Mesona chinensis neutral polysaccharide as well as extraction method and application thereof

Abstract

The invention relates to the technical field of polysaccharide preparation, in particular to mesona chinensis neutral polysaccharide and an extraction method and application thereof. The extraction method comprises the following steps: 1) preparing fresh mesona chinensis into mesona chinensis crude polysaccharide; (2) decoloring the crude mesona chinensis benth polysaccharide through macroporous resin D-900; (3) performing chromatographic purification by adopting a DEAE-52 ion exchange column; and 4) carrying out chromatographic purification, elution and freeze-drying by using a Sephadex G-100 ion exchange column to obtain the purified mesona chinensis neutral polysaccharide. The method disclosed by the invention has the beneficial effects that the decolorization rate of the D-900 macroporous resin under the same concentration can reach 88.37%, and the polysaccharide preservation rate reaches up to 84.82%. The obtained mesona chinensis neutral polysaccharide and modified cassava starch composite gel has unique gel characteristics and wide application prospects.

Description

Technical Field

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

[0002] my country boasts abundant resources and high yields of herbaceous jelly. However, current domestic utilization and development of herbaceous jelly primarily focuses on the whole plant. Research on the polysaccharides present in herbaceous jelly primarily focuses on optimizing their extraction processes and demonstrating their bioactivity in vitro and in vivo. However, limited research exists on the isolation and purification of herbaceous jelly polysaccharides and their subsequent applications.

[0003] As the functions of active polysaccharides continue to be elucidated, they are playing an increasingly important role in biological research and the production of health products. However, decolorization has always been a challenge in the purification of Herba Jellyfish polysaccharides. Due to the unique characteristics of the sample, the Herba Jellyfish polysaccharide solution is dark brown, which places higher demands on decolorization. Traditional decolorization methods cannot achieve both pigment removal and polysaccharide retention. Some reagents even destroy the polysaccharide structure, thereby affecting its biological activity.

[0004] The Chinese invention patent application with application number 202410336718.1 discloses a method for extracting jelly grass polysaccharide, the extracted polysaccharide and its application. The extraction method comprises the following process steps: (1) extraction: after the dried jelly grass material is crushed, water is added, and then anhydrous sodium carbonate is added, heated to boiling, and extracted for 2-4 hours to obtain jelly grass extract; decolorization: activated carbon is added to the jelly grass extract obtained in step (1), heated and boiled for 30-45 minutes, and then hot filtered to collect the filtrate; (3) concentration: the filtrate obtained in step (2) is concentrated, ethanol is added for precipitation, and then the crude polysaccharide is collected by filtration; the crude polysaccharide is further processed; the extraction method of the present invention can extract the polysaccharide in jelly grass to the greatest extent, with a low polysaccharide loss rate and effective removal of protein, thereby improving the quality of jelly grass polysaccharide. However, the above technical solution does not further separate the components of jelly grass polysaccharide, and the decolorization and purification effect is poor, which is not suitable for large-scale extraction production. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a neutral polysaccharide of grass jelly with good decolorization effect and high polysaccharide preservation rate, and an extraction method and application 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 neutral polysaccharides from Herba Cibotii, comprising the following steps: 1) Boil fresh Herba Jelly with water to produce alkali extraction to obtain Herba Jelly extract; The extract is added with ethanol, mixed, allowed to stand, centrifuged, and the precipitate is evaporated to remove ethanol, and freeze-dried to obtain crude polysaccharide of Herba Cibotii; 2) adding distilled water to the crude polysaccharide of Herba Jellyii to prepare a crude polysaccharide solution of Herba Jellyii, decolorizing it with macroporous resin D-900, and freeze-drying it to obtain a decolorized crude polysaccharide powder of Herba Jellyii; 3) dissolving the decolorized Herba Jelly polysaccharide powder in distilled water, centrifuging, filtering through a membrane, and purifying by DEAE-52 ion exchange column chromatography, eluting with ultrapure water, collecting the eluate, dialyzing, and concentrating to obtain preliminarily purified Herba Jelly neutral polysaccharide; 4) purifying the preliminarily purified neutral polysaccharide from the grass jelly through Sephadex G-100 ion exchange column chromatography, eluting with ultrapure water, and freeze-drying to obtain purified neutral polysaccharide from the grass jelly; The neutral polysaccharide of Herba Angelicae Dahuricae is composed of rhamnose:arabinose:galactose:glucose:xylose in a molar ratio of 0.61:3.88:4.54:11.80:79.17. The molecular weight of the neutral polysaccharide of Herba Epimedii is 2917 Da.

[0007] Another technical solution provided by the present invention is to provide a neutral polysaccharide of Herba Angelicae Sinensis prepared by a method for extracting neutral polysaccharide of Herba Angelicae Sinensis.

[0008] Another technical solution provided by the present invention is to provide the use of the above-mentioned grass jelly neutral polysaccharide in the preparation of grass jelly neutral polysaccharide composite gel.

[0009] The beneficial effects of the present invention are: 1) The present invention provides a method for extracting neutral polysaccharides from Herba Jellyfish, which exhibits excellent decolorization, high polysaccharide preservation, and guaranteed biological activity. Compared to other resins, the D-900 macroporous resin used in the present invention achieves a decolorization rate of 88.37% and a polysaccharide preservation rate of 84.82% at the same concentration.

[0010] 2) The composite gel of neutral polysaccharide of grass jelly and modified cassava starch obtained by the method of the present invention has unique gel properties and has broad application prospects in the development of low-calorie and low-calorie grass jelly and grass jelly beverage products, which is conducive to the further development and utilization of neutral polysaccharide of grass jelly.

[0011] 3) The extraction method of neutral polysaccharides from Herba Jelly of the present invention is not only simple to operate and low in cost, but also more efficient and practical. The biological activity of the neutral polysaccharides from Herba Jelly is also better guaranteed, and is worthy of promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Comparison of dynamic adsorption decolorization rate, preservation rate, and comprehensive score of different resin models in the specific embodiment of the present invention; Figure 2 Adsorption curves of D-900 macroporous resin at different flow rates for adsorption of polysaccharides and pigments of Herba Cibotii. Figure 3 DEAE-52 cellulose gradient elution curve of neutral polysaccharide of Herba Cibotii according to a specific embodiment of the present invention; Figure 4 Sephadex G-100 elution curve of the neutral polysaccharide of Herba Cibotii according to a specific embodiment of the present invention; Figure 5 The scavenging rate of the Herba Aspergillus crude polysaccharide / Herba Aspergillus neutral polysaccharide on DPPH and ABTS free radicals according to the specific embodiment of the present invention; Figure 6 Full-band UV scanning curve of neutral polysaccharide of Herba Cibotii according to a specific embodiment of the present invention; Figure 7 Infrared spectrum of neutral polysaccharide of Herba Cibotii according to a specific embodiment of the present invention; Figure 8 A chromatogram showing the molecular weight determination of neutral polysaccharides from Herba Cibotii according to a specific embodiment of the present invention; Figure 9 Chromatogram of monosaccharide standards according to a specific embodiment of the present invention; Figure 10 Chromatogram of determination of monosaccharide composition of neutral polysaccharide of Herba Cibotii according to a specific embodiment of the present invention; Figure 11 Comparison of the water holding capacity of the Herba Cibotii polysaccharides-cassava starch MCP-CS and the Herba Cibotii polysaccharides-modified cassava starch MCP-CSA composite gels according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0013] 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.

[0014] The present invention provides a method for extracting neutral polysaccharides from grass jelly, comprising the following steps: 1) Boil fresh Herba Jelly with water to produce alkali extraction to obtain Herba Jelly extract; The extract is added with ethanol, mixed, allowed to stand, centrifuged, and the precipitate is evaporated to remove ethanol, and freeze-dried to obtain crude polysaccharide of Herba Cibotii; 2) adding distilled water to the crude polysaccharide of Herba Jellyii to prepare a crude polysaccharide solution of Herba Jellyii, decolorizing it with macroporous resin D-900, and freeze-drying it to obtain a decolorized crude polysaccharide powder of Herba Jellyii; 3) dissolving the decolorized grass jelly polysaccharide powder in distilled water, centrifuging, filtering, and purifying by DEAE-52 ion exchange column chromatography, eluting with the solution, collecting the eluate, dialyzing, and concentrating to obtain preliminarily purified grass jelly neutral polysaccharide; 4) purifying the preliminarily purified neutral polysaccharide from the grass jelly through Sephadex G-100 ion exchange column chromatography, eluting, and freeze-drying to obtain purified neutral polysaccharide from the grass jelly; The neutral polysaccharide of Herba Angelicae Dahuricae is composed of rhamnose:arabinose:galactose:glucose:xylose in a molar ratio of 0.61:3.88:4.54:11.80:79.17. The obtained neutral polysaccharide of grass jelly will also contain other trace components, such as inorganic salts, but these trace components will not affect its monosaccharide composition.

[0015] The molecular weight of the neutral polysaccharide of Herba Epimedii is 2917 Da.

[0016] Furthermore, in the above-mentioned method for extracting neutral polysaccharides from grass jelly, the step 1) is specifically as follows: Fresh grass jelly is added with water at a material-liquid weight ratio of 1:40 to obtain a mixed solution, 0.15% by mass of NaOH is added to the mixed solution and boiled to obtain a grass jelly extract, ethanol is added to the grass jelly extract, the mixture is mixed at a volume ratio of 1:4, allowed to stand, and centrifuged, the precipitate is evaporated to remove ethanol, and the mixture is freeze-dried to obtain crude grass jelly polysaccharide.

[0017] Furthermore, in the above-mentioned method for extracting neutral polysaccharides from grass jelly, the cooking conditions in step 1) are 95° C. for 2 h.

[0018] Furthermore, in the above-mentioned method for extracting neutral polysaccharides from grass jelly, the step 2) is specifically as follows: The crude polysaccharide of Herba Hypogaeae obtained in step 1 was added with distilled water to prepare a 1.5 mg / mL crude polysaccharide solution of Herba Hypogaeae. The solution was decolorized with macroporous resin D-900 at a mobile phase flow rate of 2.1 mL / min and freeze-dried to obtain decolorized crude polysaccharide powder of Herba Hypogaeaee.

[0019] Furthermore, in the above-mentioned method for extracting neutral polysaccharides from grass jelly, the step 3) is specifically as follows: The decolorized Herba Jellyfish crude polysaccharide powder obtained in step 2 was dissolved in distilled water to prepare a 1.5 mg / mL crude polysaccharide solution. After centrifugation and membrane filtration, it was purified by DEAE-52 ion exchange column chromatography with a sample volume of 10 mL and a flow rate of 1 mL / min. One tube was collected every 10 minutes and eluted with ultrapure water. The eluate of the first single elution peak (to obtain Herba Jellyfish neutral polysaccharide) was collected, and after dialysis and concentration, the preliminarily purified Herba Jellyfish neutral polysaccharide was obtained.

[0020] Furthermore, in the above-mentioned method for extracting neutral polysaccharides from grass jelly, the step 4) is specifically as follows: The initially purified neutral polysaccharide from the herb jelly obtained in step 3 was loaded with a sample volume of 2 mL, a flow rate of 0.3 mL / min, and a collection time of 10 min / tube, and eluted with ultrapure water. The collected solution was identified by the elution curve (the solution corresponding to all elution peaks was collected), and the purified neutral polysaccharide from the herb jelly was obtained by vacuum freeze drying at 20 Pa to 30 Pa and a temperature of -50°C to -40°C for 48 to 72 h.

[0021] The present invention also provides a neutral polysaccharide of Herba Jelly that is prepared by the above-mentioned neutral polysaccharide extraction method of Herba Jelly.

[0022] The present invention also provides a use of the grass jelly neutral polysaccharide in preparing a grass jelly neutral polysaccharide composite gel.

[0023] Furthermore, in the above application, the method for preparing the neutral polysaccharide composite gel of grass jelly includes the following steps: a) dissolving the neutral polysaccharide of Herba Cibotii obtained above and cassava starch in distilled water, adjusting the pH to 7 with NaHCO3, and stirring in a water bath at a constant temperature to completely dissolve the starch to obtain a mucus; b) placing the mucus obtained in step a in a refrigerator at 2-6° C. and shaping the mucus to obtain a Herba Immortalis neutral polysaccharide composite gel.

[0024] Furthermore, in the above application, the method for preparing the neutral polysaccharide composite gel of Herba Aspergillus niger comprises the following steps: a) dissolving 50 mg of the neutral polysaccharide of Herba Hypogaeae (Chen's Herba) obtained according to claim 6 and 0.75 g of cassava starch in 20 mL of distilled water, adjusting the pH to 7 with 1 mol / L NaHCO3, and stirring in a water bath at 80°C and 500 rpm / min for 5-10 min to completely dissolve the starch to obtain a mucus; b) The mucus obtained in step a was placed in a refrigerator at 4° C. for 12 h to shape the mucus to obtain a Herba Cibotii neutral polysaccharide composite gel.

[0025] Furthermore, in the above application, the cassava starch is modified cassava starch.

[0026] Example 1 A method for extracting neutral polysaccharides from grass jelly comprises the following steps: 1) Extraction of Crude Grass Jelly Polysaccharides: Place a certain amount of fresh grass jelly in a cooking pot at a material-to-liquid ratio of 1:40, add 0.15% NaOH, and cook at 95°C or above for 2 hours to obtain the grass jelly extract. The extract is then diluted with anhydrous ethanol at a ratio of 1:4 and allowed to settle. The precipitate is then centrifuged, the ethanol removed by rotary evaporation, and lyophilized to obtain the crude grass jelly polysaccharide for later use.

[0027] 2) Preparation of decolorized Herba Jelly polysaccharide: The Herba Jelly polysaccharide obtained in step 1 was added to distilled water to prepare a 1.5 mg / mL Herba Jelly polysaccharide solution, which was decolorized by passing through macroporous resin D-900 at a mobile phase flow rate of 1.1-3.1 mL / min and freeze-dried to obtain a decolorized Herba Jelly polysaccharide powder.

[0028] 3) Preliminary purification of Herba Jellyfish neutral polysaccharides: The decolorized Herba Jellyfish crude polysaccharide powder obtained in step 2 was dissolved in distilled water to prepare a 1.5 mg / mL crude polysaccharide solution, the supernatant was centrifuged, passed through a 0.45 μm microporous membrane, and purified by DEAE-52 ion exchange column chromatography and eluted with ultrapure water. The elution was specifically as follows: the Herba Jellyfish crude polysaccharide solution after centrifugation through the microporous membrane was loaded, the loading volume was 10 mL, the flow rate was set to 1 mL / min, one tube was collected every 10 minutes, and the ultrapure water elution peak liquid was collected to obtain the eluate. The eluate was dialyzed and concentrated to obtain the preliminary purified neutral polysaccharide.

[0029] 4) Purification of crude polysaccharide of Herba Jellyfish: The neutral polysaccharide obtained in step 3 is purified by Sephadex G-100 ion exchange column chromatography to obtain a Herba Jellyfish neutral polysaccharide collection solution, which is then freeze-dried in vacuo to obtain a purified neutral polysaccharide.

[0030] In step 4), the fraction obtained in step 3) is used. The sample volume is 2 mL, the elution flow rate is 0.3 mL / min, the collection time is 10 min / tube, and the elution mobile phase is ultrapure water. The collected solution of the neutral polysaccharide from Herba Siegesbeckiae is confirmed by the elution curve (the solution corresponding to all elution peaks is collected). The vacuum freeze-drying process is performed at a vacuum degree of 20 Pa to 30 Pa, a temperature of -50°C to -40°C, and a drying time of 48 to 72 h.

[0031] The neutral polysaccharide of the grass jelly is composed of rhamnose, arabinose, galactose, glucose and xylose in a molar ratio of 0.61:3.88:4.54:11.80:79.17, and has a molecular weight of 2917 Da.

[0032] 5) Solution preparation: Dissolve 20-60 mg of Herba Siegesbeckiae neutral polysaccharide and 0.5-1.5 g of cassava starch in 20 mL of distilled water, adjust the pH to 7 with 1 mol / L NaHCO3, and stir in a water bath at 80°C and 500 rpm / min for 5-10 min to completely dissolve the starch.

[0033] 6) Preparation of composite gel: Pour the solution from step a into a mold with a height of 30 mm and a radius of 25 mm and place it in a refrigerator at 4°C for 12 h to shape it.

[0034] Example 2 A method for extracting crude polysaccharides from grass jelly, comprising the following steps: 1) Extraction of Crude Grass Jelly Polysaccharides: Place a certain amount of fresh grass jelly in a cooking pot at a material-to-liquid ratio of 1:40, add 0.15% NaOH, and cook at 95°C or above for 2 hours to obtain the grass jelly extract. The extract is then diluted with anhydrous ethanol at a ratio of 1:4 and allowed to settle. The precipitate is then centrifuged, the ethanol removed by rotary evaporation, and lyophilized to obtain the crude grass jelly polysaccharide for later use.

[0035] 2) Preparation of decolorized Herba Jellyfish polysaccharide: The Herba Jellyfish crude polysaccharide obtained in step 1 was added to distilled water to prepare a 1.5 mg / mL Herba Jellyfish crude polysaccharide solution, which was decolorized by using different macroporous resins at a mobile phase flow rate of 1.1-3.1 mL / min. After freeze-drying, the decolorized Herba Jellyfish crude polysaccharide powder was obtained.

[0036] Experiment on decolorization process of neutral polysaccharide of Herba Cibotii: 1.1 Screening of macroporous resins Add the resin wet-column to a chromatography column (1.6 cm × 30 cm) and fill it to a height of 20 cm ± 1 cm. Rinse with ultrapure water for 2 BV and allow to settle for 20 min. Accurately measure 10 mL of a 1.5 mg / mL solution of Herba Siegesbeckiae crude polysaccharides and pass it into the chromatography column. Once the solution has completely entered the column, replace the mobile phase with ultrapure water to balance it for 1.5 BV. Collect the sample and determine the decolorization rate and polysaccharide preservation rate.

[0037] 1.2 Screening of mobile phase flow rate Select the optimal resin screened in 1.1 and load it into the chromatography column. Use 1.1 mL / min, 1.6 mL / min, 2.1 mL / min, 2.6 mL / min, and 3.1 mL / min as the mobile phase flow rates, respectively. Adjust the automatic fraction collector to collect sample liquid per tube every 2 minutes. Detect the absorbance of each tube at 420 nm and draw the adsorption curve.

[0038] In the resin screening step, the resin used was D-900 macroporous resin.

[0039] Comparative Example 1: The difference between Comparative Example 1 and Example 2 is that in the resin screening step, the resin used is HPD-722 macroporous resin.

[0040] Comparative Example 2: The difference between Comparative Example 2 and Example 2 is that in the resin screening step, the resin used is LSA-10 macroporous resin.

[0041] Comparative Example 3: The difference between Comparative Example 3 and Example 2 is that in the resin screening step, the resin used is HA-803 macroporous resin.

[0042] Comparative Example 4: The difference between Comparative Example 4 and Example 2 is that in the resin screening step, the resin used is DM-130 macroporous resin.

[0043] The experimental results are as follows Figure 1 、 Figure 2 and as shown in Table 1.

[0044] Table 1

[0045] Depend on Figure 1 It can be seen that all five macroporous resins showed good pigment adsorption and polysaccharide preservation capabilities. Among them, D-900 had the best decolorization effect, which could reach 88.37±1.14%. The solution could change from the initial dark brown to almost colorless, and its polysaccharide preservation ability also reached a high of 84.82±0.71%. The comprehensive score was the highest at 86.60±0.93.

[0046] The polysaccharide preservation effect of DM-130 macroporous resin is the best, reaching 87.29±1.04%, but its pigment adsorption rate is lower than that of D-900 macroporous resin. After comprehensive evaluation, D-900 macroporous resin was selected for the decolorization experiment of neutral polysaccharides from Herba Siegesbeckiae.

[0047] Depend on Figure 2 The dynamic elution curves for Herba Siegesbeckiae polysaccharides and pigments exhibit a parabolic profile. As the flow rate within the column increases, the highest or lowest points of the parabola gradually shift to the left, indicating an earlier leakage point. Table 1 shows that pigment adsorption initially increases and then decreases at faster rotational speeds. The greater the adsorption, the larger the peak area. This indicates that selecting an appropriate rotational speed can increase the resin's adsorption capacity. The polysaccharide retention rate, on the other hand, exhibits an irregular, zigzag chain-like pattern. By selecting a specific flow rate, the resin can fully adsorb the solution, resulting in a leak point at an optimal location. At 40 rpm (2.1 mL / min), D-900 achieves the best pigment removal rate and a high polysaccharide retention rate. Based on these considerations, 40 rpm was selected as the optimal parameter for the dynamic adsorption process.

[0048] Example 3 A method for purifying the decolorized grass jelly polysaccharide obtained in Example 2 comprises the following steps: The decolorized Herba Angelicae Sinensis crude polysaccharide powder obtained in Example 2 was dissolved in distilled water to prepare a 1.5 mg / mL crude polysaccharide solution, and the supernatant was centrifuged and passed through a 0.45 μm microporous membrane. It was purified by DEAE-52 ion exchange column chromatography and eluted with a NaCl solution with a concentration of 0-2 mol / L. The first single elution peak (ultrapure water elution peak) solution was collected and dialyzed and concentrated to obtain a preliminarily purified neutral polysaccharide. It was then purified by Sephadex G-100 ion exchange column chromatography, eluted, and freeze-dried to obtain purified Herba Angelicae Sinensis neutral polysaccharide for subsequent characterization and gel preparation. The elution curve is shown in FIG. Figure 3 and Figure 4 shown.

[0049] Depend on Figure 3 As can be seen, the alkali-extracted crude polysaccharides from Herba Jelly were separated into three components on DEAE-52. The first peak, eluted with ultrapure water, represents the neutral polysaccharides from Herba Jelly. The second and third peaks were not collected due to their low concentrations. Therefore, the first single peak was collected for subsequent purification.

[0050] Depend on Figure 4 It can be seen that the neutral polysaccharide of Herba Jellyfish is a uniform elution peak on G-100, so a single peak was collected and freeze-dried to obtain purified neutral polysaccharide of Herba Jellyfish.

[0051] Verification Example 1 Antioxidant analysis of the neutral polysaccharide of Herba Immortadellae obtained in Example 3: DPPH: (1) Solution preparation: Reagent 1: prepare anhydrous ethanol; Reagent 2: place the powder in an EP tube in a bottle. Add 6.08 mL of Reagent 1 and shake to dissolve immediately before use. Prepare the working solution according to the required amount of Reagent 2: Reagent 1 (V:V) = 4:21. Prepare it now; Reagent 3: 10 mg / mL vitamin C. (2) Sample determination: determine the absorbance at 515 nm after adding the working solution to the sample solution.

[0052] ABTS: (1) Prepare the ABTS solution, ABTS buffer, and ABTS oxidant in a certain ratio to prepare the ABTS reagent. Prepare it immediately before use. (2) Add the ABTS reagent to the 96-well plate and allow the reaction to continue until a dark green color appears. (3) Add SDS reagent to the wells containing the sample solution to terminate the reaction. (4) Measure the absorbance at 405 nm on a microplate reader.

[0053] The experimental results are as follows Figure 5 shown.

[0054] Depend on Figure 5It can be seen that both crude and neutral polysaccharides from grass jelly showed good scavenging rates for both free radicals. With increasing concentrations of grass jelly polysaccharides, the free radical scavenging rate gradually increased and eventually leveled off. The antioxidant properties of neutral polysaccharides from grass jelly were well preserved during the decolorization and purification process.

[0055] Verification Example 2 Spectral analysis of neutral polysaccharides of Herba Immortalii obtained in Example 3: 1. UV spectrometry: Weigh 5 mg of polysaccharide sample into a 5 mL colorimetric tube, add 10 mL of distilled water to prepare a polysaccharide solution with a concentration of 2 mg / mL, pour an appropriate amount of the solution into a colorimetric dish, and measure the UV scan of the polysaccharide solution at 190 nm-750 nm. The experimental results are as follows: Figure 6 shown.

[0056] The decolorization and purification of Herba Cibotii polysaccharides in existing technologies usually adopt methods such as activated carbon adsorption, ion exchange resin or gel filtration chromatography. Literature reports that the purity of polysaccharides purified by these methods usually reaches more than 90%, the protein content is less than 0.5%, and the nucleic acid content is less than 0.1%. Ultraviolet spectroscopy is a common means of evaluating purity. Figure 6 It can be seen that the neutral polysaccharide of Herba Jellyfish has no obvious characteristic absorption peaks at 260 nm and 280 nm, indicating that the purified neutral polysaccharide of Herba Jellyfish contains essentially no nucleic acids and proteins and has an extremely low impurity content. This demonstrates that the purification method of this example is effective in removing impurities.

[0057] 2. Fourier transform infrared spectroscopy: 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 in a mortar under an infrared lamp. Then scoop an appropriate amount of the mixed powder into a tableting mold. Use a tablet press to prepare uniform and transparent thin slices. Spectral analysis: 4000 cm-1-400 cm-1 -1 Scan the sample slice within the range, set the number of scans: 64, resolution: 4 cm -1 The experimental results are as follows. Figure 7 shown.

[0058] The infrared spectrum of neutral polysaccharides from Herba Cibotii generally displays typical characteristic absorption peaks of polysaccharides, including O–H stretching vibration at 3200–3400 cm⁻¹, hydrate or carboxyl absorption peaks at 1600–1650 cm⁻¹, and C–O–C and sugar ring characteristic peaks at 1000–1200 cm⁻¹. If the purification effect is poor, impurity interference peaks may appear in the spectrum (such as the amide II band of protein at 1550 cm⁻¹). Figure 7 It can be seen that the neutral polysaccharide of Herba Cibotii at 1646 cm -1 A characteristic absorption peak appears at 1394 cm, which is the characteristic absorption peak of sugar hydrate.-1 、1352 cm -1 It shows that it has a double bond skeleton and a CH bending surface, 1182 cm -1 The weak characteristic absorption peak of the sugar ring is the absorption peak, indicating that it contains a pyranose ring, 868 cm -1 At 835 cm · The vibration peaks at indicate that it contains both α- and β-glycosidic bonds. Furthermore, the absence of impurity interference peaks in the spectrum indicates that the purified polysaccharide has a complete structure and high purity. Compared with the prior art, the infrared spectral characteristics of this example are consistent with those of reported neutral polysaccharides from Herba Cibotii, and without impurity interference peaks, demonstrating significant decolorization and purification results.

[0059] Verification Example 3 Analysis of molecular weight and monosaccharide composition of the neutral polysaccharide of Herba Immortadellae obtained in Example 3: 1. Molecular Weight Determination of Neutral Polysaccharides from Herba Hypogaeae: Polysaccharide samples were dissolved in a 0.1 M aqueous NaNO₃ 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 and then analyzed. The chromatography system used was a gel chromatography-differential refractive index multi-angle laser light scattering system using a U3000 (Thermo, USA) liquid chromatography system, an Optilab T-rEX differential refractive index detector (Wyatt Technology, CA, USA), and a DAWN HELEOS II laser light scattering detector (Wyatt Technology, CA, USA).

[0060] Gel exclusion chromatography columns (Ohpak SB-805 HQ (300 × 8 mm) and Ohpak SB-803 HQ (300 × 8 mm)) were 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.

[0061] The experimental results are as follows Figure 8 shown.

[0062] Depend on Figure 8 As can be seen, blue represents the differential signal, while red represents the light scattering signal. The portion with both red and blue signal peaks (the green box in the figure) is considered the primary portion of the molecular weight determination. A faint blue signal is observed behind the primary peak, while the red signal is zero. This indicates that a small amount of low-molecular-weight substances may be present in the neutral polysaccharide of Herba Angelicae Sinensis. This suggests that a small amount of residual low-molecular-weight substances, such as oligosaccharides, oligopeptides, or impurities, may be present in the sample. Due to their small molecular weight and low content, these substances have little impact on the overall composition of the sample.

[0063] Table 2

[0064] As shown in Table 2, the molecular weight of the neutral polysaccharide from Herba Jellyfish is 2.917 kDa, and the dispersity is close to 1, indicating that the neutral polysaccharide from Herba Jellyfish has good uniformity and high sample purity.

[0065] 2. Determination of Monosaccharide Composition of Neutral Polysaccharides from Herba Hypogaeae: Accurately weigh 13 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, and guluronic acid). Add water to prepare a 10 mg / mL stock standard solution. Then, take appropriate amounts of the stock standard solutions and mix them to prepare a standard mix with a peak concentration of 60 μg / mL, 50 μg / mL, or 40 μg / mL. Prepare the required series of standards based on the desired concentration gradient. In a clean chromatographic vial, weigh an appropriate amount of polysaccharide sample and add 1 mL of 2M TFA solution. Heat at 121°C for 2 h. Purge with nitrogen and blow dry. 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. Monosaccharide components were analyzed and detected using a Thermo ICS 5000+ ion chromatography system (ICS5000+, Thermo Fisher Scientific, USA). A Dionex™ CarboPac™ PA20 (150 x 3.0 mm, 10 μm) liquid chromatography column was used, and the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 mL / min; column temperature 30°C; elution gradient: 0 min phase A / B / C (95:5:0, V / V), 26 min phase A / B / C (85:5:10, V / V), 42 min phase A / B / C (85:5:10, V / V), 42.1 min phase A / B / C (60:0:40, V / V), 52 min phase A / B / C (60:40:0, V / V), 52.1 min phase A / B / C (95:5:0, V / V), 60 min phase A / B / C (95:5:0, V / V). The experimental results are shown in Figure 2. Figure 9 and Figure 10 shown.

[0066] Depend on Figure 10The monosaccharide composition of the neutral polysaccharide of Herba Jelly contains rhamnose, arabinose, galactose, glucose, and xylose, with a molar ratio of 0.61:3.88:4.54:11.80:79.17. Furthermore, the sample does not contain uronic acid, confirming that it is a neutral sugar of the Herba Jelly polysaccharide.

[0067] Verification Example 4 Methylation analysis of neutral polysaccharides from Herba Immortalis obtained in Example 3: Dissolve a small amount of polysaccharide sample (2-3 mg) in 500 μL 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 dry it with nitrogen. Add 100 μL of 2 mol / L TFA and react at 121°C for 90 min. Evaporate to dryness at 30°C. Add 50 μL of 2 mol / L ammonia and 50 μL of 1 mol / L NaBD4, mix well, and react at room temperature for 2.5 h. Terminate the reaction with 20 μL of acetic acid, dry it with nitrogen, wash twice with 250 μL of methanol, and dry it with nitrogen. Add 250 μL of acetic anhydride, vortex to mix, and react at 100°C for 2.5 h. Add 1 mL of water and let it stand for 10 min. Add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and repeat the water wash three times. Remove the dichloromethane phase and analyze it on a GC-MS system. The experimental results are shown in Table 3.

[0068] Table 3

[0069] Table 3 shows that grass jelly neutral polysaccharides have six different glycosidic bond types. In the grass jelly neutral polysaccharide sample, xylose exists as T-Xylp, 4-Xylp, and 3,4-linked Xylp linkages, while galactose and glucose are linked as 4-linked Glcp and 4-linked Galp, respectively. Based on the molar ratios of the various sugar residues, it is inferred that 4-linked Xylp is the primary component of the grass jelly neutral polysaccharide backbone structure, with the presence of 3,4-linked Xylp as a branch point indicating the presence of partial branching. The presence of T-Xylp and T-Araf indicates their presence at the ends of the branches, and the total terminal sugar content (T-Xylp) is approximately equal to the total sugar content of the branch point residue 3,4-linked Xylp, consistent with theoretical results. Comparing the molar ratio of monosaccharides with the molar ratio obtained by methylation, it can be seen that the glycosidic bond type of xylose contained is similar to the molar ratio of xylose in the monosaccharide (79.3:79.17), indicating that the neutral polysaccharide of Herba Cibotii was successfully methylated and the experimental conclusion is reliable.

[0070] Example 4 A method for preparing a grass jelly neutral polysaccharide composite gel comprises the following steps: 1. Determination of polysaccharide content: Weigh 1.0 g of cassava starch (CS) and modified cassava starch (CSA) and add 20 mg, 30 mg, 40 mg, 50 mg, and 60 mg of polysaccharide sample (neutral polysaccharide of Herba Cibotii obtained in Example 3) into a beaker, add 20 mL of distilled water, adjust the pH to 7 with 1 mol / L NaHCO3, and stir in a water bath at 80°C and 500 rpm / min for 5-10 min. After the starch is completely dissolved, observe whether a gelatinous substance is formed. Pour the viscous liquid into a mold with a height of 30 mm and a radius of 25 mm, place it in a refrigerator at 4°C for 12 h to shape it, and compare the state of the formed gel.

[0071] 2. Determination of cassava starch content: According to the optimal amount of polysaccharide added determined in 1, 0.5 g, 0.75 g, 1.0 g, 1.25 g, and 1.5 g of starch were added, respectively. 20 mL of distilled water was added, and the pH was adjusted to 7 with 1 mol / L NaHCO3. The mixture was stirred in a water bath at 80°C and 500 rpm / min for 5-10 min. After the starch was completely dissolved, observe whether a gelatinous substance was formed. The viscous liquid was poured into a mold with a height of 30 mm and a radius of 25 mm. The mold was refrigerated at 4°C for 12 h to shape the mixture and compare the state of the gel.

[0072] Comparative Example 5: The difference between Comparative Example 5 and Example 4 is that in the preparation process of the composite gel, the polysaccharide sample is the neutral polysaccharide of Herba Cibotii obtained in Comparative Example 1 after decolorization and purification with HPD-722 resin.

[0073] Comparative Example 6: The difference between Comparative Example 6 and Example 4 is that in the preparation process of the composite gel, the polysaccharide sample is the neutral polysaccharide of Herba Cibotii obtained in Comparative Example 2 after decolorization and purification by LSA-10 resin.

[0074] Comparative Example 7: The difference between Comparative Example 7 and Example 4 is that in the preparation process of the composite gel, the polysaccharide sample is the neutral polysaccharide of Herba Cibotii obtained in Comparative Example 3 after decolorization and purification with HZ-803 resin.

[0075] Comparative Example 8: The difference between Comparative Example 8 and Example 4 is that in the preparation process of the composite gel, the polysaccharide sample is the neutral polysaccharide of Herba Cibotii obtained in Comparative Example 4 after decolorization and purification with DM-130 resin.

[0076] Comparative Example 9: The difference between Comparative Example 9 and Example 4 is that in the preparation process of the composite gel, the polysaccharide sample is the crude polysaccharide of Herba Siegesbeckiae decolorized by HPD-722 resin in Comparative Example 1.

[0077] Comparative Example 10: The difference between Comparative Example 10 and Example 4 is that in the preparation process of the composite gel, the polysaccharide sample is the crude polysaccharide of Herba Cibotii decolorized by LSA-10 resin in Comparative Example 2.

[0078] Comparative Example 11: The difference between Comparative Example 11 and Example 4 is that in the preparation process of the composite gel, the polysaccharide sample is the crude polysaccharide of Herba Cibotii in Comparative Example 3 that has been decolorized with HZ-803 resin.

[0079] Comparative Example 12: The difference between Comparative Example 12 and Example 4 is that in the preparation process of the composite gel, the polysaccharide sample is the crude polysaccharide of Herba Cibotii in Comparative Example 4 that has been decolorized with DM-130 resin.

[0080] The experimental results are shown in Table 4.

[0081] Table 4

[0082] As shown in Table 4, at a pH of , the gelation of the MCP-CS / CSA system is proportional to both the polysaccharide content and the starch content. When the amounts of polysaccharide and starch are low, the composite gel cannot solidify and becomes a viscous liquid. When the starch and polysaccharide amounts are appropriately increased, the Herba Ceropegiae polysaccharides-starch gel system can achieve a relatively stable jelly-like state. Considering the sample usage, 50 mg of polysaccharide and 0.75 g of starch were selected for preparation.

[0083] Verification Example 5 Analysis of gel properties of the Herba Immortadellae polysaccharide composite gel obtained in Example 4 1. Determination of water holding capacity (WHC) of MCP-CS / CSA gel system: The WHC of MCP-CS / CSA composite gel was determined by centrifugation. About 2 g of sample was weighed, wrapped with double-layer filter paper, and placed in a 50 mL centrifuge tube. The sample was centrifuged at 10,000 rpm for 15 min at 4°C. The surface water was wiped off with filter paper. The WHC was calculated using the following formula:

[0084] Where: M0——mass of gel sample after centrifugation (g) M1 - mass of gel sample before centrifugation (g) The experimental results are as follows Figure 11 shown.

[0085] Depend on Figure 11 It can be seen that the neutral polysaccharide of grass jelly and cassava starch obtained by decolorization and purification with D-900 have a slightly lower water holding capacity than the gel system, but the water holding rate is above 80%, indicating that the network structure inside the neutral polysaccharide of grass jelly-CS / CSA gel system is relatively tight.

[0086] 2. Determination of the texture characteristics of the MCP-CS / CSA gel system: TPA measurement: The composite gel was scooped out of the beaker and placed on the stage to equilibrate at room temperature for 10 minutes. The test was performed using the TPA program of a texture analyzer. Specific parameters were: a P / 36R cylindrical probe, an initial force of 0.75 N, a recovery height of 15 mm, a deformation of 50%, and a detection rate of 60 mm / min. Gel strength measurement: The composite gel was scooped out of the beaker and placed on the stage to equilibrate at room temperature for 10 minutes. The test was performed using the puncture program of a texture analyzer. Specific parameters were: a 0.5 mm probe, an initial force of 0.75 N, a puncture distance of 10 mm, a recovery height of 15 mm, and a detection rate of 60 mm / min. The experimental results are shown in Table 5.

[0087] Table 5

[0088] As shown in Table 5, the grass jelly neutral polysaccharide composite gel decolorized and purified with D-900 exhibited the best gel properties. Specifically, the grass jelly neutral polysaccharide composite gel decolorized and purified with D-900, with the addition of CS, exhibited slightly lower hardness, chewiness, elasticity, cohesion, and gel strength than the composite gel with the addition of CSA. This suggests that the addition of modified starch to the grass jelly neutral polysaccharide composite gel can achieve a superior texture and has broad application prospects in the development of low-calorie and low-calorie grass jelly and grass jelly beverages.

[0089] 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 neutral polysaccharides from Herba Cibotii, characterized in that: The following steps are involved: 1) boiling fresh grass jelly with water to produce alkali extraction to obtain a grass jelly extract; adding ethanol to the extract, mixing, standing, and centrifuging; taking the precipitate and removing the ethanol by rotary evaporation; and freeze-drying to obtain a crude grass jelly polysaccharide; 2) adding distilled water to the crude polysaccharide of Herba Jellyii to prepare a crude polysaccharide solution of Herba Jellyii, decolorizing it with macroporous resin D-900, and freeze-drying it to obtain a decolorized crude polysaccharide powder of Herba Jellyii; 3) dissolving the decolorized Herba Jelly polysaccharide powder in distilled water, centrifuging, filtering through a membrane, and purifying by DEAE-52 ion exchange column chromatography, eluting with ultrapure water, collecting the eluate, dialyzing, and concentrating to obtain preliminarily purified Herba Jelly neutral polysaccharide; 4) purifying the preliminarily purified neutral polysaccharide from the grass jelly through Sephadex G-100 ion exchange column chromatography, eluting, and freeze-drying to obtain purified neutral polysaccharide from the grass jelly; The neutral polysaccharide of the herba jellyfish is composed of rhamnose:arabinose:galactose:glucose:xylose in a molar ratio of 0.61:3.88:4.54:11.80:79.17; the molecular weight of the neutral polysaccharide of the herba jellyfish is 2917 Da.

2. The method for extracting neutral polysaccharides from Herba Immortalis according to claim 1, wherein: The step 1) is specifically as follows: Fresh grass jelly is added with water at a material-liquid weight ratio of 1:40 to obtain a mixed solution, 0.15% by mass of NaOH is added to the mixed solution and boiled to obtain a grass jelly extract, ethanol is added to the grass jelly extract, the mixture is mixed at a volume ratio of 1:4, allowed to stand, and centrifuged, the precipitate is evaporated to remove ethanol, and the mixture is freeze-dried to obtain crude grass jelly polysaccharide.

3. The method for extracting neutral polysaccharides from Herba Immortalis according to claim 2, wherein: The cooking condition in step 1) is 95° C. for 2 h.

4. The method for extracting neutral polysaccharides from Herba Immortalis according to claim 1, wherein: The step 2) is specifically as follows: The crude polysaccharide of Herba Hypogaeae obtained in step 1 was added with distilled water to prepare a 1.5 mg / mL crude polysaccharide solution of Herba Hypogaeae. The solution was decolorized with macroporous resin D-900 at a mobile phase flow rate of 2.1 mL / min and freeze-dried to obtain decolorized crude polysaccharide powder of Herba Hypogaeaee.

5. The method for extracting neutral polysaccharides from Herba Immortalis according to claim 1, wherein: The step 3) is specifically as follows: The decolorized Herba Jellyfish crude polysaccharide powder obtained in step 2 was dissolved in distilled water to prepare a 1.5 mg / mL crude polysaccharide solution. After centrifugation and membrane filtration, it was purified by DEAE-52 ion exchange column chromatography with a sample volume of 10 mL and a flow rate of 1 mL / min. One tube was collected every 10 minutes and eluted with ultrapure water solution. The eluate was collected and dialyzed and concentrated to obtain the preliminarily purified Herba Jellyfish neutral polysaccharide.

6. The neutral polysaccharide of Herba Jelly prepared according to the method for extracting neutral polysaccharide of Herba Jelly according to any one of claims 1 to 5.

7. Use of the neutral polysaccharide of Herba Jellyii according to claim 6 in the preparation of neutral polysaccharide composite gel of Herba Jellyii.

8. The use according to claim 7, characterized in that The method for preparing the neutral polysaccharide composite gel of Herba Cibotii comprises the following steps: a) dissolving the neutral polysaccharide of Herba Cibotii obtained in claim 6 and cassava starch in distilled water, adjusting the pH to 7 with NaHCO 3 , and stirring in a water bath at a constant temperature to completely dissolve the starch to obtain a mucus; b) placing the mucus obtained in step a in a refrigerator at 2-6° C. and shaping the mucus to obtain a Herba Immortalis neutral polysaccharide composite gel.

9. The application according to claim 8, characterized in that: The method for preparing the neutral polysaccharide composite gel of Herba Immortals specifically comprises the following steps: a) dissolving 50 mg of the neutral polysaccharide of Herba Hypogaeae (Chen's Herba) obtained according to claim 6 and 0.75 g of cassava starch in 20 mL of distilled water, adjusting the pH to 7 with 1 mol / L NaHCO3, and stirring in a water bath at 80°C and 500 rpm / min for 5-10 min to completely dissolve the starch to obtain a mucus; b) The mucus obtained in step a was placed in a refrigerator at 4° C. for 12 h to shape the mucus to obtain a Herba Cibotii neutral polysaccharide composite gel.

10. The use according to claim 8, characterized in that: The cassava starch is modified cassava starch.

Images