苦参碱的毒理及HPLC法测定绵羊血浆苦参碱的研究
发布时间：2019-02-13 07:38
【摘要】：本文目的为研究苦豆子的主要有效成分苦参碱的毒理以及苦参碱经绵羊皮肤给药的吸收作用。试验所用到的苦豆子均采自于鄂尔多斯市鄂托克前旗,并经由内蒙古农业科学院兽医研究所化学提纯后得到苦参碱样品。实验一为苦参碱毒性试验；该实验用一定浓度的苦参碱溶液对小白鼠进行灌胃,记录小白鼠的急性中毒症状、探寻苦参碱作用的主要靶器官、测定苦参碱对小白鼠的半数致死量以及观察停止灌胃苦参碱后主要靶器官的毒性恢复程度。借鉴Bliss的公式来计算苦参碱的半数致死量。结果表明：通过眼观与病理组织学对比发现苦参碱主要作用靶器官有肺脏、肝脏、脑、肾脏和心脏。在恢复程度上肺脏的情况较差,脑组织、肝脏和肾脏造成的损害恢复情况较为理想。实验二为刺激性试验,试验从研究苦参碱对皮肤的刺激性和眼的刺激性两方面来进行。皮肤刺激性试验分为单次皮肤刺激性试验和长期皮肤刺激性试验两部分,所用药物分别是苦参碱制剂和苦参碱透皮制剂两种规格。其中苦参碱透皮制剂组中添加有透皮促进剂(氮酮和丙二醇),所用到的试验动物为大白兔。实验结果显示：未添加透皮剂的苦参碱组在单次与多次刺激试验中对皮肤与眼结膜均无刺激性；而添加了透皮促进剂的苦参碱透皮制剂组在单次与多次刺激试验中对皮肤存在中等强度刺激性,刺激作用的眼观变化表现为眼皮红肿、皮增厚、皮结痂、痂皮脱落、新皮生成等；病理切片上则表现为皮肤角质层的增生脱落,真皮层的纤维水肿断裂,刺激小血管充血、出血,炎性细胞侵润等。实验三苦参碱液相学研究,采用岛津LC-2010C液相色谱仪,建立血浆中苦参碱的HPLC检测方法,其中用槐定碱作为内标。血浆前处理以甲醇为蛋白沉淀剂,通过紫外检测器进行检测,检测波长220nm,流动相为A-0.05moL/L的磷酸二氢钾(磷酸氢二钾调pH到6.46)和B-甲醇(A/B,65：35),等浓度洗脱,流速0.8mL·min-1,建立在羊血浆检测苦参碱的高效液相色谱方法,目标生物碱色谱峰出峰良好。苦参碱的线性关系良好R20.999。其中MT使用非经脉管模型计算其药效动力学各参数。实验证明苦参碱外用时,药物的主要作用部位为体表,添加透皮促进剂也仅有极小量的药物会进入全身血液循环,因此透皮吸收进入血液的有效成分少,本实验建立了在绵羊血浆内检测苦参生物碱的方法。
[Abstract]:Objective to study the toxicology of matrine, the main active component of Sophora flavescens, and the absorption of matrine through sheep skin. The samples of Sophora flavescens collected from Qianqi, Ordos city, were purified by chemical purification from Institute of Veterinary Medicine, Inner Mongolia Academy of Agricultural Sciences, and the samples of matrine were obtained. The first experiment is matrine toxicity test. In this experiment, a certain concentration of matrine solution was used to gavage the mice, to record the acute toxic symptoms of the mice, and to explore the main target organs of matrine action. The median lethal dose of matrine on mice and the recovery of toxicity of the main target organs after the cessation of oral administration of matrine were determined. The Bliss formula is used to calculate the 50% lethal dose of matrine. The results showed that the main target organs of matrine were lung, liver, brain, kidney and heart. The recovery degree of lung was poor, and the damage caused by brain tissue, liver and kidney was better. The second experiment was an irritation test, which studied the skin irritation of matrine and the irritation of the eye. The skin irritation test is divided into two parts: single skin irritation test and long-term skin irritation test. The drugs used are matrine preparation and matrine transdermal preparation respectively. Transdermal enhancers (azone and propylene glycol) were added in matrine transdermal preparation group. The results showed that there was no irritation to the skin and conjunctiva in the matrine group without the addition of transdermal agents. In the single and multiple irritation tests, the skin was irritated with moderate intensity in the matrine transdermal preparation group. The ocular changes of the irritation were as follows: swelling of the eyelid, thickening of the skin, scabbing of the skin, exfoliation of the scab, and exfoliation of the skin. New skin formation; The histopathological sections showed the proliferation and exfoliation of the cuticular layer of the skin, the fibrous edema and rupture of the dermis, the stimulation of small blood vessel congestion, hemorrhage, inflammatory cell infiltration and so on. The HPLC method for the determination of matrine in plasma was established by using LC-2010C liquid chromatograph of Shimadzu, in which Sophorine was used as the internal standard. In plasma pretreatment, methanol was used as protein precipitator and detected by UV detector. The detection wavelength was 220 nm. The mobile phase consisted of A-0.05moL/L potassium dihydrogen phosphate (pH to 6.46) and B- methanol (A / B). 65:35), iso-concentration elution, flow rate 0.8mL min-1, was established in sheep plasma for the detection of matrine by high performance liquid chromatography, the target alkaloid chromatographic peak produced a good peak. The linear relationship of matrine is good R20.999. The pharmacokinetic parameters of MT were calculated by non-vessel model. The experiment proved that when matrine was used for external use, the main action site of the drug was the body surface, and the addition of transdermal accelerator only a minimal amount of drugs would enter the whole body blood circulation, so transdermal absorption into the blood of the effective components less, A method for the determination of alkaloids of Sophora flavescens in sheep plasma was established.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7
[Abstract]:Objective to study the toxicology of matrine, the main active component of Sophora flavescens, and the absorption of matrine through sheep skin. The samples of Sophora flavescens collected from Qianqi, Ordos city, were purified by chemical purification from Institute of Veterinary Medicine, Inner Mongolia Academy of Agricultural Sciences, and the samples of matrine were obtained. The first experiment is matrine toxicity test. In this experiment, a certain concentration of matrine solution was used to gavage the mice, to record the acute toxic symptoms of the mice, and to explore the main target organs of matrine action. The median lethal dose of matrine on mice and the recovery of toxicity of the main target organs after the cessation of oral administration of matrine were determined. The Bliss formula is used to calculate the 50% lethal dose of matrine. The results showed that the main target organs of matrine were lung, liver, brain, kidney and heart. The recovery degree of lung was poor, and the damage caused by brain tissue, liver and kidney was better. The second experiment was an irritation test, which studied the skin irritation of matrine and the irritation of the eye. The skin irritation test is divided into two parts: single skin irritation test and long-term skin irritation test. The drugs used are matrine preparation and matrine transdermal preparation respectively. Transdermal enhancers (azone and propylene glycol) were added in matrine transdermal preparation group. The results showed that there was no irritation to the skin and conjunctiva in the matrine group without the addition of transdermal agents. In the single and multiple irritation tests, the skin was irritated with moderate intensity in the matrine transdermal preparation group. The ocular changes of the irritation were as follows: swelling of the eyelid, thickening of the skin, scabbing of the skin, exfoliation of the scab, and exfoliation of the skin. New skin formation; The histopathological sections showed the proliferation and exfoliation of the cuticular layer of the skin, the fibrous edema and rupture of the dermis, the stimulation of small blood vessel congestion, hemorrhage, inflammatory cell infiltration and so on. The HPLC method for the determination of matrine in plasma was established by using LC-2010C liquid chromatograph of Shimadzu, in which Sophorine was used as the internal standard. In plasma pretreatment, methanol was used as protein precipitator and detected by UV detector. The detection wavelength was 220 nm. The mobile phase consisted of A-0.05moL/L potassium dihydrogen phosphate (pH to 6.46) and B- methanol (A / B). 65:35), iso-concentration elution, flow rate 0.8mL min-1, was established in sheep plasma for the detection of matrine by high performance liquid chromatography, the target alkaloid chromatographic peak produced a good peak. The linear relationship of matrine is good R20.999. The pharmacokinetic parameters of MT were calculated by non-vessel model. The experiment proved that when matrine was used for external use, the main action site of the drug was the body surface, and the addition of transdermal accelerator only a minimal amount of drugs would enter the whole body blood circulation, so transdermal absorption into the blood of the effective components less, A method for the determination of alkaloids of Sophora flavescens in sheep plasma was established.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7
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