CN101396422A - Red clover extract and preparation method thereof - Google Patents
Red clover extract and preparation method thereof Download PDFInfo
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- CN101396422A CN101396422A CNA2007100612188A CN200710061218A CN101396422A CN 101396422 A CN101396422 A CN 101396422A CN A2007100612188 A CNA2007100612188 A CN A2007100612188A CN 200710061218 A CN200710061218 A CN 200710061218A CN 101396422 A CN101396422 A CN 101396422A
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Abstract
The invention discloses a red clover extract and a preparation method thereof, red clover is taken, ethanol containing water is used for reflux extraction, filtrate is concentrated to obtain a liquid extract; water precipitation, placement and centrifugation are carried out on the liquid extract to obtain sediments; the ethanol containing the water is sequentially added in the sediments for gradient ethanol precipitation, the centrifugation is carried out, supernatant liquid with the corresponding ethanol concentration is taken, and the extract powder is obtained by concentrating and drying. The content of total isoflavones of the method is not less than 20 percent, the operation is simple and the cost is low, thereby being applicable to industrial production.
Description
Technical field
The present invention relates to a kind of plant extract, especially relate to a kind of Herba Trifolii Pratentis extract and preparation method thereof.
Background technology
Along with the progress and development of society, the quickening of rhythm of life, operating pressure strengthens, and the nervous life of high strength produces a series of autonomic nervous dysfunction diseases afterwards, brings great misery for patient and family as diseases such as climacteric syndromes.According to EPDML statistical estimation, climacteric women accounts for 20% of whole female population, and wherein 80～90% symptoms that have climacteric syndrome show.As the cardiovascular and cerebrovascular vessel symptom: hectic fever occurs, perspire, vasospasm type pain, hypertension, dizzy, tinnitus, dim eyesight etc.; Neural spiritual aspect: emotional instability, anxiety and anxiety, insomnia and forgetful, variation has all taken place in somebody even personality, serious all fours psychotic, most of depressive type patients.Present Chinese climacteric women is about 1.3 hundred million, wherein clinical manifestation climacteric syndrome reach 100,000,000 people, global climacteric women is about 4.5 hundred million, Chinese climacteric women occupies 28%.Past thinks that always the climacteric Hormone Replacement Therapy can treat and alleviate menopause syndrome, can also prevent the generation of diseases such as myocardial infarction, but show according to current research, climacteric, Hormone Replacement Therapy not only do not have the effect of angiocardiopathy preventing, and the aggravation women danger of suffering from cardiovascular and cerebrovascular disease and breast carcinoma.
It is reported: isoflavone can effectively alleviate involutional symptom, soybean isoflavone with estrogen-like effects can be brought into play its antioxidation of estrogen-like effect and can postpone female aging in women's body, make skin keep elasticity: isoflavone is scalable menopause women nerve also---hormonal system, make the endocrine of climacteric women reach balance, effectively prevent and treat climacteric syndrome, thereby improve the quality of life of old women, the isoflavone antioxidation can postpone female aging.Also adjustable bone metabolism of isoflavone prevents that mineral such as calcium runs off from skeleton, promote skeleton to form, thereby improve bone density, is applicable to prevention of osteoporosis.
Herba Trifolii Pratentis (Trifolium pratense L) is a leguminous plant, and another name is also referred to as red Herba Trifolii Pratentis, red clover, and red Holland Astragalus sinicuses.Herba Trifolii Pratentis contains compositions such as Flavonoid substances, protein, aminoacid, saccharide and vitamin.Contain Biochanin A (biochanin A) and B (formonetin), genistein (genistein) and big legumin (4 kinds of isoflavone such as daidzein0 as Herba Trifolii Pratentis, they mainly exist in the glycosides form in plant, are respectively sissoo glycosides (sissoirin), ononin (ononin), the wooden glycosides 9genistin of dyeing) and daiazi (daidzin).Herba Trifolii Pratentis is the highest with Biochanin A and B content, accounts for the 0.1-0.9% of dry weight, and other isoflavone and flavonoid are about 0.08%.Herba Trifolii Pratentis also contains the plain 9peclolinarigenin of trifoside, calycosin (calycosin), Herba Linariae Vulgaris), pratensein (pratensein) and baptin isoflavones components such as (pseudobaptigenin).At present, find that Herba Trifolii Pratentis has 3 kinds of flavonols materials: Quercetin (quercetin), isoquercitrin (isoquercitrin) and hyperin (hyperoside).During spending, Herba Trifolii Pratentis also contains Trifolin (trifolin), different Lee Mus element (isorhamnetin), pratol multiple flavone components such as (pratol).Other composition comprises: coumarin and phenol carboxylic acid, contain the about 15.5 μ g/g of dicoumarol (dicoumarol) in the bright grass, and contain the coumestrol (coumestrol) of trace, also have an effect of female sample, and salicylic acid, coumaric acid phenol carboxylic acid compound are arranged; Volatile oil, its content is about 0.03%, comprises kinds more than 40 such as methyl salicylate, benzyl ethyl alcohol, ethyl ester, 2 phenylethyl alcohol and ethyl ester, o-amino benzoyl ester, acetaminol and furfural very much; Protein, sugar and vitamin: with dry weight basis, proteinic mass fraction reaches 23% at later stages; Contain various essential amino acids.The saccharide mass fraction is 24.4%, and sucrose, glucose, fructose, xylose and starch etc. are wherein arranged.Carotene, Vit D and Vit E are still arranged in the herb.The mass fraction of carotene is the highest in florescence, α-and the mass fraction of beta-carotene be respectively 4.3% and 53.6% in florescence in spring, be respectively 3.2% and 45.9% in florescence in summer.
The pharmacological action of Herba Trifolii Pratentis:
(1) selectivity spasmolytic, antiinflammatory and bactericidal action: Herba Trifolii Pratentis is in the external effect (0.2% pure preserved material can make paramecium stop action in 30min) that the inhibition paramecium is arranged.Big legumin has the releasing smooth muscle spasm effect of similar papaverine sample, and daiazi and big legumin can be alleviated hyperpietic's symptoms such as headache.
(2) estrogen-like effects: genistein, Biochanin A and B and big legumin all have the effect of estrogen sample, they and diethylstilbestrol and estradiol structural similarity, and activity is about 1,/10 ten thousand of diethylstilbestrol (diethylstilbestrol), is 1,/20,000-1/50 (by weight) of estradiol (estradiol).Herba Trifolii Pratentis has its distinctive feature as a phytoestrogen: they almost can be distributed in each cell of human body and play a role, and this is that the human body self hormone is inaccessible; The two regulating actions of their tools, when the human body estrogen level is high, but the inhibitory hormone secretion because of it can combine with estrogen receptor, thereby has stoped the combination of human body self hormone.Otherwise when the human body estrogen level was low, they can provide extra estrogen-like effects.Just because of this, the premenstrual tension syndrome when Herba Trifolii Pratentis both had been used to treat the rising of human female inner estrogen level clinically, climacteric syndrome and menopause when being used to again treat the estrogen reduction.
(3) Herba Trifolii Pratentis also can be treated the estrogen disease: as hysteromyoma, endometriosis and osteoporosis.
(4) antitumaous effect: estrogen is higher in the breast cancer disease human plasma, and plant estrogen has antagonism to it, thereby suppresses breast carcinoma.
Herba Trifolii Pratentis has the multiple efficacies of the multiple disease of control women, will produce good social benefit and economic benefit.Therefore, more and more, also more and more deep to the research of Herba Trifolii Pratentis both at home and abroad.At present, following several to the general branch of the preparation method of Herba Trifolii Pratentis extract both at home and abroad:
1, column chromatography: the method adopts pure solvent to extract, and extracting solution concentrates the centrifuging and taking medicinal residues, with medicinal residues with petroleum ether or defat with n-hexane drying, use ethyl acetate extraction, after extract concentrated, crystallize obtained extract A, with the medicinal residues after the extraction with 50～90% alcohol extraction, reclaim alcohol, with mother solution behind the resulting crystallize and concentrate and also transfer pH value 8～10, separate after crossing macroporous resin, get extract B, extract A and B are merged, exquisite with alcohol, obtain high-load extract.
The method can obtain the total isoflavone extract of higher degree, but complex process, yield is low, and the cost height is not suitable for suitability for industrialized production.
2, Herba Trifolii Pratentis is adopted alcohol extraction, and the extracting solution concentrate drying adds the extraction of petroleum ether or solvent naphtha, extract dry dry thing, add alcohol reflux again in the dry thing and extract, the extracting solution concentrating under reduced pressure gets concentrated solution, add neutral alumina in the concentrated solution, absorption washes the cleaning mixture drying with water, the reuse ethyl acetate backflow, the dry total isoflavone extract that gets of extracting solution.
Though the method has been avoided the use of resin column, neutral alumina is replaced resin column absorption, it is low still to keep away unavoidable yield, extracts incomplete shortcoming; Secondly, used a large amount of organic solvents in the refabrication process, chemical contamination is more serious, and concerning the staff, internal metabolism is slow after sucking, and actual bodily harm is very big; Once more, preparation process is loaded down with trivial details, and is not easy to operate, therefore more is not suitable for suitability for industrialized production.
3, solvent method: Herba Trifolii Pratentis is adopted the extract obtained total isoflavone of alcohol extraction after drying, but content is lower than 20% more.
Summary of the invention
In order to overcome the problems referred to above, the purpose of this invention is to provide a kind of Herba Trifolii Pratentis extract; Another purpose just provides that a kind of technology is simple, steady quality, yield height, cost is low, pollutions is little, the preparation method of the Herba Trifolii Pratentis extract of suitable suitability for industrialized production; A further object of the invention just provides the preparation of this extract.
The present invention is achieved in that
The present invention adopts aquiferous ethanol successively after the gradient dissolving on the basis of conventional solvent method, and the extract total isoflavone content that obtains at last is not less than 20%.
A kind of Herba Trifolii Pratentis extract of the present invention is prepared by following steps:
(1) get Herba Trifolii Pratentis, use moisture lower alcohol extraction, filter, concentrate extractum;
(2) water precipitating is placed, the centrifugal precipitate that gets;
(3) in precipitate, add moisture lower alcohol gradient dissolving respectively successively, centrifugal, get correspondent alcohol concentration supernatant, concentrate drying promptly gets extract powder.
Extracting method described in the step 1 includes but not limited to that flooding, decocting are carried, pure lixiviate, alcohol reflux extraction, alcohol percolation etc.
Described method for concentration includes but not limited to that normal pressure concentrates, concentrating under reduced pressure, reverse osmosis concentration etc.
Described lower alcohol refers to C
1～C
3Aqueous alcohol solutions includes but not limited to methanol, ethanol, propanol, preferred alcohol.
In the described step (1), the concentration of aquiferous ethanol is 10～95%, preferred 50～95% ethanol, best 80% ethanol.
Concentration of lower alcohols increases successively in the described step (3), and being specially concentration of alcohol is 10～95%, preferred 30～70% concentration of alcohol, best 40～60%.
Further, add 10～40% dissolve with ethanols in the precipitate with above-mentioned steps (2), centrifugal, it is standby to get precipitate 1; Get the supernatant concentration drying, get extract powder a.
Further again, the dissolve with ethanol that adds 40～50% in the above-mentioned precipitate 1 is centrifugal, and it is standby to get precipitate 2; Get the dry extract powder b of getting of supernatant.
Further, the dissolve with ethanol that adds 50～70% in the above-mentioned precipitate 2 is centrifugal, and it is standby to get precipitate 3; Get the dry extract powder c of getting of supernatant.
The described precipitate that obtains successively can also dissolve with the ethanol of higher concentration, up to the effective ingredient in the precipitate is extracted fully, everyly utilizes extract that gradient dissolving of the present invention obtains all in protection scope of the present invention.As reuse 70～80% dissolve with ethanols in the precipitate 3, get extract, its total isoflavone content is 5.3～8%.
Total isoflavone comprises daidzein, genistein, Biochanin A, formoononetin in the described extract of the present invention.Containing total isoflavone among the preferred extract a of institute is 5～10%; Total isoflavone content is not less than 20% among the extract b, and best 20～30%; Total isoflavone content is not less than 20% among the extract c, and best 20～30%.
The preparation method of Herba Trifolii Pratentis of the present invention may further comprise the steps:
(1) get Herba Trifolii Pratentis, use the aquiferous ethanol reflux, extract,, filter, concentrate extractum;
(2) water precipitating is placed, the centrifugal precipitate that gets;
(3) in precipitate, add the dissolving of aquiferous ethanol gradient respectively successively, centrifugal, get the different ethanol concentration supernatant, concentrate drying promptly gets extract powder.
The preparation method of the preferred Herba Trifolii Pratentis of the present invention may further comprise the steps:
(1) get Herba Trifolii Pratentis, with 50～95% alcohol reflux 1～3 of 10～12 times of amounts this, each 1～3 hour, merging filtrate, the filtrate concentrate drying gets extractum;
(2) add water in the extractum, stir, place the centrifugal precipitate that obtains;
(3) add the dissolving of 10～90% ethanol gradients respectively successively in precipitate, 50～80 ℃ were stirred 1～3 hour, placed, centrifugal, got the different ethanol concentration supernatant concentration respectively and were drying to obtain extract.
The preparation method of the preferred Herba Trifolii Pratentis of the present invention, its step is as follows:
(1) get the Herba Trifolii Pratentis pulverizing medicinal materials and become coarse powder, with 80% alcohol reflux twice, each 2 hours, for the first time 80% ethanol consumption was 12 times of medical material, is 10 times for the second time, filters, and merging filtrate, concentrating under reduced pressure reclaim ethanol to thick extractum;
(2) thick extractum adds entry to the medical material doubling dose, stirs 2 hours down in 60 ℃, places the centrifugal precipitate that obtains;
(3) in precipitate, add 10～40% ethanol (the best is 30%) to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, centrifugal, precipitate 1 is standby, getting supernatant, to be evaporated to proportion be 1.10-1.15, drying under reduced pressure obtains extract powder a; In medicinal residues, add 40～50% (the best is 50%) ethanol to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, centrifugal, precipitate 2 is standby, getting supernatant, to be evaporated to proportion be 1.10-1.15, drying under reduced pressure obtains extract powder b; In medicinal residues, add 50～70% ethanol (the best is 60%) to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, centrifugal, precipitate 3 is standby, getting supernatant, to be evaporated to proportion be 1.10-1.15, drying under reduced pressure obtains extract powder c.
The present invention also provides the pharmaceutical composition that is prepared from as pharmaceutically active with extract of the present invention, can select for use among extract powder a of the present invention, b, the c one or more as active constituents of medicine, preferred extract powder b, c.Pharmaceutical composition of the present invention, comprise extract and as required said composition can also add the medicine acceptable carrier, can also comprise other active component.
Compositions of the present invention is the pharmaceutical dosage forms of unit dose.Described unit dosage form is meant the unit of preparation, as every of tablet, and capsular every capsules, every bottle of oral liquid, every bag of granule etc.
Compositions of the present invention, extract wherein are as active component, and its shared percentage by weight in preparation can be 0.1～99.9%, and all the other are the medicine acceptable carrier, prepare according to the preparation conventional method.
Compositions of the present invention, its pharmaceutical dosage forms can be any oral formulations or injection.Wherein oral formulations includes but not limited to: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, buccal tablet, oral cavity disintegration tablet, granule.Electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, drop pill, pellet, suppository, cream, spray etc.; Injection includes but not limited to: intravenous injection, injectable powder, subcutaneous injection agent etc.
Compositions of the present invention, when being prepared into medicament, optionally add suitable medicine acceptable carrier, described medicine acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, the EDTA disodium, EDTA calcium sodium, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and derivant thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, soil temperature 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, β-cyclodextrin, the phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
Beneficial effect of the present invention
1, the present invention adopts gradient dissolving successively, can fully active component be extracted, and total isoflavone content is not less than 20% in the Herba Trifolii Pratentis, prior art is thought and is not only adopted resin column or aluminium oxide etc. to adsorb purification according to solvent method, can't prepare total isoflavone content at the Herba Trifolii Pratentis extract more than 20%, overcome technology prejudice.
2, the present invention adopts the method for gradient precipitate with ethanol successively, and yield height, extractive content extract fully fully and be stable, controllable product quality.
3, avoid a large amount of as petroleum ether, solvent naphtha, ethyl acetate among the present invention, the use of organic solvents such as normal hexane reduces the pollution to environment, has reduced the infringement of chemical solvent to staff's body and mind.Only used water and Different concentrations of alcohol among the present invention, toxicity and hazardness have been dropped to minimum, production cost is low simultaneously, is fit to large-scale industrial production.
4, preparation method of the present invention did not adopt adsorption systems such as resin column, and just through simple dissolving, preparation process is simple, and operability is good, grasps favorable reproducibility easily.
5, different concentration ethanol extract powder of the present invention is promptly prepared the extract of different content with Same Way, can use separately also according to the technological requirement of preparation to merge use, more helps suitability for industrialized production.
6, avoid the use of adsorbing mediums such as resin or aluminium oxide in the preparation method of the present invention, reduced cost, be fit to suitability for industrialized production.
The assay method of total isoflavone in the experimental example extract
Adopt the extract of embodiment 1 preparation to carry out assay.
Chromatographic condition and system suitability test chromatograph enlightening horse C18 post (4.6mm * 200mm, 5 μ m);
Mobile phase: with 0.1%H
3PO
4Aqueous solution, acetonitrile are that mobile phase is carried out gradient elution, see Table 1; The detection wavelength is 254nm, 30 ℃ of column temperatures.
Table 1 eluent gradient table
| Time (min) | A:0.1％H 3PO 4Aqueous solution | B: acetonitrile | Flow velocity (ml/min) |
| 0 | 85 | 15 | 1.2 |
| 25.00 | 65 | 35 | 1.2 |
| 25.01 | 65 | 35 | 1.0 |
| 50.00 | 50 | 50 | 1.0 |
| 50.01 | 85 | 15 | 1.2 |
It is an amount of that the preparation precision of reference substance solution takes by weighing daidzein, genistein, formoononetin, Biochanin A reference substance, adds methanol and make the solution that contains the above-mentioned reference substance of 15 μ g among every 1mL approximately, promptly.
Three crowdes of each 25mg of Herba Trifolii Pratentis extract are got in the preparation of need testing solution, and accurate the title decides, and puts in the 25mL volumetric flask, and it is an amount of to add methanol, ultrasonic 30min, and standardize solution, promptly.
Algoscopy is got reference substance solution and each 10 μ L of need testing solution respectively, injects chromatograph of liquid, and the record chromatogram is measured, promptly.
The results are shown in Table 2
Total isoflavone content in table 2 Herba Trifolii Pratentis
Specific embodiment
Embodiment 1 preparation method of extract
The Herba Trifolii Pratentis pulverizing medicinal materials becomes coarse powder, with 80% alcohol reflux twice, and each 2 hours, for the first time 80% ethanol consumption is 12 times of medical material, is 10 times for the second time, filters, merging filtrate, concentrating under reduced pressure reclaims ethanol to thick extractum, adds entry to the medical material doubling dose, stirs 2 hours down in 60 ℃, freezer is placed and is spent the night, the centrifugal precipitate that obtains to wherein adding 30% ethanol to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder a, medicinal residues are standby, wherein add 50% ethanol to the medical material doubling dose to medicinal residues, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder b, and medicinal residues are standby; Add 60% ethanol to the medical material doubling dose in medicinal residues, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, and it is 1.10-1.15 that the centrifuging and taking supernatant is evaporated to proportion, and drying under reduced pressure obtains extract powder c.Containing total isoflavone among the above-mentioned extract powder a is 10%, and containing total isoflavone among the extract b is 30%, and containing total isoflavone among the extract c is 30.0%.
Embodiment 2 preparation method of extract
The clover pulverizing medicinal materials becomes coarse powder, with 80% alcohol reflux twice, and each 2 hours, for the first time 80% ethanol consumption is 12 times of medical material, is 10 times for the second time, filters, merging filtrate, concentrating under reduced pressure reclaims ethanol to thick extractum, adds entry to the medical material doubling dose, stirs 2 hours down in 60 ℃, freezer is placed and is spent the night, the centrifugal precipitate that obtains to wherein adding 10～40% ethanol to the medical material doubling dose, stirred 1.5 hours down in 50 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder a, medicinal residues are standby, wherein add 40～50% ethanol to the medical material doubling dose to medicinal residues, stirred 1.5 hours down in 50 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder b, and medicinal residues are standby; Add 50～70% ethanol to the medical material doubling dose in medicinal residues, stirred 1.5 hours down in 50 ℃, room temperature is placed and is spent the night, and it is 1.10-1.15 that the centrifuging and taking supernatant is evaporated to proportion, and drying under reduced pressure obtains extract powder c.Containing total isoflavone among the above-mentioned extract powder a is 9.5%, and containing total isoflavone among the extract b is 28%, and containing total isoflavone among the extract c is 26.0%.
Embodiment 3 preparation method of extract
The clover pulverizing medicinal materials becomes coarse powder, with 80% alcohol reflux twice, and each 2 hours, for the first time 80% ethanol consumption is 12 times of medical material, is 10 times for the second time, filters, merging filtrate, concentrating under reduced pressure reclaims ethanol to thick extractum, adds entry to the medical material doubling dose, stirs 2 hours down in 60 ℃, freezer is placed and is spent the night, the centrifugal precipitate that obtains to wherein adding 10～40% ethanol to the medical material doubling dose, stirred 1.5 hours down in 80 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder a, medicinal residues are standby, wherein add 40～50% ethanol to the medical material doubling dose to medicinal residues, stirred 1.5 hours down in 80 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder b, and medicinal residues are standby; Add 50～70% ethanol to the medical material doubling dose in medicinal residues, stirred 1.5 hours down in 80 ℃, room temperature is placed and spent the night, and is centrifugal, and getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder c.Containing total isoflavone among the above-mentioned extract powder a is 9.5%, and containing total isoflavone among the extract b is 15%, and containing total isoflavone among the extract c is 26.4%.
Embodiment 4 preparation method of extract
The Herba Trifolii Pratentis pulverizing medicinal materials becomes coarse powder, with 95% alcohol reflux twice, and each 1 hour, for the first time 95% ethanol consumption is 12 times of medical material, is 10 times for the second time, filters, merging filtrate, concentrating under reduced pressure reclaims ethanol to thick extractum, adds entry to the medical material doubling dose, stirs 2 hours down in 60 ℃, freezer is placed and is spent the night, the centrifugal precipitate that obtains to wherein adding 10% ethanol to the medical material doubling dose, stirred 1 hour down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder a, medicinal residues are standby, wherein add 50% ethanol to the medical material doubling dose to medicinal residues, stirred 1 hour down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder b, and medicinal residues are standby; Add 60% ethanol to the medical material doubling dose in medicinal residues, stirred 1 hour down in 60 ℃, room temperature is placed and is spent the night, and it is 1.10-1.15 that the centrifuging and taking supernatant is evaporated to proportion, and drying under reduced pressure obtains extract powder c.Containing total isoflavone among the above-mentioned extract powder a is 8%, and containing total isoflavone among the extract b is 20%, and containing total isoflavone among the extract c is 20%.
Embodiment 5 preparation method of extract
The Herba Trifolii Pratentis pulverizing medicinal materials becomes coarse powder, with 10% alcohol reflux 3 times, and each 3 hours, for the first time 50% ethanol consumption is 12 times of medical material, is 10 times for the second time, filters, merging filtrate, concentrating under reduced pressure reclaims ethanol to thick extractum, adds entry to the medical material doubling dose, stirs 2 hours down in 60 ℃, freezer is placed and is spent the night, the centrifugal precipitate that obtains to wherein adding 40% ethanol to the medical material doubling dose, stirred 3 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder a, medicinal residues are standby, wherein add 70% ethanol to the medical material doubling dose to medicinal residues, stirred 3 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder b, and medicinal residues are standby; Add 90% ethanol to the medical material doubling dose in medicinal residues, stirred 3 hours down in 60 ℃, room temperature is placed and is spent the night, and it is 1.10-1.15 that the centrifuging and taking supernatant is evaporated to proportion, and drying under reduced pressure obtains extract powder c.Containing total isoflavone among the above-mentioned extract powder a is 15%, and containing total isoflavone among the extract b is 8%, and containing total isoflavone among the extract c is 5%.
Embodiment 6 preparation method of extract
The Herba Trifolii Pratentis pulverizing medicinal materials becomes coarse powder, with 50% alcohol reflux twice, and each 2 hours, for the first time 80% ethanol consumption is 12 times of medical material, is 10 times for the second time, filters, merging filtrate, concentrating under reduced pressure reclaims ethanol to thick extractum, adds entry to the medical material doubling dose, stirs 2 hours down in 60 ℃, freezer is placed and is spent the night, the centrifugal precipitate that obtains to wherein adding 30% ethanol to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder a, medicinal residues are standby, wherein add 50% ethanol to the medical material doubling dose to medicinal residues, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder b, and medicinal residues are standby; Add 60% ethanol to the medical material doubling dose in medicinal residues, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, and it is 1.10-1.15 that the centrifuging and taking supernatant is evaporated to proportion, and drying under reduced pressure obtains extract powder c.Containing total isoflavone among the above-mentioned extract powder a is 5%, and containing total isoflavone among the extract b is 15%, and containing total isoflavone among the extract c is 15%.
Embodiment 7 preparation method of extract
The Herba Trifolii Pratentis pulverizing medicinal materials becomes coarse powder, with twice of 80% alcohol reflux, each 2 hours, for the first time 80% ethanol consumption was 12 times of medical material, is 10 times for the second time, filter, merging filtrate, concentrating under reduced pressure reclaim ethanol to thick extractum, add entry to the medical material doubling dose, stirred 2 hours down in 60 ℃, freezer is placed and is spent the night, and the centrifugal precipitate that obtains is to wherein adding 30% ethanol to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, room temperature is placed and to be spent the night, and is centrifugal, and getting supernatant, to be evaporated to proportion be 1.10-1.15, drying under reduced pressure obtains extract powder a, contains total isoflavone 10% in the extract.
Embodiment 8 preparation method of extract
Get the Herba Trifolii Pratentis pulverizing medicinal materials and become coarse powder, with 80% alcohol reflux twice, each 2 hours, for the first time 80% ethanol consumption was 12 times of medical material, it is 10 times for the second time, filter, merging filtrate, concentrating under reduced pressure reclaim ethanol to thick extractum, add entry to the medical material doubling dose, stirred 2 hours down in 60 ℃, freezer is placed and is spent the night the centrifugal precipitate that obtains, to wherein adding 30% ethanol to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, room temperature is placed and spent the night, and is centrifugal, abandoning supernatant, medicinal residues are standby, wherein add 50% ethanol to the medical material doubling dose to medicinal residues, stir 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder.Isoflavone-containing content is 25% in the extract.
Embodiment 9 preparation method of extract
The Herba Trifolii Pratentis pulverizing medicinal materials becomes coarse powder, with 80% alcohol reflux twice, and each 2 hours, for the first time 80% ethanol consumption is 12 times of medical material, is 10 times for the second time, filters, merging filtrate, concentrating under reduced pressure reclaim ethanol to thick extractum, add entry to the medical material doubling dose, stirred 2 hours down in 60 ℃, freezer is placed and is spent the night the centrifugal precipitate that obtains, to wherein adding 30% ethanol to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, room temperature is placed and spent the night, and is centrifugal, supernatant discards, medicinal residues are standby, wherein add 50% ethanol to the medical material doubling dose to medicinal residues, stir 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, supernatant discards, and medicinal residues are standby; Add 60% ethanol to the medical material doubling dose in medicinal residues, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, and it is 1.10-1.15 that the centrifuging and taking supernatant is evaporated to proportion, and drying under reduced pressure obtains extract powder.Containing total isoflavone content in the extract is 25%.
Example 10 extract formulations
Get the extract of embodiment 1, extract powder a40g, extract powder b40g, extract powder c10g behind the mix homogeneously, adds 1.5 times of amount dextrin, 0.5% sucrose, 1.5% microcrystalline Cellulose is made soft material with an amount of dissolve with ethanol, granulates, 60 ℃ of forced air dryings, granulate, granulate promptly gets granule.
Embodiment 11 extract formulations
Get the extract of embodiment 1, extract powder a50g, extract powder b30.9g, extract powder c15g, behind the uniform mixing,
Add 5% polyvinylpolypyrrolidone, 0.1% magnesium stearate, 50% microcrystalline Cellulose is made soft material with an amount of alcoholic solution, granulates, and 60 ℃ of forced air dryings are granulated, granulate, compacting promptly gets oral cavity disintegration tablet in flakes.
Embodiment 12 extract formulations
Get the extract powder 100g of embodiment 7, add 100mgPEG, mix homogeneously, fusion, last drop pill machine is made drop pill.
Embodiment 13 extract formulations
Get the extract powder 100g of embodiment 8, add 100mgPEG, mix homogeneously, fusion, last drop pill machine is made drop pill.
Embodiment 14 extract formulations
Get embodiment 7,8, any one extract powder 0.5g in 9, glucose 4.5g, sodium thiosulfate 0.9g and distilled water 1ml, behind the said components mix homogeneously, lyophilization, 500 of packing, promptly.
The preparation of embodiment 15 extracts
The Herba Trifolii Pratentis pulverizing medicinal materials becomes coarse powder, extracts twice with 80% methanol eddy, each 2 hours, for the first time 80% methanol usage is 12 times of medical material, is 10 times for the second time, filters, merging filtrate, concentrating under reduced pressure reclaims methanol to thick extractum, adds entry to the medical material doubling dose, stirs 2 hours down in 60 ℃, freezer is placed and is spent the night, the centrifugal precipitate that obtains to wherein adding 30% methanol alcohol to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder a, medicinal residues are standby, wherein add 50% methanol to the medical material doubling dose to medicinal residues, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder b, and medicinal residues are standby; Add 60% methanol to the medical material doubling dose in medicinal residues, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, and it is 1.10-1.15 that the centrifuging and taking supernatant is evaporated to proportion, and drying under reduced pressure obtains extract powder c.
Embodiment 16
The Herba Trifolii Pratentis pulverizing medicinal materials becomes coarse powder, with 80% propanol reflux, extract, twice, and each 2 hours, for the first time 80% propanol consumption is 12 times of medical material, is 10 times for the second time, filters, merging filtrate, concentrating under reduced pressure reclaims propanol to thick extractum, adds entry to the medical material doubling dose, stirs 2 hours down in 60 ℃, freezer is placed and is spent the night, the centrifugal precipitate that obtains to wherein adding 30% propanol to the medical material doubling dose, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder a, medicinal residues are standby, wherein add 50% propanol to the medical material doubling dose to medicinal residues, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, centrifugal, getting supernatant, to be evaporated to proportion be 1.10-1.15, and drying under reduced pressure obtains extract powder b, and medicinal residues are standby; Add 60% propanol to the medical material doubling dose in medicinal residues, stirred 1.5 hours down in 60 ℃, room temperature is placed and is spent the night, and it is 1.10-1.15 that the centrifuging and taking supernatant is evaporated to proportion, and drying under reduced pressure obtains extract powder c.
Claims (10)
1, the extract of a kind of Herba Trifolii Pratentis is characterized in that, is obtained by following steps:
(1) get Herba Trifolii Pratentis, use moisture lower alcohol extraction, filter, concentrate extractum;
(2) water precipitating is placed, the centrifugal precipitate that gets;
(3) in precipitate, add moisture lower alcohol gradient dissolving respectively successively, centrifugal, get correspondent alcohol concentration supernatant, concentrate drying promptly gets extract powder.
2, the extract of a kind of Herba Trifolii Pratentis as claimed in claim 1 is characterized in that, concentration of lower alcohols increases successively in the described step (3).
3, a kind of Herba Trifolii Pratentis extract as claimed in claim 1 is characterized in that: described lower alcohol is an ethanol.
4, a kind of Herba Trifolii Pratentis extract as claimed in claim 1 is characterized in that: the concentration of described lower alcohol is 10～95%.
5, the extract of a kind of Herba Trifolii Pratentis is characterized in that: add 10～40% dissolve with ethanols in the precipitate of claim 1, centrifugal, it is standby to get precipitate (1), gets dry extract powder, the called after extract (a) of getting of supernatant concentration.
6, a kind of Herba Trifolii Pratentis extract is characterized in that: the weighting profit requires 5 precipitate (1), and is centrifugal to wherein adding 40～50% dissolve with ethanols, and it is standby to get precipitate (2), gets dry extract powder, the called after extract (b) of getting of supernatant concentration.
7, a kind of Herba Trifolii Pratentis extract is characterized in that: the weighting profit requires 6 precipitate (2), and is centrifugal to wherein adding 50%～70% dissolve with ethanol, and it is standby to get precipitate (3), gets dry extract powder, the called after extract (c) of getting of supernatant concentration.
8, as claim 6 or 7 described Herba Trifolii Pratentis extract, it is characterized in that: total isoflavone content is not less than 20% in the described extract.
9, a kind of pharmaceutical composition is characterized in that: comprise any extract of claim 1～8.
10, the preparation method of Herba Trifolii Pratentis extract as claimed in claim 1, its step is as follows:
(1) get the Herba Trifolii Pratentis coarse powder, with 10～12 times, 50～95% ethanol extractions 1～3 time, each 1～3 hour, filter, merging filtrate concentrates and reclaims ethanol to thick extractum;
(2) thick extractum adds entry, stirs, and places the centrifugal precipitate that obtains;
(3) add the dissolving of 10～90% ethanol gradients respectively successively in precipitate, 50～80 ℃ were stirred 1～3 hour, placed, centrifugal, got corresponding concentration of alcohol supernatant respectively, and concentrate drying promptly gets extract.
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| CN2007100612188A CN101396422B (en) | 2007-09-28 | 2007-09-28 | Red clover extract and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2007100612188A CN101396422B (en) | 2007-09-28 | 2007-09-28 | Red clover extract and preparation method thereof |
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| CN101396422B CN101396422B (en) | 2011-12-28 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103690523A (en) * | 2012-09-27 | 2014-04-02 | 香港科技大学 | Composition used for promoting erythropoiesis, and its use |
| CN109276589A (en) * | 2018-11-08 | 2019-01-29 | 浙江中医药大学 | A kind of clover extract improves the application in sleep drug in preparation |
| CN112142706A (en) * | 2020-10-19 | 2020-12-29 | 湖南朗林生物资源股份有限公司 | Method for extracting and purifying red clover isoflavone |
-
2007
- 2007-09-28 CN CN2007100612188A patent/CN101396422B/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103690523A (en) * | 2012-09-27 | 2014-04-02 | 香港科技大学 | Composition used for promoting erythropoiesis, and its use |
| CN103690523B (en) * | 2012-09-27 | 2019-06-14 | 香港科技大学 | A kind of composition and application thereof for promoting RBC acceptor garland rate |
| CN109276589A (en) * | 2018-11-08 | 2019-01-29 | 浙江中医药大学 | A kind of clover extract improves the application in sleep drug in preparation |
| CN109276589B (en) * | 2018-11-08 | 2022-03-18 | 浙江中医药大学 | Application of herba Trifolii Pratentis extract in preparation of sleep improving medicine |
| CN112142706A (en) * | 2020-10-19 | 2020-12-29 | 湖南朗林生物资源股份有限公司 | Method for extracting and purifying red clover isoflavone |
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| CN101396422B (en) | 2011-12-28 |
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