓度达峰值,8 h后胞内已测不出黄芩苷;在50～400 mg.L-1的给药浓度下,胞内黄芩苷含量与给药剂量呈正相关;抑制剂Verapamil与Nimodipine对黄芩苷转运有明显影响;同时胞内检测出黄芩素、6-甲氧基-黄芩苷两种结构类似物。结论黄芩苷能够浓度依赖性的跨膜入胞,此过程受相关抑制剂影响,并产生代谢产物。     

不同剂量黄芩素在大鼠体内的药动学差异*

目的:比较不同给药剂量下黄芩素在大鼠体内的药动学差异。方法:采用HPLC-ECD技术,对灌胃给予黄芩素药低、中、高剂量(20,100,200 mg.kg-1)后,大鼠血浆中黄芩苷的浓度进行测定,比较了不同给药剂量下,黄芩苷的血药浓度-时间曲线图。结果:黄芩苷的血药浓度-时间曲线均存在双峰现象。黄芩素低剂量组:黄芩苷的Tm ax=1和9 h,Cm ax,1 h=3.74μg.mL-1,Cm ax,9 h=2.18μg.mL-1,AUC0~24 h=30.44μg.mL-1.h;黄芩素中剂量组:黄芩苷的Tm ax=1和13 h,Cm ax,1 h=15.32μg.mL-1,Cm ax,13 h=6.76μg.mL-1,AUC0~24 h=123.77μg.mL-1.h;黄芩素高剂量组:黄芩苷的Tm ax=1.5和9 h,Cm ax,1.5 h=14.63μg.mL-1,Cm ax,9 h=10.92μg.mL-1,AUC0~24 h=149.63μg.mL-1.h。与低剂量(20 mg.kg-1)相比,中、高剂量组的剂量分别增加了4和9倍,但AUC0~24值仅提高了3.1和4倍。结论:黄芩素给药剂量在20~100 mg.kg-1间,代谢产物黄芩苷的药动学行为呈线性关系;黄芩素给药剂量在100~200mg.kg-1间,黄芩苷的药动学行为呈非线性关系。     

黄芩中黄芩苷的提取工艺研究

目的 优化黄芩中黄芩苷的最佳水提工艺.方法 采用单因素结合正交试验设计法,以黄芩苷提取率和含量为评价指标,对影响黄芩苷制备工艺的影响因素(浸泡条件、溶剂用量、提取时间、提取次数)进行考察.结果 最佳提取工艺为10+5倍药材量的沸水,提取2次,时间分别为60 min和30 min,抽滤,合并两次提取液,用4 mol/L盐...     

6，7-双乙酰化黄芩素在大鼠体内的代谢产物研究

研究6，7-双乙酰化黄芩素在大鼠体内的代谢产物。大鼠灌胃给予6，7-X2K,酰化黄芩素后，采用HPLC—DAD和LC／MS^n方法，检测大鼠肠内和血浆中的代谢产物。6,7-双乙酰化黄芩素在大鼠肠内降解为6-单乙酰黄芩素和黄芩素；在大鼠血浆中检测到4种葡萄糖醛酸苷类代谢产物，初步鉴定为6-O-葡萄糖醛酸黄芩素，6-O-甲基-7-O-葡萄糖醛酸黄芩素，6，7-O-双葡萄糖醛酸黄芩素和6-O-葡萄糖-7-O-葡萄糖醛酸黄芩素。首次报道了6，7-双乙酰化黄芩素在大鼠体内的药物代谢，在肠内和血浆中共鉴定了六个代谢产物。     

大鼠微粒体代谢酶对黄芩素葡萄糖醛酸化代谢物影响的实验研究

目的 观察大鼠微粒体代谢酶对黄芩素(治疗呼吸道感染中药)代谢作用的影响.方法 用体外微粒体药物代谢酶孵育法;用HPLC法测定黄芩素及其葡萄糖醛酸化代谢物的含量,在不同孵育时间和不同浓度下,观察大鼠肝肠不同微粒体对黄芩素葡萄糖醛酸化代谢产物生成速率的影响.结果 5种不同微粒体药物代谢酶对黄芩素均有代谢作用,且随孵育时间延长,代谢作用也相应增加,呈较好的时间和剂量依赖关系.在5种微粒体中,十二指肠微粒体代谢作用最强;而肝代谢作用最弱.结论 黄芩素主要代谢部位是肠道.     

大鼠血清中黄芩素代谢产物的鉴定(英文)

黄芩素是黄芩苷的苷元,是黄芩的主要活性黄酮类成分之一。大鼠灌胃给予黄芩素(200mg/kg)之后,利用HPLC-DAD和LC-MS/MS法从大鼠血清中检测并鉴定出5个代谢产物(M1-M5)以及原型药(M0)。在此结果上,推测了黄芩素在大鼠血清中可能的代谢途径。     

RP—HPLC法测定7种药用黄芩中黄芩苷和汉黄芩苷的含量

目的:建立测定7种药用黄芩中黄芩苷和汉黄芩苷的反相高效液相色谱法,7种药用黄芩为黄芩,粘毛黄芩,甘肃黄芩,滇黄芩,韧黄芩,川黄芩和丽江黄芩。方法:采用Wakosil;C_(18)柱(150mm×4.6mm,5μm),以甲醇-水-冰乙酸(41:59:0.2)和(36:64:0.2)为流动相,流速为1.0mL·min~(-1),检测波长为280nm。应用二极管阵列检测器对峰的纯度进行鉴定。结果:样品中黄芩苷和汉黄芩苷平均回收率分别为98.3％和96.8％,RSD分别为0.48％和1.3％,n=5;黄芩苷在0.08～0.4μg内,r=0.9998,汉黄芩苷在0.04～0.2 μg内,r=0.9995;峰面积与浓度线性关系良好。结论:黄芩苷、汉黄芩苷与其他成分能得到很好的分离,不同品种间2种苷含量差别比较大。     

复方地黄合剂及复方逍遥合剂中黄芩苷含量测定

目的 :建立复方地黄合剂及复方逍遥合剂中黄芩苷含量的测定方法.方法 :采用高效液相色谱(HPLC)法、C18色谱柱(4.6 mm×250 mm,5μm)、流速1.2 ml/min,柱温25℃,波长280 nm测定,流动相为乙腈-0.2%磷酸溶液(27:73).结果 :在不同中药制剂中,黄芩苷峰形良好,方法专属性强.在浓度为16~320μg/ml范围内均呈良好的线性关系(r=0.9993);在50%、100%、150%的浓度下,二制剂中黄芩苷的回收率分别为99%及97%,RSD分别为1.7%和1.6%.此外,黄芩苷在8 h内含量变化的RSD分别为0.3%和0.2%,稳定性良好.结论 :该方法简便、准确、灵敏度高、重复性好,可用于复方地黄合剂及复方逍遥合剂中黄芩苷的含量的测定.     

人参皂甙Rb1的肠内菌代谢

通过离体及整体实验观察了人和大鼠肠内菌对人参皂甙Rb₁（G-Rb₁）的代谢。方法：采用薄层色谱（TLC）和电喷雾质谱（ESI-MS）检测G-Rb₁及其代谢产物。结果：离体实验表明,G-Rb₁容易被大鼠和人消化道菌群代谢,随着代谢时间的延长,相继出现Rd, Rg₃/F₂, Rh₂/C-K和Ppd 4种代谢产物。给大鼠ig G-Rb₁ 500 mg.kg^-1后收集4 h和6 h粪,提取G-Rb₁的代谢产物,证明粪中存在Rd和Rg₃/F₂两种代谢产物。结论：G-Rb₁可被人和大鼠肠内菌代谢,其代谢模式为G-Rb₁→Rd→F₂→compound K（C-K）→20（S）protopanaxadiol（Ppd）。     

Identification of in‐vivo and in‐vitro metabolites of palmatine by liquid chromatography–tandem mass spectrometry

Objectives Despite its important therapeutic value, the metabolism of palmatine is not yet clear. Our objective was to investigate its in‐vivo and in‐vitro metabolism. Methods Liquid chromatography–tandem electrospray ionization mass spectrometry (LC‐ESI/MSⁿ) was employed in this work. In‐vivo samples, including faeces, urine and plasma of rats, were collected after oral administration of palmatine (20 mg/kg) to rats. In‐vitro samples were prepared by incubating palmatine with intestinal flora and liver microsome of rats, respectively. All the samples were purified via a C₁₈ solid‐phase extraction procedure, then chromatographically separated by a reverse‐phase C₁₈ column with methanol–formic acid aqueous solution (pH 3.5, 70: 30 v/v) as mobile phase, and detected by an on‐line MSⁿ detector. The structure of each metabolite was elucidated by comparing its molecular weight, retention time and full‐scan MSⁿ spectra with those of the parent drug. Key findings The results revealed that 12 metabolites were present in rat faeces, 13 metabolites in rat urine, 7 metabolites in rat plasma, 10 metabolites in rat intestinal flora and 9 metabolites in rat liver microsomes. Except for six of the metabolites in rat urine, the other in‐vivo and in‐vitro metabolites were reported for the first time. Conclusions Seven new metabolites of palmatine (tri‐hydroxyl palmatine, di‐demethoxyl palmatine, tri‐demethyl palmatine, mono‐demethoxyl dehydrogen palmatine, di‐demethoxyl dehydrogen palmatine, mono‐demethyl dehydrogen palmatine, tri‐demethyl dehydrogen palmatine) were reported in this work.     

Comparison of in vitro preparations for semi-quantitative prediction of in vivo drug metabolism.

Various in vitro preparations were compared with respect to their ability to mimic in vivo metabolism. For this purpose, S9-liver homogenate, microsomes, cryopreserved hepatocytes, cryopreserved liver slices and fresh liver, lung, kidney, and intestinal slices were incubated with three drugs in development, which are metabolized in vivo by a wide range of biotransformation pathways. Metabolites were identified and quantified with liquid chromatography-mass spectometry/UV from the in vitro incubations and compared with metabolite patterns in feces, urine, and bile of dosed rats. In vitro systems with intact liver cells produced the same metabolites as the rat in vivo and are a valuable tool to study drug metabolism. Phase I metabolites were almost all conjugated in intact cells, whereas S9-homogenate only conjugated by sulfation and N-acetylation. Microsomes and S9-homogenate are useful to study phase I metabolism but not for the prediction of in vivo metabolism. Extra-hepatic organ slices did not form any metabolites that were not produced by liver cells, but the relative amounts of the various metabolites differed considerably. Small intestinal slices were more active than liver slices in the formation of the N-glucuronide of compound C, which is the major metabolite in vivo. When the relative contribution of liver and small intestinal slices to the metabolism of this compound was taken into account, it appeared that the in vivo metabolite pattern could be well predicted. Results indicate that for adequate prediction of in vivo metabolism, fresh or cryopreserved liver slices or hepatocytes in combination with slices of the small intestines should be used.     

Anti-pruritic effect of baicalin and its metabolites, baicalein and oroxylin A, in mice Anti-tumorPharmacology

Aim:

To explore whether intestinal microflora plays a role in anti-pruritic activity of baicalin, a main constituent of the rhizome of Scutellaria baicalensis (SB).

Methods:

Baicalin was anaerobically incubated with human fecal microflora, and its metabolites, baicalein and oroxylin A, were isolated. The inhibitory effect of baicalin and its metabolites was accessed in histamine- or compound 48/80-induced scratching behavior in mice.

Results:

Baicalin was metabolized to baicalein and oroxylin A, with metabolic activities of 40.2±26.2 and 1.2±1.1 nmol·h⁻¹·mg⁻¹ wet weight of human fecal microflora, respectively. Baicalin (20, 50 mg/kg) showed more potent inhibitory effect on histamine-induced scratching behavior when orally administered than intraperitoneally. In contrast, baicalein and oroxylin A had more potent inhibitory effect when the intraperitoneally administered. The anti-scratching behavior activity of oral baicalin and its metabolites was in proportion to their inhibition on histamine-induced increase of vascular permeability with oroxylin A more potent than baicalein and baicalin. In Magnus test using guinea pig ileum, oroxylin A is more potent than baicalein and baicalin in inhibition of histamine-induced contraction. The anti-scratching behavioral effect of oral baicalin was significantly reduced when oral antibiotics were simultaneously administered, whereas the effect of baicalein and oroxylin A were not affected.

Conclusion:

Oral baicalin may be metabolized by intestinal microflora into baicalein and oroxylin A, which ameliorate pruritic reactions through anti-histamine action.     

双黄连滴注射液提取工艺研究

目的优选双黄连滴注液的提取工艺。方法以黄芩药材黄芩苷的收率及金银花中绿原酸的含量为评价指标,考察不同提取条件对有效成分的影响,对提取工艺进行优化。结果确定双黄连滴注液最佳提取工艺。结论实验研究结果表明,以优化的工艺提取的有效成分提取率较高,有利于大生产。     

黄山药总皂苷肠内菌代谢及代谢产物吸收的研究

目的 :本文离体和整体观察人和大鼠肠内菌对黄山药总皂苷(DX)的代谢作用及整体给予DX后吸收入血的有效成分。方法 :用薄层色谱(TLC)及电喷雾质谱(ESI -MS)法检测粪中DX及其代谢产物。整体给予大鼠灌服DX900mg/kg ,于给药后不同时间采集尿及血清样品 ,用ESI -MS检测吸收入血成分。结果 :DX容易被人和大鼠消化道菌群代谢 ,随着代谢时间的延长 ,出现了各种甾体皂苷的降解产物及终产物薯蓣皂苷元 (Dio)。整体实验表明 ,在大鼠血及尿中均发现分子量为415 3的代谢产物 ,经ESI -MS二级质谱分析 ,上述分子量的化合物为Dio。结论 :DX可被人和大鼠肠内菌代谢 ,DX经口服后Dio被吸收入血。     

黄山药总皂甙肠内菌代谢及代谢产物吸收的研究

目的：本离体和整体观察人和大鼠肠内菌对黄山药总皂苷（DX）的代谢作用及整体给予DX后吸收入血的有效成分，方法：用薄层色谱（TLC）及电喷雾质谱（ESI－MS）法检测粪中DX及其代谢产物。整体给予大鼠灌服DX900mg/kg，于给药后不同时间采集尿及血清样品，用ESI－MS检测吸收入血成分。结果：DX容易被人和大鼠消化道菌群代谢，随着代谢时间的延长，出现了各种甾体皂苷的降解产物及终产物薯蓣皂苷元（Dio)。整体实验表明，在大鼠血及尿中均发现分子量为415．3的代谢产物，经ESIMS二级质谱分析，上述分子量的化合物为Dio。结论：DX可被人和大鼠肠内菌代谢，DX经口服后Dio被吸收入血。     

Metabolism of roquinimex in mouse and rat: an in vitro/in vivo comparison

1. In vitro studies with roquinimex, an immuno-modulator, in liver microsomes from mouse and rat were conducted to evaluate the primary metabolism and compare the metabolite pattern as well as the rate of metabolism with the in vivo pharmacokinetics of the compound in these two species. 2. In the presence of NADPH, roquinimex was metabolized to six primary metabolites (R1-6) by liver microsomes from mouse and rat. The formation of these metabolites was qualitatively similar in both species, and was greatly enhanced by pretreatment with PCN, an inducer of cytochrome P4503A. 3. The identification of the R1-6 demonstrated that roquinimex had been hydroxylated and demethylated. Hydroxylation at different sites of the quinoline moiety was the dominating reaction in both species. 4. Comparison of the resulting microsomal intrinsic clearance of 0.3 micromol mg(-1) protein min(-1) in mouse liver microsomes, versus 0.03 micromol mg(-1) protein min(-1) in rat liver microsomes demonstrated that the mouse possesses about a 10-fold greater metabolic capacity for roquinimex than the rat. 5. The in vivo pharmacokinetics of roquinimex demonstrated a 7-fold higher clearance in mouse than in the rat (82 ml h(-1) kg(-1) in mouse, 10.6 ml h(-1) kg(-1) in rat), which is in concordance with the in vitro findings.     

Protein binding capacity in vitro changes metabolism of substrates and influences the predictability of metabolic pathways in vivo

Large numbers of lipophilic molecules are attached to fractions of serum protein, e.g. albumin, in vivo. Cell culture medium of most in vitro hepatocyte models for the prediction of metabolism does not contain albumin. Consequently, in vitro availability and metabolism of substrates could differ significantly from the in vivo situation. The influence of albumin on the in vitro metabolism was tested on a new lipophilic compound.

Methods: Primary human and rat hepatocytes were cultured in a collagen sandwich configuration and incubated with ¹⁴C-labeled compound X127 that is known to exhibit a high propensity to bind to plastic surfaces. Groups contained either 1% (w/v) BSA or none. Substrates as well as metabolism products were determined with radio-HPLC and radioactivity levels in the medium were recorded.

Results: Quantitative differences were seen in the distribution of the compound in BSA and non BSA containing groups, thus indicating a substantial binding of the compound to polystyrol surfaces of cell culture dishes. Metabolic radio-HPLC profiles showed different patterns after 24 h of incubation between the two species as well as between the BSA- and non-BSA groups within the species.

Conclusions: With addition of albumin the adherance of lipophilic substrates and metabolites to cell culture dish surfaces can be neutralized and in vitro systems can more closely mimic the in vivo situation.     

Studies comparing in vivo:in vitro metabolism of three pharmaceutical compounds in rat, dog, monkey, and human using cryopreserved hepatocytes, microsomes, and collagen gel immobilized hepatocyte cultures.

The in vivo metabolism of three pharmaceutical compounds, EMD68843, EMD96785, and EMD128130, was compared in fresh and cryopreserved hepatocyte (CPH) suspensions and microsomes from rat, dog, monkey, and human livers and fresh human and rat hepatocyte collagen gel immobilized cultures (GICs). Half of the major in vivo metabolites was produced by phase 1 (hydroxylation, oxidation, hydrolysis, N-dealkylation) and half by phase 2 metabolism (mostly glucuronidation but also sulfation and glycine conjugation). The identity and percentage of phase 1 and 2 metabolites from each compound produced in hepatocytes compared well with that in each species in vivo. Glucuronidation was more extensive in GICs than in CPHs. In contrast, CPHs but not GICs, produced sulfate metabolites. Microsomes (supplemented with NADPH only) produced most of the phase 1 but no phase 2 metabolites. Metabolism in CPHs was the same as in fresh hepatocyte suspensions. Discrete species differences in metabolism were detected by CPHs and microsomes. Cytochrome P450 and glucuronosyl S-transferase contents of CPHs did not account for species differences in the percentage of phase 1 and 2 metabolites or the rate of disappearance of the parent compounds in these cells. These data show a good correlation between major metabolites formed in vivo and in vitro. CPHs and GICs, unlike microsomes, carried out sequential phase 1 and 2 metabolism. Each in vitro system has its own advantages, however, for short-term metabolism studies CPHs may be more useful since they are readily available, easier and quicker to prepare than GICs, and have more comprehensive enzyme systems than microsomes.