Quanzhen Yiqi decoction polysaccharide for treating chronic obstructive pulmonary disease and its preparation method and application

Abstract

The present invention discloses a Quanzhen Yiqi Decoction polysaccharide for treating chronic obstructive pulmonary disease and a preparation method thereof. The polysaccharide is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 1.2: 0.95: 0.44: 2.15: 60.72: 32.64: 1.9. Experimental results show that the Quanzhen Yiqi Decoction polysaccharide provided by the present invention can reduce the total number of white blood cells and the expression levels of inflammatory factors IL-6 and TNF-α in BALF, reduce pathological structural damage of lung tissue, and alleviate the formation of emphysema. At the same time, it may reduce the expression of MUC5AC protein and mRNA in lung tissue by regulating the IL-13 / Jak1 / Stat6 signaling pathway, inhibit the hypersecretion of airway mucus in COPD, and improve the lung function of mice.

Description

Technical Field

[0001] The invention relates to a traditional Chinese medicine compound, in particular to a Quanzhen Yiqi decoction polysaccharide for treating chronic obstructive pulmonary disease and a preparation method thereof. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by persistent airflow limitation, which is usually not completely reversible and is progressive. COPD is often caused by inhalation of toxic gases or particles, infection, genetics, etc., with cigarette smoke being the main cause. Pathogenesis Cause. Cigarette smoke, infection and other factors can damage airway epithelial cells, causing them to secrete a large number of pro-inflammatory factors, which in turn cause the airway goblet cells and submucosal glands to synthesize and release a large amount of mucus, known as airway mucus hypersecretion, which can induce and aggravate airway obstruction. At the same time, the ciliary clearance ability is impaired, making it easy for pathogens to invade repeatedly, thereby aggravating the infection and forming a vicious cycle. The main clinical manifestations of airway mucus hypersecretion are chronic cough and sputum, which are considered to be important risk factors for the onset, clinical progression and prognosis of chronic airway inflammatory diseases including COPD.

[0003] There are many studies on the pathogenesis of COPD, and its core pathogenesis has not yet been fully elucidated. The most studied are inflammation, oxidative stress, and protease-antiprotease imbalance. These three factors influence and interact with each other throughout the entire process of COPD. Persistent inflammation, oxidative stress, and protease-antiprotease imbalance in the respiratory tract can cause mucosal cell / goblet cell hyperplasia and metaplasia, airway mucus hypersecretion, fibrosis, smooth muscle cell changes, and destruction of lung tissue. Airway damage and repair are repeated, forming scar tissue and eventually leading to airflow limitation and the development of COPD.

[0004] Quanzhen Yiqi Decoction was created by Feng Zhaozhang, a famous doctor in the Qing Dynasty. It is composed of seven herbs, namely, Rehmannia glutinosa, Atractylodes macrocephala, Radix Ophiopogonis, Codonopsis pilosula, Aconite root, Schisandra chinensis and Achyranthes bidentata. Aconite root can restore yang and rescue adverse conditions, and can guide fire back to the origin; Rehmannia glutinosa nourishes yin and tonifies the kidney, Ophiopogon japonicus nourishes yin and clears the lungs, and Atractylodes macrocephala can strengthen the spleen and eliminate dampness. The combination of these three herbs can achieve the function of tonifying the lungs, spleen and kidneys, and can also restrain the warm and dry nature of Aconite root; Codonopsis pilosula can greatly replenish the original qi, Achyranthes bidentata can guide fire downward, tonify the kidney and absorb qi, and Schisandra chinensis can restrain yin and produce body fluid, and restrain the qi of the five internal organs. The whole prescription can greatly replenish the true essence, nourish yin and extinguish fire. It is a prescription for strengthening the root and curing the root. It mainly treats yin anxiety, upper fullness and lower emptiness, yin exhaustion inside and yang excess outside, maculopapular fever, extreme irritability, upper wheezing and lower diarrhea, or fever due to yin deficiency in stroke, vomiting blood, coughing and wheezing. It is mostly used to treat all kinds of severe asthenia. At present, there are no reports on the research of Quanzhen Yiqi Decoction polysaccharide and its use in the treatment of chronic obstructive pulmonary disease. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to systematically study the chemical composition of Quanzhen Yiqi Decoction polysaccharides and confirm its efficacy in treating chronic obstructive pulmonary disease through animal pharmacology experiments.

[0006] Technical solution: In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A Quanzhen Yiqi Decoction polysaccharide for treating chronic obstructive pulmonary disease. The polysaccharide is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 1.2:0.95:0.44:2.15:60.72:32.64:1.9.

[0008] The method for preparing the Quanzhen Yiqi Decoction polysaccharide for treating chronic obstructive pulmonary disease provided by the present invention comprises the following steps:

[0009] Take the raw materials of Quanzhen Yiqi Decoction, Rehmannia root, Ophiopogon japonicus, Atractylodes macrocephala, Codonopsis pilosula, prepared Aconitum root, Achyranthes bidentata, Schisandra chinensis, grind them, add 5 to 20 times the volume of the total medicinal materials into water, boil and reflux for extraction 1 to 3 times, each time for 1 to 2 hours, combine the extracts, filter, and concentrate the filtrate under reduced pressure to obtain a clear paste, add 95% ethanol, stir evenly and let stand to precipitate, filter the precipitate, vacuum dry, and grind to obtain dark brown-yellow Quanzhen Yiqi Decoction crude polysaccharide; then use macroporous resin to decolorize and purify to obtain Quanzhen Yiqi Decoction polysaccharide.

[0010] As a preferred embodiment, the method for preparing the Quanzhen Yiqi Decoction polysaccharide for treating chronic obstructive pulmonary disease of the present invention comprises the following steps:

[0011] Take the raw materials of Quanzhen Yiqi Decoction, 30g of Rehmannia glutinosa, 10g of Ophiopogon japonicus, 10g of Atractylodes macrocephala, 10g of Codonopsis pilosula, 10g of prepared Aconitum, 10g of Achyranthes bidentata, and 6g of Schisandra chinensis, grind them, add 8-10 times the volume of the total medicinal materials in water, decoct and reflux for extraction 1-2 times, each time for 1-2 hours, combine the extracts, filter, and concentrate the filtrate under reduced pressure to obtain a clear paste, add 4-8 times the volume of 95% ethanol, stir evenly, and then let it stand for precipitation for 24-48 hours, filter the precipitate, vacuum dry it at 60°C, and grind it to obtain dark brown-yellow Quanzhen Yiqi Decoction crude polysaccharide; then use macroporous resin to decolorize and purify to obtain Quanzhen Yiqi Decoction polysaccharide.

[0012] As a more preferred embodiment, the method for preparing the above-mentioned Quanzhen Yiqi Decoction polysaccharide for treating chronic obstructive pulmonary disease comprises the following steps:

[0013] (1) Take the raw materials of Quanzhen Yiqi Decoction, including 30 g of Rehmannia glutinosa, 10 g of Ophiopogon japonicus, 10 g of Atractylodes macrocephala, 10 g of Codonopsis pilosula, 10 g of Aconite root, 10 g of Achyranthes bidentata, and 6 g of Schisandra chinensis, grind them, add 10 times the volume of water, boil and reflux for extraction twice, each time for 1 h, combine the two extracts, filter, and concentrate the filtrate under reduced pressure to a clear paste with a density of about 1.05 g / mL, add 4 times the volume of 95% ethanol, stir evenly, and let it stand for 24 h to precipitate, filter the precipitate by suction, and vacuum dry it at 60°C for 36 h, and grind it to obtain the Quanzhen Yiqi Decoction crude sugar.

[0014] (2) The crude polysaccharide of Quanzhen Yiqi Decoction prepared in step (1) was added with water to prepare a 20 mg / mL crude polysaccharide solution of Quanzhen Yiqi Decoction, and the pretreated HPD100 macroporous resin was added to adjust the pH value of the solution to 5.0. The solution was placed in a shaker at 125 r / min and 80°C for 4 h for decolorization, and then centrifuged at 3000-5000 r / min for 3-10 min. The supernatant sample was taken and concentrated to prepare the Quanzhen Yiqi Decoction polysaccharide.

[0015] The invention discloses an application of the Quanzhen Yiqi Decoction polysaccharide in the preparation of medicine for preventing and treating chronic obstructive pulmonary disease.

[0016] The invention can prepare the Quanzhen Yiqi Decoction polysaccharide and a pharmaceutically acceptable carrier into a medicine in the form of tablets, capsules, granules or pills.

[0017] When the tablet is prepared, carrier lactose or corn starch is added to the Quanzhen Yiqitang polysaccharide, and lubricant magnesium stearate is added when necessary, mixed evenly, and then compressed into tablets.

[0018] When preparing the capsule, the Quanzhen Yiqitang polysaccharide and the carrier lactose or corn starch are evenly mixed, granulated, and then filled into capsules to prepare the capsule.

[0019] When preparing the granules, the Quanzhen Yiqitang polysaccharide and the diluent lactose or corn starch are uniformly mixed, granulated, and dried to prepare the granules.

[0020] Beneficial effects: The Quanzhen Yiqitang polysaccharide provided by the present invention has the following advantages compared with the prior art:

[0021] 1. The present invention obtains high-purity Quanzhen Yiqitang polysaccharide through a large number of experimental screenings, and obtains its specific monosaccharide composition and molar ratio.

[0022] 2. The present invention uses cigarette smoke exposure combined with LPS intratracheal instillation to establish a COPD mouse model. The model mice can have high secretion of airway mucus, and the expression level of IL-13 / Jak1 / Stat6 signaling pathway molecules in the lung tissue of the model mice increases, which may be involved in the regulation of mucin MUC5AC expression in COPD. QZYQ-DT intervention can reduce the total number of white blood cells in BALF and the expression levels of inflammatory factors IL-6 and TNF-α, reduce pathological structural damage in lung tissue, and alleviate the formation of emphysema. At the same time, it may regulate the IL-13 / Jak1 / Stat6 signaling pathway, reduce the expression of MUC5AC protein and mRNA in lung tissue, inhibit high secretion of airway mucus in COPD, and improve the lung function of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The general chemical characterization results of QZYQ-DT. A is the UV spectrum of QZYQ-DT; B is the infrared spectrum of QZYQ-DT; C is the standard curve of dextran molecular weight standard; D is the molecular weight distribution of QZYQ-DT; E is the PMP-HPLC diagram of monosaccharide mixed standard; F is the HPGPC diagram of QZYQ-DT.

[0024] Figure 2 The figure shows the changes in mouse body weight.

[0025] Figure 3 Mouse weight after modeling. In the figure, CTL: blank control group; CS: model group; QZYQTDT(L): Quanzhen Yiqi Decoction polysaccharide low-dose group; QZYQTDT(M): Quanzhen Yiqi Decoction polysaccharide medium-dose group; QZYQTDT(H): Quanzhen Yiqi Decoction polysaccharide high-dose group; SCMC: carbocysteine ​​group. Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05.

[0026] Figure 4 HE staining results of lung tissue pathological sections (200×).

[0027] Figure 5 The figure shows the average intercept of alveoli in each group of mice. In the figure, CTL: blank control group; CS: model group; QZYQTDT(L): low-dose group of Quanzhen Yiqi Decoction polysaccharide; QZYQTDT(M): medium-dose group of Quanzhen Yiqi Decoction polysaccharide; QZYQTDT(H): high-dose group of Quanzhen Yiqi Decoction polysaccharide; SCMC: carbocysteine ​​group. Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05.

[0028] Figure 6 The results of PAS staining of lung tissue pathological sections (200×).

[0029] Figure 7 This is a graph showing the total number of white blood cells in BALF.

[0030] Figure 8 The graph shows the expression of IL-6 in BALF.

[0031] Fig. 9 is the expression of TNF-α in BALF. In the figure, CTL: blank control group; CS: model group; QZYQTDT(L): Quanzhen Yiqi Decoction polysaccharide low-dose group; QZYQTDT(M): Quanzhen Yiqi Decoction polysaccharide medium-dose group; QZYQTDT(H): Quanzhen Yiqi Decoction polysaccharide high-dose group; SCMC: carbocysteine ​​group. Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05.

[0032] Fig.10 is the relative expression level of MUC5AC in BALF.

[0033] Fig.11 is the relative expression of MUC5AC mRNA in lung tissue. In the figure, CTL: blank control group; CS: model group; QZYQTDT(L): low-dose group of Quanzhen Yiqi Decoction polysaccharide; QZYQTDT(M): medium-dose group of Quanzhen Yiqi Decoction polysaccharide; QZYQTDT(H): high-dose group of Quanzhen Yiqi Decoction polysaccharide; SCMC: carbocysteine ​​group. Compared with the blank control group,

[0034] *P<0.05, **P<0.01, ***P<0.001; #P<0.05 compared with the model group.

[0035] Fig.12 The figure shows the changes of protein expression in lung tissue.

[0036] Fig.13 This is the relative expression of P-Stat6 in lung tissue.

[0037] Fig.14 The relative expression of IL-13 mRNA in lung tissue. In the figure, CTL: blank control group; CS: model group; QZYQTDT(L): low-dose group of Quanzhen Yiqi Decoction polysaccharide; QZYQTDT(M): medium-dose group of Quanzhen Yiqi Decoction polysaccharide; QZYQTDT(H): high-dose group of Quanzhen Yiqi Decoction polysaccharide; SCMC: carbocysteine ​​group. Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05. DETAILED DESCRIPTION

[0038] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0039] Example 1 Study on the purification of polysaccharide from Quanzhen Yiqi Decoction using macroporous resin

[0040] The crude polysaccharide of Quanzhen Yiqitang obtained by water extraction and alcohol precipitation has a relatively complex composition, including several polysaccharide components and impurities such as miscellaneous proteins and pigments, and has poor appearance and color properties. In this regard, a refining and purification process should be used to treat the crude polysaccharide to obtain a refined polysaccharide product. The present invention investigates the decolorization process of the polysaccharide of Quanzhen Yiqitang using a macroporous resin.

[0041] 1. Experimental Materials

[0042] The preparation method of the crude polysaccharide extracted from Quanzhen Yiqi Decoction is as follows: 30g of Radix Rehmanniae Preparata, 10g of Radix Ophiopogonis, 10g of Atractylodes macrocephala, 10g of Codonopsis pilosula, 10g of Radix Aconiti Lateralis Preparata, 10g of Achyranthes bidentata, and 6g of Schisandra chinensis are taken. The Chinese medicinal pieces are weighed according to the proportion in the prescription, crushed, and 10 times the volume of water of the total weight of the medicinal materials are added each time, and the decoction is refluxed and extracted twice, each time for 1 hour, and the two extracts are combined, filtered through 4 layers of gauze, and the filtrate is decompressed and concentrated to a clear paste with a density of about 1.05g / mL, and 4 times the volume of 95% ethanol is added thereto, stirred evenly, and then allowed to stand for precipitation for 24 hours, the precipitate is filtered, and vacuum dried at 60°C for 36 hours, and crushed to obtain a dark brown-yellow Quanzhen Yiqi Decoction crude polysaccharide (QZYQ-CDT) powder, which is sealed and stored in a vacuum desiccator away from light.

[0043] D101, HPD100, HPD600, NKA-2 resins (Solaibao Biotechnology Co., Ltd.); X-5, H103, AB-8, HPD750 resins (Tianjin Nankai University Resin Co., Ltd.); sulfuric acid, anthrone (China National Pharmaceutical Group); other reagents were domestic analytical grade.

[0044] Sartorius CPA225D electronic balance (Sartorius Scientific Instrument Beijing Co., Ltd.); desktop refrigerated centrifuge XIR* (Thermo Fisher Scientific Inc.); TU-1810 UV-visible spectrophotometer (Beijing Puxi General Instrument Co., Ltd.); HY-45 gas bath constant temperature biological shaker (Jintan Jincheng Guosheng Experimental Instrument Factory, Jiangsu); HH-4 digital display constant temperature water bath (Guohua Electric Co., Ltd.); acidity meter (Shanghai Leici Instrument Factory); multifunctional microplate reader (Berteng Instrument Co., Ltd., USA); Synergy UV system ultrapure water instrument (Millopore, USA).

[0045] 2. Experimental methods and results

[0046] 2.1 Resin pretreatment

[0047] Take macroporous resin X-5, H103, D101, HPD100, AB-8, HPD750, HPD600, and NKA-2 and soak them in 95% ethanol for 24 hours to swell, then wash them with ethanol several times until the washing solution is 3 times the amount of water added to the test tube and there is no turbidity. Finally, wash with double distilled water until there is no alcohol smell and set aside.

[0048] 2.2 Resin screening

[0049] Accurately measure 5 mL of the pretreated 8 kinds of resins above into a 250 mL conical flask, add 40 mL of 10 mg / mL of the above-mentioned Quanzhen Yiqi Decoction crude polysaccharide solution, place it in a shaker, oscillate at 125 r / min and 25°C for 24 h, centrifuge at 3000 r / min for 3 min, take the supernatant and determine the optical density value OD621 of the decolorized polysaccharide and the optical density value OD450 of the pigment, calculate the retention rate of Quanzhen Yiqi Decoction polysaccharide and the removal rate of the pigment, and determine the optimal resin type.

[0050] The polysaccharide retention rate detection method is as follows: the supernatant of the crude polysaccharide of Quanzhen Yiqi Decoction after centrifugation is prepared into a polysaccharide solution of a certain concentration with distilled water, 2 mL of the polysaccharide solution is placed in a stoppered test tube, 5 mL of 2 mg / mL anthrone-sulfuric acid solution is added in an ice water bath, mixed, heated in a boiling water bath for 10 minutes, taken out and quickly placed in an ice water bath to cool for 20 minutes, and then placed at room temperature for 10 minutes. At 621 nm, the optical density value OD621 of the polysaccharide solution is measured using a multifunctional microplate reader to represent the polysaccharide content. The polysaccharide retention rate formula is as follows:

[0051]

[0052] The polysaccharide decolorization rate detection method is: the supernatant of the crude polysaccharide of Quanzhen Yiqi Decoction after centrifugation is prepared into a polysaccharide solution of a certain concentration with distilled water, and scanned in the wavelength range of 400-700nm. The results show that the crude polysaccharide solution of Quanzhen Yiqi Decoction has no maximum absorption wavelength. Because the polysaccharide solution is orange-yellow before and after decolorization, according to the principle of complementary colors, the solution mainly absorbs visible light in the blue band, so 450n is selected as the detection wavelength. At 450nm, the optical density value OD450 is measured by a multifunctional microplate reader to indicate the content of the pigment. The polysaccharide decolorization rate formula is shown in the following formula:

[0053]

[0054] By comparing the decolorization rate and polysaccharide retention rate of 8 macroporous resins for Quanzhen Yiqi Tang polysaccharides (Table 1), the decolorization rates of X-5, AB-8, and NKA-2, which are cross-linked polystyrene, are relatively low, and the polysaccharide retention rate is relatively low, indicating that this type of resin has too strong adsorption capacity for Quanzhen Yiqi Tang polysaccharides and weak adsorption capacity for pigments, so this type of resin is not suitable for the decolorization of Quanzhen Yiqi Tang polysaccharides. The HPD series macroporous resins have a higher decolorization rate and a higher polysaccharide retention rate, which is probably because the particle size range, specific surface area, and average pore size of this type of resin are relatively similar. The above parameters are more suitable for adsorbing pigments and retaining polysaccharides. The polar HPD100 in this series has the highest decolorization rate and polysaccharide retention rate, so HPD100 is selected as the decolorization macroporous resin for Quanzhen Yiqi Tang polysaccharides.

[0055] Table 1 Comparison of decolorization ability of different macroporous resins on Quanzhen Yiqi Decoction polysaccharide

[0056]

[0057] 2.3 Single-factor experiment

[0058] Accurately measure 5 mL of pretreated macroporous resin, add 50 mL of a certain concentration of Quanzhen Yiqitang polysaccharide (QZYQ-CDT) solution to it, adjust the pH of the solution to a fixed value, place it in a shaker, keep decolorization at 125 r / min and a certain temperature for several hours, centrifuge at 3000 r / min for 3 minutes, take the supernatant and measure the optical density value OD of the decolorized polysaccharide 621脱色后 and the optical density of the pigment OD 450脱色后 , calculate the retention rate of Quanzhen Yiqi Decoction polysaccharides and the removal rate of pigments.

[0059] The effect of sample concentration on the decolorization effect of the macroporous resin of Quanzhen Yiqitang polysaccharide was investigated. It was found that the decolorization rate decreased with the increase of sample concentration, and the polysaccharide retention rate showed an increasing trend. When the sample concentration was greater than 15 mg / mL, the decolorization rate changed slightly, so the sample concentration of HPD100 resin used in the orthogonal experiment was set to 15, 20, and 25 mg / mL. The effect of pH on the decolorization effect of Quanzhen Yiqitang polysaccharide was investigated. It was found that the decolorization effect of HPD100 resin was best at pH 3.5-6.5, at which time the decolorization rate was the highest and the polysaccharide retention rate was also the highest, so the pH of HPD100 resin used in the orthogonal experiment was set to 4, 5, and 6. The effect of decolorization temperature on the decolorization effect of the macroporous resin of Quanzhen Yiqitang polysaccharide showed that the decolorization effect of HPD100 resin was the best at 70°C, with the highest decolorization rate and the highest polysaccharide retention rate. The decolorization rate and polysaccharide retention rate at 60 and 80°C could reach more than 73%, so the temperature of HPD100 resin for orthogonal experiments was set at 60, 70 and 80°C. The effect of decolorization time on the decolorization effect of Quanzhen Yiqitang polysaccharide showed that the decolorization effect of HPD100 resin was significantly enhanced with the extension of time, while the polysaccharide retention rate was slowly decreasing. After 8h, the decolorization rate decreased, and the pigment desorption may be greater than the adsorption at this time. Therefore, the decolorization time of HPD100 resin for orthogonal experiments was selected as 4, 6, and 8h.

[0060] 2.4 Orthogonal experiment

[0061] Based on the single factor experiment, the orthogonal experiment method was used to determine the optimal decolorization process of Quanzhen Yiqi Decoction polysaccharide. 4 )Orthogonal array design experiment, see Table 2.

[0062] The decolorization effect evaluation adopts the comprehensive weighted scoring method, with the weight coefficient being 0.5. The largest index in the test item is set as 100 points, and the other indicators are weighted and comprehensively scored according to the following formula:

[0063]

[0064] Table 2 Factor levels of orthogonal experiment of HPD100 for polysaccharide decolorization

[0065]

[0066] Table 3 Results and analysis of orthogonal test on the decolorization effect of HPD100 on the polysaccharide extract of Quanzhen Yiqi Decoction

[0067] Test No. A B C D Decolorization rate (%) Polysaccharide retention rate (%) score 1 1 1 1 1 86.58 66.55 89.99 2 1 2 2 2 84.32 81.32 97.56 3 1 3 3 3 81.09 87.93 95.41 4 2 1 2 3 84.12 73.38 88.84 5 2 2 3 1 86.01 61.50 83.14 6 2 3 1 2 82.30 84.68 94.24 7 3 1 3 2 86.73 37.22 69.74 8 3 2 1 3 79.04 82.28 91.05 9 3 3 2 1 82.40 81.39 92.43 K1 91.813 87.840 93.597 90.267 K2 87.547 94.823 87.780 96.670 K3 94.167 90.863 92.150 86.590 R 6.620 6.983 5.817 10.080

[0068] All data are the average values ​​of 3 parallel experiments, and the experimental data are expressed in the form of mean ± standard deviation (Mean ± SD), and variance analysis is performed using SPSS software. From the variance analysis table 4, it can be seen that the priority order of the four factors is D>B>A>C, and the optimal process conditions are sample concentration>sample pH>decolorization temperature>decolorization time. The optimal process conditions are A3B2C1D2, that is, the temperature is 80°C, the sample pH is 5, the decolorization time is 4h, and the sample concentration is 20mg / mL. Under these conditions, the polysaccharide decolorization rate was measured to be 80.25%, and the polysaccharide retention rate was 76.94%.

[0069] Table 4 Analysis of variance of the decolorization effect of HPD100 on the polysaccharide extract of Quanzhen Yiqi Decoction

[0070] Sources of variance Sum of squared deviations Degree of freedom F-number F(0.10) critical value Significance A 67.567 2.000 0.767 4.460 B 73.589 2.000 0.836 4.460 C 55.023 2.000 0.625 4.460 D 156.127 2.000 1.773 4.460 error 352.31 8 .

[0071] The present invention uses 8 kinds of macroporous resins, namely X-5, H103, D101, HPD100, AB-8, HPD750, HPD100 and NKA-2, to purify and decolorize the crude polysaccharide of Quanzhen Yiqi Decoction. The screening test results show that HPD100 has better comprehensive decolorization effect than the other 7 kinds of macroporous resins. Therefore, HPD100 is selected as the decolorizer of the crude polysaccharide of Quanzhen Yiqi Decoction.

[0072] HPD100 macroporous resin adsorption was used as the adsorption purification method for crude polysaccharides from Quanzhen Yiqi Decoction. Single factor optimization was performed on sample concentration, sample pH, decolorization temperature, and decolorization time. Three levels were selected for orthogonal experiments. The results showed that the optimal decolorization process parameters for crude polysaccharides from Quanzhen Yiqi Decoction were: sample concentration 20 mg / mL, sample pH 5.0, decolorization temperature 80℃, and decolorization time 4h. At this time, the measured polysaccharide decolorization rate reached 80.25%, and the polysaccharide retention rate was 76.94%. After being treated with HPD100 resin, the crude polysaccharides from Quanzhen Yiqi Decoction achieved a satisfactory decolorization effect and a high polysaccharide retention rate, and the color changed from dark brown to light yellow.

[0073] Example 2 Preparation of Quanzhen Yiqi Decoction Polysaccharide

[0074] (1) Prescription of Quanzhen Yiqi Decoction: 30g of Radix Rehmanniae Preparata, 10g of Radix Ophiopogonis, 10g of Atractylodes Macrocephalae, 10g of Codonopsis Pilosulae, 10g of Radix Aconiti Lateralis Preparata, 10g of Radix Aconiti Lateralis, and 6g of Fructus Schisandrae Chinensis. According to the proportion in the prescription, the Chinese medicinal pieces were weighed and crushed, and 10 times the volume of water of the total weight of the medicinal materials was added each time. The decoction was refluxed and extracted twice, each time for 1 hour. The two extracts were combined and filtered through 4 layers of gauze. The filtrate was concentrated under reduced pressure to a clear paste with a density of about 1.05g / mL (the volume conversion was equivalent to 2 times the weight of the raw medicinal pieces), and 4 times the volume of 95% ethanol was added to it. After stirring evenly, it was allowed to settle for 24 hours. The precipitate was filtered and vacuum dried at 60°C for 36 hours. The dark brown-yellow Quanzhen Yiqi Decoction crude polysaccharide powder (QZYQ-CDT) was crushed and stored in a sealed vacuum desiccator away from light.

[0075] (2) The crude polysaccharide of Quanzhen Yiqi Decoction prepared in step (1) was added with water to prepare a crude polysaccharide solution of Quanzhen Yiqi Decoction with a concentration of 20 mg / mL, and the pretreated HPD100 macroporous resin was added to adjust the pH value of the solution to 5.0. The solution was placed in a shaker at 125 r / min and 80°C for 4 h for decolorization, and then centrifuged at 3000 r / min for 3 min. The supernatant sample was taken and concentrated to prepare Quanzhen Yiqi Decoction polysaccharide (QZYQ-DT).

[0076] Example 2 Structural Identification of Polysaccharides

[0077] 1. Experimental Materials

[0078] Raw materials of Radix Rehmanniae Preparata, Radix Ophiopogonis, Atractylodes Macrocephalae, Codonopsis Pilosulae, Radix Aconiti Lateralis, Radix Achyranthis Bidentatae, and Fructus Schisandrae Chinensis were purchased from the National Medical Hall Clinic of Nanjing University of Chinese Medicine; glucose (Glc), rhamnose (Rha), arabinose (Ara), galactose (Gal), xylose (Xyl), mannose (Man), glucuronic acid (GlcA), and galacturonic acid (GalA) were purchased from Shanghai Yuanye Co., Ltd.; dextran molecular weight standard materials Mw of 3.05 KDa, 10.2 KDa, and 63.3 KDa were purchased from China National Institute of Metrology; dextran T-10 and dextran T-40 were purchased from Beijing Solebold Technology Co., Ltd.; chromatographic grade acetonitrile and methanol were purchased from Tedia. Other commercially available chemicals and reagents were of analytical grade.

[0079] 2. Experimental methods

[0080] (1) General chemical analysis of QZYQ-DT

[0081] The total sugar content of QZYQ-DT samples was determined by the phenol-sulfuric acid method with glucose as the standard; the impurity protein content of QZYQ-DT samples was detected by the BCA method (Blanford method) with standard bovine serum albumin as the reference; the uronic acid content of QZYQ-DT samples was determined by the m-hydroxybiphenyl method with galacturonic acid as the standard; the reducing sugar content was determined by the DNS method with glucose as the standard.

[0082] (2) UV and FITC spectral characterization of QZYQ-DT

[0083] The QZYQ-DT solution (10ug / mL) was tested for protein and nucleic acid content by full wavelength scanning UV spectrophotometer in the wavelength range of 190-350nm. QZYQ-DT was analyzed by infrared tableting. Take an appropriate amount of KBr powder in an agate mortar and grind it under an infrared lamp. After grinding until completely dry, add an appropriate amount of QZYQ-DT sample and continue grinding to make QZYQ-DT evenly distributed in it. Increase the grinding time to ensure that the water is completely removed, add an appropriate amount of completely dried QZYQ-DT to the mold, spread it evenly, and the tablet press machine presses the tablets into transparent and uniform thin slices. The infrared spectrum is scanned by Fourier transform infrared spectrometer, and the scanning range is set to 4000cm -1 -400cm -1 .

[0084] (3) Molecular weight distribution of components in QZYQ-DT

[0085] High performance gel permeation chromatography (HPGPC) was used to detect the molecular weight distribution of the components in QZYQ-DT. 10.00 mg of QZYQ-DT dried polysaccharide sample was accurately weighed, dissolved in ultrapure water, and prepared into a sugar solution with a concentration of 2 mg / mL. After filtering with an aqueous microporous filter membrane, it was measured on the machine. HPGPC analysis conditions: injection volume of 20 μL, mobile phase of 0.09 mM ammonium acetate, flow rate of 0.3 mL / min, chromatographic column of OHpak-SH-803 (5 μm, 4.6 mm × 250 mm), column temperature of 30 ° C, detector of evaporative light scattering detector (ELSD), drift tube temperature of 116 ° C, gas flow rate of 4.0 L / min. At the same time, standard dextran reference substances of equal concentrations of 3000, 10000, 123000, 400000, and 633000 were injected as references for molecular weight detection.

[0086] (4) Analysis of monosaccharide composition of QZYQ-DT

[0087] The monosaccharide composition of QZYQ-DT was analyzed by PMP pre-column derivatization. 10 mg of QZYQ-DT powder was accurately weighed, 2.0 mL of 3 mol / L trifluoroacetic acid solution was added and ultrasonically dissolved, transferred to an ampoule, sealed, hydrolyzed at 105 °C for 6 h, cooled to room temperature, centrifuged (3000 r / min, 10 min), the supernatant was evaporated under reduced pressure, dissolved in methanol and evaporated 3 times, and the residue was dissolved in ultrapure water and fixed to 1 mL to prepare the hydrolyzed sample test solution. An appropriate amount of polysaccharide hydrolysis solution was taken for monosaccharide PMP derivatization reaction. After sufficient reaction, the unreacted PMP reagent was extracted with chloroform, and the aqueous phase was fixed to volume and injected into HPLC to analyze the monosaccharide composition. HPLC conditions: mobile phase A is pH 6.7 0.1 mol / L potassium dihydrogen phosphate buffer, mobile phase B is acetonitrile; flow rate is 1.0 mL / min; column temperature is 30 °C; detection wavelength is 250 nm. The gradient elution method was as follows: 0-3 min, 13% (B); 3-8 min, 13%-16 (B); 8-15 min, 16%-23% (B); 15-30 min, 23% (B).

[0088] 3. Results and Discussion

[0089] The total sugar content of the QZYQ-DT sample was about 96.78%, the protein content was about 0.69%, the reducing sugar content was 37.31%, and the uronic acid content was about 9.23%; Figure 1 -A shows a small peak at 190-350nm, indicating that the crude QZYQ-DT may still contain trace amounts of protein and nucleic acid components; the FT-IR of the QZYQ-DT sample ( Figure 1 -B) shows 3408cm -1 、2967cm -1 The characteristic peak of polysaccharide indicates its macromolecular polysaccharide nature; 1623cm -1 , indicating that QZYQ-DT contains a small amount of uronic acid; the molecular weight distribution of QZYQ-DT samples was characterized by HPLC and it was found that the molecular weight distribution of polysaccharides was relatively wide ( Figure 1 -D), the molecular weight range is from less than 3.0kDa to 100.0kDa macromolecules, and the peak concentration in the 3-10kDa molecular weight component is the main component of the QZYQDT sample; the liquid phase analysis of the monosaccharide composition of the QZYQ-DT sample using the PMP derivatization method found that its polysaccharide is composed of mannose Man, rhamnose Rha, glucuronic acid GlcA, galacturonic acid GalA, glucose Glc, galactose Gal and arabinose Ara, and the molar percentage is 1.2:0.95:0.44:2.15:60.72:32.64:1.9.

[0090] Example 3 Drug efficacy experiment

[0091] 1. Experimental Materials

[0092] 1. Experimental animals

[0093] 63 SPF-grade male C57BL / 6J mice, aged 4 - 6 weeks and weighing 18 - 22 g, were purchased from the Guangdong Provincial Medical Experimental Animal Center. The license number is SCXK(Guangdong)2018 - 0002. They were housed in the Experimental Animal Center of Guangzhou Medical University and allowed free access to food and water for 1 week to adapt to the environment.

[0094] 2. Experimental drugs

[0095] The polysaccharide of Quanzhen Yiqi Decoction (QZYQ - DT) was prepared according to Example 1.

[0096] 2.2 Carbocisteine tablets: Purchased from the General Pharmaceutical Factory of Guangzhou Baiyunshan Pharmaceutical Holdings Co., Ltd., with a specification of 250 mg per tablet.

[0097] 2.3 Coconumber brand cigarettes: Purchased from China Tobacco Guangdong Industrial Co., Ltd., with a tar content of 11 mg, nicotine content of 1 mg, and carbon monoxide content of 12 mg per cigarette.

[0098] 3. Reagents and consumables

[0099]

[0100]

[0101]

[0102] 4. Main instruments

[0103]

[0104]

[0105] 5. Solution preparation

[0106] 5.1 Tissue lysate

[0107] RIPA 1ml PMSF 10μl Cocktail 2 10μl Cocktail 3 10μl

[0108] 5.2 WB electrophoresis buffer

[0109] Tris Base 3.03g Glycine 14.42g SDS 1g Ultrapure water Make up to 1000ml

[0110] 5.3 WB transfer buffer

[0111]

[0112]

[0113] 5.4 TBST

[0114] TBS powder 1 pack Tween-20 2ml Ultrapure water Make up to 2000ml

[0115] 5.5 WB blocking solution and secondary antibody dilution solution (5% w / v milk)

[0116] Skim milk powder 1g TBST 20ml

[0117] 5.6 WB primary antibody diluent (5% w / v BSA containing 0.02% NaN3)

[0118] BSA 1g TBST 20ml <![CDATA[NaN3]]> 0.004g

[0119] 5.7 10% SDS polyacrylamide gel (separation gel) × 2 pieces

[0120] 30% acrylamide solution 5.32ml Ultrapure water 6.36ml Tris.HCl (pH 8.8) 4ml 10% SDS 160μl 10% AP 160μl TEMED 16μl

[0121] 5.8 5% SDS polyacrylamide gel (concentrating gel) × 2 pieces

[0122] 30% acrylamide solution 0.99ml Ultrapure water 4.2ml Tris.HCl (pH 6.8) 750μl 10% SDS 60μl 10% AP 60μl TEMED 6μl

[0123] 5.9 Red blood cell lysis buffer

[0124]

[0125]

[0126] 5.10 ELISA Washing Buffer

[0127] PBS powder 1 pack Tween-20 2ml Ultrapure water Make up to 2000ml

[0128] 5.11 ELISA antibody diluent

[0129] PBS 100ml Triton X-100 300μl Tween-80 200 μl BSA 1g Thimerosal 0.02g

[0130] 5.12 ELISA sample diluent

[0131] <![CDATA[NaHCO3]]> 1.4001g <![CDATA[Na2CO3]]> 1.7665g Ultrapure water 200ml

[0132] 5.13 ELISA blocking solution

[0133] PBS 100ml BSA 1g sucrose 1g Sodium Azide 0.02g

[0134] 2. Experimental Methods

[0135] 1. Grouping and feeding of experimental animals

[0136] SPF-grade C57BL / 6J male mice, 4-6 weeks old, weighing 18-22 g, were randomly divided into 6 groups, namely blank control group, model group, QZYQ-DT low-dose group, QZYQ-DT medium-dose group, QZYQ-DT high-dose group and carbocysteine ​​group. They were raised in the Experimental Animal Center of Guangzhou Medical University and had free access to food and water for 1 week to adapt to the environment, and drinking water, feed and bedding were replaced regularly.

[0137] 2. COPD model preparation method

[0138] The stable COPD mouse model was established by cigarette smoke exposure combined with LPS intratracheal instillation. The size of the homemade fumigation box was 50cm×60cm×90cm, with a 1.5cm×1.5cm round hole on each side to communicate with the outside world, and connected to the exhaust system to exhaust cigarette smoke. At the same time, the computer PAB-S200 passive smoking animal exposure system was used to detect the changes in humidity, temperature, gas (CO, SO2, CO2), etc. in the fumigation box. The blank control group was placed in an SPF environment and fed with food and water freely; the modeling group was instilled with LPS (50μg / 50μl / mouse) in the airway with the help of a laryngoscope on the 1st and 14th day from the beginning of modeling, and no fumigation was performed on the same day. The rest were placed in a homemade fumigation box every day for passive smoking. The cigarette treatment was 2 times / d, once in the morning and afternoon, each time for 2h (15-20min ventilation every 1h), with at least 3h interval between the morning and afternoon, 10 cigarettes / h, 6d / week, and the modeling time was 12 weeks in total. Eight weeks after modeling, the corresponding drugs were given: the blank control group and the model group were gavaged with normal saline, 0.25 ml / d; the QZYQ-DT low-dose group was gavaged with 0.5 times the clinical dose of Quanzhen Yiqi Decoction (0.68 g / kg), 0.25 ml / d; the QZYQ-DT medium-dose group was gavaged with 1 times the clinical dose of Quanzhen Yiqi Decoction (1.36 g / kg), 0.25 ml / d; the QZYQ-DT high-dose group was gavaged with 2 times the clinical dose of Quanzhen Yiqi Decoction (2.72 g / kg), 0.25 ml / d; the carbocysteine ​​group was gavaged with 1 times the clinical dose of carbocysteine ​​aqueous solution (225 mg / kg), 0.25 ml / d, for a total of 4 weeks. The process is as follows:

[0139]

[0140] 3. Specimen Collection

[0141] 3.1 The mice were weighed after the last smoke treatment, anesthetized with avertin, fixed on a surgical board, incised along the trachea of ​​the neck and intubated, and placed on a Buxco small animal pulmonary function meter to collect relevant pulmonary function data.

[0142] 3.2 Prepare 6 1ml syringes for each mouse and draw 0.6ml of pre-cooled saline in advance, inject 0.6ml saline into the trachea, lavage the bilateral lung lobes (or ligate the left lung and lavage the right lung), recover after 15s, place the recovered BALF in a 2ml centrifuge tube and place it on ice, operate continuously for 3 times, the recovery rate is preferably above 80%, mark it as tube 1, take another 2ml centrifuge tube, repeat the above operation, and mark it as tube 2. Centrifuge after collection (1000 rpm, 5min), take the supernatant of tube 1 and divide it into 600μl centrifuge tubes, and store it at -80℃ for subsequent experiments; discard the supernatant of tube 2, add 500μl red blood cell lysis buffer, gently blow and resuspend, add it to tube 1, gently blow and resuspend again, and let it stand for 5min. Centrifuge tube 1 (1000 rpm, 5 min), discard the supernatant, add 1 ml of saline (the blank control group can only add 500 μl because of the small number of cells), gently blow to mix, take 10 μl and place it on the cell counting plate for cell counting (20 μl machine counting). After counting, take an appropriate amount (at least 200 μl, if the volume is insufficient, use saline to make up, containing about 10 cells 5 The cells were evenly spun (1000 rpm, 5 min) onto a glass slide. After the cells on the glass slide were naturally dried, they were fixed with methanol for 1 min (gently to prevent the cells from falling off), and then left at room temperature for the methanol to evaporate.

[0143] 3.3 After taking BALF, cut open the skin and muscle layer of the mouse abdomen, gently cut the diaphragm to expose the heart, insert a 1ml syringe into the right ventricle to collect about 500-800μl of blood, place it in a 1.5ml anticoagulant centrifuge tube containing EDTA, centrifuge (1000 turns, 5min), draw the supernatant light yellow plasma into a new 1.5ml centrifuge tube, and store it at -80℃ for later use. Continue to cut open the chest skin and muscle layer, expose the lungs, cut off the left atrial appendage, inject physiological saline from the right ventricle until the lung color becomes light and bloodless, ligate the right hilum, cut off the right lung according to the upper, middle, lower, and round lobes, and place them in different 1.5ml centrifuge tubes, and store them at -80℃ for later use. Infuse 10% paraformaldehyde fixative from the airway to the left lung, ligate the left hilum and cut off the left lung, place it in a 4ml centrifuge tube containing 10% paraformaldehyde, and then dehydrate, embed, slice, etc.

[0144] 4. Lung Tissue Protein Extraction and Concentration Determination

[0145] 4.1 Lung tissue protein extraction: The lung tissue frozen at -80℃ was taken out and weighed. 1 ml of tissue lysis buffer was added for every 100 mg of lung tissue. The tissue was homogenized using a pre-cooled mechanical homogenizer (shake = 20 s, hold = 1 min, temp = 4℃, speed = 6.0 m / s, cycle = 4). The tissue was then centrifuged for 30 min (4℃, 12000 g). The supernatant was carefully aspirated and placed in a 1.5 ml centrifuge tube for long-term storage in a -80℃ refrigerator.

[0146] 4.2 Determination of protein concentration by BCA method

[0147] ① Prepare the BSA standard included in the product according to the instructions, dilute the sample with water or other buffer that does not interfere with the color development reaction, so that the concentration of the sample to be tested is in the linear part of the standard curve. Prepare 3 parallel determinations for each reaction, and the standard curve is generally 5-6. Determine the specific concentration of each point according to the concentration of the standard. Water can be used to dilute BSA, and it is best to use a solution that is consistent or similar to the sample. For example, add 0, 2.5, 5, 10, 15, 20, 25μl of the standard (500μg / ml) in sequence, and balance it with ultrapure water to a total volume of 25μl. First add 23.75μl / well of ultrapure water to the position of the sample well to be tested, and then add 1.25μl of the sample to be tested in sequence, which is equivalent to diluting the sample to be tested 20 times. Finally, multiply the calculated sample concentration by 20 to obtain the actual sample protein concentration.

[0148] ② Prepare BCA working solution and use the following formula to determine the total volume of working solution required: (number of standards + number of protein samples to be tested) × number of experimental repetitions × volume of working solution required for each sample = total volume of working solution required.

[0149] ③ Add 200 μl of working solution (solution A: solution B = 50:1) to each reaction well, cover the well and mix for 30 seconds, and react at 37°C for 30 minutes.

[0150] ④ Cool to room temperature and measure the absorbance of each reaction well at a wavelength of 562nm. Note that the OD value should be determined within 1 hour.

[0151] ⑤ Subtract the absorbance value of the blank control well (i.e., the "0" well) from the absorbance value of each standard or sample to be tested, draw a standard curve, and calculate the protein concentration of the sample to be tested according to the standard curve.

[0152] ⑥ Pipette an appropriate amount of sample protein and prepare it with tissue lysis buffer to make a protein sample of equal concentration and volume according to the concentration of 2.5μg / μl, add the corresponding volume of 5× loading buffer, shake and centrifuge it slightly, and then boil it in boiling water for 10 minutes to denature the protein. After cooling, store it at -80℃ for a long time or store it at -20℃ after aliquoting for later use.

[0153] 5. Western Blotting (WB)

[0154] ① Electrophoresis: Thaw the protein sample in advance, take an appropriate amount of supernatant protein (about 20-50μg) after shaking and centrifugation, and add it to the concave groove of the concentrated gel. Perform electrophoresis at 80V constant voltage for about 30min until bromophenol blue is compressed into a straight line, then change to 120V constant voltage electrophoresis for 1h. When bromophenol blue reaches the bottom of the gel, the electrophoresis ends.

[0155] ②Electroporation: Activate the PVDF membrane with methanol for 30 seconds in advance, place the membrane and gel into a sandwich electroporation chuck in a certain order to avoid bubbles, and electroporate at a constant current of 300 mA for 2 hours, keeping the whole process in an ice bath.

[0156] ③ Blocking: Place the transferred membrane in an incubation box containing 5% milk blocking solution and incubate on a shaker at room temperature for 1 hour. After incubation, wash it 3 times with TBST, 5 minutes each time, and wash it thoroughly on a shaker.

[0157] ④ Primary antibody incubation: After adding the corresponding primary antibody, slowly shake it on a shaker and incubate it overnight at 4°C. The next day, recover the primary antibody and wash it three times with TBST, each time for 10 minutes, and wash it thoroughly on a shaker.

[0158] ⑤ Secondary antibody incubation: After adding the corresponding secondary antibody, slowly shake it on a shaker, incubate it at room temperature for 1 hour, recover the secondary antibody, wash it 3 times with TBST, 10 minutes each time, and wash it thoroughly on a shaker.

[0159] ⑥Luminescence development: Mix ECL chemiluminescent solution A and solution B in a ratio of 1:1, drop an appropriate amount of the prepared luminescent solution on the membrane, spread it evenly, wait for a while, then put it into the darkroom of the Tanon 5200 chemiluminescence imaging system for exposure, save the developed image on the computer, and use professional analysis tools for semi-quantitative analysis.

[0160] 6. Extraction of total RNA from lung tissue and reverse transcription PCR

[0161] 6.1 Total RNA extraction using TRIzol

[0162] ① Take an appropriate amount of lung tissue and place it in a homogenizer tube. Add 1 ml of TRIzol for every 100 mg of lung tissue and set the homogenizer parameters (shake = 20 s, hold = 1 min, temp = 4 °C, speed = 6.0 m / s, cycle = 4) for homogenization.

[0163] ② Collect the supernatant and place it in a 1.5ml centrifuge tube. Add 200μl of chloroform and shake up and down for 15s to mix thoroughly or form an emulsion. Incubate at room temperature for 5min.

[0164] ③ Centrifuge for 15 minutes (12000g, 4°C), then carefully take out the centrifuge tube and place it on ice to avoid shaking.

[0165] ④ Slowly aspirate the upper clear liquid, avoiding the middle white protein layer, and place it in a new centrifuge tube.

[0166] ⑤ Add 500 μl of isopropanol (to precipitate RNA) into the tube, mix well, incubate at room temperature for 10 min, and centrifuge for 10 min (12000 g, 4°C).

[0167] ⑥Discard the supernatant, add 1 ml of 75% ethanol (prepared with DEPC water and anhydrous ethanol) and centrifuge for 5 min (7500 g, 4°C).

[0168] ⑦ Pour off the upper layer of ethanol, fix the centrifuge tube upside down (be careful not to pour out the RNA), and wait until the ethanol evaporates completely (wait about 30 minutes. If there is a lot of residual alcohol on the wall of the tube, use a pipette to absorb it dry, but avoid sucking up the RNA precipitate).

[0169] ⑧Add 30μl DEPC water to dissolve RNA and store it at -80℃ for a long time to avoid repeated freezing and thawing.

[0170] 6.2 Reverse transcription PCR reaction

[0171] ① Measure the RNA concentration (ng / μl) at a wavelength of 260nm. 1000ng (1μg) RNA is required for reverse transcription. Xμl is calculated and then balanced with DEPC water.

[0172] ②Operate on ice and prepare the reaction mixture. The formula is shown in the table below. To ensure the accuracy of the preparation, the Master Mix should be prepared according to the standard of reaction number + 2, then dispensed into each reaction tube, and finally the RNA sample was added. Put it on the machine, gradient PCR instrument, 42℃ 2min, 4℃, take it out and put it on ice.

[0173] Reagents Usage 5×gDNA Eraser Buffer 2μl gDNA Eraser 1μl Total RNA Xμl <![CDATA[Rnase Free dH2O]]> 7-Xμl Total: 10μl

[0174] Note: X is the volume calculated based on RNA concentration and sample loading amount

[0175] ③Operate on ice and prepare the reaction mixture. The formula is shown in the table below. To ensure the accuracy of the preparation, the Master Mix should be prepared according to the standard of reaction number + 2, and then dispensed into each reaction tube. After brief centrifugation and mixing, put it on the machine, gradient PCR instrument, 37℃15min, 85℃5s, 4℃.

[0176] Reagents Usage 5×PrimeScript Buffer 2(for real time) 4μl PrimeScript RT Enzyme Mix 1μl RT Primer Mix 1μl ②Reaction solution 10μl <![CDATA[Rnase Free dH2O]]> 4μl Total: 20 μl

[0177] 6.3qPCR reaction

[0178] ①Take out the sample and place it on ice. Take out the primers of IL-13, MUC5AC target gene and 18S internal reference gene from the -20℃ refrigerator and thaw them. The primer sequences are as follows:

[0179]

[0180] ② Prepare eight tubes, with three replicate wells for each sample;

[0181] ③ Prepare the PCR reaction system on ice according to the following components:

[0182]

[0183]

[0184] After the sample is prepared, centrifuge briefly and amplify using a Bio Rad real-time fluorescence PCR instrument. The reaction conditions are:

[0185]

[0186] 18S was used as the internal reference gene, 2 -ΔΔCt The relative expression levels of IL-13 and MUC5AC mRNA were calculated by the method.

[0187] 7. Analysis of lung tissue pathomorphology

[0188] 7.1 Hematoxylin-eosin (HE) staining

[0189] ① Bake at 60℃ for 20min;

[0190] ② Dewaxing to water: xylene I 10 min → xylene II 10 min → anhydrous ethanol I 5 min → anhydrous ethanol II 5 min → 95% alcohol 3 min → 85% alcohol 2 min → 75% alcohol 1 min;

[0191] ③ Rinse in distilled water for 5 minutes;

[0192] ④Hematoxylin staining for 3 minutes;

[0193] ⑤ Rinse in distilled water for 2 minutes;

[0194] ⑥ Soak in 1% hydrochloric acid ethanol for 1-2 seconds;

[0195] ⑦ Rinse in distilled water for 5 minutes;

[0196] ⑧ 0.6% ammonia water to blue or lithium carbonate to blue for 2 minutes (this step can be omitted), rinse in distilled water for 2 minutes;

[0197] ⑨Eosin staining for 10 seconds;

[0198] ⑩ Rinse under distilled water and observe the color change after flushing several times;

[0199] Dehydration and transparency: 75% alcohol for 5 min → 85% alcohol for 5 min → 95% alcohol for 5 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → xylene I for 5 min → xylene II for 5 min;

[0200] Allow to dry and seal with neutral gum.

[0201] Note: HE staining results in blue nuclei, and different degrees of red in cytoplasm, muscle fibers, collagen fibers, and red blood cells. Use a microscope to control the quality of hematoxylin staining of the nucleus. HE staining should be bright red, with distinct red and blue colors and clear contrast. After staining, use a clean cover glass and neutral gum with moderate viscosity to seal the slide. There should be no bubbles in the cover and no overflow of glue. The sealed slides should be labeled, placed in a slice box, and stored at room temperature. Use a professional slice scanner to scan the results of HE staining and save the data.

[0202] 7.2 Calculation of the mean alveolar intercept (MLI)

[0203] After HE staining, the lung tissue pathological sections were scanned by a scanning machine. The mean linear intercept (MLI) was used to represent the size of the alveolar cavity diameter. Five fields of view were randomly selected under a 20x microscope. A cross was drawn in the middle of each field of view. The number of alveolar septa N intersecting with the cross was calculated. The total length of the cross was measured. The mean alveolar intercept was calculated according to the formula MLI = L / N. The average value was taken to represent the mean alveolar cavity diameter (unit: μm); the number of alveoli was represented by radial alveolar counts (RAC). The counting method was as follows: a vertical line was drawn from the center of the respiratory bronchioles to the distal pleura under a 20x microscope. The number of alveoli on this line was the RAC, and the average value was taken to represent the number of alveoli.

[0204] 7.3 Periodic acid-Schiff (PAS) staining

[0205] ① Bake at 60℃ for 20min;

[0206] ② Dewaxing to water: xylene I 10 min → xylene II 10 min → anhydrous ethanol I 5 min → anhydrous ethanol II 5 min → 95% alcohol 3 min → 85% alcohol 2 min → 75% alcohol 1 min;

[0207] ③ Rinse in distilled water for 5 minutes;

[0208] ④ Oxidize with periodic acid solution for 10 minutes, rinse with distilled water for 3 minutes, and shake off excess water;

[0209] ⑤Stain with Schiff's reagent for 15 minutes, rinse with distilled water for 3 minutes, and shake off excess water;

[0210] ⑥Restain with hematoxylin for 2 minutes, rinse with distilled water for 3 minutes, and examine under a microscope;

[0211] ⑦ Dehydration and transparency: 75% alcohol 5min→85% alcohol 5min→95% alcohol 5min→anhydrous ethanol I 5min→anhydrous ethanol II 5min→xylene I 5min→xylene II 5min;

[0212] ⑧ Allow to dry and seal the slides with neutral gum.

[0213] Note: The result of PAS staining is that glycogen and neutral mucus substances are red, and the nucleus is blue. Take out the Schiff reagent half an hour before use and restore it to room temperature. It cannot be used when the solution appears light red. The time of Schiff reagent staining depends on the room temperature. In summer, the room temperature is high, and 10 minutes is enough. In winter, the room temperature is low, and it can be extended to about 20 minutes. It is recommended that the opened staining solution within the validity period be used within 3 months after opening. The bottle cap should be tightened in time after each use to avoid volatilization or deterioration.

[0214] 8. White blood cell differential count in BALF

[0215] 8.1 Diff Fast Staining

[0216] ① Prepare methanol-fixed BALF sections and stain with solution A for 1 min;

[0217] ② Wash away solution A with phosphate solution and stain with solution B for 1 minute;

[0218] ③ Wash away solution B with distilled water, examine under a microscope, and flexibly adjust the staining time;

[0219] ④ Dry and seal with neutral gum;

[0220] 8.2 White blood cell differential count

[0221] Classification was performed under an optical microscope, and 200 cells were counted (5 fields of view were selected under a 40x microscope). The percentage of macrophages, lymphocytes, neutrophils, etc. was calculated, and the actual number of each type of cell was calculated based on the total number of cells.

[0222] 9. Enzyme-linked immunosorbent assay (ELISA)

[0223] 9.1 Determination of IL-6, TNF-α, and IL-13 in BALF

[0224] Prepare 1× coating solution, diluent, capture antibody, detection antibody, standard substance, and HRP-labeled avidin according to the kit instructions. The assay steps are as follows:

[0225] ① Use a 96-well plate dedicated to ELISA, add 1× capture antibody 100 μl / well, seal with sealing film to reduce volatility, and place at 4°C overnight;

[0226] ② Pour off the coating solution, add at least 250 μl / well of washing solution, soak for 1 min and then pour off, absorb the remaining liquid with absorbent paper, and repeat this step 3 times;

[0227] ③ Add 200 μl / well of diluent, seal, and incubate at room temperature for 1 hour;

[0228] ④ Pour away the diluent and add 250 μl of washing solution to wash at least once;

[0229] ⑤Add standard products as required to make standard curve;

[0230] ⑥ Add 100 μl / well of the sample to be tested and 100 μl of diluent to the blank well, seal and incubate at room temperature for 1 hour or at 4°C overnight;

[0231] ⑦ Repeat step ②, wash 3-5 times, add 100 μl / well of detection antibody, seal and incubate at room temperature for 1 hour;

[0232] ⑧Repeat step ②, wash 3-5 times, add 100 μl / well of HRP-labeled avidin, seal and incubate at room temperature for 30 min;

[0233] ⑨Repeat step ②, wash 5-7 times, add 100 μl / well of TMB solution, seal and incubate at room temperature for 15 min;

[0234] ⑩ Add 50 μl / well of stop solution, measure the absorbance at 450 nm with an enzyme-labeled instrument, and calculate the concentration of the sample to be tested based on the standard curve.

[0235] 9.2 Determination of MUC5AC in BALF

[0236] Prepare sample diluent, antibody diluent and blocking solution in advance. According to the preliminary experimental results, dilute the sample at 1:400, the primary antibody at 1:500, and the secondary antibody at 1:1000.

[0237] ①Use a 96-well plate dedicated to ELISA, add 100 μl / well of the diluted sample, seal with sealing film to reduce volatility, and place at 4°C overnight;

[0238] ② Pour out the sample, add at least 250 μl / well of washing solution, soak for 1 min and then pour out, absorb the remaining liquid with absorbent paper, and repeat this step 3 times;

[0239] ③ Add 200 μl / well of blocking solution, seal, and incubate at room temperature for 1 hour;

[0240] ④ Repeat step ②, wash 4 times, add primary antibody 100 μl / well, seal and incubate at room temperature for 2 h;

[0241] ⑤ Repeat step ②, wash 4 times, add 100 μl / well of secondary antibody, seal and incubate at room temperature for 1 hour;

[0242] ⑥ Repeat step ②, wash 5 times, add 100 μl / well of TMB solution, seal and incubate at room temperature for 15-20 min;

[0243] ⑦ Add 50μl / well of stop solution to terminate the reaction. The reaction can be terminated early depending on the color depth. Measure the absorbance at 450nm with an enzyme reader.

[0244] 3. Statistical Processing

[0245] GraphPad Prism 5.0 software was used for drawing. The measured data that conformed to the normal distribution were expressed as Mean±SD. All the results were statistically analyzed using SPSS16.0 software. The t-test was used to compare the means of two independent samples, and the one-way ANOVA was used to compare the means of multiple groups of samples. The LSD-t-test was used for further pairwise comparisons between groups. P<0.05 was considered statistically significant.

[0246] IV. Experimental Results

[0247] 1. General Condition of Mice

[0248] Before modeling, all mice were randomly assigned to groups, and there was no statistical difference in the body weight of mice in each group (P>0.05); during the modeling period, there was no death in the blank control group, the mice grew well, had shiny fur, flexible movements, quick reactions, normal food and water intake, and the weight increase showed a trend of first fast and then slow; one mouse died in the model group, QZYQ-DT low-dose, medium-dose, and high-dose groups, and two mice died in the carbocysteine ​​group. The modeling mice had dull fur, liked to pull their hair, were slow to move, slow to react, listless, and had less food and water intake. From the third week, their body weight decreased significantly, which was significantly different from the blank control group (P<0.001), and then their body weight increased slowly; after the modeling, the body weight values ​​of mice in each group were higher than those in the model group. Compared with the model group, the weight gain of the high-dose polysaccharide group was the most obvious (P<0.05), and there was no statistical difference between the low-dose and medium-dose QZYQ-DT groups and the carbocysteine ​​group (P>0.05).

[0249] Table 5 Body weight changes of mice in each group during modeling ( Weight unit / g)

[0250] Group N Day 1 Week 3 Week 6 Week 9 Week 12 Blank control group 10 23.0±0.8 26.8±1.3 29.2±0.6 29.9±1.2 30.8±1.3 Model Group 10 23.3±1.6 <![CDATA[22.7±1.9 *** ]]> <![CDATA[23.1±2.0 *** ]]> <![CDATA[23.5±1.8 *** ]]> <![CDATA[25.1±1.7 *** ]]> Low dose group 9 23.3±1.3 22.5±1.3 23.3±1.0 23.7±1.4 25.4±1.3 Medium dose group 10 23.2±1.5 22.2±1.3 23.2±1.4 23.6±1.5 25.7±1.4 High dose group 10 23.8±1.1 22.9±1.1 24.0±1.1 24.2±1.3 <![CDATA[26.7±1.7 # ]]> Carbocisteine ​​group 8 23.7±0.9 22.8±1.0 23.8±1.1 24.1±1.5 26.3±1.7

[0251] Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05.

[0252] 2. Changes in liver, spleen and kidney indexes of mice

[0253] The mice were fixed on the operating table, the abdominal cavity was opened, and the spleen, liver and right kidney of the mice were removed respectively. After the residual blood was absorbed with filter paper, they were weighed (g) and divided by the weight of the mice (g) to obtain the spleen index, liver index and kidney spleen index, that is, spleen index = spleen weight (g) / mouse weight (g); liver index = liver weight (g) / mouse weight (g); kidney index = kidney weight (g) / mouse weight (g). The liver index of the model group and each drug-treated group was higher than that of the blank control group, and the difference was statistically significant (P<0.05). There was no statistical difference in the kidney index and spleen index among the groups (P>0.05), indicating that the COPD mouse model established by cigarette smoke exposure combined with LPS airway instillation may have certain liver damage, which needs to be considered in combination with liver pathology, blood biochemical indicators such as aspartate aminotransferase and alanine aminotransferase. The reasons need further study. The data are shown in Table 6.

[0254] Table 6 Organ index of mice in each group

[0255]

[0256]

[0257] Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05.

[0258] 3. Quanzhen Yiqi Decoction polysaccharide delays the decline of lung function in COPD model mice

[0259] Compared with the blank control group, the lung function parameters FRC, TLC, RI, and Cchord of the mice in the model group were significantly increased (P<0.05), and FEV 50 / FVC and Cdyn were significantly decreased (P<0.05). Compared with the model group, RI in each drug-treated group decreased, FEV 50 / FVC increased slightly, but the difference was not statistically significant (P>0.05). TLC in each medication group decreased significantly, and the difference was statistically significant (P<0.05). FRC in the high-dose QZYQ-DT group decreased significantly, and Cdyn increased significantly, and the differences were statistically significant (P<0.05).

[0260] Table 7 Lung function parameters FRC, TLC, FEV of mice in each group50 Comparison of FVC

[0261] Group FRC(ml) TLC (ml) <![CDATA[FEV 50 / FVC(%)]]> Blank control group 0.5507±0.0852 1.4807±0.1621 0.6387±0.1652 Model Group <![CDATA[0.6601±0.0303 ** ]]> <![CDATA[2.0085±0.1383 *** ]]> <![CDATA[0.4355±0.0555 ** ]]> QZYQ-DT low dose group 0.6274±0.0205 <![CDATA[1.8446±0.1336 # ]]> <![CDATA[0.5280±0.0927 * ]]> QZYQ-DT medium dose group 0.6077±0.0529 <![CDATA[1.8406±0.0876 # ]]> <![CDATA[0.5314±0.1299 * ]]> QZYQ-DT high dose group <![CDATA[0.5836±0.0735 # ]]> <![CDATA[1.8262±0.1256 # ]]> 0.5498±0.0643 Carbocisteine ​​group 0.6232±0.0464 <![CDATA[1.8414±0.1195 # ]]> <![CDATA[0.4784±0.0633 * ]]>

[0262] Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05

[0263] Table 8 Comparison of lung function parameters RI, Cchord, Cdyn in each group of mice

[0264]

[0265]

[0266] Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05

[0267] 4. Quanzhen Yiqi Decoction polysaccharide alleviates emphysema in COPD model mice

[0268] HE staining of mouse lung tissue showed that the blank group had normal alveolar structure, no compensatory emphysema, and no inflammatory cell infiltration in the alveolar cavity; the model group had enlarged alveolar cavity, thinned alveolar septa, broken and fused to form bullae, compensatory emphysema, local inflammatory cell infiltration, and increased average alveolar spacing. Compared with the blank control group, the average alveolar spacing in the model group increased from 41.20±4.70μm to 80.01±8.38μm, with a statistically significant difference (P<0.05), which was a typical pathological change of emphysema. Compared with the model group, the emphysema of each drug group was improved, with a small amount of inflammatory cell infiltration, and the average alveolar spacing was reduced, which was most obvious in the high-dose polysaccharide group, with an average alveolar spacing of 56.88±4.92μm, with a statistically significant difference (P<0.05), indicating that QZYQ-DT can improve the pathological changes of emphysema in COPD mice, reduce the infiltration of local inflammatory cells, and thus protect lung function. N=5. One-way analysis of variance was used to compare the means of multiple groups of samples, and LSD-t test was used for pairwise comparison. α=0.05 was considered statistically significant.

[0269] Table 9 Average intercept of alveoli in each group of mice

[0270] Group Average alveolar intercept (μm) Blank control group 41.20±4.70 Model Group <![CDATA[80.01±8.38 *** ]]> Polysaccharide low dose group <![CDATA[63.95±5.66 ## ]]> Polysaccharide medium dose group <![CDATA[60.19±9.61 ### ]]> Polysaccharide high dose group <![CDATA[56.88±4.92 ### ]]> Carbocisteine ​​group <![CDATA[64.77±7.33 ## ]]>

[0271] Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001

[0272] 5. PAS staining of lung tissue

[0273] like Figure 5 PAS staining of mouse lung tissue showed no obvious positive results in the airways, and there was no significant difference between the groups. The possible reason may be that the lung tissue structure of mice is quite different from that of humans. At the same time, mucus is mainly secreted by proximal bronchial goblet cells and submucosal glands and concentrated in the large airways. However, the pathological sections in this study were mostly of the distal airways, with fewer mucus-secreting cells.

[0274] 6. Quanzhen Yiqi Decoction polysaccharide reduces lung inflammation in COPD model mice

[0275] Compared with the blank control group, the total white blood cell count in the bronchoalveolar lavage fluid of mice in the model group was significantly increased, and the difference was statistically significant (P<0.05). The cytokines IL-6 and TNF-α were significantly increased, and the difference was statistically significant (P<0.001). The white blood cell count, IL-6 and TNF-α in each drug-treated group were lower than those in the model group, among which the high-dose polysaccharide had the most obvious decrease, and the difference was statistically significant (P<0.01). The white blood cell count and TNF-α in the low-dose polysaccharide group were lower than those in the model group, but there was no statistical significance (P>0.05).

[0276] Table 10 BALF inflammation levels of mice in each group

[0277] Group <![CDATA[Total white blood cell count (10 5 cells / ml)]]> IL-6 (pg / ml) TNF-α(pg / ml) Blank Group 1.89±1.23 1.323±0.895 5.726±4.143 Model Group <![CDATA[11.72±3.20 * ]]> <![CDATA[10.178±7.087 *** ]]> <![CDATA[18.810±3.870 *** ]]> Polysaccharide low dose group 8.88±2.49 <![CDATA[1.633±1.161 ### ]]> 16.490±6.259 Polysaccharide medium dose group <![CDATA[7.78±2.59 # ]]> <![CDATA[1.552±1.233 ### ]]> 15.548±7.524 Polysaccharide high dose group <![CDATA[7.29±2.49 ## ]]> <![CDATA[1.399±1.381 ### ]]> <![CDATA[6.901±3.722 ### ]]> Carbocisteine ​​group <![CDATA[8.16±4.32 # ]]> <![CDATA[2.001±0.920 ### ]]> <![CDATA[6.263±4.286 ### ]]>

[0278] Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001

[0279] 7. Quanzhen Yiqi Decoction polysaccharide inhibits airway mucus hypersecretion in COPD model mice

[0280] ELISA results showed that compared with the blank control group, the content of mucin MUC5AC in BALF of the model group was significantly increased (P<0.001). Compared with the model group, the expression of MUC5AC in each drug-treated group was decreased, and the differences were statistically significant (P<0.05), among which the carbocysteine ​​group decreased most significantly (P<0.001), and the high-dose group decreased most significantly among all the dose groups of QZYQ-DT (P<0.001).

[0281] qPCR results showed that compared with the blank control group, the mRNA expression level of mucin MUC5AC in the lung tissue of the model group was significantly increased, but the difference was not statistically significant (P>0.05); compared with the model group, the mRNA expression level of MUC5AC in each drug-treated group was decreased, but the difference was not statistically significant (P>0.05), among which the QZYQ-DT group showed the most obvious decrease at a high dose.

[0282] 8. Effect of Quanzhen Yiqi Decoction Polysaccharide on IL-13 / Jak1 / Stat6 Signaling Pathway in COPD Model Mice

[0283] qPCR results showed that compared with the blank control group, the mRNA expression level of IL-13 in the model group was significantly increased, but the difference was not statistically significant (P>0.05); compared with the model group, the mRNA expression level of IL-13 in each drug-treated group was decreased, but the difference was not statistically significant (P>0.05); the low, medium and high dose groups of QZYQ-DT were all significantly decreased compared with the carbocysteine ​​group, among which the low dose group had the most obvious decrease.

[0284] WB results showed that compared with the blank control group, the expression level of P-Stat6 protein in the model group was significantly increased, and the difference was statistically significant (P<0.05). The expression of P-Stat6 protein could be significantly reduced after drug treatment (P<0.05), and the reduction was most obvious in the high-dose group of QZYQ-DT group. There was no significant difference in the expression levels of Jak1 and Satat6 proteins among the groups (P>0.05).

[0285] The above implementation modes are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A Quanzhen Yiqi Decoction polysaccharide for treating chronic obstructive pulmonary disease, characterized in that: The polysaccharide is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 1.2:0.95:0.44:2.15:60.72:32.64:1.9; The polysaccharide is prepared by the following method: (1) Take the raw materials of Quanzhen Yiqi Decoction, 30 g of Rehmannia glutinosa, 10 g of Radix Ophiopogonis, 10 g of Atractylodes macrocephala, 10 g of Codonopsis pilosula, 10 g of Radix Aconiti Lateralis Preparata, 10 g of Achyranthes bidentata, and 6 g of Schisandra chinensis, grind them, add 10 times the volume of the total medicinal materials into water, decoct and reflux for extraction twice, each time for 1 h, combine the two extracts, filter, and concentrate the filtrate under reduced pressure to a clear paste with a density of 1.05 g / mL, add 4 times the volume of 95% ethanol, stir evenly, and then let it stand for 24 h to precipitate, filter the precipitate, vacuum dry it at 60°C for 36 h, and grind it to obtain the crude polysaccharide of Quanzhen Yiqi Decoction; (2) The crude polysaccharide of Quanzhen Yiqi Decoction prepared in step (1) was added with water to prepare a crude polysaccharide solution of Quanzhen Yiqi Decoction with a concentration of 20 mg / mL, and the pretreated HPD100 macroporous resin was added. The pH value of the solution was adjusted to 5.0, and the solution was placed in a shaker at 125 r / min and 80°C for 4 h for decolorization, and then centrifuged at 3000-5000 r / min for 3-10 min. The supernatant was collected and concentrated to prepare the polysaccharide of Quanzhen Yiqi Decoction.

2. The method for preparing the Quanzhen Yiqi Decoction polysaccharide for treating chronic obstructive pulmonary disease according to claim 1, characterized in that: The following steps are involved: (1) Take the raw materials of Quanzhen Yiqi Decoction, 30 g of Rehmannia glutinosa, 10 g of Radix Ophiopogonis, 10 g of Atractylodes macrocephala, 10 g of Codonopsis pilosula, 10 g of Radix Aconiti Lateralis Preparata, 10 g of Achyranthes bidentata, and 6 g of Schisandra chinensis, grind them, add 10 times the volume of the total medicinal materials into water, decoct and reflux for extraction twice, each time for 1 h, combine the two extracts, filter, and concentrate the filtrate under reduced pressure to a clear paste with a density of 1.05 g / mL, add 4 times the volume of 95% ethanol, stir evenly, and then let it stand for 24 h to precipitate, filter the precipitate, vacuum dry it at 60°C for 36 h, and grind it to obtain the crude polysaccharide of Quanzhen Yiqi Decoction; (2) The crude polysaccharide of Quanzhen Yiqi Decoction prepared in step (1) was added with water to prepare a crude polysaccharide solution of Quanzhen Yiqi Decoction with a concentration of 20 mg / mL, and the pretreated HPD100 macroporous resin was added. The pH value of the solution was adjusted to 5.0, and the solution was placed in a shaker at 125 r / min and 80°C for 4 h for decolorization, and then centrifuged at 3000-5000 r / min for 3-10 min. The supernatant was collected and concentrated to prepare the polysaccharide of Quanzhen Yiqi Decoction.

3. Use of the Quanzhen Yiqi Decoction polysaccharide according to claim 1 in the preparation of a medicine for treating emphysema.

4. Use of the Quanzhen Yiqi Decoction polysaccharide according to claim 1 in the preparation of a medicine for treating chronic obstructive pulmonary disease.

5. According to the use of claim 3 or 4, the Quanzhen Yiqi Decoction polysaccharide and a pharmaceutically acceptable carrier are made into a drug in the form of tablets, capsules, granules, pills, mixtures, ointments, patches, and sprays.

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