Lumbellate lobelia herb extract, preparation method thereof and application of lobelia lobelia herb extract in preparation of anti-asthma drugs

Abstract

The invention provides a lobelia tomentosa extract, a preparation method thereof and application of the lobelia tomentosa extract in preparation of anti-asthma drugs, and belongs to the technical field of medicines. The invention provides a preparation method of a lobelia himalayana extract, which comprises the following steps: 1) crushing roots of lobelia himalayana, adding ethanol to infiltrate and extract to obtain an extracting solution, and concentrating to remove ethanol to obtain a lobelia himalayana alcohol extract; 2) adding water into the lobelia tomentosa alcohol extract, resuspending, adjusting the pH to be acidic, and reacting to obtain a crude extract acid solution; 3) extracting the crude extract acid solution with ethyl acetate, separating to obtain an acid water layer, adjusting the pH to be alkaline, and reacting to obtain a crude extract alkaline solution; and 4) extracting the alkali solution of the crude extract with dichloromethane, separating to obtain a dichloromethane layer, and concentrating to obtain the total alkaloid extract of the lobelia tomentosa. The lobelia tomentosa extract disclosed by the invention can be used for remarkably improving pathological conditions such as lung inflammatory infiltration, pulmonary parenchyma, collagenous fiber increase, thickening and the like of asthma patients.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a Lobelia pubescens extract, a preparation method thereof and application thereof in the preparation of anti-asthma drugs. Background Art

[0002] Asthma (BA) is a heterogeneous disease characterized by chronic inflammation of the airways involving multiple cells and cell components. This chronic inflammation is associated with airway hyperresponsiveness, and usually presents with extensive and variable reversible expiratory airflow limitation, leading to recurrent symptoms such as wheezing, shortness of breath, chest tightness, and / or cough, with intensity varying over time. It often attacks and worsens at night and / or in the early morning, and most patients can relieve themselves or after treatment. If bronchial asthma is not diagnosed and treated in time, it can cause irreversible airway narrowing and airway remodeling as the course of the disease prolongs. Bronchial asthma is a chronic respiratory disease that has a serious impact on people of all ages and is one of the most common chronic diseases with high morbidity and mortality. At present, in many countries, the prevalence of asthma is still on the rise, placing a serious burden on the global health care system and society. According to the guiding principles of the most authoritative global asthma initiative GINA guidelines, the goal of asthma treatment should be to achieve and maintain clinical control. Therefore, drugs for the treatment of asthma are divided into control drugs and relieve drugs. Asthma control drugs achieve the purpose of asthma control through long-term anti-inflammatory effects, mainly including inhaled and systemic glucocorticoids (the first choice for long-term control), leukotriene modifiers, long-acting β2 receptor agonists, sustained-release theophylline and anti-IgE antibodies, etc. Rapid relief drugs (relief drugs) are used to relieve the symptoms of acute asthma attacks. Relief drugs are drugs used during asthma attacks, mainly including rapid-acting inhaled short-acting β2 receptor agonists, formoterol, anticholinergic drugs, short-acting theophylline, etc. Relief drugs can directly stimulate the respiratory tract to relax it, which can immediately improve breathing conditions. Therefore, many asthma patients rely on reliever drugs for a long time. One of the major functions of reliever drugs is to block histamine, a harmful substance in asthma, but because there are antibodies in the human body, it will cause mast cells to release more histamine. Therefore, asthma patients should not overuse reliever drugs.

[0003] Through the above analysis, it can be seen that the problems and defects of the existing technology are: clinical medications are mainly drugs that combine bronchial dilation and airway inflammation. These drugs treat asthma by inhibiting airway inflammation and relaxing airway smooth muscle, but patients who take such drugs for a long time will develop drug resistance, which will weaken the efficacy of the drugs and bring serious adverse consequences. Therefore, it is urgent to develop drugs that are highly effective and low in toxicity and can target the source of asthma (sensitization and excitation stages).

[0004] Lobelia clavata E.Wimm belongs to the genus Lobelia of Campanulaceae, and its roots, leaves and whole plants are mainly used as medicine. Lobelia clavata is used to treat many diseases in some areas, including asthma with its roots. The dosage is to use 30g of roots, decocted in water, twice a day, 20mL each time, with pepper as a medicine guide. However, its pharmacological activity has not been further described. Therefore, its pharmacological effect application development can be experimentally verified to broaden the new application disease range of Lobelia plants, and provide a reliable direction and basis for the development of safe and effective new drugs for the treatment of asthma. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a Lobelia pubescens extract, so as to effectively extract the pharmacologically active ingredients of the Lobelia pubescens and improve its therapeutic effect on asthma.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a Lobelia pubescens extract, comprising the following steps:

[0008] 1) taking the root of Lobelia pubescens and crushing it, adding ethanol to soak and extract, to obtain an extract, and concentrating the extract to remove ethanol to obtain Lobelia pubescens ethanol extract;

[0009] 2) resuspending the Lobelia pubescens ethanol extract in water, adjusting the pH to be acidic, and obtaining a crude extract acid solution after acid-base neutralization reaction;

[0010] 3) extracting the crude acid solution with ethyl acetate, separating to obtain an acid water layer, adjusting the pH of the acid water layer to be alkaline, and performing double decomposition reaction to obtain a crude alkaline solution;

[0011] 4) extracting the crude extract alkaline solution with dichloromethane, separating and obtaining a dichloromethane layer, and concentrating the dichloromethane layer to obtain a total alkaloid extract of Lobelia pubescens.

[0012] Preferably, in step 1), the mass volume ratio of the roots of Lobelia pubescens to ethanol is 80-100 g:800-1000 mL, and the concentration of the ethanol is 90-95%; the infiltration time is 20-30 min, the extraction times are 2-4 times, and each extraction takes 20-40 min; the extraction is assisted by ultrasound, and the frequency of the ultrasound is 30-50 Hz.

[0013] Preferably, in step 2), the mass volume ratio of the Lobelia pubescens ethanol extract to water is 8-10 g:80-100 mL; the pH is 2-3; and the time of the acid-base neutralization reaction is 20-30 min.

[0014] Preferably, the amount of ethyl acetate used in step 3) is 1 to 2 times the volume of the crude acid solution, the number of extractions is 2 to 4 times; the pH is 9 to 11, and the double decomposition reaction time is 20 to 30 minutes.

[0015] Preferably, the amount of dichloromethane used in step 4) is 1 to 2 times the volume of the crude extraction alkaline solution, and the number of extractions is 2 to 4 times.

[0016] Preferably, the total alkaloid extract of Lobelia pubescens obtained in step 4) is further purified by alkaline alumina column chromatography.

[0017] The present invention also provides a Lobelia pubescens extract prepared by the above preparation method, wherein the Lobelia pubescens extract comprises the Lobelia pubescens ethanol extract obtained in step 1) and / or the Lobelia pubescens total alkaloid extract obtained in step 4).

[0018] Preferably, the main effective active ingredients in the Lobelia pubescens extract include: lobeline, lobeline ketone, neolobeline A, lobeline and dehydrolobeline.

[0019] The present invention also provides a use of the Lobelia pubescens extract in preparing a drug for preventing and / or treating asthma.

[0020] Preferably, the drug further contains pharmaceutically acceptable excipients or auxiliary ingredients; the drug is an oral preparation, an injection or a topical preparation.

[0021] The total alkaloid extract of Lobelia pubescens of the present invention can significantly improve the release of allergic asthma-related cytokines in the airways, improve allergic conditions, and improve pathological conditions such as inflammatory infiltration, lung consolidation, increased collagen fibers, and thickening of the lungs of asthmatic mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A is a HE staining image of the lung tissue of each group of mice in Example 4 (magnification: 200×), and B is a bar graph of tracheal wall thickness calculated by Fiji ImageJ of the lung tissue of each group of mice in Example 4;

[0023] Figure 2 A is a Masson staining image of the lung tissue of each group of mice in Example 4 (magnification: 200×), and B is a histogram of the positive area calculated by Fiji ImageJ of the lung tissue of each group of mice in Example 4;

[0024] Figure 3A is a variance analysis diagram of the results of LTB4 content in lung tissue of each group of mice in Example 4, B is a variance analysis diagram of the results of TGF-β1 content in lung tissue of each group of mice in Example 4, C is a variance analysis diagram of the results of IL-4 content in lung tissue of each group of mice in Example 4, and D is a variance analysis diagram of the results of IFN-γ content in lung tissue of each group of mice;

[0025] Figure 4 A is the electrophoresis band diagram of related proteins in the lung tissues of each group of mice in Example 4, B is the p-p38 protein expression level diagram in the lung tissues of each group of mice in Example 4, and C is the p-mTOR protein expression level diagram in the lung tissues of each group of mice in Example 4;

[0026] Figure 5 A is the electrophoresis band diagram of proteins related to the TLR4 / MyD88 / NF-κB signaling pathway in the lung tissues of each group of mice in Example 4, B is the graph of the TLR4 protein expression level in the lung tissues of each group of mice in Example 4, C is the graph of the MyD88 protein expression level in the lung tissues of each group of mice in Example 4, and D is the graph of the NF-κB protein expression level in the lung tissues of each group of mice in Example 4. DETAILED DESCRIPTION

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1

[0029] Take 100g of clean and dried Lobelia pubescens root powder, use 1000ml of water as extraction solvent, boil it in water for 0.5 hours, and concentrate it under reduced pressure at 0.08MPa to obtain Lobelia pubescens water extract (named LC-W in the experiment), with an extraction rate of 3%.

[0030] Example 2

[0031] 100 g of clean and dried Lobelia pubescens root powder was soaked in 1000 ml of 95% ethanol aqueous solution for 10 minutes, and then ultrasonically extracted three times at an ultrasonic frequency of 40 Hz for 0.5 hours each time. The extract was concentrated by a rotary evaporator at 0.08 MPa to recover ethanol, and the extract was placed in a fume hood to evaporate the remaining ethanol to obtain Lobelia pubescens ethanol extract (named LC-E in the experiment), with an extraction rate of 10%.

[0032] Example 3

[0033] According to the method in Example 2, 10 g of the alcohol extract of Lobelia pubescens was extracted and suspended in 100 ml of water, and the pH of the solution was adjusted to 2 with concentrated hydrochloric acid; after 0.5 hours of acid-base neutralization reaction, a crude acid solution was obtained. The crude acid solution was extracted three times with 1.5 times the volume of ethyl acetate in a separatory funnel, and an acid water layer and an ethyl acetate layer were obtained after standing. The acid water layer was taken and the pH of the solution was adjusted to 10 with NaOH, and a crude alkaline solution was obtained after 0.5 hours of double decomposition reaction. The crude alkaline solution was extracted three times with 1.5 times the volume of dichloromethane in a separatory funnel to obtain an alkaline water layer and a dichloromethane layer. The dichloromethane layer was concentrated under reduced pressure at 0.08 MPa to obtain a crude biological extract of total alkali, and the extraction rate was 0.1%. Finally, the crude extract of total alkaloids was chromatographed on an alkaline alumina column, washed with petroleum ether to remove small polar substances such as fatty acids, eluted with dichloromethane and methanol (5:1), desalted, concentrated and dried to obtain the purified total alkaloids (named LC-A in the experiment).

[0034] Example 4 Therapeutic effects of Lobelia pubescens water extract, Lobelia pubescens ethanol extract and Lobelia pubescens total alkaloids on asthma model

[0035] 1. Preparation of Lobelia pubescens water extract, Lobelia pubescens ethanol extract and Lobelia pubescens total alkaloids

[0036] The Lobelia pubescens water extract, Lobelia pubescens ethanol extract and Lobelia pubescens total alkaloids used in this example were prepared according to the methods of Examples 1 to 3, respectively.

[0037] 2. Experimental Animals

[0038] Forty-two healthy SPF Balb / c female mice, aged 4-5 weeks and weighing 18-20 g, were provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences and housed in the animal room of the School of Life Sciences and Technology, Kunming University of Science and Technology.

[0039] 3. Experimental methods

[0040] The mice were divided into 6 groups, with 7 mice in each group. The mouse allergic asthma model was established by intraperitoneal injection of ovalbumin (OVA). The asthma model group and the drug-treated group were sensitized by intraperitoneal injection of 0.2 ml of OVA sensitization solution [containing 2 mg of OVA (GradeⅤ) and 20 mg of aluminum hydroxide] on days 0, 7, and 14, respectively. The mice in the normal control group were sensitized with saline instead of sensitization solution, and the injection time, site, and dose were the same as those of the drug-treated group mice. From the 21st day of the experiment, the mice were placed in a 40cm×30cm×20cm homemade nebulizer, and a 1% OVA (Grade II) solution was atomized and inhaled by a 402AI ultrasonic nebulizer once a day for 30 minutes each time for 14 days. The control group was stimulated by atomization of an equal amount of saline. The Lobelia pubescens water extract group was given Lobelia pubescens water extract (100 mg / kg) by gavage, the Lobelia pubescens alcohol extract group was given Lobelia pubescens alcohol extract (100 mg / kg) by gavage, the Lobelia pubescens total alkaloid group was given Lobelia pubescens total alkaloid (100 mg / kg) by gavage, the blank control group and the asthma model group were given an equal amount of normal saline by gavage, and the positive control group was given dexamethasone (1 mg / kg) by intraperitoneal injection. As shown in Table 1.

[0041] Table 1 Grouping and dosage of Lobelia pubescens for OVA asthma animal experiment

[0042] Grouping (drug administration / time) Group Abbreviation Dosage Blank control group Group C - Asthma model group Group M - Positive drug group DEX Group 1mg / kg Lobelia pubescens water-extracted group LC-W group 100mg / kg Lobelia pubescens alcohol extract LC-E group 100mg / kg Total alkaloids from Lobelia pubescens LC-A group 100mg / kg

[0043] Results: The observation of the appearance and behavior of mice showed that 10 to 15 minutes after each aerosol inhalation, the mice in the model group continued to scratch their heads and faces, became restless, arched their backs, breathed rapidly, and had abdominal muscle spasms. After a week of aerosol inhalation, their hair stood up and lost its luster. The mice in the control group did not have obvious abnormal reactions, and the above reactions of the mice in the drug intervention groups at various concentrations were milder.

[0044] 4. Collection of tissue specimens

[0045] The lung tissues of mice killed by eye bleeding were quickly removed, and the right lung was immersed in 4% paraformaldehyde for 2 weeks, followed by conventional OCT embedding medium embedding, sectioning, HE staining, Masson staining, and observation of tissue morphology and pathological state under a microscope. The remaining lung tissues were placed in cryovials and immediately frozen in liquid nitrogen. After freezing, they were stored at -80°C for use, and stored in a -80°C refrigerator for long-term use for subsequent index determination.

[0046] 5. Lung histopathological examination

[0047] The lung tissue fixed with 4% paraformaldehyde was embedded in OCT embedding medium and cut into 5 μm thick sections for hematoxylin-eosin (HE) and Masson trichrome staining. Lung tissue pathological analysis and image acquisition were performed under a microscope, and Fiji ImageJ was used to calculate the tracheal wall thickness and positive area. The results are shown in Figure 1 and Figure 2 shown.

[0048] HE staining ( Figure 1 ) The results showed that the pathological changes of the lung tissues of mice in each group were observed by taking photos under a microscope. Compared with the blank control group, the asthma model group showed inflammatory cell infiltration around the bronchi of the lung tissues of mice, thickening of the tracheal wall, thickening of the alveolar wall, damaged and detached airway epithelial cells, and rough and uneven airway surfaces. Compared with the asthma model group, after being given different extracts of Lobelia pubescens, the worst effect was the Lobelia pubescens water extract group, and the lung pathology of mice in this group did not improve significantly; while the lung pathology of mice in the Lobelia pubescens alcohol extract group and the Lobelia pubescens total alkaloid group was significantly improved. Among them, the improvement effect of the Lobelia pubescens total alkaloid group was better.

[0049] Masson staining ( Figure 2 ) The results showed that compared with the blank control group, the asthma model group showed obvious collagen fiber tissue hyperplasia and thickening. Compared with the asthma model group, after being given different extracts of Lobelia pubescens, the worst effect was the Lobelia pubescens water extract group, and the lung pathology of mice in this group did not improve significantly; while the Lobelia pubescens alcohol extract group and Lobelia pubescens total alkaloid group, the lung pathology of mice was significantly improved. Among them, the improvement effect of the Lobelia pubescens total alkaloid group was better.

[0050] The results showed that the ethanol extract of Lobelia pubescens and the total alkaloids of Lobelia pubescens improved the pathological conditions of lung inflammatory infiltration, lung consolidation, increased collagen fibers and thickening in OVA asthma mice to varying degrees. In order to better compare the efficacy of the ethanol extract of Lobelia pubescens and the total alkaloids of Lobelia pubescens, we compared the changes in cytokines and related proteins in the lung tissues of the two groups of mice.

[0051] 6. Determination of IL-4, TGF-β1, LTB 4 and IFN-γ content in lung tissue

[0052] The blank control group, asthma model group, positive drug group, Lobelia pubescens alcohol extract group, and Lobelia pubescens total alkaloid group were divided into five groups. Frozen lung tissues of the same size were taken from each group. PBS and protease phosphatase inhibitors were added, ground into homogenate, and then centrifuged to extract tissue supernatant. The ELISA kit instructions were followed to determine the changes in the contents of IL-4, TGF-β1, LTB 4, and IFN-γ. Graphpad Prism software was used for plotting and one-way analysis of variance (ANOVA). p<0.05 indicated a significant difference. The results are shown in Figure 3 And as shown in Table 2.

[0053] Table 2 Comparison of IL-4, TGF-β1, LTB 4 and IFN-γ levels in bronchoalveolar lavage fluid of mice in each group

[0054]

[0055] Note: Compared with the blank control group, a P<0.05; compared with the asthma group, b P<0.05

[0056] Depend on Figure 3 As shown in Table 2, compared with the blank control group, the concentrations of leukotriene inflammatory factor LTB4, key factor of airway remodeling TGF-β1, and Th2 response representative factor IL-4 in the lung tissue of mice in the asthma model group were significantly increased; the concentration of Th1 response representative factor IFN-γ was significantly reduced. Compared with the asthma model group, the ethanol extract group of Lobelia pubescens and the total alkaloid group of Lobelia pubescens could significantly reduce the concentrations of LTB4, TGF-β1, and IL4 in the lungs of asthmatic mice, and increase the concentration of IFN-γ.

[0057] 7. Western Blot to detect related protein levels in lung tissue

[0058] Remove the mouse lung tissue from liquid nitrogen, add an appropriate amount of steel beads, add pre-cooled RIPA lysis buffer and protease phosphatase inhibitors to extract total tissue protein, use a tissue grinder to break the tissue, place it on ice for 10 minutes, and vortex it every 3 minutes. Centrifuge at 14000rpm for 10 minutes at 4°C, take an appropriate amount of supernatant, and measure the protein concentration. Add an appropriate amount of loading buffer to the supernatant, mix well, and heat to denature the protein (100°C, 10 minutes). Prepare 7.5% and 10% SDS-PAGE separation gel and concentrated gel, load an equal amount of protein (20μg) per well, electrophoresed at a voltage of 150V, and stop electrophoresis after the target protein is separated. Transfer the membrane in an ice bath at a constant voltage of 110V for 2 hours, and then block it in 5% skim milk at room temperature for 2 hours. After blocking, the primary antibody was incubated at 4°C overnight, washed with TBST (3 times, 10 minutes / time) the next day, incubated with secondary antibody at room temperature for 1 hour, washed with TBST (3 times, 10 minutes / time), and imaged with ECL luminescence imaging system. Image J software was used to analyze the gray value of protein bands. Graphpad Prism software was used to draw graphs and perform one-way analysis of variance (ANOVA). p<0.05 indicated a significant difference. Figure 4 and Figure 5 shown.

[0059] Depend on Figure 4 It can be seen that compared with the blank control group, the p-p8 and p-mTOR protein levels in the lung tissue of the asthma model mice were increased; compared with the asthma group, the Lobelia pubescens alcohol extract group and the Lobelia pubescens total alkaloid group could reduce the p-p38 protein level in the lungs of asthmatic mice. Lobelia pubescens total alkaloids could also reduce the p-mTOR protein level in the lungs of asthmatic mice.

[0060] Depend on Figure 5 It can be seen that compared with the blank control group, the expression levels of TLR4, MyD88 and NF-κB proteins in the lung tissue of mice in the asthma model group were significantly increased. Compared with the asthma model group, the expression levels of TLR4, MyD88 and NF-κB proteins in the lung tissue of mice in the Lobelia pubescens total alkaloids group were reduced. Therefore, we speculate that the total alkaloids of Lobelia pubescens relieve the symptoms of asthmatic mice and reduce the levels of inflammatory factors. Its mechanism of action may be related to the inhibition of the expression of the TLR4 / MyD88 / NF-κB signaling pathway.

[0061] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a Lobelia pubescens extract, characterized in that: The steps include: 1) taking the root of Lobelia pubescens and crushing it, adding ethanol to soak and extract, to obtain an extract, and concentrating the extract to remove ethanol to obtain Lobelia pubescens ethanol extract; 2) resuspending the Lobelia pubescens ethanol extract in water, adjusting the pH to be acidic, and obtaining a crude extract acid solution after acid-base neutralization reaction; 3) extracting the crude acid solution with ethyl acetate, separating to obtain an acid water layer, adjusting the pH of the acid water layer to be alkaline, and performing double decomposition reaction to obtain a crude alkaline solution; 4) extracting the crude extract alkaline solution with dichloromethane, separating and obtaining a dichloromethane layer, and concentrating the dichloromethane layer to obtain a total alkaloid extract of Lobelia pubescens.

2. The preparation method according to claim 1, characterized in that In step 1), the mass volume ratio of the roots of Lobelia pubescens to ethanol is 80-100 g: 800-1000 mL, and the concentration of the ethanol is 90-95%. The soaking time is 20-30 min, the extraction times are 2-4 times, and each extraction takes 20-40 min. The extraction is assisted by ultrasound, and the frequency of the ultrasound is 30-50 Hz.

3. The preparation method according to claim 1, characterized in that: In step 2), the mass volume ratio of the Lobelia pubescens ethanol extract to water is 8-10 g:80-100 mL; the pH is 2-3; and the time of the acid-base neutralization reaction is 20-30 min.

4. The preparation method according to claim 1, characterized in that: In step 3), the amount of ethyl acetate used is 1 to 2 times the volume of the crude acid solution, and the number of extractions is 2 to 4 times; the pH is 9 to 11, and the time for the double decomposition reaction is 20 to 30 minutes.

5. The preparation method according to claim 1, characterized in that: The amount of dichloromethane used in step 4) is 1 to 2 times the volume of the crude extraction alkaline solution, and the number of extractions is 2 to 4 times.

6. The preparation method according to claim 1, characterized in that: The total alkaloid extract of Lobelia pubescens obtained in step 4) is further purified by alkaline alumina column chromatography.

7. A Lobelia pubescens extract prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The Lobelia pubescens extract comprises the Lobelia pubescens ethanol extract obtained in step 1) and / or the Lobelia pubescens total alkaloid extract obtained in step 4).

8. The Lobelia pubescens extract according to claim 7, characterized in that The main effective active ingredients in the Lobelia pubescens extract include lobeline, lobeline ketone, neolobeline A, lobeline and dehydrolobeline.

9. Use of the Lobelia pubescens extract according to claim 7 or 8 in the preparation of a drug for preventing and / or treating asthma.

10. The use according to claim 9, characterized in that The medicine also contains pharmaceutically acceptable excipients or auxiliary ingredients; the medicine is an oral preparation, an injection or a local administration preparation.

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