Prunella vulgaris polysaccharide, preparation method and application in medicine for treating ulcerative colitis

Abstract

The present application discloses a Prunella vulgaris polysaccharide, a preparation method and an application in a drug for treating ulcerative colitis. A Prunella vulgaris polysaccharide is obtained by extracting Prunella vulgaris, graded alcohol precipitation, depigmentation, deproteinization and dialysis. The results of animal experiments show that in a mouse ulcerative colitis mucosal recovery model, the Prunella vulgaris polysaccharide of the present application can significantly improve the inflammatory damage of the colon, effectively reduce the inflammatory factors in the colon, and promote the repair of DSS-induced mouse ulcerative colitis mucosal damage. The Prunella vulgaris polysaccharide can be used to treat ulcerative colitis.

Description

Technical Field

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

[0002] Prunella vulgaris L. is a perennial herbaceous plant of the Labiatae family. The Pharmacopoeia of the People's Republic of my country stipulates that the medicinal part of Prunella vulgaris is the fruit spike. Because it withers at the end of summer, it is named "Prunella vulgaris". Prunella vulgaris is a traditional Chinese medicinal material with a long history of use. It first appeared in the Shennong's Herbal Classic, which listed it as a low-grade product. It tastes bitter and pungent, is cold in nature, and belongs to the liver and gallbladder meridians. It has the effects of clearing the liver and purging fire, improving eyesight, dispersing stagnation and reducing swelling. It is used for red and swollen eyes, night pain in the eyeballs, headache and dizziness, scrofula, goiter, mastitis, mastitis, and breast pain.

[0003] 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.

[0004] Studies have shown that Prunella vulgaris contains terpenes, phenolic acids, flavonoids, sterols, coumarins, organic acids, volatile oils and polysaccharides, which have multiple pharmacological effects such as lowering blood pressure, lowering blood sugar, antibacterial and anti-inflammatory, immunosuppressive, scavenging free radicals, anti-oxidation, anti-tumor, and inhibiting virus growth. Among them, polysaccharides, as one of the main active ingredients of Prunella vulgaris, have multiple functional activities, mainly concentrated in anti-oxidation, immunomodulation, anti-inflammatory, anti-viral, anti-fibrosis, anti-cancer and anti-complement. The main components of Prunella vulgaris for the treatment of other diseases still need to be studied. The different growth environment, processing, extraction and purification methods of Prunella vulgaris will cause differences in the content, molecular weight, monosaccharide composition and molar ratio, connection position and order of the obtained polysaccharides, thereby affecting the biological activity of the polysaccharides and their activity strength. Summary of the invention

[0005] In order to solve the above problems existing in the art, the present application aims to provide a Prunella Vulgaris polysaccharide and a preparation method thereof and application thereof in a drug for treating ulcerative colitis.

[0006] According to one aspect of the present application, there is provided a use of Prunella vulgaris polysaccharide in a drug for treating ulcerative colitis, wherein the Prunella vulgaris polysaccharide is extracted from Prunella vulgaris.

[0007] According to another aspect of the present application, the molecular weight range of the above-mentioned Prunella vulgaris polysaccharide is: 2.78×10 5 -2.5×10 9 .

[0008] According to one aspect of the present application, a method for preparing the above-mentioned Prunella vulgaris polysaccharide is also provided, comprising:

[0009] Step 1: Extraction: extracting the herb Prunella vulgaris with water to obtain a water extract;

[0010] Step 2: impurity removal: pass the water extract obtained in step 1 through a macroporous resin column, elute with pure water, combine the effluent and the water wash, and concentrate to 1 g / mL crude drug amount; let it stand, discard the precipitate, and take the supernatant for later use;

[0011] Step 3: alcohol precipitation: subjecting the supernatant obtained in step 2 to alcohol precipitation to obtain a precipitate;

[0012] Step 4: Dialysis: Wash the precipitate obtained in step 3 with ethanol and then redissolve it in water to obtain a reconstituted solution, filter the reconstituted solution, dialyze the filtrate, and concentrate the retentate to 1 g / mL crude drug;

[0013] Step 5 Drying: freeze-dry the retentate obtained in step 4 to obtain the Prunella vulgaris polysaccharide.

[0014] In some embodiments, water extraction comprises: adding 8-16 times the amount of water, decocting and extracting 2-3 times, each time for 1-2 hours.

[0015] In some embodiments, the alcohol precipitation comprises: adding 70%-95% ethanol to the supernatant, standing overnight, and collecting the precipitate.

[0016] In some embodiments, the model of the macroporous resin column is selected from D101 / HP20 macroporous resin column.

[0017] In some embodiments, the dialysis bag particle size is 3500Da-100000Da.

[0018] In some embodiments, the dialysis bag particle size is 100,000 Da.

[0019] In some embodiments, the ethanol concentration in the alcohol precipitation is 80%.

[0020] The present application also provides a Chinese medicine preparation for treating ulcerative colitis, comprising the aforementioned Prunella vulgaris polysaccharide, wherein the Prunella vulgaris polysaccharide is the only effective ingredient in the Chinese medicine preparation.

[0021] In some embodiments, the Chinese medicine preparation further comprises other active ingredients and / or drugs, wherein the other active ingredients comprise nutritional supplements.

[0022] In some embodiments, the Chinese medicine preparation can be a capsule, a pill, a powder, a syrup, a tablet, a granule, a plaster, a mixture, a pill, a suppository, an aerosol, an ointment or an injection.

[0023] Use of the above-mentioned traditional Chinese medicine preparation in the preparation of a drug for preventing and / or treating ulcerative colitis.

[0024] Compared with the prior art, this application has at least the following beneficial effects:

[0025] The present application provides an application of Prunella vulgaris polysaccharide in a drug for treating ulcerative colitis. The present application finds that Prunella vulgaris polysaccharide can significantly improve inflammatory damage in the colon, effectively reduce inflammatory factors in the colon, and promote the repair of mucosal damage in DSS-induced ulcerative colitis in mice, and can be used to treat ulcerative colitis.

[0026] Compared with other extracts in Prunella vulgaris, the Prunella vulgaris polysaccharide of the present application has a better therapeutic effect and less side effects at the same dosage. The Prunella vulgaris polysaccharide of the present application has diverse targets, is not prone to drug resistance, and has no adverse reactions or fewer adverse reactions.

[0027] The preparation method of Prunella vulgaris 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

[0028] Figure 1 This is a molecular weight distribution diagram of Prunella vulgaris polysaccharide in an exemplary embodiment of this application.

[0029] Figure 2 This is an example of the effect of each experimental group on the body weight of mice in the implementation of this application.

[0030] Figure 3 This is the effect of each experimental group on DAI in the example implementation of this application.

[0031] Figure 4 This is an example of the effect of each experimental group on colon length in the implementation of this application.

[0032] Figure 5 These are pictures of the colon of each experimental group in the exemplary implementation of this application.

[0033] Figure 6 Pathological sections of each experimental group in the exemplary implementation of this application. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] 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.

[0037] The selection scope of "and / or" and "or / and" in this application includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the said any and all combinations include any combination of any two related listed items, any more related listed items, or all related listed items.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The application is described in detail below.

[0042] This application is a study on Prunella vulgaris in order to find an active ingredient that is effective in treating ulcerative colitis.

[0043] During the experiment, it was found that the polysaccharide part of Prunella vulgaris had a good therapeutic effect; and the process and composition of this extract were further studied. Different extracts were obtained through water extraction, macroporous resin impurity removal, graded alcohol precipitation, enzymatic hydrolysis and dialysis. The water extract was concentrated and dried to obtain the water extract part of Prunella vulgaris. The macroporous resin was eluted with water and then eluted with 95% ethanol. The 95% eluate was collected, concentrated and dried to obtain the 95% elution part of the macroporous resin of Prunella vulgaris. After alcohol precipitation and dialysis, the polysaccharide part of Prunella vulgaris was dried. The physical and chemical properties of the polysaccharide part of Prunella vulgaris were measured, and the molecular weight range was: 2.78×10 5 -2.5×10 9 .

[0044] The present application provides a Prunella vulgaris polysaccharide and a preparation method and application thereof, for early treatment or intervention of the occurrence and development of ulcerative colitis and reduction of side effects.

[0045] This application proposes the use of Prunella vulgaris 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 Prunella vulgaris polysaccharide part of this application has a good therapeutic effect on ulcerative colitis.

[0046] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0047] The Herba Prunellae used in the examples is Herba Prunellae large-flowered, and the medicinal materials, reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0048] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0049] Example 1

[0050] Step 1: Extraction: Add 8 times the amount of water to the herb, decoct and extract twice, each time for 2 hours, to obtain a water extract; take one third of the water extract and concentrate and dry it.

[0051] Step 2: Impurity removal: The water extract obtained in step 1 is cooled to room temperature, passed through a HP20 macroporous resin column, eluted with 2BV pure water, the effluent and the water washing liquid are collected and combined, concentrated to 1 g / mL crude drug amount, allowed to stand overnight, the precipitate is discarded, and the supernatant is taken for later use; the macroporous resin column is continuously eluted with 95% ethanol, the eluate is collected, and concentrated to dryness.

[0052] Step 3: Alcohol precipitation: Add ethanol to the supernatant obtained in step 2, adjust the ethanol concentration to 80%, let it stand overnight, and collect the precipitate.

[0053] Step 4: Dialysis: Wash the precipitate obtained in step 3 twice with ethanol, add appropriate amount of water to re-dissolve, filter, and place the filtrate in dialysis bags with particle sizes of 3500 Da, 50000 Da, and 100000 Da, respectively, dialyze, and concentrate the retentate to 1 g / mL crude drug;

[0054] Step 5 Drying: The concentrated solution obtained in step 4 was pre-frozen at -30°C and freeze-dried to obtain extract 1 (3500 Da), extract 2 (50000 Da), and extract 3 (100000 Da).

[0055] Experimental example

[0056] 1. Molecular Weight Determination of Extract 3 in Example 1

[0057] Prunella vulgaris polysaccharide was measured by HPGPC. According to the Shodex STANDARD P-82 standard, the molecular weight retention time standard curve was log (Mw) = -0.4017T + 9.739, R² = 0.998. Figure 1 As shown, the calculated molecular weight distribution of extract 3 is 2.78×10 5 -2.5×10 9 Da.

[0058] II. Therapeutic effects of Prunella Vulgaris extracts 1-3 in Example 1 on mice with ulcerative colitis induced by dextran sulfate sodium salt

[0059] 1. Experimental Materials

[0060] 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. The breeding environment was clean, the ambient temperature was controlled at 23±2°C, 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.

[0061] Drugs and reagents: Dextran sulfate sodium salt (Shanghai McLean Biochemical Technology Co., Ltd.); Sulfasalazine (Shanghai Fuda Pharmaceutical Co., Ltd.)

[0062] 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)

[0063] 2. Dosage design

[0064] The dose for mice was 200 mg / kg / day for extract 1, 200 mg / kg / day for extract 2, and 200 mg / kg / day for extract 3.

[0065] Based on the body surface area conversion method, the equivalent dose of sulfasalazine for mice is 100 mg / kg / day.

[0066] 3. Experimental Methods

[0067] After the adaptive feeding of mice, they were randomly divided into 6 groups according to their body weight, with 10 mice in each group, namely 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) and positive drug group (sulfasalazine, 100 mg / kg / day). Except for the blank control group, the other 5 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 establish the UC model.

[0068] The weight of mice was recorded daily, and the disease activity index (DAI) was scored for mice. The drugs were administered while the model was being built. Each extract was administered by gavage. The blank control group and the model control group were gavaged with pure water, and the other groups were gavaged 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 the 9th day, blood was collected from the mice after anesthesia and centrifuged at 3000 rpm for 15 minutes. Serum was obtained and stored at -80°C for testing and analysis of inflammatory indicators.

[0069] Inflammatory markers included interleukin-6 (IL-6) and myeloperoxidase (MPO). Colonic tissues were 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 immediately frozen at -80°C for intestinal microbiota analysis. The distal colon sections were fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin (H&E), and tissue morphological changes and histological evaluation were observed using light microscopy.

[0070] GraphPad Prism 9 software was used to draw graphs for analysis of differences among the groups.

[0071] 4. Experimental Results

[0072] (1) Evaluation of drug efficacy in DSS-induced UC mice

[0073] After DSS induction, the body weight of mice decreased significantly. However, treatment with extract 3 reversed this loss (Table 1, Figure 2 ). The weight loss rate of mice (%) = (weight on the first day (g) - weight on the tenth day (g)) / (weight on the first day (g)) × 100%; DSS can significantly increase the DAI score, while extract 3 can improve this phenomenon, indicating that it has a certain alleviating effect on ulcerative enteritis (Table 2, Figure 3 ).

[0074] 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 1, Extract 2 and Extract 3 alleviated the shortening of colon length to varying degrees, with Extract 3 having the best effect ( Figure 4 and Figure 5 ).

[0075] The comprehensive results show that Extract 3, i.e. the Prunella Vulgaris polysaccharide of the present application, has the best effect on the recovery of ulcerative colitis.

[0076] Table 1 Effects of each experimental group on the body weight of mice

[0077]

[0078] Table 2 Effects of each experimental group on DAI scores of mice

[0079]

[0080] in, Figure 2-4 middle # Compared with the Control group, there is a statistical difference. ## Indicates significant difference compared with the Control group. ### Indicates that the difference compared with the Control group is extremely significant; * It indicates that there is a statistical difference compared with the Model group. ** Indicates that there is a significant difference compared with the Model group. *** Indicates that the difference compared with the Model group is extremely significant.

[0081] Compared with the blank group, the integrity of the intestinal mucosal epithelium of the model group mice was destroyed after modeling, the goblet cells decreased, and a large number of inflammatory cells aggregated into clusters, invading the muscle layer and serosal layer; compared with the model group, the colon lesions of each drug-treated group were improved, the crypt structure was restored to a certain extent, and the infiltration range of inflammatory cells in extract 3 was smaller ( Figure 6 ).

[0082] According to the above experimental data, the present application has discovered that the DSS-induced mouse ulcerative colitis model constructed by Prunella vulgaris polysaccharide can effectively improve the levels of gold indicators such as mouse weight, feces score, colon length and pathological damage. The Prunella vulgaris polysaccharide part of the present application has a good therapeutic effect on ulcerative colitis.

[0083] 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. A use of Prunella vulgaris polysaccharide in the preparation of a drug for treating ulcerative colitis, characterized in that: The Prunella Vulgaris polysaccharide is extracted from Prunella Vulgaris; The molecular weight range of the Prunella vulgaris polysaccharide is: 2.78×10 5 -2.5×10 9 ; Wherein, the preparation method of the Prunella vulgaris polysaccharide comprises: Step 1: Extraction: extracting the herb Prunella vulgaris with water to obtain a water extract; Step 2: impurity removal: pass the water extract obtained in step 1 through a macroporous resin column, elute with pure water, combine the effluent and the water washing, and concentrate to 1 g / mL crude drug amount; let it stand overnight, discard the precipitate, and take the supernatant for later use; Step 3: alcohol precipitation: subjecting the supernatant obtained in step 2 to alcohol precipitation to obtain a precipitate; Step 4: Dialysis: Wash the precipitate obtained in step 3 with ethanol and then redissolve it in water to obtain a reconstituted solution, filter the reconstituted solution, dialyze the filtrate, and concentrate the retentate to 1 g / mL crude drug; Step 5 Drying: freeze-dry the retentate obtained in step 4 to obtain the Prunella vulgaris polysaccharide.

2. The use according to claim 1, characterized in that: The water extraction comprises: adding 8-16 times the amount of water, decocting and extracting 2-3 times, each time for 1-2 hours.

3. The use according to claim 1, characterized in that: The alcohol precipitation comprises: the concentration of the supernatant is 70%-95% ethanol, standing overnight, and collecting the precipitate.

4. The use according to claim 3, characterized in that: The model of the macroporous resin column is selected from D101 / HP20 macroporous resin column.

5. The use according to claim 1, characterized in that: The particle size of the dialysis bag used for dialysis is 3500Da-100000Da.

6. The use according to claim 5, characterized in that: The particle size of the dialysis bag is 100000Da.

7. The use according to claim 3, characterized in that: The ethanol concentration in the alcohol precipitation is 80%.

8. A Chinese medicine preparation for treating ulcerative colitis, characterized in that: Including Prunella Vulgaris polysaccharides; Wherein, the Prunella Vulgaris polysaccharide is extracted from Prunella Vulgaris; The molecular weight range of the Prunella vulgaris polysaccharide is: 2.78×10 5 -2.5×10 9 ; Wherein, the preparation method of the Prunella vulgaris polysaccharide comprises: Step 1: Extraction: extracting the herb Prunella vulgaris with water to obtain a water extract; Step 2: impurity removal: pass the water extract obtained in step 1 through a macroporous resin column, elute with pure water, combine the effluent and the water washing, and concentrate to 1 g / mL crude drug amount; let it stand overnight, discard the precipitate, and take the supernatant for later use; Step 3: alcohol precipitation: subjecting the supernatant obtained in step 2 to alcohol precipitation to obtain a precipitate; Step 4: Dialysis: Wash the precipitate obtained in step 3 with ethanol and then redissolve it in water to obtain a reconstituted solution, filter the reconstituted solution, dialyze the filtrate, and concentrate the retentate to 1 g / mL crude drug; Step 5 Drying: freeze-dry the retentate obtained in step 4 to obtain the Prunella vulgaris polysaccharide.

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