Pharmaceutical composition for treating liver cancer and preparation method thereof

Abstract

The invention belongs to the field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for treating liver cancer and a preparation method thereof. The traditional Chinese medicine composition is prepared from the following raw materials: 10 parts of flos mume, 10 parts of flos albiziae, 15 parts of fructus ligustri lucidi, 15 parts of herba ecliptae, 30 parts of rhizoma polygonati, 30 parts of radix codonopsis, 15 parts of rhizoma atractylodis macrocephalae, 15 parts of poria cocos, 10 parts of radix glycyrrhizae preparata, 6 parts of rhizoma zingiberis, 10 parts of radix angelicae sinensis, 30 parts of radix salviae miltiorrhizae, 10 parts of rhizoma chuanxiong, 30 parts of herba scutellariae barbatae and 30 parts of herba hedyotis diffusae. In the formula, the monarch drugs are sculellaria barbata and oldenlandia diffusa which are used for clearing away heat and toxic materials Ministerial drugs are rhizoma polygonati, radix codonopsis, rhizoma atractylodis macrocephalae, poria cocos, glossy privet fruit, herba ecliptae, radix angelicae sinensis, radix salviae miltiorrhizae, rhizoma chuanxiong and rhizoma zingiberis, the rhizoma zingiberis has the effects of tonifying qi and nourishing blood, tonifying spleen and tonifying liver and kidney, and the rhizoma zingiberis has the effect of warming the middle warmer and can prevent the herba scutellariae barbatae and the herba hedyotidis diffusae from hurting intestines and stomach due to excessive heat clearing; the adjuvant drugs are armeniaca mume and albizia flower for soothing liver-qi stagnation and soothing nerves; and the conductant drug is honey-fried licorice root which has the effects of invigorating spleen, replenishing qi and harmonizing the drugs. The traditional Chinese medicine composition can effectively inhibit the growth of liver cancer cells and has no side effect.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular, to a pharmaceutical composition for treating liver cancer and a preparation method thereof. Background Art

[0002] Liver cancer is one of the most common malignant tumors worldwide, especially in my country, where the incidence and mortality rates of liver cancer are among the highest. The pathogenesis of liver cancer is complex and is often associated with chronic viral hepatitis, cirrhosis, environmental factors, and genetic factors. Currently, the main treatments for liver cancer include surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, since liver cancer is often discovered in the middle or late stages, the surgical resection rate is low, while radiotherapy, chemotherapy, and targeted drug therapy often have significant toxic side effects and serious drug resistance problems, resulting in a poor prognosis for patients. Therefore, finding a safe and effective treatment method has become an important topic in current medical research.

[0003] Traditional Chinese Medicine (TCM) has a long history in my country and has accumulated extensive experience in the prevention and treatment of liver cancer. TCM exerts its anti-tumor effects through holistic regulation, improving immune function, inhibiting tumor cell proliferation, and inducing apoptosis, making it an important adjunct to liver cancer treatment. In recent years, novel TCM combinations developed based on TCM principles have gained increasing attention for their use in liver cancer treatment. Rational combinations of TCM herbs can enhance efficacy while minimizing toxic side effects, ultimately improving patients' quality of life.

[0004] Currently, the treatment of liver cancer faces numerous challenges. First, few patients are diagnosed with liver cancer early enough to undergo surgical intervention, and some have already lost the opportunity for surgery by the time of diagnosis. Even those who are eligible for surgical treatment face the risk of recurrence. Currently, the main treatments used in Western medicine include chemotherapy, immunotherapy, and radiofrequency ablation. While these treatments can achieve some control effects on tumors, they cannot completely eliminate cancer cells. Furthermore, chemotherapy and immunotherapy often cause significant side effects for patients, including but not limited to mucosal damage, gastrointestinal reactions, and decreased immune function.

[0005] In addition to chemotherapy and immunotherapy, targeted therapy in Western medicine is also an option. While it is continuously developing, it still has the problem of side effects. Although targeted therapy can act on specific molecular targets and reduce the impact on normal cells, some patients still face the risk of immune system disorders, such as excessive suppression or overactivation of immune function. This requires patients to carefully weigh the risks and benefits before undergoing treatment.

[0006] In contrast, Traditional Chinese Medicine (TCM) offers advantages in mitigating the side effects of Western medicine. It can help alleviate common chemotherapy side effects such as nausea, vomiting, and fatigue, and may also inhibit tumor growth. TCM emphasizes individualized treatment and comprehensive conditioning, aiming to improve patients' quality of life.

[0007] Overall, while Western medicine has some efficacy in treating liver cancer, side effects still require attention. Traditional Chinese medicine (TCM) has demonstrated potential in mitigating these side effects and improving patients' overall health. In the treatment of liver cancer, integrating multiple approaches and adopting individualized treatment plans can help improve patients' survival rates and quality of life. Summary of the Invention

[0008] In order to overcome the problems existing in the prior art, the present invention provides a pharmaceutical composition for treating liver cancer and its application First, in the first aspect, the present invention provides a traditional Chinese medicine composition for treating liver cancer, wherein the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 10 parts of green-calyx plum, 10 parts of julibrissin flowers, 15 parts of glossy privet fruits, 15 parts of Eclipta prostrata, 30 parts of polygonatum, 30 parts of codonopsis pilosula, 15 parts of atractylodes macrocephala, 15 parts of poria, 10 parts of roasted licorice, 6 parts of dried ginger, 10 parts of angelica sinensis, 30 parts of salvia miltiorrhiza, 10 parts of ligusticum chuanxiong, 30 parts of scutellaria barbata, and 30 parts of oldenlandia diffusa.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned Chinese medicine composition, the method comprising the following steps: (1) Before decocting, soak the 15 medicinal materials in cold water, and keep the water level 2 cm above the surface of the medicinal materials; (2) First decoction: After boiling over high heat, reduce to low heat, pour out the medicinal juice and add water again for the second decoction; (3) Second decoction: Continue to decoct over low heat; combine the two decoctions, filter, and centrifuge to remove the residue.

[0010] In one embodiment, the method further comprises the step of concentrating the medicinal solution.

[0011] In a preferred embodiment, the soaking time in step (1) is 30 minutes, the boiling time in step (2) is 30 minutes, and the boiling time in step (3) is 20 minutes.

[0012] In one embodiment, excipients are added to the traditional Chinese medicine composition to prepare a corresponding dosage form.

[0013] In a preferred embodiment, the dosage form is an oral dosage form. More preferably, the oral dosage form is an oral liquid, tablet, powder, granule or capsule. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 The effect of the prescription of green calyx plum and julibrissin on the proliferation of liver cancer cells was studied. Figure 1 A: The IncuCyteZoom live cell imaging system randomly recorded the growth and proliferation of cells in the blank serum group, the Chinese medicine prescription group 1, and the Chinese medicine prescription group 2 for 12 hours. Figure 1 B: Statistical analysis compared the cell auto-confluence of the blank serum group, Chinese medicine prescription group 1, and Chinese medicine prescription group 2. Compared with the blank group, P<0.05, Compared with the blank group, P<0.01; compared with Chinese medicine prescription 1, P<0.05.

[0015] Figure 2 The tumor growth of the liver cancer-bearing mouse model, Figure 2 A: The tumor volumes of nude mice in the model group, Chinese medicine group, and positive drug group were observed with the naked eye. Figure 2 B: The tumor volume was calculated every four days and the growth of liver cancer cells was recorded. Figure 2 C: Statistical analysis comparing the tumor volumes of nude mice in the model group, Chinese medicine group, and positive drug group. *P<0.05 compared with the blank group, **P<0.01 compared with the blank group, ***P<0.001 compared with the blank group. DETAILED DESCRIPTION

[0016] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0017] Example 1: Formula of Chinese medicine composition (Meilianjunzi Decoction) Weigh the following ingredients: 10g of green-calyx plum, 10g of julibrissin flower, 15g of glossy privet fruit, 15g of Eclipta prostrata, 30g of polygonatum, 30g of codonopsis pilosula, 15g of atractylodes macrocephala, 15g of poria, 10g of roasted licorice root, 6g of dried ginger, 10g of angelica root, 30g of salvia miltiorrhiza, 10g of chuanxiong, 30g of bark lotus, 30g of white flower hedyotis diffusa, a total of 15 Chinese herbs. The main medicines are Scutellaria barbata and Hedyotis diffusa, which play the role of clearing heat and detoxifying, removing blood stasis and promoting diuresis.

[0018] The assistant medicines are Polygonatum sibiricum, Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, Ligustrum lucidum fruit, Houttuynia cordata, Angelica sinensis, Salvia miltiorrhiza, Chuanxiong and dried ginger, which play the role of replenishing qi and blood, strengthening the spleen and nourishing the liver and kidneys. In addition, the warming effect of dried ginger can prevent Scutellaria barbata and Houttuynia cordata from clearing away heat too much and damaging the stomach and intestines.

[0019] The adjuvant drugs are green-calyx plum and julibrissin, which play the role of soothing the liver, relieving depression and calming the mind.

[0020] The guiding drug is roasted licorice root, which plays the role of strengthening the spleen and replenishing qi, and harmonizing various medicines.

[0021] The decoction method of Chinese medicine is: Use a clay pot for decocting herbs. Before decocting, soak the herbs in cold water for half an hour, with the water level reaching 2 cm above the surface. For herbs like flowers, the water level may drop after soaking, so add additional cold water to the standard level.

[0022] First Decoction: After boiling over high heat, reduce to low heat (simmer) and simmer for 30 minutes. Pour out the liquid and add water for the second decoction. Second Decoction: Continue simmering over low heat for 20 minutes. Combine the two decoctions, filter, centrifuge to remove residue, and concentrate to 3.25g of crude drug / ml. Store at 4°C until ready for use.

[0023] Example 2 Comparative Example Formula (lacking green-calyx plum and julibrissin flowers) The following raw materials were weighed: 15 g of Ligustrum lucidum fruit, 15 g of Eclipta prostrata, 30 g of Polygonatum sibiricum, 30 g of Codonopsis pilosula, 15 g of Atractylodes macrocephala, 15 g of Poria cocos, 10 g of Radix Glycyrrhizae roasted, 6 g of dried ginger, 10 g of Angelica sinensis, 30 g of Salvia miltiorrhiza, 10 g of Ligusticum chuanxiong, 30 g of Scutellaria barbata, and 30 g of Hedyotis diffusa. The mixture was crushed to 50 mesh using a grinder, and then cold-soaked for more than 10 hours with 10 times the amount of 50% ethanol by weight. After removal, ultrasonic extraction was performed at room temperature for 30 min with an ultrasonic power of 30 kHz. The mixture was centrifuged at 5000 rpm for 10 minutes, and the supernatant was filtered.

[0024] Decoction of Chinese medicine: Use a clay pot for decocting herbs. Before decocting, soak the herbs in cold water for half an hour, with the water level reaching 2 cm above the surface. For herbs like flowers, the water level may drop after soaking, so add additional cold water to the standard level.

[0025] First Decoction: After boiling over high heat, reduce to low heat (simmer) and simmer for 30 minutes. Pour out the liquid and add water for the second decoction. Second Decoction: Continue simmering over low heat for 20 minutes. Combine the two decoctions, filter, centrifuge to remove residue, and concentrate to 3.25g of crude drug / ml. Store at 4°C until ready for use.

[0026] 1. Preparation of Hepatocellular Carcinoma Nude Mouse Tumor Model and Drug Treatment Experiment 1.1. Selection of experimental animals Healthy tumor-bearing nude mice, aged 6-8 weeks and weighing approximately 20-25 grams, were used. They were housed for one week prior to the experiment to maintain their health. During the experiment, the mice's health was observed, and weight changes, food intake, water intake, and activity levels were regularly recorded.

[0027] 1.2. Establishment of liver cancer model 1.2.1. Culture of liver cancer cells: HepG2 human liver cancer cell line was obtained from a standard cell bank and cultured in DMEM + 10% FBS + 1% penicillin / streptomycin in a 37°C, 5% CO2 incubator.

[0028] 1.2.2. Preparation of liver cancer cells: When the cell confluence is approximately 80%-90%, digest the cells with trypsin, collect the cells, and wash them with PBS. Then adjust the cell suspension concentration to 1×10^6 cells / ml.

[0029] 1.2.3. Subcutaneous Injection: Nude mice were first anesthetized with isoflurane to ensure sedation during the procedure. After anesthesia, the right abdomen was disinfected and the skin was cleaned with 75% alcohol to ensure sterility. The cell suspension was aspirated using a 1ml syringe and injected subcutaneously into the right abdomen. A volume of 0.1ml, containing 1 x 10^5 cells, was injected to ensure accurate delivery into the subcutaneous adipose tissue.

[0030] 1.3. Tumor formation and grouping 1.3.1. Tumor Observation: Nude mice were regularly observed after surgery to record tumor growth and weight changes. Tumor growth was typically observed 1-2 weeks after injection.

[0031] 1.3.2. Grouping: After tumor formation, the nude mice were randomly divided into three groups, namely the model group, the Chinese medicine group and the positive drug group.

[0032] 1.4. Dosage Preparation of the positive drug 5-fluorouracil: Weigh 5-fluorouracil (5-FU) powder and prepare a 4 mg / kg working solution with normal saline.

[0033] 1.4.1. Pre-drug treatment: The drug should be preheated in a 37°C water bath before administration.

[0034] 1.4.2. Grouping and drug administration: The Chinese medicine group was given 0.4 ml of the drug daily by gavage; the positive drug group was given 0.2 ml of the drug every three days by intraperitoneal injection; the model group was not given any drug.

[0035] 1.4.3. Dosing Cycle: The total dosing cycle is 21 days. During this period, tumor diameters (long and short diameters) are recorded every 4 days, and tumor volume (V = (long diameter × short diameter²) / 2) and changes in nude mouse body weight are calculated.

[0036] 1.5. Obtain materials After the administration period, the nude mice were sacrificed and the tumors and related organs were removed.

[0037] 1.6. Data Recording and Analysis The changes in tumor volume in each group were analyzed. ANOVA was used to analyze the data and compare the differences among the groups.

[0038] 2. Experiment on the Effect of Traditional Chinese Medicine on the Proliferation of Liver Cancer Cells Both Albizia julibrissin and Rhodoleia championii have medicinal value in traditional Chinese medicine and folk herbal medicine. Albizia julibrissin's benefits include calming the nerves, clearing heat and detoxifying, promoting blood circulation and removing blood stasis, and acting as a diuretic and detumescent agent. Rhodoleia championii's benefits include clearing heat and detoxifying, reducing swelling and pain, and enhancing immunity. Furthermore, both can enter the liver meridian, relieving liver qi and stagnation.

[0039] This experiment aimed to compare the inhibitory effects of Chinese herbal formula 1 (Meilianjunzi Decoction without green-calyx plum and julibrissin flowers) described in the comparative example with Chinese herbal formula 2 (Meilianjunzi Decoction) described in Example 1 on tumor cells. This experiment was designed as a cellular experiment targeting Chinese herbal formulas for the treatment of liver cancer, aiming to observe the effects of different drug-containing sera on the proliferation of HepG2 liver cancer cells and the anti-cancer effects of different formulas.

[0040] 2.1. Preparation of drug-containing serum 2.1.1 Drug Administration: Decoctions of Recipe 1 and Recipe 2 were prepared according to the decoction methods described in Examples 1 and 2. These decoctions were then administered orally to male SD rats twice daily for one week. Rats in the control group were administered an equal amount of distilled water.

[0041] 2.1.2. Serum Collection: One hour after the last dose, collect blood from the abdominal aorta. Allow the blood to clot for 2 hours. Centrifuge at 3000 rpm for 15 minutes to collect the supernatant serum. Sterilize by filtration through a 0.22 μm filter, aliquot, and store frozen at -80°C.

[0042] 2.2. Culture of Hepatocellular Carcinoma Cells HepG2 cells were cultured in DMEM at 37°C with 5% CO2 until the cells reached 80%-90% confluence. The cells were trypsinized and re-cultured to the desired cell density.

[0043] 2.3. Experimental Grouping and Drug Administration HepG2 cells were divided into three groups, each with at least three replicates: a serum group containing Chinese herbal formula 1, a serum group containing Chinese herbal formula 2, and a blank serum group. The corresponding drug-containing serum was added to each group at a concentration of 10%.

[0044] 2.4. Result detection 3000 cells were seeded per well in a 96-well plate, and cell proliferation was randomly recorded in each well for 12 hours using an IncuCyte Zoom Live Cell Imaging System (2015A, Essen Bioscience, Ann Arbor, MI). Three observation points were observed in each well, and the instrument automatically calculated the average cell confluence.

[0045] 3. Experimental results: 3.1 Effects of Chinese herbal formulas on liver cancer cell proliferation In this experiment, the IncuCyte Zoom live cell imaging system (2015A, Essen Bioscience, Ann Arbor, MI) was used to randomly record the proliferation of cells in each well of the blank serum group, the Chinese herbal formula group 1, and the Chinese herbal formula group 2 for 12 hours. As shown in the figure below, compared with the cells in the blank serum group, the cell density and aggregation degree of the Chinese herbal formula group 1 and the Chinese herbal formula group 2 were significantly lower than those in the blank serum group (see Figure 1 A); and the instrument automatically calculated the average cell confluence of each group of cells. We found that compared with the blank serum group, the confluence of the Chinese medicine prescription group 1 was significantly reduced at 12 hours (P=0.003), and the confluence of the Chinese medicine prescription group 2 was also significantly reduced (P<0.001), and the confluence of the Chinese medicine prescription group 2 was significantly lower than that of the Chinese medicine prescription group 1 (P=0.020) (see Figure 1 B). The above results all indicate that the prescription containing green-calyx plum and julibrissin flowers significantly downregulated the proliferation ability of liver cancer cells and had a certain inhibitory effect on liver cancer cells.

[0046] 3.2 Inhibitory Effect of Meilianjunzi Decoction on Tumor Development Throughout the experiment, all mice remained in good health. Their weight fluctuated within the normal range, with no significant decrease or abnormal increase. Furthermore, their activity levels did not decrease significantly, indicating that they showed no obvious signs of fatigue or weakness. This demonstrates the biosafety of the formulation used in this experiment.

[0047] In the mouse experiment, we administered drugs to the Chinese medicine group (Meilian Junzi Decoction) and the positive drug group, but not to the model group. The tumor volume of the nude mice in the Chinese medicine group and the positive drug group was significantly smaller than that of the nude mice in the model group (see Figure 2A); At the same time, the growth of liver cancer cells was recorded by calculating the tumor volume (V = (long diameter × short diameter²) / 2) every four days. The results showed that as the number of days increased, the tumor volume of the model group without drug administration became more and more significantly larger than that of the Chinese medicine group and the positive drug group (see Figure 2 B); Subsequently, the tumor volumes of each group were statistically analyzed, and the results showed that the tumor volumes of the Chinese medicine group and the positive drug group were significantly smaller than those of the model group (Chinese medicine group vs. model group, P<0.001; positive drug group vs. model group, P < 0.001, and there was no statistical difference between the Chinese medicine group and the positive drug group (see Figure 2 C). The above results all indicate that Chinese medicine can inhibit the proliferation of liver cancer cells, and its effect is the same as that of positive drugs.

[0048] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A Chinese medicine composition for treating liver cancer, characterized in that: The Chinese medicine composition is composed of the following raw materials in parts by weight: Ingredients: 10 parts of green-calyx plum, 10 parts of julibrissin flowers, 15 parts of glossy privet fruits, 15 parts of eclipta prostrata, 30 parts of polygonatum, 30 parts of codonopsis pilosula, 15 parts of atractylodes macrocephala, 15 parts of poria, 10 parts of preparatory liquorice, 6 parts of dried ginger, 10 parts of angelica sinensis, 30 parts of salvia miltiorrhiza, 10 parts of ligusticum chuanxiong, 30 parts of scutellaria barbata, and 30 parts of oldenlandia diffusa.

2. The method for preparing the Chinese medicine composition according to claim 1, wherein: The following steps are involved: (1) Before decocting, soak the 15 medicinal materials in cold water, and keep the water level 2 cm above the surface of the medicinal materials; (2) First decoction: After boiling over high heat, reduce to low heat, pour out the medicinal juice and add water again for the second decoction; (3) Second decoction: Continue to decoct over low heat; combine the two decoctions, filter, and centrifuge to remove the residue.

3. The preparation method according to claim 2, wherein The method further comprises the step of concentrating the medicinal solution.

4. The method for preparing the Chinese medicine composition according to claim 2, wherein: The soaking time in step (1) is 30 minutes, the boiling time in step (2) is 30 minutes, and the boiling time in step (3) is 20 minutes.

5. The method for preparing the Chinese medicine composition according to any one of claims 2 to 4, characterized in that: Add auxiliary materials to the traditional Chinese medicine composition to prepare corresponding dosage forms.

6. The method for preparing the Chinese medicine composition according to claim 5, wherein: The dosage form is an oral dosage form.

7. The method for preparing the Chinese medicine composition according to claim 6, wherein: The oral dosage form is oral liquid, tablet, powder, granule or capsule.

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