复元镇痛汤组分鉴定方法研究

【作者】 刘春；

【导师】 张贵君；

【作者基本信息】 北京中医药大学 ， 中药学， 2006， 硕士

【摘要】 复元镇痛汤系统研究是教育部重点课题。本文对复元镇痛汤镇痛组分的鉴定方法进行研究,主要包括镇痛组分来源鉴定,植物药组配伍关系研究,标准饮片模糊定量法探索三部分内容。为复元镇痛汤的开发奠定了基础,并在中药饮片及中药复方质量标准的制定方面进行了有益的探索。1.镇痛组分来源鉴定目的:确定复元镇痛汤中镇痛组分来源。方法:将全方分为益气补血、活血化瘀、通络止痛等6个功效组,以功效组为因素,以醋酸扭体法的平均扭体次数为统计指标,选择L16(215)正交试验设计表,进行拆方实验。结果:C的P<0.01,B、D及C×D的P<0.05,A和B×C的P<0.1,说明C对扭体次数有极显著影响,B、D及C×D有显著影响,A和B×C有较显著影响,筛选结果为:A1B1C1D1E2F2。结论:组合镇痛处方为黄芪、当归、桃仁、红花、丙、丁、戊、己8味药物,镇痛组分即来自于新处方。2.植物药组配伍关系研究目的:考察植物药组中药物在多糖、皂苷、黄芪甲苷含量方面的相互影响。方法:采用22和23析因试验设计,以苯酚-浓硫酸显色法、0.5%香草醛冰醋酸溶液-高氯酸显色法和高效液相色谱(HPLC)法为含量测定方法,研究植物药组中,当归、桃仁、红花对主药黄芪的影响。结果:3药可使多糖含量显著增加,黄芪甲苷含量显著降低,而对皂苷含量影响不明显。结论:植物药组水提溶液中,当归等3药对黄芪的多糖、黄芪甲苷含量有显著影响。3.标准饮片模糊定量法探索目的:将黄芪作为整体,探索建立标准饮片模糊定量法测定黄芪饮片和植物药组中黄芪相对标准饮片的量的可行性。方法:采用HPLC法,色谱条件为,色谱柱:依利特4.6mm×250mm,5μm,HypersilODS2,流动相:水(A)-乙腈(B)梯度,检测波长:254nm,柱温:室温(18～22℃),流速:1.0mL/min,以对羟基苯甲酸乙酯为内标物,对A、B、C共3批黄芪饮片及植物药组样品溶液绘制特征色谱图,选定1、2、3、4共4个共有特征峰,考察3批饮片及植物药组共有特征峰比例关系;以黄芪饮片B为标准饮片,采用内标对比法测定A、C饮片和3批植物药组中黄芪相对标准饮片的量。结果:饮片A、C相对标准饮片的量分别为100.7%±1.15%,79.34%±0.21%;植物药组A、B、C相对标准饮片的量分别为26.84%±0.22%,21.34%±0.10%和20.94%±0.32%,稳定性实验表明48h内样品稳定性良好。结论:在确定的HPLC实验条件下,选择黄芪未知特征成分进行相对量测定的方法基本可行。创新点:本文首次对复元镇痛汤用拆方实验的方法确定镇痛组分来源;首次以析因实验设计研究复元镇痛汤植物药组的配伍关系;首次探索建立标准饮片模糊定量法,定义为:在一定实验条件下,以未知特征组分标示供试样品相对标准饮片的量的方法。更多还原

【Abstract】 Decoction for Recovery and Relieving Pain(DRRP) is a key project of Ministry of Education to do the research on the DRRP. The article is about research on the identification methods of the paregoric constituent of DRRP, divided into three parts: identification of the source of the paregoric composition; study on the compatible relationship of the herb groups; exploration of the fuzziness quantitative method of the herb slices. The project established the basis of the development of DRRP and explored beneficially the method to institute the quality criteria of the herb slices and compound traditional Chinese medicine.1. Identification of the source of the paregoric composition. Objective: ascertain the source of the paregoric constituent of DRRP.Methods: divide the whole prescription into six effectiveness groups: replenishing qi and enriching the blood, promoting blood circulation by removing stasis, dredging collaterals to relieve pain and so on. Do the prescription-breaking experiment according to the L16(215) orthogonal experiment design table, with the average writhing frequency of the acetic acid writhing experiment as statistical index. Results: P(C)<0.01, P(B,D and C×D)<0.05, P(A and B×C)<0.1. The results show that the factors of B, C, D and C×D has conspicuous effective on the writhing frequency and the factors of A and B×C has relatively conspicuous effective on that.Conclusions: The result of screening is A1B1C1D1E2F2 and the paregoric prescription is the combination of eight Chinese medicine Radix Astragali, Radix Angelica sinensis, Senmen pruni persicae, Flos Carthami and another four chinese medicine(need to be a secret) and ascertain the source of the paregoric constituent.2. Study on the compatible relationship of the herb group.Objective: Investigate the interaction of the contents (mainly polyose, saponin and astragalosideⅣ) of the Chinese medicine in the herb group.Methods: According to 22 and 23 factorial experiment design, phenol-sulphuric acid method, vanillin-perchloric acid method and HPLC were used to study the effects of Radix Angelica sinensis, Senmen pruni persicae, Flos Carthami to Radix Astragali.Results: Radix Angelica sinensis and two other Chinese medicine could make the content of polyose higer,the content of astragalosideⅣlower;but could not influence the content of. Saponin in the extract. Conclusions: The contents of polyose and astragalosideⅣof Radix Astragali were significantly influenced by the compatibility of the three Chinese medicine in the water extract in the herb group..3. Exploration of the fuzziness quantitative method of the herb slices. Objective: Radix Astragali as a whole, explore the feasibility of the fuzziness quantitative method of the standard herb slices established to determine the relative quantity of it in the herb groups. Methods: HPLC was used for determination. The chromatographic condition: HypersilODS2(4.6mm×250mm,5μm) as Analytica Column; H2O-ACN as mobile phase gradient eluting; wave length for determination 254nm; room temperature(18～22℃) as the column temperature; flow rate 1.0mL/min; aethylparabenum as the internal standard. Draw the HPLC characteristic chromatogram of three batches of Radix Astragali (A,B,C) and the herb groups, select four common characteristic peaks (1,2,3,4) and contrast the proportion relationship the common characteristic peaks of three batches of the herb slices and the herb group. Determine the relative quantity of the Radix Astragali herb slice A, C and the Radix Astragali of three batches of herb group compared with B as a standard by mean of internal standard contrast method.Results: The relative quantity of herb slice A,C were 100.7%±1.15%, 79.34%±0.21%; The relative quantity of Radix Astragali in the herb group A,B,Cwere 26.84%±0.22% , 21.34%±0.10% and 20.94%±0.32%. The stability test showed that the sample had favorable stability in 48h. Conclusions: The method is basically feasible to determine the relative quantity after selecting the unknown characteristic components of Radix Astragali under the definite HPLC experimental condition.更多还原