CN115406985A - Method for characterizing polysaccharide structure information and method for identifying authenticity or quality of traditional Chinese medicine - Google Patents

Abstract

The invention relates to the technical field of traditional Chinese medicine quality control, and discloses a method for characterizing polysaccharide structure information and a method for identifying authenticity or quality of a traditional Chinese medicine. The characterization method comprises the following steps: respectively carrying out hydrolysis treatment and derivatization treatment on the polysaccharide sample to obtain a derivatization product, and analyzing the derivatization product by using GC-MS to obtain monosaccharide composition and relative proportion in the derivatization product; carrying out methylation treatment, hydrolysis treatment, reduction treatment and acetylation treatment on a plurality of polysaccharide samples respectively in sequence to obtain methylated sugar alcohol acetyl ester products, and analyzing the methylated sugar alcohol acetyl ester products by using GC-MS (gas chromatography-mass spectrometry) to obtain types of glycosidic bonds and relative proportion of each glycosidic bond in polysaccharides from different sources; and (3) carrying out statistical analysis on monosaccharide composition and relative proportion and/or glycosidic bond type and relative proportion, and characterizing and distinguishing polysaccharide samples. The characterization method can be used for characterizing the structural information of the polysaccharide in a diversified manner, and realizing the rapid and accurate identification of the authenticity and quality of the traditional Chinese medicine.

Description

Method for characterizing polysaccharide structure information and method for identifying authenticity or quality of traditional Chinese medicine

Technical Field

The invention relates to the technical field of traditional Chinese medicine quality control, in particular to a method for representing polysaccharide structure information and a method for identifying authenticity or quality of a traditional Chinese medicine.

Background

Polysaccharides are natural macromolecular substances formed by condensing more than 10 monosaccharide molecules, are formed by connecting aldose and/or ketose and the like through glycosidic bonds, and are one of four basic substances forming life. Polysaccharides are widely found in animal, plant, microbial cells, but due to the complexity of polysaccharides, and the past underestimation of polysaccharide contribution to life processes, the research of polysaccharides has lagged behind nucleic acids and proteins.

At present, chinese medicines with good bioactive polysaccharides are reported to be cordyceps sinensis, dendrobium officinale, lucid ganoderma, medlar, codonopsis pilosula, astragalus and the like. Because of the complexity of the structure of the polysaccharide of the traditional Chinese medicine, the characterization of the polysaccharide structure is the difficulty of polysaccharide analysis, the quality control method of the polysaccharide still takes a color development method as the main part, and the structural information of the polysaccharide cannot be reflected. However, the activity of polysaccharide is closely related to the structural characteristics of polysaccharide, so that the establishment of a quality control method based on the structural characteristics of polysaccharide is particularly important for ensuring the safety and effectiveness of traditional Chinese medicine.

The traditional pretreatment of polysaccharide is complicated and tedious, the hydrolysis and methylation process of monosaccharide takes a long time, the acid hydrolysis process usually takes 6-8h in monosaccharide composition analysis, and the methylation and subsequent dialysis, hydrolysis and other processes also take several days in glycosidic bond analysis of polysaccharide. Therefore, a rapid, accurate and efficient polysaccharide structure information analysis program is developed and searched to collect polysaccharide structure information, which is of great significance to the identification of traditional Chinese medicines, especially traditional Chinese medicines with definite saccharide component and drug effects, and is a development trend of quality control of traditional Chinese medicines.

Disclosure of Invention

The invention aims to solve the problems of complex pretreatment and long time consumption of polysaccharide in the prior art, and provides a method for representing polysaccharide structure information and a method for identifying the authenticity or quality of a traditional Chinese medicine.

In order to achieve the above object, the first aspect of the present invention provides a method for characterizing polysaccharide structural information, comprising the steps of:

(1) Respectively carrying out hydrolysis treatment I on a plurality of polysaccharide samples to obtain a hydrolysate I, carrying out derivatization treatment on the hydrolysate I to obtain a derivatization product, and analyzing the derivatization product by using GC-MS (gas chromatography-mass spectrometry) to obtain monosaccharide composition and relative proportion of each monosaccharide in the derivatization product corresponding to each polysaccharide sample;

(2) Carrying out methylation treatment, hydrolysis treatment II, reduction treatment and acetylation treatment on a plurality of polysaccharide samples respectively to obtain methylated sugar alcohol acetyl ester products, and analyzing the methylated sugar alcohol acetyl ester products by using GC-MS (gas chromatography-mass spectrometry) to obtain the types of glycosidic bonds in the methylated sugar alcohol acetyl ester products corresponding to each polysaccharide sample and the relative proportion of each glycosidic bond;

(3) And (3) carrying out statistical analysis on the monosaccharide composition obtained in the step (1) and the relative proportion of each monosaccharide and/or the glycosidic bond type obtained in the step (2) and the relative proportion of each glycosidic bond, and characterizing and distinguishing a plurality of polysaccharide samples.

Preferably, the hydrolysis treatment I in the step (1) comprises: and mixing the polysaccharide sample with an acid liquor I under the action of microwave assistance I to perform a hydrolysis reaction I.

Preferably, the power of the microwave-assisted reaction I is 300-600W, and the conditions of the hydrolysis reaction I comprise: the temperature is 75-95 deg.C, and the time is 2-5min.

Preferably, the acid solution I is selected from one of a hydrochloric acid solution, a sulfuric acid solution, a nitric acid solution and a trifluoroacetic acid solution, and more preferably, a trifluoroacetic acid solution.

Preferably, the molar amount of acid in the acid liquor I is 0.1-1mmol relative to 1mg of the polysaccharide sample.

Preferably, the process of the derivatization treatment in step (1) comprises: and (3) respectively carrying out aldose derivatization and ketose derivatization on the hydrolysis product I to obtain an aldose derivatization product and a ketose derivatization product.

Preferably, the process of aldose derivatization comprises: and mixing the hydrolysate I with pyridine and hydroxylamine hydrochloride to perform reaction I, and mixing with acetic anhydride to perform reaction II.

Preferably, the conditions of reaction I include: the temperature is 80-100 ℃, the time is 25-35min, and the conditions of the reaction II comprise: the temperature is 80-100 deg.C, and the time is 25-35min.

Preferably, the process of ketose derivatization comprises: and mixing the hydrolysate I with pyridine and methoxyamine hydrochloride, then carrying out reaction III, and mixing with acetic anhydride, and carrying out reaction IV.

Preferably, the conditions of reaction III include: the temperature is 60-80 ℃, the time is 50-70min, and the conditions of the reaction IV comprise: the temperature is 40-50 deg.C, and the time is 50-70min.

Preferably, the methylation treatment in step (2) comprises: in a reaction solvent I, mixing the polysaccharide sample with alkali for ultrasonic treatment, mixing with methyl iodide for methylation reaction to obtain a methylation reaction solution, and performing ultrafiltration and centrifugation on the methylation reaction solution to obtain a methylation product.

Preferably, the reaction solvent I is dimethyl sulfoxide, and the base is sodium hydroxide and/or potassium hydroxide.

Preferably, the time of the ultrasonic treatment is 8-12min, the number of the methylation reaction is 2-4, and the conditions of each methylation reaction comprise: the microwave-assisted power is 300-600W, and the time is 1-3min.

Preferably, the amount of the base is 2-7mg and the amount of the methyl iodide is 10-34 μ L relative to 1mg of the polysaccharide sample.

Preferably, the hydrolysis treatment II in the step (2) comprises: and under the action of microwave assistance II, mixing the methylation product with an acid solution II to perform a hydrolysis reaction II to obtain a hydrolysis product II.

Preferably, the power of the microwave-assisted II is 300-600W, and the conditions of the hydrolysis reaction II comprise: the temperature is 75-95 deg.C, and the time is 2-5min.

Preferably, the acid solution II is selected from one of a hydrochloric acid solution, a sulfuric acid solution, a nitric acid solution and a trifluoroacetic acid solution, and more preferably, a trifluoroacetic acid solution.

Preferably, the reduction treatment in step (2) comprises: and mixing the hydrolysate II with sodium borohydride for reduction reaction to obtain a reduction product.

Preferably, the conditions of the reduction reaction include: the temperature is 10-40 ℃ and the time is 1-3h.

Preferably, the acetylation treatment process comprises: and mixing the reduction product with a pyridine-acetic anhydride solution, and then carrying out acetylation reaction.

Preferably, the acetylation reaction conditions include: the temperature is 90-110 deg.C, and the time is 20-40min.

Preferably, the amount of sodium borohydride used is 4-7mg, relative to 1mg of the polysaccharide sample; the volume ratio of the pyridine to the acetic anhydride is 1.8-1.2.

Preferably, in step (3), the statistical analysis is selected from at least one of principal component analysis, factor analysis, cluster analysis and heat map analysis.

The second aspect of the invention provides the application of the above characterization method in identifying the authenticity or quality of the traditional Chinese medicine.

The third aspect of the invention provides a method for identifying the authenticity or quality of a traditional Chinese medicine, which comprises the following steps: and (3) taking polysaccharides separated from a plurality of traditional Chinese medicine samples as polysaccharide samples, and characterizing and distinguishing by adopting the characterization method.

Preferably, the process for separating and obtaining the polysaccharide from the traditional Chinese medicine sample comprises the following steps:

s1, mixing the traditional Chinese medicine sample with an extraction solvent I to extract I to obtain an extraction precipitate, and mixing the extraction precipitate with an extraction solvent II to extract II to obtain an extracting solution;

and S2, carrying out alcohol precipitation on the extracting solution to obtain a precipitate, and drying the precipitate.

Preferably, the extraction solvent I is an ethanol-water solution, and the extraction solvent II is deionized water.

Preferably, the weight volume ratio of the traditional Chinese medicine sample to the extraction solvent I is 1.

Preferably, the conditions for extracting I include: the temperature is 95-105 ℃, and the time is 1-3h; the conditions for extracting II comprise: the temperature is 80-130 ℃ and the time is 1-3h.

Through the technical scheme, the invention has the beneficial effects that:

the method for representing the polysaccharide structure information provided by the invention comprises the steps of hydrolyzing and derivatizing polysaccharide, analyzing monosaccharide components of the polysaccharide and the relative proportion of each monosaccharide by adopting a GC-MS technology, carrying out methylation treatment, hydrolysis treatment II, reduction treatment and acetylation treatment on the polysaccharide to obtain a methylated sugar alcohol acetyl ester product, analyzing the composition and the relative proportion of glycosidic bonds by adopting the GC-MS technology, further jointly applying the monosaccharide and the structural information such as the composition and the proportion of the glycosidic bonds, carrying out statistical analysis such as principal component analysis, factor analysis and cluster analysis on the monosaccharide and the structural information such as the proportion, and representing and distinguishing different polysaccharide samples. The characterization method provided by the invention is applied to the identification of the authenticity or the quality of the traditional Chinese medicine, can realize the rapid and accurate identification of the authenticity or the quality of the traditional Chinese medicine through the multi-representation of the traditional Chinese medicine polysaccharide, and provides a basis for the quality control of the traditional Chinese medicine.

According to the characterization method of the polysaccharide structure information, provided by the invention, the acid hydrolysis and methylation processes of the polysaccharide are accelerated by using a microwave-assisted technology, redundant methylation reagents are quickly removed by using ultrafiltration centrifugation, a plurality of samples can be simultaneously treated, the operation is easy, the reaction time is short, the efficiency is high, and the complexity and the time consumption of the traditional technical means are avoided.

Drawings

FIG. 1 is a scatter plot of the principal component analysis of the types and proportions of glycosidic linkages of different samples of Atractylodes lancea in example 1;

FIG. 2 is a schematic diagram of the glycosidic bond types and scale factor analysis of the crushed stones of the different rhizoma Atractylodis samples of example 1;

FIG. 3 is a dendrogram of glycosidic bond types and ratio clustering analysis of different rhizoma Atractylodis samples in example 1, wherein A is 9-variable clustering analysis and B is 3-variable clustering analysis;

FIG. 4 is a GC-MS chromatogram of an aldose (A) derivative and a ketose (B) derivative in example 2, and representative GC-MS chromatograms (C, D) of monosaccharide compositions in a cordyceps sample; wherein the aldose comprises galactose (Gla), mannose (Man), glucose (Glc), rhamnose (Rha), fucose (Fuc), ribose (Rib), arabinose (Ara), xylose (Xyl) and mannitol (Ma-ol), and the ketose is fructose (Fru);

FIG. 5 is a GC-MS chromatogram (A) of the acetyl ester product of the methylated sugar alcohol representative of example 2 and a mass spectrum (B) of the T-Araf glycosidic linkage with a peak time of 6.505 min;

FIG. 6 is a chart of the polysaccharide monosaccharide composition heatmap and cluster analysis of different Cordyceps samples of example 2;

FIG. 7 is a chart of the glycosidic bond types and scale chart and cluster analysis of different Cordyceps sinensis samples of example 2.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the present invention provides a method for characterizing polysaccharide structural information, comprising the steps of:

(1) Respectively carrying out hydrolysis treatment I on a plurality of polysaccharide samples to obtain a hydrolysate I, carrying out derivatization treatment on the hydrolysate I to obtain a derivatization product, and analyzing the derivatization product by using GC-MS (gas chromatography-mass spectrometry) to obtain monosaccharide composition and relative proportion of each monosaccharide in the derivatization product corresponding to each polysaccharide sample;

(2) Carrying out methylation treatment, hydrolysis treatment II, reduction treatment and acetylation treatment on a plurality of polysaccharide samples respectively to obtain methylated sugar alcohol acetyl ester products, and analyzing the methylated sugar alcohol acetyl ester products by using GC-MS (gas chromatography-mass spectrometry) to obtain the types of glycosidic bonds in the methylated sugar alcohol acetyl ester products corresponding to each polysaccharide sample and the relative proportion of each glycosidic bond;

(3) And (3) carrying out statistical analysis on the monosaccharide composition obtained in the step (1) and the relative proportion of each monosaccharide and/or the glycosidic bond type obtained in the step (2) and the relative proportion of each glycosidic bond, and characterizing and distinguishing a plurality of polysaccharide samples.

In the invention, the polysaccharide sample can be a polysaccharide product separated from polysaccharide-containing traditional Chinese medicines and foods, for example, traditional Chinese medicine polysaccharide extracted from traditional Chinese medicines such as rhizoma atractylodis, cordyceps sinensis and dendrobe. The relative proportion of each monosaccharide refers to that the peak area of one analyzed monosaccharide is 1, and the ratio of the peak areas of other monosaccharides to the peak area is calculated; the relative proportion of each glycosidic bond means that the peak area of one glycosidic bond is analyzed to be 1, and the ratio of the peak areas of other glycosidic bonds to the peak area is calculated.

In the present invention, in step (3), the relative ratio of the monosaccharide composition and each monosaccharide may be statistically analyzed, the type of glycosidic bond and the relative ratio of each glycosidic bond may be statistically analyzed, or the relative ratio of the monosaccharide composition and each monosaccharide, the type of glycosidic bond and the relative ratio of each glycosidic bond may be statistically analyzed. Preferably, the type of glycosidic linkages and the relative proportion of each glycosidic linkage are statistically analyzed and the analysis of the monosaccharide composition and the relative proportion are determined to match to provide a basis for methylation analysis, or preferably, the monosaccharide composition and the relative proportion of each monosaccharide, the type of glycosidic linkages and the relative proportion of each glycosidic linkage are statistically analyzed.

In the research process, the inventor of the invention unexpectedly finds that the monosaccharide component of the polysaccharide is combined with the glycosidic bond component and the proportion information to carry out statistical analysis, can quickly and accurately represent and distinguish the structural information of different polysaccharide samples, is further applied to identifying the authenticity or quality of the traditional Chinese medicine, can realize the quick and accurate identification of the authenticity or quality of the traditional Chinese medicine through the multivariate representation of the polysaccharide of the traditional Chinese medicine, and provides a basis for the quality control of the traditional Chinese medicine.

According to the present invention, the hydrolysis treatment I may be carried out by a conventional method capable of hydrolyzing polysaccharides. Preferably, the hydrolysis treatment I in the step (1) comprises: and mixing the polysaccharide sample with an acid solution I under the action of microwave assistance I to perform hydrolysis reaction I. The inventor finds that under the preferred embodiment, the hydrolysis efficiency of the polysaccharide is accelerated, and the simultaneous treatment of a plurality of samples can be realized, so that the method is simple, convenient and time-saving.

According to the present invention, preferably, the power of the microwave-assisted I is 300-600W, specifically 300W, 400W, 500W, 600W, or any value in the range formed by any two of the above values; the conditions of the hydrolysis reaction I comprise: the temperature is 75-95 deg.C, specifically 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C, or any value in the range of any two values; the time is 2-5min, specifically 2min, 3min, 4min, 5min, or any value in the range of any two of the above values. The inventors have found that in this preferred embodiment it is advantageous to further increase the efficiency of hydrolysis of the polysaccharide.

According to the present invention, preferably, the acid solution I is selected from one of a hydrochloric acid solution, a sulfuric acid solution, a nitric acid solution and a trifluoroacetic acid solution, and more preferably, a trifluoroacetic acid solution. In the present invention, the concentration of trifluoroacetic acid in the trifluoroacetic acid solution is preferably 0.5 to 3mol/L.

According to the invention, the amount of acid in the acid liquor I is such that the polysaccharide sample is completely hydrolyzed. Preferably, the molar amount of acid in the acid liquor I is 0.1-1mmol relative to 1mg of the polysaccharide sample. The inventors have found that in this preferred embodiment, it is advantageous to further shorten the time for hydrolysis of the polysaccharide and to improve the hydrolysis efficiency.

In the invention, the reaction solution obtained in the hydrolysis reaction I can be subjected to repeated cleaning for 2-4 times by removing redundant trifluoroacetic acid solution by using methanol, and then is dried by using nitrogen to obtain the hydrolysis product I.

According to the present invention, the derivatization treatment may be carried out by a conventional method capable of derivatizing the hydrolysate I of polysaccharides. Preferably, the process of the derivatization treatment in step (1) comprises: and (3) respectively carrying out aldose derivatization and ketose derivatization on the hydrolysis product I to obtain an aldose derivatization product and a ketose derivatization product. Further, the aldose derivative product and the ketose derivative product are respectively analyzed by a gas chromatography-mass spectrometer (GC-MS) to obtain the composition and relative proportion of the monosaccharide.

Exemplary working conditions for GC-MS analysis of the derivatized product in the present application are: the chromatographic column adopts Agilent HP-5MS (30 m multiplied by 0.25mm,0.25 mu m); carrier gas (He) flow rate: 1mL/min; temperature rising procedure: the initial column temperature is 150 ℃, the temperature is kept for 4min, the column temperature is increased to 180 ℃ at the temperature increasing speed of 4 ℃/min and kept for 5min, then the column temperature is increased to 210 ℃ at the temperature of 5 ℃/min, and finally the column temperature is increased to 280 ℃ at the temperature of 20 ℃/min; sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L of the solution; the split ratio is 10: 1.

According to the present invention, preferably, the process of aldose derivatization comprises: and mixing the hydrolysate I with pyridine and hydroxylamine hydrochloride to perform reaction I, and mixing with acetic anhydride to perform reaction II. The inventors have found that in this preferred embodiment, it is advantageous to increase the efficiency of derivatization with aldoses and thus to increase the accuracy of the monosaccharide quantification.

According to the present invention, preferably, the conditions of the reaction I include: the temperature is 80-100 deg.C, specifically 80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C, 100 deg.C, or any value in the range of any two above values; the time is 25-35min, specifically 25min, 27min, 29min, 31min, 33min, 35min, or any value in the range formed by any two of the above values; the conditions of the reaction II include: the temperature is 80-100 deg.C, specifically 80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C, 100 deg.C, or any value in the range of any two above values; the time is 25-35min, specifically 25min, 27min, 29min, 31min, 33min, 35min, or any value in the range of any two of the above values.

According to the present invention, preferably, the process of ketose derivatization comprises: and mixing the hydrolysate I with pyridine and methoxyamine hydrochloride, then carrying out reaction III, and mixing with acetic anhydride, and carrying out reaction IV. The inventors have found that in this preferred embodiment, it is advantageous to increase the efficiency of ketose derivatization, and thus the accuracy of monosaccharide quantification.

According to the present invention, preferably, the conditions of the reaction III include: the temperature is 60-80 deg.C, specifically 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, or any value in the range of any two above values; the time is 50-70min, specifically 50min, 55min, 60min, 65min, 70min, or any value in the range formed by any two values; the conditions of the reaction IV include: the temperature is 40-50 deg.C, specifically 40 deg.C, 42 deg.C, 44 deg.C, 46 deg.C, 48 deg.C, 50 deg.C, or any value in the range of any two above values; the time is 50-70min, specifically 50min, 55min, 60min, 65min, 70min, or any value in the range of any two of the above values.

In the invention, in the process of derivatization of aldose, the dosage of pyridine and hydroxylamine hydrochloride can meet the requirement of completely derivatizing aldose in the hydrolysate I; in the derivatization process of the ketose, the dosage of pyridine and methoxyamine hydrochloride can meet the requirement of completely derivatizing the ketose in the hydrolysate I. Illustratively, the amount of pyridine used in the derivatization of aldoses is 0.1-0.3mL, and the amount of hydroxylamine hydrochloride is 2-10mg, relative to 1mg of the polysaccharide sample; relative to 1mg of the polysaccharide sample, the amount of pyridine in ketose derivatization is 0.1-0.3mL, and the amount of methoxylamine hydrochloride is 2-10mg.

In the invention, after the aldose derivatization and the ketose derivatization are finished, the obtained reactants are respectively dried by nitrogen, added with methanol and dried again, dissolved by methanol after being repeated for 2-4 times, filtered by a 0.22 mu m microporous filter membrane and analyzed by GC-MS.

According to the present invention, preferably, the methylation treatment in step (2) comprises: in a reaction solvent I, mixing the polysaccharide sample with alkali for ultrasonic treatment, mixing with methyl iodide for methylation reaction to obtain a methylation reaction solution, and performing ultrafiltration and centrifugation on the methylation reaction solution to obtain a methylation product. The inventors have found that in this preferred embodiment it is advantageous to increase the effect of methylation on the polysaccharide sample.

In the invention, the methylation reaction liquid obtained by the methylation reaction is subjected to ultrafiltration and centrifugation to remove redundant alkali and methyl iodide, so that the time for removing redundant derivatization reagents can be greatly saved, and the efficiency of separation and purification of the methylation product is effectively improved. Illustratively, after mixing the methylation reaction solution with water, performing ultrafiltration centrifugation by using an ultrafiltration centrifugal tube with the molecular weight of 1000Da to remove excess reagent, and repeatedly washing with water for 2-4 times after centrifugation to obtain the methylated product. Compared with the traditional dialysis method, the process avoids the process of overnight dialysis and the large volume of water added by dialysis, thereby effectively shortening the consumed time. Preferably, the conditions of each ultrafiltration centrifugation include: the rotation speed is 6000-10000rpm, and the time is 20-40min.

According to the present invention, preferably, the reaction solvent I is dimethyl sulfoxide and the base is sodium hydroxide and/or potassium hydroxide.

According to the invention, preferably, the time of the ultrasonic treatment is 8-12min, the number of methylation reactions is 2-4, and the conditions of each methylation reaction comprise: the microwave-assisted power is 300-600W, specifically 300W, 400W, 500W, 600W, or any value in the range formed by any two values; the time is 1-3min, specifically 1min, 2min, 3min, or any value in the range of any two of the above values. That is, after mixing with methyl iodide, microwave-assisted methylation is carried out for 1-3min at 300-600W, and the process is repeated for 2-4 times. The inventors have found that in this preferred embodiment, the methylation process can be further accelerated by microwave assistance, which is easy to handle, short in reaction time, and efficient.

According to the present invention, preferably, the amount of the base is 2 to 7mg and the amount of the methyl iodide is 10 to 34. Mu.L, relative to 1mg of the polysaccharide sample.

According to the invention, the hydrolysis treatment II can be carried out by a conventional method capable of hydrolyzing the methylated product. Preferably, the hydrolysis treatment II in the step (2) comprises: and under the action of microwave assistance II, mixing the methylated product with acid liquor II to perform hydrolysis reaction II to obtain a hydrolysate II. The inventors have found that in this preferred embodiment, it is advantageous to accelerate the efficiency of the hydrolysis of the methylated product and to achieve simultaneous processing of multiple samples, which is simple and time-saving.

In the invention, the reaction liquid obtained by the hydrolysis reaction II can be dried by nitrogen, added with methanol and dried by blowing to remove redundant trifluoroacetic acid, and repeatedly cleaned for 2-4 times, and then dried by nitrogen to obtain the hydrolysis product II.

According to the present invention, preferably, the power of the microwave assistant II is 300-600W, specifically 300W, 400W, 500W, 600W, or any value in the range formed by any two of the above values; the conditions of the hydrolysis reaction II comprise: the temperature is 75-95 deg.C, specifically 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C, or any value in the range of any two values; the time is 2-5min, and can be 2min, 3min, 4min, 5min, or any value in the range of any two of the above values. The inventors have found that in this preferred embodiment it is advantageous to further increase the efficiency of the hydrolysis of the methylated products.

According to the present invention, preferably, the acid solution II is selected from one of a hydrochloric acid solution, a sulfuric acid solution, a nitric acid solution and a trifluoroacetic acid solution, and more preferably, a trifluoroacetic acid solution. In the present invention, the concentration of trifluoroacetic acid in the trifluoroacetic acid solution is preferably 0.5 to 3mol/L.

According to the invention, the acid in the acid liquor II is used in an amount such that the methylation product is completely hydrolyzed. Preferably, the molar amount of acid in the acid solution II is 0.1-1mmol relative to 1mg of the polysaccharide sample. The inventors have found that, in this preferred embodiment, it is advantageous to further shorten the time for the hydrolysis of the methylated product and to improve the hydrolysis efficiency.

According to the present invention, preferably, the reduction treatment in step (2) comprises: and mixing the hydrolysate II with sodium borohydride for reduction reaction to obtain a reduction product. The inventors have found that in this preferred embodiment it is advantageous to increase the efficiency of the reduction of the hydrolysate II.

In the invention, reaction liquid obtained by reduction reaction is mixed with acetic acid to neutralize redundant alkali, then methanol is added, nitrogen is used for blow drying, and washing is repeated for 2-4 times to obtain a reduction product.

According to the present invention, preferably, the conditions of the reduction reaction include: the temperature is 10-40 deg.C, specifically 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, or any value in the range of any two above values; the time is 1 to 3 hours, and specifically, the time may be 1 hour, 2 hours, 3 hours, or any value in a range formed by any two of the above numerical values.

According to the present invention, preferably, the acetylation treatment process comprises: and mixing the reduction product with a pyridine-acetic anhydride solution, and then carrying out acetylation reaction. The inventors have found that in this preferred embodiment it is advantageous to increase the efficiency of acetylation of the reduced product.

In the invention, reaction liquid obtained by acetylation reaction is dried by nitrogen, then mixed with dichloromethane to obtain a sample containing methylated sugar alcohol acetyl ester products, filtered by an organic phase microporous filter membrane, filled into a sample bottle, labeled with relevant information, and subjected to GC/MS analysis.

Exemplary working conditions for the GC-MS analysis of methylated sugar alcohol acetyl ester products herein are: the GC-MS column was an Agilent HP-5MS (30 m.times.0.25mm, 0.25 μm); temperature rising procedure: the initial column temperature is 120 ℃, the temperature is kept for 1min, the temperature is increased to 200 ℃ at the temperature increasing speed of 5 ℃/min, and finally the temperature is increased to 250 ℃ at the temperature increasing speed of 8 ℃/min, and the temperature is kept for 1min; the carrier gas (He) flow rate was: 1.0mL/min; the injection port temperature is: 250 ℃; the sample injection amount is as follows: 1 mu L of the solution; the split ratio is 10: 1.

According to the present invention, preferably, the acetylation reaction conditions include: the temperature is 90-110 deg.C, specifically 90 deg.C, 95 deg.C, 100 deg.C, 105 deg.C, 110 deg.C, or any value in the range of any two values; the time is 20-40min, specifically 20min, 25min, 30min, 35min, 40min, or any value in the range of any two values. The inventors have found that in this preferred embodiment it is advantageous to further increase the efficiency of the acetylation reaction.

According to the invention, the amount of sodium borohydride used in the reduction treatment can enable the hydrolysis product II to be completely reduced, and the amount of pyridine-acetic anhydride solution used in the acetylation treatment can enable the reduction product to be completely acetylated. According to the invention, preferably, the amount of sodium borohydride is 4-7mg and the amount of pyridine-acetic anhydride solution is 0.2-0.5mL, relative to 1mg of the polysaccharide sample; the volume ratio of pyridine to acetic anhydride in the pyridine-acetic anhydride solution is 1.8-1.2.

According to the present invention, preferably, in the step (3), the statistical analysis is selected from at least one of principal component analysis, factor analysis, cluster analysis and heat map analysis. For example, a statistical analysis combining principal component analysis and cluster analysis may be used, and a statistical analysis combining cluster analysis and heatmap analysis may also be used.

The second aspect of the invention provides the application of the above characterization method in identifying the authenticity or quality of the traditional Chinese medicine.

The third aspect of the invention provides a method for identifying the authenticity or quality of a traditional Chinese medicine, which comprises the following steps: and (3) taking polysaccharides separated from a plurality of traditional Chinese medicine samples as polysaccharide samples, and characterizing and distinguishing by adopting the characterization method.

In the invention, the polysaccharide sample can be a polysaccharide product separated from polysaccharide-containing traditional Chinese medicines and foods, for example, traditional Chinese medicine polysaccharide extracted from traditional Chinese medicines such as rhizoma atractylodis, cordyceps sinensis and dendrobe.

According to the present invention, preferably, the process of separating polysaccharides from the Chinese medicine sample comprises:

s1, mixing the traditional Chinese medicine sample with an extraction solvent I to extract I to obtain an extraction precipitate, and mixing the extraction precipitate with an extraction solvent II to extract II to obtain an extracting solution;

and S2, carrying out alcohol precipitation on the extracting solution to obtain a precipitate, and drying the precipitate.

According to the present invention, preferably, the extraction solvent I is an ethanol-water solution, and the extraction solvent II is deionized water. Wherein the ethanol content in the ethanol-water solution is preferably 60 to 80 vol%.

According to the present invention, preferably, the weight volume ratio of the traditional Chinese medicine sample to the extraction solvent I is 1.

In the present invention, the extraction II may be repeated plural times, for example, 2 times by using the extraction solvent II, and the liquids obtained by the two times are mixed to obtain an extract.

According to the present invention, preferably, the conditions for extracting I include: the temperature is 95-105 deg.C (oil bath temperature), specifically 95 deg.C, 100 deg.C, 105 deg.C, or any value in the range of any two values; the time is 1-3h, and specifically can be 1h, 2h, 3h, or any value in the range formed by any two values; the conditions for extracting II comprise: the temperature is 80-130 deg.C (oil bath temperature), specifically 80 deg.C, 90 deg.C, 100 deg.C, 110 deg.C, 120 deg.C, 130 deg.C, or any value in the range of any two values; the time is 1 to 3 hours, and specifically, the time may be 1 hour, 2 hours, 3 hours, or any value in a range formed by any two of the above numerical values.

In the invention, the alcohol precipitation process comprises the following steps: concentrating the extracting solution, mixing with ethanol overnight for alcohol precipitation, centrifuging after alcohol precipitation to remove supernatant, collecting precipitate, repeatedly washing the precipitate with n-butanol and acetone until the supernatant is colorless, and then blowing the residual organic solvent in the precipitate with a nitrogen blowing instrument for drying. The drying process comprises the following steps: mixing the precipitate obtained by alcohol precipitation with deionized water, performing vortex oscillation to completely dissolve the precipitate, and freeze drying to obtain the traditional Chinese medicine polysaccharide.

According to a particularly preferred embodiment of the present invention, the method for identifying the authenticity or quality of the traditional Chinese medicine comprises the following steps:

(1) Mixing a traditional Chinese medicine sample with an ethanol-water solution according to a weight-volume ratio of 1-25, extracting for 1-3h under the condition of a temperature of 95-105 ℃ (oil bath temperature), removing supernatant to obtain an extraction precipitate, mixing the extraction precipitate with deionized water according to a material-liquid ratio of 1-10-35, extracting for 1-3h under the condition of a temperature of 80-130 ℃ (oil bath temperature), repeatedly extracting twice with deionized water, and combining supernatants to obtain an extracting solution;

(2) Concentrating the extracting solution, mixing with ethanol for overnight alcohol precipitation, carrying out alcohol precipitation, centrifuging to remove supernatant, collecting precipitate, repeatedly washing the precipitate with n-butanol and acetone until the supernatant is colorless, blow-drying the residual organic solvent in the precipitate with a nitrogen blowing instrument, mixing the blow-dried precipitate with deionized water, carrying out vortex oscillation to completely dissolve the precipitate, and carrying out freeze drying to obtain traditional Chinese medicine polysaccharide;

(3) Mixing multiple traditional Chinese medicine polysaccharides obtained from different traditional Chinese medicine samples in the steps (1) and (2) with trifluoroacetic acid solution, and performing hydrolysis treatment for 2-5min under the microwave-assisted action of 300-600W and at the temperature of 75-95 ℃ to obtain hydrolysate I; mixing the hydrolysate I with pyridine and hydroxylamine hydrochloride, reacting at 80-100 ℃ for 25-35min, mixing with acetic anhydride, and reacting at 80-100 ℃ for 25-35min to obtain an aldose derivative product; mixing the hydrolysate I with pyridine and methoxyamine hydrochloride, reacting at 60-80 deg.C for 50-70min, mixing with acetic anhydride, and reacting at 40-50 deg.C for 50-70min to obtain ketose derivative; analyzing the aldose derivative product and the ketose derivative product by GC-MS respectively to obtain the monosaccharide composition in the derivative product corresponding to each polysaccharide sample and the relative proportion of each monosaccharide;

(4) Respectively dissolving a plurality of traditional Chinese medicine polysaccharides obtained by different traditional Chinese medicine samples in the steps (1) and (2) in dimethyl sulfoxide, mixing with alkali, carrying out ultrasonic treatment for 8-12min, mixing with iodomethane, carrying out methylation reaction for 1-3min under the assistance of microwave with the power of 300-600W, repeating for 2-4 times to obtain methylation reaction liquid, mixing the methylation reaction liquid with water, carrying out ultrafiltration centrifugation by using an ultrafiltration centrifugal tube with the molecular weight of 1000Da, and repeatedly washing with water for 2-4 times after centrifugation to obtain a methylation product; mixing the methylation product with a trifluoroacetic acid solution, and carrying out hydrolysis treatment for 2-5min under the microwave assistance of 300-600W at the temperature of 75-95 ℃ to obtain a hydrolysis product II; mixing the hydrolysate II with sodium borohydride, and carrying out reduction reaction for 1-3h at the temperature of 10-40 ℃ to obtain a reduction product; mixing the reduction product with a pyridine-acetic anhydride solution, and then carrying out acetylation reaction at the temperature of 90-110 ℃ for 20-40min to obtain methylated sugar alcohol acetyl ester products, wherein the methylated sugar alcohol acetyl ester products are analyzed by GC-MS (gas chromatography-mass spectrometry), and the type of glycosidic bonds and the relative proportion of each glycosidic bond in the methylated sugar alcohol acetyl ester products corresponding to each polysaccharide sample are obtained;

(5) And (3) carrying out statistical analysis on the monosaccharide composition obtained in the step (3) and the relative proportion of each monosaccharide and/or the glycosidic bond type obtained in the step (4) and the relative proportion of each glycosidic bond, and characterizing and distinguishing a plurality of polysaccharide samples.

The present invention will be described in detail below by way of examples.

In the following examples, the raw materials and reagents used were all conventional commercial products unless otherwise specified; the experimental procedures, for which specific conditions are not specified, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturer.

In the following examples, the working conditions for GC-MS analysis of aldose derivatization and ketose derivatization were: the chromatographic column adopts Agilent HP-5MS (30 m multiplied by 0.25mm,0.25 mu m); carrier gas (He) flow rate: 1mL/min; temperature rising procedure: the initial column temperature is 150 ℃, the temperature is kept for 4min, the column temperature is increased to 180 ℃ at the temperature increasing speed of 4 ℃/min and kept for 5min, then the column temperature is increased to 210 ℃ at the temperature of 5 ℃/min, and finally the column temperature is increased to 280 ℃ at the temperature of 20 ℃/min; sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L of the solution; the split ratio is 10: 1.

The operating conditions for the GC-MS analysis of the methylated sugar alcohol acetyl ester product were: the GC-MS column was an Agilent HP-5MS (30 m.times.0.25mm, 0.25 μm); temperature rising procedure: the initial column temperature is 120 ℃, the temperature is kept for 1min, the temperature is raised to 200 ℃ at the heating rate of 5 ℃/min, and finally the temperature is raised to 250 ℃ at the heating rate of 8 ℃/min, and the temperature is kept for 1min; the carrier gas (He) flow rate was: 1.0mL/min; the sample inlet temperature is: 250 ℃; the sample injection amount is as follows: 1 mu L of the solution; the split ratio is 10: 1.

Example 1

9 parts of rhizoma atractylodis lanceae (south) and 9 parts of rhizoma atractylodis (N1-N9), 6 parts of rhizoma atractylodis macrocephalae (B1-B6), 5 parts of rhizoma atractylodis macrocephalae (H1-H5) and 2 parts of elecampane (H6-H7) are collected from different producing areas to be used as analysis samples, detailed information of the analysis samples is shown in table 1, and the types of the medicinal materials are determined by the identification of a crude drug pharmacy method respectively.

TABLE 1 areas of origin and numbering of Atractylodes lancea

(1) Extraction of polysaccharide from rhizoma atractylodis and pseudo-mixture thereof

Respectively preparing rhizoma atractylodis samples into fine powder, precisely weighing 0.5g of rhizoma atractylodis sample, putting the fine powder into a 50mL round-bottom flask, adding 10mL of ethanol-water solution with the ethanol concentration of 60 volume percent, heating and refluxing at 100 ℃ for 1h for degreasing, removing oligosaccharide, cooling, filtering to obtain an extraction precipitate, adding 15mL of deionized water into the extraction precipitate, extracting at 80 ℃ for 1h, cooling, centrifuging at 3500rpm for 5min, taking supernatant to obtain primary supernatant, adding 15mL of deionized water into the centrifuged residue, continuing extracting at 80 ℃ for 1h, cooling, centrifuging at 3500rpm for 5min, removing medicinal material residue to obtain secondary supernatant, and combining the two supernatants to obtain an extracting solution;

(2) Concentrating the extracting solution respectively, adding 4 times of ethanol-water solution (the ethanol concentration is 95 vol%), standing overnight for alcohol precipitation, centrifuging at 3500rpm for 5min after alcohol precipitation to remove supernatant and collect precipitate, repeatedly washing the precipitate with n-butanol and acetone until the supernatant is colorless, then blowing dry the residual organic solvent in the precipitate with a nitrogen blower, adding 5mL of deionized water into each sample respectively, performing vortex oscillation to completely dissolve the deionized water, and freeze-drying for 36h to obtain rhizoma atractylodis polysaccharide powder;

(3) Analysis of monosaccharide composition in different rhizoma Atractylodis samples

Weighing 5mg of rhizoma atractylodis polysaccharide powder extracted from each rhizoma atractylodis sample, placing the rhizoma atractylodis polysaccharide powder into a sample bottle, hydrolyzing for 3min by using 1mL of trifluoroacetic acid solution (the concentration of trifluoroacetic acid is 1.5 mol/L) under the microwave assistance of 400W, controlling the hydrolysis temperature to be 90 ℃, adding methanol to remove redundant trifluoroacetic acid solution, repeating for 3 times, and drying by nitrogen to obtain a hydrolysate I;

a) Derivatization of aldoses

Adding 5mg of hydrolysate I into 0.5mL of hydroxylamine hydrochloride-pyridine solution (the concentration of hydroxylamine hydrochloride is 20 mg/mL), reacting at 90 ℃ for 30min, adding 0.5mL of acetic anhydride, continuing to react at 90 ℃ for 30min, blowing the mixture to dry by nitrogen, adding methanol and blowing the mixture to dry again, repeating the reaction for 3 times to obtain an aldose derivative product, dissolving the aldose derivative product by methanol, filtering the product by a 0.22 mu m microporous filter membrane, and analyzing by GC-MS to obtain the composition of monosaccharides in the aldose derivative product and the relative proportion of the peak area of each monosaccharide (the peak area of glucose Glc is 1), wherein the results are shown in Table 2;

b) Derivatization of ketoses

Adding 5mg of hydrolysate I into 0.5mL of methoxylamine hydrochloride-pyridine solution (the concentration of the methoxylamine hydrochloride solution is 20 mg/mL), reacting at 70 ℃ for 60min, then adding 0.5mL of acetic anhydride, reacting at 45 ℃ for 60min, blowing dry with nitrogen, adding methanol, blowing dry again, repeating for 3 times to obtain a ketose derivatization product, dissolving the ketose derivatization product with methanol, filtering with a 0.22 mu m microporous membrane, and analyzing with GC-MS to obtain the composition of monosaccharides and the relative proportion of each monosaccharide in the ketose derivatization product (the peak area of glucose Glc is 1).

The monosaccharide in the sample is compared with a standard substance to determine the type, and the proportion information of the saccharides with different compositions is obtained through peak area comparison (the peak area of glucose Glc is 1), and the monosaccharide composition and the relative proportion of each monosaccharide in the rhizoma atractylodis samples with different types and sources are shown in a table 2.

As can be seen from Table 2, different sources of Atractylodes lancea and mixed artifacts thereof all contain the same kind of monosaccharides, namely arabinose (Ara), rhamnose (Rha), fucose (Fuc), mannose (Man), glucose (Glc), galactose (Gal) and fructose (Fru).

(4) Constructing polysaccharide characteristic maps of glycosidic bond information in different rhizoma atractylodis samples

a) Methylation of

2 parts of rhizoma atractylodis polysaccharide powder extracted from each rhizoma atractylodis sample are respectively taken in parallel, 5mg of rhizoma atractylodis polysaccharide powder is precisely weighed for each part, the rhizoma atractylodis polysaccharide powder is dissolved in 1mL of dimethyl sulfoxide (DMSO), 10mg of NaOH is added, ultrasonic treatment is carried out for 10min, and 100 mu L of methyl iodide (CH) is added ₃ I) Methylating for 3min under the assistance of microwave with the power of 400W, repeating for 3 times to obtain methylation reaction liquid, adding 5mL of water into the methylation reaction liquid, removing redundant reagents by using an ultrafiltration centrifugal tube with the molecular weight of 1000Da, centrifuging for 30min at 8000rpm, mixing with water, repeating ultrafiltration, centrifuging and washing for 3 times to obtain a methylation product;

b) Hydrolysis

Fixing the volume of the methylated product to 1mL, adding trifluoroacetic acid to enable the concentration of the trifluoroacetic acid to be 1mol/L, carrying out acid hydrolysis for 3min under the assistance of microwaves with the power of 400W, controlling the temperature to be 95 ℃ to obtain hydrolysis reaction liquid, blowing nitrogen into the hydrolysis reaction liquid for drying, adding methanol for drying to remove the residual trifluoroacetic acid, and repeating for 2 times to obtain a hydrolysate II;

c) Reduction of

1mL of NaBH at a concentration of 20mg/mL was added to hydrolyzate II ₄ Reducing the solution (prepared by 0.5mol/L ammonia water) for 2 hours, then slowly dropwise adding 4mol/L acetic acid, neutralizing NaOH, adding 1mL methanol, drying by blowing with nitrogen, and repeating the step for 2 times to obtain a reduction product;

d) Acetylation

Adding 1mL of pyridine/acetic anhydride (volume ratio is 1;

as can be seen from Table 3, the compositions and the ratios of the glycosidic bonds between the Atractylodes lancea and the pseudodrug from different sources are different and have characteristic differences. 9 parts of rhizoma atractylodis lanceae (south) and 6 parts of rhizoma atractylodis lanceae (N1-N9) and 6 parts of rhizoma atractylodis macrocephalae (B1-B6) contain T-linked Araf residues in different proportions, while 5 parts of rhizoma atractylodis macrocephalae (H1-H5) and 2 parts of elecampane (H6-H7) which are pseudo-ingredients do not contain T-linked Araf residues, and meanwhile, the proportion of the 2,6linked-Galp residues in the polysaccharides of 5 parts of rhizoma atractylodis macrocephalae (H1-H5) and 2 parts of elecampane (H6-H7) is higher than that in the polysaccharides of rhizoma atractylodis (N1-N9 and B1-B6). The polysaccharide of the Atractylodes lancea (N1-N9) does not contain 6-linked Glcp residue, part of the polysaccharide of the Atractylodes lancea (N1-N3, N5, N6) does not contain 1,2,3-linked Fruf residue, the polysaccharide of the Atractylodes japonica (B1-B6) contains 1,2,3-linked Fruf residue, and part of the polysaccharide of the Atractylodes japonica (B4-B6) does not contain 6-linked Glcp residue.

(5) Construction of multi-element characterization method based on atractylodes lancea polysaccharide structure information

Analyzing the types of the glycosidic bonds obtained in the step (4) and the percentage of each glycosidic bond in all glycosidic bonds by utilizing main component analysis, factor analysis and cluster analysis in SPSS 18.0 software, wherein the results are shown in figures 1 to 3, and the types of the Atractylodes lancea (south) 9 parts (N1-N9), atractylodes lancea (south) 6 parts (B1-B6), atractylodes macrocephala (H1-H5) and Aucklandia lappa (H6-H7) can be obviously characterized so as to distinguish the Atractylodes lancea from the pseudolite with different sources.

Therefore, the composition and the proportion of glycosidic bonds are very important for the quality control of the atractylodes lancea polysaccharide, and the 2,6-linked-Galp, T-linked-Glcp and 1,2-linked-Fruf glycosidic bonds are characteristic evaluation indexes, as shown in figures 1 to 3, the method adopted in the embodiment 1 can well distinguish atractylodes lancea samples, and factor analysis results determine the types of glycosidic bonds playing a role in determining the distinction, so that important attention is needed in the quality control, the method is reasonable and feasible, the accuracy of the quality control of the atractylodes lancea polysaccharide in south and north is improved, and the work difficulty of quality control research is reduced.

TABLE 2 monosaccharide compositions and proportions of Atractylodes lancea and polysaccharide mixed with counterfeit substances thereof

TABLE 3 glycosidic bond composition and ratio of different sources of rhizoma Atractylodis and its polysaccharide

Example 2

The method comprises the steps of collecting 6 parts of cordyceps militaris (CM 1-6), 6 parts of silkworm cordyceps (SCM 1-6), 4 parts of cordyceps sinensis zymophyte powder (CSF 1-4) and 2 parts of cordyceps sinensis (CS 1-2) from different production places as cordyceps sinensis samples, wherein the detailed information of the cordyceps sinensis samples is shown in a table 4, and the types of the medicinal materials are determined by identification of a crude drug pharmacy method.

TABLE 4 production areas and numbers of Cordyceps medicinal materials from different sources

Numbering	Species of	Source	Numbering	Species of	Source
						CM1	Cordyceps militaris (L.) Link	Guangdong river	SCM4	Silkworm cordyceps	Heilongjiang iron power
CM2	Cordyceps militaris (L.) Link	Guangdong river	SCM5	Silkworm cordyceps	Guangdong New meeting
						CM3	Cordyceps militaris (L.) Link	(Jilin)	SCM6	Silkworm cordyceps	Xinjiang
CM4	Cordyceps militaris (L.) Link	Internal Mongolia red peak	CSF-1	Cordyceps sinensis fermented product	Baixin pharmacy
						CM5	Cordyceps militaris (L.) Link	Shenyang Liaoning medicine	CSF-2	Cordyceps sinensis fermented product	Baixin pharmacy
CM6	Cordyceps militaris (L.) Link	Shenyang Liaoning medicine	CSF-3	Cordyceps sinensis fermented product	Baixin pharmacy
						SCM1	Silkworm cordyceps	Shenyang Liaoning medicine	CSF-4	Cordyceps sinensis fermentation powder	Laboratory development
SCM2	Cordyceps militaris (L.) Link	Shenyang Liaoning medicine	CS1	Chinese Caterpillar fungus	(Qinghai)
						SCM3	Silkworm cordyceps	Heilongjiang iron power	CS2	Chinese Caterpillar fungus	Sichuan province

(1) Extraction of cordyceps and its mixed counterfeit polysaccharide

Respectively preparing cordyceps sinensis samples into fine powder, respectively precisely weighing 2g of cordyceps sinensis samples, placing the cordyceps sinensis samples into a 150mL round-bottom flask, adding 40mL of ethanol-water solution with the ethanol concentration of 60%, heating and refluxing for 1h by using a 100 ℃ oil bath to remove micromolecule components, centrifuging and pouring out supernatant to obtain extraction precipitate, then pouring 60mL of deionized water into the extraction precipitate, refluxing for 1h at 130 ℃ (oil bath), centrifuging at 6000rpm for 5min to obtain supernatant, adding 60mL of deionized water into the centrifugation residue, refluxing for 1h at 130 ℃ (oil bath), cooling, centrifuging at 6000rpm for 5min to remove medicinal material residue to obtain secondary supernatant, and combining the two supernatants to obtain an extracting solution;

(2) Respectively carrying out rotary evaporation and concentration on the extracting solution, adding 4 times of volume of absolute ethyl alcohol, carrying out alcohol precipitation for 12h at 4 ℃, centrifuging the solution to remove supernatant and collecting precipitate, blowing dry the mixed solution in the precipitate to obtain dry precipitated powder, adding 10mL of deionized water to completely dissolve the precipitated powder, and carrying out freeze drying for 48h to obtain cordyceps polysaccharide powder;

(3) Analysis of monosaccharide composition in different Cordyceps samples

Weighing 3mg of cordyceps polysaccharide powder extracted from each cordyceps sample, placing the cordyceps polysaccharide powder into a sample bottle, hydrolyzing the cordyceps polysaccharide powder for 5min by using 1mL of trifluoroacetic acid solution (the concentration of trifluoroacetic acid is 2 mol/L) under the microwave assistance action with the power of 300W, adding methanol to remove redundant trifluoroacetic acid solution, repeating the steps for 3 times, and drying the solution by using nitrogen to obtain a hydrolysate I;

a) Derivatization of aldoses

Adding 3mg of hydrolysate I into 0.5mL of pyridine-hydroxylamine hydrochloride solution (the concentration of hydroxylamine hydrochloride is 20 mg/mL), reacting for 30min at the temperature of a water bath kettle at 90 ℃, adding 0.5mL of acetic anhydride, reacting for 30min at 90 ℃, blowing by nitrogen, adding a proper amount of methanol, blowing a sample to obtain an aldose derivative product, dissolving the aldose derivative product by using 2mL of methanol, filtering by using an organic microporous filter membrane (0.22 mu m), and analyzing by using GC-MS to obtain the composition of monosaccharides in the aldose derivative product and the relative proportion of each monosaccharide;

b) Derivatization of ketoses

Adding 3mg of hydrolysate I into 0.5mL of pyridine-methoxyamine hydrochloride solution (concentration of methoxyamine hydrochloride is 20 mg/mL), reacting in a water bath at 70 ℃ for 60min, adding 0.5mL of acetic anhydride, reacting at 45 ℃ for 60min, drying with nitrogen, adding an appropriate amount of methanol solution, drying the sample to obtain a ketose derivative product, dissolving the ketose derivative product with 2mL of methanol, filtering through an organic microporous membrane (0.22 μm), and analyzing by GC-MS to obtain the composition of monosaccharides and the relative proportion of each monosaccharide in the ketose derivative product, as shown in FIG. 4, the GC-MS chromatograms of aldose (A) and ketose (B), and representative GC-MS chromatograms (C, D) of the composition of monosaccharides in cordyceps sinensis samples, wherein different cordyceps sinensis samples comprise galactose (Gla), mannose (Man), glucose (Glc), rhamnose (Rha), fucose (Fuc), ribose (Rib), arabinose (Ara), xylose (Xyl) and mannitol (Ma-u), and fructose (Fr);

(4) Establishing polysaccharide characteristic maps of glycosidic bond information in different cordyceps sinensis samples

a) Methylation of

2 parts of cordyceps polysaccharide powder extracted from each cordyceps sample are respectively and parallelly weighed, 3mg of cordyceps polysaccharide powder is precisely weighed for each part, dissolved in 1mL of dimethyl sulfoxide (DMSO), 10mg of NaOH is added, ultrasonic treatment is carried out for 10min, and 100 mu L of methyl iodide (CH) is added ₃ I) Methylating for 3min under the assistance of microwave with the power of 300W, repeating for 3 times to obtain methylation reaction liquid, adding 5mL of water into the methylation reaction liquid, removing redundant reagents by using an ultrafiltration centrifugal tube with the molecular weight of 1000Da, centrifuging for 40min at 8000rpm, mixing with water, repeating ultrafiltration, centrifuging and washing for 3 times to obtain a methylation product;

b) Hydrolysis

Fixing the volume of the methylated product to 1mL, adding trifluoroacetic acid to enable the concentration to be 2mol/L, performing acid hydrolysis for 5min under the assistance of microwave at the power of 300W, controlling the hydrolysis temperature to be 85 ℃ to obtain hydrolysis reaction liquid, drying the hydrolysis reaction liquid by nitrogen, adding methanol to remove the residual trifluoroacetic acid by drying, and repeating for 2 times to obtain a hydrolysate II;

c) Reduction of

1mL of NaBH at a concentration of 20mg/mL was added to hydrolyzate II ₄ The solution (prepared by 0.5mol/L ammonia water) is reduced for 2 hours, then 4mol/L acetic acid is slowly dropped to neutralize NaOH. Adding 1mL of methanol, blowing the mixture to dry by using nitrogen, and repeating the step for 2 times to obtain a reduction product;

d) Acetylation

Adding 1mL of pyridine/acetic anhydride (volume ratio is 1); as shown in fig. 5, GC-MS chromatogram (a) and mass spectrum (B) of T-Araf glycosidic bond with peak time of 6.505min for representative methylated sugar alcohol acetyl ester products, respectively;

(5) Multi-element characterization method for constructing structural information based on cordyceps polysaccharide

The monosaccharide composition and relative ratio obtained in step (3), and the type of glycosidic bond and the relative ratio of each glycosidic bond obtained in step (4) were analyzed by cluster analysis and thermographic analysis in SPSS 18.0 software, and the results are shown in fig. 6 and 7.

As can be seen from FIGS. 6 and 7, there are significant differences between the polysaccharides of fermented Cordyceps powder (CSF 1-4) and the polysaccharides of artificially cultured Cordyceps militaris (CM 1-6), cordyceps bombycis (SCM 1-6) and Cordyceps sinensis (CS 1-2) among different types of Cordyceps sinensis.

FIG. 6 shows that the mannose and galactose contents in the fermented powder of Cordyceps sinensis (CSF 1-4) are higher than those in the polysaccharides of other Cordyceps sinensis. Only 4 kinds of fermented Cordyceps powder (CSF 1-4) can be distinguished from other kinds of Cordyceps by monosaccharide composition and relative proportion. The monosaccharide composition needs further auxiliary methylation analysis to distinguish different types of cordyceps. FIG. 7 shows that the polysaccharide of Cordyceps sinensis fermentation broth powder (CSF 1-4) contains T-linked Araf residues, 4-linked Arap residues and 3,4-linked Manp residues at various ratios, while the polysaccharide of other Cordyceps sinensis (CM 1-6, SCM1-6 and CS 1-2) has no T-linked Araf residues, 4-linked Arap residues and 3,4-linked Manp residues. The ratio of 4-linked Galp residue in polysaccharide of Cordyceps militaris (SCM 1-6) is lower than that of Cordyceps militaris and Cordyceps sinensis. The polysaccharides of Cordyceps militaris (CM 1-6) and Cordyceps militaris (SCM 1-6) do not contain 4-linked Galp residues, and the polysaccharides of Cordyceps militaris (CS 1 and CS 2) do not contain 4,6-linked Manp residues and 2,3,6-linked Glcp residues.

In example 2, the types and relative proportions of glycosidic bonds relative to the monosaccharide composition and relative proportions have better discrimination for different types of cordyceps sinensis, and the combination of the multivariate method can completely discriminate different types of cordyceps sinensis, thereby providing an effective means for polysaccharide structure analysis and discrimination of polysaccharides from different sources.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (10)

1. A method for characterizing polysaccharide structural information, comprising the steps of:

(1) Respectively carrying out hydrolysis treatment I on a plurality of polysaccharide samples to obtain a hydrolysate I, carrying out derivatization treatment on the hydrolysate I to obtain a derivatization product, and analyzing the derivatization product by using GC-MS (gas chromatography-mass spectrometry) to obtain monosaccharide composition and relative proportion of each monosaccharide in the derivatization product corresponding to each polysaccharide sample;

(2) Carrying out methylation treatment, hydrolysis treatment II, reduction treatment and acetylation treatment on a plurality of polysaccharide samples respectively to obtain methylated sugar alcohol acetyl ester products, and analyzing the methylated sugar alcohol acetyl ester products by using GC-MS (gas chromatography-mass spectrometry) to obtain the types of glycosidic bonds in the methylated sugar alcohol acetyl ester products corresponding to each polysaccharide sample and the relative proportion of each glycosidic bond;

(3) And (3) carrying out statistical analysis on the monosaccharide composition obtained in the step (1) and the relative proportion of each monosaccharide and/or the glycosidic bond type obtained in the step (2) and the relative proportion of each glycosidic bond, and characterizing and distinguishing a plurality of polysaccharide samples.

2. The characterization method according to claim 1, wherein the hydrolysis treatment I in step (1) comprises: under the action of microwave assistance I, mixing the polysaccharide sample with acid liquor I to perform hydrolysis reaction I;

preferably, the power of the microwave-assisted reaction I is 300-600W, and the conditions of the hydrolysis reaction I comprise: the temperature is 75-95 deg.C, and the time is 2-5min;

preferably, the acid solution I is selected from one of a hydrochloric acid solution, a sulfuric acid solution, a nitric acid solution and a trifluoroacetic acid solution, and more preferably is a trifluoroacetic acid solution;

preferably, the molar amount of acid in the acid liquor I is 0.1-1mmol relative to 1mg of the polysaccharide sample.

3. The characterization method according to claim 1, wherein the derivatization process in step (1) comprises: respectively performing aldose derivatization and ketose derivatization on the hydrolysis product I to obtain an aldose derivatization product and a ketose derivatization product;

preferably, the process of aldose derivatization comprises: mixing the hydrolysate I with pyridine and hydroxylamine hydrochloride to perform a reaction I, and then mixing the hydrolysate I with acetic anhydride to perform a reaction II;

preferably, the conditions of reaction I include: the temperature is 80-100 ℃, the time is 25-35min, and the conditions of the reaction II comprise: the temperature is 80-100 deg.C, and the time is 25-35min;

preferably, the process of ketose derivatization comprises: mixing the hydrolysate I with pyridine and methoxyamine hydrochloride, then carrying out reaction III, and mixing with acetic anhydride to carry out reaction IV;

preferably, the conditions of reaction III include: the temperature is 60-80 ℃, the time is 50-70min, and the reaction IV conditions comprise: the temperature is 40-50 deg.C, and the time is 50-70min.

4. The characterization method according to any one of claims 1 to 3, wherein the methylation process in step (2) comprises: mixing the polysaccharide sample with alkali in a reaction solvent I, performing ultrasonic treatment, mixing with methyl iodide, performing methylation reaction to obtain a methylation reaction solution, and performing ultrafiltration and centrifugation on the methylation reaction solution to obtain a methylation product;

preferably, the reaction solvent I is dimethyl sulfoxide, and the base is sodium hydroxide and/or potassium hydroxide;

preferably, the time of the ultrasonic is 8-12min, the number of methylation reactions is 2-4, and the methylation reaction conditions comprise: the microwave-assisted power is 300-600W, and the time is 1-3min;

preferably, the amount of the base is 2-7mg and the amount of the methyl iodide is 10-34 μ L relative to 1mg of the polysaccharide sample.

5. The characterization method according to claim 4, wherein the hydrolysis treatment II in step (2) comprises: under the action of microwave assistance II, mixing the methylated product with acid liquor II to perform hydrolysis reaction II to obtain a hydrolysate II;

preferably, the power of the microwave-assisted II is 300-600W, and the conditions of the hydrolysis reaction II comprise: the temperature is 75-95 deg.C, and the time is 2-5min;

preferably, the acid solution II is selected from one of a hydrochloric acid solution, a sulfuric acid solution, a nitric acid solution and a trifluoroacetic acid solution, and more preferably, a trifluoroacetic acid solution.

6. The characterization method according to claim 5, wherein the reduction process in step (2) comprises: mixing the hydrolysate II with sodium borohydride for reduction reaction to obtain a reduction product;

preferably, the conditions of the reduction reaction include: the temperature is 10-40 ℃, and the time is 1-3h;

preferably, the acetylation treatment process comprises: mixing the reduction product with a pyridine-acetic anhydride solution and then carrying out acetylation reaction;

preferably, the acetylation reaction conditions include: the temperature is 90-110 deg.C, and the time is 20-40min;

preferably, the amount of sodium borohydride is 4-7mg, relative to 1mg of the polysaccharide sample; the volume ratio of the pyridine to the acetic anhydride is 1.8-1.2.

7. The characterization method according to any one of claims 1 to 3, wherein in step (3), the statistical analysis is selected from at least one of principal component analysis, factor analysis, cluster analysis and heat map analysis.

8. Use of the characterization method according to any one of claims 1 to 7 for identifying authenticity or quality of Chinese medicine.

9. A method for identifying the authenticity or quality of a traditional Chinese medicine is characterized by comprising the following steps:

the polysaccharide separated from a plurality of traditional Chinese medicine samples is used as a polysaccharide sample, and the polysaccharide sample is characterized and distinguished by adopting the characterization method of any one of claims 1 to 7.

10. The method of claim 9, wherein the step of isolating the polysaccharides from the herbal sample comprises:

s1, mixing the traditional Chinese medicine sample with an extraction solvent I to extract I to obtain an extraction precipitate, and mixing the extraction precipitate with an extraction solvent II to extract II to obtain an extracting solution;

s2, carrying out alcohol precipitation on the extracting solution to obtain a precipitate, and drying the precipitate;

preferably, the extraction solvent I is an ethanol-water solution, and the extraction solvent II is deionized water;

preferably, the weight volume ratio of the traditional Chinese medicine sample to the extraction solvent I is 1;

preferably, the conditions for extracting I include: the temperature is 95-105 ℃, and the time is 1-3h; the conditions for extracting II comprise: the temperature is 80-130 ℃ and the time is 1-3h.

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