New use of arrowhead extract and composition for preventing and treating copd and preparation method thereof

Abstract

The application provides a new use of a swordleaf arrowhead extract and a composition for preventing and treating COPD and a preparation method of the composition, the swordleaf arrowhead extract has a remarkable treatment effect on relieving early COPD inflammation and preventing and treating COPD, can be used for preparing a medicine for relieving early COPD inflammation or preventing and treating COPD, and has important clinical application value; the effective component of the composition is composed of a swordleaf arrowhead extract, a perilla fruit extract, a pinellia ternate extract, a liquorice extract, a tangerine peel extract, a mulberry bark extract, a cinnamon extract and a raphanus sativus seed extract, the components have a synergistic effect, and the effect of relieving early COPD inflammation or preventing and treating COPD is remarkable; the preparation method is simple, convenient to use, and suitable for the needs of large-scale industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine, in particular to a new use of an extract of sagittaria sagittifolia and a composition for preventing and treating COPD and a preparation method thereof. BACKGROUND

[0002] Chronic obstructive pulmonary disease (COPD) is a common respiratory disease characterized by persistent respiratory symptoms and airflow limitation.

[0003] At present, the treatment of COPD is not perfect, and the drugs used in the clinic for the treatment of COPD mainly include four categories: bronchodilators, glucocorticoids, phosphodiesterase inhibitors and antioxidants. The above drugs, whether used alone or in combination, have good surface disease treatment effect on COPD, but cannot change the decline of lung function, and at the same time can cause many toxic side reactions, such as nausea and vomiting, hand tremor, palpitation, arrhythmia, hypertension and bacterial infection. Therefore, it is still very important to seek new and safe and effective COPD treatment drugs.

[0004] China has rich resources of Chinese herbal medicine, and traditional Chinese medicine has the characteristics of multi-component, multi-link and multi-target in action mode. It has very important application value to extract existing Chinese herbal medicine and perform specific screening to achieve the effect of relieving early COPD inflammation. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a new use of an extract of sagittaria sagittifolia.

[0006] Another technical problem to be solved by the present application is to provide a composition for preventing and treating COPD.

[0007] Another technical problem to be solved by the present application is to provide a preparation method of the above-mentioned composition for preventing and treating COPD.

[0008] To solve the above technical problems, the technical scheme of the present application is:

[0009] The application of the extract of sagittaria sagittifolia in the preparation of a drug for preventing and treating COPD.

[0010] Preferably, in the application of the above-mentioned extract of sagittaria sagittifolia, the drug for preventing and treating COPD is a drug for treating or relieving early COPD inflammation.

[0011] Preferably, in the application of the above-mentioned extract of sagittaria sagittifolia, the COPD (chronic obstructive pulmonary disease) is non-acute COPD.

[0012] The above arrowhead extract is an extract containing arrowhead polysaccharide obtained by water extraction and alcohol precipitation, and the preparation method is described in CN201610640684.0. The extract contains active substances such as arrowhead polysaccharide.

[0013] A composition for preventing and treating COPD, the effective components of which are composed of arrowhead extract, perilla fruit extract, liquorice extract, pinellia ternate extract, dried tangerine or orange peel extract, mulberry bark extract, raphanus sativus extract and cinnamon extract, wherein the arrowhead extract is 50-70 parts, the perilla fruit extract is 5-15 parts, the liquorice extract is 5-15 parts, the pinellia ternate extract is 5-15 parts, the dried tangerine or orange peel extract is 5-15 parts, the mulberry bark extract is 5-15 parts, the raphanus sativus extract is 5-15 parts, and the cinnamon extract is 5-15 parts by weight.

[0014] Preferably, the above composition for preventing and treating COPD, the arrowhead extract is 60 parts, the perilla fruit extract is 10 parts, the liquorice extract is 10 parts, the pinellia ternate extract is 10 parts, the dried tangerine or orange peel extract is 10 parts, the mulberry bark extract is 10 parts, the raphanus sativus extract is 5 parts, and the cinnamon extract is 5 parts by weight.

[0015] Preferably, the above composition for preventing and treating COPD, the perilla fruit extract is obtained by water extraction and alcohol precipitation, and the extract contains perilla oil, flavonoids and polyphenols.

[0016] Preferably, the above composition for preventing and treating COPD, the liquorice extract is obtained by water extraction and alcohol precipitation, and the extract contains glycyrrhizin, glycyrrhizic acid, glycyrrhizic yellow copper and liquorice polysaccharide.

[0017] Preferably, the above composition for preventing and treating COPD, the pinellia ternate extract is obtained by alcohol extraction, and the extract contains succinic acid, total alkaloids and calcium oxalate.

[0018] Preferably, the above composition for preventing and treating COPD, the dried tangerine or orange peel extract is obtained by alcohol extraction, and the extract contains volatile oil, flavonoids and tangerine peel polysaccharide and other active substances.

[0019] Preferably, the above composition for preventing and treating COPD, the mulberry bark extract is obtained by alcohol extraction, and the extract contains various flavonoid active substances.

[0020] Preferably, the above composition for preventing and treating COPD, the raphanus sativus extract is obtained by water extraction and alcohol precipitation, and the extract contains alkali thiocyanate, raphanus sativus and raphanus sativus polysaccharide and other active substances.

[0021] Preferably, the composition for preventing and treating COPD, the cinnamon extract is extracted by water extraction and alcohol precipitation method, and the extract contains cinnamon oil, cinnamon residue polysaccharide and other active substances.

[0022] Preferably, the composition for preventing and treating COPD further comprises diluents, suspending agents, antioxidants, emulsifiers, disintegrants, binders and / or stabilizers.

[0023] Preferably, the composition for preventing and treating COPD is in the form of granules or capsules.

[0024] The preparation method of the composition for preventing and treating COPD is as follows:

[0025] (1) The raw materials of arrowhead extract, perilla fruit extract, liquorice extract, pinellia ternate extract, dried tangerine or orange peel extract, mulberry bark extract, raphanus sativus seed extract and cinnamon extract are weighed according to the prescription amount;

[0026] (2) The components are uniformly mixed to obtain the composition.

[0027] Preferably, the preparation method of the composition for preventing and treating COPD, the mixture obtained in step (2) is granulated, dried to obtain granules.

[0028] Preferably, the preparation method of the composition for preventing and treating COPD, lactose is used as an excipient, the weight ratio of the mixture obtained in step (2) to lactose is 1:2, the concentration of ethanol is 80% (v / v), the obtained granules have good granule forming property, fast dissolving speed, no precipitation after standing, a rest angle less than 40°, and are easy to granulate and not sticky.

[0029] Preferably, the preparation method of the composition for preventing and treating COPD, the mixture obtained in step (2) is dried, sieved and put into a capsule to obtain a capsule.

[0030] The composition for preventing and treating COPD is used in an amount of 5g per day for a 60kg adult.

[0031] The composition for preventing and treating COPD has the following advantages:

[0032] The arrowhead extract can be used for preventing and treating COPD, especially in relieving early COPD inflammation, and can be used for preparing a medicine for preventing and treating COPD and relieving early COPD inflammation, which has important clinical application value.

[0033] The composition for preventing and treating COPD has a significant therapeutic effect in relieving early COPD inflammation and preventing and treating COPD, and its preparation method is simple, convenient to use and suitable for large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The effect of the modified traditional Chinese medicine group in Example 1 on the body weight change of the COPD model mice.

[0035] Figure 2 The effect of the modified traditional Chinese medicine group in Example 1 on the lung function change of the COPD model mice.

[0036] Figure 3 The HE, AB-PAS and Masson staining results of the lung tissue of the mice in each group in Example 1 at 12w (x200).

[0037] Figure 4 The effect of the modified traditional Chinese medicine group in Example 2 on the content of the inflammatory factors in the lung tissue of the COPD model mice.

[0038] Figure 5 The effect of the modified traditional Chinese medicine group in Example 2 on the content of the oxidative stress indicators in the lung tissue of the COPD model mice.

[0039] Figure 6 The effect of the modified traditional Chinese medicine group in Example 2 on the expression of reactive oxygen species in the lung tissue of the COPD model mice (x10).

[0040] Figure 7 The effect of the modified traditional Chinese medicine group in Example 2 on the protein expression of NF-κB, MyD88 and TLR4 in the lung tissue of the COPD model mice.

[0041] Figure 8 The effect of the modified traditional Chinese medicine group in Example 2 on the protein expression of Sirt1, Sirt6 and PGC1a in the lung tissue of the COPD model mice.

[0042] Figure 9 The effect of the modified traditional Chinese medicine group in Example 2 on the protein expression of Nrf2 and HO-1 in the lung tissue of the COPD model mice (x20).

[0043] Figure 10 The effect of the experimental group in Example 3 on the body weight change of the COPD model mice.

[0044] Figure 11 The effect of the experimental group in Example 3 on the lung function change of the COPD model mice.

[0045] Figure 12 The HE staining results of the lung tissue of the mice in each group in Example 3 at 12w (x200). DETAILED DESCRIPTION

[0046] The technical solutions of the present application will be further described below in combination with specific embodiments.

[0047] The method for obtaining each component in the following examples is as follows:

[0048] The arrowhead extract is obtained by the following method (see CN201610640684.0 for details):

[0049] (1) 1 part of arrowhead extract is prepared from 1 g of arrowhead dry (slices). Fresh arrowhead produced in Yunnan is purchased on the market, peeled and sliced, and dried at 55°C. The arrowhead dry (slices) is decocted with 10 times the amount of water for 3 times (1.5, 1, and 0.5 h, respectively), and the residue is removed by filtration with an 80-mesh nylon screen. The filtrate is heated and concentrated to 15% of the volume of the decoction water, and then centrifuged to obtain the supernatant. The precipitate is boiled with water at a volume of half the volume of the supernatant for 30 min, and then centrifuged again to obtain the supernatant. The two supernatants are combined.

[0050] (2) The supernatant is heated and concentrated to 15% of the original volume, and then mixed with 5 times the weight of silica gel (80-100 mesh) and dried in a vacuum (60°C, -0.08 MPa). The dried silica gel is then packed into a column.

[0051] (3) The column is eluted with 95% ethanol (15% of the volume of the decoction water) and 85% ethanol (12% of the volume of the decoction water), respectively. The eluates are combined and dried under reduced pressure (78°C, -0.08 MPa). The residue is dissolved in water (0.2% of the volume of the decoction water), and then diluted with 95% ethanol to 12 times the volume of the aqueous solution (the ethanol content is less than 85%). The mixture is allowed to stand overnight, and the supernatant is poured out to obtain a small amount of unrefined arrowhead composition.

[0052] (4) The sample column is eluted with water (25% of the volume of the decoction water) and hot water (60-70°C, 12.5% of the volume of the decoction water), respectively. The eluates are combined and heated and concentrated to 7% of the original volume to obtain an unrefined arrowhead composition solution.

[0053] (5) The unrefined arrowhead composition solutions obtained in steps (3) and (4) are combined, placed in a separatory funnel, and mixed with chloroform at a volume of 30% of the combined volume and n-butanol at a volume of 6% of the combined volume. The mixture is allowed to stand overnight.

[0054] (6) The lower layer and the middle emulsion layer of the separatory funnel are centrifuged, and the solid (protein) is removed by filtration with an 80-mesh nylon screen. The liquid phase is again poured back into the separatory funnel and mixed, and the mixture is allowed to stand overnight (this step is repeated 3 times).

[0055] (7) The final filtrate was heated and concentrated to 40% of the volume of the combined crude polysaccharide solution, 10 volumes of 95% ethanol were added while hot and the mixture was allowed to stand overnight. The supernatant was decanted to obtain a refined arrowhead composition. The decanted supernatant was evaporated under reduced pressure (78°C, -0.08 MPa) to dryness, dissolved in water (0.2% of the volume of the decoction water), and diluted with 95% ethanol to 12 volumes of the aqueous solution (the ethanol content was less than 85%), allowed to stand overnight, and the supernatant was again decanted to obtain a small amount of refined arrowhead composition. The two portions of refined arrowhead composition were combined and vacuum dried (60°C, -0.08 MPa) to obtain arrowhead composition products (2.3% of the dry arrowhead). The content of the refined arrowhead composition was determined by the phenol-sulfuric acid method: the determination wavelength was 490 nm, the polysaccharide conversion factor f = 1.82; a good linear relationship was obtained within the range of 20 μg to 100 μg, r = 0.9992. The results showed that the test solution was stable in color development within 2 h, had good reproducibility, the average recovery rate was 104%, RSD = 4.72 (n = 5), and the polysaccharide content was 34.31%.

[0056] The perilla fruit extract was obtained by the following method:

[0057] 1 part of the perilla fruit extract was prepared from 1 g of perilla fruit. The perilla fruit was purchased from Beijing Tong Ren Tang (Miaozhou) Herbal Pieces Co., Ltd.

[0058] The extraction process was as follows:

[0059] After the perilla fruit was crushed, 10 times the amount of petroleum ether (60-90°C) was added and refluxed for 3 times, each for 2 h, to obtain a yellow transparent solution. The solutions were combined, filtered, and evaporated to dryness under reduced pressure to obtain a yellow oily liquid, which was the petroleum ether extract (the main component was fatty oil). After the residue was dried, 10 times the amount of 75% ethanol was added and refluxed for 2 times, each for 2 h, to obtain a dark yellow solution. The solutions obtained in each time were combined, filtered, and evaporated to dryness under reduced pressure to obtain a extract, which was the perilla fruit alcohol extract (the main component was flavones).

[0060] The liquorice extract was obtained by the following method:

[0061] 1 part of the liquorice extract was prepared from 1 g of liquorice. The liquorice was purchased from Beijing Tong Ren Tang (Miaozhou) Herbal Pieces Co., Ltd. The extraction process was as follows:

[0062] After the liquorice was crushed, 10 times the amount of an extraction solvent, which was a 60% (V / V) ethanol aqueous solution containing 1.0% of ammonia water, was added and heated to 80°C, and refluxed for 3 h, and filtered. The residue was added with 500 ml of the extraction solvent and subjected to secondary extraction. The extract obtained by extraction was concentrated to obtain a liquorice extract extract.

[0063] The pinellia extract was obtained by the following method:

[0064] 1 part of Pinellia extract was prepared from 1 g of Pinellia ternata. Pinellia ternata was purchased from Beijing Tong Ren Tang (Miaozhou) Herbal Pieces Co. Ltd. The extraction process was as follows:

[0065] An appropriate amount of Pinellia ternata was taken, ground into coarse powder, and decocted with 10 times the amount of water for 2 times, 2 hours for the first time and 1.5 hours for the second time. The filtrates were combined and concentrated to a relative density of 1.30 at 60°C. The solution was allowed to cool, and ethanol was added to make the alcohol content 70%. The solution was allowed to stand overnight, and the supernatant was concentrated under reduced pressure to a thick paste with a relative density of 1.25. The thick paste was dried under reduced pressure at 75°C to obtain the water extract of Pinellia ternata. An appropriate amount of Pinellia ternata was taken, ground into coarse powder, and extracted with 8 times the amount of 75% ethanol by reflux for 2 times, 2 hours for each time. The filtrates were combined, and the ethanol was recovered under reduced pressure. The residue was concentrated to a thick paste with a relative density of 1.25, and dried under reduced pressure to obtain the alcohol extract of Pinellia ternata.

[0066] The extract of Pericarpium Citri Reticulatae was obtained by the following method:

[0067] 1 part of Pericarpium Citri Reticulatae extract was prepared from 1 g of Pericarpium Citri Reticulatae. Pericarpium Citri Reticulatae was purchased from Beijing Tong Ren Tang (Miaozhou) Herbal Pieces Co. Ltd. The extraction process was as follows: Pericarpium Citri Reticulatae was added to 10 times the amount of water (70-100°C) and refluxed for 3 times, 2 hours for each time. The obtained solutions were combined, filtered, and concentrated to dryness under reduced pressure to obtain the water extract of Pericarpium Citri Reticulatae. The residue was dried, 10 times the amount of 75% ethanol was added, and refluxed for 2 times, 2 hours for each time. The obtained solutions were combined, filtered, and concentrated to dryness under reduced pressure to obtain the alcohol extract of Pericarpium Citri Reticulatae.

[0068] The extract of Morus Alba was obtained by the following method:

[0069] 1 part of Morus Alba extract was prepared from 1 g of Morus Alba. Morus Alba was purchased from Beijing Tong Ren Tang (Miaozhou) Herbal Pieces Co. Ltd. The extraction process was as follows: Morus Alba was washed and put into a decocting container for high-temperature decoction at 100°C-108°C. The mass ratio of Morus Alba to water was 1 kg of Morus Alba to 8 kg of water, and the decoction time was 15-30 minutes. 80-95% ethanol was added to the mixture of residue and liquid at a ratio of 20 g / L, and the mixture was stirred for 10-15 minutes. The residue and liquid were separated, and the liquid was clarified and concentrated at a temperature of 50°C-70°C to a thick paste. The obtained solutions were combined, filtered, and concentrated to dryness under reduced pressure to obtain the extract of Morus Alba.

[0070] The extract of Semen Raphani was obtained by the following method:

[0071] 1 part of Raphani Semen extract was prepared from 1 g of Raphani Semen decoction pieces. Raphani Semen medicinal materials were purchased from Beijing Tong Ren Tang (Miaozhou) Decoction Pieces Co., Ltd. The extraction process was as follows: Raphani Semen was cleaned and fried. When the pan bottom temperature reached 200-250°C, the medicine was added and quickly and evenly fried for 1-1.5 min. When the texture was puffy, easy to peel, and had a characteristic aroma, it was removed and allowed to cool. It was then ground into a 10-mesh powder and baked at 90°C for 1-2 h. The powder temperature was uniformly brought to 90°C. The oil press material chamber was controlled at 95°C ± 3°C. The oil press was used to gradually increase the pressure to make the fat oil overflow. When the oil overflow rate significantly slowed down, the pressure was increased again until the oil overflowed. This process was repeated until the oil yield reached 75% ± 3% of the fat oil content in the raw material. The fat oil was stored separately, and the cake powder was ground for further extraction. The physically defatted fried Raphani Semen cake was crushed into coarse powder, soaked in water for 0.5 h, and heated to reflux for three times. The water amount was 8 times, 6 times, and 6 times the amount of the medicinal material, respectively. The extraction time was 1.0 h, 0.5 h, and 0.5 h, respectively. The filtrate was combined and concentrated under reduced pressure to 1 g / ml. 95% ethanol was added while stirring quickly to make the ethanol concentration 80%. It was allowed to stand for 24 h, filtered, and the filtrate was concentrated under reduced pressure to recover the ethanol until there was no alcohol taste. It was further concentrated to near dryness and vacuum dried to a water content of 4.0%-5.0% to obtain Raphani Semen extract.

[0072] Cinnamomi Cortex extract was obtained by the following method:

[0073] 1 part of Cinnamomi Cortex extract was prepared from 1 g of Cinnamomi Cortex decoction pieces. Cinnamomi Cortex medicinal materials were purchased from Beijing Tong Ren Tang (Miaozhou) Decoction Pieces Co., Ltd. The extraction process was as follows: Cinnamomi Cortex was ground into a powder with a particle size of 8-12 cm, soaked in 10 times the amount of water (20-35°C) for 15-20 hours. After soaking and extraction, the soaking extract and the soaked Cinnamomi Cortex residue were separated by filtration; the soaked Cinnamomi Cortex residue was added with 10 times the amount of 75% ethanol and refluxed twice for 2 h each time. The solutions obtained each time were combined, filtered, and concentrated under reduced pressure to dryness to obtain Cinnamomi Cortex extract.

[0074] Example 1

[0075] Caldium Tuber extract (SSP) helps to delay the progression of cigarette smoke and lipopolysaccharide-induced COPD in mice

[0076] 1. Experimental materials

[0077] 1.1 Experimental animals and grouping

[0078] SPF grade C57BL / 6 male mice (6-8 weeks old, body weight 22 g-27 g, Beijing Vantoll Life Science and Technology Co., Ltd.) 30, were randomly divided into 3 groups, 10 in each group, the grouping was blank control group (air exposure), COPD mouse model group (smoke and lipopolysaccharide induction) and traditional Chinese medicine modification group (given Sagittaria sagittifolia extract). The feeding environment temperature was (24±2)℃, humidity 50%-60%, 12h alternating illumination, free access to standard food and water. The animal experiment was approved by the ethics committee of Beijing University of Chinese Medicine, and the ethics number was BUCM-1-2023050101-0020.

[0079] 1.2 Experimental drugs

[0080] Sagittaria sagittifolia extract.

[0081] 1.3 Experimental reagents and instruments

[0082] 1.3.1 Experimental reagents

[0083] Commercial cigarettes (tobacco tar content 10 mg), mouse carbon monoxide hemoglobin (COHb) and cotinine (COT) ELISA kit (Jiangsu Enzyme Immune Industry Co., Ltd.), lipopolysaccharide (Sigma, USA).

[0084] 1.3.2 Experimental instruments

[0085] HRH-MNE3026 small animal single concentration oral-nasal exposure system (Beijing Huironghe, China), ten-channel turntable type smoking machine (Beijing Huironghe, China), eSpira Forced Manoeuvers System (EMMS series animal lung function detection system) (EMMS, UK), iMark enzyme label analyzer.

[0086] 2 Experimental methods

[0087] 2.1 Construction of COPD mouse model

[0088] After the mice were adaptively fed for 1 week, except for the blank control group, each experimental group was instilled with lipopolysaccharide (LPS; L2880, Sigma, USA) in the airway of the mice on the 1st, 14th day, with a dosage of 7.5 μg / 20 μl (not treated with smoke exposure on the injection day), and the rest was exposed to commercial cigarettes (tobacco tar content 10 mg) twice a day, each for 2h, using an automatic smoking machine to light 10 cigarettes / h, and the average total particulate matter concentration was maintained at 40 mg / m 3 , once in the morning and once in the afternoon, with a time interval of more than 4h, 2w. After two weeks of tobacco exposure adaptation, from the 3rd week, it was still exposed twice a day, each for 2h, using an automatic smoking machine to light 13 cigarettes / h, and the average total particulate matter concentration was maintained at 150 mg / m3 The blank control group was instilled with the same amount of normal saline in the airway on the 1st and 14th day, and was exposed to filtered fresh air every day.

[0089] 2.2 Administration method

[0090] From the day of modeling, the traditional Chinese medicine modified arrowhead extract group (hereinafter referred to as the traditional Chinese medicine modified group) was given arrowhead extract liquid (800 mg / kg) by gavage 2 hours before smoke exposure. The gavage dose of the mice was equivalent to the recommended human dose. The model group and the blank control group were given the same amount of distilled water by gavage. Each group was given gavage once a day, and lasted until the end of modeling. After 12 weeks of exposure, samples were collected from 10 mice in each group for detection and analysis.

[0091] 2.3 Observation index and method

[0092] 2.3.1 General condition

[0093] The body weight was measured 2-3 times a week, and the mice in each group were observed, including but not limited to the mental state, food intake, activity and death of the mice.

[0094] 2.3.2 Phenol red sputum experiment

[0095] After 12 weeks of exposure, 0.5 mL of 7.05 mmol / L phenol red solution was injected intraperitoneally into each mouse in each group. The mice were executed by cervical dislocation 0.5 hours after injection. The trachea was separated, and a section from the annular cartilage to the tracheal bifurcation was cut off and placed in a small dish containing 4 mL of 0.9% sodium chloride solution. The solution was repeatedly washed 3 times with 0.5 mL of 1 mol / L NaOH solution, and the washing solution was injected into a colorimetric tube. The supernatant was measured at a wavelength of 546 nm to determine the absorbance. The amount of phenol red excretion was calculated according to the standard curve of phenol red, and the amount of sputum excretion was calculated.

[0096] 2.3.3 Detection of smoke exposure index

[0097] After 12 weeks of exposure, the mice were anesthetized and the eyeballs were removed to collect blood. The content of carbon monoxide hemoglobin (COHb) and cotinine (COT) was detected by Elisa kit.

[0098] 2.3.4 Detection of invasive lung function index

[0099] After 12 weeks of exposure, the airway resistance (RI), peak expiratory flow (PEF), dynamic lung compliance (Cdyn), forced expiratory volume in 90 ms (FEV90) and forced expiratory volume ratio (FVC) of the mice in each group were measured by small animal invasive lung function instrument, and FEV90 / FVC was calculated.

[0100] 2.3.5 Gross anatomy observation and lung organ index

[0101] After 12w exposure, the mouse lung with larynx and trachea was taken out by dissection, washed with pre-cooled normal saline, and the tissue was observed and photographed, the size, shape, color or texture change was recorded, weighed and calculated the organ index.

[0102] 2.3.6 Morphological observation of lung tissue

[0103] After 12w exposure, one side of the lung tissue was taken into 4% paraformaldehyde for fixation, HE staining, and the pathological morphological changes of lung tissue such as inflammation, injury and airway remodeling were observed under optical microscope. In addition, Masson staining was used to observe the distribution of lung tissue fibers, AB-PAS staining was used to observe the distribution and total number of lung tissue goblet cells, and the airway mucus secretion was evaluated.

[0104] 2.4 Statistical method

[0105] The data was input by using Office Excel software, and the experimental results were statistically analyzed by using SPSS25.0 statistical software. The experimental data was represented by , one-way ANOVA was used, and LSD test was used for pairwise comparison between groups. P<0.05 was considered statistically significant.

[0106] 3 Results

[0107] 3.1 General situation

[0108] After 12w modeling, compared with the control group, the model group mice were emaciated, with dull hair, quiet and liked to huddle and curl up. Compared with the model group, the mice in the traditional Chinese medicine modification group were in better condition.

[0109] 3.2 Body weight change

[0110] From Table 1 and Figure 1 , compared with the control group, the body weight of the model group mice decreased significantly, and the difference was statistically significant (P<0.05). Compared with the model group, the body weight of the mice in the traditional Chinese medicine modification group increased significantly (P<0.05).

[0111] Table 1 Effect of traditional Chinese medicine modification group on body weight change of COPD model mice

[0112]

[0113] Note: compared with the control group, * P<0.05; compared with the model group, # P<0.05.

[0114] 3.3 Lung organ index

[0115] From table 2, compared with the control group, the lung organ index of the model group increased significantly (P<0.05). The lung organ index of the traditional Chinese medicine improved group decreased compared with the model group, but there was no statistical significance (P>0.05).

[0116] Table 2 Effect of traditional Chinese medicine improved group on lung organ index of COPD model mice

[0117]

[0118] Note: compared with the control group, * P<0.05.

[0119] 3.4 Phenol red sputum experiment

[0120] From table 3, compared with the control group, the phenol red excretion of the model group increased significantly (P<0.05). Compared with the model group, the phenol red excretion of the traditional Chinese medicine improved group decreased significantly (P<0.05).

[0121] Table 3 Effect of traditional Chinese medicine improved group on phenol red excretion of COPD model mice

[0122]

[0123] Note: compared with the control group, * P<0.05; compared with the model group, # P<0.05.

[0124] 3.5 Detection of smoke exposure index

[0125] From table 4, compared with the control group, the cotinine content in the blood of the model group and the traditional Chinese medicine improved group increased significantly, which had statistical significance (P<0.05).

[0126] Table 4 Effect of traditional Chinese medicine improved group on smoke exposure index content of COPD model mice

[0127]

[0128] Note: compared with the control group, * P<0.05.

[0129] 3.6 Lung function detection

[0130] As shown in table 5 and Figure 2As shown in Table 5, compared with the control group, the lung function indicators FEV90 / FVC and PEF Cdyn of the model group mice were significantly decreased, and the RI of the model group was statistically significantly higher than that of the control group (P<0.05), indicating that the lung function of the model group mice was weakened. Compared with the model group, the lung function indicators FEV90 / FVC, PEF and Cdyn of the traditional Chinese medicine modified group mice increased, but the difference was not significant (P>0.05). However, the RI of the traditional Chinese medicine modified group was significantly lower than that of the model group, which was statistically significant (P<0.05), indicating that the lung function of the traditional Chinese medicine modified group mice partially recovered.

[0131] Table 5 Effect of traditional Chinese medicine modified group on lung function of COPD model mice

[0132]

[0133] Note: compared with the control group, * P<0.05; compared with the model group, # P<0.05.

[0134] 3.7 Lung tissue morphological observation of mice in each group after modeling for 12w

[0135] As shown in Table 6 and Figure 3 compared with the control group, the lung tissue of the model group was dark in color, and obvious congestion or ecchymosis could be seen. The color of the lung tissue of the traditional Chinese medicine modified group was similar to that of the normal group, and there was little congestion or ecchymosis. The HE staining results showed that the alveolar size of the control group was uniform, the structure was complete, no obvious rupture of the alveolar septum was found, and there was no exudation in the alveolar cavity, and there were occasional a small amount of inflammatory cell infiltration. The model group showed obvious alveolar cavity expansion, alveolar wall rupture, adjacent expanded alveolar cavities fused into larger cystic cavities, and a large number of inflammatory cell infiltration could be seen in the lumen and alveoli. Compared with the model group, the inflammatory cells in the lumen and alveoli of the traditional Chinese medicine modified group were significantly reduced, the alveolar cavity was slightly expanded, and the alveolar wall rupture was not obvious.

[0136] Table 6 Proportion of mice in each group meeting the pathological characteristics of COPD

[0137]

[0138] The AB-PAS staining results showed that the control group occasionally had a small amount of goblet cells, and the glycogen secretion in the small bronchus was less. The goblet cell hyperplasia of the model group was higher than that of the control group, and the glycogen secretion in the bronchus was significantly increased. The goblet cell hyperplasia and glycogen secretion of the traditional Chinese medicine modified group were reduced compared with the model group.

[0139] The Masson staining results showed that compared with the control group, the lung tissue structure of the model group changed, the degree of fibrosis significantly increased, mainly manifested as increased deposition of extracellular matrix and fibrous protein in the lung interstitium, while the degree of lung tissue fibrosis in the traditional Chinese medicine modified group was significantly reduced compared with the model group, and the deposition of fibrous protein was reduced.

[0140] Overall, 100% of the model group met the pathological characteristics of COPD, while the rate of the traditional Chinese medicine modified group meeting the pathological characteristics of COPD was significantly reduced to 40%.

[0141] Example 2

[0142] Sagittaria sagittifolia extract (SSP) helps to alleviate oxidative stress and inflammatory response in cigarette smoke and lipopolysaccharide-induced mouse COPD model

[0143] 1. Experimental materials

[0144] 1.1 Experimental animals and grouping

[0145] The same as 1.1 of Example 1.

[0146] 1.2 Experimental drugs

[0147] Sagittaria sagittifolia extract.

[0148] 1.3 Experimental reagents and instruments

[0149] 1.3.1 Experimental reagents

[0150] ELISA detection kits of TNF-α, IL-6, IL-8, IL-1β, LTB4 (Jiangsu Enzyme Immune Industry Co., Ltd.), SOD, GSH, MDA, protein carbonyl kit (Nanjing JC Bioengineering Institute, China), ROS staining solution, DAPI, anti-fluorescence quenching mounting agent, rabbit anti-mouse Nrf2, HO-1 polyclonal antibody, rabbit IgG SABC immunohistochemical staining kit, concentrated DAB kit were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; Sodium pentobarbital, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, paraformaldehyde, 100% ethanol, 95% ethanol, dimethylbenzene, solid paraffin for section, hematoxylin, concentrated hydrochloric acid, ammonia water, 30% hydrogen peroxide, sodium citrate, citric acid, Tween-20, bovine serum albumin, polylysine were all of analytical pure.TLR4, MyD88, NF-κB p65, SIRT1 antibodies were purchased from Abeam (ab13556, ab219413, ab32536, ab189494, respectively), SIRT6 Polyclonal antibody (Proteintech, 13572-1-AP), PGC1a Monoclonal antibody (Proteintech, 66369-1-Ig), Beta Actin Polyclonal antibody (Proteintech, 20536-1-AP), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech, SA00001-1), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, SA00001-2), 5x SDS Protein Loading Buffer (LABLEAD, G2527-5mL), Pre-stained Protein Marker (10-180KD) (LABLEAD, P1018-250uL), PVDF Membrane (0.45um) (Millipore, IPVH00010), Skim Milk Powder (Bioruler, RH61873), Ammonium Persulfate (Bioruler, RJ0519), 4x SDS-PAGE Concentration Gel Buffer (Aiji, S1003), 4x SDS-PAGE Separation Gel Buffer (Aiji, S1002), 30% Acrylamide (29:1) (Aiji, S1001), Electrophoresis Buffer (Dry Powder) (Servicebio, G2018-1L), Transmembrane Buffer (Dry Powder) (Servicebio, G2017-1L), TBS Buffer (Dry Powder) (Servicebio, G0001-2L), RIPA Lysis Buffer (Strong) (Servicebio, G2002-100mL), PMSF (100mM) (Servicebio, G2008-1mL), BCA Protein Quantitative Kit (Servicebio, G2026-1000T), Bovine Serum Albumin (Servicebio, WB100D).

[0151] 1.3.2 Experimental instruments

[0152] HRH-MNE3026 small animal single concentration oral-nasal exposure system (HRH-MNE3026, Beijing, China), ten-channel rotary smoking machine (HRH-MNE3026, Beijing, China), frozen high-throughput tissue grinder (Scientz-48L, Ningbo Xinzhi), constant-temperature water bath (SSW-420-2S type, Shanghai Boxun Industry), decolorization shaker (Orbital Shaker TS-2, Its Linbeier), centrifuge (Eppendorf 5418R), transfer electrophoresis tank (Bio-Rad), chemiluminescence imaging system (SH-523, Shenhua), high-throughput tissue crusher, iMark enzyme-labeled analyzer, ultraviolet spectrophotometer, freezing microtome, histochemical pen, decolorization shaker, fluorescence microscope, enzyme-labeled analyzer, electrophoresis power supply, vertical electrophoresis tank.

[0153] 2 Experimental methods

[0154] 2.1 COPD model construction

[0155] The same as 2.1 of Example 1.

[0156] 2.2 Administration method

[0157] The same as 2.2 of Example 1.

[0158] 2.3 Observation index and method

[0159] 2.3.1 ELISA method for detecting the levels of TNF-α, IL-6, IL-8, IL-1β and LTB4 in mouse lung tissue

[0160] An appropriate amount of lung tissue was cut and added with 9 times the volume of PBS, four chromium oxide grinding beads were added to each tube, and a high-throughput tissue crusher was used for frozen homogenization to prepare a 10% tissue homogenate. Centrifugation was performed at 4°C and 5000 r / min for 15 min. After standing for 30 min, the tissue supernatant was collected and frozen in a -80°C refrigerator for subsequent use.

[0161] The operation was strictly in accordance with the instructions of each Elisa kit. Briefly, the standard was gradiently diluted, and blank holes, standard holes and sample holes were set. After the sample was added, incubation was performed at 37°C for 30 min, washing was performed 5 times, and drying was performed. Enzyme-labeled reagent 50 μL (except for blank holes) was added to each hole. Incubation was again performed in a 37°C constant-temperature box for 30 min, washing was performed 5 times, and drying was performed. Color developing agents A and B were added to each hole in turn, 50 μL each, color developing was performed at 37°C for 10 min in the dark, 50 μL of stop solution was added, and the absorbance of each hole was measured at 450 nm wavelength in sequence and the concentration was calculated.

[0162] 2.3.2 Kit detection of SOD, GSH, MDA and protein carbonyl levels in mouse lung tissue

[0163] Cut the lung tissue, add 9 times volume of 0.9% saline solution, add four chromium oxide grinding beads to each tube, and use a high-throughput tissue homogenizer to prepare a 10% tissue homogenate. Centrifuge at 3000 r / min for 10 min at 4°C. After standing for 30 min, collect the tissue supernatant and freeze it in a -80°C refrigerator for later use. In addition, for the detection of protein carbonyl content, weigh an appropriate amount of lung tissue, add 9 times the reagent according to the ratio of weight (g) to volume (ml) = 1:9, homogenize, centrifuge at 2500 r / min for 10 min at 4°C, then take 10% of the supernatant homogenate and add reagent two, mix well, and stand at room temperature for 10 min, centrifuge at 11000 r / min for 10 min, and then take the supernatant for subsequent operation.

[0164] After the experiment according to the operation content of different kits, the OD value of each kit is determined by ultraviolet spectrophotometry, and the content or activity of SOD, GSH, MDA, and protein carbonyl in the lung tissue is calculated according to the formula.

[0165] 2.3.3 DCFH-DA fluorescent probe detection of ROS content in mouse lung tissue

[0166] Staining: After the frozen section is slightly shaken dry, draw a circle around the tissue with a histological pen (to prevent the antibody from flowing away), and add ROS staining solution in the circle. Incubate in a constant temperature incubator at 37°C for 30 min.

[0167] DAPI restains the cell nucleus: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 min each time. After the section is slightly shaken dry, add DAPI staining solution in the circle, and incubate at room temperature for 10 min in the dark.

[0168] Mounting: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 min each time. After the section is slightly shaken dry, mount the section with an anti-fluorescence quenching mounting agent.

[0169] Microscopy and photography: Observe the section under a fluorescence microscope and collect images.

[0170] The cell nucleus stained by DAPI is blue under ultraviolet excitation, and ROS positive expression is red light corresponding to the fluorescence label.

[0171] 2.3.4 Quantitative analysis of Nrf2 and HO-1 in mouse lung tissue by immunohistochemical method

[0172] ① Paraffin section is routinely deparaffinized to hydration.

[0173] ② Heat antigen retrieval: immerse the section in citrate buffer (adjust pH to 6.0), repeatedly heat to boiling, and then stop (3 times), with an interval of 8 min. After cooling, wash with PBS for 5 min x 3 times.

[0174] ③Blocking: Take 20 mL 30% H2O2, add 200 ml distilled water, mix well, and then incubate at room temperature for 30 min to inactivate endogenous enzymes. Wash with PBS for 5 min x 3 times.

[0175] ④Blocking: Add 5% BSA blocking solution dropwise, and incubate at room temperature for 30 min. Shake off the excess liquid, and do not wash.

[0176] ⑤Add 200-fold diluted primary antibody (rabbit IgG), and incubate overnight at 4°C. Wash with PBST for 5 min x 3 times.

[0177] ⑥Add biotinylated goat anti-rabbit IgG, and incubate at 37°C for 50 min. Wash with PBS for 5 min x 3 times.

[0178] ⑦DAB color development: Take 1 mL DAB substrate solution, add 50 μl DAB concentrate, mix well, and then prepare DAB working solution. Develop color at room temperature, control the reaction time under a microscope, and finally terminate the reaction in distilled water.

[0179] ⑧Hematoxylin re-staining, gradient alcohol dehydration, xylene transparency, and mounting.

[0180] 2.3.5 Western Blot detection of lung tissue Sirt1, Sirt6, NF-κB, Sirt1, Sirt6, and PGC-1α protein expression

[0181] ①Preparation of lung tissue homogenate: Prepare tissue lysis solution according to the ratio of RIPA lysis solution:PMSF:proteinase and phosphatase inhibitor cocktail = 98:1:1. Randomly take 3 lung tissues stored at -80°C refrigerator from each group, weigh and record, and then place in pre-cooled centrifuge tubes. According to the ratio of tissue weight (mg):lysis solution volume (μL) = 1:20, add the corresponding volume of lysis solution to the lung tissues in each group. Place two clean grinding beads in each centrifuge tube, and then transfer to a full-automatic multi-sample frozen grinder for tissue grinding, 1 minute each time, and operate twice. Incubate on ice for 30 min, and then centrifuge at 12000 rpm, 4°C, for 15 min, aspirate the supernatant into a new centrifuge tube, and store at -80°C refrigerator.

[0182] BCA method for determining the protein concentration of lung tissue homogenate: Prepare a protein standard stock solution with a concentration of 25 mg / mL: Add 1 mL of protein standard preparation solution to 25 mg of protein standard (BSA) and shake until fully dissolved. Prepare a protein standard working solution with a concentration of 0.5 mg / mL: Take 20 μL of 25 mg / mL protein standard stock solution and add to 980 μL of RIPA lysis buffer to dilute to a final concentration of 0.5 mg / mL protein standard working solution. According to the number of samples to be tested, prepare the BCA color developing working solution by mixing the BCA reagent and copper sulfate solution at a ratio of 50:1. Take 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard working solution and add to the 96-well plate, and use RIPA lysis buffer to make up the total volume of each well to 20 μL. Dilute the lung tissue protein samples to be tested 20 times with RIPA lysis buffer, 20 μL per well, and add to the 96-well plate, with 4 replicate wells per group. Add 200 μL of the prepared BCA color developing working solution to each well, mix thoroughly, and then place the 96-well plate in a 37°C incubator for 30 minutes. Use a microplate reader to read the absorbance value of the 96-well plate at 562 nm wavelength. Draw a standard curve, and according to the formula obtained from the standard curve, calculate the protein concentration of each group of samples. Use 5x SDS protein loading buffer and RIPA lysis buffer for protein quantification, and the final protein concentration for loading is 1.5 μg / μL. Denature the quantified protein sample by boiling in 100°C water for 5 min, cool to room temperature, and store in a -20°C refrigerator.

[0183] ③ Western Blot method to detect related protein expression: Prepare separation gel (8%-12%) and stacking gel according to the molecular weight of the test protein. After determining the loading order, add 5 μL of pre-stained protein marker to each lane on both sides, and the sample loading volume is 10 μL per well. After completing the loading, first electrophoresis at a constant voltage of 90V for about 20 minutes. When the sample moves to the junction of the separation gel and the stacking gel, switch to 120V electrophoresis for 70-90 minutes. When a bromophenol blue band is observed about 1 cm from the bottom, stop electrophoresis. Soak the sponge and filter paper in electrotransfer buffer in advance, cut a PVDF membrane of appropriate size, and place it in anhydrous methanol to completely soak for 1 minute for activation. Transfer the cut gel to the filter paper, cover the PVDF membrane and drive out bubbles. After the plywood is installed, place it in the electrotransfer tank, place a small ice box in the gap in the tank and add electrotransfer buffer to the highest water level. Wet transfer is performed under constant current of 300mA and 90min. The whole process is carried out on ice. Then, prepare an appropriate amount of 5% blocking buffer using TBST and skim milk powder. Remove the electroporated PVDF membrane and place it face-up in an incubation box containing blocking buffer, ensuring that the blocking buffer completely covers the membrane. Block on a rocker for 1 hour. Wash the blocked PVDF membrane three times with TBST for 5 minutes each. Dilute the corresponding primary antibody to the appropriate concentration according to the manufacturer's instructions. Place the cut PVDF membrane in the corresponding primary antibody incubation box and incubate overnight at 4°C. The next day, remove the primary antibody incubation box and rewarm at room temperature for 1 hour. After recovering the primary antibody, wash the membrane three times with TBST for 5 minutes each. Add the diluted secondary antibody and incubate at room temperature for 1 hour on a rocker at medium speed. After recovering the secondary antibody, wash the membrane three times with TBST for 5 minutes each. Visualize and photograph using a chemiluminescence imaging system. Quantify the grayscale intensity of Western blot bands using Image J software, using β-actin as a reference protein.

[0184] 3 Results

[0185] 3.1 IL-6, IL-8, TNF-α, IL-1β, and LTB4 levels in mouse lung tissue

[0186] As shown in Table 7 and Figure 4 As shown in the results, compared with the control group, the content of inflammatory factors in the lung tissue of the model group was significantly increased (P < 0.05). Compared with the model group, the content of inflammatory factors in the lung tissue of the modified Chinese medicine group was significantly reduced (P < 0.05).

[0187] Table 7 Effects of the modified Chinese medicine group on the levels of inflammatory factors in lung tissue of COPD model mice

[0188]

[0189] Note: Compared with the control group, * P<0.05; compared with the model group, #P<0.05.

[0190] 3.2 The content of SOD, GSH, MDA and protein carbonyl in lung tissue of mice

[0191] As shown in Table 8 and Figure 5 compared with the control group, the endogenous antioxidants GSH and SOD in the lung tissue of the model group were significantly reduced, and the contents of the peroxidation product indicators MDA and protein carbonyl were significantly increased (P<0.05). On the contrary, compared with the model group, the GSH and SOD in the traditional Chinese medicine modification group were significantly increased, while the contents of MDA and protein carbonyl were significantly reduced (P<0.05).

[0192] Table 8 Effect of traditional Chinese medicine modification group on the content of oxidative stress indicators in lung tissue of COPD model mice

[0193]

[0194] 3.3 The content of ROS in lung tissue of mice

[0195] As Figure 6 shown, compared with the control group, the red fluorescence in the model group was significantly enhanced, indicating that the expression of ROS in the lung tissue of the model group increased; compared with the model group, the red fluorescence intensity in the lung tissue of the traditional Chinese medicine modification group was significantly weakened, indicating that the content of ROS decreased.

[0196] 3.4 The content of TLR4, MyD88 and NF-κB in lung tissue of mice

[0197] As Figure 7 shown, compared with the control group, the expression contents of NF-κB, MyD88 and TLR4 proteins in the model group were significantly increased (P<0.05). Compared with the model group, the expression contents of NF-κB, MyD88 and TLR4 proteins in the traditional Chinese medicine modification group were significantly reduced (P<0.05).

[0198] 3.5 Protein expression of Sirt1, Sirt6 and PGC-1α in lung tissue of mice

[0199] As Figure 8 shown, compared with the control group, the expression contents of Sirt1, Sirt6 and PGC1α proteins in the model group were significantly reduced (P<0.05). Compared with the model group, the expression of Sirt1, Sirt6 and PGC1α proteins in the traditional Chinese medicine modification group was significantly increased (P<0.05).

[0200] 3.6 Protein expression of Nrf2 and HO-1 in lung tissue of mice

[0201] As Figure 9As shown, compared with the control group, the expression of Nrf2 and HO-1 in the lung tissue of the model group mice increased (P<0.05). Compared with the model group, the expression of Nrf2 and HO-1 in the lung tissue of the traditional Chinese medicine modified group mice decreased (P<0.05).

[0202] Example 3

[0203] A composition for preventing and treating COPD, the effective components of which are composed of extractum of sagittaria sagittifolia, perilla frutescens extract, glycyrrhiza extract, pinellia ternate extract, citrus reticulata extract, morus alba extract, raphanus sativus extract and cinnamomum cassia extract. Among them, the extractum of sagittaria sagittifolia is 60 parts, the perilla frutescens extract is 10 parts, the glycyrrhiza extract is 10 parts, the pinellia ternate extract is 10 parts, the citrus reticulata extract is 10 parts, the morus alba extract is 10 parts, the raphanus sativus extract is 5 parts and the cinnamomum cassia extract is 5 parts by weight.

[0204] The preparation method of the above-mentioned composition for relieving early COPD inflammation is as follows:

[0205] (1) The extractum of sagittaria sagittifolia, perilla frutescens extract, glycyrrhiza extract, pinellia ternate extract, citrus reticulata extract, morus alba extract, raphanus sativus extract and cinnamomum cassia extract are weighed according to the prescription amount as raw materials;

[0206] (2) The components are uniformly mixed to obtain the composition.

[0207] Example 4

[0208] A composition for preventing and treating COPD, the effective components of which are composed of extractum of sagittaria sagittifolia, perilla frutescens extract, glycyrrhiza extract, pinellia ternate extract, citrus reticulata extract, morus alba extract, raphanus sativus extract and cinnamomum cassia extract. Among them, the extractum of sagittaria sagittifolia is 70 parts, the perilla frutescens extract is 15 parts, the glycyrrhiza extract is 15 parts, the pinellia ternate extract is 5 parts, the citrus reticulata extract is 5 parts, the morus alba extract is 15 parts, the raphanus sativus extract is 5 parts and the cinnamomum cassia extract is 5 parts by weight.

[0209] The preparation method is the same as that in example 3.

[0210] Example 5

[0211] A composition for preventing and treating COPD, the effective components of which are composed of extractum of sagittaria sagittifolia, perilla frutescens extract, glycyrrhiza extract, pinellia ternate extract, citrus reticulata extract, morus alba extract, raphanus sativus extract and cinnamomum cassia extract. Among them, the extractum of sagittaria sagittifolia is 70 parts, the perilla frutescens extract is 15 parts, the glycyrrhiza extract is 5 parts, the pinellia ternate extract is 5 parts, the citrus reticulata extract is 5 parts, the morus alba extract is 15 parts, the raphanus sativus extract is 15 parts and the cinnamomum cassia extract is 5 parts by weight.

[0212] The preparation method is the same as that in example 3.

[0213] Example 6

[0214] A composition for preventing and treating COPD, the effective components of which are composed of extract of Sagittaria trifolia, extract of Perilla frutescens, extract of Glycyrrhiza uralensis, extract of Pinellia ternata, extract of Citrus reticulata, extract of Morus alba, extract of Raphanus sativus and extract of Cinnamomum cassia. Among them, the extract of Sagittaria trifolia is 70 parts, the extract of Perilla frutescens is 15 parts, the extract of Glycyrrhiza uralensis is 15 parts, the extract of Pinellia ternata is 15 parts, the extract of Citrus reticulata is 15 parts, the extract of Morus alba is 15 parts, the extract of Raphanus sativus is 10 parts and the extract of Cinnamomum cassia is 10 parts by weight.

[0215] The preparation method refers to Example 3, and the obtained mixture is granulated, dried to obtain granules, wherein the weight ratio of the mixture to lactose is 1:2, the ethanol concentration is 80% (v / v), the obtained granules have good granule forming property, fast dissolving speed, no precipitation after standing, an angle of repose less than 40°, and are easy to granulate and not sticky.

[0216] Example 7

[0217] A composition for preventing and treating COPD, the effective components of which are composed of extract of Sagittaria trifolia, extract of Perilla frutescens, extract of Glycyrrhiza uralensis, extract of Pinellia ternata, extract of Citrus reticulata, extract of Morus alba, extract of Raphanus sativus and extract of Cinnamomum cassia. Among them, the extract of Sagittaria trifolia is 50 parts, the extract of Perilla frutescens is 5 parts, the extract of Glycyrrhiza uralensis is 5 parts, the extract of Pinellia ternata is 5 parts, the extract of Citrus reticulata is 5 parts, the extract of Morus alba is 5 parts, the extract of Raphanus sativus is 5 parts and the extract of Cinnamomum cassia is 15 parts by weight.

[0218] The preparation method refers to Example 3, and the obtained mixture is dried, sieved and put into a capsule to obtain a capsule.

[0219] The use amount of each component in one of Examples 3-7 is increased or reduced at the same ratio, and the weight ratio of the obtained components all falls within the protection scope of the present application.

[0220] Analysis of the composition:

[0221] Sagittaria Sagittifolia L. is a dual-purpose medicinal and edible plant, which has the effects of nourishing the body, clearing heat and detoxifying, clearing the lung and relieving cough, and resolving swelling and removing stasis. It is confirmed by Examples 1 and 2 that Sagittaria Sagittifolia L. contains active ingredients for early prevention and treatment of COPD and rich antioxidant nutrients, and has significant effects in delaying the progression of COPD and relieving the inflammatory response of COPD. Perilla frutescens extract has the effects of relieving qi, clearing phlegm, moistening the lung and relieving asthma, and relaxing the intestines. Glycyrrhiza extract has the effects of regulating the middle and relieving urgency, moistening the lung, detoxifying, and regulating various drugs, and in this composition, it regulates various drugs, moistens the lung and relieves cough. Pinellia extract has the effects of drying dampness, resolving phlegm, descending adverse qi and stopping vomiting, and resolving mass and removing stasis, and can be combined with various drugs to relieve the symptoms of cough, phlegm and asthma. Orange peel extract has the effects of regulating qi, regulating the middle, drying dampness and resolving phlegm. Mulberry bark extract has the effects of relieving lung and asthma, and removing edema, and is used to relieve airway obstruction. Raphanus sativus extract has the effects of relieving qi, relieving asthma, resolving food and phlegm, and is combined with various drugs to relieve the symptoms of airway obstruction and phlegm. Cinnamon extract has the effects of tonifying primordial yang, warming the spleen and stomach, removing cold accumulation and unblocking blood vessels, and is combined with various drugs to have the effects of warming and tonifying the lower energizer, and relieving qi and asthma. The above components have synergistic effects, and have significant effects in relieving early COPD inflammation and delaying the progression of COPD.

[0222] Experimental Example

[0223] The COPD mouse model was used to determine that the Sagittaria composition helps to delay the progression of COPD induced by smoke and lipopolysaccharide in mice.

[0224] 1 Experimental materials

[0225] 1.1 Experimental animals and grouping

[0226] SPF C57BL / 6 male mice (6-8 weeks old, weighing 22-27 g, Beijing Vantoll Life Experimental Animal Technology Co., Ltd.) were randomly divided into 4 groups, 10 mice in each group, and the groups were blank control group (air exposure), COPD mouse model group (induced by smoke and lipopolysaccharide), experimental group (given Sagittaria composition), and single Sagittaria control group (given Sagittaria extract). The feeding environment temperature was (24±2) ℃, humidity 50%-60%, 12 h alternating light, and free access to standard food and water. The animal experiment was approved by the Ethics Committee of Beijing University of Chinese Medicine, and the ethics number was BUCM-1-2023050101-0020.

[0227] 1.2 Experimental drugs

[0228] The above-mentioned Sagittaria extract and the composition for preventing and treating COPD described in Example 3.

[0229] 1.3 Experimental reagents and instruments

[0230] 1.3.1 Experimental reagents

[0231] Commercial cigarettes (10 mg tar), lipopolysaccharide (Sigma, USA).

[0232] 1.3.2 Experimental instruments

[0233] HRH-MNE3026 small animal single concentration oral-nasal exposure system (Beijing Huironghe, China), ten-channel carousel smoking machine (Beijing Huironghe, China), eSpira Forced Manoeuvers System (EMMS series animal lung function detection system) (EMMS, UK), iMark enzyme label analyzer.

[0234] 2 Experimental methods

[0235] 2.1 COPD mouse model construction

[0236] After 1 week of adaptive feeding, the mice in each group were instilled with lipopolysaccharide (LPS; L2880, Sigma, USA) in the airway on days 1 and 14, with a dosage of 7.5 μg / 20 μl (no smoke exposure treatment on the injection day), and the rest were exposed to commercial cigarettes (10 mg tar) twice a day, each for 2 h, using an automatic smoking machine to light 10 cigarettes / h, with an average total particulate matter concentration of 40 mg / m 3 , twice a day, each for 2 h, using an automatic smoking machine to light 13 cigarettes / h, with an average total particulate matter concentration of 150 mg / m 3 , twice a day, each for 2 h, using an automatic smoking machine to light 10 cigarettes / h, with an average total particulate matter concentration of 40 mg / m

[0237] 2.2 Administration method

[0238] From the day of modeling, the experimental group and the single sagittaria trifolia control group were given sagittaria trifolia composition solution (800 mg / kg) and sagittaria trifolia extract solution (800 mg / kg) by gavage 2 hours before smoke exposure, and the mouse gavage dose was equivalent to the recommended human dose. The model group and the blank control group were given the same amount of distilled water by gavage, and each group was given gavage once a day, lasting until the end of modeling. After 12 weeks of exposure, samples were collected from 10 mice in each group for detection and analysis.

[0239] 2.3 Observation index and method

[0240] 2.3.1 General condition

[0241] The mice in each group were weighed 2 to 3 times a week and their status, food intake, activity, and mortality were observed.

[0242] 2.3.2 Invasive pulmonary function test

[0243] After 12 weeks of exposure, the airway resistance (RI), peak expiratory flow (PEF), dynamic lung compliance (Cdyn), forced expiratory volume in 90 ms (FEV90) and forced expiratory volume ratio (FVC) of mice in each group were measured using a small animal invasive spirometer, and FEV90 / FVC was calculated.

[0244] 2.3.3 Lung tissue morphology observation

[0245] After 12 weeks of exposure, lung tissue from one side was fixed with 4% paraformaldehyde and stained with hematoxylin and eosin. Pathological changes in the lung tissue, such as inflammation, damage, and airway remodeling, were observed under a light microscope. In addition, Masson staining was used to observe lung fiber distribution, and AB-PAS staining was used to observe the distribution and total number of goblet cells in the lung tissue, and to assess airway mucus secretion.

[0246] 2.4 Statistical methods

[0247] The data were input using Office Excel software, and SPSS 25.0 statistical software was used to perform statistical analysis on the experimental results. The data were analyzed by one-way analysis of variance, and the LSD test was used for pairwise comparison between groups. P < 0.05 was considered statistically significant.

[0248] 3 Results

[0249] 3.1 General

[0250] Twelve weeks after modeling, the mice in the model group were thinner, had gray fur, and were quiet and preferred to huddle together. The mice in the experimental group and the arrowhead control group were in better condition than the model group.

[0251] 3.2 Weight changes

[0252] Depend on Figure 10 It can be seen that compared with the control group, the weight of mice in the model group decreased significantly, and the difference was statistically significant (P < 0.05). Compared with the model group, the weight of mice in the experimental group and the single arrowhead control group increased significantly (P < 0.05), and the weight increase of mice in the experimental group was slightly higher than that in the single arrowhead control group.

[0253] 3.3 Pulmonary function test

[0254] like Figure 11As shown in the figure, compared with the control group, the lung function indicators FEV90 / FVC, PEF, and PIF of the mice in the model group were significantly decreased, with statistical significance (P < 0.05), indicating that the lung function of the mice in the model group was weakened. Compared with the model group, the lung function indicators FEV90 / FVC, PEF, and PIF of the mice in the experimental group and the single arrowhead control group increased, with statistical significance (P < 0.05), indicating that the lung function of the mice in the experimental group and the single arrowhead control group was partially restored, and the lung function recovery of the mice in the experimental group was better than that in the single arrowhead control group.

[0255] 3.4 Observation of lung tissue morphology of mice in each group 12 weeks after model establishment

[0256] like Figure 12 HE staining revealed that compared with the control group, the lung tissue in the model group was often darker in color, with visible congestion or ecchymosis. The color of the lung tissue in the single-sagittaria control group was similar to that of the control group, with minor congestion or ecchymosis. The color of the lung tissue in the experimental group was essentially the same as that of the control group, with minimal congestion or ecchymosis. Furthermore, the alveoli in the control group were uniform in size and structurally intact, with no apparent rupture of the alveolar septa, no exudate within the alveolar cavity, and occasional small amounts of inflammatory cell infiltration. In the model group, significant alveolar cavities were observed to be dilated, with alveolar wall ruptures, and adjacent dilated alveolar cavities fused into larger cysts. Numerous inflammatory cell infiltration was observed within the lumina and alveoli. Compared with the model group, the experimental and single-sagittaria control groups showed significantly fewer inflammatory cells within the lumina and alveoli, with slightly dilated alveolar cavities and less obvious alveolar wall ruptures. The experimental group showed more pronounced recovery from inflammation than the single-sagittaria control group.

[0257] As shown in Table 9, overall, the model group met the pathological characteristics of COPD at 100%, the rate of the single arrowhead control group meeting the pathological characteristics of COPD was 40%, and the rate of the experimental group meeting the pathological characteristics of COPD was 30%.

[0258] Table 9 The proportion of mice in each group that met the pathological characteristics of COPD

[0259]

[0260] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of an extract of Sagittaria sagittifolia L. in the preparation of a medicament for preventing and treating chronic obstructive pulmonary disease, wherein the extract of Sagittaria sagittifolia L. is obtained by the following method: (1) Fresh Sagittaria sagittifolia L. produced in Yunnan is purchased and peeled and sliced, and then dried at 55°C. The dried Sagittaria sagittifolia L. slices are decocted in 10 times the amount of water for 3 times, each for 1.5, 1 and 0.5 hours, and then filtered with a 80-mesh nylon screen to remove the residue. The filtrate is heated and concentrated to 15% of the volume of the decoction water, and then centrifuged to obtain the supernatant. The precipitate is dissolved in half the volume of water of the supernatant, and then boiled for 30 minutes, and then centrifuged again to obtain the supernatant. The two supernatants are combined; (2) The combined supernatant is heated and concentrated to 15% of the original volume, and then mixed with 5 times the weight of 80-100 mesh silica gel, and then dried under vacuum at 60°C and -0.08 MPa. The dried product is then packed into a column; (3) The column is eluted with 95% ethanol at 15% of the volume of the decoction water, and then eluted with 85% ethanol at 12% of the volume of the decoction water. The eluate is combined and then dried under reduced pressure at 78°C and -0.08 MPa. The dried product is dissolved in 0.2% water of the volume of the decoction water, and then diluted with 95% ethanol to 12 times the volume of the aqueous solution, and then allowed to stand overnight. The supernatant is decanted to obtain a small amount of unrefined Sagittaria sagittifolia L. composition; (4) The sample column is eluted with water at 25% of the volume of the decoction water, and then eluted with hot water at 60-70°C at 12.5% of the volume of the decoction water. The eluate is combined and then heated and concentrated to 7% of the original volume to obtain a solution of the unrefined Sagittaria sagittifolia L. composition; (5) The solutions of the unrefined Sagittaria sagittifolia L. composition obtained in steps (3) and (4) are combined, and then placed in a separatory funnel. Chloroform is added to 30% of the volume of the combined solution, and then n-butanol is added to 6% of the volume of the combined solution. The mixture is shaken and allowed to stand overnight; (6) The lower layer and the middle emulsion layer of the separatory funnel are centrifuged, and then filtered with a 80-mesh nylon screen to remove solid proteins. The liquid phase is again placed in the separatory funnel, shaken and allowed to stand overnight. This step is repeated 3 times; (7) The final filtrate is heated and concentrated to 40% of the volume of the combined solution of crude polysaccharides. While hot, 10 times the volume of 95% ethanol is added, and then allowed to stand overnight. The supernatant is decanted to obtain the refined Sagittaria sagittifolia L. extract. The decanted supernatant is dried under reduced pressure at 78°C and -0.08 MPa. The dried product is dissolved in 0.2% water of the volume of the decoction water, and then diluted with 95% ethanol to 12 times the volume of the aqueous solution, and then allowed to stand overnight. The supernatant is again decanted to obtain a small amount of refined Sagittaria sagittifolia L. extract. The two portions of the refined Sagittaria sagittifolia L. extract are combined, and then dried under vacuum at 60°C and -0.08 MPa.

2. The use of the Sagittaria sagittifolia L. extract according to claim 1, characterized in that: The medicament for preventing and treating chronic obstructive pulmonary disease is a medicament for treating or relieving inflammation of early chronic obstructive pulmonary disease.

3. The use of the Sagittaria sagittifolia extract according to claim 1 or 2, characterized in that: The chronic obstructive pulmonary disease is non-acute chronic obstructive pulmonary disease.

4. A composition for the prophylaxis and treatment of chronic obstructive pulmonary disease, characterized by, Is made by the following weight parts of raw materials: 50-70 parts of arrowhead extract, 5-15 parts of perilla fruit extract, 5-15 parts of liquorice extract, 5-15 parts of pinellia ternate extract, 5-15 parts of dried tangerine or orange peel extract, 5-15 parts of mulberry bark extract, 5-15 parts of raphanus sativus extract, 5-15 parts of cinnamon extract; the arrowhead extract is obtained by water extraction and alcohol precipitation method, the perilla fruit extract is obtained by water extraction and alcohol precipitation method, the liquorice extract is obtained by water extraction and alcohol precipitation method, the pinellia ternate extract is obtained by alcohol extraction method, the dried tangerine or orange peel extract is obtained by alcohol extraction method, the mulberry bark extract is obtained by alcohol extraction method, the raphanus sativus extract is obtained by water extraction and alcohol precipitation method, and the cinnamon extract is obtained by water extraction and alcohol precipitation method.

5. The composition for preventing and treating chronic obstructive pulmonary disease according to claim 4, wherein Is made by the following weight parts of raw materials: 60 parts of arrowhead extract, 10 parts of perilla fruit extract, 10 parts of liquorice extract, 10 parts of pinellia ternate extract, 10 parts of dried tangerine or orange peel extract, 10 parts of mulberry bark extract, 5 parts of raphanus sativus extract, and 5 parts of cinnamon extract.

6. The composition for preventing and treating chronic obstructive pulmonary disease according to claim 4 or 5, characterized by: Also includes diluents, suspending agents, antioxidants, emulsifiers, disintegrants, binders, and / or stabilizers.

7. The method of claim 4 or 5 for the preparation of a composition for the prevention and treatment of chronic obstructive pulmonary disease, characterized in that: The specific preparation steps are as follows: (1) The arrowhead extract, perilla fruit extract, liquorice extract, pinellia ternate extract, dried tangerine or orange peel extract, mulberry bark extract, raphanus sativus extract, and cinnamon extract are weighed according to the prescription amount as raw materials; (2) The components are mixed uniformly, and the granules are obtained.

8. The method for preparing the composition for preventing and treating chronic obstructive pulmonary disease according to claim 7, characterized in that: The mixture obtained in step (2) is granulated, dried, and screened to obtain granules.

9. The method for preparing the composition for preventing and treating chronic obstructive pulmonary disease according to claim 7, characterized in that: The mixture obtained in step (2) is dried, screened, and placed in a capsule to obtain a capsule.

Images