CN107632096A - A kind of method for the monose composition for determining sposknikovan - Google Patents
A kind of method for the monose composition for determining sposknikovan Download PDFInfo
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- CN107632096A CN107632096A CN201710983391.7A CN201710983391A CN107632096A CN 107632096 A CN107632096 A CN 107632096A CN 201710983391 A CN201710983391 A CN 201710983391A CN 107632096 A CN107632096 A CN 107632096A
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- sposknikovan
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Abstract
The invention discloses a kind of method for the monose composition for determining sposknikovan, it comprises the following steps：1) sposknikovan hydrolyzation sample is prepared；2) mixing monose standard items are prepared；3) pre-column derivatization is handled：Mixing monose hydrolysis standard items obtained by sposknikovan hydrolyzation sample obtained by step 1) and step 2) are mixed with 0.5mol/L PMP methanol solutions and 0.3mol/L NaOH solution respectively, shake well, 25min is reacted under 65 DEG C of water bath conditions, after be cooled to room temperature, the hydrochloric acid for adding 0.3mol/L is neutralized, chloroform is added to be extracted, centrifugation, discard organic layer, extraction 2~3 times is repeated, upper strata aqueous is obtained, through 0.22 μm of filtering with microporous membrane, sposknikovan derivatization treatment solution and mixing monose derivatization treatment solution are obtained respectively, it is standby；4) high-efficient liquid phase chromatogram technique analysis：Determine the monose composition and its content of sposknikovan.High sensitivity of the present invention, amount of samples is few, stability is good, detects the monose composition in sposknikovan with realizing simple to operate and lower cost.
Description
Technical field
The present invention relates to assay method field, the monose for more particularly to determining polysaccharide forms.
Background technology
Herbal polysaccharide has the extensive biological activity such as strengthen immunity, resisting stress, antiviral, is led in animal and veterinary
Domain is widely used, and the focus of novel chiral synthon research.But current present situation is the quality control mark of veterinary Chinese medicine polysaccharide
Accurate imperfection (only detecting total sugar content), the polyose for causing some false, bad occur repeatedly, endanger the development of aquaculture.
Therefore carry out the research of veterinary Chinese medicine polysaccharide quality control, be the important prerequisite for formulating novel polysaccharide medicine or feed addictive.
In general, pharmacological activity possessed by polysaccharide is often closely related with its monose composition, therefore in detection total sugar content
When also need to its monose form carry out qualitative analysis.Because polysaccharide and monose all do not have UV absorption, directly it can not be entered
Row measure.When with high-efficient liquid phase chromatogram technique analysis polysaccharide composition and its content, typically gel chromatographic columnses and differential refraction are used
The instrument such as detector or EISD, not only sensitivity is low for these methods, baseline noise is big, column temperature influences on result
Monose conversion easily occurs in larger, continuous mode and these instruments and chromatographic column price are high.
The content of the invention
It is an object of the invention to for above-mentioned the deficiencies in the prior art, there is provided a kind of side of the monose composition of sposknikovan
Method, it is realized detects to the monose composition of sposknikovan efficiently, simply and at low cost.
The technical solution used in the present invention is：A kind of method for the monose composition for determining sposknikovan, it includes as follows
Step：
1) sposknikovan hydrolyzation sample is prepared：Windproof refined polysaccharide is taken to be mixed with 2mol/L trifluoroacetic acid solution after 92
Water bath processing 6h at DEG C, neutrality is neutralized to 4mol/L sodium hydroxide solution, centrifugation goes supernatant to obtain sposknikovan hydrolysis sample
Product, it is standby；
2) mixing monose standard items are prepared：Take D-MANNOSE, rhamnose, D-Glucose, D-ribose, D- galacturonic acids,
D- galactolipins and arabinose, which are codissolved in water, must mix monose standard items, standby；
3) pre-column derivatization is handled：Respectively by mixing monose obtained by sposknikovan hydrolyzation sample obtained by step 1) and step 2)
Hydrolysis standard items mix with 0.5mol/L PMP methanol solutions and 0.3mol/L NaOH solution, shake well, 65 DEG C of water-bath bars
React 25min under part, after be cooled to room temperature, the hydrochloric acid for adding 0.3mol/L is neutralized, and is added chloroform and is extracted, from
The heart, organic layer is discarded, repeat extraction 2~3 times, obtain upper strata aqueous, through 0.22 μm of filtering with microporous membrane, obtained sposknikovan respectively and spread out
Biochemical treatment solution and mixing monose derivatization treatment solution, it is standby；
4) high-efficient liquid phase chromatogram technique analysis：The sposknikovan obtained by step 3) is derived respectively as high performance liquid chromatography
The chromatogram for changing processing solution and mixing monose derivatization treatment solution carries out, to when analyzing, determining the monose group of sposknikovan
Into and its content.
Specifically, the temperature in step 1) hydrolytic process and water-curing treatment duration are particularly important, and the present invention is through having been surprisingly found that
Sposknikovan can be fully hydrolyzed at 92 DEG C after water bath processing 6h, to improve the accuracy of late detection data.Similarly, step 3)
In pre-column derivatization processing procedure, 65 DEG C are will be defined in, water-curing treatment duration is limited to 25min so that sposknikovan derivatization
Processing is more abundant.
As the further improvement of such scheme, the windproof refined polysaccharide described in step 1) is windproof Thick many candies through macropore
Through obtained by vacuum freeze drying after purifying resin concentration.
As the further improvement of such scheme, windproof Thick many candies described in step 1) are to get it filled with windproof by feed liquid weight
Than for 1：14th, extraction time 1.5h, 85 DEG C of extraction temperature, extraction time 3 times, by suction filtration, concentrated extracting solution, absolute ethyl alcohol sinks
Form sediment concentration 12h, the vacuum dried gained of taking precipitate.Specifically, the present invention obtains to the medicinal windproof windproof Thick many candies process of extraction
The restriction of solid-liquid ratio, extraction time, extraction temperature and extracting times substantially increases extracting efficiency, avoids unnecessary former material
Material wastes.
As the further improvement of such scheme, high performance liquid chromatography is by the sposknikovan derivatization in step 4)
Processing solution and mixing monose derivatization treatment solution are pressed respectively through chromatogram post separation with phosphate buffer solution and acetonitrile solution
Weight part ratio is 85：15 mixed liquor is eluted, and controls 40 DEG C, flow velocity 1mL/min of column temperature, the μ L Detection wavelengths of sample size 20
254nm。
The beneficial effects of the invention are as follows：The present invention is by establishing PMP pre-column derivatizations-high performance liquid chromatography to windproof more
The monosaccharide component and content of sugar are analyzed, and its high sensitivity, amount of samples are few, stability is good, and method of the invention accurately may be used
Lean on, each monosaccharide component can be separated well, detect the monose group in sposknikovan with realizing simple to operate and lower cost
Into.
Brief description of the drawings
Fig. 1 is the high-efficient liquid phase analysis chromatogram that monose derivatization treatment solution is mixed in embodiment 1 in the present invention；
Fig. 2 is the high-efficient liquid phase analysis chromatogram of sposknikovan derivatization treatment solution in embodiment 1 in the present invention.
Embodiment
The present invention is specifically described with reference to embodiment, in order to art personnel to the present invention
Understand.It is necessary that herein the present invention will be further described it is emphasized that embodiment is only intended to, it is impossible to be interpreted as to this
The limitation of invention protection domain, art person skilled in the art, the non-intrinsically safe made according to foregoing invention content to the present invention
The modifications and adaptations of property, should still fall within protection scope of the present invention.Simultaneously following mentioned raw materials are unspecified, are
Commercially available prod；The processing step or preparation method not referred in detail be processing step known to a person skilled in the art or
Preparation method.
Embodiment
A kind of method for the monose composition for determining sposknikovan, it comprises the following steps：
1) windproof Thick many candies are prepared：Get it filled with it is windproof by feed liquid weight ratio be 1：14th, extraction time 1.5h, extraction temperature 85
DEG C, extraction time 3 times, by filter, concentrated extracting solution, absolute ethyl alcohol precipitation concentration 12h, taking precipitate is vacuum dried, obtains
Windproof Thick many candies；
2) windproof refined polysaccharide is prepared：Take windproof Thick many candies obtained by step 1) cold through vacuum after macroporous resin purification concentrates
It is lyophilized dry, obtain windproof refined polysaccharide；
3) sposknikovan hydrolyzation sample is prepared：Windproof refined polysaccharide 50mg and 2mol/L trifluoroacetic acid solution 2mL is taken to mix
Close after water bath processing 6h at 92 DEG C, be neutralized to neutrality with 4mol/L sodium hydroxide solution, it is windproof more that centrifugation goes supernatant to obtain
Sugared hydrolyzation sample, it is standby；
4) mixing monose standard items are prepared：Take D-MANNOSE 12mg, rhamnose 13.3mg, D-Glucose 12mg, D-ribose
12mg, D- galacturonic acid 10.9mg, D- galactolipin 11.5mg and arabinose 11.2mg constant volumes obtain mixed in 25mL volumetric flasks
Monose standard items are closed, it is standby；
5) pre-column derivatization is handled：Respectively by sposknikovan hydrolyzation sample obtained by 100 μ L steps 1) and 100 μ L steps 2) institutes
Monose hydrolysis standard items must be mixed to mix with 50 μ L 0.5mol/L PMP methanol solutions and 50 μ L 0.3mol/L NaOH solution
Close, shake well, react 25min under 65 DEG C of water bath conditions, after be cooled to room temperature, the hydrochloric acid for adding 50 μ L 0.3mol/L is carried out
Neutralize, add 1mL chloroforms and extracted, centrifuge, discard organic layer, repeat extraction 2 times, obtain upper strata aqueous, it is micro- through 0.22 μm
Hole membrane filtration, sposknikovan derivatization treatment solution and mixing monose derivatization treatment solution are obtained respectively, it is standby；
6) high-efficient liquid phase chromatogram technique analysis：By the sposknikovan derivatization treatment solution and mixing monose derivatization treatment
Solution is respectively through Wondasil C18 (250mm × 4.6mm, 5 μm) chromatogram post separation, with phosphate buffer solution and acetonitrile solution
It is 85 by weight：15 mixed liquor is eluted, and controls 40 DEG C, flow velocity 1mL/min of column temperature, the μ L Detection wavelengths of sample size 20
254nm, each chromatographic peak obtains good separation after sample introduction determines, wherein the efficient liquid phase point of mixing monose derivatization treatment solution
Analyse chromatogram such as accompanying drawing 1, the high-efficient liquid phase analysis chromatogram such as accompanying drawing 2 of sposknikovan derivatization treatment solution.Monose will be mixed
Derivatization treatment solution distinguishes the μ L of sample introduction 1,2,4,6,8,10, the mass concentration of each monose is returned with peak area, linear result
And equation of linear regression is as shown in table 1 below.Take the continuous 6 sample introductions measure of sposknikovan derivatization treatment solution in replica test
Each monose peak area result, obtain average value, substitute into the equation of linear regression of each monose and obtain a monose mol ratio, as a result
It is as shown in table 2 below, D-MANNOSE in sposknikovan, D-ribose, rhamnose, D- galacturonic acids, D-Glucose, D- is calculated
Galactolipin, the mol ratio of arabinose are 360:42:259:467:18422:207:2589.
The monose calibration curve equation of table 1
The mol ratio result of calculation of each monose in the sposknikovan of table 2
Above-described embodiment is the preferred embodiments of the present invention, all with similar technique of the invention and the equivalence changes made,
The protection category of the present invention all should be belonged to.
Claims (4)
- A kind of 1. method for the monose composition for determining sposknikovan, it is characterised in that comprise the following steps：1) sposknikovan hydrolyzation sample is prepared：Windproof refined polysaccharide is taken to be mixed with 2mol/L trifluoroacetic acid solution after at 92 DEG C Water bath processing 6h, neutrality is neutralized to 4mol/L sodium hydroxide solution, centrifugation goes supernatant to obtain sposknikovan hydrolyzation sample, standby With；2) mixing monose standard items are prepared：Take D-MANNOSE, rhamnose, D-Glucose, D-ribose, D- galacturonic acids, D- half Lactose and arabinose, which are codissolved in water, must mix monose standard items, standby；3) pre-column derivatization is handled：Respectively by mixing monose hydrolysis obtained by sposknikovan hydrolyzation sample obtained by step 1) and step 2) Standard items mix with 0.5mol/L PMP methanol solutions and 0.3mol/L NaOH solution, shake well, under 65 DEG C of water bath conditions React 25min, after be cooled to room temperature, the hydrochloric acid for adding 0.3mol/L is neutralized, and is added chloroform and is extracted, centrifugation, abandon Organic layer is removed, extraction 2~3 times is repeated, obtains upper strata aqueous, through 0.22 μm of filtering with microporous membrane, obtains sposknikovan derivatization respectively Processing solution and mixing monose derivatization treatment solution, it is standby；4) high-efficient liquid phase chromatogram technique analysis：As high performance liquid chromatography respectively to the sposknikovan derivatization obtained by step 3) at Reason solution and mix the chromatogram of monose derivatization treatment solution and carry out to when analyzing, determine sposknikovan monose composition and Its content.
- A kind of 2. method of monose composition for determining sposknikovan according to claim 1, it is characterised in that：In step 1) Described windproof refined polysaccharide be windproof Thick many candies after macroporous resin purification concentrates through obtained by vacuum freeze drying.
- A kind of 3. method of monose composition for determining sposknikovan according to claim 2, it is characterised in that：In step 1) The windproof Thick many candies for get it filled with it is windproof by feed liquid weight ratio be 1：14th, extraction time 1.5h, 85 DEG C of extraction temperature, extraction time Number 3 times, by suction filtration, concentrated extracting solution, absolute ethyl alcohol precipitation concentration 12h, the vacuum dried gained of taking precipitate.
- A kind of 4. method of monose composition for determining sposknikovan according to claim 1, it is characterised in that：In step 4) High performance liquid chromatography is respectively through color by the sposknikovan derivatization treatment solution and mixing monose derivatization treatment solution Post separation is composed, is 85 by weight with phosphate buffer solution and acetonitrile solution：15 mixed liquor is eluted, and controls column temperature 40 DEG C, flow velocity 1mL/min, the μ L Detection wavelengths 254nm of sample size 20.
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Cited By (4)
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| WO2019149284A1 (en) * | 2018-02-05 | 2019-08-08 | 上海绿谷制药有限公司 | Separated saposhnikovia divaricata polysaccharide and use thereof |
| CN111195231A (en) * | 2020-02-07 | 2020-05-26 | 佛山科学技术学院 | Preparation method of radix sileris polysaccharide liposome immunopotentiator |
| CN113109489A (en) * | 2021-05-21 | 2021-07-13 | 夏永刚 | Method for simultaneously qualitatively and quantitatively analyzing aldose, ketose, sugar alcohol, sugar acid and aminosugar in traditional Chinese medicine polysaccharide |
| WO2022143716A1 (en) * | 2020-12-30 | 2022-07-07 | 上海医药集团股份有限公司 | Method for measuring content of ganoderma lucidum polysaccharide |
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2017
- 2017-10-19 CN CN201710983391.7A patent/CN107632096A/en active Pending
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| 朱雪琼: "黑木耳多糖的提取、功能及单糖组成的研究", 《广西大学硕士学位论文》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019149284A1 (en) * | 2018-02-05 | 2019-08-08 | 上海绿谷制药有限公司 | Separated saposhnikovia divaricata polysaccharide and use thereof |
| JP2021512997A (en) * | 2018-02-05 | 2021-05-20 | シャンハイ、グリーン、バレー、ファーマスーティカル、カンパニー、リミテッドＳｈａｎｇｈａｉ Ｇｒｅｅｎ Ｖａｌｌｅｙ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｃｏ．， Ｌｔｄ． | Separated windproof polysaccharides and their uses |
| CN111195231A (en) * | 2020-02-07 | 2020-05-26 | 佛山科学技术学院 | Preparation method of radix sileris polysaccharide liposome immunopotentiator |
| WO2022143716A1 (en) * | 2020-12-30 | 2022-07-07 | 上海医药集团股份有限公司 | Method for measuring content of ganoderma lucidum polysaccharide |
| CN113109489A (en) * | 2021-05-21 | 2021-07-13 | 夏永刚 | Method for simultaneously qualitatively and quantitatively analyzing aldose, ketose, sugar alcohol, sugar acid and aminosugar in traditional Chinese medicine polysaccharide |
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Application publication date: 20180126 |