CN1769888A - Soybean saponins detection method - Google Patents
Soybean saponins detection method Download PDFInfo
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- CN1769888A CN1769888A CN 200410009732 CN200410009732A CN1769888A CN 1769888 A CN1769888 A CN 1769888A CN 200410009732 CN200410009732 CN 200410009732 CN 200410009732 A CN200410009732 A CN 200410009732A CN 1769888 A CN1769888 A CN 1769888A
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- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 63
- 244000068988 Glycine max Species 0.000 title claims abstract description 62
- 238000001514 detection method Methods 0.000 title claims description 26
- 229930182490 saponin Natural products 0.000 title claims description 22
- 150000007949 saponins Chemical class 0.000 title claims description 22
- 235000017709 saponins Nutrition 0.000 title claims description 22
- 238000000034 method Methods 0.000 claims abstract description 79
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- 235000008696 isoflavones Nutrition 0.000 claims abstract description 43
- 150000002515 isoflavone derivatives Chemical class 0.000 claims abstract description 30
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 82
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- 239000000243 solution Substances 0.000 claims description 39
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- 238000005238 degreasing Methods 0.000 claims description 33
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- 238000000605 extraction Methods 0.000 claims description 31
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
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- 239000000469 ethanolic extract Substances 0.000 claims description 12
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- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 description 3
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- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 description 3
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 description 3
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- 108010073771 Soybean Proteins Proteins 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical group Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
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- PWAOOJDMFUQOKB-WCZZMFLVSA-N ginsenoside Re Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@](C)(CCC=C(C)C)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O PWAOOJDMFUQOKB-WCZZMFLVSA-N 0.000 description 1
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- BTFQKIATRPGRBS-UHFFFAOYSA-N o-tolualdehyde Chemical compound CC1=CC=CC=C1C=O BTFQKIATRPGRBS-UHFFFAOYSA-N 0.000 description 1
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a detecting method for soybean saponine which comprises steps of: employing conventional method to measure the saponine content of soybean and relevant products, in the measure result including soybean isoflavones; then employing the known HPLC method to measure the soybean isoflavones content and subtracting the content value to get the content of soybean saponine. The inventive method has the advantages of being accurate, less error and simple.
Description
[technical field]
The present invention relates to a kind of chemical detection method, specifically, the present invention relates to a kind of detection method of soybean saponins.
[background technology]
Soyasaponins (Soybean Saponin) belongs to the acid saponin in triterpene five rings, has pungent and bitter taste.At present, the soyasaponins component of having found reaches 22 kinds more than, has 11 kinds as final basicly stable component.Wherein, 6 kinds of the A family saponins of disaccharide chain, 5 kinds of the B family saponins of monose chain
[3], be that the class that exists in the soybean has the material than strong biological activity.Soyasaponins has lipopenicillinase, antiobesity action; Anticoagulation, antithrombotic and antidiabetic effect; Has antioxidation; Has the effect of antiviral enhance immunity power etc.Therefore, the Application and Development of soyasaponins has broad prospects.The extraction of soybean biological active substance and application are comprising soyasaponins, isoflavones and soyabean oligosaccharides etc., the fashionable in recent years world.Its reason is them separately to the obvious useful effect of human body, has no side effect and raw material is easily got.The research of soyasaponins raw material and application facet is a lot, but manufacturer is less relatively, and its basic reason is that many producers can not produce, but how the soyasaponins product of producing quantitatively becomes the key factor of its development of restriction.
According to reported in literature, Frenwik and Oakenful survey that saponin content is 4.3% in the soybean, and Price surveys that soyasaponins content is 0.65% in the soybean, and Kitagawa etc. utilize TLC method and HPLC method to survey saponin content in the soybean on average about 0.3%.Such result be on the one hand since the different institutes of conditions such as soybean varieties, the place of production and cultural method extremely, but its basic reason still is the difference of its analytical approach
[1]
We measure the summation that is meant soyasaponins by soybean saponins described herein.
Soyasaponins analytical approach in the existing documents and materials is summarized as follows:
1, high pressure lipuid chromatography (HPLC) (HPLC method), this method can be analyzed 11 kinds of key components of soyasaponins.Its shortcoming is must divide secondary to measure for (1) A family and B family saponin.(2) must there be 11 kinds of standard models just now can be accurately quantitative.But still not having soyasaponins component standard sample at present both at home and abroad can buy.Though so the method is better, impracticable
[4](3) someone use ginsenoside Re standard items etc. as object of reference carry out the molecular weight weighting convert with result's (being not that 11 kinds of soyasaponins standard items are arranged).Obviously, because different soyasaponins components difference structurally
[8], its light absorption response (OD) can be not identical yet, and the ratio between different its components of different cultivars soybean and cultivation and processing conditions also has evident difference
[7], so the result also is a rough result.
2, thin-layered chromatography (TLC method), these method relative merits are roughly identical with above-mentioned HPLC method.This method can be made the result by once analyzing, but can make 7 components at most, still lacks 4 solvents, and used object of reference is generally the oleanolic acid standard items
[5]Obviously, the TLC method is not as good as the HPLC method.
3, spectrophotometric method (UV method)
The principle of the method: soyasaponins plays color reaction in vanillic aldehyde, glacial acetic acid, perchloric acid solution, is that standard items carry out colorimetric estimation with the oleanolic acid.Calculate with present known soyasaponins weight average molecular weight then.
The shortcoming of this method is conspicuous: (1) weighted mean value is 2.2.It draws like this, soyasaponins " average molecular mass according to document is 1157.5; with oleanolic acid relative molecular mass 456 contrast; the two theoretic amount is 2.54 than coefficient; consider the comparative analysis of losing the high-pressure liquid chromatography result of sample 2 in sugar and the early-stage Study in leaching process, is decided to be 2.2 than coefficient and suits so will measure "
[5]Obviously, this also is a very thick result.Because soybean varieties, the place of production and cultivation condition and process is different, the ratio of the various components of soyasaponins has very big-difference, so this amount definite than coefficient is inapplicable in many cases.(2) ultraviolet spectrophotometry is right aspect principle, but because the complicacy of soyasaponins composition, particularly contain the interfering component-isoflavones of can not ignore, the result who has determined soyasaponins that this method is surveyed is very thick, can be described as obsolete.
Mr. Li Lite asserted, " spectrophotometric method is utilized soyasaponins and vanillic aldehyde or methyl benzaldehyde generation color reaction is measured; because reaction is not unique sometimes; the compound resemble the isoflavones also can produce has colour substance, calculates soyasaponins content so this method is impractical in."
[4]We have also proved this point in practice.
We analyze certain defatted soybean ethanol extract, and wherein isoflavones, soyabean oligosaccharides and sucrose use the HPLC method to measure, and soybean protein and ash content, moisture use National Standard Method to measure its result following (over dry result):
Isoflavones 41.2%
Soyabean oligosaccharides 6.9%
Sucrose 6.5%
Protein 6.8%
Ash content 4.5%
Subtotal: 65.9%
This subtotal result should be reliably, judges all the other about 34.1% content (is 33.98% with detection method measured result of the present invention) that should be soyasaponins according to the production technology result.
Survey this sample with spectrophotometric method, its soyasaponins value is 64.3%.This value and above-mentioned other composition summation of surveying are 130.2%, and obviously this result is because of serious higher and unavailable.
The pre-treating method that it is worth mentioning this detection is to use " the soyasaponins extract that normal butyl alcohol redistribution procedure purifying makes, yellow powder "
[6]Analyze.This pre-treating method does not lose the soyasaponins solvent, should be best pre-treating method.
According to corresponding data
[5]The TLC spectrum data that provides increases the sample of macroporous resin treatment step in the pre-treatment process, obviously lack 3 components, can not individual at last good method so increase the method that macroreticular resin carries out pre-treatment.
4, gravimetric method (Weight), according to specialty book data such as " Chinese herbal medicine effective ingredients extract and separate " aspect: " saponin usually from aqueous solution with butanols or amylalcohol extracting; make it to separate with hydrophilic compositions such as carbohydrate and protein ", weighing after the drying thus to get saponin content (being gravimetric method)." research of genseng " (Wang Benxiang) in content of ginsenoside the report of operating weight method is promptly arranged
[6]
Facts have proved, use water saturated butanols or amylalcohol to make with extra care saponin,, therefore also be mixed among the saponin, and its content can not be ignored, so traditional gravimetric method is not suitable for the detection of soyasaponins content because isoflavone glycoside also belongs to glucoside.Because macroreticular resin is used in pre-treatment, easily loses component, so also be not rigorous using common gravimetric determination soyasaponins thereafter.Do not measure the report of soyasaponins so also there is the operating weight method at present.
[summary of the invention]
The purpose of this invention is to provide a kind of high pressure lipuid chromatography (HPLC) (HPLC) and thin-layer chromatography (TLC) of both having got around measures soyasaponins and can buy unpractical shortcoming because of domestic and international no standard items, do not adopt the ultraviolet spectrometry degree method (UV) that soyasaponins is measured that is not suitable for again, simultaneously, also avoided common gravimetric method not to be suitable for the soybean saponins detection method of the shortcoming of measuring the soyasaponins content detection.The advantage of this method is that measured result is relatively actual, accurate, error is little, and repeatable high, sample preparation is relatively simple, and does not use soyasaponins component standard items.The method is real to be " subtracting poor method ", is applicable to the mensuration of the soyasaponins total content in soybean, big dregs of beans and the extract of soybean.
The detection method of soybean saponins provided by the present invention is characterized in that, this method may further comprise the steps:
(1) sample preparation
Take by weighing over dry powder or fluid sample, weight is G1;
(2) degreasing
According to the standard method that crude fat content is measured, degreasing in fatty extractor;
(3) extract
The sample of filter paper packet dry after the above-mentioned degreasing after together with degreasing moved in another fatty extractor, add ethanol or methanol extraction then, extraction conditions is identical with above-mentioned degreasing condition;
(4) water-soluble
With above-mentioned ethanol or methanol extract liquid vacuum concentrate drying, after water dissolving and the washing, change in the separating funnel standby then;
(5) refining
Add in the above-mentioned standby separating funnel standing demix after the shaking out with the butanols of 1: 1 volume or amyl alcohol solution, take off butanols or the amyl alcohol solution that layer water layer add 1: 1 volume more then and extract 3-4 time repeatedly, get whole upper layer of extraction liquid;
(6) above-mentioned extract being placed weight is that the vacuum drying of G2 is circled round and concentrated bottle and be concentrated into driedly, and the cooling of dry back is weighed in baking oven then, and the weight of gained evaporate to dryness thing is G3;
(7) fully dissolve above-mentioned evaporate to dryness thing with ethanol or methanol solution, carry out centrifugal treating after, it is standby to get supernatant;
(8) get above-mentioned standby supernatant, inject high pressure liquid chromatograph, measure isoflavone content in the evaporate to dryness thing, the weight that gets isoflavones among the G3 is G4;
(9) calculate: concrete calculating formula is as follows:
Wherein said absolute dried sample is soybean and dregs of rice sample or soybean germ and dregs of rice sample thereof.
The present invention also provides a kind of preferred detection method, it is characterized in that, this method may further comprise the steps:
(1) sample preparation
Take by weighing over dry powder or fluid sample, weight is G1.Wherein, (a) soybean and dregs of rice samples weighing are: 10-20g; (b) soybean germ and dregs of rice samples weighing thereof are: 1-2g; (c) soyasaponins, isoflavones and soyabean oligosaccharides powder are weighed as: 0.1-0.2g; (d) soyasaponins, isoflavones and soyabean oligosaccharides powder reprocessing goods are weighed as: 0.3-0.5g; (e) aqueous solution of extract of soybean is weighed as: 1-3g (mL) is standby;
(2) degreasing
With the standard method that above-mentioned standby pulverized specimen is measured according to crude fat content, degreasing in fatty extractor;
(3) extract
The sample of filter paper packet dry after the above-mentioned degreasing after together with degreasing moved in another fatty extractor, add ethanol or the methanol extraction of 70-95% then, extraction conditions is identical with above-mentioned degreasing condition, and it is standby to get extract;
(4) water-soluble
With above-mentioned standby ethanol or methanol extract liquid vacuum concentrate drying, after substep fully dissolves also wash-bottle with total amount 200mL water then, change in the separating funnel standby;
(5) refining
Add shake well extraction back standing demix in the above-mentioned standby separating funnel with butanols or amyl alcohol solution 200mL, take off then that a layer 200mL water layer adds the 200mL butanols again or amyl alcohol solution extracts 3-4 time repeatedly, whole upper layer of extraction liquid are merged, and 600-800mL is standby altogether;
(6) above-mentioned 600-800mL extract being placed weight is that the dry vacuum of G2 is circled round and concentrated bottle and be concentrated into driedly, and dry 2h in 105 ℃ of baking ovens weighs after the cooling then, and the weight of gained evaporate to dryness thing is G3;
(7) fully dissolve above-mentioned evaporate to dryness thing with 80% ethanol/methanol solution 50mL, the supernatant of getting after the 1mL centrifugal treating is standby;
(8) get above-mentioned standby supernatant 10-20 μ L, inject high pressure liquid chromatograph, measure isoflavone content among the evaporate to dryness thing G3, draw the isoflavones weight G4 among the G3.
(9) calculate: concrete calculating formula is as follows:
Be G0 wherein as if isoflavone content in known certain product, the isoflavones weight G4=G1 * G0 among then measured soyasaponins and the isoflavones general assembly (TW) G3.
For the known respective sample that does not contain crude fat, can omit defatting step, directly enter extraction step.
This detection method also is applicable to the fluid product of corresponding extract of soybean, gets the certain quantity of fluid sample and directly enter water-soluble step or will detect by the present invention behind this sample evaporate to dryness in testing process.
The methyl alcohol of indication or ethanol content are concentration of volume percent in this method.
Described butanols or amyl alcohol solution are with water saturated butanols or amyl alcohol solution.
Described degreasing method is meant the remaining method in the respective standard method that crude fat content measures.
High-pressure liquid phase chromatograph measuring isoflavones general conditions is: (1) high pressure liquid chromatograph, attached column incubator and UV-detector; (2) chromatographic column: C18 post, Φ 4.6 * 250mm; (3) column temperature: 40 ℃; (4) flowing: 0.1% glacial acetic acid aqueous solution: second is fine=and 85: 15; (5) flow: 1.5mL/min; (6) detect wavelength: 254nm.
The real a kind of new method that combines with the HPLC method for gravimetric method of " a kind of soybean saponins detection method " that the present invention proposes.Traditional and present for containing isoflavone glycoside in the used gravimetric method measured result of people.Because in butanols or the amylalcohol extraction process, glucoside is blanket wherein comprising soyasaponins and the isoflavone glycoside of can not ignore, so gravimetric method is not used for the detection of soyasaponins for people.The detection of the various compositions of isoflavones uses the HPLC method very ripe at present.So using traditional gravimetric determination to go out " soyasaponins " content, real is the summation of soyasaponins and isoflavone glycoside.Determine the content of the isoflavone glycoside in this " summation " with the HPLC method, obviously, availablely subtract the content that poor method is tried to achieve soyasaponins.What be worth explanation a bit is: correct gravimetric method is most basic detection method.
Pre-treating method of the present invention only uses the refining extraction of butanols or amylalcohol, does not use macroreticular resin to purify, to reduce the disappearance of some component of saponin.
The advantage of this detection method is that measured result is relatively actual, accurate, error is little, repeatability is high, and sample preparation is relatively simple, and does not use soyasaponins component standard items.The method is applicable to the mensuration of the soyasaponins total content in soybean, big dregs of beans, soybean ethanol extract and the goods thereof.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment 1:
Soyasaponins Determination on content in certain kind soya seeds.
(1) in Soxhlet extractor, carries out degreasing according to crude fat content determination method (remaining method GB2906-82).Wherein take by weighing and pulverize the dry soy meal 15.213g (G1) in back, be distributed into 15 filter paper packet as specimen;
(2) 15 filter paper packet (having removed the ether organic solvent) after the above-mentioned degreasing are put in another Soxhlet extractor, add 80% ethanol after, carry out extracting according to the degreasing condition.After extracting finishes, extract is concentrated into dried, with total amount 200mL moisture step fully after dissolving and the wash bottle, changes in the separating funnel standby then;
(3) will add shake well extraction back standing demix in the above-mentioned standby separating funnel with water saturated butanol solution 200mL, the 200mL water layer that takes off layer then is standby, and it is standby to collect upper strata (normal butyl alcohol phase) 200mL simultaneously;
(4) use water saturated butanol solution 200mL respectively according to the above-mentioned steps method, continue to abandon it behind 2 above-mentioned standby lower aqueous solutions of extraction, it is standby to merge three collected normal butyl alcohol phase 600mL then;
(5) vacuum is circled round after the eggplant-shape bottle of concentrator places 105 ℃ of dry 2h of baking box, place the vacuum dryer cooling after, it is standby to be weighed as 133.258g (G2);
(6) (4) step is collected standby normal butyl alcohol phase 600mL moves in the eggplant-shape bottle of above-mentioned drying for standby, and is concentrated into dried in vacuum is circled round concentrator.After then this eggplant-shape bottle being placed 105 ℃ of dry 2h of baking oven together with the evaporate to dryness material, put in the vacuum dryer cooling into after, it is standby to be weighed as 133.334g (G3);
(7) 50mL 80% ethanolic solution is poured in the above-mentioned standby eggplant-shape bottle into fully dissolving after, it is centrifugal under 10000rpm, 10min condition to get 1mL, it is standby to get supernatant;
(8) get above-mentioned standby supernatant 20 μ L and inject high pressure liquid chromatograph, measure that isoflavones weight is 0.023g (G4) in this sample;
(9) result calculates:
＝0.35(％)
(10) result describes
Contain soyasaponins 0.35g in the 100g over dry soybean.
Embodiment 2:
Certain contains soybean saponins Determination on content in the 65% soybean germ degreasing dregs of rice raw material.
(1) in the Soxhlet extracting, carries out degreasing according to crude fat content determination method (remaining method GB2906-82).Wherein take by weighing and pulverize the dry soybean germ powder 2.011g (G1) in back, be distributed into 4 filter paper packet as specimen;
(2) 4 filter paper packet (having removed the ether organic solvent) after the above-mentioned degreasing are put in another Soxhlet extractor, add 90% ethanol after, carry out extracting according to the degreasing condition.After extracting finishes, extract is concentrated into dried, with total amount 200mL moisture step fully after dissolving and the wash bottle, changes in the separating funnel standby then;
(3) will add shake well extraction back standing demix in the above-mentioned standby separating funnel with water saturated butanol solution 200mL, the 200mL water layer that takes off layer then is standby, and it is standby to collect upper strata (normal butyl alcohol phase) 200mL simultaneously;
(4) according to the above-mentioned steps method with water saturated butanol solution 200mL, continue to abandon it behind 3 standby lower aqueous solutions of extraction, it is standby to merge four collected normal butyl alcohol phase 800mL then;
(5) vacuum is circled round after the eggplant-shape bottle of concentrator places 105 ℃ of dry 2h of baking box, place the vacuum dryer cooling after, it is standby to be weighed as 128.356g (G2);
(6) (4) step is collected standby normal butyl alcohol phase 800mL moves in the eggplant-shape bottle of above-mentioned drying for standby, and is concentrated into dried in vacuum is circled round concentrator.After then this eggplant-shape bottle being placed 105 ℃ of dry 2h of baking oven together with the evaporate to dryness material, put in the vacuum dryer cooling into after, it is standby to be weighed as 128.452mg (G3);
(7) 50mL 80% ethanolic solution is poured in the above-mentioned standby eggplant-shape bottle into fully dissolving after, it is centrifugal under 10000rpm, 10min condition to get 1mL, it is standby to get supernatant;
(8) get above-mentioned standby supernatant 20 μ L and inject high pressure liquid chromatograph, the mensuration isoflavone content is 0.021g (G4);
(9) result calculates:
＝3.73(％)
(10) result describes
Contain the 3.73g soyasaponins in the 100g over dry soybean germ degreasing dregs of rice.
Embodiment 3:
Soyasaponins Determination on content in the soyasaponins sheet.
(1) get the even sample 3-5g that pulverized 40 above sieve meshes and be put in the glass dish and shakeout, put in 105 ℃ of baking ovens behind the 1h, it is standby to move in the vacuum dryer cooling back;
(2) it is standby to take by weighing the standby sample 0.305g (G1) in above-mentioned dry back;
(3) above-mentioned standby sample is wrapped with filter paper after, in the apparatus,Soxhlet's of packing into, the alcohol extract with 90%, its condition is identical with Soxhlet method degreasing condition.It is standby to get ethanol extract;
(4) get above-mentioned standby ethanol extract, evaporate to dryness in the vacuum concentration evaporator with after abundant dissolving of total amount 200mL moisture step and the wash bottle, changes in the separating funnel standby then;
(5) will add shake well extraction back standing demix in the above-mentioned standby separating funnel with water saturated butanol solution 200mL, the 200mL water layer that takes off layer then is standby, and it is standby to collect upper strata (normal butyl alcohol phase) 200mL simultaneously;
(6) continue to abandon it behind 3 above-mentioned standby lower aqueous solutions of extraction with water saturated butanol solution 200mL respectively according to the above-mentioned steps method, it is standby to merge four collected normal butyl alcohol phase 800mL then;
(7) vacuum is circled round after the eggplant-shape bottle of concentrator places 105 ℃ of dry 2h of baking box, place the vacuum dryer cooling after, it is standby to be weighed as 131.326g (G2);
(8) (6) step is collected standby normal butyl alcohol phase 800mL moves in the eggplant-shape bottle of above-mentioned drying for standby, and places the vacuum concentrator that circles round to be concentrated into dried.After then this eggplant-shape bottle being placed 105 ℃ of dry 2h of baking oven together with the evaporate to dryness material, put in the vacuum dryer cooling into after, it is standby to be weighed as 131.427g (G3);
(9) 50mL 80% ethanolic solution is poured in the above-mentioned standby eggplant-shape bottle into fully dissolving after, it is centrifugal under 10000rpm, 10min condition to get 1mL, it is standby to get supernatant;
(10) get above-mentioned standby supernatant 20 μ L and inject high pressure liquid chromatograph, the mensuration isoflavone content is 0.014g (G4);
(11) result calculates:
＝28.52(％)
(12) result describes
Contain soyasaponins 28.52g in the 100g over dry soyasaponins sheet.
Embodiment 4:
Soyasaponins Determination on content in the isoflavones sheet.
(1) get the even sample 3-5g that pulverized 40 above sieve meshes and be put in the glass dish and shakeout, put in 105 ℃ of baking ovens behind the 1h, it is standby to move in the vacuum dryer cooling back;
(2) it is standby to take by weighing the standby sample 0.452g (G1) in above-mentioned dry back;
(3) above-mentioned standby sample is wrapped with filter paper after, in the apparatus,Soxhlet's of packing into, the alcohol extract with 80%, its condition is identical with Soxhlet method degreasing condition.It is standby to get ethanol extract;
(4) get above-mentioned standby ethanol extract, evaporate to dryness in the vacuum concentration evaporator with after abundant dissolving of total amount 200mL moisture step and the wash bottle, changes in the separating funnel standby then;
(5) will add shake well extraction back standing demix in the above-mentioned standby separating funnel with water saturated butanol solution 200mL, the 200mL water layer that takes off layer then is standby, and it is standby to collect upper strata (normal butyl alcohol phase) 200mL simultaneously;
(6) continue to abandon it behind 2 above-mentioned standby lower aqueous solutions of extraction with water saturated butanol solution 200mL respectively according to the above-mentioned steps method, it is standby to merge three collected normal butyl alcohol phase 600mL then;
(7) vacuum is circled round after the eggplant-shape bottle of concentrator places 105 ℃ of dry 2h of baking box, place the vacuum dryer cooling after, it is standby to be weighed as 133.258g (G2);
(8) (6) step is collected standby normal butyl alcohol phase 600mL moves in the eggplant-shape bottle of above-mentioned drying for standby, and places the vacuum concentrator that circles round to be concentrated into dried.After then this eggplant-shape bottle being placed 105 ℃ of dry 2h of baking oven together with the evaporate to dryness material, put in the vacuum dryer cooling into after, it is standby to be weighed as 133.306g (G3);
(9) to show accurate isoflavone content be 12.1% (G0) to isoflavones sheet inspection report, claims sample 0.103g (G1), so isoflavones weight is 0.012g (G4) in the sample;
(10) result calculates:
＝133.306-133.258-0.012×100
0.452
＝7.96(％)
(11) result describes
Contain soyasaponins 7.96g in the 100g over dry isoflavones sheet.
Embodiment 5:
Soyasaponins Determination on content in the soyabean oligosaccharides sheet.
(1) get the even sample 3-5g that pulverized 40 above sieve meshes and be put in the glass dish and shakeout, put in 105 ℃ of baking ovens behind the 1h, it is standby to move in the vacuum dryer cooling back;
(2) it is standby to take by weighing the standby sample 0.502g (G1) in above-mentioned dry back;
(3) above-mentioned standby sample is wrapped with filter paper after, in the apparatus,Soxhlet's of packing into, the alcohol extract with 70%, its condition is identical with Soxhlet method degreasing condition.It is standby to get ethanol extract;
(4) get above-mentioned standby ethanol extract, evaporate to dryness in the vacuum concentration evaporator with after abundant dissolving of total amount 200mL moisture step and the wash bottle, changes in the separating funnel standby then;
(5) will add shake well extraction back standing demix in the above-mentioned standby separating funnel with water saturated butanol solution 200mL, the 200mL water layer that takes off layer then is standby, and it is standby to collect upper strata (normal butyl alcohol phase) 200mL simultaneously;
(6) continue to abandon it behind 2 above-mentioned standby lower aqueous solutions of extraction with water saturated butanol solution 200mL respectively according to the above-mentioned steps method, it is standby to merge three collected normal butyl alcohol phase 600mL then;
(7) vacuum is circled round after the eggplant-shape bottle of concentrator places 105 ℃ of dry 2h of baking box, place the vacuum dryer cooling after, it is standby to be weighed as 128.356g (G2);
(8) (6) step is collected standby normal butyl alcohol phase 800mL moves in the eggplant-shape bottle of above-mentioned drying for standby, and places the vacuum concentrator that circles round to be concentrated into dried.After then this eggplant-shape bottle being placed 105 ℃ of dry 2h of baking oven together with the evaporate to dryness material, put in the vacuum dryer cooling into after, it is standby to be weighed as 128.451g (G3);
(9) 50mL 80% ethanolic solution is poured in the above-mentioned standby eggplant-shape bottle into fully dissolving after, it is centrifugal under 10000rpm, 10min condition to get 1mL, it is standby to get supernatant;
(10) get above-mentioned standby supernatant 20 μ L and inject high pressure liquid chromatograph, the mensuration isoflavone content is 0.020g (G4);
(11) result calculates:
＝14.94(％)
(12) result describes
Contain soyasaponins 14.94g in the 100g over dry soyabean oligosaccharides sheet.
Embodiment 6:
Soyasaponins Determination on content in the soyasaponins powder.
(1) get even sample 3-5g and be put in the glass dish and shakeout, put in 105 ℃ of baking ovens behind the 1h, it is standby to move in the vacuum dryer cooling back;
(2) take by weighing the above-mentioned dry standby sample 0.102g (G1) in back, after abundant dissolving of total amount 200mL moisture step and wash bottle, change in the separating funnel standby;
(3) will add shake well extraction back standing demix in the above-mentioned standby separating funnel with water saturated butanol solution 200mL, the 200mL water layer that takes off layer then is standby, and it is standby to collect upper strata (normal butyl alcohol phase) 200mL simultaneously;
(4) continue to abandon it behind 3 above-mentioned standby lower aqueous solutions of extraction with water saturated butanol solution 200mL respectively according to the above-mentioned steps method, it is standby to merge four collected normal butyl alcohol phase 800mL then;
(5) vacuum is circled round after the eggplant-shape bottle of concentrator places 105 ℃ of dry 2h of baking box, place the vacuum dryer cooling after, it is standby to be weighed as 132.224g (G2);
(6) (4) step is collected standby normal butyl alcohol phase 800mL moves in the eggplant-shape bottle of above-mentioned drying for standby, and places the vacuum concentrator that circles round to be concentrated into dried.After then this eggplant-shape bottle being placed 105 ℃ of dry 2h of baking oven together with the evaporate to dryness material, put in the vacuum dryer cooling into after, it is standby to be weighed as 132.305g (G3);
(7) 50mL 80% ethanolic solution is poured in the above-mentioned standby eggplant-shape bottle into fully dissolving after, it is centrifugal under 10000rpm, 10min condition to get 1mL, it is standby to get supernatant;
(8) get above-mentioned standby supernatant 20 μ L and inject high pressure liquid chromatograph, the mensuration isoflavone content is 0.008g (G4);
(9) result calculates:
＝71.57(％)
(10) result describes
Contain soyasaponins 71.57g in the 100g over dry soyasaponins powder.
Embodiment 7:
Soyasaponins Determination on content in the 40% isoflavones powder.
(1) get even sample 3-5g and be put in the glass dish and shakeout, put in 105 ℃ of baking ovens behind the 1h, it is standby to move in the vacuum dryer cooling back;
(2) take by weighing the above-mentioned dry standby sample 0.103g (G1) in back, after abundant dissolving of total amount 200mL moisture step and wash bottle, change in the separating funnel standby;
(3) will add shake well extraction back standing demix in the above-mentioned standby separating funnel with water saturated butanol solution 200mL, the 200mL water layer that takes off layer then is standby, and it is standby to collect upper strata (normal butyl alcohol phase) 200mL simultaneously;
(4) continue to abandon it behind 2 above-mentioned standby lower aqueous solutions of extraction with water saturated butanol solution 200mL respectively according to the above-mentioned steps method, it is standby to merge three collected normal butyl alcohol phase 600mL then;
(5) vacuum is circled round after the eggplant-shape bottle of concentrator places 105 ℃ of dry 2h of baking box, place the vacuum dryer cooling after, it is standby to be weighed as 131.326g (G2);
(6) (4) step is collected standby normal butyl alcohol phase 600mL moves in the eggplant-shape bottle of above-mentioned drying for standby, and places the vacuum concentrator that circles round to be concentrated into dried.After then this eggplant-shape bottle being placed 105 ℃ of dry 2h of baking oven together with the evaporate to dryness material, put in the vacuum dryer cooling into after, it is standby to be weighed as 131.403g (G3);
It is 41.2% (G0) that (7) 40% isoflavones powder inspection reports show accurate isoflavone content, claims sample 0.103g (G1), so isoflavones weight is 0.042g (G4) in the sample;
(8) result calculates:
＝33.98(％)
(10) result describes
Contain soyasaponins 33.98g in the 100g over dry isoflavones powder.
Embodiment 8:
Certain is soyasaponins Determination on content in the oral liquid produced of raw material with the soybean.
(1) the standby G1 of extracting sample solution 1.0g (mL);
(2) with above-mentioned standby sample solution with total amount 200mL moisture step fully after the dilution, move in the separating funnel standby;
(3) will add shake well extraction back standing demix in the above-mentioned standby separating funnel with water saturated butanol solution 200mL, the 200mL water layer that takes off layer then is standby, and it is standby to collect upper strata (normal butyl alcohol phase) 200mL simultaneously;
(4) continue to abandon it behind 2 above-mentioned standby lower aqueous solutions of extraction with water saturated butanol solution 200mL respectively according to the above-mentioned steps method, it is standby to merge three collected normal butyl alcohol phase 600mL then;
(5) vacuum is circled round after the eggplant-shape bottle of concentrator places 105 ℃ of dry 2h of baking box, place the vacuum dryer cooling after, it is standby to be weighed as 133.224g (G2);
(6) (4) step is collected standby normal butyl alcohol phase 600mL moves in the eggplant-shape bottle of above-mentioned drying for standby, and places the vacuum concentrator that circles round to be concentrated into dried.After then this eggplant-shape bottle being placed 105 ℃ of dry 2h of baking oven together with the evaporate to dryness material, put in the vacuum dryer cooling into after, it is standby to be weighed as 133.256g (G3);
(7) 50mL 80% ethanolic solution is poured in the above-mentioned standby eggplant-shape bottle into fully dissolving after, it is centrifugal under 10000rpm, 10min condition to get 1mL, it is standby to get supernatant;
(8) get above-mentioned standby supernatant 20 μ L and inject high pressure liquid chromatograph, the mensuration isoflavone content is 0.001g (G4);
(9) result calculates:
＝3.1(％)
(10) result describes
Contain soyasaponins 3.1g in the 100g oral liquid.
Reference:
[1] soybean science the 15th volume the 1st phases 1996.2 P76
" soyasaponins progress " Wang Xuezhang
[2] " exploitation of soybean biological active substance and application " chief editor Cui Hongbin P117
[3] " soyasaponins current research overview " Tang Chuanhe etc.
Soybean science the 20th volume the 1st phases 2001.2 P60
[4] top grade P324 in " functional soy food " Lee
[5] " health food check and technical manual enforcement manual " electronic publishing society of Tsing Hua Tong Fang
The assay method of P1135＜soybean saponins in 2003＞(spectrophotometric method)
[6] this auspicious P79 of " research of genseng " king
[7] the remaining method (GB2906-82) of the mensuration of crude fat
[8] Huaiyingong College journal 2004.13 (1)/80-82 such as the difference of the soyasaponins in soybean germ and the Soybean Leaves " research " Zhu Kerui
Claims (9)
1, a kind of detection method of soybean saponins is characterized in that, this method may further comprise the steps:
(1) sample preparation
Take by weighing over dry powder or fluid sample, weight is G1;
(2) degreasing
According to the standard method that crude fat content is measured, degreasing in fatty extractor;
(3) extract
The sample of filter paper packet dry after the above-mentioned degreasing after together with degreasing moved in another fatty extractor, add ethanol or methanol extraction then, extraction conditions is identical with above-mentioned degreasing condition;
(4) water-soluble
With above-mentioned ethanol or methanol extract liquid vacuum concentrate drying, after water dissolving and the washing, change in the separating funnel standby then;
(5) refining
Add in the above-mentioned standby separating funnel standing demix after the shaking out with the butanols of 1: 1 volume or amyl alcohol solution, take off butanols or the amyl alcohol solution that layer water layer add 1: 1 volume more then and extract 3-4 time repeatedly, get whole upper layer of extraction liquid;
(6) above-mentioned extract being placed weight is that the vacuum drying of G2 is circled round and concentrated bottle and be concentrated into driedly, and the cooling of dry back is weighed in baking oven then, and the weight of gained evaporate to dryness thing is G3;
(7) fully dissolve above-mentioned evaporate to dryness thing with ethanol or methanol solution, carry out centrifugal treating after, it is standby to get supernatant;
(8) get above-mentioned standby supernatant, inject high pressure liquid chromatograph, measure isoflavone content in the evaporate to dryness thing, the weight that gets isoflavones among the G3 is G4;
(9) calculate: concrete calculating formula is as follows:
2, detection method as claimed in claim 1, it is characterized in that, described sample is soybean and dregs of rice sample, soybean germ and dregs of rice sample thereof, and soyasaponins, isoflavones and soyabean oligosaccharides powder and reprocessing goods thereof, or the aqueous solution of extract of soybean.
3, detection method as claimed in claim 1 is characterized in that, this method may further comprise the steps:
(1) sample preparation
Take by weighing over dry powder or fluid sample, weight is G1.Wherein, (a) soybean and dregs of rice samples weighing are: 10-20g; (b) soybean germ and dregs of rice samples weighing thereof are: 1-2g; (c) soyasaponins, isoflavones and soyabean oligosaccharides powder are weighed as: 0.1-0.2g; (d) soyasaponins, isoflavones and soyabean oligosaccharides powder reprocessing goods are weighed as: 0.3-0.5g; (e) aqueous solution of extract of soybean is weighed as: 1-3g (mL) is standby;
(2) degreasing
With the standard method that above-mentioned standby pulverized specimen is measured according to crude fat content, degreasing in fatty extractor;
(3) extract
The sample of filter paper packet dry after the above-mentioned degreasing after together with degreasing moved in another fatty extractor, add ethanol or the methanol extraction of 70-95% then, extraction conditions is identical with above-mentioned degreasing condition, and it is standby to get extract;
(4) water-soluble
With above-mentioned standby ethanol or methanol extract liquid vacuum concentrate drying, after substep fully dissolves also wash-bottle with total amount 200mL water then, change in the separating funnel standby;
(5) refining
Add shake well extraction back standing demix in the above-mentioned standby separating funnel with butanols or amyl alcohol solution 200mL, take off then that a layer 200mL water layer adds the 200mL butanols again or amyl alcohol solution extracts 3-4 time repeatedly, whole upper layer of extraction liquid are merged, and 600-800mL is standby altogether;
(6) above-mentioned 600-800mL extract being placed weight is that the dry vacuum of G2 is circled round and concentrated bottle and be concentrated into driedly, behind the dry 2h, weighs after the cooling in 105 ℃ of baking ovens then, and the weight of gained evaporate to dryness thing is G3;
(7) fully dissolve above-mentioned evaporate to dryness thing with 80% ethanol/methanol solution 50mL, the supernatant of getting after the 1mL centrifugal treating is standby;
(8) get above-mentioned standby supernatant 10-20 μ L, inject high pressure liquid chromatograph, measure isoflavone content among the evaporate to dryness thing G3, draw the isoflavones weight G4 among the G3;
(9) calculate: concrete calculating formula is as follows:
4, as claim 1 or 3 described detection methods, it is characterized in that,, can omit defatting step, directly enter extraction step for the known respective sample that does not contain crude fat.
5, as claim 1 or 3 described detection methods, it is characterized in that, when sample is fluid product, in testing process, gets the certain quantity of fluid sample and directly enter water-soluble step or will use behind this sample evaporate to dryness.
6, as claim 1 or 3 described detection methods, it is characterized in that, if isoflavone content is G0 in known certain product, the isoflavones weight G4=G1 * G0 among then measured soyasaponins and the isoflavones general assembly (TW) G3.
As claim 1 or 3 described detection methods, it is characterized in that 7, described butanols or amyl alcohol solution are with water saturated butanols or amyl alcohol solution.
As claim 1 or 3 described detection methods, it is characterized in that 8, the methyl alcohol of indication or ethanol content are concentration of volume percent in this method.
As claim 1 or 3 described detection methods, it is characterized in that 9, described degreasing method is meant the remaining method in the respective standard method that crude fat content measures.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102721626A (en) * | 2012-06-29 | 2012-10-10 | 中国科学院宁波材料技术与工程研究所 | Method for detecting content of chemical fiber oil |
| CN103048403A (en) * | 2012-12-11 | 2013-04-17 | 浙江德清博诚生物科技有限公司 | Method for detecting lactic acid in fermented soybean meal |
| CN103323557A (en) * | 2013-06-16 | 2013-09-25 | 中国科学院成都生物研究所 | Method for analyzing isoflavone in soybeans and products thereof |
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| CN1096456C (en) * | 2000-06-08 | 2002-12-18 | 蒋宇扬 | Process for continuously extracting glycitin, olegose and saponin |
| JP4388289B2 (en) * | 2003-02-20 | 2009-12-24 | 株式会社ファンケル | Skin aging prevention / improving agent and / or rough skin prevention / improving agent kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102721626A (en) * | 2012-06-29 | 2012-10-10 | 中国科学院宁波材料技术与工程研究所 | Method for detecting content of chemical fiber oil |
| CN103048403A (en) * | 2012-12-11 | 2013-04-17 | 浙江德清博诚生物科技有限公司 | Method for detecting lactic acid in fermented soybean meal |
| CN103048403B (en) * | 2012-12-11 | 2015-04-15 | 浙江德清博诚生物科技有限公司 | Method for detecting lactic acid in fermented soybean meal |
| CN103323557A (en) * | 2013-06-16 | 2013-09-25 | 中国科学院成都生物研究所 | Method for analyzing isoflavone in soybeans and products thereof |
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