CN114010718A

CN114010718A - Preparation method and application of fritillaria cirrhosa extract

Info

Publication number: CN114010718A
Application number: CN202111342525.XA
Authority: CN
Inventors: 叶本贵; 贺喜; 雷龙
Original assignee: Sichuan Lvbeibei Biotechnology Co ltd; Sichuan Miaoying Biotechnology Co ltd; Sichuan University
Current assignee: Sichuan Lvbeibei Biotechnology Co ltd; Sichuan Miaoying Biotechnology Co ltd; Sichuan University
Priority date: 2021-11-12
Filing date: 2021-11-12
Publication date: 2022-02-08

Abstract

The invention discloses a preparation method and application of a fritillaria cirrhosa extract, wherein the method comprises the following steps: pulverizing the bulbus fritillariae cirrhosae into powder, soaking the bulbus fritillariae cirrhosae in an alcohol solution overnight, continuously performing reflux extraction at the temperature of 60-90 ℃, collecting an extraction concentrated solution, removing a solvent to obtain a concentrated extract, and performing vacuum drying under reduced pressure to obtain the bulbus fritillariae cirrhosae extract; the alkaloid in the fritillaria cirrhosa extract comprises: the medicinal composition comprises sipeimine, peimine, sipeimine glycoside, peimine, peiminine, peimine, ibimine glycoside A and dehydroibeiiminine. The fritillaria cirrhosa extract can be used as a medicine for treating chronic respiratory system diseases, and comprises the following components: chronic obstructive pneumonia, chronic pharyngitis, pulmonary fibrosis, and can activate MAPKs and Nrf2 signaling pathways.

Description

Preparation method and application of fritillaria cirrhosa extract

Technical Field

The invention relates to extraction of fritillaria cirrhosa, and particularly relates to a preparation method and application of a fritillaria cirrhosa extract.

Background

The pharynx refers to the cavity behind the oral cavity and nasal cavity and above the esophagus, is the common passage for food and breath, and is composed of muscles and mucous membrane. The lung is part of the respiratory system and functions to exchange gas, and good lung function is the guarantee of life support. Both are important components of the respiratory tract and are important functional sites for the body to exchange gases.

Chronic Obstructive Pulmonary Disease (COPD) is a group of lung diseases characterized by airflow limitation, which develops progressively in association with an increased chronic inflammatory response of the airways and lungs to toxic particles or gases, often manifested as progressive exacerbation of chronic cough, expectoration, dyspnea. Chronic pharyngitis is a chronic inflammation of the pharyngeal mucosa, submucosa and lymphoid tissues. The acute pharyngolaryngitis is converted into chronic pharyngolaryngitis due to repeated attack caused by incomplete treatment of acute pharyngolaryngitis, or due to various rhinopathy, nasal orifice obstruction, long-term mouth opening and breathing, and frequent stimulation to pharynx by physical and chemical factors, neck radiotherapy and the like. Pulmonary Fibrosis (IPF) is a very serious pulmonary disorder, an abnormal structure resulting from abnormal repair after normal alveolar tissue is damaged. Pulmonary fibrosis seriously affects the respiratory function of human body, manifested as dry cough and progressive dyspnea, and the respiratory function of patients is continuously worsened with the aggravation of illness and lung injury.

Bulbus Fritillariae Cirrhosae is widely used for treating respiratory system diseases due to its good antitussive, expectorant, and antiasthmatic effects. Recent pharmacological research also indicates that the bulbus fritillariae cirrhosae has certain effects of resisting inflammation and relieving oxidative stress. The modern chronic respiratory system diseases mainly comprise chronic obstructive lung disease, pulmonary fibrosis and chronic pharyngitis, the three diseases have obvious inflammatory reaction and oxidative stress, and the bulbus fritilariae has obvious effects on two aspects. The safety and toxicity of the fritillaria cirrhosa as a precious traditional Chinese medicinal material are proved definitely, but no clear method and curative effect information exist for the treatment and application of the three diseases, and the fritillaria cirrhosa has great potential and application prospect.

Disclosure of Invention

The invention aims to provide a preparation method and application of a fritillaria cirrhosa extract, wherein the fritillaria cirrhosa extract can be used as a medicine for treating chronic respiratory diseases and can activate MAPKs and Nrf2 signal pathways.

In order to achieve the above object, the present invention provides a fritillaria cirrhosa extract, wherein alkaloids in the fritillaria cirrhosa extract comprise: the medicinal composition comprises sipeimine, peimine, sipeimine glycoside, peimine, peiminine, peimine, ibimine glycoside A and dehydroibeiiminine.

Preferably, the fritillaria cirrhosa extract is obtained by extracting fritillaria cirrhosa bulbs with alcohol and then concentrating.

Preferably, the fritillaria cirrhosa extract is obtained by performing reflux extraction on fritillaria cirrhosa bulbs with alcohol at 60-90 ℃ and then concentrating.

Another object of the present invention is to provide a method for preparing a fritillaria cirrhosa extract, which comprises: pulverizing the bulbs of the fritillaria cirrhosa, soaking the bulbs of the fritillaria cirrhosa in an alcohol solution overnight, continuously performing reflux extraction at the temperature of 60-90 ℃, collecting an extracted concentrated solution, removing a solvent to obtain a concentrated extract, and performing vacuum drying under reduced pressure to obtain an extract of the fritillaria cirrhosa; the alkaloid in the fritillaria cirrhosa extract comprises: the medicinal composition comprises sipeimine, peimine, sipeimine glycoside, peimine, peiminine, peimine, ibimine glycoside A and dehydroibeiiminine.

Preferably, the alcohol is selected from ethanol.

Preferably, the ethanol is selected from 85% ethanol.

The invention also aims to provide application of the fritillaria cirrhosa extract in preparing a medicament for treating chronic respiratory diseases.

Preferably, the chronic respiratory disease comprises: chronic obstructive pneumonia, chronic pharyngitis, and pulmonary fibrosis.

Preferably, the extract of Sichuan fritillary bulb is capable of down-regulating the expression of phosphorus-Erk 1/2and phosphorus-JNK.

Preferably, the extract of fritillaria cirrhosa is capable of upregulating Nrf2 expression.

The preparation method and the application of the fritillaria cirrhosa extract have the following advantages:

the fritillaria cirrhosa extract can obviously relieve the phosphorylation increase level of Erk1/2and JNK, and is in positive correlation with the administration concentration. Compared with a model group, the expression of phospho-Erk1/2andphospho-JNK is obviously reduced in the low-dose fritillaria cirrhosa extract administration treatment group, and meanwhile, the thickening and the materialization of the alveolar space are relieved, a small amount of inflammatory cell infiltration is still seen, and the alveolar collapse condition is relieved; the levels of phospho-Erk1/2and phospho-JNK were significantly reduced in the medium and high dose groups, indicating that the fritillaria cirrhosa extract reduced the inflammatory response of the lungs by activating MAPKs signaling pathways in the COPD rat model. Nrf2 expression was significantly elevated with increasing doses administered, with concomitant relief of inflammatory responses and oxidative stress.

Drawings

FIG. 1 is a total ion flow diagram of LC-MS/MS measurement of 9 alkaloids in Bulbus Fritillariae Cirrhosae extract.

FIG. 2 shows the body weight change of male (A) and female (B) rats within 6 weeks of administration.

FIG. 3 is a graph showing HE staining of lung tissue (magnification: 40X).

FIG. 4 is an HE staining pattern (magnification: 100X) of liver tissue, spleen tissue, kidney tissue and heart tissue at 2 weeks.

FIG. 5 is an HE staining pattern (magnification: 100X) of liver tissue, spleen tissue, kidney tissue and heart tissue at 4 weeks.

FIG. 6 is an HE staining pattern (magnification: 100X) of liver tissue, spleen tissue, kidney tissue and heart tissue at 6 weeks.

FIG. 7 shows the levels of TNF- α (A), IL-6(B), IL-1 β (C) and HO-1(D) in rat alveolar lavage fluid.

FIG. 8 is the levels of MDA (A) and GSH/GSSG (B) in rat lung tissue.

Fig. 9 is an immunohistochemical image of rat lung tissue.

FIG. 10 shows changes in p-p38 (400X), p-JNK (400X), p-Erk (400X) and Nrf2 (400X) levels in the treatment with fritillary bulb extract.

FIG. 11 shows the results of the TNF-. alpha.content in the serum of each group.

FIG. 12 shows the results of the IL-1. beta. content in the serum of each group.

FIG. 13 shows the hydroxyproline content in lung tissues of each group.

FIG. 14 shows the TGF-. beta.content in 10% lung homogenates of each group.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A method for preparing a Bulbus Fritillariae Cirrhosae extract, the method comprising:

pulverizing Bulbus Fritillariae Cirrhosae 6kg into powder, soaking in 85% ethanol overnight, placing in large and medium-sized continuous reflux concentration extraction tank, continuously reflux extracting at 70-80 deg.C for 8 hr, collecting concentrated extractive solution, removing residual solvent with rotary evaporator to obtain concentrated extract, placing in vacuum drying oven, and drying at 50 deg.C for 24 hr to obtain Bulbus Fritillariae Cirrhosae extract, weighing 581.4g, and obtaining extract yield of 9.69%.

Experimental example 1 determination of alkaloid content in Bulbus Fritillariae Cirrhosae extract

For the determination of the alkaloid content in the fritillaria cirrhosa extract prepared in example 1, the alkaloid content was determined by LC-MS:

chromatographic conditions for LC-MS: the column was Shim-pack XR-ODS (2.2 μm,2.00 mm. times.100 mm I.D.) at a column temperature of 40 ℃. Mobile phase: 0.1% aqueous formic acid (A), acetonitrile (B), gradient elution,0.00 → 1.00min, B% 0.0 → 15.0; 1.00 → 3.00min, B% 15.0 → 25.0; 3.00 → 4.00min, B% 25.0 → 25.0; 4.00 → 5.00min, B% 25.0 → 95.0; 5.00 → 5.50min, B% 95.0 → 95.0; 5.50 → 5.60min, B% 95.0 → 15.0; 5.60 → 7.90min, B% 15.0 → 15.0; 8.00min, stop. Flow rate: 0.40 mL/min^-1The amount of the sample was 5. mu.L.

Mass spectrum conditions: an ESI source; positive ion mode monitoring mode, the injection voltage is set to 5500V; the ion source temperature was 500 ℃. Atomizing Gas (Gas1)50.0psi, heating Gas (Gas2)50.0psi, curtain Gas (CUR)40.0psi, and nitrogen Gas is introduced all the time. The scanning mode adopts Multiple Reaction Monitoring (MRM); collision gas (CAD) pressure was 9.0; both Q1 and Q3 resolutions are UNITs.

As shown in figure 1, the total alkaloid content is 0.117% as determined by LC-MS/MS determination of total ion flow diagram of 9 alkaloids in Bulbus Fritillariae Cirrhosae extract, wherein the content of Siberine, peiminine B, peiminine A, Siberine glycoside, Fritillarine, peimisine, Fritillarionene, ibesine A, and dehydroebeibeidine is 38.31 μ g/g, 3.49 μ g/g, 6.32 μ g/g, 16.52 μ g/g, 1.51 μ g/g, 83.06 μ g/g, 31.27 μ g/g, 7.59 μ g/g, and 7.24 μ g/g in sequence.

Experimental example 2 preparation of Experimental animal model

1. Grouping of laboratory animals

100 SD rats were housed separately in the Wasp institute of pharmacy, Sichuan university for one week and were allowed free access to water and food to adapt to the environment. After one week, the body weights were weighed and recorded, and divided into a blank control group (C), a COPD model group (P), a low dose administration group (L), a medium dose administration group (M), and a high dose administration group (H) in such a manner that the body weights were randomized, 20 in each group, and half in each male and female.

2. Establishment and administration treatment of COPD rat disease model

A rat COPD disease model is established by adopting a mode of combining Lipopolysaccharide (LPS) tracheal instillation and smoking, and the rat COPD disease model is established by adopting a cycle of 6 weeks. Rats in the experimental group were subjected to abdominal anesthesia with 4% chloral hydrate (10mL/kg) on days 1 and 15, the upper teeth of the rats were fixed with a thin wire, the tongue root was pulled out, a scalp needle thin tube with a needle removed was slowly inserted into the airway of the rats, a LPS (1mg/mL, 0.2mL) solution was instilled through the trachea, and a blank control group was replaced with an equal amount of physiological saline. On days 2-14 and 16-42, after administration for 4h each day, the experimental rats were put into a self-made smoking box (85cm × 40cm × 45cm) for smoking, and 6 small holes were opened on the box cover to allow air to enter. 4 cigarettes were lit each time, and smoked with a 50mL syringe 5 times/5 min, with each puff of smoke injected into the box through a small hole, leaving the remaining cigarettes free to burn. Smoking for 20min every 4 cigarettes, opening the box cover to ventilate for 10min, repeating the smoking operation once, namely smoking for 8 cigarettes each day for 40min, 6 days each week, and leaving the blank control group untreated.

Except for the blank control group and the COPD model group, distilled water is given for intragastric administration every day, and rats in the administration group are continuously administered for 6 weeks according to crude drug amount according to low dose of 3.15g/kg/d, medium dose of 9.45g/kg/d and high dose of 18.9g/kg/d respectively.

Hair color, behavior, diet, feces, etc. were observed and recorded daily, and body weight changes were recorded weekly. At the end of

weeks

2, 4 and 6, 30 and 40 experimental rats were sacrificed and fasted for 12h before sacrifice, but without water deprivation, respectively.

The body weight of the male mice in the COPD model group was substantially unaffected in the first 3 weeks and showed a significant decline starting at week 4 compared to the blank control group; the female rats in the model group showed a significantly slower trend in weight gain from week 2 than the blank group. The rats treated with fritillary bulb extract had improved weight loss compared to the COPD model group. Wherein, the weight gain of the male administration group from the 4 th week and the weight gain of the female administration group from the 2 nd week are obviously higher than that of the COPD model group; the body weight improvement effect of the high, medium and low doses in male rats was not much different, while the low dose > high dose > medium dose trend was shown in female rats (see fig. 2).

Along with the increase of modeling time, symptoms such as cough, wheeze, accelerated and deepened breathing, abdominal muscle twitch and the like appear in the model rats from 2 weeks, the model rats are most obvious, the hair color is dull, the actions are reduced, the diet is reduced, the drinking water is increased, the breathing of the most serious rats is slowed down, the behaviors are delayed, and the limbs are flaccid. The condition of the rats in the administration group is improved along with the increase of the administration time, the symptoms such as cough, dyspnea and the like are gradually relieved, the drinking water and diet are basically normal, and the behavioral activities are increased.

Experimental example 3 Collection and measurement of COPD rat disease model specimen

1. Blood sample collection

After the administration of the drug for 2 hours, the rat to be killed is injected with 4% chloral hydrate for anesthesia, the abdominal cavity is cut open to expose abdominal aorta blood vessels, a proper amount of blood is taken by a disposable blood taking needle into a vacuum blood collection tube containing anticoagulant, the blood is immediately shaken and evenly shaken gently to prevent clotting, and the routine blood determination is completed within 2 hours.

At the end of week 2, there was no statistical difference in blood normothermia between the model group and rats in each of the administered groups compared to the blank control group. Compared with the model group, the blood routine change of rats in each administration group is not statistically different.

At the end of week 4, the model rats had increased White Blood Cells (WBC), Hemoglobin (HGB), Hematocrit (HCT), width of red blood cell distribution (CV/RDWR, SD/RDWA), decreased median cell percentage (MPR), and significant differences compared to the blank control group. The average volume of red blood cells (MCV), the average hemoglobin amount (MCH) and the distribution width of red blood cells (CV/RDWR, SD/RDWA) of the rats in the low-dose administration group are increased; the average volume of red blood cells and the distribution width of red blood cells (CV/RDWR, SD/RDWA) of the rats in the medium-dose administration group are increased; the hemoglobin, hematocrit and erythrocyte distribution width (CV/RDWR, SD/RDWA) of the rats in the high dose group were increased, and the mean hemoglobin concentration (MCHC) was decreased. Compared with the model group, the blood routine index of rats in each group of the administration group has no statistical difference.

After 6 weeks, the model rats had increased Red Blood Cells (RBC), Hematocrit (HCT), red blood cell distribution width (CV/RDWR, SD/RDWA) and decreased mean hemoglobin concentration compared to the blank control group; the hematocrit, the mean volume of the red blood cells and the red blood cell distribution width (SD/RDWA) of the rats in the low-dose administration group are increased, and the mean hemoglobin concentration is reduced; the indexes of the rats in the medium-dose administration group have no statistical difference; the high dose group rats had an increased width of red blood cell distribution (SD/RDWA). Compared with the model group, the blood routine index of rats in each group of the administration group has no statistical difference.

In summary, the model rats showed an increase in both White Blood Cell (WBC) and neutrophil percentage (GPR) and a decrease in Lymphocyte Percentage (LPR) compared to the blank control group, and there was no statistical difference except for a significant difference in WBC at 4 weeks. There was a certain reduction in White Blood Cells (WBC) in rats of each group administered compared to the model group.

2. Bronchoalveolar lavage fluid collection and cell differential enumeration

(1) The experimental rat after exsanguination through abdominal aorta is cut open by operating scissors to separate the right main bronchus and ligate, the neck part is provided with a free air outlet pipe, a T-shaped opening is cut at the upper part, a No. 12 stomach filling needle is inserted into the cavity of the tube and is fixed by cotton thread ligation. Sucking 5mL of precooled physiological saline by using a 5mL syringe, slowly injecting the precooled physiological saline into the lumen, allowing the liquid to enter the left lung, repeatedly sucking for 3 times to perform lavage for 1 time, repeatedly performing lavage for 3 times (the recovery rate is more than 80%), and collecting and combining the lavage liquid to obtain alveolar lavage liquid (BALF).

(2) Centrifuging the obtained alveolar lavage fluid at low temperature, collecting the supernatant, and storing at-80 deg.C. Taking the precipitate, adding a proper amount of physiological saline for resuspension, transferring the precipitate into a 1.5mL centrifuge tube, placing the centrifuge tube in a high-speed refrigerated centrifuge, centrifuging the centrifuge tube at 15000rpm and 4 ℃ for 10min, removing supernatant, adding 0.5mL PBS solution for resuspension, preparing a cell smear, staining the cell smear by using Rie's-Giemsa, observing cell classification and counting.

Referring to tables 1-3, the number of leukocytes, the percentage of lymphocytes decreased, and the percentage of neutrophils increased in the BALF of rats in the model groups at

weeks

2, 4, and 6, compared to the blank control group. There was a significant increase in the percentage of macrophages at weeks 2and 4. Compared with the model group rats, the number of the white blood cells in the administration treatment group is obviously reduced. The high dose group showed a significant decrease in the percentage of neutrophils, a significant increase in the percentage of lymphocytes at weeks 2and 6, and a significant increase in the percentage of macrophages in BALF at week 4. The percentages of lymphocytes, neutrophils and macrophages in the BALF of the rats of the other administration groups were not statistically different from those of the rats of the model group.

Table 1 shows the cell count and classification of alveolar lavage fluid (BALF) of 2-week-old rats

Note: compared with the blank control group, the composition of the composition,^#P<0.05，^###P<0.001; comparison with model group<0.05，**P<0.01。

Table 2 shows the cell count and classification of alveolar lavage fluid (BALF) of 4-week-old rats

Note: compared with the blank control group, the composition of the composition,^#P<0.05，^##P<0.01，^###P<0.001; comparison with model group<0.05，**P<0.01，***P<0.001。

Table 3 shows the cell count and classification of alveolar lavage fluid (BALF) of 6-week-old rats

Note: compared with the blank control group, the composition of the composition,^###P<0.001; comparison with model group<0.05，**P<0.01，***P<0.001。

3. Collection of tissue specimens

(1) After the abdominal aorta is bled, the heart, the liver, the right kidney and the spleen are quickly separated and picked up, the pre-cooled physiological saline is used for rinsing to remove blood, and the filter paper is used for absorbing excessive water and then weighing. After the completion of alveolar lavage, the right lung was immediately taken and the same procedure was performed, and the organ indexes were weighed and recorded, and the organ indexes of heart, liver, spleen, lung and kidney of each group of rats were not statistically different.

(2) After weighing, a part of right lung tissue is taken and quickly frozen in liquid nitrogen for measuring subsequent indexes. Fixing the rest organs and tissues in 4% paraformaldehyde for 48h, selecting tissue samples with appropriate sizes, performing conventional paraffin embedding, slicing, HE staining, observing tissue forms and changes of heart, liver, right kidney, spleen and left lung, and mainly observing integrity of alveolar structure, thickness of alveolar space, size of bronchiole lumen, infiltration condition of airway inflammatory cells and the like in lung tissues.

(3) HE staining and immunohistochemical staining

HE staining: after the abdominal aorta is bled, the heart, the liver, the right kidney and the spleen are quickly separated and picked up, the pre-cooled physiological saline is used for rinsing to remove blood, and the filter paper is used for absorbing excessive water and then weighing. After completion of alveolar lavage, the lungs were immediately removed and the same procedure was followed, weighed and recorded, and the organ index was calculated. Fixing organs and tissues in 4% paraformaldehyde for 48h, selecting tissue samples with proper sizes, carrying out conventional paraffin embedding, carrying out HE staining on the sections, observing tissue morphology and change, and mainly observing integrity of alveolar structures in lung tissues, thickness of alveolar spaces, sizes of bronchiole lumens, infiltration conditions of airway inflammatory cells and the like.

Immunohistochemical staining: putting the dewaxed slices into a staining jar, washing with 3% methanol and hydrogen peroxide at room temperature for 10min, soaking the washed PBS in 0.01M citrate buffer solution (PH6.0), heating to boil twice with high fire in a microwave oven, washing with the cooled PBS, adding goat serum (ZLI-9021, Beijing Zhongshan Jinqiao biology, Ltd.), sealing for 20min, and adding primary anti-Nrf 2 (concentration: 1); p-SAPK, 1:50 (concentration); P-P38, 1:200 (concentration); P-P44, 1:200 (concentration) overnight at 4 ℃, biotinylated secondary goat anti-rabbit working solution (SP-9001, Kingshan Fuqiao Bio Inc. in Beijing) was added dropwise and incubated at 37 ℃ for 30min, a concentrated DAB kit (K135925C, Kingshijin Bio Inc. in Beijing) was used for color development, and a stained section was imaged using a microscope (BA400Digital, Miaodi practice group Co., Ltd.).

As shown in fig. 3 (2W, 4W, and 6W in the figure indicate 2 weeks, 4 weeks, and 6 weeks, respectively), as the modeling cycle increased, inflammatory cell infiltration broke through the basement membrane, smooth muscle destruction became prominent, inflammatory cell infiltration such as a large number of lymphocytes was observed in the lung interstitium, and the lung became more parenchymal, and thin and small bronchi were in the form of rosettes, manifesting as atelectasis and difficulty in ventilation, as compared to the blank group. Compared with the model group, the lung conditions of the rats in the fritillaria cirrhosa administration treatment group are all improved, and the effect is best in the high-dose group. Compared with the same time node, the alveolar spaces of low, medium and high dose groups are obviously thickened and partially materialized at 2 weeks, inflammatory cell infiltration can be seen in lung interstitium, a large amount of inflammation is seen near a basement membrane or in the vicinity of the basement membrane, and part of alveoli are shrunk and collapsed; the high dose group had a relatively weak increase in marginal alveolar septa. At 4 weeks, the alveolar spaces of the low-dose groups are thickened, and inflammatory cell infiltration can be seen in part of the lung interstitium; the alveolar space of the medium-dose group is not thickened and thinned, part of alveoli collapse, and the lung interstitium is not infiltrated by inflammatory cells; in the high-dose group, the alveolar septal thickening and the lung interstitial inflammatory cell infiltration are not seen, part of the alveolar septal thinning, part of the alveolar atrophy and collapse and the edge alveoli are intact. At 6 weeks, the alveolar septa of the low-dose groups are thickened, the pulmonary interstitium and the basement membrane of the smooth muscle are locally subjected to inflammatory infiltration, and the annuliform of the thin small bronchial tubes is relieved; the medium-dose administration group occasionally has basement membrane inflammatory infiltration, pulmonary interstitium does not have inflammatory infiltration, alveolar space obviously does not have thickening or thinning, alveoli are complete and do not have atrophy collapse, and lung parenchyma and flower-ring-shaped thin small bronchus are not seen; inflammatory cell infiltration, thickening of alveolar space, complete most alveoli with little atrophy, and no change in parenchyma and rosette are observed in the high-dose administration group. With the increase of the administration time, the alveolar septum of the medium and high dose groups gradually returns to normal, most alveoli are intact without collapse and materialization, inflammatory cell infiltration is also reduced, the alveolar interstitium does not have inflammatory cell infiltration, and the rosette of the thin small bronchus is not changed.

As shown in FIGS. 4-6, the heart, liver, spleen and kidney tissues of the rats in each group were not abnormal, which indicates that the Bulbus Fritillariae Cirrhosae extract has no toxicity for affecting the function of organs.

4. Determination of IL-6, TNF-alpha, IL-1 beta and HO-1 in alveolar lavage fluid (BALF)

The alveolar lavage fluid supernatant of the sacrificed experimental animals after the 6 th week is taken and placed at room temperature for melting, and the content changes of IL-6, TNF-alpha, IL-1 beta and HO-1 (heme oxygenase) are determined according to the instruction operation of an ELISA kit.

As shown in FIG. 7, TNF-. alpha.IL-6 and IL-1. beta. levels were significantly increased in BALF of the model group rats at week 6 as compared with the blank control group; compared with the rats in the model group, the levels of TNF-alpha, IL-6 and IL-1 beta in the high-dose group and the medium-dose group in the administration group are obviously reduced; while the levels of TNF-alpha, IL-6 and IL-1 beta in BALF of the rats in the low dose group were not significantly changed. As shown in D in FIG. 7, the mice in the model group at week 6 did not have a significant increase in HO-1 levels in BALF compared to the blank control group; compared with the model group rats, the administration group BALF has the advantage that the HO-1 level is increased, wherein the HO-1 level of the high-dose administration group is obviously increased, and the medium-dose administration group and the low-dose administration group have no statistical difference.

5. Determination of Malondialdehyde (MDA) content in lung tissue

(1) Taking a proper amount of lung tissue, and carrying out the following steps: lysate RIPA was added at a ratio of 9 and homogenized on an ice bath. Thereafter, the mixture was centrifuged at 4 ℃ at 10000g for 10min, and the supernatant was collected for measurement.

(2) And taking a proper amount of sample, and determining the protein concentration according to the BCA kit method for calculating the MDA content in the subsequent unit protein weight tissue.

(3) And performing subsequent operations according to the instruction of the MDA detection kit.

(4) Finally, the absorbance at 540nm is measured by a microplate reader, the MDA content in the sample is calculated, and the MDA content in the original sample is expressed by the protein content per unit weight, namely, the mu mol/mg protein.

As shown in a in fig. 8, MDA levels in lung tissue of rats of the week 6 model group were significantly increased compared to the blank control group. Compared with the rats in the model group, the MDA level in the high-dose group and the medium-dose group in the administration group is obviously reduced; while the MDA level of the rats in the low-dose administration group is not obviously reduced.

6. Determination of GSH/GSSG content in Lung tissue

20mg of lung tissue was homogenized thoroughly with 200ul of protein removal M reagent using a glass homogenizer at 4 ℃ for 10 min. The supernatant was centrifuged at 10000g,4 ℃ for 10min for subsequent measurements. Following the instructions in the GSH and GSSG detection kits (purchased from Beyotime, nanjing, china). The absorbance at 405nm was measured at a single point by a microplate reader. And calculating the content of the total glutathione and the glutathione. GSH content-total GSH-GSSG × 2, calculated from the standard curve.

As shown in B of fig. 8, GSH/GSSG levels were significantly reduced in the 6 th week model group rats compared to the blank control group. The GSH/GSSG level of the high dose group and the middle dose group is obviously increased compared with that of the model group rats; while the GSH/GSSG levels of the rats in the low-dose group were not statistically different.

7. Activation of fritillaria cirrhosa on MAPKs and Nrf2 signal pathways of COPD model rat

As can be seen from fig. 9 and 10, after being stimulated by LPS and cigarette, the model group induces and activates the MAPKs pathway to cause the development of lung injury, while the fritillaria cirrhosa extract can significantly relieve the phosphorylation increase level of Erk1/2and JNK, and is positively correlated with the administration concentration. Compared with a model group, the expression of phospho-Erk1/2andphospho-JNK is obviously reduced in the low-dose fritillaria cirrhosa extract administration treatment group, and meanwhile, the thickening and the materialization of the alveolar space are relieved, a small amount of inflammatory cell infiltration is still seen, and the alveolar collapse condition is relieved; the levels of phospho-Erk1/2and phospho-JNK were significantly reduced in the medium and high dose groups, apparently in a rat model of COPD, in which Bulbus Fritillariae Cirrhosae extract reduced the inflammatory response of the lung by activating the MAPKs signaling pathway. Nrf2 is an important transcription factor that regulates cellular oxidative stress, and is also a central regulator of maintaining intracellular redox homeostasis. It can be seen from figure 10 that Nrf2 expression increased significantly with increasing dose administered, with concomitant relief of inflammatory responses and oxidative stress. Bulbus Fritillariae Cirrhosae is activated by two signal pathways, MAPKs and Nrf 2.

Experimental example 4 preparation of model of chronic pharyngitis in Experimental animal

1. Grouping of laboratory animals

50 SD rats were housed separately in the Wasp institute of pharmacy, Sichuan university for one week and were allowed free access to water and food to adapt to the environment. After one week, the body weights were weighed and recorded, and divided into a blank control group (C), a chronic pharyngitis model group (P), a low dose administration group (L), a medium dose administration group (M), and a high dose administration group (H) in such a manner that the body weights were randomized, 10 each for each group, and half for each male and female.

2. Establishment of chronic pharyngitis rat disease model

Spraying prepared ammonia water with concentration of 2.5% into the pharynx of the model group rat by a throat sprayer at a ratio of 10:00 and 15:00 every day for 4 weeks at a fixed time of 1 time and 3 times per press, and spraying equivalent amount of 0.9% NaCl to prepare a chronic pharyngitis rat model. Taking abdominal aorta blood of rats in the normal group and the model group after 4 weeks, killing 10 rats in the normal group and 10 rats in the model group, taking pharyngeal mucosa of 2 groups of rats to send pathology, observing pharyngeal mucosa pathological changes, and evaluating whether modeling is successful by combining symptom signs of the rats.

And (3) molding results: after the 1 st week of model building, the experimental rat has obvious oral scratching behavior, increased water intake, decreased feed intake, and found in the 3 rd week of model building, the rat has obviously decreased diet and water intake, listlessness, salivation, increased urine volume, weight reduction, increased hair loss, erosion of part of oral mucosa of the rat, loose stool, dark red throat mucosa color deepening, and other symptoms and signs, and the blank group does not have the above conditions compared with the model group. Comparing the blank group, the pathology of the model group rats is found: the boundary of each layer of tissue under the pharyngeal mucosa is unclear, the mucosal layer is continuously destroyed, necrotic cells and inflammatory cells are infiltrated locally, and the nail prominence is prolonged, which indicates that the molding is successful.

3. After the molding is successful, the medicine is applied

Experiments prove that the model can be successfully formed by the method, the model is formed on the rest 3 groups of SD rats by the same model forming method, the treatment is carried out by the fritillaria cirrhosa extract in the 5 th week, and the stomach is continuously filled for 3 weeks. The administration time is 1 intragastric administration per day, and the administration is continued for 3 weeks at low dose of 3.15g/kg/d, medium dose of 9.45g/kg/d and high dose of 18.9 g/kg/d.

Experimental example 5 Collection and measurement of model specimen of chronic pharyngitis

1. Blood sample collection and measurement

After 3 weeks of administration, the rats are anesthetized, abdominal aortic blood is extracted, the blood is naturally coagulated for 10-20 min at room temperature, then the sample is centrifuged for about 20min (2000-3000 r/min), and the supernatant is collected. Serum CRP (C-reactive protein) and ICAM-1 levels were determined by ELISA.

TABLE 4 comparison of ICAM-1 content in serum among groups

Note: compared with the blank group, the P is less than 0.05, and the difference has statistical significance; the above table shows that the extract of Sichuan fritillary bulb can reduce ICAM-1 in serum to different degrees.

TABLE 5 comparison of CRP content in serum between groups

Note: compared with the blank group, the P is less than 0.05, and the difference has statistical significance; the above table shows that the fritillaria cirrhosa extract can reduce the level of CRP in serum to different degrees.

As can be seen from the above tables 4 and 5, the ICAM-1 expression level in the serum of the rat in the model group is the highest, and the ICAM-1 expression level in the fritillaria cirrhosa extract group is obviously reduced; the CRP expression level in the serum of the rat of the model group is the highest; CRP expression level of high-dose Bulbus Fritillariae Cirrhosae extract is reduced significantly.

2. Collection of pharyngeal mucosal tissue samples and TNF-alpha, IL-1 beta, IL-6, TRAF6, NF-kappa Bp65mRNA

TABLE 6 groups of rat pharyngeal mucosa tissues TNF-alpha, IL-1 beta and IL-6

As can be seen from the above Table 6, the levels of TNF-alpha, IL-1 beta and IL-6 in the pharyngeal mucosa tissues of the rats in the model group are all increased compared with those in the normal group; compared with the model group, the fritillaria cirrhosa extract has the advantages that the TNF-alpha, IL-1 beta and IL-6 levels of each dose group of the fritillaria cirrhosa extract are all reduced, the IL-1 beta and IL-6 levels of the medium dose group and the low dose group are all increased, the TNF-alpha, IL-6 and TNF-alpha, IL6 and IL-1 beta differences of the medium dose group and the high dose group are not statistically significant (P is more than 0.05), compared with the low dose group, the TNF-alpha, IL-1 beta and IL-6 levels of the high dose group are all reduced (P is less than 0.05), and the TNF-alpha, IL-1 beta and IL-6 levels of the high dose group and the medium dose group are not statistically significant (P is more than 0.05).

TABLE 7 groups of rat pharyngeal mucosal tissues TRAF6, NF- κ Bp65mRNA

As can be seen from the above Table 7, compared with the normal group, the model group has the advantages that the expression of TRAF6 and NF-kappa Bp65mRNA in pharyngeal mucosa tissues of rats is increased (P is less than 0.05); compared with the model group, TRAF6 and NF-kappa Bp65mRNA expression in the medium, high and low dose groups were all reduced (P < 0.05).

Experimental example 6 preparation of pulmonary fibrosis model of experimental animal

1. Grouping of laboratory animals

48 male SD rats were housed in the animal room of the Sichuan university college of Western medicine for one week, and were allowed free access to water and food for acclimation. After one week, the body weights were weighed and recorded, and divided into a blank control group (C), a pulmonary fibrosis disease model group (M), a low dose administration group (D), a medium dose administration group (Z), and a high dose administration group (G) in a random manner, and 8 each of the pirfenidone positive control group (P).

2. Establishment and administration treatment of pulmonary fibrosis SD rat disease model

The SD rat pulmonary fibrosis disease model is established by adopting a mode of bleomycin perfusion through tracheotomy, and the model making dose is 5 mg/kg. The blank control group (C) was replaced with an equal amount of physiological saline.

The treatment drugs are given on the second day of model building, except that the blank control group and the pulmonary fibrosis model group are administrated with the same amount of physiological saline every day for intragastric administration, rats in the administration group are continuously administrated for 3 weeks respectively according to the crude drug amount by the low dose of 0.45g/kg/d, the middle dose of 0.9g/kg/d and the high dose of 1.8 g/kg/d.

Hair color, behavior, diet, feces, etc. were observed and recorded daily, and body weight changes were recorded weekly. At the end of week 3, 8 experimental rats were sacrificed and fasted for 12h before sacrifice.

Experimental example 7 Collection and measurement of pulmonary fibrosis model specimen

1. Collection of serum samples

And (3) taking a rat to be killed, administering the drug for 24 hours, weighing the rat, injecting 1% sodium pentobarbital into the abdominal cavity for anesthesia, splitting the thoracic cavity, taking blood from the heart, centrifuging 3000-4000 g at 4 ℃ for 10-20 min. Subpackaging and storing at-80 deg.C.

2. Collection of tissue specimens

(1) After the heart exsanguinates, quickly separating and picking lung tissues, rinsing the lung tissues in precooled physiological saline to remove blood, sucking excess water by using filter paper, weighing the lung tissues and calculating the index of the visceral organs.

(2) After weighing, the right lung tissue is taken and quickly frozen at-80 ℃ for the measurement of subsequent indexes. The content of TNF-alpha and IL-1 beta in serum and the content of TGF-beta in lung tissue are measured by an Elisa method, and the content of Hydroxyproline (HYP) in the lung tissue is measured by an alkaline water method.

3. TNF-alpha content in serum of each group

As shown in fig. 11, the TNF- α content results in each group of sera, where P <0.01 compared to the blank group; # denotes P <0.05 compared to model group; # indicates P <0.01 compared to model group; ns indicates no significant difference compared to the model group. The results show that the content of the inflammatory factor TNF-alpha in the model group is obviously increased compared with that in the blank group, the model is successfully established, the content of the TNF-alpha in the medium-dose and high-dose three-group drug treatment group is obviously reduced compared with that in the model group, and the inflammation water level is reduced.

4. Content of IL-1. beta. in serum of each group

As shown in fig. 12, the results of the content of IL-1 β in each group of serum are indicated by P <0.01 compared to the blank group; # denotes P <0.05 compared to model group; # indicates P <0.01 compared to the model group. The results show that the content of the inflammatory factor IL-1 beta in the model group is obviously increased compared with that in the blank group, the model is successfully established, the content of the IL-1 beta in the low, medium and high drug treatment groups is obviously reduced compared with that in the model group, and the inflammation water level is reduced.

5. Hydroxyproline content in lung tissue of each group

As shown in figure 13, the Hydroxyproline (HYP) content in lung tissue of each group is indicated by P <0.01 compared to the blank group; # denotes P <0.05 compared to model group; # indicates P <0.01 compared to the model group. Hydroxyproline is one of the main components of collagen of the body and is an important index of collagen tissue metabolism. The HYP in the lung tissue of the model group is obviously higher than that of the blank group, which indicates that the lung tissue of the model group animals has pulmonary fibrosis. The lung tissue HYP content of the low, medium and high administration groups is obviously reduced after treatment, which shows that the drug can obviously reduce the degree of pulmonary fibrosis induced by bleomycin.

6. TGF-beta content in 10% lung homogenate in each group

As shown in figure 14, TGF- β content in 10% lung homogenate of each group is indicated by P <0.01 compared to the blank group; # denotes P <0.05 compared to model group; # indicates P <0.01 compared to the model group. TGF-beta is an important induction factor in the course of pulmonary fibrosis, can attract fibroblasts and stimulate the proliferation capacity of the fibroblasts, and can also induce epithelial-mesenchymal transition. The content of TGF-beta in the lung tissue of the model group induced by bleomycin is obviously increased compared with that of the blank group. The TGF-beta content of lung tissues of the low, medium and high administration groups is obviously reduced, which shows that the drug of the invention can obviously reduce the level of pulmonary fibrosis induced by bleomycin.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Claims

1. A fritillaria extract, wherein the alkaloids in the fritillaria extract comprise: the medicinal composition comprises sipeimine, peimine, sipeimine glycoside, peimine, peiminine, peimine, ibimine glycoside A and dehydroibeiiminine.

2. The Bulbus Fritillariae Cirrhosae extract as claimed in claim 1, wherein the Bulbus Fritillariae Cirrhosae extract is obtained by extracting Bulbus Fritillariae Cirrhosae with alcohol and concentrating.

3. The fritillaria cirrhosa extract as claimed in claim 2, wherein the fritillaria cirrhosa extract is obtained by extracting fritillaria cirrhosa bulbs with alcohol under reflux at 60-90 ℃ and concentrating.

4. A preparation method of a fritillaria cirrhosa extract is characterized by comprising the following steps:

pulverizing the bulbs of the fritillaria cirrhosa, soaking the bulbs of the fritillaria cirrhosa in an alcohol solution overnight, continuously performing reflux extraction at the temperature of 60-90 ℃, collecting an extracted concentrated solution, removing a solvent to obtain a concentrated extract, and performing vacuum drying under reduced pressure to obtain an extract of the fritillaria cirrhosa;

the alkaloid in the fritillaria cirrhosa extract comprises: the medicinal composition comprises sipeimine, peimine, sipeimine glycoside, peimine, peiminine, peimine, ibimine glycoside A and dehydroibeiiminine.

5. Use of an extract of fritillaria cirrhosa as claimed in any of claims 1 to 3 for the preparation of a medicament for the treatment of chronic respiratory diseases.

6. The use of claim 5, wherein the chronic respiratory disease comprises: chronic obstructive pneumonia, chronic pharyngitis, and pulmonary fibrosis.