WO2021093088A1 - 一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法 - Google Patents

一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法 Download PDF

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WO2021093088A1
WO2021093088A1 PCT/CN2019/126537 CN2019126537W WO2021093088A1 WO 2021093088 A1 WO2021093088 A1 WO 2021093088A1 CN 2019126537 W CN2019126537 W CN 2019126537W WO 2021093088 A1 WO2021093088 A1 WO 2021093088A1
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parts
chinese medicine
traditional chinese
shichangpu
water
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PCT/CN2019/126537
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French (fr)
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邹圣灿
张曾亮
李鑫
王尚龙
宗磊
宗建成
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美国琛蓝营养制品股份有限公司
青岛琛蓝医药科技发展有限公司
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Publication of WO2021093088A1 publication Critical patent/WO2021093088A1/zh
Priority to US17/743,414 priority Critical patent/US20220326200A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material

Definitions

  • the invention relates to the field of traditional Chinese medicine analysis, and in particular to a method for establishing a fingerprint of a traditional Chinese medicine compound or its preparation with a cognition-improving effect.
  • MCI mild cognitive impairment
  • VCI vascular cognitive impairment
  • the present invention provides a traditional Chinese medicine compound with cognition-improving effect.
  • the Chinese medicine compound is made of the following raw materials in parts by weight: 1-20 parts of Gastrodia, 1-15 parts of Polygala, 1-30 parts of Shichangpu, 0.1-10 parts of Cistanche, 0.1-10 parts of Rehmannia glutinosa, 0.01-1 part of curcumin.
  • the present invention provides a method for establishing a fingerprint of a traditional Chinese medicine compound or its preparation with a cognitive improvement effect.
  • the fingerprint pattern obtained by the method for establishing the fingerprint pattern comprehensively reflects the quality information of the traditional Chinese medicine compound, so as to achieve the purpose of more comprehensively and effectively controlling the quality of the traditional Chinese medicine compound product.
  • the present invention provides a method for establishing a fingerprint of a traditional Chinese medicine compound with improving cognition or its preparation.
  • the traditional Chinese medicine compound is made of the following raw materials: 1-20 parts of Gastrodia and 1-15 parts of Polygala , 1-30 parts of Shichangpu, 0.1-10 parts of Cistanche, 0.1-10 parts of Rehmannia glutinosa, 0.01-1 parts of curcumin;
  • the test solution was constructed by high performance liquid chromatography.
  • the chromatographic conditions were as follows: the column was octadecylsilane bonded silica gel column; the column temperature was 25 ⁇ 35°C; the flow rate was 0.9 ⁇ 1.1mL/min; the detection wavelengths were respectively 210 ⁇ 230nm, 310 ⁇ 330nm; mobile phase A is acetonitrile, mobile phase B is water, the gradient elution procedure is:
  • the detection wavelengths are 220 and 320 nm, respectively.
  • the chromatographic column is InertSustain AQ-C18, the specification is: 4.6 ⁇ 250mm, 5 ⁇ m.
  • the column temperature is 30°C.
  • the flow rate is 1.0 mL/min.
  • the preparation method of the test solution is: take the traditional Chinese medicine compound or its preparation and mix with the acetonitrile aqueous solution, in g/mL, the ratio of the traditional Chinese medicine compound or its preparation to the acetonitrile aqueous solution is (0.5 ⁇ 1.5): 50, Ultrasound treatment, filter, and take additional filtrate.
  • the volume percentage concentration of the acetonitrile aqueous solution is 40% to 60%.
  • the volume percentage concentration of the acetonitrile aqueous solution is 50%.
  • the ratio of the traditional Chinese medicine complex or its preparation to the aqueous acetonitrile solution is 1:50.
  • the ultrasonic treatment time is 40 minutes.
  • the traditional Chinese medicine compound is made from the following raw materials: 16-20 parts Gastrodia, 10-14 parts Polygala, 16-20 parts Shichangpu, 5-7 parts Cistanche, 5-7 parts Rehmannia glutinosa, Curcumin 0.05 ⁇ 0.15 parts.
  • the traditional Chinese medicine compound in parts by weight, is made of the following raw materials: 18 parts of Tianma, 12 parts of Polygala, 18 parts of Shichangpu, 6 parts of Cistanche, 6 parts of Rehmannia glutinosa, and 0.1 parts of curcumin. Copies.
  • the preparation method of the traditional Chinese medicine complex includes the following steps:
  • Alcohol extraction Gastrodia, Polygala and ethanol aqueous solution are heated to reflux, and alcohol extraction liquid and alcohol extraction residue are collected;
  • Water extraction add water and heat reflux for Shichangpu, collect Shichangpu medicinal solution, Shichangpu medicinal residue and Shichangpu volatile oil; Cistanche, Rehmannia glutinosa, alcohol extract medicinal residue, Shichangpu medicinal residue, add water and heat to reflux, and collect water extract liquid;
  • Filtration and concentration Mix the alcohol extraction liquid, Shichangpu liquid and water extraction liquid, and filter and concentrate to obtain the extract;
  • Mixing Mix the extract and pellets.
  • the volume percent concentration of the ethanol aqueous solution is 50% to 80%; the amount of the ethanol aqueous solution is 8 to 20 times the total weight of Gastrodia and Polygala; the number of heating and refluxing is 1 to 5 times, each time 0.5 ⁇ 3 hours;
  • the amount of the ethanol aqueous solution is 10 times the total weight of Gastrodia and Polygala; the number of heating and refluxing is 3 times, each time is 1 hour.
  • Shichangpu is heated and refluxed with 8-30 times the weight of water, the number of heating and refluxing is 1 to 3 times, each time is 3 to 5 hours, and Shichangpu medicinal solution, Shichangpu medicinal residue and stone are collected.
  • Volatile oil of Acorus; Cistanche, Rehmannia glutinosa, alcohol-extracted medicinal residue, Acorus medicinal residue and 8-30 times the total weight of the medicinal materials are heated to reflux, the number of heating and refluxing is 1 to 5 times, 0.5 to 3 hours each time, and the water is collected and extracted Liquid medicine
  • the Shichangpu is heated to reflux with 14 times the weight of water, and the number of heating and refluxing is once, each time is 5 hours.
  • the cistanche, radix rehmanniae, the alcohol-extracted medicinal residue, the calamus medicinal residue plus the water 10 times the total weight of the medicinal materials are heated and refluxed, and the number of heating and refluxing is 2 Times, 1.5 hours each time.
  • the weight ratio of the mixture of calamus calamus volatile oil and curcumin to ⁇ -cyclodextrin is 1: (1-5); the grinding time during inclusion is 1 to 4 hours, and the drying temperature is 40 ⁇ 60°C.
  • the weight ratio of the mixture of Acorus calamus volatile oil and curcumin to ⁇ -cyclodextrin is 1:1.5; the grinding time during inclusion is 2 hours, and the drying temperature It is 45°C.
  • the present invention provides a method for establishing a fingerprint of a traditional Chinese medicine compound or its preparation with the function of improving cognition.
  • the traditional Chinese medicine compound is made of the following raw materials: 1-20 parts of Gastrodia, 1-15 parts Polygala 1-15 parts, Acorus 1-30 parts, Cistanche cistanche 0.1-10 parts, Rehmannia glutinosa 0.1-10 parts, and curcumin 0.01 parts ⁇ 1 copy;
  • the test solution uses high performance liquid chromatography to construct the atlas, the chromatographic conditions are: chromatographic column adopts octadecylsilane bonded silica gel column; column temperature 25 ⁇ 35°C; flow rate 0.9 ⁇ 1.1mL/min;
  • the detection wavelengths are respectively 210-230nm and 310-330nm; mobile phase A is acetonitrile, mobile phase B is water, gradient elution.
  • the technical effects of the present invention are:
  • the method for establishing a fingerprint of a traditional Chinese medicine compound with improved cognition comprehensively reflects the quality information of the traditional Chinese medicine compound, so as to achieve a more comprehensive and effective control of the quality of the traditional Chinese medicine compound product. purpose.
  • the method for establishing fingerprints of traditional Chinese medicine complexes with improved cognition adopts the Chinese medicine chromatographic fingerprint similarity evaluation system provided by the National Pharmacopoeia Commission to identify the measured fingerprints, and the operation is convenient and fast; moreover, The relative degree results obtained are used to evaluate the fingerprint of the preparation, and the conclusion is relatively objective and accurate.
  • Figure 1 shows the fingerprint of the Chinese medicine complex in Example 1
  • Figure 2 shows the fingerprint of the extract of Acorus calamus in Example 2
  • Figure 3 shows the fingerprint of Polygala extract in Example 3.
  • Figure 4 shows the fingerprint of the Chinese medicine complex of Comparative Example 1
  • Figure 5 shows the fingerprint of the Chinese medicine complex of Comparative Example 2
  • Figure 6 shows the fingerprint of the traditional Chinese medicine complex in Example 4.
  • Figure 7 shows the fingerprint of the traditional Chinese medicine complex in Example 4.
  • Figure 8 shows the fingerprint of the Chinese medicine complex of Example 4.
  • the present invention discloses a method for establishing a fingerprint of a traditional Chinese medicine compound or its preparation with an effect of improving cognition.
  • Those skilled in the art can learn from the content of this article and appropriately improve the process parameters.
  • all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the reagents or instruments used in the method for establishing the fingerprint of the traditional Chinese medicine complex with cognition improvement or its preparation provided by the present invention can be purchased from the market.
  • Gastrodia 18g Polygala 12g, Shichangpu 18g, Cistanche cistanche 6g, Rehmannia glutinosa 6g, and curcumin 0.1g.
  • the preparation method is:
  • step (2) the volatile oil is mixed with curcumin, and then encapsulated by cyclodextrin to synthesize pellets.
  • the weight ratio of the mixture of volatile oil and curcumin to ⁇ -cyclodextrin is 1:1.5, the grinding time is 2 hours, and the drying temperature is 45°C.
  • step (6) Mix the extract of step (4) with the pellets of step (5) to obtain.
  • Octadecyl-bonded silica gel is used as the packing, and the column model is: InertSustain AQ-C18 (4.6 ⁇ 250mm, 5 ⁇ m); use acetonitrile as mobile phase A, water as mobile phase B, and perform gradient washing as specified in the following table
  • the column temperature is 30°C
  • the flow rate is 1mL/min
  • the detection wavelength is 220nm, 320nm.
  • chromatographic peaks with better resolution and larger content are selected from the spectrum as characteristic peaks.
  • the peaks are labeled 1-13 in turn, and their retention times are 27.30, 28.56, 36.18, 43.70, 44.67, 47.37, 50.57, 56.32, 70.08, 71.00, 79.94, 100.89, 105.195.
  • the peak areas are 462683, 466606, 5751435, 1019754, 2121885, 1037280, 4017599, 837200, 450141, 571265, 950998, 1003678, 11177198, respectively.
  • Figure 2 shows the spectrum of the injection of 10 microliters.
  • chromatographic peaks with better resolution and larger content are selected from the spectrum as characteristic peaks.
  • the peaks are labeled 1-4 in sequence, and their retention times are 46.28, 58.77, 67.45, 72.52, respectively.
  • the peak areas are 52868, 32769, 30682, 25271, respectively.
  • Figure 3 shows the spectrum of the injection of 10 microliters.
  • chromatographic peaks with better resolution and larger content are selected from the spectrum as characteristic peaks. They are labeled as peaks 1-7 in sequence, and their retention times are 27.05, 28.38, 50.34, 51.81, 56.15, 70.08, 71.01, respectively.
  • the peak areas are 1960057, 1448764, 10550417, 1685443, 3468449, 1390382, 1978811, respectively.
  • Example 1 The only difference from the method in Example 1 is that the mobile phase elution gradient is different.
  • Figure 4 shows the spectrum of the injection of 10 microliters.
  • the only difference from the method in Example 1 is that the fingerprint detection wavelength is different.
  • Figure 5 shows the spectrum of the injection of 10 microliters.
  • Example 2 The method is the same as in Example 1, and 3 batches of traditional Chinese medicine complexes are extracted respectively. Compare the retention time and peak area of the peaks labeled 1-13 in sequence. The results are as follows (see 6, 7, 8 for the spectrum).
  • n is the number of fingerprint peaks of the fingerprint
  • n is the peak area of the j-th fingerprint peak of the fingerprint of the mixed extract
  • the peak area of the jth fingerprint peak of the fingerprint is the average of the fingerprint peak area of the fingerprint of the standard extract.
  • the similarity is 0.9982 with that in Example 1, and the compound specified S should be ⁇ 0.9.
  • the retention time deviation is ⁇ 0.2min. It shows that the fingerprint method is stable and feasible.

Abstract

一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法。以重量份计，该中药复合物由如下原料制成：天麻1～20份，远志1～15份，石菖蒲1～30份，肉苁蓉0.1～10份，熟地黄0.1～10份，姜黄素0.01～1份；供试品溶液采用高效液相色谱法构建图谱，色谱条件为：色谱柱采用十八烷基硅烷键合硅胶色谱柱；柱温25～35℃；流速0.9～1.1mL/min；检测波长分别为210～230nm、310～330nm；流动相A为乙腈，流动相B为水，梯度洗脱。该方法所得指纹图谱全面反映出该中药复合物质量信息，从而能够达到更加全面、有效地控制此中药复合物产品质量的目的。

Description

一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法
本申请要求于2019年11月14日提交中国专利局、申请号为201911112565.8、发明名称为“一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及中药分析领域，特别涉及一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法。
背景技术
随着世界老龄化趋势逐年增加，作为正常老龄化向痴呆过渡状态的轻度认知功能障碍(mild cognitive impaiment,MCI)、血管性认知功能障碍(Vascular cognitive impairment,VCI)越来越受到人们的关注。在发达国家，老年痴呆的死亡率仅次于心脏病、肿瘤、中风位居第四。随着世界人口日趋老龄化，该病已经成为当前老年医学面临的最严峻的问题之一。而老年痴呆的前期轻度的认知障碍被公认为是一种高度流行和可以预防的综合征。目前，针对老年痴呆患者还没有特效治疗方法。美国FDA批准的药物可短期改善痴呆症状但不能减缓病情发展。积极开展对MCI、VCI的研究和防治工作，对提高患者的生存质量有着重要的意义。
对轻度认知功能障碍积极进行干预是延缓认知功能进一步衰退的有效措施，但目前关于轻度认知功能障碍干预措施的报道较少，药物也仅有个别的多中心研究报道。因此，就目前国内外对轻度认知功能障碍的治疗现状及进展进行系统性研究。由于最多见的轻度认知功能障碍亚型是遗忘型认知功能障碍即阿尔茨海默病的前期，故而关于轻度认知功能障碍的干预策略多沿袭了阿尔茨海默病的治疗方案。
中药复方如调心方、补肾方等具有保护神经，提高学习记忆作用。脑认知作用退化与“肾虚”密切相关，肾亏之年、精髓乏源，髓海失养，神机失用，则渐发痴呆。同时年老脏腑虚衰，生理或病理产物不能及时排除，蓄积体内，生痰生瘀，痰蒙清窍和/或淤阻脑络，使脑气与脏器不相连接，神机失用，便发痴呆。临床研究表明，脑认知功能降低中的肾气不足、痰浊蒙窍和脑络淤阻症 候分别占97％、78％、76％。本发明提供了一种具有改善认知作用的中药复合物，以重量份计，该中药复合物由如下原料制成：天麻1～20份，远志1～15份，石菖蒲1～30份，肉苁蓉0.1～10份，熟地黄0.1～10份，姜黄素0.01～1份。
采用中药指纹图谱方法，通过可量化的鉴定手段，建立在中药化学成分系统研究的基础上，评价单味提取物。其控制方法质量真实、优良且稳定。中药及其制剂均为多组分复杂体系，因此建立中药指纹图谱将能较为全面地反映中药及其制剂中所含化学成分的种类与数量，能提供丰富鉴别信息的检测方法，进而对药品质量进行整体描述和评价，提高中药质量，促进中药现代化具有重要意义。目前缺乏对此改善认知作用的中药配方复合物系统的定性研究。因此，有必要建立此中药配方复合物的指纹图谱，能够完成对此中药配方复合物的定性分析，以便对此中药配方复合物有效的质量控制。
发明内容
有鉴于此，本发明提供了一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法。该指纹图谱的建立方法所得的指纹图谱全面反映出该中药复合物质量信息，从而能够达到更加全面、有效地控制此中药复合物产品质量的目的。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法，以重量份计，该中药复合物由如下原料制成：天麻1～20份，远志1～15份，石菖蒲1～30份，肉苁蓉0.1～10份，熟地黄0.1～10份，姜黄素0.01～1份；
供试品溶液采用高效液相色谱法构建图谱，色谱条件为：色谱柱采用十八烷基硅烷键合硅胶色谱柱；柱温25～35℃；流速0.9～1.1mL/min；检测波长分别为210～230nm、310～330nm；流动相A为乙腈，流动相B为水，梯度洗脱程序为：
Figure PCTCN2019126537-appb-000001
Figure PCTCN2019126537-appb-000002
作为优选，检测波长分别为220、320nm。
作为优选，色谱柱为InertSustain AQ-C18，规格为：4.6×250mm，5μm。
作为优选，柱温为30℃。
作为优选，流速1.0mL/min。
作为优选，供试品溶液的制备方法为：取中药复合物或其制剂与乙腈水溶液混合，以g/mL计，中药复合物或其制剂与乙腈水溶液的比例为(0.5～1.5)：50，超声处理，滤过，取续滤液。
作为优选，乙腈水溶液的体积百分浓度为40％～60％。
在本发明提供的具体实施例中，乙腈水溶液的体积百分浓度为50％。
在本发明提供的具体实施例中，以g/mL计，中药复合物或其制剂与乙腈水溶液的比例为1：50。
在本发明提供的具体实施例中，超声处理的时间为40分钟。
作为优选，以重量份计，该中药复合物由如下原料制成：天麻16～20份，远志10～14份，石菖蒲16～20份，肉苁蓉5～7份，熟地黄5～7份，姜黄素0.05～0.15 份。
在本发明提供的具体实施例中，以重量份计，该中药复合物由如下原料制成：天麻18份，远志12份，石菖蒲18份，肉苁蓉6份，熟地黄6份，姜黄素0.1份。
作为优选，该中药复合物的制备方法包括如下步骤：
醇提：天麻、远志加乙醇水溶液加热回流，收集醇提药液、醇提药渣；
水提：石菖蒲加水加热回流，收集石菖蒲药液、石菖蒲药渣以及石菖蒲挥发油；肉苁蓉、熟地黄、醇提药渣、石菖蒲药渣加水加热回流，收集水提药液；
过滤浓缩：将醇提药液、石菖蒲药液、水提药液混合，过滤浓缩得浸膏；
包合：将石菖蒲挥发油和姜黄素混合，经环糊精包合得微丸；
混合：将浸膏与微丸混合。
作为优选，醇提步骤中，乙醇水溶液的体积百分浓度为50％～80％；乙醇水溶液的用量为天麻、远志总重量的8～20倍；加热回流的次数为1～5次，每次0.5～3小时；
在本发明提供的具体实施例中，醇提步骤中，乙醇水溶液的用量为天麻、远志总重量的10倍；加热回流的次数为3次，每次1小时。
作为优选，水提步骤中，石菖蒲加8～30倍重量的水加热回流，加热回流的次数为1～3次，每次3～5小时，收集石菖蒲药液、石菖蒲药渣以及石菖蒲挥发油；肉苁蓉、熟地黄、醇提药渣、石菖蒲药渣加药材总重8～30倍的水加热回流，加热回流的次数为1～5次，每次0.5～3小时，收集水提药液；
在本发明提供的具体实施例中，石菖蒲加14倍重量的水加热回流，加热回流的次数为1次，每次5小时。
在本发明提供的具体实施例中，水提步骤中，肉苁蓉、熟地黄、所述醇提药渣、所述石菖蒲药渣加药材总重10倍的水加热回流，加热回流的次数为2次，每次1.5小时。
作为优选，包合步骤中，石菖蒲挥发油和姜黄素的混合物与β-环糊精的重量比为1：(1～5)；包合时的研磨时间为1～4小时，烘干温度为40～60℃。
在本发明提供的具体实施例中，包合步骤中，石菖蒲挥发油和姜黄素的混合物与β-环糊精的重量比为1：1.5；包合时的研磨时间为2小时，烘干温度为 45℃。
在本发明提供的具体实施例中，
本发明提供了一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法。以重量份计，该中药复合物由如下原料制成：天麻1～20份，远志1～15份，石菖蒲1～30份，肉苁蓉0.1～10份，熟地黄0.1～10份，姜黄素0.01～1份；供试品溶液采用高效液相色谱法构建图谱，色谱条件为：色谱柱采用十八烷基硅烷键合硅胶色谱柱；柱温25～35℃；流速0.9～1.1mL/min；检测波长分别为210～230nm、310～330nm；流动相A为乙腈，流动相B为水，梯度洗脱。本发明具有的技术效果为：
1、本发明提供的具有改善认知作用的中药复合物指纹图谱的建立方法所得的指纹图谱全面反映出该中药复合物质量信息，从而能够达到更加全面、有效地控制此中药复合物产品质量的目的。
2、本发明提供的具有改善认知作用的中药复合物指纹图谱的建立方法采用国家药典委员会提供的中药色谱指纹图谱相似度评价系统对所测指纹图谱的辨认，操作方便、快捷；而且，以此得出的相以度结果对制剂指纹图谱进行评价，结论较为客观、准确。
附图说明
图1示实施例1中药复合物的指纹图谱；
图2示实施例2中石菖蒲提取物指纹图谱；
图3示实施例3中远志提取物指纹图谱；
图4示对比例1中药复合物的指纹图谱；
图5示对比例2中药复合物的指纹图谱；
图6示实施例4中药复合物的指纹图谱；
图7示实施例4中药复合物的指纹图谱；
图8示实施例4中药复合物的指纹图谱。
具体实施方式
本发明公开了一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别 需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法中所用试剂或仪器均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1 中药复合物指纹图谱的构建
中药复合物及其制备方法：
1、本实施例配方为：
天麻18g，远志12g，石菖蒲18g，肉苁蓉6g，熟地黄6g，姜黄素0.1g。
2、制备方法为：
(1)将天麻、远志加10倍量70％乙醇加热回流3次，每次1小时，分别收集药液、药渣；
(2)将石菖蒲加14倍量水加热回流1次，5小时，分别收集药液，药渣以及挥发油；
(3)将肉苁蓉、熟地黄以及步骤(1)、(2)收集的中药渣加10倍量水加热回流2次，每次1.5小时，分别收集药液；
(4)将步骤(1)、(2)、(3)中所得全部药液过滤浓缩得浸膏；
(5)步骤(2)中挥发油与姜黄素混合，经环糊精包合成微丸。挥发油和姜黄素的混合物与β-环糊精的重量比为1：1.5，研磨时间为2小时，烘干温度为45℃。
(6)将步骤(4)浸膏与步骤(5)微丸混合即得。
3、供试品制备：
取1g复合物混悬于50mL 1:1乙腈-水中，超声处理40min，滤纸滤过，取续滤液，过0.22微孔滤膜即得复合物样品。
4、指纹图谱参考色谱条件：
十八烷基键合硅胶为填料，色谱柱型号为：InertSustain AQ-C18(4.6×250mm，5μm)；以乙腈为流动相A，以水为流动相B，按下表中的规定进行梯度洗脱，柱温30℃，流速为1mL/min；检测波长为220nm、320nm。
表1洗脱梯度程序
Figure PCTCN2019126537-appb-000003
进样10微升得图谱，见图1。
根据指纹图谱的要求，从图谱中选择分离度较好且含量较大的13个色谱峰作为特征峰。依次标记为1-13号峰，其保留时间分别为27.30、28.56、36.18、43.70、44.67、47.37、50.57、56.32、70.08、71.00、79.94、100.89、105.195。其峰面积分别为462683、466606、5751435、1019754、2121885、1037280、4017599、837200、450141、571265、950998、1003678、11177198。
实施例2 石菖蒲提取物指纹图谱的构建
石菖蒲提取物制备方法：
取石菖蒲100g，浸泡3小时。8倍水加热回流提取3次，每次2h，2h，1.5h。合并提取液，过滤浓缩干燥后，即得石菖蒲提取物。
精密称取1.00g石菖蒲提取物，精密加入50％乙腈溶液20mL；超声提取40min，滤纸滤过，续滤液以0.22微孔滤膜滤过，取续滤液，即得石菖蒲提取物样品。
石菖蒲提取物指纹图谱参考色谱条件：十八烷基键合硅胶为填料，色谱柱型号为：InertSustain AQ-C18(4.6×250mm，5μm)；以乙腈为流动相A，以水为流动相B，按表1中的规定进行梯度洗脱，柱温30℃，流速为1mL/min；检测波长为220nm。
进样10微升得图谱见图2。
根据指纹图谱的要求，从图谱中选择分离度较好且含量较大的4个色谱峰作为特征峰。依次标记为1-4号峰，其保留时间分别为46.28、58.77、67.45、72.52。其峰面积分别为52868、32769、30682、25271。
实施例3 远志提取物指纹图谱的构建
远志提取物制备方法：
取远志药材100g，80％乙醇，加热回流提取3次，每次2h，液料比为8、6、6倍。合并提取液，过滤浓缩干燥后，即得远志提取物。
精密称取1.00g远志提取物，精密加入50％乙腈溶液20mL；超声提取40min，滤纸滤过，续滤液以0.22微孔滤膜滤过，取续滤液，即得远志提取物样品。
远志提取物指纹图谱参考色谱条件：十八烷基键合硅胶为填料，色谱柱型号为：InertSustain AQ-C18(4.6×250mm，5μm)；以乙腈为流动相A，以水为流动相B，按表1中的规定进行梯度洗脱，柱温30℃，流速为1mL/min；检测波长为280nm。
进样10微升得图谱见图3。
根据指纹图谱的要求，从图谱中选择分离度较好且含量较大的7个色谱峰作为特征峰。依次标记为1-7号峰，其保留时间分别为27.05、28.38、50.34、51.81、56.15、70.08、71.01。其峰面积分别为1960057、1448764、10550417、1685443、3468449、1390382、1978811。
对比例1 中药复合物指纹图谱的构建
与实施例1方法唯一不同的是流动相洗脱梯度不同。
取1g复合物(未含姜黄素)混悬于20mL 1:1乙腈-水中，超声处理40min， 滤纸滤过，续滤液以0.22微孔滤膜滤过，取续滤液，即得复合物样品。
复合物指纹图谱参考色谱条件：十八烷基键合硅胶为填料，色谱柱型号为：InertSustain AQ-C18(4.6×250mm，5μm)；以乙腈为流动相A，以水为流动相B，按下表中的规定进行梯度洗脱，柱温30℃，流速为1mL/min；检测波长为220nm。
表2洗脱梯度程序
Figure PCTCN2019126537-appb-000004
进样10微升得图谱见图4。
如图4可以看出，未经优化的对比例1中，没能将主要的峰成功的分离，只有少数峰有较高的分离度，可以用作指纹峰的参照。而优化后的实施例1中，较好的分离了13个峰，达到了指纹图谱的要求。
对比例2 中药复合物指纹图谱的构建
与实施例1方法唯一不同的是指纹图谱检测波长不同。
取1g复合物混悬于50mL 1:1乙腈-水中，超声处理40min，滤纸滤过，续滤液以0.22微孔滤膜滤过，取续滤液，即得复合物样品。
复合物指纹图谱参考色谱条件：十八烷基键合硅胶为填料，色谱柱型号为：InertSustain AQ-C18(4.6×250mm，5μm)；以乙腈为流动相A，以水为流动相B，按下表中的规定进行梯度洗脱，柱温30℃，流速为1mL/min；检测波长为 360nm。
表3洗脱梯度程序
Figure PCTCN2019126537-appb-000005
进样10微升得图谱见图5。
如图5，当波长超出权利要求给出的范围后，吸收信号降低，峰面积减少，不能够呈现出良好的指纹图谱效果。
实施例4 中药复合物指纹图谱的稳定性
与实施例1方法相同，分别提取3批次中药复合物。对依次标记为1-13号峰，其保留时间、以及峰面积进行比较。其结果如下(图谱见6、7、8)。
表4 3批次中药复合物的保留时间以及峰面积
Figure PCTCN2019126537-appb-000006
Figure PCTCN2019126537-appb-000007
相似度公式：
Figure PCTCN2019126537-appb-000008
其中S为指纹图谱相似度，n为指纹图谱指纹峰的个数，为混合提取物指纹图谱第j个指纹峰的峰面积，为混合提取物指纹图谱指纹峰峰面积的均值，为标准提取物指纹图谱第j个指纹峰峰面积，为标准提取物指纹图谱指纹峰峰面积的均值。
根据相似度公式得出，其相似度与实施例1中相似度为0.9982，复合规定的S应≥0.9。并且保留时间偏差≤0.2min。说明指纹图谱方法稳定可行。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种具有改善认知作用的中药复合物或其制剂指纹图谱的建立方法，其特征在于，以重量份计，所述中药复合物由如下原料制成：天麻1～20份，远志1～15份，石菖蒲1～30份，肉苁蓉0.1～10份，熟地黄0.1～10份，姜黄素0.01～1份；
    供试品溶液采用高效液相色谱法构建图谱，色谱条件为：色谱柱采用十八烷基硅烷键合硅胶色谱柱；柱温25～35℃；流速0.9～1.1mL/min；检测波长分别为210～230nm、310～330nm；流动相A为乙腈，流动相B为水，梯度洗脱程序为：
    Figure PCTCN2019126537-appb-100001
    Figure PCTCN2019126537-appb-100002
  2. 根据权利要求1所述的建立方法，其特征在于所述检测波长分为220nm、320nm。
  3. 根据权利要求1所述的建立方法，其特征在于，所述色谱柱为InertSustain AQ-C18，规格为：4.6×250mm，5μm。
  4. 根据权利要求1所述的建立方法，其特征在于，所述柱温为30℃。
  5. 根据权利要求1所述的建立方法，其特征在于，所述流速1.0mL/min。
  6. 根据权利要求1所述的建立方法，其特征在于，所述供试品溶液的制备方法为：取中药复合物或其制剂与乙腈水溶液混合，以g/mL计，中药复合物或其制剂与乙腈水溶液的比例为(0.5～1.5)：50，超声处理，滤过，取续滤液。
  7. 根据权利要求1所述的建立方法，其特征在于，所述乙腈水溶液的体积百分浓度为40％～60％。
  8. 根据权利要求1所述的建立方法，其特征在于，以重量份计，所述中药复合物由如下原料制成：天麻16～20份，远志10～14份，石菖蒲16～20份，肉苁蓉5～7份，熟地黄5～7份，姜黄素0.05～0.15份。
  9. 根据权利要求1所述的建立方法，其特征在于，所述中药复合物的制备方法包括如下步骤：
    醇提：天麻、远志加乙醇水溶液加热回流，收集醇提药液、醇提药渣；
    水提：石菖蒲加水加热回流，收集石菖蒲药液、石菖蒲药渣以及石菖蒲挥发油；肉苁蓉、熟地黄、所述醇提药渣、所述石菖蒲药渣加水加热回流，收集水提药液；
    过滤浓缩：将所述醇提药液、所述石菖蒲药液、所述水提药液混合，过滤浓缩得浸膏；
    包合：将所述石菖蒲挥发油和姜黄素混合，经环糊精包合得微丸；
    混合：将所述浸膏与所述微丸混合。
  10. 根据权利要求9所述的建立方法，其特征在于，醇提步骤中，所述乙醇水溶液的体积百分浓度为50％～80％；所述乙醇水溶液的用量为天麻、远志总重量的8～20倍；加热回流的次数为1～5次，每次0.5～3小时；
    水提步骤中，石菖蒲加8～30倍重量的水加热回流，加热回流的次数为1～3次，每次3～5小时，收集石菖蒲药液、石菖蒲药渣以及石菖蒲挥发油；肉苁蓉、熟地黄、所述醇提药渣、所述石菖蒲药渣加药材总重8～30倍的水加热回流，加热回流的次数为1～5次，每次0.5～3小时，收集水提药液；
    包合步骤中，所述石菖蒲挥发油和姜黄素的混合物与β-环糊精的重量比为1：(1～5)；所述包合时的研磨时间为1～4小时，烘干温度为40～60℃。
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