CN102977066A - Method for extracting and purifying isoflavones in soybean - Google Patents
Method for extracting and purifying isoflavones in soybean Download PDFInfo
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- CN102977066A CN102977066A CN2012105222786A CN201210522278A CN102977066A CN 102977066 A CN102977066 A CN 102977066A CN 2012105222786 A CN2012105222786 A CN 2012105222786A CN 201210522278 A CN201210522278 A CN 201210522278A CN 102977066 A CN102977066 A CN 102977066A
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Abstract
The invention belongs to the technical field of plant extraction, and particularly relates to a method for extracting and purifying isoflavones in soybean. The method for extracting and purifying the isoflavones in soybean, provided by the invention, mainly aims at obtaining soybean isoflavone products which are consistent with natural soybean in terms of components and can be in line with the requirements for national traditional Chinese medicines, thus traditional Chinese medicine products are developed. The method is simple in extraction and has low requirements on equipment; the extraction rate of the soybean isoflavones is more than 50% by adopting the steps of alcohol extraction, extraction and silica-gel column chromatography according to the method; and the method is a new approach for extracting isoflavone compounds in soybean and has important significances on taking full advantage of plant resources and developing pure natural medicines.
Description
Technical field
The invention belongs to technical field of plant extraction, relate in particular to a kind of from soybean the method for extraction and purification isoflavones.
Background technology
Soybean isoflavones is the secondary metabolite in the soybeans they grow, is present in and comprises isoflavone glycoside and soybean isoflavone aglycones in soybean germ and the cotyledon.Through years of researches, find that soybean isoflavones has multiple important physiological function and trophism, and become gradually a main direction of research, particularly to prevention and the restraining effect of many illnesss.Epidemiological investigation shows that some diseases such as mammary cancer, prostatitis, colitis, heart trouble and osteoporosis etc. are higher than oriental countries far away at Hesperian sickness rate.Scientist thinks after this phenomenon has been carried out analyzing: the diet difference in east, west is to cause the major cause of this disease incidence.The Aisa people is edible bean product in a large number, and its consumption per head is Hesperian 20-40 times, and find to contain a large amount of isoflavone like substance in Aisa people's food.Isoflavone like substance in Asia human blood or the urine and the level of meta-bolites thereof all are more than 20 times of westerner.After having done large quantities of control experiments, they propose: the isoflavone like substance in the diet has the effect of anti-cancer and the diseases such as treatment mammary cancer, prostatitis, colorectal carcinoma, cardiovascular diseases and osteoporosis.Confirm that isoflavone like substance is the reason that causes this species diversity.
The research of relevant Extraction Technique of Soybean Isoflavones, method such as US Patent No. 5679806 disclosed isolation and purification soybean isoflavones, the method is take soy molasses or defatted soyflour as raw material, obtain respectively purer isoflavone glycoside and soybean isoflavone aglycones through three-step reaction, do not obtain the higher total soybean isoflavones of purity.And this technique has the following disadvantages:
1. carry out lixiviate with methyl alcohol, soyabean waste dreg can not be done feed or his usefulness again after the lixiviate.
2. make eluent with methyl alcohol during column chromatography, residual methanol is harmful.
3. carry out column chromatography with polymethylmethacrylate, price is higher, is not easy to suitability for industrialized production.
Publication number is that the Chinese patent of CN 1174839A discloses the method that reclaims isoflavones from soy molasses.The method main purpose is to obtain soybean isoflavone aglycones, but this process using centrifugation concentrates the enzymic hydrolysis that usefulness is expensive, the difficult realization of industrialization.Publication number is that the Chinese patent of CN 1211573 discloses a kind of method of extracting soybean isoflavones, the method is take soyflour as raw material, slough fat with normal hexane, obtain isoflavone glycoside and soybean isoflavone aglycones through two one-step hydrolysis again, main purpose is to obtain different glucosides and glucoside unit, but the soybean isoflavones purity that the method obtains remains further to be improved.
Summary of the invention:
The present invention relates generally to and extract the also method of purifying isoflavones from soybean, and main purpose is to obtain the soybean isoflavones product consistent with crude soy composition, can meet the requirement that national Chinese medicine is produced, and then develop tcm product.
Method of the present invention may further comprise the steps:
(1) raw material is prepared: getting dry soyflour, to be broken into particle diameter be 40-80 orders;
(2) alcohol extracting: the ratio refluxing extraction 2 ~ 4h in per 2000 ~ 4000mL, 50 ~ 80% ethanol adding 10kg raw material, cross 80 ~ 500 order aperture sieve, get filtrate, above-mentioned extraction repeats 2 ~ 5 times;
(3) merging filtrate is concentrated into alcohol concn≤2.5% concentrated solution;
(4) extraction: concentrated solution is extracted 2 ~ 4h in the ethyl acetate of 2 ~ 5 times of concentrated volumes, cross 80 ~ 500 order aperture sieve, above-mentioned extraction repeats 2 ~ 5 times;
(5) the gained extraction liquid is concentrated into ethyl acetate concentration≤2.5%;
(6) silica gel column chromatography: measure acetic acid ethyl acetate extract through silica gel column chromatography by 8 ~ 12 times of silica gel cylinder volumes; Described silica gel particle diameter 100-300 order;
(7) wash-out: be 10 ~ 50% sherwood oil-acetone mixed solution wash-out with acetone content;
(8) collect elutriant, through ODS high performance liquid phase preparative column, resulting moving phase is removed sherwood oil and acetone namely obtain isoflavonoid in the soybean.
(9) extraction yield of drying, calculating soybean isoflavones.
Wherein the silica gel particle diameter in the above-mentioned steps (6) is preferably the 100-200 order.
Wherein in the above-mentioned steps (8), described preparative column diameter and length are respectively: diameter is that 1.5cm length is 70cm
Wherein the ODS in the above-mentioned steps (8) is the octadecylsilane chemically bonded silica post, generally directly writes ODS.
Beneficial effect of the present invention is:Extracting method of the present invention is simple, equipment requirements is low, after alcohol extracting, extract and through silica gel column chromatography, can make the extraction yield of soybean isoflavones surpass 50%, provide a kind of new way for from soybean, extracting isoflavone compounds, significant for taking full advantage of plant resources and developing high natural drug.
Embodiment
Below in conjunction with specific embodiment technical scheme of the present invention is described in detail.
Embodiment 1
The soybean particle of getting dry pulverizing is raw material, ethanol by every 3000mL60% adds 10kg raw material refluxing extraction 2.0h, cross 80 purpose aperture sieve, get filtered liquid, the proportion of ethanol that trapped substance is pressed 10kg/2500mL60% again repeats refluxing extraction 2 times, merging filtrate, concentrating under reduced pressure and dealcoholysis to alcohol concn 2% gets concentrated solution, concentrated solution is extracted 2h in the ethyl acetate of 2 times of concentrated volumes, cross 140 mesh sieves, trapped substance repeats 2 extractions, the gained extraction liquid is concentrated into ethyl acetate concentration 2%, measuring acetic acid ethyl acetate extract through 100 order silica gel column chromatographies by 8 times of silica gel cylinder volumes, is 15% sherwood oil-acetone mixed solution wash-out with acetone content, collects elutriant, through anti-phase ODS high performance liquid phase preparative column, stationary phase is silica gel, and granularity is 325 orders, resulting moving phase is removed sherwood oil and acetone namely obtain isoflavonoid in the soybean.Extraction yield dry, that calculate soybean isoflavones is 55%, and it extracts quality is 13.75g.
Embodiment 2
The soybean particle of getting dry pulverizing is raw material, ethanol by every 3000mL65% adds 10kg raw material refluxing extraction 2.5h, cross 80 purpose aperture sieve, get filtered liquid, the proportion of ethanol that trapped substance is pressed 10kg/2500mL65% again repeats refluxing extraction 2 times, merging filtrate, concentrating under reduced pressure and dealcoholysis to alcohol concn 2% gets concentrated solution, concentrated solution is extracted 2.5h in the ethyl acetate of 2 times of concentrated volumes, cross 500 mesh sieves, trapped substance repeats 2 extractions, the gained extraction liquid is concentrated into ethyl acetate concentration 2%, measuring acetic acid ethyl acetate extract through 120 order silica gel column chromatographies by 10 times of silica gel cylinder volumes, is 30% sherwood oil-acetone mixed solution wash-out with acetone content, collects elutriant, through anti-phase ODS high performance liquid phase preparative column, stationary phase is silica gel, and granularity is 500 orders, resulting moving phase is removed sherwood oil and acetone namely obtain isoflavonoid in the soybean.Extraction yield dry, that calculate soybean isoflavones is 63%, and it extracts quality is 15.75g.
Embodiment 3
The soybean particle of getting dry pulverizing is raw material, ethanol by every 3000mL70% adds 10kg raw material refluxing extraction 2.5h, cross 80 purpose aperture sieve, get filtered liquid, the proportion of ethanol that trapped substance is pressed 10kg/2500mL70% again repeats refluxing extraction 3 times, merging filtrate, concentrating under reduced pressure and dealcoholysis to alcohol concn 2% gets concentrated solution, concentrated solution is extracted 3h in the ethyl acetate of 2.5 times of concentrated volumes, cross 140 mesh sieves, trapped substance repeats 2 extractions, the gained extraction liquid is concentrated into ethyl acetate concentration 2%, measuring acetic acid ethyl acetate extract through 120 order silica gel column chromatographies by 10 times of silica gel cylinder volumes, is 50% sherwood oil-acetone mixed solution wash-out with acetone content, collects elutriant, through anti-phase ODS high performance liquid phase preparative column, stationary phase is silica gel, and granularity is 800 orders, resulting moving phase is removed sherwood oil and acetone namely obtain isoflavonoid in the soybean.Extraction yield dry, that calculate soybean isoflavones is 75%, and it extracts quality is 15.75g.
Embodiment 4
The soybean particle of getting dry pulverizing is raw material, ethanol by every 3000mL75% adds 10kg raw material refluxing extraction 3.0h, cross 100 purpose aperture sieve, get filtered liquid, the proportion of ethanol that trapped substance is pressed 10kg/2500mL75% again repeats refluxing extraction 3 times, merging filtrate, concentrating under reduced pressure and dealcoholysis to alcohol concn 2% gets concentrated solution, concentrated solution is extracted 3.5h in the ethyl acetate of 2.5 times of concentrated volumes, cross 140 mesh sieves, trapped substance repeats 2 extractions, the gained extraction liquid is concentrated into ethyl acetate concentration 2%, measuring acetic acid ethyl acetate extract through 120 order silica gel column chromatographies by 10 times of silica gel cylinder volumes, is 50% sherwood oil-acetone mixed solution wash-out with acetone content, collects elutriant, through anti-phase ODS high performance liquid phase preparative column, stationary phase is silica gel, and granularity is 800 orders, resulting moving phase is removed sherwood oil and acetone namely obtain isoflavonoid in the soybean.Extraction yield dry, that calculate soybean isoflavones is 90%, and it extracts quality is 22.50g.
Embodiment 5
The soybean particle of getting dry pulverizing is raw material, ethanol by every 3000mL80% adds 10kg raw material refluxing extraction 3.0h, cross 200 purpose aperture sieve, get filtered liquid, the proportion of ethanol that trapped substance is pressed 10kg/2500mL80% again repeats refluxing extraction 3 times, merging filtrate, concentrating under reduced pressure and dealcoholysis to alcohol concn 2% gets concentrated solution, concentrated solution is extracted 4h in the ethyl acetate of 2.5 times of concentrated volumes, cross 200 mesh sieves, trapped substance repeats 2 extractions, the gained extraction liquid is concentrated into ethyl acetate concentration 2%, measuring acetic acid ethyl acetate extract through 120 order silica gel column chromatographies by 8 times of silica gel cylinder volumes, is 25% sherwood oil-acetone mixed solution wash-out with acetone content, collects elutriant, through anti-phase ODS high performance liquid phase preparative column, stationary phase is silica gel, and granularity is 200 orders, resulting moving phase is removed sherwood oil and acetone namely obtain isoflavonoid in the soybean.Extraction yield dry, that calculate soybean isoflavones is 89%, and it extracts quality is 22.25g.
?
Claims (3)
1. the extraction and purification method of isoflavone from soybean is characterized in that: may further comprise the steps:
(1) raw material is prepared: getting dry soyflour, to be broken into particle diameter be 40-80 orders;
(2) alcohol extracting: the ratio refluxing extraction 2 ~ 4h in per 2000 ~ 4000mL, 50 ~ 80% ethanol adding 10kg raw material, cross 80 ~ 500 order aperture sieve, get filtrate, above-mentioned extraction repeats 2 ~ 5 times;
(3 merging filtrates are concentrated into alcohol concn≤2.5% concentrated solution;
(4) extraction: concentrated solution is extracted 2 ~ 4h in the ethyl acetate of 2 ~ 5 times of concentrated volumes, cross 80 ~ 500 order aperture sieve, above-mentioned extraction repeats 2 ~ 5 times;
(5) the gained extraction liquid is concentrated into ethyl acetate concentration≤2.5%;
(6) silica gel column chromatography: measure acetic acid ethyl acetate extract through silica gel column chromatography by 8 ~ 12 times of silica gel cylinder volumes; Described silica gel particle diameter 100-300 order;
(7) wash-out: be 10 ~ 50% sherwood oil-acetone mixed solution wash-out with acetone content;
(8) collect elutriant, through ODS high performance liquid phase preparative column, resulting moving phase is removed sherwood oil and acetone namely obtain isoflavonoid in the soybean;
(9) extraction yield of drying, calculating soybean isoflavones.
2. the extraction and purification method of isoflavone from soybean as claimed in claim 1, it is characterized in that: the silica gel particle diameter in the described step (6) is preferably the 100-200 order.
3. the extraction and purification method of isoflavone from soybean as claimed in claim 1, it is characterized in that: the ODS in the described step (8) is the octadecylsilane chemically bonded silica post.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105585546A (en) * | 2015-12-29 | 2016-05-18 | 成都华罗生物科技有限公司 | Soy isoflavone refining method |
| CN108503675A (en) * | 2018-07-11 | 2018-09-07 | 黑龙江八农垦大学 | The method for extraction and purification and application thereof of Soybean Leaves glycitin |
| CN110200174A (en) * | 2019-07-12 | 2019-09-06 | 山东农业大学 | A kind of production method of artificial bee bread pollen spleen |
| CN114018666A (en) * | 2021-11-04 | 2022-02-08 | 山东东宇生态科技有限公司 | Detection method of soybean isoflavone |
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| WO1998049153A1 (en) * | 1997-04-28 | 1998-11-05 | Novogen Inc. | Preparation of isoflavones from legumes |
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| CN1284503A (en) * | 2000-08-04 | 2001-02-21 | 清华大学 | Method of separating and refining soybean isoflavone from soybean dregs |
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| WO1998049153A1 (en) * | 1997-04-28 | 1998-11-05 | Novogen Inc. | Preparation of isoflavones from legumes |
| WO1999006057A1 (en) * | 1997-07-30 | 1999-02-11 | Indena S.P.A. | Soya extract, process for its preparation and pharmaceutical composition |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105585546A (en) * | 2015-12-29 | 2016-05-18 | 成都华罗生物科技有限公司 | Soy isoflavone refining method |
| CN108503675A (en) * | 2018-07-11 | 2018-09-07 | 黑龙江八农垦大学 | The method for extraction and purification and application thereof of Soybean Leaves glycitin |
| CN110200174A (en) * | 2019-07-12 | 2019-09-06 | 山东农业大学 | A kind of production method of artificial bee bread pollen spleen |
| CN114018666A (en) * | 2021-11-04 | 2022-02-08 | 山东东宇生态科技有限公司 | Detection method of soybean isoflavone |
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Application publication date: 20130320 |