CN1935195B - Method for extracting female sex hormone from polygonum cuspidatum - Google Patents
Method for extracting female sex hormone from polygonum cuspidatum Download PDFInfo
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- CN1935195B CN1935195B CN2005100472588A CN200510047258A CN1935195B CN 1935195 B CN1935195 B CN 1935195B CN 2005100472588 A CN2005100472588 A CN 2005100472588A CN 200510047258 A CN200510047258 A CN 200510047258A CN 1935195 B CN1935195 B CN 1935195B
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- ethyl acetate
- rhizoma polygoni
- polygoni cuspidati
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Abstract
The present invention relates to plant estrogen in pharmaceutical field. In the concrete, it relates to an extraction method of estrogen from Chinese medicinal material bushy knotweed root. Said extraction method includes the following steps: (1), placing the Chinese medicinal material bushy knotweed root in aqueous solution of ethyl alcohol to make normal temperature extraction to obtain bushy knotweed extract; (2), concentrating and drying the bushy knotweed extract, using water to make suspension, then using ethyl acetate to make extraction; (3), making obtained ethyl acetate region be passed through silica gel column to make chromatographic separation, using petroleum ether: ethyl acetate: formic acid=2:8:0.5 as developing agent and collecting the fraction whose Rf is greater than 0.66and less than or equal to 1; and (4), utilizing HPLC to refine the above-mentioned fraction so as to obtain the target product.
Description
Technical field
The present invention relates to the phytoestrogen in the pharmaceutical field, estrogenic extracting method in specifically a kind of Rhizoma Polygoni Cuspidati.
Background technology
Phytoestrogen be meant some can in conjunction with and activate mammal and the mankind estrogen receptor, thereby have estrogen-like and/or the active plant component of estrogen antagonist, have wide biological activity.Phytoestrogen is being carried out in the process of broad research, it is found that the chemical constituent that is associated with phytoestrogen is rich among some Chinese medicine, and be the biological active component that Chinese medicine is brought into play some drug action.Particularly in recent years discover phytoestrogen except osteoporosis, climacteric syndrome, the cardiovascular disease that causes because of estrogen decrease behind the postmenopausal women had the obvious curative effects, to some hormone dependence property tumor, also have excellent prevention and therapeutical effect as breast carcinoma, carcinoma of endometrium, carcinoma of prostate etc., thereby caused drug research person's deepest concern.Therefore, the phytoestrogen composition in the screening Chinese medicine will have very significant meaning.
The Chinese medicine Rhizoma Polygoni Cuspidati extensively is used to prevent and treat a series of diseases that produce after menoxenia and the menopause, has stronger estrogen activity.At present, the main estrogen activity composition of Rhizoma Polygoni Cuspidati is the abundant emodin of content wherein, and studies show that some of them micro constitutent might have high estrogen activity.
Summary of the invention
The invention provides estrogenic extracting method in a kind of Rhizoma Polygoni Cuspidati, the present invention utilizes preparative high-performance liquid chromatographic that the estrogen activity position in the Rhizoma Polygoni Cuspidati is made with extra care, and has obtained having the chemical constituent of higher estrogen activity.
For achieving the above object, the present invention can obtain having the active chemical constituent of high estrogen by separating solvent extraction, extraction, silica gel column chromatography and the high performance liquid preparative chromatography of Rhizoma Polygoni Cuspidati pharmaceutical decocting piece are smart.
The technical scheme that adopts is as follows: the extracting method of the Rhizoma Polygoni Cuspidati that the present invention proposes is the ethanol cold-maceration.The principle that the total extract that extraction is obtained adopts solvent to distribute extracts.At first adopt silicagel column to realize crude separation to active site-ethyl acetate extract, target components is carried out preliminary enrichment after, utilize high performance liquid preparative chromatography that target components is carried out fly-cutting again.Whole process adopts high performance liquid chromatography and mass spectrum to monitor.
Concrete leaching process is as follows,
1) with Rhizoma Polygoni Cuspidati room temperature lixiviate in ethanol water (being to adopt the ethanol cold-maceration to carry out lixiviate under the room temperature);
2) after the Rhizoma Polygoni Cuspidati extracting solution that step (1) is obtained was concentrated into fully and does, the water suspendible extracted with ethyl acetate then;
3) the ethyl acetate extract applying silicon plastic column chromatography that step (2) is obtained, petroleum ether: ethyl acetate: formic acid=be developing solvent at 2: 8: 0.5, collect the fraction of 0.66＜Rf≤1;
4) utilize high performance liquid chromatography to make with extra care to the fraction in the step (3), it is the target components at m/z 286,206 and 270 places to collect Mass Spectrometer Method.
Ethanol water is 60～80% ethanol/waters (volume ratio) solution in the step (1), and the volume ratio of medical material Rhizoma Polygoni Cuspidati and solvent is 1: 3～1: 5; Repeat to extract 3～5 times;
Water in the extract in the step (2): the volume ratio of ethyl acetate is 1: 1～1: 3;
The chromatography column packing is a silica gel in the step (3), and the particle diameter of silica gel is not limit, and the particle diameter of filler is 5～10 μ m usually; Mobile phase is selected methanol and formic acid water two-phase system, and the concentration range of wherein adding formic acid is 0.1%～0.5% (volume ratio);
Used column packing is C18 in the step (4); Mobile phase A is a methanol, and B is 0.5% aqueous formic acid; Used flow velocity is 120～150mL/min; Molecular ion peak 286,206 and 270 according to the target components of Mass Spectrometer Method cuts according to chromatographic peak; Merge, concentrate, drain;
Described target components is to detect by the transgenic yeast method, and the enzyme of beta galactosidase is lived and two indexs of EC50 value of sample determine that this chemical constituent has higher estrogen activity.
Compared with prior art, this method has the following advantages:
(1) preparation method is controlled, and component is formed simply clear, favorable reproducibility.Because the present invention adopts high performance liquid preparative chromatography that the fraction of silica gel column chromatography is made with extra care, using mass spectrum simultaneously monitors active component, with common column chromatography technology or alkali gradient extraction phase ratio, whole process controllability is higher, and the target components composition is clear, has higher repeatability.
(2) the estrogen activity height of target components derives from plant simultaneously, has lower toxic and side effects.
(3) the present invention uses high performance liquid preparative chromatography and makes with extra care separation on the basis of Mass Spectrometer Method, filters out to have the active chemical constituent of high estrogen from the Chinese medicine Rhizoma Polygoni Cuspidati, tentatively meets national Chinese crude drug two kind new medicine standards.
The specific embodiment
The estrogen activity test of experimental example 1. Rhizoma Polygoni Cuspidati active components
A. estrogen activity method of testing:
Utilize estrogen activity (the document 1:Zhang of the method test Rhizoma Polygoni Cuspidati sample of transgenic yeast, C.Z., Wang, S.X., Zhang, Y., Chen, J.P., Liang, X.M., 2005.In vitro estrogenicactivities of Chinese medicinal plants traditionally used for the management ofmenopausal symptoms.Journal of Ethnopharmology 98,295-300.).It is transgenic yeast strain (the Saccharomyces cerevisiae strain that will contain female hormone receptor gene and beta galactosidase reporter gene, goods number BJ3505) liquid medium within (SC culture medium, it consists of: leucine, 3.96 ‰; Histidine, 0.05 ‰; Yeast nitrogen base (YNB), 1.7 ‰; Ammonium sulfate, 5 ‰; Glucose 20 ‰) in, 30 ℃ of constant temperature shaking table overnight incubation.Optical density at the 595nm place after diluting 10 times is between the 0.15-0.2 time, with containing 10 μ M CuSO
4Fluid medium yeast liquid is diluted to OD
595=0.75 as working solution.The sample that 5 μ L is diluted to variable concentrations (is respectively 50mg/ml, 5mg/ml, 0.5mg/ml, 0.05mg/ml, 0.005mg/ml 0.0005mg/ml 0.00005mg/ml) is added to respectively in the centrifuge tube that 995 μ L yeast liquid are housed, draw 100 μ L behind the mixing in 96 orifice plates, parallel three times of the sample of each variable concentrations.Under the concussion condition, in 30 ℃ of constant temperature shaking tables, expose 2 hours, measure OD
595(TECAN microplate reader).Add enzyme reaction solution (60mM Na
2HPO
4, 40mM NaH
2PO
4, 10mM KCl, 1mM MgSO
4, 2mg/mlONPG, 38mM β-mercaptoethanol, 0.01 ‰ triton X-100,15U/mL lyticase) 100 μ L/well, treat the solution flavescence after, add stop buffer (1MNa
2CO
3) 100 μ L/well, measure OD
420
One of the evaluation index of the estrogen activity size of sample is the enzyme activity of beta galactosidase, and another one is the EC50 value of sample.The former reflects the size of the estrogen activity of foreign substance by indirect mensuration beta galactosidase to the conversion capability of substrate, and the latter is then directly reflected the bonded ability size of test substance and estrogen receptor, promptly as the function of part.In the middle of system of the present invention, estradiol (E2) is as constant positive reference, and the enzyme work of its maximum beta galactosidase is considered as 100%; Dimethyl sulfoxide (DMSO) is as negative control.Betagalactosidase activity and EC50 value calculating method are shown in formula (1) and (2).
In the formula, u is an enzyme activity, A
420Be the absorbance of yeast reaction system at the 420nm place, A
595Be the absorbance of yeast reaction system at the 595nm place, t is the response time, and 50 is the E2 response time.In the data handling procedure of the present invention the E2 maximum enzyme is lived by 100% calculating.
In the formula, Y is a betagalactosidase activity, and X is the concentration of sample in reaction system, and A is a beta galactosidase peak response activity, and C is the slope of the linear segment in concentration-response curve, and D is a lowest detectable limit.
B. Rhizoma Polygoni Cuspidati chemical constituent estrogen activity test result
The fraction that obtains after Rhizoma Polygoni Cuspidati ethyl acetate part silicagel column separates, it is refining to use high performance liquid preparative chromatography, and gained target components estrogen activity test result shows: its EC50 value can reach 10
-4(EC50 of E2 is 10 to g/L
-7G/L).
The mass spectrum monitoring of experimental example 2. Rhizoma Polygoni Cuspidati active components
The used ion source of mass spectrum monitoring is the APCI source, and temperature of vaporization chamber is 350 ℃, and the heated capillary temperature is 260 ℃, and assist gas pressure power is 10～20au, and the sheath atmospheric pressure is 40～60psi, and collision induced dissociation (CID) is 30～40V.The main molecules quasi-molecular ions of active component is 286,206 and 270.
Provide following examples in conjunction with content of the present invention:
The preparation of estrogen activity component in embodiment 1. Rhizoma Polygoni Cuspidati
A.1 kilogram Rhizoma Polygoni Cuspidati medical material is 60% ethanol merceration extraction three times according to 1: 3 volume ratio volumetric concentration after crushed, each 24 hours.Behind the total extract water suspendible of draining, according to water: the volume ratio of ethyl acetate=1: 2 extracts with ethyl acetate, continued operation three times, ethyl acetate extract 82 grams, carry out crude separation with silicagel column; With volume ratio, petroleum ether: ethyl acetate: formic acid=be developing solvent at 2: 8: 0.5, collect the fraction of 0.66＜Rf≤1.
B. the fraction that step a is collected utilizes the high performance liquid chromatogram preparing instrument to make with extra care.Used column packing is C18 (100A, 10 μ m).Mobile phase A is a methanol, and B is 0.2% aqueous formic acid.Methanol concentration was raised to 82% from 50% with linear gradient in 17 minutes, be raised to 90% then in 9 minutes, was raised to 100% again in 14 minutes.Flow velocity is 150mL/min, and the detection wavelength is 254nm, and sampling volume is 10mL, and column temperature is a room temperature.Molecular ion peak 286,206 and 270 according to the target components of Mass Spectrometer Method cuts according to chromatographic peak.Merge, concentrate, drain.
The preparation of estrogen activity component in embodiment 2. Rhizoma Polygoni Cuspidati
A.1 kilogram Rhizoma Polygoni Cuspidati medical material is 60% ethanol merceration extraction three times according to 1: 4 volume ratio volumetric concentration after crushed, each 24 hours.Behind the total extract water suspendible of draining, according to water: the volume ratio of ethyl acetate=1: 1 extracts with ethyl acetate, continued operation three times, get ethyl acetate extract 95 grams, carry out crude separation with silicagel column, petroleum ether: ethyl acetate: formic acid=be developing solvent at 2: 8: 0.5, collect the fraction of 0.66＜Rf≤1.
B. the fraction that step a is collected utilizes the high performance liquid chromatogram preparing instrument to make with extra care.Used column packing is C18 (100A, 5 μ m).Mobile phase A is a methanol, and B is 0.5% aqueous formic acid.Methanol concentration was raised to 85% from 45% with linear gradient in 25 minutes, kept 3 minutes, was raised to 100% again in 14 minutes.Flow velocity is 120mL/min, and the detection wavelength is 254nm, and sampling volume is 8mL, and column temperature is a room temperature.Molecular ion peak 286,206 and 270 according to the target components of Mass Spectrometer Method cuts according to chromatographic peak.Merge, concentrate, drain.
The preparation of estrogen activity component in embodiment 3. Rhizoma Polygoni Cuspidati
A.1 kilogram Rhizoma Polygoni Cuspidati medical material is 70% ethanol merceration extraction three times according to 1: 5 volume ratio volumetric concentration after crushed, each 24 hours.Behind the total extract water suspendible of draining, according to water: the volume ratio of ethyl acetate=1: 3 extracts with ethyl acetate, continued operation three times, get ethyl acetate extract 80 grams, carry out crude separation with silicagel column, petroleum ether: ethyl acetate: formic acid=be developing solvent at 2: 8: 0.5, collect the fraction of 0.66＜Rf≤1.
B. the fraction that step a is collected utilizes the high performance liquid chromatogram preparing instrument to make with extra care.Used column packing is C18 (100A, 10 μ m).Mobile phase A is a methanol, and B is 0.5% aqueous formic acid.Methanol concentration was raised to 80% from 45% with linear gradient in 16 minutes, be raised to 100% then in 24 minutes.Flow velocity is 120mL/min, and the detection wavelength is 254nm, and sampling volume is 10mL, and column temperature is a room temperature.Molecular ion peak 286,206 and 270 according to the target components of Mass Spectrometer Method cuts according to chromatographic peak.Merge, concentrate, drain.
The preparation of estrogen activity component in embodiment 4. Rhizoma Polygoni Cuspidati:
A.1 kilogram Rhizoma Polygoni Cuspidati medical material is 80% ethanol merceration extraction three times according to 1: 3 volume ratio volumetric concentration after crushed, each 24 hours.Behind the total extract water suspendible of draining, according to water: the volume ratio of ethyl acetate=1: 2 extracts with ethyl acetate, continued operation three times, get ethyl acetate extract 82 grams, carry out crude separation with silicagel column, petroleum ether: ethyl acetate: formic acid=be developing solvent at 2: 8: 0.5, collect the fraction of 0.66＜Rf≤1.
B. the fraction that step a is collected utilizes the high performance liquid chromatogram preparing instrument to make with extra care.Used column packing is C18 (100A, 10 μ m).Mobile phase A is a methanol, and B is 0.1% aqueous formic acid.Methanol concentration was raised to 90% from 30% with linear gradient in 40 minutes, be raised to 100% again in 15 minutes.Flow velocity is 150mL/min, and the detection wavelength is 254nm, and sampling volume is 9mL, and column temperature is a room temperature.Molecular ion peak 286,206 and 270 according to the target components of Mass Spectrometer Method cuts according to chromatographic peak.Merge, concentrate, drain.
The preparation of estrogen activity component in embodiment 5. Rhizoma Polygoni Cuspidati:
A.1 kilogram Rhizoma Polygoni Cuspidati medical material is 70% ethanol merceration extraction three times according to 1: 4 volume ratio volumetric concentration after crushed, each 24 hours.Behind the total extract water suspendible of draining, according to water: the volume ratio of ethyl acetate=1: 2 extracts with ethyl acetate, continued operation three times, get ethyl acetate extract 82 grams, carry out crude separation with silicagel column, petroleum ether: ethyl acetate: formic acid=be developing solvent at 2: 8: 0.5, collect the fraction of 0.66＜Rf≤1.
B. the fraction that step a is collected utilizes the high performance liquid chromatogram preparing instrument to make with extra care.Used column packing is C18 (100A, 5 μ m).Mobile phase A is a methanol, and B is 0.2% aqueous formic acid.Methanol concentration was raised to 82% from 50% with linear gradient in 20 minutes, be raised to 90% then in 6 minutes, was raised to 100% again in 14 minutes.Flow velocity is 120mL/min, and the detection wavelength is 254nm, and sampling volume is 10mL, and column temperature is a room temperature.Molecular ion peak 286,206 and 270 according to the target components of Mass Spectrometer Method cuts according to chromatographic peak.Merge, concentrate, drain.
Claims (4)
1. estrogenic extracting method in the Rhizoma Polygoni Cuspidati is characterized in that:
1) with Rhizoma Polygoni Cuspidati room temperature lixiviate in ethanol water; Ethanol water is 60～80% ethanol/water solution, and the volume ratio of medical material Rhizoma Polygoni Cuspidati and solvent is 1: 3～1: 5; Repeat to extract 3～5 times;
2) after the Rhizoma Polygoni Cuspidati extracting solution that step (1) is obtained was concentrated into fully and does, the water suspendible extracted with ethyl acetate then;
3) the ethyl acetate extract applying silicon plastic column chromatography that step (2) is obtained, petroleum ether: ethyl acetate: formic acid=be developing solvent at 2: 8: 0.5, collect the fraction of 0.66＜Rf≤1;
4) utilize high performance liquid chromatography to make with extra care to the fraction in the step (3), it is the target components at m/z 286,206 and 270 places to collect Mass Spectrometer Method;
Used column packing is C18; Mobile phase A is a methanol, and B is 0.5% aqueous formic acid; Used flow velocity is 120～150mL/min; Molecular ion peak 286,206 and 270 according to the target components of Mass Spectrometer Method cuts according to chromatographic peak; Merge, concentrate, drain.
2. according to estrogenic extracting method in the described Rhizoma Polygoni Cuspidati of claim 1, it is characterized in that: water in the extract in the step (2): the volume ratio of ethyl acetate is 1: 1～1: 3.
3. according to estrogenic extracting method in the described Rhizoma Polygoni Cuspidati of claim 1, it is characterized in that: the chromatography column packing is a silica gel in the step (3), particle diameter 5～10 μ m of filler.
4. according to estrogenic extracting method in the described Rhizoma Polygoni Cuspidati of claim 1, it is characterized in that: described target components is to detect by the transgenic yeast method, and the enzyme of beta galactosidase two indexs of EC50 value alive and sample are determined the estrogen activity of this chemical constituent.
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| CN1935195B true CN1935195B (en) | 2010-04-28 |
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| CN114588179A (en) * | 2020-12-03 | 2022-06-07 | 中国科学院大连化学物理研究所 | Method for separating and purifying conjugated estrogens from pregnant mare urine by using preparative chromatography |
| CN115184483B (en) * | 2022-06-08 | 2024-05-03 | 浙江工业大学 | Two-dimensional screening method for active ingredients of traditional Chinese medicine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1323776A (en) * | 2001-05-31 | 2001-11-28 | 北京天纯维通生物技术有限公司 | Prepn. of resveratrol and resveratrol glycoside |
| CN1384088A (en) * | 2002-05-21 | 2002-12-11 | 杨建文 | Extraction process of resveratrol from giant knotweed |
| CN1546503A (en) * | 2003-12-12 | 2004-11-17 | 深圳海王药业有限公司 | Method for preparing polygonin and resveratrol |
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- 2005-09-21 CN CN2005100472588A patent/CN1935195B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1323776A (en) * | 2001-05-31 | 2001-11-28 | 北京天纯维通生物技术有限公司 | Prepn. of resveratrol and resveratrol glycoside |
| CN1384088A (en) * | 2002-05-21 | 2002-12-11 | 杨建文 | Extraction process of resveratrol from giant knotweed |
| CN1546503A (en) * | 2003-12-12 | 2004-11-17 | 深圳海王药业有限公司 | Method for preparing polygonin and resveratrol |
Non-Patent Citations (2)
| Title |
|---|
| 伍晓春,陆豫.虎杖的药理作用及临床应用研究进展.中医药信息22 2.2005,22(2),22-25. |
| 伍晓春,陆豫.虎杖的药理作用及临床应用研究进展.中医药信息22 2.2005,22(2),22-25. * |
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