A traditional Chinese medicine composition for advanced primary liver cancer and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for advanced primary liver cancer, a medicine comprising the traditional Chinese medicine composition and a preparation method of the medicine. The application also provides application of the traditional Chinese medicine composition and the medicine in treatment of advanced primary liver cancer, in particular, in treatment of advanced primary liver cancer which cannot be resected by operation. The traditional Chinese medicine composition comprises Ginkgo biloba 1-50 parts, Phyllanthus emblica 1-35 parts, Aeginetia indica 1-30 parts, Artemisia annua 1-30 parts, Citrus reticulata 1-35 parts, Citrus aurantium 1-35 parts, Citrus aurantium 1-20 parts, Citrus reticulata 1-30 parts and Citrus aurantium 1-35 parts. The application provides a new choice for clinical treatment of patients with advanced primary liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a traditional Chinese medicine composition for advanced primary liver cancer and its application. Background Technology

[0002] Primary liver cancer (PLC) refers to malignant tumors that occur in hepatocytes or intrahepatic bile duct cells, with hepatocellular carcinoma accounting for the vast majority of primary liver cancers. Primary liver cancer is one of the most common and most dangerous malignant tumors. In recent years, the incidence of primary liver cancer has been increasing worldwide, and its incidence and mortality rates rank 5th and 3rd among malignant tumors, respectively (Sung H, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021; 71(3):209-249. doi:10.3322 / caac.21660). Modern medicine treats primary liver cancer primarily based on its stage, including surgical treatment (hepatectomy, liver transplantation), ablation therapy, transarterial chemoembolization (TACE), molecularly targeted therapy, antiviral therapy and biotherapy, radiotherapy, comprehensive chemotherapy, and various rapidly developing biotechnologies such as immunotherapy, gene therapy, and endocrine therapy. Surgical treatment is the preferred and most effective method. However, most patients with primary liver cancer show no significant symptoms in the early stages, and once diagnosed, the disease often progresses to the middle or late stages, at which point most patients have lost the opportunity for surgery and cannot achieve a radical cure. Therefore, non-surgical drug therapy is an important treatment option for patients who cannot undergo surgery or have already undergone surgery. Although drugs for advanced primary liver cancer have always been a hot topic in medical research, clinical needs remain far from being met.

[0003] In traditional Chinese medicine (TCM) theory, primary liver cancer falls under the categories of "abdominal masses, accumulations, ascites, lumps, scrofula, and obesity." Most physicians believe that its most important and fundamental pathogenesis is deficiency leading to accumulation and internal accumulation of toxins and blood stasis. This is often caused by factors such as deficiency of vital energy, exposure to pathogenic toxins, emotional distress, dietary damage, and pre-existing conditions, which lead to dysfunction of the internal organs, abnormal circulation of qi, blood, and body fluids, resulting in pathological changes such as qi stagnation, blood stasis, phlegm, dampness, and heat toxins. These accumulate in the internal organs, interacting and binding with each other, gradually forming a type of malignant disease characterized by deficiency in the root and excess in the branch.

[0004] Given the complex and intertwined nature of liver cancer patients, characterized by deficiency of the root cause and excess of the symptoms, Traditional Chinese Medicine (TCM) treatment for liver cancer often adopts the principle of supporting the body's resistance and eliminating pathogenic factors, addressing both the root cause and the symptoms. Common methods for treating the symptoms include soothing the liver and regulating qi, promoting blood circulation and removing blood stasis, clearing heat and dampness, purging fire and detoxifying, and resolving stagnation and nodules. Common methods for treating the root cause include strengthening the spleen and replenishing qi, nourishing blood and softening the liver, and tonifying yin fluids. For example, Chinese invention patent application CN 105853895A discloses a traditional Chinese medicine composition for adjuvant treatment of mid-to-late stage primary liver cancer, which is prepared from the following traditional Chinese medicine raw materials in parts by weight: Curcuma zedoaria 120-200 parts, Fritillaria thunbergii 100-150 parts, Sparganium stoloniferum 120-200 parts, Scutellaria barbata 150-280 parts, Patrinia scabiosaefolia 150-180 parts, Atractylodes macrocephala 120-150 parts, Coix lacryma-jobi 50-120 parts, Hirudo medicinalis 10-50 parts, Astragalus membranaceus 100-300 parts, Panax ginseng 20-60 parts, Angelica sinensis 100-200 parts, Ligustrum lucidum 100-200 parts, and Glycyrrhiza uralensis 30-100 parts. For example, Chinese invention patent application CN 107468949A also discloses a traditional Chinese medicine composition for treating advanced primary liver cancer, made from 10-30 parts of Codonopsis pilosula, 30-50 parts of Coix lacryma-jobi, 15-30 parts of Ligusticum chuanxiong, 6-15 parts of Cinnamomum cassia seed, 6-15 parts of Myrica rubra bark, 15-30 parts of Coptis chinensis, 5-35 parts of Myrica rubra root, 10-35 parts of Starfruit leaf, and 3-9 parts of Hirudo medicinalis. Both of these compositions embody the principles of promoting blood circulation, removing blood stasis, detoxifying, resolving masses, strengthening the spleen, replenishing qi, nourishing yin, and softening the liver.

[0005] Although drug researchers and clinicians are dedicated to the research and development of drugs for the treatment of liver cancer, the persistently high mortality rate of primary liver cancer, especially advanced primary liver cancer, still indicates that clinical treatment needs are far from being met. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a pharmaceutical composition for treating advanced primary liver cancer. This traditional Chinese medicine composition features a novel formulation and rigorous composition. Preliminary experiments show that it significantly inhibits the growth of human liver cancer cell lines (BEL-7402, MHCC-97H, and SMMC-7721) and mouse liver cancer cell lines (H22 and Hepa 1-6), suggesting its potential for treating advanced primary liver cancer. Preliminary clinical observations indicate that this traditional Chinese medicine composition can benefit patients with intermediate to advanced primary liver cancer. Therefore, this invention holds promise for providing another option for the clinical treatment of primary liver cancer, especially inoperable advanced primary liver cancer.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0008] A traditional Chinese medicine composition for advanced primary liver cancer, comprising the following medicinal materials in parts by weight:

[0009] Ginkgo biloba 1-50 parts, Phyllanthus emblica 1-35 parts, Centipeda minima 1-30 parts, Artemisia annua 1-30 parts, Citrus reticulata 1-35 parts, Citrus aurantium 1-35 parts, Citrus reticulata 1-20 parts, Citrus reticulata 1-30 parts, Citrus aurantium 1-35 parts.

[0010] Preferably, the traditional Chinese medicine composition for advanced primary liver cancer comprises the following raw materials in parts by weight:

[0011] Ginkgo biloba 10-50 parts, Phyllanthus emblica 5-35 parts, Centipeda minima 5-30 parts, Artemisia annua 5-30 parts, Citrus reticulata 5-35 parts, Citrus aurantium 5-35 parts, Citrus reticulata 2-20 parts, Citrus reticulata 5-30 parts, Citrus aurantium 5-35 parts.

[0012] Preferably, the traditional Chinese medicine composition for advanced primary liver cancer comprises the following raw materials in parts by weight:

[0013] Ginkgo biloba 24-50 parts, Phyllanthus emblica 14-35 parts, Centipeda minima 15-30 parts, Artemisia annua 15-30 parts, Citrus reticulata 14-35 parts, Citrus aurantium 18-35 parts, Citrus reticulata 9-20 parts, Citrus reticulata 5-30 parts, Citrus aurantium 5-35 parts.

[0014] More preferably, the traditional Chinese medicine composition for advanced primary liver cancer comprises the following medicinal materials in parts by weight:

[0015] Ginkgo biloba 32-50 parts, Phyllanthus emblica 22-35 parts, Centipeda minima 20-30 parts, Artemisia annua 20-30 parts, Citrus reticulata 22-35 parts, Citrus aurantium 24-35 parts, Citrus reticulata 12-20 parts, Citrus reticulata 20-30 parts, Citrus aurantium 22-35 parts.

[0016] More preferably, the traditional Chinese medicine composition for advanced primary liver cancer comprises the following medicinal materials in parts by weight:

[0017] Ginkgo biloba 42-45 parts, Phyllanthus emblica 26-30 parts, Centipeda minima 24-28 parts, Artemisia annua 21-25 parts, Citrus reticulata 26-30 parts, Citrus aurantium 26-30 parts, Citrus reticulata 15-20 parts, Citrus reticulata 21-25 parts, Citrus aurantium 26-30 parts.

[0018] As a preferred embodiment, the present invention provides a traditional Chinese medicine composition for advanced primary liver cancer, the raw materials comprising the following medicinal materials in parts by weight:

[0019] Ginkgo biloba 45 parts, Phyllanthus emblica 28 parts, Centipeda minima 25 parts, Artemisia annua 25 parts, Citrus reticulata 28 parts, Citrus aurantium 30 parts, Citrus reticulata 16 parts, Citrus reticulata 25 parts, Citrus aurantium 28 parts.

[0020] As a preferred embodiment, the traditional Chinese medicine composition for advanced primary liver cancer provided by the present invention consists of ginkgo, amla, centipeda minima, artemisia annua, tangerine peel, immature bitter orange, tangerine peel, green tangerine peel, and immature bitter orange peel, with the weight parts of each medicinal material as defined above.

[0021] Another objective of this invention is to provide a medicine comprising the above-mentioned traditional Chinese medicine composition, and may include or exclude pharmaceutically acceptable excipients.

[0022] As an alternative embodiment, the drug is made from the traditional Chinese medicine composition of the present invention and, optionally, pharmaceutically acceptable excipients.

[0023] Preferably, the drug is any clinically acceptable formulation, and more preferably an oral formulation.

[0024] Preferably, the oral preparation is selected from decoctions, powders, capsules, tablets, honey pills, water-honey pills, water pills, concentrated pills, paste pills, wax pills, granules, oral liquids, or drop pills; more preferably, it is a decoction, tablet, granule, or capsule.

[0025] A third objective of this invention is to provide a method for preparing the aforementioned drug, comprising preparing each medicinal material according to the stated weight proportions, and, according to conventional methods in the art, adding or omitting pharmaceutically acceptable excipients to prepare a clinically acceptable formulation.

[0026] The pharmaceutically acceptable excipients described in this invention include, but are not limited to: (1) diluents, such as starch, powdered sugar, dextrin, lactose, pregelatinized starch, microcrystalline fiber, inorganic calcium salts (such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical calcium carbonate, etc.), mannitol, vegetable oil, polyethylene glycol, etc.; (2) binders, such as distilled water, ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose, hydroxypropyl methyl cellulose, etc.; (3) disintegrants, such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium, etc.; (4) lubricants, such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, magnesium lauryl sulfate, etc.; (5) solvents, such as water, ethanol solutions, etc.

[0027] As an exemplary embodiment, the present invention provides a method for preparing a decoction, comprising preparing each medicinal material of the traditional Chinese medicine composition of the present invention according to the stated weight proportions, decocting with water two to three times, combining the decoctions, filtering, and obtaining the final product.

[0028] The preparation method of the above-mentioned decoction, the amount of water added for each decoction and the time of each decoction, these parameters can be easily determined by those skilled in the art based on the formulation rules of traditional Chinese medicine decoctions and the actual dosage of medicinal materials.

[0029] As an exemplary embodiment, the present invention also provides a method for preparing granules, comprising: preparing each medicinal material of the traditional Chinese medicine composition of the present invention according to the stated weight parts; adding water at 10 times the total weight of the medicinal materials; soaking for 1-2 hours; extracting volatile oil; collecting the volatile oil separately for later use; collecting the distilled liquid separately; taking β-cyclodextrin at 10 times the weight of the volatile oil; adding an appropriate amount of water; dissolving in an aqueous solution at 50-70°C; cooling; adding the volatile oil; stirring at 300-500 r / min for 1-2 hours; and refrigerating. After 24 hours, remove the residue, filter it, dry it at 40°C, grind it into a fine powder to obtain the β-cyclodextrin inclusion complex, and set it aside. Continue to decoct the residue twice with water, adding 8 times the weight of water each time, and decoct for 1-2 hours each time. Filter it while hot, combine the filtrates, add the distilled liquid and concentrate it to an extract with a relative density of 1.20-1.25 (50°C). Dry it, pulverize it into a fine powder, add the β-cyclodextrin inclusion complex, with or without pharmaceutically acceptable excipients, mix evenly, dry granulate, dry, and granulate to obtain the final product.

[0030] As another exemplary embodiment, the present invention also provides a method for preparing capsules, comprising preparing each medicinal material of the traditional Chinese medicine composition of the present invention according to the stated weight parts, pulverizing each medicinal material separately into fine powder, mixing them, and filling them into capsules; or mixing each medicinal material, pulverizing it into fine powder, and filling it into capsules.

[0031] As another exemplary embodiment, the present invention provides a method for preparing tablets, comprising preparing each medicinal material of the traditional Chinese medicine composition of the present invention according to the stated weight parts, mixing the medicinal materials, pulverizing them into fine powder, adding an appropriate amount of dextrin, and directly compressing them into tablets.

[0032] In addition, the present invention also provides the application of the above-mentioned traditional Chinese medicine composition, the above-mentioned drug, and the drug prepared according to the above-mentioned preparation method in the preparation of a drug for treating advanced primary liver cancer; symptoms include rapid progression of cancer, abdominal distension or abdominal pain with tenderness, swollen mass, emaciation, dizziness and tinnitus, low-grade fever or high fever and thirst, red tongue with little saliva, thin yellow coating, and wiry and rapid pulse.

[0033] In particular, the present invention also provides the use of the aforementioned traditional Chinese medicine composition, the aforementioned drug, and the drug prepared according to the aforementioned preparation method in the preparation of a drug for treating unresectable advanced primary liver cancer.

[0034] The traditional Chinese medicine composition, the drug, and the drug prepared according to the preparation method of the present invention can improve the quality of life of patients with advanced primary liver cancer, protect liver function, and prolong survival.

[0035] It is readily understood that the traditional Chinese medicine composition, the drug, and the drug prepared according to the preparation method of the present invention can also be used for patients with intermediate-stage primary liver cancer or patients with liver cancer after surgery, and can also benefit the patients.

[0036] In this instruction manual, the "parts by weight" of each component refers to the ratio of the amounts of each component, not the actual unit of mass. Depending on the actual situation, a unit of weight can be any mass, such as 1 part by weight can be 1g, 500g, or 1kg, or even 15g, 30g, etc.

[0037] In the traditional Chinese medicine composition of the present invention:

[0038] Ginkgo biloba is the dried, mature seed of the Ginkgo biloba plant (Ginkgo biloba L.). It tastes sweet, bitter, and astringent, and is neutral in nature. It enters the lung and kidney meridians and has the effects of astringing the lungs and relieving asthma, astringing and stopping leukorrhea, and reducing urination. It is used for asthma, leukorrhea, spermatorrhea, and frequent urination.

[0039] Phyllanthus emblica L., the dried, ripe fruit of the plant in the Euphorbiaceae family, is sweet, sour, and astringent in taste, and cool in nature. It enters the lung and stomach meridians. It has the effects of clearing heat and cooling blood, promoting digestion and strengthening the stomach, and generating fluids and relieving cough. It is often used for blood heat and blood stasis, indigestion, abdominal distension, cough, sore throat, and dry mouth.

[0040] The whole herb of *Centipeda minima* (L.) A. Braun et Aschers., a plant in the Asteraceae family, is pungent and warm in nature. It enters the lung meridian and has the functions of dispersing wind-cold, clearing nasal passages, relieving cough, and detoxifying. It is used for colds due to wind-cold, nasal congestion, cough and asthma due to cold phlegm, and carbuncles and boils.

[0041] Artemisia annua L., a plant in the Asteraceae family, is a dried aerial part. It tastes bitter and pungent, is cold in nature, and enters the liver and gallbladder meridians. It has the effects of clearing deficiency heat, eliminating bone steaming fever, relieving summer heat, stopping malaria, and reducing jaundice. It is often used to treat diseases such as yin deficiency due to warm pathogens, night fever and morning coolness, yin deficiency fever, bone steaming fever, summer heat fever, malaria with chills and fever, and damp-heat jaundice.

[0042] Dried tangerine peel is the dried, mature peel of Citrus reticulata Blanco and its cultivated varieties, belonging to the Rutaceae family. It has a bitter and pungent taste, is warm in nature, and enters the lung and spleen meridians. It has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm. It is used for abdominal distension, poor appetite, vomiting and diarrhea, and cough with excessive phlegm.

[0043] Citrus aurantium L., a plant of the Rutaceae family, is the dried young fruit of Citrus aurantium L. and its cultivated varieties or Citrus sinensis Osbeck. It has a bitter, pungent, and sour taste, is slightly cold in nature, and enters the spleen and stomach meridians. It has the effects of breaking up stagnation, resolving phlegm, and dispersing masses. It is used for internal stagnation, abdominal distension and pain, tenesmus after diarrhea, constipation, phlegm stagnation and qi obstruction, chest pain, chest tightness, and organ prolapse.

[0044] Huazhou tangerine peel is the dried outer peel of immature or nearly mature Citrus grandis Tomentosa or Citrus grandis (L.) Osbeck, belonging to the Rutaceae family. It has a pungent and bitter taste, is warm in nature, and enters the lung and spleen meridians. It has the effects of regulating qi and relieving chest tightness, drying dampness and resolving phlegm. It is used for cough with excessive phlegm, food stagnation and alcohol poisoning, nausea and vomiting and chest tightness.

[0045] Green peel is the dried young or immature fruit peel of Citrus reticulata Blanco and its cultivated varieties, belonging to the Rutaceae family. It tastes bitter and pungent, and is warm in nature. It enters the liver, gallbladder, and stomach meridians. It has the effects of soothing the liver and breaking up qi stagnation, eliminating food stagnation and resolving stagnation. It is used for chest and rib pain, hernia pain, mastopathy, mastitis, food stagnation and qi stagnation, and abdominal distension and pain.

[0046] Citrus aurantium L., a plant of the Rutaceae family, is a dried, immature fruit. It has a bitter, pungent, and sour taste, is slightly cold in nature, and enters the spleen and stomach meridians. It has the effects of regulating qi and relieving chest tightness, promoting blood circulation and relieving bloating. It is used for chest and rib stagnation, abdominal distension and pain, indigestion, phlegm retention, and organ prolapse.

[0047] Of the nine medicinal herbs mentioned above, ginkgo is the principal herb. Ginkgo is sweet, bitter, astringent, and neutral in nature. The Compendium of Materia Medica states that cooked ginkgo "warms the lungs and benefits qi, relieves asthma and cough, reduces urination, and stops leukorrhea; raw ginkgo reduces phlegm, disinfects, and kills parasites." It can both detoxify cancer and eliminate the cause of the disease, and also resolve phlegm. *Centipeda minima* is pungent and dispersing, helping ginkgo detoxify cancer. *Eupatorium fortunei* is sweet, sour, astringent, and cool, clearing heat, generating fluids, cooling blood, promoting blood circulation, and aiding digestion and strengthening the stomach. *Citrus reticulata* (green tangerine peel) soothes the liver, breaks up qi stagnation, and eliminates stagnation. *Citrus reticulata* (dried tangerine peel) regulates qi, strengthens the spleen, dries dampness, and resolves phlegm. *Citrus aurantium* (immature bitter orange) breaks up qi stagnation, eliminates stagnation, and resolves phlegm and dissipates masses. These five herbs together serve as assistant herbs. *Citrus reticulata* and *Citrus aurantium* combined with *Eupatorium fortunei* enhance the blood-activating power of *Eupatorium fortunei*, and also aid in strengthening the stomach, aiding digestion, regulating qi, and eliminating stagnation. *Citrus reticulata* combined with ginkgo enhances the phlegm-resolving effect of ginkgo. In late-stage liver cancer, internal heat arises, manifesting as dry mouth, bitter taste, irritability, and restlessness. Artemisia annua, bitter and pungent, clears away internal heat, cools the blood, and relieves irritability. Citrus reticulata peel, combined with dried tangerine peel, assists ginkgo in resolving phlegm. Citrus aurantium promotes qi circulation, opens the chest, relieves abdominal distension, and, combined with Citrus aurantium fruit and Citrus reticulata peel, enhances the qi-regulating effect. These three herbs serve as adjuvants. The entire formula primarily uses qi-regulating herbs, combined with detoxifying, blood-activating, and phlegm-resolving herbs. This allows qi to circulate and toxins to be eliminated, phlegm to be cleared, and blood stasis to be removed, thus alleviating various symptoms. Together, they support the body's resistance, eliminate pathogenic factors, detoxify, and disperse nodules.

[0048] The traditional Chinese medicine composition for treating advanced primary liver cancer described in this invention has a novel formulation and significantly inhibits the growth of xenografts of human liver cancer cell lines (BEL-7402, MHCC-97H, SMMC-7721). Therefore, this suggests that the traditional Chinese medicine composition described in this invention can be used to treat advanced primary liver cancer. Clinical observation of 9 patients with intermediate-to-advanced liver cancer (7 advanced and 2 intermediate) showed that at the end of the observation period, 3 patients survived (without progression), and 6 died. Furthermore, there was a certain correlation between the number of days of medication and survival time; that is, the longer the medication time, the longer the survival time. Moreover, the patients' relevant liver function indicators showed a trend of improvement. This demonstrates that the traditional Chinese medicine composition of this invention can benefit patients with advanced liver cancer. Attached Figure Description

[0049] The present invention will be further described below with reference to the accompanying drawings.

[0050] Figure 1 The figure shows the changes in tumor volume in female tumor-bearing nude mice (human liver cancer MHCC-97H cells) in each group in Experiment Example 2.

[0051] Figure 2 The figure shows the survival time curves of male tumor-bearing nude mice (human liver cancer MHCC-97H cells) in each group in Experiment Example 2.

[0052] Figure 3 The figure shows the changes in tumor volume in female tumor-bearing nude mice (human liver cancer SMMC-7721 cells) in each group in Experiment Example 2.

[0053] Figure 4 The figures shown are the survival time curves of female tumor-bearing nude mice (human liver cancer SMMC-7721 cells) in each group in Experiment Example 2.

[0054] Figure 5 The figures shown are the survival time curves of male tumor-bearing nude mice (human liver cancer SMMC-7721 cells) in each group in Experiment Example 2.

[0055] Figure 6 The figure shows the survival curves of patients over days in a clinical observation case.

[0056] Figure 7 The figure shows the monthly survival curves of patients in the clinical observation cases. Detailed Implementation

[0057] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the medicinal materials and reagents used in the following examples are commercially available products.

[0059] The following are the English abbreviations that may appear in this article and their Chinese meanings:

[0060] ALT: Alanine aminotransferase;

[0061] AST: Aspartate aminotransferase;

[0062] TBIL: Total bilirubin;

[0063] DBIL: Direct bilirubin;

[0064] ALB: Albumin;

[0065] AKP: Alkaline phosphatase;

[0066] r-GGT: Gamma-glutamyl transpeptidase;

[0067] AFP: Alpha-fetoprotein;

[0068] BUN: Blood urea nitrogen;

[0069] CR: Creatinine.

[0070] Example 1 A traditional Chinese medicine composition for advanced primary liver cancer

[0071] The raw material composition of the traditional Chinese medicine composition described in this embodiment is (1 part = 1 kg):

[0072] Ginkgo biloba 32 parts, Phyllanthus emblica 22 parts, Centipeda minima 20 parts, Artemisia annua 20 parts, Citrus reticulata 22 parts, Citrus aurantium 24 parts, Citrus reticulata peel 12 parts, Citrus reticulata peel 20 parts, Citrus aurantium 22 parts

[0073] Example 2 A traditional Chinese medicine composition for advanced primary liver cancer

[0074] The raw material composition of the traditional Chinese medicine composition described in this embodiment is (1 part = 1 kg):

[0075] Ginkgo biloba 24 parts, Phyllanthus emblica 14 parts, Centipeda minima 15 parts, Artemisia annua 15 parts, Citrus reticulata 14 parts, Citrus aurantium 18 parts, Citrus reticulata peel 9 parts, Citrus reticulata peel 15 parts, Citrus aurantium 14 parts

[0076] Example 3 A traditional Chinese medicine composition for advanced primary liver cancer

[0077] The raw material composition of the traditional Chinese medicine composition described in this embodiment is (1 part = 1 kg):

[0078] Ginkgo biloba 45 parts, Phyllanthus emblica 28 parts, Centipeda minima 25 parts, Artemisia annua 25 parts, Citrus reticulata 28 parts, Citrus aurantium 30 parts, Citrus reticulata 16 parts, Citrus reticulata 25 parts, Citrus aurantium 28 parts.

[0079] Example 4 A capsule

[0080] Take the traditional Chinese medicine composition from Example 1, mix it, grind it into a fine powder, pass it through a 100-mesh sieve, and fill it into capsules, 0.4g of raw medicine per capsule, and you will get the product.

[0081] Dosage and administration: Take 2-5 capsules three times a day with warm water.

[0082] Example 5 A type of granule (Jiuwei Zhengxiao Granules)

[0083] The traditional Chinese medicine composition prescription for preparing granules in this embodiment is (12g = 1 part by weight):

[0084] Ginkgo biloba 523g, Phyllanthus emblica 349g, Centipeda minima 314g, Artemisia annua 279g, Citrus reticulata 349g, Citrus aurantium 349g, Citrus reticulata peel 209g, Citrus reticulata peel 279g, Citrus aurantium 349g.

[0085] Preparation method: Soak the above nine ingredients in water (10 times their total weight) for 1-2 hours, extract the volatile oil, and collect the volatile oil separately for later use; collect the distilled liquid separately; take 10 times the weight of β-cyclodextrin from the volatile oil, add an appropriate amount of water, dissolve in an aqueous solution at 50-70℃, let cool, add the volatile oil, stir at 300-500 rpm for 1-2 hours, refrigerate for 24 hours, remove, filter, dry the residue at 40℃, grind finely, and obtain β-cyclodextrin inclusion complex. Prepare the medicine; continue to decoct the dregs with water twice, adding 8 times the weight of water each time, and decoct for 1-2 hours each time. Filter while hot, combine the filtrates, add the distilled medicine liquid and concentrate to an extract with a relative density of 1.20-1.25 (50℃), dry, pulverize into a fine powder, add the β-cyclodextrin inclusion complex, with or without pharmaceutically acceptable excipients, mix evenly, dry granulate, dry, and granulate to obtain 1000g; package into 7g bags.

[0086] Dosage and administration: Take 7g three times a day, dissolved in boiling water.

[0087] Example 6 A tablet

[0088] Take the traditional Chinese medicine composition from Example 3, mix it, grind it into a fine powder, pass it through an 80-100 mesh sieve, add dextrin and directly compress it into tablets, 0.3g of raw medicine / tablet, and you have obtained the product.

[0089] Dosage and administration: Take 3-6 tablets three times a day with warm water.

[0090] Example 7 A type of honey pill

[0091] Take the traditional Chinese medicine composition from Example 1, mix it, grind it into a fine powder, pass it through a 60-100 mesh sieve, add an appropriate amount of refined honey, and prepare small honey pills according to conventional methods, 3g / 10 pills, and you have obtained the product.

[0092] Dosage and administration: Take 3 grams three times a day with warm water.

[0093] Example 8 A decoction

[0094] Weigh each medicinal material according to the following mass measurements:

[0095] Ginkgo biloba 15g, Phyllanthus emblica 10g, Centipeda minima 9g, Artemisia annua 8g, Citrus reticulata peel 10g, Citrus aurantium 10g, Citrus reticulata peel 6g, Citrus reticulata peel 8g, Citrus aurantium 10g

[0096] Mix all the medicinal herbs together, add 5-10 times their weight of water, heat to a boil and simmer for 25 minutes, then filter while hot. Add 5-10 times their weight of water to the dregs, heat to a boil and simmer for 20-30 minutes, then filter while hot. Combine the two decoctions to obtain the final product. Divide into 2-3 doses and consume within one day.

[0097] Experimental Example 1 In vitro pharmacological experiments

[0098] 1. Materials

[0099] 1.1 Experimental drugs:

[0100] ① Jiuwei Zhengxiao Granules, prepared according to the prescription and method of Example 5; batch number: 20200101, content: 3.70g crude drug / g extract powder, provided by the Institute of Clinical Basic Research of China Academy of Chinese Medical Sciences, received on January 21, 2020, and stored in a cool, dry cabinet.

[0101] ② Hepatoprotective Capsules: Batch No.: 20190817, Kangpu Pharmaceutical Co., Ltd.

[0102]

Ingredients

[0103]

Specifications

[0104] 1.2 Experimental reagents and cell lines:

[0105] DMEM high glucose medium, specification: 500mL / bottle, batch number: AF29498408, HyClone; RPMI-1640 medium, specification: 500mL / bottle, batch number: AF29498408, HyClone; fetal bovine serum (FBS), specification: 100mL / bottle, batch number: M009-6, Sciencell.

[0106] Human hepatocellular carcinoma cells (BEL-7402), human hepatocellular carcinoma cells (SMMC-7721, poorly differentiated, HBV positive), mouse hepatocellular carcinoma cells (H22, ascites type), and mouse hepatocellular carcinoma cells (Hepa 1-6) were all obtained from Wuhan Pronosei Biotechnology Co., Ltd.; human hepatocellular carcinoma cells (MHCC-97H, poorly differentiated, highly metastatic type) were obtained from Shanghai Fuxiang Biotechnology Co., Ltd.

[0107] BEL-7402 cells, H22 cells, and SMMC-7721 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum at 37°C, 5% CO2, and saturated humidity. Hepa 1-6 cells and MHCC-97H cells were cultured in DMEM medium with 10% fetal bovine serum at 37°C, 5% CO2, and saturated humidity.

[0108] 1.3 Experimental Apparatus:

[0109] CJ-1F Medical Cleanroom Workbench, Center No.: 176, Suzhou Fengshi Experimental Animal Equipment Co., Ltd.; 3111 CO2 Incubator, Center No.: 024, Thermo Fisher Scientific, USA; DMIL Inverted Fluorescence Microscope, Center No.: 027, Leica, Germany; Spectra Max i3x Multifunctional Microplate Reader, Center No.: 525, MD International, USA.

[0110] 2. Methods

[0111] 2.1 Grouping and Concentration Settings:

[0112] Based on the solubility of Jiuwei Zhengxiao Granules and Ganfule Capsules, six concentration gradients were sequentially set up: 10000, 3000, 1000, 300, 100, and 30 μg (granule or capsule contents) / mL, with corresponding prepared concentrations of 20000 (maximum soluble concentration), 6000, 2000, 600, 200, and 60 μg / mL. A solvent control group was also included. Each concentration had five replicates, and four zeroing wells were set up with samples but no cells. The experiment was repeated three times. Based on the results of the first batch of experiments, the concentrations of the test substances in the second and third batches were adjusted to 1000, 300, 100, 30, 10, and 3 μg / mL. See Table 1 for details of the grouped administration.

[0113] Table 1 Grouping and Dosing Table

[0114]

[0115]

[0116] 2.2 Cell Culture:

[0117] BEL-7402, H22, and SMMC-7721 cell suspensions were seeded at 100 μL / well into 96-well plates, and 200 μL / well of PBS was added. Two 96-well plates were seeded for each cell type, and the cells were cultured overnight until they adhered. The seeding order for the 96-well plates is detailed in Table 2.

[0118] Table 2. Example diagram of sample loading for 96-well plate

[0119] 1 2 3 4 5 6 7 8 9 10 11 12 A PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS B PBS cell cell cell cell cell cell blank blank blank blank PBS C PBS cell cell cell cell cell cell blank blank blank blank PBS D PBS cell cell cell cell cell cell blank blank blank blank PBS E PBS cell cell cell cell cell cell blank blank blank blank PBS F PBS cell cell cell cell cell cell blank blank blank blank PBS G PBS cell cell cell cell cell cell blank blank blank blank PBS H PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS

[0120] 2.3 Processing of test sample / reference sample:

[0121] Prepare Jiuwei Zhengxiao granules or Ganfule capsules at various concentrations and add 100 μL / well to the wells of the 96-well plate prepared in step 2.2. Add the test sample / control sample to the cell-inoculated culture wells or blank wells (columns 3-11, 9 wells in total) according to the arrangement in Table 2. Add 100 μL / well of complete culture medium to the second column of wells. The final concentrations are 10000, 3000, 1000, 300, 100, and 30 μg / mL / well / concentration. Perform 5 replicates per group and 4 zeroing wells with samples but no cells. After intervention with the corresponding liver cancer cells using the test sample or control sample, incubate the 96-well plate at 37℃ and 5% CO2 saturated humidity for 72 h. Each cell experiment is repeated three times. Adjust the concentrations of the second and third batches to 1000, 300, 100, 30, 10, and 3 μg / mL based on the results of the first batch.

[0122] 2.4 Cell viability detection:

[0123] After 72 hours of cell culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were cultured for another 4 hours. Upon termination of culture, the culture medium was aspirated from the wells, and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the crystals. The absorbance at OD492 nm was measured using a multi-mode microplate reader, and the cell growth inhibition rate (%) was calculated as (OD solvent – ​​OD experimental group) / (OD solvent – ​​OD background) × 100%, and the half-maximal inhibitory concentration (IC50) was also calculated.

[0124] 2.5 Statistical Methods:

[0125] The significant figures in this experiment were rounded to the nearest whole number. The cell inhibition rate was expressed as a percentage, and the half-maximal inhibitory concentration (IC50) was calculated using SPSS 19.0.

[0126] 3. Experimental Results

[0127] 3.1 Results of the first batch of tests:

[0128] As shown in Table 3, the IC50 of Jiuwei Zhengxiao Granules on human hepatocellular carcinoma BEL-7402 cells, MHCC-97H cells, SMMC-7721 cells, and mouse H22 and Hepa1-6 cells... 50 The values ​​were 517.919, 16.782, 782.321, <30.000, and 161.348 μg / mL, respectively, and a 95% confidence interval could not be calculated. The IC50 values ​​of Ganfule capsules against human hepatocellular carcinoma BEL-7402 cells, MHCC-97H cells, SMMC-7721 cells, and mouse H22 and Hepa1-6 cells were [not specified in the original text]. 50 The effective concentrations of Jiuwei Zhengxiao Granules and Ganfule Capsules against hepatocellular carcinoma cells in vitro were 72.919, 17.720, 396.233, <30.000, and 3.201 μg / mL, respectively. The 95% confidence intervals for human hepatocellular carcinoma cells BEL-7402, MHCC-97H, and SMMC-7721 were 0.835–261.626, 0.118–55.727, and 45.824–2531.187 μg / mL, respectively. Based on these results, the effective concentrations of Jiuwei Zhengxiao Granules and Ganfule Capsules against hepatocellular carcinoma cells in vitro were significantly less than 10000 and 3000 μg / mL, respectively. To obtain more accurate IC50 values, the concentrations of the test substances were adjusted to 1000, 300, 100, 30, 10, and 3 μg / mL in the second and third batches of experiments.

[0129] Table 3. Effects of Jiuwei Zhengxiao Granules on the proliferation of different liver cancer cells (First Batch)

[0130]

[0131] 3.2 Results of the second and third batches of tests: The results are shown in Tables 4 and 5.

[0132] The data in Tables 4 and 5 show the IC50 of Jiuwei Zhengxiao Granules on BEL-7402 cells. 50 The concentrations were 243.429 μg / mL and 24.397 μg / mL, respectively; the IC50 concentrations against H22 cells were... 50 The IC50 values ​​for Hepa 1-6 cells were 0.151 μg / mL and <3 μg / mL, respectively. 50 The concentrations were 8.772 μg / mL and 2.590 μg / mL, respectively; the IC50 concentrations against MHCC-97H cells were... 50The concentrations were 0.971 μg / mL and 1.841 μg / mL, respectively; the IC50 concentrations against SMMC-7721 cells were... 50 The values ​​were >4.922 μg / mL and 116.252 μg / mL, respectively.

[0133] Hepatoprotective capsules on the IC50 of BEL-7402 cells 50 The concentrations were 600.121 μg / mL and 65.660 μg / mL, respectively; the IC50 concentrations against H22 cells were... 50 The concentrations were 0.001 μg / mL and 0.018 μg / mL, respectively; the IC50 concentrations against Hepa1-6 cells were... 50 The IC50 concentrations were 2.950 μg / mL and 7.729 μg / mL, respectively; for MHCC-97H cells... 50 The concentrations were 0.041 μg / mL and 0.229 μg / mL, respectively; the IC50 concentrations against SMMC-7721 cells were... 50 The values ​​were 21215.318 μg / mL and 2659.064 μg / mL, respectively.

[0134] 4. Experimental conclusions:

[0135] The above results indicate that both Jiuwei Zhengxiao granules and Ganfule capsules have significant inhibitory effects on the in vitro proliferation of liver cancer cells (BEL-7402, H22, Hepa 1-6, MHCC-97H, SMMC-7721) from different sources. In the range of 3–1000 μg / mL, the inhibitory effect of Jiuwei Zhengxiao granules is superior to that of Ganfule capsules.

[0136] Table 4. Effects of Jiuwei Zhengxiao Granules on the proliferation of different liver cancer cells (Second batch)

[0137]

[0138] Table 5. Effects of Jiuwei Zhengxiao Granules on the proliferation of different liver cancer cells (Third batch)

[0139]

[0140] Experimental Example 2 In vivo pharmacodynamic studies

[0141] Human hepatocellular carcinoma SMMC-7721 cells (poorly differentiated, HBV positive) and MHCC-97H cells (poorly differentiated, highly metastatic) were used to construct a BALB / c nude mouse subcutaneous xenograft model. The in vivo antitumor efficacy of the traditional Chinese medicine composition of this invention was evaluated by observing indicators such as tumor volume and survival time.

[0142] 1. Experimental Materials

[0143] 1.1 Test sample

[0144] Jiuwei Zhengxiao Granules, prepared according to the method of Example 5, batch number: 20200101, content: 3.70 g of crude drug / g of extract powder.

[0145] 1.2 Positive control drug

[0146] Ganfule Capsules, specification: 0.5 g / capsule, batch number: 20190817 / 20201006, expiration date: August 12, 2021 / October 2022, production unit: Kangpu Pharmaceutical Co., Ltd.

[0147] 1.3 Experimental animals

[0148] 20 SPF-grade in vivo passage BALB / c nude mice, 10 males and 10 females, animal quality certificate numbers: No.1107272011004623, No.430727201101206635; 140 SPF-grade BALB / c nude mice, 70 males and 70 females, animal quality certificate number: No.430727211100012884, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., experimental animal production license number: SCXK(Xiang)2019-0004. Raised in Area A of the barrier environment animal laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., experimental animal use license number: SYXK(Xiang)2015-0016, used for the influence test on the subcutaneous transplanted tumor model of human hepatocellular carcinoma MHCC-97H cells.

[0149] 20 SPF-grade in vivo passage BALB / c nude mice, 10 males and 10 females, animal quality certificate number: No.430727221100833945; 120 SPF-grade BALB / c nude mice, 60 males and 60 females, animal quality certificate number: No.430727221101058843, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., experimental animal production license number: SCXK(Xiang)2019-0004. Raised in Area A of the barrier environment animal laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., experimental animal use license number: SYXK(Xiang)2020-0015, used for the influence test on the subcutaneous transplanted tumor model of human hepatocellular carcinoma SMMC-7721 cells.

[0150] 1.4 Cell lines

[0151] Human hepatocellular carcinoma cells (SMMC-7721, poorly differentiated, HBV positive), purchased from Wuhan Punosai Life Science Co., Ltd.; human hepatocellular carcinoma cells (MHCC-97H, poorly differentiated, highly metastatic), purchased from Shanghai Fuxiang Biotechnology Co., Ltd.

[0152] 1.5 Main reagents

[0153] DMEM high glucose medium (batch number: 081012-UY, Corning product); fetal bovine serum (batch number: NYL1009, HyClone product).

[0154] 1.6 Main Instruments

[0155] 3111 CO2 incubator (Thermo, USA); SHHW21.420A three-in-one electric thermostatic water bath (Beijing Kewei Yongxing Instrument Co., Ltd.); DMIL inverted microscope (Leica, Germany); LDZX-50KBS vertical pressure sterilizer (Shanghai Shenan Medical Instrument Factory); TD4 benchtop low-speed centrifuge (Hunan Keda Industrial Development Co., Ltd.); PL1501-S electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); digital display vernier calipers (Yongkang Zhengfeng Hardware Co., Ltd.).

[0156] 2. Experimental Methods

[0157] 2.1 Tumor cell resuscitation culture

[0158] The SMMC-7721 or MHCC-97H human hepatocellular carcinoma cell lines were placed in DMEM high-glucose medium containing 10% fetal bovine serum and cultured and passaged at 37°C in a 5% CO2 incubator for future use.

[0159] 2.2 In vivo passage of tumor cells

[0160] Twenty SPF-grade BALB / c nude mice (half male and half female) were subcutaneously injected with 5.54 × 10⁵ human hepatocellular carcinoma MHCC-97H cells via the right axilla. 7 Each mouse was passaged in vivo, and tumor-bearing mice that had been passaged to at least the second generation were selected. Tumor homogenates were prepared from the excised tumors for later use. Twenty SPF-grade BALB / c nude mice (half male, half female) were subcutaneously injected with 5.4 × 10⁴ human hepatocellular carcinoma SMMC-7721 cells via the right axilla. 7 Each mouse was passaged in vivo, and tumor-bearing mice that had been passaged to the second generation or higher were selected. The tumors were then removed and a tumor homogenate was prepared for later use.

[0161] 2.3 Modeling, Grouping and Drug Administration

[0162] 2.3.1 Effects of Jiuwei Zhengxiao Granules on a human hepatocellular carcinoma MHCC-97H cell subcutaneous xenograft model

[0163] Seventy SPF-grade female and 70 male BALB / c nude mice (quarantined and certified) were selected. The mouse tumor homogenate (MHCC-97H cells) from section "2.2" was subcutaneously injected into the right axilla of each mouse to establish a mouse subcutaneous xenograft model. Fifty female and 50 male mice with tumor formation were selected, with tumor volumes ranging from 156.1 to 2247.0 mm² in females and males, respectively. 3 and 146.5~10238.6mm 3 Mice were randomly divided into 5 groups based on tumor volume: a model control group, a Ganfule capsule group (1.2g capsule contents / kg, the clinically equivalent dose), and low, medium, and high dose Jiuwei Zhengxiao granules groups (4.1, 8.2, and 16.4g crude drug / kg, equivalent to 0.5, 1, and 2 times the clinically equivalent dose, respectively). Ten mice per group, per sex, were administered each day. Before daily administration, the dry extract powder of Jiuwei Zhengxiao granules and Ganfule capsules were prepared to the appropriate concentration using pure water. Each group was administered the corresponding concentration of the drug solution orally at a dose of 20mL / kg, while the model control group was administered an equal volume of pure water orally. Administration was repeated once daily for 4 weeks. The time of death and survival days were observed for each group. The major diameter (a) and minor diameter (b) of the tumor were measured twice weekly using calipers, and the tumor volume V (mm²) was calculated. 3 ) = 1 / 2 × a × b 2 .

[0164] 2.3.2 Effects of Jiuwei Zhengxiao Granules on a human hepatocellular carcinoma SMMC-7721 cell subcutaneous xenograft model

[0165] Seventy SPF-grade female and 70 male BALB / c nude mice (quarantined and certified) were selected. The mouse tumor homogenate (SMMC-7721 cells) from section 2.2 was inoculated at 0.2 mL per mouse into the right axilla to replicate a subcutaneous xenograft model. Fifty female and 50 male mice with tumor formation were selected, with tumor volumes ranging from 1138.23 to 4669.2 mm² in females and males, respectively. 3 and 1378.62~3786.32mm 3 Mice were randomly divided into 5 groups based on tumor volume: a model control group, a Ganfule capsule group (1.2g capsule contents / kg), and low, medium, and high dose Jiuwei Zhengxiao granules groups (4.1, 8.2, and 16.4g crude drug / kg, respectively), with 10 mice per group per sex. Before daily administration, the dry extract powder of Jiuwei Zhengxiao granules and Ganfule capsules were prepared into corresponding concentrations using pure water. Each group was administered the corresponding concentration of the drug solution orally at 20mL / kg, while the model control group was administered an equal volume of pure water orally, once daily for 4 consecutive weeks. The time to death and survival days were observed and calculated for each group. The major diameter (a) and minor diameter (b) of the tumor were measured using calipers twice weekly, and the tumor volume V (mm²) was calculated. 3) = 1 / 2 × a × b 2 .

[0166] 2.5 Statistical Methods

[0167] The data in this experiment were rounded to the nearest whole number and statistical analysis was performed according to the standard operating procedure (SOP). SPSS 22.0 was used for statistical analysis. Quantitative data are expressed as mean ± standard deviation. The results indicate that Leven's test was used to test for normality and homogeneity of variance. If there was no statistical significance (P>0.05), one-way ANOVA was used for statistical analysis. If the ANOVA was statistically significant (P≤0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P≤0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P≤0.05), Dunnett's test (nonparametric method) was used for comparative analysis. The statistical results were presented with α = 0.05 as the test limit, where P≤0.05 indicated statistical significance, and P≤0.01 indicated a highly significant difference.

[0168] 3. Experimental Results

[0169] 3.1 Effect on tumor volume of human hepatocellular carcinoma MHCC-97H cell subcutaneous xenograft model 3.1.1 Effect on tumor volume of female mouse subcutaneous xenograft model

[0170] The results are shown in Table 6 and Figure 1 As shown in the figure. Compared with the model control group, the tumor volume of Ganfule capsules was significantly reduced on D17, D21, D24 and D27 after administration (P≤0.05 or P≤0.01). The tumor volume of Jiuwei Zhengxiao granules was significantly reduced on D27 after administration (P≤0.01). The tumor volume of Jiuwei Zhengxiao granules was significantly reduced on D21, D24 and D27 after administration (P≤0.05 or P≤0.01). The tumor volume of Jiuwei Zhengxiao granules was significantly reduced on D17, D21, D24 and D27 after administration (P≤0.05 or P≤0.01).

[0171] Table 6. Effects of Jiuwei Zhengxiao Granules on Tumor Volume in Female Mouse Subcutaneous Xenograft Tumor Model ( n=10)

[0172]

[0173] Note: Compared with the model control group + P≤0.05, ++ P≤0.01.

[0174] 3.1.2 Effect on tumor volume in male mouse subcutaneous xenograft tumor model

[0175] The results are shown in Table 7. Due to the successive deaths of male animals in the model group after drug administration, the number of animals was small and therefore they were not included in the tumor volume statistics. However, the mortality of the animals was statistically analyzed, as detailed in section 3.2.

[0176] Table 7. Effects of Jiuwei Zhengxiao Granules on Tumor Volume in Male Animals

[0177]

[0178]

[0179] 3.2 Effect on survival time of human hepatocellular carcinoma MHCC-97H cell subcutaneous xenograft model

[0180] The results are shown in Table 8 and... Figure 2 As shown, compared with the model control group, the number of surviving animals in the medium and high dose groups of Jiuwei Zhengxiao granules was significantly increased (P≤0.05 or P≤0.01), and the survival time in the high dose group was significantly prolonged (P≤0.01). Among them, the number of surviving animals in the Ganfule capsule group and the low dose group of Jiuwei Zhengxiao granules showed an increasing trend, but there was no statistical difference. In addition, no deaths were observed in female mice during the administration period.

[0181] Table 8. Effects of Jiuwei Zhengxiao Granules on Survival Time and Mortality in Male Xenograft Tumor Models

[0182]

[0183] Note: Compared with the model control group + P≤0.05, ++ P≤0.01.

[0184] 3.3 Effect on tumor volume in a human hepatocellular carcinoma SMMC-7721 cell subcutaneous xenograft model 3.3.1 Effect on tumor volume in female mice

[0185] The results are shown in Table 9 and... Figure 3 As shown, compared with the model control group, the tumor volume of D14 was significantly reduced in the medium-dose group of Jiuwei Zhengxiao Granules after administration (P≤0.05). Although the tumor volume of the low- and high-dose groups showed a decreasing trend, there was no significant statistical difference.

[0186] Table 9. Effects of Jiuwei Zhengxiao Granules on Tumor Volume in Female Animals

[0187]

[0188]

[0189] Note: Compared with the model control group + P≤0.05, " / " indicates that all animals have died and there is no data.

[0190] 3.3.2 Effect on tumor volume in male mice

[0191] The results are shown in Table 10 and... Figure 4 As shown, compared with the model control group, there was no significant difference in tumor volume among male animals in the Ganfule capsule group and the Jiuwei Zhengxiao granule group at each dose after administration.

[0192] Table 10. Effects of Jiuwei Zhengxiao Granules on Tumor Volume in Male Animals

[0193]

[0194] 3.4 Effect on survival days of human hepatocellular carcinoma SMMC-7721 cell subcutaneous xenograft model 3.4.1 Effect on lifespan of female animals

[0195] The results are shown in Table 11 and... Figure 4 As shown, compared with the model control group, the survival time of female animals in the high-dose group of Jiuwei Zhengxiao Granules was significantly prolonged (P≤0.01).

[0196] Table 11 Effects of Jiuwei Zhengxiao Granules on the Survival Days of Female Animals

[0197]

[0198]

[0199] Note: Compared with the model control group ++ P≤0.01.

[0200] 3.4.2 Effects on the life cycle of male animals

[0201] The results are shown in Table 12 and... Figure 5 As shown, compared with the model control group, the survival time of male animals in the low, medium and high dose groups of Jiuwei Zhengxiao Granules tended to be prolonged, but there was no statistical difference.

[0202] Table 12 Effects of Jiuwei Zhengxiao Granules on Survival Days in Male Animals

[0203]

[0204] 4. Experiment Summary

[0205] The above results indicate that low, medium, and high doses of Jiuwei Zhengxiao granules (4.1, 8.2, and 16.4 g crude drug / kg) significantly inhibited tumor growth in female mice with MHCC-97H human hepatocellular carcinoma transplanted into them, significantly reduced mortality in male mice with advanced hepatocellular carcinoma, and prolonged survival. A medium dose (8.2 g crude drug / kg) significantly inhibited tumor growth in female mice with SMMC-7721 human hepatocellular carcinoma transplanted into them, while a high dose (16.4 g crude drug / kg) significantly prolonged survival. These results suggest that Jiuwei Zhengxiao granules have a significant inhibitory effect on tumors in mice with MHCC-97H and SMMC-7721 human hepatocellular carcinoma transplanted into them.

[0206] Clinical observation cases Clinical observation case report of the granules of the present invention

[0207] From April 2019 to June 2020, the clinical efficacy of Jiuwei Zhengxiao Granules prepared in Example 5 in treating advanced liver cancer was observed at Hubei Provincial Hospital of Traditional Chinese Medicine.

[0208] 1. Patient's basic information, see Table 13.

[0209] Table 13 Basic information of clinically observed cases

[0210]

[0211]

[0212] 2. Dosing regimen

[0213] Jiuwei Zhengxiao Granules (prepared in Example 5) are taken 3 times a day, 7g each time, dissolved in boiling water.

[0214] 3. Evaluation of therapeutic effect

[0215] 3.1 Overall therapeutic effect

[0216] The treatment duration was 3-90 days, and the patient survival time was 8-263 days; survival curves by day and month are shown below. Figure 6 and Figure 7 .

[0217] Figure 6 and Figure 7 The results show that there is a certain correlation between the number of days of medication and survival time, that is, the longer the medication time, the longer the survival time (medication time of 3-90 days, survival time of 8-263 days), with a correlation coefficient of 0.5669.

[0218] 3.2 Changes in physiological and biochemical indicators before and after treatment are shown in Table 14.

[0219] Table 14 Changes in relevant physiological and biochemical indicators before and after treatment

[0220]

[0221]

[0222] Table 14 shows that Jiuwei Zhengxiao Granules have a trend of reducing AFP, r-GGT, ALT, AST, etc.

[0223] In summary, this invention provides a traditional Chinese medicine composition that can be used for advanced primary liver cancer. The composition includes ginkgo, emblica, centipeda minima, artemisia annua, tangerine peel, immature bitter orange, tangerine peel (processed with Citrus reticulata peel), green tangerine peel, and immature bitter orange peel. The combined effects of these herbs are to regulate qi, resolve phlegm, detoxify, and remove blood stasis. Pharmacological experiments have demonstrated that the traditional Chinese medicine composition of this invention has a significant inhibitory effect on the growth of human liver cancer cell lines (BEL-7402, MHCC-97H, SMMC-7721) and mouse liver cancer cell lines (H22, Hepa 1-6). Clinical observations show that the traditional Chinese medicine composition of this invention can benefit patients. Therefore, this invention provides a new clinical treatment option for patients with advanced primary liver cancer and for physicians.

Claims

1. A traditional Chinese medicine composition for advanced primary liver cancer, comprising the following raw materials in parts by weight: Ginkgo biloba 32-50 parts, Phyllanthus emblica 22-35 parts, Centipeda minima 20-30 parts, Artemisia annua 20-30 parts, Citrus reticulata 22-35 parts, Citrus aurantium 24-35 parts, Citrus reticulata 12-20 parts, Citrus reticulata 20-30 parts, Citrus aurantium 22-35 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following raw materials in parts by weight: Ginkgo biloba 42-45 parts, Phyllanthus emblica 26-30 parts, Centipeda minima 24-28 parts, Artemisia annua 21-25 parts, Citrus reticulata 26-30 parts, Citrus aurantium 26-30 parts, Citrus reticulata 15-20 parts, Citrus reticulata 21-25 parts, Citrus aurantium 26-30 parts.

3. The traditional Chinese medicine composition according to claim 2, characterized in that, The traditional Chinese medicine composition comprises the following raw materials in parts by weight: Ginkgo biloba 45 parts, Phyllanthus emblica 28 parts, Centipeda minima 25 parts, Artemisia annua 25 parts, Citrus reticulata 28 parts, Citrus aurantium 30 parts, Citrus reticulata 16 parts, Citrus reticulata 25 parts, Citrus aurantium 28 parts.

4. A medicament made from the traditional Chinese medicine composition according to any one of claims 1 to 3 and pharmaceutically acceptable excipients.

5. The drug according to claim 4, characterized in that, The drug is any clinically acceptable formulation.

6. The drug according to claim 5, characterized in that, The preparation is an oral preparation.

7. The drug according to claim 6, characterized in that, The oral preparations are selected from decoctions, powders, capsules, tablets, honey pills, water-honey pills, water pills, concentrated pills, paste pills, wax pills, granules, oral liquids, or drop pills.

8. The medicament according to claim 7, characterized in that, The oral preparations are decoctions, tablets, granules, or capsules.

9. A method for preparing a drug according to any one of claims 4 to 8, comprising preparing each medicinal material of the traditional Chinese medicine composition according to the weight proportions of any one of claims 1 to 3, adding pharmaceutically acceptable excipients according to conventional methods in the art, and preparing a clinically acceptable formulation.

10. A method for preparing a decoction, comprising preparing each medicinal material of the traditional Chinese medicine composition according to the weight proportions of any one of claims 1 to 3, decocting with water two to three times, combining the decoctions, filtering, and obtaining the decoction.

11. A method for preparing granules, comprising preparing each medicinal material of the traditional Chinese medicine composition according to the weight proportions of any one of claims 1 to 3; adding water at 10 times the total weight of the medicinal materials; soaking for 1-2 hours; extracting volatile oil; collecting the volatile oil separately for later use; collecting the distilled liquid separately; taking β-cyclodextrin at 10 times the weight of the volatile oil; adding an appropriate amount of water; dissolving in an aqueous solution at 50-70°C; cooling; adding the volatile oil; stirring at 300-500 r / min for 1-2 hours; and refrigerating for 24 hours. Remove the residue, filter it, dry it at 40°C, grind it into a fine powder to obtain the β-cyclodextrin inclusion complex, and set it aside. Continue to decoct the residue twice with water, adding 8 times the weight of water each time, and decoct for 1-2 hours each time. Filter it while hot, combine the filtrates, add the distilled liquid and concentrate it to an extract with a relative density of 1.20-1.25 at 50°C. Dry it, pulverize it into a fine powder, add the β-cyclodextrin inclusion complex, with or without pharmaceutically acceptable excipients, mix evenly, dry granulate, dry, and granulate to obtain the final product.

12. The use of the traditional Chinese medicine composition according to any one of claims 1 to 3, the drug according to any one of claims 4 to 8, and the drug prepared according to the preparation method according to any one of claims 9 to 11 in the preparation of a drug for treating advanced primary liver cancer; symptoms include distending pain in the hypochondrium, lumps under the hypochondrium, poor appetite, fatigue, dizziness, dry mouth and bitter taste, irritability, red tongue, yellow coating, and wiry and rapid pulse.

13. The use of the traditional Chinese medicine composition according to any one of claims 1 to 3, the drug according to any one of claims 4 to 8, and the drug prepared according to the preparation method according to any one of claims 9 to 11 in the preparation of a drug for treating unresectable advanced primary liver cancer.

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