CN1799572A - Method for extracting biological active substances from bean embryo and its application - Google Patents

Abstract

The invention relates to a process for extracting biologically active substances from soybean embryos, which comprises extracting soybean embryo oil through supercritical CO2 method, using degreased soybean embryo obtained through extraction separation as the raw material, extracting soybean isoflavones by utilizing thermal backflow extraction-concentrator.

Description

From bean embryo, extract the method and the application thereof of bioactive substance

Technical field

The present invention relates to from bean embryo, extract the technology and the application thereof of bioactive substance.

Background technology

In numerous vegetable oil, soybean embryo fat/oil is to take the course of its own, and alpha-linolenic acid content is 8 times of soybean oil in the bean embryo oil, and vitamin content is 6 times of soybean oil, is very effective hypolipidemic raw material, the natural V of high concentration _EBe other equal health care oil as Semen Tritici aestivi germ oil and Fructus Maydis oil incomparable, therefore develop a kind of like this health care oil and have great realistic meaning.

Supercritical fluid extraction is a special nature of utilizing supercritical fluid, condition of high voltage solid or liquid mixture following and to be separated contacts, regulating system operating pressure and temperature, owing to solute dissolubility in supercritical fluid increases along with the increase of critical fluids density, extract desired material, by the method for blood pressure lowering or intensification, reduce the density of supercritical fluid subsequently, make extract obtain separating.Be in the above fluid of critical pressure and critical temperature and generally can reach several magnitude the increase of organic compound dissolubility.CO wherein ₂Be first-selected industrial extractant, because of series of advantages such as its critical condition easily reaches, low toxicity, few residual, inertia, to thermal sensitivity and easily the extraction of the product of oxidation have more captivation.CO ₂Be a kind of non-polar solven, polarity is suitable for this apolar substance extraction of bean embryo oil between normal hexane and chloroform.The factor that influences the supercritical extraction effect mainly is extraction conditions-pressure, temperature, time, flow etc., determines that these factors are the key points that realize the soybean isoflavone extraction.Many experimentatioies both domestic and external show utilizes supercritical CO ₂Abstraction technique can be from multiple different natural plants oils chemical compound like the extraction phase, as Corn germ oil, Semen Tritici aestivi germ oil, Semen Vitis viniferae wet goods, but also find to utilize this technology extraction bean embryo oil so far.

Soybean isoflavone (soy isoflavone) has the chemical mother nucleus structure of the heterocycle phenol similar to estrogen, thereby shows estrogen activity and estrogen antagonist activity; The chemical constitution of its polyphenol hydroxyl has also determined it to have non-oxidizability, thus, soybean isoflavone has many important physical functions, as to reducing serum cholesterol, treatment climacteric syndrome, senile osteoporosis control, carcinoma of prostate, cardiovascular disease, breast carcinoma etc.These are all confirmed in Epidemiological study, vivo and vitro experimentation in recent years.Along with the effect of soybean isoflavone progressively is familiar with by people, with and the research of physiological function constantly make further progress, the soybean isoflavone production of articles is subjected to the extensive concern of researcher.

From soybean meal, extract isoflavone and generally can pass through methods extractions such as water extraction, alcoholic solution extraction method and weak caustic solution extraction method.Wherein alkaline extraction is operated because of using a large amount of alkali liquor, and the impurity of extraction is more, and pollution is big and processing cost is high, and the bean cake quality is influenced also bigger.With water is in the heating leaching process of solvent, and soybean meal is easily imbibition and gelatinizing in hot water, causes solid-liquid isolated by filtration difficulty, water content height in the dregs of rice, and production efficiency descends, and extraction ratio is also had a strong impact on; And in follow-up drying course, need to consume a large amount of energy, increase production cost.Therefore, the most frequently used extracting method is an alcohol extracting method in laboratory method and the actual production technology, and extraction solvent commonly used in the alcohol extracting method is an ethanol.The principal element that influences extraction efficiency in the ethanol extraction process is the concentration of alcohol solvent, extracts solid-liquid ratio, extraction time and extract temperature etc. that there is the deficiency that extraction efficiency is low, production cost is high in existing ethanol extraction method.

Summary of the invention

In view of also not finding to utilize supercritical CO so far ₂Technology extraction bean embryo oil, there is the deficiency that extraction efficiency is low, production cost is high in the method for existing ethanol extraction soybean isoflavone simultaneously, the invention provides a kind of method and application thereof of extracting bioactive substance from bean embryo.Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The method of extracting bioactive substance from bean embryo is carried out according to following step: with Semen sojae atricolor bean embryo is raw material, adopts supercritical CO ₂Method extraction bean embryo germ oil, control supercritical extraction pressure is 30～40MPa, and extraction temperature is 43～47 ℃, and the extraction time is 1.2～1.6 hours.The bean embryo oil that this method obtains is used to prepare blood fat reducing health products.

Extracting the method for bioactive substance from bean embryo carries out according to following step: a, being raw material with Semen sojae atricolor bean embryo, is 30～40MPa at pressure, and extraction temperature is to carry out supercritical CO under 43～47 ℃ the condition ₂Extraction, the extraction time is 1.2～1.6 hours; B, the defat bean embryo that obtains with a step extract and separate are raw material, utilize hot reflux extracting-concentrator to extract soybean isoflavone, extract solid-liquid ratio and be 1: 5, ethanol volumetric concentration and be 75%, extraction time is 4 hours, lixiviate 1 time, obtains soybean isoflavone.The soybean isoflavone that this method obtains can be respectively applied for preparation osteosporosis resistant medicament, prevention climacteric syndrome medicine and prevention prostatosis health product.

The present invention is a raw material with the bean embryo, gives full play to the effect of specific nutrient and bioactive substance in the bean embryo, utilizes supercritical CO ₂Technology and soybean isoflavone extractive technique develop have blood fat reducing, osteoporosis, alleviation climacteric syndrome etc. be rich in bean embryo germ oil and soybean isoflavone health food, to the comprehensive utilization soybean resource, the nutrition health-care functions of performance Semen sojae atricolor, the guarantee people's health acquires a special sense, and is embodied in: (1) adopts suitable supercritical CO ₂Technology (extraction conditions-pressure, temperature, time, flow) extracts high-quality bean embryo oil from bean embryo, the extraction yield is more than 90%.(2) experimental selection goes out best soybean isoflavone extraction conditions, from bean embryo embryo slag, obtain to have multiple bioactive soybean isoflavone crude product, utilizing hot reflux extracting-concentrator to extract soybean isoflavone can save time, save a large amount of solvents that extract, improve and extract workshop condition, and the extraction efficiency that is reached and other alcoholic solution extract phase are worked as.(3) the present invention inquires into suitable extraction conditions by experiment and successfully utilizes the supercritical technology of " green technology " to obtain high-quality germ oil.(4) be raw material with bean embryo oil, soybean isoflavone crude product, develop four kinds of guarantors and build food (blood fat reducing, osteoporosis, control climacteric syndrome, anti-prostatosis) with special health care.

Description of drawings

Fig. 1 is a supercritical CO ₂Technology extraction process flow figure, wherein 1:CO ₂Steel cylinder, 2: depurator, 3: liquefaction groove, 4: hydraulic pump, 5: preheater, 6: depurator, 7: extraction steel, 8: separator, 9: separator, 10: depurator; Fig. 2 is pressure-extraction yield curve chart; Fig. 3 is temperature-residual oil content curve chart; Fig. 4 be time-extraction yield curve chart; Fig. 5 changes results of comparison figure, a for bone density before and after taking medicine: before matched group is taken medicine, and b: after matched group is taken medicine, c: before the dosage group is taken medicine, d: after the dosage group is taken medicine; Fig. 6 is the result of variations figure of dosage group at the duration of test sex hormone level, LH,

FSH,

E2; Fig. 7 is the result of variations figure of matched group at the duration of test sex hormone level, LH,

FSH,

E2; Fig. 8 is the experimental result picture of Gen processing PC3 cell, Genistein,

Cisplatin; Fig. 9 is the PC-3 cellular morphology figure of matched group under the inverted phase contrast microscope; Figure 10 is the PC-3 cellular morphology figure that 5 μ g/ml Gen handle under the inverted phase contrast microscope; Figure 11 is matched group PC-3 cell ultrastructure figure; Figure 12 is the PC-3 cell ultrastructure figure that 5 μ g/ml Gen handle.

The specific embodiment

The specific embodiment one: the method for extracting bioactive substance in the present embodiment from bean embryo is carried out according to following step: decortication according to a conventional method, separate the bean embryo, and be raw material with Semen sojae atricolor bean embryo, adopt supercritical CO ₂Method extraction bean embryo oil, control supercritical extraction pressure is 30～40MPa, and extraction temperature is 43～47 ℃, and the extraction time is 1.2～1.6 hours.The bean embryo oil that this method obtains is used to prepare blood fat reducing health products.

The specific embodiment two: present embodiment is with supercritical CO ₂Technology extraction bean embryo oil is that example is elaborated:

The extraction process of present embodiment adopts FA121-50-01 type supercritical extraction equipment (Huaan, Nantong supercritical extraction company limited), and process chart is seen Fig. 1.

Principal element (pressure, temperature, time, flow velocity) in the examination supercritical extraction is to the influence of extraction, and the factorial analysis result is shown in Fig. 2～4.On above monofactorial basis, further carry out response surface analysis with three factors, three levels, to seek optimum process condition, determine that finally the optimum condition of supercritical extraction soybean embryo fat/oil is: 34MPa, 45 ℃, 1.5h, extraction yield 90%.Extraction oil quality and protein quality all meet the requirements.

The specific embodiment three: present embodiment is that example describes with the experimentation of bean embryo oil hyperlipidemia disease.

1. materials and methods:

With bean embryo oil the healthy adult male Wistar rat is carried out zoopery (body weight 150～200g, totally 48).Automatic biochemistry analyzer, desk type high speed refrigerated centrifuge, serum total cholesterol, triglyceride and high-density LP determination reagent box.

Feedstuff is formed and is divided into normal diet component and high lipid food component.

The animal group technology:

Rat is got tail blood raise 6d under experimental situation after, measures serum total cholesterol (TC), triglyceride (TG), HDL-C (HDL-C) level.Carry out random packet according to serum total cholesterol (TC) level.Rat is divided into 4 groups at random, 12 every group, i.e. hyperlipidemia model matched group, low dose group, middle dosage group, high dose group.

Lipid-lowering test:

When each experimental group is given high lipid food, give the given the test agent of various dose through irritating stomach, the hyperlipidemia model matched group gives water, and low dose group gives 0.17g/kg sample, and middle dosage group gives 0.33g/kg sample, and high dose group gives 0.67g/kg sample.Body weight of weighing is irritated the stomach amount by the change calculations of body weight weekly, the fasting in the 30th day of testing 16 hours, surveys serum TC, TG, HDL-C level.Index determining method: body weight special electronic animal balance weighing.Serum total cholesterol (TC), triglyceride (TG), HDL-C (HDL-C) is measured on automatic biochemistry analyzer with the corresponding test kit of measuring.Statistical analysis: set up the FOXBASE data base, measurement data is carried out variance analysis with the SPSS statistical software.The results are shown in Table 1 and table 2, the result shows the rat body weight no significant difference.Bean embryo oil promotes the generation of high density cholesterol (HDL) in the rat body, and HDL has and transports cholesterol to effect that liver decomposes its metabolism, thereby further promotes the metabolism of cholesterol and bring into play the effect of its blood fat reducing.

Each treated animal experimental session changes of body mass of table 1

Group	The 0th day	The 7th day	The 14th day	The 21st day	The 30th day
						Dosage group high dose group in the high fat contrast low dose group	179.3±9.7 176.8±10.2 180.3±10.8 181.9±8.69	194.9±9.4 196.9±11.5 204.2±14.6 200.2±9.4	218.3±9.9 219.3±11.4 224.3±13.7 221.3±15.0	237.6±8.2 234.6±10.8 243.4±15.8 242.6±16.2	253.2±8.7 259.6±10.7 256.6±16.5 264.6±16.6

Each treated animal serum serum lipid concentrations of table 2. changes

Group	TC mmol/L		TG mmol/L		HDL mmol/L
							The 0th day	The 30th day	The 0th day	The 30th day	The 0th day	The 30th day
	Dosage group high dose group in the high fat contrast low dose group	2.18±0.73 2.16±0.55 2.17±0.79 2.15±0.74	3.90±0.31 3.58±0.75 3.17±0.50 3.08±0.68*	1.87±0.51 1.76±0.55 1.99±0.37 1.70±0.41	3.17±0.87 2.69±0.93 3.31±0.95 3.44±0.98	0.47±0.12 0.43±0.11 0.44±0.11 0.54±0.12							0.26±0.07 0.32±0.07 0.36±0.06* 0.35±0.08*

Annotate: each organizes the Mus number average is 10.Compare with matched group with before irritating stomach ^*P＜0.05

The specific embodiment four: the method for extracting bioactive substance in the present embodiment from bean embryo is carried out according to following step: a, being raw material with Semen sojae atricolor bean embryo, is 30～40MPa at pressure, and extraction temperature is to carry out supercritical CO under 43～47 ℃ the condition ₂Extraction, the extraction time is 1.2～1.6 hours; B, the defat bean embryo that obtains with a step extract and separate are raw material, utilize hot reflux extracting-concentrator to extract soybean isoflavone, extract solid-liquid ratio and be 1: 5, ethanol volumetric concentration and be 75%, extraction time is 4 hours, lixiviate 1 time, obtains soybean isoflavone, extracting yield is 81.5%.The soybean isoflavone that this method obtains can be respectively applied for preparation osteosporosis resistant medicament, prevention climacteric syndrome medicine and prevention prostatosis health product.

The specific embodiment five: the soybean isoflavone that present embodiment adopts Semen sojae atricolor bean embryo embryo slag to extract carries out crowd's experiment, observes improvement and the function of resisting osteoporosis of soybean isoflavone to the climacteric syndrome symptom.

One, the screening of object of study:

The first step: carry out primary dcreening operation according to the unified in the world kupperman scoring form of filling in that the climacteric syndrome symptom is evaluated, select score greater than the climacteric women between 20 minutes (promptly reach moderate climacteric syndrome symptom performance) 45～55 years old in principle, and get rid of and have following symptom:

A. menolipsis person or person more than 55 years old more than 5 years;

B. gestation or women breast-feeding their children, allergic constitution person;

C. late deformity, maimed person, disability;

D. be associated with serious primary disease such as cardiovascular, cerebrovascular, liver, kidney and hemopoietic system, psychotic;

E. take other related drugs, health food person that can not stop using immediately for a long time;

F. do not meet the standard of including in, edible in accordance with regulations, can't judge curative effect and data not umbra sound effect and safety judgement person.

Second step: the test-meal object that just filters out is carried out a medical examination, comprise the mensuration of bone density, serum sex hormone level, blood lipids index etc.Select the test-meal object that bone density is low, the serum estradiol level is low as far as possible.

Comprehensive first two steps result determines this development test object 90 people.

Two, grouping and method:

At random subjects is divided into groups according to estrogen level, be divided into two groups, take soybean isoflavone capsule dosage group and take the Cebo-Caps matched group.Soybean isoflavone dosage group test-meal object oral soybean isoflavone capsule every day 2 times, each 2 (promptly taking in soybean isoflavone 90mg for each person every day altogether).Equally, placebo group test-meal object is taken the Cebo-Caps of equivalent every day.

Three, result:

Matched group has 33 people to meet the requirements, and holds on to the last, and wherein menopause number is 11 people; The dosage group has 37 people to hold on to the last, and the menopause number is 14 people, and the menopause time limit is all in 5 years.Experimental result is as follows:

1, menopause syndrome is improved situation (Kupperman scoring):

Take soybean isoflavone after six months, from self relatively, whole 12 indexs of dosage group subjects all improve, and scoring reduces statistical significance, and matched group other indexs except Tidal fever with perspiration, insomnia and urinary system infection also make moderate progress; From take soybean isoflavone organize after 6 months between relatively, except that depressed, dizzy, tired and have a headache these several, the dosage group is all improved obviously than matched group on other 8 indexs, and statistical significance is arranged.

From table 3 and table 4 as can be seen, take soybean isoflavone after, aspect most of symptoms the dosage group all than matched group improve good, wherein hectic fever hyperhidrosis, insomnia, irritated irritability, tired, headache, these several of urinary system infections improve obviously.In addition, from feedback information, dosage group discrete trial object also has otherwise improvement: hyper-menorrhea, sxs in menstrual period (2 example), and appetite increases (1 example), and skin improves, good, the skin smooth (2 example) of the colour of skin, energetic (2 example) etc.

Table 3 matched group take soybean isoflavone after six months every doing well,improving number account for total number of persons percentage ratio (n=33)

Symptom	Tidal fever with perspiration	Insomnia	Irritated irritability	Depressed suspicion	Dizzy	Tired	Osteoarthrosis pain	Headache	Cardiopalmus	Formication	Urinary system infection	The sexual life situation
													Number %	16 48.4	16 48.4	11 33.3	3 9.1	7 21.2	10 30.3	14 42.4	3 9.1	6 18.2	4 12.1	1 3	2 6

Table 4 dosage group take soybean isoflavone after six months every doing well,improving number account for total number of persons percentage ratio (n=37)

Symptom	Tidal fever with perspiration	Insomnia	Irritated irritability	Depressed suspicion	Dizzy	Tired	Osteoarthrosis pain	Headache	Cardiopalmus	Formication	Urinary system infection	The sexual life situation
													Number %	27 72.9	22 59.5	12 32.4	2 5.4	9 24.3	19 51.3	20 54.1	9 24.3	13 35.1	2 5.4	0 0	3 8.1

2, bone densitometry result: take the soybean isoflavone capsule after six months, dosage group study subject bone density all has the trend of increase than basic value, tibial bone density has significant difference (as Fig. 5), but the bone mineral density and bone there was no significant difference (sees Table 3,4, Fig. 5).And the bone mineral density and bone of matched group study subject and tibial bone density and basic value relatively do not increase, and all do not have significant difference.

3, serum gonadal hormone measurement result: from this result of the test as can be seen, take soybean isoflavone after 6 months, the LH of two groups of subjects, FSH level are all on a declining curve, and the LH level of dosage group descends obviously, and difference has statistical significance.Two groups E2 level all descends obviously (p＜0.05), but matched group decline scope bigger (seeing Fig. 6, Fig. 7).From the downward trend of dosage group estradiol level, first trimester E ₂Descend obviously, compared statistical significance with basic value, three months E2 levels in back descend and are tending towards relaxing, compared the difference not statistically significant in six months during with three months, analyzing this may be that estrogenic effect needs a period of time to human body owing to soybean isoflavone, along with the prolongation of action time, the effect that improves estrogen level is also obvious more.On the contrary, the matched group estradiol level is not seen this trend, but presents the straight line downward trend.

4, bone metabolism index determining result: take the soybean isoflavone capsule after six months, the serum calcium of two groups of study subjects all rises, and significant difference is arranged; Serum paraoxonase changes little; Serum alkaline phosphatase has downward trend, and the dosage group descends obviously, and significant difference (p＜0.05) is arranged.

The bone metabolism index is quick on the draw, non-invasive, not only can understand the speed of bone loss and conversion, and aspect the therapeutic evaluation after the patients with osteoporosis medication, curative effect monitoring its important function is arranged also.In the osteoplastic metabolic index of reflection, the activity of serum neutral and alkali phosphatase can reflect osteoplastic active degree, some scholars study the relation between bone formation and bone resorption marker and the osteoporosis in recent years, bone formation index ALP sharply rises after finding menopause, about 50% alkali phosphatase derives from bone in the serum, second half derives from liver, and the alkali phosphatase of minute quantity derives from small intestinal and Placenta Hominis.The shared ratio of serum neutral and alkali phosphatase can be increased to 77% in 10 years after menopause, rising that it is generally acknowledged serum alkaline phosphatase is to be accompanied by the hyperfunction of bone resorption and the compensatory bone formation increase that occurs causes, in this experiment, the serum alkaline phosphatase of dosage group subjects obviously reduces, statistical significance is arranged, reflected the inhibitory action of soybean isoflavone bone resorption.

The specific embodiment six: trihydroxy-isoflavone suppresses the effect research of prostate gland cancer cell (PC-3):

Material:

(1) cell culture: the PC-3 cell draws from the Shanghai Inst. of Tumor, and cell culture contains 10% inactivated fetal bovine serum, 100k μ/l penicillin, 100mg/l streptomycin in F12-DMEF (Gibco company) culture medium, put 37 ℃, 5%CO ₂Cultivate in the incubator.

(2) drug treating: soybean isoflavone monomer trihydroxy isoflavone purity is 98%.With dimethyl sulfoxide (DMSO) Gen is mixed with the storing solution that concentration is 10mmol/L, is diluted to desired concn with the F12-DMEF culture fluid then.The final concentration of DMSO in each group culture fluid is 5%.

(3) main agents: SABC detects S-P (streptomycete avidin-peroxidase connects) test kit (Beijing Zhong Shan biological reagent company), the anti-people Bax of rabbit, iNOS (inducible nitric oxide synthase) polyclonal antibody (Beijing Zhong Shan biological reagent company), Trizol test kit (GIBCOBRL).

MTT test: be inoculated in 96 well culture plates, every hole 5 * 10 after being in the PC-3 cell routine digestion of exponential phase ³Individual.After cultivating 24h respectively, add Gen and make that final concentration is respectively 0.1,0.5,1,2,5,10mmol/L, every concentration is established 4 parallel holes, other establishes the blank group, and cultivation 1,2,3,4,5,6,7d add 5g/L MTT 20 μ l, cultivate behind the 4h the culture fluid in the plate to the greatest extent again, each hole adds 150 μ lDMSO, and optical density (OD) value in each hole is measured with microplate reader (wavelength 490m) in vibration back gently, with the meansigma methods of every group of 4 the hole OD average OD value as each group.

Cellular morphology changes observes: administration 24,48h various dose group PC-3 cell and cellular control unit are directly placed under the common inverted phase contrast microscope, and the change of observation of cell form and growth characteristics, and take a picture.Cell is handled 48h, EDTA/ trypsinization, phosphate buffer through Gen 5mmol/L, and (transmission electron microscope is observed down for PBS, pH74) washing, fixing, ethanol dehydration, resin embedding, ultrathin section, the plumbous double staining of uranium, and observed result is seen Fig. 9～12.

The phytoestrogen Gen that is rich in the bean product is the non-nutrient composition that is subjected to Food Science circle extensive concern in recent years.The massive epidemiology investigation shows that Asian countries resident's breast carcinoma, carcinoma of prostate sickness rate significantly are lower than western developed country, the consumption per head that is Asian countries resident bean product is 20～50 times of western developed country, think that Semen sojae atricolor has antitumaous effect, mainly because of wherein soybean isoflavone composition, particularly tri hydroxy isoflavone Gen.Gen suppresses tumor cell proliferation, realizes by cell death inducing.Apoptosis is the main mode of cell physiological death, at body growth, grow and keep in the homeostasis and play an important role.Increasing data shows: apoptosis and tumor have substantial connection, because tumor is not only the disease of propagation and disdifferentiation, also are the diseases of abnormal apoptosis simultaneously, so the research apoptosis provides new thinking for tumor treatment.This experiment shows by morphological observation: Gen has inhibitory action to human prostate gastric cancer PC-3 cell proliferation, and its effect is relevant with cell death inducing.Under the Electronic Speculum behind this experiment 5mmol/L Gen effect 48h visible nuclear chromatin pyknosis become morphological changes such as the block aggregation of several high electron densities, Cytoplasm concentrate, endochylema vacuolation.These changes show that effect is owing to excited the apoptosis of PC-3 cell to Gen to the PC-3 cell inhibitory effect.

Claims (8)

1, extract the method for bioactive substance from bean embryo, it is characterized in that the described method of extracting bioactive substance from bean embryo carries out according to following step: with Semen sojae atricolor bean embryo is raw material, adopts supercritical CO ₂Method extraction bean embryo oil, control supercritical extraction pressure are that 30～40MPa, extraction temperature are that 43～47 ℃, extraction time are 1.2～1.6 hours.

2,, it is characterized in that supercritical extraction pressure is that 34MPa, extraction temperature are that 45 ℃, extraction time are 1.5 hours according to the described method of from bean embryo, extracting bioactive substance of claim 1.

3, claim 1 is described extracts the application that the bean embryo oil that obtains in the method for bioactive substance is used to prepare blood fat reducing health products from bean embryo.

4, from bean embryo, extract the method for bioactive substance, it is characterized in that the described method of extracting bioactive substance from bean embryo carries out according to following step: a, being raw material with Semen sojae atricolor bean embryo, is that 30～40MPa, extraction temperature are to carry out supercritical CO under 43～47 ℃ the condition at pressure ₂Extraction, the extraction time is 1.2～1.6 hours; B, the defat bean embryo that obtains with a step extract and separate are raw material, utilize hot reflux extracting one concentrator to extract soybean isoflavone, extract solid-liquid ratio and be 1: 5, ethanol volumetric concentration and be 75%, extraction time is 4 hours, lixiviate 1 time, obtains soybean isoflavone.

5, the method for extracting bioactive substance from bean embryo according to claim 4 is characterized in that described supercritical extraction pressure is that 34MPa, extraction temperature are that 45 ℃, extraction time are 1.5 hours.

6, claim 4 is described extracts the application that the soybean isoflavone that obtains in the method for bioactive substance is used to prepare the osteoporosis health product from bean embryo.

7, the described soybean isoflavone that obtains in the method for bioactive substance that extracts from bean embryo of claim 4 is used to prepare the application that prevents the climacteric syndrome health product.

8, the described soybean isoflavone that obtains in the method for bioactive substance that extracts from bean embryo of claim 4 is used to prepare the application that prevents the prostatosis health product.

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