A Glehnia littoralis polysaccharide, a preparation method thereof, and an application thereof in the preparation of a product for preventing and treating ulcerative colitis

Abstract

The present invention relates to the technical field of traditional Chinese medicine polysaccharides, and particularly relates to a Glehnia littoralis polysaccharide, a preparation method thereof, and an application thereof in the preparation of products for preventing and treating ulcerative colitis. The Glehnia littoralis polysaccharide provided by the present invention is a polysaccharide mixture, and its hydrolyzed monosaccharides include galacturonic acid, L-rhamnose, D-glucose, D-galactose, and L-arabinose. Through experimental verification, the Glehnia littoralis polysaccharide can treat DSS-induced ulcerative colitis in rats, has the potential to treat human ulcerative colitis, and regulate intestinal disorders, and is expected to be developed into a polysaccharide drug for preventing and treating ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine polysaccharides, and particularly to a Glehnia littoralis polysaccharide, a preparation method thereof, and an application thereof in the preparation of products for preventing and treating ulcerative colitis. Background Art

[0002] The World Health Organization has pointed out that ulcerative colitis has become a global public health problem. The first-line medications for treating ulcerative colitis clinically mainly include Western medicine (such as salicylic acids, glucocorticoids, immunosuppressants, etc.), biological agent treatments, and surgical therapies. These treatment methods have significant drawbacks: salicylic acid drugs can relieve symptoms but have limited effects; glucocorticoids cannot be used for a long time to avoid producing more side effects; immunosuppressants achieve the purpose of treating ulcerative colitis by inhibiting cellular immunity and humoral immunity, and are also prone to causing adverse reactions of varying degrees; biological agents are usually not recommended for long-term use to avoid drug resistance; surgical treatment is not the first choice and is usually only used in severe cases.

[0003] Traditional Chinese medicine has shown unique advantages in the treatment of chronic diseases, such as clear effects, treating both the symptoms and the root causes, treating both inside and outside, the concept of wholism, and small toxic and side effects. However, the components of traditional Chinese medicine are complex, and not any traditional Chinese medicine can produce curative effects on ulcerative colitis. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides a Glehnia littoralis polysaccharide, a preparation method thereof, and an application thereof in the preparation of products for preventing and treating ulcerative colitis. Through experimental verification, the Glehnia littoralis polysaccharide can alleviate various symptoms of ulcerative colitis and can be used for the prevention and treatment of ulcerative colitis.

[0005] To achieve the above invention purpose, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a Glehnia littoralis polysaccharide, including polysaccharides with molecular weights of 3.648×10 6 Da, 2.983×10 4 Da, 1.644×10 3 Da, and 1.510×10 3 Da. Its hydrolyzed monosaccharides include galacturonic acid (GalA), L-rhamnose (Rha), D-glucose (Glc), D-galactose (Gal), and L-arabinose (Ara). The molar ratio of the galacturonic acid, L-rhamnose, D-glucose, D-galactose, and L-arabinose is (1.26 - 1.54):(8.28 - 10.12):(29.97 - 36.63):(2.25 - 2.75):(2.61 - 3.19).

[0007] The structural characteristics of this Glehnia littoralis polysaccharide are different from those of other Glehnia littoralis polysaccharides that have been publicly disclosed. Through experimental verification, this Glehnia littoralis monosaccharide can reduce the disease activity index, reduce colon bleeding, repair the colon structure, reduce inflammatory cell infiltration, increase mucus secretion, reduce the level of inflammatory factors, and restore the intestinal flora structure. It can be used for the prevention and treatment of ulcerative colitis, providing a safer, more reliable, and long-term active ingredient for the development of products for the prevention and treatment of ulcerative colitis.

[0008] Combined with the first aspect, the molar ratio of the galacturonic acid, L-rhamnose, D-glucose, D-galactose, and L-arabinose is 1.4:9.2:33.3:2.5:2.9.

[0009] In the second aspect, the present invention provides a preparation method for the above-mentioned Glehnia littoralis polysaccharide, which specifically includes the following steps:

[0010] S1. After cleaning, drying, and pulverizing the fresh Glehnia littoralis medicinal materials, extract them by refluxing with water. Centrifuge the obtained extract, take the supernatant, and concentrate the supernatant to obtain a water extract concentrate;

[0011] S2. Add ethanol to the water extract concentrate until the alcohol content is 75% - 85% v / v, let it stand for at least 12 h, then centrifuge and take the centrifugal precipitate;

[0012] S3. Dry the centrifugal precipitate, then dissolve it in water, add Sevage reagent, mix well and centrifuge, take the supernatant, dialyze it with a dialysis bag with a molecular weight cut-off of 3500 Da for at least 48 h, and dry the obtained retentate to obtain the Glehnia littoralis polysaccharide.

[0013] This preparation method is different from the extraction processes of Glehnia littoralis polysaccharides that have been publicly disclosed. The monosaccharide composition, molecular weight, glycosidic bond morphological characteristics, etc. of the Glehnia littoralis polysaccharide prepared by this preparation method are all different from those of the publicly disclosed Glehnia littoralis polysaccharides, thus being able to produce biological activities for the prevention and treatment of ulcerative colitis that have not been reported in the prior art.

[0014] Combined with the second aspect, the pulverization in S1 is to pulverize to a particle size less than or equal to 0.25 mm.

[0015] Combined with the second aspect, the extraction time of refluxing with water in S1 is 50 - 70 min. Further preferably 60 min.

[0016] Combined with the second aspect, the rotation speed of centrifugation in S1 is 3000 - 4000 rpm, and the time is 5 - 10 min. The further preferably centrifugation rotation speed is 3500 rpm, and the time is 5 min.

[0017] Combined with the second aspect, the alcohol content in S2 is 80% v / v.

[0018] In combination with the second aspect, the rotation speed of centrifugation in S2 is 3000 - 4000 rpm, and the time is 5 - 10 min. Further preferably, the rotation speed of centrifugation is 3500 rpm and the time is 5 min.

[0019] In combination with the second aspect, the water addition amount for reconstitution with water in S3 is 4.5 - 5.5 times the mass after drying the centrifugal precipitate. Further preferably, it is 5 times.

[0020] In combination with the second aspect, the addition amount of Sevage reagent in S3 is 0.8 - 1.2 times the water addition amount for reconstitution with water. Further preferably, it is 1 time.

[0021] In combination with the second aspect, the rotation speed of centrifugation in S3 is 3500 - 4500 rpm, and the time is 10 - 15 min. Further preferably, the rotation speed of centrifugation is 4000 rpm and the time is 10 min.

[0022] In a third aspect, the present invention also provides the application of the above-mentioned Glehnia littoralis polysaccharide or the Glehnia littoralis polysaccharide prepared by the above-mentioned preparation method in the preparation of products for preventing and treating ulcerative colitis.

[0023] In combination with the third aspect, the product is a product for reducing colonic hemorrhage.

[0024] In combination with the third aspect, the product is a product for repairing the colonic structure.

[0025] In combination with the third aspect, the product is a product for restoring the intestinal flora structure.

[0026] In combination with the third aspect, the product includes drugs for treating ulcerative colitis and health foods and drugs for preventing ulcerative colitis.

[0027] Preferably, the dosage form of the drug is an oral preparation.

[0028] The beneficial effects of the present invention are as follows: The present invention provides a Glehnia littoralis polysaccharide with novel structural characteristics and a preparation method. This Glehnia littoralis polysaccharide can reduce the disease activity index, reduce colonic hemorrhage, repair the colonic structure, reduce inflammatory cell infiltration, increase mucus secretion, reduce the level of inflammatory factors, and restore the intestinal flora structure. It can be used for the prevention and treatment of ulcerative colitis, is safe and effective, can be taken for a long time without developing drug resistance, makes up for the defects of western medicine preparations such as salicylic acid, glucocorticoids, immunosuppressants, and biological agents, and can be used for the preparation of products for preventing and treating ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following will further illustrate the present invention in conjunction with the drawings and embodiments. In the drawings:

[0030] Figure 1It is the HPGPC chart of Glehnia littoralis polysaccharide (GLP) in Example 1 of the present invention;

[0031] Figure 2 It is the HPLC chart of the monosaccharide composition of Glehnia littoralis in Example 1 of the present invention;

[0032] Figure 3 It is the DAI index change curve in Example 4 of the present invention;

[0033] Figure 4 It is the change in colon length in Example 4 of the present invention;

[0034] Figure 5 It is the change in the inner wall of the colon in Example 4 of the present invention;

[0035] Figure 6 It is the result of histopathological analysis of hematoxylin-eosin (H&E) stained colon tissue in Example 4 of the present invention (magnification ×100);

[0036] Figure 7 It is the result of histopathological analysis of alcian blue (AB) stained colon tissue in Example 4 of the present invention (magnification ×100) and the average optical density value (magnification ×400);

[0037] Figure 8 It is the level of pro-inflammatory cytokine TNF-α in colon tissues of each group in Example 4 of the present invention. Each point is expressed as mean ± standard deviation (n = 4 - 6). Compared with the control group, *** p < 0.001, ** p < 0.01, * p < 0.05; compared with the model group, ### p < 0.001, ## p < 0.01, # p < 0.05;

[0038] Figure 9 It is the level of pro-inflammatory cytokine IL-6 in colon tissues of each group in Example 4 of the present invention. Each point is expressed as mean ± standard deviation (n = 4 - 6). Compared with the control group, *** p < 0.001, ** p < 0.01, * p < 0.05; compared with the model group, ### p < 0.001, ## p < 0.01, # p < 0.05;

[0039] Figure 10 It is the level of pro-inflammatory cytokine IL-1β in colon tissues of each group in Example 4 of the present invention. Each point is expressed as mean ± standard deviation (n = 4 - 6). Compared with the control group, ***p < 0.001, ** p < 0.01, * p < 0.05 compared with the model, ### p < 0.001, ## p < 0.01, # p < 0.05;

[0040] Figure 11 It is the effect of the aqueous extract of Glehniae Radix and Glehniae Radix polysaccharide in regulating the intestinal flora structure at the phylum level (relative abundance at the phylum level) in Example 4 of the present invention;

[0041] Figure 12 It is the effect of the aqueous extract of Glehniae Radix and Glehniae Radix polysaccharide in regulating the intestinal flora structure at the genus level (relative abundance at the genus level) in Example 4 of the present invention. Detailed implementation manners

[0042] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] Glehniae Radix is derived from the dried roots of the umbelliferous plant Glehnia littoralis Fr. Schmidt ex Miq. The Glehniae Radix included in the Chinese Pharmacopoeia (2020 Edition) has a sweet taste, a slightly bitter taste, a slightly cold nature, and belongs to the lung and stomach meridians. It has the effects of nourishing yin and clearing the lung, and promoting the production of body fluid to quench thirst, and is used for dry cough due to lung heat, chronic cough with blood-streaked sputum, insufficiency of stomach yin, injury of body fluid caused by febrile diseases, dryness and thirst in the throat, etc. Glehniae Radix is rich in chemical components such as polysaccharides, saponins, and flavonoids. Studies have shown that Glehniae Radix polysaccharide can be degraded into small molecular weight polysaccharides under the action of intestinal flora and improve the proliferation of macrophages and lymphocytes. Macrophages are important immune cells in the intestine, and their activation will cause inflammatory reactions of cells such as neutrophils and lymphocytes, and macrophages themselves can also recognize pathogenic microorganisms and secrete inflammatory factors. For ulcerative colitis, the inflammatory reaction caused by macrophage hyperactivation is the main factor for the onset and continuous development of ulcerative colitis, which will aggravate the intestinal inflammatory reaction. Therefore, there has been no report on the application of Glehniae Radix polysaccharide in the prevention and treatment of ulcerative colitis so far.

[0044] The present invention has obtained a Glehniae Radix polysaccharide through a specific extraction process, which includes four polysaccharides with different molecular weights, and its hydrolyzed monosaccharides include galacturonic acid, L-rhamnose, D-glucose, D-galactose, and L-arabinose in a specific ratio.

[0045] The present invention also provides a preparation method for the above-mentioned Glehniae Radix polysaccharide, which specifically includes the following steps:

[0046] S1. After cleaning, drying and pulverizing the fresh Glehnia littoralis roots, extract them by refluxing with water. Centrifuge the obtained extract, take the supernatant, and concentrate the supernatant to obtain a water extract concentrate.

[0047] S2. Add ethanol to the water extract concentrate to make the alcohol content 75% - 85% v / v. Let it stand for at least 12 h and then centrifuge. Take the centrifugal precipitate.

[0048] S3. Dry the centrifugal precipitate, then redissolve it with water. Add Sevage reagent, mix well and centrifuge. Take the supernatant and dialyze it with a dialysis bag with a molecular weight cut-off of 3500 Da for at least 48 h. Dry the obtained retentate to obtain the Glehnia littoralis polysaccharide.

[0049] Since its extraction process is different from the publicly disclosed extraction processes in the prior art, the Glehnia littoralis polysaccharide obtained thereby has structural characteristics different from those of the publicly disclosed Glehnia littoralis polysaccharides, and there are significant differences in terms of monosaccharide composition, molecular weight, glycosidic bond morphological characteristics, etc., thus resulting in different biological activities. The present invention unexpectedly discovers through experimental research that the Glehnia littoralis polysaccharide provided by the present invention has an obvious therapeutic effect on DSS-induced ulcerative colitis rats, can reduce the disease activity index, reduce colon bleeding, repair the colon structure, reduce inflammatory cell infiltration, increase mucus secretion, reduce the level of inflammatory factors, and restore the intestinal flora structure. Based on this research, the present invention also provides the application of the above-mentioned Glehnia littoralis polysaccharide in the preparation of products for preventing and treating ulcerative colitis.

[0050] The following will be further described with specific examples.

[0051] The reagents and samples used in the following examples:

[0052] Ethanol (Shanghai Titan Scientific Co., Ltd.), dichloromethane, chloroform, n-butanol (Tianjin Beichen Fangzheng Reagent Factory), ultrapure water and pure water were prepared using a Milli-Q purification water system (Millipore Corporation, USA); 1-phenyl-3-methyl-5-pyrazolone (1-pheny-3-methyl-5-pyrazolone, PMP), ammonium acetate (Ammonium acetate, AA), acetic acid, trifluoroacetic acid (Trifluoroacetic Acid, TFA), 9 kinds of monosaccharide reference standards - mannose (D-Mannose, Man), rhamnose (L-Rhamnose, Rha), glucose (D-Glucose, Glc), galactose (D-Glactose, Gal), xylose (D-Xylose, Xyl), arabinose (D / L-Arabinose, Ara), glucuronic acid (D-Glucuronic Acid, GlcA), galacturonic acid (D-Galacturonic acid monohydrate, GalA), fucose (Fucose, Fuc) were purchased from Shanghai Merck Chemical Technology Co., Ltd.; pullulan (molecular weights of 642,000 Da, 334,000 Da, 494,00 Da, 22,000 Da, 6,300 Da, Showa Denko K.K., Japan), dextran sodium sulfate (Dextran sodium sulfate, DSS, Dalian Meilun Biotechnology Co., Ltd.), isoflurane (Rewod Life Science Co., Ltd.); SPF-grade 6-week-old male SD rats, weighing 140 - 160 g, were provided by Liaoning Changsheng Biotechnology Co., Ltd., and the animal license number was SCXK (Ji) 2020-002. This animal experiment followed the Guidelines for the Care and Use of Laboratory Animals and was approved by the Laboratory Animal Ethics Committee of Hebei Medical University, approval number IACUA-Hebmu-P2023083.

[0053] The following instruments and equipment were used in the following examples:

[0054] N-1100 Rotary Evaporator: Tokyo Rika Kikai Co., Ltd.; MIX-25P Mini Mixer: Hangzhou Mio Instruments Co., Ltd.; TG16-WS Desktop High-Speed Centrifuge: Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; -80°C Ultra-Low Temperature Refrigerator, 200μL / 1000μL Pipette: Eppendorf AG, Germany; ATS.B5-2MB Freeze Dryer: Beijing Sihuan Qihang Technology Co., Ltd.; Milli-Q Purified Water System: Millipore Corporation, USA; 3500Da Dialysis Membrane: Union Carbide (Viskase), USA; High Performance Gel Permeation Chromatography-Refractive Index Detector (HPGPC-RID) equipped with RID-20A Refractive Index Detector and Lab Solutions GPC V5.93 Processing Software: Shimadzu Corporation, Japan; 1260 Infinity II High Performance Liquid Chromatograph (equipped with DAD Detector): Agilent Technologies, USA; DHG-9030A Forced Air Drying Oven: Shanghai Yiheng Scientific Instrument Co., Ltd.; BT125D Electronic Analytical Balance: Sartorius AG, Germany; UV-2450 UV-Visible Spectrophotometer: Shimadzu Corporation, Japan; WD-2102B Automatic Microplate Reader: Beijing Liuyi Biotechnology Co., Ltd.

[0055] Example 1

[0056] The embodiment of the present invention provides a Glehnia littoralis polysaccharide and a preparation method thereof.

[0057] 1. Preparation of Glehnia littoralis polysaccharide

[0058] The fresh Glehnia littoralis medicinal materials are purified (removing impurities such as sediment and fibrous roots, washing, and slightly drying), and then dried for later use. Take the dried sample, crush it with a pulverizer, and pass through a 60-mesh sieve. The sieving rate (m after sieving / m before crushing) > 90%, and then it is reserved for later use.

[0059] Take 150 g of the powder and place it in a 5 L round-bottom flask. Add 2 L of purified water and mix well. Then place it in an electric heating mantle for extraction. After reflux extraction for 1 h, use a centrifuge to centrifuge the extract at 3500 rpm for 5 min. Concentrate it at 60 °C using a rotary evaporator until the volume of the concentrated solution is approximately 750 mL (aqueous extract concentrate). Take 450 mL of it and add absolute ethanol to make the alcohol content 80% (v / v, stir well). Let the alcohol precipitation solution stand for 12 h and then centrifuge it at 3500 rpm for 5 min. The obtained precipitate is dried thoroughly in an oven at 60 °C. The dried crude polysaccharide sample is redissolved in 5 times the amount of purified water, and then 1 time the amount of Sevage reagent (chloroform∶n-butanol = 4∶1 (v / v)) is added. After vortex mixing thoroughly, centrifuge it at 4000 rpm for 10 min. The supernatant is dialyzed and retained using a dialysis bag with a molecular weight cut-off of 3500 Da. After dialysis for 48 h, the retained solution is dried using a freeze dryer to obtain Glehnia littoralis polysaccharide (GLP).

[0060] 2. Determination of polysaccharide content

[0061] Preparation of reference solution: Weigh accurately 10 mg of anhydrous glucose reference substance, dissolve it in water and make up the volume to 100 mL in a volumetric flask to prepare a stock solution of 100 mg / L.

[0062] Drawing of standard curve: Accurately pipette 0 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL of the glucose solution stock solution into 10 mL stoppered test tubes respectively, add water to 500 μL, add 500 μL of 4% phenol solution, quickly add 2 mL of concentrated sulfuric acid, shake well, cool to room temperature, and measure the absorbance A at 482 nm. Draw the standard curve and the linear regression equation.

[0063] Sample determination: Take an appropriate amount of the Glehnia littoralis polysaccharide component sample, weigh it accurately, dissolve it in water and prepare a test solution of 50 mg / L. Take 500 μL of the solution and place it in a 10 mL stoppered test tube, add water to 500 μL, add 500 μL of 4% phenol solution, quickly add 2 mL of concentrated sulfuric acid, shake well, place it in a water bath at 80 °C for 15 min, cool to room temperature, and measure the absorbance A at 482 nm.

[0064] Substitute the absorbance value of the sample into the standard curve to calculate the sugar content of the polysaccharide in the Glehnia littoralis sample.

[0065] After detection, the sugar content of the Glehnia littoralis polysaccharide prepared in this experiment is 97%.

[0066] 2. Determination of protein content

[0067] Refer to the instruction manual of the test kit, use an enzyme-linked immunosorbent assay reader to determine the protein content in the polysaccharide, and use BAS (bovine serum albumin, BSA) as the standard product (R 2(= 0.9973): Mix 20 μL (2 mg / mL) of the diluted polysaccharide sample solution with 200 μL of the BCA working solution, incubate at 37 °C for 30 min, then measure the absorbance A at 470 nm using a WD-2102B full-automatic microplate reader, and calculate the protein content (%).

[0068] After detection, the protein content in the Glehnia littoralis polysaccharide prepared in this experiment was 1.17%, proving that the prepared polysaccharide had a high purity.

[0069] 3. Determination of polysaccharide molecular weight

[0070] Precisely weigh the prepared Glehnia littoralis polysaccharide, add the polysaccharide sample to the mobile phase to prepare a test solution at 10 mg / mL, and centrifuge at 14,000 rpm for 10 min to obtain the test solution.

[0071] Chromatographic conditions: The experimental equipment used was a Shimadzu HPGPC-RID liquid chromatograph; chromatographic column: TOSOH TSKgel GMPW XL (7.8×300 mm, 13 μm); the mobile phase used was 0.1 M NaNO3 + 0.05% NaN3, the chromatographic column temperature: 35 °C; flow rate: 0.6 mL / min; injection volume: 20 μL; isocratic elution. Use an HPGPC system equipped with an RID detector to collect and analyze data on the prepared polysaccharide components, record the chromatogram, as Figure 1 shown. Use the Lab Solutions GPC V5.93 processing software to calculate the molecular weight distribution of the sample based on the experimental results (using a narrow-distribution Pullulan polysaccharide standard sample to make a standard curve and perform relative calibration of the Mark-Houwink parameters).

[0072] Calculated from Figure 1 : The total Glehnia littoralis polysaccharide was mainly composed of four polysaccharides, with molecular weights of 3.648×10 6 Da (PDI = 2.35), 2.983×10 4 Da (PDI = 3.05), 1.644×10 3 Da (PDI = 1.29), 1.510×10 3 Da (PDI = 1.28).

[0073] 4. Analysis of monosaccharide composition based on the PMP pre-column derivatization method

[0074] Preparation of hydrolyzed monosaccharides: Weigh accurately 1.0 mg of Glehnia littoralis polysaccharide powder, dissolve it in 500 μL of ultrapure water to prepare a polysaccharide solution with a concentration of 2 mg / mL, and centrifuge it at 14,000 rpm for 10 min using a centrifuge. Take 200 μL of the centrifuged solution, add 200 μL of TFA (4.0 M, to make the final concentration of TFA 2.0 M), and hydrolyze it in an oven at 110 °C for 2 h. Remove TFA from the hydrolyzed sample under the condition of a 60 °C water bath (during the water bath process, add methanol to increase the evaporation rate).

[0075] PMP derivatization: Add 200 μL of ultrapure water to the dried sample, vortex it thoroughly to dissolve it completely, add 50 μL of 0.6 M sodium hydroxide solution and 100 μL of 0.5 M PMP methanol solution to the solution. Mix the reaction solution and react it in an oven at 70 °C for 30 min. After the reaction, neutralize the reaction solution by adding 100 μL of HCl (0.3 M) solution. Then, extract the unreacted PMP completely with 400 μL of chloroform. After mixing well for 10 s, carefully remove the bottom chloroform layer, repeat the above operation 3 times, and take the upper aqueous phase as the test sample for monosaccharide composition analysis. Monosaccharide reference standards do not need to be completely acid-hydrolyzed, and the remaining operations are the same as those of the sample.

[0076] Chromatographic conditions: The experimental equipment uses an Agilent 1260 high-performance liquid chromatograph; chromatographic column: ZORBAX SB-C18 (4.6×250 mm, 3.5 μm); the mobile phase uses 50 mM AA-water (A) / ACN (B), the chromatographic column temperature: 30 °C; flow rate: 0.6 mL / min; injection volume: 5 μL; isocratic elution with 20% (B).

[0077] Calculation of the molar ratio of each monosaccharide: Since the relative molecular masses of monosaccharides are different, simple peak area ratios cannot be directly converted into molar ratios and need to be corrected. Calculate the molar ratio (n) of the monosaccharides contained in Glehnia littoralis polysaccharide determined by HPLC according to the following formula.

[0078]

[0079] Among them: n1~n x respectively represent the amounts of substances of each monosaccharide in the test sample (Glehnia littoralis polysaccharide) solution;

[0080] A1'~A x ' are respectively the peak areas of the derivatives of each monosaccharide in the test sample (Glehnia littoralis polysaccharide) solution;

[0081] A1~A x are respectively the peak areas of the derivatives of each monosaccharide in the mixed reference standard solution;

[0082] m1~mx are the masses of the individual monosaccharide reference substances in the mixed reference substance solution, respectively;

[0083] M1 to M x are the relative molecular masses of the individual monosaccharide reference substances in the mixed reference substance solution, respectively.

[0084] By calculation, it was determined that Glehnia littoralis polysaccharide is mainly composed of five monosaccharides, namely GalA / Rha / Glc / Gal / Ara, and their molar ratio is 1.4:9.2:33.3:2.5:2.9.

[0085] Example 2

[0086] The embodiment of the present invention provides a Glehnia littoralis polysaccharide and a preparation method thereof.

[0087] The fresh Glehnia littoralis medicinal materials are purified (removing impurities such as sediment and fibrous roots, washing, and slightly drying), and then dried for later use. Take the dried sample, pulverize it with a pulverizer, and pass through a 60-mesh sieve. The sieving rate (m after sieving / m before pulverizing) > 90%, and it is reserved for later use.

[0088] Take 150 g of the powder and place it in a 5-L round-bottom flask. Add 2 L of purified water and mix well, then place it in an electric heating mantle for extraction. After reflux extraction for 70 min, centrifuge the extract at 3000 rpm for 10 min, and concentrate it at 60 °C using a rotary evaporator until the volume of the concentrated solution is about 750 mL. Take 450 mL of it and add anhydrous ethanol to make the alcohol content 75% (v / v, stir well). Let the alcohol precipitation solution stand for 16 h and then centrifuge it at 3000 rpm for 10 min. The obtained precipitate is dried thoroughly in an oven at 60 °C. The dried crude polysaccharide sample is redissolved with 4.5 times the amount of purified water, and then 0.8 times the amount of Sevage reagent (chloroform∶n-butanol = 4∶1 (v / v)) is added. After vortex mixing well, centrifuge it at 35000 rpm for 15 min. The supernatant is dialyzed and retained using a dialysis bag with a molecular weight cut-off of 3500 Da. After dialysis for 50 h, the retained solution is dried with a freeze dryer to obtain Glehnia littoralis polysaccharide.

[0089] Example 3

[0090] The embodiment of the present invention provides a Glehnia littoralis polysaccharide and a preparation method thereof.

[0091] The fresh Glehnia littoralis medicinal materials are purified (removing impurities such as sediment and fibrous roots, washing, and slightly drying), and then dried for later use. Take the dried sample, pulverize it with a pulverizer, and pass through a 60-mesh sieve. The sieving rate (m after sieving / m before pulverizing) > 90%, and it is reserved for later use.

[0092] Take 150 g of the powder and place it in a 5-L round-bottom flask. Add 2 L of purified water and mix well. Then place it in an electric heating mantle for extraction. After reflux extraction for 50 min, centrifuge the extract at 4000 rpm for 5 min using a centrifuge. Concentrate it at 60 °C using a rotary evaporator until the volume of the concentrated solution is approximately 750 mL. Take 450 mL of it and add absolute ethanol to make the alcohol content 85% (v / v, stir well). Let the alcohol-precipitated solution stand for 14 h and then centrifuge it at 4000 rpm for 5 min. The obtained precipitate is dried thoroughly in an oven at 60 °C. The dried crude polysaccharide sample is redissolved in 5.5 times the amount of purified water, and then 1.2 times the amount of Sevage reagent (chloroform∶n-butanol = 4∶1 (v / v)) is added. After vortex mixing well, centrifuge it at 4500 rpm for 10 min. The supernatant is dialyzed and retained using a dialysis bag with a molecular weight cut-off of 3500 Da. After dialysis for 52 h, the retained solution is dried using a freeze dryer to obtain Glehnia littoralis polysaccharide.

[0093] Example 4

[0094] In this example of the present invention, the therapeutic effect of Glehnia littoralis polysaccharide on DSS-induced ulcerative colitis rats was investigated.

[0095] 1) Animal feeding

[0096] Take SPF-grade male SD rats (body weight 150 ± 10 g). Raise them in an IVC-type independent air supply isolation cage system with an environmental temperature of (22 ± 1) °C, a humidity of 55% ± 10%, a 12 h / 12 h light-dark cycle, and sufficient drinking water and food.

[0097] 2) Grouping and modeling administration of experimental animals

[0098] After 7 days of adaptive feeding, the animals were randomly divided into a blank group (C), a model group (M), a water extract group of Glehnia littoralis (GLA), and a polysaccharide group of Glehnia littoralis (GLP), with 6 rats in each group. The blank group drank normal distilled water, and the animals in other groups were given 3% DSS aqueous solution instead of daily drinking water for 7 days to induce a classical ulcerative colitis model. At the same time of inducing the model, the water extract group of Glehnia littoralis and the polysaccharide group of Glehnia littoralis were respectively intragastrically administered the water extract of Glehnia littoralis and Glehnia littoralis polysaccharide at a dose of 1 g (calculated as crude drug) / kg body weight. The blank group and the model group were intragastrically administered an equal amount of distilled water every day for 7 days, once a day. On the 8th day, after anesthetizing the rats by intraperitoneal injection of 3% (w / v) sodium pentobarbital at a dose of 0.2 mL / 100 g, the samples were taken.

[0099] The Glehnia littoralis polysaccharide used in this experiment was the Glehnia littoralis polysaccharide prepared in Example 1, and the Glehnia littoralis extract was obtained by thoroughly drying the remaining water extract concentrated solution in Example 1 at 60 °C.

[0100] 3) Evaluation of disease activity index

[0101] During the experiment, the body weight, stool viscosity, and blood in the stool of the rats were observed and recorded, and the three indicators were quantitatively scored.

[0102] Scoring criteria: The body weight of the rats decreased (0 points, < 0 g; 1 point, 0 - 5 g; 2 points, 5 - 10 g; 3 points, 10 - 15 g; 4 points, > 15 g);

[0103] Stool viscosity (0 points, normal; 2 points, loose; 4 points, loose stool);

[0104] Blood in the stool (0 points, normal; 2 points, occult blood in the stool; 4 points, visible blood in the stool with the naked eye).

[0105] The disease activity index (DAI) was the average of the above three scores.

[0106] As Figure 3 shown, by statistically analyzing the disease activity index (DAI), colon length, and the morphology of the colon internal section of the blank group, model group, aqueous extract of Glehnia littoralis group, and polysaccharide of Glehnia littoralis group, it can be determined that after modeling, the DAI index of the rats increased significantly. When the rats were intragastrically administered with the polysaccharide of Glehnia littoralis, the DAI index decreased significantly.

[0107] 4) Measurement of colon length, hematoxylin-eosin (H&E) staining, and alcian blue (AB) staining

[0108] Colon tissues were taken from each group of rats. After measuring the length, they were fixed in paraformaldehyde fixative. After 24 hours, they were dehydrated, cleared, infiltrated with wax, embedded, and sectioned into 5-μm-thick sections using a microtome. After hematoxylin-eosin staining, they were stained according to the procedure of the alcian blue staining kit and observed under a microscope.

[0109] As Figure 4 shown, when the rats were intragastrically administered with the polysaccharide of Glehnia littoralis, the colon length was extended; in addition, the bleeding condition of the colon section improved, as Figure 5 shown. This result proves that the polysaccharide of Glehnia littoralis has a good effect in treating colon inflammation.

[0110] The results of H&E staining of the rat colon were as Figure 6 shown. In the blank group, the colon structure was complete, the crypts were arranged neatly, and the epithelial cells were arranged tightly; while in the model group, the colon structure was severely damaged, the crypt structure was damaged, almost no goblet cells were seen, and a large area of inflammatory infiltration occurred; compared with the model group, in the administration group, the crypt structure was complete, the number of goblet cells increased, and the degree of inflammatory cell infiltration decreased.

[0111] The results of alcian blue staining were as Figure 7As shown, compared with the blank group, the color of the colonic mucus area in the model group rats was lighter, the mucus area became smaller, indicating a decrease in mucus secretion; after treatment with the aqueous extract and polysaccharide, the mucus staining deepened, proving that the mucus secretion amount increased significantly.

[0112] 5) Effects of ELISA method on measuring the levels of TNF-α, IL-6 and IL-1β in intestinal cells

[0113] According to the steps of the ELISA method, measure the content levels of TNF-α, IL-6 and IL-1β in the supernatant.

[0114] The results are as Figure 8 、 9 、Figure 10 shown. Compared with the model group, the protein levels of TNF-α, IL-6 and IL-1β in the colon tissues of the GLA group and GLPH group were significantly decreased.

[0115] 6) DNA sequencing and intestinal flora analysis

[0116] Use a DNA extraction kit to extract the genomic DNA of fecal samples. Amplify the genes in the V3V4 region, and the collected products are used to sequence the intestinal flora structure. Use the online website of the Magigene Cloud Platform (http: / / cloud.magigene.com / resource) to perform α-diversity, β-diversity and linear discriminant analysis effect size (LEfSe) analysis to evaluate the differences in the intestinal flora composition of different groups.

[0117] The regulatory effects of the aqueous extract of Glehnia littoralis and Glehnia littoralis polysaccharide on the intestinal flora structure at the phylum level are as Figure 11 shown. At the phylum level, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria are the most abundant taxa among all taxa in fecal samples. Compared with the control group, the abundances of Bacteroidetes and Proteobacteria in DSS-induced colitis rats were significantly increased, while the abundances of Firmicutes and Actinobacteria were significantly decreased. After administration of the aqueous extract of Glehnia littoralis and Glehnia littoralis polysaccharide, the intestinal flora structure can be restored, and Glehnia littoralis polysaccharide can make the flora structure closer to the control group.

[0118] The regulatory effects of the aqueous extract of Glehnia littoralis and Glehnia littoralis polysaccharide on the intestinal flora structure at the genus level are as Figure 12As shown, at the genus level, Lactobacillus, Bacteroides, Romboutsia, Lachnospiraceae_NK4A136_group, Faecalibaculum, Desulfovibrio, Parabacteroides, and Bifidobacterium are the genera with the largest numbers among all genera in fecal samples. Compared with the control group, the abundances of Bacteroides, Lachnospiraceae_NK4A136_group, Faecalibaculum, and Parabacteroides in DSS-induced colitis rats were significantly increased, while the abundances of Lactobacillus, Romboutsia, Desulfovibrio, and Bifidobacterium were significantly decreased. After administration of the aqueous extract of Glehnia littoralis and Glehnia littoralis polysaccharide, the intestinal flora structure could be restored, and Glehnia littoralis polysaccharide could make the flora structure closer to that of the control group.

[0119] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of Glehnia littoralis polysaccharide in preparing medicine for preventing and treating ulcerative colitis, characterized in that, The preparation method of the Glehnia littoralis polysaccharide comprises the following steps: S1. After cleaning, drying and pulverizing the fresh Glehnia littoralis medicinal materials, perform water reflux extraction, centrifuge the obtained extract, take the supernatant, and concentrate the supernatant to obtain a water extract concentrate; S2. Add ethanol to the water extract concentrate until the alcohol content is 75% - 85% v / v, let stand for at least 12 h and then centrifuge, take the centrifugal precipitate; S3. Dry the centrifugal precipitate, then redissolve it with water, add Sevage reagent, mix well and centrifuge, take the supernatant, dialyze with a dialysis bag having a molecular weight cut-off of 3500 Da for at least 48 h, and dry the obtained retentate to obtain the Glehnia littoralis polysaccharide.

2. The application according to claim 1, wherein The Glehnia littoralis polysaccharide includes polysaccharides with molecular weights of 3.648×10 6 Da, 2.983×10 4 Da, 1.644×10 3 Da, and 1.510×10 3 Da. Its hydrolyzed monosaccharides include galacturonic acid, L-rhamnose, D-glucose, D-galactose, and L-arabinose. The molar ratio of galacturonic acid, L-rhamnose, D-glucose, D-galactose, and L-arabinose is (1.26~1.54):(8.28~10.12):(29.97~36.63):(2.25~2.75):(2.61~3.19).

3. The application according to claim 2, wherein The molar ratio of the galacturonic acid, L-rhamnose, D-glucose, D-galactose and L-arabinose is 1.4:9.2:33.3:2.5:2.

9.

4. The application according to claim 1, characterized in that, The pulverizing in S1 is pulverizing to a particle size less than or equal to 0.25 mm; and / or The extraction time of the water reflux extraction in S1 is 50 - 70 min; and / or The rotation speed of the centrifuge in S1 is 3000 - 4000 rpm, and the time is 5 - 10 min.

5. The application according to claim 1, wherein The alcohol content in S2 is 80% v / v; and / or The rotation speed of the centrifuge in S2 is 3000 - 4000 rpm, and the time is 5 - 10 min.

6. The application according to claim 1, characterized in that, The water addition amount for redissolving with water in S3 is 4.5 - 5.5 times the mass of the dried centrifugal precipitate; and / or The addition amount of the Sevage reagent in S3 is 0.8 - 1.2 times the water addition amount for redissolving with water; and / or The rotation speed of the centrifuge in S3 is 3500 - 4500 rpm, and the time is 10 - 15 min.

7. The application according to claim 1, characterized in that, The medicament is a medicament for reducing colonic bleeding; and / or The medicament is a medicament for repairing the colonic structure; and / or The medicament is a medicament for restoring the intestinal flora structure.

8. The application according to claim 1 or 7, characterized in that The medicament includes a medicament for treating ulcerative colitis and a medicament for preventing ulcerative colitis.

9. The application according to claim 8, wherein The dosage form of the medicament is an oral preparation.

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