CN117164737A - Radix glehniae polysaccharide, preparation method thereof and application thereof in preparation of products for preventing and treating ulcerative colitis - Google Patents

Abstract

The invention relates to the technical field of traditional Chinese medicine polysaccharides, in particular to glehnia littoralis polysaccharide, a preparation method thereof and application thereof in preparation of products for preventing and treating ulcerative colitis. The glehnia root polysaccharide provided by the invention is a polysaccharide mixture, and hydrolyzed monosaccharides comprise galacturonic acid, L-rhamnose, D-glucose, D-galactose and L-arabinose. Experiments prove that the glehnia littoralis polysaccharide can treat rat ulcerative colitis induced by DSS, has the potential effects of treating human ulcerative colitis and regulating intestinal disorder, and is expected to be developed into a polysaccharide medicament for preventing and treating ulcerative colitis.

Description

A kind of Adenophora polysaccharide, its preparation method and its use in preventing and treating ulcerative colitis Applications in products

Technical field

The present invention relates to the technical field of traditional Chinese medicine polysaccharides, and in particular relates to a polysaccharide from Radix Adenophora, its preparation method and its application in the preparation of products for the prevention and treatment of ulcerative colitis.

Background technique

The World Health Organization states that ulcerative colitis has become a global public health problem. The first-line clinical treatment of ulcerative colitis mainly includes Western medicine (such as salicylates, glucocorticoids, immunosuppressants, etc.), biological agents, and surgery. These treatment methods have significant disadvantages: salicylates It can relieve symptoms but has limited effect; glucocorticoids cannot be used for a long time to avoid causing more side effects; immunosuppressants achieve the purpose of treating ulcerative colitis by suppressing cellular immunity and humoral immunity, and can also easily cause varying degrees of adverse reactions; Biological agents are usually not recommended for long-term use to avoid the development of drug resistance; surgical treatment is not the first choice and is usually only used in severe cases.

Traditional Chinese medicine has shown unique advantages in the treatment of chronic diseases, such as clear effects, treating both symptoms and root causes, treating both internal and external diseases, a holistic concept, and low toxic and side effects. However, the ingredients of traditional Chinese medicine are complex, and not all traditional Chinese medicine can be effective in treating ulcerative colitis.

Contents of the invention

In view of the above technical problems, the present invention provides a Radix Adenophora polysaccharide, its preparation method and its application in preparing products for preventing and treating ulcerative colitis. It has been experimentally verified that the Adenophora polysaccharide can alleviate various symptoms of ulcerative colitis and can be used for the prevention and treatment of ulcerative colitis.

In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a polysaccharide of Radix Adenophora, 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, respectively, and its hydrolyzed monosaccharides include half Lacturonic acid (GalA), L-rhamnose (Rha), D-glucose (Glc), D-galactose (Gal) and L-arabinose (Ara), the galacturonic acid, L-rhamnose The molar ratio of sugar, 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).

The structural characteristics of this Adenophora polysaccharide are different from other disclosed Adenophora polysaccharides. It has been experimentally verified that this Adenophora monosaccharide can reduce disease activity index, reduce colon bleeding, repair colon structure, reduce inflammatory cell infiltration, increase mucus secretion, reduce inflammatory factor levels, restore intestinal flora structure, and can be used for ulcerative colon It provides safer, more reliable and long-term active ingredients for the development of products to prevent and treat ulcerative colitis.

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

In a second aspect, the present invention provides a method for preparing the above-mentioned Adenophora polysaccharide, which specifically includes the following steps:

S1. Clean the fresh Radix Adenophora medicinal materials, dry and pulverize them, extract with water reflux, centrifuge the obtained extract, take the supernatant, and concentrate the supernatant to obtain a water-extract concentrate;

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

S3. Dry the centrifugal precipitate, then redissolve it with water, add Sevage reagent, mix thoroughly and centrifuge, take the supernatant, and dialyze it with a dialysis bag with a molecular weight cutoff of 3500 Da for at least 48 hours, and dry the obtained cutoff liquid to obtain The Adenophora polysaccharide.

This preparation method is different from the extraction process of Adenophora polysaccharides that has been disclosed. The monosaccharide composition, molecular weight, glycosidic bond morphological characteristics, etc. Ginseng polysaccharide can produce biological activities for preventing and treating ulcerative colitis that have not been reported in the prior art.

Combined with the second aspect, the crushing described in S1 is crushing to a particle size of less than or equal to 0.25 mm.

Combined with the second aspect, the extraction time of water reflux extraction described in S1 is 50 to 70 minutes. 60 minutes is further preferred.

Combined with the second aspect, the centrifugal speed described in S1 is 3000-4000 rpm and the time is 5-10 minutes. A further preferred centrifugation speed is 3500 rpm and the centrifugation time is 5 minutes.

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

Combined with the second aspect, the centrifugal speed described in S2 is 3000-4000 rpm and the time is 5-10 minutes. A further preferred centrifugation speed is 3500 rpm and the centrifugation time is 5 minutes.

Combined with the second aspect, the amount of water added when reconstituting S3 with water is 4.5 to 5.5 times the mass of the centrifugal precipitation after drying. More preferably, it is 5 times.

Combined with the second aspect, the added amount of Sevage reagent described in S3 is 0.8 to 1.2 times the amount of water added when reconstituting it with water. More preferably, 1 time is used.

Combined with the second aspect, the centrifugal speed described in S3 is 3500-4500 rpm and the time is 10-15 minutes. A further preferred centrifugation speed is 4000 rpm and the centrifugation time is 10 minutes.

In a third aspect, the present invention also provides the use of the above-mentioned Adenophora polysaccharide or the Adenophora polysaccharide prepared according to the above preparation method in the preparation of products for preventing and treating ulcerative colitis.

In conjunction with the third aspect, the product is a product for reducing colon bleeding.

In conjunction with the third aspect, the product is a product for repairing colon structure.

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

In conjunction with the third aspect, the products include medicines for treating ulcerative colitis and health foods and medicines for preventing ulcerative colitis.

Preferably, the dosage form of the medicine is an oral preparation.

The beneficial effects of the present invention are: the present invention provides a polysaccharide of Radix Adenophora with brand-new structural characteristics and a preparation method. The polysaccharide of Radix Adenophora can reduce disease activity index, reduce colon bleeding, repair colon structure, reduce inflammatory cell infiltration, It can increase mucus secretion, reduce the level of inflammatory factors, and restore the structure of intestinal flora. It can be used to prevent and treat ulcerative colitis. It is safe and effective. It can be taken for a long time without developing drug resistance. It makes up for the salicylates, glucocorticoids, The shortcomings of western medicine preparations such as immunosuppressants and biological preparations can be used to prepare products for the prevention and treatment of ulcerative colitis.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and examples. In the accompanying drawings:

Figure 1 is the HPGPC diagram of Radix Adenophora polysaccharide (GLP) in Example 1 of the present invention;

Figure 2 is an HPLC diagram of the monosaccharide composition of Radix Adenophora in Example 1 of the present invention;

Figure 3 is a DAI index change curve in Embodiment 4 of the present invention;

Figure 4 shows the changes in colon length in Embodiment 4 of the present invention;

Figure 5 shows the changes in the inner wall of the colon in Embodiment 4 of the present invention;

Figure 6 is the result of hematoxylin-eosin (H&E) staining of colon histopathology in Example 4 of the present invention (magnification × 100 times);

Figure 7 is the histopathological analysis results of alcian blue (AB) staining of the colon in Example 4 of the present invention (magnification × 100 times) and the average optical density value (magnification × 400);

Figure 8 shows the levels of the pro-inflammatory cytokine TNF-α in the colon tissues of each group in Example 4 of the present invention. Each point is expressed as the 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;

Figure 9 shows the levels of pro-inflammatory cytokine IL-6 in the colon tissues of each group in Example 4 of the present invention. Each point is expressed as the 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;

Figure 10 shows the levels of pro-inflammatory cytokine IL-1β in colon tissues of each group in Example 4 of the present invention. Each point is expressed as the 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;

Figure 11 is the effect of the water extract of Adenophora miltiorrhiza and the polysaccharide of Adenophora miltiorrhiza in regulating the intestinal flora structure at the phylum level in Example 4 of the present invention (relative abundance at the phylum level);

Figure 12 shows the effects of the water extract of Adenophora miltiorrhiza and Adenophora miltiorrhiza polysaccharide in regulating the intestinal flora structure at the genus level in Example 4 of the present invention (relative abundance at the genus level).

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, 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 here are only used to explain the present invention and are not intended to limit the present invention.

Glehniae Radix is derived from the dried roots of Glehnia littoralis Fr.Schmidtex Miq. According to the 2020 edition of the "Chinese Pharmacopoeia", Adenophora ginseng has a sweet taste, slightly bitter taste, slightly cold nature, and returns to the lung and stomach meridians. It has the effect of nourishing yin and clearing the lungs, replenishing the stomach and promoting fluid production. It is used for dryness and cough due to lung heat, labored cough with phlegm and blood, insufficient stomach yin, heat disease and fluid damage, dry throat and thirst. North Adenophora is rich in polysaccharides, saponins, flavonoids and other chemical components. Studies have shown that Adenophora polysaccharides can degrade into small molecular weight polysaccharides under the action of intestinal flora and increase the proliferation of macrophages and lymphocytes. Macrophages are important immune cells in the intestine. Their activation can cause inflammatory reactions in cells such as neutrophils and lymphocytes. Macrophages themselves can also recognize pathogenic microorganisms and secrete inflammatory factors. For ulcerative colitis, macrophages The inflammatory response caused by hyperactivation of phagocytes is the main factor in the onset and development of ulcerative colitis and will aggravate the intestinal inflammatory response. Therefore, there have been no reports on the application of Adenophora polysaccharides in the prevention and treatment of ulcerative colitis.

The present invention obtains a Radix Adenophora polysaccharide through a specific extraction process, which includes four polysaccharides with different molecular weights, and its hydrolyzed monosaccharides include a special proportion of galacturonic acid, L-rhamnose, D-glucose, and D-glucose. Galactose and L-arabinose.

The invention also provides a method for preparing the above-mentioned Adenophora polysaccharide, which specifically includes the following steps:

S1. Clean the fresh Radix Adenophora medicinal materials, dry and pulverize them, extract with water reflux, centrifuge the obtained extract, take the supernatant, and concentrate the supernatant to obtain a water-extract concentrate;

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

S3. Dry the centrifugal precipitate, then redissolve it with water, add Sevage reagent, mix thoroughly and centrifuge, take the supernatant, and dialyze it with a dialysis bag with a molecular weight cutoff of 3500 Da for at least 48 hours, and dry the obtained cutoff liquid to obtain The Adenophora polysaccharide.

Since its extraction process is different from the extraction process disclosed in the prior art, the thus obtained Adenophora polysaccharide has structural characteristics different from the disclosed Adenophora polysaccharides in terms of monosaccharide composition, molecular weight, and glycosidic bond morphology. There are significant differences in characteristics and other aspects, resulting in different biological activities. Through experimental research, the present invention unexpectedly discovered that the polysaccharide of Radix Adenospermum provided by the present invention has a significant therapeutic effect on DSS-induced ulcerative colitis rats, and can reduce disease activity index, reduce colon bleeding, repair colon structure, and reduce inflammatory cell infiltration. , increase mucus secretion, reduce inflammatory factor levels, and restore intestinal flora structure. Based on this research, the present invention also provides the application of the above-mentioned Adenophora polysaccharide in the preparation of products for preventing and treating ulcerative colitis.

Further description will be given below with reference to specific embodiments.

Reagents and samples used in the following examples:

Ethanol (Shanghai Titan Technology Co., Ltd.), methylene chloride, chloroform, n-butanol (Tianjin Beichen Founder Reagent Factory), ultrapure water and pure water were prepared using Milli-Q purified water system (Millipore Company of the United States); 1-phenyl-3-methyl-5-pyrazolone (PMP), ammonium acetate (AA), acetic acid, trifluoroacetic acid, TFA), 9 kinds of monosaccharide reference substances - 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 monohydrate (GalA), fucose Sugar (Fucose, Fuc) was purchased from Shanghai Merrill Chemical Technology Co., Ltd.; pullulan (molecular weight: 642,000Da, 334,000Da, 494,00Da, 22,000Da, 6,300Da, Japan Showa Denko Co., Ltd.) dextran sulfate sodium salt ( Dextran sodium sulfate, DSS, Dalian Meilun Biotechnology Co., Ltd.), isoflurane (Reward Life Technology Co., Ltd.); SPF grade 6-week-old male SD rats, body weight 140-160g, Liaoning Changsheng Biotechnology Co., Ltd. , Animal license number: SCXK (Ji) 2020-002. This animal experiment followed the guidelines for the care and use of experimental animals and was approved by the Laboratory Animal Ethics Committee of Hebei Medical University, approval number IACUA-Hebmu-P2023083.

Instruments and equipment used in the following examples:

N-1100 rotary evaporator: Tokyo Rika Instruments Co., Ltd.; MIX-25P mini mixer: Hangzhou Miou Instrument Co., Ltd.; TG16-WS desktop high-speed centrifuge: Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; -80℃ ultra-low temperature refrigerator , 200μL/1000μL pipette: Eppendorf Company of Germany; ATS.B5-2MB freeze dryer: Beijing Sihuan Qihang Technology Co., Ltd.; Milli-Q purified water system: Millipore Company of the United States; 3500Da dialysis bag: Union Carbide (Viskase) of the United States ; High Performance Gel Permeation Chromatography-Refractive Index Detector (HPGPC-RID) equipped with RID-20A Refractive Index Detector, Lab Solutions GPC V5.93 processing software: Shimadzu ); 1260Infinity II high performance liquid chromatograph (equipped with DAD detector): American Agilent; DHG-9030A blast drying oven: Shanghai Yiheng Scientific Instrument Co., Ltd.; BT125D electronic balance, Sartorius, Germany; UV-2450 UV-visible spectrophotometer: Shimadzu; WD-2102B automatic microplate reader: Beijing Liuyi Biotechnology Co., Ltd.

Example 1

The embodiments of the present invention provide a Radix Adenophora polysaccharide and a preparation method thereof.

1. Preparation of Adenophora polysaccharide

Clean the fresh Adenophora ginseng medicinal materials (remove sediment, fibrous roots and other impurities, wash them, and dry them briefly), dry them and set them aside for later use. Take the dry sample, crush it with a pulverizer, and then pass it through a 60-mesh sieve. The sieving rate (m after sieving/m before crushing) is >90% and set aside.

Take 150g of the powder and place it in a 5L round-bottomed flask. Add 2L of purified water and mix thoroughly. Place it in an electric heating mantle for extraction. After reflux extraction for 1 hour, use a centrifuge to centrifuge the extract at 3500 rpm for 5 minutes. Use a rotary evaporator to Concentrate at 60°C until the volume of the concentrated solution is approximately 750mL (water-extracted concentrated solution). Take 450 mL of it and add absolute ethanol until the alcohol content is 80% (v/v, stir well). Let the alcohol precipitate stand for 12 hours and then centrifuge at 3500 rpm for 5 minutes. The resulting precipitate is fully dried in an oven at 60°C. The dried crude polysaccharide sample is reconstituted with 5 times the amount of purified water, and then 1 times the amount of Sevage reagent (chloroform: normal) is added. Butanol = 4:1 (v/v)), after thorough vortex mixing, centrifuge at 4000 rpm for 10 min. The supernatant is dialyzed and retained using a dialysis bag with a molecular weight cutoff of 3500 Da. After dialysis for 48 hours, the retentate is dried using a freeze dryer. , to obtain Radix Adenophora Polysaccharide (GLP).

2. Determination of polysaccharide content

Preparation of reference substance solution: Precisely weigh 10 mg of anhydrous glucose reference substance, add water to dissolve and dilute to volume in a 100 mL volumetric flask to prepare a 100 mg/L mother solution.

Draw a standard curve: Precisely pipette 0 μL, 100 μL, 200 μL, 300 μL, 400 μL, and 500 μL of the glucose solution mother solution respectively, 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, and shake well. , cool to room temperature, measure the absorbance A at 482nm, and draw the standard curve and linear regression equation.

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

The absorbance value of the sample was brought into the standard curve to calculate the sugar content of the polysaccharide of the Radix Adenophora sample.

After testing, the sugar content of the Adenophora polysaccharide prepared in this experiment was 97%.

2. Determination of protein content

Refer to the instructions of the test kit, use a microplate reader to measure the protein content in the polysaccharide, and use BAS (bovine serum albumin, BSA) as the standard (R ² =0.9973): mix 20 μL (2 mg/mL) of the diluted polysaccharide sample solution with 200 μL of BCA working solution was mixed and incubated at 37°C for 30 min, and then the absorbance A was measured at 470 nm using a WD-2102B automatic microplate reader, and the protein content (%) was calculated.

After testing, the protein content in the polysaccharide of Adenophora miltiorrhiza prepared in this experiment was 1.17%, which proved that the purity of the polysaccharide prepared was relatively high.

3. Determination of polysaccharide molecular weight

Precisely weigh the prepared polysaccharide from Adenophora ginseng, add the polysaccharide sample to the mobile phase to prepare a test solution of 10 mg/mL, and centrifuge at 14,000 rpm for 10 minutes to obtain the test solution.

Chromatographic conditions: The experimental equipment used Shimadzu HPGPC-RID liquid chromatograph; chromatographic column: TOSOH TSKgelGMPW _XL (7.8×300mm, 13μm); the mobile phase used 0.1M NaNO ₃ + 0.05% NaN ₃ , column temperature: 35°C; Flow rate: 0.6mL/min; injection volume: 20μL; isocratic elution. An HPGPC system equipped with a RID detector was used to collect and analyze data on the prepared polysaccharide components, and record the chromatogram, as shown in Figure 1. Use Lab Solutions GPC V5.93 processing software to calculate the molecular weight distribution of the sample based on the experimental results (use a narrow distribution Pullulan polysaccharide standard sample to make a standard curve and perform relative correction of Mark-Houwink parameters).

It is calculated from Figure 1 that the total polysaccharide of Radix Adenophora is mainly composed of four polysaccharides, whose molecular weights are 3.648×10 ⁶ Da (PDI=2.35), 2.983×10 ⁴ Da (PDI=3.05), and 1.644×10 ³ Da respectively. (PDI=1.29), 1.510×10 ³ Da (PDI=1.28).

4. Monosaccharide composition analysis based on PMP pre-column derivatization method

Preparation of hydrolyzed monosaccharides: Precisely weigh 1.0 mg of Adenophora polysaccharide powder, dissolve it in 500 μL of ultrapure water, prepare a 2 mg/mL polysaccharide solution, and centrifuge at 14,000 rpm for 10 min. Take 200 μL of the centrifuged solution and add 200 μL of TFA (4.0 M, so that the final concentration of TFA is 2.0 M) and hydrolyze in a 110°C oven for 2 hours. TFA was removed from the hydrolyzed samples in a 60°C water bath (methanol was added during the water bath process to increase the evaporation to dryness rate).

PMP derivatization: Add 200 μL of ultrapure water to the dried sample, vortex thoroughly to dissolve completely, and add 50 μL of 0.6M sodium hydroxide solution and 100 μL of 0.5M PMP methanol solution to the solution. Mix the reaction solution and react in an oven at 70°C for 30 minutes. After the reaction, the reaction solution was neutralized by adding 100 μL HCl (0.3M) solution. Then, 400 μL of chloroform was used to extract the unreacted PMP. After mixing thoroughly for 10 seconds, carefully remove the chloroform layer at the bottom, repeat the above operation three times, and take the upper water phase as the test sample for monosaccharide composition analysis. The monosaccharide reference substance does not need to be completely acid hydrolyzed, and the remaining operations remain the same as for the sample.

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

Calculation of the molar ratio of each monosaccharide: Due to the difference in the relative molecular mass of the monosaccharides, the simple peak area ratio cannot be directly converted into a molar ratio and needs to be corrected. According to the following formula, the polysaccharides in the polysaccharides measured by HPLC are calculated. The molar ratio of monosaccharides (n) is calculated.

Among them: n ₁ ~ n _x respectively represent the amount of each monosaccharide in the solution of the test product (Northern Adenophora polysaccharide);

A ₁ '～A _x ' are respectively the peak areas of each monosaccharide derivative in the test sample (Northern Adenophora polysaccharide) solution;

A ₁ to A _x are the peak areas of each monosaccharide derivative in the mixed reference solution;

m ₁ ~ m _x are the masses of each monosaccharide reference substance in the mixed reference substance solution;

M ₁ to M _x are the relative molecular masses of each monosaccharide reference substance in the mixed reference substance solution.

Through calculation, it was determined that the polysaccharide of Radix Adenophora polysaccharide is mainly composed of five monosaccharides including GalA/Rha/Glc/Gal/Ara, and its molar ratio is 1.4:9.2:33.3:2.5:2.9.

Example 2

The embodiments of the present invention provide a Radix Adenophora polysaccharide and a preparation method thereof.

Clean the fresh Adenophora ginseng medicinal materials (remove sediment, fibrous roots and other impurities, wash them, and dry them briefly), dry them and set them aside for later use. Take the dry sample, crush it with a pulverizer, and then pass it through a 60-mesh sieve. The sieving rate (m after sieving/m before crushing) is >90% and set aside.

Take 150g of the powder and place it in a 5L round-bottomed flask. Add 2L of purified water and mix thoroughly. Place it in an electric heating mantle for extraction. After reflux extraction for 70 minutes, use a centrifuge to centrifuge the extract at 3000 rpm for 10 minutes. Use a rotary evaporator to Concentrate at 60°C until the volume of the concentrated solution is approximately 750mL. Take 450 mL of it and add absolute ethanol until the alcohol content is 75% (v/v, stir well). Let the alcohol precipitate stand for 16 hours and then centrifuge at 3000 rpm for 10 min. The resulting precipitate is fully dried in an oven at 60°C. The dried crude polysaccharide sample is reconstituted with 4.5 times the amount of purified water, and then 0.8 times the amount of Sevage reagent (chloroform: normal) is added. Butanol = 4:1 (v/v)), after thorough vortex mixing, centrifuge at 35000 rpm for 15 minutes, the supernatant is dialyzed and retained using a dialysis bag with a molecular weight cutoff of 3500 Da. After dialysis for 50 hours, the retentate is dried using a freeze dryer. , get Adenophora polysaccharide.

Example 3

The embodiments of the present invention provide a Radix Adenophora polysaccharide and a preparation method thereof.

Clean the fresh Adenophora ginseng medicinal materials (remove sediment, fibrous roots and other impurities, wash them, and dry them briefly), dry them and set them aside for later use. Take the dry sample, crush it with a pulverizer, and then pass it through a 60-mesh sieve. The sieving rate (m after sieving/m before crushing) is >90% and set aside.

Take 150g of the powder and place it in a 5L round-bottomed flask. Add 2L of purified water and mix thoroughly. Place it in an electric heating mantle for extraction. After reflux extraction for 50 minutes, use a centrifuge to centrifuge the extract at 4000 rpm for 5 minutes. Use a rotary evaporator to Concentrate at 60°C until the volume of the concentrated solution is approximately 750mL. Take 450 mL of it and add absolute ethanol until the alcohol content is 85% (v/v, stir well). Let the alcohol precipitate stand for 14 hours and then centrifuge at 4000 rpm for 5 minutes. The resulting precipitate is fully dried in an oven at 60°C. The dried crude polysaccharide sample is reconstituted with 5.5 times the amount of purified water, and then 1.2 times the amount of Sevage reagent (chloroform: normal) is added. Butanol = 4:1 (v/v)), after thorough vortex mixing, centrifuge at 4500 rpm for 10 minutes. The supernatant is dialyzed and retained using a dialysis bag with a molecular weight cutoff of 3500 Da. After dialysis for 52 hours, the retentate is dried using a freeze dryer. , get Adenophora polysaccharide.

Example 4

The embodiments of the present invention examined the therapeutic effect of Radix Adenophora polysaccharide on rats with DSS-induced ulcerative colitis.

1) Animal feeding

Take SPF grade male SD rats (weight 150±10g). They were kept in an IVC-type independent air supply isolation cage system with an ambient temperature of (22±1)℃, a humidity of 55%±10%, a 12h/12h light and dark alternation, and sufficient drinking water and food.

2) Grouping of experimental animals and modeling and drug administration

After adaptive feeding for 7 days, the animals were randomly divided into a blank group (C), a model group (M), a water extract of Adenophora miltiorrhizae group (GLA), and a ginseng root polysaccharide group (GLP), with 6 animals in each group. The blank group drank normal distilled water, and the animals in the other groups used 3% DSS aqueous solution instead of daily drinking water to induce a classic ulcerative colitis model for 7 days. During the induction of the model, the water extract group of Adenophora miltiorrhizae and the polysaccharide group of Adenophora miltiorrhizae were treated respectively. The water extract of Adenophora miltiorrhiza and Adenophora miltiorrhiza polysaccharide were administered intragastrically at a rate of 1g (calculated as crude drug)/kg body weight. The blank group and the model group were administered an equal amount of distilled water daily for 7 consecutive days, once a day. On the 8th day, the rats were anesthetized by intraperitoneal injection of 3% (w/v) sodium pentobarbital at a dose of 0.2 mL/100 g and then harvested.

The Adenophora polysaccharide used in this experiment was the Adenophora polysaccharide prepared in Example 1, and the Adenophora extract was obtained by fully drying the remaining aqueous concentrate in Example 1 at 60°C.

3) Disease activity index assessment

During the experiment, the rats' weight, stool viscosity and blood in the stool were observed and recorded, and the three indicators were quantitatively scored.

Scoring criteria: Rat weight loss (0 points, <0g; 1 point, 0~5g; 2 points, 5~10g; 3 points, 10~15g; 4 points, >15g);

Stool consistency (0 points, normal; 2 points, loose stools; 4 points, loose stools);

Blood in the stool (0 points, normal; 2 points, occult blood in the stool; 4 points, visible blood in the stool).

The disease activity index (DAI) is the average of the above three scores.

As shown in Figure 3, by counting the disease activity index (DAI), colon length and intracolonic cross-section morphology of the blank group, model group, Adenophora miltiorrhiza aqueous extract group, and Adenophora miltiorrhiza polysaccharide group, it can be determined that after modeling , the DAI index of rats increased significantly. When the rats were intragastrically administered with Adenophora polysaccharide, the DAI index decreased significantly.

4) Colon length measurement, hematoxylin-eosin (H&E) staining and alcian blue (AB) staining

Colon tissue was taken from rats in each group, and the length was measured and then fixed in paraformaldehyde fixative. After 24 hours, the colon tissue was dehydrated and made transparent, embedded in wax, and sliced into 5 μm thick sections using a paraffin microtome. After staining with hematoxylin-eosin, and Perform staining using the Alcian blue staining kit procedure and observe under a microscope.

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

The H&E staining results of the rat colon are shown in Figure 6. The colon structure of the rats in the blank group was intact, the crypts were neatly arranged, and the epithelial cells were tightly arranged; while the colon structure of the rats in the model group was severely damaged, the crypt structure was destroyed, and the epithelial cells were almost intact. Goblet cells were seen, and large areas of inflammatory infiltration appeared. Compared with the model group, the crypt structure was intact, the number of goblet cells increased, and the infiltration of inflammatory cells in the drug group was reduced.

The results of Alcian blue staining are shown in Figure 7. Compared with the blank group, the colon mucus area of the rats in the model group was lighter in color and the mucus area was smaller, indicating reduced mucus secretion. After treatment with water extracts and polysaccharides, the mucus staining Deepening proves that the amount of mucus secretion increases significantly.

5) ELISA method to determine the effects of TNF-α, IL-6 and IL-1β levels in intestinal cells

The levels of TNF-α, IL-6 and IL-1β in the supernatant were determined according to the ELISA method.

The results are shown in Figures 8, 9, and 10. Compared with the model group, the protein levels of TNF-α, IL-6, and IL-1β in the colon tissue of the GLA group and GLPH group were significantly reduced.

6)DNA sequencing and intestinal flora analysis

Extract genomic DNA from stool samples using a DNA extraction kit. The V3V4 region genes were amplified, and the collected products were used to sequence the intestinal flora structure. Use the Magigene Cloud Platform online website (http://cloud.magigene.com/resource) to conduct alpha-diversity, beta-diversity and linear discriminant analysis effect size (LEfSe) analysis to evaluate different groups of intestinal flora Differences in composition.

At the phylum level, the regulatory effects of Adenophora aqueous extract and Adenophora polysaccharides on the intestinal flora structure are shown in Figure 11. At the phylum level, Firmicutes, Bacteroidetes, Proteobacteria, and Proteobacteria and Actinobacteria are the most abundant of all groups in fecal samples. Compared with the control group, the abundance of Bacteroidetes and Proteobacteria in DSS-induced colitis rats was significantly increased, and the abundance of Firmicutes and Actinobacteria was significantly decreased. The structure of intestinal flora can be restored after administration of Adenophora aqueous extract and Adenophora polysaccharide, and Adenophora polysaccharide can make the bacterial flora structure closer to that of the control group.

At the genus level, the regulatory effects of Adenophora aqueous extract and Adenophora polysaccharides on the intestinal flora structure are shown in Figure 12. At the genus level, Lactobacillus, Bacteroides, Roma Romboutsia, Lachnospiraceae_NK4A136_group, Faecalibaculum, Desulfovibrio, Parabacteroides, and Bifidobacterium are the number among all genera in fecal samples The most common genus. Compared with the control group, the abundance of Bacteroides, Lachnospiraceae_NK4A136_group, Faecalibaculum, and Parabacteroides in rats with DSS-induced colitis was significantly increased, and the abundance of Lactobacillus, Romboutsia, Desulfovibrio, and Bifidobacterium was significantly decreased. The structure of intestinal flora can be restored after administration of Adenophora aqueous extract and Adenophora polysaccharide, and the Adenophora polysaccharide can make the bacterial flora structure closer to that of the control group.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

1. A glehnia littoralis polysaccharide is characterized by comprising the components with molecular weights of 3.648 multiplied by 10 respectively ⁶ Da、2.983×10 ⁴ Da、1.644×10 ³ Da and 1.510×10 ³ Da polysaccharide, the hydrolysis monosaccharide of which comprises galacturonic acid, L-rhamnose, D-glucose, D-galactose and L-arabinose, wherein 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).

2. The coastal glehnia root polysaccharide of claim 1, wherein the molar ratio of galacturonic acid, L-rhamnose, D-glucose, D-galactose and L-arabinose is 1.4:9.2:33.3:2.5:2.9.

3. The preparation method of the glehnia littoralis polysaccharide according to claim 1 or 2, which is characterized by comprising the following steps:

s1, cleaning fresh radix glehniae, drying, crushing, extracting with water under reflux, centrifuging the obtained extracting solution, taking supernatant, and concentrating the supernatant to obtain water-extracted concentrated solution;

s2, adding ethanol into the water extraction concentrated solution until the ethanol content is 75-85% v/v, standing for at least 12h, centrifuging, and taking centrifugal precipitation;

s3, drying the centrifugal precipitate, re-dissolving the precipitate with water, adding a Sevage reagent, fully mixing, centrifuging, taking supernatant, dialyzing for at least 48 hours by using a dialysis bag with the molecular weight cutoff of 3500Da, and drying the obtained cut-off liquid to obtain the glehnia littoralis polysaccharide.

4. The method for preparing glehnia littoralis polysaccharide according to claim 3, wherein the grinding in S1 is to a particle size of 0.25mm or less; and/or

The extraction time of the reflux extraction with water in the step S1 is 50-70 min; and/or

The rotational speed of the centrifugation in the step S1 is 3000-4000 rpm, and the time is 5-10 min; and/or.

5. A method for preparing glehnia littoralis polysaccharide according to claim 3, wherein the alcohol content in S2 is 80% v/v; and/or

The rotational speed of the centrifugation in the S2 is 3000-4000 rpm, and the time is 5-10 min; and/or

The alcohol content in S2 is 80% v/v.

6. The preparation method of glehnia littoralis polysaccharide according to claim 3, wherein the water adding amount in the re-dissolution of water in S3 is 4.5-5.5 times of the mass of the dried centrifugal precipitate; and/or

The adding amount of the Sevage reagent in the S3 is 0.8-1.2 times of the adding amount of water when water is redissolved; and/or

The rotational speed of the centrifugation in the step S3 is 3500-4500 rpm, and the time is 10-15 min.

7. Use of a glehnia littoralis polysaccharide according to claim 1 or 2 or a glehnia littoralis polysaccharide prepared by a preparation method according to any one of claims 3 to 6 for the preparation of a product for the prevention and treatment of ulcerative colitis.

8. The use according to claim 7, wherein the product is a product for reducing colon bleeding; and/or

The product is a product for repairing colon structure; and/or

The product is a product for recovering the intestinal flora structure.

9. The use according to claim 7 or 8, characterized in that the product comprises a medicament for the treatment of ulcerative colitis and a health food and a medicament for the prevention of ulcerative colitis.

10. The use according to claim 9, wherein the pharmaceutical product is in the form of an oral formulation.