阅读说明：本技术 一种鲜地黄多糖及其制备方法和应用 (Fresh rehmannia root polysaccharide and preparation method and application thereof ) 是由 周艳 黄勇 张红瑞 张江山 夏至 李贺敏 于 2021-10-21 设计创作，主要内容包括：本发明公开了一种鲜地黄多糖及其制备方法和应用,属于天然药物和天然药物提取技术领域。所述鲜地黄多糖中的单糖单元包括鼠李糖、阿拉伯糖、半乳糖、葡萄糖,其分子量为17.1KDa。其中鼠李糖：阿拉伯糖：半乳糖：葡萄糖的摩尔比为25.1：26.2：28.7：20.0。所述鲜地黄多糖的主链为→3,6)-β-D-Galp-(1→6)-β-D-Galp-(1→,支链为→5)-α-L-Araf-(1→2,5)-α-L-Araf-(1→的阿拉伯糖链,并通过→3,6)-β-D-Galp-(1→的O-3连接在主链上。本发明公开的鲜地黄多糖,具有免疫调节和抗炎活性。(The invention discloses a fresh rehmannia root polysaccharide and a preparation method and application thereof, and belongs to the technical field of natural medicines and natural medicine extraction. Monosaccharide units in the fresh rehmannia root polysaccharide comprise rhamnose, arabinose, galactose and glucose, and the molecular weight of the monosaccharide units is 17.1 KDa. Wherein the weight ratio of rhamnose: arabinose: galactose: the molar ratio of glucose is 25.1:26.2:28.7: 20.0. the main chain of the fresh rehmannia root polysaccharide is an arabinose chain which is → 3,6) -beta-D-Galp- (1 → and the branched chain is → 5) -alpha-L-Araf- (1 → 2,5) -alpha-L-Araf- (1 → and is connected with the main chain through → 3,6) -beta-D-Galp- (1 → O-3). The fresh rehmannia root polysaccharide disclosed by the invention has the immunoregulation and anti-inflammatory activities.)

1. The fresh rehmannia root polysaccharide is characterized in that monosaccharide units in the fresh rehmannia root polysaccharide comprise rhamnose, arabinose, galactose and glucose; the molecular weight of the fresh rehmannia root polysaccharide is 17.1 KDa.

2. The fresh rehmannia root polysaccharide of claim 1, wherein the rhamnose: arabinose: galactose: the molar ratio of glucose is 25.1:26.2:28.7: 20.0.

3. a method for preparing the fresh rehmannia glutinosa polysaccharide of claim 1 or 2, comprising the steps of:

(1) carrying out enzymolysis on the fresh rehmannia by using compound enzyme, and precipitating enzymolysis liquid by using alcohol to obtain total polysaccharide of the fresh rehmannia;

(2) removing protein in the total polysaccharide of the fresh rehmannia root obtained in the step (1) by utilizing a Sevag method to obtain a crude polysaccharide solution of the fresh rehmannia root;

(3) separating the crude polysaccharide solution of the fresh rehmannia root obtained in the step (3) by an ion exchange chromatographic column; and performing gel column series chromatography to obtain fresh rehmanniae radix polysaccharide.

4. The preparation method according to claim 3, wherein the compound enzyme in the step (1) is obtained by compounding pectinase and papain in a mass ratio of 1: 1; the mass ratio of the compound enzyme to the fresh rehmannia is 0.15: 100; the volume ratio of the enzymolysis liquid to the fresh rehmannia is (5-10): 1.

5. The preparation method of claim 3, wherein the specific operation of enzymolysis in step (1) is to add complex enzyme, adjust the pH value to 4-5, carry out enzymolysis for 1h at 50 ℃, and then carry out ultrasonic treatment for 1h at 50 ℃ with the ultrasonic power of 80W.

6. The method according to claim 3, wherein the alcohol precipitation in the step (1) is performed to adjust the alcohol concentration of the enzymatic hydrolysate to 90%.

7. The method according to claim 3, wherein the extraction liquid used in the Sevag method in the step (2) is a mixture of chloroform and n-butanol at a volume ratio of 4: 1.

8. The method according to claim 3, wherein the chromatographic conditions for the ion exchange chromatography column separation in step (3) are:

a chromatographic column: DEAE-Sepharose FF column;

flow rate: 50 mL/h;

elution procedure: elution was carried out with water, 0.2M NaCl, 0.5M NaCl, and 2M NaCl in this order.

9. The method according to claim 3, wherein the gel column tandem chromatography conditions in step (3) are:

a chromatographic column: gel Superdex-200;

detection conditions are as follows: LC-10A high performance liquid chromatograph, RI-502 differential detector;

mobile phase: 0.05M NaCl solution, flow rate: 0.6mL/min, column temperature: 40 ℃;

sample introduction amount: 20 μ L.

10. Use of the fresh rehmannia glutinosa polysaccharide of claim 1 or 2 for the preparation of an immunomodulatory or anti-inflammatory drug.

Technical Field

The invention relates to the technical field of natural medicines and natural medicine extraction, in particular to a fresh rehmannia root polysaccharide and a preparation method and application thereof.

Background

The polysaccharide is a chemical component commonly existing in traditional Chinese medicines, almost all plant traditional Chinese medicines contain the component, and the polysaccharide is an important material basis for the traditional Chinese medicines to play the drug effect and has the effects of reducing blood fat, reducing blood sugar, resisting tumors, enhancing the immune function and the like. In recent years, with the further development of scientific research, the unique pharmacological activity of polysaccharide components is gradually recognized, and the traditional Chinese medicine polysaccharide has become the focus of common attention of modern medicine and food functional chemistry. Many researches show that the traditional Chinese medicine polysaccharide generally has a good effect on immune function, and the change of the immune function and inflammatory reaction are accompanied in the occurrence of most diseases. Macrophages, which are important effector cells of the immune system, are considered as target cells of various immunomodulatory and anti-inflammatory drugs, and have the effects of secreting various cytokines, activating immune response, participating in inflammatory cascade, and the like. In the treatment of diseases, traditional Chinese medicines often play an anti-inflammatory role by regulating the immune process of an organism and reduce the injury of the organism, thereby intervening the occurrence and development of diseases.

The rehmanniae radix is fresh root tuber or dried root tuber of Rehmannia glutinosa Libosch of Scrophulariaceae family. Fresh, or slowly bake rehmannia glutinosa until about eighty percent dry. Since Shen nong Ben Cao Jing (Shen nong's herbal), its clinical application history is long, it is mainly divided into fresh rehmannia root, Sheng Di Huang and Shu Di Huang, all having the effect of tonifying, and the fresh herb is called fresh rehmannia root. Fresh rehmannia root, sweet in taste, bitter and cold in nature, enters heart, liver and kidney channels, and has the effects of clearing heat, promoting fluid, cooling blood and stopping bleeding; can be used for treating yin impairment due to febrile disease, crimson tongue with dipsosis, toxic heat, macula, hematemesis, epistaxis, and sore throat. The actions of Xian Di Huang for clearing heat and cooling blood, such as treating high fever acute syndrome and recklessly blood flow due to blood heat, are superior to those of dry product, and they cannot be used together. The fresh rehmannia root has a long history of administration, and the ancient herbal medical records the use characteristics of pounding (grinding) rehmannia root juice for drinking or coating. Modern researches show that rehmannia contains abundant polysaccharide components, and is an important active component of rehmannia for playing the drug effect. The rehmanniae radix polysaccharide has biological activities of improving immunity, resisting tumor, relieving fatigue, and resisting anxiety. The fresh rehmannia root juice is rich in polysaccharide components, but because the fresh rehmannia root is not easy to store, is sensitive to temperature and is easy to oxidize, the clinical application and activity research are less, and the immunoregulation effect of the fresh rehmannia root is not clear.

Disclosure of Invention

The invention aims to provide a fresh rehmannia root polysaccharide and a preparation method and application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

one of the technical schemes of the invention is as follows: providing a fresh rehmannia root polysaccharide, wherein monosaccharide units in the fresh rehmannia root polysaccharide comprise rhamnose (Rha), arabinose (Ara), galactose (Gal) and glucose (Glc); the molecular weight of the fresh rehmannia root polysaccharide is 17.1 KDa.

Preferably, the rhamnose: arabinose: galactose: the molar ratio of glucose was 25.1:26.2:28.7: 20.0.

Preferably, the main chain of the fresh rehmannia root polysaccharide is an arabinose chain which is → 3,6) -beta-D-Galp- (1 → 5) -alpha-L-Araf- (1 → 2,5) -alpha-L-Araf- (1 → and is connected to the main chain through → 3,6) -beta-D-Galp- (1 → O-3;

the bond → 6) -beta-D-Galp- (1 → the anomeric carbon of the polysaccharide of fresh rehmannia glutinosa is linked with → 6) -beta-D-Galp- (1 → H6; the glycosidic linkage → the anomeric carbon of 3,6) - β -D-Galp- (1 → is linked to H6 of → 6) - β -D-Galp- (1 →; the anomeric hydrogen of → 5) - α -L-Araf- (1 → is linked to C5 of → 2,5) - α -L-Araf- (1 →; the anomeric H1 of → 2,5) -alpha-L-Araf- (1 → is linked with C3 of → 3,6) -beta-D-Galp- (1 → is linked with the anomeric hydrogen of → 2,5) -alpha-L-Araf- (1 → C2 of → 2, 5).

The second technical scheme of the invention is as follows: provides a preparation method of the fresh rehmannia root polysaccharide, which comprises the following steps:

(1) carrying out enzymolysis on the fresh rehmannia by using compound enzyme, and precipitating enzymolysis liquid by using alcohol to obtain total polysaccharide of the fresh rehmannia;

(2) removing protein in the total polysaccharide of the fresh rehmannia root obtained in the step (1) by utilizing a Sevag method to obtain a crude polysaccharide solution of the fresh rehmannia root;

(3) separating the crude polysaccharide solution of the fresh rehmannia root obtained in the step (3) by an ion exchange chromatographic column; and performing gel column series chromatography to obtain fresh rehmanniae radix polysaccharide.

Preferably, the complex enzyme in the step (1) is obtained by compounding pectinase and papain in a mass ratio of 1: 1; the mass ratio of the compound enzyme to the fresh rehmannia is 0.15: 100; the volume ratio of the enzymolysis liquid to the fresh rehmannia is (5-10): 1.

Preferably, the specific operation of the enzymolysis in the step (1) is to add complex enzyme, adjust the pH value to 4-5, perform enzymolysis for 1h at 50 ℃, and perform ultrasonic treatment for 1h at 50 ℃ with the ultrasonic power of 80W.

Preferably, the alcohol precipitation in the step (1) is performed to adjust the alcohol concentration of the enzymatic hydrolysate to 90%.

Preferably, the extract used in the Sevag method in step (2) is a mixture of chloroform and n-butanol at a volume ratio of 4: 1.

More preferably, the number of times of extraction by the Sevag method is 3 to 5.

Preferably, the chromatographic conditions of the ion exchange chromatographic column separation in the step (3) are as follows:

a chromatographic column: DEAE-Sepharose FF column;

flow rate: 50 mL/h;

elution procedure: elution was sequentially carried out with water, 0.2M NaCl, 0.5M NaCl, 2M NaCl.

The invention utilizes phenol-sulfuric acid method to trace and detect the absorbance value of the eluent, and determines the elution time according to the detection result, the fresh rehmannia root polysaccharide selected by the invention is a sample contained in the eluent obtained by 0.2M NaCl elution.

Preferably, the gel column tandem chromatography conditions in step (3) are as follows:

a chromatographic column: gel Superdex-200;

detection conditions are as follows: LC-10A high performance liquid chromatograph, RI-502 differential detector;

mobile phase: 0.05M NaCl solution, flow rate: 0.6mL/min, column temperature: 40 ℃;

sample introduction amount: 20 μ L.

The third technical scheme of the invention is as follows: provides an application of the fresh rehmannia root polysaccharide in preparing an immunoregulation medicament or an anti-inflammatory medicament.

The invention has the following beneficial technical effects:

the invention provides a new fresh rehmannia root polysaccharide and a preparation method thereof. The preparation method provided by the invention is suitable for industrial production and provides a direction for quality control and standardized production of the fresh rehmannia root polysaccharide.

The method comprises the steps of extracting fresh rehmannia root polysaccharide by enzymolysis, combining ion exchange column chromatography and gel column chromatography, purifying and separating to obtain the fresh rehmannia root polysaccharide, determining the molecular weight by adopting high performance gel filtration chromatography (HPGPC), analyzing monosaccharide composition by an ion spectrometer, analyzing glycoside bond configuration and functional group information by an infrared spectrometer, analyzing the type of acetyl ester of sugar alcohol by GC-MS after methylation, judging monosaccharide residue, carrying out nuclear magnetic spectrum analysis on the monosaccharide residue, and determining the glycoside bond connection mode to obtain the new fresh rehmannia root polysaccharide.

The fresh rehmannia root polysaccharide provided by the invention has the immunoregulation and anti-inflammatory activities, and provides a direction for developing potential new medicines for immunoregulation and anti-inflammatory.

Drawings

FIG. 1 is a scattergram prepared by plotting the absorbance of an eluate eluted at a gradient in example 1.

FIG. 2 shows the results of the primary purified rehmannia glutinosa polysaccharide of example 1 passing through Sephadex Superdex-200 column and then detecting with a differential detector.

FIG. 3 is an HPGPC chart of fresh rehmannia glutinosa polysaccharide prepared in example 1.

FIG. 4 is an infrared spectrum of the polysaccharide of fresh rehmannia glutinosa prepared in example 1.

FIG. 5 is the HH-COSY spectrum of fresh rehmannia glutinosa polysaccharide prepared in example 1.

FIG. 6 is an HSQC spectrum of fresh rehmannia glutinosa polysaccharide prepared in example 1.

FIG. 7 is an HMBC profile of the fresh rehmannia glutinosa polysaccharide prepared in example 1.

FIG. 8 is a NOESY spectrum of the polysaccharides of fresh rehmannia glutinosa prepared in example 1.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The fresh rehmannia root used in the embodiment of the invention is selected from fresh radix rehmanniae tuberous roots of Huai-Dihuang, which is prepared from Henan Job's wort.

Example 1

Preparing fresh rehmannia root polysaccharide:

cutting 500g of fresh rehmannia root, adding 5 times of volume of distilled water, adding 75g of pectinase and papain (mass ratio of 1:1), adjusting the pH value to 4.5, uniformly stirring, carrying out enzymolysis at constant temperature of 50 ℃ for 1h, transferring the enzymolysis solution of the fresh rehmannia root into an ultrasonic extractor, carrying out ultrasonic extraction for 1h at 50 ℃ and power of 80W to obtain about 2.5L of extracting solution, filtering the extracting solution, concentrating by using a rotary thin-film evaporator to obtain fluid extract, dispersing and dissolving the fluid extract by using 100mL of distilled water, adding 95% industrial ethanol until the alcohol concentration reaches 90%, standing for 24h, centrifuging to obtain precipitate, and volatilizing the ethanol to obtain the total polysaccharide of the fresh rehmannia root.

Adding distilled water 5 times the volume of the obtained total polysaccharides of radix rehmanniae, adding 1/5 solution amount of chloroform-n-butanol (4:1) mixed solution according to Sevag method, extracting, shaking in shaking table for 20min, transferring to separating funnel, standing, collecting upper layer liquid, centrifuging for 1min to remove residual protein precipitate. Repeating the steps for 3 times to obtain crude polysaccharide solution of fresh rehmannia root after protein removal, and concentrating and drying under reduced pressure to obtain about 40g of crude polysaccharide of fresh rehmannia root.

Loading the obtained crude polysaccharide of rehmanniae radix on column (DEAE-Sepharose FF column), and connecting with flow collector and peristaltic pump. Sequentially eluting with distilled water, 0.2M NaCl, 0.5M NaCl and 2M NaCl, measuring absorbance at 490nm of the obtained eluates by phenol-sulfuric acid method, eluting each eluent until absorbance at 490nm is reduced to the minimum, replacing the next eluent, and drawing a scatter diagram (see FIG. 1) according to the measurement result. Collecting 0.2M NaCl eluate, concentrating, dialyzing with 3500Da dialysis bag, and freeze drying to obtain once-purified fresh rehmanniae radix polysaccharide, about 10.8 g.

Dissolving 100mg of once purified fresh rehmannia root polysaccharide in 3mL of distilled water, centrifuging by using a centrifuge (12000rpm) for 10min, further separating and purifying the supernatant by using a sephadex Superdex-200 column, and performing online detection by combining a differential detector under the detection conditions that: LC-10A high performance liquid chromatograph, RI-502 differential detector; mobile phase: 0.05M NaCl solution, flow rate: 0.6mL/min, column temperature: 40 ℃; sample introduction amount: 20 mu L, the detection result is shown in figure 2, the component of the third characteristic peak is collected (the component of the symmetrical peak is collected for 130-160 min in the experiment), the collected solution is concentrated by a rotary evaporator, and the fresh rehmannia root polysaccharide, about 15mg, is obtained by freeze drying.

Example 2

The fresh rehmannia root polysaccharide prepared in example 1 is subjected to structural verification:

(1) HPGPC measurement of molecular weight of fresh rehmannia glutinosa polysaccharide of example 1 (see FIG. 3)

Measuring with Shimadzu LC-10A high performance liquid chromatograph, RI-502 differential detector, and series gel chromatographic column (8 × 300 mm); mobile phase 0.05M NaCl solution, flow rate: 0.6mL/min, column temperature: 40 ℃; sample introduction amount: 20 μ L. Establishing a regression curve by using dextran standards with different molecular weights, and calculating the molecular weight. The dextran standard substance (molecular weight 1152, 5000, 11600, 23800, 48600, 80900, 148000, 273000) is prepared into 5mg/mL solution before mobile phase use, lg value of molecular weight is used as abscissa, retention time RT is used as ordinate to make standard curve, lgMp-RT correction curve equation is: Y-0.1933X +12.336 (R)²0.9968); the lgMw-RT calibration curve equation is: -0.2064X +13.013, (R)²0.9971); the lgMn-RT calibration curve equation is: -0.178X +13.033 (R)²0.9969); the molecular weight of the fresh rehmannia root polysaccharide is calculated by a standard curve regression equation, and the measurement result is 17.1 KDa.

(2) Ion chromatography for measuring the content of each monosaccharide of the fresh rehmannia root polysaccharide in example 1

Separately, 4mg of polysaccharide was sampled, 1mL of 2M trifluoroacetic acid solution (TFA) was added, the mixture was sealed with nitrogen, and hydrolyzed at 120 ℃ for 3 hours. Precisely sucking 200 μ L of acid hydrolysis solution into a 1.5mL LEP tube, and blowing with nitrogen. 1mL of distilled water was added to the EP tube and dissolved by vortexing. The supernatant was centrifuged at 12000rpm for 5min and assayed by ion chromatography (ICS 5000). The chromatographic conditions are as follows: dionex CarbopacTM PA20 column (3X 150mm), column temperature 30 ℃, sample size 5. mu.L, flow rate: 0.3mL/min, mobile phase: a is H₂O；B:250mM NaOHC:50mM NaOH&500mM NaOAC, detector: an electrochemical detector. The standard substance of mixed monosaccharide of rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, fucose, glucosamine hydrochloride, N-acetylglucosamine, glucuronic acid and galacturonic acid is used as a reference. The monosaccharide composition of the fresh rehmannia glutinosa polysaccharide was obtained and the results are shown in Table 1.

TABLE 1 monosaccharide composition of polysaccharides of fresh rehmannia glutinosa

(3) Methylation reaction to determine monosaccharide residues

Accurately weighing 3mg purified polysaccharide in a glass reaction bottle, adding 1mL anhydrous DMSO, rapidly adding NaOH powder, and adding N in a nitrogen blowing instrument₂Blocking, dissolving with ultrasound, and adding 1mL iodomethane (CH)₃I) The reaction was carried out for 60min in a magnetic stirring 30 ℃ water bath, and the methylation reaction was terminated by adding 2mL of ultrapure water. The methylated product was hydrolyzed by adding 2mol/L of LTFA1mL for 1.5h, and concentrated under reduced pressure on a rotary evaporator. After reduction of the hydrolysate, the residue was taken up in 2mL of double distilled water and washed with 60mg of sodium borohydride (NaBH)₄) And reducing for 8 h. Then adding 0.1mL of glacial acetic acid for neutralization, concentrating by a rotary evaporator, drying by an oven at 101 ℃, then adding acetic anhydride, reacting at 1mL of 100 ℃ for 1h for acetylation, and cooling. 3mL of toluene was added, concentrated under reduced pressure to dryness, and repeated 5 times to remove excess acetic anhydride. The acetylated product was treated with 3mL of CH₂Cl₂Dissolve and extract 4 times with a distilled water separatory funnel. Removing the upper distilled water layer, CH₂Cl₂Drying with appropriate amount of anhydrous sodium sulfate, diluting to 10mL, filtering with 0.22 μm microporous membrane, and measuring and analyzing by GC-MS, the measurement results are shown in Table 2.

TABLE 2 GC-MS measurement results of methylated glycal acetyl ester of fresh rehmannia root polysaccharide

(3) Infrared spectrum of fresh rehmannia glutinosa polysaccharide prepared in example 1 (see FIG. 4)

As can be seen from FIG. 4, the absorption band of fresh rehmannia root polysaccharide is 3600-^-1Is a stretching vibration absorption peak of-OH, this regionThe absorption peak of (2) is a characteristic peak of the glucide. 3363cm^-1Is the absorption peak of stretching vibration of O-H and is the characteristic peak of saccharide. At 2933cm^-1There is an absorption peak, which is attributed to C-H stretching vibration. At 1650cm^-1Absorption peak, attributed to stretching vibration of C ═ O. 1421cm^-1Has an absorption peak which is attributed to C-O stretching vibration. At 1043cm^-1Has an absorption peak belonging to O-H variable angle vibration.

(4) NMR spectra of the fresh rehmannia glutinosa polysaccharide prepared in example 1, wherein HH-COSY spectrum is shown in FIG. 5, HSQC spectrum is shown in FIG. 6, HMBC spectrum is shown in FIG. 7, and NOESY spectrum is shown in FIG. 8.

From FIG. 5, it can be observed that the anomeric carbon signal is δ 104.69, the corresponding anomeric hydrogen signal in FIG. 5 is δ 4.47, and from FIG. 6, it can be observed that the signal of H1-2 is 4.47/3.57; the signal of H2-3 is 3.57/3.68; signal 3.68/4.05 of H3-4; the signal of H4-5 is 4.05/3.87; the signal of H5-6a is 3.87/3.96; it can further be concluded that H1, H2, H3, H4, H5, and H6a are δ 4.47, 3.57, 3.68, 4.05, 3.87, and 3.96, respectively. Corresponding C1-C6 are 104.69, 71.31, 81.5, 69.82, 74.81, 70.76; therefore, the signal should be assigned to the glycosidic bond → 3,6) -Galp- (1 →.

From FIG. 5, it can be observed that the anomeric carbon signal is δ 108.81, the corresponding anomeric hydrogen signal in FIG. 5 is δ 5.12, and from FIG. 6, it can be observed that the signal for H1-2 is 5.12/4.04; the signal of H2-3 is 4.04/3.87; the signal of H3-4 is 3.87/4.01; the signal of H4-5a is 4.01/3.76; it can further be concluded that H1, H2, H3, H4, and H5a are δ 5.12, 4.04, 3.87, 4.01, and 3.76, respectively. The corresponding C1-C5 are 108.81, 82.62, 77.97, 85.22 and 62.64; therefore, the signal should be assigned to the glycosidic linkage α -L-Araf- (1 →.

From FIG. 5, it can be observed that the anomeric carbon signal is δ 108.8, and the corresponding anomeric hydrogen signal in FIG. 5 is δ 5.11, and from FIG. 6, it can be observed that the signal of H1-2 is 5.11/4.16; the signal of H2-3 is 4.16/4.09; signal 4.09/3.93 of H3-4; the signal of H4-5a is 3.93/3.80; it can further be concluded that H1, H2, H3, H4, and H5a are δ 5.11, 4.16, 4.09, 3.93, and 3.80, respectively. The corresponding C1-C5 is 108.8, 88.62, 77.1, 83.85, 67.81; therefore, the signal should be assigned to the glycosidic bond → 2,5) - α -L-Araf- (1 →.

From FIG. 5, an anomeric carbon signal of δ 108.8 can be observed, and the corresponding anomeric hydrogen signal in FIG. 5 is δ 5.01, and from FIG. 6, the signal of H1-2 is 5.01/4.05; the signal of H2-3 is 4.05/3.88; signal 3.88/4.16 of H3-4; the signal of H4-5a is 4.16/3.80; it can further be concluded that H1, H2, H3, H4, and H5a are δ 5.01, 4.05, 3.88, 4.16, and 3.80, respectively. The corresponding C1-C5 are 108.8, 82.52, 77.93, 83.6 and 67.81; thus, the signal should be assigned to the glycosidic bond → 5) - α -L-Araf- (1 →.

According to the nuclear magnetic one-dimensional two-dimensional maps in fig. 7 and 8, the glycosidic bond signals of the polysaccharides can be assigned;

the glycosidic bond → 6) - β -D-Galp- (1 → the anomeric carbon → 6) - β -D-Galp- (1 → H6 has a signal peak; indicating the presence of the → 6) - β -D-Galp- (1 → 6) - β -D-Galp- (1 → the linkage.

Glycosidic bond → 3,6) - β -D-Galp- (1 → the anomeric carbon → 6) - β -D-Galp- (1 → H6 has a signal peak; indicating the presence of the → 3,6) - β -D-Galp- (1 → 6) - β -D-Galp- (1 → a linkage.

The anomeric hydrogen → 5) - α -L-Araf- (1 → has a correlation peak with C5 → 2,5) - α -L-Araf- (1 → indicating the presence → 5) - α -L-Araf- (1 → 2,5) - α -L-Araf- (1 →.

The anomeric hydrogen of α -L-Araf- (1 → correlates with C2 of → 2,5) - α -L-Araf- (1 → indicating the presence of α -L-Araf- (1 → 2,5) - α -L-Araf- (1 →).

→ 2,5) - α -L-Araf- (1 → anomeric H1 and → 3,6) - β -D-Galp- (1 → C3 have related signal peaks; indicating the presence of the → 2,5) - α -L-Araf- (1 → 3,6) - β -D-Galp- (1 → linkage.

In summary, the backbone chain of the polysaccharide of fresh rehmannia glutinosa prepared in example 1 is → 3,6) - β -D-Galp- (1 →, and the side chain is → 5) - α -L-Araf- (1 → 2,5) - α -L-Araf- (1 → arabinose chain, and is connected to the backbone chain via → 3,6) - β -D-Galp- (1 → O-3.

All glycosidic bond signals were assigned according to the information in FIGS. 5-8, and the results are shown in Table 3.

TABLE 3 carbon and hydrogen signal attribution of fresh rehmannia root polysaccharides

Example 3

Pharmacological Activity assay of fresh rehmannia glutinosa polysaccharide prepared in example 1

(1) Apparatus and materials

Carbon dioxide incubator (Thermo corporation, usa); 5920R high speed centrifuge (eppendorf, germany); an Epoch2 microplate reader (BIOTEK, USA); SW-CJ-2F clean bench (Suzhou clean bench Equipment Co., Ltd.).

Mouse mononuclear macrophage RAW264.7 was purchased from shanghai cell bank of chinese academy of sciences; MTT and LPS were purchased from Sigma; penicillin, streptomycin, Fetal Bovine Serum (FBS), DMEM from Gibco; neutral red staining solution was purchased from solibao corporation; mouse Lysozyme (LZM), IL-1 beta, IL-6, TNF-alpha ELISA kits and NO kits were purchased from Wuhan Eleret Biotech GmbH.

The polysaccharide was the fresh rehmannia glutinosa polysaccharide prepared from example 1.

(2) Experimental methods

Influence of fresh rehmannia root polysaccharide on normal RAW264.7 cell biological function

Screening of safe dose: culturing RAW264.7 cells in DMEM medium containing 10% FBS and 1% streptomycin, collecting cells in logarithmic growth phase, and culturing at 4 × 10⁴Cell density per mL was seeded in 96 well cell culture plates, and 200 μ L of cell suspension was added per well. After 24h of culture, the culture solution is sucked out, the FBS-free culture medium is replaced, and a control group and a fresh rehmannia root polysaccharide administration group with series concentration are set: 1, 10, 20, 50, 100. mu.g/mL, 6 duplicate wells, and incubation was continued for 24 h. The MTT method measures the absorbance value of each group of cells at 490nm and calculates the cell survival rate. The results are shown in Table 3.

Neutral red phagocytosis assay: taking cells in logarithmic growth phase at 4X 10⁴The cells are inoculated in 96-well cell culture plates at a cell density of one/mL, and 200. mu.L of cell suspension is added to each well for 24h of culture. The experiments were grouped into blank control, positive control (LPS, 1. mu.g/mL) and concentrationsPolysaccharide administration group, at the concentration of 2.2.1. After 24h of administration and incubation, the stock culture was aspirated, washed with PBS 1 time, 100. mu.L of 0.1% neutral red solution was added to each well, and after 30min, the neutral red solution was completely aspirated and washed with PBS 1 time. Add 200. mu.L of cell lysis buffer (containing 50% ethanol, 1% acetic acid, 49% water) to each well to extract neutral red, and measure the absorbance value of the solution at 540nm with a microplate reader. The results are shown in Table 4.

Effect of fresh rehmannia glutinosa polysaccharide on RAW264.7 cell lysozyme activity, cytokines (IL-1 beta, IL-6, TNF-alpha): taking the concentration of 4 × 10⁴one/mL of the cell suspension in the logarithmic growth phase was inoculated into 24-well plates, 1mL was added to each well, and the cells were cultured in an incubator for 24 hours. Then the experiment is divided into a control group, a positive control group (LPS, 1 mu g/mL) and a fresh rehmannia root polysaccharide group with the concentration of 1, 20, 50 mu g/mL, and the wells are repeated for 4 times, and the treatment is continued for 24 hours. The culture fluid of each group is collected, centrifuged by a centrifuge for 10min (12000r/min) to remove the precipitate, and the determination is carried out according to the mouse lysozyme activity and the kit instructions of the cell factors (IL-1 beta, IL-6 and TNF-alpha). The results are shown in Table 5.

Effect of fresh rehmannia glutinosa polysaccharide on LPS-stimulated RAW264.7 cell biological function: taking the concentration of 4 × 10⁴one/mL of the cell suspension in the logarithmic growth phase was inoculated into 24-well plates, 1mL was added to each well, and the cells were cultured in an incubator for 24 hours. The experiment was divided into a control group, an LPS model group (LPS, 1. mu.g/mL), and 1. mu.g/mLLPS + administration group (1, 20, 50. mu.g/mL) of fresh rehmannia glutinosa polysaccharide at each concentration, and the treatment was continued for 24 hours in 4 wells. The culture fluid of each group is collected, centrifuged by a centrifuge for 10min (12000r/min) to remove the precipitate, and the determination is carried out according to the mouse lysozyme activity and the kit instructions of the cell factors (IL-1 beta, IL-6 and TNF-alpha). The results are shown in Table 6.

TABLE 4 cell proliferation and phagocytic Activity of fresh rehmannia root polysaccharides

Note: p < 0.05, P < 0.01, in contrast to Control group

As can be seen from Table 4, compared with the control group, the cell viability of the polysaccharide group of fresh rehmannia root has no significant change (P is more than 0.05) in the concentration range of 1, 20 and 50 mug/mL, and the cell activity is significantly reduced (P is less than 0.01) at the concentration of 100 mug/mL, which indicates that the high dose has inhibition effect on macrophages; the phagocytosis of neutral red is obviously increased within the concentration range of 1-10 mug/mL (P is less than 0.05 or 0.01), and the phagocytosis capacity is obviously reduced within the concentration range of 50-100 mug/mL (P is less than 0.05), which shows that the fresh rehmannia root polysaccharide has the function of promoting normal macrophages to phagocytize foreign matters within the range of 1-10 mug/mL, and is inhibited within the range of 50-100 mug/mL.

TABLE 5 Effect of fresh rehmannia glutinosa polysaccharide on cytokine and lysozyme secretion from normal macrophages

Note: p < 0.05, P < 0.01, in contrast to Control group

TABLE 6 Effect of fresh rehmannia glutinosa polysaccharide on secretion of cytokines and lysozyme by LPS-stimulated macrophages

Note: in comparison to the set of models,^#P＜0.05，^##P＜0.01

as can be seen from tables 5 and 6, compared with the control group, the fresh rehmannia root polysaccharide significantly promotes the activity of normal macrophage lysozyme (P is less than 0.01) within the concentration range of 1-50 mug/mL; after LPS stimulation, the content of lysozyme in macrophages is enhanced rapidly, and the lysozyme (P is less than 0.01) can be reduced remarkably by the fresh rehmannia polysaccharide; the fresh rehmannia polysaccharide has no obvious influence on the secretion of TNF-alpha (P is more than 0.05) of normal cells at the concentration of 1 mu g/mL, and obviously promotes normal macrophages to secrete TNF-alpha (P is less than 0.05) at the concentration of 20-50 mu g/mL; the secretion of IL-6 by normal cells is not obviously influenced (P is more than 0.05) when the concentration is 1-20 mu g/mL, and the secretion of normal macrophages and IL-6 are obviously promoted (P is less than 0.01) when the concentration is 50 mu g/mL; the secretion of IL-1 beta by normal macrophages is not obviously influenced (P is more than 0.01) when the concentration is 1-50 mu g/mL; when LPS activates macrophages to secrete cytokines TNF-alpha, IL-6 and IL-1 beta, the fresh rehmannia root polysaccharide inhibits the macrophages to secrete cytokines (P is less than 0.05) within the concentration range of 1-50 mu g/mL. The fresh rehmannia root polysaccharide has a bidirectional immunoregulation effect, improves the phagocytic capacity of macrophages in the early stage of inflammation, promotes the release of lysozyme and cytokines to enhance the immunocompetence of an organism, has certain dose dependence, and improves the excessive release of the lysozyme and the cytokines in inflammatory cells under an inflammatory environment.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.