Blood replenishing activity evaluation method for different polar parts of Chinese angelica and caulis spatholobi decoction

Abstract

The invention provides a blood replenishing activity evaluation method for different polar parts of Chinese angelica and caulis spatholobi decoction. The blood replenishing activity evaluation method for different polar parts of the Chinese angelica and caulis spatholobi decoction comprises the following steps: step 1, preparing different extraction parts of DJD (Chinese angelica and caulis spatholobi decoction): taking Chinese angelica, caulis spatholobi, prepared rehmannia root, white peony root, red sage root and longan aril, and crushing; the preparation method comprises the following steps: crushing the medicinal materials, adding 8 times by volume of purified water, soaking for 1 hour, heating to boil, boiling for 30 minutes, pouring out decoction, adding 6 times by volume of purified water into decoction dregs, continuously boiling for 30 minutes, combining the two decoctions, filtering, and carrying out rotary evaporation to obtain concentrated extract; according to the blood replenishing activity evaluation method for the different polar parts of the angelica sinensis and caulis spatholobi decoction, a blank group, a model group, a DJD original formula group, a petroleum ether group, an ethyl acetate group and an n-butyl alcohol group are set for experimental comparison, the difference has statistical significance, and it is verified that the angelica sinensis and caulis spatholobi decoction exerts the bone marrow hematopoiesis recovery function; the symptoms of the blood-deficiency rats can be relieved.

Description

Technical Field

[0001] The invention relates to the technical field of drug research, and in particular to a method for evaluating the blood-tonifying activity of different polarity parts of Angelica Sinensis Millettia Soup. Background Art

[0002] The incidence of cancer is increasing every year, and it is often accompanied by side effects of treatment. In the current medical field, chemotherapy is one of the common anti-cancer treatments. However, the problem of bone marrow suppression due to its toxic side effects remains a serious challenge. Myelosuppression after chemotherapy (MAC) not only reduces the quality of life of patients, but may also pose a threat to health. Although biological drugs and blood products can significantly improve MAC, they have many limitations, such as a single target and many side effects. Therefore, finding effective drugs to reduce bone marrow damage is of great significance for improving the quality of life and treatment effect of patients. From the perspective of traditional Chinese medicine, this situation can also be understood as a manifestation of blood deficiency. Blood deficiency is a common syndrome in traditional Chinese medicine, which refers to insufficient blood or poor quality in the body. Blood deficiency syndrome is common in cases of weakness, long-term illness, chemotherapy toxicity and malnutrition. Traditional Chinese medicine believes that blood is one of the important substances for health preservation. It flows in the body, provides nutrients and oxygen to various tissues of the body, and maintains the normal function of the body. Traditional Chinese medicine can use some drugs and conditioning methods to nourish blood and nourish essence and blood to regulate the body's qi and blood state, help restore bone marrow function and promote the recovery of hematopoietic function. Traditional Chinese medicine prescriptions often involve multiple targets, which may increase drug efficacy, reduce toxicity, relieve clinical symptoms and improve quality of life.

[0003] Danggui Jixueteng Decoction (DJD), originated from the Traumatology of Traditional Chinese Medicine, is composed of six Chinese medicinal materials: Angelica sinensis, Millettia rehmanniae, Rehmannia glutinosa, Longan meat, White Peony Root, and Salvia miltiorrhiza. It is mainly used to treat patients with late-stage Qi and blood deficiency in bone injuries; patients with leukocytopenia and thrombocytopenia during chemotherapy or radiotherapy for tumors. It has the effects of replenishing Qi and activating blood circulation, activating blood circulation and removing blood stasis, activating collaterals and relieving pain. It is widely used to treat periarthritis of the shoulder, cervical spondylosis and heel pain. The drugs in the DJD prescription have different degrees of tonic and blood circulation effects, and Angelica sinensis and Salvia miltiorrhiza are particularly prominent in relieving the clinical symptoms and indicators of anemia. In our previous pharmacodynamic experiments, we found that the water extract of DJD had a good therapeutic effect on the blood deficiency rat model established by acetylphenylhydrazine combined with cyclophosphamide, and effectively improved the state of blood deficiency rats. However, for prescriptions with complex ingredients, the effective components and mechanisms of blood tonification are not very clear.

[0004] Therefore, the present invention will evaluate the blood-tonifying activity of different polar parts of DJD based on the rat blood deficiency model, explore the mechanism of the pharmacological effect, and provide a method for evaluating the blood-tonifying activity of different polar parts of Danggui Millettia reticulata decoction. Summary of the invention

[0005] Based on the above content, the present invention provides a method for evaluating the blood-tonifying activity of different polar parts of Angelica Sinensis Millettia Soup.

[0006] The method for evaluating the blood-tonifying activity of different polarity parts of Angelica Sinensis Millettia Soup provided by the present invention comprises the following steps:

[0007] Step 1: Preparation of different extraction parts of DJD (Danggui Millettia Spatholobi Decoction):

[0008] Take Chinese angelica, Millettia reticulata, Rehmannia root, White peony root, Salvia miltiorrhiza, and Longan pulp, and crush them;

[0009] After the medicinal materials are crushed, add 8 times the volume of pure water to soak for 1 hour, heat to boiling and then boil for 30 minutes, pour out the decoction, add 6 times the volume of pure water to the residue and continue boiling for 30 minutes, combine the two decoctions, filter, and rotary evaporate to obtain a concentrated extract;

[0010] The extract is extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and then the solvent is removed by rotary evaporation, and the corresponding lyophilized powder is obtained by freeze drying. The lyophilized powder is dissolved with purified water before use;

[0011] Step 2: Animal model establishment and intervention:

[0012] After one week of adaptive feeding, SD rats were randomly divided into a blank group, a model group, and a DJD original formula group (6.21 g·kg -1 ), petroleum ether group (0.8 g kg -1 ), ethyl acetate group (0.8 g·kg -1 ) and n-butanol group (0.8 g·kg -1 ), 5 in each group;

[0013] Except for the normal group, the other groups were subcutaneously injected with 80 and 30 mg kg -1 Acetylphenylhydrazine solution;

[0014] On days 4 to 7, 50 mg / kg was injected intraperitoneally. -1 Cyclophosphamide solution was used to prepare the blood deficiency rat model. Each group was given the drug by gavage at the set dose at the same time of modeling. The blank group and the model group were given the same amount of 0.9% NaCl solution once a day for 15 consecutive days.

[0015] Step 3: Detect blood routine with automatic biochemical analyzer:

[0016] Blood was collected from the medial canthal venous plexus of rats, anticoagulated with EDTA-2K, and the red blood cell count (RBC), white blood cell (WBC), hemoglobin (HGB), and platelet (PLT) levels in peripheral blood were determined using an automatic biochemical analyzer.

[0017] Step 4: Detection of IFN-γ, IL-3, SCF and MMP-2 in serum by ELISA:

[0018] Blood was collected from the medial canthal venous plexus of rats and placed at 4°C for 2 h. 4 r min -1 After centrifugation for 10 min, the supernatant was collected and the contents of IFN-γ, IL-3, SCF and MMP-2 in rat serum were detected according to the instructions of the ELISA detection kit;

[0019] Step 5: Bone marrow tissue pathology observation:

[0020] Rats were killed by cervical dislocation, and the right fibula was immediately removed under sterile conditions and immersed in a fixative (containing 100 mL of paraformaldehyde and 5.5 g of EDTA). The fixative was changed once a week. After decalcification at 4°C for 15 days, the fibula was embedded in paraffin and stained with HE and Masson, respectively, and observed under an optical microscope.

[0021] Step 6. Detection of PI3K and AKT protein expression in bone marrow cells by Western blot:

[0022] Rats in the blank group, model group, ethyl acetate group, n-butanol group, and DJD group were taken and killed by cervical dislocation. The left fibula was immediately removed under sterile conditions, and the contents of the fibula cavity were repeatedly flushed out with sterile PBS. -1 Centrifuge for 10 min and discard the supernatant;

[0023] Add RIPA lysis buffer to the precipitate and shake it for lysis at 12000 r / min at 4°C. -1 Centrifuge for 10 min and collect the supernatant;

[0024] The protein concentration of the supernatant was measured using the BCA protein quantification method;

[0025] The sample was added with reduced protein loading buffer, denatured in boiling water for 10 min, and frozen for later use;

[0026] The samples were added to the gel wells for SDS PAGE gel electrophoresis, transferred to PVDF (0.45 μm) membrane, and sealed with 5% skim milk at room temperature for 30 minutes. The corresponding primary antibodies PI3K, p-PI3K, AKT and p-AKT (1:3000) and β-actin (1:5000) were added and kept at 4°C overnight; the next day, the membranes were washed with TBST 3 times, 5 minutes each time, and then the secondary antibodies (1:5000) were added, shaken slowly on a shaker, incubated at room temperature for 30 minutes, and the membranes were quickly rinsed three times with TBST, imaged with a chemiluminescence imaging system, and photographed;

[0027] The protein bands were scanned, and the grayscale values ​​were analyzed using Image J software. β-actin was used as an internal reference to perform semi-quantitative analysis of the target protein.

[0028] Step 7: Statistical processing:

[0029] SPSS17.0 software was used for statistical analysis. The experimental data were expressed as x±s. One-way analysis of variance (ANOVA) was used when the variance was in accordance with normal distribution. Nonparametric tests were used when it was not. P<0.05 was considered statistically significant.

[0030] The above steps indicate that the treatment with Angelica Sinensis Millettia Decoction can significantly increase the level of protein phosphorylation. Angelica Sinensis Millettia Decoction regulates the expression of proteins related to the PI3K / AKT signaling pathway, restores bone marrow hematopoietic function, and thus alleviates the symptoms of blood deficiency in rats.

[0031] Preferably, in the step 2, male SPF-grade SD rats of 7 weeks of age and weighing 180-220 g are used.

[0032] Preferably, the weight of each component in the Angelica and Millettia reticulata soup is 15g of Angelica sinensis, 15g of Millettia reticulata, 15g of Rehmannia root, 9g of White Peony Root, 9g of Salvia miltiorrhiza, and 6g of Longan pulp.

[0033] Preferably, the equipment for rotary evaporation of the filtrate and the extract in step 1 is a rotary evaporator produced by Japan EYELA Company.

[0034] Preferably, the analyzer used in step three is the XN9500 ​​fully automatic blood analyzer produced by Sysmex Corporation of Japan.

[0035] Preferably, the microscope used in step five is a BX53 Olympus biological microscope produced by Olympus Corporation of Japan.

[0036] Preferably, the equipment used to heat the sample in step six is ​​the HH-4 digital display constant temperature water bath produced by Changshu Aohua Instrument Co., Ltd.

[0037] Compared with the related art, the method for evaluating the blood-tonifying activity of different polar parts of Angelica Sinensis Millettia Soup provided by the present invention has the following beneficial effects:

[0038] The present invention provides a method for evaluating the blood-tonifying activity of different polarity parts of Angelica Sinensis Millettia Soup:

[0039] 1. By setting up blank group, model group, DJD original formula group, petroleum ether group, ethyl acetate group and n-butanol group, the differences were statistically significant;

[0040] 2. After treatment with Danggui Jixueteng decoction, the protein phosphorylation level increased significantly, and compared with the control group, the increase in protein phosphorylation level was the most significant;

[0041] 3. The research using the evaluation method of the present invention shows that DJD regulates the expression of proteins related to the PI3K / AKT signaling pathway, restores bone marrow hematopoietic function, and thus alleviates the symptoms of blood deficiency rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the result diagram of the effect of Angelica Sinensis Millettia Decoction on the bone marrow of blood-deficiency rats (HE, ×200);

[0043] Figure 2 This is a graph showing the effect of DJD on the protein expression of rat bone marrow cells (x±s, n=3) in the present invention. DETAILED DESCRIPTION

[0044] The materials used in the evaluation method of the blood-tonifying activity of different polar parts of Angelica Sinensis Millettia Soup are:

[0045] (1) Animals: Male, 7-week-old, SPF-grade SD rats weighing 180–220 g were purchased from Weitonglihua Experimental Animal Co., Ltd. Animal license number: SCXK (Zhejiang) 2019-0001;

[0046] (2) Drugs and reagents:

[0047] Angelica and Millettia reticulata decoction (composed of Angelica sinensis, Millettia reticulata, Rehmannia root, White Peony Root, Salvia miltiorrhiza, and Longan pulp), cyclophosphamide, acetylphenylhydrazine, interferon-γ (IFN-γ), interleukin 3 (IL-3), rat stem cell factor (SCF), matrix metalloproteinase-2 (MMP-2), enzyme-linked immunosorbent assay (ELISA) kit, petroleum ether, ethyl acetate, and n-butanol;

[0048] (3) Instruments:

[0049] BX53 Olympus biological microscope, XN9500 ​​fully automatic blood analyzer, qTOWER 2.2 PCR instrument, rotary evaporator, HH-4 digital constant temperature water bath.

[0050] The present invention will be further described below in conjunction with the embodiments.

[0051] The method for evaluating the blood-tonifying activity of different polarity parts of Angelica Sinensis Millettia Soup comprises the following steps:

[0052] Step 1: Preparation of different extraction parts of DJD (Danggui Millettia Spatholobi Decoction):

[0053] Weigh the various medicinal materials of Danggui Millettia Soup, which are 15g of Danggui, 15g of Millettia, 15g of Rehmannia glutinosa, 9g of White Peony Root, 9g of Salvia miltiorrhiza, and 6g of Longan Meat, and grind them;

[0054] After the medicinal materials are crushed, add 8 times the volume of pure water to soak for 1 hour, heat to boiling and then boil for 30 minutes, pour out the decoction, add 6 times the volume of pure water to the residue and continue boiling for 30 minutes, combine the two decoctions, filter, and rotary evaporate to obtain a concentrated extract;

[0055] The extract is extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and then the solvent is removed by rotary evaporation, and the corresponding lyophilized powder is obtained by freeze drying. The lyophilized powder is dissolved with purified water before use;

[0056] Step 2: Animal model establishment and intervention:

[0057] After one week of adaptive feeding, SD rats were randomly divided into a blank group, a model group, and a DJD original formula group (6.21 g·kg -1 ), petroleum ether group (0.8 g kg -1 ), ethyl acetate group (0.8 g·kg -1 ) and n-butanol group (0.8 g·kg -1 ), 5 in each group;

[0058] Except for the normal group, the other groups were subcutaneously injected with 80 and 30 mg kg -1 Acetylphenylhydrazine solution;

[0059] On days 4 to 7, 50 mg / kg was injected intraperitoneally. -1 Cyclophosphamide solution was used to prepare the blood deficiency rat model. Each group was given the drug by gavage at the set dose at the same time of modeling. The blank group and the model group were given the same amount of 0.9% NaCl solution once a day for 15 consecutive days.

[0060] Step 3: Detect blood routine with automatic biochemical analyzer:

[0061] Blood was collected from the medial canthal venous plexus of rats, anticoagulated with EDTA-2K, and the red blood cell count (RBC), white blood cell (WBC), hemoglobin (HGB), and platelet (PLT) levels in peripheral blood were determined using an automatic biochemical analyzer.

[0062] Step 4: Detection of IFN-γ, IL-3, SCF and MMP-2 in serum by ELISA:

[0063] Blood was collected from the medial canthal venous plexus of rats and placed at 4°C for 2 h. 4 r min -1 After centrifugation for 10 min, the supernatant was collected and the contents of IFN-γ, IL-3, SCF and MMP-2 in rat serum were detected according to the instructions of the ELISA detection kit;

[0064] Step 5: Bone marrow tissue pathology observation:

[0065] Rats were killed by cervical dislocation, and the right fibula was immediately removed under sterile conditions and immersed in a fixative (containing 100 mL of paraformaldehyde and 5.5 g of EDTA). The fixative was changed once a week. After decalcification at 4°C for 15 days, the fibula was embedded in paraffin and stained with HE and Masson, respectively, and observed under an optical microscope.

[0066] Step 6. Detection of PI3K and AKT protein expression in bone marrow cells by Western blot:

[0067] Rats in the blank group, model group, ethyl acetate group, n-butanol group, and DJD group were taken and killed by cervical dislocation. The left fibula was immediately removed under sterile conditions, and the contents of the fibula cavity were repeatedly flushed out with sterile PBS. -1 Centrifuge for 10 min and discard the supernatant;

[0068] Add RIPA lysis buffer to the precipitate and shake it for lysis at 12000 r / min at 4°C. -1 Centrifuge for 10 min and collect the supernatant;

[0069] The protein concentration of the supernatant was measured using the BCA protein quantification method;

[0070] The sample was added with reduced protein loading buffer, denatured in boiling water for 10 min, and frozen for later use;

[0071] The samples were added to the gel wells for SDS PAGE gel electrophoresis, transferred to PVDF (0.45 μm) membrane, and sealed with 5% skim milk at room temperature for 30 minutes. The corresponding primary antibodies PI3K, p-PI3K, AKT and p-AKT (1:3000) and β-actin (1:5000) were added and kept at 4°C overnight; the next day, the membranes were washed with TBST 3 times, 5 minutes each time, and then the secondary antibodies (1:5000) were added, shaken slowly on a shaker, incubated at room temperature for 30 minutes, and the membranes were quickly rinsed three times with TBST, imaged with a chemiluminescence imaging system, and photographed;

[0072] The protein bands were scanned, and the grayscale values ​​were analyzed using Image J software. β-actin was used as an internal reference to perform semi-quantitative analysis of the target protein.

[0073] Step 7: Statistical processing:

[0074] SPSS17.0 software was used for statistical analysis. The experimental data were expressed as x±s. One-way analysis of variance (ANOVA) was used when the variance conformed to the normal distribution. Non-parametric test was used when the variance did not conform to the normal distribution. P<0.05 was considered statistically significant.

[0075] The following is an analysis of the experimental results using the above evaluation method:

[0076] (1) Effects of DJD on the general condition and blood count of blood-deficient rats:

[0077] Compared with the normal group, the rats in the model group and petroleum ether group had a worse mental state, reduced activity and food intake, fluffy fur, pale eyes, lips and limbs, significantly decreased body weight, and anemia. Compared with the model group, the mental state of the DJD group, ethyl acetate group and n-butanol group was significantly improved, and their body weight began to gradually increase.

[0078] Compared with the normal group, the levels of WBC, RBC, HGB and PLT of the rats in the model group were significantly decreased, and the differences were statistically significant (all P<0.01); compared with the model group, the levels of WBC, RBC and PLT of the rats in the DJD group, ethyl acetate group, n-butanol group and polysaccharide group were significantly different (P<0.05, P<0.01); compared with the model group, the level of PLT of the rats in the petroleum ether group was significantly different (P<0.01), while the levels of WBC, RBC and HGB were not significantly different (P>0.05); compared with the model group, the levels of HGB of the rats in the DJD group and crude polysaccharide group were significantly different (P<0.01), the results are shown in Table 1.

[0079] The results showed that DJD and its ethyl acetate and n-butanol fractions could significantly improve the general condition, body weight and peripheral blood indexes of blood deficiency rats.

[0080] Table 1. Effects of different polarity parts of Danggui Jixueteng decoction on peripheral blood cells in blood deficiency rats

[0082]

[0083] Compared with the blank group, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0084] (2) Effects of DJD on serum IFN-γ, IL-3, SCF and MMP-2 levels in blood deficiency rats:

[0085] Compared with the normal group, the levels of IL-3, SCF and MMP-2 in the serum of the rats in the model group were significantly decreased, and the level of IFN-γ was significantly increased, and the differences were statistically significant (P<0.01); compared with the model group, the levels of IL-3, SCF and MMP-2 in the serum of the rats in the DJD group, ethyl acetate group and n-butanol group were significantly increased, and the level of IFN-γ was significantly decreased (P<0.05, P<0.01); compared with the model group, the levels of IL-3 and MMP-2 in the serum of the rats in the petroleum ether group were significantly different (P<0.05), while the levels of IFN-γ and SCF were not significantly different (P>0.05), the results are shown in Table 2.

[0086] Table 2 Effects of DJD on serum IFN-γ, IL-3, SCF and MMP-2 levels in blood deficiency rats

[0087]

[0088] Compared with the blank group, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0089] (3) Effect of DJD on bone marrow of blood deficiency rats:

[0090] like Figure 1 As shown in the figure, the bone marrow of rats in the model group was significantly damaged, with large and extra-large lipid droplet vacuoles and obvious fatification. The bone marrow injury scores of the low-, medium-, and high-dose experimental groups, the positive control group, the model group, and the blank group were (2.38±0.30), (1.31±0.18), (1.96±0.09), (1.49±20.42), (3.76±0.19), and (0.05±0.04), respectively; the bone marrow injury scores of the low-, medium-, and high-dose experimental groups, the positive control group, and the blank group were significantly different from those of the model group (all P<0.01).

[0091] (4) Effect of DJD on PI3K / AKT protein expression in bone marrow cells:

[0092] like Figure 2 As shown in the results, compared with the control group, the levels of p-PI3K and p-AKT in the bone marrow cells of the rats in the model group were lower, but after treatment, the levels of protein phosphorylation were significantly increased (P < 0.01). The increase in the DJD group was the most significant, followed by the n-butanol group and the ethyl acetate group. The results are shown in Table 3.

[0093] These results indicate that DJD regulates the expression of proteins related to the PI3K / AKT signaling pathway, restores bone marrow hematopoietic function, and thus alleviates the symptoms of blood deficiency in rats.

[0094] Table 3 Effects of different polarity parts of Danggui Jixueteng decoction on peripheral blood cells in blood deficiency rats

[0096]

[0097] Compared with the blank group, ** P<0.01; compared with the model group, ## P<0.01.

[0098] Among them, in the experiment of the present invention,

[0099] Weigh 15 doses of DJD, with a total weight of 1035g of medicinal materials. According to the operation in "Step 1", 436.25g of total extract was obtained, with a yield of 4.88%. The mass of freeze-dried powder of the petroleum ether part was 33.92g, with a yield of 7.77%; the mass of freeze-dried powder of the ethyl acetate part was 27.86g, with a yield of 6.39%; the mass of freeze-dried powder of the n-butanol part was 21.38g, with a yield of 4.88%.

[0100] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for evaluating the blood-tonifying activity of different polar parts of Angelica Sinensis Millettia Soup, characterized in that: The following steps are involved: Step 1: Preparation of different extraction parts of DJD (Danggui Millettia Spatholobi Decoction): Take Chinese angelica, Millettia reticulata, Rehmannia root, White peony root, Salvia miltiorrhiza, and Longan pulp, and crush them; After the medicinal materials are crushed, add 8 times the volume of pure water to soak for 1 hour, heat to boiling and then boil for 30 minutes, pour out the decoction, add 6 times the volume of pure water to the residue and continue boiling for 30 minutes, combine the two decoctions, filter, and rotary evaporate to obtain a concentrated extract; The extract is extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and then the solvent is removed by rotary evaporation, and the corresponding lyophilized powder is obtained by freeze drying. The lyophilized powder is dissolved with purified water before use; Step 2: Animal model establishment and intervention: After one week of adaptive feeding, SD rats were randomly divided into a blank group, a model group, and a DJD original formula group (6.21 g·kg -1 ), petroleum ether group (0.8 g·kg -1 ), ethyl acetate group (0.8 g·kg -1 ) and n-butanol group (0.8 g·kg -1 ), 5 in each group; Except for the normal group, the other groups were subcutaneously injected with 80 and 30 mg kg -1 Acetylphenylhydrazine solution; On days 4 to 7, 50 mg / kg was injected intraperitoneally. -1 Cyclophosphamide solution was used to prepare the blood deficiency rat model. Each group was given the drug by gavage at the set dose at the same time of modeling. The blank group and the model group were given the same amount of 0.9% NaCl solution once a day for 15 consecutive days. Step 3: Detect blood routine with automatic biochemical analyzer: Blood was collected from the medial canthal venous plexus of rats, anticoagulated with EDTA-2K, and the red blood cell count (RBC), white blood cell (WBC), hemoglobin (HGB), and platelet (PLT) levels in peripheral blood were determined using an automatic biochemical analyzer. Step 4: Detection of IFN-γ, IL-3, SCF and MMP-2 in serum by ELISA: Blood was collected from the medial canthal venous plexus of rats and placed at 4°C for 2 h. 4 r min -1 After centrifugation for 10 min, the supernatant was collected and the contents of IFN-γ, IL-3, SCF and MMP-2 in rat serum were detected according to the instructions of the ELISA detection kit; Step 5: Bone marrow tissue pathology observation: Rats were killed by cervical dislocation, and the right fibula was immediately removed under sterile conditions and immersed in a fixative (containing 100 mL of paraformaldehyde and 5.5 g of EDTA). The fixative was changed once a week. After decalcification at 4°C for 15 days, the fibula was embedded in paraffin and stained with HE and Masson, respectively, and observed under an optical microscope. Step 6. Detection of PI3K and AKT protein expression in bone marrow cells by Western blot: Rats in the blank group, model group, ethyl acetate group, n-butanol group, and DJD group were taken and killed by cervical dislocation. The left fibula was immediately removed under sterile conditions, and the contents of the fibula cavity were repeatedly flushed out with sterile PBS. -1 Centrifuge for 10 min and discard the supernatant; Add RIPA lysis buffer to the precipitate and shake it for lysis at 12000 r / min at 4°C. -1 Centrifuge for 10 min and collect the supernatant; The protein concentration of the supernatant was measured using the BCA protein quantification method; The sample was added with reduced protein loading buffer, denatured in boiling water for 10 min, and frozen for later use; The samples were added to the gel wells for SDS PAGE gel electrophoresis, transferred to PVDF (0.45 μm) membrane, and sealed with 5% skim milk at room temperature for 30 min; the corresponding primary antibodies PI3K, p-PI3K, AKT and p-AKT (1:3000) and β-actin (1:5000) were added and incubated at 4°C overnight; the membranes were washed with TBST three times every other day for 5 min each time, and then the secondary antibodies (1:5000) were added, and the membranes were shaken slowly on a shaker and incubated at room temperature for 30 min, and the membranes were quickly rinsed three times with TBST, imaged with a chemiluminescence imaging system, and photographed; The protein bands were scanned, and the grayscale values ​​were analyzed using Image J software. β-actin was used as an internal reference to perform semi-quantitative analysis of the target protein. Step 7: Statistical processing: SPSS17.0 software was used for statistical analysis. The experimental data were expressed as x±s. One-way analysis of variance (ANOVA) was used when the variance was in accordance with normal distribution. Nonparametric tests were used when it was not. P<0.05 was considered statistically significant. The above steps indicate that the treatment with Angelica Sinensis Millettia Decoction can significantly increase the level of protein phosphorylation. Angelica Sinensis Millettia Decoction regulates the expression of proteins related to the PI3K / AKT signaling pathway, restores bone marrow hematopoietic function, and thus alleviates the symptoms of blood deficiency in rats.

2. The method for evaluating the blood-tonifying activity of different polarity parts of Angelica Sinensis Millettia Soup according to claim 1, characterized in that: In the step 2, male SPF SD rats of 7 weeks old and weighing 180-220 g are used.

3. The method for evaluating the blood-tonifying activity of different polar parts of Angelica Sinensis Millettia Soup according to claim 1, characterized in that: The weight of each component in the Angelica and Millettia reticulata soup is respectively composed of 15g of Angelica sinensis, 15g of Millettia reticulata, 15g of Rehmannia root, 9g of White Peony Root, 9g of Salvia miltiorrhiza, and 6g of Longan pulp.

4. The method for evaluating the blood-tonifying activity of different polarity parts of Angelica Sinensis Millettia Soup according to claim 1, characterized in that: The equipment for rotary evaporation of the filtrate and the extract in step 1 is a rotary evaporator from Japan's EYELA company.

5. The method for evaluating the blood-tonifying activity of different polarity parts of Angelica Sinensis Millettia Soup according to claim 1, characterized in that: The analyzer used in step 3 is the XN9500 ​​fully automatic blood analyzer produced by Sysmex Corporation of Japan.

6. The method for evaluating the blood-tonifying activity of different polarity parts of Angelica Sinensis Millettia Soup according to claim 1, characterized in that: The microscope used in step 5 is a BX53 Olympus biological microscope produced by Olympus Corporation of Japan.

7. The method for evaluating the blood-tonifying activity of different polarity parts of Angelica Sinensis Millettia Soup according to claim 1, characterized in that: The equipment used to heat the sample in step six is ​​the HH-4 digital display constant temperature water bath produced by Changshu Aohua Instrument Co., Ltd.

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