A method for extracting and separating houttuynia cordata extract and its application

Abstract

The invention discloses an extraction and separation method of houttuynia cordata extract and its application. The extraction and separation method of houttuynia cordata extract comprises extracting houttuynia cordata with ethanol aqueous solution to obtain ethanol extract, recovering ethanol and concentrating, extracting with ethyl acetate, taking water phase to obtain ethyl acetate water part, and obtaining houttuynia cordata extract. The houttuynia cordata extract can be used to prepare drugs for preventing and treating chronic obstructive pulmonary disease, reduce the secretion of airway epithelial cell mucin MUC5AC, increase the activity of pulmonary microvascular endothelial cells, inhibit the apoptosis of pulmonary microvascular endothelial cells, reduce the number of lung tissue macrophages, and improve lung function.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to an extraction and separation method of houttuynia cordata extract and application thereof. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is one of the major chronic diseases that seriously endangers the health of residents. It is characterized by persistent airflow limitation and corresponding respiratory symptoms. In recent years, more and more studies have revealed the important role of apoptosis and excessive airway mucus secretion in the pathogenesis of COPD.

[0003] First, the study by Demedts et al. (Demedts IK, Demoor T, Bracke KR, et al. Role of apoptosis in the pathogenesis of COPD and pulmonary emphysema. Respir Res. 2006; 7(53): 1–10. doi: 10.1186 / 1465-9921-7-53) showed that the apoptotic alveolar epithelial cells and endothelial cells in the lungs of COPD patients increased, and these pathological changes could not be offset by the proliferation of structural cells, leading to the destruction of lung tissue and the development of emphysema, suggesting that apoptosis of pulmonary structural cells may be an important upstream event in the pathogenesis of COPD. Further studies have found that apoptosis of pulmonary vascular endothelial cells is not only an initiating event in the onset of COPD, but also plays an important role in the progression of the disease. Peng et al. (Peng H, Yang M, Chen ZY, Chen P, Guan CX, Xiang XD, et al. Expression and methylation of mitochondrial transcriptionfactor a in chronic obstructive pulmonary disease patients with lung cancer. PLoS ONE. 2013;8(12):e82739. doi: 10.1371 / journal.pone.0082739) examined lung tissues of 11 COPD patients and 10 non-COPD patients. The results showed that the apoptosis rate of pulmonary vascular endothelial cells in COPD patients was significantly increased. Cigarette smoking (CS) is one of the main causes of COPD and can accelerate the occurrence and development of COPD by inducing apoptosis of pulmonary vascular endothelial cells (Song Q, Chen P, Liu XM. The role of cigarette smoke-induced pulmonary vascular endothelial cell apoptosis in COPD. Respir Res. 2021 Feb 5;22(1):39. doi: 10.1186 / s12931-021-01630-1. PMID: 33546691; PMCID:PMC7866753.). Therefore, inhibiting cigarette smoke-induced endothelial cell apoptosis may delay or reverse the progression of COPD.

[0004] Secondly, the mucus and cilia on the surface of the airway epithelium together form the cilia-mucus barrier, which serves as the first line of defense against the invasion of bacteria, viruses and harmful substances. However, in COPD patients, excessive airway mucus secretion is another important pathological feature of COPD. Excessive mucus secretion leads to airway obstruction, restricts airflow, reduces lung function, and causes repeated airway infections, thereby aggravating the progression of COPD. (Chen Yumei, Tong Jin. Research progress on the mechanism of airway mucus hypersecretion [J]. Basic Medicine and Clinic, 2016, 36(11): 1573-1577). There is mucus retention and even mucus blockage in the lung tissue of patients who died of COPD. MUC5AC is the main component of mucin, accounting for 95% of the total mucin secretion of airway epithelium. MUC5AC is over-secreted in COPD patients. Studies have shown that mucus with high MUC5AC content is prone to form embolism in the airways, resulting in obstruction of mucus clearance, promoting the colonization of pathogens and the persistence of infection, and further exacerbating the disease. External stimuli (such as air pollution, smoking, and biomass fuels) can significantly increase the expression of MUC5AC, thereby exacerbating mucus secretion and infection in the lower respiratory tract. Therefore, the secretion level of MUC5AC is a key evaluation indicator of COPD, and reducing the secretion of MUC5AC has become an important goal in the treatment of COPD. (Hou Hongdou, Zhang Liying, Li Yangyang, Niu Fan, Li Jing, Yun Chengcai, Yong Wenxing. Research progress on the mechanism of increased mucin 5AC production in chronic obstructive pulmonary disease and intervention with traditional Chinese medicine [J]. Journal of Traditional Chinese Medicine, 2024, 52(2): 108-115).

[0005] In summary, the regulation of apoptosis of lung structural cells and the intervention of excessive airway mucus secretion are of great significance in the prevention and treatment of COPD, and drug development methods targeting these two mechanisms have significant clinical value. Summary of the invention

[0006] In view of the above technical status, the present invention provides a method for extracting and separating Houttuynia cordata extract, and further provides the extract obtained by the method and application thereof.

[0007] In a first aspect, the present invention provides a method for extracting and separating a Houttuynia cordata extract, comprising the following steps:

[0008] (1) extracting Houttuynia cordata with an ethanol aqueous solution to obtain an ethanol extract;

[0009] (2) recovering ethanol from the ethanol extract to obtain a concentrated solution;

[0010] (3) Extracting the concentrated solution with ethyl acetate, taking the aqueous phase to obtain the ethyl acetate water portion.

[0011] In some embodiments, the extraction and separation method further comprises: (4) concentrating and drying the ethyl acetate water portion to obtain a dry paste.

[0012] In some embodiments, in step (1), the volume concentration of the ethanol aqueous solution is 55-65%, preferably 58-62%, and more preferably 60%;

[0013] In some embodiments, in step (1), in step (1), the extraction temperature is such that the ethanol aqueous solution is in a reflux state;

[0014] In some embodiments, in step (1), the number of extractions is 1-5 times, preferably 2-3 times; the time for each extraction is 1-3 hours, preferably 1.5-2 hours;

[0015] In some embodiments, in step (1), during each extraction, the mass volume ratio of Houttuynia cordata to the ethanol aqueous solution used is 1:5-15 (g / ml), preferably 1:10-12 (g / ml).

[0016] In some embodiments, in step (3), the number of extractions is 1-5 times, preferably 2-4 times, and more preferably 3 times;

[0017] In some embodiments, in step (3), the volume of ethyl acetate used for each extraction is 0.5 to 3 times, preferably 1 to 1.5 times, the volume of the concentrated solution.

[0018] In some embodiments, in step (4), the ethyl acetate extract is concentrated under reduced pressure at 40-80°C and a vacuum degree of 0.04-0.09 MPa to obtain an extract, and the extract is dried at 50-80°C and a vacuum degree of 0.04-0.09 MPa to obtain a dry paste.

[0019] In a second aspect, the present invention provides a Houttuynia cordata extract prepared by the above extraction and separation method.

[0020] In a third aspect, the present invention provides an application of the Houttuynia cordata extract, specifically, the application is an application for preparing a drug for preventing and treating chronic obstructive pulmonary disease.

[0021] In some embodiments, the application is the application for preparing a drug for reducing the secretion of airway epithelial cell mucin MUC5AC.

[0022] In some embodiments, the application is for preparing a drug for increasing the activity of pulmonary microvascular endothelial cells and / or inhibiting apoptosis of pulmonary microvascular endothelial cells.

[0023] In a fourth aspect, the present invention provides a composition comprising the Houttuynia cordata extract.

[0024] In some embodiments, the composition is in the form of capsules, powders, granules, tablets, suspensions or injections.

[0025] The present invention studies the extraction method of Houttuynia cordata, and the obtained extract has obvious effect in treating COPD, especially reducing the secretion of airway epithelial cell mucin MUC5AC and inhibiting the apoptosis of pulmonary microvascular endothelial cells, which has a significant effect compared with other extracts and is expected to be developed into a drug for preventing and treating COPD. At the same time, Houttuynia cordata is a medicinal material for both medicinal and edible purposes, and the extract can also be prepared into health food. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention provides a preparation process for the Houttuynia cordata extract of Example 1 and Comparative Examples 2-7.

[0027] Figure 2 The immunofluorescence staining results of the Houttuynia cordata extract of the present invention on MUC5AC are shown; the first row and the second row are immunofluorescence staining images of different fields of view, respectively.

[0028] Figure 3 The results of hematoxylin-eosin (HE) staining of lung tissue by the Houttuynia cordata extract of the present invention are shown in the figure, wherein the first row is a 200-fold magnification and the second row is a 400-fold magnification; the red arrows indicate inflammatory cells. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Example 1

[0030] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0031] Take 1 kg of Houttuynia cordata, add 12 L 60% (v / v) ethanol, boil and reflux for extraction for 2 h, filter and collect the filtrate, add 10 L 60% (v / v) ethanol to the residue, boil and reflux for extraction for 1.5 h, filter and collect the filtrate, combine the two filtrates, recover ethanol under reduced pressure, concentrate until there is no alcohol taste, and add water to adjust the volume to obtain 800 ml of extract solution.

[0032] Take 200 ml of the above extract solution, add 300 ml of ethyl acetate for extraction, separate the phases, add 200 ml of ethyl acetate to the aqueous phase for extraction again, separate the phases, add 200 ml of ethyl acetate to the aqueous phase for extraction three times, separate the phases, take the aqueous phase, and concentrate under reduced pressure at a temperature of 40°C-80°C and a vacuum degree of 0.04-0.09Mpa, and dry to obtain a dry paste of the ethyl acetate water portion.

[0033] Comparative Example 1

[0034] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0035] Take 1 kg of Houttuynia cordata, add 12 L of water, boil and reflux for 2 h, filter and collect the filtrate, add 10 L of water to the residue, boil and reflux for 1.5 h, filter and collect the filtrate, combine the two filtrates, and concentrate under reduced pressure at a temperature of 40°C-80°C and a vacuum degree of 0.04-0.09 MPa to obtain an extract, and dry the extract at 50-80°C and a vacuum degree of 0.04~0.09 MPa to obtain a water extract dry paste.

[0036] Comparative Example 2

[0037] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0038] Take 200 ml of the extract solution of Example 1, recover ethanol under reduced pressure and concentrate to obtain an extract, and dry the extract at 50-80° C. and a vacuum degree of 0.04-0.09 MPa to obtain an alcohol extract dry paste.

[0039] Comparative Example 3

[0040] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0041] The ethyl acetate phases extracted three times in Example 1 are combined, and reduced pressure concentration is performed at a temperature of 40°C-80°C and a vacuum degree of 0.04-0.09Mpa to obtain an extract, and the extract is dried at 50-80°C and a vacuum degree of 0.04~0.09Mpa to obtain a dry paste of the ethyl acetate part.

[0042] Comparative Example 4

[0043] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0044] Take 200 ml of the extract solution of Example 1, add 300 ml of n-butanol for extraction, separate the phases, add 200 ml of n-butanol to the aqueous phase for extraction again, separate the phases, add 200 ml of n-butanol to the aqueous phase for extraction three times, separate the phases, take the aqueous phase, and concentrate under reduced pressure at a temperature of 40°C-80°C and a vacuum degree of 0.04-0.09 MPa to obtain an extract, and the extract is dried at 50-80°C and a vacuum degree of 0.04~0.09 MPa to obtain a dry paste of the n-butanol water portion.

[0045] Comparative Example 5

[0046] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0047] The n-butanol phases extracted three times in Comparative Example 4 were combined, and reduced pressure concentration was performed at a temperature of 40°C-80°C and a vacuum degree of 0.04-0.09Mpa to obtain an extract, and the extract was dried at 50-80°C and a vacuum degree of 0.04~0.09Mpa to obtain a dry paste of the n-butanol part.

[0048] Comparative Example 6

[0049] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0050] Take 200 ml of the extract solution of Example 1, add 300 ml of petroleum ether for extraction, separate the phases, add 200 ml of petroleum ether to the aqueous phase for extraction again, separate the phases, add 200 ml of petroleum ether to the aqueous phase for extraction three times, separate the phases, take the aqueous phase, and concentrate under reduced pressure at a temperature of 40°C-80°C and a vacuum degree of 0.04-0.09 MPa to obtain an extract, and dry the extract at 50-80°C and a vacuum degree of 0.04~0.09 MPa to obtain a petroleum ether water portion dry paste.

[0051] Comparative Example 7

[0052] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0053] The petroleum ether phases extracted three times in Comparative Example 6 were combined, and reduced pressure concentration was performed at a temperature of 40°C-80°C and a vacuum degree of 0.04-0.09 MPa to obtain an extract, and the extract was dried at 50-80°C and a vacuum degree of 0.04-0.09 MPa to obtain a dry paste of the petroleum ether part.

[0054] The preparation process of Example 1 and Comparative Examples 2-7 is shown in Figure 1 .

[0055] Application Example 1 Effect of Houttuynia cordata extract on mucus secretion of H292 cells

[0056] 1 Experimental Materials

[0057] 1.1 Instruments and Equipment

[0058]

[0059] 1.2 Main Reagents

[0060]

[0061] 2 Experimental methods

[0062] 2.1 Preparation of main solution:

[0063] Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 4, Comparative Example 6: Weigh an appropriate amount of dry paste, dissolve it in RPMI 1640 culture medium to prepare a 10 mg / mL stock solution, filter it with a 0.22 μM filter membrane, store it in aliquots at -20°C, and dilute it with RPMI 1640 culture medium before use.

[0064] Comparative Example 3, Comparative Example 5, Comparative Example 7: Weigh an appropriate amount of dry paste, dissolve it in DMSO to prepare a 50 mg / mL stock solution, store it in aliquots at -20°C, and dilute it with RPMI 1640 culture medium before use.

[0065] The Houttuynia cordata injection was diluted in a 3-fold gradient to obtain 8 concentration samples.

[0066] The corresponding parts of each group are as follows:

[0067]

[0068] 2.2 Cell culture:

[0069] Quickly remove the cryopreservation tube containing cells from liquid nitrogen, immediately put it into a 37℃ water bath for rapid thawing for about 1 minute, centrifuge it at 800rpm for 2 minutes, wipe the cryopreservation tube with 75% alcohol, move it to the clean bench, remove the cryopreservation solution, add RPMI 1640 culture medium containing 10% fetal bovine serum, blow it evenly, transfer it to a culture dish, and culture it in a 37℃, 5% CO2 constant temperature cell culture incubator. When the cell growth and fusion reach about 80% under an inverted microscope, pass it at a ratio of 1:3 to 1:8. Take the cells with good growth status after passaging for experiments.

[0070] 2.3 Experimental grouping, intervention methods and indicator detection:

[0071] Cells in logarithmic growth phase were taken and inoculated into cell culture plates (cell inoculation density 1×10 5 When the cells were fused to 80%, the old culture medium was discarded and the cells were divided into the following groups:

[0072] 2.3.1 Determination of the effects of extracts from different parts on the viability of H292 cells - Determining the safe dose range

[0073] 2.3.1.1 Experimental groups and intervention methods

[0074] (1) Normal group: cells were cultured in serum-free medium for 24 h.

[0075] (2) Vehicle group (DMSO): Cultured in serum-free culture medium containing 0.1% DMSO for 24 h.

[0076] (3) Drug group: Cultured in serum-free medium containing various concentrations of drugs (see experimental results) for 24 h.

[0077] 2.3.1.2 Index detection: cell viability

[0078] After the experiment, discard the old culture medium, add 100 μL of culture medium containing MTS detection reagent to each well, incubate at 37 °C for 1 h, and measure the absorbance OD at 490 nm using a microplate reader. 490 , with OD 630 The value is used as a reference. The experimental operation was carried out strictly in accordance with the MTS reagent test instructions. The formula for calculating the cell viability of each well is as follows:

[0079]

[0080] 2.3.2 Determination of the effects of extracts from different parts on the hypersecretion of mucin MUC5AC in H292 cells induced by smoke extract combined with lipopolysaccharide

[0081] 2.3.2.1 Experimental groups and intervention methods

[0082] (1) Normal group: cells were cultured in serum-free medium for 24 h.

[0083] (2) Model group (CSE+LPS): cells were cultured in serum-free medium containing 25 μg / mL CSE and 1 μg / mL LPS for 24 h.

[0084] (3) Drug group: The cells were cultured in serum-free medium containing corresponding concentrations of drugs, 25 μg / mL CSE and 1 μg / mL LPS for 24 h.

[0085] 2.3.2.2 Index detection: immunofluorescence staining of MUC5AC protein

[0086] After the experiment, the medium was discarded and the cells were washed 3 times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes and washed 3 times with PBS. Then they were blocked with immunofluorescence staining blocking solution for 1 hour. They were incubated with MUC5AC primary antibody at 4°C overnight. The next day, they were washed 3 times with PBS and incubated with goat anti-mouse secondary antibody at room temperature in the dark for 50 minutes, and then stained with DAPI. The detection was observed under a Zeiss confocal microscope. The average immunofluorescence intensity of MUC5AC was evaluated and quantified using the software ImageJ.

[0087] 3 Experimental results

[0088] 3.1 Determination of the effect of Houttuynia cordata extract on the viability of H292 cells - Determining the safe dose range

[0089] The experimental results show that each drug administration group has certain toxicity to H292 cells at high concentrations, and the drug concentration with a cell survival rate of about 80% is selected for subsequent experiments. The subsequent drug concentrations are as follows: Example 1 (ethyl acetate water part) -78μg / ml, Comparative Example 1 (water extract) -78μg / ml, Comparative Example 2 (ethanol extract) -78μg / ml, Comparative Example 3 (ethyl acetate part) -12.5μg / ml, Comparative Example 4 (n-butanol water part) -39μg / ml, Comparative Example 5 (n-butanol part) -12.5μg / ml, Comparative Example 6 (petroleum ether water part) -78μg / ml, Comparative Example 7 (petroleum ether part) -3.125μg / ml, Houttuynia cordata injection diluted 27 times.

[0090] Table 1 Cell viability in each group in water phase

[0091]

[0092] Table 2 Cell viability in each group in the organic phase

[0093]

[0094] Table 3 Cell viability of Houttuynia cordata at different concentrations

[0095]

[0096] 3.2 Effect of Houttuynia cordata extract on the hypersecretion of mucin MUC5AC in H292 cells induced by smoke extract combined with lipopolysaccharide

[0097] The experimental results are as follows Figure 2 As shown in Table 4, CSE+LPS can significantly increase the expression level of MUC5AC in H292 cells; Houttuynia injection, Comparative Example 2, Comparative Example 4, and Comparative Example 5 can reduce the expression level of MUC5AC in model cells, and Example 1 and Comparative Example 3 can significantly reduce the expression level of MUC5AC in model cells, and the level reduced by Example 1 is significantly better than that by Comparative Example 3 (P < 0.05).

[0098] Table 4 Results of H292 cell mucin MUC5AC secretion experiment

[0099]

[0100] 4 Conclusion

[0101] Extracts from different parts of Houttuynia cordata can reduce the expression level of mucus protein MUC5AC in H292 cells induced by CSE combined with LPS. Example 1 and Comparative Example 3 can significantly reduce the expression level of MUC5AC in model cells, among which Example 1 has the most significant effect.

[0102] Application Example 2 Effects of Houttuynia cordata extract on proliferation and apoptosis of HPMEC cells damaged by CSE

[0103] 1 Experimental Materials

[0104] 1.1 Instruments and Equipment

[0105]

[0106] 1.2 Main Reagents

[0107]

[0108] 2 Experimental methods

[0109] 2.1 Preparation of main solution:

[0110] Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 4, Comparative Example 6: Weigh an appropriate amount of dry paste, dissolve it in RPMI 1640 culture medium to prepare a 10 mg / mL stock solution, filter it with a 0.22 μM filter membrane, store it in aliquots at -20°C, and dilute it with ECM culture medium before use.

[0111] Comparative Example 3, Comparative Example 5, Comparative Example 7: Weigh an appropriate amount of dry paste, dissolve it in DMSO to prepare a 50 mg / mL stock solution, store it in aliquots at -20°C, and dilute it with ECM culture medium before use.

[0112] The Houttuynia cordata injection was diluted in a 3-fold gradient to obtain 8 concentration samples.

[0113] 2.2 Cell culture:

[0114] Quickly remove the cryovial containing HPMEC cells (human pulmonary microvascular endothelial cells) from liquid nitrogen, immediately put it into a 37°C water bath for rapid thawing for about 1 minute, centrifuge it at 800rpm for 2 minutes, wipe the cryovial with 75% alcohol, move it to the clean bench, remove the cryofluid, add ECM culture medium containing 10% fetal bovine serum, blow it evenly, transfer it to a culture dish, and culture it in a 37°C, 5% CO2 constant temperature cell culture incubator. When the cell growth and fusion reach about 80% under an inverted microscope, subculture at a ratio of 1:3 to 1:8. Take the cells with good growth status after subculture for the experiment.

[0115] 2.3 Experimental grouping, intervention methods and indicator detection:

[0116] Cells in logarithmic growth phase were taken and inoculated into cell culture plates (cell inoculation density 1×10 5 When the cells were fused to 80%, the old culture medium was discarded and the cells were divided into the following groups:

[0117] 2.3.1 Determination of the effects of extracts from different parts on the survival activity of HPMEC cells damaged by CSE - Determining the subsequent action concentration of the drug

[0118] 2.3.1.1 Experimental groups and intervention methods

[0119] (1) Normal group: cells were cultured with ECM medium for 24 h.

[0120] (2) Model group: Cultured with ECM medium containing 62.5 μg / mL CSE for 24 h.

[0121] (3) Drug group: The cells were cultured in ECM medium containing various concentrations of drugs (see experimental results; Houttuynia cordata injection was diluted 3 times in series) for 24 h.

[0122] 2.3.1.2 Index detection: cell viability

[0123] After the experiment, add 100 μL of CellTiter-Glo Reagent to each well and incubate the plate at room temperature for 10 minutes. Use an ELISA reader to detect the absorbance of each well. Calculate the cell activity at each drug concentration according to the following formula.

[0124]

[0125] 2.3.2 Determination of the effects of extracts from different parts on apoptosis of HPMEC cells damaged by smoke extract

[0126] 2.3.2.1 Experimental groups and intervention methods

[0127] (1) Normal group: cells were cultured with ECM medium for 24 h.

[0128] (2) Model group: Cultured with ECM medium containing 62.5 μg / mL CSE for 24 h.

[0129] (3) Drug group: Cultured with ECM culture medium containing various concentrations of drugs (see experimental results) for 24 h.

[0130] 2.3.2.2 Index detection: flow cytometry detection of cell apoptosis

[0131] After the experiment, the culture medium was discarded, the cells were washed by centrifugation with pre-cooled PBS, and 1-10 × 1 05cells (including cells in the culture supernatant). Dilute 5× Binding Buffer with double distilled water to 1× working solution, and resuspend the cells in 500μl 1× Binding Buffer. Add 5μl AnnexinV-FITC and 10 μlPI to each tube. After gently vortexing, incubate at room temperature in the dark for 5 minutes. Perform flow analysis according to the experimental method. On the flow cytometer, detect AnnexinV-FITC (Ex=488nm;Em=530nm) through the FITC detection channel and detect PI through the PI detection channel (Ex=535nm;Em=615nm).

[0132] 3 Experimental results

[0133] 3.1 Determination of the effect of Houttuynia cordata extract on the survival activity of CSE-injured HPMEC cells - Determination of drug concentration

[0134] The experimental results show that CSE can significantly reduce the survival activity of HPMEC cells; each medication group can increase the survival activity of the model cells to a certain extent, among which Houttuynia cordata injection, Comparative Example 5 (n-butanol fraction), and Example 1 (ethyl acetate water fraction) can significantly increase the survival activity of the model cells, and Example 1 has the largest increase and the best effect.

[0135] The best drug concentration was selected for subsequent tests. The subsequent drug concentrations were as follows: Example 1 (ethyl acetate water fraction) -625 μg / ml, Comparative Example 1 (water extract) -625 μg / ml, Comparative Example 2 (ethanol extract) -625 μg / ml, Comparative Example 3 (ethyl acetate fraction) -25 μg / ml, Comparative Example 4 (n-butanol water fraction) -312.5 μg / ml, Comparative Example 5 (n-butanol fraction) -12.5 μg / ml, Comparative Example 6 (petroleum ether water fraction) -312.5 μg / ml, Comparative Example 7 (petroleum ether fraction) -25 μg / ml, Houttuynia cordata injection diluted 27 times.

[0136] Table 5 Effects of each water phase group on the viability of HPMEC cells

[0137]

[0138] Table 6 Effects of organic phase groups on HPMEC cell viability

[0139]

[0140] Table 7 Effects of different concentrations of Houttuynia cordata injection on the viability of HPMEC cells

[0141]

[0142] 3.2 Effect of Houttuynia cordata extract on apoptosis of HPMEC cells induced by smoke extract

[0143] The experimental results are shown in Table 8. CSE can significantly increase the apoptosis level of HPMEC cells; Houttuynia injection can reduce the apoptosis rate of model cells; Houttuynia injection, Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 6, and Comparative Example 7 can significantly reduce the apoptosis rate of model cells, among which Example 1 has a significantly better reduction than the other groups (P < 0.01), and has the best effect.

[0144] Table 8 Effects of each group on HPMEC cell apoptosis

[0145]

[0146] A comprehensive analysis of the effects of different extraction parts of Houttuynia cordata on the activity of model cells and the level of apoptosis showed that the ethyl acetate water part of Example 1 could significantly increase the survival activity of model cells and significantly reduce the level of apoptosis, and was superior to the extracts from other parts.

[0147] Application Example 3 Effect of Houttuynia cordata extract on smoke-induced COPD model mice

[0148] 1. Experimental Materials

[0149] 1.1 Reagents and Materials

[0150] Lipopolysaccharide (sigma), mouse IL-6 ELISA kit (Lianke Biotechnology), mouse TNFα ELISA kit (Lianke Biotechnology).

[0151] 1.2 Experimental system

[0152] Animal strain: C57BL / 6J.

[0153] Animal grade: SPF grade.

[0154] Animal sex and number: Male, 40.

[0155] Animal age: approximately 8 weeks old.

[0156] Animal weight: 20-30 g.

[0157] Quarantine: Newly received animals are quarantined for 3 days. During this period, the animals' drinking water, food intake, health status, and signs of disease and death are observed.

[0158] Identification: Animal ear tag.

[0159] 2 Experimental methods

[0160] 2.1 Grouping and Dosage

[0161] Forty C57BL / 6J mice were randomly divided into a normal group (normal), a model group (model), a low-dose drug group (YXC-L, 2.8 g crude drug / kg of the dry paste of Example 1), and a high-dose drug group (YXC-H, 5.6 g crude drug / kg of the dry paste of Example 1) according to their body weight, with 10 animals in each group.

[0162] 2.2 Experimental procedures

[0163] The COPD mouse model was established by fumigation combined with intratracheal instillation of LPS in the model group and the drug group. Lipopolysaccharide (25 μg / 50 μL) was instilled into the trachea on the 1st and 14th days of modeling, and passive smoking was performed on the 2nd-30th day (except the 14th day). The mice were placed in a fumigation device, 20 cigarettes each time, twice a day, each time for 2 hours (more than 4 hours between two times), and smoked for a total of 28 days. The control group was instilled with the same amount of physiological saline through the airway on the 1st and 14th days, and no fumigation was performed. The mice in the low- and high-dose drug treatment groups were gavaged once a day with the Houttuynia extract of Example 1 (2.8 g crude drug / kg, 5.6 g crude drug / kg), and the control group and the model group were gavaged with the same volume of CMC once a day.

[0164] 2.3 Detection indicators

[0165] The animals were anesthetized with 2% sodium pentobarbital before the test, and the trachea was intubated after fixation. The main pulmonary function indicators were tested, including pulmonary resistance (RI), dynamic pulmonary compliance (Cdyn), and forced expiratory volume in 20 milliseconds (FEV 20 ), forced vital capacity (FVC), FEV 20 / FVC, functional residual capacity (FRC), inspiratory capacity (IC), vital capacity (VC), static lung compliance (Cchord), etc.

[0166] After the lung function test, blood was collected from the animals. A portion of the animals in each group were perfused with 10% neutral formalin fixative in the lungs, and hematoxylin-eosin (HE) staining was performed to observe the effect of the test substance on COPD. A certain amount of the remaining lungs were taken and prepared into a 10% homogenate in an ice bath. After the lung homogenate was centrifuged at 12000r / min for 10 minutes, the supernatant was taken for testing, and the TNF-α and IL-6 levels in the lung tissue homogenate were detected by enzyme-linked immunosorbent assay. The detection method was strictly carried out in accordance with the instructions of the kit.

[0167] 2.4 Statistical methods

[0168] The experimental data were analyzed and processed using SPSS18.0 statistical software, with the test level of 0.05 and 0.01. The measurement data results were expressed as mean ± standard deviation ( ) indicates that a normality test was performed first. For data that conformed to normal distribution, one-way ANOVA was used for mean comparison. If the variances were equal, the least significant difference (LSD) method was used for pairwise comparisons. If the variances were unequal, Dunnett's T3 test was used for pairwise comparisons. If the data did not conform to normal distribution, nonparametric tests were used.

[0169] 3. Experimental results

[0170] 3.1 Effects of Houttuynia cordata extract on lung function

[0171] From the lung function test results in Table 9, it can be seen that compared with the normal group, the FRC level of mice in the model group was significantly increased, and FEV20 / FVC was significantly decreased; while the YXC-L and YXC-H groups were able to significantly inhibit the increase of FRC and significantly increase the FEV20 / FVC level.

[0172] Table 9 Effects of Houttuynia cordata extract on lung function

[0173]

[0174] 3.2 Effect of Houttuynia cordata extract on the number of inflammatory cells in lung tissue

[0175] HE staining results Figure 3 As shown in Table 10, the number of inflammatory cells in the model group increased significantly compared with the normal group, and the YXC-L and YXC-H groups were able to reduce the inflammatory infiltration caused by smoke. The results of the number of inflammatory cells (macrophages) are shown in Table 10. The number of inflammatory cells in the model group increased significantly compared with the normal group, and the YXC-L and YXC-H groups were able to significantly reduce the number of inflammatory cells.

[0176] Table 10 Effects of Houttuynia cordata extract on the number of inflammatory cells in lung tissue

[0177]

[0178] 3.3 Effects of Houttuynia cordata extract on the levels of inflammatory factors in lung tissue

[0179] The results of the levels of inflammatory factors in lung tissue are shown in Table 11. The results showed that compared with the normal group, the levels of TNF-α and IL-6 in the lung tissue homogenate of the model group mice were significantly increased, and Houttuynia cordata extract could significantly inhibit the increase of TNF-α and IL-6.

[0180] Table 11 Effects of Houttuynia cordata extract on the levels of inflammatory factors in lung tissue

[0181]

[0182] It can be seen from Application Examples 1-3 that the Houttuynia cordata extract of the present invention can improve the pathological characteristics of COPD animal models, reduce lung inflammation, improve lung function, reduce airway mucus production, increase endothelial cell proliferation, and reduce endothelial cell apoptosis, showing significant activity in treating COPD.

[0183] Example 2

[0184] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0185] Take 1 kg of Houttuynia cordata, add 5 L 60% (v / v) ethanol, boil and reflux for extraction for 1 hour, filter and collect the filtrate, add 5 L 60% (v / v) ethanol to the residue, boil and reflux for extraction for 1 hour, filter and collect the filtrate, add 5 L 60% (v / v) ethanol to the residue, boil and reflux for extraction for 1 hour, filter and collect the filtrate, combine the three filtrates, recover the ethanol under reduced pressure and concentrate to a volume of 200 ml, extract four times with ethyl acetate, each time using 150 ml of ethyl acetate, separate the phases, take the aqueous phase, concentrate under reduced pressure at 40-80°C and vacuum degree of 0.04-0.09 MPa, and dry to obtain a dry paste of the aqueous part of ethyl acetate.

[0186] Example 3

[0187] A herb of houttuynia cordata extract, the specific extraction method is as follows:

[0188] Take 1 kg of Houttuynia cordata, add 15 L of 60% (v / v) ethanol, boil and reflux for extraction for 2 hours, filter and collect the filtrate, add 15 L of 60% (v / v) ethanol to the residue, boil and reflux for extraction for 2 hours, filter and collect the filtrate, combine the two filtrates, recover the ethanol under reduced pressure and concentrate to a volume of 200 ml, add 300 ml of ethyl acetate for extraction, separate the phases, add 300 ml of ethyl acetate to the aqueous phase for extraction again, separate the phases, take the aqueous phase, concentrate under reduced pressure at 40-80 ℃ and vacuum degree of 0.04-0.09 MPa, and dry to obtain a dry paste of the ethyl acetate water portion.

[0189] Example 4

[0190] A houttuynia cordata extract tablet is prepared by wet granulation or dry granulation to form a compressed tablet from the dry paste of Example 1 and auxiliary materials such as dextrin, sodium carboxymethyl cellulose, and magnesium stearate.

[0191] Example 5

[0192] A houttuynia cordata extract granule is prepared from the dry paste of Example 1 and auxiliary materials such as lactose by wet granulation or dry granulation.

[0193] Example 6

[0194] A houttuynia cordata extract injection is prepared by dissolving the dry paste of Example 1 with water for injection, filtering, and sterilizing according to conventional methods.

[0195] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A use of a houttuynia cordata extract in the preparation of a drug for preventing or treating chronic obstructive pulmonary disease, characterized in that: The extraction and separation method of the Houttuynia cordata extract comprises the following steps: (1) extracting Houttuynia cordata with an ethanol aqueous solution, wherein the volume concentration of the ethanol aqueous solution is 55-65%, to obtain an ethanol extract; (2) recovering ethanol from the ethanol extract to obtain a concentrated solution; (3) Extracting the concentrated solution with ethyl acetate, taking the aqueous phase to obtain the ethyl acetate water portion.

2. The use according to claim 1, characterized in that: The extraction and separation method of the Houttuynia cordata extract also includes the following steps: (4) Concentrating and drying the ethyl acetate water portion to obtain a dry paste.

3. The use according to claim 2, characterized in that: In step (1), the extraction temperature is such that the ethanol aqueous solution is in a reflux state; and / or: In step (1), the number of extractions is 1-5 times; the time for each extraction is 1-3 hours; and / or: In step (1), during each extraction, the mass volume ratio of Houttuynia cordata to the ethanol aqueous solution used is 1:5-15 g / ml; and / or: In step (3), the number of extractions is 1 to 5 times; and / or: In step (3), the volume of ethyl acetate used for each extraction is 0.5 to 3 times the volume of the concentrated solution; and / or: In step (4), the ethyl acetate water fraction is concentrated under reduced pressure at 40-80°C and a vacuum degree of 0.04-0.09 MPa to obtain an extract, and the extract is dried at 50-80°C and a vacuum degree of 0.04-0.09 MPa to obtain a dry paste.

4. The use according to any one of claims 1 to 3, characterized in that: The Houttuynia cordata extract reduces the secretion of airway epithelial cell mucin MUC5AC.

5. The use according to any one of claims 1 to 3, characterized in that: The houttuynia cordata extract improves the activity of pulmonary microvascular endothelial cells.

6. The use according to any one of claims 1 to 3, characterized in that: The houttuynia cordata extract inhibits apoptosis of pulmonary microvascular endothelial cells.

7. Use of a composition in the preparation of a drug for preventing or treating chronic obstructive pulmonary disease, characterized in that: The composition comprises the Houttuynia cordata extract according to any one of claims 1 to 3.

8. The use according to claim 7, characterized in that: The dosage form of the composition is capsule, powder, granule, tablet, suspension or injection.

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