Evaluation method of analgesic effect of angelica sinensis stomachache dropping pill

Abstract

The invention discloses a method for evaluating the analgesic effect of angelica sinensis stomachache dropping pills, belongs to the technical field of efficacy evaluation, and aims to solve the problem that no scientific method is provided for efficacy verification of dysmenorrhea traditional Chinese medicine preparations in the formula of angelica sinensis. The method comprises the steps of experimental mouse selection, acetic acid body twisting model test, hot plate pain model test and xylene inflammation model test. Selecting an experimental rat, and testing the primary dysmenorrhea model constructed by estradiol and oxytocin. The method comprises the following steps: constructing an analgesic effect evaluation model, and verifying analgesic and anti-inflammatory effects of angelica sinensis stomachache relieving; pharmacodynamic mechanism analysis is further carried out on the angelica sinensis stomachache dropping pill for simulating primary dysmenorrhea, it is revealed that the content of serum E2, IL-6, PROG, beta-EP and TNF-alpha of primary dysmenorrhea model rats treated by the angelica sinensis stomachache dropping pill has an adjusting effect, the effect of relieving dysmenorrhea can be achieved, the inflammation effect can be relieved by inhibiting NF-kappa B and MAPK channels, and the pain behavior can be relieved.

Description

Technical Field

[0001] This invention belongs to the field of drug efficacy evaluation technology, specifically relating to a method for evaluating the analgesic effect of Danggui Futongning Dripping Pills. Background Technology

[0002] Dysmenorrhea refers to lower abdominal pain and distension, accompanied by backache or other discomfort, occurring before, during, or after menstruation. Epidemiological studies show that over 60% of women of reproductive age may experience dysmenorrhea symptoms to varying degrees. It is mainly divided into primary dysmenorrhea (PD) and secondary dysmenorrhea (SD), with PD accounting for approximately 90%. Modern medical treatment for dysmenorrhea primarily uses oral nonsteroidal anti-inflammatory drugs (NSAIDs), with ibuprofen being the most common. Surgical treatment is also available, but it has a high recurrence rate and significant side effects.

[0003] Traditional Chinese medicine compositions and related extracts have also been widely used in the treatment of dysmenorrhea. In particular, Angelica sinensis, which is warm in nature, sweet and pungent in taste, and enters the liver, heart and spleen meridians, has been used clinically for more than two thousand years as a traditional blood-tonifying and blood-activating drug.

[0004] There are many drugs in the existing technology that contain Angelica sinensis, with Angelica sinensis abdominal pain relief pills as a representative drug. Its main component is Angelica sinensis oil, which contains active ingredients such as phthalides (ligustilide, n-butenylphthalide), organic acids (ferulic acid, vanillic acid), polysaccharides (ASDP-1) and volatile oil (0.4-0.7%).

[0005] However, there is currently a lack of methods for evaluating the efficacy and mechanism of action of Angelica sinensis-containing drugs, such as Danggui Futongning Dripping Pills, both domestically and internationally. There is a problem that many patients recognize the efficacy, but there is a lack of methods to verify the efficacy, which is very detrimental to the development of Angelica sinensis-based traditional Chinese medicine preparations.

[0006] Therefore, researchers proposed an evaluation method for the analgesic effect of Danggui Futongning Dripping Pills. Using Danggui Futongning Dripping Pills as the research object, the efficacy verification of other Chinese herbal preparations for dysmenorrhea with Danggui in the formula was extended to other Chinese herbal preparations. This provides a new method for verifying the efficacy of therapeutic drugs for dysmenorrhea patients. Summary of the Invention

[0007] The purpose of this invention is to effectively and specifically evaluate the significant therapeutic effect of Danggui Futongning on primary dysmenorrhea, and also to clearly verify its definite analgesic effect on common pain induced by various experimental models, so as to solve the problem of lack of scientific methods for verifying the efficacy of dysmenorrhea-related traditional Chinese medicine preparations containing Danggui.

[0008] To solve the above problems, the technical solution of the present invention is as follows: The evaluation method for the analgesic effect of Danggui Futongning Dripping Pills consists of the following steps: S1. Selection of experimental mice; Select laboratory mice and feed them using standard methods; S2, Acetic acid torsional model test; After one week of acclimatization, the mice were divided into the following groups: control group, low-dose Angelica sinensis abdominal pain relief pill group, medium-dose Angelica sinensis abdominal pain relief pill group, high-dose Angelica sinensis abdominal pain relief pill group, and aspirin tablet group. Seven days after administration of the drugs as described in the above grouping, each group of mice was intraperitoneally injected with an analgesic agent. The latency period was observed, the number of writhing movements within 30 minutes was recorded, and the writhing inhibition rate was calculated. By comparing the number of writhing episodes and the writhing inhibition rate in each group, a low number of writhing episodes and a high writhing inhibition rate were defined as having a good analgesic effect, and the analgesic effect of different dosages was evaluated accordingly. S3, Hot Plate Pain Model Test; S3.1 Adaptive feeding transition; After the S2 experiment was completed and the effects of the drug wore off, the mice were put into an acclimatization period for one week. S3.2 Mouse screening and grouping; Mice were initially screened from those that had undergone adaptive feeding according to the screening criteria in S2; Before the hot plate test, mice were screened to meet the pain threshold, and mice that did not meet the pain threshold were eliminated. Mice with a qualified pain threshold were randomly grouped into groups, such as the group in the acetic acid writhing test in the S2 step, and then subjected to a hot plate test after drug administration. S3.3 Hot plate pain test after drug administration; After grouping, continue to administer the medication for seven consecutive days according to the grouping method in S2; On the seventh day after administration, the pain threshold of mice was tested using a hot plate tester set at 55.0±0.1℃. The pain threshold of each mouse was tested at different administration time points. By comparing the pain threshold of each group of mice before drug administration with the pain threshold after a period of time after drug administration, the effect of drug dosage on the pain threshold of mice and the effect of the duration of drug effect after drug administration on the pain threshold were evaluated. S4, Xylene-induced inflammation model test; S4.1 Adaptive feeding transition and mouse grouping; After the S3 experiment was completed and the drug effect was eliminated, the mice were acclimatized for one week. Mice that passed the screening criteria of S2 were randomly grouped again, with the groups being the same as those in the acetic acid writhing test in S2. After grouping, administer the medication as described in the above group for 7 days to allow the drug effect to accumulate in the body; The mice were fasted for 12 hours before the last administration. 30 minutes after the last administration, 20 μL of xylene was immediately applied to both sides of the right ear of the mice using a pipette to induce inflammation. The left ear was left untreated as a control. Mice were euthanized by cervical dislocation 30 minutes later; both ears were cut off along the auricular baseline, and circular ear pieces were punched out at the same location using a 6mm diameter punch and weighed. The weight difference between the left and right ear pieces was used as the degree of auricular swelling. Data were recorded, and the swelling inhibition rate was calculated based on the degree of auricular swelling. The anti-inflammatory effect of different dosages was evaluated by comparing the degree of ear swelling and the swelling inhibition rate of mice in each group before drug administration.

[0009] Furthermore, the dosages for each group in S2 are as follows: the control group is given distilled water, the low-dose group of Danggui Futongning Dripping Pills is given a single dose of 22.5 mg / Kg, the medium-dose group of Danggui Futongning Dripping Pills is given a single dose of 45 mg / Kg, the high-dose group of Danggui Futongning Dripping Pills is given a single dose of 90 mg / Kg, and the aspirin tablet group is given a single dose of 35 mg / Kg.

[0010] Furthermore, the pain-inducing agent in S2 is a 0.8% glacial acetic acid solution.

[0011] Furthermore, in S3.2, mice were screened for pain threshold compliance. The process was as follows: Each time, one mouse was placed on a hot plate apparatus preheated to 55.0±0.1℃. The time from when the mouse was placed on the hot plate until it began to lick its paws due to heat stimulation was taken as the pain threshold of the mouse before drug administration. Mice with a pain threshold <5s or >30s, or mice that jumped during the experiment, were eliminated.

[0012] Furthermore, the selection criteria for the experimental mice in S1 are: female Kunming mice with a growth cycle of 6 weeks, a weight of 220±25g, and SPF grade.

[0013] Furthermore, it also includes the following steps: S5. Selection of experimental rats; In addition, experimental rats were selected and fed using standard methods; S6, a primary dysmenorrhea model constructed with estradiol and oxytocin; After one week of acclimatization, the rats were randomly divided into groups: Control group, model group, low-dose Angelica sinensis abdominal pain relief pills group, medium-dose Angelica sinensis abdominal pain relief pills group, high-dose Angelica sinensis abdominal pain relief pills group, and Leonurus japonicus granules group; Except for the control group, the other groups received subcutaneous injections of estradiol valerate 2.0 mg / Kg on days 1 and 10 of the experiment, and subcutaneous injections of estradiol valerate 1.0 mg / Kg on days 2-9, to simulate the fluctuation of estrogen in the human menstrual cycle. Starting on the fourth day after subcutaneous injection of estradiol valerate, estradiol valerate was injected daily according to the prescribed dose. Two hours later, 2 mL of Dang Gui Fu Tong Ning Dripping Pills were administered by gavage to each group for 7 consecutive days. The control group and the model group were administered the same volume of physiological saline by gavage. On the seventh day after administration of Danggui Futongning Dripping Pills, half an hour after administration, except for the control group, the other groups were injected with 2U of oxytocin per animal. The effects of writhing were observed and the indicators were measured within 30 minutes. S2.1 Observe the effect of torsion; Within 30 minutes of oxytocin injection, latency, number of writhing episodes, and writhing inhibition rate were observed. The indicators of each group were compared to evaluate the analgesic effect of different dosages. S2.2, Index Measurement; The following parameters were measured in SD rats within 30 minutes of oxytocin injection: S2.2.1, Serum biochemical index determination; The serum levels of E2, IL-6, PROG, TNF-α, and β-EP in SD model rats were measured using enzyme-linked immunosorbent assay (ELISA). S2.2.2, Histopathological evaluation; Histopathological evaluation included HE staining of uterine and ovarian tissues from rats with a primary dysmenorrhea model. S2.2.3 Evaluation of the mechanism of action; qPCR and Western blot analysis were performed on rat uterine tissue to detect changes in the expression levels of key genes and proteins related to inflammatory response, apoptosis, and the MAPK signaling pathway.

[0014] Furthermore, the dosages for each group were as follows: no medication was administered to the control group; distilled water was administered to the model group; the single dose of the low-dose group of Danggui Futongning Dripping Pills was 16 mg / Kg; the single dose of the medium-dose group of Danggui Futongning Dripping Pills was 32 mg / Kg; the single dose of the high-dose group of Danggui Futongning Dripping Pills was 64 mg / Kg; and the single dose of the Yimucao Granules group was 3.5 mg / Kg.

[0015] The beneficial effects of this invention are as follows: This invention first verifies the analgesic and anti-inflammatory effects of Danggui Futongning by constructing an analgesic effect evaluation model. Further, it constructs a model to analyze the pharmacodynamic mechanism of Danggui Futongning dripping pills in simulated primary dysmenorrhea. The results reveal that Danggui Futongning dripping pills regulate the serum levels of E2, IL-6, PROG, β-EP, and TNF-α in rats with primary dysmenorrhea. It also shows that Danggui Futongning dripping pills can alleviate dysmenorrhea by improving related histopathological changes and can reduce inflammation and pain by inhibiting the NF-κB and MAPK pathways.

[0016] The method of this invention can also serve as a reference for evaluating the efficacy of similar drugs for treating dysmenorrhea. Attached Figure Description

[0017] Figure 1 Image A shows HE staining of pathological tissue from rats with primary dysmenorrhea model in Experiment Example 2; Image B shows HE staining of uterine tissue from rats with primary dysmenorrhea model; Image C shows HE staining of ovarian tissue from rats with primary dysmenorrhea model. Figure 2 The effects of Danggui Futongning Dripping Pills on serum biochemical indicators E2, IL-6, PROG, TNF-α, and β-EP in rats with primary dysmenorrhea in Experiment Example 2; Figure 3 The effect of Danggui Futongning Dripping Pills on NFκB and NFκB-p65 proteins in Experiment Example 2; Figure 4 The effect of Danggui Futongning Dripping Pills on Bcl-2, BAX, and Cap-3 proteins in Experiment Example 2.

[0018] Figure 5 The effect of Danggui Futongning Dripping Pills on ERK, p-ERK, JNF, p-JNK, p38, and p-p38 proteins in Experiment Example 2. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Therefore, the following detailed description of embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0021] The drugs and animal sources used in the following examples are as follows: Drug source: The Angelica Abdominal Pain Relief Dripping Pills used in this experiment were purchased from Lanzhou Heshengtang Pharmaceutical Co., Ltd., with a specification of 15 pills / bag × 4 bags / barrel and a pill core weight of 20mg (Z62020027). The motherwort granules used in this experiment were purchased from Beijing Tongrentang Natural Medicine (Tangshan) Co., Ltd., with a specification of 15g / bag × 8 bags / box (Z13020660). Aspirin enteric-coated tablets were purchased from CSPC Ouyi Pharmaceutical Co., Ltd., with a specification of 100mg×36 tablets (H220153035). Prednisolone acetate tablets were purchased from Zhejiang Xianju Pharmaceutical Co., Ltd., with a specification of 5mg×100 tablets (H33021207). Animal sources: 72 SPF-grade female nonpregnant SD rats were purchased from Beijing Sibeifu Experimental Animal Center; 72 SPF-grade female Kunming mice were purchased from Huiji District Experimental Animal Center, Zhengzhou City, Henan Province. The animal experimental protocol was approved by the Ethics Committee of the Affiliated Hospital of Shihezi University.

[0022] Example 1 The evaluation method for the analgesic effect of Danggui Futongning Dripping Pills consists of the following steps: S1. Selection of experimental mice; Seventy-two SPF-grade female Kunming mice, with a growth cycle of 6 weeks and a weight of 220±25g, were selected and raised using standard methods. A clean-grade environment was maintained, with automatic light switching every 12 hours, an indoor temperature of 22±2℃, and a relative humidity of 40%±5%. S2, Acetic acid torsional model test; After one week of acclimatization, 60 healthy mice weighing 20-30g, exhibiting agility, and possessing thick, smooth, and glossy fur were selected and randomly divided into 5 groups of 12 mice each. The groups were as follows: The control group, the low-dose group of Danggui Futongning Dripping Pills (single dose 22.5 mg / Kg), the medium-dose group of Danggui Futongning Dripping Pills (single dose 45 mg / Kg), the high-dose group of Danggui Futongning Dripping Pills (single dose 90 mg / Kg), and the aspirin tablet group (single dose 35 mg / Kg).

[0023] Mice in the control group were administered an equal volume of distilled water by gavage, while mice in the other groups were given the drugs as described in the grouping instructions above, allowing the drug effect to accumulate in the body. On the seventh day, 30 minutes after administration, mice in each group were intraperitoneally injected with 0.8% glacial acetic acid (0.1 mL / 10 g) to induce pain. The latency period (the time from the injection of acetic acid to the first writhing) was observed, and the number of writhing episodes within 30 minutes was recorded (based on the degree of abdominal concavity, hind limb extension, and hip elevation). The writhing inhibition rate was calculated. The specific results are shown in Table 1.

[0024] Table 1. Effects of Danggui Futongning Dripping Pills on Acetic Acid Writhing Response in Mice (mean±SD, n=12) Compared with the control group: * P<0.05, ** P<0.01 Table 1 shows that, compared with the control group, administration of Danggui Futongning Dripping Pills at low doses reduced the number of writhing movements in mice with pain (P < 0.05); medium and high doses significantly reduced the number of writhing movements (P < 0.01) and significantly increased the inhibition rate. Furthermore, the analgesic effect became more pronounced with increasing drug dosage. These results suggest that Danggui Futongning Dripping Pills have analgesic effects, with the high-dose group showing superior analgesic efficacy compared to the positive control group (aspirin).

[0025] S3, Hot Plate Pain Model Test; S3.1 Adaptive feeding transition; After the S2 experiment concluded and the drug's effects wore off, all purchased mice underwent an acclimatization period of one week. This step was taken into consideration that the main component of Danggui Futongning Dripping Pills is Danggui oil, including ligustilide, with a half-life of 2h±30min, meaning it can be completely eliminated (98%) in about 10 hours; the half-life of the original aspirin drug is 15-20min, and the half-life of its salicylic acid metabolite is 2-3h, meaning it can be completely eliminated (98%) in about 12 hours; and considering that the drug was administered continuously for seven days, the mice participating in the S2 experiment were acclimatized to other female Kunming mice for one week.

[0026] S3.2 Mouse screening and grouping; According to the screening criteria of S2, qualified mice were initially screened from Kunming mice that had undergone adaptive feeding in S3.1. Considering that some mice may have recorded an initial pain threshold that was not qualified, the number of mice in the initial screening was greater than 60, and a portion of the mice were reserved for selection.

[0027] Before the hot plate test, mice need to be screened to meet the pain threshold requirement. The process is as follows: One mouse was placed on a hot plate apparatus preheated to (55.0±0.1)℃ each time. The time from when the mouse was placed on the hot plate until it began licking its paw in response to the heat stimulus was recorded as the mouse's pain threshold before drug administration. The data is shown in the first column of Table 1, which represents the average pain threshold of all mice in the corresponding group before drug administration. After the mouse exhibited the licking paw response, it was quickly removed to prevent burns. Mice with a pain threshold <5s or >30s, or those that jumped, were culled.

[0028] Sixty mice with qualified pain thresholds were randomly divided into 5 groups, similar to the groups in the acetic acid writhing test in the S2 step, and then a hot plate test was performed after drug administration.

[0029] S3.3 Hot plate pain test after drug administration; After grouping, continue to administer the drugs according to the grouping method in S2 for 7 consecutive days, once a day. The control group mice were given an equal volume of distilled water by gavage.

[0030] On the seventh day after drug administration, the pain threshold was tested using a constant-temperature hot plate tester at (55.0±0.1)℃, following the same testing method used during the screening for qualified pain thresholds. The pain threshold of each mouse was tested at 15 min, 30 min, 60 min, and 90 min, and the data were recorded, with the average value for each group of mice being calculated. The results are summarized in Table 2. Specific test results are shown in Table 2.

[0031] Table 2. Effect of Danggui Futongning Dripping Pills on the Mean Pain Threshold in Mice (mean±SD, n=12) Compared with the control group: * P<0.05, ** P<0.01 Table 2 shows that compared with the control group, the pain threshold of all treatment groups was increased, and the increase was more significant with increasing dosage. The percentage increase in pain threshold reached its peak at 90 minutes for high-dose Danggui Futongning Dripping Pills. This suggests that Danggui Futongning Dripping Pills have a sustained analgesic effect, and the effect of the normal medium dose reaches its maximum at 60 minutes. In contrast, the aspirin group showed rapid onset of action 15 minutes after administration, at which point the percentage increase in pain threshold reached its highest level, but then gradually decreased. This suggests that aspirin has a strong and rapid analgesic effect; while Danggui Futongning Dripping Pills, at medium and high doses, not only have a stronger analgesic effect but also a more sustained and stable effect.

[0032] S4, Xylene-induced inflammation model test; S4.1 Adaptive feeding transition and mouse grouping; After the S3 experiment was completed and the effects of the drug wore off, all the purchased mice were acclimatized for one week, and 60 mice were selected according to the S2 screening criteria.

[0033] They were then randomly divided into 5 groups of 12 each. The groups were as follows: The control group, the low-dose group of Danggui Futongning Dripping Pills (single dose 22.5 mg / Kg), the medium-dose group of Danggui Futongning Dripping Pills (single dose 45 mg / Kg), the high-dose group of Danggui Futongning Dripping Pills (single dose 90 mg / Kg), and the prednisone acetate tablets (single dose 35 mg / Kg).

[0034] After grouping, the mice were administered the drugs as described in the grouping for 7 days to allow the drug effect to accumulate in the body. The control group mice were given an equal volume of distilled water by gavage.

[0035] Mice were fasted for 12 hours before the last administration. Thirty minutes after the last administration, 20 μL of xylene was immediately applied to both sides of the right ear using a pipette to induce inflammation. The left ear was left untreated as a control.

[0036] Mice were euthanized by cervical dislocation 30 minutes later. Both ears were cut off along the auricular baseline, and circular ear pieces were punched out at the same location using a 6mm diameter punch. The weights of the ear pieces were then measured, and the difference in weight between the left and right ear pieces was used as the degree of swelling. Data were recorded, and the average value for each group was calculated to determine the swelling inhibition rate. Specific results are shown in Table 3.

[0037] Table 3. Effects of Danggui Futongning Dripping Pills on Ear Swelling in Mice (n=12) Compared with the control group: * P<0.05, ** P<0.01 Table 3 shows that, compared with the control group, the low, medium, and high doses of Danggui Futongning Dripping Pills all significantly inhibited xylene-induced ear swelling in mice (P < 0.01). The results indicate that Danggui Futongning Dripping Pills have anti-inflammatory effects, and the high dose of Danggui Futongning Dripping Pills showed better anti-inflammatory effects than the positive control drug prednisolone group.

[0038] Example 2 The difference from Example 1 is as follows: A primary dysmenorrhea model constructed with S2, estradiol, and oxytocin was tested. After one week of acclimatization, healthy female SD rats were randomly divided into 6 groups of 12 rats each.

[0039] They are respectively: The control group, model group, low-dose Danggui Futongning Dripping Pills (single dose 16 mg / Kg), medium-dose Danggui Futongning Dripping Pills (single dose 32 mg / Kg), high-dose Danggui Futongning Dripping Pills (single dose 64 mg / Kg), and Yimucao Granules (single dose 3.5 g / kg) group consisted of 12 animals in each group.

[0040] Except for the control group, the other groups received subcutaneous injections of estradiol valerate 2.0 mg / kg on days 1 and 10 of the experiment, and subcutaneous injections of estradiol valerate 1.0 mg / kg on days 2-9, to simulate the fluctuations of estrogen during the human menstrual cycle.

[0041] Starting on the fourth day after subcutaneous injection of estradiol valerate, estradiol valerate was injected daily according to the prescribed dose. Two hours later, 2 mL of Dang Gui Fu Tong Ning Dripping Pills were administered by gavage to each group for 7 consecutive days. The control group and the model group were administered the same volume of physiological saline by gavage.

[0042] On the seventh day after administration of Danggui Futongning Dripping Pills, half an hour after administration, except for the control group, the other groups were injected with oxytocin 2U / animal. The effects of writhing were observed and the indicators were measured within 30 minutes.

[0043] S2.1 Observe the effect of torsion; The results of the effects of oxytocin injection on body writhing within 30 minutes are shown in Table 4.

[0044] Table 4. Effects of Danggui Futongning Dripping Pills on Oxytocin-Induced Painful Writhing in SD Rats (mean±SD, n=12) Compared with the control group: * P<0.05, ** P<0.01 As shown in Table 4, compared with the control group, the number of writhing movements in the model group increased significantly, indicating that the combination of oxytocin and estradiol can successfully induce a dysmenorrhea model.

[0045] After administration of Danggui Futongning Dripping Pills, compared with the model group, both medium and high doses significantly reduced (P<0.01) the number of writhing movements in the dysmenorrhea model rats, and the inhibition rate was significantly increased. The results indicate that Danggui Futongning Dripping Pills can effectively relieve dysmenorrhea, and the analgesic effect of medium and high doses of Danggui Futongning Dripping Pills is superior to that of Yimucao Granules.

[0046] S2.2, Index Measurement; The following parameters were measured in SD rats within 30 minutes of oxytocin injection: S2.2.1, Serum biochemical index determination; The serum levels of E2, IL-6, PROG, TNF-α, and β-EP in SD model rats were measured using enzyme-linked immunosorbent assay (ELISA). The results are shown below. Figure 2 As shown, compared with the control group, the levels of β-EP and PROG in the model group rats were significantly decreased (P<0.01), while the levels of E2, IL-6, and TNF-α were significantly increased (P<0.01). Compared with the model group, the rats treated with high doses of Danggui Futongning Dripping Pills showed a significant recovery in β-EP and PROG levels (P<0.01); and a significant downregulation in the levels of E2, IL-6, and TNF-α.

[0047] S2.2.2, Histopathological evaluation; Histopathological evaluation included HE staining of uterine and ovarian tissues from rats with a primary dysmenorrhea model. The results are as follows: Figure 1 As shown: Figure A is an HE staining image of uterine tissue from a rat model of primary dysmenorrhea; Figure B is an HE staining image of ovarian tissue from a rat model of primary dysmenorrhea. Combination Figure 1 It can be seen that: in the normal control group, the endometrial mucosal epithelial cells of rats were low columnar with occasional apoptosis, and the glandular cavities were not dilated or secreted; the ovarian medulla had a large number of stromal cells, and the follicles at each stage were arranged regularly.

[0048] In the model group rats treated with estradiol, the endometrial thickness increased, vacuolation was observed, some endometrial cell nuclei were condensed, fragmented and dissolved, the uterine wall thickness decreased, and the myometrium and endometrium were accompanied by a large number of inflammatory cell infiltrations.

[0049] In the model group rats treated with estradiol, the number of primary follicles in the ovaries increased, but the number of secondary follicles and oocytes decreased significantly. Furthermore, the follicles at each level were arranged in a disordered manner, the follicular cavity was enlarged and filled with necrotic tissue, and the volume of the corpus luteum decreased.

[0050] In the high-dose group of rats treated with Danggui Futongning Dripping Pills, the arrangement of uterine mucosal epithelial cells returned to a low columnar shape, and the thickness of the uterine wall returned to normal. The ovaries showed a slight decrease in size, the follicles at all levels were arranged in a more orderly manner, and the corpus luteum showed slight changes.

[0051] S2.2.3 Evaluation of the mechanism of action; To further elucidate the pharmacodynamic mechanism of Danggui Futongning Dripping Pills in treating primary dysmenorrhea, this study used qPCR and Western blot analysis on rat uterine tissue to detect changes in the expression levels of key genes and proteins related to inflammatory response (NFκB signaling pathway), apoptosis (Bcl-2 / BAX / Caspase-3), and MAPK signaling pathway.

[0052] Depend on Figure 3 It was found that, compared with the control group, the expression of p-NFκB-65 in the model group was significantly increased (P<0.01), indicating that the NFκB signaling pathway was activated in the dysmenorrhea model. Compared with the model group, the expression of p-NFκB-65 in the low, medium, and high dose groups of Danggui Futongning Dripping Pills was significantly decreased (P<0.01), suggesting that Danggui Futongning Dripping Pills may exert its therapeutic effect by inhibiting the activation of the NFκB signaling pathway.

[0053] Depend on Figure 4 Compared with the control group, the expression of BAX and Cap-3 in the model group was significantly increased (P<0.01), while the expression of Bcl-2 was significantly decreased, indicating that the apoptosis signaling pathway was activated in the dysmenorrhea model. Compared with the model group, after high-dose treatment with Danggui Futongning Dripping Pills, the expression of Cap-3 and BAX was significantly decreased (P<0.01), while the expression of Bcl-2 significantly increased, suggesting that Danggui Futongning Dripping Pills may exert its therapeutic effect by maintaining the dynamic balance of Bcl-2 and BAX in apoptosis and accelerating nerve cell apoptosis.

[0054] Depend on Figure 5 The results showed that, compared with the control group, the expression of p-ERK, p-JNK, and p-P38 proteins in the model group was significantly increased (P<0.01), indicating that the MAPK signaling pathway was activated in the dysmenorrhea model. In contrast to the model group, the expression of these proteins gradually decreased with increasing dosage of Danggui Futongning Dripping Pills in the treatment group. This suggests that Danggui Futongning Dripping Pills may exert its therapeutic effect by inhibiting the activation of the MAPK signaling pathway.

[0055] Based on the above two embodiments and their results, it can be concluded that: This invention provides an evaluation method for the analgesic effect of Angelica sinensis abdominal pain relief dripping pills, which clearly demonstrates that Angelica sinensis abdominal pain relief dripping pills have significant analgesic effects in classic analgesia models (acetic acid writhing test, hot plate test), and confirms that they have good inhibitory effects on acute visceral pain and central pain sensitivity, thus verifying that the drug has basic analgesic efficacy.

[0056] A primary dysmenorrhea model was constructed using estradiol and oxytocin to simulate primary dysmenorrhea, and the pharmacodynamic mechanism of Danggui Futongning Dripping Pills was analyzed. The effects of Danggui Futongning Dripping Pills on E2, IL-6, PROG, β-EP, TNF-α levels, histopathological changes in rat uterine and ovarian tissues, and the effects on NFκB, MAPK, and apoptosis pathways were observed. The therapeutic effect of Danggui Futongning Dripping Pills on dysmenorrhea was clarified, providing a new therapeutic drug for dysmenorrhea.

[0057] Verification using the standardized pharmacodynamic evaluation system established by this invention shows that Danggui Futongning Dripping Pills not only exhibit a specific therapeutic effect on primary dysmenorrhea, but also demonstrate significant analgesic activity in classic acute pain models (such as the acetic acid writhing test and hot plate test), confirming that it possesses a clear broad-spectrum basic analgesic efficacy.

Claims

1. A method for evaluating the analgesic effect of Danggui Futongning dripping pills, characterized in that, The method comprises the following steps: S1, selection of experimental mice; Select experimental mice and raise them in a standard manner; S2, acetic acid writhing model test; After adaptive feeding for one week, the mice are grouped into: control group, low-dose Danggui Fentongning drop pill group, medium-dose Danggui Fentongning drop pill group, high-dose Danggui Fentongning drop pill group, and aspirin tablet group; After seven days of drug administration according to the above grouping, each group of mice is injected intraperitoneally with a pain-causing agent, the latency period is observed, the number of writhing within 30 minutes is recorded, and the writhing inhibition rate is calculated; Compare the writhing frequency and writhing inhibition rate of each group, define low writhing frequency and high writhing inhibition rate as good analgesic effect, and evaluate the analgesic effect of different dosages in this way; S3, hot plate pain model test; S3.1 adaptive feeding transition; After the experiment in S2 is completed and the drug effect is eliminated, the mice are adaptively fed for one week; S3.2 mouse screening and grouping; Select qualified mice from the mice adaptively fed in S3.1 according to the screening criteria in S2; Select mice with a qualified pain threshold and randomly group them, and the groups are as in the acetic acid writhing experiment in S2; S3.3 hot plate pain test after administration; After grouping, continue to administer the drugs according to the grouping in S2 for seven consecutive days; On the seventh day after administration, the pain threshold test is performed again using the constant temperature hot plate instrument set at 55.0±0.1℃; the pain threshold of each mouse at different administration time periods is tested; Compare the pain threshold of each group of mice before administration with the pain threshold after a period of administration to evaluate the effect of the administration amount on the pain threshold of the mice and the effect of the duration of the drug effect on the pain threshold; S4, dimethylbenzene-induced inflammation model test; S4.1 adaptive feeding transition and mouse grouping; After the experiment in S3 is completed and the drug effect is eliminated, the mice are adaptively fed for one week, and the mice that meet the screening criteria in S2 are selected; the mice are randomly grouped again, and the groups are as in the acetic acid writhing experiment in S2; After grouping, administer the drugs as described in the above grouping for seven days to allow the drug effect to accumulate in the body; Fast for 12 hours before the last administration, and 30 minutes after the last administration, immediately apply 20μL of dimethylbenzene to the right ear of the mouse to induce inflammation, and do not treat the left ear as a control; Kill the mice by cervical dislocation after 30 minutes; cut off the ears along the baseline, punch two circular ear pieces with a diameter of 6mm at the same site, and weigh them to obtain the ear swelling degree; record the data, and calculate the swelling inhibition rate; Compare the ear swelling degree and swelling inhibition rate of each group of mice before administration to evaluate the anti-inflammatory effect of different dosages. ​ 2. The method for evaluating the analgesic effect of Danggongning dripping pills according to claim 1, characterized in that, The administration amount of the group in S2 is as follows: distilled water for the control group, 22.5 mg / Kg for the low-dose group, 45 mg / Kg for the middle-dose group, 90 mg / Kg for the high-dose group, and 35 mg / Kg for the aspirin tablet group.

3. The method for evaluating the analgesic effect of the Danggongning dripping pills according to claim 1, characterized in that, The pain-causing agent in S2 is 0.8% ice acetic acid solution.

4. The method for evaluating the analgesic effect of the Danggongning dripping pills according to claim 1, characterized in that, In S3.2, the mice are screened for pain threshold qualification, and the process is as follows: one mouse is placed on a constant temperature hot plate instrument preheated to 55.0±0.1℃ at a time, and the time from the mouse being placed on the hot plate to licking the foot due to heat stimulation is recorded as the pain threshold of the mouse before administration. Mice with a pain threshold of <5 s or >30 s, or mice that jump during the experiment, are eliminated.

5. The method for evaluating the analgesic effect of Angelicae Gigantis Nodum Pill according to claim 1, characterized in that, The selection criteria for the experimental mice in S1 are as follows: 6-week-old, 220±25 g, SPF female Kunming mice.

6. The method for evaluating the analgesic effect of the Danggongning dripping pills according to claim 1, characterized in that, The method further comprises the following steps: S5, selection of experimental rats; In addition, the experimental rats are selected and raised in a standard manner. S6, testing of the primary dysmenorrhea model constructed by estradiol and oxytocin; After one week of adaptive feeding, the rats are randomly divided into the following groups: control group, model group, low-dose Angelicae Gigantis Nodum Pill group, middle-dose Angelicae Gigantis Nodum Pill group, high-dose Angelicae Gigantis Nodum Pill group, and motherwort granule group; Except for the control group, the remaining groups are subcutaneously injected with estradiol valerate 2.0 mg / Kg on the 1st and 10th days of the experiment, and subcutaneously injected with estradiol valerate 1.0 mg / Kg from the 2nd to the 9th day to simulate the fluctuation of estrogen in the human menstrual cycle. On the fourth day of subcutaneous injection of estradiol valerate, estradiol valerate is injected according to the dose, and two hours later, Angelicae Gigantis Nodum Pill 2 mL is given by gavage according to the group, for 7 consecutive days. The control group and the model group are given the same volume of normal saline by gavage. On the seventh day of administration of Angelicae Gigantis Nodum Pill, half an hour after administration, the remaining groups are injected with oxytocin 2 U per rat, and the writhing effect and index determination are observed within 30 min. S2.1, observation of the writhing effect; The latency period, writhing frequency, and writhing inhibition rate are observed within 30 min after injection of oxytocin, and the indexes of different groups are compared to evaluate the analgesic effect of different administration amounts. S2.2, index determination; The following index determination is performed on the SD rats within 30 min after injection of oxytocin: S2.2.1, determination of serum biochemical indexes; The levels of E2, IL-6, PROG, TNF-α, and β-EP in the serum of the SD model rats are determined by enzyme-linked immunosorbent assay (ELISA). S2.2.2, histopathological evaluation; Histopathological evaluation includes HE staining of the uterine tissue and ovarian tissue of the primary dysmenorrhea model rats. S2.2.3, evaluation of the pharmacodynamic mechanism; qPCR and Western blot analysis are performed on the uterine tissue of the rats to detect the changes in the expression levels of key genes and proteins related to inflammatory response, cell apoptosis, and the MAPK signaling pathway.

7. The method for evaluating the analgesic effect of the Danggongning dripping pills according to claim 6, characterized in that, The dosages for the groups described in S6 were as follows: no medication was administered to the control group; distilled water was administered to the model group; the single dose of the low-dose group of Danggui Futongning Dripping Pills was 16 mg / Kg; the single dose of the medium-dose group of Danggui Futongning Dripping Pills was 32 mg / Kg; the single dose of the high-dose group of Danggui Futongning Dripping Pills was 64 mg / Kg; and the single dose of the Yimucao Granules group was 3.5 mg / Kg.

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