水红花子药效学及毒性实验研究

【作者】 程飞；

【导师】 翟延君；

【作者基本信息】 辽宁中医药大学 ， 中药鉴定， 2011， 硕士

【摘要】 目的:本论文是国家自然基金项目“水红花子炮制前后功用的改变与物质基础变化相关性研究(项目编号:30873437)”课题的部分内容。水红花子自1977年版药典开始收载,具有“散血消癥,消积止痛”的功效,临床生用或炒至爆花使用,有文献报道水红花子具有抗肿瘤的作用,对原发性肝癌有较好疗效。本文针对水红花子“散血消癥、消积止痛”功效,对其生品与制品不同溶媒提取物及单体化合物进行了体外抗肿瘤作用、S-180腹水瘤小鼠生命延长作用、S-180荷瘤小鼠的抑制作用、花旗松素对人宫颈癌Hela荷瘤裸鼠的抑制作用及消积止痛作用的药效学实验,并进行了抗肿瘤作用机理、抗氧化作用及急性毒性实验研究。通过本研究探讨生品与制品药理作用的相关性,以及传统中医药理论的科学性,为临床合理用药提供科学依据;通过探索其抗氧化活性成分的有效部位、抗肿瘤作用机理及急性毒性,为水红花子的药效学研究及新药开发提供科学参考。方法:1.生制品不同溶媒提取物及单体化合物体外抗肿瘤作用药效筛选:采用结晶紫法,检测水红花子生、制品不同溶媒提取物及单体化合物对人宫颈癌HeLa细胞、人胃癌MGC细胞、人肝癌细胞HepG-2细胞和人盲肠癌Hce-8693细胞的增殖抑制作用,计算抑制率。2.生制品不同溶媒提取物及单体化合物对S-180腹水瘤小鼠生命延长作用:建立S-180肉瘤腹水型小鼠模型,观察水红花子生、制品不同溶媒提取物及单体化合物对S-180腹水瘤小鼠的生命延长作用,计算生命延长率。3.生制品不同溶媒提取物及单体化合物对S-180荷瘤小鼠的抑制作用:建立S-180肉瘤实体瘤小鼠模型,观察水红花子生、制品不同溶媒提取物及单体化合物对S180荷瘤小鼠的抑制作用,计算抑瘤率。4.花旗松素对人宫颈癌Hela荷瘤裸鼠的抑制作用:建立人宫颈癌Hela荷瘤裸鼠模型,检测花旗松素对人宫颈癌Hela荷瘤裸鼠的抑制作用,计算瘤体积及抑瘤率。5.花旗松素抗肿瘤作用机理研究:通过倒置显微镜及电镜观察花旗松素对人宫颈癌HeLa细胞形态变化及DNA片段的影响;采用RT-PCR法,检测花旗松素对人宫颈癌HeLa细胞Bcl-2 mRNA、Bax mRNA及P53 mRNA、P21 mRNA基因表达的影响。6.“消积”功效药效学实验研究:采用家兔离体肠管法,检测水红花子生、制品不同溶媒提取物对家兔离体肠管张力的影响;采用小鼠小肠炭末推进法,检测水红花子生、制品不同溶媒提取物对小鼠胃肠功能的影响,计算炭末推进率。7.“止痛”功效药效学实验研究:采用热板法,检测水红花子生、制品不同溶媒提取物对热刺激所致小鼠疼痛的影响,痛阈提高百分率;采用扭体法,检测水红花子生、制品不同溶媒提取物对冰醋酸所致小鼠扭体次数的影响,计算扭体抑制百分率。8.水红花子抗氧化作用初步实验:采用DPPH法,测定水红花子生、制品不同溶媒提取物的抗氧化活性。9.水红花子急性毒性实验:按《新药审批办法》的有关规定,测定LD50及进行最大给药量实验,检测水红花子生、制品不同溶媒提取物及单体化合物的急性毒性。结果:1.生制品不同溶媒提取物及单体化合物体外抗肿瘤作用药效筛选:水红花子生、制品不同溶媒提取部分对Hela细胞生长的抑制呈现浓度和时间的依赖性,且生品比制品的抑制作用强,其中生品乙酸乙酯部分的抑制作用最强;水红花子生、制品不同溶媒提取部分,除生品水溶部分外,对MGC细胞生长的抑制呈现浓度和时间的依赖性,其中生品乙酸乙酯部分的抑制作用最强;水红花子生、制品不同溶媒提取部分,对HepG-2细胞生长的抑制呈现浓度和时间的依赖性,其中生品乙酸乙酯部分的抑制作用最强;水红花子生、制品不同溶媒提取部分,对Hce-8693细胞生长的抑制呈现浓度和时间的依赖性,其中生品乙酸乙酯部分的抑制作用最强;3,3′-二甲氧基鞣花酸-4-O-β-D-吡喃葡萄糖苷和花旗松素在1~500μg/ml浓度范围内对人宫颈癌HeLa细胞、人胃癌MGC细胞、人肝癌细胞HepG-2细胞和人盲肠癌Hce-8693细胞有增殖抑制作用,随着剂量的增大和作用时间的延长,呈现较好的剂量-时间-效应关系,同等条件下花旗松素比3,3′-二甲氧基鞣花酸-4-O-β-D-吡喃葡萄糖苷的抑制作用强。2.生制品不同溶媒提取物及单体化合物对S-180腹水瘤小鼠生命延长作用:生品水提组、生品乙酸乙酯组、槲皮素组、花旗松素组对小鼠均有较好的生命延长作用,其强弱顺序为:花旗松素>槲皮素>生品乙酸乙酯提取物>生品水提物>其他3.生制品不同溶媒提取物及单体化合物对S-180荷瘤小鼠的抑制作用:生品乙酸乙酯组,槲皮素组,花旗松素组对S-180小鼠有一定的抑制作用,其强弱顺序为:花旗松素>生品乙酸乙酯提取物>槲皮素>生品水提物>其他4.花旗松素对人宫颈癌Hela荷瘤裸鼠的抑制作用:花旗松素在给药8d开始,裸鼠肿瘤体积开始小于生理盐水组,花旗松素组肿瘤质量明显低于生理盐水组,抑瘤率为45.45%,大于体内抑瘤试验实体瘤疗效判定标准的30%,表明花旗松素对人宫颈癌Hela移植瘤有一定的抑制作用。5.花旗松素抗肿瘤作用机理研究:花旗松素对人宫颈癌Hela细胞处理12h后,细胞变圆并逐渐从培养瓶壁上脱落下来,诱导24h后细胞膜发泡并呈现细胞核迸裂,大部分细胞逐渐形成凋亡小体;花旗松素能够诱导HeLa细胞产生200 bp倍数的DNA片段,进一步说明花旗松素诱导HeLa细胞死亡与细胞凋亡相关;花旗松素对mRNA和Bax mRNA的表达均没有变化,但能够诱导P53 mRNA和P21 mRNA的表达增加。6.“消积”功效药效学实验研究:水红花子各提取物均对家兔离体肠管张力产生了不同程度的影响,其中水提生、制品均能兴奋家兔离体肠管平滑肌,使其收缩加快,但水提生品使张力变小,而水提制品作用时间持久,振幅基本一致;醇提生品作用效果与水提生品相当,醇提制品作用时间极短,加大剂量时呈现抑制作用;水红花子各提取物对小鼠胃肠功能均有不同程度的促进作用,制品提取物比生品提取物作用强,具体作用强弱顺序为:制品醇提物>制品水提物>生品水提物>生品醇提物。7.“止痛”功效药效学实验研究:热板法实验中,生品与制品水提物均能提高小鼠痛阈值,而醇提物止痛效果不明显,其止痛效果强弱顺序为:制品水提物>生品水提物>生品与制品醇提物;扭体法实验中,制品水提物、生品与制品醇提物均能减少小鼠扭体次数,产生不同程度的止痛效果,生品水提物使小鼠扭体次数增大,止痛效果不明显,各提取物止痛效果强弱顺序为:制品水提物>生品醇提物>制品醇提物>生品水提物。8.水红花子抗氧化作用初步实验:水红花子生品与制品不同溶媒提取物均有不同程度的清除自由基作用,其石油醚部分(生、制),乙酸乙酯部分(生、制),正丁醇部分(生、制),水溶部分(生、制)EC50值分别为189.3、196.4;134.3、155.6;189.2、194.2;161.6、195.4mg/L。9.水红花子急性毒性实验:给药后小鼠表现出活动减少,神情倦怠现象,给药1h后此现象逐渐消失,无异常跳跃行为,无刺激敏感征象,口、眼、鼻未出现异常分泌物,饮食、饮水、排便均正常,呼吸活动正常未见死亡,未能测出LD50值;最大给药量实验中,连续观察7d内发现,给药后小鼠活动减少,神情倦怠,给药3h后逐渐消失,全部小鼠健存,动物的食欲、外观、行为活动、排泄等均为见异常,第7天体重平均增长27.61%,处死后解剖发现组织器官未见异常。结论:1.生制品不同溶媒提取物及单体化合物体外抗肿瘤作用药效筛选:水红花子生品乙酸乙酯提取物对4种肿瘤细胞均有较好的抑制作用,为水红花子抗肿瘤的有效抑制物质组;3,3′-二甲氧基鞣花酸-4-O-β-D-吡喃葡萄糖苷和花旗松素均能抑制4种肿瘤细胞的生长,同等条件下花旗松素抑制作用较强,可能是水红花子中主要的抗肿瘤有效物质成分。2.生制品不同溶媒提取物及单体化合物对S-180腹水瘤小鼠生命延长作用:花旗松素、槲皮素、生品水提物及生品乙酸乙酯提取物对小鼠均有较好的生命延长作用。3.生制品不同溶媒提取物及单体化合物对S-180荷瘤小鼠的抑制作用:花旗松素、槲皮素及生品乙酸乙酯提取物对S-180小鼠有一定的抑制作用。4.花旗松素对人宫颈癌Hela荷瘤裸鼠的抑制作用:花旗松素对人宫颈癌Hela移植瘤有一定的抑制作用。5.花旗松素抗肿瘤作用机理研究:花旗松素能诱导HeLa细胞形成凋亡小体,200 bp倍数的DNA片段,说明花旗松素诱导HeLa细胞死亡与细胞凋亡相关;花旗松素诱导HeLa细胞细胞凋亡,可能与通过诱导P53 mRNA和P21 mRNA的表达增加有关。6.“消积”功效药效学实验研究:各提取物均能兴奋家兔离体肠管平滑肌,制品水提物作用效果强于其他各组;并对对小鼠胃肠功能均有不同程度的促进作用,制品醇提物作用效果强于其他各组。结果显示“消积”作用制品优于生品。7.“止痛”功效药效学实验研究:对热板及冰醋酸所致疼痛,生、制品提取物均有不同程度的止痛效果,制品水提物均强于其他各组,有较好的止痛效果。8.水红花子抗氧化作用初步实验:生品乙酸乙酯提取物有较好的清除自由基作用。9.水红花子急性毒性实验:本实验按照《新药审批办法》的有关规定,未能测出LD50值,说明水红花子没有急性毒性作用。更多还原

【Abstract】 Purpose:This paper is part of the National Natural Science Foundation project "Fructus Polygoni Orientalis function changes before and after processing and the material basis of change-related research (Project No: 30873437) ". Fructus Polygoni Orientalis(FPO)began accepting set on Chinese Pharmacopoeia since 1977, with "dispersing stasis blood and mass gathering in the abdomen, relieving food retention and alleviating pain" effect, clinical students to use with or fry popcorn. FPO has been reported with anti-tumor effect on the primary Liver cancer has a good effect. This paper accords to the " dispersing stasis blood and mass gathering in the abdomen, relieving food retention and alleviating pain" effect of FPO, its raw materials and processed products of different solvent extracts and single compounds in vitro anti-tumor effect, S-180 ascites tumor effect in mice life span, S -180 inhibition of tumor-bearing mice, Taxifolin factor on human cervical cancer Hela inhibition of tumor-bearing nude mice and negatively analgesic efficacy experiments, and conducted anti-tumor mechanism of antioxidant effect and acute toxicity Experimental study. Through this study, pharmacological effects of raw materials and processed products, as well as traditional Chinese medicine theory of science, to provide a scientific basis for clinical therapy; by exploring its antioxidant components of the effective parts, Mechanism of antitumor effect and acute toxicity, can provide a scientific reference Polygonum and Pharmacodynamic Study of Drug Development about FPO.Methods:1.Its raw materials and processed products with different solvent extracts and compounds were screened in vitro efficacy of anti-tumor effects: using crystal violet to detect the difference of its raw materials and processed products with different solvent extracts and purified compounds on Hela cells, MGC cells, HepG-2 cells and Hce-8693 cells proliferation, inhibition rate calculated.2.Its raw materials and processed products with different solvent extracts and compounds on S-180 ascites of mice extend the role of life: the establishment of S-180 ascites sarcoma in mice was observed as its raw materials and processed products with different solvent extracts and purified compounds on S-180 to extend the life of mice with ascites tumor effect, calculated life extension rate.3.Its raw materials and processed products with different solvent extracts and compounds on S-180 inhibition of tumor-bearing mice: the establishment of solid tumor S-180 sarcoma in mice was observed as its raw materials and processed products with different solvent extracts and purified compounds on the S-180 inhibition of tumor-bearing mice, inhibitory rate was calculated.4.Taxifolin on human cervical cancer Hela inhibition of tumor-bearing nude mice: to establish nude mouse model of cervical cancer Hela, testing human cervical carcinoma Hela nude mice inhibited tumor volume calculation and the antitumor rate.5.Taxifolin mechanism of antitumor effect of factors: by inverted microscope and electron microscope,texting the morphological changes and the impact of DNA fragments of HeLa cell with Taxifolin; by RT-PCR method to detect the influence on human Bcl-2 mRNA, Bax mRNA and P53 mRNA, P21 mRNA gene expression.6."Relieving food retention" effect of Pharmacodynamics Study: the isolated rabbit intestine method to detect the influence of its raw materials and processed products with different solvent extracts and compounds on isolated rabbit intestinal tension; mouse small intestine by promoting carbon powder used to detect its raw materials and processed products with different solvent extracts and compounds on the gastrointestinal function of mice was calculated forward rate of carbon powder.7."Alleviating pain" effect of Pharmacodynamics Study: hot plate used to detect its raw materials and processed products with different solvent extracts and compounds from mice stimulated by the thermal effects of pain, pain threshold increased percentage; by writhing, detected acetic acid induced writhing times of mice to calculate the percentage inhibition of writhing.8.Preliminary antioxidant experiment of FPO: DPPH method used to determine the antioxidant activity of its raw materials and processed products with different solvent extracts and compounds.9.Acute toxicity test of FPO: Press the part of "Approval of New Pharmaceuticals", test the determination of LD50 and the maximum amount of test administration, determine acute toxicity of its raw materials and processed products with different solvent extracts and compounds.Results:1.Its raw materials and processed products with different solvent extracts and compounds were screened in vitro efficacy of anti-tumor effects:its raw materials and processed products with different solvent extracts and compounds on the growth inhibition of Hela cells in a concentration and time dependent, and inhibition of raw materials than processed products stronger, raw materials of which the strongest inhibition is ethyl acetate; in addition to raw materials soluble part, the inhibition of cell growth on the MGC cell in a dose and time dependent, in which raw materials the strongest part of the inhibitory effect is ethyl acetate; HepG-2 cell growth inhibition in a dose and time dependent, in which raw materials the strongest inhibition is ethyl acetate; Hce-8693 cell growth inhibition in a dose and time dependent, in which raw materials the strongest inhibition is ethyl acetate; 3,3 ’- dimethoxy tanning Spent acid-4-O-β-D-glucopyranoside and taxifolin in 1 ~ 500μg/ml concentration of elements in the range of Hela cells, MGC cells, HepG-2 cells and Hce-8693 cell proliferation, as the dose increases, and the role of time, showing a good dose - time - effect relationship, the same factors under taxifolin than 3,3 ’- dimethoxy tanning Spent acid-4-O-β-D-glucopyranoside stronger inhibition.2.Its raw materials and processed products with different solvent extracts and compounds on S-180 ascites of mice extend the role of life: raw water extraction group, raw ethyl acetate group materials, quercetin and taxifolin were extend the role of mice a good life, the strength of the order: taxifolin > quercetin> raw ethyl acetate extract> raw water extract materials> Other.3.Its raw materials and processed products with different solvent extracts and compounds on S-180 inhibition of tumor-bearing mice:raw ethyl group, quercetin, taxifolin on the S-180 study group had a certain inhibition in mice, the strength of the order: taxifolin > raw ethyl acetate extract > quercetin> raw water extract materials > Other.4.Taxifolin on human cervical cancer Hela inhibition of tumor-bearing nude mice: factors in the administration of taxifolin 8d starting tumor volume in nude mice than the saline group started, the quality of taxifolin group was significantly lower than the saline group, suppression Tumor rate was 45.45%, greater than in vivo antitumor efficacy test criteria of solid tumors, 30%, indicating that the certain extent on Hela cell with taxifolin.5.Taxifolin mechanism of antitumor effect of factors: taxifolin hormone treatment of human cervical carcinoma Hela cells after 12h, cells became round and gradually detached from the culture bottle wall down, 24h after the induction of foam cell and present nuclear burst, most of the cells gradually formation of apoptotic bodies; taxifolin prime HeLa cells inducing a multiple of 200 bp DNA fragments, and further description of HeLa cells death induced by apoptosis; taxifolin on the expression of mRNA and Bax mRNA did not change, but can induce P53 mRNA and P21 mRNA increased expression.6."Relieving food retention" effect of Pharmacodynamics Study: the extract of FPO are on isolated rabbit intestinal tension produced different degrees of impact, which provide raw water, the product can excited rabbit intestinal smooth muscle, it contraction accelerated, but the tension of aqueous raw materials become smaller, and the role of long-lasting water extraction products, basically the same amplitude; ethanol effect of raw materials provide raw materials and water equivalent to the role of ethanol products, a very short time, increase the dose showing inhibition; FPO extract on the gastrointestinal promote varying degrees, products, raw materials extraction extract than the stronger, the specific role of the strength of the order: ethanol extract of processed products > water extract of processed products > water extract of raw materials> ethanol extract of raw materials.7."Alleviating pain" effect of Pharmacodynamics Study: hot plate test, the raw materials and products water extract could enhance the pain threshold in mice, while the ethanol extract of analgesic effect was not obvious, the strength of its analgesic effect of the order: water extract of processed products>water extract of raw materials>alcohol extract of raw materials and processed products; writhing test, the water extract products, raw materials and products can reduce the ethanol extract of writhing in mice, resulting in varying degrees of pain effect of water extract of raw materials to increase the number of writhing, analgesic effect was not obvious, the strength of the analgesic effect of extract of the order: water extract of processed products> ethanol extract of raw materials >ethanol extract of processed products > water extract of raw materials.8.Preliminary antioxidant experiment of FPO: raw materials and processed products of FPO extracts from different solvents have different levels of free radical scavenging effect.9.Acute toxicity test of FPO: mice after administration showed reduced activity, and looked burnout, 1h after administration of this phenomenon gradually disappeared, no abnormal jumping behavior, no stimulus-sensitive signs, mouth, eyes, nose, abnormal secretion does not appear , food, water, defecation, breath were normal, no death, could not measure the LD50 value; the maximum amount of drug experiments, the continuous observation 7d found, reduced activity in mice after administration, and looked tired, administration 3h Disappears, all the mice were kept healthy, animal appetite, appearance, behavior, excretion and so are the exception, see, the 7 day average weight increase of 27.61%, were killed after dissection found no abnormal tissues and organs.Conclusions:1.Its raw materials and processed products with different solvent extracts and compounds were screened in vitro efficacy of anti-tumor effects: ethyl acetate extract of raw materials for 4 inhibition of tumor cells are better than others, is the effective inhibition tumor Material group of FPO; 3,3’-dimethoxy ellagic acid-4-O-β-D- glucopyranoside and taxifolin can inhibit the growth of 4 tumor cells ,under the same conditions inhibited prime taxifolin is stronger, probably as FPO the main anti-tumor effective substance.2.Its raw materials and processed products with different solvent extracts and compounds on S-180 ascites of mice extend the role of life: taxifolin, quercetin, water extract of raw materials and ethyl acetate extract of raw materials were well prolongation of life.3.Its raw materials and processed products with different solvent extracts and compounds on S-180 inhibition of tumor-bearing mice: taxifolin, quercetin, water extract of raw materials and ethyl acetate extract of raw materials exhibited a potent inhibition on the proliferation of S-180 mice.4.Taxifolin on human cervical cancer Hela inhibition of tumor-bearing nude mice: taxifolin exhibited a potent inhibition on the proliferation of Hela nude mice.5.Taxifolin mechanism of antitumor effect of factors: the mechanism of cell death induced by taxifolin was related to cell apoptosis. With regard to the molecular mechanism of apoptosis induced by taxifolin, upregulation of P53 mRNA and P21 mRNA might be involved and no relation between Bcl-2 mRNA/Bax mRNA and apoptosis was observed.6."Relieving food retention" effect of Pharmacodynamics Study: the excitement can extract isolated rabbit intestinal smooth muscle, effect of water extract products is stronger than the other groups; and gastrointestinal function of mice have different levels of promotion effect, effect of ethanol extract of processed products stronger than other groups. The results showed that about "consumer product" effect processed products is stronger than raw materials.7."Alleviating pain" effect of Pharmacodynamics Study: hot plate and acetic acid induced pain, both processed products and raw materials extract varying degrees of analgesic effect, water extract of processed products is stronger than the other groups, has a better analgesic effect .8.Preliminary antioxidant experiment of FPO: raw ethyl acetate extract of a better free radical scavenging effect.9.Acute toxicity test of FPO: the test in accordance with the "approval of new pharmaceuticals", the relevant provisions of the LD50 value not detected, indicating that FPO has no acute toxic effects.更多还原