A compound preparation of cinobufagin and a preparation method thereof

Abstract

This invention discloses a compound preparation of toad skin and its preparation method, which is made by processing dried toad skin, astragalus, and tortoise shell powder in a certain proportion. Compared with the prior art, this invention has the effects of invigorating qi and tonifying the kidney, detoxifying and relieving pain. The formulation of this invention is more reasonable, and it has significant curative effects on mid-to-late stage tumors, chronic hepatitis B and other diseases, without any toxic side effects.

Description

Technical Field

[0001] This invention relates to a compound preparation of bufotalin and its preparation method, characterized by its effects of invigorating qi and tonifying the kidneys, detoxifying and relieving pain, and is used for mid-to-late stage tumors, chronic hepatitis B, and other conditions. It belongs to the field of pharmaceutical technology. Technical Background

[0002] Prior Art: 1. Chinese Patent Gazette published a patent application entitled "A New Drug for Treating Tumors and Its Preparation Method" (publication number CN112870267A) on June 1, 2021. The weight composition of this invention's drug is as follows: Astragalus membranaceus 15-30%, Ganoderma lucidum 10-20%, Hedyotis diffusa 10-60%, Scutellaria barbata 15-30%, Lonicera japonica 10-15%, Salvia miltiorrhiza 10-15%, Coptis chinensis 8-10%, Toad skin 20-40%, Rabdosia rubescens 30-80%, and Panax notoginseng powder 30-50%. The applicant, after years of clinical and pharmacodynamic research, has found that the above prior art has only moderate efficacy in treating tumors. Through extensive prescription screening and pharmacological research, the inventors developed a traditional Chinese medicine formula composed of dried toad skin, astragalus, and tortoise shell powder. This formula has shown significant pharmacodynamic effects in treating mid-to-late stage tumors. It possesses the functions of invigorating qi and tonifying the kidneys, detoxifying and relieving pain. It is remarkably effective for mid-to-late stage tumors and chronic hepatitis B, with no toxic side effects. We have manufactured it into capsules, tablets, and granules using conventional processes. Summary of the Invention

[0003] The purpose of this invention is to provide a more effective bufotalin compound preparation and its preparation method. Its characteristic is that the composition of each raw material in the bufotalin compound preparation of this invention is as follows:

[0004] 2000-3000g dried toad skin, 300-450g astragalus, 80-160g tortoise shell powder;

[0005] The preferred composition of each raw material in the Huachansu compound preparation of this invention is as follows:

[0006] 2500g dried toad skin, 360g astragalus root, 120g tortoise shell powder;

[0007] The traditional Chinese medicine composition of this invention can be formulated into capsules, tablets, and granules. The specific preparation methods are as follows:

[0008] 1. Preparation method of the Huachansu compound preparation capsule of the present invention:

[0009] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, and fill into enteric-coated capsules to make 1000 capsules, thus obtaining the capsule formulation.

[0010] 2. Preparation method of the Huachansu compound tablet of the present invention:

[0011] For the above three ingredients, take dried toad skin, wash it, and extract it twice with 80% ethanol under reflux. The first extraction is with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is with 10 times the amount of water for 2 hours, and the second decoction is with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, add appropriate amount of lubricant, mix well, and compress into 1000 tablets to obtain tablets.

[0012] 3. Preparation method of the Huachansu compound preparation granules of the present invention:

[0013] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder, dextrin, and flavoring agent, mix well, granulate, dry, and shape into 1000g granules, then package them to obtain granules.

[0014] Beneficial effects of this invention: Through extensive prescription screening and pharmacological research, the inventors have developed a traditional Chinese medicine formula composed of dried toad skin, astragalus, and tortoise shell powder. This formula demonstrates significant pharmacodynamic effects in treating mid-to-late stage tumors. It possesses the effects of invigorating qi and tonifying the kidneys, detoxifying and relieving pain. It is remarkably effective for mid-to-late stage tumors and chronic hepatitis B, with no toxic side effects. We have manufactured it into capsules, tablets, and granules using conventional processes. The intended use of this Huachansu compound preparation is in the treatment of mid-to-late stage tumors.

[0015] Pharmacodynamic studies:

[0016] I. Prescription Screening Test of the Invention: The ratio of active pharmaceutical ingredients in the prescription of the present invention is the optimal ratio of active pharmaceutical ingredients obtained through rigorous pharmacological screening tests.

[0017] Preparation of drugs for prescription screening experiments:

[0018] Group A: Formulation ratio:

[0019] 2500g dried toad skin, 360g astragalus, 120g tortoise shell powder.

[0020] Preparation method:

[0021] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, and fill into enteric-coated capsules to make 1000 capsules, thus obtaining the capsule formulation.

[0022] Group B: Formulation ratio:

[0023] 1500g dried toad skin, 1320g astragalus, 160g tortoise shell powder.

[0024] Preparation method:

[0025] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, and fill into enteric-coated capsules to make 1000 capsules, thus obtaining the capsule formulation.

[0026] Group C: Formulation ratio:

[0027] 2000g dried toad skin, 810g astragalus, 170g tortoise shell powder.

[0028] Preparation method:

[0029] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, and fill into enteric-coated capsules to make 1000 capsules, thus obtaining the capsule formulation.

[0030] Group d: Formula ratio:

[0031] 2760g dried toad skin, 160g astragalus, 60g tortoise shell powder.

[0032] Preparation method:

[0033] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, and fill into enteric-coated capsules to make 1000 capsules, thus obtaining the capsule formulation.

[0034] Prescription screening experiment process:

[0035] Experimental objective: To conduct pharmacological studies on Lewis lung cancer tumors, physical endurance, cellular immunity, and non-specific immunity using groups a, b, c, and d, and to compare the pharmacological effects of group a with those of groups b, c, and d to observe the relative strength of their pharmacological effects.

[0036] Experimental methods: The inhibitory effects of groups a, b, c, and d on tumor growth in Lewis lung cancer mice; the effects on mouse endurance; the effects on delayed-type hypersensitivity in mice; and the effects on phagocytic function of mouse reticuloendothelial cells.

[0037] I. Inhibitory effect on tumor growth in Lewis lung cancer mice

[0038] Experimental materials

[0039] 1. Animal: Kunming mouse, both male and female, weighing 18-20g.

[0040] 2. Drugs: Four prescription groups: group a, group b, group c, and group d. The drugs were prepared with distilled water and administered by gavage before the experiment.

[0041] Experimental methods

[0042] Modeling: Lewis lung cancer mice resuscitated and passaged for 14 days were euthanized by cervical dislocation. Tumor tissue was aseptically excised from the subcutaneous tissue of the axilla, minced, ground, filtered, and prepared into a tumor cell suspension at a ratio of 1g tumor tissue to 3ml physiological saline. A batch of Kunming mice, half male and half female, weighing 18-20g, were routinely inoculated subcutaneously with 0.2ml of tumor cell suspension in the right axilla of each mouse. Fifty successfully inoculated mice, half male and half female, were randomly divided into 5 groups of 10 mice each. Administration of the drug via gavage began 24 hours after inoculation. The control group was administered the same volume of physiological saline via gavage; groups a, b, c, and d were administered 3.2g / kg via gavage. The drug was administered once daily for 20 consecutive days. The mice were euthanized the day after drug withdrawal, and the tumor masses were excised and weighed. Experimental results: See Table 1. Table 1: Inhibitory effect on tumor growth in Lewis lung cancer mice.

[0043]

[0044] Compared with the control group, **P<0.01, *P<0.05; compared with group a, △P<0.05.

[0045] The results showed that groups a, b, c, and d significantly inhibited tumor growth in Lewis lung cancer mice. Group a showed a highly significant difference compared to the control group (P < 0.01); groups b, c, and d showed significant differences compared to the control group (P < 0.05); and groups b, c, and d showed significant differences compared to group a (P < 0.05). Therefore, group a had a stronger inhibitory effect on lung cancer tumor growth than groups b, c, and d.

[0046] II. Effects on mouse endurance

[0047] Experimental materials

[0048] 1. Animal: Kunming mouse, both male and female, weighing 18-20g.

[0049] 2. Drugs: Four prescription groups: group a, group b, group c, and group d. The drugs were prepared with distilled water and administered by gavage before the experiment.

[0050] Experimental methods

[0051] Fifty Kunming mice (half male and half female, weighing 18-20g) were randomly divided into 5 groups of 10 mice each. The control group was administered the same volume of physiological saline by gavage; groups a, b, c, and d were administered the same drug by gavage at a dose of 3.2g / kg. The drugs were administered once daily for 10 consecutive days. One hour after the last administration, the mice were placed in a 220ml wide-mouthed bottle at room temperature (20℃), sealed, and the time of death was recorded. Cardiac arrest was defined as the criterion for death. Experimental results: See Table 2.

[0052] Table 2 Effects on mouse endurance

[0053]

[0054] Compared with the control group, **P<0.01, *P<0.05; compared with group a, △P<0.05.

[0055] The results showed that groups a, b, c, and d significantly prolonged the survival time of mice in a normal-pressure closed environment. Group a showed a highly significant difference compared with the control group (P < 0.01); groups b, c, and d showed significant differences compared with the control group (P < 0.05); and groups b, c, and d showed significant differences compared with group a (P < 0.05). Therefore, group a showed stronger tolerance to hypoxia than groups b, c, and d.

[0056] III. Effects on delayed-type hypersensitivity in mice

[0057] Experimental materials

[0058] 1. Animals: Kunming mice, half male and half female, weighing 18-22g.

[0059] 2. Drugs: Four prescription groups: group a, group b, group c, and group d. The drugs were prepared with distilled water and administered by gavage before the experiment.

[0060] Experimental methods

[0061] Fifty Kunming mice (half male and half female, weighing 18-22g) were randomly divided into 5 groups of 10 mice each. The control group received the same volume of physiological saline via gavage. Groups a, b, c, and d received the same dose of the drug via gavage at 3.2g / kg. The drugs were administered once daily for one day. The following day, the abdomen of each mouse was shaved, and a 3cm × 3cm area was sensitized by applying 50μl of a 1% dinitrochlorophenylacetone-sesame oil solution. The drug was administered once daily for another 6 days. 0.5 hours after the last administration, 10μl of a 1% dinitrochlorophenylacetone solution was applied evenly to the right ear of each mouse as a challenge. Twenty-four hours after the challenge, the mice were euthanized by cervical dislocation. Ear flaps were punched from the same location on both ears using an 8mm diameter punch, and their weight was measured. The difference in weight between the left and right ear flaps was used to determine the degree of swelling. The results are shown in Table 3.

[0062] Table 3 Effects on delayed-type hypersensitivity in mice

[0063]

[0064] Compared with the control group, **P<0.01, *P<0.05; compared with group a, △P<0.05.

[0065] The results showed that groups a, b, c, and d significantly inhibited the delayed-type hypersensitivity reaction induced by dinitrochlorobenzene in mice. Group a showed a highly significant difference compared to the control group (P < 0.01); groups b, c, and d showed significant differences compared to the control group (P < 0.05); and groups b, c, and d showed significant differences compared to group a (P < 0.05). Therefore, group a significantly enhanced cellular immunity than groups b, c, and d.

[0066] IV. Effects on phagocytic function of mouse reticuloendothelial cells

[0067] Experimental materials

[0068] 1. Animal: Kunming mouse, both male and female, weighing 18-22g.

[0069] 2. Drugs: Four prescription groups: group a, group b, group c, and group d. The drugs were prepared with distilled water and administered by gavage before the experiment.

[0070] Experimental methods

[0071] Fifty Kunming mice (half male and half female, weighing 18–22 g) were randomly divided into five groups of ten each. The control group received the same volume of physiological saline via gavage; groups a, b, c, and d received the same dose of the drug via gavage at a concentration of 3.2 g / kg. The drugs were administered once daily for 10 consecutive days. One hour after the last administration, 0.2 ml of Indian ink was injected via the tail vein. Fifteen minutes after injection, 20 μl of blood was collected from the retroorbital venous plexus, added to a 2 ml 1% Na₂CO₃ test tube, and the optical density was measured at 680 nm. The clearance index (K) was calculated. Results are shown in Table 4.

[0072] Table 4 Effects on phagocytic function of mouse reticuloendothelial cells

[0073]

[0074] Compared with the control group, **P<0.01, *P<0.05; compared with group a, △P<0.05.

[0075] The results showed that groups a, b, c, and d significantly enhanced the phagocytic function of mouse reticuloendothelial cells. Group a showed a highly significant difference compared to the control group (P < 0.01); groups b, c, and d showed significant differences compared to the control group (P < 0.05); and groups b, c, and d showed significant differences compared to group a (P < 0.05). Therefore, group a had a stronger effect on enhancing non-specific immunity than groups b, c, and d.

[0076] Experimental results: Groups a, b, c, and d significantly inhibited tumor growth in Lewis lung cancer mice; significantly prolonged the survival time of mice in a normal pressure closed environment; significantly inhibited delayed-type hypersensitivity reactions induced by dinitrochlorobenzene in mice and improved the function of cellular immunity; and significantly enhanced the phagocytic function of mouse reticuloendothelial cells and improved the effect of non-specific immunity.

[0077] Conclusion: Group A showed stronger pharmacological effects than Groups B, C, and D in inhibiting the growth of Lewis lung cancer tumors, improving hypoxia tolerance, and enhancing cellular and non-specific immunity. Therefore, Group A had better clinical efficacy in treating mid-to-late stage tumors than Groups B, C, and D, and the prescription for Group A was determined to be the prescription of this invention.

[0078] II. Pharmacodynamic test of the present invention: Compared with the prior art, the pharmacodynamic test results of the Huachansu compound preparation of the present invention are significantly improved.

[0079] According to the patent search, the closest prior art comparison document is: Chinese Patent Gazette published on June 1, 2021, a patent application entitled "A new drug for treating tumors and its preparation method" with publication number CN112870267A. The weight composition of the drug in this invention is as follows: Astragalus membranaceus 15-30%, Ganoderma lucidum 10-20%, Hedyotis diffusa 10-60%, Scutellaria barbata 15-30%, Lonicera japonica 10-15%, Salvia miltiorrhiza 10-15%, Coptis chinensis 8-10%, dried toad skin 20-40%, Rabdosia rubescens 30-80%, and Panax notoginseng powder 30-50%.

[0080] Compared with the closest prior art and capsules made from single-ingredient dried toad skin, the Huachansu compound preparation of this invention has advantages in H... 22 Significantly improved efficacy in pharmacodynamic tests for solid tumors, Lewis lung cancer, liver injury, physical endurance, and cellular immunotherapy.

[0081] The main pharmacodynamic studies are as follows:

[0082] (I) Preparation of experimental drugs:

[0083] 1. The Huachansu compound preparation group of the present invention: capsules prepared according to the method of Example 1 of the present invention.

[0084] 2. Group a: Capsules prepared according to the method of the embodiments in the prior art document Group 1 (Publication No. CN112870267A) (paragraphs 0020-0025 of the specification).

[0085] 3. Group B: Prescription: 2980g of dried toad skin;

[0086] Preparation method: The above dried toad skin is washed and extracted twice by reflux with 80% ethanol. The first extraction is carried out by reflux with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is carried out by reflux with 6 times the amount of 80% ethanol for 1 hour. The extracts are combined, filtered, and the filtrate is concentrated to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. The extract is spray-dried into a fine powder, and an appropriate amount of starch is added, mixed well, granulated, dried, and sized. The granules are then filled into enteric-coated capsules to make 1000 capsules, which are the capsule dosage form.

[0087] (II) Pharmacodynamic experimental procedure:

[0088] Experimental objective: To investigate the effects of H on the effects of the Huachansu compound preparation group, group a, and group b. 22 Pharmacological experiments were conducted on solid tumors, Lewis lung cancer tumors, liver injury, physical endurance, and cellular immunity. The Huachansu compound preparation group was compared with group a and group b to observe the strength of its pharmacological effects.

[0089] Experimental methods: The Huachansu compound preparation group, group a, and group b were used to treat H in mice. 22 Inhibition of solid tumor growth; inhibition of tumor growth in Lewis lung cancer mice; effect on liver damage induced by thioacetamide; effect on mouse endurance; effect on delayed-type hypersensitivity in mice.

[0090] I. Effects on mouse H 22 Inhibitory effect on solid tumor growth

[0091] Experimental materials

[0092] 1. Animal: Kunming mouse, both male and female, weighing 18-20g.

[0093] 2. Drugs: Three preparation groups were established: Huachansu compound preparation group, group a, and group b. The drugs were prepared with distilled water and administered by gavage before the experiment.

[0094] Experimental methods

[0095] Modeling: Aseptically extract tumor-bearing H cells from passaged cells on day 8. 22 Ascites fluid from mice was diluted 1:3 with sterile physiological saline to adjust the cell concentration to 5.0 × 10⁻⁶. 7 / ml, take a batch of Kunming mice, half male and half female, weighing 18-20g, and administer 1.0×10 ml of the solution intraperitoneally to each mouse. 4 Forty mice, half male and half female, were successfully inoculated and randomly divided into four groups of ten mice each. Drug administration began 24 hours after inoculation. The control group received the same volume of physiological saline via gavage; the Huachansu compound preparation group, group a, and group b received 3.2 g / kg via gavage. Drug administration continued for 20 days, once daily. Twenty-four hours after the last administration, the mice were sacrificed, and the tumor was dissected and weighed.

[0096] Experimental results: See Table 1

[0097] Table 1. Effects of H on mouse H 22 Inhibitory effect on solid tumor growth

[0098]

[0099] Compared with the control group, **P<0.01, *P<0.05; compared with the Huachansu compound preparation group, △P<0.05.

[0100] The results showed that the Huachansu compound preparation group, group a, and group b could significantly inhibit H in mice. 22 The growth of solid tumors showed a highly significant difference between the Huachansu compound preparation group and the control group (P < 0.01); significant differences between groups a and b and the control group (P < 0.05); and significant differences between groups a and b and the Huachansu compound preparation group (P < 0.05). This indicates that the Huachansu compound preparation group has a stronger inhibitory effect on tumor growth than groups a and b.

[0101] II. Inhibitory effect on tumor growth in Lewis lung cancer mice

[0102] Experimental materials

[0103] 1. Animal: Kunming mouse, both male and female, weighing 18-20g.

[0104] 2. Drugs: Three preparation groups were established: Huachansu compound preparation group, group a, and group b. The drugs were prepared with distilled water and administered by gavage before the experiment.

[0105] Experimental methods

[0106] Modeling: Lewis lung cancer mice resuscitated and passaged for 14 days were euthanized by cervical dislocation. Tumor tissue was aseptically excised from the subcutaneous tissue of the axilla, minced, ground, filtered, and prepared into a tumor cell suspension at a ratio of 1g tumor tissue to 3ml physiological saline. A batch of Kunming mice, half male and half female, weighing 18-20g, were routinely inoculated subcutaneously with 0.2ml of tumor cell suspension in the right axilla of each mouse. Forty mice that were successfully inoculated, half male and half female, were randomly divided into 4 groups of 10 mice each. Administration of the drug via gavage began 24 hours after inoculation. The control group was administered the same volume of physiological saline via gavage; the Huachansu compound preparation group, group a, and group b were administered 3.2g / kg via gavage. Administration continued for 20 days, once daily. The mice were euthanized the day after drug withdrawal, the tumor masses were excised, and their weight was measured. Experimental results: See Table 2.

[0107] Table 2. Inhibitory effect on tumor growth in Lewis lung cancer mice.

[0108]

[0109] Compared with the control group, **P<0.01, *P<0.05; compared with the Huachansu compound preparation group, △P<0.05.

[0110] The results showed that the Huachansu compound preparation group, group a, and group b all significantly inhibited the growth of Lewis lung cancer tumors in mice. The Huachansu compound preparation group showed a highly significant difference compared to the control group (P < 0.01); groups a and b showed significant differences compared to the control group (P < 0.05); and groups a and b showed significant differences compared to the Huachansu compound preparation group (P < 0.05). Therefore, the Huachansu compound preparation group had a stronger inhibitory effect on lung cancer tumor growth than groups a and b.

[0111] III. Effects of Thioacetamide on Liver Damage

[0112] Experimental materials

[0113] 1. Animal: Kunming mouse, both male and female, weighing 20-23g.

[0114] 2. Drugs: Three preparation groups were established: Huachansu compound preparation group, group a, and group b. The drugs were prepared with distilled water and administered by gavage before the experiment.

[0115] Experimental methods

[0116] Forty Kunming mice (half male and half female, weighing 20-23g) were randomly divided into four groups of ten mice each. The control group received the same volume of physiological saline via gavage; the Huachansu compound preparation group, group a, and group b received the same amount of the drug via gavage at a dose of 3.2g / kg. The drugs were administered once daily for 10 consecutive days. One hour after the last administration, each group of mice was intraperitoneally injected with 50mg / kg of thioacetamide. Sixteen hours after the injection, blood was collected from the orbital sinus, and serum was separated by centrifugation to measure the serum alanine aminotransferase level. Experimental results: See Table 3.

[0117] Table 3 Effects of thioacetamide on liver damage

[0118]

[0119]

[0120] Compared with the control group, **P<0.01, *P<0.05; compared with the Huachansu compound preparation group, △P<0.05.

[0121] The results showed that the Huachansu compound preparation group, group a, and group b significantly reduced the serum alanine aminotransferase levels in mice with thioacetamide-induced liver injury. The Huachansu compound preparation group showed a highly significant difference compared to the control group (P < 0.01); groups a and b showed significant differences compared to the control group (P < 0.05); and groups a and b showed significant differences compared to the Huachansu compound preparation group (P < 0.05). Therefore, the Huachansu compound preparation group had a stronger effect in preventing liver injury than groups a and b.

[0122] IV. Effects on mouse endurance

[0123] Experimental materials

[0124] 1. Animal: Kunming mouse, both male and female, weighing 18-20g.

[0125] 2. Drugs: Three preparation groups were established: Huachansu compound preparation group, group a, and group b. The drugs were prepared with distilled water and administered by gavage before the experiment.

[0126] Experimental methods

[0127] Forty Kunming mice (half male and half female, weighing 18-20g) were randomly divided into four groups of ten mice each. The control group received the same volume of physiological saline via gavage; the Huachansu compound preparation group, group a, and group b received the same amount of the drug via gavage at a dose of 3.2g / kg. The drugs were administered once daily for 10 consecutive days. One hour after the last administration, the mice were placed in a 220ml wide-mouthed bottle at room temperature (20℃), sealed, and the time of death was recorded. Cardiac arrest was defined as the criterion for death. Experimental results: See Table 4.

[0128] Table 4 Effects on mouse endurance

[0129]

[0130] Compared with the control group, **P<0.01, *P<0.05; compared with the Huachansu compound preparation group, △P<0.05.

[0131] The results showed that the Huachansu compound preparation group, group a, and group b significantly prolonged the survival time of mice in a normal pressure closed environment. The Huachansu compound preparation group showed a highly significant difference compared to the control group (P < 0.01); groups a and b showed significant differences compared to the control group (P < 0.05); and groups a and b showed significant differences compared to the Huachansu compound preparation group (P < 0.05). Therefore, the Huachansu compound preparation group exhibited stronger hypoxia tolerance than groups a and b.

[0132] V. Effects on delayed-type hypersensitivity in mice

[0133] Experimental materials

[0134] 1. Animals: Kunming mice, half male and half female, weighing 18-22g.

[0135] 2. Drugs: Three preparation groups were established: Huachansu compound preparation group, group a, and group b. The drugs were prepared with distilled water and administered by gavage before the experiment.

[0136] Experimental methods

[0137] Forty Kunming mice (half male and half female, weighing 18-22g) were randomly divided into four groups of ten mice each. The control group received the same volume of physiological saline via gavage. The Huachansu compound preparation group, group a, and group b received the same dose via gavage at 3.2g / kg. Administration was repeated once daily for one day. The following day, the abdomen of each mouse was shaved, and a 3cm × 3cm area was sensitized by applying 50μl of a 1% dinitrochlorophenylacetone sesame oil solution. Administration continued for 6 days, once daily. 0.5h after the last administration, 10μl of a 1% dinitrochlorobenzene solution was applied evenly to the right ear of each mouse as a challenge. Twenty-four hours after the challenge, the mice were euthanized by cervical dislocation. Ear flaps were punched from the same location on both ears using an 8mm diameter punch, and their weight was measured. The difference in weight between the left and right ear flaps was used to determine the degree of swelling. The results are shown in Table 5.

[0138] Table 5 Effects on delayed-type hypersensitivity in mice

[0139]

[0140] Compared with the control group, **P<0.01, *P<0.05; compared with the Huachansu compound preparation group, △P<0.05.

[0141] The results showed that the Huachansu compound preparation group, group a, and group b significantly inhibited the delayed-type hypersensitivity reaction induced by dinitrochlorobenzene in mice. The Huachansu compound preparation group showed a highly significant difference compared to the control group (P < 0.01); groups a and b showed significant differences compared to the control group (P < 0.05); and groups a and b showed significant differences compared to the Huachansu compound preparation group (P < 0.05). Therefore, the Huachansu compound preparation group has a stronger effect on enhancing cellular immunity than groups a and b.

[0142] Experimental results: The Huachansu compound preparation group, group a, and group b could significantly inhibit H in mice. 22 It inhibits the growth of solid tumors; it significantly inhibits the growth of Lewis lung cancer tumors in mice; it significantly reduces the serum alanine aminotransferase content in mice with liver injury induced by thioacetamide; it significantly prolongs the survival time of mice in a normal pressure closed environment; it significantly inhibits the delayed-type hypersensitivity reaction induced by dinitrochlorobenzene in mice and enhances the function of cellular immunity.

[0143] Conclusion: The compound preparation of bufotoxin in this invention showed better inhibition of H compared to groups a and b.22 The compound preparation of Huachansu has strong pharmacological effects such as inhibiting the growth of solid tumors and Lewis lung cancer, preventing liver damage, improving hypoxia tolerance, and enhancing the body's cellular immunity. Therefore, the Huachansu compound preparation group has better clinical efficacy in treating intermediate and advanced tumors than group a and group b. Detailed Implementation

[0144] Example 1: Preparation of the capsule formulation of the present invention:

[0145] Formulation ratio:

[0146] 2500g dried toad skin, 360g astragalus root, 120g tortoise shell powder;

[0147] Preparation method:

[0148] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, and fill into enteric-coated capsules to make 1000 capsules, thus obtaining the capsule formulation.

[0149] Example 2: Preparation of the tablets of the present invention:

[0150] Formulation ratio:

[0151] 2500g dried toad skin, 360g astragalus root, 120g tortoise shell powder;

[0152] Preparation method:

[0153] For the above three ingredients, take dried toad skin, wash it, and extract it twice with 80% ethanol under reflux. The first extraction is with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is with 10 times the amount of water for 2 hours, and the second decoction is with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, add appropriate amount of lubricant, mix well, and compress into 1000 tablets to obtain tablets.

[0154] Example 3: Preparation of the granules of the present invention:

[0155] Formulation ratio:

[0156] 2500g dried toad skin, 360g astragalus root, 120g tortoise shell powder;

[0157] Preparation method:

[0158] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder, dextrin, and flavoring agent, mix well, granulate, dry, and shape into 1000g granules, then package them to obtain granules.

[0159] Example 4: Preparation of the capsule formulation of the present invention:

[0160] Formulation ratio:

[0161] 2000g dried toad skin, 300g astragalus root, 80g tortoise shell powder;

[0162] Preparation method:

[0163] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, and fill into enteric-coated capsules to make 1000 capsules, thus obtaining the capsule formulation.

[0164] Example 5: Preparation of the tablets of the present invention:

[0165] Formulation ratio:

[0166] 2000g dried toad skin, 300g astragalus root, 80g tortoise shell powder;

[0167] Preparation method:

[0168] For the above three ingredients, take dried toad skin, wash it, and extract it twice with 80% ethanol under reflux. The first extraction is with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is with 10 times the amount of water for 2 hours, and the second decoction is with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, add appropriate amount of lubricant, mix well, and compress into 1000 tablets to obtain tablets.

[0169] Example 6: Preparation of the granules of the present invention:

[0170] Formulation ratio:

[0171] 2000g dried toad skin, 300g astragalus root, 80g tortoise shell powder;

[0172] Preparation method:

[0173] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder, dextrin, and flavoring agent, mix well, granulate, dry, and shape into 1000g granules, then package them to obtain granules.

[0174] Example 7: Preparation of the capsule formulation of the present invention:

[0175] Formulation ratio:

[0176] 3000g dried toad skin, 450g astragalus root, 160g tortoise shell powder;

[0177] Preparation method:

[0178] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, and fill into enteric-coated capsules to make 1000 capsules, thus obtaining the capsule formulation.

[0179] Example 8: Preparation of the tablets of the present invention:

[0180] Formulation ratio:

[0181] 3000g dried toad skin, 450g astragalus root, 160g tortoise shell powder;

[0182] Preparation method:

[0183] For the above three ingredients, take dried toad skin, wash it, and extract it twice with 80% ethanol under reflux. The first extraction is with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is with 10 times the amount of water for 2 hours, and the second decoction is with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, add appropriate amount of lubricant, mix well, and compress into 1000 tablets to obtain tablets.

[0184] Example 9: Preparation of the granules of the present invention:

[0185] Formulation ratio:

[0186] 3000g dried toad skin, 450g astragalus root, 160g tortoise shell powder;

[0187] Preparation method:

[0188] For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is done with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is done with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.08. For Astragalus membranaceus, decoct it twice with water. The first decoction is done with 10 times the amount of water for 2 hours, and the second decoction is done with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder, dextrin, and flavoring agent, mix well, granulate, dry, and shape into 1000g granules, then package them to obtain granules.

Claims

1. A bufotalin capsule, characterized in that, The active pharmaceutical ingredient composition of the capsules is as follows: 2000-3000g of dried toad skin Astragalus 300-450g 80-160g of turtle shell powder; Prepared according to the following method: For the above three ingredients, take dried toad skin, wash it, and extract it twice by refluxing with 80% ethanol. The first extraction is with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.

08. For Astragalus membranaceus, decoct it twice with water. The first decoction is with 10 times the amount of water for 2 hours, and the second decoction is with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.

08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, and fill into enteric-coated capsules to make 1000 capsules, thus obtaining the capsule dosage form.

2. The Huachansu capsules according to claim 1, characterized in that, The active pharmaceutical ingredient composition of the capsules is as follows: 2500g dried toad skin Astragalus 360g 120g of turtle powder.

3. The use of the Huachansu capsules according to any one of claims 1-2 in the preparation of drugs for treating lung cancer.

4. A bufotalin tablet, characterized in that, The active pharmaceutical ingredient in the tablets is composed of: 2000-3000g of dried toad skin Astragalus 300-450g 80-160g of turtle shell powder; Prepared according to the following method: For the above three ingredients, take dried toad skin, wash it, and extract it twice with 80% ethanol under reflux. The first extraction is with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.

08. For Astragalus membranaceus, decoct it twice with water. The first decoction is with 10 times the amount of water for 2 hours, and the second decoction is with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.

08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder and starch, mix well, granulate, dry, granulate, add appropriate amount of lubricant, mix well, and compress into 1000 tablets to obtain tablets.

5. The bufotalin tablets according to claim 4, characterized in that, The active pharmaceutical ingredient in the tablets is composed of: 2500g dried toad skin Astragalus 360g 120g of turtle powder.

6. The use of the Huachansu tablets according to any one of claims 4-5 in the preparation of a drug for treating lung cancer.

7. A bufotalin granule, characterized in that, The active pharmaceutical ingredient composition of the granules is as follows: 2000-3000g of dried toad skin Astragalus 300-450g 80-160g of turtle shell powder; Prepared according to the following method: For the above three ingredients, take dried toad skin, wash it, and extract it twice with 80% ethanol under reflux. The first extraction is with 8 times the amount of 80% ethanol for 2 hours, and the second extraction is with 6 times the amount of 80% ethanol for 1 hour. Combine the extracts, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.05-1.

08. For Astragalus membranaceus, decoct it twice with water. The first decoction is with 10 times the amount of water for 2 hours, and the second decoction is with 8 times the amount of water for 1 hour. Combine the decoctions, filter, and concentrate the filtrate to 85℃-90℃ to obtain a clear extract with a relative density of 1.06-1.

08. Combine this extract with the above clear extract, mix well, spray dry into a fine powder, add appropriate amounts of tortoise shell powder, dextrin, and flavoring agent, mix well, granulate, dry, and shape into 1000g granules, then package to obtain granules.

8. The bufotalin granules according to claim 7, characterized in that, The active pharmaceutical ingredient composition of the granules is as follows: 2500g dried toad skin Astragalus 360g 120g of turtle powder.

9. The use of the Huachansu granules according to any one of claims 7-8 in the preparation of drugs for treating lung cancer.

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