Application of pigeon pea leaves in rats with chronic myelosuppression caused by MTX

Abstract

The invention relates to a Chinese herbal medicine pigeon pea leaf. Research shows that the pigeon pea leaf can improve chronic myelosuppression caused by methotrexate (MTX). Results of animal experiments show that blood analysis results show that liver and kidney functions are normal after administration of pigeon pea leaves; after treatment, the number of red blood cells and white blood cells and the level of hemoglobin are remarkably improved, and after treatment of a pigeon pea leaf high-dose group (0.81 g / ml), the level of platelets (PLT) is remarkably increased. In addition, a thighbone marrow smear experiment result shows that compared with a blank group, hematopoietic cells are increased after the pigeon pea leaves are used for treatment. In general, the pigeon pea leaves can significantly improve bone marrow injury caused by MTX, and the curative effect of the pigeon pea leaves is superior to that of folic acid (FA) in a positive control group. Therefore, the pigeon pea leaf has an obvious protective effect on chronic myelosuppression caused by methotrexate, and shows relatively high safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of pigeon pea leaves in rats with chronic bone marrow suppression induced by MTX. Background Art

[0002] Methotrexate (MTX) is the first choice disease-modifying antirheumatic drug (DMARDs) for the treatment of rheumatoid arthritis (RA). Although it can effectively relieve symptoms, it also has some side effects and potential risks.

[0003] First, methotrexate may cause gastrointestinal discomfort, including nausea, vomiting and diarrhea. These symptoms not only affect the patient's daily life, but may also interfere with nutrient absorption, leading to malnutrition and electrolyte imbalance.

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

[0005] In addition, methotrexate may damage liver function, leading to liver cell damage, jaundice, increased liver enzymes and other problems. Long-term use may cause liver cell necrosis, fatty liver and fibrosis, and even develop into cirrhosis in severe cases.

[0006] Methotrexate may also cause skin lesions, including hair loss, rash, and itching. In patients with psoriasis, methotrexate may inhibit keratinocyte DNA synthesis and promote hyperkeratosis, thereby aggravating skin lesions, manifested as erythema, increased scaling, and increased itching. In severe cases, pustules or arthritis may occur.

[0007] In addition, methotrexate enhances the phagocytic ability of macrophages, but at the same time inhibits the immune response and reduces the body's resistance. This makes patients more susceptible to infection by bacteria, fungi or other microorganisms, manifested as fever, local redness, swelling and pain.

[0008] In addition to the above side effects, bone marrow suppression is a common adverse reaction when using MTX for treatment, manifested as leukopenia, pancytopenia and anemia. Although its risk can be reduced through reasonable treatment plans (such as taking folic acid weekly) and regular monitoring (such as peripheral blood testing), it is an important scientific issue to find drugs with good efficacy and few toxic side effects to treat MTX-induced bone marrow suppression. Summary of the invention

[0009] In order to solve the above problems, the present application proposes a preparation method and application of pigeon pea leaves for treating chronic bone marrow suppression caused by MTX.

[0010] In one aspect, the present application provides a pigeon pea leaf for treating chronic bone marrow suppression caused by MTX, wherein the main components of the pigeon pea leaf are as follows:

[0011] Pinostrobin, cajanin stilbene acid, pratensein, longistylin A, apigenin, longistylin C, genistein, cajanolactone A, ononin, cajanol, prunetin, cajanin, and luteolin.

[0012] On the other hand, the present application provides a method for preparing the above-mentioned pigeon pea leaves, comprising the following steps:

[0013] Weigh the raw materials according to the amount: 60-90g of pigeon pea leaves.

[0014] The raw materials are divided into multiple batches, washed, dried, selected, sliced ​​and crushed into particles with a particle size not exceeding 50-80 mesh;

[0015] The particles 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;

[0016] 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;

[0017] filtering the soaking liquid, and centrifuging the soaking liquid to obtain a formula solution;

[0018] Evaporating and concentrating the formula solution to obtain a formula concentrate;

[0019] 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 spleen-activating, detoxifying, collateral-draining and dampness-removing pigeon bean particles;

[0020] Package;

[0021] Quality inspection;

[0022] Storage in warehouse.

[0023] As an optional embodiment of the present application, optionally, the solvent is one or more of the following solvents:

[0024] Methanol, ethanol, acetone, ether, chloroform, ethyl acetate or ethylene dichloride.

[0025] As an optional embodiment of the present application, optionally, the conditions for immersion stirring are:

[0026] Stir at 20-28°C for 1-5 hours.

[0027] Technical effects of the present invention:

[0028] The present application provides a Chinese herbal medicine for treating chronic bone marrow suppression, namely, pigeon pea leaf (Folium Cajani), the effective doses of which are 0.54 mg / ml and 0.81 g / ml respectively. As a Chinese herbal medicine, pigeon pea leaf has the advantages of being easy to carry, having a long-lasting efficacy, stable quality and accurate dosage.

[0029] After verification by animal experiments, the results showed that there was no statistical difference in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (CREA) levels between the MDY group and the blank group, indicating that the administration of pigeon pea leaves would not significantly affect liver and kidney function. In addition, compared with the blank group, methotrexate (MTX) significantly reduced the levels of red blood cells (RBC), white blood cells (WBC) and hemoglobin (HGB) in peripheral blood, while after pigeon pea leaf treatment, the number of red blood cells and white blood cells and hemoglobin levels were improved, and there was no statistical difference in platelet (PLT) levels among the groups.

[0030] In the femoral bone marrow smear experiment, 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. After treatment with pigeon pea leaves, the hematopoietic cells increased and the bone marrow morphology was significantly restored. 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 pigeon pea leaf treatment, indicating that pigeon pea leaves improved the erythroid and granulocyte colony formation ability of hematopoietic cells to a certain extent.

[0031] These results show that MTX modeling causes significant bone marrow damage, and pigeon pea leaf treatment can significantly improve MTX-induced bone marrow damage, and its efficacy is better than that of the positive control group of folic acid (FA). Therefore, pigeon pea leaf (folium cajani) has a significant protective effect on rats with chronic bone marrow suppression caused by methotrexate and is highly safe.

[0032] 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

[0033] 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.

[0034] Figure 1 It is a schematic diagram showing the working principle of the dissolution device of the present invention;

[0035] FIG. 2 shows the experimental comparative data of peripheral blood routine of the present invention;

[0036] Figure 3 Shown is an experimental comparison diagram of the bone marrow smear of the present invention;

[0037] Figure 4 Shown is an experimental comparison diagram of the bone marrow cell erythroid colony experiment of the present invention;

[0038] Figure 5 Shown is an experimental comparison diagram of the bone marrow cell granulocyte colony experiment of the present invention. 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 Chinese medicinal decoction piece for treating chronic bone marrow suppression.

[0044] In one aspect, the present application provides a pigeon pea leaf for treating chronic bone marrow suppression caused by MTX, wherein the pigeon pea leaf comprises the following main active ingredients:

[0045] Pinostrobin, cajanin stilbene acid, pratensein, longistylin A, apigenin, longistylin C, genistein, cajanolactone A, ononin, cajanol, prunetin, cajanin, luteolin.

[0046] After being made into Chinese medicinal pieces, pigeon pea leaves will be used to relieve or treat chronic bone marrow suppression caused by MTX.

[0047] Chinese herbal medicines 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 found in his long-term clinical experience that the main components of pigeon pea leaves have a significant therapeutic effect on chronic bone marrow suppression caused by MTX.

[0049] Pinus globulus is a common flavonoid compound with anti-inflammatory and antioxidant properties. Studies have shown that pinus globulus may affect bone marrow function by inhibiting the release of cytokines and regulating immune responses. It may have a bone marrow protective effect under certain conditions.

[0050] Cinnamomum camphora has significant antioxidant properties and can reduce the damage of oxidative stress to bone marrow cells. Cinnamomum camphora may protect bone marrow hematopoietic cells by scavenging free radicals and enhancing the activity of antioxidant enzymes. Secondly, Cinnamomum camphora can promote the proliferation and differentiation of hematopoietic stem cells in the bone marrow and increase the production of red blood cells and white blood cells.

[0051] Clover may stimulate the proliferation and differentiation of hematopoietic stem cells and increase the production of red and white blood cells. This has a positive effect on bone marrow suppression caused by chemotherapy, radiotherapy or other factors. In animal experiments and clinical studies, the supplementation of clover may be associated with improvements in hematological indicators, such as increased red blood cell counts, hemoglobin concentrations, and white blood cell counts, indicating its potential in improving bone marrow suppression. In some animal experiments, the use of clover was found to increase the red blood cell count, hemoglobin concentration, and white blood cell count in the blood, reflecting the improvement of bone marrow hematopoietic function.

[0052] Apigenin is a widely distributed natural flavonoid with antioxidant and anti-inflammatory effects. Studies have shown that apigenin may affect bone marrow function by regulating immune response and cytokine expression, and has a potential bone marrow protective effect.

[0053] Genistein is an isoflavone in soybeans that has anti-inflammatory, antioxidant and immunomodulatory effects. Studies have shown that genistein may have an effect by regulating the bone marrow microenvironment and the proliferation and differentiation of hematopoietic stem cells.

[0054] In some studies, caesalpinol A has been found to have anti-tumor and anti-inflammatory effects. Although relatively few studies have examined its myelosuppressive effects, it may indirectly affect the health and function of the bone marrow by affecting the activity of immune cells and the secretion of cytokines.

[0055] Formononetin may directly affect hematopoietic stem cells in the bone marrow, promoting their proliferation and differentiation. By activating related signaling pathways, formononetin may enhance the hematopoietic capacity of the bone marrow and improve bone marrow suppression caused by chemotherapy, radiotherapy or other factors.

[0056] Cajanol is a compound found in legumes that has some biological activity. Its direct effect on bone marrow suppression has not been fully studied, but it may indirectly affect the hematopoietic function of the bone marrow due to its possible influence on immune response.

[0057] Sakuraflavin is a polyphenolic compound with antioxidant and anti-inflammatory effects. It may affect bone marrow function by inhibiting inflammation and regulating immune responses.

[0058] Pigeon pea isoflavones are compounds related to pigeon pea that have antioxidant, anti-inflammatory and immunomodulatory properties. Research suggests that at low doses, they may exhibit bone marrow protective effects and promote cell growth and repair.

[0059] Luteolin is a widely-occurring flavonoid with anti-inflammatory, antioxidant and anti-tumor properties. Studies have shown that luteolin may affect the bone marrow by inhibiting the release of pro-inflammatory cytokines and regulating the activity of immune cells.

[0060] In summary, Chinese herbal medicine ingredients such as pinocybin and caesalpin have the potential to alleviate or treat bone marrow suppression in traditional Chinese medicine theory. However, when used specifically, it is necessary to make a diagnosis and treatment based on the patient's specific condition and constitution, and use them reasonably under the guidance of a doctor.

[0061] Therefore, the above ingredients are all main ingredients of pigeon pea leaves and can be used to alleviate or treat chronic bone marrow suppression caused by MTX.

[0062] 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 is a method for preparing pigeon pea leaves with chronic bone marrow suppression caused by MTX, comprising the following steps:

[0063] Weigh the raw materials according to the amount (which can be the amount of the above-mentioned proportion components): 60-90g pigeon pea leaves;

[0064] 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;

[0065] 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;

[0066] 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;

[0067] filtering the soaking liquid, and centrifuging the soaking liquid to obtain a formula solution;

[0068] Evaporating and concentrating the formula solution to obtain a formula concentrate;

[0069] 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 spleen-activating, detoxifying, collateral-draining and dampness-removing pigeon bean particles;

[0070] Package;

[0071] Quality inspection;

[0072] Storage in warehouse.

[0073] 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:

[0074] 1. Preliminary preparation

[0075] 1. Raw material selection and processing:

[0076] 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.

[0077] 2. Extraction

[0078] 1.Solvent selection:

[0079] Select a suitable solvent, such as water, ethanol, etc., according to the characteristics of the medicinal materials and extraction requirements.

[0080] 2. Extraction operation:

[0081] The pretreated Chinese medicinal materials are soaked in a solvent, and the effective ingredients are fully dissolved in the solvent through heating, stirring, etc.

[0082] 3. Extraction liquid treatment:

[0083] After the extraction is completed, the extract is filtered or centrifuged to remove impurities and drug residues.

[0084] 3. Concentration

[0085] 1. Concentration process:

[0086] The solvent in the extract is evaporated to increase the concentration of the active ingredient to form a concentrated solution or extract.

[0087] 2.Quality Control:

[0088] 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.

[0089] 4. Drying

[0090] 1. Drying method:

[0091] The concentrated liquid or extract is dried into solid powder or granules by using drying technology such as drying and spray drying.

[0092] 2. Drying conditions:

[0093] 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.

[0094] 5. Granulation

[0095] 1. Granulation equipment:

[0096] Use swing granulator, spray drying granulator and other equipment to make uniform Chinese medicine granules from the dried solid powder or granules.

[0097] 2. Granulation process:

[0098] 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.

[0099] 6. Packaging and quality inspection

[0100] 1. Packaging:

[0101] The prepared Chinese medicine granules are packaged according to certain specifications and dosages for easy storage and transportation.

[0102] 2. Quality inspection:

[0103] 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.

[0104] 7. Finished product warehousing and storage

[0105] 1. Finished product storage:

[0106] The Chinese herbal medicine granules that have passed the quality inspection shall be stored in the warehouse and properly labeled and recorded.

[0107] 2. Storage conditions:

[0108] According to product characteristics and storage requirements, set appropriate storage temperature and humidity conditions to ensure product stability and safety.

[0109] The preparation of the particles can refer to the above steps, but in the preparation process of the present invention, it is necessary to make certain restrictions on the preparation process of the pigeon pea leaves in order to better adapt to the preparation and application of the particles of this formula, so that the preparation process is more controllable and process quantitatively controlled, avoid blindly waiting for the process preparation time, and make the preparation process more node-based.

[0110] Among them, during the heating, stirring and soaking process:

[0111] 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;

[0112] 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.

[0113] 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.

[0114] Because Chinese medicinal materials will dissolve their effective ingredients in the solvent, the content of the effective ingredients in the solvent can be detected and monitored in real time to determine whether the effective ingredients of the Chinese medicinal materials are fully dissolved in the solvent.

[0115] 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.

[0116] 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.

[0117] Start filtering the soaking liquid and enter the concentration process.

[0118] For composition and content analysis of gas chromatograph, just refer to its functions.

[0119] The solvent of the present invention is one or more of the following solvents:

[0120] 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.

[0121] As an optional embodiment of the present application, optionally, the conditions for immersion stirring are:

[0122] Stir at 20-28°C for 1-5 hours.

[0123] Combined with Figure 1 As shown, the dissolving equipment has the functions of sealing (to prevent solvent volatilization), heating stirring and temperature sensing, which is convenient for better coordination in the preparation of Chinese medicine granules.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] The following will combine the action model to verify the experimental mechanism of alleviating or treating chronic bone marrow suppression caused by MTX.

[0128] 1. First, construct an animal model. All animal experiments were conducted 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: 20240527-03). 25 SPF female Wistar 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.

[0129] After one week of adaptive feeding, the rats were randomly divided into 4 groups: CONTROL group (blank group), MTX group (fed with methotrexate), FA group (fed with folic acid solution), MDY-L group (low-dose pigeon pea leaf group) and MDY-H group (high-dose pigeon pea leaf group), 5 rats in each group, and gavage was performed for 4 weeks. On the 35th day, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital 40 mg / kg and euthanized by 100% CO2 for 12 minutes. Peripheral blood, bone marrow cells of femur and tibia of both lower limbs were collected for experiments.

[0130] 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.

[0131] MDY (pigeon pea leaf gavage): According to the clinical dosage and rat dosage converted by a coefficient of 6.3, weigh 60g of pigeon pea leaves, soak the pigeon pea leaves in double distilled water for 12 hours, then decoct and concentrate the pigeon pea leaves and their soaking liquid to a concentration of 0.54g / ml, which is the concentration required for the low-dose pigeon pea leaf group. Weigh 90g of pigeon pea leaves, soak the pigeon pea leaves in double distilled water for 12 hours, then decoct and concentrate the pigeon pea leaves and their soaking liquid to a concentration of 0.81g / ml, which is the concentration required for the high-dose pigeon pea leaf group. The volume of the drug solution for gavage is 2ml per 200g rat, and it is gavaged for four weeks, once a day.

[0132] FA (folic acid solution by oral gavage): The adult dose is converted into a rat dose of 0.045 mg / ml. The oral gavage volume is 1 ml per 200 g rat. The dose is administered by oral gavage for 4 weeks, twice a week.

[0133] 2. Experiment:

[0134] 2.1 Bone marrow hematopoietic function, comparison of expression levels of components in the blood of each mouse

[0135] As shown in Figure 2, animal experiments show that pigeon pea leaves have a significant protective effect on rats with chronic bone inhibition caused by methylamine and are highly safe.

[0136] Combined with Figures 2A-2D As shown in Figure 2, there was no statistical difference in serum alanine aminotransferase (ALT) levels among the groups before and after modeling ( Figure 2A ), and there was no statistical difference in aspartate aminotransferase (AST) levels ( Figure 2B ), and there was no statistical difference in the level of urea nitrogen (BUN) ( Figure 2C ), compared with the blank group, the creatinine (CREA) level in the MTX model group showed an upward trend, and the creatinine (CREA) level in the high-dose pigeon pea leaf group (0.81 g / ml) decreased slightly after treatment, but was still within the normal range, indicating that it is highly safe ( Figure 2D ).

[0137] Combined with Figure 2E-2H As shown in the figure, compared with the blank group, MTX significantly reduced the number of white blood cells (WBC) in peripheral blood, while the number of white blood cells (WBC) in the low-dose group (0.54 g / ml) of pigeon pea leaves increased after treatment, and the number of white blood cells (WBC) in the high-dose group (0.81 g / ml) of pigeon pea leaves increased significantly after treatment ( Figure 2E ). Compared with the blank group, MTX significantly reduced the number of red blood cells (RBC) in peripheral blood, while the number of red blood cells (RBC) in the low-dose group (0.54 g / ml) of pigeon pea leaves increased after treatment, and the number of red blood cells (RBC) in the high-dose group (0.81 g / ml) of pigeon pea leaves increased significantly after treatment ( Figure 2F ). Compared with the blank group, MTX significantly reduced the level of hemoglobin (HGB) in peripheral blood, while the level of hemoglobin (HGB) in the low-dose group (0.54 g / ml) of pigeon pea leaves increased after treatment, and the level of hemoglobin (RBC) in the high-dose group (0.81 g / ml) of pigeon pea leaves increased significantly after treatment ( Figure 2G ). Compared with the blank group, MTX had no significant effect on the platelet (PLT) level in peripheral blood. After treatment with the low-dose pigeon pea leaf group (0.54 g / ml), there was no statistical difference in the platelet (PLT) level, while after treatment with the high-dose pigeon pea leaf group (0.81 g / ml), the platelet (PLT) level was significantly increased ( Figure 2H ).

[0138] Comparison of blood biochemistry and routine blood tests in different groups of Wistar 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<0.01, "***" represents P<0.001, "****" represents P<0.0001, and there are five samples in each group.

[0139] 2.2 Bone marrow smear analysis

[0140] 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.

[0141] Compared with the blank group ( Figure 3 Compared with A), the bone marrow hematopoietic structure of rats after MTX modeling was disordered and the number of nucleated cells was significantly reduced ( Figure 3 B), folic acid ( Figure 3 C) After treatment, no significant changes were observed in bone marrow nucleated cells. After treatment with the low-dose pigeon pea leaf group (0.54 g / ml), hematopoietic cells increased and bone marrow image was restored ( Figure 3 D), after treatment with high-dose pigeon pea leaves (0.81 g / ml), hematopoietic cells increased significantly and bone marrow was restored ( Figure 3 E).

[0142] 2.3 Colony formation assay

[0143] Combined with Figure 4 Single cell suspensions were prepared from bone marrow cells of bilateral tibiae at a concentration of 5×10 6 cells / sample (repeated 3 times / sample). The suspension was cultured in a 24-well plate according to an appropriate culture system. After 7-10 days, typical colonies were photographed under a light microscope according to morphological standards.

[0144] Bone marrow cell erythroid colony formation assay showed that compared with the blank group ( Figure 4 A), MTX caused a significant decrease in the number of erythroid colonies ( Figure 4 B), erythroid colonies showed an upward trend after folic acid (FA) treatment ( Figure 4 C), the erythroid colonies showed an upward trend after treatment with the low-dose pigeon pea leaf group (0.54 g / ml) ( Figure 4D), after treatment with high-dose pigeon pea leaves (0.81 g / ml), the erythroid colonies increased significantly ( Figure 4 E), indicating that pigeon pea leaves can improve the erythroid colony-forming ability of hematopoietic cells to a certain extent.

[0145] Combined with Figure 5 Single cell suspensions were prepared from bone marrow cells of bilateral tibiae at a concentration of 5×10 6 cells / sample (repeated 3 times / sample). The suspension was cultured in a 24-well plate according to an appropriate culture system. After 7-10 days, typical colonies were photographed under a light microscope according to morphological standards.

[0146] Bone marrow cell granulocyte colony formation assay showed that compared with the blank group ( Figure 5 A), MTX caused a significant decrease in the number of granulocyte colonies ( Figure 5 B), there is an upward trend in granulocyte colonies after folic acid (FA) treatment ( Figure 5 C), the number of granulocyte colonies increased after treatment with low-dose pigeon pea leaves (0.54 g / ml) ( Figure 5 D), the granulocyte colonies increased significantly after treatment with high-dose pigeon pea leaves (0.81 g / ml) Figure 5 E), indicating that pigeon pea leaves can improve the granulocyte colony-forming ability of hematopoietic cells to a certain extent.

[0147] Therefore, animal experiments show that pigeon pea leaves (Folium Cajani) have a significant protective effect on rats with chronic bone marrow suppression caused by methotrexate, and are highly safe. There was no statistical difference in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (CREA) levels between the MDY group and the blank group, proving that liver and kidney function was normal after pigeon pea leaf administration). 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 pigeon pea leaf treatment improved the number of red and white blood cells and hemoglobin levels. Compared with the blank group, MTX had no significant effect on the platelet (PLT) level in peripheral blood. After treatment with the low-dose group (0.54 g / ml) of pigeon pea leaves, there was no statistical difference in the platelet (PLT) level, while after treatment with the high-dose group (0.81 g / ml) of pigeon pea leaves, the platelet (PLT) level increased significantly. The results of femoral bone marrow smear experiments showed that compared with the blank group, the hematopoietic structure of the rat bone marrow was disordered and the number of nucleated cells was significantly reduced after MTX modeling, while the hematopoietic cells increased and the bone marrow image was restored after pigeon pea leaf 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 erythroid and granulocyte colonies increased after pigeon pea leaf treatment, indicating that pigeon pea leaves can improve the erythroid and granulocyte colony formation ability of hematopoietic cells to a certain extent.

[0148] In conclusion, MTX modeling will cause obvious bone marrow damage, while treatment with pigeon pea leaves 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.

[0149] 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. An application of pigeon pea leaves in rats with chronic bone marrow suppression induced by MTX, characterized in that: The pigeon pea leaves include the following effective ingredients: Pinus pinifolia, cajanin, prunin, cajanin A, apigenin, cajanin C, genistein, cajanolide A, formononetin, cajanol, prismatin, cajanin isoflavones and luteolin.

2. The method for preparing pigeon pea leaves according to claim 1, characterized in that: The steps include: Weigh the following ingredients: 60-90g pigeon pea leaves; 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 spleen-activating, detoxifying, collateral-draining and dampness-removing pigeon bean particles; Package; Quality inspection; Storage in warehouse.

3. The preparation method according to claim 1, characterized in that: The solvent is one or more of the following solvents: Methanol, ethanol, acetone, ether, chloroform, ethyl acetate or ethylene dichloride.

4. The preparation method according to claim 2, characterized in that: The conditions of the immersion stirring are: Stir at 20-28°C for 1-5 hours.

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