CN100366628C - Effective parts of fructus sophorae flavone production and use thereof - Google Patents

Abstract

The present invention relates to effective parts of highly purified fructus sophorae flavone, namely the isoflavone extractive, the flavonol extractive and the total flavonoid extractive of fructus sophorae, and relates to the industrialized preparing method of the effective parts and application thereof to medicine or functional food or skin-protecting products. The effective parts of fructus sophorae flavone of the present invention are used for treating or curing osteoporosis, female climacteric syndromes, senile dementia, tumor cancer, hyperlipidemia, hypertension, coronary disease, cerebral thrombosis, etc., and can be used for resisting aging and preventing skin aging and face color spots or freckles, etc.

Description

Fructus Sophorae flavone effective part, production method and application thereof

Technical field

The present invention relates to Fructus Sophorae flavone effective part, production method and application thereof.More particularly, the present invention relates to Fructus Sophorae isoflavone extract, flavonol extract, extractive of general flavone and production method and purposes, also relate to the pharmaceutical preparation, functional foodstuff and the skin care product that contain the said extracted thing simultaneously.

Background technology

Controversies in hormone replacement in the elderly is the therapy of the known a kind of effective prevention of people or treatment and estrogen deficiency relative disease, particularly prevention or treatment osteoporosis, female dimacteric syndrome.In addition, controversies in hormone replacement in the elderly also has the evidence of coronary heart diseases of reduction, prevention or effects such as treatment senile dementia, dysthymia disorders and memory loss.But the oestrogenic hormon of human body Excessive Intake can bring some side effects, for example can increase the sickness rate of mammary cancer and carcinoma of endometrium.Therefore in this field, people have good estrogenic activity in searching always but do not have the phytoestrogen of oestrogenic hormon side effect.

Flavones ingredient in the plant is exactly a class phytoestrogen.Chromocor compound in the plant has more research and report.In recent years, owing to find that these compounds have some good biological activitys for human body, especially found the biological activity of isoflavonoid, thereby caused the very big concern of people human body.Flavonoid compound is to the biological activity of human body, comprise antioxygenation, antibacterial and anti-inflammation functions, decreasing cholesterol effect, glutamate pyruvate transaminase lowering effect, prevent and treat cerebral thrombosis and coronary heart disease effect, to the prevention of cardiovascular system diseases and therapeutic action or the like, known by people.The big quantity research of nearlyer years, found that this compounds has good estrogenic activity, thereby be called " phytoestrogen " by scientific circles, in osteoporosis, female dimacteric syndrome and anticancer and antitumor, especially there are a lot of useful effects aspects such as mammary cancer, uterus carcinoma and ovarian cancer.

In the prior art, in some pharmaceutical preparations that use clinically, contain the plant flavone composition, a large amount of reference that in Foods or drinks, adds vegetable flavonoid also occurs.

In the people WO0064276 patents such as Chen, a kind of food of calcic, VITAMIN and plant flavone composition with health role has been described.

In the people US5858449 patents such as Crank, product and their manufacture method of containing daizeol and soybean protein have been narrated.The product of Crank can be put into infant food, nutritional drink, milk, sour milk, frozen product, spread food and carnivorous matrix.

Narrated in people's such as Gorbach the US5498631 patent a kind of by taking significant quantity isoflavones treatment menopause disease, premenstrual syndrome or reduce diseases associated with estrogen level.Described flavones can absorb by the form of chocolate candy, biscuit, cereal food or beverage is edible.

A kind of method for the treatment of presenile dementia or the cognitive decrease disease relevant with the age is disclosed in people's such as Gorbach the US5733926 patent, comprising specific isoflavones and flavonol.

Disclose secretion and minimizing bone resorption in people's such as Barnes the US5506211 patent, supplied with the isoflavone aglycone composition with the various forms that comprises food such as soybean for suppressing osteoclast.

People's such as Jackson United States Patent (USP) 5654011 discloses a kind of nutrition-fortifying agent that increases premenopausal and postmenopausal women nutrition, wherein comprises the phytoestrogen of 8-50 milligram.

The United States Patent (USP) 5424331 of Shylankevvich discloses the material of a kind of treatment and preventing osteoporosis disease, comprising one or more phytoestrogen compounds, can provide with food supplement or with the form of medicine.

The United States Patent (USP) 5569459 of Shylankevvich has been narrated the appliable plant estrogen compound and has been regulated estrogenic secretion.

The present production of Schouten U.S. limited liability company is a kind of to be called Soylife from the isolating product of soybean, can comprise this material in various food and food-drink.

In above-mentioned many reference, all there are certain weak point in described flavones that obtains from soybean and isoflavone extract or enriched material, the taste that their existence are made us disliking.This mainly is because the isoflavones product that obtains from soybean in the prior art is that isoflavone content is the product of 5%-40%, mostly is 10%-20% greatly, contains a large amount of non-flavone components and exists, and causes the finished product that taste beastly is arranged.If further purifying, its cost will be a height very, and the productive rate of isoflavones also can be very low.

The chemical ingredients of the Fructus Sophorae is very complicated, except that containing flavonoid compound, also contains a large amount of carbohydrates and other composition.Flavonoid compound in the Fructus Sophorae is a class important " phytoestrogen ", has very high exploitation and is worth.The sophoricoside extracting method of enzymolysis, alkali extraction and acid precipitation is passed through in record in Chinese patent 1417220A, and the purposes of record sophoricoside in diseases such as coronary heart disease, cerebral thrombosis.Because the chemical ingredients in the Fructus Sophorae is very complicated, before the present invention, also do not have the high purity flavonoid extracts that from the Fructus Sophorae, obtains of record in the prior art, but do not put down in writing the method for from the Fructus Sophorae, extracting the high purity flavonoid extracts of the commercial scale production of comparative maturity yet.Therefore, this is unfavorable for developing Fructus Sophorae resource.Obtain Fructus Sophorae total flavone valid target, for the Fructus Sophorae utilization of resources, developing new drug, functional foodstuff and beverage etc. are very necessary, have significant values.

In addition, osajin composition contained in the Fructus Sophorae and flavonols composition purified respectively obtains highly purified separately purification thing, thereby can satisfy the requirement of using respectively according to different situations, also be very valuable.

Summary of the invention

The Fructus Sophorae of the present invention, refer to the fruit of pulse family (Leguminosae) Sophora (Sophora Linn.) plant, comprise for example Chinese scholartree (Sophorajaponica L.), Root of Vetchleaf Sophora Chinese scholartree (Sophora viciifolia Hunce.), Sophora moocroftiana(Wall (Sophora moorcroftiana), fine hair Chinese scholartree (Sophora tomentosa), spend Chinese scholartree (Sophorasecundiflora) partially, Australia Chinese scholartree (Sophora fraseri), big fruit Chinese scholartree (Sophora macrocarpa), Fujian Chinese scholartree (Sophora franchetiana), kowhai (Sophora tetraptera), silk hair Chinese scholartree (Sophoranuttaliana), mamani (Sophora chrysophylla), thick fruit Chinese scholartree (Sophora pachycarpa), leaflet Chinese scholartree (Sophora nicrophylla), Isabel Fern Chinese scholartree (Sophora fernandeziana), Chile Chinese scholartree (Sophora masafuerana), rarely see Chinese scholartree (Sophora exigua), hard ridge Chinese scholartree (Sophora mollis var.griffthii), the fruit of willow leaf Chinese scholartree sophora plants such as (Sophora dunnii); Be preferably the fruit of pulse family (Leguminosae) Sophora (Sophora Linn.) plant Chinese scholartree (Sophorajaponica L.).The Fructus Sophorae of the present invention, promptly fruit comprises immature young fruit, also comprises mellow fruit.The Fructus Sophorae of the present invention, i.e. fruit can be the product of giving birth to, also can be through the process of preparing Chinese medicine, as stir-fry process product, product processed by wine or vinegar is processed product etc.The Fructus Sophorae of the present invention comprises the pericarp and/or the seed of fruit.

By the present invention, the contriver finds pleasantly surprisedly, can the highly purified Fructus Sophorae isoflavones of plant-scale acquisition from the Fructus Sophorae, Fructus Sophorae flavonol and Fructus Sophorae total flavones, highly purified Fructus Sophorae isoflavones, Fructus Sophorae flavonol or Fructus Sophorae total flavones are prepared into pharmaceutical preparation, or add in general food, beverage, the milk-product, or add in the general skin care product, have prevention or the treatment disease relevant with estrogen deficiency, have prevention or treatment and oxygenizement diseases associated.Can prevent or treat osteoporosis, female dimacteric syndrome, hyperplasia of prostate, tumor and cancer etc., also have chronic degenerative disorders, dysthymia disorders, hypomnesis and skin aging, color spot or the freckle etc. of prevention or treatment senile dementia, arteriosclerosis, cerebral thrombosis, coronary heart disease, stenocardia, myocardial ischemia and cardiovascular systems.

Therefore, one of purpose of the present invention provides the highly purified Fructus Sophorae extractive of general flavone of a kind of highly purified Fructus Sophorae isoflavone extract, highly purified Fructus Sophorae flavonol extract and main isoflavone-containing and flavonol;

Two of purpose of the present invention provides a kind of method for preparing the said extracted thing;

Three of purpose of the present invention provides a kind of pharmaceutical preparation that contains the said extracted thing;

Four of purpose of the present invention provides a kind of functional foodstuff that contains the said extracted thing;

Five of purpose of the present invention provides a kind of skin care product that contain the said extracted thing;

Six of purpose of the present invention provides the purposes of said extracted thing.

Purpose of the present invention realizes by following technical scheme:

A kind of Fructus Sophorae isoflavone extract mainly contains the isoflavones with formula (I) structure, with 5, and 7-dihydroxyl-isoflavones-4 '-calculating of O-neohesperidoside, the weight percentage that isoflavones accounts for extract is 20%～100%, but is not 100%; Preferred 50%～100%, but be not 100%; More preferably 90%～100%, but be not 100%;

Wherein, R ₁, R ₂Be selected from hydrogen, methyl, glucose and neohesperidose respectively, R ₁And R ₂Be same to each other or different to each other.

Preferred above-mentioned Fructus Sophorae isoflavone extract contains 5,7-dihydroxyl-isoflavones-4 '-O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-O-glucoside and 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside.

A kind of Fructus Sophorae flavonol extract mainly contains the flavonol with formula (II) structure, with 4 ', 5,7-trihydroxy--flavonol-3-O-sophoroside calculates, and the weight percentage that flavonol accounts for extract is 20%～100%, but is not 100%; Preferred 50%～100%, but be not 100%; More preferably 90%～100%, but be not 100%;

Wherein, R ¹, R ²And R ³The disaccharide or the trisaccharide that are selected from hydrogen, methyl, glucose, rhamnosyl respectively and form by glucose and/or rhamnosyl, R ¹, R ²And R ³Be same to each other or different to each other; R ⁴Be selected from hydrogen, hydroxyl and methyl.

Preferred above-mentioned Fructus Sophorae flavonol extract contains 4 ', 5,7-trihydroxy--flavonol-3-O-sophoroside, 4 ', 5,7-trihydroxy--flavonol-3-O-rutinoside and 3 ', 4 ', 5,7-tetrahydroxy-flavonol-3-O-rutinoside.

A kind of Fructus Sophorae extractive of general flavone, mainly contain isoflavones with formula (I) structure and flavonol with formula (II) structure, with 5,7-dihydroxyl-isoflavones-4 '-isoflavones that the O-neohesperidoside calculates and with 4 ', 5, the total flavones that obtains after the flavonol addition that 7-trihydroxy--flavonol-3-O-sophoroside calculates, the weight percentage that accounts for extract is 20%～100%, but is not 100%; Preferred 50%～100%, but be not 100%; More preferably 90%～100%, but be not 100%;

Wherein, R ₁, R ₂Be selected from hydrogen, methyl, glucose and neohesperidose respectively, R ₁And R ₂Be same to each other or different to each other;

Wherein, R ¹, R ²And R ³Be selected from hydrogen, methyl, glucose, rhamnosyl respectively and form disaccharide or trisaccharide, R by glucose and/or rhamnosyl ¹, R ²And R ³Be same to each other or different to each other; R ⁴Be selected from hydrogen, hydroxyl and methyl.

Preferred above-mentioned Fructus Sophorae extractive of general flavone contains 5,7-dihydroxyl-isoflavones-4 '-O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 4 ', 5,7-trihydroxy--flavonol-3-O-sophoroside, 4 ', 5,7-trihydroxy--flavonol-3-O-rutinoside and 3 ', 4 ', 5,7-tetrahydroxy-flavonol-3-O-rutinoside.

Above-mentioned Fructus Sophorae isoflavone extract, Fructus Sophorae flavonol extract and Fructus Sophorae extractive of general flavone, by method for hydrolysis conventional in this area, methods such as for example acid hydrolysis, basic hydrolysis, enzymolysis or heating, make flavonoid glycoside generation hydrolysis, obtain the product of partial hydrolysis or the product that complete hydrolysis becomes aglycon.

Above-mentioned Fructus Sophorae isoflavone extract, Fructus Sophorae flavonol extract and Fructus Sophorae extractive of general flavone with the chemical reaction of pharmaceutically useful alkali by routine, can make the salifiable form of shape between isoflavones, flavonol and the alkali.Described pharmaceutically acceptable alkali is preferably sodium hydroxide, potassium hydroxide, yellow soda ash, salt of wormwood, sodium bicarbonate, saleratus, sodium-acetate, Potassium ethanoate etc.For example, above-mentioned extract under normal pressure, with the sodium hydroxide solution reaction of 0.01-5N, can obtain the sodium salt derivative of corresponding isoflavones, flavonol.

Above-mentioned Fructus Sophorae isoflavone extract, Fructus Sophorae flavonol extract and Fructus Sophorae extractive of general flavone, with metal cation salt, for example between the metal cation salts such as zinc, magnesium, chromium, iron, aluminium, copper, calcium, cobalt, barium, strontium or zirconium, through the routine reaction, obtain the metal composite of Fructus Sophorae isoflavones or flavonol.Described metal composite also has other new purposes except that the drug effect with Fructus Sophorae isoflavones or flavonol with using.For example described molysite mixture also has iron replenishing function, and described zinc salt mixture also has the zinc supplementation function, and described calcium salt composition also has calcium-enriching function, thereby can be used for respectively and iron deficiency, scarce zinc or calcium deficiency diseases associated; Again for example, described chromic salts mixture prevents in addition, treats or improves purposes such as diabetes.In the aqueous solution or acidic solution of Fructus Sophorae isoflavone extract, flavonol extract or extractive of general flavone, the above-mentioned metal ion salt solution that adds 0.01-5N, stirring reaction under proper temperature just obtains the metal composite salt of Fructus Sophorae isoflavones or flavonol.

Fructus Sophorae isoflavone extract of the present invention, flavonol extract or extractive of general flavone can be produced by following method and obtain, and this method comprises:

A. with water-soluble solvent as extracting solvent extraction Fructus Sophorae raw material, extracting solution is concentrated into to be done or is concentrated in right amount, obtain extracting enriched material, described water-soluble solvent is selected from a kind of solvent in water, ethanol, methyl alcohol, the acetone or the mixed solvent of two or more solvent compositions;

B. the extraction enriched material that above-mentioned a obtains is suspended in the ethanol that volume ratio is 0-50: 100-50: water or methyl alcohol: water or acetone: in the water solvent, after the placement, separate out solid, filter, obtain solid and filtrate, promptly obtain extract A behind the solid drying;

C. after the filtrate that above-mentioned b obtains concentrates in right amount, separate with chromatography column, with the volume ratio is the ethanol of 0-100: 100-0: water or methyl alcohol: water or acetone: water carries out gradient elution, the Fractional Collections effluent liquid, wherein said chromatography column is selected from macroporous resin column, polyamide column or sephadex column;

D. will be in the above-mentioned c effluent liquid main effluent liquid that contains formula (I) isoflavones merges, and reclaims solvent, is concentrated into driedly, obtains extract B after the drying;

E. will be in the above-mentioned c effluent liquid main effluent liquid that contains formula (II) flavonol merges, and reclaims solvent, is concentrated into driedly, obtains extract C after the drying;

Perhaps above-mentioned d and e are united two into one, become:

F. will be in the above-mentioned c effluent liquid main effluent liquid that contains formula (I) isoflavones and/or formula (II) flavonol merges, and reclaims solvent, is concentrated into driedly, obtains extract D after the drying;

Wherein, above-mentioned described extract A, B are Fructus Sophorae isoflavone extract of the present invention; Described extract C is Fructus Sophorae flavonol extract of the present invention; Described extract D or the mixture that is formed by extract A and/or B and extract C are Fructus Sophorae extractive of general flavone of the present invention.

If desired, with above-mentioned resulting extract A, B, C, D,, just can obtain the higher extract of purity again through treating process, process for purification for example is to carry out the separation and purification one or many again with above-mentioned described column chromatography method respectively, just can obtain more highly purified extract as required; Or with above-mentioned resulting extract A, B, C, D, the solvent with suitable polarity extracts respectively, and extracting solution reclaims solvent to the greatest extent, is concentrated into driedly, after the drying, just can obtain more highly purified extract as required.The solvent of described suitable polarity is selected from the methyl alcohol that volume ratio is 75-100: 25-0: water, ethanol: water, acetone: water and ethyl acetate, chloroform.

The extraction solvent that the present invention is used, when being mixed solvent, the ratio between each solvent can be proportioning commonly used in this area, for example the ethanol water of 30%, 60%, 70%, 90%% (volume) or methanol-water or acetone water

Extraction of the present invention can be an extracting method conventional in this area, as refluxing extraction, supersound extraction, diacolation extraction, supercritical carbon dioxide extraction etc.

When using water as when extracting solvent, adopt the suitably potass extraction of alkalescence, for example liming of PH8-10 or sodium hydrogen carbonate solution can improve the extraction yield of flavone component.To those skilled in the art, this is conspicuous.

The used macroporous resin of the present invention, polyamide resin, dextrane gel resin etc. can be respectively resin materials commonly used in this area.For example macroporous resin is the D101 type macroporous resin of Tianjin insecticide factory, and the dextrane gel resin is a Sephadex LH-20 resin etc.The pre-treatment of various resins and manipulation of regeneration etc. all are well known to those of ordinary skill in the art.

Described chromatography column lock out operation is that the skilled person is known in this area; Described gradient elution, the gradient that determining alcohol changes in the eluent is that the skilled person is easy to determine according to the situation of resin column in this area.

During Fractional Collections, can watch the colour band situation of resin column to carry out Fractional Collections by naked eyes, after also can carrying out the equal-volume collection, evaluation by routine is after for example thin layer plate is identified, contain merging of same composition, also can carry out effluent liquid and follow the tracks of discriminating, contain same composition and merge collection etc. by the means of routines such as thin-layer chromatography technology.This is well-known to one skilled in the art.The thin-layer chromatography technology can be referring to " Xu Rensheng " natural product chemistry " Science Press publishes the 589th page of June in 1997." for a person skilled in the art, this is conspicuous.

Drying of the present invention can be a drying means conventional in this area, as usually press dry dry, drying under reduced pressure, spraying drying or lyophilize etc.

The assay of flavones ingredient has the lot of documents report, thereby is one of ordinary skill in the art are easy to carry out flavones in the extract of the present invention according to prior art assay in the prior art.Isoflavones, flavonol assay in the extract of the present invention, can use content assaying method conventional in this area, for example colorimetry, ultraviolet spectrophotometry, thin layer chromatography scanning, acid base titration, high performance liquid chromatography etc. are measured, and preferably adopt high performance liquid chromatography to carry out assay.Isoflavones, flavonol content in the high effective liquid chromatography for measuring extract, its measuring method, those skilled in the art are easy to measure according to prior art.

Here describe a kind of high performance liquid chromatography (HPLC) and measure the method for flavones content in the extract of the present invention, but to content of the present invention without any the qualification effect.

Tianjin, island high performance liquid chromatograph, SPD-10AV type ultraviolet-visible detector, LC-10AD type solvent delivery pump unit; 25 microlitre samplers; 100,000/analytical balance.

Chromatographic separation condition: ODS-C ₁₈Post, 5um, 250mm * 4.6mm, column temperature: 22 ± 2 ℃,

Moving phase: methanol gradient elution

Sample size: 10ul

Flow velocity: 1ml/min

Detect wavelength: 260nm, 365nm

Reference substance is self-control, and process wave spectrum and high performance liquid chromatography are identified, purity detecting, meet the requirements.

With extract with acid hydrolysis after, by the HPLC method measure 4 ', 5,7-trihydroxy-isoflavone amount is converted into 5 again, 7-dihydroxyl-isoflavones-4 '-O-neohesperidoside amount, to calculate isoflavone levels; Measure 4 ', 3,5, the 7-kaempferol is pure and mild 3 ', 4 ', 3,5,7-pentahydroxyflavone alcohol amount, be converted into 4 again ', 5,7-trihydroxy--flavonol-3-O-sophoroside amount is to calculate the content of flavonol; After the amount addition of the amount of isoflavones and flavonol, obtain the amount of total flavones; The conversion of the content between described Flavone aglycone and the flavonoid glycoside converts by their molecular weight ratio.

Fructus Sophorae flavonoid extracts of the present invention has estrogenic activity.Be specially adapted to treatment, prevention or improvement and estrogen deficiency diseases associated.

Fructus Sophorae flavonoid extracts of the present invention also has very strong antioxygenation, can prevent, improve or treatment and oxidizing reaction diseases associated.

Fructus Sophorae flavonoid extracts of the present invention, treatment, prevent or improve the especially postclimacteric osteoporosis of osteoporosis, female dimacteric syndrome or menopausal syndrome (having symptoms such as hectic fever, anxiety, depression, night sweat, headache or emotional instability), menopause after paresthesia of skin, menoxenia especially have special effect and effectiveness in aspects such as premenstrua syndromes (have dysmenorrhoea, periodically symptoms such as mazalgia, liquid retention), senile dementia, aging, thunder Nader (Reynaud) syndromes, Buerger's disease.

Fructus Sophorae flavonoid extracts of the present invention treating, prevent or improving cancer aspects such as tumour or cancer, especially mammary cancer, uterus carcinoma, ovarian cancer, carcinoma of endometrium, large bowel cancer, prostate cancer, has good medical effect.

Fructus Sophorae flavonoid extracts of the present invention, in treatment, prevent or improve aspect the diseases of cardiovascular and cerebrovascular systems, especially to the chronic degenerative disorders aspect of hyperlipidemia, arteriosclerosis, hypertension, cerebral thrombosis, cerebral infarction, coronary heart disease, myocardial ischemia and cardiovascular systems, have good preventing, improvement or result of treatment.

Fructus Sophorae flavonoid extracts of the present invention has excellent prevention, improvement or result of treatment for paresthesia of skin after skin aging, color spot, freckle, the women's menopause etc.

Fructus Sophorae flavonoid extracts of the present invention at aspects such as male prostatic hyperplasia disease, acne or comedo, alopecias especially male sex's alopecia areata, hereditary alopecia, rheumatosis especially rheumatoid arthritis, has very ideal treatment, prevents or improves effect.

In the present invention, described Fructus Sophorae isoflavone extract, Fructus Sophorae flavonol extract and Fructus Sophorae extractive of general flavone, the dosage that uses is relevant with many factors, comprises that the mode of application, the mode of administration, patient's disease situation, patient's individual state etc. have relation.For example, take Fructus Sophorae flavonoid extracts of the present invention by functional foodstuff or beverage mode, to reach the effect of preventing or improving described disease, per daily dose can be smaller so, can take for a long time; If take by the pharmaceutical preparation mode for reaching the specific medical purpose, per daily dose can be greatly so, and can lack the course of treatment of taking.The dosage of extract of the present invention can calculate by contained flavones amount, and usually, single patient's per daily dose can be 0.5mg-5g; Preferred dose is 0.05g-2g; 0.25g-1g more preferably.Extract of the present invention can use with dosage in a usual manner, for example referring to Goodmanand Gilman, and the pharmacological basis of therapeutica, p1299 (the 7th edition, 1985).Used concrete dosage is relevant with the state of the disease for the treatment of, curee's situation, route of administration and other known facts.

Fructus Sophorae flavonoid extracts of the present invention, toxicity is very little.With the extract of Kou Shi improved method intravenous injection gained of the present invention, when the dosed administration of 50mg-2000mg/Kg scope and mouse, do not show tangible toxicity yet; Mouse oral administration LD ₅₀＞5g/kg/ day is equivalent to 5000 times of human body consumption, and is nontoxic.With Salmonella reversion test branch test method(s) and plate test method(s) research mutagenic activity, the result shows that Fructus Sophorae flavonoid extracts of the present invention does not all demonstrate mutagenic activity.

Especially, Fructus Sophorae flavonoid extracts of the present invention does not have effect to the uterus, and the target organ of its effect is or not the uterus, does not have general oestrogenic hormon to the toxic side effect due to the uterus.

Preparation is used to prevent, treat or improve the pharmaceutical preparation of described disease or indication, be (for simplicity with Fructus Sophorae isoflavone extract of the present invention, Fructus Sophorae flavonol extract or Fructus Sophorae extractive of general flavone, hereinafter referred to as " efficient part "), with one or more pharmaceutically useful carrier known in the art and/or mixed with excipients.

Certainly, carrier must be that medicine is acceptable, just and chemical reaction does not take place between the effective constituent in the preparation, and safe and harmless to the curee.Carrier or vehicle can be that solid or liquid or the two all are, and preferably make the preparation that efficient part is a dose unit, for example, a kind of tablet, the efficient part that wherein can contain the 0.5%-60% weight ratio, or up to the efficient part of 100% weight ratio.Pharmaceutical preparation of the present invention can use drug preparation technique well known in the art to prepare.

Preparation of the present invention comprises being suitable for oral, rectum, oral cavity (for example hypogloeeis), parenteral (for example subcutaneous, intramuscular, intravenous injection etc.) and Percutaneously administrable preparation etc.In concrete medication, only route of administration has relation with the disease characteristic of being treated and curee's situation etc.

Be suitable for the pharmaceutical preparation of oral administration, can make one unitary dose, for example capsule, tablet, soft capsule, pill, oral liquid, extractum or the like wherein contain the Fructus Sophorae efficient part of the present invention of predetermined dose in the per unit dosage.These preparations can be prepared with appropriate formulation technology known in the art, comprising with efficient part of the present invention and suitable pharmaceutical carrier and/or this step of mixed with excipients.For example, tablet can be by will containing efficient part of the present invention powder or particle compressing tablet or mold prepare, contain one or more suitable carriers or vehicle in wherein said powder or the particle, as inert diluent, tackiness agent, lubricant and/or show promoting agent (dispersion agent) etc.

The preparation that is suitable for oral cavity (hypogloeeis) administration comprises efficient part of the present invention and suitable inert base or flavoured base is mixed that flavoured base is sucrose or gum arabic etc. normally, and inert base is gelatin, glycerine or gum arabic etc. normally.

The preparation that is suitable for administered parenterally among the present invention generally includes the sterilized water injection that contains efficient part of the present invention, powder injection, intravenous infusion agent etc.Can be by efficient part of the present invention be mixed with water or glycine buffer, then the solution that obtains is transferred to ooze and sterilize and is just obtained these preparations with blood etc.According to injection formulations of the present invention, contain the efficient part of 0.1%-80%w/w usually.

The preparation that is suitable for rectal administration is preferably made the suppository of unitary dose.These suppositorys can be with active compound and one or more conventional carriers, and for example Oleum Cocois mixes, and typing just obtains then.

Be suitable for Percutaneously administrable preparation and be preferably emulsifiable paste, sprays, aerosol, ointment etc.Spendable carrier comprises Vaseline, lanolin, polyoxyethylene glycol etc., and the content of efficient part is 0.1%-20%w/w normally, and these preparations also comprise the skin cosmetic emulsifiable paste.

The preparation that is suitable for transdermal administration also can pass through the iontophoresis release of active compounds, can be referring to " pharmaceutical research, 1986,3 (6): 318 ", and typical type of service is the optional aqueous buffer solution of active compound.Comprise citrate buffer (PH6) or ethanol/water in the appropriate formulation, and contain the efficient part of 0.05M-1M.

Functional foodstuff of the present invention, refer to contain the similar food products such as food, beverage or milk-product of Fructus Sophorae flavonoid extracts of the present invention (being efficient part), efficient part can be added in the food with adding, mixing, dressing, combination or other approach and go, or to dissolve, to suspend or to be suspended in beverage, the milk-product.The food, beverage or the milk-product that contain Fructus Sophorae flavone effective part of the present invention can the establishing criteria method easily prepare.

Therefore Fructus Sophorae efficient part of the present invention has anti-oxidant activity, also can be widely used in the skin care product, for example prevents in the middle of the similar articles such as skin care emulsifiable paste, sun-screening agent, cleansing milk of skin aging, color spot, freckle.These skin care product can establishing criteria method prepare at an easy rate.

In addition, product of the present invention can be united use to produce synergistic effect with other material.For example, when the treatment tumour, product of the present invention can combine with conventional chemotherapy, when obtaining inhibition or kill tumor cell, the oxidative damage that again can ameliorate tumor chemotherapeutics (as some anthracycline compounds or metal platinum compound etc.) brings, thus double dominant had.

Again for example, in other situations such as arteriosclerosis, product of the present invention and other antioxidant share, as carotenoid, particularly Lyeopene and zeaxanthin etc., to bring into play collaborative antioxygenation.Equally, at aspects such as osteoporosis, female dimacteric syndromes, product of the present invention can be united use with other Chinese medicine or Western medicine with treatment or preventing osteoporosis, female dimacteric syndrome, to obtain the effect of synergy.

Embodiment

Following embodiment will help further to understand the present invention, but not limit content of the present invention.

Embodiment 1

The preparation of Fructus Sophorae isoflavone extract, flavonol extract

Exsiccant Fructus Sophorae fine powder 2Kg uses 6000ml water, and heating keeps little boiling, extracted 2 hours, and filtered through gauze, the dregs of a decoction extract each 1.5 hours 2 times with little the boiling of 2 * 4000ml water again; United extraction liquid, filter paper filtering; Filtrate is concentrated into the dried enriched material that obtains, and enriched material is added in 20%% the ethanol water (V/V) of 3000ml, and stirring and dissolving after the placement, is separated out white solid, filters, and the leaching solid after the low-temperature atmosphere-pressure drying, obtains extract A-1;

After filtrate boils off ethanol, be concentrated in right amount, join the D101 macroporous resin column of having handled well, with Different concentrations of alcohol water gradient elution, flow velocity is about 10-15ml/min.With the about 5 liters of wash-outs of deionized water, being 10% the about 4 liters of wash-outs of ethanol again with concentration earlier, is 30% ethanol elution again with concentration, is come out by wash-out fully until ribbon colour band I, spends about 2 liters of 30% ethanol; Be 50%% about 5 liters of wash-outs of ethanol again with concentration; Be 70% ethanol elution again with concentration, come out by wash-out fully, spend about 2 liters of 70% ethanol until ribbon colour band II.Be 95% ethanol elution regenerating resin post again with concentration.

The flow point that merges colour band I, decompression recycling ethanol is concentrated into driedly, obtains extract B-1;

The flow point that merges colour band II, decompression recycling ethanol is concentrated into driedly, obtains extract C-1;

Composition analysis: silica gel G F ₂₅₄The thin layer plate thin-layer chromatography; Developping agent is chloroform/methanol (4: 1), contains little acetic acid; Developer is 2% aluminum chloride ethanolic soln; Observe fluorescence under the 365nm UV-light.Assay adopts above-mentioned HPLC method.

The result:

Extract A-1 contains: 5,7-dihydroxyl-isoflavones-4 '-the O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-the O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-neohesperidoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-glucoside;

The weight percent that isoflavones accounts for extract is 82.35%.

Extract B-1 contains: 5,7-dihydroxyl-isoflavones-4 '-the O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-the O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-neohesperidoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-glucoside, 4 '-methoxyl group-5-hydroxyl-isoflavones-7-O-glucoside, 4 ', 5, the 7-trihydroxy-isoflavone;

The weight percent that isoflavones accounts for extract is 58.23%.

Extract C-1 contains: 4 ', 5,7-trihydroxy--flavonol-3-O-sophoroside, 4 ', 5,7-trihydroxy--flavonol-3-O-rutinoside, 3 ', 4 ', 5,7-tetrahydroxy-flavonol-3-O-rutinoside, 4 ', 5,7-trihydroxy--flavonol-3-O-[α-L-rhamanopyranosyl (1 → 6)-O-β-D-glucosyl group (1 → 2)-O-β-D-glucoside], 4 ', 3,5-trihydroxy--flavonol-7-O-rhamnoside, 4 ', 3,5, the 7-tetrahydroxyflavonol, 3 ', 4 ', 3,5,7-pentahydroxyflavone alcohol;

The weight percent that flavonol accounts for extract is 53.05%.

Embodiment 2

The preparation of Fructus Sophorae isoflavone extract

Get 10g embodiment 1 resulting extract A-1, add 95% ethanol of 100ml, reflux 40min, filtered while hot is concentrated into driedly behind the filtrate recycling ethanol, after the resistates cryodrying, obtain extract A-1a.

Extract A-1a is through the thin-layer chromatography composition analysis, contain 5,7-dihydroxyl-isoflavones-4 '-the O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-the O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-neohesperidoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-glucoside;

The weight percent that HPLC method assay result, isoflavones account for extract is 93.77%.

Embodiment 3

The preparation of Fructus Sophorae isoflavone extract

Get 20g embodiment 1 resulting extract B-1, add an amount of water dissolution after, join the D101 macroporous resin column of having handled well, with Different concentrations of alcohol water gradient elution, flow velocity is about 5-8ml/min.Earlier being 10% the about 0.7 liter of wash-out of ethanol with concentration, is 60% ethanol elution again with concentration, is come out by wash-out fully until the ribbon colour band, spends about 0.5 liter of 60% ethanol; Be 95% ethanol elution regenerating resin post again with concentration.

The colour band effluent liquid that merges main isoflavone-containing, decompression recycling ethanol is concentrated into driedly, obtains extract B-1a.

Extract B-1a contains 5 through the thin-layer chromatography composition analysis, 7-dihydroxyl-isoflavones-4 '-the O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-the O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-neohesperidoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-glucoside, 4 '-methoxyl group-5-hydroxyl-isoflavones-7-O-glucoside, 4 ', 5, the 7-trihydroxy-isoflavone;

HPLC method assay result shows that it is 77.46% that extract B-1a institute isoflavone-containing accounts for extract weight per-cent.

Embodiment 4

The preparation of Fructus Sophorae isoflavone extract

Get 10g embodiment 1 resulting extract B-1, add 95% ethanol of 100ml, reflux 60min, filtered while hot is concentrated into driedly behind the filtrate recycling ethanol, after the resistates cryodrying, obtain extract B-1b.

Extract B-1b contains 5 through the thin-layer chromatography composition analysis, 7-dihydroxyl-isoflavones-4 '-the O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-the O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-neohesperidoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-glucoside, 4 '-methoxyl group-5-hydroxyl-isoflavones-7-O-glucoside, 4 ', 5, the 7-trihydroxy-isoflavone;

The weight percent that HPLC method assay result, isoflavones account for extract is 69.77%.

Embodiment 5

The preparation of Fructus Sophorae flavonol extract

Get 20g embodiment 1 resulting extract C-1, add an amount of water dissolution after, join the D101 macroporous resin column of having handled well, with Different concentrations of alcohol water gradient elution, flow velocity is about 5-8ml/min.Earlier with the about 1 liter of wash-out of the ethanol of concentration about 10%, again with the ethanol elution of concentration about 75%, come out by wash-out fully until the ribbon colour band, spend about 0.5 liter of 75% ethanol; Be 95% ethanol elution regenerating resin post again with concentration.

The colour band effluent liquid that merges main isoflavone-containing, decompression recycling ethanol is concentrated into driedly, obtains extract C-1a.

Extract C-1a is through the thin-layer chromatography composition analysis, contain 4 ' and, 5,7-trihydroxy--flavonol-3-O-sophoroside, 4 ', 5,7-trihydroxy--flavonol-3-O-rutinoside, 3 ', 4 ', 5,7-tetrahydroxy-flavonol-3-O-rutinoside, 4 ', 5,7-trihydroxy--flavonol-3-O-[α-L-rhamanopyranosyl (1 → 6)-O-β-D-glucosyl group (1 → 2)-O-β-D-glucoside], 4 ', 3,5-trihydroxy--flavonol-7-O-rhamnoside, 4 ', 3,5, the 7-tetrahydroxyflavonol, 3 ', 4 ', 3,5,7-pentahydroxyflavone alcohol;

HPLC method assay result shows that it is 80.46% that the contained flavonol of extract C-1a accounts for extract weight per-cent.

Embodiment 6

The preparation of Fructus Sophorae flavonol extract

Get 10g embodiment 1 resulting extract C-1, add 95% ethanol of 100ml, reflux 30min, filtered while hot is concentrated into driedly behind the filtrate recycling ethanol, after the resistates cryodrying, obtain extract C-1b.

Extract C-1b is through the thin-layer chromatography composition analysis, contain 4 ' and, 5,7-trihydroxy--flavonol-3-O-sophoroside, 4 ', 5,7-trihydroxy--flavonol-3-O-rutinoside, 3 ', 4 ', 5,7-tetrahydroxy-flavonol-3-O-rutinoside, 4 ', 5,7-trihydroxy--flavonol-3-O-[α-L-rhamanopyranosyl (1 → 6)-O-β-D-glucosyl group (1 → 2)-O-0-D-glucoside], 4 ', 3,5-trihydroxy--flavonol-7-O-rhamnoside, 4 ', 3,5, the 7-tetrahydroxyflavonol, 3 ', 4 ', 3,5,7-pentahydroxyflavone alcohol;

HPLC method assay result, the weight percent that flavonol accounts for extract is 66.13%.

Embodiment 7

The preparation of Fructus Sophorae isoflavone extract, flavonol extract

Exsiccant Fructus Sophorae fine powder 1Kg is 60% ethanol with 3000ml concentration, heating and refluxing extraction 1.5 hours, and filtered through gauze, the dregs of a decoction are that 60% alcohol heating reflux extracts each 1 hour 2 times with 2 * 150%0ml concentration again; United extraction liquid, filter paper filtering; Be concentrated into driedly behind the decompression filtrate recycling ethanol, obtain enriched material; Enriched material is added in 30% the acetone water (V/V) of 1000ml, stirring and dissolving after the placement, is separated out white solid, filters, and the leaching solid after the low-temperature atmosphere-pressure drying, obtains extract A-2;

After filtrate is reclaimed acetone, be concentrated into driedly, obtain enriched material.Get enriched material 20%g, add an amount of dissolve with methanol after, admix Silon after, decompressing and extracting, levigate, join on the polyamide column of having handled well, with Different concentrations of alcohol water gradient elution, flow velocity is about 8-10ml/min.With the about 1000ml wash-out of deionized water,, with the ethanol elution of concentration about 40%, come out by complete wash-out more earlier, spend 40% the about 1000ml of ethanol until ribbon colour band I again with the about 800ml wash-out of the ethanol of concentration about 10%; With the ethanol elution of concentration about 70%, come out by complete wash-out again, spend 60% the about 150%0ml of ethanol until ribbon colour band II.Be 95% ethanol, 5% sodium hydroxide, deionized water wash-out polyamide column with concentration again, with the regeneration polyamide column.

The flow point that merges colour band I, decompression recycling ethanol is concentrated into driedly, obtains extract B-2;

The flow point that merges colour band II, decompression recycling ethanol is concentrated into driedly, obtains extract C-2;

Composition analysis: silica gel G F ₂₅₄The thin layer plate thin-layer chromatography; Developping agent is chloroform/methanol (4: 1), contains little acetic acid; Developer is 2% aluminum chloride ethanolic soln; Observe fluorescence under the 365nm UV-light.Assay adopts above-mentioned HPLC method.

The result:

Extract A-2 contains: 5, and 7-dihydroxyl-isoflavones-4 '-the O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-the O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-glucoside;

The weight percent that isoflavones accounts for extract is 91.07%.

Extract B-2 contains: 5,7-dihydroxyl-isoflavones-4 '-the O-neohesperidoside, 5,7-dihydroxyl-isoflavones-'-the O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-neohesperidoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-glucoside, 5-hydroxyl-isoflavones-7-O-rhamnoside-4 '-the O-sophoroside, 4 '-methoxyl group-5-hydroxyl-isoflavones-7-O-glucoside;

The weight percent that isoflavones accounts for extract is 67.43%.

Extract C-2 contains: 4 ', 5,7-trihydroxy--flavonol-3-O-sophoroside, 4 ', 5,7-trihydroxy--flavonol-3-O-rutinoside, 3 ', 4 ', 5,7-tetrahydroxy-flavonol-3-O-rutinoside, 4 ', 5,7-trihydroxy--flavonol-3-O-[α-L-rhamanopyranosyl (1 → 6)-O-β-D-glucosyl group (1 → 2)-O-β-D-glucoside], 4 ', 5-dihydroxyl-flavonol-3-O-glucoside-7-O-glucoside, 4 ', 3,5-trihydroxy--flavonol-7-O-rhamnoside, 4 ', 3,5, the 7-tetrahydroxyflavonol, 3 ', 4 ', 3,5,7-pentahydroxyflavone alcohol;

The weight percent that flavonol accounts for extract is 62.72%.

Embodiment 8

The preparation of Fructus Sophorae isoflavone extract

Get 0.5g embodiment 7 resulting extract B-2, add the ethyl acetate of 150ml, reflux 90min is put cold after-filtration, and filtrate is reclaimed to the greatest extent and to be concentrated into after the ethyl acetate driedly, after the resistates cryodrying, obtains extract B-2a.

Extract B-2a is through the thin-layer chromatography composition analysis, contain 5,7-dihydroxyl-isoflavones-4 '-the O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-the O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-neohesperidoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-glucoside, 5-hydroxyl-isoflavones-7-O-rhamnoside-4 '-the O-sophoroside, 4 '-methoxyl group-5-hydroxyl-isoflavones-7-O-glucoside;

HPLC method assay result, to account for the weight percent of extract be 90.21% to isoflavones among extract B-2a.

Embodiment 9

The preparation of Fructus Sophorae extractive of general flavone

Get the enriched material 40g of embodiment 7, add an amount of dissolve with methanol after, admix macroporous resin, the decompressing and extracting solvent joins on the D101 macroporous resin column of having handled well, with Different concentrations of alcohol water gradient elution, the about 5-8ml/min of flow velocity.Earlier with the ethanol elution of concentration about 10% to the effluent liquid look very light till, spend 10% ethanol and be about 1 liter, with the ethanol elution of the about 50%-75% of concentration, till the effluent liquid look very light, be 95% ethanol elution regenerating resin post again with concentration again.Merge the effluent liquid contain flavones, amount to 1.5 liters, be concentrated into driedly behind the decompression recycling ethanol, after the resistates cryodrying, obtain extract D-1.

Extract D-1 is respectively with chloroform, after ethyl acetate and the propyl carbinol segmentation, each section carries out the thin-layer chromatography composition analysis respectively, the result shows that extract D-1 contains 5,7-dihydroxyl-isoflavones-4 '-the O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-the O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-neohesperidoside, 5-hydroxyl-isoflavones-7-O-glucoside-4 '-the O-glucoside, 5-hydroxyl-isoflavones-7-O-rhamnoside-4 '-the O-sophoroside, 5-hydroxyl-isoflavones-7-O-rhamnoside-4 '-the O-neohesperidoside, 4 '-methoxyl group-5-hydroxyl-isoflavones-7-O-glucoside, 4 ', 5,7-trihydroxy--flavonol-3-O-sophoroside, 4 ', 5,7-trihydroxy--flavonol-3-O-rutinoside, 3 ', 4 ', 5,7-tetrahydroxy-flavonol-3-O-rutinoside, 4 ', 5,7-trihydroxy--flavonol-3-O-[α-L-rhamanopyranosyl (1 → 6)-O-0-D-glucosyl group (1 → 2)-O-β-D-glucoside], 4 ', 5-dihydroxyl-flavonol-3-O-glucoside-7-O-glucoside, 4 ', 3,5-trihydroxy--flavonol-7-O-rhamnoside, 4 ', 5, the 7-trihydroxy-isoflavone, 4 ', 3,5, the 7-tetrahydroxyflavonol, 3 ', 4 ', 3,5,7-pentahydroxyflavone alcohol;

HPLC method assay result, the weight percent that the total flavones that contains among the extract D-1 accounts for extract is 59.51%.

Get the extract D-1 of 0.5g, press embodiment 8 methods, make solvent refluxing with acetone and extract, obtain extract D-1a at last.Through HPLC method assay, general flavone content is 74.34% among the extract D-1a.

Embodiment 10

Useful in preparing drug formulations

1. the gelatine capsule that contains Fructus Sophorae isoflavone extract

The Fructus Sophorae isoflavone extract (extract B-2) of isoflavone-containing 3g is mixed with 8g Microcrystalline Cellulose, 0.3g silica gel, 0.8g Xylo-Mucine and 0.1g Magnesium Stearate.The mixture that forms is packed in 100 hard capsule.

2. the coated tablet that contains Fructus Sophorae extractive of general flavone

The Fructus Sophorae extractive of general flavone (extract A-2 and extract C-2 is even by 1: 1 mixed) that will contain total flavones 4g mixes with 5g Microcrystalline Cellulose, 3.3g calcium phosphate,dibasic, 0.3g silica gel, 1.2g Xylo-Mucine and 0.2g Magnesium Stearate, and this mixed powder is pressed 100; The tablet that forms carries out dressing with containing 0.45g Vltra tears, 0.09g polyoxyethylene glycol, 0.2g titanium dioxide and 0.1g talcum powder, obtains coating tablet.

Embodiment 11

Preparation contains the functional foodstuff of Fructus Sophorae flavonoid extracts

1. preparation contains the food of Fructus Sophorae extractive of general flavone

The Fructus Sophorae extractive of general flavone (extract D-1) of capacity is joined in the wheat-flour, make the wheat-flour mixture that every 100g contains the about 5g of total flavones.Adopt this wheat-flour mixture to prepare bread, cake, cookies, crispbread, noodles, instant noodles etc., thereby obtain to contain the functional foodstuff of Fructus Sophorae activeconstituents.

2. preparation contains the milk preparation of Fructus Sophorae isoflavone extract

(extract B-1b) join in the milk makes the mixed milk of the about 2-5g of isoflavone-containing in every 100g milk, prepares various milk preparations by it then, as sour milk, icecream, cheese etc. with the Fructus Sophorae isoflavone extract of capacity.

3. preparation contains the beverage of Fructus Sophorae extractive of general flavone

The Fructus Sophorae extractive of general flavone (extract D-1) of capacity is joined in vegetables juice, fruit juice or the soda pop, obtain to comprise the total flavones of 0.1-10g in every 100ml beverage, thereby obtain containing the functional foodstuff of Fructus Sophorae activeconstituents.

Embodiment 12

Preparation contains the skin care product of Fructus Sophorae flavonoid extracts

Cosmetic emulsion

The Fructus Sophorae extractive of general flavone that contains total flavones 5g (extract D-1a) with capacity, PEG-20 anhydrosorbitol monostearate, lg anhydrosorbitol monostearate, 10g mineral oil, 5g three octanoates, 0.5g trolamine, 5g glycerine, 3g propylene glycol, an amount of sanitas, an amount of spices and surplus pure water with 1g stearic acid, 2g hexadecanol, 1g, mix, make the emulsion that prevents skin aging, facial colour spot or freckle of 100g.

Embodiment 13

Anti-tumor activity test

Test principle: the succinodehydrogenase in the viable cell plastosome can make ectogenic MTT be reduced to the bluish voilet crystallisate of insoluble and be deposited in the cell, and dead cell does not have this function.Purple crystal thing in dimethyl sulfoxide (DMSO) (DMSO) the energy dissolved cell is measured its absorption value with the enzyme linked immunological instrument at 490nm wavelength place, can reflect viable cell quantity indirectly.This method can be referring to: E Zheng chief editor's " tissue culture and molecular cell learn a skill ", Beijing Publishing House; Han Rui chief editor's " cancer therapy drug research and experiment technology ", Beijing Medical University and combined publication society of China Concord Medical Science University.

Instrument: Bechtop; Enzyme-linked immunosorbent assay instrument; Inverted biologic microscope; Dull and stereotyped shaking table; Constant temperature CO ₂Incubator.

Reagent: RPMI1640 (GIBCO); Trypsin SIGMA); Calf serum (HYCLONE); MTT (SIGMA); DMSO (SIGMA).

Cell strain: the PD adenocarcinoma of stomach cell of people (BGC), be incubated in the RPMI1640 substratum, contain 10% calf serum, 37 degrees centigrade, 5%CO ₂

Be subjected to the reagent thing: Fructus Sophorae isoflavone extract (extract A-1a); Fructus Sophorae flavonol extract (extract C-1a); Fructus Sophorae extractive of general flavone (extract D-1a); The acid hydrolysis products of Fructus Sophorae isoflavone extract (hydrolysate); 5 FU 5 fluorouracil (5-FU, positive control drug).

Method:

1. get and be in one bottle in cell in good condition exponential phase of growth, add 0.25% tryptic digestive juice, Digestive system makes and takes off parietal cell and come off, and makes cell suspension;

2. cell counting, and cell density is diluted to 5 * 10 ⁴Individual/ml.

3. obtained cell suspension is inoculated on 96 orifice plates, and the 180ul/ hole is put in the constant temperature CO2 incubator and cultivated 24 hours.

4. prepared by the reagent thing.

5. change liquid, adding is subjected to the reagent thing, and cultivated 48 hours in the 20ul/ hole.

6. MTT is added in 96 orifice plates, the 20ul/ hole, reaction is 4 hours in the incubator.

7. inhale and remove supernatant liquor, add DMSO, 150ul/ hole, jolting 5min on the dull and stereotyped shaking table.

8. be the absorbancy that the 490nm place measures every hole with enzyme-linked immunosorbent assay instrument at wavelength, and calculate cell inhibitory rate.

The results are shown in Table 1.

Table 1 cell inhibitory rate (BGC)

Be subjected to the reagent thing	Concentration (ug/ml)	Cell inhibitory rate %		The result
					Average	SD
		Extract A-1a extract C-1a extract D-1a hydrolysate 5 FU 5 fluorouracil	1 10 100 1 10 100 1 10 100 1 10 100 1 10 100				29.91 41.98 69.88 30.53 36.32 61.43 24.15 39.33 64.72 37.14 40.25 67.33 34.26 69.38 84.76	0.03 0.03 0.02 0.03 0.04 0.03 0.03 0.02 0.02 0.05 0.03 0.02 0.1 0.05 0.06	+ + + + ++

Test-results has shown Fructus Sophorae flavonoid extracts of the present invention, has antineoplastic action.

Embodiment 14

The effect of protect against osteoporosis

6 month female Wistar rats, be divided into 4 groups: model group (the two ovaries of surveying of excision), sham operated rats (row sham-operation), model adds the Fructus Sophorae flavones group of Fructus Sophorae flavonoid extracts (extract A-1), and model adds the nilestriol group (positive controls) of Nilestriol Tablets.

1. the influence of femoral bmd

The result shows that the bone density of model group obviously reduces, and has compared utmost point significant difference (p＜0.001) with sham operated rats; Fructus Sophorae flavones group and nilestriol group all obviously increase than model group bone density, obvious difference (p＜0.05); Difference is not obvious between Fructus Sophorae flavones group and the nilestriol group, but Fructus Sophorae flavones group bone density increased value is generally a little more than nilestriol group bone density increased value.

2. the variation of serum osteocalcin content

The result shows that model group serum osteocalcin content is higher than sham operated rats, has compared utmost point significant difference (p＜0.001) with sham operated rats; Fructus Sophorae flavones group and nilestriol group all significantly reduce than model group serum osteocalcin, significant difference (p＜0.01); The serum osteocalcin content of Fructus Sophorae flavones group is obviously low than the nilestriol group, between obvious difference (p＜0.05).

3. biomechanics test

Test-results:

The numerical value that lotus numerical value and largest deformation numeric ratio nilestriol group and sham operated rats are loaded with in 3 folding tests of femur, elastic deformation numerical value, the maximum of Fructus Sophorae flavones group is slightly high, and these numerical value are all apparently higher than model group, and obvious difference (p＜0.05);

Fracture deformation, the fracture deformation numerical value of Fructus Sophorae flavones group, nilestriol group and sham operated rats all is significantly higher than model group, and significant difference (p＜0.01); The fracture deformation numerical value of Fructus Sophorae flavones group is apparently higher than nilestriol group and sham operated rats, and obvious difference (p＜0.05);

Toughness, the toughness of Fructus Sophorae flavones group, nilestriol group and sham operated rats all is significantly higher than model group, and significant difference (p＜0.01); The toughness of Fructus Sophorae flavones group is apparently higher than nilestriol group and sham operated rats, and obvious difference (p＜0.05);

Maximum stress, quite, they are compared with model group, and there were significant differences (p＜0.05) between Fructus Sophorae flavones group, nilestriol group and the sham operated rats;

Elastic stress quite, compares with model group that there were significant differences (p＜0.01) between Fructus Sophorae flavones group and the sham operated rats, has compared notable difference (p＜0.05) with the nilestriol group

Lumbar vertebrae compression tests: quite, compare with model group that there were significant differences (p＜0.01) between Fructus Sophorae flavones group, nilestriol group and the sham operated rats.

Above-mentioned protect against osteoporosis test shows: Fructus Sophorae flavonoid extracts of the present invention has obvious prevention and therapeutic action to the removal ovary osteoporosis rat, can significantly improve the biomechanical property of removal ovary rat compact bone and spongy bone.

Embodiment 15

Uterus influence test

Rudimentary female Wistar rats (3 age in week) uses Fructus Sophorae flavonoid extracts of the present invention (extract D-1a) through one week of oral cavity gastric infusion, every day 1 time.As a result, the weight in wet base and the dry weight of rat uterus all do not have considerable change before and after the medication, and pathological observation does not see that uterus shape is to the change of reaching maturity yet.

The test explanation, Fructus Sophorae flavonoid extracts of the present invention does not have effect to the uterus, and the target organ of its effect is not or not the uterus.This does not provide test basis for Fructus Sophorae flavonoid compound has general oestrogenic hormon to side effects such as uterus.

Embodiment 16

To the hyperplasia of prostate therapeutic test

The preparation of rat prostate model of hyperplasia: extract two SD male white rats of surveying testis, with the intramuscular injection of refining sesame oil dilution androlin, successive administration one month makes the rat prostate hyperplasia.

Test method: male SD big white mouse, be divided into 4 groups: model group (surgical removal bilateral testes), sham operated rats (row sham-operation), model adds the Fructus Sophorae flavones group of Fructus Sophorae flavonoid extracts (extract D-1a), and model adds the estradiol group (positive controls) of estradiol.Model group, Fructus Sophorae flavones group and estradiol group are injected androlin every day, and Fructus Sophorae flavones group and estradiol group are irritated stomach respectively and given extract of the present invention and estradiol simultaneously.

Test-results:

1. model group is compared with sham operated rats, and prostatic volume and weight all obviously increases, and difference is (p＜0.001) extremely significantly; The estradiol group is compared with model group, and prostatic volume and weight is all obviously little, and difference is (p＜0.001) extremely significantly.This explanation rat prostate model of hyperplasia manufacturing is reasonable, and positive control drug is selected from also correct.

2. the volume and weight of Fructus Sophorae flavones group rat prostate obviously alleviates than model group, and alleviates manyly more with the increase of dosage, demonstrates certain dose relationship; Fructus Sophorae flavones group is compared with the estradiol group, and effect is approaching, and difference is not obvious.

Above-mentioned test-results shows that product of the present invention has certain prevention and therapeutic action to hyperplasia of prostate.

Embodiment 17

Fructus Sophorae flavonoid extracts reducing blood-fat test

The hyperlipemia rat molds, methods of making: rat is raised high lipid diet (2% cholesterol), gavages 20% day continuously, and blood lipid level is raise.

SD rat with the normal lipid level, be divided into 4 groups: model group (gavaging high lipid diet), model adds the Fructus Sophorae flavones group of Fructus Sophorae flavonoid extracts (extract D-1a), and model adds the zhibituo group (positive controls) of zhibituo, blank group (normal diet feed).The rat eye socket is got the numerical value that blood is surveyed rat blood serum cholesterol, serum triglyceride, serum high-density LP and serum low-density LP.

Test-results:

1. the serum cholesterol of model group, triglyceride level and low-density lipoprotein white level are apparently higher than blank group (p＜0.01), and the serum high-density LP value is starkly lower than blank group (p＜0.01).The above results shows hyperlipemia model of rats manufacturing success.

2. compare with model group, the serum cholesterol of Fructus Sophorae flavones group and positive controls, triglyceride level and low-density lipoprotein white level significantly reduce (p＜0.01), high density lipoprotein level of serum significantly raise (p＜0.01); Fructus Sophorae flavones group is compared with positive controls, and lipid-lowering effect is approaching, but Fructus Sophorae flavones group is slightly inferior to positive controls, the two difference not statistically significant.

Above-mentioned test shows that product of the present invention has sure reducing blood lipid to hyperlipidaemia, thereby can prevent or treat hyperlipidemia.

Embodiment 18

Fructus Sophorae flavonoid extracts Study on antioxidation

Test reagent: NO, SOD, LPO and NOS testing cassete and protein quantification (biuret method) testing cassete; D-semi-lactosi etc.

The healthy Kunming kind female mice at 3-4 monthly age is divided into 4 groups: model group (subcutaneous injection D-semi-lactosi), model adds the Fructus Sophorae flavones group of Fructus Sophorae flavonoid extracts (extract D-1a), the dimension E group (positive controls) of model vitaminize E, control group.Control group administered physiological saline.Behind the continuous use 30 days, get brain and weigh, make brain homogenate, get supernatant and measure every index with physiological saline.

The results are shown in Table 2.

The anti-oxidant test of table 2

Group	SOD(NU/ml)	LPO(umol/L)	NO(umol/L)	NOS(U/ml)
					Control group model group Fructus Sophorae flavones group positive controls	2.15±0.23 0.98±0.24 3.15±0.40 2.64±0.51	23.15±4.01 31.14±4.23 20％.28±3.98 21.05±4.27	0.64±0.03 0.45±0.05 0.75±0.09 0.69±0.07	0.95±0.03 0.65±0.04 1.02±0.05 0.93±0.03

Above-mentioned test shows:

1. the SOD activity of model group, NO amount and NOS activity significantly are lower than control group (p＜0.01), and the LPO amount is significantly higher than control group (p＜0.01).This explanation D-semi-lactosi is made the mice aging model success.

2. the SOD activity of Fructus Sophorae flavones group, positive controls, NO amount and NOS activity are significantly higher than model group (p＜0.01), and the LPO amount significantly is lower than model group (p＜0.01), and test shows that Fructus Sophorae flavonoid extracts of the present invention has antioxygenation.

3. the SOD activity of Fructus Sophorae flavones group, NO amount and NOS activity are apparently higher than dimension E positive controls (p＜0.05), and the LPO amount is starkly lower than dimension E positive controls (p＜0.05), and test card is understood the obviously strong and vitamin-E of Fructus Sophorae flavonoid extracts antioxygenation of the present invention.

Above-mentioned anti-oxidant test-results shows, Fructus Sophorae flavonoid extracts of the present invention has good antioxygenation, can treat and the oxidation diseases associated, product for example of the present invention can delay senility, prevents and treat senile dementia, control skin aging, bread color spot or freckle etc., and effect obviously is better than antioxidant vitamin E commonly used in the market.

Clinical implementation example 1

Climacteric syndrome effect after the Fructus Sophorae flavonoid extracts treatment women menopause

Select age 45-65 year, menopause more than 1 year, have that Tidal fever with perspiration in various degree, dizzy insomnia, irritated irritability, palpitaition are had a headache, depressed, weak, vaginal dryness or dyspareunia, back or symptoms such as joint pain, paresthesia of skin 30 examples.Take the capsule that contains product of the present invention (extract D-1a) every day, and cut out other estrogens or with treatment above-mentioned symptom relevant medicine or healthcare products; 10 days was 1 course of treatment.Take 3-4 after the course of treatment, have 22 routine above-mentioned symptoms be improved significantly, wherein 18 routine clinical symptom disappear substantially; There are 3 routine clinical symptom to alleviate to some extent; All the other 5 routine DeGrains.30 all routine users take product of the present invention, do not observe any clinical toxicity, side effect.

Test card is understood, Fructus Sophorae flavonoid extracts of the present invention, the effect of climacteric syndrome after very ideal prevention, improvement being arranged or treating postmenopausal women, not only effective, prior advantage, be that Fructus Sophorae flavonoid extracts of the present invention does not have general estrogenic toxic side effect, take for a long time, also do not have the danger of aspect disease incidences such as increasing mammary gland, uterine endometrium.

Those of ordinary skills obviously can recognize; in the above-described embodiment, allow to make conventional change, therefore; the present invention not only comprises the technical characterictic of mentioning in the application documents, also comprises variation or change that all do not exceed aim of the present invention and protection domain.

Claims (18)

1. a Fructus Sophorae isoflavone extract mainly contains the isoflavones with formula (I) structure, with 5, and 7-dihydroxyl-isoflavones-4 '-calculating of O-neohesperidoside, the weight percentage that isoflavones accounts for extract is 50%～100%, but is not 100%,

Wherein, R ₁, R ₂Be selected from hydrogen, methyl, glucose and neohesperidose respectively, R ₁And R ₂Mutually the same or different.

2. Fructus Sophorae isoflavone extract according to claim 1 is characterized in that the weight percentage that isoflavones accounts for extract is 90%～100%, but is not 100%.

3. Fructus Sophorae isoflavone extract according to claim 1 and 2 is characterized in that containing 5,7-dihydroxyl-isoflavones-4 '-O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside.

4. a Fructus Sophorae flavonol extract mainly contains the flavonol with formula (II) structure, with 4 ', 5,7-trihydroxy--flavonol-3-O-sophoroside calculates, and the weight percentage that flavonol accounts for extract is 50%～100%, but is not 100%,

Wherein, R ¹, R ²And R ³The disaccharide or the trisaccharide that are selected from hydrogen, methyl, glucose, rhamnosyl respectively, form by glucose and/or rhamnosyl, R ¹, R ²And R ³Be same to each other or different to each other; R ⁴Be selected from hydrogen, hydroxyl and methyl.

5. Fructus Sophorae flavonol extract according to claim 4 is characterized in that the weight percentage that flavonol accounts for extract is 90%～100%, but is not 100%.

6. according to claim 4 or 5 described Fructus Sophorae flavonol extracts, it is characterized in that containing 4 ', 5,7-trihydroxy--flavonol-3-O-sophoroside, 4 ', 5,7-trihydroxy--flavonol-3-O-rutinoside, 3 ', 4 ', 5,7-tetrahydroxy-flavonol-3-O-rutinoside.

7. Fructus Sophorae extractive of general flavone, mainly contain isoflavones with formula (I) structure and flavonol with formula (II) structure, with 5,7-dihydroxyl-isoflavones-4 '-isoflavones that the O-neohesperidoside calculates and with 4 ', 5, obtain total flavones after the flavonol addition that 7-trihydroxy--flavonol-3-O-sophoroside calculates, the weight percentage that described total flavones accounts for extract is 20%～100%, but be not 100%

Wherein, R ₁, R ₂Be selected from hydrogen, methyl, glucose and neohesperidose respectively, R ₁And R ₂Mutually the same or different;

Wherein, R ¹, R ²And R ³The disaccharide or the trisaccharide that are selected from hydrogen, methyl, glucose, rhamnosyl respectively, form by glucose and/or rhamnosyl, R ¹, R ²And R ³Be same to each other or different to each other; R ⁴Be selected from hydrogen, hydroxyl and methyl.

8. Fructus Sophorae extractive of general flavone according to claim 7 is characterized in that the weight percentage that total flavones accounts for extract is 50%～100%, but is not 100%.

9. Fructus Sophorae extractive of general flavone according to claim 7 is characterized in that the weight percentage that total flavones accounts for extract is 90%～100%, but is not 100%.

10. according to any described Fructus Sophorae extractive of general flavone of claim 7-9, it is characterized in that containing 5,7-dihydroxyl-isoflavones-4 '-O-neohesperidoside, 5,7-dihydroxyl-isoflavones-4 '-O-glucoside, 4 ', 5-dihydroxyl-isoflavones-7-O-glucoside, 4 ', 5,7-trihydroxy--flavonol-3-O-sophoroside, 4 ', 5,7-trihydroxy--flavonol-3-O-rutinoside, 3 ', 4 ', 5,7-tetrahydroxy-flavonol-3-O-rutinoside.

11. any described extract of claim 1-10, flavonoid glycoside wherein forms hydrolyzed product through hydrolysis reaction, and described hydrolysis reaction is selected from acid-catalyzed hydrolysis reaction, alkali catalyzed hydrolysis reaction and enzymatic hydrolysis reaction.

12. any described extract of claim 1-11, flavonoid compound wherein and metal ion have formed flavones metal-salt mixture, and described metal ion is selected from a kind of or several metal ion in sodium, potassium, zinc, magnesium, chromium, iron, aluminium, copper, calcium, cobalt, barium, strontium, selenium and the zirconium.

13. any described preparation method of extract of claim 1-10, this method comprises:

A. with water-soluble solvent as extracting solvent extraction Fructus Sophorae raw material, extracting solution is concentrated into to be done or is concentrated in right amount, obtain extracting enriched material, described water-soluble solvent is selected from a kind of solvent in water, ethanol, methyl alcohol, the acetone or the mixed solvent of two or more solvent compositions;

B. the extraction enriched material that above-mentioned a obtains is suspended in the ethanol that volume ratio is 0-50: 100-50: water or methyl alcohol: water or acetone: in the water solvent, after the placement, separate out solid, filter, obtain solid and filtrate, promptly obtain extract A behind the solid drying;

C. after the filtrate that above-mentioned b obtains concentrates in right amount, separate with chromatography column, with the volume ratio is the ethanol of 0-100: 100-0: water or methyl alcohol: water or acetone: water carries out gradient elution, the Fractional Collections effluent liquid, wherein said chromatography column is selected from macroporous resin column, polyamide column or sephadex column;

D. will be in the above-mentioned c effluent liquid main effluent liquid that contains formula (I) isoflavones merges, and reclaims solvent, is concentrated into driedly, obtains extract B after the drying;

E. will be in the above-mentioned c effluent liquid main effluent liquid that contains formula (II) flavonol merges, and reclaims solvent, is concentrated into driedly, obtains extract C after the drying;

Perhaps above-mentioned d and e are united two into one, become:

F. will be in the above-mentioned c effluent liquid main effluent liquid that contains formula (1) isoflavones and/or formula (II) flavonol merges, and reclaims solvent, is concentrated into driedly, obtains extract D after the drying;

Wherein, above-mentioned described extract A, B are any described Fructus Sophorae isoflavone extract of claim 1-3; Described extract C is any described Fructus Sophorae flavonol extract of claim 4-6; Described extract D or the mixture that is formed by extract A and/or B and extract C are any described Fructus Sophorae extractive of general flavone of claim 7-10.

14. method according to claim 13, the extract A that wherein obtains, B, C or D are again through treating process, just can obtain the higher extract of purity, described treating process is for pressing same column chromatography method repurity one or many, perhaps for extract A, B, C or D are extracted with the solvent of suitable polarity, extracting solution reclaims solvent to the greatest extent, concentrate, promptly obtain the higher extract of purity after the drying, the solvent of described suitable polarity is selected from the methyl alcohol that volume ratio is 75-100: 25-0: water, ethanol: water, acetone: water and ethyl acetate, chloroform.

15. a pharmaceutical preparation, functional foodstuff or skin care product is characterized in that containing any described extract of claim 1-12.

16. any described extract of claim 1-12 is used for preventing, improve or the application of treatment and pharmaceutical preparation estrogen-deficiency related complaints disease or that have antioxygenation, functional foodstuff or skin care product in preparation.

17. any described extract of claim 1-12 is used for preventing, improving or treat the pharmaceutical preparation of osteoporosis, female dimacteric syndrome, female irregular menstruation, senile dementia, aging, tumor and cancer, male prostatic hyperplasia disease, hyperlipidemia, vascular hypertension, arteriosclerosis, cerebral thrombosis or coronary heart disease or the application of functional foodstuff in preparation.

18. the application of any described extract of claim 1-12 pharmaceutical preparation, functional foodstuff or skin care product of skin syndromes after preparation is used for preventing, improve or treat skin aging, facial colour spot freckle or women's menopause.