Application of Xinlikangfang preparation in drug preparation

Abstract

The present invention discloses the application of Xinlikangfang preparation in the preparation of drugs for treating leukemia, lipid metabolism disorder or complications of the two. The present invention constructs an in vitro and in vivo model of metabolic-related fatty liver disease, and after screening, it is found that Xinlikangfang preparation can reduce the accumulation of lipids in hepatocytes of mice in the leukemia group, reduce the content of inflammatory factors (IL-6, TNF-α) and blood sugar in serum, reduce the content of free fatty acids in serum to inhibit lipid metabolism pathway disorders, effectively inhibit the infiltration of leukocytes in the spleen of mice and fatty degeneration of the liver of mice, and repair the damage of leukemia cells to the spleen of mice to a certain extent, showing that Xinlikangfang preparation can effectively inhibit the further occurrence and development of leukemia combined with metabolic disorders; in the future, it will be applied to the treatment of leukemia combined with metabolic disorders, providing new medication options for patients with leukemia combined with metabolic disorders, and has important effects and significance for the treatment of patients with leukemia combined with metabolic disorders in the current world.

Description

Technical Field

[0001] The present invention belongs to the technical field of new uses of traditional Chinese medicines, and specifically relates to the use of Xinli Kangfu prescription preparations in the preparation of medicines for treating leukemia, lipid metabolism disorders or complications thereof. Background Art

[0002] Xinlikang is a traditional Chinese medicine formula, manufactured by Guiyang Xintian Pharmaceutical Co., Ltd. It has the effects of replenishing qi and blood, removing blood stasis and detoxifying, and is clinically used as an adjuvant treatment for radiotherapy and chemotherapy of breast cancer, lung cancer, bladder cancer, etc. The main raw materials include astragalus, angelica, nightshade, turmeric, red ginseng, snakeberry, snow lotus, cynarostemon root, and salvia miltiorrhiza.

[0003] Leukemia is a malignant clonal tumor of hematopoietic stem cells or hematopoietic progenitor cells. Traditional Chinese medicine believes that it is caused by internal deficiency of vital energy and invasion of warm and toxic pathogens. It is a malignant tumor of the hematopoietic system with fever, bleeding, anemia, and enlarged liver, spleen, and lymph nodes as the main clinical manifestations. Due to malignant proliferation, apoptosis disorders, and blocked differentiation, leukemia cells proliferate, accumulate, and infiltrate extensively in tissues and organs such as the bone marrow, spleen, and liver, and inhibit the normal hematopoietic function of the bone marrow. According to statistics, in 2018, there were 437,000 new cases of leukemia and 309,000 deaths worldwide. Moreover, leukemia accounts for 29% of all childhood cancers (including benign and borderline malignant brain tumors), which seriously threatens human health. The development of drugs that can effectively treat leukemia is an urgent hope of leukemia patients around the world. If not controlled, the rapid proliferation of leukemia cells and the damage to immune function can further lead to the appearance of complications such as lipid metabolism disorders in leukemia patients, such as diabetes and non-alcoholic fatty liver disease. Lipid metabolism disorders further aggravate the progression of the disease, the difficulty of treatment and the risk of death in leukemia patients. According to statistics from relevant institutions, 60% of leukemia patients have leukemia combined with lipid metabolism disorders. Therefore, regulating lipid metabolism abnormalities while inhibiting the development of leukemia is an important way to treat leukemia in the future. Therefore, the development of therapeutic drugs for leukemia combined with lipid metabolism disorders has important social significance and medical research value.

[0004] At present, there is still a lack of ideal therapeutic drugs for the treatment of leukemia. In the past, clinical treatments for leukemia mainly included chemotherapy, differentiation therapy, targeted therapy, monoclonal antibody therapy and stem cell transplantation, and chemotherapy is still the first choice for most leukemia patients. Chemotherapy drugs have strong toxic side effects on leukemia patients, which leads to a serious decline in the quality of life of patients. In addition, there are disadvantages such as a single target and easy relapse. In addition, leukemia combined with lipid metabolism disorders is caused by factors such as malignant proliferation of cancer cells, metabolic disorders and immune system damage. It is difficult for Western medicine to effectively cure leukemia combined with lipid metabolism disorders through a single target. Therefore, it has become an urgent task to find an effective and safe drug for the treatment of leukemia combined with lipid metabolism disorders. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the Xinlife Kangfang prescription preparation in the preparation of a drug for treating leukemia combined with lipid metabolism disorders; the preparation significantly reduces the size and weight of the spleen of leukemia model mice, and at the same time significantly inhibits the infiltration of a large number of basophilic leukemia cells in the spleen of leukemia mice, and the normal structure of the spleen is restored.

[0006] To achieve the above object, the technical solution of the present invention is:

[0007] The Xinlife Kangfang preparation is used in the preparation of drugs for treating leukemia, lipid metabolism disorders or their complications. The Xinlife Kangfang preparation is prepared from 300 parts by weight of astragalus, 300 parts by weight of angelica, 250 parts by weight of Solanum nigrum, 250 parts by weight of Curcuma aromatica, 250 parts by weight of red ginseng, 200 parts by weight of Duchesnea indica, 200 parts by weight of Snow Lotus, 200 parts by weight of Cynarostegia cyrtonema roots and 200 parts by weight of Salvia miltiorrhiza as raw materials.

[0008] In the application of the aforementioned Xinlifen Kangfang prescription preparation, the leukemia is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia; the lipid metabolism disorder disease is non-alcoholic fatty liver disease or diabetes.

[0009] The application of the aforementioned Xinlife Kangfang prescription preparation, the preparation method of the Xinlife Kangfang prescription preparation is: weigh astragalus, angelica, Solanum nigrum, curcuma, red ginseng, snakeberry, snow lotus, cyperus rotundus root, and salvia miltiorrhiza, cut into sections, mix well, add water and boil twice, the first time the amount of water added is 4 times the weight of the medicinal materials, and boil for 3 hours, the second time the amount of water added is 3 times the weight of the medicinal materials, and boil for 1 hour, combine the decoctions, filter, and concentrate the filtrate under reduced pressure to 80°C with a relative density of 1.25, spray dry into a dry powder extract, and then add or no excipients to make various oral preparations.

[0010] The application of the aforementioned Xinlifen Kangfang prescription preparation is that the Xinlifen Kangfang prescription preparation is prepared by adding appropriate amounts of starch, dextrin, sucrose, and ethylparaben as auxiliary materials to the extract, mixing, making granules, drying, and packaging to make capsule preparations.

[0011] Traditional Chinese medicine is an important part of Chinese medicine. Under the guidance of the theory of yin and yang and the five elements, traditional Chinese medicine studies the relationship between the human body's physiology, pathology and natural environment from a dynamic and holistic perspective, and involves pharmacology and the search for methods to prevent and treat diseases. In the works of doctors of traditional Chinese medicine from generation to generation, myelodysplastic syndrome belongs to the category of "consumption" and "blood syndrome". Modern doctors and scholars inherited and absorbed modern medical knowledge, combined with clinical characteristics and general manifestations, and innovatively named myelodysplastic syndrome "marrow toxicity and fatigue" in 2008. "Marrow" mainly represents the location of the disease, "toxicity" mainly represents the nature of the disease, and "labor" mainly represents the symptoms, realizing the dialectic of TCM disease names and clinical characteristics, pathogenesis, and location of the disease. Doctors of all generations believed that "toxicity" played an important role in the occurrence and development of malignant blood diseases, and in the works of doctors of all generations, "toxicity" involved the occurrence, development and even dialectical treatment of malignant blood diseases. In the discussion of the six evil spirits in the Yellow Emperor's Classic of Internal Medicine, it is believed that only when the excessive qi invades the body can it be transformed into poison. The Treatise on the Causes and Symptoms of Various Diseases points out: "This is caused by the interaction of wind and qi, which turns into heat poison." "This is caused by the exterior being solid and the interior being empty, and the heat taking advantage of the emptiness to enter and attack the spleen and stomach, which causes yellow and red juice to flow out. This is caused by heat poison." In the Neijing, toxic evil refers to a special cause of disease that has a strong pathogenic effect and is different from the six evils in poisoning the human body. In traditional Chinese medicine, the manifestations of toxic evil infection in malignant blood diseases include leukemia, lymphoma, etc., and the onset of these diseases all involve factors of virus infection. Therefore, in clinical practice, traditional Chinese medicine uses methods such as dispelling cold and detoxification, dispelling wind and resolving phlegm, clearing dampness and toxins, replenishing essence and qi, strengthening the spleen and kidney, and clearing heat and removing blood stasis to treat malignant blood diseases according to the characteristics of malignant blood diseases.

[0012] The main effects of Xinlikang prescription preparations are: replenishing qi and nourishing blood, removing blood stasis and detoxifying. Red ginseng in the prescription is sweet, bitter and warm, replenishing vital energy, restoring pulse and consolidating deficiency, replenishing qi and blood, and is the main medicine. Salvia miltiorrhiza is bitter and cold, promoting blood circulation and removing blood stasis, dredges menstruation and relieves pain, clears the heart and relieves vexation, cools blood and eliminates carbuncle, and is the minister medicine. Curcuma is pungent and cold, promoting qi and removing blood stasis, clears the heart and relieves depression, and promotes bile and relieves jaundice, and is the minister medicine. Astragalus is sweet and warm, invigorates the spleen and replenishes the middle, raises yang and lifts the sunken, benefits the defense and consolidates the exterior, promotes urination, supports toxins and promotes muscle regeneration, and is the adjuvant medicine. Angelica sinensis is pungent, sweet and warm, replenishes blood and activates blood circulation, regulates menstruation and relieves pain, and moisturizes the intestines and promotes bowel movements, and is the adjuvant medicine. Duchesnea oleracea is sweet, bitter and cold, clears heat, cools blood, reduces swelling, and detoxifies, and is the adjuvant medicine. Solanum nigrum is bitter and cold, clears heat, detoxifies, promotes blood circulation, and reduces swelling, and is the guiding medicine. Snow lotus is sweet and warm, removes cold, strengthens yang, regulates menstruation, and stops bleeding, and is the guiding medicine. The root of Cynarostegium wilfordii is bitter and cold, clears away heat and detoxifies, promotes diuresis and relieves stranguria, dispels wind and relieves pain, and is used as a guiding drug. This prescription promotes blood circulation and dredges collaterals, removes blood stasis and relieves pain, protects the liver and strengthens the spleen, and has the effects of reducing swelling and removing blood stasis, anti-cancer, and relieving pain. Under the guidance of the holistic concept and the theory of syndrome differentiation and treatment, Chinese medicine has the characteristics of multiple pathways, multiple targets, and overall regulation. It has obvious advantages such as significant clinical treatment effect on leukemia models, few adverse reactions, low price, and low resistance. It has become an important means of treating the disease and also provides a new direction for the development of new drugs for the treatment of leukemia combined with lipid metabolism disorders.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] The present invention discloses for the first time the therapeutic effect of Xinlikang prescription on leukemia, lipid metabolism disorder or their complications. The present invention screened the activity of multiple Guizhou ethnic medicine compound preparations through animal models and cell models of leukemia, lipid metabolism disorder and their complications, and for the first time found that the Guizhou ethnic medicine Miao medicine compound preparation "Xinlikang" has a new use. It can not only treat single leukemia or lipid metabolism disorder, but also has a significant therapeutic effect on leukemia combined with lipid metabolism disorder. The experiment found that Xinlikang prescription has strong inhibitory activity on leukemia cells Jurkat, HEL and K562. After 48 hours of treatment, the IC of Jurkat 50 The value was 193.64±15.13μg / ml, and the IC 50 The value was 231.69±16.93μg / ml, and the IC 50 The value was 245.50±5.67μg / ml; Xinlikang prescription can inhibit the progression of leukemia and its induced abnormal lipid metabolism complications by acting on multiple targets. After administration, the size and weight of the spleen of leukemia model mice were significantly reduced, and the infiltration of a large number of basophilic leukemia cells in the spleen of leukemia mice was significantly inhibited, and the normal structure of the spleen was restored; in addition, the Xinlikang administration group significantly inhibited the macrovesicular fatty degeneration of the liver of leukemia model mice, and reduced the liver weight and lipid accumulation of leukemia model mice; in addition, the administration group reduced the content of inflammatory factors (IL-6, TNF-α), blood sugar, and lipid content in serum, and the expression level of lipid synthesis genes was downregulated. It is expected that Xinlikang prescription can be used in the treatment of leukemia combined with lipid metabolism disorders in the future, providing a new drug option for patients with leukemia combined with lipid metabolism disorders, and it has an important role and significance in the treatment of the current world malignant blood disease-leukemia and its induced lipid metabolism disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Inhibitory effects of different concentrations of Xinlikangfang preparations on the proliferation of leukemia cells Jurkat, HEL and K562 after 48 hours of treatment; A- leukemia cells Jurkat; B- leukemia cells HEL; C- leukemia cells K562;

[0016] Figure 2 Flow cytometry was used to analyze the effects of different concentrations of Xinlifengfang prescription on apoptosis of Jurkat leukemia cells;

[0017] Figure 3 Flow cytometry was used to analyze the effects of different concentrations of Xinlifenkangfang preparations on apoptosis of leukemia cells HEL;

[0018] Figure 4Flow cytometry was used to analyze the effects of different concentrations of Xinlifengfang prescription on apoptosis of leukemia cells K562;

[0019] Figure 5 H&E staining was used to observe the protective effect of Xinlikangfang preparation on the liver and the repair effect of spleen structure in mice with leukemia combined with lipid metabolism disorder; XLK: Xinlikangfang preparation treatment group, PRS: prednisone group, M: leukemia combined with lipid metabolism disorder model group, CN: normal group mice;

[0020] Figure 6 Effects of Xinlifekangfang preparation on the weight of heart, liver, spleen, lung and kidney in mice with leukemia and lipid metabolism disorder;

[0021] Figure 7 Effect of Xinlifekangfang preparation on blood volume ratio in mice with leukemia and lipid metabolism disorder;

[0022] Figure 8 Effects of Xinlifekang prescription on serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC) and blood glucose (GLU) levels in mice; XLK: Xinlifekang prescription treatment group, PRS: prednisone group, M: leukemia combined with lipid metabolism disorder model group, CN: normal group;

[0023] Fig. 9 The regulatory effect of Xinlikangfang preparation on inflammatory factors (IL-6 and TNF-α) in mouse serum; XLK: Xinlikangfang preparation treatment group, PRS: prednisone group, M: leukemia combined with lipid metabolism disorder model group, CN: normal group;

[0024] Fig.10 Effects of Xinlikang prescription drug-containing serum on lipid content in lipid metabolism disorder cell model; 5%, 10%, 15%: different concentrations of Xinlikang prescription drug-containing serum in rats, M: lipid metabolism disorder cell model, CN: normal group;

[0025] Fig.11 The inhibitory effect of Xinlikang preparation rat drug-containing serum on the proliferation of leukemia cells Jurkat, HEL and K562 after 48 hours of treatment; 5%, 10%, 15%: different concentrations of Xinlikang preparation rat drug-containing serum, CN: normal group. DETAILED DESCRIPTION

[0026] Embodiment 1 of the present invention: Application of Xinlikangfang preparation:

[0027] Xinlikang capsules can be purchased on the market, and the obtained capsules can be directly used to treat leukemia (leukemia is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia) combined with lipid metabolism disorders (non-alcoholic fatty liver or diabetes). Usage and dosage: Oral, 3-5 capsules at a time, 3 times a day.

[0028] Example 2: Application of Xinlikangfang preparation:

[0029] Weigh 300g of Astragalus, 300g of Angelica, 250g of Solanum nigrum, 250g of Curcuma, 250g of Red Ginseng, 200g of Duchesnea, 200g of Snow Lotus, 200g of Cynarostemon Root and 200g of Salvia miltiorrhiza, chop them into pieces, mix them evenly, add water and boil them twice. The first time, the amount of water added is 5 times the weight of the medicinal materials, and the medicine is boiled for 4 hours. The second time, the amount of water added is 4 times the weight of the medicinal materials, and the medicine is boiled for 2 hours. The decoctions are combined, filtered, and the filtrate is concentrated under reduced pressure to a relative density of 1.25 at 80°C. It is microwave-dried into a clear dry paste, crushed into 100 mesh powder, and then the auxiliary materials starch and dextrin are added. The mixture is granulated, dried, and put into capsules to obtain the product.

[0030] The obtained capsule is directly used for treating leukemia (acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia) combined with lipid metabolism disorder (non-alcoholic fatty liver or diabetes). Usage and Dosage: Oral, 3-5 capsules at a time, 3 times a day.

[0031] Example 3: Application of Xinlikangfang preparation:

[0032] Weigh 300g of Astragalus, 300g of Angelica, 250g of Solanum nigrum, 250g of Curcuma, 250g of Red Ginseng, 200g of Duchesnea indica, 200g of Saussurea involucrata, 200g of Cynanchum wilfordii root and 200g of Salvia miltiorrhiza, and prepare various oral preparations by conventional extraction and preparation molding processes.

[0033] The obtained oral preparation is directly used for treating metabolic-related fatty liver disease (non-alcoholic fatty liver or diabetes). Usage and dosage: Oral, 1.5-2.5 g each time, 3 times a day.

[0034] Example 4: Application of Xinlikangfang preparation:

[0035] Weigh 300g of Astragalus, 300g of Angelica, 250g of Solanum nigrum, 250g of Curcuma, 250g of Red Ginseng, 200g of Duchesnea, 200g of Snow Lotus, 200g of Cynarocephala Root and 200g of Salvia miltiorrhiza, cut into sections, mix well, add water and boil twice, the first time adding water amount is 4 times the weight of the medicinal materials, and boiling is carried out for 2 hours, the second time adding water amount is 3 times the weight of the medicinal materials, and boiling is carried out for 1.5 hours, the decoctions are combined, and allowed to stand for 24 hours, the supernatant is concentrated to 70% of the total amount, and the obtained medicinal solution is added with or without excipients to prepare various oral preparations.

[0036] The obtained oral preparation is directly used for treating leukemia (acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia). Usage and dosage: Oral administration, 1.5-2.5 g each time, 3 times a day.

[0037] Experimental example:

[0038] 1. Experimental Materials

[0039] 1.1 Materials and methods

[0040] SPF BALBc mice were injected with Friend virus to induce leukemia 48 hours after birth, a total of 40 mice; 10 normal mice; BALBc mice were bred in the laboratory; SD rats, male mice, weighing 200±20g, a total of 20, were purchased from Nanjing Model Animal Research Institute. Mice were fed with growth and reproduction feed and high-fat feed, and rats were fed with growth and reproduction feed, all produced by Shanghai Huayasichuang Biological Company. Drinking water was sterilized tap water. During the experiment, animals were free to eat and drink. The laboratory temperature was 21±2℃, the humidity was 40-70%, and the laboratory license number was: SYXK (Qian) 2018-0001. HepG2 cells and DMEM high-glucose medium for the experiment were purchased from Wuhan Punosai Life Science Technology Co., Ltd. Gibco fetal bovine serum was purchased from Thermo Fisher Scientific.

[0041] 1.2 Instruments

[0042] Table centrifuge (Beckman), biological safety cabinet (Thermo Scientific), cell culture incubator (ThermoScientific), fluorescence inverted microscope (Olympus), constant temperature water bath (Grant), high pressure steam sterilizer (Shenan), Milli-Q water machine (BioGen), multifunctional microplate reader (Bio tek).

[0043] 1.3 Grouping and establishment of leukemia model

[0044] Six weeks after virus injection, the mice were divided into cages. The 40 mice were randomly divided into 4 groups, with 10 mice in each group. They were: model group, positive control group, and Xinlikang preparation group. All groups of mice were fed with normal breeding feed and sterilized water for 8 weeks.

[0045] 1.4 Grouped Dosing

[0046] The experiment started with oral administration of the drug to mice at 6 weeks of age. The Xinlikang preparation of the present invention was administered by oral administration at 3.37 g crude drug / kg, and the positive drug group was given 10 mg / kg of prednisone; the normal group and the model group were gavaged with an equal volume of normal saline, once a day, for 8 consecutive weeks. During the administration period, except for the normal group, all groups of mice continued to model. The mice were weighed once a week, and the dosage was changed according to the body weight. SD rats were divided into two groups (normal group and drug administration group, 5 in each group), and the dosage was 2.35 g / kg, and the drug administration was continued for 7 consecutive days.

[0047] 1.5 Experimental Animal Treatment

[0048] At the end of the 8th week of drug administration, the animals were fasted but not watered for 12 hours and weighed. After each group of mice was anesthetized with 10% chloral hydrate, they were killed by dislocation, and blood was collected from the eye sockets to collect mouse serum. The blood volume ratio of each group of mice was also determined. The mouse liver was frozen in a -80℃ refrigerator for subsequent analysis and testing.

[0049] 1.6 Detection indicators

[0050] 1.6.1 Liver and spleen samples

[0051] After the administration, partial liver and spleen samples of the mice were taken and sent to Wuhan Saiweier Biotechnology Co., Ltd. for pathological analysis of the liver tissue and spleen (H&E staining).

[0052] 1.6.2 Detection of serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), and blood glucose (Glu) in the serum samples of each group of mice by microplate reader

[0053] For the detection of mouse blood biochemical indices, the mouse serum samples prepared in 1.5 were used to detect the contents of ALT, AST, TG, TC, Glu and other indicators using an ELISA kit produced by Nanjing Detection Biological Products Co., Ltd.

[0054] 1.6.3 ELISA to detect the levels of IL-6 and TNF-α in the serum of mice in each group

[0055] For the detection of mouse blood biochemical indices, the mouse serum samples prepared in 1.5 were used to detect IL-6, TNF-α and other indicators using an ELISA kit and an enzyme marker.

[0056] 1.7 Effects of Xinlikangfang prescription-containing serum on abnormal lipid metabolism model of hepatocytes and leukemia cells

[0057] (1) Establishment of an in vitro model of abnormal hepatocyte lipid metabolism: HepG2 cells were treated with 0.5 mM oleic acid for 24 h to establish a model of abnormal hepatocyte lipid metabolism. The model of abnormal hepatocyte lipid metabolism was then treated with the drug-containing serum of rats prepared with the Xinlikang prescription to detect the effect of the drug-containing serum on lipid metabolism in the model of abnormal hepatocyte lipid metabolism.

[0058] (2) The leukemia cells Jurkat, K562 and HEL were treated with Xinlikang rat serum, and the effect of Xinlikang rat serum on the proliferation of leukemia cells was detected by MTT method and flow cytometry.

[0059] Preparation of drug-containing serum of rats for Xinlikangfang prescription:

[0060] a. Normal 6-week-old SD rats weighing 200±20g were gavaged with the total extract of Xinlikangfang prescription at a dose of 2.35g / kg, once a day for 7 consecutive days.

[0061] b. After the last dose, serum was collected after 4-8 hours of fasting but not water restriction.

[0062] c. Before blood sampling, SD rats were anesthetized with chloral hydrate.

[0063] d. After the anesthetic takes effect, the abdominal cavity of the SD rat is opened and the rat serum is collected into a procoagulant tube through the abdominal aorta.

[0064] e. Place the coagulation tube at room temperature for 1-2 hours. After the blood is completely coagulated, place the tube in a centrifuge at 4000 rpm for 15 minutes and collect the supernatant, which is the drug-containing serum.

[0065] f. After the serum is collected, it is inactivated in a 56°C water bath for 30 minutes. The inactivated drug-containing serum is filtered through a 0.22 μm microporous filter membrane and stored in a -20°C refrigerator.

[0066] 1.8 Statistical methods

[0067] The experimental results were analyzed using SPSS 22.0 statistical software. All data were expressed as mean ± standard deviation. Pairwise comparisons were performed using t-test, and differences were considered statistically significant when p < 0.05.

[0068] 2 Experimental results

[0069] 2.1 Effects of Xinlikangfang prescription treatment group on the body weight of mice

[0070] like Figure 1 As shown in the figure, by comparing the experimental results of each group, it can be seen that after 48 hours of treatment, the inhibitory effect of Xinlikangfang preparation on the activity of Jurkat, HEK and K562 cells showed a gradually increasing trend with the increase of concentration ( Figure 1A, B and C), IC 50 The values ​​were 193.64±15.13μg / ml, 231.69±16.93μg / ml and 245.50±5.67μg / ml respectively (Table 1). In addition, the results of flow cytometry also showed that the apoptosis rates of Jurkat, HEK and K562 cells after 48h of treatment with Xinlikangfang preparation showed a dose-dependent effect with the increase of concentration ( Figure 2 , Figure 3 , Figure 4 ).

[0071] Table 1 IC values ​​of Xinlikangfang preparations on leukemia cells Jurkat, HEL and K562 50

[0072]

[0073] 2.2 Effect of Xinlikangfang preparation treatment group on macrovesicular fatty degeneration in leukemia model mice

[0074] H&E staining showed that the high-fat-induced leukemia model mice had punctate necrosis and inflammatory cell infiltration, as well as varying degrees of fatty degeneration and vacuolar degeneration. At the same time, the liver of the leukemia model group mice was significantly larger than that of the Xinlikang prescription (XLK) group and the prednisone (PRS) group ( Figure 5 ), and their livers are milky white to varying degrees ( Figure 5 Oil red O staining results showed that the fat content in the liver of mice in the XLK and PRS groups was significantly reduced compared with that in the model group ( Figure 5 The Xinlikang prescription (XLK) significantly improved fatty degeneration, vacuolar degeneration and lipid accumulation in the liver of mice after treatment, and the morphology of the liver of mice was consistent with that of the normal group ( Figure 5 In addition, H&E staining revealed that compared with the model group, the number of basophilic leukemia cells in the spleen of mice in the PRS and XLK groups was significantly reduced, and the structure of the spleen was repaired ( Figure 5 ). The above results show that Xinlikang prescription effectively inhibits the infiltration of leukocytes in the spleen of mice and repairs the damage of leukemia cells to the spleen of mice to a certain extent.

[0075] 2.3 Analysis of the effects of Xinlikang prescription on the heart, liver, spleen, lung and kidney of each group of mice

[0076] like Figure 6 As shown, compared with the model group, the XLK and PRS groups significantly reduced the weight of the mouse liver (p<0.001) and the weight of the mouse spleen (p<0.01 and p<0.001) ( Figure 6 B and C), but had no effect on the weight of the heart, lung, and kidney of mice ( Figure 6A, D and E). The above data results show that Xinlikang prescription can effectively reduce the weight of liver and spleen of model group mice, and its effect is comparable to that of positive drug PRS.

[0077] 2.4 Effect of Xinlikang prescription on hematocrit ratio in mice

[0078] like Figure 7 As shown in the figure, compared with the model group, the XLK and PRS groups both improved the hematocrit of mice, and the hematocrit of mice in the XLK group was significantly higher than that in the model group (p<0.05). The hematocrit data analysis showed that the improvement effect of Xinlikang prescription on leukemia mice was significantly better than that of the positive drug group PRS.

[0079] 2.5 Effects of Xinlikang prescription treatment group on serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), and blood glucose (Glu) levels in leukemia combined with metabolic disorder model mice

[0080] In order to further analyze the therapeutic effect of Xinlikangfang preparation on leukemia combined with metabolic disorder model mice, the experiment also measured the levels of biochemical indicators such as ALT, AST, TG, TC, and Glu in the serum of each group of mice. Figure 8 As shown, compared with the normal group, the Xinlifekang prescription preparation reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC) and blood glucose (Glu) in the serum of mice, and the effect of increasing the levels of TC and TG in the serum of mice was significantly better than that of the positive drug PRS group.

[0081] 2.6 Effect of Xinlikangfang preparation treatment group on the levels of inflammatory factors IL-6 and TNF-α in the serum of mice with leukemia and metabolic disorders

[0082] To further explore whether Xinlikangfang preparation can inhibit the inflammatory response in mice caused by lipid peroxidation, the contents of inflammatory factors IL-6 and TNF-α in mouse serum were measured by Elisa method. Fig. 9 As shown, from the experimental results of the control groups, it can be seen that the Xinlife Kangfang prescription preparation reduced the levels of inflammatory factors IL-6 and TNF-α in mouse serum and inhibited the inflammatory response in the leukemia combined with metabolic disorder model mice.

[0083] 2.7 Effects of Xinlikangfang prescription-containing serum on lipid disorder cell model and leukemia cell activity

[0084] The in vitro cell model of lipid metabolism disorder model was established by treating human liver cells with oleic acid, and then the leukemia cell model was treated with the drug-containing serum of the Xinlikangfang preparation rats to clarify the effect of the drug-containing serum of the Xinlikangfang preparation on the human liver cell lipid metabolism disorder model. Fig.10 As shown in the figure, from the experimental results of the control groups, it can be seen that the Xinlikangfang preparation significantly reduced the accumulation of lipids in the leukemia model cell model. In addition, the MTT assay showed that the activity of Jurkat, HEK and K562 cells showed a gradually decreasing trend with the increase of the serum concentration of the Xinlikangfang preparation (5%, 10%, 15%). Fig.11 ).

Claims

1. Use of a compound preparation in the preparation of a drug for treating leukemia or complications of leukemia and lipid metabolism disorders, the compound preparation being prepared from 300 parts by weight of astragalus, 300 parts by weight of angelica, 250 parts by weight of solanum nigrum, 250 parts by weight of curcuma, 250 parts by weight of red ginseng, 200 parts by weight of duchesnea, 200 parts by weight of snow lotus, 200 parts by weight of cycas revoluta root and 200 parts by weight of salvia miltiorrhiza as raw materials.

2. The use of the compound preparation according to claim 1, characterized in that: The leukemia is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia; the lipid metabolism disorder is non-alcoholic fatty liver disease or diabetes.

3. The use of the compound preparation according to claim 1, characterized in that: The preparation method of the compound preparation is as follows: astragalus, angelica, Solanum nigrum, turmeric, red ginseng, snakeberry, snow lotus, cyperus rotundus root, and salvia miltiorrhiza are weighed, cut into sections, mixed, and then decocted twice with water, the first time the amount of water added is 4 times the weight of the medicinal materials, and the decocting is carried out for 3 hours, the second time the amount of water added is 3 times the weight of the medicinal materials, and the decocting is carried out for 1 hour, the decoctions are combined, filtered, the filtrate is concentrated under reduced pressure to 80° C. with a relative density of 1.25, spray-dried into a dry powder extract, and then various oral preparations are prepared with or without the addition of auxiliary materials.

4. The use of the compound preparation according to claim 3, characterized in that: The compound preparation is prepared by adding appropriate amounts of starch, dextrin, sucrose and ethylparaben as auxiliary materials to the extract, mixing them evenly, making them into granules, drying them and packaging them into capsule preparations.

Images