CN108088927A - A kind of method of triterpene glucoside V content in measure Siraitia grosvenorii - Google Patents

A kind of method of triterpene glucoside V content in measure Siraitia grosvenorii Download PDF

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Publication number
CN108088927A
CN108088927A CN201711367024.0A CN201711367024A CN108088927A CN 108088927 A CN108088927 A CN 108088927A CN 201711367024 A CN201711367024 A CN 201711367024A CN 108088927 A CN108088927 A CN 108088927A
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content
siraitia grosvenorii
triterpene glucoside
measuring
extraction
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陈学松
钟水桥
梁峰
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Wuzhou Institutes for Food and Drug Control
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Wuzhou Institutes for Food and Drug Control
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/047—Standards external
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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Abstract

The invention discloses a kind of methods for measuring triterpene glucoside V content in Siraitia grosvenorii, belong to technical field of chemical detection.It is contemplated that improve the quality control standard of Siraitia grosvenorii, it is high to provide a kind of analysis efficiency, the assay method that testing result has been reappeared, and this method uses the Siraitia grosvenorii that ASE extraction process crushed and prepares hexamethylene alkane extract；Triterpene glucoside V content in extract liquor is detected using HPLC methods.The present invention can be used for the measure of triterpene glucoside V content.

Description

A kind of method of triterpene glucoside V content in measure Siraitia grosvenorii
Technical field
The invention belongs to technical field of chemical detection, especially a kind of side for measuring triterpene glucoside V content in Siraitia grosvenorii Method.
Background technology
Siraitia grosvenorii is China Guangxi specialty Chinese medicine,《The south of the Five Ridges is gathered medicinal herbs record》It records：" fruit is sweet in flavor, manages phlegm-fire cough and lean meat Soup takes it." among the people for removing heat from the lung to relieve cough, cool blood ease constipation.It is separated from Lo Han Guo fruit at present and identifies 11 kinds of cucurbitanes Triterpene glycosides compound, such compound are main active substances in Lo Han Guo fruit, are had been reported that using one perchloric acid of vanillic aldehyde The content that reagent determines triterpenoid saponin in Siraitia grosvenorii with carrying out colorimetric analysis after triterpenoid saponin colour developing.
Wherein triterpene glucoside V is content and the higher ingredient of sugariness in Lo Han Guo fruit, and Chinese Pharmacopoeia is only at present There is the measure item to Siraitia grosvenorii extract, therefore be conducive to improve the quality control of Siraitia grosvenorii to the measure of triterpene glucoside V content Standard processed.
The content of the invention
Against the above deficiency, it is high it is an object of the invention to provide a kind of analysis efficiency, the measure that testing result has been reappeared The method of triterpene glucoside V content in Siraitia grosvenorii.
In order to realize above-mentioned technique effect, technical solution provided by the invention is such：Sieve in a kind of measure Siraitia grosvenorii The method of Chinese fruit saponin(e V content, comprises the following steps successively：
Step 1：The Siraitia grosvenorii that crushed using ASE extraction process simultaneously prepares hexamethylene alkane extract；
Step 2：Triterpene glucoside V content in extract liquor is detected using HPLC methods.
Further, the step 1 includes following sub-steps：
Step S1：Siraitia grosvenorii is smashed, No. four sieves is crossed, the powder 0.5g after sieving is taken to be uniformly mixed with 1g diatomite；
Step S2：Add 2g silica gel in the 10mlASE abstraction pools for being placed with fibrous filter membrane, add above-mentioned uniformly mixed sample Product add diatomite extremely parallel with pond mouthful；
Step S3：It is extracted with hexamethylene, collect and 25ml is settled to using hexamethylene, filtered, obtain hexamethylene alkane extract.
Further, the extraction parameters described in step S3 are：Extraction temperature is 100 DEG C, and the static extracting time is 5min, is extracted It is 1 time to take number, flush volume 100%, purge time 60s, pressure position 1500psi.
Further, parameter setting is as follows when the HPLC methods described in step 2 are analyzed：
Mobile phase：Volume ratio is 23:77 acetonitrile-water；Flow velocity：0.3mL/min；Column temperature：40℃；Chromatographic column：Thermo Syncronis C18 columns；Detection wavelength：203nm.
Further, the specification of the chromatographic column is 3 μm, 3mm*100mm.
Further, instrument used in the extraction is：The ASE350 Accelerate solvent extractions of DIONEX companies of the U.S. Instrument.
Further, instrument used in the HPLC is Thermo U3000 UHPLC liquid chromatographs.
Further, the chromatographic column octadecylsilane chemically bonded silica is filler
Further, calculated using following formula：
C in formulaR- reference substance solution concentration, unit are micro- gram per liter；AXThe peak area of-test sample；AR- reference substance peak Area.
Compared with prior art, the present invention has the following advantages：
《Chinese Pharmacopoeia》Large pore resin absorption column AB-8 is used in versions in 2015 as cleanser, it is contemplated that sugared in Siraitia grosvenorii Class impurity is more, we used PSA, neutral alumina, silica gel as cleanser, finds to add in 2g silica gel in extractor bottom Content of the carbohydrate impurity without influencing triterpene glucoside can be effectively removed, therefore uses silica gel as cleanser；
Extraction temperature, we using 80,100,120 DEG C be used as extraction temperature, the results showed that 100 and 120 effects preferably and It is almost equal, therefore use 100 DEG C to be used as extraction temperature；
Extraction time we using 3,5,8min, it is found that 5min extraction effects are good, the time is longer result is influenced it is little, And extraction time influences result unobvious；
The method analysis efficiency of triterpene glucoside V content is high in measure Siraitia grosvenorii provided by the present invention, testing result weight It is now good.
Description of the drawings
Fig. 1 is the chemical structural formula figure of triterpene glucoside V；
Concentration (μ g/ml)-peak area that Fig. 2 is triterpene glucoside V carries out linear regression graph.
Specific embodiment
With reference to embodiment, the claim of the present invention is described in further detail, but do not formed pair Any restrictions of the present invention, the modification of any limited number of time made in the claims in the present invention protection domain, still the present invention's Within claims.
Embodiment 1
1. instrument and equipment and reagent
Electronic analytical balance (XA205DU), ASE350 Accelerate solvent extractions instrument (DIONEX companies of the U.S.), Thermo U3000 UHPLC liquid chromatographs；
Reagent：
Hexamethylene (chromatographically pure)；Hexamethylene (analysis is pure)；Large pore resin absorption column AB-8；Ethyl alcohol (analysis is pure)；Diatomite (grain size about 2mm)；Silica gel.
The chemical structural formula of triterpene glucoside V is as shown in Figure 1.
2. method
The preparation of 2.1 reference substance solutions：
Take triterpene glucoside V reference substances appropriate, it is accurately weighed, add mobile phase be made solution of every 1ml containing 0.2mg to get；
The preparation of 2.2 test solutions：
Step S1：Sample through pulverizer is crushed, No. three sieves is crossed, 0.5g is taken to be uniformly mixed with 1g diatomite, obtains sample 1；
Step S2：Filter membrane is put into 10ml abstraction pools, sequentially adds 2g silica gel and the sample 1 obtained by step S1, is added in Diatomite to itself and the horizontal saliva in pond mouthful is put down；
Step S3：The sample obtained by step S2 is extracted with hexamethylene, the extraction temperature is 100 DEG C, static Extraction time is 5min, and extraction times are 1 time, flush volume for 100% to get.
2.3 it measures：
It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, high performance liquid chromatograph is injected, is measured.
By external standard method：
It calculates to get C in formulaR- reference substance solution concentration, unit are micro- gram per liter；AXThe peak area of-test sample； AR- reference substance peak area；
Parameter setting is as follows when 2.4 high performance liquid chromatographies are analyzed：
Mobile phase：Volume ratio is 23:77 acetonitrile-water,
Flow velocity：0.3mL/min,
Column temperature：40 DEG C,
Chromatographic column：Thermo Syncronis C18 columns, 3 μm, 3mm*100mm,
Detection wavelength：203nm.
3. measure and result
3.1 linear relationship
Take triterpene glucoside V reference substances appropriate, it is accurately weighed, it puts in brown measuring bottle, adds mobile phase that every 1ml is made containing arhat The solution of 880.4 μ g of fruit saponin(e V, then accurate 0.1 μ l of the solution, 0.2 μ l, 0.5 μ l, 0.8 μ l, the 1.0 μ l of drawing enter respectively LC is measured, and is measured according to the above method, the results detailed in Table 1：
Table 1
Linear regression is carried out with concentration (μ g/ml)-peak area, acquires regression equation：Y=0.83977x-22.351, R2= 0.99967.Triterpene glucoside V, in good linear relationship, refers to Fig. 2 in the range of 88~880 μ g/ml.
3.2 reappearance test
Take the sample (lot number of identical lot number：1510180) 0.5g, it is totally 6 parts, accurately weighed, it extracts and supplies by ASE extracting methods Test sample solution, sample size are 1 μ L, and with above-mentioned chromatographic condition parallel test, the content for measuring triterpene glucoside V in sample is shown in Table 2, RSD 1.5%, experiments have shown that ASE extracting methods repeatability is good：
Table 2
3.3 precision and stability test
Triterpene glucoside V reference substance solutions (0.8804mg/ml) are taken, continuous sample introduction 6 times records peak area, peak area RSD For 1.5%, show that instrument precision is good.
Same test solution is taken, is placed at room temperature, is measured in accordance with the law respectively at 0,1,3,6,12h.
As a result the RSD of triterpene glucoside V peak areas is 1.6%, shows that test solution is stablized in 12h.
The different instruments extraction results of (3 batches, lot number is respectively 1508166,1510180,150723), sample and with pharmacopeia side Method results contrast is shown in Table 3：
Table 3
4. discussion of results：
The selection of 4.1 ASE Extraction solvents：
In experiment, once the solvent that Accelerate solvent extraction uses was investigated,《Chinese Pharmacopoeia》Version uses hexamethylene within 2015 Alkane extraction triterpene glucoside V relative amounts are higher, therefore to hexamethylene, acetonitrile and ethyl alcohol is used to carry out solvent investigation, as a result show Show that hexamethylene extraction triterpene glucoside V is consistent with Chinese Pharmacopoeia method content, and impurity peaks are essentially identical, therefore use and pharmacopeia one The Extraction solvent of cause.
The optimization of 4.2 ASE extraction conditions：
《Chinese Pharmacopoeia》Large pore resin absorption column AB-8 is used in versions in 2015 as cleanser, it is contemplated that sugared in Siraitia grosvenorii Class impurity is more, we used PSA, neutral alumina, silica gel as cleanser, finds to add in 2g silica gel in extractor bottom Content of the carbohydrate impurity without influencing triterpene glucoside can be effectively removed, therefore uses silica gel as cleanser.
Extraction temperature, we using 80,100,120 DEG C be used as extraction temperature, the results showed that 100 and 120 effects preferably and It is almost equal, therefore use 100 DEG C to be used as extraction temperature；Extraction time we using 3,5,8min, hair 5min extraction effects are good, Time is longer little on result influence, and extraction time influences result unobvious.
Finally definite condition is that extraction solution is hexamethylene, 100 DEG C of extraction temperature, extraction time 5min, cycle-index 1 It is secondary.
Above-described is only presently preferred embodiments of the present invention, all timess made in the range of the spirit and principles in the present invention What modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.

Claims (8)

  1. A kind of 1. method for measuring triterpene glucoside V content in Siraitia grosvenorii, which is characterized in that comprise the following steps successively：
    Step 1：The Siraitia grosvenorii that crushed using ASE extraction process simultaneously prepares hexamethylene alkane extract；
    Step 2：Triterpene glucoside V content in extract liquor is detected using HPLC methods.
  2. 2. the method according to claim 1 for measuring triterpene glucoside V content in Siraitia grosvenorii, which is characterized in that described Step 1 includes following sub-steps：
    Step S1：Siraitia grosvenorii is smashed, No. four sieves is crossed, the powder 0.5g after sieving is taken to be uniformly mixed with 1g diatomite；
    Step S2：Add 2g silica gel in the 10mlASE abstraction pools for being placed with fibrous filter membrane, add above-mentioned uniformly mixed sample, Add diatomite extremely parallel with pond mouthful；
    Step S3：It is extracted with hexamethylene, collect and 25ml is settled to using hexamethylene, filtered, obtain hexamethylene alkane extract.
  3. 3. the method according to claim 2 for measuring triterpene glucoside V content in Siraitia grosvenorii, which is characterized in that step S3 The extraction parameters are：Extraction temperature is 100 DEG C, and the static extracting time is 5min, and extraction times are 1 time, and flush volume is 100%, purge time 60s, pressure position 1500psi.
  4. 4. the method according to claim 1 for measuring triterpene glucoside V content in Siraitia grosvenorii, which is characterized in that step 2 institute Parameter setting is as follows when the HPLC methods stated are analyzed：
    Mobile phase：Volume ratio is 23:77 acetonitrile-water；Flow velocity：0.3mL/min；Column temperature：40℃；Chromatographic column：Thermo Syncronis C18 columns；Detection wavelength：203nm.
  5. 5. the method according to claim 4 for measuring triterpene glucoside V content in Siraitia grosvenorii, which is characterized in that described The specification of chromatographic column is 3 μm, 3mm*100mm.
  6. 6. the method according to claim 3 for measuring triterpene glucoside V content in Siraitia grosvenorii, which is characterized in that described Instrument is used in extraction：The ASE350 Accelerate solvent extraction instrument of DIONEX companies of the U.S..
  7. 7. the method according to claim 4 for measuring triterpene glucoside V content in Siraitia grosvenorii, which is characterized in that described Instrument used in HPLC is Thermo U3000 UHPLC liquid chromatographs.
  8. 8. the method according to claim 5 for measuring triterpene glucoside V content in Siraitia grosvenorii, which is characterized in that described Chromatographic column octadecylsilane chemically bonded silica is filler.
CN201711367024.0A 2017-12-18 2017-12-18 A kind of method of triterpene glucoside V content in measure Siraitia grosvenorii Pending CN108088927A (en)

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Application publication date: 20180529