Radix rehmanniae polysaccharides with a novel structure and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a kind of novel structure of rehmannia polysaccharide and application thereof. The crude polysaccharide of raw rehmannia is obtained by water extraction and fractional alcohol precipitation, and the crude polysaccharide of raw rehmannia is further separated and purified after impurity removal to obtain the rehmannia polysaccharide, and the obtained rehmannia polysaccharide is determined to have a novel structure by combining infrared spectroscopy, complete methylation GC-MS and NMR spectrum analysis. In-vitro experiments prove that the obtained rehmannia polysaccharide has a blood glucose lowering effect and anti-inflammatory activity, and provides a direction for developing a natural medicine with small toxic and side effects and good pharmacological activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. More specifically, it relates to a novel type of Rehmannia glutinosa polysaccharide and its applications. Background Technology

[0002] Rehmannia glutinosa (RG) is the fresh or dried rhizome of the plant Rehmannia glutinosa, belonging to the Scrophulariaceae family. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it exists primarily in three forms: fresh Rehmannia, raw Rehmannia, and processed Rehmannia, each possessing significant medicinal effects. Raw Rehmannia is the dried slice of fresh Rehmannia after soaking, washing, slicing, or cutting into strips. It is cold in nature and has the effects of clearing heat, cooling the blood, nourishing yin, and promoting body fluid production. Currently, various compounds have been isolated from Rehmannia, with the main active ingredients being iridoid glycosides and sugars.

[0003] Rehmannia glutinosa polysaccharides (RGP) are extracted and purified from the rhizome of Rehmannia glutinosa. Based on the number of monosaccharides, they can be divided into oligosaccharides (2 ≤ monosaccharide count ≤ 9) and polysaccharides (≥ 10 monosaccharide count). Rehmannia glutinosa polysaccharides have immune-enhancing and tumor-inhibiting effects; oligosaccharides can enhance hematopoietic function, promote immunity, and lower blood sugar. They can also promote the proliferation of Bifidobacteria in the human gut and can be used to treat acute and chronic diarrhea and hepatic encephalopathy, leading to their increasing recognition and utilization. However, the source and structure greatly affect the efficacy of polysaccharides; chain length, glycosidic bond type, linkage mode, and molecular weight all influence their overall efficacy. For example, Chinese patent application CN113896807A discloses a fresh Rehmannia glutinosa polysaccharide with a specific structure, obtained through enzymatic hydrolysis, alcohol precipitation, and further purification. The resulting fresh Rehmannia glutinosa polysaccharide exhibits immunomodulatory and anti-inflammatory activities. For example, Chinese patent application CN110075181A discloses that Rehmannia glutinosa solution and Rehmannia glutinosa polysaccharide solution obtained by water extraction and alcohol precipitation have anti-anxiety effects and can be effectively used to prepare anti-anxiety drugs. It is evident that Rehmannia glutinosa polysaccharides obtained by different extraction methods have different effects, but the efficacy of Rehmannia glutinosa polysaccharides from different sources and with different structures cannot be confirmed at present. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art, which is that polysaccharides obtained by different extraction methods have different effects, and the efficacy of polysaccharides from different sources and with different structures cannot be confirmed for the time being. The present invention provides a new type of polysaccharide with a novel structure.

[0005] Another object of the present invention is to provide the application of the aforementioned Rehmannia polysaccharide.

[0006] Another object of the present invention is to provide a hypoglycemic or anti-inflammatory drug comprising the aforementioned Rehmannia polysaccharide.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention protects a Rehmannia glutinosa polysaccharide, named RGP-70-2-1, with a molecular weight of 5.7 kDa. Its monosaccharide composition consists of mannose, glucose, rhamnose, galactose, and arabinose, exhibiting 14 linkage modes: L-Araf-(1→, L-Rhap-(1→, →3)-L-Araf-(1→, →5)-L-Araf-(1→, D-Glcp-(1→, D-Galp-(1→, →3,5)-L-Araf-(1→, →2,5)- L-Araf-(1→、→2)-D-Manp-(1→、→2)-D-Galp-(1→、→4)-D-Galp-(1→、→3)-D-Galp-(1→、→6)-D-Glcp-(1→ and →3,6)-D-Galp-(1→, with a molar ratio of 6.3:1.7:1.0:5.1:1.2:4.5:2.9:3.0:1.7:1.4:1.3:2.6:1.8:8.7, and its repeating unit structure is shown below:

[0009]

[0010] Furthermore, the preparation method of the Rehmannia glutinosa polysaccharide RGP-70-2-1 includes the following steps:

[0011] SI-1. After deproteinizing and decolorizing the crude polysaccharide RG70 from Rehmannia glutinosa, it was subjected to anion exchange column chromatography with 0-0.01M sodium chloride solution as the eluent. The eluent with a sodium chloride solution concentration of 0.05M was collected, dialyzed, concentrated, and dried. The obtained part was denoted as RG70-2.

[0012] SI-2. The RG70-2 obtained in step SI-1 is subjected to anion exchange column chromatography again, using a 0-0.05M sodium chloride solution as the eluent. The sodium chloride solution with a concentration of 0.01M is collected, concentrated, and dried. The obtained portion is recorded as RG70-2-a2.

[0013] SI-3. Perform gel column chromatography on the RG70-2-a2 obtained in step SI-2, using 0.01M sodium chloride solution as the eluent. Collect the eluent with an elution time of 5.75 to 6.5 h, and dry it to obtain the final product.

[0014] Preferably, in steps SI-1 and SI-2, the anion exchange column is DEAE-Cellulose 52.

[0015] Further, in step SI-1, the dialysis is performed through a dialysis bag with a molecular weight cutoff of 100 Da, and the retentate is retained.

[0016] Preferably, in step SI-3, the gel column is Sephacryl S-100.

[0017] Furthermore, the deproteinization is performed using the Sevag method.

[0018] Furthermore, the decolorization process involves using AB-8 macroporous adsorption resin to decolorize the sugar solution.

[0019] Furthermore, the preparation method of the crude polysaccharide RG70 from Rehmannia glutinosa includes the following steps: fully extracting Rehmannia glutinosa with water, concentrating the water extract, performing a first alcohol precipitation and filtering, taking the supernatant, performing a second alcohol precipitation and filtering, and taking the precipitate obtained from the second alcohol precipitation.

[0020] Furthermore, the water extraction temperature is 80-90℃ (preferably 85℃), and the water extraction time is 2-4 hours (preferably 3 hours).

[0021] Furthermore, the process of fully extracting Rehmannia glutinosa with water involves 3 to 5 water extractions (preferably 3 times), and the ratio of Rehmannia glutinosa to water during the water extraction is 1:8 to 12 (kg / L) (preferably 1:10 (kg / L)).

[0022] Furthermore, the first alcohol precipitation involves concentrating the aqueous extract and then adding 80-85% ethanol (preferably 85% ethanol) while stirring until the ethanol concentration of the solution reaches 45%-50% (preferably 50%), and then allowing it to stand for 36-60 hours (preferably 48 hours).

[0023] Furthermore, the second alcohol precipitation involves concentrating the supernatant obtained after filtration following the first alcohol precipitation, adding 85-90% ethanol (preferably 90% ethanol) until the ethanol concentration of the solution reaches 65-70% (preferably 70%), and allowing it to stand for 36-60 hours (preferably 48 hours).

[0024] Specifically, as a preferred and feasible method, the preparation method of the crude polysaccharide RG70 from Rehmannia glutinosa includes the following steps: Add water to Rehmannia glutinosa at a material-to-liquid ratio of 1:10 (kg / L), heat to 85℃, extract for 3 hours, and repeat the extraction three times. Combine the aqueous extracts. Filter the aqueous extract through gauze, concentrate under reduced pressure, and centrifuge at a low speed (3500 r / min). Combine the supernatants and slowly add 85% ethanol while stirring until the ethanol concentration reaches 50%. Let stand for 48 hours, collect the supernatant, concentrate, add 90% ethanol to the concentrate until the ethanol concentration reaches 70%, let stand for 48 hours, and the resulting precipitate is crude polysaccharide RG70 from Rehmannia glutinosa.

[0025] This invention protects a Rehmannia glutinosa polysaccharide, named RGP-90-1-1, with a molecular weight of 13.2 kDa. Its monosaccharide composition consists of arabinose, xylose, and glucose, exhibiting eight linkage modes: L-Araf-(1→, →3)-L-Araf-(1→, →5)-L-Araf-(1→, →3,5)-L-Araf-(1→, →2,5)-L-Araf-(1→, D-Glcp-(1→, →6)-D-Glcp-(1→ and →2,3,4)-D-Xylp(1→), with a molar ratio of 18.2:1.0:10.2:2.5:18.3:2.4:2.6:2.7. The repeating unit structure is shown below:

[0026]

[0027] The structural formula of R is as follows:

[0028]

[0029] Furthermore, the preparation method of the Rehmannia glutinosa polysaccharide RGP-90-1-1 includes the following steps:

[0030] SII-1. After deproteinizing the crude polysaccharide RG90 from Rehmannia glutinosa, anion exchange column chromatography was performed using 0-0.1M sodium chloride solution as the eluent. The eluent with a sodium chloride solution concentration of 0M was collected, dried, and the obtained portion was denoted as RG90-1.

[0031] SII-2. Perform gel column chromatography on the RG90-1 obtained in step SII-1, using water as the eluent. Collect the eluent with an elution time of 4.25 to 5 hours, and dry it to obtain the final product.

[0032] Preferably, in step SII-1, the anion exchange column is DEAE-Cellulose 52.

[0033] Preferably, in step SII-2, the gel column is Sephadex G-75.

[0034] Further, the crude polysaccharide RG90 of Rehmannia glutinosa is prepared by the following steps: fully extracting Rehmannia glutinosa with water, concentrating the water extract, performing a first alcohol precipitation and filtering, taking the supernatant for a second alcohol precipitation and filtering, taking the supernatant for a third alcohol precipitation and filtering, and taking the precipitate obtained from the third alcohol precipitation.

[0035] Furthermore, in the preparation steps of the crude polysaccharide RG90 from Rehmannia glutinosa, the water extraction, the first alcohol precipitation, and the second alcohol precipitation are all the same as those for the preparation of RG70.

[0036] Furthermore, the third alcohol precipitation involves concentrating the supernatant obtained after filtration following the second alcohol precipitation, adding anhydrous ethanol until the ethanol concentration of the solution reaches 85-90% (preferably 90%), and allowing it to stand for 36-60 hours (preferably 48 hours).

[0037] Specifically, as a preferred and feasible method, the preparation method of the crude polysaccharide RG90 from Rehmannia glutinosa includes the following steps: Add water to Rehmannia glutinosa at a material-to-liquid ratio of 1:10 (kg / L), heat to 85℃, extract for 3 hours, and repeat the extraction three times. Combine the aqueous extracts. Filter the aqueous extract through gauze, concentrate under reduced pressure, and centrifuge at a low speed (3500 r / min). Combine the supernatants and slowly add 85% ethanol while stirring until the ethanol concentration reaches 50%. Let stand for 48 hours, collect the supernatant, concentrate it, add 90% ethanol to the concentrate until the ethanol concentration reaches 70%, let stand for 48 hours, collect the supernatant, concentrate it, add anhydrous ethanol until the ethanol concentration reaches 90%, let stand for 48 hours, and the resulting precipitate is crude polysaccharide RG90 from Rehmannia glutinosa.

[0038] This invention protects a Rehmannia glutinosa polysaccharide, named RGP-90-1-2, with a molecular weight of 6.2 kDa. Its monosaccharide composition consists of arabinose and glucose, exhibiting five linkage schemes: L-Araf-(1→, →3)-L-Araf-(1→, →5)-L-Araf-(1→, →3,5)-L-Araf-(1→ and →6)-D-Glcp-(1→), with a molar ratio of 13.1:1.2:11.7:12.5:1.0. The repeating unit structure is shown below:

[0039]

[0040] Further, the Rehmannia glutinosa polysaccharide RGP-90-1-2 is prepared by the following steps:

[0041] SIII-1. After deproteinizing the crude polysaccharide RG90 from Rehmannia glutinosa, anion exchange column chromatography was performed using 0-0.1M sodium chloride solution as the eluent. The eluent with a sodium chloride solution concentration of 0M was collected and the resulting fraction was denoted as RG90-1.

[0042] SIII-2. Perform gel column chromatography on the RG90-1 obtained in step SIII-1, using water as the eluent. Collect the eluent with an elution time of 8.5 to 9.25 hours, and dry it to obtain the final product.

[0043] Furthermore, the preparation steps and parameters of the crude Rehmannia glutinosa polysaccharide RGP90-1-2 are basically the same as those of the Rehmannia glutinosa polysaccharide RGP-90-1-1. Both involve sequentially performing anion exchange column chromatography and gel column chromatography to separate and purify RG-90-1. The difference lies in the final collection stage. For Rehmannia glutinosa polysaccharide RGP-90-1-1, the eluent with an elution time of 4.25 to 5 hours is collected and dried, while for Rehmannia glutinosa polysaccharide RGP-90-1-2, the eluent with an elution time of 8.5 to 9.25 hours is collected and dried.

[0044] Preferably, all of the above drying methods are freeze drying.

[0045] Furthermore, all the collected eluents exhibited high absorbance values ​​at a wavelength of 490 nm, indicating that the collected eluents had a high sugar content.

[0046] Alpha-amylase inhibitors can inhibit the activity of salivary and pancreatic amylases in the gastrointestinal tract, hindering or delaying the hydrolysis and digestion of the main carbohydrates in food, reducing the breakdown and absorption of starchy sugars in food, thereby lowering blood sugar and blood lipids, inhibiting the rise in blood glucose concentration, and thus benefiting the dietary treatment of diabetic patients; Alpha-glucosidase inhibitors, by reversibly inhibiting Alpha-glucosidase on the small intestinal mucosa, slow down the rate at which carbohydrates are broken down into glucose, thereby lowering blood sugar.

[0047] The three Rehmannia glutinosa polysaccharides extracted in this invention all exhibited inhibitory effects on the activities of α-glucosidase and α-amylase. In terms of inhibiting α-glucosidase activity, all three polysaccharides were significantly superior to acarbose. Regarding the inhibition of α-amylase activity, RGP90-1-2 and RGP70-2-1 showed greater inhibition rates than acarbose, with an IC50 concentration of [missing value]. 50 The value was significantly lower than that of acarbose.

[0048] In addition, this invention constructs an LPS-induced RAW264.7 inflammation model. RGP70-2-1 dose-dependently reduces TNF-α expression in the LPS-induced RAW264.7 inflammation model, and RGP90-1-2 dose-dependently inhibits nitric oxide secretion and iNOS expression in the LPS-induced RAW264.7 inflammation model. Furthermore, PGP70-2-1 and RGP90-1-2 exert significant anti-inflammatory effects through the NF-κB / IκBα pathway.

[0049] Therefore, this invention protects the use of the Rehmannia polysaccharide in the preparation of hypoglycemic or anti-inflammatory drugs.

[0050] This invention protects a hypoglycemic or anti-inflammatory drug comprising one or more of the above-mentioned Rehmannia polysaccharides RGP70-2-1, RGP90-1-1, and RGP90-1-2.

[0051] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses water extraction and fractional alcohol precipitation to obtain crude polysaccharide from Rehmannia glutinosa. After removing impurities, the crude polysaccharide is further separated and purified to obtain Rehmannia glutinosa polysaccharide. Combined with infrared spectroscopy, fully methylated GC-MS, and NMR spectroscopy analysis, it is determined that the obtained Rehmannia glutinosa polysaccharide has a novel structure. In vitro experiments have shown that the obtained Rehmannia glutinosa polysaccharide has hypoglycemic effect and anti-inflammatory activity, providing a direction for the development of natural drugs with low toxicity and good pharmacological activity. Attached Figure Description

[0052] Figure 1 The following are elution curves of Rehmannia glutinosa polysaccharides: (A) is the elution curve of RG70 by DEAE-52 column chromatography; (B) is the elution curve of RG70-2 by DEAE-52 column chromatography; (C) is the elution curve of RG70-2-a2 by Sephacryl S-100 column chromatography; (D) is the elution curve of RG90 by DEAE-52 column chromatography; and (E) is the elution curve of RG90-1 by Sephadex G-75 column chromatography.

[0053] Figure 2 The chromatograms are of HPGPC, a polysaccharide of Rehmannia glutinosa, where (A) is the chromatogram of RGP70-2-1; (B) is the chromatogram of RGP90-1-1; and (C) is the chromatogram of RGP90-1-2.

[0054] Figure 3 The HPLC chromatograms of the PMP derivatization products of RGP70-2-1 monosaccharide are shown, where (A) is the mixed monosaccharide standard and (B) is the RGP70-2-1 hydrolysate.

[0055] Figure 4 The HPLC chromatograms are of the PMP derivatized products of the monosaccharides RGP90-1-1 and RGP90-1-2, where (A) is the mixed monosaccharide standard; (B) is the hydrolysate of RGP90-1-1; and (C) is the hydrolysate of RGP90-1-2.

[0056] Figure 5 The infrared spectra of Rehmannia glutinosa polysaccharides are shown below. (A) is the infrared spectrum of RGP70-2-1; (B) is the infrared spectrum of RGP90-1-1; and (C) is the infrared spectrum of RGP90-1-2.

[0057] Figure 6The 1H-NMR (A), 13C-NMR (B), 1H-1H COSY (C), HSQC (D) and HMBC (E) spectra of RGP70-2-1 are shown.

[0058] Figure 7 The 1H-NMR (A), 13C-NMR (B), 1H-1H COSY (C), HSQC (D), and HMBC (E) spectra of RGP90-1-1 are shown.

[0059] Figure 8 The 1H-NMR (A), 13C-NMR (B), 1H-1H COSY (C), HSQC (D), and HMBC (E) spectra of RGP90-1-2 are shown.

[0060] Figure 9 It is a repeating unit structure of RGP70-2-1.

[0061] Figure 10 It is a repeating unit structure for RGP90-1-1.

[0062] Figure 11 It is a repeating unit structure of RGP90-1-2.

[0063] Figure 12 This is a statistical chart showing the inhibitory effect of Rehmannia glutinosa polysaccharides on α-glycosidase activity.

[0064] Figure 13 Statistical chart showing the inhibitory effect of Rehmannia glutinosa polysaccharide on α-amylase activity.

[0065] Figure 14 Statistical graph showing the effect of RGP90-1-2 on NO secretion (#: compared with the control group, ##P<0.001; *: compared with the model group, *P<0.05, **P<0.001).

[0066] Figure 15The following are statistical graphs of Western blot (WB) results for the LPS-induced RAW264.7 inflammation model using RGP70-2-1 and PGP90-1-2: A and E are WB results for RGP70-2-1 and RGP90-1-2, respectively; B is a statistical graph of TNFα expression after RGP70-2-1 treatment; C is a statistical graph of NF-κB expression after RGP70-2-1 treatment; D is a statistical graph of IKB-α expression after RGP70-2-1 treatment; F is a statistical graph of iNOS expression after RGP90-1-2 treatment; G is a statistical graph of NF-κB expression after RGP90-1-2 treatment; and H is a statistical graph of IκBα expression after RGP90-1-2 treatment. Detailed Implementation

[0067] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0068] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0069] PMP: 1-Phenylon-3-methyl-5-pyrazolone

[0070] RAW264.7 cells: derived from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0071] Example 1: Extraction and Identification of Rehmannia Polysaccharides

[0072] I. Extraction of Rehmannia glutinosa polysaccharides

[0073] (1) Preparation method

[0074] 19.85 kg of raw Rehmannia glutinosa slices and deionized water were added to an extraction tank and heated to 85°C for 3 hours. The material-to-liquid ratio was 1:10 (m / v). The extraction was repeated three times, and the aqueous extracts were combined. The aqueous extracts were filtered through gauze, concentrated under reduced pressure, and centrifuged at a low speed (3500 r / min). The supernatants were combined, and 85% ethanol was slowly added while stirring until the ethanol concentration reached 50%. The mixture was allowed to stand for 48 hours, filtered, and the supernatant was collected and concentrated. 90% ethanol was added to the concentrate until the ethanol concentration reached 70%. The mixture was allowed to stand for 48 hours, filtered, and the supernatant and precipitate (the precipitate is crude Rehmannia glutinosa polysaccharide RG70) were obtained. The supernatant was collected, concentrated, and anhydrous ethanol was added until the ethanol concentration reached 90%. The mixture was allowed to stand for 48 hours, filtered, and the precipitate obtained was crude Rehmannia glutinosa polysaccharide RG90. Subsequently, RG70 and RG90 were deproteinized using the Sevag method, and the sugar solution was decolorized using AB-8 macroporous adsorption resin. After further elution and purification by DEAE-Cellulose 52 column chromatography and gel chromatography, the polysaccharides with uniform molecular weight distribution were collected, concentrated, and lyophilized to obtain RGP-70-2-1, RGP-90-1-1, and RGP90-1-2.

[0075] Taking RG70 as an example, the steps for removing proteins using the Sevag method are as follows:

[0076] Add 670g of RG70 to an appropriate amount of deionized water, and sonicate to accelerate the dissolution of the precipitate. After there is no obvious insoluble matter in the beaker, centrifuge at low speed to remove the insoluble matter. Take the supernatant and mix it with Sevag reagent (chloroform: n-butanol = 4:1) at a volume ratio of 5:1. Shake vigorously and let stand. After obvious separation, separate the liquid to remove the protein layer. Repeat the above steps 2-3 times. Concentrate the solution after the last protein removal under reduced pressure to remove the organic solvent. Then, take an appropriate amount of solution and pour it into a dialysis bag (1000 Da) and dialyze with running water for 48 hours. Collect the dialysate, concentrate it, centrifuge at low speed to remove the insoluble matter, concentrate the supernatant again, and freeze-dry it to obtain deproteinized RG70.

[0077] Taking RG70 as an example, the steps for decolorizing sugar solutions using AB-8 macroporous adsorption resin are as follows:

[0078] Weigh 20 g of AB-8 macroporous adsorption resin, soak it in ethanol to swell, then wash it with deionized water until the solution is clear and odorless. Filter (to remove water) and set aside. Prepare a 20 mg / mL sample solution, centrifuge to remove insoluble matter, and collect the supernatant. Add 15 mL of the supernatant to the AB-8 macroporous adsorption resin, allow it to stand in a water bath at 60°C for 1 hour, filter, collect the filtrate, dialyze (molecular weight cutoff: 1000 Da), and lyophilize to obtain the decolorized RG70.

[0079] (2) Measurement results

[0080] RG70 separation and purification results:

[0081] The crude polysaccharide RG70 after deproteinization and pigment removal was eluted with DEAE-Cellulose 52. The eluent concentration and elution results are as follows: Figure 1 As shown in Figure A, the eluent with high sugar content (i.e., the portion with high absorbance at 490 nm) was collected to obtain RG70-1, RG70-2, and RG70-3 (yields of 2.15%, 37.89%, and 1.05%, respectively). Because eluent RG70-2 was dark in color and had a high pigment content, it was dialyzed through a dialysis bag with a molecular weight cutoff of 100 Da, concentrated, lyophilized, and then loaded onto a DEAE-52 chromatography column. RG70-2 was eluted using a DEAE-Cellulose 52 anion exchange column. The eluent concentration and elution results are shown below. Figure 1 As shown in Figure B, the eluents with high sugar content were collected and named RG70-2-a1, RG70-2-a2, and RG70-2-a3, respectively (yields of 0.4%, 4.58%, and 5.40%, respectively). RG70-2-a2 was further purified by elution using a Sephacryl S-100 chromatography column. After purity verification by GPC, the polysaccharides with uniform molecular weight distribution were collected, concentrated, and lyophilized. RG70-2-a2 eluted with an S-100 column yielded a single polysaccharide, and the number of collection tubes is shown in Figure B. Figure 1 As shown in C, it is named RGP70-2-1, with a yield of 2.65%.

[0082] RG90 separation and purification results:

[0083] The crude polysaccharide RG90 after deproteinization was eluted using a DEAE-Cellulose 52 anion exchange column. The eluent concentration and elution results are as follows: Figure 1 As shown in D, the eluents with high sugar content were collected and named RG90-1, RG90-2, and RG90-3 (yields of 26.63%, 0.67%, and 1.24%, respectively). RG90-1 was further purified by elution using a Sephadex G-75 column. After purity verification by HPGPC, polysaccharides with uniform molecular weight distribution and identical composition were collected, concentrated, and lyophilized. Two polysaccharides were obtained after elution of RG90-1 using a G-75 column, and the number of collection tubes is shown in Figure D. Figure 1 As shown in E, they were named RGP90-1-1 and RGP90-1-2 (yields of 4.01% and 4.84%, respectively).

[0084] II. Identification of Rehmannia glutinosa polysaccharides

[0085] 1. High-performance gel permeation chromatography (HPGPC)

[0086] (1) Measurement method

[0087] A 2 mg / mL dextran standard solution and a 3 mg / mL Rehmannia glutinosa polysaccharide solution were prepared. Using the mobile phase as the solvent, the solutions were completely dissolved and filtered through a 0.45 μm aqueous filter membrane. The molecular weight was determined using the HPGPC method. A standard curve was plotted with retention time on the x-axis and the logarithm of the relative molecular mass (logMw) of different dextran standards on the y-axis. A regression equation was established, and the relative molecular weight of the samples was calculated based on the regression equation and the sample retention time. The conditions for determining molecular weight using the HPGPC method are shown in Table 1.

[0088] Table 1 Conditions for determining molecular weight by HPGPC method

[0089] mobile phase <![CDATA[0.02 M KH2PO4 solution]]> gel column Ultrahydrogel 1000 and Ultrahydrogel 500 in series Injection volume 20μL Flow rate 0.8 mL / min detector Waters 2414 differential detector Column temperature 35℃

[0090] (2) Measurement results

[0091] The polysaccharides from Rehmannia glutinosa were detected by HPGPC method, and the results are as follows: Figure 2 The results show that the three polysaccharides RGP70-2-1, RGP90-1-1, and RGP90-1-2 all have uniformly distributed (i.e., symmetrical peaks) single peaks, indicating that these three polysaccharides are homogeneous. According to the standard curve y = -3.9304 + 0.1733X... 2 -0.0027X 3 The molecular weight of RGP70-2-1 was calculated to be 5.7 kDa based on +35.8703; according to the standard curve y = -3.9184 + 0.1725X 2 -0.0027X 3 +35.8247, the molecular weights of RGP90-1-1 and RGP90-1-2 are calculated to be 13.2 kDa and 6.2 kDa, respectively.

[0092] 2. Determination of monosaccharide composition

[0093] (1) Measurement method

[0094] Prepare a 2M trifluoroacetic acid solution in a 10mL volumetric flask. Add 5.0mg of Rehmannia glutinosa polysaccharide sample and 2mL of trifluoroacetic acid solution to a 10mL stoppered round-bottom glass test tube, respectively. Then place the tube in a 120℃ oil bath for high-temperature acid hydrolysis for 5h while heating and stirring. After hydrolysis, transfer the sample to a 50mL round-bottom flask, add 6mL of methanol and concentrate under reduced pressure to dryness. Repeat this step at least 3 times until the residual trifluoroacetic acid is removed. Add 1 mL of deionized water to the polysaccharide sample that has been concentrated and dried under reduced pressure to remove residual trifluoroacetic acid. Then, take 200 μL of the solution and place it in a 5 mL sealed reaction tube. Add equal volumes of 0.3 M NaOH solution and 0.5 M PMP methanol solution (PMP methanol solution is prepared with methanol as the solvent). Mix thoroughly and react in a 70 °C water bath for 0.5 h. After cooling, add 212 μL of 0.3 M HCl solution. After neutralizing the solution, dilute it with 100 μL of deionized water using a pipette. Extract three times with 1 mL of chloroform. Take the upper layer (i.e., the aqueous layer), filter it through a 0.45 μm organic filter membrane, and analyze it by HPLC.

[0095] PMP derivatization reaction of mixed monosaccharide standards: Prepare 9 2mM monosaccharide standard solutions in 10mL volumetric flasks. The volume ratio and reaction steps of the mixed monosaccharide standard solution, NaOH solution, PMP methanol solution, and HCl solution are the same as those of the standard PMP derivatization reaction.

[0096] HPLC chromatographic conditions: Agilent ZORBAX Eclipse XDB-C 18 (5μm, 4.6×250mm); mobile phase (A: water, C: acetonitrile, D: 0.05M phosphate buffer at pH 6.72), elution program is shown in Table 2; flow rate: 1mL / min; injection volume: 10μL; detection wavelength: 250nm.

[0097] Table 2 Gradient elution procedure for HPLC method

[0098] 0 - 17 83 24 - 17 83 25 - 19 81 30 - 18 82 31 5 14 81 32 5 14 81 33 - 18 82 34 5 14 81 36 - 18 82

[0099] (2) Measurement results

[0100] Depend on Figure 3 It is known that RGP70-2-1 contains derivatized products of mannose, rhamnose, glucose, galactose, and arabinose, indicating that the Rehmannia glutinosa polysaccharide RGP70-2-1 is composed of mannose, rhamnose, glucose, galactose, and arabinose. Figure 4It can be seen that RGP90-1-1 contains derivatized products of glucose, xylose, and arabinose, indicating that Rehmannia glutinosa polysaccharide RGP90-1-1 is composed of glucose, xylose, and arabinose. RGP90-1-2 contains derivatized products of glucose and arabinose, indicating that Rehmannia glutinosa polysaccharide RGP90-1-2 is composed of glucose and arabinose.

[0101] 3. Infrared spectroscopy determination

[0102] The infrared spectra of Rehmannia glutinosa polysaccharides RGP70-2-1, RGP90-1-1, and RGP90-1-2 were determined, and the results are as follows: Figure 5 As shown, taking RGP70-2-1 as an example, 3367cm -1 The wide and strong stretching bands on both sides belong to the stretching vibration of OH, 2931cm. -1 The weak peak at 1611 cm⁻¹ is caused by the CH stretching vibration. -1 and 1411cm -1 The absorption peaks at 1038 cm⁻¹ are attributed to the C-C stretching vibration and the CH bending vibration, respectively. -1 The strong absorption peaks around 610 cm⁻¹ are attributed to the COC asymmetric stretching vibrations of the sugar ring and glycosidic bonds. -1 The absorption peak at that point is attributed to the out-of-plane bending vibration of OH.

[0103] 4. Methylation reaction and GC-MS determination

[0104] The polysaccharides from Rehmannia glutinosa underwent methylation. After complete methylation, the polysaccharides were subjected to complete acid hydrolysis, reduction, and acetylation. The resulting products were analyzed by GC-MS, and the results are shown in Tables 3-5.

[0105] Table 3. Methylation analysis results of RGP90-1-1

[0106]

[0107] Note: PMMA refers to Partially Methylated Alditol Acetate.

[0108] Table 4. Results of RGP90-1-2 methylation analysis

[0109]

[0110]

[0111] Table 5. Methylation analysis results of RGP70-2-1

[0112]

[0113] After complete methylation of Rehmannia glutinosa polysaccharide RGP90-1-1, the product was subjected to complete acid hydrolysis, reduction, and acetylation. The results were analyzed by GC-MS, as shown in Table 3. Eight acetylated sugar alcohols were detected in the product, with the relative proportions of retention times from shortest to longest being: 18.2:1.0:10.2:2.5:18.3:2.4:2.6:2.7. The analysis indicates that Rehmannia glutinosa polysaccharide RGP90-1-1 contains eight linkages: L-Araf-(1→、→3)-L-Araf-(1→、→5)-L-Araf-(1→、→3,5)-L-Araf-(1→、→2,5)-L-Araf-(1→), D-Glcp-(1→、→6)-D-Glcp-(1→), and →2,3,4)-D-Xylp(1→).

[0114] After complete methylation of Rehmannia glutinosa polysaccharide RGP90-1-2, the product was subjected to complete acid hydrolysis, reduction, and acetylation. The resulting product was analyzed by GC-MS, and the results are shown in Table 4. Five acetylated sugar alcohols were detected in the product, with the relative proportions of retention times from shortest to longest being 13.1:1.2:11.7:12.5:1.0. The analysis indicates that Rehmannia glutinosa polysaccharide RGP90-1-2 contains five linkages: L-Araf-(1→, →3)-L-Araf-(1→, →5)-L-Araf-(1→, →3,5)-L-Araf-(1→ and →6)-D-Glcp-(1→).

[0115] After complete methylation of Rehmannia glutinosa polysaccharide RGP70-2-1, the product was subjected to complete acid hydrolysis, reduction, and acetylation. The product was analyzed by GC-MS, and the results are shown in Table 5. Fourteen acetylated sugar alcohols were detected in the product. The relative proportions of the products with the longest retention times were: 6.3:1.7:1.0:5.1:1.2:4.5:2.9:3.0:1.7:1.4:1.3:2.6:1.8:8.7. The results showed that there are 14 linkage modes in the Rehmannia glutinosa polysaccharide RGP70-2-1, including L-Araf-(1→, L-Rhap-(1→, →3)-L-Araf-(1→, →5)-L-Araf-(1→, D-Glcp-(1→, D-Galp-(1→, →3,5)-L-Araf-(1→, →2,5)-L-Araf-(1→, →2)-D-Manp-(1→, →2)-D-Galp-(1→, →4)-D-Galp-(1→, →3)-D-Galp-(1→, →6)-D-Glcp-(1→ and →3,6)-D-Galp-(1→).

[0116] 5. Nuclear magnetic resonance (NMR) measurement

[0117] (1) Measurement method

[0118] Weigh out the freeze-dried Rehmannia glutinosa polysaccharide sample and add D2O to prepare an 80 mg / mL polysaccharide sample solution. Heat and sonicate to dissolve as completely as possible, then centrifuge at 10000 rpm for 5 minutes. Filter the sample solution through a 0.45 μm aqueous filter membrane and transfer it to an NMR tube. Add acetone as an internal standard and mix well. Finally, detect with a 500 MHz NMR spectrometer to obtain a one-dimensional ( 1 H and 13 CNMR spectrum and two-dimensional nuclear magnetic resonance spectrum 1 H- 1 HCOSY spectrum, HSQC spectrum, and HMBC spectrum).

[0119] (1) Measurement results

[0120] Depend on Figure 7 It can be seen that all of RGP90-1-1 I The 1H NMR peaks were all between 1 and 6 ppm, which is typical for polysaccharides. From the HSQC spectrum, eight terminal hydrogen and terminal carbon related signals were clearly observed, at 5.09 / 107.3 ppm, 5.03 / 107.6 ppm, 5.05 / 107.4 ppm, 5.18 / 106.7 ppm, 5.12 / 107.1 ppm, 5.01 / 104.2 ppm, 5.08 / 103.8 ppm, and 5.07 / 100.1 ppm, named sugar residues A, B, D, E, F, G, I, and J, respectively. The relative configuration of the sugar was determined based on the chemical shifts of the terminal hydrogens, coupling constants, and chemical shifts of the terminal carbons. Since the α-configuration of L-furan ring arabinose has a lower field ratio to the β-configuration, the relative configuration of the sugar was determined. 13 Sugars with chemical shifts of 107.3 ppm, 107.6 ppm, 107.4 ppm, 106.7 ppm, and 107.1 ppm in the C NMR spectrum are classified as α-configured L-furan-ring arabinoses. Similarly, the terminal carbons of β-configured D-pyranose glucose are located at a lower field, and 3 J H1,H2 Since the chemical signal of the terminal carbon of D-pyranose is >5Hz, the D-pyranose with a chemical shift of 103.8ppm and 104.1ppm is classified as the β configuration. Since the chemical signal of the terminal carbon of D-pyranose with an α configuration is at a higher field, the D-pyranose with a chemical shift of 100.1ppm is classified as the α configuration.

[0121] The chemical shift of H2 is determined based on its correlation with H-1. This is based on relevant reference books, literature reports, and... 1 H- 1 The resonance peak δ in the HCOSY spectrum HFrom 5.09 / 4.22, the H2 signal of residue A is estimated to be 4.22 ppm. Similarly, based on the good correlation between H2 and H3, the H3 of residue A is assigned to δ. H The signal at 3.96, δ H 3.96 / 3.97 and δ H 3.97 and 3.66 are respectively assigned to H3 / H4 and H4 / H5 of residual A, and then combined with 13 The C and H signals of residue A were assigned using C NMR and HSQC spectra. The relevant peaks 5.09 / 107.3ppm, 4.22 / 81.5ppm, 3.96 / 76.7ppm, 3.97 / 83.9ppm, 3.66, and 3.76 / 61.1ppm correspond to H1 / C1, H2 / C2, H3 / C3, H4 / C4, and H5 / C5 of residue A, respectively. For residue B, 1 H- 1 The resonance peak δ in the HCOSY spectrum H 5.03 / 4.06, δ H 4.24 / 3.82 correspond to H1 / H2 and H4 / H5 respectively, while H2 and C3 show a good correlation. C3 is substituted on the L-furan ring arabinose, and the C3 chemical shift shifts to a lower field. Combined with the resonance signal 4.06 / 81.4 ppm on the HMBC spectrum, the chemical shift of C3 can be inferred to be 81.4 ppm. Further, combined with... 1 H NMR and HSQC spectra were used to assign the C and H signals of residue B. The relevant peaks 5.03 / 107.6ppm, 4.06 / 81.2ppm, 4.24 / 81.4ppm, 4.24 / 82.2ppm, 3.73, and 3.82 / 66.4ppm correspond to H1 / C1, H2 / C2, H3 / C3, H4 / C4, and H5 / C5 of residue B, respectively. Similarly, combined with... 1 H- 1 H COSY, HSQC, and HMBC spectral analyses were performed to assign the C and H signals of the remaining sugar residues. The C and H signal assignment results are shown in Table 6. 13 No obvious chemical signal was observed in the δ170-180ppm range of the C NMR spectrum, indicating that the polysaccharide does not contain uronic acid, which is consistent with the results of the monosaccharide composition.

[0122] The connection sites and connection order between various sugar residues were analyzed based on the resonance peaks in the HMBC spectrum. The correlation peak 5.07 / 76.5ppm (J H1 / J C2) indicates that the O-1 of residue J is connected to the C-2 of residue J, indicating the existence of a repeating unit, residue J; the correlation peak 3.83 / 100.1ppm (E H5 / J C1) indicates that the O-5 of residue E is connected to the C-1 of residue J; the correlation peak 5.05 / 81.4ppm (D H1 / B C3) indicates that the O-1 of residue D is connected to the C-3 of residue B; the correlation peak 5.01 / 66.5ppm (G H1 / I C6) indicates that the O-1 of residue G is connected to the C-6 of residue I; the correlation peak 3.88 / 81.4ppm (J H4 / B C3) indicates that the O-4 of residue J is connected to the C-3 of residue B; the correlation peak 4.21 / 107.4ppm (JH3 / D C1) indicates that the O-3 of residue J is connected to the C-2 of residue J. The correlation peak 3.75 / 107.6ppm (D H5 / B C1) indicates that the O-5 of residue D is connected to the C-1 of residue B; the correlation peak 5.18 / 103.8ppm (E H1 / I C1) indicates that the O-1 of residue E is connected to the C-1 of residue I; the correlation peak 4.06 / 66.2ppm (J H2 / E C5) indicates that the O-2 of residue J is connected to the C-5 of residue E; the correlation peak 5.03 / 85.0ppm (B H1 / E C2) indicates that the O-1 of residue B is connected to the C-2 of residue E; the correlation peak 4.24 / 79.6ppm (E H2 / F C3) indicates that the O-2 of residue E is connected to the C-3 of residue F; the correlation peak 5.12 / 81.4ppm (F H1 / B C3) indicates that the O-1 of residue F is connected to the C-3 of residue E. C-3 of B; the relevant peaks 5.03 / 81.4ppm (B H1 / B C3) and 4.24 / 107.6ppm (B H3 / B C1) indicate that the O-1 of residue B is connected to the C-3 of residue B, or the O-3 of residue B is connected to the C-1 of residue B, indicating the existence of a repeating unit, residue B; the relevant peak 3.82 / 107.3ppm (B H5 / AC1) indicates that the O-5 of residue B is connected to the C-1 of residue A; the relevant peak 3.90 / 81.4ppm (EH5 / B C3) indicates that the O-5 of residue E is connected to the C-3 of residue B; the relevant peak 5.05 / 66.2 ppm (D H1 / D C5 and E C5) indicates that the O-1 of residue D connects to the C-5 of residue D and the C-5 of residue E, meaning that a repeating unit, residue D, exists.

[0123] Table 6 Chemical shift values ​​of sugar residues in RGP90-1-1

[0124]

[0125] Based on structural characterization analysis, the primary structure of RGP90-1-1 was deduced, and its repeating structural units are as follows: Figure 10 As shown. RGP70-2-1, RGP90-1-2 1 H, 13 CNMR, 1 H- 1 HCOSY, HSQC, and HMBC spectra are as follows: Figure 6 and 8 As shown. Similar to RGP90-1-1, RGP70-2-1 ( Figure 9 RGP90-1-2 Figure 11 The structure of ) can be determined using the methods described above and spectral information.

[0126] Example 2: Inhibition test of Rehmannia glutinosa polysaccharide on α-glucosidase

[0127] (1) Results of the determination method

[0128] First, prepare a 0.1M potassium phosphate buffer solution with a pH of 6.8. Then, use this buffer to prepare an α-glucosidase solution with a concentration of 0.2 U / mL. Mix the α-glucosidase solution and the sample solution thoroughly and incubate at 37°C for 5 min. Next, add 10 mM of substrate (pNPG solution, the substrate for colorimetric β-D-glucosidase), react at 37°C for 30 min, and terminate the reaction by adding sodium carbonate. Measure the absorbance at 405 nm. Acarbose was used as a positive control. The experiment was repeated in triplicate. Samples without α-glucosidase were designated as control blanks; samples containing the sample but without α-glucosidase were designated as sample blanks; and samples containing α-glucosidase but without the sample were designated as controls. The inhibition rate was calculated using the following formula:

[0129]

[0130] Where A0 and A3 are the absorbances of the control blank and sample blank, respectively, and A1 and A4 are the absorbances of the control and sample, respectively.

[0131] (2) Measurement results

[0132] Table 7. Half-inhibitory concentration of Rehmannia glutinosa polysaccharides against α-glucosidase

[0133] Acarbose 6.918±0.378c RGP70-2-1 0.369±0.044e

[0134] Note: Different letters indicate significant differences (p<0.05) between the data groups.

[0135] from Figure 12 As shown in Table 7, all three isolated and purified heteropolysaccharides inhibited α-glucosidase activity. With increasing concentration, acarbose and Rehmannia glutinosa polysaccharides showed stronger inhibitory effects on α-glucosidase activity. Within the concentration range of 0.3375–10 mM, the minimum inhibition rates of RGP90-1-1 and RGP90-1-2 were both greater than 50%, indicating that these three Rehmannia glutinosa polysaccharides were superior to acarbose in inhibiting α-glucosidase activity, especially RGP70-2-1, which showed a significantly higher IC50 value. 50 The value was significantly lower than that of acarbose.

[0136] Example 3: Inhibition test of Rehmannia glutinosa polysaccharide on α-amylase

[0137] (1) Measurement method

[0138] Prepare a 1.0 mg / mL amylase solution using the prepared potassium phosphate buffer. Take equal volumes of the sample solution and α-amylase solution, mix well, and incubate at 37°C for 10 min. Then add starch solution and incubate at 37°C for 10 min. Add DNS reagent, mix well, and heat in a water bath at 100°C for 5 min, then cool to room temperature. Use acarbose as a positive control and measure absorbance at 540 nm. Perform the experiment in triplicate. A control blank is defined as the sample without α-amylase, a sample blank is defined as the sample containing the sample but without α-amylase, and a control is defined as the sample containing α-amylase but without the sample. The inhibition rate is calculated using the same formula as in Equation 1.

[0139] (2) Measurement results

[0140] Table 8. Half-inhibitory concentration of Rehmannia glutinosa polysaccharides against α-amylase

[0141]

[0142]

[0143] Note: Different letters indicate significant differences (p<0.05) between the data groups.

[0144] from Figure 13 As shown in Table 8, all three Rehmannia glutinosa polysaccharides inhibited α-amylase activity. At concentrations below 0.25 mM, RGP90-1-2 and RGP70-2-1 showed greater inhibition rates against α-amylase than acarbose. 50The values ​​were significantly lower than those of acarbose, indicating that polysaccharides RGP70-2-1 and RGP90-1-2 had a significantly stronger inhibitory effect on α-amylase activity than acarbose.

[0145] Example 4: Inhibitory effect of RGP90-1-2 on LPS-induced RAW264.7 inflammation model

[0146] (1) Measurement method

[0147] RAW 264.7 cells were cultured at 1.2 × 10⁻⁶. 4 Cells were seeded at a final density of cells / well in 96 culture plates for evaluation of anti-inflammatory effects. Lipopolysaccharide (LPS) and RGP90-1-2 were dissolved in the culture medium. Cells were then treated with LPS (0.5 μg / mL) for 13 h as the model group, and without LPS as the control group. Cells were co-incubated with LPS and RGP90-1-2 (250, 500, and 1000 μg / mL) as the experimental group, and co-incubated with LPS (0.5 μg / mL) and minocycline (20 μM) as the positive group. After 13 h of stimulation, the cell culture supernatant was collected, and the nitrite oxide (NO) content was determined using a NO detection kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The absorbance of the sample solution was measured at 540 nm, and the NO secretion was calculated based on the standard curve to evaluate the anti-inflammatory activity of RGP90-1-2.

[0148] (2) Measurement results

[0149] NO is a major inflammatory mediator. To evaluate the anti-inflammatory activity of RGP90-1-2, we measured NO production. Figure 14 As shown, LPS-induced NO production in RAW264.7 cells increased, while high doses of RGP90-1-2 inhibited NO secretion, indicating that RGP90-1-2 can suppress LPS-induced RAW inflammation.

[0150] Example 5: Inhibitory effect of RG70-2-1 and RG90-1-2 on LPS-induced RAW264.7 inflammation model

[0151] (1) Measurement method

[0152] RAW 264.7 cells were fed at a rate of 4.2 × 10⁻⁶. 5Cells were seeded at a density of cells / well in 6-well plates. The model group was incubated with LPS (0.5 μg / mL) for 13 h, while the control group received only culture medium. LPS was co-incubated with RGP70-2-1 (1000 μg / mL) and RGP90-1-2 (250, 500, and 1000 μg / mL) in 6-well plates as experimental groups. After 13 h of incubation, cells were collected for Western blotting (WB). Lysis buffer was added to the cells, and the plates were incubated on ice for 20 min. Cell lysates were collected by scraping, centrifuged, quantified, and denatured at 100 °C for 10 min. Equal volumes of 30 mg protein were separated by molecular weight on a 10% SDS gel and then transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature, the membrane was incubated with primary antibody overnight at 4 °C. The membrane was washed five times with TBST buffer and then incubated with secondary antibody for 1.5 h. The blots were visualized using an ECL kit, and the band density was quantified using ImageJ analysis software.

[0153] (2) Measurement results

[0154] The results are as follows Figure 15 As shown in the figure, LPS induction increased the inflammatory factor TNFα, but this trend was reversed by the addition of RGP70-2-1. LPS induction increased the level of the cellular inflammatory marker iNOS, but RGP90-1-2 treatment significantly decreased iNOS levels. NF-κB is a transcription factor that promotes the transcription of inflammation-related genes; therefore, we investigated the effects of RGP70-2-1 and RGP90-1-2 on the LPS-induced NF-κB signaling pathway. As shown in the figure, LPS treatment led to an increase in NF-κB levels, while RGP70-2-1 and RGP90-1-2 treatments significantly counteracted the LPS-induced upregulation of NF-κB expression. LPS treatment decreased IκBα expression in RAW264.7 cells, but the addition of RGP70-2-1 and RGP90-1-2 treatments increased IκBα expression, especially at high doses. These results indicate that RGP70-2-1 and RGP90-1-2 significantly inhibit LPS-induced inflammatory damage via the NF-κB / IκBα pathway.

[0155] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Rehmannia glutinosa polysaccharide, characterized in that, The polysaccharide from Rehmannia glutinosa has a molecular weight of 5.7 kDa and a monosaccharide composition of mannose, glucose, rhamnose, galactose, and arabinose, exhibiting 14 linkage modes: L-Ara f -(1→、L-Rha p -(1→、→3)-L-Ara f -(1→、→5)-L-Ara f -(1→、D-Glc p -(1→、D-Gal p -(1→、→3,5)-L-Ara f -(1→、→2,5)-L-Ara f -(1→、→2)-D-Man p -(1→、→2)-D-Gal p -(1→、→4)-D-Gal p -(1→、→3)-D-Gal p -(1→、→6)-D-Glc p -(1→and→3,6)-D-Gal p -(1→, its molar ratio is: 6.3:1.7:1.0:5.1:1.2:4.5:2.9:3.0:1.7:1.4:1.3:2.6:1.8:8.7, and its repeating unit structure is shown below:

2. The Rehmannia glutinosa polysaccharide according to claim 1, characterized in that, The preparation method of the Rehmannia glutinosa polysaccharide includes the following steps: SI-1. After deproteinizing and decolorizing the crude polysaccharide RG70 from Rehmannia glutinosa, it was subjected to anion exchange column chromatography with 0-0.01 M sodium chloride solution as the eluent. The eluent with a sodium chloride solution concentration of 0.05 M was collected, dialyzed, concentrated, and dried. The obtained portion was denoted as RG70-2. SI-2. The RG70-2 obtained in step SI-1 is subjected to anion exchange column chromatography again, using a 0~0.05 M sodium chloride solution as the eluent. The eluent with a sodium chloride solution concentration of 0.01 M is collected, concentrated, and dried. The obtained portion is recorded as RG70-2-a2. SI-3. Perform gel column chromatography on the RG70-2-a2 obtained in step SI-2, using 0.01 M sodium chloride solution as the eluent. Collect the eluent with an elution time of 5.75~6.5 h, and dry it to obtain the final product. The preparation method of the crude polysaccharide RG70 of Rehmannia glutinosa includes the following steps: fully extracting raw Rehmannia glutinosa with water, concentrating the water extract, performing a first alcohol precipitation and filtering, taking the supernatant, performing a second alcohol precipitation and filtering, and taking the precipitate obtained from the second alcohol precipitation. The first alcohol precipitation involves concentrating the aqueous extract and then adding 80% to 85% ethanol while stirring until the ethanol concentration of the solution reaches 45% to 50%. The second alcohol precipitation involves concentrating the supernatant obtained after the first alcohol precipitation by filtration, and then adding 85% to 90% ethanol until the ethanol concentration of the solution reaches 65% to 70%. In steps SI-1 and SI-2, the anion exchange column is DEAE-Cellulose 52; In step SI-3, the gel column is Sephacryl S-100.

3. A Rehmannia glutinosa polysaccharide, characterized in that, The polysaccharide from Rehmannia glutinosa has a molecular weight of 13.2 kDa, and its monosaccharide composition consists of arabinose, xylose, and glucose, exhibiting eight linkage modes: L-Ara f -(1→、→3)-L-Ara f -(1→、→5)-L-Ara f -(1→、→3, 5)-L-Ara f -(1→、→2, 5)-L-Ara f -(1→、D-Glc p -(1→、→6)-D-Glc p- (1→ and →2, 3, 4)-D-Xyl p (1→, its molar ratio is: 18.2:1.0:10.2:2.5:18.3:2.4:2.6:2.7, and its repeating unit structure is shown below: ； The structural formula of R is as follows: 。 4. The Rehmannia glutinosa polysaccharide according to claim 3, characterized in that, The preparation method of the Rehmannia glutinosa polysaccharide includes the following steps: SII-1. After deproteinizing the crude polysaccharide RG90 from Rehmannia glutinosa, anion exchange column chromatography was performed using 0-0.1 M sodium chloride solution as the eluent. The eluent with a sodium chloride solution concentration of 0 M was collected, dried, and the obtained portion was denoted as RG90-1. SII-2. Perform gel column chromatography on the RG90-1 obtained in step SII-1, using water as the eluent, collect the eluent after 4.25~5 h of elution, and dry it to obtain the final product. The crude polysaccharide RG90 from Rehmannia glutinosa is prepared by the following steps: fully extracting Rehmannia glutinosa with water, concentrating the water extract, performing a first alcohol precipitation and filtering, taking the supernatant for a second alcohol precipitation and filtering, taking the supernatant for a third alcohol precipitation and filtering, and taking the precipitate obtained from the third alcohol precipitation. The first alcohol precipitation involves concentrating the aqueous extract and then adding 80% to 85% ethanol while stirring until the ethanol concentration of the solution reaches 45% to 50%. The second alcohol precipitation involves concentrating the supernatant obtained after the first alcohol precipitation by filtration, and then adding 85% to 90% ethanol until the ethanol concentration of the solution reaches 65% to 70%. The third alcohol precipitation is achieved by concentrating the supernatant obtained after the second alcohol precipitation and then adding anhydrous ethanol until the ethanol concentration of the solution reaches 85%~90%. In step SII-1, the anion exchange column is DEAE-Cellulose 52; In step SII-2, the gel column is Sephadex G-75.

5. A Rehmannia glutinosa polysaccharide, characterized in that, The polysaccharide from Rehmannia glutinosa has a molecular weight of 6.2 kDa, and its monosaccharide composition consists of arabinose and glucose, exhibiting five linkage modes: L-Ara... f -(1→、→3)-L-Ara f -(1→、→5)-L-Ara f -(1→、→3,5)-L-Ara f -(1→and→6)-D-Glc p -(1→, its molar ratio is: 13.1:1.2:11.7:12.5:1.0, and its repeating unit structure is shown below: 。 6. The Rehmannia glutinosa polysaccharide according to claim 5, characterized in that, The Rehmannia polysaccharide is prepared by the following steps: SIII-1. After deproteinizing and decolorizing the crude polysaccharide RG90 from Rehmannia glutinosa, it was subjected to anion exchange column chromatography with 0-0.1 M sodium chloride solution as the eluent. The eluent with a sodium chloride concentration of 0 M was collected, concentrated, and dried. The obtained portion was denoted as RG90-1. SIII-2. Perform gel column chromatography on the RG90-1 obtained in step SII-1, using water as the eluent. Collect the eluent after elution for 8.5 to 9.5 hours, and dry it to obtain the final product. The crude polysaccharide RG90 from Rehmannia glutinosa is prepared by the following steps: fully extracting Rehmannia glutinosa with water, concentrating the water extract, performing a first alcohol precipitation and filtering, taking the supernatant for a second alcohol precipitation and filtering, taking the supernatant for a third alcohol precipitation and filtering, and taking the precipitate obtained from the third alcohol precipitation. The first alcohol precipitation involves concentrating the aqueous extract and then adding 80% to 85% ethanol while stirring until the ethanol concentration of the solution reaches 45% to 50%. The second alcohol precipitation involves concentrating the supernatant obtained after the first alcohol precipitation by filtration, and then adding 85% to 90% ethanol until the ethanol concentration of the solution reaches 65% to 70%. The third alcohol precipitation is achieved by concentrating the supernatant obtained after the second alcohol precipitation and then adding anhydrous ethanol until the ethanol concentration of the solution reaches 85%~90%. In step SII-1, the anion exchange column is DEAE-Cellulose 52; In step SII-2, the gel column is Sephadex G-75.

7. The use of Rehmannia polysaccharide according to any one of claims 1 to 6 in the preparation of hypoglycemic drugs.

8. The use of the Rehmannia polysaccharide according to claim 1, 2, 5 or 6 in the preparation of anti-inflammatory drugs.

9. A hypoglycemic drug, characterized in that, It includes one or more of the Rehmannia polysaccharides described in any one of claims 1 to 6.

10. An anti-inflammatory drug, characterized in that, It contains one or more of the Rehmannia polysaccharides described in claims 1, 2, 5 or 6.

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