A pharmaceutical composition for treating liver cancer and a preparation method thereof

Abstract

The application belongs to the field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating liver cancer and a preparation method thereof. The traditional Chinese medicine composition is composed of the following raw materials: green calyx plum 10 parts, Chinese scholartree flower 10 parts, privet fruit 15 parts, eclipta 15 parts, yellow ginseng 30 parts, Chinese wolfberry root 30 parts, white atractylodes 15 parts, poria 15 parts, fried licorice 10 parts, dried ginger 6 parts, angelica 10 parts, red sage root 30 parts, Szechuan lovage 10 parts, half branch lotus 30 parts, and white flower snake tongue grass 30 parts. The half branch lotus and the white flower snake tongue grass are the monarch drugs, which clear heat and detoxify and resolve stasis and promote diuresis; the yellow ginseng, the Chinese wolfberry root, the white atractylodes, the poria, the privet fruit, the eclipta, the angelica, the red sage root, the Szechuan lovage, and the dried ginger are the ministerial drugs, which tonify qi and nourish blood, invigorate the spleen and tonify the liver and kidney, and the dried ginger is used for warming the middle, which can prevent the half branch lotus and the white flower snake tongue grass from being too hot to hurt the stomach and intestines; the green calyx plum and the Chinese scholartree flower are the auxiliary drugs, which soothe the liver, relieve stagnation, and calm the mind; and the fried licorice is the ministerial drug, which invigorates the spleen and tonifies qi and harmonizes all the drugs. The traditional Chinese medicine composition can effectively inhibit the growth of liver cancer cells and has no side effects.

Description

TECHNICAL FIELD

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

[0002] Liver cancer is one of the most common malignant tumors worldwide, especially in China, the incidence and mortality of liver cancer are among the top. The pathogenesis of liver cancer is complex, and is related to chronic viral hepatitis, cirrhosis, environmental factors and genetic factors. At present, the main treatment methods of liver cancer include surgical resection, radiotherapy, chemotherapy, targeted therapy and immunotherapy. However, due to the fact that liver cancer is often found in the middle and late stages, the surgical resection rate is low, and radiotherapy and chemotherapy and targeted drug therapy often have serious side effects, and drug resistance is a serious problem, and the prognosis of patients is poor. Therefore, finding safe and efficient treatment methods has become an important topic in current medical research.

[0003] Traditional Chinese medicine has a long history in China, and has accumulated rich experience in the prevention and treatment of liver cancer. Traditional Chinese medicine plays an important role in the treatment of liver cancer by regulating the whole body, improving immune function, inhibiting tumor cell proliferation, inducing apoptosis and other ways. In recent years, the application of new traditional Chinese medicine compositions developed based on traditional Chinese medicine theory in the treatment of liver cancer has gradually attracted attention. Reasonable compatibility of traditional Chinese medicine can enhance the efficacy while reducing side effects and improve the quality of life of patients.

[0004] At present, the treatment of liver cancer is facing many challenges. First, there are not many patients who can be diagnosed early and can be surgically intervened, and some patients have lost the opportunity for surgery at the time of diagnosis. Even if surgical treatment is possible, a considerable number of patients still face the risk of recurrence. The main treatment methods currently used by Western medicine include chemotherapy, immunotherapy and radiofrequency ablation. Although these treatment methods can produce certain control effect on tumors, they cannot completely eliminate cancer cells. In addition, chemotherapy and immunotherapy often bring obvious side effects to patients, including but not limited to mucosal damage, gastrointestinal reactions, decreased immune function, etc.

[0005] In addition to chemotherapy and immunotherapy, targeted therapy of Western medicine is also an option, although it is constantly developing, but there are still problems of side effects. Targeted therapy can act on specific molecular targets, reduce the impact on normal cells, but some patients may still face the risk of immune system disorders, such as excessive inhibition or excessive activation of immune function. This makes patients have to carefully weigh the risks and benefits when receiving treatment.

[0006] In contrast, traditional Chinese medicine has certain advantages in reducing the side effects of Western medicine. Traditional Chinese medicine can help alleviate common side effects of chemotherapy such as nausea, vomiting, fatigue, and may have a certain inhibitory effect on tumor growth. Traditional Chinese medicine emphasizes individualized treatment and comprehensive conditioning, aiming to improve the quality of life of patients.

[0007] Overall, Western medicine has certain efficacy in the treatment of liver cancer, but the problem of side effects needs to be taken seriously, while traditional Chinese medicine shows potential in reducing these side effects and improving the overall health of patients. In the treatment of liver cancer, a combination of various treatment methods and individualized treatment plans can help improve the survival rate and quality of life of patients. SUMMARY

[0008] In order to overcome the problems in the prior art, the present application provides a pharmaceutical composition for treating liver cancer and its application

[0009] First, in the first aspect, the present application provides a traditional Chinese medicine composition for treating liver cancer, wherein the traditional Chinese medicine composition is composed of the following raw materials by weight: green calyx 10 parts, sweet-scented osmanthus 10 parts, female privet fruit 15 parts, spider lily 15 parts, yellow ginseng 30 parts, Chinese wolfberry root 30 parts, white atractylodes 15 parts, poria 15 parts, fried licorice 10 parts, dried ginger 6 parts, angelica 10 parts, salvia miltiorrhiza 30 parts, chuanxiong 10 parts, half branch orchid 30 parts, white flower snake tongue grass 30 parts.

[0010] In the second aspect, the present application provides a preparation method of the above-mentioned traditional Chinese medicine composition, which comprises the following steps:

[0011] (1) Before decocting, 15 kinds of medicinal materials are soaked with cold water, and the water amount is maintained to be more than 2 cm above the surface of the medicinal materials after soaking;

[0012] (2) First decoction: after boiling with large fire, change to small fire decoction, pour out the medicinal juice and add water for second decoction;

[0013] (3) Second decoction: continue to decoct with small fire; mix and filter the medicinal liquid of two times of decoction, and centrifuge to remove residue.

[0014] In one embodiment, the method further comprises the step of concentrating the medicinal liquid.

[0015] In a preferred embodiment, the soaking time in step (1) is 30 min, the decoction time in step (2) is 30 min, and the decoction time in step (3) is 20 min.

[0016] In one embodiment, an auxiliary material is added to the traditional Chinese medicine composition to form a corresponding dosage form.

[0017] 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 DRAWINGS

[0018] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0019] Figure 1 Effects of the prescription with green calyx plum and sweet-scented olive on the proliferation of hepatoma cells, wherein, Figure 1 A: IncuCyteZoom live cell imaging system randomly records the growth and proliferation of cells in the blank serum group, the traditional Chinese medicine prescription 1 group and the traditional Chinese medicine prescription 2 group for 12 hours. Figure 1 B: Statistical analysis and comparison of the automatic convergence degree of cells in the blank serum group, the traditional Chinese medicine prescription 1 group and the traditional Chinese medicine prescription 2 group. *P<0.05 compared with the blank group, **P<0.01 compared with the blank group, #P<0.05 compared with the traditional Chinese medicine prescription 1 group.

[0020] Figure 2 Effects of the prescription with green calyx plum and sweet-scented olive on the proliferation of hepatoma cells, wherein, Figure 2 A: Macroscopically observe the tumor volume of nude mice in the model group, the traditional Chinese medicine group and the positive drug group. Figure 2 B: Calculate the tumor volume every four days to record the growth of hepatoma cells. Figure 2 C: Statistical analysis and comparison of the tumor volume of nude mice in the model group, the traditional Chinese medicine group and the 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

[0021] The preferred embodiments of the application will be described below with reference to the accompanying drawings, and it should be understood that the preferred embodiments described herein are only used to explain and illustrate the application, and do not limit the application.

[0022] The prescription of the traditional Chinese medicine composition in Example 1 (Meilianjunzi Decoction)

[0023] The weights of each raw material are as follows: green calyx plum 10g, sweet-scented olive 10g, privet fruit 15g, eclipta 15g, polygonatum 30g, radix codonopsis 30g, atractylodes 15g, poria 15g, fried licorice 10g, dried ginger 6g, angelica 10g, salvia 30g, chuanxiong 10g, barbork 30g, and white flower snake tongue 30g, a total of 15 kinds of traditional Chinese medicines. Among them,

[0024] The monarch drugs are barbork and white flower snake tongue, which play the roles of clearing heat and resolving toxicity, removing blood stasis and promoting diuresis.

[0025] The main ingredients are Polygonatum sibiricum, Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, Ligustrum lucidum, Eclipta prostrata, Angelica sinensis, Salvia miltiorrhiza, Ligusticum chuanxiong, and dried ginger. These ingredients work to replenish qi and blood, strengthen the spleen, and nourish the liver and kidneys. In addition, the warming effect of dried ginger can prevent the excessive heat-clearing effects of Scutellaria barbata and Hedyotis diffusa from damaging the stomach and intestines.

[0026] The adjuvant herbs, green plum blossom and mimosa flower, played a role in soothing the liver, relieving depression, and calming the mind.

[0027] The herb used is roasted licorice root, which plays a role in strengthening the spleen and replenishing qi, as well as harmonizing the effects of other herbs.

[0028] The method for decocting traditional Chinese medicine is as follows:

[0029] Use an earthenware pot as the utensil for decocting the medicine; before decocting, soak the medicinal materials in cold water for half an hour, with the water level 2 cm above the surface of the medicinal materials. Because there are herbal medicinal materials, the water level drops after soaking, so add more cold water to the standard water level.

[0030] First decoction: After bringing to a boil over high heat, reduce to a simmer and cook for 30 minutes. Pour out the decoction and add fresh water for the second decoction. Second decoction: Continue to simmer over low heat for 20 minutes. Combine the two decoctions, filter, centrifuge to remove residue, and then concentrate to a concentration of 3.25g of raw herb / ml. Store at 4℃ for later use.

[0031] Example 2 Comparative formulation (lacking green plum blossom and mimosa flower)

[0032] Weigh the following ingredients according to the following weights: 15g Ligustrum lucidum, 15g Eclipta prostrata, 30g Polygonatum sibiricum, 30g Codonopsis pilosula, 15g Atractylodes macrocephala, 15g Poria cocos, 10g Glycyrrhiza uralensis (processed), 6g Zingiber officinale (dried), 10g Angelica sinensis, 30g Salvia miltiorrhiza, 10g Ligusticum chuanxiong, 30g Scutellaria barbata, and 30g Hedyotis diffusa, totaling 15 Chinese herbs. Grind them to 50 mesh using a grinder, add 10 times their weight of 50% ethanol and soak for at least 10 hours. After soaking, extract by ultrasonication at room temperature for 30 minutes at an ultrasonic power of 30kHz. Centrifuge at 5000rpm for 10 minutes and filter the supernatant to obtain the final product.

[0033] Decoction of Chinese herbs:

[0034] Use an earthenware pot as the utensil for decocting the medicine; before decocting, soak the medicinal materials in cold water for half an hour, with the water level 2 cm above the surface of the medicinal materials. Because there are herbal medicinal materials, the water level drops after soaking, so add more cold water to the standard water level.

[0035] First decoction: After bringing to a boil over high heat, reduce to a simmer and cook for 30 minutes. Pour out the decoction and add fresh water for the second decoction. Second decoction: Continue to simmer over low heat for 20 minutes. Combine the two decoctions, filter, centrifuge to remove residue, and then concentrate to a concentration of 3.25g of raw herb / ml. Store at 4℃ for later use.

[0036] Example 3: Validation of antitumor activity (cell experiments and animal experiments)

[0037] 1. Preparation of Hepatocellular Carcinoma Nude Mouse Model and Drug Treatment Experiment

[0038] 1.1. Selection of Experimental Animals

[0039] Healthy tumor-bearing nude mice, aged 6-8 weeks, weighing about 20-25 grams, were used to ensure adaptive feeding for 1 week before the experiment to maintain their health. During the experiment, the health status of the mice was observed, and indicators such as body weight change, food intake, water intake, and activity level were recorded regularly.

[0040] 1.2. Establishment of Hepatocellular Carcinoma Model

[0041] 1.2.1. Culture of Hepatocellular Carcinoma Cells: HepG2 human hepatocellular carcinoma cell line was obtained from a standard cell bank and cultured in culture medium (DMEM + 10% FBS + 1% penicillin / streptomycin) in a 37°C, 5% CO2 incubator.

[0042] 1.2.2. Preparation of Hepatocellular Carcinoma Cells: When the cell confluence was about 80%-90%, trypsin was used for digestion, and the cells were collected and washed with PBS. Then the cell suspension concentration was adjusted to 1×10^6 cells / ml.

[0043] 1.2.3. Subcutaneous Injection: First, isoflurane was used to anesthetize the nude mice to ensure their sedation during the operation. After anesthesia, the right abdominal area was disinfected, and the skin was cleaned with 75% alcohol to ensure the sterility of the operation. A 1ml syringe was used to draw the cell suspension, and a subcutaneous injection was made on the right side of the abdomen, with an injection volume of 0.1ml containing 1×10^5 cells to ensure accurate injection of the cells into the subcutaneous adipose tissue.

[0044] 1.3. Tumor Formation and Grouping

[0045] 1.3.1. Tumor Observation: After the operation, the nude mice were observed regularly, and the tumor growth and body weight changes were recorded. Generally, tumor growth can be observed 1-2 weeks after injection.

[0046] 1.3.2. Grouping: After tumor formation, the nude mice were randomly divided into 3 groups, namely the model group, the traditional Chinese medicine group, and the positive drug group.

[0047] 1.4. Drug Administration Method

[0048] Preparation of 5-Fluorouracil (5-FU): Weigh 5-FU powder and prepare a 4mg / kg working solution with normal saline.

[0049] 1.4.1. Pre-treatment before administration: The drug was preheated in a 37°C water bath before administration.

[0050] 1.4.2. Group administration: The traditional Chinese medicine group was administered 0.4 ml per day by gavage; the positive drug group was administered 0.2 ml every three days by intraperitoneal injection; the model group was not administered.

[0051] 1.4.3. Administration period: The total administration period was 21 days. During this period, the diameters (long diameter and short diameter) of the tumors were recorded every 4 days, and the tumor volume (V = (long diameter x short diameter2) / 2) and the body weight changes of the nude mice were calculated.

[0052] 1.5. Sample collection

[0053] After the end of the administration period, the nude mice were sacrificed. The tumors and related organs were removed.

[0054] 1.6. Data recording and analysis

[0055] The tumor volume changes of each group were analyzed. ANOVA was used to analyze the data and compare the differences between groups.

[0056] 2. Experiment on the effect of traditional Chinese medicine prescriptions on the proliferation of liver cancer cells

[0057] Albizia julibrissin and Rhodoleia championii (flower material) have certain medicinal value in traditional Chinese medicine and folk herbal medicine. The effects of Albizia julibrissin include calming the mind, clearing heat and detoxifying, promoting blood circulation and removing blood stasis, and diuresis and swelling reduction; the effects of Rhodoleia championii include clearing heat and detoxifying, swelling and pain relief, and immune enhancement. In addition, both of them can enter the liver channel,

[0058] and play the role of soothing the liver and relieving depression.

[0059] This experiment aims to compare the inhibitory effect of traditional Chinese medicine prescription 1 (Meilianjunzi Decoction without Rhodoleia championii and Albizia julibrissin) described in the comparative example, and

[0060] traditional Chinese medicine prescription 2 (Meilianjunzi Decoction) described in Example 1 on tumor cells. This experiment is a cell experiment designed for the treatment of liver cancer by traditional Chinese medicine, aiming to observe the effects of different prescriptions on the proliferation of HepG2 liver cancer cells and the anti-cancer effects of different prescriptions.

[0061] 2.1. Preparation of drug-containing serum

[0062] 2.1.1. Drug administration: According to the decoction method of traditional Chinese medicine described in Examples 1 and 2, traditional Chinese medicine prescription 1 and traditional Chinese medicine prescription 2 were prepared into decoctions, and male SD rats were administered by gavage, twice a day, for 1 week.

[0063] The control group of rats was administered with the same amount of distilled water by gavage.

[0064] 2.1.2. Serum collection: Blood was taken from abdominal aorta 1 hour after the last administration, and left to clot for 2 hours. Centrifugation was performed at 3000 rpm for 15 minutes to obtain the supernatant. Filtration sterilization was performed through a 0.22 μm filter, aliquoted and stored at -80°C.

[0065] 2.2. Culture of liver cancer cells

[0066] HepG2 cells were cultured in DMEM medium at 37°C, 5% CO2 until the cell confluence reached 80-90%. The cells were treated with trypsin and re-cultured to the required cell density.

[0067] 2.3. Experimental grouping and administration

[0068] HepG2 cells were divided into 3 groups, each group with at least 3 replicates: TCM prescription 1 serum group, TCM prescription 2 serum group and blank serum group. The corresponding drug-containing serum was added to each group, with a concentration of 10%.

[0069] 2.4. Result detection

[0070] Each well was seeded with 3000 cells, and the IncuCyte Zoom live cell imaging system (2015A, Essen Bioscience, Ann Arbor, MI) was used to randomly record the proliferation of each well at 12 hours. Three observation points were used per well, and the average cell confluence was automatically calculated by the instrument.

[0071] 3. Experimental results:

[0072] 3.1 Effect of TCM prescription on liver cancer cell proliferation

[0073] In this experiment, the IncuCyte Zoom live cell imaging system (2015A, Essen Bioscience, Ann Arbor, MI) was used to randomly record the proliferation of each well of the blank serum group, TCM prescription 1 group and TCM prescription 2 group at 12 hours, as shown in the following figure. Compared with the blank serum group, the cell density and aggregation degree of the TCM prescription 1 group and the TCM prescription 2 group were significantly lower than those of the blank serum group (see Figure 1 A); and the average cell confluence of each group was automatically calculated by the instrument, and we found that compared with the blank serum group, the 12h confluence of the TCM prescription 1 group was significantly reduced (P=0.003), and the confluence of the TCM prescription 2 group was also significantly reduced (P<0.001), and the confluence of the TCM prescription 2 group was significantly lower than that of the TCM prescription 1 group (P=0.020) (see Figure 1 B). The above results all show that the prescription with green calyx and sweet-scented osmanthus significantly down-regulates the proliferation ability of liver cancer cells and has a certain inhibitory ability on liver cancer cells.

[0074] 3.2 Inhibitory effect of Meilianjunzi Decoction on tumor development

[0075] During the entire experiment, the health status of all mice remained good. Body weight fluctuated within the normal range, with no significant decrease or abnormal increase. In addition, the activity level of mice did not decrease significantly, indicating that they did not exhibit obvious symptoms of fatigue or weakness. This proves that the prescription used in this experiment has good biological safety.

[0076] In the mouse experiment, we administered the Chinese medicine group (Meilianjunzi Decoction) and the positive drug group, respectively, and did not administer the model group. Naked mice in the Chinese medicine group and the positive drug group had significantly smaller tumor volumes than naked mice in the model group (see Figure 2 A); at the same time, we recorded the growth of hepatocarcinoma cells by calculating the tumor volume every four days (V = (long diameter x short diameter2) / 2), and the results showed that the tumor volume of the model group without administration was significantly larger than that of the Chinese medicine group and the positive drug group as the number of days increased (see Figure 2 B); then we statistically analyzed the recorded tumor volumes of each group, and the results showed that the tumor volumes of the Chinese medicine group and the positive drug group were significantly smaller than that 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 hepatocarcinoma cells, and its effect is the same as that of positive drugs.

[0077] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A traditional Chinese medicine composition for treating liver cancer, characterized in that, The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: green apricot 10 parts, sweet-scented osmanthus 10 parts, female privet fruit 15 parts, spider lily 15 parts, yellow ginseng 30 parts, Chinese wolfberry root 30 parts, white atractylodes 15 parts, poria 15 parts, fried licorice 10 parts, dried ginger 6 parts, angelica 10 parts, red sage root 30 parts, Szechuan lovage 10 parts, half branch lotus 30 parts, white flower snake tongue grass 30 parts.

2. The preparation method of the traditional Chinese medicine composition according to claim 1, characterized in that, The method comprises the following steps: (1) before decocting, 15 kinds of medicinal materials are soaked with cold water, and the water level is kept 2 cm higher than the surface of the medicinal materials after soaking; (2) first decoction: after boiling with large fire, small fire is used for decocting, the medicinal juice is poured out and water is added for second decoction; (3) second decoction: continue to decoct with small fire; the medicinal liquid of two times of decoction is mixed, filtered and centrifuged to remove residue.

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

4. The preparation method of the traditional Chinese medicine composition according to claim 2, characterized in that, The soaking time in step (1) is 30 min, the decocting time in step (2) is 30 min, and the decocting time in step (3) is 20 min.

5. The preparation method of the traditional Chinese medicine composition according to any one of claims 2-4, characterized in that, Auxiliary materials are added to the traditional Chinese medicine composition to prepare corresponding dosage forms.

6. The preparation method of the traditional Chinese medicine composition according to claim 5, characterized in that, The dosage form is an oral dosage form.

7. The preparation method of the traditional Chinese medicine composition according to claim 6, characterized in that, The oral dosage form is an oral liquid, a tablet, a powder, a granule or a capsule. The oral dosage form is an oral liquid, a tablet, a powder, a granule or a capsule.

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