Spleen activating, detoxifying, collateral dredging and dampness clearing treatment formula for chronic myelosuppression caused by MTX

Abstract

The invention relates to a spleen-activating, detoxifying, collateral-dredging and dampness-clearing treatment formula for chronic myelosuppression caused by MTX. Animal experiments show that blood analysis shows that liver and kidney functions are normal after YJTQ particles are administered; after YJTQ particle treatment, the number of leukocytes and the hemoglobin level are improved, and meanwhile, the platelet (PLT) level has no statistical difference in each group; femoral bone marrow smear experiment results show that compared with a blank group, hematopoietic cells are increased and bone marrow images are recovered after YJTQ particle treatment. A bone marrow cell apoptosis flow cytometry result shows that the apoptosis rate is reduced after the YJTQ prescription is used for treatment. After the YJTQ particles are used for treatment, bone marrow injury caused by MTX can be obviously improved, and the medicine effect of the YJTQ particles is basically equivalent to that of erythropoietin (EPO) in a positive control group. Therefore, the YJTQ granules for activating spleen, detoxifying, dredging collaterals and clearing damp have an obvious protective effect on rats with chronic myelosuppression caused by methotrexate, and are high in safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a spleen-regulating, detoxifying, collateral-draining and dampness-removing therapeutic formula for MTX-induced chronic bone marrow suppression, and a preparation method and application thereof. Background Art

[0002] Methotrexate (MTX) is the first choice DMARDs drug for the treatment of rheumatoid arthritis (RA). Although it can effectively relieve symptoms, it also has some disadvantages:

[0003] First of all, methotrexate may cause gastrointestinal symptoms such as nausea, vomiting, diarrhea, etc. These uncomfortable symptoms may affect the patient's daily life and nutrient absorption, and even lead to malnutrition and electrolyte imbalance.

[0004] Secondly, methotrexate has a certain toxic effect on the blood system, which may cause leukopenia, anemia and other problems, and then symptoms such as fatigue and dizziness. If it is not treated in time for a long time, it may cause infection or bleeding tendency, posing a threat to the patient's health.

[0005] In addition, methotrexate may also cause damage to liver function, leading to symptoms such as liver cell damage, jaundice, and increased liver enzymes. Long-term oral administration may also lead to liver cell necrosis, fatty fibrosis, and even serious consequences such as cirrhosis.

[0006] In addition, methotrexate can also cause some skin lesions, such as hair loss, rash, itching, etc. For patients with psoriasis, methotrexate may inhibit keratinocyte DNA synthesis and promote hyperkeratosis, thereby aggravating the symptoms of skin lesions, manifested as erythema, increased scaling, increased itching, etc. In severe cases, pustules or arthritis may occur.

[0007] In addition, methotrexate can enhance the phagocytic ability of macrophages, but it can also inhibit the immune response and reduce the body's resistance, which can easily induce colonization or infection by bacteria, fungi or other microorganisms, resulting in symptoms such as fever, local redness, swelling, heat and pain.

[0008] In addition to the above-mentioned medication drawbacks, bone marrow suppression is a common adverse reaction when using MTX for treatment. The use of MTX can lead to chronic bone marrow suppression. Clinical bone marrow suppression manifests as leukopenia, and in severe cases, bone marrow suppression, pancytopenia, anemia, etc. occur. Although the risk can be reduced through reasonable treatment plans (such as taking folic acid weekly) and monitoring (such as regular peripheral blood testing), finding drugs with good efficacy and few toxic side effects to treat MTX-induced bone marrow suppression is an important scientific issue. Summary of the invention

[0009] In order to solve the above problems, the present application proposes a spleen-regulating, detoxifying, collateral-draining and dampness-removing therapeutic formula for MTX-induced chronic bone marrow suppression, as well as a preparation method and application thereof.

[0010] In one aspect, the present application provides a spleen-activating, detoxifying, collateral-activating and dampness-removing therapeutic formula for MTX-induced chronic bone marrow suppression, wherein the spleen-activating, detoxifying, collateral-activating and dampness-removing therapeutic formula comprises the following components in the following proportions:

[0011] 15g of Atractylodes lancea, 15g of honeysuckle, 1 centipede, 15g of Polygonum cuspidatum, 9g of stir-fried white peony root, 10g of Angelica sinensis, 12g of Cuscuta australis, 18g of stir-fried Coix seeds, and 6g of roasted Licorice root.

[0012] As an optional embodiment of the present application, optionally, a spleen-activating, detoxifying, dredging and dampness-removing therapeutic formula for MTX-induced chronic bone marrow suppression is characterized in that the spleen-activating, detoxifying, dredging and dampness-removing therapeutic formula comprises the following components in the following proportions:

[0013] Atractylodes 10-20g, honeysuckle 10-20g, centipedes 1-3, Polygonum cuspidatum 12-18g, stir-fried white peony root 5-13g, angelica 7-12g, Cuscuta australis 10-16g, stir-fried coix seed 11-23g, and roasted licorice root 3-8g.

[0014] On the other hand, the present application provides a method for preparing a spleen-activating, detoxifying, collateral-draining and dampness-removing therapeutic formula for MTX-induced chronic bone marrow suppression, comprising the following steps:

[0015] Weigh the following raw materials: Atractylodes lancea, honeysuckle, centipede, Polygonum cuspidatum, stir-fried white peony root, angelica, dodder seed, stir-fried coix seed and roasted licorice root;

[0016] The raw materials are divided into multiple batches, washed, dried, selected, sliced ​​and crushed into formulated particles with a particle size not exceeding 50-80 mesh;

[0017] The formula granules are immersed in a pre-prepared solvent, and a first content value of the effective components of the traditional Chinese medicine is detected by a gas chromatograph and the immersion and stirring are started;

[0018] Real-time detection of a second content value of the effective ingredient of the traditional Chinese medicine until the second content value does not change, and the soaking is finished;

[0019] filtering the soaking liquid, and centrifuging the soaking liquid to obtain a formula solution;

[0020] Evaporating and concentrating the formula solution to obtain a formula concentrate;

[0021] The formula concentrated liquid is dried into solid particles by using spray drying technology, and then put into a spray drying granulator to make uniform Chinese medicine particles to obtain Yunpi Jiedu Tongluo Qushi granules;

[0022] Package;

[0023] Quality inspection;

[0024] Storage in warehouse.

[0025] As an optional embodiment of the present application, optionally, the solvent is one or more of the following solvents:

[0026] Methanol, ethanol, acetone, ether, chloroform, ethyl acetate or ethylene dichloride.

[0027] As an optional embodiment of the present application, optionally, the conditions for immersion stirring are:

[0028] Stir at 20-28°C for 1-5 hours.

[0029] On the other hand, the present application also proposes the use of a spleen-regulating, detoxifying, collateral-clearing and dampness-removing therapeutic formula for chronic bone marrow suppression induced by MTX, which is used to treat or alleviate bone marrow suppression caused by MTX.

[0030] Technical effects of the present invention:

[0031] The present application provides a granule formula for treating chronic bone marrow suppression, according to the Chinese medicine pieces of Atractylodes 15g, Honeysuckle 15g, Centipede 1, Polygonum cuspidatum 15g, Stir-fried White Peony 9g, Angelica 10g, Cuscuta 12g, Stir-fried Coix Seed 18g, and Roasted Licorice 6g. The granules are easy to carry, have long-lasting efficacy, stable quality and accurate dosage. Animal experiments have shown that there is no statistical difference in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (CREA) levels between the YJTQ group and the blank group, proving that liver and kidney function is normal after administration of YJTQ granules). Compared with the blank group, MTX significantly reduced the levels of red blood cells (RBC), white blood cells (WBC) and hemoglobin (HGB) in peripheral blood, while YJTQ granules improved the number of red and white blood cells and hemoglobin levels after treatment, and there was no statistical difference in the level of platelets (PLT) among the groups; the results of femoral bone marrow smear experiments showed that compared with the blank group, the hematopoietic structure of rat bone marrow was disordered and the number of nucleated cells was significantly reduced after MTX modeling, while the number of hematopoietic cells increased and the bone marrow image was restored after YJTQ granules treatment. The erythroid and granulocyte colony formation experiment of bone marrow cells showed that MTX caused a significant decrease in the number of erythroid and granulocyte colonies, while the number of erythroid and granulocyte colonies increased after YJTQ granules treatment, indicating that YJTQ granules can improve the erythroid and granulocyte colony formation ability of hematopoietic cells to a certain extent. The results of bone marrow cell apoptosis flow cytometry showed that compared with the blank group, the apoptosis rate in the MTX group increased, while the apoptosis rate decreased after YJTQ treatment. In conclusion, MTX modeling will cause significant bone marrow damage, while treatment with YJTQ granules can significantly improve the bone marrow damage caused by MTX, and its efficacy is basically equivalent to that of erythropoietin (EPO) in the positive control group.

[0032] Therefore, Yunpijiedutongluoqushi granules (YJTQ) have obvious protective effects on rats with chronic myelosuppression induced by methotrexate and are highly safe.

[0033] Further features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments, features, and aspects of the disclosure and, together with the description, serve to explain the principles of the disclosure.

[0035] Figure 1 It is a schematic diagram showing the working principle of the dissolution device of the present invention;

[0036] FIG. 2 shows the comparative experimental data of peripheral blood routine of the present invention;

[0037] Figure 3 Shown is an experimental comparison diagram of the bone marrow smear and bone marrow cell colony experiment of the present invention;

[0038] FIG. 4 shows the experimental comparison data of bone marrow cell apoptosis of the present invention ( Figures 4A-4H , respectively for the apoptosis data of each group). DETAILED DESCRIPTION

[0039] Various exemplary embodiments, features and aspects of the present disclosure will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0040] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0041] In addition, in order to better illustrate the present disclosure, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present disclosure can also be implemented without certain specific details. In some examples, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present disclosure.

[0042] In this embodiment, the equipment and process flow used in the preparation can be understood and implemented in combination with existing pharmaceutical processes, and only need to be implemented according to the preparation principle of the present invention.

[0043] Aiming at the disadvantages of high cost and long period in the treatment of mild and moderate chronic bone marrow suppression caused by MTX, the present invention provides a granule formula for treating chronic bone marrow suppression.

[0044] In one aspect, the present application provides a spleen-activating, detoxifying, collateral-activating and dampness-removing therapeutic formula for MTX-induced chronic bone marrow suppression, wherein the spleen-activating, detoxifying, collateral-activating and dampness-removing therapeutic formula comprises the following components in the following proportions:

[0045] 15g of Atractylodes lancea, 15g of honeysuckle, 1 centipede, 15g of Polygonum cuspidatum, 9g of stir-fried white peony root, 10g of Angelica sinensis, 12g of Cuscuta australis, 18g of stir-fried Coix seeds, and 6g of roasted Licorice root.

[0046] According to the above formula, the corresponding Chinese medicine granules, namely Yunpi Jiedu Tongluo Qushi Granules (abbreviated as YJTQ), are prepared, which will be used to alleviate or treat chronic bone marrow suppression caused by MTX.

[0047] The granules are easy to carry, have long-lasting efficacy, stable quality and accurate dosage, and are designed to relieve or treat chronic bone suppression caused by methylamine.

[0048] Professor Wen Chengping established the Yunpi Jiedu Tongluo Qushi Granules based on long-term clinical experience, which uses Atractylodes macrocephala to dry out dampness and strengthen the spleen, honeysuckle, Polygonum cuspidatum, and Coix seed to detoxify and remove dampness, centipede to unblock the meridians and relieve pain, stir-fried white peony root, Chinese angelica, and Cuscuta australis to strengthen the body and nourish the vital energy, and all the medicines are harmonized with licorice to treat both the symptoms and the root cause.

[0049] In relieving or treating bone marrow suppression, Atractylodes lancea mainly strengthens the spleen to replenish the acquired foundation, strengthens stomach qi, and thus helps to replenish qi and blood and improve the patient's blood cell level. Modern pharmacological research shows that the chemical components in Atractylodes lancea have the effects of inhibiting gastric acid secretion, promoting gastrointestinal motility and gastric emptying, and antibacterial and anti-inflammatory effects. These effects may indirectly help alleviate the adverse reactions caused by bone marrow suppression.

[0050] Honeysuckle has many functions, such as clearing away heat and detoxifying, cooling blood and stopping diarrhea, antibacterial and anti-inflammatory, and strengthening immune function. Its antibacterial effect can inhibit a variety of pathogens, including some that may cause bone marrow suppression. At the same time, honeysuckle can also enhance the phagocytic function of immune cells, which helps to improve the patient's overall immunity, thereby alleviating bone marrow suppression.

[0051] Although centipedes are slightly poisonous, they are used in traditional Chinese medicine to treat a variety of diseases, including osteomyelitis. Their anti-tumor effects, antispasmodic effects, and immunity-enhancing functions may have a positive effect on alleviating bone marrow suppression. However, centipedes should be used with caution and under the guidance of a traditional Chinese medicine practitioner.

[0052] Polygonum cuspidatum has multiple functions such as promoting blood circulation and removing blood stasis, cooling blood and detoxifying, and antibacterial and antiviral. Its blood circulation and blood stasis removal effect helps improve bone marrow microcirculation and promote the recovery of bone marrow hematopoietic function. At the same time, the antibacterial and antiviral effects of Polygonum cuspidatum can inhibit some bacteria that may cause bone marrow suppression, thereby alleviating the condition.

[0053] Fried white peony root has multiple effects such as analgesia, liver protection, inhibition of gastrointestinal spasm, and immune enhancement. These effects may help relieve the pain and discomfort caused by bone marrow suppression and improve the patient's overall immunity.

[0054] Angelica sinensis is a holy medicine for blood tonic, with multiple functions such as blood tonic and blood circulation, regulating menstruation and relieving pain, and laxative. In the treatment of bone marrow suppression, Angelica sinensis can improve the patient's blood cell level through blood tonic effect. At the same time, its blood circulation effect also helps to improve bone marrow microcirculation and promote the recovery of bone marrow hematopoietic function.

[0055] In traditional Chinese medicine theory, Chinese medicinal materials such as Cuscuta chinensis, stir-fried Coix seeds and roasted Licorice root are believed to have certain effects on alleviating or treating bone marrow suppression.

[0056] Cuscuta seeds are sweet and warm in nature, and belong to the liver, kidney, and spleen meridians. They have the effects of nourishing the liver and kidney, consolidating essence and reducing urine, improving eyesight and stopping diarrhea. Modern pharmacological studies have shown that Cuscuta seeds contain a variety of active ingredients, such as cuscuta glycosides and flavonoid compounds, which have significant effects on promoting bone marrow hematopoietic function and enhancing the body's immunity. In terms of alleviating or treating bone marrow suppression, Cuscuta seeds can promote the proliferation and differentiation of bone marrow cells by nourishing the liver and kidneys, replenishing essence and nourishing marrow, thereby improving the symptoms of bone marrow suppression.

[0057] Fried coix seeds are sweet, mild and slightly cold in nature. They belong to the spleen, stomach and lung meridians and have the effects of promoting diuresis and removing dampness, strengthening the spleen and stopping diarrhea, removing numbness and pus, and detoxifying and dispersing nodules. The coix seed oil and coix ester in coix seeds have multiple pharmacological effects such as anti-tumor and immune enhancement. In terms of relieving or treating bone marrow suppression, fried coix seeds can improve the body's nutritional status and enhance the body's resistance by strengthening the spleen and removing dampness, thereby helping to relieve the symptoms of bone marrow suppression.

[0058] Licorice root is sweet and mild in nature, and it enters the heart, lung, spleen, and stomach meridians. It has the effects of tonifying the spleen and stomach, replenishing qi and restoring the pulse, clearing away heat and detoxifying, and harmonizing various medicines. The glycyrrhizin, glycyrrhetinic acid and other ingredients in licorice root have multiple pharmacological effects such as anti-inflammatory, antiviral, and anti-tumor. In terms of alleviating or treating bone marrow suppression, licorice root can enhance the body's immune function and promote the regeneration and repair of bone marrow cells by tonifying the spleen and replenishing qi, thereby helping to improve the symptoms of bone marrow suppression.

[0059] In summary, Chinese medicinal materials such as Cuscuta chinensis, stir-fried coix seeds and roasted licorice have the potential to alleviate or treat bone marrow suppression in traditional Chinese medicine theory. However, when using them, they should be treated according to the patient's specific condition and constitution, and used reasonably under the guidance of a doctor.

[0060] Therefore, the Yunpi Jiedu Tongluo Qushi Granules (abbreviated as YJTQ) prepared by the above formula can be used to alleviate or treat chronic bone marrow suppression caused by MTX.

[0061] The formula of the present invention can be made into granules or ointments according to the conventional method of Chinese medicine. The present invention preferably prepares it into granules. The following describes a method for preparing a formula for treating chronic bone marrow suppression caused by MTX, which includes the following steps:

[0062] Weigh the raw materials according to the amount (which can be the amount of the above-mentioned components): Atractylodes lancea, honeysuckle, centipede, Polygonum cuspidatum, stir-fried white peony root, angelica, Cuscuta australis, stir-fried coix seed and roasted licorice root;

[0063] The raw materials are divided into multiple batches, washed, dried, selected, sliced ​​and crushed into formulated particles with a particle size not exceeding 50-80 mesh;

[0064] The formula granules are immersed in a pre-prepared solvent, and a first content value of the effective components of the traditional Chinese medicine is detected by a gas chromatograph and the immersion and stirring are started;

[0065] Real-time detection of a second content value of the effective ingredient of the traditional Chinese medicine until the second content value does not change, and the soaking is finished;

[0066] filtering the soaking liquid, and centrifuging the soaking liquid to obtain a formula solution;

[0067] Evaporating and concentrating the formula solution to obtain a formula concentrate;

[0068] The formula concentrated liquid is dried into solid particles by using spray drying technology, and then put into a spray drying granulator to make uniform Chinese medicine particles to obtain Yunpi Jiedu Tongluo Qushi granules;

[0069] Package;

[0070] Quality inspection;

[0071] Storage in warehouse.

[0072] The preparation process of Chinese medicine granules is a complex and delicate process that aims to transform traditional Chinese medicine into granular dosage forms that are easy to carry and take. The following are the main steps of the Chinese medicine granule preparation process:

[0073] 1. Preliminary preparation

[0074] 1. Raw material selection and processing:

[0075] Select Chinese medicinal materials that meet the standards and carry out pre-processing such as cleaning, slicing or crushing to ensure the purity and suitability of the medicinal materials.

[0076] 2. Extraction

[0077] 1.Solvent selection:

[0078] Select a suitable solvent, such as water, ethanol, etc., according to the characteristics of the medicinal materials and extraction requirements.

[0079] 2. Extraction operation:

[0080] The pretreated Chinese medicinal materials are soaked in a solvent, and the effective ingredients are fully dissolved in the solvent through heating, stirring, etc.

[0081] 3. Extraction liquid treatment:

[0082] After the extraction is completed, the extract is filtered or centrifuged to remove impurities and drug residues.

[0083] 3. Concentration

[0084] 1. Concentration process:

[0085] The solvent in the extract is evaporated to increase the concentration of the active ingredient to form a concentrated solution or extract.

[0086] 2.Quality Control:

[0087] During the concentration process, parameters such as temperature and pressure need to be controlled to ensure the stability of the active ingredients and the concentration effect.

[0088] 4. Drying

[0089] 1. Drying method:

[0090] The concentrated liquid or extract is dried into solid powder or granules by using drying technology such as drying and spray drying.

[0091] 2. Drying conditions:

[0092] According to the characteristics of medicinal materials and the performance of drying equipment, select the appropriate drying temperature and time to ensure the drying effect and product quality.

[0093] 5. Granulation

[0094] 1. Granulation equipment:

[0095] Use swing granulator, spray drying granulator and other equipment to make uniform Chinese medicine granules from the dried solid powder or granules.

[0096] 2. Granulation process:

[0097] The dried extract is mixed evenly with an appropriate amount of auxiliary materials (such as powdered sugar, dextrin, etc.), and then made into granules through a granulating device.

[0098] 6. Packaging and quality inspection

[0099] 1. Packaging:

[0100] The prepared Chinese medicine granules are packaged according to certain specifications and dosages for easy storage and transportation.

[0101] 2. Quality inspection:

[0102] Carry out quality inspection on packaged Chinese herbal medicine granules, including inspection of appearance, particle size, content and other indicators, to ensure that product quality meets the standards.

[0103] 7. Finished product warehousing and storage

[0104] 1. Finished product storage:

[0105] The Chinese herbal medicine granules that have passed the quality inspection shall be stored in the warehouse and properly labeled and recorded.

[0106] 2. Storage conditions:

[0107] According to product characteristics and storage requirements, set appropriate storage temperature and humidity conditions to ensure product stability and safety.

[0108] The preparation of the granules can refer to the above steps, but in the preparation process of the present invention, it is necessary to make certain limitations on the preparation process of the Yunpi Jiedu Tongluo Qushi Granules (abbreviated as YJTQ) in order to better adapt to the preparation and application of the granules of this formula, make the preparation process more controllable and process quantitatively controlled, avoid blindly waiting for the process preparation time, and make the preparation process more node-based.

[0109] Among them, during the heating, stirring and soaking process:

[0110] The formula granules are immersed in a pre-prepared solvent, and a first content value of the effective components of the traditional Chinese medicine is detected by a gas chromatograph and the immersion and stirring are started;

[0111] The second content value of the effective ingredient of the traditional Chinese medicine is detected in real time until the second content value does not change, and the soaking is completed.

[0112] This part controls the dissolution efficiency of Chinese medicine components and medicinal properties when they are dissolved in the solvent, and feedback controls the dissolution time by detecting the content of the effective ingredients of the Chinese medicine in the solvent.

[0113] Because Chinese medicinal materials will dissolve their active ingredients in the solvent, the content of the active ingredients in the solvent can be detected and monitored in real time to determine whether the active ingredients of the Chinese medicinal materials are fully dissolved in the solvent.

[0114] Therefore, during the initial soaking, sampling can be performed through the sampling port 8 of the dissolution device, and the composition and content can be detected and analyzed through a gas chromatograph to obtain the first content value of the effective ingredient of the traditional Chinese medicine before stirring and record it on the terminal.

[0115] After stirring begins, the medicinal materials, Chinese medicines, organic ingredients, etc., dissolve in the solvent. After a period of time, the dissolution is complete or almost complete. At this time, the Chinese medicine ingredients contained in the solvent reach a certain content value, and after multiple subsequent measurements, the content value will no longer change or change slightly (negligible), and the soaking is considered to be over. The technician can test it every 15-30 minutes, firstly to allow the ingredients to be fully dissolved, and secondly to record the corresponding experimental data, which is convenient for the preparation of Chinese medicine and dosage ratio.

[0116] Start filtering the soaking liquid and enter the concentration process.

[0117] For composition and content analysis of gas chromatograph, just refer to its functions.

[0118] The solvent of the present invention is one or more of the following solvents:

[0119] Methanol, ethanol, acetone, ether, chloroform, ethyl acetate or dichloroethane. It is preferred to use a combination of two or more solvents. The combination can be made by the user.

[0120] As an optional embodiment of the present application, optionally, the conditions for immersion stirring are:

[0121] Stir at 20-28°C for 1-5 hours.

[0122] Combined with Figure 1 As shown, the dissolving equipment has the functions of sealing (to avoid solvent volatilization), heating stirring and temperature sensing, which is convenient for better coordination in the preparation of Chinese medicine granules.

[0123] The dissolving device comprises: a dissolving barrel 5, the top of which can be matched with a sealing cover 2 through a sealing ring 3, a heating module 6 (such as a resistance heating wire or other electric heating module) is arranged at the bottom of the barrel for heating the solvent in the barrel, and a temperature sensor 7 is arranged on the inner wall of the barrel for sensing the heating temperature and feeding back to a controller 9, and the controller 9 adjusts the temperature value of the heating module 6 according to the feedback temperature. A stepper motor 1 is arranged above the barrel, and its driving shaft cooperates with and passes through the sealing cover and a stirring fork 4 is arranged at the lower end thereof for stirring.

[0124] The stepper motor, heating module and temperature sensor are all electrically connected to the controller. The controller has its own control chip MCU (single-chip microcomputer or PLC controller, etc.), which can control the heating module to heat and the motor to drive the stirring according to the preset program. The MCU will collect the temperature value fed back by the temperature sensor in real time and determine whether the current stirring temperature reaches the preset value range, and control the heating module to adjust the temperature.

[0125] Therefore, the dissolving device of the present invention can facilitate the dissolution of Chinese medicinal materials. After the dissolution is completed, the cover can be opened, or the liquid can be directly discharged from the sampling port, which is more convenient. The sampling port has a matching seal, which can be sealed after sampling. When taking liquid, the problem of liquid spilling and volatilization can also be avoided.

[0126] The following will combine the action model to verify the experimental mechanism of alleviating or treating chronic bone marrow suppression caused by MTX.

[0127] 1. First, construct an animal model. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) and approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University (approval number: 20210809-10). 20 SPF female Wi star rats (220 ± 20 g) were obtained from SLAC Animal Co., Ltd., Shanghai, China (production license number: SCXK (Zhejiang, China) 2017-0005). The ambient temperature was 22 ± 1 °C, the humidity was 50% to 60%, and the light / dark cycle was 12 h.

[0128] After one week of adaptive feeding, the rats were randomly divided into 4 groups: blank group (Control group, normal group), MTX group (fed with methotrexate), YJTQ group (fed with Yunpi Jiedu Tongluo Qushi granules) and EPO positive control group (fed with EPO solution, erythropoietin (EPO) is also known as erythrocyte stimulating factor, erythropoietin), 5 rats in each group, gavage for 4 weeks. On the 35th day, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital 40 mg / kg and euthanized with 100% CO2 for 12 minutes. Peripheral blood, femur, and lower limb bone marrow cells were collected for experiments.

[0129] MTX (oral administration of methotrexate): According to the clinical guidelines for RA, the weekly adult dose of 30 mg / 70 kg is converted into a rat dose of 2.7 mg / kg. The oral administration volume is 1 ml per 200 g rat. The administration is oral for 4 weeks, twice a week.

[0130] YJTQ (granule gavage): Based on the clinical dosage and rat dosage converted by a coefficient of 6.3, the concentration of YJTQ granules for rat gavage is 0.2178 g / ml, and the gavage volume is 2 ml per 200 g rat. The gavage is administered once a day for four weeks.

[0131] EPO (EPO solution gavage): The adult dose is converted into a rat dose of 200U / kg. The gavage volume is 2ml per 200g rat. The gavage is for 4 weeks, twice a week.

[0132] 2. Experiment:

[0133] 2.1 Bone marrow hematopoietic function, comparison of expression levels of components in the blood of each mouse

[0134] As shown in Figure 2, animal experiments show that Yunpai Jiedu Tongluo Qushi Granules (YJTQ) has a significant protective effect on rats with chronic bone inhibition caused by methylamine and is highly safe.

[0135] Combined with Figures 2A-2D As shown, compared with the MTX modeling group, there was no statistical difference in the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CREA) levels of each group, and the liver and kidney functions were normal after YITQ granules were administered, indicating its high safety.

[0136] Combined with Figure 2E-2G As shown in the results, compared with the blank group, MTX significantly reduced the levels of red blood cells (RBC), white blood cells (WBC) and hemoglobin (HGB) in peripheral blood, while YJTQ granules significantly improved the number of red and white blood cells and hemoglobin levels in peripheral blood after treatment. In addition, there was no statistical difference in platelet (PLT) levels among the groups.

[0137] Comparison of blood biochemistry and routine blood tests in different groups of Wi star rats. Blood was collected from the heart on the 35th day to obtain whole blood for routine blood tests and serum for blood biochemistry tests. Alanine aminotransferase ALT (A), alanine aminotransferase AST (B), urea nitrogen BUN (C), creatinine (CREA), white blood cell count WBC (E), red blood cell count RBC (F), hemoglobin HGB concentration (G) and platelet count PLT (H). The data given are mean ± standard error and analyzed by one-way ANOVA. "ns" represents P>0.05, "*" represents P<0.05. "**" represents P<0.01, "***" represents P<0.001, and "****" represents P<0.0001. There are five samples in each group.

[0138] 2.2 Bone marrow smear analysis

[0139] Combined with Figure 3 As shown, the right femur of each group of rats was selected for bone marrow smear, the two ends of the femur were cut open and the bone marrow was picked out, diluted 2 times with normal saline and then dropped onto a glass slide, the slide was pushed quickly, Swiss Guisam staining was performed, and the staining was observed using a microscope X400.

[0140] Compared with the blank group, the bone marrow hematopoietic structure of rats after MTX modeling was disordered and the number of nucleated cells was significantly reduced, while the hematopoietic cells increased and the bone marrow image was restored after YJTQ granule treatment.

[0141] 2.3 Colony formation assay

[0142] Combined with Figure 3 The erythroid and granulocyte colony formation experiment of bone marrow cells showed that MTX caused a significant decrease in the number of erythroid and granulocyte colonies, while the erythroid and granulocyte colonies increased after YJTQ granule treatment, indicating that YJTQ granules can improve the erythroid and granulocyte colony formation ability of hematopoietic cells to a certain extent.

[0143] 2.4 Apoptosis rate

[0144] Combined with Figure 4, the results of bone marrow cell apoptosis flow cytometry showed that the apoptosis rate of the MTX group was high, while the apoptosis rate decreased after treatment with the YJTQ prescription. Combined with Figure 4, according to the Black tube ( Figure 4A ), PI single dye tube ( Figure 4B ) and FI TC single staining tube ( Figure 4C ) to determine the gate of apoptosis experiment. Figure 4C ontro l group Figure 4D ), the proportion of early withering Q1-LR was 0.22%, and the proportion of late withering Q1-UR was 0.19%. Combined with the attached figure 4 MTX group ( Figure 4E ), the proportion of early withering Q1-LR was 3.16%, and the proportion of late withering Q1-UR was 0.77%. Combined with Figure 4YJTQ group ( Figure 4F ), the proportion of early withering Q1-LR was 0.45%, and the proportion of late withering Q1-UR was 0.34%. Figure 4E PO group ( Figure 4G ), the proportion of early apoptosis Q1-LR was 1.17%, and the proportion of late apoptosis Q1-UR was 0.74%. Combined with the apoptosis rate of bone marrow cells in each group ( Figure 4H ), compared with the Control group, the apoptosis rate of the MTX group was the highest, and after YJTQ granule treatment or EPO treatment, the apoptosis rate of bone marrow cells was significantly reduced and approached the apoptosis rate level of the normal group. Therefore, this formula has good efficacy and small toxic side effects, and has a positive effect on the treatment of bone marrow suppression caused by MTX.

[0145] Comparison of cell apoptosis rates in different groups of Wi star rats. After cardiac blood sampling on day 35, bone marrow cell samples were collected for cell apoptosis rate detection using flow cytometry. The data presented are mean ± standard error and analyzed by one-way ANOVA. "***" represents P < 0.001, five samples per group.

[0146] Therefore, animal experiments show that: Yunpi Jiedu Tongluo Qushi Granule (YJTQ) has a significant protective effect on rats with chronic bone marrow suppression caused by methotrexate, and it is highly safe. There was no statistical difference in the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (CREA) between the YJTQ group and the blank group, proving that liver and kidney function was normal after administration of YJTQ granules). Compared with the blank group, MTX significantly reduced the levels of red blood cells (RBC), white blood cells (WBC) and hemoglobin (HGB) in peripheral blood, while YJTQ granules improved the number of red and white blood cells and hemoglobin levels after treatment, and there was no statistical difference in platelet (PLT) levels among the groups; the results of the femoral bone marrow smear experiment showed that: compared with the blank group, the bone marrow hematopoietic structure of rats after MTX modeling was disordered and the number of nucleated cells was significantly reduced, while the hematopoietic cells increased and the bone marrow image was restored after YJTQ granules were treated. The results of bone marrow cell apoptosis flow cytometry showed that compared with the blank group, the apoptosis rate in the MTX group increased, while the apoptosis rate decreased after YJTQ treatment. The bone marrow cell erythroid and granulocyte colony formation experiment showed that MTX caused a significant decrease in the number of erythroid and granulocyte colonies, while the number of erythroid and granulocyte colonies increased after YJTQ granules treatment, indicating that YJTQ granules can improve the erythroid and granulocyte colony formation ability of hematopoietic cells to a certain extent.

[0147] In conclusion, MTX modeling will cause significant bone marrow damage, while treatment with YJTQ granules can significantly improve the bone marrow damage caused by MTX, and its efficacy is basically equivalent to that of erythropoietin (EPO) in the positive control group.

[0148] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A formula for treating chronic bone marrow suppression caused by MTX, characterized in that: The spleen-regulating, detoxifying, collateral-draining and dampness-removing therapeutic formula comprises the following components in the following proportions: Atractylodes 10-20g, honeysuckle 10-20g, centipedes 1-3, Polygonum cuspidatum 12-18g, stir-fried white peony root 5-13g, angelica 7-12g, Cuscuta australis 10-16g, stir-fried coix seed 11-23g, and roasted licorice root 3-8g.

2. A formula for treating chronic bone marrow suppression caused by MTX, characterized in that: The spleen-regulating, detoxifying, collateral-draining and dampness-removing therapeutic formula comprises the following components in the following proportions: 15g of Atractylodes lancea, 15g of honeysuckle, 1 centipede, 15g of Polygonum cuspidatum, 9g of stir-fried white peony root, 10g of Angelica sinensis, 12g of Cuscuta australis, 18g of stir-fried Coix seeds, and 6g of roasted Licorice root.

3. A method for preparing a spleen-regulating, detoxifying, collateral-draining and dampness-removing therapeutic formula for treating chronic bone marrow suppression induced by MTX according to any one of claims 1 or 2, characterized in that: The steps include: Weigh the following raw materials: Atractylodes lancea, honeysuckle, centipede, Polygonum cuspidatum, stir-fried white peony root, angelica, Cuscuta australis, stir-fried coix seed and roasted licorice root; The raw materials are divided into multiple batches, washed, dried, selected, sliced ​​and crushed into formulated particles with a particle size not exceeding 50-80 mesh; The formula granules are immersed in a pre-prepared solvent, and a first content value of the effective components of the traditional Chinese medicine is detected by a gas chromatograph and the immersion and stirring are started; Real-time detection of a second content value of the effective ingredient of the traditional Chinese medicine until the second content value does not change, and the soaking is finished; filtering the soaking liquid, and centrifuging the soaking liquid to obtain a formula solution; Evaporating and concentrating the formula solution to obtain a formula concentrate; The formula concentrated liquid is dried into solid particles by using spray drying technology, and then put into a spray drying granulator to make uniform Chinese medicine particles to obtain Yunpi Jiedu Tongluo Qushi granules; Package; Quality inspection; Storage in warehouse.

4. The preparation method according to claim 3, characterized in that: The solvent is one or more of the following solvents: Methanol, ethanol, acetone, ether, chloroform, ethyl acetate or ethylene dichloride.

5. Use of the spleen-activating, detoxifying, collateral-activating and dampness-removing therapeutic formula for MTX-induced chronic bone marrow suppression as described in any one of claims 1 or 2 for treating or alleviating bone marrow suppression caused by MTX.

6. The preparation method according to claim 3, characterized in that: The conditions of the immersion stirring are: Stir at 20-28°C for 1-5 hours.

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