Traditional Chinese medicine polysaccharide, preparation method and application thereof in preparing medicine for treating ulcerative colitis

Abstract

The present application discloses a Chinese medicine polysaccharide, a preparation method and its application in preparing a drug for treating ulcerative colitis. The Chinese medicine polysaccharide includes a Rhodiola rosea polysaccharide extracted from Rhodiola rosea. The molecular weight range of the Rhodiola rosea polysaccharide is 2.2×10 6 ‑7.7×10 8 Da; the content of Rhodiola rosea polysaccharide is 56wt.%-85wt.%, and the protein content is 14.5wt.%-17.8wt.%. The Rhodiola rosea polysaccharide can significantly improve the inflammatory damage of the colon, effectively reduce the inflammatory factors in the colon, and promote the repair of mucosal damage in DSS-induced ulcerative colitis mice. At the same time, Rhodiola rosea polysaccharide regulates the intestinal microbial composition and diversity of colitis mice, reduces the ratio of Firmicutes to Bacteroidetes (F / B), and maintains the level of intestinal short-chain fatty acids (SCFA).

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine polysaccharide, a preparation method and application thereof in the preparation of a medicine for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific intestinal disease of unknown etiology characterized by continuous, diffuse inflammatory changes in the colorectal mucosa. Clinical manifestations include persistent or recurrent abdominal pain, diarrhea, bloody and mucous stools, tenesmus, or varying degrees of systemic symptoms. Bloody and mucous stools are the most common symptoms of UC. The disease often recurs and is difficult to cure. Currently, its etiology is unknown, there is no radical treatment, and there is a clear risk of cancer.

[0003] At present, the research on the etiology of UC is still a bottleneck, with different opinions, and the more recognized factors are environmental, infection, genetic and immune factors. The clinical treatment of UC is mainly based on salicylic acid (ASA), glucocorticoids (GC), and immunosuppressants (ISD) drugs, but long-term or overdose use has serious side effects and is prone to recurrence. ASA is prone to cause kidney damage and aplastic anemia; GC is prone to cause electrolyte disorders and full moon face; ISD is prone to cause adverse reactions such as bone marrow suppression and pancreatitis. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the art, the present application aims to provide a traditional Chinese medicine polysaccharide, a preparation method and an application in the preparation of a drug for treating ulcerative colitis.

[0005] According to one aspect of the present application, there is provided a use of a traditional Chinese medicine polysaccharide in the preparation of a drug for treating ulcerative colitis, wherein the traditional Chinese medicine polysaccharide is Rhodiola rosea polysaccharide.

[0006] According to another aspect of the present application, a Chinese medicine polysaccharide for the above application is provided, wherein the Chinese medicine polysaccharide is a Rhodiola rosea polysaccharide extracted from Rhodiola rosea;

[0007] The molecular weight range of Rhodiola rosea polysaccharide is 2.2×10 6 -7.7×10 8 Da;

[0008] The polysaccharide content of Rhodiola rosea is 56wt.%-85wt.%, and the protein content is 14.5wt.%-17.8wt.%.

[0009] According to some embodiments of the present application, Rhodiola rosea is Rhodiola rosea.

[0010] According to another aspect of the present application, a method for preparing a traditional Chinese medicine polysaccharide is provided, comprising:

[0011] Step 1: Extraction: extracting the Rhodiola rosea medicinal material with alcohol to obtain alcohol extraction residue and alcohol extraction filtrate;

[0012] Step 2: removing impurities: extracting the alcohol extraction residue in step 1 with water to obtain a water extract;

[0013] Step 3: Enzymatic hydrolysis: passing the aqueous extract in step 2 through a column and eluting, collecting and combining the effluent and the eluate, and concentrating to obtain a concentrate;

[0014] Step 4: alcohol precipitation: enzymatically hydrolyze the concentrated solution in step 3 and take the supernatant;

[0015] Step 5: Dialysis and drying: The supernatant in step 4 is subjected to alcohol precipitation, and the precipitate is collected, dialyzed, and freeze-dried to obtain the Chinese medicine polysaccharide.

[0016] According to some embodiments of the present application, column passing and elution are as follows: the water extract is cooled to room temperature, passed through a macroporous resin column, and eluted with 2BV pure water;

[0017] The macroporous resin model is selected from one of D312, LSA-30, LX-68G, AB-8 and D101.

[0018] According to some embodiments of the present application, the volume ratio of the loading amount of the water extract to the macroporous resin is 1:(1-3);

[0019] According to some embodiments of the present application, the loading flow rate is 2-3 BV / h.

[0020] According to some embodiments of the present application, the concentration is concentration to a crude drug concentration of 1 g / mL.

[0021] According to some embodiments of the present application, enzymatic hydrolysis is performed by adding one of papain, pepsin, trypsin and endopeptidase, performing enzymatic hydrolysis at 37-60° C. for 1-1.5 h, cooling and centrifuging to obtain a supernatant;

[0022] According to some embodiments of the present application, the dosage of the enzyme is 0.0004-0.001 g / g crude drug amount.

[0023] According to some embodiments of the present application, alcohol precipitation includes: adjusting the ethanol concentration to 40% by the first alcohol precipitation, and adjusting the ethanol concentration to 80% by the second alcohol precipitation.

[0024] According to some embodiments of the present application, the dialysis comprises: washing the precipitate twice with ethanol, adding an appropriate amount of water to redissolve, filtering, transferring the filtrate to a dialysis bag, dialysis, and concentrating the retentate to a crude drug concentration of 1 g / mL;

[0025] According to some embodiments of the present application, the particle size of the dialysis bag is 3500Da-90000Da, and can be optionally 90000Da.

[0026] According to some embodiments of the present application, the alcohol extraction uses 60 vol%-95 vol% ethanol aqueous solution.

[0027] The present application also provides a traditional Chinese medicine preparation for treating ulcerative colitis comprising the above-mentioned traditional Chinese medicine polysaccharide.

[0028] Compared with the prior art, this application has at least the following beneficial effects:

[0029] The present application provides a traditional Chinese medicine polysaccharide - Rhodiola rosea polysaccharide, which can significantly improve inflammatory damage to the colon, effectively reduce inflammatory factors in the colon, and promote the repair of mucosal damage in DSS-induced ulcerative colitis in mice. At the same time, Rhodiola rosea polysaccharide regulates the intestinal microbial composition and diversity of colitis mice, reduces the ratio of Firmicutes to Bacteroidetes (F / B), and maintains the level of intestinal short-chain fatty acids (SCFA). Therefore, the Rhodiola rosea polysaccharide can be used to treat ulcerative colitis.

[0030] This application proposes the use of Rhodiola rosea polysaccharide in the preparation of ulcerative colitis drugs. The constructed model is a DSS-induced mouse ulcerative colitis model, which can effectively improve the levels of gold indicators such as mouse weight, fecal score, colon length and pathological damage. The Rhodiola rosea polysaccharide part of this application has a good therapeutic effect on ulcerative colitis.

[0031] The present application also provides a method for preparing the above-mentioned Chinese medicine polysaccharide, by removing small molecule compounds including salidroside through the steps of alcohol extraction followed by water extraction, macroporous resin column separation, graded alcohol precipitation and dialysis, and comparing the activities of the extracts obtained under dialysis bags with different particle sizes on an animal model, the activity of the polysaccharide part with a larger particle size in the dialysis bag was significantly higher than that of the other two extracts, therefore, compared with other Rhodiola rosea extracts, the Rhodiola rosea polysaccharide of the present application has a more significant therapeutic effect and fewer side effects at the same dosage, and at the same time, the Chinese medicine polysaccharide has diverse targets, is not prone to drug resistance, and has no adverse reactions or fewer adverse reactions.

[0032] The preparation method of the Chinese medicine polysaccharide of the present application is simple to operate and has high implementation efficiency. The raw materials used in the operation process are safe, low in cost, and environmentally friendly, and have wide application potential and good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the analysis spectrum of the extract of the Chinese medicine polysaccharide in this application.

[0034] Figure 2 This is the effect of each experimental group on body weight in the exemplary embodiments of this application.

[0035] Figure 3 This is the effect of each experimental group on DAI in the exemplary embodiment of this application.

[0036] Figure 4 This is the effect of each experimental group on colon length in the exemplary embodiments of this application.

[0037] Figure 5 These are colon pictures of each experimental group in the exemplary embodiments of this application.

[0038] Figure 6 These are pathological sections of each experimental group in the exemplary embodiments of this application.

[0039] Figure 7 This is the effect of each experimental group on the inflammatory factor interleukin-6 in the exemplary embodiments of this application.

[0040] Figure 8 This is the effect of the Rhodiola rosea polysaccharide group on short-chain fatty acids in DSS-induced colitis in mice in the exemplary embodiments of this application. DETAILED DESCRIPTION

[0041] The technical solution of the present application will be clearly and completely described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application.

[0042] It is particularly important to point out that similar substitutions and modifications made to the present application are obvious to those skilled in the art, and they are all deemed to be included in the present application. Relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application to implement and apply the technology of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments.

[0043] If no specific conditions are specified in this application, the preparation shall be carried out under conventional conditions or the conditions recommended by the manufacturer. The raw materials or excipients used, as well as the reagents or instruments used, for which the manufacturers are not specified, are all conventional products that can be obtained commercially.

[0044] In the present application, the terms “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0045] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0046] In the present application, the percentage content, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0047] In this application, when it comes to percentage concentration, unless otherwise specified, it refers to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0048] In the present application, wt.% means weight percentage, and vol% means volume percentage.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0050] The application is described in detail below.

[0051] Rhodiola rosea L. is a perennial herbaceous plant of the Crassulaceae family, with the Tibetan name Saoromabul. Its dried rhizomes are used as medicine. It is widely distributed in Qinghai, Tibet, Xinjiang, Sichuan and other places. It was recorded as early as in the Four Medical Classics that its roots are used as medicine, and it is both a medicine and a food, and has the effects of clearing heat and activating blood circulation. The Ming Dynasty's Compendium of Materia Medica records that "Rhodiola rosea is a top-grade plant in this book, which can remove evil spirits and make up for deficiencies."

[0052] Modern research shows that Rhodiola rosea contains a variety of active ingredients, including polysaccharides, glycosides, tyrosol, salidroside, phenols, flavonoids, amino acids, coumarins, organic acids, mineral elements, steroidal compounds and alkaloids. It has many potential benefits, especially in terms of human athletic ability and anti-aging, which has attracted widespread attention. Among them, Rhodiola rosea polysaccharides have the effects of anti-fatigue, anti-aging, anti-virus, hypoglycemic, immune regulation, antibacterial, anti-oxidation, hypoglycemic, regulating sugar and lipid metabolism, anti-injury, and anti-adverse stimulation.

[0053] This application is a study on Rhodiola rosea in order to find an active ingredient that is effective in treating ulcerative colitis. During the experiment, it was found that the polysaccharide from Rhodiola rosea has a good therapeutic effect; and further research is conducted on the extraction process and ingredients.

[0054] The traditional Chinese medicine polysaccharide of the present application is extracted from Rhodiola rosea, wherein the polysaccharide content is 56wt.%-85wt.%, and the protein content is 14.5wt.%-17.8wt.%.

[0055] The molecular weight range of Chinese medicine polysaccharides is: 2.2×10 6 -7.7×10 8 Da;

[0056] Optionally, the Rhodiola rosea is Rhodiola rosea.

[0057] The present application also provides a method for preparing the above-mentioned traditional Chinese medicine polysaccharide, comprising:

[0058] Step 1: Extraction: extracting the Rhodiola rosea medicinal material with alcohol to obtain alcohol extraction residue and alcohol extraction filtrate; and extracting the alcohol extraction residue with water.

[0059] Step 2: Impurity removal: The water extract is cooled to room temperature, passed through a macroporous resin column, eluted with 2BV of pure water, the effluent and the water wash are collected and combined, and concentrated to 1 g / mL of crude drug.

[0060] The macroporous resin model is selected from one of D312, LSA-30, LX-68G, AB-8 and D101.

[0061] Step 3: Enzymatic hydrolysis: add papain, pepsin, trypsin, or endopeptidase to the concentrate, perform enzymatic hydrolysis at 37-60°C, boil, inactivate the enzyme for 10 minutes, cool to room temperature, centrifuge, and take the supernatant.

[0062] Step 4: alcohol precipitation: first alcohol precipitation, add ethanol to the supernatant obtained in the previous step, adjust the ethanol concentration to 30%-60%, let the alcohol precipitation stand overnight, centrifuge, collect the supernatant, and perform a second alcohol precipitation. Concentrate the supernatant obtained from the first alcohol precipitation until there is no alcohol taste, add ethanol to adjust the concentration to 70%-95%, let it stand overnight, and collect the precipitate.

[0063] Step 5: Dialysis and drying: Wash the precipitate twice with ethanol, add appropriate amount of water to re-dissolve, filter, transfer the filtrate to a dialysis bag, dialyze, concentrate the retentate to 1 g / mL crude drug, pre-freeze at -30°C, and freeze-dry to obtain the product.

[0064] The ratio of the sample loading amount to the macroporous resin in the impurity removal step is 1:(1-3); and the sample loading flow rate in the impurity removal step is 2-3 BV / h.

[0065] Optionally, the ethanol concentration is adjusted to 40% by the first alcohol precipitation, and the ethanol concentration is adjusted to 80% by the second alcohol precipitation.

[0066] The particle size of the dialysis bag is 3500Da-90000Da; optionally, the particle size of the dialysis bag is 90000Da.

[0067] The above preparation method also includes:

[0068] The alcohol extraction uses 60vol%-95vol% ethanol aqueous solution;

[0069] In the alcohol extraction step, the ethanol solution is refluxed for extraction at least twice, each time for 0.5-3 hours; the solid-liquid ratio of each alcohol extraction is 1: (8-20), and the ratio relationship is g / mL;

[0070] In the water extraction step, water is added and boiled at least twice, each time for 0.5-2 hours; the material-liquid ratio of each water extraction is 1:(10-20), and the ratio relationship is g / mL;

[0071] The enzymatic hydrolysis adopts papain; in the enzymatic hydrolysis step, the dosage of papain is 0.0004-0.001g / g crude drug amount; in the enzymatic hydrolysis step, the enzymatic hydrolysis temperature is 55-60℃, and the enzymatic hydrolysis time is 1-1.5h.

[0072] After the centrifugation step, the solid is washed at least once with an ethanol solution to obtain a precipitate; the concentration of the ethanol solution is 80 vol%-95 vol%.

[0073] The pharmaceutical preparation of the present application comprises a preventive and / or therapeutically effective amount of a Chinese medicinal polysaccharide;

[0074] Optionally, the Chinese medicine polysaccharide is the only active ingredient in the Chinese medicine preparation; or the medicine preparation also includes other active ingredients or drugs; other active ingredients include nutritional supplements.

[0075] The Chinese medicine preparation can be in the form of capsules, pills, powders, syrups, tablets, granules, plasters, mixtures, pills, suppositories, aerosols, ointments or injections.

[0076] The technical solution of the present application is further described below in conjunction with specific embodiments.

[0077] Example 1 Preparation of the traditional Chinese medicine polysaccharide of the present application.

[0078] Step 1: Extraction: Take 500 g of Rhodiola rosea, crush it through a No. 1 sieve, add 10 times the amount of 95% ethanol, extract twice, each time for 1 hour, filter, leave the residue for 24 hours, add 12 times the amount of water and boil twice, each time for 1 hour, filter, collect the filtrate, and discard the residue; take 1 / 3 of the water extract, concentrate it, and dry it under reduced pressure.

[0079] Step 2: Impurity removal: The filtrate in step 1 was cooled to room temperature, passed through an LSA-30 macroporous resin column (500 g wet resin) at a flow rate of 2 BV / h, and the effluent was concentrated to 1 g / mL crude drug amount; the LSA-30 macroporous resin was continuously eluted with 4 BV of 95% ethanol, 95% of the eluate was collected, concentrated and dried under reduced pressure.

[0080] Step 3: Enzymatic hydrolysis: add 0.2 g papain to the concentrated solution of 1 g / mL crude drug, perform enzymatic hydrolysis at 60°C for 1 h, boil, inactivate the enzyme for 10 min, cool to room temperature, centrifuge at 4000 r / min for 10 min, and take the supernatant.

[0081] Step 4: alcohol precipitation: add ethanol to the supernatant obtained in step 3 for alcohol precipitation, adjust the ethanol concentration to 40%, let the alcohol precipitation stand overnight, centrifuge, and collect the supernatant; after concentrating the supernatant until there is no alcohol taste, add ethanol to adjust the concentration to 80%, let it stand overnight, and collect the precipitate.

[0082] Step 5: Dialysis and drying: Wash the precipitate obtained in step 4 twice with ethanol; re-dissolve in water, filter, and place the filtrate in dialysis bags with particle sizes of 3500Da, 8500 Da, 35000 Da, and 90000 Da, respectively, dialyze for 24 hours, concentrate to 1g / mL crude drug, pre-freeze at -30°C, and freeze-dry to obtain Extract 1, Extract 2, Extract 3, and Extract 4.

[0083] Example 2

[0084] Analysis of Extract 4 in Example 1

[0085] The phenol-sulfuric acid method was used to determine: D-glucose was used as the standard monosaccharide composition analysis of extract 4. The proportion of polysaccharides in extract 4 was 78.15±0.016%, and the standard curve was y=9.3195x+0.0281, R²=0.9915. By HPGPC measurement, according to the Shodex STANDARD P-82 standard, the molecular weight retention time standard curve was log (Mw) = -0.4396T +11.401, R² = 0.999, and the calculated molecular weight distribution of extract 4 was 2.2×10 6 ~7.7×10 6 Da (such as Figure 1 A).

[0086] The monosaccharide composition of extract 4 was measured using pre-column PMP derivatization HPLC ( Figure 1 B). The results showed that extract 4 was composed of Man, Rha, GlcA, GalA, Glc, Gal, and Ara with a relative molar ratio of 0.22:1:0.07:7.03:2.88:0.64:4.12, indicating that extract 4 was a heteropolysaccharide.

[0087] Example 3

[0088] Therapeutic effects of different extracts in Example 1 on mice with ulcerative colitis induced by dextran sulfate sodium salt

[0089] Experimental Materials

[0090] Animals: SPF grade 8-week-old male C57BL / 6 mice weighing 22-24 g were purchased from the Experimental Animal Center of Hangzhou Medical College (production license number: SCXK (Zhejiang) 2019-0002). The breeding environment was clean, the ambient temperature was controlled at 23±2℃, the humidity was maintained at 50%-60%, and the light was kept at 12 hours of day and night. Food was available at will, and drinking water was used after filtration and autoclaving.

[0091] Drugs and reagents: dextran sulfate sodium salt (Shanghai McLean Biochemical Technology Co., Ltd.); sulfasalazine (Shanghai Fuda Pharmaceutical Co., Ltd.); extract 1, extract 2, extract 3, and extract 4 were prepared according to the method of Example 1.

[0092] Instruments: Refrigerated centrifuge (ThermoFisher Scientific, USA); vortex mixer (Qilin Bell Instrument Manufacturing Co., Ltd.); electronic balance (Startorius, USA); pathology slicer (Shanghai Leica Instrument Co., Ltd.); upright optical microscope (Nikon, Japan)

[0093] Dosage design: The dose of extract 1 for mice is 200 mg / kg / day, the dose of extract 2 for mice is 200 mg / kg / day, the dose of extract 3 for mice is 200 mg / kg / day, and the dose of extract 4 for mice is 200 mg / kg / day. Based on the body surface area conversion method, the equivalent dose of sulfasalazine in mice is 100 mg / kg / day.

[0094] Experimental methods: After the adaptive feeding of mice, they were randomly divided into 7 groups according to their body weight, with 10 mice in each group, which were set as blank control group (Control), model group (Model), extract group 1 (200 mg / kg / day), extract group 2 (200 mg / kg / day), extract group 3 (200 mg / kg / day), extract group 4 (200 mg / kg / day) and positive drug group (sulfasalazine, 100 mg / kg / day). Except for the blank control group, the other 6 groups were given 2.5% DSS free drinking water for 7 days (fresh 2.5% DSS solution was replaced on days 1, 3, and 5) to construct the UC model. The weight of mice was recorded daily, and the DAI score of mice was performed. The drugs were administered by gavage while the model was established. Each extract group was administered by gavage, the blank control group and the model control group were administered by gavage with pure water, and the other groups were administered with the corresponding drug solution once a day for 8 consecutive days. After the last administration, at 8 pm, the mice were fasted but not watered for more than 12 hours. On day 9, blood was collected from mice after anesthesia and centrifuged at 3000 rpm for 15 min, and then serum was obtained and stored at -80 °C for the analysis of inflammatory indicators, including interleukin-6 (IL-6) and myeloperoxidase (MPO). Colonic tissue was resected, and the length of the colon from the cecum to the anus was measured and recorded. Samples of colonic contents were collected in plastic tubes and then immediately frozen at -80 °C for intestinal microbiota analysis. The distal colon section was fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin (H&E), and the tissue morphological changes were observed by light microscopy and histological evaluation was performed.

[0095] GraphPad Prism 9 software was used to draw graphs for analysis of differences among the groups.

[0096] Experimental results:

[0097] (1) Evaluation of drug efficacy in DSS-induced UC mice

[0098] After DSS induction, the body weight of mice decreased significantly, but treatment with extract 4 reversed this loss (Table 1 and Figure 2 DSS can also significantly increase DAI, while extract 4 can improve this phenomenon, indicating that it has a certain alleviating effect on ulcerative colitis (as shown in Table 2 and Figure 3 Effects of each experimental group on DAI). By measuring the colon length of each group of mice, the colon length of the model group was significantly shorter than that of the control group, while Extract 4, Extract 2 and Extract 1 alleviated the shortening of colon length to varying degrees, with Extract 4 having the best effect. The comprehensive results showed that Extract 4 had the best effect on the recovery of ulcerative colitis.

[0099] Table 1 Effects of each experimental group on the body weight of mice

[0100]

[0101] Table 2 Effects of each experimental group on DAI scores of mice

[0102]

[0103] in, Figure 4 The effect of each experimental group on colon length, # indicates statistical difference compared with the Control group, ## indicates significant difference compared with the Control group, ### indicates extremely significant difference compared with the Control group; * indicates statistical difference compared with the Model group, ** indicates significant difference compared with the Model group, *** indicates extremely significant difference compared with the Model group. Figure 5 The following are pictures of the colon in each experimental group. Compared with the blank group, the integrity of the intestinal mucosal epithelium in the model group was destroyed after modeling, the goblet cells were reduced, and a large number of inflammatory cells aggregated into clusters, invading the muscle layer and serosa layer. Compared with the model group, the colon lesions in each drug-treated group were improved, the crypt structure was restored to a certain extent, and the infiltration range of inflammatory cells in extract 4 was smaller.

[0104] Figure 6 The pathological sections of each experimental group show the effects of the extract of the present application on the inflammatory indicators of UC mice induced by DSS. The results are as follows Figure 6 shown. Figure 7 The effects of each extract on the inflammatory factor interleukin-6 (IL-6) in UC model mice, data are expressed as mean ± standard deviation, n = 4. Compared with the control group, ***P<0.001, **P<0.01, *P<0.05.

[0105] In this study, experimental data were expressed as Mean ± SEM. Data statistics and graphing were performed using GraphPadPrism 9. The significant differences between the control group and the treatment group were analyzed by analysis of variance (ANOVA). P values ​​less than 0.05 were considered statistically significant. The extract 4 administration group can significantly correct the changes in various inflammatory indicators under the UC model, and its effect is better than other extracts and positive drugs.

[0106] 3. Short-chain fatty acid detection

[0107] like Figure 8 Shown are the effects of extract 4 on the SCFA content of DSS-induced colitis in mice, including acetate, propionate, isobutyrate, butyrate, isovaleric acid, and valeric acid. Data (n = 6 per group) are expressed as median (IQR) #p < 0.05, ##p < 0.01 compared with the NC group *p < 0.05, **p < 0.01 compared with the MC group.

[0108] In the above-mentioned preparation example 1, through the polysaccharide preparation process of the present application, the content of small molecule compounds including salidroside in extracts 1-3 gradually decreases through the steps of alcohol extraction followed by water extraction, macroporous resin column separation, graded alcohol precipitation and dialysis. It can be seen from the above-mentioned experimental example data that the activity of extract 4 of the present application (which does not contain small molecule compounds including salidroside) is the best.

[0109] The description of the above embodiments is only used to help understand the method and core idea of ​​the present application. It should be noted that, for ordinary technicians in this technical field, several improvements and modifications can be made to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. Use of a traditional Chinese medicine polysaccharide in preparing a drug for treating ulcerative colitis, characterized in that: The Chinese medicine polysaccharide includes Rhodiola rosea polysaccharide extracted from Rhodiola rosea; The molecular weight range of the Rhodiola rosea polysaccharide is 2.2×10 6 -7.7×10 8 Da; The preparation method of Rhodiola rosea polysaccharide comprises: Step 1: Extraction: extracting the Rhodiola rosea medicinal material with alcohol to obtain alcohol extraction residue and alcohol extraction filtrate; Step 2: removing impurities: extracting the alcohol extraction residue in step 1 with water to obtain a water extract; Step 3: Enzymatic hydrolysis: passing the aqueous extract in step 2 through a column and eluting, collecting and combining the effluent and the eluate, and concentrating to obtain a concentrate; Step 4: alcohol precipitation: enzymatically hydrolyze the concentrated solution in step 3 and take the supernatant; Step 5: Dialysis and drying: The supernatant in step 4 is subjected to alcohol precipitation, and the precipitate is collected, dialyzed, and freeze-dried to obtain the Chinese medicine polysaccharide.

2. The use according to claim 1, characterized in that: The Rhodiola rosea is Rhodiola rosea.

3. The use according to claim 1, characterized in that: The alcohol extraction uses 60vol%-95vol% ethanol aqueous solution.

4. The use according to claim 1, characterized in that: The column and elution are as follows: the water extract is cooled to room temperature, passed through a macroporous resin column, and eluted with 2BV pure water; The volume ratio of the water extract to the macroporous resin is 1:(1-3); The sample flow rate is 2-3 BV / h; The concentration is to concentrate to a crude drug concentration of 1 g / mL; The macroporous resin model is selected from one of D312, LSA-30, LX-68G, AB-8 and D101.

5. The use according to claim 1, characterized in that: The enzymatic hydrolysis comprises adding one of papain, pepsin, trypsin and endopeptidase, performing enzymatic hydrolysis at 37-60° C. for 1-1.5 hours, cooling and centrifuging to obtain a supernatant; Wherein, the enzyme dosage of the enzymolysis is 0.0004-0.001g / g crude drug amount.

6. The use according to claim 1, characterized in that: The alcohol precipitation comprises: adjusting the ethanol concentration to 40% in the first alcohol precipitation and adjusting the ethanol concentration to 80% in the second alcohol precipitation.

7. The use according to claim 1, characterized in that: The dialysis comprises: washing the precipitate twice with ethanol, adding an appropriate amount of water to redissolve, filtering, transferring the filtrate to a dialysis bag, dialysis, and concentrating the retentate to a crude drug concentration of 1 g / mL; Wherein, the particle size of the dialysis bag is 3500Da-90000Da.

8. The use according to claim 7, characterized in that: The particle size of the dialysis bag is 90000Da.

9. A Chinese medicine preparation for treating ulcerative colitis, characterized in that: The Chinese medicine preparation comprises Rhodiola rosea polysaccharide extracted from Rhodiola rosea; The molecular weight range of the Rhodiola rosea polysaccharide is 2.2×10 6 -7.7×10 8 Da; The preparation method of Rhodiola rosea polysaccharide comprises: Step 1: Extraction: extracting the Rhodiola rosea medicinal material with alcohol to obtain alcohol extraction residue and alcohol extraction filtrate; Step 2: removing impurities: extracting the alcohol extraction residue in step 1 with water to obtain a water extract; Step 3: Enzymatic hydrolysis: passing the aqueous extract in step 2 through a column and eluting, collecting and combining the effluent and the eluate, and concentrating to obtain a concentrate; Step 4: alcohol precipitation: enzymatically hydrolyze the concentrated solution in step 3 and take the supernatant; Step 5: Dialysis and drying: The supernatant in step 4 is subjected to alcohol precipitation, and the precipitate is collected, dialyzed, and freeze-dried to obtain the Rhodiola rosea polysaccharide.

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