Traditional Chinese medicine preparation for treating liver cancer

Abstract

The invention provides a traditional Chinese medicine preparation for treating liver cancer, and relates to the technical field of traditional Chinese medicine preparation. The traditional Chinese medicine preparation is prepared from radix fici simplicissimae, euphorbia helioscopia, herba ardisiae japonicae, pollen typhae, ginseng, radix gentianae macrophyllae, cortex eucommiae, citrus aurantium, sambucus chinensis, hypericum japonicum and fructus ziziphi jujubae. In the formula, radix fici simplicissimae and euphorbia helioscopia are used as monarch drugs, herba ardisiae japonicae, pollen typhae, ginseng and gentiana macrophylla are used as ministerial drugs, citrus aurantium, sambucus chinensis, hypericum japonicum and eucommia ulmoides are used as adjuvant drugs, Chinese dates are used as conductant drugs, and the medicines in the whole formula are reasonably matched in monarch, ministerial, adjuvant and conductant drugs, give consideration to body resistance strengthening and evil eliminating, and jointly achieve the effects of tonifying qi, strengthening body resistance, dispersing blood stasis, detoxifying, clearing heat, promoting diuresis, soothing liver and regulating qi. The traditional Chinese medicine preparation has a remarkable inhibiting effect on proliferation, migration and invasion of hepatoma carcinoma cells, can effectively reduce the tumor volume and weight of tumor-bearing mice, slows down the tumor growth speed, can effectively improve the immune organ index and immune cell factors of the tumor-bearing mice, has an immunological enhancement effect, and can be used for treating liver cancer. The traditional Chinese medicine preparation is expected to serve as a new choice of medicines for treating liver cancer.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine pharmaceutical technology, specifically to a traditional Chinese medicine preparation for treating liver cancer. Background Technology

[0002] Liver cancer is one of the most common malignant tumors in China, divided into secondary liver cancer (SLC) and primary liver cancer (PLC), with hepatocellular carcinoma (HCC) being the most common type of PLC. The onset of liver cancer is associated with risk factors such as viral hepatitis, chronic alcohol consumption, and metabolic syndrome. Currently, the main treatments include surgical resection, radiotherapy and chemotherapy, targeted therapy, and liver transplantation. However, due to the high malignancy, rapid metastasis, and high postoperative recurrence rate of liver cancer, the overall survival rate after surgery has not significantly improved. In traditional Chinese medicine (TCM) theory, liver cancer is categorized as "liver accumulation" or "aggregation." TCM believes that the basic pathogenesis of liver cancer is insufficient vital energy (Qi), leading to invasion of the body and the production of pathological products such as heat, blood stasis, and phlegm accumulation, which eventually accumulate into a tumor. TCM treatment for liver cancer combines strengthening the body's resistance with eliminating pathogenic factors, regulating the body's immune function to achieve an anti-tumor effect. The combined treatment of TCM and Western medicine can effectively improve the patient's immunity, improve quality of life, and prolong survival. Currently, several studies have reported that traditional Chinese medicine (TCM) preparations for treating relapsed liver cancer have shown significant efficacy. Therefore, this invention aims to develop a TCM compound preparation with definite efficacy and significant anti-tumor effects, providing new ideas and options for the treatment of liver cancer. Summary of the Invention

[0003] (a) Technical problems to be solved

[0004] In view of the shortcomings of the prior art, the present invention provides a traditional Chinese medicine preparation for the treatment of liver cancer.

[0005] (II) Technical Solution

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] First, the present invention provides a traditional Chinese medicine preparation for treating liver cancer, which is made from the following traditional Chinese medicinal materials in parts by weight: 20-40 parts of *Ficus hirta*, 20-40 parts of *Euphorbia helioscopia*, 20-40 parts of *Tetrapanax papyriferus*, 20-40 parts of *Pyrola rotundifolia*, 20-40 parts of *Gentiana macrophylla*, 20-40 parts of *Eucommia ulmoides*, 10-30 parts of *Citrus aurantium*, 10-30 parts of *Hedyotis diffusa*, 10-30 parts of *Gynostemma pentaphyllum*, 10-30 parts of *Gynostemma pentaphyllum*, and 5-20 parts of jujube.

[0008] Furthermore, the traditional Chinese medicine preparation for treating liver cancer is made from the following parts by weight of Chinese medicinal materials: 25-35 parts of *Ficus hirta*, 25-35 parts of *Euphorbia helioscopia*, 20-30 parts of *Tetrapanax papyriferus*, 20-30 parts of *Typha orientalis*, 25-35 parts of *Gentiana macrophylla*, 25-35 parts of *Gentiana macrophylla*, 15-25 parts of *Eucommia ulmoides*, 15-25 parts of *Citrus aurantium*, 15-25 parts of *Hedyotis diffusa*, 15-25 parts of *Gynostemma pentaphyllum*, and 5-15 parts of jujube.

[0009] Furthermore, the traditional Chinese medicine preparation for treating liver cancer is made from the following parts by weight of Chinese medicinal materials: 32 parts of five-finger peach, 30 parts of Euphorbia helioscopia, 24 parts of dwarf ground jasmine, 24 parts of cattail pollen, 28 parts of Asarum heterotropoides, 26 parts of Gentiana macrophylla, 18 parts of Eucommia ulmoides, 22 parts of Citrus aurantium, 20 parts of Lu Ying, 18 parts of Di Er Cao, and 10 parts of jujube.

[0010] Furthermore, the traditional Chinese medicine preparation for treating liver cancer provided by the present invention is made from the above-mentioned proportions of Chinese medicinal materials by weight, supplemented with pharmaceutically acceptable excipients, and prepared into any one of oral liquid, granules, powders, pills, tablets, capsules, and decoctions.

[0011] The relevant descriptions of each Chinese medicinal material in the traditional Chinese medicine preparation of this invention are as follows:

[0012] Five-finger fig (Ficus simplicissima Lour.): This is the root of the plant *Ficus simplicissima* Lour., belonging to the Moraceae family. It has a sweet taste and neutral properties, entering the spleen, lung, and liver meridians. It is used to treat spleen deficiency edema, poor appetite and weakness, pulmonary tuberculosis cough, night sweats, leukorrhea, postpartum agalactia, rheumatic pain, edema, ascites due to cirrhosis, hepatitis, and traumatic injuries.

[0013] Euphorbia helioscopia: The dried whole herb of Euphorbia helioscopia, a plant of the Euphorbiaceae family. It is pungent, bitter, and slightly cold in nature, and is poisonous. It has the effects of promoting diuresis and reducing swelling, resolving phlegm and relieving cough, and dispersing nodules. Clinically, it is often used for edema, cough and asthma with phlegm, scrofula and sores.

[0014] Lowland tea: The dried whole plant of Ardisia crenata, a plant of the Myrsinaceae family; it is neutral in nature and has a pungent and slightly bitter taste; it can resolve phlegm and stop cough, promote diuresis and invigorate blood; it is used for cough, hemoptysis, chronic bronchitis, damp-heat jaundice, and traumatic injuries.

[0015] Typha pollen: the dried pollen of Typha angustifolia L., Typha orientalis Presl, or related species of the Typhaceae family; sweet and neutral in nature, it enters the liver and pericardium meridians; it has the effects of stopping bleeding, removing blood stasis, and promoting urination; it is used for hematemesis, epistaxis, hemoptysis, metrorrhagia, traumatic bleeding, amenorrhea, dysmenorrhea, chest and abdominal pain, traumatic swelling and pain, and painful hematuria.

[0016] Xin Shen: The tuberous root of *Phlomis betonicoides* Diels, a plant in the Lamiaceae family; it is sweet and bitter in taste, and cool in nature; it has the effects of regulating qi and strengthening the spleen, detoxifying and reducing swelling. It is commonly used for indigestion, abdominal distension and pain, diarrhea, cough and asthma, chest tightness, colds, sore throat, drug poisoning, and traumatic injuries with swelling and bruises.

[0017] Gentiana macrophylla: the dried root of Gentiana macrophylla Pall., Gentiana straminea Maxim., Gentiana crassicaulis Duthie ex Burk., or Gentianadahurica Fisch. (Gentianaceae family); pungent and bitter in taste, neutral in nature; enters the stomach, liver, and gallbladder meridians; has the functions of dispelling wind and dampness, clearing damp heat, relieving pain, and reducing deficiency heat; used for rheumatic pain, hemiplegia due to stroke, muscle spasms, joint pain, damp-heat jaundice, bone steaming fever, and infantile malnutrition with fever.

[0018] Eucommia ulmoides: The dried bark of Eucommia ulmoides, a plant in the Eucommiaceae family. Also known as eucommia bark. It is sweet and warm in nature, and enters the liver and kidney meridians. It contains lignans, iridoids, and various amino acids and trace elements. It has the effects of tonifying the liver and kidneys, strengthening tendons and bones, and calming the fetus. It is mainly used to treat symptoms such as lower back and knee pain, fetal restlessness, and high blood pressure.

[0019] Bitter orange blossom: Derived from the dried flower buds or newly opened flowers of the bitter orange tree (Bitter Orange), a plant in the Rutaceae family. It is slightly bitter and neutral in nature, and enters the liver and stomach meridians. It has the effects of soothing the liver and stomach, regulating qi and relieving depression, and is often used to alleviate symptoms such as abdominal distension, loss of appetite, and liver qi stagnation. Modern research shows that bitter orange blossom contains volatile oils, flavonoids, and other components, which have antioxidant, anti-inflammatory, and gastrointestinal regulating effects.

[0020] Lu Ying: The dried aerial parts of the plant Lu Ying (Sambucus chinensis) of the Caprifoliaceae family. It is sweet, bland, slightly bitter, and neutral in nature. It has the effects of dispelling wind and dampness, promoting blood circulation and removing blood stasis, and promoting diuresis and reducing swelling. It is often used to treat rheumatic pain, traumatic injuries, edema, and other symptoms.

[0021] Hypericum perforatum, a plant of the genus Hypericum in the family Clusiaceae, is also known as field sedge. It is bitter and cool in nature, and enters the liver and gallbladder meridians. It has the effects of clearing heat and detoxifying, promoting diuresis and relieving jaundice, reducing swelling and dissipating nodules. Clinically, it is often used for diseases such as damp-heat jaundice, carbuncles and boils, and traumatic injuries.

[0022] Jujube: The mature fruit of the jujube plant (Ziziphus jujuba), belonging to the Rhamnaceae family; sweet and warm in nature, it enters the spleen, stomach, and heart meridians; its functions and indications include: tonifying the spleen and stomach, replenishing qi and generating fluids, regulating the body's defenses, and detoxifying. It is used to treat conditions such as poor appetite due to stomach deficiency, loose stools due to spleen weakness, insufficient qi, blood, and body fluids, disharmony between the body's defenses, palpitations, and hysteria in women.

[0023] (III) Beneficial Effects

[0024] In Traditional Chinese Medicine (TCM), liver cancer is classified as "liver accumulation" or "accumulation," primarily manifested as spleen deficiency and impaired transport, phlegm and blood stasis, and damp-heat stagnation. Based on this pathogenesis, this invention presents a self-formulated TCM preparation for treating liver cancer, composed of five-finger peach, eupatorium fortunei, dwarf groundcherry, cattail pollen, asarum, gentian, eucommia, bitter orange blossom, romaine lettuce, ground lily, and jujube. Five-finger peach in the formula has the effects of invigorating qi and strengthening the spleen, promoting qi circulation and eliminating dampness, and relaxing muscles and tendons. It can replenish the spleen and stomach to strengthen the acquired constitution, and its qi-regulating and dampness-eliminating properties help to clear stagnation, thus supporting the body's resistance. Eupatorium fortunei, being bitter and cold, can clear away blood stasis and toxins; its pungent and dispersing properties can break up stagnation and resolve phlegm; its diuretic effect can expel dampness and turbidity, thus eliminating pathogenic factors. Five-finger peach and eupatorium fortunei work synergistically as the principal herbs, working together to invigorate qi and strengthen the spleen, resolve phlegm and detoxify, and support the body's resistance while eliminating pathogenic factors. Low-lying tea has the effects of clearing heat and dampness, resolving phlegm and dissipating nodules, and promoting blood circulation and removing blood stasis. When combined with the principal herb Euphorbia helioscopia, it enhances the overall effect of resolving phlegm and removing blood stasis. Typha pollen can remove blood stasis and stop bleeding, removing blood stasis without harming the body's vital energy. Asarum sibiricum regulates qi, strengthens the spleen, detoxifies and reduces swelling. When combined with the principal herb Ficus hirta, it enhances the effect of strengthening the spleen. Its cool nature can clear latent heat in the spleen and stomach, and its detoxifying effect can clear blood stasis and toxins. Gentiana macrophylla clears damp heat, reduces deficiency heat, unblocks meridians, and relieves pain. When combined with Euphorbia helioscopia and low-lying tea, it works together to clear damp heat, unblock meridians and remove blood stasis. Low-lying tea, Typha pollen, Asarum sibiricum, and Gentiana macrophylla are all assistant herbs, working together to clear heat, promote blood circulation, remove dampness, and unblock meridians, assisting the principal herb in eliminating the pathogenic products of phlegm, blood stasis, and toxins. Then, bitter orange blossom, ground ivy, ground ivy, and eucommia bark are used as adjuvant herbs. Bitter orange blossom soothes the liver, regulates qi, harmonizes the stomach, and stops vomiting, helping the principal and assistant herbs to achieve the same qi and blood regulation. Ground ivy invigorates blood, removes blood stasis, detoxifies, and reduces swelling, helping the principal and assistant herbs to enhance their ability to remove blood stasis and detoxify. Ground ivy clears heat, promotes diuresis, detoxifies, and reduces swelling, helping the principal herb, Euphorbia helioscopia, to break up masses and enhance the overall formula's ability to expel pathogens. Eucommia bark tonifies the liver and kidneys, strengthens tendons and bones, and protects the innate foundation, forming a tonifying effect with the principal herb, five-finger peach, which strengthens the spleen. Finally, jujube is used as the guiding herb to tonify the middle jiao, replenish qi, nourish blood, calm the mind, and harmonize the effects of all the herbs. The combination of all the herbs in the formula is reasonable, balancing the tonifying effect with the expelling pathogens, achieving the effects of tonifying qi, removing blood stasis, detoxifying, clearing heat, promoting diuresis, and soothing the liver and regulating qi.

[0025] This invention comprehensively evaluated the anti-liver cancer effect of the traditional Chinese medicine preparation through in vivo and in vitro experiments. In vitro experimental results showed that the traditional Chinese medicine preparation of this invention significantly inhibited the proliferation, migration, and invasion of liver cancer cells. Furthermore, in vivo experiments demonstrated that the traditional Chinese medicine preparation of this invention exhibited good anti-tumor effects, effectively reducing tumor volume and weight in tumor-bearing mice, slowing tumor growth, and effectively improving immune organ indices and immune cytokines in tumor-bearing mice, thus possessing an immune-enhancing effect. Especially when used in combination with DOX, it significantly enhanced both the anti-tumor and immune-enhancing effects. The traditional Chinese medicine preparation of this invention shows promise as a new option for treating liver cancer. Attached Figure Description

[0026] Figure 1 The change in body weight of mice in each group during the experiment.

[0027] Figure 2Tumor volume changes in each group of mice during the experiment. Note: Compared with the Mod group, *P<0.05, **P<0.01, ***P<0.001; compared with the DOX group, # P<0.05, ## P<0.01.

[0028] Figure 3 The spleen index and thymus index of mice in each group were measured 24 hours after the end of drug administration. Note: *P<0.05, **P<0.01, ***P<0.001;

[0029] Figure 4 The CD4 count in peripheral blood of mice in each group was measured 24 hours after the end of drug administration. + CD8 + and CD4 + / CD8 + Level. Note: ns P>0.05, *P<0.05, **P<0.01, ***P<0.001; Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] A traditional Chinese medicine preparation for treating liver cancer is made from the following medicinal materials in parts by weight: 32 parts of five-finger peach, 30 parts of Euphorbia helioscopia, 24 parts of dwarf ground jasmine, 24 parts of cattail pollen, 28 parts of Asarum heterotropoides, 26 parts of Gentiana macrophylla, 18 parts of Eucommia ulmoides, 22 parts of Citrus aurantium, 20 parts of Lu Ying, 18 parts of Di Er Cao, and 10 parts of jujube.

[0033] The above-mentioned traditional Chinese medicine preparations for treating liver cancer are made from the above-mentioned proportions of Chinese medicinal materials by weight, supplemented with pharmaceutically acceptable excipients, and prepared into any one of the following: oral liquid, granules, powders, pills, tablets, capsules, and decoctions.

[0034] Example 2

[0035] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicinal materials: 20 parts of five-finger peach, 20 parts of Euphorbia helioscopia, 20 parts of dwarf tea, 20 parts of cattail pollen, 20 parts of ginseng, 20 parts of Gentiana macrophylla, 10 parts of Eucommia ulmoides, 10 parts of bitter orange flower, 10 parts of Lu Ying, 10 parts of lichen, and 5 parts of jujube.

[0036] Example 3

[0037] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicinal materials: 40 parts of five-finger peach, 40 parts of Euphorbia helioscopia, 40 parts of dwarf ground tea, 40 parts of cattail pollen, 40 parts of ginseng, 40 parts of Gentiana macrophylla, 30 parts of Eucommia ulmoides, 30 parts of bitter orange flower, 30 parts of Lu Ying, 30 parts of lichen, and 20 parts of jujube.

[0038] Example 4

[0039] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicinal materials: 38 parts of five-finger peach, 36 parts of Euphorbia helioscopia, 32 parts of dwarf ground jasmine, 32 parts of cattail pollen, 36 parts of Asarum heterotropoides, 36 parts of Gentiana macrophylla, 27 parts of Eucommia ulmoides, 26 parts of Citrus aurantium, 28 parts of Lu Ying, 26 parts of Di Er Cao, and 18 parts of jujube.

[0040] Example 5

[0041] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation for treating liver cancer is made from the following parts by weight of traditional Chinese medicinal materials: 35 parts of five-finger peach, 35 parts of Euphorbia helioscopia, 30 parts of dwarf ground jasmine, 30 parts of cattail pollen, 35 parts of Asarum heterotropoides, 35 parts of Gentiana macrophylla, 25 parts of Eucommia ulmoides, 25 parts of Citrus aurantium, 25 parts of Lu Ying, 25 parts of Di Er Cao, and 15 parts of jujube.

[0042] Example 6

[0043] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation for treating liver cancer is made from the following parts by weight of Chinese medicinal materials: 25 parts of five-finger peach, 25 parts of Euphorbia helioscopia, 20 parts of dwarf ground jasmine, 20 parts of cattail pollen, 25 parts of Asarum heterotropoides, 25 parts of Gentiana macrophylla, 15 parts of Eucommia ulmoides, 15 parts of Citrus aurantium, 15 parts of Lu Ying, 15 parts of Di Er Cao, and 5 parts of jujube.

[0044] Example 7

[0045] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation for treating liver cancer is made from the following parts by weight of traditional Chinese medicinal materials: 27 parts of five-finger peach, 25 parts of Euphorbia helioscopia, 21 parts of dwarf tea, 21 parts of cattail pollen, 26 parts of ginseng, 26 parts of gentian root, 18 parts of eucommia bark, 21 parts of bitter orange flower, 18 parts of dandelion, 21 parts of ground lily, and 9 parts of jujube.

[0046] Example 8

[0047] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation for treating liver cancer is made from the following parts by weight of traditional Chinese medicinal materials: 30 parts of five-finger peach, 30 parts of Euphorbia helioscopia, 25 parts of dwarf ground jasmine, 25 parts of cattail pollen, 27 parts of Asarum heterotropoides, 27 parts of Gentiana macrophylla, 20 parts of Eucommia ulmoides, 20 parts of Citrus aurantium, 20 parts of Lu Ying, 20 parts of Di Er Cao, and 10 parts of jujube.

[0048] Experimental Example 1

[0049] Evaluation of the in vitro anti-hepatocellular carcinoma effect of the traditional Chinese medicine preparation of this invention

[0050] 1. Experimental drug

[0051] Weigh out the following Chinese medicinal materials according to the following weight proportions: 32 parts of Five-Finger Peach, 30 parts of Euphorbia helioscopia, 24 parts of Dwarf Tea, 24 parts of Typha pollen, 28 parts of Asarum sieboldii, 26 parts of Gentiana macrophylla, 18 parts of Eucommia ulmoides, 22 parts of Bitter Orange Flower, 20 parts of Lu Ying, 18 parts of Earth Ear Grass, and 10 parts of Jujube. Each weight proportion is 1g. Wash the herbs, add 3 times the amount of water and decoct for 40 minutes. Repeat the decoction at least twice. Collect the decoction liquid and concentrate it to 1g / mL (1mL contains 1g of raw herbs) to obtain the Chinese herbal extract. Store at 4℃ for later use.

[0052] 2 Experimental Methods

[0053] 2.1 Preparation of drug-containing serum from rats

[0054] Sixteen 6-8 week old male SD rats were acclimatized for 3 days and then randomly divided into a control group, a low-dose traditional Chinese medicine (TCM) group (5 g / kg), a medium-dose TCM group (10 g / kg), and a high-dose TCM group (15 g / kg). The low, medium, and high-dose TCM groups were administered the corresponding doses of TCM extract by gavage, while the control group was administered an equal volume of physiological saline by gavage. After 7 consecutive days of gavage, the rats were anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta. The supernatant was collected by centrifugation at 3500 r / min for 10 min to obtain the corresponding drug-containing serum. The serum was inactivated at 56℃, filtered through a 0.22 µm microporous membrane, and stored at -20℃ for later use.

[0055] 2.2 Cell proliferation experiment

[0056] Select HepG2 cells in logarithmic growth phase, at a rate of 1×10⁻⁶. 5 Cells were seeded per well in 96-well plates. After 24 hours of full cell adhesion, four groups were established: a control group (NC), a low-dose group (L group), a medium-dose group (M group), and a high-dose group (H group). 50 μL of the drug-containing serum prepared in section 2.1 was added to each well, with the remainder added to basal medium to a final volume of 200 μL per well. Six replicates were used per group. After 48 hours of culture, the medium was replaced with fresh medium, MTT was added, and the cells were cultured for another 4 hours. DMSO was then added, and the absorbance was measured. The cell proliferation inhibition rate was calculated as: Cell proliferation inhibition rate = (1 - OD value of drug-containing serum group / OD value of control group) × 100%.

[0057] 2.3 Transwell Invasion Experiment

[0058] HepG2 cells in logarithmic growth phase were harvested at a rate of 1×10⁻⁶. 5Cells were seeded per well in 96-well plates. After 24 hours of full cell adhesion, four groups were established: control group (NC), low-dose group (L group), medium-dose group (M group), and high-dose group (H group). 50 μL of the drug-containing serum prepared in section 2.1 was added to each group, with 6 replicates per well. The matrix gel was mixed with the basal medium, and the cells were cultured in serum-free medium for 24 hours to obtain 2.5 × 10⁶ cells / well plates. 5 100 μL of cell suspension was added to the upper chamber, and 500 μL of complete culture medium was added to the lower chamber. After standing for 15 min, the cells were incubated for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min, washed twice with PBS, stained with 0.1% crystal violet for 10 min, washed with ultrapure water, observed under a microscope, analyzed with ImageJ software, and the number of invasive cells in each group was counted.

[0059] 2.4 Cell Scratch Assay

[0060] HepG2 cells in logarithmic growth phase were harvested at a rate of 1×10⁻⁶. 5 Cells were seeded per well in 96-well plates. After 24 hours of full cell adhesion, four groups were established: control (NC), low-dose (L), medium-dose (M), and high-dose (H). 50 μL of the drug-containing serum prepared in section 2.1 was added to each well, with the remainder added to basal culture medium to a final volume of 200 μL per well, with 6 replicates per group. Once the cells had reached confluence with the bottom of the culture dish, a 200 μL pipette tip was used to make a line perpendicular to the central axis of symmetry of the culture dish. The drawn cells were washed away with PBS. Cells were observed and photographed under a microscope at 0, 24, and 48 hours, and the location of the drawn lines was recorded. ImageJ software was used to analyze the area not covered at 48 hours, and the change in area was used to assess healing efficiency. Cell migration rate was calculated as follows: Cell migration rate = (0h drawn line area - 24h / 48h drawn line area) / 0h drawn line area × 100%

[0061] 2.4 Statistical Analysis

[0062] Data were analyzed and statistically processed using GraphPad Prism v.8.0. Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0063] 3 Results

[0064] As shown in Table 1, the traditional Chinese medicine preparation of this invention achieved a 67.62% inhibition rate against HepG2 cell proliferation. Furthermore, compared to the control group, the traditional Chinese medicine preparation effectively reduced the number of invasive HepG2 cells and the cell migration rate at 24h and 48h, with significant differences (P<0.01). These results demonstrate that the traditional Chinese medicine preparation of this invention has a significant inhibitory effect on the proliferation, migration, and invasion of HepG2 cells in vitro, providing strong evidence that it can be used as a drug for treating liver cancer.

[0065] Table 1. Effects of the traditional Chinese medicine preparation of this invention on the proliferation, invasion, and migration abilities of HepG2 cells.

[0066] Group Cell proliferation inhibition rate / % Cell invasion count / cell 24h cell migration rate / % 48h cell migration rate / % NC Group — 96.17±12.73 28.98±2.97 68.84±7.85 Group L 37.77±4.16 50.33±6.71** 15.43±2.36** 39.52±5.65** Group M 48.67±2.85 23.17±4.62** 11.05±1.87** 30.05±3.61** Group H 67.26±4.48 9.00±4.20** 8.15±1.64** 17.85±1.74**

[0067] Note: Compared with the NC group, *P<0.05, **P<0.01.

[0068] Experimental Example 2

[0069] Evaluation of the in vivo anti-liver cancer effect of the traditional Chinese medicine preparation of this invention

[0070] 1. Experimental Materials

[0071] 1.1 Test Drug

[0072] Weigh out the following Chinese medicinal materials according to the following weight proportions: 32 parts of Five-Finger Peach, 30 parts of Euphorbia helioscopia, 24 parts of Dwarf Tea, 24 parts of Typha pollen, 28 parts of Asarum sieboldii, 26 parts of Gentiana macrophylla, 18 parts of Eucommia ulmoides, 22 parts of Bitter Orange Flower, 20 parts of Lu Ying, 18 parts of Earth Ear Grass, and 10 parts of Jujube. Each weight proportion is 1g. Wash the herbs, add 3 times the amount of water and decoct for 40 minutes. Repeat the decoction at least twice. Collect the decoction liquid and concentrate it to 1g / mL (1mL contains 1g of raw herbs) to obtain the Chinese herbal extract. Store at 4℃ for later use.

[0073] 1.2 Laboratory Animals

[0074] Fifty-six male KM mice, weighing 18–20 g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0075] 2 Experimental Methods

[0076] 2.1 Animal modeling and grouping

[0077] Eight mice were randomly selected as the blank group (NC), and the remaining mice were used to construct a subcutaneous xenograft animal model of liver cancer. The method was as follows: mouse liver cancer H22 cells were routinely cultured and inoculated into the liver of adult male mice. After inoculation, the growth of subcutaneous tumors in nude mice was observed regularly. The formation of tumors could be observed with the naked eye within 7-10 days after inoculation. When the tumor grew to 6 mm, it indicated that the model was successfully established. Nude mice bearing tumors that had successfully developed the tumor model were randomly divided into a model group (Mod group), a positive control group (DOX group, doxorubicin, 1.2 mg / kg), a low-dose group (Low group, 2 g / kg of traditional Chinese medicine extract), a high-dose group (High group, 4 g / kg of traditional Chinese medicine extract), a combined drug group 1 (DOX+Low group, 1.2 mg / kg doxorubicin + 2 g / kg of traditional Chinese medicine extract), and a combined drug group 2 (DOX+High group, 1.2 mg / kg doxorubicin + 4 g / kg of traditional Chinese medicine extract), with 8 mice in each group. Except for the model group and the blank group, which were given physiological saline, each drug group was administered the drug once daily by gavage for 14 consecutive days.

[0078] 2.2 Observation Indicators

[0079] 2.2.1 Changes in body weight of liver cancer model mice

[0080] During the drug administration period, the general condition of the mice was observed, including their weight, coat cleanliness, food and water intake, activity level, and mental state. The body weight of mice in each group was measured before drug administration and on days 3, 7, 10, and 14 after drug administration, and the changes in average body weight were observed and calculated.

[0081] 2.2.2 Tumor Changes in Liver Cancer Model Mice

[0082] The volume of subcutaneous tumors in mice in each group was measured before drug administration and at 3, 7, 10 and 14 days after drug administration. The changes in the average volume of subcutaneous tumors were observed and calculated.

[0083] Twenty-four hours after the end of drug administration, mice were euthanized by cervical dislocation, the intact tumor was removed, the surface blood was blotted dry with filter paper, and the weight was recorded. The average tumor volume, average tumor mass, and tumor inhibition rate were calculated.

[0084] Tumor volume inhibition rate = (mean tumor volume in the model group - mean tumor volume in the treatment group) / mean tumor volume in the model group × 100%

[0085] Tumor mass inhibition rate = (Average tumor mass in the model group - Average tumor mass in the treatment group) / Average tumor volume in the model group × 100%

[0086] 2.2.3 Immune organ index

[0087] Twenty-four hours after the administration of the drug, the mice were euthanized by cervical dislocation, and the thymus and spleen were removed, weighed, and recorded. The thymus and spleen indices were calculated.

[0088] Organ Index = Organ Mass (mg) / Body Mass (g)

[0089] 2.2.4 Serum levels of immune cytokines

[0090] 24 hours after the end of drug administration, blood was collected from the eyeballs, centrifuged, and the supernatant was used to obtain serum. CD4 count was then performed using flow cytometry. + CD8 + The level of expression.

[0091] 2.3 Statistical Analysis

[0092] Data were analyzed and statistically processed using GraphPad Prism v.8.0. Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0093] 3 Results

[0094] 3.1 Changes in body weight and tumor volume in each group of mice

[0095] During the experiment, no mice died in any group, and the overall body weight of mice in each group showed an increasing trend, with no statistically significant difference in body weight among the groups (P>0.05). The results are as follows: Figure 1 As shown.

[0096] Tumor growth in each group of mice is as follows: Figure 2 As shown in the figure, the tumors in the model group mice grew the fastest and had the largest volume. Compared with the model group, on day 3 of administration, the DOX group, DOX+Low group, and DOX+High group already showed an inhibitory effect on tumor growth, with significant differences (P<0.05); on day 7 of administration, the DOX group, High group, DOX+Low group, and DOX+High group all showed significant inhibitory effects on tumor growth (P<0.01); on day 10 of administration, all administration groups showed significant inhibitory effects on tumor growth (P<0.001); after day 14 of administration, the tumor volume in the DOX+Low group and DOX+High group was significantly lower than that in the DOX group. These results indicate that the traditional Chinese medicine preparation of this invention synergistically enhances the anti-tumor effect with DOX.

[0097] 3.2 Tumor inhibition rate of mice in each group

[0098] As shown in Table 2, compared with the model group, the average tumor volume and average tumor mass of mice in each treatment group were significantly reduced (P<0.01); among them, compared with the DOX group, the tumor volume and mass of mice in the DOX+Low and DOX+High groups were significantly lower than those in the DOX group (P<0.05, P<0.01). These results indicate that the traditional Chinese medicine preparation of this invention can effectively inhibit tumor growth in tumor-bearing mice, and its combination with DOX can significantly enhance the anti-tumor effect.

[0099] Table 2. Average tumor volume, average tumor mass, and tumor inhibition rate in mice of each group.

[0100] Group <![CDATA[Average tumor volume / mm 2 > Average tumor mass / g Tumor volume inhibition rate / % Tumor quality inhibition rate / % Mod 777.75±32.98 0.8054±0.0412 — — DOX <![CDATA[387.25±29.65 ** ]]> <![CDATA[0.4758±0.0126 ** ]]> 50.21 40.92 Low <![CDATA[498.38±41.45 **# ]]> <![CDATA[0.5420±0.0337 **# ]]> 35.92 32.70 High <![CDATA[427.13±49.09 ** ]]> <![CDATA[0.4858±0.0253 ** ]]> 45.08 39.68 DOX+Low <![CDATA[326.75±47.16 **# ]]> <![CDATA[0.3415±0.0257 **# ]]> 57.99 57.60 DOX+High <![CDATA[293.50±47.76 **## ]]> <![CDATA[0.2758±0.214 **## ]]> 62.26 62.76

[0101] Note: Compared with the Mod group, **P<0.01; compared with the DOX group, # P<0.05, ## P<0.01.

[0102] 3.3 Immune organ indices and immune cytokines in each group of mice

[0103] like Figure 3 As shown, compared with the NC group, the thymus index and spleen index of the Mod group mice were significantly decreased (P<0.05, P<0.001); compared with the model group, the thymus index and spleen index of the DOX group mice were further decreased (P<0.001); compared with the DOX group, the thymus index and spleen index of the Low group, High group, DOX+Low group and DOX+High group mice were all increased to varying degrees, with significant differences (P<0.05, P<0.01). The above results indicate that the traditional Chinese medicine preparation of the present invention can effectively improve the immune organ index of mice and improve the damage of DOX to immune organs.

[0104] like Figure 4 As shown, compared to the NC group, the Mod group has higher CD4 counts. + CD8 + The percentage of T lymphocytes was significantly decreased (P<0.001), CD4 + / CD8 + The CD4 count was significantly lower (P<0.001), indicating a decline in cellular immune function in tumor-bearing mice. Compared with the Mod group, the CD4 count in the DOX and Low groups was significantly lower. + / CD8 + There was an increase, but the difference was not statistically significant (P>0.05). CD4 levels were observed in the High group, DOX+Low group, and DOX+High group. + / CD8 + Compared to the model group, the CD4 count increased significantly (P<0.001). Specifically, the Low, High, DOX+Low, and DOX+High groups showed significantly higher CD4 counts. + / CD8 + All levels were higher than those in the DOX group to varying degrees. These results indicate that the traditional Chinese medicine preparation of this invention can improve cellular immune function in tumor-bearing mice, and its combination with DOX significantly enhances immune function.

[0105] In summary, the experimental results of this invention demonstrate that the traditional Chinese medicine preparation exhibits good anti-tumor effects, effectively reducing tumor volume and weight in tumor-bearing mice, slowing tumor growth, and effectively improving immune organ indices and immune cytokines in tumor-bearing mice, thus possessing an immune-enhancing effect. In particular, its combination with DOX significantly enhances both anti-tumor and immune-enhancing effects. This traditional Chinese medicine preparation shows promise as a new option for treating liver cancer.

[0106] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine preparation for treating liver cancer, characterized in that, The traditional Chinese medicine preparation is made from the following weight parts of traditional Chinese medicinal materials: Ficus tikoua 20-40 parts, Jatropha curcas 20-40 parts, Kalmia latifolia 20-40 parts, Typha angustifolia 20-40 parts, Panacis quinquefolii radix 20-40 parts, Gentiana macrophylla 20-40 parts, Eucommia ulmoides 10-30 parts, Dalbergia hupeana 10-30 parts, Humulus scandens 10-30 parts, Galium verum 10-30 parts, Ziziphus jujuba 5-20 parts.

2. The traditional Chinese medicine preparation for treating liver cancer according to claim 1, characterized in that, The traditional Chinese medicine preparation is made from the following weight parts of traditional Chinese medicinal materials: Ficus tikoua 25-35 parts, Jatropha curcas 25-35 parts, Kalmia latifolia 20-30 parts, Typha angustifolia 20-30 parts, Panacis quinquefolii radix 25-35 parts, Gentiana macrophylla 25-35 parts, Eucommia ulmoides 15-25 parts, Dalbergia hupeana 15-25 parts, Humulus scandens 15-25 parts, Galium verum 15-25 parts, Ziziphus jujuba 5-15 parts.

3. The traditional Chinese medicine preparation for treating liver cancer according to claim 1, characterized in that, The traditional Chinese medicine preparation is made from the following weight parts of traditional Chinese medicinal materials: Ficus tikoua 32 parts, Jatropha curcas 30 parts, Kalmia latifolia 24 parts, Typha angustifolia 24 parts, Panacis quinquefolii radix 28 parts, Gentiana macrophylla 26 parts, Eucommia ulmoides 18 parts, Dalbergia hupeana 22 parts, Humulus scandens 20 parts, Galium verum 18 parts, Ziziphus jujuba 10 parts.

4. The traditional Chinese medicine preparation for treating liver cancer according to any one of claims 1 to 3 is made from the above weight parts of traditional Chinese medicinal materials as raw materials, supplemented with pharmaceutically acceptable adjuvants, into any one of oral liquid, granules, powder, pills, tablets, capsules, and decoction.

Images