Application of cistanche glycoside F in preparation of antitumor drugs

Abstract

The invention provides an application of cistanche glycoside F or pharmaceutically acceptable salts thereof in preparation of antitumor drugs. In-vitro experiments show that the cistanoside F has remarkable inhibitory activity on various tumor strains such as human lung cancer cells A549, human gastric adenocarcinoma cells AGS-CAS9, human liver cancer cells SK-HEP-1, human bladder cancer cells RT112, human cervical cancer cells Hela, human prostate cancer cells PC-3, human neuroblastoma cells SH-SY5Y and the like. Animal tests show that cistanche glycoside F has a good in-vivo anti-tumor effect and has a remarkable treatment effect on various tumors such as bladder cancer and liver cancer. The cistanche glycoside F is high in anti-tumor activity and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and relates to an anti-tumor compound and a use thereof, and specifically to the medical use of cistancheside F in the preparation of anti-tumor drugs. Background Art

[0002] Malignant tumor is one of the major diseases that seriously threatens human health and life.

[0003] Bladder cancer is one of the most common malignant tumors of the urinary system. The common clinical manifestations of bladder cancer are painless hematuria, followed by frequent urination, urgency, pain, dysuria, low back pain, etc. Its diagnosis mainly relies on cystoscopy, renal function assessment, and upper urinary tract imaging (including urinary system color Doppler ultrasound, CT, MRI, angiography, etc.). Currently, modern medicine divides it into non-muscle-invasive bladder cancer and muscle-invasive bladder cancer. 70%-80% of patients with newly diagnosed bladder cancer show non-muscle-invasive disease, and their treatment mainly includes transurethral bladder tumor resection and postoperative intravesical chemotherapy; the treatment of muscle-invasive bladder cancer mainly includes radical cystectomy and related neoadjuvant chemotherapy.

[0004] Although China has achieved certain results in the prevention and treatment of liver cancer through the promotion of hepatitis B vaccines in recent years, according to statistics, the incidence of liver cancer in my country is still only lower than lung cancer, colorectal cancer, and gastric cancer, and the mortality rate is second only to lung cancer. Therefore, the treatment of liver cancer still occupies an important position. At present, there are many treatment methods according to the different stages of liver cancer development, such as surgery, liver transplantation, radiotherapy, chemotherapy and traditional Chinese medicine treatment.

[0005] Although the emergence of new drugs and treatments in the past 10 years has improved the survival rate of cancer patients, cancer is still the second leading cause of death in the world. Currently, chemotherapy is the main method of cancer treatment. However, chemotherapy drugs have low specificity. While killing tumor cells, they also affect the biological functions of normal cells and produce "off-target" toxicity. In addition, chemotherapy drugs are prone to drug resistance, which will significantly reduce the effectiveness of cancer treatment.

[0006] Cistanoside F (CF) is a phenylethanol glycoside compound existing in nature. It is currently mainly derived from traditional Chinese medicines with kidney-tonifying effects, such as Cistanche deserticola, Rehmannia glutinosa, Scrophularia ningpoensis, and Ophiopogon japonicus.

[0007] Ma Mengling (Ma Mengling, Tang Zeling, Zhu Weiliang, et al. Cistancheside F inhibits adipogenic differentiation of C2C12 cells via AMPK / ACC pathway [J]. Journal of Traditional Chinese Medicine, 2023, 34(12): 2822-2825) found that Cistancheside F may inhibit adipogenic differentiation of C2C12 cells by regulating the AMPK / ACC pathway. Chinese patent CN116966193A discloses the medical use of Cistancheside F, specifically the use of Cistancheside F in the preparation of drugs for preventing and treating skeletal muscle atrophy, which can be used to prevent and treat skeletal muscle atrophy.

[0008] At present, there is no report on the use of Cistancheside F in the preparation of anti-tumor drugs. Summary of the invention

[0009] The object of the present invention is to provide the use of Cistancheside F or its pharmaceutically acceptable salt in the preparation of anti-tumor drugs. The Cistancheside F or its pharmaceutically acceptable salt of the present invention has a broad anti-tumor spectrum, high anti-tumor activity and high safety.

[0010] In order to achieve the above-mentioned purpose of the invention, the following technical solutions are provided:

[0011] The structural formula of Cistancheside F is as follows:

[0012]

[0013] The present invention provides use of cistancheside F or a pharmaceutically acceptable salt thereof in preparing a drug for preventing and / or treating tumors.

[0014] In vitro experiments showed that the IC of Cistanche deserticola F of the present invention on 7 human tumor cells was 50 The mean range is: 0.73-4.55μg / ml, among which the IC values ​​for human bladder cancer cell RT112, human liver cancer cell SK-HEP-1, and human lung cancer cell A549 are 50 The value is the lowest.

[0015] The results of in vivo tests show that the Cistancheside F of the present invention has a high tumor inhibition rate on RT112 human bladder cancer and SK-HEP-1 human liver cancer. Cistancheside F has a good in vivo anti-tumor effect.

[0016] Based on this, the present invention requests to protect the use of Cistancheside F in the preparation of anti-tumor drugs.

[0017] Preferably, the tumor is lung cancer, gastric adenocarcinoma, liver cancer, bladder cancer, cervical cancer, prostate cancer, or neuroblastoma.

[0018] More preferably, the tumor is bladder cancer or liver cancer.

[0019] The administration routes of the Cistancheside F or its pharmaceutically acceptable salt described in the present invention include, but are not limited to, oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0020] Preferably, the administration route is oral administration.

[0021] For oral administration, the pharmaceutical composition can be prepared by mixing Cistancheside F with a pharmaceutically acceptable carrier well known in the art. These carriers enable the compound of the present invention to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients. For example, a pharmaceutical composition for oral administration can be obtained in the following manner: the active ingredient is combined with one or more solid carriers, the resulting mixture is granulated if necessary, and a small amount of excipients is added if necessary to process into a mixture or granules to form a tablet or a core. The core can be combined with an optionally enteric coating material to be processed into a coated preparation form that is more conducive to absorption by an organism (e.g., a human).

[0022] A pharmaceutical composition, characterized in that it comprises cistancheside F or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0023] The beneficial effects of the present invention are:

[0024] The invention discloses the anti-tumor activity of Cistanche deserticola F. In vitro experiments show that Cistanche deserticola F has significant inhibitory activity on various tumor strains such as human lung cancer cells A549, human gastric adenocarcinoma cells AGS-CAS9, human liver cancer cells SK-HEP-1, human bladder cancer cells RT112, human cervical cancer cells Hela, human prostate cancer cells PC-3, and human neuroblastoma cells SH-SY5Y, and inhibits tumor cell proliferation. 50 The value is low.

[0025] Animal experiments have shown that Cistancheside F has a good anti-tumor effect in vivo and has significant therapeutic effects on various tumors such as bladder cancer and liver cancer. Cistancheside F has strong anti-tumor activity and has broad application prospects.

[0026] Definition of terms

[0027] In the present invention, unless otherwise specified, the terms used have the following meanings:

[0028] The term "pharmaceutically acceptable" means that salts, solvents, excipients, etc. are generally non-toxic, safe, and suitable for use by patients. The "patient" is preferably a mammal, more preferably a human.

[0029] The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound of the present invention and a relatively nontoxic, pharmaceutically acceptable acid. When the compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting a neutral form of such compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acid includes an inorganic acid, including but not limited to hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, phosphorous acid, sulfuric acid, hydrogen sulfate, and the like. The pharmaceutically acceptable acid includes organic acids, including but not limited to acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, bitartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthoic acid)), amino acids (e.g., glutamic acid, arginine), etc. When the compound of the present invention contains a relatively basic functional group, it can be converted into an acid addition salt.

[0030] The term "treatment" as used herein means any treatment of a disease in a mammal, including: (1) preventing the disease, i.e., causing clinical symptoms of the disease not to develop; (2) inhibiting the disease, i.e., preventing the development of clinical symptoms; and (3) alleviating the disease, i.e., causing the regression of clinical symptoms.

[0031] As used herein, "prevention" refers to a reduction in the risk of acquiring or developing a disease or disorder.

[0032] The "pharmaceutical composition" of the present invention refers to a preparation of one or more compounds of the present invention or their salts and a carrier generally accepted in the art for delivering biologically active compounds to an organism (e.g., human). The purpose of a pharmaceutical composition is to facilitate administration and delivery to an organism.

[0033] The term "pharmaceutically acceptable carrier" refers to a substance that is co-administered with an active ingredient and is conducive to the administration of the active ingredient, including but not limited to any glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that is acceptable for human or animal (e.g. livestock) and approved by the State Food and Drug Administration. Examples include but are not limited to calcium carbonate, calcium phosphate, various sugars and various starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycol. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1The inhibitory activity of the Cistanche deserticola F on tumor cells (IC 50 value, μg / ml). DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to fully understand the present invention, the present invention is further described below through specific examples, but those skilled in the art should be aware that the examples of the present invention do not limit the present invention in any way.

[0036] Example 1 In vitro antitumor test of the compounds of the present invention

[0037] In vitro experiments are mainly used to screen candidate compounds, to gain a preliminary understanding of the sensitive tumor types and action strength of the test compound, and to provide a reference for subsequent in vivo experiments. This experiment referred to the "Guidelines for Nonclinical Studies of Cytotoxic Antitumor Drugs", selected human tumor cells, and evaluated the in vitro antitumor effect of Cistancheside F, to gain a preliminary understanding of the antitumor effect strength of Cistancheside F, and to provide a reference for subsequent in vivo experiments.

[0038] 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt

[0039] The anti-tumor activity of the compounds of the present invention was tested by colorimetry (CCK-8 method).

[0040] Instruments: clean bench (SW-CJ-1FD, AIRTECH, Sujing Antai), constant temperature CO2 incubator (3111, Thermo, USA), inverted biological microscope (IX71, OLYMPUS, Japan), enzyme-linked immunosorbent assay (Model 680, BIO-RAD, USA), flat shaker (Kylin-bell lab Instruments), high pressure sterilizer (YXO.SG41.280, Shanghai Huaxian), centrifuge (SIGMA).

[0041] Reagents: DMEM (GIBCO), fetal bovine serum (GIBCO), trypsin (SIGMA), DMSO (SIGMA).

[0042] 1.1 Experimental Materials

[0043] Cell lines: human lung cancer cells A549, human gastric adenocarcinoma cells AGS-CAS9, human liver cancer cells SK-HEP-1, human bladder cancer cells RT112, human cervical cancer cells Hela, human prostate cancer cells PC-3, and human neuroblastoma cells SH-SY5Y.

[0044] 1.2 Experimental methods

[0045] Take cells in logarithmic growth phase and adjust the cell concentration to 4.0×10 4 / ml, inoculated in a 96-well plate, 100μl per well, about 4,000 cells / well. After 24 hours of incubation in the incubator, the complete medium was replaced with 100μl of complete medium containing different concentrations of drugs. 100μl of complete medium without drugs was added to each well of the control group. Three replicates were set for the control group and the drug group. In addition, a blank group (no cells but only culture medium) was set as the zeroing group. After 48 hours of incubation in a 37°C, 5% CO2 incubator, 10μl of CCK-8 solution was added to each well. After continuing to culture for 1.5 hours, the OD value was measured at 450nm by an enzyme reader.

[0046] Cell inhibition rate % = [(OD value of drug administration group blank - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%.

[0047] IC was calculated according to Koch's modified formula 50 Value (μg / ml).

[0048] 1.3 Results Figure 1 .

[0049] The IC50 average range of Cistanoside F (CF) of the present invention for 7 human tumor cells is: 0.73-4.55 μg / ml, among which the IC50 for human bladder cancer cell RT112, human liver cancer cell SK-HEP-1, and human lung cancer cell A549 is the highest. 50 The value is the lowest.

[0050] Example 2 Inhibitory effect of Cistanche deserticola F of the present invention on human bladder cancer cell RT112 tumor growth

[0051] Animal in vivo tests reflect the comprehensive results of drug efficacy and toxicity, and are the decisive link in the drug screening process that cannot be replaced by in vitro detection methods and models. The transplanted tumor model is currently the most studied tumor model. It is easy to manipulate artificially, and the animal tumor formation is stable, uniform, and has a high incidence rate. The difference in tumor formation time is not large, and it is easy to add interference factors. In vivo tests usually use animal tumor transplantation models and human cancer xenograft tumor models.

[0052] 1 Experimental animals and materials

[0053] 1.1 SPF BALB / c nude mice, male, 18-22g, 48 pieces.

[0054] 1.1 Human bladder cancer cell RT112 was purchased from the Wuhan University Collection Center and preserved by the College of Life Sciences of Inner Mongolia Agricultural University.

[0055] 2 Establishment of RT112 bladder cancer mouse model

[0056] 2.1 Take out the frozen RT112 cell line from liquid nitrogen and shake it in a 40℃ water bath to dissolve it quickly. Then centrifuge at 800rpm for 3min, spray the outer surface of the cryopreservation tube with 75% alcohol, and move the cryopreservation tube into the clean bench. Discard the supernatant and inoculate the cells in a 10cm cell culture dish with complete culture medium (DMEM culture medium containing 10% FBS) for subculture. Before inoculation, digest the cells with trypsin (0.25% trypsin + 0.02% EDTA), blow them apart, suspend them with serum-free DMEM, and adjust the cell concentration to 1.0×10 7 The cells / mL were inoculated subcutaneously in the right axilla of BALB / c nude mice (0.2 mL / mouse).

[0057] 2.2 Grouping and Dosing

[0058] There were 8 animals in each group.

[0059]

[0060]

[0061] 3. Detection indicators:

[0062] 3.1 General observation: Observe the animal behavior, reaction, diet and death after administration, and record the body weight every other day.

[0063] 3.2 Tumor weight and tumor transplantation rate (%): After blood collection, the mice were killed by cervical dislocation, the tumor was removed and weighed on an analytical balance, the tumor weight was recorded, and the tumor inhibition rate (%) was calculated according to the following formula.

[0064] Tumor inhibition rate (%) = (tumor weight of model group - tumor weight of drug-treated group) / tumor weight of model group × 100%.

[0065] 3.3 Effects on immune organs

[0066] Take out the thymus and spleen, weigh them on an analytical balance, record the organ weight, and calculate the organ index according to the following formula.

[0067] Organ coefficient = organ weight (mg) / body weight (10g).

[0068] 4 The test results were expressed as mean ± standard deviation, and the t test was used for comparison between groups.

[0069] 5 Results

[0070] 5.1 General Observations

[0071] As can be seen from the table below, compared with the model group, the body weight of mice in the Cistancheside F group, the echinacoside group, and the verbascoside group were significantly reduced, which was statistically significant.

[0072] Compared with the model group, the tumor weights of mice in the Cistancheside F group, echinacoside group, and verbascoside group were significantly reduced, which was statistically significant; but at the same dose, the tumor inhibition effects of the echinacoside group and verbascoside group were weaker than that of the Cistancheside F group.

[0073] Table 1 Effects of Cistancheside F of the present invention on body weight, tumor weight and tumor inhibition rate of RT112 bladder cancer-bearing mice

[0074]

[0075] * Compared with the model group, P<0.05, # P and Cistancheside F-high dose group compared with P < 0.05.

[0076] 5.2 Effects of Cistanche deserticola F on organ indexes of RT112 bladder cancer-bearing mice

[0077] Table 2 Effect of Cistanche deserticola F of the present invention on thymus index and spleen index of RT112 bladder cancer-bearing mice

[0078]

[0079] * Compared with the model group, P<0.05, # P and Cistancheside F-high dose group compared with P < 0.05.

[0080] Compared with the model group, the thymus index and spleen index of mice in the Cistancheside F group, echinacoside group, and verbascoside group were significantly reduced, which was statistically significant; but at the same dose, the thymus index and spleen index of mice in the echinacoside group and verbascoside group were higher than those in the Cistancheside F group.

[0081] The test results show that the Cistanoside F (CF) of the present invention has a high tumor inhibition rate on RT112. Compared with echinacoside and verbascose, Cistanoside F shows better in vivo anti-tumor effect.

[0082] Example 3 Inhibitory effect of Cistanche deserticola F of the present invention on human liver cancer cell SK-HEP-1 tumor growth

[0083] 1 Experimental animals and materials

[0084] 1.1 SPF BALB / c nude mice, male, 18-22g, 48 pieces.

[0085] 1.1 Human hepatoma cell line SK-HEP-1 was purchased from the Wuhan University Collection Center and preserved by the College of Life Sciences of Inner Mongolia Agricultural University.

[0086] 2 Establishment of SK-HEP-1 liver cancer tumor-bearing mouse model

[0087] 2.1 SK-HEP-1 cells cultured in RPMI-1640 medium (containing 10% FBS) were digested with trypsin (0.25% trypsin + 0.02% EDTA), dispersed with serum-free RPMI-1640 medium, and the cell concentration was adjusted to 7.5×10 6 The cells / mL were inoculated subcutaneously in the right axilla of BALB / c nude mice (0.2 mL / mouse).

[0088] 2.2 Grouping and Dosing

[0089] There were 8 animals in each group.

[0090]

[0091]

[0092] 3. Detection indicators:

[0093] 3.1 General observation: Observe the animal behavior, reaction, diet and death after administration, and record the body weight every other day.

[0094] 3.2 Tumor weight and tumor transplantation rate (%): After blood collection, the mice were killed by cervical dislocation, the tumor was removed and weighed on an analytical balance, the tumor weight was recorded, and the tumor inhibition rate (%) was calculated according to the following formula.

[0095] Tumor inhibition rate (%) = (tumor weight of model group - tumor weight of drug-treated group) / tumor weight of model group × 100%.

[0096] 3.3 Effects on immune organs

[0097] Take out the thymus and spleen, weigh them on an analytical balance, record the organ weight, and calculate the organ index according to the following formula.

[0098] Organ coefficient = organ weight (mg) / body weight (10g).

[0099] 4 The test results were expressed as mean ± standard deviation, and the t test was used for comparison between groups.

[0100] 5 Results

[0101] 5.1 General Observations

[0102] As can be seen from the table below, compared with the model group, the body weight of mice in the Cistancheside F group, the echinacoside group, and the verbascoside group were significantly reduced, which was statistically significant.

[0103] Compared with the model group, the tumor weights of mice in the Cistancheside F group, echinacoside group, and verbascoside group were significantly reduced, which was statistically significant; but at the same dose, the tumor inhibition effects of the echinacoside group and verbascoside group were weaker than that of the Cistancheside F group.

[0104] Table 3 Effects of Cistancheside F of the present invention on body weight, tumor weight and tumor inhibition rate of SK-HEP-1 liver cancer-bearing mice

[0105]

[0106] * Compared with the model group, P<0.05, # P and Cistancheside F-high dose group compared with P < 0.05.

[0107] 5.2 Effects of Cistanche deserticola F on organ indexes of SK-HEP-1 liver cancer-bearing mice

[0108] Table 4 Effect of Cistanche deserticola F of the present invention on thymus index and spleen index of SK-HEP-1 liver cancer-bearing mice

[0109]

[0110] * Compared with the model group, P<0.05, # P and Cistancheside F-high dose group compared with P < 0.05.

[0111] Compared with the model group, the thymus index and spleen index of mice in the Cistancheside F group, echinacoside group, and verbascoside group were significantly reduced, which was statistically significant; but at the same dose, the thymus index and spleen index of mice in the echinacoside group and verbascoside group were higher than those in the Cistancheside F group.

[0112] The test results show that the Cistancheside F of the present invention has a high tumor inhibition rate on SK-HEP-1. Compared with echinacoside and verbascoside, Cistancheside F shows better in vivo anti-tumor effect.

[0113] The above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description, and the obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. Use of Cistancheside F or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating tumors.

2. The use according to claim 1, characterized in that: The tumor is lung cancer, gastric adenocarcinoma, liver cancer, bladder cancer, cervical cancer, prostate cancer or neuroblastoma.

3. The use according to claim 1, characterized in that: The tumor is bladder cancer.

4. The use according to claim 1, characterized in that: The tumor is liver cancer.

5. The use according to claim 1, characterized in that: The administration route of the Cistancheside F or its pharmaceutically acceptable salt is oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, or intravenous administration.

6. The use according to claim 5, characterized in that: The administration route is oral administration.

7. A pharmaceutical composition, characterized in that It comprises the Cistancheside F or a pharmaceutically acceptable salt thereof as claimed in claim 1, and a pharmaceutically acceptable carrier.

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