Method for analyzing oligosaccharide composition of american ginseng polysaccharides
By degrading American ginseng polysaccharides into oligosaccharide fragments and using liquid chromatography-mass spectrometry, the problem of identifying the structure of American ginseng polysaccharides was solved, enabling accurate analysis of the structure and content of American ginseng polysaccharides, guiding the optimization of the steaming process, and ensuring the quality and bioactivity of American ginseng.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- SHANDONG ANALYSIS AND TEST CENTER
- Filing Date
- 2025-06-26
- Publication Date
- 2026-05-07
AI Technical Summary
Existing technologies are insufficient to effectively identify structural changes in American ginseng polysaccharides, and impurities during the extraction process affect the analytical results, making it difficult to optimize steaming conditions to ensure quality.
American ginseng polysaccharides were degraded into oligosaccharide fragments, and the types and proportions of oligosaccharides were detected by liquid chromatography-mass spectrometry. The extraction and degradation steps were optimized, and specific mobile phases and mass spectrometry parameters were used to remove interference from impurities, thus guiding the optimization of the steaming process.
This improves the accuracy and clarity of the analysis of American ginseng polysaccharide structures, guides the optimization of steaming conditions, and ensures the quality and bioactivity of American ginseng.
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Figure CN2025103820_07052026_PF_FP_ABST
Abstract
Description
An analytical method for the oligosaccharide composition of American ginseng polysaccharides Technical Field
[0001] This invention belongs to the field of pharmaceutical analytical chemistry technology, specifically relating to an analytical method for the oligosaccharide composition of American ginseng polysaccharides. Background Technology
[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] American ginseng is the dried root of *Panax quinquefolium* L., a plant in the Araliaceae family. It has the effects of replenishing qi and nourishing yin, clearing heat and promoting body fluid production. It is mainly used for qi and yin deficiency, fatigue due to deficiency heat, cough with phlegm and blood, internal heat and thirst, and dry mouth and throat. Since the Qing Dynasty, American ginseng has been used in China for over 300 years and cultivated for over 40 years. Compared with ginseng, American ginseng is milder and less likely to cause internal heat. American ginseng is both a high-quality traditional Chinese medicine and a nourishing tonic, widely used in medicinal, health care, and edible fields.
[0004] Steaming American ginseng can enhance its medicinal value and improve its properties. During steaming, the chemical composition of American ginseng changes, such as the transformation of ginsenosides and changes in the structure and content of polysaccharides. Steaming conditions, especially steaming time and temperature, have a significant impact on the production of polysaccharides in American ginseng. The length of steaming time and the temperature can alter the degradation, repositioning, and conformational inversion of polysaccharides, thus affecting the polysaccharide content and bioactivity in the final product. Therefore, in actual processing, it is necessary to strictly control the steaming conditions to ensure the quality and efficacy of American ginseng.
[0005] Polysaccharides are macromolecular compounds with important biological activities found in American ginseng. They are composed of more than 10 monosaccharides condensed through glycosidic bonds. Studies have shown that polysaccharides possess anti-tumor, antioxidant, hypoglycemic, and immune-enhancing effects. Different structures of American ginseng polysaccharides significantly influence their biological activity, with different structures potentially exhibiting superior performance in specific activities. The polysaccharide structures of American ginseng differ after steaming from ginseng from different origins, and variations in steaming conditions also affect the polysaccharide structure.
[0006] Because of the complex structure and large molecular weight of American ginseng polysaccharides, direct identification and analysis of their structure are very difficult. 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 of American ginseng polysaccharides and a method for identifying different parts of American ginseng by degrading polysaccharides into monosaccharides and identifying different parts of American ginseng by the relative content of different monosaccharides.
[0007] However, during the steaming process of American ginseng, the polysaccharide structure of American ginseng changes, while the types and relative contents of its monosaccharides change only slightly. It is difficult to use the above methods to identify the quality of American ginseng. In the processing of a specific batch of American ginseng, it is necessary to optimize and improve the steaming conditions according to the specific effects. The above methods are also difficult to distinguish and identify the quality of American ginseng.
[0008] In addition, during the extraction of polysaccharides and oligosaccharides, the ginseng and the extraction solution inevitably introduce a certain amount of impurities into the polysaccharides and oligosaccharides, which will affect the analysis and detection, and affect the peak shape of the obtained liquid chromatography-mass spectra. Furthermore, the choice of mobile phase and related parameters of liquid chromatography-mass spectrometry will also affect the peak shape of the spectrum. Summary of the Invention
[0009] To address the shortcomings of existing technologies, the purpose of this invention is to provide an analytical method for the oligosaccharide composition in American ginseng polysaccharides. This invention degrades the polysaccharides in American ginseng into smaller oligosaccharide fragments, and uses liquid chromatography-mass spectrometry to detect the types and proportions of oligosaccharide fragments, thereby characterizing the structural changes of steamed American ginseng polysaccharides. The identification results can also guide the optimization of the steaming process of American ginseng.
[0010] This invention provides a method for analyzing the oligosaccharide composition of American ginseng polysaccharides, comprising:
[0011] 1) American ginseng was initially extracted using methanol / methyl tert-butyl ether solution, and the supernatant was removed to obtain American ginseng after the initial extraction;
[0012] 2) Add pure water to the American ginseng after the first extraction for a second extraction, take the supernatant, add anhydrous ethanol to the supernatant, let it stand and then centrifuge to obtain the precipitate;
[0013] 3) Dissolve the precipitate in water, add n-butanol / chloroform solution, mix and shake, and dry the aqueous solution to obtain crude polysaccharide;
[0014] 4) Dissolve the crude polysaccharide in water, add trifluoroacetic acid to react, and centrifuge and concentrate to obtain oligosaccharides;
[0015] 5) Dissolve the oligosaccharides in an aqueous acetonitrile solution, take the sample to be tested and detect it by liquid chromatography-mass spectrometry to obtain the types and proportions of oligosaccharides in the sample to be tested.
[0016] In some embodiments, in step 1), the mass-to-volume ratio of American ginseng and the methanol / methyl tert-butyl ether solution 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℃ and the steaming time is 2-12h.
[0018] In some embodiments, in step 1), the American ginseng is American ginseng powder, and methanol / methyl tert-butyl ether solution is added to the American ginseng powder and ultrasonically extracted 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); more 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 ginseng after the initial extraction, and ultrasonic extraction is performed at 80-90°C for 0.5-1.5 hours. The extracted mixture is centrifuged for 10-30 minutes, the supernatant is collected, 2-4 times the amount of anhydrous ethanol is added, and the mixture is allowed to stand for 8-16 hours before centrifugation to obtain a precipitate. Preferably, the ratio of ginseng to pure water is 1 mg:(400-600) μL.
[0021] In some embodiments, in step 2), pure water is added to the ginseng after the initial extraction, and ultrasonic extraction is performed at 80-90°C for 1 hour. The extracted mixture is centrifuged for 20 minutes, the supernatant is collected, 3 times the amount of anhydrous ethanol is added, and the mixture is allowed to stand for 12 hours before centrifugation to obtain the precipitate. The ratio of ginseng to pure water is 1 mg: 500 μL.
[0022] In some embodiments, in step 4), the crude polysaccharide obtained in step 3) is reconstituted with water, centrifuged, and the supernatant is collected. Trifluoroacetic acid is added to the supernatant for reaction, followed by centrifugation and concentration to obtain oligosaccharide solids. The solids are then washed with methanol to obtain oligosaccharides. Preferably, the reaction conditions between the supernatant and trifluoroacetic acid are 75-85°C and ultrasonic-assisted reaction for 0.8-1.5 h. More preferably, the reaction conditions between the supernatant and trifluoroacetic acid are 80°C and ultrasonic-assisted reaction for 1 h.
[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 a Waters ACQUITY UPLCBEH Amide (2.1 × 100 mm, 1.7 μm); mobile phase A is an aqueous solution containing 20 mM ammonium formate, mobile phase B is a 95% acetonitrile / aqueous 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 as follows: 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; total time was 34 min.
[0026] Mass spectrometry conditions: In negative ion mode, the oligosaccharide mass number scan range was 50-1200 m / z, the capillary voltage was 3000V, the dry gas flow rate was 8L / min, the nebulizer pressure was 2.0 bar, the dry gas temperature was 200℃, the collision cell RF voltage amplitude was 750Vpp, the pulse wait time was 5s, the transmission time was 60s, and the collision energy was 5-70eV.
[0027] The beneficial effects of this invention are as follows:
[0028] 1. This invention extracts and degrades polysaccharides from American ginseng, and uses liquid chromatography-mass spectrometry to determine the types and relative contents of oligosaccharides. By purifying the polysaccharides during the extraction process, interfering impurities are removed to improve the accuracy of detection.
[0029] 2. This invention degrades the polysaccharides in American ginseng into smaller oligosaccharide fragments. The types and relative contents of the degraded oligosaccharides are detected by liquid chromatography-mass spectrometry. The structure and content of polysaccharides in American ginseng are indirectly characterized by the types and relative contents of oligosaccharides. This characterization can guide the steaming conditions of American ginseng, such as steaming time and steaming temperature.
[0030] 3. This invention obtains the sample to be tested by steaming, extracting, degrading and purifying polysaccharides in American ginseng, and then performs liquid chromatography-mass spectrometry detection. The column temperature and flow rate of liquid chromatography-mass spectrometry detection are further optimized, and a mobile phase containing ammonium formate is used to improve the response intensity of the color peak and optimize the peak shape. Attached Figure Description
[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0032] Figure 1 shows the extraction ion chromatograms of rhamnose-xylose in the sample to be tested under different mobile phases, where A is the extraction ion chromatogram of rhamnose-xylose in the formic acid mobile phase and B is the extraction ion chromatogram of rhamnose-xylose in the ammonium formate mobile phase.
[0033] Figure 2 shows the extraction ion chromatograms of rhamnose-xylose in the sample to be tested in this invention at different flow rates, where A is the extraction ion chromatogram of rhamnose-xylose at a flow rate of 0.2 mL / min and B is the extraction ion chromatogram of rhamnose-xylose at a flow rate of 0.27 mL / min.
[0034] Figure 3 shows the extraction ion chromatograms of rhamnose-xylose in the sample to be tested in this invention at different column temperatures, where A is the extraction ion chromatogram of rhamnose-xylose at a column temperature of 40℃ and B is the extraction ion chromatogram of rhamnose-xylose at a column temperature of 50℃.
[0035] Figure 4 shows the extraction ion chromatograms of the tetrasaccharide glucose-glucose-xylose-xylose in the sample to be tested under different hydrolysis conditions. Among them, A is the extraction ion chromatogram of ultrasonic-assisted hydrolysis for 30 minutes, B is the extraction ion chromatogram of ultrasonic-assisted hydrolysis for 1 hour, C is the extraction ion chromatogram of ultrasonic-assisted hydrolysis for 2 hours, and D is the extraction ion chromatogram of hydrolysis in a 100°C water bath for 2 hours.
[0036] Figure 5 shows the mass spectrum of the sample to be tested in Example 1 of the present invention.
[0037] Figure 6 is a thermogram showing the analysis of oligosaccharides after degradation of American ginseng polysaccharides at different steaming times according to the present invention.
[0038] Figure 7 is a thermogram showing the analysis of oligosaccharides after degradation of American ginseng polysaccharides at different steaming temperatures according to the present invention. Detailed Implementation
[0039] To address the challenges of identifying and analyzing the complex polysaccharide structure and large molecular weight of American ginseng, this invention proposes an analytical method for the oligosaccharide composition of American ginseng polysaccharides. This method degrades the polysaccharides in American ginseng into smaller molecular weight oligosaccharide fragments. By optimizing the extraction and degradation steps and related parameters of liquid chromatography-mass spectrometry (LC-MS), the types and relative contents of different oligosaccharide fragments are detected, thereby identifying the quality of American ginseng. The identification results can also guide the optimization of the steaming process for American ginseng.
[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0041] All materials used in the following examples are commercially available.
[0042] Example 1
[0043] This embodiment provides a method for analyzing the oligosaccharide composition in American ginseng polysaccharides. The specific steps are as follows:
[0044] Extraction of crude polysaccharides from steamed American ginseng:
[0045] Steamed American ginseng (steaming conditions: 8 hours, 100℃) was dried and ground to 100 mesh. 500 mg of the powder was weighed and added to 5 mL of a methanol / methyl tert-butyl ether mixture (v / v, 1:4). The mixture was ultrasonically extracted for 30 minutes to remove fat-soluble components and small-molecule organic compounds. After removing the supernatant, 10 mL of pure water was added. Extraction was performed at 85℃ using ultrasound-assisted extraction for 1 hour. The mixture was then centrifuged for 20 minutes. After centrifugation, the supernatant was collected, and three volumes of anhydrous ethanol were added. The mixture was allowed to stand at 4℃ for 12 hours. After centrifugation, the precipitate was dissolved in water to form a polysaccharide solution. An equal volume of a n-butanol / chloroform mixture (v / v, 1:4) was added to the polysaccharide solution, and the mixture was shaken thoroughly for 30 minutes. Centrifugation was performed to separate the aqueous and organic phases. This process was repeated several times to remove proteins. The crude polysaccharide was then freeze-dried.
[0046] Oligosaccharide preparation:
[0047] Dissolve 1 mg of crude polysaccharide in 500 μL of water in a 1.5 mL centrifuge tube. Centrifuge at 14000 rpm for 5 min, collect 200 μL of the supernatant, and add 200 μL of 1 mol / L trifluoroacetic acid to the supernatant. Incubate the reaction at 80 °C with ultrasound for 1 h. After the reaction is complete, transfer the mixture to a centrifuge to obtain oligosaccharide solid. Wash the solid solid five times with 400 μL of methanol to obtain the oligosaccharide. Redissolve the oligosaccharide in 70% acetonitrile aqueous solution (v / v, 7:3, the same below) for later use.
[0048] LC-MS analysis:
[0049] American ginseng oligosaccharides were analyzed using a UPLC-Q-TOF-MS system. The chromatographic column was a 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 as follows: 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] Mass spectrometry data were acquired in negative ion mode. The oligosaccharide mass number scan range was 50-1200 m / z. The capillary voltage in negative ion mode was 3000 V, the dry gas flow rate was 8 L / min, the nebulizer pressure was 2.0 bar, the dry gas temperature was 200 °C, the collision cell RF voltage amplitude was 750 Vpp, the pulse wait time was 5 s, the transmission time was 60 s, and the collision energy was set to 5-70 eV.
[0052] Example 2
[0053] This embodiment provides an analytical method for 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: 8 hours, 100℃) was ground to 100 mesh. 500 mg of the powder was weighed and added to 5 mL of a methanol / methyl tert-butyl ether mixture (v / v, 1:4). The mixture was ultrasonically extracted for 20 min to remove fat-soluble components and small-molecule organic compounds. After removing the supernatant, 10 mL of pure water was added. Extraction was performed at 80℃ using ultrasound-assisted extraction for 1.5 h. The mixture was then centrifuged for 10 min. After centrifugation, the supernatant was collected, and two volumes of anhydrous ethanol were added. The mixture was allowed to stand at 4℃ for 8 h. After centrifugation, the precipitate was dissolved in water to form a polysaccharide solution. An equal volume of a n-butanol / chloroform mixture (v / v, 1:4) was added to the polysaccharide solution, and the mixture was shaken thoroughly for 30 min. Centrifugation was performed to separate the aqueous and organic phases. This process was repeated several times to remove proteins. The crude polysaccharide was then freeze-dried.
[0056] Oligosaccharide preparation:
[0057] Dissolve 1 mg of crude polysaccharide in 400 μL of water in a 1.5 mL centrifuge tube. Centrifuge at 14000 rpm for 5 min, collect 200 μL of the supernatant, and add 200 μL of 1 mol / L trifluoroacetic acid to the supernatant. Incubate the reaction at 75 °C with ultrasound for 1.5 h. After the reaction is complete, concentrate the solution in a centrifuge to obtain oligosaccharide solids. Wash the solids five times with 400 μL of methanol to obtain the oligosaccharides. Redissolve the oligosaccharides in 70% acetonitrile aqueous solution for later use.
[0058] Example 3
[0059] This embodiment provides an analytical method for 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: 8 hours, 100℃) was ground to 100 mesh. 500 mg of the powder was weighed and added to 5 mL of a methanol / methyl tert-butyl ether mixture (v / v, 1:4). The mixture was ultrasonically extracted for 40 min to remove fat-soluble components and small-molecule organic compounds. After removing the supernatant, 10 mL of pure water was added. Extraction was performed at 90℃ using ultrasound-assisted extraction for 0.5 h. The mixture was then centrifuged for 30 min. After centrifugation, the supernatant was collected and 4 volumes of anhydrous ethanol were added. The mixture was allowed to stand at 4℃ for 16 h. After centrifugation, the precipitate was dissolved in water to form a polysaccharide solution. An equal volume of n-butanol / chloroform mixture (v / v, 1:4) was added to the polysaccharide solution, and the mixture was shaken thoroughly for 30 min. Centrifugation was performed to separate the aqueous and organic phases. This process was repeated several times to remove proteins. The crude polysaccharide was then freeze-dried.
[0062] Oligosaccharide preparation:
[0063] Dissolve 1 mg of crude polysaccharide in 500 μL of water in a 1.5 mL centrifuge tube. Centrifuge at 14000 rpm for 5 min, collect 200 μL of the supernatant, and add 200 μL of 1 mol / L trifluoroacetic acid to the supernatant. Incubate the reaction at 85 °C with sonication for 0.8 h. After the reaction is complete, concentrate the mixture in a centrifuge to obtain oligosaccharide solids. Wash the solids five times with 400 μL of methanol to obtain the oligosaccharides. Redissolve the oligosaccharides in 70% acetonitrile aqueous solution for later use. Perform three replicates for each sample.
[0064] Example 4
[0065] This embodiment provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Embodiment 1 is that the steaming time of the steamed American ginseng is changed to 2 hours.
[0066] Example 5
[0067] This embodiment provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Embodiment 1 is that the steaming time of the steamed American ginseng is changed to 4 hours.
[0068] Example 6
[0069] This embodiment provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Embodiment 1 is that the steaming time of the steamed American ginseng is changed to 6 hours.
[0070] Example 7
[0071] This embodiment provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Embodiment 1 is that the steaming time of the steamed American ginseng is changed to 10 hours.
[0072] Example 8
[0073] This embodiment provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Embodiment 1 is that the steaming time of the steamed American ginseng is changed to 12 hours.
[0074] Example 9
[0075] This embodiment provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Embodiment 1 is that the steaming conditions for steaming American ginseng are changed to a steaming time of 2 hours and a steaming temperature of 110°C.
[0076] Example 10
[0077] This embodiment provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Embodiment 1 is that the steaming conditions for steaming American ginseng are changed to a steaming time of 2 hours and a steaming temperature of 120°C.
[0078] Example 11
[0079] This embodiment provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Embodiment 1 is that the steaming conditions for steaming American ginseng are changed to a steaming time of 2 hours and a steaming temperature of 130°C.
[0080] Comparative Example 1
[0081] This comparative example provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Example 1 is that the mobile phase A in the liquid chromatography-mass spectrometry detection conditions is a 0.1% formic acid aqueous solution, and the mobile phase B is a 95% acetonitrile aqueous solution (v / v, 95:5).
[0082] As shown in Figure 1, in Comparative Example 1, the formic acid mobile phase resulted in severe peak bifurcation, with multiple peaks appearing for the same substance (Figure 1A). The salt mobile phase used in Example 1, however, improved this phenomenon (Figure 1B).
[0083] Comparative Example 2
[0084] This comparative example provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Example 1 is that the flow rate of the liquid chromatography-mass spectrometry detection conditions is 0.2 mL / min.
[0085] As shown in Figure 2, at a flow rate of 0.2 mL / min in Comparative Example 2, the oligosaccharide peak shape was poor, with peak bifurcation (Figure 2A). In Example 1, at a flow rate of 0.27 mL / min, the peak shape was sharp (Figure 2B).
[0086] Comparative Example 3
[0087] This comparative example provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Example 1 is that the column temperature for liquid chromatography-mass spectrometry detection is 40°C.
[0088] As shown in Figure 3, the peak shape of oligosaccharides was poor at a column temperature of 40°C in Comparative Example 3 (Figure 3A). When the column temperature was increased to 55°C as in Example 1, the peak shape improved (Figure 3B).
[0089] Comparative Example 4
[0090] This comparative example provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Example 1 is that after adding trifluoroacetic acid, the ultrasonic-assisted water bath hydrolysis time is 30 min.
[0091] Comparative Example 5
[0092] This comparative example provides an analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Example 1 is 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 analytical method for the oligosaccharide composition in American ginseng polysaccharides. The difference from Example 1 is that after adding trifluoroacetic acid, the mixture is hydrolyzed in a water bath for 2 hours.
[0095] As shown in Figure 4, taking the tetrasaccharide glucose-glucose-xylose-xylose as an example, in Comparative Example 4, when the ultrasonic-assisted water bath hydrolysis time was 30 min, there was no signal in the extracted ion chromatogram (Figure 4A), indicating that the reaction was incomplete and no oligosaccharide was produced. In Comparative Example 5, when ultrasonic-assisted water bath hydrolysis was performed for 2 h, there was also no signal (Figure 4C), indicating that the reaction time was excessive and the tetrasaccharide was further hydrolyzed. In Example 1, ultrasonic-assisted water bath hydrolysis for 1 h resulted in a strong signal for the tetrasaccharide (Figure 4B). Comparative Example 6, with water bath hydrolysis for 2 h, showed comparable results to ultrasonic-assisted hydrolysis for 1 h (Figure 4D). Therefore, appropriate ultrasonic-assisted hydrolysis can accelerate the reaction time and promote the hydrolysis of polysaccharides.
[0096] The steamed American ginseng in Examples 1-3, through extraction steps and parameter settings of chromatography-mass spectrometry, can identify the types and proportions of the above-mentioned oligosaccharides in the degraded samples of American ginseng polysaccharides. By observing the types and proportions of oligosaccharides in different samples, the differences in the structure and content of polysaccharides in American ginseng can be indirectly characterized.
[0097] Taking Example 1 as an example, 17 oligosaccharides were identified in the hydrolyzed ginseng sample, including 6 disaccharides, 4 trisaccharides, and 7 tetrasaccharides (see Table 1). The oligosaccharides were identified by secondary mass spectrometry. The identification of oligosaccharides was based on characteristic ions and neutral loss. Taking the identification of tetrasaccharides (Figure 5) as an example, using glucose-glucose-xylose-xylose as an example, the molecular formula of the tetrasaccharide is C0. 22 H 38 O 19 In negative mode, the tetrasaccharide has an m / z of 605 ([M +CH3COO] - When the glycosidic bond connecting one glucose molecule in the tetrasaccharide glucose-glucose-xylose-xylose breaks, 162 molecules are neutrally lost, resulting in a mass-to-charge ratio (m / z) of 443. Then, another glucose molecule is lost, resulting in a m / z ... - ). 341 is the mass-to-charge ratio of glucose-to-glucose.
[0098] Table 1. Detection results of oligosaccharides in Example 1
[0099]
[0100] As shown in Examples 1 and 4-8, the inventors analyzed and verified the polysaccharides in steamed American ginseng under different steaming conditions (changing the steaming time). Figure 6 is a heatmap showing the content of different oligosaccharides after the degradation of American ginseng polysaccharides at different steaming times when the steaming temperature is 100℃. 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 8h. With the extension of steaming time, the content of disaccharides increases, while the content of trisaccharides and tetrasaccharides decreases, indicating that the structure of polysaccharides has changed.
[0101] As shown in Examples 1 and 9-11, the inventors analyzed and verified the polysaccharides in steamed American ginseng under different steaming conditions (changing the steaming temperature). Figure 7 is a heatmap showing the content of different oligosaccharides after the 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 polysaccharides has changed.
[0102] The results show that this oligosaccharide analysis method can indirectly characterize the changes in the types and structures of polysaccharides in steamed American ginseng. By comparing the oligosaccharide analysis results of American ginseng and the target American ginseng under different steaming conditions, the best steaming conditions can be selected to guide the optimization of the American ginseng steaming process.
[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for analyzing the oligosaccharide composition of American ginseng polysaccharides, characterized in that, include: 1) American ginseng was initially extracted using methanol / methyl tert-butyl ether solution, and the supernatant was removed to obtain American ginseng after the initial extraction; 2) Add pure water to the American ginseng after the first extraction for a second extraction, take the supernatant, add anhydrous ethanol to the supernatant, let it stand and then centrifuge to obtain the precipitate; 3) Dissolve the precipitate in water, add n-butanol / chloroform solution, mix and shake, and dry the aqueous solution to obtain crude polysaccharide; 4) Dissolve the crude polysaccharide in water, add trifluoroacetic acid to react, and centrifuge and concentrate to obtain oligosaccharides; 5) Dissolve the oligosaccharides in an aqueous acetonitrile solution, take the sample to be tested and detect it by liquid chromatography-mass spectrometry to obtain the types and proportions of oligosaccharides in the sample to be tested.
2. The method for analyzing the oligosaccharide composition of American ginseng polysaccharides as described in claim 1, characterized in that, In step 1), the mass-to-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 polysaccharides as described in claim 1, characterized in that, In step 1), the American ginseng is steamed American ginseng; preferably, the steaming temperature is 100-130℃ and the steaming time is 2-12h.
4. The analytical method for the oligosaccharide composition in American ginseng polysaccharides as described in claim 1, characterized in that, In step 1), the American ginseng is American ginseng powder. A 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); more 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 polysaccharides as described in claim 1, characterized in that, In step 2), pure water is added to the American ginseng after the initial extraction, and ultrasonic extraction is performed at 80-90℃ for 0.5-1.5 hours. The extracted mixture is centrifuged for 10-30 minutes, the supernatant is collected, 2-4 times the amount of anhydrous ethanol is added, and the mixture is allowed to stand for 8-16 hours before centrifugation to obtain the precipitate.
6. The method for analyzing the oligosaccharide composition in American ginseng polysaccharides as described in claim 5, characterized in that, Preferably, the ratio of physalis to pure water is 1 mg:(400-600) μL.
7. The method for analyzing the oligosaccharide composition in American ginseng polysaccharides as described in claim 1, characterized in that, In step 2), pure water is added to the initial extracted American ginseng, and ultrasonic extraction is performed at 80-90℃ for 1 hour. The extracted mixture is centrifuged for 20 minutes, and the supernatant is collected. Three times the amount of anhydrous ethanol is added, and the mixture is allowed to stand for 12 hours before centrifugation to obtain the precipitate. The ratio of American ginseng to pure water is 1 mg: 500 μL.
8. The method for analyzing the oligosaccharide composition in American ginseng polysaccharides as described in claim 1, characterized in that, In step 4), the crude polysaccharide obtained in step 3) is reconstituted with water, centrifuged, and the supernatant is collected. Trifluoroacetic acid is added to the supernatant for reaction, followed by centrifugation and concentration to obtain oligosaccharide solid. The solid is then washed with methanol to obtain oligosaccharide. Preferably, the reaction conditions of the supernatant and trifluoroacetic acid are 75-85℃ and ultrasonic-assisted reaction for 0.8-1.5h. More preferably, the reaction conditions of the supernatant and trifluoroacetic acid are 80℃ and ultrasonic-assisted reaction for 1h.
9. The method for analyzing the oligosaccharide composition of American ginseng polysaccharides as described in 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 of American ginseng polysaccharides as described in claim 1, characterized in that, The liquid chromatography-mass spectrometry (LC-MS) detection conditions were as follows: the chromatographic column was a Waters ACQUITY UPLC BEH Amide (2.1 × 100 mm, 1.7 μm); mobile phase A was an aqueous solution containing 20 mM ammonium formate, mobile phase B was a 95% acetonitrile / aqueous 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; 10-14 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; Total time: 34 min. Mass spectrometry conditions: In negative ion mode, the oligosaccharide mass number scan range was 50-1200 m / z, the capillary voltage was 3000V, the dry gas flow rate was 8L / min, the nebulizer pressure was 2.0 bar, the dry gas temperature was 200℃, the collision cell RF voltage amplitude was 750Vpp, the pulse wait time was 5s, the transmission time was 60s, and the collision energy was 5-70eV.