CN112266399B - High-purity separation and extraction method of epimedium extract - Google Patents
High-purity separation and extraction method of epimedium extract Download PDFInfo
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- 238000000605 extraction Methods 0.000 title claims abstract description 22
- 238000000926 separation method Methods 0.000 title claims abstract description 17
- 235000020696 epimedium extract Nutrition 0.000 title claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 285
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 75
- 238000000034 method Methods 0.000 claims abstract description 65
- 239000003480 eluent Substances 0.000 claims abstract description 46
- 241000893536 Epimedium Species 0.000 claims abstract description 41
- 235000018905 epimedium Nutrition 0.000 claims abstract description 41
- 239000000284 extract Substances 0.000 claims abstract description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- IYCPMVXIUPYNHI-UHFFFAOYSA-N Icariside I Natural products C1=CC(OC)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(OC3C(C(O)C(O)C(CO)O3)O)C(CC=C(C)C)=C2O1 IYCPMVXIUPYNHI-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000001914 filtration Methods 0.000 claims abstract description 32
- 238000001179 sorption measurement Methods 0.000 claims abstract description 31
- TZJALUIVHRYQQB-XLRXWWTNSA-N icariin Chemical compound C1=CC(OC)=CC=C1C1=C(O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)C(=O)C2=C(O)C=C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C(CC=C(C)C)=C2O1 TZJALUIVHRYQQB-XLRXWWTNSA-N 0.000 claims abstract description 29
- TZJALUIVHRYQQB-UHFFFAOYSA-N icariine Natural products C1=CC(OC)=CC=C1C1=C(OC2C(C(O)C(O)C(C)O2)O)C(=O)C2=C(O)C=C(OC3C(C(O)C(O)C(CO)O3)O)C(CC=C(C)C)=C2O1 TZJALUIVHRYQQB-UHFFFAOYSA-N 0.000 claims abstract description 29
- TZJALUIVHRYQQB-XFDQAQKOSA-N Icariin Natural products O(C)c1ccc(C2=C(O[C@H]3[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O3)C(=O)c3c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O4)c(C/C=C(\C)/C)c3O2)cc1 TZJALUIVHRYQQB-XFDQAQKOSA-N 0.000 claims abstract description 28
- NGMYNFJANBHLKA-SENBMHEBSA-N Icariside II Natural products O(C)c1ccc(C2=C(O[C@H]3[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O3)C(=O)c3c(O)cc(O)c(C/C=C(\C)/C)c3O2)cc1 NGMYNFJANBHLKA-SENBMHEBSA-N 0.000 claims abstract description 27
- NGMYNFJANBHLKA-LVKFHIPRSA-N icariside II Chemical compound C1=CC(OC)=CC=C1C1=C(O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)C(=O)C2=C(O)C=C(O)C(CC=C(C)C)=C2O1 NGMYNFJANBHLKA-LVKFHIPRSA-N 0.000 claims abstract description 27
- 239000011347 resin Substances 0.000 claims abstract description 27
- 229920005989 resin Polymers 0.000 claims abstract description 27
- 238000001035 drying Methods 0.000 claims abstract description 13
- 239000000843 powder Substances 0.000 claims abstract description 13
- 238000002386 leaching Methods 0.000 claims abstract description 11
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 claims abstract description 10
- 229930003944 flavone Natural products 0.000 claims abstract description 10
- 150000002212 flavone derivatives Chemical class 0.000 claims abstract description 10
- 235000011949 flavones Nutrition 0.000 claims abstract description 10
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000012043 crude product Substances 0.000 claims abstract description 6
- 239000013078 crystal Substances 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 15
- 238000002425 crystallisation Methods 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 230000008025 crystallization Effects 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000002198 insoluble material Substances 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 36
- 235000019441 ethanol Nutrition 0.000 description 74
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 150000004676 glycans Chemical class 0.000 description 13
- 229920001282 polysaccharide Polymers 0.000 description 13
- 239000005017 polysaccharide Substances 0.000 description 13
- 238000001953 recrystallisation Methods 0.000 description 12
- 229930003935 flavonoid Natural products 0.000 description 8
- 150000002215 flavonoids Chemical class 0.000 description 8
- 235000017173 flavonoids Nutrition 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000000178 monomer Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 239000004952 Polyamide Substances 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 229930182721 icariside Natural products 0.000 description 6
- 229920002647 polyamide Polymers 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
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- 239000000463 material Substances 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 238000004945 emulsification Methods 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 238000010298 pulverizing process Methods 0.000 description 3
- 239000012047 saturated solution Substances 0.000 description 3
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229930183477 epimedin Natural products 0.000 description 2
- 230000003054 hormonal effect Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
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- 239000003440 toxic substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000133570 Berberidaceae Species 0.000 description 1
- 241000893531 Epimedium koreanum Species 0.000 description 1
- 241001660849 Epimedium pubescens Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 238000009835 boiling Methods 0.000 description 1
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- 230000002124 endocrine Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
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- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
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- 238000002844 melting Methods 0.000 description 1
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- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 229920006122 polyamide resin Polymers 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
Abstract
A high purity separation and extraction method of herba Epimedii extract comprises: leaching herba Epimedii coarse powder with 80% ethanol, and filtering to obtain crude product; dissolving the crude product with water, filtering, and adsorbing with DM301 macroporous resin adsorption column; eluting the adsorption column with water until the adsorption column is colorless, eluting the adsorption column with 20%, 45% and 60% ethanol sequentially, and respectively collecting ethanol eluents with corresponding concentrations; concentrating 45% ethanol eluent and 60% ethanol eluent respectively; adjusting pH of the concentrated solution to be=5, extracting with ethyl acetate, discarding water phase, and concentrating; recrystallizing the extract with hot ethanol, and drying to obtain icariin and icariside II respectively; concentrating the 20% ethanol eluent, and drying to obtain epimedium flavone. The application adopts the same process to separate icariin and icariside II at the same time, wherein the purity of the icariin is higher than 98%.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicine extraction, in particular to an extraction method of epimedium.
Background
The herba Epimedii medicinal materials are dry leaves of herba Epimedii of berberidaceae, and include herba Epimedii Epinedinbreviconru Maxim, herba Epimedii Epimedium saittatum (Sieb. Et Zucc.) Maxin, herba Epimedii Epimediumpubescens Maxim and herba Epimedii Epimedium koreanum Makai. Herba Epimedii has effects of invigorating kidney yang, strengthening tendons and bones, and dispelling pathogenic wind and dampness.
Icariin is one of the monomer components, has the effects of improving cardiovascular system, regulating endocrine, enhancing immunity, sex hormone-like and the like, and has positive therapeutic effects on resisting tumor, resisting liver toxicity and the like. Its molecular formula is C 33 H 40 O 15 The molecular weight is 676.65, the light yellow needle crystal (pyridine with water) has a melting point of 231-232 ℃, is dissolved in water, ethanol, methanol and ethyl acetate, and is insoluble in ether, benzene and chloroform.
Icariside II is a monomer substance newly developed from epimedium in recent years, and has not only the same pharmacological actions (bone-strengthening, sex hormone-like, etc.) as those of icariine, but also antioxidant actions such as lipid peroxidation inhibition, etc. in addition to improving vascular endothelial cell function. The current method for preparing the high-purity epimedium monomer is less, complex in process and high in cost, and is mostly limited to small-scale laboratory preparation. The separation and purification process of the epimedium monomer is optimized, the simple and efficient mass production is realized, and the method is the basis for deeply researching the pharmacological functions of the epimedium and improving the effective utilization rate of the epimedium medicinal material.
In the prior art, for extracting the effective components in epimedium herb, a macroporous adsorption resin method or a recrystallization method is generally adopted.
FIG. 1 is a technical route of a macroporous adsorption resin method, which has the advantages of fast adsorption, large adsorption capacity, good adsorption selectivity, mild desorption condition, high elution rate, high physical and chemical stability, no influence of inorganic matters, simple regeneration, long service cycle, easy formation of closed cycle, cost saving and the like, and is suitable for industrial production. The macroporous adsorption resin is a separation material combining adsorption and screening, when the separation condition is determined, firstly, proper resin pore diameter is selected according to the size of the molecular volume of the separated compound, and secondly, the model and the separation condition of the resin are determined according to whether the molecular contains phenolic hydroxyl, carboxyl or basic nitrogen atoms. Common eluents are water, methanol, acetone, ethyl acetate, ethanol, etc. For nonpolar macroporous resin, the smaller the polarity of the eluent is, the stronger the eluting capability is; for polar macroporous resin and compounds with larger polarity, the polar solvent can be used for better separation.
The method has the following defects: (1) the purity of the product is lower only through the separation and purification of a resin column; (2) the extract contains other effective components, which cannot be effectively utilized, and the utilization rate of raw materials is low.
FIG. 2 is a general technical route for recrystallization: firstly, selecting a proper solvent, heating and dissolving the mixture to form a saturated solution of an active ingredient, filtering the saturated solution while the saturated solution is hot to remove insoluble impurities, standing the filtrate at a low temperature or steaming part of the solvent and then standing at a low temperature to enable most of the active ingredient to precipitate crystals, wherein the crystals which are initially precipitated always contain some impurities, so-called recrystallization is needed to obtain crystals with high purity by repeated crystallization. The choice of the solvent for crystallization is one of the most important factors, and generally it has a high solubility in the heat and a low solubility in the cold of the component to be crystallized, and it is not dissolved in the impurities or in the cold or in the hot, and it is not chemically reacted with the component to be crystallized, and the boiling point is not so high. Due to the solubility differences of flavonoids in cold or hot methanol, ethanol, acetone and water. Thus, the separation can be performed by recrystallization.
The method for extracting the epimedium herb by recrystallization has the following defects: (1) similar to the macroporous resin method, the purity is lower than that of the macroporous resin method by only purifying through a crystallization method; (2) the finished product contains more components, and the yield of icariin is not high.
Disclosure of Invention
The process for extracting flavone monomer components from epimedium and separating icariside and icariside II by using macroporous resin has not been reported. The application combines the related documents, adopts reflux extraction, macroporous adsorption resin separation and purification, and extraction and recrystallization of organic solvent, and realizes the separation of two monomer substances of icariin and icariside II by adopting the same separation method, and the purity of the two monomer substances is more than 90 percent. The method greatly simplifies experimental process, fully utilizes herba Epimedii medicinal material resource, greatly reduces cost, and is suitable for industrial production.
The application aims at: the icariin and icariside II are separated simultaneously by adopting the same process, wherein the purity of the icariin is higher than 98%, and the extracted residues are fully utilized, so that the economic effect is improved, and the application space is expanded. The application has high utilization rate of raw materials and can greatly save production cost.
The application adopts the technical means that: a high purity separation and extraction method of herba Epimedii extract comprises the following steps:
(1) Leaching herba Epimedii coarse powder with 80% ethanol, filtering, concentrating, and recovering ethanol to obtain herba Epimedii extract crude product;
(2) Dissolving crude herba Epimedii extract with water, filtering, and adsorbing supernatant with DM301 macroporous resin adsorption column;
(3) Eluting the adsorption column with water until the adsorption column is colorless, eluting the adsorption column with 20% ethanol, 45% ethanol and 60% ethanol sequentially, and respectively collecting ethanol eluents with corresponding concentrations;
(4) Concentrating the 45% ethanol eluent to recover ethanol; adjusting pH of the concentrated solution to be=5, extracting with ethyl acetate, removing water phase, concentrating and recovering ethyl acetate to obtain extract; recrystallizing the extract with hot ethanol, and drying to obtain icariin;
(5) Concentrating the 60% ethanol eluent to recover ethanol; adjusting pH of the concentrated solution to be=5, extracting with ethyl acetate, removing water phase, concentrating and recovering ethyl acetate to obtain extract; recrystallizing the extract with hot ethanol, and drying to obtain icariside II;
(6) Concentrating the 20% ethanol eluent, recovering ethanol, and drying to obtain epimedium flavone.
The 20% ethanol, 45% ethanol, 60% ethanol and 80% ethanol are ethanol-water mixed solutions with volume percentages of 20%, 45%, 60% and 80%.
Further, when the epimedium coarse powder is leached by adopting 80 percent ethanol, leaching is carried out for 3 times at 70 ℃ according to the feed liquid ratio of 1:10, leaching is carried out for 90 minutes each time, and filtering is carried out, and then the filtrates are combined.
Further, the crude epimedium extract is dissolved with water according to the ratio of 7.45g/L, insoluble substances are removed by filtration, and the crude epimedium extract is applied to a DM301 macroporous resin adsorption column with the flow rate of 3BV/h.
Further, when the adsorption column is eluted, eluting the column by water until the column is colorless; eluting impurities with 20% ethanol; then eluting with 45% ethanol at a flow rate of 1.5BV/h, and eluting with 4.5BV of eluent; finally eluting with 60% ethanol, wherein the eluting agent dosage is 4.5BV at the flow rate of 1.5 BV/h; collecting 20% ethanol eluent, 45% ethanol eluent and 60% ethanol eluent respectively.
Further, when treating 45% ethanol eluent, firstly concentrating the eluent under reduced pressure, adjusting the pH to 5 with hydrochloric acid, extracting 2 times with 2 times of ethyl acetate, discarding the water layer, concentrating under reduced pressure, and collecting ethyl acetate; dissolving the extract with anhydrous ethanol under heating, filtering while the extract is hot, standing for crystallization, and suction filtering after no crystals are generated; recrystallizing the crystal with absolute ethanol, filtering, and vacuum drying to obtain icariin.
Further, when treating 60% ethanol eluent, firstly concentrating the eluent under reduced pressure, adjusting the pH to 5 with hydrochloric acid, extracting 2 times with 2 times of ethyl acetate, discarding the water layer, concentrating under reduced pressure, and collecting ethyl acetate; dissolving the extract with anhydrous ethanol under heating, filtering while the extract is hot, standing for crystallization, and suction filtering after no crystals are generated; recrystallizing the crystal with absolute ethanol, filtering, and vacuum drying to obtain icariside II pure product.
The beneficial effects of the application are as follows:
(1) the purity of icariin can be improved to more than 95% and is close to 98% by the extraction and purification of the process.
(2) The process not only achieves high-purity icariside, but also separates high-purity icariside II with similar functional effects as icariside, the purity of which is more than 98 percent and higher than that of icariside.
(3) The whole process does not use toxic reagents, and is high in safety and environment-friendly.
(4) The process can also produce flavonoids of epimedium, mainly epimedin.
Drawings
FIG. 1 shows a process flow of a conventional macroporous adsorption resin method.
FIG. 2 shows a conventional recrystallization process.
FIG. 3 is a process flow of the method for extracting herba Epimedii of the present application.
FIG. 4 shows a process flow of extracting polysaccharide from the residue obtained in the process of the present application.
FIG. 5 is a process flow for obtaining icariin by the polyamide column-extraction-recrystallization method in comparative example.
FIG. 6 is a process flow of extracting total flavonoids of herba Epimedii by water decoction in comparative example.
Description of the embodiments
The application is further illustrated below with reference to examples.
Referring to fig. 3, fig. 3 is a process flow diagram of the extraction process of the present application for high-purity epimedium herb.
Extracting coarse powder of epimedium herb in 80% ethanol for 3 times, concentrating the obtained filtrate to obtain extract, removing insoluble substances after washing, continuously treating supernatant, washing to be colorless after passing through a DM301 macroporous resin adsorption column, eluting with 20% ethanol, 45% ethanol and 60% ethanol in sequence, wherein 20% ethanol eluent is treated to obtain epimedium flavone, 45% ethanol eluent is treated to obtain icariin, and 60% ethanol eluent is treated to obtain icariside II.
Examples
(1) Pulverizing dried herba Epimedii into 30 mesh powder;
(2) Weighing 20g of epimedium herb powder in a reflux device according to a feed liquid ratio of 1:10 adding 80% ethanol, extracting at 70deg.C for 90min, filtering, extracting for 3 times, and mixing filtrates.
(3) Concentrating the filtrate under reduced pressure until no alcohol smell exists to obtain crude product, and collecting alcohol.
(4) Dissolving the crude product into 7.45g/L liquid with pure water, filtering to remove insoluble substances, and passing through DM301 macroporous resin adsorption column with flow rate of 3BV/h.
(5) Eluting the column with water until colorless, eluting impurities with 20% ethanol, eluting with 45% ethanol at a flow rate of 1.5BV/h, eluting with 4.5BV of eluent, eluting with 60% ethanol, eluting with 4.5BV of eluent at a flow rate of 1.5BV/h, and collecting 20%, 45% and 60% ethanol eluents respectively.
(6) Concentrating 45% and 60% eluate under reduced pressure to a certain volume, adjusting pH to about 5 with hydrochloric acid solution, extracting with 2 times of ethyl acetate, discarding water layer, concentrating under reduced pressure, and collecting ethyl acetate.
(7) Dissolving the extracts with anhydrous ethanol under heating, filtering, standing for crystallization, filtering after no crystal is generated, recrystallizing, and filtering to obtain crystal.
(8) And (5) drying the crystals in vacuum to obtain corresponding pure products.
(9) Concentrating the 20% ethanol eluate, and drying to obtain epimedium flavone mainly containing epimedin.
Examples 2 to 5
This example further discusses the preferred processes and reasons for the separation and purification methods of the present application, and the following examples are carried out with reference to example 1, with the specific differences shown in Table 1:
TABLE 1 examples 1-5 Process and Effect
As can be seen from the comparison of the process flow of FIG. 3 and examples 1-3, the DM301 macroporous resin adsorption column is eluted by 20% ethanol, and the obtained total flavonoids have the highest purity and yield, and are the best process for extracting flavonoids. Compared with other ethanol concentrations, the ethanol concentration can reduce the dosage of eluent and shorten the time consumption of the process; and when ethanol is recovered by subsequent concentration and drying, the consumed energy consumption is lower, and the ethanol loss is relatively lower.
In the process of eluting the 18-22% ethanol eluent, a small amount of eluent is taken, and thin layer (silica gel plate) chromatographic analysis is carried out by taking petroleum ether/ethyl acetate as developing agent, so as to verify whether the elution of the 18-22% ethanol eluent is complete. After the elution is completed, the icariin in the DM301 macroporous resin adsorption column is continuously eluted by using 42 to 48 percent of ethanol. The results showed that 45% ethanol eluent was the optimal ratio for icariin elution. When the concentration of the ethanol is lower, the using amount of the eluent is larger, the eluting time is longer, and the purity and the yield of icariin in the product are lower; when the concentration of ethanol is higher, the purity and the yield of icariside are slightly reduced, but icariside II is easily introduced into the product. The icariside II introduced during the process of the application is not separated when the icariside II is eluted by ethanol with higher concentration because the icariside and the icariside II are extracted and recrystallized by adopting the same process, thus slightly reducing the purity.
After that, thin layer (silica gel plate) chromatography was performed still using petroleum ether/ethyl acetate as developing agent to verify whether 42-48% ethanol eluent was completely eluted. After the elution is completed, 58-63% ethanol is used for eluting icariside II. The results showed that 60-63% ethanol eluent was the preferred ratio of eluting icariside II. When the concentration of ethanol is 58%, the yield of icariside II is poor, which indicates that the concentration has poor elution capacity for icariside II; the purity of the ethanol product eluted by 63% is close to 60% and is more than 98%, which shows that the selectivity to icariside II is better under the concentration. Example 3 icariside II yields are inferior to example 1, possibly, 48% ethanol in the second eluent is not good in selectivity, and some eluting capacity is provided for icariside II, so that the subsequent process icariside II yields are reduced.
The yields, purities, etc. of icariin, icariside II, flavone, etc. as the main extracts under the preferred process of example 1 were all superior to those of the control group (examples 2, 3). The DM301 macroporous resin is eluted in batches by adopting 20 percent ethanol, 45 percent ethanol and 60 percent ethanol, and the method is the optimal process for obtaining the epimedium extract.
In the example 4, the traditional methanol is adopted to recrystallize the extract, and comparison shows that the ethanol method has better recrystallization effect, only one recrystallization is needed, the crystallization times are reduced, and the process operation is simplified. The methanol is adopted to recrystallize icariin and icariside II once, the yield and purity of the obtained product are not as good as those of absolute ethyl alcohol, and the methanol method can achieve ideal effects by repeatedly recrystallizing the product for many times.
In the embodiment 5, the pH value of the solution is not adjusted in the extraction process, and in the extraction process, both icariin and icariside II extracting solutions generate emulsification, and the emulsification enables the organic phase and the water phase to form a mixed phase, which is unfavorable for the separation of the two phases, and the emulsion needs to be demulsified by adopting an additional process. Even after demulsification, the emulsification phenomenon in the extraction process causes a decrease in extraction efficiency, which finally manifests itself in a significant decrease in product yield.
EXAMPLE 6 extraction of Epimedium polysaccharide Using residue
The whole process of the epimedium extraction process does not involve toxic reagents, and the process is high in safety and environment-friendly. Referring to fig. 4, this example provides a method for further utilization of epimedium herb residue, and evaluates the resulting epimedium polysaccharide poisoning pest residue.
(1) Drying the residues after extracting the total flavonoids of herba Epimedii, and pulverizing for use;
(2) Taking a certain amount of dregs, wherein the ratio of the dregs to the liquid is 1:10 adding water, extracting at 90deg.C for 2 times each for 30min;
(3) Filtering, mixing filtrates, and concentrating on rotary evaporator to a certain volume;
(4) Adding absolute ethanol into the concentrated solution until the alcohol concentration is 90%, stirring uniformly, standing for 12h, and filtering to obtain a precipitate;
(5) Dissolving the precipitate with water, stirring, standing for 4 hr, filtering, concentrating the filtrate, and repeating the ethanol precipitation process once again to obtain precipitate;
(6) Dissolving the precipitate in water, removing protein by Sevage method, dialyzing water layer against running water for 24 hr, and dialyzing in distilled water for 36 hr;
(7) Concentrating water in dialysis bag, and lyophilizing to obtain herba Epimedii polysaccharide.
The obtained epimedium polysaccharide was measured by high performance liquid chromatography, and as a result, no organic solvent residue was detected. Based on analysis of extraction process, toxic substances are not introduced in the process of obtaining epimedium herb residues from epimedium coarse powder, and the epimedium polysaccharide extraction process is studied at home and abroad. No significant difference was found between the polysaccharide obtained in this example and the epimedium polysaccharide directly extracted from epimedium meal by water-sedimentation alcohol extraction. Because the application adopts ethanol to extract the epimedium coarse powder, no toxic substances are introduced, and the loss of the epimedium polysaccharide is avoided, and the obtained dregs can be further treated to obtain the epimedium polysaccharide. The example shows that the epimedium herb residue obtained by the process is also green and environment-friendly, and the epimedium polysaccharide with medicinal value can be obtained by simple treatment.
Through detection, the partial free radical scavenging capability of the epimedium polysaccharide extracted from the traditional Chinese medicine residues is reduced, but the in-vitro antioxidant activity is still very high, and the epimedium polysaccharide has higher medical and health care values.
Comparative example 1
Referring to fig. 5, the current mature epimedium extraction process adopts a polyamide column-extraction-recrystallization method to extract icariin. The method mainly comprises the steps of eluting an epimedium extract through a polyamide column, extracting with chloroform, extracting with ethyl acetate, and finally carrying out pure crystallization with 60% ethanol to obtain a product. The comparative evaluation was carried out in this example using the polyamide column-extraction-recrystallization method as a comparative example, which comprises the following steps:
(1) Pulverizing dried herba Epimedii into 30 mesh powder;
(2) Extracting herba epimedii whole herb powder by adopting 80% ethanol, wherein the feed liquid ratio is 1:10, extracting for 3 times to obtain leaching liquor;
(3) Concentrating the leaching solution to obtain an extract, removing insoluble substances after water dissolution, and passing through a polyamide column with an aqueous solution;
(4) Eluting the polyamide column with 50% ethanol to obtain eluent, and concentrating to obtain extract;
(5) Extracting the extract by using a chloroform/ethyl acetate mixed solvent, and collecting the solvent under reduced pressure;
(6) Dissolving the extract with 60% ethanol, filtering while the extract is hot, standing for crystallization, and taking a filter cake part for drying after suction filtration to obtain the product.
Through inspection, the product is mainly icariin, and the purity of the product is also over 90 percent.
The different types of chromatographic columns are applied to the same process in this example, and icariin is extracted by comparing the various chromatographic columns.
TABLE 2 Selectivity of icariin extraction by different chromatographic columns
| Chromatographic column | Icariin purity | Icariin yield |
| DM301 macroporous resin | 97.5% | 92.1% |
| PA03060 polyamide resin | 90.7% | 82.3% |
| D1400 macroporous resin | 96.8% | 88.6% |
| HZ-841 macroporous resin | 93.4% | 85.1% |
| D101 macroporous resin | 96.2% | 88.2% |
Comparative example 2
Referring to fig. 6, the conventional process mainly adopts a water extraction method to extract total flavonoids contained in epimedium, and the control group extracts the total flavonoids by water to obtain a total flavone extract. The subsequent total flavone purification process was the same as in example 1, and purification was performed in the same manner.
The result shows that the water leaching of the epimedium coarse powder can achieve the purpose of extracting the total flavone, the extracts of icariin and the like are difficult to obtain, and the yield of the method is lower.
Claims (6)
1. A high-purity separation and extraction method of an epimedium extract is characterized by comprising the following steps of:
leaching herba Epimedii coarse powder with 80% ethanol, filtering, concentrating, and recovering ethanol to obtain herba Epimedii extract crude product;
dissolving crude herba Epimedii extract with water, filtering, and adsorbing supernatant with DM301 macroporous resin adsorption column;
eluting the adsorption column with water until the adsorption column is colorless, eluting the adsorption column with 20% ethanol, 45% ethanol and 60% ethanol sequentially, and respectively collecting ethanol eluents with corresponding concentrations;
concentrating the 45% ethanol eluent to recover ethanol; adjusting pH of the concentrated solution to be=5, extracting with ethyl acetate, removing water phase, concentrating and recovering ethyl acetate to obtain extract; recrystallizing the extract with hot ethanol, and drying to obtain icariin;
concentrating the 60% ethanol eluent to recover ethanol; adjusting pH of the concentrated solution to be=5, extracting with ethyl acetate, removing water phase, concentrating and recovering ethyl acetate to obtain extract; recrystallizing the extract with hot ethanol, and drying to obtain icariside II;
concentrating the 20% ethanol eluent, recovering ethanol, and drying to obtain epimedium flavone.
2. The method of claim 1, wherein when 80% ethanol is used for leaching the epimedium coarse powder, leaching is carried out for 3 times at 70 ℃ with a feed liquid ratio of 1:10, leaching is carried out for 90min each time, and filtering is carried out, and filtrates are combined.
3. The method of claim 1, wherein the crude epimedium extract is dissolved in water at a rate of 7.45g/L before adsorption by the adsorption column, insoluble materials are removed by filtration, and the flow rate is 3BV/h through a DM301 macroporous resin adsorption column.
4. The method of claim 1, wherein the adsorption column is eluted with water to colorless; eluting impurities with 20% ethanol; then eluting with 45% ethanol at a flow rate of 1.5BV/h, and eluting with 4.5BV of eluent; finally eluting with 60% ethanol, wherein the eluting agent dosage is 4.5BV at the flow rate of 1.5 BV/h; collecting 20% ethanol eluent, 45% ethanol eluent and 60% ethanol eluent respectively.
5. The method according to claim 1, wherein the 45% ethanol eluent is treated by concentrating the eluent under reduced pressure, adjusting the pH to 5 with hydrochloric acid, extracting 2 times with 2 times of ethyl acetate, discarding the aqueous layer, concentrating under reduced pressure, and collecting ethyl acetate; dissolving the extract with anhydrous ethanol under heating, filtering while the extract is hot, standing for crystallization, and suction filtering after no crystals are generated; recrystallizing the crystal with absolute ethanol, filtering, and vacuum drying to obtain icariin.
6. The method according to claim 1, wherein the 60% ethanol eluent is treated by first concentrating the eluent under reduced pressure, adjusting the pH to 5 with hydrochloric acid, extracting 2 times with 2 volumes of ethyl acetate, discarding the aqueous layer, concentrating under reduced pressure and collecting ethyl acetate; dissolving the extract with anhydrous ethanol under heating, filtering while the extract is hot, standing for crystallization, and suction filtering after no crystals are generated; recrystallizing the crystal with absolute ethanol, filtering, and vacuum drying to obtain icariside II pure product.
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| CN103275148A (en) * | 2013-05-13 | 2013-09-04 | 张洋 | Preparation method of icariin |
| CN106589020A (en) * | 2016-12-31 | 2017-04-26 | 北京颐方生物科技有限公司 | Method for extracting icariin from epimedium |
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| CN103275148A (en) * | 2013-05-13 | 2013-09-04 | 张洋 | Preparation method of icariin |
| CN106589020A (en) * | 2016-12-31 | 2017-04-26 | 北京颐方生物科技有限公司 | Method for extracting icariin from epimedium |
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