A traditional Chinese medicine preparation for treating solid tumor by activating blood and resolving stasis

Abstract

The application relates to the field of traditional Chinese medicine, in particular to a traditional Chinese medicine compound preparation for treating solid tumors and promoting blood circulation to remove blood stasis and a preparation method and application thereof. Under the guidance of traditional Chinese medicine theory, according to the syndrome manifestations of patients with solid tumors (early, middle and late stages), the traditional Chinese medicine compound preparation prepared from the animal traditional Chinese medicines gecko, sea squirts and leeches and the plant traditional Chinese medicines notoginseng, madder, safflower and licorice in a certain proportion is used to treat patients with blood stasis syndrome of malignant tumors, can obviously improve the life quality of the patients, reduce the toxic and side effects of radiotherapy and chemotherapy, prolong the survival period of the patients and improve the symptoms of blood stasis syndrome of the patients.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically to a traditional Chinese medicine compound preparation for treating solid tumors that promotes blood circulation and removes blood stasis, its preparation method, and its application. Background Technology

[0002] Clinically, common symptoms of solid tumors (such as esophageal cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, colorectal cancer, kidney cancer, prostate cancer, and breast cancer) include abnormal masses, decreased appetite, weight loss, low-grade fever, fatigue, pain, purplish lips and nails, and tortuous sublingual veins. These symptoms are similar to those described in traditional Chinese medicine as blood stasis syndrome and can be categorized under this syndrome. The connection between solid tumors and blood stasis has been extensively discussed in historical literature. The *Neijing* (Inner Canon of Medicine) records that "all diseases arise from qi stagnation," "blood stagnation leads to obstruction," "blood congealing leads to stagnation," and "irregular emotions...uneven temperature...accumulation and retention." This is the earliest discussion of the relationship between masses, lumps, and stones and blood stasis syndrome. *Gujin Yitong* (Comprehensive Medical Treatise of Ancient and Modern Times) states that "when food is difficult to swallow, with a feeling of twisting and slight pain, there must be dead blood," indicating that the pathological factor of esophageal cancer is blood stasis. *Yilin Gaicuo* (Corrections of Errors in Medical Classics) states that "lumps in the abdomen must contain tangible blood."

[0003] Blood stasis syndrome exists and persists throughout the entire pathological process of solid tumors, meaning that microscopic blood stasis exists from the non-invasive metastasis to the invasive metastasis of tumors. Modern oncology research shows that individuals with blood stasis constitution have a tendency to develop cancer, and after cancer develops, the rate of malignant metastasis is significantly higher, the recurrence rate is also higher, and the prognosis is poor. Numerous clinical observations show that abnormal blood rheology is a common pathological change in cancer patients. The higher the blood viscosity, the more obvious the tumor blood stasis, and the blood is in a viscous and aggregated state. The presence of hyperviscosity syndrome has an important influence on the formation of tumor metastasis. Another piece of evidence for the presence of blood stasis syndrome in malignant tumors is abnormal blood coagulation, characterized by activated coagulation mechanisms, platelet activation, and weakened anticoagulation function, leading to a hypercoagulable state of blood, which is prone to thrombosis. Lung cancer patients have enhanced platelet aggregation and adhesion function, especially after lung cancer metastasis. Early tumor coagulation function is enhanced, resulting in a hypercoagulable state, a tendency to form thrombi, and a poor prognosis.

[0004] Both traditional Chinese medicine and Western medicine recognize that blood stasis or hypercoagulable states are common pathogenesis and clinical manifestations of tumors and cancerous metastases, and that promoting blood circulation and removing blood stasis should be a fundamental strategy for preventing and treating cancer and malignant metastases. However, no reports have yet been found regarding a traditional Chinese medicine compound preparation that can effectively address blood stasis syndrome in patients with solid tumors. Summary of the Invention

[0005] The purpose of this invention is to provide a traditional Chinese medicine compound preparation for treating blood stasis syndrome in solid tumors, which can effectively solve the problem of blood stasis syndrome in patients with solid tumors in clinical practice. In particular, it has a significant therapeutic effect on patients with blood stasis syndrome in mid-to-late stage solid malignant tumors (such as esophageal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, pancreatic cancer, colorectal cancer, kidney cancer, prostate cancer, bladder cancer, etc.).

[0006] Through long-term clinical practice and basic research, our research group has confirmed that traditional Chinese medicine for promoting blood circulation and removing blood stasis has a good therapeutic effect on tumors, which may be related to mechanisms such as inhibiting tumor cell proliferation, promoting tumor cell apoptosis, reversing tumor cell drug resistance, regulating tumor cell immunity, inhibiting tumor angiogenesis, and preventing tumor metastasis.

[0007] In the traditional Chinese medicine compound preparation of the present invention:

[0008] Gecko: dispels wind and calms the nerves, detoxifies and dissipates nodules;

[0009] Panax notoginseng: disperses blood stasis, stops bleeding, reduces swelling, and relieves pain;

[0010] Cuttlebone: Astringent and hemostatic, astringent and stops seminal emission, neutralizes acid and relieves pain, astringes moisture and heals sores;

[0011] Madder root: Cools the blood and removes blood stasis, stops bleeding and regulates menstruation;

[0012] Leeches: break up blood stasis, promote menstruation, remove blood stasis and eliminate masses;

[0013] Safflower: Promotes blood circulation, regulates menstruation, disperses blood stasis, and relieves pain;

[0014] Licorice: Tonifies the spleen and stomach, replenishes qi and generates fluids, regulates the body's defenses, and detoxifies.

[0015] This formula uses gecko and cuttlebone as the principal ingredients, Panax notoginseng and Rubia cordifolia as the assistant ingredients, and leeches, safflower, and licorice to harmonize the other ingredients. When these seven ingredients are used together, they work together to regulate blood circulation, remove blood stasis, disperse nodules, and relieve pain.

[0016] In a first aspect, the present invention provides a traditional Chinese medicine compound preparation for treating solid tumors by promoting blood circulation and removing blood stasis, which is made from the following raw materials in parts by weight:

[0017] Geckos: 30-500 servings

[0018] Panax notoginseng: 30-500 doses

[0019] Cuttlebone: 30-500 servings

[0020] Madder: 30-500 parts

[0021] Leeches: 30-500 portions

[0022] Safflower: 30-500 portions

[0023] Licorice: 30-500 parts.

[0024] Furthermore, the aforementioned traditional Chinese medicine compound preparation for treating solid tumors by promoting blood circulation and removing blood stasis is made from the following parts by weight of traditional Chinese medicine raw materials:

[0025] Geckos: 50-300 servings

[0026] Panax notoginseng: 50-300 doses

[0027] Cuttlebone: 50-300 servings

[0028] Madder: 50-300 parts

[0029] Leeches: 50-300 portions

[0030] Safflower: 50-300 portions

[0031] Licorice: 50-300 parts.

[0032] In a preferred embodiment of the present invention, the traditional Chinese medicine compound preparation for treating solid tumors by promoting blood circulation and removing blood stasis is made from the following traditional Chinese medicine raw materials in parts by weight:

[0033] Gecko: 300 copies

[0034] Panax notoginseng: 100 doses

[0035] Cuttlebone: 300 servings

[0036] Madder: 100 portions

[0037] Leeches: 300 portions

[0038] Safflower: 100 portions

[0039] Licorice: 200 parts.

[0040] In a preferred embodiment of the present invention, the traditional Chinese medicine compound preparation for treating solid tumors by promoting blood circulation and removing blood stasis is made from the following traditional Chinese medicine raw materials in parts by weight:

[0041] Gecko: 200 copies

[0042] Panax notoginseng: 100 doses

[0043] Cuttlebone: 200 servings

[0044] Madder: 100 portions

[0045] Leeches: 200 servings

[0046] Safflower: 100 portions

[0047] Licorice: 200 parts.

[0048] In a preferred embodiment of the present invention, the traditional Chinese medicine compound preparation for treating solid tumors by promoting blood circulation and removing blood stasis is made from the following traditional Chinese medicine raw materials in parts by weight:

[0049] Gecko: 100 copies

[0050] Panax notoginseng: 100 doses

[0051] Cuttlebone: 100 servings

[0052] Madder: 100 portions

[0053] Leeches: 100 servings

[0054] Safflower: 100 portions

[0055] Licorice: 200 parts.

[0056] In a preferred embodiment of the present invention, the traditional Chinese medicine compound preparation for treating solid tumors by promoting blood circulation and removing blood stasis is made from the following traditional Chinese medicine raw materials in parts by weight:

[0057] Gecko: 100 copies

[0058] Panax notoginseng: 200 doses

[0059] Cuttlebone: 100 servings

[0060] Madder: 200 portions

[0061] Leeches: 100 servings

[0062] Safflower: 200 portions

[0063] Licorice: 200 parts.

[0064] In a preferred embodiment of the present invention, the traditional Chinese medicine compound preparation for treating solid tumors by promoting blood circulation and removing blood stasis is made from the following traditional Chinese medicine raw materials in parts by weight:

[0065] Gecko: 100 copies

[0066] Panax notoginseng: 300 doses

[0067] Cuttlebone: 100 servings

[0068] Madder: 300 portions

[0069] Leeches: 100 servings

[0070] Safflower: 300 portions

[0071] Licorice: 200 parts.

[0072] Furthermore, the aforementioned traditional Chinese medicine compound preparation can be prepared using conventional preparation methods in the field, supplemented with conventional pharmaceutical excipients, into a common dosage form in pharmaceutical science.

[0073] Furthermore, the dosage form of the traditional Chinese medicine compound preparation is powder, granule, tablet, capsule, oral liquid or pill.

[0074] Furthermore, the traditional Chinese medicine compound preparation also includes pharmaceutically acceptable excipients.

[0075] In a second aspect, the present invention provides a method for preparing a traditional Chinese medicine compound preparation for treating solid tumors by promoting blood circulation and removing blood stasis, as described above, comprising the following steps:

[0076] (A) Weigh the Chinese herbal raw materials according to the weight proportions;

[0077] (B) Alcohol extraction and water precipitation: The herbal medicines (Panax notoginseng, cuttlebone, Rubia cordifolia, safflower, and licorice) in the above-mentioned Chinese herbal raw materials are mixed together in proportion and placed in a Chinese herbal decoction pot for decoction three times. For the first decoction, add 8 times the weight of the medicines in 95% ethanol, boil, and simmer for 50 minutes, then filter out the liquid. For the second decoction, add 7 times the weight of the medicines in 95% ethanol, boil, and simmer for 45 minutes, then filter out the liquid. For the third decoction, add 6 times the weight of the medicines in 95% ethanol, boil, and simmer for 40 minutes, then filter out the liquid. Combine the three liquids and evaporate and concentrate them to a thick consistency over a low heat. Add an equal amount of water, mix thoroughly, and let stand overnight to allow precipitation. The next day, filter the precipitate completely, take the supernatant, distill to recover the ethanol, concentrate, and let stand for 24 hours. Concentrate the liquid after standing in a rotary evaporator at 80°C in a water bath to a thick paste (relative specific gravity 1.4 / 25°C).

[0078] (C) Water extraction and alcohol precipitation: The animal drugs (gecko and leech) in the Chinese herbal raw materials are mixed together in proportion and placed in a Chinese herbal decoction pot for decoction three times. For the first decoction, add 8 times the weight of the drugs and water, boil, and simmer for 50 minutes. Filter the liquid. For the second decoction, add 7 times the weight of the drugs and water, boil, and simmer for 45 minutes. Filter the liquid. For the third decoction, add 6 times the weight of the drugs and water, boil, and simmer for 40 minutes. Filter the liquid. Combine the three decoctions and evaporate and concentrate them to a thick consistency over a low heat. Add an equal amount of anhydrous ethanol, mix thoroughly, and let stand overnight to allow precipitation. The next day, filter the precipitate completely, take the supernatant, distill to recover the ethanol, concentrate, and let stand for 24 hours. Concentrate the liquid after standing in a rotary evaporator at 80°C in a water bath to a thick paste (relative specific gravity 1.4 / 25°C).

[0079] (D) Finally, the alcohol-precipitated thick paste and the water-precipitated thick paste are vacuum dried at -50°C to obtain a powdered Chinese herbal medicine compound extract, which is the Chinese herbal medicine compound preparation.

[0080] A third aspect of the present invention provides the application of the traditional Chinese medicine compound preparation described above in the preparation of a medicament for treating solid tumors.

[0081] Furthermore, the aforementioned drug is a drug for treating blood stasis syndrome in solid tumors.

[0082] Furthermore, the solid tumors mentioned include colorectal cancer, esophageal cancer, lung cancer, breast cancer, liver cancer, stomach cancer, pancreatic cancer, colorectal cancer, kidney cancer, prostate cancer, bladder cancer, etc.

[0083] Furthermore, the solid tumor is an intermediate-to-late stage solid malignant tumor.

[0084] The advantages of this invention are:

[0085] Guided by traditional Chinese medicine theory, this invention utilizes a compound Chinese medicine preparation made from animal-derived Chinese medicinal herbs (gecko, cuttlebone, and leech) and plant-derived Chinese medicinal herbs (Panax notoginseng, Rubia cordifolia, Carthamus tinctorius, and Glycyrrhiza uralensis) in a specific ratio to treat patients with blood stasis syndrome in malignant tumors. This preparation can significantly improve patients' quality of life, reduce the toxic side effects of radiotherapy and chemotherapy, prolong patients' survival, and alleviate the symptoms of blood stasis syndrome. Attached Figure Description

[0086] Figure 1 .CCK-8 broken line plot of HCT116 cells;

[0087] Figure 2 .HepG2 cell CCK-8 broken line plot;

[0088] Figure 3 A549 cell CCK-8 broken line plot;

[0089] Figure 4 Schematic diagram of animal experiments;

[0090] Figure 5 Subcutaneous tumor changes diagram;

[0091] Figure 6 Line graph showing changes in subcutaneous tumors. Detailed Implementation

[0092] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0093] Example 1: Preparation of the traditional Chinese medicine compound preparation of the present invention

[0094] Weigh out the Chinese herbal raw materials (grams) according to the following weight proportions:

[0095] 300 parts gecko, 100 parts Panax notoginseng, 300 parts cuttlebone, 100 parts madder root, 300 parts leech, 100 parts safflower, and 200 parts licorice.

[0096] Alcohol extraction and water precipitation: The herbal medicines (Panax notoginseng, cuttlebone, Rubia cordifolia, safflower, and licorice) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low heat until thick. An equal amount of water was added, and the mixture was thoroughly mixed and left to stand overnight to allow precipitation. The next day, the precipitate was completely filtered out, and the supernatant was collected. The ethanol was recovered by distillation, and the mixture was concentrated and allowed to stand for 24 hours. The liquid after standing was then concentrated into a thick paste (relative specific gravity 1.4 / 25℃) using a rotary evaporator in an 80℃ water bath.

[0097] Water extraction and alcohol precipitation: The animal-derived Chinese medicines (gecko and leech) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low flame until thick. An equal amount of anhydrous ethanol was added, mixed thoroughly, and left to stand overnight to allow precipitation. The next day, the supernatant was collected, the ethanol was recovered by distillation, concentrated, and allowed to stand for 24 hours to allow the precipitate to completely settle. The precipitate was then filtered out and concentrated in a rotary evaporator at 80°C in a water bath to a thick paste (relative specific gravity 1.4 / 25°C).

[0098] Finally, the alcohol-precipitated thick paste and the water-precipitated thick paste were vacuum dried at -50°C to obtain a powdered traditional Chinese medicine compound extract, which is the traditional Chinese medicine compound preparation of the present invention.

[0099] Example 2: Preparation of the traditional Chinese medicine compound preparation of the present invention

[0100] Weigh out the Chinese herbal raw materials (grams) according to the following weight proportions:

[0101] 200 parts gecko, 100 parts Panax notoginseng, 200 parts cuttlebone, 100 parts madder root, 200 parts leech, 100 parts safflower, and 200 parts licorice.

[0102] Alcohol extraction and water precipitation: The herbal medicines (Panax notoginseng, cuttlebone, Rubia cordifolia, safflower, and licorice) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low heat until thick. An equal amount of water was added, and the mixture was thoroughly mixed and left to stand overnight to allow precipitation. The next day, the precipitate was completely filtered out, and the supernatant was collected. The ethanol was recovered by distillation, and the mixture was concentrated and allowed to stand for 24 hours. The liquid after standing was then concentrated into a thick paste (relative specific gravity 1.4 / 25℃) using a rotary evaporator in an 80℃ water bath.

[0103] Water extraction and alcohol precipitation: The animal-derived Chinese medicines (gecko and leech) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low flame until thick. An equal amount of anhydrous ethanol was added, mixed thoroughly, and left to stand overnight to allow precipitation. The next day, the precipitate was completely filtered out, and the supernatant was collected. The ethanol was recovered by distillation, concentrated, and allowed to stand for 24 hours. The liquid after standing was then concentrated to a thick paste (relative specific gravity 1.4 / 25℃) using a rotary evaporator in an 80℃ water bath.

[0104] Finally, the alcohol-precipitated thick paste and the water-precipitated thick paste were vacuum dried at -50°C to obtain a powdered traditional Chinese medicine compound extract, which is the traditional Chinese medicine compound preparation of the present invention.

[0105] Example 3: Preparation of the traditional Chinese medicine compound preparation of the present invention

[0106] Weigh out the Chinese herbal raw materials (grams) according to the following weight proportions:

[0107] 100 parts gecko, 100 parts Panax notoginseng, 100 parts cuttlebone, 100 parts madder root, 100 parts leech, 100 parts safflower, and 200 parts licorice.

[0108] Alcohol extraction and water precipitation: The herbal medicines (Panax notoginseng, cuttlebone, Rubia cordifolia, safflower, and licorice) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low heat until thick. An equal amount of water was added, and the mixture was thoroughly mixed and left to stand overnight to allow precipitation. The next day, the precipitate was completely filtered out, and the supernatant was collected. The ethanol was recovered by distillation, and the mixture was concentrated and allowed to stand for 24 hours. The liquid after standing was then concentrated into a thick paste (relative specific gravity 1.4 / 25℃) using a rotary evaporator in an 80℃ water bath.

[0109] Water extraction and alcohol precipitation: The animal-derived Chinese medicines (gecko and leech) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low flame until thick. An equal amount of anhydrous ethanol was added, mixed thoroughly, and left to stand overnight to allow precipitation. The next day, the precipitate was completely filtered out, and the supernatant was collected. The ethanol was recovered by distillation, concentrated, and allowed to stand for 24 hours. The liquid after standing was then concentrated to a thick paste (relative specific gravity 1.4 / 25℃) using a rotary evaporator in an 80℃ water bath.

[0110] Finally, the alcohol-precipitated thick paste and the water-precipitated thick paste were vacuum dried at -50°C to obtain a powdered traditional Chinese medicine compound extract, which is the traditional Chinese medicine compound preparation of the present invention.

[0111] Example 4: Preparation of the traditional Chinese medicine compound preparation of the present invention

[0112] Weigh out the Chinese herbal raw materials (grams) according to the following weight proportions:

[0113] 100 parts gecko, 200 parts Panax notoginseng, 100 parts cuttlebone, 200 parts madder root, 100 parts leech, 200 parts safflower, and 200 parts licorice root.

[0114] Alcohol extraction and water precipitation: The herbal medicines (Panax notoginseng, cuttlebone, Rubia cordifolia, safflower, and licorice) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low heat until thick. An equal amount of water was added, and the mixture was thoroughly mixed and left to stand overnight to allow precipitation. The next day, the precipitate was completely filtered out, and the supernatant was collected. The ethanol was recovered by distillation, and the mixture was concentrated and allowed to stand for 24 hours. The liquid after standing was then concentrated into a thick paste (relative specific gravity 1.4 / 25℃) using a rotary evaporator in an 80℃ water bath.

[0115] Water extraction and alcohol precipitation: The animal-derived Chinese medicines (gecko and leech) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low flame until thick. An equal amount of anhydrous ethanol was added, mixed thoroughly, and left to stand overnight to allow precipitation. The next day, the precipitate was completely filtered out, and the supernatant was collected. The ethanol was recovered by distillation, concentrated, and allowed to stand for 24 hours. The liquid after standing was then concentrated to a thick paste (relative specific gravity 1.4 / 25℃) using a rotary evaporator in an 80℃ water bath.

[0116] Finally, the alcohol-precipitated thick paste and the water-precipitated thick paste were vacuum dried at -50°C to obtain a powdered traditional Chinese medicine compound extract, which is the traditional Chinese medicine compound preparation of the present invention.

[0117] Example 5: Preparation of the traditional Chinese medicine compound preparation of the present invention

[0118] Weigh out the Chinese herbal raw materials (grams) according to the following weight proportions:

[0119] 100 parts gecko, 300 parts Panax notoginseng, 100 parts cuttlebone, 300 parts madder root, 100 parts leech, 300 parts safflower, and 200 parts licorice.

[0120] Alcohol extraction and water precipitation: The herbal medicines (Panax notoginseng, cuttlebone, Rubia cordifolia, safflower, and licorice) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the herbs in 95% ethanol were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low heat until thick. An equal amount of water was added, and the mixture was thoroughly mixed and left to stand overnight to allow precipitation. The next day, the precipitate was completely filtered out, and the supernatant was collected. The ethanol was recovered by distillation, and the mixture was concentrated and allowed to stand for 24 hours. The liquid after standing was then concentrated into a thick paste (relative specific gravity 1.4 / 25℃) using a rotary evaporator in an 80℃ water bath.

[0121] Water extraction and alcohol precipitation: The animal-derived Chinese medicines (gecko and leech) in the above-mentioned Chinese medicine were mixed together in the specified proportions and decocted three times in a decoction pot. For the first decoction, 8 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 50 minutes and filtered out. For the second decoction, 7 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 45 minutes and filtered out. For the third decoction, 6 times the weight of the medicines in water were added, and after boiling, the decoction was simmered for 40 minutes and filtered out. The three decoctions were combined and concentrated by evaporation over a low flame until thick. An equal amount of anhydrous ethanol was added, mixed thoroughly, and left to stand overnight to allow precipitation. The next day, the precipitate was completely filtered out, and the supernatant was collected. The ethanol was recovered by distillation, concentrated, and allowed to stand for 24 hours. The liquid after standing was then concentrated to a thick paste (relative specific gravity 1.4 / 25℃) using a rotary evaporator in an 80℃ water bath.

[0122] Finally, the alcohol-precipitated thick paste and the water-precipitated thick paste were vacuum dried at -50°C to obtain a powdered traditional Chinese medicine compound extract, which is the traditional Chinese medicine compound preparation of the present invention.

[0123] Example 6: Preparation of tablets / capsules

[0124] The powdered traditional Chinese medicine compound extract prepared according to the above embodiments is then added with pharmaceutical excipients and compressed into tablets or filled capsules.

[0125] Example 7: Preparation of powders / granules

[0126] The powdered compound extract of traditional Chinese medicine was prepared according to the above embodiments, and pharmaceutical excipients were added to make it into powder, granule or granule.

[0127] Example 8: Preparation of pills

[0128] The powdered compound extract of traditional Chinese medicine was prepared according to the above embodiments, and then appropriate pharmaceutical excipients (such as white sugar, honey, benzopropionic acid or ethylparaben) were added to make pills.

[0129] Example 9: The inhibitory effect of the traditional Chinese medicine compound preparation of the present invention on the growth of colorectal cancer cell line HCT116

[0130] Test samples: HCT116 cells, purchased from the Cell Bank of the Chinese Academy of Sciences and preserved by the Tumor Laboratory of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine; the traditional Chinese medicine compound preparation of this invention (see Example 3 for prescription and preparation method); RPMI 1640 complete culture medium; CCK-8 assay kit, Beyotime;

[0131] Experimental method: HCT116 cells were cultured at 8×10⁻⁶ cells per cell line. 3 100 μL of the compound preparation was seeded into 96-well plates and cultured at 37°C and 5% CO2 for monolayer growth for 24 h. After complete cell adhesion, complete culture medium containing different concentrations of the traditional Chinese medicine compound preparation of this invention (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mg·L⁻¹) was added. -1 The effects of the traditional Chinese medicine compound preparation of the present invention on the proliferation of HCT116 cells were observed using both drug-free and untreated complete culture media, and the half-inhibitory concentration (IC50) was determined. 50 Each group had 6 replicates. After incubating cells for 24 h and 48 h, 100 μL of complete culture medium and 10% CCK-8 reagent were added to each well, and the cells were incubated at 37°C for 1 h. The optical density (OD) of each well at 450 nm was measured using an automated microplate reader, the survival rate was calculated, and the effect curve was plotted. The experiment was repeated 3 times.

[0132] like Figure 1 As shown, compared with the untreated complete culture medium group, the number of HCT116 cells observed under a microscope in the drug-treated group was significantly reduced, and the HCT116 cell survival rate was significantly reduced after ELISA reader analysis. Furthermore, with increasing concentration of the traditional Chinese medicine compound preparation of this invention, the survival rate of HCT116 cells significantly decreased in a dose-dependent manner.

[0133] Compared with 24h, the number of HCT116 cells decreased significantly at 48h, and the survival rate of HCT116 cells was lower, showing a time-dependent effect.

[0134] Experimental results show that the traditional Chinese medicine compound preparation of the present invention can inhibit the growth of colorectal cancer cell line HCT116, and further inhibits the growth of HCT116 cells after extending the treatment time of the traditional Chinese medicine compound preparation of the present invention. The concentrations of the traditional Chinese medicine compound preparation of the present invention at 24h and 48h were 0.7961 mg·L⁻¹, respectively. -1 0.519 mg·L -1 At that time, it could inhibit HCT116 cells by half.

[0135] Example 10: The inhibitory effect of the traditional Chinese medicine compound preparation of the present invention on the growth of the HepG2 liver cancer cell line.

[0136] Test samples: HepG2 cells, purchased from the Cell Bank of the Chinese Academy of Sciences and preserved by the Tumor Laboratory of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine; the traditional Chinese medicine compound preparation of this invention (see Example 3 for prescription and preparation method); RPMIDMEM complete culture medium; Gibco MEM NEAA nutrient solution; CCK-8 assay kit, Beyotime;

[0137] Experimental method: HepG2 cells were cultured at 5 × 10⁻⁶ cells / year. 3 100 μL of the compound preparation was seeded into 96-well plates and cultured at 37°C and 5% CO2 for monolayer growth for 24 h. After complete cell adhesion, complete culture medium containing different concentrations of the traditional Chinese medicine compound preparation of this invention (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mg·L⁻¹) was added. -1 The effects of the traditional Chinese medicine compound preparation of the present invention on the proliferation of HCT116 cells were observed using both drug-free and untreated complete culture media, and the half-inhibitory concentration (IC50) was determined. 50 Each group had 6 replicates. After incubating cells for 24 h and 48 h, 100 μL of complete culture medium and 10% CCK-8 reagent were added to each well, and the cells were incubated at 37°C for 1 h. The optical density (OD) of each well at 450 nm was measured using an automated microplate reader, the survival rate was calculated, and the effect curve was plotted. The experiment was repeated 3 times.

[0138] like Figure 2 As shown, compared with the untreated complete culture medium group, the number of HepG2 cells observed under a microscope in the drug-treated group was significantly reduced, and the survival rate of HepG2 cells was significantly reduced after ELISA reader analysis. Furthermore, with the increase of the concentration of the traditional Chinese medicine compound preparation of this invention, the survival rate of HepG2 cells significantly decreased in a dose-dependent manner.

[0139] Compared with 24h, the number of HepG2 cells decreased significantly at 48h, and the survival rate of HepG2 cells was lower, showing a time-dependent effect.

[0140] Experimental results show that the traditional Chinese medicine compound preparation of the present invention can inhibit the growth of HepG2 liver cancer cell line, and further inhibits the growth of HepG2 cells after extending the treatment time of the traditional Chinese medicine compound preparation of the present invention. The concentrations of the traditional Chinese medicine compound preparation of the present invention at 24h and 48h were 2.549 mg·L⁻¹, respectively. -1 0.2998 mg·L -1 It can inhibit half of HepG2 cells.

[0141] Example 11: The inhibitory effect of the traditional Chinese medicine compound preparation of the present invention on the growth of lung cancer cell line A549

[0142] Test samples: A549 cells, purchased from the Cell Bank of the Chinese Academy of Sciences and preserved by the Tumor Laboratory of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine; the traditional Chinese medicine compound preparation of this invention (see Example 3 for prescription and preparation method); RPMI 1640 complete culture medium; CCK-8 assay kit, Beyotime;

[0143] Experimental method: A549 cells were cultured at a rate of 4 × 10⁻⁶. 3 100 μL of the compound preparation was seeded into 96-well plates and cultured at 37°C and 5% CO2 for monolayer growth for 24 h. After complete cell adhesion, complete culture medium containing different concentrations of the traditional Chinese medicine compound preparation of this invention (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mg·L⁻¹) was added. -1 The effects of the traditional Chinese medicine compound preparation of the present invention on the proliferation of HCT116 cells were observed using both drug-free and untreated complete culture media, and the half-inhibitory concentration (IC50) was determined. 50 Each group had 6 replicates. After incubating cells for 24 h and 48 h, 100 μL of complete culture medium and 10% CCK-8 reagent were added to each well, and the cells were incubated at 37°C for 1 h. The optical density (OD) of each well at 450 nm was measured using an automated microplate reader, the survival rate was calculated, and the effect curve was plotted. The experiment was repeated 3 times.

[0144] like Figure 3 As shown, compared with the untreated complete culture medium group, the number of A549 cells observed under a microscope in the drug-treated group was significantly reduced, and the survival rate of A549 cells was significantly reduced after ELISA reader analysis. Furthermore, the survival rate of A549 cells decreased significantly with increasing concentration of the traditional Chinese medicine compound preparation of this invention, exhibiting a dose-dependent effect.

[0145] Compared with 24h, the number of A549 cells decreased significantly at 48h, and the survival rate of A549 cells was lower, showing a time-dependent effect.

[0146] Experimental results show that the traditional Chinese medicine compound preparation of the present invention can inhibit the growth of lung cancer cell line A549, and further inhibits the growth of A549 cells after extending the treatment time of the traditional Chinese medicine compound preparation of the present invention. The concentrations of the traditional Chinese medicine compound preparation of the present invention at 24h and 48h were 1.386 mg·L⁻¹, respectively. -1 0.8345 mg·L -1 It can inhibit A549 cells by half.

[0147] Example 12: Inhibitory effect of the present invention on subcutaneous tumors of colorectal cancer cell line HCT116 in mice

[0148] Test sample: Model group: physiological saline, commercially available;

[0149] The low-dose group of the traditional Chinese medicine compound preparation of the present invention: the prescription and preparation method are as described in Example 3, and the concentration of the traditional Chinese medicine compound in the low-dose group is 1.035 g / ml;

[0150] The high-dose group of the traditional Chinese medicine compound preparation of the present invention: the prescription and preparation method are as described in Example 3, and the concentration of the traditional Chinese medicine compound in the high-dose group is 2.07 g / ml;

[0151] Test animals: 15 female nude mice (6-8 weeks BALB / c), weighing 18-20g, 12 of which were randomly divided into 3 groups of 4 mice each for the experiment, and the remaining 3 mice were used for tumor passage. The mice were purchased from Jihui.

[0152] Tumor cell line: KRAS-mutant human colon cancer cell line HCT116, purchased from the Chinese Academy of Sciences Cell Bank and preserved by the Tumor Laboratory of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine.

[0153] Experimental method: Human colorectal cancer HCT116 cell line PBS suspension (1×10⁻⁶) was inoculated into the left axilla of a mouse. 7 / mL, inoculation dose 0.1mL / mouse). Seven days after inoculation, the size of the tumor was observed. When the tumor reached 12cm×12cm, it was dissected from the subcutaneous tissue of the mice, and two 1cm×1cm round tumors without blood vessels were separated. These were placed into small incisions made subcutaneously in the left axilla of two anesthetized mice, and finally sutured with 5-0 absorbable sutures. Seven days after tumor passage, the size of the tumors in the axilla of the two mice was observed. When the tumor reached 12cm×12cm, it was dissected from the subcutaneous tissue of the two mice, and 14 1cm×1cm round tumors without blood vessels were separated. These were placed into small incisions made subcutaneously in the left axilla of 14 anesthetized mice, and finally sutured with 5-0 absorbable sutures. The day after tumor passage, the mice were randomly divided into groups, weighed, and administered the drug by gavage (e.g. Figure 4 Each mouse was administered 0.2 ml of the traditional Chinese medicine compound by gavage once daily for 14 consecutive days. The mouse's body weight and subcutaneous tumor size were recorded every other day and plotted as a line graph (experimental method see [link to experimental method]). Figure 4 ).

[0154] like Figure 5 and Figure 6 As shown, compared with the model group, the low-dose and high-dose groups of the traditional Chinese medicine compound preparation of the present invention can significantly inhibit the growth of subcutaneous HCT116 tumors in mice. Furthermore, the high-dose group of mice showed a more significant reduction in subcutaneous HCT116 tumor size compared to the low-dose group, exhibiting a dose-dependent effect.

[0155] Experimental results show that the traditional Chinese medicine compound preparation of the present invention can inhibit the growth of subcutaneous HCT116 tumors in mice, and the effect is related to the dosage, with the best effect at high doses.

[0156] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A traditional Chinese medicine compound preparation for treating colorectal cancer by promoting blood circulation and removing blood stasis, characterized in that, Made from the following parts by weight of active pharmaceutical ingredient: Geckos: 30-500 servings Panax notoginseng: 30-500 doses Cuttlebone: 30-500 servings Madder: 30-500 parts Leeches: 30-500 portions Safflower: 30-500 portions Licorice: 30-500 parts.

2. The traditional Chinese medicine compound preparation for treating colorectal cancer by promoting blood circulation and removing blood stasis according to claim 1, characterized in that, Made from the following parts by weight of traditional Chinese medicine raw materials: Geckos: 50-300 servings Panax notoginseng: 50-300 doses Cuttlebone: 50-300 servings Madder: 50-300 parts Leeches: 50-300 portions Safflower: 50-300 portions Licorice: 50-300 parts.

3. The traditional Chinese medicine compound preparation for treating colorectal cancer by promoting blood circulation and removing blood stasis according to claim 1, characterized in that, Made from the following parts by weight of traditional Chinese medicine raw materials: Gecko: 300 copies Panax notoginseng: 100 doses Cuttlebone: 300 servings Madder: 100 portions Leeches: 300 portions Safflower: 100 portions Licorice: 200 parts.

4. The traditional Chinese medicine compound preparation for treating colorectal cancer by promoting blood circulation and removing blood stasis according to claim 1, characterized in that, Made from the following parts by weight of traditional Chinese medicine raw materials: Gecko: 200 copies Panax notoginseng: 100 doses Cuttlebone: 200 servings Madder: 100 portions Leeches: 200 servings Safflower: 100 portions Licorice: 200 parts.

5. The traditional Chinese medicine compound preparation for treating colorectal cancer by promoting blood circulation and removing blood stasis according to claim 1, characterized in that, Made from the following parts by weight of traditional Chinese medicine raw materials: Gecko: 100 copies Panax notoginseng: 100 doses Cuttlebone: 100 servings Madder: 100 portions Leeches: 100 servings Safflower: 100 portions Licorice: 200 parts.

6. The traditional Chinese medicine compound preparation for treating colorectal cancer by promoting blood circulation and removing blood stasis according to claim 1, characterized in that, Made from the following parts by weight of traditional Chinese medicine raw materials: Gecko: 100 copies Panax notoginseng: 200 doses Cuttlebone: 100 servings Madder: 200 portions Leeches: 100 servings Safflower: 200 portions Licorice: 200 parts.

7. The traditional Chinese medicine compound preparation for treating colorectal cancer by promoting blood circulation and removing blood stasis according to claim 1, characterized in that, Made from the following parts by weight of traditional Chinese medicine raw materials: Gecko: 100 copies Panax notoginseng: 300 doses Cuttlebone: 100 servings Madder: 300 portions Leeches: 100 servings Safflower: 300 portions Licorice: 200 parts.

8. A method for preparing a traditional Chinese medicine compound preparation for treating colorectal cancer with blood-activating and stasis-removing properties as described in any one of claims 1-7, characterized in that, Includes the following steps: (A) Weigh the Chinese herbal raw materials according to the weight proportions; (B) Alcohol extraction and water precipitation: The herbal raw materials Panax notoginseng, cuttlebone, madder root, safflower, and licorice root are mixed together in proportion and placed in a decoction pot for decoction three times. For the first decoction, add 8 times the weight of the herbs in 95% ethanol, boil, and simmer for 50 minutes. Filter the liquid. For the second decoction, add 7 times the weight of the herbs in 95% ethanol, boil, and simmer for 45 minutes. Filter the liquid. For the third decoction, add 6 times the weight of the herbs in 95% ethanol, boil, and simmer for 40 minutes. Filter the liquid. Combine the three decoctions and evaporate and concentrate them to a thick consistency over a low heat. Add an equal amount of water, mix thoroughly, and let stand overnight to allow precipitation. The next day, filter the precipitate completely, take the supernatant, distill to recover the ethanol, concentrate, and let stand for 24 hours. Concentrate the liquid after standing in a rotary evaporator at 80°C in a water bath to obtain a thick paste with a specific gravity of 1.4 at 25°C. (C) Water extraction and alcohol precipitation: The animal drugs gecko and leech in the Chinese herbal raw materials are mixed together in proportion and placed in a Chinese herbal decoction pot for decoction three times. For the first time, add 8 times the weight of the drugs in water, boil, and simmer for 50 minutes, then filter out the liquid. For the second time, add 7 times the weight of the drugs in water, boil, and simmer for 45 minutes, then filter out the liquid. For the third time, add 6 times the weight of the drugs in water, boil, and simmer for 40 minutes, then filter out the liquid. Combine the three liquids and evaporate and concentrate them to a thick consistency over a low heat. Add an equal amount of anhydrous ethanol, mix thoroughly, and let stand overnight to allow precipitation. The next day, filter the precipitate completely, take the supernatant, distill to recover the ethanol, concentrate, and let stand for 24 hours. Concentrate the liquid after standing in a rotary evaporator at 80°C in a water bath to a thick paste with a relative gravity of 1.4 at 25°C. (D) Finally, the alcohol-precipitated thick paste and the water-precipitated thick paste are vacuum dried at -50°C to obtain a powdered Chinese herbal medicine compound extract, which is the Chinese herbal medicine compound preparation.

9. The use of a traditional Chinese medicine compound preparation as described in any one of claims 1-7 in the preparation of a medicament for treating colorectal cancer.

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