基于地榆鞣质与皂苷治疗白细胞减少症相恶关系的欣生胶囊成药性研究
摘要
目的寻找地榆升白物质基础,以地榆鞣质与皂苷相恶关系为理论依据,研制药效显著、物质基础清楚、作用机理明确、无明显毒副作用的现代中药制剂,为临床提供升白兼具骨髓造血功能保护双重功效的新药,提升中药国际地位与影响。
方法物质基础研究：采用现代化学分离技术制备地榆鞣质和皂苷提取物,并运用环磷酰胺致白细胞减少症模型对二者生物活性进行了研究以探明地榆升白物质基础。
地榆鞣质与皂苷相恶关系研究：建立地榆鞣质提取物的LC-MS指纹图谱,初步揭示地榆鞣质提取物主要含有的化学成分,并通过血清移行成分指认,判断单用与合用对地榆鞣质各组分入血情况的影响,从而揭示地榆鞣质与皂苷相恶的组分入血规律。通过造血因子GM-CSF、EPO、TPO的ELISA检测,造血干细胞的流式细胞术检测,以及骨髓造血微环境的形态学评价,从影响造血功能的三个主要方面土壤、种子和化肥,系统地揭示地榆鞣质与皂苷相恶的药效学规律。
欣生胶囊的成药性研究：采用主成分分析和指纹图谱综合评价提取液质量,筛选地榆鞣质的提取方法,并采用Box-behken设计、均匀设计、混料设计等方法对制剂工艺路线以及处方进行优化,得到稳定、可控、科学、合理的制剂工艺路线以及参数；采用两种动物模型,充分评价地榆鞣质提取物对环磷酰胺和6。Co-Y射线所代表的放化疗因素所致的白细胞减少症的药效作用,观察地榆鞣质提取物对大剂量60Co-γ射线照射大鼠的食量、体重、状态、存活率的影响。
结果物质基础研究：分别制备了地榆鞣质和皂苷提取物,含量高达90%,生物活性筛选研究显示鞣质对白细胞减少症具有明显的治疗作用,揭示地榆升白的物质基础为鞣质,而皂苷无明显作用,且与鞣质合用后产生相恶。
地榆鞣质与皂苷相恶关系研究：鞣质组分入血规律研究显示大鼠经灌胃单独给予地榆鞣质提取物后,有没食子儿茶素、原花青素B2、原花青素B2-3’-没食子酸酯,还有没食子酸、鞣花酸进入了血液。然而,与皂苷合用后仅能检测到原花青素B2、原花青素B2-3’-没食子酸酯,揭示地榆鞣质与皂苷合用可导致没食子儿茶素、没食子酸、鞣花酸等成分的入血行为改变。药效学规律研究显示地榆鞣质升白作用是通过提高G2期细胞比例以及DNA含量,拮抗环磷酰胺所导致的细胞凋亡实现,与促进造血因子和保护造血微环境无关。然而,地榆鞣质与皂苷合用后,其增加G2期细胞比例以及DNA含量,拮抗环磷酰胺所导致的细胞凋亡作用明显减弱(P<0.05),表明地榆鞣质与皂苷相恶的药效学规律与此有关。
此外,地榆鞣质与皂苷的相恶是具体的、有条件的,故不能离开剂量配比而孤立地谈二者相恶,当鞣质与皂苷的剂量配比在1：1至4：1这个范围内时,鞣质与皂苷出现相恶,但二者相恶具有相对性,随着剂量配比变化而改变,当鞣质与皂苷的剂量配比高于4：1时,二者相恶减弱,并随着皂苷的剂量减少而呈减小趋势,直至8：1以上时,其相恶几乎消失,与单用鞣质无显著性差异(P>0.05),揭示皂苷的剂量为鞣质1/8以上就与鞣质相恶。地榆鞣质较rhG-CSF、鲨肝醇、升白胺、地榆升白片市售同类制剂作用更全面,在提高骨髓DNA含量方面,地榆鞣质明显优于rhG-CSF、鲨肝醇、升白胺、地榆升白片(P<0.05),在升高WBC方面,地榆鞣质不如rhG-CSF,但显著强于鲨肝醇、升白胺、地榆升白片(P<0.05)。急性毒性试验研究结果表明地榆鞣质提取物毒性较小,肿瘤刺激试验研究显示地榆鞣质提取物对肿瘤无催生作用,且有一定抑制趋势,并且地榆鞣质提取物联合环磷酰胺给药不会影响环磷酰胺的治疗作用,然而,皂苷对肿瘤治疗有一定副作用。
欣生胶囊的成药性研究：主成分分析显示闪式提取的综合主成分值为2.323084,而回流提取的综合主成分值为-2.32172,表明闪式提取比回流提取更有利于提取液整体质量的提高。Box-Benhnken优化提取工艺为：溶媒用量12倍、乙醇浓度60%、提取时间3分钟,转移率可达Y195.25%、Y296.03%,其快速(3分钟)、高效(基本转移完全)的提取效能进一步揭示地榆鞣质的闪式提取工艺的科学、合理、可行。均匀设计优化纯化工艺为：取地榆提取液,浓缩至0.5g生药／m1,置于离心机内,4000／转离心10min,取上清液,以流速2BV/h缓缓上样于D-101型大孔吸附树脂,径高比1：5,静态吸附30min后,用2BV水以2BV/h洗涤,再用1BV10%乙醇以2BV/h洗涤,再用2BV60%乙醇以1BV/h洗涤,收集洗脱液,浓缩,干燥即得。干燥工艺为：采用真空干燥箱干燥,控制温度在45℃。三批提取物中鞣质与皂苷配比约为16：1,表明优化后的工艺参数进一步提高了地榆鞣质含量,确保了鞣质与皂苷配比在8：1的相恶剂量配比范围之外。D-最优混料设计优化辅料配比为：淀粉和乳糖比例30%：60%,确定制剂处方为地榆鞣质提取物10g,淀粉30g,乳糖60g,共制成1000粒胶囊。此外,还对上述制剂工艺路线及参数进行了放大验证,并建立中间体以及制剂质量标准,初步考察了制剂稳定性。药效结果显示,与模型组比较,地榆鞣质组WBC、PLT、脾脏系数、骨髓DNA含量四个指标均显著性升高(P<0.05),说明地榆鞣质对放化疗所致的小鼠白细胞减少症有良好治疗作用。阳性药在WBC、PLT、脾脏系数方面有明显升高作用(P<0.05),但对骨髓DNA含量起到一定反作用。此外,观察地榆鞣质对大剂量60Co-γ射线照射大鼠的整体状态的影响,发现地榆鞣质高、中、低剂量组均有一定作用,且高剂量组效果较显著,能明显改善大剂量60Co-γ射线对大鼠食量、体重、状态的影响,能明显提高大鼠存活率。
结论单用地榆鞣质可提高G2期细胞比例以及DNA含量,拮抗环磷酰胺所导致的细胞凋亡,而与皂苷合用则上述作用减弱,当皂苷的剂量为鞣质1/8以上就与鞣质相恶,并且地榆鞣质与皂苷的相恶是具体的、有条件的,故不能离开剂量配比而孤立地谈二者相恶。因此,基于地榆鞣质与皂苷相恶关系,将鞣质从地榆药材中分离出来制备五类中药欣生胶囊,避免了鞣质与其所含地榆皂苷类成分的相恶以及皂苷类成分对肿瘤治疗干扰等副作用,增强了安全性,其药效显著、物质基础清楚、作用机理明确、无明显毒副作用、不刺激肿瘤生长,强力升白兼具骨髓造血功能保护,它的研制将为临床提供升白兼具骨髓造血功能保护双重功效的新药,将有利于单味药的组分配伍理论的完善,为继承与发扬配伍理论提供思路,将有利于全面认识中药鞣质类成分的生物活性,将有利于提升中药国际地位与影响。
Objective:With the relationship of mutual inhibition of tannins and saponins in medication principle as the theoretical basis,finding the material basis of Burnet in terms of rising white blood cells and developing modern medicine preparation,in these respects,significant effect,clear material basis, explicit mechanism, no obvious side effects. Provide dual function of innovative drugs in rising white blood cells and bone marrow function to enhance the international status and influence of traditional chinese medicine.
Method:Material foundation research:prepare tannins and saponins extract of Burnet by using the modern chemical separation technology and utilize the leukopenia disease model caused by cyclophosphamide to study the biological activity of Burnet tannins and saponins extract, for the purpose of verifing the material base in rising white blood cells.
The research result of the mutual inhibition of tannins and saponins in medication relationship as follows,to establish the tannins of Burnet LC-MS fingerprint, through identifing the serum from components and proclaiming the chemical composition into the blood, preliminary revealed the main chemical components of Burnet tannins and the related behaviour of chemical composition in the body and the influence of Burnet tannins used alone or in combination in blood. As a result,revealed the blood medicine dynamic rules of mutual inhibition of tannins and saponins.Through the hematopoietic factor GM-CSF, EPO, TPO detection of ELISA, and the flow cytometry detection of hematopoietic stem cells, and the morphology evaluation of bone marrow hematopoietic microenvironment, evaluating the influence of hemopoietic function mainly from three aspects of soil, the seeds and fertilizer,revealing the pharmacodynamic rules of mutual inhibition of tannins and saponins systematically.
The study of the druggability of Xinsheng capsule shows that comprehensive evaluation the extract quality by applying the principal component analysis and fingerprint comprehensive, screen extraction method of tannins of Burnet, and use the Box-behken design, uniform design, mixing design method and so on.To optimize the preparation process route and prescription and obtain a stable,controllable, scientific and reasonable preparation process route and parameters; Evaluate pharmacodynamic effects adequately of the capsule in the protection of bone marrow and rise white blood cells by using two kinds of animal models.leukopenia disease models are caused by cyclophosphamide and60Co-y rays represent chemoradiation.Observe the function of the Burnet tannins for large dose of60Co-γ rays, illuminating the rat appetite, weight, condition, the survival rate.
Consequence:Material foundation research:the preparation of as much as90%of the content of tannins and saponins extract.The pharmacological research shows the obvious curative effect about the tannins of Burnet to reduce leukopenia disease.However, the Burnet's saponins have no obvious function to leukopenia. Reveals, the material base to rise white blood cells is tannins.Furthermore,the combination of tannins and saponins give rise to mutual inhibition.
The research result of the mutual inhibition of tannins and saponins in medication relationship showed,in terms of tannins components into the blood rules,gavage Burnet tannins extract alone into rat's stomach,gallic catechin, procyanidins B2, procyanidins B2-3'-the gallic acid ester, as well as gallic acid, ellagic acid into the blood. However, only can be detected procyanidins B2, procyanidins B2-3'the gallic acid ester after the combination with saponins.The result revealed Burnet tannins and saponins in combination can lead to the change of behavior of gallic catechin, gallic acid and other ingredients into blood.The studies of pharmacodynamics rules show that Burnet tannins rise white cells by improving the G2period, DNA content, and antagonising cyclophosphamide in the cell apoptosis,and having no reference to promote hematopoiesis factor and protect hematopoietic microenvironment. However, the combination of Burnet tannins and saponins, the effect of increasing the G2period cells proportion, DNA content, antagonist cyclophosphamide in the cell apoptosis phase decreased significantly (P<0.05), shows that the pharmacodynamics rules of mutual inhibition of Burnet tannins and saponins have reference to above results.
In addition,the mutual inhibition of tannins and saponins is specific and conditional,so it can't leave dose ratio and discuss the mutual inhibition of tannins and saponins in isolation.when the range of tannins and saponins dose ratio is1:1to4:1,the mutual inhibition of tannins and saponins will appear. But the mutual inhibition relationship has relativity,it will vary as the dose ratio change.When tannins and saponins dose ratio higher than4:1,the mutual inhibition relationship will weaken and with the dose of saponins reducing,a decreasing trend will appear until above8:1,the mutual inhibition relationship almost disappear,which has no significant difference from tannins used alone (P>0.05).It reveals that saponins and tannins exist the mutual inhibition relationship when the saponins dose is more than1/8dose of tannins. Burnet tannins have more comprehensive function than rhG-CSF,shark liver alcohol,rise white amines,Burnet leukopoietic tablets commercially available similar preparations.with respect to improve, the DNA content in the bone marrow, Burnet tannins are better than rhG-CSF, batifol, rise white amines, Burnet leukopoietic tablets(P<0.05); with respect to elevate WBC,Burnet tannins are better than shark liver alcohol, rise white amines, Burnet leukopoietic tablets(P<0.05),but worse than rhG-CSF. Acute toxicity test results show that tannins have lower toxicity. Antitumor experimental study shows that tannins have no role of promoting tumor growth,and have a certain trend in inhibitory action, and the combination of drug delivery system about the Burnet tannins and cyclophosphamide will not affect the treatment function of cyclophosphamide. However, in a certain extent,there are side effects in cancer therapy to saponins.
The study of the druggability of Xinsheng capsule shows that flash type extraction is more conducive to improve the overall quality of extraction than reflux extractionThe result of principal component analysis is that the comprehensive component of extraction value is2.323084, and the comprehensive component of reflux extraction value is-2.32172.The optimization of Box-Benhnken extraction process as follows:solvent consumption12times,60%of ethanol concentration extraction time3minutes, transfer rate can amount to Y195.25%, Y296.03%, its fast (3minutes), efficient (basic transfer completely) extraction efficiency further reveal the flash type extraction technology of scientific, reasonable and feasible to tannins.Uniform design to optimize the purification process as follows:the Burnet extraction was concentrated to0.5g crude drug/ml,placed in a centrifuge,4000/transferred to centrifuge10min,the supernatant at a flow rate2BV/h slowly on the kind of D-101macroporous resin, diameter to height ratio is1:5, after static adsorption30min, at a rate of2BV/h washed with2BV water, and then at a rate of2BV/h washed with1BV10%ethanol,and then at a rate of1BV/h washed with2BV60%ethanol,collect the eluent,concentration, drying.The drying process:use the vacuum oven drying and control the temperature at45℃. tannins and saponins ratio is about16:1in three batches of extracts,the result shows that the optimization of process parameters further improve the Burnet tannins content,and ensures that the mutual inhibition relationship between tannins and saponins dose is beyond 1/8.D-optimal mixture design to optimize the auxiliary material ratio:30%of starch and60%of lactose. Making sure the formula was Burnet tannins extraction10g, starch30g and were made into1000capsules. In addition, the process routes and parameters of the above formulations were enlarge verified, and established the intermediates and formulations quality standards and initial observed the stability of preparation.Pharmacodynamic research results show that the four indexes of WBC, PLT, spleen and bone marrow coefficient DNA content, the Burnet tannins groups increase more significantly than model groups(P<0.05).So the Burnet tannins have a good treatment function to leukopenia disease in mice in radiation and chemotherapy.Positive medicines have an obvious function in the WBC, PLT, spleen (P<0.05),but play a counteraction function for the content of bone marrow DNA.High, medium and low dose groups of extraction from Burnet have a certain improvement to the mice by large dose the illuminate of60Co-γ rays in the overall state, and high dose groups effect is significant, and can improve the high-dose60Co-y rays to the influence of rat appetite, weight, state, and can obviously improve the survival rate of the rats.
Conclusion:Using the Burnet tannins alone can improve the G2period cells proportion and DNA content,antagonist cyclophosphamide induced apoptosis,but these functions are reduced when tannins and saponins are used together.saponins and tannins exist the mutual inhibition relationship when the saponins dose is more than1/8dose of tannins as well as the mutual inhibition of tannins and saponins is specific and conditional, so it can't leave dose ratio and discuss the mutual inhibition of tannins and saponins in isolatioaSo,according to the mutual inhibition of tannins and saponins in medication relationship,extract the Burnet tannins and prepare five kinds of traditional Chinese medicine of Xinsheng capsule in the protection of bone marrow and rise white blood cells, avoid the opposite effect of tannins and its contain ingredients like saponins and the side effects of saponins ingredients in cancer treatment.Achieve these aims:enhance security, efficacy significant,clear physical foundation, clear mechanism, no obvious side effects, not stimulate tumor growth, rise white blood cells stronger and hematopoiesis function protectionIn clinic, it will provide for double functions in rising white blood cells and medullary hematopoiesis function to the development of new drugs, which will improve the theory of single herb component compatibility,and provide ideas to inherit and carry forward the theory of compatibility,and benefit a comprehensive understanding of the biological activity of traditional chinese medicine tannins ingredients, and promote the international status and influence of traditional chinese medicine.
方法物质基础研究：采用现代化学分离技术制备地榆鞣质和皂苷提取物,并运用环磷酰胺致白细胞减少症模型对二者生物活性进行了研究以探明地榆升白物质基础。
地榆鞣质与皂苷相恶关系研究：建立地榆鞣质提取物的LC-MS指纹图谱,初步揭示地榆鞣质提取物主要含有的化学成分,并通过血清移行成分指认,判断单用与合用对地榆鞣质各组分入血情况的影响,从而揭示地榆鞣质与皂苷相恶的组分入血规律。通过造血因子GM-CSF、EPO、TPO的ELISA检测,造血干细胞的流式细胞术检测,以及骨髓造血微环境的形态学评价,从影响造血功能的三个主要方面土壤、种子和化肥,系统地揭示地榆鞣质与皂苷相恶的药效学规律。
欣生胶囊的成药性研究：采用主成分分析和指纹图谱综合评价提取液质量,筛选地榆鞣质的提取方法,并采用Box-behken设计、均匀设计、混料设计等方法对制剂工艺路线以及处方进行优化,得到稳定、可控、科学、合理的制剂工艺路线以及参数；采用两种动物模型,充分评价地榆鞣质提取物对环磷酰胺和6。Co-Y射线所代表的放化疗因素所致的白细胞减少症的药效作用,观察地榆鞣质提取物对大剂量60Co-γ射线照射大鼠的食量、体重、状态、存活率的影响。
结果物质基础研究：分别制备了地榆鞣质和皂苷提取物,含量高达90%,生物活性筛选研究显示鞣质对白细胞减少症具有明显的治疗作用,揭示地榆升白的物质基础为鞣质,而皂苷无明显作用,且与鞣质合用后产生相恶。
地榆鞣质与皂苷相恶关系研究：鞣质组分入血规律研究显示大鼠经灌胃单独给予地榆鞣质提取物后,有没食子儿茶素、原花青素B2、原花青素B2-3’-没食子酸酯,还有没食子酸、鞣花酸进入了血液。然而,与皂苷合用后仅能检测到原花青素B2、原花青素B2-3’-没食子酸酯,揭示地榆鞣质与皂苷合用可导致没食子儿茶素、没食子酸、鞣花酸等成分的入血行为改变。药效学规律研究显示地榆鞣质升白作用是通过提高G2期细胞比例以及DNA含量,拮抗环磷酰胺所导致的细胞凋亡实现,与促进造血因子和保护造血微环境无关。然而,地榆鞣质与皂苷合用后,其增加G2期细胞比例以及DNA含量,拮抗环磷酰胺所导致的细胞凋亡作用明显减弱(P<0.05),表明地榆鞣质与皂苷相恶的药效学规律与此有关。
此外,地榆鞣质与皂苷的相恶是具体的、有条件的,故不能离开剂量配比而孤立地谈二者相恶,当鞣质与皂苷的剂量配比在1：1至4：1这个范围内时,鞣质与皂苷出现相恶,但二者相恶具有相对性,随着剂量配比变化而改变,当鞣质与皂苷的剂量配比高于4：1时,二者相恶减弱,并随着皂苷的剂量减少而呈减小趋势,直至8：1以上时,其相恶几乎消失,与单用鞣质无显著性差异(P>0.05),揭示皂苷的剂量为鞣质1/8以上就与鞣质相恶。地榆鞣质较rhG-CSF、鲨肝醇、升白胺、地榆升白片市售同类制剂作用更全面,在提高骨髓DNA含量方面,地榆鞣质明显优于rhG-CSF、鲨肝醇、升白胺、地榆升白片(P<0.05),在升高WBC方面,地榆鞣质不如rhG-CSF,但显著强于鲨肝醇、升白胺、地榆升白片(P<0.05)。急性毒性试验研究结果表明地榆鞣质提取物毒性较小,肿瘤刺激试验研究显示地榆鞣质提取物对肿瘤无催生作用,且有一定抑制趋势,并且地榆鞣质提取物联合环磷酰胺给药不会影响环磷酰胺的治疗作用,然而,皂苷对肿瘤治疗有一定副作用。
欣生胶囊的成药性研究：主成分分析显示闪式提取的综合主成分值为2.323084,而回流提取的综合主成分值为-2.32172,表明闪式提取比回流提取更有利于提取液整体质量的提高。Box-Benhnken优化提取工艺为：溶媒用量12倍、乙醇浓度60%、提取时间3分钟,转移率可达Y195.25%、Y296.03%,其快速(3分钟)、高效(基本转移完全)的提取效能进一步揭示地榆鞣质的闪式提取工艺的科学、合理、可行。均匀设计优化纯化工艺为：取地榆提取液,浓缩至0.5g生药／m1,置于离心机内,4000／转离心10min,取上清液,以流速2BV/h缓缓上样于D-101型大孔吸附树脂,径高比1：5,静态吸附30min后,用2BV水以2BV/h洗涤,再用1BV10%乙醇以2BV/h洗涤,再用2BV60%乙醇以1BV/h洗涤,收集洗脱液,浓缩,干燥即得。干燥工艺为：采用真空干燥箱干燥,控制温度在45℃。三批提取物中鞣质与皂苷配比约为16：1,表明优化后的工艺参数进一步提高了地榆鞣质含量,确保了鞣质与皂苷配比在8：1的相恶剂量配比范围之外。D-最优混料设计优化辅料配比为：淀粉和乳糖比例30%：60%,确定制剂处方为地榆鞣质提取物10g,淀粉30g,乳糖60g,共制成1000粒胶囊。此外,还对上述制剂工艺路线及参数进行了放大验证,并建立中间体以及制剂质量标准,初步考察了制剂稳定性。药效结果显示,与模型组比较,地榆鞣质组WBC、PLT、脾脏系数、骨髓DNA含量四个指标均显著性升高(P<0.05),说明地榆鞣质对放化疗所致的小鼠白细胞减少症有良好治疗作用。阳性药在WBC、PLT、脾脏系数方面有明显升高作用(P<0.05),但对骨髓DNA含量起到一定反作用。此外,观察地榆鞣质对大剂量60Co-γ射线照射大鼠的整体状态的影响,发现地榆鞣质高、中、低剂量组均有一定作用,且高剂量组效果较显著,能明显改善大剂量60Co-γ射线对大鼠食量、体重、状态的影响,能明显提高大鼠存活率。
结论单用地榆鞣质可提高G2期细胞比例以及DNA含量,拮抗环磷酰胺所导致的细胞凋亡,而与皂苷合用则上述作用减弱,当皂苷的剂量为鞣质1/8以上就与鞣质相恶,并且地榆鞣质与皂苷的相恶是具体的、有条件的,故不能离开剂量配比而孤立地谈二者相恶。因此,基于地榆鞣质与皂苷相恶关系,将鞣质从地榆药材中分离出来制备五类中药欣生胶囊,避免了鞣质与其所含地榆皂苷类成分的相恶以及皂苷类成分对肿瘤治疗干扰等副作用,增强了安全性,其药效显著、物质基础清楚、作用机理明确、无明显毒副作用、不刺激肿瘤生长,强力升白兼具骨髓造血功能保护,它的研制将为临床提供升白兼具骨髓造血功能保护双重功效的新药,将有利于单味药的组分配伍理论的完善,为继承与发扬配伍理论提供思路,将有利于全面认识中药鞣质类成分的生物活性,将有利于提升中药国际地位与影响。
Objective:With the relationship of mutual inhibition of tannins and saponins in medication principle as the theoretical basis,finding the material basis of Burnet in terms of rising white blood cells and developing modern medicine preparation,in these respects,significant effect,clear material basis, explicit mechanism, no obvious side effects. Provide dual function of innovative drugs in rising white blood cells and bone marrow function to enhance the international status and influence of traditional chinese medicine.
Method:Material foundation research:prepare tannins and saponins extract of Burnet by using the modern chemical separation technology and utilize the leukopenia disease model caused by cyclophosphamide to study the biological activity of Burnet tannins and saponins extract, for the purpose of verifing the material base in rising white blood cells.
The research result of the mutual inhibition of tannins and saponins in medication relationship as follows,to establish the tannins of Burnet LC-MS fingerprint, through identifing the serum from components and proclaiming the chemical composition into the blood, preliminary revealed the main chemical components of Burnet tannins and the related behaviour of chemical composition in the body and the influence of Burnet tannins used alone or in combination in blood. As a result,revealed the blood medicine dynamic rules of mutual inhibition of tannins and saponins.Through the hematopoietic factor GM-CSF, EPO, TPO detection of ELISA, and the flow cytometry detection of hematopoietic stem cells, and the morphology evaluation of bone marrow hematopoietic microenvironment, evaluating the influence of hemopoietic function mainly from three aspects of soil, the seeds and fertilizer,revealing the pharmacodynamic rules of mutual inhibition of tannins and saponins systematically.
The study of the druggability of Xinsheng capsule shows that comprehensive evaluation the extract quality by applying the principal component analysis and fingerprint comprehensive, screen extraction method of tannins of Burnet, and use the Box-behken design, uniform design, mixing design method and so on.To optimize the preparation process route and prescription and obtain a stable,controllable, scientific and reasonable preparation process route and parameters; Evaluate pharmacodynamic effects adequately of the capsule in the protection of bone marrow and rise white blood cells by using two kinds of animal models.leukopenia disease models are caused by cyclophosphamide and60Co-y rays represent chemoradiation.Observe the function of the Burnet tannins for large dose of60Co-γ rays, illuminating the rat appetite, weight, condition, the survival rate.
Consequence:Material foundation research:the preparation of as much as90%of the content of tannins and saponins extract.The pharmacological research shows the obvious curative effect about the tannins of Burnet to reduce leukopenia disease.However, the Burnet's saponins have no obvious function to leukopenia. Reveals, the material base to rise white blood cells is tannins.Furthermore,the combination of tannins and saponins give rise to mutual inhibition.
The research result of the mutual inhibition of tannins and saponins in medication relationship showed,in terms of tannins components into the blood rules,gavage Burnet tannins extract alone into rat's stomach,gallic catechin, procyanidins B2, procyanidins B2-3'-the gallic acid ester, as well as gallic acid, ellagic acid into the blood. However, only can be detected procyanidins B2, procyanidins B2-3'the gallic acid ester after the combination with saponins.The result revealed Burnet tannins and saponins in combination can lead to the change of behavior of gallic catechin, gallic acid and other ingredients into blood.The studies of pharmacodynamics rules show that Burnet tannins rise white cells by improving the G2period, DNA content, and antagonising cyclophosphamide in the cell apoptosis,and having no reference to promote hematopoiesis factor and protect hematopoietic microenvironment. However, the combination of Burnet tannins and saponins, the effect of increasing the G2period cells proportion, DNA content, antagonist cyclophosphamide in the cell apoptosis phase decreased significantly (P<0.05), shows that the pharmacodynamics rules of mutual inhibition of Burnet tannins and saponins have reference to above results.
In addition,the mutual inhibition of tannins and saponins is specific and conditional,so it can't leave dose ratio and discuss the mutual inhibition of tannins and saponins in isolation.when the range of tannins and saponins dose ratio is1:1to4:1,the mutual inhibition of tannins and saponins will appear. But the mutual inhibition relationship has relativity,it will vary as the dose ratio change.When tannins and saponins dose ratio higher than4:1,the mutual inhibition relationship will weaken and with the dose of saponins reducing,a decreasing trend will appear until above8:1,the mutual inhibition relationship almost disappear,which has no significant difference from tannins used alone (P>0.05).It reveals that saponins and tannins exist the mutual inhibition relationship when the saponins dose is more than1/8dose of tannins. Burnet tannins have more comprehensive function than rhG-CSF,shark liver alcohol,rise white amines,Burnet leukopoietic tablets commercially available similar preparations.with respect to improve, the DNA content in the bone marrow, Burnet tannins are better than rhG-CSF, batifol, rise white amines, Burnet leukopoietic tablets(P<0.05); with respect to elevate WBC,Burnet tannins are better than shark liver alcohol, rise white amines, Burnet leukopoietic tablets(P<0.05),but worse than rhG-CSF. Acute toxicity test results show that tannins have lower toxicity. Antitumor experimental study shows that tannins have no role of promoting tumor growth,and have a certain trend in inhibitory action, and the combination of drug delivery system about the Burnet tannins and cyclophosphamide will not affect the treatment function of cyclophosphamide. However, in a certain extent,there are side effects in cancer therapy to saponins.
The study of the druggability of Xinsheng capsule shows that flash type extraction is more conducive to improve the overall quality of extraction than reflux extractionThe result of principal component analysis is that the comprehensive component of extraction value is2.323084, and the comprehensive component of reflux extraction value is-2.32172.The optimization of Box-Benhnken extraction process as follows:solvent consumption12times,60%of ethanol concentration extraction time3minutes, transfer rate can amount to Y195.25%, Y296.03%, its fast (3minutes), efficient (basic transfer completely) extraction efficiency further reveal the flash type extraction technology of scientific, reasonable and feasible to tannins.Uniform design to optimize the purification process as follows:the Burnet extraction was concentrated to0.5g crude drug/ml,placed in a centrifuge,4000/transferred to centrifuge10min,the supernatant at a flow rate2BV/h slowly on the kind of D-101macroporous resin, diameter to height ratio is1:5, after static adsorption30min, at a rate of2BV/h washed with2BV water, and then at a rate of2BV/h washed with1BV10%ethanol,and then at a rate of1BV/h washed with2BV60%ethanol,collect the eluent,concentration, drying.The drying process:use the vacuum oven drying and control the temperature at45℃. tannins and saponins ratio is about16:1in three batches of extracts,the result shows that the optimization of process parameters further improve the Burnet tannins content,and ensures that the mutual inhibition relationship between tannins and saponins dose is beyond 1/8.D-optimal mixture design to optimize the auxiliary material ratio:30%of starch and60%of lactose. Making sure the formula was Burnet tannins extraction10g, starch30g and were made into1000capsules. In addition, the process routes and parameters of the above formulations were enlarge verified, and established the intermediates and formulations quality standards and initial observed the stability of preparation.Pharmacodynamic research results show that the four indexes of WBC, PLT, spleen and bone marrow coefficient DNA content, the Burnet tannins groups increase more significantly than model groups(P<0.05).So the Burnet tannins have a good treatment function to leukopenia disease in mice in radiation and chemotherapy.Positive medicines have an obvious function in the WBC, PLT, spleen (P<0.05),but play a counteraction function for the content of bone marrow DNA.High, medium and low dose groups of extraction from Burnet have a certain improvement to the mice by large dose the illuminate of60Co-γ rays in the overall state, and high dose groups effect is significant, and can improve the high-dose60Co-y rays to the influence of rat appetite, weight, state, and can obviously improve the survival rate of the rats.
Conclusion:Using the Burnet tannins alone can improve the G2period cells proportion and DNA content,antagonist cyclophosphamide induced apoptosis,but these functions are reduced when tannins and saponins are used together.saponins and tannins exist the mutual inhibition relationship when the saponins dose is more than1/8dose of tannins as well as the mutual inhibition of tannins and saponins is specific and conditional, so it can't leave dose ratio and discuss the mutual inhibition of tannins and saponins in isolatioaSo,according to the mutual inhibition of tannins and saponins in medication relationship,extract the Burnet tannins and prepare five kinds of traditional Chinese medicine of Xinsheng capsule in the protection of bone marrow and rise white blood cells, avoid the opposite effect of tannins and its contain ingredients like saponins and the side effects of saponins ingredients in cancer treatment.Achieve these aims:enhance security, efficacy significant,clear physical foundation, clear mechanism, no obvious side effects, not stimulate tumor growth, rise white blood cells stronger and hematopoiesis function protectionIn clinic, it will provide for double functions in rising white blood cells and medullary hematopoiesis function to the development of new drugs, which will improve the theory of single herb component compatibility,and provide ideas to inherit and carry forward the theory of compatibility,and benefit a comprehensive understanding of the biological activity of traditional chinese medicine tannins ingredients, and promote the international status and influence of traditional chinese medicine.
引文
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