WO2019184194A1 - 地黄素的制备、分析和细胞免疫治疗用途 - Google Patents

地黄素的制备、分析和细胞免疫治疗用途 Download PDF

Info

Publication number
WO2019184194A1
WO2019184194A1 PCT/CN2018/100123 CN2018100123W WO2019184194A1 WO 2019184194 A1 WO2019184194 A1 WO 2019184194A1 CN 2018100123 W CN2018100123 W CN 2018100123W WO 2019184194 A1 WO2019184194 A1 WO 2019184194A1
Authority
WO
WIPO (PCT)
Prior art keywords
extract
phase
pyroxene
dichloromethane
ethanol
Prior art date
Application number
PCT/CN2018/100123
Other languages
English (en)
French (fr)
Inventor
马海周
Original Assignee
马海周
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201810249755.3A external-priority patent/CN108220240B/zh
Priority claimed from CN201810249721.4A external-priority patent/CN108250209B/zh
Priority claimed from CN201810249739.4A external-priority patent/CN108267524B/zh
Application filed by 马海周 filed Critical 马海周
Publication of WO2019184194A1 publication Critical patent/WO2019184194A1/zh

Links

Images

Classifications

    • 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)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • A61K36/804—Rehmannia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/06—Peri-condensed systems
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient

Definitions

  • the invention belongs to the field of medicine, and particularly relates to the preparation, analysis and cellular immunotherapy use of emodin.
  • the digoxigenin B, digoxigenin D, pyroxene A and pyroxene B are natural products isolated from Rehmannia glutinosa.
  • the chemical structures are very similar, and the chemical information is shown in the following table.
  • a method for preparing retinoic acid B, digesterin D and pyroxene A comprises the following steps:
  • step S1 the fresh yellow sage is washed and sliced and dried to obtain fresh radix; the fresh radix is extracted by hot reflux with an aqueous solution of ethanol, and the extract is concentrated under reduced pressure to obtain an extract;
  • Step S2 the extract is dissolved with an appropriate amount of water, first extracted with a volume of petroleum ether, and then extracted with dichloromethane, and the dichloromethane extract is combined and concentrated to obtain a dichloromethane extract;
  • Step S3 the dichloromethane extract is dissolved and applied to the XDA-1B macroporous adsorption resin column, first eluted with 30% ethanol, and then eluted with 75% ethanol, and the 8-10 BV eluate is collected and concentrated and dried. Obtaining a crude extract of Rehmannia glutinosa;
  • Step S4 the crude extract of Rehmannia glutinosa extract is applied to a normal phase silica gel column, and eluted with a mixed solvent of dichloromethane/methanol/isopropanol at a volume ratio of 10:1:0.2, and the 6-7BV eluate is collected and concentrated and dried.
  • Step S5 preparing liquid phase separation: separating the obtained extract of Rehmannia glutinosa by preparative liquid chromatography, and collecting the corresponding fractions of digoxigenin B, digesterin D, and pyroxene A according to a chromatogram;
  • Step S6 desalting: concentrating the corresponding fractions of digoxigenin B, digoxigen D and pyroxene A to dryness, and then sonicating with methanol, sodium N-hexadecanoyl-L-serine is insoluble in methanol, and the filtrate is concentrated by filtration. Drying gives digoxigen B, digesterin D, and pyroflavin A.
  • the preparative liquid chromatography parameters are as follows:
  • Mobile phase A phase acetonitrile/water in a volume ratio of 1:9, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • Mobile phase B phase acetonitrile/water in a volume ratio of 9:1, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • step S1 the fresh yellow tablets are extracted by hot refluxing with a 95% aqueous solution of ethanol for 3 times for 2 hours, and the solid-liquid ratio is 1:20.
  • the extracts are combined and concentrated under reduced pressure to obtain an extract.
  • step S2 the extract is dissolved with an appropriate amount of water, firstly extracted by volumetric extraction with petroleum ether three times, and then extracted three times with dichloromethane, and the dichloromethane extract is combined and concentrated to dryness.
  • step S3 the dichloromethane extract is dissolved in a 30% aqueous solution of ethanol, and the resin is applied to an XDA-1B macroporous adsorption resin column.
  • the resin diameter ratio of the XDA-1B macroporous adsorption resin column is 1:10, and the mixing resin accounts for 1/10 of the total amount of the resin.
  • step S3 is first eluted with 12 BV of 30% ethanol, and eluted with 75% ethanol at a flow rate of 10 BV/h to collect an 8-10 BV eluate.
  • the aspect ratio of the silica gel is 1:10, and the mixed silica gel accounts for 1/10 of the total amount of the silica gel, and the mixed solvent of dichloromethane/methanol/isopropanol in a volume ratio of 10:1:0.2 is 10 BV. Elution at /h speed, 6-7 BV eluate was collected and concentrated to dryness.
  • An HPLC method for detecting pyroxene B in pyroxene A the mobile phase containing sodium N-hexadecayl-L-serine; specifically, the mobile phase A phase is acetonitrile/water according to a volume ratio of 1:9 a mixed solution containing 5 ppm of sodium N-hexadecanoyl-L-serine; a mobile phase B phase of acetonitrile/water in a volume ratio of 9:1, containing 5 ppm of sodium N-hexadecanoyl-L-serine .
  • Example 1 is an HPLC chromatogram of the extract of Rehmannia glutinosa prepared in Example 2;
  • Figure 3 is a liquid chromatogram prepared in Example 3.
  • 4-6 are HPLC chromatograms of retinoic acid B, digoxigen D, and pyroxene A obtained in Example 3;
  • step S7 is an HPLC chromatogram of the extract of Rehmannia glutamate obtained in step S4, if XDA-1 macroporous adsorption resin is used in place of XDA-1B macroporous adsorption resin in step S3 of Example 3;
  • Figure 8 is an HPLC chromatogram of the sample solution of Example 4.
  • Figure 9 is a separation diagram of Example 4 using a flow-relative mixing solution without the addition of sodium N-hexadecayl-L-serine.
  • Example 1 Effect of emodin B, digoxigen D, pyroxene A and pyroflavin B on proliferation of CIK cells in vitro
  • the retinoic acid B, digoxigen D, pyroxene A and pyroxene B were self-made, and the purity was above 95%.
  • PBMC peripheral blood of healthy volunteers by Ficoll-Hypaque density gradient centrifugation, and 4 mL of whole blood was diluted with 4 mL of PBS (1:1 ratio). The diluted blood was removed and slowly added to a 4 mL Ficoll-Hypaque centrifuge tube. The liquid surface interface was centrifuged at 2000 r/min for 20 min, and single nuclear cells were gently aspirated into another centrifuge tube. 5 mL of physiological saline was added, and the cells were gently blown and washed twice by centrifugation.
  • CIK initial culture solution to PBMC suspended in physiological saline, adjust the cell concentration to 5 ⁇ 10 5 /mL, plant in 24-well plate, 200 ⁇ L per well, first add RPMI1640 medium containing 1000 U/mL IFN- ⁇ .
  • the cells were cultured in a 5% CO 2 and 37 ° C incubator; after 24 h, 100 ng/mL anti-CD3, 500 U/mL IL-2 was added; after 4 days of culture, the cell concentration was adjusted to 1 ⁇ 10 6 /mL, and 500 U was added every 2 days. /mL IL-2.
  • the CIK cells were harvested by day 10 amplification.
  • the CIK cells cultured for 10 days were collected, and the number of cells of 1 ⁇ 10 5 /mL was inoculated into a 96-well plate at 200 ⁇ L/well, and 20 ⁇ M of emodin B, digoxigen D, pyroxene A or pyroflavin B were added respectively (control group addition, etc.) Volumetric medium), after incubation for 48 h, CCK8 was added, and after incubation, the absorbance (OD) of each well was measured at 450 nm.
  • the preparation method comprises the following steps:
  • Step S1 the fresh yellow radix is washed and sliced, dried at 55 ° C to obtain fresh rehmannia tablets; the fresh rehmannia tablets are extracted by hot reflux with a 95% ethanol aqueous solution for 3 times, each time for 2 h, the solid-liquid ratio is 1:20, and the extracts are combined. Concentrated under reduced pressure to obtain an extract;
  • Step S2 the extract is dissolved with an appropriate amount of water, first extracted with petroleum ether in equal volume for 3 times, and then extracted with dichloromethane for 3 times, and the dichloromethane extract is combined and concentrated to obtain a dichloromethane extract;
  • Step S3 the dichloromethane extract is dissolved in a 30% aqueous solution of ethanol, and the resin is applied to the XDA-1B macroporous adsorption resin column, the resin diameter ratio is 1:10, and the mixed resin accounts for 1/10 of the total resin.
  • step S4 the crude extract of Rehmannia glutinosa extract is applied to a normal phase silica gel column with a silica gel diameter ratio of 1:10, and the sampled silica gel accounts for 1/10 of the total amount of the silica gel; and the volume ratio is 10:1:0.2 of dichloromethane.
  • the /methanol/isopropanol mixed solvent was eluted at a rate of 10 BV/h, and the 6-7 BV eluate was collected and concentrated to obtain a rehmannia extract rich in digoxigenin B, digoxigen D, and pyroxicillin A.
  • Mobile phase A phase acetonitrile/water in a volume ratio of 1:9, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • Mobile phase B phase acetonitrile/water in a volume ratio of 9:1, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • Injection volume 10 ⁇ L.
  • Figure 1 is the HPLC chromatogram of the above extract of Rehmannia glutinosa, the main components are emodin B, digoxigen D and pyroxene A, quantified by external standard method, emodin B, digesterin D and pyroxene A account for the total weight of the extract 90.2%. Since pyroxene B does not have an activity for promoting the proliferation of CIK cells, and it is very close to the structure of pyroflavin A, in order to exclude pyroxene B from the above extract, step S4 is used in the eluent of silica gel column chromatography. A small amount of isopropanol was added. If the mobile phase is not added with isopropanol, the other operations are unchanged, and the HPLC chromatogram of the obtained extract is shown in Fig. 2, which contains more pyroflavin B and other impurities.
  • the promoting activity of the extract of Rehmannia glutinosa L. on the proliferation of CIK cells in vitro was tested according to the method of Example 1.
  • the proliferation rate of the extract of 5 ⁇ g/mL rehmannia extract after 48 h intervention was (26.18 ⁇ 1.07)%, which was significantly better than the control group.
  • Example 3 Separation and preparation of retinoic acid B, digoxigen D and pyroxene A monomer
  • the separation preparation method comprises the following steps:
  • Step S1 the fresh yellow radix is washed and sliced, dried at 55 ° C to obtain fresh rehmannia tablets; the fresh rehmannia tablets are extracted by hot reflux with a 95% ethanol aqueous solution for 3 times, each time for 2 h, the solid-liquid ratio is 1:20, and the extracts are combined. Concentrated under reduced pressure to obtain an extract;
  • Step S2 the extract is dissolved with an appropriate amount of water, first extracted with petroleum ether in equal volume for 3 times, and then extracted with dichloromethane for 3 times, and the dichloromethane extract is combined and concentrated to obtain a dichloromethane extract;
  • Step S3 the dichloromethane extract is dissolved in a 30% aqueous solution of ethanol, and the resin is applied to the XDA-1B macroporous adsorption resin column, the resin diameter ratio is 1:10, and the mixed resin accounts for 1/10 of the total resin.
  • step S4 the crude extract of Rehmannia glutinosa extract is applied to a normal phase silica gel column with a silica gel diameter ratio of 1:10, and the sampled silica gel accounts for 1/10 of the total amount of the silica gel; and the volume ratio is 10:1:0.2 of dichloromethane. /Methanol / isopropanol mixed solvent eluted at a rate of 10BV / h, 6-7BV eluate was collected, concentrated and dried to obtain rehmannia extract rich in digoxigenin B, digoxigen D, pyroflavin A;
  • Step S5 preparing liquid phase separation: separating the obtained extract of Rehmannia glutinosa by preparative liquid chromatography, preparing liquid chromatography parameters as follows, and collecting digoxigen B, digesterin D, pyroflavin according to a chromatogram (as shown in FIG. 3) A corresponding stream;
  • Mobile phase A phase acetonitrile/water in a volume ratio of 1:9, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • Mobile phase B phase acetonitrile/water in a volume ratio of 9:1, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • Injection volume 100 ⁇ L
  • Step S6 desalting: concentrating the corresponding fractions of digoxigenin B, digoxigen D and pyroxene A to dryness, and then sonicating with methanol, sodium N-hexadecanoyl-L-serine is insoluble in methanol, and the filtrate is concentrated by filtration. Drying gives digoxigen B, digoxigen D, pyroflavin A monomer.
  • Mobile phase A phase acetonitrile/water in a volume ratio of 1:9, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • Mobile phase B phase acetonitrile/water in a volume ratio of 9:1, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • Injection volume 10 ⁇ L.
  • Figure 4-6 shows the HPLC chromatograms of the prepared emodin B, digoxigen D, and pyroxene A. It can be seen that the purity is high, and the quantification by external standard method is above 95%.
  • the impurity component in the extract of Rehmannia glutinosa obtained in step S4 is significantly more (as shown in FIG. 7), especially digoxigenin B.
  • the impurity between the target and the pyroflavin A has a great influence on the separation and purification of the retinoic acid B and the pyroxene A monomer.
  • Example 4 Method for detecting pyroxene B in pyroxene A
  • Mobile phase A phase acetonitrile/water in a volume ratio of 1:9, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • Mobile phase B phase acetonitrile/water in a volume ratio of 9:1, containing 5 ppm of sodium N-hexadecanoyl-L-serine;
  • Injection volume 10 ⁇ L.
  • Figure 8 is an HPLC chromatogram of the mixed solution. It can be seen that pyroflavin A and pyroflavin B can be effectively separated under the above chromatographic conditions, and the baseline separation is achieved, and the resolution is greater than 2.0.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种地黄素的制备、分析和细胞免疫治疗用途。地黄素B、地黄素D和焦地黄素A可以显著提高CIK细胞体外增殖，而焦地黄素B不具有这种作用。焦地黄素B与焦地黄素A相比，仅Cl连接的碳原子构型不同，地黄素B和地黄素D中该碳原子构型与焦地黄素A相同，证明该碳原子的构型对地黄素类化合物的促进CIK细胞增殖活性至关重要。一种制备地黄素B、地黄素D和焦地黄素A的方法以及一种检测焦地黄素A中焦地黄素B的方法。

Description

地黄素的制备、分析和细胞免疫治疗用途 技术领域
本发明属于医药领域，具体涉及地黄素的制备、分析和细胞免疫治疗用途。
背景技术
地黄素B、地黄素D、焦地黄素A和焦地黄素B为从地黄中分离得到的天然产物，化学结构非常相似，化学信息如下表所示。
Figure PCTCN2018100123-appb-000001
目前尚未见上述四种地黄素及以该四种地黄素为主要成分的提取物在CIK细胞体外增殖方面的研究报道。
发明内容
本发明目的在于提供地黄素的制备、分析和细胞免疫治疗用途。
上述目的通过如下技术方案实现：
一种制备地黄素B、地黄素D和焦地黄素A的方法，包括如下步骤：
步骤S1，将鲜地黄洗净切片、干燥得到鲜地***；将鲜地***用乙醇水溶液热回流提取，提取液减压浓缩得浸膏；
步骤S2，将浸膏用适量水溶解，先用石油醚等体积萃取除杂，再用二氯甲烷萃取，合并二氯甲烷萃取液，浓缩干燥得二氯甲烷萃取物；
步骤S3，将二氯甲烷萃取物溶解上样于XDA-1B大孔吸附树脂柱，先用30％乙醇洗脱除杂，再用75％乙醇洗脱，收集8-10BV洗脱液，浓缩干燥得到地黄提取物粗品；
步骤S4，将地黄提取物粗品上样于正相硅胶柱，用体积比为10:1:0.2的二氯甲烷/甲醇/异丙醇混合溶剂洗脱，收集6-7BV洗脱液，浓缩干燥得到富含地黄素B、地黄素D、焦地黄素A的地黄提取物；
步骤S5，制备液相分离：将所得地黄提取物用制备液相色谱法分离，根据色谱图收集地黄素B、地黄素D、焦地黄素A对应的流份；
步骤S6，脱盐：将地黄素B、地黄素D、焦地黄素A对应的流份分别浓缩至干后用甲醇超声，N-十六碳酰-L-丝氨酸钠不溶于甲醇，过滤收集滤液浓缩干燥即得地黄素B、地黄素D、焦地黄素A。
优选地，制备液相色谱参数如下：
色谱柱：XBridge Prep C18柱(19mm×250mm，5μm)；
流动相A相：乙腈/水按照体积比1:9的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
流动相B相：乙腈/水按照体积比9:1的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
洗脱程序：0-5min，15％B相；5-15min，15％→55％B相；15-30min，55％→65％B相；
流速：16.0mL/min；
柱温：30℃；
检测波长：207nm。
优选地，步骤S1将鲜地***用95％乙醇水溶液热回流提取3次，每次2h，固液比为1:20，合并提取液，减压浓缩得浸膏。
优选地，步骤S2将浸膏用适量水溶解，先用石油醚等体积萃取3次除杂，再用二氯甲烷萃取3次，合并二氯甲烷萃取液，浓缩干燥。
优选地，步骤S3将二氯甲烷萃取物用30％乙醇水溶液溶解，拌树脂上样于XDA-1B大孔吸附树脂柱。
优选地，步骤S3中XDA-1B大孔吸附树脂柱的树脂径高比为1:10，拌样树脂占树脂总量的1/10。
优选地，步骤S3先用12BV的30％乙醇洗脱除杂，再用75％乙醇以10BV/h的流速洗脱，收集8-10BV洗脱液。
优选地，步骤S4中硅胶径高比为1:10，拌样硅胶占硅胶总量的1/10，用体积比为10:1:0.2的二氯甲烷/甲醇/异丙醇混合溶剂以10BV/h的速度洗脱，收集6-7BV洗脱液，浓缩干燥。
地黄素B、地黄素D和焦地黄素A在促进CIK细胞体外增殖方面的应用。
一种检测焦地黄素A中焦地黄素B的HPLC方法，流动相中含有N-十六碳酰-L-丝氨酸钠；具体地，流动相A相为乙腈/水按照体积比1:9的混合液，含5ppm的N-十六碳酰-L-丝氨 酸钠；流动相B相为乙腈/水按照体积比9:1的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠。
附图说明
图1为实施例2制备的地黄提取物的HPLC图谱；
图2为实施例2步骤S4硅胶柱层析洗脱剂不添加异丙醇所得提取物的HPLC图谱；
图3为实施例3制备液相色谱图；
图4-6分别为实施例3所得地黄素B、地黄素D、焦地黄素A的HPLC图谱；
图7为实施例3中步骤S3若使用XDA-1大孔吸附树脂替代XDA-1B大孔吸附树脂，步骤S4所得地黄提取物的HPLC图谱；
图8为实施例4混标溶液的HPLC色谱图；
图9为实施例4使用不添加N-十六碳酰-L-丝氨酸钠的流动相对混标溶液的分离图。
具体实施方式
下面结合实施例具体介绍本发明的实质性内容，但本领域技术人员应当知道，不应将本发明的保护范围局限于这些具体实施例。
实施例1：地黄素B、地黄素D、焦地黄素A和焦地黄素B对CIK细胞体外增殖的影响
一、实验材料
地黄素B、地黄素D、焦地黄素A和焦地黄素B自制，纯度在95％以上。
二、实验方法
1、分离单个细胞
用Ficoll-Hypaque密度梯度离心法从健康志愿者外周血分离出PBMC，加4mL PBS稀释4mL全血(比例为1:1)，取出稀释血缓慢加入装有4mLFicoll-Hypaque离心管中，注意不要打乱液面界面，2000r/min离心20min，轻轻吸取单个核细胞至另一支离心管中。加入生理盐水5mL，轻轻吹匀细胞，离心洗涤2次。
2、CIK细胞的培养
在生理盐水悬浮的PBMC中加CIK初始培养液，调整细胞浓度为5×10 5/mL，种植于24孔板中，每孔200μL，首先加入含1000U/mL IFN-γ的RPMI1640培养液，置于5％CO 2、37℃恒温培养箱中培养；24h后加100ng/mL抗CD3、500U/mL IL-2；培养4d后调整细胞浓度为1×10 6/mL，每隔2d补加500U/mL IL-2。照此法扩增至第10天收获CIK细胞。
3、CCK-8法检测地黄素B、地黄素D、焦地黄素A和焦地黄素B对CIK细胞增殖影响
收集培养10d的CIK细胞，1×10 5/mL细胞数接种于96孔板，200μL/孔，分别加入20μM地黄素B、地黄素D、焦地黄素A或焦地黄素B(对照组添加等体积溶媒)，孵育48h后，加入CCK8，孵育后，于酶标仪450nm处检测各孔吸光值(OD)。
4、统计学方法
采用SPSS 19.0统计软件。计量资料以均值±标准差表示，组间比较采用t检验。P＜0.05为差异有统计学意义。
三、实验结果
与对照组相比，地黄素B组、地黄素D组、焦地黄素A组48h增殖率显著升高(P＜0.05)，焦地黄素B组增殖率无明显差异(P＞0.05)。结果如下表。
Figure PCTCN2018100123-appb-000002
上述结果表明，地黄素B、地黄素D和焦地黄素A可以显著提高CIK细胞体外增殖，而焦地黄素B不具有这种作用。焦地黄素B与焦地黄素A相比，仅Cl连接的碳原子构型不同，地黄素B和地黄素D中该碳原子构型与焦地黄素A相同，证明该碳原子的构型对地黄素类化合物的促进CIK细胞增殖活性至关重要，这种构效关系首次发现。
实施例2：以地黄素B、地黄素D、焦地黄素A为主要成分的地黄提取物的制备及活性
制备方法包括如下步骤：
步骤S1，将鲜地黄洗净切片，55℃干燥，得到鲜地***；将鲜地***用95％乙醇水溶液热回流提取3次，每次2h，固液比为1:20，合并提取液，减压浓缩得浸膏；
步骤S2，将浸膏用适量水溶解，先用石油醚等体积萃取3次除杂，再用二氯甲烷萃取3次，合并二氯甲烷萃取液，浓缩干燥得二氯甲烷萃取物；
步骤S3，将二氯甲烷萃取物用30％乙醇水溶液溶解，拌树脂上样于XDA-1B大孔吸附树脂柱，树脂径高比为1:10，拌样树脂占树脂总量的1/10，先用12BV的30％乙醇洗脱除杂，再用75％乙醇以10BV/h的流速洗脱，收集8-10BV洗脱液，浓缩干燥得到地黄提取物粗品；
步骤S4，将地黄提取物粗品上样于正相硅胶柱，硅胶径高比为1:10，拌样硅胶占硅胶总量的1/10；用体积比为10:1:0.2的二氯甲烷/甲醇/异丙醇混合溶剂以10BV/h的速度洗脱，收集6-7BV洗脱液，浓缩干燥得到富含地黄素B、地黄素D、焦地黄素A的地黄提取物。
富含地黄素B、地黄素D、焦地黄素A的地黄提取物的HPLC分析：
色谱仪：岛津LC-20AT高效液相色谱仪；
色谱柱：Shim-packVP-ODS(4.6mm×250mm，5μm)；
流动相A相：乙腈/水按照体积比1:9的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
流动相B相：乙腈/水按照体积比9:1的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
洗脱程序：0-5min，15％B相；5-15min，15％→55％B相；15-30min，55％→65％B相；
流速：1.0mL/min；
柱温：30℃；
检测波长：207nm；
进样量：10μL。
图1为上述地黄提取物的HPLC图谱，主要成分为地黄素B、地黄素D和焦地黄素A，经外标法定量，地黄素B、地黄素D和焦地黄素A占提取物总重的90.2％。由于焦地黄素B不具有促进CIK细胞增殖的活性，而其与焦地黄素A结构非常接近，为了将焦地黄素B排除在上述提取物之外，步骤S4硅胶柱层析的洗脱剂中增加了少量异丙醇。若流动相不添加异丙醇，其他操作不变，所得提取物的HPLC图谱如图2所示，含有较多焦地黄素B和其他杂质。
按照实施例1的方法测试上述地黄提取物对CIK细胞体外增殖的促进活性，5μg/mL地黄提取物干预48h后的增殖率为(26.18±1.07)％，显著优于对照组。
实施例3：地黄素B、地黄素D、焦地黄素A单体的分离制备
分离制备方法包括如下步骤：
步骤S1，将鲜地黄洗净切片，55℃干燥，得到鲜地***；将鲜地***用95％乙醇水溶液热回流提取3次，每次2h，固液比为1:20，合并提取液，减压浓缩得浸膏；
步骤S2，将浸膏用适量水溶解，先用石油醚等体积萃取3次除杂，再用二氯甲烷萃取3次，合并二氯甲烷萃取液，浓缩干燥得二氯甲烷萃取物；
步骤S3，将二氯甲烷萃取物用30％乙醇水溶液溶解，拌树脂上样于XDA-1B大孔吸附树脂柱，树脂径高比为1:10，拌样树脂占树脂总量的1/10，先用12BV的30％乙醇洗脱除杂，再用75％乙醇以10BV/h的流速洗脱，收集8-10BV洗脱液，浓缩干燥得到地黄提取物粗品；
步骤S4，将地黄提取物粗品上样于正相硅胶柱，硅胶径高比为1:10，拌样硅胶占硅胶总量的1/10；用体积比为10:1:0.2的二氯甲烷/甲醇/异丙醇混合溶剂以10BV/h的速度洗脱，收集6-7BV洗脱液，浓缩干燥得到富含地黄素B、地黄素D、焦地黄素A的地黄提取物；
步骤S5，制备液相分离：将所得地黄提取物用制备液相色谱法分离，制备液相色谱参数如下，根据色谱图(如图3所示)收集地黄素B、地黄素D、焦地黄素A对应的流份；
制备液相色谱参数：
色谱***：Waters Prep 150制备液相色谱***
色谱柱：XBridge Prep C18柱(19mm×250mm，5μm)
流动相A相：乙腈/水按照体积比1:9的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
流动相B相：乙腈/水按照体积比9:1的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
洗脱程序：0-5min，15％B相；5-15min，15％→55％B相；15-30min，55％→65％B相；
流速：16.0mL/min；
柱温：30℃；
检测波长：207nm；
进样量：100μL；
步骤S6，脱盐：将地黄素B、地黄素D、焦地黄素A对应的流份分别浓缩至干后用甲醇超声，N-十六碳酰-L-丝氨酸钠不溶于甲醇，过滤收集滤液浓缩干燥即得地黄素B、地黄素D、焦地黄素A单体。
地黄素B、地黄素D、焦地黄素A单体的HPLC分析：
色谱仪：岛津LC-20AT高效液相色谱仪；
色谱柱：Shim-pack VP-ODS(4.6mm×250mm，5μm)；
流动相A相：乙腈/水按照体积比1:9的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
流动相B相：乙腈/水按照体积比9:1的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
洗脱程序：0-5min，15％B相；5-15min，15％→55％B相；15-30min，55％→65％B相；
流速：1.0mL/min；
柱温：30℃；
检测波长：207nm；
进样量：10μL。
图4-6分别为制备得到的地黄素B、地黄素D、焦地黄素A的HPLC图谱，可见其纯度均较高，经外标法定量均在95％以上。
上述制备方法中，步骤S3若使用XDA-1大孔吸附树脂替代XDA-1B大孔吸附树脂，步骤S4所得地黄提取物中杂质成分明显较多(如图7所示)，尤其是地黄素B和焦地黄素A之间的杂质对于地黄素B和焦地黄素A单体的分离纯化影响很大。
实施例4：检测焦地黄素A中焦地黄素B的方法
HPLC分析参数：
色谱仪：岛津LC-20AT高效液相色谱仪；
色谱柱：Shim-packVP-ODS(4.6mm×250mm，5μm)；
流动相A相：乙腈/水按照体积比1:9的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
流动相B相：乙腈/水按照体积比9:1的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
洗脱程序：0-5min，15％B相；5-15min，15％→55％B相；15-30min，55％→65％B相；
流速：1.0mL/min；
柱温：30℃；
检测波长：207nm；
进样量：10μL。
单标溶液：分别配置浓度为0.1mg/mL的焦地黄素A、焦地黄素B甲醇溶液。
混标溶液：配置焦地黄素A、焦地黄素B浓度分别为0.1mg/mL的甲醇溶液。
图8为混标溶液的HPLC色谱图。可见焦地黄素A、焦地黄素B在上述色谱条件下可以有效分离，实现基线分离，分离度大于2.0。
若使用常规的乙腈/水作为流动相(流动相不添加N-十六碳酰-L-丝氨酸钠，其他参数不变)，混标溶液的HPLC色谱图如图9所示，焦地黄素A、焦地黄素B分离难度大。这表明，流动相中添加N-十六碳酰-L-丝氨酸钠有助于增加焦地黄素A、焦地黄素B这对手性异构体在反相色谱柱上的区分度。

Claims (10)

  1. 一种制备地黄素B、地黄素D和焦地黄素A的方法，其特征在于，包括如下步骤：
    步骤S1，将鲜地黄洗净切片、干燥得到鲜地***；将鲜地***用乙醇水溶液热回流提取，提取液减压浓缩得浸膏；
    步骤S2，将浸膏用适量水溶解，先用石油醚等体积萃取除杂，再用二氯甲烷萃取，合并二氯甲烷萃取液，浓缩干燥得二氯甲烷萃取物；
    步骤S3，将二氯甲烷萃取物溶解上样于XDA-1B大孔吸附树脂柱，先用30％乙醇洗脱除杂，再用75％乙醇洗脱，收集8-10BV洗脱液，浓缩干燥得到地黄提取物粗品；
    步骤S4，将地黄提取物粗品上样于正相硅胶柱，用体积比为10:1:0.2的二氯甲烷/甲醇/异丙醇混合溶剂洗脱，收集6-7BV洗脱液，浓缩干燥得到富含地黄素B、地黄素D、焦地黄素A的地黄提取物；
    步骤S5，制备液相分离：将所得地黄提取物用制备液相色谱法分离，根据色谱图收集地黄素B、地黄素D、焦地黄素A对应的流份；
    步骤S6，脱盐：将地黄素B、地黄素D、焦地黄素A对应的流份分别浓缩至干后用甲醇超声，N-十六碳酰-L-丝氨酸钠不溶于甲醇，过滤收集滤液浓缩干燥即得地黄素B、地黄素D、焦地黄素A。
  2. 根据权利要求1所述的方法，其特征在于，制备液相色谱参数如下：
    色谱柱：XBridge Prep C18柱(19mm×250mm，5μm)；
    流动相A相：乙腈/水按照体积比1:9的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
    流动相B相：乙腈/水按照体积比9:1的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；
    洗脱程序：0-5min，15％B相；5-15min，15％→55％B相；15-30min，55％→65％B相；
    流速：16.0mL/min；
    柱温：30℃；
    检测波长：207nm。
  3. 根据权利要求1所述的方法，其特征在于：步骤S1将鲜地***用95％乙醇水溶液热回流提取3次，每次2h，固液比为1:20，合并提取液，减压浓缩得浸膏。
  4. 根据权利要求1所述的方法，其特征在于：步骤S2将浸膏用适量水溶解，先用石油醚等体积萃取3次除杂，再用二氯甲烷萃取3次，合并二氯甲烷萃取液，浓缩干燥。
  5. 根据权利要求1所述的方法，其特征在于：步骤S3将二氯甲烷萃取物用30％乙醇水溶液溶解，拌树脂上样于XDA-1B大孔吸附树脂柱。
  6. 根据权利要求1所述的方法，其特征在于：步骤S3中XDA-1B大孔吸附树脂柱的树脂径高比为1:10，拌样树脂占树脂总量的1/10。
  7. 根据权利要求6所述的方法，其特征在于：步骤S3先用12BV的30％乙醇洗脱除杂，再用75％乙醇以10BV/h的流速洗脱，收集8-10BV洗脱液。
  8. 根据权利要求1所述的方法，其特征在于：步骤S4中硅胶径高比为1:10，拌样硅胶占硅胶总量的1/10，用体积比为10:1:0.2的二氯甲烷/甲醇/异丙醇混合溶剂以10BV/h的速度洗脱，收集6-7BV洗脱液，浓缩干燥。
  9. 地黄素B、地黄素D和焦地黄素A在促进CIK细胞体外增殖方面的应用。
  10. 一种检测焦地黄素A中焦地黄素B的HPLC方法，其特征在于：流动相中含有N-十六碳酰-L-丝氨酸钠；具体地，流动相A相为乙腈/水按照体积比1:9的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠；流动相B相为乙腈/水按照体积比9:1的混合液，含5ppm的N-十六碳酰-L-丝氨酸钠。
PCT/CN2018/100123 2018-03-26 2018-08-11 地黄素的制备、分析和细胞免疫治疗用途 WO2019184194A1 (zh)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN201810249755.3A CN108220240B (zh) 2018-03-26 2018-03-26 地黄素在促进cik细胞体外增殖方面的应用
CN201810249755.3 2018-03-26
CN201810249739.4 2018-03-26
CN201810249721.4A CN108250209B (zh) 2018-03-26 2018-03-26 一种制备地黄素b、地黄素d和焦地黄素a的方法
CN201810249721.4 2018-03-26
CN201810249739.4A CN108267524B (zh) 2018-03-26 2018-03-26 一种检测焦地黄素a中焦地黄素b的方法

Publications (1)

Publication Number Publication Date
WO2019184194A1 true WO2019184194A1 (zh) 2019-10-03

Family

ID=68062084

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/100123 WO2019184194A1 (zh) 2018-03-26 2018-08-11 地黄素的制备、分析和细胞免疫治疗用途

Country Status (1)

Country Link
WO (1) WO2019184194A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0283389A (ja) * 1988-09-21 1990-03-23 Tsumura & Co 新規イリドイド化合物
CN108186790A (zh) * 2018-03-26 2018-06-22 马海周 一种地黄提取物、制备方法及在促进cik细胞体外增殖方面的应用
CN108220240A (zh) * 2018-03-26 2018-06-29 马海周 地黄素在促进cik细胞体外增殖方面的应用
CN108250209A (zh) * 2018-03-26 2018-07-06 马海周 一种制备地黄素b、地黄素d和焦地黄素a的方法
CN108267524A (zh) * 2018-03-26 2018-07-10 马海周 一种检测焦地黄素a中焦地黄素b的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0283389A (ja) * 1988-09-21 1990-03-23 Tsumura & Co 新規イリドイド化合物
CN108186790A (zh) * 2018-03-26 2018-06-22 马海周 一种地黄提取物、制备方法及在促进cik细胞体外增殖方面的应用
CN108220240A (zh) * 2018-03-26 2018-06-29 马海周 地黄素在促进cik细胞体外增殖方面的应用
CN108250209A (zh) * 2018-03-26 2018-07-06 马海周 一种制备地黄素b、地黄素d和焦地黄素a的方法
CN108267524A (zh) * 2018-03-26 2018-07-10 马海周 一种检测焦地黄素a中焦地黄素b的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KITAGAVA ISAO ET AL.: "Absolute stereostructures of rehmaglutins A, B, and D three new iridoids isolated from Chinese rehmanniae radix", CHEM. PHARM. BULL., vol. 34, no. 3, 31 December 1986 (1986-12-31), pages 1399 - 1402, XP055640027 *
LIU, ET AL.: "Chemical constituents of Radix Rehmanniae", CHINESE HERBAL MEDICINES, vol. 45, no. 1, 31 January 2014 (2014-01-31), pages 16 - 22 *
MOROTA TAKASHI ET AL.: "Five cyclopentanoid monoterpenes from Rehmannia glutinosa", PHYTOCHEMISTRY, vol. 28, no. 9, 31 December 1989 (1989-12-31), pages 2385 - 2391, XP026632878, DOI: 10.1016/S0031-9422(00)97989-5 *

Similar Documents

Publication Publication Date Title
US11739113B2 (en) Method for extracting and separating flavonoids from Lindera aggregata leaves
JP7305870B2 (ja) テトラガロイルグルコースの製造方法
CN111141850B (zh) 对乙酰氨基酚原料药有关物质的液相检测分离方法
CN109942649B (zh) 一种吲哚苷类化合物及其提取分离方法和应用
CN109912604B (zh) 喹唑啉酮生物碱类化合物及其制备方法和在制备肝x受体激动剂中的应用
WO2017219510A1 (zh) 一种具有抗肿瘤活性和抗炎活性的醌式查尔酮碳苷二聚体化合物及其制备方法
CN111704641B (zh) 一种环烯醚萜苷类化合物及其制备方法和应用
CN110746421B (zh) 吲哚单萜类化合物的提取方法及应用
CN109796511B (zh) 一种新的环烯醚萜类化合物及其制备方法和医药用途
WO2019184194A1 (zh) 地黄素的制备、分析和细胞免疫治疗用途
CN108186790B (zh) 一种地黄提取物、制备方法及在促进cik细胞体外增殖方面的应用
CN102688261A (zh) 一种凤尾草提取物及其制备方法和用途
CN109912582A (zh) 从芒果叶中提取芒果苷的方法
CN111303225A (zh) 一种环烯醚萜苷类化合物及其制备方法和应用
CN102304164B (zh) 远志皂苷元衍生物及其制备方法和应用
CN111732619B (zh) 一种环烯醚萜苷类化合物及其制备方法和应用
CN107721857A (zh) 一种从平卧菊三七中制备高纯度绿原酸的方法
CN106822071B (zh) 一种用于治疗冠心病、高脂血症的中药有效部位、制备方法及从中分离有效成分的方法
CN108267524B (zh) 一种检测焦地黄素a中焦地黄素b的方法
CN107375557B (zh) 三叶青提取物TH-b部分在制备肿瘤免疫治疗药物及人树突状细胞增殖剂中的应用
CN111647031A (zh) 一类新生物碱及其提取分离方法和应用
CN105968120B (zh) 一种汉防己甲素、乙素的制备方法
CN114805465B (zh) 三萜类化合物及其制备方法和应用
CN114957362B (zh) 一种昆仑雪菊抗炎活性成分的分离方法
CN113754677B (zh) 一种新的香豆素二聚体及其制备方法与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18911452

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18911452

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 18911452

Country of ref document: EP

Kind code of ref document: A1