WO2022143716A1 - Method for measuring content of ganoderma lucidum polysaccharide - Google Patents

Method for measuring content of ganoderma lucidum polysaccharide Download PDF

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WO2022143716A1
WO2022143716A1 PCT/CN2021/142290 CN2021142290W WO2022143716A1 WO 2022143716 A1 WO2022143716 A1 WO 2022143716A1 CN 2021142290 W CN2021142290 W CN 2021142290W WO 2022143716 A1 WO2022143716 A1 WO 2022143716A1
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solution
add
monosaccharide
concentration
prepare
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Chinese (zh)
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叶冠
李坤
陈俞宇
王婷婷
房庆伟
杨旭艳
朱亚楠
徐政
陈旭
应旭辉
刘雳
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上海医药集团股份有限公司
正大青春宝药业有限公司
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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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient

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  • the invention relates to the field of chemical detection, in particular to a method for detecting the content of Ganoderma lucidum polysaccharide.
  • Ganoderma lucidum is a kind of fungus that can be used for both medicine and food. It is a traditional Chinese medicine in my country. According to the "Shen Nong's Materia Medica", Ganoderma lucidum has the functions of tonifying qi and soothing the nerves, relieving cough and asthma, and prolonging life.
  • Ganoderma lucidum polysaccharide is one of the main active ingredients of Ganoderma lucidum, and it is also one of the most in-depth researches on the active ingredients of Ganoderma lucidum.
  • Ganoderma lucidum polysaccharide has a large molecular weight and a complex structure.
  • the detection of polysaccharide content is an item that needs to be investigated in the quality control of polysaccharide drugs, which is the basis for its stability and a difficulty in current research.
  • the standard curve was made with glucose as the reference substance (comparison of the polysaccharide content of Ganoderma lucidum by the anthrone sulfate method and the sulfuric acid phenol method, Chinese Journal of Traditional Chinese Medicine, 2010, 28, 9, 2000). -2002); The 2015 edition of the Pharmacopoeia of the People's Republic of China used anthrone sulfuric acid to develop color, and UV spectrophotometry was used to measure the absorbance value, and a standard curve was made with a glucose standard solution to detect the content of polysaccharides in Ganoderma lucidum. This method is a detection method under the quality identification of Ganoderma lucidum Chinese medicinal materials, and is suitable for the quality control of Ganoderma lucidum.
  • Ganoderma lucidum polysaccharide is a heteropolysaccharide composed of a variety of monosaccharides. Only glucose is used as a reference substance to make a standard curve, and the content of Ganoderma lucidum polysaccharide cannot be accurately determined. Therefore, the selection of the reference substance is particularly important when measuring the polysaccharide content by colorimetry. important.
  • the commonly used detection methods for polysaccharide content mainly use a monosaccharide, such as glucose, as the standard, but the detection results are often inconsistent with the actual situation.
  • the object of the present invention is to provide a kind of detection method of Ganoderma lucidum polysaccharide content.
  • This method improves the current detection method.
  • the sugar composition of Ganoderma lucidum polysaccharide is determined by high performance liquid chromatography (HPLC), and then the standard solution is prepared according to the composition sugar and composition ratio.
  • HPLC high performance liquid chromatography
  • the phenol-sulfuric acid method and anthrone- The content of Ganoderma lucidum polysaccharide was detected by the sulfuric acid method.
  • This method can be used for qualitative and quantitative analysis of Ganoderma lucidum polysaccharide, which provides a reference for the quality analysis method of Ganoderma lucidum polysaccharide.
  • the present invention provides a method for detecting the content of Ganoderma lucidum polysaccharide, comprising: (1) determining the monosaccharide composition of Ganoderma lucidum polysaccharide by HPLC; (2) using phenol-sulfuric acid method or anthrone-sulfuric acid method to measure Ganoderma lucidum
  • the polysaccharide content preferably, in step (2), is measured using a mixed monosaccharide solution containing at least glucose, galactose, glucuronic acid and mannose prepared according to the monosaccharide ratio determined in step (1) as a reference solution.
  • step (1) uses following method one or method two:
  • Method one which includes the following steps:
  • aqueous solutions of glucose, galactose, glucuronic acid, mannose, ribose, rhamnose, galacturonic acid, arabinose, and fucose were prepared respectively as single-label storage solutions, and the single-label storage solutions were mixed to obtain monosaccharides.
  • Mixed aqueous solution wherein each mL of solution contains 5mg glucose, 0.3mg galactose, 0.2mg mannose, 0.1mg glucuronic acid, 0.05mg ribose, 0.05mg fucose, 0.02mg galacturonic acid, 0.015mg arabinose, 0.01mg rhamnose,
  • the second method includes the following steps:
  • the preparation contains 0.02-0.4mg/mL (preferably 0.08mg/mL) mannose, 0.0125-0.25mg/mL (preferably 0.05mg/mL) glucuronic acid, 0.3125-6.25mg/mL (preferably 1.25mg/mL) glucose, 0.0375-0.6mg/mL (preferably 0.15mg/mL) galactose, 0.0125-0.25mg/mL (preferably 0.05mg/mL) fucose, 0.00625-0.125mg/mL (preferably 0.025mg/mL) ribose, 0.001375-
  • An aqueous solution of 0.0275mg/mL (0.0055mg/mL) rhamnose and 0.0025-0.05mg/mL (preferably 0.01mg/mL) arabinose is a mixed monosaccharide stock solution,
  • step (1) in the detection method of Ganoderma lucidum polysaccharide content of the present invention, in step (1), in the sugar composition analysis, measure the HPLC chromatogram under the following chromatographic conditions:
  • step (1) uses following method A or method B:
  • Method A which includes the following steps:
  • aqueous solutions of glucose, galactose, glucuronic acid, mannose, ribose, rhamnose, galacturonic acid, arabinose, and fucose were prepared respectively as single-label storage solutions, and the single-label storage solutions were mixed to obtain monosaccharides.
  • Mixed aqueous solution wherein each mL of solution contains 5mg glucose, 0.3mg galactose, 0.2mg mannose, 0.1mg glucuronic acid, 0.05mg ribose, 0.05mg fucose, 0.02mg galacturonic acid, 0.015mg arabinose, 0.01mg rhamnose,
  • Method B which includes the following steps:
  • the aqueous solution of sugar and 0.01 mg/mL arabinose is the mixed monosaccharide stock solution
  • the residue was added with 500 ⁇ L of water, 600 ⁇ L of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, and the reaction flask was sealed and heated at 70°C for 60 min. Take out the ice-water bath for 2 minutes, add 600 ⁇ L of 0.15M hydrochloric acid, mix well, take 500 ⁇ L of the solution, add 500 ⁇ L of chloroform, and centrifuge for extraction.
  • the preparation of the HPLC reference solution comprises the following steps:
  • the preparation concentration is 50mg/mL glucose stock solution
  • step (1) the preparation of HPLC reference substance solution comprises the following steps:
  • the preparation concentration is 50mg/mL glucose stock solution
  • the mixed monosaccharide solution is an aqueous solution containing glucose, galactose, glucuronic acid and mannose, wherein, by weight percentage, based on Total weight of monosaccharides in solution containing 60%-85% glucose, 8%-17% galactose, 3%-15% glucuronic acid, 3%-7% mannose, or 60% -90% glucose, 6%-17% galactose, 1%-15% glucuronic acid, 3%-8% mannose, or the mixed monosaccharide solution is determined by high performance liquid chromatography The monosaccharide ratio was used to prepare the reference solution required for content detection, and the total concentration was 0.1 mg/mL.
  • the phenol-sulfuric acid method comprises the following steps:
  • a mixed monosaccharide solution containing at least glucose, galactose, glucuronic acid and mannose is prepared as a reference solution, and the total concentration is 0.1 mg/mL.
  • the mixed monosaccharide solution is prepared as a reference solution, the mixed monosaccharide solution contains an aqueous solution of glucose, galactose, glucuronic acid and mannose, wherein, by weight percentage, based on the total weight of the monosaccharides in the solution, contains 60%-85% of Glucose, 8%-17% galactose, 3%-15% glucuronic acid, 3%-7% mannose, or with 60%-90% glucose, 6%-17% galactose, 1 %-15% glucuronic acid, 3%-8% mannose;
  • step (2) Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the reference solution in step (2), put them in test tubes with stoppers, add water to make up to 2.0mL, and add water to step (1) precisely.
  • 1mL of medium phenol solution shake well, quickly and accurately add 5mL of concentrated sulfuric acid, shake well, place for 10 minutes, place in a 40°C water bath for 15 minutes, take out, ice bath for 2 minutes, blank without reference solution, measure the absorbance at 490nm , take the absorbance as the ordinate and the concentration of the sugar reference substance as the abscissa, draw the phenol-sulfuric acid method standard curve;
  • the anthrone-sulfuric acid method comprises the following steps:
  • the mixed monosaccharide solution contains an aqueous solution of glucose, galactose, glucuronic acid and mannose, wherein, by weight percentage, based on the total weight of the monosaccharides in the solution, contains 60%-85% of Glucose, 8%-17% galactose, 3%-15% glucuronic acid, 3%-7% mannose, or with 60%-90% glucose, 6%-17% galactose, 1 %-15% glucuronic acid, 3%-8% mannose;
  • step (6) Accurately draw 1.0 mL of the test solution in step (6), add water to 2.0 mL, and follow the method under step (5) preparation of the standard curve of anthrone-sulfuric acid method, starting from "adding 6 mL of anthrone sulfate solution", according to the law. The absorbance of the test sample was measured, and the polysaccharide content was obtained according to the standard curve of the anthrone-sulfuric acid method.
  • the detection method of Ganoderma lucidum polysaccharide content of the present invention comprises the following steps:
  • Figure 1 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200825-05) after hydrolysis.
  • Figure 2 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200902-05) after hydrolysis.
  • Figure 3 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200903-08) after hydrolysis.
  • Figure 4 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21001) after hydrolysis.
  • Figure 5 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21002) after hydrolysis.
  • Figure 6 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21003) after hydrolysis.
  • Figure 7 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21004) after hydrolysis.
  • UV5Bio UV-Vis Photometer Metaltler-Toledo International Trading Shanghai Co., Ltd.
  • Ganoderma lucidum polysaccharide (batch number: 20200825-05, after degreasing Ganoderma lucidum medicinal materials with 95% ethanol, boiling water extraction, concentrating the extract, ethanol precipitation with 95% ethanol, the obtained Ganoderma lucidum crude polysaccharide.
  • Batch number: 20200902-05 Ganoderma lucidum medicinal materials with 95% ethanol After degreasing, extracting with boiling water, concentrating the extract, first ethanol precipitation with 20% ethanol, and then ethanol precipitation with 80% ethanol, to obtain Ganoderma lucidum crude polysaccharide.
  • Trifluoroacetic acid (batch number: 20200821; Sinopharm Chemical Reagent; AR)
  • Monosaccharide composition analysis of polysaccharides is the basis for studying the structure and characteristics of polysaccharides.
  • Pre-column derivatization HPLC method is a common analytical method for sugar composition analysis. It has high sensitivity and can simultaneously determine neutral sugars and acidic sugars.
  • the hydrolysis and derivatization conditions of polysaccharides have a direct impact on the detection results.
  • the release of monosaccharides is affected by different reaction conditions. Acidic sugars are easy to release but unstable. Neutral sugars are more stable than acidic sugars but not easy to release.
  • the reaction conditions were used for saccharide composition analysis of polysaccharides.
  • the inventors conducted a detailed investigation and optimization of the hydrolysis and derivatization conditions for the determination of the monosaccharide composition of Ganoderma lucidum polysaccharides by 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization HPLC method.
  • the hydrolysis and derivatization time, temperature, concentration of trifluoroacetic acid and concentration of 1-phenyl-3-methyl-5-pyrazolone were investigated.
  • the time of acid hydrolysis was 1h, 2h, 4h, 6h; the temperature was 90°C, 100°C, 110°C, 120°C; the concentration of trifluoroacetic acid was 1M, 2M, 4M, 6M.
  • the derivatization time was investigated for 30min, 60min and 90min; the concentration of 1-phenyl-3-methyl-5-pyrazolone was investigated as 0.1M, 0.2M, 0.4M and 0.6M.
  • the peak areas of the HPLC results were compared, and the following conditions were optimized by orthogonal analysis, the acid hydrolysis time was 4h, the heating temperature was 120°C, the concentration of trifluoroacetic acid was 6M, the derivatization time was 60min, the 1-phenyl-3 -The concentration of methyl-5-pyrazolone is 0.2M.
  • the sugar composition analysis results of Ganoderma lucidum polysaccharide (20200903-08) showed that the optimized reaction conditions significantly increased the types of monosaccharide composition and total peak area, and the optimized reaction conditions could better reflect the real sugar composition of Ganoderma lucidum polysaccharides.
  • Example 2 Ganoderma lucidum polysaccharide composition analysis
  • the optimal hydrolysis and derivatization conditions were determined by orthogonal experiments, and the monosaccharide composition of Ganoderma lucidum polysaccharides was determined by comparing the single monosaccharide standard one by one, and the mixed monosaccharide solution of corresponding concentration was prepared according to the experimental results. As a reference solution, determine the sugar composition and composition ratio of Ganoderma lucidum polysaccharide.
  • the residue was added with 250 ⁇ L of water, 250 ⁇ L of internal standard solution, 600 ⁇ L of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone.
  • the closed reaction flask was heated at 70°C for 60min and cooled to room temperature. Add 600 ⁇ L of 0.15M hydrochloric acid, mix well, and pass through a 0.45 ⁇ m nylon filter to obtain the test solution.
  • Figure 1 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200825-05) after hydrolysis.
  • Figure 2 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200902-05) after hydrolysis.
  • Figure 3 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200903-08) after hydrolysis.
  • the reference solution required for content detection was prepared with the monosaccharide ratio determined by high performance liquid chromatography, and the total concentration was 0.1 mg/mL.
  • the glucose content is about 62.94%
  • the galactose content is about 62.94% in weight percentage.
  • the content is about 16.07%
  • the content of glucuronic acid is about 14.04%
  • the content of mannose is about 6.96%.
  • the glucose content is about 74.02%
  • the galactose content is about 74.02%.
  • the content is about 12.70%
  • the content of glucuronic acid is about 7.03%
  • the content of mannose is about 6.25%.
  • step 3 Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step 3, put them in test tubes with stoppers, add water to make up to 2.0mL, and add each precision into step 1.
  • 1mL of the prepared phenol solution was shaken, quickly and precisely added 5mL of concentrated sulfuric acid, shaken well, placed for 10 minutes, placed in a 40°C water bath for 15 minutes, taken out, ice bathed for 2 minutes, blank without reference solution, and measured at 490nm. Take the absorbance as the ordinate and the concentration of the sugar reference substance as the abscissa to draw a standard curve.
  • step 3 Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step 3, place them in test tubes with stoppers, respectively add water to make up to 2.0mL, and add each precision to step 2.
  • 6mL of anthrone sulfate solution prepared in The concentration is the abscissa, and the standard curve is drawn.
  • the standard curve was made with the mixed monosaccharide reference solution corresponding to the test product, and the content of Ganoderma lucidum polysaccharide was detected by the phenol-sulfuric acid method and the anthrone-sulfuric acid method.
  • the experimental results show that when anhydrous glucose is the reference substance, the phenol-sulfuric acid method is used to detect the absorbance (Y) of the solution and the concentration of monosaccharide reference substance (X, ⁇ g/mL) in a linear relationship.
  • the absorbance (Y) of the solution has a linear relationship with the concentration of monosaccharide reference substance (X, ⁇ g/mL).
  • the slope of the standard curve of different monosaccharides is different, so the selection of the reference substance is more important in the colorimetric determination of the polysaccharide content.
  • Ganoderma lucidum polysaccharide is composed of a variety of monosaccharides, and the content of each monosaccharide is also different. The situation is more complicated. A single monosaccharide standard curve cannot accurately detect the content of Ganoderma lucidum polysaccharide.
  • the corresponding mixed polysaccharide reference solution is prepared to make a standard curve.
  • the content of Ganoderma lucidum polysaccharides obtained by the two classical colorimetric methods is higher. , can more accurately reflect the total sugar content of Ganoderma lucidum polysaccharides. Therefore, the reference solution prepared with the ratio of each monosaccharide constituting Ganoderma lucidum polysaccharide can obtain data that is closer to the real situation.
  • the residue was added with 500 ⁇ L of water, 600 ⁇ L of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone.
  • the closed reaction flask was heated at 70 °C for 60 min, and the ice-water bath was taken out for 2 min.
  • Figure 4 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21001) after hydrolysis.
  • Figure 5 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21002) after hydrolysis.
  • Figure 6 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21003) after hydrolysis.
  • Figure 7 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21004) after hydrolysis.
  • Embodiment 5 Detection of Ganoderma lucidum polysaccharide content
  • the reference solution required for content detection was prepared with the monosaccharide ratio determined by high performance liquid chromatography, and the total concentration was 0.1 mg/mL.
  • the mixed monosaccharide reference solution of Ganoderma lucidum polysaccharide (21001) accurately weigh different monosaccharides, and prepare the solution containing 4.41 ⁇ g/mL mannose, 3.89 ⁇ g/mL glucuronic acid, 81.72 ⁇ g/mL glucose, 9.98 ⁇ g/mL galactose
  • the aqueous solution is the mixed monosaccharide control solution.
  • the mixed monosaccharide reference solution of Ganoderma lucidum polysaccharide (21002) accurately weigh different monosaccharides, and prepare the solution containing 3.89 ⁇ g/mL mannose, 3.60 ⁇ g/mL glucuronic acid, 84.31 ⁇ g/mL glucose, 8.20 ⁇ g/mL galactose
  • the aqueous solution is the mixed monosaccharide control solution.
  • step 3 Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step 3, put them in test tubes with stoppers, add water to make up to 2.0mL, and add each precision into step 1.
  • 1mL of the prepared phenol solution was shaken, quickly and precisely added 5mL of concentrated sulfuric acid, shaken well, placed for 10 minutes, placed in a 40°C water bath for 15 minutes, taken out, ice bathed for 2 minutes, blank without reference solution, and measured at 490nm. Take the absorbance as the ordinate and the concentration of the sugar reference substance as the abscissa to draw a standard curve.
  • step 3 Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step 3, place them in test tubes with stoppers, respectively add water to make up to 2.0mL, and add each precision to step 2.
  • 6mL of anthrone sulfate solution prepared in The concentration is the abscissa, and the standard curve is drawn.
  • the experimental results show that when the mixed monosaccharide is the reference substance, the content of Ganoderma lucidum polysaccharide (21001) is detected by the phenol-sulfuric acid method, and the absorbance (Y) of the solution is linearly related to the concentration (X, ⁇ g/mL) of the mixed monosaccharide reference substance,
  • the absorbance (Y) of the solution has a linear relationship with the concentration of the mixed monosaccharide reference substance (X, ⁇ g/mL).
  • the liquid phase analysis is mainly optimized in terms of column type, pH value of mobile phase, and increased extraction of samples, so that it can more accurately detect the sugar composition of Ganoderma lucidum polysaccharides.

Abstract

A method for measuring the content of Ganoderma lucidum polysaccharide. The method comprises determining the sugar composition of Ganoderma lucidum polysaccharide by using high-performance liquid chromatography, then preparing a standard substance solution according to the composition sugars and composition proportion thereof, and measuring the content of Ganoderma lucidum polysaccharide by using a phenol-sulfuric acid method and/or an anthrone-sulfuric acid method. The method can be used for the qualitative and quantitative analysis of Ganoderma lucidum polysaccharide, and provides a reference for a quality analysis method for Ganoderma lucidum polysaccharide.

Description

一种灵芝多糖含量的检测方法A kind of detection method of Ganoderma lucidum polysaccharide content 技术领域technical field
本发明涉及化学检测领域，具体而言，涉及一种灵芝多糖含量的检测方法。The invention relates to the field of chemical detection, in particular to a method for detecting the content of Ganoderma lucidum polysaccharide.
背景技术Background technique
灵芝为一种药食两用的真菌，是我国传统中药，据《神农本草经》记载，灵芝具有补气安神、止咳平喘、延年益寿等功效。灵芝多糖是灵芝的主要活性成分之一，也是目前灵芝活性成分研究中较深入的一类成分。Ganoderma lucidum is a kind of fungus that can be used for both medicine and food. It is a traditional Chinese medicine in my country. According to the "Shen Nong's Materia Medica", Ganoderma lucidum has the functions of tonifying qi and soothing the nerves, relieving cough and asthma, and prolonging life. Ganoderma lucidum polysaccharide is one of the main active ingredients of Ganoderma lucidum, and it is also one of the most in-depth researches on the active ingredients of Ganoderma lucidum.
灵芝多糖的分子量大，结构组成也较为复杂。多糖含量的检测是多糖类药物质量控制需要考察的项目，是其稳定性的依据，也是目前研究中的一个难点。Ganoderma lucidum polysaccharide has a large molecular weight and a complex structure. The detection of polysaccharide content is an item that needs to be investigated in the quality control of polysaccharide drugs, which is the basis for its stability and a difficulty in current research.
已发表的文献研究显示，张志军等采用葡聚糖为对照品，使用苯酚-硫酸显色法对灵芝多糖含量检测方法进行了研究(灵芝多糖含量的苯酚硫酸法检测研究，食品工业科技，2006，2，193-195)；温守功等则运用了葡萄糖为对照品(灵芝制品中粗多糖含量测定影响因素的研究，中国卫生检验杂志，2020，30，5，547-550)；郭晓蕾等在比较硫酸蒽酮法和硫酸苯酚法测定灵芝多糖含量时，都以葡萄糖为对照品制作标准曲线(硫酸蒽酮法与硫酸苯酚法测定灵芝多糖含量比较，中华中医药学刊，2010，28，9，2000-2002)；2015版中华人民共和国药典用蒽酮硫酸显色，紫外分光光度法测定吸光度值，用葡萄糖标准溶液制作标准曲线，用来检测灵芝中多糖的含量。该法为灵芝中药材质量鉴别项下的检测方法，对灵芝的质量控制具有适宜性。Published literature studies have shown that Zhang Zhijun and others used dextran as a reference substance, and used the phenol-sulfuric acid color development method to study the detection method of Ganoderma lucidum polysaccharide content (Phenol-sulfuric acid detection method of Ganoderma lucidum polysaccharide content, Food Industry Science and Technology, 2006, 2, 193-195); Wen Shougong et al. used glucose as the reference substance (Research on factors affecting the determination of crude polysaccharide content in Ganoderma lucidum products, Chinese Journal of Hygiene Inspection, 2020, 30, 5, 547-550); Guo Xiaolei et al. When measuring the polysaccharide content of Ganoderma lucidum by the anthrone method and the sulfuric acid phenol method, the standard curve was made with glucose as the reference substance (comparison of the polysaccharide content of Ganoderma lucidum by the anthrone sulfate method and the sulfuric acid phenol method, Chinese Journal of Traditional Chinese Medicine, 2010, 28, 9, 2000). -2002); The 2015 edition of the Pharmacopoeia of the People's Republic of China used anthrone sulfuric acid to develop color, and UV spectrophotometry was used to measure the absorbance value, and a standard curve was made with a glucose standard solution to detect the content of polysaccharides in Ganoderma lucidum. This method is a detection method under the quality identification of Ganoderma lucidum Chinese medicinal materials, and is suitable for the quality control of Ganoderma lucidum.
但在实际运用中，简单采用上述方法得到的检测结果，常与实际情况存在很大的偏差。灵芝多糖是一种杂多糖，由多种单糖组成，只用葡萄糖作为对照品制作标准曲线，并不能准确测定灵芝多糖的含量，因此对照品的选择在比色法测定多糖含量时就显得尤为重要。However, in practical application, the detection results obtained by simply adopting the above method often have great deviations from the actual situation. Ganoderma lucidum polysaccharide is a heteropolysaccharide composed of a variety of monosaccharides. Only glucose is used as a reference substance to make a standard curve, and the content of Ganoderma lucidum polysaccharide cannot be accurately determined. Therefore, the selection of the reference substance is particularly important when measuring the polysaccharide content by colorimetry. important.
目前，常用的多糖含量检测方法，主要以某一单糖，如葡萄糖为标准品，但其检测结果常与实际情况不符。At present, the commonly used detection methods for polysaccharide content mainly use a monosaccharide, such as glucose, as the standard, but the detection results are often inconsistent with the actual situation.
发明内容SUMMARY OF THE INVENTION
针对上述现有检测方法中所存在的问题，本发明的目的是提供一种灵 芝多糖含量的检测方法。本方法对目前的检测方法进行了完善，用高效液相色谱(HPLC)确定了灵芝多糖的糖组成情况，再以其组成糖及组成比例配制标准品溶液，运用苯酚-硫酸法和蒽酮-硫酸法对灵芝多糖的含量进行了检测，本方法能够对灵芝多糖进行定性和定量分析，为灵芝多糖质量分析方法提供参考。For the problems existing in the above-mentioned existing detection methods, the object of the present invention is to provide a kind of detection method of Ganoderma lucidum polysaccharide content. This method improves the current detection method. The sugar composition of Ganoderma lucidum polysaccharide is determined by high performance liquid chromatography (HPLC), and then the standard solution is prepared according to the composition sugar and composition ratio. The phenol-sulfuric acid method and anthrone- The content of Ganoderma lucidum polysaccharide was detected by the sulfuric acid method. This method can be used for qualitative and quantitative analysis of Ganoderma lucidum polysaccharide, which provides a reference for the quality analysis method of Ganoderma lucidum polysaccharide.
为达到以上发明目的，本发明提供了一种灵芝多糖含量的检测方法，包括：(一)用HPLC确定灵芝多糖的单糖组成；(二)使用苯酚-硫酸法或蒽酮-硫酸法测定灵芝多糖含量，优选地，步骤(二)中，使用根据步骤(一)确定的单糖比例配制的至少含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的混合单糖溶液作为对照品溶液进行测定。In order to achieve the above purpose of the invention, the present invention provides a method for detecting the content of Ganoderma lucidum polysaccharide, comprising: (1) determining the monosaccharide composition of Ganoderma lucidum polysaccharide by HPLC; (2) using phenol-sulfuric acid method or anthrone-sulfuric acid method to measure Ganoderma lucidum The polysaccharide content, preferably, in step (2), is measured using a mixed monosaccharide solution containing at least glucose, galactose, glucuronic acid and mannose prepared according to the monosaccharide ratio determined in step (1) as a reference solution.
在一个实施方式中，本发明的灵芝多糖含量的检测方法中，步骤(一)使用以下方法一或方法二：In one embodiment, in the detection method of Ganoderma lucidum polysaccharide content of the present invention, step (1) uses following method one or method two:
方法一，其包括以下步骤：Method one, which includes the following steps:
(1)HPLC对照品溶液的制备(1) Preparation of HPLC reference solution
配制浓度为0.5mg/mL的来苏糖水溶液，作为内标溶液，An aqueous solution of lyxose with a concentration of 0.5 mg/mL was prepared as the internal standard solution,
分别配制葡萄糖、半乳糖、葡萄糖醛酸、甘露糖、核糖、鼠李糖、半乳糖醛酸、阿拉伯糖、岩藻糖的水溶液作为单标储存液，将各单标储存液混合后得到单糖混合水溶液，其中每mL溶液中含有5mg葡萄糖、0.3mg半乳糖、0.2mg甘露糖、0.1mg葡萄糖醛酸、0.05mg核糖、0.05mg岩藻糖、0.02mg半乳糖醛酸、0.015mg阿拉伯糖、0.01mg鼠李糖，The aqueous solutions of glucose, galactose, glucuronic acid, mannose, ribose, rhamnose, galacturonic acid, arabinose, and fucose were prepared respectively as single-label storage solutions, and the single-label storage solutions were mixed to obtain monosaccharides. Mixed aqueous solution, wherein each mL of solution contains 5mg glucose, 0.3mg galactose, 0.2mg mannose, 0.1mg glucuronic acid, 0.05mg ribose, 0.05mg fucose, 0.02mg galacturonic acid, 0.015mg arabinose, 0.01mg rhamnose,
取250μL上述单糖混合的水溶液，加入250μL内标溶液，再加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭60℃-80℃加热30min-90min，冷却至室温，加入0.15M盐酸600μL，混匀，过滤膜，即得HPLC对照品溶液；Take 250μL of the above monosaccharide mixed aqueous solution, add 250μL of internal standard solution, then add 600μL of 0.15M sodium hydroxide aqueous solution, and 1.0mL of 0.1M-0.6M 1-phenyl-3-methyl-5-pyridine The methanol solution of oxazolinone is sealed and heated at 60℃-80℃ for 30min-90min, cooled to room temperature, added with 600μL of 0.15M hydrochloric acid, mixed well, and filtered through membrane to obtain the HPLC reference solution;
(2)HPLC供试品溶液的制备(2) Preparation of HPLC test solution
配制浓度为4mg/mL的灵芝多糖样品溶液，Prepare a sample solution of Ganoderma lucidum polysaccharide with a concentration of 4 mg/mL,
精密吸取500μL样品溶液至反应瓶中，加入500μL 1M-6M的三氟乙酸；混匀，密闭，90℃-120℃加热1h-6h，挥干溶剂后，加入适量甲醇，挥干，再重复此操作两次，Precisely pipet 500μL of sample solution into the reaction bottle, add 500μL of 1M-6M trifluoroacetic acid; mix well, seal, heat at 90℃-120℃ for 1h-6h, evaporate the solvent, add appropriate amount of methanol, evaporate to dryness, and repeat this process operate twice,
残渣加250μL水，250μL内标溶液，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液；密闭60℃-80℃加热30min-90min，冷却至室温；加入0.15M盐酸600μL，混匀，过滤膜，即得供试品溶液；To the residue, add 250 μL of water, 250 μL of internal standard solution, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.1M-0.6M methanol solution of 1-phenyl-3-methyl-5-pyrazolone; sealed Heating at 60°C-80°C for 30min-90min, cooling to room temperature; adding 600μL of 0.15M hydrochloric acid, mixing, and filtering the membrane to obtain the test solution;
(3)糖组成分析(3) Analysis of sugar composition
测量供试品与对照品的HPLC色谱图，Measure the HPLC chromatograms of the test substance and reference substance,
将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比对，确定对应的单糖特征峰，以来苏糖为内标，用峰面积计算各单糖含量，Compare the retention time of the HPLC chromatogram of the test product with the HPLC chromatogram of the reference substance, determine the corresponding monosaccharide characteristic peak, use threose as the internal standard, and use the peak area to calculate the content of each monosaccharide,
方法二，其包括以下步骤：The second method includes the following steps:
(1)HPLC对照品溶液的制备(1) Preparation of HPLC reference solution
配制含0.02-0.4mg/mL(优选0.08mg/mL)甘露糖、0.0125-0.25mg/mL(优选0.05mg/mL)葡萄糖醛酸、0.3125-6.25mg/mL(优选1.25mg/mL)葡萄糖、0.0375-0.6mg/mL(优选0.15mg/mL)半乳糖、0.0125-0.25mg/mL(优选0.05mg/mL)岩藻糖、0.00625-0.125mg/mL(优选0.025mg/mL)核糖、0.001375-0.0275mg/mL(0.0055mg/mL)鼠李糖、0.0025-0.05mg/mL(优选0.01mg/mL)阿拉伯糖的水溶液为混合单糖储备液，The preparation contains 0.02-0.4mg/mL (preferably 0.08mg/mL) mannose, 0.0125-0.25mg/mL (preferably 0.05mg/mL) glucuronic acid, 0.3125-6.25mg/mL (preferably 1.25mg/mL) glucose, 0.0375-0.6mg/mL (preferably 0.15mg/mL) galactose, 0.0125-0.25mg/mL (preferably 0.05mg/mL) fucose, 0.00625-0.125mg/mL (preferably 0.025mg/mL) ribose, 0.001375- An aqueous solution of 0.0275mg/mL (0.0055mg/mL) rhamnose and 0.0025-0.05mg/mL (preferably 0.01mg/mL) arabinose is a mixed monosaccharide stock solution,
取500μL上述混合单糖储备液，再加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭60℃-80℃加热30min-90min，冷却至室温，加入0.15M盐酸600μL，混匀，取500μL溶液，加500μL氯仿，离心进行萃取，萃取结束后取上层溶液再次离心，上清液作为HPLC对照品溶液；Take 500 μL of the above mixed monosaccharide stock solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.1M-0.6M methanol solution of 1-phenyl-3-methyl-5-pyrazolone. , sealed at 60℃-80℃, heated for 30min-90min, cooled to room temperature, added 600μL of 0.15M hydrochloric acid, mixed, take 500μL of solution, add 500μL of chloroform, centrifuge for extraction, after the extraction, take the upper layer solution and centrifuge again, the supernatant as HPLC reference solution;
(2)HPLC供试品溶液的制备(2) Preparation of HPLC test solution
配制4mg/mL的灵芝多糖样品溶液，Prepare 4 mg/mL Ganoderma lucidum polysaccharide sample solution,
精密吸取500μL灵芝多糖样品溶液至反应瓶中，加入500μL 1M-6M的三氟乙酸；混匀，密闭，90℃-120℃加热1h-6h，挥干溶剂后，加入适量甲醇，挥干，再重复此操作两次，Precisely pipet 500μL of Ganoderma lucidum polysaccharide sample solution into the reaction bottle, add 500μL of 1M-6M trifluoroacetic acid; mix well, seal, heat at 90℃-120℃ for 1h-6h, evaporate the solvent, add appropriate amount of methanol, evaporate to dryness, and then Repeat this twice,
残渣加500μL水，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液；密闭60℃-80℃加热30min-90min，冷却至室温；加入0.15M盐酸600μL，混匀，取500μL溶液，加500μL氯仿，离心进行萃取，萃取结束后取上层溶液离心，上清液作为供试品溶液；Add 500 μL of water to the residue, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.1M-0.6M methanol solution of 1-phenyl-3-methyl-5-pyrazolone; sealed at 60℃-80℃ Heating for 30min-90min, cooling to room temperature; adding 600μL of 0.15M hydrochloric acid, mixing well, taking 500μL solution, adding 500μL chloroform, centrifuging for extraction, after the extraction, take the upper layer solution and centrifuge, and the supernatant is used as the test solution;
(3)糖组成分析(3) Analysis of sugar composition
测量供试品与对照品的HPLC色谱图，Measure the HPLC chromatograms of the test substance and reference substance,
将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比对，确定对应的单糖特征峰，用峰面积计算各单糖含量。Compare the retention time of the HPLC chromatogram of the test product and the HPLC chromatogram of the reference substance to determine the corresponding monosaccharide characteristic peaks, and use the peak area to calculate the content of each monosaccharide.
在一个实施方式中，本发明的灵芝多糖含量的检测方法中，步骤(一)中，在所述糖组成分析中，在以下色谱条件下测量HPLC色谱图：In one embodiment, in the detection method of Ganoderma lucidum polysaccharide content of the present invention, in step (1), in the sugar composition analysis, measure the HPLC chromatogram under the following chromatographic conditions:
高效液相色谱仪：Agilent 1260High Performance Liquid Chromatograph: Agilent 1260
色谱柱：Agilent Zorbax SB-C18(4.6mm x 250mm；5μm)Column: Agilent Zorbax SB-C18 (4.6mm x 250mm; 5μm)
柱温：35℃Column temperature: 35℃
检测波长：250nmDetection wavelength: 250nm
流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝6)，B相：乙腈Mobile phase: A phase: 0.05M phosphate buffer solution (pH=6), B phase: acetonitrile
洗脱条件：0-20min，A(％)：84-83；20-40min，A(％)：83-82.5；40-65min，A(％)：82.5-81，1mL/min的流速，Elution conditions: 0-20min, A(%): 84-83; 20-40min, A(%): 83-82.5; 40-65min, A(%): 82.5-81, flow rate of 1 mL/min,
或者在以下色谱条件下测量HPLC色谱图：Or measure HPLC chromatograms under the following chromatographic conditions:
高效液相色谱仪：Agilent 1260；High performance liquid chromatograph: Agilent 1260;
色谱柱：C6-Phenyl(4.6mm x 25cm；5μm)；Chromatographic column: C6-Phenyl (4.6mm x 25cm; 5μm);
柱温：35℃；Column temperature: 35℃;
检测波长：250nm；Detection wavelength: 250nm;
流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝7)，B相：乙腈；Mobile phase: A phase: 0.05M phosphate buffer solution (pH=7), B phase: acetonitrile;
洗脱条件：B(％)：15-16梯度洗脱；Elution conditions: B (%): 15-16 gradient elution;
流速：0.8mL/min。Flow rate: 0.8 mL/min.
在一个实施方式中，本发明的灵芝多糖含量的检测方法中，步骤(一)使用以下方法A或方法B：In one embodiment, in the detection method of Ganoderma lucidum polysaccharide content of the present invention, step (1) uses following method A or method B:
方法A，其包括以下步骤：Method A, which includes the following steps:
(1)HPLC对照品溶液的制备(1) Preparation of HPLC reference solution
配制浓度为0.5mg/mL的来苏糖水溶液，作为内标溶液，An aqueous solution of lyxose with a concentration of 0.5 mg/mL was prepared as the internal standard solution,
分别配制葡萄糖、半乳糖、葡萄糖醛酸、甘露糖、核糖、鼠李糖、半乳糖醛酸、阿拉伯糖、岩藻糖的水溶液作为单标储存液，将各单标储存液混合后得到单糖混合水溶液，其中每mL溶液中含有5mg葡萄糖、0.3mg半乳糖、0.2mg甘露糖、0.1mg葡萄糖醛酸、0.05mg核糖、0.05mg岩藻糖、0.02mg半乳糖醛酸、0.015mg阿拉伯糖、0.01mg鼠李糖，The aqueous solutions of glucose, galactose, glucuronic acid, mannose, ribose, rhamnose, galacturonic acid, arabinose, and fucose were prepared respectively as single-label storage solutions, and the single-label storage solutions were mixed to obtain monosaccharides. Mixed aqueous solution, wherein each mL of solution contains 5mg glucose, 0.3mg galactose, 0.2mg mannose, 0.1mg glucuronic acid, 0.05mg ribose, 0.05mg fucose, 0.02mg galacturonic acid, 0.015mg arabinose, 0.01mg rhamnose,
取250μL上述单糖混合的水溶液，加入250μL内标溶液，再加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭70℃加热60min，冷却至室温，加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得HPLC对照品溶液；Take 250μL of the above monosaccharide mixed aqueous solution, add 250μL of internal standard solution, then add 600μL of 0.15M sodium hydroxide aqueous solution, and 1.0mL of 0.2M 1-phenyl-3-methyl-5-pyrazolone The methanol solution was sealed at 70°C for 60min, cooled to room temperature, added with 600μL of 0.15M hydrochloric acid, mixed well, and passed through a 0.45μm nylon filter to obtain the HPLC reference solution;
(2)HPLC供试品溶液的制备(2) Preparation of HPLC test solution
配制浓度为4mg/mL的灵芝多糖样品溶液，Prepare a sample solution of Ganoderma lucidum polysaccharide with a concentration of 4 mg/mL,
精密吸取500μL样品溶液至反应瓶中，加入500μL 6M的三氟乙酸；混匀，密闭，120℃加热4h，挥干溶剂后，加入1mL甲醇，挥干，再重复 此操作两次，Precisely pipet 500 μL of the sample solution into the reaction bottle, add 500 μL of 6M trifluoroacetic acid; mix well, seal, heat at 120 °C for 4 h, evaporate the solvent, add 1 mL of methanol, evaporate to dryness, repeat this operation twice,
残渣加250μL水，250μL内标溶液，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液；密闭70℃加热60min，冷却至室温；加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得供试品溶液；To the residue, add 250 μL of water, 250 μL of internal standard solution, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone; sealed and heated at 70°C Cool to room temperature for 60 min; add 600 μL of 0.15M hydrochloric acid, mix well, and pass through a 0.45 μm nylon filter to obtain the test solution;
(3)糖组成分析(3) Analysis of sugar composition
在以下色谱条件下测量供试品与对照品的HPLC色谱图：Measure the HPLC chromatograms of the test article and reference substance under the following chromatographic conditions:
高效液相色谱仪：Agilent 1260High Performance Liquid Chromatograph: Agilent 1260
色谱柱：Agilent Zorbax SB-C18(4.6mm x 250mm；5μm)Column: Agilent Zorbax SB-C18 (4.6mm x 250mm; 5μm)
柱温：35℃Column temperature: 35℃
检测波长：250nmDetection wavelength: 250nm
流动相：A相：0.05M的磷酸盐缓冲溶液，pH＝6，B相：乙腈Mobile Phase: Phase A: 0.05M Phosphate Buffer, pH=6, Phase B: Acetonitrile
洗脱条件：0-20min，A(％)：84-83；20-40min，A(％)：83-82.5；40-65min，A(％)：82.5-81，1mL/min的流速Elution conditions: 0-20min, A(%): 84-83; 20-40min, A(%): 83-82.5; 40-65min, A(%): 82.5-81, flow rate of 1 mL/min
将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比对，确定对应的单糖特征峰，以来苏糖为内标，用峰面积计算各单糖含量，Compare the retention time of the HPLC chromatogram of the test product with the HPLC chromatogram of the reference substance, determine the corresponding monosaccharide characteristic peak, use threose as the internal standard, and use the peak area to calculate the content of each monosaccharide,
方法B，其包括以下步骤：Method B, which includes the following steps:
(1)HPLC对照品溶液的制备(1) Preparation of HPLC reference solution
配制含0.08mg/mL甘露糖、0.05mg/mL葡萄糖醛酸、1.25mg/mL葡萄糖、0.15mg/mL半乳糖、0.05mg/mL岩藻糖、0.025mg/mL核糖、0.0055mg/mL鼠李糖、0.01mg/mL阿拉伯糖的水溶液为混合单糖储备液，Formulated to contain 0.08mg/mL mannose, 0.05mg/mL glucuronic acid, 1.25mg/mL glucose, 0.15mg/mL galactose, 0.05mg/mL fucose, 0.025mg/mL ribose, 0.0055mg/mL rhamnose The aqueous solution of sugar and 0.01 mg/mL arabinose is the mixed monosaccharide stock solution,
取500μL混合单糖储备液，加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭70℃加热60min，取出冰水浴2min，加入0.15M盐酸600μL，混匀，取500μL溶液，加500μL氯仿，离心进行萃取，萃取结束后取上层溶液离心，上清液作为HPLC对照品溶液；Take 500 μL of mixed monosaccharide stock solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, sealed and heated at 70°C for 60min , take out the ice-water bath for 2 min, add 600 μL of 0.15M hydrochloric acid, mix well, take 500 μL of the solution, add 500 μL of chloroform, centrifuge for extraction, and centrifuge the upper layer solution after extraction, and the supernatant is used as the HPLC reference solution;
(2)HPLC供试品溶液的制备(2) Preparation of HPLC test solution
配制浓度为4mg/mL的灵芝多糖样品溶液，Prepare a sample solution of Ganoderma lucidum polysaccharide with a concentration of 4 mg/mL,
精密吸取灵芝多糖样品溶液500μL至反应瓶中，加入500μL 6M的三氟乙酸，混匀，密闭，120℃加热4h，挥干溶剂后，加入1mL甲醇，挥干，再重复此操作两次，Precisely pipet 500 μL of Ganoderma lucidum polysaccharide sample solution into the reaction bottle, add 500 μL of 6M trifluoroacetic acid, mix well, seal, heat at 120 °C for 4 h, evaporate the solvent, add 1 mL of methanol, evaporate to dryness, repeat this operation twice,
残渣加500μL水，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭反应瓶70℃加热60 min，取出冰水浴2min，加入0.15M盐酸600μL，混匀，取500μL溶液，加500μL氯仿，离心进行萃取，萃取结束后取上层溶液离心，上清液作为供试品溶液；The residue was added with 500 μL of water, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, and the reaction flask was sealed and heated at 70°C for 60 min. Take out the ice-water bath for 2 minutes, add 600 μL of 0.15M hydrochloric acid, mix well, take 500 μL of the solution, add 500 μL of chloroform, and centrifuge for extraction.
(3)糖组成分析(3) Analysis of sugar composition
在以下色谱条件下测量供试品与对照品的HPLC色谱图，Measure the HPLC chromatograms of the test substance and reference substance under the following chromatographic conditions,
高效液相色谱仪：Agilent 1260；High performance liquid chromatograph: Agilent 1260;
色谱柱：C6-Phenyl(4.6mm x 25cm；5μm)；Chromatographic column: C6-Phenyl (4.6mm x 25cm; 5μm);
柱温：35℃；Column temperature: 35℃;
检测波长：250nm；Detection wavelength: 250nm;
流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝7)，B相：乙腈；Mobile phase: A phase: 0.05M phosphate buffer solution (pH=7), B phase: acetonitrile;
洗脱条件：B(％)：15-16梯度洗脱；Elution conditions: B (%): 15-16 gradient elution;
流速：0.8mL/min，Flow rate: 0.8mL/min,
将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比对，确定对应的单糖特征峰，用峰面积计算各单糖含量。Compare the retention time of the HPLC chromatogram of the test product and the HPLC chromatogram of the reference substance to determine the corresponding monosaccharide characteristic peaks, and use the peak area to calculate the content of each monosaccharide.
优选地，本发明的灵芝多糖含量的检测方法中，步骤(一)方法一或方法A中，HPLC对照品溶液的制备包括以下步骤：Preferably, in the detection method of Ganoderma lucidum polysaccharide content of the present invention, in step (1) method 1 or method A, the preparation of the HPLC reference solution comprises the following steps:
配制浓度为50mg/mL葡萄糖储备液；The preparation concentration is 50mg/mL glucose stock solution;
配制浓度为6mg/mL的半乳糖储备液；Prepare a galactose stock solution with a concentration of 6 mg/mL;
配制浓度为1mg/mL的葡萄糖醛酸储备液；Prepare a glucuronic acid stock solution with a concentration of 1 mg/mL;
配制浓度为2mg/mL的甘露糖储备液；Prepare a mannose stock solution with a concentration of 2 mg/mL;
配制浓度为0.5mg/mL的核糖储备液；Prepare a ribose stock solution with a concentration of 0.5 mg/mL;
配制浓度为2mg/mL的鼠李糖储备液；Prepare a rhamnose stock solution with a concentration of 2 mg/mL;
配制浓度为10mg/mL的半乳糖醛酸储备液；Prepare a galacturonic acid stock solution with a concentration of 10 mg/mL;
配制浓度为15mg/mL的阿拉伯糖储备液；Prepare an arabinose stock solution with a concentration of 15 mg/mL;
配制浓度为0.5mg/mL的岩藻糖储备液；Prepare a fucose stock solution with a concentration of 0.5 mg/mL;
配制浓度为0.5mg/mL的来苏糖内标溶液，Prepare a lyxose internal standard solution with a concentration of 0.5 mg/mL,
分别精密吸取1000体积核糖储备液、1000体积岩藻糖储备液、1000体积葡萄糖醛酸储备液、1000体积甘露糖储备液、1000体积葡萄糖储备液、500体积半乳糖储备液、50体积鼠李糖储备液、20体积半乳糖醛酸储备液、10体积阿拉伯糖储备液，加水定容至10000体积，即得混合溶液；Precisely draw 1000 volumes of ribose stock solution, 1000 volumes of fucose stock solution, 1000 volumes of glucuronic acid stock solution, 1000 volumes of mannose stock solution, 1000 volumes of glucose stock solution, 500 volumes of galactose stock solution, and 50 volumes of rhamnose Stock solution, 20 volumes of galacturonic acid stock solution, and 10 volumes of arabinose stock solution, add water to make up to 10,000 volumes to obtain a mixed solution;
取250μL上述混合溶液，250μL内标溶液，分别加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭60℃-80℃加热30min-90min，冷却至室温，加入0.15 M盐酸600μL，混匀，过滤膜，即得HPLC对照品溶液。Take 250 μL of the above mixed solution, 250 μL of the internal standard solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.1M-0.6M 1-phenyl-3-methyl-5-pyrazolone. Methanol solution, sealed at 60℃-80℃, heated for 30min-90min, cooled to room temperature, added 600μL of 0.15 M hydrochloric acid, mixed well, filtered through membrane, to obtain HPLC reference solution.
更优选地，本发明的灵芝多糖含量的检测方法中，步骤(一)方法一或方法A中，HPLC对照品溶液的制备包括以下步骤：More preferably, in the detection method of Ganoderma lucidum polysaccharide content of the present invention, in step (1) method 1 or method A, the preparation of HPLC reference substance solution comprises the following steps:
配制浓度为50mg/mL葡萄糖储备液；The preparation concentration is 50mg/mL glucose stock solution;
配制浓度为6mg/mL的半乳糖储备液；Prepare a galactose stock solution with a concentration of 6 mg/mL;
配制浓度为1mg/mL的葡萄糖醛酸储备液；Prepare a glucuronic acid stock solution with a concentration of 1 mg/mL;
配制浓度为2mg/mL的甘露糖储备液；Prepare a mannose stock solution with a concentration of 2 mg/mL;
配制浓度为0.5mg/mL的核糖储备液；Prepare a ribose stock solution with a concentration of 0.5 mg/mL;
配制浓度为2mg/mL的鼠李糖储备液；Prepare a rhamnose stock solution with a concentration of 2 mg/mL;
配制浓度为10mg/mL的半乳糖醛酸储备液；Prepare a galacturonic acid stock solution with a concentration of 10 mg/mL;
配制浓度为15mg/mL的阿拉伯糖储备液；Prepare an arabinose stock solution with a concentration of 15 mg/mL;
配制浓度为0.5mg/mL的岩藻糖储备液；Prepare a fucose stock solution with a concentration of 0.5 mg/mL;
配制浓度为0.5mg/mL的来苏糖内标溶液，Prepare a lyxose internal standard solution with a concentration of 0.5 mg/mL,
分别精密吸取1000体积核糖储备液、1000体积岩藻糖储备液、1000体积葡萄糖醛酸储备液、1000体积甘露糖储备液、1000体积葡萄糖储备液、500体积半乳糖储备液、50体积鼠李糖储备液、20体积半乳糖醛酸储备液、10体积阿拉伯糖储备液，加水定容至10000体积，即得混合溶液；Precisely draw 1000 volumes of ribose stock solution, 1000 volumes of fucose stock solution, 1000 volumes of glucuronic acid stock solution, 1000 volumes of mannose stock solution, 1000 volumes of glucose stock solution, 500 volumes of galactose stock solution, and 50 volumes of rhamnose Stock solution, 20 volumes of galacturonic acid stock solution, and 10 volumes of arabinose stock solution, add water to make up to 10,000 volumes to obtain a mixed solution;
取250μL上述混合溶液，250μL内标溶液，分别加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭70℃加热60min，冷却至室温，加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得HPLC对照品溶液。Take 250 μL of the above mixed solution, 250 μL of the internal standard solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, respectively, Sealed and heated at 70°C for 60min, cooled to room temperature, added 600μL of 0.15M hydrochloric acid, mixed well, and passed through a 0.45μm nylon filter to obtain the HPLC reference solution.
优选地，本发明的灵芝多糖含量的检测方法中，步骤(三)中，所述混合单糖溶液为含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的水溶液，其中，以重量百分比计，基于溶液中的单糖的总重量，含有60％-85％的葡萄糖、8％-17％的半乳糖、3％-15％的葡萄糖醛酸、3％-7％的甘露糖，或者含有60％-90％的葡萄糖、6％-17％的半乳糖、1％-15％的葡萄糖醛酸、3％-8％的甘露糖，或者，所述混合单糖溶液为以高效液相色谱确定的单糖比例配制含量检测需要的对照品溶液，总浓度为0.1mg/mL。Preferably, in the method for detecting the polysaccharide content of Ganoderma lucidum of the present invention, in step (3), the mixed monosaccharide solution is an aqueous solution containing glucose, galactose, glucuronic acid and mannose, wherein, by weight percentage, based on Total weight of monosaccharides in solution containing 60%-85% glucose, 8%-17% galactose, 3%-15% glucuronic acid, 3%-7% mannose, or 60% -90% glucose, 6%-17% galactose, 1%-15% glucuronic acid, 3%-8% mannose, or the mixed monosaccharide solution is determined by high performance liquid chromatography The monosaccharide ratio was used to prepare the reference solution required for content detection, and the total concentration was 0.1 mg/mL.
在一个所述方式中，本发明的灵芝多糖含量的检测方法中，所述苯酚-硫酸法，包括以下步骤：In one described mode, in the detection method of Ganoderma lucidum polysaccharide content of the present invention, the phenol-sulfuric acid method comprises the following steps:
(1)苯酚溶液的配制(1) Preparation of phenol solution
配制0.05g/mL的苯酚水溶液；Prepare 0.05g/mL aqueous solution of phenol;
(2)含量检测对照品溶液的制备(2) Preparation of content detection reference solution
根据步骤(一)测定的单糖比例配制至少含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的混合单糖溶液作为对照品溶液，总浓度为0.1mg/mL，优选地，配制混合单糖溶液作为对照品溶液，所述混合单糖溶液含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的水溶液，其中，以重量百分比计，基于溶液中的单糖的总重量，含有60％-85％的葡萄糖、8％-17％的半乳糖、3％-15％的葡萄糖醛酸、3％-7％的甘露糖，或者含有60％-90％的葡萄糖、6％-17％的半乳糖、1％-15％的葡萄糖醛酸、3％-8％的甘露糖；According to the monosaccharide ratio determined in step (1), a mixed monosaccharide solution containing at least glucose, galactose, glucuronic acid and mannose is prepared as a reference solution, and the total concentration is 0.1 mg/mL. Preferably, the mixed monosaccharide solution is prepared As a reference solution, the mixed monosaccharide solution contains an aqueous solution of glucose, galactose, glucuronic acid and mannose, wherein, by weight percentage, based on the total weight of the monosaccharides in the solution, contains 60%-85% of Glucose, 8%-17% galactose, 3%-15% glucuronic acid, 3%-7% mannose, or with 60%-90% glucose, 6%-17% galactose, 1 %-15% glucuronic acid, 3%-8% mannose;
(3)苯酚-硫酸法标准曲线的制作(3) Preparation of standard curve of phenol-sulfuric acid method
精密量取步骤(2)中对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置具塞试管中，分别加水补至2.0mL，各精密加入步骤(1)中苯酚溶液1mL，摇匀，迅速精密加入浓硫酸5mL，摇匀，放置10分钟，置40℃水浴中保温15分钟，取出，冰浴2min，空白不加对照品溶液，测定在490nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制苯酚-硫酸法标准曲线；Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the reference solution in step (2), put them in test tubes with stoppers, add water to make up to 2.0mL, and add water to step (1) precisely. 1mL of medium phenol solution, shake well, quickly and accurately add 5mL of concentrated sulfuric acid, shake well, place for 10 minutes, place in a 40°C water bath for 15 minutes, take out, ice bath for 2 minutes, blank without reference solution, measure the absorbance at 490nm , take the absorbance as the ordinate and the concentration of the sugar reference substance as the abscissa, draw the phenol-sulfuric acid method standard curve;
(4)含量检测供试品的制备(4) Preparation of test sample for content detection
准确称取待测灵芝多糖样品10mg，于100mL的容量瓶中，用纯水定容，充分溶解混匀，即得供试品溶液；Accurately weigh 10 mg of the Ganoderma lucidum polysaccharide sample to be tested, put it in a 100 mL volumetric flask, dilute to volume with pure water, fully dissolve and mix to obtain the test solution;
(5)多糖含量的检测(5) Detection of polysaccharide content
精密吸取步骤(4)中供试品溶液1.0mL，加水至2.0mL，照步骤(3)苯酚-硫酸法标准曲线的制作项下的方法，自“加入苯酚溶液1mL”起，依法测定供试品的吸光度，根据苯酚-硫酸法标准曲线得到多糖含量；Accurately draw 1.0 mL of the test solution in step (4), add water to 2.0 mL, and follow the method under step (3) Preparation of the phenol-sulfuric acid method standard curve, starting from "adding 1 mL of phenol solution", determine the test according to the law The absorbance of the product was obtained, and the polysaccharide content was obtained according to the standard curve of the phenol-sulfuric acid method;
所述蒽酮-硫酸法包括以下步骤：The anthrone-sulfuric acid method comprises the following steps:
(1)蒽酮硫酸溶液的配制(1) Preparation of anthrone sulfuric acid solution
配制0.001g/mL的硫酸蒽酮溶液；Prepare 0.001g/mL solution of anthrone sulfate;
(2)含量检测对照品溶液的制备(2) Preparation of content detection reference solution
以步骤(一)确定的单糖比例配制至少含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的混合单糖溶液作为对照品溶液，总浓度为0.1mg/mL，优选地，配制混合单糖溶液作为对照品溶液，所述混合单糖溶液含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的水溶液，其中，以重量百分比计，基于溶液中的单糖的总重量，含有60％-85％的葡萄糖、8％-17％的半乳糖、3％-15％的葡萄糖醛酸、3％-7％的甘露糖，或者含有60％-90％的葡萄糖、6％-17％的半乳糖、1％-15％的葡萄糖醛酸、3％-8％的甘露糖；Prepare a mixed monosaccharide solution containing at least glucose, galactose, glucuronic acid and mannose with the monosaccharide ratio determined in step (1) as a reference solution, and the total concentration is 0.1 mg/mL. Preferably, the mixed monosaccharide solution is prepared As a reference solution, the mixed monosaccharide solution contains an aqueous solution of glucose, galactose, glucuronic acid and mannose, wherein, by weight percentage, based on the total weight of the monosaccharides in the solution, contains 60%-85% of Glucose, 8%-17% galactose, 3%-15% glucuronic acid, 3%-7% mannose, or with 60%-90% glucose, 6%-17% galactose, 1 %-15% glucuronic acid, 3%-8% mannose;
(3)蒽酮-硫酸法标准曲线的制作(3) Preparation of standard curve of anthrone-sulfuric acid method
精密量取步骤(2)中对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置于具塞试管中，分别加水补至2.0mL，各精密加入步骤(1)中硫酸蒽酮溶液6mL，摇匀，放置15min后，置于冰浴中冷却15min，取出，空白不加对照品溶液，测定在625nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制蒽酮-硫酸法标准曲线；Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the reference solution in step (2), place them in test tubes with stoppers, add water to make up to 2.0mL, and add water to 2.0mL for each precise addition step (1). ) in 6mL of anthrone sulfate solution, shake well, put it for 15min, cool it in an ice bath for 15min, take it out, blank without the reference solution, measure the absorbance at 625nm, take the absorbance as the ordinate, and the concentration of the sugar reference is Abscissa, draw the standard curve of anthrone-sulfuric acid method;
(4)含量检测供试品的制备(4) Preparation of test sample for content detection
准确称取待测灵芝多糖样品10mg，于100mL的容量瓶中，用纯水定容，充分溶解混匀，即得供试品溶液；Accurately weigh 10 mg of the Ganoderma lucidum polysaccharide sample to be tested, put it in a 100 mL volumetric flask, dilute to volume with pure water, fully dissolve and mix to obtain the test solution;
(5)多糖含量的检测(5) Detection of polysaccharide content
精密吸取步骤(6)中供试品溶液1.0mL，加水至2.0mL，照步骤(5)蒽酮-硫酸法标准曲线的制作项下的方法，自“加入硫酸蒽酮溶液6mL”起，依法测定供试品的吸光度，根据蒽酮-硫酸法标准曲线得到多糖含量。Accurately draw 1.0 mL of the test solution in step (6), add water to 2.0 mL, and follow the method under step (5) preparation of the standard curve of anthrone-sulfuric acid method, starting from "adding 6 mL of anthrone sulfate solution", according to the law. The absorbance of the test sample was measured, and the polysaccharide content was obtained according to the standard curve of the anthrone-sulfuric acid method.
在另一个实施方式中，本发明的灵芝多糖含量的检测方法包括以下步骤：In another embodiment, the detection method of Ganoderma lucidum polysaccharide content of the present invention comprises the following steps:
(1)苯酚溶液的配制(1) Preparation of phenol solution
配制0.05g/mL的苯酚水溶液；Prepare 0.05g/mL aqueous solution of phenol;
(2)硫酸蒽酮溶液的配制(2) Preparation of anthrone sulfate solution
配制0.001g/mL的硫酸蒽酮溶液；Prepare 0.001g/mL solution of anthrone sulfate;
(3)混合单糖对照品溶液的制备(3) Preparation of mixed monosaccharide reference solution
确定供试品中的单糖组成，并计算各单糖含量，以高效液相色谱确定的单糖比例配制含量检测需要的对照品溶液，总浓度为0.1mg/mL；Determine the monosaccharide composition in the test product, calculate the content of each monosaccharide, and prepare the reference solution required for content detection with the monosaccharide ratio determined by high performance liquid chromatography, with a total concentration of 0.1 mg/mL;
(4)苯酚-硫酸法标准曲线的制作(4) Preparation of standard curve of phenol-sulfuric acid method
精密量取步骤(3)中混合单糖对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置具塞试管中，分别加水补至2.0mL，各精密加入步骤1中制得的苯酚溶液1mL，摇匀，迅速精密加入浓硫酸5mL，摇匀，放置10分钟，置40℃水浴中保温15min，取出，冰浴2min，空白不加对照品溶液，测定在490nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制标准曲线：Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step (3), put them in test tubes with stoppers, and add water to make up to 2.0mL respectively. 1 mL of the phenol solution prepared in 1, shake well, quickly and accurately add 5 mL of concentrated sulfuric acid, shake well, place for 10 minutes, place in a 40°C water bath for 15 minutes, take out, ice bath for 2 minutes, blank without reference solution, measure at 490nm Take the absorbance as the ordinate and the concentration of the sugar reference substance as the abscissa to draw the standard curve:
(5)蒽酮-硫酸法标准曲线的制作(5) Preparation of standard curve of anthrone-sulfuric acid method
精密量取步骤(3)中混合单糖对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置于具塞试管中，分别加水补至2.0mL，各精密加入步骤2中制得的硫酸蒽酮溶液6mL，摇匀，放置15min后，置于冰浴中冷却15min，取出，空白不加对照品溶液，测定在625nm处 的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制标准曲线：Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step (3), place them in test tubes with stoppers, respectively add water to make up to 2.0mL, and add each precision The anthrone sulfate solution prepared in step 2 was 6mL, shaken up, placed for 15min, cooled in an ice bath for 15min, taken out, blank without the reference solution, and measured the absorbance at 625nm, taking the absorbance as the ordinate, sugar The concentration of the reference substance is the abscissa, and the standard curve is drawn:
(6)供试品的制备(6) Preparation of the test product
准确称取灵芝多糖样品10mg，于100mL的容量瓶中，用纯水定容，充分溶解混匀，即得；Accurately weigh 10mg of Ganoderma lucidum polysaccharide sample, put it in a 100mL volumetric flask, dilute to volume with pure water, fully dissolve and mix to get it;
(7)供试品的检测(7) Detection of the test product
a、苯酚-硫酸法a. Phenol-sulfuric acid method
精密吸取步骤(6)中供试品溶液1.0mL，加水至2.0mL，照(4)步骤苯酚-硫酸法标准曲线的制作项下的方法，自“加入苯酚溶液1mL”起，依上述方法测定供试品的吸光度，根据苯酚-硫酸法标准曲线得到多糖含量；Accurately draw 1.0 mL of the test solution in step (6), add water to 2.0 mL, and measure according to the above method according to the method under the preparation of the standard curve of the phenol-sulfuric acid method in step (4), starting from "adding 1 mL of phenol solution" The absorbance of the test product, the polysaccharide content is obtained according to the standard curve of the phenol-sulfuric acid method;
b、蒽酮-硫酸法b. Anthrone-sulfuric acid method
精密吸取供试品溶液1.0mL，加水至2.0mL，照(5)步骤蒽酮-硫酸法标准曲线的制作项下的方法，自“加入硫酸蒽酮溶液6mL”起，依法测定供试品的吸光度，根据蒽酮-硫酸法标准曲线得到多糖含量。Accurately draw 1.0 mL of the solution of the test sample, add water to 2.0 mL, and follow the method under the preparation of the standard curve of the anthrone-sulfuric acid method in step (5). Absorbance, polysaccharide content was obtained according to the standard curve of anthrone-sulfuric acid method.
附图说明Description of drawings
图1为灵芝多糖(20200825-05)水解后衍生物的HPLC色谱图。Figure 1 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200825-05) after hydrolysis.
图2为灵芝多糖(20200902-05)水解后衍生物的HPLC色谱图。Figure 2 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200902-05) after hydrolysis.
图3为灵芝多糖(20200903-08)水解后衍生物的HPLC色谱图。Figure 3 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200903-08) after hydrolysis.
图4为灵芝多糖(21001)水解后衍生物的HPLC色谱图。Figure 4 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21001) after hydrolysis.
图5为灵芝多糖(21002)水解后衍生物的HPLC色谱图。Figure 5 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21002) after hydrolysis.
图6为灵芝多糖(21003)水解后衍生物的HPLC色谱图。Figure 6 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21003) after hydrolysis.
图7为灵芝多糖(21004)水解后衍生物的HPLC色谱图。Figure 7 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21004) after hydrolysis.
具体实施方式Detailed ways
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于示例性地对本发明进行说明，并不用于限制本发明。Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate the present invention by way of example, and are not used to limit the present invention.
实施例Example
实验材料和设备Experimental Materials and Equipment
1、仪器1. Instrument
Agilent 1260型高效液相色谱仪(美国安捷伦科技有限公司)Agilent 1260 High Performance Liquid Chromatograph (Agilent Technologies Co., Ltd., USA)
UV5Bio型紫外可见光光度计(梅特勒-托利多国际贸易上海有限公司)UV5Bio UV-Vis Photometer (Mettler-Toledo International Trading Shanghai Co., Ltd.)
Satorius SQP电子分析天平(赛多利斯科学仪器北京有限公司)Satorius SQP Electronic Analytical Balance (Satorius Scientific Instruments Beijing Co., Ltd.)
DK-S24型电热恒温水浴锅(上海精宏实验设备有限公司)DK-S24 type electric heating constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.)
R-300型旋转蒸发仪(步琦实验室设备贸易上海有限公司)R-300 Rotary Evaporator (Buchi Laboratory Equipment Trading Shanghai Co., Ltd.)
Pico 17型高速离心机(赛默飞世尔科技中国有限公司)Pico 17 high-speed centrifuge (Thermo Fisher Scientific China Co., Ltd.)
DRY BLOCK HEATER 3干浴器(艾卡广州仪器设备有限公司)DRY BLOCK HEATER 3 Dry Bath (Aika Guangzhou Instrument Equipment Co., Ltd.)
fiveEasy实验室pH计(梅特勒-托利多国际贸易上海有限公司)fiveEasy laboratory pH meter (METTLER TOLEDO International Trading Shanghai Co., Ltd.)
2、试剂2. Reagents
灵芝多糖(批号：20200825-05，灵芝药材用95％乙醇脱脂后，沸水提取，浓缩提取液，用95％乙醇醇沉，得到的灵芝粗多糖。批号：20200902-05，灵芝药材用95％乙醇脱脂后，沸水提取，浓缩提取液，先用20％乙醇醇沉，再用80％乙醇醇沉，得到灵芝粗多糖。批号：20200903-08，灵芝药材用95％乙醇脱脂后，沸水提取，浓缩提取液，用95％乙醇醇沉后，粗多糖过DEAE离子交换纤维素层析柱，收集并浓缩水洗部位，再用Sephadex S-300葡聚糖凝胶层析柱纯化，得到灵芝多糖)Ganoderma lucidum polysaccharide (batch number: 20200825-05, after degreasing Ganoderma lucidum medicinal materials with 95% ethanol, boiling water extraction, concentrating the extract, ethanol precipitation with 95% ethanol, the obtained Ganoderma lucidum crude polysaccharide. Batch number: 20200902-05, Ganoderma lucidum medicinal materials with 95% ethanol After degreasing, extracting with boiling water, concentrating the extract, first ethanol precipitation with 20% ethanol, and then ethanol precipitation with 80% ethanol, to obtain Ganoderma lucidum crude polysaccharide. Batch number: 20200903-08, Ganoderma lucidum medicinal materials are degreased with 95% ethanol, extracted with boiling water, concentrated The extract was precipitated with 95% ethanol, and the crude polysaccharide was passed through a DEAE ion-exchange cellulose chromatography column, and the water-washed parts were collected and concentrated, and then purified by a Sephadex S-300 Sephadex column to obtain Ganoderma lucidum polysaccharide)
D-(+)-无水葡萄糖(批号：BCCC7504；西格玛奥德里奇上海贸易有限公司；≥99.5％)D-(+)-Dextrose Anhydrous (Lot No.: BCCC7504; Sigma-Aldrich Shanghai Trading Co., Ltd.; ≥99.5%)
L-(+)-阿拉伯糖(批号：WXBD0839V；西格玛奥德里奇上海贸易有限公司；≥99％)L-(+)-Arabinose (Lot No.: WXBD0839V; Sigma-Aldrich Shanghai Trading Co., Ltd.; ≥99%)
D-(+)-半乳糖(批号：WXBD1824V；西格玛奥德里奇上海贸易有限公司；≥99％)D-(+)-Galactose (Lot No.: WXBD1824V; Sigma-Aldrich Shanghai Trading Co., Ltd.; ≥99%)
D-(+)-甘露糖(批号：WXBC8934V；西格玛奥德里奇上海贸易有限公司；≥99％)D-(+)-Mannose (Lot No.: WXBC8934V; Sigma-Aldrich Shanghai Trading Co., Ltd.; ≥99%)
D-(-)-核糖(批号：WXBB0660；西格玛奥德里奇上海贸易有限公司；99％)D-(-)-ribose (Lot No.: WXBB0660; Sigma-Aldrich Shanghai Trading Co., Ltd.; 99%)
L-鼠李糖(批号：WXBD0644V；西格玛奥德里奇上海贸易有限公司；≥99％)L-Rhamnose (Lot No.: WXBD0644V; Sigma-Aldrich Shanghai Trading Co., Ltd.; ≥99%)
D-葡萄糖醛酸(批号：SLCF7105；西格玛奥德里奇上海贸易有限公司；≥98％)D-glucuronic acid (Lot number: SLCF7105; Sigma-Aldrich Shanghai Trading Co., Ltd.; ≥98%)
D-(+)-半乳糖醛酸(批号：BCCB4778；西格玛奥德里奇上海贸易有限公司；≥95.0％)D-(+)-Galacturonic acid (Lot number: BCCB4778; Sigma-Aldrich Shanghai Trading Co., Ltd.; ≥95.0%)
L-(-)-岩藻糖(批号：WXBD3473V；西格玛奥德里奇上海贸易有限公司；≥99％)L-(-)-Fucose (Lot No.: WXBD3473V; Sigma-Aldrich Shanghai Trading Co., Ltd.; ≥99%)
D-(-)-来苏糖(批号：A11S11L124094；上海源叶生物科技有限公司；99％)D-(-)-lyxose (batch number: A11S11L124094; Shanghai Yuanye Biotechnology Co., Ltd.; 99%)
甲醇(批号：TCJG1H；Honeywell霍尼韦尔；HPLC)Methanol (Lot: TCJG1H; Honeywell Honeywell; HPLC)
乙腈(批号：TA5A1H；Honeywell霍尼韦尔；HPLC)Acetonitrile (Lot: TA5A1H; Honeywell; HPLC)
氢氧化钠(批号：20160922；国药集团化学试剂；AR)Sodium hydroxide (batch number: 20160922; Sinopharm Chemical Reagent; AR)
磷酸二氢钾(批号：20200617；国药集团化学试剂；GR)Potassium dihydrogen phosphate (batch number: 20200617; Sinopharm Chemical Reagent; GR)
三氟乙酸(批号：20200821；国药集团化学试剂；AR)Trifluoroacetic acid (batch number: 20200821; Sinopharm Chemical Reagent; AR)
盐酸(批号：20200928；国药集团化学试剂；AR)Hydrochloric acid (batch number: 20200928; Sinopharm Chemical Reagent; AR)
浓硫酸(批号：20191111；国药集团化学试剂；AR)Concentrated sulfuric acid (batch number: 20191111; Sinopharm Chemical Reagent; AR)
1-苯基-3-甲基-5-吡唑啉酮(批号：20200805；国药集团化学试剂；CP)1-Phenyl-3-methyl-5-pyrazolone (batch number: 20200805; Sinopharm Chemical Reagent; CP)
苯酚(批号：20150327；国药集团化学试剂；AR)Phenol (batch number: 20150327; Sinopharm Chemical Reagent; AR)
蒽酮(批号：20200312；国药集团化学试剂；AR)Anthrone (batch number: 20200312; Sinopharm Chemical Reagent; AR)
实施例1：糖组成条件优化Example 1: Optimization of sugar composition conditions
多糖的单糖组成分析是研究多糖结构及其特征的基础，柱前衍生化HPLC法是糖组成分析常用分析方法，具有较高的灵敏度，可同时测定中性糖和酸性糖。多糖水解和衍生化条件对检测结果具有直接的影响，单糖的释放度受到不同反应条件的影响，酸性糖易释放但不稳定，中性糖较酸性糖稳定但不易释放，需优选最佳的反应条件用于多糖的糖组成分析。Monosaccharide composition analysis of polysaccharides is the basis for studying the structure and characteristics of polysaccharides. Pre-column derivatization HPLC method is a common analytical method for sugar composition analysis. It has high sensitivity and can simultaneously determine neutral sugars and acidic sugars. The hydrolysis and derivatization conditions of polysaccharides have a direct impact on the detection results. The release of monosaccharides is affected by different reaction conditions. Acidic sugars are easy to release but unstable. Neutral sugars are more stable than acidic sugars but not easy to release. The reaction conditions were used for saccharide composition analysis of polysaccharides.
因此发明人对1-苯基-3-甲基-5-吡唑啉酮柱前衍生化HPLC法测定灵芝多糖的单糖组成的水解和衍生化条件进行了详细的考察和优化，采用正交实验以单糖峰面积和总峰面积为指标，考察了水解和衍生化的时间、温度、三氟乙酸浓度及1-苯基-3-甲基-5-吡唑啉酮浓度。酸水解的时间考察了1h、2h、4h、6h；温度考察了90℃、100℃、110℃、120℃；三氟乙酸的浓度考察了1M、2M、4M、6M。衍生化时间考察了30min、60min、90min；1-苯基-3-甲基-5-吡唑啉酮的浓度考察了0.1M、0.2M、0.4M、0.6M，对不同条件反应的多糖衍生化后HPLC结果的峰面积进行了比较，并通过正交分析优选了以下条件，酸水解时间4h，加热温度120℃，三氟乙酸浓度为6M时，衍生化时间60min，1-苯基-3-甲基-5-吡唑啉酮的浓度为0.2M，分析发现在优化后的反应条件下，单糖峰面积和总峰面积较优化前(美国药典USP 41，Volume 3，4629-4632)明显增加，表明优选后的条件对多糖的水解和衍生化效果更好。Therefore, the inventors conducted a detailed investigation and optimization of the hydrolysis and derivatization conditions for the determination of the monosaccharide composition of Ganoderma lucidum polysaccharides by 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization HPLC method. Using the monosaccharide peak area and total peak area as indicators, the hydrolysis and derivatization time, temperature, concentration of trifluoroacetic acid and concentration of 1-phenyl-3-methyl-5-pyrazolone were investigated. The time of acid hydrolysis was 1h, 2h, 4h, 6h; the temperature was 90℃, 100℃, 110℃, 120℃; the concentration of trifluoroacetic acid was 1M, 2M, 4M, 6M. The derivatization time was investigated for 30min, 60min and 90min; the concentration of 1-phenyl-3-methyl-5-pyrazolone was investigated as 0.1M, 0.2M, 0.4M and 0.6M. The peak areas of the HPLC results were compared, and the following conditions were optimized by orthogonal analysis, the acid hydrolysis time was 4h, the heating temperature was 120℃, the concentration of trifluoroacetic acid was 6M, the derivatization time was 60min, the 1-phenyl-3 -The concentration of methyl-5-pyrazolone is 0.2M. It is found that under the optimized reaction conditions, the monosaccharide peak area and total peak area are better than those before optimization (USP 41, Volume 3, 4629-4632) Significantly increased, indicating that the optimized conditions had better effect on the hydrolysis and derivatization of polysaccharides.
1、对照品溶液和内标溶液的制备1. Preparation of reference solution and internal standard solution
准确称取葡萄糖90mg，置于容量瓶中，用纯水定容，配制成浓度为45mg/mL葡萄糖储备液；Accurately weigh 90 mg of glucose, place it in a volumetric flask, dilute to volume with pure water, and prepare a glucose stock solution with a concentration of 45 mg/mL;
准确称取半乳糖10mg，置于容量瓶中，用纯水定容，配制成浓度为5mg/mL的半乳糖储备液；Accurately weigh 10 mg of galactose, place it in a volumetric flask, and dilute to volume with pure water to prepare a galactose stock solution with a concentration of 5 mg/mL;
准确称取葡萄糖醛酸8mg，置于容量瓶中，用纯水定容，配制成浓度为0.8mg/mL的葡萄糖醛酸储备液；Accurately weigh 8 mg of glucuronic acid, place it in a volumetric flask, dilute to volume with pure water, and prepare a glucuronic acid stock solution with a concentration of 0.8 mg/mL;
准确称取甘露糖25mg，置于容量瓶中，用纯水定容，配制成浓度为2.5mg/mL的甘露糖储备液；Accurately weigh 25 mg of mannose, place it in a volumetric flask, dilute to volume with pure water, and prepare a mannose stock solution with a concentration of 2.5 mg/mL;
准确称取核糖12mg，置于容量瓶中，用纯水定容，配制成浓度为0.6mg/mL的核糖储备液；Accurately weigh 12 mg of ribose, place it in a volumetric flask, dilute to volume with pure water, and prepare a ribose stock solution with a concentration of 0.6 mg/mL;
准确称取鼠李糖25mg，置于容量瓶中，用纯水定容，配制成浓度为2.5mg/mL的鼠李糖储备液；Accurately weigh 25 mg of rhamnose, place it in a volumetric flask, dilute to volume with pure water, and prepare a rhamnose stock solution with a concentration of 2.5 mg/mL;
准确称取半乳糖醛酸16mg，置于容量瓶中，用纯水定容，配制成浓度为8mg/mL的半乳糖醛酸储备液；Accurately weigh 16 mg of galacturonic acid, place it in a volumetric flask, dilute to volume with pure water, and prepare a galacturonic acid stock solution with a concentration of 8 mg/mL;
准确称取阿拉伯糖40mg，置于容量瓶中，用纯水定容，配制成浓度为20mg/mL的阿拉伯糖储备液；Accurately weigh 40 mg of arabinose, place it in a volumetric flask, and dilute to volume with pure water to prepare an arabinose stock solution with a concentration of 20 mg/mL;
准确称取岩藻糖12mg，置于容量瓶中，用纯水定容，配制成浓度为0.6mg/mL的岩藻糖储备液。Accurately weigh 12 mg of fucose, place it in a volumetric flask, dilute to volume with pure water, and prepare a fucose stock solution with a concentration of 0.6 mg/mL.
准确称取来苏糖12.5mg，置于25mL的容量品中，用纯水定容，混匀，即为来苏糖水溶液，即内标溶液。Accurately weigh 12.5mg of lyxose, put it in a 25mL volume product, dilute to volume with pure water, and mix well, which is the lyxose aqueous solution, that is, the internal standard solution.
将上述各储备液分别混匀，分别准确吸取1.0mL核糖储备液、1.0mL岩藻糖储备液、1.0mL葡萄糖醛酸储备液、1.0mL甘露糖储备液、1.0mL葡萄糖储备液、500μL半乳糖储备液、50μL鼠李糖储备液、20μL半乳糖醛酸储备液、10μL阿拉伯糖储备液，加水定容至10mL，即得混合单糖储备液。Mix the above-mentioned stock solutions respectively, and accurately pipette 1.0 mL ribose stock solution, 1.0 mL fucose stock solution, 1.0 mL glucuronic acid stock solution, 1.0 mL mannose stock solution, 1.0 mL glucose stock solution, and 500 μL galactose. Stock solution, 50 μL rhamnose stock solution, 20 μL galacturonic acid stock solution, 10 μL arabinose stock solution, add water to make up to 10 mL, to obtain mixed monosaccharide stock solution.
2、供试品的配制2. Preparation of the test product
准确称取灵芝多糖(20200903-08)样品40mg，至10mL容量瓶中，用水定容至刻度，混匀，得到样品溶液。Accurately weigh 40 mg of Ganoderma lucidum polysaccharide (20200903-08) sample into a 10 mL volumetric flask, make up to the mark with water, and mix well to obtain a sample solution.
3、优化前水解条件3. Optimize the hydrolysis conditions before
精密吸取灵芝多糖样品溶液500μL至反应瓶中，加入500μL 4M的三氟乙酸。混匀，密闭，110℃加热4h，挥干溶剂后，加入1mL甲醇，挥干，再重复此操作两次。Precisely pipette 500 μL of Ganoderma lucidum polysaccharide sample solution into the reaction flask, and add 500 μL of 4M trifluoroacetic acid. Mix well, seal, heat at 110 °C for 4 h, evaporate the solvent, add 1 mL of methanol, evaporate to dryness, and repeat this operation twice.
4、优化前衍生化条件4. Derivatization conditions before optimization
取250μL混合单糖储备液，250μL内标溶液，分别加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液。密闭70℃加热30min，冷却至室温。加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得优化前对照品溶液。Take 250 μL of mixed monosaccharide stock solution, 250 μL of internal standard solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.1M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, respectively . Sealed and heated at 70°C for 30min, cooled to room temperature. Add 600 μL of 0.15M hydrochloric acid, mix well, and pass through a 0.45 μm nylon filter to obtain the reference solution before optimization.
灵芝多糖水解后残渣加250μL水，250μL内标溶液，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液。密闭70℃加热30min，冷却至室温；加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得优化前供试品溶液。To the residue after hydrolysis of Ganoderma lucidum polysaccharide, add 250 μL of water, 250 μL of internal standard solution, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.1M methanol solution of 1-phenyl-3-methyl-5-pyrazolone. Sealed and heated at 70°C for 30min, cooled to room temperature; added 600μL of 0.15M hydrochloric acid, mixed well, and passed through a 0.45μm nylon filter to obtain the test solution before optimization.
5、优化后水解条件5. Optimized hydrolysis conditions
分别精密吸取灵芝多糖样品溶液500μL至反应瓶中，加入500μL 6M的三氟乙酸。混匀，密闭，120℃加热4h，挥干溶剂后，加入1mL甲醇，挥干，再重复此操作两次。Precisely pipette 500 μL of Ganoderma lucidum polysaccharide sample solution into the reaction flask, and add 500 μL of 6M trifluoroacetic acid. Mix well, seal, heat at 120°C for 4 hours, evaporate the solvent, add 1 mL of methanol, evaporate to dryness, and repeat this operation twice.
6、优化后衍生化条件6. Derivatization conditions after optimization
取250μL混合单糖储备液，250μL内标溶液，分别加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液。密闭70℃加热60min，冷却至室温。加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得优化后对照品溶液。Take 250 μL of mixed monosaccharide stock solution, 250 μL of internal standard solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, respectively . Sealed and heated at 70°C for 60min, cooled to room temperature. Add 600 μL of 0.15M hydrochloric acid, mix well, and pass through a 0.45 μm nylon filter to obtain the optimized reference solution.
灵芝多糖水解后残渣加250μL水，250μL内标溶液，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液。密闭反应瓶70℃加热60min，冷却至室温。加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得优化后供试品溶液。To the residue after hydrolysis of Ganoderma lucidum polysaccharide, add 250 μL of water, 250 μL of internal standard solution, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone. The closed reaction flask was heated at 70°C for 60min and cooled to room temperature. Add 600 μL of 0.15M hydrochloric acid, mix well, and pass through a 0.45 μm nylon filter to obtain the optimized test solution.
用以下色谱条件下分析上述优化前后两种条件下处理的供试品溶液。Use the following chromatographic conditions to analyze the test solution treated under the two conditions before and after the above optimization.
高效液相色谱仪：Agilent 1260；High performance liquid chromatograph: Agilent 1260;
色谱柱：Agilent Zorbax SB-C18(4.6mm x 250mm；5μm)；Chromatographic column: Agilent Zorbax SB-C18 (4.6mm x 250mm; 5μm);
柱温：35℃；Column temperature: 35℃;
检测波长：250nm；Detection wavelength: 250nm;
流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝6)，B相：乙腈；Mobile phase: A phase: 0.05M phosphate buffer solution (pH=6), B phase: acetonitrile;
洗脱条件：0-20min，A(％)：84-83；20-40min，A(％)：83-82.5；40-65min，A(％)：82.5-81；Elution conditions: 0-20min, A(%): 84-83; 20-40min, A(%): 83-82.5; 40-65min, A(%): 82.5-81;
流速：1mL/min。Flow rate: 1 mL/min.
分析结果见表1。The analysis results are shown in Table 1.
表1反应条件优化前后测量的灵芝多糖的糖组成对比Table 1 Comparison of sugar composition of Ganoderma lucidum polysaccharides measured before and after optimization of reaction conditions
Figure PCTCN2021142290-appb-000001
Figure PCTCN2021142290-appb-000001
灵芝多糖(20200903-08)的糖组成分析结果显示，优化后的反应条件，其单糖组成种类和总峰面积明显增加，优化后的反应条件更能反应灵芝多糖真实的糖组成情况。The sugar composition analysis results of Ganoderma lucidum polysaccharide (20200903-08) showed that the optimized reaction conditions significantly increased the types of monosaccharide composition and total peak area, and the optimized reaction conditions could better reflect the real sugar composition of Ganoderma lucidum polysaccharides.
实施例2：灵芝多糖糖组成分析Example 2: Ganoderma lucidum polysaccharide composition analysis
前期实验中，运用正交试验确定了最优的水解和衍生化条件，运用单个单糖标准品逐一比对，确定了灵芝多糖的单糖组成情况，根据实验结果配制对应浓度的混合单糖溶液作为对照品溶液，确定灵芝多糖的糖组成及组成比例。In the previous experiments, the optimal hydrolysis and derivatization conditions were determined by orthogonal experiments, and the monosaccharide composition of Ganoderma lucidum polysaccharides was determined by comparing the single monosaccharide standard one by one, and the mixed monosaccharide solution of corresponding concentration was prepared according to the experimental results. As a reference solution, determine the sugar composition and composition ratio of Ganoderma lucidum polysaccharide.
1、HPLC对照品溶液和内标溶液的制备1. Preparation of HPLC reference solution and internal standard solution
准确称取葡萄糖100mg，置于容量瓶中，用纯水定容，配制成浓度为50mg/mL葡萄糖储备液；Accurately weigh 100 mg of glucose, place it in a volumetric flask, and dilute to volume with pure water to prepare a glucose stock solution with a concentration of 50 mg/mL;
准确称取半乳糖12mg，置于容量瓶中，用纯水定容，配制成浓度为6mg/mL的半乳糖储备液；Accurately weigh 12 mg of galactose, place it in a volumetric flask, dilute to volume with pure water, and prepare a galactose stock solution with a concentration of 6 mg/mL;
准确称取葡萄糖醛酸10mg，置于容量瓶中，用纯水定容，配制成浓度为1mg/mL的葡萄糖醛酸储备液；Accurately weigh 10 mg of glucuronic acid, place it in a volumetric flask, dilute to volume with pure water, and prepare a glucuronic acid stock solution with a concentration of 1 mg/mL;
准确称取甘露糖20mg，置于容量瓶中，用纯水定容，配制成浓度为2mg/mL的甘露糖储备液；Accurately weigh 20 mg of mannose, place it in a volumetric flask, dilute to volume with pure water, and prepare a mannose stock solution with a concentration of 2 mg/mL;
准确称取核糖12.5mg，置于容量瓶中，用纯水定容，配制成浓度为0.5mg/mL的核糖储备液；Accurately weigh 12.5 mg of ribose, place it in a volumetric flask, and dilute to volume with pure water to prepare a ribose stock solution with a concentration of 0.5 mg/mL;
准确称取鼠李糖20mg，置于容量瓶中，用纯水定容，配制成浓度为2 mg/mL的鼠李糖储备液；Accurately weigh 20 mg of rhamnose, place it in a volumetric flask, dilute to volume with pure water, and prepare a rhamnose stock solution with a concentration of 2 mg/mL;
准确称取半乳糖醛酸20mg，置于容量瓶中，用纯水定容，配制成浓度为10mg/mL的半乳糖醛酸储备液；Accurately weigh 20 mg of galacturonic acid, place it in a volumetric flask, and dilute to volume with pure water to prepare a galacturonic acid stock solution with a concentration of 10 mg/mL;
准确称取阿拉伯糖30mg，置于容量瓶中，用纯水定容，配制成浓度为15mg/mL的阿拉伯糖储备液；Accurately weigh 30 mg of arabinose, place it in a volumetric flask, and dilute to volume with pure water to prepare a 15 mg/mL arabinose stock solution;
准确称取岩藻糖12.5mg，置于容量瓶中，用纯水定容，配制成浓度为0.5mg/mL的岩藻糖储备液。Accurately weigh 12.5 mg of fucose, place it in a volumetric flask, dilute to volume with pure water, and prepare a fucose stock solution with a concentration of 0.5 mg/mL.
准确称取来苏糖12.5mg，置于25mL的容量品中，用纯水定容，混匀，即为来苏糖水溶液，即内标溶液。Accurately weigh 12.5mg of lyxose, put it in a 25mL volume product, dilute to volume with pure water, and mix well, which is the lyxose aqueous solution, that is, the internal standard solution.
将上述各储备液分别混匀，分别准确吸取1.0mL核糖储备液、1.0mL岩藻糖储备液、1.0mL葡萄糖醛酸储备液、1.0mL甘露糖储备液、1.0mL葡萄糖储备液、500μL半乳糖储备液、50μL鼠李糖储备液、20μL半乳糖醛酸储备液、10μL阿拉伯糖储备液，加水定容至10mL，即得混合单糖储备液。Mix the above stock solutions, respectively, accurately pipette 1.0 mL ribose stock solution, 1.0 mL fucose stock solution, 1.0 mL glucuronic acid stock solution, 1.0 mL mannose stock solution, 1.0 mL glucose stock solution, and 500 μL galactose. Stock solution, 50 μL rhamnose stock solution, 20 μL galacturonic acid stock solution, and 10 μL arabinose stock solution, add water to make up to 10 mL, to obtain mixed monosaccharide stock solution.
取250μL混合单糖储备液，250μL内标溶液，分别加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液。密闭70℃加热60min，冷却至室温。加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得HPLC对照品溶液。Take 250 μL of mixed monosaccharide stock solution, 250 μL of internal standard solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, respectively . Sealed and heated at 70°C for 60min, cooled to room temperature. Add 600 μL of 0.15M hydrochloric acid, mix well, and pass through a 0.45 μm nylon filter to obtain the HPLC reference solution.
2、供试品溶液的制备2. Preparation of the test solution
精密称取灵芝多糖样品(20200825-05；20200902-05；20200903-08)各40mg，至10mL容量瓶中，用水定容至刻度，混匀，得到样品溶液。Precisely weigh 40 mg of Ganoderma lucidum polysaccharide samples (20200825-05; 20200902-05; 20200903-08) into a 10 mL volumetric flask, make up to the mark with water, and mix well to obtain a sample solution.
分别精密吸取灵芝多糖样品溶液500μL至反应瓶中，加入500μL 6M的三氟乙酸。混匀，密闭，120℃加热4h，挥干溶剂后，加入1mL甲醇，挥干，再重复此操作两次。Precisely pipette 500 μL of Ganoderma lucidum polysaccharide sample solution into the reaction flask, and add 500 μL of 6M trifluoroacetic acid. Mix well, seal, heat at 120°C for 4 hours, evaporate the solvent, add 1 mL of methanol, evaporate to dryness, and repeat this operation twice.
残渣加250μL水，250μL内标溶液，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液。密闭反应瓶70℃加热60min，冷却至室温。加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得供试品溶液。The residue was added with 250 μL of water, 250 μL of internal standard solution, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone. The closed reaction flask was heated at 70°C for 60min and cooled to room temperature. Add 600 μL of 0.15M hydrochloric acid, mix well, and pass through a 0.45 μm nylon filter to obtain the test solution.
3、糖组成分析3. Analysis of sugar composition
在以下色谱条件下测量供试品与对照品的HPLC色谱图。Measure the HPLC chromatograms of the test substance and reference substance under the following chromatographic conditions.
高效液相色谱仪：Agilent 1260；High performance liquid chromatograph: Agilent 1260;
色谱柱：Agilent Zorbax SB-C18(4.6mm x 250mm；5μm)；Chromatographic column: Agilent Zorbax SB-C18 (4.6mm x 250mm; 5μm);
柱温：35℃；Column temperature: 35℃;
检测波长：250nm；Detection wavelength: 250nm;
流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝6)，B相：乙腈；Mobile phase: A phase: 0.05M phosphate buffer solution (pH=6), B phase: acetonitrile;
洗脱条件：0-20min，A(％)：84-83；20-40min，A(％)：83-82.5；40-65min，A(％)：82.5-81；Elution conditions: 0-20min, A(%): 84-83; 20-40min, A(%): 83-82.5; 40-65min, A(%): 82.5-81;
流速：1mL/min。Flow rate: 1 mL/min.
图1为灵芝多糖(20200825-05)水解后衍生物的HPLC色谱图。Figure 1 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200825-05) after hydrolysis.
图2为灵芝多糖(20200902-05)水解后衍生物的HPLC色谱图。Figure 2 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200902-05) after hydrolysis.
图3为灵芝多糖(20200903-08)水解后衍生物的HPLC色谱图。Figure 3 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (20200903-08) after hydrolysis.
4、糖组成结果分析4. Analysis of sugar composition results
将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比对，确定对应的单糖特征峰，以来苏糖为内标，用峰面积计算各单糖含量，结果见表2-表4。Compare the retention time of the HPLC chromatogram of the test product with the HPLC chromatogram of the reference substance, determine the corresponding monosaccharide characteristic peak, use threose as the internal standard, and calculate the content of each monosaccharide with the peak area, the results are shown in Table 2 -Table 4.
表2 HPLC分析灵芝多糖(2020825-05)的单糖组成及含量Table 2 Monosaccharide composition and content of Ganoderma lucidum polysaccharide (2020825-05) analyzed by HPLC
Figure PCTCN2021142290-appb-000002
Figure PCTCN2021142290-appb-000002
表3 HPLC分析灵芝多糖(2020902-05)的单糖组成及含量Table 3 Monosaccharide composition and content of Ganoderma lucidum polysaccharide (2020902-05) analyzed by HPLC
Figure PCTCN2021142290-appb-000003
Figure PCTCN2021142290-appb-000003
表4 HPLC分析灵芝多糖(2020903-08)的单糖组成及含量Table 4 Monosaccharide composition and content of Ganoderma lucidum polysaccharide (2020903-08) analyzed by HPLC
Figure PCTCN2021142290-appb-000004
Figure PCTCN2021142290-appb-000004
实施例3：灵芝多糖含量的检测Example 3: Detection of Ganoderma lucidum polysaccharide content
1、苯酚溶液的配制1. Preparation of phenol solution
称取5g的苯酚，加蒸馏水溶解，定容至100mL，即得。Weigh 5g of phenol, dissolve in distilled water, and dilute to 100mL.
2、硫酸蒽酮溶液的配制2. Preparation of anthrone sulfate solution
称取0.1g的蒽酮，于100mL容量瓶中，用浓硫酸定容，充分溶解混匀，即得。Weigh 0.1g of anthrone, put it in a 100mL volumetric flask, dilute to volume with concentrated sulfuric acid, fully dissolve and mix to get it.
3、混合单糖对照品溶液的制备3. Preparation of mixed monosaccharide reference solution
确定三种供试品(20200825-05，20200902-05，20200903-08)中的单糖组成，并运用内标法计算各单糖含量。以高效液相色谱确定的单糖比例配制含量检测需要的对照品溶液，总浓度为0.1mg/mL。Determine the monosaccharide composition in the three test products (20200825-05, 20200902-05, 20200903-08), and use the internal standard method to calculate the content of each monosaccharide. The reference solution required for content detection was prepared with the monosaccharide ratio determined by high performance liquid chromatography, and the total concentration was 0.1 mg/mL.
制备灵芝多糖(20200825-05)的混合单糖对照品溶液，准确称取葡萄糖12.58mg，用纯水定容，配制成浓度为6.294mg/mL的葡萄糖储备液；准确称取半乳糖16.07mg，用纯水定容，配制成浓度为1.607mg/mL的半乳糖储备液；准确称取葡萄糖醛酸14.04mg，用纯水定容，配制成浓度为1.404mg/mL的葡萄糖醛酸储备液；准确称取甘露糖17.40mg，置于容量瓶中，用纯水定容，配制成浓度为0.696mg/mL的甘露糖储备液。将上述各储备液分别混匀，分别精密吸取1.0mL，加水定容至100mL，即得，其中，以重量百分比计，基于溶液中的单糖的总重量，葡萄糖含量约为62.94％，半乳糖含量约为16.07％，葡萄糖醛酸的含量约为14.04％，甘露糖的含量约为6.96％。Prepare the mixed monosaccharide reference solution of Ganoderma lucidum polysaccharide (20200825-05), accurately weigh 12.58mg of glucose, dilute to volume with pure water, and prepare a glucose stock solution with a concentration of 6.294mg/mL; accurately weigh 16.07mg of galactose, Dilute to volume with pure water to prepare a galactose stock solution with a concentration of 1.607mg/mL; accurately weigh 14.04mg of glucuronic acid, dilute to volume with pure water, and prepare a glucuronic acid stock solution with a concentration of 1.404mg/mL; Accurately weigh 17.40 mg of mannose, place it in a volumetric flask, dilute to volume with pure water, and prepare a mannose stock solution with a concentration of 0.696 mg/mL. Mix the above-mentioned stock solutions respectively, accurately draw 1.0 mL respectively, and add water to the volume to 100 mL, to obtain, wherein, based on the total weight of the monosaccharides in the solution, the glucose content is about 62.94%, the galactose content is about 62.94% in weight percentage. The content is about 16.07%, the content of glucuronic acid is about 14.04%, and the content of mannose is about 6.96%.
制备灵芝多糖(202000902-05)的混合单糖对照品溶液，准确称取葡萄糖14.80mg，用纯水定容，配制成浓度为7.402mg/mL的葡萄糖储备液； 准确称取半乳糖12.70mg，用纯水定容，配制成浓度为1.270mg/mL的半乳糖储备液；准确称取葡萄糖醛酸17.57mg，用纯水定容，配制成浓度为0.703mg/mL的葡萄糖醛酸储备液；准确称取甘露糖15.62mg，置于容量瓶中，用纯水定容，配制成浓度为0.625mg/mL的甘露糖储备液。将上述各储备液分别混匀，分别精密吸取1.0mL，加水定容至100mL，即得，其中，以重量百分比计，基于溶液中的单糖的总重量，葡萄糖含量约为74.02％，半乳糖含量约为12.70％，葡萄糖醛酸的含量约为7.03％，甘露糖的含量约为6.25％。Prepare the mixed monosaccharide reference solution of Ganoderma lucidum polysaccharide (202000902-05), accurately weigh 14.80mg of glucose, dilute to volume with pure water, and prepare a glucose stock solution with a concentration of 7.402mg/mL; accurately weigh 12.70mg of galactose, Dilute to volume with pure water to prepare a galactose stock solution with a concentration of 1.270 mg/mL; accurately weigh 17.57 mg of glucuronic acid, dilute to volume with pure water, and prepare a glucuronic acid stock solution with a concentration of 0.703 mg/mL; Accurately weigh 15.62 mg of mannose, place it in a volumetric flask, dilute to volume with pure water, and prepare a mannose stock solution with a concentration of 0.625 mg/mL. Mix the above-mentioned stock solutions, respectively, accurately draw 1.0 mL, and add water to make the volume to 100 mL, that is, in a percentage by weight, based on the total weight of the monosaccharides in the solution, the glucose content is about 74.02%, and the galactose content is about 74.02%. The content is about 12.70%, the content of glucuronic acid is about 7.03%, and the content of mannose is about 6.25%.
制备灵芝多糖(20200903-08)的混合单糖对照品溶液，准确称取葡萄糖16.77mg，用纯水定容，配制成浓度为8.385mg/mL的葡萄糖储备液；准确称取半乳糖20.85mg，用纯水定容，配制成浓度为0.895mg/mL的半乳糖储备液；准确称取葡萄糖醛酸16.65mg，用纯水定容，配制成浓度为0.333mg/mL的葡萄糖醛酸储备液；准确称取甘露糖19.35mg，置于容量瓶中，用纯水定容，配制成浓度为0.387mg/mL的甘露糖储备液。将上述各储备液分别混匀，分别精密吸取1.0mL，加水定容至100mL，即得，其中，以重量百分比计，基于溶液中的单糖的总重量，葡萄糖含量约为83.85％，半乳糖含量约为8.95％，葡萄糖醛酸的含量约为3.33％，甘露糖的含量约为3.87％。To prepare the mixed monosaccharide reference solution of Ganoderma lucidum polysaccharide (20200903-08), accurately weigh 16.77mg of glucose, dilute to volume with pure water, and prepare a glucose stock solution with a concentration of 8.385mg/mL; accurately weigh 20.85mg of galactose, Dilute to volume with pure water to prepare a galactose stock solution with a concentration of 0.895mg/mL; accurately weigh 16.65mg of glucuronic acid, dilute to volume with pure water, and prepare a glucuronic acid stock solution with a concentration of 0.333mg/mL; Accurately weigh 19.35 mg of mannose, place it in a volumetric flask, dilute to volume with pure water, and prepare a mannose stock solution with a concentration of 0.387 mg/mL. Mix the above-mentioned stock solutions, respectively, accurately draw 1.0 mL, and add water to make up to 100 mL, to obtain, wherein, based on the total weight of the monosaccharides in the solution, the glucose content is about 83.85%, the galactose content is about 83.85%, and the galactose The content is about 8.95%, the content of glucuronic acid is about 3.33%, and the content of mannose is about 3.87%.
4、苯酚-硫酸法标准曲线的制作4. Preparation of standard curve of phenol-sulfuric acid method
精密量取步骤3中混合单糖对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置具塞试管中，分别加水补至2.0mL，各精密加入步骤1中制得的苯酚溶液1mL，摇匀，迅速精密加入浓硫酸5mL，摇匀，放置10分钟，置40℃水浴中保温15min，取出，冰浴2min，空白不加对照品溶液，测定在490nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制标准曲线。Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step 3, put them in test tubes with stoppers, add water to make up to 2.0mL, and add each precision into step 1. 1mL of the prepared phenol solution was shaken, quickly and precisely added 5mL of concentrated sulfuric acid, shaken well, placed for 10 minutes, placed in a 40°C water bath for 15 minutes, taken out, ice bathed for 2 minutes, blank without reference solution, and measured at 490nm. Take the absorbance as the ordinate and the concentration of the sugar reference substance as the abscissa to draw a standard curve.
5、蒽酮-硫酸法标准曲线的制作5. Preparation of standard curve of anthrone-sulfuric acid method
精密量取步骤3中混合单糖对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置于具塞试管中，分别加水补至2.0mL，各精密加入步骤2中制得的硫酸蒽酮溶液6mL，摇匀，放置15min后，置于冰浴中冷却15min，取出，空白不加对照品溶液，测定在625nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制标准曲线。Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step 3, place them in test tubes with stoppers, respectively add water to make up to 2.0mL, and add each precision to step 2. 6mL of anthrone sulfate solution prepared in The concentration is the abscissa, and the standard curve is drawn.
6、供试品的制备6. Preparation of the test product
准确称取三批灵芝多糖样品(20200825-05；20200902-05；20200903-08) 各10mg，于100mL的容量瓶中，用纯水定容，充分溶解混匀，即得。Accurately weigh three batches of Ganoderma lucidum polysaccharide samples (20200825-05; 20200902-05; 20200903-08), each 10mg, in a 100mL volumetric flask, dilute to volume with pure water, fully dissolve and mix to obtain.
7、供试品的检测7. Detection of the test product
a、苯酚-硫酸法a. Phenol-sulfuric acid method
精密吸取供试品溶液1.0mL，加水至2.0mL，照(4)步骤苯酚-硫酸法标准曲线的制作项下的方法，自“加入苯酚溶液1mL”起，依上述方法测定供试品的吸光度，根据苯酚-硫酸法标准曲线得到多糖含量。Accurately draw 1.0mL of the test solution, add water to 2.0mL, and measure the absorbance of the test product according to the above method according to the method under the preparation of the standard curve of the phenol-sulfuric acid method in step (4), starting from "adding 1mL of the phenol solution" , and the polysaccharide content was obtained according to the standard curve of the phenol-sulfuric acid method.
b、蒽酮-硫酸法b. Anthrone-sulfuric acid method
精密吸取供试品溶液1.0mL，加水至2.0mL，照(5)步骤蒽酮-硫酸法标准曲线的制作项下的方法，自“加入硫酸蒽酮溶液6mL”起，依法测定供试品的吸光度，根据蒽酮-硫酸法标准曲线得到多糖含量。Accurately draw 1.0 mL of the solution of the test sample, add water to 2.0 mL, and follow the method under the preparation of the standard curve of the anthrone-sulfuric acid method in step (5). Absorbance, polysaccharide content was obtained according to the standard curve of anthrone-sulfuric acid method.
8、灵芝多糖的含量检测结果8. Content test results of Ganoderma lucidum polysaccharide
以与供试品对应的混合单糖对照品溶液制作标准曲线，用苯酚-硫酸法和蒽酮-硫酸法法检测了灵芝多糖的含量。The standard curve was made with the mixed monosaccharide reference solution corresponding to the test product, and the content of Ganoderma lucidum polysaccharide was detected by the phenol-sulfuric acid method and the anthrone-sulfuric acid method.
实验结果表明，混合单糖为对照品时，用苯酚-硫酸法检测灵芝多糖(20200825-05)的含量，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝5.9077X+0.011(R＝0.99915)。用蒽酮-硫酸法法检测时，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝4.0061X-0.0054(R＝0.99930)，结果见表5。The experimental results show that when the mixed monosaccharide is the reference substance, the content of Ganoderma lucidum polysaccharide (20200825-05) is detected by the phenol-sulfuric acid method, and the absorbance (Y) of the solution is linear with the concentration of the mixed monosaccharide reference substance (X, μg/mL) relationship, the regression equation is: Y=5.9077X+0.011 (R=0.99915). When detected by the anthrone-sulfuric acid method, the absorbance (Y) of the solution has a linear relationship with the concentration of the mixed monosaccharide reference substance (X, μg/mL). The regression equation is: Y=4.0061X-0.0054 (R=0.99930), The results are shown in Table 5.
表5灵芝多糖(20200825-05)含量检测结果(混合单糖为对照品)Table 5 Ganoderma lucidum polysaccharide (20200825-05) content detection results (mixed monosaccharide is the reference substance)
Figure PCTCN2021142290-appb-000005
Figure PCTCN2021142290-appb-000005
实验结果表明，混合单糖为对照品时，用苯酚-硫酸法检测灵芝多糖(20200902-05)的含量，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝6.7115X-0.0035(R＝0.99940)。用蒽酮-硫酸法检测时，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝4.2649X-0.005(R＝0.99895)，结果见表6。The experimental results show that when the mixed monosaccharide is the reference substance, the content of Ganoderma lucidum polysaccharide (20200902-05) is detected by the phenol-sulfuric acid method, and the absorbance (Y) of the solution is linear with the concentration (X, μg/mL) of the mixed monosaccharide reference substance relationship, the regression equation is: Y=6.7115X-0.0035 (R=0.99940). When detected by anthrone-sulfuric acid method, the absorbance (Y) of the solution has a linear relationship with the concentration of mixed monosaccharide reference substance (X, μg/mL). The regression equation is: Y=4.2649X-0.005 (R=0.99895), the result See Table 6.
表6灵芝多糖(20200902-05)含量检测结果(混合单糖为对照品)Table 6 Ganoderma lucidum polysaccharide (20200902-05) content detection results (mixed monosaccharide is the reference substance)
Figure PCTCN2021142290-appb-000006
Figure PCTCN2021142290-appb-000006
实验结果表明，混合单糖为对照品时，用苯酚-硫酸法检测灵芝多糖(20200903-08)的含量，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝7.4965X-0.0004(R＝0.99965)。用蒽酮-硫酸法检测时，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝5.1263X+0.001(R＝0.99945)，结果见表7。The experimental results show that when the mixed monosaccharide is the reference substance, the content of Ganoderma lucidum polysaccharide (20200903-08) is detected by the phenol-sulfuric acid method, and the absorbance (Y) of the solution is linear with the concentration of the mixed monosaccharide reference substance (X, μg/mL) relationship, the regression equation is: Y=7.4965X-0.0004 (R=0.99965). When detected by anthrone-sulfuric acid method, the absorbance (Y) of the solution has a linear relationship with the concentration of mixed monosaccharide reference substance (X, μg/mL). The regression equation is: Y=5.1263X+0.001 (R=0.99945), the result See Table 7.
表7灵芝多糖(20200903-08)含量检测结果(混合单糖为对照品)Table 7 Ganoderma lucidum polysaccharide (20200903-08) content test results (mixed monosaccharide is the reference substance)
Figure PCTCN2021142290-appb-000007
Figure PCTCN2021142290-appb-000007
对比例1：灵芝多糖含量的检测Comparative Example 1: Detection of Ganoderma lucidum polysaccharide content
除了在3中采用葡萄糖制备单糖对照品溶液并在4和5中以单糖对照品溶液制备标准曲线以外，其他操作与实施例3相同。The procedure was the same as Example 3, except that glucose was used to prepare the monosaccharide reference solution in 3 and the standard curve was prepared with the monosaccharide reference solution in 4 and 5.
单糖对照品溶液的制备Preparation of monosaccharide reference solution
准确称取无水葡萄糖10mg，于100mL的容量瓶中，用纯水定容，充分溶解混匀，即得。Accurately weigh 10 mg of anhydrous glucose, put it in a 100-mL volumetric flask, dilute to volume with pure water, fully dissolve and mix, and that’s it.
实验结果表明，无水葡萄糖为对照品时，用苯酚-硫酸法检测，溶液的吸光度(Y)与单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝8.6243X-0.0439(R＝0.99859)。用蒽酮-硫酸法检测，溶液的吸光度(Y)与单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝5.0501X+0.0134(R＝0.99925)，结果见表8-表10。The experimental results show that when anhydrous glucose is the reference substance, the phenol-sulfuric acid method is used to detect the absorbance (Y) of the solution and the concentration of monosaccharide reference substance (X, μg/mL) in a linear relationship. The regression equation is: Y=8.6243X -0.0439 (R=0.99859). Detected by anthrone-sulfuric acid method, the absorbance (Y) of the solution has a linear relationship with the concentration of monosaccharide reference substance (X, μg/mL). The regression equation is: Y=5.0501X+0.0134 (R=0.99925), the results are shown in the table 8-Table 10.
表8灵芝多糖(20200825-05)含量检测结果(无水葡萄糖为对照品)Table 8 Ganoderma lucidum polysaccharide (20200825-05) content detection results (anhydrous glucose is the reference substance)
Figure PCTCN2021142290-appb-000008
Figure PCTCN2021142290-appb-000008
表9灵芝多糖(20200902-05)含量检测结果(无水葡萄糖为对照品)Table 9 Ganoderma lucidum polysaccharide (20200902-05) content detection results (anhydrous glucose is the reference substance)
Figure PCTCN2021142290-appb-000009
Figure PCTCN2021142290-appb-000009
表10灵芝多糖(20200903-08)含量检测结果(无水葡萄糖为对照品)Table 10 Ganoderma lucidum polysaccharide (20200903-08) content detection results (anhydrous glucose is the reference substance)
Figure PCTCN2021142290-appb-000010
Figure PCTCN2021142290-appb-000010
在用比色法检测糖含量时，不同的单糖标准曲线斜率是不同的，因此对照品的选择在比色法测定多糖含量中就显得比较重要。灵芝多糖由多种单糖组成，且各单糖的含量也不同，情况较为复杂，单一的单糖标准曲线并不能准确检测灵芝多糖的含量。When the colorimetric method is used to detect the sugar content, the slope of the standard curve of different monosaccharides is different, so the selection of the reference substance is more important in the colorimetric determination of the polysaccharide content. Ganoderma lucidum polysaccharide is composed of a variety of monosaccharides, and the content of each monosaccharide is also different. The situation is more complicated. A single monosaccharide standard curve cannot accurately detect the content of Ganoderma lucidum polysaccharide.
在明确灵芝多糖的糖组成及含量的情况下，配制对应的混合多糖对照品溶液制作标准曲线，与单一单糖为对照品的相比，两种经典比色法得到的灵芝多糖含量都更高，能更准确的反应灵芝多糖的总糖含量。因此以组成灵芝多糖的各单糖比配成的对照品溶液，能得到更接近真实情况的数据。When the sugar composition and content of Ganoderma lucidum polysaccharides are clarified, the corresponding mixed polysaccharide reference solution is prepared to make a standard curve. Compared with the single monosaccharide as the reference substance, the content of Ganoderma lucidum polysaccharides obtained by the two classical colorimetric methods is higher. , can more accurately reflect the total sugar content of Ganoderma lucidum polysaccharides. Therefore, the reference solution prepared with the ratio of each monosaccharide constituting Ganoderma lucidum polysaccharide can obtain data that is closer to the real situation.
实施例4：灵芝多糖糖组成分析Example 4: Ganoderma lucidum polysaccharide composition analysis
1、HPLC对照品溶液的制备1. Preparation of HPLC reference solution
准确称取不同单糖，配制含0.08mg/mL甘露糖、0.05mg/mL葡萄糖醛酸、1.25mg/mL葡萄糖、0.15mg/mL半乳糖、0.05mg/mL岩藻糖、0.025mg/mL核糖、0.0055mg/mL鼠李糖、0.01mg/mL阿拉伯糖的水溶液为混合单糖储备液。Accurately weigh different monosaccharides, prepare 0.08mg/mL mannose, 0.05mg/mL glucuronic acid, 1.25mg/mL glucose, 0.15mg/mL galactose, 0.05mg/mL fucose, 0.025mg/mL ribose , 0.0055mg/mL rhamnose, 0.01mg/mL arabinose aqueous solution as mixed monosaccharide stock solution.
取500μL混合单糖储备液，分别加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液。密闭70℃加热60min，取出冰水浴2min。加入0.15M盐酸600μL，混匀，取500μL溶液，加500μL氯仿，以5000rpm离心3min进行萃取，萃取结束后取上层溶液以12000rpm离心10min，上清液作为HPLC对照品溶液。Take 500 μL of mixed monosaccharide stock solution, add 600 μL of 0.15M sodium hydroxide aqueous solution and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, respectively. Sealed and heated at 70°C for 60min, took out the ice-water bath for 2min. Add 600 μL of 0.15M hydrochloric acid, mix well, take 500 μL of the solution, add 500 μL of chloroform, and centrifuge at 5000 rpm for 3 min for extraction.
2、供试品溶液的制备2. Preparation of the test solution
精密称取灵芝多糖样品(21001；21002；21003；21004)各40mg，至10mL容量瓶中，用水定容至刻度，混匀，得到样品溶液。Precisely weigh 40 mg of Ganoderma lucidum polysaccharide samples (21001; 21002; 21003; 21004) into a 10 mL volumetric flask, make up to the mark with water, and mix well to obtain a sample solution.
分别精密吸取灵芝多糖样品溶液500μL至反应瓶中，加入500μL 6M的三氟乙酸。混匀，密闭，120℃加热4h，挥干溶剂后，加入1mL甲醇，挥干，再重复此操作两次。Precisely pipette 500 μL of Ganoderma lucidum polysaccharide sample solution into the reaction flask, and add 500 μL of 6M trifluoroacetic acid. Mix well, seal, heat at 120°C for 4 hours, evaporate the solvent, add 1 mL of methanol, evaporate to dryness, and repeat this operation twice.
残渣加500μL水，600μL的0.15M的氢氧化钠水溶液，以及1.0mL 的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液。密闭反应瓶70℃加热60min，取出冰水浴2min。加入0.15M盐酸600μL，混匀，取500μL溶液，加500μL氯仿，以5000rpm离心3min进行萃取，萃取结束后取上层溶液以12000rpm离心10min，上清液作为供试品溶液。The residue was added with 500 μL of water, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone. The closed reaction flask was heated at 70 °C for 60 min, and the ice-water bath was taken out for 2 min. Add 600 μL of 0.15M hydrochloric acid, mix well, take 500 μL of the solution, add 500 μL of chloroform, and extract by centrifugation at 5000 rpm for 3 min.
3、糖组成分析3. Analysis of sugar composition
在以下色谱条件下测量供试品与对照品的HPLC色谱图。Measure the HPLC chromatograms of the test substance and reference substance under the following chromatographic conditions.
高效液相色谱仪：Agilent 1260；High performance liquid chromatograph: Agilent 1260;
色谱柱：C6-Phenyl(4.6mm x 25cm；5μm)；Chromatographic column: C6-Phenyl (4.6mm x 25cm; 5μm);
柱温：35℃；Column temperature: 35℃;
检测波长：250nm；Detection wavelength: 250nm;
流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝7)，B相：乙腈；Mobile phase: A phase: 0.05M phosphate buffer solution (pH=7), B phase: acetonitrile;
洗脱条件：B(％)：15-16梯度洗脱；Elution conditions: B (%): 15-16 gradient elution;
流速：0.8mL/min。Flow rate: 0.8 mL/min.
图4为灵芝多糖(21001)水解后衍生物的HPLC色谱图。Figure 4 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21001) after hydrolysis.
图5为灵芝多糖(21002)水解后衍生物的HPLC色谱图。Figure 5 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21002) after hydrolysis.
图6为灵芝多糖(21003)水解后衍生物的HPLC色谱图。Figure 6 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21003) after hydrolysis.
图7为灵芝多糖(21004)水解后衍生物的HPLC色谱图。Figure 7 is the HPLC chromatogram of the derivative of Ganoderma lucidum polysaccharide (21004) after hydrolysis.
4、糖组成结果分析4. Analysis of sugar composition results
将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比对，确定对应的单糖特征峰，用峰面积计算各单糖含量，结果见表11-表13。Compare the retention time of the HPLC chromatogram of the test product and the HPLC chromatogram of the reference substance, determine the corresponding monosaccharide characteristic peak, and calculate the content of each monosaccharide by the peak area. The results are shown in Table 11-Table 13.
表11 HPLC分析灵芝多糖(21001)的单糖组成及含量Table 11 Monosaccharide composition and content of Ganoderma lucidum polysaccharide (21001) analyzed by HPLC
Figure PCTCN2021142290-appb-000011
Figure PCTCN2021142290-appb-000011
表12 HPLC分析灵芝多糖(21002)的单糖组成及含量Table 12 Monosaccharide composition and content of Ganoderma lucidum polysaccharide (21002) analyzed by HPLC
Figure PCTCN2021142290-appb-000012
Figure PCTCN2021142290-appb-000012
表13 HPLC分析灵芝多糖(21003)的单糖组成及含量Table 13 Monosaccharide composition and content of Ganoderma lucidum polysaccharide (21003) analyzed by HPLC
Figure PCTCN2021142290-appb-000013
Figure PCTCN2021142290-appb-000013
表14 HPLC分析灵芝多糖(21004)的单糖组成及含量Table 14 Monosaccharide composition and content of Ganoderma lucidum polysaccharide (21004) analyzed by HPLC
Figure PCTCN2021142290-appb-000014
Figure PCTCN2021142290-appb-000014
实施例5：灵芝多糖含量的检测Embodiment 5: Detection of Ganoderma lucidum polysaccharide content
1、苯酚溶液的配制1. Preparation of phenol solution
称取5g的苯酚，加蒸馏水溶解，定容至100mL，即得。Weigh 5g of phenol, dissolve in distilled water, and dilute to 100mL.
2、硫酸蒽酮溶液的配制2. Preparation of anthrone sulfate solution
称取0.1g的蒽酮，于100mL容量瓶中，用浓硫酸定容，充分溶解混匀，即得。Weigh 0.1g of anthrone, put it in a 100mL volumetric flask, dilute to volume with concentrated sulfuric acid, fully dissolve and mix to get it.
3、混合单糖对照品溶液的制备3. Preparation of mixed monosaccharide reference solution
确定四种供试品(21001；21002；21003；21004)中的单糖组成，并计算各单糖含量。以高效液相色谱确定的单糖比例配制含量检测需要的对照品溶液，总浓度为0.1mg/mL。Determine the monosaccharide composition in the four test products (21001; 21002; 21003; 21004), and calculate the content of each monosaccharide. The reference solution required for content detection was prepared with the monosaccharide ratio determined by high performance liquid chromatography, and the total concentration was 0.1 mg/mL.
制备灵芝多糖(21001)的混合单糖对照品溶液，准确称取不同单糖，配制含4.41μg/mL甘露糖、3.89μg/mL葡萄糖醛酸、81.72μg/mL葡萄糖、9.98μg/mL半乳糖的水溶液，为混合单糖对照溶液。Prepare the mixed monosaccharide reference solution of Ganoderma lucidum polysaccharide (21001), accurately weigh different monosaccharides, and prepare the solution containing 4.41 μg/mL mannose, 3.89 μg/mL glucuronic acid, 81.72 μg/mL glucose, 9.98 μg/mL galactose The aqueous solution is the mixed monosaccharide control solution.
制备灵芝多糖(21002)的混合单糖对照品溶液，准确称取不同单糖，配制含3.89μg/mL甘露糖、3.60μg/mL葡萄糖醛酸、84.31μg/mL葡萄糖、8.20μg/mL半乳糖的水溶液，为混合单糖对照溶液。Prepare the mixed monosaccharide reference solution of Ganoderma lucidum polysaccharide (21002), accurately weigh different monosaccharides, and prepare the solution containing 3.89 μg/mL mannose, 3.60 μg/mL glucuronic acid, 84.31 μg/mL glucose, 8.20 μg/mL galactose The aqueous solution is the mixed monosaccharide control solution.
制备灵芝多糖(21003)的混合单糖对照品溶液，准确称取准确称取不同单糖，配制含3.91μg/mL甘露糖、3.83μg/mL葡萄糖醛酸、84.40μg/mL葡萄糖、7.87μg/mL半乳糖的水溶液，为混合单糖对照溶液。Prepare the mixed monosaccharide reference solution of Ganoderma lucidum polysaccharide (21003), accurately weigh different monosaccharides, and prepare the solution containing 3.91 μg/mL mannose, 3.83 μg/mL glucuronic acid, 84.40 μg/mL glucose, 7.87 μg/mL The aqueous solution of galactose in mL is the mixed monosaccharide control solution.
制备灵芝多糖(21004)的混合单糖对照品溶液，准确称取准确称取不同单糖，配制含4.09μg/mL甘露糖、1.87μg/mL葡萄糖醛酸、87.28μg/mL葡萄糖、6.67μg/mL半乳糖的水溶液，为混合单糖对照溶液。Prepare the mixed monosaccharide reference solution of Ganoderma lucidum polysaccharide (21004), accurately weigh different monosaccharides, and prepare the solution containing 4.09 μg/mL mannose, 1.87 μg/mL glucuronic acid, 87.28 μg/mL glucose, 6.67 μg/mL The aqueous solution of galactose in mL is the mixed monosaccharide control solution.
4、苯酚-硫酸法标准曲线的制作4. Preparation of standard curve of phenol-sulfuric acid method
精密量取步骤3中混合单糖对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置具塞试管中，分别加水补至2.0mL，各精密加入步骤1中制得的苯酚溶液1mL，摇匀，迅速精密加入浓硫酸5mL，摇匀，放置10分钟，置40℃水浴中保温15min，取出，冰浴2min，空白不加对照品溶液，测定在490nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制标准曲线。Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step 3, put them in test tubes with stoppers, add water to make up to 2.0mL, and add each precision into step 1. 1mL of the prepared phenol solution was shaken, quickly and precisely added 5mL of concentrated sulfuric acid, shaken well, placed for 10 minutes, placed in a 40°C water bath for 15 minutes, taken out, ice bathed for 2 minutes, blank without reference solution, and measured at 490nm. Take the absorbance as the ordinate and the concentration of the sugar reference substance as the abscissa to draw a standard curve.
5、蒽酮-硫酸法标准曲线的制作5. Preparation of standard curve of anthrone-sulfuric acid method
精密量取步骤3中混合单糖对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置于具塞试管中，分别加水补至2.0mL，各精密加入步骤2中制得的硫酸蒽酮溶液6mL，摇匀，放置15min后，置于冰浴中冷却15min，取出，空白不加对照品溶液，测定在625nm处 的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制标准曲线。Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step 3, place them in test tubes with stoppers, respectively add water to make up to 2.0mL, and add each precision to step 2. 6mL of anthrone sulfate solution prepared in The concentration is the abscissa, and the standard curve is drawn.
6、供试品的制备6. Preparation of the test product
准确称取四批灵芝多糖样品(21001；21002；21003；21004)各10mg，于100mL的容量瓶中，用纯水定容，充分溶解混匀，即得。Accurately weigh four batches of Ganoderma lucidum polysaccharide samples (21001; 21002; 21003; 21004), each 10 mg, in a 100-mL volumetric flask, dilute to volume with pure water, fully dissolve and mix to obtain.
7、供试品的检测7. Detection of the test product
a、苯酚-硫酸法a. Phenol-sulfuric acid method
精密吸取供试品溶液1.0mL，加水至2.0mL，照(4)步骤苯酚-硫酸法标准曲线的制作项下的方法，自“加入苯酚溶液1mL”起，依上述方法测定供试品的吸光度，根据苯酚-硫酸法标准曲线得到多糖含量。Accurately draw 1.0mL of the test solution, add water to 2.0mL, and measure the absorbance of the test product according to the above method according to the method under the preparation of the standard curve of the phenol-sulfuric acid method in step (4), starting from "adding 1mL of the phenol solution" , and the polysaccharide content was obtained according to the standard curve of the phenol-sulfuric acid method.
b、蒽酮-硫酸法b. Anthrone-sulfuric acid method
精密吸取供试品溶液1.0mL，加水至2.0mL，照(5)步骤蒽酮-硫酸法标准曲线的制作项下的方法，自“加入硫酸蒽酮溶液6mL”起，依法测定供试品的吸光度，根据蒽酮-硫酸法标准曲线得到多糖含量。Accurately draw 1.0 mL of the solution of the test sample, add water to 2.0 mL, and follow the method under the preparation of the standard curve of the anthrone-sulfuric acid method in step (5). Absorbance, polysaccharide content was obtained according to the standard curve of anthrone-sulfuric acid method.
实验结果表明，混合单糖为对照品时，用苯酚-硫酸法检测灵芝多糖(21001)的含量，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝7.3845X-0.0186(R＝0.99790)。用蒽酮-硫酸法法检测时，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝4.9719X+0.0098(R＝0.99875)，结果见表14。The experimental results show that when the mixed monosaccharide is the reference substance, the content of Ganoderma lucidum polysaccharide (21001) is detected by the phenol-sulfuric acid method, and the absorbance (Y) of the solution is linearly related to the concentration (X, μg/mL) of the mixed monosaccharide reference substance, The regression equation is: Y=7.3845X-0.0186 (R=0.99790). When detected by the anthrone-sulfuric acid method, the absorbance (Y) of the solution has a linear relationship with the concentration of the mixed monosaccharide reference substance (X, μg/mL). The regression equation is: Y=4.9719X+0.0098 (R=0.99875), The results are shown in Table 14.
表15灵芝多糖(21001)含量检测结果(混合单糖为对照品)Table 15 Ganoderma lucidum polysaccharide (21001) content detection results (mixed monosaccharide is the reference substance)
Figure PCTCN2021142290-appb-000015
Figure PCTCN2021142290-appb-000015
实验结果表明，混合单糖为对照品时，用苯酚-硫酸法检测灵芝多糖(21002)的含量，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝7.6372X-0.024(R＝0.99900)。用蒽酮-硫酸法检测时，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝5.2707X+0.0114(R＝0.99935)，结果见表15。The experimental results show that when the mixed monosaccharide is the reference substance, the content of Ganoderma lucidum polysaccharide (21002) is detected by the phenol-sulfuric acid method, and the absorbance (Y) of the solution is linearly related to the concentration of the mixed monosaccharide reference substance (X, μg/mL), The regression equation is: Y=7.6372X-0.024 (R=0.99900). When detected by anthrone-sulfuric acid method, the absorbance (Y) of the solution has a linear relationship with the concentration of mixed monosaccharide reference substance (X, μg/mL). The regression equation is: Y=5.2707X+0.0114 (R=0.99935), the result See Table 15.
表16灵芝多糖(21002)含量检测结果(混合单糖为对照品)Table 16 Ganoderma lucidum polysaccharide (21002) content detection results (mixed monosaccharide is the reference substance)
Figure PCTCN2021142290-appb-000016
Figure PCTCN2021142290-appb-000016
实验结果表明，混合单糖为对照品时，用苯酚-硫酸法检测灵芝多糖(21003)的含量，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝7.5912X+0.014(R＝0.99955)。用蒽酮-硫酸法检测时，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝5.3253X+0.0187(R＝0.99960)，结果见表16。The experimental results show that when the mixed monosaccharide is the reference substance, the content of Ganoderma lucidum polysaccharide (21003) is detected by the phenol-sulfuric acid method, and the absorbance (Y) of the solution is linearly related to the concentration of the mixed monosaccharide reference substance (X, μg/mL), The regression equation is: Y=7.5912X+0.014 (R=0.99955). When detected by the anthrone-sulfuric acid method, the absorbance (Y) of the solution has a linear relationship with the concentration of the mixed monosaccharide reference substance (X, μg/mL). The regression equation is: Y=5.3253X+0.0187 (R=0.99960), the result See Table 16.
表17灵芝多糖(21003)含量检测结果(混合单糖为对照品)Table 17 Ganoderma lucidum polysaccharide (21003) content detection results (mixed monosaccharide is the reference substance)
Figure PCTCN2021142290-appb-000017
Figure PCTCN2021142290-appb-000017
实验结果表明，混合单糖为对照品时，用苯酚-硫酸法检测灵芝多糖(21004)的含量，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝7.3812X-0.0042(R＝0.99865)。用蒽酮-硫酸法检测时，溶液的吸光度(Y)与混合单糖对照品浓度(X，μg/mL)呈线性关系，回归方程为：Y＝4.9732X+0.0206(R＝0.99760)，结果见表16。The experimental results show that when the mixed monosaccharide is the reference substance, the content of Ganoderma lucidum polysaccharide (21004) is detected by the phenol-sulfuric acid method, and the absorbance (Y) of the solution is linearly related to the concentration of the mixed monosaccharide reference substance (X, μg/mL), The regression equation is: Y=7.3812X-0.0042 (R=0.99865). When detected by the anthrone-sulfuric acid method, the absorbance (Y) of the solution has a linear relationship with the concentration of the mixed monosaccharide reference substance (X, μg/mL). The regression equation is: Y=4.9732X+0.0206 (R=0.99760), the result See Table 16.
表18灵芝多糖(21004)含量检测结果(混合单糖为对照品)Table 18 Ganoderma lucidum polysaccharide (21004) content detection results (mixed monosaccharide is the reference substance)
Figure PCTCN2021142290-appb-000018
Figure PCTCN2021142290-appb-000018
根据对新制备不同批次灵芝多糖的分析，对多糖糖组成的方法进行了优化。液相分析主要在色谱柱类型、流动相pH值及增加对样品的萃取处理等方面进行了优化，使其能更准确的检测灵芝多糖的糖组成情况。Based on the analysis of newly prepared Ganoderma lucidum polysaccharides from different batches, the method of polysaccharide composition was optimized. The liquid phase analysis is mainly optimized in terms of column type, pH value of mobile phase, and increased extraction of samples, so that it can more accurately detect the sugar composition of Ganoderma lucidum polysaccharides.

Claims (10)

  1. 一种灵芝多糖含量的检测方法，包括：(一)用HPLC确定灵芝多糖的单糖组成；(二)使用苯酚-硫酸法或蒽酮-硫酸法测定灵芝多糖含量，优选地，步骤(二)中，使用根据步骤(一)确定的单糖比例配制的至少含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的混合单糖溶液作为对照品溶液进行测定。A detection method for Ganoderma lucidum polysaccharide content, comprising: (1) determining the monosaccharide composition of Ganoderma lucidum polysaccharide by HPLC; (2) using phenol-sulfuric acid method or anthrone-sulfuric acid method to measure the content of Ganoderma lucidum polysaccharide, preferably, step (2) , use the mixed monosaccharide solution containing at least glucose, galactose, glucuronic acid and mannose prepared according to the monosaccharide ratio determined in step (1) as the reference solution for determination.
  2. 根据权利要求1所述的检测方法，其中，步骤(一)使用以下方法一或方法二：detection method according to claim 1, wherein, step (1) uses following method one or method two:
    方法一，其包括以下步骤：Method one, which includes the following steps:
    (1)HPLC对照品溶液的制备(1) Preparation of HPLC reference solution
    配制浓度为0.5mg/mL的来苏糖水溶液，作为内标溶液，An aqueous solution of lyxose with a concentration of 0.5 mg/mL was prepared as the internal standard solution,
    分别配制葡萄糖、半乳糖、葡萄糖醛酸、甘露糖、核糖、鼠李糖、半乳糖醛酸、阿拉伯糖、岩藻糖的水溶液作为单标储存液，将各单标储存液混合后得到单糖混合水溶液，其中每mL溶液中含有5mg葡萄糖、0.3mg半乳糖、0.2mg甘露糖、0.1mg葡萄糖醛酸、0.05mg核糖、0.05mg岩藻糖、0.02mg半乳糖醛酸、0.015mg阿拉伯糖、0.01mg鼠李糖，The aqueous solutions of glucose, galactose, glucuronic acid, mannose, ribose, rhamnose, galacturonic acid, arabinose, and fucose were prepared respectively as single-label storage solutions, and the single-label storage solutions were mixed to obtain monosaccharides. Mixed aqueous solution, wherein each mL of solution contains 5mg glucose, 0.3mg galactose, 0.2mg mannose, 0.1mg glucuronic acid, 0.05mg ribose, 0.05mg fucose, 0.02mg galacturonic acid, 0.015mg arabinose, 0.01mg rhamnose,
    取250μL上述单糖混合的水溶液，加入250μL内标溶液，再加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭60℃-80℃加热30min-90min，冷却至室温，加入0.15M盐酸600μL，混匀，过滤膜，即得HPLC对照品溶液；Take 250μL of the above monosaccharide mixed aqueous solution, add 250μL of internal standard solution, then add 600μL of 0.15M sodium hydroxide aqueous solution, and 1.0mL of 0.1M-0.6M 1-phenyl-3-methyl-5-pyridine The methanol solution of oxazolinone is sealed and heated at 60℃-80℃ for 30min-90min, cooled to room temperature, added with 600μL of 0.15M hydrochloric acid, mixed well, and filtered through membrane to obtain the HPLC reference solution;
    (2)HPLC供试品溶液的制备(2) Preparation of HPLC test solution
    配制4mg/mL的灵芝多糖样品溶液，Prepare 4 mg/mL Ganoderma lucidum polysaccharide sample solution,
    精密吸取500μL样品溶液至反应瓶中，加入500μL 1M-6M的三氟乙酸；混匀，密闭，90℃-120℃加热1h-6h，挥干溶剂后，加入适量甲醇，挥干，再重复此操作两次，Precisely pipet 500μL of sample solution into the reaction bottle, add 500μL of 1M-6M trifluoroacetic acid; mix well, seal, heat at 90℃-120℃ for 1h-6h, evaporate the solvent, add appropriate amount of methanol, evaporate to dryness, and repeat this process operate twice,
    残渣加250μL水，250μL内标溶液，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液；密闭60℃-80℃加热30min-90min，冷却至室温；加入0.15M盐酸600μL，混匀，过滤膜，即得供试品溶液；To the residue, add 250 μL of water, 250 μL of internal standard solution, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.1M-0.6M methanol solution of 1-phenyl-3-methyl-5-pyrazolone; sealed Heating at 60℃-80℃ for 30min-90min, cooling to room temperature; adding 600μL of 0.15M hydrochloric acid, mixing well, filtering the membrane to obtain the test solution;
    (3)糖组成分析(3) Analysis of sugar composition
    测量供试品与对照品的HPLC色谱图，Measure the HPLC chromatograms of the test substance and reference substance,
    将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比 对，确定对应的单糖特征峰，以来苏糖为内标，用峰面积计算各单糖含量，方法二，其包括以下步骤：Compare the retention time of the HPLC chromatogram of the test product with the HPLC chromatogram of the reference substance, determine the corresponding monosaccharide characteristic peak, use threose as the internal standard, and use the peak area to calculate the content of each monosaccharide. Method 2, its Include the following steps:
    (1)HPLC对照品溶液的制备(1) Preparation of HPLC reference solution
    配制含0.02-0.4mg/mL(优选0.08mg/mL)甘露糖、0.0125-0.25mg/mL(优选0.05mg/mL)葡萄糖醛酸、0.3125-6.25mg/mL(优选1.25mg/mL)葡萄糖、0.0375-0.6mg/mL(优选0.15mg/mL)半乳糖、0.0125-0.25mg/mL(优选0.05mg/mL)岩藻糖、0.00625-0.125mg/mL(优选0.025mg/mL)核糖、0.001375-0.0275mg/mL(0.0055mg/mL)鼠李糖、0.0025-0.05mg/mL(优选0.01mg/mL)阿拉伯糖的水溶液为混合单糖储备液，The preparation contains 0.02-0.4mg/mL (preferably 0.08mg/mL) mannose, 0.0125-0.25mg/mL (preferably 0.05mg/mL) glucuronic acid, 0.3125-6.25mg/mL (preferably 1.25mg/mL) glucose, 0.0375-0.6mg/mL (preferably 0.15mg/mL) galactose, 0.0125-0.25mg/mL (preferably 0.05mg/mL) fucose, 0.00625-0.125mg/mL (preferably 0.025mg/mL) ribose, 0.001375- An aqueous solution of 0.0275mg/mL (0.0055mg/mL) rhamnose and 0.0025-0.05mg/mL (preferably 0.01mg/mL) arabinose is a mixed monosaccharide stock solution,
    取500μL上述混合单糖储备液，再加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭60℃-80℃加热30min-90min，冷却至室温，加入0.15M盐酸600μL，混匀，取500μL溶液，加500μL氯仿，离心进行萃取，萃取结束后取上层溶液再次离心，上清液作为HPLC对照品溶液；Take 500 μL of the above mixed monosaccharide stock solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.1M-0.6M methanol solution of 1-phenyl-3-methyl-5-pyrazolone. , sealed at 60℃-80℃, heated for 30min-90min, cooled to room temperature, added 600μL of 0.15M hydrochloric acid, mixed, take 500μL of solution, add 500μL of chloroform, centrifuge for extraction, after the extraction, take the upper layer solution and centrifuge again, the supernatant as HPLC reference solution;
    (2)HPLC供试品溶液的制备(2) Preparation of HPLC test solution
    配制4mg/mL的灵芝多糖样品溶液，Prepare 4 mg/mL Ganoderma lucidum polysaccharide sample solution,
    精密吸取500μL灵芝多糖样品溶液至反应瓶中，加入500μL 1M-6M的三氟乙酸；混匀，密闭，90℃-120℃加热1h-6h，挥干溶剂后，加入适量甲醇，挥干，再重复此操作两次，Precisely pipet 500μL of Ganoderma lucidum polysaccharide sample solution into the reaction bottle, add 500μL of 1M-6M trifluoroacetic acid; mix well, seal, heat at 90℃-120℃ for 1h-6h, evaporate the solvent, add appropriate amount of methanol, evaporate to dryness, and then Repeat this twice,
    残渣加500μL水，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液；密闭60℃-80℃加热30min-90min，冷却至室温；加入0.15M盐酸600μL，混匀，取500μL溶液，加500μL氯仿，离心进行萃取，萃取结束后取上层溶液离心，上清液作为供试品溶液；Add 500 μL of water to the residue, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.1M-0.6M methanol solution of 1-phenyl-3-methyl-5-pyrazolone; sealed at 60℃-80℃ Heating for 30min-90min, cooling to room temperature; adding 600μL of 0.15M hydrochloric acid, mixing well, taking 500μL solution, adding 500μL chloroform, centrifuging for extraction, after the extraction, take the upper layer solution and centrifuge, and the supernatant is used as the test solution;
    (3)糖组成分析(3) Analysis of sugar composition
    测量供试品与对照品的HPLC色谱图，Measure the HPLC chromatograms of the test substance and reference substance,
    将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比对，确定对应的单糖特征峰，用峰面积计算各单糖含量。Compare the retention time of the HPLC chromatogram of the test product and the HPLC chromatogram of the reference substance to determine the corresponding monosaccharide characteristic peaks, and use the peak area to calculate the content of each monosaccharide.
  3. 根据权利要求1或2所述的检测方法，其中，步骤(一)中，在以下色谱条件下测量HPLC色谱图：detection method according to claim 1 or 2, wherein, in step (1), measure HPLC chromatogram under following chromatographic conditions:
    高效液相色谱仪：Agilent 1260High Performance Liquid Chromatograph: Agilent 1260
    色谱柱：Agilent Zorbax SB-C18(4.6mm x 250mm；5μm)Column: Agilent Zorbax SB-C18 (4.6mm x 250mm; 5μm)
    柱温：35℃Column temperature: 35℃
    检测波长：250nmDetection wavelength: 250nm
    流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝6)，B相：乙腈Mobile phase: A phase: 0.05M phosphate buffer solution (pH=6), B phase: acetonitrile
    洗脱条件：0-20min，A(％)：84-83；20-40min，A(％)：83-82.5；40-65min，A(％)：82.5-81，1mL/min的流速，Elution conditions: 0-20min, A(%): 84-83; 20-40min, A(%): 83-82.5; 40-65min, A(%): 82.5-81, flow rate of 1 mL/min,
    或者在以下色谱条件下测量HPLC色谱图：Or measure HPLC chromatograms under the following chromatographic conditions:
    高效液相色谱仪：Agilent 1260；High performance liquid chromatograph: Agilent 1260;
    色谱柱：C6-Phenyl(4.6mm x 25cm；5μm)；Chromatographic column: C6-Phenyl (4.6mm x 25cm; 5μm);
    柱温：35℃；Column temperature: 35℃;
    检测波长：250nm；Detection wavelength: 250nm;
    流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝7)，B相：乙腈；Mobile phase: A phase: 0.05M phosphate buffer solution (pH=7), B phase: acetonitrile;
    洗脱条件：B(％)：15-16梯度洗脱；Elution conditions: B (%): 15-16 gradient elution;
    流速：0.8mL/min。Flow rate: 0.8 mL/min.
  4. 根据权利要求1所述的检测方法，其中，步骤(一)使用以下方法A或方法B：detection method according to claim 1, wherein, step (1) uses following method A or method B:
    方法A，其包括以下步骤：Method A, which includes the following steps:
    (1)HPLC对照品溶液的制备(1) Preparation of HPLC reference solution
    配制浓度为0.5mg/mL的来苏糖水溶液，作为内标溶液，An aqueous solution of lyxose with a concentration of 0.5 mg/mL was prepared as the internal standard solution,
    分别配制葡萄糖、半乳糖、葡萄糖醛酸、甘露糖、核糖、鼠李糖、半乳糖醛酸、阿拉伯糖、岩藻糖的水溶液作为单标储存液，将各单标储存液混合后得到单糖混合水溶液，其中每mL溶液中含有5mg葡萄糖、0.3mg半乳糖、0.2mg甘露糖、0.1mg葡萄糖醛酸、0.05mg核糖、0.05mg岩藻糖、0.02mg半乳糖醛酸、0.015mg阿拉伯糖、0.01mg鼠李糖，The aqueous solutions of glucose, galactose, glucuronic acid, mannose, ribose, rhamnose, galacturonic acid, arabinose, and fucose were prepared respectively as single-label storage solutions, and the single-label storage solutions were mixed to obtain monosaccharides. Mixed aqueous solution, wherein each mL of solution contains 5mg glucose, 0.3mg galactose, 0.2mg mannose, 0.1mg glucuronic acid, 0.05mg ribose, 0.05mg fucose, 0.02mg galacturonic acid, 0.015mg arabinose, 0.01mg rhamnose,
    取250μL上述单糖混合的水溶液，加入250μL内标溶液，再加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭70℃加热60min，冷却至室温，加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得HPLC对照品溶液；Take 250μL of the above monosaccharide mixed aqueous solution, add 250μL of internal standard solution, then add 600μL of 0.15M sodium hydroxide aqueous solution, and 1.0mL of 0.2M 1-phenyl-3-methyl-5-pyrazolone The methanol solution was sealed at 70°C for 60min, cooled to room temperature, added with 600μL of 0.15M hydrochloric acid, mixed well, and passed through a 0.45μm nylon filter to obtain the HPLC reference solution;
    (2)HPLC供试品溶液的制备(2) Preparation of HPLC test solution
    配制浓度为4mg/mL的灵芝多糖样品溶液，Prepare a sample solution of Ganoderma lucidum polysaccharide with a concentration of 4 mg/mL,
    精密吸取500μL样品溶液至反应瓶中，加入500μL 6M的三氟乙酸；混匀，密闭，120℃加热4h，挥干溶剂后，加入1mL甲醇，挥干，再重复此操作两次，Precisely pipet 500 μL of sample solution into the reaction bottle, add 500 μL of 6M trifluoroacetic acid; mix well, seal, heat at 120 °C for 4 h, evaporate the solvent, add 1 mL of methanol, evaporate to dryness, repeat this operation twice,
    残渣加250μL水，250μL内标溶液，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液；密闭70℃加热60min，冷却至室温；加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得供试品溶液；To the residue, add 250 μL of water, 250 μL of internal standard solution, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone; sealed and heated at 70°C Cool to room temperature for 60 min; add 600 μL of 0.15M hydrochloric acid, mix well, and pass through a 0.45 μm nylon filter to obtain the test solution;
    (3)糖组成分析(3) Analysis of sugar composition
    在以下色谱条件下测量供试品与对照品的HPLC色谱图：Measure the HPLC chromatograms of the test article and reference substance under the following chromatographic conditions:
    高效液相色谱仪：Agilent 1260High Performance Liquid Chromatograph: Agilent 1260
    色谱柱：Agilent Zorbax SB-C18(4.6mm x 250mm；5μm)Column: Agilent Zorbax SB-C18 (4.6mm x 250mm; 5μm)
    柱温：35℃Column temperature: 35℃
    检测波长：250nmDetection wavelength: 250nm
    流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝6)，B相：乙腈Mobile phase: A phase: 0.05M phosphate buffer solution (pH=6), B phase: acetonitrile
    洗脱条件：0-20min，A(％)：84-83；20-40min，A(％)：83-82.5；40-65min，A(％)：82.5-81，1mL/min的流速Elution conditions: 0-20min, A(%): 84-83; 20-40min, A(%): 83-82.5; 40-65min, A(%): 82.5-81, flow rate of 1 mL/min
    将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比对，确定对应的单糖特征峰，以来苏糖为内标，用峰面积计算各单糖含量，方法B，其包括以下步骤：Compare the retention time of the HPLC chromatogram of the test product and the HPLC chromatogram of the reference substance, determine the corresponding monosaccharide characteristic peak, use threose as the internal standard, and use the peak area to calculate the content of each monosaccharide. Method B, its Include the following steps:
    (1)HPLC对照品溶液的制备(1) Preparation of HPLC reference solution
    配制含0.08mg/mL甘露糖、0.05mg/mL葡萄糖醛酸、1.25mg/mL葡萄糖、0.15mg/mL半乳糖、0.05mg/mL岩藻糖、0.025mg/mL核糖、0.0055mg/mL鼠李糖、0.01mg/mL阿拉伯糖的水溶液为混合单糖储备液，Formulated to contain 0.08mg/mL mannose, 0.05mg/mL glucuronic acid, 1.25mg/mL glucose, 0.15mg/mL galactose, 0.05mg/mL fucose, 0.025mg/mL ribose, 0.0055mg/mL rhamnose The aqueous solution of sugar and 0.01 mg/mL arabinose is the mixed monosaccharide stock solution,
    取500μL混合单糖储备液，加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭70℃加热60min，取出冰水浴2min，加入0.15M盐酸600μL，混匀，取500μL溶液，加500μL氯仿，离心进行萃取，萃取结束后取上层溶液离心，上清液作为HPLC对照品溶液；Take 500 μL of mixed monosaccharide stock solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, sealed and heated at 70°C for 60min , take out the ice-water bath for 2 min, add 600 μL of 0.15M hydrochloric acid, mix well, take 500 μL of the solution, add 500 μL of chloroform, centrifuge for extraction, and centrifuge the upper layer solution after extraction, and the supernatant is used as the HPLC reference solution;
    (2)HPLC供试品溶液的制备(2) Preparation of HPLC test solution
    配制浓度为4mg/mL的灵芝多糖样品溶液，Prepare a sample solution of Ganoderma lucidum polysaccharide with a concentration of 4 mg/mL,
    精密吸取灵芝多糖样品溶液500μL至反应瓶中，加入500μL 6M的三氟乙酸，混匀，密闭，120℃加热4h，挥干溶剂后，加入1mL甲醇，挥干，再重复此操作两次，Precisely pipet 500 μL of Ganoderma lucidum polysaccharide sample solution into the reaction bottle, add 500 μL of 6M trifluoroacetic acid, mix well, seal, heat at 120 °C for 4 h, evaporate the solvent, add 1 mL of methanol, evaporate to dryness, repeat this operation twice,
    残渣加500μL水，600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M 1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭反应瓶70℃加热60min，取出冰水浴2min，加入0.15M盐酸600μL，混匀，取500μL溶液， 加500μL氯仿，离心进行萃取，萃取结束后取上层溶液离心，上清液作为供试品溶液；To the residue, add 500 μL of water, 600 μL of 0.15M aqueous sodium hydroxide solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, seal the reaction flask and heat at 70°C for 60min, take out Ice water bath for 2min, add 600μL of 0.15M hydrochloric acid, mix well, take 500μL of solution, add 500μL of chloroform, centrifuge for extraction, after extraction, take the upper layer solution and centrifuge, and the supernatant is used as the test solution;
    (3)糖组成分析(3) Analysis of sugar composition
    在以下色谱条件下测量供试品与对照品的HPLC色谱图，Measure the HPLC chromatograms of the test substance and reference substance under the following chromatographic conditions,
    高效液相色谱仪：Agilent 1260；High performance liquid chromatograph: Agilent 1260;
    色谱柱：C6-Phenyl(4.6mm x 25cm；5μm)；Chromatographic column: C6-Phenyl (4.6mm x 25cm; 5μm);
    柱温：35℃；Column temperature: 35℃;
    检测波长：250nm；Detection wavelength: 250nm;
    流动相：A相：0.05M的磷酸盐缓冲溶液(pH＝7)，B相：乙腈；Mobile phase: A phase: 0.05M phosphate buffer solution (pH=7), B phase: acetonitrile;
    洗脱条件：B(％)：15-16梯度洗脱；Elution conditions: B (%): 15-16 gradient elution;
    流速：0.8mL/min；Flow rate: 0.8mL/min;
    将供试品的HPLC色谱图与对照品的HPLC色谱图的保留时间进行比对，确定对应的单糖特征峰，用峰面积计算各单糖含量。Compare the retention time of the HPLC chromatogram of the test product and the HPLC chromatogram of the reference substance to determine the corresponding monosaccharide characteristic peaks, and use the peak area to calculate the content of each monosaccharide.
  5. 根据权利要求2或4所述的检测方法，其中，步骤(一)方法一或方法A中，HPLC对照品溶液的制备包括以下步骤：detection method according to claim 2 or 4, wherein, in step (1) method one or method A, the preparation of HPLC reference substance solution comprises the following steps:
    配制浓度为50mg/mL葡萄糖储备液；The preparation concentration is 50mg/mL glucose stock solution;
    配制浓度为6mg/mL的半乳糖储备液；Prepare a galactose stock solution with a concentration of 6 mg/mL;
    配制浓度为1mg/mL的葡萄糖醛酸储备液；Prepare a glucuronic acid stock solution with a concentration of 1 mg/mL;
    配制浓度为2mg/mL的甘露糖储备液；Prepare a mannose stock solution with a concentration of 2 mg/mL;
    配制浓度为0.5mg/mL的核糖储备液；Prepare a ribose stock solution with a concentration of 0.5 mg/mL;
    配制浓度为2mg/mL的鼠李糖储备液；Prepare a rhamnose stock solution with a concentration of 2 mg/mL;
    配制浓度为10mg/mL的半乳糖醛酸储备液；Prepare a galacturonic acid stock solution with a concentration of 10 mg/mL;
    配制浓度为15mg/mL的阿拉伯糖储备液；Prepare an arabinose stock solution with a concentration of 15 mg/mL;
    配制浓度为0.5mg/mL的岩藻糖储备液；Prepare a fucose stock solution with a concentration of 0.5 mg/mL;
    配制浓度为0.5mg/mL的来苏糖内标溶液，Prepare a lyxose internal standard solution with a concentration of 0.5 mg/mL,
    分别精密吸取1000体积核糖储备液、1000体积岩藻糖储备液、1000体积葡萄糖醛酸储备液、1000体积甘露糖储备液、1000体积葡萄糖储备液、500体积半乳糖储备液、50体积鼠李糖储备液、20体积半乳糖醛酸储备液、10体积阿拉伯糖储备液，加水定容至10000体积，即得混合溶液；Precisely draw 1000 volumes of ribose stock solution, 1000 volumes of fucose stock solution, 1000 volumes of glucuronic acid stock solution, 1000 volumes of mannose stock solution, 1000 volumes of glucose stock solution, 500 volumes of galactose stock solution, and 50 volumes of rhamnose Stock solution, 20 volumes of galacturonic acid stock solution, and 10 volumes of arabinose stock solution, add water to make up to 10,000 volumes to obtain a mixed solution;
    取250μL上述混合溶液，250μL内标溶液，分别加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.1M-0.6M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭60℃-80℃加热30min-90min，冷却至室温，加入0.15 M盐酸600μL，混匀，过滤膜，即得HPLC对照品溶液。Take 250 μL of the above mixed solution, 250 μL of the internal standard solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.1M-0.6M 1-phenyl-3-methyl-5-pyrazolone. Methanol solution, sealed at 60℃-80℃, heated for 30min-90min, cooled to room temperature, added 600μL of 0.15 M hydrochloric acid, mixed well, filtered through membrane, to obtain HPLC reference solution.
  6. 根据权利要求2或4所述的检测方法，其中，步骤(一)方法一或方法A中，HPLC对照品溶液的制备包括以下步骤：detection method according to claim 2 or 4, wherein, in step (1) method one or method A, the preparation of HPLC reference substance solution comprises the following steps:
    配制浓度为50mg/mL葡萄糖储备液；The preparation concentration is 50mg/mL glucose stock solution;
    配制浓度为6mg/mL的半乳糖储备液；Prepare a galactose stock solution with a concentration of 6 mg/mL;
    配制浓度为1mg/mL的葡萄糖醛酸储备液；Prepare a glucuronic acid stock solution with a concentration of 1 mg/mL;
    配制浓度为2mg/mL的甘露糖储备液；Prepare a mannose stock solution with a concentration of 2 mg/mL;
    配制浓度为0.5mg/mL的核糖储备液；Prepare a ribose stock solution with a concentration of 0.5 mg/mL;
    配制浓度为2mg/mL的鼠李糖储备液；Prepare a rhamnose stock solution with a concentration of 2 mg/mL;
    配制浓度为10mg/mL的半乳糖醛酸储备液；Prepare a galacturonic acid stock solution with a concentration of 10 mg/mL;
    配制浓度为15mg/mL的阿拉伯糖储备液；Prepare an arabinose stock solution with a concentration of 15 mg/mL;
    配制浓度为0.5mg/mL的岩藻糖储备液；Prepare a fucose stock solution with a concentration of 0.5 mg/mL;
    配制浓度为0.5mg/mL的来苏糖内标溶液，Prepare a lyxose internal standard solution with a concentration of 0.5 mg/mL,
    分别精密吸取1000体积核糖储备液、1000体积岩藻糖储备液、1000体积葡萄糖醛酸储备液、1000体积甘露糖储备液、1000体积葡萄糖储备液、500体积半乳糖储备液、50体积鼠李糖储备液、20体积半乳糖醛酸储备液、10体积阿拉伯糖储备液，加水定容至10000体积，即得混合溶液；Precisely draw 1000 volumes of ribose stock solution, 1000 volumes of fucose stock solution, 1000 volumes of glucuronic acid stock solution, 1000 volumes of mannose stock solution, 1000 volumes of glucose stock solution, 500 volumes of galactose stock solution, and 50 volumes of rhamnose Stock solution, 20 volumes of galacturonic acid stock solution, and 10 volumes of arabinose stock solution, add water to make up to 10,000 volumes to obtain a mixed solution;
    取250μL上述混合溶液，250μL内标溶液，分别加入600μL的0.15M的氢氧化钠水溶液，以及1.0mL的0.2M的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液，密闭70℃加热60min，冷却至室温，加入0.15M盐酸600μL，混匀，过0.45μm尼龙滤膜，即得HPLC对照品溶液。Take 250 μL of the above mixed solution, 250 μL of the internal standard solution, add 600 μL of 0.15M sodium hydroxide aqueous solution, and 1.0 mL of 0.2M methanol solution of 1-phenyl-3-methyl-5-pyrazolone, respectively, Sealed and heated at 70°C for 60min, cooled to room temperature, added 600μL of 0.15M hydrochloric acid, mixed well, and passed through a 0.45μm nylon filter to obtain the HPLC reference solution.
  7. 根据权利要求1所述的检测方法，其中，步骤(二)中，所述混合单糖溶液为含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的水溶液，其中，以重量百分比计，基于溶液中的单糖的总重量，含有60％-85％的葡萄糖、8％-17％的半乳糖、3％-15％的葡萄糖醛酸、3％-7％的甘露糖，或者含有60％-90％的葡萄糖、6％-17％的半乳糖、1％-15％的葡萄糖醛酸、3％-8％的甘露糖，The detection method according to claim 1, wherein, in step (2), the mixed monosaccharide solution is an aqueous solution containing glucose, galactose, glucuronic acid and mannose, wherein, by weight percentage, based on the amount in the solution The total weight of monosaccharides containing 60%-85% glucose, 8%-17% galactose, 3%-15% glucuronic acid, 3%-7% mannose, or 60%-90% % glucose, 6%-17% galactose, 1%-15% glucuronic acid, 3%-8% mannose,
    或者，所述混合单糖溶液为以高效液相色谱确定的单糖比例配制含量检测需要的对照品溶液，总浓度为0.1mg/mL。Alternatively, the mixed monosaccharide solution is a reference substance solution required for content detection prepared with the monosaccharide ratio determined by high performance liquid chromatography, and the total concentration is 0.1 mg/mL.
  8. 根据权利要求1-7任一项所述的检测方法，其中，The detection method according to any one of claims 1-7, wherein,
    所述苯酚-硫酸法包括以下步骤：Described phenol-sulfuric acid method comprises the following steps:
    (1)苯酚溶液的配制(1) Preparation of phenol solution
    配制0.05g/mL的苯酚水溶液；Prepare 0.05g/mL aqueous solution of phenol;
    (2)含量检测对照品溶液的制备(2) Preparation of content detection reference solution
    根据步骤(一)确定的单糖比例配制至少含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的混合单糖溶液作为对照品溶液，总浓度为0.1mg/mL；According to the monosaccharide ratio determined in step (1), a mixed monosaccharide solution containing at least glucose, galactose, glucuronic acid and mannose is prepared as a reference solution, and the total concentration is 0.1 mg/mL;
    (3)苯酚-硫酸法标准曲线的制作(3) Preparation of standard curve of phenol-sulfuric acid method
    精密量取步骤(2)中对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置具塞试管中，分别加水补至2.0mL，各精密加入步骤(1)中苯酚溶液1mL，摇匀，迅速精密加入浓硫酸5mL，摇匀，放置10分钟，置40℃水浴中保温15分钟，取出，冰浴2min，空白不加对照品溶液，测定在490nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制苯酚-硫酸法标准曲线；Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the reference solution in step (2), put them in test tubes with stoppers, add water to make up to 2.0mL, and add water to step (1) precisely. 1mL of medium phenol solution, shake well, quickly and accurately add 5mL of concentrated sulfuric acid, shake well, place for 10 minutes, place in a 40°C water bath for 15 minutes, take out, ice bath for 2 minutes, blank without reference solution, measure the absorbance at 490nm , take the absorbance as the ordinate and the concentration of the sugar reference substance as the abscissa, draw the phenol-sulfuric acid method standard curve;
    (4)含量检测供试品的制备(4) Preparation of test sample for content detection
    准确称取待测灵芝多糖样品10mg，于100mL的容量瓶中，用纯水定容，充分溶解混匀，即得供试品溶液；Accurately weigh 10 mg of the Ganoderma lucidum polysaccharide sample to be tested, put it in a 100 mL volumetric flask, dilute to volume with pure water, fully dissolve and mix to obtain the test solution;
    (5)多糖含量的检测(5) Detection of polysaccharide content
    精密吸取步骤(4)中供试品溶液1.0mL，加水至2.0mL，照步骤(3)苯酚-硫酸法标准曲线的制作项下的方法，自“加入苯酚溶液1mL”起，依法测定供试品的吸光度，根据苯酚-硫酸法标准曲线得到多糖含量；Accurately draw 1.0 mL of the test solution in step (4), add water to 2.0 mL, and follow the method under step (3) Preparation of the phenol-sulfuric acid method standard curve, starting from "adding 1 mL of phenol solution", determine the test according to the law The absorbance of the product was obtained, and the polysaccharide content was obtained according to the standard curve of the phenol-sulfuric acid method;
    所述蒽酮-硫酸法包括以下步骤：The anthrone-sulfuric acid method comprises the following steps:
    (1)蒽酮硫酸溶液的配制(1) Preparation of anthrone sulfuric acid solution
    配制0.001g/mL的硫酸蒽酮溶液；Prepare 0.001g/mL solution of anthrone sulfate;
    (2)含量检测对照品溶液的制备(2) Preparation of content detection reference solution
    根据步骤(一)确定的单糖比例配制至少含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的混合单糖溶液作为对照品溶液，总浓度为0.1mg/mL；According to the monosaccharide ratio determined in step (1), a mixed monosaccharide solution containing at least glucose, galactose, glucuronic acid and mannose is prepared as a reference solution, and the total concentration is 0.1 mg/mL;
    (3)蒽酮-硫酸法标准曲线的制作(3) Preparation of standard curve of anthrone-sulfuric acid method
    精密量取步骤(2)中对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置于具塞试管中，分别加水补至2.0mL，各精密加入步骤(1)中硫酸蒽酮溶液6mL，摇匀，放置15min后，置于冰浴中冷却15min，取出，空白不加对照品溶液，测定在625nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制蒽酮-硫酸法标准曲线；Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the reference solution in step (2), place them in test tubes with stoppers, add water to make up to 2.0mL, and add water to 2.0mL for each precise addition step (1). ) in 6mL of anthrone sulfate solution, shake well, put it for 15min, cool it in an ice bath for 15min, take it out, blank without the reference solution, measure the absorbance at 625nm, take the absorbance as the ordinate, and the concentration of the sugar reference is Abscissa, draw the standard curve of anthrone-sulfuric acid method;
    (4)含量检测供试品的制备(4) Preparation of test sample for content detection
    准确称取待测灵芝多糖样品10mg，于100mL的容量瓶中，用纯水定容，充分溶解混匀，即得供试品溶液；Accurately weigh 10 mg of the Ganoderma lucidum polysaccharide sample to be tested, put it in a 100 mL volumetric flask, dilute to volume with pure water, fully dissolve and mix to obtain the test solution;
    (5)多糖含量的检测(5) Detection of polysaccharide content
    精密吸取步骤(6)中供试品溶液1.0mL，加水至2.0mL，照步骤(5)蒽酮-硫酸法标准曲线的制作项下的方法，自“加入硫酸蒽酮溶液6mL”起，依法测定供试品的吸光度，根据蒽酮-硫酸法标准曲线得到多糖含量。Accurately draw 1.0 mL of the test solution in step (6), add water to 2.0 mL, and follow the method under step (5) preparation of the standard curve of anthrone-sulfuric acid method, starting from "adding 6 mL of anthrone sulfate solution", according to the law. The absorbance of the test sample was measured, and the polysaccharide content was obtained according to the standard curve of the anthrone-sulfuric acid method.
  9. 根据权利要求8所述的检测方法，其中，所述苯酚-硫酸法和所述蒽酮-硫酸法的步骤(2)中，所述混合单糖溶液为含有葡萄糖、半乳糖、葡萄糖醛酸和甘露糖的水溶液，其中，以重量百分比计，基于溶液中的单糖的总重量，含有60％-85％的葡萄糖、8％-17％的半乳糖、3％-15％的葡萄糖醛酸、3％-7％的甘露糖，或者含有60％-90％的葡萄糖、6％-17％的半乳糖、1％-15％的葡萄糖醛酸、3％-8％的甘露糖。The detection method according to claim 8, wherein, in the step (2) of the phenol-sulfuric acid method and the anthrone-sulfuric acid method, the mixed monosaccharide solution contains glucose, galactose, glucuronic acid and An aqueous solution of mannose, wherein, by weight percentage, based on the total weight of the monosaccharides in the solution, containing 60%-85% glucose, 8%-17% galactose, 3%-15% glucuronic acid, 3%-7% mannose, or 60%-90% glucose, 6%-17% galactose, 1%-15% glucuronic acid, 3%-8% mannose.
  10. 根据权利要求1-7任一项所述的检测方法，其中，所述灵芝多糖含量的检测方法包括以下步骤：The detection method according to any one of claims 1-7, wherein the detection method of the Ganoderma lucidum polysaccharide content comprises the following steps:
    (1)苯酚溶液的配制(1) Preparation of phenol solution
    配制0.05g/mL的苯酚水溶液；Prepare 0.05g/mL aqueous solution of phenol;
    (2)硫酸蒽酮溶液的配制(2) Preparation of anthrone sulfate solution
    配制0.001g/mL的硫酸蒽酮溶液；Prepare 0.001g/mL solution of anthrone sulfate;
    (3)混合单糖对照品溶液的制备(3) Preparation of mixed monosaccharide reference solution
    确定供试品中的单糖组成，并计算各单糖含量，以高效液相色谱确定的单糖比例配制含量检测需要的对照品溶液，总浓度为0.1mg/mL；Determine the monosaccharide composition in the test product, calculate the content of each monosaccharide, and prepare the reference solution required for content detection with the monosaccharide ratio determined by high performance liquid chromatography, with a total concentration of 0.1 mg/mL;
    (4)苯酚-硫酸法标准曲线的制作(4) Preparation of standard curve of phenol-sulfuric acid method
    精密量取步骤(3)中混合单糖对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置具塞试管中，分别加水补至2.0mL，各精密加入步骤1中制得的苯酚溶液1mL，摇匀，迅速精密加入浓硫酸5mL，摇匀，放置10分钟，置40℃水浴中保温15min，取出，冰浴2min，空白不加对照品溶液，测定在490nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制标准曲线：Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step (3), put them in test tubes with stoppers, and add water to make up to 2.0mL respectively. 1 mL of the phenol solution prepared in 1, shake well, quickly and accurately add 5 mL of concentrated sulfuric acid, shake well, place for 10 minutes, place in a 40°C water bath for 15 minutes, take out, ice bath for 2 minutes, blank without reference solution, measure at 490nm Take the absorbance as the ordinate and the concentration of the sugar reference substance as the abscissa to draw the standard curve:
    (5)蒽酮-硫酸法标准曲线的制作(5) Preparation of standard curve of anthrone-sulfuric acid method
    精密量取步骤(3)中混合单糖对照品溶液0.2mL、0.4mL、0.6mL、0.8mL、1.0mL、1.2mL，分别置于具塞试管中，分别加水补至2.0mL， 各精密加入步骤2中制得的硫酸蒽酮溶液6mL，摇匀，放置15min后，置于冰浴中冷却15min，取出，空白不加对照品溶液，测定在625nm处的吸光度，以吸光度为纵坐标，糖对照品浓度为横坐标，绘制标准曲线：Precisely measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the mixed monosaccharide reference solution in step (3), place them in test tubes with stoppers, respectively add water to make up to 2.0mL, and add each precision The anthrone sulfate solution prepared in step 2 was 6mL, shaken up, placed for 15min, cooled in an ice bath for 15min, taken out, blank without the reference solution, and measured the absorbance at 625nm, taking the absorbance as the ordinate, sugar The concentration of the reference substance is the abscissa, and the standard curve is drawn:
    (6)供试品的制备(6) Preparation of the test product
    准确称取灵芝多糖样品10mg，于100mL的容量瓶中，用纯水定容，充分溶解混匀，即得；Accurately weigh 10mg of Ganoderma lucidum polysaccharide sample, put it in a 100mL volumetric flask, dilute to volume with pure water, fully dissolve and mix to get it;
    (7)供试品的检测(7) Detection of the test product
    a、苯酚-硫酸法a. Phenol-sulfuric acid method
    精密吸取步骤(6)中供试品溶液1.0mL，加水至2.0mL，照(4)步骤苯酚-硫酸法标准曲线的制作项下的方法，自“加入苯酚溶液1mL”起，依上述方法测定供试品的吸光度，根据苯酚-硫酸法标准曲线得到多糖含量；Accurately draw 1.0 mL of the test solution in step (6), add water to 2.0 mL, and measure according to the above method according to the method under the preparation of the standard curve of the phenol-sulfuric acid method in step (4), starting from "adding 1 mL of phenol solution" The absorbance of the test product, the polysaccharide content is obtained according to the standard curve of the phenol-sulfuric acid method;
    b、蒽酮-硫酸法b. Anthrone-sulfuric acid method
    精密吸取供试品溶液1.0mL，加水至2.0mL，照(5)步骤蒽酮-硫酸法标准曲线的制作项下的方法，自“加入硫酸蒽酮溶液6mL”起，依法测定供试品的吸光度，根据蒽酮-硫酸法标准曲线得到多糖含量。Accurately draw 1.0 mL of the solution of the test sample, add water to 2.0 mL, and follow the method under the preparation of the standard curve of the anthrone-sulfuric acid method in step (5). Absorbance, polysaccharide content was obtained according to the standard curve of anthrone-sulfuric acid method.
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