Traditional Chinese medicine composition for treating malignant tumors as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention provides a traditional Chinese medicine composition for treating malignant tumors as well as a preparation method and application thereof, and is characterized in that the traditional Chinese medicine composition comprises verdigris and Chinese gall leaven in a mass ratio of 1: 10; or the traditional Chinese medicine composition comprises copper gluconate and Chinese gall leaven according to the mass ratio of 1: 5. According to the traditional Chinese medicine composition provided by the invention, the Chinese gall leaven has an active component which forms a complex with copper ions, so that the Chinese gall leaven is compatible with the verdigris or the copper gluconate, and the active component of the Chinese gall leaven can chelate the copper ions contained in the verdigris or the copper gluconate, so that the copper ions contained in the verdigris or the copper gluconate can be removed; and carrying out a coordination chemical reaction to form the copper complex with an anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine pharmacy, and particularly to a traditional Chinese medicine composition for treating malignant tumors, its preparation method and uses. Background Art

[0002] Malignant tumors are one of the diseases that seriously threaten human health globally. Current treatment methods mainly include surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy. However, various currently used anti-tumor drugs generally have problems such as large toxic and side effects and strong drug resistance, which limit their clinical application effects. Therefore, finding safer and more efficient anti-tumor drugs has become one of the key research directions currently.

[0003] Traditional Chinese medicine has a long history in the field of tumor treatment and has shown good curative effects in clinical applications. Research shows that a variety of traditional Chinese medicines and their active ingredients can play anti-tumor roles through mechanisms such as regulating the immune system, inhibiting tumor cell proliferation, and inducing apoptosis.

[0004] In recent years, the modernization and standardization process of traditional Chinese patent medicines has accelerated, and a variety of anti-tumor traditional Chinese medicine formulas have entered the clinic in the form of traditional Chinese patent medicines, such as Xihuang Pills, Compound Taxus Capsules, Compound Cantharidin Capsules and Fuzheng Xiaoji Granules, etc., which have shown outstanding performance in helping patients control tumor progression, improve quality of life, and reduce adverse reactions of radiotherapy and chemotherapy. However, currently, anti-tumor traditional Chinese patent medicines on the market still have problems such as weak targeting and unstable curative effects. Inventing and creating new anti-tumor traditional Chinese medicine formulas has become a key link in anti-tumor traditional Chinese medicine.

[0005] Copper complexes have always attracted the attention of medical researchers due to their unique anti-tumor mechanisms. Research has found that copper complexes can not only damage tumor cell DNA and proteins, but also induce the "copper death" mechanism, that is, after copper ions accumulate in tumor cells, they interfere with mitochondrial function, trigger oxidative stress and lead to cell apoptosis. This mechanism has great application potential for drug-resistant tumors.

[0006] Traditional Chinese medicine traditional medications include various copper drugs, such as malachite (mainly composed of basic copper carbonate) and chalcanthite (mainly composed of copper sulfate), both of which are included in the Chinese Pharmacopoeia and widely used. However, the toxicity of copper drugs limits their direct use, so it is necessary to find a suitable compatibility method to reduce toxicity and enhance curative effects. Based on the theory of "sovereign, ministerial, adjuvant, and guiding" in traditional Chinese medicine, by reasonably compatibly using traditional Chinese medicines and copper drugs, copper toxicity can be reduced and anti-tumor activity can be improved, providing new ideas for the development of new anti-tumor traditional Chinese patent medicines. Summary of the Invention

[0007] Aiming at the defects of the prior art, the purpose of the present invention is to provide a traditional Chinese medicine composition for treating malignant tumors and its preparation method.

[0008] The technical solution of the present invention is as follows: In the first aspect of the present invention, a traditional Chinese medicine composition for treating malignant tumors is provided. The traditional Chinese medicine composition includes malachite green and myrobalan fruit with a mass ratio of 1:10; alternatively, the traditional Chinese medicine composition includes copper gluconate and myrobalan fruit with a mass ratio of 1:5.

[0009] Further preferably, the traditional Chinese medicine composition further includes malachite green, myrobalan fruit, propolis, lacca, frankincense, and myrrh with a mass ratio of 1:10:6:2:2:2.

[0010] In the second aspect of the present invention, a preparation method of the above traditional Chinese medicine composition is provided. The preparation method includes the following steps: S1. Grind malachite green, myrobalan fruit, propolis, lacca, frankincense, and myrrh into powder respectively, sieve them, and store for later use; S2. Weigh the malachite green and myrobalan fruit in step S1 according to the ratio, add water, mix well and put them into a container. Place the container in a pressure cooker, heat at 100 - 120 °C for 30 minutes, take out the container after cooling, and mix the medicine in the container to obtain a black paste; S3. Mix the black paste in step 2 with the propolis, lacca, frankincense, and myrrh in step S1 added according to the ratio, mix well and put them into a container. Place the container in the pressure cooker again, heat at 100 - 120 °C for 30 minutes, take out after cooling, and stir evenly again to obtain a traditional Chinese medicine composition for treating malignant tumors; Alternatively, the preparation method includes the following steps: S11. Grind copper gluconate and myrobalan fruit into powder respectively, sieve them, and store for later use; S22. Weigh the copper gluconate and myrobalan fruit in step S1 according to the ratio, add water, mix well and put them into a container. Place the container in a pressure cooker, heat at 100 - 120 °C for 30 minutes, take out the container after cooling, and mix the medicine in the container to obtain a black paste, and the black paste is the traditional Chinese medicine composition for treating malignant tumors.

[0011] Further preferably, in step 1, after grinding the malachite green, myrobalan fruit, propolis, lacca, frankincense, and myrrh into powder, sieve them through a 100 - mesh sieve and store for later use; alternatively, in S11, after grinding the copper gluconate into powder, sieve it through a 100 - mesh sieve and store for later use.

[0012] Further preferably, the heating temperature in the pressure cooker is 120 °C.

[0013] Further preferably, in step S3, after adding conventional excipients to the obtained traditional Chinese medicine composition for treating malignant tumors, it is prepared into a clinically acceptable preparation according to the conventional process.

[0014] Further preferably, the preparation includes at least one of ointment, capsule, granule, external patch, injection, mixture, pill.

[0015] Further preferably, the preparation is an ointment / pill.

[0016] Further preferably, the dosage of the pill is 100 - 10000 mg; the dosage of the ointment is 10 - 10000 mg.

[0017] In the third aspect of the present invention, there is provided the above-mentioned traditional Chinese medicine composition for treating malignant tumors or the traditional Chinese medicine composition for treating malignant tumors prepared by the above-mentioned preparation method, which has at least the following uses: a. Use in the preparation of drugs for treating oral cancer; b. Use in the preparation of drugs for treating pancreatic cancer.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. According to the theory of monarch, minister, assistant and guide in traditional Chinese medicine prescription science and combined with the principle of modern traditional Chinese medicine coordination chemistry, the traditional Chinese medicine composition provided by the present invention selects malachite (or copper gluconate instead of malachite) as the monarch drug, myrobalan fruit as the minister drug, and then selects propolis, lacca, frankincense and myrrh as the assistant and guide drugs to form a traditional Chinese medicine composition for treating malignant tumors. Myrobalan fruit has the active ingredient gallic acid that can form a complex with copper ions. When myrobalan fruit is compatible with malachite or copper gluconate, the active ingredient of myrobalan fruit can complex with the copper ions contained in malachite or copper gluconate, and a coordination chemical reaction occurs to form a copper complex with anti-tumor effect. The results of cell experiments confirm that the traditional Chinese medicine composition provided by the present invention has a significant anti-tumor effect; the results of animal experiments confirm that using the traditional Chinese medicine composition provided by the present invention has no obvious toxicity and can effectively inhibit the growth of transplanted tumors in animals.

[0019] 2. The traditional Chinese medicine composition for treating malignant tumors provided by the present invention has reasonable compatibility, synergizes with each other, has clear medicinal effects, simple composition, and low medication cost. The preparation method of the traditional Chinese medicine composition for treating malignant tumors provided by the present invention is simple to operate and can be applied to industrial production, providing a new medication option for the clinical treatment of oral cancer and pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] By reading the detailed description of the non-restrictive embodiments with reference to the following drawings, other features, purposes and advantages of the present invention will become more obvious: Figure 1 It is a schematic diagram showing the results of the inhibition of cell viability by the Wujin Huacai Pills provided in the experimental examples of the present invention ( Figure 1 In A: After culturing with cell culture media containing different concentrations of Wujin Huacai Pills for 48 h, the cell state display diagram; Figure 1In B: Bar chart comparing the data results of the inhibitory rate of Wujin Huaiwan on the viability of human pancreatic cancer SW1990 cells after culturing human pancreatic cancer SW1990 cells in cell culture media containing different concentrations of Wujin Huaiwan for 48 hours; Figure 1 In C: Bar chart comparing the data results of the inhibitory rate of Wujin Huaiwan on the viability of human oral cancer CAL27 cells after culturing human oral cancer CAL27 cells in cell culture media containing different concentrations of Wujin Huaiwan for 48 hours); Figure 2 Schematic diagram showing the results of the inhibitory effect of Baoyaojian and copper green on cell viability provided by the experimental examples of the present invention ( Figure 2 In A: Diagram showing the state of cells after culturing cells in a medium containing Baoyaojian and a medium containing copper green for 48 hours; Figure 2 In B: Bar chart comparing the data results of the inhibitory rate of a medium containing Baoyaojian and a medium containing copper green on the viability of human pancreatic cancer SW1990 cells after culturing human pancreatic cancer SW1990 cells for 48 hours; Figure 2 In C: Bar chart comparing the data results of the inhibitory rate of a medium containing Baoyaojian and a medium containing copper green on the viability of human oral cancer CAL27 cells after culturing human oral cancer CAL27 cells for 48 hours); Figure 3 Physical display diagram of oral squamous cell carcinoma tumors in the control group and the experimental group on the 22nd day after intragastric administration of Wujin Huaiwan to mice provided by the experimental examples of the present invention (Control group: Mice bearing oral squamous cell carcinoma xenografts were intragastrically administered with normal saline; Experimental group: Mice bearing oral squamous cell carcinoma xenografts were intragastrically administered with Wujin Huaiwan for treatment); Figure 4 Physical display diagram of oral squamous cell carcinoma tumors in the control group and the experimental group on the 22nd day after intragastric administration of Baoyaojian combined with copper gluconate paste to mice provided by the experimental examples of the present invention (Control group: Mice bearing oral squamous cell carcinoma xenografts were intragastrically administered with normal saline; Experimental group: Mice bearing oral squamous cell carcinoma xenografts were intragastrically administered with Baoyaojian combined with copper gluconate paste for treatment); Figure 5 Schematic diagram of the mass spectrometry results of the presence of gallic acid-copper complex in mice after treatment with Wujin Huaiwan provided by the experimental examples of the present invention. Detailed implementation mode

[0021] The present invention will be described in detail below with reference to specific embodiments.

[0022] The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several changes and improvements can still be made, and these all belong to the protection scope of the present invention.

[0023] First embodiment This embodiment provides a traditional Chinese medicine composition for treating malignant tumors and a preparation method thereof.

[0024] In this embodiment, the traditional Chinese medicine composition for treating malignant tumors includes copper green, Baiyaojian, propolis, lithospermum officinale, frankincense, and myrrh in a mass ratio of 1:10:6:2:2:2.

[0025] In this embodiment, the monarch drug is albino apatinol, and the minister drug is Baiyaojian. Baiyaojian has an active ingredient that forms a complex with copper. Baiyaojian is compatible with albino apatinol. The active ingredient of Baiyaojian can chelate the copper ions contained in albino apatinol. Baiyaojian and albino apatinol form a copper complex (gallic acid-copper complex) with anti-tumor effect through a Chinese medicine coordination chemical reaction. According to traditional Chinese medicine, albino apatinol is used as a monarch drug for detoxification and anti-cancer. Baiyaojian helps its anti-cancer effect and also reduces the toxicity of albino apatinol, and is used as a minister. Cancer is an accumulation of blood. Frankincense and myrrh promote blood circulation and break up blood. Lithospermum erythrorhizon is astringent and stops bleeding. The three drugs enter the blood together and also have the function of guiding the meridians and reporting the symptoms. Propolis clears away heat and detoxifies, eliminating the ulcer symptoms caused by cancer. Therefore, Lithospermum erythrorhizon, frankincense, myrrh, and propolis are all adjuvants.

[0026] In this embodiment, the preparation method of the Chinese medicine composition for treating malignant tumors comprises the following steps: S1. Use a mortar to grind the verdigris into a fine powder, use a high-speed grinder to grind the Baiyaojian, lithospermum, frankincense, and myrrh into powder, sieve with a 100-mesh sieve, and store in aliquots for later use; S2. Weigh 10g of copper green and 100g of Baiyaojian, add 100mL of water, mix well and place in a ceramic pot. Cover the pot and place it in a pressure cooker at 120°C for 30 minutes. After cooling, remove the pot and continue to mix the drug in the pot to obtain a black paste. S3. The black paste in step S2 is mixed with 60g propolis, 20g lithospermum, 20g frankincense, and 20g myrrh, and then placed in a ceramic pot. The ceramic pot is placed in a pressure cooker again, heated at 120°C for 30 minutes, cooled, taken out, and stirred evenly to obtain a traditional Chinese medicine composition for treating malignant tumors.

[0027] In this embodiment, the container for preparing the Chinese medicine composition can be a ceramic pot or other high-temperature resistant containers. Metal containers should be avoided to prevent metal ions from complexing with the gallic acid contained in Baiyaojian and interfering with the effect of verdigris. Those skilled in the art can freely choose according to actual conditions, and no other restrictions are made in this embodiment.

[0028] Baiyaojian is a block made from the fermentation of gallnut, distiller's grains or koji, and tea leaves. Gallnut (Gallachinensis, GC), also known as clam, hundred insect warehouse, and wood aconite, is a gall formed by aphids parasitizing on the leaves of host plants such as red bran poplar, green bran poplar, and saltwort.

[0029] In this embodiment, the Chinese gallnut can also be replaced by Chinese gall, but the dosage of Chinese gall is twice that of the Chinese gallnut. This embodiment preferably uses the Chinese gallnut.

[0030] Second Embodiment This embodiment provides a traditional Chinese medicine composition preparation and its preparation method.

[0031] In this embodiment, the traditional Chinese medicine composition provided by the first embodiment is selected and prepared into pills, and the pills in this embodiment are named "Wujin Huaiwan Pills". The preparation method is as follows: S1. Use a mortar to grind malachite into fine powder, use a high-speed pulverizer to grind the Chinese gallnut, gromwell, frankincense, and myrrh into fine powders respectively, sieve them with a 100-mesh sieve, store them separately, and reserve for use; S2. Weigh 10 g of malachite and 100 g of Chinese gallnut, add 100 mL of water, mix well and put them into a clay pot. After covering the pot, place the clay pot in a pressure cooker, heat at 120 °C for 30 minutes. After cooling, take out the clay pot and continue to mix the medicines in the pot to obtain a black paste; S3. After mixing the black paste in step S2 with 60 g of propolis, 20 g of gromwell, 20 g of frankincense, and 20 g of myrrh, put them into a clay pot, place the clay pot in the pressure cooker again, heat at 120 °C for 30 minutes, take it out after cooling, and stir evenly to obtain a traditional Chinese medicine composition for treating malignant tumors.

[0032] S4. Knead the traditional Chinese medicine composition for treating malignant tumors obtained in step S3 into thin strips, divide them into 100 portions, knead them into pill shapes, and dry them in an oven at 45 °C for 48 hours, or dry them in natural ventilation conditions for 1 week to obtain clinically available traditional Chinese medicine pills, namely "Wujin Huaiwan Pills".

[0033] For the traditional Chinese medicine composition for treating malignant tumors obtained in step S3, adding conventional excipients such as honey, starch, dextrin, etc., various clinically acceptable preparations can be prepared according to conventional processes, including but not limited to paste agents, capsules, granules, external patches, injections, mixtures, pills. In this embodiment, the pill form is specifically used for illustration. Those skilled in the art can flexibly select other dosage forms according to clinical needs. This embodiment does not limit the selection of specific dosage forms.

[0034] Third Embodiment This embodiment provides a traditional Chinese medicine composition for treating malignant tumors and its preparation method.

[0035] In this example, copper gluconate is used instead of malachite to make a paste agent with the Chinese gallnut. Copper gluconate is a clinically approved copper supplement, which is more soluble and has lower toxicity than malachite. The copper ions it contains can also be complexed with gallic acid contained in the Chinese gallnut to form gallic acid-copper chelate, reflecting the application thinking of the modernization of traditional Chinese medicine.

[0036] In this embodiment, copper gluconate replaces apperurus as the main drug, and the auxiliary drug is Baiyaojian. Baiyaojian has an active ingredient that forms a complex with copper. Baiyaojian is combined with copper gluconate. The active ingredient of Baiyaojian can chelate the copper ions contained in copper gluconate. Baiyaojian and copper gluconate form a copper complex (gallic acid-copper complex) with anti-tumor effects through a Chinese medicine coordination chemical reaction. According to traditional Chinese medicine, copper gluconate replaces apperurus for detoxification and anti-cancer as the main drug; Baiyaojian helps its anti-cancer effect and also reduces copper toxicity, and is used as the auxiliary drug.

[0037] In this embodiment, the traditional Chinese medicine composition for treating malignant tumors includes copper gluconate and Baiyaojian in a mass ratio of 1:5.

[0038] In this embodiment, the preparation method of the Chinese medicine composition for treating malignant tumors comprises the following steps: S1. Grind copper gluconate into a fine powder using a mortar and pestle, grind the herbal extract into a fine powder using a high-speed grinder, sieve using a 100-mesh sieve, and store in aliquots for later use; S2. Weigh 20 g of copper gluconate and 100 g of Baiyaojian, add 100 mL of water, mix well and place in a ceramic jar, cover with a lid, place the ceramic jar in a pressure cooker, heat at 120 ° C for 30 minutes, remove the ceramic jar after cooling, continue to mix the drugs in the ceramic jar to obtain a black paste. The black paste in this embodiment is a traditional Chinese medicine composition for treating malignant tumors.

[0039] The traditional Chinese medicine composition for treating malignant tumors obtained in step S2 of this embodiment is named "Baiyaojian combined with copper gluconate paste".

[0040] In this embodiment, the container for preparing the Chinese medicine composition can be a ceramic pot or other high-temperature resistant containers. Metal containers should be avoided to prevent metal ions from complexing with the gallic acid contained in Baiyaojian and interfering with the effect of copper gluconate. Those skilled in the art can freely choose according to actual conditions, and no other restrictions are made in this embodiment.

[0041] Baiyaojian is a block made from the fermentation of gallnut, distiller's grains or koji, and tea leaves. Gallnut (Gallachinensis, GC), also known as clam, hundred insect warehouse, and wood aconite, is a gall formed by aphids parasitizing on the leaves of host plants such as red bran poplar, green bran poplar, and saltwort.

[0042] The Baiyaojian in this embodiment can also be replaced by Gallnut, but the amount of Gallnut is twice that of Baiyaojian. Therefore, it is more preferable to use Baiyaojian in this embodiment.

[0043] Application Examples This application example aims to illustrate the specific uses of the traditional Chinese medicine composition provided in the first embodiment and the third embodiment. The above-provided traditional Chinese medicine composition has at least the following uses: a. Use in the preparation of drugs for treating oral cancer; b. Use in the preparation of drugs for treating pancreatic cancer.

[0044] Experimental example 1. Reagents and raw materials used in this experimental example Culture medium (RPMI 1640 medium + 10% fetal bovine serum); CCK-8 (purchased from Tongren Chemical Company); In this experimental example, the Wujin Huaiwan provided in the second embodiment and the Baoyaojian combined with copper gluconate paste provided in the third embodiment were selected for the experiment.

[0045] 2. Cell experiment 2.1 Preliminary preparation stage Prepare the Wujin Huaiwan medicinal liquid: Crush the Wujin Huaiwan, accurately weigh 100 mg, place it in a 15 ml sterile tube, add pure water to the 10 ml volume scale line, heat it in a water bath at 100 °C for 30 minutes, after high-speed centrifugation (3000 rpm, 10 min), absorb the supernatant as the Wujin Huaiwan medicinal liquid and calibrate its concentration to 10000 mg / L (Note: For traditional Chinese medicine solid preparations, calculating the concentration according to the input amount and the total volume after adding the extraction solution is a recognized concentration measurement method in traditional Chinese medicine research, the same below).

[0046] Prepare the culture medium containing Wujin Huaiwan: Absorb the above-prepared Wujin Huaiwan medicinal liquid with a concentration of 10000 mg / L, use the cell culture medium (RPMI 1640 medium + 10% fetal bovine serum) as the solvent, and gradually dilute it with the cell culture medium (RPMI 1640 medium + 10% fetal bovine serum) to prepare a series of culture media containing Wujin Huaiwan with different concentrations. According to the content of Wujin Huaiwan in the cell culture medium, the culture media containing Wujin Huaiwan are sequentially calibrated as 100 mg / L, 50 mg / L, 20 mg / L, 15 mg / L, 10 mg / L, 5 mg / L, 1 mg / L, 0.5 mg / L, and the culture medium without Wujin Huaiwan (0 mg / L).

[0047] Prepare the Baoyaojian medicinal liquid: Crush the Baoyaojian, pass it through a 100-mesh sieve, weigh 15 mg of the sieved Baoyaojian powder and place it in a 15 ml sterile tube, add pure water to the 10 ml volume scale line, heat it in a water bath at 100 °C for 30 minutes, after high-speed centrifugation (3000 rpm, 10 min), absorb the supernatant as the Baoyaojian medicinal liquid and calibrate its concentration to 1500 mg / L.

[0048] Prepare the medium containing myrobalan: Pipette the myrobalan medicinal liquid with a concentration of 1500 mg / L prepared above, using the cell medium (RPMI 1640 medium + 10% fetal bovine serum) as the solvent, and dilute it with the cell medium (RPMI 1640 medium + 10% fetal bovine serum) to prepare a medium containing myrobalan with a myrobalan concentration of 15 mg / L.

[0049] Prepare the aeruginosa medicinal liquid: Grind aeruginosa into fine powder, sieve it through a 100-mesh sieve, weigh 1.5 mg of the sieved aeruginosa powder and place it into a 15-ml sterile tube, add pure water to the 10-ml volume scale line, heat it in a water bath at 100 °C for 30 minutes, after high-speed centrifugation (3000 rpm, 10 min), pipette the supernatant as the aeruginosa medicinal liquid and calibrate its concentration to 150 mg / L.

[0050] Prepare the medium containing aeruginosa: Pipette the 150 mg / L aeruginosa medicinal liquid prepared above, using the cell medium (RPMI1640 medium + 10% fetal bovine serum) as the solvent, and dilute it with the cell medium (RPMI 1640 medium + 10% fetal bovine serum) to prepare a medium containing aeruginosa with an aeruginosa concentration of 1.5 mg / L.

[0051] CAL27 human oral squamous carcinoma cells and SW1990 human pancreatic carcinoma cells are respectively cultured in the medium (RPMI 1640 medium + 10% fetal bovine serum), and routinely cultured in a 5% CO2, 37 °C cell incubator. Use a 96-well culture plate, implant 5000 cells in each well, and pre-culture for 12 hours to allow the cells to adhere and grow.

[0052] 2.2 Cell drug administration stage After the cells are pre-cultured for 12 hours or overnight, discard the original cell medium (RPMI 1640 medium + 10% fetal bovine serum), and add the medium containing Wu Jin Hua Ai Wan to continue the culture. The operation is as follows: Add the medium containing Wu Jin Hua Ai Wan prepared above to the cell culture wells of the 96-well plate (200 μl / well), set 3 replicates for each concentration of 100 mg / L, 50 mg / L, 20 mg / L, 15 mg / L, 10 mg / L, 5 mg / L, 1 mg / L, 0.5 mg / L, 0 mg / L, and routinely culture in a 5% CO2, 37 °C cell incubator for 48 hours.

[0053] Referring to the above cell drug administration and culture method, add the medium containing myrobalan and the medium containing aeruginosa prepared in 2.1 to the cell culture wells of the 96-well plate (200 μl / well) respectively, set 3 replicates respectively, and routinely culture in a 5% CO2, 37 °C cell incubator for 48 hours.

[0054] 2.3 Cell viability test stage Cell viability was assessed using CCK-8 reagent (Tongren Chemical Co., Ltd.) according to the manufacturer's instructions. Three blank wells were also set up (each containing only 200 µl of cell culture medium (RPMI 1640 medium + 10% fetal bovine serum and CCK-8 reagent). The absorbance (OD) of each well was measured using a microplate reader at a wavelength of 450 nm. The tumor cell growth inhibition rate and cell viability were calculated according to the following formula to evaluate the inhibitory effect of the drug on tumor cells.

[0055] Cell viability = [(OD experimental - OD blank) / (OD control - OD blank)] × 100% Tumor cell growth inhibition rate = [(cell viability of the control group - cell viability of the treatment group) / cell viability of the control group] × 100% OD experiment: cells were cultured in culture medium containing Wujin Huaai Pills for 48 hours, and then the absorbance was measured after adding CCK-8; OD blank: absorbance value obtained by adding only 200 µl of cell culture medium (RPMI 1640 medium + 10% fetal bovine serum) and CCK-8 reagent to each well without cells; OD control: absorbance value measured after adding CCK-8 reagent after culturing cells in drug-free medium (RPMI 1640 medium + 10% fetal bovine serum) for 48 hours; Control group cell viability: set to 100%; Cell viability in the treatment group: The calculation formula is [(OD experimental - OD blank) / (OD control - OD blank)] × 100%, which is consistent with the calculation formula of cell viability.

[0056] After co-cultured with cells for 48 hours with culture medium containing Wujin Huaai Pills (100 mg / L, 50 mg / L, 20 mg / L, 10 mg / L, 5 mg / L, 1 mg / L, 0.5 mg / L, 0 mg / L), GraphPad Prism software was used to calculate the half inhibitory concentration (IC50 value) of Wujin Huaai Pills on tumor cells based on the numerical value of tumor cell growth inhibition rate. The half inhibitory concentration (IC50 value) of Wujin Huaai Pills on CAL27 cells was 11.7 mg / L, and the half inhibitory concentration (IC50 value) of Wujin Huaai Pills on SW 1990 cells was 13.9 mg / L.

[0057] After culturing cells in culture medium containing Wujin Huaai Pills (Wujin Huaai Pills concentrations of 5mg / L, 10mg / L, and 15mg / L) for 48 hours, the results of inhibition on cell viability are as follows: Figure 1As shown, cell viability decreased with the increasing concentration of Wu Jin Hua Ai Wan, indicating that the inhibitory effect of Wu Jin Hua Ai Wan on cells was concentration-dependent, suggesting that the traditional Chinese medicine composition for treating malignant tumors provided by the present invention could significantly inhibit the proliferation viability of human oral cancer CAL27 cells and human pancreatic cancer SW 1990 cells.

[0058] After culturing cells for 48 hours in the medium containing gallic acid and the medium containing verdigris, the inhibitory results on cell viability were as Figure 2 shown. Compared with the blank control group, gallic acid and verdigris did not significantly inhibit the proliferation activity of the cell line, indicating that under the experimental concentration conditions of 15 mg / L of gallic acid and 1.5 mg / L of verdigris, using gallic acid or verdigris alone could not inhibit the proliferation viability of human oral cancer CAL27 cells and human pancreatic cancer SW 1990 cells.

[0059] Comparing Figure 1 with Figure 2 the data in

[0060] Blank control group: Cells were cultured in a medium without adding any drugs (RPMI 1640 medium + 10% fetal bovine serum); 5 mg / L: Cells were cultured in a medium containing Wu Jin Hua Ai Wan with a concentration of 5 mg / L of Wu Jin Hua Ai Wan; 10 mg / L: Cells were cultured in a medium containing Wu Jin Hua Ai Wan with a concentration of 10 mg / L of Wu Jin Hua Ai Wan; 15 mg / L: Cells were cultured in a medium containing Wu Jin Hua Ai Wan with a concentration of 15 mg / L of Wu Jin Hua Ai Wan; Gallic acid 15 mg / L: Cells were cultured in a medium containing gallic acid with a concentration of 15 mg / L of gallic acid; Verdigris 1.5 mg / L: Cells were cultured in a medium containing verdigris with a concentration of 1.5 mg / L of verdigris.

[0061] 3. Animal experiments 3.1 Inhibition of the growth of transplanted oral squamous cell carcinoma tumors in mice by Wu Jin Hua Ai Wan Wu Jin Hua Ai Wan was crushed, and 10 g (10000 mg) was placed into a 50 - milliliter sterile tube, and water was added to a volume of 50 ml. It was heated in a water bath at 100 °C for 30 minutes and stirred into a paste - like liquid for standby.

[0062] 5 - week - old BCLB / C thymus - immunodeficient nude mice were raised in a SPF environment.

[0063] First, 3 nude mice were taken, and 1×10 7 human oral cancer CAL27 cells per mouse were injected subcutaneously into the right scapular region. After 3 weeks, the tumor grew to 500 mm 3, The nude mice were sacrificed conventionally, and the tumor masses were taken. Among the three removed tumor masses, the two with the largest volume were selected and cut into 1 mm×1 mm×1 mm square fragments, which were implanted subcutaneously under the scapulas of another 10 nude mice. When the nude mice had tumors growing for 2 weeks, the nude mice were randomly divided into a control group (n = 5) and a treatment group (n = 5) for drug intervention: Control group: gavaged with 0.4 ml of normal saline once a day; Treatment group: gavaged with 0.4 ml of the prepared Wujin Huaiwan paste once a day.

[0064] The tumor length (L) and width (W) were measured every 3 days, and the tumor volume (V) was calculated according to the formula: V = (L × W 2 ) × 0.5. The tumor volume was recorded every 3 days, the tumor sizes were compared, and the body weights of the mice were measured. The nude mice were continuously observed for 22 days and sacrificed on the 22nd day. The tumor volumes of the mice in the control group (n = 5) and the treatment group (n = 5) were as Figure 3 shown. The numerical changes in the tumors and body weights of the mice are shown in Table 1 in detail.

[0065] The tumor inhibition rate was calculated according to the following formula: Tumor inhibition rate = [(tumor volume of the control group - tumor volume of the treatment group) / tumor volume of the control group] × 100% After calculation, the daily drug dosage for the animals in the treatment group was 4 g / kg of Wujin Huaiwan. Under the condition of this dosage, there was a difference in the tumor volume between the treatment group and the control group starting from the 13th day, and there was a significant difference compared with the control group (P < 0.001). According to the tumor volume on the 22nd day, the inhibition rate was calculated to be 71.2%. The body weight of the treatment group decreased slightly compared with the control group, but the difference was not significant, indicating that Wujin Huaiwan did not cause a significant impact on the growth and development of the mice, and the daily physiological activities of the mice were not significantly disturbed, indicating that Wujin Huaiwan did not produce obvious toxicity to the mice. The above data comprehensively show that Wujin Huaiwan can significantly inhibit the growth of transplanted tumors of oral squamous cell carcinoma in mice during the experiment and does not produce significant toxicity.

[0066] 3.2 Inhibition of the growth of transplanted tumors of oral squamous cell carcinoma in mice by Baiguojian and copper gluconate paste Weigh 10 g (10000 mg) of Baiguojian and copper gluconate paste, place it in a 50 ml centrifuge tube, add pure water to a volume of 50 ml, take it out after heating in a water bath at 100 °C for 30 minutes, and stir it into a paste for standby.

[0067] 5-week-old BCLB / C thymus immunodeficient nude mice were raised in a SPF environment.

[0068] First, take 3 nude mice and inject 1×10 human oral cancer CAL27 cells subcutaneously into the right scapula7 / only, after 3 weeks, the tumor grew to 500 mm 3 , the nude mice were sacrificed conventionally, the tumor masses were taken, and the two largest tumor masses were selected from the 3 removed tumor masses and cut into 1 mm×1 mm×1 mm square fragments, which were implanted subcutaneously under the scapulas of another 10 nude mice. When the nude mice were raised with tumors for 2 weeks, they were randomly divided into a control group (n = 6) and a treatment group (n = 6) for drug intervention: Control group: gavaged with 0.2 ml of normal saline once a day; Treatment group: gavaged with 0.2 ml of the paste of Galla Chinensis combined with copper gluconate once a day.

[0069] The tumor length (L) and width (W) were measured every 3 days, and the tumor volume (V) was calculated according to the formula: V = (L×W 2 ) ×0.5. The tumor sizes were compared, and the body weights of the mice were weighed. Observation was continued for 22 days. The mice were sacrificed on the 22nd day. The tumor volumes of the mice in the control group (n = 6) and the treatment group (n = 6) were as Figure 4 shown, and the numerical changes of the mouse tumors and body weights are shown in Table 2 in detail.

[0070] The tumor inhibition rate was calculated according to the following formula: Tumor inhibition rate = [(tumor volume of the control group - tumor volume of the treatment group) / tumor volume of the control group]×100% The average body weight of the tumor-bearing nude mice used in the experiment was 20 g. After calculation, the daily drug dosage for the animals was: Galla Chinensis 1 g / Kg, copper gluconate 0.2 g / Kg. Under this dosage condition, there was a difference between the treatment group and the control group on the 10th day, and there was a significant difference from the control group (P<0.001). According to the tumor volume on the 22nd day, the inhibition rate was 67.9%. The body weight of the treatment group was slightly reduced compared with that of the control group, but the difference was not significant. This indicates that the paste of Galla Chinensis combined with copper gluconate did not cause significant effects on the growth and development of mice, and the daily physiological activities of the mice were not significantly disturbed, indicating that the paste of Galla Chinensis combined with copper gluconate did not produce obvious toxicity to mice. The above data comprehensively show that the paste of Galla Chinensis combined with copper gluconate significantly inhibited the growth of transplanted tumors of oral squamous cell carcinoma in mice in the experiment and did not produce significant toxicity.

[0071] When comparing the tumor inhibition rate of Wujin Huaiwan with that of Baoyaojian combined with copper gluconate paste, 71.2% > 67.9%. The inhibitory effect of Baoyaojian combined with copper gluconate paste is slightly lower. Therefore, copper green is selected as the source of copper ions. When combined with propolis, lacca, frankincense, and myrrh, it can enhance the anti-tumor effects of Baoyaojian and copper green, making Wujin Huaiwan have more excellent anti-cancer effects. Wujin Huaiwan is prepared through further processing based on the traditional Chinese medicine composition for treating malignant tumors provided in the third embodiment. Therefore, it indirectly proves that the traditional Chinese medicine composition for treating malignant tumors provided in the third embodiment also has excellent anti-cancer effects and is more suitable for clinical treatment.

[0072] Although there is no assistance from propolis, lacca, frankincense, and myrrh, the Baoyaojian combined with copper gluconate paste prepared by combining Baoyaojian and copper gluconate can still achieve a tumor inhibition rate of 67.9%, still showing a significant anti-tumor effect. More importantly, the toxicity of copper gluconate is slightly lower than that of copper green, which better meets the clinical requirements for the safety of traditional Chinese medicine and provides a better choice for tumor treatment.

[0073] After treatment with Wujin Huaiwan, there is gallic acid-copper complex in the animal model body Wujin Huaiwan is crushed, and 10 g (10000 mg) is taken into a 50-ml sterile tube, and water is added to a volume of 50 ml, and heated at 100 °C for 30 minutes and stirred into a paste-like liquid.

[0074] Three 4-week-old mice are taken. Each mouse is gavaged with 0.2 ml of the Wujin Huaiwan paste-like liquid once a day for 3 consecutive days. Two hours after the last gavage, blood is collected from the orbital cavity. The blood sample and methanol are diluted according to a ratio of 1:10, and the collected blood sample is added to methanol for dilution, and then sent to the Analysis and Testing Center of Shanghai Jiao Tong University for mass spectrometry detection. The sample is centrifuged at high speed to separate the serum, and the methanol-diluted serum is analyzed by ultra-high performance liquid chromatography (UPLC) / ultra-critical fluid chromatography (UPC²)-quadrupole time-of-flight mass spectrometry (QTOF-MS) (Waters Acquity 2D-UPLC / Acquity UPC² / Xevo G2-XS QTOF).

[0075] The molecular weight of gallic acid is 170.12, and the atomic weight of copper is 63.55. It is speculated that in the mouse body, two gallic acid molecules can complex with 1 copper ion to form a five-membered ring chelate structure, and the mass-to-charge ratio (m / z) corresponding to the peak in the mass spectrum is: 170.12×2 + 63.55 = 403.79 After treatment with Wujin Huaiwan, the mass spectrometry detection results of mouse blood are as follows Figure 5As shown, a characteristic peak with a mass-to-charge ratio (m / z) of 404 can be clearly observed in the mass spectrum, and this result is highly consistent with the theoretical value of the ion peak of the gallic acid-copper complex molecule predicted according to the principle of coordination chemistry.

[0076] In the above mass spectrum ( Figure 5 ), the signal intensity of the characteristic peak at 404 is significant, indicating that the five-membered ring chelate structure formed by catechol and copper ions in the gallic acid-copper complex can still maintain a stable conformation under the metabolic environment in animals. The gallic acid content of Baoyaojian is greater than 20%, and the tannins contained in Baoyaojian may be further decomposed into gallic acid at temperatures above 100°C. Under normal conditions, two gallic acid molecules chelate one copper ion. Therefore, in the design of experiments and the preparation method of the complex, gallic acid is in a sufficient excess for the chelation reaction with copper ions. Therefore, the active ingredient gallic acid in Baoyaojian can undergo a sufficient chelation reaction with Cu²⁺ released by verdigris to form a gallic acid-copper chelate with a stable structure. This chelate exerts an anti-tumor effect through its unique chelate structure and is the core material basis for the efficacy of the composition. The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A traditional Chinese medicine composition for treating malignant tumors, characterized in that, The traditional Chinese medicine composition comprises malachite green and myrobalan fruit in a mass ratio of 1:10; alternatively, the traditional Chinese medicine composition comprises cupric gluconate and myrobalan fruit in a mass ratio of 1:

5.

2. The traditional Chinese medicine composition for treating malignant tumors according to claim 1, wherein, The traditional Chinese medicine composition comprises malachite green, myrobalan fruit, propolis, lacca, frankincense, and myrrh in a mass ratio of 1:10:6:2:2:

2.

3. The preparation method of the traditional Chinese medicine composition according to any one of claims 1-2, characterized in that, The preparation method comprises the following steps: S1. Grind malachite green, myrobalan fruit, propolis, lacca, frankincense, and myrrh into powders respectively, sieve them, and store for later use; S2. Weigh the malachite green and myrobalan fruit in step S1 according to the ratio, add water, mix well, place them in a container, put the container in a pressure cooker, heat at 100 - 120°C for 30 minutes, take out the container after cooling, and mix the medicines in the container to obtain a black paste; S3. Mix the black paste in step 2 with the propolis, lacca, frankincense, and myrrh in step S1 added according to the ratio, place them in a container, put the container in the pressure cooker again, heat at 100 - 120°C for 30 minutes, take out after cooling, and stir evenly again to obtain the traditional Chinese medicine composition for treating malignant tumors; Alternatively, the preparation method comprises the following steps: S11. Grind cupric gluconate and myrobalan fruit into powders respectively, sieve them, and store for later use; S22. Weigh the cupric gluconate and myrobalan fruit in step S11 according to the ratio, add water, mix well, place them in a container, put the container in a pressure cooker, heat at 100 - 120°C for 30 minutes, take out the container after cooling, and mix the medicines in the container to obtain a black paste, and the black paste is the traditional Chinese medicine composition for treating malignant tumors.

4. The preparation method of the traditional Chinese medicine composition for treating malignant tumors according to claim 3, wherein In step S1, after grinding the malachite green, myrobalan fruit, propolis, lacca, frankincense, and myrrh into powders, sieve them through a 100 - mesh sieve and store for later use; or in S11, sieve the cupric gluconate and myrobalan fruit through a 100 - mesh sieve and store for later use.

5. The preparation method of the traditional Chinese medicine composition for treating malignant tumors according to claim 3, characterized in that, The heating temperature in the pressure cooker is 120°C.

6. The preparation method of the traditional Chinese medicine composition for treating malignant tumors according to claim 3, characterized in that, In step S3, after adding conventional excipients to the obtained traditional Chinese medicine composition for treating malignant tumors, prepare it into a clinically acceptable preparation according to the conventional process.

7. The preparation method of the traditional Chinese medicine composition for treating malignant tumors according to claim 6, characterized in that, The preparation comprises at least one of ointment, capsule, granule, external patch, injection, mixture, and pill.

8. The preparation method of the traditional Chinese medicine composition for treating malignant tumors according to claim 7, characterized in that, The preparation is an ointment / pill.

9. The preparation method of the traditional Chinese medicine composition for treating malignant tumors according to claim 8, characterized in that, The dosage of the pill is 100 - 10000 mg; the dosage of the ointment is 10 - 10000 mg.

10. The traditional Chinese medicine composition for treating malignant tumors according to any one of claims 1 - 2 or the traditional Chinese medicine composition for treating malignant tumors prepared by the preparation method according to any one of claims 3 - 9 has at least the following uses: a. Use in the preparation of drugs for treating oral cancer; b. Use in the preparation of drugs for treating pancreatic cancer.

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