WO2023138172A1 - 一种壳寡糖肠溶胶囊及其制备方法和应用 - Google Patents

一种壳寡糖肠溶胶囊及其制备方法和应用 Download PDF

Info

Publication number
WO2023138172A1
WO2023138172A1 PCT/CN2022/130809 CN2022130809W WO2023138172A1 WO 2023138172 A1 WO2023138172 A1 WO 2023138172A1 CN 2022130809 W CN2022130809 W CN 2022130809W WO 2023138172 A1 WO2023138172 A1 WO 2023138172A1
Authority
WO
WIPO (PCT)
Prior art keywords
enteric
coated
chitosan oligosaccharide
capsule
solution
Prior art date
Application number
PCT/CN2022/130809
Other languages
English (en)
French (fr)
Inventor
苏政权
曹华
白研
贺锦灿
Original Assignee
广东药科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广东药科大学 filed Critical 广东药科大学
Publication of WO2023138172A1 publication Critical patent/WO2023138172A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1664—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of pharmaceutical preparations, in particular to a chitosan oligosaccharide enteric-coated capsule and a preparation method and application thereof.
  • Obesity is a relatively complex chronic endocrine disease, the main cause and manifestation of which is excessive accumulation of body fat, which is closely related to metabolic diseases such as diabetes, non-alcoholic fatty liver, and hyperlipidemia.
  • Adverse effects caused by obesity are mainly divided into two categories: one is symptoms caused by excessive accumulation of adipose tissue in the body (such as osteoarthritis, obstructive sleep apnea, stigma caused by appearance), and the other is diseases caused by excessive increase in the number of adipocytes in other tissues and organs under obesity (T2DM, cardiovascular disease, non-alcoholic fatty liver disease).
  • T2DM cardiovascular disease
  • non-alcoholic fatty liver disease the strength of the association between obesity and various complications varies.
  • the treatment of obesity mainly includes three methods: lifestyle intervention (regulation of energy intake and consumption), surgical treatment (operation on the tissues and organs of the digestive system) and drug treatment (inhibition of lipid synthesis and promotion of lipid metabolism).
  • Lifestyle intervention is the healthiest way to lose weight, but at the same time, it is easy to regain weight and it is difficult for patients to persist for a long time, which leads to final weight loss failure.
  • clinicians are more inclined to use drugs and surgery as the main treatment, and life intervention as an auxiliary treatment.
  • Surgical treatment is the most effective treatment for weight loss, but at the same time, it will bring relatively large adverse reactions. The morbidity and mortality rate during bariatric surgery are still as high as 4.5% and 0.3%, respectively.
  • Gut flora can affect the development of obesity through small and tight regulation, and the composition of gut microbes can affect human metabolism and eventually cause weight changes. More and more obesity-associated flora have been discovered, and changes in the abundance of these bacteria can promote/inhibit the development of obesity.
  • the idea and method of directional modification of intestinal flora are considered by many scholars to be a new direction for the future treatment of obesity.
  • Many polysaccharides derived from natural products can exhibit good lipid-lowering activity through gut flora.
  • Chitooligosaccharides (COST) are natural products formed by deacetylation and hydrolysis of chitin.
  • Chitosan oligosaccharides with specific molecular weights are formed by glucosamine and N-acetylglucosamine linked by ⁇ -1,4 glycosidic bonds, and the degree of polymerization ranges from 2 to 10.
  • chitosan has high solubility in water and low viscosity, is easily absorbed by the body, and has better bioavailability. It is widely used in agriculture, forestry, medicine, material chemistry, food science and other fields.
  • Oligochitosan has good anti-obesity activity, can effectively reduce the serum lipid level of obese animals, increase lipid excretion, reduce body fat accumulation, relieve liver lipid metabolism disorder, can also effectively regulate the intestinal flora of obese mice, reduce the F/B ratio, restore the damaged intestinal barrier, reduce the release of LPS from Gram-negative bacteria (G-) into the blood, reduce the level of inflammatory factors, and improve obesity caused by endotoxemia. Therefore, it is of great significance to study an anti-obesity drug with chitosan oligosaccharide. In the production of oligochitosaccharide preparations, stratification, turbidity, precipitation, uneven color, etc.
  • oligochitosan oral liquid and its application in the preparation of weight-loss drugs on September 4, 2020.
  • a chitosan oligosaccharide oral solution and its preparation method were disclosed. It is a solution prepared from 25-40 parts of chitosan oligosaccharide, 1-3 parts of flavoring agent, 2-4 parts of preservative, and 1.5-2 parts of stabilizer. Therefore, it is of great significance to prepare a "stomach-insoluble, enteric-instant" chitosan oligosaccharide preparation.
  • the invention provides an oligochitosan enteric-coated capsule, which includes contents and a capsule shell, and the contents include enteric-coated auxiliary materials, oligochitosan and a release regulator.
  • the enteric-coated auxiliary material is Eudragit L100
  • the release regulator is hydroxypropyl methylcellulose
  • the capsule shell is an acid-resistant vegetable capsule shell.
  • the mass ratio of the chitosan oligosaccharide to the enteric adjuvant is 1:(4-5).
  • the mass ratio of the enteric-coated excipient, chitosan oligosaccharide and release regulator is (3-5):1:1.
  • the present invention also provides a preparation method of oligochitosan enteric-coated capsule, comprising the following steps:
  • Dissolution Weigh the enteric-coated auxiliary material, dissolve it with water and let it swell to obtain the enteric-coated auxiliary material solution; weigh the release regulator, dissolve it with water and leave it to stand to obtain the release regulator solution; weigh the chitosan oligosaccharide, dissolve it with water, and obtain the chitosan oligosaccharide solution;
  • Ultrasonic treatment put the mixed medicinal solution into an ultrasonic instrument, and perform ultrasonic treatment;
  • Freeze-drying freeze-solidify the mixed drug solution after ultrasonic treatment, and then carry out vacuum freeze-drying to obtain a solid drug;
  • stirring is vigorous stirring with a magnetic stirrer for 0.5 h.
  • the temperature of the ultrasonic treatment is 35-40°C.
  • freeze solidification temperature is -80°C.
  • the vacuum freeze-drying time is 11-13 hours.
  • Another object of the present invention is to provide the application of the chitosan oligosaccharide enteric-coated capsule prepared by the above-mentioned preparation method of the chitosan oligosaccharide enteric-coated capsule in weight-loss drugs.
  • the oligochitosaccharide capsules prepared by the present invention use enteric-coated capsules. Compared with other enteric-coated types, enteric-coated capsules use less auxiliary materials than commonly used enteric-coated tablets, which can minimize the volume of pharmaceutical adjuvants, and can well cover the odor of chitosan oligosaccharides to improve patient compliance, and can reduce its degradation in gastric acid and increase the release and absorption of its molecular prototype in the intestinal tract;
  • the acid-resistant plant capsule shell (Drcaps) used in the capsule shell of the oligochitosaccharide enteric-coated capsule prepared by the present invention has the highest content of hydroxypropyl methylcellulose (HPMC), which is difficult to dissolve in gastric juice, protects the contents of the oligochitosaccharide enteric-coated capsule from gastric acid damage, has obvious enteric properties, and can cover taste and smell, preventing bad mouth aftertaste and nausea;
  • HPMC hydroxypropyl methylcellulose
  • Eudragit L100 is selected as the best enteric-coated carrier for the oligochitosan enteric-coated capsules prepared by the present invention, and the capsule content granules made are insoluble in the stomach, but can be dissolved in a buffer solution with a pH above 6, which is safe and non-toxic, and can better cover up the iron smell of oligochitosan;
  • the oligochitosaccharide enteric-coated capsule prepared by the present invention selects hydroxypropyl methylcellulose (HPMC) as a release regulator, which can regulate the release degree of oligochitosaccharide in the intestinal tract and promote the drug effect of the oligochitosaccharide enteric-coated capsule;
  • HPMC hydroxypropyl methylcellulose
  • the oligochitosan enteric-coated capsules prepared by the present invention can be applied to weight-loss drugs to indirectly inhibit adipocyte hypertrophy caused by high-fat and high-sugar diets, regulate adipocyte growth and expansion, reduce fat accumulation, and improve obesity and other symptoms.
  • Embodiment 1 chitosan oligosaccharide enteric-coated capsule of the present invention and its preparation
  • Dissolution dissolve Eudragit L100 in water and let it swell for 0.5 hours to obtain Eudragit L100 solution; dissolve HPMC in water and let it stand for 0.5 hours to obtain HPMC solution; completely dissolve chitosan oligosaccharide in water to obtain chitosan oligosaccharide solution;
  • Ultrasonic treatment put the mixed medicinal solution into an ultrasonic instrument, and perform ultrasonic treatment at the highest frequency for 0.5 h at 37°C;
  • Freeze-drying seal the ultrasonically treated mixed drug solution with plastic wrap, and quickly place it in a -80°C ultra-low temperature refrigerator to freeze and solidify overnight, then take out the completely condensed and solidified mixed drug solution, use a syringe needle to pierce the plastic wrap, and leave a certain number of ventilation holes. Put the mixed drug solid in a pre-cooled freezing device, and carry out vacuum freeze-drying for 12 hours to obtain a solid drug;
  • Embodiment 2 oligochitosaccharide enteric-coated capsule of the present invention and its preparation
  • Dissolution dissolve Eudragit L100 in water and let it swell for 0.5 hours to obtain Eudragit L100 solution; dissolve HPMC in water and let it stand for 0.5 hours to obtain HPMC solution; completely dissolve chitosan oligosaccharide in water to obtain chitosan oligosaccharide solution;
  • Ultrasonic treatment put the mixed drug into an ultrasonic instrument, and perform ultrasonic treatment at the highest frequency at 37°C for 0.5h;
  • Freeze-drying seal the ultrasonically treated mixed drug solution with plastic wrap, and quickly place it in a -80°C ultra-low temperature refrigerator to freeze and solidify overnight, then take out the completely condensed and solidified mixed drug solution, use a syringe needle to pierce the plastic wrap, and leave a certain number of ventilation holes. Put the mixed drug solid in a pre-cooled freezing device, and carry out vacuum freeze-drying for 12 hours to obtain a solid drug;
  • Embodiment 3 oligochitosaccharide enteric-coated capsule of the present invention and its preparation
  • Dissolution dissolve Eudragit L100 in water and let it swell for 0.5 hours to obtain Eudragit L100 solution; dissolve HPMC in water and let it stand for 0.5 hours to obtain HPMC solution; completely dissolve chitosan oligosaccharide in water to obtain chitosan oligosaccharide solution;
  • Ultrasonic treatment put the mixed medicinal solution into an ultrasonic instrument, and perform ultrasonic treatment at the highest frequency for 0.5 h at 37°C;
  • Freeze-drying seal the ultrasonically treated mixed drug solution with a plastic wrap, quickly place it in a -80°C ultra-low temperature refrigerator to freeze and solidify overnight, then take out the completely condensed and solidified mixed drug, use a syringe needle to pierce the plastic wrap, leave a certain number of ventilation holes, put the mixed drug solid in a pre-cooled freezing device, and perform vacuum freeze-drying for 12 hours to obtain a solid drug;
  • Example 1 The difference between this comparative example and Example 1 is that the release modifier of this comparative example is Eudragit NM30D, and the rest of the steps, ingredients and parameters are the same.
  • Preparation process Completely dissolve chitosan oligosaccharides with water to obtain a chitosan oligosaccharide solution, put it into an ultrasonic instrument, and conduct ultrasonic treatment at the highest frequency at 37°C for 0.5h; the ultrasonically treated chitosan oligosaccharide solution is sealed with a plastic wrap, and quickly placed in a -80°C ultra-low temperature refrigerator to freeze and solidify overnight, then take out the completely condensed and solidified mixed drug, use a syringe needle to pierce the plastic wrap, and leave a certain number of ventilation holes.
  • chitosan oligosaccharide drug solid in a pre-cooled freezing device, and carry out vacuum freeze-drying for 12 hours; after drying, take it out, grind it on a dry and clean ultra-clean table, sieve it with an 80-mesh sieve, then fill it into the Drcaps capsule shell, and pack it to obtain chitosan oligosaccharide enteric-coated capsules.
  • Example 1 The difference between this comparative example and Example 1 is that the oligochitosan enteric-coated capsule of this comparative example has no enteric-coated adjuvant and release regulator.
  • Example 1 The difference between this comparative example and Example 1 is that this comparative example uses a common gelatin capsule shell as the capsule shell, and the rest of the components, parameters and steps are the same as in Example 1.
  • Example 1 The difference between this comparative example and Example 1 is that this comparative example uses Eudragit L100-55 as an enteric-coated auxiliary material, and the rest of the ingredients, parameters and steps are the same as those of Example 1.
  • Test example one the loading difference of the chitosan oligosaccharide capsule prepared by the present invention, main drug content inspection
  • Test sample oligochitosan enteric-coated capsules prepared in Examples 1-3.
  • Test method randomly select 20 enteric-coated capsules prepared in Examples 1-3, weigh the weight of each enteric-coated capsule and the weight of the empty capsule shell, calculate the weight of the contents of each capsule with the weight reduction method, and calculate the average loading and the difference limit of the loading according to the weighing results of 20 enteric-coated capsules; randomly select 6 enteric-coated capsules prepared in Examples 1-3, and detect the COST content in the sample.
  • Sugar which is dehydrated to form furfural derivatives, and the derivatives react with phenol to form an orange-red substance with the largest absorption peak. The quality of polysaccharides is determined by colorimetry, thereby detecting the content of chitosan oligosaccharides in the sample;
  • the average loading capacity of the chitosan oligosaccharide enteric-coated capsules prepared by the invention is 0.2445g. Comparing the maximum value of the average loading with the average value, the difference in loading is 8.82%; comparing the minimum value of the average loading with the average value, the difference in loading is -7.34%. The test results of the difference in filling volume did not exceed the limit of the difference in loading volume, which met the relevant requirements of the "Chinese Pharmacopoeia" (2020 edition);
  • the average main drug content of the chitosan oligosaccharide enteric-coated capsules prepared in Example 1 of the present invention is 16.66%, and the relative standard deviation is 1.3%.
  • the experimental results of its content test did not exceed the limit of content difference, which met the relevant requirements of "Chinese Pharmacopoeia” (2020 edition).
  • Test Example 2 Quality inspection of chitosan oligosaccharide enteric-coated capsules
  • Test method observe whether there is precipitation or uneven grinding during the preparation process of Examples 1-3 and Comparative Examples 4-5, and observe the appearance of the prepared capsules.
  • the chitosan oligosaccharide-soluble capsules prepared in Examples 1-3 of the present invention are clean and odorless, no deformation is found, and the capsule body is smooth, without moisture absorption and adhesion, and can maintain a dry state without becoming brittle or leaking and cracking.
  • Phenomenon The enteric-coated capsules prepared in Comparative Example 5 used Eudragit L100-55 as the enteric-coated auxiliary material, and the contents of the capsules had particles and uneven color, which did not meet the requirements of "Chinese Pharmacopoeia" (2020 edition).
  • Test sample oligochitosan enteric-coated capsules prepared in Examples 1-3 and Comparative Example 3
  • Test method According to the relevant operation requirements of "Chinese Pharmacopoeia” (2020 edition), its disintegration in artificial gastric juice and artificial intestinal juice was detected.
  • the oligochitosan capsule prepared in Comparative Example 3 can disintegrate within 15 minutes in artificial gastric juice and intestinal juice, and the disintegration time limit in artificial gastric juice is shorter than that in intestinal juice, because what Comparative Example 3 uses is ordinary gelatin capsule shell, which is contrary to the characteristics of "stomach insoluble and intestinal instant dissolution" required by enteric-coated capsules.
  • the oligochitosaccharide enteric-coated capsules prepared in Examples 1-3 of the present invention can remain in the artificial gastric juice for more than 2 hours without disintegrating, but disintegrate rapidly in the alkaline intestinal juice, and the disintegration time limit is 8 minutes, and can be completely disintegrated within 1 hour, which meets the characteristics of "insoluble in the stomach and instant in the intestine", and also meets the relevant requirements of "Chinese Pharmacopoeia” (2020 edition).
  • Test method operate according to the regulations of "Chinese Pharmacopoeia” (2020 edition), and test the cumulative release degree of total sugar in artificial gastric juice and artificial intestinal juice
  • the degree of release is only 45.69%, which does not meet the relevant requirements of "Chinese Pharmacopoeia” (2020 edition), because the enteric-coated auxiliary material Eudragit NM 30D greatly inhibits the dissolution and release of chitosan oligosaccharides in intestinal juice; while the cumulative release degree of the capsules prepared in Examples 1-3 in intestinal juice within 120min has little influence, more than 90% can be dissolved within 30min, and 100% dissolution can be achieved within 120min, which meets the requirements of "Chinese Pharmacopoeia” (2020 edition). ) and meet the enteric-coated properties, therefore, the oligochitosan enteric-coated capsules prepared in Examples 1-3 possess enteric-coated properties.
  • Test Example 5 Oligochitosan acts on intestinal flora
  • Pseudo-sterile mouse model establishment Select 36 healthy, 7-week-old male C57BL/6J mice without specific pathogens (SPF grade), purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., experimental animal quality certificate No.
  • the blank control group (Standard Laboratory Diet, STD, 6 mice) fed with common feed, the pseudo-aseptic mouse model group (Pseudoaseptic Model, MOD, 30 mice) fed with high-fat and high-sugar feed and mixed antibiotics; the mice in the three groups were fed with different feeds respectively, the STD group and the HFS group were given ordinary sterile drinking water, and the MOD group was given free drinking water containing mixed antibiotics; the mixed antibiotic water was prepared according to the following ratio: Lin 1g, neomycin sulfate 1g, metronidazole 1g, vancomycin 0.5g, dissolved in 1L of sterile water, replaced once a day; modeling 28 days, free access to water and food, weighed once a week, and pay attention to observe the mental and activity state of the mice.
  • Fecal flora donor animal pretreatment In the SPF environment of the experimental animal center with a temperature of 24 ⁇ 2°C, a relative humidity of 50%-60%, and a 12-hour light-dark rhythm, 30 healthy, non-specific pathogen (SPF grade) 7-week-old male mice were adaptively fed with standard laboratory feed for 1 week, and then they were randomly divided into two groups: the blank control group (6) was fed with laboratory standard feed, and the obesity model group (24) was fed with high-fat and high-sugar D1 2327 diet, after 8 weeks of feeding, the obese mice were sorted according to body weight, and the obesity-resistant mice with less weight gain were eliminated.
  • SPF grade non-specific pathogen
  • the obese mice whose body weight is 20% higher than the normal diet healthy mice were identified as the successful establishment of the obesity model mice.
  • the obesity model mice were divided into four groups: the obesity model group (6), the drug group high, middle and low dose groups (6 each); except the blank control group which was fed with common standard feed, the other mice were given D12327 feed; according to the human-mouse dose conversion formula, the dosage of the mice was determined.
  • the high, medium and low doses of the drug group were 1700 mg/kg, 850 mg/kg, and 425 mg/kg, respectively.
  • mice in the drug group were given the corresponding dose of medicine by intragastric administration every day, and the blank group and the model group were given the same amount of normal saline by intragastric administration; after 8 weeks of intragastric administration, the fresh feces of the mice in each group were collected in a sterile environment, and each mouse collected about 300mg of feces.
  • Extraction of fecal flora Process the mouse feces collected in each group separately, weigh 150-180mg of feces, add sterile 1mL phosphate buffered saline buffer solution and sterile ceramic beads, and suspend with a vortex shaker in an anaerobic environment to obtain a suspension; at 25°C, 500rpm, centrifuge for 10min, use a pipette gun to quickly absorb the supernatant into another EP tube; centrifuge at 4000rpm for 5min, and remove the supernatant , keep the bacterial precipitate; add sterile phosphate-buffered saline buffer solution again, and wash twice in the same way; add 60% sterile glycerol according to the ratio of 1:2, combine and mix, and store for a short time in an anaerobic environment for later use, and obtain 5 groups of fecal bacterial extracts for later use.
  • Fecal flora transplantation experiment 28 days after modeling, the mice in the pseudo-sterile mouse model group were randomly divided into 5 groups: Control, Model, COST-H, COST-M, and COST-L, with 6 mice in each group; 6 mice in the STD group continued to be given ordinary standard feed; 5 groups of pseudo-sterile mice plus the STD group of healthy control mice, a total of 6 groups, and then the 5 groups of pseudo-sterile model mice were gavaged with the corresponding 5 groups of fecal bacterial extracts, 100 ⁇ L/10g Once a day, for 6 weeks, the STD group was given the same amount of 0.9% NaCl solution for injection for intragastric gavage.
  • Body weight During the FMT experiment, observe and record the body weight of the mice during the experiment period, and weigh the mice once a week;
  • Histopathological section Dissect and collect the liver and subcutaneous adipose tissue of each group of mice, wash with phosphate buffered saline solution to remove residual blood, and fix in 10% PFA tissue fixative solution; after taking out the tissue, wash it with 75% absolute ethanol, and then use gradient concentration ethanol to carry out automatic dehydration step by step in an automatic dehydrator; Freeze and harden on a freezing platform at -20°C; after the tissue wax block is completely hardened, quickly slice it with a thickness of 5 ⁇ m, and quickly place the wax slice on warm water at 43°C for spreading, and bake at 52°C; stain the cytoplasm and nucleus of the tissue with a hematoxylin and eosin (HE) staining kit, and then observe.
  • HE hematoxylin and eosin
  • Serum biochemical indicators after taking blood from the mouse orbit, let it stand for 30 minutes, centrifuge at 3000rpm for 15 minutes, collect the upper serum, and perform a second centrifugation under the same conditions; determine the contents of total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), free fatty acid (NEFA), total bile acid (TBA) and blood glucose (GLU) in the serum according to the instructions of the kit.
  • TC total cholesterol
  • TG triglyceride
  • HDL-C high-density lipoprotein
  • LDL-C low-density lipoprotein
  • NEFA free fatty acid
  • TAA total bile acid
  • GLU blood glucose
  • mice in COST-H, COST-M, and COST-L groups still have a good effect of inhibiting weight gain after transplanting fecal bacterial extracts, indicating that the preparation of chitosan oligosaccharide enteric-coated capsules in the present invention can effectively regulate the intestinal flora of mice, so that they can still effectively resist the obesity-promoting effect of high-fat and high-sugar diet after transplantation.
  • the COST-H, COST-M, and COST-L group mice had less lipid accumulation in the liver tissue, smaller fat cells, and uniform fat size, and the disorder and damage of the ileal villi of the small intestine tissue were also well improved, indicating that the chitosan oligosaccharide enteric-coated capsules prepared by the present invention can interfere with intestinal flora, indirectly inhibit fat cell hypertrophy caused by a high-fat and high-sugar diet, regulate the growth and expansion of fat cells, reduce fat accumulation, and improve symptoms such as obesity.
  • Bacteria can reduce the intake of dietary fat in high-fat and high-sugar diets, reduce total cholesterol, triglycerides, low-density lipoproteins, free fatty acids and total bile acids in serum, increase high-density lipoprotein content, thereby realizing anti-obesity effect; blood sugar (ALT) is also significantly reduced, showing a good auxiliary blood sugar regulation effect, so the intestinal flora after the influence of the chitosan oligosaccharide enteric-coated capsules prepared by the present invention can reduce the intake of dietary polysaccharides in high-fat and high-sugar diets, thereby achieving the effect of weight loss.
  • ALT blood sugar

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Botany (AREA)
  • Zoology (AREA)
  • Child & Adolescent Psychology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)

Abstract

本发明公开了一种壳寡糖肠溶胶囊，包括内容物和胶囊壳，内容物包括肠溶辅料、壳寡糖和释放调节剂，其中肠溶辅料、释壳寡糖和释放调节剂的质量比为(3-5):1:1；本发明利用冷冻干燥法制备壳寡糖肠溶胶囊内容物，相对于传统制剂工艺，可很大程度减少辅料用量，工艺合理稳定，并能良好掩盖壳寡糖的气味提高患者顺应性，拥有"胃不溶，肠速溶"的肠溶制剂特性，并能良好掩盖壳寡糖的气味提高患者顺应性，并且可以降低其在胃酸中的降解并增加其分子原型在肠道内的释放与吸收。

Description

一种壳寡糖肠溶胶囊及其制备方法和应用 技术领域
本发明涉及药物制剂技术领域，具体为一种壳寡糖肠溶胶囊及其制备方法和应用。
背景技术
肥胖症是一种相对复杂的慢性内分泌疾病，其主要原因和表现是体内脂肪过度积累，与糖尿病、非酒精性脂肪肝、高脂血症等代谢性疾病密切相关。由肥胖造成的不良影响主要分为两类：一类是由于体内脂肪组织过度蓄积而引起的症状(如骨关节炎，阻塞性睡眠呼吸暂停，形态外观引起的耻辱感)，另一类是由于肥胖症状态下其他组织器官中脂肪细胞数量过度增加而引起的疾病(T2DM，心血管疾病，非酒精性脂肪肝病)。另外，肥胖与各种并发症之间联系强度也是不同的。肥胖症与T2DM之间联系最为紧密，64％男性T2DM患者及77％女性T2DM患者均存在体内脂肪过度蓄积。体内脂肪过度积累会影响机体对胰岛素敏感性，甚至导致发生胰岛素抵抗现象，同时使人体处于长期慢性促炎状态。最近的临床病理学数据统计结果表明，肥胖会增加新型冠状病毒肺炎患病风险。由此可见，肥胖症作为一种相对复杂的内分泌病种，会使其他疾病患病风险大大提高。同时国内外肥胖症患病率越趋严峻，越来越成为人类生活中又一健康威胁因素。
现今对于肥胖症的治疗，主要有生活方式干预(能量摄入与消耗的调节)、手术治疗(对消化系统组织器官进行手术)及药物治疗(抑制脂质合成及促进脂质代谢)三种方式。生活方式干预是减肥最健康的方法，但同时亦存在体重容易反弹及患者难以长期坚持导致最终减肥失败。面对较为严重的肥胖症患者，临床医生更倾向于选择使用药物和手术作为主要治疗方式，生活干预作为辅助治疗手段。手术治疗是减肥最有效的治疗方法，但同时会带来较大的不良反应，减肥手术期间发病率及死亡率仍分别高达4.5％和0.3％，因此，相对于手术及生活方式干预，结合治疗效果、治疗范围及不良反应，最佳选择应该是安全的药物治疗。药物治疗效果显著，但其安全性成为治疗药物应用的制约性因素。有记录以来共有15种抗肥胖药物获得FDA批准上市使用，但由于其毒副作用，已有11种药物被撤市。在剩下四个药物中，只有Orlistat成为唯一OTC抗肥胖药物。但现已因肝肾毒性而被FDA警告。因此，迫切需要找到一种新的安全、有效的抗肥胖药物。
肠道菌群可通过微小而紧密的调节作用影响肥胖症发展，肠道微生物结构组成可影响人体代谢最终引起体重变化。越来越多肥胖症相关菌群被发现，这些细菌丰度变化可促进/抑制肥胖症发展。肠道菌群进行定向改造的思路及方法被很多学者认为是未来治疗肥胖症的新方向。许多来自于天然产物的多糖可通过肠道菌群表现出良好的降脂活性。壳寡糖(COST)是 由甲壳素脱乙酰化和水解而成的天然产物，特定分子量的壳寡糖(chitosan oligosaccharides，平均Mw≤1000Da，壳寡糖)由氨基葡萄糖和N-乙酰氨基葡萄糖通过β-1,4糖苷键链接而成，聚合度范围为2-10。与壳聚糖相比，壳寡糖在水中溶解度高且粘度较低，容易被机体吸收，生物利用度更好，在农林、医学、材料化学、食品科学等领域有广泛应用。壳寡糖具有良好抗肥胖活性，能有效减少肥胖动物血清脂质水平，增加脂质排泄，减少体内脂肪堆积，缓解肝脏脂质代谢紊乱状态，还能有效调节肥胖小鼠肠道菌群，降低F/B比值，恢复受损肠屏障，减少革兰氏阴性细菌(G-)释放LPS入血，降低炎症因子水平，改善内毒素血症导致的肥胖。因此研究一种带有壳寡糖的抗肥胖药物，具有十分重要的意义。在壳寡糖制剂生产中，容易出现分层、浑浊、沉淀、颜色不均匀、等现象，使得壳寡糖制剂稳定性不佳；中国专利文献CN111617030A在2020年9月4日公开了壳寡糖口服液及其在制备减肥药物中的应用，壳寡糖口服溶液容易被胃酸降解，导致作用于肠道菌群的壳寡糖量减少，降低了药效；中国专利文献CN108653317A在2018年10月16日公开了一种壳寡糖口服溶液及其制备方法，其是由壳寡糖25-40份，矫味剂1-3份、防腐剂2-4份、稳定剂1.5-2份制备而成的溶液，大量辅料的加入会导致同体积下主药含量降低，同时也会容易被胃部降解。因此制备一种“胃不溶，肠速溶”的壳寡糖制剂具有重大的意义。
发明内容
为了克服现有技术中的不足，本发明提供一种壳寡糖肠溶胶囊，包括内容物和胶囊壳，所述内容物包括肠溶辅料、壳寡糖和释放调节剂。
进一步的，所述肠溶辅料为尤特奇L100，所述释放调节剂为羟丙基甲基纤维素，所述胶囊壳为耐酸型植物胶囊壳。
进一步的，所述壳寡糖与肠溶辅料的质量比为1：(4-5)。
进一步的，所述肠溶辅料、壳寡糖和释放调节剂的质量比为(3-5)：1：1。
本发明还提供一种壳寡糖肠溶胶囊的制备方法，包括以下步骤：
S1、溶解：称量肠溶辅料，用水溶解并静置溶胀，得到肠溶辅料溶液；称量释放调节剂，用溶解并静置，得到释放调节剂溶液；称量壳寡糖，用水溶解，得到壳寡糖溶液；
S2、搅拌：将壳寡糖溶液、肠溶辅料溶液和释放调节剂溶液混合，然后搅拌，得混合药液；
S3、超声处理：将所述混合药液放入超声仪中，超声处理；
S4、冷冻干燥：将超声处理后的混合药液进行冷冻固化后，再进行真空冷冻干燥，得到固体药物；
S5、研磨过筛：将所述固体药物进行研磨，过筛，获得胶囊内容物颗粒，然后填充胶囊 壳，包装，即得。
进一步的，所述的搅拌为磁力搅拌器剧烈搅拌0.5h。
进一步的，所述的超声处理的温度为35-40℃。
进一步的，所述的冷冻固化温度为-80℃。
进一步的，所述的真空冷冻干燥时间为11-13h。
本发明的另一目的在于提供由上述的壳寡糖肠溶胶囊的制备方法制备的壳寡糖肠溶胶囊在减肥药物中的应用。
与现有技术相比，本发明的有益效果是：
1、本发明制备的壳寡糖胶囊使用肠溶胶囊，相对其他肠溶剂型，肠溶胶囊比常用的肠溶片剂使用的辅料更少，能最大程度地减小药物辅料的体积，并能良好掩盖壳寡糖的气味提高患者顺应性，并且可以降低其在胃酸中的降解并增加其分子原型在肠道内的释放与吸收；
2、本发明制备的壳寡糖肠溶胶囊的胶囊壳使用的耐酸型植物胶囊壳(Drcaps)，含量最高的材料是羟丙基甲基纤维素(HPMC)，在胃液中不易溶解，保护壳寡糖肠溶胶囊内容物不受胃酸损害，具有明显的肠溶特性，并且可掩盖味道和气味，防止不良口腔余味和反胃；
3、本发明制备的壳寡糖肠溶胶囊选择尤特奇L100作为最佳的肠溶载体，制成的胶囊内容物颗粒，在胃中不溶解，但在pH6以上缓冲液中可以溶解，安全无毒，并且更好的掩盖住壳寡糖的铁腥味；
4、本发明制备的壳寡糖肠溶胶囊选择羟丙基甲基纤维素(HPMC)作为释放调节，可以调节壳寡糖在肠道中的释放度，促进壳寡糖肠溶胶囊的药效发挥；
5、本发明制备的壳寡糖肠溶胶囊可应用于减肥药物，间接抑制高脂高糖饮食引起的脂肪细胞肥大，调节脂肪细胞生长扩大，减少脂肪堆积，改善肥胖等症状。
实施例1、本发明壳寡糖肠溶胶囊及其制备
配方：壳寡糖10g、尤特奇L10040g、HPMC10g、Drcaps胶囊壳
制备过程：
S1、溶解：用水溶解尤特奇L100并静置溶胀0.5h，得尤特奇L100溶液；用水溶解HPMC并静置0.5h，得HPMC溶液；用水将壳寡糖完全溶解，得壳寡糖溶液；
S2、搅拌：将壳寡糖溶液边搅拌边缓慢加入到尤特奇L100溶液中，然后加入HPMC溶液，再用磁力搅拌器剧烈搅拌0.5h，得混合药液；
S3、超声处理：将混合药液放入超声仪中，并在37℃下以最高频率超声处理0.5h；
S4、冷冻干燥：将超声处理后的混合药液，使用保鲜膜封口，迅速置于-80℃超低温冰箱中冷冻固化过夜，然后取出完全凝结固化的混合药物，使用注射器针头刺穿保鲜膜，留置一 定数量的的通气孔。将混合药物固体放在预先冷却的冷冻设备中，进行12h的真空冷冻干燥，得到固体药物；
S5、研磨过筛：将固体药物在干燥洁净的超净台上进行研磨，用80目筛子过筛，获得胶囊内容物颗粒，然后填充进Drcaps胶囊壳，包装得到壳寡糖肠溶胶囊。
实施例2、本发明壳寡糖肠溶胶囊及其制备
配方：壳寡糖20g、尤特奇L10080g、HPMC 20g、Drcaps胶囊壳
制备过程：
S1、溶解：用水溶解尤特奇L100并静置溶胀0.5h，得尤特奇L100溶液；用水溶解HPMC并静置0.5h，得HPMC溶液；用水将壳寡糖完全溶解，得壳寡糖溶液；
S2、搅拌：将壳寡糖溶液边搅拌边缓慢加入到尤特奇L100溶液中，然后加入HPMC溶液，再用磁力搅拌器剧烈搅拌0.5h，得混合药液；
S3、超声处理：将混合药物放入超声仪中，并在37℃下以最高频率超声处理0.5h；
S4、冷冻干燥：将超声处理后的混合药液，使用保鲜膜封口，迅速置于-80℃超低温冰箱中冷冻固化过夜，然后取出完全凝结固化的混合药物，使用注射器针头刺穿保鲜膜，留置一定数量的的通气孔。将混合药物固体放在预先冷却的冷冻设备中，进行12h的真空冷冻干燥，得到固体药物；
S5、研磨过筛：把固体药物在干燥洁净的超净台上进行研磨，用80目筛子过筛，获得胶囊内容物颗粒，然后填充进Drcaps胶囊壳，包装得到壳寡糖肠溶胶囊。
实施例3、本发明壳寡糖肠溶胶囊及其制备
配方：壳寡糖30g、尤特奇L100120g、HPMC 30g、Drcaps胶囊壳
制备过程：
S1、溶解：用水溶解尤特奇L100并静置溶胀0.5h，得尤特奇L100溶液；用水溶解HPMC并静置0.5h，得HPMC溶液；用水将壳寡糖完全溶解，得壳寡糖溶液；
S2、搅拌：将壳寡糖溶液边搅拌边缓慢加入到尤特奇L100溶液中，然后加入HPMC溶液，再用磁力搅拌器剧烈搅拌0.5h，得混合药液；
S3、超声处理：将混合药液放入超声仪中，并在37℃下以最高频率超声处理0.5h；
S4、冷冻干燥：将超声处理后的混合药液，使用保鲜膜封口，迅速置于-80℃超低温冰箱中冷冻固化过夜，然后取出完全凝结固化的混合药物，使用注射器针头刺穿保鲜膜，留置一定数量的的通气孔，将混合药固体放在预先冷却的冷冻设备中，进行12h的真空冷冻干燥，得到固体药物；
S5、研磨过筛：将固体药物在干燥洁净的超净台上进行研磨，用80目筛子过筛，获得胶 囊内容物颗粒，然后填充进Drcaps胶囊壳，包装得到壳寡糖肠溶胶囊。
对比例1
配方：壳寡糖10g、尤特奇L10040g、尤特奇NM30D 10g、Drcaps胶囊壳
制备过程：与实施例1相同
本对比例与实施例1的区别在于，本对比例的释放调节剂为尤特奇NM30D，其余步骤和成分、参数都一样。
对比例2
配方：壳寡糖10g、Drcaps胶囊壳
制备过程：用水将壳寡糖完全溶解，得壳寡糖溶液，放入超声仪中，并在37℃下以最高频率超声处理0.5h；超声处理后的壳寡糖溶液，使用保鲜膜封口，迅速置于-80℃超低温冰箱中冷冻固化过夜，然后取出完全凝结固化的混合药物，使用注射器针头刺穿保鲜膜，留置一定数量的通气孔。将壳寡糖药物固体放在预先冷却的冷冻设备中，进行12h的真空冷冻干燥；干燥后，将其取出，在干燥洁净的超净台上研磨，用80目筛子过筛，然后填充进Drcaps胶囊壳，包装得到壳寡糖肠溶胶囊。
本对比例与实施例1的区别在于，本对比例的壳寡糖肠溶胶囊没有肠溶辅料和释放调节剂。
对比例3
配方：壳寡糖10g、尤特奇L10040g、HPMC10g、普通明胶胶囊壳
本对比例与实施例1的区别在于，本对比例使用普通明胶胶囊壳作为囊壳，其余组分、参数和步骤与实施例1相同。
对比例4
配方：壳寡糖10g、尤特奇L10060g、HPMC 10g、Drcaps胶囊壳
本对比例与实施例1的区别在于，本对比例的制成的肠溶胶囊内容物：壳寡糖：尤特奇L100：HPMC＝1：6：1；其余成分、参数和步骤与实施例1相同。
对比例5
配方：壳寡糖10g、尤特奇L100-55 50g、HPMC 10g；Drcaps胶囊壳
本对比例与实施例1的区别在于，本对比例使用尤特奇L100-55作为肠溶辅料，其余成分、参数和步骤与实施例1相同。
试验例一、本发明制备的壳寡糖胶囊的装量差异、主药含量检查
1、试验样品：实施例1-3制备的壳寡糖肠溶胶囊。
2、试验方法：随机抽取实施例1-3制备所得的肠溶胶囊共20粒，称定每粒肠溶胶囊重量及空胶囊壳重量，用减重法计算每粒胶囊内容物的重量，根据20粒肠溶胶囊的称量结果计算其平均装量及装量差异限度；随机抽取实施例1-3所制备的肠溶胶囊共6粒，检测样品中的COST含量，采用苯酚-硫酸法，利用多糖在过量浓硫酸作用下水解成单糖，将其脱水形成糠醛衍生物，衍生物与苯酚反应形成具有最大吸收峰的橙红色物质，采用比色法进行多糖质量的测定，从而检测样品中壳寡糖的含量；
3、试验结果：
本发明制备的壳寡糖肠溶胶囊的平均装量为0.2445g。平均装量最大值与平均值比较，装量差异为8.82％；平均装量最小值与平均值比较，装量差异为-7.34％。装量差异实验结果均未超出装量差异限度，符合《中国药典》(2020版)的相关要求；
本发明实施例1制备的壳寡糖肠溶胶囊的平均主药含量为16.66％，相对标准偏差为1.3％。其含量测试实验结果均未超出含量差异限度，符合《中国药典》(2020版)的相关要求。
试验例二：壳寡糖肠溶胶囊质量检查
1、试验方法：观察实施例1-3、对比例4-5制备过程中是否有沉淀或者研磨不均匀现象，观察制备好的胶囊的外形。
2、试验结果：
表1、壳寡糖肠溶胶囊的质量检测
Figure PCTCN2022130809-appb-000001
由表1可知，本发明实施例1-3制备的壳寡糖溶胶囊洁净无异味，没有发现变形的现象，且囊体光滑，无吸湿黏连的现象，能保持干燥状态，并无变脆或出现渗漏破裂的现象，其外观检查符合《中国药典》(2020版)的相关要求；而对比例4制备的胶囊由于辅料比例过大， 辅料析出沉淀，且会使胶囊填充物体积增大，而导致胶囊外观有破裂、变形、渗漏现象；对比例5制备的肠溶胶囊由于使用尤特奇L100-55作为肠溶辅料，胶囊的内容物有颗粒且颜色不均匀，也不符合《中国药典》(2020版)的要求。
试验例三、壳寡糖胶囊的崩解时限检查
1、试验样品：实施例1-3和对比例3所制备的壳寡糖肠溶胶囊
2、试验方法：根据《中国药典》(2020版)的相关操作要求，检测其在人工胃液中及人工肠液中内的崩解情况。
表2、胶囊在不同介质中的崩解时限
Figure PCTCN2022130809-appb-000002
根据表2可知，对比例3制备的壳寡糖胶囊在人工胃液及肠液中15min内即可崩解，且在人工胃液中的崩解时限比在肠液中短，因为对比例3使用的是普通明胶胶囊壳，与肠溶胶囊所要求的“胃不溶，肠速溶”的特性相悖。相反的，本发明实施例1-3制备的壳寡糖肠溶胶囊在人工胃液中能保持2h以上不崩解，而在碱性的肠液中迅速崩解，崩解时限为8min，即可在1h内完全崩解,符合“胃不溶，肠速溶”的特性，也符合《中国药典》(2020版)的相关要求。
试验例四、壳寡糖胶囊的释放度检测
1、试验方法：根据《中国药典》(2020版)规定进行操作，测试人工胃液和人工肠液中的总糖累计释放度
2、试验结果：
表3、人工胃液中的总糖累计释放度(％)
样品 0.25h 0.5h 1h 2h 3h 4h 5h 6h
实施例1 0.57 1.12 2.86 5.35 15.83 21.37 27.56 35.66
实施例2 0.56 1.10 2.71 5.32 15.68 21.28 26.78 35.24
实施例3 0.52 1.13 2.68 5.41 15.72 21.41 27.12 34.89
对比例1 0.45 0.75 1.62 2.53 12.81 16.33 25.61 29.52
对比例2 95.68 100 100 100 100 100 100 100
表4、人工肠液中的总糖累计释放度(％)
样品 5min 10min 15min 20min 30min 60min 90min 120min
实施例1 33.54 59.84 76.61 83.23 91.07 95.61 99.54 100
实施例2 33.25 58.47 75.89 83.54 91.01 94.99 99.05 100
实施例3 35.97 60.35 76.44 84.68 91.25 95.95 100 100
对比例1 0.75 1.06 1.93 3.51 15.33 25.61 31.57 45.69
对比例2 95.72 98.86 100 100 100 100 100 100
由表3-4可知，对比例2制备的胶囊，由于没有肠溶辅料和释放调节剂，壳寡糖在人工胃液和人工肠液中都很快100％溶解，不符合肠溶胶囊“胃不溶，肠难溶”的特性；而实施例1-3和对比例1制备的胶囊在人工胃液中对累计释放度的影响效果相似，但在人工肠液中调节效果则出现较大差异，其中对比例1制备的胶囊在肠液中120min内累计释放度只有45.69％，不符合《中国药典》(2020版)的相关要求，因为肠溶辅料尤特奇NM 30D极大地抑制了壳寡糖在肠液中的溶出和释放；而实施例1-3制备的胶囊在肠液中120min内的累计释放度影响较小，在30min内即可溶出90％以上，在120min内达到100％溶出，符合《中国药典》(2020 版)的相关要求，符合肠溶特性，因此，实施例1-3制备的壳寡糖肠溶胶囊是具备肠溶特性的。
试验例五、壳寡糖作用于肠道菌群
1、试验方法：
伪无菌小鼠模型建立：选取健康、无特异性病原体(SPF级)的7周龄雄性C57BL/6J小鼠36只，购自湖南斯莱克景达实验动物有限公司，实验动物质量证书号NO.110727201003661，实验动物饲养于广东药科大学实验动物中心SPF级实验室(NO：SPF2017288)，小鼠适应性喂养1周后，随机分为2组：给与实验室标准普通饲料喂养的空白对照组(Standard Laboratory Diet,STD,6只)、给与高脂高糖饲料喂养并给与混合抗生素的伪无菌小鼠模型组(Pseudoaseptic Model,MOD,30只)；三组小鼠分别给与不同饲料进行喂养，STD组与HFS组给与普通无菌饮用水自由饮用，MOD组给与含有混合抗生素的无菌饮用水自由饮用；混合抗生素水按以下比例配制：氨苄西林1g、硫酸新霉素1g、甲硝唑1g、万古霉素0.5g，溶于1L无菌水中，每天更换一次；造模28天，自由进水进食，每周称量体重一次，并注意观察小鼠的精神及活动状态。
粪便菌群供体动物预处理：在温度为24±2℃，相对湿度为50％-60％，12小时明暗节律的实验动物中心的SPF环境中，健康、无特异性病原体(SPF级)的7周龄雄性小鼠30只，适应性地喂食标准实验室饲料1周，然后将它们随意地分成2个组：空白对照组(6只)饲喂实验室的标准普通饲料，肥胖模型组(24只)饲喂高脂高糖D12327饲料，喂养8周后，按照体重对肥胖小鼠进行排序，并淘汰体重增加较少的肥胖抵抗小鼠。根据啮齿类肥胖动物模型的造模的体重标准，将体量高于普通饮食健康小鼠体重20％的肥胖小鼠认定为成功建立肥胖模型小鼠。将肥胖模型小鼠分为四组：肥胖模型组(6只)、药物组高、中、低剂量组(各6只)；除空白对照组给与普通标准饲料喂养外，其他小鼠均给与D12327饲料；根据人体-小鼠剂量转换公式，确定小鼠的给药剂量，药物组高、中、低剂量分别为1700mg/kg、850mg/kg、425mg/kg。药物组小鼠每天灌胃给与对应剂量的药，空白组和模型组灌胃给与等量生理盐水；灌胃8周后，在无菌环境下，收集每组小鼠的新鲜粪便，每只小鼠收集约300mg粪便。
粪便菌群的提取：把每组收集所得的小鼠粪便分别进行处理，称取150-180mg的粪便，加入无菌1mL磷酸缓冲盐溶液缓冲液和无菌陶瓷珠，并在无氧环境下用涡旋振荡器混悬得到悬浊液；在25℃下，500rpm，离心10min，用移液枪迅速吸取上清液至另一EP管中；再以4000rpm的速度离心5min，吸除上清液，保留细菌沉淀；再次加入无菌磷酸缓冲盐溶液缓冲 液，以同样的方式再清洗2次；按照1:2的比例加入60％无菌甘油，合并混匀，于无氧环境下短时间保存备用，得到5组粪便细菌提取液,备用。
粪便菌群移植实验(FMT实验)：造模28天后，将伪无菌小鼠模型组小鼠随机分成5组：Control、Model、COST-H、COST-M、COST-L，每组各6只；STD组6只小鼠继续给与普通标准饲料；伪无菌小鼠5个组加健康对照组小鼠STD组，共6个组，然后5组伪无菌模型小鼠分别灌胃对应的5组粪便细菌提取液，100μL/10g的量，每天一次，持续6周，STD组给与等量注射用的0.9％NaCl溶液进行灌胃。
体重：FMT实验期间，观察并记录实验期内小鼠的体重、每周一次称量小鼠体重；
组织病理学切片：解剖收集每组小鼠肝，皮下脂肪组织，用磷酸缓冲盐溶液清洗去除残留血污后，于10％PFA组织固定液中进行固定；取出组织后，先用75％无水乙醇冲洗，再用梯度浓度乙醇于全自动脱水机中进行自动逐级脱水；脱水完成后，用Leica高熔点石蜡(60℃)于Leica EG1160一体式石蜡包埋机中进行包埋，将包埋好的石蜡模具块置于-20℃冷冻台上冷冻硬化；待组织蜡块完全硬化后，以5μm为厚度迅速进行切片，并将蜡片迅速置于43℃温水上进行摊片，52℃下烤片；采用苏木素伊红(HE)染色试剂盒对组织的细胞质及细胞核等进行染色，然后观察。
血清生化指标：从小鼠眼眶取血后，静置30min，以3000rpm离心15min，收集上层血清，以同样的条件进行二次离心；根据试剂盒的说明书测定血清中总胆固醇(TC)，甘油三酯(TG)，高密度脂蛋白(HDL-C)，低密度脂蛋白(LDL-C)，游离脂肪酸(NEFA)、总胆汁酸(TBA)和血糖(GLU)的含量。
2、试验结果：
(1)小鼠体重
表5、FMT实验期间小鼠体重变化
组别 FMT实验前体重(g) FMT实验后体重(g) 体重增加(g)
STD 26.58 28.84 2.26
Control 27.68 29.85 2.17
Model 27.67 32.49 4.82
COST-H 26.95 26.19 -0.76
COST-M 27.15 27.38 0.23
COST-L 27.19 27.88 0.69
由表5可知，COST-H、COST-M、COST-L组的小鼠，在移植粪便细菌提取液后仍拥有良好的抑制体重增重效果，说明本发明制备壳寡糖肠溶胶囊可以有效调节小鼠肠道菌群，使其移植后仍能够有效对抗高脂高糖饮食带来的促肥胖效应。
(2)FMT实验小鼠组织病理学切片
表6、小鼠组织病理学切片情况表
Figure PCTCN2022130809-appb-000003
由表6数据可知，相对于肥胖模型Model组小鼠，COST-H、COST-M、COST-L组小鼠的肝脏组织脂质积累少，脂肪细胞体积小，且脂肪大小均匀，小肠组织的回肠肠绒毛的紊乱及损伤状态也得到了很好的改善，说明本发明制备的壳寡糖肠溶胶囊能干预肠道菌群，间接抑制高脂高糖饮食引起的脂肪细胞肥大，调节脂肪细胞生长扩大，减少脂肪堆积，改善肥胖等症状。
(3)FMT实验小鼠血清生化指标
表7、小鼠血清生化指标表
Figure PCTCN2022130809-appb-000004
由表7中数据可知，相对于Model组小鼠，COST-H、COST-M、COST-L组小鼠的血液中的总胆固醇(TC)，甘油三酯(TG)，低密度脂蛋白(LDL-C)，游离脂肪酸(NEFA)、总胆汁酸(TBA)和血糖(GLU)显著降低，而高密度脂蛋白(HDL-C)含量增高，其中COST-H组的效果更显著，说明有壳寡糖干预后的肠道菌群能减少高脂高糖饮食中膳食脂肪的摄入，减少血清中的总胆固醇、甘油三酯、低密度脂蛋白、游离脂肪酸和总胆汁酸，增加了高密度脂蛋白含量，从而实现抗肥胖的效果；血糖(ALT)也显著降低，表现出良好辅助的血糖调节作用，所以本发明制备的壳寡糖肠溶胶囊影响后的肠道菌群能减少高脂高糖饮食中膳食多糖的摄入，从而达到减肥的作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种壳寡糖肠溶胶囊，其特征在于，包括内容物和胶囊壳，所述内容物包括肠溶辅料、壳寡糖和释放调节剂。
  2. 如权利要求1所述的壳寡糖肠溶胶囊，其特征在于，所述肠溶辅料为尤特奇L100，所述释放调节剂为羟丙基甲基纤维素，所述胶囊壳为耐酸型植物胶囊壳。
  3. 如权利要求1所述的壳寡糖肠溶胶囊，其特征在于，所述壳寡糖与肠溶辅料的质量比为1:(4-5)。
  4. 如权利要求1所述的壳寡糖肠溶胶囊，其特征在于，所述肠溶辅料、壳寡糖和释放调节剂的质量比为(3-5)：1：1。
  5. 一种如权利要求1-4所述的壳寡糖肠溶胶囊的制备方法，其特征在于，包括以下步骤：
    S1、溶解：称量肠溶辅料，用水溶解并静置溶胀，得到肠溶辅料溶液；称量释放调节剂，用水溶解并静置，得到释放调节剂溶液；称量壳寡糖，用水溶解，得到壳寡糖溶液；
    S2、搅拌：将壳寡糖溶液、肠溶辅料溶液和释放调节剂溶液混合，然后搅拌，得混合药液；
    S3、超声处理：将所述混合药液放入超声仪中，超声处理；
    S4、冷冻干燥：将超声处理后的混合药液进行冷冻固化后，再进行真空冷冻干燥，得到固体药物；
    S5、研磨过筛：将所述固体药物进行研磨，过筛，获得胶囊内容物颗粒，然后填充胶囊壳，包装，即得。
  6. 如权利要求5所述的壳寡糖肠溶胶囊的制备方法，其特征在于，所述搅拌为磁力搅拌器剧烈搅拌0.5h。
  7. 如权利要求5所述的壳寡糖肠溶胶囊的制备方法，其特征在于，所述超声处理温度为35-40℃。
  8. 如权利要求5所述的壳寡糖肠溶胶囊的制备方法，其特征在于，所述冷冻固化温度为-80℃。
  9. 如权利要求5所述的壳寡糖肠溶胶囊的制备方法，其特征在于，所述真空冷冻干燥时间为11-13h。
  10. 如权利要求1-4任一项所述的壳寡糖肠溶胶囊在制备减肥药物中的应用。
PCT/CN2022/130809 2022-01-18 2022-11-09 一种壳寡糖肠溶胶囊及其制备方法和应用 WO2023138172A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210054442.9A CN114376982B (zh) 2022-01-18 2022-01-18 一种壳寡糖肠溶胶囊及其制备方法和应用
CN202210054442.9 2022-01-18

Publications (1)

Publication Number Publication Date
WO2023138172A1 true WO2023138172A1 (zh) 2023-07-27

Family

ID=81204148

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/130809 WO2023138172A1 (zh) 2022-01-18 2022-11-09 一种壳寡糖肠溶胶囊及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN114376982B (zh)
WO (1) WO2023138172A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114376982B (zh) * 2022-01-18 2023-06-30 广东药科大学 一种壳寡糖肠溶胶囊及其制备方法和应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005068094A (ja) * 2003-08-26 2005-03-17 Nof Corp 腸溶性製剤およびその製造方法
CN105380924A (zh) * 2015-12-24 2016-03-09 广东药学院 一种壳寡糖胶囊剂及其制备方法
CN108553425A (zh) * 2018-06-01 2018-09-21 广东药科大学 一种壳寡糖滴丸及其制备方法
CN111617030A (zh) * 2020-05-22 2020-09-04 广东药科大学 壳寡糖口服液及其在制备减肥药物中的应用
CN112956699A (zh) * 2021-01-27 2021-06-15 上海海洋大学 一种降血糖肽肠溶固体分散体及其制备方法
CN114376982A (zh) * 2022-01-18 2022-04-22 广东药科大学 一种壳寡糖肠溶胶囊及其制备方法和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005068094A (ja) * 2003-08-26 2005-03-17 Nof Corp 腸溶性製剤およびその製造方法
CN105380924A (zh) * 2015-12-24 2016-03-09 广东药学院 一种壳寡糖胶囊剂及其制备方法
CN108553425A (zh) * 2018-06-01 2018-09-21 广东药科大学 一种壳寡糖滴丸及其制备方法
CN111617030A (zh) * 2020-05-22 2020-09-04 广东药科大学 壳寡糖口服液及其在制备减肥药物中的应用
CN112956699A (zh) * 2021-01-27 2021-06-15 上海海洋大学 一种降血糖肽肠溶固体分散体及其制备方法
CN114376982A (zh) * 2022-01-18 2022-04-22 广东药科大学 一种壳寡糖肠溶胶囊及其制备方法和应用

Also Published As

Publication number Publication date
CN114376982A (zh) 2022-04-22
CN114376982B (zh) 2023-06-30

Similar Documents

Publication Publication Date Title
AU2002342641B2 (en) Prebiotic and probiotic compositions and methods for their use in gut-based therapies
AU2002342641A1 (en) Prebiotic and probiotic compositions and methods for their use in gut-based therapies
CN1939296B (zh) 一种茶多酚组合物及其制备方法
JP4626752B2 (ja) 炭水化物および組成物の新規な使用
CN114558038B (zh) 一种用于减肥减脂的益生菌组合物及其制备方法和应用
WO2023138172A1 (zh) 一种壳寡糖肠溶胶囊及其制备方法和应用
EP2266582B1 (en) Pharmaceutical composition for preventing dysbiosis associated with enteral administration of antibiotics
Zhang et al. Fabrication, characterization, and lipid-lowering effects of naringenin-zein-sodium caseinate-galactosylated chitosan nanoparticles
CN112168876A (zh) 一种具有降血脂作用的组合物及其制备方法和应用
CN115025101B (zh) 一种添加石胆酸的防治霉菌毒素中毒药物及其制备方法和应用
CN110432500A (zh) 一种含菊粉的益生元组合物及制备方法和用途
CN106551915A (zh) 肠溶组合物及其制备方法
CN108619283B (zh) 一种用于改善脂质代谢、减少内脏脂肪的组合物
Zhao et al. Recent research advances on oral colon-specific delivery system of nature bioactive components: A review
TWI725030B (zh) 降低食品/飲料消化率/吸收率或減少消化液之方法及組合物
EP2739270B1 (en) Non-digestible capsules for the delivery of fluid absorbing materials
CN110448569A (zh) 一种具有止泻功效的组合物及其制备方法和其应用
US11918602B2 (en) Methods for reducing cholesterol with superabsorbent materials
US11925660B2 (en) Treatment for obesity with superabsorbent materials
WO2022162649A1 (en) Delayed release composition comprising enteric coated drug loaded in psyllium husk matrix
CN107669738B (zh) 一种活血化瘀、益气强心的中药制剂及其制备方法
CN118267374A (zh) 一种调节血脂水平的组合物、制剂及制备方法
Wu et al. Oral quercetin nanoparticles in hydrogel microspheres alleviate high-altitude sleep disturbance based on the gut-brain axis
CN117481342A (zh) 一种降低药物呕吐副作用的组合物及其制备方法和应用
CN118320052A (zh) 一种用于痛风调理的组合物、制备方法及用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22921596

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE