CN109776474A - A kind of method for separating and purifying flavonoids from wild horse chasing - Google Patents
A kind of method for separating and purifying flavonoids from wild horse chasing Download PDFInfo
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- CN109776474A CN109776474A CN201910238987.3A CN201910238987A CN109776474A CN 109776474 A CN109776474 A CN 109776474A CN 201910238987 A CN201910238987 A CN 201910238987A CN 109776474 A CN109776474 A CN 109776474A
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- 238000000034 method Methods 0.000 title claims abstract description 27
- 229930003935 flavonoid Natural products 0.000 title abstract description 3
- 150000002215 flavonoids Chemical class 0.000 title abstract description 3
- 235000017173 flavonoids Nutrition 0.000 title abstract description 3
- 241001600609 Equus ferus Species 0.000 title 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 54
- 239000000469 ethanolic extract Substances 0.000 claims abstract description 26
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000000284 extract Substances 0.000 claims abstract description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000002904 solvent Substances 0.000 claims abstract description 9
- 239000003208 petroleum Substances 0.000 claims abstract description 8
- 238000000926 separation method Methods 0.000 claims abstract description 8
- 239000012153 distilled water Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 241000735527 Eupatorium Species 0.000 claims description 29
- 150000001875 compounds Chemical class 0.000 claims description 26
- FHHSEFRSDKWJKJ-UHFFFAOYSA-N nepetin Chemical compound C=1C(=O)C2=C(O)C(OC)=C(O)C=C2OC=1C1=CC=C(O)C(O)=C1 FHHSEFRSDKWJKJ-UHFFFAOYSA-N 0.000 claims description 26
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- 229930003944 flavone Natural products 0.000 claims description 19
- 235000011949 flavones Nutrition 0.000 claims description 19
- 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 description 18
- 239000007900 aqueous suspension Substances 0.000 claims description 15
- YFZSQPRYLBGYKE-UHFFFAOYSA-N Jaceoside Natural products C1=C(O)C(OC)=CC(C=2OC3=CC(OC4C(C(O)C(O)C(CO)O4)O)=C(OC)C(O)=C3C(=O)C=2)=C1 YFZSQPRYLBGYKE-UHFFFAOYSA-N 0.000 claims description 14
- GLAAQZFBFGEBPS-UHFFFAOYSA-N jaceosidin Chemical compound C1=C(O)C(OC)=CC(C=2OC3=CC(O)=C(OC)C(O)=C3C(=O)C=2)=C1 GLAAQZFBFGEBPS-UHFFFAOYSA-N 0.000 claims description 14
- AOAPCDXPLWGVGI-UHFFFAOYSA-N jaceosidin Natural products COc1c(O)cc2OC(=CC(=O)c2c1O)c3ccc(O)c(C)c3 AOAPCDXPLWGVGI-UHFFFAOYSA-N 0.000 claims description 14
- SEBFKMXJBCUCAI-DBMPWETRSA-N silybin Chemical compound C1=C(O)C(OC)=CC(C2C(OC3=CC=C(C=C3O2)[C@@H]2[C@H](C(=O)C3=C(O)C=C(O)C=C3O2)O)CO)=C1 SEBFKMXJBCUCAI-DBMPWETRSA-N 0.000 claims description 14
- XRHHDQSPFPQKMS-UHFFFAOYSA-N sudachitin Natural products C1=C(O)C(OC)=CC(C=2OC3=C(OC)C(O)=C(OC)C(O)=C3C(=O)C=2)=C1 XRHHDQSPFPQKMS-UHFFFAOYSA-N 0.000 claims description 14
- 150000002212 flavone derivatives Chemical class 0.000 claims description 13
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- 238000010262 high-speed countercurrent chromatography Methods 0.000 claims description 11
- 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 claims description 7
- 230000005526 G1 to G0 transition Effects 0.000 claims description 6
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 claims description 6
- HZQXXYJHLCSUGQ-UHFFFAOYSA-N ethyl acetate hexane methanol hydrate Chemical compound O.OC.CCCCCC.CCOC(C)=O HZQXXYJHLCSUGQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 5
- 230000005484 gravity Effects 0.000 claims description 5
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- 239000002021 butanolic extract Substances 0.000 abstract 2
- VEVZSMAEJFVWIL-UHFFFAOYSA-O cyanidin cation Chemical compound [O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC=C(O)C(O)=C1 VEVZSMAEJFVWIL-UHFFFAOYSA-O 0.000 abstract 2
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- WHKUVVPPKQRRBV-UHFFFAOYSA-N Trasan Chemical compound CC1=CC(Cl)=CC=C1OCC(O)=O WHKUVVPPKQRRBV-UHFFFAOYSA-N 0.000 abstract 1
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- 229960003083 cymarin Drugs 0.000 abstract 1
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- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 2
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- LUJAXSNNYBCFEE-UHFFFAOYSA-N Quercetin 3,7-dimethyl ether Natural products C=1C(OC)=CC(O)=C(C(C=2OC)=O)C=1OC=2C1=CC=C(O)C(O)=C1 LUJAXSNNYBCFEE-UHFFFAOYSA-N 0.000 description 1
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明涉及一种从野马追中分离纯化黄酮类化合物的方法，取干燥的野马追粉碎，加入80‑95％乙醇溶液加热回流或常温浸提，将提取液过滤后减压浓缩，得乙醇提取物，将乙醇提取物分散于蒸馏水中，依次用萃取溶剂石油醚、乙酸乙酯和正丁醇进行萃取，最后真空浓缩正丁醇萃取相，得到正丁醇萃取物，对其采用高速逆流色谱仪进行分离，根据色谱图收集相应峰组分，减压浓缩干燥即可得到高纯度的泽兰叶黄素、线蓟素和棕矢车菊素。本发明方法具有高效快速、成本低廉、分离量大、样品损失小、产品纯度高等优点，并且操作简单、重现性好，具有很好的推广使用价值。
The invention relates to a method for separating and purifying flavonoids from Yemachai, which comprises the following steps: taking dried Yemachai and pulverizing, adding 80-95% ethanol solution for heating under reflux or leaching at room temperature, filtering the extract and then concentrating under reduced pressure to obtain ethanol extraction. The ethanol extract was dispersed in distilled water, followed by extraction with the extraction solvent petroleum ether, ethyl acetate and n-butanol, and finally the n-butanol extract was concentrated in vacuo to obtain the n-butanol extract, which was subjected to a high-speed countercurrent chromatograph Separation is carried out, the corresponding peak components are collected according to the chromatogram, and concentrated and dried under reduced pressure to obtain high-purity Zelan lutein, cymarin and cyanidin. The method of the invention has the advantages of high efficiency, rapidity, low cost, large separation amount, small sample loss, high product purity, simple operation, good reproducibility, and good popularization and use value.
Description
Technical field
The method of the present invention relates to a kind of from eupatorium lindleynun var. trifoliolatum separating and purifying flavone class compound, it is especially a kind of from eupatorium lindleynun var. trifoliolatum
In isolate and purify the method for eupafolin, line silibin and Jaceosidin, belong to field of natural medicinal chemistry.
Background technique
Eupatorium Lindleyanum DC is the drying overground part of compositae plant Eupatorium lindleyanum (Eupatorium lindleyanum DC.)
Point, bitter is mild-natured, there is clearing lung and relieving cough, resolving sputum and relieving asthma, antibacterial anti-inflammatory, inducing diuresis to remove edema, blood pressure lowering and other effects, clinical main use
In illnesss such as treatment tracheitis, coughing with a lot of sputum, hypertension.Chemical component contained by eupatorium lindleynun var. trifoliolatum herb based on flavone compound,
There are also alkaloids, volatile oil and cumarins, sesquiterpenoids ester etc..Flavone compound has significant physiology and pharmacology living
Property, such as free radical resisting, antitumor, antiviral, anti-inflammatory, protection cardiovascular system, improvement memory, antidepression and protection nerveous system
System and other effects, the preparation of preparation have treatment valuable the diseases such as cardiovascular and cerebrovascular, artery sclerosis, " three high " and take for a long time
It has no toxic side effect.Sufficiently to develop and use this valuable natural resources of Chinese medicinal materials comprehensively, so extraction purification goes out flavones list from eupatorium lindleynun var. trifoliolatum
Body compound has very important significance.
Currently, separating and purifying flavone compound is mainly using column chromatography method from eupatorium lindleynun var. trifoliolatum.For example, Wu Shuanqing etc.
[eupatorium lindleynun var. trifoliolatum chemical component, CHINA JOURNAL OF CHINESE MATERIA MEDICA, the 7th phase in 2012] is repeatedly using column chromatographys such as silica gel, Sephadex LH-20
Method isolates and purifies the Ethyl acetate fraction of 95% ethanol extract of eupatorium lindleynun var. trifoliolatum, isolates eupafolin, palm fibre arrow
16 compounds such as Che Jusu, Quercetin, line silibin；[research of flavonoids of Eupatorium lindleyanum, Chinese medicine are miscellaneous by Qian Shihui etc.
Will, the 1st phase in 2004] pass through the methods of solvent extraction, silica gel column chromatography and recrystallization, isolated Jaceosidin, quercitrin
Six kinds of compounds such as element.The above method requires that column chromatography is repeated, and complicated for operation, time-consuming, and solvent-oil ratio is big, and
Sample loss is big.
High speed adverse current chromatogram (High-Speed Counter-Current Chromatography, HSCCC) is a kind of company
Continue efficient liquid-liquid chromatography isolation technics, since HSCCC is not required to any solid support as stationary phase, the separation of substance according to
It is realized according to the difference of its distribution coefficient in two-phase, therefore absorption, pollution, denaturation and the peak shape hangover etc. that avoid sample are asked
Topic, have the advantages that efficiently, quickly with preparation greatly, expense it is low etc., thus be increasingly subject to the concern of people in recent years, answer extensively
For fields such as biological medicine, bioengineering, plant, chemical industry, environment, foods and cosmetics.The present inventor utilizes high-speed counter-current
Chromatography separation from eupatorium lindleynun var. trifoliolatum prepares the flavone compounds such as Jaceosidin, eupafolin, line silibin, has not yet to see state
It is inside and outside in relation to document and patent report.
Summary of the invention
It is an object of the invention to solve in the prior art using column chromatography separating and purifying flavone compound from eupatorium lindleynun var. trifoliolatum
Deficiency existing for method, provide it is a kind of it is easy to operate, low in cost, fractional dose is big, product purity is high, sample loss is small therefrom
The method of eupafolin, line silibin and Jaceosidin is isolated and purified in medicine eupatorium lindleynun var. trifoliolatum.
Technical solution
A method of the separating and purifying flavone class compound from eupatorium lindleynun var. trifoliolatum includes the following steps:
(1) it takes dry eupatorium lindleynun var. trifoliolatum to crush, is then carried out using the ethanol solution of volumetric concentration 80-95% as Extraction solvent
It extracts, obtains extracting solution, extracting solution is filtered, then reprocess filter residue 2-4 times, merging filtrate is simultaneously concentrated under reduced pressure, and obtains
Ethanol extract；
(2) it disperses ethanol extract in distilled water, obtains the water suspension solution of ethanol extract, then successively with extraction
It takes solvent petroleum ether, ethyl acetate and n-butanol to be extracted, is finally concentrated in vacuo extracting n-butyl alcohol phase, obtains extracting n-butyl alcohol
Object；
(3) use n-hexane-ethyl acetate-methanol-water of volume ratio 3:4:3:4 for dicyandiamide solution, upper phase is stationary phase,
Lower phase is mobile phase, carries out HSCCC separation to n-butyl alcohol extract, UV detector on-line monitoring collects different fractions respectively
It is dried under reduced pressure to get eupafolin, line silibin and Jaceosidin.
Further, in step (1), the extracting method is to be added to smashed eupatorium lindleynun var. trifoliolatum with the solid-to-liquid ratio of 1:8-12
In Extraction solvent, heating and refluxing extraction or soak at room temperature 2-3 days are carried out.
Further, in step (2), the specific gravity of the water suspension solution of the ethanol extract is 1.1-1.2.
Further, in step (2), when extraction, the water suspension solution of ethanol extract and the volume ratio of extractant are 1:
2-3。
Further, in step (2), when using extractant petroleum ether, ethyl acetate and extracting n-butyl alcohol, every kind of extraction is molten
Agent extracts 2-4 times.
Further, in step (3), when carrying out HSCCC separation, flow rate of mobile phase 2-3mL/min, splitter revolving speed is
800-900rpm。
Beneficial effects of the present invention: the side of the present invention provides a kind of from eupatorium lindleynun var. trifoliolatum separating and purifying flavone class compound
Method, this method has many advantages, such as that efficiently quick, low in cost, fractional dose is big, sample loss is small, product purity is high, and operates
Simply, favorable reproducibility, can industrialized production.
Detailed description of the invention
Fig. 1 is the n-hexane-ethyl acetate-methanol-water (V:V:V:V=3:4:3:4) that uses of embodiment 1 for dicyandiamide solution
When high speed adverse current chromatogram isolate and purify the chromatogram of Herba Eupatorii Lindleyani extract；
Fig. 2 is the chromocor compound Jaceosidin that embodiment 1 is isolated1H-NMR map；
Fig. 3 is the chromocor compound eupafolin that embodiment 1 is isolated1H-NMR map；
Fig. 4 is the chromocor compound line silibin that embodiment 1 is isolated1H-NMR map.
Specific embodiment
Further illustrate the present invention in the following with reference to the drawings and specific embodiments.
Embodiment 1
A method of the separating and purifying flavone class compound from eupatorium lindleynun var. trifoliolatum includes the following steps:
(1) dry eupatorium lindleynun var. trifoliolatum 10.0kg is taken, 100L 95v% ethanol solution is used to be mentioned after crushing as Extraction solvent
It takes and (impregnates 3 days at room temperature), obtain extracting solution, extracting solution is filtered, then repeat to impregnate 4 times by filter residue, merging filtrate simultaneously subtracts
Pressure concentration, obtains ethanol extract；
(2) it disperses ethanol extract in distilled water, obtains the water suspension solution of ethanol extract, make ethanol extract
Water suspension solution specific gravity between 1.1-1.2, then successively extracted with extractant petroleum ether, ethyl acetate and n-butanol
It takes, the water suspension solution and extractant volume ratio of ethanol extract are 1:2, and each solvent extraction 3 times is finally concentrated in vacuo positive fourth
Alcohol extraction phase, obtains n-butyl alcohol extract；
(3) use n-hexane-ethyl acetate-methanol-water of volume ratio 3:4:3:4 for dicyandiamide solution, upper phase is stationary phase,
Lower phase is mobile phase, and flow rate of mobile phase 2mL/min, splitter revolving speed is 900rpm, carries out HSCCC points to n-butyl alcohol extract
From, UV detector on-line monitoring, chromatogram is shown in Fig. 1, collects different target fraction respectively according to chromatogram, after being dried under reduced pressure,
Up to corresponding high-purity compound.
Through1H-NMR identification, gained monomeric compound is respectively that eupafolin, line silibin and Jaceosidin, Fig. 2 are
Isolate chromocor compound Jaceosidin1H-NMR map；Fig. 3 is the chromocor compound eupafolin isolated1H-
NMR spectra；Fig. 4 isolates chromocor compound line silibin1H-NMR map.With the calculating of chromatographic peak area normalization method, Herba Lycopi
Lutein purity is 96.5%, and line silibin purity is 97.3%, and Jaceosidin purity is 98.9%.
Embodiment 2
A method of the separating and purifying flavone class compound from eupatorium lindleynun var. trifoliolatum includes the following steps:
(1) dry eupatorium lindleynun var. trifoliolatum 500g is taken, uses 6L 90v% ethanol solution to extract after crushing as Extraction solvent and (adds
Heat reflux 90min), extracting solution is obtained, extracting solution is filtered, is then repeated filter residue refluxing extraction 2 times, merging filtrate simultaneously depressurizes
Concentration, obtains ethanol extract；
(2) it disperses ethanol extract in distilled water, obtains the water suspension solution of ethanol extract, make ethanol extract
Water suspension solution specific gravity between 1.1-1.2, then successively extracted with extractant petroleum ether, ethyl acetate and n-butanol
It takes, the water suspension solution and extractant volume ratio of ethanol extract are 1:3, and each solvent extraction 3 times is finally concentrated in vacuo positive fourth
Alcohol extraction phase, obtains n-butyl alcohol extract；
(3) use n-hexane-ethyl acetate-methanol-water of volume ratio 3:4:3:4 for dicyandiamide solution, upper phase is stationary phase,
Lower phase is mobile phase, and flow rate of mobile phase 3mL/min, splitter revolving speed is 900rpm, carries out HSCCC points to n-butyl alcohol extract
From UV detector on-line monitoring collects different target fraction according to chromatogram, to get corresponding height after being dried under reduced pressure respectively
Pure compound.
Through1H-NMR identification, gained monomeric compound is respectively eupafolin, line silibin and Jaceosidin, with chromatography
Areas of peak normalization method calculates, and eupafolin purity is 95.8%, and line silibin purity is 96.6%, and Jaceosidin purity is
98.3%.
Embodiment 3
A method of the separating and purifying flavone class compound from eupatorium lindleynun var. trifoliolatum includes the following steps:
(1) dry eupatorium lindleynun var. trifoliolatum 2kg is taken, uses 20L 85v% ethanol solution to extract after crushing as Extraction solvent and (adds
Heat reflux 3h), extracting solution is obtained, extracting solution is filtered, is then repeated filter residue refluxing extraction 3 times, merging filtrate simultaneously depressurizes dense
Contracting, obtains ethanol extract；
(2) it disperses ethanol extract in distilled water, obtains the water suspension solution of ethanol extract, make ethanol extract
Water suspension solution specific gravity between 1.1-1.2, then successively extracted with extractant petroleum ether, ethyl acetate and n-butanol
It takes, the water suspension solution and extractant volume ratio of ethanol extract are 1:2, and each solvent extraction 3 times is finally concentrated in vacuo positive fourth
Alcohol extraction phase, obtains n-butyl alcohol extract；
(3) use n-hexane-ethyl acetate-methanol-water of volume ratio 3:4:3:4 for dicyandiamide solution, upper phase is stationary phase,
Lower phase is mobile phase, and flow rate of mobile phase 2mL/min, splitter revolving speed is 800rpm, carries out HSCCC points to n-butyl alcohol extract
From UV detector on-line monitoring collects different target fraction according to chromatogram, to get corresponding height after being dried under reduced pressure respectively
Pure compound.
Through1H-NMR identification, gained monomeric compound is respectively eupafolin, line silibin and Jaceosidin, with chromatography
Areas of peak normalization method calculates, and eupafolin purity is 97.2%, and line silibin purity is 97.9%, and Jaceosidin purity is
98.5%.
Claims (6)
1. a kind of method of the separating and purifying flavone class compound from eupatorium lindleynun var. trifoliolatum, which comprises the steps of:
(1) it takes dry eupatorium lindleynun var. trifoliolatum to crush, is then mentioned using the ethanol solution of volumetric concentration 80-95% as Extraction solvent
It takes, obtains extracting solution, extracting solution is filtered, then reprocess filter residue 2-4 times, merging filtrate is simultaneously concentrated under reduced pressure, and obtains second
Alcohol extracting thing；
(2) it disperses ethanol extract in distilled water, obtains the water suspension solution of ethanol extract, it is then successively molten with extracting
Agent petroleum ether, ethyl acetate and n-butanol are extracted, and are finally concentrated in vacuo extracting n-butyl alcohol phase, are obtained n-butyl alcohol extract；
(3) use n-hexane-ethyl acetate-methanol-water of volume ratio 3:4:3:4 for dicyandiamide solution, upper phase is stationary phase, lower phase
For mobile phase, HSCCC separation is carried out to n-butyl alcohol extract, UV detector on-line monitoring collects different fractions decompression respectively
Drying is to get eupafolin, line silibin and Jaceosidin.
2. as described in claim 1 from eupatorium lindleynun var. trifoliolatum separating and purifying flavone class compound method, which is characterized in that step (1)
In, the extracting method is that smashed eupatorium lindleynun var. trifoliolatum is added in Extraction solvent with the solid-to-liquid ratio of 1:8-12, is heated back
Flow extraction or soak at room temperature 2-3 days.
3. as described in claim 1 from eupatorium lindleynun var. trifoliolatum separating and purifying flavone class compound method, which is characterized in that step (2)
In, the specific gravity of the water suspension solution of the ethanol extract is 1.1-1.2.
4. as described in claim 1 from eupatorium lindleynun var. trifoliolatum separating and purifying flavone class compound method, which is characterized in that step (2)
In, when extraction, the water suspension solution of ethanol extract and the volume ratio of extractant are 1:2-3.
5. as described in claim 1 from eupatorium lindleynun var. trifoliolatum separating and purifying flavone class compound method, which is characterized in that step (2)
In, when using extractant petroleum ether, ethyl acetate and extracting n-butyl alcohol, every kind of extractant is extracted 2-4 times.
6. as described in claims 1 or 2 or 3 or 4 or 5 from eupatorium lindleynun var. trifoliolatum separating and purifying flavone class compound method, feature
It is, in step (3), when carrying out HSCCC separation, flow rate of mobile phase 2-3mL/min, splitter revolving speed is 800-900rpm.
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