CN113788904B - Laminarin compound with intestinal barrier repairing effect and preparation method thereof - Google Patents
Laminarin compound with intestinal barrier repairing effect and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and particularly discloses laminarin compounds with intestinal barrier repairing effect, a preparation method and application thereof, wherein the laminarin compounds are laminarin modified by carboxylic acid groups; the preparation method comprises the following steps: adding laminarin, pyridine and/or pyridine derivative into organic solvent, and drying after reaction to obtain solid product; adding the solid product into an organic solvent for dissolution to obtain a dissolution solution; adding an organic matter containing a carboxylic acid group into an organic solvent, and then adding a dissolving solution to mix to obtain a reaction solution; heating the reaction solution, cooling, pouring into an organic solvent, and drying to obtain the laminarin compound. The pyridine laminarin carboxylate modified by carboxylic acid groups has good effect on intestinal barrier repair, and can improve intestinal fibrosis and regulate intestinal dysbacteriosis.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to laminarin compounds with an intestinal barrier repairing effect, and a preparation method and application thereof.
Background
With the continuous improvement of the physical living standard of people, the selection of people in the aspect of diet is continuously increased, and due to the wide popularization and abuse of fast food, food additives and the like, the diet structure and habit of people are continuously changed, and high-salt, high-sugar and high-fat foods are increasingly appeared on dining tables. Such changes in dietary structure have also led to the development of many intestinal diseases, which severely threatens intestinal health and stability, thereby affecting people's normal life and work. According to epidemiological studies, inflammatory enteritis is mainly caused by unreasonable dietary structure, which is a group of gastrointestinal diseases characterized by chronic inflammation and dysbacteriosis of the intestinal tract, including crohn's disease and ulcerative colitis. At present, inflammatory enteritis is mainly treated by adopting inflammatory drugs, immunosuppressants and biological drugs, but the drugs have larger toxic and side effects.
The intestines contain a microbial ecological system, and balanced microbial groups play an important role in the healthy development of the intestinal mucosa immune system. Intestinal microbiota is closely linked to intestinal diseases. Clinical and experimental data indicate that the composition and abundance of intestinal microbiota vary in patients with intestinal disease. This phenomenon is known as intestinal biological disorders. Intestinal microbiota of patients with intestinal disease is characterized by low microbial diversity, reduced growth of probiotics (including bifidobacteria, lactobacilli and enterococci), and expansion of inflammation-associated bacteria (e.g. enterobacteriaceae) compared to healthy individuals. These intestinal dysbiosis can enhance inflammatory responses, affecting the health of the intestine. Given the impact of dysregulation of intestinal flora in intestinal diseases, intestinal flora is considered as a new area of drug-targeted therapy.
Therefore, there is a need to develop a drug with barrier repair effect on the intestinal tract, which has important significance for intestinal health.
Disclosure of Invention
The invention provides laminarin compounds with intestinal barrier repairing effect, a preparation method and application thereof, which are used for solving one or more technical problems in the prior art and at least providing a beneficial selection or creation condition.
To overcome the above problems, a first aspect of the present invention provides a laminarin compound or a pharmaceutically acceptable salt thereof.
Specifically, a laminarin compound represented by formula (1) or a pharmaceutically acceptable salt thereof:
wherein: n is an integer between 1 and 100; preferably, n is an integer between 5 and 25.
The invention takes laminarin as a main raw material, and pyridine laminarin carboxylate is obtained by adding a pyridine group to laminarin molecules and adding a carboxylic acid group to a pyridine ring. The laminaria japonica is a leaf-shaped body of laminaria japonica of laminariaceae or black laminaria japonica of pterophyceae, has the effects of softening hardness to dissipate stagnation, dissolving phlegm and promoting diuresis, and the main components of the laminaria japonica are polysaccharide, natural protein, fat, cellulose, mineral substances, nucleic acid and the like, has obvious pharmacological effects, the laminarin is one of main components of laminaria japonica, the biological activity exerted by the laminarin is closely related to the structural characteristics (such as group types and quantity, molecular weight, composition sugar, glycosidic bond types, connection sequence and the like) of the laminarin, and the biological activities of the laminarin with different structures are also different to a certain extent. The invention modifies laminarin by carboxylic acid groups on the basis of laminarin molecules, changes the spatial structure of laminarin, initiates activity change of laminarin, and confirms that the laminarin carboxylate with the structure of formula (1) not only has the effect of improving the fibrosis degree of intestinal cells, but also has the effects of regulating intestinal microbial structures, promoting the growth of probiotics and inhibiting the proliferation of pathogenic bacteria by analyzing the repairing effect of the laminarin carboxylate on intestinal barriers.
As a further improvement of the above-mentioned scheme, the starting materials for preparing the laminarin compound or a pharmaceutically acceptable salt thereof include laminarin, pyridine and/or a derivative of pyridine and an organic substance containing a carboxylic acid group.
Preferably, the pyridine and/or derivative of pyridine comprises methyl isonicotinate;
preferably, the organic matter containing carboxylic acid groups comprises gamma-butyrolactone or propiolic acid.
As a further improvement of the above scheme, the mass to volume ratio of laminarin to pyridine and/or pyridine derivative is 1g: (10-20) mL.
Further preferably, the mass to volume ratio of laminarin to pyridine and/or pyridine derivative is 1g:15mL.
In a second aspect, the present invention provides a process for preparing laminarin compound or a pharmaceutically acceptable salt thereof.
Specifically, the preparation method of laminarin compound or pharmaceutically acceptable salt thereof comprises the following steps:
(1) Adding laminarin, pyridine and/or pyridine derivative into organic solvent, and drying after reaction to obtain solid product;
(2) Adding the solid product prepared in the step (1) into an organic solvent for dissolution to obtain a dissolution solution;
(3) Adding an organic matter containing a carboxylic acid group into an organic solvent, and then adding the dissolved solution prepared in the step (2) to mix to obtain a reaction solution;
(4) And (3) heating the reaction liquid obtained in the step (3), cooling, pouring the reaction liquid into an organic solvent, and drying to obtain the laminarin compound.
Specifically, in the step (1), when laminarin and methyl isonicotinate are used as the raw materials, the synthesis process of the reaction is as follows, wherein: laminarin-Pyridine (LPY) is Laminarin:
in the step (3), when the preparation raw material is gamma-butyrolactone and the solution prepared in the step (2), the synthesis process of the reaction solution is as follows, wherein: laminarin Zwitterion Carboxylate (LZC) is a salt of a laminarin carboxylate of pyridine:
as a further improvement of the above scheme, in step (1), the process conditions of the reaction are: stirring and refluxing at 110-130 deg.c for 36-54 hr.
Preferably, in step (1), the drying process conditions are: spin-drying at 60-70deg.C.
Preferably, in step (1), the solvent comprises toluene or absolute ethanol.
As a further improvement of the above scheme, in step (2), the mass volume of the solid product and the organic solvent is 1g: (5-20) mL.
Preferably, in step (2), the mass volume of the solid product and the organic solvent is 1g:10mL.
Preferably, in step (2), the solvent comprises dimethyl sulfone or dimethylformamide.
As a further improvement of the above scheme, in the step (3), the volume ratio of the organic matter containing carboxylic acid groups to the organic solvent is 1: (1-10).
Preferably, in the step (3), the volume ratio of the organic matter containing carboxylic acid groups to the organic solvent is 1:5.
preferably, in step (3), the solvent comprises dimethyl sulfone or dimethylformamide.
As a further improvement of the above scheme, in the step (4), the heating process conditions are as follows: stirring at 70-90 deg.c for 36-60 hr;
preferably, in step (4), the heating process conditions are: stirring at 80 ℃ for 48 hours;
preferably, in step (4), the volume ratio of the reaction solution to the organic solvent is 1: (1-10).
Further preferably, in step (4), the volume ratio of the reaction solution to the organic solvent is 1:4.
preferably, in step (4), the solvent comprises acetone or absolute ethanol.
Further preferably, in step (4), the temperature of the solvent is 4 ℃.
In a third aspect, the invention provides the use of a laminarin compound or a pharmaceutically acceptable salt thereof.
In particular to application of laminarin compound or pharmaceutically acceptable salt thereof in preparing medicines for repairing intestinal barrier.
The invention uses a sodium dextran sulfate (DSS) induced colonitis mouse model to carry out intestinal tissue morphology observation and intestinal flora structure composition change test, and the result proves that: the laminarin carboxylate has good effect on intestinal barrier repair, can improve intestinal fibrosis and maintain the stability of intestinal structure; can also regulate intestinal flora imbalance and maintain intestinal flora stability.
Further, the intestinal barrier repair includes improving a colon injury status of the mouse; improving the structural integrity of colon villus, the arrangement condition of epithelial cells and the histological score. The modulation of intestinal dysbacteriosis refers to an increase in the number of beneficial and a decrease in the number of detrimental flora. Wherein the beneficial bacteria include lactobacillus, bifidobacterium, lactococcus and the like; the harmful bacteria include Escherichia, helicobacter, and Bacteroides.
Compared with the prior art, the technical scheme of the invention has at least the following technical effects or advantages:
according to the invention, a pyridine group is introduced into laminarin molecules to obtain an intermediate product, and then a carboxylic acid group is introduced to prepare laminarin carboxylate, so that laminarin modified by the carboxylic acid group changes the spatial structure of laminarin and causes activity change of laminarin. The results of the morphological observation of intestinal tissues and the structural change test of intestinal flora by using a sodium dextran sulfate (DSS) induced colonitis mouse model prove that: the laminarin carboxylate has good effect on intestinal barrier repair, can improve intestinal fibrosis and maintain the stability of intestinal structure; can also regulate intestinal flora imbalance, promote intestinal probiotics flora proliferation, inhibit growth of intestinal harmful bacteria, and maintain intestinal flora stability. Provides a theoretical basis for physiological function research of laminarin carboxylate, is beneficial to promoting development and popularization of laminarin industry, and provides a treatment basis for clinical treatment of intestinal diseases. Meanwhile, the preparation method is simple, does not need complex chemical synthesis reaction, has single reaction product and high yield, and is suitable for industrialized mass production.
Drawings
FIG. 1 is an infrared spectrum of laminarin and laminarin carboxylate;
FIG. 2 is a flow chart of mouse feeding;
FIG. 3 is a graph of H & E staining results of colon tissue of mice;
FIG. 4 is a graph showing the results of masson staining of colon tissue of mice;
FIG. 5 is a graph of the relative abundance of enterobacteria at the portal level;
FIG. 6 is a graph of the ratio of the relative abundance of the Thick-walled/Bacteroides flora.
Detailed Description
The present invention is specifically described below by way of examples to facilitate the understanding of the present invention by those skilled in the art, and it is necessary to specifically point out that the examples are provided for further illustration only and are not to be construed as limiting the scope of the present invention, and that insubstantial modifications and adjustments of the present invention according to the above teachings should still fall within the scope of the present invention, and that the raw materials mentioned below are not specifically described, but are commercially available products, and that the process steps or preparation methods not specifically mentioned are those known to those skilled in the art.
Example 1
A preparation method of laminarin compound comprises the following steps:
(1) Laminarin and methyl isonicotinate were added to toluene, wherein: the mass volume ratio of laminarin to methyl isonicotinate is 1g:10mL, the temperature is 130 ℃, nitrogen is filled as a protective gas, stirring reflux is carried out for 36 hours, and then rotary evaporation is carried out at 70 ℃ to obtain a solid product;
(2) Adding the solid product obtained in the step (1) into dimethyl sulfoxide for dissolution, wherein: the mass volume ratio of the solid product to the dimethyl sulfoxide is 1g:15mL, stirring uniformly at room temperature to obtain a solution;
(3) Adding gamma-butyrolactone to dimethyl sulfoxide, wherein: the volume ratio of the gamma-butyrolactone to the dimethyl sulfoxide is 1:4, then dropwise adding the mixture into the solution prepared in the step 2, and uniformly mixing to obtain a reaction solution;
(4) Stirring the reaction solution prepared in the step (3) for 36 hours at 90 ℃, cooling to room temperature, and pouring the reaction solution into acetone at 4 ℃, wherein the volume ratio of the reaction solution to the acetone is 1:10, drying to obtain the laminarin carboxylate of this example.
The yield of the laminarin carboxylate prepared in this example was 73%, and the structural formula thereof was formula (1), wherein: n has a value of 20.
Example 2
A preparation method of laminarin compound comprises the following steps:
(1) Laminarin and methyl isonicotinate were added to toluene, wherein: the mass volume ratio of laminarin to methyl isonicotinate is 1g:20mL, the temperature is 110 ℃, nitrogen is filled as a protective gas, stirring reflux is carried out for 54 hours, and then rotary evaporation is carried out at 60 ℃ to obtain a solid product;
(2) Adding the solid product obtained in the step (1) into dimethyl sulfoxide for dissolution, wherein: the mass volume ratio of the solid product to the dimethyl sulfoxide is 1g:5mL, stirring uniformly at room temperature to obtain a solution;
(3) Adding gamma-butyrolactone to dimethyl sulfoxide, wherein: the volume ratio of the gamma-butyrolactone to the dimethyl sulfoxide is 1:10, then dropwise adding the mixture into the solution prepared in the step (2), and uniformly mixing to obtain a reaction solution;
(4) Stirring the reaction solution prepared in the step (3) for 60 hours at 70 ℃, cooling to room temperature, and pouring the reaction solution into acetone at 4 ℃, wherein the volume ratio of the reaction solution to the acetone is 1:4, after drying, the laminarin carboxylate of this example was obtained.
The yield of the laminarin carboxylate prepared in this example was 71%, and the structural formula thereof was formula (1), wherein: n has a value of 25.
Example 3
A preparation method of laminarin compound comprises the following steps:
(1) Laminarin and methyl isonicotinate were added to toluene, wherein: the mass volume ratio of laminarin to methyl isonicotinate is 1g:15mL, the temperature is 120 ℃, nitrogen is filled as a protective gas, stirring reflux is carried out for 48 hours, and then rotary evaporation is carried out at 65 ℃ to obtain a solid product;
(2) Adding the solid product obtained in the step (1) into dimethyl sulfoxide for dissolution, wherein: the mass volume ratio of the solid product to the dimethyl sulfoxide is 1g:10mL, stirring uniformly at room temperature to obtain a solution;
(3) Adding gamma-butyrolactone to dimethyl sulfoxide, wherein: the volume ratio of the gamma-butyrolactone to the dimethyl sulfoxide is 1:5, then dropwise adding the mixture into the solution prepared in the step 2, and uniformly mixing to obtain a reaction solution;
(4) Stirring the reaction solution prepared in the step (3) at 80 ℃ for 48 hours, cooling to room temperature, and pouring the reaction solution into acetone at 4 ℃, wherein the volume ratio of the reaction solution to the acetone is 1:5, drying to obtain the laminarin carboxylate of this example.
The yield of the laminarin carboxylate prepared in this example was 67%, and the structural formula thereof was formula (1), wherein: n has a value of 18.
The laminarin carboxylates used in examples 4-7 were all those prepared in example 1.
Example 4
This example shows structural analysis of laminarin carboxylate.
The dried laminarin carboxylate prepared in example 1, 0.5mg, was mixed thoroughly with 50mg of dried potassium bromide (KBr) powder and ground, and the powder was compressed into tablets using a die and a tablet press. Background scanning is carried out to remove interference factors before sample measurement, and the distance between 400 cm and 4000cm is reached -1 Infrared spectral scan (FT-IR) analysis was performed.
The infrared spectrum of laminarin is shown in FIG. 1, wherein LAMINARIN is laminarin as control group; LZC is laminarin carboxylate, and is shown in the abscissa as WavenumbersWavenumber. Wherein: LAMINARIN at 3382cm -1 The characteristic absorption peak of O-H stretching vibration is provided, and the absorption peak is widened due to intramolecular or intramolecular hydrogen bond association, which indicates that intramolecular or intermolecular hydrogen bonds exist; 1200cm -1 -950cm -1 Characteristic absorption peaks of ether bond (C-O-C) and hydroxyl (C-O-H) of pyranose ring; 2893cm -1 with-CH at 3 or-CH 2 C-H stretching vibration absorption peaks of the groups, these structural regions are one of the important characterizations of laminarin.
LZC group is 1635cm based on laminarin group -1 And 1373cm -1 Characteristic absorption peaks are shown at these positions, indicating the telescoping vibration of carboxylate c=o and carboxylate C-O, which coincide with the expected absorption peak of the target product, and also confirm the presence of laminarin carboxylate.
Example 5
This example is the establishment of a mouse model for colitis.
Subject mice (C57 BL/6 mice) were randomly divided into 3 groups (no significant differences between mice). Five mice were included in each group, and the three groups were a blank control group, a DSS colitis model group, and a laminarin compound group, respectively.
The feeding flow in the experimental process is shown as 2, and A in the figure 2 is a blank control group; b is DSS colitis group; c is laminarin compound group. The experimental period is about 21 days, and the control group feeds physiological saline to the mice in the whole experimental period; DSS group, feeding right amount of dextran sulfate sodium salt at 7-14 days, and feeding physiological saline to mice in other time period; and the laminarin compound group is fed with laminarin compound prepared in the present invention at 7-14 days, and physiological saline is fed for the rest of the time.
During the experiment, the mice were observed daily for weight, diet, mental state, etc., for 14 consecutive days. Fasted for 24 hours after last feeding, sacrificed and the colon, colon contents and serum were collected for later use.
Example 6
This example is a morphological observation of intestinal tissue.
1. Paraffin section preparation:
(1) Sampling, namely taking colon tissue of a mouse, wherein the selected tissue blocks meet the conditions of proper size, uniform thickness, regular shape and the like as much as possible, and the action is gentle when the colon tissue is clamped, so that the selected tissue is prevented from being mechanically damaged;
(2) Fixing: selecting 10% neutral formalin solution as a fixing solution, and placing colon tissues of the mice into the fixing solution for fixing for 24-48 hours;
(3) Dehydrating: dehydrating the fixed tissue with alcohol of different alcohol concentrations, comprising dehydrating with 50%, 70%, 80%, 90% and 95% alcohol for 2 hours each; dehydrating 100% I and 100% II alcohol for 1.5 hr respectively;
(4) And (3) transparency: allowing the tissue mass to be stored in 100% alcohol + xylene (1:1) for 20min, and in xylene I, II for 15min each;
(5) Wax dipping: controlling the temperature at about 58 ℃, and then immersing the tissue blocks in a mixed solution of paraffin and toluene for 30min respectively, wherein the paraffin I and the paraffin II are immersed for 2-3h respectively;
(6) Embedding: embedding the tissue into paraffin at 60 ℃ and gradually solidifying the tissue into blocks;
(7) Slicing: fixing the wax block on a slicing machine, and cutting into tissue slices with uniform thickness by using a sharp blade;
(8) Sticking and baking the sheet: spreading the slice on warm water, taking out the slice from the water by using a glass slide after spreading the slice, standing the glass slide with the slice attached to the slice to remove redundant water, and baking the glass slide at 65 ℃ for about 20 min.
H & e staining:
(1) Dyeing: the slices are immersed in xylene I and II for 15min respectively, xylene+100% alcohol (1:1), 100% alcohol I and II with concentrations of 95%, 90%, 80%, 70% alcohol and 50% alcohol respectively, distilled water for 2min respectively, hematoxylin dye solution for 5-10min, water-washed and differentiated into 1% hydrochloric acid alcohol for 5s, tap water, 1% ammonia bluing, tap water, 50% alcohol, 70% alcohol, 80% alcohol, 90% alcohol and 95% alcohol respectively for 2min,1% eosin dye solution for 5min,95% alcohol, 100% alcohol I and II, 100% alcohol+xylene (1:1), xylene I and II respectively for 2min.
(2) Sealing piece: sealing with neutral gum, and baking in an incubator for about 2 hr.
The results of H & E staining (hematoxylin-eosin staining) of the colon tissue of mice are shown in fig. 3, wherein: a in fig. 3 is a control group; b is DSS mediated group; c is a group of laminarin carboxylates. As can be seen from fig. 3: compared with a control group, the goblet cells in the DSS colitis group slice have a large area, infiltrate a large amount of mucus among cells, and separate muscle fibers; the laminarin carboxylate compound group cells were more densely packed and filled than the DSS group cells, and also had more similarity in morphology and arrangement to the control group.
3. Maron dyeing
(1) Dewaxing slices to water;
(2) Dyeing with the prepared Weibert hematoxylin dye solution for 5-10min;
(3) Differentiating the acidic ethanol differentiation liquid for 5-15s, and washing with water;
(4) The Masson bluing liquid returns to blue for 3-5min, and is washed with water. Washing with distilled water for 1min;
(5) Dyeing with ponceau dye liquor for 5-10min;
(6) Washing with glacial acetic acid for 1min;
(7) Rapidly dehydrating 95% ethanol for 2-3s, and dehydrating absolute ethanol for 3 times each for 5-10s;
(8) The xylene is transparent for 3 times, each time for 1-2min, and the neutral gum is sealed.
The results of the masson staining of colon tissue of mice are shown in fig. 4, wherein: a in fig. 4 is a control group; b is DSS mediated group; c is a group of laminarin carboxylates. As can be seen from fig. 4: the higher blue fraction (collagen fiber fraction) in the colon tissue in the DSS-induced group compared to the placebo group, indicated an increased degree of fibrosis of the collagen fibers in the intestinal tissue; in the laminarin compound group, the blue collagen fiber component is reduced compared with the DSS group, which proves that the laminarin compound can improve intestinal fibrosis and has an important effect on maintaining the stability of intestinal barrier structure.
Example 7
This example shows the measurement of structural changes in intestinal flora.
DNA (deoxyribonucleic acid) extraction and sequencing: and (3) taking out thalli which are separated from the mouse feces in advance and frozen in a refrigerator at 80 ℃, extracting genome DNA of the sample by using a DNA extraction kit, and detecting the concentration of the DNA by using agarose gel electrophoresis and a micro-ultraviolet spectrophotometer (Nanodrop 2000). PCR (polymerase chain reaction) was performed using a specific primer with barcode according to the selection of a sequencing region using Tks Gflex DNA Polymerase polymerase from Takara company, thereby ensuring amplification efficiency and accuracy;
PCR amplification, namely, selecting the V3-V4 region of 16S rRNA for PCR amplification, wherein the primer is 343F (5 '-TACGGRAGGCAGCAG-3') at the front end and 798R (5'-AGGGTATCTAATCCT-3') at the rear end of the bacteria 16S. 30. Mu.L of amplification system comprising 15. Mu.L of 2 XGflex PCR buffer, 3.2. Mu.L of 2.5mmol/L dNTP (deoxyribonucleoside triphosphate), 0.6. Mu.L of 1.25U/. Mu.L of Tks Gflex DNA polymerase, 5 pmol/. Mu.L of each of front and rear primers, 50ng of template DNA, ddH 2 O (double distilled water). Reaction conditions: pre-denaturing at 94 ℃ for 5min, then denaturing at 94 ℃ for 1min, renaturating at 5 ℃ for 45s, extending at 72 ℃ for 1min, cycling the above steps for 30 times, and extending at 72 ℃ for 10min.
3.16S rRNA sequencing, high throughput sequencing of PCR amplified products using an Illumina sequencing System, quality screening of raw data using QIIME software to obtain effective data, and analysis of data results using bioinformatics means
4. Bioinformatics analysis: the original double-ended sequence was stripped using trimmatic software. The impurity removal parameters are as follows: detecting and truncating the ambiguous base N; and the average base quality is checked by a sliding window method, and when the quality is lower than 20, the previous high quality sequence is intercepted. The double-end sequence after impurity removal is performed by using FLASH software. The splicing parameters are as follows: the smallest Overlap is 10bp, the largest Overlap is 200bp, and the largest mismatch is 20%. In order to ensure the accuracy of the result, precise impurity removal can be performed to remove sequences containing ambiguous bases (ambiguos) and single base high repetition regions (homologo) and sequences with too short length. The parameters of accurate impurity removal are as follows: the sequence containing N bases is removed, and the sequence with the mass fraction of the bases Q20 reaching at least 75% is reserved. At the same time, the chimaeric sequence in the sequence was detected and removed using UCHIME. After the sequencing data are preprocessed to generate high-quality sequences, vsearch software is adopted to classify the sequences into a plurality of OTUs according to the similarity of the sequences. The parameters are that the sequence similarity of 97% or more is classified as an OTU unit. The representative sequences of each OTU were picked using the QIIME software package and all representative sequences were aligned to the database for annotation. 16S was aligned using Greengenes or Silva (version 123) databases, species alignment annotation was performed using RDP classifier software, and annotation results with confidence intervals greater than 0.7 were retained. ITS uses Unite database alignment. The species alignment annotation uses blast software.
5. Phylum level flora composition and species differential analysis various taxonomic level flora composition analyses of bacteroides phylum were performed on mouse fecal samples, at the phylum level bacteroides phylum (bacterioides) and Firmicutes (Firmicutes) are usually dominant; meanwhile, the ratio of the thick-wall fungus door to the bacteroides is analyzed through a Stamp software Tukey-Kramer algorithm.
The relative abundance of bacteroides at the enterobacteria level is shown in fig. 5, where: NC is a control group; DSS is dextran sulfate sodium salt mediated group; LLZC is a low concentration laminarin carboxylate group (1 mg/mL); MLZC is a medium-concentration laminarin carboxylate group (5 mg/mL); HLZC is a high concentration laminarin carboxylate group (10 mg/mL). As can be seen from fig. 5: with the use of laminarin, the growth of probiotics is promoted, while the growth of pathogenic bacteria is inhibited. The concrete steps are as follows: along with the use of laminarin compounds, the relative abundance of probiotics such as bacteroides in intestinal bacteria of mice is obviously increased compared with that of a DSS group, and the relative abundance of bacteria of the phylum Firmicutes is obviously reduced compared with that of the DSS group. Meanwhile, since Firmics/bacterioides can reflect the degree of disturbance of intestinal flora.
The ratio of the relative abundance of the firmicutes/bacteroides flora is shown in figure 6, where: NC is a control group; DSS is dextran sulfate sodium salt mediated group; LLAM is a low concentration laminarin group (1 mg/mL); MLAM is medium-concentration laminarin group (5 mg/mL); HLAM is a high concentration laminarin group (10 mg/mL); LLZC is a low concentration laminarin carboxylate group (1 mg/mL); MLZC is a medium-concentration laminarin carboxylate group (5 mg/mL); HLZC is a high concentration laminarin carboxylate group (10 mg/mL); F/B ratio is the relative abundance of the Thick-wall and Bacteroides flora. As can be seen from fig. 6: the ratio of the relative abundance of the Phanerochaete (Firmics) and the Bacteroides (Bacteroides) in the laminarin compound group is drastically reduced compared with the DSS-mediated group, and is also reduced to a certain extent compared with the blank group and the laminarin group, which shows that the laminarin compound can regulate the imbalance of intestinal flora and has important effect on maintaining the stability of the intestinal flora.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While obvious variations or modifications are contemplated as falling within the scope of the present invention.
Claims (6)
1. A laminarin compound represented by formula (1) or a pharmaceutically acceptable salt thereof:
(1)
Wherein: n is an integer between 1 and 100;
the laminarin compound is prepared from laminarin, organic matter containing carboxylic acid group and pyridine derivative;
the organic matter containing carboxylic acid groups is gamma-butyrolactone; the derivative of pyridine is methyl isonicotinate;
the mass volume ratio of laminarin to pyridine derivative is 1g: (10-20) mL;
the preparation method of laminarin compound comprises the following steps:
(1) Adding laminarin and pyridine derivatives into organic solvent, reacting at 110-130deg.C, and drying to obtain solid product;
(2) Adding the solid product prepared in the step (1) into an organic solvent for dissolution to obtain a dissolution solution;
(3) Adding an organic matter containing a carboxylic acid group into an organic solvent, and then adding the dissolved solution prepared in the step (2) to mix to obtain a reaction solution;
(4) Heating the reaction liquid obtained in the step (3) at 70-90 ℃, cooling, pouring into an organic solvent, and drying to obtain the laminarin compound.
2. The laminarin compound or pharmaceutically acceptable salt thereof according to claim 1, wherein in step (1), the process conditions of the reaction are: stirring and refluxing for 36-54 hours by filling inert gas; the drying process conditions are as follows: spin-drying at 60-70deg.C.
3. The laminarin compound or pharmaceutically acceptable salt thereof according to claim 1, wherein in step (2), the mass volume of the solid product and the organic solvent is 1g: (5-20) mL.
4. A process for the preparation of laminarin compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, comprising the steps of:
(1) Adding laminarin and pyridine derivatives into organic solvent, reacting at 110-130deg.C, and drying to obtain solid product;
(2) Adding the solid product prepared in the step (1) into an organic solvent for dissolution to obtain a dissolution solution;
(3) Adding an organic matter containing a carboxylic acid group into an organic solvent, and then adding the dissolved solution prepared in the step (2) to mix to obtain a reaction solution;
(4) Heating the reaction liquid prepared in the step (3) at 70-90 ℃, cooling, pouring into an organic solvent, and drying to obtain the laminarin compound;
in the step (3), the volume ratio of the organic matter containing the carboxylic acid group to the organic solvent is 1: (1-10).
5. A process for the preparation of laminarin compound or pharmaceutically acceptable salt thereof according to claim 4, wherein in step (4), the heating process conditions are: heating and stirring for 36-60 hours; the volume ratio of the reaction liquid to the organic solvent is 1: (1-10).
6. Use of a laminarin compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for intestinal barrier repair.
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