A preparation method of Curcuma zedoaria polysaccharide and its use in preparing liver protection medicine

Abstract

The invention belongs to the field of traditional Chinese medicinal material processing and application, and particularly relates to a method for preparing a Curcuma zedoaria polysaccharide and a use thereof in preparing a liver protection medicine. The preparation method comprises the following steps: taking raw Curcuma zedoaria or vinegar Curcuma zedoaria medicinal materials, crushing them, performing defatting and small molecule removal treatments with petroleum ether to obtain defatted medicine powder; adding ethanol, performing water bath reflux extraction, repeating 1-3 times, filtering, collecting filter residues, evaporating ethanol from the filter residues, drying, and setting aside; adding 30-50 times the weight of water, performing reflux extraction, repeating 1-4 times, filtering, combining filtrates, evaporating and concentrating, and cooling to room temperature; adding 3-5 times the volume of ethanol, standing at 1-6°C for 18-36 hours; collecting precipitation by centrifugation, dissolving the precipitation in pure water, and evaporating ethanol; and freeze-drying to obtain the Curcuma zedoaria polysaccharide. In addition, the method also includes separation and purification of the obtained Curcuma zedoaria polysaccharide, using DEAE-52 cellulose column and Sephadex G-100 polysaccharide gel column for chromatographic separation and purification in sequence, which has the advantages of high extraction and purification efficiency, excellent purity and good medicinal activity.

Description

Technical Field

[0001] The invention relates to the processing and application of traditional Chinese medicinal materials, in particular to a preparation method of Curcuma zedoaria polysaccharide and the use of the polysaccharide in preparing liver protection medicine, and belongs to the field of biomedicine. Background Art

[0002] Curcuma acuminata is a traditional Chinese medicinal material with a long history and wide application. Modern research shows that Curcuma acuminata polysaccharide has multiple biological activities, such as anti-tumor, antioxidant, anti-inflammatory, etc. In recent years, with the in-depth study of Curcuma acuminata, its liver protection effect has gradually attracted attention.

[0003] However, the extraction components of Curcuma zedoaria polysaccharide are currently quite different due to different preparation methods, resulting in the pharmaceutical application effect of Curcuma zedoaria polysaccharide not being very ideal. In particular, the more complex preparation methods used in previous studies made the preparation of Curcuma zedoaria polysaccharide more difficult, and the cost of obtaining the components was high, which failed to give full play to the application value of Curcuma zedoaria polysaccharide in the medical field.

[0004] Chinese invention patent application CN107089992A discloses a total sesquiterpene and three sesquiterpene monomers of turmeric plants, as well as a preparation method and use thereof. The patent application uses turmeric plants as raw materials, extracts them through supercritical carbon dioxide, and then extracts them through low-polarity organic solvents to obtain a low-polarity total sesquiterpene extract, and further separates them through chromatography to obtain pure products of three sesquiterpenes, namely, curcuminol, curcuma dione, and curcuma epoxide. Activity studies have shown that the total sesquiterpene and curcuminol, curcuma dione, and curcuma epoxide have anti-diabetic activity, and can be used to prepare anti-diabetic drugs, anti-diabetic health foods, functional foods, and the like.

[0005] Chinese invention patent application CN114732888A discloses a Curcuma zedoaria extract and its application in the preparation of drugs for the prevention and treatment of coronary heart disease. The invention patent application heats raw or vinegar Curcuma zedoaria slices with ethanol for extraction; extracts the drug residue with water; combines the extracts, adds ethanol to a concentration of 40-60% alcohol, and then stands, and after filtering, decompresses and concentrates the filtrate to a raw drug content of 0.5-1.0 g / mL. The resulting Curcuma zedoaria extract is used to reduce the risk of coronary heart disease, improve blood lipid indicators, reduce vascular cholesterol deposition and inflammation, and regulate lipid metabolism abnormalities in coronary heart disease by inhibiting sphingolipid metabolism and promoting glyceride metabolism and fatty acyl metabolism.

[0006] Existing studies have found that Curcuma has certain application value in the treatment of diabetes and coronary heart disease, but there is a lack of research on the differences in the properties of Curcuma caused by the processing methods of Curcuma, as well as its application in the treatment of other diseases. Summary of the invention

[0007] The purpose of the present invention is to overcome the problems in the prior art of insufficient quality research of Curcuma zedoaria polysaccharide and relatively single downstream application, and to provide a preparation method of Curcuma zedoaria polysaccharide and its liver protection use.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A method for preparing Curcuma zedoaria polysaccharide comprises the following steps:

[0010] S1. Crush raw Curcuma zedoaria or vinegar Curcuma zedoaria, and perform defatting treatment with petroleum ether to obtain defatted medicinal powder.

[0011] S2. Add 8-13 times amount of 90vol%-95vol% ethanol to the defatted powder, reflux extract in a water bath at 65℃-80℃ for 2-5h, repeat 1-3 times, filter, collect the residue, evaporate the ethanol from the residue, dry at 30-60℃ to obtain the Curcuma zedoaria solid powder for later use.

[0012] S3. Take the solid powder of Curcuma zedoaria, add 30-50 times the weight of water, reflux extraction at 80℃-95℃ for 2-5 hours, repeat 1-4 times, filter, combine the filtrate, evaporate and concentrate to 1 / 7-1 / 4 times the volume, cool to room temperature, and obtain a concentrated solution.

[0013] S4. Take the concentrated solution, add 3 to 5 times the volume of 75v% or more ethanol, and let it stand at 1°C-6°C for 18-36 hours; collect the precipitate by centrifugation, dissolve the precipitate in pure water, and evaporate the ethanol; freeze-dry, collect the lyophilized powder, and obtain the Curcuma zedoaria polysaccharide.

[0014] The preparation method of the curcuma zedoaria polysaccharide of the present invention optimizes the extraction process, and is subjected to petroleum ether degreasing and ethanol purification treatment to fully defatted and remove the fat-soluble components on the surface of the raw curcuma zedoaria pulverized medicinal material, and then adopts a water extraction and alcohol precipitation method to prepare the curcuma zedoaria polysaccharide product. The preparation method of the present invention has the characteristics of high extraction efficiency and excellent extraction purity, the yield of curcuma zedoaria polysaccharide can reach more than 2.3%, and the obtained curcuma zedoaria polysaccharide has good medicinal activity, and the development value of the production and preparation of drugs is high. The curcuma zedoaria used can be raw curcuma zedoaria or vinegar curcuma zedoaria. The vinegar curcuma zedoaria is prepared by a patented method, and the patent number is: ZL 201310400255.2. Preferably, take an appropriate amount of raw Curcuma zedoaria, use 20 kg of 9° rice vinegar for every 100 kg of raw product, mix well and simmer for 2 hours, cook until the vinegar liquid is dry, place at 120°C to dry for 30 minutes, take out and cool, and dry to obtain the vinegar Curcuma zedoaria slices. The vinegar Curcuma zedoaria is used as a raw material for extracting Curcuma zedoaria polysaccharides.

[0015] Further, in step S1, the petroleum ether is used for degreasing treatment as follows: add 10 times the weight of petroleum ether of the raw Curcuma zedoaria medicinal material, extract under reflux in a water bath at 70°C-80°C for 2-3h, such as 2.5h, extract 1-3 times, such as 2 times, filter, collect the filter residue, and dry at 30°C-50°C, such as 40°C. The degreasing treatment method can also achieve the removal of small molecules, and a single degreasing treatment can achieve multiple purposes.

[0016] Further, in step S2, after defatting, the defatted powder is added with 9-11 times the amount of 90vol%-97vol% ethanol, extracted under reflux in a water bath at 67°C-75°C for 2-3h, extracted 1-2 times, filtered, and the residue is collected. The ethanol is evaporated from the residue and dried at 35°C-45°C to obtain a solid powder of Curcuma zedoaria for later use.

[0017] Further, in step S3, take the solid powder of Curcuma zedoaria, add 35-45 times the weight of water, reflux extraction at 85°C-95°C for 2-4 hours, repeat 1-3 times, filter, combine the filtrate, evaporate and concentrate to 1 / 6-1 / 4 times the volume, cool to room temperature, and obtain a concentrated solution.

[0018] Preferably, a rotary evaporator is used for evaporation and concentration.

[0019] Preferably, cooling to room temperature refers to cooling to ambient temperature, and the ambient temperature is controlled at 15°C-28°C.

[0020] Further, in step S4, the concentrated solution is taken, 4-5 times the amount of 80 vol% ethanol is added, and the solution is placed in a refrigerator at 3-4°C for 24 hours. Centrifugation is then performed to collect the precipitate. The precipitate is dissolved in pure water, the ethanol is evaporated, and the solution is pre-frozen overnight in a refrigerator at -20°C, transferred to a freeze dryer for freeze drying, and the freeze-dried powder is collected for later use. Preferably, when adding 80 vol% ethanol, the stirring is preferably slowed down. Preferably, the solution is centrifuged at 4000 rpm for 10 minutes.

[0021] Further, in step S4,

[0022] S4. Take the concentrated solution, add 3 to 5 times the volume of ethanol with a concentration of more than 80v%, and let it stand at 1°C-6°C for 20-30 hours; collect the precipitate by centrifugation, dissolve the precipitate in pure water, and evaporate the ethanol; freeze-dry and collect the lyophilized powder to obtain the Curcuma zedoaria polysaccharide.

[0023] Preferably, the centrifugal speed is 4000-12000 rpm, and the centrifugal time is 5-20 min, such as 10 min.

[0024] Furthermore, the method further comprises step S5 of separating and purifying the obtained Curcuma zedoaria polysaccharide.

[0025] Further, DEAE-52 cellulose column chromatography is first used for separation and purification to obtain a crude product of Curcuma zedoaria polysaccharide; then, Sephadex G-100 dextran gel column chromatography is used for separation and purification to obtain pure Curcuma zedoaria polysaccharide. Preferably, during the DEAE-52 cellulose column chromatography separation and purification process, water and sodium chloride aqueous solution are used for elution in sequence, and the crude product of Curcuma zedoaria polysaccharide of the water elution product is collected. According to the raw materials used: raw Curcuma zedoaria and vinegar Curcuma zedoaria are named RCPP-0 and VCPP-0 respectively.

[0026] Further, the separation and purification step comprises:

[0027] ① DEAE-52 cellulose filler pretreatment;

[0028] Weigh 100g of DEAE-52 cellulose resin dry powder filler, soak it in 20 times the volume of pure water for 24h, remove suspended matter and impurities after the filler is fully swollen, and remove the filtrate by suction. After vacuum filtration, soak the filler in 0.5mol / L NaOH solution for 30min, discard the upper layer of liquid, and rinse it repeatedly with pure water several times until the pH is neutral. Soak it in 0.5mol / L HCl solution for 30min, discard the upper layer of liquid, and rinse it repeatedly with pure water several times until the pH is neutral, and set aside.

[0029] ② Column loading:

[0030] The pretreated filler is loaded into a glass chromatography column of 2.5×60cm in size by wet column loading. After the chromatography column is fixed vertically, a small amount of pure water is added to the lower end of the column to remove the air in the column and avoid bubbles during column loading. Add an appropriate amount of pure water to the pretreated DEAE-52 cellulose ester filler, stir evenly, slowly pour it into the chromatography column, let it sink naturally, open the lower switch to allow the pure water to flow out naturally, ensure that the filler is uniform, the column surface is flat without bubbles and faults, and when the filler reaches 2 / 3 of the column height, use pure water to balance the DEAE-52 cellulose chromatography column for 30 minutes.

[0031] ③ Loading: Take 500 mg of crude polysaccharides of Curcuma zedoaria before and after vinegar processing (raw Curcuma zedoaria polysaccharide, vinegar Curcuma zedoaria polysaccharide), dissolve it in 20 mL of distilled water, centrifuge (8000 rpm, 4°C, 10 min), remove the precipitate, use a pipette to absorb the supernatant and add it slowly in a circle along the inner wall of the chromatography column.

[0032] ④ Elution and collection;

[0033] Purified components were obtained by eluting with pure water, 0.1, 0.2, 0.3, and 0.4 mol / L NaCl solution on a DEAE-52 cellulose chromatography column, respectively, at a flow rate of 0.6 mL / min. The eluate was collected in a 5 mL test tube. The absorbance value was measured at a UV absorption wavelength of 490 nm using a phenol-sulfuric acid method, and an elution curve was drawn. Each collected fraction was concentrated and dialyzed for 48 hours using a 3500 Da dialysis bag to remove salt. The resulting dialyzate was freeze-dried to obtain the crude raw Curcuma zedoaria polysaccharide of each component.

[0034] Further, the separation and purification step also includes:

[0035] Sephadex G-100 column chromatography of polysaccharides from Curcuma zedoariae before and after vinegar processing

[0036] That is, the crude product of Curcuma zedoaria polysaccharide obtained by treating with the DEAE-52 cellulose chromatography column is selected, and the crude product of Curcuma zedoaria polysaccharide RCPP-0 and / or VCPP-0 of the water elution product is selected and separated and purified by Sephadex G-100 dextran gel column chromatography.

[0037] ① Pretreatment of Sephadex G-100 dextran gel column: Pretreatment of Sephadex G-100 filler: soak in ultrapure water, fully swell, pour off the gel fragments, and repeat several times.

[0038] ② Column packing and column balance:

[0039] Use the wet column packing method to load the pretreated filler into a glass chromatography column of 2.5×60cm in size. After fixing the chromatography column vertically, add a small amount of pure water to the lower end of the column to remove the air in the column and avoid bubbles when loading the column. Add an appropriate amount of pure water to the pretreated Sephadex G-100 filler, stir evenly, slowly pour it into the chromatography column, let it sink naturally, open the lower switch to allow the pure water to flow out naturally, ensure that the filler is uniform, the column surface is flat without bubbles and faults, and when the filler reaches 2 / 3 of the column height, use pure water to balance the Sephadex G-100 filler chromatography column for 30 minutes;

[0040] ③ Loading: Prepare 10 mg / mL RCPP-0 and VCPP-0 sugar solutions respectively, take 50 mL and filter through a 0.45 μm filter membrane, and apply the sample with a rubber-tipped dropper. Elute with ultrapure water at a flow rate of 0.4 mL / min, and collect 4 mL of eluate in each tube. Determine the total sugar by the phenol-sulfuric acid method until no polysaccharide is washed out of the eluate.

[0041] The present invention also provides application of the Curcuma zedoaria polysaccharide obtained by separation.

[0042] Application of the raw Curcuma zedoaria polysaccharide and / or the vinegar Curcuma zedoaria polysaccharide obtained by the above method in the preparation of drugs for protecting the liver.

[0043] Furthermore, the drug is a drug that protects the liver from damage.

[0044] Preferably, the drug is a drug that reduces the activity of ALT and AST.

[0045] Preferably, the drug is a drug that increases SOD levels.

[0046] Furthermore, the drug is a drug that has the function of protecting the intestinal mechanical barrier.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1. The preparation method of the Curcuma zedoaria polysaccharide of the present invention determines the optimal extraction process through orthogonal test research, and obtains a high extraction recovery rate of the Curcuma zedoaria polysaccharide component, and the corresponding Curcuma zedoaria polysaccharide component has a high proportion of effective active ingredients and great medicinal value.

[0049] 2. The preparation method of Curcuma zedoaria polysaccharide of the present invention comprises the following steps: subjecting raw Curcuma zedoaria to petroleum ether degreasing and ethanol purification treatment to fully degreasing and removing fat-soluble components on the surface of crushed raw Curcuma zedoaria medicinal materials, and then obtaining a high-purity Curcuma zedoaria polysaccharide product through water extraction and alcohol precipitation, with high yield; then, subjecting the product to chromatography separation and purification on a DEAE-52 cellulose chromatography column and a Sephadex G-100 polysaccharide gel column in sequence to obtain a purified Curcuma zedoaria polysaccharide product, which has the characteristics of good quality, higher sugar content, and better liver protection effect.

[0050] 3. The application of raw Curcuma zedoaria polysaccharide in the preparation of liver protection drugs can significantly reduce the activities of ALT and AST (r < 0.0001), significantly increase the level of SOD (r < 0.001), and significantly reduce the level of MDA (r < 0.0001), thereby protecting the liver from damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a curve chart of the single factor experimental results of the polysaccharide extraction process of Curcuma zedoaria.

[0052] Figure 2 3D model diagram (A; B; C) and contour diagram (A'; B'; C') of the effect of the interaction of various factors on the yield of polysaccharides from Curcuma zedoaria.

[0053] Figure 3 It is the DEAE-52 cellulose purification elution curve of raw Curcuma zedoaria polysaccharide (A) and Curcuma acetozea polysaccharide (B).

[0054] Figure 4It is the elution curve of the raw Curcuma zedoaria polysaccharide (A) and the Curcuma acetozea polysaccharide (B) purified by Sephadex G-100.

[0055] Figure 5 These are the calibration curves of RCPP lg Mw-RT (A) and lg Mn-RT (B).

[0056] Figure 6 These are the calibration curves of lg Mw-RT (A) and lg Mn-RT (B) of VCPP.

[0057] Figure 7 It is the molecular weight diagram of RCPP (A) and VCPP (B).

[0058] Figure 8 It is the FT-IR graph of RCPP and VCPP.

[0059] Fig. 9 are scanning electron microscope images of RCPP and VCPP (100×, 500×, 1000×, 2000×).

[0060] Fig.10 This is the GS-MS total ion current diagram of RCPP.

[0061] Fig.11 This is the GS-MS total ion current of VCPP.

[0062] Fig.12 It is the purified polysaccharide RCPP from Curcuma zedoaria 1 H NMR spectrum.

[0063] Fig.13 It is the purified polysaccharide VCPP from Curcuma zedoariae 1 H NMR spectrum.

[0064] Fig.14 It is the purified polysaccharide RCPP from Curcuma zedoaria 13 C NMR spectrum.

[0065] Fig.15 It is the VCPP of Curcuma acetosporum polysaccharide 13 C NMR spectrum.

[0066] Fig.16 This is the design drawing of the animal experiment plan.

[0067] Fig.17 It is the graph of the weight change of mice (n=10).

[0068] Fig.18 These are photos comparing the morphology and appearance of the livers of mice in each group.

[0069] Fig.19This is a comparison chart of the expression levels of ALT and AST in mouse serum (n=10).

[0070] Fig. 20 This is a comparison chart of the expression levels of SOD and MDA in mouse serum (n=10).

[0071] Fig.21 The following are the comparison photos of the pathological results of the liver tissues of mice in each group (400 times).

[0072] Fig. 22 These are the comparative photographs of H&E stained pathological sections of ileum tissues of mice in each group (200 times).

[0073] Fig.23 This is a comparison photograph of the sections showing the positive expression of Occludin in the ileal mucosal tissues of each group (200 times).

[0074] Fig.24 The following are comparison photos of the sections showing the positive expression of ZO-1 in the ileal mucosal tissues of each group (200 times).

[0075] Fig.25 This is a comparative chart of the expression results of Occludin and ZO-1 in the ileal mucosal tissue of each group (n=10). DETAILED DESCRIPTION

[0076] The present invention is further described in detail below in conjunction with the test examples and specific embodiments. However, this should not be understood as the scope of the above-mentioned subject of the present invention being limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention. The percentage of ethanol in the present invention refers to the volume percentage, written as v% or vol%; the percentage of the remaining solid raw materials is the weight percentage, written as % or wt%.

[0077] Example 1

[0078] A method for preparing raw Curcuma zedoaria polysaccharide comprises the following steps:

[0079] S1. Pretreatment of medicinal materials: Take the raw Curcuma zedoaria medicinal materials, grind them, pass through a No. 2 sieve, and use petroleum ether to defat and remove small molecules. That is, add 10 times the amount of petroleum ether, reflux extraction in a 70℃ water bath for 2.5h, extract twice, filter, collect the filter residue, and dry at 40℃.

[0080] After defatting, use defatted powder, add 10 times amount of 95% ethanol, extract under reflux in a 70°C water bath for 2.5 hours, extract twice, filter, collect the residue, evaporate the ethanol from the residue, dry at 40°C for later use.

[0081] S2. Extraction of Curcuma zedoaria polysaccharide: The Curcuma zedoaria polysaccharide was extracted by the classic water extraction and alcohol precipitation method. That is, 20.00 g of the treated Curcuma zedoaria solid powder was weighed, and the reflux extraction operation was performed according to the experimental scheme, respectively, with the extraction temperature of 90°C, the extraction time of 3h, the water addition amount of 40 times, and the extraction times of 3 times. The obtained extract was filtered with gauze, concentrated to 1 / 5 of the original volume by a rotary evaporator at 60°C, and cooled for use.

[0082] Alcohol precipitation: Take the above concentrate, add 4 times the amount of 80% ethanol, and stir slowly. Let it stand in a 4℃ refrigerator for 24 hours. Centrifuge it at 4000rpm for 10 minutes and collect the precipitate. Dissolve the precipitate in pure water, evaporate the ethanol, pre-freeze it in a -20℃ refrigerator overnight, transfer it to a freeze dryer for freeze drying, collect the freeze-dried powder, and you will get the raw Curcuma zedoaria polysaccharide, which is the crude product of raw Curcuma zedoaria polysaccharide, named Cru-RCPP, for later use.

[0083] The same method as above was used to prepare Curcuma zedoaria polysaccharide, except that the raw material was replaced with Curcuma zedoaria prepared with vinegar (Curcuma zedoaria), and the obtained Curcuma zedoaria polysaccharide was a crude product of Curcuma zedoaria polysaccharide, named Cru-VCPP.

[0084] Example 2

[0085] On the basis of Example 1, raw Curcuma zedoaria was used as raw material, and the experiment was conducted with the extraction rate of Curcuma zedoaria polysaccharide as an index, and the effects of five factors on the yield of Curcuma zedoaria polysaccharide were investigated, including extraction times (1, 2, 3, 4, 5 times), extraction time (1, 2, 3, 4, 5 h), extraction temperature (60, 70, 80, 90, 100 ° C), solid-liquid ratio (10, 20, 30, 40, 50 g / mL) and alcohol precipitation concentration (60%, 70%, 80%, 90%, anhydrous ethanol). Wherein, alcohol precipitation concentration refers to the concentration of the ethanol solution used, and compared with the 95% ethanol used in Example 1, the experiment compared the precipitation effects of 60%, 70%, 80%, 90% purity ethanol and anhydrous ethanol.

[0086] When only a single factor in the preparation method is changed, the experimental results are shown in Figure 1 , the optimal parameters under single factor conditions of some basic factors in the preparation method can be determined.

[0087] Example 3

[0088] Based on the single factor experimental results in Example 2 ( Figure 1 ), selected extraction time of 2h, 3h, and 4h, extraction temperature of 80℃, 90℃, and 100℃, solid-liquid ratio of 30g / mL, 40g / mL, and 50g / mL, and extraction times of 3 times, and used response surface methodology to further optimize the key technical processes for extracting polysaccharides from Curcuma zedoaria.

[0089] The Box-Behnken design-double response surface method principle was adopted, and the yield of Curcuma zedoaria polysaccharide was taken as the response value. The three-factor three-level response surface analysis scheme was designed using Design-Expert13.0.1 software, as shown in Table 1.

[0090] Table 1. Response surface methodology experimental design factors and levels

[0091] factor level -1 0 1 A Extraction time / (h) 2 3 4 B Extraction temperature / (℃) 80 90 100 C Solid-liquid ratio / (g / mL) 30 40 50

[0092] The Box-Behnken central composite experimental design principle was adopted, and the extraction time (A), extraction temperature (B), and solid-liquid ratio (C) were selected as optimization factors. The yield of polysaccharides from Curcuma zedoariae (Y) was used as the response value. The experiment was designed using Design-Expert 10.0.1 software to obtain the optimal extraction process. The response surface experimental design and results are shown in Table 2.

[0093] Response surface regression analysis was performed using Design-Expert 10.0.1 software, with the yield of Curcuma zedoaria polysaccharide as the response value. The data were analyzed to obtain the regression fitting equation:

[0094] Y=2.31+0.035A-0.099B-0.029C+0.0075AB+0.027AC-0.075BC-0.072A 2 -0.25B 2 -0.039C 2

[0095] The significance of the regression model and the results of variance analysis are shown in Table 3. The results show that the model is extremely significant (P < 0.0001), the lack of fit term is not significant (P > 0.05), and the determination coefficient of the model is R 2 =0.9832, Corrected determination coefficient R 2 Adj =0.9616, indicating that the model has a good fitting degree and the experimental error is relatively small. The model can be used to analyze and predict the yield of polysaccharides from Curcuma zedoaria. From the F value, it can be concluded that within the experimental range, the influence of various factors on the yield of polysaccharides from Curcuma zedoaria is extraction temperature> extraction time> solid-liquid ratio.

[0096] Table 2 Response surface analysis experimental design and the yield of polysaccharides from Curcuma zedoaria (%)

[0097] Experiment No. A(h) B(℃) C(g / mL) Experimental value Predicted value 1 3 90 40 2.31 2.308 2 3 90 40 2.35 2.308 3 3 90 40 2.27 2.308 4 3 90 40 2.3 2.308 5 3 90 40 2.31 2.308 6 3 100 50 1.81 1.8175 7 3 80 50 2.14 2.165 8 3 100 30 2.05 2.025 9 3 80 30 2.08 2.0725 10 4 90 50 2.26 2.23125 11 2 90 50 2.11 2.10625 12 4 90 30 2.23 2.23375 13 2 90 30 2.19 2.21875 14 4 100 40 1.91 1.93125 15 2 100 40 1.85 1.84625 16 4 80 40 2.11 2.11375 17 2 80 40 2.08 2.05875

[0098] Table 3. Significance of test results and variance analysis

[0099]

[0100] (2) Response surface interaction analysis

[0101] The results of the response surface interaction analysis are shown in Figure 2 As shown by Figure 2 It can be seen that the strongest interaction is between extraction temperature and solid-liquid ratio, followed by the interaction between extraction time and solid-liquid ratio, and the weakest interaction is between extraction time and extraction temperature.

[0102] After the polysaccharide precipitate collected under the above investigation conditions was dissolved, the polysaccharide aqueous solution was deproteinized using Sevage reagent. That is, Sevage reagent (n-butanol: chloroform = 1:4) was prepared, 1 / 3 volume of Sevage reagent was added to the polysaccharide aqueous solution, and after sufficient shaking, centrifugation (4000rpm, 5min) was performed to collect the upper polysaccharide aqueous solution, and the middle protein emulsion and the lower organic liquid were discarded. The obtained polysaccharide aqueous solution was concentrated by rotary evaporation at 50°C to remove the residual organic reagent, and then 3% activated carbon particles were added for decolorization. The crude polysaccharide aqueous solution was dried by a vacuum freeze dryer. The polysaccharide content was determined by the phenol-sulfuric acid method using D-anhydrous glucose as the standard. Each group was measured in parallel 3 times. The yield of the polysaccharide from Curcuma zedoaria was calculated using the following formula.

[0103] Y=cW1 / W2×100%

[0104] Wherein, Y is the polysaccharide yield, c is the polysaccharide content (%), W1 is the crude polysaccharide mass (g), and W2 is the original medicinal material mass (g).

[0105] The extraction conditions with the highest predicted value of the yield of Curcuma zedoaria polysaccharide are: extraction time 3.211h, extraction temperature 88.240℃, solid-liquid ratio 38.740g / mL. Considering operability, the extraction conditions were adjusted to: extraction time 3h, extraction temperature 90℃, solid-liquid ratio 1:40g / mL. Under this condition, the actual yield of Curcuma zedoaria polysaccharide was 2.305±0.01% (n=3), which is close to the theoretical value of 2.322%, indicating that the model is suitable for predicting the extraction conditions of Curcuma zedoaria polysaccharide.

[0106] The best preparation method of Curcuma zedoaria polysaccharide was determined through the research of the above Examples 1-3. After petroleum ether degreasing and ethanol purification, the fat-soluble components on the surface of the crushed raw Curcuma zedoaria medicinal materials were fully defatted and removed, and then the Curcuma zedoaria polysaccharide product was prepared by water extraction and alcohol precipitation.

[0107] The parameter selection ranges for each specific process step are as follows:

[0108] S1. Crush raw Curcuma zedoariae, and use petroleum ether to perform defatting and small molecule removal treatments to obtain defatted medicinal powder.

[0109] S2. Add 8-13 times amount of 90-95% ethanol to the defatted powder, extract under reflux in a water bath at 65-80°C for 2-5h, repeat 1-3 times, filter, collect the residue, evaporate the ethanol from the residue, dry at 30-60°C to obtain the Curcuma zedoaria solid powder for later use.

[0110] S3. Take the solid powder of Curcuma zedoaria, add 30-50 times the weight of water, reflux extraction at 80-95°C for 2-5 hours, repeat 1-4 times, filter, combine the filtrate, evaporate and concentrate to 1 / 7-1 / 4 times the volume, cool to room temperature, and obtain a concentrated solution.

[0111] S4. Take the concentrated solution, add 3 to 5 times the volume of 75v% or more ethanol, and let it stand at 1-6°C for 18-36 hours; collect the precipitate by centrifugation, dissolve the precipitate in pure water, and evaporate the ethanol; freeze-dry and collect the lyophilized powder to obtain the Curcuma zedoaria polysaccharide.

[0112] The patented method (patent number: ZL 201310400255.2) is used to prepare the vinegar-processed Curcuma zedoaria, that is, take an appropriate amount of raw Curcuma zedoaria, use 20 kg of 9° rice vinegar for every 100 kg of raw product, mix well and simmer for 2 hours, cook until the vinegar liquid is dry, place it at 120°C to dry for 30 minutes, take it out and cool it, and dry it to obtain the vinegar Curcuma zedoaria slices. Then, extract and obtain the purified polysaccharide of vinegar Curcuma zedoaria by the same method as in Example 1, and the crude product (named Cru-VCPP) is set aside.

[0113] Example 4

[0114] Isolation and purification of polysaccharide from Curcuma zedoaria

[0115] (1) First, the polysaccharides of Curcuma zedoariae before and after vinegar processing were separated and purified by DEAE-52 cellulose column chromatography.

[0116] The specific process steps are as follows:

[0117] ① Pretreatment of DEAE-52 cellulose filler: Weigh 100g of DEAE-52 cellulose resin dry powder filler, soak it in 20 times the volume of pure water for 24 hours, remove suspended matter and impurities after the filler is fully swollen, and remove the filtrate by suction. After vacuum filtration, soak the filler in 0.5mol / L NaOH solution for 30min, discard the upper layer of liquid, and rinse it repeatedly with pure water several times until the pH is neutral. Soak it in 0.5mol / L HCl solution for 30min, discard the upper layer of liquid, and rinse it repeatedly with pure water several times until the pH is neutral, and set aside.

[0118] ② Column loading: Use the wet column loading method to load the pretreated filler into a glass chromatography column of 2.5×60cm in size. After fixing the chromatography column vertically, add a small amount of pure water to the lower end of the column to remove the air in the column and avoid bubbles when loading the column. Add an appropriate amount of pure water to the pretreated DEAE-52 cellulose resin filler, stir evenly, slowly pour it into the chromatography column, let it sink naturally, open the lower switch to allow the pure water to flow out naturally, ensure that the filler is uniform, the column surface is flat, there are no bubbles, and there are no faults. When the filler reaches 2 / 3 of the column height, use pure water to balance the DEAE-52 cellulose chromatography column for 30 minutes.

[0119] ③ Loading: Take 500 mg of crude polysaccharides of Curcuma zedoariae before and after vinegar processing extracted by the best extraction process in Example 3, dissolve it in 20 mL of distilled water, centrifuge (8000 rpm, 4°C, 10 min), remove the precipitate, use a pipette to absorb the supernatant and slowly add it in a circle along the inner wall of the chromatography column.

[0120] ④ Elution and collection: Use pure water to elute on a DEAE-52 cellulose chromatography column with pure water, 0.1, 0.2, 0.3, and 0.4 mol / L NaCl solution to obtain purified components, eluting at a flow rate of 0.6 mL / min. Collect the eluate in a 5 mL test tube. Use the phenol-sulfuric acid method to measure the absorbance at a UV absorption wavelength of 490 nm and draw an elution curve.

[0121] The results of DEAE-52 cellulose purification elution curve are shown in Figure 3 , elution curves of DEAE-52 cellulose purification of raw Curcuma zedoaria polysaccharide (A) and Curcuma acetozea polysaccharide (B).

[0122] Each collected fraction was concentrated and dialyzed for 48 hours using a 3500Da dialysis bag to remove salt. The resulting dialysate was freeze-dried to obtain each component of raw Curcuma zedoaria polysaccharide (RCPP-0, RCPP-0.1, RCPP-0.2, RCPP-0.3, RCPP-0.4), and each component of vinegar Curcuma zedoaria polysaccharide (VCPP-0, VCPP-0.1, VCPP-0.2, VCPP-0.3, VCPP-0.4).

[0123] In the nomenclature, RCPP-0 represents the pure water elution component, and the elution peak is significant and symmetrical, indicating that the raw Curcuma zedoaria polysaccharide mainly contains neutral polysaccharide (RCPP-0), and the yield of RCPP-0 is 7.454±0.6549% compared with the mass of the sample. RCPP-0.1, -0.2, etc. represent the elution products of different concentrations of sodium chloride solution, -0.1 corresponds to the elution product of 0.1M sodium chloride solution, 0.2 corresponds to the elution product of -0.2M sodium chloride solution, -0.3 corresponds to the elution product of 0.3M sodium chloride solution, and 0.4 corresponds to the elution product of 0.4M sodium chloride solution. RCPP represents raw Curcuma zedoaria polysaccharide, VCPP represents vinegar Curcuma zedoaria polysaccharide, and VCPP-0 represents the pure water elution component. The elution peak is significant and symmetrical, indicating that vinegar Curcuma zedoaria polysaccharide mainly contains neutral polysaccharide (VCPP-0). The yield of VCPP-0 is 8.005±0.4937% based on its mass and sample amount.

[0124] (2) Then, the polysaccharide of Curcuma zedoariae before and after vinegar treatment was chromatographed on Sephadex G-100 gel column

[0125] ① Pretreatment of Sephadex G-100 dextran gel column: Pretreatment of Sephadex G-100 filler: soak in ultrapure water, fully swell, pour off the gel fragments, and repeat several times.

[0126] ② Column packing and column balancing: refer to the column packing and column balancing method of “(1) Chromatography of Curcuma zedoaria polysaccharide before and after vinegar treatment on DEAE-52 cellulose column”.

[0127] ③ Loading: Prepare 10 mg / mL RCPP-0 and VCPP-0 sugar solutions respectively (using the above-mentioned DEAE-52 cellulose column chromatography separation and purification products), take 50 mL to pass through a 0.45 μm filter membrane, and apply the sample with a rubber-tipped dropper. Elute with ultrapure water at a flow rate of 0.4 mL / min, and collect 4 mL of eluate in each tube. Determine the total sugar by the phenol-sulfuric acid method until no polysaccharide is washed out of the eluate. Use the absorbance value of each tube as the ordinate and the number of tubes as the abscissa to draw an elution curve and merge the same components. Mark the elution component of raw Curcuma zedoaria as RCPP, and the elution component of vinegar Curcuma zedoaria as VCPP. Concentrate under reduced pressure, dialyze, and freeze-dry for later use. See the results of the SephadexG-100 purification elution curve. Figure 4 , wherein (A) is the purification elution curve of raw Curcuma zedoaria polysaccharide, and (B) is the Sephadex G-100 purification elution curve of Curcuma acetozea polysaccharide (B).

[0128] The results of Sephadex G-100 purification showed that the separation degree of RCPP-0 and VCPP-0 obtained by DEAE-52 cellulose column chromatography was high. After further purification by Sephadex G-100 gel column chromatography, a uniform purified polysaccharide RCPP from Curcuma zedoaria was obtained. Figure 4 Middle (A); and purified polysaccharide VCPP from Curcuma acetosporum, see Figure 4 Middle (B).

[0129] The yield of RCPP was 71.07±1.164% of the mass and the yield of VCPP was 70.13±1.635% of the mass and the amount of sample loaded.

[0130] Example 5

[0131] Determination of molecular weight: HPGPC was used to determine the molecular weight and purity of RCPP and VCPP. RCPP and VCPP sample solutions (2 mg / mL) and 8 Shodex STANDARD standard solutions (dextran, 2 mg / mL) were prepared respectively, with molecular weights of 5900, 9600, 21 100, 47100, 107000, 200000, 344000, and 708000 Da. BRT105-103-101 series gel column (8×300mm) was used, with 0.2mol / L NaCl solution as the mobile phase, a flow rate of 0.8mL / min, a column temperature of 40°C, an injection volume of 25μL, and a differential detector RID-10A for analysis for 50min.

[0132] Molecular weight determination results: HPGPC was used to determine the molecular weight of RCPP and VCPP, and the standard curves of weight average molecular weight and number average molecular weight were drawn. Figure 5 As shown, the molecular weight of RCPP was calculated by regression equations lgMw = -0.2237x + 11.391 (R2 = 0.9953) and lgMn = -0.2237x + 11.383 (R2 = 0.9994). Figure 6 As shown, by lg Mw = -0.2004x + 10.872 (R 2 =0.9998) and lg Mn = -0.201x + 10.876 (R 2 =0.9996) to calculate the molecular weight of VCPP (see Table 4). Figure 7 (A) shows that the elution time of RCPP mobile phase is about 33.767min, and the water peak is at 37.825min. Figure 7(B) shows that the elution time of VCPP mobile phase is about 35.54min. Both RCPP and VCPP are single symmetrical peaks. From the results in Table 4, it can be seen that RCPP and VCPP have lower molecular weights, which are 7244Da and 5621Da, respectively. The Mw / Mn of RCPP and VCPP are both less than 2, indicating that the molecular weight distribution is relatively uniform.

[0133] Table 4 Molecular weight determination results of RCPP and VCPP

[0134] sample Mw(Da) Mn(Da) Mw / Mn RCPP 7244 7079 1.02 VCPP 5621 5401 1.04

[0135] Example 6

[0136] Determination of monosaccharide composition: HPIC analysis was used to determine the monosaccharide composition of RCPP and VCPP. Fucose (Fuc), glucose (Glc), rhamnose (Rha), arabinose (Ara), galactose (Gal), mannose (Man), fructose (Fru), xylose (Xyl), ribose (Rib), galacturonic acid (GalA), and glucuronic acid (GlcA) were used as monosaccharide standards. 5 mg of RCPP and VCPP samples were weighed, 2 mL of trifluoroacetic acid (TFA) (3 mol / L) was added, and hydrolyzed at 120°C for 3 h. A Dionex Carbopac TM PA10 (4*250 mm) column was used with the following mobile phases: A: H2O; B: 500 mM NaOH & 50 mM NaOAc; C: 20 mM NaOH. The flow rate was 1.0 mL / min and the samples were analyzed at 30°C.

[0137] Results of monosaccharide composition: HPGPC was used to analyze the monosaccharide composition of RCPP and VCPP. The results are shown in Table 5. RCPP is mainly composed of 94.8% Glc, followed by 3% Ara and 2.2% Gal. VCPP is mainly composed of 92.2% Glc, followed by 4.9% Ara and 2.9% Gal. The types of monosaccharides in Curcuma zedoaria polysaccharide did not change before and after vinegar processing, but there was a difference in the proportion of monosaccharides.

[0138] Table 5 Monosaccharide composition determination results of RCPP and VCPP

[0139] sample Ara Gal Glc RCPP 3 2.2 94.8 VCPP 4.9 2.9 92.2

[0140] Example 7

[0141] Fourier transform infrared spectroscopy: FT-IR analysis was used to identify the functional groups of RCPP and VCPP. The FT-IR instrument was used at 4000-400 cm -1 The infrared spectrum is detected in the wavelength range.

[0142] Measurement results: The infrared absorption spectra of RCPP and VCPP are as follows: Figure 8 As shown. RCPP is at about 3280cm -1 At 3260cm, VCPP -1 A strong and broad absorption band was observed at 2930 cm -1 The peak of the band corresponds to the stretching vibration of carbon-hydrogen bonds, indicating the existence of intermolecular hydrogen bonds. -1 and 2930cm -1 The two bands at 1650cm are the characteristic absorption peaks of polysaccharides. -1 The peak at 1150cm indicates the stretching vibration of the carboxyl group. This peak is a common peak of RCPP and VCPP, suggesting that the polysaccharide sample may contain uronic acid. -1 and 1010cm -1 At 1150cm, VCPP -1 and 998cm -1 The two absorption peaks observed at 852 cm-1 are characteristic absorption peaks of COC of glycosidic bond, indicating the possible existence of pyranose ring. -1 At 847cm, VCPP -1 The weak absorption bands are shown at , which are the characteristics of α-configuration pyranose and β-configuration arabinose in the polysaccharide chain, indicating that it contains glucose and arabinose, which is consistent with the monosaccharide composition results. The infrared spectra of RCPP and VCPP both show typical polysaccharide absorption peaks, and there is no significant difference in the characteristic peak spectra.

[0143] Example 8

[0144] Determination of apparent structure: SEM was used to study the apparent structure of RCPP and VCPP. 2.0 mg of RCPP and VCPP samples were weighed, the surface was treated with gold spraying, the apparent structure of RCPP and VCPP was observed, and images (100×-2000×) were collected.

[0145] Measurement results: The apparent structures of RCPP and VCPP were observed using SEM. Fig. 9As shown in the figure, RCPP shows a rough surface, and at a higher magnification, it presents irregular granular aggregates. VCPP shows a loose and porous surface, and at a higher magnification, it is distributed with holes of different sizes, presenting a loose honeycomb shape. RCPP and VCPP have different apparent structures, indicating that vinegar processing affects the morphological characteristics of Curcuma zedoaria polysaccharides. These differences may cause different biological activities of RCPP and VCPP.

[0146] Example 9

[0147] Methylation analysis: Referring to the analysis method of Guangjing Chen @2018, the methylation analysis of RCPP and VCPP was slightly modified. First, the free hydroxyl groups in the polysaccharide sample were methylated, TFA was added for hydrolysis, and then acetic anhydride was added for acetylation to obtain the product sample, which was finally determined by gas chromatography-mass spectrometry (GC-MS).

[0148] GC-MS chromatographic conditions: RXI-5SIL MS column (30*0.25mm*0.25μm), starting temperature: 120℃, heating rate: 3℃ / min, heating to 250℃, maintaining for 5min; injection port and detector temperature: 250℃, carrier gas: helium (He), flow rate: 1 mL / min.

[0149] Results: The glycosidic bond connection mode of monosaccharide residues of RCPP and VCPP was studied by methylation analysis, and the connection mode of monosaccharides was obtained by GC-MS analysis. Fig.10 and Fig.11 is the GS-MS total ion current result, where Fig.10 is the GS-MS total ion current of RCPP; Fig.11 The GS-MS total ion current of VCPP is shown in Table 6. There are 9 identifiable methylated glycoside residues in RCPP. The main monosaccharide residue connection modes in RCPP are →4)-Glcp-(1→(34.3%) and Glcp-(1→(13%).

[0150] Table 6 Glycosidic bond composition of RCPP

[0151]

[0152] As shown in Table 7, there are 8 identifiable methylated glycoside residues in VCPP. The main monosaccharide residue connection modes in VCPP are →3,4)-Glcp-(1→(43.3%) and →4)-Glcp-(1→(33.9%). The methylation results of RCPP and VCPP showed that both RCPP and VCPP were mainly composed of glucose, galactose and arabinose, which was consistent with the structure of monosaccharide composition. The main monosaccharide residue connection mode of Curcuma zedoaria polysaccharide changed after vinegar treatment.

[0153] Table 7 Glycosidic bond composition of VCPP

[0154]

[0155] Example 10

[0156] Nuclear magnetic resonance spectroscopy determination: About 50 mg of the RCPP and VCPP samples finally separated by Sephadex G-100 dextran gel column in Example 4 were weighed, dissolved in 1 mL of deuterium oxide (D2O), freeze-dried three times, transferred into nuclear magnetic resonance tubes, and the RCPP and VCPP spectrometer was used to record the 1 H NMR and 13 C NMR spectrum.

[0157] Result: Passed 1 H NMR and 13 C NMR spectroscopy was used to preliminarily analyze the structural characteristics of RCPP and VCPP. Fig.12 and Fig.13 As shown, Fig.12 It is the purified polysaccharide RCPP from Curcuma zedoaria 1 H NMR spectroscopy; Fig.13 It is the purified polysaccharide VCPP from Curcuma zedoariae 1 H NMR spectroscopy.

[0158] RCPP 1 The H NMR spectrum shows obvious multiple anomeric proton signals at δ3.26-5.41ppm and VCPP at δ3.27-5.40ppm, which are typical signals of polysaccharides. The anomeric hydrogen proton signals of α-configuration sugar residues are in the range of δ5.0-6.0ppm, while the hydrogen proton signals of β-configuration sugar residues are in the range of δ5.0-4.50ppm. The signals in the range of δ4.50-3.20ppm are due to the protons of C2 to C6 in the glycoside ring. RCPP has proton signals in the range of δ5.41-4.65ppm, and VCPP has proton signals in the range of δ5.40-4.56ppm, indicating that both RCPP and VCPP contain α- and β-configuration sugar residues. In addition, RCPP and VCPP show strong signals in the range of δ3.26-3.50ppm and δ3.27-3.44ppm, respectively, which may be due to the presence of more -OCH3 in polysaccharides.

[0159] RCPP and VCPP 13 C NMR spectrum results are shown in Fig.14 and Fig.15 ,in, Fig.14 It is the purified polysaccharide RCPP from Curcuma zedoaria 13 C NMR spectroscopy; Fig.15 It is the VCPP of Curcuma acetosporum polysaccharide 13 C NMR spectroscopy.

[0160] From the figure, we know that RCPP has anomeric carbon signals at δ109.23ppm, and VCPP has anomeric carbon signals at δ106.75ppm and δ109.51ppm, which are β-type pyranose, and the peak of α-type pyranose is less than δ101ppm. This shows that both RCPP and VCPP contain α- and β-type pyranose, which is consistent with the infrared spectrum, 1 The results of H NHM were consistent. δ110~90ppm was attributed to terminal carbon, and δ90~60ppm was attributed to non-terminal carbon. Both RCPP and VCPP showed dense response signals in the range of δ85.00~70.00ppm, proving that RCPP and VCPP were heteropolysaccharides, which was consistent with the results of GC-MS analysis.

[0161] Embodiment 11

[0162] Determination of total sugar content of Curcuma zedoaria polysaccharide: The total sugar content of RCPP and VCPP was determined by phenol-sulfuric acid method. Weigh 10 mg of D-anhydrous glucose standard and prepare a 1 mg / mL glucose standard solution with deionized water. Accurately pipette 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 mL of glucose standard solution, add deionized water to make the volume 2 mL, and then pipette 0.2 mL of glucose standard solution respectively, and add 0.2 mL of deionized water to prepare a series of glucose standard solutions. Using the phenol-sulfuric acid method, take 1 mL of the standard solution, add 1 mL of 6% phenol, immediately add 5 mL of concentrated sulfuric acid, shake evenly, boil in a boiling water bath for 15 minutes, ice in an ice water bath for 10 minutes, cool to room temperature, measure the absorbance value A at 490 nm, and draw a glucose standard curve. Use an enzyme reader to measure the absorbance value A at 490 nm, and draw a glucose standard curve y = 4.9419x + 0.0018 (R 2 =0.994), and a good linear relationship was observed in the range of 0.01-0.1902 mg / mL.

[0163] Determination of samples: Cru-RCPP, Cru-VCPP, RCPP and VCPP were weighed and prepared into a solution with a concentration of 1.0 mg / mL with pure water. 1.0 mL of RCPP and VCPP were taken in glass test tubes respectively, and each sample was paralleled in 3 groups. The color was developed according to the above-mentioned gradient glucose standard solution colorimetric method, and the absorbance value A was determined by the phenol-sulfuric acid method. The total sugar content of Cru-RCPP, Cru-VCPP, RCPP and VCPP was calculated according to the glucose standard curve.

[0164] Example 12

[0165] Determination of uronic acid content: Using galacturonic acid as the standard, the uronic acid content of Cru-RCPP, Cru-VCPP, RCPP and VCPP was determined by the m-hydroxybiphenyl colorimetric method. Prepare sodium tetraborate / sulfuric acid solution (reagent A) and sodium hydroxide / m-hydroxybiphenyl solution (reagent B) according to the literature method. Weigh 10.0 mg of galacturonic acid in a 25 mL volumetric flask, dilute with pure water to prepare a 0.4 mg / mL galacturonic acid standard solution. Accurately pipette 0.05, 0.1, 0.2, 0.3, 0.8, 1.0 mL of galacturonic acid standard solution, add pure water, and prepare a gradient uronic acid standard solution of 0.02, 0.04, 0.08, 0.12, 0.32, 0.40 mg / mL. Place the test tubes of each standard solution in ice water. Add 3 mL of reagent A to each test tube, shake and mix well, then react in a boiling water bath for 5 minutes, take out, and quickly put it in an ice water bath to cool. After cooling completely, add 50 μL of the reagent, shake and mix, and use an enzyme reader to measure the absorbance value A at 530 nm to draw a standard curve of uronic acid y=4.7754x-0.0144 (R 2 =0.9983), and a good linear relationship was observed in the range of 0.02005-0.2005 mg / mL.

[0166] Determination of samples: Take Cru-RCPP, Cru-VCPP, RCPP and VCPP, prepare a polysaccharide sample solution with a concentration of 1.0 mg / mL with pure water, draw 1.0 mL of the sample solution into a test tube, and each sample is paralleled in 3 groups. Develop the color according to the above-mentioned gradient uronic acid standard solution colorimetric method, determine the absorbance value A by the m-hydroxybiphenyl colorimetric method, and calculate the uronic acid content of Cru-RCPP, Cru-VCPP, RCPP and VCPP according to the uronic acid standard curve.

[0167] Example 13

[0168] Protein content determination: The protein content of Cru-RCPP, Cru-VCPP, RCPP and VCPP was determined using a BCA microprotein assay kit, and the protein content was calculated (see Formula 2).

[0169]

[0170] Determination of samples: Take Cru-RCPP, Cru-VCPP, RCPP and VCPP, prepare a solution with a concentration of 1 mg / mL with pure water, take 25 μL RCPP and VCPP solution in 96 wells respectively, set up 3 parallel wells for each sample, add 200 μL BCA working solution to each well, shake for 30 seconds to mix thoroughly, cover the microplate, incubate at 37°C for 30 minutes, and cool to room temperature. The absorbance value A was determined by the BCA method, and the protein content of Cru-RCPP, Cru-VCPP, RCPP and VCPP was calculated according to formula 2.

[0171] The total sugar content, uronic acid and protein content of Cru-RCPP, Cru-VCPP, RCPP and VCPP measured in the above Examples 11-13 are shown in Table 8.

[0172] Table 8 Results of total sugar content, uronic acid and protein content of Cru-RCPP, Cru-VCPP, RCPP and VCPP

[0173]

[0174] Note: Each value is expressed as an average value (n=3).

[0175] The above examples respectively perform quality detection and analysis of the prepared Curcuma zedoaria polysaccharide in multiple dimensions to determine the optimal extraction process, specifically, the extraction time is 3.2h, the extraction temperature is 90°C, the solid-liquid ratio is 40g / mL, the number of extractions is 3 times, the alcohol precipitation concentration is 80%, and the yield of Curcuma zedoaria polysaccharide reaches 2.305±0.01%. DEAE-52 cellulose resin and SephadexG-100 dextran gel are used to separate and purify the crude Curcuma zedoaria polysaccharide before and after vinegar processing, respectively, to obtain raw Curcuma zedoaria purified polysaccharide (RCPP) and vinegar Curcuma zedoaria purified polysaccharide (VCPP).

[0176] The total sugar contents of Cru-RCPP, Cru-VCPP, RCPP and VCPP were determined by phenol-sulfuric acid method, m-hydroxybiphenyl colorimetric method and BCA protein detection method, which were 22.77±0.03799%, 42.59±0.03767%, 79.81±0.227% and 87.07±0.1769%, respectively. The total sugar content increased after purification, and the total sugar content of VCPP was higher than that of RCPP. The uronic acid contents of Cru-RCPP, Cru-VCPP, RCPP and VCPP were 6.866±0.03799%, 42.59±0.03767%, 79.81±0.227% and 87.07±0.1769%, respectively. ±0.0590%, 10.1±0.04225%, 3.702±0.01971% and 7.145±0.2301%, the uronic acid content of VCPP was higher than that of RCPP; the protein contents of Cru-RCPP, Cru-VCPP, RCPP and VCPP were 26.97±0.1784%, 25.49±0.1692%, 3.978±0.0731% and 3.533±0.06824%, respectively. The protein content decreased after purification, and the protein contents of RCPP and VCPP were both low.

[0177] Embodiment 14

[0178] Liver injury mouse experiment

[0179] 1.1 Preparation of Curcuma zedoaria polysaccharide before and after vinegar treatment

[0180] The Curcuma zedoaria polysaccharide used the Curcuma zedoaria polysaccharide separated and purified in Example 4: crude Curcuma zedoaria polysaccharide (Cru-RCPP), crude Curcuma zedoaria polysaccharide with vinegar (Cru-VCPP), purified Curcuma zedoaria polysaccharide (RCPP), purified Curcuma zedoaria polysaccharide with vinegar (VCPP).

[0181] The patented method for making Curcuma zedoaria vinegar (patent number: ZL 201310400255.2) developed by the research team was used for preparation, that is, an appropriate amount of raw Curcuma zedoaria was taken, 20 kg of 9° rice vinegar was used for every 100 kg of raw product, mixed well and simmered for 2 hours, boiled until the vinegar liquid was dry, placed at 120℃ to dry for 30 minutes, taken out and cooled, and dried to obtain the vinegar Curcuma zedoaria decoction pieces.

[0182] 1.2 Preparation of Curcuma zedoaria polysaccharide solution before and after vinegar treatment

[0183] Preparation of drug solution containing Curcuma zedoaria polysaccharide: Cru-RCPP, Cru-VCPP, RCPP and VCPP were dissolved in pure water respectively, and high and low dose (400 mg / kg / d, 200 mg / kg / d) polysaccharide drug solutions were prepared for oral administration to mice.

[0184] Preparation of modeling drugs: Accurately weigh an appropriate amount of thioacetamide, fully dissolve it in physiological saline, and prepare a drug solution with a concentration of 10 mg / mL for use.

[0185] Preparation of positive drug: Accurately weigh an appropriate amount of silybin meglumine tablets, fully dissolve them in normal saline, and prepare a drug solution with a concentration of 6 mg / mL for use.

[0186] 1.3 Establishment of TAA-induced liver injury model in mice and drug intervention

[0187] A total of 110 male Kunming mice were randomly divided into 11 groups, with 10 mice in each group, including control group, model group, positive group, high-dose purified polysaccharide from raw Curcuma zedoaria (RCPP-H), low-dose purified polysaccharide from raw Curcuma zedoaria (RCPP-L), high-dose purified polysaccharide from vinegar Curcuma zedoaria (VCPP-H), low-dose purified polysaccharide from vinegar Curcuma zedoaria (VCPP-L), high-dose crude polysaccharide from raw Curcuma zedoaria (Cru-RCPP-H), low-dose crude polysaccharide from raw Curcuma zedoaria (Cru-RCPP-L), high-dose crude polysaccharide from vinegar Curcuma zedoaria (Cru-VCPP-H), low-dose crude polysaccharide from vinegar Curcuma zedoaria (Cru-VCPP-L).

[0188] The experimental plan is shown in Fig.16 Each group was given the drug by gavage once a day for 4 consecutive days, and the weight was weighed every day. One hour after the drug administration on the 4th day, all groups except the Control group were intraperitoneally injected with TAA at a dose of 100 mg / kg / d to establish a liver injury model. The control group was intraperitoneally injected with the same dose of normal saline.

[0189] Weigh the mice 24 hours after modeling, remove the eyeballs to collect blood, and kill them by dislocating the neck. Remove the complete liver of the mice and weigh and record. After the mouse blood was allowed to stand at room temperature for 0.5 hours, centrifuge (3000rpm, 4℃, 10min), take the serum supernatant, freeze it at -20℃ for later use. Open the abdominal cavity, separate the terminal ileum tissue of about 5cm in length, and wash the intestinal contents with pre-cooled (4℃) PBS. One part was fixed in 4% paraformaldehyde and stored for later use; the other part was placed in a cryopreservation tube and frozen in a -80℃ refrigerator for later use. Collect mouse feces in a sterile EP tube and store it in a -80℃ refrigerator for later use.

[0190] 1.4 Weight gain of mice

[0191] Fig.17 Figure 2 is the weight change of mice. During the 1-4 days of drug administration, the weight of mice in each group showed a uniform growth trend, and there was no significant difference in the weight of mice between the groups. The weight loss on the 5th day was due to the fasting of mice in each group after modeling on the 4th day.

[0192] 1.5 Effects of Curcuma zedoaria polysaccharide before and after vinegar treatment on the liver of mice with liver injury

[0193] 1.5.1 Mouse liver morphology and liver index determination

[0194] The appearance of the whole liver just removed from each group of mice was observed and compared. The size of the liver, surface color, touch, and the presence of fine particles inside were the key points of observation and recorded. The livers of the mice in the Control group were of moderate size, with a tender and smooth surface without granularity, sharp edges, and blood red color. Compared with the Control group, the livers of the Model group were enlarged, with a rough surface, and the color turned gray-red. Compared with the Model group, the pathological morphology of the livers in the Positive group and the groups treated with Curcuma zedoaria polysaccharide before and after vinegar processing (RCPP-H group, RCPP-L group, VCPP-H group, VCPP-L group, Cru-RCPP-H group, Cru-RCPP-L group, Cru-VCPP-H group, and Cru-VCPP-L group) were significantly improved, with smoother surfaces and more rosy colors. There were no significant differences in the morphological appearance of the livers of mice between the treatment groups. The results showed that Curcuma zedoaria polysaccharide before and after vinegar processing could alleviate the pathological changes in the appearance of the livers of mice with acute liver injury induced by TAA to a certain extent. The results of the morphological appearance of the livers of mice are shown in Table 1. Fig.18 .

[0195] The weight of the mouse and the weight of the fresh whole liver were weighed before surgery, and the liver index was calculated according to the following formula 3.

[0196] Liver index = liver weight (g) / mouse body weight (g) × 100%

[0197] Compared with the Control group, the liver index of the mice in the Model group increased by 31.38%, with a significant difference (P < 0.0001), indicating that the liver of the mice in the Model group was significantly damaged and swollen. Compared with the Model group, the liver index of the mice in each drug-treated group decreased to varying degrees, with a decrease of 5.19% in the RCPP-H group, 13.17% in the RCPP-L group (P < 0.5), 7.58% in the VCPP-H group, 5.54% in the VCPP-L group, 3.73% in the Cru-RCPP-H group, 4.37% in the Cru-RCPP-L group; 4.80% in the Cru-VCPP-H group; 7.42% in the Cru-VCPP-L group; the groups of vinegar Curcuma zedoaria polysaccharide were closer to the normal level of the control group than the groups of raw Curcuma zedoaria polysaccharide, and the purified polysaccharide showed better ability to alleviate liver damage than the crude polysaccharide. The above research results show that both the curcuma zedoaria polysaccharide and the vinegar-processed curcuma zedoaria polysaccharide can protect the liver in mice with thioacetamide-induced liver injury, and the VCPP-H group has a better efficacy in alleviating liver injury. The results of the effects of curcuma zedoaria polysaccharide and the vinegar-processed curcuma zedoaria polysaccharide on the liver index of mice are shown in Table 9.

[0198] Table 9 Effects of Curcuma zedoaria polysaccharide on liver index of mice before and after vinegar treatment (±SD, n=10)

[0199] Group Liver index / % Control group 4.626±0.3024 Model group <![CDATA[6.078±0.2136 **** <!-- 15 -->]]> Positive group 5.647±0.2724 High-dose group of purified polysaccharide from Curcuma zedoariae (RCPP-H group) 5.762±0.3008 Low-dose group of purified polysaccharide from Curcuma zedoariae (RCPP-L group) <![CDATA[5.271±0.3966 # ]]> High-dose group of purified polysaccharide from Curcuma acetosporum (VCPP-H group) 5.617±0.4708 Low-dose group of purified polysaccharide from Curcuma acetosporum (VCPP-L group) 5.741±0.4782 High-dose group of crude polysaccharide from Curcuma zedoariae (Cru-RCPP-H group) 5.851±0.4857 Low-dose group of crude polysaccharide from Curcuma zedoariae (Cru-RCPP-L group) 5.812±0.5022 High-dose group of crude polysaccharide from Curcuma acetosporum (Cru-VCPP-H group) 5.786±0.6461 Low-dose group of crude polysaccharide from Curcuma acetosporum (Cru-VCPP-L group) 5.627±0.3873

[0200] (Note: ****P<0.0001, vs. control group; #P<0.5, vs. model group)

[0201] 1.5.2 Determination of liver function indicators

[0202] According to the instructions of the kit, the liver function indexes were determined by an automatic biochemical analyzer. Compared with the Control group, the serum ALT (P < 0.0001) and AST (P < 0.0001) levels of the mice in the Model group were significantly increased. Compared with the Model group, the ALT and AST levels of each drug-treated group were reduced to varying degrees, and showed a certain dose dependence. However, there were certain differences in the ALT and AST levels among the drug-treated groups. Among them, the VCPP-H group significantly reduced the ALT (P < 0.0001) and AST levels (P < 0.0001). Compared with the groups of equal doses of raw Curcuma zedoaria polysaccharide, there was no significant difference in the groups of vinegar Curcuma zedoaria polysaccharide. The above results show that Curcuma zedoaria polysaccharide before and after vinegar preparation can play a liver-protective role in mice with TAA-induced liver injury, among which the VCPP-H group had the best improvement effect. The results of the measurement of liver function indexes of mice in each group, the expression levels of ALT and AST in serum are shown in Fig.19 , where the left side shows the ALT expression level, and the right side shows the AST expression level in serum.

[0203] 1.5.3 Determination of SOD and MDA biochemical indicators

[0204] Take an appropriate amount of frozen mouse liver tissue, add physiological saline (4°C) as a homogenization medium at a mass ratio of 1:9, prepare a 10% tissue homogenate in an ice bath environment, centrifuge (5000rpm, 4°C, 10min), and take the supernatant for use. Use ELISA method to detect the total protein content of liver tissue according to the instructions of the kit, measure the OD value of SOD at 550nm, measure the OD value of MDA at 532nm, and calculate according to formula 4 and formula 5 respectively.

[0205]

[0206]

[0207] Compared with the Control group, the SOD level in the Model group was significantly decreased (P<0.001), while the MDA level was significantly increased (P<0.0001). Compared with the Model group, the SOD and MDA levels of each drug-treated group were reversed to varying degrees, and were dose-dependent. The SOD level of the RCPP-H group was significantly increased (P<0.05), and the SOD level of the VCPP-H group was significantly increased (P<0.001). The MDA level of each group of purified Curcuma zedoaria polysaccharide before and after vinegar processing was significantly decreased (P<0.0001), the MDA level of the Cru-RCPP-H group was significantly decreased (P<0.001), the MDA level of the Cru-VCPP-L group was significantly decreased (P<0.05), and the MDA level of the vinegar Curcuma zedoaria crude polysaccharide group was significantly decreased (P<0.01). Compared with the groups of raw Curcuma zedoaria polysaccharide with equal doses, there was no significant difference in the groups of vinegar Curcuma zedoaria polysaccharide. The above research results show that both the curcuma polysaccharide and the vinegar-processed curcuma polysaccharide can protect the liver in mice with thioacetamide-induced liver damage, and the curcuma polysaccharide with vinegar has better anti-oxidative damage efficacy, among which the VCPP-H group has the most significant improvement effect. The results of the determination of SOD and MDA biochemical indicators are shown in Fig. 20 , where the left side is the comparison of SOD biochemical indicators, and the right side is the comparison of MDA biochemical indicators.

[0208] 1.5.4 Results of H&E staining pathological evaluation of liver tissue

[0209] Small pieces of liver tissue were cut and immersed in 4% paraformaldehyde for fixation, dehydration, embedding, dewaxing, staining, sectioning, and drying. Finally, an optical microscope was used to observe and collect images for analysis. The results of liver sections of mice in the Control group showed that it had a complete cell structure, clear liver lobule structure, neat and regular arrangement of liver plates, hepatocytes and hepatic sinusoids, clear and round nuclei, and no obvious inflammatory cell infiltration and cell necrosis. The results of liver sections of mice in the Model group showed that the liver lobule structure was damaged, hepatocytes and hepatic sinusoids were irregularly arranged, cell nuclei were deformed, and inflammatory cells were infiltrated. Among the drug-treated groups, the VCPP-H group had a relatively clear and complete liver lobule structure, with rare swelling and enlargement of hepatocytes and no obvious inflammatory cell infiltration; while the liver tissues of the VCPP-L group, RCPP-H group, and RCPP-L group had more irregular cavities and inflammatory cell infiltration. The pathological sections of the Cru-RCPP-H group, Cru-RCPP-L group, Cru-VCPP-H group and Cru-VCPP-L group showed that the hepatocytes were watery and swollen, with loose cytoplasm, and had more irregular cavities and inflammatory cell infiltration. The above results show that the curcuma polysaccharide before and after vinegar treatment can alleviate the liver damage of mice with thioacetamide-induced liver damage to varying degrees, among which the VCPP-H group had the most significant effect. The pathological results of the liver tissue HE staining of mice in each group are shown in Fig.21 .

[0210] 1.6 Effects of Curcuma zedoaria polysaccharide before and after vinegar treatment on the intestinal mechanical barrier of mice with liver injury

[0211] 1.6.1 Results of H&E staining pathological evaluation of ileum tissue

[0212] An appropriate amount of intestinal tissue was cut and immersed in 4% paraformaldehyde for 24 hours. After HE staining, an optical microscope was used to observe and collect images for analysis. The mice in the Control group had a relatively complete ileal mucosal structure and a thicker intestinal endothelium, with neatly arranged villi and normal epithelial cell morphology; the mice in the Model group had severe ileal damage, destroyed tissue structure, broken villi, and widened tight junction gaps of epithelial cells. The ileal tissue damage in each group of Curcuma zedoaria polysaccharide administration was alleviated to varying degrees, and the ileal section results of the VCPP-H group were closest to the normal level. The ileal tissue structure was relatively complete, the villi morphology was improved and arranged neatly, the inflammatory cell infiltration was less, and the intestinal mucosal epithelium was intact and normal in morphology. The above results show that Curcuma zedoaria polysaccharides before and after vinegar processing can improve the ileal pathological changes of mice with thioacetamide-induced liver damage, among which the VCPP-H group had the most significant effect, and had a good protective effect on the ileal structure. The pathological results of H&E staining of ileal tissue of mice in each group are shown in Fig. 22 .

[0213] 1.6.2 Results of immunohistochemical staining of occludin and ZO-1 in ileum tissue

[0214] Immunohistochemical staining was used to detect the expression of occludin and ZO-1 in the ileum. Occludin and ZO-1 proteins were positively expressed in the intestinal epithelial and glandular epithelial cell membranes and the cytoplasmic area proximal to the membrane in the Control group, Positive group, and each group of Curcuma zedoaria polysaccharide administration before and after vinegar processing, with a large and relatively uniform brown-yellow staining area. In the Model group, the brown-yellow area decreased, and the expression of occludin and ZO-1 proteins showed a downward trend, indicating that the expression of occludin and ZO-1 proteins in the ileum would be reduced after the mouse modeling liver injury. Compared with the Control group, the expression of occludin protein in the Model group was significantly reduced (P<0.0001), and the expression of ZO-1 protein was significantly reduced (P<0.001). Compared with the Model group, the expression of occludin and ZO-1 proteins in each administration group increased to varying degrees. Among them, the expression of occludin protein in each group of Curcuma zedoaria polysaccharide administration before and after vinegar processing was significantly increased (P<0.0001). The expression of ZO-1 protein in the VCPP-H group was significantly increased (P < 0.05). There was no significant difference in the expression of Occludin and ZO-1 proteins between the different dose groups of Curcuma zedoaria polysaccharide before and after vinegar treatment. Compared with the groups treated with the same dose of raw Curcuma zedoaria polysaccharide, the high-dose groups of vinegar Curcuma zedoaria polysaccharide (VCPP-H group, Cru-VCPP-H group) showed a stronger trend of increasing the expression of Occludin and ZO-1 proteins, and the protective effect on the ileum of mice with liver injury was the most obvious.

[0215] The positive expression results of occludin in the ileal mucosal tissue of each group are shown in Fig.23 The positive expression results of ZO-1 in the ileal mucosa of each group are shown in Fig.24 The expression results of occludin and ZO-1 in the ileal mucosa of each group are shown in Fig.25 , where the left side shows the comparison of Occludin expression results, and the right side shows the comparison of ZO-1 expression results.

[0216] Animal experiments investigated the improvement of Curcuma zedoaria polysaccharide on TAA-induced liver injury mouse model from the aspects of mouse liver protection, intestinal barriers (mechanical barriers, biological barriers, immune barriers), and the impact on the "intestinal-liver axis". The liver function indexes and liver tissue pathological changes of liver injury mice before and after administration were detected by automatic biochemical analyzer, ELISA method and H&E staining method. Combined with the liver index, it was found that all doses of Curcuma zedoaria polysaccharide before and after vinegar treatment could improve the liver phenotype of liver injury mice and reduce the liver index. Among them, the purified polysaccharide of Curcuma zedoaria vinegar could more significantly alleviate the liver pathological changes of mice, significantly reduce the activities of ALT and AST (P < 0.0001), significantly increase the level of SOD (P < 0.001), and significantly reduce the level of MDA (P < 0.0001). Curcuma zedoaria vinegar polysaccharide can better play a protective role in the liver of TAA-induced liver injury mice.

[0217] H&E staining and immunohistochemistry were used to detect the changes in ileal histopathology and occludin and ZO-1 protein expression in ileal tissue before and after administration. All doses of Curcuma zedoaria polysaccharide before and after vinegar treatment can improve the pathological changes in the structure of each layer of the ileal mucosa in mice with liver injury. The expression of occludin protein in all administration groups of Curcuma zedoaria polysaccharide before and after vinegar treatment was significantly increased (P<0.0001). The expression of ZO-1 protein in the VCPP-H group was significantly increased (P<0.05). It shows that Curcuma zedoaria polysaccharide before and after vinegar treatment can improve liver injury by protecting the intestinal mechanical barrier. However, there was no significant difference in the expression of occludin and ZO-1 proteins between the different dose groups of Curcuma zedoaria polysaccharide before and after vinegar treatment. Curcuma zedoaria polysaccharide before and after vinegar treatment can improve the disease state of liver injury in mice by exerting anti-inflammatory activity based on the "intestinal-liver axis".

Claims

1. A method for preparing Curcuma zedoaria polysaccharide, characterized in that: The following steps are involved: S1, grinding the Curcuma zedoariae and defatting it with petroleum ether to obtain defatted powder; The method of degreasing with petroleum ether is as follows: add petroleum ether 10 times the weight of the medicinal material, extract under reflux at 70℃-80℃ water bath for 2-3h, extract 1-3 times, filter, collect the residue, and dry at 30℃-50℃; The vinegar curcuma zedoaria medicinal material is obtained by the following method: taking an appropriate amount of raw curcuma zedoaria, using 20 kg of 9° rice vinegar for every 100 kg of raw product, mixing well and steaming for 2 hours, boiling until the vinegar liquid is dry, placing at 120° C. to dry for 30 minutes, taking out and cooling, and drying to obtain the vinegar curcuma zedoaria medicinal material; S2, adding 8-13 times the amount of 90vol%-95vol% ethanol to the defatted powder, extracting under reflux in a water bath at 65℃-80℃ for 2-5h, repeating 1-3 times, filtering, collecting the residue, evaporating the ethanol from the residue, drying at 30℃-60℃, obtaining a solid powder of Curcuma zedoaria, and setting aside; S3, take the solid powder of Curcuma zedoaria, add 30-50 times the weight of water, reflux extraction at 80°C-95°C for 2-5 hours, repeat 1-4 times, filter, combine the filtrate, evaporate and concentrate to 1 / 7~1 / 4 times the volume, cool to room temperature, and obtain a concentrated solution; S4, taking the concentrated solution, adding 3 to 5 times the volume of 75v% or more ethanol, and standing at 1°C-6°C for 18-36 hours; collecting the precipitate by centrifugation, dissolving the precipitate in pure water, and evaporating the ethanol; freeze-drying, collecting the lyophilized powder, and obtaining the Curcuma zedoaria polysaccharide; S5, separating and purifying the obtained Curcuma zedoaria polysaccharide: firstly separating and purifying by DEAE-52 cellulose column chromatography, during the separation and purification process, eluting with water and sodium chloride aqueous solution in sequence, collecting the water elution product, and obtaining a crude product of Curcuma zedoaria polysaccharide; Then, Sephadex G-100 dextran gel column chromatography was used for separation and purification to obtain pure Curcuma zedoaria polysaccharide.

2. The method for preparing a Curcuma zedoaria polysaccharide according to claim 1, characterized in that: In step S2, after defatting, the defatted powder is added with 90%-97% ethanol in an amount of 9-11 times, and extracted in a water bath under reflux at 67-75° C. for 2-3 hours, and the extraction is repeated 1-2 times. The residue is filtered, the ethanol is evaporated from the residue, and the residue is dried at 35° C.-45° C. for later use.

3. The method for preparing a Curcuma zedoaria polysaccharide according to claim 2, characterized in that: In step S3, take the solid powder of Curcuma zedoaria, add 35-45 times the weight of water, reflux extraction at 85° C.-95° C. for 2-4 hours, repeat 1-3 times, filter, combine the filtrate, evaporate and concentrate to 1 / 6-1 / 4 times the volume, cool to room temperature, and obtain a concentrated solution.

4. The method for preparing a Curcuma zedoaria polysaccharide according to claim 3, characterized in that: The product was concentrated by evaporation using a rotary evaporator.

5. The method for preparing a Curcuma zedoaria polysaccharide according to claim 1, characterized in that: In step S4, the concentrated solution is taken, 4-5 times the amount of 80 vol% or more ethanol is added, and the solution is placed in a refrigerator at 3-4°C for 24 hours; the solution is centrifuged again, and the precipitate is collected; the precipitate is dissolved in pure water, and after evaporating the ethanol, the solution is pre-frozen in a refrigerator at -20°C overnight, and the solution is transferred to a freeze dryer for freeze drying, and the freeze-dried powder is collected for later use.

6. The method for preparing Curcuma zedoaria polysaccharide according to claim 5, characterized in that: Take the concentrated solution, add 3-5 times the volume of ethanol with a concentration of more than 80v%, and let it stand at 1-6℃ for 20-30 hours; collect the precipitate by centrifugation, dissolve the precipitate in pure water, evaporate the ethanol; freeze-dry, collect the lyophilized powder, and obtain the Curcuma zedoaria polysaccharide.

7. Use of Curcuma pandurata polysaccharide in the preparation of a drug for protecting the liver, wherein the Curcuma pandurata polysaccharide is Curcuma pandurata acetoin polysaccharide obtained by the preparation method according to any one of claims 1 to 6.

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