CN117756955A - Application of Lycium barbarum polysaccharide in inhibiting P.azotoformans - Google Patents
Application of Lycium barbarum polysaccharide in inhibiting P.azotoformans Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, and particularly provides an application of lycium barbarum polysaccharide in inhibiting P.azotoformans. The invention provides a matrimony vine polysaccharide which can relieve intestinal damage caused by immunodeficiency by inhibiting the abundance of P.azotoformans bacteria in the intestinal tract of a mouse. Specifically, after the matrimony vine polysaccharide is ingested by an immunodeficient mouse, the relative abundance of P.azotoformans in the intestinal tract is reduced, jejunum tissue morphology is improved, short chain fatty acids in the intestinal tract are enriched and serum metabolites are changed, and the matrimony vine polysaccharide is proved to be capable of relieving intestinal damage caused by immunodeficiency by inhibiting the abundance of P.azotoformans bacteria.
Description
Technical Field
The invention relates to the field of biotechnology, and particularly provides an application of lycium barbarum polysaccharide in inhibiting P.azotoformans.
Background
Fructus Lycii (Lycium barbarum L.) is widely used as a traditional Chinese herbal medicine and functional food in China for thousands of years, and the 'Ben Cao gang mu' records that fructus Lycii is sweet and flat and moist, can tonify kidney, moisten lung, promote vital essence generation and tonify qi, and is a tonic. The wolfberry polysaccharide is water-soluble polysaccharide extracted from wolfberry, is a kind of protein heteropolysaccharide which is mainly formed by connecting various monosaccharides such as galactose (Gal), arabinose (Ara), glucose (Glc), galacturonic acid (GalA), glucuronic acid (GlcA) and the like through different forms of glycosidic bonds, and contains various microelements and amino acids. It is reported that Lycium barbarum polysaccharide has various biological activities of enhancing immunity, resisting tumor, resisting aging, reducing blood sugar and blood lipid, resisting oxidation, resisting radiation, etc., and is considered as one of the most important active ingredients in Lycium barbarum.
Pseudomonas azotoformans (Pseudomonas azotoformans) is a bacterium associated with nitrogen fixation and has the main function of converting nitrogen in the air into plant available ammoniacal nitrogen. However, under certain environmental conditions, especially where the animal immune system is impaired or the environmental conditions are poor, p.azoformans may cause infections that may affect different organs, leading to the appearance of symptoms such as fever, inflammation, tissue damage, etc.
Disclosure of Invention
The invention provides application of lycium barbarum polysaccharide in inhibiting P.azoformans, which is used for solving intestinal tract injury caused by immunodeficiency.
In a first aspect, the present invention provides a lycium barbarum polysaccharide, wherein the lycium barbarum polysaccharide has a carbohydrate content of 42% -62% and a protein content of 29% -42%.
More specifically, the invention provides four Chinese wolfberry polysaccharide components, wherein the four Chinese wolfberry polysaccharide components can inhibit the P.azoformans abundance from rising caused by intestinal injury, and the carbohydrate content in the four Chinese wolfberry polysaccharide components is 50.23+/-0.61 percent and the protein content is 35.44+/-1.58 percent; or a carbohydrate content of 43.70+ -0.87% and a protein content of 40.50+ -1.17%; or carbohydrate content of 43.01+ -0.98%, protein content of 33.81 + -1.18%; or the carbohydrate content is 60.21+/-0.53% and the protein content is 31.06+/-1.94%.
The molecular weight of the wolfberry polysaccharide provided by the invention is 1.46 multiplied by 10 4 Da-1.07×10 5 Da。
More specifically, the four types of wolfberry polysaccharide components provided by the invention are wolfberry crude polysaccharide, wolfberry polysaccharide component 1, wolfberry polysaccharide component 2 and wolfberry polysaccharide component 3.
Wherein the fructus Lycii crude polysaccharide comprises arabinose (48.18%), galactose (30.20%), glucose (10.22%), rhamnose (5.23%) and galacturonic acid (2.57%), and has molecular weight of 6.04×10 4 Da。
The main components of the polysaccharide component 1 comprise arabinose (36.43%), galactose (16.85%), glucose (13.53%), rhamnose (13.16%) and galacturonic acid (11.36%), and the molecular weight is 1.67×10 4 Da。
The fructus Lycii polysaccharide component 2 comprises arabinose (24.09%), galactose (31.85%), glucose (18.79%), rhamnose (9.81%) and galacturonic acid (7.29%) as main components, and has molecular weight of 1.07×10 5 Da and 1.46×10 4 Da, present two symmetrical peaks.
The polysaccharide component 3 of fructus Lycii contains arabinose (44.25%) and galactose (43.81%) as main components, and has molecular weight of 8.83×10 4 Da。
The invention provides application of the lycium barbarum polysaccharide in inhibiting P.azotoformans.
And the application of the matrimony vine polysaccharide or the raw material taking the matrimony vine polysaccharide as a main component in preparing a product for inhibiting P.azotoformans.
And application of the matrimony vine polysaccharide or raw materials taking the matrimony vine polysaccharide as main components in preparing medicines for treating intestinal injuries.
In the provided application, the intestinal injury is an immunodeficiency-caused dysbacteriosis of the intestinal tract.
In a second aspect, the present invention provides a therapeutic agent for intestinal injury, which comprises the above-mentioned matrimony vine polysaccharide as a main ingredient.
The medicine for treating intestinal injury provided by the invention comprises the following components: adding pharmaceutically acceptable adjuvants into the Lycium barbarum polysaccharide, and making into capsule, tablet, granule, injection, delayed release agent, oral liquid or dripping pill.
In the medicine for treating intestinal injury provided by the invention, the dosage of the lycium barbarum polysaccharide is 100mg/kg/d.
The invention has the beneficial effects that:
according to the invention, through researching the inhibition effect of different components of matrimony vine polysaccharide on intestinal tract harmful bacteria, the relative abundance of P.azotoformans in intestinal tract is reduced after an immunodeficient mouse ingests the matrimony vine polysaccharide, jejunum tissue morphology is improved, and short chain fatty acid in intestinal tract is enriched and serum metabolite is changed. Based on the above, the invention provides the application of the lycium barbarum polysaccharide in relieving intestinal damage caused by immunodeficiency by inhibiting the abundance of P.azotoformans bacteria in the intestinal tract of a mouse.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the body weight and immune organ index change of an immunodeficient mouse, (A) body weight; and (B) thymus and spleen index.
FIG. 2 shows changes in intestinal flora and P.azooformans abundance in immunodeficient mice: (a) a wien (Venn) plot; (B) a principal coordinate analysis (PCoA) map; (C) LDA analysis map; (D) P.azoformans relative abundance map
FIG. 3 shows changes in jejunal tissue morphology in immunodeficient mice: (a) HE staining; (B) AB-PAS staining.
FIG. 4 shows the variation of short chain fatty acid content in the feces of immunodeficient mice. In the figures, "ns" indicates no significant difference from the immunodeficiency group (P < 0.05), "indicates significant difference from the immunodeficiency group (P < 0.01)," indicates significant difference from the immunodeficiency group (P < 0.005), "" indicates significant difference from the immunodeficiency group (P < 0.001).
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The BALB/c mice to which the present invention relates were purchased from Experimental animal technologies, inc. of Beijing veteh.
The dried fruits of the medlar are purchased from Ningxia Zhongning Jiabao medlar commercial limited company.
In the invention, the related P.azoformans relative quantitative detection method is 16SrRNA flora detection, and comprises the following specific steps:
(1) DNA extraction: the total genomic DNA of the sample was extracted by CTAB/SDS method. The DNA concentration and purity were monitored on a 1% agarose gel. The DNA was diluted to 1 ng/. Mu.L with sterile water depending on the concentration.
(2) And (3) PCR amplification: the 16S rRNA/18SrRNA/ITS genes (16 SV4/16S V3-V4/16S V4-V5, 18S V4/18S V9, ITS1/ITS2, arc V4) of the different regions were amplified using specific primers (e.g., 16S V4:515F-806R,18S V4:528F-706R,18S V9:1380F-1510R, etc.).
(3) Data analysis: samples were sequenced on the Illumina HiSeq platform. FLASH (version 1.2.8) is used to allocate, truncate and merge pairs of end reads. Chimeric sequences were screened and 97% similarity was assigned to the same Operational Taxon (OTU) using ≡using Vsearch software (v 2.3.4). A representative sequence is selected for each OTU and classification data is assigned to each representative sequence using a ribosomal database entry (RDP) classifier. The difference in dominant species in the different populations was determined using the mafft software (V7.310).
The method for determining the short-chain fatty acid in the intestinal tract of the mice comprises the following steps:
about 100mg of fecal sample was taken into a 2mL centrifuge tube, one steel ball was added, 10. Mu.L of dilute sulfuric acid was added for acidification (50%, v/v), 1000. Mu.L of diethyl ether was added, and the mixture was vortexed and milled for 3 minutes twice. 12000g was centrifuged for 10min (4 ℃ C.), the supernatant was transferred to a 1.5mL centrifuge tube, 250mg anhydrous sodium sulfate was added, vortexed, 12000g was centrifuged for 10min (4 ℃ C.), and the supernatant was filtered through a 0.22 μm filter membrane and analyzed on the machine.
In the method for determining the short-chain fatty acid in the intestinal tract of the mice, besides extracting the short-chain fatty acid in the sample, a solution for detecting a standard curve is required to be prepared: adding 9.52 mu L of acetic acid, 10.10 mu L of propionic acid, 10.10 mu L of butyric acid, 10.54 mu L of isobutyric acid, 10.66 mu L of n-valeric acid and 10.80 mu L of isovaleric acid, adding 9938 mu L of diethyl ether, loading into a 15mL centrifuge tube, and carrying out vortex mixing to obtain a mixed standard stock solution A of 6 short-chain fatty acids. The stock solution concentrations were 100. Mu.g/mL.
Preparing standard curves with different concentrations: the standard was diluted in a gradient according to Table 1, with a total volume of 1mL. GC parameter settings are shown in table 2.
TABLE 1 short chain fatty acid mix gradient concentration information (μg/mL)
| Chinese name | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Acetic acid | 5 | 50 | 100 | 200 | 300 | 400 | 500 |
| Propionic acid | 5 | 50 | 100 | 200 | 300 | 400 | 500 |
| Butyric acid | 5 | 50 | 100 | 200 | 300 | 400 | 500 |
| Isobutyric acid | 5 | 50 | 100 | 200 | 300 | 400 | 500 |
| Valeric acid | 5 | 50 | 100 | 200 | 300 | 400 | 500 |
| Isopentanoic acid | 5 | 50 | 100 | 200 | 300 | 400 | 500 |
Table 2GC parameter settings
EXAMPLE 1 Effect of crude Lycium barbarum polysaccharide on intestinal injury in immunodeficient mice
The invention provides the influence of wolfberry crude polysaccharide on intestinal tract injury of an immunodeficiency mouse, which comprises the following specific steps:
1. the preparation method of the medlar crude polysaccharide aqueous solution comprises the following steps:
the wolfberry polysaccharide is prepared by the following steps: squeezing the dried fruits of the Chinese wolfberry by a juicer, and mixing the Chinese wolfberry juice with deionized water 1: extracting for 2h at 75 ℃, filtering with gauze, concentrating the filtrate by rotary evaporation at 50 ℃, precipitating with 4 times volume of absolute ethyl alcohol for 12h, dissolving and precipitating with 60 ℃ hot water, deproteinizing by Sevag method, dialyzing to obtain aqueous solution of fructus Lycii crude polysaccharide, and gradient freeze-drying under the following conditions to obtain fructus Lycii crude polysaccharide: -40 ℃ for 3 hours; -30 ℃ for 3 hours; -20 ℃ for 3 hours; -10 ℃ for 3 hours; 4 hours at 0 ℃; 3h at 5 ℃; 3h at 10 ℃; 3h at 15 ℃; and 3h at 25 ℃.
Mixing the wolfberry crude polysaccharide with deionized water, and performing vortex oscillation to dissolve the wolfberry crude polysaccharide, wherein the concentration of the wolfberry crude polysaccharide in the wolfberry crude polysaccharide aqueous solution is as follows: 10mg/mL.
The content of carbohydrate in the used fructus Lycii crude polysaccharide is 50.23+ -0.61%, and the content of protein is 35.44+ -1.58%. Mainly comprises arabinose (48.18%), galactose (30.20%), glucose (10.22%), rhamnose (5.23%) and galacturonic acid (2.57%), and has a molecular weight of 6.04×10 4 Da。
2. Animal experiment
After 30 7-week-old male BALB/c mice were self-adapted for 1 week, they were randomly divided into 3 groups (n=10), and the remaining 20 mice except 10 mice as a blank control group (NC) were intraperitoneally injected with 80mg/kg/d CTX on days 1,2,3, respectively, to induce immunodeficiency. Immunodeficient mice were randomly divided into model control group (CTX) and wolfberry crude polysaccharide group (ctx+crude polysaccharide). NC group and CTX group mice were perfused with 100mg/kg/d physiological saline for the next 25 days, and CTX+crude polysaccharide group mice were perfused with 100mg/kg/d Lycium barbarum crude polysaccharide.
The experimental period amounted to 28 days, namely 3 days of molding and 25 days of gastric lavage, and the body weight of the mice was measured every 3 days.
After the experiment is finished, the jejunum is fixed by paraformaldehyde, and the steps of embedding, slicing, HE/AB-PAS staining, sealing and the like are carried out subsequently for jejunum histological evaluation. The fresh mouse feces is collected in a sterile EP tube and stored in a refrigerator at-80 ℃ for determining the intestinal flora and the short chain fatty acid content in the mouse feces.
3. Effect of intake of Lycium barbarum crude polysaccharide on intestinal tract of immunodeficient mice
(1) Influence of Lycium barbarum crude polysaccharide on body weight and immune organ index of immunodeficiency mice
1) Influence on body weight
As shown in FIG. 1A, after 3 days of intraperitoneal injection of CTX, the CTX group body weight (20.52.+ -. 0.84 g) showed a significant decrease compared to the CTX+ crude polysaccharide group body weight (20.47.+ -. 0.59 g) compared to the NC group body weight (23.43.+ -. 0.62 g), indicating successful modeling of the immunodeficiency model.
After 25 days of wolfberry crude polysaccharide intervention, the body weight (24.95+/-0.95 g) of the NC mice is significantly higher than that of the CTX mice (23.24+/-1.00 g), and the body weight (24.17+/-0.52 g) of the CTX+ crude polysaccharide mice is also significantly higher than that of the CTX mice, and has no significant difference from that of the NC mice. This indicates that the intervention of the wolfberry crude polysaccharide can effectively relieve the weight reduction of mice caused by immunodeficiency.
2) Effects on immune organ index
As shown in B of FIG. 1, after the experiment is finished, the thymus index (1.62+/-0.41 mg/g) and the spleen index (3.04+/-0.15 mg/g) of the CTX group are obviously lower than those of the NC group (2.13+/-0.12 mg/g) and the spleen index (3.59+/-0.34 mg/g), however, after the wolfberry crude polysaccharide is dried, the thymus index (2.03+/-0.09 mg/g) and the spleen index (3.45+/-0.13 mg/g) of the CTX+ crude polysaccharide group are obviously higher than those of the CTX group, and are not obviously different from those of the NC group, so that the ingestion of the wolfberry crude polysaccharide can effectively relieve the reduction of the immune organ index of mice caused by immunodeficiency.
(2) Effect on intestinal flora and relative quantitative analysis of p.azooforms
The feces of each group of mice were subjected to 16S rRNA assay, and the results are shown in Table 3 and FIG. 2.
TABLE 3 horizontal colony count of intestinal harmful bacteria
As shown in fig. 2, the number of unique units (OTUs) of CTX group was significantly reduced compared to NC group, and furthermore, principal Component Analysis (PCA) found that there was a significant separation between NC and CTX group, and CTX resulted in a change in intestinal flora of mice in combination with LEfSe figure and table 3.
Compared with the CTX group, the unique unit number (OTUs) of the wolfberry crude polysaccharide is obviously increased after the wolfberry crude polysaccharide is dried, the PCA is used for finding that obvious separation occurs between the CTX+ crude polysaccharide group and the CTX group, and the wolfberry crude polysaccharide is close to the NC group, so that the wolfberry crude polysaccharide is interfered and the changes of intestinal flora of mice induced by the CTX are relieved.
In addition, the relative abundance of P.azotoformans in the intestinal tract of the CTX mice (0.165+/-0.041%) is increased by about 10.66 times compared with that of NC mice (0.015+/-0.008%), and the abundance of P.azotoformans is reduced after taking the wolfberry crude polysaccharide.
After the dry prognosis of the wolfberry crude polysaccharide, the relative abundance (0.026+/-0.006%) of P.azotoformans bacteria in the intestinal tract of the CTX+ crude polysaccharide group mice is reduced by about 6.29 times compared with that of the CTX group mice, which shows that the wolfberry crude polysaccharide can relieve the intestinal damage caused by immunodeficiency by inhibiting the enrichment of P.azotoformans bacteria in the intestinal tract.
(3) Effect of jejunal tissue morphology
FIG. 3 shows jejunal tissue morphology after staining with HE and AB-PAS. Compared with NC group, CTX group mice have incomplete jejunum villus structure, broken villus length, and low mucin content.
After the wolfberry fruit crude polysaccharide is dried, CTX+ crude polysaccharide groups show complete and orderly jejunum villus structures, gaps among villi are relatively compact, and mucin content is increased, so that the wolfberry fruit crude polysaccharide can positively influence jejunum tissue structures, and the intestinal absorption capacity and mucous membrane barrier function can be improved.
(4) Influence on short chain fatty acids in mouse faeces
Short chain fatty acid determination was performed by taking mouse faeces of NC group, CTX group and ctx+ crude polysaccharide group, respectively, and the results are shown in fig. 4:
the results show that there is a significant decrease in the concentration of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid in CTX compared to NC. However, after the wolfberry crude polysaccharide is dried, compared with the CTX group, the content of each short chain fatty acid in the CTX+ crude polysaccharide group is obviously increased, and the content of each short chain fatty acid is not obviously different from that in the NC group, which indicates that the ingestion of the wolfberry crude polysaccharide can effectively relieve the decrease of the concentration of the short chain fatty acid in the mouse feces caused by the enrichment of P.azoformans bacteria.
EXAMPLE 2 Effect of Lycium barbarum polysaccharide component 1 on intestinal injury in immunodeficient mice
The invention provides an effect of a lycium barbarum polysaccharide component 1 on intestinal injury of an immunodeficiency mouse, which comprises the following specific steps:
1. preparation of aqueous solution of Lycium barbarum polysaccharide component 1
Adding 215mL of absolute ethyl alcohol into the aqueous solution of the crude wolfberry polysaccharide obtained in the example 1, centrifuging at 4000rpm/min, collecting supernatant, dissolving precipitate in hot water at 60 ℃, centrifuging at 4000rpm/min, and carrying out gradient freeze-drying on the supernatant under the following conditions to obtain a wolfberry polysaccharide component 1: -40 ℃ for 3 hours; -30 ℃ for 3 hours; -20 ℃ for 3 hours; -10 ℃ for 3 hours; 4 hours at 0 ℃; 3h at 5 ℃; 3h at 10 ℃; 3h at 15 ℃; and 3h at 25 ℃.
Mixing the wolfberry polysaccharide component 1 with deionized water, and performing vortex oscillation to dissolve the wolfberry polysaccharide component 1, wherein the concentration of the wolfberry polysaccharide component 1 in the wolfberry polysaccharide component 1 aqueous solution is as follows: 10mg/mL.
The content of carbohydrate in the polysaccharide component 1 of the Chinese wolfberry is 43.70+/-0.87%, and the content of protein is 40.50+/-1.17%; mainly comprises arabinose (36.43%), galactose (16.85%), glucose (13.53%), rhamnose (13.16%) and galacturonic acid (11.36%), and has a molecular weight of 1.67×10 4 Da。
2. Animal experiment
After 30 7-week-old male BALB/c mice were self-adapted for 1 week, they were randomly divided into 3 groups (n=10), and the remaining 20 mice except 10 mice as a blank control group (NC) were intraperitoneally injected with 80mg/kg/d CTX on days 1,2,3, respectively, to induce immunodeficiency. Immunodeficient mice were randomly assigned to model control group (CTX) and Lycium barbarum polysaccharide fraction 1 group (ctx+fraction 1). NC group and CTX group mice were perfused with 100mg/kg/d saline for the next 25 days, and CTX+ group 1 mice were perfused with 100mg/kg/d Lycium barbarum polysaccharide component 1.
The experimental period amounted to 28 days, namely 3 days of molding and 25 days of gastric lavage, and the body weight of the mice was measured every 3 days.
After the experiment is finished, the jejunum is fixed by paraformaldehyde, and the steps of embedding, slicing, HE/AB-PAS staining, sealing and the like are carried out subsequently for jejunum histological evaluation. The fresh mouse feces is collected in a sterile EP tube and stored in a refrigerator at-80 ℃ for determining the intestinal flora and the short chain fatty acid content in the mouse feces.
3. Effect of Lycium barbarum polysaccharide component 1 intake on intestinal tract of immunodeficient mice
(1) Effect of Lycium barbarum polysaccharide component 1 on body weight and immune organ index of immunodeficient mice
1) Influence on body weight
As shown in FIG. 1A, after 3 days of CTX intraperitoneal injection, the CTX group body weight (20.52+ -0.84 g) showed a significant decrease compared to the NC group body weight (23.43+ -0.62 g) with the CTX+group 1 body weight (20.55+ -0.52 g), indicating that the immunodefect model molding was successful.
After 25 days of intervention with Lycium barbarum polysaccharide component 1, NC group mice body weight (24.95+ -0.95 g) was significantly higher than CTX group (23.24+ -1.00 g), CTX+ component 1 group mice body weight (24.11+ -0.91 g) was also significantly higher than CTX group, and there was no significant difference from NC group mice body weight. This indicates that the intervention of the polysaccharide component 1 of the medlar can effectively relieve the weight reduction of mice caused by immunodeficiency.
2) Effects on immune organ index
As shown in B of FIG. 1, after the experiment is finished, the thymus index (1.62+/-0.41 mg/g) and the spleen index (3.04+/-0.15 mg/g) of the CTX group are obviously lower than those of the NC group (2.13+/-0.12 mg/g) and the spleen index (3.59+/-0.34 mg/g), however, after the wolfberry polysaccharide component 1 is dried, the thymus index (1.91+/-0.24 mg/g) and the spleen index (3.19+/-0.14 mg/g) of the CTX+ component 1 are obviously higher than those of the CTX group, so that the ingestion of the wolfberry polysaccharide component 1 can effectively relieve the reduction of the immune organ index of the mice caused by immunodeficiency.
(2) Effect on intestinal flora and relative quantitative analysis of p.azooforms
The feces of each group of mice were subjected to 16S rRNA assay, and the results are shown in Table 4 and FIG. 2.
TABLE 4 horizontal colony count of intestinal harmful bacteria
The results show that: in addition, the number of unique units (OTUs) in the CTX group was significantly reduced compared to the NC group, and in addition, the Principal Component Analysis (PCA) found that there was a significant separation between NC and CTX groups, and CTX resulted in a change in the intestinal flora of mice, as collectively illustrated by the LEfSe map and table 4.
As shown in fig. 2, the number of unique units (OTUs) of the matrimony vine polysaccharide component 1 was significantly increased after dry prognosis compared to CTX group, and a significant separation between ctx+component 1 group and CTX group was found by PCA, and close to NC group, indicating that the matrimony vine polysaccharide component 1 intervention alleviated the changes in intestinal flora of mice induced by CTX.
In addition, the relative abundance of P.azotoformans bacteria in the intestinal tract of the CTX group mice (0.165+/-0.041%) is increased by about 10.66 times compared with that of NC group mice (0.015+/-0.008%), and the abundance of P.azotoformans bacteria is reduced after the Chinese wolfberry polysaccharide component 1 is ingested.
After the dry prognosis of the lycium barbarum polysaccharide component 1, the relative abundance of the P.azotoformans bacteria in the intestinal tract of the mice in the CTX+component 1 group (0.034+/-0.014%) is reduced by about 4.85 times relative to the mice in the CTX group, which indicates that the lycium barbarum polysaccharide component 1 can relieve the intestinal damage caused by immunodeficiency by inhibiting the enrichment of the P.azotoformans bacteria in the intestinal tract.
(3) Effect of jejunal tissue morphology
FIG. 3 shows jejunal tissue morphology after staining with HE and AB-PAS. Compared with NC group, CTX group mice have incomplete jejunum villus structure, broken villus length, and low mucin content.
After the wolfberry polysaccharide component 1 is dried, the CTX+ component 1 presents a complete and orderly arranged jejunum villus structure, gaps among villi are relatively compact, and the mucin content is increased, so that the wolfberry polysaccharide component 1 can positively influence jejunum tissue structure, and is beneficial to improving the absorption capacity of intestinal tracts and mucous membrane barrier function.
(4) Influence on short chain fatty acids in mouse faeces
Short chain fatty acid determination was performed by taking mouse faeces of NC group, CTX group and ctx+ group 1 group respectively, and the results are shown in fig. 4:
the results show that there is a significant decrease in the concentration of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid in CTX compared to NC. However, after the matrimony vine polysaccharide component 1 is dried, the content of each short chain fatty acid in the CTX+component 1 group is obviously increased compared with that in the CTX group, which indicates that the ingestion of the matrimony vine polysaccharide component 1 can effectively relieve the decrease of the concentration of the short chain fatty acid in the mouse feces caused by the enrichment of P.azoformans bacteria.
EXAMPLE 3 Effect of Lycium barbarum polysaccharide component 2 on intestinal injury in immunodeficient mice
The invention provides an effect of a lycium barbarum polysaccharide component 2 on intestinal injury of an immunodeficiency mouse, which comprises the following specific steps:
1. preparation of aqueous solution of Lycium barbarum polysaccharide component 2
Adding 285mL of absolute ethanol into the supernatant obtained in the example 2, centrifuging at 4000rpm/min, collecting the supernatant, dissolving the precipitate in hot water at 60 ℃, centrifuging at 4000rpm/min, and gradient freeze-drying the supernatant obtained by centrifugation under the following conditions to obtain a Lycium barbarum polysaccharide component 2: -40 ℃ for 3 hours; -30 ℃ for 3 hours; -20 ℃ for 3 hours; -10 ℃ for 3 hours; 4 hours at 0 ℃; 3h at 5 ℃; 3h at 10 ℃; 3h at 15 ℃; and 3h at 25 ℃. Mixing the wolfberry polysaccharide component 2 with deionized water, and performing vortex oscillation to dissolve the wolfberry polysaccharide component 2, wherein the concentration of the wolfberry polysaccharide component 2 in the wolfberry polysaccharide component 2 aqueous solution is as follows: 10mg/mL.
The carbohydrate content in the polysaccharide component 2 of the medlar is 43.01 plus or minus 0.98 percent, and the protein content is 33.81 plus or minus 1.18 percent; mainly comprises arabinose (24.09%), galactose (31.85%), glucose (18.79%), rhamnose (9.81%) and galacturonic acid (7.29%), and has molecular weight of 1.07×10 respectively 5 Da and 1.46×10 4 Da, present two symmetrical peaks.
2. Animal experiment
After 30 7-week-old male BALB/c mice were self-adapted for 1 week, they were randomly divided into 3 groups (n=10), and the remaining 20 mice except 10 mice as a blank control group (NC) were intraperitoneally injected with 80mg/kg/d CTX on days 1,2,3, respectively, to induce immunodeficiency. Immunodeficient mice were randomly assigned to model control group (CTX) and Lycium barbarum polysaccharide fraction 2 group (ctx+fraction 2). NC group and CTX group mice were perfused with 100mg/kg/d saline for the next 25 days, and CTX+ group 2 mice were perfused with 100mg/kg/d Lycium barbarum polysaccharide component 2.
The experimental period amounted to 28 days, namely 3 days of molding and 25 days of gastric lavage, and the body weight of the mice was measured every 3 days.
After the experiment is finished, the jejunum is fixed by paraformaldehyde, and the steps of embedding, slicing, HE/AB-PAS staining, sealing and the like are carried out subsequently for jejunum histological evaluation. The fresh mouse feces is collected in a sterile EP tube and stored in a refrigerator at-80 ℃ for determining the intestinal flora and the short chain fatty acid content in the mouse feces.
3. Effect of matrimony vine polysaccharide fraction 2 intake on the intestinal tract of immunodeficient mice
(1) Effect of Lycium barbarum polysaccharide fraction 2 on body weight and immune organ index of immunodeficient mice
1) Influence on body weight
As shown in FIG. 1A, after 3 days of CTX intraperitoneal injection, the CTX group body weight (20.52.+ -. 0.84 g) showed a significant decrease compared to the CTX+group 2 body weight (20.54.+ -. 0.40 g) compared to the NC group body weight (23.43.+ -. 0.62 g), indicating that the immunodeficiency model was successfully modeled.
After 25 days of intervention with Lycium barbarum polysaccharide component 2, NC group mice body weight (24.95+ -0.95 g) was significantly higher than CTX group (23.24+ -1.00 g), CTX+ component 2 group mice body weight (24.26+ -0.30 g) was also significantly higher than CTX group, and there was no significant difference from NC group mice body weight. This indicates that the intervention of the polysaccharide component 2 of the medlar can effectively relieve the weight reduction of mice caused by immunodeficiency.
2) Effects on immune organ index
As shown in B of FIG. 1, after the experiment is finished, the thymus index (1.62+/-0.41 mg/g) and the spleen index (3.04+/-0.15 mg/g) of the CTX group are obviously lower than those of the NC group (2.13+/-0.12 mg/g) and the spleen index (3.59+/-0.34 mg/g), however, after the intervention of the Lycium barbarum polysaccharide component 2, the thymus index (2.04+/-0.16 mg/g) and the spleen index (3.36+/-0.24 mg/g) of the CTX+ component 2 are obviously higher than those of the CTX group, and are not obviously different from those of the NC group, so that the ingestion of the Lycium barbarum polysaccharide component 2 can effectively relieve the reduction of the immune organ index of mice caused by immunodeficiency.
(2) Effect on intestinal flora and relative quantitative analysis of p.azooforms
The feces of each group of mice were subjected to 16S rRNA assay, and the results are shown in Table 5 and FIG. 2.
TABLE 5 horizontal colony count of intestinal harmful bacteria
The results show that:
as shown in fig. 2, the number of unique units (OTUs) of CTX group was significantly reduced compared to NC group, and furthermore, principal Component Analysis (PCA) found that there was a significant separation between NC and CTX group, and CTX resulted in a change in intestinal flora of mice in combination with LEfSe figure and table 5.
Compared with the CTX group, the unique unit number (OTUs) of the Chinese wolfberry polysaccharide component 2 is obviously increased after intervention, and the PCA finds that obvious separation occurs between the CTX+component 2 group and the CTX group and the Chinese wolfberry polysaccharide component 2 is close to the NC group, so that the Chinese wolfberry polysaccharide component 2 is proved to be intervened to relieve the change of the intestinal flora of the mice induced by the CTX.
In addition, the relative abundance of P.azotoformans bacteria in the intestinal tract of the CTX group mice (0.165+/-0.041%) is increased by about 10.66 times compared with that of NC group mice (0.015+/-0.008%), and the abundance of P.azotoformans bacteria is reduced after the Chinese wolfberry polysaccharide component 2 is ingested.
After the dry prognosis of the lycium barbarum polysaccharide component 2, the relative abundance of P.azotoformans bacteria (0.033+/-0.011%) in the intestinal tract of the mice in the CTX+ component 2 is reduced by about 4.97 times relative to the mice in the CTX component, which indicates that the lycium barbarum polysaccharide component 2 can relieve intestinal damage caused by immunodeficiency by inhibiting the enrichment of P.azotoformans bacteria in the intestinal tract.
(3) Effect of jejunal tissue morphology
FIG. 3 shows jejunal tissue morphology after staining with HE and AB-PAS. Compared with NC group, CTX group mice have incomplete jejunum villus structure, broken villus length, and low mucin content.
After intervention of the wolfberry polysaccharide component 2, the CTX+ component 2 presents a complete and orderly arranged jejunum villus structure, gaps among villi are relatively compact, and the mucin content is obviously increased, which indicates that the wolfberry polysaccharide component 2 can positively influence jejunum tissue structure, and is beneficial to improving the absorption capacity of intestinal tracts and the mucous membrane barrier function.
(4) Influence on short chain fatty acids in mouse faeces
Short chain fatty acid determination was performed by taking mouse faeces of NC group, CTX group and ctx+ group 2 groups respectively, and the results are shown in fig. 4:
the results show that there is a significant decrease in the concentration of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid in CTX compared to NC. However, after the matrimony vine polysaccharide component 2 is dried, the content of each short chain fatty acid in the CTX+component 2 group is obviously increased compared with that in the CTX group, which indicates that the ingestion of the matrimony vine polysaccharide component 2 can effectively relieve the decrease of the concentration of the short chain fatty acid in the mouse feces caused by the enrichment of P.azoformans bacteria.
EXAMPLE 4 Effect of Lycium barbarum polysaccharide component 3 on intestinal injury in immunodeficient mice
The invention provides an effect of a lycium barbarum polysaccharide component 3 on intestinal injury of an immunodeficiency mouse, which comprises the following specific steps:
1. preparation of aqueous solution of Lycium barbarum polysaccharide component 3
Adding 670mL of absolute ethyl alcohol into the supernatant obtained in the example 3, centrifuging at 4000rpm/min, collecting the supernatant, dissolving the precipitate in hot water at 60 ℃, centrifuging at 4000rpm/min, and gradient freeze-drying the supernatant obtained by centrifugation under the following conditions to obtain a lycium barbarum polysaccharide component 3: -40 ℃ for 3 hours; -30 ℃ for 3 hours; -20 ℃ for 3 hours; -10 ℃ for 3 hours; 4 hours at 0 ℃; 3h at 5 ℃; 3h at 10 ℃; 3h at 15 ℃; and 3h at 25 ℃.
Mixing the wolfberry polysaccharide component 3 with deionized water, and performing vortex oscillation to dissolve the wolfberry polysaccharide component 3, wherein the concentration of the wolfberry polysaccharide component 3 in the wolfberry polysaccharide component 3 aqueous solution is as follows: 10mg/mL.
The carbohydrate content in the polysaccharide component 3 of fructus Lycii is 60.21+ -0.53%, the protein content is 31.06+ -1.94%, the polysaccharide component 3 of fructus Lycii mainly comprises arabinose (44.25%) and galactose (43.81%), and its molecular weight is 8.83×10 4 Da。
2. Animal experiment
After 30 7-week-old male BALB/c mice were self-adapted for 1 week, they were randomly divided into 3 groups (n=10), and the remaining 20 mice except 10 mice as a blank control group (NC) were intraperitoneally injected with 80mg/kg/d CTX on days 1,2,3, respectively, to induce immunodeficiency. Immunodeficient mice were randomly assigned to model control group (CTX) and Lycium barbarum polysaccharide fraction 3 group (ctx+fraction 3). NC group and CTX group mice were perfused with 100mg/kg/d saline for the next 25 days, and CTX+ group 3 mice were perfused with 100mg/kg/d Lycium barbarum polysaccharide component 3.
The experimental period amounted to 28 days, namely 3 days of molding and 25 days of gastric lavage, and the body weight of the mice was measured every 3 days.
After the experiment is finished, the jejunum is fixed by paraformaldehyde, and the steps of embedding, slicing, HE/AB-PAS staining, sealing and the like are carried out subsequently for jejunum histological evaluation. The fresh mouse feces is collected in a sterile EP tube and stored in a refrigerator at-80 ℃ for determining the intestinal flora and the short chain fatty acid content in the mouse feces.
3. Effect of matrimony vine polysaccharide component 3 intake on the intestinal tract of immunodeficient mice
(1) Effect of Lycium barbarum polysaccharide fraction 3 on body weight and immune organ index of immunodeficient mice
1) Influence on body weight
As shown in FIG. 1A, after 3 days of CTX intraperitoneal injection, the CTX group body weight (20.52.+ -. 0.84 g) showed a significant decrease compared to the CTX+3 group body weight (20.49.+ -. 0.66 g) compared to the NC group body weight (23.43.+ -. 0.62 g), indicating that the immunodeficiency model was successfully modeled.
After 25 days of intervention with Lycium barbarum polysaccharide component 3, the NC group mice body weight (24.95+ -0.95 g) was significantly higher than the CTX group (23.24+ -1.00 g), and the CTX+ component 3 group mice body weight (24.58+ -0.43 g) was also significantly higher than the CTX group, and there was no significant difference from the NC group mice body weight. This indicates that the intervention of the polysaccharide component 3 of the medlar can effectively relieve the weight reduction of mice caused by immunodeficiency.
2) Effects on immune organ index
As shown in B of FIG. 1, after the experiment is finished, the thymus index (1.62+/-0.41 mg/g) and the spleen index (3.04+/-0.15 mg/g) of the CTX group are obviously lower than those of the NC group (2.13+/-0.12 mg/g) and the spleen index (3.59+/-0.34 mg/g), however, after the wolfberry polysaccharide component 3 is dried, the thymus index (2.18+/-0.32 mg/g) and the spleen index (3.43+/-0.21 mg/g) of the CTX+wolfberry polysaccharide component 3 are obviously higher than those of the CTX group, and are not obviously different from those of the NC group, so that the ingestion of the wolfberry polysaccharide component 3 can effectively relieve the reduction of the immune organ index of mice caused by immunodeficiency.
(2) Effect on intestinal flora and relative quantitative analysis of p.azooforms
The feces of each group of mice were subjected to 16S rRNA assay, and the results are shown in Table 6 and FIG. 2.
TABLE 6 horizontal colony count of intestinal harmful bacteria
The results show that:
as shown in fig. 2, the number of unique units (OTUs) of CTX group was significantly reduced compared to NC group, and furthermore, principal Component Analysis (PCA) found that there was a significant separation between NC and CTX group, and CTX resulted in a change in intestinal flora of mice in combination with LEfSe figure and table 6.
Compared with the CTX group, the unique unit number (OTUs) of the Lycium barbarum polysaccharide component 3 is obviously increased after the interference, the CTX+component 3 and the CTX group are obviously separated through PCA, and the CTX+component 3 and the CTX group are close to each other, so that the interference of the Lycium barbarum polysaccharide component 3 relieves the change of the intestinal flora of the mice induced by the CTX.
In addition, the relative abundance of P.azotoformans bacteria in the intestinal tract of the CTX group mice (0.165+/-0.041%) is increased by about 10.66 times compared with that of NC group mice (0.015+/-0.008%), and the abundance of P.azotoformans bacteria is reduced after the Chinese wolfberry polysaccharide component 3 is ingested.
After the dry prognosis of the lycium barbarum polysaccharide component 3, the relative abundance of P.azotoformans bacteria (0.021+/-0.011%) in the intestinal tract of the mice in the CTX+ component 3 is reduced by about 7.76 times compared with the mice in the CTX group, and the relative abundance of P.azotoformans bacteria in the intestinal tract is not significantly different from that in the NC group, which indicates that the lycium barbarum polysaccharide component 3 can relieve the intestinal tract injury caused by immunodeficiency by inhibiting the enrichment of P.azotoformans bacteria in the intestinal tract.
(3) Effect of jejunal tissue morphology
FIG. 3 shows jejunal tissue morphology after staining with HE and AB-PAS. Compared with NC group, CTX group mice have incomplete jejunum villus structure, broken villus length, and low mucin content.
After the wolfberry polysaccharide component 3 is dried, the CTX+ component 3 presents a complete and orderly arranged jejunum villus structure, gaps among villi are relatively compact, and the mucin content is obviously increased, which indicates that the wolfberry polysaccharide component 3 can positively influence jejunum tissue structure, and is beneficial to improving the absorption capacity of intestinal tracts and the mucous membrane barrier function.
(4) Influence on short chain fatty acids in mouse faeces
Short chain fatty acid determination was performed by taking mouse faeces of NC group, CTX group and ctx+ group 3 groups respectively, and the results are shown in fig. 4:
the results show that there is a significant decrease in the concentration of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid in CTX compared to NC. However, after the matrimony vine polysaccharide component 3 is dried, the content of each short chain fatty acid in the CTX+component 3 group is obviously increased compared with that in the CTX group, which indicates that the ingestion of the matrimony vine polysaccharide component 3 can effectively relieve the decrease of the concentration of the short chain fatty acid in the mouse feces caused by the enrichment of P.azoformans bacteria.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
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