Traditional Chinese medicine composition and traditional Chinese medicine extract for preventing and treating lung injury and preparation method of traditional Chinese medicine composition and traditional Chinese medicine extract

Abstract

The invention discloses a traditional Chinese medicine composition for preventing and treating lung injury, a traditional Chinese medicine extract and a preparation method thereof. The traditional Chinese medicine composition comprises the following components in percentage by weight: 25-75% of eclipta and 25-75% of portulaca grandiflora. The preparation method comprises the following steps: uniformly mixing medicinal material powder of eclipta and sculellaria barbata according to a mass ratio of (1-3): (3-1), adding 8-12 times (V / W) of distilled water, carrying out reflux extraction twice, merging filtrate, adding absolute ethyl alcohol for precipitation, and centrifuging. And carrying out SP825 macroporous resin column chromatography purification on the supernatant, collecting a 70% (V / V) acidic ethanol elution part, and carrying out freeze drying to obtain composition powder. The extraction composition provided by the invention can effectively reduce the injury of lung epithelial cells caused by cigarette smoke, improve the cell survival rate and achieve the effect of protecting lung tissues. The invention provides an optional extract for clinically developing products for preventing and treating diseases related to cigarette smoke exposure.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of Chinese medicine compositions, and in particular to a Chinese medicine composition for preventing and treating lung injury, a Chinese medicine extract and a preparation method thereof. The Chinese medicine composition consists of Eclipta prostrata and Eclipta prostrata. Background Art

[0002] A large number of studies have shown that cigarette smoke (CS) contains a variety of chemical components. Long-term stimulation of CS in bronchial epithelial cells (NHBE) can lead to irregular cell proliferation and morphological changes, deterioration, and even lung cancer. Active and passive smoking can affect the vitality of NHBE and cause lung tissue lesions. Due to the large consumption of tobacco products, the number of people worldwide who suffer from lung lesions due to exposure to CS pollution is increasing. Most lung diseases caused by CS exposure develop slowly and require a long course of treatment.

[0003] Chinese patent CN113018372A uses Ganoderma lucidum as the main drug, Polygonatum odoratum, Fritillaria cirrhosa, Momordica grosvenori, Honeysuckle, Platycodon grandiflorum, Licorice, and Houttuynia cordata as auxiliary drugs, and prepares a Ganoderma lucidum Chinese medicine composition through the steps of mixing, crushing, alcohol extraction, water extraction, extract concentration and drying, and extract merging, which can reduce the lung injury induced by cigarette smoke. However, the compound contains more medicinal flavors, and the preparation process is only a simple extraction, and the effective components are not further separated and purified, and the effective components in the composition are unclear; Chinese patent CN112870240A discloses the preventive and protective effects of volatile components of coltsfoot on lung injury caused by cigarette smoke exposure. When used, the whole coltsfoot powder is used to produce volatile components through aromatherapy to play a role, and the types and contents of its volatile components are not clarified. Therefore, it is of great significance to develop a Chinese medicine active ingredient extract that can effectively prevent or alleviate lung injury caused by CS pollution. Summary of the invention

[0004] The present invention is based on a large number of experimental studies and aims to provide a Chinese medicine composition, a Chinese medicine extract and a preparation method thereof for preventing and treating lung injury.

[0005] The present invention first provides a traditional Chinese medicine composition for preventing and treating lung injury, which comprises, by weight percentage: 25-75% of Eclipta prostrata and 25-75% of Herba Scutellariae. That is, by weight, it is composed of the following two components: 10-30 parts of Eclipta prostrata and 10-30 parts of Herba Scutellariae.

[0006] Furthermore, the composition comprises 50-75% of Eclipta prostrata and 25-50% of Scutellaria barbata by weight.

[0007] Scutellaria barbata is the dried whole herb of Scutellaria barbata, a plant of the genus Scutellaria in the family Lamiaceae. It has the effects of strengthening the immune system, resisting free radical oxidation, and anti-tumor. At present, the effective ingredients separated from Scutellaria barbata include polyphenols, flavonoids, alkaloids, polysaccharides and volatile oils, among which polyphenols and flavonoids have attracted much attention; Eclipta prostrata is the dried aerial part of the plant Channa chussima in the family Asteraceae. It has the activities of cooling blood and stopping bleeding, anti-oxidation, anti-bacteria, anti-hyperlipidemic, and anti-tumor.

[0008] Eclipta prostrata is rich in flavonoids, saponins, coumarin and polyphenols. Modern pharmacological studies have shown that polyphenols and flavonoids have significant antioxidant and anti-tumor effects. Previous studies in our laboratory have also found that coumarin components in Eclipta prostrata have a good protective effect on NHBE cells (Comparison of the protective effects of 8 Eclipta prostrata ethyl acetate extracts on NHBE cells based on the "component structure" theory [J]. Chinese Journal of Traditional Chinese Medicine, 2014, 39(16):3136-3141.). Therefore, the present invention designs an extraction and purification process, extracts phenolic acids and flavonoids from Scutellaria barbata and Eclipta prostrata medicinal materials, and purifies the extract to obtain a composition containing polyphenols, flavonoids and coumarin components in the two medicinal materials.

[0009] The present invention also provides a Chinese medicine extract for preventing and treating lung injury, which is prepared by one or more methods of decoction, maceration, percolation, reflux extraction, continuous reflux extraction, ultrasonic extraction, flash extraction, microwave extraction, supercritical fluid extraction, etc.

[0010] The present invention further provides a method for preparing the above-mentioned Chinese medicine extract for preventing and treating lung injury, comprising the following specific steps: (1) Preparation of water extract of the combination of Eclipta prostrata and Scutellaria barbata: Take the dried aerial part of Eclipta prostrata, remove impurities, crush, and pass through a 20-mesh sieve; take the dried aerial part of Scutellaria barbata, remove impurities, crush, and pass through a 20-mesh sieve. Mix the powders of the two at a mass ratio of 1:3 to 3:1, add a 1% hydrochloric acid solution to wet the surface of the medicinal material, place in a round-bottom flask, and seal for 10 minutes. Add 8-12 times (V / W) of distilled water, reflux extraction twice, each time for 30 minutes, filter, and combine the filtrate.

[0011] (2) Alcohol precipitation: Take the filtrate obtained from the extraction part and decompress it to recover the solvent until the concentration of the concentrate is 1.0 g crude drug / mL. Slowly add anhydrous ethanol to the concentrate to make the final ethanol concentration in the concentrate reach 70-80% (V / V). Let it stand for 12-30 hours and centrifuge it at a speed of 9000 rpm and a centrifugation time of 5 minutes. Take the supernatant and recover the solvent until there is no alcohol taste.

[0012] (3) Macroporous resin purification: Take the upper centrifuge liquid and purify it by SP825 macroporous resin column chromatography. The loading ratio is: V 离心液 / W 大孔树脂 =3:1, the centrifuge was repeatedly loaded twice, and after loading, it was left to stand for 20-60 minutes. First, 1 column volume was eluted with distilled water and discarded; then 2 column volumes were eluted with 20% (V / V) ethanol and discarded; then 4 column volumes were eluted with 70-80% (V / V) acidic ethanol (0.1 mol / L hydrochloric acid was added to adjust the pH to 2-5 before use), the eluate was collected, the solvent was recovered, and the concentrated product was placed in a freeze dryer for freeze drying to obtain a dry powder of a Chinese herbal medicine extract containing the effective ingredients of Eclipta prostrata and Scutellaria barbata.

[0013] As one of the preferred embodiments of the method for preparing the above-mentioned Chinese herbal medicine extract, the final ethanol concentration in the concentrated solution in step (2) is 70% by volume, and the standing time is 12 to 24 hours.

[0014] As one of the preferred embodiments of the method for preparing the above-mentioned Chinese herbal medicine extract, in step (3), after the loading is completed, the mixture is allowed to stand for 30 to 60 minutes; more preferably, the mixture is allowed to stand for 30 minutes.

[0015] As one of the preferred embodiments of the method for preparing the above-mentioned Chinese herbal medicine extract, in step (3), the volume fraction of acidic ethanol is 70%, and the pH is 3-5; more preferably, the pH is 3.

[0016] The Chinese herbal medicine extract prepared by the method has, by percentage, a total flavonoid content of more than 6%, a coumarin content of more than 4%, and a total phenolic acid content of more than 1.5%.

[0017] The normal human bronchial epithelial (NHBE) cell injury model was induced by cigarette smoke extract (CSE). The experimental results confirmed that the Chinese medicine extract prepared by the present invention can reduce the NHBE cell toxicity caused by CSE to varying degrees and has a protective effect on lung epithelial cells. Its mechanism of action may be related to scavenging oxygen free radicals and improving antioxidant capacity. Compared with the single-ingredient Ecliptae Scutellariae extract or Scutellaria barbata extract, the compound Chinese medicine extract of the present invention has a synergistic anti-lung injury effect. It is suggested that the Chinese medicine composition or Chinese medicine extract provided by the present invention can be used to prevent and treat lung injury-related diseases.

[0018] The present invention also provides the use of the above-mentioned Chinese medicine composition and Chinese medicine extract in the preparation of medicines for preventing and treating diseases related to lung injury.

[0019] Furthermore, the lung damage is lung damage caused by cigarette smoke.

[0020] The present invention further provides a traditional Chinese medicine preparation, which comprises the traditional Chinese medicine extract as one of the main active ingredients and a pharmaceutically acceptable carrier.

[0021] Furthermore, the Chinese medicine preparation can be an injection, a solid oral preparation, a spray or an atomized inhalation preparation, etc.

[0022] The present invention provides a composition of effective ingredients of Eclipta prostrata and Scutellaria barbata, which can effectively prevent and reduce NHBE damage caused by CS, and provides an extraction and purification process route for effective components in Eclipta prostrata and Scutellaria barbata, which can remove impurities and ineffective components in the medicinal materials while retaining the effective ingredients to the greatest extent, so that when the composition is used in clinical practice, the quality is more controllable and the efficacy is more clear, and convenience is provided for further research on pharmacological mechanisms.

[0023] The present invention is designed to mix two medicinal materials, use water as solvent, and reflux extract together. Because the chlorophyll content in the two medicinal materials is relatively high, water is used as solvent to avoid the dissolution of chlorophyll and interfere with the extraction of effective components. At the same time, when the saponin components in Eclipta prostrata are refluxed together, due to the active effect of saponin surface, the dissolution of polyphenols, flavonoids and coumarin in the two medicinal materials can be increased, thereby improving efficiency. Since the target effective components in the process are mostly acidic, the present invention is designed to wet the surface with hydrochloric acid before the medicinal material is extracted to ensure that the effective components are free. In order to achieve the purpose of separation and purification, the present invention purifies the extract with a macroporous resin. When collecting the eluent, a method of eluting with acidic ethanol is specially designed, because most of the target components are acidic, and when purified by macroporous resin, some components are desorbed slowly, and eluted with acidic ethanol, thereby improving the elution efficiency.

[0024] When used in combination, the protective effect on NHBE is better than that when used alone, indicating that the two extracts have a synergistic effect after combination. The prepared composition can not only effectively prevent and reduce the damage of lung epithelial cells caused by cigarette smoke, but also the extraction process is green, safe, and environmentally friendly, providing favorable conditions for the further development of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the activity diagram of NHBE in each group of Example 1 after CSE intervention (n=3). In the figure, compared with the blank group, ## P<0.01; compared with 10% CSE group, *P<0.05, **P<0.01.

[0026] Figure 2 This is a comparison of SOD activity in NHBE of each group in Example 1 (n=3). In the figure, compared with the blank group, ## P<0.01; compared with 10% CSE group, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with embodiments, but is not limited thereto. Example 1

[0028] A composition containing effective ingredients of Eclipta prostrata and Scutellaria barbata, the preparation method of which is as follows: (1) Extraction: Take the dried aerial part of Eclipta prostrata, remove impurities, crush, and pass through a 20-mesh sieve; take the dried aerial part of Scutellaria barbata, remove impurities, crush, and pass through a 20-mesh sieve. Mix the two powders in a mass ratio of 1:1, add a 1% hydrochloric acid solution to wet the surface of the medicinal material, place it in a round-bottom flask, and seal it for 10 minutes. Add 10 times the volume (V / W) of distilled water to the round-bottom flask, reflux and extract twice, each time for 30 minutes, filter, and combine the filtrate.

[0029] (2) Alcohol precipitation: The filtrate obtained above is decompressed to recover the solvent until the concentration of the concentrate is 1.0 g crude drug / mL. Anhydrous ethanol is slowly added to the concentrate to make the final ethanol concentration in the concentrate reach 70% (V / V). The concentrate is allowed to stand for 12 hours and centrifuged at a speed of 9000 rpm and a centrifugal time of 5 minutes. The supernatant is taken and the solvent is recovered until there is no alcohol taste.

[0030] (3) Macroporous resin purification: Take the upper centrifuge liquid and purify it by SP825 macroporous resin column chromatography. The loading ratio is: V 离心液 / W 大孔树脂 =3:1, the centrifuge was repeatedly loaded twice, and the sample was allowed to stand for 30 minutes after loading. First, 1 column volume was eluted with distilled water and discarded; then 2 column volumes were eluted with 20% (V / V) ethanol and discarded; then 4 column volumes were eluted with 70% (V / V) acidic ethanol (0.1 mol / L hydrochloric acid was added to adjust the pH to 3 before use), the eluate was collected, the solvent was recovered, and the mixture was freeze-dried in a freeze dryer to obtain a dry powder of a composition containing the effective ingredients of Eclipta prostrata and Scutellaria barbata.

[0031] Efficacy evaluation: 1. Effect of the composition containing the active ingredients of Eclipta prostrata and Scutellaria barbata on the survival ability of normal human bronchial epithelial (NHBE) cells after CSE intervention The method disclosed in the reference (Comparison of the protective effects of 8 kinds of Eclipta prostrata ethyl acetate extracts on NHBE cells based on the "component structure" theory [J]. Chinese Journal of Traditional Chinese Medicine, 2014, 39(16):3136-3141.) was used to investigate the effect of a composition containing the effective ingredients of Eclipta prostrata and Scutellaria barbata on the viability of NHBE cells after CSE intervention. Take an appropriate amount of the powder of the composition of Example 1 and dissolve it in 70% ethanol for later use. The drug administration groups were added with the solution of the composition of Example 1 to make the final concentration of the drug 6.25, 12.5, 25, 50, and 100 μg / mL, respectively, and cultured for 2 h, then added with 10% CSE and continued to culture for 24 h; the model group was directly added with 10% CSE and continued to culture for 24 h; the blank control group was added with 100 μL of serum-free culture medium and continued to culture for 24 h. The cell viability was detected by MTT method. The viability of NHBE in each group after CSE intervention was calculated according to the following formula (1); the protection rate of the composition on NHBE after CSE intervention was calculated according to formula (2). The activity of NHBE in the model group, blank group and drug-treated group was shown in Figure 1 The protection rate of the combination against NHBE after CSE intervention is shown in Table 1.

[0032] Cell viability% (1) Protection rate% (2) Depend on Figure 1 It can be seen that compared with the blank group, the model group with a concentration of 10% CSE can significantly inhibit cell growth after acting on NHBE cells, indicating that CSE has a killing effect on NHBE, that is, it has a certain toxic effect on cells. Figure 1 As shown in Table 1, after 24 hours of exposure to 10% CSE on NHBE, the cell viability was only 42.8%. However, administration of the composition extracted from Example 1 at different concentrations significantly reduced the cytotoxicity caused by 10% CSE and increased the cell viability of NHBE (see Table 1). Figure 1 ). Combined Figure 1 As shown in the data of Table 1, when the final concentration of the composition of Example 1 reached 100 μg / mL, the viability of NHBE cells was significantly improved compared with the model group, and the cell protection rate reached 77.8%. This indicates that the composition extracted by the present invention can reduce the toxicity of NHBE cells caused by CSE and has a protective effect on lung epithelial cells.

[0033] Table 1 The activity of NHBE in each group and the protection rate of the combination on NHBE after CSE intervention

[0034] 2. Effect of the combination containing the active ingredients of Eclipta prostrata and Scutellaria barbata on the antioxidant capacity of NHBE cells after CSE intervention CSE was prepared in the same manner as in the item “Effect of the composition on the viability of NHBE cells after CSE intervention”, and the sample NHBE cells of Example 1 were administered. After 24 hours of continuous culture, the supernatant of the above cells was removed, 200 μL of cell lysis solution was added to each well, lysed at 4°C for 30 min, centrifuged in a refrigerated centrifuge (15000 r / min, 5 min), and the cell supernatant was collected. The protein concentration was determined, and the SOD content in the cells was determined according to the method recommended by the kit. The activity of SOD in each group of cells is shown in Table 2 and Figure 2 .

[0035] Table 2 SOD activity of NHBE cells in each group after CSE intervention

[0036] Antioxidant effect has always been an important mechanism of Chinese medicine in preventing and curing diseases. The body has an antioxidant defense system that can remove free radicals and maintain the body's normal oxidative balance, including superoxide dismutase (SOD), which can reflect the strength of the body's antioxidant capacity. Figure 2 As shown in Table 2, 24 hours after administration, compared with the blank control group, the SOD level in the 10% CSE group (model group) cells decreased significantly, reaching 0.48 U / mL; while when the cells were given 10% CSE and the composition extracted in Example 1, the SOD activity in the cells increased, especially in the high-dose group. This shows that when damaged by smoke, the composition in Example 1 can regulate the SOD activity in NHBE cells to normal levels in a dose-dependent manner. These results show that the composition extracted by the present invention has a protective effect in NHBE oxidative damage induced by CSE intervention. Example 2

[0037] The preparation method of this embodiment is the same as that of Example 1, except that when distilled water is added to reflux and extract the medicinal materials, the solid-liquid ratio of the solvent used is different, and the specific extraction conditions are different. When the administration concentration of the obtained composition is 100 μg / mL, the protection rate of NHBE after CSE intervention is as shown in Table 3.

[0038] Table 3 Cell protection rate of the compositions obtained with different solid-liquid ratios (concentration 100 μg / mL)

[0039] As can be seen from Table 3, when the mixed medicinal materials are extracted with solvents of different solid-liquid ratios, the ratios are different, and the obtained compositions have different protection rates for NHBE after CSE intervention. When the solid-liquid ratio (W / V) is 1:7, the cell protection rate of the obtained composition is relatively low. As the solvent ratio increases, the cell protection rate of the composition continues to increase. When the solid-liquid ratio (W / V) is 1:13, the protection rate is not significantly improved, and the use of a large amount of solvent has a certain impact on the operation and cost. Therefore, the present invention determines the solid-liquid ratio (W / V) during extraction to be 1:8-1:12. Example 3

[0040] The preparation method of this example is the same as that of Example 1, except that during alcohol precipitation, the final ethanol concentration in the concentrate after adding ethanol is different, the standing time is also different, and the specific different extraction conditions, when the administration concentration of the obtained composition is 100 μg / mL, the protection rate of NHBE after CSE intervention is as shown in Table 4.

[0041] Table 4 Cell protection rate of each group of compositions (concentration 100 μg / mL)

[0042] As can be seen from Table 4, when the alcohol precipitation concentration is 60%, the cell protection rate of the obtained composition is not high, when the alcohol precipitation concentration is 70%, the cell protection rate of the composition is significantly improved, and when the alcohol precipitation concentration reaches 80%, the cell protection rate of the composition is not significantly improved, and it brings higher costs to the process, so the alcohol precipitation concentration is set to 70% in the present invention; on the other hand, when the sedimentation time is less than 12 hours, the cell protection rate is not high, and when the sedimentation time reaches 30 hours, the improvement of the cell protection rate is not obvious relative to 24 hours, and the operation time is long, so the alcohol precipitation standing time is set to 12 to 24 hours in the present invention. Example 4

[0043] The preparation method of this example is the same as that of Example 1. The difference is that when performing macroporous resin purification, the concentration and pH of the acidic ethanol eluent selected for the collection part are different. Under different extraction conditions, when the administration concentration of the obtained composition is 100 μg / mL, the protection rate of NHBE after CSE intervention is shown in Table 5.

[0044] Table 5 Cell protection rate of each group of compositions (concentration 100 μg / mL)

[0045] As can be seen from Table 5, when the concentration of the ethanol eluent is 60%, the cell protection rate of the obtained composition is not high. When the concentration of the ethanol precipitate is 80%, the cell protection rate of the composition is reduced compared with the 70% ethanol elution. It is possible that the high concentration ethanol elution introduces some impurities; when the pH of the ethanol solution is 2, the cell protection rate is not much different from that of the pH of 3, and the acidic solution causes some structural changes of the components, while when the pH of the ethanol solution is 6, the cell protection rate of the composition is low, indicating that the content of the effective ingredient in the eluent is not high. In summary, the present invention sets the acidic ethanol elution condition to 70% ethanol and pH 3-5 during column chromatography operation. Example 5

[0046] The preparation method of this example is the same as that of Example 1, except that when performing macroporous resin purification, the loading ratio, the standing time, and the distilled water elution volume are different. When the administration concentration of the obtained composition is 100 μg / mL, the protection rate of NHBE after CSE intervention is as shown in Table 6.

[0047] Table 6 Cell protection rate of each group of compositions (concentration 100 μg / mL)

[0048] As can be seen from Table 6, when the ratio of centrifugal liquid: macroporous resin (V: W) is 3: 1, the cell protection rate of the obtained composition is the highest. This may be because too little sample loading results in more dead adsorbed components, while too much sample loading will result in overload; if the sample is not allowed to stand or the standing time is too short after loading, the effective components are not completely adsorbed, and the cell protection rate of the obtained composition is not high. The protection rate is not much different between standing for 60 minutes and standing for 30 minutes. Considering the economic benefits, the standing time is determined to be 30 minutes in this process; after standing, not using distilled water for elution will also affect the protection rate. When eluting with distilled water for 2 column volumes, the cell protection rate of the composition is not much different from that of 1 column volume. Considering the convenience of operation and cost, the column volume of distilled water elution is determined to be 1 column volume in this process. Example 6

[0049] The preparation method of this example is the same as that of Example 1, except that the mass ratio of the raw materials Eclipta prostrata to Scutellaria barbata powder is 3:1. When the administration concentration of the obtained composition is 100 μg / mL, the protection rate of NHBE after CSE intervention is as shown in Table 7. Comparative Example 1

[0050] The preparation method of this comparative example is the same as that of Example 1, except that the extract is not subjected to ethanol precipitation treatment and macroporous resin purification treatment. Comparative Example 2

[0051] The preparation method of this comparative example is the same as that of Example 1, except that the extracted medicinal material raw materials are not wetted with a hydrochloric acid solution with a mass concentration of 1%. Comparative Example 3

[0052] The preparation method of this comparative example is the same as that of Example 1, except that the only raw material for extraction is Eclipta prostrata. Comparative Example 4

[0053] The preparation method of this comparative example is the same as that of Example 1, except that the only raw material for extraction is Scutellaria barbata. Comparative Example 5

[0054] The preparation method of this comparative example is the same as that of Example 1, except that when the extract is not purified by macroporous resin, hydrochloric acid is not added to the ethanol solution for acidification when eluting with 70% ethanol.

[0055] The compositions obtained in Comparative Examples 1-5 and Example 6, when administered at a concentration of 100 μg / mL, had NHBE protection rates after CSE intervention, as shown in Table 7. It can be seen that the compositions obtained in Comparative Examples 1-5 had relatively low NHBE protection rates.

[0056] Table 7 Cell protection rate of the compositions of Example 6 and Comparative Examples 1-5 (concentration 100 μg / mL) Example 7

[0057] 1. Determination of total phenolic acid content in the composition The total phenolic acid content in each composition was determined by the Folin-phenol method. Accurately weigh an appropriate amount of the composition sample, place it in a 250 mL volumetric flask, add an appropriate amount of 60% methanol to dissolve, shake well, and add 60% methanol to the scale to serve as the test solution.

[0058] Accurately weigh 0.010 g of gallic acid standard into a 10 mL volumetric flask, add 60% methanol to the mark, and shake well. Pipette 0.5 mL, 1.0 mL, 1.2 mL, 1.5 mL, and 2.0 mL into a 50 mL volumetric flask, add 60% methanol to the mark, and shake well to serve as a series of concentrations of reference solution.

[0059] Accurately measure 1 mL of the test solution and the reference solution respectively, transfer them to a 50 mL beaker, add 5 mL of Folin-phenol reagent respectively, mix thoroughly, let stand for 5 min, and then add 7.5% Na 2 CO 3 4 mL of the solution was shaken and placed in a dark place for 1.5 h. Using distilled water as a blank control, the absorbance values ​​of the standard and test samples at each concentration were measured at a wavelength of 765 nm. [The mass concentration of gallic acid standard (μg / mL) is used as the horizontal axis ( X） , the absorbance value is the vertical axis ( Y ) Draw a standard curve. Calculate the total phenolic acid content in the sample according to the linear regression equation of the gallic acid standard curve.

[0060] 2. Determination of total flavonoid content in the composition The total flavonoid content in the composition was determined by the sodium nitrite-aluminum nitrate colorimetric method. Accurately weigh the rutin standard and add anhydrous methanol solution to prepare a standard solution with a concentration of 100 µg / mL. Accurately pipette 5, 6, 7, 8, 9, and 10 mL of the rutin standard solution into a 25 mL volumetric flask, add 1.5 mL of 5% sodium nitrite solution, mix well, and place for 6 min, then add 2 mL of 10% aluminum nitrate solution, shake well, and place for 6 min, add 2 mL of 4% sodium hydroxide solution, make up to volume with 70% ethanol solution, shake well, and place for 15 min, measure the absorbance at a wavelength of 510 nm, and draw a standard curve.

[0061] Take an appropriate amount of the powder of the composition of the extract of the present invention and place it in a 25 mL volumetric flask, dissolve it with 5 mL of 70% ethanol solution, add 1.5 mL of 5% sodium nitrite solution, mix well, and let it stand for 6 min, then add 2 mL of 10% aluminum nitrate solution, shake well, let it stand for 6 min, add 2 mL of 4% sodium hydroxide solution, make up to volume with 70% ethanol solution, shake well, let it stand for 15 min, measure the absorbance at a wavelength of 510 nm, substitute it into the standard curve equation, and calculate the total flavonoid content.

[0062] 3. Determination of total content of coumarin in the composition The total content of coumarin was determined by high performance liquid chromatography. The instrument used was an Agilent 1200 high performance liquid chromatograph, which included a diode array detector (DAD), an automatic sampler, a quaternary pump, a column heater-cooler, and Agilent CSCATSO software. The analysis conditions were as follows: Agilent ZOBAX 5 TC-C18 (250 mm×4.6 mm, 5μm), column temperature: 30℃, detection wavelength: 350 nm; flow rate: 1 mL / min; injection volume: 10μL. The mobile phase consisted of acetonitrile (A) and 0.1% acetic acid aqueous solution (B). Gradient elution conditions: 0-10 min, 12-20% A; 11-20 min, 20-35% A; 21-30 min, 35-80% A. Isodemethylwedeholide and wedeholide were selected as mixed reference substances, and the contents of the two indicator components in the samples were calculated using the standard curve method. The sum of the contents of the two components was taken as the total content of the coumarin component.

[0063] The contents of total phenolic acid, total flavonoids and coumarin in each group of the compositions of the examples and comparative examples are shown in Table 8.

[0064] Table 8 Contents of phenolic acid, flavonoids and coumarin in each group of samples (mg / g composition)

[0065] 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 Chinese medicine composition for preventing and treating lung injury, characterized in that: The weight percentage includes: 25-75% Eclipta prostrata and 25-75% Hemiptera:

2. A Chinese medicine extract for preventing and treating lung injury, characterized in that: It is obtained by extracting the Chinese medicine composition according to claim 1.

3. A method for preparing the Chinese medicine extract for preventing and treating lung injury as claimed in claim 2, characterized in that: The steps include: (1) Grinding Eclipta prostrata and Scutellaria barbata respectively, sieving to obtain Eclipta prostrata powder and Scutellaria barbata powder; mixing the two powders evenly, adding a hydrochloric acid solution with a mass concentration of 1% to wet the surface of the powders, and sealing for 10 minutes; adding 8-12 times the amount of distilled water, reflux extraction, and combining the filtrates; (2) The filtrate obtained in step (1) is concentrated under reduced pressure to a concentrate concentration of 1.0 g crude drug / mL; anhydrous ethanol is slowly added to the concentrate to make the final ethanol concentration in the concentrate reach 70-80% by volume, and the concentrate is allowed to stand for 12-30 hours, centrifuged, and the supernatant is taken to recover the solvent until there is no alcohol smell, and marked as the centrifuged liquid; (3) Purify the centrifuge liquid obtained in step (2) by macroporous resin column chromatography: the loading ratio is: V 离心液 / W 大孔树脂 =3mL:1g, the centrifuge liquid was repeatedly loaded with sample for 2 times, and then allowed to stand for 20-60 minutes; firstly, 1 column volume was eluted with distilled water, and then discarded; then, 2 column volumes were eluted with 20% ethanol by volume, and then discarded; then, 4 column volumes were eluted with acidic ethanol with a pH of 2-5 and a volume fraction of 70-80%, and the eluate was collected and concentrated by rotary evaporation. The concentrated solution was placed in a freeze dryer and freeze-dried to obtain the Chinese medicine extract powder containing the effective ingredients of Eclipta prostrata and Scutellaria barbata.

4. The method for preparing the Chinese medicine extract for preventing and treating lung injury according to claim 3, characterized in that: The reflux extraction in step (1) is performed twice, each time for 30 minutes.

5. The method for preparing the Chinese medicinal extract for preventing and treating lung injury according to claim 3, characterized in that: In step (3), let the sample stand for 30 to 60 minutes after loading.

6. The method for preparing the Chinese medicinal extract for preventing and treating lung injury according to claim 3, characterized in that: In step (3), the pH of the acidic ethanol is 3-5, and the volume fraction is 70%.

7. The method for preparing the Chinese medicinal extract for preventing and treating lung injury according to claim 3, characterized in that: The macroporous resin described in step (3) is SP825 macroporous resin.

8. Use of the Chinese medicine composition according to claim 1, the Chinese medicine extract according to claim 2, or the Chinese medicine extract prepared by the method according to any one of claims 3 to 7 in the preparation of drugs for preventing and treating diseases related to lung injury.

9. The use according to claim 8, characterized in that: The lung damage described is lung damage caused by cigarette smoke.

10. A Chinese medicine preparation, characterized in that: The invention comprises the Chinese herbal extract according to claim 2 or the Chinese herbal extract prepared by the method according to any one of claims 3 to 7 as one of the main active ingredients, and a pharmaceutically acceptable carrier.

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