基于炮制产物的比较研究探寻何首乌肝毒性的物质基础

【作者】 王蕾；

【导师】 李飞；

【作者基本信息】 北京中医药大学 ， 中药炮制学， 2017， 硕士

【摘要】 何首乌来源于蓼科植物何首乌Polygonum multiflorum Thunb.的干燥块根[1]。何首乌具有生熟异治的特点,生品具有解毒,消痈,截疟,润肠通便的功效,制品是临床常用的滋补之品,具有补肝肾,益精血,乌须发,强筋骨,化浊降脂的功效,且鲜有医家认为何首乌具有毒性。目前,何首乌致肝损伤的问题日益突出,其毒性的物质基础尚不明确,但炮制可降低何首乌的毒性,故炮制与何首乌饮片的质量、临床用药的安全有效紧密相关。本研究对四十余部古籍著作、建国至今的9版药典及16部不同省市炮制规范等文献资料进行了整理、分析,发现何首乌的古今炮制方法差异明显。现行药典何首乌项下收载了清蒸、黑豆汁蒸、黑豆汁炖三种湿热炮制方法。各省市炮制规范中收载的炮制方法均为湿热法,多为蒸、炖法。企业在实际生产中则多采用高压蒸制。该方法与炖法均为密闭式加热,且水蒸气不直接作用于何首乌,蒸法则与之相反。现行的何首乌的炮制方法主要为净制、水处理、蒸或炖等。古代,各医家多采用蒸法或煮法炮制何首乌,而浸泡是其中一道重要的工序,且加热炮制的时间长、次数多;现今基本省略浸泡这一步骤,且加热炮制次数仅为一次,炮制时间长短不一。2015版《中华人民共和国药典》(一部,以下简称《药典》)以二苯乙烯苷、结合型蒽醌或游离型蒽醌含量的低限作为何首乌生品和制品的质量评价标准。目前,研究认为二苯乙烯苷、大黄素等游离型蒽醌、大黄素甲醚-8-O-β-吡喃葡萄糖苷等结合型蒽醌是何首乌致肝损伤的潜在的物质基础。因此,药典中何首乌的质量评价标准不能有效地控制其毒性,保证临床用药的安全。此外,有研究表明何首乌蒸制汁液的急性毒性大,而何首乌蒸制汁液拌回制品是否具有科学性有待进一步研究。物质基础决定药物的功效、毒性,故本研究从何首乌古今炮制的差异切入,按照常压清蒸、高压清蒸、高压黑豆汁蒸、黑豆汁炖及浸泡等炮制方法制备何首乌样品,并收集炮制过程产物,比较不同样品及炮制过程产物间化学成分的差异,结合其肝细胞毒性的大小及变化规律,探寻何首乌致肝损伤的物质基础。本研究收集了河南产地、贵州产地生品何首乌,共计两批。观察、记录两个产地生品的外观性状,两批何首乌的规格均大小或厚薄不一,鲜有饮片符合《药典》对厚片或块的规定。本研究分别以河南、贵州两个产地生品为原料,采用高压清蒸、高压黑豆汁蒸、常压清蒸、黑豆汁炖等方法分别加热炮制一系列时间的制品,并收集常压蒸制何首乌的蒸制余液;以河南生品为原料,采用不同辅料浸泡何首乌,并收集浸泡余液,以上述炮制品及炮制过程产物作为本研究的实验样品。本研究对实验室自制的以上何首乌样品分别进行传统性状和现代检测相结合的多指标质量考察,全面了解何首乌饮片的质量现状,分析何首乌外观性状、化学成分随炮制程度等因素的变化,为探寻何首乌肝毒性的物质基础提供科学依据。观察并记录何首乌及炮制过程产物的外观性状。结果发现:不同原料产地、按照不同炮制方法、不同炮制时间炮制的何首乌样品在饮片的气味、颜色、质地、粉末颜色等方面均有差异。分别以两个产地何首乌生品为原料炮制的制品的苦涩味均随炮制时间的延长而变淡,饮片颜色、粉末颜色均逐渐加深而后变为红褐色。何首乌分别采用清水、米泔水、黑豆等不同辅料浸泡后气味变淡、颜色变浅,浸泡余液颜色不一。水浸泡余液呈橙黄色到橙红色,且沉淀相对较少;米泔水浸泡余液呈黄色,且黄色沉淀较多;黑豆浸泡余液呈黑紫色,且沉淀较少,这可能与黑豆中的色素溶出有关。采用高效液相色谱法测定所有自制何首乌样品中二苯乙烯苷的含量,两批生品何首乌均符合《药典》的规定,即生品中二苯乙烯苷≥1.0%,河南产地生品的含量为3.92%,贵州产地生品含量为3.73%;何首乌采用不同加热炮制方法炮制的过程中,其二苯乙烯苷含量均随炮制时间的延长而逐渐下降。不符合《药典》规定的制何首乌分别是以贵州生品为原料的高压蒸制10h、12h、黑豆汁炖56h。故原料药的质量、炮制时间等均能影响何首乌制品的质量。此外,不同辅料浸泡品的含量均低于生品,但高于《药典》对生品的含量要求,三者中二苯乙烯苷的含量与(河南)高压炮制3～4h、(河南)常压炮制8～16小时接近。按照《药典》(通则2202鞣质含量测定法)方法,测定何首乌样品中鞣质的含量,加热炮制品、浸泡品中的整体变化趋势与二苯乙烯苷的变化趋势基本一致,浸泡品中鞣质的含量与河南高压蒸制3～4h接近。采用薄层色谱的方法比较何首乌各样品中化学成分的变化,结果:生品中有7个斑点,炮制后制品中除6、7号的斑点稍有增强外,其余斑点均随着炮制时间的延长而逐渐减弱或消失。采用水、米泔、黑豆等辅料浸泡后的何首乌样品TLC的斑点数目和斑点大小变化与制品基本一致。1、3、4、5号斑点基本消失,2号斑点显著变小,6、7号斑点稍有增大。上述变化情况与何首乌长时间加热炮制前后的变化情况较为相似。HPLC法比较何首乌炮制前后的变化需要115min,耗时过长,为了节约时间,提高效率,采用UPLC法进行检测。两个产地何首乌生品有13各共有峰。以上13个色谱峰在炮制前后有明显的变化,随炮制时间延长1～11号峰峰面积逐渐降低或消失;12(大黄素)、13(大黄素甲醚)号峰峰面积明显增加。其中,生品中的2～6号峰可能是茋类成分;7～11号峰可能是结合型蒽醌类成分,经过炮制后变为大黄素、大黄素甲醚等游离型蒽醌类成分。炮制至一定时间,样品在2min左右出现了的一个新的A峰,其峰面积随炮制时间延长而增加。不同辅料浸泡品1～11号峰均峰面积降低或基本消失,12、13峰峰面积明显增加。通过不同辅料浸泡余液的验证,并未发现何首乌生品浸泡后基本消失的9、10、11号峰。此外,生品浸泡后,其浸泡余液的图谱在前6min出现了多个新的色谱峰。故推测何首乌不同辅料浸泡后消失的色谱峰可能是在水和自身含有的具有水解作用的酶的作用下分解成大黄素、大黄素甲醚等游离型化合物或小分子化合物。加热炮制及不同辅料浸泡何首乌的图谱较生品均出现明显的色谱峰数目或峰面积的变化。生品中色谱峰数目较为丰富,且2号峰(二苯乙烯苷)的峰高高于12号峰(大黄素),加热炮制后色谱峰的数目减少,且2号峰与12号峰的相对峰高出现反转现象,说明炮制前后二苯乙烯苷与大黄素的含量出现了反转现象。不同辅料浸泡后何首乌的图谱的色谱峰数目及相对峰高与长时间加热炮制的制品何首乌较为相似。采用超高效液相与线性离子阱轨道组合高分辨质谱联用技术系统指认何首乌炮制前后变化明显的特征峰,并分析炮制前后物质基础的差异。目前,鉴定出生品何首乌中36个成分,制品何首乌中34个成分。已鉴定出的化学成分中,生品与制品共有成分28个,生品特有成分8个,制品特有成分6个。本研究通过LTQ-Orbitrap多级质谱技术,解析出UPLC谱图中保留时间为8～15min主要的色谱峰的成分,该保留时间段的色谱峰主要为蒽醌苷类及决明柯酮苷类。该类成分的降低或消失可能与何首乌生熟异治及炮制减毒有关。采用CCK-8法测定何首乌制品及浸泡样品的肝细跑毒性,结果炮制后毒性均降低。贵州、河南两个产地的常压清蒸品的毒性均随着炮制时间的延长而降低,且在32h后河南常压清蒸品的毒性降低迅速。河南黑豆汁炖的制品的毒性随着炮制时间的延长先降低后升高,然后再降低,呈波动变化的趋势;在32h毒性增大,且高于与清蒸品,其后毒性降低速率缓慢。高压黑豆汁蒸1-6h,其毒性随炮制时间的延长而降低;高压清蒸何首乌的毒性在前1h显著降低,2～4h毒性稍有升高,4～10h毒性基本稳定,10h后开始再次降低。高压蒸制品毒性的整体变化趋势与黑豆汁炖品基本一致。何首乌不同辅料浸泡后的毒性均降低,黑豆浸泡何首乌的毒性与常压蒸制56h相近。综上所述,本研究立足于炮制,着眼于古今何首乌炮制方法对物质基础的影响,并分析主要炮制过程产物的成分,采用TLC、UPLC、液质联用技术对何首乌炮制品进行成分分析,探寻何首乌炮制前后、炮制过程物质基础变化及肝细胞毒性的相关性,结果表明:炮制的确能够降低何首乌的毒性,而大黄素-8-O-β-D-葡萄糖苷、大黄甲醚素-8-O-β-D-葡萄糖苷等蒽醌苷类、大黄素等游离蒽醌类、鞣质、二苯乙烯苷类等成分可能影响何首乌毒性,其中二苯乙烯苷与蒽醌类成分的比例对其毒性有重大影响。以上推测尚需进一步的毒理或药理实验研究确认。更多还原

【Abstract】 Polygonum multiflorum is a dry root of Polygonum multiflorum Thunb.Polygonum multiflorum has the characteristics of raw and cooked with different treatment,raw materials with detoxification,carbuncle,malaria,laxative effect,products are commonly used in clinical nourishing products,with nourishing liver and kidney,essence and blood,black hair,strengthening the bones and muscles,reducing blood lipid efficacy,and few physicians believe that Polygonum multiflorum has toxicity.At present,the problem of liver injury caused by Polygonum multiflorum is becoming more and more prominent.The material basis of its toxicity is not clear,but the concoction can reduce the toxicity of Polygonum multiflorum,so the processing closely related to the quality of fleece-flower root slices and the safety and efficacy of clinical medicine.In this study,more than forty books of ancient books,9 version of the Pharmacopoeia and 16 concocted literature and other materials of different provinces and cities were sorted out and analyzed.It was found that there were significant differences in the processing methods of Polygoni multiflori in ancient and modern.The method of Polygonum multiflorum in the current pharmacopoeia contains the steamed,steamed,stewed three damp heat processing method.The provincial and municipal processing standards contained in are wet heat method,mostly steaming,stewing method.Enterprises adopt high pressure steaming method in the actual production.The method and stewing method are closed heating,and water vapor does not directly act on Polygonum multiflorum,then steaming is the opposite.The current processing methods of Polygonum multiflorum are mainly net,water treatment,steaming or stewing.In ancient times,the doctors used cooking or steaming to processing Polygonum multiflorum many times,while soaking is one of an important process,and the heating time is long;this step of soaking today without and heating times is only one.2015 version of the "People’s Republic of China Pharmacopoeia"(the first part,hereinafter referred to as "Pharmacopoeia")puts stilbene glucoside(THSG),combined with anthraquinone or free anthraquinone content of the lower limit as the quality evaluation standards of the raw and the processed product.At present,the study suggests that THSG,emodin and other free anthraquinone,emodin-8-O-β-glucopyranoside and other combined anthraquinone are the potential material basis of liver damage caused by Polygonum multiflorum.Therefore,Polygonum multiflorum quality evaluation criteria in pharmacopoeia can not effectively control its toxicity and ensure the safety of clinical medication.The material basis determines the efficacy and toxicity of the drug.In addition,some studies have shown that the acute toxicity of Polygonum multiflorum steamed juice is large,and that juice mixed with the product whether the scientific need for further study to confirm.Therefore,this study is based on the differences of the processing method in ancient and modern.Then prepared different products according to steaming at normal pressure,high pressure steaming,high pressure black bean juice steaming,black bean juice stew and soaking processing methods,and collect the processing products of process,then compared the differences of chemical composition between different samples,and combined with the size and variation of hepatocyte toxicity,to explore the material basis of liver damage caused by Polygonum multiflorum.This study collected Henan,Guizhou origin raw fleece-flower roots,a total of two groups.Observe and record two origin raw product appearance,all of the specifications of the two batches of fleece-flower root size or thickness is differ,few Pieces comply with the provisions of the Pharmacopoeia on thick slices or blocks.In this study,the two products of Henan and Guizhou were used as raw materials,and the products were prepared by high pressure steaming,high pressure black bean juice steaming,atmospheric steaming and black bean stew,and the steamed residual liquid was collected;The raw product of Henan was used as raw materials,using different materials to immerse fleece-flower root,and collect the soaking liquid.The above-mentioned processed samples and processing products were used as experimental samples of this study.This study examined the quality indexes of home-made samples above Polygonum traditional characters and modern combination of detection,a comprehensive understanding of the quality status of Polygonum multiflorum slices,analysis the changes of appearance and chemical composition of Polygonum multiflorum with the processing level,and provide a scientific basis for the exploration of the material basis for liver toxicity of Polygonum multiflorum.The appearance and traits of the product of Polygonum multiflorum and the process were observed and found.The results showed that there were differences in the smell,color,texture and color of the Polygonum multiflorum samples prepared by different processing methods and different processing times.The bitter taste of the products made from two kinds of raw materials were reduced with the extension of the processing time,the color of the pieces and the color of the powder were deepened gradually and then turned into red brown.Polygonum multiflorum using water,rice swill,black beans and other different materials soaked,the smell fades,lighter color.The color of the remaining liquid after soaking is different.Water immersion raffinate was orange yellow to orange red,and the precipitation was relatively less;the rice water immersion remaining liquid was yellow,and yellow precipitatation more;black bean with water immersion raffinate was dark purple,with little precipitation,which may be associated with the dissolution of pigment in black beans,The content of THSG in all samples of Polygonum multiflorum was determined by high performance liquid chromatography(HPLC).The two batches of Polygonum multiflorum were all in accordance with the provisions of Pharmacopoeia,that is,THSG in raw materials is more than 1%,and the content of stilbene glucoside in Henan origin was 3.92%,the content of raw material in Guizhou was 3.73%;Polygonum multiflorum was treated with different heating methods,the content of stilbene glycosides gradually decreased with the processing time.Polygonum multiflorum products a total of 54 batches,THSG content do not meet the"Pharmacopoeia" provisions of the Polygonum multiflorum products are Guizhou raw materials for the high-pressure steaming 10h,high pressure steaming 12h,high pressure black bean juice steam for 10h,high pressure black bean juice steamed 12h,black bean juice stew 56h.So the quality of raw materials,processing time can affect the quality of Polygonum multiflorum products.In addition,the content of different excipients is lower than the raw materials,but higher than the "Pharmacopoeia" on the content of raw materials requirements,the content of THSG in the three groups was close to that of the high pressure processing 3～4h and the normal pressure processing 8～16h.According to the "Pharmacopoeia" method,the content of tannin in the samples of Radix Polygoni Multiflori was determined.The trend of the whole change in the processed products and the soaking products was consistent with that of the THSG.The content of tannin in the soaking product is close to that of Henan high pressure steaming 3～4h.The method of thin layer chromatography was used to compare the chemical composition of Polygonum multiflorum with different heating and processing methods.The results showed that there were seven spots in the raw materials.In addition,the spots of No.6 and No.7 were slightly enhanced in the processed products,and the others were gradually weakened or disappeared with the extension of processing time.After immersion,the number of spots and spot size of TLC were similar to that of the processed product.No.1,No.3,No.4,No.5 spots disappeared,No.2 spots significantly smaller,No.6 and No.7 spots slightly increased.The above changes and fleece-flower root changes before and after heating up for a long time are similar.HPLC method was used to compare the changes of Polygonum multiflorum before and after processing,and needed about 115min.In order to save time and improve efficiency,UPLC method was used for detection.There are 13 peaks in the two producing areas of Polygonum multiflorum,and the above 13 peaks have obvious changes before and after the processing,and the peak area of 1～llis gradually reduced or disappears with the processing time.12(emodin),13(physcion)peak area was significantly increased.Among them,the crude 2～6 in the peak may be stilbenes composition;7～11 peak is probably a combination of anthraquinones,processed into emodin and physcion free anthraquinones.After processing for a certain time,a new A peak appeared around 2min,and the peak area increased with the increase of processing time.The peak area of number of different excipients soaked in 1～11 decreased or disappeared,and peak areas of No.12 and No.13 increased significantly.Therefore,it is speculated that the chromatographic peaks disappeared after the immersion of different excipients of Polygonum multiflorum may be decomposed into free compounds or small molecule compounds such as emodin,emodin and the like under the combined action of water and its own hydrolyzable enzyme.The relative peak height of peaks 2 and 12 peaks was reversed after the heat treatment or soaking,and the results showed that there was a significant change in the number of peaks or peaks in the samples.The number of peaks and the relative peak height of the fleece-flower root soaking product were similar to that of Polygonum multiflorum that heating for a long time.The high-resolution liquid-phase and linear ion-well orbital combined highresolution mass spectrometry system was used to identify the characteristic peaks before and after the processing of Polygonum multiflorum,and the difference of material basis before and after processing was analyzed.At present,36 ingredients of the raw product,34 ingredients of the processed product has been identified.Among them,raw materials and products with a total of 28 ingredients in common,8 unique ingredients in the raw,6 unique ingredients in the processed.In this study,the LTQ-Orbitrap multistage mass spectrometry was used to successfully analyze the main chromatographic peaks in the UPLC spectra with a retention time of 8 to 15 min.The chromatographic peaks of the retention period were anthraquinone glycosides class.The reduction or disappearance of these components may be related to the treatment of He Shouwu and other diseases.The CCK-8 method was used to determine the liver toxicity of the processed product and the soaked samples.The results showed that the toxicity was reduced in processed products.The toxicity of Guizhou and Henan two origin of atmospheric steaming products decreased with the extension of processing time,and the toxicity of Henan atmospheric steaming products decreased rapidly after 32h.The toxicity of Henan black bean juice stewed products decreased first and then decreased with the extension of processing time,and then decreased,showing a trend of fluctuation.After 32 hours,the toxicity of the product was increased,and higher than that of steamed product,and the rate of toxicity decreased slowly after that.Under the condition of high pressure steaming 1-6h with black bean juice,the toxicity of autoclaved products showed the trend of decreasing firstly and then increasing with the extension of the processing time.The toxicity of the high pressure steameing products was significantly decreased in the first 1h,the toxicity of 2～4h was slightly increased,the toxicity was stable at 4～10h,and then decreased again after 10h.The overall trend of the toxicity of high pressure steam products is basically the same as that of Henan black bean juice stewed products.The toxicity of Radix Polygoni Multiflori was decreased after soaking with different excipients,and the toxicity of black bean with water immersion is close to that of atmospheric pressure steaming for 56h.In summary,this research is based on the processing,focusing on the effect of ancient and modern concoction method on material basis,and analyzes the composition of the main process products.TLC,UPLC and LC/MS are used to analyze the composition of Polygonum multiflorum,to explore the changes of material basis and the correlation between material basis and hepatocellular toxicity before and after the processing of Polygonum multiflorum.The results showed that the concoction could reduce the toxicity of Polygonum multiflorum,and anthraquinone glycosides such as emodin-8-O-β-D-glucoside,physcion-8-O-β-D-glucoside,emodin Free anthraquinones,tannins and THSG may affect the toxicity of Polygonum multiflorum,in which the ratio of stilbene glycosides and anthraquinones have a significant impact on its toxicity.All of the above speculations require further toxicological or pharmacological experimental to studies.更多还原