A traditional Chinese medicine compound preparation for treating lung cancer and use thereof

Abstract

The application discloses a traditional Chinese medicine compound preparation for treating lung cancer, which is prepared from the following raw materials in parts by weight: Ginkgo biloba leaves 6-9 parts, Schisandra chinensis 6-9 parts, Astragalus membranaceus 15-45 parts, Atractylodes macrocephala 9-15 parts, Coix lachryma-jobi 15-30 parts, Dioscorea opposita 15-30 parts, Prunus davidiana 12-24 parts, Triadica cochinchinensis 15-30 parts, Berberis dictyota 15-30 parts, Spermacoce latifolia 15-30 parts. The application finds that the traditional Chinese medicine compound preparation can inhibit lung cancer cell invasion and metastasis through a TGF-beta / STAT3 / PD-L1 signal pathway, and reveals that the traditional Chinese medicine compound preparation can kill lung cancer tumor cells and also has a significant inhibiting effect on tumor metastasis and recurrence, and the anti-tumor effect is clear and reliable.

Description

Technical Field

[0001] This invention belongs to the field of research, development and utilization of traditional Chinese medicine, specifically relating to a traditional Chinese medicine compound preparation for the treatment of lung cancer and its uses. Background Technology

[0002] Lung cancer is one of the world's deadliest and most dangerous cancers, causing numerous deaths in men and women each year. Despite advances in chemotherapy, immunotherapy, and targeted therapy, lung cancer remains a leading cause of cancer-related deaths. Distant metastasis is the primary cause of most cancer deaths, and preventing tumor recurrence and distant metastasis is a hallmark of effective cancer treatment.

[0003] Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule that inhibits immune responses, and it is closely related to the immune escape process of tumor cells. Targeting PD-L1 may be a promising treatment for cancer patients; we can also inhibit PD-L1 expression by inhibiting upstream STAT3 signaling. Compared with the TNM staging system, p-STAT3 and PD-L1 can serve as excellent predictors of postoperative prognosis in cancer patients. Inhibiting STAT3 activation to reduce PD-L1 expression can suppress tumor cell invasion and metastasis. Chemotherapy and targeted therapies are accompanied by significant adverse reactions, and immune checkpoint inhibitors have low efficacy. Despite significant progress in the diagnosis and treatment of lung cancer in recent years, the goal of basic disease control remains far from being achieved. Therefore, exploring new treatment approaches remains an urgent priority. Summary of the Invention

[0004] In view of the above-mentioned shortcomings of the prior art, according to embodiments of the present invention, it is desirable to provide a compound preparation for treating lung cancer that has the effect of significantly improving the general condition of lung cancer patients and inhibiting lung cancer metastasis and recurrence.

[0005] Based on the clinical practice and research foundation of traditional Chinese medicine in the treatment of lung cancer, this invention uses the improvement of the general condition of lung cancer patients or animal models and the inhibition of lung cancer metastasis and recurrence as efficacy indicators. Through dialectical analysis of the pathogenesis of lung cancer and sorting out and screening the efficacy of traditional Chinese medicine, the compound formula of this invention is combined.

[0006] According to the embodiments, the present invention provides a compound preparation for treating lung cancer, the innovation of which is that it is made from the following raw materials in parts by weight: 6-9 parts of Ginkgo biloba leaf, 6-9 parts of Schisandra chinensis, 15-45 parts of Astragalus membranaceus, 9-15 parts of Atractylodes macrocephala, 15-30 parts of Coix lacryma-jobi, 15-30 parts of Dioscorea opposita, 12-24 parts of Citrus aurantium, 15-30 parts of Sedum aizoon, 15-30 parts of Selaginella tamariscina, and 15-30 parts of Hedyotis diffusa.

[0007] According to one embodiment, in the aforementioned compound preparation for treating lung cancer of the present invention, the raw materials and their weight parts are preferably: 9 parts of Ginkgo biloba leaf, 9 parts of Schisandra chinensis, 30 parts of Astragalus membranaceus, 9 parts of Atractylodes macrocephala, 30 parts of Coix lacryma-jobi, 15 parts of Dioscorea opposita, 15 parts of Gynostemma pentaphyllum, 30 parts of Hedyotis diffusa, 30 parts of Selaginella tamariscina, and 30 parts of Hedyotis diffusa.

[0008] According to one embodiment, in the aforementioned compound preparation for treating lung cancer of the present invention, the compound preparation is a decoction, tablet, granule, capsule or oral liquid.

[0009] According to one embodiment, in the aforementioned compound preparation for treating lung cancer of the present invention, the lung cancer is non-small cell lung cancer.

[0010] There are no special requirements for the preparation method of the compound preparation for treating lung cancer mentioned above in this invention. It can be prepared into any dosage form as defined in pharmaceutics, such as decoction, granules, tablets, capsules or oral liquid, using conventional methods of traditional Chinese medicine preparation.

[0011] The properties of the active pharmaceutical ingredients included in this invention are as follows:

[0012] Ginkgo leaves: sweet, bitter, and astringent; neutral in nature. They enter the heart and lung meridians. They have the effects of astringing the lungs and relieving asthma, promoting blood circulation and removing blood stasis, clearing the channels and relieving pain, and reducing turbidity and lipids.

[0013] Schisandra chinensis: Sour and sweet in taste, warm in nature. It enters the lung, heart, and kidney meridians. It has the effects of astringing and consolidating, replenishing qi and promoting body fluids, tonifying the kidneys and calming the mind.

[0014] Astragalus: Sweet in taste and slightly warm in nature. It enters the spleen and lung meridians. It has the effects of strengthening the spleen and replenishing the middle jiao, raising yang and lifting prolapse, benefiting wei qi and consolidating the exterior, promoting diuresis, and promoting tissue regeneration and detoxification.

[0015] Atractylodes macrocephala: Bitter and sweet in taste, warm in nature. It enters the spleen and stomach meridians. It has the effects of tonifying qi and strengthening the spleen, drying dampness and promoting diuresis, stopping sweating, and calming the fetus.

[0016] Job's tears: sweet and bland in taste, cool in nature. It enters the spleen, stomach, and lung meridians. It has the effects of promoting diuresis and eliminating dampness, strengthening the spleen and stopping diarrhea, relieving numbness, draining pus, detoxifying and dispersing nodules.

[0017] Yam: Sweet in taste and neutral in nature; it enters the spleen, lung, and kidney meridians. It has the effects of strengthening the spleen, nourishing the lungs, tonifying the kidneys, and replenishing essence.

[0018] Yuzhizi: Sweet and cold in nature. It enters the liver, spleen, and kidney meridians. It has the effects of soothing the liver and regulating qi, promoting blood circulation, dispersing blood stasis and relieving pain, eliminating irritability and promoting urination.

[0019] Stone-like herb: pungent, bitter, slightly cold. Meridians: Liver and Spleen. Functions: clears heat and detoxifies, reduces swelling and dissipates nodules.

[0020] Sedum aizoon: Sweet, slightly bitter, and astringent in taste, and cool in nature. It enters the lung and liver meridians. It has the effects of clearing heat and detoxifying, anti-cancer, and hemostasis.

[0021] Oldenlandia diffusa: Slightly bitter in taste, cold in nature. It enters the stomach, large intestine, and small intestine meridians. It has the effects of clearing heat and detoxifying, promoting diuresis and relieving strangury.

[0022] The theoretical basis of this compound medicine is as follows: the overall pathogenesis of lung cancer is deficiency of the root and excess of the branch, with deficiency of the ancestral qi being the root cause, and stagnation of qi and blood stasis and toxic pathogens being the branch cause. Ginkgo biloba leaf is the principal herb, entering the heart and lung meridians, and has the effects of astringing the lungs and relieving asthma, and promoting blood circulation and removing blood stasis. Schisandra chinensis and Astragalus membranaceus are the assistant herbs, assisting Ginkgo biloba leaf in astringing the lungs and replenishing qi. Atractylodes macrocephala, Coix lacryma-jobi, and Dioscorea opposita are added to promote diuresis and strengthen the spleen, and promote the generation of qi and blood. Anemarrhena asphodeloides is added to soothe the liver, regulate qi and promote blood circulation, so as to regulate the ancestral qi and promote the circulation of qi. Hedyotis diffusa, Selaginella tamariscina, and Hedyotis diffusa are added to detoxify and dissipate nodules, so as to remove masses and lumps.

[0023] Studies of the components of traditional Chinese medicine show that the raw materials in this invention contain monomers such as ginkgo biflavonoids, ursolic acid, and luteolin, which have inhibitory effects on tumor metastasis. Subsequent examples and experimental cases will demonstrate that the compound preparation of this invention can improve the general condition of lung cancer patients, regulate tumor development and progression, inhibit tumor metastasis and recurrence, and can also enhance anti-tumor immune responses by regulating PD-L1 expression, thus preventing tumor immune escape. Attached Figure Description

[0024] Figure 1 The Ginkgo Biloba Decoction Granules provided in this embodiment of the invention inhibit the cell proliferation of PC-9 and A549 cells. Figure 1 middle: Figure 1 (A) The effects of Ginkgo biloba extract granules at concentrations of 0, 0.75, 1.5, 3, 6, 12, 24, 48, 96, and 192 mg / ml on the growth and proliferation of PC-9 and A549 cells were detected by CCK-8 assay. The results showed that after treatment with different concentrations of Ginkgo biloba extract granules for 24 h, the granules at 12 mg / ml had an inhibitory effect on the growth of lung cancer cells, and the inhibitory effect was more obvious with the increase of drug concentration. Figure 1 (B) Compared with the control group, when the concentration of Ginkgo biloba extract granules was 6 mg / ml, cell proliferation did not change significantly (P > 0.05); when the concentration of Ginkgo biloba extract granules was 12 mg / ml, cell proliferation was inhibited (P < 0.05). To eliminate the influence of the cytotoxic effect of Ginkgo biloba extract granules on PC-9 and A549 cells, the drug concentrations for subsequent in vitro studies on the anti-lung cancer cell metastasis effects of Ginkgo biloba extract granules were selected as 1.5, 3, and 6 mg / ml.

[0025] Figure 2 This is a scratch assay diagram showing the effect of Ginkgo biloba extract granules provided in this embodiment of the invention on inhibiting lung cancer cell migration. The results of the 24-hour scratch assay show... Figure 2(A) shows that in PC-9 cells, compared with the Ginkgo biloba extract granules group, the scratch repair rate of the control group was significantly increased and the migration ability was significantly enhanced (P<0.01). The Ginkgo biloba extract granules group inhibited the metastasis of lung cancer cells in a drug concentration gradient-dependent manner. Figure 2 (B) is a bar chart showing the statistical area of ​​scratches on PC-9 cells. Figure 2 (C) shows that in A549 cells, compared with the Ginkgo biloba extract granules group, the scratch repair rate of the control group was significantly increased and the migration ability was significantly enhanced (P<0.01). The Ginkgo biloba extract granules group inhibited the metastasis of lung cancer cells in a drug concentration gradient-dependent manner. Figure 2 (D) is a bar chart showing the statistical area of ​​scratches on A549 cells.

[0026] Figure 3 The image shown is a Western blot result of the effect of Ginkgo biloba extract granules on lung cancer cells provided in this embodiment of the invention. Figure 3 (A) is a diagram showing the changes in protein expression in PC-9 and A549 cells regulated by Ginkgo biloba extract granules; Figure 3 (B) is a statistical graph of the results of Western Blot analysis of PC-9 and A549 cells.

[0027] Figure 4 (A) Effect of scratch assay on the migration of Ginkgo biloba extract granules on TGF-β-induced PC-9 lung cancer cells. Figure 4 (B) is a bar chart showing the statistical area of ​​scratches on PC-9 cells. Figure 4 (C) Scratch test: Effect of Ginkgo biloba extract granules on TGF-β-induced migration of lung cancer cells A549. Figure 4 (D) is a bar chart showing the statistical area of ​​scratches on A549 cells.

[0028] Figure 5 (A) Western Blot results of protein changes in PC-9 and A549 cells induced by TGF-β induced by Ginkgo biloba extract granules. Figure 5 (B) is a statistical graph of the results of Western Blot analysis of PC-9 and A549 cells.

[0029] Figure 6 (A) is a Western blotting result of SiPD-L1 in PC-9 cells. Figure 6 (B) is a statistical graph of the results of Western Blot on PC-9 cells. Figure 6 (C) is a Western blot result of SiPD-L1 in A549 cells. Figure 6 (D) is a statistical graph of the results of Western Blot on PC-9 cells. Figure 6(E) is a diagram showing the scratch test results after PC-9 cells were Si PD-L1. Figure 6 (F) Statistical graph of the results of the Si PD-L1 scratch experiment on PC-9 cells. Figure 6 (G) is a scratch assay result of A549 cells after Si PD-L1 treatment. Figure 6 Statistical chart of Si PD-L1 scratch assay results for (H)A549 cells. Figure 6 (I) is a figure showing the results of a scratch assay induced by TGF-β after SiPD-L1 in PC-9 cells. Figure 6 (J) is a statistical graph of the scratch test results induced by TGF-β after SiPD-L1 in PC-9 cells. Figure 6 (K) is a graph showing the results of a scratch assay induced by TGF-β after Si PD-L1 in A549 cells. Figure 6 (L) is a statistical graph of the scratch test results induced by TGF-β after SiPD-L1 in A549 cells.

[0030] Figure 7 (A) is an in vivo experiment of Ginkgo biloba extract granules provided in the embodiments of the present invention. Figure 7 (A) Results show that, compared with the Control group, the tumor burden of lung tissue in mice in the YXGJD1 (Ginkgo biloba extract granules 3.3 g / kg), YXGJD2 (Ginkgo biloba extract granules 6.6 g / kg) and Cisplatin groups was significantly different. Figure 7 (B) shows the HE results of lung tissue from each group of mice. Figure 7 (C) is a statistical chart of lung tissue weight in each group of mice. Figure 7 (DF) is a statistical graph of liver and kidney function in each group of mice. Figure 7 (G) is a statistical graph of the body weight of mice in each group. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments should be understood as illustrative only and not as limiting the scope of protection of the present invention. After reading the description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0032] Example 1

[0033] Weigh out 9g of Ginkgo biloba leaves, 9g of Schisandra chinensis, 30g of Astragalus membranaceus, 9g of Atractylodes macrocephala, 30g of Coix lacryma-jobi, 15g of Dioscorea opposita, 15g of Citrus aurantium, 30g of Hedyotis diffusa, 30g of Selaginella tamariscina, and 30g of Hedyotis diffusa. Prepare granules (Ginkgo Biloba Decoction Granules) using conventional methods for traditional Chinese medicine preparations. Each packet contains 11g of granules, and each 1g of granules is equivalent to 9.4g of crude drugs. Dilute with pure water to 0.75, 1.5, 3, 6, 12, 24, 48, 96, and 192mg / ml for use in cells; or dilute with pure water to 3.3g / kg and 6.6g / kg for oral administration to mice.

[0034] In the following test examples 1-4 of this invention, Ginkgo biloba extract granules were purchased from Wanshicheng Pharmaceutical Products Co., Ltd., and the raw materials, their proportions, and preparation methods were the same as in Example 1.

[0035] Ginkgo biloba extract granules are an experienced formula developed by Chief Physician Deng Haibin of Longhua Hospital. Does it have the effect of inhibiting the invasion and metastasis of lung cancer cells? A thorough explanation of the anti-lung cancer cell invasion and metastasis effect and safety of Ginkgo biloba extract granules is key to expanding the medicinal value of Ginkgo biloba extract granules and is also a challenge in the development of anticancer drugs. The technical solution of this invention will be further described below with reference to experimental examples 1-5.

[0036] S101, Cell Culture: Cell resuscitation, cell culture, and cell passage. The effects of different concentrations of Ginkgo biloba extract granules on the growth and proliferation of PC-9 and A549 cells were detected using the CCK-8 (cellcounting kit 8) assay.

[0037] S102, the inhibitory effect of Ginkgo biloba extract granules on the migration of PC-9 and A549 cells was detected by scratch assay.

[0038] S103, Western blot experiment was conducted to detect the effect of Ginkgo biloba extract granules on the expression of MMP2 and MMP9 proteins related to the invasion and metastasis of PC-9 and A549 lung cancer cells, and the expression of STAT3, p-STAT3 and PD-L1 proteins related to the TGF-β / STAT3 / PD-L1 signaling pathway was also detected.

[0039] S104, using transforming growth factor-β1 (TGF-β1) to induce invasion and metastasis in PC-9 and A549 cells; the effect of Ginkgo biloba extract granules on TGF-β1-induced PC-9 and A549 cell migration was observed using a scratch assay; Western blot was used to detect the effect of Ginkgo biloba extract granules on the expression of invasion and metastasis-related proteins and the TGF-β / STAT3 / PD-L1 signaling pathway in TGF-β1-induced PC-9 and A549 cells. After knocking down PD-L1 in PC-9 and A549 cells, the effect of PD-L1 on lung cancer cell invasion and metastasis was observed using a scratch assay. TGF-β induced invasion and metastasis in PD-L1-knockdown PC-9 and A549 cells, further investigating the effect of PD-L1 on invasion and metastasis in lung cancer cells.

[0040] S105 was used to establish an in vivo lung cancer metastasis model in BALB / c nude mice by injecting A549 cells into the tail vein; different concentrations of Ginkgo biloba extract granules were administered to the mice, and the lung weight of the mice was observed; the liver and kidney function and body weight of the mice were tested to observe the toxic side effects of the drugs.

[0041] Experimental Example 1 - Effect of Ginkgo Biloba Decoction Granules on the Growth and Proliferation of PC-9 and A549 Lung Cancer Cells.

[0042] 1. Experimental Materials

[0043] 1.1 Experimental Cells

[0044] Human non-small cell lung cancer PC-9 cells and A549 cells were both purchased from Wuhan Pronosei Life Sciences Co., Ltd. PC-9 and A549 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin dual antibodies. Both cell types were cultured in a constant temperature incubator at 37°C and 5% CO2 saturated humidity.

[0045] 1.2 Experimental Drugs

[0046] Ginkgo Biloba Decoction Granules, purchased from Wanshicheng Pharmaceutical Products Co., Ltd., contain 9g of ginkgo leaves, 9g of schisandra fruit, 30g of astragalus root, 9g of atractylodes macrocephala, 30g of coix seed, 15g of yam, 15g of euphorbia fruit, 30g of sclerotium truncatum, 30g of cypress bark, and 30g of oldenlandia diffusa. Each packet contains 11g granules, and each 1g granule is equivalent to 9.4g of raw herbs.

[0047] 1.3 Main reagents for the experiment

[0048]

[0049] 1.4 Main Experimental Instruments

[0050]

[0051] 2. Cell Culture

[0052] 2.1 Cell resuscitation

[0053] Remove the frozen cells from the -80℃ freezer and quickly place the cryovials into a preheated 37℃ water bath. Shake the cryovials until the liquid in them thaws. Spray the outside with alcohol for disinfection and place them in a clean bench. Carefully aspirate the cell suspension into a centrifuge tube containing 10mL of culture medium. Gently pipette the cells and centrifuge at 1000rpm for 5 minutes. Discard the supernatant and pipette the cells again with 8-10mL of culture medium. Transfer the cells to a culture dish and incubate at 37℃ in a 5% CO2 incubator.

[0054] 2.2 Cell Culture

[0055] Cells were cultured in a complete culture medium containing 10% fetal bovine serum, 1% penicillin-streptomycin mixture, and 89% DMEM, and then incubated at 37°C in a constant temperature incubator containing 5% CO2.

[0056] 2.3 Cell passage

[0057] When the cells have cultured to approximately 80-90% of the bottom area of ​​a 10cm culture dish, they can be passaged. Take 2ml of PBS buffer and gently rinse the adherent cells in the culture dish twice. Using a 1ml pipette, add 1ml of 0.25% phenol red-free trypsin to the 10cm culture dish, shake the dish to ensure the 1ml of trypsin fully covers the bottom, and gently pipette away any remaining cells at the bottom of the 10cm culture dish to form a cell suspension. Collect all the suspension into a 5ml EP tube, balance it, and centrifuge. Centrifuge EP tubes at 1300 rpm for 3 minutes, discard the supernatant (be careful not to aspirate the cells), add 1 ml of complete culture medium, gently blow and mix well. After centrifuging the cell suspension, pre-add 7 ml of complete culture medium to each 10 cm culture dish. Distribute the cell suspension evenly to each 10 cm culture dish at a 1:3 passage ratio, label the cells with their names, passage dates, and names, and place them in an incubator for continuous culture until the cells are completely adhered. Passage the cells 3-4 times per week.

[0058] 3. Cell proliferation experiment

[0059] PC-9 and A549 cells in good growth condition and in the logarithmic growth phase were collected and processed according to the passage procedure. After digestion, centrifugation, and remixing, 20 μl of the cell suspension was vertically injected into a cell counting chamber for cell counting. Complete culture medium was placed in a disposable sample container, and the cells were diluted to the required concentration. After remixing, the cells were seeded into each well of a 96-well plate. During each layering process, the cells were remixed several times using a pipette to ensure a consistent cell count in each well. 100 μl of PBS solution was added to each of the four wells of the 96-well plate. After layering, the cell type, name, time, and experimental type were labeled, and the plates were incubated until the cells were fully adhered. The drug concentration was pre-set before drug addition: Ginkgo biloba extract granules were set at concentrations of 0, 0.75, 1.5, 3, 6, 12, 24, 48, 96, and 192 mg / ml. Each concentration was used in triplicate. 24 hours after plating, observe for cell adhesion. If cell growth is good, proceed with the drug intervention procedure. Discard the old culture medium using a multi-channel pipette, then aspirate the remaining medium using a 100μl pipette tip. Prepare the solution according to the drug concentration mentioned above in a multi-channel sample well, and mix thoroughly using a 1ml pipette. After 24 hours of drug-treated culture, use a CCK-8 proliferation kit to detect cell growth and proliferation after intervention. The entire procedure must be performed in the dark. After thoroughly aspirating the culture medium with a pipette tip, remove the remaining culture medium again. Add 100 μl of FBS-free DMEM medium containing 10% CCK-8 solution to each well. Place the plate in an incubator to allow the CCK-8 reagent to react fully. After 1 hour, remove the 96-well plate and measure the absorbance (OD value) of each solution under a multi-mode microplate reader at a wavelength of 450 nm. Calculate the cell viability using the equation [(As(experimental wells) - Ab(blank wells) / Ac(control wells) - Ab(blank wells)] × 100%. Save the data and use Graph Pad Prism 9.0 for data analysis and graphing.

[0060] 4. Experimental results: Ginkgo biloba extract granules inhibited the cell proliferation of PC-9 and A549 cells. Figure 1 middle: Figure 1 (A) The effects of Ginkgo biloba extract granules at concentrations of 0, 0.75, 1.5, 3, 6, 12, 24, 48, 96, and 192 mg / ml on the growth and proliferation of PC-9 and A549 cells were detected by CCK-8 assay. The results showed that after treatment with different concentrations of Ginkgo biloba extract granules for 24 h, the granules at 12 mg / ml had an inhibitory effect on the growth of lung cancer cells, and the inhibitory effect was more obvious with the increase of drug concentration. Figure 1(B) Compared with the control group, when the concentration of Ginkgo biloba extract granules was 6 mg / ml, cell proliferation did not change significantly (P > 0.05); when the concentration of Ginkgo biloba extract granules was 12 mg / ml, cell proliferation was inhibited (P < 0.05). To eliminate the influence of the cytotoxic effect of Ginkgo biloba extract granules on PC-9 and A549 cells, the drug concentrations for subsequent in vitro studies on the anti-lung cancer cell metastasis effects of Ginkgo biloba extract granules were selected as 1.5, 3, and 6 mg / ml.

[0061] Experimental Example 2 - Effect of Ginkgo Biloba Decoction Granules on the Migration Ability of PC-9 and A549 Cells.

[0062] 1. Experimental Materials

[0063] 1.1 Experimental Cells

[0064] Same as the experimental cells in Experiment 1.1.

[0065] 1.2 Experimental Drugs

[0066] Ginkgo Biloba Decoction Granules, purchased from Wanshicheng Pharmaceutical Products Co., Ltd., contain 9g of ginkgo leaves, 9g of schisandra chinensis, 30g of astragalus membranaceus, 9g of atractylodes macrocephala, 30g of coix seed, 15g of yam, 15g of euphorbia pekinensis, 30g of sclerotium affine, 30g of selaginella tamariscina, and 30g of hedyotis diffusa. Each packet contains 11g granules, and each 1g granule is equivalent to 9.4g of raw herbs. The granules are diluted to concentrations of 1.5, 3, and 6mg / ml to target lung cancer cells.

[0067] 1.3 Main reagents and instruments used in the experiment

[0068] The main reagents and instruments used in experiments 1.3 and 1.4 of Experiment 1 are the same.

[0069] 2. Cell Culture

[0070] Same as in Experiment 1, Section 2. Cell culture method.

[0071] 3. Scratch test

[0072] This section is divided into groups with different concentrations of Ginkgo biloba extract granules. PC-9 and A549 cells in the logarithmic growth phase were trypsinized and centrifuged, then mixed with serum-free DMEM medium to adjust the cell concentration to 2.5 × 10⁻⁶ cells / year. 5Cells were seeded at a density of 100 cells / ml into 6-well plates, with 3ml of culture medium added to each well. After 24 hours of seeding, when the cells reached 80%-90% cellularity and were in good growth condition, a scratching procedure was performed. A 100μL pipette tip was used to make a scratch perpendicular to the plane of the 6-well plate. The bottom of the plate was gently washed twice with PBS solution to clean the scratched area as much as possible. Then, 3ml of serum-free DMEM culture medium was added to the 6-well plate. Images were taken under a microscope, and the plates were then incubated. The repair of the scratches was observed and photographed under a microscope at 24 hours. Experimental data were analyzed using ImageJ software and computation was performed using Graphpad.

[0073] Increased migration ability is a decisive characteristic of tumor cell metastasis, and the scratch assay can detect the migration ability of tumor cells over a certain period of time. Experimental results show that the 24-hour scratch assay results... Figure 2 (A) shows that in PC-9 cells, compared with the Ginkgo biloba extract granules group, the scratch repair rate of the control group was significantly increased and the migration ability was significantly enhanced (P<0.01). The Ginkgo biloba extract granules group inhibited the metastasis of lung cancer cells in a drug concentration gradient-dependent manner. Figure 2 (B) is a bar chart showing the statistical area of ​​scratches on PC-9 cells. Figure 2 (C) shows that in A549 cells, compared with the Ginkgo biloba extract granules group, the scratch repair rate of the control group was significantly increased and the migration ability was significantly enhanced (P<0.01). The Ginkgo biloba extract granules group inhibited the metastasis of lung cancer cells in a drug concentration gradient-dependent manner. Figure 2 (D) is a bar chart showing the statistical area of ​​scratches on A549 cells.

[0074] Experimental Example 3 - Protein Level Detection: The Inhibition of Lung Cancer Invasion and Metastasis by Ginkgo Biloba Decoction Granules.

[0075] 1. Western blot analysis was conducted to detect the effects of Ginkgo biloba extract granules on the expression of MMP2 and MMP9 proteins, which are related to the invasion and metastasis of PC-9 and A549 lung cancer cells, and on the inhibition of TGF-β / STAT3 / PD-L1.

[0076] Cells were digested and plated, then cultured in an incubator. The cell culture medium in the culture dish was aspirated, and the cells were washed twice with pre-cooled PBS. An appropriate amount of lysis buffer (RIPA lysis buffer, PMSF, protease inhibitor, phosphatase inhibitor) was added, and the dish was gently agitated to ensure complete contact between the bottom and the lysis buffer. The dish was then placed on ice for 25 min to lyse. Cells were scraped from one end of the dish bottom and collected into a labeled EP tube. The tubes were centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was transferred to a newly labeled EP tube, taking care not to aspirate cell debris from the bottom of the dish. When the BSA standard was diluted to 2 mg / ml, it was halved according to the required number of wells, and 20 μl was added to each well of a 96-well plate. Take 4 μl of protein sample and 16 μl of ddH2O, add them to a 96-well plate, mix thoroughly, then add 200 μl of BCA working solution to each well and incubate at 37°C for 30 min. Use a microplate reader to read the absorbance at 562 nm to create a standard curve and calculate the protein concentration in the sample. Mix 5× loading buffer with the protein sample at a ratio of 1:4, vortex to mix thoroughly, and cook the protein at 100°C for 10 min. Open the cap once before cooking to prevent the EP tube cap from bursting during cooking. After cooling to room temperature, store in a -20°C freezer for later use.

[0077] Arrange clean, thick and thin glass plates neatly, paying attention to both sides, and then vertically clamp them onto the holder, ready for gel pouring. Prepare the gel according to the instructions and mix thoroughly. After preparing the separating gel, add an appropriate amount of anhydrous ethanol to seal it. When a clear boundary line appears (about 15 minutes), pour off the ethanol and absorb any excess ethanol. Add the stacking gel and insert a 10-well comb, slowly and vertically, avoiding air bubbles. After the protein sample dissolves, vortex to mix. Heat at 100°C for 5 minutes, then allow it to return to room temperature. Centrifuge briefly before loading the sample. Place the sample in the electrophoresis tank, filling the inner tank with 1× running buffer, completely covering the loading wells. Ensure there is no leakage before loading. Load the marker first, then the protein. Add the remaining 1× running buffer to the outer electrophoresis tank, close the cap, and pay attention to the positive and negative electrodes. Turn on the power. Set the upper gel to 80V. After the marker runs out of the upper gel (about 25 minutes), change the voltage to 120V and continue running the lower gel. Once the marker in the protein sample reaches the bottom of the lower gel, electrophoresis is complete. Then proceed with the transfer.

[0078] Pre-cool the prepared transfer buffer at 4°C. Immerse the PVDF membrane (5.5cm × 8.5cm) in methanol for 10 seconds, then transfer it along with the sponge to the transfer buffer. After electrophoresis, pry open the glass plate and remove all the stacking gel, creating a sandwich structure. Following the sequence of "sponge-filter paper-PVDF membrane-gel-filter paper-sponge" and adhering to the "black gel, white membrane" principle, tighten the clamps and place the membrane in the transfer tank. Pour the pre-cooled transfer buffer up to the blotting line, and close the lid, ensuring electrode alignment. Place the transfer tank in a bowl and wrap it with ice to prevent overheating of the PVDF membrane. Select different transfer currents and transfer times according to the molecular weight of the antibody. After the transfer, put the PVDF membrane into the prepared skim milk in advance and block it on a shaker for 2 hours. In winter, the blocking time can be appropriately extended. After blocking, wash with 1×TBST 3 times for 5 minutes each time. Cut the membrane according to the molecular weight of the target protein. Prepare the primary antibody by mixing the primary antibody with the primary antibody at a ratio of 1:1000. After washing, place the band in an incubation box and add the primary antibody for overnight incubation.

[0079] Wash the membrane three times a day for 10 minutes each time with TBST on a shaker. Prepare the secondary antibody at a ratio of 1:10000 (TBST to secondary antibody). After washing, incubate the secondary antibody on a shaker at room temperature for 1 hour. After incubation, wash the membrane three times a day for 10 minutes each time with TBST on a shaker. Prepare ECL chromogenic solution in advance (approximately 300 μl / membrane), and wrap it with aluminum foil to protect it from light. Remove the membrane from the TBST, gently pat dry with a paper towel, place it in the center of the imaging system, and add the pre-prepared developing solution, ensuring the membrane is completely covered without overflowing. After 30 seconds of developing reaction, develop the membrane using the machine. Once a clear immunoblot band appears on the membrane, click "save" to save the results to your folder. Analyze the experimental data using ImageJ software and perform data analysis and graphing using GraphPad Prism 9.0.

[0080] Western blot analysis was used to detect changes in the expression of invasion and metastasis-related marker proteins in lung cancer cells (PC-9 and A549 cells) in different groups after intervention with Ginkgo biloba extract granules. The results showed that... Figure 3 (A) Different concentrations of Ginkgo biloba extract granules regulate changes in PC-9 and A549 cell invasion and metastasis-related proteins (MMP2, MMP9) and inhibit the TGF-β / STAT3 / PD-L1 signaling pathway. Figure 3 (B) is a statistical graph of the results of Western Blot analysis of PC-9 and A549 cells.

[0081] Experimental Example 4 - Effect of Ginkgo Biloba Decoction Granules on TGF-β-induced lung cancer cell migration.

[0082] 1. Inducing invasion and metastasis in PC-9 and A549 cells with transforming growth factor-β1 (TGF-β1).

[0083] Preparation of TGF-β1 working solution: Take an appropriate amount of sterile sodium citrate solution, dissolve TGF-β1 lyophilized powder, prepare a working stock solution of 2.5ug / ml, and store at -20℃.

[0084] 1.1 The effect of Ginkgo biloba extract granules on TGF-β1-induced migration of PC-9 and A549 cells was observed by scratch assay.

[0085] The scratch assay was divided into four groups: a blank control group, a Ginkgo biloba extract granule group, a TGF-β group, and a TGF-β + Ginkgo biloba extract granule group (Ginkgo biloba extract granule concentration was 6 mg / ml). PC-9 and A549 cells in logarithmic growth phase were trypsinized, centrifuged, and then plated. After 24 hours of plate formation, the cell saturation was observed to be 80%-90%. Under conditions of good cell growth, the scratch assay was performed. A 100 μl sterile pipette tip was used to make a scratch perpendicular to the plane of a six-well plate. The cells were gently washed twice with PBS to remove the scratched cells. Ginkgo biloba extract granules, TGF-β, and Ginkgo biloba extract granules + TGF-β were added to serum-free medium, then to six-well plates. After photographing under a microscope, the plates were incubated for another 24 hours. Changes in migration ability in each group were observed under a microscope and photographed. The experimental data were analyzed using ImageJ software, and graphs were generated using Graph PadPrism 9.0.

[0086] The scratch assay was used to examine the changes in cell migration ability in four groups of lung cancer cells (PC-9 and A549 cells): NC group, Ginkgo biloba extract granules, TGF-β group, and TGF-β + Ginkgo biloba extract granules group. Figure 4 (A) Effect of scratch assay on the migration of Ginkgo biloba extract granules on TGF-β-induced PC-9 lung cancer cells. Figure 4 (B) is a bar chart showing the statistical area of ​​scratches on PC-9 cells. Figure 4 (C) Scratch test: Effect of Ginkgo biloba extract granules on TGF-β-induced migration of lung cancer cells A549. Figure 4 (D) is a bar chart showing the statistical area of ​​scratch wounds on A549 cells. Ginkgo biloba extract granules significantly inhibited TGF-β-induced lung cancer cell metastasis.

[0087] 1.2 The effects of Ginkgo biloba extract granules on the expression of TGF-β1-induced PC-9 and A549 invasion and metastasis-related proteins and the TGF-β / STAT3 / PD-L1 signaling pathway were detected by Western blot experiment.

[0088] The mechanism by which Ginkgo Biloba Extract Granules inhibit the invasion and metastasis of lung cancer cells (PC-9 and A549 cells) was investigated using Western blot experiments. Results showed: Figure 5 (A) Ginkgo biloba extract granules inhibited TGF-β-induced changes in PC-9 and A549 cell invasion and metastasis-related proteins (MMP2, MMP9) and suppressed the expression of proteins related to the TGF-β / STAT3 / PD-L1 signaling pathway, as well as the expression of p-STAT3 and PD-L1.

[0089] Figure 5 (B) is a statistical graph of the results of Western Blot analysis of PC-9 and A549 cells. Figure 6 (A) is a Western blotting result of SiPD-L1 in PC-9 cells. Figure 6 (B) is a statistical graph of the results of Western Blot on PC-9 cells. Figure 6 (C) is a Western blot result of SiPD-L1 in A549 cells. Figure 6 (D) is a statistical graph of the results of Western Blot on PC-9 cells. Figure 6 (E) is a diagram showing the scratch test results after PC-9 cells were Si PD-L1. Figure 6 (F) Statistical graph of the results of the Si PD-L1 scratch experiment on PC-9 cells. Figure 6 (G) is a scratch assay result of A549 cells after Si PD-L1 treatment. Figure 6 Statistical chart of Si PD-L1 scratch assay results for (H)A549 cells. Figure 6 (I) is a figure showing the results of a scratch assay induced by TGF-β after SiPD-L1 in PC-9 cells. Figure 6 (J) is a statistical graph of the scratch test results induced by TGF-β after SiPD-L1 in PC-9 cells. Figure 6 (K) is a graph showing the results of a scratch assay induced by TGF-β after Si PD-L1 in A549 cells. Figure 6 (L) is a statistical graph showing the results of a TGF-β-induced scratch assay in A549 cells after Si PD-L1 inoculation. The experimental results indicate that inhibiting PD-L1 expression can suppress the invasion and metastasis of lung cancer cells.

[0090] Experimental Example 5 – In vivo animal experiments verify that Ginkgo biloba extract granules inhibit lung cancer cell metastasis.

[0091] 1. An in vivo lung cancer metastasis model was established by injecting A549 cells into BALB / c nude mice via tail vein.

[0092] When the A549 cell density in the culture dish reaches 80% or higher, the cells are digested with trypsin, counted, and then resuspended in PBS to achieve a cell concentration of 4 × 10⁻⁶. 8 Cells / ml; Fix the mouse, thoroughly mix and disperse the cell suspension using a pipette to prevent cell aggregation and embolism. Draw 100 μl of the cell suspension using an insulin needle. Wipe the blood vessels on both sides of the mouse's tail with alcohol to make the blood vessels in the tail full. Insert the needle at 3 / 4 of the way from the base of the tail. When inserting the needle, hold the upper end with the index and middle fingers of the left hand for fixation, and hold the lower end with the thumb and ring finger. The needle tip and blood vessel should be at approximately a 30° angle, with the bevel of the needle facing upwards. Gently prick the skin and the needle tip should immediately be parallel to the blood vessel. Smooth injection indicates successful insertion. After injecting 100 μl of suspension, press a dry cotton ball on the injection site to stop bleeding and lift the tail for 30 seconds.

[0093] 2. Different concentrations of Ginkgo biloba extract granules were administered to mice, and changes in lung tissue and toxic side effects were observed.

[0094] Male BALB / c nude mice were randomly divided into four groups of six each: a model control group, a low-dose Ginkgo biloba extract granule group (YXGJD1), a high-dose Ginkgo biloba extract granule group (YXGJD2), and a cisplatin group. Each mouse was injected with 4 × 10⁴ ginkgo biloba extract granules via the tail vein. 7 A mouse lung metastasis model was established using A549 cells. Fourteen days after modeling, drug treatment was initiated. The Control group was administered physiological saline solution by gavage daily. The YXGJD1 and YXGJD2 groups were administered Ginkgo biloba extract granules (3.3 g / kg and 6.6 g / kg) by gavage daily, respectively (dosage selection was based on our preliminary experimental results, dose conversion criteria, and relevant studies; the low-dose Ginkgo biloba extract granule group (3.3 g / kg) is equivalent to nine times the human dose, and each gram of granules is equivalent to 9.4 g of crude drug). The Cisplatin group received 2 mg / kg of cisplatin every other day. Each BALB / c nude mouse was weighed every two days. Euthanasia was performed according to animal welfare ethics. Lung tissue was removed from the mice, photographed, and the number of lung nodules was counted. The tissue was then aliquoted into PBS and 10% neutral formalin and stored at -80°C for subsequent experiments.

[0095] After treatment with Bouin fixative for 24 hours, white deposits were visible on the surface of the lung tissue. Photographs of mouse lung tissue were taken, and the number of lung nodules was recorded. The lung tissue was then fixed by soaking in 10% neutral formalin. After 24 hours of fixation in 10% formalin, the lung tissue was dehydrated, routinely embedded in paraffin, and sectioned. The paraffin sections were baked in an oven at 60°C for 1-2 hours, dewaxed with xylene and ethanol, stained with hematoxylin for 10 minutes, rinsed with sterile water to remove residual staining, differentiated with 0.7% hydrochloric acid and ethanol, rinsed with sterile water, and the sections turned blue for about 15 minutes. They were then soaked in 95% ethanol for 30 seconds, followed by staining with alcoholic eosin for 30 seconds. The sections were then dehydrated with different concentrations of ethanol, thoroughly soaked in xylene, mounted with neutral resin, and three fields of view were randomly photographed under an optical microscope.

[0096] Observations revealed alterations in the morphology of lung tissue in the Control, YXGJD1, YXGJD2, and Cisplatin groups, with several irregular, visible growths of varying sizes on the surface. After 24 hours of treatment with Bouin fixative, white deposits were observed on the lung tissue surface. Compared to the Control group, the white deposits were reduced in the YXGJD1, YXGJD2, and Cisplatin groups. Microscopic examination of HE-stained lung tissue sections from each group showed that in the Control group, alveolar and interstitial cancer cells occupied normal tissue, losing their normal structure, and most alveolar cells were atrophied and disorganized. Compared to the Control group, the alveolar morphology was more normal in the YXGJD1, YXGJD2, and Cisplatin groups, with a reduced area of ​​cancerous cells. The tumor burden in each group was calculated using ImageJ software. The results showed that compared to the Control group, the average tumor burden in the lung tissues of the YXGJD1, YXGJD2, and Cisplatin groups was reduced, with statistically significant differences (P < 0.01). Experimental results: In vivo experiments showed that Ginkgo biloba extract granules had a concentration gradient-dependent effect on lung cancer cell metastasis, and the difference was statistically significant compared with the control group (P < 0.01). Figure 7 (A) is an in vivo experiment of Ginkgo biloba extract granules provided in the embodiments of the present invention. Figure 7 (A) Results show that, compared with the Control group, the tumor burden of lung tissue in mice in the YXGJD1 (Ginkgo biloba extract granules 3.3 g / kg), YXGJD2 (Ginkgo biloba extract granules 6.6 g / kg) and Cisplatin groups was significantly different. Figure 7 (B) shows the HE results of lung tissue from each group of mice. Figure 7 (C) is a statistical chart of lung tissue weight in each group of mice. Figure 7 (DF) is a statistical graph of liver and kidney function in each group of mice. Figure 7(G) is a statistical graph of the body weight of mice in each group. Blood tests on liver and kidney function in mice in each group showed that there was no significant liver and kidney function damage in the Ginkgo Biloba Decoction Granules group compared with the control group.

[0097] Metastasis in lung cancer signifies reduced survival time and decreased quality of life, and existing treatments have limited efficacy. Traditional Chinese medicine (TCM) possesses multi-target effects, and the innovation of this invention lies in the discovery that Ginkgo biloba extract granules inhibit lung cancer cell invasion and metastasis through the TGF-β / STAT3 / PD-L1 signaling pathway. Therefore, this invention provides a potential novel anti-tumor drug approach for the treatment of lung cancer, which has not yet been reported in the published literature. Its specific mechanism of anti-cancer action is clearly defined, and it has significant implications for the prevention and treatment of malignant tumors, showing broad prospects.

[0098] In summary, the traditional Chinese medicine compound preparation of this invention has a concentration-dependent inhibitory effect on lung cancer cell proliferation; its inhibitory effect on migration also increases in a concentration-dependent manner. The compound preparation is effective, convenient to use, safe, and has no toxic side effects, making it suitable for all stages of lung cancer. This invention reveals that Ginkgo biloba extract granules not only kill lung cancer tumor cells but also significantly inhibit tumor metastasis and recurrence, demonstrating a clear and reliable anti-tumor effect.

Claims

1. A traditional Chinese medicine compound preparation for treating lung cancer, characterized in that, It is made from the following raw materials in parts by weight: 9 parts ginkgo leaves, 9 parts schisandra fruit, 30 parts astragalus root, 9 parts atractylodes rhizome, 30 parts coix seed, 15 parts yam, 15 parts eucommia fruit, 30 parts hedyotis diffusa, 30 parts selaginella tamariscina, 30 parts hedyotis diffusa, and 30 parts oldenlandia diffusa.

2. The traditional Chinese medicine compound preparation for treating lung cancer according to claim 1, characterized in that, The compound preparation is a decoction, granules, tablets, capsules, or oral liquid.

3. The traditional Chinese medicine compound preparation for treating lung cancer according to claim 1, characterized in that, The lung cancer mentioned is non-small cell lung cancer.

4. The use of the traditional Chinese medicine compound preparation according to claim 1 or 2 in the preparation of a drug for treating lung cancer.

5. The use according to claim 4, characterized in that, The lung cancer is non-small cell lung cancer.

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