决明子配方颗粒制备及提取过程动力学研究
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摘要
决明子是临床上具有降脂、降压、通便、明目等功效的常用中药,本论文对其配方颗粒的制备工艺、药理药效、制备过程中成分的变化规律以及提取过程的动力学机理进行研究。
(1)本实验首先建立了决明子总蒽醌的紫外-可见分光光度测定方法,该方法简便、准确、可靠。以总蒽醌为考察指标,用正交试验设计结合单因素试验优化了决明子水提、乙醇提取和微波辅助-乙醇提取工艺。比较了不同提取溶剂最佳工艺下决明子总蒽醌的提取率,乙醇提取为94.91%,水提为27.55%。比较了乙醇提取过程中采用不同加热方式对决明子总蒽醌提取率的影响,其中普通加热方式的乙醇回流提取为94.91%,微波辅助提取为79.11%。选择普通加热方式下乙醇回流提取作为决明子配方颗粒制备的提取方法,其最佳提取工艺为70%乙醇,提取温度80℃,提取1.5小时,液固比8,药材粒度40目,提取2次。
(2)本实验得到了决明子总蒽醌大孔树脂纯化的工艺路线。从AB-8、D-101-I、DA-201、DM-130、DM-301、NKA-Ⅱ、NKA-9七种大孔树脂中筛选出DM-130大孔树脂作为纯化决明子总蒽醌的树脂。分别从pH值、提取液浓度、流速、不同种类的洗脱剂、不同浓度乙醇洗脱剂等方面考察并优化了该树脂的吸附和洗脱工艺条件。最佳的吸附、解吸条件是：提取液pH值7-8、提取液浓度0.4g药材／mL、吸附流速2BV／h、上柱液体积与树脂重量比为10：1、洗脱液为0.02500M氢氧化钠-70%乙醇溶液、洗脱流速2BV／h。在最佳纯化工艺条件下,决明子总蒽醌的转移率可达到近60%,并且工艺稳定。
(3)本实验建立了决明子乙醇提取物浸膏的喷雾、真空和冷冻干燥工艺,比较了干燥后产品的吸湿性和溶解性,并对喷雾、真空和冷冻三种干燥工艺下决明子配方颗粒的蒽醌含量进行测定。在吸湿性上,以冷冻干燥颗粒最易吸湿,其次是真空干燥,喷雾干燥比较稳定。在溶解性上,以喷雾干燥颗粒最易溶解,其次是冷冻干燥,真空干燥颗粒不易溶解。真空干燥和冷冻干燥的总蒽醌和结合蒽醌含量相当并高于喷雾干燥,游离蒽醌以喷雾干燥最高。
(4)本实验研究了决明子总蒽醌在其配方颗粒制备过程中不同阶段的转移率和原药材及不同干燥工艺下配方颗粒的成分变化规律。决明子总蒽醌从原药材到配方颗粒制备过程中各阶段的转移率分别为：醇提液为94.91%,醇沉后为68.15%,真空干燥后为69.19%,冷冻干燥后为62.35%,喷雾干燥后为59.68%。其中总蒽醌在醇沉阶段的损失最大,损失率可达近30%,各干燥工艺阶段总蒽醌的损失以喷雾干燥最大,其次是冷冻干燥,真空干燥几乎没有影响。采用高效液相色谱法对原药材及三种干燥工艺配方颗粒的指纹图谱进行保留时间的相似度评价,原药材、真空干燥配方颗粒和冷冻干燥配方颗粒的相似度在0.98以上,喷雾干燥的相似度为0.7046,真空干燥和冷冻干燥对成分的破坏较小,喷雾干燥对成分有一定的破坏作用。
(5)本实验选择通便和调脂两个药理指标,研究不同干燥工艺下决明子配方颗粒与传统水煎液的药效差别。在对正常小鼠的肠推进实验中,三种配方颗粒均能保留饮片促进正常小鼠肠运动的功能,且作用略优于传统饮片。在对便秘模型小鼠的实验中,决明子醇提物经喷雾、冷冻、真空干燥获得的三种配方颗粒均能保留饮片的泻下功能,并有优于传统水提液的趋势,其中以冷冻干燥的效果最好,其次是喷雾干燥和真空干燥。在对高脂血症模型小鼠的实验中,三种配方颗粒均能保留饮片的降血脂功能,即具有降低TC、TG和LDL-C,升高HDL-C的功效,并有优于传统水提液的趋势,其中以冷冻干燥的效果最好,其次是喷雾干燥和真空干燥。考虑到冷冻干燥技术的不足及三种配方颗粒的性状、蒽醌的含量,选择喷雾干燥作为决明子配方颗粒制备的干燥工艺。
(6)根据中药材提取的非稳态扩散过程,以Fick第二扩散定律为基础建立了决明子总蒽醌和大黄酚乙醇回流提取的一级一项动力学数学模型。该模型能较好地描述决明子总蒽醌和大黄酚提取的动态过程。决明子总蒽醌和大黄酚的提取均符合一级动力学方程特征,说明该提取过程属于内部扩散控制的相内反应过程。计算获得了决明子总蒽醌和大黄酚提取的表观速率常数、相对萃余率、活化能及半衰期等一系列有价值的动力学参数。在两区质量传递模型的基础上对一级一项动力学方程进行优化,建立了一级两项动力学方程,数据拟合效果较好,快速项和慢速项的相应参数随温度变化有一定的物理意义。一级两项动力学方程能更好地反映决明子总蒽醌和大黄酚提取过程的动力学机制,对决明子蒽醌类成分的提取有重要的理论意义。
本论文在中医药学基本理论的指导下开展实验研究,将分析化学、物理化学、中药化学、中药制剂、中药药理、统计学等多个学科领域相结合,探讨了决明子配方颗粒制备工艺、成分变化规律和提取过程的动力学机理等内容,为配方颗粒的研究提供了实践和理论依据。
With lowering lipid and blood pressure, catharsis, eyesight and other effects, Semen Cassiae is a traditional Chinese medicine commonly used in clinical. In this paper, preparation technology of Semen Cassiae prescription granules, pharmacological activities of granules, changes of composition during preparation process and kinetic mechanism of the extraction process were studied.
(1) In this part, the UV-vis spectrophotometric determination method on total anthraquinones of Semen Cassia was established. This method is simple, accurate and reliable. Indicated by the content of total anthraquinones, water extraction, ethanol extraction and microwave-assisted extraction were optimized respectively by orthogonal tests based on their single factor tests. The yields of total anthraquinones by the best extraction technologies of the different solvents extraction methods were compared. Ethanol extraction is the best, the extraction rate is 94.91%, the rate of water extraction is 27.55%. The yields of total anthraquinones by different heating modes in ethanol extraction process were compared. The extraction rate of ethanol extraction by normal heating mode is 94.91%, the rate of microwave-assisted extraction is 79.11%. Ethanol extraction by normal heating mode is choosed as the extraction method in the preparation process of Semen Cassiae prescription granules. The optimum extraction process is as follows:extraction temperature was 80℃, reflux extraction with 8-fold 70% alcohol for 2 times,1.5h every time, the granularity of Semen Cassiae power was 40 mesh sieve.
(2) The route of separation and purification of total anthraquinones in Semen Cassiae by macroporous resin was obtained. In this part, from the AB-8, D-101-I, DA-201, DM-130, DM-301, NKA-II and NKA-9 seven macroporous resins, DM-130 was choosed to separate and purify the total anthraquinones of Semen Cassiae. Extract concentration, pH, flow rate, different eluants, eluants of different ethanol concentration and other factors were investigated. The adsorption and desorption crafts of DM-130 macroporous resin were optimized. The appropriate adsorption conditions is:the extract concentration 0.4g/mL, pH 7～8, flow rate 2BV/h, the liquid volume and the resin weight ratio of 10:1; the appropriate desorption conditions is:the 70% ethanol used as eluant and the concentration of NaOH is 0.025 mol/L, and the flow rate is 2BV/h. By DM-130 macroporous resin confirmatory test under the best adsorption and desorption conditions, the total anthraquinones of Semen Cassiae transfer rate can reach nearly 60%, and the process is stable.
(3) The technologies of spray drying, vacuum drying and freeze drying was established. The water absorption and water soluble capability of products treated with the three drying methods were investigated. The total, dissociative and conjugated anthraquinones of the three kinds of products were determined. The order of water absorption capability is:freeze drying, vacuum drying and spray drying. The order of soluble capability is:spray drying, freeze drying and vacuum drying. Compared the anthraquinone content of the three kinds of drying products, the total and conjugated anthraquinones content of vacuum drying approach freeze drying, and they are higher than spray drying. The content of dissociative anthraquinone in spray drying product is the highest.
(4) The transfer rate changes of total anthraquinones in the preparation process of Semen Cassiae prescription granules were studied. The transfer rates of total anthraquinones during the preparation process of Semen Cassiae prescription granules were:the yield of ethanol extraction was 94.91%, the yield was 68.15% after alcohol precipitation, the yields were 69.19%,62.35% and 59.68% by vacuum, freeze, and spray drying respectively. The stage of alcohol precipitation caused the biggest loss of total anthraquinones, the loss rate was up to nearly 30%. Among the three drying methods, spray drying caused the biggest loss of total anthraquinones, followed by freeze drying, vacuum drying had little impact. The chromatographic fingerprints of original medicine and granules treated with the three drying methods were obtained by RP-HPI-C. According to retention time, data were analysed by fingerprint similarity evaluation software to compare the similarity of the samples. The similarity of original herbs, vacuum-dried and freeze-dried granules was above 0.98, the similarity of spray-dried granules was 0.7046. The results showed that vacuum drying and freeze drying methods have little destruction on the composition of the samples, spray drying method has a certain degree of destructive effect on the samples.
(5) The pharmacodynamic of Semen Cassiae prescription granules and its traditional decoction was compared by laxative test and lipid test. In the intestinal propulsion experiments, the three kinds of Semen Cassiae prescription granules can increase the intestinal movement in normal mice, and they have similar effect with the traditional water extraction. The three kinds of granules were used in constipation mice. The results showed that the spray, freeze, vacuum drying granules can be reserved the purgative function, and have a trend of being better than the traditional water extraction group. Among these granules, the purgative effect of freeze drying is best, followed by spray drying and vacuum drying. The three kinds of drying granules were used in hyperlipidemic mice. The results show that all of them have the function of reducing blood lipid, and have a trend of being better than the water extraction group. They can reduce TC, TG and LDL-C, increase HDL-C. Among these granules, the regulating blood lipid effect of freeze drying is best, followed by spray drying and vacuum drying. Considering the lack of freeze drying technology, the characteristics of Semen Cassiae prescription granules and the content of anthraquinones, we choose spray drying in the preparation technology of Semen Cassiae prescription granules, and create its quality standard.
(6) According to the non-steady state diffusion process of traditional Chinese medicine extraction, the Fick's second law of diffusion was used to establish the one-component kinetic model(OKM) equation of extracting total anthraquinones and chrysophanol from Semen Cassiae by ethanol. The model can describe the dynamic extraction process of total anthraquinone and chrysophanol from Semen Cassiae. The results showed that the kinetics on the extraction of total anthraquinone and chrysophanol match the first-order rate equation, and the rate-determining step for extraction is the diffusion of total anthraquinone and chrysophanol through particles. The kinetics parameters, such as k(rate constant), Ea (activation energy), y (residual rate of extraction) and t(?)(half life) are obtained from the calculation. Based on two-region mass transfer model, the one-component kinetic model (OKM) equation was optimized and the two-component kinetic model (TKM) equation was developed. The research showed that the regression results of the TKM fit the experimental data well, and the changes of the parameters with temperature have some physical meaning. The TKM can reflect the dynamic mechanism better of the extraction process of total anthraquinone and chrysophanol, which has an important theoretical significance for extracting anthraquinones from Semen Cassiae.
This research was conducted under the basic theory of traditional Chinese medicine. It combined analytical chemistry, physical chemistry, Chinese medicinal chemistry, Chinese medicine preparation, pharmacology, statistics and other disciplines to explore the preparation technology, composition changes and kinetic mechanism of the extraction process of the Semen Cassiae prescription granules. It can provide a practical and theoretical basis for prescription granules research.
(1)本实验首先建立了决明子总蒽醌的紫外-可见分光光度测定方法,该方法简便、准确、可靠。以总蒽醌为考察指标,用正交试验设计结合单因素试验优化了决明子水提、乙醇提取和微波辅助-乙醇提取工艺。比较了不同提取溶剂最佳工艺下决明子总蒽醌的提取率,乙醇提取为94.91%,水提为27.55%。比较了乙醇提取过程中采用不同加热方式对决明子总蒽醌提取率的影响,其中普通加热方式的乙醇回流提取为94.91%,微波辅助提取为79.11%。选择普通加热方式下乙醇回流提取作为决明子配方颗粒制备的提取方法,其最佳提取工艺为70%乙醇,提取温度80℃,提取1.5小时,液固比8,药材粒度40目,提取2次。
(2)本实验得到了决明子总蒽醌大孔树脂纯化的工艺路线。从AB-8、D-101-I、DA-201、DM-130、DM-301、NKA-Ⅱ、NKA-9七种大孔树脂中筛选出DM-130大孔树脂作为纯化决明子总蒽醌的树脂。分别从pH值、提取液浓度、流速、不同种类的洗脱剂、不同浓度乙醇洗脱剂等方面考察并优化了该树脂的吸附和洗脱工艺条件。最佳的吸附、解吸条件是：提取液pH值7-8、提取液浓度0.4g药材／mL、吸附流速2BV／h、上柱液体积与树脂重量比为10：1、洗脱液为0.02500M氢氧化钠-70%乙醇溶液、洗脱流速2BV／h。在最佳纯化工艺条件下,决明子总蒽醌的转移率可达到近60%,并且工艺稳定。
(3)本实验建立了决明子乙醇提取物浸膏的喷雾、真空和冷冻干燥工艺,比较了干燥后产品的吸湿性和溶解性,并对喷雾、真空和冷冻三种干燥工艺下决明子配方颗粒的蒽醌含量进行测定。在吸湿性上,以冷冻干燥颗粒最易吸湿,其次是真空干燥,喷雾干燥比较稳定。在溶解性上,以喷雾干燥颗粒最易溶解,其次是冷冻干燥,真空干燥颗粒不易溶解。真空干燥和冷冻干燥的总蒽醌和结合蒽醌含量相当并高于喷雾干燥,游离蒽醌以喷雾干燥最高。
(4)本实验研究了决明子总蒽醌在其配方颗粒制备过程中不同阶段的转移率和原药材及不同干燥工艺下配方颗粒的成分变化规律。决明子总蒽醌从原药材到配方颗粒制备过程中各阶段的转移率分别为：醇提液为94.91%,醇沉后为68.15%,真空干燥后为69.19%,冷冻干燥后为62.35%,喷雾干燥后为59.68%。其中总蒽醌在醇沉阶段的损失最大,损失率可达近30%,各干燥工艺阶段总蒽醌的损失以喷雾干燥最大,其次是冷冻干燥,真空干燥几乎没有影响。采用高效液相色谱法对原药材及三种干燥工艺配方颗粒的指纹图谱进行保留时间的相似度评价,原药材、真空干燥配方颗粒和冷冻干燥配方颗粒的相似度在0.98以上,喷雾干燥的相似度为0.7046,真空干燥和冷冻干燥对成分的破坏较小,喷雾干燥对成分有一定的破坏作用。
(5)本实验选择通便和调脂两个药理指标,研究不同干燥工艺下决明子配方颗粒与传统水煎液的药效差别。在对正常小鼠的肠推进实验中,三种配方颗粒均能保留饮片促进正常小鼠肠运动的功能,且作用略优于传统饮片。在对便秘模型小鼠的实验中,决明子醇提物经喷雾、冷冻、真空干燥获得的三种配方颗粒均能保留饮片的泻下功能,并有优于传统水提液的趋势,其中以冷冻干燥的效果最好,其次是喷雾干燥和真空干燥。在对高脂血症模型小鼠的实验中,三种配方颗粒均能保留饮片的降血脂功能,即具有降低TC、TG和LDL-C,升高HDL-C的功效,并有优于传统水提液的趋势,其中以冷冻干燥的效果最好,其次是喷雾干燥和真空干燥。考虑到冷冻干燥技术的不足及三种配方颗粒的性状、蒽醌的含量,选择喷雾干燥作为决明子配方颗粒制备的干燥工艺。
(6)根据中药材提取的非稳态扩散过程,以Fick第二扩散定律为基础建立了决明子总蒽醌和大黄酚乙醇回流提取的一级一项动力学数学模型。该模型能较好地描述决明子总蒽醌和大黄酚提取的动态过程。决明子总蒽醌和大黄酚的提取均符合一级动力学方程特征,说明该提取过程属于内部扩散控制的相内反应过程。计算获得了决明子总蒽醌和大黄酚提取的表观速率常数、相对萃余率、活化能及半衰期等一系列有价值的动力学参数。在两区质量传递模型的基础上对一级一项动力学方程进行优化,建立了一级两项动力学方程,数据拟合效果较好,快速项和慢速项的相应参数随温度变化有一定的物理意义。一级两项动力学方程能更好地反映决明子总蒽醌和大黄酚提取过程的动力学机制,对决明子蒽醌类成分的提取有重要的理论意义。
本论文在中医药学基本理论的指导下开展实验研究,将分析化学、物理化学、中药化学、中药制剂、中药药理、统计学等多个学科领域相结合,探讨了决明子配方颗粒制备工艺、成分变化规律和提取过程的动力学机理等内容,为配方颗粒的研究提供了实践和理论依据。
With lowering lipid and blood pressure, catharsis, eyesight and other effects, Semen Cassiae is a traditional Chinese medicine commonly used in clinical. In this paper, preparation technology of Semen Cassiae prescription granules, pharmacological activities of granules, changes of composition during preparation process and kinetic mechanism of the extraction process were studied.
(1) In this part, the UV-vis spectrophotometric determination method on total anthraquinones of Semen Cassia was established. This method is simple, accurate and reliable. Indicated by the content of total anthraquinones, water extraction, ethanol extraction and microwave-assisted extraction were optimized respectively by orthogonal tests based on their single factor tests. The yields of total anthraquinones by the best extraction technologies of the different solvents extraction methods were compared. Ethanol extraction is the best, the extraction rate is 94.91%, the rate of water extraction is 27.55%. The yields of total anthraquinones by different heating modes in ethanol extraction process were compared. The extraction rate of ethanol extraction by normal heating mode is 94.91%, the rate of microwave-assisted extraction is 79.11%. Ethanol extraction by normal heating mode is choosed as the extraction method in the preparation process of Semen Cassiae prescription granules. The optimum extraction process is as follows:extraction temperature was 80℃, reflux extraction with 8-fold 70% alcohol for 2 times,1.5h every time, the granularity of Semen Cassiae power was 40 mesh sieve.
(2) The route of separation and purification of total anthraquinones in Semen Cassiae by macroporous resin was obtained. In this part, from the AB-8, D-101-I, DA-201, DM-130, DM-301, NKA-II and NKA-9 seven macroporous resins, DM-130 was choosed to separate and purify the total anthraquinones of Semen Cassiae. Extract concentration, pH, flow rate, different eluants, eluants of different ethanol concentration and other factors were investigated. The adsorption and desorption crafts of DM-130 macroporous resin were optimized. The appropriate adsorption conditions is:the extract concentration 0.4g/mL, pH 7～8, flow rate 2BV/h, the liquid volume and the resin weight ratio of 10:1; the appropriate desorption conditions is:the 70% ethanol used as eluant and the concentration of NaOH is 0.025 mol/L, and the flow rate is 2BV/h. By DM-130 macroporous resin confirmatory test under the best adsorption and desorption conditions, the total anthraquinones of Semen Cassiae transfer rate can reach nearly 60%, and the process is stable.
(3) The technologies of spray drying, vacuum drying and freeze drying was established. The water absorption and water soluble capability of products treated with the three drying methods were investigated. The total, dissociative and conjugated anthraquinones of the three kinds of products were determined. The order of water absorption capability is:freeze drying, vacuum drying and spray drying. The order of soluble capability is:spray drying, freeze drying and vacuum drying. Compared the anthraquinone content of the three kinds of drying products, the total and conjugated anthraquinones content of vacuum drying approach freeze drying, and they are higher than spray drying. The content of dissociative anthraquinone in spray drying product is the highest.
(4) The transfer rate changes of total anthraquinones in the preparation process of Semen Cassiae prescription granules were studied. The transfer rates of total anthraquinones during the preparation process of Semen Cassiae prescription granules were:the yield of ethanol extraction was 94.91%, the yield was 68.15% after alcohol precipitation, the yields were 69.19%,62.35% and 59.68% by vacuum, freeze, and spray drying respectively. The stage of alcohol precipitation caused the biggest loss of total anthraquinones, the loss rate was up to nearly 30%. Among the three drying methods, spray drying caused the biggest loss of total anthraquinones, followed by freeze drying, vacuum drying had little impact. The chromatographic fingerprints of original medicine and granules treated with the three drying methods were obtained by RP-HPI-C. According to retention time, data were analysed by fingerprint similarity evaluation software to compare the similarity of the samples. The similarity of original herbs, vacuum-dried and freeze-dried granules was above 0.98, the similarity of spray-dried granules was 0.7046. The results showed that vacuum drying and freeze drying methods have little destruction on the composition of the samples, spray drying method has a certain degree of destructive effect on the samples.
(5) The pharmacodynamic of Semen Cassiae prescription granules and its traditional decoction was compared by laxative test and lipid test. In the intestinal propulsion experiments, the three kinds of Semen Cassiae prescription granules can increase the intestinal movement in normal mice, and they have similar effect with the traditional water extraction. The three kinds of granules were used in constipation mice. The results showed that the spray, freeze, vacuum drying granules can be reserved the purgative function, and have a trend of being better than the traditional water extraction group. Among these granules, the purgative effect of freeze drying is best, followed by spray drying and vacuum drying. The three kinds of drying granules were used in hyperlipidemic mice. The results show that all of them have the function of reducing blood lipid, and have a trend of being better than the water extraction group. They can reduce TC, TG and LDL-C, increase HDL-C. Among these granules, the regulating blood lipid effect of freeze drying is best, followed by spray drying and vacuum drying. Considering the lack of freeze drying technology, the characteristics of Semen Cassiae prescription granules and the content of anthraquinones, we choose spray drying in the preparation technology of Semen Cassiae prescription granules, and create its quality standard.
(6) According to the non-steady state diffusion process of traditional Chinese medicine extraction, the Fick's second law of diffusion was used to establish the one-component kinetic model(OKM) equation of extracting total anthraquinones and chrysophanol from Semen Cassiae by ethanol. The model can describe the dynamic extraction process of total anthraquinone and chrysophanol from Semen Cassiae. The results showed that the kinetics on the extraction of total anthraquinone and chrysophanol match the first-order rate equation, and the rate-determining step for extraction is the diffusion of total anthraquinone and chrysophanol through particles. The kinetics parameters, such as k(rate constant), Ea (activation energy), y (residual rate of extraction) and t(?)(half life) are obtained from the calculation. Based on two-region mass transfer model, the one-component kinetic model (OKM) equation was optimized and the two-component kinetic model (TKM) equation was developed. The research showed that the regression results of the TKM fit the experimental data well, and the changes of the parameters with temperature have some physical meaning. The TKM can reflect the dynamic mechanism better of the extraction process of total anthraquinone and chrysophanol, which has an important theoretical significance for extracting anthraquinones from Semen Cassiae.
This research was conducted under the basic theory of traditional Chinese medicine. It combined analytical chemistry, physical chemistry, Chinese medicinal chemistry, Chinese medicine preparation, pharmacology, statistics and other disciplines to explore the preparation technology, composition changes and kinetic mechanism of the extraction process of the Semen Cassiae prescription granules. It can provide a practical and theoretical basis for prescription granules research.
引文
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