CN117159733A - Polydopamine modified montmorillonite and preparation method and application thereof - Google Patents
Polydopamine modified montmorillonite and preparation method and application thereof Download PDFInfo
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- CN117159733A CN117159733A CN202310047607.4A CN202310047607A CN117159733A CN 117159733 A CN117159733 A CN 117159733A CN 202310047607 A CN202310047607 A CN 202310047607A CN 117159733 A CN117159733 A CN 117159733A
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- montmorillonite
- sodium
- polydopamine
- suspension
- calcium
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- GUJOJGAPFQRJSV-UHFFFAOYSA-N dialuminum;dioxosilane;oxygen(2-);hydrate Chemical class O.[O-2].[O-2].[O-2].[Al+3].[Al+3].O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O GUJOJGAPFQRJSV-UHFFFAOYSA-N 0.000 title claims abstract description 250
- 229920001690 polydopamine Polymers 0.000 title claims abstract description 76
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 58
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims abstract description 34
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Classifications
-
- 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
Abstract
The invention provides polydopamine modified montmorillonite, and a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The preparation method of the invention comprises the following steps: (1) Crushing sodium-based montmorillonite clay to prepare sodium-based montmorillonite suspension; (2) And (3) after regulating the pH value of the sodium-based montmorillonite suspension, adding dopamine for reaction, and obtaining the polydopamine modified montmorillonite after the reaction is finished. The polydopamine modified montmorillonite has a special microstructure, has good colloid stability when being dispersed in aqueous solution, is not easy to settle, is convenient for dosage control, and reduces constipation caused by excessive administration. And the composition has the properties of montmorillonite and polydopamine, can absorb excessive intestinal juice in intestinal tracts to form a pasty protective layer, can control diarrhea symptoms of enteritis, can resist oxidation and inflammation in situ, and can promote the treatment of enteritis.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to polydopamine modified montmorillonite, and a preparation method and application thereof.
Background
Montmorillonite is a natural clay, which is named montmorillonite because it is originally produced in Mongolian De City in Mediterranean, france. Montmorillonite has long been used for treating acute and chronic enteritis, gastritis and canker sore due to its unique ability to detoxify and adsorb and protect mucous membranes. Mature products such as fine-particle powders of montmorillonite (montmorillonite powder) are already available on the global market. Montmorillonite powder has been used for treating acute diarrhea and other gastrointestinal diseases for nearly 50 years, and 150 hundred million bags have been sold worldwide by 2019, and the biological safety of nearly 7 hundred million patients in 80 countries worldwide is fully verified. In recent years, montmorillonite has attracted great interest in the biomedical field due to its excellent cation exchange capacity, adsorption capacity, biocompatibility and other advantages.
The montmorillonite powder mainly comprises montmorillonite, wherein the montmorillonite consists of a layered structure of 2:1, and two silicon tetrahedra sandwich one alumina octahedron. The silicon element and the aluminum element in the molecule are replaced by metal ions with lower price, i.e. isomorphous replacement occurs, so that stable net negative charges are formed. The montmorillonite absorbs a large amount of moisture in the body, the layered structure of the montmorillonite expands and disperses, the layers slide and open, and are connected with each other through electrostatic adsorption between the sheets, the layers are not scattered and separated, and a continuous montmorillonite protective layer is formed on the surface of the digestive tract. Thanks to the huge specific surface area of montmorillonite, one gram of montmorillonite powder can cover the digestive tract surface of basketball court size of 110 square meters. In addition, the montmorillonite has the characteristics of uneven charge, the basic layer is negatively charged, and the interlayer is positively charged, so that the montmorillonite has strong electrostatic adsorption capacity, can adsorb and fix mycotoxin and harmful bacteria, and does not generate drug resistance to any bacterial virus. Meanwhile, montmorillonite can provide various microelements, and has the functions of nourishing, stopping bleeding and resisting stress.
The intestinal tract of enteritis patient has massive tissue fluid extravasation, increased intestinal fluid, massive intestinal bacteria reproduction and toxin release, and the patient has abdominal pain and diarrhea. The above properties of montmorillonite can solve various problems of enteritis intestinal tract. The montmorillonite can absorb excessive intestinal juice in intestinal tract, control diarrhea symptom, and correct intestinal electrolyte disorder. The montmorillonite forms a temporary protective barrier on the surface of the intestinal canal which is broken by inflammation or infection, adsorbs and fixes intestinal germs, germs and toxic products thereof, improves the defending function of the mucous membrane barrier on the attack factors, is also beneficial to balancing normal flora and improves the immune function of the digestive tract. And montmorillonite does not enter blood, and is completely discharged out of the body through excrement after the absorption is cleared.
However, when montmorillonite is used for enteritis treatment, the dosage of montmorillonite is difficult to control, and the simple use of montmorillonite for detoxification adsorption and barrier protection often causes excessive intake of montmorillonite to cause serious constipation, aggravate intestinal injury, and is unfavorable for enteritis treatment and recovery. Meanwhile, researches in the last twenty years show that a clear relation exists between the oxidative stress and the enteritis, and the regulation and control of the oxidative stress can become an important treatment target for the enteritis treatment. Therefore, the montmorillonite medicament provides a new way for regulating and controlling oxidative stress and is a new way for treating enteritis. But no related studies have been made in the art.
Disclosure of Invention
The invention aims to provide polydopamine modified montmorillonite, and a preparation method and application thereof. The polydopamine modified montmorillonite has a special microstructure, has good colloid stability when being dispersed in aqueous solution, is not easy to settle, is convenient for dosage control, and reduces constipation caused by excessive administration. And the composition has the properties of montmorillonite and polydopamine, can absorb excessive intestinal juice in intestinal tracts to form a pasty protective layer, can control diarrhea symptoms of enteritis, can resist oxidation and inflammation in situ, and can promote the treatment of enteritis.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of polydopamine modified montmorillonite, which comprises the following steps:
(1) Crushing sodium-based montmorillonite clay to prepare sodium-based montmorillonite suspension;
(2) And (3) after regulating the pH value of the sodium-based montmorillonite suspension, adding dopamine for reaction, and obtaining the polydopamine modified montmorillonite after the reaction is finished.
Preferably, the particle size of the crushed sodium montmorillonite clay is 100nm-2000nm.
Preferably, the sodium-based montmorillonite suspension has a concentration of 5-20wt%.
Preferably, the pH of the sodium-based montmorillonite suspension is 6-10 after adjustment.
Preferably, the solute concentration of the dopamine after being added is 2-40mg/ml.
Preferably, the temperature of the reaction is 20-25 ℃; the reaction time is 2-4h; the rotational speed during the reaction is 100-140rpm.
The invention also provides the polydopamine modified montmorillonite prepared by the preparation method.
Preferably, the polydopamine modified montmorillonite is in a suspension form.
The invention also provides application of the polydopamine modified montmorillonite in preparing a medicine for treating enteritis.
The invention provides polydopamine modified montmorillonite, and a preparation method and application thereof. The polydopamine modified montmorillonite is formed by depositing the polydopamine modified layer on the surface of the sodified montmorillonite, the polydopamine modified reaction condition is green and safe, complex chemical substances and synthesis processes are not needed, toxic and harmful substances are not introduced, the operation is simple, the control is efficient, and the mass production is easy to realize. The produced polydopamine modified montmorillonite has a special microstructure, as shown in figure 1, the polydopamine modified montmorillonite particles are in a polygonal lamellar shape, the particle size is between 100nm and 2000nm, and the thickness is between 5nm and 50 nm. The polydopamine modified montmorillonite is dispersed in an aqueous solution, has good colloid stability, the sedimentation volume ratio of 2h is not more than 20%, fine lamellar powder is formed after freeze-drying, the property of the aqueous solution is unchanged after re-suspension, the dosage control is convenient, and the constipation problem caused by excessive administration is reduced.
The polydopamine modified montmorillonite is fine in taste, good in oral compliance, capable of resisting damage of gastric acid after oral administration or clysis, capable of keeping the aggregation of lumps in the stomach, stable in performance, gradually dispersed in a neutral/weak alkaline intestinal environment along with gastrointestinal peristalsis, capable of absorbing excessive intestinal juice to form a pasty protective layer, capable of controlling diarrhea symptoms of enteritis, capable of promoting secretion of mucosal proteins, capable of enhancing strength and quality of the mucosal layer, capable of up-regulating expression of tight junction proteins, and capable of improving defense function of endogenous mucosal barriers against attack factors. After covering the surface of the digestive tract, the polydopamine modified montmorillonite can remove excessive toxic free radicals, resist oxidation and inflammation in situ, regulate and control the expression of oxidative stress and inflammatory factors, recover oxidation reduction and immune homeostasis of the intestinal tract, and promote non-fibrosis repair of the intestinal tract.
Drawings
FIG. 1 is a schematic diagram of the structure of the polydopamine-modified montmorillonite of the invention.
FIG. 2 is a transmission electron microscope comparison of groups 1, 3, 5 of polydopamine modified montmorillonite and unmodified montmorillonite of comparative example 2 of example 1 of the present invention.
FIG. 3 is a graph showing the comparison of the ability of the suspensions of the polydopamine-modified montmorillonite of groups 1, 3 and 5 and the suspensions of the unmodified montmorillonite of comparative example 2 to scavenge endogenous free radicals of Caco-2 cells in test example 2 according to the present invention.
FIG. 4 is a graph showing the comparative therapeutic effects of the suspensions of group 3 polydopamine-modified montmorillonite and unmodified montmorillonite of comparative example 2 in experimental example 3 of the present invention on mice models of enteritis.
FIG. 5 is a graph showing comparative statistics of constipation side effects of the group 3 polydopamine modified sodium montmorillonite suspensions of test example 4, the unmodified calcium montmorillonite suspensions of comparative example 1, the unmodified sodium montmorillonite suspensions of comparative example 2, and the polydopamine modified calcium montmorillonite suspensions of comparative example 4 on enteritis mice models, according to the present invention.
Detailed Description
The invention provides a preparation method of polydopamine modified montmorillonite, which comprises the following steps:
(1) Crushing sodium-based montmorillonite clay to prepare sodium-based montmorillonite suspension;
(2) And (3) after regulating the pH value of the sodium-based montmorillonite suspension, adding dopamine for reaction, and obtaining the polydopamine modified montmorillonite after the reaction is finished.
Sodium montmorillonite clay is crushed to prepare sodium montmorillonite suspension.
In the present invention, the sodium-based montmorillonite clay is preferably obtained by sodium-curing calcium-based montmorillonite.
In the present invention, the calcium-based montmorillonite is preferably a pharmaceutically acceptable calcium-based montmorillonite.
In the present invention, the pharmaceutical calcium-based montmorillonite is preferably purified prior to sodium modification.
In the present invention, the purification process is preferably: mixing the medicinal calcium-based montmorillonite with deionized water, magnetically stirring, standing for precipitation, and removing mixed soluble impurities in the medicinal calcium-based montmorillonite to obtain purified calcium-based montmorillonite solid.
In the invention, the mass volume ratio of the medicinal calcium-based montmorillonite to deionized water is preferably 1g: (5-15) ml, further preferably 1g:10ml.
In the present invention, the rotation speed of the stirring is preferably 100 to 200rpm, and more preferably 120rpm.
In the present invention, the stirring time is preferably 1 to 3 hours, more preferably 2 hours.
In the present invention, the temperature of the standing is preferably 20 to 25 ℃, and more preferably 25 ℃.
In the present invention, the time for the standing is preferably 20 to 32 hours, more preferably 24 hours.
In the present invention, the sodium treatment process is preferably: re-suspending the purified calcium-based montmorillonite solid into suspension, adding sodium chloride and stirring to fully mix sodium ions and montmorillonite; standing to enable sodium ions to replace calcium ions in montmorillonite molecules through the cation exchange capacity of montmorillonite; centrifuging after standing, discarding the supernatant, adding deionized water with the volume equal to that of the discarded supernatant into the precipitate, and repeating the above centrifuging steps until the centrifuged supernatant is free of chloride ions, thereby obtaining the sodium montmorillonite clay.
In the present invention, the weight percentage of the suspension is preferably 5 to 20wt%, and more preferably 20wt%.
In the present invention, the solute concentration after the sodium chloride addition is preferably 0.5 to 2mol/L, more preferably 0.1mol/L.
In the present invention, the rotation speed of the stirring is preferably 100 to 200rpm, and more preferably 120rpm.
In the present invention, the stirring time is preferably 10 to 15 hours, more preferably 12 hours.
In the present invention, the time for the standing is preferably 8 to 15 hours, more preferably 12 hours.
In the present invention, the rotational speed of the centrifugation is preferably 3000 to 5000rpm, more preferably 4000rpm.
In the present invention, the time for the centrifugation is preferably 25 to 40min, more preferably 35min.
In the invention, the crushing is preferably carried out by firstly carrying out wet grinding on sodium-based montmorillonite clay to obtain a sodium-based montmorillonite coarse dispersion system, and then carrying out ultrasonic crushing on the sodium-based montmorillonite coarse dispersion system.
In the present invention, the wet milling is preferably carried out using a mill, and more preferably a KZ-III-F cryogenic mill (Wohai Vir Biotech Co., ltd.).
In the present invention, the parameters of the mill are preferably 50Hz.
In the invention, the frequency of grinding is preferably 40-60s, and the intermittent time is 10-20s; further preferably, the polishing time is 45s and the batch time is 15s.
In the present invention, the mass ratio of the grinding medium to the sodium montmorillonite clay at the time of the grinding is preferably 1: (1-3), further preferably 1:2.
the particle size of the grinding medium is preferably 0.4 to 0.6mm, more preferably 0.4mm.
In the present invention, the total time of the grinding is preferably 40 to 60 minutes, more preferably 50 minutes.
In the present invention, the ultrasonic disruption is preferably carried out by using an ultrasonic disruptor, more preferably a scientific-IID ultrasonic cell disruptor (Ningbo New Zhi Biotech Co., ltd.).
In the present invention, the ultrasonic breaker parameter is preferably 500J/ml.
In the present invention, the time of the ultrasonic disruption is preferably 20 to 40 minutes, more preferably 30 minutes.
In the present invention, the particle size of the crushed sodium montmorillonite clay is preferably 100nm to 2000nm.
In the present invention, the concentration of the sodium-based montmorillonite suspension is preferably 5 to 20wt%, and more preferably 10wt%.
After the pH value of the sodium-based montmorillonite suspension is regulated, adding dopamine for reaction, and obtaining the polydopamine modified montmorillonite after the reaction is finished.
In the present invention, the pH of the sodium montmorillonite suspension after adjustment is preferably 6 to 10, more preferably 8.
In the present invention, the solute concentration after the addition of dopamine is preferably 2 to 40mg/ml, and more preferably 20mg/ml.
In the present invention, the temperature of the reaction is preferably 20 to 25 ℃, and more preferably 25 ℃.
In the present invention, the reaction time is preferably 2 to 4 hours, more preferably 3 hours.
In the present invention, the rotational speed at the time of the reaction is preferably 100 to 140rpm, more preferably 120rpm.
The invention also provides the polydopamine modified montmorillonite prepared by the preparation method.
In the present invention, the dosage form of the polydopamine modified montmorillonite is preferably a suspension.
The invention also provides application of the polydopamine modified montmorillonite in preparing a medicine for treating enteritis.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The invention provides a polydopamine modified montmorillonite suspension, which is prepared by the following steps:
according to the pharmaceutically acceptable calcium montmorillonite with deionized water=1 g: mixing in a proportion of 10ml, magnetically stirring at 120rpm for 2h, standing at room temperature (25 ℃) for natural precipitation for 24h, and removing mixed soluble impurities in the medical calcium-based montmorillonite to obtain the purified calcium-based montmorillonite solid.
The purified calcium-based montmorillonite solid is resuspended into suspension with the weight percentage of 20wt%, sodium chloride is added according to the solute concentration of 1mol/L, stirring and dissolution are carried out at 120rpm for 12 hours, so that sodium ions and montmorillonite are fully mixed, and standing is carried out for 12 hours, and the sodium ions replace calcium ions in montmorillonite molecules through the cation exchange capacity of montmorillonite. After the standing is finished, centrifuging at 4000rpm for 35min, discarding the supernatant, adding deionized water equivalent to the discarded supernatant into the precipitate, repeating the centrifugation steps until detecting that the centrifuged supernatant is free of chloride ions by using a silver nitrate solution, and completing the sodium modification of the calcium-based montmorillonite to obtain the sodium-based montmorillonite clay.
Wet grinding sodium montmorillonite clay, wherein the mass of grinding medium is as follows: sodium montmorillonite clay mass=1: 2, 0.4mm grinding media (zirconium beads) was added in a proportion, the grinding parameters were set to 50Hz, the grinding time 45s, the batch time 15s, the total working time 50min. Grinding to obtain a coarse sodium montmorillonite dispersion system. The coarse dispersion of sodium montmorillonite was further particle size optimized using ultrasonication. The crude dispersion of sodium montmorillonite was placed in an ultrasonic breaker with parameters set at 500J/ml and run for 30min. The above crushing treatment (wet grinding and ultrasonic crushing method) is helpful for forming sodium-based montmorillonite clay with more uniform particle size, and the particle size range of the crushed sodium-based montmorillonite clay is 100nm-2000nm.
The crushed nanoclay was resuspended to a sodium-based montmorillonite suspension having a concentration of 5-20wt% according to the technical parameters set forth in groups 1-19 of Table 1. The sodium montmorillonite suspension was adjusted to pH 8 with Tris (hydroxymethyl) aminomethane, dopamine (DA) was added according to the solute concentration shown in Table 1, stirred and dissolved at 120rpm, reacted for a certain period of time, centrifuged at 4000rpm for 20min, and the supernatant was discarded. Adding deionized water with the same volume as the supernatant to the precipitate, repeating the centrifugation steps until the concentration of dopamine in the supernatant after centrifugation is lower than 5 mug/ml by using an ultraviolet-visible light spectrophotometer at 280nm, and obtaining the polydopamine modified montmorillonite of groups 1-19.
The polydopamine modified montmorillonite of each group was resuspended to a polydopamine modified montmorillonite suspension with a concentration of 10wt% for use.
Comparative example 1
According to the pharmaceutically acceptable calcium montmorillonite with deionized water=1 g: mixing in a proportion of 10ml, magnetically stirring at 120rpm for 2h, standing at room temperature, naturally precipitating for 24h, and removing mixed soluble impurities in medicinal calcium-based montmorillonite to obtain purified calcium-based montmorillonite solid.
Wet grinding the purified calcium-based montmorillonite solid, wherein the mass of the grinding medium is as follows: calcium-based montmorillonite solid mass=1: 2, 0.4mm grinding media (zirconium beads) was added in a proportion, the grinding parameters were set to 50Hz, the grinding time 45s, the batch time 15s, the total working time 50min. Grinding to obtain coarse calcium montmorillonite dispersion system. The coarse dispersion system of the calcium-based montmorillonite is further optimized in particle size by using an ultrasonic crushing method. The coarse dispersion of calcium-based montmorillonite was placed in an ultrasonic crusher with parameters set to 500J/ml and run for 30min. Detecting the crushed calcium-based montmorillonite clay, wherein the particle size range is 500-2000 nm.
The crushed calcium-based montmorillonite clay is resuspended into a 10wt% calcium-based montmorillonite suspension for later use.
Comparative example 2
According to the pharmaceutically acceptable calcium montmorillonite with deionized water=1 g: mixing in a proportion of 10ml, magnetically stirring at 120rpm for 2h, standing at room temperature, naturally precipitating for 24h, and removing mixed soluble impurities in medicinal calcium-based montmorillonite to obtain purified calcium-based montmorillonite solid.
The purified calcium-based montmorillonite solid is resuspended into suspension with the weight percentage of 20wt%, sodium chloride is added according to the solute concentration of 1mol/L, stirring and dissolution are carried out at 120rpm for 12 hours, so that sodium ions and montmorillonite are fully mixed, and standing is carried out for 12 hours, and the sodium ions replace calcium ions in montmorillonite molecules through the cation exchange capacity of montmorillonite. After the standing is finished, centrifuging at 4000rpm for 35min, discarding the supernatant, adding deionized water with the same volume as the supernatant, and repeating the centrifugation steps until the centrifugal supernatant is detected to be free of chloride ions by using a silver nitrate solution, thereby completing the sodium modification of the calcium-based montmorillonite and obtaining the sodium-based montmorillonite clay solid.
Wet grinding sodium montmorillonite clay, wherein the mass of grinding medium is as follows: sodium montmorillonite clay mass=1: 2, 0.4mm grinding media (zirconium beads) was added in a proportion, the grinding parameters were set to 50Hz, the grinding time 45s, the batch time 15s, the total working time 50min. Grinding to obtain a coarse sodium montmorillonite dispersion system. The coarse dispersion of sodium montmorillonite was further particle size optimized using ultrasonication. The crude dispersion of sodium montmorillonite was placed in an ultrasonic breaker with parameters set at 500J/ml and run for 30min. Detecting the crushed sodium montmorillonite clay, wherein the particle size range is 100nm-2000nm.
The crushed sodium montmorillonite clay is resuspended into 10wt% sodium montmorillonite suspension for standby.
Comparative example 3
The calcium-based montmorillonite suspension prepared in comparative example 1 was adjusted to pH 8 with Tris (hydroxymethyl) aminomethane, dopamine (DA) was added at a solute concentration of 2mg/ml, stirred and dissolved, stirred at 120rpm, oxidized for 3h, centrifuged at 4000rpm for 20min, and the supernatant was discarded. Adding deionized water with the same volume as the supernatant to the precipitate, repeating the centrifugation steps until the concentration of dopamine in the supernatant after centrifugation is detected to be lower than 5 mug/ml by an ultraviolet-visible light spectrophotometer (280 nm of characteristic absorption wavelength of dopamine), and obtaining the polydopamine modified calcium montmorillonite.
The polydopamine modified calcium-based montmorillonite is resuspended to a polydopamine modified calcium-based montmorillonite suspension with the concentration of 10 weight percent for standby.
Comparative example 4
The calcium-based montmorillonite suspension prepared in comparative example 1 was adjusted to pH 8 with Tris (hydroxymethyl) aminomethane, dopamine (DA) was added at a solute concentration of 20mg/ml, stirred and dissolved, stirred at 120rpm, oxidized for 3h, centrifuged at 4000rpm for 20min, and the supernatant was discarded. Adding deionized water with the same volume as the supernatant to the precipitate, repeating the centrifugation steps until the concentration of dopamine in the supernatant after centrifugation is detected to be lower than 5 mug/ml by an ultraviolet-visible light spectrophotometer (280 nm of characteristic absorption wavelength of dopamine), and obtaining the polydopamine modified calcium montmorillonite.
The polydopamine modified calcium-based montmorillonite is resuspended to a polydopamine modified calcium-based montmorillonite suspension with the concentration of 10 weight percent for standby.
Comparative example 5
The calcium-based montmorillonite suspension prepared in comparative example 1 was adjusted to pH 8 with Tris (hydroxymethyl) aminomethane, dopamine (DA) was added at a solute concentration of 40mg/ml, stirred and dissolved, stirred at 120rpm, oxidized for 3h, centrifuged at 4000rpm for 20min, and the supernatant was discarded. Adding deionized water with the same volume as the supernatant to the precipitate, repeating the centrifugation steps until the concentration of dopamine in the supernatant after centrifugation is detected to be lower than 5 mug/ml by an ultraviolet-visible light spectrophotometer (280 nm of characteristic absorption wavelength of dopamine), and obtaining the polydopamine modified calcium montmorillonite.
The polydopamine modified calcium-based montmorillonite is resuspended to a polydopamine modified calcium-based montmorillonite suspension with the concentration of 10 weight percent for standby.
Table 1 Process parameters of montmorillonite suspensions and polydopamine content of each group
Note that: the polydopamine content is calculated using the following formula:
polydopamine total mass= (initial solute concentration of dopamine-first supernatant dopamine concentration) x supernatant volume;
polydopamine content (%) = [ (polydopamine total mass/(polydopamine total mass+montmorillonite total mass) ]×100%.
Test example 1
The performance of each group of montmorillonite suspensions in example 1, and montmorillonite suspensions in comparative examples 1 to 5 was examined and analyzed. The specific analysis process is as follows:
1. determination of structural characteristics of montmorillonite
The structural characteristics of the smectites of each group of example 1 and comparative examples 1 to 5 were examined by transmission electron microscopy. The transmission electron microscope contrast diagram of the polydopamine-modified montmorillonite of the groups 1, 3, and 5 in example 1 and the unmodified montmorillonite of the comparative example 2 is shown in fig. 2.
2. Determination of colloidal stability of montmorillonite suspension
The colloidal stability of the montmorillonite suspension was evaluated based on the sedimentation volume ratio of montmorillonite particles in the suspension system per unit time.
Taking 10ml of uniformly dispersed montmorillonite suspension, loading into a glass measuring cylinder, standing on a horizontal table, measuring the sedimentation surfaces of montmorillonite particles at 1h and 2h respectively, and calculating the sedimentation volume ratio according to the following formula.
Volume ratio of sedimentation = (initial liquid level height-sedimentation surface height after sedimentation)/(initial liquid level height) ×100%
3. Broad-spectrum antioxidant capacity determination of montmorillonite suspension
The degree of scavenging DPPH free radical, hydroxyl free radical, superoxide free radical and hydrogen peroxide by montmorillonite suspensions of each group was examined, and the broad-spectrum antioxidant ability of the anti-montmorillonite suspensions was evaluated.
3.1DPPH radical scavenging Rate
DPPH free radical is a stable nitrogen center free radical, is an important index of the oxidation resistance of a sample, and has purple color and strong absorption at 515 nm. The absorbance of the solution at 515nm decreased to a degree proportional to the degree to which DPPH radicals were scavenged after DPPH was scavenged by the antioxidant.
Test, control and blank groups were set.
Test group: 20ml of DPPH radical (1, 1-diphenyl-2-picrylhydrazyl) solution was taken, 2ml of montmorillonite suspension was added, the mixture was vortexed and allowed to stand for 30min in the dark after vortexing, and the supernatant was taken after centrifugation at 12000rpm for 10min and was examined for absorbance at 515nm (A test).
Control group: 20ml of the aqueous solution was taken and the rest of the procedure was carried out in the same test group, and the absorbance at 515nm (A control) was measured.
Blank group: 20ml of DPPH radical solution was taken, 2ml of an aqueous solution determined to have no antioxidant ability was added, and the rest was conducted in the same manner as in the test group to detect absorbance at 515nm (A blank).
The absorbance data of the three groups are synthesized, and the free radical clearance is calculated according to the following formula:
DPPH radical clearance = [ a blank- (a test-a control) ] ×100%
3.2 superoxide anion (O) 2 - ) Clearance rate of
Superoxide anion (.o) 2 - ) Is determined by measuring the inhibition of NBT (nitro blue tetrazolium) oxidation. As a sensitive superoxide anion indicator, NBT can be reduced by superoxide anion to generate blue formazan, and the absorbance at 530nm is changed. The inhibition of the rate of decrease in absorbance at 530nm by montmorillonite suspensions reflects their ability to scavenge superoxide anions.
Test, control and blank groups were set.
Test group: 9910. Mu.L of the superoxide radical solution was taken, 90. Mu.L of montmorillonite suspension was added, the mixture was vortexed and allowed to stand in a dark place for 60 minutes, and after centrifugation at 12000rpm for 10 minutes, the supernatant was taken and detected for absorbance at 530nm (A test).
Control group: 9910. Mu.L of the aqueous solution was taken and the rest was run in the same test group to detect absorbance at 530nm (A control).
Blank 1 group: 9910. Mu.L of a superoxide radical solution was added to 90. Mu.L of an aqueous solution determined to have no antioxidant ability, and the rest of the procedure was the same as that of the test group to detect absorbance at 530nm (A1 blank).
Blank 2 groups: 9910. Mu.L of an aqueous solution was added to 90. Mu.L of an aqueous solution determined to have no antioxidant ability, and the rest was conducted in the same manner as in the test group to detect absorbance at 530nm (A2 blank).
Delta aassay = a assay-a control, delta a blank = A1 blank-A2 blank.
The removal rate of superoxide anions was calculated according to the following formula:
superoxide anion (.o) 2 - ) Radical clearance = (Δa blank- Δa measurement)/Δa blank×100%
3.3 hydroxy radical (. OH) clearance
By measuring Fe 2+ Inhibition of oxidation to evaluate the scavenging activity of hydroxyl radicals (.OH). The test system contains a metal chelating agent phenanthroline and Fe 2+ After chelation, the 536nm absorbance values are concentration dependent. The hydroxy radical being formed by H 2 O 2 And Fe (Fe) 2+ Generated by Fenton reaction interaction, fe in aqueous solution 2+ Oxidation to Fe 3+ This resulted in a decrease in absorbance at 536 nm.
Test, control and blank groups were set.
Test group: 100. Mu.L of a hydroxyl radical solution was taken and 50. Mu.L of Fe was added 2+ The mixture was vortexed, left to stand in the dark for 60min, and the supernatant was centrifuged at 12000rpm for 10min, whereupon the absorbance at 536nm was measured (A test).
Control group: 100. Mu.L of a hydroxyl radical solution was taken and 50. Mu.L of Fe was added 2+ Phenanthroline solution and 50. Mu.L of aqueous solution, the rest of the procedure being the same as the test group, the absorbance at 530nm (control A) was measured.
Blank group: 100. Mu.L of the aqueous solution was taken and 50. Mu.L of Fe was added 2+ O-phenanthroline solution and 50. Mu.L of montmorillonite suspension, the rest of the procedure was the same as for the test group, and the absorbance at 530nm (A blank) was measured.
The removal rate of superoxide anions was calculated according to the following formula:
hydroxyl radical (·oh) clearance = [ (a test-a control) - (a blank-a control) ] ×100%
3.4H 2 O 2 Clearance rate of
H 2 O 2 Is an important free radical in vivo, H 2 O 2 A yellow titanium peroxide complex forms with titanium sulfate, with a characteristic absorption at 415 nm.
Test, control and blank groups were set.
Test group: 100 mu L of H is taken 2 O 2 The solution was added with 50. Mu.L of a titanium sulfate solution and 50. Mu.L of a montmorillonite suspension, vortexed and mixed uniformly, left in a dark place for 60min, centrifuged at 12000rpm for 10min, and the supernatant was taken and detected for absorbance at 415nm (A test).
Control group: 100. Mu.L of the aqueous solution was taken, 50. Mu.L of a titanium sulfate solution and 50. Mu.L of a montmorillonite suspension were added, and the remaining operations were the same as those of the test group, to thereby detect absorbance at 415nm (control A).
Blank group: 100 mu L of H is taken 2 O 2 The solution was added with 50. Mu.L of a titanium sulfate solution and 50. Mu.L of an aqueous solution, and the remaining operations were the same as those of the test group, and absorbance at 415nm (A blank) was measured.
The removal rate of superoxide anions was calculated according to the following formula:
H 2 O 2 clearance = [ (a blank-a test) - (a blank-a control)]×100％
4. Determination of the binding Strength of montmorillonite suspension and mucus
Mucin is the main component of mucus, and the binding strength of anti-montmorillonite suspensions to mucus was evaluated by the binding rate of montmorillonite suspensions to mucin in solution.
Taking 1ml of a pig intestine-derived mucin solution with a known concentration, adding 1ml of a montmorillonite suspension, magnetically stirring (60 rpm,60 min) to enable montmorillonite particles and mucin molecules to be fully mixed and contacted, centrifuging at 12000rpm for 10min, taking a supernatant, detecting the mucin concentration in the supernatant by using a BCA protein concentration detection kit, and calculating the mucin binding rate according to the following formula:
mucin binding ratio = [ (initial mucin concentration/2-supernatant protein concentration)/(initial mucin concentration/2) ]. Times.100%
The test results are shown in Table 2.
TABLE 2 analysis of Performance of montmorillonite suspensions of examples, groups, comparative examples 1-5
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From the results in table 2, it is understood that the ionic type of montmorillonite has an important influence on the colloidal stability (sedimentation volume ratio) of montmorillonite and its modified products. The sedimentation volume ratio of the calcium-based montmorillonite and the polydopamine modified calcium-based montmorillonite for 2h is within 50-60%. When the calcium ions of the calcium-based montmorillonite are replaced with sodium ions by cation exchange, sodium-based montmorillonite is formed. The sedimentation volume ratio of the sodium-based montmorillonite and the polydopamine modified sodium-based montmorillonite is within 10-20%, which shows that the colloid stability of the sodium-based montmorillonite is obviously superior to that of the calcium-based montmorillonite, and the surface modification of polydopamine does not influence the colloid stability.
The ionic type of montmorillonite also has an important effect on the antioxidant capacity of the modified montmorillonite product, the radical scavenging rate of the unmodified calcium-based montmorillonite suspension (comparative example 1) is 2.3%, and even after polydopamine modification, the radical scavenging rate of the prepared modified montmorillonite products (comparative examples 3 to 5) is only 18.3 to 20.0%.
The ionic type of montmorillonite also has an important effect on the mucoadhesive strength of the modified montmorillonite product, and the mucin binding rate of unmodified sodium-based montmorillonite (comparative example 2) is 63.2%; in contrast, the mucin binding rate of unmodified calcium-based montmorillonite (comparative example 1) was 47.3%, and the radical scavenging rate of the prepared modified montmorillonite products (comparative examples 3 to 5) was only 48.6 to 50.1% even after polydopamine modification, even lower than that of unmodified sodium-based montmorillonite suspension (comparative example 2).
The polydopamine modification has an important influence on the antioxidant capacity of the modified montmorillonite product: the free radical scavenging rate of the unmodified sodium-based montmorillonite suspension (comparative example 2) was only 6.6%; when the weight percentage of the sodium-based montmorillonite is 10wt%, and the concentration of the dopamine solute is 2-40mg/ml, the free radical clearance of the prepared modified montmorillonite suspension is 48.6-80.4%.
The radical scavenging ability of polydopamine-modified montmorillonite is mainly derived from surface-modified polydopamine, while its mucin binding ability is derived from the affinity of montmorillonite itself for mucin on the one hand and also from surface-modified polydopamine on the other hand. Because the polydopamine molecule is rich in catechol groups, the polydopamine has extremely strong oxidation-reduction activity and can remove free radicals. And the catechol structure is also a substance basis for strong adhesion of mussels, so that the mucin binding rate of the modified montmorillonite increases with the increase of the modification amount of the surface polydopamine. However, the polydopamine modified layer is not absolutely stable, as the reaction times for group 12, group 6, group 1, group 9 are sequentially 1h, 2h, 3h, 4h, the remaining reaction conditions are the same, the free radical scavenging capacity gradually increases with increasing reaction time, and the free radical scavenging capacity decreases significantly when the reaction time reaches 5h (group 14). In addition, the polydopamine content with the dopamine solute concentration of 40mg/ml and the reaction time of 5h (group 15) is lower than that of 3 or 4h (group 5, group 11), which indicates that too long reaction time plus too high dopamine concentration can lead to exfoliation of the surface modified polydopamine layer, and the corresponding free radical scavenging capacity and mucin binding capacity can be reduced, which indicates that the optimal modification condition of polydopamine is dopamine feeding concentration of 2-40mg/ml and reaction time of 2-4h.
Thus, by adjusting the ionic type, dopamine solute concentration and reaction time of the montmorillonite in the modified montmorillonite formulation, the colloid stability, antioxidant capacity and mucin binding strength of the modified montmorillonite can be adjusted.
Test example 2
The ability of the montmorillonite suspensions of groups 1, 3, 5 of example 1, and the montmorillonite suspension of comparative example 2 to scavenge endogenous free radicals from human colorectal adenocarcinoma cells was verified using in vitro experiments. The specific process is as follows:
human colorectal adenocarcinoma cells (Caco-2 cells, available from Shanghai Fu He biological Co., ltd.) were placed in 12-well plates at a density of 10 ten thousand cells/well, incubator (5% CO) 2 Pre-incubation was carried out for 12 hours at 37 ℃. Adding modified montmorillonite suspension (0.4 ml,50 mg/ml) with different formulations into culture solution, culturing for 24 hr, washing off extracellular modified montmorillonite with PBS,rosup (available from Shanghai Biyun) was added to stimulate free radical production of cells and after 30 minutes of stimulation, PBS was used for washing. DCFH-DA dye solution (purchased from Shanghai Biyun) was added and incubated for 20 minutes. DCFH-DA itself is not fluorescent and can freely pass through the cell membrane into the cell, and then is hydrolyzed by intracellular lipase to generate DCFH. Whereas DCFH cannot permeate the cell membrane and is loaded into the cell, endogenous free radicals in the cell can oxidize non-fluorescent DCFH to produce fluorescent DCF. After washing the cells twice with PBS, the fluorescence of the cells was observed.
The results are shown in FIG. 3, where normal Caco-2 cells not stimulated with Rosup do not have DCF fluorescence, whereas strong DCF fluorescence occurs after Rosup stimulation, indicating endogenous free radical production. The montmorillonite suspensions of group 3 and group 5 play a role in removing endogenous free radicals, and remarkably reduce the intensity of DCF fluorescence. In contrast, the DCF fluorescence decrease was not evident in comparative example 2 and group 1, and it is understood from the content of polydopamine in table 2 that sufficient polydopamine modification is the basis for the modified montmorillonite to scavenge free radicals and exert antioxidant effect.
Test example 3
The therapeutic effect of the montmorillonite suspension of group 3 in example 1 and the montmorillonite suspension of comparative example 2 on dextran sodium sulfate enteritis mice model was verified by animal experiments. The specific process is as follows:
the Dextran Sodium Sulfate (DSS) enteritis model is one of the most widely used enteritis models at present, and diarrhea, hematochezia, weight loss and other enteritis symptoms similar to human pathological features can be rapidly formed by continuously feeding a DSS solution (2.5 wt%) to mice. Mice were continuously fed DSS solution (2.5 wt%) for 5 days, and a dextran sodium sulfate enteritis mice model was constructed. 9 mice models were taken, each 3 groups on average, control group, experimental group 1 and experimental group 2. Another 3 healthy mice were taken as normal group. Wherein, the normal group and the control group are continuously infused with PBS solution of 0.2ml for 5 days once a day; experimental group 1 was given a dose of 3.9mg/10g body weight, and the montmorillonite suspension of group 3 was administered parenterally, once a day, for 5 consecutive days; experiment group 2 was given a montmorillonite suspension of comparative example 2 by gavage at a dose of 3.9mg/10g body weight for 5 consecutive days, once a day.
After the experiment was completed, mice were euthanized humanizedly, the colon was photographed and section stained to observe intestinal epithelium microform and barrier integrity. As shown in fig. 4, the unmodified montmorillonite suspension of comparative example 2 was unable to effectively scavenge free radicals and bind mucomucin, so that the repair of the epithelial barrier of the digestive tract and the insufficient antioxidant capacity were unable to effectively restore the shortening of the intestinal tract due to enteritis, apoptosis of epithelial cells in HE staining, barrier defect, crypt necrosis, inflammatory cell infiltration. In contrast, the polydopamine modified montmorillonite suspension of group 3 can treat enteritis by resisting oxidation and repairing the epithelial barrier of the digestive tract, the colon length is obviously recovered, the HE staining epithelial cells and barrier are nearly normal, the crypt is complete, and no obvious inflammation exists.
Test example 4
Animal experiments were performed to verify constipation after administration of the montmorillonite suspensions of group 3, comparative example 1, comparative example 2, and comparative example 4, respectively, to dextran sodium sulfate enteritis mice. The specific process is as follows:
mice were continuously fed DSS solution (2.5 wt%) for 5 days, and a dextran sodium sulfate enteritis mice model was constructed. 12 mice models were taken for successful construction, and the mice were equally divided into 4 groups of 3 mice each, which were group 3 (polydopamine modified sodium montmorillonite suspension), comparative example 1 (calcium montmorillonite suspension), comparative example 2 (sodium montmorillonite suspension) and comparative example 4 (polydopamine modified calcium montmorillonite suspension), respectively. Each group was given an excess dose (8 mg/10g mouse body weight) for 5 consecutive days, and the corresponding montmorillonite suspension was given by gavage once a day. Five days later, the mice were observed for bowel movements, with particular attention to abdominal distension due to constipation. The number of constipation mice was counted, and the constipation rate was calculated as constipation rate= (number of constipation mice/3) ×100%. The results are shown in FIG. 5.
As can be seen from fig. 5, 3 constipation cases were observed in comparative example 1 (constipation rate=100%), 3 constipation cases were observed in comparative example 2 (constipation rate=100%), 1 constipation case was observed in comparative example 4 (constipation rate=33.3%), and 0 constipation case was observed in group 3 (constipation rate=0%). It was shown that constipation rate of the polydopamine modified group 3 and comparative example 4 constipation mice was lower than that of the polydopamine unmodified comparative examples 1 and 2.
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 (9)
1. The preparation method of the polydopamine modified montmorillonite is characterized by comprising the following steps of:
(1) Crushing sodium-based montmorillonite clay to prepare sodium-based montmorillonite suspension;
(2) And (3) after regulating the pH value of the sodium-based montmorillonite suspension, adding dopamine for reaction, and obtaining the polydopamine modified montmorillonite after the reaction is finished.
2. The method according to claim 1, wherein the crushed sodium montmorillonite clay has a particle size of 100nm to 2000nm.
3. The method of preparation according to claim 2, wherein the concentration of the sodium-based montmorillonite suspension is 5-20wt%.
4. A process according to claim 3, wherein the pH of the sodium montmorillonite suspension after adjustment is between 6 and 10.
5. The method according to claim 4, wherein the solute concentration after the addition of dopamine is 2-40mg/ml.
6. The method of claim 5, wherein the temperature of the reaction is 20-25 ℃; the reaction time is 2-4h; the rotational speed during the reaction is 100-140rpm.
7. A polydopamine-modified montmorillonite prepared by the preparation method of any one of claims 1 to 6.
8. The polydopamine-modified montmorillonite according to claim 7 wherein the polydopamine-modified montmorillonite is in the form of a suspension.
9. Use of the polydopamine-modified montmorillonite according to claim 7 or 8 for the preparation of a medicament for the treatment of enteritis.
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