CN110151771B

CN110151771B - High-rheological-property 25-hydroxy vitamin D for repairing colitis3Preparation

Info

Publication number: CN110151771B
Application number: CN201910606725.8A
Authority: CN
Inventors: 王宝维; 刘亚楠; 谢玉娥; 葛文华; 张名爱; 岳斌; 孙京新
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2019-07-06
Filing date: 2019-07-06
Publication date: 2021-05-11
Anticipated expiration: 2039-07-06
Also published as: CN110151771A

Abstract

The invention provides a composition comprising a duck oil diglyceride and 25OHV₃(ii) a The diglyceride of duck oil and 25OHV₃The mass ratio of (1: 1.5) - (3.0) (g/mug); the composition of the invention is used for preparing a restoration preparation for treating the ecological colitis. The invention also provides a colitis repair preparation, which is prepared by using the composition; one specific preparation method is that the diglyceride of the duck oil is dissolved, and the temperature is adjusted to be 25-40 ℃; according to duck oil diglyceride and 25OHV₃The mass ratio g/mug of the raw materials is 1: 1.5-3.0, and the raw materials are mixed for 0.5-2 min to complete the preparation. The composition has the effects of improving physiological indexes of colitis rats, reducing TNF-alpha and IL-6 intestinal inflammatory factors, reducing D-LA to weaken the generation of intestinal endotoxin and bacterial translocation, and regulating intestinal flora. Is expected to develop a novel and efficient health care product for treating ulcerative colitis.

Description

High-rheological-property 25-hydroxy vitamin D for repairing colitis3Preparation

Technical Field

The invention belongs to the technical field of enteritis treatment preparations, and particularly relates to high-rheological-property 25-hydroxy vitamin D for repairing colitis₃And (4) preparing the preparation.

Background

Ulcerative Colitis (UC), also called chronic nonspecific ulcerative colitis, is a disease that is mainly characterized by superficial and nonspecific inflammatory diseases of the rectum and colon, and is also susceptible to damage to multiple organs outside the intestine. The disease is more common in Europe and America, the Asia is higher than that in Asia, the incidence rate in China is low, so that the importance is low, and the prevalence rate is increased year by year in recent years.

Colitis is a difficult and complicated disease with unknown reasons, and can be caused by various factors, such as poor eating habits such as long-term heavy drinking, autoimmune system disorder, smoking environment, even emotional stimulation and the like, which are all easy to cause colitis, wherein autoimmunity is the root. The clinical manifestations of colitis are diarrhea, abdominal pain, mucous stool and bloody purulent stool, often accompanied by emaciation, hypodynamia, etc., with infectivity, long course of disease, and many repeated attacks and afflictions. In recent years, the incidence of ulcerative colitis in China is on the rise, the ulcerative colitis is common to people of all ages, the life of people cannot be influenced in the initial stage of incidence, and people pay attention to the colitis, so that the colitis is difficult to cure when discovered. The colon has large mobility and is connected with important parts such as the caecum, the pancreas, the pelvic cavity, the ureter and the like, and the operation and the treatment are difficult, so the protection of the colon is paid attention to people. In addition, the livestock and poultry ulcerative colitis is more caused by feed mould and escherichia coli infection and the like, and the effective concentration of the drug in the rear section of the intestinal tract is greatly reduced, so that the livestock and poultry ulcerative colitis is difficult to cure by antibiotic treatment, and therefore, the livestock and poultry breeding loss is great.

At present, more drugs are clinically used for ulcerative colitis, antacids, proton pump inhibitors, H2-receptor antagonists and the like are the main drugs for resisting peptic ulcer at present in China, but the drugs are easy to lose efficacy at the rear end of the intestinal tract, generally have adverse reactions and drug residues with different degrees, have high recurrence rate after healing and can not obtain satisfactory treatment effect on the peptic ulcer. Therefore, people pay attention to research and develop therapeutic drugs for treating the peptic ulcer diseases for a long time, and the therapeutic drugs have very important significance for reducing drug residues, improving drug effects and improving the life quality of patients.

Disclosure of Invention

The invention provides an ecological colitis restoration preparation, namely a high rheological property 25-hydroxy vitamin D for colitis restoration₃Preparation prepared from duck oil diglyceride and 25-hydroxy vitamin D₃(25HVD₃) The composition is used for inhibiting bacteria, and regulating intestinal immunity and microorganism system of ulcerative enteritis; thereby making up for the deficiencies of the prior art.

The invention firstly provides a composition which comprises the diglyceride of duck oil and 25OHV₃；

The diglyceride of duck oil and 25OHV₃The mass ratio of (1: 1.5) - (3.0) (g/mug);

the composition is used for preparing a repairing preparation for treating ulcerative colitis.

The invention also provides a colitis repair preparation, which is prepared by using the composition; the specific preparation method comprises the steps of dissolving the diglyceride of the duck oil, and adjusting the temperature to be 25-40 ℃; according to duck oil diglyceride and 25OHV₃The mass ratio g/mug of the raw materials is 1: 1.5-3.0, and the raw materials are mixed for 0.5-2 min to complete the preparation.

The composition has the effects of improving physiological indexes of colitis rats, reducing TNF-alpha and IL-6 intestinal inflammatory factors, reducing D-LA to weaken the generation of intestinal endotoxin and bacterial translocation, and regulating intestinal flora. Is expected to develop a novel and efficient health care product for treating ulcerative colitis.

Drawings

FIG. 1: rheological profiles of different greases;

FIG. 2: graph for analysis of Beta diversity of intestinal flora in rats at different treatment levels, wherein Colon 3: group iii (repair group); colon 4: group iv (repair group); colon 5: group v (repair group); colon 6: group vi (repair group); colon 7: group vii (repair group); colon 8: group VIII; ColonD (normal group), ColonM (model inflammation group);

FIG. 3: optimal level composition versus E.coli morphology change plot, wherein A: blank control (× 15,000); b: soybean oil group (. times.15,000); c: composition group (× 15,000); d: enrofloxacin group (× 15,000);

FIG. 4: optimal level composition versus salmonella morphological change profile, wherein a: blank control (× 15,000); b: soybean oil group (. times.15,000); c: composition group (× 15,000); d: enrofloxacin group (× 15,000);

FIG. 5: graph of optimal composition versus morphological changes of staphylococcus aureus.

FIG. 6: the optimal composition is plotted against the morphological changes of Staphylococcus epidermidis.

Detailed Description

Diglyceride is a lipid with a fatty acid-substituted structure of triglyceride, has the property of lipid dissolving fat-soluble vitamins, is a Generally Recognized As Safe (GRAS) food ingredient, and has the functions of reducing blood fat, losing weight, improving solubility and liquid stability and the like. It is known that diacylglycerol of duck oil can be extracted from duck oil by an enzymatic method. The invention converts vitamin D into vitamin D₃In vivo active form of 25-hydroxyvitamin D₃(25-OH-VD₃) The compound can be used together with solvent of the duck oil diglyceride, and can effectively inhibit bacteria, inhibit the release of inflammatory factors and adjust the intestinal flora structure.

The present invention will be described in detail with reference to examples.

Example 1: determination of rheological Properties of Duck oil diglycerides

Pouring duck oil, soybean oil, duck oil diglyceride and lard into a beaker, detecting by using a rheometer, wherein the shear rate is as follows: 2.509-100/s, temperature 25 ℃.

As can be seen from FIG. 1, the shear stress of the duck oil, the soybean oil and the duck oil diglyceride is not significantly different, but the shear stress of the lard is significantly increased compared with the shear stress of the duck oil, the soybean oil and the duck oil diglyceride, which indicates that the rheological property of the duck oil diglyceride is better.

Example 2: compounding of compositions

The dissolution temperature range of the diglyceride of the duck oil is 25-40 ℃; mass ratio: duck oil diglyceride 1g, 25 hydroxyl vitamin D₃1.5 to 3.0. mu.g. The mixing and stirring time of the two is 0.5-2 min.

Example 3: detection of Effect of the composition

Test animals and groups: the test is carried out in two stages:

the first stage is as follows: and (5) establishing a colitis model. Dividing the rat into a model group and a control group, wherein the control group has 2 repetitions, and the model group has 16 repetitions, each repetition is 5, and the number of the rats is 100; model group rats were fed with 5% Dextran Sodium Sulfate (DSS) solution daily for 7 consecutive days, and the control group was fed with distilled water.

After the model building is finished, 10 rats are respectively extracted from the model group and the control group, fasting is carried out for 12h, serum and organs are taken, and growth indexes and blood biochemical indexes are detected, namely whether the model building is successful or not is verified.

And a second stage: duck oil diglyceride and 25OHV₃Synergistic colitis rat intervention effect test

70 rats successfully modeled in the first stage were selected and randomly divided into 7 experimental groups, namely a model group and a treatment group. Another 10 normal rats were selected as a control group. Each group had 2 replicates, 5 replicates each, and the feeding was as shown in table 1.

Table 1: duck oil diglyceride and 25OHV₃Design of synergistic intervention effect test on rats

Material taking and processing: after 24h fasting, each rat was weighed. Taking blood from rat orbit, removing neck, killing, centrifuging blood at 4 deg.C and 3000r/min for 15min, taking supernatant, and placing into 1.5mL centrifuge tube, and storing in refrigerator at-40 deg.C for detection; dissecting the rat, taking the colon and the spleen, washing with physiological saline at 4 ℃, and weighing the length and the weight of the colon and the spleen. The organs and blood were stored in a-80 ℃ refrigerator for examination.

Detection indexes are as follows:

1) the weight, colon length, spleen length and weight of each rat were weighed.

2) The contents of TNF-alpha, IL-6 and D-LA are determined according to the kit.

3) The structure and the number of the colon microbial flora are determined by a 16SrRNA sequencing technology based on an Illumina HiSeq sequencing platform and a double-End sequencing (Paired-End) method.

And (3) test results:

1) influence of groups on physiological growth of rats with ulcerative enteritis

The change of physiological indexes of rats in each group after administration is shown in table 2, compared with a normal group, the weight and the colon length of the rats in the model group are obviously reduced, and the spleen weight and the spleen length are obviously increased, which indicates that the rats have dyspepsia in vivo due to enteritis, the weight is reduced, the colon is shortened, immune organs excessively respond, and the spleen length and the spleen weight are increased. Compared with the model group, the treatment groups (III, IV, V, VI, VII and VIII) all obviously increase the weight and the colon length, and obviously reduce the spleen weight and the spleen length, which indicates that the composition has obvious function of repairing inflammation.

Table 2: duck oil diglyceride and 25OHV₃Synergistic effect on physiological index of enteritis rat

2) Influence of each group on inflammatory factors and intestinal permeability of rats with ulcerative enteritis

The change of physiological indexes of rats in each group after administration is shown in table 3, compared with the normal group, the serum TNF-alpha, IL-6 and D-LA of rats in the model group are obviously increased, which shows that enteritis has the function of obviously promoting the increase of inflammatory factors and permeability factors in rats. Compared with the model group, the treatment groups (III, IV, V, VI, VII and VIII)) all significantly reduce serum inflammatory factors and endotoxin permeability, which indicates that the composition has a significant effect of repairing inflammation.

Table 3: duck oil diglyceride and 25OHV₃Synergistic effect on inflammatory factors and intestinal permeability of enteritis rats

3)4.1Alpha diversity analysis

At the 97% similarity level, the Alpha diversity index values for each sample are statistically shown in table 4 below. Alpha diversity is responsive to species abundance, mainly represented by OUTs, Ace, Chao, and diversity, represented by Shannon index. The test result shows that the OTU number, the Ace index, the Chao index and the Shannon index of the colon flora of the rat in the model group are all smaller than those of the normal group and have significant difference. After the intestinal tract protective agent is given, the OTU number, the Ace index, the Chao index and the Shannon index of the colon flora of the rat are increased, which shows that DSS induces intestinal inflammation to reduce the abundance and diversity of the intestinal tract flora of the rat, and after the intestinal tract protective agent is added, the abundance and diversity of the intestinal tract flora of the enteritis rat are increased, and the intestinal tract flora is recovered to be normal. The results show that compared with the normal group of rats, the diversity of the intestinal bacteria and the abundance change of the bacterial genes of the rats in the model group are reduced, and the diversity of the intestinal microorganisms is improved to a certain extent after the composition is administered. The Coverage index of colon content flora of each group is more than 0.998, so that the sequencing quality of samples is high, and the probability of undetected samples is very low.

Table 4: diversity analysis of rat colonic flora Alpha at different levels

4) Beta diversity analysis

To further demonstrate the comparison of species diversity among samples, the diversity Analysis among samples was demonstrated by the Principal Component Analysis (Principal Component Analysis). As shown in fig. 2, the principal axis analysis graph shows a clear connection between the bacterial combinations of the healthy control group and the enteritis group along the principal axis, the PCA graph shows that the enteritis mice treated with the composition are significantly different from the DSS model group, and the principal components of the iv (4) group are similar to those of the healthy control group. The results show that the composition can recover the DSS-induced intestinal flora principal component analysis towards a normal control group, and the imbalance of the intestinal flora is adjusted.

FIG. 2 is a graph of the Beta diversity analysis of the intestinal flora of rats at different levels, wherein the abscissa represents the first principal component, a contribution of 8.52% to the difference of the samples; the ordinate represents the contribution of the second principal component to 17.03% of the sample difference. ColonD colon control group, ColonM colon model group

Example 4: duck oil diglyceride-25 OHV₃Effect on the phylum class Classification level of the intestinal microflora of rats with ulcerative colitis

Table 5: duck oil diglyceride-25 OHV₃Synergistic effect on species composition at the level of the enterobacteria phylum of rats with ulcerative colitis

Table 5 shows the diglyceride-25 OHV of duck oil₃The effect of different levels on the level of the dominant phylum of colonic microbiota in enteritis rats. In the case of a similarity of 97%, the content of the dominant microflora at the level of the colonic phylum was calculated. Among them, Firmicutes, Bacteroidetes and Proteobacteria are the main dominant phyla.

The relative abundance of Firmicutes was lowest in group I and highest in group II, which was significantly higher than group I (P < 0.05). Treatment groups III-VIII all showed significant reductions (P < 0.05) compared to group II, with group IV being lower than the other five groups and closer to group I. Bacteroides (Bacteroides) was highest in relative abundance in group I and lowest in group II, which was significantly lower (P < 0.05) than group I. The treatment groups III-VIII all showed significant increases (P < 0.05) compared to group II, with group IV being significantly higher than the other treatment groups (P < 0.05). Proteobacteria (Proteobacteria) relative abundance was lowest in group I and highest in group II, which was significantly higher than group I (P < 0.05). Treatment groups III-VIII all showed significant reductions (P < 0.05) compared to group II, with group IV being lower than the other four groups.

The results showed that the addition of diglyceride-25 OHV₃The relative abundance of the firmicutes and the proteobacteria of the later pathogenic bacteria is low, the relative abundance of the bacteroidetes of beneficial bacteria is high, the composition has a beneficial regulation effect on the intestinal flora structure, and the group IV has the best effect.

Table 6: duck oil diglyceride-25 OHV₃Synergistic effect on the composition of species at the level of the enterobacteria of rats with ulcerative colitis

Table 6 shows the diglyceride-25 OHV of duck oil₃Different levels of colonic microbiota in enteritis ratsInfluence of the horizontally dominant phylum. In the case of a similarity of 97%, the content of the dominant flora at the level of colonic microbiota was calculated. Wherein Bacteroidia (Bacteroidia); clostridia (clostridium); group C of Proteobacteria (Gamma); the erysipelomyces (Erysipelotrichia) is the main predominant class.

The relative abundance of Bacteroides (Bacteroides) was highest in group I and lowest in group II, which was significantly lower than group I (P < 0.05). The treatment groups III-VIII are all significantly higher (P < 0.05) than the treatment group II, wherein the group IV is higher than the other five groups and is closer to the group I. The relative abundance of Clostridia (clostridium) was highest in group ii and lowest in group i, with no significant difference between groups (P > 0.05). The relative abundance of group I of the group C of the Proteobacteria (Gamma proteobacteria) is the lowest, the relative abundance of group II of the Proteobacteria is the highest, and the relative abundance of group II of the Proteobacteria is obviously increased compared with that of group I (P < 0.05). Treatment groups III-VIII all showed significant reductions (P < 0.05) compared to group II, with group IV being lower than the other four groups. The relative abundance of Erysipelotrichia (Erysipelotrichia) was lowest in group I, highest in group II, and significantly higher in group II compared to group I (P < 0.05). The treatment groups III-VIII are all reduced significantly compared with the group II (P is less than 0.05), and the groups III, IV, VI and VII are not significantly different from the group I (P is more than 0.05), wherein the group IV is closer to the control group than the other three groups.

The results showed that the addition of diglyceride-25 OHV₃The relative abundance of the later beneficial bacteroides becomes high, the relative abundance of the pathogenic bacteria class C and erysipelothrix becomes low, and the composition has a beneficial regulation effect on the intestinal flora structure, wherein the effect of the group IV is the best.

Example 5: duck oil diglyceride-25 OHV₃Observation of in vitro bacteriostatic effect

The effect of the composition on the morphology of escherichia coli, salmonella, staphylococcus aureus and staphylococcus epidermidis was observed with a Scanning Electron Microscope (SEM). Blank thallus group and soybean oil group are used as negative control group, and enrofloxacin is used as positive control group. Firstly, respectively taking 2mL of the bacterial suspension of the test group and the bacterial suspension of the control group, placing the bacterial suspension and the bacterial suspension in a centrifuge tube, and centrifuging the mixture for 5min at 4000r/min to collect thalli. Then fixing the somatic cells for 4h by using a 4% glutaraldehyde solution,residual glutaraldehyde was washed with PBS and the cells were treated for 15min by dehydration with 50%, 70%, 90%, 100% gradient volume fraction ethanol, respectively. Finally, coating the dehydrated thallus on a clean copper table, naturally drying, performing surface gold plating on the thallus by using a sputtering instrument, placing a sample into an electron microscope chamber, deflating the sample chamber, and pumping to high vacuum of 1.0 multiplied by 10^-3After Pa, the surface morphology of the cells was observed by scanning electron microscopy.

From FIG. 3, it can be seen that the E.coli morphology of the untreated bacteria (FIG. 3A) and the soybean oil treatment (FIG. 3B) had an intact short rod shape, an intact membrane structure and a smooth cell surface. The surface structure and morphology of the bacterial cells were significantly destroyed after treatment with the composition (fig. 3C), the morphology of the bacterial cells was reduced, and the integrity of the cells was lost, close to that of the drug treated group (fig. 3D). The composition has damage effect on Escherichia coli cell membrane.

As can be seen in FIG. 4, the untreated bacteria (FIG. 4A) and the soybean oil treatment (FIG. 4B) exhibited Salmonella morphology, long rods, intact membrane structure and smooth texture. The surface structure and morphology of the bacterial cells were significantly destroyed after treatment with the composition (fig. 4C), the morphology of the bacterial cells was reduced, and the integrity of the cells was lost, similar to the drug treated group (fig. 4D). The composition has injury effect on salmonella cell membrane.

As can be seen in FIG. 5, the untreated bacteria (FIG. 5A) and the soybean oil treatment (FIG. 5B) showed the form of Staphylococcus aureus, which was spherical, had an intact membrane structure and had a smooth texture. The surface structure and morphology of the bacterial cells were significantly destroyed after treatment with the composition (fig. 5C), the morphology of the bacterial cells was reduced, and the integrity of the cells was lost, close to that of the drug treated group (fig. 5D). The composition has injury effect on Staphylococcus aureus cell membrane.

As can be seen in FIG. 6, the untreated bacteria (FIG. 6A) and the soybean oil treatment (FIG. 6B) have Staphylococcus epidermidis morphology, cylindrical shape, intact membrane structure and smooth texture. The surface structure and morphology of the bacterial cells were significantly destroyed after treatment with the composition (fig. 6C), the morphology of the bacterial cells was reduced, and the integrity of the cells was lost, similar to the drug treated group (fig. 6D). The composition has injury effect on Staphylococcus epidermidis cell membrane.

The invention combines the diglyceride of the duck oil with 25OHV₃The repairing agent is prepared by combination, so that the cell structure of bacteria can be damaged, and the growth of the bacteria can be inhibited; the repair of the ulcerative colitis is carried out, the release of inflammatory factors is obviously reduced, the intestinal permeability is reduced, so that the release of endotoxin and bacterial displacement are reduced, the intestinal flora is regulated, the intestinal pathogenic bacteria are reduced, and the repair effect on the ulcerative colitis is better.

Claims

1. A composition with effect of repairing ulcerative enteritis comprises diglyceride of duck oil and 25OHV₃Wherein the diglyceride of duck oil and 25OHV₃The mass ratio g/mug is 1: 1.5-3.0.

2. Use of the composition of claim 1 for the preparation of a colitis repair preparation.