Application of tremella aurantialba, active substance for improving chronic obstructive pulmonary disease and preparation method and product of active substance

Abstract

The invention belongs to the technical field of microorganism extraction and application, and particularly relates to application of tremella aurantialba, an active substance for improving chronic obstructive pulmonary disease, a preparation method of the active substance and a product. The tremella auramtialba is obtained by culturing tremella auramtialba with the preservation number of CGMCC (China General Microbiological Culture Collection Center) 5.506 or tremella auramtialba with the preservation number of CCTCC HF (China Center for Type Culture Collection) 20081008. Wherein the tremella aurantialba comprises a tremella aurantialba extract, and the obtained tremella aurantialba extract can effectively and obviously relieve pathological changes of lung tissues, improve the levels of FVC, FEV0.1 and PEF of rats with chronic obstructive pulmonary diseases and obviously reduce the levels of inflammatory factors such as LTB4, IL-6 and NF-kB in serum; the protein expression of NF-kB and IL-6 can be reduced, and a new choice is provided for treating and improving COPD by natural products.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial extraction and application, and relates to an application of Tremella fuciformis, an active substance for improving chronic obstructive pulmonary disease, and a preparation method and product thereof. Background Art

[0002] There are a large number of smokers and passive smokers in my country. Exposure to cigarette smoke can lead to various chronic diseases, among which chronic obstructive pulmonary disease is the leading cause of death from smoking. Chronic obstructive pulmonary disease (COPD) is a disease characterized by respiratory symptoms and airflow limitation caused by a large amount of exposure to harmful particles or gases. my country is a high-incidence area for COPD. The prevalence of COPD in adults aged 20 and above is 8.6%, and that in people aged 40 and above is 13.7%, with about 100 million people suffering from the disease.

[0003] There is currently no safe, effective and low-toxic treatment for COPD, and there is an urgent need to find new therapeutic drugs. Existing COPD treatment drugs all improve certain aspects of COPD or target certain proteins and pathways, and none of them can significantly slow the progression of COPD. The complexity of COPD means that multi-target treatments are more needed. Natural products are the most widely used form of drug for treating various human diseases, and many countries have medicinal plants for the treatment of COPD. In view of the potential of natural products to treat COPD, more and more research is being conducted on the therapeutic effects of natural product active ingredients on COPD.

[0004] Golden ear [Naematelia aurantialba (Bandoni & M. Zang) Millanes & Wedin], commonly known as brain ear, golden wood ear, yellow and white white ear, etc., is a rare and precious edible and medicinal fungus with unique characteristics in my country. It belongs to the Tremellales, Naemateliaceae, and Naematelia Fr. Golden ear is very precious and is the best among the three ears (golden ear, white ear, and wood ear). The fruiting body is large and the whole is tough and colloid. The colloid is fine, refreshing and delicious, fragrant and beautiful. Traditional Chinese medicine believes that golden ear is warm and cold in nature, sweet in taste, can resolve phlegm, relieve cough, calm asthma, regulate qi, and calm liver yang. "Compendium of Materia Medica" records: "The golden color can cure the accumulation of drinking, abdominal pain and wounds", and has the effects of "tonifying the body and mind", "turning weakness into strength", and "prolonging life". It was a treasure and tribute in ancient times, along with ginseng and deer antler. There is no report that Tremella fuciformis can improve chronic obstructive pulmonary disease. Summary of the invention

[0005] To this end, the purpose of the present invention is to provide the use of a specific Tremella fuciformis strain in the preparation of an active substance for improving the symptoms of chronic obstructive pulmonary disease, so that it can significantly improve the physiological and biochemical indicators of chronic obstructive pulmonary disease;

[0006] Another object of the present invention is to provide the active substance and a preparation method thereof, as well as related products based on the active substance.

[0007] As a first aspect of the present invention, provided is the use of Tremella fuciformis in preparing an active substance for improving symptoms of chronic obstructive pulmonary disease, wherein the Tremella fuciformis comprises Tremella fuciformis with a preservation number of CGMCC5.506 and / or Tremella fuciformis with a preservation number of CCTCC HF20081008.

[0008] Optionally, the Tremella fuciformis comprises Tremella fuciformis fruiting body extract.

[0009] As a second aspect of the present invention, a method for preparing an active substance for improving the symptoms of chronic obstructive pulmonary disease is provided, comprising water extraction of Tremella fuciformis fruiting bodies, solid-liquid separation, and the obtained supernatant is the Tremella fuciformis water extract active substance for improving the symptoms of chronic obstructive pulmonary disease; the Tremella fuciformis comprises Tremella fuciformis with a preservation number of CGMCC5.506 and / or Tremella fuciformis with a preservation number of CCTCC HF20081008.

[0010] Optionally, the solid-liquid ratio of the Tremella fuciformis fruiting body and water is 1:(100-200).

[0011] Optionally, the water extraction adopts ultrasonic extraction; further optionally, the parameters of the ultrasonic extraction include ultrasonic temperature of 70-80°C, ultrasonic time of 2-3h, ultrasonic power of 30-50kHz, and ultrasonic power of 400-600W.

[0012] Optionally, the preparation method further comprises: removing protein from the Tremella fuciformis water extract and subjecting it to alcohol precipitation, and the obtained precipitate is Tremella fuciformis polysaccharide active substance that improves the symptoms of chronic obstructive pulmonary disease.

[0013] As a third aspect of the present invention, provided is an active substance for improving symptoms of chronic obstructive pulmonary disease prepared by the preparation method.

[0014] As a fourth aspect of the present invention, a product for improving symptoms of chronic obstructive pulmonary disease is provided, comprising the active substance of the present invention and excipients permitted by food and pharmaceuticals.

[0015] Optionally, the product further comprises other functional ingredients that do not interfere with the active substance of the present invention.

[0016] Compared with the prior art, the present invention has the following advantages: the present invention provides a new use of a specific Tremella fuciformis strain in the preparation of active substances for improving symptoms of chronic obstructive pulmonary disease and related products thereof. The present invention has studied and obtained that the Tremella fuciformis strain with a deposit number of CGMCC5.506 or the extract prepared from the Tremella fuciformis fruiting body obtained by culturing with a deposit number of CCTCC HF 20081008 has a good effect of improving chronic obstructive pulmonary disease. The experimental results show that the Tremella fuciformis fruiting body extract can effectively reduce the pathological changes of lung tissue, improve the FVC, FEV0.1, and PEF levels of rats with chronic obstructive pulmonary disease, and significantly reduce the levels of inflammatory factors such as LTB4, IL-6, and NF-kB in serum; it can reduce the protein expression of NF-kB and IL-6, providing a new idea for the treatment and improvement of chronic obstructive pulmonary disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the lung tissue morphology of the normal control group; Figure 2 This is the lung tissue morphology of the model control group; Figure 3 This is the lung tissue morphology of group 1 of Tremella fuciformis freeze-dried powder; Figure 4 This is the lung tissue morphology of the two groups of Tremella fuciformis freeze-dried powder; Figure 5 This is the lung tissue morphology of Tremella fuciformis polysaccharide freeze-dried powder group 1; Figure 6 This is the lung tissue morphology of the two groups treated with Tremella fuciformis polysaccharide freeze-dried powder. DETAILED DESCRIPTION

[0018] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0019] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0020] The present invention is further described below by way of specific examples. Where specific experimental steps or conditions are not specified in the examples, the operations or conditions of the conventional steps described in the literature in the art can be carried out. Where the manufacturers of all reagents are not specified, they can be obtained through conventional commercial channels. In each group of comparative experiments provided by the present invention, unless otherwise specified, other experimental conditions, materials, etc. are kept consistent, so as to be comparable.

[0021] Auricularia auricula 1 was purchased from China General Microbiological Culture Collection Center with the collection number of CGMCC No.5.506;

[0022] Auricularia auriculariae 2 was purchased from China Center for Type Culture Collection with the collection number of CCTCC HF 20081008.

[0023] Golden ear [Naematelia aurantialba (Bandoni & M. Zang) Millanes & Wedin], commonly known as brain ear, golden wood ear, yellow and white white ear, etc., is a rare and precious edible and medicinal fungus belonging to the order Tremellales, family Naemateliaceae, genus Naematelia Fr. Golden ear is very precious and is the best among the three ears (golden ear, white ear, wood ear). The fruiting body is large and the whole is tough and colloid. The colloid is fine, refreshing and delicious, fragrant and beautiful. "Compendium of Materia Medica" records: "The golden color can cure the accumulation of drinking, abdominal pain and wounds", and has the effects of "tonifying the body and mind", "turning weakness into strength" and "prolonging life". At present, golden ear is mostly used in cosmetics, as well as anti-oxidation and blood sugar improvement products. There is no report that golden ear can improve chronic obstructive pulmonary disease.

[0024] Therefore, in a first aspect of the present invention, there is provided a use of Tremella fuciformis in preparing an active substance for improving symptoms of chronic obstructive pulmonary disease, wherein the Tremella fuciformis comprises Tremella fuciformis with a deposit number of CGMCC5.506 and / or Tremella fuciformis with a deposit number of CCTCCHF 20081008.

[0025] In certain embodiments of the present invention, the Tremella fuciformis comprises Tremella fuciformis fruiting body extract, such as aqueous extract of Tremella fuciformis fruiting body, Tremella fuciformis polysaccharide and the like.

[0026] In a second aspect of the present invention, a method for preparing an active substance for improving symptoms of chronic obstructive pulmonary disease is provided, comprising:

[0027] After the Tremella fuciformis fruiting bodies are extracted with water, the solid and liquid are separated, and the obtained supernatant is the Tremella fuciformis water extract active substance for improving the symptoms of chronic obstructive pulmonary disease; the Tremella fuciformis includes Tremella fuciformis with a preservation number of CGMCC5.506 and / or Tremella fuciformis with a preservation number of CCTCC HF20081008.

[0028] In some embodiments of the present invention, the solid-liquid ratio of Tremella fuciformis fruiting bodies to water is 1:(100-200). In other embodiments of the present invention, when preparing Tremella fuciformis water extract active substances, the solid-liquid ratio of Tremella fuciformis fruiting bodies to water is 1:100.

[0029] In certain embodiments of the present invention, the water extraction adopts ultrasonic extraction, that is, the Tremella fuciformis fruiting body and water are mixed and then heated with the assistance of ultrasonic means; in other embodiments of the present invention, the parameters of the ultrasonic extraction include ultrasonic temperature of 70-80°C, ultrasonic time of 2-3h, ultrasonic power of 30-50kHz, and ultrasonic power of 400-600W.

[0030] In certain embodiments of the present invention, the Tremella fuciformis water extract obtained is freeze-dried to obtain Tremella fuciformis lyophilized powder.

[0031] In certain embodiments of the present invention, based on the Tremella fuciformis water extract, Tremella fuciformis polysaccharide active substance can be further prepared, including: removing protein from the Tremella fuciformis water extract and ethanol precipitation, and the obtained precipitate is Tremella fuciformis polysaccharide active substance for improving the symptoms of chronic obstructive pulmonary disease.

[0032] In certain embodiments of the present invention, the method for removing proteins adopts a chemical method to remove proteins, that is, chemical reagents are used to react with proteins to precipitate or denature them. Commonly used chemical reagents include organic solvents (such as acetonitrile, methanol), neutral salts (such as ammonium sulfate), acidic precipitants (such as trichloroacetic acid, perchloric acid), etc., which dehydrate proteins, form insoluble salts or directly precipitate them.

[0033] In certain embodiments of the present invention, after removing the protein, ethanol is added for alcohol precipitation to precipitate the polysaccharide. In other embodiments of the present invention, the obtained Tremella fuciformis polysaccharide can also be freeze-dried to obtain Tremella fuciformis polysaccharide freeze-dried powder.

[0034] In the third aspect of the present invention, an active substance for improving the symptoms of chronic obstructive pulmonary disease prepared by the preparation method is provided. The present invention conducts physiological and biochemical and animal model efficacy experiments on the prepared Tremella fuciformis water extract and freeze-dried powder of Tremella fuciformis polysaccharide, and the results show that Tremella fuciformis water extract and Tremella fuciformis polysaccharide can significantly improve the lung function FVC, FEV0.1, PEF levels, significantly reduce serum LTB4, NF-kB, IL-6, HSP60 levels, significantly reduce the area of ​​pulmonary tissue fibrosis, and significantly reduce the expression levels of NF-kB and IL-6 in lung tissue. The above experimental results fully demonstrate that Tremella fuciformis has significant efficacy in improving and treating the symptoms of chronic obstructive pulmonary disease.

[0035] In the fourth aspect of the present invention, a product for improving symptoms of chronic obstructive pulmonary disease is provided, comprising the active substance of the present invention and food and pharmaceutically acceptable excipients, so that the active ingredients can be prepared into any form of food or medicine, such as oral liquid, tablet, pill, granule, powder, paste, spray, injection, drop, etc. The food and pharmaceutically acceptable excipients include but are not limited to wetting agents, disintegrants, antioxidants, preservatives, flavors, thickeners, fillers, pigments, coatings / encapsulations, flavoring agents, stabilizers, pH regulators, etc., and the addition of these excipients is intended to ensure that the active substance presents the best active state and the best flavor and taste.

[0036] In some other embodiments of the present invention, the product also includes other functional ingredients that do not affect the active ingredients of the present invention, such as functional ingredients that improve blood sugar, blood lipids, and blood pressure, functional ingredients with antioxidant effects, functional ingredients that improve myocardial function, liver and kidney function, etc., and other functional ingredients that improve chronic obstructive pulmonary disease may also be added.

[0037] The following further describes the application of Tremella fuciformis provided in the present application, as well as the active substance for improving chronic obstructive pulmonary disease, and its preparation method and product.

[0038] Example 1 Cultivation of Auricularia auricularia

[0039] The cultivation of the golden ear fungus with a preservation number of CGMCC No.5.506 and the golden ear fungus with a preservation number of CCTCC HF 20081008 refers to Gao Yuxin. Research on key technologies for bagged cultivation of golden ear. [D] Northwest: Northwest Agriculture and Forestry University, 2021, and the golden ear fruiting body is cultivated by the cultivation method.

[0040] Specifically, cultivation: Cultivation method: culture medium formula: 30% cottonseed hulls, 56% sawdust, 12% wheat bran, 1% potassium dihydrogen phosphate, 1% calcium sulfate, and 60% water content. Make fungus bags according to the culture medium formula, and put 2.00 kg (wet weight) of evenly stirred culture medium into polypropylene fungus bags (15 cm×55 cm). Sterilize with high-temperature and high-pressure steam at 121 ℃ and 0.103MPa for 90 minutes before inoculation. When inoculating, a small amount of golden ear fruiting bodies and mycelium will be inoculated together to the surface of the matrix in the bag. Fungus conditions: temperature 20-25℃, dark conditions, relative air humidity 70-75%, generally after 20-30 days, the mycelium can fill the bag.

[0041] Management of ear emergence: After the mycelium grows to full physiological maturity, the surface of the substrate begins to differentiate into fruiting bodies. The outer protective bag of the long bag should be torn open and water should be sprayed regularly; the ring of the short bag should be untied to ensure ventilation. The temperature of the culture room should be controlled at 18-22℃, and the relative humidity of the air should be 85%-95%. The water in the bag should be removed in time to prevent contamination by foreign bacteria and "ear rot". Pay attention to indoor ventilation. When the fruiting body grows to a diameter of about 40 mm, increase the light intensity to 500-600 lx to promote the color change of the fruiting body.

[0042] Harvest: Golden ear fungus can be harvested after 50-70 days of fermentation. When the fruiting body is expanded into a brain shape, elastic, golden yellow or orange yellow, it should be harvested in time to obtain Golden ear fungus fruiting body 1 (obtained by culturing Golden ear fungus 1 with a preservation number of CGMCC No.5.506) and Golden ear fruiting body 2 (obtained by culturing Golden ear fungus 2 with a preservation number of CCTCC HF 20081008).

[0043] Example 2 Preparation of Tremella fuciformis freeze-dried powder

[0044] The Tremella fuciformis fruiting bodies 1 and 2 prepared in Example 1 were dried, 30 g of the dried Tremella fuciformis fruiting bodies were weighed, ground into powder and passed through an 80-mesh sieve, pure water was added at a ratio of 1:100 (w:v), ultrasonic extraction was performed continuously for 3 h at a temperature of 80°C, an ultrasonic frequency of 40 kHz, and an ultrasonic power of 500 W, and the mixture was centrifuged at 7000 r / min for 15 min in a high-speed centrifuge, and the supernatant was collected to obtain a Tremella fuciformis water extract.

[0045] The Tremella fuciformis water extract was freeze-dried, and the Tremella fuciformis water extract was first precooled at -40°C for 6 h. After precooling, it was placed in a vacuum freeze dryer and frozen for 12 h at a cold trap temperature of -45°C and a pressure of 30 Pa, and then crushed and sieved to obtain Tremella fuciformis freeze-dried powder 1 and Tremella fuciformis freeze-dried powder 2.

[0046] Example 3 Preparation of Tremella fuciformis polysaccharide freeze-dried powder

[0047] The golden ear fruiting bodies 1 and 2 prepared in Example 1 were dried, weighed, ground and sieved through an 80-mesh sieve, and pure water was added at a solid-liquid ratio of 1:200 (w / v). The mixture was ultrasonically treated at a power of 500 W, a frequency of 40 kHz, and a temperature of 70°C for 2 h. The mixture was filtered and the filtrate was collected to obtain a golden ear water extract.

[0048] 10% trichloroacetic acid was added to the Tremella fuciformis water extract at a ratio of 1:1 (v:v), and the extract was allowed to stand at 4°C for 12 h to remove protein. After standing, the extract was centrifuged at 8000 r / min for 10 min, and the supernatant was concentrated to 10 mL by rotary evaporation at 70°C, 40 mL of ethanol was added, the extract was allowed to stand at 4°C for 12 h, and centrifuged at 8000 r / min for 10 min to collect the precipitate to obtain Tremella fuciformis polysaccharide; the Tremella fuciformis polysaccharide precipitate was freeze-dried, and the freeze-dried Tremella fuciformis polysaccharide was crushed and sieved to obtain Tremella fuciformis polysaccharide freeze-dried powder 1 and Tremella fuciformis polysaccharide freeze-dried powder 2.

[0049] Experimental Example 1

[0050] 1. Experimental Methods

[0051] 1. Construction of animal model of chronic obstructive pulmonary disease

[0052] Animal source: healthy male SD rats, average weight 160±10g; provided by Hunan Slake Jingda Experimental Animal Co., Ltd., animal qualification certificate number: SCXK (Beijing) 2009-0007, in line with SPF grade conditions. 70 healthy male SD rats, 4-6 weeks old, average weight 160±10g, were selected and raised in cages in a breeding room with natural light and artificial light under the environment of 24±2℃ and relative humidity of 45%-65%. Animals were free to eat and drink water; adaptive feeding for one week. The experimental animals were randomly divided into normal control group, model control group, Tremella fuciformis lyophilized powder experimental group (Tremella fuciformis lyophilized powder group 1, Tremella fuciformis lyophilized powder group 2), Tremella fuciformis polysaccharide lyophilized powder experimental group (Tremella fuciformis lyophilized powder group 1, Tremella fuciformis polysaccharide lyophilized powder group 2), 10 rats in each group.

[0053] 2. Model Preparation

[0054] On the 1st and 15th days after the start of the experiment, rats in the model control group, Tremella fuciformis lyophilized powder experimental group, and Tremella fuciformis polysaccharide lyophilized powder experimental group were anesthetized with 10% chloral hydrate 4 ml / kg by intraperitoneal injection, and 0.2 ml of lipopolysaccharide (LPS, produced by Sigma, USA, product number L2880) aqueous solution containing 200 ug was slowly injected into the rats' airways, while 0.2 ml of normal saline was injected into the normal control group.

[0055] After the operation, the rats were returned to their original cages and fed and drank water normally. The model control group, the Tremella fuciformis lyophilized powder experimental group, and the Tremella fuciformis polysaccharide lyophilized powder experimental group were not smoked on the day of surgery (i.e., the 1st and 15th days). The rats were placed in a homemade smoking box (about 50*50*35cm in size, made of plexiglass, with a small door that can be opened in the center of the top, 4 small air holes around, and a smoking room for cigarettes at the lower right foot of the box. The smoke in the small room can directly enter the dyeing box through the hole) at other times. They were given passive smoking at 9:00-10:10 in the morning and 15:00-16:10 in the afternoon. Five cigarettes were given each time (Daqianmen brand cigarettes: (tar content 11mg / stick, smoke nicotine content 0.8mg / stick, smoke CO content 13mg / stick, Shanghai Tobacco Group Co., Ltd.)) for 0.5 hours. After 10 minutes, when the smoke was gone, the second passive smoking was given. After passive smoking was completed, the experimental animals were returned to their original cages and fed and watered normally. In the afternoon, passive smoking was continued for 120 days in the same way.

[0056] The normal control group was not fumigated during the experiment.

[0057] Model evaluation: Rats developed shortness of breath, frequent coughing, arched backs, sluggish reactions, slow movements, less activity, listlessness, messy hair, hair loss, and reduced appetite. Lung function decreased, and pathological sections of the lungs and trachea showed an increase in inflammatory cells and infiltration, incomplete or shedding bronchial mucosa, and dilation or fusion of alveoli, proving that the COPD model was successfully established.

[0058] 3. Dosage method:

[0059] The animals in the Tremella fuciformis lyophilized powder experimental group and the Tremella fuciformis polysaccharide lyophilized powder experimental group were gavaged once a day after modeling was completed, for a total of 28 days; the rats in the Tremella fuciformis lyophilized powder experimental group were gavaged with Tremella fuciformis lyophilized powder, and the rats in the Tremella fuciformis polysaccharide lyophilized powder group were gavaged with Tremella fuciformis polysaccharide lyophilized powder, once a day; the normal control group and the model group were gavaged with equal amounts of normal saline.

[0060] 4 Dosage

[0061] The proposed clinical dosage of Auricularia australis for adults is 30 g / d. The clinical equivalent dosage for rats calculated based on the body surface area conversion method is 30*0.018*1000 / 200=2.7 g·kg-1.

[0062] 5. Rat lung function test

[0063] One hour after the last administration, the animals were anesthetized with intraperitoneal injection of 1% sodium pentobarbital solution (50 mg kg-1), the trachea was surgically exposed, and a three-way cannula was inserted, one end of which was connected to a physiological recording machine. After the animals woke up, the forced vital capacity (FVC), forced expiratory volume in one second (FEV0.1), and peak expiratory flow (PEF) were measured.

[0064] 6 Specimen collection and preparation

[0065] After the pulmonary function test, blood was collected from the abdominal aorta and centrifuged (3000 r·min-1, 15 min), and the supernatant was stored at -80°C for later use. The chest cavity was opened surgically, and the lung tissue was completely removed. The right lung was frozen in liquid nitrogen for later use; the left lung was fixed in 4% paraformaldehyde solution for later use.

[0066] 7 Serum related factor detection

[0067] After thawing the frozen serum samples at -20℃ and 4℃, the kit was equilibrated to room temperature, and washing solution, standard working solution, biotinylated antibody working solution, and enzyme conjugate working solution were prepared. Samples were added, incubated, plates were washed, and changes in NF-kB, IL-6, HPS60, and LTB4 levels in serum were detected according to the instructions of the ELISA kit.

[0068] 8 Lung Histopathological Examination

[0069] Paraffin sections were made from the left part of the lung tissue. The dewaxed sections were placed on a staining rack, stained with hematoxylin, and separated with 1% hydrochloric acid. Washed with water, blued with warm water or a weakly alkaline aqueous solution, stained with eosin, dehydrated, and sealed. The morphological changes of lung tissue were observed under an optical microscope. The main manifestations of successful modeling of experimental animals are the infiltration of inflammatory cells around the bronchi, the presence or absence of exudates in the lumen, changes in alveolar wall interstitial inflammation and alveolar emphysema, etc. The degree of bronchial inflammatory infiltration: Observe the infiltration of all bronchial inflammatory cells in the sections, count the number of inflammatory cell-infiltrated bronchi and non-infiltrated bronchi, and make statistical comparisons.

[0070] 9 Immunofluorescence detection of NF-kB and IL-6 protein expression

[0071] Lung tissue sections were taken, permeabilized with 0.3% TtitonX100 for 5 min, blocked with 5% BSA for 30 min, and then incubated with VCAM1 (1:200) and CD31 (1:100) at 4°C overnight. After washing, they were incubated with FITC-conjugated goat anti-rabbit IgG (1:200) or Cy-3-conjugated rabbit anti-goat IgG (1:200) for 1 h at room temperature. After washing three times, the cell nuclei were stained with DAPI for 5 min. Fluorescence microscopy was used for observation and photography, and the average fluorescence intensity of positive staining was quantitatively analyzed by Image J software.

[0072] 10 Result calculation and statistics

[0073] SPSS17.0 was used for statistical analysis. The measurement data were expressed as mean ± standard deviation (X ± S). The t-test was used for comparison between the two groups when the data met the normality and variance homogeneity requirements. The Mann-Whitney Test was used when the data did not meet the normality or variance homogeneity requirements. P ≤ 0.05 indicated statistical significance, and P ≤ 0.01 indicated that the difference tested was very significant.

[0074] 2. Experimental Results

[0075] 1 The effects of each experimental group on the lung function of rats with chronic obstructive pulmonary disease model are shown in Table 1. Compared with the blank control group, the FVC, FEV0.1, and PEF levels of rats in the model control group were significantly reduced, and the difference was statistically significant (P < 0.01); compared with the model control group, the FVC, FEV0.1, and PEF levels of rats in the Tremella fuciformis freeze-dried powder group 1, Tremella fuciformis freeze-dried powder group 2, Tremella fuciformis freeze-dried powder group 1, and Tremella fuciformis freeze-dried powder group 2 were significantly increased, and the difference was statistically significant (P < 0.05), among which the Tremella fuciformis freeze-dried powder group 1 had the best effect.

[0076] Table 1 Effects on lung function in rats with chronic obstructive pulmonary disease

[0077]

[0078] Note: Different letters in the same column represent significant differences, p < 0.05

[0079] 2 The effects of each experimental group on serum LTB4, NF-kB, IL-6 and HSP60 in rats with chronic obstructive pulmonary disease are shown in Table 2. Compared with the blank control group, the serum LTB4, NF-kB and IL-6 levels of rats in the model control group were significantly increased, and the difference was statistically significant (P < 0.05 or P < 0.01); compared with the model control group, the serum LTB4, NF-kB and IL-6 levels of rats in the Tremella fuciformis freeze-dried powder group 1, Tremella fuciformis freeze-dried powder group 2, Tremella fuciformis freeze-dried powder group 1 and Tremella fuciformis freeze-dried powder group 2 were significantly reduced, and the difference was statistically significant (P < 0.05 or P < 0.01), among which the Tremella fuciformis freeze-dried powder group 1 had the best effect.

[0080] Table 2 Effects on serum-related factors in rats with chronic obstructive pulmonary disease

[0081]

[0082] Note: Different letters in the same column represent significant differences, p < 0.05

[0083] 3 Masson staining to observe the effect of Auricularia auriculariae powder on the lung tissue morphology of rats with chronic obstructive pulmonary disease (see Table 3 and Figure 1-6 Compared with the blank control group, the pulmonary fibrosis area of ​​the rats in the model control group increased significantly, and the difference was statistically significant (P < 0.05); compared with the model control group, the pulmonary fibrosis area of ​​the rats in the Tremella fuciformis freeze-dried powder 1 group, Tremella fuciformis freeze-dried powder 2 group, Tremella fuciformis polysaccharide freeze-dried powder 1 group, and Tremella fuciformis polysaccharide freeze-dried powder 2 group was significantly reduced, and the difference was statistically significant (P < 0.05), among which the Tremella fuciformis freeze-dried powder 1 group had the best effect.

[0084] Table 3 Masson staining to observe the effects of each group of experiments on the lung fiber area of ​​rats with chronic obstructive pulmonary disease

[0085]

[0086] Note: Different letters in the same column represent significant differences, p < 0.05

[0087] 4 The effects of each experimental group on the fluorescence intensity of NF-kB, IL-6, and HSP60 in the lung tissue of rats with chronic obstructive pulmonary disease model are shown in Table 4. Compared with the blank control group, the fluorescence intensity (protein expression) of NF-kB and IL-6 in the lung tissue of rats in the model control group was significantly increased, and the difference was statistically significant (P < 0.05); compared with the model control group, the fluorescence intensity of NF-kB and IL-6 in the lung tissue of rats in the Tremella fuciformis freeze-dried powder group 1, Tremella fuciformis freeze-dried powder group 2, Tremella fuciformis freeze-dried powder group 1, and Tremella fuciformis freeze-dried powder group 2 were significantly reduced, and the difference was statistically significant (P < 0.05 or P < 0.01), among which the Tremella fuciformis freeze-dried powder group 1 had the best effect.

[0088] Table 4 Effects on the expression of NF-kB, IL-6 and HSP60 proteins in lung tissue of rats with chronic obstructive pulmonary disease

[0089]

[0090] Note: Different letters in the same column represent significant differences, p < 0.05

[0091] COPD is a common disease characterized by progressive airflow limitation. Its pathogenesis is centered on airway inflammation, involving many factors such as oxidative stress, mitochondrial dysfunction, aging, and genetic polymorphism. Smoking is the main risk factor for COPD. Animal models are usually established by combining smoke exposure with airway instillation of lipopolysaccharide. This method is also the most commonly used animal model of COPD, which can more realistically simulate the occurrence and development of human COPD.

[0092] In this study, FVC and FEV0.1 were used to observe ventilation function, and PEF was used to investigate the degree of airflow limitation. This study found that the lung function indicators FVC, FEV0.1, and PEF levels in the model group were significantly lower than those in the blank control group; these results showed that the rat model of chronic obstructive pulmonary disease was successfully established. After the intervention of Tremella fuciformis, the pathological changes in lung tissue of the model animals were significantly alleviated, and the levels of FVC, FEV0.1, and PEF improved toward normal, revealing that Tremella fuciformis has a good therapeutic effect on chronic obstructive pulmonary disease. In recent years, many studies have found that the inflammatory cells in the lungs of chronic obstructive pulmonary disease are mainly macrophages and neutrophils, and the activated inflammatory cells release a variety of inflammatory mediators, including LTB4, IL-6, TNF-α, etc. These inflammatory mediators can destroy the structure of the lungs and promote neutrophil inflammatory response. The results of this study showed that Tremella fuciformis can significantly reduce the levels of inflammatory factors such as LTB4, IL-6, and NF-kB in serum; it can reduce the protein expression of NF-kB and IL-6, suggesting that the mechanism of action of Tremella fuciformis in treating chronic obstructive pulmonary disease is related to the inhibition of the NF-kB / IL-6 / MMP-1 signaling pathway.

[0093] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of Tremella fuciformis in preparing an active substance for improving symptoms of chronic obstructive pulmonary disease, wherein the Tremella fuciformis comprises Tremella fuciformis with a preservation number of CGMCC5.506 and / or Tremella fuciformis with a preservation number of CCTCC HF 20081008.

2. The use according to claim 1, characterized in that: The Tremella fuciformis comprises Tremella fuciformis fruiting body extract.

3. A method for preparing an active substance for improving symptoms of chronic obstructive pulmonary disease, characterized in that: include: After the Tremella fuciformis fruiting bodies are extracted with water, the solid and liquid are separated, and the obtained supernatant is the Tremella fuciformis water extract active substance for improving the symptoms of chronic obstructive pulmonary disease; the Tremella fuciformis includes Tremella fuciformis with a preservation number of CGMCC5.506 and / or Tremella fuciformis with a preservation number of CCTCC HF20081008.

4. The preparation method according to claim 3, characterized in that: The solid-liquid ratio of the Tremella fuciformis fruiting body to water is 1:(100-200).

5. The preparation method according to claim 3, characterized in that: The water extraction adopts ultrasonic extraction.

6. The preparation method according to claim 5, characterized in that: The parameters of the ultrasonic extraction include: ultrasonic temperature of 70-80°C, ultrasonic time of 2-3h, ultrasonic power of 30-50kHz, and ultrasonic power of 400-600W.

7. The preparation method according to any one of claims 3 to 6, characterized in that: Also includes: The Tremella fuciformis water extract is protein-free and alcohol-precipitated, and the obtained precipitate is Tremella fuciformis polysaccharide active substance which can improve the symptoms of chronic obstructive pulmonary disease.

8. An active substance for improving symptoms of chronic obstructive pulmonary disease prepared by the preparation method according to any one of claims 3 to 7.

9. A product for improving symptoms of chronic obstructive pulmonary disease, characterized in that: The invention comprises the active substance as claimed in claim 8 and excipients permitted by food and medicine.

10. The product according to claim 9, characterized in that It also includes other functional ingredients that do not affect the active ingredients described in claim 8.

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