CN113398073A - Salvianic acid A sodium self-assembled liposome for treating ulcerative colitis and preparation method thereof - Google Patents
Salvianic acid A sodium self-assembled liposome for treating ulcerative colitis and preparation method thereof Download PDFInfo
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- CN113398073A CN113398073A CN202110535833.8A CN202110535833A CN113398073A CN 113398073 A CN113398073 A CN 113398073A CN 202110535833 A CN202110535833 A CN 202110535833A CN 113398073 A CN113398073 A CN 113398073A
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- sodium
- liposome
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- ulcerative colitis
- salvianic acid
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to an oral salvianic acid A sodium self-assembly liposome for treating ulcerative colitis and a preparation method thereof. The salvianic acid A sodium self-assembly liposome for treating ulcerative colitis, which is prepared by the method, is prepared from salvianic acid A sodium, phospholipid, cholesterol, chitosan and sodium alginate by adopting a reverse phase evaporation method and an LbL self-assembly technology. The oral salvianic acid A sodium self-assembly liposome prepared by the invention has the effects of improving clinical symptoms of colitis mice, reducing the contents of proinflammatory factors TNF-alpha, IF-6 and IF-1 beta in serum of the ulcerative colitis mice, increasing the content of anti-inflammatory factors IL-10 and improving the intestinal flora imbalance of the ulcerative colitis mice, so that the oral salvianic acid A sodium self-assembly liposome has a better drug effect of treating the ulcerative colitis.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an oral salvianic acid A sodium self-assembly liposome for treating ulcerative colitis and a preparation method thereof.
Background
Inflammatory Bowel Disease (IBD), is a chronic and progressive inflammatory disease of the gastrointestinal tract, including Ulcerative Colitis (UC) and Crohn's Disease (CD). The incidence of inflammatory bowel disease is currently high worldwide, presenting an increasing trend, particularly in asian regions, with the incidence increasing two to three times in some countries. Ulcerative colitis is a common and refractory disease, the pathogenesis is not completely clear at present, and the cause of the disease is generally considered to be related to factors such as heredity and intestinal dysbacteriosis, environment and the like (Ingrid Ord & lt & gt, Lars Eckmann, Mark Talamini, Daniel CBaumgart, & William J Sandborn. (2012) & Ulcerative colitis. Lancet,380(9853), 1606-1619.). Generally, the diseased part of ulcerative colitis is in the mucosal layer of colon, mostly affects rectum and far-end colon, and can also spread over the whole colon, and its clinical symptoms include abdominal pain, diarrhea, mucopurulent bloody stool, etc., and the disease course is long, and it is easy to recur, and it is classified as one of the modern difficult diseases by the world organization, and the normal life of the patient is greatly influenced.
At present, the treatment means of the ulcerative colitis mainly comprise drug treatment and surgical treatment. Aminosalicylic acids are common medicines for treating ulcerative colitis, wherein sulfasalazine (SASP) is a main medicine for treating mild and moderate cases of UC and is also the most effective medicine for maintaining and relieving UC, but the aminosalicylic acids have more adverse reactions, such as nausea, vomiting, headache, reversible male infertility, autoimmune hemolysis, aplastic anemia and the like, so hemograms must be checked regularly during the medicine taking period. Another type of drug commonly used to treat UC is corticosteroids, but these drugs are prone to produce more side effects and relapse after long-term use, and therefore need to be reduced in dose and stopped after symptoms improve. The development of new formulations for the treatment of UC is therefore imperative.
Danshensu is a water-soluble substance derived from dry root of red sage root, has definite chemical structure, and has therapeutic effect on cardiovascular diseases (such as myocardial infarction, myocardial ischemia/reperfusion injury, arrhythmia, cardiac hypertrophy, myocardial fibrosis, atherosclerosis and hypertension), brain injury (such as ischemia, cognitive function decline and anxiety) and other health problems (such as thrombosis, tumorigenesis, pancreatitis). Salvianic acid A also directly inhibits the secretion of TNF, IL-1, IL-6 and IL-8, and inhibits the inflammatory activation of macrophages. It has been reported that after TNBS is used to make mouse ulcerative colitis model, the 10.24% tanshinol extract can be used to improve ulcer symptom obviously and regulate the content change of IL-1 beta and TNF-alpha.
Liposomes, one of the main carriers of drug delivery systems, can successfully and effectively load various drugs. Has important effects in the fields of medicine, biology and medicine, such as anticancer, antibiotic, anti-inflammatory, gene and antifungal.
The surface coating not only is a component of LbL (biomacromolecule layer-by-layer self-assembly technology) functionalized liposome, but also is an interface where the liposome meets and interacts with an external biological environment, and the stability and the administration efficiency of the liposome can be increased by properly optimizing the coating performance and the procedure. LbL functionalized liposomes must pass through different intestinal processes before entering the systemic circulation. By incorporating polymers with mucoadhesive and mucopenetrating properties into LbL functionalization, the mucoadhesion of LbL liposomes to intestinal epithelium can be enhanced, thereby extending the elimination half-life of the liposomes in the small intestine. Mucoadhesion can be achieved by ionic interactions between positively charged polymers and negatively charged components of mucus, including sialic acid and sulfonic acid residues. Modification of the participating LbL functionalization with these polymers may be a viable approach to enhance the mucus penetrating ability of the coated liposomes.
Common materials include Chitosan, sodium alginate and the like, and Chitosan (Chitosan, CH) is biocompatible cationic polysaccharide with low toxicity. Studies have shown that coating CH on the surface of liposomes can act as a stability enhancer and bioadhesive material. However, the main limitation of CH as a carrier is that it readily dissolves at the low pH of the stomach, resulting in denaturation of the encapsulated components. Sodium Alginate (Alginate, Alg) is a water-soluble anionic polymer that is widely used as a thickener, emulsifier, and stabilizer. The main disadvantage of coating Alg in liposomes for delivery is that it dissolves at high pH, subsequently loses protection and releases the drug. The liposome can effectively improve the stability and the drug release characteristic of the liposome in the gastrointestinal tract after the liposome and the liposome are coated layer by layer.
No researchers at home and abroad combine the danshensu sodium and the self-assembly liposome technology for treating the ulcerative colitis by consulting domestic and foreign documents and patents. In conclusion, the development of the oral liposome with small toxic and side effects and the function of treating the ulcerative colitis has important significance in the field of ulcerative colitis treatment.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides an oral self-assembled liposome for treating ulcerative colitis and a preparation method thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
a salvianic acid A sodium self-assembly liposome for treating ulcerative colitis is composed of phospholipid, cholesterol, salvianic acid A sodium, chitosan, sodium alginate and a solvent, wherein the solvent is a two-phase mixed solvent of an organic solvent and water.
Preferably, the mass ratio of the total mass of the phospholipid and the cholesterol to the mass of the medicine salvianic acid A sodium is (20-5): 1, more preferably 10: 1.
preferably, the mass ratio of the phospholipid to the cholesterol is (2-5): 1, more preferably 4: 1.
preferably, the volume ratio of the aqueous phase to the organic phase in the solvent is (1-5): 1, more preferably 3: 1.
preferably, the phospholipid is soybean phospholipid, and the purity is not lower than 95%.
Preferably, the organic solvent is chloroform and/or diethyl ether, and the water is water commonly used for formulation, such as purified water.
The invention also provides a preparation method of the salvianic acid A sodium self-assembly liposome for treating ulcerative colitis, which comprises the following steps:
(1) preparing an aqueous phase: dissolving sodium danshensu in water to obtain water phase;
(2) preparing an organic phase: adding phospholipid and cholesterol into trichloromethane for dissolving, removing a solvent to form a film, and adding diethyl ether for dissolving to obtain an organic phase;
(3) preparing liposome: mixing organic phase and water phase, performing ultrasonic treatment in ice bath with cell crusher until uniform emulsion is formed, removing organic phase, adding water for hydration for a certain time, and filtering with microporous membrane to obtain sodium salvianic acid A liposome;
(4) preparing self-assembled liposome: slowly dropwise adding the salvianic acid A sodium liposome DS-Lip into the prepared chitosan CH solution with the same volume under the stirring state, and continuously stirring for 1h after the liposome is dropwise added to fully wrap CH on the surface of the liposome; slowly dropwise adding a sodium alginate solution with the same volume as the chitosan solution, and continuously stirring for 1h after dropwise adding to fully wrap the sodium alginate on the surface of the chitosan; excessive chitosan and sodium alginate can generate cross-linking flocculation to generate precipitation, so that flocs are removed by centrifugation after layer-by-layer deposition, and the supernatant is collected to be the chitosan-sodium alginate double-layer modified sodium danshensu self-assembled liposome (Alg-CH-DS-Lip).
Preferably, the concentration of the danshensu sodium in the pure water in the step (1) is 1.5-3 mg/mL.
Preferably, the ultrasonic method of the cell disruptor in the step (3) is ultrasonic for 3s and stopping for 5s under the power of 200W, and the ultrasonic time is 10 min.
Preferably, the organic solvent is removed by rotary evaporation in step (3) at a temperature of 25-40 ℃.
Preferably, the hydration is carried out by rotary evaporation in the step (3), the hydration temperature is 25-40 ℃, and the hydration time is 1-2 h.
Preferably, the pore size of the microporous filter membrane in the step (3) is not more than 0.45 μm.
Preferably, the amount of water used in the step (1) is 2-4 mL; the dosage of the diethyl ether in the step (2) is 4-6 mL; water was added to 4mL in step (3).
Preferably, the ice bath temperature in the step (3) is-5 ℃ to 0 ℃.
Preferably, the concentration of the chitosan solution and the concentration of the sodium alginate solution in the step (4) are both 0.05-0.6%, and more preferably both 0.2% (w/v).
Preferably, the dropping speed in the step (4) is 0.1-1.0 mL/min.
Preferably, the encapsulation efficiency of the sodium danshensu self-assembly liposome obtained in the step (4) is determined as follows: taking a certain amount of prepared liposome, adding 9 times of methanol, performing ultrasonic demulsification, performing ultraviolet absorbance detection at 280nm, substituting the detected absorbance value into the standard curve of sodium danshensu, and calculating to obtain the amount of free medicine; encapsulation ratio (total drug amount-free drug amount)/total drug amount.
Compared with the prior art, the invention has the following beneficial effects: the salvianic acid A sodium self-assembly liposome for treating ulcerative colitis, which is prepared by the method, is prepared from salvianic acid A sodium, phospholipid, cholesterol, chitosan and sodium alginate by adopting a reverse phase evaporation method and an LbL self-assembly technology. The oral salvianic acid A sodium self-assembly liposome prepared by the invention is proved to have the efficacy of treating ulcerative colitis in a DSS (dextran sulfate sodium salt) -induced ulcerative colitis mouse experiment, and the oral salvianic acid A sodium self-assembly liposome specifically has the following beneficial effects:
(1) the salvianic acid A sodium self-assembly liposome prepared by the invention has the effect of improving the clinical symptoms of a colitis mouse.
(2) The sodium danshensu self-assembly liposome prepared by the invention has the functions of reducing the contents of proinflammatory factors TNF-alpha, IF-6 and IF-1 beta in the serum of an ulcerative colitis mouse and increasing the content of an anti-inflammatory factor IL-10.
(3) The salvianic acid A sodium self-assembly liposome prepared by the invention has the effect of improving the imbalance of the intestinal flora of mice with ulcerative colitis.
In conclusion, the oral salvianic acid A sodium self-assembly liposome prepared by the invention has better effect of treating ulcerative colitis.
Drawings
FIG. 1 is disease Activity index score (DAI) for each group of mice from day 1 to day 14 in the efficacy experiment of example 4;
FIG. 2 is the average concentration of IL-1 β, IL-6, IL-10, TNF- α in each group of mouse serum samples;
FIG. 3 shows the distribution of intestinal flora in mice in each group.
Detailed Description
The technical solution of the present invention is further specifically described by specific embodiments with reference to the accompanying drawings.
Example 1:
a. dissolving phospholipid 40mg and cholesterol 20mg in 10mL of chloroform, performing rotary evaporation under reduced pressure, recovering and removing the organic solvent chloroform to form a film, and dissolving with 6mL of diethyl ether to obtain an organic phase.
b. Salvianic acid A sodium 6mg was dissolved in 2mL of purified water as an aqueous phase.
c. And c, adding the water phase obtained in the step b into the organic phase obtained in the step a, and carrying out ultrasonic treatment in an ice bath at 0 ℃ by using a cell disruptor, wherein the ultrasonic treatment is carried out for 3s and 5s with the ultrasonic power of 200W for 10min to form a uniform emulsion.
d. And c, evaporating the emulsion obtained in the step c under reduced pressure at room temperature, volatilizing the organic solvent, adding a proper amount of purified water after reaching the colloidal state, adding the purified water to 4mL, continuing to evaporate under reduced pressure for hydration, and filtering by using 0.45 and 0.22 mu m microporous filter membranes in sequence to obtain the liposome.
e. And d, slowly dropwise adding the liposome prepared in the step d into an equal volume of CH solution (0.2%, w/v) under a stirring state, continuously stirring for 1h after the liposome is dropwise added, then slowly dropwise adding an equal volume of Alg solution (0.2%, w/v), and continuously stirring for 1h after the liposome is dropwise added. After the layer-by-layer deposition, the mixture is centrifuged (3000rpm, 10min) to remove flocculate, and the supernatant is collected to be the salvia miltiorrhiza sodium liposome (Alg-CH-DS-Lip) modified by the chitosan-sodium alginate double layer.
f. Taking a proper amount of liposome, demulsifying and determining that the entrapment rate is 45.25 +/-3.25%. And (3) determining the encapsulation efficiency: taking a certain amount of prepared liposome, adding 9 times of methanol, performing ultrasonic demulsification, performing ultraviolet absorbance detection at 280nm, substituting the detected absorbance value into the standard curve of sodium danshensu, and calculating to obtain the amount of free medicine; encapsulation ratio (total drug amount-free drug amount)/total drug amount. The method for measuring the content of the sodium danshensu comprises the following steps: the chromatographic conditions are as follows: the chromatographic column is Agilent TC-C18(150 × 4.6mm, 5 μm), the mobile phase is methanol-0.5% acetic acid aqueous solution (10:90), the flow rate is 1.0mL/min, the detection wavelength is 280nm, the column temperature is 40 ℃, and the sample injection amount is 10 μ L.
Example 2:
a. after dissolving 50mg of phospholipid and 10mg of cholesterol in 10mL of chloroform, the organic phase was recovered under reduced pressure and removed, and then 6mL of ether was added to dissolve the mixture to obtain an organic phase.
b. Salvianic acid A sodium 6mg was dissolved in 2mL of purified water as an aqueous phase.
c. And c, adding the water phase obtained in the step b into the organic phase obtained in the step a, and carrying out ultrasonic treatment in an ice bath by using a cell disruptor, wherein the ultrasonic treatment is carried out for 3s and 5s, and the ultrasonic treatment is carried out at the ultrasonic power of 200W for 10min to form a uniform emulsion.
d. And c, evaporating the emulsion obtained in the step c under reduced pressure at room temperature, volatilizing the organic solvent, adding a proper amount of purified water after the emulsion reaches a colloidal state, adding the purified water to 4mL, continuing to evaporate under reduced pressure for hydration, and filtering by using 0.45 and 0.22 mu m microporous filter membranes respectively to obtain the liposome.
e. And d, slowly dropwise adding the liposome prepared in the step d into an equal volume of CH solution (0.2%, v/w) under a stirring state, continuously stirring for 1h after the liposome is dropwise added, then slowly dropwise adding an equal volume of Alg solution (0.2%, v/w), and continuously stirring for 1h after the liposome is dropwise added. After the layer-by-layer deposition, the mixture is centrifuged (3000rpm, 10min) to remove flocculate, and the supernatant is collected to be the salvia miltiorrhiza sodium liposome (Alg-CH-DS-Lip) modified by the chitosan-sodium alginate double layer.
f. Taking a proper amount of liposome, demulsifying and determining that the entrapment rate is 31.25 +/-3.25%.
Example 3:
a. phospholipid 48mg and cholesterol 12mg were added to 5mL of chloroform to prepare an organic phase.
b. Salvianic acid A sodium 6mg was dissolved in 2mL of purified water as an aqueous phase.
c. And c, adding the water phase obtained in the step b into the organic phase obtained in the step a, and carrying out ultrasonic treatment in an ice bath by using a cell disruptor, wherein the ultrasonic treatment is carried out for 3s and 5s, and the ultrasonic treatment is carried out at the ultrasonic power of 200W for 10min to form a uniform emulsion.
d. And c, evaporating the emulsion obtained in the step c under reduced pressure at room temperature, volatilizing the organic solvent, adding a proper amount of purified water after the emulsion reaches a colloidal state, adding the purified water to 4mL, continuing to evaporate under reduced pressure for hydration, and filtering by using 0.45 and 0.22 mu m microporous filter membranes respectively to obtain the liposome.
e. And d, slowly dropwise adding the liposome prepared in the step d into an equal volume of CH solution (0.2%, v/w) under a stirring state, continuously stirring for 1h after the liposome is dropwise added, then slowly dropwise adding an equal volume of Alg solution (0.2%, v/w), and continuously stirring for 1h after the liposome is dropwise added. After the layer-by-layer deposition, the mixture is centrifuged (3000rpm, 10min) to remove flocculate, and the supernatant is collected to be the salvia miltiorrhiza sodium liposome (Alg-CH-DS-Lip) modified by the chitosan-sodium alginate double layer.
f. Taking a proper amount of liposome, demulsifying and determining that the entrapment rate is 33.68 +/-3.25%.
Example 4: effect of inulin liposomes on proinflammatory factors in serum of mice with ulcerative colitis
BALB/c mice are randomly divided into healthy groups and model building groups; preparing 3% (w/v) DSS solution with purified water, allowing the mice of the model building group to freely drink water for 7 days, replacing the mice with the purified water after the model building is finished, and allowing the mice of the healthy group to fully take the purified water for comparison. After the start of molding, mice were weighed and evaluated daily. The administration dosage of the positive drug SASP group is 400mg/kg, the administration dosage of the high-low dose Alg-CH-DS-Lip group is 40mg/kg and 20mg/kg (calculated by danshensu sodium) respectively, the administration dosage of the high-low dose danshensu sodium group is 40mg/kg and 20mg/kg respectively, the administration dosage of the blank carrier group (self-assembled liposome without drug) and the administration dosage of the physical mixed PM group are physical mixing of the blank carrier and the danshensu sodium raw material drug, and the administration dosage of the model group and the health group are equivalent physiological saline, wherein the specific administration scheme is shown in Table 1. From day 1 to day 14, the mice were recorded daily for weight change, mental status, fecal traits, dietary status and hair shine after DSS induction. Disease Activity Index (DAI) was measured blindly and evaluated according to the DAI criteria described in table 2.
TABLE 1 ulcerative colitis mouse DSS modeling and dosing regimen
TABLE 2 DAI scoring criteria
The experimental results show that the healthy mice have good conditions in the experimental process, the weight and the DAI score are basically kept in a stable state after stably increasing to reach the maximum weight in the initial stage, and the healthy mice have the conditions of hair luster, stool formation, no diarrhea, hematochezia and the like. The body weight of mice given 3% DSS for modeling began to significantly decrease at 4-5 days, the DAI score was significantly increased, and the mice were listened, shrunken in fur, contracted in body, and suffered from diarrhea, hematochezia, and death to various degrees. The weight of the mice in the model group reaches the lowest value in 7-8 days, the weight is obviously different from that of the mice in the healthy group, the DSS is replaced by pure water and then administration is started, the control group is only administered with the same amount of normal saline, positive medicines SASP, a danshensu sodium raw material medicine, Alg-CH-DS-Lip and a physical mixture improve the weight reduction condition of the mice with the DSS-induced ulcerative colitis in the treatment stage, the mental state of the mice in each administration group is gradually improved, the conditions of diarrhea, hematochezia and the like are relieved, and the DAI score is gradually reduced. Although the DAI and body weight conditions of the mice with the positive drug group are gradually good, the mice have the phenomenon of yellowing and rough hair in the administration process.
The DAI score is shown in figure 1, the DAI score of the healthy mice does not generate, the DAI of the model mice slowly increases during modeling, the DAI score reaches the highest value at 7-8 days of modeling similarly to the weight trend, the DAI score starts to improve and slowly decreases after administration, the decrease degree of the positive drug and the liposome high-low dose group is similar, the DAI score decreases to about 1-2 at 13-14 days, and the significant difference (p is less than 0.01) is compared with the DSS group, so that the liposome group and the positive drug have similar drug effects and have good effect of treating ulcerative colitis. The DAI scores of the high-dose group and the physical mixture group of the salvianic acid A sodium bulk drug are also obviously different from those of the control group (p is less than 0.01), which indicates that the salvianic acid A sodium also has certain effect of treating ulcerative colitis.
Example 5: effect of sodium danshensu self-assembled liposomes on proinflammatory and anti-inflammatory factors in ulcerative colitis
BALB/c mice are randomly divided into healthy groups and model building groups; preparing 3% (w/v) DSS solution with purified water, allowing the mice of the model building group to freely drink water for 7 days, replacing the mice with the purified water after the model building is finished, and allowing the mice of the healthy group to fully take the purified water for comparison. After the start of molding, mice were weighed and evaluated daily. The administration is started on day 8, the administration dose of the positive drug SASP group is 400mg/kg, the administration doses of the high and low dose Alg-CH-DS-Lip groups are respectively 40mg/kg and 20mg/kg (calculated by salvianic acid A sodium), the administration doses of the high and low dose salvianic acid A sodium groups are respectively 40mg/kg and 20mg/kg, the blank carrier group and the physical mixture are blank carriers and salvianic acid A sodium bulk drugs, and the model group and the healthy group are administered with the same amount of physiological saline. Mice were sacrificed on day 15, blood from each group of animals was collected by eye-picking and blood-drawing methods before the mice were sacrificed, after standing at room temperature for 30 minutes, centrifuged at 3000rpm at 4 ℃, supernatant serum was collected, and the levels of cytokines (TNF- α, IL-6, IL-1 β, IL-10) were quantitatively determined using ELISA kits according to the procedure required by the manufacturer. The statistical method comprises the following steps: t-tests were performed with GraphPad Prism 7 software. (in comparison with healthy groups, treatment groups,. beta.. p:<0.05, 0.01; in comparison to the model set,#，##：p＜0.05，0.01)。
the significant difference between the proinflammatory cytokine levels IL-1 beta, IL-6 and TNF-alpha (p < 0.05, 0.01 and 0.05 respectively) and the anti-inflammatory factor IL-10 (p < 0.05) exists in the plasma of the DSS-induced colitis of the mice and the mice in the healthy group, which indicates that each cytokine is likely to be affected by inflammation during the modeling period. After SASP, the salvianic acid A sodium bulk drug, Alg-CH-DS-Lip and a physical mixture are used for treatment, proinflammatory factors can be inhibited and anti-inflammatory factors can be promoted to be produced, wherein the IL-1 beta, IL-6 and TNF-alpha factor levels of the SASP as a positive drug and the Alg-CH-DS-Lip as a high dose are remarkably different from those of a DSS group (p is less than 0.05), and the phenomenon of cytokine secretion disorder after the SASP and the Alg-CH-DS-Lip as the high dose are used for treatment can be proved to be remarkably improved; in IL-1 beta data, there was also some difference between high and low doses of Alg-CH-DS-Lip and the blank vector group (p < 0.05, 0.01, respectively), and the blank vector may not exert therapeutic effect during the whole treatment process; compared with DSS, the IL-1 beta and IL-alpha of the bulk drug of the low-dose Alg-CH-DS-Lip and the high-low dose sodium danshensu have significant difference (p is less than 0.05), which shows that the treatment effect trends of the Alg-CH-DS-Lip, the DSSSA and the SASP are the same, and the antiulcer effect of the high-dose Alg-CH-DS-Lip is similar to that of the SASP, so that the level of the cell factor can be effectively adjusted to develop towards the normal trend.
Example 6: the influence of the salvianic acid A sodium self-assembled liposome on intestinal flora in ulcerative colitis BALB/c mice is randomly divided into a healthy group and a modeling group; preparing 3% (w/v) DSS solution with purified water, allowing the mice of the model building group to freely drink water for 7 days, replacing the mice with the purified water after the model building is finished, and allowing the mice of the healthy group to fully take the purified water for comparison. After the start of molding, mice were weighed and evaluated daily. The administration is started on day 8, the administration dose of the positive drug SASP group is 400mg/kg, the administration doses of the high and low dose Alg-CH-DS-Lip groups are respectively 40mg/kg and 20mg/kg (calculated by salvianic acid A sodium), the administration doses of the high and low dose salvianic acid A sodium groups are respectively 40mg/kg and 20mg/kg, the blank carrier group and the physical mixture are blank carriers and salvianic acid A sodium bulk drugs, and the model group and the healthy group are administered with the same amount of physiological saline. At day 15, the mice were sacrificed and the colon contents were removed for intestinal flora analysis.
Several species with a greater tendency to change in induced colitis by screening versus DSS belong to two phyla of dominance: bacteroidetes and Firmicutes, wherein Bacteroidetes includes S24-7, Bacteroides; the firmicutes include: clostridium, Ruminococcus, Lachnospiraceae, Lactobacillus and Clostridium. As a result, it was found that: there are some obvious differences in the species Clostridium, Bacteroides, Bacterodales (S24-7), Akkermansia, Lactobacillus, etc. after molding and administration of DSS; the difference between Bacteroides, Ruminococcus and Lachnospiraceae in the DSS group and other administration groups is large, the Bacteroides bacteria are analyzed to find that the Bacteroides bacteria and other administration groups have significant difference (p is less than 0.05), the Bacteroides bacteria belong to Bacteroides, have complex and delicate interaction relationship with other flora and hosts, and play an important role in the health of the hosts. Bacteroides is also a opportunistic pathogen that may trigger endogenous interference when its normal microbial homeostasis is disrupted.
The Akkermansia bacteria belong to the genus Exkermansia, and as a result, it was observed that the ratio of SASP to the presence of the bacteria in mice of the healthy group was low, while the other administration groups all had a certain ratio of the bacteria. It has been shown that Akkermansia muciniphila (muciniphila-Ackermansia) is an enterobacteria that has some effect on host metabolism and PD-1 immunotherapy and induces both mouse immunoglobulin G1(IgG1) antibody and antigen-specific T cell responses. Muciniphilic-akkermansia-specific responses are environmentally dependent and during homeostasis, can be influenced by other bacteria in the gut to react with the T cell microbiota and modulate host immune function.
The above-described embodiments are merely preferred embodiments of the present invention, which is not intended to be limiting in any way, and other variations and modifications are possible without departing from the scope of the invention as set forth in the appended claims.
Claims (10)
1. The salvianic acid A sodium self-assembly liposome for treating ulcerative colitis is characterized by consisting of phospholipid, cholesterol, salvianic acid A sodium, chitosan, sodium alginate and a solvent, wherein the solvent is a two-phase mixed solvent of an organic solvent and water.
2. The self-assembled salvianic acid A sodium liposome for treating ulcerative colitis according to claim 1, wherein the mass ratio of the total mass of phospholipid and cholesterol to the mass of the medicine salvianic acid A sodium is (20-5): 1.
3. the sodium danshensu self-assembled liposome for treating ulcerative colitis according to claim 1, wherein the mass ratio of phospholipid to cholesterol is (2-5): 1.
4. the sodium danshensu self-assembled liposome for treating ulcerative colitis according to claim 1, wherein the volume ratio of the aqueous phase to the organic phase in the solvent is (1-5): 1; the organic solvent is chloroform and/or diethyl ether.
5. The sodium danshensu self-assembled liposome for treating ulcerative colitis according to claim 1, wherein the phospholipid is soybean phospholipid with purity of not less than 95%.
6. A method for preparing the salvianic acid A sodium self-assembled liposome for treating ulcerative colitis according to claim 1, comprising the steps of:
(1) preparing an aqueous phase: dissolving sodium danshensu in water to obtain water phase;
(2) preparing an organic phase: adding phospholipid and cholesterol into trichloromethane for dissolving, removing a solvent to form a film, and adding diethyl ether for dissolving to obtain an organic phase;
(3) preparing liposome: mixing organic phase and water phase, performing ultrasonic treatment in ice bath with cell crusher until uniform emulsion is formed, removing organic phase, adding water for hydration for a certain time, and filtering with microporous membrane to obtain sodium salvianic acid A liposome;
(4) preparing self-assembled liposome: slowly dropwise adding the salvianic acid A sodium liposome into the prepared chitosan solution with the same volume under the stirring state, and continuously stirring for 1h after the liposome is dropwise added so as to fully wrap the chitosan on the surface of the liposome; slowly dropwise adding a sodium alginate solution with the same volume as the chitosan solution, and continuously stirring for 1h after dropwise adding to fully wrap the sodium alginate on the surface of the chitosan; centrifuging, and collecting the supernatant to obtain the chitosan-sodium alginate double-layer modified sodium danshensu self-assembled liposome.
7. The preparation method of the salvianic acid A sodium self-assembled liposome for treating ulcerative colitis according to claim 6, wherein the concentration of the salvianic acid A sodium in pure water in step (1) is 1.5-3 mg/mL.
8. The method for preparing the sodium danshensu self-assembled liposome for treating ulcerative colitis according to claim 6, wherein the ultrasonic method of the cell disruptor in step (3) is ultrasonic at 200W for 3s and 5s for 10 min.
9. The method for preparing the salvianic acid A sodium self-assembled liposome for the treatment of ulcerative colitis according to claim 6, wherein the step (3) is carried out by rotary evaporation, the hydration temperature is 25-40 ℃, and the hydration time is 1-2 h; the aperture of the microporous filter membrane in the step (3) is not more than 0.45 μm.
10. The method for preparing the sodium danshensu self-assembled liposome for treating ulcerative colitis according to claim 6, wherein the concentrations of the chitosan solution and the sodium alginate solution in step (4) are 0.05-0.6% respectively.
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