CN114376982B

CN114376982B - Chitosan oligosaccharide enteric-coated capsule as well as preparation method and application thereof

Info

Publication number: CN114376982B
Application number: CN202210054442.9A
Authority: CN
Inventors: 苏政权; 曹华; 白研; 贺锦灿
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2022-01-18
Filing date: 2022-01-18
Publication date: 2023-06-30
Anticipated expiration: 2042-01-18
Also published as: WO2023138172A1; CN114376982A

Abstract

The invention discloses a chitosan oligosaccharide enteric capsule, which comprises a content and a capsule shell, wherein the content comprises enteric auxiliary materials, chitosan oligosaccharide and a release regulator, and the mass ratio of the enteric auxiliary materials to the release chitosan oligosaccharide to the release regulator is (3-5) 1:1; compared with the traditional preparation process, the preparation method can greatly reduce the dosage of auxiliary materials, has reasonable and stable process, can well mask the smell of the chitosan oligosaccharide to improve the compliance of patients, has the characteristics of enteric preparation of 'insoluble stomach and instant intestine', can well mask the smell of the chitosan oligosaccharide to improve the compliance of the patients, and can reduce the degradation of the chitosan oligosaccharide in gastric acid and increase the release and absorption of molecular prototypes in intestinal tracts.

Description

Chitosan oligosaccharide enteric-coated capsule as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of pharmaceutical preparations, in particular to a chitosan oligosaccharide enteric capsule and a preparation method and application thereof.

Background

Obesity is a relatively complex chronic endocrine disease, the main cause and manifestation of which is excessive accumulation of fat in the body, and is closely related to metabolic diseases such as diabetes, non-alcoholic fatty liver disease, hyperlipidemia, etc. Adverse effects caused by obesity are largely divided into two categories: one is symptoms caused by excessive accumulation of adipose tissue in the body (such as osteoarthritis, obstructive sleep apnea, and stigma caused by morphological appearance), and the other is diseases caused by excessive increase of the number of adipocytes in other organs and tissues in an obese state (T2 DM, cardiovascular disease, nonalcoholic fatty liver disease). In addition, the strength of the link between obesity and various complications is also different. The most closely related obesity and T2DM are found in 64% of male T2DM patients and 77% of female T2DM patients, and excessive accumulation of fat in the body is found. Excessive accumulation of fat in the body can affect the body's sensitivity to insulin and even cause insulin resistance to occur, while leaving the body in a chronic, pro-inflammatory state. Recent statistics of clinical pathology data indicate that obesity increases the risk of developing new coronavirus pneumonia. It follows that obesity, a relatively complex endocrine disease, greatly increases the risk of other diseases. Meanwhile, the prevalence of obesity at home and abroad becomes more severe, and becomes a health threat factor in human life.

Today there are three main ways of treatment of obesity, lifestyle intervention (regulation of energy intake and consumption), surgical treatment (surgery on tissues and organs of the digestive system) and drug treatment (inhibition of lipid synthesis and promotion of lipid metabolism). Lifestyle interventions are the healest method of weight loss, but at the same time there is also a tendency for weight regain and for patients to persist for long periods of time leading to eventual weight loss failure. In the face of more severe obese patients, clinicians prefer to choose drugs and surgery as the primary treatment modality, and life intervention as an adjunct treatment modality. The surgical treatment is the most effective treatment method for losing weight, but simultaneously brings great adverse reaction, and the morbidity and mortality rate during the weight-losing operation still reach 4.5 percent and 0.3 percent respectively, so the optimal selection should be safe drug treatment relative to the surgical and life-style intervention and combined treatment effect, treatment range and adverse reaction. The therapeutic effect of the drug is remarkable, but the safety of the drug becomes a limiting factor for the application of the therapeutic drug. There are 15 anti-obesity drugs recorded since the FDA approval for use on the market, but 11 drugs have been withdrawn from the market due to their toxic side effects. Of the four remaining drugs, only Orlistat was the sole OTC anti-obesity drug. But are now alerted by the FDA due to hepatorenal toxicity. Therefore, there is an urgent need to find a new safe and effective anti-obesity drug.

Intestinal flora can influence the development of obesity through a small and compact regulation effect, and the structural composition of intestinal microorganisms can influence the metabolism of a human body to finally cause weight change. An increasing number of obesity-related flora is found, and these changes in bacterial abundance can promote/inhibit the development of obesity. The concept and method of directed modification of intestinal flora is considered by many scholars as a new direction for the future treatment of obesity. Many polysaccharides from natural products may exhibit good lipid lowering activity through the intestinal flora. The Chitosan Oligosaccharide (COST) is a natural product formed by deacetylation and hydrolysis of chitin, the chitosan oligosaccharide (chitosan oligosaccharides, average Mw is less than or equal to 1000Da, and the chitosan oligosaccharide) with specific molecular weight is formed by linking glucosamine and N-acetylglucosamine through beta-1, 4 glycosidic bonds, and the polymerization degree is 2-10. Compared with chitosan, the chitosan oligosaccharide has high solubility in water, low viscosity, easy absorption by organism and better bioavailability, and can be widely applied in the fields of agriculture and forestry, medicine, material chemistry, food science and the like. The chitosan oligosaccharide has good anti-obesity activity, can effectively reduce serum lipid level of obese animals, increase lipid excretion, reduce fat accumulation in vivo, relieve liver lipid metabolism disorder state, effectively regulate intestinal flora of obese mice, reduce F/B ratio, recover damaged intestinal barrier, reduce LPS released by gram-negative bacteria (G-) into blood, reduce inflammatory factor level, and improve obesity caused by endotoxemia. Therefore, research on an anti-obesity drug with chitosan oligosaccharide has very important significance. In the production of the chitosan oligosaccharide preparation, layering, turbidity, precipitation, uneven color and the like are easy to occur, so that the stability of the chitosan oligosaccharide preparation is poor; chinese patent document CN111617030A discloses chitosan oligosaccharide oral liquid and application thereof in preparing weight-losing medicaments in 9 and 4 months in 2020, wherein the chitosan oligosaccharide oral liquid is easy to be degraded by gastric acid, so that the chitosan oligosaccharide amount acting on intestinal flora is reduced, and the drug effect is reduced; the Chinese patent document CN108653317A discloses a chitosan oligosaccharide oral solution and a preparation method thereof in 10 months and 16 days of 2018, wherein the chitosan oligosaccharide oral solution is prepared from 25-40 parts of chitosan oligosaccharide, 1-3 parts of a flavoring agent, 2-4 parts of a preservative and 1.5-2 parts of a stabilizer, and the content of main drugs in the same volume is reduced due to the addition of a large amount of auxiliary materials, and meanwhile, the chitosan oligosaccharide oral solution is easy to be degraded by stomach. Therefore, the preparation of the 'stomach insoluble and intestine instant' chitosan oligosaccharide preparation has great significance.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a chitosan oligosaccharide enteric capsule which comprises a content and a capsule shell, wherein the content comprises enteric auxiliary materials, chitosan oligosaccharide and a release regulator.

Furthermore, the enteric adjuvant is Eudragit L100, the release regulator is hydroxypropyl methylcellulose, and the capsule shell is acid-resistant plant capsule shell.

Further, the mass ratio of the chitosan oligosaccharide to the enteric auxiliary material is 1: (4-5).

Further, the mass ratio of the enteric auxiliary material, the chitosan oligosaccharide and the release regulator is (3-5): 1:1.

the invention also provides a preparation method of the chitosan oligosaccharide enteric capsule, which comprises the following steps:

s1, dissolving: weighing enteric auxiliary materials, dissolving with water, standing for swelling to obtain enteric auxiliary material solution; weighing release regulator, dissolving and standing to obtain release regulator solution; weighing chitosan oligosaccharide, and dissolving the chitosan oligosaccharide with water to obtain a chitosan oligosaccharide solution;

s2, stirring: mixing the chitosan oligosaccharide solution, the enteric adjuvant solution and the release regulator solution, and stirring to obtain a mixed liquid medicine;

s3, ultrasonic treatment: placing the mixed liquid medicine into an ultrasonic instrument for ultrasonic treatment;

s4, freeze drying: freezing and solidifying the mixed liquor after ultrasonic treatment, and then performing vacuum freeze drying to obtain a solid medicine;

s5, grinding and sieving: grinding the solid medicine, sieving to obtain capsule content particles, filling capsule shells, and packaging.

Further, the stirring is that a magnetic stirrer vigorously stirs for 0.5h.

Further, the temperature of the ultrasonic treatment is 35-40 ℃.

Further, the freezing and solidifying temperature is-80 ℃.

Further, the vacuum freeze drying time is 11-13h.

The invention also aims to provide the application of the chitosan oligosaccharide enteric capsule prepared by the preparation method of the chitosan oligosaccharide enteric capsule in weight-losing medicines.

Compared with the prior art, the invention has the beneficial effects that:

1. compared with other enteric-coated preparations, the enteric-coated capsule has fewer auxiliary materials than the common enteric-coated tablets, can reduce the volume of the auxiliary materials to the greatest extent, can well cover the smell of the chitosan oligosaccharide to improve the compliance of patients, and can reduce the degradation of the chitosan oligosaccharide in gastric acid and increase the release and absorption of the molecular prototype in intestinal tracts;

2. the acid-resistant plant capsule shell (dragcaps) used for the capsule shell of the chitosan oligosaccharide enteric capsule prepared by the invention has the highest content of hydroxypropyl methylcellulose (HPMC), is not easy to dissolve in gastric juice, protects the content of the chitosan oligosaccharide enteric capsule from being damaged by gastric acid, has obvious enteric characteristic, can mask taste and smell, and prevents bad oral cavity aftertaste and regurgitation;

3. the chitosan oligosaccharide enteric capsule prepared by the invention selects Eudragit L100 as the optimal enteric carrier, and the prepared capsule content particles are insoluble in the stomach, but can be dissolved in buffer above pH6, so that the chitosan oligosaccharide enteric capsule is safe and nontoxic, and better covers the iron fishy smell of chitosan oligosaccharide;

4. the chitosan oligosaccharide enteric capsule prepared by the invention selects hydroxypropyl methylcellulose (HPMC) as release regulation, so that the release degree of chitosan oligosaccharide in intestinal tracts can be regulated, and the drug effect of the chitosan oligosaccharide enteric capsule is promoted to be exerted;

5. the chitosan oligosaccharide enteric capsule prepared by the invention can be applied to weight-losing medicines, indirectly inhibit fat cell hypertrophy caused by high-fat and high-sugar diet, regulate growth expansion of fat cells, reduce fat accumulation, improve symptoms such as obesity and the like.

EXAMPLE 1 Chitosan oligosaccharide enteric-coated capsules of the invention and preparation thereof

The formula comprises the following components: chitosan oligosaccharide 10g, eudragit L100 g, HPMC10g, dracaps capsule shell

The preparation process comprises the following steps:

s1, dissolving: dissolving the Eudragit L100 with water, standing and swelling for 0.5h to obtain Eudragit L100 solution; dissolving HPMC with water and standing for 0.5h to obtain HPMC solution; completely dissolving chitosan oligosaccharide with water to obtain chitosan oligosaccharide solution;

s2, stirring: slowly adding the chitosan oligosaccharide solution into the Eudragit L100 solution while stirring, then adding the HPMC solution, and vigorously stirring for 0.5h by using a magnetic stirrer to obtain a mixed liquid medicine;

s3, ultrasonic treatment: placing the mixed liquid medicine into an ultrasonic instrument, and carrying out ultrasonic treatment at the highest frequency for 0.5h at 37 ℃;

s4, freeze drying: sealing the ultrasonic treated mixed liquid medicine with preservative film, freeze-curing overnight in-80 deg.c ultralow temperature refrigerator, taking out the completely coagulated and cured mixed medicine, penetrating the preservative film with the needle of the injector and maintaining the vent holes. Placing the mixed medicine solid in pre-cooled refrigeration equipment, and performing vacuum freeze drying for 12 hours to obtain a solid medicine;

s5, grinding and sieving: grinding the solid medicine on a dry clean super clean bench, sieving with a 80-mesh sieve to obtain capsule content particles, filling into a dragaps capsule shell, and packaging to obtain the chitosan oligosaccharide enteric capsule.

EXAMPLE 2 Chitosan oligosaccharide enteric-coated capsules of the invention and preparation thereof

The formula comprises the following components: chitosan oligosaccharide 20g, eudragit L100 80g, HPMC 20g and Drcaps capsule shell

The preparation process comprises the following steps:

s3, ultrasonic treatment: placing the mixed medicine into an ultrasonic instrument, and carrying out ultrasonic treatment at the highest frequency for 0.5h at 37 ℃;

EXAMPLE 3 Chitosan oligosaccharide enteric-coated capsules of the invention and preparation thereof

The formula comprises the following components: chitosan oligosaccharide 30g, eudragit L100 120g, HPMC 30g and Drcaps capsule shell

The preparation process comprises the following steps:

s4, freeze drying: sealing the ultrasonic treated mixed liquid medicine by using a preservative film, rapidly placing the mixed liquid medicine in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for freezing and solidifying overnight, taking out the completely coagulated and solidified mixed medicine, penetrating the preservative film by using a syringe needle, retaining a certain number of vent holes, placing the mixed medicine solid in a precooled freezing device, and performing vacuum freeze drying for 12 hours to obtain the solid medicine;

Comparative example 1

The formula comprises the following components: chitosan oligosaccharide 10g, eudragit L100 g, eudragit NM30D 10g, drcaps capsule shell

The preparation process comprises the following steps: the same as in example 1

The difference between this comparative example and example 1 is that the release modifier of this comparative example is Uttky NM30D, and the rest steps, components and parameters are the same.

Comparative example 2

The formula comprises the following components: chitosan oligosaccharide 10g, dragcaps capsule shell

The preparation process comprises the following steps: completely dissolving chitosan oligosaccharide with water to obtain chitosan oligosaccharide solution, placing into an ultrasonic instrument, and performing ultrasonic treatment at 37 ℃ for 0.5h at the highest frequency; the chitosan oligosaccharide solution after ultrasonic treatment is sealed by using a preservative film, is rapidly placed in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for freezing and solidifying overnight, then the completely coagulated and solidified mixed medicine is taken out, the preservative film is pierced by using a syringe needle, and a certain number of vent holes are reserved. Placing the chitosan oligosaccharide drug solid in pre-cooling refrigeration equipment, and performing vacuum freeze drying for 12 hours; and (3) after drying, taking out, grinding on a clean and dry super clean bench, sieving with a 80-mesh sieve, filling into a dragaps capsule shell, and packaging to obtain the chitosan oligosaccharide enteric capsule.

The present comparative example differs from example 1 in that the chitosan oligosaccharide enteric capsules of the present comparative example are free of enteric adjuvants and release modifiers.

Comparative example 3

The formula comprises the following components: chitosan oligosaccharide 10g, eudragit L100 g, HPMC10g and common gelatin capsule shell

This comparative example differs from example 1 in that it uses a common gelatin capsule shell as the capsule shell, and the remaining components, parameters and steps are the same as those of example 1.

Comparative example 4

The formula comprises the following components: chitosan oligosaccharide 10g, eudragit L100 60g, HPMC10g, dracaps capsule shell

The difference between this comparative example and example 1 is that the content of the finished enteric capsule of this comparative example: chitosan oligosaccharide: special L100: hpmc=1: 6:1, a step of; the remaining ingredients, parameters and steps were the same as in example 1.

Comparative example 5

The formula comprises the following components: 10g of chitosan oligosaccharide, 100-55 50g of Eudragit L and 10g of HPMC; dracaps capsule shell

This comparative example differs from example 1 in that the comparative example uses Eudragit L100-55 as an enteric adjuvant, and the remaining ingredients, parameters and steps are the same as in example 1.

Test example one, inspection of the content difference and the content of the main drug of the chitosan oligosaccharide capsules prepared by the invention

1. Test sample: the chitosan oligosaccharide enteric capsules prepared in examples 1-3.

2. The test method comprises the following steps: randomly extracting 20 enteric capsules prepared in the examples 1-3, weighing the weight of each enteric capsule and the weight of empty capsule shells, calculating the weight of the content of each capsule by a weight reduction method, and calculating the average loading and the loading difference limit according to the weighing result of the 20 enteric capsules; randomly extracting 6 enteric capsules prepared in the examples 1-3, detecting the COST content in a sample, hydrolyzing polysaccharide into monosaccharide by using a phenol-sulfuric acid method under the action of excessive concentrated sulfuric acid, dehydrating the monosaccharide to form a furfural derivative, reacting the derivative with phenol to form an orange-red substance with a maximum absorption peak, and measuring the polysaccharide mass by using a colorimetric method so as to detect the chitosan content in the sample;

3. test results:

the average loading of the chitosan oligosaccharide enteric capsule prepared by the invention is 0.2445g. Comparing the maximum value of the average loading with the average value, wherein the loading difference is 8.82%; the average loading minimum was compared to the average and the loading difference was-7.34%. The experimental results of the difference of the loading amount do not exceed the limit of the difference of the loading amount, and meet the related requirements of Chinese pharmacopoeia (2020 edition);

the average main drug content of the chitosan oligosaccharide enteric capsule prepared in the embodiment 1 of the invention is 16.66%, and the relative standard deviation is 1.3%. The content test experiment results do not exceed the content difference limit, and meet the related requirements of Chinese pharmacopoeia (2020 edition).

Test example two: chitosan oligosaccharide enteric-coated capsule quality inspection

1. The test method comprises the following steps: the appearance of the prepared capsules was observed by observing whether or not there was precipitation or uneven grinding during the preparation of examples 1 to 3 and comparative examples 4 to 5.

2. Test results:

TABLE 1 quality detection of Chitosan oligosaccharide enteric-coated capsules

As can be seen from Table 1, the chitosan oligosaccharide sol capsules prepared in examples 1-3 of the invention are clean and odorless, have no deformation phenomenon, have smooth capsule bodies, have no moisture absorption and adhesion phenomena, can be kept in a dry state, have no embrittlement or leakage and rupture phenomena, and have appearance inspection meeting the relevant requirements of Chinese pharmacopoeia (2020 edition); the capsule prepared in comparative example 4 has excessive proportion of auxiliary materials, and the auxiliary materials are precipitated and precipitated, so that the filling volume of the capsule is increased, and the appearance of the capsule is broken, deformed and leaked; the enteric capsule prepared in comparative example 5 has particles and nonuniform color of the content of the capsule due to the use of the Uttky L100-55 as an enteric auxiliary material, and also does not meet the requirements of Chinese pharmacopoeia (2020 edition).

Test example three disintegration time limit examination of Chitosan oligosaccharide Capsule

1. Test sample: chitosan oligosaccharide enteric-coated capsules prepared in examples 1-3 and comparative example 3

2. The test method comprises the following steps: according to the related operation requirements of Chinese pharmacopoeia (2020 edition), the disintegration conditions of the medicine in artificial gastric juice and artificial intestinal juice are detected.

TABLE 2 disintegration time of capsules in different media

As is clear from Table 2, the chitosan oligosaccharide capsule prepared in comparative example 3 disintegrated in artificial gastric juice and intestinal juice within 15 minutes, and the disintegration time period in artificial gastric juice was shorter than that in intestinal juice, because the comparative example 3 used a common gelatin capsule shell, contrary to the "stomach insoluble, intestine fast dissolving" characteristics required for enteric capsules. In contrast, the chitosan oligosaccharide enteric-coated capsules prepared in the embodiments 1-3 of the invention can be kept not disintegrated for more than 2 hours in artificial gastric juice, but can be rapidly disintegrated in alkaline intestinal juice, the disintegration time limit is 8min, and the chitosan oligosaccharide enteric-coated capsules can be completely disintegrated within 1 hour, thereby meeting the characteristics of 'gastric insolubility and enteric instant' and also meeting the related requirements of 'Chinese pharmacopoeia' (2020 edition).

Test example four, release degree detection of Chitosan oligosaccharide Capsule

1. The test method comprises the following steps: operating according to the specification of Chinese pharmacopoeia (2020 edition), testing total sugar cumulative release degree in artificial gastric juice and artificial intestinal juice

2. Test results:

TABLE 3 cumulative sugar Release in Artificial gastric juice (%)

Sample of	0.25h	0.5h	1h	2h	3h	4h	5h	6h
									Example 1	0.57	1.12	2.86	5.35	15.83	21.37	27.56	35.66
Example 2	0.56	1.10	2.71	5.32	15.68	21.28	26.78	35.24
									Example 3	0.52	1.13	2.68	5.41	15.72	21.41	27.12	34.89
Comparative example 1	0.45	0.75	1.62	2.53	12.81	16.33	25.61	29.52
									Comparative example 2	95.68	100	100	100	100	100	100	100

TABLE 4 cumulative sugar release in artificial intestinal juice (%)

Sample of	5min	10min	15min	20min	30min	60min	90min	120min
									Example 1	33.54	59.84	76.61	83.23	91.07	95.61	99.54	100
Example 2	33.25	58.47	75.89	83.54	91.01	94.99	99.05	100
									Example 3	35.97	60.35	76.44	84.68	91.25	95.95	100	100
Comparative example 1	0.75	1.06	1.93	3.51	15.33	25.61	31.57	45.69
									Comparative example 2	95.72	98.86	100	100	100	100	100	100

As can be seen from tables 3-4, the capsules prepared in comparative example 2, due to the absence of enteric adjuvants and release modifiers, the chitosan oligosaccharide was rapidly 100% dissolved in both artificial gastric juice and artificial intestinal juice, and did not meet the "gastric insoluble, enteric insoluble" characteristics of enteric capsules; the capsules prepared in examples 1-3 and comparative example 1 have similar effect on the cumulative release degree in artificial gastric juice, but have larger difference in the adjusting effect in artificial intestinal juice, wherein the cumulative release degree of the capsule prepared in comparative example 1 in the intestinal juice is only 45.69% within 120min, which does not meet the related requirements of Chinese pharmacopoeia (2020 edition), because the enteric auxiliary material, namely, the UTeqi NM30D, greatly inhibits the dissolution and release of chitosan oligosaccharide in the intestinal juice; the capsules prepared in examples 1-3 have less influence on the cumulative release degree in 120min in intestinal juice, can be dissolved out for more than 90% in 30min, reach 100% dissolution in 120min, meet the related requirements of Chinese pharmacopoeia (2020 edition) and meet the enteric characteristic, so the chitosan oligosaccharide enteric capsule prepared in examples 1-3 has the enteric characteristic.

Test example five, action of Chitosan oligosaccharide on intestinal flora

1. The test method comprises the following steps:

establishing a pseudo-sterile mouse model: 36 healthy 7-week-old male C57BL/6J mice without specific pathogen (SPF grade) were selected and purchased from Hunan Srilek's laboratory animals Limited, laboratory animal quality certificate No.110727201003661, laboratory animals were fed to the laboratory animal center of Guangdong university of medical science (NO: SPF 2017288), and after 1 week of adaptive feeding, the mice were randomly divided into 2 groups: a blank group (Standard Laboratory Diet, STD, 6) fed with laboratory standard normal feed, a pseudo-sterile mouse model group (Pseudoaseptic Model, MOD, 30) fed with high-fat high-sugar feed and fed with antibiotics mixed therewith; the three groups of mice are respectively fed with different feeds, the STD group and the HFS group are freely drunk with common sterile drinking water, and the MOD group is freely drunk with the sterile drinking water containing mixed antibiotics; the mixed antibiotic water is prepared according to the following proportion: ampicillin 1g, neomycin sulfate 1g, metronidazole 1g and vancomycin 0.5g are dissolved in 1L of sterile water and replaced once a day; modeling was performed for 28 days, feeding with free water, weighing once a week, and observing the mental and active status of the mice.

Pretreatment of fecal flora donor animals: in an SPF environment at the laboratory animal center with 24.+ -. 2 ℃ and 50% -60% relative humidity for 12 hours of bright and dark rhythms, 30 healthy, 7 week old male mice without specific pathogens (SPF grade) were fed standard laboratory feed adaptively for 1 week, and then they were randomly divided into 2 groups: the blank control group (6) was fed with laboratory standard normal diet, the obese model group (24) was fed with high fat and high sugar D12327 diet, and after 8 weeks of feeding, obese mice were ranked according to body weight and less weight-gain obese resistant mice were eliminated. Obese mice with a body weight 20% higher than that of normal diet healthy mice were considered as mice with a successfully established obesity model according to the modeling weight standard of rodent obesity animal models. Model mice for obesity were divided into four groups: obesity model group (6), drug group high, medium, low dose group (6 each); other mice were given D12327 feed except for the blank group given normal standard feed; according to the human-mouse dose conversion formula, the administration dose of the mouse is determined, and the high, medium and low doses of the drug group are 1700mg/kg, 850mg/kg and 425mg/kg respectively. The mice in the drug group are irrigated with corresponding doses of drugs every day, and the blank group and the model group are irrigated with equivalent physiological saline; after 8 weeks of gavage, fresh faeces were collected from each group of mice, each mouse collecting approximately 300mg faeces, under sterile conditions.

Extraction of fecal flora: treating each group of collected mouse faeces respectively, weighing 150-180mg of faeces, adding sterile 1mL of phosphate buffer solution and sterile ceramic beads, and suspending by a vortex oscillator under an anaerobic environment to obtain suspension; centrifuging at 25deg.C and 500rpm for 10min, and rapidly sucking the supernatant into another EP tube with a pipette; centrifuging at 4000rpm for 5min, sucking supernatant, and retaining bacterial precipitate; adding sterile phosphate buffer solution again, and cleaning for 2 times in the same way; 60% sterile glycerin is added according to the ratio of 1:2, and the mixture is mixed uniformly and stored for standby in a short time under an anaerobic environment, so that 5 groups of fecal bacteria extract are obtained for standby.

Fecal flora transplantation experiments (FMT experiments): after 28 days of molding, the pseudo-sterile mouse model group mice were randomly divided into 5 groups: control, model, COST-H, COST-M, COST-L, 6 each; the STD group 6 mice continue to be given normal standard feed; the STD group of 5 mice of the pseudo sterile mice and the healthy control group of mice are added for 6 groups, then the corresponding 5 groups of fecal bacteria extract liquid of 5 groups of pseudo sterile model mice are respectively irrigated, the amount of 100 mu L/10g is carried out once a day for 6 weeks, and the STD group is irrigated with 0.9% NaCl solution for equivalent injection.

Weight of: during the FMT experiment, the weight of the mice in the experiment period is observed and recorded, and the weight of the mice is weighed once a week;

histopathological section: dissecting and collecting liver and subcutaneous adipose tissues of each group of mice, washing with phosphate buffer solution to remove residual blood stains, and fixing in 10% PFA tissue fixing solution; after the tissue is taken out, washing the tissue with 75% absolute ethyl alcohol, and then automatically dehydrating the tissue step by step in a full-automatic dehydrator by using gradient concentration ethanol; after dehydration, embedding the dehydrated paraffin blocks in a Leica EG1160 integrated paraffin embedding machine by using Leica high-melting-point paraffin (60 ℃), and placing the embedded paraffin mold blocks on a-20 ℃ refrigeration table for refrigeration hardening; after the tissue wax block is completely hardened, slicing rapidly with the thickness of 5 mu m, and rapidly placing the wax sheet on warm water at 43 ℃ for spreading, and baking at 52 ℃; the cytoplasm and the nucleus of the tissue are stained with Hematoxylin Eosin (HE) staining kit and then observed.

Serum biochemical index: after blood is taken from the eyebox of the mice, standing for 30min, centrifuging for 15min at 3000rpm, collecting upper serum, and performing secondary centrifugation under the same conditions; the content of Total Cholesterol (TC), triglycerides (TG), high density lipoproteins (HDL-C), low density lipoproteins (LDL-C), free fatty acids (NEFA), total Bile Acids (TBA) and blood Glucose (GLU) in the serum was determined according to the instructions of the kit.

2. Test results:

(1) Body weight of mice

Table 5, change in body weight of mice during FMT experiments

As shown in Table 5, the mice in COST-H, COST-M, COST-L group still have good weight gain inhibiting effect after being transplanted with fecal bacteria extract, which indicates that the enteric chitosan oligosaccharide capsule prepared by the invention can effectively regulate intestinal flora of the mice, and can effectively resist obesity promotion effect caused by high-fat and high-sugar diet after being transplanted.

(2) FMT experimental mouse histopathological section

TABLE 6 Table of the histopathological section of mice

As shown in Table 6, compared with the Model mice of obesity Model, the liver tissue lipid accumulation of COST-H, COST-M, COST-L mice is less, the fat cell volume is small, the fat size is uniform, and the disorder and damage state of ileal intestinal villus of small intestinal tissues are well improved, so that the chitosan oligosaccharide enteric capsule prepared by the invention can interfere with intestinal flora, indirectly inhibit fat cell hypertrophy caused by high-fat high-sugar diet, regulate the growth expansion of fat cells, reduce fat accumulation, and improve symptoms such as obesity.

(3) Serum biochemical index of FMT experimental mouse

TABLE 7 Biochemical index Table of mouse serum

As can be seen from the data in table 7, total Cholesterol (TC), triglycerides (TG), low density lipoproteins (LDL-C), free fatty acids (NEFA), total Bile Acids (TBA) and blood Glucose (GLU) were significantly reduced and high density lipoproteins (HDL-C) were increased in blood relative to Model group mice, and the effect of the COST-H group was more remarkable, demonstrating that the intestinal flora with chitosan oligosaccharide dry prognosis was able to reduce the intake of dietary fat in a high-fat high-sugar diet, reduce total cholesterol, triglycerides, low density lipoproteins, free fatty acids and total bile acids in serum, and increase the high density lipoprotein content, thereby achieving an anti-obesity effect; blood sugar (ALT) is also obviously reduced, and a good auxiliary blood sugar regulating effect is shown, so that intestinal flora after the influence of the chitosan oligosaccharide enteric capsule prepared by the invention can reduce the intake of dietary polysaccharide in high-fat high-sugar diet, thereby achieving the effect of losing weight.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The chitosan oligosaccharide enteric capsule is characterized by comprising a content and a capsule shell, wherein the content comprises enteric auxiliary materials, chitosan oligosaccharide and a release regulator;

the enteric adjuvant is Eudragit L100, the release regulator is hydroxypropyl methylcellulose, and the capsule shell is Dracaps capsule shell;

the mass ratio of the enteric auxiliary materials to the chitosan oligosaccharide to the release regulator is 4:1:1, a step of;

the preparation method of the chitosan oligosaccharide enteric capsule comprises the following steps:

s1, dissolving: weighing enteric auxiliary materials, dissolving with water, standing for swelling to obtain enteric auxiliary material solution; weighing a release regulator, dissolving with water, and standing to obtain a release regulator solution; weighing chitosan oligosaccharide, and dissolving the chitosan oligosaccharide with water to obtain a chitosan oligosaccharide solution;

2. The chitosan oligosaccharide enteric capsule of claim 1, wherein the stirring is vigorous stirring for 0.5h with a magnetic stirrer.

3. The chitosan oligosaccharide enteric capsule according to claim 1, wherein the ultrasonic treatment temperature is 35-40 ℃.

4. The chitosan oligosaccharide enteric capsule of claim 1, wherein the freeze-cure temperature is-80 ℃.

5. The chitosan oligosaccharide enteric capsule according to claim 1, wherein the vacuum freeze-drying time is 11-13h.

6. Use of the chitosan oligosaccharide enteric-coated capsule according to any one of claims 1-5 in the preparation of a weight-loss medicament.