CN112485354A - Ganoderma lucidum index detection method and authenticity identification method - Google Patents
Ganoderma lucidum index detection method and authenticity identification method Download PDFInfo
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
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- 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
- G01N30/14—Preparation by elimination of some components
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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Abstract
The disclosure relates to the technical field of fingerprint spectrums, and particularly provides a method for detecting various indexes of lucid ganoderma and a method for identifying authenticity. The method for detecting each index of the lucid ganoderma comprises the following steps: identifying the content of total polysaccharides in the ganoderma lucidum, decomposing the total polysaccharides into oligosaccharides and monosaccharides, wherein the monosaccharides are rhamnose, fucose, xylose, arabinose, mannose, glucose and galactose respectively, and establishing fingerprint spectrums of the oligosaccharides and the monosaccharides by using a HILIC-electrospray mass spectrometry. The method solves the problems that the error of the method for identifying the authenticity of the ganoderma lucidum by using the fingerprint map in the prior art is large, and the one-sidedness problem exists in the method for identifying the polysaccharide content in the ganoderma lucidum medicinal material.
Description
Technical Field
The disclosure relates to the technical field of fingerprint spectrums, and particularly provides a method for detecting various indexes of lucid ganoderma and a method for identifying authenticity.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Ganoderma lucidum (Ganoderma lucidum) is named as Ruicao, which is recorded in Shen nong's herbal Jing, is a traditional famous and precious traditional Chinese medicine for strengthening body resistance, consolidating constitution, nourishing and strengthening in China, and has two functions of food and medicine. Ganoderma polysaccharides are one of main effective components of Ganoderma, and have effects of reducing blood lipid, lowering blood sugar, resisting oxidation, scavenging free radicals, resisting aging, resisting tumor, and improving immunity. And is also the main quality control index of ganoderma lucidum medicinal material in the Chinese pharmacopoeia. The polysaccharide is formed by combining monosaccharides through glycosidic bonds, and the pharmacological action of the polysaccharide is related to the composition and content of the polysaccharide, the structural characteristics such as the connection mode and the connection sequence among the monosaccharides, the physicochemical properties and the like.
In recent years, ganoderma lucidum and extracts thereof are increasingly applied in the fields of Chinese herbal medicines, health products and foods, so that the phenomena of misuse and adulteration of confused products of ganoderma lucidum and extracts thereof occur. The present inventors found that the fingerprint spectrum method in the prior art mainly extracts and identifies the main components in the ganoderma lucidum, and then compares the main components with the standard spectrum in a database, thereby identifying the authenticity of the ganoderma lucidum. However, there are many kinds of ganoderma lucidum, and ganoderma lucidum as one kind of fungi also has many similar strains, and some components have consistency, so deviation may occur in the actual judgment process.
In addition, the inventor finds that the existing content determination method aiming at the ganoderma lucidum polysaccharide cannot comprehensively reflect the quality of the ganoderma lucidum medicinal material and has the one-sidedness. Therefore, the research on the specificity and the overall quality evaluation method of the ganoderma lucidum polysaccharide has important practical significance.
Disclosure of Invention
Aiming at the problems that the method for identifying the authenticity of the lucid ganoderma by using the fingerprint has larger error and the method for identifying the polysaccharide content in the lucid ganoderma medicinal material has one-sidedness in the prior art.
In one or some embodiments of the present disclosure, a method for detecting various indexes of ganoderma lucidum is provided, which includes the following steps: identifying the content of total polysaccharides in the ganoderma lucidum, decomposing the total polysaccharides into oligosaccharides and monosaccharides, wherein the monosaccharides are rhamnose, fucose, xylose, arabinose, mannose, glucose and galactose respectively, and establishing fingerprint spectrums of the oligosaccharides and the monosaccharides by using a HILIC-electrospray mass spectrometry.
In one or some embodiments of the present disclosure, a method for identifying authenticity of ganoderma is provided, where the total polysaccharide content and the fingerprint obtained by the above-mentioned methods for detecting each index of ganoderma are entered into a database, an object to be detected is processed according to the above-mentioned methods for detecting each index of ganoderma, and the total polysaccharide content and the fingerprint of the object to be detected are compared with the values in the database.
One or some of the above technical solutions have the following advantages or beneficial effects:
1) the present disclosure develops a method for analyzing and characterizing ganoderan based on hydrophilic chromatography-electrospray detector/electrospray mass spectrometry (HILIC-CAD-ESI-MS), and combines monosaccharide composition, characteristic fingerprint spectrum and statistical analysis for Ganoderma lucidum and confounders (such as: ganoderma applanatum) improves the accuracy of the authenticity identification and quality evaluation of ganoderma lucidum polysaccharide.
2) The analysis and comparison of the content of the polysaccharide in different types of ganoderma lucidum shows that: the polysaccharide content of Ganoderma applanatum is higher than that of Ganoderma lucidum. The ganoderma lucidum and ganoderma applanatum polysaccharide are respectively composed of fucose, rhamnose, arabinose, xylose, mannose, glucose and galactose, but the monosaccharide content and proportion of the polysaccharides are different. The characteristic fingerprint spectrum of partial acid hydrolysate of ganoderma lucidum polysaccharide is established, and the ganoderma lucidum and the confuse (ganoderma applanatum) thereof can be correctly distinguished by combining similarity analysis and cluster analysis.
3) The decomposed oligosaccharide and monosaccharide are respectively researched to establish respective fingerprint spectrums, and the configuration difference of the oligosaccharide of the ganoderan is large, so that the residual polysaccharide after the monosaccharide is decomposed from different positions is different, the polysaccharide component is measured, namely the polysaccharide component is reflected integrally, and the component is measured, so that the one-sidedness of the content detection of the ganoderan is avoided in a manner of combining macroscopicity and microcosmicity, and the accuracy of true and false identification of the ganoderan is further improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the disclosure and, together with the description, serve to explain the disclosure and not to limit the disclosure.
FIG. 1 is a comparison of monosaccharide contents of different types of Ganoderma lucidum in the prior art.
FIG. 2 is an HPLC-CAD chromatogram of a control (A) and a Ganoderma lucidum sample (B) in example 2, wherein 3. rhamnose; 4. fucose; 5,10. arabinose; 6,7 xylose; 13. mannose; 14,16. glucose; 15,17, galactose.
FIG. 3 is the HPLC fingerprint of different batches of ganoderan in example 2.
FIG. 4 is a chromatogram of crude polysaccharides of Ganoderma lucidum, Astragalus membranaceus, Polygonum multiflorum, Morinda officinalis, Lycium barbarum, and Lentinus edodes of example 2.
FIG. 5 is a graph of cluster analysis of Ganoderma lucidum samples in example 2.
Detailed Description
The technical solutions in the embodiments of the present disclosure will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present disclosure, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the disclosure without making any creative effort, shall fall within the protection scope of the disclosure.
Aiming at the problems that the method for identifying the authenticity of the lucid ganoderma by using the fingerprint has larger error and the method for identifying the polysaccharide content in the lucid ganoderma medicinal material has one-sidedness in the prior art.
In one or some embodiments of the present disclosure, a method for detecting various indexes of ganoderma lucidum is provided, which includes the following steps: identifying the content of total polysaccharides in the ganoderma lucidum, decomposing the total polysaccharides into oligosaccharides and monosaccharides, wherein the monosaccharides are rhamnose, fucose, xylose, arabinose, mannose, glucose and galactose respectively, and establishing fingerprint spectrums of the oligosaccharides and the monosaccharides by using a HILIC-electrospray mass spectrometry.
By oligosaccharide is meant a non-broken down polysaccharide of the total polysaccharide except for several monosaccharides.
According to the method, decomposed oligosaccharides and monosaccharides are respectively researched to establish respective fingerprint spectrums, and as the configuration of the oligosaccharides of the ganoderan is greatly different, the remaining polysaccharides after the monosaccharides are decomposed from different positions are different, so that the method adds the measurement of polysaccharide components, and further improves the accuracy of the authenticity identification of the ganoderan.
Preferably, the total polysaccharide content is identified by the following steps: extracting crude polysaccharide in the ganoderma, preparing a standard solution, drawing a standard curve, and measuring the content of the polysaccharide;
preferably, the extraction of the crude polysaccharide comprises the steps of: putting a ganoderma lucidum sample into vacuum drying and drying, slicing, crushing, sieving, accurately weighing sample powder, adding a proper amount of distilled water according to a certain material-liquid ratio, carrying out ultrasonic extraction for a certain time at a certain temperature, rapidly cooling the extracted sample to room temperature, and centrifuging the extracting solution. Filtering the supernatant, adding ethanol into the supernatant, precipitating with ethanol for a period of time, centrifuging, drying the precipitate in water bath, re-dissolving with hot water, centrifuging again, and diluting to desired volume;
further preferably, vacuum drying is adopted, the vacuum drying temperature is 55-65 ℃, preferably 60 ℃,
further preferably, the sieving is 38-42 mesh sieve, preferably 40 mesh sieve,
further preferably, the feed-to-liquid ratio of the sample to the distilled water is 1-2:15-20, unit g: ml, preferably 1:15, unit g: ml,
further preferably, the ultrasonic temperature is 90 ℃,
further preferably, the ultrasonic power is 300W,
further preferably, the ultrasonic extraction time is 30-40min, preferably 30min,
further preferably, 95% by volume ethanol is added to the supernatant, so that the final concentration of ethanol in the solution is 80% by volume,
further preferably, the alcohol precipitation temperature is 4 ℃,
further preferably, the alcohol precipitation time is 12h,
further preferably, the centrifugation time after alcohol precipitation is 15min,
further preferably, the water bath drying temperature is 80 ℃,
further preferably, the re-centrifugation parameter is the rotation speed of 5000r/min and the time of 15 min.
Preferably, the standard solution is a glucose solution and a phenol solution, preferably, the concentration of the glucose solution is 0.1mg/mL, and preferably, the mass fraction of the phenol solution is 5%;
or, the step of drawing the standard curve comprises the following steps: preparing a series of glucose standard solutions with different concentrations by using distilled water, mixing and shaking the standard solution with phenol respectively, adding concentrated sulfuric acid solution, shaking and placing, heating in a boiling water bath, rapidly cooling to room temperature, measuring with a 1cm cuvette at a wavelength of 487nm, drawing a standard curve by using the concentration value of glucose in the unit of mu g/mL as an abscissa and the absorbance value as an ordinate,
preferably, the water bath time is 8-15min, preferably 10 min.
Preferably, the polysaccharide content is determined by a phenol-sulfuric acid method, which comprises the following steps: diluting the crude polysaccharide solution, mixing with phenol, adding concentrated sulfuric acid solution, shaking, standing, heating in boiling water bath for a period of time, rapidly cooling to room temperature, measuring at 487nm with 1cm cuvette, measuring absorbance value on a standard curve to obtain sample concentration, and calculating total polysaccharide content in each sample according to regression equation;
preferably, the crude polysaccharide solution is diluted to 1/10;
preferably, the volume ratio of the polysaccharide to the phenol to the concentrated sulfuric acid is 1-2:1-2: 5-10; preferably 1:1: 5;
preferably, the concentrated sulfuric acid solution is shaken for 6-10min and kept standing for 5-10min, and further preferably shaken for 6min and kept standing for 5 min.
Preferably, the heating time of the water bath is 10-15min, preferably 10 min.
The percentage content w of the total ganoderma lucidum polysaccharide is (c multiplied by d multiplied by v/m) multiplied by 100 percent, wherein c is the glucose concentration in the sample liquid, d is the dilution multiple, v is the total volume of the extracting solution, and m is the weight of the sample.
Preferably, the decomposition of the total polysaccharide into oligosaccharides and several monosaccharides comprises the following steps: the crude polysaccharide solution was treated with trifluoroacetic acid solution, placed in a constant temperature ultrasonic cleaner, and the hydrolysate washed with methanol and blown dry with nitrogen.
Preferably, the concentration of the trifluoroacetic acid is 4 mol/L;
preferably, the incubation is carried out in a constant temperature ultrasonic cleaner at 65-75 ℃ for 1-2h, and further preferably at 70 ℃ for 1.0 h.
Preferably, the chromatographic conditions for HILIC-electrospray mass spectrometry are as follows: a chromatographic column: xbridge BEH Amide column (2.1X 150mm, 2.5 μm), mobile phase A is acetonitrile solution, and mobile phase B is water. Elution gradient: 0-13 min, 91% -90% A; 13-20 min, 90% A-80% A; 20-40 min, 80% A; flow rate: 0.3 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: at 25 ℃. CAD detection parameters: the gas source is N2Pressure 61.2Psi, Filter 5.0sec, atomizer temperature 35 ℃.
Preferably, the method further comprises the step of identifying the total polysaccharide decomposition product by chromatography,
preferably, the chromatographic conditions are column: ACQUITY
BEH Amide column (3.0X 100mm, 1.7 μm), mobile phase A was acetonitrile solution, and mobile phase B was water (0.8% formic acid). Elution gradient: 0-6 min, 85% A; 6-8 min, 85% A-72% A; 8-12 min, 72% A. Flow rate: 0.4 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: at 25 ℃. CAD detection parameters: the gas source was N2, pressure 61.2Psi, Filter 5.0sec, atomizer temperature 35 ℃.
Preferably, the method also comprises the step of investigating various indexes of the ganoderma lucidum, namely performing precision, stability and repeatability experiments.
In one or some embodiments of the present disclosure, a method for identifying authenticity of ganoderma is provided, where the total polysaccharide content and the fingerprint obtained by the above-mentioned methods for detecting each index of ganoderma are entered into a database, an object to be detected is processed according to the above-mentioned methods for detecting each index of ganoderma, and the total polysaccharide content and the fingerprint of the object to be detected are compared with the values in the database.
Preferably, the database records data of a plurality of different varieties of lucid ganoderma.
Example 1
1.1 materials
The dry fruiting bodies of Ganoderma lucidum were purchased from Yaowanglou (Table 1) of Jinan city.
TABLE 1 Ganoderma lucidum test samples
1.2 reagents
95% ethanol (analytical purity, Shandong Yuwang Industrial Co., Ltd.), phenol (analytical purity, Tianjin City Mao chemical reagent factory), acetonitrile (chromatographic purity, Oceanpak reagent Co., Sweden), hydrofluoric acid (chromatographic purity, Shanghai Arlatin Biotechnology Co., Ltd.), trifluoroacetic acid, concentrated hydrochloric acid, ammonium formate (analytical purity, Tianjin Kogyou chemical reagent Co., Ltd.), D- (+) -glucose, L-rhamnose, D-galactose, D-mannose, D-arabinose, D- (+) -xylose, L- (+) -fucose (purity is more than 98%, Shanghai Yuan leaf Biotechnology Co., Ltd.).
1.3 instruments
One in ten thousand electronic analytical balance (SARTOURIUS BSA, USA), SBL-10DT type constant temperature ultrasonic cleaner (Ningbo Xinzhi Biotech Co., Ltd.), TG16-WS type desk type high speed centrifuge (Hunan Xiang instruments laboratory development Co., Ltd.), GENESYS 10S ultraviolet visible spectrophotometer (Germany ThermoFisher Co., Ltd.), HH-S4Digital display constant temperature water bath (jin Tan city medical instruments factory), Sammerfei Ultimate high performance liquid chromatograph (Germany ThermoFisher Co.).
1.4 measurement of polysaccharide content
1.4.1 extraction of crude polysaccharide
Placing the ganoderma lucidum sample in a vacuum drying oven, drying at 60 ℃, slicing, crushing, and sieving with a 40-mesh sieve. Accurately weighing 2g of sample powder, placing the sample powder in a conical flask, adding a proper amount of distilled water according to a certain material-liquid ratio (1:15, g/mL), and carrying out ultrasonic extraction (with the power of 300W) at a certain temperature (90 ℃) for a certain time (30 min). The extracted sample was rapidly cooled to room temperature, and then the extract was centrifuged. Filtering the supernatant, adding 95% ethanol to obtain a final concentration of 80% (v/v) ethanol, precipitating at 4 deg.C for 12 hr, and centrifuging (15 min). Drying the precipitate in 80 deg.C water bath, re-dissolving with hot water, centrifuging again (5000r/min, 15min), and diluting to 5 mL.
1.4.2 preparation of solutions
Glucose standard solution: accurately weighing 1.0mg of the anhydrous glucose standard substance in a 10mL volumetric flask, adding water to dilute the anhydrous glucose standard substance to a scale mark, and shaking up to prepare a 0.1mg/mL standard substance solution.
Phenol solution: accurately weighing 5.0g of phenol in a 50mL volumetric flask, adding water to scale marks to prepare a phenol solution with the mass fraction of 5%, shaking uniformly, and transferring the solution to a brown reagent bottle for later use.
1.4.3 drawing of Standard Curve
Accurately measuring glucose standard solutions (0.1mg/mL)0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 and 4.0mL in a 10mL volumetric flask, adding water to the scale mark, and shaking up to prepare a series of glucose standard solutions. Respectively putting 1.0mL series of standard solutions into a dry test tube, adding 1.0mL of 5% phenol solution into each standard solution, shaking uniformly, slowly adding 5mL of concentrated sulfuric acid solution along the wall of the test tube, shaking for 6min, paying attention to uncover, deflating, placing for 5min, placing in a boiling water bath for 10min, rapidly cooling to room temperature, measuring by using a 1cm cuvette at a wavelength of 487nm, and drawing a standard curve by taking the concentration value (unit: mu g/mL) of glucose as an abscissa (x) and the absorbance value as an ordinate (y).
1.4.4 determination of polysaccharide content in samples
The content of the ganoderma lucidum polysaccharide of different varieties is measured by adopting an improved phenol-sulfuric acid method. Accurately measuring 0.3mL of each crude polysaccharide solution in a 10mL volumetric flask, adding water to a constant volume to a scale mark, and shaking up. Accurately sucking 1.0mL of diluted crude polysaccharide solution into a test tube, adding 1.0mL of 5% phenol solution, shaking up, adding 5.0mL of concentrated sulfuric acid solution along the wall, shaking up for 6min, standing for 5min, heating in a boiling water bath for 10min, rapidly cooling to room temperature, measuring at 487nm with a 1cm cuvette, obtaining the concentration of the sample on a standard curve according to the measured absorbance value, and calculating the content of total polysaccharide in each sample according to a regression equation. The percentage content w of the total ganoderma lucidum polysaccharide is (c × d × v/m) × 100% (c is the glucose concentration in the sample liquid, d is the dilution factor, v is the total volume of the extracting solution, and m is the weight of the sample).
1.5 monosaccharide composition of ganoderan
1.5.1 hydrolysis of polysaccharides
The crude polysaccharide solution was treated with a 4mol/L solution of trifluoroacetic acid (TFA). Placing in a constant temperature ultrasonic cleaner, and incubating at 70 deg.C for 1.0 h. The hydrolyzate was washed with methanol and blown dry with nitrogen.
1.5.2 preparation of solutions
Preparing a test solution: and (3) fixing the volume of the dried sample to 1.0mL by using water, and filtering the sample by using a 0.22-micrometer microporous membrane to prepare a test solution.
Control solution: accurately weighing 1.0mg of rhamnose, fucose, xylose, arabinose, mannose, glucose and galactose reference substances respectively, placing in a 1mL volumetric flask, adding water to scale mark, and preparing into reference substance solution with mass concentration of 1.0 mg/mL.
1.5.3 chromatographic conditions
A chromatographic column: ACQUITY
BEH Amide column (3.0X 100mm, 1.7 μm), mobile phase A was acetonitrile solution, and mobile phase B was water (0.8% formic acid). Elution gradient: 0-6 min, 85% A; 6-8 min, 85% A-72% A; 8-12 min, 72% A. Flow rate: 0.4 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: at 25 ℃. CAD detection parameters: the gas source is N2Pressure 61.2Psi, Filter 5.0sec, atomizer temperature 35 ℃.
1.6 characteristic fingerprint of ganoderan
1.6.1 hydrolysis of polysaccharides
To the crude polysaccharide solution was added a 4moL/L solution of trifluoroacetic acid (TFA). Placing in a water bath, hydrolyzing at 80 deg.C for 5h, and cooling to room temperature. The hydrolyzate was washed with methanol and blown dry with nitrogen.
1.6.2 preparation of solutions
Preparing a test solution: and (3) fixing the volume of the dried sample to 1.0mL by using water, and filtering the sample by using a 0.22-micrometer microporous membrane to prepare a test solution.
Mixing the reference solution: respectively accurately weighing 1.0mg of rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose reference substances, dissolving the reference substances into 1mL of water, and preparing a reference substance solution with the mass concentration of 1.0 mg/mL. Precisely sucking 100 mu L of each reference substance solution into a 1mL volumetric flask, and diluting the solution to scale marks by using water to obtain a mixed reference substance solution.
1.6.3 chromatographic conditions
A chromatographic column: xbridgeTMBEH Amide column (2.1X 150mm, 2.5 μm), mobile phase A was acetonitrile solution, and mobile phase B was water. Elution gradient: 0-13 min, 91% -90% A; 13-20 min, 90% A-80% A; 20-40 min, 80% A; flow rate: 0.3 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: at 25 ℃. CAD detection parameters: the gas source is N2Pressure 61.2Psi, Filter 5.0sec, atomizer temperature 35 ℃.
Example 2
2.1 comparative analysis of different Ganoderma polysaccharides
TABLE 2 comparison of the content of ganoderan of different species (mean,%)
The content of polysaccharide in different Ganoderma lucidum (Ganoderma lucidum, Ganoderma applanatum) is determined by the analysis method under item "1.4.4" by calculation according to the method described in 1.4.3. As shown in Table 2, the polysaccharide content of Pinggu ganoderma lucidum is significantly higher than that of Chi ganoderma lucidum. Wherein, the average value of the polysaccharide content of the ganoderma applanatum is 2.68 percent; the average content of Ganoderma lucidum is 1.67%. The difference between the contents of ganoderan in different varieties may be the change of secondary metabolites among different varieties of ganoderma due to the influence of natural hybridization, natural selection, artificial cultivation and other factors in the long-term evolution process. In addition, the interaction between genetic variation and habitat variation causes the phenomenon of good and bad quality of the ganoderma lucidum. As can be seen from the above, the content of the ganoderma lucidum polysaccharide of different varieties shows a certain difference, which indicates that the determination of the polysaccharide content has important significance for the evaluation of the quality of ganoderma lucidum medicinal materials.
2.2 monosaccharide composition analysis of ganoderan
The extracted ganoderma lucidum crude polysaccharide sample is hydrolyzed by the method under the item 1.5.1, then the sample solution is prepared by the method under the item 1.5.2, and the content of 7 monosaccharides in the sample is respectively determined according to the chromatographic conditions under the item 1.5.3, and the result is shown in the table 3.
TABLE 3 measurement of monosaccharide content after hydrolysis of crude polysaccharide
Polysaccharides are natural biological macromolecules, and are formed by polymerizing a large number of monosaccharide groups and glycosidic bonds, and monosaccharide compositions are complex and have large differences. The research on the composition and the content of monosaccharide in the polysaccharide can provide information reference for the variety identification of the ganoderma lucidum. As shown in Table 3, the hydrolysate of polysaccharide of Ganoderma lucidum and Ganoderma Applanatum consists of 7 monosaccharides, including rhamnose, fucose, xylose, arabinose, mannose, glucose and galactose, but the contents and distribution of the monosaccharides are similar and different (FIG. 1). First, the general contents of monosaccharides in Ganoderma lucidum and Ganoderma applanatum are approximately the same. Wherein, xylose content is highest, arabinose is next, and monosaccharide with lowest content are slightly different (Ganoderma lucidum has lowest mannose content, Ganoderma applanatum has lowest galactose content). Secondly, the composition ratios of monosaccharides of various ganoderma lucidum have certain differences, and the content of arabinose and mannose in the ganoderma lucidum is slightly higher than that of ganoderma applanatum; the ganoderma applanatum has obviously low galactose content and slightly high rhamnose and xylose content. The monosaccharide composition difference of different ganoderma lucidum polysaccharides indicates that the polysaccharide structure has complexity, thereby leading to the diversity of the functions.
2.3 Ganoderma lucidum polysaccharide characteristic fingerprint and confuse distinguishing research
2.3.1 chromatographic analysis of ganoderan
Preparing Ganoderma polysaccharide sample hydrolyzed under different conditions into test solution under item "1.6.2", and analyzing by sample injection under chromatographic condition under item "1.6.3", wherein HPLC chromatogram of the obtained Ganoderma polysaccharide hydrolysate is shown in FIG. 2.
2.3.2 methodology investigation
2.3.2.3 precision test
Taking the same sample solution, continuously injecting sample for 6 times under the chromatographic condition of '1.6.3', and respectively measuring the retention time RSD of each monosaccharide peak to be 0.85%, 0.47%, 0.43%, 0.39%, 0.85%, 0.39%, 0.79%, 0.69%, 0.30%, 0.26% and 0.34%, wherein the retention time RSD is less than 1%; the peak areas RSD are respectively 2.94%, 3.09%, 1.55%, 0.88%, 1.63%, 2.66%, 4.14%, 3.94%, 4.41%, 1.46% and 1.29%, and are all less than 5%. The results show that the precision of the instrument is good.
2.3.2.4 stability test
Sampling the same sample solution under chromatographic conditions of '1.6.3' for 0, 4, 8, 12, 16 and 24h respectively, and analyzing to obtain monosaccharide peaks with retention times RSD of 0.74%, 0.24%, 0.45%, 0.61%, 0.88%, 0.61%, 0.55%, 0.62%, 0.37%, 0.30% and 0.35%, all of which are less than 1%; the peak areas RSD are respectively 3.81%, 3.12%, 2.93%, 2.89%, 2.77%, 3.25%, 2.91%, 2.50%, 1.74%, 4.38% and 1.41%, and are all less than 5%. The test solution is stable within 24 hours.
2.3.2.5 repeatability test
Taking the same batch of crude polysaccharide samples, preparing 6 parts of test solution according to the method under item 1.6.2, measuring according to the chromatographic condition under item 1.6.3, and respectively measuring the retention time RSD of each monosaccharide peak to be 0.89%, 0.29%, 0.55%, 0.33%, 0.38%, 0.78%, 0.52%, 0.72%, 0.21%, 0.20% and 0.40%, wherein the retention time RSD is less than 1%; the peak areas RSD are respectively 4.00%, 3.91%, 3.08%, 4.32%, 2.80%, 3.86%, 3.53%, 3.56%, 3.25%, 4.62% and 0.81%, and are all less than 5%. The results show that the method has good repeatability.
2.3.4 construction of characteristic fingerprint
Processing multiple batches of crude Ganoderma polysaccharide samples of different species into test solution according to the method under item "1.6.2", respectively determining according to the chromatographic conditions under item "1.6.3", and selecting 30 chromatogram maps of common characteristic chromatogram peaks as fingerprint of partial acidolysis of Ganoderma. The HPLC finger print of different batches of ganoderan is shown in figure 3.
2.3.7 fingerprint specificity verification
Processing crude polysaccharide samples of different samples (Ganoderma, radix astragali, Polygoni Multiflori radix, radix Morindae officinalis, fructus Lycii and Lentinus Edodes) into test solution under item "1.6.2", and measuring respectively under chromatographic conditions under item "1.6.3", with chromatogram shown in FIG. 4. As can be seen from the figure, the characteristic fingerprints of the ganoderma lucidum and other samples have certain difference, particularly the distribution of the chromatographic peaks in the picture frame region has obvious difference, which shows that the method has certain specificity in analyzing and characterizing the ganoderma lucidum polysaccharide.
2.3.5 evaluation of similarity
A traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004A version) is used for analyzing and evaluating the fingerprint of the ganoderan, and the similarity of each sample is shown in a table 4. As can be seen from Table 4, the similarity of the characteristic fingerprint of ganoderma lucidum polysaccharide is 0.9249-0.9547; the similarity of the 10 batches of ganoderma applanatum characteristic fingerprints is between 0.8449 and 0.8735, which indicates that polysaccharide components contained in different ganoderma lucidum samples have certain difference. Therefore, the intraspecies similarity of ganoderma lucidum and ganoderma applanatum is relatively close, and the interspecies difference is relatively large. These differences can provide basis for the identification of different varieties of ganoderan and the quality control of products thereof.
TABLE 4 Ganoderma lucidum polysaccharide fingerprint similarity evaluation
2.3.6 Cluster analysis
And analyzing the ganoderma lucidum and ganoderma applanatum by using the area of each characteristic peak as a variable and adopting a system clustering analysis technology. As can be seen from FIG. 5, the Ganoderma lucidum samples have slightly greater intraspecific variation but can better be grouped into one class; the Ganoderma applanatum samples have small intraspecific differences and are grouped into one type. The method is expected to realize the discrimination of adulterated ganoderma lucidum by the technical set of the ganoderma lucidum polysaccharide characteristic fingerprint spectrum and cluster analysis.
The disclosure of the present invention is not limited to the specific embodiments, but rather to the specific embodiments, the disclosure is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A method for detecting each index of lucid ganoderma is characterized by comprising the following steps: identifying the content of total polysaccharides in the ganoderma lucidum, decomposing the total polysaccharides into oligosaccharides and monosaccharides, wherein the monosaccharides are rhamnose, fucose, xylose, arabinose, mannose, glucose and galactose respectively, and establishing fingerprint spectrums of the oligosaccharides and the monosaccharides by using a HILIC-electrospray mass spectrometry.
2. The method for detecting each index of ganoderma lucidum as claimed in claim 1, wherein the step of identifying the total polysaccharide content comprises the steps of: extracting crude polysaccharide in the ganoderma, preparing a standard solution, drawing a standard curve, and measuring the content of the polysaccharide;
preferably, the extraction of the crude polysaccharide comprises the steps of: putting a ganoderma lucidum sample into vacuum drying and drying, slicing, crushing, sieving, accurately weighing sample powder, adding a proper amount of distilled water according to a certain material-liquid ratio, carrying out ultrasonic extraction for a certain time at a certain temperature, rapidly cooling the extracted sample to room temperature, and centrifuging the extracting solution. Filtering the supernatant, adding ethanol into the supernatant, precipitating with ethanol for a period of time, centrifuging, drying the precipitate in water bath, re-dissolving with hot water, centrifuging again, and diluting to desired volume;
further preferably, vacuum drying is adopted, the vacuum drying temperature is 55-65 ℃, preferably 60 ℃,
further preferably, the sieving is 38-42 mesh sieve, preferably 40 mesh sieve,
further preferably, the feed-to-liquid ratio of the sample to the distilled water is 1-2:15-20, unit g: ml, preferably 1:15, unit g: ml,
further preferably, the ultrasonic temperature is 90 ℃,
further preferably, the ultrasonic power is 300W,
further preferably, the ultrasonic extraction time is 30-40min, preferably 30min,
further preferably, 95% by volume ethanol is added to the supernatant, so that the final concentration of ethanol in the solution is 80% by volume,
further preferably, the alcohol precipitation temperature is 4 ℃,
further preferably, the alcohol precipitation time is 12h,
further preferably, the centrifugation time after alcohol precipitation is 15min,
further preferably, the water bath drying temperature is 80 ℃,
further preferably, the re-centrifugation parameter is the rotation speed of 5000r/min and the time of 15 min.
3. The method for detecting each index of ganoderma lucidum as claimed in claim 2, wherein the standard solution is a glucose solution and a phenol solution, preferably, the concentration of the glucose solution is 0.1mg/mL, and preferably, the mass fraction of the phenol solution is 5%;
or, the step of drawing the standard curve comprises the following steps: preparing a series of glucose standard solutions with different concentrations by using distilled water, mixing and shaking the standard solution with phenol respectively, adding concentrated sulfuric acid solution, shaking and placing, heating in a boiling water bath, rapidly cooling to room temperature, measuring with a 1cm cuvette at a wavelength of 487nm, drawing a standard curve by using the concentration value of glucose in the unit of mu g/mL as an abscissa and the absorbance value as an ordinate,
preferably, the water bath time is 8-15min, preferably 10 min.
4. The method for detecting each index of ganoderma lucidum as claimed in claim 1, wherein the step of measuring the content of polysaccharide is performed by a phenol-sulfuric acid method, comprising the steps of: diluting the crude polysaccharide solution, mixing with phenol, adding concentrated sulfuric acid solution, shaking, standing, heating in boiling water bath for a period of time, rapidly cooling to room temperature, measuring at 487nm with 1cm cuvette, measuring absorbance value on a standard curve to obtain sample concentration, and calculating total polysaccharide content in each sample according to regression equation;
preferably, the crude polysaccharide solution is diluted to 1/10;
preferably, the volume ratio of the polysaccharide to the phenol to the concentrated sulfuric acid is 1-2:1-2: 5-10; preferably 1:1: 5;
preferably, the concentrated sulfuric acid solution is shaken for 6-10min and kept standing for 5-10min, and further preferably shaken for 6min and kept standing for 5 min.
Preferably, the heating time of the water bath is 10-15min, preferably 10 min.
The percentage content w of the total ganoderma lucidum polysaccharide is (c multiplied by d multiplied by v/m) multiplied by 100 percent, wherein c is the glucose concentration in the sample liquid, d is the dilution multiple, v is the total volume of the extracting solution, and m is the weight of the sample.
5. The method for detecting each index of ganoderma lucidum according to claim 1, wherein the step of decomposing the total polysaccharides into oligosaccharides and monosaccharides comprises the steps of: the crude polysaccharide solution was treated with trifluoroacetic acid solution, placed in a constant temperature ultrasonic cleaner, and the hydrolysate washed with methanol and blown dry with nitrogen.
Preferably, the concentration of the trifluoroacetic acid is 4 mol/L;
preferably, the incubation is carried out in a constant temperature ultrasonic cleaner at 65-75 ℃ for 1-2h, and further preferably at 70 ℃ for 1.0 h.
6. The method for detecting each index of ganoderma lucidum as claimed in claim 1, wherein the chromatographic conditions of HILIC-electrospray mass spectrometry are as follows: a chromatographic column: xbridge BEH Amide column (2.1X 150mm, 2.5 μm), mobile phase A is acetonitrile solution, and mobile phase B is water. Elution gradient: 0-13 min, 91% -90% A; 13-20 min, 90% A-80% A; 20-40 min, 80% A; flow rate: 0.3 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: at 25 ℃. CAD detection parameters: the gas source is N2Pressure 61.2Psi, Filter 5.0sec, atomizer temperature 35 ℃.
7. The method of detecting each index of Ganoderma lucidum according to claim 1, further comprising the step of identifying the total polysaccharide decomposition product by chromatography,
preferably, the chromatographic conditions are column: ACQUITY
BEH Amide column (3.0X 100mm, 1.7 μm), mobile phase A was acetonitrile solution, and mobile phase B was water (0.8% formic acid). Elution gradient: 0-6 min, 85% A; 6-8 min, 85% A-72% A; 8-12 min, 72% A. Flow rate: 0.4 mL/min; sample introduction amount: 10 mu L of the solution; column temperature: at 25 ℃. CAD detection parameters: the gas source was N2, pressure 61.2Psi, Filter 5.0sec, atomizer temperature 35 ℃.
8. The method for detecting each index of Ganoderma lucidum according to claim 1, further comprising the step of examining each index of Ganoderma lucidum by performing precision, stability and repeatability tests.
9. A method for identifying the authenticity of lucid ganoderma is characterized in that the total polysaccharide content and the fingerprint obtained by each index detection method of lucid ganoderma according to any one of claims 1 to 8 are input into a database, an object to be detected is processed according to each index detection method of lucid ganoderma according to any one of claims 1 to 8, and the total polysaccharide content and the fingerprint of the object to be detected are compared with numerical values in the database.
10. The method for authenticating authenticity of ganoderma lucidum according to claim 9, wherein a plurality of different varieties of ganoderma lucidum data are entered in the database.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113092653A (en) * | 2021-03-31 | 2021-07-09 | 山东省分析测试中心 | Method for measuring monosaccharide composition in American ginseng polysaccharide and method for identifying different parts |
| JP7487987B1 (en) | 2023-12-21 | 2024-05-21 | 伊那食品工業株式会社 | Analytical Method for Agro-oligosaccharides |
| JP7496657B1 (en) | 2023-12-21 | 2024-06-07 | 伊那食品工業株式会社 | Analytical Method for Agro-oligosaccharides |
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