Traditional Chinese medicine composition for treating pulmonary nodule as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for treating pulmonary nodules and a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following traditional Chinese medicine raw materials in parts by weight: 10 to 20 parts of lucid ganoderma, 10 to 15 parts of radix astragali seu hedysari, 10 to 15 parts of rhizoma dioscoreae, 5 to 10 parts of herba houttuyniae, 5 to 10 parts of herba taraxaci, 5 to 10 parts of herba hedyotidis diffusae, 5 to 10 parts of radix ginseng, 5 to 10 parts of radix notoginseng, 5 to 10 parts of radix ophiopogonis and 5 to 10 parts of bulbus lilii. The composition is based on traditional Chinese medicine theories, is combined with a modern extraction process, is suitable for adjuvant therapy of pulmonary nodules, and has an effect equivalent to that of positive drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, in particular to a traditional Chinese medicine composition for treating pulmonary nodule and a preparation method and application thereof. BACKGROUND

[0002] With the gradual improvement of people's awareness of lung nodule screening, more and more lung nodules are found in clinical practice. Pulmonary nodules can be divided into solitary and multiple pulmonary nodules according to the number, and can be divided into inflammatory nodules and neoplastic nodules according to the nature of the nodules. Most of the pulmonary nodules in clinical practice are inflammatory. According to the density, pulmonary nodules can be divided into solid nodules, mixed ground glass nodules and pure ground glass nodules. If not treated in time or long-term persistence, it may develop into a tumor or even cancer.

[0003] In the prior art, the treatment of pulmonary nodules mainly uses antibiotics to reduce inflammation. However, long-term and large-scale use of antibiotics to treat pulmonary nodules has the risk of antibiotic abuse, and the therapeutic effect is not good. At present, the main treatment methods for pulmonary nodules in clinical practice are regular follow-up, biopsy or surgical resection. However, invasive diagnosis and treatment methods bring high trauma risk to patients, decrease lung function reserve and cause great economic burden, resulting in low patient compliance. Although there are many reports on the treatment of pulmonary nodules by traditional Chinese medicine prescriptions at present, such as Chinese patent application CN115814019A discloses a traditional Chinese medicine composition for treating pulmonary nodules and a preparation method and application thereof. The traditional Chinese medicine composition is composed of Herba Sedi Simplex, Radix Astragali, Houttuynia cordata, Pericarpium Citri Reticulatae, Radix Codonopsis, Solanum nigrum L., Rhizoma Dioscoreae, Radix Bupleuri, Radix Glycyrrhizae, Rhizoma Atractylodis Macrocephalae, Radix Ophiopogonis, Radix Angelicae Sinensis, Radix Scrophulariae, Radix Paeoniae Alba and Herba Patriniae. After taking the traditional Chinese medicine composition for 2 months, the total effective rate of treating pulmonary nodules is more than 76%, and the highest is 86.6%, and the size of the nodules is also significantly reduced. Since the composition of the above-mentioned patent application contains many medicinal ingredients, and the effect needs to be further improved, in addition, even if the mature fruit of Solanum nigrum L. contains a small amount of solamum nigrum L. which may cause discomfort to the human body, the safety of the above-mentioned traditional Chinese medicine composition needs to be further improved.

[0004] Therefore, it is of great significance to develop a traditional Chinese medicine composition with better curative effect, high safety and stable process. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a traditional Chinese medicine composition for treating pulmonary nodules and a preparation method and application thereof. The raw materials of the composition are within a specific dosage range, and the composition obtained by the preparation process of partial alcohol extraction and partial water extraction has an effect comparable to that of positive drugs.

[0006] The present application is realized by the following technical solutions: The first aspect of the present application relates to a traditional Chinese medicine composition for treating pulmonary nodule, which comprises the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 10-20 parts, Astragalus 10-15 parts, Dioscorea 10-15 parts, Houttuynia cordata 5-10 parts, Taraxacum 5-10 parts, Oldenlandia 5-10 parts, Panax 5-10 parts, Sanchi 5-10 parts, Ophiopogon 5-10 parts, Lily 5-10 parts.

[0007] Preferably, the traditional Chinese medicine composition comprises the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 12-18 parts, Astragalus 10-15 parts, Dioscorea 10-15 parts, Houttuynia cordata 6-9 parts, Taraxacum 6-9 parts, Oldenlandia 5-10 parts, Panax 5-8 parts, Sanchi 5-8 parts, Ophiopogon 8-10 parts, Lily 8-10 parts.

[0008] Further preferably, the traditional Chinese medicine composition comprises the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 15 parts, Astragalus 12 parts, Dioscorea 12 parts, Houttuynia cordata 9 parts, Taraxacum 9 parts, Oldenlandia 9 parts, Panax 6 parts, Sanchi 6 parts, Ophiopogon 9 parts, Lily 9 parts.

[0009] The second aspect of the present application relates to a preparation method of the above-mentioned traditional Chinese medicine composition for treating pulmonary nodule, which comprises the following preparation steps: (1) Ganoderma lucidum, Astragalus, Dioscorea, Ophiopogon, Lily, Panax, Sanchi, and Oldenlandia are decocted with water for 1-3 times, each time for 1-2 hours, the filtrates are combined, concentrated, and a water extract is obtained; (2) Houttuynia cordata and Taraxacum are extracted with an aqueous ethanol solution for 1-2 times, each time for 1-2 hours, the filtrates are combined, ethanol is recovered, and an alcohol extract is obtained by concentration; (3) The water extract and the alcohol extract are combined, concentrated, and dried to obtain dry extract powder; (4) The dry extract powder is mixed with an appropriate amount of excipients to granulate, and the granules are obtained.

[0010] Preferably, in step (1), the decoction is performed twice, the amount of water added each time is 6-10 times the amount of the medicinal materials, and each time is 1.5 hours, and the concentration is concentrated to a relative density of 1.10-1.15 at 60°C.

[0011] Preferably, in step (2), the volume fraction of ethanol in the aqueous ethanol solution is 70%, the amount of aqueous ethanol solution used is 6-10 times the amount of medicinal materials, the extraction is performed for 2 times, each time for 1 hour, and the concentration is concentrated to a relative density of 1.10-1.15 at 60°C.

[0012] Preferably, in step (3), the concentration is concentrated to a relative density of 1.25-1.30 at 60°C, and the drying method is spray drying or vacuum drying.

[0013] Preferably, the adjuvant of step (4) is at least one of microcrystalline cellulose, starch, lactose and magnesium stearate.

[0014] The third aspect of the present application relates to the use of the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above preparation method in the preparation of a drug for treating pulmonary nodules.

[0015] Preferably, the drug is a capsule or a tablet.

[0016] Preferably, the daily dose of the drug is 1.5-3.0g of dry extract powder, taken 2-3 times.

[0017] The legislative core of the present application is "supporting the healthy qi, eliminating the pathogenic factors, and resolving toxins and nodules". Ganoderma lucidum, Astragalus and Panax ginseng are used as monarch drugs to support the healthy qi and tonify the spleen and lung; Houttuynia cordata, Dandelion, Oldenlandia and Sanguis Draconis are used as ministerial drugs to clear heat and resolve toxins, activate blood and resolve stasis, and dredge collaterals; and Dioscorea, Ophiopogon and Lily are used as auxiliary drugs to nourish yin and moisten the lung. The present application not only tonifies the lung, but also pays more attention to tonifying the spleen, which is the acquired constitution. The combination of the above drugs has a strict structure and treats the symptoms and the root cause.

[0018] The present application has the following advantages: (1) Based on the theory of traditional Chinese medicine and the etiology and pathogenesis of pulmonary nodules, the present application selects tonifying qi and yin drugs such as Ganoderma lucidum, Astragalus and Dioscorea, and combines with heat-clearing and toxin-resolving drugs such as Houttuynia cordata and Dandelion, which has the characteristics of supporting the healthy qi, eliminating the pathogenic factors, and treating the symptoms and the root cause; (2) By replacing Cordyceps sinensis with high cost and Kadsura japonica with toxic side effects, the present application optimizes the prescription composition and improves the safety and economy; (3) The present application uses water extraction for Ganoderma lucidum, Astragalus, Dioscorea, Ophiopogon, Lily, Panax ginseng, Sanguis Draconis and Oldenlandia, and uses alcohol extraction for Houttuynia cordata and Dandelion, which maximizes the retention of effective components of each drug. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : CCK-8 method for screening the concentration of the drug in Example 1; Figure 2 : A549 cell proliferation experiment results of different groups; Figure 3 : Flow cytometry of A549 cell apoptosis in different groups, wherein A is the blank control group, B is the positive drug cisplatin group, C is the low-dose group of Example 1, D is the medium-dose group of Example 1, and E is the high-dose group of Example 1; Q3-1 in each figure represents mechanical damage, Q3-2 represents late apoptosis, Q3-3 represents negative, and Q3-4 represents early apoptosis; Figure 4 : A549 cell apoptosis column chart of different groups; Figure 5 : Crystal violet staining of cell migration in different groups Figure 6Different groups of cell migration crystal violet column chart Figure 7 WB method for detecting different groups of protein expression chart; Figure 8 WB method for detecting different groups of protein expression column chart. DETAILED DESCRIPTION

[0020] The present application will be further described below in conjunction with specific examples, and the advantages and features of the present application will become more apparent as the description proceeds. However, these examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

[0021] Example 1 A traditional Chinese medicine composition for treating pulmonary nodules, the traditional Chinese medicine composition is prepared from the following weight parts of traditional Chinese medicine raw materials: Ganoderma 15g, Astragalus 12g, Chinese yam 12g, Houttuynia cordata 9g, dandelion 9g, Oldenlandia 9g, ginseng 6g, Sanchi 6g, Ophiopogon 9g, Lily 9g.

[0022] Preparation method The preparation method comprises the following steps: (1) decoct Ganoderma, Astragalus, Chinese yam, Ophiopogon, Lily, ginseng, Sanchi, and Oldenlandia with water twice, each time adding 8 times the amount of water, each time for 1.5 hours, combine the filtrates, and concentrate to a relative density of 1.10 (60°C) to obtain a water extract; (2) reflux extract Houttuynia cordata and dandelion with 70% ethanol twice, the single ethanol solution is 8 times the amount, each time for 1 hour, combine the filtrates, recover ethanol, and concentrate to a relative density of 1.10 (60°C) to obtain an alcohol extract; (3) combine the water extract and the alcohol extract, concentrate to a relative density of 1.30 (60°C), and spray dry to obtain dry extract powder; and (4) mix the dry extract powder with microcrystalline cellulose and starch to obtain the product.

[0023] Example 2 A traditional Chinese medicine composition for treating pulmonary nodules, the traditional Chinese medicine composition is prepared from the following weight parts of traditional Chinese medicine raw materials: Ganoderma 10g, Astragalus 15g, Chinese yam 10g, Houttuynia cordata 10g, dandelion 5g, Oldenlandia 10g, ginseng 5g, Sanchi 10g, Ophiopogon 5g, Lily 10g.

[0024] The preparation method is the same as that in Example 1.

[0025] Example 3 A traditional Chinese medicine composition for treating pulmonary nodule is prepared from the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 20 g, Astragalus 10 g, Chinese yam 15 g, Houttuynia cordata 5 g, dandelion 10 g, Speranskia 5 g, ginseng 10 g, Sanchi 5 g, Ophiopogon 10 g, lily 5 g.

[0026] The preparation method is the same as that in Example 1.

[0027] Comparative Example 1 A traditional Chinese medicine composition for treating pulmonary nodule is prepared from the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 15 g, Astragalus 12 g, Chinese yam 9 g, Houttuynia cordata 12 g, dandelion 9 g, Speranskia 9 g, ginseng 6 g, Sanchi 6 g, Ophiopogon 9 g, lily 9 g.

[0028] The difference from Example 1 is that the amounts of Chinese yam and Houttuynia cordata are different, and in Comparative Example 1, Chinese yam is 9 g and Houttuynia cordata is 12 g, which are not within the amount range of Chinese yam and Houttuynia cordata in the application.

[0029] The preparation method is the same as that in Example 1.

[0030] Comparative Example 2 A traditional Chinese medicine composition for treating pulmonary nodule is prepared from the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 15 g, Astragalus 9 g, Chinese yam 12 g, Houttuynia cordata 9 g, dandelion 9 g, Speranskia 9 g, ginseng 6 g, Sanchi 6 g, Ophiopogon 12 g, lily 9 g.

[0031] The difference from Example 1 is that the amounts of Astragalus and Ophiopogon are different, and in Comparative Example 2, Astragalus is 9 g and Ophiopogon is 12 g, which are not within the amount range of Astragalus and Ophiopogon in the application.

[0032] The preparation method is the same as that in Example 1.

[0033] Comparative Example 3 A traditional Chinese medicine composition for treating pulmonary nodule is prepared from the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 15 g, Astragalus 12 g, Chinese yam 12 g, Houttuynia cordata 9 g, dandelion 9 g, Speranskia 9 g, ginseng 6 g, Sanchi 6 g, Ophiopogon 9 g, lily 9 g.

[0034] The difference from Example 1 is that the preparation method is different, and all raw materials are alcohol extracted. The specific preparation method is as follows: all raw materials are refluxed with 70% ethanol for 2 times, 8 times the amount of 70% ethanol solution is added each time, 1 hour each time, the filtrates are combined, the ethanol is recovered, concentrated to a relative density of 1.30 (60°C), and then spray dried to prepare dry extract powder; (4) the dry extract powder is mixed with microcrystalline cellulose and starch to obtain the product.

[0035] Comparative Example 4: A traditional Chinese medicine composition for treating pulmonary nodules, the composition being prepared from the following traditional Chinese medicine raw materials in parts by weight: Ganoderma lucidum 15g, Astragalus membranaceus 12g, Dioscorea opposita 12g, Houttuynia cordata 9g, Taraxacum mongolicum 9g, Hedyotis diffusa 9g, Panax ginseng 6g, Panax notoginseng 6g, Ophiopogon japonicus 9g, and Lilium brownii 9g.

[0036] The difference from Example 1 lies in the preparation method; all raw materials are extracted with water. The specific preparation method is as follows: all raw materials are decocted twice with water, adding 8 times the amount of water each time, for 1.5 hours each time. The filtrates are combined and concentrated to a relative density of 1.30 (60°C). The filtrate is then spray-dried to obtain a dry extract powder. The dry extract powder is then mixed with microcrystalline cellulose and starch to obtain the final product.

[0037] Experiment Example 1: Drug Efficacy Experiment The main reagents and equipment used in the experiment are shown in Table 1 and Table 2.

[0038] Table 1: Main Cells and Reagents

[0039] Table 2: Main Equipment

[0040] Cell culture process: (1) Recovery and Cultivation A549 and BEAS-2B cells were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. The cell suspension was transferred to centrifuge tubes containing 5 mL of complete culture medium (DMEM / Ham's F-12K + 10% FBS + 1% antibiotics), centrifuged (1000 rpm, 5 min), and the supernatant was discarded. The cells were resuspended in fresh complete culture medium, seeded in T25 culture flasks, and cultured in a 37°C, 5% CO2 humidified incubator.

[0041] (2) Fluid replacement and passage Replace the culture medium with fresh complete medium every 2-3 days. When the cell confluence reaches 80%-90%, passage the cells. Discard the old culture medium and wash the cells once with PBS. Add an appropriate amount of trypsin for digestion (37°C, 1 min). When the cells become rounded and the gaps between them increase under a microscope, immediately add serum-containing complete culture medium to stop the digestion. Gently pipette to completely detach the cells, forming a single-cell suspension. After centrifugation, discard the supernatant, resuspend the cells in fresh culture medium, and seed them into new culture flasks at a ratio of 1:2 or 1:3 for continued culture.

[0042] The above-mentioned cells were used to conduct the following experiments. The experimental results were analyzed using statistical software such as GraphPad Prism 9.0. A p-value < 0.05 was considered statistically significant.

[0043] 1. Drug intervention concentration screening experiment (CCK-8 method) (1) Inoculation of cells Logarithmic growth phase A549 and BEAS-2B cells were taken, trypsinized and resuspended into a single cell suspension, counted with a cell counting plate, and the cell density was adjusted to 5x10 4 Each well was inoculated with 200 μL (about 10,000 cells per well). Zero adjustment holes (only medium), control holes (cells + medium), and drug administration holes (cells + drug-containing medium) were set.

[0044] (2) Drug treatment The culture plate was placed in the incubator for 24 hours to allow the cells to adhere completely. A series of concentrations of traditional Chinese medicine extract (traditional Chinese medicine composition prepared in Example 1) working solution concentration gradients (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, 1000 μg / mL, 1200 μg / mL, 2500 μg / mL, 5000 μg / mL, 10000 μg / mL, 12500 μg / mL, 25000 μg / mL, 50000 μg / mL, 75000 μg / mL, 100000 μg / mL) were prepared. The original culture medium was discarded, and fresh culture medium containing different concentrations of drugs was added to the experimental group, and the control group was replaced with the same volume of fresh culture medium without drugs. Each concentration was set with 6 replicate wells.

[0045] (3) Incubation and detection The culture plate was placed back in the incubator for 48 hours. After the time, 10 μL of CCK-8 solution was added to each well, the culture plate was gently shaken to mix, and then placed in the incubator for 2 hours.

[0046] Determination of absorbance: The absorbance (OD value) of each well was determined at 450 nm wavelength using an enzyme marker.

[0047] Calculation: Calculate cell viability (%) = (OD drug group - OD zero adjustment group) / (OD control group - OD zero adjustment group) x 100%. Use software such as GraphPad Prism to draw a dose-effect curve and calculate the IC 50 value.

[0048] The results are shown in Figure 1 .

[0049] The Chinese medicine extract can cause half of the A549 cells to die when the drug concentration reaches 2500 μg / mL. The BEAS-2B cells can be half dead when the drug concentration reaches 100000 μg / mL. The drug has very low toxicity to normal lung epithelial cells, and the drug may have high safety.

[0050] 2. Cell proliferation experiment (CCK-8 method) (1) Grouping and inoculation: Take the logarithmic growth period of A549 cells, inoculate in 96-well plates (2x10 4 cells per well, 200 μL). Set groups: blank control group, low, medium and high concentration of traditional Chinese medicine group (IC 50 ), positive drug control group (cisplatin 40 μM). Each group has 6 replicates.

[0051] (2) Drug treatment: Place the culture plate in the incubator for 24 hours to allow the cells to adhere completely. Prepare high (2500 μg / mL), medium (1200 μg / mL) and low concentration (600 μg / mL) of traditional Chinese medicine extract working solution. Add 40 uM of cisplatin solution to the positive control group.

[0052] (3) Incubation and detection: Put the culture plate back into the incubator and continue to culture for 0, 24, 48 and 72 hours. After the time, add 10 μL of CCK-8 solution to each well (make sure no bubbles are generated), gently shake the culture plate to mix, and then put it into the incubator for 2 hours. Detect at 450.

[0053] (4) Draw the curve: Take time as the horizontal axis and cell survival rate as the vertical axis to draw the cell survival curve of each group.

[0054] The results are shown in Table 3, Figure 2 .

[0055] Table 3: Survival curve table

[0056] According to the results of the chart, the drug in Example 1 showed a significant time-dependent and concentration-dependent inhibitory effect on the proliferation of A549 cells. That is, with the increase of drug concentration and the extension of action time, the cell survival rate gradually decreased. The cisplatin treatment group had a strong proliferation inhibition effect on A549 cells. After 48 hours of treatment, the cell survival rate decreased significantly, and by 72 hours, the survival rate further decreased by 33.51%. The low-dose group (600 μg / mL) had a slight inhibitory effect after 72 h, with a decrease in activity to 76.41%. The medium dose had a significant inhibition at 48 h, with a decrease in survival rate to 66.39%, and reached 55.56% at 72 h. The high dose showed the strongest inhibitory effect, with a trend closest to the positive drug cisplatin. At 48 hours, the survival rate decreased significantly to 56.33%, and at 72 hours, the anti-proliferation effect (survival rate 41.62%) exhibited a significant potential to inhibit the proliferation of A549 cells.

[0057] 3. Apoptosis experiment (Annexin V-FITC / PI double staining method) (1) Grouping and treatment: A549 cells were seeded in a 6-well plate, with about 5 x 10 5 cells per well. After the cells adhered, they were divided into groups and treated with drug-free medium, low, medium, and high concentration drug-containing medium for 48 hours.

[0058] (2) Cell collection: Cells were collected by trypsin digestion without EDTA (including floating cells in the culture medium), and centrifuged and washed twice with pre-cooled PBS (3) Staining: Resuspend the cells with 500 μL of 1 x Binding Buffer. Add 5 μL of Annexin V-FITC and 5 μL of PI staining solution, mix gently. Incubate at room temperature for 15 minutes in the dark.

[0059] (4) Detection: Within 1 hour, use a flow cytometer for detection. Analyze the cell population through the FITC and PI channels to distinguish between viable cells (Annexin V － / PI － ), early apoptotic cells (Annexin V + / PI － ), and late apoptotic / necrotic cells (Annexin V + / PI + ).

[0060] Results are shown in Tables 4, 5, Figure 3 and Figure 4 .

[0061] Table 4: Percentage of late apoptosis

[0062] Table 5: Percentage of early apoptosis

[0063] Compared with the blank control group, the positive drug cisplatin (40 μM) and high and medium doses of traditional Chinese medicine extracts can significantly induce apoptosis of A549 cells (Total Apoptosis Rate, early apoptosis + late apoptosis), and have a concentration-dependent effect. The early apoptosis of the low-dose group is slightly higher than that of the control group, and the late apoptosis has no significant increase, and the effect is weak. The early apoptosis (18.76%) and late apoptosis (6.69%) of the medium-dose group and the early apoptosis (44.33%) and late apoptosis (11.67%) of the high-dose group show that the drug mainly starts the programmed death pathway, and most of the cells in the high-dose group enter the early stage of programmed death. Cisplatin treatment mainly leads to late apoptosis and necrosis of cells (44.93%), which is consistent with the mechanism of action of cisplatin as a DNA damaging agent. It causes a large number of DNA cross-linking, which eventually leads to cell membrane rupture, directly entering the late apoptosis or necrosis stage.

[0064] 4. Cell migration experiment (1) Preparation of cell suspension: trypsin digestion, resuspension with serum-free medium and counting, and adjustment of cell density to 2.5 x 10 5 / mL.

[0065] (2) Add 500 μL of medium containing 10% FBS and containing corresponding high, medium and low concentrations of traditional Chinese medicine extracts and positive drugs to the lower chamber (the bottom of the 24-well plate). Put the Transwell chamber into the hole. Take 300 μL of cell suspension and carefully add it to the upper chamber.

[0066] (3) Incubation: place the culture plate in a 37°C, 5% CO2 incubator for 24 hours.

[0067] (4) Fixation and staining: remove the chamber and wash the chamber bottom membrane twice with PBS. Put the chamber into the cell fixation solution for 15-30 minutes, then stain with 0.1% crystal violet solution for 20 minutes. Gently wipe off the un-migrated cells inside the upper chamber with a cotton swab.

[0068] (5) Washing and counting: gently wash off the excess dye with PBS and air dry. Randomly select multiple fields under an inverted microscope and count the number of cells migrated under the membrane surface, and take the average value for statistical analysis.

[0069] The results are shown in Table 6, Figure 5 , Figure 6 .

[0070] Table 6: Cell migration number table

[0071] The blank control group cells showed very strong migration ability, with a migration cell number of 1213, indicating that A549 cells themselves had high migration potential. The migration cell number of the low-dose group decreased to 737, the migration cell number of the medium-dose group decreased significantly to 164, and the migration cell number of the high-dose group decreased to 69.00. The drug had a significant effect on inhibiting cell migration.

[0072] 5. Protein expression detection (1) Total protein extraction: collect cells treated with drugs, wash twice with pre-cooled PBS. Add appropriate amount of RIPA lysis buffer (containing PMSF protease inhibitor and phosphatase inhibitor) to each well, lyse on ice for 30 minutes, and shake from time to time. Collect the lysate with a cell scraper, centrifuge (4°C, 12000 rpm, 15 min), and take the supernatant as the total protein solution.

[0073] (2) Protein quantification (BCA method): according to the BCA kit instructions, prepare a standard curve, and measure the protein concentration of each sample. Adjust the protein concentration of all samples to be consistent, add 5xLoading Buffer, and boil for 10 minutes in a 100°C metal bath to denature the protein.

[0074] (3) SDS-PAGE electrophoresis: according to the target protein molecular weight, prepare the appropriate concentration of separation gel (10%) and concentration gel (5%). Add the protein to the sample well in turn, and perform constant voltage electrophoresis (concentration gel 80V, separation gel 120V).

[0075] (4) Membrane transfer: after electrophoresis, cut the gel block in the range of the target protein, and use the "sandwich" structure (negative electrode-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-positive electrode) to transfer the membrane in the transfer solution at a constant current (200mA) for 45 minutes.

[0076] (5) Blocking and antibody incubation Blocking: immerse the membrane in 5% skim milk or BSA blocking solution, and block on a shaker at room temperature for 1 hour.

[0077] Primary antibody incubation: dilute the primary antibody with the blocking solution according to the instructions. Incubate the membrane with the primary antibody at 4°C overnight.

[0078] Wash the membrane: wash the membrane with TBST solution on a shaker for 3 times, 10 minutes each time.

[0079] Secondary antibody incubation: dilute the HRP-labeled secondary antibody (such as goat anti-rabbit or goat anti-mouse) with the blocking solution, and incubate at room temperature for 1 hour.

[0080] Wash the membrane: wash the membrane with TBST again for 3 times, 10 minutes each time.

[0081] Chemiluminescence development: mix equal volume of ECL chemiluminescence reagent A and B, drop onto the membrane, after 30 seconds reaction, in chemiluminescence imaging system.

[0082] Results are shown in Figure 7 , Figure 8 .

[0083] Compared with the blank control group, the protein expression level of E-cadherin in each administration group was significantly increased, and the Chinese medicine extract could up-regulate the expression of E-cadherin protein in a concentration-dependent manner, but the effect of the medium dose group was the most significant. The Chinese medicine extract could inhibit the epithelial-mesenchymal transition (EMT) process and promote the cells to maintain epithelial characteristics, thereby reducing the migration ability. Compared with the blank control group, the protein expression level of PCNA in each administration group was significantly decreased, and the proliferation activity of the cells was inhibited. Compared with the blank control group, the expression level of cleaved-Caspase-3 protein, a key execution protein of apoptosis, in each administration group was significantly changed, directly proving from the molecular level that the Chinese medicine extract could induce A549 cell death by activating the Caspase-3-dependent apoptosis pathway. This is completely consistent with the phenotype results of the flow cytometry detecting the increase of early apoptosis rate.

[0084] The above detailed description is a specific description of one of the feasible embodiments of the present application, which is not used to limit the patent scope of the present application, and any equivalent implementation or change without departing from the present application shall be included in the scope of the technical solutions of the present application.

Claims

1. A traditional Chinese medicine composition for treating lung nodules, characterized in that, The traditional Chinese medicine composition comprises the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 10-20 parts, Astragalus 10-15 parts, Chinese yam 10-15 parts, Houttuynia cordata 5-10 parts, dandelion 5-10 parts, Oldenlandia 5-10 parts, ginseng 5-10 parts, Sanchi 5-10 parts, Ophiopogon 5-10 parts, lily 5-10 parts.

2. The traditional Chinese medicine composition for treating lung nodules according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 12-18 parts, Astragalus 10-15 parts, Chinese yam 10-15 parts, Houttuynia cordata 6-9 parts, dandelion 6-9 parts, Oldenlandia 5-10 parts, ginseng 5-8 parts, Sanchi 5-8 parts, Ophiopogon 8-10 parts, lily 8-10 parts.

3. The traditional Chinese medicine composition for treating lung nodules according to claim 2, characterized in that, The traditional Chinese medicine composition comprises the following weight parts of traditional Chinese medicine raw materials: Ganoderma lucidum 15 parts, Astragalus 12 parts, Chinese yam 12 parts, Houttuynia cordata 9 parts, dandelion 9 parts, Oldenlandia 9 parts, ginseng 6 parts, Sanchi 6 parts, Ophiopogon 9 parts, lily 9 parts.

4. A preparation method of the traditional Chinese medicine composition for treating pulmonary nodules according to any one of claims 1-3, the preparation method comprising the following preparation steps: (1) decoct Ganoderma lucidum, Astragalus, Chinese yam, Ophiopogon, lily, ginseng, Sanchi, Oldenlandia 1-3 times, each time adding 6-10 times the amount of water, each time for 1-2 hours, combining the filtrates, concentrating to obtain a water extract; (2) reflux extract Houttuynia cordata and dandelion with an aqueous ethanol solution 1-2 times, each time for 1-2 hours, combining the filtrates, recovering ethanol, concentrating to obtain an alcohol extract; (3) combine the water extract and the alcohol extract, concentrate and dry to obtain dry extract powder; (4) mix the dry extract powder with appropriate excipients to granulate, and obtain the traditional Chinese medicine composition.

5. The preparation method according to claim 4, characterized in that, In step (1), the water decoction is performed 2 times, each time for 1.5 hours, and the concentration is concentrated to a relative density of 1.10-1.15 at 60°C; in step (2), the volume fraction of ethanol in the aqueous ethanol solution is 70%, the amount added is 6-10 times the amount of medicinal materials, and the reflux extraction is performed 2 times, each time for 1 hour, and the concentration is concentrated to a relative density of 1.10-1.15 at 60°C; in step (3), the concentration is concentrated to a relative density of 1.25-1.30 at 60°C, and the drying is performed by spray drying or vacuum drying.

6. The production method according to claim 4 or 5, characterized by, In step (4), the excipients are selected from at least one of microcrystalline cellulose, starch, lactose and magnesium stearate.

7. Use of the traditional Chinese medicine composition according to any one of claims 1-3 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 4-6 in the preparation of a drug for treating pulmonary nodules.

8. Use according to claim 7, characterized in that, The drug is a capsule or a tablet.

9. Use according to claim 7, characterized in that, The daily dose of the drug is 1.5-3.0 g of dry extract powder, taken 2-3 times a day.

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