Mesona chinensis acidic polysaccharide and extraction method thereof

Abstract

The invention relates to the technical field of polysaccharide preparation, in particular to mesona chinensis acidic polysaccharide and an extraction method thereof. The extraction method comprises the following steps: 1) crushing, degreasing and drying dried mesona chinensis, boiling and performing alkali extraction to obtain a mesona chinensis extracting solution; macroporous resin D-900 is adopted for adsorption to remove pigments, and crude mesona chinensis benth polysaccharide is obtained; carrying out primary purification by adopting DEAE-52 ion exchange column chromatography; and performing chromatographic purification through a Sephadex G-100 ion exchange column, collecting components, and performing freeze-drying to obtain the mesona chinensis benth acidic polysaccharide. The extraction method of the mesona chinensis benth acidic polysaccharide has the beneficial effects that the operation is simple, the cost is low, the characteristics of good decoloring effect and high polysaccharide preservation rate are also realized, and the biological activity of the mesona chinensis benth acidic polysaccharide can be guaranteed. Compared with other resins, the D-900 macroporous resin is used for selectively adsorbing melanin, the adsorption capacity of the D-900 macroporous resin to mesona melanin is remarkably higher than that of other resins, the adsorption rate of the D-900 macroporous resin to polysaccharide is lower than 2%, and the D-900 macroporous resin has the advantages of being good in decolorization effect and high in polysaccharide preservation rate.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharide preparation, and in particular to a grass thunbergia acidic polysaccharide and an extraction method thereof. Background Art

[0002] Polysaccharides are sugar chains linked by glycosidic bonds. They are polymeric sugars composed of at least 10 monosaccharides and are high molecular carbohydrates. They can be expressed in the following ways: 10 O5)n represents. Polysaccharides are not pure chemical substances, but rather mixtures of substances at different polymerization levels. They are generally insoluble in water, have no sweetness, and do not form crystals, exhibit no reducing properties, or exhibit mutarotation. As the functions of active polysaccharides continue to be elucidated, polysaccharides are playing an increasingly important role in biological research and the production of health products. Currently, the most common extraction techniques for polysaccharides include hot water extraction and alkaline extraction. These techniques are simple to operate and relatively inexpensive, but they will, to some extent, affect the molecular weight distribution, structural properties, and biological activity of the polysaccharides.

[0003] Mesona chinesis Benth., a plant of the genus Mesona in the Lamiaceae family, is an important medicinal and edible plant resource, known for its summer heat-clearing, antipyretic, and diuretic properties. Mesona chinesis Benth contains significant amounts of melanin and protein, making it difficult to effectively remove these impurities using traditional extraction methods, resulting in insufficient polysaccharide purity. Furthermore, Mesona chinesis polysaccharides have a complex structure and contain multiple uronic acid components, making the active components susceptible to loss during the purification process. The existing art lacks efficient purification methods tailored to the specific properties of Mesona chinesis polysaccharides, particularly those that can simultaneously remove color and retain the active components.

[0004] Chinese invention patent application publication number CN104387488A, entitled "A method for extracting jelly grass polysaccharides through microbial fermentation," discloses a microbial fermentation method for extracting jelly grass polysaccharides, including pulverization, primary extraction, inoculation, fermentation, secondary extraction, separation, concentration, alcohol precipitation, and drying. However, the method described in this patent is complex and requires a long extraction cycle, resulting in uneven composition and low purity of the jelly grass polysaccharides. Chinese invention patent application publication number CN102911282A, entitled "A method for decolorizing and deproteinizing a jelly grass polysaccharide solution," discloses a method for purifying jelly grass polysaccharides through decolorization and deproteinization. However, this method does not further separate the polysaccharides into their components, resulting in low purity and yield, making it unsuitable for large-scale extraction and production. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a herba syringae acidic polysaccharide with good decolorization effect and high polysaccharide preservation rate and an extraction method thereof.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: to provide a method for extracting acidic polysaccharides from angelica sinensis, comprising the following steps: 1) Crush the dried grass jelly, defatted, dried, boiled and alkali extracted to obtain the grass jelly extract 2) precipitating the Herba Jelly Extract with alcohol, centrifuging, discarding the supernatant, re-dissolving the precipitate with distilled water, removing protein by sevage method, centrifuging, discarding the precipitate, repeating the process until no more precipitate remains after centrifugation, removing pigment by adsorption using macroporous resin D-900, concentrating under reduced pressure, and freeze-drying to obtain Herba Jelly Extract crude polysaccharide; 3) dissolving the crude polysaccharide of Herba Angelicae Dahuricae in distilled water, centrifuging and filtering, and then initially purifying it by DEAE-52 ion exchange column chromatography. Gradient elution was performed sequentially with salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl; the sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 minutes; the elution peak liquid of the 0.5 mol / L NaCl solution was collected to obtain the eluate; The eluate is dialyzed, concentrated, and freeze-dried to obtain preliminarily purified herba schoenopsis acidic polysaccharide; 4) purifying the preliminarily purified herba schoenopsis acidic polysaccharide by Sephadex G-100 ion exchange column chromatography, collecting the fractions, concentrating, and freeze-drying to obtain herba schoenopsis acidic polysaccharide; The herb acidic polysaccharide is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid and guluronic acid in a molar ratio of 10.85:1.72:2.98:18.22:24.70:19.13:20.02:1.94:2.15:2.38; The molecular weight of the herba scutellariae acidic polysaccharide is 7587 Da.

[0007] Another technical solution provided by the present invention is to provide a herba schoenopsis acidic polysaccharide prepared by a method for extracting herba schoenopsis acidic polysaccharide.

[0008] The beneficial effects of the present invention are: 1) This invention provides a method for extracting acidic polysaccharides from Herba Cibotii. This method exhibits excellent decolorization, high polysaccharide preservation, and guaranteed biological activity. Compared to other resins, the present invention utilizes D-900 macroporous resin for selective adsorption of melanin. Its adsorption capacity for Herba Cibotii melanin (158 mg / g) is significantly higher than other resins, while its adsorption rate for polysaccharides is less than 2%. This method offers the advantages of excellent decolorization and high polysaccharide preservation.

[0009] 2) The DEAE-52 ion exchange method of the present invention uses a combination of a NaCl elution gradient and Sephadex G100 to reduce the molecular weight distribution coefficient of the herba syringae acidic polysaccharide from >2.0 in conventional processes to below 1.38, thereby obtaining herba syringae acidic polysaccharides with high purity (total sugar content of more than 71%) and more uniform molecular weight (average molecular weight of more than 7587 Da). BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A diagram of a chromatography column device according to a specific embodiment of the present invention; Figure 2 Glucose standard curve of the herba scutellariae acidic polysaccharide according to a specific embodiment of the present invention; Figure 3 DEAE-52 cellulose gradient elution curve of the herbaceous acid polysaccharide according to a specific embodiment of the present invention; Figure 4 Sephadex elution curve of the herba scutellariae acidic polysaccharide according to a specific embodiment of the present invention; Figure 5 Chromatogram of monosaccharide composition determination of the herbaceous celery acid polysaccharide according to a specific embodiment of the present invention; Figure 6 A chromatogram showing the molecular weight determination of the herbaceous celery acid polysaccharide according to a specific embodiment of the present invention; Figure 7 GC / MS reconstructed total ion current spectrum of the methylated fragments of the herbaceous acid polysaccharide according to a specific embodiment of the present invention; Figure 8 Infrared spectrum of the herba sylvestris acidic polysaccharide according to a specific embodiment of the present invention. DETAILED DESCRIPTION To illustrate the technical content, achieved objectives and effects of the present invention in detail, the following description is given in conjunction with the embodiments and accompanying drawings.

[0011] The present invention provides a method for extracting acidic polysaccharides from angelica sinensis, comprising the following steps: 1) Crush the dried grass jelly, defatted, dried, boiled and alkali extracted to obtain the grass jelly extract 2) precipitating the Herba Jelly Extract with alcohol, centrifuging, discarding the supernatant, re-dissolving the precipitate with distilled water, removing protein by sevage method, centrifuging, discarding the precipitate, repeating the process until no more precipitate remains after centrifugation, removing pigment by adsorption using macroporous resin D-900, concentrating under reduced pressure, and freeze-drying to obtain Herba Jelly Extract crude polysaccharide; 3) dissolving the crude polysaccharide of Herba Angelicae Dahuricae in distilled water, centrifuging and filtering, and then initially purifying it by DEAE-52 ion exchange column chromatography. Gradient elution was performed sequentially with salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl; the sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 minutes; the elution peak liquid of the 0.5 mol / L NaCl solution was collected to obtain the eluate; The eluate is dialyzed, concentrated, and freeze-dried to obtain preliminarily purified herba schoenopsis acidic polysaccharide; 4) purifying the preliminarily purified herba schoenopsis acidic polysaccharide by Sephadex G-100 ion exchange column chromatography, eluting, and freeze-drying to obtain herba schoenopsis acidic polysaccharide; The herb acidic polysaccharide is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid and guluronic acid in a molar ratio of 10.85:1.72:2.98:18.22:24.70:19.13:20.02:1.94:2.15:2.38; The molecular weight of the herba scutellariae acidic polysaccharide is 7587 Da.

[0012] Furthermore, in the above-mentioned method for extracting the acidic polysaccharide of Herba Cibotii, the step 1) is specifically as follows: The dried grass jelly was crushed, defatted, and dried, and then subjected to alkaline extraction at a solid-liquid ratio of 1:30-1:40 g / mL, a temperature of 95-100°C, and an extraction time of 2-3 h to obtain the grass jelly extract.

[0013] Furthermore, in the above-mentioned method for extracting the acidic polysaccharide from Herba Cibotii, the degreasing in step 1) is performed by reflux degreasing with petroleum ether 2-3 times.

[0014] Furthermore, in the above-mentioned method for extracting the acidic polysaccharide of Herba Aspergillus, the alcohol used in the alcohol precipitation in step 2) is anhydrous ethanol, and the added weight of the anhydrous ethanol is 4 times that of the Herba Aspergillus extract.

[0015] Furthermore, in the above-mentioned method for extracting the acidic polysaccharide of Herba Cibotii, the polysaccharide groups are labeled with carboxylated quantum dots during the elution process with NaCl solution in step 3), and the elution process is monitored in real time by combining fluorescence resonance energy transfer technology; The carboxylation quantum dots labeling polysaccharide groups specifically comprises: performing a coupling reaction between the carboxylation quantum dots and the polysaccharide groups under EDC / NHS catalysis.

[0016] As can be seen from the above description, this invention innovatively applies quantum dot-FRET technology to polysaccharide purification. By labeling polysaccharide groups with CdSe / ZnS quantum dots, it enables real-time monitoring and precise control of the polysaccharide chromatography purification process. This technology is compatible with existing chromatography systems and requires only the addition of a fluorescence detection module.

[0017] Furthermore, in the above-mentioned method for extracting the acidic polysaccharide of Herba Cibotii, the step 3) is specifically as follows: 10 g of the crude polysaccharide of Herba Immortalis was dissolved in distilled water to a concentration of 1.5 mg / mL, centrifuged at 4000 rpm for 10 min, and the supernatant was taken and passed through a 0.45 μm microporous membrane; preliminary purification was performed by DEAE-52 ion exchange column chromatography, and gradient elution was performed sequentially with salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl; the sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 min; the elution peak liquid of the 0.5 mol / L NaCl solution was collected to obtain the eluate; The eluate was placed in a dialysis bag with a molecular weight of 8000 Da and dialyzed in distilled water for 36 hours, with the water being changed every 3 hours. The eluate was concentrated and freeze-dried to obtain preliminarily purified herba schizonepetae acidic polysaccharide.

[0018] Furthermore, in the above-mentioned method for extracting the acidic polysaccharide of Herba Cibotii, the step 4) is specifically as follows: The purified celery acid polysaccharide was further purified by Sephadex G100 glucose gel chromatography. The preliminarily purified celery acid polysaccharide was dissolved in distilled water. The mobile phase was ultrapure water at a flow rate of 0.3 mL / min. The fractions (tubes 30 to 50) were collected and concentrated and freeze-dried to obtain celery acid polysaccharide.

[0019] The present invention also provides a herba sylvestris acidic polysaccharide prepared by the herba sylvestris acidic polysaccharide extraction method.

[0020] Example 1 A method for extracting grass thunbergia acidic polysaccharide comprises the following steps: 1) Crush 1 kg of dried grass jelly and defatted three times with petroleum ether reflux. Dry the defatted grass jelly and extract it in a water bath with 40 L of 100°C alkali solution for 2 hours to obtain the grass jelly extract. 2) The Herba Jellyfish extract was centrifuged with 4 volumes of anhydrous ethanol, the supernatant was discarded, and the precipitate was redissolved in distilled water. Protein was removed by sevage method, centrifuged, and the precipitate was discarded three times until no more precipitate was present. The pigment was removed by adsorption using macroporous resin D-900, and the extract was concentrated under reduced pressure and freeze-dried to obtain 210 g of Herba Jellyfish crude polysaccharide.

[0021] 3) The crude polysaccharide of Herba Cibotii was initially purified by DEAE-52 ion exchange column chromatography (separation pattern as shown in FIG. Figure 3 As shown, the second single elution peak sample collected is the preliminarily purified herba schoenopsis acidic polysaccharide). 10 g of the herba schoenopsis crude polysaccharide was dissolved in distilled water to a concentration of 1.5 mg / mL. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and passed through a 0.45 μm microporous membrane. Gradient elution was performed using salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl in sequence; the sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 minutes; Collect the elution peak liquid of 0.5 mol / L NaCl solution; collect the eluate; The NaCl solution elution process uses carboxylated quantum dots to label polysaccharide groups, and fluorescence resonance energy transfer technology is used to monitor the elution process in real time. The eluate is tracked and detected at 490 nm, and an elution curve is drawn to achieve real-time monitoring and precise control of the polysaccharide chromatography purification process.

[0022] 10 g of the crude polysaccharide of Herba Immortalis was dissolved in distilled water to a concentration of 1.5 mg / mL, centrifuged at 4000 rpm for 10 min, and the supernatant was taken and passed through a 0.45 μm microporous membrane; preliminary purification was performed by DEAE-52 ion exchange column chromatography, and gradient elution was performed sequentially with salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl; the sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 min; the elution peak liquid of the 0.5 mol / L NaCl solution was collected to obtain the eluate; The eluate was placed in a dialysis bag with a molecular weight of 8000 Da and dialyzed in distilled water for 36 hours, with the water being changed every 3 hours. The eluate was concentrated and freeze-dried to obtain preliminarily purified herba schizonepetae acidic polysaccharide.

[0023] The carboxylation quantum dots labeling polysaccharide groups specifically include: performing a coupling reaction between the carboxylation quantum dots and the polysaccharide groups under EDC / NHS catalysis.

[0024] 1. Quantum dot activation: Take 1 mL of carboxylated CdSe / ZnS quantum dots (10 mg / mL); add EDC (1 mg) and NHS (0.3 mg); shake at room temperature in the dark for 30 min 2. Polysaccharide treatment: Dissolve 20 mg of Herba Cibotii polysaccharides in 2 mL of pH 7.4 PBS buffer. 3. Directed coupling: Mix the activated quantum dots with the polysaccharide solution (volume ratio 1:2) and react at 4°C with shaking for 12 hours; See also Figure 1 As shown, this invention innovatively applies quantum dot-FRET technology to polysaccharide purification. By labeling polysaccharide groups with CdSe / ZnS quantum dots, it enables real-time monitoring and precise control of the polysaccharide chromatography purification process. This technology is compatible with existing chromatography systems and only requires the addition of a fluorescence detection module.

[0025] The eluate was placed in a dialysis bag with a molecular weight of 8000 Da and dialyzed in distilled water for 36 h, with the water changed every 3 h. The eluate was concentrated and freeze-dried.

[0026] 4) Further purification using Sephadex G100 glucose gel chromatography column (separation pattern as shown in Figure 4 The polysaccharide obtained from the preliminary purification was dissolved in distilled water. The mobile phase was ultrapure water at a flow rate of 0.3 mL / min. Fractions were collected (if there were impurities, collect tubes 30-50). After concentration and freeze-drying, 0.5 g of scutellaria baicalensis acidic polysaccharide was obtained. The yield was 5%.

[0027] Example 2 A method for extracting grass thunbergia acidic polysaccharide comprises the following steps: 1) Crush 1 kg of dried grass jelly and defatted twice with petroleum ether reflux. Dry the defatted grass jelly and extract it in a water bath with 30 L of 95°C alkaline solution for 3 h.

[0028] 2) The Herba Jellyfish extract was centrifuged with 4 volumes of anhydrous ethanol, the supernatant was discarded, and the precipitate was redissolved in distilled water. Protein was removed by sevage method, centrifuged, and the precipitate was discarded three times until no more precipitate was present. The pigment was removed by adsorption using macroporous resin D-900, and the extract was concentrated under reduced pressure and freeze-dried to obtain 190 g of Herba Jellyfish crude polysaccharide.

[0029] 3) Preliminary purification of the Herba Angelicae Sinensis crude polysaccharide by DEAE-52 ion exchange column chromatography: 10 g of the Herba Angelicae Sinensis crude polysaccharide was dissolved in distilled water to a concentration of 1.5 mg / mL, centrifuged at 4000 rpm for 10 min, and the supernatant was collected and passed through a 0.45 μm microporous membrane. Gradient elution was performed using salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl, in sequence; the sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 minutes; Collect the elution peak liquid of 0.5 mol / L NaCl solution; collect the eluate; The NaCl solution elution process uses carboxylated quantum dots to label polysaccharide groups, and fluorescence resonance energy transfer technology is used to monitor the elution process in real time. The eluate is tracked and detected at 490 nm, and an elution curve is drawn to achieve real-time monitoring and precise control of the polysaccharide chromatography purification process.

[0030] 10 g of the crude polysaccharide of Herba Immortalis was dissolved in distilled water to a concentration of 1.5 mg / mL, centrifuged at 4000 rpm for 10 min, and the supernatant was taken and passed through a 0.45 μm microporous membrane; preliminary purification was performed by DEAE-52 ion exchange column chromatography, and gradient elution was performed sequentially with salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl; the sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 min; the elution peak liquid of the 0.5 mol / L NaCl solution was collected to obtain the eluate; The eluate was placed in a dialysis bag with a molecular weight of 8000 Da and dialyzed in distilled water for 24 h, with the water changed every 3 h. The eluate was concentrated and freeze-dried.

[0031] 4) Further purification was performed using a Sephadex G100 glucose gel chromatography column. The polysaccharide obtained from the preliminary purification was dissolved in distilled water with ultrapure water as the mobile phase at a flow rate of 0.3 mL / min. Fractions (tubes 30–50) were collected, concentrated, and freeze-dried to obtain 0.4 g of scutellaria baicalensis acidic polysaccharide. The yield was 4%.

[0032] Example 3 A method for extracting grass thunbergia acidic polysaccharide comprises the following steps: 1) Crush 1 kg of dried grass jelly and defatted three times with petroleum ether reflux. Dry the defatted grass jelly and extract it in a water bath with 35 L of 95°C alkaline solution for 2.5 h.

[0033] 2) The Herba Jellyfish extract was centrifuged with 4 volumes of anhydrous ethanol, the supernatant was discarded, and the precipitate was redissolved in distilled water. Protein was removed by sevage method, centrifuged, and the precipitate was discarded. This was repeated four times until no more precipitate was present. Pigment was removed by adsorption using macroporous resin D-900, and the product was concentrated under reduced pressure and freeze-dried to obtain 200 g of crude Herba Jellyfish polysaccharide.

[0034] 3) Preliminary purification of the Herba Angelicae Sinensis crude polysaccharide by DEAE-52 ion exchange column chromatography: 10 g of the Herba Angelicae Sinensis crude polysaccharide was dissolved in distilled water to a concentration of 1.5 mg / mL, centrifuged at 5000 rpm for 10 min, and the supernatant was collected and passed through a 0.45 μm microporous membrane. Gradient elution was performed using salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl, in sequence; the sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 minutes; Collect the elution peak liquid of 0.5 mol / L NaCl solution; collect the eluate; The NaCl solution elution process is carried out by labeling the polysaccharide groups with carboxylated quantum dots, and the elution process is monitored in real time by combining fluorescence resonance energy transfer technology. The eluate is tracked and detected at 490 nm, and the elution curve is drawn (e.g. Figure 3 ), to achieve real-time monitoring and precise control of the polysaccharide chromatography purification process.

[0035] 10 g of the crude polysaccharide of Herba Immortalis was dissolved in distilled water to a concentration of 1.5 mg / mL, centrifuged at 4000 rpm for 10 min, and the supernatant was taken and passed through a 0.45 μm microporous membrane; preliminary purification was performed by DEAE-52 ion exchange column chromatography, and gradient elution was performed sequentially with salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl; the sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 min; the elution peak liquid of the 0.5 mol / L NaCl solution was collected to obtain the eluate; The eluate was placed in a dialysis bag with a molecular weight of 8000 Da and dialyzed in distilled water for 30 h, with the water changed every 3 h. The eluate was concentrated and freeze-dried.

[0036] 4) Further purification was performed using Sephadex G100 glucose gel chromatography. The polysaccharide obtained from the preliminary purification was dissolved in distilled water with ultrapure water as the mobile phase at a flow rate of 0.3 mL / min. Fractions (tubes 30–50) were collected, concentrated, and freeze-dried to obtain 0.45 g of scutellaria baicalensis acidic polysaccharide. The yield was 4.5%.

[0037] Comparative Example 1 Single-factor authentication using quantum dot labeling technology (1) Experimental group setup (using the extraction method of Example 3 of the present invention, the experimental group and the control group differ only in monitoring the elution process): Experimental group: carboxylated quantum dots (CdSe / ZnS) labeling + FRET monitoring Control group 1: Traditional UV monitoring (280nm) Control group 2: phenol-sulfuric acid method offline detection Control group 3: unlabeled quantum dots, FRET monitoring only (2) Detection indicators: elution peak recognition accuracy (%), polysaccharide recovery rate (%), target component purity (%) and operation time (h) (3) The results are shown in Table 1. Comparison of the effects of different monitoring technologies; Table 1 (4) Conclusion: Quantum dot labeling combined with FRET technology significantly improved the monitoring accuracy of the elution process (p<0.01), increased the polysaccharide recovery rate by 8.5-13.2%, and the purity by 6.6-10.1%, while shortening the operation time.

[0038] Comparative Example 2 Single factor verification of the decolorization effect of D-900 resin (1) Experimental design: Using the extraction method of Example 3 of the present invention, each experimental group and control group only changed the type of decolorizing resin; Experimental group: D-900 resin Control group 1: AB-8 resin (commonly used polysaccharide decolorization resin) Control group 2: activated carbon decolorization Control group 3: HPD-100 resin (2) Testing indicators: Decolorization rate (420nm absorbance), polysaccharide retention rate, uronic acid retention rate and appearance score of the decolorized solution (1-10 points) (3) The results are shown in Table 2, which compares the effects of different decolorization materials.

[0039] Table 2 (4) Conclusion: While maintaining a high decolorization rate (>93%), D-900 resin has significantly higher polysaccharide and uronic acid retention rates than other materials (p<0.01). In particular, the uronic acid retention rate is 6.2% higher than that of the commonly used AB-8 resin, solving the technical problem of easy loss of uronic acid during the purification of acidic polysaccharides.

[0040] Comparative Example 3 Purification process combination optimization and verification (1) Experimental design: Using the extraction method of Example 3 of the present invention, each experimental group and control group only changed the following purification combination parts; Experimental group: DEAE-52 (0.5M NaCl) + Sephadex G100 (Example 3) Control group 1: DEAE-52 purification only Control group 2: DEAE-52 (0.3M NaCl) + Sephacryl S-200 Control group 3: DEAE-52 (0.7M NaCl) + Sephadex G100 Control 4: Direct Sephadex G100 purification (2) Testing indicators: Polysaccharide yield, molecular weight distribution coefficient (Mw / Mn), uronic acid content and protein residue (3) The results are shown in Table 3. Comparison of the effects of different purification combinations; Table 3 (4) Conclusion: The combination of DEAE-52 (0.5M NaCl) and Sephadex G100 with specific parameters resulted in the narrowest polysaccharide molecular weight distribution (Mw / Mn=1.38), the highest uronic acid retention rate, and the best protein removal effect, demonstrating the specific advantage of this combination in the purification of herba schizonepetae acidic polysaccharides.

[0041] The following structural identification of the herb acidic polysaccharide was performed using the herb acidic polysaccharide obtained in Example 3 as an example.

[0042] Test Example 1 Determination of polysaccharide content in Herba Cibotii acidic polysaccharides Determination method: phenol-sulfuric acid method (1) Preparation of 6% phenol solution: Accurately weigh 15 g of phenol solid, add 250 mL of distilled water and fully dissolve in a 60°C water bath. Store in a brown ground-mouth bottle away from light until ready for use.

[0043] (2) Preparation of 0.1 mg / mL solution of scutellaria baicalensis acidic polysaccharide: Accurately weigh 10 mg of scutellaria baicalensis acidic polysaccharide and add distilled water to a 100 mL volumetric flask to prepare a 0.1 mg / mL solution of scutellaria baicalensis acidic polysaccharide for testing.

[0044] (3) Preparation of 0.1 mg / mL glucose standard solution: Accurately weigh 10 mg of glucose standard and add distilled water to a 100 mL volumetric flask to make a 0.1 mg / mL glucose standard solution for later use.

[0045] (4) Drawing of glucose standard curve: Use a pipette to measure 200 μL, 400 μL, 600 μL, 800 μL, and 1000 μL of glucose standard solution into 10 mL glass test tubes, add 1 mL of distilled water, and repeat 3 times for each concentration. Next, add 1 mL of 6% phenol solution to each test tube in an ice water bath, and then quickly add 5 mL of concentrated sulfuric acid. After mixing, place in a 100℃ water bath for color development for 30 minutes, cool naturally until the color change stabilizes, and then measure its absorbance at a wavelength of 490 nm. With the mass of the glucose standard as the horizontal axis and the absorbance A as the vertical axis, draw a standard curve ( Figure 2 ).

[0046] (5) Determination of polysaccharide content: Take 1 mL of 0.1 mg / mL scutellaria acidic polysaccharide solution, add 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid solution, and measure the absorbance at a wavelength of 490 nm. Calculate the content of scutellaria acidic polysaccharide based on the standard curve.

[0047] (6) The results are shown in Table 4. The polysaccharide content of the herba schizonepetae acidic polysaccharide prepared in Example 3 was determined by the phenol-sulfuric acid method. It can be seen that the polysaccharide content prepared in this example is 71.10%.

[0048] Table 4 Test Example 2 Identification of Monosaccharide Composition of Herba Cibotii Acidic Polysaccharides Identification method: Ion chromatography Abe method (1) Preparation of standard substances: Accurately weigh 13 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, guluronic acid), add water to prepare 10 mg / mL standard solution stock solution, then take appropriate amount of standard stock solution stock solution to prepare the highest index concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL standard solution mixture, and prepare the series of standards required for the instrument according to the required concentration gradient.

[0049] (2) Sample Preparation: Take a clean chromatographic vial, weigh an appropriate amount of the herbaceous sage polysaccharide obtained in Example 3, add 1 mL of 2M TFA solution, and heat at 121°C for 2 hours. Blow dry with nitrogen. Rinse with 99.99% methanol, blow dry again, and repeat the methanol wash 2-3 times. Dissolve in sterile water and transfer to a chromatographic vial for analysis.

[0050] (3) Chromatographic conditions: The chromatographic system used was the Thermo ICS 5000+ ion chromatography system (ICS 5000+, ThermoFisher Scientific, USA), and the monosaccharide components were analyzed and detected using an electrochemical detector.

[0051] (4) Results: After comparing the retention times with the monosaccharide standards ( Figure 5 ), it can be determined that the monosaccharide composition it contains is fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid and guluronic acid, with the molar ratios of 10.85:1.72:2.98:18.22:24.70:19.13:20.02:1.94:2.15:2.38 respectively.

[0052] Test Example 3 Determination of molecular weight of celery acid polysaccharide Determination method: Gel permeation chromatography-multi-angle laser light scattering (1) Sample pretreatment: The herba scutellariae acidic polysaccharide obtained in Example 3, i.e., the polysaccharide sample, was dissolved in a 0.1 M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL. The solution was filtered through a 0.45 μm filter membrane and then tested on an instrument.

[0053] (2) Instrumental parameters: The chromatographic system used was a gel chromatography-differential refractive index multi-angle laser light scattering system, the liquid chromatography system was a U3000 (Thermo, USA), the differential refractive index detector was an Optilab T-rEX (Wyatt Technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS II (Wyatt Technology, CA, USA). The gel exclusion chromatography columns used were Ohpak SB-805 HQ (300 × 8 mm) and Ohpak SB-803 HQ (300 × 8 mm) connected in series. The column temperature was 45°C, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1 M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0054] (3) Analysis: The molecular weight of the acidic polysaccharide of Herba Cibotii was determined in the chromatogram ( Figure 6), blue represents the differential signal, while red represents the light scattering signal. A red signal first appears in the chromatogram, and at the same time, the corresponding blue signal is zero. This indicates the presence of a higher molecular weight substance in the sample, but its actual content is almost zero. The portion with both red and blue signal peaks (the green box in the figure) is considered the main portion of the molecular weight determination. Behind the main peak, a weak blue signal is observed, while the red signal value is zero. This indicates that a small amount of low-molecular-weight substance may be present in the sample. This indicates that a small amount of low-molecular-weight substance, such as oligosaccharides, oligopeptides, or some impurities, may remain in the sample. Due to the small molecular weight and low content of these substances, they have little impact on the overall composition of the sample.

[0055] (4) Results: As shown in Table 5, the molecular weight of the polysaccharide prepared in this example is 7587 Da, and the polydispersity is similar and close to 1. This indicates that the homogeneity of the acidic polysaccharide of Herba Cibotii is good and the purity of the polysaccharide sample is high.

[0056] Table 5 shows the molecular weight of the herba schizonepetae acidic polysaccharide prepared in Example 3 determined by gel permeation chromatography-multi-angle laser light scattering method. Table 5 Experimental Example 4: Methylation Analysis of Herba Cibotii Acidic Polysaccharides Dissolve a small amount of polysaccharide sample (2-3 mg) in 500 μL of DMSO. Add 1 mg of NaOH and incubate for 30 min. Add 50 μL of iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times. Aspirate the lower dichloromethane phase and blow dry with nitrogen.

[0057] Add 100 μL 2 mol / L TFA and react at 121°C for 90 min. Evaporate to dryness at 30°C. Add 50 μL 2 mol / L ammonia water and 50 μL 1 mol / L NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μL acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL methanol, and blow dry with nitrogen. Add 250 μL acetic anhydride, vortex mix well, and react at 100°C for 2.5 h. Add 1 mL of water and let stand for 10 min. Add 500 μL dichloromethane, vortex mix well, centrifuge, discard the aqueous phase, and repeat the water washing 3 times. Remove the lower dichloromethane phase and detect it on a GC-MS. The sample solution was injected and detected according to the above-mentioned chromatographic and mass spectrometric conditions to obtain characteristic fragments after polysaccharide methylation. The GC / MS reconstructed total ion current spectrum of the methylated fragments is shown in Figure 7 The bonding mode was confirmed by comparison with the existing database, and the results are shown in Table 6: Acidic polysaccharide derivatives of fennel and their bonding modes.

[0058] Table 6 Test Example 5 Infrared Spectrum Analysis of Herba Cibotii Acidic Polysaccharides Weigh 2-5 mg of polysaccharide sample and place it in an agate mortar. Mix it with KBr at a ratio of 1:100. Grind it into fine powder with the mortar under infrared light. Then scoop an appropriate amount of mixed powder into a tableting mold. Use a tablet press to prepare uniform and transparent thin slices. -1 -400 cm -1 Scan the sample slice within the range, set the number of scans: 64, resolution: 4 cm -1 .

[0059] Result analysis: In the infrared spectrum of celery acid polysaccharide ( Figure 8 ), the acidic polysaccharide of Herba Cibotii at 1617 cm -1 It shows a strong absorption at 1455 cm, which is the stretching vibration peak of carboxyl group (COO-), indicating that it contains uronic acid. -1 The absorption peak is the bending vibration signal peak in the CH plane. -1 There is a weak vibration peak on the left and right, which is caused by CO stretching vibration. Therefore, COH and COC structures exist in the acidic polysaccharide of angelica dahurica, which are attributed to the pyranose ring. -1 and 838 cm -1 The signal peak at indicates that the herba scutellariae acidic polysaccharide contains both α-glycosidic bonds and β-glycosidic bonds.

[0060] In summary, the grass jelly acidic polysaccharide obtained in this invention can be used to make foods such as jelly and grass jelly. Grass jelly acidic polysaccharide is a key ingredient in these foods. The acidic polysaccharide in grass jelly dissolves readily in alkaline solutions. Adding a small amount of edible alkali during the production process helps release the gelatin. Upon cooling, it solidifies into a jelly-like form, creating a delicious summer-relief food. Compared to intact grass jelly polysaccharide, the isolated and purified grass jelly acidic polysaccharide exhibits superior or more specific biological activities and effects in its applications.

[0061] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's description and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for extracting acidic polysaccharides from Herba Cibotii, characterized in that: The following steps are involved: 1) Crush the dried grass jelly, defatted, dried, boiled and alkali extracted to obtain the grass jelly extract 2) precipitating the Herba Jelly Extract with alcohol, centrifuging, discarding the supernatant, re-dissolving the precipitate with distilled water, removing protein by sevage method, centrifuging, discarding the precipitate, repeating the process until no more precipitate remains after centrifugation, removing pigment by adsorption using macroporous resin D-900, concentrating under reduced pressure, and freeze-drying to obtain Herba Jelly Extract crude polysaccharide; 3) The crude polysaccharide of Herba Angelicae Dahuricae was dissolved in distilled water, centrifuged, and initially purified by DEAE-52 ion exchange column chromatography. Gradient elution was performed using salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl, in sequence. The sample volume was 10 mL, the elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 minutes. Collect the elution peak liquid of 0.5 mol / L NaCl solution to obtain the eluate; The eluate is dialyzed, concentrated, and freeze-dried to obtain preliminarily purified herba schoenopsis acidic polysaccharide; 4) purifying the preliminarily purified herba schoenopsis acidic polysaccharide by Sephadex G-100 ion exchange column chromatography, collecting fractions, and freeze-drying to obtain herba schoenopsis acidic polysaccharide; The herba syringae acidic polysaccharide is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid and guluronic acid in a molar ratio of 10.85:1.72:2.98:18.22:24.70:19.13:20.02:1.94:2.15:2.38; the herba syringae acidic polysaccharide has a molecular weight of 7587 Da.

2. The method for extracting the acidic polysaccharide from Herba Cibotii according to claim 1, wherein: The step 1) is specifically as follows: The dried grass jelly was crushed, defatted, and dried, and then subjected to alkaline extraction at a solid-liquid ratio of 1:30-1:40 g / mL, a temperature of 95-100°C, and an extraction time of 2-3 h to obtain the grass jelly extract.

3. The method for extracting the acidic polysaccharide from Herba Cibotii according to claim 1, wherein: The degreasing in step 1) is as follows: degreasing by refluxing petroleum ether 2-3 times.

4. The method for extracting the acidic polysaccharide from Herba Cibotii according to claim 1, wherein: In the alcohol precipitation in step 2), the alcohol used is anhydrous ethanol, and the added weight of the anhydrous ethanol is 4 times that of the herbal jelly extract.

5. The method for extracting the acidic polysaccharide from Herba Cibotii according to claim 1, wherein: In the step 3), the polysaccharide groups are labeled with carboxylated quantum dots during the NaCl solution elution process, and the elution process is monitored in real time using fluorescence resonance energy transfer technology; The carboxylation quantum dots labeling polysaccharide groups specifically comprises: performing a coupling reaction between the carboxylation quantum dots and the polysaccharide groups under EDC / NHS catalysis.

6. The method for extracting the acidic polysaccharide of Herba Cibotii according to claim 1, wherein: The step 3) is specifically as follows: 10 g of the crude polysaccharide of Herba Immortalis was dissolved in distilled water to a concentration of 1.5 mg / mL, centrifuged at 4000 rpm for 10 min, and the supernatant was taken and passed through a 0.45 μm microporous membrane; preliminary purification was performed by DEAE-52 ion exchange column chromatography, and gradient elution was performed sequentially with salt solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl; the sample volume was 10 mL, elution was 2 times the column volume, the flow rate was set at 1 mL / min, and one tube was collected every 10 min; the elution peak liquid of the 0.5 mol / L NaCl solution was collected to obtain the eluate; The eluate was placed in a dialysis bag with a molecular weight of 8000 Da and dialyzed in distilled water for 36 hours, with the water being changed every 3 hours. The eluate was concentrated and freeze-dried to obtain preliminarily purified herba schizonepetae acidic polysaccharide.

7. The method for extracting the acidic polysaccharide from Herba Cibotii according to claim 1, wherein: The step 4) is specifically as follows: The product was further purified by Sephadex G100 glucose gel chromatography. The preliminarily purified herba schoenopsis acidic polysaccharide was dissolved in distilled water. The mobile phase was ultrapure water at a flow rate of 0.3 mL / min. The fractions were collected and concentrated and freeze-dried to obtain herba schoenopsis acidic polysaccharide.

8. The herbaceous celery acidic polysaccharide prepared according to the method for extracting herbaceous celery acidic polysaccharide according to any one of claims 1 to 7.

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