【作者】 杜建平；

【导师】 涂瑶生；

【作者基本信息】 广州中医药大学 ， 中药学， 2010， 博士

【摘要】 积雪草(Cent el la asiatica (L.) Urb.)为伞形科积雪草属植物积雪草的干燥全草,在许多国家和地区的传统医药领域里中的应用已有几千年历史,我国中医药对积雪草的内服和外用也已有两千多年的历史,广东民间现在仍常用之制备凉茶。目前临床上主要用于治疗烧伤、抑制瘢痕增生作用、抗抑郁作用等。积雪草抗胃溃疡作用已有报导,但国内目前尚无积雪草用于治疗胃溃疡的药物上市。积雪草药材为《中国药典》2005版首次收录,在《中国药典》2010版中又新增积雪草总苷条目。本积雪草总苷缓释微丸以现代制药技术为主要手段,将积雪草药材的有效部位积雪草总苷研制成具有明确治疗胃溃疡作用的新药。本文主要开展了以下几方面的研究工作,取得了较满意的结果。1.针对积雪草总苷这一目标成分,建立了TLC定性鉴别方法,UV和HPLC的定量分析方法,方法准确可靠,简便快速,为积雪草总苷缓释微丸的研究提供了有效的分析手段。在TLC色谱研究中,经过对比研究采用了氯仿-甲醇-水(13：7：2)展开系统,显色剂为10%硫酸乙醇溶液,经过实验研究证实更为有效。药材前处理经对比性实验研究选择甲醇为提取溶剂,提取方法为超声提取。利用高效液相色谱仪中二极管阵列检测器的波长扫描功能,选择205nm作为检测波长,与《中国药典》2010版积雪草总苷条目下高效液相色谱法中的检测波长一致。色谱条件为,色谱柱：KromasilC18柱(4.6×250mm,5μm)；流动相：乙腈：水溶液=26：74；流速：1ml／min；检测波长：205nm；柱温：25℃。2.积雪草总苷提取精制工艺研究中,进行了以积雪草总苷含量、积雪草总苷提取率和干浸膏得率为监测指标,对提取、纯化工艺优化。单因素考察提取溶媒对积雪草总苷含量的影响。采用正交设计以乙醇浓度、溶剂用量、提取时间和提取次数这四个工艺参数为因素,优选出提取工艺为：取积雪草总苷药材,用8倍量50%乙醇煮沸回流提取3次。采用不同AB-8、D-101、HPD200、HPD100大孔树脂法优选纯化工艺,结果以AB-8大孔树脂法最好,除杂速度快,指标性成分转移率高,精制度达到328.81%。积雪草总苷在干浸膏中的含量为56.21%。积雪草提取液纯化工艺为：取积雪草0.0625g生药／mL的浓缩液,按生药与树脂比例为1：1上AB-8大孔吸附树脂柱,药液以1ml／min的流速上柱吸附,先用1BV 10%乙醇溶液以1ml／min的流速进行洗脱,再用2BV70%乙醇进行洗脱,洗脱流速为1ml／min,收集洗脱液,70℃以下旋转蒸发仪减压回收乙醇,直至浓缩至相对密度1.15g／ml(70℃),70℃真空干燥,干膏以粉碎机粉碎,备用。3.对积雪草总苷的主要理化性质进行了测试。结果表明纯化水和稀盐酸均可作为积雪草总苷在体外溶出试验的溶媒。积雪草总苷提取物粉末的孔隙率相对较大,平均堆密度为0.652 g／ml。该药粉休止角大于40°,流动性不好。相对湿度在54%下药粉的吸水率较小,而在54%以上药粉吸湿性明显增加,较易吸湿。积雪草总苷对光、热、湿稳定。采用TLC法研究积雪草总苷提取物与部分拟采用辅料的相互作用,结果表明积雪草总苷与HPMC、EC、卡波姆934P、乳糖、硬脂酸未发生配伍变化。在人工胃液和人工肠液测试条件下,卡波姆(carbomer)均能在比其它成分更低的浓度下,提供药物的控制释放作用。因此,拟选用卡波姆934P为主要辅料。4.在参考文献的基础上,通过考察各种影响成丸的因素,结合动物体内外胃黏膜表面实验和体外释放度测定结果,设计出了合适的制丸工艺,确定了最佳处方,由该处方在确定的工艺条件下制得的积雪草总苷素丸圆整度好,收率较高,具有缓释功能,达到了预期的目标。本积雪草总苷缓释微丸的处方确定如下：积雪草总苷50%、MCC25.5%、卡波姆934P13%、硬脂酸6.5、乙基纤维素5%、润湿剂为水。积雪草总苷缓释微丸的制备工艺：将积雪草总苷提取物与辅料分别过80目筛,取13%(w)的卡波姆934P和硬脂酸6.5%(w)高速搅拌混匀,于60℃的恒温水浴中加热熔融后立即放入冰箱中冷却,2h后取出置于干燥器中放至室温,取出粉碎过80目筛；另取积雪草总苷提取物占总量50%的量和EC(5%)、MCC(25.5%),使之混合后,以水为润湿剂,通过挤出滚圆法制备微丸。制备好的微丸首先置于40℃干燥2h后再置于80℃热处理2h即得。筛分,得20-40目的目标微丸。5.应用多功能制粒包衣机对挤出滚圆法制备的素丸进行缓释包衣。包衣液主要采用Killcoat SR30D。分别考察了处方因素和工艺因素,并采用中心复合设计-效应面优化法对包衣处方进行了条件优化。确定积雪草总苷缓释微丸的最终包衣工艺为：聚合物中Kollicoat SR30D比例为43.86%,包衣增重为20%。处方为：KollicoatSR30D43.86%、丙二醇2%、乳糖0.5%、水46.14%、二氧化钛0.5%、滑石粉2%、水5%。包衣条件参数：进风温度为60度；喷雾压力为0.08Mpm保护气压力为0.08Mpa；恒流泵流速调节为0.5-0.8ml／min；包衣增重10%；包衣后40℃下熟化12h6.通过药效学研究证实,积雪草总苷在一定程度上可以清除氧自由基、提高机体抗氧化能力,刺激胃粘膜肉芽组织生长,促进炎症修复和溃疡愈合,对于胃溃疡具有一定的预防及治疗作用。实验结果显示,积雪草总苷高、中剂量组和雷贝拉唑钠组大鼠乙酸致慢性胃溃疡溃疡指数极显著低于模型组(P<0.05),积雪草总苷高、中剂量组显著低于猴头菌组(P<0.05),与雷贝拉唑钠组无显著性差异(P>0.05)。积雪草总苷高、中剂量均能显著降低大鼠溃疡指数,并呈一定的量效关系,说明积雪草总苷对于大鼠慢性乙酸型溃疡同样具有显著的修复作用。对乙酸法致胃溃疡模型胃组织SOD活性和MDA含量的影响方面,积雪草总苷高、中剂量组强于雷贝拉唑钠组。积雪草总苷高剂量组病理组织学主要表现：炎症细胞浸润以黏膜层为主,可见黏膜缺损区及其下炎性肉芽组织,可见少量纤维瘢痕组织增生修复。更多还原

【Abstract】 Centella asiatica (Centella asiatica (L.) Urb.) is the whole dry plant of the Umbelliferae Centella asiatica-Centella asiatica, and was used as a traditional medicine for thousands of years in many countries and regions, in Chinese medicine it also has been used for oral and external for 2000 years,it is still used as one of the main raw material of Herbal tea in Guangdong too. Currently, Centella asiatica are used in clinical to treat burns, inhibite scar proliferation and antidepressant. Although it was reported that Centella asiatica could be used to cure gastric ulcer, there were no Centella asiatica for the treatment of gastric ulcer drug on domestic market yet.Centella asiatica were included in Chinese Pharmacopoeia 2005 edition for the first time, and its active ingredients Asiaticosides added in the Chinese Pharmacopoeia 2010 edition as a new item.In this thesis use modern pharmaceutical technology to develop the effective parts of Centella asiatica herbs Asiaticosides to a clear treatment of gastric ulcer drug, and the the purpose of this thesis is to explore the best preparation process, quality standards and preliminary observe its anti-ulcer effects of Asiaticosides sustained-release pellets.1. TLC qualitative distinction, UV and HPLC quantitative analysis methods were established for the target constituents measurement of Asiaticosides, those methods mentioned above are accurate and reliable, simple and rapid, and provides an effective analytical method of the studying Asiaticosides sustained-release pellets. In the TLC chromatograph research, after a comparative study, chloroform-methanol-water (13:7:2) were selected as developing systems,10% acid solution as developer sulfuric, and experiments proved its effective. A compared experiment founded that ultrasound with extraction solvent methyl alcohol is a best extraction method. High performance liquid chromatography with diode array detector and wavelength scanning function, and 205nm were used as detection wave length, which consistent with the highly effective liquid phase chromatography’s examination wave length in Asiaticosides item in "Chinese Pharmacopoeia" 2010 Edition. The chromatograph conditions are:chromatographic column: KromasilC18 column (4.6×250mm,5μm); Flowing:Methyl cyanide:Peroxide solution= 26:74; Speed of flow:1ml/min; Examination wave length:205nm; The column is warm: 25℃.2.In the refining research of Asiaticosides, the content of Asiaticosides extraction rate and yield of dry extract were used as monitoring indicators, to optimize the extraction and purification process. And observed the single factor effect of extraction solvent on Asiaticosides content. Orthogonal design was used to optimized the four factors related to extraction process:ethanol concentration, solvent volume, extraction time and number of process.The optimized extraction process are:Asiaticosides herbs were extract 3 times with eight times the volume of 50% ethanol boils backset. AB-8 macroporous resin was selected in purification process from AB-8, D-101, HPD200, HPD100, for its maximum cleaning speed, the highest transfer rate of the index components. The rate of extracting and the content of Asiaticosides in the dry extract is 328.81%,56.21%. respectively.Centella asiatica extract purification process described as follows:the concentrated solution (the grass of Centella asiatica 0.0625g in every 1mL solution), according to the ratio of raw herb and resin 1:1, was added in AB-8 macroporous resin column at first, 1ml/min,10% ethanol solution eluted, then lml/min,2BV 70% ethanol elution, the eluent was collected in the following rotary evaporator vacuum for the recovery of ethanol at 70℃, at last, when the relative density of the eluent reached 1.15g/ml (70℃), the eluent was dried in Vacuum drier at the degrade of 70℃, and the dry paste was smashed and reserved.3.The main physical and chemical propertie of the Asiaticosides’s were tested. The results indicated that the purified water and dilute hydrochloric acid can be used as Asiaticosides solvent in the dissolution test. The porosity of Asiaticosides extract powder is relatively large, the average bulk density was 0.652 g/ml. The angle of repose of Asiaticosides extract powder is more than 40°, the fluidity is not good, and the relative humidity is small in 54% medicinal powder’s water-absorptivity, but in 54% above the medicinal powder water absorbability increases obviously. Asiaticosides shows stability in light, heat, moisture conditions. The interaction between Asiaticosides extract and some accessories to be adopted,by TLC, and the results show that no changes occurred in Asiaticosides combined with HPMC, EC, carbomer 934P, lactose, stearic acid. In artificial gastric juice and intestinal fluid, Kabo (carbomer) could provide the controlled release of drug effect, in a lower density than other ingredient, as a result, Carbomer 934P was selected as the main accessories.4. Based on the references studying, both the appropriate suitable pill preparative technology and the best prescription were designed, by inspecting each factors related to pill formation craft, combined with the results of the stomach mucous membrane surface experiment in vivo and release test in vitro. The prescription determination of the micro pill is as follows:Asiaticosides(50% of the total amount), MCC, (25.5%, of the total amount), carbomer 934P(13% of the total amount), stearic acid (6.5% of the total amount), ethyl cellulose(5% of the total amount),, wetting agent are the water。The cat’s foot total glucoside 50%, MCC25.5%, Kabo 934P13%, stearic acid 6.5, ethyl cellulose 5%,.This Asiaticoside extended-release pellets show better pill roundness, higher yield,and the extended-release satisfied the anticipated demand, under selected pill preparative technology total glucoside releases slowly The preparation craft of Asiaticosides sustained-release pellets are as follows:Asiaticosides extracts and accessories were filtrated by 80 mesh filter, and mixed with 13%(w) Carbopol 934P and stearic acid 6.5%(w) at a high-speed stiring, refrigeraeted 2h immediately after melted in a constant temperature water bath at 60℃, then were put in a dryer to room temperature, crushed and the grinding through 80 mesh sieve at last; More Asiaticosides mixed with MCC, (25.5%, of the total amount), carbomer 934P(13% of the total amount), stearic acid (6.5% of the total amount), ethyl cellulose(5% of the total amount), and water(the wetting agent),were prepared the micro pellets through the Extrusion spheronization system, the prepared micro pellets first put in a desiccator drying 2h,at the 40℃, then heated 80℃for 2h, the purpose micro pellets were Picked out form a 20～40 mesh sieve.5. The purpose micro pellets were coated with the Release coating fluid:Killcoat SR30D, by using the multifunctional Coating Machine Granulator. Prescription and process factors were investigated, and the Prescription of Release coating fluid were optimizated with the method of central composite design-response surface. The final coating process of Asiaticosides sustained-release pellets are:the polymer contains 43.86% Kollicoat SR30D, and the coating weight add by 20%.. Prescription are:43.86% Kollicoat SR30D, 2% propylene glycol,0.5% lactose,46.14%water,0.5% titanium dioxide,2% talc,5% water. Parameters of:the coating conditions:60℃inlet air temperature,0.08Mpa spray pressure,0.08Mpa protective air pressure,0.5-0.8ml/min constant pump flow rate,10% added coating weight, and the coating curing for curing 12h at the temperature of 40℃.6. Pharmacodynamic studies Confirmed that:To some extent Asiaticosides could scavenge oxygen free radicals, improve antioxidant capacity, stimulate the growth of granulation tissue mucosa, and promote inflammation damages and ulcer, all of these prove that the anti-gastric ulcer effects of Asiaticosides.The Animal experiments showed that compared with modle group, the ulcer indexies of rabeprazole sodium acetic acid and the asiaticosides high and middle dose group were significantly lower (P<0.05), the ulcer indexies of asiaticosides high and middle groups were significantly lower than Hericium group (P<0.05) and was not significant different from rabeprazole group(P> 0.05), all of these result reveals Asiaticosides show the anti-gastric ulcer effects in a dose-dependent manner, and could be used to treat the experimental ulcer in rats induced by acetic acid. SOD activity and MDA content of asiaticosides high and middle dose was stronger than rabeprazole sodium in Gastric tissue from Acetic acid induced gastric ulcer animals. Asiaticosides high dose histological examination showed that the inflammatory cell infiltration mainly infiltrated in mucous layer, a small amount of fibrous scar tissue and inflammatory granulation tissue around the mucosal were in visible.更多还原