Pharmaceutical composition (Rhododendron muricatum series) for treating cancer and preparation method and application of pharmaceutical composition (Rhododendron muricatum series)

Abstract

The invention relates to a pharmaceutical composition for treating cancers (Ligusticum wallichii series) as well as a preparation method and application of the pharmaceutical composition. The pharmaceutical composition comprises a ginseng extract, a coix seed extract and a marsdenia tenacissima extract. The pharmaceutical composition disclosed by the invention has a synergistic effect in the aspect of treating cancers, can still have a synergistic effect when being combined with mannatide and berberine hydrochloride, and also has a synergistic effect and toxicity reducing effect when being combined with cis-platinum or gefitinib.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a (Shengmingshu series) pharmaceutical composition for treating cancer, and a preparation method and use thereof. Background Art

[0002] Malignant tumors are what people call cancer. They are a general term for more than 100 related diseases, including breast cancer, lung cancer, colorectal cancer, prostate cancer, bladder cancer, nasopharyngeal cancer, esophageal cancer, stomach cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, etc. When cells in the body mutate, they will continue to divide, out of the control of the body, and eventually form cancer. Malignant tumor cells can invade and destroy adjacent tissues and organs. Moreover, cancer cells can pass out of the tumor and enter the blood or lymphatic system. This is how cancer spreads from the original site to other organs to form new tumors. This process is called cancer metastasis. Most cancers are named according to the organ or cell type where they start.

[0003] Lung cancer is a common malignant tumor, and its etiology is still unclear. A large amount of data shows that the risk factors for lung cancer include smoking (including second-hand smoke), asbestos, radon, arsenic, ionizing radiation, halogenated olefins, polycyclic aromatic compounds, nickel, etc. Lung cancer has no special symptoms in the early stage, and only has the common symptoms of general respiratory diseases, such as cough, blood in sputum, low fever, chest pain, shortness of breath, etc., which are easy to ignore. It is not easy to be discovered until the middle and late stages of lung cancer.

[0004] Currently, chemotherapy with cisplatin is the main treatment for patients with advanced lung cancer. The main side effect of cisplatin chemotherapy is dose-related renal insufficiency. If patients use multiple courses of treatment repeatedly, the toxicity to the kidneys will become more serious. About 30% of patients treated with cisplatin may experience tinnitus or high-pitched hearing loss, which is more serious in children. The neurotoxicity of cisplatin is mainly manifested as peripheral neuropathy, including abnormalities of sensory and motor nerves. Bone marrow suppression can also occur in patients using cisplatin. Other side effects of cisplatin include hyperuricemia, which is mainly caused by drug-induced nephrotoxicity, hypomagnesemia and hypocalcemia, which can manifest as muscle irritation or twitching, paroxysmal spasms, tremors, etc. Allergic reactions caused by cisplatin can manifest as facial edema, sneezing, tachycardia, hypotension, etc. Moreover, the treatment effect decreases gradually after multiple chemotherapy. The main reason for the failure of chemotherapy in clinical practice is that lung cancer cells develop resistance to anticancer drugs. Drug resistance is the most troublesome problem for lung cancer patients with epidermal growth factor receptor (EGFR) gene mutations.

[0005] Gefitinib is suitable for the treatment of locally advanced or metastatic lung cancer that has previously received chemotherapy or is not suitable for chemotherapy. Its inhibition rate on A549 cells is only 12%, and the therapeutic effect is very insignificant.

[0006] Although ginseng extract, coix seed extract and Rhizoma Dioscoreae extract are auxiliary drugs for treating cancer, their inhibitory effects on tumors are not obvious and the effects are poor when used alone.

[0007] Therefore, it is urgent to find a drug therapy that can effectively overcome the drug resistance of tumor cells and has few toxic side effects. Summary of the invention

[0008] In order to overcome the defects of the prior art, the present invention provides a (Shengmingshu series) pharmaceutical composition for treating cancer and its preparation method and use. The pharmaceutical composition of the present invention comprises ginseng extract, coix seed extract and clematis extract. Ginseng extract has the effect of relieving physical fatigue, coix seed extract has the effects of promoting water and dampness, strengthening the spleen and stopping diarrhea, removing numbness, draining pus, detoxifying and dispersing knots, and clematis extract can be used for wheezing, coughing and sputum, postpartum milk stagnation, rheumatism, swelling and pain, sores and carbuncle. The three effects are not obvious in the treatment of cancer, but the inhibition rate of cancer cells after the three are used in combination is as high as 75.6%. The pharmaceutical composition of the present invention has a synergistic effect in treating cancer, and can still have a synergistic effect in combination with mannan peptide and berberine hydrochloride, and also has a synergistic effect of synergistic enhancement and toxicity reduction in combination with cisplatin or gefitinib.

[0009] In one aspect, the present invention provides a pharmaceutical composition comprising a ginseng extract, a coix seed extract and a clematis tongguancao extract.

[0010] Preferably, the pharmaceutical composition (Shengmingshu composition 1) consists of ginseng extract, coix seed extract and clematis extract.

[0011] Preferably, the pharmaceutical composition consists of 300-3000 parts of ginseng extract, 1700-15000 parts of coix seed extract and 1000-10000 parts of clematis tongguan tung extract, based on parts by mass.

[0012] Preferably, the pharmaceutical composition consists of 1000-2000 parts of ginseng extract, 5000-10000 parts of coix seed extract and 3000-8000 parts of clematis tongguan tung extract, based on parts by mass.

[0013] Preferably, the pharmaceutical composition consists of 1700 parts of ginseng extract, 8300 parts of coix seed extract and 5000 parts of clematis tongguan tung extract, calculated by mass.

[0014] Preferably, the pharmaceutical composition consists of 1,300 parts of ginseng extract, 6,700 parts of coix seed extract and 5,000 parts of clematis tongguan tung extract, calculated by weight.

[0015] Preferably, the pharmaceutical composition further comprises one or more of the following: mannan peptide, berberine hydrochloride.

[0016] Preferably, the pharmaceutical composition comprises ginseng extract, coix seed extract, clematis tongguan tung extract, mannan peptide, and berberine hydrochloride.

[0017] Preferably, the pharmaceutical composition (Shengmingshu composition 2) is composed of ginseng extract, coix seed extract, clematis tongguanteng extract, mannan peptide, and berberine hydrochloride.

[0018] Preferably, the pharmaceutical composition consists of 300-3000 parts of ginseng extract, 1700-15000 parts of coix seed extract, 1000-10000 parts of clematis tongguan tung extract, 20-160 parts of mannan peptide, and 7-35 parts of berberine hydrochloride, calculated by mass.

[0019] Preferably, the pharmaceutical composition consists of 1000-2000 parts of ginseng extract, 5000-10000 parts of coix seed extract, 3000-8000 parts of clematis tongguan tung extract, 70-120 parts of mannan peptide, and 10-15 parts of berberine hydrochloride, calculated by mass.

[0020] Preferably, the pharmaceutical composition consists of 1700 parts of ginseng extract, 8300 parts of coix seed extract, 5000 parts of clematis tongguan teng extract, 100 parts of mannan peptide, and 14.9 parts of berberine hydrochloride, calculated by mass.

[0021] Preferably, the pharmaceutical composition consists of 1300 parts of ginseng extract, 6700 parts of coix seed extract, 5000 parts of clematis tongguan teng extract, 100 parts of mannan peptide, and 14.9 parts of berberine hydrochloride, calculated by mass.

[0022] Preferably, the pharmaceutical composition further comprises cisplatin or gefitinib.

[0023] Preferably, the pharmaceutical composition further comprises 0.2-1.5 parts of cisplatin or 0.1-3 parts of gefitinib, calculated by weight.

[0024] Preferably, the pharmaceutical composition further comprises 0.5-1.0 parts of cisplatin or 0.3-1.0 parts of gefitinib, calculated by weight.

[0025] Preferably, the pharmaceutical composition further comprises 0.745 parts of cisplatin or 0.558 parts of gefitinib, calculated by weight.

[0026] Preferably, the pharmaceutical composition (Shengmingshu composition 3) is composed of ginseng extract, coix seed extract, clematis tongguanteng extract, mannan peptide, berberine hydrochloride, cisplatin or gefitinib.

[0027] Preferably, the pharmaceutical composition consists of 300-3000 parts of ginseng extract, 1700-15000 parts of coix seed extract, 1000-10000 parts of clematis tongguan tung extract, 20-160 parts of mannan peptide, 7-35 parts of berberine hydrochloride, 0.2-1.5 parts of cisplatin or 0.1-3 parts of gefitinib, in parts by mass.

[0028] Preferably, the pharmaceutical composition consists of 1000-2000 parts of ginseng extract, 5000-10000 parts of coix seed extract, 3000-8000 parts of clematis tongguan tung extract, 70-120 parts of mannan peptide, 10-15 parts of berberine hydrochloride, 0.5-1.0 parts of cisplatin or 0.3-1.0 parts of gefitinib, in parts by mass.

[0029] Preferably, the pharmaceutical composition consists of 1700 parts of ginseng extract, 8300 parts of coix seed extract, 5000 parts of clematis tongguan teng extract, 100 parts of mannan peptide, 14.9 parts of berberine hydrochloride, 0.745 parts of cisplatin or 0.558 parts of gefitinib, in parts by mass.

[0030] Preferably, the pharmaceutical composition consists of 1300 parts of ginseng extract, 6700 parts of coix seed extract, 5000 parts of clematis tongguan tung extract, 100 parts of mannan peptide, 14.9 parts of berberine hydrochloride, 0.745 parts of cisplatin or 0.558 parts of gefitinib, in parts by mass.

[0031] In a specific embodiment, the composition consists of 0.3-3 mg / ml of ginseng extract, 1.7-15 mg / ml of coix seed extract, 1-10 mg / ml of clematis tongguan tung extract, 20-160 μg / ml of mannan peptide, and 7-35 μg / ml of berberine hydrochloride;

[0032] Preferably, the composition consists of 1.0-2.0 mg / ml of ginseng extract, 5.0-10 mg / ml of coix seed extract, 3-8 mg / ml of clematis tongguan tung extract, 70-120 μg / ml of mannan peptide, and 10-15 μg / ml of berberine hydrochloride;

[0033] Preferably, the composition consists of 1.7 mg / ml of ginseng extract, 8.3 mg / ml of coix seed extract, 5 mg / ml of clematis tongguan tung extract, 100 μg / ml of mannan peptide, and 14.9 μg / ml of berberine hydrochloride;

[0034] Preferably, the composition consists of 1.3 mg / ml of ginseng extract, 6.7 mg / ml of coix seed extract, 5 mg / ml of clematis tongguan tung extract, 100 μg / ml of mannan peptide, and 14.9 μg / ml of berberine hydrochloride;

[0035] Preferably, the composition consists of 0.3-3 mg / ml of ginseng extract, 1.7-15 mg / ml of coix seed extract, 1-10 mg / ml of clematis tongguan tung extract, 20-160 μg / ml of mannan peptide, 7-35 μg / ml of berberine hydrochloride, 0.2-1.5 μg / ml of cisplatin or 0.1-3 μg / ml of gefitinib;

[0036] Preferably, the composition consists of 1.0-2.0 mg / ml of ginseng extract, 5.0-10 mg / ml of coix seed extract, 3-8 mg / ml of clematis tongguan tung extract, 70-120 μg / ml of mannan peptide, 10-15 μg / ml of berberine hydrochloride, 0.5-1.0 μg / ml of cisplatin or 0.3-1.0 μg / ml of gefitinib;

[0037] Preferably, the composition consists of 1.7 mg / ml of ginseng extract, 8.3 mg / ml of coix seed extract, 5 mg / ml of clematis tongguan tung extract, 100 μg / ml of mannan peptide, 14.9 μg / ml of berberine hydrochloride, 0.745 μg / ml of cisplatin or 0.558 μg / ml of gefitinib;

[0038] Preferably, the composition consists of 1.3 mg / ml of ginseng extract, 6.7 mg / ml of coix seed extract, 5 mg / ml of clematis tongguan tung extract, 100 μg / ml of mannan peptide, 14.9 μg / ml of berberine hydrochloride, 0.745 μg / ml of cisplatin or 0.558 μg / ml of gefitinib;

[0039] Ginseng extract is mainly suitable for coronary heart disease, angina pectoris, bradycardia, tachycardia, ventricular premature beats, blood pressure disorders, neurasthenia, menopausal syndrome, excessive fatigue, physical weakness after illness, childbirth, and surgery.

[0040] Coix seed extract can strengthen the spleen and eliminate dampness, eliminate numbness and stop diarrhea, clear away heat and discharge pus. It can promote the metabolism of blood and water in the body, has diuretic and edema-relieving effects, and can help defecation, so it can help lose weight.

[0041] The extract of Rhizoma Dioscoreae has anti-cancer, anti-inflammatory and anti-asthmatic effects. It is used for esophageal cancer, gastric cancer, lung cancer, colorectal cancer, cervical cancer, leukemia and other malignant tumors. It also has a certain effect. It can also be used in conjunction with radiotherapy, chemotherapy and postoperative treatment, and is used to treat chronic tracheitis and bronchial asthma.

[0042] Mannan peptide is used to treat various diseases caused by low immune function; it can be used to treat various viruses and pathogenic bacteria infections; it can treat recurrent respiratory infections and chronic bronchitis; it can be used to treat aplastic anemia and has the effect of increasing white blood cells and platelets.

[0043] Berberine hydrochloride has inhibitory effects on Shigella dysenteriae, Escherichia coli, Pneumococcus, Staphylococcus aureus, Streptococcus, Salmonella typhi and amoeba.

[0044] Cisplatin is a first-line drug for the treatment of various solid tumors. Combination with VP-16 (EP regimen) is the first-line treatment for SCLC or NSCLC. Combination with MMC, IFO (IMP regimen), or NVB is a common regimen for the treatment of NSCLC. Combination chemotherapy based on DDP is also the main treatment for advanced ovarian cancer, osteosarcoma and neuroblastoma. Combination with ADM, CTX, etc. is effective for squamous cell carcinoma and transitional cell carcinoma in multiple sites, such as head and neck, cervical, esophageal and urinary system tumors.

[0045] Gefitinib, also known as Iressa, is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor used to treat locally advanced or metastatic non-small cell lung cancer (NSCLC) that has been previously treated with chemotherapy or is not suitable for chemotherapy.

[0046] In the present invention, ginseng extract and coix seed extract cooperate with clematis tongguan twig extract to strengthen the body and eliminate evil, and increase immune surveillance. Caulis tongguan twig extract inhibits the proliferation of cancer cells, mediates cell apoptosis, promotes cell differentiation and other direct anti-tumor effects, and alleviates drug resistance by regulating tumor cell angiogenesis, and has the effect of strengthening the body and eliminating evil. Ginseng extract and coix seed extract improve the internal environment, improve the living environment of tumor cells, and reduce factors that stimulate mutations. The active ingredients in ginseng extract and clematis tongguan twig extract (ginsenosides, clematis tongguan twig C21 steroidal saponins) control cancer cells, inhibit cancer cell proliferation, and promote apoptosis of cancer cells A549 cells while inhibiting cancer cells, playing a synergistic role. At the same time, the signal channel is blocked by anti-angiogenesis (RH2 and C21 steroidal saponins) to delay drug resistance.

[0047] Mannan peptides work together with the above three extracts to exert anti-tumor effects. Mannan peptides induce and activate the body's cellular immunity, re-establish cellular immune balance, inhibit gene mutations, and synergistically inhibit tumors.

[0048] Berberine hydrochloride can synergistically exert anti-tumor effects through its anti-tumor effects and reversal of tumor multidrug resistance.

[0049] The composition of the present invention has the purpose of effectively inhibiting the proliferation of tumor cells, and can also have the effect of synergistically enhancing efficacy and reducing toxicity when used in combination.

[0050] The pharmaceutical composition (Mingshu composition 1) composed of ginseng extract, coix seed extract and clematis tongguan twig extract of the present invention has an inhibition rate of up to 75.6% on A549 cells; the pharmaceutical composition (Mingshu composition 2) composed of ginseng extract, coix seed extract, clematis tongguan twig extract, mannan peptide and berberine hydrochloride combined with cisplatin has an inhibition rate on A549 cells increased from 38% when cisplatin is used alone to 91.4%; the inhibition rate of A549 cells combined with gefitinib is increased from 29% when gefitinib is used alone to 89.6%.

[0051] Ginseng extract, coix seed extract, Thunbergia oxyphylla extract, mannan peptide, and berberine hydrochloride are combined. Among them, the combination of ginseng extract, coix seed extract and Thunbergia oxyphylla extract can exert a synergistic effect when used in combination with other drugs.

[0052] Cisplatin is often used as the main therapeutic drug in combined chemotherapy, but its repeated use can cause A549 cells to develop drug resistance. The combination of ginseng extract, coix seed extract, clematis extract, mannan peptide, and berberine hydrochloride can reduce the resistance of tumor cells to cisplatin and enhance the inhibitory effect of cisplatin on tumor cell migration, reduce the dosage of the drug, improve the therapeutic effect of the drug, reduce the occurrence of toxic and side effects, and the effect of increasing efficacy and reducing toxicity is very obvious.

[0053] The composition of the present invention can be prepared by directly mixing the components. In another aspect, the present invention provides the use of the pharmaceutical composition in preparing a drug for preventing and / or treating cancer.

[0054] Preferably, the cancer is selected from small cell and non-small cell lung cancer, breast cancer, liver cancer, esophageal cancer, gastric cancer, testicular cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, bladder cancer, melanoma, sarcoma, head and neck tumors, etc., preferably small cell and non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Effect of the combination and cisplatin on the body weight of mice with lung cancer;

[0056] Figure 2 Effect of the combination in combination with cisplatin on tumors in mice with lung cancer, wherein A: effect of the combination in combination with cisplatin on tumor weight in mice with lung cancer; B: effect of the combination in combination with cisplatin on tumor inhibition rate in mice with lung cancer;

[0057] Figure 3 Effects of the combination and cisplatin on liver index of lung cancer mice;

[0058] Figure 4 Effects of the combination and cisplatin on the renal index of mice with lung cancer;

[0059] Figure 5Effect of the combination and cisplatin on the spleen index of mice with lung cancer;

[0060] Figure 6 CD4 and CD4 / CD8 levels were determined by flow cytometry, where CD4+ indicates CD4 immune cell positive, CD4- indicates CD4 immune cell negative, CD8+ indicates CD8 lymphocyte positive, and CD8- indicates CD8 lymphocyte negative. A: blank group; B: model group; C: cisplatin group; D: combination-low-dose group; E: cisplatin + combination-low-dose group; F: combination-high-dose group; G: cisplatin + combination-high-dose group;

[0061] Figure 7 Effect of the combination and cisplatin on spleen IL-2 level in mice with lung cancer;

[0062] Figure 8 Effect of the combination and cisplatin on spleen IFN-γ level in mice with lung cancer;

[0063] Fig. 9 The effect of the combination and cisplatin on the spleen IgG level in mice with lung cancer;

[0064] Fig.10 Effect of the combination and cisplatin on spleen IgA level in mice with lung cancer;

[0065] Fig.11 The effect of the combination and cisplatin on the white blood cell (WBC) level in mice with lung cancer;

[0066] Fig.12 The effect of the combination and cisplatin on the red blood cell (RBC) level in mice with lung cancer;

[0067] Fig.13 Effect of the combination and cisplatin on the hemoglobin (HGB) level in mice with lung cancer;

[0068] Fig.14 The effect of the combination and cisplatin on the platelet (PLT) level in mice with lung cancer;

[0069] Fig.15 The effect of the combination and cisplatin on the lymphocyte percentage (Lym%) level in mice with lung cancer;

[0070] Fig.16 Effect of the combination and cisplatin on the neutrophil percentage (GR%) level in mice with lung cancer;

[0071] Fig.17 The effect of the combination and cisplatin on the ALT activity level of liver function in mice with lung cancer;

[0072] Fig.18 The effect of the combination and cisplatin on the activity level of AST in liver function of mice with lung cancer;

[0073] Fig.19 Effect of the combination and cisplatin on the ALB level in liver function of mice with lung cancer;

[0074] Fig. 20 The effect of the combination and cisplatin on the BUN activity level of renal function in lung cancer mice;

[0075] Fig.21 The effect of the combination and cisplatin on the renal function and CRE activity level of lung cancer mice;

[0076] Fig. 22 Effects of the combination and cisplatin on the level of SOD in the liver of mice with lung cancer;

[0077] Fig.23 Effects of the combination and cisplatin on liver GSH levels in mice with lung cancer;

[0078] Fig.24 Effects of the combination and cisplatin on the MDA level in the liver of mice with lung cancer;

[0079] Fig.25 Effects of the combination and cisplatin on the level of SOD in the kidneys of mice with lung cancer;

[0080] Fig.26 Effects of the combination and cisplatin on the level of GSH in the kidneys of mice with lung cancer;

[0081] Fig. 27 Effect of the combination synergistically with cisplatin on the MDA level in the kidneys of mice with lung cancer. DETAILED DESCRIPTION

[0082] The technical solution of the present invention is further described below in conjunction with specific examples. The examples are only for illustrating the present invention, not for limiting the scope of the present invention, and the embodiments of the present invention are not limited thereto. Any other changes, substitutions, modifications, simplifications, etc. made without departing from the technical ideas and method principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0083] Unless otherwise specified, the drugs and devices used in the following examples are commercially available.

[0084] Embodiment 1: Extract preparation method

[0085] 1. Preparation method of ginseng extract

[0086] (1) Weighing

[0087] Wash the ginseng and slice it. Weigh 100 g of the dried ginseng slices and set aside.

[0088] (2) Decoction and filtration

[0089] Soak the ginseng in water for 2 hours, then add 15 times the amount of water to the ginseng and boil for 2 hours, filter, take the filtered ginseng and add 12 times the amount of water to boil for 2 hours, filter, combine the filtrate and set aside;

[0090] (3) Concentration and alcohol precipitation

[0091] The filtrate was transferred to a vacuum-reducing jacketed concentrator and concentrated to a relative density of 1.15 at 48°C. Three times the volume of the concentrated medicinal solution was added with ethanol, and the mixture was allowed to stand for 5 hours. The supernatant was taken and the ethanol was recovered until there was no alcohol taste. The ginseng extract was obtained.

[0092] 2. Preparation method of coix seed extract

[0093] (1) Weighing

[0094] Wash and dry the coix seed medicinal material for later use. Weigh 100g of the dried medicinal material and set aside.

[0095] (2) Decoction and filtration

[0096] Place coix seeds in an extraction tank, add 13 times the amount of water to the medicinal material and boil for 3 hours, filter, take the filtered medicinal material and add 13 times the amount of water to boil for 2 hours, filter, combine the filtrate and set aside;

[0097] (3) Concentration and alcohol precipitation

[0098] The filtrate was transferred to a vacuum-reducing jacketed concentration tank and concentrated to a relative density of 1.18 at 50°C. Three times the volume of ethanol was added to the concentrated solution and allowed to stand for 5 hours. The supernatant was taken and the ethanol was recovered until there was no alcohol taste. This was to obtain the coix seed extract.

[0099] 3. Preparation method of scutellaria baicalensis extract

[0100] (1) Weighing

[0101] Weigh 100 g of Radix Glehniae and set aside.

[0102] (2) Decoction and filtration

[0103] Take Tongguanteng and soak it in water for 12 hours. Add 8 times the amount of water of the medicinal material and boil it three times, 2 hours for the first time, 1.5 hours for the second time, and 1 hour for the third time. Combine the decoctions, filter them and set aside.

[0104] (3) Concentration

[0105] The filtrate is transferred to a vacuum-reduced jacketed concentration tank and concentrated to a clear paste with a relative density of 1.23 (70-80° C.) to obtain the Thunbergia clematis extract.

[0106] Embodiment 2: Preparation of the composition of the present invention

[0107] Example 2.1

[0108] 1700 g of ginseng extract (obtained from Example 1), 8300 g of coix seed extract (obtained from Example 1) and 5000 g of clematis tongguan tung extract (obtained from Example 1) were mixed.

[0109] Example 2.2

[0110] 1300 g of ginseng extract (obtained from Example 1), 6700 g of coix seed extract (obtained from Example 1) and 5000 g of clematis tongguan tung extract (obtained from Example 1) were mixed.

[0111] Example 2.3

[0112] Ginseng extract (obtained from Example 1) 1700g, coix seed extract (obtained from Example 1) 8300g and clematis extract (obtained from Example 1) 5000g, mannan peptide 100g, berberine hydrochloride 14.9g were mixed

[0113] Example 2.4

[0114] 1300 g of ginseng extract (obtained from Example 1), 6700 g of coix seed extract (obtained from Example 1), 5000 g of clematis tongguan tung extract (obtained from Example 1), 100 g of mannan peptide, and 14.9 g of berberine hydrochloride were mixed.

[0115] Example 2.5

[0116] The mixture is prepared by mixing 1700 g of ginseng extract (obtained from Example 1), 8300 g of coix seed extract (obtained from Example 1), 5000 g of clematis extract (obtained from Example 1), 100 g of mannan peptide, 14.9 g of berberine hydrochloride, 0.745 μg of cisplatin or 0.558 μg of gefitinib.

[0117] Example 2.6

[0118] The mixture is prepared by mixing 1300 g of ginseng extract (obtained from Example 1), 6700 g of coix seed extract (obtained from Example 1), 5000 g of clematis extract (obtained from Example 1), 100 g of mannan peptide, 14.9 g of berberine hydrochloride, 0.745 μg of cisplatin or 0.558 μg of gefitinib.

[0119] Example 2.7

[0120] The mixture is prepared by mixing 1700 g of ginseng extract (obtained from Example 1), 8300 g of coix seed extract (obtained from Example 1), 5000 g of clematis extract (obtained from Example 1), 100 g of mannan peptide, 14.9 g of berberine hydrochloride, 0.373 μg of cisplatin or 1.17 μg of gefitinib.

[0121] Example 2.8

[0122] The mixture is prepared by mixing 1300 g of ginseng extract (obtained from Example 1), 6700 g of coix seed extract (obtained from Example 1), 5000 g of clematis extract (obtained from Example 1), 100 g of mannan peptide, 14.9 g of berberine hydrochloride, 0.373 μg of cisplatin or 1.17 μg of gefitinib.

[0123] The pharmaceutical composition used in the following examples of the present invention is prepared by mixing the raw materials of Example 1 with commercially available mannan peptide, berberine hydrochloride, cisplatin and gefitinib.

[0124] Embodiment 3: Inhibitory effect of single drug on A549 cells

[0125] 1. Experimental Methods

[0126] 1.1 Preparation of main solutions

[0127] ① Complete culture medium: Add 500ml of fetal bovine serum (FBS) to 1 bottle of DMEM culture medium, then add 5ml of penicillin-streptomycin (100X), shake gently, and place in a 4°C refrigerator for use.

[0128] ②Cell freezing solution: Take 10mL FBS, add it to 35mL culture medium, then add 5mL cell-grade DMSO, use a pipette to gently blow and mix, divide into portions, and store in a -20℃ refrigerator for later use.

[0129] ③MTT solution: weigh 500 mg of MTT powder, add 100 mL of PBS, and sterilize with a 0.22 μm filter membrane after fully dissolving, and store at -20°C in a dark environment.

[0130] ④ Gefitinib solution: Grind gefitinib tablets into powder, weigh 111.725 mg using a precision electronic balance, add 5 mL DMSO and shake thoroughly to dissolve, and finally make a 50 mmol / ml stock solution, and store it in a light-proof environment at -20°C. When using, dilute the stock solution to the required concentration with DMEM complete medium and sterilize it through a 0.22 μm filter membrane. The final DMSO concentration should be less than 0.1% when acting on cells. Store in a 4°C refrigerator for future use.

[0131] ⑤PBS solution: weigh 0.81g potassium dihydrogen phosphate, 4.26g disodium hydrogen phosphate and 8g sodium chloride, pour into a 3000mL beaker filled with double distilled water, stir to dissolve, and obtain 0.01mol / LPBS solution. Dispense into 500mL glass bottles, sterilize in an autoclave, and store in a 4℃ refrigerator for later use.

[0132] 1.2 In vitro cell culture

[0133] ① Cell recovery: adjust the temperature of the water bath to 37°C, wait for the water temperature to stabilize, take out the required cell cryopreservation tube from the liquid nitrogen tank, quickly transfer it to a 37°C water bath for heating, and shake it to melt the cryopreservation solution in the cryopreservation tube as quickly as possible; after the cryopreservation solution melts into liquid, transfer the cryopreservation tube to a biosafety cabinet, open the tube cover, transfer the liquid in the tube to 10 times the volume of complete culture medium for dilution, and mix thoroughly; the mixed cell suspension is centrifuged at a speed of 1000r / min for 5min, remove the supernatant, add 1ml of complete culture medium to resuspend the cells, and gently blow evenly, then add 5ml of complete culture medium to blow evenly and inoculate it into the culture bottle, shake the culture bottle to make the cells evenly distributed at the bottom of the bottle; finally, place the culture bottle marked with the cell name, passage number, operator and operation date in a cell culture incubator at 37°C and 5% CO2 for culture.

[0134] ② Cell culture medium replacement and subculture: The revived cells should be observed every other day to determine the cell status and adhesion, and the cell culture medium should be replaced as needed. When the cells grow to 70%-90% of the bottom of the bottle, in order to prevent contact inhibition between cells from affecting the cell status, the cells need to be subcultured. After taking the cell culture bottle out of the incubator, carefully discard the old culture medium, add sterile PBS to rinse twice, and then suck out the remaining PBS; add trypsin containing EDTA to the culture bottle (EDTA: 0.01%, trypsin: 0.125%; every 25cm 2 Add 1ml of trypsin to the culture flask with the same area, shake it evenly and place it in a cell culture incubator at 37℃ for 3min, then add complete culture medium equal to trypsin to neutralize the trypsin to stop the digestion, and use a pipette to gently blow the cells at the bottom of the flask to blow off the cells still attached to the wall, then transfer the obtained cell suspension into a centrifuge tube and centrifuge it at 1000r / min for 5min. After the centrifugation, discard the supernatant and gently blow the cells at the bottom of the tube with 1ML complete culture medium to resuspend them; after counting the cells using a cell counting plate, transfer the suspension to culture flasks of different specifications marked with cell name, passage number, operator and operation date, and place them in a cell culture incubator at 37℃ and 5% CO2 for continued culture for subsequent experiments.

[0135] ③Cell freezing: When the cells are passaged to the 2nd or 3rd generation and grow to the logarithmic growth phase, or when the cells are in good condition, the cells can be frozen according to experimental needs. The treated cell suspension should be divided into cryopreservation tubes marked with cell type, generation, operator and operation time, and quickly moved to a programmed cooling box at room temperature, placed in an ultra-low temperature freezer at -80°C, and after freezing for 24 hours, the cryopreservation tubes should be moved to a liquid nitrogen tank for long-term storage.

[0136] 1.3 MTT assay to detect cellular toxicity to drugs

[0137] In order to detect the toxic effects of the drugs to be tested (i.e., gefitinib, mannan peptide, berberine hydrochloride, clematis tongguanteng extract (obtained from Example 1), ganoderma lucidum and astragalus extract (extracted by Letai Pharmaceutical Co., Ltd.), ginseng extract (obtained from Example 1), coix seed extract (obtained from Example 1), cisplatin) on human non-small cell lung cancer A549 cells, and to measure the half inhibitory concentration (IC50) of various drugs on lung cancer cells, and to provide working concentrations for subsequent experiments, this part of the experiment was conducted. Using the MTT method, when the MTT solution is added to the cell growth environment, the succinate dehydrogenase and cytochrome C in the living cells can reduce the MTT to form blue-purple crystals. According to the amount of crystals, the number of living cells after medication is judged, thereby inferring the toxicity of the drug to the cells. The detailed experimental steps are as follows:

[0138] ① Cell inoculation

[0139] Take cells in the logarithmic growth phase, discard the old culture medium, rinse twice with sterile PBS, add trypsin containing EDTA, and transfer to a 37°C cell culture incubator for digestion; then add a complete culture medium equal to the amount of trypsin to neutralize the trypsin, and centrifuge at 1000r / min for 5min; discard the supernatant, add 5ml of culture medium and gently blow to resuspend the cells, use a cell counting plate to count the cells of the obtained cell suspension under an inverted microscope, and finally adjust the A594 cell concentration to 4×10^4 cells / mL. After gently blowing and evenly dispersing the cell suspension, inoculate it in a 96-well plate with a volume of 100μL per well, and fill the edge holes of the plate with sterile PBS.

[0140] ②Cell culture

[0141] The 96-well plate was moved into a cell culture incubator at 37°C and 5% CO2 and cultured for 24 hours until the cells were completely attached.

[0142] ③ Grouping and drug administration

[0143] The groups were divided into: negative control group (containing only cells and culture medium); gefitinib group: set the concentration to 0.558μg / ml, 1.117μg / ml, 2.234μg / ml, 4.469μg / ml, 8.938μg / ml; mannan peptide group: set the concentration to 100μg / ml, 200μg / ml, 300μg / ml, 400μg / ml, 500μg / ml; clematis extract group: set the concentration to 5mg / ml, 10mg / ml, 20mg / ml, 40mg / ml, 80mg / ml; ganoderma lucidum astragalus extract group: set the concentration to 80μg / ml, 160μg / ml, 320μg / ml, 640μg / ml, 1280μg / ml; berberine hydrochloride group: set the concentration to Ginseng extract group: set the concentration to 1000μg / ml, 1300μg / ml, 1700μg / ml, 2000μg / ml, 2300μg / ml; Coix seed extract group: set the concentration to 5000μg / ml, 6700μg / ml, 8300μg / ml, 10000μg / ml, 11000μg / ml; Cisplatin group: set the concentration to 0.373μg / ml, 0.745μg / ml, 1.49μg / ml, 2.98μg / ml, 5.96μg / ml; set 10 replicate wells for each drug concentration.

[0144] ④ Method of administration

[0145] After 24 hours of cells completely adhering to the wall, carefully remove the original culture medium in the well (suck along the edge of the well bottom, be careful not to touch the cells at the bottom of the well), then add 200 μL of drug-containing culture medium along the well wall according to the group, and add DMEM complete culture medium to the blank control well; cover the plate tightly, move the plate to a cell culture incubator at 37°C and 5% CO2 and continue to culture for 48 hours.

[0146] ⑤ Color

[0147] 48 hours after administration, add 20 μL MTT solution (5 mg / ml) prepared in advance to each well in a light-proof environment, and move to a 37°C incubator for further incubation for 4 hours; after 4 hours, carefully aspirate the supernatant in the well (aspirate along the edge of the well bottom, and be careful not to touch the purple crystals at the bottom of the well), and then add 150 μL DMSO to each well; remove the plate cover, move the plate to a multi-function microplate reader, and set the program to oscillate for 1 minute to completely dissolve the purple crystals.

[0148] ⑥ Colorimetry

[0149] The absorbance (OD) value of each well sample was measured at a wavelength of 490 nm using a multifunctional microplate reader, and the cell survival rate and proliferation inhibition rate were calculated using the OD value. The specific calculation formula is as follows:

[0150] Cell survival rate (%) = OD value of drug well / OD value of control well × 100%. Inhibition rate (%) = (OD value of control group - OD value of drug group / OD value of control group - OD value of blank group) × 100%. Graphpad9.0 software was used to calculate the IC50 value of each group of drugs and gefitinib on A549 cells based on the cell survival rate. CompuSyn software was used to verify the synergistic effect of the two drugs.

[0151] 2. Experimental results

[0152] 2.1 Inhibition rate and IC of gefitinib on lung cancer A549 50 As shown in the table

[0153] Table 1 OD value, inhibition rate and IC of gefitinib on A549 at 24h and 48h 50

[0154]

[0155] 2.2 Inhibitory rate and IC of mannan peptide against lung cancer A549 50 As shown in the table

[0156] Table 2 OD values, inhibition rates and IC50 of mannan peptide on A549 at 24h and 48h

[0157]

[0158] 2.3 Inhibitory effect and IC of berberine hydrochloride on lung cancer A549 50 As shown in the table

[0159] Table 3 OD value, inhibition rate and IC50 of berberine hydrochloride on A549 at 24h and 48h

[0160]

[0161] 2.4 Inhibition rate and IC of Rhizoma Cyperi extract on lung cancer A549 50 As shown in the table

[0162] Table 4 OD values, inhibition rates and IC50 of the extract of Rhizoma Cyperi on A549 at 24h and 48h

[0163]

[0164]

[0165] 2.5 Inhibition rate and IC of Ganoderma lucidum and Astragalus extract on lung cancer A549 50 As shown in the table

[0166] Table 5 OD values, inhibition rates and IC50 of Ganoderma lucidum and Astragalus extracts on A549 for 24h and 48h

[0167]

[0168] 2.6 Inhibition rate and IC of ginseng extract on lung cancer A549 50 As shown in the table

[0169] Table 6 OD values, inhibition rates and IC values ​​of ginseng extract on A549 at 24h and 48h 50

[0170]

[0171] 2.7 Inhibition rate and IC of coix seed extract on lung cancer A549 50 As shown in the table

[0172] Table 7 OD values, inhibition rates and IC values ​​of coix seed extract on A549 at 24h and 48h 50

[0173]

[0174] 2.8 The inhibition rate and IC50 of cisplatin on lung cancer A549 are shown in the table

[0175] Table 8 OD value, inhibition rate and IC50 of cisplatin on A549 for 48h

[0176]

[0177] According to the above results, it can be seen that gefitinib, mannan peptide, berberine hydrochloride, clematis tongguan teng extract, ganoderma lucidum and astragalus extract, ginseng extract, coix seed extract, and cisplatin all have a certain inhibitory effect on lung cancer A549, and the higher the concentration, the better the inhibitory effect. However, the inhibitory effect cannot be doubled when the dosage is doubled, indicating that the drugs have obvious drug resistance when used in high concentrations, and long-term use cannot achieve the expected therapeutic effect.

[0178] Embodiment 4: The inhibitory effect of the combination of the two drugs on A549 cells

[0179] 1. Experimental Methods

[0180] The experiment was carried out according to the method described in Example 3, and the grouping and dosing conditions are reflected in the results.

[0181] 2. Experimental results

[0182] 2.1 Effects of gefitinib combined with clematis extract

[0183] The dose of gefitinib in the combination of gefitinib and Rhizoma Cyperi extract was 2.234 μg / ml, and the Rhizoma Cyperi extract was selected at two doses of 5000 and 10000 μg / ml. After 48 hours of combined action, CI <1, and both groups had synergistic effects.

[0184] Table 9 OD value, inhibition rate and CI value of gefitinib combined with clematis tongguanyi extract on A549 for 48h

[0185]

[0186] 2.2 Effect of gefitinib combined with mannan peptide

[0187] In the combination of gefitinib and mannan peptide, the dose of gefitinib was 2.234 μg / ml, and two doses of mannan peptide were selected, 100 and 200 μg / ml. After 48 hours of combined action, the CI of the gefitinib 2.234 μg / ml + mannan peptide 200 μg / ml group was <1, which had a synergistic effect.

[0188] Table 10 OD value, inhibition rate and CI value of gefitinib combined with mannan peptide on A549 for 48h

[0189]

[0190] 2.3 Effect of gefitinib combined with berberine hydrochloride

[0191] In the combination of gefitinib and berberine hydrochloride, the dose of gefitinib was 2.234 μg / ml and two doses of berberine hydrochloride were selected, 14.912 and 29.825 μg / ml. After 48 hours of combined action, the CI of the gefitinib 2.234 μg / ml + berberine hydrochloride 29.825 μg / ml group was <1, indicating a synergistic effect.

[0192] Table 11 OD value, inhibition rate and CI value of gefitinib combined with berberine hydrochloride on A549 for 48 hours

[0193]

[0194] 2.4 Effects of gefitinib combined with Ganoderma lucidum and Astragalus extract

[0195] The dose of gefitinib in the combination of gefitinib and Ganoderma lucidum and Astragalus extract was 2.234 μg / ml, and the Ganoderma lucidum and Astragalus extract was selected at two doses of 2800 and 5600 μg / ml. After 48 hours of combined action, CI>1, and there was no combined effect.

[0196] Table 12 OD value, inhibition rate and CI value of gefitinib combined with Ganoderma lucidum and Astragalus extract on A549 for 48 hours

[0197]

[0198] 2.5 Gefitinib combined with ginseng extract

[0199] The dose of gefitinib in gefitinib combined with ginseng extract was 2.234 μg / ml, and two doses of ginseng extract were selected, 1300 and 1700 μg / ml. After 48 hours of combined action, CI>1, and there was no combined effect.

[0200] Table 13 OD value, inhibition rate and CI value of gefitinib combined with ginseng extract on A549 for 48 hours

[0201]

[0202] 2.6 Gefitinib combined with coix seed extract

[0203] The dose of gefitinib in the combination of gefitinib and coix seed extract was 2.234 μg / ml, and two doses of coix seed extract were selected, 6700 and 8300 μg / ml. After 48 hours of combined action, CI>1, and there was no combined effect.

[0204] Table 14 OD value, inhibition rate and CI value of gefitinib combined with coix seed extract on A549 for 48 hours

[0205]

[0206] 2.7 Cisplatin combined with Radix Achyranthis Bidentatae extract

[0207] The dose of cisplatin in the combination of cisplatin and Rhizoma Cyperi extract was 1.49 μg / ml, and the Rhizoma Cyperi extract was selected in two doses of 5000 and 10000 μg / ml. After 48 hours of combined action, CI <1, and both groups had synergistic effects.

[0208] Table 15 OD value, inhibition rate and CI value of cisplatin combined with scutellaria baicalensis extract on A549 for 48 hours

[0209]

[0210] 2.8 Cisplatin combined with mannan peptide

[0211] In the combination of cisplatin and mannan peptide, the cisplatin dose was 1.49 μg / ml, and two doses of mannan peptide were selected, 100 and 200 μg / ml. After 48 hours of combined action, CI>1, and there was no synergistic effect.

[0212] Table 16 OD value, inhibition rate and CI value of cisplatin combined with mannan peptide on A549 for 48h

[0213]

[0214] 2.9 Cisplatin combined with berberine hydrochloride

[0215] In the combination of cisplatin and berberine hydrochloride, the cisplatin dose was 1.49 μg / ml and the berberine hydrochloride was selected in two doses of 14.912 and 29.825 μg / ml. After 48 hours of combined action, the CI of the cisplatin 1.49 μg / ml + berberine hydrochloride 29.825 μg / ml group was <1, which had a synergistic effect.

[0216] Table 17 OD value, inhibition rate and CI value of cisplatin combined with berberine hydrochloride on A549 for 48h

[0217]

[0218] 2.10 Cisplatin combined with Ganoderma lucidum and Astragalus extract

[0219] The cisplatin dose in the cisplatin combined with Ganoderma lucidum and Astragalus extract was 1.49 μg / ml, and two doses of 2800 and 5600 μg / ml were selected for Ganoderma lucidum and Astragalus. After 48 hours of combined action, CI>1, and there was no combined effect.

[0220] Table 18 OD value, inhibition rate and CI value of cisplatin combined with Ganoderma lucidum and Astragalus on A549 for 48 hours

[0221]

[0222] 2.11 Cisplatin combined with ginseng extract

[0223] The cisplatin dose in cisplatin combined with ginseng extract was 1.49 μg / ml, and two doses of ginseng extract were selected, 1300 and 2000 μg / ml. After 48 hours of combined action, CI>1, and there was no combined effect.

[0224] Table 19 OD value, inhibition rate and CI value of cisplatin combined with ginseng extract on A549 for 48 hours

[0225]

[0226] 2.12 Cisplatin combined with coix seed extract

[0227] The cisplatin dose in the combination of cisplatin and coix seed extract was 1.49 μg / ml, and the coix seed extract was selected at two doses of 6700 and 8300 μg / ml. After 48 hours of combined action, CI>1, and there was no combined effect.

[0228] Table 20 OD value, inhibition rate and CI value of cisplatin combined with coix seed extract on A549 for 48 hours

[0229]

[0230] 2.13 Mannan peptide combined with berberine hydrochloride

[0231] After 48h of combined action of 200μg / ml of mannan peptide and 14.912μg / ml and 29.825μg / ml of berberine hydrochloride, CI>1, there was an antagonistic effect.

[0232] Table 21 OD value, inhibition rate and CI value of mannan peptide combined with berberine hydrochloride on A549 for 48 hours

[0233]

[0234] 2.14 Mannan peptide combined with Radix Achyranthis Bidentatae extract

[0235] After 48h of combined action of 200μg / ml of mannan peptide and 5000μg / ml and 10000μg / ml of Psoralea corylifolia extract, CI <1, there was a synergistic effect.

[0236] Table 22 OD value, inhibition rate and CI value of mannan peptide combined with clematis extract on A549 for 48h

[0237]

[0238] 2.15 Berberine hydrochloride combined with Rhizoma Cynanchum extract

[0239] After 48h of combined action of berberine hydrochloride 29.825μg / ml and Rhizoma Atractylodis Macrocephalae extract 5000μg / ml and 10000μg / ml, the CI was <1, which showed a synergistic effect.

[0240] Table 23 OD value, inhibition rate and CI value of berberine hydrochloride combined with clematis extract on A549 for 48h

[0241]

[0242] 2.16 Berberine hydrochloride combined with ginseng extract

[0243] After 48h of combined action of berberine hydrochloride 29.825μg / ml and ginseng extract 1300μg / ml and 1700μg / ml, CI <1, there was a synergistic effect.

[0244] Table 24 OD value, inhibition rate and CI value of berberine hydrochloride combined with ginseng extract on A549 for 48 hours

[0245]

[0246] 2.17 Berberine hydrochloride combined with coix seed extract

[0247] After 48h of combined action of berberine hydrochloride 29.825μg / ml and coix seed extract 6700μg / ml and 8300μg / ml, CI <1, there was a synergistic effect.

[0248] Table 25 OD value, inhibition rate and CI value of berberine hydrochloride combined with coix seed extract on A549 for 48 hours

[0249]

[0250] 2.18 Ginseng Extract Combined with Coix Seed Extract

[0251] After 48h of combined action of 1700μg / ml ginseng extract and 6700μg / ml and 8300μg / ml coix seed extract, CI < 1, which showed synergistic effect.

[0252] Table 26 OD value, inhibition rate and CI value of ginseng extract combined with coix seed extract on A549 for 48 hours

[0253]

[0254] 2.19 Ginseng Extract Combined with Radix Achyranthis Bidentatae Extract

[0255] After 48h of combined action of 1700μg / ml ginseng extract and 5000μg / ml and 10000μg / ml Rhizoma Cyperi extract, CI < 1, there was a synergistic effect.

[0256] Table 27 OD value, inhibition rate and CI value of ginseng extract combined with coix seed extract on A549 for 48 hours

[0257]

[0258] 2.20 Ginseng extract combined with mannan peptide extract

[0259] After 48h of combined action of 1700μg / ml ginseng extract and 100μg / ml and 200μg / ml mannan peptide, CI<1, there was a synergistic effect.

[0260] Table 28 OD value, inhibition rate and CI value of ginseng extract combined with coix seed extract on A549 for 48 hours

[0261]

[0262] According to the above results, it can be seen that:

[0263] Ginseng extract has a synergistic effect when used in combination with coix seed extract and clematis tongguanteng extract; it has an additive effect when used in combination with mannan peptide and berberine hydrochloride; and it has an antagonistic effect with gefitinib and cisplatin.

[0264] The combination of Rhizoma Cyperi extract and gefitinib, cisplatin, ginseng extract, mannan peptide and berberine hydrochloride has a synergistic effect.

[0265] Mannan peptide has a synergistic effect when used in combination with gefitinib, an additive effect when used in combination with ginseng extract, a weaker antagonistic effect when used in combination with cisplatin, and an antagonistic effect when used in combination with berberine hydrochloride.

[0266] Berberine hydrochloride has a synergistic effect when used in combination with gefitinib and cisplatin, and has an additive effect when used in combination with ginseng extract and coix seed extract.

[0267] Ganoderma lucidum and Astragalus extracts combined with other drugs exert antagonistic effects.

[0268] Although the combination of the two drugs has a synergistic effect, the tumor inhibition effect can still be improved. At the same time, the dosage of a single drug is still large, and long-term use will still make cancer cells drug-resistant.

[0269] Embodiment 5: Inhibitory effect of multi-drug combination on A549 cells

[0270] 1. Experimental Methods

[0271] The experiment was carried out according to the method described in Example 3, and the grouping and dosing conditions are reflected in the results.

[0272] 2. Experimental results

[0273] 2.1 Ginseng extract, coix seed extract combined with gefitinib

[0274] The dose of gefitinib was 2.234 μg / ml, and the doses of ginseng extract and coix seed extract were 1300 μg / ml, 6700 μg / ml and 1700 μg / ml, 8300 μg / ml, respectively. After 48 hours of combined action, CI < 1, indicating a synergistic effect.

[0275] Table 29 OD value, inhibition rate and CI value of ginseng extract, coix seed extract combined with gefitinib on A549 for 48 hours

[0276]

[0277] 2.2 Ginseng extract, coix seed extract combined with cisplatin

[0278] Cisplatin was 1.49 μg / ml, and the doses of ginseng extract and coix seed extract were 1300 μg / ml, 6700 μg / ml and 1700 μg / ml, 8300 μg / ml, respectively. After 48 hours of combined action, CI < 1, indicating a synergistic effect.

[0279] Table 30 OD value, inhibition rate and CI value of ginseng extract, coix seed extract combined with cisplatin on A549 for 48 hours

[0280]

[0281] 2.3 Ginseng extract, coix seed extract and cyperus rotundus extract

[0282] The doses of Radix Glehniae extract were 10000μg / ml, ginseng extract and coix seed extract were 1300μg / ml, 6700μg / ml and 1700μg / ml, 8300μg / ml respectively. After 48h of combined action, CI <1, indicating a synergistic effect.

[0283] Table 31 OD value, inhibition rate and CI value of ginseng extract, coix seed extract and clematis extract on A549 after 48 hours

[0284]

[0285] 2.4 Ginseng extract, coix seed extract combined with mannan peptide

[0286] Mannan peptide 200μg / ml, ginseng extract, coix seed extract doses were 1300μg / ml, 6700μg / ml and 1700μg / ml, 8300μg / ml, after 48h of combined action, CI = 1, which has an additive effect.

[0287] Table 32 OD value, inhibition rate and CI value of ginseng extract, coix seed extract and mannan peptide on A549 for 48 hours

[0288]

[0289] 2.5 Ginseng extract, coix seed extract and berberine hydrochloride

[0290] Berberine hydrochloride was 29.825 μg / ml, and the doses of ginseng extract and coix seed extract were 1300 μg / ml, 6700 μg / ml, and 1700 μg / ml, 8300 μg / ml, respectively. After 48 hours of combined action, the CI value was around 1, which can be considered an additive effect.

[0291] Table 33 OD value, inhibition rate and CI value of ginseng extract, coix seed extract and berberine hydrochloride on A549 for 48 hours

[0292]

[0293] 2.6 Ginseng extract, coix seed extract combined with Ganoderma lucidum and Astragalus extract

[0294] The dose of Ganoderma lucidum and Astragalus extract was 11200μg / ml, the dose of ginseng extract and coix seed extract was 1300μg / ml, 6700μg / ml and 1700μg / ml, 8300μg / ml respectively. After 48h of combined action, CI>1 had antagonistic effect.

[0295] Table 34 OD value, inhibition rate and CI value of ginseng extract, coix seed extract and Ganoderma lucidum and Astragalus extract on A549 after 48 hours

[0296]

[0297] 2.7 Ginseng extract, coix seed extract combined with cyperus rotundus extract, mannan peptide, berberine hydrochloride

[0298] Table 35 OD value, inhibition rate and CI value of 1300 μg / ml ginseng extract, 6700 μg / ml coix seed extract combined with 5000 μg / ml clematis extract, 100 μg / ml mannan peptide and 14.912 μg / ml berberine hydrochloride on A549 after 48 hours

[0299]

[0300]

[0301] Table 36 OD value, inhibition rate and CI value of 1700 μg / ml ginseng extract, 8300 μg / ml coix seed extract combined with 5000 μg / ml clematis extract, 100 μg / ml mannan peptide and 14.912 μg / ml berberine hydrochloride on A549 after 48 hours

[0302]

[0303]

[0304] Conclusion: Ginseng extract, coix seed extract, Thunbergia tongguanyi extract, mannan peptide and berberine hydrochloride are combined. Among them, the combination of ginseng extract, coix seed extract and Thunbergia tongguanyi extract can exert synergistic effects when used in combination with other drugs.

[0305] 2.8 Combination of cisplatin with ginseng extract, coix seed extract, clematis extract, mannan peptide, and berberine hydrochloride

[0306] Table 37 48h OD value, inhibition rate, CI value of the combined effect of cisplatin 0.373 μg / ml with ginseng extract 1300 μg / ml, coix seed extract 6700 μg / ml, tongguanteng extract 5000 μg / ml, mannan peptide 100 μg / ml, berberine hydrochloride 14.912 μg / ml on A549

[0307]

[0308]

[0309] Table 38 48hOD value, inhibition rate, CI value of cisplatin 0.745 μg / ml combined with ginseng extract 1300 μg / ml, coix seed extract 6700 μg / ml, tongguanteng extract 5000 μg / ml, mannan peptide 100 μg / ml, berberine hydrochloride 14.912 μg / ml on A549

[0310]

[0311]

[0312]

[0313] Conclusion: Cisplatin combined with ginseng extract, coix seed extract, clematis tongguan tung extract, mannan peptide, and berberine hydrochloride exerts a synergistic effect regardless of whether it is a three-drug combination or a multi-drug combination.

[0314] 2.9 Gefitinib combined with ginseng extract, coix seed extract, clematis extract, mannan peptide, and berberine hydrochloride

[0315] Table 39 48h OD value, inhibition rate, CI value of gefitinib 0.558 μg / ml combined with ginseng extract 1300 μg / ml, coix seed extract 6700 μg / ml, tongguanteng extract 5000 μg / ml, mannan peptide 100 μg / ml, berberine hydrochloride 14.912 μg / ml on A549

[0316]

[0317]

[0318] Table 40 OD value, inhibition rate and CI value of 1.117 μg / ml of gefitinib combined with 1300 μg / ml of ginseng extract, 6700 μg / ml of coix seed extract, 5000 μg / ml of clematis extract, 100 μg / ml of mannan peptide and 14.912 μg / ml of berberine hydrochloride on A549 for 48 hours

[0319]

[0320]

[0321]

[0322] Conclusion: The synergistic effect of gefitinib combined with other drugs is obvious. Gefitinib 0.558 μg / ml combined with other drugs can exert a good synergistic effect.

[0323] 2.10 Combination of Rhizoma Cyperi extract with mannan peptide and berberine hydrochloride

[0324] Table 41 OD value, inhibition rate, and CI value of the combination of 5000 μg / ml of Pleurotus eryngii extract, 100 μg / ml of mannan peptide, and 14.912 μg / ml of berberine hydrochloride on A549 for 48 hours

[0325]

[0326] Table 42 OD value, inhibition rate, CI value of the combination of 10000 μg / ml of 100 μg / ml of berberine hydrochloride and 14.912 μg / ml on A549 for 48 hours

[0327]

[0328] Conclusion: The synergistic effect of the combination of Thunb. tongguanyi extract with mannan peptide and berberine hydrochloride is not obvious. The CI value of the combination of 10000μg / ml Thunb. tongguanyi extract with mannan peptide and berberine hydrochloride is <1, but the inhibition rate is only 53.8%.

[0329] According to the above experimental results, it can be seen that:

[0330] Ginseng extract and coix seed extract combined with gefitinib, cisplatin or Phellodendron chinense extract have a synergistic effect, combined with mannan peptide or berberine hydrochloride have an additive effect, and combined with Ganoderma lucidum and Astragalus extract have an antagonistic effect.

[0331] Ginseng extract, coix seed extract, Thunbergia oxyphylla extract, mannan peptide, and berberine hydrochloride are combined. Among them, the combination of ginseng extract, coix seed extract and Thunbergia oxyphylla extract can exert a synergistic effect when used in combination with other drugs.

[0332] Cisplatin is used in combination with ginseng extract, coix seed extract, clematis tongguan vine extract, mannan peptide, and berberine hydrochloride, and it exerts a synergistic effect regardless of whether it is a three-drug combination or a multi-drug combination.

[0333] The synergistic effect of gefitinib combined with other drugs is obvious. Gefitinib 0.558μg / ml combined with other drugs can exert a good synergistic effect.

[0334] The synergistic effect of the combination of Thunb. tongguan tung extract with mannan peptide and berberine hydrochloride was not obvious. However, the combination of ginseng extract, coix seed extract, and Thunb. tongguan tung extract with mannan peptide, berberine hydrochloride, cisplatin or gefitinib had a good synergistic effect. The inhibition rate of A549 cancer cells by cisplatin 0.373μg / ml combined with ginseng extract 1300μg / ml, coix seed extract 6700μg / ml, Thunb. tongguan tung extract 5000μg / ml, mannan peptide 100μg / ml, and berberine hydrochloride 14.912μg / ml reached 87.6%, and cisplatin 0.745μg / ml combined with the above drugs The inhibition rate of A549 cancer cells by the combined action of 0.558μg / ml of gefitinib, 1300μg / ml of ginseng extract, 6700μg / ml of coix seed extract, 5000μg / ml of tongguanteng extract, 100μg / ml of mannan peptide, and 14.912μg / ml of berberine hydrochloride was 85.5%, and the inhibition rate of A549 cancer cells by 1.117μg / ml of gefitinib combined with the above drugs was 89.6%. The dosage can be adjusted according to the different stages of the patient and the effect of the action to achieve the best effect of tumor inhibition. For this reason, the (shengmingshu series) pharmaceutical composition of the present invention is finally obtained.

[0335] Embodiment 6: Enhancement and reduction of toxicity of cisplatin

[0336] Shengmingshu composition 2 (hereinafter referred to as the composition): 1700g of ginseng extract (obtained from Example 1), 8300g of coix seed extract (obtained from Example 1), 5000g of clematis extract (obtained from Example 1), 100g of mannan peptide, and 14.9g of berberine hydrochloride, which are mixed and used.

[0337] 1. Animal modeling

[0338] Healthy male C57BL / 6J mice were selected as experimental animals. The cultured Lewis cells were collected with sterile 1× PBS and made into a cell culture medium with a density of 1×10 7 / mL of cell suspension, 0.2 mL was inoculated into the right subcutaneous axilla of each mouse.

[0339] 2. Grouping and Dosing

[0340] The tumor growth at the inoculation site of the mice was observed daily after inoculation. Subcutaneous nodules could be felt around the 4th day, indicating that the model was successful. The mice were randomly divided into 6 groups, 8 mice in each group.

[0341] Control group: 5 mg / kg normal saline was given by intraperitoneal injection daily.

[0342] Cisplatin DDP group: DDP was injected intraperitoneally every 3 days, 5 mg / kg.

[0343] Combined group: clinical dose as low dose, 10 times clinical dose as high dose

[0344] Combination + DDP group: DDP + combination dose group, DDP + combination high dose group

[0345] 3. Test indicators:

[0346] (1) Verify the efficacy of the drug by measuring biochemical indicators (body weight, blood routine, tumor weight and tumor inhibition rate, liver and kidney indicators)

[0347] (2) Pathological tissue sections of tumor tissue and liver and kidney tissue to explore whether it enhances efficacy and reduces toxicity

[0348] (3) To explore whether the drug is attenuated by antioxidant indicators of the liver and kidneys (SOD, GSH and MDA content)

[0349] (4) To explore whether it enhances efficacy through immunological indicators (changes in splenic lymphocyte CD4+ and CD8+ T subsets, immunoglobulin IgA, IgG content, IL-2 and IFN-γ protein content).

[0350] 4. Specific experimental steps:

[0351] 4.1 Mouse tumor modeling

[0352] (1) Experimental Materials

[0353] ①Tumor cell line: mouse tumor line LEWIS

[0354] ② Experimental animals: 50 healthy 6-week-old male C57BL / 6 mice, weighing 18-22 g.

[0355] ③ Reagents: DMEM complete medium, trypsin-EDTA digestion solution

[0356] ④Instruments: pipette, centrifuge, microscope, cell counting plate, 1mL syringe

[0357] (2) Experimental methods

[0358] ① Use trypsin-EDTA digestion solution to digest the cells in the logarithmic growth phase to a cell suspension state. Centrifuge the cell suspension and discard the supernatant, resuspend it with PBS and blow it evenly, take 10μl and drop it into the cell counting plate to adjust the live cell concentration to 1x10 7 cel1s / mL.

[0359] ② Keep the animals in a clean animal house and allow them to adapt to the environment for one week.

[0360] ③ After wiping the skin of the right forelimb of the mouse with an alcohol cotton ball for disinfection, gently shake the centrifuge tube containing the cell suspension until the cells are evenly dispersed. After aspirating the cell suspension with a syringe, inject 0.2mL subcutaneously into the disinfected area. Observe and record the mouse status and tumor formation, and measure the size of the visible tumor (>3mm) with a vernier ruler. A total of 40 cancer mouse subcutaneous transplant tumor models were established.

[0361] 4.2 Grouped Dosing

[0362] (1) Experimental Materials

[0363] ① Experimental animals: healthy 6-week-old male C57BL / 6J mice, weighing 18-22 g.

[0364] ② Reagents: cisplatin, DMSO, PBS buffer solution, composition (ginseng extract, coix seed extract, clematis ternata extract, mannan peptide, berberine hydrochloride).

[0365] Preparation of cisplatin (0.5 mg / ml): Dissolve 1 mg of cisplatin in 1 mL of DMSO, then add 1 mL of PBS buffer solution. Prepare it just before the experiment to ensure freshness.

[0366] 5. Experimental Results

[0367] 5.1 Synergistic effect of drug combination on cisplatin - determination of tumor inhibition rate

[0368] 5.1.1 Effect of the combination and cisplatin on the body weight of mice with lung cancer

[0369] According to the above experimental method, the blank group and the other 6 groups of mice were given a drug administration experiment. After 18 days, the body weight of the mice was measured. The test results are shown in Table 43 and Figure 1 shown.

[0370] Table 43 Effect of the combination of cisplatin on the body weight of lung cancer mice (Mean±SD, n=8)

[0371]

[0372] ** p<0.01, there was a very significant difference compared with the model group.

[0373] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0374] Conclusion: As shown in Table 36, Figure 1As shown in the figure, compared with the blank group, the body weight of the model group increased significantly (P < 0.01), and the axillary tumor was larger than 3 mm, indicating that the Lewis lung cancer model of mice was successfully replicated. Compared with the model group, the body weight of the drug-treated groups decreased significantly (P < 0.01), and compared with the cisplatin group, the body weight of the other four drug-treated groups increased (P < 0.01). After tumor implantation, the tumor volume increased and the body weight increased, so tumor weight analysis was also required.

[0375] 5.1.2 Effect of the combination of cisplatin and lung cancer on tumor weight and tumor inhibition rate in mice

[0376] The experimental results are shown in Table 44. Figure 2 a. Figure 2 As shown in Figure b, compared with the model group, the tumor weight of the cisplatin group decreased significantly (P < 0.01), and the tumor inhibition rate reached 33.78%. Compared with the model group, each drug administration group can significantly reduce the tumor weight (P < 0.01), and the tumor inhibition rates of the low-dose combination group and the high-dose combination group are 38.23% and 42.34%, respectively, which are slightly higher than the tumor inhibition effect of cisplatin. Compared with the cisplatin group, the tumor weight of the cisplatin + low-dose combination group and the cisplatin + high-dose combination group decreased significantly (P < 0.01), and the tumor inhibition rates can reach 79.18% and 85.27%, respectively. The combination significantly improves the anti-tumor effect of cisplatin.

[0377] Table 44 Effect of the combination of cisplatin on tumor weight and tumor inhibition rate in lung cancer mice (Mean±SD, n=8)

[0378]

[0379] Tumor inhibition rate % = (1-average tumor weight of drug administration group / average tumor weight of model group) × 100%

[0380] ** p<0.01, there was a very significant difference compared with the model group.

[0381] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0382] 5.1.3 Effects of the combination and cisplatin on liver weight and liver index in lung cancer mice

[0383] The experimental results are shown in Table 45. Figure 3 As shown, there was no significant change in liver weight and liver index of mice in each group.

[0384] Table 45 Effect of the combination of cisplatin and lung cancer mice on liver weight and liver index (Mean±SD, n=8)

[0385]

[0386]

[0387] Liver index = liver weight (mg) / body weight (g)

[0388] 5.1.4 Effects of the combination and cisplatin on kidney weight and renal index in lung cancer mice

[0389] The experimental results are shown in Table 46. Figure 4 As shown, the kidney weight of the model group was significantly reduced compared with the blank group. Compared with the model group and the cisplatin group, the kidney weight of the cisplatin + low-dose combination group decreased significantly after administration, p<0.01, and the kidney weight of the high-dose combination group increased significantly compared with the model group, p<0.05. The changes in the kidney index, after administration, except for the cisplatin + low-dose combination group, the other groups were significantly different from the model group, p<0.01. Compared with the cisplatin group, except for the high-dose combination group, the other groups could reduce the kidney index. It can be seen from the kidney weight and kidney index that the effects of each medication group on the kidney are different, and the effects are clarified through subsequent indicator tests.

[0390] Table 46 Effect of the combination of cisplatin on kidney weight and renal index of lung cancer mice (Mean±SD, n=8)

[0391]

[0392] Renal index = kidney weight (mg) / body weight (g)

[0393] * p<0.01, there was a significant difference compared with the model group.

[0394] ** p<0.01, there was a very significant difference compared with the model group.

[0395] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0396] 5.1.5 Effect of the combination of cisplatin and lung cancer on spleen weight and spleen index in mice

[0397] The experimental results are shown in Table 47. Figure 5As shown, compared with the blank group, the spleen weight and spleen index of the model group were significantly increased (P < 0.01), indicating that the immune function of the mouse Lewis lung cancer model was abnormal. Compared with the model group, the spleen weight and spleen of the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin inhibited the immune function of the mouse Lewis lung cancer model. Compared with the model group, the spleen weight and spleen index of the composition-low-dose group and the composition-high-dose group were significantly increased (P < 0.01), indicating that the composition can effectively improve the immune function of the mouse Lewis lung cancer model. Compared with the cisplatin group, the spleen weight and spleen index of the cisplatin + composition-low-dose group and the cisplatin + composition-high-dose group were significantly increased (P < 0.01), indicating that the composition can effectively improve the inhibitory effect of cisplatin on the immune function of mice with lung cancer model. Compared with the model group, the spleen weight of the cisplatin + combination-low-dose group was significantly increased (P < 0.01), and the spleen index of the cisplatin + combination-low-dose group was significantly increased (P < 0.01), indicating that the combination can reverse the toxic side effects caused by cisplatin.

[0398] Table 47 Effect of the combination of cisplatin on spleen weight and spleen index of lung cancer mice (Mean±SD, n=8)

[0399]

[0400] Spleen index = spleen weight (mg) / body weight (g)

[0401] ** p<0.01, there was a very significant difference compared with the model group.

[0402] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0403] in conclusion

[0404] After the model group was implanted with the tumor, the body weight increased with the increase of tumor weight. After administration, the tumor growth was inhibited, the tumor weight decreased, and the body weight decreased. The tumor inhibition rate of the cisplatin + combination group was much higher than that of cisplatin alone, and the tumor weight decreased significantly, but the body weight was higher than that of the cisplatin group, indicating that the combination of cisplatin and the combination not only reduced the tumor weight, but also had a repairing effect on the damage of the body. There was no significant change in the weight of the liver and kidney, but a significant change in the weight of the spleen. Cisplatin significantly inhibited the immune organs. After the combination of cisplatin and the combination activated the immune tissue, the spleen weight increased, indicating that the combination synergized cisplatin through the immune pathway to exert an anti-tumor effect.

[0405] 5.2 Synergistic effect of the combination on cisplatin - immune function

[0406] 5.2.1 Effects of the combination and cisplatin on CD4 and CD4 / CD8 levels in mice with lung cancer

[0407] CD4+T cells are a type of white blood cell and are the command center of the human immune system. They are responsible for eliminating and controlling a variety of infections and fighting invading pathogens. The number of CD4+T cells in the body can be understood by testing the CD4+T count in the blood.

[0408] As shown in Table 48, Figure 6 As shown, compared with the blank group, the levels of CD4 and CD4 / CD8 in the model group decreased, indicating abnormal immune indicators in the Lewis lung cancer model in mice. Compared with the model group, the levels of CD4 and CD4 / CD8 in the cisplatin group decreased significantly, indicating that cisplatin caused a reduction in immune factors in mice with lung cancer. Compared with the model group, the levels of CD4 and CD4 / CD8 in the low-dose group and the high-dose group of the composition increased, indicating that the composition reduced the abnormal immune indicators caused by the mouse lung cancer model. Compared with the cisplatin group, the levels of CD4 and CD4 / CD8 in the cisplatin+composition-low-dose group and the cisplatin+composition-high-dose group increased, indicating that the high-dose composition can reverse the toxic and side effects caused by cisplatin.

[0409] Table 48 Effect of the combination of cisplatin on CD4 and CD4 / CD8 levels in mice with lung cancer

[0410]

[0411] 5.2.2 Effect of the combination of cisplatin and splenic IL-2 levels in mice with lung cancer

[0412] Basic research shows that IL-2 indirectly exerts anti-tumor effects, and its mechanism is to stimulate and activate effector cells to achieve the purpose of treating tumors. Figure 7 As shown, compared with the blank group, the IL-2 level in the model group was significantly decreased (P < 0.01), indicating that the spleen immune function of the mouse Lewis lung cancer model was decreased.

[0413] Compared with the model group, the IL-2 level in the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin produced immunosuppression in mice with lung cancer. The IL-2 levels in the low-dose combination group and the low-dose cisplatin + combination group increased significantly, with significant differences compared with the model group and the cisplatin group (P < 0.01), indicating that the low-dose combination can effectively reduce the immunosuppression of the lung cancer model, enhance the body's immune function, and reverse the immunosuppression caused by cisplatin. There was no significant difference between the high-dose combination group and the model group, and the cisplatin + combination-high-dose group caused a significant decrease in IL-2 levels, with a significant difference compared with the model group (P < 0.01), and IL-2 increased compared with the cisplatin group (P < 0.05), indicating that the high-dose combination has a repairing effect on the immunosuppression caused by cisplatin, but the effect is not as good as the low-dose coix seed oral liquid.

[0414] Table 49 Effect of the combination of cisplatin on the level of IL-2 in the spleen of lung cancer mice (Mean±SD, n=8)

[0415]

[0416] **p<0.01, there is a very significant difference compared with the model group.

[0417] # p<0.01, compared with the cisplatin group, there was a significant difference.

[0418] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0419] 5.2.3 Effect of the combination of cisplatin and splenic IFN-γ levels in mice with lung cancer

[0420] As shown in Table 50, Figure 8 As shown, compared with the blank group, the IFN-γ level in the model group decreased significantly (P < 0.01), indicating that the immune function of the spleen in the Lewis lung cancer model of mice decreased. Compared with the model group, the IFN-γ level in the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin produced immunosuppression in mice with lung cancer. The low-dose combination group had significant differences compared with the model group and the cisplatin group (P < 0.01), indicating that the combination activated cellular immunity and had a significant immune enhancement effect. There was no significant difference compared with the model group after combining with cisplatin, and there was a significant difference compared with the cisplatin group (P < 0.01), indicating that the low-dose combination enhanced the immune system suppressed by cisplatin. The high-dose combination group and the cisplatin + combination-high-dose group can significantly increase the IFN-γ level (P < 0.01), and the IFN-γ level is higher than that of the blank group, indicating that the high-dose combination has a stronger ability to enhance the body's immunity.

[0421] Table 50 Effect of the combination of cisplatin on the level of IFN-γ in the spleen of lung cancer mice (Mean±SD, n=8)

[0422]

[0423] ** p<0.01, there was a very significant difference compared with the model group.

[0424] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0425] 5.2.4 Effect of the combination of cisplatin and splenic IgG levels in mice with lung cancer

[0426] A decrease in the CD4 / CD8 ratio indicates a disorder in cellular immune function, which can suppress normal B lymphocytes, causing low immunoglobulins, and subsequently reducing serum IgA and IgG levels, reducing the body's immune protection. As shown in Table 51, Fig. 9 As shown, compared with the blank group, the IgG level in the model group was significantly decreased (P < 0.01), indicating that the spleen immune function of the mouse Lewis lung cancer model was decreased.

[0427] Compared with the model group, the IgG level in the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin produced immunosuppression in mice with lung cancer. The IgG level in the low-dose combination group was significantly increased compared with the model group and the cisplatin group (P < 0.01). There was no difference between the high-dose combination group, the cisplatin + combination - low-dose group, and the cisplatin + combination - high-dose group and the model group, but there was a significant difference compared with the cisplatin group (P < 0.01), indicating that the effect of the low-dose combination in enhancing the body's immunity is more obvious than the high-dose combination. After combined use with cisplatin, both the high-dose and low-dose combinations can enhance the immunity suppressed by cisplatin.

[0428] Table 51 Effect of the combination of cisplatin on the level of spleen IgG in lung cancer mice (Mean±SD, n=8)

[0429]

[0430] ** p<0.01, there was a very significant difference compared with the model group.

[0431] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0432] 5.2.5 Effect of the combination of cisplatin and lung cancer on spleen IgA levels in mice

[0433] As shown in Table 52, Fig.10 As shown, compared with the blank group, the IgA level in the model group decreased significantly (P < 0.01), indicating that the immune function of the spleen in the Lewis lung cancer model of mice decreased. Compared with the model group, the IgA level in the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin produced immunosuppression in mice with lung cancer. There was no difference in the IgA levels of the low-dose and high-dose combination groups compared with the model group, which were significantly higher than the cisplatin group (P < 0.01). There was no difference between the cisplatin + low-dose combination group and the model group, but there was a difference with the cisplatin group (P < 0.05). The cisplatin + high-dose combination group was lower than the model group (P < 0.05) and had no difference with the cisplatin group. It shows that the low-dose combination can not only play a better role in enhancing immunity, but also has a regulatory effect on the immunosuppression of cisplatin, and the effect is better than the high-dose combination.

[0434] Table 52 Effect of the combination of cisplatin on the level of spleen IgA in lung cancer mice (Mean±SD, n=8)

[0435]

[0436]

[0437] *p<0.01, compared with the model group, there is a significant difference.

[0438] **p<0.01, there is a very significant difference compared with the model group.

[0439] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0440] in conclusion

[0441] 1. After modeling, the CD4+ content was significantly reduced, and the CD4 / CD8 ratio was reduced. The combination alone significantly increased the CD4+ and CD4 / CD8 ratios. The low-dose group had a more obvious effect than the high-dose group. After combined use with cisplatin, the inhibitory effect of cisplatin on CD4+T cells was significantly improved, and the changes in IL-2 were consistent with the changes in CD4+T cells.

[0442] 2. The IFN-γ content was significantly reduced after modeling, and the content in the cisplatin group was further reduced after administration. The IFN-γ content in the combination group was significantly increased. The high-dose combination was more effective than the low-dose group. IFN-γ is also an important activator of macrophages, which directly induces anti-tumor effects.

[0443] 3. After modeling, the levels of IgG and IgA were significantly reduced. After administration, the levels in the cisplatin group were further reduced. The levels in the combination group were significantly increased. The low-dose group had better effects than the high-dose group.

[0444] 4. Through the determination of immune-related indicators, it was found that the composition exerted humoral immunity at a low dose and cellular immunity at a high dose.

[0445] 5.3 The attenuation effect of the combination on cisplatin - blood routine test

[0446] 5.3.1 Effect of the combination and cisplatin on the white blood cell (WBC) level in lung cancer mice

[0447] As shown in Table 53, Fig.11As shown, compared with the blank group, the white blood cell (WBC) level in the model group increased significantly (P < 0.01), and the normal reference value was 0.8-6.8, indicating that the blood routine indicators of the mouse Lewis lung cancer model were abnormal. Compared with the model group, the white blood cell (WBC) level of all drug-treated groups decreased significantly (P < 0.05). This shows that the abnormal increase in white blood cells caused by tumor docking has a repairing effect after administration. Compared with the cisplatin group, there was no significant difference in the cisplatin + combination-low-dose group, and the white blood cell (WBC) level of the combination-low-dose group, the combination-high-dose group, and the cisplatin + combination-high-dose group was higher than that of the cisplatin group (P < 0.01).

[0448] Table 53 Effect of the combination of cisplatin on the level of white blood cells (WBC) in lung cancer mice (Mean±SD, n=8)

[0449]

[0450]

[0451] *p<0.01, compared with the model group, there is a significant difference.

[0452] **p<0.01, there is a very significant difference compared with the model group.

[0453] ##p<0.01, there is a very significant difference compared with the cisplatin group.

[0454] 5.3.2 Effect of the combination and cisplatin on the red blood cell (RBC) level in lung cancer mice

[0455] As shown in Table 54, Fig.12 As shown, compared with the blank group, the red blood cell (RBC) level in the model group decreased significantly (P < 0.01), and the normal reference value was 6.36-9.42 (10^12 / L), indicating that the blood routine indicators of the Lewis lung cancer model in mice were abnormal. Compared with the model group, the red blood cell (RBC) level in the cisplatin group decreased more significantly (P < 0.01), indicating that cisplatin caused a decrease in the number of red blood cells in lung cancer mice and that cisplatin had significant blood toxicity. The red blood cell (RBC) levels in the low-dose group and the high-dose group of the combination increased significantly and returned to the level of the blank group (P < 0.01), indicating that the combination had a repairing effect on the erythrocyte toxicity of the lung cancer mouse model.

[0456] The red blood cell (RBC) levels in the cisplatin + combination-low-dose group and the cisplatin + combination-high-dose group were lower than those in the model group, but higher than those in the cisplatin group (P < 0.01), indicating that the combination can reverse the erythrocyte toxicity caused by cisplatin.

[0457] Table 54 Effect of the combination of cisplatin on the red blood cell (RBC) level in lung cancer mice (Mean±SD, n=8)

[0458]

[0459]

[0460] **p<0.01, there is a very significant difference compared with the model group.

[0461] #p<0.01, compared with the cisplatin group, there is a significant difference.

[0462] ##p<0.01, there is a very significant difference compared with the cisplatin group.

[0463] 5.3.3 Effect of the combination and cisplatin on hemoglobin (HGB) levels in mice with lung cancer

[0464] As shown in Table 55, Fig.13 As shown, compared with the blank group, the hemoglobin (HGB) level in the model group decreased significantly (P < 0.01), and the normal reference value was 110-143 (g / L), indicating that the blood routine index of the Lewis lung cancer model in mice was abnormal. Compared with the model group, the hemoglobin (HGB) level in the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin caused a decrease in the number of hemoglobin in lung cancer mice, and the hemoglobin (HGB) levels in the low-dose group and the high-dose group of the composition increased significantly (P < 0.01), indicating that the composition has the ability to repair the abnormal hemoglobin index caused by the lung cancer mouse model. Compared with the cisplatin group, the hemoglobin (HGB) level in the cisplatin + composition-high-dose group increased (P < 0.05), indicating that the composition can reduce the toxic side effects caused by cisplatin.

[0465] Table 55 Effect of the combination of cisplatin and hemoglobin (HGB) level in mice with lung cancer (Mean±SD, n=8)

[0466]

[0467] **p<0.01, there is a very significant difference compared with the model group.

[0468] #p<0.01, compared with the cisplatin group, there is a significant difference.

[0469] ##p<0.01, there is a very significant difference compared with the cisplatin group.

[0470] 5.3.4 Effect of the combination and cisplatin on platelet (PLT) levels in mice with lung cancer

[0471] As shown in Table 56, Fig.14As shown, compared with the blank group, the platelet (PLT) level in the model group decreased significantly (P < 0.01), and the normal reference value was 450-1590 (g / L), indicating that the blood routine index of the mouse Lewis lung cancer model was abnormal. Compared with the model group, the platelet (PLT) level in the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin caused a decrease in the number of platelets in lung cancer mice, and the platelet (PLT) level in the low-dose group and the high-dose group of the composition increased significantly (P < 0.01), indicating that the composition repaired the abnormal platelet index caused by the mouse lung cancer model. Compared with the cisplatin group, the platelet (PLT) level in the cisplatin + composition - low-dose group and the cisplatin + composition - high-dose group increased significantly (P < 0.01), indicating that the composition can reduce the toxic side effects caused by cisplatin.

[0472] Table 56 Effect of the combination of cisplatin on platelet (PLT) levels in mice with lung cancer (Mean±SD, n=8)

[0473]

[0474] ** p<0.01, there was a very significant difference compared with the model group.

[0475] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0476] 5.3.5 Effect of the combination and cisplatin on the lymphocyte percentage (Lym%) level in lung cancer mice

[0477] As shown in Table 57, Fig.15 As shown, compared with the blank group, the level of lymphocyte percentage (Lym%) in the model group decreased significantly (P < 0.01), and the normal reference value was 55.8-90.6%, indicating that the blood routine indicators of the Lewis lung cancer model in mice were abnormal. Compared with the model group, the level of lymphocyte percentage (Lym%) in the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin caused a decrease in the number of lymphocytes in lung cancer mice, and there was no significant difference between the other drug-administered groups and the model group. Compared with the cisplatin group, the level of lymphocyte percentage (Lym%) in the cisplatin + combination-low-dose group and the cisplatin + combination-high-dose group increased significantly (P < 0.05), indicating that the combination can reduce the toxic side effects caused by cisplatin.

[0478] Table 57 Effect of the combination of cisplatin on the lymphocyte percentage (Lym%) level in lung cancer mice (Mean±SD, n=8)

[0479]

[0480] **p<0.01, there was a very significant difference compared with the model group.

[0481] # p<0.01, compared with the cisplatin group, there was a significant difference.

[0482] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0483] 5.3.6 Effect of the combination and cisplatin on the neutrophil percentage (GR%) level in lung cancer mice

[0484] As shown in Table 58, Fig.16 As shown, compared with the blank group, the neutrophil percentage (GR%) level in the model group increased significantly (P < 0.01), and the normal reference value was 8.6-38.9%, indicating that the blood routine indicators of the mouse Lewis lung cancer model were abnormal. Compared with the model group, the neutrophil percentage (GR%) level of all the drug-administered groups decreased significantly (P < 0.01), indicating that the number of neutrophils in lung cancer mice was reduced after administration. Compared with the cisplatin group, the neutrophil percentage (GR%) level of the combination-low-dose group and the combination-high-dose group increased (P < 0.05), indicating that the combination alleviated the abnormal blood routine indicators caused by the mouse lung cancer model.

[0485] Table 58 Effect of the combination of cisplatin on the neutrophil percentage (GR%) level in lung cancer mice (Mean±SD, n=8)

[0486]

[0487]

[0488] ** p<0.01, there was a very significant difference compared with the model group.

[0489] # p<0.01, compared with the cisplatin group, there was a significant difference.

[0490] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0491] in conclusion

[0492] 1. The composition alone or in combination with cisplatin can reduce the leukocyte level of tumor-implanted mice. When used in combination with cisplatin, it has a significant restorative effect on the decrease in leukocytes caused by cisplatin.

[0493] 2. The combination alone can increase the number of red blood cells and hemoglobin to the level of the blank group, and can be used in combination with cisplatin to restore anemia caused by cisplatin.

[0494] 3. The composition alone increases the platelet count and has a restorative effect on the decrease in platelet count caused by tumor docking. When used in combination with cisplatin, it has a restorative effect on the decrease in platelet count caused by cisplatin, indicating that the composition has the function of restoring blood coagulation.

[0495] 4. There was no obvious change in the lymphocytes of mice after tumor implantation, but cisplatin significantly reduced the number of lymphocytes. The combination alone and in combination with cisplatin had no effect on lymphocytes, but compared with the cisplatin group, the low lymphocyte count caused by cisplatin was significantly increased.

[0496] 5. The number of neutrophils increased significantly after tumor implantation and decreased after drug administration, with the decrease being most significant in the cisplatin group. The combination of the composition and cisplatin had no significant effect on the recovery of neutrophils.

[0497] 5.4 The combination reduces toxicity of cisplatin - liver and kidney damage

[0498] ALT and AST are currently the most commonly used liver function indicators. If liver cells are damaged, the activity of ALT and AST in serum will increase, and the degree of increase is positively correlated with the degree of liver cell damage. ALT is mainly distributed in the liver cytoplasm. Increased ALT reflects damage to the liver cell membrane, indicating acute liver cell damage; AST is mainly distributed in the liver cytoplasm and liver cell mitochondria. Its increase indicates that liver cell damage has reached the level of organelles, indicating chronic liver damage. Both are also present in other tissue cells. Liver function ALB refers to albumin, which is an important indicator in liver function testing. It is an important protein in plasma and an important nutrient in the body. Albumin is mainly synthesized in the liver, and its main functions include maintaining plasma osmotic pressure and running small molecules in the body.

[0499] 5.4.1 Effect of the combination and cisplatin on the ALT activity level of liver function in lung cancer mice

[0500] Alanine aminotransferase (ALT) is mainly present in various cells, especially in liver cells. The level of transaminase in the entire liver is about 100 times that in the blood. Under normal circumstances, as long as a small amount is released into the blood, the activity of the enzyme in the serum can be significantly increased. In the acute phase of various viral hepatitis and when liver cells are necrotic due to drug toxicity, ALT is released into the blood in large quantities. Therefore, it is an important indicator for the diagnosis of viral hepatitis and toxic hepatitis. The concentration of alanine aminotransferase in liver cells is 1000 to 3000 times higher than that in serum. As long as 1% of liver cells are necrotic, the enzyme activity in the blood can be increased by 1 times. Therefore, transaminase (especially ALT) is a sensitive marker of acute liver cell damage. As shown in Table 59, Fig.17 As shown, compared with the blank group, the ALT activity level in the model group was significantly increased (P < 0.05), indicating that liver damage occurred in the Lewis lung cancer model in mice.

[0501] Compared with the model group, the ALT activity level in the cisplatin group increased significantly (P < 0.01), indicating that cisplatin aggravated liver damage in mice with lung cancer. The ALT activity level in the low-dose combination group and the low-dose cisplatin + combination group decreased significantly (P < 0.01). The low-dose combination significantly improved the liver function of tumor-transmitted mice and repaired the liver damage caused by cisplatin, bringing the ALT value to the level of the blank group. There was no significant difference in the ALT activity level between the high-dose combination group and the high-dose cisplatin + combination group and the model group, indicating that the low-dose combination has a stronger effect on repairing liver ALT than the high-dose combination.

[0502] Compared with the cisplatin group, the ALT activity levels of the combination-low-dose group, cisplatin + combination-low-dose group, combination-high-dose group, and cisplatin + combination-high-dose group were significantly decreased (P < 0.01), indicating that the combination can effectively repair the liver damage caused by cisplatin to lung cancer model mice.

[0503] Table 59 Effect of the combination of cisplatin on the liver function and ALT activity level of lung cancer mice (Mean±SD, n=8)

[0504]

[0505]

[0506] * p<0.01, there was a significant difference compared with the model group.

[0507] ** p<0.01, there was a very significant difference compared with the model group.

[0508] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0509] 5.4.2 Effect of the combination of drugs and cisplatin on the activity level of AST in liver function of mice with lung cancer

[0510] 80% of aspartate aminotransferase (AST) in liver cells is present in the mitochondria of liver cells. When serum aspartate aminotransferase increases, it generally indicates mitochondrial damage and persistent condition, which is usually positively correlated with the severity of liver disease. As shown in Table 60, Fig.18 As shown, compared with the blank group, the AST activity level in the model group was significantly increased (P < 0.01), indicating that liver damage occurred in the Lewis lung cancer model in mice.

[0511] Compared with the model group, the AST activity level in the cisplatin group increased significantly (P < 0.01), indicating that cisplatin aggravated liver damage in mice with lung cancer. The AST activity levels in the low-dose combination group, high-dose combination group, and combination group with cisplatin all decreased significantly (P < 0.01), indicating that the combination alone and in combination with cisplatin can repair liver cells, effectively improve the degree of liver damage in the lung cancer model, and reduce the AST level to normal levels.

[0512] Compared with the cisplatin group, the AST activity levels in the low-dose combination group, high-dose combination group and the combination with cisplatin group were significantly decreased (P < 0.01), indicating that the combination effectively repaired the liver damage caused by cisplatin in lung cancer model mice. The high-dose combination had a stronger repair ability.

[0513] Table 60 Effect of the combination of cisplatin on the activity level of AST in liver function of lung cancer mice (Mean±SD, n=8)

[0514]

[0515]

[0516] ** p<0.01, there was a very significant difference compared with the model group.

[0517] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0518] 5.4.3 Effect of the combination of drugs and cisplatin on the ALB level in liver function of mice with lung cancer

[0519] Albumin level represents the synthetic function of the liver. Fig.19 As shown, compared with the blank group, the ALB level in the model group was significantly decreased (P < 0.01), indicating that liver damage occurred in the Lewis lung cancer model in mice.

[0520] Compared with the model group, the ALB level in the cisplatin group decreased significantly (P < 0.01). Cisplatin reduced the synthesis of ALB, indicating that liver damage in mice with lung cancer worsened. The ALB levels in the low-dose and high-dose groups of the combination increased significantly (P < 0.01), indicating that the combination has the effect of repairing liver damage in lung cancer models. After the low-dose combination was combined with cisplatin, there was no significant difference compared with the model group. After the high-dose combination was combined with cisplatin, the ALB level increased significantly (P < 0.01), which was not only significantly higher than the model group, but also significantly higher than the blank group, indicating that the high-dose combination has a stronger repair effect on the liver than the low-dose combination.

[0521] Compared with the cisplatin group, the ALB levels of the combination-low-dose group, cisplatin + combination-low-dose group, combination-high-dose group, and cisplatin + combination-high-dose group were significantly increased (P < 0.01), indicating that the combination can effectively repair the liver damage caused by cisplatin to lung cancer model mice.

[0522] Table 61 Effect of the combination of cisplatin on the ALB level in liver function of lung cancer mice (Mean±SD, n=8)

[0523]

[0524]

[0525] ** p<0.01, there was a very significant difference compared with the model group.

[0526] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0527] 5.4.4 Effect of the combination and cisplatin on the renal function and BUN activity level of lung cancer mice

[0528] BUN is an indicator of the glomerular filtration rate in the kidney. BUN is urea nitrogen, which is the end product of protein metabolism. It is produced in the liver through the guanine cycle. After entering the blood circulation, it is mainly filtered through the glomerulus and excreted in the urine. When renal function is damaged, it can lead to an increase in blood BUN levels. As shown in Table 62, Fig. 20 As shown, compared with the blank group, the BUN activity level in the model group was significantly increased (P < 0.01), indicating that the mouse Lewis lung cancer model had renal damage. Compared with the model group, the BUN activity level in the cisplatin group was significantly increased (P < 0.01), indicating that cisplatin aggravated the renal damage of lung cancer mice. There was no significant difference between the low-dose combination group and the low-dose cisplatin + combination group and the model group, but there was a significant difference compared with the cisplatin group (P < 0.01), indicating that low-dose coix seed oral liquid had no damaging effect on the kidneys, and combined with cisplatin repaired the renal damage of cisplatin. The high-dose combination group and the high-dose cisplatin + combination group could significantly reduce BUN, and the BUN level was lower than that of the model group but did not reach the level of the blank group. There were significant differences compared with the model group and the cisplatin group (P < 0.01), indicating that the high-dose combination could reverse the toxic side effects caused by cisplatin, and even effectively improve the renal damage caused by its lung cancer model and restore it to normal activity levels.

[0529] Table 62 Effect of the combination of cisplatin on the BUN activity level of renal function in lung cancer mice (Mean±SD, n=8)

[0530]

[0531] ** p<0.01, there was a very significant difference compared with the model group.

[0532] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0533] 5.4.5 Effect of the combination of cisplatin and renal function of lung cancer mice on CRE activity level

[0534] Creatinine (Cre) is a product of muscle metabolism in the human body and is mainly excreted from the body through glomerular filtration. When the filtration capacity decreases, the creatinine concentration increases. An increase in blood creatinine means kidney damage. Blood creatinine can more accurately reflect the damage to the renal parenchyma, but it is not a sensitive indicator. As shown in Table 63, Fig.21 As shown, compared with the blank group, the CRE activity level in the model group was significantly increased (P < 0.01), indicating that renal damage occurred in the Lewis lung cancer model in mice.

[0535] Compared with the model group, the CRE activity level in the cisplatin group was significantly increased (P < 0.01), indicating that cisplatin aggravated the renal damage of mice with lung cancer. There was no significant difference between the low-dose combination group and the low-dose cisplatin + combination group and the model group, indicating that the low-dose combination had no renal damage effect, and there was a significant difference compared with the cisplatin group (P < 0.01), indicating that it had a repair effect on the renal damage caused by cisplatin. The CRE in the high-dose combination group was significantly reduced, and there was a significant difference from the model group and the cisplatin group (P < 0.01), indicating that the high-dose combination repaired the renal damage of the lung cancer model. There was no significant difference between the cisplatin + combination-high-dose group and the model group, and there was a significant difference from the cisplatin group (P < 0.01), indicating that the high-dose combination reduced the renal toxicity of cisplatin and reached the model level.

[0536] Table 63 Effect of the combination of cisplatin on the renal function of lung cancer mice CRE activity level (Mean ± SD, n = 8)

[0537]

[0538] ** p<0.01, there was a very significant difference compared with the model group.

[0539] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0540] in conclusion

[0541] 1. After modeling, ALT and AST were significantly increased. After administration, the damage in the cisplatin group was aggravated, and ALT and AST were further increased. The ALT and AST of the combination alone and the combination with cisplatin group decreased. ALT had a better effect in the low-dose group and the combination with cisplatin group, and AST had a good effect in the high-dose group and the combination with cisplatin group.

[0542] 2. Albumin ALB is synthesized in the liver. When liver function is severely damaged, ALB content decreases. ALB content decreases after modeling. After administration, ALB in the cisplatin group decreased more significantly, indicating that cisplatin further damaged liver function. The composition alone and in combination with cisplatin increased ALB, indicating that the composition has the effect of repairing liver function. The high-dose composition and the combination with cisplatin group have more obvious effects.

[0543] 3. Urea nitrogen BUN and creatinine Cre reflect the degree of renal function damage. After modeling, BUN and Cre increased significantly, indicating kidney damage after tumor grafting. After administration, BUN and Cre in the cisplatin group increased further, and the damage worsened, especially the Cre value doubled. The combination alone and in combination with cisplatin reduced BUN and Cre, and the high-dose group of the combination and the group combined with cisplatin had the best effect.

[0544] 5.5 Combination of drugs to reduce cisplatin toxicity - oxidative damage

[0545] Oxidative stress is the main cause of cisplatin DDP hepato-renal toxicity. GSH is one of the most important molecules for cell defense against toxic compounds or oxidative stress chemical reactions. SOD is a metalloprotein that can scavenge oxygen free radicals produced in the body, thereby protecting cells from damage by superoxide free radicals, and has similar functions to GSH. Therefore, the detection of SOD and GSH content can reflect the antioxidant capacity of cells. Studies have shown that DDP can cause a serious loss of GSH content in the body by binding to GSH, and can also inhibit the activity of glutathione peroxide (GSH-Px), SOD, etc., thereby reducing the body's antioxidant capacity.

[0546] 5.5.1 Effect of the combination of cisplatin and shampoxetine on the level of SOD in the liver of mice with lung cancer

[0547] Superoxide dismutase (SOD) has anti-oxidation and anti-aging effects, and its mechanism of action is mainly to remove superoxide anion free radicals (O2-) that are harmful to the body. As shown in Table 64, Fig. 22 As shown, compared with the blank group, the SOD level in the model group was significantly decreased (P < 0.01), indicating that the liver of the Lewis lung cancer model in mice was damaged by oxidative damage.

[0548] Compared with the model group, the SOD level in the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin aggravated the oxidative damage to the liver of lung cancer mice; the SOD level in the low-dose combination group increased significantly (P < 0.01), indicating that the low-dose combination can effectively improve the degree of liver oxidative damage in the lung cancer model; the SOD level after the low-dose combination of the combination and cisplatin was lower than that in the model group (P < 0.01); the SOD level in the high-dose combination group increased significantly (P < 0.01); the SOD level in the high-dose combination of the combination and cisplatin group decreased (P < 0.01).

[0549] Compared with the cisplatin group, the SOD levels of the combination-low dose, cisplatin + combination-low dose group, combination-high dose group, and cisplatin + combination-high dose group were significantly increased (P < 0.01), indicating that the combination can effectively improve the liver oxidative damage caused by cisplatin to lung cancer model mice. The improvement effect of the low-dose combination is particularly obvious when administered in combination, indicating that the combination can repair the oxidative damage caused by cisplatin, and the repair ability of the clinical dose is stronger than 10 times the amount.

[0550] The low-dose combination can exert an antioxidant effect, and the effect is better than 10 times the amount. It has a repairing effect on cisplatin oxidative damage, but it does not reach the level of the model group.

[0551] Table 64 Effect of the combination of cisplatin on the level of SOD in the liver of lung cancer mice (Mean±SD, n=8)

[0552]

[0553] ** p<0.01, there was a very significant difference compared with the model group.

[0554] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0555] 5.5.2 Effect of the combination of cisplatin and cisplatin on liver GSH levels in mice with lung cancer

[0556] Glutathione can help maintain normal immune system function, and has antioxidant and integrated detoxification effects. As an important antioxidant in the body, it can remove free radicals in the human body. Since GSH itself is susceptible to oxidation by certain substances, it can protect the sulfhydryl groups in many proteins and enzymes in the body from being oxidized by harmful substances, thereby ensuring the normal physiological functions of proteins and enzymes. As shown in Table 65, Fig.23 As shown, compared with the blank group, the GSH level in the model group was significantly decreased (P < 0.01), indicating that the liver of the Lewis lung cancer model in mice was oxidatively damaged, which was consistent with SOD.

[0557] Compared with the model group, the GSH level in the cisplatin group decreased, but there was no significant difference, indicating that cisplatin had no effect on GSH. The combination-low-dose group and the combination-low-dose and cisplatin group increased the GSH content (P < 0.01) and the combination-high-dose group (P < 0.05), indicating that the combination can significantly reduce liver oxidative damage in lung cancer models. There was no change in GSH compared with the combination-high-dose and cisplatin groups and the model group.

[0558] Compared with the cisplatin group, the GSH levels in the combination-low-dose group, cisplatin + combination-low-dose group, combination-high-dose group, and cisplatin + combination-high-dose group were significantly increased (P < 0.01), indicating that the combination can effectively improve the liver oxidative damage caused by cisplatin to lung cancer model mice, and the improvement effect of the low-dose combination is particularly obvious when administered in combination.

[0559] Both the low-dose and high-dose compositions can exert antioxidant effects and repair the oxidative damage in lung cancer model mice to the level of the blank group. The repair effect on the oxidative damage caused by cisplatin is also quite obvious, and the GSH level exceeds that of the model group. Only the high-dose composition combined with cisplatin group reaches the level of the model group.

[0560] Table 65 Effect of the combination of cisplatin on liver GSH levels in lung cancer mice (Mean±SD, n=8)

[0561]

[0562] * p<0.01, there was a significant difference compared with the model group.

[0563] ** p<0.01, there was a very significant difference compared with the model group.

[0564] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0565] 5.5.3 Effect of the combination of cisplatin and cisplatin on the level of MDA in the liver of mice with lung cancer

[0566] Malondialdehyde (MDA) is the final product of lipid peroxidation, and its concentration can reflect the degree of lipid peroxidation. Fig.24 As shown, compared with the blank group, the MDA level in the model group was significantly increased (P < 0.01), indicating that the liver of the Lewis lung cancer model in mice was damaged by lipid peroxides.

[0567] Compared with the model group, the MDA level in the cisplatin group increased significantly (P < 0.01), indicating that cisplatin increased the production of lipid peroxides and aggravated the oxidative damage to the liver of mice with lung cancer; the MDA level in the high-dose combination group and the high-dose cisplatin combination group decreased significantly (P < 0.01), indicating that the high-dose combination reduced the production of lipid peroxides and effectively improved the liver oxidative damage of the lung cancer model. There was no significant difference between the low-dose combination group and the model group, and the cisplatin combination low-dose group increased the MDA level, but it was lower than that of the cisplatin group, indicating that the low-dose combination can weaken the oxidative damage of cisplatin.

[0568] Compared with the cisplatin group, the combination-low-dose group, cisplatin + combination-low-dose group, combination-high-dose group, and cisplatin + combination-high-dose group could all significantly decrease the MDA level (P < 0.01), indicating that the combination can effectively improve the liver oxidative damage caused by cisplatin to lung cancer model mice. The improvement effect of the high-dose combination was particularly obvious when administered in combination, and the MDA level was lower than that of the blank group.

[0569] Cisplatin induced the production of a large amount of MDA. The other drug-administered groups significantly reduced the content of MDA. The high-dose group of the combination and the group combined with cisplatin had a more obvious reduction effect, reaching the level of the blank group.

[0570] Table 66 Effect of the combination of cisplatin on the MDA level in the liver of lung cancer mice (Mean±SD, n=8)

[0571]

[0572] ** p<0.01, there was a very significant difference compared with the model group.

[0573] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0574] After the mice were implanted with tumors, oxidative damage was induced, SOD and GSH were significantly reduced, and MDA was significantly increased. After cisplatin was used, the damage was aggravated, SOD was further reduced, GSH remained unchanged, and MDA levels increased. The combination alone exerted a significant antioxidant effect, and the levels of SOD and GSH were better at low doses than at high doses, and the effect of MDA was more obvious at high doses. Combination with cisplatin can repair the oxidative damage of cisplatin.

[0575] 5.5.4 Effect of the combination of drugs and cisplatin on the level of SOD in the kidneys of mice with lung cancer

[0576] As shown in Table 67, Fig.25 As shown, compared with the blank group, the SOD level in the model group was significantly decreased (P < 0.01), indicating that the kidneys of the Lewis lung cancer model mice were oxidatively damaged.

[0577] Compared with the model group, the SOD level in the cisplatin group decreased significantly (P < 0.01), indicating that cisplatin aggravated the oxidative damage to the kidneys of mice with lung cancer. There was no difference between the low-dose combination group and the model group, and no effect on the SOD in the kidneys. The SOD level in the combination group with cisplatin was significantly lower than that in the model group (P < 0.05), indicating that the oxidative damage was still very serious after the clinical dose of the combination and cisplatin, and there was no significant change compared with the cisplatin group, indicating that the clinical dose of the combination had no repair effect on the SOD of the kidney oxidative damage caused by cisplatin. The SOD levels in the high-dose combination group and the cisplatin + combination-high-dose group increased significantly (P < 0.01), indicating that the high-dose combination can increase the level of antioxidant SOD, not only has a repair effect on the kidney oxidative damage of the lung cancer model, but also has a very obvious repair effect on the oxidative damage caused by cisplatin. After medication, the SOD level reached the level of the blank group.

[0578] Table 67 Effect of the combination of cisplatin on the level of SOD in the kidney of mice with lung cancer (Mean±SD, n=8)

[0579]

[0580] * p<0.01, there was a significant difference compared with the model group.

[0581] ** p<0.01, there was a very significant difference compared with the model group.

[0582] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0583] 5.5.5 Effect of the combination of cisplatin and shampoxetine on the level of GSH in the kidneys of mice with lung cancer

[0584] As shown in Table 68, Fig.26 As shown, compared with the blank group, the GSH level in the model group was significantly decreased (P < 0.01), indicating that the kidneys of the Lewis lung cancer model in mice were damaged by oxidation.

[0585] Compared with the model group, the GSH level in the cisplatin group decreased significantly (P < 0.05), indicating that cisplatin aggravated the oxidative damage to the kidneys of mice with lung cancer. The GSH levels in the low-dose combination group, the low-dose cisplatin + combination group, the high-dose combination group, and the high-dose cisplatin + combination group increased significantly, and there were very significant differences compared with the model group and the cisplatin group (P < 0.01), indicating that the clinical dose of the combination repairs the oxidative damage to the kidney through GSH, not only has an effect on the oxidative damage to the kidneys of the lung cancer model, but also has a repairing effect on the oxidative damage to the kidneys caused by cisplatin, restoring the GSH content to normal levels.

[0586] Table 68 Effect of the combination of cisplatin on the level of GSH in the kidney of mice with lung cancer (Mean±SD, n=8)

[0587]

[0588] * p<0.01, there was a significant difference compared with the model group.

[0589] ** p<0.01, there was a very significant difference compared with the model group.

[0590] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0591] 5.5.6 Effect of the combination of cisplatin and shampoxetine on the level of MDA in the kidneys of mice with lung cancer

[0592] As shown in Table 69, Fig. 27 As shown, compared with the blank group, the MDA level in the model group was significantly increased (P < 0.01), indicating that the kidneys of the Lewis lung cancer model mice were oxidatively damaged.

[0593] Compared with the model group, the MDA level in the cisplatin group increased significantly (P < 0.01), indicating that cisplatin induced an increase in the content of lipid peroxidation products, causing oxidative damage to the kidneys of mice with lung cancer. The MDA levels in the low-dose and high-dose combination groups decreased significantly, significantly lower than those in the model group (P < 0.01), indicating that the combination can reduce the MDA level and improve the degree of kidney oxidative damage in the lung cancer model. After combining with cisplatin, the MDA level was significantly lower than that of cisplatin alone, indicating that the combination can improve the kidney oxidative damage of cisplatin-induced lung cancer model mice by reducing the content of MDA.

[0594] Table 69 Effect of the combination of cisplatin on the level of MDA in the kidney of mice with lung cancer (Mean±SD, n=8)

[0595]

[0596] ** p<0.01, there was a very significant difference compared with the model group.

[0597] ## p<0.01, compared with the cisplatin group, there was a very significant difference.

[0598] in conclusion

[0599] 1. Oxidative damage to the liver: After modeling, the levels of SOD and GSH decreased significantly, and MDA increased significantly, indicating that oxidative free radicals and oxidative products such as MDA were produced, consuming the antioxidants SOD and GSH. After administration, the SOD in the cisplatin group further decreased, and the MDA content further increased, indicating that the oxidative damage worsened. The low-dose group of the combination significantly increased SOD and GSH, and the high-dose group significantly reduced the MDA content, indicating that the low-dose combination exerts an antioxidant effect by stimulating the production of antioxidants, and the high-dose exerts its effect by directly eliminating oxidative products.

[0600] 2. Oxidative damage to the kidney: After modeling, the SOD and GSH contents were significantly reduced, and the MDA content was significantly increased. After administration, the SOD and GSH contents in the cisplatin group further decreased, and the MDA content further increased. The low-dose group and high-dose group of the composition increased the SOD content, and the effect of the high-dose group was more obvious. When used in combination with cisplatin, it had a reversal effect on the decrease in SOD content caused by cisplatin. Both the low-dose group and the high-dose group of the composition can increase the GSH content, and the low-dose effect is more obvious, and it has a reversal effect on the decrease in GSH content caused by cisplatin. Both the low-dose group and the high-dose group of the composition can significantly reduce the MDA content. After combined use with cisplatin, the MDA content is lower than when cisplatin is used alone. The results of kidney antioxidant effects show that the composition can play an antioxidant role at low doses and in combination with cisplatin.

[0601] In summary, by studying the synergistic and toxicity-reducing effects of the drug combination on cisplatin, the following conclusions can be drawn:

[0602] 1. Synergistic effect of the combination on cisplatin:

[0603] 1. The tumor inhibition rate of the combination of the combination and cisplatin far exceeds that of cisplatin alone, and the synergistic effect is obvious. At the same time, the spleen organ coefficient results show that weight gain is obvious and the immune system is activated.

[0604] 2. Immune-related indicators (CD4+, IL-2, IFN-Y, IgG, IgA) showed that the composition exerted anti-tumor effects by activating humoral immunity at low doses and exerted effects through cellular immunity at high doses. After combined use with cisplatin, the low-dose composition had a better effect in activating immune function than the high-dose group.

[0605] 2. The toxicity-reducing effect of the combination on cisplatin:

[0606] 1. Blood toxicity: The experimental results show that cisplatin significantly reduces blood routine related indicators (white blood cells, red blood cells, hemoglobin, platelets, lymphocytes, neutrophils), and has obvious blood toxicity. The combination and its combination with cisplatin can increase blood routine related indicators and have a significant improvement effect on the blood toxicity of cisplatin.

[0607] 2. Hepatotoxicity: Cisplatin significantly increased the liver function indicators ALT and AST, and significantly decreased the ALB content, indicating that cisplatin caused serious damage to the liver. The composition has the effect of repairing liver function. After combined use with cisplatin, ALT and AST decreased, and ALB increased. The high-dose combination of the composition and cisplatin had better effects than the low-dose group.

[0608] 3. Nephrotoxicity: Cisplatin significantly increases the levels of BUN and Cre, causing severe kidney damage. The combination can significantly reduce the levels of BUN and Cre. The combination of a high dose of the combination and cisplatin has the best effect.

[0609] 4. Oxidative damage: Cisplatin causes oxidative damage to the liver and kidneys, which is manifested by decreased SOD and GSH levels and increased MDA content, that is, a decrease in the content of antioxidants and an increase in the content of oxidation products. Oxidative damage aggravates liver and kidney toxicity. The combination has a significant antioxidant effect and can exert an antioxidant effect at a low dose. When used in combination with cisplatin, the antioxidant effect is outstanding.

[0610] Through all the indicators, it can be seen that the combination has a clear synergistic and toxicity-reducing effect on cisplatin, and the high and low doses show different performances among different indicators. However, through comprehensive comparison, a low dose of the combination can meet the synergistic and toxicity-reducing effect of combination with cisplatin.

Claims

1. A pharmaceutical composition comprising ginseng extract, coix seed extract and clematis tongguan truncatum extract.

2. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition consists of ginseng extract, coix seed extract and clematis tongguan truncatum extract.

3. The pharmaceutical composition according to claim 1 or 2, wherein The pharmaceutical composition is composed of 300-3000 parts of ginseng extract, 1700-15000 parts of coix seed extract and 1000-10000 parts of clematis tongguan tung extract in parts by mass; Preferably, the pharmaceutical composition consists of 1000-2000 parts of ginseng extract, 5000-10000 parts of coix seed extract and 3000-8000 parts of clematis tongguan tung extract in parts by mass; Preferably, the pharmaceutical composition consists of 1700 parts of ginseng extract, 8300 parts of coix seed extract and 5000 parts of clematis tongguan tung extract, by weight. Preferably, the pharmaceutical composition consists of 1,300 parts of ginseng extract, 6,700 parts of coix seed extract and 5,000 parts of clematis tongguan tung extract, calculated by weight.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The pharmaceutical composition further comprises one or more of the following: mannan peptide, berberine hydrochloride; Preferably, the pharmaceutical composition comprises ginseng extract, coix seed extract, clematis tongguan twig extract, mannan peptide, and berberine hydrochloride; Preferably, the pharmaceutical composition consists of ginseng extract, coix seed extract, clematis tongguan tung extract, mannan peptide, and berberine hydrochloride.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein In parts by mass, the pharmaceutical composition consists of 300-3000 parts of ginseng extract, 1700-15000 parts of coix seed extract, 1000-10000 parts of clematis tongguan teng extract, 20-160 parts of mannan peptide, and 7-35 parts of berberine hydrochloride; Preferably, the pharmaceutical composition is composed of 1000-2000 parts of ginseng extract, 5000-10000 parts of coix seed extract, 3000-8000 parts of clematis tongguan teng extract, 70-120 parts of mannan peptide, and 10-15 parts of berberine hydrochloride, in parts by mass; Preferably, the pharmaceutical composition consists of 1700 parts of ginseng extract, 8300 parts of coix seed extract, 5000 parts of clematis tongguan teng extract, 100 parts of mannan peptide, and 14.9 parts of berberine hydrochloride, in parts by mass; Preferably, the pharmaceutical composition consists of 1300 parts of ginseng extract, 6700 parts of coix seed extract, 5000 parts of clematis tongguan teng extract, 100 parts of mannan peptide, and 14.9 parts of berberine hydrochloride, calculated by mass.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein The pharmaceutical composition further comprises cisplatin or gefitinib; Preferably, the pharmaceutical composition further comprises 0.2-1.5 parts of cisplatin or 0.1-3 parts of gefitinib, by weight; Preferably, the pharmaceutical composition further comprises 0.5-1.0 parts of cisplatin or 0.3-1.0 parts of gefitinib, by weight; Preferably, the pharmaceutical composition further comprises 0.745 parts of cisplatin or 0.558 parts of gefitinib, calculated by weight.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein The pharmaceutical composition consists of ginseng extract, coix seed extract, clematis tongguan twig extract, mannan peptide, berberine hydrochloride, cisplatin or gefitinib.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein The pharmaceutical composition is composed of 300-3000 parts of ginseng extract, 1700-15000 parts of coix seed extract, 1000-10000 parts of clematis tongguan tung extract, 20-160 parts of mannan peptide, 7-35 parts of berberine hydrochloride, 0.2-1.5 parts of cisplatin or 0.1-3 parts of gefitinib in parts by mass; Preferably, the pharmaceutical composition consists of 1000-2000 parts of ginseng extract, 5000-10000 parts of coix seed extract, 3000-8000 parts of clematis tongguan tung extract, 70-120 parts of mannan peptide, 10-15 parts of berberine hydrochloride, 0.5-1.0 parts of cisplatin or 0.3-1.0 parts of gefitinib, in parts by mass.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein In parts by mass, the pharmaceutical composition consists of 1700 parts of ginseng extract, 8300 parts of coix seed extract, 5000 parts of clematis tongguan twig extract, 100 parts of mannan peptide, 14.9 parts of berberine hydrochloride, 0.745 parts of cisplatin or 0.558 parts of gefitinib; Preferably, the pharmaceutical composition consists of 1300 parts of ginseng extract, 6700 parts of coix seed extract, 5000 parts of clematis tongguan tung extract, 100 parts of mannan peptide, 14.9 parts of berberine hydrochloride, 0.745 parts of cisplatin or 0.558 parts of gefitinib, in parts by mass.

10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a drug for preventing and / or treating cancer; Preferably, the cancer is selected from small cell and non-small cell lung cancer, breast cancer, liver cancer, esophageal cancer, gastric cancer, testicular cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, bladder cancer, melanoma, sarcoma, head and neck tumors, etc., preferably small cell and non-small cell lung cancer.

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