CN101216463A - Sea pumpkin saponin content determination method - Google Patents
Sea pumpkin saponin content determination method Download PDFInfo
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- CN101216463A CN101216463A CNA200810013642XA CN200810013642A CN101216463A CN 101216463 A CN101216463 A CN 101216463A CN A200810013642X A CNA200810013642X A CN A200810013642XA CN 200810013642 A CN200810013642 A CN 200810013642A CN 101216463 A CN101216463 A CN 101216463A
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Abstract
The invention discloses a method for determining holothurin content, which is characterized in that the method comprises the following steps of: hydrolyzing a sample with a trifluoroacetic acid aqueous solution, neutralizing the hydrolysate, filtering, adding water to fixed volume, adding 1-pheny-3-methy-5-pyrazolone derivative agent to carry out derivation reaction, determining the content of quinose in the derivatives by high-performance liquid chromatographic analysis, and calculating the holothurin content in the sample by multiplying the content of quinose by a conversion coefficient. The invention has the advantages of simple operation, high sensitivity, less sample consumption and good stability, and is suitable for content determination of glycosides of various sea cucumbers.
Description
Technical field
The present invention relates to a kind of assay method of bioactivator, particularly relate to a kind of selenka Determination on content method.
Background technology
Selenka is a kind of important biological material in the sea cucumber, and the selenka of having found at present has kind more than 100.Saponins compound complex structure in the sea cucumber, and difference is arranged because of sea cucumber kind and growing environment different.Saponin(e structure difference in different types of sea cucumber is with containing several in a kind of sea cucumber usually even tens kinds of structural similarities, the trickle saponin component of difference.In addition, also contain complicated ingredients such as amounts of protein, polysaccharide, fat, trace element in the sea cucumber, this brings a lot of difficulties will inevitably for the mensuration of saponin content in the sea cucumber.
Application number is the assay method that discloses a kind of momordica saponins in 200510014471.9 the patent, this method is not strong to the selectivity of target-finding thing, impurity component in the sample easily causes bigger error at measurment, usually the sample pretreatment process that needs more complicated, and column chromatography processes such as macroreticular resin, silica gel will inevitably cause the loss of sample, easily bring further error to measurement result.Application number is to disclose ginsenoside Rb 1 Determination on content method in the Chinese medicine in the patent of 200510013275.X "; this method is sensitive and accurate; but also exist the problem of complex pretreatment simultaneously; and this method be applicable to usually a kind of or a few saponin(e component quantitatively; contain tens kinds even tens kinds of saponin(e components in the sea cucumber usually; their complex structures and difference are minimum, separate also relatively difficulty with high performance liquid chromatography.In addition, selenka is big at sea cucumber differences not of the same race, can't determine that a kind of suitable saponin monomer carries out the content of various saponin(es in spectrophotometric or the liquid chromatogram measuring sea cucumber as standard items.Raising along with people's living standard, the food relevant with sea cucumber, health products and medicine become the focus and the main flow of market comsupton day by day, selenka is as main active component, its content influences very big to its edible medical value that reaches, be indispensable sea cucumber nutrient evaluation index, therefore press for a kind of simple and feasible selenka Determination on content method of setting up.
Summary of the invention
The purpose of this invention is to provide a kind of selenka Determination on content method, it as quantitative criterion, need not complicated pre-treatment process by distinctive quinoline promise sugar in the selenka, can accurately quantitative various sea cucumbers in the content of saponin(e.
A kind of selenka Determination on content method, it is characterized in that the following step is arranged: (1) takes by weighing the sea cucumber sample, sample is pulverized or be cut into small pieces, placing concentration is the trifluoroacetic acid aqueous solution of 2mol/L-2.5mol/L, at 110 ℃ of-120 ℃ of following hydrolysis 2-3 hours,, filter the hydrolyzate neutralization, constant volume is as test liquid; (2) take by weighing quinoline promise sugar, be mixed with 6-8 concentration becomes concentration gradient between 0.01mmol/L-1mmol/L standard water solution; (3) draw above-mentioned standard water solution, 1-phenyl-3-methyl-5-pyrazolones ketone derivating agent and the 0.3mol/L-0.35mol/L sodium hydrate aqueous solution of equal volume respectively, at 65-70 ℃ of following derivatization reaction 25-35min, cooling 8-10min neutralizes with aqueous hydrochloric acid solution; Use chloroform extraction, inhale and to abandon lower floor, repeats 3 times,, carry out liquid-phase chromatographic analysis, the concentration of quinoline promise sugar is carried out linear regression, obtain typical curve with the peak area of quinoline promise sugar derivatives with the water layer filtration; (4) get above-mentioned test liquid and carry out derivatization reaction and liquid-phase chromatographic analysis, obtain the content of quinoline promise sugar in the sample in the above-mentioned typical curve of peak area substitution with quinoline promise sugar derivatives; The content of quinoline promise sugar multiply by the content that transformation ratio promptly gets saponin(e in the sea cucumber sample.
The invention has the advantages that: (1) is easy and simple to handle, need not sample is extracted pre-service such as purifying, can directly be hydrolyzed.(2) highly sensitive, quinoline promise sugar derivatives has very strong absorption at 250nm, and detectability can reach 0.804 μ M.(3) sampling is few, and required sea cucumber sample only is less than 1 gram dry weight.(4) lack detection time, single mensuration is consuming time only to be about 3h; Because therefore steps such as can being hydrolyzed simultaneously, deriving also is applicable to mensuration in enormous quantities.(5) good stability, quinoline promise sugar derivatives peak area is stable in 24 hours, RSD＜3.5%.(6) this method is applicable to the mensuration of saponin content in the multiple sea cucumber.
Embodiment
Further describe the present invention below by embodiment.
Embodiment one
(1) take by weighing commercially available dried sea-cucumber A (stichopus japonicus) 500.8mg, pulverize, adding 1mL concentration is the trifluoroacetic acid aqueous solution of 2mol/L, and in 120 ℃ of hydrolysis 2 hours, the gained hydrolyzate filtered with the neutralization of 5mo l/L sodium hydrate aqueous solution, is settled to 5mL as test liquid.
(2) take by weighing quinoline promise sugar water and be made into the solution of 1mmol/L as storing solution.Further be diluted to 0.01mmol/L, 0.05mmol/L, 0.1mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L successively, make (the standard water solution of 8 concentration gradients.
(3) draw each 250 μ L of standard water solution of above-mentioned concentration respectively, add concentration and be the sodium hydrate aqueous solution of the 0.3mol/L of the methanol solution 250 μ L of 1-phenyl-3-methyl-5-pyrazolones ketone of 0.5mol/L and 250 μ L, 70 ℃ of following derivatization reaction 30min, cooling 10min neutralizes with 250 μ L 0.3mol/L aqueous hydrochloric acid solutions; Add the 1mL chloroform extraction, fully concussion is inhaled and is abandoned lower floor, repeats 3 times, and water layer is made efficient liquid phase chromatographic analysis after filtering.The liquid phase chromatogram condition that is adopted is: Agilent 1100HPLC (U.S. Agilent company), Agilent ZORBAXEclipse XDB-C18 (4.6 * 150mm, 5 μ m) chromatographic column, 25 ℃ of column temperatures, with 22% acetonitrile-0.05mol/L potassium dihydrogen phosphate buffer solution as moving phase, flow velocity 1.0mL/mi n, the detection wavelength is 250nm.The liquid of deriving of the standard water solution of above-mentioned 8 concentration gradients is through liquid-phase chromatographic analysis, and the appearance time that obtains quinoline promise sugar is 19.86min, with quinoline promise sugar peak area quinoline promise Standard for Sugars solution concentration is carried out linear regression, obtains typical curve y=10219x+9.0095 (R
2=0.9995), wherein x is a quinoline promise Standard for Sugars solution concentration, and y is a quinoline promise sugar peak area.
(4) draw sea cucumber A sample test liquid 250 μ L, carry out derivatization reaction, the gained liquid of deriving carries out liquid-phase chromatographic analysis by above-mentioned condition, obtaining wherein, the peak area of quinoline promise sugar is 360.02, with quinoline promise sugar peak area bring into working curve calculate in the sample content of quinoline promise sugar be 0.00562%, multiply by the transformation ratio of sea cucumber A (stichopus japonicus), get the content of saponin(e among the sea cucumber A.Because sea cucumber A is stichopus japonicus (Stichopus japonicus), its saponin constituent of bibliographical information is mainly holotoxin A
1(molecular weight 1392), holotoxin B
1(molecular weight 1378), holotoxinA (molecular weight 1422) and holotoxin B (molecular weight 1408), its mean molecular weight is about 1400, so the ratio of saponin(e mean molecular weight 1400 and quinoline promise glycan molecule amount 164 is 8.5, and promptly transformation ratio is 8.5; Calculate sea cucumber A (stichopus japonicus) saponin content 0.0477%.
Embodiment two
(1) take by weighing commercially available dried sea-cucumber B (leather Pi Shi sea cucumber) 75.4mg, pulverize, add the 0.2mL2mol/L trifluoroacetic acid aqueous solution, in 120 ℃ of hydrolysis 2 hours, hydrolyzate was settled to 10mL as test liquid with the neutralization of 5mol/L sodium hydroxide solution.
(2) obtain working curve y=10219x+9.0095 (R by the method identical with embodiment one
2=0.9995), wherein x is a quinoline promise Standard for Sugars solution concentration, and y is a quinoline promise sugar peak area.
(3) the sample test liquid 250 μ L of absorption sea cucumber B, carry out derivatization reaction and liquid-phase chromatographic analysis according to the method identical with embodiment one, recording quinoline promise sugar peak area is 2784.27, bring into working curve calculate in the sample content of quinoline promise sugar be 0.591%, multiply by the transformation ratio of sea cucumber B (leather Pi Shi sea cucumber), can get saponin content among the sea cucumber B.Sea cucumber B is leather Pi Shi sea cucumber
(Pearsonothuria graeffei) still has nothing to do in the report of this kind selenka composition, and selenka is the quite similar compounds of a series of structures, its mean molecular weight is about 1200, therefore saponin(e mean molecular weight (1200) is 7.3 with the ratio of quinoline promise glycan molecule amount (164), so its transformation ratio can calculate by 7.3.Sea cucumber B (leather Pi Shi sea cucumber) saponin content is calculated to such an extent that be 4.312%.
Selenka described in the present invention is the lanostane-type triterpenoid saponin, and aglycon is 18 (20) lactonic rings, and the C-3 of aglycon links to each other with oligonucleotide chain by β-0-glycosidic bond, and quinoline promise sugar is one of monose of forming oligonucleotide chain.Described sea cucumber example weight is 0.05g-1g, and the amount that adds trifluoroacetic acid aqueous solution is the 2-3mL/g sea cucumber.Described derivating agent is the 1-phenyl-3-methyl-5-pyrazolones ketone methanol solution of 0.45mol/L-0.5mol/L.
Claims (5)
1. selenka Determination on content method, it is characterized in that the following step is arranged: (1) takes by weighing the sea cucumber sample, sample is pulverized or be cut into small pieces, placing concentration is the trifluoroacetic acid aqueous solution of 2mol/L-2.5mol/L, at 110 ℃ of-120 ℃ of following hydrolysis 2-3 hours,, filter the hydrolyzate neutralization, constant volume is as test liquid; (2) take by weighing quinoline promise sugar, be mixed with 6-8 concentration becomes concentration gradient between 0.01mmol/L-1mmol/L standard water solution; (3) draw above-mentioned standard water solution, 1-phenyl-3-methyl-5-pyrazolones ketone derivating agent and the 0.3mol/L-0.35mol/L sodium hydrate aqueous solution of equal volume respectively, at 65-70 ℃ of following derivatization reaction 25-35min, cooling 8-10min neutralizes with aqueous hydrochloric acid solution; Use chloroform extraction, inhale and to abandon lower floor, repeats 3 times,, carry out liquid-phase chromatographic analysis, the concentration of quinoline promise sugar is carried out linear regression, obtain typical curve with the peak area of quinoline promise sugar derivatives with the water layer filtration; (4) get above-mentioned test liquid and carry out derivatization reaction and liquid-phase chromatographic analysis, obtain the content of quinoline promise sugar in the sample in the above-mentioned typical curve of peak area substitution with quinoline promise sugar derivatives; The content of quinoline promise sugar multiply by the content that transformation ratio promptly gets saponin(e in the sea cucumber sample.
2. by the described selenka Determination on content of claim 1 method, it is characterized in that described selenka is the lanostane-type triterpenoid saponin, aglycon is 18 (20) lactonic rings, and the C-3 of aglycon links to each other with oligonucleotide chain by β-O-glycosides key, and quinoline promise sugar is one of monose of forming oligonucleotide chain.
3. by the described selenka Determination on content of claim 1 method, it is characterized in that described sea cucumber example weight is 0.05g-1g, the amount that adds trifluoroacetic acid aqueous solution is the 2-3mL/g sea cucumber.
4. by the described selenka Determination on content of claim 1 method, it is characterized in that described derivating agent is the 1-phenyl-3-methyl-5-pyrazolones ketone methanol solution of 0.45mol/L-0.5mol/L.
5. by the described selenka Determination on content of claim 1 method, it is characterized in that the separation condition of described liquid chromatography is: ZORBAX Eclipse XDB-C18 type chromatographic column; 22% acetonitrile-0.05mol/L potassium dihydrogen phosphate buffer solution is as moving phase; 25 ℃ of column temperatures; Flow velocity 1.0mL/min, the detection wavelength is 250nm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507760A (en) * | 2011-10-17 | 2012-06-20 | 浙江大学 | Method for determining content of pectin |
| CN107796889A (en) * | 2017-10-13 | 2018-03-13 | 西北大学 | The different two functional group reagents derivatization of amino-pyrazol quinoline ketone and method for separating and analyzing of reproducibility sugar chain and glycoprotein O sugar chains |
| CN109430854A (en) * | 2018-09-18 | 2019-03-08 | 广东精核生物科技发展有限公司 | A kind of health care product and preparation method thereof |
| CN110702843A (en) * | 2019-09-30 | 2020-01-17 | 佛山市欧若拉生物科技有限公司 | Non-calibration amount determination kit and determination method for soapberry saponin standard substance |
-
2008
- 2008-01-14 CN CNA200810013642XA patent/CN101216463A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507760A (en) * | 2011-10-17 | 2012-06-20 | 浙江大学 | Method for determining content of pectin |
| CN107796889A (en) * | 2017-10-13 | 2018-03-13 | 西北大学 | The different two functional group reagents derivatization of amino-pyrazol quinoline ketone and method for separating and analyzing of reproducibility sugar chain and glycoprotein O sugar chains |
| CN109430854A (en) * | 2018-09-18 | 2019-03-08 | 广东精核生物科技发展有限公司 | A kind of health care product and preparation method thereof |
| CN109430854B (en) * | 2018-09-18 | 2021-10-26 | 广东精核生物科技发展有限公司 | Health product and preparation method thereof |
| CN110702843A (en) * | 2019-09-30 | 2020-01-17 | 佛山市欧若拉生物科技有限公司 | Non-calibration amount determination kit and determination method for soapberry saponin standard substance |
| CN110702843B (en) * | 2019-09-30 | 2022-05-17 | 东莞微领生物科技有限公司 | Non-calibration amount determination kit and determination method for soapberry saponin standard substance |
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Open date: 20080709 |