Preparation method and application of pachysandra chinensis polysaccharide

Abstract

The preparation method comprises the following steps: step 1, extraction of crude polysaccharide of the pachysandra chinensis, step 2, deproteinization and impurity removal treatment of the crude polysaccharide, step 3, preliminary purification treatment, step 4, further purification treatment to prepare the polysaccharide of the pachysandra chinensis, and step 5, column chromatography purification of the polysaccharide of the pachysandra chinensis. According to the method, a water extraction and alcohol precipitation method and a column chromatography method are used for extracting and purifying the pachyrhizus erosus polysaccharide, the operation method is simple, stable and reliable, the purity of the purified pachyrhizus erosus polysaccharide is high and reaches 90% or above, a scientific basis is provided for development and application of the pachyrhizus erosus polysaccharide, and meanwhile the method is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of plant extraction, and in particular to a preparation method and application of radix scutellariae polysaccharide. Background Art

[0002] The Chinese name of pallet root is "Rubus" or "Niu Die Du". Pallet root belongs to the Rosaceae family. As a common Chinese herbal medicine, it is widely distributed in northern my country, east China and Central Plains. It has high nutritional and medicinal value. In China, its fruits and roots have long been used as folk prescriptions for treating various diseases.

[0003] Modern pharmacological studies have shown that the extracts or active ingredients of the pallet root have multiple biological activities, such as antioxidant, antibacterial, liver protection, immunomodulatory, anti-tumor, etc.; the active natural products include flavonoids, steroids, phenylpropionic acid, saponins and polysaccharides.

[0004] Prior art research shows that the water-soluble components in the pallet root may be the main components of the pallet root that exert pharmacological effects, but the research on the water-soluble polysaccharides in the pallet root, especially the separation and purification of polysaccharides, structural analysis and biological activity research reports are still very limited; currently there are patents and related research on the purification method and application of pallet roots and polysaccharides of the same genus. It is reported in the literature that the water-soluble polysaccharide was purified by hot water extraction, alcohol precipitation, destarch, freeze-thaw centrifugation, protease hydrolysis, Sevag method deproteinization, ultrafiltration, and gel column chromatography, and its purity reached 81.0%. The specific rotation measurement and high performance liquid chromatography (HPLC) analysis proved that RCP was a uniform component with a molecular weight of about 7000; gas chromatography (GC) analysis showed that its monosaccharide composition was mainly glucose and trace amounts of rhamnose, indicating that RCP is a neutral heteropolysaccharide. The molecular structure of RCP was determined by partial acid hydrolysis, periodate oxidation, Smith degradation, methylation, GC-MS analysis, IR analysis, NMR and other methods: the terminal and core parts are composed of Glc, the main chain is composed of 1→4Glc, with 1→4,6Glc as branch points, and on average there is one 1→4,6 branch point for every 9 1→4Glc. 1→6Glc and trace Rha may be in the side chain.

[0005] The polysaccharide components extracted from the pallet root in the prior art have low purity and the preparation and purification methods are complicated, which is not suitable for large-scale industrial production. Therefore, the present invention proposes a preparation method and application of pallet root polysaccharide with liver protection effect to solve the problems existing in the prior art. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to propose a preparation method and application of pallet root polysaccharide. The preparation method and application of pallet root polysaccharide use water extraction and alcohol precipitation method and column chromatography method to extract and purify pallet root polysaccharide. The operation method is simple and stable and reliable. The purity of the purified pallet root polysaccharide is high, reaching more than 90%, which provides a scientific basis for the development and application of pallet root polysaccharide and is suitable for industrial production.

[0007] To achieve the purpose of the present invention, the present invention is implemented by the following technical scheme: a method for preparing polysaccharide from the root of pallet root, comprising the following steps:

[0008] Step 1: first crush the dried pallet root and pass it through a 60-mesh sieve to obtain fine medicinal material particles, then weigh 80g of the fine medicinal material particles into a 2000ml round-bottom flask, add 15-30 times the volume of distilled water, reflux in a 90°C water bath for 2-3h, filter, repeat the operation 2-3 times, combine the filtrate and concentrate under reduced pressure, add 3-4 times the volume of 95% ethanol to the concentrate, precipitate with alcohol for 12h, filter and obtain the supernatant, then perform rotary evaporation to remove ethanol and freeze-dry to obtain pallet root crude polysaccharide and weigh it;

[0009] Step 2: Dissolve the crude polysaccharide of the root of the radix serrata in ultrapure water, add Sevag reagent to remove the protein in the solution to obtain a polysaccharide aqueous solution without protein, and then dialyze for three days with a bag of 3000Da molecular weight cutoff to remove low molecular weight components, and finally freeze-dry to obtain the crude polysaccharide with small molecular impurities removed;

[0010] Step 3: Prepare the crude polysaccharide into an aqueous solution, first use a DEAE-52 cellulose chromatographic column for preliminary purification, then use water and NaCl solution as eluents for gradient elution to obtain a preliminary purified eluent, and sample to obtain 7 types of polysaccharides;

[0011] Step 4: Load the preliminary purified eluate onto a Sephadex G-100 column for the next step of purification, using deionized water as the eluent to elute, obtaining a purified eluate, and taking samples to obtain four purified radix serrata polysaccharides;

[0012] Step 5. Finally, use a UV-visible spectrometer to determine the purity of the purified pallet root polysaccharide.

[0013] A further improvement is that the Sevag reagent used in the step 2 is prepared from chloroform and n-butanol in a volume ratio of 4:1; the NaCl concentrations used in the gradient elution in the step 3 are 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L and 1 mol / L, respectively, and the gradient elution flow rate is 1 ml / min.

[0014] A further improvement is that after obtaining the preliminary purified eluate in step three, 10 ml of the eluate is collected in a tube, and the polysaccharide content in each component is determined by the phenol-sulfuric acid method, and then 7 polysaccharides are obtained after concentration, dialysis and freeze-drying, namely D-RCP-1, D-RCP-2, D-RCP-3, D-RCP-4, D-RCP-5, D-RCP-6 and D-RCP-7.

[0015] A further improvement is that in the step 4, the elution flow rate is 0.5 ml / min, and after obtaining the purified eluate, a 5 ml tube of the purified eluate is collected and the absorbance is measured until all the polysaccharides are collected. Using the same fraction and the same freeze-drying method, four purified pallet root polysaccharides are obtained, namely RCP-1, RCP-3, RCP-4 and RCP-5.

[0016] A further improvement is that the concentration determination in step 5 is specifically:

[0017] S1. Take 20 mg of glucose and place it in a 100 ml volumetric flask, add water to make up to volume, and obtain a glucose solution which is used as the reference solution;

[0018] S2, then take 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml and 1.2ml of glucose solution and put them in test tubes, add water to make up to 2ml respectively, then add 1ml of 4% phenol solution respectively and shake well, quickly add 7ml of concentrated H2SO4, let it stand for 5min, then heat it in a 40℃ water bath for 30min, react to room temperature, measure the absorbance at a wavelength of 490nm, and then use absorbance as the ordinate and concentration as the abscissa to obtain the standard curve, and the linear regression equation is A=8.415x+0.1773, R 2 =0.9994, where A represents absorbance, x represents glucose mass concentration in mg / ml;

[0019] S3. Take 20 mg of the purified pallet root polysaccharide sample and place it in a 25 ml volumetric flask, add water to make up the volume, then take 2 ml of the fixed solution into a test tube, add 1 ml of 4% phenol solution and shake well, quickly add 7 ml of concentrated H2SO4, let it stand for 5 minutes, then place it in a 40°C water bath and heat for 30 minutes, react to room temperature, measure the absorbance at a wavelength of 490 nm, and substitute it into the above linear regression equation to calculate the pallet root polysaccharide concentration.

[0020] The application of 4 kinds of purified radix ophiopogonis polysaccharides prepared by the aforementioned preparation method of radix ophiopogonis polysaccharides in liver protection.

[0021] The beneficial effects of the present invention are as follows: the present invention uses a water extraction and alcohol precipitation method and a column chromatography method to extract and purify the polysaccharide from the root of pallet, the operation method is simple, stable and reliable, the purity of the polysaccharide from the root of pallet after purification is high, reaching more than 90%, providing a scientific basis for the development and application of the polysaccharide from the root of pallet, and at the same time being suitable for industrial production, and having a good application prospect;

[0022] The prepared pallet root polysaccharide contains a triple helical conformation, has good antioxidant activity, and has high application value in the development of liver protective drugs and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the elution curve of the crude polysaccharide in Examples 1-4 of the present invention on a DEAE-52 cellulose column.

[0024] Figure 2 This is a Sephadex G-100 column chromatography elution curve during further purification in Examples 1-4 of the present invention.

[0025] Figure 3 This is a scanning electron micrograph of four purified pallet root polysaccharides in Example 5 of the present invention.

[0026] Figure 4 This is the UV scanning spectrum of 4 purified pallet root polysaccharides in Example 5 of the present invention.

[0027] Figure 5 This is the infrared scanning spectrum of four purified pallet root polysaccharides in Example 5 of the present invention.

[0028] Figure 6 This is a liquid chromatogram for molecular weight determination of four purified pallet root polysaccharides in Example 5 of the present invention.

[0029] Figure 7 This is a liquid chromatogram of the monosaccharide standard and four purified pallet root polysaccharides from Example 5 of the present invention.

[0030] Figure 8 The four purified pallet root polysaccharides in Example 5 of the present invention are 1 H-NMR spectrum and 13 C-NMR spectrum of Congo red experimental results.

[0031] Fig. 9 This is a graph of the maximum absorption wavelengths of Congo red and four purified radix palletis polysaccharides in Example 5 of the present invention in sodium hydroxide solutions of different concentrations.

[0032] Fig.10 This is the XRD scanning spectrum of 4 purified pallet root polysaccharides in Example 5 of the present invention.

[0033] Fig.11This is a bar graph showing the effects of four purified polysaccharides of the present invention on the survival rate of HepG2 cells damaged by H2O2.

[0034] Fig.12 The bar graphs are the effects of RCP-1 pretreatment on AST levels of HepG2 cells induced by H2O2 (a) and the effects of RCP-1 pretreatment on ALT levels of HepG2 cells induced by H2O2 (b) according to the application examples of the present invention.

[0035] Fig.13 The bar graphs are of the application examples of the present invention, showing the effect of RCP-1 on the SOD activity of HepG2 cells induced by H2O2 (a), the effect of RCP-1 on the CAT activity of HepG2 cells induced by H2O2 (b), the effect of RCP-1 on the GSH activity of HepG2 cells induced by H2O2 (c), and the effect of RCP-1 on the MDA activity of HepG2 cells induced by H2O2 (d).

[0036] Fig.14 This is an application example of the present invention. The effects of different concentrations of RCP-1 on the ROS level of HepG2 cells induced by H2O2 (a, b). The Heochst33342 staining method was used to observe the effect of RCP-1 on the morphology of HepG2 cells. c is the blank group, d is the H2O2 treatment group, e is the silybin treatment group, and f is the RCP-1 treatment group.

[0037] Fig.15 Schematic diagram (a, b) of the effect of RCP-1 on the levels of Bax and Bcl-2 proteins in HepG2 cells induced by H2O2 in the application example of the present invention.

[0038] Fig.16 Schematic diagram (a, b) of the effect of RCP-1 on the protein levels of NF-κB and TLR-4 in HepG2 cells induced by H2O2 in the application example of the present invention. DETAILED DESCRIPTION

[0039] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0040] Example 1

[0041] This embodiment provides a method for preparing polysaccharide from the root of Psoralea corylifolia, comprising the following steps:

[0042] Step 1. First, crush the dried pallet root and pass it through a 60-mesh sieve to obtain fine medicinal particles. Then weigh 80g of the fine medicinal particles into a 2000ml round-bottom flask and add 20 times the volume of distilled water. Reflux in a 90°C water bath for 2h and filter. Repeat the operation twice. Combine the filtrate and concentrate under reduced pressure. Add 4 times the volume of 95% ethanol to the concentrate. After alcohol precipitation for 12h, filter and obtain the supernatant. Then, perform rotary evaporation to remove the ethanol and freeze-dry to obtain the pallet root crude polysaccharide and weigh it.

[0043] Step 2: Dissolve the crude polysaccharide of the pallet root in ultrapure water, add Sevag reagent prepared by mixing chloroform and n-butanol in a volume ratio of 4:1, remove the protein in the solution, and obtain a polysaccharide aqueous solution with the protein removed, then dialyze with 3000Da cutoff for three days to remove low molecular weight components, and finally freeze-dry to obtain the crude polysaccharide with small molecular impurities removed.

[0044] Step 3: Prepare the crude polysaccharide in an aqueous solution, first use a DEAE-52 cellulose chromatographic column for preliminary purification, and then use water and NaCl solutions with concentrations of 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L and 1 mol / L as eluents for gradient elution, with an elution flow rate of 1 ml / min to obtain a preliminary purified eluate, and collect 10 ml of the eluate in a tube, and use the phenol-sulfuric acid method to determine the polysaccharide content of each group, and then concentrate, dialyze and freeze-dry to obtain 7 polysaccharides, namely D-RCP-1, D-RCP-2, D-RCP-3, D-RCP-4, D-RCP-5, D-RCP-6 and D-RCP-7.

[0045] Step 4: Load the preliminary purified eluate onto a Sephadex G-100 column for the next step of purification, using deionized water as the eluent for elution at a flow rate of 0.5 ml / min to obtain a purified eluate, and collect a 5 ml tube of the purified eluate, and measure the absorbance until all polysaccharides are collected. Using the same fraction and the same freeze-drying method, four purified pallet root polysaccharides are obtained, namely RCP-1, RCP-3, RCP-4 and RCP-5;

[0046] Step 5. Finally, use a UV-visible spectrometer to measure the purity of the purified pallet root polysaccharide, specifically:

[0047] S1. Take 20 mg of glucose and place it in a 100 ml volumetric flask, add water to make up to volume, and obtain a glucose solution which is used as the reference solution;

[0048] S2, then take 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml and 1.2ml of glucose solution and put them in test tubes, add water to make up to 2ml respectively, then add 1ml of 4% phenol solution respectively and shake well, quickly add 7ml of concentrated H2SO4, let it stand for 5min, then heat it in a 40℃ water bath for 30min, react to room temperature, measure the absorbance at a wavelength of 490nm, and then use absorbance as the ordinate and concentration as the abscissa to obtain the standard curve, and the linear regression equation is A=8.415x+0.1773, R 2 =0.9994, where A represents absorbance, x represents glucose mass concentration in mg / ml;

[0049] S3. Take 20 mg of the purified pallet root polysaccharide sample and place it in a 25 ml volumetric flask, add water to make up the volume, then take 2 ml of the fixed solution into a test tube, add 1 ml of 4% phenol solution and shake well, quickly add 7 ml of concentrated H2SO4, let it stand for 5 minutes, then place it in a 40°C water bath and heat for 30 minutes, react to room temperature, measure the absorbance at a wavelength of 490 nm, and substitute it into the above linear regression equation to calculate the pallet root polysaccharide concentration.

[0050] Example 2

[0051] This embodiment provides a method for preparing polysaccharide from the root of Psoralea corylifolia, comprising the following steps:

[0052] Step 1. First, crush the dried pallet root and pass it through a 60-mesh sieve to obtain fine medicinal particles. Then weigh 80g of the fine medicinal particles into a 2000ml round-bottom flask and add 20 times the volume of distilled water. Reflux in a 90°C water bath for 3h and filter. Repeat the operation twice. Combine the filtrate and concentrate under reduced pressure. Add 4 times the volume of 95% ethanol to the concentrate. After alcohol precipitation for 12h, filter and obtain the supernatant. Then, perform rotary evaporation to remove the ethanol and freeze-dry to obtain the pallet root crude polysaccharide and weigh it.

[0053] Step 2: Dissolve the crude polysaccharide of the pallet root in ultrapure water, add Sevag reagent prepared by mixing chloroform and n-butanol in a volume ratio of 4:1, remove the protein in the solution, and obtain a polysaccharide aqueous solution with the protein removed, then dialyze with 3000Da cutoff for three days to remove low molecular weight components, and finally freeze-dry to obtain the crude polysaccharide with small molecular impurities removed.

[0054] Step 3: Prepare the crude polysaccharide in an aqueous solution, first use a DEAE-52 cellulose chromatographic column for preliminary purification, and then use water and NaCl solutions with concentrations of 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L and 1 mol / L as eluents for gradient elution, with an elution flow rate of 1 ml / min to obtain a preliminary purified eluate, and collect 10 ml of the eluate in a tube, and use the phenol-sulfuric acid method to determine the polysaccharide content of each group, and then concentrate, dialyze and freeze-dry to obtain 7 polysaccharides, namely D-RCP-1, D-RCP-2, D-RCP-3, D-RCP-4, D-RCP-5, D-RCP-6 and D-RCP-7.

[0055] Step 4: Load the preliminary purified eluate onto a Sephadex G-100 column for the next step of purification, using deionized water as the eluent for elution at a flow rate of 0.5 ml / min to obtain a purified eluate, and collect a 5 ml tube of the purified eluate, and measure the absorbance until all polysaccharides are collected. Using the same fraction and the same freeze-drying method, four purified pallet root polysaccharides are obtained, namely RCP-1, RCP-3, RCP-4 and RCP-5;

[0056] Step 5. Finally, use a UV-visible spectrometer to measure the purity of the purified pallet root polysaccharide, specifically:

[0057] S1. Take 20 mg of glucose and place it in a 100 ml volumetric flask, add water to make up to volume, and obtain a glucose solution which is used as the reference solution;

[0058] S2, then take 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml and 1.2ml of glucose solution and put them in test tubes, add water to make up to 2ml respectively, then add 1ml of 4% phenol solution respectively and shake well, quickly add 7ml of concentrated H2SO4, let it stand for 5min, then heat it in a 40℃ water bath for 30min, react to room temperature, measure the absorbance at a wavelength of 490nm, and then use absorbance as the ordinate and concentration as the abscissa to obtain the standard curve, and the linear regression equation is A=8.415x+0.1773, R 2 =0.9994, where A represents absorbance, x represents glucose mass concentration in mg / ml;

[0059] S3. Take 20 mg of the purified pallet root polysaccharide sample and place it in a 25 ml volumetric flask, add water to make up the volume, then take 2 ml of the fixed solution into a test tube, add 1 ml of 4% phenol solution and shake well, quickly add 7 ml of concentrated H2SO4, let it stand for 5 minutes, then place it in a 40°C water bath and heat for 30 minutes, react to room temperature, measure the absorbance at a wavelength of 490 nm, and substitute it into the above linear regression equation to calculate the pallet root polysaccharide concentration.

[0060] Example 3

[0061] This embodiment provides a method for preparing polysaccharide from the root of Psoralea corylifolia, comprising the following steps:

[0062] Step 1. First, crush the dried pallet root and pass it through a 60-mesh sieve to obtain fine medicinal particles. Then weigh 80g of the fine medicinal particles into a 2000ml round-bottom flask and add 30 times the volume of distilled water. Reflux in a 90°C water bath for 2h and filter. Repeat the operation 3 times. Combine the filtrate and concentrate under reduced pressure. Add 4 times the volume of 95% ethanol to the concentrate. After alcohol precipitation for 12h, filter and obtain the supernatant. Then, perform rotary evaporation to remove the ethanol and freeze-dry to obtain the pallet root crude polysaccharide and weigh it.

[0063] Step 2: Dissolve the crude polysaccharide of the pallet root in ultrapure water, add Sevag reagent prepared by mixing chloroform and n-butanol in a volume ratio of 4:1, remove the protein in the solution, and obtain a polysaccharide aqueous solution with the protein removed, then dialyze with 3000Da cutoff for three days to remove low molecular weight components, and finally freeze-dry to obtain the crude polysaccharide with small molecular impurities removed.

[0064] Step 3: Prepare the crude polysaccharide in an aqueous solution, first use a DEAE-52 cellulose chromatographic column for preliminary purification, and then use water and NaCl solutions with concentrations of 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L and 1 mol / L as eluents for gradient elution, with an elution flow rate of 1 ml / min to obtain a preliminary purified eluate, and collect 10 ml of the eluate in a tube, and use the phenol-sulfuric acid method to determine the polysaccharide content of each group, and then concentrate, dialyze and freeze-dry to obtain 7 polysaccharides, namely D-RCP-1, D-RCP-2, D-RCP-3, D-RCP-4, D-RCP-5, D-RCP-6 and D-RCP-7.

[0065] Step 4: Load the preliminary purified eluate onto a Sephadex G-100 column for the next step of purification, using deionized water as the eluent for elution at a flow rate of 0.5 ml / min to obtain a purified eluate, and collect a 5 ml tube of the purified eluate, and measure the absorbance until all polysaccharides are collected. Using the same fraction and the same freeze-drying method, four purified pallet root polysaccharides are obtained, namely RCP-1, RCP-3, RCP-4 and RCP-5;

[0066] Step 5. Finally, use a UV-visible spectrometer to measure the purity of the purified pallet root polysaccharide, specifically:

[0067] S1. Take 20 mg of glucose and place it in a 100 ml volumetric flask, add water to make up to volume, and obtain a glucose solution which is used as the reference solution;

[0068] S2, then take 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml and 1.2ml of glucose solution and put them in test tubes, add water to make up to 2ml respectively, then add 1ml of 4% phenol solution respectively and shake well, quickly add 7ml of concentrated H2SO4, let it stand for 5min, then heat it in a 40℃ water bath for 30min, react to room temperature, measure the absorbance at a wavelength of 490nm, and then use absorbance as the ordinate and concentration as the abscissa to obtain the standard curve, and the linear regression equation is A=8.415x+0.1773, R 2 =0.9994, where A represents absorbance, x represents glucose mass concentration in mg / ml;

[0069] S3. Take 20 mg of the purified pallet root polysaccharide sample and place it in a 25 ml volumetric flask, add water to make up the volume, then take 2 ml of the fixed solution into a test tube, add 1 ml of 4% phenol solution and shake well, quickly add 7 ml of concentrated H2SO4, let it stand for 5 minutes, then place it in a 40°C water bath and heat for 30 minutes, react to room temperature, measure the absorbance at a wavelength of 490 nm, and substitute it into the above linear regression equation to calculate the pallet root polysaccharide concentration.

[0070] Example 4

[0071] This embodiment provides a method for preparing polysaccharide from the root of Psoralea corylifolia, comprising the following steps:

[0072] Step 1: first crush the dried pallet root and pass it through a 60-mesh sieve to obtain fine medicinal material particles, then weigh 80g of the fine medicinal material particles into a 2000ml round-bottom flask and add 15 times the volume of distilled water, reflux in a 90°C water bath for 3h and filter, repeat the operation twice, combine the filtrate and concentrate under reduced pressure, add 3 times the volume of 95% ethanol to the concentrate, precipitate with alcohol for 12h, filter and obtain the supernatant, then perform rotary evaporation to remove the ethanol and freeze-dry to obtain the pallet root crude polysaccharide and weigh it.

[0073] Step 2: Dissolve the crude polysaccharide of the pallet root in ultrapure water, add Sevag reagent prepared by mixing chloroform and n-butanol in a volume ratio of 4:1, remove the protein in the solution, and obtain a polysaccharide aqueous solution with the protein removed, then dialyze with 3000Da cutoff for three days to remove low molecular weight components, and finally freeze-dry to obtain the crude polysaccharide with small molecular impurities removed.

[0074] Step 3: Prepare the crude polysaccharide in an aqueous solution, first use a DEAE-52 cellulose chromatographic column for preliminary purification, and then use water and NaCl solutions with concentrations of 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L and 1 mol / L as eluents for gradient elution, with an elution flow rate of 1 ml / min to obtain a preliminary purified eluate, and collect 10 ml of the eluate in a tube, and use the phenol-sulfuric acid method to determine the polysaccharide content of each group, and then concentrate, dialyze and freeze-dry to obtain 7 polysaccharides, namely D-RCP-1, D-RCP-2, D-RCP-3, D-RCP-4, D-RCP-5, D-RCP-6 and D-RCP-7.

[0075] Step 4: Load the preliminary purified eluate onto a Sephadex G-100 column for the next step of purification, using deionized water as the eluent for elution at a flow rate of 0.5 ml / min to obtain a purified eluate, and collect a 5 ml tube of the purified eluate, and measure the absorbance until all polysaccharides are collected. Using the same fraction and the same freeze-drying method, four purified pallet root polysaccharides are obtained, namely RCP-1, RCP-3, RCP-4 and RCP-5;

[0076] Step 5. Finally, use a UV-visible spectrometer to measure the purity of the purified pallet root polysaccharide, specifically:

[0077] S1. Take 20 mg of glucose and place it in a 100 ml volumetric flask, add water to make up to volume, and obtain a glucose solution which is used as the reference solution;

[0078] S2, then take 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml and 1.2ml of glucose solution and put them in test tubes, add water to make up to 2ml respectively, then add 1ml of 4% phenol solution respectively and shake well, quickly add 7ml of concentrated H2SO4, let it stand for 5min, then heat it in a 40℃ water bath for 30min, react to room temperature, measure the absorbance at a wavelength of 490nm, and then use absorbance as the ordinate and concentration as the abscissa to obtain the standard curve, and the linear regression equation is A=8.415x+0.1773, R 2=0.9994, where A represents absorbance, x represents glucose mass concentration in mg / ml;

[0079] S3. Take 20 mg of the purified pallet root polysaccharide sample and place it in a 25 ml volumetric flask, add water to make up the volume, then take 2 ml of the fixed solution into a test tube, add 1 ml of 4% phenol solution and shake well, quickly add 7 ml of concentrated H2SO4, let it stand for 5 minutes, then place it in a 40°C water bath and heat for 30 minutes, react to room temperature, measure the absorbance at a wavelength of 490 nm, and substitute it into the above linear regression equation to calculate the pallet root polysaccharide concentration.

[0080] In step 3 of Example 1-4, polysaccharides were separated by anion exchange chromatography using a DEAE-52 cellulose column, and the elution curve is shown in the attached manual. Figure 1 As shown, 7 independent peaks were obtained by elution in water, 0.1mol / L, 0.2mol / L, 0.3mol / L, 0.4mol / L, 0.5mol / L and 1mol / L NaCl solution, respectively named D-RCP-1, D-RCP-2, D-RCP-3, D-RCP-4, D-RCP-5, D-RCP-6 and D-RCP-7, and the 4 main peaks were D-RCP-1, D-RCP-3, D-RCP-4 and DRCP-5; in step 4, the product was purified by Sephadex G-100 column chromatography and eluted with deionized water. The purification results are shown in the attached manual. Figure 2 As shown in the figure, a, b, c, and d are the elution curves of D-RCP-1, D-RCP-3, D-RCP-4, and DRCP-5 on Sephadex G-100 column chromatography, respectively. The single-peak symmetrical peak indicates that the purified polysaccharide is a relatively uniform sample. After concentration and freeze-drying, RCP-1, RCP-3, RCP-4, and RCP-5 powders are obtained. The purity of the purified pallet root polysaccharide is more than 90%.

[0081] The prepared RCP-1, RCP-3, RCP-4 and RCP-5 have a high degree of purification, and do not contain impurities such as nucleic acids and proteins. RCP-4 may contain trace amounts of glycoproteins. RCP-1, RCP-3, RCP-4 and RCP-5 are all polypyranoses, RCP-1 contains uronic acid and α-pyranose, and RCP-4 contains uronic acid and S=O bonds.

[0082] Example 5

[0083] according to Figure 3-Figure 10 As shown, this example uses scanning electron microscopy, X-ray diffraction, ultraviolet spectroscopy, Fourier transform infrared spectroscopy, liquid chromatography, nuclear magnetic resonance spectroscopy and Congo red experiment to analyze the structure of the pallet root polysaccharide prepared in Examples 1-4.

[0084] 1. Scanning electron microscope analysis

[0085] Scanning electron microscopy can be used to observe the ultramicroscopic morphology and structural characteristics of macromolecular polysaccharide particles and explore the microscopic morphological changes and characteristic laws of polysaccharides. The microstructures of RCP-1, RCP-3, RCP-4 and RCP-5 at ×500 (a, d, g, j), ×2000 (b, e, h, k), and ×5000 (c, f, i, l) are shown in the attached manual. Figure 3 As shown, a, b, c are RCP-1, d, e, f are RCP-3, g, h, i are RCP-4, j, k, l are RCP-5. As can be seen from the figure, under low-power electron microscope, the polysaccharide surface of RCP-1 presents a smooth, irregular flaky structure, and under high-power electron microscope, a large number of depressions or holes can be seen, and the intermolecular attraction is small. Under low-power electron microscope, the polysaccharide flaky structure of RCP-3, RCP-4 and RCP-5 is messy and of different sizes. Some flaky structures are curved, with a flat surface and a rough oblique or longitudinal surface. Under medium-power electron microscope, the polysaccharide is loose and porous, with a long chain structure. Under high-power electron microscope, the polysaccharide structure is dense, with different surface levels, and spherical particles can be seen, which indicates that there is no repulsion of impurities such as proteins in the polysaccharide, and the degree of purification is high.

[0086] 2. UV full wavelength scanning

[0087] The UV full wavelength scanning spectra of RCP-1, RCP-3, RCP-4 and RCP-5 are as shown in the attached manual. Figure 4 As shown, a is RCP-1, b is RCP-3, c is RCP-4, and d is RCP-5. As can be seen from Figures a, b, and d, RCP-1, RCP-3, and RCP-5 have no obvious absorption peaks in the range of 260-280nm, and the spectral curves are relatively smooth, which indicates that impurities such as nucleic acids and proteins in these three components have been basically eliminated. As can be seen from Figure c, RCP-4 has an obvious ultraviolet absorption peak near 280nm, which indicates that the polysaccharide of this component contains trace proteins. It is inferred that glycoproteins may exist in the pallet root polysaccharide of this component. The ultraviolet absorption spectra of the polysaccharides of these four components show an overall downward trend, which is consistent with the characteristic properties of polysaccharides.

[0088] 3. Infrared spectroscopy analysis

[0089] Fourier transform infrared spectroscopy can analyze the functional group structure in polysaccharides, distinguish between furanose and pyranose, and determine the various glycosidic bond configurations in polysaccharides. It is a conventional method for analyzing polysaccharide structure.

[0090] Instructions attached Figure 5 The infrared scanning spectra of RCP-1 (a), RCP-3 (b), RCP-4 (c) and RCP-5 (d) show that these four components are at 3410-3420 cm-1 There is a strong and broad peak at 2915-2930cm, which is the stretching vibration peak of OH in polysaccharide. -1 There is a medium-strong absorption at 1420cm, which indicates the presence of a characteristic peak of sugars in the component, which is the stretching vibration of sugars -CH2 or -CH3. -1 The absorption peaks at 1100-1010 indicate that the component is pyranose. In addition, at 604cm -1 The stretching vibration of pyranose skeleton was also detected at 1, which indicates that RCP-1, RCP-3, RCP-4 and RCP-5 are polypyranose. -1 and 1426cm -1 The absorption detected around 850cm -1 α-Glycosidic bond was detected near RCP-4 at 1730 cm -1 The absorption of uronic acid was detected at 1410 cm -1 Carbonyl absorption was detected at 1252 cm -1 The stretching vibration of S=O bond was detected at 1064cm -1 There are stretching vibrations of the C-O-C bond and the C-O-H bond on the left and right.

[0091] 4. Liquid chromatography determination of molecular weight and monosaccharide composition

[0092] Molecular weight

[0093] The molecular weight of polysaccharides affects their solubility and viscosity, and is closely related to the size of the molecular chain and biological activity. The number average molecular weights of RCP-1, RCP-3, RCP-4 and RCP-5 are approximately 1.70×10 6 Da, 5.56×10 6 Da, 4.97×10 6 Da, 9.80×10 6 Da, the molecular weight order of the four components of pallet root polysaccharides is RCP-5>RCP-3>RCP-4>RCP-1.

[0094] Monosaccharide composition

[0095] The ratio of different monosaccharide components in polysaccharides will affect their antioxidant activity, so the study of monosaccharide composition is an important part of exploring the structure-activity relationship of polysaccharides. The monosaccharide composition of RCP-1, RCP-3, RCP-4 and RCP-5 was determined by PMP derivatization method, high performance liquid chromatography combined with ultraviolet detector, and Man, GluN, Rib, Rha, GluA, GalA, Glu, Gal, Xyl, Fuc were used as standards to draw standard curves and calculate the molar ratio of monosaccharides in the four components. The results are shown in Table 1 below.

[0096] Table 1 Standard curve of monosaccharide standards

[0097]

[0098] Instructions attached Figure 7 In the figure, ae are the monosaccharide liquid chromatograms of monosaccharide standard, RCP-1, RCP-3, RCP-4 and RCP-5 respectively. It can be seen from the figure that the peak times of 11 standard monosaccharides are mannose (16.417min), glucosamine (18.850min), ribose (21.483min), rhamnose (23.157min), glucuronic acid (24.667min), galacturonic acid (27.997min), glucose (33.073min), galactose (37.300min), xylose (40.093min), arabinose (41.450min) and fucose (48.980min). The molar ratio of monosaccharides in polysaccharides was calculated by the monosaccharide standard curve, and the results are shown in Table 2 below.

[0099] Table 2 Monosaccharide composition and molar ratio of RCP-1, RCP-3, RCP-4 and RCP-5

[0100]

[0101] It can be seen that the polysaccharide of the pallet root is mainly composed of GluN, GlcA and Gal, and also contains a small amount of GalA, Arab and Glu, as well as trace amounts of Rib, Xyl and Rha. RCP-1 contains 6 monosaccharides, the main monosaccharide is Gal. The types of monosaccharides in RCP-3, RCP-4 and RCP-5 are the same, with only slight differences in content.

[0102] 5. Nuclear Magnetic Resonance Spectroscopy Analysis

[0103] RCP-1 1 H-NMR and 13 C-NMR spectrum is attached in the instruction manual Figure 8As shown in a and b, in the 1H-NMR spectrum, δ4.5-5.9ppm is the anomeric hydrogen proton signal region. Usually, signals greater than 4.95ppm are α-configuration sugar residues, and signals less than 4.95ppm are β-configuration sugar residues. 1 In the H-NMR spectrum, δ4.4, 4.5, 4.6, 5.0, 5.1, 5.2, 5.3, and 5.5 are signals of sugar anomeric protons, and most of the signals are greater than 4.95 ppm, indicating that the structure is mainly α-type. δ3.2, 3.3, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.4, and 4.5 are signals of sugar ring non-anomeric protons. Since the chemical shift values ​​are close, signals overlap. δ8.5 is the signal of H on the amino group of glucosamine. The signal peak at δ1.2-1.4 ppm is the signal peak of rhamnose methyl, indicating that rhamnose may exist. The signal peak at around δ2.0 ppm is generally the signal peak of methyl in acetylamino, indicating that acetylamino may exist.

[0104] RCP-3 1 H-NMR and 13 C-NMR spectrum is attached in the instruction manual Figure 8 As shown in c and d, 1 In the H-NMR spectrum, the signal of the anomeric proton of RCP-1 appears at δ4.4-5.7ppm, indicating that there are two sugar residue configurations, α and β, in the structure. The remaining proton signals in the sugar mostly appear at δ3.2-4.2ppm. The signal peak at δ1.0-1.5 is the methyl signal peak of rhamnose. The signal at δ2.0ppm indicates that there may be an acetylamino group in the structure. 13 In the C-NMR spectrum, no carbonyl signal peak appeared, which may be related to the low content of uronic acid. The anomeric carbon signals in the polysaccharide are δ107.0, 100.7, and 97.9, indicating that there are two sugar residue configurations, α and β, in the structure. 1 The results of H-NMR analysis were consistent. The remaining carbon signals (C2-C6) appeared at δ61.2-81.5ppm. The methyl signals of rhamnose appeared at δ15.3 and 16.7.

[0105] RCP-4 1 H-NMR and 13 C-NMR spectrum is attached in the instruction manual Figure 8 As shown in e and f, 1In the H-NMR spectrum, seven anomeric proton signals appeared at chemical shifts of δ4.6-5.9ppm, indicating the presence of α and β configuration sugar residues in RCP-4. The proton signals from C-2 to C-6 appeared at δ3.4-4.1ppm. Due to the large molecular weight of the polysaccharide and the high degree of polymerization, the signals overlapped and were difficult to distinguish. The δ1.2-1.4ppm peak that appeared in the high field was the methyl signal peak in rhamnose. The δ1.9, 2.0ppm peaks were generally the methyl signal peaks in acetylamino, indicating that acetylamino may exist in the structure of RCP-4. 13 In the C-NMR spectrum, the peaks at δ176.260 and 175.141 ppm are the carbonyl signals of glucuronic acid and galacturonic acid. The signals of the anomeric carbon at δ104.1, 100.8 and 99.5 indicate the presence of α-type glycosidic bonds and β-type glycosidic bonds in the structure. The remaining carbon signals in the structure appear at δ61-81 ppm. 1 The H-NMR analysis was the same, the signal at δ15.4 indicated the presence of rhamnose, and the peak at δ25.4 was probably the methyl signal of the acetylamino group.

[0106] RCP-5 1 H-NMR and 13 C-NMR spectrum is attached in the instruction manual Figure 8 As shown in g and h, 13 In the C-NMR spectrum, δ181.671, 176.260, 175.141 are carbonyl signals of glucuronic acid and galacturonic acid, which are consistent with the analysis results of infrared spectrum and monosaccharide composition. At δ95-105ppm, it is the anomeric carbon signal area. Generally, the chemical shift value greater than 103ppm indicates a β-type glycosidic bond, and less than 103ppm indicates an α-type glycosidic bond. In the 1H-NMR spectrum of RCP-5, δ98.5, 99.5, 100.8, 102.8, 107.6 are anomeric carbon signals, which also indicates that there are α-type glycosidic bonds and β-type glycosidic bonds in the polysaccharide, and the α configuration is dominant. The signals of 70-77 in the carbon spectrum signal are the signals of C-2, C-3 and C4 that have not been substituted in the polysaccharide, while the signal of δ81.5 indicates that substitution has occurred at the C-2, C-3 and C-4 positions. The signal at δ68-70 indicates that C-6 has been substituted. The signal at δ25.3 indicates that there is an acetylamino substitution in the structure. δ15.4-17.6 also indicates that rhamnose may be present.

[0107] 6. Congo Red Test

[0108] Congo red is an acidic dye that can be used to determine whether a triple helix structure exists in polysaccharides by detecting whether the maximum absorption wavelength is complexed with the triple helix structure of the polysaccharide to produce a red shift. Fig. 9As shown, the maximum absorption wavelength of pure Congo red decreases with the increase of sodium hydroxide concentration, and the maximum absorption wavelength of the complexes of Congo red and four components of pallet root polysaccharides increases with the increase of sodium hydroxide concentration, proving that there is a triple helix structure in these four components of pallet root polysaccharides. The triple helix structure is more active than the irregular coil chain structure and has more value for resource development.

[0109] 7. X-ray diffraction experiment

[0110] As the instruction manual Fig.10 As shown in the figure, the X-ray diffraction patterns of RCP-1 (a), RCP-3 (b), RCP-4 (c) and RCP-5 (d) are shown. It can be seen from the figure that RCP-3, RCP-4 and RCP-5 have small diffraction peaks at diffraction angles 2θ of about 10°, 20° and 30°, indicating that the polysaccharides of these three components are not highly crystalline and are semi-crystalline polymers. RCP-1 has strong diffraction peaks at diffraction angles 2θ of about 32° and 75°, indicating that this component is a high molecular polymer with a high degree of crystallinity.

[0111] Application Examples

[0112] This application example provides an application experiment of the protective effect of the pallet root polysaccharide prepared in Examples 1-4 on HepG2 cell damage induced by H2O2.

[0113] 1. Cell culture:

[0114] HepG2 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture conditions were: a temperature of 37° C. and an environment of 5% CO 2 .

[0115] 2. Cell survival rate experiment:

[0116] The cell survival rate was determined by 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) method. Diluted HepG2 cells (2×105 / well) were transferred to 24-well plates. 1 mL per well was incubated for 24 hours. After drug treatment for 12 hours, H2O2 was added for 4 hours to create the model, and then MTT solution was added for 4 hours. The absorbance was detected at a wavelength of 570 nm to calculate the cell survival rate.

[0117] Attached to the instruction manual Fig.11It can be seen that HepG2 cells were pretreated with four purified polysaccharides of corresponding concentrations for 12 hours, and then incubated with H2O2. The survival rate of HepG2 cells induced by H2O2 was detected by MTT. The experiment showed that the survival rate of the model group cells treated with H2O2 for 4 hours was significantly reduced. Within the experimental concentration range, the survival rate of HepG2 cells damaged by oxidation was significantly increased after treatment with four different concentrations of polysaccharides (RCP-1, RCP-3, RCP-4, RCP-5), and the cell survival rate gradually increased with the increase of concentration. Among them, RCP-1 has a higher protective effect on the damage of HepG2 cells, so RCP-1 was selected for subsequent experiments.

[0118] 3. Elisa method to detect cellular liver function indicators AST and ALT:

[0119] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are medical indicators for determining whether the liver is damaged. They are two common glycosidases, and the increase or decrease in their values ​​can reflect the degree of cell damage to a certain extent.

[0120] The logarithmically growing HepG2 cells were centrifuged at 12000g for 10 min, and the supernatant was taken for later use. The AST and ALT contents were determined according to the instructions of the Elisa kit, and the absorbance of the supernatant was measured at 450 nm.

[0121] The experiment showed that the best effect of drug intervention was to reduce the activities of ALT and AST, which were (355.22±7.39)U / L and (239.79±25.43)U / L respectively, indicating that RCP-1 has a good repair effect on cell damage.

[0122] 4. Elisa method to detect the levels of SOD, CAT, GSH and MDA in cell serum:

[0123] HepG2 cells were cultured as described above, centrifuged at 12000 g for 10 minutes, the precipitate was collected, and the cells were crushed on ice for 5 minutes by ultrasonic crusher, and finally centrifuged to collect the supernatant for use. According to the instructions of the Elisa kit, the absorbance of the supernatant was measured at 450 nm.

[0124] By the attached Fig.13 It can be seen from the experiment that after treatment with 50.0 μg / mL of RCP-1, the SOD activity of the control group increased significantly (P<0.05), indicating that RCP-1 can improve the antioxidant activity of cells by increasing the intracellular SOD activity;

[0125] Silybin can effectively improve the decrease in CAT activity caused by H2O2 oxidative damage. When RCP-1 is used, the CAT activity of damaged HepG2 cells gradually increases with the increase in concentration, and the best effect is achieved at 50μg / ml, indicating that RCP-1 can effectively increase the CAT activity of oxygen-damaged HepG2 cells.

[0126] After adding H2O2 and without RCP-1 intervention, the enzyme activity value decreased significantly (P<0.05), indicating that the activity of the antioxidant enzyme GSH in the cells was destroyed. After adding different concentrations of RCP-1 intervention, the content of GSH-Px increased significantly (p<0.05). With the increase of RCP-1 concentration, the level of GSH became closer to the normal value, and the best performance was achieved at a concentration of 50μg / ml, indicating that RCP-1 can increase the activity of GSH to a certain extent, which is beneficial to blocking lipid peroxidation in the body and protecting cells from oxidative damage.

[0127] The MDA content produced by cells treated with H2O2 alone was the highest. When different concentrations of RCP-1 were added to the cells and intervened for 12 hours, the intracellular MDA level was significantly reduced, indicating that the cell membrane of HepG2 cells was severely damaged after H2O2 treatment. It was significantly increased after pretreatment with 50μg / mL RCP-1; thus, RCP-1 alleviated the H2O2-induced decrease in cell survival rate and the decrease in enzyme levels such as GSH, SOD and CAT.

[0128] 5. Flow cytometry to detect intracellular ROS levels:

[0129] DCFH-DA is a cell-permeable compound that is enzymatically hydrolyzed within cells to produce DCFH, a non-fluorescent compound. The fluorescent product dichlorofluorescein (DCF) is generated by the oxidation of DCFH by intracellular ROS, especially H2O2; increased levels of ROS indicate increased oxidative stress.

[0130] Logarithmically growing HepG2 cells were seeded in 24-well plates, 1 mL of drug was added to each well and incubated for 24 h, treated with different concentrations of pallet root extract for 12 h, and damaged with H2O2 for 4 h. Then, 1 mL of 10 μmol / L DCFH-DA was added to each well in the dark and incubated for 30 min. All cells in the wells were scraped off with a scraper, and the cells were collected and placed in a centrifuge tube. Finally, the fluorescence intensity was measured by flow cytometry (Ex = 488 nm, FL1EM = 525 nm).

[0131] By the attached Fig.14 It can be seen that the experiment showed that with the increase of RCP-1 concentration, the ROS level in the damaged HepG2 cells gradually decreased, and RCP-1 effectively improved the oxidative damage of H2O2 to HepG2 cells, showing a good protective effect.

[0132] 6. Observation of cell Hochest 33342 staining under fluorescence microscope:

[0133] HepG2 cells were cultured as described above, and the diluted HepG2 cells (2.5×105 / well) were transferred to a 6-well plate. 2 mL per well was incubated for 24 h, drug treatment for 12 h, H2O2 treatment for 4 h, and then 10 μg / mL Hoechst333421 mL was added for 5 min in a dark environment, the dye solution was discarded, and the cells were washed 3 times with PBS. Cell growth was observed under a fluorescence microscope.

[0134] The experiment showed that Fig.15 As shown, intervention with 50 μg / mL of RCP-1 can alleviate cell apoptosis to a certain extent, just like Silybin.

[0135] VII. Mechanism of the protective effect of RCP-1 on H2O2-induced HepG2 cell damage

[0136] Effects of RCP-1 on Bax and Bcl-2 pathways

[0137] In this study, Fig.15 As shown in the results, compared with untreated cells, H2O2 treatment downregulated the expression of Bcl-2 in HepG2 cells, while increasing the expression of the pro-apoptotic protein Bax (p<0.05). Pretreatment with different concentrations of D-RCP-1 significantly reduced the expression of Bax and increased the expression of Bcl-2. The results showed that the protective effect of RCP-1 from the root of R. rapae on apoptosis of HepG2 cells treated with H2O2 can be attributed to the regulation of Bax and Bcl-2 family proteins. RCP-1 can exert its inhibitory effect on H2O2-induced cellular oxidative stress and apoptosis by regulating Bax and Bcl-2.

[0138] This experiment further explored whether TLR-4 receptor is involved in the protection against H2O2-induced apoptosis. Western blot analysis was performed. Fig.16 The results showed that H2O2 treatment significantly increased the expression levels of TLR-4 and NF-κB proteins (p<0.05). Compared with the H2O2 treatment group, pretreatment with 25 and 50 μg / mL RCP-1 could significantly reduce the expression of TLR-4 and NF-κB proteins, while the expression levels of TLR-4 and NF-κB proteins in cells treated with low concentrations were not significantly different from those in the model group. The results showed that the protective effect of RCP-1 on cell apoptosis is related to the regulation of TLR-4 and NF-κB protein expression.

[0139] In summary, the purified polysaccharide (RCP-1) from the root of pallet showed a significant protective effect on the oxidative stress of HepG2 cells stimulated by H2O2 in a dose-dependent manner at a concentration of 6.25-50μg / mL. It can inhibit the levels of ALT and AST in HepG2 cells induced by H2O2, reduce the production of ROS in oxidatively damaged cells, increase the activities of antioxidant enzymes CAT, SOD and GSH, and reduce the content of MDA. The mechanism of action is that RCP-1 can regulate the expression of Bax / Bcl-2 protein, upregulate the expression level of TLR-4 protein, and upregulate the expression of NF-KB protein, indicating that the homogeneous polysaccharide RCP-1 from the root of pallet can reduce the oxidative damage induced by H2O2 in HepG2 cells, and the polysaccharide RCP-1 from the root of pallet has a good application prospect in the prevention and treatment of liver protection.

[0140] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing polysaccharide from the root of the Chinese hollyhock root, characterized in that: The following steps are involved: Step 1: first crush the dried pallet root and pass it through a 60-mesh sieve to obtain fine medicinal material particles, then weigh 80g of the fine medicinal material particles into a 2000ml round-bottom flask, add 15-30 times the volume of distilled water, reflux in a 90°C water bath for 2-3h, filter, repeat the operation 2-3 times, combine the filtrate and concentrate under reduced pressure, add 3-4 times the volume of 95% ethanol to the concentrate, precipitate with alcohol for 12h, filter and obtain the supernatant, then perform rotary evaporation to remove ethanol and freeze-dry to obtain pallet root crude polysaccharide and weigh it; Step 2: Dissolve the crude polysaccharide of the root of the radix serrata in ultrapure water, add Sevag reagent to remove the protein in the solution to obtain a polysaccharide aqueous solution without protein, then dialyze for three days with a 3000Da cutoff to remove low molecular weight components, and finally freeze-dry to obtain the crude polysaccharide with small molecular impurities removed; Step 3: Prepare the crude polysaccharide into an aqueous solution, first use a DEAE-52 cellulose chromatographic column for preliminary purification, then use water and NaCl solution as eluents for gradient elution to obtain a preliminary purified eluent, and sample to obtain 7 types of polysaccharides; Step 4: Load the preliminary purified eluate onto a Sephadex G-100 column for the next step of purification, using deionized water as the eluent to elute, obtaining a purified eluate, and taking samples to obtain four purified radix serrata polysaccharides; Step 5. Finally, use a UV-visible spectrometer to determine the purity of the purified pallet root polysaccharide.

2. The method for preparing polysaccharide from the root of the Chinese pallet root according to claim 1, characterized in that: The Sevag reagent used in the step 2 is prepared from chloroform and n-butanol in a volume ratio of 4:1; the NaCl concentrations used in the gradient elution in the step 3 are 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L and 1 mol / L, respectively, and the gradient elution flow rate is 1 ml / min.

3. The method for preparing polysaccharide from the root of Psoralea corylifolia according to claim 1, characterized in that: After obtaining the preliminary purified eluate in step three, collect a tube of 10 ml of the eluate, and use the phenol-sulfuric acid method to determine the polysaccharide content in each component, and then concentrate, dialyze and freeze-dry to obtain 7 polysaccharides, namely D-RCP-1, D-RCP-2, D-RCP-3, D-RCP-4, D-RCP-5, D-RCP-6 and D-RCP-7.

4. The method for preparing polysaccharide from the root of Psoralea corylifolia according to claim 1, characterized in that: In the step 4, the elution flow rate is 0.5 ml / min. After obtaining the purified eluate, a 5 ml tube of the purified eluate is collected and the absorbance is measured until all the polysaccharide components are collected. Using the same fraction and the same freeze-drying method, four purified pallet root polysaccharides are obtained, namely RCP-1, RCP-3, RCP-4 and RCP-5.

5. The method for preparing polysaccharide from the root of Psoralea corylifolia according to claim 1, characterized in that: The concentration determination in step 5 is specifically as follows: S1. Take 20 mg of glucose and place it in a 100 ml volumetric flask, add water to make up to volume, and obtain a glucose solution which is used as the reference solution; S2, then take 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml and 1.2ml of glucose solution and put them in test tubes, add water to make up to 2ml respectively, then add 1ml of 4% phenol solution respectively and shake well, quickly add 7ml of concentrated H2SO4, let it stand for 5min, then heat it in a 40℃ water bath for 30min, react to room temperature, measure the absorbance at a wavelength of 490nm, and then use absorbance as the ordinate and concentration as the abscissa to obtain the standard curve, and the linear regression equation is A=8.415x+0.1773, R 2 =0.9994, where A represents absorbance, x represents glucose mass concentration in mg / ml; S3. Take 20 mg of the purified pallet root polysaccharide sample and place it in a 25 ml volumetric flask, add water to make up the volume, then take 2 ml of the fixed solution into a test tube, add 1 ml of 4% phenol solution and shake well, quickly add 7 ml of concentrated H2SO4, let it stand for 5 minutes, then place it in a 40°C water bath and heat for 30 minutes, react to room temperature, measure the absorbance at a wavelength of 490 nm, and substitute it into the above linear regression equation to calculate the pallet root polysaccharide concentration.

6. Use of four kinds of purified radix trechomae polysaccharides prepared according to the preparation method of radix trechomae polysaccharides according to any one of claims 1 to 5 in liver protection.

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