摘 要
目的：建立杜仲炮制工艺的质量标准，研究盐炙前后杜仲中化学成分组群的变化特征，结合血清药物化学及体内代谢动力学阐明杜仲盐炙入肾的炮制机理。
方法：采用UV、HPLC等分析方法建立盐制杜仲主要化学部位总含量，分析炮制过程对杜仲化学成分群组成结构的影响；在此基础上，结合血清药物化学的研究方法，考察盐制杜仲在肾阳虚证模型大鼠的时空动态HPLC血清指纹图谱，在肾阳虚证模型大鼠肾组织的分布特征；测定血清中血清皮质醇、睾酮、雌二醇、环磷酸腺苷、环磷酸鸟苷、17-羟类固醇随时间变化情况。
结果：（1）分别以芦丁、松脂醇二葡萄糖苷、谷氨酸、D-葡萄糖为对照品，采用紫外分光光度法测定杜仲盐炙前后的总黄酮、总木脂素、氨基酸和多糖的含量，数据显示生品总黄酮的含量为2.76%，制品总黄酮含量为2.31%，炮制后含量明显下降（p﹤0.05）；生品总木质素的含量为10.59%，制品总木脂素的含量为9.51%，炮制后含量明显下降（p﹤0.05）；生品氨基酸的含量为0.09%，制品氨基酸含量为0.12%，炮制后含量明显上升（p﹤0.05）；生品多糖的含量为0.26%，制品多糖的含量为0.36%，炮制后含量明显上升（p﹤0.05）。
（2）分别以京尼平酸、绿原酸、京尼平苷、松脂醇二葡萄糖苷为对照品，采用高效液相色谱法测定杜仲生品、盐炙品中京尼平苷酸、绿原酸、京尼平苷、松脂醇二葡萄糖苷的含量，数据分析显示，京尼平苷酸生品含量为0.38%，制品含量为0.19%，炮制后含量明显下降（p﹤0.05）；绿原酸生品含量0.37%，为制品含量为0.29%，炮制后含量明显下降（p﹤0.05）；京尼平苷生品含量为0.24%，制品含量为0.20%，炮制后含量明显下降；松脂醇二葡萄糖苷生品含量为0.24%，制品含量为0.18%，炮制后含量明显下降。
（3）以氢化可的松造成肾阳虚模型，将杜仲生品及盐炙品灌胃给药，观察给药前后负重游泳时间、各脏器指数、血清中睾酮、雌二醇、环磷酸腺苷、环磷酸鸟苷、皮质醇和17-羟类固醇的变化，数据显示对于氢化可的松所致肾阳虚模型，模型与空白组比较有显著性差异（p﹤0.01）；盐炙杜仲与生品杜仲比较有显著性差异（p﹤0.05），给药组与模型组有显著性差异（p﹤0.05）；空白组与给药组无明显差异。
（4）取SD大鼠24只，随机分为6组，按成人用量的40倍量灌胃给予杜仲生品和盐制品浓缩液（浓度2.5g生药/mL/100g体重），采用HPLC法测定入血成分，分析杜仲生品和盐制品化学成分的变化规律。结果显示，共有入血成分京尼平苷酸和松脂醇二葡萄糖苷，二者含量与液相测得数据变化规律类似，都是盐炙后成分略有减少；肝脏的分布京尼平苷酸含量变化不显著，而松脂醇二葡萄糖苷的含量盐炙后明显大于生品；肾脏的分布京尼平苷酸虽差异不大但二者吸收都较高，松脂醇二葡萄糖苷的吸收也是制品大于生品，吸收较高且差异显著。
结论：结合各检测结果分析，杜仲炮制品总黄酮和总木脂素含量下降明显，多糖与氨基酸显著上升，而HPLC检测结果显示京尼平苷酸、绿原酸、京尼平苷及松脂醇二葡萄糖苷含量都下降，只是下降程度不同；但药效学结果显示盐炙品效果优于生品；在血清药物化学成分中，入血成分京尼平苷酸和松脂醇二葡萄糖苷含量较高，且在肝脏和肾脏中分布盐炙品均大于生品；综合上述，有效成分的单项含量增加不能等同于药效结果的优劣，或者说入肾治疗肾阳虚的有效化学成分群不完全是文章中所述的全部，就本文而言，炮制后杜仲化学成分组群不论在质还是量上都发生了一定的改变，也在一定范围内迎合了本文所提出的化学成分配比关系的效应最大化假说，所以，以中医理论为指导，杜仲炮制是以各种炮制方法为间接手段，旨在使其化学成分组群在配比关系上达到最佳，从而发挥其入肾而治疗肾阳虚的最佳疗效。
Abstract
Objective: To establish the quality standard of Eucommia ulmoides Oliv processed, study the variation characteristics of chemical group after stir fried with salt water. Clarify the Processing mechanism of Eucommia ulmoides Oliv transfer into the kidney by serum pharmaceutical chemistry and pharmacokinetics.
Methods: Establish the total content testing method of main chemical parts of Eucommia ulmoides Oliv by UV, HPLC and other analysis methods, Analysis the effect of processing to the composition structure of Eucommia group chemical. By the method of serum pharmacochemistry, we studied the spatio-temporal dynamics of HPLC serum fingerprint of Eucommia ulmoides Oliv stir fried with salt water , and the distribution characteristics in renal tissue in in sufficiency of kidney-YANG rat model. Determined the content variation of serum cortisol, testosterone, estradiol, cAMP, cGMP, 17-hydroxysteroid .
Results: (1) Using rutin, pinoresinol diglucoside, glutamic acid, D-glucose as standard respectively, tested the total flavonoids, total lignans, amino acid and polysaccharide of Eucommia ulmoides Oliv by UV method, tested the samples before stir fried with salt and after stir fried with salt respectively. The results showed that content of total flavonoids was 2.76% in the raw products, while the content of total flavonoids was 2.31% after processed. The content decreased significantly after processed (P < 0.05). The content of total lignans was 10.59% in the raw products, the total lignans content productsis 9.51%after processed,The content decreased significantly after processed (P < 0.05). The content of amino acids in raw products was 0.09%, while the content was 0.12% after processed, The content increased significantly (P < 0.05).The content of polysaccharide in raw products was 0.26%, while the content was 0.36% after processed, The content increased significantly (P < 0.05).
(2) Using geniposidic acid, chlorogenic acid, geniposide, pinoresinol diglucoside as standard respectively, tested the contents of them in both raw product and the product after processed. The analysis showed, the content of geniposidic acid was 0.38%,while the content of the product after processed was 0.19%.The content decreased significantly after processed(P < 0.05); the content of chlorogenic acid in raw product was 0.37%, as the product after processed was 0.29%.The content decreased significantly after processed(P < 0.05); the content of geniposide in raw product was 0.24%, while the content of product after processed was 0.20%, The content decreased significantly after processed. The content of pinoresinol diglucoside in raw product was 0.24%, as the content of product after processed was 0.18%, The content decreased significantly after processed.
(3) the insufficiency of kidney-YANG model was built with hydrocortisone, the raw material and salt frying of Eucommia ulmoides Oliv were taken by intragastric administration, observe the variation of the swimming time, Viscera index, serum testosterone, estradiol, cAMP, cGMP, cortisol and 17-hydroxysteroid before and after administration. The result showed, for hydrogenation kidney yang deficiency model induced by hydrocortisone, there was significant differences (P < 0.01) between model group and the control group; compared with the raw Eucommia ulmoides Oliv, the processed product showed significantly different (P<0.05), the drug group and model group were significantly different (P < 0.05); control group and administration group had no evident difference.
(4) (4) 24 SD rats were randomly divided into 6 groups, 40 times of adult dosage was taken by intragastric administration, both the raw Eucommia ulmoides Oliv and processed product concentrated solution (concentration 2.5g /mL/100g body weight) was experiment. HPLC was used for the determination of components transfer into blood, analysis the chemical composition variation of Eucommia ulmoides Oliv raw materials and of processed products. The results showed that, there were two components transfer into blood, they were geniposidic acid and pinoresinol diglucoside, these components change similar to the HPLC result studied before. They both decreased slightly after processed; content of geniposidic acid distributed in liver did not change significantly. The content of pinoresinol diglucoside of Eucommia ulmoides Oliv that stir fried with salt was significantly greater than raw product; geniposidic acid in renal distribution has little difference between the raw product and the processed one, the absorption were both high. Pinoresinol diglucoside in kidney distribution of Eucommia ulmoides Oliv that stir fried with salt was greater than the crude product too, the absorption was high and the difference was significant.
Conclusion: all the result showed , the content of total flavonoids and total lignans of Eucommia ulmoides Oliv Processed products have obviously decline, and Polysaccharide and andamino acid have obviously rise. On the other hand, the content of geniposidic acid, geniposide, chlorogenic acid, pinoresinol diglucoside have differently decline. Also, the pharmacodynamics study indicated that, the effect of the Salt roast product is better than the crude, and the distribution both in liver and in kidney, the Salt roast product are more than the crude. Above all, the increase of only one effective component’s content can not indicate all the efficacy, namely, the effective chemical compositions, treaing insufficiency of kidney-YANG into the kidney, are incompletely all included in the article.In this article, after processed, the Chemical compositions of Eucommia ulmoides Oliv both quality and quantity have been changed,which in accordance with the Effect maximization hypothesis of chemical composition, mentioned in this article. So, Guided by TCM theory, the processing method of Eucommia ulmoides Oliv is combined with different kinds of processing method ,to make the groups of chemical composition in combination achieve to the best, for the best curative effect through treating insufficiency of kidney-YANG into the kidney.
Abstract
Objective: to establish the quality standard of Eucommia ulmoides Oliver process, before and after the study on saltsunburn Eucommia chemical element in the groupcharacteristics, processing mechanism combined with serum pharmaceutical chemistry and pharmacokinetics of Eucommia ulmoides stir fried with salt into the kidney to elucidate the.
Methods: to establish the main chemical parts of Eucommia ulmoides Oliv total content using UV, HPLC and other analysis methods, analysis on the effect of chemical composition of processing Eucommia group structure; on this basis, combining with the research method of serum pharmacochemistry, Eucommia ulmoides Oliv on spatio-temporal dynamics of HPLC serum fingerprint of kidney yang deficiency rat model, distribution characteristics inrenal tissue of rats with kidney yang deficiency model;Determination of serum cortisol, testosterone, estradiol,cyclic adenosine monophosphate, cGMP, 17-hydroxysteroid variation with time.
Results: (1) respectively with rutin, pinoresinol glucoside,glutamic acid, two D- glucose as standard, thedetermination of total flavonoids of Eucommia ulmoides, stir fried with salt before and after the spectrophotometry of total lignans, amino acid and polysaccharide content of UV,data displays content of total flavonoids was 2.76%, total flavonoids the content of product is 2.31%, after processingcontent significantly decreased (P < 0.05); the content ofcrude lignin was 10.59%, the total lignans content productsis 9.51%, after processing content significantly decreased(P < 0.05); content of raw products of amino acids was 0.09%, product amino acid content was 0.12%, after processing content increased significantly (P < 0.05);students content polysaccharide was 0.26%, the content of polysaccharide was 0.36% products, processed content increased significantly (P < 0.05).
(2) respectively to geniposidic acid, geniposide,chlorogenic acid, pinoresinol two glucoside as control sample, using high performance liquid content determination Eucommia raw materials, salt fryinggeniposidic acid, geniposide, chlorogenic acid, pinoresinoltwo glucoside chromatography, data analysis display,geniposidic acid content is 0.38%, product content is 0.19%, after processing content significantly decreased (P < 0.05); chlorogenic acid product content of 0.37%, as theproduct was 0.29%, after processing content significantly decreased (P < 0.05); genipin Gansheng product content is 0.24%, the content of products 0.20%, after processingdecreased two; pinoresinol glucoside product content is 0.24%, product content is 0.18%, after processingdecreased.
(3) with hydrocortisone causes the kidney yang deficiency model, the Eucommia raw materials and salt fryingintragastric administration, observe the change before and after administration of the swimming time, Viscera index,serum testosterone, estradiol, cyclic adenosine monophosphate, cGMP, cortisol and 17- hydroxysteroid,data display for hydrogenation kidney yang deficiency induced by hydrocortisone model, model and the control group had significant differences (P < 0.01); stir fried with salt and crude Eucommia Eucommia ulmoides were significantly different (P < 0.05), drug group and modelgroup were significantly different (P < 0.05); control group and administration group had no evident difference.
(4) 24 SD rats, were randomly divided into 6 groups,according to 40 times of adult dosage by intragastric administration Eucommia raw goods and salt productsconcentrated solution (concentration 2.5g / /mL/100g body weight), using HPLC method for the determination of blood components, variation analysis of Eucommia raw materials and chemical composition of salt products. The resultsshowed that, there were blood component of geniposidic acid and pinoresinol glucoside content of two, two and the liquid phase changes similar to measured data, areprocessed with salt composition decreased slightly; liverdistribution geniposidic acid content did not change significantly, and pinoresinol glucoside content two stir fried with salt was significantly greater than products; renal distribution geniposidic acid has little difference but the twoabsorption is higher, pinoresinol two glucoside absorption is greater than the crude products, absorption is higherand the difference was significant.
Conclusion: combined with the analysis of the test results of total flavonoids of Eucommia ulmoides, processed productsand the total lignans content decreased significantly,significantly increased polysaccharide and amino acid, andHPLC detection showed geniposidic acid, geniposide,chlorogenic acid, pinoresinol glucoside content decreasedtwo, just falling degree but different; pharmacodynamicresults show that salt broiled product effect is better than that of raw products; in the chemical composition of serum drug, blood component of geniposidic acid and pinoresinoltwo glucoside content is higher, and in the liver and kidney in the distribution of salt processed products were higher than products; above all, effective single content increaseis not equal to pharmacodynamics result, or into the kidneyin treatment of kidney yang deficiency of effective chemical component groups is not entirely the article in full, this thesis, after processing of chemical composition of Eucommia ulmoides group both in quality and quantity have undergone some changes, but also cater to the effects of chemical composition between the the maximization hypothesis, within a certain range, so, to Chinese medicine theory, Eucommia ulmoides Oliver in various processing methods for indirect means, in order to make the chemicalgroup in combination to achieve the best, The best curative effect in the treatment of kidney yang deficiency in kidney.
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