Application of cynomorium songaricum total polysaccharide in preparation of medicine for treating asthma
阅读说明：本技术 锁阳总多糖在制备治疗哮喘药物中的应用 (Application of cynomorium songaricum total polysaccharide in preparation of medicine for treating asthma ) 是由 辛贵忠 陈琳 刘丽芳 于 2019-11-22 设计创作，主要内容包括：本发明公开了锁阳总多糖在制备治疗哮喘药物中的应用,实验证明锁阳总多糖能够明显改善屋尘螨致敏哮喘小鼠肺组织的病理变化,抑制炎性因子的增加,可用于制备预防或治疗哮喘药物。锁阳总多糖用于哮喘的防治,安全无毒副作用,有良好的应用前景。(The invention discloses application of cynomorium songaricum total polysaccharide in preparation of a medicine for treating asthma, and experiments prove that the cynomorium songaricum total polysaccharide can obviously improve pathological changes of lung tissues of house dust mite allergic asthma mice and inhibit increase of inflammatory factors, and can be used for preparing medicines for preventing or treating asthma. The cynomorium songaricum total polysaccharide is used for preventing and treating asthma, is safe, has no toxic or side effect, and has good application prospect.)
1. Application of herba Cynomorii total polysaccharide in preparing medicine for preventing and/or treating asthma is provided.
2. The use according to claim 1, characterized in that the total polysaccharides of Cynomorium songaricum are total polysaccharides of Cynomorium songaricum or a formulation containing total polysaccharides of Cynomorium songaricum.
3. The use according to claim 2, characterized in that the preparation is prepared from cynomorium songaricum total polysaccharides with pharmaceutically acceptable excipients.
4. Use according to claim 2, characterized in that the formulation is an oral formulation.
5. The use according to claim 3, characterized in that the pharmaceutically acceptable excipients are selected from one or more of fillers, binders, disintegrants, solubilizers, solvents or flavoring agents.
6. The use according to claim 4, characterized in that the oral formulation is a tablet, capsule, pill, granule, oral liquid.
7. The use according to claim 1, wherein the total polysaccharide of Cynomorium songaricum is the main component of the total polysaccharide extract of Cynomorium songaricum, and the sugar content in the total polysaccharide extract of Cynomorium songaricum is above 70%.
8. Application of herba Cynomorii total polysaccharide extract in preparing medicine for preventing and/or treating asthma is provided.
9. The preparation method of the cynomorium songaricum total polysaccharide extract according to claim 8, which is characterized by comprising the following steps:
weighing sieved herba Cynomorii medicinal powder, adding ethanol, refluxing for degreasing, filtering, precipitating, oven drying, adding water into the dried precipitate, heating, refluxing for extraction, repeatedly extracting for several times, mixing extractive solutions, concentrating, adding ethanol, precipitating overnight, centrifuging to obtain precipitate solid, and lyophilizing to obtain herba Cynomorii crude polysaccharide; dissolving the crude cynomorium songaricum polysaccharide in water, filtering and collecting filtrate; reacting the filtrate with papain, collecting supernatant and H2O2Reacting, dialyzing with running water, concentrating, adding ethanol for overnight precipitation, centrifuging to obtain precipitate solid, and lyophilizing to obtain herba Cynomorii total polysaccharide extract solid.
10. The method for preparing the cynomorium songaricum total polysaccharide extract according to claim 9, wherein the molecular weight range of polysaccharides in the cynomorium songaricum total polysaccharide extract is 36464-24 KDa.
Technical Field
The invention belongs to the application of natural medicines, and particularly relates to application of cynomorium songaricum total polysaccharide in preparation of a medicine for treating asthma.
Background
Bronchial Asthma (Asthma ) is a chronic airway disease, which is mainly characterized by reversible airway obstruction, airway hyperresponsiveness, eosinophilic infiltration and CD4+ T helper (Th)2 type cell inflammatory infiltration and airway remodeling, and has the symptoms of wheezing, dyspnea, chest distress, cough and the like. Asthma is an autoimmune disease, and in recent years, "endogenous metabolites" produced by host organisms and intestinal flora play an important role in the occurrence and development of asthma, especially lipid metabolites. Short chain fatty acids, such as acetate, propionate and butyrate, are produced by fermentation of undigested or partially digested dietary fiber by large intestine microorganisms and have a wide range of effects on the development and function of the host immune system. The pathogenesis of asthma is not clarified up to now, the anti-inflammatory therapy is mainly clinically carried out on chronic airway inflammation, the anti-inflammatory therapy is mainly western medicines, but the western medicines have obvious limitations in treating the asthma disease in consideration of the characteristic of multi-factor pathogenesis of asthma, and the traditional Chinese medicines have unique advantages in the aspect of prevention and treatment of the asthma due to the action characteristics of multiple components, multiple targets and integral regulation.
Dietary fiber is an edible part of a plant or similar carbohydrate, an edible carbohydrate polymer having three or more monomeric units, including polysaccharides, oligosaccharides, lignin and related plant matter. It is resistant to digestion and absorption in the human small intestine, but can be fermented by the intestinal flora. It has now been found that dietary fiber has a great influence on the composition, diversity and abundance of the intestinal flora, and that intestinal microorganisms and their metabolites protect the intestinal epithelium and muscles from pathogens, playing an important role in many physiological processes, chronic diseases. The introduction of dietary fiber into clinic as diet-assisted intervention therapy is also a current research hotspot, and a new meaning of 'homology of medicine and food' is given.
Cynomorium songaricum is derived from dried succulent stems of Cynomorium songaricum Songaricum Ruhr, is a variety accepted in Chinese pharmacopoeia, and has the effects of invigorating kidney yang, replenishing essence and blood, and loosening bowel to relieve constipation. The water extract is rich in various chemical components, such as polysaccharide, flavone, triterpenes, steroids, alkaloids, and saponin. Polysaccharide substances are macromolecular substances with rich content in cynomorium songaricum, and studies on cynomorium songaricum polysaccharide are shallow so far, so that a refining process of water-soluble cynomorium songaricum polysaccharide is established and optimized to obtain a cynomorium songaricum total polysaccharide component, and the cynomorium songaricum total polysaccharide component is found to have the activity of relieving asthma for the first time, so that a literature report about the control of asthma by using cynomorium songaricum polysaccharide is not found so far.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide application of water-soluble cynomorium songaricum total polysaccharide obtained by refining in preparation of medicines for preventing or treating asthma or auxiliary treatment medicines.
The technical scheme is as follows: application of herba Cynomorii total polysaccharide in preparing medicine for preventing and/or treating asthma is provided.
The cynomorium songaricum total polysaccharide is cynomorium songaricum total polysaccharide or a preparation containing the cynomorium songaricum total polysaccharide.
The preparation is prepared by adding pharmaceutically acceptable auxiliary materials into cynomorium songaricum total polysaccharide.
The preparation is an oral preparation.
The pharmaceutically acceptable auxiliary materials are selected from one or more of fillers, adhesives, disintegrants, solubilizers, solvents or flavoring agents.
The oral preparation is tablet, capsule, pill, granule, or oral liquid.
The cynomorium songaricum total polysaccharide is the main component of the cynomorium songaricum total polysaccharide extract, and the sugar content in the cynomorium songaricum total polysaccharide extract is more than 70 percent.
Application of herba Cynomorii total polysaccharide extract in preparing medicine for preventing and/or treating asthma is provided.
The preparation method of the cynomorium songaricum total polysaccharide extract specifically comprises the following steps:
weighing sieved herba Cynomorii medicinal powder, adding ethanol, refluxing for degreasing, filtering, precipitating, oven drying, adding water into the dried precipitate, heating, refluxing for extraction, repeatedly extracting for several times, mixing extractive solutions, concentrating, adding ethanol, precipitating overnight, centrifuging to obtain precipitate solid, and lyophilizing to obtain herba Cynomorii crude polysaccharide; dissolving the crude cynomorium songaricum polysaccharide in water, filtering and collecting filtrate; reacting the filtrate with papain, collecting supernatant and H2O2Reacting, dialyzing with running water, concentrating, adding ethanol for overnight precipitation, centrifuging to obtain precipitate solid, and lyophilizing to obtain herba Cynomorii total polysaccharide extract solid.
The molecular weight range of polysaccharide in the cynomorium songaricum total polysaccharide extract is 36464-24 KDa.
Has the advantages that: the invention discloses cynomorium songaricum total polysaccharide which can obviously improve the pathological changes of lung tissues of house dust mite allergic asthma mice and inhibit the increase of inflammatory factors, and can be used for preparing medicines for preventing or treating asthma. The cynomorium songaricum total polysaccharide is used for preventing and treating asthma, is safe, has no toxic or side effect, and has good application prospect.
Drawings
FIG. 1 is the result of single factor investigation of the deproteinization experiment of cynomorium songaricum total polysaccharide;
FIG. 2 is a single factor investigation result of a cynomorium songaricum total polysaccharide decolorization experiment;
FIG. 3 is a high performance size exclusion chromatography of total polysaccharides from Cynomorium songaricum;
FIG. 4 shows the differential count of inflammatory cells in mouse BALF;
FIG. 5 shows the results of HE staining of mouse lung tissue sections;
FIG. 6 shows the result of IgE detection in mouse serum.
Detailed Description
Example 1: preparation method of cynomorium songaricum total polysaccharide extract
Weighing 50g of the cynomorium songaricum medicinal material which is crushed and sieved by a 80-mesh sieve into an extraction container, adding 5 times of ethanol by volume, carrying out reflux degreasing for 3 hours, filtering to remove supernatant, and drying a sample. Placing the dried sample into a round-bottom flask, adding 1.25L of single distilled water, heating and refluxing at 100 ℃ for extraction for 4 hours, and repeatedly extracting for 4 times. Mixing the four extractive solutions, concentrating to 150mL, adding 600mL of ethanol, precipitating at 4 deg.C overnight, centrifuging to obtain precipitate solid, lyophilizing to obtain polysaccharide solid, and weighing to obtain crude polysaccharide 5.65 g. Weighing 1g of crude cynomorium songaricum polysaccharide, dissolving in 200ml of distilled water, heating and stirring at 70 ℃, dissolving, filtering and collecting filtrate. Adjusting pH to 7, reacting with 10% (V/V) papain at 55 deg.C for 2.5 hr, boiling in water bath for 5min, cooling to room temperature, centrifuging to remove excessive enzyme (4000rpm/min,5min), and collecting supernatant. Adjusting the pH of the supernatant to 8 with 15% (V/V) of 30% H2O2Reacting at 55 deg.C for 1.5 hr, loading the sample solution into dialysis bag, dialyzing with running water for 36 hr, concentrating to 10mL, adding ethanol 40mL, precipitating at 4 deg.C overnight, centrifuging to obtain precipitate solid, lyophilizing to obtain herba Cynomorii total polysaccharide extract solid, weighing to obtain 0.468g, and sugar content of 71.4%.
Relative molecular weight analysis was performed by high performance liquid chromatography using Shodex SUGAR KS-805(8 mmID. times.300 mm) as a high performance liquid chromatography column using a differential refractometer detector. The chromatographic conditions are as follows: h2O is a mobile phase, the flow rate is 1ml/min, the sample concentration is 1mg/ml, 35 mul is injected each time, and the detection time is 30 min. Sequentially using standard molecular weight polysaccharide DexAnd drawing a standard curve of the logarithmic relation between the retention time and each molecular weight by tranT-5, T-10, T-20, T40 and T70, measuring the retention time of the sample, and obtaining the molecular weight of the sample according to the standard curve. The results are shown in FIG. 3, and the molecular weight of the obtained polysaccharide ranges from 36464 KDa to 24 KDa.
The process parameters of the invention were determined by single factor experiments (fig. 1, fig. 2) and orthogonal experiments (table 1, table 3), and the results are shown in tables 2 and 4. According to the analysis of the results, the factors influencing the deproteinization efficiency of the total polysaccharide of the cynomorium songaricum are analyzed, wherein the main sequence and the secondary sequence are that the pH is more than the enzyme content and more than the temperature is more than the time, namely the enzyme content is 10 percent, the reaction temperature is 55 ℃, the pH is 7, and the deproteinization efficiency is highest when the reaction time is 2.5 hours. The factors influencing the decolorizing efficiency of the cynomorium songaricum total polysaccharide have the primary and secondary sequence that the pH is more than the temperature, the time is approximately equal to the time and the time is more than H2O2Content of H2O2The content is 15%, the reaction temperature is 65 ℃, the pH value is 8, and the decolorizing efficiency is highest when the reaction time is 1.5 h.
TABLE 1 Deproteinization test factor levels
TABLE 2 Suo-cynomorium songaricum total polysaccharide deproteinization orthogonal experimental results
TABLE 3 levels of Experimental factors for decolorization
TABLE 4 decolorization orthogonal experiment results for cynomorium songaricum total polysaccharide
Example 2: activity study of cynomorium songaricum total polysaccharide on asthma
First, experimental material
1.1 Experimental instruments
Centrifuge, positive biological microscope, microplate reader, grinder, digital pathological section scanner, and ultralow temperature refrigerator
1.3 Experimental animals
60 clean-grade C57BL/6J female mice, 6-8 weeks old, 18-20g in weight, Shanghai Si Laike laboratory animals, Inc., license number: SCXK (Shanghai) 2017-.
1.4 preparation of test solutions
(1)0.01M PBS solution: 0.2g of KCl, 7.9g of NaCl, 1.44g of Na were weighed out2HPO4And 1.8gK2HPO4Dissolving in 800mL deionized water, adjusting the pH of the solution to 7.4 with HCl, finally using deionized water to 1L, and refrigerating for later use.
(2) Dexamethasone (DXM) solution (positive drug): dexamethasone was weighed precisely at 1.3mg and dissolved in 13ml of physiological saline (0.02mg/20 g/200. mu.l).
(3) Cynomorium songaricum total polysaccharide solution: dissolving the total polysaccharide extract of herba Cynomorii in purified water at a dose of 53 mg/kg/day, wherein the volume of purified water is calculated based on 5mL per mouse per day, and the purified water is prepared on site daily.
(4) 1% sodium pentobarbital (anesthetic): pentobarbital sodium (0.35 g) was precisely weighed and dissolved in 35ml of physiological saline (100. mu.l/piece).
(5) Sensitizing agent: an HDM stock solution of 8mg/ml was prepared and diluted 40-fold with physiological saline, i.e., 0.2mg/ml was the desired concentration. 0.4ml of the mother solution was removed precisely, and 15.6ml of physiological saline was added to prepare 16ml of a diluent (10. mu.g/50. mu.l/piece), which was stored at-20 ℃.
Second, Experimental methods
2.1 establishment of HDM-induced asthma model
After the mice were adaptively fed for 7 days, they were randomly divided into a normal Control group (Control), a Model group (Model), a polysaccharide group (CSP), and a positive drug group (DXM). Except for the blank group, all groups use HDM to induce the asthma model on the 0 th, 3 th, 5 th, 10 th, 12 th and 14 th days, the specific operation is to inject 100 mul of anesthetic into the abdominal cavity, after the mice are anesthetized, 50 mul of sensitizer is taken, and nose dropping and trachea instillation double-effect sensitization are carried out. The polysaccharide group is administrated by drinking water every day, and the positive medicine group is injected with 200 μ l dexamethasone solution in abdominal cavity in 10-16 days.
2.2 mouse model Observation of asthma
During the experiment, the respiratory state of the mice was observed daily.
2.3 sample Collection
After fasting for 12h, the eyeball is picked to draw blood, the blood of the mouse is collected, after standing for 30min, the supernatant is obtained by centrifugation at 3000rpm for 10min and is subpackaged in a 200 mu L EP tube and stored at
2.4 statistical treatment
Statistical analysis was performed using SPSS software, and two independent samples were compared using t-test, expressed as mean ± standard deviation (X ± SD). about.p<0.05 indicates that the sample differed significantly from the control group by p<0.01 indicates that the difference is very significant compared with the control group sample,#p<0.05 indicates that the sample difference was significant compared to the model group,##p<0.01 indicates that the sample difference is very significant compared to the model group.
2.4 items tested
2.4.1 differential cell count: the lower layer cells after BALF centrifugation are added with 15 mu LPBS, vortexed or blown by a pipette to be resuspended, 10 mu L of the supernatant is pipetted and spotted on a glass slide, and the supernatant is dried at 37 ℃. The slides were placed on a staining cassette and subjected to Reishi-Giemsa staining. Dripping 150 μ L of solution A into each piece of the tablet, dyeing for 1min, adding 450 μ L of solution B, dyeing for 10min, continuously blowing ear washing ball uniformly, washing with water for 15s, and air drying. 400 cells were observed under an upright microscope, and the proportion of macrophages, eosinophils and lymphocytes was calculated.
2.4.2HE staining:
a. fixing: fixing 4% paraformaldehyde for at least more than 24 h;
b. dehydrating and transparent: sequentially adding 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol and 100% ethanol for 1 hr respectively, and finally performing xylene transparent treatment;
c. wax dipping and embedding: soaking in soft wax for 1h, soaking in hard wax for 30min, cutting into 5 μm slices after embedding, and dyeing.
d. Hematoxylin-eosin (HE) staining: soaking in xylene for dewaxing for 10min × 2 times, sequentially soaking in 100% ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 1min respectively, hydrating, and washing with clear water; staining with hematoxylin for 10min, and washing with clear water; soaking in 1% hydrochloric acid ethanol for 10s, and washing with clear water; eosin staining for 30s, and rinsing with clear water; gradient ethanol dehydration (85% ethanol, 95% ethanol, 100% ethanol each for 2 min); soaking in xylene I for 5min and xylene II for 5min, and slicing; and finally, sealing the neutral gum.
Observation by a Nanozomer digital pathological section scanner: observing whether the alveolar wall of the lung tissue section is congested or not and inflammatory cell infiltration exists; presence or absence of pulmonary emphysema; infiltration of inflammatory cells around blood vessels and bronchi in lung; the number of small bronchial wall goblet cells in the lung is increased, and inflammatory exudation in the lumen is avoided.
2.4.3 determination of serum sample IgE: the procedures were as described for the ELISA kit.
Third, experimental results
3.1 respiratory State Change in asthmatic mice
After the mouse is instilled with the sensitizer each time, a strong cough and asthma reaction is shown, which indicates that the air passage instillation is successful, and the states of tachypnea, cough and asthma, arch back and the like of the mouse can be observed from the 10 th day, which is the clinical manifestation of asthma and indicates that the model building is successful.
3.2 proportion of macrophages, eosinophils and lymphocytes in BALF
As can be seen from FIG. 4, the percentage of eosinophils and lymphocytes in BALF is significantly higher than that of the blank control group (p < 0.01), indicating that the asthma is successfully established, the percentage of inflammatory cells, such as lymphocytes and eosinophils, is increased, and the total number of cells is obviously increased, indicating that the lung is undergoing an inflammatory reaction. The percentage of eosinophils and lymphocytes was significantly reduced in the glycan group and the positive drug group compared to the model group (p < 0.01). The cynomorium songaricum total polysaccharide has a relieving effect on pulmonary inflammation of asthma.
3.3 asthma mouse Lung tissue HE outcomes
The lung tissue HE staining results showed that the blank control mice showed clear bronchial and alveolar structure and no inflammation (FIG. 5-a). The mice in the model group showed inflammatory reaction compared with the blank control group, inflammatory cell infiltration was observed around the bronchi, monocytes were mainly used, eosinophils were observed, the structures of bronchi and alveoli were not clear, epithelial cells were exfoliated and necrotic, and exudates were present in the bronchial lumen (fig. 5-b). The polysaccharide group and the positive drug group both show reduced inflammation (fig. 5-c, d), which shows that the cynomorium songaricum total polysaccharide has a relieving effect on inflammation of asthma lung.
3.4 serum IgE detection results
As can be seen from FIG. 6, the serum IgE of the model group mice is abnormally increased and is obviously higher than that of the blank control group (P is less than 0.01), and the IgE levels of the mice of the positive drug group and the polysaccharide group are both obviously reduced (P is less than 0.05), which indicates that the cynomorium songaricum total polysaccharide has the function of relieving airway inflammation.
Fourth, conclusion of experiment
The successful establishment of an asthma model can be proved by integrating the morphological expression of the mice and the results of various pathological indexes, and the cynomorium songaricum total polysaccharide has a relieving effect on the inflammatory reaction of asthma.
Example 3: preparation of cynomorium songaricum total polysaccharide preparation
20mg of herba Cynomorii total polysaccharide extract solid, mixing with 50mg of starch and 50mg of dextrin, adding appropriate amount of 30% ethanol as humectant, making into soft mass, granulating by conventional method, adding magnesium stearate, mixing, and making into tablet.
The herba Cynomorii total polysaccharide extract solid is 20mg, and is mixed with common adjuvants such as dextrin to make into granule by conventional method, and then filled into hard capsule shell to make into capsule.
The herba Cynomorii total polysaccharide extract solid 20mg, sugar powder 20mg, and water 5mg by conventional method to obtain syrup, and adding starch 70mg to make into pill by conventional method.
20mg of cynomorium songaricum total polysaccharide extract solid, 70mg of starch, 10mg of dextrin and 10mg of powdered sugar are mixed, 30% ethanol is used as a wetting agent to prepare soft materials, and wet granulation is carried out to prepare granules.
The herba Cynomorii total polysaccharide extract solid is 20mg, and is mixed with starch 50mg and water 50mg to make into oral liquid by conventional method.
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