CN114522150B - Preparation method and application of pH-sensitive plant microcapsule nano extruder - Google Patents
Preparation method and application of pH-sensitive plant microcapsule nano extruder Download PDFInfo
- Publication number
- CN114522150B CN114522150B CN202210181718.XA CN202210181718A CN114522150B CN 114522150 B CN114522150 B CN 114522150B CN 202210181718 A CN202210181718 A CN 202210181718A CN 114522150 B CN114522150 B CN 114522150B
- Authority
- CN
- China
- Prior art keywords
- pollen
- solution
- filtering
- dissolving
- stirring
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
-
- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
- A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
-
- 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
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Endocrinology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Emergency Medicine (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Organic Chemistry (AREA)
- Nutrition Science (AREA)
- Physiology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Botany (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a preparation method of a pH sensitive plant microcapsule nano extruder and application thereof, which can effectively solve the problem of diabetes treatment.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a preparation method and application of a pH sensitive plant microcapsule nano extruder.
Background
According to the data published by the international diabetes union (IDF) on the global diabetes map of 2021IDF (10 th edition), the number of people with global adult diabetes in 2021 reached 5.37 billions (10.5%), a reduced proportion, about one tenth of adults was affected. The health burden imposed by diabetes is an absolute major challenge to individuals, families and society. Although blood glucose control has been performed by subcutaneous insulin injection to diabetics for the past several decades, normal blood glucose has been maintained for a long period of time by such painful injection, seriously affecting the daily life of diabetics, and in addition, insulin moves in the body through peripheral circulation upon subcutaneous injection, which can produce peripheral hyperinsulinemia, so it is necessary to explore a noninvasive insulin delivery system for treating diabetes. Drug delivery by oral administration of polypeptides remains a challenge, however, particularly given the low oral bioavailability of these peptides. Despite these challenges, the advantages of the oral route are significant and improve upon the disadvantages of the parenteral route. This is especially true in the treatment of chronic diseases such as type 2 diabetes (T2 DM), where oral therapeutic insulin can significantly improve the quality of life of diabetics who must regularly receive injections of insulin; moreover, insulin is more convenient to take orally, and can enter the liver through the hepatic portal vein at a higher concentration, avoiding extensive systemic exposure and its related side effects.
However, in practice oral insulin administration has been difficult to achieve for two main reasons, we discuss in turn the order of clinical transformation complexity. Firstly, because of the acidic environment of the gastrointestinal tract and the abundant peptidases, proteases, which are prone to denaturating or degrading protein drugs, oral insulin needs to be protected in the gastrointestinal tract environment; the oral bioavailability of biomolecules, including insulin, in its functionally active state is at most 1-2%. Second, oral administration requires overcoming the intestinal mucus barrier and epithelial cell barrier into the blood stream, which requires frequent doses or sustained bioavailability.
Currently, oral polypeptide drug delivery systems have received extensive attention, and many oral insulin polymer drug delivery systems and polymers of different formulations have been developed. Currently, most of effective insulin oral administration technologies utilize a cationic natural polymer Chitosan (CS), which is popular because of its low toxicity, good biocompatibility and availability, and the like, and can adhere to mucus on the surface of the intestinal tract, thereby prolonging the residence time of the intestinal tract, and opening tight connection between intestinal epithelial cells to deliver insulin to the blood circulation, so that chitosan is receiving a great deal of attention as an oral insulin delivery vehicle. However, chitosan nanoformulations adsorb a large amount of proteins and other substances while passing through the stomach tissue, so that the mucous retention effect of chitosan and the intestinal epithelial cell gap opening effect are masked when reaching the small intestine. Therefore, the realization of the specific release of the chitosan nanoparticle in the small intestine has important significance for oral insulin delivery.
Natural plant pollen, which is a rich natural renewable biological resource, has been increasingly paid attention to in recent years in the biomedical field, and is recognized as a safe material by the FDA of the U.S. food and drug administration. The Cheng Moguo allergen pollen shell is extracted from natural plant pollen by a chemical treatment method, the outer layer is composed of sporopollen, the inner wall is composed of cellulose, and the natural plant pollen has uniform structure, mechanical and chemical stability, monodisperse size, porosity and large inner cavity, and has great potential in microencapsulation and drug controlled release. Many studies have demonstrated the great potential of pollen as a drug delivery vehicle in vivo, because of their large lumen and strong exostructural spore biopolymers, considered as novel vehicles encapsulating materials such as drugs, proteins, vaccines, oils, etc. The pollen has various shapes, and the wild chrysanthemum flower pollen with spike-shaped structures on the surface is selected to have better residence in the small intestine than the common spherical pollen. In addition, we found that natural pollen itself contains carbohydrate, although the components are removed through a series of chemical treatments, pollen can still adsorb carbohydrate as a natural sugar loader to reduce absorption of sugar in intestinal tract, which can directly reduce sugar intake from root source to reduce blood sugar, so that it is imperative to invent a drug-loaded pH-sensitive plant microcapsule nano-extruder.
Disclosure of Invention
Aiming at the situation, the invention aims to solve the defects in the prior art, and the invention aims to provide a preparation method and application of a drug-loaded pH-sensitive plant microcapsule nano extruder, which can effectively solve the problem of treatment of diabetes.
The invention solves the technical scheme that the preparation method of the drug-loaded pH sensitive plant microcapsule nano extruder adopts natural green pollen grains with high biocompatibility and sugar adsorptivity, the allergen-free pollen shell is extracted by a chemical treatment method as a basic material, the strong electrostatic acting force between free carboxyl rich in gamma-polyglutamic acid (gamma-PGA) and a large amount of amino in Chitosan (CS) is utilized by ultrasonic stirring under an acidic condition, insulin is selected as a hypoglycemic drug, self-assembly is carried into nano gel which is loaded in the pollen shell, and the pH sensitive plant microcapsule nano extruder is constructed.
The method specifically comprises the following steps:
1) Weighing 20-30 g of plant pollen, dissolving in 300-400 ml of acetone, adding into a 1000ml round bottom flask, stirring and refluxing in an oil bath at 70 ℃ for 12 hours, cooling the solution, and vacuum filtering to obtain recovered pollen;
2) Recovering pollen in the step 1), dissolving with 400-500 ml of 85% (w/w) phosphoric acid, adding into a 1000ml round bottom flask, stirring and refluxing in a 60 ℃ oil bath for 12 hours, cooling and filtering the pollen solution after phosphoric acid treatment, and washing and filtering the pollen solution with water, acetone, 2M HCl, 2M NaOH, water, acetone and 70% ethanol in sequence to obtain primary filtered pollen;
3) Dissolving the pollen obtained in the step 2) by using 300-400 ml of 6% (w/v) KOH solution, adding the solution into a 1000ml round bottom flask, refluxing the solution in an 80 ℃ oil bath for 6 hours, cooling the pollen solution subjected to suction filtration, diluting and filtering the pollen solution by using water, and washing and suction-filtering the pollen solution by using water, acetone and 70% ethanol to obtain secondary pollen subjected to suction filtration;
4) Drying the secondarily filtered pollen in the step 3) in a baking oven at 60 ℃ for 24 hours, and determining the weight of the finally dried pollen, wherein all the dried pollen is stored at room temperature before experimental use;
5) Under stirring (600 r/min) at room temperature, dissolving 20-30 mg of Chitosan (CS) in 10-15 ml of 1% acetic acid solution, then adding 200-500 mg of dry pollen obtained in the step 4) into the chitosan solution, stirring for 10min after ultrasonic treatment, centrifuging at 1500rpm for 2min, discarding the supernatant, and re-suspending with 1% acetic acid solution; and then dissolving 20-30 mg of gamma-polyglutamic acid (gamma-PGA) and 10-20 mg of insulin in 5mL of deionized water, adding acetic acid solution accounting for 1% of the total volume, slowly dripping the gamma-PGA acetic acid solution into the pollen chitosan solution under stirring, stirring for 10-15 min to naturally settle the solution, washing the supernatant by 1% acetic acid solution for 3 times, centrifuging to remove the free matters, and thus obtaining the pH sensitive plant microcapsule nano extruder for encapsulating the insulin.
The plant pollen is one or a mixture of more than two of natural wild chrysanthemum, motherwort and sunflower in any proportion.
The particle size of the pH sensitive plant microcapsule nano extruder for encapsulating the insulin is about 20 mu m.
The pH sensitive plant microcapsule nano extruder for encapsulating insulin is applied to the preparation of an oral drug delivery system for diabetes.
The preparation method is simple, the prepared drug-loaded pH sensitive plant microcapsule nano extruder can effectively protect drugs from passing through rugged stomach environment and can rapidly transport the drugs to intestinal environment, the pH sensitive plant microcapsule does not expand in the acidic stomach environment, chitosan nanoparticles can be continuously extruded from pollen shell apertures along with the peristalsis of pollen grains in small intestines under the condition of entering alkaline intestinal cell gaps, so that the drugs can be accurately positioned in the small intestines and insulin can be continuously released to play a role in treatment, and the drug-loaded pH sensitive plant microcapsule nano extruder is a great innovation on the microcapsule nano extruder.
Drawings
FIG. 1 is a graph showing the effect of glucose adsorption capacity of the pH sensitive plant microcapsule nano extruder of the insulin entrapped in the present invention.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples.
Example 1
When the invention is concretely implemented, the preparation method of the drug-loaded pH sensitive plant microcapsule nano extruder specifically comprises the following steps:
1) Weighing 20g of natural wild chrysanthemum flower pollen, dissolving in 300ml of acetone, adding into a 1000ml round bottom flask, stirring and refluxing in an oil bath at 70 ℃ for 12 hours, and vacuum filtering after cooling the solution to obtain recovered pollen;
2) Recovering pollen in the step 1), dissolving with 400ml 85% (w/w) phosphoric acid, adding into a 1000ml round bottom flask, stirring and refluxing in a 60 ℃ oil bath for 12 hours, cooling and filtering the pollen solution after phosphoric acid treatment, and washing and filtering the pollen solution with water, acetone, 2M HCl, 2M NaOH, water, acetone and 70% ethanol in sequence to obtain primary filtered pollen;
3) Filtering pollen once in the step 2), dissolving with 300ml of 6% (w/v) KOH solution, adding into a 1000ml round bottom flask, refluxing in an 80 ℃ oil bath for 6 hours, cooling the pollen solution, diluting and filtering with water, washing and filtering with water, acetone and 70% ethanol to obtain secondary filtered pollen;
4) Drying the secondarily filtered pollen in the step 3) in a baking oven at 60 ℃ for 24 hours, and determining the weight of the finally dried pollen, wherein all the dried pollen is stored at room temperature before experimental use;
5) Under stirring (600 r/min) at room temperature, dissolving 20-30 mg of Chitosan (CS) in 10-15 ml of 1% acetic acid solution, adding 200mg of dry pollen obtained in the step 4) into the chitosan solution, stirring for 10min after dissolving by ultrasound, centrifuging at 1500rpm for 2min, discarding the supernatant, and re-suspending with 1% acetic acid solution; then 20mg of gamma-polyglutamic acid (gamma-PGA) and 10mg of insulin are taken and dissolved in 5mL of deionized water, acetic acid solution accounting for 1% of the total volume is added, the gamma-PGA acetic acid solution is slowly dripped into the pollen chitosan solution under stirring, the solution is naturally settled after stirring for 10min, the supernatant is discarded, the solution is washed for 3 times by 1% of acetic acid solution, and the dissociation is removed by centrifugation, so that the pH sensitive plant microcapsule nano extruder for encapsulating the insulin is obtained.
Example 2
When the invention is concretely implemented, the preparation method of the drug-loaded pH sensitive plant microcapsule nano extruder specifically comprises the following steps:
1) Weighing 25g of natural wild chrysanthemum flower and motherwort flower powder, dissolving in 300-400 ml of acetone, adding into a 1000ml round bottom flask, stirring and refluxing in an oil bath at 70 ℃ for 12h, cooling the solution, and vacuum filtering to obtain recovered pollen;
2) Recovering pollen in the step 1), dissolving with 450ml of 85% (w/w) phosphoric acid, adding into a 1000ml round bottom flask, stirring and refluxing in a 60 ℃ oil bath for 12 hours, cooling and filtering the pollen solution after phosphoric acid treatment, and washing and filtering the pollen solution with water, acetone, 2M HCl, 2M NaOH, water, acetone and 70% ethanol in sequence to obtain primary filtered pollen;
3) Filtering pollen once in the step 2), dissolving with 350ml of 6% (w/v) KOH solution, adding into a 1000ml round bottom flask, refluxing in an 80 ℃ oil bath for 6 hours, cooling the pollen solution, diluting and filtering with water, washing and filtering with water, acetone and 70% ethanol to obtain secondary filtered pollen;
4) Drying the secondarily filtered pollen in the step 3) in a baking oven at 60 ℃ for 24 hours, and determining the weight of the finally dried pollen, wherein all the dried pollen is stored at room temperature before experimental use;
5) Under stirring (600 r/min) at room temperature, dissolving 25mg of Chitosan (CS) in 12mL of 1% acetic acid solution, adding 350mg of dry pollen in step 4) into the chitosan solution, stirring for 10min after ultrasonic dissolution, centrifuging at 1500rpm for 2min, discarding the supernatant, and re-suspending with 1% acetic acid solution; then 25mg of gamma-polyglutamic acid (gamma-PGA) and 15mg of insulin are taken and dissolved in 5mL of deionized water, acetic acid solution accounting for 1% of the total volume is added, the gamma-PGA acetic acid solution is slowly dripped into the pollen chitosan solution under stirring, the solution is naturally settled after stirring for 12min, the supernatant is discarded, the solution is washed for 3 times by 1% of acetic acid solution, and the dissociation is removed by centrifugation, so that the pH sensitive plant microcapsule nano extruder for encapsulating the insulin is obtained.
Example 3
When the invention is concretely implemented, the preparation method of the drug-loaded pH sensitive plant microcapsule nano extruder specifically comprises the following steps:
1) Weighing 30g of natural wild chrysanthemum flower, motherwort herb and sunflower pollen, dissolving in 400ml of acetone, adding into a 1000ml round bottom flask, stirring and refluxing in an oil bath at 70 ℃ for 12 hours, cooling the solution, and vacuum filtering to obtain recovered pollen;
2) Recovering pollen in the step 1), dissolving with 500ml 85% (w/w) phosphoric acid, adding into a 1000ml round bottom flask, stirring and refluxing in a 60 ℃ oil bath for 12 hours, cooling and filtering the pollen solution after phosphoric acid treatment, and washing and filtering the pollen solution with water, acetone, 2M HCl, 2M NaOH, water, acetone and 70% ethanol in sequence to obtain primary filtered pollen;
3) Filtering pollen once in the step 2), dissolving with 400ml of 6% (w/v) KOH solution, adding the solution into a 1000ml round bottom flask, refluxing the solution in an 80 ℃ oil bath for 6 hours, cooling the pollen solution, diluting and filtering the pollen solution with water, washing and filtering the pollen with water, acetone and 70% ethanol to obtain secondary filtered pollen;
4) Drying the secondarily filtered pollen in the step 3) in a baking oven at 60 ℃ for 24 hours, and determining the weight of the finally dried pollen, wherein all the dried pollen is stored at room temperature before experimental use;
5) Under stirring (600 r/min) at room temperature, dissolving 20-30 mg of Chitosan (CS) in 15mL of 1% acetic acid solution, then adding 500mg of dry pollen into the chitosan solution, stirring for 10min after ultrasonic dissolution, centrifuging at 1500rpm for 2min, discarding the supernatant, and re-suspending with 1% acetic acid solution; then 30mg of gamma-polyglutamic acid (gamma-PGA) and 20mg of insulin are taken and dissolved in 5mL of deionized water, acetic acid solution accounting for 1% of the total volume is added, the gamma-PGA acetic acid solution is slowly dripped into the pollen chitosan solution under stirring, the solution is naturally settled after stirring for 15min, the supernatant is discarded, the solution is washed for 3 times by 1% of acetic acid solution, and the dissociation is removed by centrifugation, so that the pH sensitive plant microcapsule nano extruder for encapsulating the insulin is obtained.
From the above, it can be seen that the invention adopts natural green pollen grains with high biocompatibility and sugar adsorptivity, extracts allergen-free pollen shells as basic materials through a chemical treatment method, ultrasonically stirs and utilizes strong electrostatic force between free carboxyl rich in gamma-polyglutamic acid (gamma-PGA) and a large amount of amino groups of Chitosan (CS), selects insulin as hypoglycemic drugs, self-assembles to prepare nano gel to load the nano gel into pollen shells, constructs a pH sensitive plant microcapsule nanoparticle extruder delivery system with double-tube simultaneous oral treatment function for diabetes, and obtains very good beneficial technical effects through experiments, and related experimental data are as follows (taking example 1):
1. particle size of pH sensitive plant microcapsule nano extruder for encapsulating insulin
The plant microcapsule nano extruder prepared by the method described in the example 1 is observed by a scanning electron microscope and a transmission electron microscope, and the result shows that the particle size is uniform, and the average particle size is about 20 mu m.
2. Morphology of pH sensitive plant microcapsule nano extruder for encapsulating insulin
The pH-sensitive plant microcapsule nano-extruder prepared by the method described in example 1 was examined by scanning electron microscopy under pH2 and pH7.2 conditions, respectively, and showed that pH-sensitive chitosan nano-gel was loaded inside pollen shells under acidic conditions, and chitosan nanoparticles were extruded through pollen pores under alkaline conditions.
3. Toxicity of insulin-entrapped pH-sensitive plant microcapsule nano extruder to human colon cancer cells
In vitro cytotoxicity test uses human colon cancer cells as research object, sets blank control group and pollen group, treats cells with pollen of different concentrations, continues to cultivate for 24 hours, and tests toxicity to vascular endothelial cells by CCK-8 method, and results show that pollen shells have little influence on activity of human colon cancer cells.
4. Investigation of metabolic capability of pH sensitive plant microcapsule nano extruder for encapsulating insulin in small intestine
The ability of pollen shell microcapsules to enter the small intestine was examined by gavage administration using blank mice as an animal model for administration. Three groups of experiments were set up: 1. blank IR783; 2. motherwort @ IR783; 3. wild chrysanthemum flower @ IR783. Mice in each group were given gavage at a dose of 20 mg/dose. After the administration is finished, the small intestine of the light-shielding dissected mice is taken after 2 hours, 4 hours, 8 hours, 12 hours and 24 hours respectively, small intestine tissues are gently clamped by forceps and are cleaned by normal saline, then the fluorescence intensity of the small intestine is observed by a living body imager, and the fluorescence intensity of the wild chrysanthemum group with obvious spike shape is strong.
5. Investigation of sugar adsorption capability of pH sensitive plant microcapsule nano extruder for encapsulating insulin
In order to investigate the effect of pollen adsorption on sugars to avoid their enzymatic hydrolysis in the intestinal lumen, in vitro processes were simulated in PBS. For sucrose, 10ml of pollen solution (10 mg/ml), 10ml disaccharide solution (10 mg/ml) and 5ml of alpha-glucosidase solution (70U/ml) were prepared and mixed with PBS as a solvent, respectively. In contrast, the control group, disaccharide and α -glucosidase were mixed, PBS solution was free of pollen, the mixed solution of pollen and sugar was incubated on a shaker at 37℃for 4 hours at 200 rpm, and then glucose levels in each solution were measured by glucose oxidase-peroxidase method using a glucose test kit, and the experimental results are shown in FIG. 1.
Through repeated experiments, other embodiments can obtain the same or similar experimental results as those of embodiment 1, and the detailed description is not repeated, and meanwhile, it should be pointed out that the above embodiments are only preferred embodiments of the present invention, and are not limited in any way, and any person skilled in the art can make modifications or modifications to equivalent embodiments using the above-disclosed technical disclosure without departing from the scope of the present invention.
According to the invention, by utilizing strong electrostatic acting force between free carboxyl rich in gamma-polyglutamic acid (gamma-PGA) and a large amount of amino groups of Chitosan (CS), insulin is selected as a hypoglycemic drug, self-assembly is carried into the nano gel which is loaded in pollen shells, and as the pH is gradually increased, the amino groups of CS are gradually deprotonated and uncharged, and the electrostatic action between the amino groups and the carboxyl groups of gamma-PGA is reduced, so that the nano gel in pollen expands, and nano particles with irregular sizes are extruded from pollen holes. The pollen utilizes the advantages of the pollen as a pH sensitive nanoparticle extruder, so that the release of insulin in an acidic stomach environment can be avoided, but the insulin is gradually released by continuously extruding nanoparticles after the expansion in an alkaline small intestine environment, the coated insulin is effectively protected from being damaged by gastric acid and is accurately positioned in the small intestine to continuously exert a therapeutic effect, meanwhile, the pollen is a natural sugar loader and can adsorb carbohydrates so as to reduce the absorption of sugar in intestinal tracts, and the pH sensitive plant microcapsule prepared by utilizing the unique advantages of the pollen not only can adsorb sugar to reduce the absorption of sugar from sources, but also can be used as a protective carrier to transport insulin to exert a hypoglycemic effect, and is used for treating diabetes in double-tube.
The preparation method is simple, the prepared drug-loaded pH-sensitive plant microcapsule nano extruder can effectively protect drugs from passing through rugged stomach environment and can be rapidly transported to intestinal environment, the pH-sensitive plant microcapsule is not expanded in acidic stomach environment, chitosan nanoparticles can be continuously extruded from pollen shell apertures along with the peristalsis of pollen grains in small intestines under the condition of entering alkaline intestinal cell gaps, so that the drugs can be accurately positioned in small intestines and continuously release insulin to play a role in treatment, and meanwhile, pollen is a natural sugar loader and can adsorb carbohydrates so as to reduce the absorption of sugar in intestinal tracts, so that the drug-loaded pH-sensitive plant microcapsule nano extruder has a great innovation on the microcapsule nano extruder and has good application and popularization values.
Claims (3)
1. The preparation method of the pH sensitive plant microcapsule nano extruder is characterized by comprising the following steps of:
1) Weighing 20-30 g of plant pollen, dissolving in 300-400 ml of acetone, adding into a 1000ml round bottom flask, stirring and refluxing in an oil bath at 70 ℃ for 12 hours, cooling the solution, and vacuum filtering to obtain recovered pollen;
2) Recovering pollen in the step 1), dissolving with 400-500 ml of 85% phosphoric acid, adding into a 1000ml round bottom flask, stirring and refluxing for 12 hours in a 60 ℃ oil bath, cooling and filtering the pollen solution after phosphoric acid treatment, and washing and filtering the pollen solution with water, acetone, 2M HCl, 2M NaOH, water, acetone and 70% ethanol in sequence to obtain primary filtered pollen;
3) Dissolving the pollen obtained in the step 2) by using 300-400 ml of 6% KOH solution, adding the solution into a 1000ml round bottom flask, refluxing the solution in an 80 ℃ oil bath for 6 hours, cooling the plant pollen solution, diluting and filtering the solution by using water, and washing and filtering the solution by using water, acetone and 70% ethanol to obtain secondary filtered pollen;
4) Drying the secondarily filtered pollen in the step 3) in a baking oven at 60 ℃ for 24 hours, and determining the weight of the finally dried pollen, wherein all the dried pollen is stored at room temperature before experimental use;
5) Under stirring at room temperature, dissolving 20-30 mg of chitosan in 10-15 ml of 1% acetic acid solution, then adding 200-500 mg of dry pollen obtained in the step 4) into the chitosan solution, stirring for 10min after ultrasonic dissolving, centrifuging at 1500rpm for 2min, discarding the supernatant, and re-suspending with 1% acetic acid solution; then, dissolving 20-30 mg of gamma-polyglutamic acid and 10-20 mg of insulin in 5mL of deionized water, adding acetic acid solution accounting for 1% of the total volume, slowly dripping the gamma-PGA acetic acid solution into the pollen chitosan solution under stirring, stirring for 10-15 min to naturally settle the solution, washing 3 times by using 1% acetic acid solution after removing the supernatant, and centrifuging to remove the free matters to obtain the pH sensitive plant microcapsule nano extruder for encapsulating the insulin;
the plant pollen is natural flos Chrysanthemi Indici pollen.
2. The method for preparing the pH-sensitive plant microcapsule nano extruder according to claim 1, which is characterized by comprising the following steps:
1) Weighing 20g of natural wild chrysanthemum flower pollen, dissolving in 300ml of acetone, adding into a 1000ml round bottom flask, stirring and refluxing in an oil bath at 70 ℃ for 12 hours, and vacuum filtering after cooling the solution to obtain recovered pollen;
2) Recovering pollen in the step 1), dissolving with 400ml of 85% phosphoric acid, adding into a 1000ml round bottom flask, stirring and refluxing for 12h in a 60 ℃ oil bath, cooling and filtering the pollen solution after phosphoric acid treatment, and washing and filtering with water, acetone, 2M HCl, 2M NaOH, water, acetone and 70% ethanol in sequence to obtain primary filtered pollen;
3) Filtering pollen once in the step 2), dissolving with 300ml of 6% KOH solution, adding into a 1000ml round bottom flask, refluxing in an 80 ℃ oil bath for 6 hours, cooling the pollen solution, diluting and filtering with water, washing and filtering with water, acetone and 70% ethanol to obtain secondary filtered pollen;
4) Finally, drying the secondarily filtered pollen in the step 3) in a baking oven at 60 ℃ for 24 hours, and determining the weight of the finally dried pollen, wherein all the dried pollen is stored at room temperature before experimental use;
5) Under stirring at room temperature, dissolving 20mg of chitosan in 10mL of 1% acetic acid solution, adding 200mg of dry pollen obtained in the step 4) into the chitosan solution, stirring for 10min after ultrasonic dissolution, centrifuging at 1500rpm for 2min, discarding the supernatant, and re-suspending with 1% acetic acid solution; then 20mg of gamma-polyglutamic acid and 10mg of insulin are dissolved in 5mL of deionized water, acetic acid solution accounting for 1% of the total volume is added, gamma-PGA acetic acid solution is slowly dripped into the pollen chitosan solution under stirring, the solution is naturally settled after stirring for 10min, the supernatant is discarded, the solution is washed for 3 times by 1% acetic acid solution, and the dissociation is removed by centrifugation, so that the pH sensitive plant microcapsule nano extruder for encapsulating the insulin is obtained.
3. Use of an insulin-entrapped pH-sensitive plant microcapsule nanoextruder prepared by the method of any one of claims 1-2 in the preparation of an oral drug delivery system for diabetes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210181718.XA CN114522150B (en) | 2022-02-25 | 2022-02-25 | Preparation method and application of pH-sensitive plant microcapsule nano extruder |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210181718.XA CN114522150B (en) | 2022-02-25 | 2022-02-25 | Preparation method and application of pH-sensitive plant microcapsule nano extruder |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114522150A CN114522150A (en) | 2022-05-24 |
| CN114522150B true CN114522150B (en) | 2023-06-23 |
Family
ID=81624491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210181718.XA Active CN114522150B (en) | 2022-02-25 | 2022-02-25 | Preparation method and application of pH-sensitive plant microcapsule nano extruder |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114522150B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114747569B (en) * | 2022-03-15 | 2023-02-03 | 中国农业科学院蔬菜花卉研究所 | Preparation method of sporopollenin-based copper preparation pesticide carrier |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7608270B2 (en) * | 2003-06-27 | 2009-10-27 | University Of Hull | Dosage form |
| CN104941544B (en) * | 2015-05-29 | 2018-01-09 | 蔡文 | A kind of cryptogam microcapsules and its preparation method and application |
| CN108434119B (en) * | 2018-03-07 | 2020-10-09 | 华中农业大学 | Preparation method of protein oral micro-capsule preparation |
-
2022
- 2022-02-25 CN CN202210181718.XA patent/CN114522150B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| Chitosan/sporopollenin microcapsules: Preparation, characterisationand application in heavy metal removal;˙Idris Sargın等;《International Journal of Biological Macromolecules》;第75卷;第230-238页 * |
| Effect of glutaraldehyde cross-linking degree of chitosan/sporopollenin microcapsules on removal of copper(II) from aqueous solution;I˙ dris Sargin等;《Desalination and Water Treatment》;第1-13页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114522150A (en) | 2022-05-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Panos et al. | New drug delivery systems based on chitosan | |
| Sailaja et al. | Different techniques used for the preparation of nanoparticles using natural polymers and their application | |
| Rani et al. | Chitosan based hydrogel polymeric beads–As drug delivery system | |
| JP5175017B2 (en) | Particulate carrier for improving oral absorption of active ingredients | |
| CN111265533A (en) | Preparation method of core-shell nanoparticles based on lipid membrane and metal organic framework | |
| JP2013177434A (en) | Nanoparticle for protein drug delivery | |
| Asal et al. | Controlled synthesis of in-situ gold nanoparticles onto chitosan functionalized PLGA nanoparticles for oral insulin delivery | |
| CN105056212B (en) | A kind of chitosan nano and preparation method for improving oral insulin colonic absorption | |
| Zhang et al. | pH-sensitive O-carboxymethyl chitosan/sodium alginate nanohydrogel for enhanced oral delivery of insulin | |
| CN110623918A (en) | Carboxymethyl chitosan/sodium alginate nano hydrogel and preparation method and application thereof | |
| CN105617362A (en) | Novel insulin-phospholipid-chitosan self-assembled microparticle carrier and preparation thereof | |
| CN102908627A (en) | pH-sensitive nanoparticles for oral insulin delivery | |
| CN114522150B (en) | Preparation method and application of pH-sensitive plant microcapsule nano extruder | |
| KR20200132584A (en) | Stabilized astaxanthin nanoparticles and its manufacturing method | |
| Ngwuluka et al. | Natural polymers in micro-and nanoencapsulation for therapeutic and diagnostic applications: part II-polysaccharides and proteins | |
| JP2008297288A (en) | Particulate and production method | |
| CN109568601B (en) | Protein polypeptide drug dual microsphere, preparation method thereof and insulin dual microsphere | |
| KR102386223B1 (en) | Albumin layer-polyelectrolyte multi-layers particle encapsulating poorly water-soluble drug | |
| Das et al. | Chitosan-based systems for oral drug delivery applications | |
| Yurdasiper et al. | An overview of modified release chitosan, alginate and eudragit RS microparticles | |
| De Anda-Flores et al. | Polysaccharides nanoparticles as oral drug delivery systems | |
| CN109675020B (en) | Oral GLP-1 polypeptide nano preparation and preparation method and application thereof | |
| CN107823178B (en) | Niflumic acid colon targeted preparation for treating irritable bowel syndrome and preparation method thereof | |
| EP3666264B1 (en) | Biodegradable covalent matrices for the oral delivery of insulin directed to the colon, activated by microbiota, and production method thereof | |
| CN113244189A (en) | Preparation method of ultra-small bionic nanoparticles based on erythrocyte membranes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |