Analysis method of oligosaccharide composition in American ginseng polysaccharide

Abstract

The invention discloses a method for analyzing oligosaccharide composition in American ginseng polysaccharide, and belongs to the technical field of pharmaceutical analytical chemistry, and the method comprises the following steps: 1) carrying out primary extraction on American ginseng through a methanol / methyl tert-butyl ether solution, and removing supernate to obtain American ginseng subjected to primary extraction; (2) adding pure water into the American ginseng subjected to primary extraction, extracting again, taking supernate, adding absolute ethyl alcohol into the supernate, standing, and centrifuging to obtain precipitate; 3) dissolving the precipitate with water, adding an n-butyl alcohol / chloroform solution, mixing and oscillating, and drying the aqueous solution to obtain crude polysaccharide; 4) adding water into the crude polysaccharide for redissolving, adding trifluoroacetic acid for reaction, and centrifugally concentrating to obtain oligosaccharide; and 5) dissolving the oligosaccharides in an acetonitrile aqueous solution, taking a sample to be detected, and detecting by using liquid chromatography-mass spectrometry to obtain the variety and proportion of the oligosaccharides in the sample to be detected. The problem that the structure of American ginseng polysaccharide is difficult to identify and analyze in the prior art is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analytical chemistry, and particularly relates to a method for analyzing the oligosaccharide composition in American ginseng polysaccharide. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] American ginseng is the dried root of Panaxquinquefolium L., a plant of the Araliaceae family. It has the effects of replenishing qi and nourishing yin, clearing away heat and promoting body fluid. It is mainly used for qi deficiency and yin deficiency, deficiency heat and fatigue, cough, asthma, sputum and blood, internal heat and thirst, dry mouth and throat. Since the Qing Dynasty, American ginseng has been used in China for more than 300 years and cultivated for more than 40 years. Compared with ginseng, American ginseng is more peaceful and less likely to cause internal heat. American ginseng is not only a top-grade Chinese medicine, but also a good tonic. It is widely used in the fields of medicine, health care, and food.

[0004] Steaming American ginseng can enhance its medicinal value and improve its medicinal properties. During the steaming process, the chemical composition of American ginseng will be affected by steaming and change, such as the conversion of ginsenosides, changes in the structure and content of polysaccharides. Steaming conditions, especially steaming time and temperature, have a significant effect on the production of polysaccharides in American ginseng. The length of steaming time and the temperature can change the degradation, position change and configuration inversion of polysaccharides, thereby affecting the content and biological activity of polysaccharides in the final product. Therefore, in the actual processing process, the steaming conditions need to be strictly controlled to ensure the quality and efficacy of American ginseng.

[0005] Polysaccharide is a macromolecular compound with important biological activity in American ginseng. It is composed of more than 10 monosaccharides condensed through glycosidic bonds. Studies have shown that polysaccharides have anti-tumor, antioxidant, hypoglycemic, and immune-enhancing effects. American ginseng polysaccharides with different structures have an important influence on their biological activity, and polysaccharides with different structures may perform better in specific activities. After steaming American ginseng from different origins, the structure of American ginseng polysaccharides is different, and changes in the steaming conditions of American ginseng will affect the structure of American ginseng polysaccharides.

[0006] Due to the complex structure and large molecular weight of American ginseng polysaccharides, it is very difficult to directly identify and analyze their structure. Therefore, some patents often use the method of degrading American ginseng polysaccharides into monosaccharides for analysis. For example, patent CN 113092653 A discloses a method for determining the monosaccharide composition in American ginseng polysaccharides and a method for identifying different parts. The polysaccharides are degraded into monosaccharides, and different parts of American ginseng are identified by the relative content of different monosaccharides.

[0007] However, during the steaming process of American ginseng, the polysaccharide structure of American ginseng changes, and the types and relative contents of its monosaccharides change little, making it difficult to identify the quality of American ginseng using the above method. During the processing of a specific batch of American ginseng, the steaming conditions need to be optimized and improved according to specific effects, and the above method is also difficult to distinguish and identify the quality of American ginseng.

[0008] In addition, during the extraction process of polysaccharides and oligosaccharides, American ginseng and the extraction solution will inevitably add a certain amount of impurities to the polysaccharides and oligosaccharides, which will affect the analysis and detection and the peak shape of the obtained liquid chromatography-mass spectrometry. In addition, the choice of mobile phase and liquid chromatography-mass spectrometry-related parameters will also affect the peak shape of the spectrum. Summary of the invention

[0009] In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a method for analyzing the oligosaccharide composition in American ginseng polysaccharides. The present invention degrades the polysaccharides in American ginseng into smaller oligosaccharide fragments, and detects the types and proportions of the oligosaccharide fragments by liquid chromatography-mass spectrometry, thereby characterizing the changes in the structure of steamed American ginseng polysaccharides. The identification results can also guide the optimization of the American ginseng steaming process.

[0010] The present invention provides a method for analyzing the oligosaccharide composition in American ginseng polysaccharide, comprising:

[0011] 1) performing a primary extraction on American ginseng using a methanol / methyl tert-butyl ether solution, removing the supernatant, and obtaining American ginseng after the primary extraction;

[0012] 2) Adding pure water to the ginseng after the initial extraction to perform a second extraction, taking the supernatant, adding anhydrous ethanol to the supernatant, standing it and then centrifuging it to obtain a precipitate;

[0013] 3) dissolving the precipitate in water, adding n-butanol / chloroform solution, mixing and shaking, and drying the aqueous solution to obtain crude polysaccharide;

[0014] 4) taking the crude polysaccharide and re-dissolving it in water, adding trifluoroacetic acid to react, and centrifuging and concentrating to obtain oligosaccharides;

[0015] 5) Dissolve the oligosaccharide in acetonitrile aqueous solution, take the sample to be tested and detect it by liquid chromatography-mass spectrometry to obtain the type and proportion of oligosaccharides in the sample to be tested.

[0016] In some embodiments, in step 1), the mass volume ratio of American ginseng and the solution of methanol / methyl tert-butyl ether is 500 mg:(4-6) mL; preferably 500 mg:5 mL.

[0017] In some embodiments, in step 1), the American ginseng is steamed American ginseng; preferably, the steaming temperature is 100-130° C., and the steaming time is 2-12 h.

[0018] In some embodiments, in step 1), the American ginseng is American ginseng powder, methanol / methyl tert-butyl ether solution is added to the American ginseng powder, and ultrasonic extraction is performed for 20-40 minutes;

[0019] Preferably, in the methanol / methyl tert-butyl ether solution, the volume ratio of methanol to methyl tert-butyl ether is 1:(3-5); further preferably, the ultrasonic extraction time is 30 min, and the volume ratio of methanol to methyl tert-butyl ether is 1:4.

[0020] In some embodiments, in step 2), pure water is added to the Western ginseng after the initial extraction, and ultrasonic-assisted extraction is performed at 80-90°C for 0.5-1.5 hours. The extracted mixed solution is centrifuged for 10-30 minutes, the supernatant is taken, 2-4 times of anhydrous ethanol is added, and the mixture is allowed to stand for 8-16 hours and then centrifuged to obtain a precipitate; preferably, the ratio of Western ginseng to pure water is 1 mg: (400-600) μL.

[0021] In some embodiments, in step 2), pure water is added to the Western ginseng after the initial extraction, and ultrasonic-assisted extraction is performed at 80-90°C for 1 hour. The extracted mixed solution is centrifuged for 20 minutes, the supernatant is taken, 3 times of anhydrous ethanol is added, and the mixture is allowed to stand for 12 hours and then centrifuged to obtain a precipitate, wherein the ratio of Western ginseng to pure water is 1 mg:500 μL.

[0022] In some embodiments, in step 4), the crude polysaccharide obtained in step 3) is redissolved in water and centrifuged to obtain the supernatant. Trifluoroacetic acid is added to the supernatant for reaction, and then centrifuged and concentrated to obtain an oligosaccharide solid, which is then washed with methanol to obtain the oligosaccharide; preferably, the reaction conditions of the supernatant and trifluoroacetic acid are 75-85°C, and the ultrasonic-assisted reaction is performed for 0.8-1.5h; further preferably, the reaction conditions of the supernatant and trifluoroacetic acid are 80°C, and the ultrasonic-assisted reaction is performed for 1h.

[0023] In some embodiments, in step 5), the acetonitrile aqueous solution is a 70% acetonitrile aqueous solution.

[0024] In some embodiments, the liquid chromatography-mass spectrometry detection conditions are as follows: the chromatographic column is Waters ACQUITY UPLC BEH Amide (2.1×100 mm, 1.7 μm); the mobile phase A is an aqueous solution containing 20 mM ammonium formate, the mobile phase B is a 95% acetonitrile / water solution containing 20 mM ammonium formate, the column temperature is 55° C., and the flow rate is 0.27 mL / min;

[0025] The gradient elution conditions were: 0-5 min, 95-93% B; 5-10 min, 93-92% B; 10-14 min, 92-70% B; 14-24 min, 70-50% B; 24-29 min, 50-50% B; 29-29.1 min, 50-95% B; 29.1-34 min, 95-95% B; the total elution time was 34 min;

[0026] Mass spectrometry conditions: In negative ion mode, the oligosaccharide mass number scan range m / z was 50-1200, the capillary voltage was 3000 V, the drying gas flow rate was 8 L / min, the nebulizer pressure was 2.0 bar, the drying gas temperature was 200 °C, the collision cell RF voltage amplitude was 750 Vpp, the pulse waiting time was 5 s, the transmission time was 60 s, and the collision energy was 5-70 eV.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention extracts and degrades polysaccharides in American ginseng, determines the types and relative contents of oligosaccharides by liquid chromatography-mass spectrometry, and purifies the polysaccharides during the polysaccharide extraction process to remove interfering impurities, thereby improving the accuracy of detection.

[0029] 2. The present invention degrades polysaccharides in American ginseng into smaller oligosaccharide fragments, and detects the types and relative contents of the oligosaccharides after degradation through liquid chromatography-mass spectrometry. The structure and content of the polysaccharides in American ginseng are indirectly characterized by the types and relative contents of the oligosaccharides. The characterization can guide the steaming conditions of American ginseng, such as steaming time and steaming temperature.

[0030] 3. The present invention obtains a sample to be tested for liquid chromatography-mass spectrometry detection by steaming, extracting, degrading, and purifying the polysaccharides in American ginseng, further optimizes the column temperature and flow rate of liquid chromatography-mass spectrometry detection, and adopts a mobile phase containing ammonium formate to improve the response intensity of the color peak and optimize the peak shape. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 The extracted ion chromatograms of rhamnose-xylose in the sample to be tested of the present invention under different mobile phases, wherein A is the extracted ion chromatogram of rhamnose-xylose under formic acid mobile phase, and B is the extracted ion chromatogram of rhamnose-xylose under ammonium formate mobile phase.

[0033] Figure 2] are extracted ion chromatograms of rhamnose-xylose in the sample to be tested of the present invention at different flow rates, wherein A is the extracted ion chromatogram of rhamnose-xylose at a flow rate of 0.2 mL / min, and B is the extracted ion chromatogram of rhamnose-xylose at a flow rate of 0.27 mL / min.

[0034] Figure 3 The extracted ion current diagrams of rhamnose-xylose in the sample to be tested at different column temperatures of the present invention, wherein A is the extracted ion current diagram of rhamnose-xylose at a column temperature of 40°C, and B is the extracted ion current diagram of rhamnose-xylose at a column temperature of 50°C.

[0035] Figure 4 The extracted ion current graphs of the tetrasaccharide glucose-glucose-xylose-xylose in the test sample of the present invention under different hydrolysis conditions, wherein A is the extracted ion current graph of ultrasonic-assisted hydrolysis for 30 minutes, B is the extracted ion current graph of ultrasonic-assisted hydrolysis for 1 hour, C is the extracted ion current graph of ultrasonic-assisted hydrolysis for 2 hours, and D is the extracted ion current graph of 100°C water bath hydrolysis for 2 hours.

[0036] Figure 5 This is the mass spectrum of the sample to be tested in Example 1 of the present invention.

[0037] Figure 6 This is a heat map of oligosaccharide analysis after degradation of American ginseng polysaccharide at different steaming times of the present invention.

[0038] Figure 7 This is a heat map of oligosaccharide analysis after degradation of American ginseng polysaccharide at different steaming temperatures of the present invention. DETAILED DESCRIPTION

[0039] In view of the fact that the polysaccharide structure of American ginseng is complex and the molecular weight is large, and it is difficult to directly identify and analyze its structure, the present invention proposes an analysis method for the oligosaccharide composition in American ginseng polysaccharides. The present invention degrades the polysaccharides in American ginseng into oligosaccharide fragments with smaller molecular weights, optimizes the extraction and degradation steps, and optimizes the relevant parameters of liquid phase mass spectrometry-chromatography through liquid phase mass spectrometry-chromatography, detects the types and relative contents of different oligosaccharide fragments, and thus identifies the quality of American ginseng. The identification results can also guide the optimization of the American ginseng steaming process.

[0040] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0041] The materials used in the following examples are all commercially available.

[0042] Example 1

[0043] This embodiment provides a method for analyzing the oligosaccharide composition in American ginseng polysaccharide, and the specific steps are:

[0044] Extraction of crude polysaccharides from steamed American ginseng:

[0045] Steamed American ginseng (steaming conditions: steaming time 8h, steaming temperature 100℃) was dried and powdered to 100 mesh. 500mg of the American ginseng powder was weighed, and 5mL of methanol / methyl tert-butyl ether mixed solution (v / v, 1:4) was added. Ultrasonic extraction was performed for 30 minutes to remove fat-soluble components and small molecular organic compounds. After removing the supernatant, 10ml of pure water was added. Ultrasonic assisted extraction was performed at 85℃ for 1h. The mixed solution was then centrifuged for 20min, and after taking the supernatant after centrifugation, 3 times the volume of anhydrous ethanol was added and allowed to stand at 4℃ for 12h. After centrifugation, the precipitate was dissolved in water to form a polysaccharide solution, and an equal volume of n-butanol / chloroform mixed solution (v / v, 1:4) was added to the polysaccharide solution, fully shaken for 30min, centrifuged to separate the aqueous phase from the organic phase, and the protein was removed repeatedly, and then the crude polysaccharide was freeze-dried.

[0046] Oligosaccharide preparation:

[0047] Take 1 mg of crude polysaccharide in a 1.5 mL centrifuge tube and add 500 μL of water to dissolve it. Centrifuge at 14000 rpm / min for 5 min, take 200 μL of supernatant, and add 200 μL of 1 mol / L trifluoroacetic acid to the supernatant. At 80 ° C, ultrasonic-assisted reaction for 1 hour, after the reaction is completed, put it into a centrifugal concentrator to obtain oligosaccharide solids, wash it with 400 μL of methanol 5 times to obtain oligosaccharides. Redissolve the oligosaccharides with 70% acetonitrile aqueous solution (v / v, 7:3, the same below) for later use.

[0048] LC-MS analysis:

[0049] American ginseng oligosaccharides were analyzed by UPLC-Q-TOF-MS system. The chromatographic column was Waters ACQUITY UPLCBEH Amide (2.1×100 mm, 1.7 μm). Mobile phase A was an aqueous solution containing 20 mM ammonium formate, and mobile phase B was a 95% acetonitrile / water solution (v / v, 95:5) containing 20 mM ammonium formate. The column temperature was 55°C and the flow rate was 0.27 mL / min.

[0050] The gradient elution conditions were: 0-5 min, 95-93% B; 5-10 min, 93-92% B; 10-14 min, 92-70% B; 14-24 min, 70-50% B; 24-29 min, 50-50% B; 29-29.1 min, 50-95% B; 29.1-34 min, 95-95% B. The total elution time was 34 min.

[0051] The mass spectrometry data were obtained in negative ion mode with an oligosaccharide mass number scan range of m / z 50-1200, a capillary voltage of 3000 V, a drying gas flow rate of 8 L / min, a nebulizer pressure of 2.0 bar, a drying gas temperature of 200 °C, a collision cell RF voltage amplitude of 750 Vpp, a pulse waiting time of 5 s, a transmission time of 60 s, and a collision energy set to 5-70 eV.

[0052] Example 2

[0053] This example provides a method for analyzing the oligosaccharide composition in American ginseng polysaccharides, which differs from Example 1 in that:

[0054] Extraction of crude polysaccharides from American ginseng:

[0055] Steamed American ginseng (steaming conditions: steaming time 8h, steaming temperature 100℃) was powdered to 100 mesh, 500mg of the above American ginseng powder was weighed, 5mL of methanol / methyl tert-butyl ether mixed solution (v / v, 1:4) was added, and ultrasonic extraction was performed for 20min to remove fat-soluble components and small molecular organic compounds. After removing the supernatant, 10ml of pure water was then added. Ultrasonic assisted extraction was performed at 80℃ for 1.5h. The mixed solution was then centrifuged for 10min, and after taking the supernatant after centrifugation, 2 times the volume of anhydrous ethanol was added and allowed to stand at 4℃ for 8h. After centrifugation, the precipitate was dissolved in water to form a polysaccharide solution, and an equal volume of n-butanol / chloroform mixed solution (v / v, 1:4) was added to the polysaccharide solution, fully shaken for 30min, centrifuged to separate the aqueous phase from the organic phase, and the protein was removed repeatedly, and then the crude polysaccharide was freeze-dried.

[0056] Oligosaccharide preparation:

[0057] Take 1 mg of crude polysaccharide in a 1.5 mL centrifuge tube and add 400 μL of water to dissolve it. Centrifuge at 14000 rpm / min for 5 min, take 200 μL of supernatant, and add 200 μL of 1 mol / L trifluoroacetic acid to the supernatant. At 75 ° C, ultrasonic-assisted reaction for 1.5 hours, after the reaction is completed, put it into a centrifugal concentrator to obtain oligosaccharide solids, wash it with 400 μL of methanol 5 times, and obtain oligosaccharides. Redissolve the oligosaccharides with 70% acetonitrile aqueous solution for later use.

[0058] Example 3

[0059] This example provides a method for analyzing the oligosaccharide composition in American ginseng polysaccharides, which differs from Example 1 in that:

[0060] Extraction of crude polysaccharides from American ginseng:

[0061] Steamed American ginseng (steaming conditions: steaming time 8h, steaming temperature 100℃) was powdered to 100 mesh, 500mg of the above American ginseng powder was weighed, 5mL of methanol / methyl tert-butyl ether mixed solution (v / v, 1:4) was added, and ultrasonic extraction was performed for 40min to remove fat-soluble components and small molecular organic compounds. After removing the supernatant, 10ml of pure water was then added. Ultrasonic assisted extraction was performed at 90℃ for 0.5h. The mixed solution was then centrifuged for 30min, and after taking the supernatant after centrifugation, 4 times the volume of anhydrous ethanol was added, and it was allowed to stand at 4℃ for 16h. After centrifugation, the precipitate was dissolved in water to form a polysaccharide solution, and an equal volume of n-butanol / chloroform mixed solution (v / v, 1:4) was added to the polysaccharide solution, fully shaken for 30min, centrifuged to separate the aqueous phase from the organic phase, and the protein was removed repeatedly, and then the crude polysaccharide was freeze-dried.

[0062] Oligosaccharide preparation:

[0063] Take 1 mg of crude polysaccharide in a 1.5 mL centrifuge tube and add 500 μL of water to dissolve it. Centrifuge at 14000 rpm / min for 5 min, take 200 μL of supernatant, and add 200 μL of 1 mol / L trifluoroacetic acid to the supernatant. At 85 ° C, ultrasonic-assisted reaction for 0.8 h, after the reaction is completed, put it into a centrifugal concentrator to obtain oligosaccharide solids, wash it with 400 μL of methanol 5 times to obtain oligosaccharides. Redissolve the oligosaccharides with 70% acetonitrile aqueous solution for standby use, and repeat three times for each sample.

[0064] Example 4

[0065] This embodiment provides an analysis method for the oligosaccharide composition in American ginseng polysaccharide, which is different from Example 1 in that the steaming time of American ginseng is changed to 2 hours.

[0066] Example 5

[0067] This embodiment provides an analysis method for the oligosaccharide composition in American ginseng polysaccharide, which is different from Embodiment 1 in that the steaming time of American ginseng is changed to 4 hours.

[0068] Example 6

[0069] This embodiment provides a method for analyzing the oligosaccharide composition in American ginseng polysaccharides, which is different from Embodiment 1 in that the steaming time of American ginseng is changed to 6 hours.

[0070] Example 7

[0071] This embodiment provides a method for analyzing the oligosaccharide composition in American ginseng polysaccharides, which is different from Example 1 in that the steaming time of American ginseng is changed to 10 hours.

[0072] Example 8

[0073] This embodiment provides an analysis method for the oligosaccharide composition in American ginseng polysaccharides, which is different from Example 1 in that the steaming time of steamed American ginseng is changed to 12 hours.

[0074] Example 9

[0075] This embodiment provides an analysis method for the oligosaccharide composition in American ginseng polysaccharide, which is different from Example 1 in that the steaming conditions for steaming American ginseng are changed to a steaming time of 2 h and a steaming temperature of 110°C.

[0076] Example 10

[0077] This embodiment provides an analysis method for the oligosaccharide composition in American ginseng polysaccharide, which is different from Embodiment 1 in that the steaming conditions for steaming American ginseng are changed to a steaming time of 2 h and a steaming temperature of 120°C.

[0078] Embodiment 11

[0079] This embodiment provides an analysis method for the oligosaccharide composition in American ginseng polysaccharide, which is different from Embodiment 1 in that the steaming conditions for steaming American ginseng are changed to a steaming time of 2 h and a steaming temperature of 130°C.

[0080] Comparative Example 1

[0081] This comparative example provides an analysis method for the oligosaccharide composition in American ginseng polysaccharides, which differs from Example 1 in that the mobile phase A of the liquid chromatography-mass spectrometry detection conditions is 0.1% formic acid aqueous solution, and the mobile phase B is 95% acetonitrile aqueous solution (v / v, 95:5).

[0082] Depend on Figure 1 It can be seen that in comparative example 1, under the formic acid mobile phase, the peak bifurcation is more serious, and the same substance has multiple peaks ( Figure 1 A). The salt mobile phase used in Example 1 can improve this phenomenon ( Figure 1 B).

[0083] Comparative Example 2

[0084] This comparative example provides an analysis method for the oligosaccharide composition in American ginseng polysaccharides, which is different from Example 1 in that the flow rate of the liquid chromatography-mass spectrometry detection condition is 0.2 mL / min.

[0085] Depend on Figure 2 It can be seen that at a flow rate of 0.2 mL / min in Comparative Example 2, the peak shape of the oligosaccharide is very poor, with peak bifurcation ( Figure 2 A). When the flow rate of Example 1 is 0.27 mL / min, the peak is sharp ( Figure 2 B).

[0086] Comparative Example 3

[0087] This comparative example provides an analysis method for the oligosaccharide composition in American ginseng polysaccharide, which is different from Example 1 in that the column temperature of the liquid chromatography-mass spectrometry detection condition is 40°C.

[0088] Depend on Figure 3 It can be seen that at the column temperature of 40°C in Comparative Example 3, the peak shape of oligosaccharides is poor ( Figure 3 A). When the column temperature was raised to 55°C in Example 1, the peak shape became better ( Figure 3 B).

[0089] Comparative Example 4

[0090] This comparative example provides an analysis method for the oligosaccharide composition in American ginseng polysaccharide, which is different from Example 1 in that after adding trifluoroacetic acid, the ultrasonic-assisted water bath hydrolysis time is 30 minutes.

[0091] Comparative Example 5

[0092] This comparative example provides an analysis method for the oligosaccharide composition in American ginseng polysaccharide, which is different from Example 1 in that after adding trifluoroacetic acid, the ultrasonic-assisted water bath hydrolysis time is 2 hours.

[0093] Comparative Example 6

[0094] This comparative example provides an analysis method for the oligosaccharide composition in American ginseng polysaccharide, which is different from Example 1 in that after adding trifluoroacetic acid, hydrolysis is carried out in a water bath for 2 hours.

[0095] Depend on Figure 4 It can be seen that, taking the tetrasaccharide glucose-glucose-xylose-xylose as an example, it can be seen that when the ultrasonic-assisted water bath hydrolysis time of Comparative Example 4 is 30 min, there is no signal in the extracted ion current diagram ( Figure 4 A), indicating that the reaction was not sufficient and no oligosaccharides were produced. Comparative Example 5 also had no signal when ultrasonic-assisted water bath hydrolysis was performed for 2 h ( Figure 4 C), indicating that the reaction time is too long and the tetrasaccharide is further hydrolyzed. Example 1 is an ultrasonic-assisted water bath hydrolysis for 1h, and the tetrasaccharide has a strong signal ( Figure 4 B). Comparative Example 6, water bath hydrolysis for 2 h, had the same effect as ultrasound-assisted hydrolysis for 1 h ( Figure 4 D). It can be seen that appropriate ultrasound-assisted hydrolysis can speed up the reaction time and promote the hydrolysis of polysaccharides.

[0096] The steamed American ginseng of Examples 1-3, after extraction steps and chromatographic mass spectrometry parameter settings, can identify the types and proportions of the above-mentioned different oligosaccharides in the degradation samples of American ginseng polysaccharides, and indirectly characterize the differences in the structure and content of polysaccharides in American ginseng through the types and proportions of oligosaccharides in different samples.

[0097] Taking Example 1 as an example, 17 oligosaccharides were identified in the hydrolyzed sample of American ginseng, including 6 disaccharides, 4 trisaccharides and 7 tetrasaccharides, as shown in Table 1. Oligosaccharides were identified by secondary mass spectrometry. Oligosaccharides were identified by characteristic ions and neutral loss. Figure 5 ), taking glucose-glucose-xylose-xylose as an example, the molecular formula of the tetrasaccharide is C 22 H 38 O 19 , tetrasaccharide in negative mode, m / z 605 ([M + CH3COO] - ). When the glycosidic bond connecting one glucose in the tetrasaccharide glucose-glucose-xylose-xylose is broken, it will lose 162 neutrally, resulting in a mass-to-charge ratio of m / z 443, and then it will continue to lose one glucose, resulting in a mass-to-charge ratio of m / z 281. In the negative mode, xylose-xylose has an m / z of 281 ([MH] - ). 341 is the mass-to-charge ratio of glucose-glucose.

[0098] Table 1 Detection results of oligosaccharides in Example 1

[0099]

[0100]

[0101] As shown in Examples 1, 4-8, the inventors analyzed and verified the polysaccharides in steamed American ginseng under different steaming conditions (changing the steaming time). Figure 6 This is a heat map of the content analysis of different oligosaccharides after degradation of American ginseng polysaccharides at different steaming times at a steaming temperature of 100°C. It can be seen that the types and relative contents of oligosaccharides change with the change of steaming time. The content of oligosaccharides is the most abundant at 8 hours. As the steaming time increases, the content of disaccharides increases, while the content of trisaccharides and tetrasaccharides decreases, indicating that the structure of the polysaccharide has changed.

[0102] As shown in Examples 1, 9-11, the inventors analyzed and verified the polysaccharides in steamed American ginseng under different steaming conditions (changing the steaming temperature). Figure 7 This is a heat map of the content analysis of different oligosaccharides after degradation of American ginseng polysaccharides at different steaming temperatures when the steaming time is 2 hours. As the steaming temperature increases, the contents of disaccharides, trisaccharides, and tetrasaccharides all change, indicating that the structure of the polysaccharide has changed.

[0103] The results showed that the oligosaccharide analysis method could indirectly characterize the changes in the types and structures of polysaccharides in steamed American ginseng, compare the oligosaccharide analysis results of American ginseng under different steaming conditions and the target American ginseng, and select the best steaming conditions to guide the optimization of the American ginseng steaming process.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for analyzing the oligosaccharide composition in American ginseng polysaccharides, characterized in that: include: 1) performing a primary extraction on American ginseng using a methanol / methyl tert-butyl ether solution, removing the supernatant, and obtaining American ginseng after the primary extraction; 2) Adding pure water to the ginseng after the initial extraction to extract again, taking the supernatant, adding anhydrous ethanol to the supernatant, standing it and then centrifuging it to obtain a precipitate; 3) dissolving the precipitate in water, adding n-butanol / chloroform solution, mixing and shaking, and drying the aqueous solution to obtain crude polysaccharide; 4) taking the crude polysaccharide and re-dissolving it in water, adding trifluoroacetic acid to react, and centrifuging and concentrating to obtain oligosaccharides; 5) Dissolve the oligosaccharide in an acetonitrile aqueous solution, take the sample to be tested and detect it by liquid chromatography-mass spectrometry to obtain the type and proportion of the oligosaccharide in the sample to be tested.

2. The method for analyzing the oligosaccharide composition in American ginseng polysaccharide according to claim 1, characterized in that: In step 1), the mass volume ratio of American ginseng to methanol / methyl tert-butyl ether solution is 500 mg:(4-6) mL; preferably 500 mg:5 mL.

3. The method for analyzing the oligosaccharide composition in American ginseng polysaccharide according to claim 1, characterized in that: In step 1), the American ginseng is steamed American ginseng; preferably, the steaming temperature is 100-130° C., and the steaming time is 2-12 hours.

4. The method for analyzing the oligosaccharide composition in American ginseng polysaccharide according to claim 1, characterized in that: In step 1), the American ginseng is American ginseng powder, methanol / methyl tert-butyl ether solution is added to the American ginseng powder, and ultrasonic extraction is performed for 20-40 minutes; Preferably, in the methanol / methyl tert-butyl ether solution, the volume ratio of methanol to methyl tert-butyl ether is 1:(3-5); further preferably, the ultrasonic extraction time is 30 min, and the volume ratio of methanol to methyl tert-butyl ether is 1:

4.

5. The method for analyzing the oligosaccharide composition in American ginseng polysaccharide according to claim 1, characterized in that: In step 2), pure water is added to the ginseng after the initial extraction, and ultrasonic-assisted extraction is performed at 80-90° C. for 0.5-1.5 hours. The extracted mixed solution is centrifuged for 10-30 minutes, the supernatant is taken, 2-4 times of anhydrous ethanol is added, and the mixture is allowed to stand for 8-16 hours and then centrifuged to obtain a precipitate.

6. The method for analyzing the oligosaccharide composition in American ginseng polysaccharide according to claim 5, characterized in that: Preferably, the ratio of American ginseng to pure water is 1 mg: (400-600) μL.

7. The method for analyzing the oligosaccharide composition in American ginseng polysaccharide according to claim 1, characterized in that: In step 2), pure water is added to the Western ginseng after the initial extraction, and ultrasonic-assisted extraction is performed at 80-90°C for 1 hour. The extracted mixed solution is centrifuged for 20 minutes, the supernatant is taken, 3 times of anhydrous ethanol is added, and the mixture is allowed to stand for 12 hours and then centrifuged to obtain a precipitate, wherein the ratio of Western ginseng to pure water is 1 mg:500 μL.

8. The method for analyzing the oligosaccharide composition in American ginseng polysaccharide according to claim 1, characterized in that: In step 4), the crude polysaccharide obtained in step 3) is redissolved in water, centrifuged, the supernatant is taken, trifluoroacetic acid is added to the supernatant for reaction, and then centrifuged and concentrated to obtain oligosaccharide solids, which are then washed with methanol to obtain oligosaccharides; preferably, the reaction conditions of the supernatant and trifluoroacetic acid are 75-85° C., and the ultrasonic-assisted reaction is 0.8-1.5 h; further preferably, the reaction conditions of the supernatant and trifluoroacetic acid are 80° C., and the ultrasonic-assisted reaction is 1 h.

9. The method for analyzing the oligosaccharide composition in American ginseng polysaccharide according to claim 1, characterized in that: In step 5), the acetonitrile aqueous solution is a 70% acetonitrile aqueous solution.

10. The method for analyzing the oligosaccharide composition in American ginseng polysaccharide according to claim 1, characterized in that: The liquid chromatography-mass spectrometry detection conditions were as follows: the chromatographic column was Waters ACQUITY UPLC BEH Amide (2.1×100 mm, 1.7 μm); the mobile phase A was an aqueous solution containing 20 mM ammonium formate, the mobile phase B was a 95% acetonitrile / water solution containing 20 mM ammonium formate, the column temperature was 55° C., and the flow rate was 0.27 mL / min; The gradient elution conditions were: 0-5 min, 95-93% B; 5-10 min, 93-92% B; 10-14min, 92-70% B; 14-24min, 70-50% B; 24-29min, 50-50% B; 29-29.1min, 50-95% B; 29.1-34min, 95-95% B; total time is 34min; Mass spectrometry conditions: In negative ion mode, the oligosaccharide mass number scan range m / z was 50-1200, the capillary voltage was 3000 V, the drying gas flow rate was 8 L / min, the nebulizer pressure was 2.0 bar, the drying gas temperature was 200 °C, the collision cell RF voltage amplitude was 750 Vpp, the pulse waiting time was 5 s, the transmission time was 60 s, and the collision energy was 5-70 eV.

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