Preparation method of composition for treating ulcerative colitis

Abstract

The invention discloses a method for preparing a composition for treating ulcerative colitis, and relates to a method for preparing a composition for treating ulcerative colitis. The present invention is to solve the problem that the existing method for treating ulcerative colitis is difficult to reach the lesion site intact. Method: one, carbonization of a mixture of sanguisorba officinalis and black plum; two, extraction and purification of the carbonized product; three, preparation of palm charcoal-purslane; four, preparation of gallnut and pomegranate peel fermented extract; five, preparation of gel micropills; six, preparation of gallnut and pomegranate peel fermented product colon-localized micropills; mixing the gel micropills with gallnut and pomegranate peel fermented product colon-localized micropills, to obtain. In an in vitro release test, the double colon micropills prepared in the present application can basically not release in an environment of simulated gastric acid for 2h, release less in an environment of simulated small intestinal fluid for 3h, only about 6%, and can release more than 90% in an environment of simulated colon fluid for 7h. The present invention belongs to the field of preparation of drugs for treating ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to a preparation method of a composition for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a common gastrointestinal inflammatory disease with complex etiology and pathogenesis. Lesions are often located in the sigmoid colon and rectum, and in severe cases can extend to the descending colon or the entire colon. Key symptoms include abdominal pain, diarrhea, thick mucus, bloody stools, and tenesmus. The disease primarily affects the intestinal mucosa and submucosa, often in the sigmoid colon and rectum. Relapses are common and the disease course is long, significantly impacting both the patient's physical and mental health and financial well-being. In recent years, with the increasing research on traditional Chinese medicine (TCM), the potential role of TCM in the treatment of gastrointestinal diseases has been widely recognized worldwide. During the course of UC, localized colonic tissue damage, exogenous pathogenic factors, and endogenous factors can lead to significant immune abnormalities in patients. These include the infiltration of numerous immune cells into the intestinal mucosa; fluctuations in proinflammatory cytokines such as IL-18, IL-2, IL-1β, IL-5, and TNF-α; and a significant increase in T and B lymphocytes, monocytes, and natural killer (NK) cells. These antibodies, cytokines and inflammatory mediators released into the intestinal mucosal tissue trigger severe reactions around the intestine, directly destroying the intestinal mucosal epithelial tissue and causing long-term inflammatory lesions.

[0003] Tannins constrict wound capillaries and can bind to proteins in wound exudates to form macromolecules, causing proteins on the surface of wound tissue to coagulate, reducing secretion and plasma exudation. They can also form lumps at damaged blood vessels, exerting a hemostatic effect. Therefore, tannins are a key component of traditional Chinese medicine for hemostasis. Traditional Chinese medicine charcoal has a proven astringent and hemostatic effect. More importantly, it has also been shown to possess multiple benefits, including anti-inflammatory, sedative, and analgesic properties. Black plums are the dried, nearly ripe fruit of the Rosaceae plant, Prunus mume (Sieb) Sibe. et Zucc. It is sour, astringent, and neutral in nature, and enters the liver, spleen, lung, and large intestine meridians, boasting lung astringency, intestinal astringency, fluid production, and anti-intestinal properties. Black plum charcoal is made from processed black plums. The black plums are placed in a stir-fry container and heated over high heat until the skin and flesh swell and the surface turns black. Remove and cool to create black plum charcoal. It has a sour, slightly astringent flavor and a neutral nature. It enters the liver, spleen, lung, stomach, and large intestine meridians, and has the effects of astringing the lungs and relieving coughs, astringing the intestines and relieving diarrhea, promoting fluid production and quenching thirst, and stopping bleeding and relieving pain. Sanguisorba officinalis is a plant in the Rosaceae family and is the dried root of Sanguisorba officinalis or Sanguisorba longifolia. It has the effects of cooling blood, stopping bleeding, and detoxifying and healing sores. The purpose of carbonization is primarily to enhance or produce this hemostatic effect.

[0004] Palm is the dried petiole of Trachycarpus fortunei (HooK.f) H.Wendl, a palm plant of the Arecaceae family. When harvesting palm, cut off the lower part of the old petiole and the sheath, remove the fibrous palm hair, and dry it in the sun. This product has a mild medicinal property, bitter and astringent taste, and is an important medicine for astringency and hemostasis. Due to its strong astringency, it is suitable for treating bleeding without blood stasis. Sanguisorba officinalis charcoal (SRC) is the product of high-temperature carbonization of Sanguisorba officinalis. It has the effects of cooling blood and stopping bleeding, detoxifying and healing sores. It is clinically used to treat blood diseases, as well as pain caused by various diseases such as burns caused by water and fire, carbuncles, sores, etc., with a definite effect. Portulaca oleracea is a plant of the Portulacaceae family, Portulaca oleracea L., and the whole plant is used as medicine. It has the effects of clearing heat and dampness, cooling blood and detoxifying.

[0005] The oral colon-targeted targeting drug delivery system (OCTDDS) refers to a new type of drug delivery system that, after oral administration, does not release the drug in the upper gastrointestinal tract, allowing the drug to pass smoothly through the stomach and small intestine and reach the ileocecal region or colon before releasing the drug to exert local or systemic therapeutic effects. It can be divided into several types, including pH-dependent, time-delay-dependent, enzyme-triggered, and bioadhesive types. Its application in the treatment of colonic diseases has broad significance.

[0006] Hydrogels are a type of gel with a three-dimensional network structure composed of hydrophilic polymer chains held together by crosslinks. They swell rapidly in water and can retain a large volume of water in this swollen state. Sodium alginate [C6H7O6Na]n is a byproduct of the extraction of iodine and mannitol from brown algae, such as Sargassum or kelp. It is a linear natural polysaccharide polymer composed of β-1,4-D-mannuronic acid (M) units and α-1,4-L-guluronic acid (G) units.

[0007] Since the lesions of ulcerative colitis are mainly in the colon, with the sigmoid colon being the most common, its pathological manifestation is that the intestinal mucosa is infiltrated by inflammatory cells such as neutrophils for a long time, leading to epithelial cell apoptosis, thereby causing damage to the intestinal mucosa and further causing bleeding and other phenomena. Therefore, the key to treatment is to accurately deliver the drug to the lesion site, prolong the drug retention time, and repair the ulcer surface. Clinically, suppositories or enemas are mainly used. Although the efficacy is significant, patient compliance is not high. Conventional ordinary oral preparations lack the colon positioning characteristics and it is difficult to reach the lesion site intact. Even if they can reach the site, the remaining amount of the drug is very little, and it is difficult to achieve the treatment purpose. Summary of the Invention

[0008] The purpose of the present invention is to solve the technical problems of low patient compliance and difficulty in reaching the lesion site intact in existing methods for treating ulcerative colitis, and to provide a method for preparing a composition for treating ulcerative colitis.

[0009] Preparation method of a composition for treating ulcerative colitis:

[0010] 1. Carbonization of the mixture of Sanguisorba officinalis and Plum

[0011] Weigh 100.0 g of dried raw Sanguisorba officinalis and 100 g of dried plum, then place the raw Sanguisorba officinalis and plum in a clean crucible, cover the crucible mouth with clean tin foil, seal it with a lid, and place it stably in a muffle furnace. Set the muffle furnace to two heating gradients: the first gradient is to heat to 80 ° C within 10 min and hold for 35 min; the second gradient is to heat to 260 ° C-380 ° C within 45 min and hold for 1.5 h, and collect the carbonized mixture powder of Sanguisorba officinalis and plum;

[0012] 2. Extraction and purification of carbonized products

[0013] Weigh 100 g of the carbonized mixture powder of Sanguisorba officinalis and Ebony plum, add 8 to 10 times the weight of the carbonized mixture powder of Sanguisorba officinalis and Ebony plum, place in a 1L beaker to soak, then add silica, stir evenly with a glass rod, and let stand for 30 minutes to obtain a mixture with a silica concentration of 0.1%;

[0014] Ultrasonic extraction was performed twice at 65°C, each extraction time was 35 min, and the extract was filtered through gauze to remove larger particles. The combined filtrate was collected and finally the filtrate was filtered through a 0.45 μm microporous membrane under negative pressure for three times to obtain the aqueous extract.

[0015] The aqueous extract was concentrated using a rotary evaporator, and then placed in a dialysis bag with a molecular weight cutoff of 3000 Da and sealed for dialysis. The dialysis fluid was deionized water, and the dialysis time was not less than 96 hours, until the solution outside the bag was colorless and transparent. During this period, the dialysis bag was changed every 6 hours to ensure that the small molecule residues in the dialysis bag were completely exuded. The exudate solution was taken out and then centrifuged on a rotary evaporator at 14000 r / min for 15 minutes to obtain a supernatant. The supernatant was freeze-dried to obtain a freeze-dried powder of a carbonized Sanguisorba officinalis and Prunus mume mixture;

[0016] 3. Preparation of Palm Charcoal-Purslane

[0017] Take 100g of purslane, add 8-10 times the weight of ethanol, reflux extract twice, combine the filtrate, and rotate until there is no alcohol smell to obtain the purslane extract;

[0018] Soak 150g of clean palm in purslane extract for 6 hours, place it in a pot, put a smaller pot on top of the pot, seal the joint of the two pots with yellow mud, and press a heavy object on the smaller pot. Heat it over high heat until it is thoroughly calcined. After cooling, take it out, cut it into small pieces, put it in a hot pot, and stir-fry it over high heat until the surface is dark brown and the inside is burnt brown. Spray it with clean water to extinguish sparks, take it out, spread it out in time to cool thoroughly, and you will get palm charcoal powder.

[0019] 100 g of palm charcoal crude powder was extracted with 95% ethanol under reflux for 2 h, and the residue was extracted with 75% ethanol for 2 h. The filtrate was filtered while hot and combined, extracted with ethyl acetate, and the filtrate was concentrated by rotary evaporation to remove residual ethanol, and freeze-dried to obtain palm charcoal freeze-dried powder;

[0020] Preparation of Gallnut and Pomegranate Peel Fermented Extracts

[0021] Take 50g of gallnut, dry in an oven at 60℃ for 4h, grind and pass through a 40-mesh sieve to obtain gallnut powder;

[0022] Take 50g of pomegranate peel, dry it in an oven at 60℃ for 6h, and grind it through a 40-mesh sieve to obtain pomegranate peel powder;

[0023] On a sterile operating table, use a loop to dip frozen bacterial suspensions of Lactobacillus rhamnosus, Lactobacillus plantarum, and Bacillus coagulans, streak inoculate onto MRS solid medium, and incubate in a constant temperature incubator for 24 hours. Then, pick colonies and inoculate onto MRS liquid medium. Subculture continuously for 3 days to obtain a mixed bacterial strain, which is stored at 4°C for later use.

[0024] The activated mixed bacteria were inoculated into a 500 mL Erlenmeyer flask containing gallnut powder, pomegranate peel powder, and 300 mL of fermentation liquid medium at a 3% inoculum rate, and cultured in a shaking incubator at 35°C and 180 rpm for 48 hours to obtain a fermentation liquid and a fermentation product of the gallnut and pomegranate peel;

[0025] The fermentation product was extracted with 75% ethanol at a solid-liquid ratio of 1:8, each extraction was 4 hours, and a total of 2 extractions were performed. The combined extracts were freeze-dried to obtain a fermentation product extract;

[0026] 5. Preparation of Gel Micropellets

[0027] The carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder are mixed to obtain a mixture of the carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder, and the mixture is added to an oil phase, wherein each gram of the oil phase contains 5 mg of the carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder. After vortexing, the mixture is sonicated for 20 to 25 minutes to dissolve the mixture, thereby obtaining an oil phase containing a charcoal powder extract;

[0028] The mass ratio of the carbonized Sanguisorba officinalis and Prunus mume mixture freeze-dried powder to the palm charcoal freeze-dried powder is 1:1, and the oil phase is composed of propylene glycol monocaprylate (Capryol 90), corn oil and caprylic / capric triglyceride in a mass ratio of 1:0.5:0.05;

[0029] Heat the emulsifier in an oven at 70°C, and after it is completely melted, place it in a small beaker, add an equal amount of co-emulsifier polyethylene glycol 600 (PEG-600), and use a cell disruptor to disrupt the cells for 60 seconds to obtain a mixed emulsifier.

[0030] The emulsifier is composed of polyethylene glycol 15-hydroxystearate (Kolliphor HS15) and Tween 20 (Tween 20) in a mass ratio of 1:1;

[0031] Adding 1.5% oleic acid to the oil phase containing the charcoal powder extract, stirring and mixing with a magnetic stirrer for 5 minutes, adding a mixed emulsifier constant at 30°C, continuing stirring for 30 minutes, and then disrupting the cells with a cell disrupter for 60 seconds, and then adding distilled water dropwise on a magnetic stirrer constant at 30°C, wherein the amount of distilled water added is 35.5% of the total weight of oleic acid, the oil phase containing the charcoal powder extract and the distilled water, to obtain a charcoal powder extract mixture nanoemulsion;

[0032] Weigh 0.5 g of sodium alginate and dissolve it in 30 mL of deionized water. Stir at room temperature to obtain a uniform sodium alginate solution.

[0033] Weigh 0.05 g of sodium hyaluronate solution and dissolve it in 10 mL of deionized water. After sufficient swelling, a sodium hyaluronate solution is obtained.

[0034] Weigh 0.2 g of Carbomer 940, dissolve it in 20 mL of deionized water, and place it at room temperature for 24 hours until it is fully swollen to obtain a Carbomer solution.

[0035] The sodium alginate solution, the sodium hyaluronate solution and the carbomer solution were mixed in a mass ratio of 1:0.2:1 and stirred evenly to obtain a mixed gel solution;

[0036] The charcoal powder extract mixture nanoemulsion was added to the mixed gel solution, wherein the mass ratio of the charcoal powder extract mixture nanoemulsion to the mixed gel solution was 1:1.5, and the mixture was first disrupted by a cell disruptor for 30 seconds at room temperature, and magnetically stirred at room temperature for 30 minutes until uniformly mixed. The mixture was then added dropwise to a mixed solution of CaCl2 and CaSO4 using a 5 mL syringe with a 0.45 mm needle and stirred for 15 minutes. The cross-linked nanoemulsion / gel complex was collected by filtration, rinsed with distilled water to remove the unreacted mixed solution of CaCl2 and CaSO4, and then freeze-dried to obtain gel pellets.

[0037] The mass concentrations of CaCl2 and CaSO4 in the mixed solution of CaCl2 and CaSO4 are 5% respectively;

[0038] The carbonized Sanguisorba officinalis and Prunus mume freeze-dried powder mixture and the palm charcoal freeze-dried powder mixture account for 22% of the gel micropill mass;

[0039] The emulsifier accounts for 40.0% of the mass of the gel pellets;

[0040] The mass concentration of 1.5% oleic acid accounts for 2.5% of the mass of the gel pellets;

[0041] Preparation of colon-targeted micropellets from the fermentation products of gallnut and pomegranate peel

[0042] Drug layer:

[0043] Add the fermented liquor of gallnut and pomegranate peel prepared in step 4 to water, with the mass ratio of the fermented liquor of gallnut and pomegranate peel to water being 3:7, and dissolve by ultrasonication to prepare a gallnut and pomegranate peel fermented aqueous solution;

[0044] Preparation of time-lag coating solution:

[0045] The fermentation extract prepared in step 4 was dispersed in an acetic acid aqueous solution (EC-PEG4000) with a mass concentration of 1.5% and a temperature of 60°C, wherein the mass ratio of ethyl cellulose to acetic acid in the acetic acid aqueous solution was 3:1, and stirred until fully hydrated. Then, plasticizer triethyl citrate, micropowder silica gel, and fumed silica were added in sequence and stirred continuously to obtain a time-lag layer coating solution;

[0046] The acetic acid aqueous solution is a coating solution and accounts for 7% of the weight of the gallnut and pomegranate peel fermented product colon-targeted micropills;

[0047] The triethyl citrate accounts for 15% of the weight of the time-lag layer coating solution;

[0048] The micro powder silica gel accounts for 10% of the weight of the time lag layer coating solution;

[0049] The fumed silica accounts for 2% of the weight of the time-lag layer coating solution;

[0050] Preparation of pH-dependent outer coating solution:

[0051] Under magnetic stirring, methacrylic acid, methyl acrylate and methyl methacrylate copolymer (FS30D), methacrylic acid and methyl methacrylate copolymer (Eudragit S100), and ethyl cellulose (EC) were added to ethanol and completely dissolved, and then triethyl citrate and talc were added in sequence and stirred thoroughly to obtain a pH-dependent outer coating solution;

[0052] The methacrylic acid and methyl methacrylate copolymer is prepared from methacrylic acid and methyl methacrylate in a mass ratio of 1:2;

[0053] The methacrylic acid, methyl acrylate and methyl methacrylate copolymer is prepared from methacrylic acid, methyl acrylate and methyl methacrylate in a mass ratio of 1:1:1;

[0054] The mass ratio of the methacrylic acid, the copolymer of methyl acrylate and methyl methacrylate, the copolymer of methacrylic acid and methyl methacrylate, and the ethyl cellulose is 1:5:1;

[0055] The triethyl citrate accounts for 3% of the mass of the pH-dependent outer coating solution;

[0056] The talc powder accounts for 0.5% of the mass of the pH-dependent outer coating solution;

[0057] The gallnut pomegranate peel fermentation aqueous solution, the time-lag layer coating solution, and the pH-dependent outer layer coating solution are preheated to 40-42°C in a fluidized bed, and then 3 / h, inlet air temperature 60-70°C, atomization pressure 120-140kPa, and spray volume flow rate 4-8mL / min, coating the drug layer, time lag layer, and pH-dependent layer in sequence to obtain gallnut and pomegranate peel fermentation product colon-targeted micropellets;

[0058] The time-delay layer accounts for 7% of the weight of the colon-targeted micropellets of gallnut and pomegranate peel ferment;

[0059] The pH-dependent layer accounts for 20% to 35% of the weight of the colon-targeted micropellets of gallnut and pomegranate peel ferments;

[0060] The gel pellets prepared in step 5 are mixed with the colon-targeted pellets of gallnut and pomegranate peel fermentation product prepared in step 6 in a mass ratio of 1:1 to obtain a composition for treating ulcerative colitis;

[0061] Alternatively, the gel pellets prepared in step five are mixed with the colon-targeted pellets of gallnut and pomegranate peel fermentation product prepared in step six at a mass ratio of 1:2 to obtain a composition for treating ulcerative colitis;

[0062] Alternatively, the gel micropellets prepared in step five are mixed with the colon-targeted micropellets of gallnut and pomegranate peel fermentation product prepared in step six in a mass ratio of 2:1 to obtain a composition for treating ulcerative colitis.

[0063] The clean palm oil in step 3 is obtained by removing impurities from palm oil, washing it and drying it.

[0064] Traditional Chinese medicine (TCM) has a history of thousands of years in my country and has demonstrated its irreplaceable advantages in treating diarrheal illnesses. Compared to antibiotics, TCM has relatively few side effects and exhibits certain antibacterial and anti-inflammatory properties. TCM contains a variety of macromolecular organic compounds, such as cellulose and pectin. During the fermentation process, probiotics produce a variety of extracellular enzymes that hydrolyze the cellulose and pectin in the TCM cell walls, thereby effectively releasing the active ingredients. Many ingredients are difficult to digest and absorb directly after entering the digestive tract and require the influence of intestinal microorganisms to exert their effects. During this process, probiotics ferment TCM to produce a variety of secondary metabolites, promoting the transformation of biomass within the TCM, thereby enhancing the efficacy of the TCM. Furthermore, the combination of TCM and probiotics can better promote the growth and reproduction of probiotics, significantly enhancing their efficacy. Fermentation by probiotics can inhibit pathogens and regulate the intestinal microbiome, achieving rapid therapeutic effects. Studies have shown that fermented TCM preparations alter the composition or molecular structure of the TCM, enabling the full release of its active ingredients. Probiotics have multiple mechanisms of action in improving intestinal barrier function and maintaining homeostasis.

[0065] Bacillus coagulans can reduce multiple toxins in the intestines through four comprehensive mechanisms: acid production, antibacterial activity, decomposition, and adsorption. Bacillus coagulans produces L-lactic acid and acetic acid, organic acids that combine with amines and ammonia in the intestines to form insoluble salts, reducing intestinal absorption of ammonia and protecting the liver.

[0066] Because the mixed extracts of Sanguisorba officinalis, Prunus mume, and palm charcoal powder have high density and poor solubility, the traditional coating process cannot achieve the purpose of complete encapsulation. Therefore, nanoemulsion gel micropellets are used to encapsulate the three charcoal powders.

[0067] Alginate is stable in acidic environments and has increased solubility in alkaline environments. It neither dissolves nor degrades in gastric juice, but slowly dissolves in the colon. Therefore, carbomer and sodium alginate were chosen as the gel material before pelleting.

[0068] This invention combines Prunus mume and Sanguisorba officinalis, breaking away from conventional research approaches for carbonized drugs. The method ultimately selected for preparing the carbonized drug is calcination in a muffle furnace. The carbonized product is then extracted. Silicon dioxide is added during the extraction process for enrichment. After ultrasonic extraction, the product is filtered and dialyzed in a 3000 Da molecular weight cutoff dialysis bag to obtain a small molecule dialyzate. The resulting carbonized mixture is then freeze-dried to obtain a freeze-dried powder.

[0069] In an in vitro release test, the double colon micropellets prepared by the present invention showed that there was basically no release in a simulated gastric acid environment for 2 hours, and less release (only about 6%) in a simulated small intestinal fluid environment for 3 hours, and the release reached more than 90% in a simulated colon fluid environment for 7 hours. DETAILED DESCRIPTION

[0070] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0071] Specific embodiment 1: Preparation method of the composition for treating ulcerative colitis in this embodiment:

[0072] 1. Carbonization of the mixture of Sanguisorba officinalis and Plum

[0073] Weigh 100.0 g of dried raw Sanguisorba officinalis and 100 g of dried plum, then place the raw Sanguisorba officinalis and plum in a clean crucible, cover the crucible mouth with clean tin foil, seal it with a lid, and place it stably in a muffle furnace. Set the muffle furnace to two heating gradients: the first gradient is to heat to 80 ° C within 10 min and hold for 35 min; the second gradient is to heat to 260 ° C-380 ° C within 45 min and hold for 1.5 h, and collect the carbonized mixture powder of Sanguisorba officinalis and plum;

[0074] 2. Extraction and purification of carbonized products

[0075] Weigh 100 g of the carbonized mixture powder of Sanguisorba officinalis and Ebony plum, add 8 to 10 times the weight of the carbonized mixture powder of Sanguisorba officinalis and Ebony plum, place in a 1L beaker to soak, then add silica, stir evenly with a glass rod, and let stand for 30 minutes to obtain a mixture with a silica concentration of 0.1%;

[0076] Ultrasonic extraction was performed twice at 65°C, each extraction time was 35 min, and the extract was filtered through gauze to remove larger particles. The combined filtrate was collected and finally the filtrate was filtered through a 0.45 μm microporous membrane under negative pressure for three times to obtain the aqueous extract.

[0077] The aqueous extract was concentrated using a rotary evaporator, and then placed in a dialysis bag with a molecular weight cutoff of 3000 Da and sealed for dialysis. The dialysis fluid was deionized water, and the dialysis time was not less than 96 hours, until the solution outside the bag was colorless and transparent. During this period, the dialysis bag was changed every 6 hours to ensure that the small molecule residues in the dialysis bag were completely exuded. The exudate solution was taken out and then centrifuged on a rotary evaporator at 14000 r / min for 15 minutes to obtain a supernatant. The supernatant was freeze-dried to obtain a freeze-dried powder of a carbonized Sanguisorba officinalis and Prunus mume mixture;

[0078] 3. Preparation of Palm Charcoal-Purslane

[0079] Take 100g of purslane, add 8-10 times the weight of ethanol, reflux extract twice, combine the filtrate, and rotate until there is no alcohol smell to obtain the purslane extract;

[0080] Soak 150g of clean palm in purslane extract for 6 hours, place it in a pot, put a smaller pot on top of the pot, seal the joint of the two pots with yellow mud, and press a heavy object on the smaller pot. Heat it over high heat until it is thoroughly calcined. After cooling, take it out, cut it into small pieces, put it in a hot pot, and stir-fry it over high heat until the surface is dark brown and the inside is burnt brown. Spray it with clean water to extinguish sparks, take it out, spread it out in time to cool thoroughly, and you will get palm charcoal powder.

[0081] 100 g of palm charcoal crude powder was extracted with 95% ethanol under reflux for 2 h, and the residue was extracted with 75% ethanol for 2 h. The filtrate was filtered while hot and combined, extracted with ethyl acetate, and the filtrate was concentrated by rotary evaporation to remove residual ethanol, and freeze-dried to obtain palm charcoal freeze-dried powder;

[0082] Preparation of Gallnut and Pomegranate Peel Fermented Extracts

[0083] Take 50g of gallnut, dry in an oven at 60℃ for 4h, grind and pass through a 40-mesh sieve to obtain gallnut powder;

[0084] Take 50g of pomegranate peel, dry it in an oven at 60℃ for 6h, and grind it through a 40-mesh sieve to obtain pomegranate peel powder;

[0085] On a sterile operating table, use a loop to dip frozen bacterial suspensions of Lactobacillus rhamnosus, Lactobacillus plantarum, and Bacillus coagulans, streak inoculate onto MRS solid medium, and incubate in a constant temperature incubator for 24 hours. Then, pick colonies and inoculate onto MRS liquid medium. Subculture continuously for 3 days to obtain a mixed bacterial strain, which is stored at 4°C for later use.

[0086] The activated mixed bacteria were inoculated into a 500 mL Erlenmeyer flask containing gallnut powder, pomegranate peel powder, and 300 mL of fermentation liquid medium at a 3% inoculum rate, and cultured in a shaking incubator at 35°C and 180 rpm for 48 hours to obtain a fermentation liquid and a fermentation product of the gallnut and pomegranate peel;

[0087] The fermentation product was extracted with 75% ethanol at a solid-liquid ratio of 1:8, each extraction was 4 hours, and a total of 2 extractions were performed. The combined extracts were freeze-dried to obtain a fermentation product extract;

[0088] 5. Preparation of Gel Micropellets

[0089] The carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder are mixed to obtain a mixture of the carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder, and the mixture is added to an oil phase, wherein each gram of the oil phase contains 5 mg of the carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder. After vortexing, the mixture is sonicated for 20 to 25 minutes to dissolve the mixture, thereby obtaining an oil phase containing a charcoal powder extract;

[0090] The mass ratio of the carbonized Sanguisorba officinalis and Prunus mume mixture freeze-dried powder to the palm charcoal freeze-dried powder is 1:1, and the oil phase is composed of propylene glycol monocaprylate (Capryol 90), corn oil and caprylic / capric triglyceride in a mass ratio of 1:0.5:0.05;

[0091] Heat the emulsifier in an oven at 70°C, and after it is completely melted, place it in a small beaker, add an equal amount of co-emulsifier polyethylene glycol 600 (PEG-600), and use a cell disruptor to disrupt the cells for 60 seconds to obtain a mixed emulsifier.

[0092] The emulsifier is composed of polyethylene glycol 15-hydroxystearate (Kolliphor HS15) and Tween 20 (Tween 20) in a mass ratio of 1:1;

[0093] Adding 1.5% oleic acid to the oil phase containing the charcoal powder extract, stirring and mixing with a magnetic stirrer for 5 minutes, adding a mixed emulsifier constant at 30°C, continuing stirring for 30 minutes, and then disrupting the cells with a cell disrupter for 60 seconds, and then adding distilled water dropwise on a magnetic stirrer constant at 30°C, wherein the amount of distilled water added is 35.5% of the total weight of oleic acid, the oil phase containing the charcoal powder extract and the distilled water, to obtain a charcoal powder extract mixture nanoemulsion;

[0094] Weigh 0.5 g of sodium alginate and dissolve it in 30 mL of deionized water. Stir at room temperature to obtain a uniform sodium alginate solution.

[0095] Weigh 0.05 g of sodium hyaluronate solution and dissolve it in 10 mL of deionized water. After sufficient swelling, a sodium hyaluronate solution is obtained.

[0096] Weigh 0.2 g of Carbomer 940, dissolve it in 20 mL of deionized water, and place it at room temperature for 24 hours until it is fully swollen to obtain a Carbomer solution.

[0097] The sodium alginate solution, the sodium hyaluronate solution and the carbomer solution were mixed in a mass ratio of 1:0.2:1 and stirred evenly to obtain a mixed gel solution;

[0098] The charcoal powder extract mixture nanoemulsion was added to the mixed gel solution, wherein the mass ratio of the charcoal powder extract mixture nanoemulsion to the mixed gel solution was 1:1.5, and the mixture was first disrupted by a cell disruptor for 30 seconds at room temperature, and magnetically stirred at room temperature for 30 minutes until uniformly mixed. The mixture was then added dropwise to a mixed solution of CaCl2 and CaSO4 using a 5 mL syringe with a 0.45 mm needle and stirred for 15 minutes. The cross-linked nanoemulsion / gel complex was collected by filtration, rinsed with distilled water to remove the unreacted mixed solution of CaCl2 and CaSO4, and then freeze-dried to obtain gel pellets.

[0099] The mass concentrations of CaCl2 and CaSO4 in the mixed solution of CaCl2 and CaSO4 are 5% respectively;

[0100] The carbonized Sanguisorba officinalis and Prunus mume freeze-dried powder mixture and the palm charcoal freeze-dried powder mixture account for 22% of the gel micropill mass;

[0101] The emulsifier accounts for 40.0% of the mass of the gel pellets;

[0102] The mass concentration of 1.5% oleic acid accounts for 2.5% of the mass of the gel pellets;

[0103] Preparation of colon-targeted micropellets from the fermentation products of gallnut and pomegranate peel

[0104] Drug layer:

[0105] Add the fermented liquor of gallnut and pomegranate peel prepared in step 4 to water, with the mass ratio of the fermented liquor of gallnut and pomegranate peel to water being 3:7, and dissolve by ultrasonication to prepare a gallnut and pomegranate peel fermented aqueous solution;

[0106] Preparation of time-lag coating solution:

[0107] The fermentation extract prepared in step 4 was dispersed in an acetic acid aqueous solution (EC-PEG4000) with a mass concentration of 1.5% and a temperature of 60°C, wherein the mass ratio of ethyl cellulose to acetic acid in the acetic acid aqueous solution was 3:1, and stirred until fully hydrated. Then, plasticizer triethyl citrate, micropowder silica gel, and fumed silica were added in sequence and stirred continuously to obtain a time-lag layer coating solution;

[0108] The acetic acid aqueous solution is a coating solution and accounts for 7% of the weight of the gallnut and pomegranate peel fermented product colon-targeted micropills;

[0109] The triethyl citrate accounts for 15% of the weight of the time-lag layer coating solution;

[0110] The micro powder silica gel accounts for 10% of the weight of the time lag layer coating solution;

[0111] The fumed silica accounts for 2% of the weight of the time-lag layer coating solution;

[0112] Preparation of pH-dependent outer coating solution:

[0113] Under magnetic stirring, methacrylic acid, methyl acrylate and methyl methacrylate copolymer (FS30D), methacrylic acid and methyl methacrylate copolymer (Eudragit S100), and ethyl cellulose (EC) were added to ethanol and completely dissolved, and then triethyl citrate and talc were added in sequence and stirred thoroughly to obtain a pH-dependent outer coating solution;

[0114] Eudragit S100 and FS30D are two models of Eudragit.

[0115] The methacrylic acid and methyl methacrylate copolymer is prepared from methacrylic acid and methyl methacrylate in a mass ratio of 1:2;

[0116] The methacrylic acid, methyl acrylate and methyl methacrylate copolymer is prepared from methacrylic acid, methyl acrylate and methyl methacrylate in a mass ratio of 1:1:1;

[0117] The mass ratio of the methacrylic acid, the copolymer of methyl acrylate and methyl methacrylate, the copolymer of methacrylic acid and methyl methacrylate, and the ethyl cellulose is 1:5:1;

[0118] The triethyl citrate accounts for 3% of the mass of the pH-dependent outer coating solution;

[0119] The talc powder accounts for 0.5% of the mass of the pH-dependent outer coating solution;

[0120] The gallnut pomegranate peel fermentation aqueous solution, the time-lag layer coating solution, and the pH-dependent outer layer coating solution are preheated to 40-42°C in a fluidized bed, and then 3 / h, inlet air temperature 60-70°C, atomization pressure 120-140kPa, and spray volume flow rate 4-8mL / min, coating the drug layer, time lag layer, and pH-dependent layer in sequence to obtain gallnut and pomegranate peel fermentation product colon-targeted micropellets;

[0121] The time-delay layer accounts for 7% of the weight of the colon-targeted micropellets of gallnut and pomegranate peel ferment;

[0122] The pH-dependent layer accounts for 20% to 35% of the weight of the colon-targeted micropellets of gallnut and pomegranate peel ferments;

[0123] The gel pellets prepared in step 5 are mixed with the colon-targeted pellets of gallnut and pomegranate peel fermentation product prepared in step 6 in a mass ratio of 1:1 to obtain a composition for treating ulcerative colitis;

[0124] Alternatively, the gel pellets prepared in step five are mixed with the colon-targeted pellets of gallnut and pomegranate peel fermentation product prepared in step six at a mass ratio of 1:2 to obtain a composition for treating ulcerative colitis;

[0125] Alternatively, the gel micropellets prepared in step five are mixed with the colon-targeted micropellets of gallnut and pomegranate peel fermentation product prepared in step six in a mass ratio of 2:1 to obtain a composition for treating ulcerative colitis.

[0126] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that the clean palm in step 3 is prepared by removing impurities from palm, washing it, and drying it. Other aspects are the same as those of specific embodiment 1.

[0127] Specific embodiment 3: This embodiment differs from specific embodiment 1 or 2 in that 9 times the weight of ethanol of purslane is added in step 3, and reflux extraction is performed twice. Other aspects are the same as specific embodiment 1 or 2.

[0128] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the pH-dependent layer in step 6 accounts for 30% of the weight of the gallnut and pomegranate peel fermented product colon-targeted micropellets. Other aspects are the same as specific embodiments 1 to 3.

[0129] The following examples are used to verify the effects of the present invention:

[0130] Example 1:

[0131] Carbonization of the Sanguisorba officinalis and Plum Blossom Mixture:

[0132] First, weigh 100.0g of dried Sanguisorba officinalis and 100g of Prunus mume. Place them in a clean crucible, cover the crucible with clean tin foil, seal it tightly, and place it steadily in a muffle furnace for firing. First, set the muffle furnace to three temperature ramps: the first ramp is to increase the temperature to 80°C within 10 minutes and hold it for 35 minutes; the second ramp is to increase the temperature to 260°C within 45 minutes and hold it for 1.5 hours. Collect the powder of the carbonized mixture of Sanguisorba officinalis and Prunus mume.

[0133] Example 2:

[0134] Carbonization of the Sanguisorba officinalis and Plum Blossom Mixture:

[0135] First, weigh 100.0g of dried Sanguisorba officinalis and 100g of Prunus mume. After mixing, place the mixture in a clean crucible, cover the crucible with clean tin foil, seal it tightly, and place it steadily in a muffle furnace for firing. First, set the muffle furnace to three temperature ramps: the first ramp is to increase the temperature to 80°C within 10 minutes and hold it for 35 minutes; the second ramp is to increase the temperature to 380°C within 45 minutes and hold it for 1.5 hours. Collect the carbonized mixture powder of Sanguisorba officinalis and Prunus mume.

[0136] Example 3:

[0137] Carbonization of the Sanguisorba officinalis and Plum Blossom Mixture:

[0138] First, weigh 100.0g of dried Sanguisorba officinalis and 100g of Prunus mume. Place them in a clean crucible, cover the crucible with clean tin foil, seal it tightly, and place it steadily in a muffle furnace for firing. First, set the muffle furnace to three temperature ramps: the first ramp is to increase the temperature to 80°C within 10 minutes and hold it for 35 minutes; the second ramp is to increase the temperature to 460°C within 45 minutes and hold it for 1.5 hours. Collect the powder of the carbonized mixture of Sanguisorba officinalis and Prunus mume.

[0139] Table 1 Output rate of carbonized mixture of Sanguisorba officinalis and Prunus mume at various temperatures in various embodiments

[0140] Example Firing temperature Weight of crude drug (g) Carbon weight (g) Output rate (%) Example 1 260℃ 200.0 138.7 67.8 Example 2 380℃ 200.0 123.2 61.6 Example 3 460℃ 200.0 89.6 44.8

[0141] The mixture was carbonized in a muffle furnace at three different temperatures: 260°C, 380°C, and 460°C. After the carbonization was complete, the carbonized material was removed and the SRC yield at different preparation temperatures was calculated. It was found that the carbonized material yield decreased with increasing preparation temperature.

[0142] Example 4:

[0143] 1. Carbonization of the mixture of Sanguisorba officinalis and Plum

[0144] Weigh 100.0 g of dried raw Sanguisorba officinalis and 100 g of dried plum, then place the raw Sanguisorba officinalis and plum in a clean crucible, cover the crucible mouth with clean tin foil, seal it with a lid, and place it stably in a muffle furnace. Set the muffle furnace to two heating gradients: the first gradient is to heat to 80 ° C within 10 min and hold for 35 min; the second gradient is to heat to 260 ° C within 45 min and hold for 1.5 h, and collect the carbonized mixture powder of Sanguisorba officinalis and plum;

[0145] 2. Extraction and purification of carbonized products

[0146] Weigh 100 g of the carbonized mixture powder of Sanguisorba officinalis and Ebony plum, add 8 to 10 times the weight of the carbonized mixture powder of Sanguisorba officinalis and Ebony plum, place in a 1L beaker to soak, then add silica, stir evenly with a glass rod, and let stand for 30 minutes to obtain a mixture with a silica concentration of 0.1%;

[0147] Ultrasonic extraction was performed twice at 65°C, each extraction time was 35 min, and the extract was filtered through gauze to remove larger particles. The combined filtrate was collected and finally the filtrate was filtered through a 0.45 μm microporous membrane under negative pressure for three times to obtain the aqueous extract.

[0148] The aqueous extract was concentrated using a rotary evaporator, and then placed in a dialysis bag with a molecular weight cutoff of 3000 Da and sealed for dialysis. The dialysis fluid was deionized water, and the dialysis time was not less than 96 hours, until the solution outside the bag was colorless and transparent. During this period, the dialysis bag was changed every 6 hours to ensure that the small molecule residues in the dialysis bag were completely exuded. The exudate solution was taken out and then centrifuged on a rotary evaporator at 14000 r / min for 15 minutes to obtain a supernatant. The supernatant was freeze-dried to obtain a freeze-dried powder of a carbonized Sanguisorba officinalis and Prunus mume mixture;

[0149] 3. Preparation of Palm Charcoal-Purslane

[0150] Take 100g of purslane, add 8-10 times the weight of ethanol, reflux extract twice, combine the filtrate, and rotate until there is no alcohol smell to obtain the purslane extract;

[0151] Soak 150g of clean palm in purslane extract for 6 hours, place it in a pot, put a smaller pot on top of the pot, seal the joint of the two pots with yellow mud, and press a heavy object on the smaller pot. Heat it over high heat until it is thoroughly calcined. After cooling, take it out, cut it into small pieces, put it in a hot pot, and stir-fry it over high heat until the surface is dark brown and the inside is burnt brown. Spray it with clean water to extinguish sparks, take it out, spread it out in time to cool thoroughly, and you will get palm charcoal powder.

[0152] 100 g of palm charcoal crude powder was extracted with 95% ethanol under reflux for 2 h, and the residue was extracted with 75% ethanol for 2 h. The filtrate was filtered while hot and combined, extracted with ethyl acetate, and the filtrate was concentrated by rotary evaporation to remove residual ethanol, and freeze-dried to obtain palm charcoal freeze-dried powder;

[0153] Preparation of Gallnut and Pomegranate Peel Fermented Extracts

[0154] Fermentation time and inoculum selection: The appropriate inoculum size and time can ensure bacterial growth and achieve maximum fermentation efficiency. In this experiment, the inoculum size was set to 3% and two groups of compound Chinese medicine were fermented. After 48 hours, the fermented samples were removed.

[0155] Take 50g of gallnut, dry in an oven at 60℃ for 4h, grind and pass through a 40-mesh sieve to obtain gallnut powder;

[0156] Take 50g of pomegranate peel, dry it in an oven at 60℃ for 6h, and grind it through a 40-mesh sieve to obtain pomegranate peel powder;

[0157] On a sterile operating table, use a loop to dip frozen bacterial suspensions of Lactobacillus rhamnosus, Lactobacillus plantarum, and Bacillus coagulans, streak inoculate onto MRS solid medium, and incubate in a constant temperature incubator for 24 hours. Then, pick colonies and inoculate onto MRS liquid medium. Subculture continuously for 3 days to obtain a mixed bacterial strain, which is stored at 4°C for later use.

[0158] The activated mixed bacteria were inoculated into a 500 mL Erlenmeyer flask containing gallnut powder, pomegranate peel powder, and 300 mL of fermentation liquid medium at a 3% inoculum rate, and cultured in a shaking incubator at 35°C and 180 rpm for 48 hours to obtain a fermentation liquid and a fermentation product of the gallnut and pomegranate peel;

[0159] The fermentation product was extracted with 75% ethanol at a solid-liquid ratio of 1:8, each extraction was 4 hours, and a total of 2 extractions were performed. The combined extracts were freeze-dried to obtain a fermentation product extract;

[0160] 5. Preparation of Gel Micropellets

[0161] The carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder are mixed to obtain a mixture of the carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder, and the mixture is added to an oil phase, wherein each gram of the oil phase contains 5 mg of the carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder. After vortexing, the mixture is sonicated for 20 to 25 minutes to dissolve the mixture, thereby obtaining an oil phase containing a charcoal powder extract;

[0162] The mass ratio of the carbonized Sanguisorba officinalis and Prunus mume mixture freeze-dried powder to the palm charcoal freeze-dried powder is 1:1, and the oil phase is composed of propylene glycol monocaprylate, corn oil and caprylic / capric triglyceride in a mass ratio of 1:0.5:0.05;

[0163] Heat the emulsifier in an oven at 70°C, and after it is completely melted, place it in a small beaker, add an equal amount of co-emulsifier polyethylene glycol 600, and use a cell disruptor to disrupt the cells for 60 seconds to obtain a mixed emulsifier.

[0164] The emulsifier is composed of polyethylene glycol 15-hydroxystearate and Tween 20 in a mass ratio of 1:1;

[0165] Adding 1.5% oleic acid to the oil phase containing the charcoal powder extract, stirring and mixing with a magnetic stirrer for 5 minutes, adding a mixed emulsifier constant at 30°C, continuing stirring for 30 minutes, and then disrupting the cells with a cell disrupter for 60 seconds, and then adding distilled water dropwise on a magnetic stirrer constant at 30°C, wherein the amount of distilled water added is 35.5% of the total weight of oleic acid, the oil phase containing the charcoal powder extract and the distilled water, to obtain a charcoal powder extract mixture nanoemulsion;

[0166] Weigh 0.5 g of sodium alginate and dissolve it in 30 mL of deionized water. Stir at room temperature to obtain a uniform sodium alginate solution.

[0167] Weigh 0.05 g of sodium hyaluronate solution and dissolve it in 10 mL of deionized water. After sufficient swelling, a sodium hyaluronate solution is obtained.

[0168] Weigh 0.2 g of Carbomer 940, dissolve it in 20 mL of deionized water, and place it at room temperature for 24 hours until it is fully swollen to obtain a Carbomer solution.

[0169] The sodium alginate solution, the sodium hyaluronate solution and the carbomer solution were mixed in a mass ratio of 1:0.2:1 and stirred evenly to obtain a mixed gel solution;

[0170] The charcoal powder extract mixture nanoemulsion was added to the mixed gel solution, wherein the mass ratio of the charcoal powder extract mixture nanoemulsion to the mixed gel solution was 1:1.5, and the mixture was first disrupted by a cell disruptor for 30 seconds at room temperature, and magnetically stirred at room temperature for 30 minutes until uniformly mixed. The mixture was then added dropwise to a mixed solution of CaCl2 and CaSO4 using a 5 mL syringe with a 0.45 mm needle and stirred for 15 minutes. The cross-linked nanoemulsion / gel complex was collected by filtration, rinsed with distilled water to remove the unreacted mixed solution of CaCl2 and CaSO4, and then freeze-dried to obtain gel pellets.

[0171] The mass concentrations of CaCl2 and CaSO4 in the mixed solution of CaCl2 and CaSO4 are 5% respectively;

[0172] The carbonized Sanguisorba officinalis and Prunus mume freeze-dried powder mixture and the palm charcoal freeze-dried powder mixture account for 22% of the gel micropill mass;

[0173] The emulsifier accounts for 40.0% of the mass of the gel pellets;

[0174] The mass concentration of 1.5% oleic acid accounts for 2.5% of the mass of the gel pellets;

[0175] Preparation of colon-targeted micropellets from the fermentation products of gallnut and pomegranate peel

[0176] Drug layer:

[0177] Add the fermented liquor of gallnut and pomegranate peel prepared in step 4 to water, with the mass ratio of the fermented liquor of gallnut and pomegranate peel to water being 3:7, and dissolve by ultrasonication to prepare a gallnut and pomegranate peel fermented aqueous solution;

[0178] Preparation of time-lag coating solution:

[0179] The fermentation extract prepared in step 4 is dispersed in an acetic acid aqueous solution having a mass concentration of 1.5% and a temperature of 60°C, wherein the mass ratio of ethyl cellulose to acetic acid in the acetic acid aqueous solution is 3:1, and stirred until fully hydrated. Then, plasticizer triethyl citrate, micropowder silica gel, and fumed silica are added in sequence and stirred uniformly to obtain a time-lag layer coating solution;

[0180] The acetic acid aqueous solution is a coating solution and accounts for 7% of the weight of the gallnut and pomegranate peel fermented product colon-targeted micropills;

[0181] The triethyl citrate accounts for 15% of the weight of the time-lag layer coating solution;

[0182] The micro powder silica gel accounts for 10% of the weight of the time lag layer coating solution;

[0183] The fumed silica accounts for 2% of the weight of the time-lag layer coating solution;

[0184] Preparation of pH-dependent outer coating solution:

[0185] Under magnetic stirring, methacrylic acid, methyl acrylate and methyl methacrylate copolymer, methacrylic acid and methyl methacrylate copolymer, and ethyl cellulose are added to ethanol and completely dissolved, and then triethyl citrate and talc are added in sequence and stirred thoroughly to obtain a pH-dependent outer coating solution;

[0186] The methacrylic acid and methyl methacrylate copolymer is prepared from methacrylic acid and methyl methacrylate in a mass ratio of 1:2;

[0187] The methacrylic acid, methyl acrylate and methyl methacrylate copolymer is prepared from methacrylic acid, methyl acrylate and methyl methacrylate in a mass ratio of 1:1:1;

[0188] The mass ratio of the methacrylic acid, the copolymer of methyl acrylate and methyl methacrylate, the copolymer of methacrylic acid and methyl methacrylate, and the ethyl cellulose is 1:5:1;

[0189] The triethyl citrate accounts for 3% of the mass of the pH-dependent outer coating solution;

[0190] The talc powder accounts for 0.5% of the mass of the pH-dependent outer coating solution;

[0191] The gallnut pomegranate peel fermentation aqueous solution, the time-lag layer coating solution, and the pH-dependent outer layer coating solution are preheated to 40-42°C in a fluidized bed, and then 3 / h, inlet air temperature 60-70°C, atomization pressure 120-140kPa, and spray volume flow rate 4-8mL / min, coating the drug layer, time lag layer, and pH-dependent layer in sequence to obtain gallnut and pomegranate peel fermentation product colon-targeted micropellets;

[0192] The time-delay layer accounts for 7% of the weight of the colon-targeted micropellets of gallnut and pomegranate peel ferment;

[0193] The pH-dependent layer accounted for 30% of the weight of the colon-targeted micropellets of Gallnut and Pomegranate peel ferments.

[0194] Example 5:

[0195] The gel pellets prepared in step 5 of Example 4 were mixed with the colon-targeted pellets of gallnut and pomegranate peel fermentation product prepared in step 6 at a mass ratio of 1:1 to obtain a composition for treating ulcerative colitis.

[0196] Example 6:

[0197] The gel pellets prepared in step 5 of Example 4 were mixed with the colon-targeted pellets of gallnut and pomegranate peel fermentation product prepared in step 6 at a mass ratio of 1:2 to obtain a composition for treating ulcerative colitis.

[0198] Example 7:

[0199] The gel pellets prepared in step 5 of Example 4 were mixed with the colon-targeted pellets of gallnut and pomegranate peel fermentation product prepared in step 6 at a mass ratio of 2:1 to obtain a composition for treating ulcerative colitis.

[0200] Example 8:

[0201] The gel pellets prepared in step 5 of Example 4 were used as a composition for treating ulcerative colitis.

[0202] Example 9:

[0203] The colon-targeted micropellets of gallnut and pomegranate peel fermentation product prepared in step 6 of Example 4 were used as a composition for treating ulcerative colitis.

[0204] Animal Experiment Section:

[0205] 7-8 week-old female C57BL / 6 mice were used as experimental subjects, SPF grade, and purchased from Harbin Medical University.

[0206] Modeling method: 7-8 week old female C57BL / 6 mice were used as experimental subjects. After 7 days of adaptive feeding, the mice were allowed to drink sterile water and 3.0% DSS solution freely for 7 consecutive days (DSS solution was changed every 3 days). 。

[0207] Preparation method of medicine: Examples 5-7

[0208] Animal Grouping: 48 mice were randomly divided into 8 groups, each consisting of 6 mice: a normal group, a model group, a positive control group, and Examples 5, 6, 7, 8, and 9. The positive control group was gavaged with mesalamine enteric-coated tablets at a dose of 400 mg / kg, and Examples 5-9 were gavaged with 400 mg / kg. Care was taken to prevent the mice from biting the pellets. The normal and model groups were gavaged with equal volumes of sterile water for 7 days. The model group, the positive control group, and Examples 5-9 were all administered 3.0% DSS for modeling. The normal group mice received regular drinking water.

[0209] Dextran sulfate sodium DSS (molecular weight 4000), KINGBIO Biotechnology Co., Ltd.;

[0210] Tumor necrosis factor (TNF-α) ELASA kit, Shanghai ELISA Biotechnology Co., Ltd.;

[0211] Mouse interleukin-18 (IL-18) ELISA kit, Shanghai ELISA Biotechnology Co., Ltd.;

[0212] Mouse interleukin-1β (IL-1β) ELISA kit, Shanghai ELISA Biotechnology Co., Ltd.;

[0213] Animal grouping: After receiving the experimental animals, they were acclimated and raised in the experimental environment for 7 days and then randomly divided into 8 groups, namely, a normal control group (K), a 3% dextran sulfate sodium group, a positive control group (Example 5 group), an Example 6 group, an Example 7 group, an Example 8 group, and an Example 9 group;

[0214] Observation indicators: ① Disease activity index (DAI) of mice. From the first day of modeling, the mental state of mice was observed and weighed every day; after the end of the experiment, DAI was calculated based on the scores of body weight, stool characteristics, and occult blood / gross blood in stool (see Table 1 for scoring criteria), DAI = body weight loss score + stool viscosity score + stool blood score. ② Mouse colon length. After the experiment, colon tissues of mice in each group were collected and their lengths were measured and recorded. Expression of inflammatory factors in colon tissue. ELISA method was used to determine the levels of IL-6, IL-4, IL-8, IL-10 and TNF-α in serum. The serum to be used was taken out of the -20℃ refrigerator and placed in a 4℃ refrigerator to dissolve for 24 hours. It was brought to room temperature 1 hour before the experiment until it was completely dissolved and gently shaken to mix to avoid precipitation. The enzyme-linked immunosorbent assay kit was strictly followed in operation. After the operation, the absorbance value at 450nm was measured using an automated microplate reader spectrophotometer to calculate the concentrations of IL-6, IL-4, IL-8, IL-10 and TNF-α in serum.

[0215] Table 2 Scoring criteria for weight loss, stool characteristics and blood in stool in mice

[0216]

[0217]

[0218] Note: Normal stool: formed stool; Loose stool: Pasty, semi-formed stool that does not stick to the anus; Loose stool: Loose, watery stool that can stick to the anus.

[0219] Table 3 Comparison of DAI scores among 8 groups of mice

[0220] Group Number of rats DAI Normal group 6 0.0±0.0 Model Group 6 <![CDATA[9.6±0.3 ① ]]> Positive control group 6 <![CDATA[6.8±0.1 ② ]]> Example 5 6 <![CDATA[6.7±0.2 ② ]]> Example 6 6 <![CDATA[6.9±0.5 ② ]]> Example 7 6 <![CDATA[7.4±0.1 ② ]]> Example 8 6 8.8±0.2 Example 9 6 8.9±0.6

[0221] ①Compared with the normal group, P<0.05②Compared with the model group, P<0.05③Compared with the model group, P<0.1

[0222] The DAI score of mice in the model group was significantly higher than that in the normal group (P < 0.05). Compared with the model group, the DAI scores of the positive control group and Examples 5 to 7 were significantly decreased (P < 0.05), while the DAI scores of Examples 8 to 9 changed (P < 0.1).

[0223] Table 4 Comparison of UC severity in 8 groups of mice

[0224]

[0225]

[0226] ①Compared with the normal group, P<0.05②Compared with the model group, P<0.05③Compared with the model group, P<0.1

[0227] After the experiment, the colon length of the mice in the model group was shorter than that in the normal group (P < 0.05). Compared with the model group, the colon length of the mice in the positive control group and Examples 5 to 7 increased significantly (P < 0.05), while the length of the mice in Examples 7 to 8 increased slightly, which was statistically significant (P < 0.1).

[0228] The cause of UC is not fully understood, but it is certain that an abnormal immune response plays a crucial role in the development of UC. The dual-micropellet combination significantly inhibited IL-6 mRNA expression in inflammatory animal models. This abnormal immune response activates the reactive oxygen / nitrogen species (ROS / RNS) production system, a marker of UC pathogenesis, leading to excessive production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-8 in the serum, which promotes the progression of UC inflammation. IL-4 and IL-10, two major anti-inflammatory factors, play a crucial role in inhibiting UC inflammation and promoting disease outcomes. Decreased levels of the anti-inflammatory factors IL-4 and IL-10 amplify the inflammatory response.

[0229] Inflammatory factors like IL-6, IL-8, and TNF-α are pro-inflammatory factors that promote the progression of UC inflammation. IL-4 and IL-10, two major anti-inflammatory factors, play a crucial role in inhibiting UC inflammation and promoting disease progression.

[0230] Table 5 Expression of several cytokine levels in serum after drug administration (x±s; ng / L)

[0231]

[0232] ①Compared with the normal group, P<0.05②Compared with the model group, P<0.05③Compared with the model group, P<0.1

[0233] The levels of inflammatory factors IL-6, IL-8, and TNF-α in the serum of mice in the UC model control group were higher than those in the normal control group, while the levels of IL-4 and IL-10 were lower than those in the normal control group. After treatment, it was found that the levels of cytokines IL-6, IL-8, and TNF-α decreased, and the levels of IL-4 and IL-10 increased in the mice in Examples 5 to 9 and the mesalazine group compared with the mice in the model control group, respectively. Among them, 5 to 6 were statistically significant.

[0234] The following examples simulate the gastrointestinal environment in the human body to carry out in vitro release tests. The test was carried out according to the 2020 edition of the Chinese Pharmacopoeia, Part IV, to prepare different pH dissolution media, and according to the first method of the 0931 dissolution and release determination method of Part IV of the Chinese Pharmacopoeia (2020 edition), the blue method was adopted, and the dissolution media were 750 ml of artificial gastric juice (pH = 1.2), artificial small intestinal juice (pH = 6.8), and artificial colonic juice (pH = 7.4), respectively. The temperature was 37 ° C ± 0.5 ° C, the speed was 50 rpm, and the dissolution medium was in artificial gastric juice for 2 hours, artificial small intestinal juice for 3 hours, and artificial colonic juice for 7 hours. The dissolution content was determined according to the previously established dissolution content determination method, and the average cumulative release was calculated.

[0235] Artificial gastric juice (pH 1.2): Measure 16.4 ml of dilute hydrochloric acid and add ultrapure water to 1000 ml, then mix thoroughly. (Preparation of dilute hydrochloric acid: Take 234 ml of hydrochloric acid, add water to 1000 ml and mix thoroughly.)

[0236] Artificial small intestinal fluid (pH 6.8): Take 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, add 118 ml of 0.2 mol / L sodium hydroxide solution, dilute with water to 1000 ml, shake well, and obtain.

[0237] Artificial colonic fluid (pH 7.6): Weigh 27.22 g of potassium dihydrogen phosphate, add water to dissolve it into 1000 ml, take 50 ml, add 42.4 ml of 0.2 mol / L sodium hydroxide solution, and then add water to dilute to 200 ml.

[0238] Table 6

[0239]

[0240] In the in vitro release test, the double colon micropellets prepared in this application showed that there was basically no release in the environment of simulated gastric acid for 2 hours, and the release was very small, only about 6%, in the environment of simulated small intestinal fluid for 3 hours. In the environment of simulated colonic fluid, the release reached more than 90% in the environment of simulated colonic fluid for 7 hours.

Claims

1. A method for preparing a composition for treating ulcerative colitis, characterized in that Preparation method of the composition for treating ulcerative colitis:

1. Carbonization of the mixture of Sanguisorba officinalis and Plum Weigh 100.0 g of dried raw Sanguisorba officinalis and 100 g of dried plum, then place the raw Sanguisorba officinalis and plum in a clean crucible, cover the crucible mouth with clean tin foil, seal it with a lid, and place it stably in a muffle furnace. Set the muffle furnace to two heating gradients: the first gradient is to heat to 80 ° C within 10 min and hold for 35 min; the second gradient is to heat to 260 ° C-380 ° C within 45 min and hold for 1.5 h, and collect the carbonized mixture powder of Sanguisorba officinalis and plum; 2. Extraction and purification of carbonized products Weigh 100 g of the carbonized mixture powder of Sanguisorba officinalis and Ebony plum, add 8 to 10 times the weight of the carbonized mixture powder of Sanguisorba officinalis and Ebony plum, place in a 1L beaker to soak, then add silica, stir evenly with a glass rod, and let stand for 30 minutes to obtain a mixture with a silica concentration of 0.1%; Ultrasonic extraction was performed twice at 65°C, each extraction time was 35 min, and the extract was filtered through gauze to remove larger particles. The combined filtrate was collected and finally the filtrate was filtered through a 0.45 μm microporous membrane under negative pressure for three times to obtain the aqueous extract. The aqueous extract was concentrated using a rotary evaporator, and then placed in a dialysis bag with a molecular weight cutoff of 3000 Da and sealed for dialysis. The dialysis fluid was deionized water, and the dialysis time was not less than 96 hours, until the solution outside the bag was colorless and transparent. During this period, the dialysis bag was changed every 6 hours, and the exudate solution was taken out and then centrifuged on a rotary evaporator at 14000 r / min for 15 minutes to obtain a supernatant. The supernatant was freeze-dried to obtain a freeze-dried powder of a carbonized Sanguisorba officinalis and Prunus mume mixture; 3. Preparation of Palm Charcoal-Purslane Take 100g of purslane, add 8-10 times the weight of ethanol, reflux extract twice, combine the filtrate, and rotate until there is no alcohol smell to obtain the purslane extract; Soak 150g of clean palm in purslane extract for 6 hours, place it in a pot, put a smaller pot on top of the pot, seal the joint of the two pots with yellow mud, and press a heavy object on the smaller pot. Heat it over high heat until it is thoroughly calcined. After cooling, take it out, cut it into small pieces, put it in a hot pot, and stir-fry it over high heat until the surface is dark brown and the inside is burnt brown. Spray it with clean water to extinguish sparks, take it out, spread it out in time to cool thoroughly, and you will get palm charcoal powder. 100 g of palm charcoal crude powder was extracted with 95% ethanol under reflux for 2 h, and the residue was extracted with 75% ethanol for 2 h. The filtrate was filtered while hot and combined, extracted with ethyl acetate, and the filtrate was concentrated by rotary evaporation to remove residual ethanol, and freeze-dried to obtain palm charcoal freeze-dried powder; Preparation of Gallnut and Pomegranate Peel Fermented Extracts Take 50g of gallnut, dry in an oven at 60℃ for 4h, grind and pass through a 40-mesh sieve to obtain gallnut powder; Take 50g of pomegranate peel, dry it in an oven at 60℃ for 6h, and grind it through a 40-mesh sieve to obtain pomegranate peel powder; On a sterile operating table, use a loop to dip frozen bacterial suspensions of Lactobacillus rhamnosus, Lactobacillus plantarum, and Bacillus coagulans, streak inoculate onto MRS solid medium, and incubate in a constant temperature box for 24 hours. Then, pick a colony and inoculate into MRS liquid medium. Subculture continuously for 3 days to obtain a mixed bacteria. The activated mixed bacteria were inoculated into a 500 mL Erlenmeyer flask containing gallnut powder, pomegranate peel powder, and 300 mL of fermentation liquid medium at a 3% inoculum rate, and cultured in a shaking incubator at 35°C and 180 rpm for 48 hours to obtain a fermentation liquid and a fermentation product of the gallnut and pomegranate peel; The fermentation product was extracted with 75% ethanol at a solid-liquid ratio of 1:8, each extraction was 4 hours, and a total of 2 extractions were performed. The combined extracts were freeze-dried to obtain a fermentation product extract; 5. Preparation of Gel Micropellets The carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder are mixed to obtain a mixture of the carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder, and the mixture is added to an oil phase, wherein each gram of the oil phase contains 5 mg of the carbonized Sanguisorba officinalis and Ebony plum freeze-dried powder and palm charcoal freeze-dried powder. After vortexing, the mixture is sonicated for 20 to 25 minutes to dissolve the mixture, thereby obtaining an oil phase containing a charcoal powder extract; The mass ratio of the carbonized Sanguisorba officinalis and Prunus mume mixture freeze-dried powder to the palm charcoal freeze-dried powder is 1:1, and the oil phase is composed of propylene glycol monocaprylate, corn oil and caprylic / capric triglyceride in a mass ratio of 1:0.5:0.05; Heat the emulsifier in an oven at 70°C, and after it is completely melted, place it in a small beaker, add an equal amount of co-emulsifier polyethylene glycol 600, and use a cell disruptor to disrupt the cells for 60 seconds to obtain a mixed emulsifier. The emulsifier is composed of polyethylene glycol 15-hydroxystearate and Tween 20 in a mass ratio of 1:1; Adding 1.5% oleic acid to the oil phase containing the charcoal powder extract, stirring and mixing with a magnetic stirrer for 5 minutes, adding a mixed emulsifier constant at 30°C, continuing stirring for 30 minutes, and then disrupting the cells with a cell disrupter for 60 seconds, and then adding distilled water dropwise on a magnetic stirrer constant at 30°C, wherein the amount of distilled water added is 35.5% of the total weight of oleic acid, the oil phase containing the charcoal powder extract and the distilled water, to obtain a charcoal powder extract mixture nanoemulsion; Weigh 0.5 g of sodium alginate and dissolve it in 30 mL of deionized water. Stir at room temperature to obtain a uniform sodium alginate solution. Weigh 0.05 g of sodium hyaluronate solution and dissolve it in 10 mL of deionized water. After sufficient swelling, a sodium hyaluronate solution is obtained. Weigh 0.2 g of Carbomer 940, dissolve it in 20 mL of deionized water, and place it at room temperature for 24 hours until it is fully swollen to obtain a Carbomer solution. The sodium alginate solution, the sodium hyaluronate solution and the carbomer solution were mixed in a mass ratio of 1:0.2:1 and stirred evenly to obtain a mixed gel solution; The charcoal powder extract mixture nanoemulsion was added to the mixed gel solution, wherein the mass ratio of the charcoal powder extract mixture nanoemulsion to the mixed gel solution was 1:1.5, and the mixture was first disrupted by a cell disruptor for 30 seconds at room temperature, and magnetically stirred at room temperature for 30 minutes until uniformly mixed. The mixture was then added dropwise to a mixed solution of CaCl2 and CaSO4 using a 5 mL syringe with a 0.45 mm needle and stirred for 15 minutes. The cross-linked nanoemulsion / gel complex was collected by filtration, rinsed with distilled water to remove the unreacted mixed solution of CaCl2 and CaSO4, and then freeze-dried to obtain gel pellets. The mass concentrations of CaCl2 and CaSO4 in the mixed solution of CaCl2 and CaSO4 are 5% respectively; The carbonized Sanguisorba officinalis and Prunus mume freeze-dried powder mixture and the palm charcoal freeze-dried powder mixture account for 22% of the gel micropill mass; The emulsifier accounts for 40.0% of the mass of the gel pellets; The mass concentration of 1.5% oleic acid accounts for 2.5% of the mass of the gel pellets; Preparation of colon-targeted micropellets from the fermentation products of gallnut and pomegranate peel Drug layer: Add the fermented liquor of gallnut and pomegranate peel prepared in step 4 to water, with the mass ratio of the fermented liquor of gallnut and pomegranate peel to water being 3:7, and dissolve by ultrasonication to prepare a gallnut and pomegranate peel fermented aqueous solution; Preparation of time-lag coating solution: The fermentation extract prepared in step 4 is dispersed in an acetic acid aqueous solution having a mass concentration of 1.5% and a temperature of 60°C, wherein the mass ratio of ethyl cellulose to acetic acid in the acetic acid aqueous solution is 3:1, and stirred until fully hydrated. Then, plasticizer triethyl citrate, micropowder silica gel, and fumed silica are added in sequence and stirred uniformly to obtain a time-lag layer coating solution; The acetic acid aqueous solution is a coating solution and accounts for 7% of the weight of the gallnut and pomegranate peel fermented product colon-targeted micropills; The triethyl citrate accounts for 15% of the weight of the time-lag layer coating solution; The micro powder silica gel accounts for 10% of the weight of the time lag layer coating solution; The fumed silica accounts for 2% of the weight of the time-lag layer coating solution; Preparation of pH-dependent outer coating solution: Under magnetic stirring, methacrylic acid, methyl acrylate and methyl methacrylate copolymer, methacrylic acid and methyl methacrylate copolymer, and ethyl cellulose are added to ethanol and completely dissolved, and then triethyl citrate and talc are added in sequence and stirred thoroughly to obtain a pH-dependent outer coating solution; The methacrylic acid and methyl methacrylate copolymer is prepared from methacrylic acid and methyl methacrylate in a mass ratio of 1:2; The methacrylic acid, methyl acrylate and methyl methacrylate copolymer is prepared from methacrylic acid, methyl acrylate and methyl methacrylate in a mass ratio of 1:1:1; The mass ratio of the methacrylic acid, the copolymer of methyl acrylate and methyl methacrylate, the copolymer of methacrylic acid and methyl methacrylate, and the ethyl cellulose is 1:5:1; The triethyl citrate accounts for 3% of the mass of the pH-dependent outer coating solution; The talc powder accounts for 0.5% of the mass of the pH-dependent outer coating solution; The gallnut pomegranate peel fermentation aqueous solution, the time-lag layer coating solution, and the pH-dependent outer layer coating solution are preheated to 40-42°C in a fluidized bed, and then 3 / h, inlet air temperature 60-70°C, atomization pressure 120-140kPa, and spray volume flow rate 4-8mL / min, coating the drug layer, time-lag layer, and pH-dependent layer in sequence to obtain gallnut and pomegranate peel fermentation product colon-targeted micropellets; The time-delay layer accounts for 7% of the weight of the colon-targeted micropellets of gallnut and pomegranate peel ferment; The pH-dependent layer accounts for 20% to 35% of the weight of the colon-targeted micropellets of gallnut and pomegranate peel ferments; The gel pellets prepared in step 5 are mixed with the colon-targeted pellets of gallnut and pomegranate peel fermentation product prepared in step 6 in a mass ratio of 1:1 to obtain a composition for treating ulcerative colitis; Alternatively, the gel pellets prepared in step five are mixed with the colon-targeted pellets of gallnut and pomegranate peel fermentation product prepared in step six at a mass ratio of 1:2 to obtain a composition for treating ulcerative colitis; Alternatively, the gel micropellets prepared in step five are mixed with the colon-targeted micropellets of gallnut and pomegranate peel fermentation product prepared in step six in a mass ratio of 2:1 to obtain a composition for treating ulcerative colitis.

2. The method for preparing the composition for treating ulcerative colitis according to claim 1, characterized in that The clean palm oil in step 3 is obtained by removing impurities from palm oil, washing it and drying it.

3. The method for preparing the composition for treating ulcerative colitis according to claim 1, characterized in that Step 3: Add 9 times the weight of ethanol to the purslane and reflux extract twice.

4. The method for preparing the composition for treating ulcerative colitis according to claim 1, characterized in that The pH-dependent layer in step six accounts for 30% of the weight of the colon-targeted micropellets of the Chinese gallnut and pomegranate peel fermentation product.

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