Traditional Chinese medicine compound granules and preparation method thereof

Abstract

The present invention discloses a traditional Chinese medicine compound granule and its preparation method. The compound granule is made from excipients and a traditional Chinese medicine extract as an active ingredient. The excipients contain a filler, and the traditional Chinese medicine extract is a full-ingredient extract extracted from a traditional Chinese medicine composition consisting of the following components in parts by weight: 5-8 parts of cornus fruit; 3-5 parts of prepared rehmannia root; 2-5 parts of salt-fleshed psoralea corylifolia; 2-4 parts of roasted astragalus root; 2-4 parts of angelica sinensis; 0.5-3 parts of donkey-hide gelatin; 2-4 parts of millettia reticulata; 2-4 parts of codonopsis pilosula; 3-5 parts of vinegar-cured turtle shell; and 0.5-2 parts of amomum villosum. The compound granule can provide good patient compliance, is easy to carry, store, transport, and take, is more durable, safer to use, has fewer toxic side effects, and exhibits excellent therapeutic efficacy for thalassemia.

Description

Technical Field

[0001] The present invention relates to a traditional Chinese medicine compound granule and a preparation method thereof. Background Art

[0002] Thalassemia, also known as thalassemia, is a hereditary blood disorder caused by a congenital genetic defect that prevents the body from synthesizing hemoglobin and globin. It is the most common and devastating single-gene genetic disorder worldwide and a difficult-to-treat hereditary hemolytic anemia, named after the Mediterranean region where it was first discovered. Its pathogenesis is due to mutations or deletions in genes regulating globin synthesis on chromosomes 11 and 16, resulting in ineffective red blood cell production and ultimately hemolytic anemia. This cumulative effect can affect the function of vital organs such as the heart and spleen. Thalassemia is characterized by distinct thalassemia features: a sallow complexion, a square head, prominent cheekbones, a sunken nose, puffy eyelids, a sallow complexion, pale lips and tongue, and an enlarged liver and spleen. According to the WHO Secretariat, approximately 100 million people worldwide carry the thalassemia gene, and approximately 300,000 babies are born with the thalassemia syndrome each year. my country has a high incidence of thalassemia. Two main types of thalassemia are most prevalent in my country: α- and β-thalassemia, with a higher incidence in southern provinces. In Guangxi, Guangdong, and Hainan provinces alone, over 20 million people are carriers of the thalassemia gene. The disease primarily affects infants and adolescents, and presents with symptoms such as fatigue, dizziness, palpitations, jaundice, abdominal scarring, and delayed growth and development. Patients present with severe anemia and thalassemia-like features. Blood tests reveal hypochromic erythrocyte anemia, with the presence of target cells, increased reticulocytes, and decreased nuclear red blood cells and red blood cell osmotic pressure. In addition to the aforementioned characteristics, patients with β-thalassemia may also experience symptoms such as weakness in the lower back and knees, dizziness, palpitations, shortness of breath, fatigue, hair loss, tinnitus, pale complexion, lips, and nails, hot palms and soles, and night sweats.

[0003] Internationally, the treatment principles for thalassemia remain primarily based on blood transfusions and iron chelation. Except for thalassemia minor, which does not require specific treatment (folic acid and vitamin E supplementation is appropriate), α- and β-thalassemia intermedia generally utilizes small-volume red blood cell transfusions, while β-thalassemia major utilizes high-dose blood transfusions or transfusions of concentrated red blood cells combined with iron chelators. These transfusions should be initiated early to maintain normal growth and development, reduce intestinal iron absorption, inhibit splenomegaly, and prevent bone lesions. The approach is to initially administer repeated transfusions of concentrated red blood cells to achieve a hemoglobin level of 120-150 g / L. Subsequently, 10-15 ml / kg of concentrated red blood cells are transfused every two to four weeks to maintain a hemoglobin level above 90-105 g / L. However, this approach can easily lead to hemosiderosis, so concurrent iron chelation (e.g., deferoxamine mesylate for injection, deferiprone tablets) should be administered. These increase iron excretion in the urine and feces but do not prevent gastrointestinal absorption. Long-term use can also cause cataracts and bone developmental disorders, and excessive doses can cause vision and hearing loss. However, due to the massive hemolysis in thalassemia patients, splenomegaly and hyperfunction can worsen anemia and lead to iron accumulation, leading to damage to other organs. Therefore, this treatment can only be effective for a decade or so. Splenectomy: Splenectomy is effective for α-thalassemia and β-thalassemia intermedia, but less effective for β-thalassemia major. Splenectomy can weaken immune function and have very serious sequelae. It should be performed after the age of 5 to 6 years and only for strictly defined indications. Bone marrow and stem cell transplantation: Allogeneic hematopoietic stem cell transplantation is currently the only treatment that can cure β-thalassemia major. If an HLA-matched hematopoietic stem cell donor is available, it should be the preferred treatment for β-thalassemia major. However, limited access to bone marrow and matching makes this approach difficult to widespread and expensive. Gene therapy: Gene therapy can increase γ gene expression or decrease α gene expression to improve β-thalassemia symptoms. While a promising approach, clinical application remains uncertain due to the unresolved homologous recombination rate. Additionally, exploratory treatments have been attempted domestically and internationally with chemotherapy drugs such as hydroxyurea, 5-azacytidine, cytarabine, myleran, and isoniazid. However, their clinical application is limited by their uncertain efficacy and significant side effects. Therefore, effective treatment for thalassemia remains a global challenge, and few effective traditional Chinese or Western medicines exist.

[0004] CN200610078866.X discloses a drug for treating thalassemia and a preparation process thereof. However, the process route is complex, and the resulting ointment contains a Polygonum multiflorum extract and organic solvent residues, which is unsafe for long-term use. In addition, the organic solvent used in the ethanol extraction process is prone to explosion, which is a significant production safety hazard. In addition, only some types of active ingredients are extracted from Angelica sinensis and Amomum villosum in the form of volatile oils. Some types of active ingredients do not enter the final product at all, and only a portion of some active ingredients enter the final product, affecting the efficacy of the final drug. The resulting ointment is heavy, difficult to store and transport, easily corrupted, and inconvenient to take. In addition, the process requires complex volatile oil inclusion and drying steps, and the volatile oil inclusion compound is significantly lost during the drying process, which also increases production costs.

[0005] Therefore, there is still a need for an improved drug for treating thalassemia and a preparation process thereof. Summary of the Invention

[0006] In order to overcome the problems of the prior art, an object of the present invention is to provide a Chinese medicine compound granule, which can provide patients with good medication compliance, is convenient to carry, store, transport and take, is more resistant to storage, safer to use, has fewer toxic and side effects, and has good therapeutic efficacy for thalassemia.

[0007] Another object of the present invention is to provide a method for preparing a Chinese medicine compound granule, which can avoid the safety hazard of organic solvent explosion, reduce production costs, and can extract the effective ingredients of each Chinese medicine component with all ingredients. This method can provide patients with good medication compliance, easy to carry, store, transport, and take, more storable, safer to use, with less toxic and side effects, and at the same time have good therapeutic efficacy for thalassemia.

[0008] To achieve the above-mentioned purpose of the present invention, one aspect of the present invention provides a Chinese medicine compound granule, which is made of excipients and Chinese medicine extracts as active ingredients, wherein the excipients contain fillers, and the Chinese medicine extracts are full-ingredient extracts extracted from a Chinese medicinal material composition consisting of the following components in parts by weight: 5 to 8 parts of cornus fruit; 3 to 5 parts of prepared rehmannia root; 2 to 5 parts of salt psoralea corylifolia; 2 to 4 parts of roasted astragalus; 2 to 4 parts of angelica; 0.5 to 3 parts of donkey-hide gelatin; 2 to 4 parts of Millettia reticulata; 2 to 4 parts of Codonopsis pilosula; 3 to 5 parts of vinegar-cured turtle shell; and 0.5 to 2 parts of Amomum villosum.

[0009] According to the Chinese herbal compound granules of the present invention, further, the Chinese herbal medicine composition is composed of the following components in parts by weight: 6 parts of cornus fruit; 4 parts of Rehmannia root; 3 parts of salt-cured Psoralea corylifolia; 3 parts of roasted Astragalus root; 3 parts of Chinese angelica; 1 part of donkey-hide gelatin; 3 parts of Millettia reticulata; 3 parts of Codonopsis pilosula; 4 parts of vinegar-cured turtle shell; and 1 part of Amomum villosum.

[0010] According to the Chinese herbal compound granules of the present invention, further, based on the total weight of the Chinese herbal compound granules, the amount of the filler is 10 to 70 weight %.

[0011] According to the traditional Chinese medicine compound granules of the present invention, further, the filler is selected from sucrose, soluble starch or a combination thereof.

[0012] According to the Chinese herbal compound granules of the present invention, further, the excipients further contain a flavoring agent, and the amount of the flavoring agent is 0.1 to 10 weight % based on the total weight of the Chinese herbal compound granules.

[0013] Another aspect of the present invention provides a method for preparing any of the above-mentioned traditional Chinese medicine compound granules, the method comprising the following steps: steam distilling Amomum villosum and collecting volatile oil and the distilled aqueous solution; decocting a premix of Cornus officinalis, Rehmannia root, Psoralea corylifolia, Radix Astragali, Angelica sinensis, Codonopsis pilosula, Millettia reticulata and Carapax tricuspidata with water to obtain an aqueous extract; mixing the distilled aqueous solution with the aqueous extract and concentrating them into a thick paste; drying the thick paste to obtain a dry paste; grinding donkey-hide gelatin and the dry paste and adding auxiliary materials containing fillers to dry-mix them to obtain a dry mixture; dry-granulating the dry mixture to obtain semi-finished granules; sizing the semi-finished granules; spraying the volatile oil into the sizing semi-finished granules and sealing and moistening them to obtain the traditional Chinese medicine compound granules.

[0014] According to the method of the present invention, further, the method also includes the step of re-dry granulating the semi-finished granules removed after granulation.

[0015] According to the method of the present invention, further, the step of mixing the distilled aqueous solution with the water extract and concentrating them into a thick paste is carried out at a temperature of 40 to 100° C. and under reduced pressure.

[0016] According to the method of the present invention, further, the step of drying the thick paste to obtain a dry paste is carried out at a temperature of 40 to 100° C. and under reduced pressure.

[0017] According to the method of the present invention, the method further comprises the step of drying the semi-finished granules after granulation so that the moisture content of the granules does not exceed 13% by weight.

[0018] Beneficial effects

[0019] The method according to the present invention has a simple process route and does not adopt an organic solvent extraction process such as ethanol. Since there is no need to use organic solvents, the safety production hazard of organic solvent explosion is avoided, the cost is reduced, and a complicated Amomum villosum volatile oil inclusion process is not required, which further reduces the cost. In addition, Amomum villosum adopts a full-component distillation extraction process to extract its full components (including volatile oil and distilled water solution), while Angelica sinensis adopts a water decoction process to extract its full components. The full components are used to enter the final preparation product, and the medicinal effect is better, which is more in line with the clinical effect of traditional Chinese medicine decoctions; moreover, the method according to the present invention adopts a dry granulation process, and the obtained Chinese medicine compound preparation is a granule, which is convenient for patients to carry, store, transport, and take, has good compliance, high drug loading, is more resistant to storage, not easy to corrupt, does not contain hepatotoxic Polygonum multiflorum extract and organic solvent residues, is safer to use, and has less toxic and side effects. In addition, by further combining the use of salt psoralea corylifolia, whose active ingredients are more easily dissolved and has a stronger kidney-tonifying effect, and vinegar turtle shell, whose active ingredients are more easily dissolved and has a stronger softening and dispersing effect, the effects of this Chinese medicine compound granule on nourishing yin and tonifying the kidney, benefiting the marrow and producing blood, and replenishing qi and strengthening the spleen are enhanced, and it is suitable for the treatment of α- and β-thalassemia.

[0020] Animal toxicity experiments showed that this Chinese herbal compound granule has better safety and lower toxic side effects. Its maximum dosage can reach 162.36g of crude drug / kg, which is equivalent to 104.75 times the clinical dosage. No delayed drug toxicity reaction was observed after administration within the dosage range of 9-36g / kg / d for 6 months, indicating that the safe dosage of the granule in SD rats is at least 36g / kg.

[0021] The results of the study on the effect of the Chinese herbal compound granules on the proliferation and differentiation of early hematopoietic progenitor cells in mouse bone marrow showed that each dose group could increase the 60 Co-γ ray irradiation induced the number of peripheral blood leukocytes in the blood of mice with kidney deficiency and myeloid damage, promoted the proliferation of CFU-GM, CFU-E, and CFU-Meg, and increased the number of CD34+ cells in the mouse bone marrow. The high-dose group had a significant effect on peripheral blood leukocytes, the high and medium-dose groups had a significant effect on bone marrow hematopoietic stem / progenitor cells, and the low-dose group had a significant effect on 0D34+ cells. This Chinese medicine compound granule is used for the prevention and treatment of 60 The results of the pharmacodynamic study on Co-γ ray-induced aplastic anemia in rats showed that 60 Co-γ ray induced aplastic anemia in rats was effective. DETAILED DESCRIPTION

[0022] The Chinese herbal compound granules according to the present invention are prepared from excipients and Chinese herbal extracts as active ingredients, wherein the excipients contain fillers, and the Chinese herbal extracts are full-ingredient extracts extracted from a Chinese herbal material composition consisting of the following components in parts by weight: 5 to 8 parts of cornus fruit; 3 to 5 parts of prepared rehmannia root; 2 to 5 parts of salt psoralea corylifolia; 2 to 4 parts of roasted astragalus root; 2 to 4 parts of angelica sinensis; 0.5 to 3 parts of donkey-hide gelatin; 2 to 4 parts of millettia reticulata; 2 to 4 parts of codonopsis pilosula; 3 to 5 parts of vinegar-coated turtle shell; and 0.5 to 2 parts of amomum villosum.

[0023] This traditional Chinese medicine preparation uses Fructus Corni as the main ingredient, nourishing yin and tonifying the kidneys. It aims to nourish the true yin (true essence) in the kidneys, replenishing the sea of marrow and providing a source of blood, thus benefiting the marrow, producing essence, and transforming it into blood. Rehmannia root, roasted Astragalus root, Codonopsis pilosula, Angelica sinensis, Psoralea corylifolia (salt-soaked), and donkey-hide gelatin serve as assistant ingredients. Rehmannia root and Psoralea corylifolia assist the main ingredients in producing essence and transforming it into blood, strengthening the kidney-tonifying effect. Furthermore, Psoralea corylifolia (salt-soaked) nourishes kidney yang and generates yang energy. Compared to Psoralea corylifolia, its active ingredients are more easily dissolved, resulting in a stronger kidney-tonifying effect. Roasted Astragalus root nourishes the middle and replenishes qi, while donkey-hide gelatin nourishes yin and replenishes blood, together tonifying qi and blood. Kidney-tonifying drugs are fundamentally effective in treating thalassemia, treating the root cause, while blood-tonifying drugs treat the symptoms. Treating both the root cause and the symptoms together enhances each other. This disease is caused by kidney deficiency, which is insufficient to warm the spleen and earth. The spleen governs qi, and qi controls blood. Spleen deficiency weakens qi, failing to control blood circulation, leading to blood stasis and the development of scars. Roasted Astragalus and Codonopsis nourish Qi, and Qi is the commander of blood, so when Qi flows, blood flows. Millettia reticulata and Vinegar-cured Turtle Shell serve as adjuvants. Millettia reticulata and Vinegar-cured Turtle Shell nourish Yin and blood, softening and dispersing lumps, and dissipating abdominal scars. Compared to Turtle Shell, the active ingredients of Vinegar-cured Turtle Shell are more easily dissolved, and the softening and dispersing effect is stronger. Amomum villosum is used as an adjuvant to reduce the greasy feeling of Yin-nourishing and blood-tonifying drugs, so that the drug will not affect the spleen and stomach if taken for a long time. This Chinese medicine preparation uses the four ingredients of monarch, minister, adjuvant, and adjuvant together to nourish Yin and tonify the kidney, benefit the marrow and produce blood, and tonify Qi and strengthen the spleen. It treats both the root cause and the symptoms, the main disease and the accompanying symptoms, and is very consistent with the etiology, pathogenesis, and syndrome differentiation and treatment of thalassemia.

[0024] In the present invention, the term "full-ingredient extract" means that all the effective ingredients of each component of the Chinese herbal medicine composition are extracted during extraction and enter into the final Chinese herbal compound granules.

[0025] In the Chinese herbal compound granules according to the present invention, preferably, the Chinese herbal medicine composition is composed of the following components in parts by weight: 6 parts of cornus fruit; 4 parts of Rehmannia root; 3 parts of salt-cured Psoralea corylifolia; 3 parts of roasted Astragalus root; 3 parts of Chinese angelica; 1 part of donkey-hide gelatin; 3 parts of Millettia reticulata; 3 parts of Codonopsis pilosula; 4 parts of vinegar-cured turtle shell; and 1 part of Amomum villosum.

[0026] The Chinese herbal compound granules according to the present invention contain excipients. The excipients should contain fillers. In some more specific embodiments, in addition to the fillers, the excipients may also contain flavoring agents. The fillers may be selected from sucrose, soluble starch, or a combination thereof, and the flavoring agents may be selected from stevia, etc. When the Chinese herbal compound granules contain fillers, the amount of the fillers may be 10 to 70% by weight based on the total weight of the Chinese herbal compound granules. When the Chinese herbal compound granules contain flavoring agents, the amount of the flavoring agents may be 0.1 to 10% by weight based on the total weight of the Chinese herbal compound granules.

[0027] The above-mentioned traditional Chinese medicine compound granules are prepared by the preparation method of traditional Chinese medicine compound granules according to the present invention. The method may include the following steps: steam distilling Amomum villosum and collecting volatile oil and the distilled aqueous solution; decocting a premix of Cornus officinalis, Rehmannia root, Psoralea corylifolia, Radix Astragali, Angelica sinensis, Codonopsis pilosula, Millettia reticulata, and Carapax tricuspidata with water to obtain an aqueous extract; mixing the distilled aqueous solution with the aqueous extract and concentrating them into a thick paste; drying the thick paste to obtain a dry paste; grinding donkey-hide gelatin and the dry paste, adding an auxiliary material containing a filler, and dry-mixing them to obtain a dry blend; dry-granulating the dry blend to obtain semi-finished granules; sizing the semi-finished granules; spraying the volatile oil into the sizing semi-finished granules and sealing and moistening them to obtain the traditional Chinese medicine compound granules.

[0028] In the method according to the present invention, Amomum villosum can be subjected to steam distillation and the volatile oil and the aqueous solution after distillation can be collected. Specifically, Amomum villosum can be put into a volatile oil extraction tank, water can be added, heated, steam distilled, and the volatile oil can be collected (sealed). The aqueous solution after distillation can be collected in another device and purified by centrifugation or filtration. Before putting Amomum villosum into the volatile oil extraction tank, the Amomum villosum can be crushed using a grinder or a pulverizer, and the water added can be 6 to 10 times the amount of Amomum villosum. The Amomum villosum can be soaked in water for 0 to 4 hours before distillation, or it can be not soaked. The steam distillation time can be 6 hours. It is most preferred to add 8 times the amount of water for distillation and extraction for 6 hours, because the volatile oil extraction amount is the largest.

[0029] In the method according to the present invention, a premix of cornus fruit, Rehmannia root, salt psoralea corylifolia, roasted astragalus root, Chinese angelica root, Codonopsis pilosula, Millettia reticulata and vinegar turtle shell can be decocted with water to obtain an aqueous extract. This step can be performed using an extraction unit. The decocting can be performed multiple times. The decocting time can be 0.5 to 4 hours. In some specific embodiments, water can be added and decocted three times, each for 1 hour. The amount of water added can be 6 to 8 times the amount of the premix. In some more specific embodiments, water is added and decocted three times, each for 1.0 hour, with the amount of water added being 8 times (first time), 6 times (second time), and 6 times (third time) of the premix. After decocting, the aqueous extract can be purified and clarified by centrifugation or filtration.

[0030] The distilled aqueous solution of Amomum villosum can then be mixed with the water extract and concentrated into a thick paste. The concentration can be performed at a temperature of 40-100° C. and under reduced pressure (e.g., vacuum). The relative density of the concentrated thick paste is typically 1.34-1.38 (at 70-80° C.).

[0031] After concentration, the thick paste can be dried to form a dry paste. The drying can be performed at a temperature of 40 to 100° C. and under reduced pressure (e.g., vacuum). The drying time can be more than 12 hours.

[0032] According to the method of the present invention, after obtaining the dry paste, the donkey-hide gelatin and the dry paste can be ground and dry-mixed with an auxiliary material containing a filler to obtain a dry blend. The donkey-hide gelatin and the dry paste can be ground into fine powder (e.g., 80 mesh) separately, and then mixed with the auxiliary material containing the filler to obtain the dry blend; or the donkey-hide gelatin and the dry paste can be ground into fine powder together, and then mixed with the auxiliary material containing the filler to obtain the dry blend. Grinding can be performed using a pulverizer or a grinder. The weight ratio of the dry paste powder to the filler can be 9:1 to 3:7, preferably 1:1 to 3:1.

[0033] The dry blend can then be dry granulated to obtain semi-finished granules. Dry granulation can be performed using a dry granulator. The roller speed can be 10-20 rpm, the feed paddle speed can be 20-40 rpm, the roller pressure can be in the range of 4-8 MPa, and the side seal pressure can be in the range of 1-3 MPa.

[0034] According to the present invention, after dry granulation, the semi-finished granules can be sized. The granulation can be performed by screening with a 12-mesh sieve, then selecting the granules with 10-mesh and 30-mesh sieves, and collecting the granules that pass through the 10-mesh sieve but not the 30-mesh sieve.

[0035] After granulation, the Amomum villosum volatile oil can be sprayed into the semi-finished granules after granulation, and then sealed and steamed to obtain the traditional Chinese medicine compound granules. After spraying the Amomum villosum volatile oil, the mixture can be mixed and steamed in a sealed container. The steaming time is generally more than 2 hours.

[0036] The method according to the present invention may further include the step of re-dry granulating the semi-finished granules removed after granulation. Specifically, the granules that cannot pass through a 10-mesh sieve and can pass through a 30-mesh sieve can be re-granulated. The conditions for re-granulation are as described above.

[0037] Typically, the moisture content of the semi-finished granules obtained by dry granulation does not exceed 13%, preferably 5.0%. If the moisture content exceeds the standard, it is necessary to dry the semi-finished granules below 80°C until the moisture content returns to the standard. Therefore, the method according to the present invention may also include the step of drying the semi-finished granules after granulation to reduce the moisture content of the granules to no more than 5.0% by weight. The drying temperature can be 50-80°C.

[0038] In addition, the method according to the present invention may further comprise the step of mixing the Chinese medicine compound granules. The mixing time may be 15 minutes.

[0039] Hereinafter, the present invention will be specifically described by way of some examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the present invention in any way.

[0040] Example 1

[0041] Preparation of Traditional Chinese Medicine Compound Granules

[0042] First, remove impurities and non-medicinal parts from Amomum villosum, then crush it using a grinder. Place 62.5g of the crushed Amomum villosum into a volatile oil extraction tank set, add 8 times the amount of water, heat, and steam distill for 6 hours. Collect the volatile oil (sealed), and collect the distilled aqueous solution in another container and filter (filter pore size not less than 200 mesh).

[0043] 375g of cornus fruit, 250g of prepared rehmannia root, 187.5g of roasted astragalus root, 187.5g of codonopsis pilosula, 187.5g of angelica root, 187.5g of salt psoralea corylifolia, 187.5g of millettia reticulata, and 250g of vinegar turtle shell were put into the extraction unit, decocted three times with water (8 times the amount of water was added for the first time, and 6 times the amount of water was added for the second and third times), each time for 1 hour, the decoction was filtered (the filter pore size was not less than 200 mesh), the filtrate was combined with the aqueous solution after distillation of amomum villosum, and vacuum concentrated at 70°C to a thick paste with a relative density of 1.34 to 1.38 (70 to 80°C). The thick paste was vacuum dried at 80°C for 12 hours in a vacuum drying oven to form a dry paste, and the dried dry paste was cooled. The dry paste was crushed into fine powder (80 mesh) using a grinder to obtain dry paste powder.

[0044] Use a grinder to grind 62.5g of donkey-hide gelatin into fine powder (80 mesh). Then, add dry paste powder, donkey-hide gelatin powder, 10g of stevia and sucrose powder (the weight ratio of dry paste powder: sucrose is about 1:1) into the mixer in sequence and dry mix for 5 minutes. Use a dry granulator (granulation conditions: roller speed of 15rpm, feed paddle speed of 30rpm, roller pressure of 6MPa, side seal pressure of 2MPa) to granulate with a 20-mesh screen to obtain semi-finished granules. Use a 12-mesh sieve for granulation, and use 10-mesh and 30-mesh sieves for granulation. Collect granules that pass through the 10-mesh sieve but not the 30-mesh sieve. Re-granulate the granules that cannot pass through the 10-mesh sieve and can pass through the 30-mesh sieve. Combine the granules obtained after granulation with the granules after granulation.

[0045] Finally, spray the Amomum villosum volatile oil into the granules while stirring in a mixer, dry mix for 5 minutes, transfer to a material barrel, seal and humidify for 2 hours. Finally, mix the granules for 15 minutes and bag them.

[0046] Example 2

[0047] First, remove impurities and non-medicinal parts from Amomum villosum, then crush it using a grinder. Place 62.5g of the crushed Amomum villosum into a volatile oil extraction tank set, add 8 times the amount of water, heat, and steam distill for 6 hours. Collect the volatile oil (sealed), and collect the distilled aqueous solution in another container and filter (filter pore size not less than 200 mesh).

[0048] 500g of cornus fruit, 250g of prepared rehmannia root, 187.5g of roasted astragalus root, 187.5g of codonopsis pilosula, 125g of angelica root, 187.5g of salt psoralea corylifolia, 125g of millettia reticulata, and 250g of vinegar turtle shell were put into the extraction unit, decocted three times with water (8 times the amount of water was added for the first time, and 6 times the amount of water was added for the second and third times), each time for 1 hour, and the decoction was filtered (the filter pore size was not less than 200 mesh), and the filtrate was combined with the aqueous solution after distillation of amomum villosum, and concentrated at 80°C in vacuum to a thick paste with a relative density of 1.34 to 1.38 (70 to 80°C). The thick paste was vacuum dried at 90°C for 12 hours in a vacuum drying oven to form a dry paste, and the dried dry paste was cooled. The dry paste was crushed into fine powder (80 mesh) using a grinder to obtain dry paste powder.

[0049] Use a grinder to grind 187.5g of donkey-hide gelatin into fine powder (80 mesh). Then, add dry paste powder, donkey-hide gelatin powder, 10g of stevia and sucrose powder (the weight ratio of dry paste powder to sucrose is about 3:1) into the mixer in sequence and dry mix for 5 minutes. Use a dry granulator (granulation conditions: roller speed of 15rpm, feed paddle speed of 30rpm, roller pressure of 6MPa, side seal pressure of 2MPa) to granulate with a 20-mesh screen to obtain semi-finished granules. Use a 12-mesh sieve for granulation, and use 10-mesh and 30-mesh sieves for granulation. Collect granules that pass through the 10-mesh sieve but not the 30-mesh sieve. Re-granulate the granules that cannot pass through the 10-mesh sieve and can pass through the 30-mesh sieve. Combine the granules obtained after granulation with the granules after granulation.

[0050] Finally, spray the Amomum villosum volatile oil into the granules while stirring in a mixer, dry mix for 5 minutes, transfer to a material barrel, seal and humidify for 2 hours. Finally, mix the granules for 15 minutes and bag them.

[0051] Example 3

[0052] First, remove impurities and non-medicinal parts from Amomum villosum, then crush it using a grinder. Place 62.5g of the crushed Amomum villosum into a volatile oil extraction tank set, add 8 times the amount of water, heat, and steam distill for 6 hours. Collect the volatile oil (sealed), and collect the distilled aqueous solution in another container and filter (filter pore size not less than 200 mesh).

[0053] 375g of cornus fruit, 312.5g of prepared rehmannia root, 187.5g of roasted astragalus root, 125g of codonopsis pilosula, 187.5g of angelica root, 187.5g of salt psoralea corylifolia, 187.5g of millettia reticulata, and 250g of vinegar turtle shell were put into the extraction unit, decocted three times with water (8 times the amount of water was added for the first time, and 6 times the amount of water was added for the second and third times), each time for 1 hour, and the decoction was filtered (the filter pore size was not less than 200 mesh), and the filtrate was combined with the aqueous solution after distillation of amomum villosum, and vacuum concentrated at 70°C to form a thick paste with a relative density of 1.34 to 1.38 (70 to 80°C). The thick paste was vacuum dried at 80°C for 12 hours in a vacuum drying oven to form a dry paste, and the dried dry paste was cooled. The dry paste was crushed into fine powder (80 mesh) using a grinder to obtain dry paste powder.

[0054] Use a grinder to grind 62.5g of donkey-hide gelatin into fine powder (80 mesh). Then put dry paste powder, donkey-hide gelatin powder and 10g of stevia and soluble starch (the weight ratio of dry paste powder to soluble starch is about 5:1) into the mixer in sequence and dry mix for 5 minutes. Use a dry granulator (granulation conditions: roller speed of 15rpm, feed paddle speed of 30rpm, roller pressure of 6MPa, side seal pressure of 2MPa) to granulate with a 20-mesh screen to obtain semi-finished granules. Use a 12-mesh sieve for granulation, and use 10-mesh and 30-mesh sieves for granulation. Collect the granules that pass through the 10-mesh sieve but not the 30-mesh sieve. Re-granulate the granules that cannot pass through the 10-mesh sieve and can pass through the 30-mesh sieve. Combine the granules obtained after granulation with the granules after granulation.

[0055] Finally, spray the Amomum villosum volatile oil into the granules while stirring in a mixer, dry mix for 5 minutes, transfer to a material barrel, seal and humidify for 2 hours. Finally, mix the granules for 15 minutes and bag them.

[0056] Example 4

[0057] Related animal toxicity tests of the Chinese herbal compound granules of Examples 1 to 3

[0058] 1. Animal acute toxicity test

[0059] Take 40 Kunming mice (clean grade, body weight 19-21g, half male and half female, provided by Beijing Huafukang Biotechnology Co., Ltd.), half male and half female, 20 each in the normal control group and the drug administration group. Mice were raised for 2 days under experimental conditions, fasted for 16 hours before administration, and the embodiment drug was mixed with 1.353g crude drug / ml with distilled water, and the administration volume was 0.4ml / 10g. The control group was given the same volume of distilled water. The drug solution was given 3 times by gavage in one day, each time with an interval of 6 hours. The dosage of each dose was 54.12g crude drug / kg, and the cumulative dosage in one day was 162.36g crude drug / kg. After administration, the animal behavior and reaction, appearance, limb activity, food intake, drinking water, excretion and other conditions were observed immediately, and observed once a day for 14 consecutive days, and the reaction of each animal was recorded in detail. Record daily food intake and body weight at the same time.

[0060] Results showed that the experimental mice were observed to be curled up and less active on the day of dosing, and some mice developed diarrhea. Food intake decreased significantly on the first day after dosing, but gradually recovered thereafter. Male mice experienced slow weight gain one day after dosing, showing a significant difference compared to the normal control group (P < 0.05), but returned to normal after two days. No other significant abnormalities were observed. No animal deaths were observed at the end of the 14-day observation period.

[0061] The drug of the present invention is administered orally three times a day at a maximum permissible concentration of 1.353 g crude drug / ml and a maximum oral volume of 0.4 ml / 10 g for mice, with a cumulative dosage of 162.36 g crude drug / kg. After 14 days of observation, except for the mice curling up and moving less on the day of administration, diarrhea in some mice, a significant decrease in food intake on the first day after administration, and slow weight gain in male mice one day after administration, no other obvious abnormalities were observed, and no mice died during the observation period. The results showed that when the cumulative dosage of the mice was 162.36 g crude drug / kg in one day, no deaths or serious adverse reactions due to drug toxicity were observed. The recommended daily dosage for humans in clinical trials is 93 g crude drug. Based on an average human weight of 60 kg, the clinical human daily dosage is 1.55 g crude drug / kg. Therefore, the maximum dosage for mice is equivalent to 104.75 times the daily dosage for humans in clinical trials.

[0062] 2. Long-term toxicity test in animals

[0063] The experiment used SPF-grade SD rats as the rodent species for long-term toxicity testing. They were provided by the Experimental Animal Center of the Academy of Military Medical Sciences. There were 160 rats, 5 to 6 weeks old, half male and half female, with 30 rats per group, weighing 120 to 160g. After one week of acclimatization, the experimental animals were divided into groups. The animals' body weight, food intake, and water intake were measured. They were then divided into groups based on the principle of balanced and randomized distribution of sex and body weight, with 30 rats per group, half male and half female. Three treatment groups and one control group were set up, with 15 animals of each sex in each group. Based on the maximum dose, the dosage of each long-term toxicity group of rats was set as 9g / kg for the low-dose group, 18g / kg for the medium-dose group, and 36g / kg for the high-dose group. The route of administration for the rat experiment was oral gavage. Drugs were administered six times per week between 8:30 and 10:30 AM daily, with an initial gavage volume of 1.56 ml / 100 g body weight. After 40 days of the study, due to the animals' increased weight, the drug concentration was adjusted to 1.25 ml / 100 g body weight. The control group received a corresponding volume of distilled water. The drug was administered for six months, followed by 30 days of observation after drug withdrawal. The animals' general condition, including coat appearance, nasal and anal discharge, and activity, was observed daily. Body weights were measured weekly, along with food and water intake. Forty rats (10 per group, half male and half female) were sacrificed at three, six, and 30 days after drug withdrawal. Urine biochemical analysis, peripheral blood formed element indices, coagulation parameters, serum biochemical parameters, gross autopsy, organ weights, organ coefficients, and histopathological examinations were performed.

[0064] Results showed that after six months of oral administration of the granules to rats, the low-dose group (9g / kg, equivalent to 13.75 times the recommended daily clinical dose) was a safe dose. Although a few animals in the medium-dose group (18g / kg, equivalent to 27.50 times the recommended daily clinical dose) and the high-dose group (36g / kg, equivalent to 55.00 times the recommended daily clinical dose) showed abnormalities in urine biochemical analysis (blood, protein, urea nitrogen (URE), and creatine kinase (CK) levels after administration (as did the control group), these were normal reactions to the high dose. Furthermore, one month after discontinuation of the drug, except for the significantly lower CK levels in female rats in the high-dose group, no significant differences were observed in other indicators compared with the control group. This indicates that the granules of the present invention exhibited no delayed toxic effects within a six-month dose range of 9-36g / kg / day, indicating that the safe dose of the granules in SD rats is at least 36g / kg.

[0065] After six months of oral administration of the test compound granules to SD rats, changes in all observed parameters, except body weight and creatine kinase (CK) levels, returned to normal one month after drug withdrawal, indicating that the drug's toxic effects are reversible. Oral administration of low, medium, and high doses of the compound granules to SD rats for three and six months, followed by one month of withdrawal, resulted in mild to moderate enhancement of extramedullary hematopoiesis in the spleen and erythroid hematopoiesis in the bone marrow, representing therapeutic effects beyond the therapeutic effect, indicating a significant clinical therapeutic effect. The drug also demonstrated no damage to organs, structures, or tissues of the hematopoietic and immune systems.

[0066] Example 5

[0067] Pharmacological Experiments on Traditional Chinese Medicine Compound Granules

[0068] 1. Study on the effects of Chinese herbal compound granules on the proliferation and differentiation of early hematopoietic progenitor cells in mouse bone marrow

[0069] 1.1 Experimental Materials

[0070] 1.1.1 Experimental animals

[0071] 132 Kunming mice, secondary, 102 females and 30 males. Among them, peripheral blood index detection was performed in 10 mice per group, a total of 60 mice, half male and half female; hematopoietic progenitor cell culture was performed in 3 mice per group, a total of 36 mice at two time points, female; CD34 + There were 6 cells in each group for cell testing, for a total of 36 female cells.

[0072] Mice aged 6 to 8 weeks and weighing 18 to 22 g were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of the PLA.

[0073] 1.1.2 Experimental drugs

[0074] Example 1 Granules, drug concentration: Each gram of Chinese herbal compound granules is equivalent to 2.368g of crude drug.

[0075] Positive drug: recombinant human granulocyte-stimulating factor injection (G-CSF): Beijing Shuanglu Pharmaceutical Co., Ltd.

[0076] 1.1.3 Main instruments and reagents

[0077] F820 hematology analyzer: Sysmex Co., Ltd., Japan. CO2 incubator (model: HEPA class 100): Thermo Forma Co., Ltd.

[0078] RPMI 1640 was purchased from Gibco BRL. Fetal bovine serum and horse serum were purchased from the Beijing Military Region Veterinary Prevention and Control Center. Methylcellulose was purchased from Whatman. FITC-labeled rat anti-mouse CD34 antibody and control antibody were purchased from Becton Dickinson. Epo, IL-3, IL-11, and L-glutamine were purchased from Sigma. Hemolysin, diluent, and washing solution were all produced by Shandong Lanqiao Technology Co., Ltd.

[0079] 1.2 Test methods

[0080] 1.2.1 Model making

[0081] The experiment used 3.5Gy 60 Co-γ irradiation, dose 3.5 Gy, dose rate 1.31 Gy.min -1 .

[0082] 1.2.2 Peripheral blood test

[0083] 1.2.2.1 Grouping and Dosing

[0084] The experiment was divided into 6 groups: normal control group, model control group, positive drug control group, high-dose Chinese herbal compound granules group, medium-dose Chinese herbal compound granules group, and low-dose Chinese herbal compound granules group. Each group consisted of 10 mice, half male and half female.

[0085] Two days after oral administration of the Chinese herbal compound granules group, 60 Irradiation with 3.5 Gy of Co-γ rays suppressed bone marrow hematopoiesis and reduced peripheral blood components in mice. After model establishment, the drug was administered once daily for 14 consecutive days. The normal and model groups were gavaged with an equal volume of distilled water, while the positive control group was subcutaneously injected with recombinant human granulocyte-stimulating factor (G-CSF) once daily for 14 consecutive days.

[0086] Normal control group: administered with equal volume of distilled water by gavage.

[0087] Model control group: the same amount of distilled water was given by gavage.

[0088] G-CSF positive drug control group: 30 μg / kg (injection volume: 0.1 ml / 10 g).

[0089] High-dose group of Chinese herbal compound granules: the dosage was 10.0 g / kg body weight.

[0090] Medium-dose group of Chinese herbal compound granules: dosage 5.0 g / kg body weight.

[0091] Low-dose group of Chinese herbal compound granules: the dosage was 2.5 g / kg body weight.

[0092] 1.3.3.2 Observation indicators and detection methods

[0093] Before irradiation and 1, 3, 5, 7, 11, 13, 15, 17, and 21 days after irradiation, 20 μl of blood was collected from the tail vein of each mouse, and peripheral blood count was measured using a Sysmex-800 automatic blood cell counter.

[0094] 1.2.3 Hematopoietic progenitor cell detection experiment

[0095] 1.2.3.1 Grouping and Dosing

[0096] The experiment was divided into 6 groups: normal control group, model control group, positive drug control group, high-dose Chinese herbal compound granules group, medium-dose Chinese herbal compound granules group, and low-dose Chinese herbal compound granules group. Each group consisted of 6 female mice.

[0097] Two days after oral administration of the Chinese herbal compound granules group, 60 Models were established by irradiation with 3.5 Gy of Co-γ rays, and then the drug was administered once a day for 7 consecutive days. The normal group and the model group were gavaged with an equal volume of distilled water, and the positive control group was subcutaneously injected with recombinant human granulocyte-stimulating factor injection (G-CSF) once a day for 7 consecutive days.

[0098] Normal control group: administered with equal volume of distilled water by gavage.

[0099] Model control group: the same amount of distilled water was given by gavage.

[0100] G-CSF positive drug control group: 30 μg / kg (injection volume: 0.1 ml / 10 g).

[0101] High-dose group of Chinese herbal compound granules: the dosage was 10.0 g / kg body weight.

[0102] Medium-dose group of Chinese herbal compound granules: dosage 5.0 g / kg body weight.

[0103] Low-dose group of Chinese herbal compound granules: the dosage was 2.5 g / kg body weight.

[0104] 1.2.3.2 Colony culture

[0105] (1) Three mice were taken from each group and killed by cervical dislocation at two time points, 3 days and 7 days after modeling. The mice were killed by cervical dislocation and immersed in 75% alcohol for a while for disinfection. The femurs of the mice were removed and the bone marrow cells were flushed out with RPMI 1640 culture medium. The cells were passed through a No. 4 needle to make a single cell suspension. The cells were counted and the amount of cell suspension to be inoculated was calculated. The number of nucleated cells inoculated was 1×10 5 / ml system calculation.

[0106] (2) Granulocyte culture system (horse serum 2 ml; G-CSF 100 ng; cells 1×10 5 / ml; 1640 to 7.6ml). Preheat in a constant temperature water bath at 37°C for 10 minutes, then add 0.5ml of 5% boiled agar and mix immediately. Use a 5ml syringe to quickly add and shake to mix. Add 1ml of this solution to each plate. Place in a plastic box with several water-filled plates, incubate at 37°C, 5% CO2 for 6 days, and count cells. A colony of more than 50 cells is considered a cell colony.

[0107] (3) Erythroid, mixed and megakaryocytic culture systems (mercaptoethanol 1×10 -4 M 0.2 ml; 3% L-glutamine 0.03 ml; horse serum 0.7 ml; Epo 100 U; IL-3 60 ng; IL-11 50 ng; 2.7% methylcellulose 0.7 ml; cells 1×10 5 / ml), place it on a shaker and shake for a while, add it to a 24-well plate, 0.2 ml per well, and culture it at 37°C, 5% CO2 for 3 days to observe and count CFU-E and CFU-Meg; culture it for 6 days to observe and count BFU-E and CFU-mix.

[0108] 1.2.4 CD34 + Cell detection experiments

[0109] 1.2.4.1 Grouping and Dosing

[0110] The experiment was divided into 6 groups: normal control group, model control group, positive drug control group, high-dose Chinese herbal compound granules group, medium-dose Chinese herbal compound granules group, and low-dose Chinese herbal compound granules group. Each group consisted of 6 female mice.

[0111] Two days after oral administration of the Chinese herbal compound granules group, 60Models were established by irradiation with 3.5 Gy of Co-γ rays, and then the drug was administered once a day for 7 consecutive days. The normal group and the model group were gavaged with an equal volume of distilled water, and the positive control group was subcutaneously injected with recombinant human granulocyte-stimulating factor injection (G-CSF) once a day for 7 consecutive days.

[0112] Normal control group: administered with equal volume of distilled water by gavage.

[0113] Model control group: the same amount of distilled water was given by gavage.

[0114] G-CSF positive drug control group: 30 μg / kg (injection volume: 0.1 ml / 10 g).

[0115] High-dose group of Chinese herbal compound granules: the dosage was 10.0 g / kg body weight.

[0116] Medium-dose group of Chinese herbal compound granules: dosage 5.0 g / kg body weight.

[0117] Low-dose group of Chinese herbal compound granules: the dosage was 2.5 g / kg body weight.

[0118] 1.2.4.2 Observation indicators and detection methods

[0119] Seven days after modeling, mice were killed by cervical dislocation, and femoral bone marrow cells were flushed out with PBS buffer containing 0.2% bovine serum albumin. 1×10 6 Add 30 μl of mouse serum to each cell and incubate for 10 minutes to block nonspecific binding sites. Then, add 10 μl of FITC-labeled rat anti-mouse CD34 antibody. Add the corresponding control antibody to a control tube and incubate at 4°C in the dark for 30 minutes. Add 2 ml of red blood cell lysis buffer and incubate for 5 minutes. Wash the cells twice with PBS containing 0.2% bovine serum albumin and 0.1% sodium azide. Add PI stain to a final concentration of 3 μg / ml and analyze on an analyzer.

[0120] 1.2.5 Data processing and statistics

[0121] The measurement results of all animals at different times were input, and the mean and standard deviation of each group and the significance t test of the means between groups were calculated.

[0122] 1.3 Test results

[0123] 1.3.1 Peripheral blood count

[0124] 1.3.1.1 Peripheral blood leukocytes

[0125] Compared with the normal control group, the irradiated control group showed a significant decrease in white blood cell count from day 1 to 21 after modeling (P < 0.01 to 0.001), indicating successful modeling. Compared with the irradiated control group, the positive drug group showed a significant increase in white blood cell count from day 1 to 17 (except day 3 and 11) after modeling (P < 0.05 to 0.001). All dose groups of the Chinese herbal compound granules were able to increase white blood cell counts. The high, medium, and low dose groups significantly promoted the increase in white blood cell count on days 5, 7, and 13, day 13, and day 13 and 17 after modeling, respectively (P < 0.05 to 0.01). The results are shown in Table 1.

[0126] Table 1 Effects of Chinese herbal compound granules on 3.5 Gy 60 Co-γ ray irradiated mouse peripheral blood leukocytes (WBC 1×10 9 / L) (n=10, )

[0127]

[0128] Note: Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the irradiated control group, #P<0.05, ##P<0.01, ###P<0.001

[0129] 1.3.1.2 Peripheral blood red blood cells and hemoglobin

[0130] Compared with the normal control group, the peripheral blood red blood cell and hemoglobin levels in the irradiated control group decreased to a certain extent after model establishment. The red blood cell and hemoglobin levels decreased significantly from days 3 to 21 (P < 0.05-0.001), and the hemoglobin levels decreased significantly from days 3 to 21 (except on day 17, P < 0.05-0.001), indicating that the model establishment was successful. The red blood cell and hemoglobin levels in the positive drug group and the various dose groups of the Chinese herbal compound granules were significantly higher than those in the irradiated control group on days 15 or 17 (P < 0.05-0.01). The results are shown in Tables 2 and 3.

[0131] Table 2 Effects of Chinese herbal compound granules on 3.5 Gy 60 Co-γ ray irradiated mouse peripheral blood red blood cells (RBC 1×10 12 / L) (n=10, )

[0132]

[0133]

[0134] Note: Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the irradiated control group, #P<0.05, ##P<0.01, ###P<0.001

[0135] Table 3 Effects of Chinese herbal compound granules on 3.5 Gy 60 Effects of Co-γ ray irradiation on peripheral blood hemoglobin (HGBg / L) in mice (n=10, )

[0136]

[0137] Note: Compared with the normal control group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the irradiated control group, #P < 0.05, ##P < 0.01

[0138] 1.3.1.3 Platelet count

[0139] Platelet counts in both the irradiated control group and the medication group decreased one day after irradiation, then rose three to five days before declining again. Platelet counts reached their lowest value seven days after irradiation and then slowly recovered. However, starting from day five after irradiation, platelet counts in the various dose groups of the Chinese herbal compound granules recovered faster than those in the control group. Platelet counts in the high-dose group were significantly higher than those in the irradiated control group on days 5, 11, and 13, and in the medium-dose group on day 13 (P < 0.05-0.001). The results are shown in Table 4.

[0140] Table 4 Effects of Chinese herbal compound granules on 3.5 Gy 60 Co-γ ray irradiated mouse peripheral blood platelets (PLT1×10 9 L) (n = 10, )

[0141]

[0142] Note: Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the irradiated control group, #P<0.05, ##P<0.01, ###P<0.001

[0143] 1.3.2 Hematopoietic Progenitor Cells

[0144] Compared with the irradiation control group, the positive drug and each dose group of the Chinese herbal compound granules promoted the proliferation of CFU-GM and CFU-E 3 and 7 days after irradiation (P < 0.05 ~ 0.001); the high and medium dose groups of the Chinese herbal compound granules also promoted the proliferation of CFU-Meg and CFU-Mix 3 and 7 days after the drug (P < 0.05 ~ 0.01). The results are shown in Table 5.

[0145] Table 5 Effects of Chinese herbal compound granules on 3.5 Gy 60 Effects of Co-γ irradiation on mouse hematopoietic progenitor cells (n=3, )

[0146]

[0147]

[0148] Note: Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the irradiated control group, #P<0.05, ##P<0.01, ###P<0.001

[0149] 1.3.3 CD34 + cell

[0150] Compared with the normal control group, the bone marrow CD34 + The number of cells in the irradiated model group was significantly decreased (P<0.01), indicating that the model was successfully established. + The number of cells decreased significantly after positive drug treatment. + The number of cells increased significantly, and each dose group of the Chinese medicine compound granules could increase the number of CD34 in the mouse bone marrow + The number of cells in the low-dose group was significantly different from that in the irradiated model control group (P < 0.05). The results are shown in Table 6.

[0151] Table 6 Effects of Chinese herbal compound granules on 3.5 Gy 60 Effects of Co-γ irradiation on CD34+ cells in bone marrow of mice for 7 days (n=6, )

[0152]

[0153] Note: Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001; compared with the irradiated control group, #P<0.05, ##P<0.01, ###P<0.001

[0154] The results showed that each dose group of the Chinese herbal compound granules could increase the number of peripheral blood leukocytes, promote the proliferation of CFU-GM, CFU-E, and CFU-Meg, and increase the expression of CD34 in the mouse bone marrow. + The high-dose group had a significant effect on peripheral blood leukocytes, the high- and medium-dose groups had a significant effect on bone marrow hematopoietic stem / progenitor cells, and the low-dose group had a significant effect on CD34 + The cellular effect is significant.

[0155] 2. Prevention and treatment of traditional Chinese medicine compound granules 60 Pharmacodynamics of Co-γ ray-induced aplastic anemia in rats

[0156] 2.1 Test materials

[0157] Rats were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0158] Example 1 Granules, drug concentration: Each gram of Chinese herbal compound granules is equivalent to 2.368g of crude drug.

[0159] Cyclophosphamide for injection: Shanxi Pude Pharmaceutical Co., Ltd., 0.2g / vial.

[0160] Positive drugs: Stanozolol tablets (Stanozolol, clinically used to treat aplastic anemia, may improve bone marrow hematopoietic function, especially erythropoiesis), Guangxi Nanning Baihui Pharmaceutical Group Co., Ltd., 2 mg / tablet.

[0161] 2.2 Test methods

[0162] 2.2.1 Experimental animals and adaptive feeding

[0163] 80 SD rats, weighing 180-200 g, were purchased and placed in an animal room with a constant temperature (24±0.5°C) and constant humidity (50±10%) for 1 week of adaptive feeding with free access to water and food.

[0164] 2.2.2 Trial Grouping and Dosing

[0165] After 1 week of adaptive feeding, 10 SD rats were randomly divided into normal control group according to their body weight. The remaining 70 rats were used in the cobalt source room of the Institute of Chemistry, Peking University. 60 After irradiation with 4.0 Gy of Co-γ rays, the rats showed poor condition, curled up, and reduced activity. 60 After irradiation with 4.0 Gy of Co-γ rays, the surviving rats were randomly divided into 5 groups according to their body weight. This experiment was divided into 6 groups in total: normal control group, model control group, positive drug control group, low-dose Chinese herbal compound granules group, medium-dose Chinese herbal compound granules group, and high-dose Chinese herbal compound granules group.

[0166] Cyclophosphamide 25.0 mg·kg was given starting from the 4th day -1 Intraperitoneal injection was performed for 3 consecutive days to prepare aplastic anemia rat model.

[0167] Cyclophosphamide was injected continuously for 3 days. On the day of the last injection, the positive drug group was given 2.8 mg / kg of body weight, and the Chinese medicine compound granules small, medium and large doses were given by gavage at a dose of 7.2 g / kg, 4.16 g / kg and 2.4 g / kg respectively. The drug was dissolved in deionized water and the rats were gavaged at a dose of 1 ml / 100 g of body weight. The normal control group and the model control group were given the same deionized water by gavage.

[0168] 2.2.3 Observation indicators

[0169] (1) Effects on the general condition of rats with aplastic anemia: activity, diet, defecation, fur, diarrhea, weight loss, development, and mental state

[0170] (2) Effects on peripheral blood counts of rats with aplastic anemia: changes in blood WBC, RBC, HGB, and Pt

[0171] (3) Effect on the number of nucleated cells in the bone marrow of rats with aplastic anemia: The number of nucleated cells in the bone marrow of each femur was counted.

[0172] (4) Effects on the immune function of rats with aplastic anemia

[0173] ① Flow cytometry detection: CD3, CD4, CD8 and CD4 / CD8.

[0174] ②Radioimmunoassay detection: TGF-β1, IL-1, IFNγ, TNFα, etc.

[0175] 2.3 Test results

[0176] 2.3.1 Effects on the general condition of rats with aplastic anemia

[0177] Rats in the model control group showed decreased activity and diet, poor mental state, loose stools, blood in urine, erect fur, emaciated and stunted growth, weight loss, pale ears, lips, and nails, and significant anemia. All doses of the TCM compound granules improved these symptoms to varying degrees, with anemia particularly improving and weight gain evident. The best results were achieved in the medium-dose group.

[0178] Some animals died during modeling and treatment. The survival status of rats in each group is shown in Table 7. Peripheral blood counts were measured in all surviving animals. Considering the feasibility of the experimental operation and meeting statistical requirements, 6 animals were randomly selected from each surviving group to measure bone marrow nucleated cell count, CD4, CD8, CD3, CD4 / CD8 ratio, TGF-β1, and IL-1.

[0179] 2.3.2 Effects on peripheral blood counts of rats with aplastic anemia (Table 7)

[0180] Table 7 Chinese herbal compound granules 6 0 Effects of Co-γ ray-induced peripheral blood counts on aplastic anemia in rats

[0181]

[0182] Note: Compared with the normal control group, # P<0.05, ## P<0.01; compared with the model control group, *P<0.05, **P<0.01; compared with the positive drug group, △P<0.05, △△ P<0.01.

[0183] During the experiment 60 Co-γ ray irradiation 4.0 Gy combined with cyclophosphamide 25.0 mg kg -1 Intraperitoneal injection of aplastic rat models revealed a decrease in peripheral blood counts of whole cells (WBC), RBC, and HGB, with statistically significant differences compared to the normal control group (P < 0.01), indicating successful modeling. After treatment, peripheral blood counts increased significantly in all dose groups of the Chinese herbal compound granules, with significant differences compared to the model control group (P < 0.01). Comparisons between the high- and medium-dose groups and the positive drug group revealed significant differences in WBC counts (P < 0.05), and in HGB counts (P < 0.05), in the low-dose group. This suggests that the Chinese herbal compound granules are superior to stanozolol tablets and exhibit a dose-dependent therapeutic effect.

[0184] 2.3.3 Effects on the number of nucleated cells in the bone marrow of rats with aplastic anemia (Table 8)

[0185] Table 8 Chinese herbal compound granules 60 Effect of Co-γ irradiation on the number of bone marrow nucleated cells in rats with aplastic anemia

[0186] Group n <![CDATA[Bone marrow nucleated cell count × 10 7 > Normal control group 6 6.510±0.191 Model control group 6 <![CDATA[1.042±0.322 ## <!-- 17 -->]]> Positive drug group 6 4.632±0.473** Low-dose group 6 4.734±0.171** Medium dose group 6 4.470±0.369** High-dose group 6 5.042±0.354**

[0187] Note: Compared with the normal control group, # P<0.05, ## P<0.01; compared with the model control group, *P<0.05, **P<0.01; compared with the positive drug group, △ P<0.05, △△ P<0.01.

[0188] In the model control group, the number of bone marrow nucleated cells decreased significantly, and bone marrow proliferation was significantly reduced, with statistically significant differences compared with the normal control group (P < 0.01), indicating that the model was successfully established. After treatment with the Chinese herbal compound granules at all doses, the number of bone marrow nucleated cells increased significantly, with significant differences compared with the model control group (P < 0.01).

[0189] 2.3.4 Effects on CD4, CD8, CD3, and CD4 / CD8 in rats with aplastic anemia (Table 9)

[0190] Table 9 Chinese herbal compound granules 60 Effects of Co-γ irradiation on CD4, CD8, CD3 and CD4 / CD8 in rats with aplastic anemia

[0191]

[0192] Note: Compared with the normal control group, #P<0.05, ##P<0.01; compared with the model control group, *P<0.05, **P<0.01; compared with the positive drug group, △P<0.05, △△P<0.01.

[0193] Compared with the normal control group, the peripheral blood CD8, CD3, and CD4 / CD8 of the model control group increased to varying degrees, with statistically significant differences (P < 0.05), indicating successful modeling. The TCM compound granules significantly decreased after treatment, with significant differences compared with the model control group (P < 0.05 or P < 0.01), but no significant differences compared with the positive drug group. CD4 significantly increased after treatment in all TCM compound granules groups, with significant differences compared with the model control group (P < 0.05 or P < 0.01), except for the low-dose group. The therapeutic effect of TCM compound granules was dose-dependent.

[0194] 2.3.5 Effects on serum TGF-β1 and IL-1 in rats with aplastic anemia (Table 10)

[0195] Table 10 Chinese herbal compound granules 60 Effects of Co-γ irradiation on serum TGF-β1 and IL-1 in rats with aplastic anemia

[0196] Group n <![CDATA[TGF-β1]]> IL~1 Normal control group 6 13.839±0.405 0.267±0.012 Model control group 6 <![CDATA[28.411±0.714 ## ]]> <![CDATA[0.150±0.009 ## ]]> Positive drug group 6 17.646±0.605** 0.214±0.005** Granule low-dose group 6 18.777±0.257** <![CDATA[0.153±0.002 △ ]]> Medium dose granule group 6 <![CDATA[13.421±1.277** △ ]]> 0.195±0.008* Granule high-dose group 6 16.658±1.080** 0.209±0.006**

[0197] Note: Compared with the normal control group, #P<0.05, ##P<0.01; compared with the model control group, *P<0.05, **P<0.01; compared with the positive drug group, △P<0.05, △△P<0.01.

[0198] Compared with the normal control group, the serum TGF-β1 level of rats in the model control group was significantly increased, with statistically significant differences (P < 0.01). Compared with the normal control group, the serum IL-1 level was significantly decreased, with statistically significant differences (P < 0.01), indicating that the model was successfully established. After treatment with the Chinese herbal compound granules, TGF-β1 was significantly decreased in all dose groups, with statistically significant differences (P < 0.01) compared with the model control group, and the medium dose group was significantly different from the positive drug group (P < 0.05). After treatment with the Chinese herbal compound granules, IL-1 was significantly increased, with statistically significant differences (P < 0.05 or P < 0.01) in the medium and high dose groups compared with the model control group, and statistically significant differences (P < 0.05) in the low dose group compared with the positive drug group. The therapeutic effect of the Chinese herbal compound granules was dose-dependent.

[0199] 2.3.6 Effects on serum IFN-γ and TNF-α in rats with aplastic anemia are shown in Table 11

[0200] Table 11 Chinese herbal compound granules 60 Effects of Co-γ irradiation on serum IFN-γ and TNF-α in rats with aplastic anemia

[0201]

[0202]

[0203] Note: Compared with the normal control group, #P<0.05, ##P<0.01; compared with the model control group, *P<0.05, **P<0.01; compared with the positive drug group, △P<0.05, △△P<0.01.

[0204] Compared with the normal control group, the serum IFN-γ and TNF-d of the rats in the model control group increased significantly, and there was a significant difference after statistical processing (P < 0.01), indicating that the model was successfully established. Compared with the model control group, the IFN-γ and TNF-α levels of the Chinese herbal compound granules in each dose group were significantly reduced after treatment. significant There was no significant difference in the efficacy of the compound granules (P < 0.01). Compared with the positive drug group, the high-dose group of the Chinese herbal compound granules had a significant difference (P < 0.05), and the therapeutic effect of the compound granules was dose-dependent.

[0205] Similar pharmacological tests on the granules of Examples 2-3 showed no significant difference from the granules of Example 1.

[0206] The above embodiments are merely illustrative of the present invention and are not intended to limit the present invention. Those skilled in the art will appreciate that any modifications and variations made to the embodiments of the present invention without departing from the spirit and scope of the present invention are intended to fall within the scope of the present invention. The scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A Chinese medicine compound granule, the Chinese medicine compound granule being prepared from an auxiliary material and a Chinese medicine extract as an active ingredient, the auxiliary material containing a filler, the Chinese medicine extract being a full-ingredient extract extracted from a Chinese medicine composition consisting of the following components in parts by weight: 5-8 parts of cornus fruit; 3-5 parts of prepared rehmannia root; 2-5 parts of salt psoralea corylifolia; 2-4 parts of roasted astragalus root; 2-4 parts of angelica sinensis; 0.5-3 parts of donkey hide gelatin; 2-4 parts of millettia reticulata; 2-4 parts of codonopsis pilosula; 3-5 parts of vinegar-cured turtle shell; and 0.5-2 parts of amomum villosum; the Chinese medicine compound granule being prepared by a method comprising the following steps: steam distilling the amomum villosum and collecting the volatiles The invention discloses a method for preparing a Chinese herbal compound granule comprising the steps of: preparing an oil and a distilled aqueous solution; decocting a premix of cornus fruit, prepared rehmannia root, salt-fleshed psoralea corylifolia, roasted astragalus root, angelica root, codonopsis pilosula, millettia reticulata and turtle shell with vinegar to obtain an aqueous extract; mixing the distilled aqueous solution with the aqueous extract and concentrating the mixture into a thick paste; drying the thick paste to obtain a dry paste; grinding donkey-hide gelatin and the dry paste, adding an auxiliary material containing a filler, and dry-mixing the mixture to obtain a dry mixture; dry-granulating the dry mixture to obtain semi-finished granules; sizing the semi-finished granules; spraying the volatile oil into the sizing semi-finished granules, and performing airtight steaming to obtain the Chinese herbal compound granules.

2. The Chinese medicine compound granules according to claim 1, characterized in that The Chinese medicinal material composition is composed of the following components in parts by weight: 6 parts of cornus fruit; 4 parts of prepared rehmannia root; 3 parts of salt psoralea corylifolia; 3 parts of roasted astragalus root; 3 parts of angelica root; 1 part of donkey-hide gelatin; 3 parts of millettia reticulata; 3 parts of codonopsis pilosula; 4 parts of vinegar-cured turtle shell; and 1 part of amomum villosum.

3. The Chinese medicinal compound granules according to claim 1 or 2, characterized in that: Based on the total weight of the traditional Chinese medicine compound granules, the amount of the filler is 10 to 70 weight %.

4. The Chinese medicinal compound granules according to claim 1 or 2, characterized in that: The filler is selected from sucrose, soluble starch or a combination thereof.

5. The Chinese medicinal compound granules according to claim 1 or 2, characterized in that: The auxiliary material further contains a flavoring agent, and based on the total weight of the traditional Chinese medicine compound granules, the amount of the flavoring agent is 0.1 to 10 weight percent.

6. The Chinese medicinal compound granules according to claim 1, characterized in that: The method further comprises the step of re-dry granulating the semi-finished granules removed after granulation.

7. The Chinese medicinal compound granules according to claim 1 or 6, characterized in that: The step of mixing the distilled aqueous solution with the water extract and concentrating the mixture into a thick paste is carried out at a temperature of 40 to 100° C. and under reduced pressure.

8. The Chinese medicinal compound granules according to claim 1 or 6, characterized in that: The step of drying the thick paste to obtain a dry paste is performed at a temperature of 40 to 100° C. and under reduced pressure.

9. The Chinese medicinal compound granules according to claim 1 or 6, characterized in that: The method further comprises the step of drying the semi-finished granules after granulation so that the moisture content of the granules does not exceed 13% by weight.

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