WO2020113450A1 - 一种提取三七总皂苷的纯化制备方法 - Google Patents

一种提取三七总皂苷的纯化制备方法 Download PDF

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WO2020113450A1
WO2020113450A1 PCT/CN2018/119264 CN2018119264W WO2020113450A1 WO 2020113450 A1 WO2020113450 A1 WO 2020113450A1 CN 2018119264 W CN2018119264 W CN 2018119264W WO 2020113450 A1 WO2020113450 A1 WO 2020113450A1
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panax notoginseng
column
purification
total saponins
chromatography
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French (fr)
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梁鑫淼
叶贤龙
郭志谋
杨小平
王超然
申一波
万瑛
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泰州医药城国科化物生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation

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  • the invention relates to a preparation process of traditional Chinese medicine, in particular to a refined Panax notoginseng saponin component and a method for extracting and preparing high-purity total saponins from Panax notoginseng, in particular to a technique using high-efficiency liquid phase preparation and ion exchange chromatography Method for preparing Panax notoginseng saponins.
  • Panax notoginseng is a Panax ginseng plant with a medicinal history of more than 600 years. Its roots, stems, leaves and flowers can be used as medicine. It is commonly used in compound Danshen dripping pills, Yunnan Baiyao, Pianzaihuang, compound Sanqi oral liquid, etc.
  • One of the main ingredients of traditional Chinese medicine preparations At present, more than 20 preparations have been entered into the "National Essential Medicines List” and "National Traditional Medicines Protection Variety List” with Sanqi as a formula. Traditionally, Sanqi has the effect of stopping bleeding and removing blood stasis, reducing swelling and pain.
  • Saponin compounds are the main chemical constituents of Panax notoginseng, and they are also the effective ingredients for Panax notoginseng to exert its main functions. So far, nearly 100 kinds of saponin compounds have been discovered from Panax notoginseng, and new saponin compounds have been discovered continuously.
  • Panax notoginseng saponins Panax Notoginseng Saponins, PNS
  • PNS Panax Notoginseng saponins
  • the main components of PNS are panax notoginseng saponins R1, ginsenosides Rg1, Rb1, Rd, Re, etc., which are the main medicinal ingredients of panax notoginseng.
  • Notoginsenoside R1 has shown certain pharmacological effects in maintaining blood circulation, improving myocardial ischemia, anti-arrhythmia, anti-shock, sedation, improving intelligence, anti-aging, anti-oxidation, anti-cell proliferation and anti-tumor.
  • the macroporous resin adsorption separation process is easy to operate and has low cost.
  • the resin can be used repeatedly, which is suitable for industrial production.
  • the macroporous resin adsorption separation technology has shortcomings: (1) There are certain requirements for the purity of the extraction liquid, and the extraction liquid with more impurity content needs to be pre-treated before it can be put on the column. (2) It is only suitable for coarse separation of products. If higher content is needed, it needs to be reprocessed. Therefore, it is necessary to overcome the shortcomings of the traditional process. After analysis and comparison, we find a time-saving, simple operation, low equipment requirements, low cost, environmental protection and suitable for large-scale industrial production extraction and separation and purification methods.
  • the invention mainly provides a method for preparing high-purity panax notoginseng saponins using high-performance liquid chromatography and ion exchange chromatography technology, which is used to solve the current low extraction yield of panax notoginseng saponins extraction preparation process, low content in purified products, and a large amount of organic solvents Use causes environmental pollution and high production costs.
  • the steps of the purification preparation method used in the present invention to prepare a panax notoginseng are as follows:
  • Ion exchange chromatography technology is used to decolorize and remove impurities of the purified Panax notoginseng, and the column flow-through liquid is collected to obtain the total saponin component with higher purity; it is concentrated and dried to obtain Panax notoginseng Soap pure powder.
  • the packing matrix of the reversed-phase chromatography column is silica gel or a polymer; the packing ligand has 1-30 carbon atoms in the normal chain alkyl group
  • chromatographic column inner diameter is 20mm ⁇ 1600mm, chromatographic column bed height is 100mm ⁇ 1000mm;
  • chromatographic equilibrium liquid contains organic solvents and acids, organic solvents include methanol, ethanol or acetonitrile, the content of organic solvents is 5% ⁇ 10%, the acid includes phosphoric acid, formic acid or acetic acid, the concentration of the acid is 0.01% ⁇ 0.5%;
  • the loading capacity of the chromatographic column is 0.01% ⁇ 20% of the weight of the stationary phase; the loading and elution flow rate of the chromatography is 140cm /h ⁇ 350cm/h; chromatographic purification
  • the organic solvent concentration of the total saponin target component is 30-90%, and the elution mode can be isocratic
  • the matrix of the separation material used in the ion exchange chromatography technique used in step 3 is agarose, silica gel or high molecular polymer; the particle size is 40-100 ⁇ m; the structural formula of the ligand may be
  • the sample loading volume is 10-50 times the column volume; the sample loading and elution flow rate of the chromatographic operation can be 150-500 cm/h; the method of chromatographic purification of the target component can be the flow-through and elution modes.
  • the concentrated drying method used in steps 1 and 3 may be microwave drying, freeze drying, vacuum drying or spray drying.
  • the applicant found that the preparation of total saponins of Panax notoginseng using high-performance liquid phase preparation and ion exchange chromatography technology can significantly improve the purity of the target substance and the content of active ingredients.
  • This method saves the cost of medicinal materials and can provide more industrial benefits Production and preparation conditions, and ensure the stability and safety of the process, so that Sanqi medicinal materials can exert their effects as much as possible.
  • the characteristics of the present invention are: the combination of efficient preparation of liquid phase and ion exchange chromatography technology can effectively remove ineffective components such as lipids, pigments, heavy metals, and pesticide residues, while avoiding the use of organic reagents and reducing industrial production pollution; It is important to retain the active ingredients to maximize the medicinal value of Panax notoginseng.
  • the present invention also proposes a refined Panax notoginseng saponin component.
  • the raw material is mainly composed of Panax notoginseng. It is refined by the preparation methods in the above embodiments. Compared with the refined Panax notoginseng powder obtained in steps 2 and 3, The crude dry powder obtained in step 1 has a uniform pink color, smooth surface, and delicate texture; the Panax notoginseng refined powder and the final pure powder are quickly dissolved after adding purified water, and the solution is clear and translucent, while the crude dry powder dissolves slowly and the solution turbid.
  • the high-performance liquid chromatography separation technology adopted by the method of the present invention can effectively separate impurities such as saponins and their structural analogs, and realize the efficient purification and preparation of panax notoginseng saponins.
  • the ion exchange chromatography separation technology adopted in the method of the present invention can effectively remove toxic and harmful substances such as pigments, pesticide residues, heavy metals, endotoxins, etc., and realize the efficient purification and preparation of Panax notoginseng saponins. More suitable for industrial production.
  • Figure 1 is a HPLC analysis chromatogram of Sanqi crude extract powder prepared in the present invention
  • Fig. 3 is a HPLC analysis chromatogram of the pure product of Panax notoginseng prepared in the present invention.
  • first and second are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features.
  • first and second features defined may explicitly or implicitly include one or more of the features.
  • at least means one or more than one, unless otherwise specifically Limit.
  • the invention provides a purification preparation method for extracting total saponins of Panax notoginseng, the specific steps are as follows:
  • the invention provides a purification preparation method for extracting total saponins of Panax notoginseng, the specific steps are as follows:
  • the invention provides a purification preparation method for extracting total saponins of Panax notoginseng, the specific steps are as follows:
  • the invention provides a method for preparing total saponins in Panax notoginseng, the specific steps are as follows:
  • HPLC analysis conditions are as follows:
  • the present invention also proposes a refined Panax notoginseng saponin component.
  • the raw material is mainly composed of Panax notoginseng. It is refined by the preparation methods in the above embodiments. Compared with the refined Panax notoginseng powder obtained in steps 2 and 3, The crude dry powder obtained in step 1 has a uniform pink color, smooth surface, and delicate texture; the Panax notoginseng refined powder and the final pure powder are quickly dissolved after adding purified water, and the solution is clear and translucent, while the crude dry powder dissolves slowly and the solution turbid.
  • the HPLC analysis results in each step show that the target peak in the analysis spectrum of the sample before and after purification does not change significantly. It is calculated based on the total volume, integrated peak area and weight. The content of the main components in the refined and pure products of total saponins has not decreased significantly. The yield of total saponins has reached more than 95% and the purity has reached more than 85%, and the prior art is shown in Table 1.
  • the above experimental results indicate that the Panax notoginseng total saponins obtained by using the purification method of the Panax notoginseng total saponins refined and pure products in the present invention have the advantages of high yield and high purity.

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Abstract

一种提取三七总皂苷的纯化制备方法，具体以三七药材为原料，采用乙醇提取、浓缩干燥、粉末溶解配制成三七粗提液，三七粗提液经过滤后，通过反相液相制备色谱进行精制，最后采用离子交换色谱脱色除杂。该制备方法实现了三七总皂苷的高效纯化制备，易于产业，制备获得的三七总皂苷含量及收率高。

Description

一种精制的三七总皂苷组分及提取三七中总皂苷的纯化制备方法 技术领域
本发明涉及一种中药制备工艺，尤其涉及一种精制的三七总皂苷组分及从三七药材中提取制备高纯度总皂苷的方法，具体涉及一种应用高效液相制备和离子交换色谱技术制备三七总皂苷的方法。
背景技术
三七为五加科人参属植物，已有600年以上的药用历史，其根、茎、叶、花均可入药，是复方丹参滴丸、云南白药、片仔癀、复方三七口服液等常见中药制剂的主要成分之一。目前以三七为配方进入《国家基本药物目录》和《国家中药保护品种目录》的制剂达20余种。传统认为三七具有止血散瘀、消肿定痛的功效，现代药理学表明其具有止血、保护心肌细胞、保护脑组织、调血脂、抗血栓、增强免疫力、抗炎、抗纤维化、抗肿瘤、清除氧自由基、抗氧化等作用。
皂苷类化合物是三七的主要化学成分，也是三七发挥主要功效的有效成分。迄今为止，从三七中发现的皂苷类化合物己经将近百种，而且不断有新的皂苷类化合物被发现。三七总皂苷(Panax Notoginseng Saponins,PNS)是从三七中提取的二十多种皂苷活性物质的总称。PNS的主要成分有三七皂苷R1、人参皂苷Rg1、Rb1、Rd、Re等，是三七的主要药效成分。三七皂苷R1在维持血液循环、改善心肌缺血、抗心律失常、抗休克、镇静、提高智力、抗衰老、抗氧化、抗细胞增殖和抗肿瘤等方面均显示出一定的药理作用。
三七总皂苷分离纯化工艺种类繁多，它包括有：溶剂法、沉淀法、柱层析法(如硅胶、氧化铝、大孔吸附树脂)和结晶法等。其中大孔树脂吸附分离工艺操作简便，成本较低，树脂可反复使用，适合工业生产。但大孔树脂吸附分离技术存在缺点：(1)对提取液纯度有一定要求，杂质含量较多的提取液需经过前处理方能上柱。(2)只适用于产品的粗分离，如需更高含量的产品还需要再处理。故需克服传统工艺的不足，经过分析比较寻找到一种省时、操作简单、设备要求低、成本低廉、环保并适用于大规模工业生产的提取及分离纯化方法。
发明内容
本发明主要提供一种应用高效液相色谱和离子交换色谱技术制备高纯度三七总皂苷的方法，用于解决目前三七总皂苷提取制备工艺收率低、纯化产品中含量低、大量有机溶剂使用造成环境污染和生产成本高等问题。
为了实现上述发明目的，本发明采用的制备一种三七总皂苷的纯化制备方法步骤如下：
(1)提取和浓缩干燥：将三七药材洗净、干燥、切片，加20-80％乙醇进行煎煮，滤取药液，药渣再加20-80％煎煮，滤取药液，弃去药渣；合并两次药液，进行旋蒸浓缩、干燥，即得三七粗提干粉；
(2)精纯：用水溶解三七粗提干粉后过滤膜去颗粒物，采用高效液相色谱技术进行精制制备，收集柱洗脱液，得精制三七总皂苷组分；
(3)脱色除杂：采用离子交换色谱技术对精制三七总皂苷组分进行脱色除杂，收集柱流穿液，得纯度较高的总皂苷组分；将其浓缩干燥，即得三七总皂纯品粉末。
在本发明的一些具体实施方案中，优选地，在步骤2中，反相色谱柱的填料基质为硅胶或高分子聚合物；填料配基的碳原子数为1-30的正链烷基中的一种或多种；色谱柱内径为20mm～1600mm，色谱柱床高度为100mm～1000mm；色谱平衡液中含有有机溶剂和酸，有机溶剂包括甲醇、乙醇或乙腈，有机溶剂的含量为5％～10％，酸包括为磷酸、甲酸或乙酸，酸的浓度为0.01％～0.5％；色谱柱的载样量为固定相重量的0.01％～20％；色谱的上样和洗脱流速为140cm/h～350cm/h；色谱纯化洗脱总皂苷目标组分的有机溶剂浓度为30-90％，洗脱方式可以是等度或线性梯度。
优选的，步骤3中所用得离子交换色谱技术所用分离材料的基质为琼脂糖、硅胶或高分子聚合物；粒径为40-100μm；配基的结构式可为
Figure PCTCN2018119264-appb-000001
Figure PCTCN2018119264-appb-000002
上样体积为柱体积的10-50倍；色谱操作的上样和洗脱流速可以为150-500cm/h；色谱纯化目标组分的方式可以为流穿和洗脱模式。
优选地，在步骤1、3中所用浓缩干燥方式可以为微波干燥、冷冻干燥、真空干燥或喷雾干燥。
基于上述技术方案，申请人发现，采用高效液相制备及离子交换色谱技术制备三七总皂苷可显著提高目标物的纯度及有效成分的含量，该方法节约了药材成本，能够提供更有利于工业生产制备条件，并确保工艺的稳定性和安全性，使三七药材尽可能的发挥其功效。本发明的特点为：采用高效制备液相及离子交换色谱技术相结合的方法，可有效去除脂类、色素、重金属、农残等无效成分，同时避免有机试剂的使用、减少工业生产污染；更重要的是保留了活性成分，能最大化提高三七药材的药用价值。
本发明还提出一种精制的三七总皂苷组分，原料主要组成为三七药材，经上述各实施例中的制备方法精制，步骤2和3中获得的精制三七总皂苷粉相比于步骤1中获得的粗提干燥粉色泽均匀，表面光洁，质地细腻；三七精制粉和最终纯品粉末加纯化水后迅速溶解，且其溶液澄清透亮，而粗提干燥粉溶解速度缓慢且溶液浑浊。
与现有技术相比，本发明的优点为：
(1)本发明方法整个三七总皂苷制备过程均可以在室温下进行，减少了能源消耗，显著提高三七总皂苷终产品的质量和收率。
(2)本发明方法采用的高效液相色谱分离技术，可以有效的分离皂苷与其结构类似物等杂质，实现了三七总皂苷的高效纯化制备。
(3)本发明方法采用的离子交换色谱分离技术，可以有效的去除色素、农药残留、重金属、内毒素等有毒有害物质，实现三七总皂苷的高效纯化制备。更适于工业化生产。
(4)本发明方法整个操作过程操作简单，洗脱剂和再生剂均可回收重复利用，节约溶剂，可用于大规模生产。
附图说明
图1为本发明中制备的三七粗提粉末的HPLC分析色谱图；
图2为本发明中制备的三七总皂苷精制液的HPLC分析色谱图；
图3为本发明中制备的三七总皂纯品的HPLC分析色谱图。
具体实施方式
下面将结合示意图对本发明的三七总皂苷精制物和纯品及其制备方法进行更详细的描述，其中表示了本发明的优选实施例，应该理解本领域技术人员可以修改在此描述的本发明，而仍然实现本发明的有利效果。因此，下列描述应当被理解为对于本领域技术人员的广泛知道，而并不作为对本发明的限制。
为了清楚，不描述实际实施例的全部特征。在下列描述中，不详细描述公知的功能和结构，因为它们会使本发明由于不必要的细节而混乱。应当认为在任何实际实施例的开发中，必须做出大量实施细节以实现开发者的特定目标，例如按照有关系统或有关商业的限制，由一个实施例改变为另一个实施例。另外，应当认为这种开发工作可能是复杂和耗费时间的，但是对于本领域技术人员来说仅仅是常规工作。
在本发明中，除另有明确规定和限定，如有术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或隐含指明技术特征的数量。由此，限定有“第一”、“第二”特征可以明示或者隐含包括一个或者多个该特征，在本发明描述中，“至少”的含义是一个或一个以上，除非另有明确具体的限定。
在下列段落中参照附图以举例方式更具体地描述本发明。根据下面说明，本发明的优点和特征将更清楚。需说明的是，附图均采用非常简化的形式且均使用非精准的比例，仅用以方便、明晰地辅助说明本发明实施例的目的。
实施例1
本发明提出一种提取三七总皂苷的纯化制备方法，具体步骤如下：
称取预处理好的三七药材500g，加65％乙醇5L放入煎药机中煮(120℃)0.5 h，纱布过滤，药渣加65％乙醇2.5L再次放入煎药机中煮(120℃)0.3h，纱布过滤，合并滤液，旋蒸浓缩后经喷雾干燥得提取物干燥粉。将提取物干燥粉溶解的样品进行HPLC分析，色谱分析结果如图1所示。
将C18反相色谱柱(填料重量300g，粒径30μm)连接到高压液相系统上，设计流速60ml/min(190cm/h)，采用5％乙醇平衡缓冲液冲洗色谱柱2BV，将平衡缓冲液溶解的60g三七提取物干燥粉(使用0.22μm滤膜去除颗粒性不溶物)通过系统泵流经色谱柱纯化，通过15％乙醇淋洗杂质，60％乙醇洗脱目标物，收集各馏分进行HPLC分析，合格馏分合并，即为精制液。将精制液旋蒸去乙醇、真空干燥直至水分全部去除，收集干燥粉末，即为精制三七总皂苷粉末。精制液的HPLC分析结果见图2所示。
取20g精制干燥粉用纯化水溶解，配置浓度100mg/mL。将Q Sepharose 6FF离子交换色谱柱(柱体积20mL)连接到层析系统上，设计流速10ml/min(300cm/h)，将样品通过系统泵过平衡好的色谱柱，上样完毕后，纯水淋洗色谱柱，采用0.5M NaOH洗脱结合到色谱柱上的杂质。收集柱穿出液和淋洗液进行HPLC分析，即为高纯度三七总皂苷组分。将该馏分冷冻后放入冻干机中，抽真空干燥直至水分全部去除，收集冻干粉末，即为三七总皂苷纯品粉末。称重分析计算总皂苷含量。高纯度三七总皂苷组分的HPLC分析结果见图3所示，皂苷含量约为87％，收率约为95％。
实施例2
本发明提出一种提取三七总皂苷的纯化制备方法，具体步骤如下：
称取预处理好的三七药材500g，加60％乙醇10L放入煎药机中煮(120℃)1h，纱布过滤，药渣加50％乙醇5L放入煎药机中煮(120℃)0.5h，纱布过滤，合并滤液，旋蒸浓缩后经喷雾干燥得提取物干燥粉。
将C18反相色谱柱(填料重量300g，粒径60μm)连接到高压液相系统上，设计流速60ml/min(190cm/h)，采用10％乙醇平衡缓冲液冲洗色谱柱2BV，将平衡缓冲液溶解的50g三七提取物干燥粉(使用0.22μm滤膜去除颗粒性不溶物)通过系统泵流经色谱柱纯化，通过20％乙醇淋洗杂质，70％乙醇洗脱目标物，收集各馏分进行HPLC分析，合格馏分合并，即为精制液。将精制液旋蒸去除乙醇 后冷冻，抽真空干燥直至水分全部去除，收集冻干粉末，即为精制三七总皂苷粉末。
取30g精制干燥粉用纯化水溶解，配置浓度100mg/mL。将WorkBeads 40TREN离子交换色谱柱(柱体积20mL)连接到层析系统上，设计流速12ml/min(360cm/h)，将样品通过系统泵过平衡好的色谱柱，上样完毕后，纯水淋洗色谱柱，采用0.5M NaOH和0.1M HCl洗脱结合到色谱柱上的杂质。收集柱穿出液和淋洗液进行HPLC分析，即为高纯度三七总皂苷组分。将该馏分冷冻后放入冻干机中，抽真空干燥直至水分全部去除，收集冻干粉末，即为三七总皂苷纯品粉末。称重分析计算总皂苷含量约为85％，收率约为96％。
实施例3
本发明提出一种提取三七总皂苷的纯化制备方法，具体步骤如下：
称取预处理好的三七药材500g，加65％乙醇5L放入煎药机中煮(120℃)1h，纱布过滤，药渣加入50％乙醇5L再次放入煎药机中煮(120℃)0.5h，纱布过滤，合并滤液，旋蒸浓缩后经喷雾干燥得提取物干燥粉。
将C18反相色谱柱(填料重量300g，粒径15μm)连接到高压液相系统上，设计流速50ml/min(150cm/h)，采用10％乙醇平衡缓冲液冲洗色谱柱2BV，将平衡缓冲液溶解的50g三七提取物干燥粉(使用0.22μm滤膜去除颗粒性不溶物)通过系统泵流经色谱柱纯化，通过线性梯度增加的方式(10-70％乙醇，40min)洗脱目标物，根据峰型收集各馏分进行HPLC分析，合格馏分合并，即为精制液。将精制液旋蒸浓缩后，抽真空干燥直至水分全部去除，收集冻干粉末，即为精制三七总皂苷粉末。
取40g精制干燥粉用纯化水溶解，配置浓度100mg/mL。将WorkBeads40TREN离子交换色谱柱(柱体积20mL)连接到层析系统上，设计流速12ml/min(360cm/h)，将样品通过系统泵过平衡好的色谱柱，上样完毕后，纯水淋洗色谱柱，采用0.5M NaOH和0.1MHCl洗脱结合到色谱柱上的杂质。收集柱穿出液和淋洗液进行HPLC分析，即为高纯度三七总皂苷组分。将该馏分冷冻后放入冻干机中，抽真空干燥直至水分全部去除，收集冻干粉末，即为三七总皂苷纯品粉末。 称重分析计算总皂苷含量约为90％，收率约为96％。
实施例4
本发明提出一种三七药材中总皂苷的制备方法，具体步骤如下：
称取预处理好的三七药材500g，加65％乙醇5L放入煎药机中煮(120℃)1.5h，纱布过滤，药渣50％乙醇2.5L再次放入煎药机中煮(120℃)0.5h，纱布过滤，合并滤液，旋蒸浓缩后经喷雾干燥得提取物干燥粉。
将C18反相色谱柱(填料重量300g，粒径30μm)连接到高压液相系统上，设计流速60ml/min(190cm/h)，采用10％乙醇平衡缓冲液冲洗色谱柱2BV，将平衡缓冲液溶解的50g三七提取物干燥粉(使用0.22μm滤膜去除颗粒性不溶物)通过系统泵流经色谱柱纯化，通过15％乙醇淋洗杂质，65％乙醇洗脱目标物，收集各馏分进行HPLC分析，合格馏分合并，即为精制液。将精制液理性浓缩干燥直至水分全部去除，收集干燥粉末，即为精制三七总皂苷粉末。
取30g精制干燥粉用纯化水溶解，配置浓度100mg/mL。将WorkBeads 40Q离子交换色谱柱(柱体积20mL)连接到层析系统上，设计流速12ml/min(360cm/h)，将样品通过系统泵过平衡好的色谱柱，上样完毕后，纯水淋洗色谱柱，采用0.5M NaOH和0.1M HCl洗脱结合到色谱柱上的杂质。收集柱穿出液和淋洗液进行HPLC分析，即为高纯度三七总皂苷组分。将该馏分冷冻后放入冻干机中，抽真空干燥直至水分全部去除，收集冻干粉末，即为三七总皂苷纯品粉末。称重分析计算总皂苷含量约为86％，收率约为95％。
上述各实施例中，HPLC分析条件如下：
(1)色谱柱填料：Unitary C18(5μm，4.6*150mm)；
(2)流动相A：水，流动相B：乙腈；
(3)流速：1.5ml/min；
(4)梯度条件：0-20min 20％B相，20-45min从20-46％B相，45-55min从46-55％B相，55-60min 55％B相。
(5)柱温：25℃；
(6)检测波长：203nm；
(7)进样体积：2μl。
本发明还提出一种精制的三七总皂苷组分，原料主要组成为三七药材，经上述各实施例中的制备方法精制，步骤2和3中获得的精制三七总皂苷粉相比于步骤1中获得的粗提干燥粉色泽均匀，表面光洁，质地细腻；三七精制粉和最终纯品粉末加纯化水后迅速溶解，且其溶液澄清透亮，而粗提干燥粉溶解速度缓慢且溶液浑浊。
如图1、2、3所示，各步骤中的HPLC分析结果表明，纯化前后样品的分析谱图中目标峰没有显著变化，根据总体积、积分峰面积及重量计算得知，纯化后三七总皂苷精制物和纯品中主要成分的含量没有明显下降，总皂苷类物质收率达95％以上，纯度可达85％以上，和现有技术对比如表1所示。上述实验结果表明了，采用本发明中三七总皂苷精制物及纯品的纯化方法制备，获得的三七总皂苷具有高收率和高纯度的优势。
上述仅为本发明的优选实施例而已，并不对本发明起到任何限制作用。任何所属技术领域的技术人员，在不脱离本发明的技术方案的范围内，对本发明揭露的技术方案和技术内容做任何形式的等同替换或修改等变动，均属未脱离本发明的技术方案的内容，仍属于本发明的保护范围之内。
表1本发明实施例和现有技术制备方法得到的总皂苷含量和收率比较
制备方法 总皂苷含量(％) 皂苷总收率(％)
实施例1 87 95
实施例2 85 96
实施例3 90 96
实施例4 86 95
现有技术 70-80 60-85
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 一种提取三七总皂苷的纯化制备方法，其特征在于，步骤如下：
    (1)提取和浓缩干燥：将三七药材洗净、干燥、切片，加20-80％乙醇进行煎煮，滤取药液，药渣再加20-80％煎煮，滤取药液，弃去药渣；合并两次药液，进行旋蒸浓缩、干燥，即得三七粗提干粉；
    (2)精纯：用水溶解三七粗提干粉后过滤膜去颗粒物，采用高效液相色谱技术进行精制制备，收集柱洗脱液，得精制三七总皂苷组分；
    (3)脱色除杂：采用离子交换色谱技术对精制三七总皂苷组分进行脱色除杂，收集柱流穿液，得纯度较高的总皂苷组分；将其浓缩干燥，即得三七总皂纯品粉末。
  2. 根据权利要求1所述一种提取三七总皂苷的纯化制备方法，其特征在于，在步骤2中，反相色谱柱的填料基质为硅胶或高分子聚合物；填料配基的碳原子数为1-30的正链烷基中的一种或多种；色谱柱内径为20mm～1600mm，色谱柱床高度为100mm～1000mm；色谱平衡液中含有有机溶剂和酸，有机溶剂包括甲醇、乙醇或乙腈，有机溶剂的含量为5％～10％，酸包括为磷酸、甲酸或乙酸，酸的浓度为0.01％～0.5％；色谱柱的载样量为固定相重量的0.01％～20％；色谱的上样和洗脱流速为140cm/h～350cm/h；色谱纯化洗脱总皂苷目标组分的有机溶剂浓度为30-90％，洗脱方式可以是等度或线性梯度。
  3. 根据权利要求1所述一种提取三七总皂苷的纯化制备方法，其特征在于，在步骤2中，反相色谱柱的填料基质为硅胶或高分子聚合物；填料配基的碳原子数为1-30的正链烷基中的一种或多种；色谱柱内径为20mm～1600mm，色谱柱床高度为100mm～1000mm；色谱平衡液中含有有机溶剂和酸，色谱柱的载样量为固定相重量的0.01％～20％；色谱的上样和洗脱流速为140cm/h～350cm/h；色谱纯化洗脱总皂苷目标组分的有机溶剂浓度为30-90％，洗脱方式可以是等度或线性梯度。
  4. 根据权利要求3所述一种提取三七总皂苷的纯化制备方法，其特征在于，所述有机溶剂包括甲醇、乙醇或乙腈，有机溶剂的含量为5％～10％。
  5. 根据权利要求3所述一种提取三七总皂苷的纯化制备方法，其特征在于，所述酸可以为磷酸、甲酸、乙酸，浓度为0.01-0.5％。
  6. 根据权利要求1所述一种提取三七总皂苷的纯化制备方法，其特征在于，步骤3中所用得离子交换色谱技术所用分离材料的基质为琼脂糖、硅胶或高分子聚合物；粒径 为40-100μm；配基的结构式可为
    Figure PCTCN2018119264-appb-100001
    上样体积为柱体积的10-50倍；色谱操作的上样和洗脱流速可以为150-500cm/h；色谱纯化目标组分的方式可以为流穿和洗脱模式。
  7. 根据权利要求1所述一种提取三七总皂苷的纯化制备方法，其特征在于，在步骤1、3中所用浓缩干燥方式可以为微波干燥、冷冻干燥、真空干燥或喷雾干燥。
  8. 一种采用上述制备方法精制的三七总皂苷组分，其特征在于，原料主要组成为三七药材。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1334267A (zh) * 2001-06-28 2002-02-06 中国科学院昆明植物研究所 三七总甙的制备方法
CN1634970A (zh) * 2004-11-18 2005-07-06 张平 一种三七总皂苷的制备方法
CN103948659A (zh) * 2014-05-23 2014-07-30 云南维和药业股份有限公司 三七叶总皂苷的制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101084962A (zh) * 2007-06-18 2007-12-12 云南植物药业有限公司 三七总皂苷制备中的吸附澄清方法
CN101575357B (zh) * 2008-05-09 2011-11-09 澳门大学 一种三七皂苷R1、人参皂苷Rg1、Re、Rb1和Rd的制备方法
WO2013089402A1 (ko) * 2011-12-14 2013-06-20 (주)셀트리온 돌외의 지페노사이드 추출물을 포함하는 제2형 당뇨병, 비만 또는 고지혈증의 치료 또는 예방용 조성물

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1334267A (zh) * 2001-06-28 2002-02-06 中国科学院昆明植物研究所 三七总甙的制备方法
CN1634970A (zh) * 2004-11-18 2005-07-06 张平 一种三七总皂苷的制备方法
CN103948659A (zh) * 2014-05-23 2014-07-30 云南维和药业股份有限公司 三七叶总皂苷的制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MENG, QIONG ET AL.: "Non-official translation: Research Progress on Extraction, Separation and Analytical Method of Total Saponins of Panaxnotoginseng", JOURNAL OF CHENGDE MEDICAL COLLEGE, vol. 27, no. 2, 28 February 2010 (2010-02-28), DOI: 20190823150241A *

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