CN115894729A - Porphyra haitanensis polysaccharide degradation product and preparation method and application thereof - Google Patents
Porphyra haitanensis polysaccharide degradation product and preparation method and application thereof Download PDFInfo
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- porphyra haitanensis
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- 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
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Abstract
The invention belongs to the technical field of biochemistry, and particularly discloses a porphyra haitanensis polysaccharide degradation product and a preparation method and application thereof. The porphyra haitanensis polysaccharide degradation product is prepared by acidolysis of porphyra haitanensis polysaccharide and processes of alcohol precipitation, standing, centrifugation, redissolution, dialysis, freeze-drying and the like, and the porphyra haitanensis polysaccharide degradation product subjected to acidolysis has a typical structure of the porphyra haitanensis polysaccharide, and the molecular weight of the porphyra haitanensis polysaccharide degradation product is far smaller than that of the porphyra haitanensis polysaccharide. The inventor finds that tests such as intestinal mucus layer permeability, intestinal intercellular junction and intestinal tissue tight junction protein expression quantity are carried out by utilizing a colitis mouse model induced by sodium dextran sulfate (DSS), and the results prove that: the porphyra haitanensis polysaccharide degradation product can reduce the permeability of intestinal mucosa, has a repairing effect on a tight junction structure among intestinal epithelial cells, can also improve the expression quantity of tight junction protein in intestinal tissues, increases the thickness of a mucus layer, and is favorable for further preparing intestinal mucosa barrier repairing medicaments.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to porphyra haitanensis polysaccharide degradation product and a preparation method and application thereof.
Background
In the modern society with high economic development, the intestinal dysfunction of people is caused by excessive intake of high-sugar, high-salt and high-fat foods, so that a series of intestinal problems are caused, and the intestinal health of people is seriously threatened. These intestinal inflammatory diseases also bring economic, physiological and psychological stresses to patients themselves, and seriously affect the quality of life of individuals.
The intestinal mucosal barrier is an important ring in the maintenance of the intestinal environment, and the structural integrity of the intestinal mucosal barrier is closely related to the generation and development of intestinal diseases. The mucous layer in the large intestine is divided into an inner layer and an outer layer, the inner layer mucous layer is attached to epithelial cells, the pore size is small, and bacteria cannot penetrate through the inner layer mucous layer; the outer layer is covered on the inner layer, has larger aperture, and can be used for field planting of normal intestinal flora. Therefore, when intestinal inflammation occurs due to various causes, the thickness of the mucus layer becomes thinner; the tight junction protein between cells is destroyed, the original uniform and continuous tight junction protein (such as Occludin and ZO-1) becomes disordered and even breaks, the gap between cells is enlarged, the permeability of intestinal mucosa is increased, and bacteria pass through the mucus layer and directly contact the epithelial cells of the intestinal tract, thereby causing inflammatory reaction. The intact intestinal mucosal barrier can secrete various proteins including mucin, and the mucus layer formed by the proteins can be used as a barrier to prevent bacteria in the intestinal cavity from directly contacting intestinal epithelium, so that the intestinal mucosal barrier has a very important role in maintaining the intestinal environment steady state.
Therefore, the development of a medicament which has small side effect and proper price, is derived from natural products, has a repairing effect on intestinal mucosal barriers and can increase the expression of the tight junction protein is urgently needed. Porphyra haitanensis is taken as a common edible red alga in coastal zones of southeast China, and active substances extracted from porphyra haitanensis are known to have various biological activities including anti-tumor, anti-oxidation and immunoregulation, so that the related biological activity of porphyra haitanensis is further known, and the method has important significance in the aspects of promoting the development of porphyra haitanensis related food and medicine processing industries, maintaining human health and the like.
Disclosure of Invention
The present invention has been made to solve at least one of the above-mentioned problems occurring in the prior art. Therefore, the invention provides porphyra haitanensis polysaccharide degradation product and a preparation method and application thereof, and aims to further improve the biological activity of porphyra haitanensis polysaccharide and promote the application of porphyra haitanensis in intestinal barrier repair medicines.
In order to overcome the technical problems, the invention provides a porphyra haitanensis polysaccharide degradation product in a first aspect.
Specifically, the porphyra haitanensis polysaccharide degradation product has a chemical formula shown in a formula (1):
wherein: n is an integer between 8 and 20.
The porphyra haitanensis polysaccharide degradation product has the structural characteristics conforming to the porphyra haitanensis polysaccharide structure, the main structure of the porphyra haitanensis polysaccharide degradation product is composed of two parts, wherein one part is a structural unit I composed of beta-D-galactopyranose and 4-connected alpha-L-galactose-6-sulfate, the other part is a structural unit II composed of 3-connected beta-D-galactopyranose and 4-connected 3, 6-dehydrated-alpha-D-galactopyranose, and the composition ratio of the two structural units is 2:1, and the value of n is 8-20, and the molecular weight of n is far less than that of porphyra haitanensis polysaccharide. Therefore, the porphyra haitanensis polysaccharide degradation product not only has the basic functions of porphyra haitanensis polysaccharide, but also has more excellent bioactivity compared with the porphyra haitanensis polysaccharide due to smaller molecular weight.
Preferably, in formula (1), n is an integer of 10 to 15.
The second aspect of the invention provides a preparation method of porphyra haitanensis polysaccharide degradation products.
Specifically, the preparation method of porphyra haitanensis polysaccharide degradation product is used for preparing the porphyra haitanensis polysaccharide degradation product of the first aspect of the invention, and comprises the following steps:
(1) Porphyra haitanensis is taken as a raw material, and porphyra haitanensis polysaccharide is obtained by extraction;
(2) Dissolving porphyra haitanensis polysaccharide prepared in the step (1), and adding an acid solution for reaction to obtain a porphyra haitanensis polysaccharide degradation product solution;
(3) And (3) adding ethanol into the porphyra haitanensis polysaccharide degradation product solution prepared in the step (2) for alcohol precipitation, standing, centrifuging, taking precipitate, adding water for redissolution, dialyzing, and freeze-drying to obtain the porphyra haitanensis polysaccharide degradation product.
The invention firstly takes porphyra haitanensis as raw material, and porphyra haitanensis polysaccharide is firstly extracted from the porphyra haitanensis; then, degrading porphyra haitanensis polysaccharide by adopting an acidolysis method to prepare porphyra haitanensis polysaccharide degradation product solution; and then carrying out processes such as alcohol precipitation, standing, centrifugation, redissolution, dialysis, freeze-drying and the like to prepare the porphyra haitanensis polysaccharide degradation product. The degraded porphyra haitanensis polysaccharide still has a typical structure of porphyra haitanensis polysaccharide, but the degraded porphyra haitanensis polysaccharide has smaller molecular weight, can relieve the representation of a colitis mouse on the histomorphology level, reduce the permeability of an intestinal mucosa barrier, improve the expression quantity of closely-connected complex protein, increase the thickness of the mucus layer and is beneficial to further preparing intestinal mucosa barrier repair medicines.
As a further improvement of the above scheme, in the step (2), the process conditions of the reaction are as follows: the temperature is 80-90 ℃ and the time is 2-3 hours.
As a further improvement of the scheme, in the step (2), the concentration of the solution obtained after the porphyra haitanensis polysaccharide is dissolved is 0.1-0.2g/mL, and the porphyra haitanensis polysaccharide is dissolved by water.
As a further improvement of the scheme, in the step (2), the acid solution is hydrochloric acid, and the final concentration of the hydrochloric acid is 0.05-0.15mol/L.
Preferably, in the step (2), the final concentration of the hydrochloric acid is 0.05-0.10mol/L.
As a further improvement of the above scheme, in the step (3), the volume ratio of the porphyra haitanensis polysaccharide degradation product solution to the ethanol is 1:2.5-4.
Preferably, in the step (3), the volume ratio of the porphyra haitanensis polysaccharide degradation product solution to the ethanol is 1:3.5-4.
As a further improvement of the scheme, in the step (3), the standing temperature is 4-8 ℃, the standing time is 12-24 hours, the rotating speed of the centrifugation is 4000-8000rpm, and the time of the centrifugation is 5-15 minutes.
As a further improvement of the scheme, in the step (1), the preparation steps of the porphyra haitanensis polysaccharide are as follows:
(11) Adding a developing agent into porphyra haitanensis, oscillating and centrifuging, and drying precipitates to obtain precipitates;
(12) Adding water into the precipitate obtained in the step (11), reacting, centrifuging, and taking supernatant to obtain porphyra haitanensis polysaccharide solution;
(13) And (4) adding ethanol into the porphyra haitanensis polysaccharide solution obtained in the step (12) for alcohol precipitation, standing, centrifuging, taking precipitate, adding water for redissolution, dialyzing, and freeze-drying to obtain the porphyra haitanensis polysaccharide.
The invention takes fresh porphyra haitanensis as raw material, adopts a water extraction method, and prepares porphyra haitanensis powder after drying, crushing and decoloring; and then carrying out water extraction, alcohol precipitation, redissolution, dialysis, freeze-drying and other processes to prepare porphyra haitanensis polysaccharide. The porphyra haitanensis polysaccharide has a chemical formula shown in a formula (2):
wherein: n is an integer between 150 and 400. Namely, the porphyra haitanensis polysaccharide has the same structural unit as the porphyra haitanensis polysaccharide degradation product, but the molecular weight is far larger than that of the porphyra haitanensis polysaccharide degradation product.
As a further improvement of the scheme, in the step (11), the developing agent comprises 4-5 parts of methanol, 2-3 parts of dichloromethane and 1-2 parts of water by weight; the mass volume ratio of the porphyra haitanensis powder to the developing agent is 1: (10-20) g/mL.
Preferably, the mass volume ratio of the porphyra haitanensis powder to the developing agent is 1: (10-15) g/mL.
As a further improvement of the above scheme, in the step (12), the mass-to-volume ratio of the precipitate to the water is 1: (20-40) g/mL; the process conditions of the reaction are as follows: the temperature is 90-100 ℃, and the time is 2-4 hours;
preferably, the mass volume ratio of the porphyra haitanensis powder to the developing agent is 1: (30-40) g/mL.
As a further improvement of the scheme, the volume ratio of the porphyra haitanensis polysaccharide solution to the ethanol is 1:2.5-4.
Preferably, the volume ratio of the porphyra haitanensis polysaccharide solution to the ethanol is 1:3.5-4.
The third aspect of the invention provides an application of porphyra haitanensis polysaccharide degradation products.
Specifically, the porphyra haitanensis polysaccharide degradation product disclosed by the invention is applied to preparation of a medicine for repairing intestinal barrier.
As a further improvement of the scheme, the intestinal barrier repair comprises the reduction of the permeability of intestinal mucosa and the improvement of the expression level of tight junction protein in the intestinal tract, wherein the tight junction protein comprises ZO-1, occludin and Claudin-1.
Specifically, the test such as intestinal mucus layer permeability, intestinal intercellular junction and intestinal tissue tight junction protein expression level is carried out by using a dextran sulfate (DSS) induced colitis mouse model, and the results prove that: the porphyra haitanensis polysaccharide degradation product can reduce the permeability of intestinal mucosa, repair the tight junction structure among intestinal epithelial cells, and improve the expression quantity of tight junction proteins ZO-1, occludin and Claudin-1 in intestinal tissues.
Compared with the prior art, the technical scheme of the invention at least has the following technical effects or advantages:
(1) The porphyra haitanensis polysaccharide degradation product is prepared by acidolysis of porphyra haitanensis polysaccharide and processes of alcohol precipitation, standing, centrifugation, redissolution, dialysis, freeze-drying and the like, and the porphyra haitanensis polysaccharide degradation product subjected to acidolysis has a typical structure of porphyra haitanensis polysaccharide, and the molecular weight of the porphyra haitanensis polysaccharide degradation product is far smaller than that of the porphyra haitanensis polysaccharide. The discovery that tests such as intestinal mucus layer permeability, intestinal intercellular junction and intestinal tissue tight junction protein expression quantity are carried out by utilizing a colitis mouse model induced by sodium dextran sulfate (DSS), and the results prove that: the porphyra haitanensis polysaccharide degradation product can reduce the permeability of intestinal mucosa, has a repairing effect on a tight junction structure among intestinal epithelial cells, can also improve the expression quantity of tight junction protein in intestinal tissues, increases the thickness of a mucus layer, and is favorable for further preparing intestinal mucosa barrier repairing medicaments.
(2) The invention provides theoretical basis for the research on the biological activity of porphyra haitanensis polysaccharide degradation products, is favorable for promoting the development of porphyra haitanensis related food and medicine processing industries, and provides more theoretical basis for the clinical treatment of intestinal diseases. Meanwhile, the preparation method of the target product is simple, does not need to undergo complex chemical synthesis reaction, is single in target product, is high in yield, and is suitable for industrial mass production.
Drawings
FIG. 1 is the NMR chart of the degraded Porphyra haitanensis polysaccharide obtained in example 1;
FIG. 2 is the NMR spectrum of the degraded Porphyra haitanensis polysaccharide obtained in example 1;
FIG. 3 is a flow chart of a mouse experiment;
FIG. 4 is a graph showing the results of in situ hybridization of mouse colon mucosa;
FIG. 5 is a transmission electron microscope image of colon tissue of mouse;
FIG. 6 is a graph showing the results of tight junction protein in mouse colon tissue.
Detailed Description
The present invention is specifically described below with reference to examples in order to facilitate understanding of the present invention by those skilled in the art. It should be particularly noted that the examples are given solely for the purpose of illustration and are not to be construed as limitations on the scope of the invention, as non-essential improvements and modifications to the invention may occur to those skilled in the art, which fall within the scope of the invention as defined by the appended claims. Meanwhile, the raw materials mentioned below are not specified in detail and are all commercial products; the process steps or preparation methods not mentioned in detail are all process steps or preparation methods known to the person skilled in the art.
Example 1
A preparation method of porphyra haitanensis polysaccharide degradation product comprises the following steps:
(1) Taking porphyra haitanensis, crushing the porphyra haitanensis in a constant-temperature oven by using a crusher, and sieving to obtain porphyra haitanensis powder;
(2) Weighing a proper amount of porphyra haitanensis powder prepared in the step (1), and adding a developing agent (methanol: dichloromethane: water mass ratio of 4: the mass volume ratio of the porphyra haitanensis powder to the developing agent is 1: shaking the mixture for 12 hours at a shaker with the concentration of 10g/mL, centrifuging the mixture (4000rpm, 5 minutes), taking a precipitate, and drying the precipitate to obtain a precipitate;
(3) Adding a proper amount of distilled water into the precipitate prepared in the step (2) for decoloring, wherein: the mass volume ratio of the porphyra haitanensis powder precipitate to the distilled water is 1:40g/mL, the reaction temperature is 90 ℃, the centrifugation is carried out after the reaction is carried out for 2 hours (4000rpm, 5 minutes), and supernate is taken to obtain porphyra haitanensis polysaccharide solution;
(4) Adding 95% ethanol with 4 times volume of the porphyra haitanensis polysaccharide solution prepared in the step (3) for alcohol precipitation, standing for 12 hours at 4 ℃, centrifuging (4000rpm, 5 minutes), taking the precipitate, adding water for redissolution, dialyzing for 48 hours, and freeze-drying to obtain porphyra haitanensis polysaccharide;
(5) Dissolving 10g of porphyra haitanensis polysaccharide obtained in the step (4) in 100mL of water, and then adding HCl, wherein the reaction temperature is 80 ℃, the reaction is carried out for 2 hours, and the final concentration of the HCl is 0.05mol/L, so as to obtain a porphyra haitanensis polysaccharide degradation product solution;
(6) And (4) adding 95% ethanol with the volume being 4 times that of the porphyra haitanensis polysaccharide degradation product solution prepared in the step (5) for alcohol precipitation, standing at 4 ℃ for 12 hours, centrifuging (4000 rpm,5 minutes), taking the precipitate, adding water for redissolution, dialyzing for 48 hours, and freeze-drying to obtain the porphyra haitanensis polysaccharide degradation product sample.
The structural formula of the porphyra haitanensis polysaccharide degradation product prepared in the embodiment is shown as formula (1), wherein: the value of n is 10.
The porphyra haitanensis polysaccharide prepared in experimental example 1 is taken for reducingSample lysate, dissolved in D 2 In O, freeze-drying, repeating for three times, and completely replacing hydrogen in the sample with deuterium hydrogen; then dissolving the sample in tetramethylsilane; transferring into nuclear magnetic resonance tube, detecting on spectrometer, and recording at room temperature 1 H NMR (hydrogen nuclear magnetic resonance) and 13 c NMR (nuclear magnetic resonance carbon spectrum) data are shown in FIG. 1-2, wherein the abscissa fl (ppm) in FIG. 1-2 represents chemical shift. Detailed degradation product of Porphyra haitanensis polysaccharide 1 H NMR (hydrogen nuclear magnetic resonance) and 13 the shift of the chemical bond in C NMR is shown in Table 1.
Table 1: 1 h NMR and 13 shift of chemical bond in C NMR
| Displacement of chemical bonds | H1/C1 | H2/C2 | H3/C3 | H4/C4 | H5/C5 | H6/C6 |
| G (Red mark) | 4.30/103.00 | 3.63/70.54 | 4.10/78.46 | 4.00/68.70 | 3.61/75.07 | 3.70/69.23 |
| L6S (blue mark) | 5.16/100.52 | 3.72/69.16 | 3.65/70.40 | 3.61/80.10 | 3.63/75.21 | 3.69/68.55 |
| LA (Green mark) | 4.50/96.39 | 3.52/72.45 | 3.80/72.73 | 4.09/78.2 | 4.04/71.72 | 3.71/68.60 |
Wherein: g represents 3-linked beta-D-galactopyranose;
L6S represents 4-linked α -L-galactose-6-sulfate;
LA: represents 4 linked 3, 6-anhydro-alpha-D-galactopyranose.
As can be seen from FIGS. 1-2 and Table 1, the structural characteristics of the degraded Porphyra haitanensis polysaccharide conform to the structure of Porphyra haitanensis polysaccharide, and the main structural composition thereof is composed of two parts, one part is a structural unit I composed of beta-D-galactopyranose and 4-linked alpha-L-galactose-6-sulfate, the other part is a structural unit II composed of 3-linked beta-D-galactopyranose and 4-linked 3, 6-anhydro-alpha-D-galactopyranose, and the composition ratio of the two structural units is 2:1.
example 2
A preparation method of porphyra haitanensis polysaccharide degradation product comprises the following steps:
(1) Taking porphyra haitanensis, crushing the porphyra haitanensis in a constant-temperature oven by using a crusher, and sieving to obtain porphyra haitanensis powder;
(2) Weighing a proper amount of porphyra haitanensis powder prepared in the step (1), and adding a developing agent (methanol: dichloromethane: water mass ratio of 4: the mass volume ratio of the porphyra haitanensis powder to the developing agent is 1:15g/mL, shaking for 12 hours by a shaking table, centrifuging (4000rpm, 5 minutes), taking a precipitate, and drying the precipitate to obtain a precipitate;
(3) Adding a proper amount of distilled water into the precipitate prepared in the step (2) for decoloring, wherein: the mass volume ratio of the porphyra haitanensis powder precipitate to the distilled water is 1:30g/mL, the reaction temperature is 90 ℃, the centrifugation is carried out after the reaction is carried out for 2 hours (4000rpm, 5 minutes), and supernate is taken to obtain porphyra haitanensis polysaccharide solution;
(4) Adding 95% ethanol with 4 times volume of the porphyra haitanensis polysaccharide solution prepared in the step (3) to carry out alcohol precipitation, standing for 12 hours at 4 ℃, centrifuging (4000rpm, 5 minutes), taking the precipitate, adding water to carry out redissolution, dialyzing for 48 hours, and freeze-drying to obtain porphyra haitanensis polysaccharide;
(5) Dissolving 10g of porphyra haitanensis polysaccharide obtained in the step (4) in 100mL of water, and then adding HCl, wherein the reaction temperature is 80 ℃, the reaction time is 2 hours, and the final concentration of HCl is 0.15mol/L, so as to obtain a porphyra haitanensis polysaccharide degradation product solution;
(6) And (3) adding 95% ethanol with the volume of 4 times that of the porphyra haitanensis polysaccharide degradation product solution prepared in the step (5) to carry out alcohol precipitation, standing at 4 ℃ for 12 hours, centrifuging (4000 rpm for 5 minutes), taking the precipitate, adding water to carry out redissolution, dialyzing for 48 hours, and freeze-drying to obtain the porphyra haitanensis polysaccharide degradation sample.
The structural formula of the porphyra haitanensis polysaccharide degradation product prepared in the embodiment is shown as formula (1), wherein: the value of n is 12.
Example 3
A preparation method of porphyra haitanensis polysaccharide degradation product comprises the following steps:
(1) Pulverizing Porphyra haitanensis in a constant temperature oven, and sieving to obtain Porphyra haitanensis powder;
(2) Weighing a proper amount of porphyra haitanensis powder prepared in the step (1), and adding a developing agent (methanol: dichloromethane: water mass ratio of 4: the mass volume ratio of the porphyra haitanensis powder to the developing agent is 1:20g/mL, shaking for 12 hours by a shaking table, centrifuging (4000rpm, 5 minutes), taking a precipitate, and drying the precipitate to obtain a precipitate;
(3) Adding a proper amount of distilled water into the precipitate prepared in the step (2) for decoloring, wherein: the mass volume ratio of porphyra haitanensis powder precipitate to distilled water is 1:20g/mL, the reaction temperature is 90 ℃, the centrifugation is carried out after the reaction is carried out for 2 hours (4000rpm, 5 minutes), and supernate is taken to obtain porphyra haitanensis polysaccharide solution;
(4) Adding 95% ethanol with 4 times volume of the porphyra haitanensis polysaccharide solution prepared in the step (3) to carry out alcohol precipitation, standing for 12 hours at 4 ℃, centrifuging (4000rpm, 5 minutes), taking the precipitate, adding water to carry out redissolution, dialyzing for 48 hours, and freeze-drying to obtain porphyra haitanensis polysaccharide;
(5) Dissolving 10g of porphyra haitanensis polysaccharide obtained in the step (4) in 100mL of water, and then adding HCl, wherein the reaction temperature is 80 ℃, the reaction time is 2 hours, and the final concentration of HCl is 0.1mol/L, so as to obtain a porphyra haitanensis polysaccharide degradation product solution;
(6) And (4) adding 95% ethanol with the volume being 4 times that of the porphyra haitanensis polysaccharide degradation product solution prepared in the step (5) for alcohol precipitation, standing at 4 ℃ for 12 hours, centrifuging (4000 rpm,5 minutes), taking the precipitate, adding water for redissolution, dialyzing for 48 hours, and freeze-drying to obtain the porphyra haitanensis polysaccharide degradation product sample.
The structural formula of the porphyra haitanensis polysaccharide degradation product prepared in the embodiment is shown as formula (1), wherein: the value of n is 15.
Example 4
This example is the establishment of a mouse model of colitis:
mice (Kunming mice) were randomly divided into 4 groups (no significant difference in body weight among mice in each group), which were a blank control group, a Dextran Sulfate Sodium (DSS) colitis model group, a Porphyra haitanensis polysaccharide group and a Porphyra haitanensis polysaccharide degradation product group, respectively. The number of mice per group was 5.
The experimental flow is shown in figure 3: wherein CON is a control group; DSS is dextran sulfate sodium mediated group; PHP is Porphyra haitanensis polysaccharide group; PHP-D is porphyra haitanensis polysaccharide degradation product group. At the first Week (Week 1), all groups of mice had free access to distilled water; at Week two (Week 2), the blank control group was intragastrically administered daily with normal saline, sodium dextran sulfate (DSS) colitis model group, porphyra haitanensis polysaccharide group and porphyra haitanensis polysaccharide degradation product group daily with distilled water containing 2.5% sodium dextran sulfate (DSS); in the third Week (Week 3), mice in the blank control group and the Dextran Sulfate Sodium (DSS) colitis model group were gavaged with normal saline every day, and mice in the porphyra haitanensis polysaccharide group and the porphyra haitanensis degradation product group were gavaged with 100 mg/kg of normal saline every day, respectively -1 Porphyra haitanensis polysaccharide and porphyra haitanensis polysaccharide degradants (relative to the weight of the mouse) are continuously used for one week.
Throughout the experiment, as described above, the weight, diet, mental state, stool frequency, stool characteristics and occult blood of the mice were observed daily for 14 consecutive days. Fasting for 24 hours after the last feeding, sacrifice (sacrifice), and collection of colon, colon contents and serum for use.
Example 5
This example is a morphological observation of intestinal tract tissue.
1. Preparing a paraffin section:
(1) Material taking: selecting mouse colon tissue, wherein the tissue block has proper size, uniform thickness and regular shape, and the tissue clamping action is soft, so that the mechanical damage of the tissue is avoided;
(2) Fixing: selecting a 10% neutral formalin solution as a fixing solution, and fixing for 24 hours;
(3) And (3) dehydrating: dehydrating the fixed tissue at room temperature, and gradually increasing the alcohol concentration by 50%, 70%, 80%, 90% and 95% of alcohol for 2 hours respectively; 100% I and 100% II alcohol each for 1.5 hours;
(4) And (3) transparency: performing transparent tissue blocks by using xylene at room temperature, storing the tissue blocks in 100% alcohol + xylene (1;
(5) Wax dipping: the temperature is controlled to be about 58 ℃, the tissue block is soaked in toluene and paraffin (1) for 30 minutes, and the temperature of the tissue block is respectively soaked in the paraffin I and the paraffin II for 2 hours;
(6) Embedding: embedding the tissues into paraffin at the temperature of 60 ℃ and solidifying the tissues into blocks;
(7) Slicing: fixing the wax block on a slicer by using a sharp blade, and cutting into complete tissue slices with uniform thickness;
(8) Chip mounting and baking: spreading the sliced glass on warm water with the glossy surface facing downwards, taking out the sliced glass from the water by using a clean glass slide after the sliced glass is unfolded, aligning the sliced glass slide, vertically placing the sliced glass slide on a dyeing rack for draining water, immediately baking the sliced glass slide, and baking the sliced glass slide at the temperature of 65 ℃ for about 30 minutes.
2. Fluorescence in situ hybridization
(1) Paraffin section dewaxing to water: putting the slices into xylene I for 15 minutes, xylene II for 15 minutes, absolute ethyl alcohol I for 5 minutes, absolute ethyl alcohol II for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and washing with distilled water.
(2) And (3) protease K digestion: PBST was used to prepare 10. Mu.g/mL proteinase K solutions, appropriate amounts of proteinase K solutions were added dropwise to the tissues, incubated at 37 ℃ for 5 minutes, and then washed 3 times with PBS for 5 minutes each.
(3) Pre-hybridization of the probe: according to the following steps of 1: diluting salmon sperm DNA with hybridization buffer solution according to the dilution ratio of 100 to prepare a prehybridization solution, uniformly mixing, dripping the prehybridization solution on tissues, and incubating for 60 minutes at 37 ℃.
(4) And (3) probe hybridization: according to the following steps of 1: diluting the probe with hybridization buffer solution at a dilution ratio of 100 to prepare hybridization solution, uniformly mixing, throwing away the prehybridization solution on the tissue, then dropwise adding the hybridization solution, and covering with a cover glass to prevent the liquid from being evaporated to dryness in the overnight process. The incubation cassette was placed in a 42 ℃ incubator and hybridized overnight.
(5) Washing the slices: the overnight hybridized slide was placed in 2 XSSC solution, washed at 37 ℃ for 5 minutes, transferred to 1 XSSC solution, washed at 37 ℃ for 5 minutes, and finally transferred to 0.2 XSSC solution, washed at 37 ℃ for 5 minutes.
(6) DAPI counterstained nuclei: slides were washed 3 times in PBST (pH 7.4) for 5 minutes each on a destaining shaker with shaking. After the section is slightly dried, DAPI dye solution is dripped into the circle, and the section is incubated for 10 minutes at room temperature in a dark place.
(7) And (3) sealing: slides were washed 3 times in PBST (pH 7.4) for 5 minutes each on a destaining shaker with shaking. The slices were slightly spun dry and mounted with an anti-fluorescent quenching mounting agent.
(8) And (5) microscopic examination and photographing: the sections were observed under a Nikon inverted fluorescence microscope and images were collected.
The fluorescence in situ hybridization results of the colon tissues of the mice are shown in FIG. 4, wherein CON is a control group; DSS is dextran sulfate sodium mediated group; PHP is Porphyra haitanensis polysaccharide group; PHP-D is porphyra haitanensis polysaccharide degradation product group. As can be seen from fig. 4: intestinal bacteria (red fraction) in the DSS group had infiltrated into colon tissue epithelial cells (blue fraction) compared to the control group, and the spacing between the two was no longer evident; after the porphyra haitanensis polysaccharide and the porphyra haitanensis polysaccharide degradation product are treated, the permeability of a mucus layer is reduced, intestinal bacteria and intestinal epithelial tissues are separated by a complete intestinal mucosa barrier structure, the intestinal infection phenomenon is prevented, the effect of the porphyra haitanensis polysaccharide degradation product is more obvious, and the porphyra haitanensis polysaccharide degradation product has an obvious improvement effect on the intestinal mucosa permeability and the structural integrity.
Example 6
This example is a measure of the tight junction structure between intestinal cells.
A tabletting step:
1. material drawing and fixing: fresh tissue is used for determining the material-taking part, sampling is carried out within 1-3 minutes, and the tissue sampling is 1mm 3 Size. Before material drawing, a culture dish filled with electron microscope stationary liquid can be prepared in advance, the small tissue blocks are taken off in vitro and immediately put into the culture dish, and a scalpel is used for cutting the small tissue blocks into 1mm in the stationary liquid of the culture dish 3 Small tissue blocks. And transferring the cut small tissue blocks into an EP tube filled with a new electron microscope fixing solution for continuous fixation, and carrying out fixed storage and transportation at 4 ℃.0.1M phosphate buffer PB (pH 7.4) was rinsed 3 times for 15 minutes each.
2. Post-fixing: 1% osmic acid in 0.1M phosphate buffered PBS (pH 7.4) was fixed at room temperature for 2 hours in the dark. 0.1M phosphate buffer PB (pH7.4) was rinsed 3 times for 15 minutes each.
3. Dehydrating at room temperature: the tissue is sequentially added with 30% -50% -70% -80% -95% -100% -100% alcohol to be dehydrated for 20 minutes each time, and 100% acetone is added twice for 15 minutes each time.
4. And (3) infiltration embedding: acetone 812 embedding medium = 1:1 ℃ for 2-4 hours, acetone 812 embedding medium = 1:2 ℃ for overnight permeation, pure 812 embedding medium 37 ℃ for 5-8 hours. The pure 812 embedding medium was poured into the embedding plate and the samples were inserted into the embedding plate and then oven left overnight at 37 ℃.
5. Polymerization: the embedded plate was polymerized in an oven at 60 ℃ for 48 hours, and the resin block was taken out for use.
6. Ultrathin slicing: the resin block is subjected to 60-80nm ultrathin section by an ultrathin slicer, and the sheet is fished by a 150-mesh diamond film copper net.
7. Dyeing: dyeing the copper mesh in a 2% uranium acetate saturated alcohol solution for 8 minutes in a dark place; cleaning with 70% ethanol for 3 times; cleaning with ultrapure water for 3 times; 2.6 percent lead citrate solution avoids carbon dioxide dyeing for 8 minutes; the ultrapure water was washed 3 times and the filter paper was slightly blotted dry. The copper mesh slices are placed in a copper mesh box and dried overnight at room temperature.
8. Observing under a transmission electron microscope, and collecting and analyzing images.
The results of the transmission electron microscope for the colon tissues of the mice are shown in fig. 5, wherein CON is a control group; DSS is dextran sulfate sodium mediated group; PHP is Porphyra haitanensis polysaccharide group; PHP-D is Porphyra haitanensis polysaccharide degradation product. As can be seen from fig. 5: compared to the control group, cell junctions between intestinal epithelial cells were not apparent in the DSS group (indicated by white arrows); after the porphyra haitanensis polysaccharide degradation product is treated, intercellular connection on the surface of intestinal cells in the porphyra haitanensis polysaccharide degradation product group is more clear and visible, and the tight connection compounds close extracellular gaps among the cells, are favorable for maintaining the activity of cadherin and promoting the stability of an intestinal epithelial barrier structure, and have more obvious effect relative to the porphyra haitanensis polysaccharide group, so that the porphyra haitanensis polysaccharide degradation product has an obvious repairing effect on the tight connection structure among the intestinal epithelial cells.
Example 7
This example is the determination of the amount of claudin in intestinal tissue
1. Extraction of total colon protein:
(1) Shearing the tissue into fine pieces; wrapping with tinfoil paper, adding a small amount of liquid nitrogen, quickly knocking, and further crushing; repeating for multiple times; collecting the mixture by using a 2mL centrifuge tube (precooling in a centrifuge tube) for later use;
(2) Appropriate amounts of RIPA lysate were taken and mixed according to a 50:1, adding a 50X protease phosphatase inhibitor mixture;
(3) Adding lysis solution at a ratio of 150-250 (200) (1: 10) microliter of lysis solution per 20mg of tissue, and lysing for 15-30 minutes on ice;
(4) After full lysis, centrifuging at 12000 rpm for 20-30 min at 4 deg.C, collecting supernatant, transferring to a new centrifuge tube, and placing in a refrigerator at 4 deg.C for use.
2. Determination of total protein content:
(1) Adding 0.8mL of protein standard preparation solution into a tube of protein standard (20 mg BSA), and fully dissolving to prepare 25mg/mL of protein standard solution;
(2) Preparing a BCA working solution: according to the number of samples, preparing a proper amount of BCA working solution by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50;
(3) Protein concentration determination: adding the standard substance into standard substance wells of a 96-well plate according to 0, 1, 2, 4, 8, 12, 16 and 20 μ L, and adding standard substance diluent to make up to 20 μ L; adding an appropriate volume of sample to the sample wells of a 96-well plate; adding 200 mu L of BCA working solution into each hole, and standing at 37 ℃ for 20-30 minutes; measuring absorbance at 562nm wavelength by using an enzyme-labeling instrument; the protein concentration of the sample was calculated from the standard curve and the sample volume used.
3. Protein denaturation: according to the protein concentration measured by the BCA kit, PBS is added to dilute the protein to a proper concentration, then 5X protein loading buffer solution is added, water bath is carried out for 5-15 minutes at 99 ℃, and the protein is centrifuged for standby use and is stored at-20 ℃.
4. Electrophoresis:
(1) SDS-PAGE gel preparation: installing an electrophoresis tank glass plate, detecting leakage, preparing glue pouring, preparing 8% separation glue, and sealing the glue by using absolute ethyl alcohol after glue pouring. Standing at room temperature for 30-60 min, polymerizing the separation gel, removing the upper layer of anhydrous ethanol, sucking the excessive liquid with absorbent paper, injecting 2mL of 5% concentrated gel into the upper layer of the separation gel, rapidly inserting into a comb, and standing at room temperature for 15-20 min to polymerize the concentrated gel;
(2) Loading: installing an electrophoresis tank, placing the prepared rubber plate in the electrophoresis tank, adding electrophoresis buffer solution to submerge a sample adding hole, slowly pulling out a comb, adding 5 mu L of pre-dyed protein marker into the sample adding hole on one side, and sequentially adding 10 mu L of protein sample;
(3) Electrophoresis: after the sample loading is finished, the electrophoresis program is set to 60V electrophoresis for 30 minutes, then the voltage is adjusted to 80V, electrophoresis is carried out for 90 minutes, and the bromophenol blue reaches the lowest position of the gel.
5. Film transfer: carefully prying off the electrophoresis gel plate, cutting a whole piece of separation gel, putting a precooled wet-conversion buffer solution, selecting a PVDF (polyvinylidene fluoride) membrane with a proper size, sequentially putting 2 layers of sponges, 2 pieces of filter paper, the separation gel and the PVDF membrane from top to bottom according to a sandwich form, assembling a wet-conversion system, filling a precooled 1 multiplied wet-conversion buffer solution, immersing the sandwich, and wet-converting at room temperature of 100V for 60 minutes.
6. Preparing 5% skimmed milk by using 1X TBST solution, taking out PVDF membrane from wet-transferring system, rinsing with TBST for 5 min for 2-3 times, adding appropriate amount of 5% skimmed milk solution, and sealing on shaking table at room temperature for 60 min.
7. Primary antibody incubation
According to the following steps of 1: diluting primary antibody with WB primary antibody diluent at a ratio of 1000; removing the blocking solution by suction, adding diluted primary antibody, and incubating overnight at 4 deg.C under slow shaking; after overnight incubation, the washes were repeated 3-5 times with 5-10 minutes each by adding wash (TBST).
8. Incubation with a second antibody
Selecting corresponding secondary antibody, diluting the secondary antibody diluent, adding the diluted secondary antibody, and incubating on a shaking table for 1 hour at room temperature; rinsing with TBST solution for 3-5 times, and discarding the rinsing solution to prepare for protein color development.
9. Developing and photographing
Preparing a developing solution according to instructions of a hypersensitive ECL chemiluminescence kit, mixing the solution A and the solution B uniformly in equal volume, dripping the mixture on a PVDF membrane which is placed with the front surface facing upwards to ensure that the mixture is uniformly distributed on the surface of the membrane, carrying out exposure imaging by using an Amersham Imager 600 gel imaging system, preserving a photo, and quantifying gray values of all bands in Image J software.
The results of the expression level of claudin in colon tissues of mice are shown in FIG. 6, wherein CON is a control group; DSS is dextran sulfate sodium mediated group; PHP is Porphyra haitanensis polysaccharide group; PHP-D is porphyra haitanensis polysaccharide degradation product group. As can be seen from fig. 6: compared with the control group, the expression level of the closely-linked complex related protein in the DSS group is obviously reduced, and after the DSS group is treated by the porphyra haitanensis polysaccharide degradation product, the expression level of the related closely-linked protein is obviously increased, and the effect is more obvious compared with the porphyra haitanensis polysaccharide group. Therefore, the porphyra haitanensis polysaccharide degradation product has a remarkable promotion effect on the expression of zon-1, occludin and Claudin-1 in intestinal tract tissues and has an obvious effect on maintaining the stability of the intestinal tract structure.
It will be obvious to those skilled in the art that many simple derivations or substitutions can be made without inventive effort without departing from the inventive concept. Therefore, simple modifications to the present invention by those skilled in the art based on the present disclosure should be within the scope of the present invention. The above embodiments are preferred embodiments of the present invention, and all similar processes and equivalent variations to those of the present invention should fall within the scope of the present invention.
Claims (10)
1. The porphyra haitanensis polysaccharide degradation product is characterized by having a chemical formula shown as a formula (1):
wherein: n is an integer between 8 and 20.
2. A preparation method of porphyra haitanensis polysaccharide degradation product, which is used for preparing the porphyra haitanensis polysaccharide degradation product of claim 1, and comprises the following steps:
(1) Porphyra haitanensis is taken as a raw material, and porphyra haitanensis polysaccharide is obtained by extraction;
(2) Dissolving porphyra haitanensis polysaccharide prepared in the step (1), and adding an acid solution for reaction to obtain a porphyra haitanensis polysaccharide degradation product solution;
(3) And (3) adding ethanol into the porphyra haitanensis polysaccharide degradation product solution prepared in the step (2) for alcohol precipitation, standing, centrifuging, taking precipitate, adding water for redissolution, dialyzing, and freeze-drying to obtain the porphyra haitanensis polysaccharide degradation product.
3. The method for preparing porphyra haitanensis polysaccharide degradation product according to claim 2, wherein in the step (2), the reaction process conditions are as follows: the temperature is 80-90 ℃ and the time is 2-3 hours.
4. The method for preparing porphyra haitanensis polysaccharide degradation product according to claim 2, wherein in the step (2), the concentration of the solution obtained after the porphyra haitanensis polysaccharide is dissolved is 0.1-0.2g/mL; the acid solution is hydrochloric acid, and the final concentration of the hydrochloric acid is 0.05-0.15mol/L.
5. The method for preparing porphyra haitanensis polysaccharide degradation product according to claim 2, wherein in the step (3), the volume ratio of the porphyra haitanensis polysaccharide degradation product solution to the ethanol is 1:2.5-4.
6. The method for preparing porphyra haitanensis polysaccharide degradation product according to claim 2, wherein in the step (3), the standing temperature is 4-8 ℃, the standing time is 12-24 hours, the rotation speed of the centrifugation is 4000-8000rpm, and the centrifugation time is 5-15 minutes.
7. The method for preparing porphyra haitanensis polysaccharide degradation product according to claim 2, wherein in the step (1), the porphyra haitanensis polysaccharide is prepared by the following steps:
(11) Adding a developing agent into porphyra haitanensis, vibrating and centrifuging, and drying precipitates to obtain precipitates;
(12) Adding water into the precipitate obtained in the step (11), reacting, centrifuging, and taking supernatant to obtain porphyra haitanensis polysaccharide solution;
(13) And (4) adding ethanol into the porphyra haitanensis polysaccharide solution obtained in the step (12) for alcohol precipitation, standing, centrifuging, taking precipitate, adding water for redissolution, dialyzing, and freeze-drying to obtain the porphyra haitanensis polysaccharide.
8. The method for preparing porphyra haitanensis polysaccharide degradation product according to claim 7, wherein in the step (11), the developing agent comprises 4-5 parts by weight of methanol, 2-3 parts by weight of dichloromethane and 1-2 parts by weight of water; the mass volume ratio of the porphyra haitanensis powder to the developing agent is 1: (10-20) g/mL;
in the step (12), the mass-to-volume ratio of the precipitate to the water is 1: (20-40) g/mL; the process conditions of the reaction are as follows: the temperature is 90-100 ℃, and the time is 2-4 hours;
in the step (13), the volume ratio of the porphyra haitanensis polysaccharide solution to the ethanol is 1:2.5-4.
9. Use of porphyra haitanensis polysaccharide degradation product according to claim 1 for preparing a medicament for intestinal barrier repair.
10. The use according to claim 9, wherein the intestinal barrier repair comprises reducing permeability of intestinal mucosa and increasing expression of Claudin in the intestinal tract, wherein the Claudin comprises ZO-1,Occludin and Claudin-1.
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| CN109232762A (en) * | 2018-10-10 | 2019-01-18 | 汕头大学 | A kind of Porphyra haitanensis polysaccharide and preparation method thereof is applied with prebiotic activity |
| CN111436613A (en) * | 2020-04-23 | 2020-07-24 | 汕头大学 | Application of porphyra haitanensis polysaccharide in stomach strengthening and gastrointestinal injury prevention or repair |
| CN113278088A (en) * | 2021-05-27 | 2021-08-20 | 华南理工大学 | Sargassum fusiforme polysaccharide with obvious intestinal mucosa repair activity and preparation method and application thereof |
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| CN109232762A (en) * | 2018-10-10 | 2019-01-18 | 汕头大学 | A kind of Porphyra haitanensis polysaccharide and preparation method thereof is applied with prebiotic activity |
| CN111436613A (en) * | 2020-04-23 | 2020-07-24 | 汕头大学 | Application of porphyra haitanensis polysaccharide in stomach strengthening and gastrointestinal injury prevention or repair |
| CN113278088A (en) * | 2021-05-27 | 2021-08-20 | 华南理工大学 | Sargassum fusiforme polysaccharide with obvious intestinal mucosa repair activity and preparation method and application thereof |
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| CN116903762A (en) * | 2023-06-13 | 2023-10-20 | 广东海洋大学 | Fucoidan with intestinal mucosa barrier repairing function and preparation method thereof |
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