WO2024055459A1 - 一种高纯千金藤素的分离方法 - Google Patents

一种高纯千金藤素的分离方法 Download PDF

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WO2024055459A1
WO2024055459A1 PCT/CN2022/139271 CN2022139271W WO2024055459A1 WO 2024055459 A1 WO2024055459 A1 WO 2024055459A1 CN 2022139271 W CN2022139271 W CN 2022139271W WO 2024055459 A1 WO2024055459 A1 WO 2024055459A1
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stephanatine
purity
silica gel
cepharanthine
chromatography
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李挥
王洪宇
王俊帅
王雪仪
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Hebei Institute for Drug and Medical Device Control
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Hebei Institute for Drug and Medical Device Control
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings

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  • the invention belongs to the technical field of separation and purification, and is specifically a method for separating high-purity stephanatine.
  • Cepharanthine is a leukocyte proliferating drug that can promote the proliferation of bone marrow tissue and thereby increase white blood cells.
  • leukopenia can increase leukocytes in peripheral blood. Animal experiments have found that it can significantly prevent leukopenia caused by mitomycin, but it does not inhibit the anti-cancer effect of mitomycin. Its mechanism of action is to promote bone marrow tissue proliferation, thereby increasing white blood cells. It is used for leukopenia caused by agranulocytosis caused by tumor chemotherapy, radiotherapy and other reasons.
  • the separation and purification method of Stephania spp. is complex and not easy to automate industrial production.
  • the present invention provides a method for isolating high-purity stephanotis. It is characterized in that the raw materials are mountain turtle slices, Diphenosa or Fangjiaceae Chinese medicinal materials containing stephenathin, and extracted with an extractant. Through silica gel column chromatography, elution, and preparative liquid chromatography separation, purified stephanatine is obtained;
  • the chromatography material is loaded in the adsorption column.
  • the chromatography material is organic hydrocarbon bonded silica gel or organic hydrocarbon bonded silica gel containing polar organic functional groups;
  • the adsorption column material is a physical mixture of two or more bonded silica gels or two. More than one type of organic hydrocarbon bonded silica gel;
  • the polar organic functional group is selected from the group consisting of ester group, amino group, amide group and urea group.
  • step B Pass the extract obtained in step A through a silica gel column for chromatography, and obtain crude stephanatine after elution;
  • step D Rotary evaporate the fraction obtained in step C under reduced pressure to remove methanol, and then freeze-dry to obtain a white solid, which is purified stephanatine.
  • the extraction agent is one or more mixed liquids of methanol, ethanol, n-propanol or isopropanol.
  • the particle size of the chromatography material is 5-100um, and the pore size is
  • the elution flow rate is 5-30 times column volume/hour.
  • the eluent is selected from any one of the organic solvents methanol, acetonitrile, acetone, tetrahydrofuran, ethanol, isopropanol, dioxane, or a mixture of more than one organic solvent and water. Mixing, or a mixture of more than one organic solvent and an aqueous solution with a modifier added.
  • the viscous proportion of water or aqueous solution in the eluent is 10%-60%.
  • the modifier is one or more of formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, phosphoric acid, ammonium bicarbonate, ammonia, triethylamine, diethylamine, ammonium acetate or ammonium formate. the mix of.
  • the invention discloses a method for separating high-purity stephanatine, which includes extraction of stephanatine, chromatographic separation, collection and chromatographic purity detection.
  • a method for separating high-purity stephanatine which includes extraction of stephanatine, chromatographic separation, collection and chromatographic purity detection.
  • Figure 1 is a typical online detection chart of chromatographic separation of stephanatine extract
  • Figure 2 is the LC-MS detection chart of stephanatine extraction
  • Figure 3 is the LC/MS detection chart of purified stephanatine.
  • any reference to “one embodiment” means that the specific features, structures or parameters, steps, etc. described in the embodiment are included in at least one embodiment according to the present invention. Therefore, in the description of the present invention, if terms such as “according to one embodiment of the present invention” and “in an embodiment” are used, they are not used to specifically refer to the same embodiment. If terms such as “in an embodiment” are used, Terms such as “in other embodiments”, “according to different embodiments of the present invention”, “according to other embodiments of the present invention” are not used to specifically indicate that the mentioned features can only be included in specific different embodiments. . Those skilled in the art should understand that each specific feature, structure or parameter, step, etc. disclosed in one or more embodiments of the present invention may be combined in any suitable manner.
  • step B Pass the extract obtained in step A through a bonded silica gel column for chromatography, and obtain crude stephanatine after elution;
  • the chromatography material is octadecyl bonded silica gel (organic alkane bonded silica gel) packed in the adsorption column; the eluent is methanol;
  • the filler Bioseps-Flash C18, 20-45 ⁇ m,
  • step D Rotary evaporate the fraction obtained in step C under reduced pressure to remove methanol, and freeze-dry to obtain 37.5 mg of white solid, which is purified stephanatine.
  • Chromatographic analysis After diluting the extract 2 times with methanol, pass it through a 0.45um membrane and enter it into a liquid mass spectrometer for chromatographic analysis.
  • the chromatographic conditions are as follows:
  • Mass spectrometer voltage 3000V, scanning range 150 ⁇ 1500m/z, Pos-TIC mode
  • Purified stephanatine was detected using liquid chromatography-mass spectrometry.
  • the liquid chromatography column was an octadecyl-bonded silica matrix, 2.1 ⁇ 100mm, with a pore diameter of The particle size is 3 ⁇ m, the mobile phase is a mixture of methanol and 0.1% triethylamine phosphate aqueous solution at a volume ratio of 70:30, the detection wavelength is 282nm, and the mass spectrum uses the positive mode full scan to detect the peak corresponding to the quasi-molecular ion peak of 607.7 That is Stephania vinesulin.
  • step B Pass the extract obtained in step A through a bonded silica gel column for chromatography, and obtain crude stephanatine after elution;
  • the chromatography material is octyl-bonded silica gel packed in the adsorption column; the eluent is methanol;
  • the filler Bioseps-Flash C8, 20-45 ⁇ m,
  • step D Rotary evaporate the fraction obtained in step C under reduced pressure to remove methanol, and freeze-dry to obtain 37.4 mg of white solid, which is purified stephanatine.
  • step B Pass the extract obtained in step A through a bonded silica gel column for chromatography, and obtain crude stephanatine after elution;
  • the chromatography material is NH 2 -modified octadecyl bonded silica gel packed in the adsorption column; the eluent is methanol;
  • the filler Bioseps-Flash C18-N, 20-45 ⁇ m,
  • step D Rotary evaporate the fraction obtained in step C under reduced pressure to remove methanol, and freeze-dry to obtain 37.5 mg of white solid, which is purified stephanatine.
  • step B Pass the extract obtained in step A through a bonded silica gel column for chromatography, and obtain crude stephanatine after elution;
  • the chromatography material is octadecyl-bonded silica gel with amide modification and is packed in the adsorption column;
  • the eluent is an acetone/methanol mixture with a volume ratio of 20:80;
  • the filler Bioseps-Flash C18-NC, 20-45 ⁇ m,
  • step D Rotary evaporate the fraction obtained in step C under reduced pressure to remove methanol, and freeze-dry to obtain 37.2 mg of white solid, which is purified stephanatine.
  • step B Pass the extract obtained in step A through a bonded silica gel column for chromatography, and obtain crude stephanatine after elution;
  • the filler Bioseps-Flash C18-N, 20-45 ⁇ m,
  • step D Rotary evaporate the fraction obtained in step C under reduced pressure to remove methanol, and freeze-dry to obtain 37.3 mg of white solid, which is purified stephanatine.
  • step B Pass the extract obtained in step A through a silica gel column for chromatography, and obtain the crude stephanatine after elution;
  • the chromatography material is octadecyl bonded silica gel modified with propyl ester group, which is packed in the adsorption column;
  • the eluent is methanol and an aqueous solution with formic acid added, and the volume ratio of the aqueous solution with formic acid added is 10%.
  • the filler Bioseps-Flash C18, 20-45 ⁇ m,
  • step D Rotary evaporate the fraction obtained in step C under reduced pressure to remove methanol, and freeze-dry to obtain 37.4 mg of white solid, which is purified stephanatine.
  • the stephanotis separated by the present invention has high purity, the separation process is simple and controllable, and it is very easy to automate industrial production.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

本发明属于分离纯化技术领域，提供了一种高纯千金藤素的分离方法，包括以下步骤：A、将山乌龟片、地不容或含有千金藤素的防己科中药材为原料用研钵捣碎至粉末，加入提取剂超声提取20min后减压旋蒸浓缩除去提取剂；B、将步骤A中得到的提取液过硅胶柱进行层析，经洗脱后得到千金藤素粗品；C、将千金藤素粗品，通过0.45μm的膜后，经制备液相色谱进行纯化；D、将步骤C中得到的馏分经35℃下减压旋蒸除去甲醇，冷冻干燥，得白色固体，即纯化千金藤素。本发明的方法千金藤素纯度高，分离工艺简单可控，非常易于工业化生产的自动化。

Description

一种高纯千金藤素的分离方法 技术领域
本发明属于分离纯化技术领域，具体是一种高纯千金藤素的分离方法。
背景技术
千金藤素(Cepharanthine)，白细胞增生药，能促进骨髓组织增生，从而升高白细胞。用于白细胞减少症。可使外周血白细胞增多，动物实验发现，可明显预防丝裂霉素引起的白细胞减少，但不抑制丝裂霉素的抗癌作用。其作用机制是促进骨髓组织增生，从而产生升高白细胞作用。用于因肿瘤化疗、放疗引起的粒细胞缺乏症和其他原因引起的白细胞减少症。现有技术中，千金藤素主的分离纯化方法工艺复杂，不易于工业化生产的自动化。
发明内容
本发明针对以上问题，提供了一种高纯千金藤素的分离方法，其特征在于，以山乌龟片、地不容或含有千金藤素的防己科中药材为原料，用提取剂提取，提取液过硅胶柱层析、洗脱，制备液相色谱分离，得到纯化千金藤素；
所述层析材料装填在吸附柱中，层析材料为有机烃类键合硅胶或含有极性有机官能团的有机烃类键合硅胶；吸附柱材料为两种以上键合硅胶的物理混合或两种以上有机烃类键合硅胶；
所述极性有机官能团选自酯基、氨基、酰胺基、脲基。
优选的，包括以下步骤：
A、将中药材山乌龟片用研钵捣碎至粉末，加入提取剂超声提取后减压旋蒸浓缩除去提取剂；
B、将步骤A中得到的提取液过硅胶柱进行层析，经洗脱后得到千金藤素粗品；
C、将千金藤素粗品过滤后，经制备液相色谱进行纯化；
D、将步骤C中得到的馏分经减压旋蒸除去甲醇，冷冻干燥，得白色固体，即纯化千金藤素。
优选的，提取剂为甲醇、乙醇、正丙醇或异丙醇的一种或一种以上的混合液。
优选的，所述层析材料的粒径为5-100um，孔径为
Figure PCTCN2022139271-appb-000001
优选的，所述步骤B中，洗脱流速为5-30倍柱体积/小时。
优选的，所述步骤B中，洗脱液选自有机溶剂甲醇、乙腈、丙酮、四氢呋喃、乙醇、异丙醇、二氧六环的任意一种，或一种以上有机溶剂的混合液与水混合，或一种以上有机溶剂的混合液与添加改性剂的水溶液混合。
优选的，所述洗脱液中水或水溶液粘的比例为10％-60％。
优选的，所述改性剂为甲酸、乙酸、三氟乙酸、三氯乙酸、磷酸、碳酸氢铵、氨水、三乙胺、二乙胺、乙酸铵或甲酸铵中的一种或一种以上的混合。
本发明的有益效果在于：
本发明公开了一种高纯千金藤素的分离方法，包括千金藤素的提取，色谱分离，收集和色谱纯度检测。取山乌龟、地不容或含有千金 藤素的防己科中药材，捣碎，经甲醇提取后，过键合硅胶层析柱，所得馏分浓缩后，结晶，可得纯度达到98％的千金藤素纯品。采用本发明所述的方法，千金藤素纯度高，分离工艺简单可控，非常易于工业化生产的自动化。
附图说明
图1为典型千金藤素提取液层析分离在线检测图；
图2为千金藤素提取液质联用检测图；
图3为纯化千金藤素液质联用检测图。
具体实施方式
为让本发明的上述特征和优点能更明显易懂，下文特举实施例，并配合附图，作详细说明如下，但本发明并不限于此。
在本发明的说明书中，提及“一个实施例”时均意指在该实施例中描述的具体特征、结构或者参数、步骤等至少包含在根据本发明的一个实施例中。因而，在本发明的说明书中，若采用了诸如“根据本发明的一个实施例”、“在一个实施例中”等用语并不用于特指在同一个实施例中，若采用了诸如“在另外的实施例中”、“根据本发明的不同实施例”、“根据本发明另外的实施例”等用语，也并不用于特指提及的特征只能包含在特定的不同的实施例中。本领域的技术人员应该理解，在本发明说明书的一个或者多个实施例中公开的各具体特征、结构或者参数、步骤等可以以任何合适的方式组合。
实施例1
A、将中药材山乌龟片用研钵捣碎至粉末，用研钵捣碎至粉末，称取50g于具塞锥形瓶中，加入1000ml甲醇，超声提取20min后，静置冷却减压旋蒸浓缩除去提取剂；
B、将步骤A中得到的提取液过键合硅胶柱进行层析，经洗脱后得到千金藤素粗品；
其中，层析材料为十八烷基键合硅胶(有机烷烃键合硅胶)装填在吸附柱中；洗脱液为甲醇；
C、将千金藤素粗品过0.45μm的膜后，经制备液相色谱进行纯化；
其中填料：Bioseps-Flash C18,20-45μm，
Figure PCTCN2022139271-appb-000002
填装规格：30倍柱体积/小时；
进样体积：500ml
洗脱液：1)甲醇：水＝85:15(V/V)保持30min，2)25min内，比例线性调整为甲醇：水＝95:5(V/V)；3)保持步骤2)的比例5min.
洗脱流速：6倍柱体积/小时；
收集方式：紫外检测282nm；
D、将步骤C中得到的馏分经减压旋蒸除去甲醇，冷冻干燥，得37.5mg白色固体，即纯化千金藤素。
色谱分析：将提取液用甲醇稀释2倍后，过0.45um膜，进液质联用仪进行色谱分析，色谱条件如下：
色谱柱：Bioseps AQC18,2.1*100mm,3um,
Figure PCTCN2022139271-appb-000003
流动相：甲醇：0.1％TEA＝80:20(V/V)
检测波长：282nm
流速：0.3ml/min；
柱温：35℃
进样量：1ul
质谱电压：3000V,扫描范围150～1500m/z,Pos-TIC模式
纯化检测，经LCMS检测，纯化千金藤素样品纯度为98.5％。
纯化千金藤素采用液相色谱质谱联用检测，液相色谱柱为十八烷基键合硅胶基质，2.1×100mm，孔径为
Figure PCTCN2022139271-appb-000004
粒径为3μm，流动相为甲醇与0.1磷酸三乙胺水溶液以70：30体积比的混合液，检测波长为282nm,质谱采用正模式全扫描方式下检测到607.7的准分子离子峰对应的峰即为千金藤素。
实施例2
A、将中药材山乌龟片用研钵捣碎至粉末，用研钵捣碎至粉末，称取50g于具塞锥形瓶中，加入1000ml甲醇，超声提取20min后，静置冷却减压旋蒸浓缩除去提取剂；
B、将步骤A中得到的提取液过键合硅胶柱进行层析，经洗脱后得到千金藤素粗品；
其中，层析材料为辛烷基键合硅胶装填在吸附柱中；洗脱液为甲醇；
C、将千金藤素粗品过0.45μm的膜后，经制备液相色谱进行纯化；
其中填料：Bioseps-Flash C8,20-45μm，
Figure PCTCN2022139271-appb-000005
填装规格：50×250mm；
进样体积：500ml
洗脱液：四氢呋喃：水＝78:22(V/V)
洗脱流速：10倍柱体积/小时；
收集方式：紫外检测282nm；
D、将步骤C中得到的馏分经减压旋蒸除去甲醇，冷冻干燥，得37.4mg白色固体，即纯化千金藤素。
纯化检测，经LCMS检测，纯化千金藤素样品纯度为98.3％。
实施例3
A、将中药材山乌龟片用研钵捣碎至粉末，用研钵捣碎至粉末，称取50g于具塞锥形瓶中，加入1000ml甲醇，超声提取20min后，静置冷却减压旋蒸浓缩除去提取剂；
B、将步骤A中得到的提取液过键合硅胶柱进行层析，经洗脱后得到千金藤素粗品；
其中，层析材料为有NH 2-修饰的十八烷基键合硅胶装填在吸附柱中；洗脱液为甲醇；
C、将千金藤素粗品过0.45μm的膜后，经制备液相色谱进行纯化；
其中填料：Bioseps-Flash C18-N,20-45μm，
Figure PCTCN2022139271-appb-000006
填装规格：50×250mm；
进样体积：500ml
洗脱液：乙腈：水＝85:15(V/V)
洗脱流速：15倍柱体积/小时；
收集方式：紫外检测282nm；
D、将步骤C中得到的馏分经减压旋蒸除去甲醇，冷冻干燥，得37.5mg白色固体，即纯化千金藤素。
纯化检测，经LCMS检测，纯化千金藤素样品纯度为97.1％。
实施例4
A、将中药材山乌龟片用研钵捣碎至粉末，用研钵捣碎至粉末，称取50g于具塞锥形瓶中，加入1000ml提取剂(体积比为20:80的丙酮/甲醇混合液)，超声提取20min后，静置冷却减压旋蒸浓缩除去提取剂；
B、将步骤A中得到的提取液过键合硅胶柱进行层析，经洗脱后得到千金藤素粗品；
其中，层析材料为有酰胺基修饰的十八烷基键合硅胶装填在吸附柱中；洗脱液为体积比为20:80的丙酮/甲醇混合液；
C、将千金藤素粗品过0.45μm的膜后，经制备液相色谱进行纯化；
其中填料：Bioseps-Flash C18-NC,20-45μm，
Figure PCTCN2022139271-appb-000007
填装规格：50×250mm；
进样体积：500ml
洗脱液：丙酮：甲醇：水(含0.05％三氟乙酸和0.01％三乙胺)＝10:50:40(V/V)
洗脱流速：25倍柱体积/小时；
收集方式：紫外检测282nm；
D、将步骤C中得到的馏分经减压旋蒸除去甲醇，冷冻干燥，得37.2mg白色固体，即纯化千金藤素。
纯化检测，经LCMS检测，纯化千金藤素样品纯度为97.8％。
实施例5
A、将中药材山乌龟片用研钵捣碎至粉末，用研钵捣碎至粉末，称取50g于具塞锥形瓶中，加入1000ml甲醇，超声提取20min后，静置冷却减压旋蒸浓缩除去提取剂；
B、将步骤A中得到的提取液过键合硅胶柱进行层析，经洗脱后得到千金藤素粗品；
其中，层析材料为有NH 2-修饰的十八烷基键合硅胶装填在吸附柱中；洗脱液为甲醇：水＝85：15(V/V)
C、将千金藤素粗品过0.45μm的膜后，经制备液相色谱进行纯化；
其中填料：Bioseps-Flash C18-N,20-45μm，
Figure PCTCN2022139271-appb-000008
填装规格：50×250mm；
进样体积：500ml
洗脱液：甲醇：乙醇：水(含0.1％甲酸)＝68:17:15(V/V)
洗脱流速：5倍柱体积/小时；
收集方式：紫外检测282nm；
D、将步骤C中得到的馏分经减压旋蒸除去甲醇，冷冻干燥，得37.3mg白色固体，即纯化千金藤素。
纯化检测，经LCMS检测，纯化千金藤素样品纯度为98.1％。
实施例6
A、将中药材山乌龟片用研钵捣碎至粉末，用研钵捣碎至粉末，称取50g于具塞锥形瓶中，加入1000ml甲醇，超声提取20min后，静置冷却减压旋蒸浓缩除去提取剂；
B、将步骤A中得到的提取液过硅胶柱进行层析，经洗脱后得到 千金藤素粗品；
其中，层析材料为有丙酯基修饰的十八烷基键合硅胶装填在吸附柱中；洗脱液为甲醇和添加甲酸的水溶液，其中添加甲酸的水溶液占的体积比例为10％。
C、将千金藤素粗品过0.45μm的膜后，经制备液相色谱进行纯化；
其中填料：Bioseps-Flash C18,20-45μm，
Figure PCTCN2022139271-appb-000009
填装规格：50×250mm；
进样体积：500ml
洗脱液：甲醇：水＝85:15(V/V)
洗脱流速：20倍柱体积/小时；
收集方式：紫外检测282nm；
D、将步骤C中得到的馏分经减压旋蒸除去甲醇，冷冻干燥，得37.4mg白色固体，即纯化千金藤素。
纯化检测，经LCMS检测，纯化千金藤素样品纯度为98.5％。
综上所述，本发明分离的千金藤素纯度高，分离工艺简单可控，非常易于工业化生产的自动化。

Claims (8)

  1. 一种高纯千金藤素的分离方法，其特征在于，以山乌龟片、地不容或含有千金藤素的防己科中药材为原料，用提取剂提取，提取液过硅胶柱层析、洗脱，制备液相色谱分离，得到纯化千金藤素；
    所述层析材料装填在吸附柱中，层析材料为有机烃类键合硅胶或含有极性有机官能团的有机烃类键合硅胶；吸附柱材料为两种以上键合硅胶的物理混合或两种以上有机烃类键合硅胶；
    所述极性有机官能团选自酯基、氨基、酰胺基、脲基。
  2. 根据权利要求1所述的一种高纯千金藤素的分离方法，其特征在于，包括以下步骤：
    A、将山乌龟片、地不容或含有千金藤素的防己科中药材为原料用研钵捣碎至粉末，加入提取剂超声提取后减压旋蒸浓缩除去提取剂；
    B、将步骤A中得到的提取液过硅胶柱进行层析，经洗脱后得到千金藤素粗品；
    C、将千金藤素粗品过滤后，经制备液相色谱进行纯化；
    D、将步骤C中得到的馏分经减压旋蒸除去甲醇，冷冻干燥，得白色固体，即纯化千金藤素。
  3. 根据权利要求1所述的一种高纯千金藤素的分离方法，其特征在于，提取剂为甲醇、乙醇、正丙醇或异丙醇的一种或一种以上的混合液。
  4. 根据权利要求1所述的一种高纯千金藤素的分离方法，其特征在于，所述层析材料的粒径为5-100um，孔径为
    Figure PCTCN2022139271-appb-100001
  5. 根据权利要求2所述的一种高纯千金藤素的分离方法，其特 征在于，所述步骤B中，洗脱流速为5-30倍柱体积/小时。
  6. 根据权利要求2所述的一种高纯千金藤素的分离方法，其特征在于，所述步骤B中，洗脱液选自有机溶剂甲醇、乙腈、丙酮、四氢呋喃、乙醇、异丙醇、二氧六环的任意一种，或一种以上有机溶剂的混合液与水混合，或一种以上有机溶剂的混合液与添加改性剂的水溶液混合。
  7. 根据权利要求6所述的一种高纯千金藤素的分离方法，其特征在于，所述洗脱液中水或添加改性剂的水溶液占的比例为10％-60％。
  8. 根据权利要求6所述的一种高纯千金藤素的分离方法，其特征在于，所述改性剂为甲酸、乙酸、三氟乙酸、三氯乙酸、磷酸、碳酸氢铵、氨水、三乙胺、二乙胺、乙酸铵或甲酸铵中的一种或一种以上的混合。
PCT/CN2022/139271 2022-09-14 2022-12-15 一种高纯千金藤素的分离方法 Ceased WO2024055459A1 (zh)

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