CN117618367A - New traditional Chinese medicine monomer paeonol drop pill for treating premenstrual dysphoric disorder liver qi reverse syndrome and irritability subtype - Google Patents

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a paeonol dripping pill which is a novel traditional Chinese medicine monomer for treating premenstrual dysphoric disorder liver qi reverse syndrome and irritability subtype. The invention prepares paeonol drop pills reaching the standard of quality and three batches of pilot scale tests through process optimization. Compared with diazepam, the paeonol dripping pill has obvious pharmacological actions of resisting PMDD core symptom anxiety for a C57BL/6J mouse triple experiment and a social interaction experiment; compared with fluoxetine, the paeonol dripping pill has the pharmacological action mechanism of obviously improving the main symptoms of anger emotion and aggressive behavior, correcting the changes of related indexes of brain and serum which produce the symptoms and up-regulating the content of GABA, NE, P and E2 in the brain region of the Hippocampus for a Wistar rat PMDD-PWD rat model and a PMDD-PWD liver qi reverse syndrome irritability subtype rat model. The invention further discloses a clinical test scheme of the medicine for treating the liver qi reverse syndrome irritability subtype of PMDD. The invention provides a paeonol dripping pill which is a novel traditional Chinese medicine monomer for treating the liver qi reverse syndrome and irritability subtype of PMDD for the research and development of medicines for treating the PMDD.

Description

New traditional Chinese medicine monomer paeonol drop pill for treating premenstrual dysphoric disorder liver qi reverse syndrome and irritability subtype

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a paeonol dripping pill which is a novel traditional Chinese medicine monomer for treating premenstrual dysphoric disorder liver qi reverse syndrome and irritability subtype.

Background

Premenstrual dysphoric disorder (PMDD) is a severe premenstrual syndrome (PMS), which refers to a mental disorder syndrome in which women of childhood have abnormal emotion, somatic symptoms, behavioral changes and reduced working ability before menstruation (14-7 days), and the symptoms are relieved and disappeared after menstruation, and the mental disorder syndrome is concerned by the mental society due to the periodic premenstrual morbidity of the women per month, causing the patients to feel painful to their heart and affect their work and interpersonal relationship.

The international treatment PMDD first-line medicine is fluoxetine hydrochloride, sertraline and the like, and the journal of the medicine journal analysis of the central nervous in 2006 shows that the total effective rate of the medicine is less than or equal to 60 percent, and the total effective rate of the medicine is possibly improved to the upper limit of about 60 percent at present if the treatment is aimed at a definite patient subgroup. The clinical curative effect of treating PMDD is greatly sampled by the American medical institute journal of psychiatry in 2016, and the multi-center observation study shows that: sertraline has significantly better efficacy on the irritable subtype (Irritability subtype) distinguished by the DRSP scale in PMDD patients than the depressive subtype (Depressive mood subtype).

In PMS/PMDD subtype studies: PMDD has been proposed in 2007 abroad to be classified into four subtypes, anxiety type, depression type, edema type, and diet craving type; later, researchers have also proposed three types of symptoms-based emotional symptoms, body symptoms, and mixed emotional and body, but none have gained acceptance by the same party. The inventor team discovers that liver qi adverse flow, liver qi stagnation, liver fire flaming and heart and spleen deficiency four syndromes account for 95 percent of the total PMS syndrome through the investigation of the people in Sandi city in Shandong province and the people in six provinces in China and clinical epidemiology, wherein the liver qi adverse flow accounts for 58.9 percent, and the invention is characterized by high premenstrual emotion, irritability, breast distending pain and easy ignition; the liver qi depression accounts for 27.5 percent, and is characterized by low emotion, depression, chest distress, sigh and easy retraction when meeting things. Therefore, the two symptoms of liver qi inversion and liver qi depression are two main symptoms of PMS/PMDD; ICD-11 defines that PMDD is mainly characterized by Irritability and depressed mood, and the two emotion subtypes respectively correspond to the liver qi adverse qi and liver qi depression.

In the aspect of developing effective therapeutic drugs, the research on the pathogenesis of PMDD is mainly focused on the aspects of hormone secretion regulated by progesterone (P) and its metabolite tetrahydroprogesterone (allopregnanolone, ALLO) and receptor expression changes such as gamma-aminobutyric acid (gamma aminobutyric acid, GABA), but the pathological mechanism is not clear at present. However, for the syndrome of liver qi inversion of PMDD, selective 5-hydroxytryptamine reuptake inhibitors (SSRIs), such as fluoxetine, sertraline and the like, and hormonal drugs, which are commonly used in clinic, have limited effects as first-line therapeutic drugs. The inventor has developed a new drug Baixiangdan capsule (drug clinical test batch number: 2007L 05105) for treating PMDD liver qi adverse disease in earlier stage, and found that paeonol which is a main effective component in Bai Xiangdan capsules plays a main pharmacological role. Later researches show that paeonol can penetrate through the blood brain barrier to mediate the action of central brain regions, and the paeonol provides clues for researching the drug effect and pharmacological mechanism of paeonol for treating PMDD liver qi adverse disease.

Currently, the national treatment of PMDD is mainly carried out by western medicines such as international first-line medicine sertraline; the traditional Chinese medicine is mainly 'Jingping granule' which is a compound new medicine for treating PMS liver qi adverse disease and is developed and marketed in the early stage of team, and the new medicine is added with 'Xiangshao granule' for treating the indication climacteric syndrome yin deficiency and liver hyperactivity. So far, no western medicine and no Chinese medicine monomer component new medicine aiming at PMDD disease symptoms are found.

Thereby causing the scientific and technological problem of common attention of both domestic and foreign Chinese and Western doctors: how to develop a new medicine aiming at the liver qi reverse syndrome irritability subtype of the main syndrome type of PMDD and solve the problem becomes an urgent need for improving the diagnosis and treatment effect of the traditional Chinese and western medicine.

In view of the above, there is an urgent need in the art to develop new drugs that can be used by both traditional Chinese and western medicine for treating the liver-qi reverse syndrome and irritability subtype of PMDD, and are easy for patients to take.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine monomer danshenyiyiyishenphenol dripping pill for treating PMDD liver qi reverse syndrome and irritability subtype.

The term "traditional Chinese medicine monomer" refers to a single active ingredient extracted from traditional Chinese medicine, and has definite chemical structure and various pharmacological activities, such as anti-inflammatory and antioxidant stress. Paeonol is a single active ingredient extracted from such a traditional Chinese medicine.

The term "new drug" refers to a drug that is not marketed in China for chemical structure, drug composition, and pharmacological effect different from those of existing drugs. The paeonol dripping pill belongs to a new medicine pointed by the definition.

The term "paeonol dripping pill" refers to a dripping pill preparation containing paeonol as a single component.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

the invention provides a preparation method and a process flow of paeonol dripping pills for treating PMDD liver qi reverse syndrome irritability subtype, wherein the paeonol dripping pills comprise the following components in parts by mass: 3-7 parts of paeonol, 6000 20-30 parts of PEG, 5-10 parts of poloxamer and 3-5 parts of polyoxyl 40 stearate.

Specifically, the inventor finds that the paeonol dripping pill prepared by taking PEG4000 as a matrix has low hardness and is easy to crush in the research process; the paeonol dripping pill prepared by taking PEG6000 as a matrix has moderate hardness; after the PEG6000 is added with the poloxamer, the hardness is increased along with the increase of the adding proportion of the poloxamer, and when the base is prepared by only adopting the PEG6000 and the poloxamer (poloxamer 188), more foam is easy to generate in the feed liquid; after PEG6000 is added into polyoxyl 40 stearate, the hardness is reduced along with the increase of the adding proportion of polyoxyl 40 stearate, and when the base is prepared by only adopting the two substances, the matrix feed liquid is thinner.

In terms of dissolution, poloxamer and polyoxyl 40 stearate have the effect of promoting paeonol dissolution, and the polyoxyl 40 stearate has a stronger dissolution promoting effect.

Furthermore, the inventor adjusts the proportion of PEG6000, poloxamer and polyoxyl 40 stearate, so that the prepared matrix and paeonol medicine are easier to mix, the paeonol dripping pill is better in molding, the hardness is more suitable for industrial production requirements, and the dissolution rate is suitable.

In some embodiments, the paeonol dripping pill comprises the following components in parts by mass: 3-6 parts of paeonol, 6000 23-27 parts of PEG, 6-8 parts of poloxamer and 3-5 parts of polyoxyl 40 stearate.

Further, the paeonol dripping pill comprises the following components in parts by mass: 4-6 parts of paeonol, 6000 24-26 parts of PEG, 6-7 parts of poloxamer, and 3-4 parts of polyoxyl 40 stearate.

In some embodiments, the extraction process of paeonol is: soaking cortex moutan coarse powder in water for 20-40min, distilling with steam, collecting distillate, cooling and crystallizing to obtain paeonol crude product;

in the soaking process of the cortex moutan coarse powder in water, the feed liquid ratio (w/w) is 1:10-15, preferably 1:12.

Dissolving paeonol crude product with 4-6BV of 90-95% ethanol at 40-50deg.C, vacuum filtering, adding distilled water to reduce alcohol concentration below 5%, cooling, crystallizing, separating solid and liquid, and drying at 40-45deg.C under reduced pressure.

In another aspect of the present invention, the present invention provides a preparation method of the paeonol dripping pill, comprising the following steps: mixing the raw materials in proportion, heating to 70-90 ℃, and dripping, wherein the coolant is dimethyl silicone oil.

In some embodiments, the temperature of the coolant is 4-15 ℃.

Preferably, the temperature of the coolant is 4-10 ℃.

In some embodiments, the drop distance is 2-12cm.

In some embodiments, the viscosity of the coolant is 80-200 mPa-s.

Preferably, the viscosity of the coolant is 80-150 mpa.s.

Further preferably, the viscosity of the coolant is 80 to 120mpa·s.

In some embodiments, the drip speed is 20-40 dmin ^-1 。

In a second aspect, the invention provides pharmacological studies of paeonol dripping pill efficacy on two experimental animals and one experimental animal according to the requirements of Chinese medicine registration classification and reporting data.

Specifically, the C57BL/6J mice are subjected to gastric lavage administration, and OFT, EPM and LDB anxiety triple and social interaction experiments prove that 17.5mg/kg of paeonol dripping pills have the optimal anxiolytic effect.

Furthermore, an intervention effect of paeonol dripping pills on a model of a Wistar female rat PMDD-PWD and a pharmacological mechanism experiment thereof are provided, a progestogen withdrawal method is adopted for experimental rats, a model of the PMDD-PWD rat is copied, and the administration intervention result shows that: paeonol dripping pill 6.05mg/kg can significantly improve anxiety and dysphoria-like emotional behavior of PMDD-PWD model rat, and the pharmacological mechanism of the therapeutic effect is to effectively improve steroid hormones P, ALLO and E in hippocampal brain region of PMDD-PWD model rat ₂ Levels thus mediate up-regulation of central GABA and NE levels expression to correct anxiety, irritability-like emotional behavior in animal behavioral tests.

Furthermore, the paeonol dripping pill PMDD-PWD liver qi reverse syndrome irritability subtype rat model intervention effect and action mechanism experiment thereof are provided, and the living invasion method is adopted for the PMDD-PWD rats to replicate the PMDD-PWD liver qi reverse syndrome irritability subtype rat model, and the administration intervention result proves that: the paeonol drop 12.11mg/kg can effectively improve the dysphoria and irritability attack behaviors of rats with PMDD liver qi reverse syndrome models, and the pharmacological mechanism of the curative effect is that the paeonol drop 12.11mg/kg can up-regulate the content of GABA, NE and P, E2 in the brain region of the hippocampus so as to correct the dysphoria and irritability attack behaviors of rats with PMDD liver qi reverse syndrome models.

In a third aspect of the invention, the invention provides clinical studies of paeonol particles and dripping pills.

Specifically, the observation of clinical efficacy of paeonol particles for treating patients with PMS/PMDD liver qi adverse symptoms is provided, 117 patients meeting the inclusion standard of PMS/PMDD liver qi adverse symptoms are included, 93 patients with complete checking data are checked, 46 patients in paeonol particle treatment group, 34 patients in pre-flat particle control group and 13 patients in placebo group. The results show that: the clinical cure rate of paeonol in the treatment group on PMS/PMDD liver qi adverse disease is 84.2%, and the total effective rate is 96.5%; the cure rate of 3 menstrual cycles after follow-up is 68.48%, and the total effective rate is 91.96%.

Furthermore, the invention provides a clinical test and research scheme of paeonol dripping pills for treating PMDD liver qi reverse syndrome and irritability subtype drugs, wherein the research scheme is from case selection to PMDD and PMDD liver qi reverse syndrome, irritability subtype diagnosis, case inclusion and exclusion criteria thereof, and clear and complete regulation is made up to treatment course and curative effect evaluation and statistical method. The method provides necessary declaration data for registering and applying for new traditional Chinese medicine; furthermore, a research scheme is provided for developing a clinical test of paeonol dripping pills for treating PMDD liver qi reverse syndrome irritability subtype medicines.

Compared with the prior art, the one or more technical schemes at least achieve the following beneficial effects:

1. the invention prepares paeonol drop pills reaching the standard of quality and three batches of pilot scale tests through process optimization.

The paeonol dripping pills prepared by the method determine the quality standard of paeonol extracts and the quality standard of paeonol dripping pills, and three batches of paeonol dripping pill samples which reach the two quality standards in pilot scale are prepared, so that the paeonol dripping pill is easy to dissolve out and has high bioavailability; the volume is small, and the administration, the storage and the carrying are convenient; and has the advantages of being suitable for industrial production and low in production cost.

2. The invention provides a research scheme of a PMDD liver qi reverse syndrome irritability subtype animal model and a drug clinical test for PMDD drug research and development.

Pharmaceutical research and development of PMDD requires proper animal model of diseases and clinical test research prescription of pharmaceutical

In the scheme, the research shows that the PMDD liver qi reverse syndrome irritability subtype is the most main syndrome and clinical subtype of the PMDD, but the prior art does not see an animal model and a clinical test research scheme of the PMDD liver qi reverse syndrome irritability subtype, and the invention initiates a progestogen withdrawal and living invasion method to copy a rat model with clinical characteristics and pathological changes of the PMDD-PWD liver qi reverse syndrome irritability subtype; the clinical test scheme of the PMDD liver qi reverse syndrome irritability subtype drug of the structural integrity evaluation index system is disclosed for the first time, and an animal model and test scheme which are worthy of reference application are provided for the research and development of new PMDD drugs.

3. The invention creates a new paeonol drop pill of traditional Chinese medicine monomer, which is an innovation for the research and development of traditional Chinese and western medicines for treating PMDD disease syndrome subtype.

The invention proves that the paeonol dripping pill medicine efficacy experiment proves that the curative effect of treating the liver-qi reverse syndrome irritability subtype of the PMDD is definite, and the pharmacological action mechanism is definite; clinical curative effect observation of paeonol particles shows that the medicine has obvious curative effect on treating PMS/PMDD liver qi adverse disease. Therefore, the invention creates a new paeonol drop pill of the traditional Chinese medicine monomer, which is an innovation for the research and development of the traditional Chinese medicine and western medicine for treating the subtype of the PMDD disease.

4. The paeonol dripping pill is registered for applying the traditional Chinese medicine innovative medicine, is hopeful to obtain clinical test parts of the medicine, and is incorporated into the research and development planning of the applicant's cooperation medicine enterprise.

Other advantages and features of the present invention will be in part apparent from the following description of the drawings.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a process flow diagram of an embodiment of the present invention.

FIG. 2 is a photograph of a pilot plant test of the present invention for producing the resulting paeonol product.

FIG. 3 is a thin layer chromatogram of a paeonol product prepared in accordance with an embodiment of the present invention.

Fig. 4 is a physical diagram of paeonol dripping pill (a) and packaging bottle (b) prepared in the embodiment of the invention.

Fig. 5 is a thin-layer chromatogram of paeonol dripping pills according to an embodiment of the invention.

Fig. 6 is the effect of paeonol drop pills of the invention administration of 1d, 3d, 7d on the open field behavior model of mice (n=9); (A) total distance of 1dOFT movement. (B) 1dOFT central zone distance was administered. (C) administration of 1dOFT for a central zone residence time. (D) The administration was performed 1dOFT for the number of erections (the mice front paws were simultaneously lifted off the ground, suspended upright or attached upright). (E) administration of 3dOFT moves total distance. (F) administration of 3dOFT central zone distance. (G) administration of 3dOFT Central zone residence time. (H) 3dOFT was administered for an upright number of times. (I) total distance of 7dOFT movement. (J) administration of 7dOFT central zone distance. (K) administration of 7dOFT Central zone residence time. (L) number of erections administered 7 dOFT. * p <0.05, < p <0.01, < p <0.0001 vs control group; #p <0.05, #p <0.01 vs positive drug group (diazepam).

Fig. 7 is the effect of paeonol drop pill administration of the invention 1d, 3d, 7d on mice overhead maze behavioral model (n=9); (A) total distance traveled by 1 dEPM. (B) percent of times 1dEPM was administered into the open arm. (C) percent time to enter the open arm for administration of 1 dEPM. (D) total distance traveled by 3 dEPM. (E) percent of the number of times 3dEPM was administered into the open arm. (F) percent time to 3dEPM into open arms. (G) total distance traveled by 7 dEPM. (H) percent of the number of times 7dEPM was dosed into the open arm. (I) percent time to 7dEPM into open arms. * p <0.05, < p <0.01 vs. control group; #p <0.05, #p <0.01 vs positive drug group (diazepam).

Fig. 8 is the effect of paeonol drop pills of the invention administration 1d, 3d, 7d on mice bright-dark box behavior model (n=9); (A) total distance traveled by 1 dLDB. (B) 1dLDB bright field movement path of drug administration. (C) 1dLDB open area residence time of administration. (D) Dosing 1 ldb was entered into the open box the number of times (standard for the first two paws of the mice to all enter the open area). (E) total distance of 3dLDB movement of administration. (F) administering 3dLDB bright field movement path. (G) administration of 3dLDB open area residence time. (H) The total movement path of the administration 3dLDB and the administration 7dLDB enters the open box. (J) administration of 7dLDB bright field movement path. (K) administration of 7dLDB open area residence time. (L) number of times 7dLDB was dosed into the open bin. * p <0.05, < p <0.01 vs. control group; #p <0.05, #p <0.01 vs positive drug group (diazepam).

Fig. 9 is the effect of paeonol drop pill administration of the present invention 1d, 3d, 7d on the social behavior model of mice (n=9); (A) The 1dSIT first stage social preferred time coefficient percentage was administered. (B) The social interaction test of 1dSIT was administered as a percentage of the second phase social time coefficient. (C) The 3dSIT first stage social preferred time coefficient percentage was administered. (D) The social time coefficient percentage of the second phase of the 3dSIT social interaction experiment was dosed. (E) The 7dSIT first stage social preferred time coefficient percentage was administered. (F) The social interaction experiment of 7dSIT was dosed as a percentage of the second phase social time coefficient. * p <0.05, < p <0.001 vs. control group; # p <0.01, # p <0.001 vs positive drug group (diazepam).

Fig. 10 is a flow chart of the experimental effect and mechanism of paeonol dripping pills of the invention on the progestin withdrawal PMDD-PWD rat model intervention.

Fig. 11 is the effect of paeonol drop pills of the present invention on the open field behavior of rats in the PMDD-PWD model (n=6); (A) Open field experimental blank group and model group open field experimental motion trail diagram. (B) the total movement distance of the open field experiment between the administration groups. (C) The central area distance of the open field experiment between each administration group is compared. (D) Comparison of residence time in the central zone of open field experiments between the various dosing groups. (E) The number of animals standing during the open field experiment between each dosing group was compared (mice front paws were simultaneously lifted off the ground, suspended standing or attached standing). (F) the four-week movement path of the open field experiment between each administration group. * p <0.05 ratio blank; #p <0.05 ratio model group. And (3) injection: +: a progestogen withdrawal group; -: blank group.

Fig. 12 is the effect of paeonol drop pills of the present invention on the elevated plus maze behavior of a PMDD-PWD model rat (n=6); (A) An overhead cross maze blank experimental track diagram and a model experimental track diagram. (B) And comparing the total movement path of the overhead plus maze experiment among all the administration groups. (C) And comparing the movement path of the closed arm of the overhead plus maze experiment among all the administration groups. (D) The total number of times of the overhead plus maze experiments between each dosing group was compared. (E) The number of times of entry into the closed arm was compared for the overhead plus maze experiment between each dosing group. (F) The percentage of open arm entries for the overhead plus maze experiments between each dosing group was compared. (G) The percentage of time that the overhead plus maze experiment entered the open arm between each dosing group was compared. * p <0.05, < p <0.01 ratio blank; #p <0.05, #p <0.01 ratio model group. And (3) injection: +: a progestogen withdrawal group; -: blank group.

Fig. 13 is the effect of paeonol drop pills of the present invention on the behavior of the bright-dark box of PMDD-PWD model rats (n=6); (A) And (3) a blank group and model group motion trail roadmap for the light and dark box experiment. (B) And comparing the total path of experimental movement of the light and dark boxes among the administration groups. (B) And comparing the movement path of the bright and dark box experiment open area among the administration groups. (D) And comparing the movement path of dark areas of the light and dark box experiments among the administration groups. (E) Comparison of bright and dark box experimental open area residence time between each dosing group. (F) Dark box experiment dark area residence time comparison between each administration group. (G) The number of times of entering open boxes in the light and dark box experiments among the administration groups is compared (the two paws of the mice enter open areas as standard). (H) And comparing the times of entering the dark box in the light and dark box experiments among the administration groups. * p <0.05 ratio blank; #p <0.05, #p <0.01, #p <0.005 vs model group. And (3) injection: +: a progestogen withdrawal group; -: blank group

FIG. 14 shows the serum and the P, ALLO, E of the paeonol dripping pill of the present invention for the PMDD-PWD model ₂ Kit experimental results (n=4); (A) serum P content. (B) P content in hippocampal brain region. (C) the content of ALLO in serum. (D) ALLO content in the hippocampal brain region. (E) Serum E ₂ Is contained in the composition. (F) Sea horse brain region E ₂ Is contained in the composition. * P is p<0.05 ratio blank; #p (1)<0.05、##p<0.01、###p<0.005、####p<Model group 0.001 ratio. And (3) injection: +: a progestogen withdrawal group; -: blank group.

FIG. 15 shows the results of experiments (n=4) of detection of GABA and NE in the hippocampal brain region of a PMDD-PWD model by using paeonol dripping pills of the present invention; (A) GABA content in the hippocampal brain region. (B) NE content in hippocampal brain region. * p <0.05 ratio blank; #p <0.05 ratio model group. And (3) injection: +: a progestogen withdrawal group; -: blank group.

FIG. 16 shows the result of immunofluorescence expression experiment of paeonol dripping pill of the present invention on the hippocampal GABAAR alpha 4 protein of PMDD-PWD model; (n=3) note: +: a progestogen withdrawal group; -: blank group; (A) results of immunofluorescent labeling in hippocampal brain region. (B) average intensity of immunofluorescence results in hippocampal brain region. And (3) injection: +: a progestogen withdrawal group; -: blank group. Integral multiple of hippocampal region: 4X, scale: 500um; local multiples: 40X, scale: 50um; the GABAAR α4 protein positive response was a red marker.

FIG. 17 shows the experimental results (n=3) of the paeonol dripping pill of the present invention on the GABAAR α4 gene expression in the hippocampal brain region of the PMDD-PWD model; and (3) injection: +: a progestogen withdrawal group; -: blank group.

Fig. 18 is a graph showing the experimental results (n=3) of paeonol dripping pills according to the present invention on the protein content of GABAAR α4 in the hippocampal brain region of PMDD-PWD model; and (3) injection: +: a progestogen withdrawal group; -: blank group.

Fig. 19 is a flow chart of the experimental process of the intervention effect and the mechanism of the paeonol dripping pill on the rat model with the PMDD liver qi adverse symptoms.

Fig. 20 is a graph showing the test result of the attack behavior of paeonol dripping pill of the present invention on PMDD liver qi adverse type rat model (n=6); (A) fight performance in model and blank groups. (B) model group and blank group baseline period fight scores. (C) a fight score for the pre-and post-dosing blank. (D) a fight score between each stress group before and after dosing. * P <0.001 vs. blank; # p <0.01, # p <0.001 ratio model group.

Fig. 21 is the results of an OFT test at NR stage (n=6) before administration of paeonol drops of the present invention to a rat model with PMDD liver qi adverse side effects; and (A) a blank group and model group OFT motion trail graph. (B) total distance of OFT movement before administration. (C) total distance traveled by the central zone of OFT prior to administration. (D) pre-dosing OFT central zone residence time. * p <0.05, < p <0.01 vs. blank.

Fig. 22 is the results of an OFT test at NR stage (n=6) after administration of paeonol drops of the present invention to a rat model with PMDD liver qi adverse symptoms; and (A) a blank group and model group OFT motion trail graph. (B) total distance of OFT movement after administration. (C) total distance traveled by the central zone of OFT after administration. (D) central zone residence time of OFT after administration. * p <0.05, < p <0.01 ratio blank; #p <0.05 ratio model group.

Fig. 23 is the result of EPM test of paeonol dripping pill of the present invention at NR stage (n=6) before administration to rat model with PMDD liver qi adverse side effect; and (A) a blank group and stress group EPM motion path track diagram. (B) total distance traveled by EPM prior to administration. (C) percent of open arm access times of EPM prior to dosing. (D) percent of open arm time of EPM prior to dosing. * p <0.05 ratio blank.

Fig. 24 is the result of EPM test at NR stage (n=6) after administration of paeonol drop pills of the present invention to a rat model with PMDD liver qi adverse symptoms; and (A) a blank group and model group EPM motion path track diagram. (B) EPM total distance of exercise after dosing (C) percentage of EPM open arm access times after dosing. (D) percent of open arm time of EPM after dosing. * p <0.05, < p <0.005 vs. blank; #p <0.05, #p <0.01, #p <0.005 vs model group.

FIG. 25 shows serum, hippocampal region P, ALLO, E before and after administration of paeonol dripping pill of the present invention to PMDD liver qi adverse side effect rat model ₂ Test results (n=4); (A) P content between the peripheral serum pre-and post-dosing experimental groups. (B) P content between the various experimental groups after administration to the hippocampal brain region. (C) ALLO content between the peripheral serum pre-and post-dosing groups. (D) ALLO content between the various experimental groups after administration in the hippocampal brain region. (E) E between the groups of experiments before and after administration of peripheral serum ₂ The content is as follows. (F) E between the experimental groups after administration in the hippocampal brain region ₂ The content is as follows. * P is p<0.05、**p<A 0.01 ratio blank; #p (1)<0.05、##p<0.01、###p<0.005 ratio model group.

FIG. 26 shows the results of paeonol dripping pills of the present invention on GABA and NE in the hippocampal brain region of a rat model with PMDD liver qi adverse effect (n=4); (A) GABA content between the various experimental groups in the hippocampal brain region following administration. (B) NE content between the various experimental groups of the hippocampal brain region after administration. * p <0.05 ratio blank; #p <0.05 ratio model group.

FIG. 27 shows the results of immunofluorescence test (n=3) of paeonol dripping pills of the present invention on GABAAR alpha 4 protein of the hippocampal brain region of a rat in a model of PMDD liver qi reverse syndrome; (A) average intensity of immunofluorescence results in hippocampal brain region. (B) results of immunofluorescent labeling in hippocampal brain region. Integral multiple of hippocampal region: 4X, scale: 500um; local multiples: 40X, scale: 50um; the GABAAR α4 protein positive response was a red marker.

FIG. 28 shows the results of RT-PCR (n=3) of paeonol dripping pills in hippocampal brain regions of a rat model with PMDD liver qi reverse syndrome; * P <0.01 ratio blank.

Fig. 29 is a graph showing WB test results (n=3) of paeonol dripping pills of the present invention on a rat model of PMDD liver qi adverse side; * p <0.05 ratio blank; #p <0.05 ratio model group.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The invention is further illustrated by the following examples, which are given for the purpose of illustration only and are not intended to be limiting. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the sales company; the present invention is not particularly limited and can be commercially available.

Example 1

Paeonol drop pill pharmaceutical study

1. Paeonol extraction and purification process research

The paeonol steam distillation extraction process is optimized by an orthogonal test method, and the test result shows that: the size order of the influence of the granularity, the water adding amount and the collection volume on the paeonol extraction process is that the collection volume > granularity > water adding amount. The granularity and the collection volume are both significant, i.e. have significant influence on paeonol extraction. The average value of the coarse powder (40 meshes) and the coarse powder (80 meshes) is obviously larger than that of decoction pieces, the crystal yield of the coarse powder is higher than that of the coarse powder, but the influence is not very obvious, and in actual industrial production, the problem of blockage during filtration of waste residues and waste liquid can be caused, and potential safety hazards are brought to production, so that the coarse powder which is more convenient to pre-treat and safer to produce and operate is selected as the optimal medicinal material granularity. According to the result, the optimal paeonol steam distillation extraction process is determined to be the coarse powder, 12BV of water is added, and 6BV of distillate is collected. According to the result of the orthogonal experiment, 3 batches of verification tests are carried out on the orthogonal optimal process condition, and the average transfer rate of three batches of paeonol is 84.12%, so that the process is proved to be good in reproducibility, stable and feasible.

According to the solubility difference of paeonol in different temperatures and different alcohol concentrations, the paeonol crystal yield is taken as an evaluation index, the paeonol purification process is inspected, and experimental results show that: the temperature can influence the dissolution condition of paeonol in ethanol, in order to save production cost, the ethanol addition amount when paeonol is dissolved at different temperatures and the crystallization condition of direct refrigeration crystallization at corresponding temperatures are examined, the higher the temperature is, the lower the amount of ethanol is added when paeonol is dissolved, the higher the crystallization rate is, but the crystallization caking condition occurs, the analysis reason is probably due to the fact that the ethanol addition amount is in a critical range, and paeonol is greatly separated out after cooling.

Then, under the same temperature, the influence of different alcohol addition amounts on the paeonol crystal crystallization condition is examined, and the paeonol crystal crystallization condition is best under the condition that 5BV of 95% ethanol is dissolved. However, since the crystallization rate is low and the loss is excessive, it is considered to decrease the alcohol concentration and increase the crystallization amount. With the decrease of the alcohol concentration, the crystallization rate of paeonol increases, when the alcohol concentration is reduced to 5%, the crystallization rate reaches 91.89%, and the difference from the crystallization rate of 1% is not large, in terms of crystallization effect, crystals are needle-shaped when the alcohol concentration is reduced to 50%, crystals are mostly needle-shaped when the alcohol concentration is reduced to 30%, and a small part of crystals are fine powder, and when the alcohol concentration is reduced to 10%, 5% and 1%, the crystals are powdery.

Considering the crystal yield and crystallization condition, considering the gradient to reduce the alcohol concentration to 5%, and then carrying out suction filtration to obtain the crystal. Therefore, 5BV ethanol is adopted to dissolve the crude product at 45 ℃, the crude product is placed at room temperature after being filtered while the crude product is hot, distilled water is added to be reduced to 5 percent in gradient, cold storage crystallization, filtration and decompression drying at 45 ℃ are carried out, and paeonol crystals are obtained.

TABLE 1 optimization orthogonal and visual analysis tables for paeonol extraction process

TABLE 2 analysis of variance table

Factors of	Sum of squares of deviation	Degree of freedom	F ratio	Critical value F	Significance of the invention
						Particle size	352.213	2	19.241	19.000	*
Water adding quantity BV	60.461	2	3.303	19.000
						Collection volume BV	530.305	2	28.970	19.000	*
Error of	18.30	2

TABLE 3 extraction process validation test results

TABLE 4 ethanol consumption and corresponding crystallization of paeonol in 95% ethanol at different temperatures

TABLE 5 influence of different 95% ethanol amounts on paeonol crystallization at 45℃

	3BV	5BV	7BV
				Crystallization condition	Fine needle-like crystals with agglomerates	Needle-like fine crystals	Needle-like coarse crystals
Yield of crystal	50.20％	41.87％	40.52％

TABLE 6 influence of alcohol concentration on paeonol crystallization

2. Pilot experiment research for paeonol extraction and purification

3 pilot tests were performed according to the established process route and experimental conditions. Weighing proper amount of the coarse powder of cortex moutan, soaking the medicinal materials in 12 times of water for 30min, extracting by steam distillation, and collecting distillate of 6BV. And (3) standing the distillate to room temperature, refrigerating and crystallizing for 12 hours, and carrying out suction filtration to obtain a paeonol wet crystal crude product.

The crude product is completely dissolved by 5BV ethanol at 45 ℃, is placed at room temperature after being filtered while the crude product is hot, is added with distilled water to reduce the alcohol concentration to below 5 percent in a gradient way, is refrigerated and crystallized for 12 hours, is filtered by suction, is dried for 6 hours at 45 ℃ under reduced pressure, and thus the dry crystal is obtained. The average transfer rate of paeonol of three pilot-scale products is 78.00%, and the average paeonol content is 99.22%.

TABLE 7 results of pilot plant experiments on paeonol extraction

3. Research on paeonol dripping pill preparation process

Because paeonol has poor water solubility and low dissolution rate, the paeonol is used as a dripping pill to improve the dissolution performance of the medicine and the dissolution rate. The type and the amount of the matrix, the type of the coolant, the cooling temperature, the drop distance, the dropping speed and the feed liquid temperature in the prescription are inspected through a single factor experiment; taking the roundness, the pill weight difference and the dissolution rate as indexes to examine the performance of the dripping pill.

(1) Matrix proportion and medicine auxiliary proportion research

The matrix screening result shows that the paeonol dripping pill prepared by taking PEG4000 as the matrix has low hardness and is easy to crush; the paeonol dripping pill prepared by taking PEG6000 as a matrix has moderate hardness; after the PEG6000 is added with the poloxamer, the hardness is increased along with the increase of the adding proportion of the poloxamer; after PEG6000 is added into polyoxyl 40 stearate, the hardness is reduced along with the increase of the adding proportion of polyoxyl 40 stearate, so that the PEG6000: poloxamer = 8:2, the hardness of the dripping pill is the largest, and PEG6000 comprises poloxamer: polyoxyl 40 stearate = 7:2: under condition 1, the dripping pill is harder, and the hardness of the dripping pill is moderate under other matrix proportions. From the aspect of dissolution, both poloxamer and polyoxyl 40 stearate have the effect of promoting paeonol dissolution, and the polyoxyl 40 stearate has stronger dissolution promoting effect. Considering the three aspects of the roundness, the hardness and the dissolution rate, when the matrix ratio is PEG 6000:poloxamer: polyoxyl 40 stearate = 7:2:1, the matrix and the medicine are easier to mix, the molding is better, the hardness is more suitable for the industrial production, the dissolution rate is proper, so PEG6000 is selected from poloxamer: polyoxyl 40 stearate = 7:2: 1.

The research result of the medicine auxiliary ratio shows that in the roundness investigation, the medicine auxiliary ratio is 1:1, the dripping pill has poor roundness, the hardness measurement does not meet the requirement, and the data is meaningless; medicine auxiliary ratio 1: 3. 1: 5. 1: 7. 1:9, the roundness is obviously improved from 1: and 3, the roundness is good, and the requirement of the dripping pill on the roundness is met.

Hardness investigation shows that with the increase of the proportion of the auxiliary material matrix, the hardness of the dripping pill is increased, and the medicine auxiliary ratio is 1:9, the hardness of the dripping pill reaches the maximum, and the medicine auxiliary ratio is 1: 5. 1: 7. 1: the hardness of 9 meets the industrial production requirement. In terms of dissolution, as the proportion of auxiliary materials increases, the dissolution speed of the dripping pill is increased, and the medicine auxiliary ratio is 1:1, the dissolution rate of the dripping pill is smaller than that of paeonol monomer components, 1: and 3, the dissolution rate of the dripping pill is similar to that of the paeonol monomer, 1: 5. 1: 7. 1:9, the dissolution speed is rapidly increased, and the dissolution promoting requirement of the dripping pill is met.

Considering the three aspects of roundness, hardness and dissolution rate, the medicine auxiliary ratio is 1: 7. 1:9 meets the preparation requirement of the dripping pill, and from the viewpoint of reducing the dosage of auxiliary materials, the medicine auxiliary ratio is 1: and 7, preparing the dripping pills according to the proportion. Determining the prescription of paeonol dripping pills: 5g of paeonol, 24.5g of PEG6000, 7g of poloxamer and 3.5g of polyoxyl 40 stearate.

(2) Research on dripping technology

The viscosity investigation result of the coolant shows that the simethicone 20 has low viscosity, good fluidity, quick dropping of the dripping pill in the coolant and poor roundness; the viscosity of the simethicone 50 is slightly higher, the roundness is slightly improved, the uniformity is poor, the difference among single dropping pills is large, the viscosity of the simethicone 100, 350 and 500 is large, the dropping speed of the dropping pills is slow, the roundness is ensured, the viscosity of the simethicone 350 and 500 is too large, the fluidity is poor, the dropping speed of the dropping pills is too slow, and the simethicone 100 is selected as a coolant of the dropping pills in consideration of the production time cost. The cooling temperature examination results show that when the temperature of the coolant is higher than 10 ℃, the dripping pills are easy to adhere, the non-adhered dripping pills are ellipsoidal, the roundness is low, and the pill weight difference is large; when the temperature of the coolant is less than 4 ℃, the tailing of the dripping pill part has an influence on the roundness; when the temperature of the coolant is between 4 and 10 ℃, the roundness is good, the weight difference of the dripping pills is reduced, and the effect is optimal at 10 ℃, so the optimal temperature of the coolant of the dripping pills is determined at 10 ℃.

The investigation result of the dripping distance shows that the roundness of the dripping pill is reduced along with the increase of the dripping distance, the dripping pill has better roundness when the dripping distance is 2-12cm, the dripping pill has poor roundness when the dripping distance is 17-22cm, and part of the dripping pill is elliptical and has fine particles; meanwhile, the increase of the drop distance increases the pill weight difference of the dropping pill, and when the drop distance is 17-22cm, the pill weight difference of the dropping pill is larger. So the dripping pill has better quality when the dripping distance is 2-12 cm.

The examination results of paeonol and matrix melting temperature show that the matrix is not melted at 50 ℃; the matrix is partially melted at 60 ℃, is sticky after being stirred, and does not flow in a slurry state; the matrix melted at 70 ℃, but bubbles appeared. Then, as the temperature increases, the fluidity of the feed liquid is enhanced, the roundness of the dripping pills is increased, and the pill weight difference is reduced. Considering that paeonol has volatility, too high temperature can cause the loss of paeonol in the stirring process, so the temperature group of 100 ℃ is omitted. The pill has good roundness and weight difference when the temperature of the feed liquid is 70-90 ℃.

The dripping speed investigation result shows that the dripping speed influences the roundness and the pill weight difference of the dripping pill, when the dripping speed is 10-40d/min, the dripping pill has good roundness, when the dripping speed reaches 50d/min, the dripping pill starts to adhere, some of the dripping pill adheres to form big pills, some of the dripping pill tails, the roundness is reduced, and the fine particles are generated. The dripping speed is too large or too small, which can influence the weight difference of the dripping pills, and the dripping pills have better roundness and weight difference when the dripping speed is 20-40 d/min.

Determining a final preparation process: prescription: paeonol 5g, PEG6000 24.5g, poloxamer 188.5 g, polyoxyl 40 stearate 3.5g.

And (3) a cooling agent: simethicone (100 mPas). Cooling temperature: 10 ℃. Drop distance: 2-12cm. The temperature of the feed liquid is 70-90 ℃. Dropping speed: 10-40d/min.

Table 8 matrix formulation survey experiment results overall evaluation table

Table 9 general evaluation table of results of drug-assisted ratio investigation experiment

Medicine auxiliary ratio	Roundness degree	Hardness of	Dissolution conditions
				1：1	Molding difference	—	Slow dissolution
1：3	Is well formed	Low and low	Slow dissolution
				1：5	Is well formed	Moderate to moderate	The dissolution is faster
1：7	Is well formed	Harder and harder	Quick dissolution
				1：9	Is well formed	Hard	Quick dissolution

Table 10 coolant survey experiment results overall evaluation table

viscosity/mPa.s	Roundness degree	Speed of movementDegree of
			20	Difference of difference	Too fast
50	Preferably, it is	Quick-acting toy
			100	Good (good)	Moderate to moderate
350	Good (good)	Slow down
			500	Good (good)	Too slow

Table 11 total evaluation table of test results for cooling temperature investigation

Table 12 drip distance investigation experiment result general evaluation table

Cooling temperature/°c	Roundness degree	Pill weight difference
			0	Poor quality	Difference of difference
4	Moderate to moderate	Preferably, it is
			10	Moderate to moderate	Preferably, it is
20	Difference of difference	Difference of difference

TABLE 13 general evaluation Table of results of feed liquid temperature investigation

Feed liquid temperature/°c	Viscosity of feed liquid	Roundness degree	Pill weight difference
				50	The feed liquid is not melted	—	—
60	Partial melting	—	—
				70	Melting with a small amount of bubbles	Good formability	Smaller size
80	Melting	Good formability	Smaller size
				90	Melting	Good formability	Smaller size
100	Melting	Good formability	Small size

Table 14 drip speed investigation experiment result general evaluation table

Dripping speed/d.min -1	Roundness degree	Pill weight difference
			10	Preferably, it is	Difference of difference
20	Preferably, it is	Preferably, it is
			30	Preferably, it is	Preferably, it is
40	Preferably, it is	Preferably, it is
			50	Difference of difference	Difference of difference

4. Pilot plant test research of paeonol drop pills

3 pilot tests were performed according to the established process route and experimental conditions.

Paeonol drop pill prescription: paeonol 5g, PEG6000 24.5g, poloxamer 188.5 g, polyoxyl 40 stearate 3.5g.

The preparation method comprises the following steps: heating polyethylene glycol 6000, poloxamer 188 and polyoxyl 40 stearate to melt, adding paeonol crystal, mixing, dripping into cooled dimethyl silicone oil, and making into dripping pill or film coating.

The paeonol content in the three pilot-scale products is 92.89%, 90.40% and 90.76% of theoretical content respectively, and the average content is 91.35%.

Table 15 paeonol dripping pills pilot plant test results (average paeonol-containing quality per pill)

5. Standard study of paeonol extract quality

The quality standard of paeonol is formulated by referring to the standard of a traditional Chinese medicine extract in the Chinese pharmacopoeia of 2020 edition, and the content of the formulated standard is as follows:

the name is Paeonol, paeonol, which is an extract obtained by extracting dried root bark of Paeonia suffruticosa Paeonia Suffruticosa Andr. Of Ranunculaceae by steam distillation, purifying and drying.

The preparation method comprises the following steps: taking proper amount of the coarse powder of the cortex moutan, soaking the medicinal materials in 12 times of water for 30min, extracting by a steam distillation method, and collecting the distillate of 6BV. Standing the distillate at normal temperature for 1h, refrigerating and crystallizing for 12h, and performing suction filtration to obtain paeonol wet crystal crude products. And (3) completely dissolving the crude product with 5BV ethanol at 45 ℃, standing at room temperature after suction filtration while the crude product is hot, adding distilled water to reduce the alcohol concentration to below 5% in a gradient manner, refrigerating for crystallization for 12h, suction filtration, and drying at 45 ℃ under reduced pressure for 6h to obtain paeonol dry crystals.

Traits: the product is needle crystal or crystalline powder with white or light orange color; has special fragrance; slightly spicy. The thin layer identification refers to the method under the item of a part of cortex moutan medicinal material and cortex moutan extract in the 2020 edition of Chinese pharmacopoeia, and spots with the same color appear on the position corresponding to the reference substance chromatogram in the sample chromatogram.

And (3) moisture inspection: the water content of the tentative paeonol crystal is not more than 4.0 percent by referring to the 2020 edition of Chinese pharmacopoeia four 0831 dry weight loss determination method.

Glowing residues: the residue content of the glowing paeonol crystal is not more than 0.3% measured by referring to the residue inspection method of the four 0841 glowing parts of the 2020 edition of Chinese pharmacopoeia.

And (3) content measurement: the purity of paeonol is not lower than 98% based on dry product by referring to the high performance liquid chromatography (general rule 0512) of four parts of the 2020 edition Chinese pharmacopoeia.

TABLE 16 moisture measurement results

Lot number	2020081301	2020081402	2020081803	Average of
					Moisture content/%	2.35	2.48	2.30	2.37

TABLE 17 glowing residue inspection results

Lot number	2020081301	2020081402	2020081803	Average of
					Ignition residue content/%	0.10	0.12	0.12	0.11

Table 18. Paeonol content determination of pilot plant extracts of batch 3

6. Quality standard research of paeonol drop pills

The quality standard of paeonol dripping pills is formulated by referring to the standard of a traditional Chinese medicine preparation in the Chinese pharmacopoeia of 2020 edition, and the content of the formulated standard is as follows: prescription: paeonol 5g. The preparation method comprises the following steps: heating polyethylene glycol 6000, poloxamer 188, and polyoxyl 40 stearate to melt, adding paeonol crystal, mixing, dripping into cooled dimethyl silicone oil, and making into dripping pill or film coating. Traits: the product is white dripping pill or film coated dripping pill, and is white after removing film; fragrant smell, slightly spicy. The thin layer identification refers to the identification of a part of cortex moutan medicinal materials and cortex moutan extract under the 2020 edition of Chinese pharmacopoeia, and spots with the same color appear on the position corresponding to the reference substance chromatogram in the sample chromatogram. The weight difference inspection, the loading difference inspection and the dissolution time limit inspection meet the regulations of the dripping pill under the pill item (general rule 0108). And (3) content measurement: and (3) determining by referring to the high performance liquid chromatography (general rule 0512) of four parts of the 2020 edition Chinese pharmacopoeia, and tentatively taking a mark amount as a reference, wherein the paeonol content is not lower than 90%.

Table 19 weight difference check results for three pilot products (n=20)

Lot number	Average weight/mg	Weight difference/%
			21DP01	40.255	-4.00～+5.75
21DP02	40.300	-4.50～+5.25
			21DP03	0.369	-3.75～+5.25

Table 20 paeonol drop pill difference examination (n=10)

Lot number	Average loading/g	Load difference/%
			21DP01	3.984	-0.58～+1.94
21DP02	4.006	-0.94～+2.41
			21DP03	4.016	-0.88～+2.11

Table 21 pilot-scale test three-batch extract paeonol drop pill content determination

Sample number	21DP01	21DP02	21DP03
				Average mass/mg of paeonol in each pill	4.64	4.54	4.57

Example 2

Pharmacological study of paeonol drop pill effect: anti-anxiety and dysphoria effects of paeonol drop pills

The premenstrual anxiety depression and dysphoria are one of common main symptoms of PMDD patients, and in order to confirm the main pharmacological effects of paeonol dripping pills on PMDD and lay the pharmacological and pharmacodynamic basis of the paeonol dripping pills on the subtype of PMDD, experimental study is firstly carried out.

1.1. Experimental materials

(1) Experimental animal

C57BL/6J mice, 6-8 weeks old, 18-22g, male, 80. The production license number is provided by Beijing Vetolihua laboratory animal technology Co., ltd: SCXK (jing) 2016-0006. After the mice are transported, the mice are numbered by using a tail marking instrument, and 5 mice are fed for adapting to the environment for one week in one cage. After entering a laboratory, the light and shade period of 12h/12h is kept

(7:00 on lamp, 19:00 off lamp), free drinking water and feeding, keeping room of the fed mice at 21+ -1deg.C and humidity 45%. In order to eliminate the influence of artificial stimulus factors in the later experimental process, each animal carries out the grasping operation which is stimulated by the same gastric lavage administration every day, so that the animal is familiar with the environment and experimental personnel, and the influence of non-controlled variable factors is eliminated. All animal experimental procedures were followed by the national institute of laboratory animal care and use guide issued by NIH in the united states and approved by the laboratory animal ethics committee of the university of traditional Chinese medicine in the Shandong (approval No. DWSY 201703013).

(2) Medicine and instrument

1.2. Experimental method

(1) Design of experiment

Paeonol was given in 5 dose groups: 8.75, 17.5, 35, 70, 140mg/kg, positive control: diazepam 2mg/kg. The mice were given a dose of 10ml/kg by gavage, 1d, 3d, 7d respectively, and OFT, EPM, LDB, SIT behavioural test was performed after the last administration for 1 h. Each of the administration groups was formulated with a 0.5% sodium carboxymethylcellulose (CMC-Na) solution, and the control group was given an equal amount of solvent.

In the same batch of experiments, other behavioral tests were grouped by their body weight according to the randomized block design method, except that the OFT behavioral tests required baseline leveling for multiple exposures. Animals in different batches are used, so that the animals are prevented from being exposed to the behavior test stress environment for many times to form memory, and the experimental test result is prevented from being influenced.

All behavioural test times were fixed at 9:00am-17:00pm, tested under dim red (< 25 lux). The behavior test is according to the experimental design of the random block, so that the experimenter does not know the group of animals and the errors caused by different test time among the uniform groups of animals.

The test for screening the anxiolytic effective dose of the paeonol dripping pill is arranged as follows:

experiment one: the EPM, LDB, SIT test was performed on each of doses 1d, 3d, and 7d after weight-group.

Experiment II: the SIT, EPM, LDB test was performed on each of doses 1d, 3d, and 7d after weight-group.

Experiment III: the LDB, SIT, EPM test was performed on each of doses 1d, 3d, and 7d after weight-group.

Experiment IV: the OFT test was performed on each of doses 1d, 3d, and 7d after grouping with the total course of OFT exercise.

(2) Open field behavior test (OFT)

Referring to the method of Choles E et al, an XR-Super size animal behavior experiment video analysis tracking system is used for detection, an open field box is composed of acrylic plastic plates, the size L multiplied by W multiplied by H=40 cm multiplied by 60cm multiplied by 50cm (mice), the open field box bottom is 9 grids, and the shooting and sampling speed is high: 15 frames/second; the test time of each experimental animal is 6min, the motion trail of the animal is recorded by a computer through an infrared camera system and a video synthesizer, and the following behavior changes are analyzed at the same time: total distance of movement (Total distance), central zone distance (Center area distance), and central zone residence time (Time in center area). The experimenters after uniform standard training observe the experimental videos and record the number of erections (reporting).

After the experiment is finished, the feces of each animal are firstly cleaned by a cleaning cloth, then the bottom of the box is sprayed by 75% ethanol and is cleaned by gauze, the odor secreted by the animal is removed, and then the next animal experiment is carried out. So as to prevent the residual smell of the motion trail of the previous animal from affecting the next test mouse.

(3) Overhead plus maze behavioral testing (EPM)

With reference to the method of Pellow S et al, XR-Super size animal behavior video tracking analysis system was used for detection. The main body observation box is arranged in "+" and consists of two Open arms (Open arms), two closed arms (Close arms), a Central plane and a base. The open arm dimensions l×w were 70cm×5cm, the closed arm dimensions l×w×h were 35cm×5cm×15cm, the central block was a square of 5cm×5cm, and the base (h=50 cm) was a stainless steel bracket.

The head of the mouse is placed in the central area crossing the elevated plus maze in the direction facing any open arm, video recording is carried out through a camera after the release, then the movement track of the mouse is recorded in 5min by a video tracking analysis system, and meanwhile, related behavior indexes are analyzed. The evaluation index is as follows: (1) open Arm Entry times (OE): the number of times that more than half of the mouse body enters any open arm; (2) number of closed arms Entry (CE): the number of times that more than 2/3 of the body of the mouse enters any closed arm; (3) open Arm Time (Time in Open Arm, OT): the residence time of the mouse body at more than 2/3 of the body part entering any open arm; (4) closing arm time (Time in Close Arm, CT): the time for the more than 2/3 parts of the mouse body to enter any closed arm. (5) Total distance of exercise (Total distance); (6) the closing arm movement path (Distance in close arm). OE% and OT% were calculated from (1) to (4). The calculation method comprises the following steps:

OE％＝OE/(OE+CE)×100％，OT％＝OT/(OT+CT)×100％。

To avoid the interference of animal odor in different groups, each mouse is cleaned with towel, sprayed with 75% alcohol, wiped clean and tested for the next time. The action of grabbing the mice is required to be particularly gentle in the experimental test process, and the mice are far away from the test box body, so that the interference of smell and limb stimulation on experimental results is avoided.

(4) Light and shade box behavior test (LDB)

With reference to the Bourin M et al method, XR-Super size animal behavior video tracking was applied and simultaneously analyzed for system detection. The light and dark box is divided into two identical sizes by a whole box body and a middle inserting door (6.5 cm multiplied by 6.5 cm)

(lxwxh=25 cm×25cm×30 cm) compartment (bright area illuminated by white light and dark area illuminated by red light). At the beginning of the experiment, each mouse was placed in the center of the open area facing the partition, and the partition door was pulled away. The four paws of the animal were entered once, and were noted as one entry. The computer records the movement track of the mouse within 5min to enter the Total movement distance (Total distance), the open area residence time (Time in the light area), the open area entering times (Entries in the light area) and the open area distance (Distance in the light area) as the indexes of movement and anxiety.

(5) Social interaction behavior test (SIT)

Referring to the method of Kaidanovich-Beilin O et al, the SuperMaze animal behavior video tracking system was used for analysis and detection. The SIT experiments were performed in two stages, each stage for 10min testing, with the following parameters recorded by a computer analysis system.

In the first stage of the experiment, the experimental animal socializes with a strange mouse 1 and an empty constraint cage; in the second stage of the experiment, the experimental mice are social to strange mice 1 and 2.

The first stage of experiment:

a) Before the formal experiment starts, the mice are put into a room to be tested to adapt to the environment at least 30min in advance;

b) The shuttle door of the three-box social box is closed by a small transparent glass resin plate, and the experimental mouse is put into the middle box for 5 minutes;

c) Randomly placing strange animals A of the same species in a constraint cage in the leftmost or rightmost box of the social box, wherein animals are not in the constraint cage of the box at the other side;

d) The shuttle door baffle of the social box is taken away, so that the laboratory mouse can freely move back and forth in the three boxes for ten minutes;

e) Relevant parameter criteria: calculating social system percentage= (1)/((1) + (2))

(1) The duration of direct contact between the laboratory and strange rats was in the range of 3-5 cm.

(2) The duration of direct contact between the rats into the empty metal cages was in the range of 3-5 cm. And (3) testing a second stage: and after the first-stage experiment is finished, taking out the experimental mouse. In the first stage experiment, another constraint cage is hollow, a strange mouse B of a second same strain is placed in the constraint cage, the experimental mouse is placed in a middle social box, and then ten minutes of animal exercise behaviors are recorded. The time of contact between the experimental mice and the "familiar mice" a and "strange mice" B was observed, and the social percentage=b/(a+b) was calculated.

(6) Statistical method

Statistical analysis was performed using Graph Pad Prism 7.0 software. Normal distribution and variance alignment were checked with parameter tests, group-to-group comparisons were performed using One-Way ANOVA, and two-by-two comparisons were performed using Bonferroni's multiple comparisons test; both sets of comparisons were tested with a single tail unpaired t-test, with a significance level set at p <0.05.

1.3. Results of the study

(1) Influence of paeonol drop pill administration on open field behavior model of mice

In the OFT experiment for dosing 1 d: as shown in fig. 6A, the diazepam-dosed group was significantly elevated (p < 0.05) compared to the control group over the total course of exercise; as shown in fig. 6C, the diazepam-dosed group was significantly reduced (p < 0.05) compared to the control group during the central zone residence time; as shown in fig. 6B, 6D, there was no significant difference (p > 0.05) between the experimental groups between the central zone distance and the number of erections.

In the OFT experiment for dosing 3 d: as shown in fig. 6E, there was no significant difference (p > 0.05) between each experimental group in the total course of exercise. As shown in FIG. 6F, paeonol drops 17.5mg/kg and 70mg/kg were significantly increased (p <0.05 ) compared to diazepam group during the central zone movement path. As shown in fig. 6G, in the central zone residence time results, diazepam dosed groups were significantly reduced compared to control groups (p < 0.05); paeonol dripping pills 17.5mg/kg and 70mg/kg are remarkably increased compared with diazepam administration group (p <0.05 and p < 0.05). As shown in fig. 6H, in the results of the number of erections, the significance of diazepam administration group, 35mg/kg of paeonol was reduced (p <0.01, p < 0.05) compared to the normal group, and the significance of paeonol dripping pills 17.5mg/kg, 70mg/kg was increased (p <0.01, p < 0.05) compared to the diazepam administration group.

In the OFT experiment for dosing 7 d: as shown in fig. 6I, there was no significant difference between the individual dosing groups (p < 0.05) over the total course of exercise. As shown in fig. 6J, paeonol dripping pills 140mg/kg were significantly reduced (p < 0.01) compared with the control group in the movement path of the central zone. As shown in fig. 6K, in the central zone residence time, the diazepam-administered group, paeonol 140mg/kg was significantly reduced compared to the control group (p <0.05 ), paeonol drops 17.5mg/kg, 35mg/kg, 70mg/kg were significantly increased compared to the diazepam-administered group (p <0.05, p <0.01, p < 0.05). As shown in fig. 6L, in the number of erections, the diazepam-administered group was significantly reduced (p < 0.005) compared to the control group, and paeonol dripping pills 8.75mg/kg, 17.5mg/kg, 35mg/kg, 70mg/kg were significantly increased (p <0.01 ) compared to the diazepam-administered group.

(2) Influence of paeonol drop pill administration on mice overhead maze behavior model

In EPM experiments with 1 d: as shown in fig. 7A, 7B, 7C, there was no significant difference (p > 0.05) between each experimental group in total distance of movement, percentage of times to open arms, percentage of time to open arms.

In EPM experiments with 3d dosing: as shown in fig. 7D, there was no significant difference (p > 0.05) between the individual experimental groups in the total course of the movement. As shown in fig. 7E, in the percentage of open arm times, diazepam administration group was significantly increased (p < 0.05) compared to the control group, and 35mg/kg of paeonol drop pills was significantly decreased (p <0.05, p < 0.01) compared to the control group and the diazepam administration group. As shown in fig. 7F, 17.5mg/kg of diazepam-dosed groups and paeonol drops showed a significant increase (p <0.05 ) compared to the control group and 35mg/kg of paeonol drops showed a significant decrease (p <0.05 ) compared to the control group and the diazepam-dosed groups in the percentage of time that the open arms entered.

In EPM experiments with 7d dosing: as shown in fig. 7G, the diazepam-dosed group was significantly elevated (p < 0.01) compared to the control group over the total course of exercise. As shown in fig. 7H, 17.5mg/kg of diazepam-dosed groups and paeonol drops showed a significant increase (p <0.05 ) compared to the control group and 8.75mg/kg of paeonol drops showed a significant decrease (p < 0.05) compared to the diazepam-dosed group in the percentage of open arm entries. As shown in fig. 7I, there was no significant difference between each dosing group in the open arm entry time percentage (p < 0.05).

(3) Influence of paeonol drop pill administration on behavior model of light and dark box of mice

In LDB experiments with dosing 1 d: as shown in fig. 8A, 8B, 8C, 8D, there was no significant difference (p > 0.05) between each administration group in total movement distance, bright field residence time, bright field entry times.

In LDB experiments with 3d dosing: as shown in fig. 8E, there was no significant difference (p > 0.05) between each dosing group over the total course of exercise. As shown in fig. 8F, 35mg/kg, 140mg/kg of paeonol drops were significantly reduced (p <0.05 ) compared to diazepam-administered groups over the total course of open-range exercise. As shown in FIG. 8G, paeonol dripping pills of 8.75mg/kg, 35mg/kg, 140mg/kg were significantly reduced compared to diazepam-administered groups (p <0.05 ). As shown in fig. 8H, 17.5mg/kg of diazepam-administered group, paeonol drop pills, was significantly elevated (p <0.01, p < 0.05) compared to the control group in the open area entry times; paeonol dripping pills 8.75mg/kg, 35mg/kg and 70mg/kg are remarkably reduced compared with diazepam administration group (p <0.01, p <0.05 and p < 0.05). In LDB experiments with dosing 7 d: as shown in fig. 8I, the diazepam-dosed group was significantly elevated (p < 0.01) compared to the control group over the total course of exercise. As shown in fig. 8J, diazepam dosing group, paeonol drop pills 17.5mg/kg, 35mg/kg were significantly elevated compared to control group (p <0.01, p < 0.05) during the open area exercise path; paeonol dripping pills 8.75mg/kg, 70mg/kg and 140mg/kg are remarkably reduced compared with diazepam administration groups (p <0.01, p <0.05 and p < 0.05). As shown in fig. 8K, 17.5mg/kg of diazepam-administered group, paeonol drop pills, was significantly elevated compared to the control group in the open-zone residence time (p <0.05 ). As shown in fig. 8L, in the open area entry times, the diazepam administration group and paeonol 17.5mg/kg were significantly increased (p <0.05 ) compared to the control group, and paeonol dripping pills 8.75mg/kg, 70mg/kg, 140mg/kg were significantly decreased (p <0.01, p < 0.05) compared to the diazepam administration group.

(4) Influence of paeonol dripping pill administration on social interaction behavior model of mice

In the SIT experiments with 1 d: as shown in fig. 9A, 9B, there was no significant difference in the percentage of social preference time between the groups of animals administered during the first stage of SIT and the second stage of SIT for 1d (p > 0.05).

In the SIT experiments with 3d dosing: as shown in fig. 9C, 35mg/kg, 70mg/kg, 140mg/kg of paeonol dripping pills were significantly reduced compared to the social interaction percentage of the control group, diazepam administration group (p <0.005 ) in the first stage of SIT. As shown in fig. 9D, there was no significant difference in the percentage of social interactions (p > 0.05) between the individual dosing groups during the SIT second phase.

In the SIT experiments with 7d dosing: as shown in FIG. 9E, paeonol drops 8.75mg/kg, 17.5mg/kg, 35mg/kg, 140mg/kg were significantly elevated in the first stage of SIT compared to the control, diazepam-administered groups (p <0.05, p < 0.05; p <0.01, p <0.005, p < 0.01). As shown in fig. 9F, there was no significant difference (p > 0.05) between the individual dosing groups in the SIT second phase.

1.4 conclusion of the study

We found that 17.5mg/kg paeonol drop pills had anxiolytic effect in 3d and 7d dosing results in the anxiety triple and social interaction model; 35mg/kg of paeonol dripping pill shows anxiety-like effect at administration 3d, but has anxiolytic effect at administration 7 d; the paeonol dripping pill 140mg/kg shows anxiety-inducing effect in OFT test of 7d administration.

OFT, EPM and LDB are used as classical anxiety triple models to be sensitive to most anxiolytic drugs, the operation is simple and quick, the method is widely applied to the primary screening of the anxiolytic drugs, and the SIT models better simulate social phobia of patients suffering from clinical anxiety. The anxiety triple and social interaction model is used as a measuring tool to find the dose-effect relationship of the anti-anxiety effect of the paeonol dripping pill: the anxiolytic dose effect is bell-shaped, consistent with different effects of different concentrations of alcohol anxiolytic, similar to the ALLO anxiolytic type U dose response curve. 17.5mg/kg of paeonol drop pills has the best anxiolytic effect, has no obvious side effect on animal exercise capacity compared with diazepam, and provides a preliminary pharmacodynamic basis for the application of paeonol drop pill anxiolytic drugs in PMDD disease model.

Example 3

Pharmacological study of paeonol drop pill effect: paeonol dripping pill for treating PMDD-PWD rat model intervention effect and pharmacological mechanism thereof

Annotation: PMD is an english abbreviation for progestogen withdrawal.

1.1 Experimental materials

(1) Experimental animal

SPF-class female healthy non-pregnant 6-7 week-old Wistar rats with body mass of 140-160g and 100. The production license number is provided by Beijing Vetolihua laboratory animal technology Co., ltd: SCXK (jing) 2016-0006.

After entering the laboratory, the 12h/12h bright-dark period (8:00 on lamp, 20:00 off lamp) was maintained, and the drinking water was free to eat. The room of the raised rat is kept at 21+/-1 ℃ and the humidity is 45%. After the rats were transferred, animals were numbered using a rat tail scale and 5 animals per cage were acclimatized for one week, and then entered into the experiment.

In order to eliminate the influence of artificial stimulus factors in the later experimental process, each animal carries out the grasping operation which is stimulated by the same gastric lavage administration every day, so that the animal is familiar with the environment and experimental personnel, and the influence of non-controlled variable factors is eliminated. All experimental runs were performed under a dim red light (< 25 lux). All animal experimental procedures were followed by the national institute of laboratory animal care and use guide issued by NIH in the united states and approved by the laboratory animal ethics committee of the university of traditional Chinese medicine in the Shandong (approval No. DWSY 201703013).

1.2 Experimental methods

(1) Design of experiment

a. Grouping and administration

Animals were randomly grouped into a blank group, a model group, fluoxetine group (2.7 mg/kg) after 7d of adaptive rearing, 24.23mg/kg, 12.11mg/kg, 6.05mg/kg of paeonol, and then entered the experiment. The dose of paeonol to be administered to rats was set to be a medium dose for administration to rats using the equivalent dose coefficient algorithm=17.5 mg/kg (pre-mouse behavioural screening effective dose)/9.1×6.3. The small dose is set to 1/2 times of 6.05mg/kg of the dose in paeonol, and the large dose is set to 2 times of 24.23mg/kg of the medium dose. All drugs were fixed to volume with 0.5% CMC-Na, and the equivalent solution was administered to the blank and model groups, with the amount of 1ml/100g administered to animals by gavage.

b. Design of behavioral experiments

The PMDD-PWD rat model is prepared by injecting 7d of progestogen to 6 mg/mouse intraperitoneally and then performing gastric lavage administration. After 21d of progestin induction, progestin injection was stopped, resulting in abrupt withdrawal to induce PMDD animal model (PMDD-PWD) while dosing continued. Behavioural tests were performed and the materials were taken within 48h-72h of withdrawal, as shown in figure 10.

21d total progestogen intraperitoneal injection (6 mg/patient), 7d total progestogen intraperitoneal injection, and then grouping gastric lavage administration till the end of the experiment. OFT, EPM, LDB assays were performed and material obtained within 48-72 hours of stopping progestin injection at 21 d.

c. Microscopic index experimental detection design

Rats were randomly divided into two groups for sampling after the end of the last behavioral test. The animals were first anesthetized with 2% sodium pentobarbital (60 mg/kg), and after the palpebral reflex detection, the abdominal aorta was bled. Standing the obtained animal blood plasma at room temperature for 30min, centrifuging at 3500rpm/15min, collecting supernatant, storing in refrigerator at-80deg.C for detection, and detecting hormone index in peripheral serum with the later-stage kit.

After the first batch of animals are bloodletting, the head of the animals is broken by a mouse head breaker to peel off the skull, and the whole brain tissue is taken out by using a hemostatic forceps on a sterile workbench. And separating the hippocampal brain tissue by using sterile ophthalmic forceps, weighing the extracted tissue, rapidly placing the tissue into an EP tube with a marked number, and rapidly placing the tissue into dry ice for freezing so as to prevent the tissue from self-thawing. After the material is obtained, all tissues are stored in a refrigerator at the temperature of minus 80 ℃ for standby detection, and detection is carried out by a later-stage kit (ELISA), ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS), western immunoblotting (WB) and reverse transcription-polymerase chain reaction (RT-PCR).

The second animals were perfused with heart. The thoracic cavity is cut off from two sides of the forelimb of the rat, the heart is gently lifted by the left hand, after the ascending of the main vessel is found, the right hand uses the blunt needle to enter the right atrium from the left ventricle of the rat heart in a 45-degree left upper direction, after the needle is seen to enter the main vessel, the needle is fixed by the aortic clamp so as to prevent falling, and then the right auricle of the rat is cut off. After rapid rinsing with 120ml of physiological saline within 1min, the blood was slowly instilled with 200ml-250ml of physiological saline until the liver of the rat became white, and finally fixed with 4% paraformaldehyde solution at 4 ℃. As the blood washing method, the method is rapid and slow until the limbs of the rat are stiff. Then taking the whole brain after head breaking, placing the whole brain in a 50ml centrifuge tube filled with 4% paraformaldehyde, placing the whole brain in a refrigerator at 4 ℃ for fixing for 24 hours, and dehydrating to prepare paraffin sections for immunofluorescence detection preparation.

ELISA detection of peripheral serum and P, ALLO, E in hippocampal brain region ₂ Content changes, changes in the brain and peripheral hormonal indicators were probed.

UHPLC-MS/MS detects NE and GABA content changes in the hippocampal brain region, and detects changes of central neurotransmitter indexes.

RT-PCR, immunofluorescence and Western Blot to detect GABAAR alpha 4 subunit and protein expression in hippocampal brain region, and to probe the gene and protein changes in hippocampal brain region.

(2) Testing of open field behavior (OFT)

The experimental method is the same as the test of open field behavior.

(3) Test of elevated plus maze behavior (EPM)

The experimental method is the same as the test of the overhead plus maze behavior.

(4) Testing of light and dark box behavior (LDB)

The experimental method is the same as the test of the behavior of the light and dark box.

(5) ELISA experiments

(6) UHPLC-MS/MS detection of GABA and NE in hippocampal brain region of PMDD-PWD model rat

(7) Immunofluorescence assay

(8) RT-PCR detection of PMDD-PWD model rat hippocampal brain region regulation GABAAR alpha 4mRNA expression experiment

(9) Western Blot experiments

1.3 experimental results

(1) Influence of paeonol dripping pill on open field behavior of PMDD-PWD model rat

As shown in fig. 11B, there was no significant difference (p > 0.05) between the individual dosing groups over the total course of OFT exercise. As shown in fig. 11D, the model group was significantly reduced compared to the blank (p < 0.05) in the OFT central zone residence time. As shown in fig. 11E, in the comparison of the groups of the number of the OFT animals, the significance of the model group to the blank group was reduced (p < 0.05), and the significance of 24.23mg/kg of paeonol dripping pills was reduced (p < 0.05) compared with the model group.

As shown in fig. 11F, the fluoxetine group and paeonol drop pill 24.23mg/kg dose group were significantly reduced (p <0.05 ) compared to the model group during the OFT peripheral movement path.

(2) Influence of paeonol dripping pill on elevated plus maze behavior of PMDD-PWD model rat

As shown in fig. 12B, there was no significant difference (p > 0.05) between the individual experimental groups over the total distance traveled by the EPM. As shown in fig. 12C, the model group was significantly elevated (p < 0.05) compared to the blank group during the EPM closed arm movement path; the fluoxetine group, paeonol drop pills 24.23mg/kg dose group and 6.05mg/kg dose group have significantly reduced significance compared with the model group (p <0.05 ).

As shown in fig. 12E, the model group was significantly elevated (p < 0.05) compared to the blank group in EPM closed arm entry times; the fluoxetine group and paeonol 6.05mg/kg were significantly reduced compared to the model group (p <0.05 ). As shown in fig. 12F, 12G, the model group was significantly reduced (p < 0.05) compared to the blank group in the EPM open arm entry times percentage and entry time percentage; the fluoxetine group and paeonol dripping pill 6.05mg/kg are significantly increased compared with the model group (p <0.05 ).

(3) Influence of paeonol dripping pill on behavior of bright and dark box of rats with PMDD-PWD model

As shown in fig. 13B, there was no significant difference (p > 0.05) between the experimental groups in the LDB total course of motion.

As shown in fig. 13C, 13G, 24.23mg/kg of paeonol drop pills was significantly reduced (p <0.01, p < 0.001) compared with the model group in LDB open area movement path and open area entrance times; whereas paeonol dripping pills 6.05mg/kg dose group were significantly elevated compared to model group (p <0.05 ).

As shown in fig. 13D, the model group was significantly elevated (p < 0.05) compared to the blank group during the LDB dark space movement path; the fluoxetine group and paeonol drop pills 12.11mg/kg and 6.05mg/kg have significantly reduced significance (p <0.05 and p < 0.05) compared with the model group.

As shown in fig. 13E, paeonol drop pills 6.05mg/kg were significantly elevated (p < 0.05) compared to model group in LDB open area residence time.

As shown in fig. 13F, paeonol drop pills 6.05mg/kg were significantly reduced (p < 0.05) compared to model group in LDB dark space residence time.

As shown in fig. 13H, paeonol drop pills 24.23mg/kg dose group were significantly elevated (p < 0.01) compared to model group in LDB dark space entry times; whereas paeonol dripping pills 6.05mg/kg dose group showed significantly reduced significance (p < 0.001) compared to the model group.

(4) ELISA detection of serum and hippocampal brain regions P, ALLO and E of PMDD-PWD model rats ₂ Results

As shown in fig. 14A, 14C, and 14E, P, ALLO, and E between the respective administration groups were found in the peripheral serum ₂ There was no significant difference in hormone content (p>0.05)。

As shown in fig. 14B, in the detection of P content in hippocampal brain region, fluoxetine group and paeonol drop pill 6.05mg/kg were significantly improved compared with model group (P <0.05 ).

As shown in fig. 14D, fluoxetine group, paeonol drop pills 12.11mg/kg, 6.05mg/kg were significantly elevated compared to model group (p <0.05, p < 0.01) in the detection of ALLO content in hippocampal brain region.

As shown in fig. 14F, in the content detection of E2 in the hippocampal brain region, the model group was significantly reduced compared to the blank group (p < 0.05); the fluoxetine group and paeonol dripping pills have the significance increased 24.23mg/kg, 12.11mg/kg and 6.05mg/kg compared with the model group (p <0.01, p <0.005, p <0.01 and p < 0.001).

(5) UHPLC-MS/MS detection of GABA and NE results in hippocampal brain region of rat in PMDD-PWD model

As shown in fig. 15A, in the GABA content test in the hippocampal brain region, the model group was significantly reduced compared to the blank group (p < 0.05); the fluoxetine group and paeonol dripping pill 6.05mg/kg are obviously increased compared with the model group (p < 0.05).

As shown in fig. 15B, in the NE content test in the hippocampal brain region, the model group was significantly reduced compared to the blank group (p < 0.05); the fluoxetine group and paeonol drop pills 12.11mg/kg and 6.05mg/kg are obviously improved compared with the model group (p <0.05 and p < 0.05).

(6) Immunofluorescence localization of GABAAR alpha 4 protein results in hippocampal brain regions of PMDD-PWD model rats

As shown in fig. 16A, there was no significant difference (p > 0.05) between groups of GABAAR α4 protein content within the hippocampal brain region.

As shown in fig. 16B, in the GABAAR α4 protein localization distribution in the hippocampal brain region in the model group compared to the blank group, a significant increase in the amount and distribution range of GABAAR α4 protein can be seen in the model group compared to the blank group. The amount of GABAAR alpha 4 in the dripping pills of fluoxetine and paeonol of 6.05mg/kg is obviously reduced compared with that in the model group.

(7) RT-PCR detection of PMDD-PWD model rat hippocampal brain region regulatory GABAAR alpha 4mRNA expression results

As shown in fig. 17, there was no significant difference (p > 0.05) between groups of GABAAR α4 gene content within the hippocampal brain region.

(8) Western Blot detection of the results of the GABAAR alpha 4 protein in the hippocampal brain region of the rat of the PMDD-PWD model

As shown in fig. 18, there was no significant difference (p > 0.05) between the groups of GABAAR α4 protein content in the hippocampal brain region.

1.4 conclusion of experiment

Because the anxiety-like emotion behaviors expressed in the progestin withdrawal animal model are similar to the pathogenesis of the dysphoria and irritability emotion of a clinical PMDD patient, the research uses the progestin withdrawal preparation PMDD model to research the change of paeonol dripping pills on the anxiety-like emotion behaviors of the PMDD model to investigate the possible effect of the paeonol dripping pills on improving the anxiety-like emotion of a PMDD model rat, and discovers that 6.05mg/kg of paeonol dripping pills can obviously improve the anxiety and irritability-like emotion of the PMDD-PWD model rat, and 24.23mg/kg of paeonol dripping pills show anxiety-like effects in behavioral tests.

Progestin withdrawal induced PMDD rat model in peripheral serum E caused by abrupt withdrawal of progestin ₂ Reduced levels mediate upregulation of GABA and NE levels in the hippocampal brain region to exhibit anxiety-like mood. Fluoxetine and paeonol dripping pill 6.05mg/kg can be used for effectively improving steroid hormone P, ALLO and E in hippocampal brain region of rat in PMDD-PWD model ₂ Levels thus mediate up-regulation of central GABA and NE levels expression and correct anxiety-like emotional behavior in animal behavioral tests. Paeonol dripping pill 24.23mg/kg for upregulation of E in hippocampal brain region ₂ And NE content, but behaviorally exhibit anxiety-like effects, possibly associated with a high dose of paeonol, which is metabolically slowed in the central nervous system, as a GABA-negative modulator.

Example 4

Pharmacological study of paeonol drop pill effect: intervention effect and action mechanism of liver qi reverse syndrome irritability subtype rat model of PMDD-PWD

Based on the pharmacological effect of paeonol dripping pill on rats of PMDD-PWD model, the above experiment proves that the paeonol dripping pill has the efficacy of mainly treating the liver-qi reverse syndrome irritability subtype of PMDD and the efficacy action mechanism is illustrated, living invasion method is adopted for PMDD-PWD rats, and a model of PMDD-PWD liver qi reverse syndrome irritability subtype rats is copied, so that an intervention effect of paeonol dripping pills and an action mechanism experiment are developed.

1.1 Experimental materials

(1) Experimental animal

SPF-class female healthy non-pregnant 6-8 week-old Wistar rats with a mass of 120-160g, 100; SPF-class female healthy non-pregnant 4-6 week-old Wistar rats with body mass of 80-120g, 50. The production license number is provided by Beijing Vetolihua laboratory animal technology Co., ltd: SCXK (jing) 2016-0006.

After entering the laboratory, the 12h/12h bright-dark period (8:00 on lamp, 20:00 off lamp) was maintained, and the drinking water was free to eat. The room of the raised rat is kept at 21+/-1 ℃ and the humidity is 45%. After the rats are transferred, the animals are numbered by using a rat tail marker instrument, and 1 animal per cage is suitable for the raising environment for one week and then enters the experiment.

In order to eliminate the influence of artificial stimulus factors in the later experimental process, each animal carries out the grasping operation which is stimulated by the same gastric lavage administration every day, so that the animal is familiar with the environment and experimental personnel, and the influence of non-controlled variable factors is eliminated. All experimental runs were performed under a dim red light (< 25 lux). All animal experimental procedures were followed by the national institute of laboratory animal care and use guide issued by NIH in the united states and approved by the laboratory animal ethics committee of the university of traditional Chinese medicine in the Shandong (approval No. DWSY 201703013).

1.2 Experimental methods

(1) Design of experiment

a. Grouping and administration

The animals are subjected to ovariectomy after being adaptively raised for 7d, and then exogenous hormone supplementation is performed to induce normal estrus cycle, so that the animals are in the same estrus cycle time period, and the parallelism between experimental tests is ensured. The dose of paeonol to be administered to rats was set to be a medium dose for administration to rats using the equivalent dose coefficient algorithm=17.5 mg/kg (pre-mouse behavioural screening effective dose)/9.1×6.3. The small dose is set to 1/2 times of 6.05mg/kg of the dose in paeonol, and the large dose is set to 2 times of 24.23mg/kg of the medium dose. All drugs were fixed to volume with 0.5% CMC-Na, and the equivalent solution was administered to the blank and model groups, with the amount of 1ml/100g administered to animals by gavage.

Living intrusion tests of the R phase and the NR phase are carried out before animals are grouped, the fight score difference value of the R phase is subtracted from the fight score of the NR phase, the front 30% of the score difference value is divided into stress groups by a random grouping method, the rear 30% of the score difference value is divided into blank groups, and the rest animals kick out the experiment. The medicine is divided into a blank group, a stress +fluoxetine group (2.7 mg/kg), and 24.23mg/kg, 12.11mg/kg and 6.05mg/kg of the drop pills of stress +paeonol, and then the medicine enters an experiment.

b. Design of behavioral experiments

The ovariectomized rats (invasive rats) were kept in another laboratory (same feeding environment as normal rats) to ensure that the invasive and resident rats were strange prior to challenge behavior testing. Only in the living intrusion test (RIT) is the intrusion mouse temporarily placed in the living mouse feeding environment.

OFT and EPM were performed in NR phase before administration, and OFT, EPM, RIT detection was performed in NR phase in the third estrus cycle after administration for 3 estrus cycles. In order to better improve the exploring behavior of animals, the animal was first 3min OFT and then 5min EPM test.

All behavioural test times were fixed at 9:00am-17:00pm, tested under dim red (< 25 lux). The behavior test after the last administration for 1h is according to the random block experimental design, so that an experimenter does not know the group of animals and the errors caused by different test time among the uniform groups of animals.

After 7d of animal-adaptive rearing, ovariectomy was performed on resident mice and invasive mice. One week after the operation, the normal estrus cycle was induced by injecting hormone subcutaneously into the living mice until the end of the experiment. RIT tests were performed during the receptive and non-receptive phases of the hormone-induced second estrus cycle, with groupings being made with RIT scores (non-receptive phase). OFT, EPM, jugular vein blood sampling were performed in the non-receiving period prior to dosing, OFT, EPM, RIT testing was performed and the material was taken in the non-receiving period after 3 estrus cycles of dosing.

c. Microcosmic index detection design

(1) Jugular vein blood sampling

After behavioral testing in the pre-dosing NR phase, animals were removed after anesthesia in the exchange box using a small animal anesthesia machine and maintained under anesthesia with a respiratory mask. The rat is laid on the rat board in the supine position, the two front paws of the rat are stuck and fixed by 30-45 degrees downwards by using a medical adhesive tape, the running position of the jugular vein blood vessel is fully exposed, and the operation position is wiped and disinfected by using an alcohol cotton ball. The blood vessel was seen to be blurry in color at 0.5cm from the cervical midline in the supraclavicular fossa of the rat, and was penetrated into the blood vessel by a vertical needle penetration of about 2mm with a 1ml syringe at the pulse of the blood vessel, with the sensation of penetration felt under the hand. The needle penetration depth is too deep or too shallow, otherwise it is easy to penetrate the blood vessel or not reach the vascular site and no blood is withdrawn. Fixing the needling position, slowly drawing 0.6-0.8ml of blood on the premise of not affecting the normal physiological function of the rat, pressing a needling port by using a sterile cotton swab, immediately loosening the fixed rat forepaws, and enabling the animal to wake up immediately after closing the breathing mask. The extracted plasma was allowed to stand at room temperature for 30min and centrifuged at 3500rpm/10min to obtain a supernatant.

(2) Experimental materials

The experimental material obtaining method is the same as that of the previous experiment.

Preparation of rat model with PMDD liver qi adverse syndrome

(1) Ovariectomy

Specific methods of ovariectomy: all surgical instruments are soaked in 75% ethanol solution and then sterilized at high temperature for later use. The rats are fasted for 12 hours before the operation without water inhibition, 2% pentobarbital sodium is injected into the abdominal cavity of the rats for anesthesia (60 mg/kg), the rats are fixed on a rat plate after losing consciousness, and a small amount of physiological saline is dipped by using ophthalmic small surgical scissors to prevent fly. The method is characterized in that the position, 2.5cm away from the vaginal orifice, of the midline of the abdomen is taken as the center, the hair of a rat is cut off by scissors within the radius of 1.5cm, the skin preparation position is disinfected by iodophor, the position, which is about 1cm away from the upper connecting line of the anterior upper ridge of the rear limb, of the abdomen is cut by a surgical knife at the position, which is 0.5-1.0cm away from the midline of the abdomen, the incision is gradually and passively separated into the abdominal cavity, and the incision is prevented from too much, and the viscera of the abdominal cavity are damaged. The ophthalmic surgical curved forceps are used for finding the uterine horn from the incision to the lower part of the abdominal cavity, and the fat is slowly pulled out along the left and right sides of the uterus to wrap the uterine horn into pink mulberry shape, such as soybean ovary. Then ligating the crossing part of the rat ovary and the oviduct by using a medical suture line at the position of 0.5-1cm, shearing the rat ovary after ligating, dripping 0.1ml of penicillin potassium into the ligating part, slightly lifting the abdominal skin upwards by using ophthalmic forceps, placing the uterus back to the original position, suturing the wound of the rat, and further 0.1ml of penicillin potassium around the wound. The blood around the wound is wiped clean by a sterile cotton ball, the rats are fed in a single cage, the awakening condition of the animals is observed 1-2 hours after operation, and the animals are fed after 4 hours. Every mouse is subjected to intraperitoneal injection of 2 ten thousand units of penicillin every day 3 days after operation/wound inflammation is eliminated, infection is prevented, and the defecation and wound recovery condition of the rat is observed.

(2) Exogenous hormone induces normal estrus cycle

After 7d recovery from ovariectomy, the normal estrus cycle was induced by the artificial hormone. Each rat was subcutaneously injected with 0.5ug/0.1ml of estradiol benzoate on day 11:30 (0 h), 0.5ug/0.1ml of estradiol on day 19:30 (32 h), and 0.5mg/0.1ml of progesterone on day 7:30 (44 h).

The estrus cycle of rats is divided into non-receiving and receiving periods (four periods): non-accepted (postestrus, estrus 1, estrus 2), accepted (pre-estrus/estrus), 3d, 1d.

The first day of exogenous hormone injection belongs to estrus interval 1, the second day belongs to estrus interval 2, and the third day belongs to receiving period. All behavioral tests were tested at interval 1.

(3) PMDD liver qi reverse syndrome rat model-Resident Invasion (RIT)

Rat model was prepared by living intrusion method: residential intrusion experiments in rats estrus (R-phase) and interphase 1 (NR-phase) at 12:30 to 15:30 light was dim (< 2 lux) i.e. rat night. After 15 minutes of adaptation period, a female whose ovaries are removed is stained with black odorless vegetable dye and placed in the cage of living mice, and the cage is recorded by video equipment, fighted for 10 minutes and archived.

And then uniformly training video analysts, watching video, scoring by using attack behavior scoring software, and recording attack behavior scores-attack time, bite, climbing and climbing time. The standard for distinguishing the attack behavior and the mutual chase play between the living mice and the invasive mice is that the living mice at least show one behavior of side attack, biting and vertical hair. The hybrid attack score calculation formula is as follows:

composite aggression score = number of attacks +0.2 time of attack(s) + number of bites +0.2 time of climb(s). When the invasive mice are subjected to the challenge test, latin square design is adopted, so that different invasive mice are faced by the living mice each time.

(2) Attack behavior test (RIT)

The experimental method is the same as the attack behavior test.

(3) Open field behavior test (OFT)

The experimental method is the same as the open field behavior test.

(4) Overhead plus maze behavioral testing (EPM)

The experimental method is the same as the overhead plus maze behavior test.

(5) Kit experiment

(6) UHPLC-MS/MS experiment

(7) Immunofluorescence assay

(8) RT-PCR experiments

(9) Western Blot experiments

(10) Statistical method

Statistical analysis was performed using Graph Pad Prism 7.0 software. Normal distribution and variance alignment were checked with parameter tests, group-to-group comparisons were performed using One-Way ANOVA, and two-by-two comparisons were performed using Bonferroni's multiple comparisons test; both sets of comparisons were tested with a single tail unpaired t-test, with a significance level set at p <0.05.

1.3 experimental results

(1) Test result of paeonol dripping pill on PMDD liver qi adverse syndrome rat model aggression

As shown in fig. 20B, the baseline stress group had a significant difference (p < 0.001) compared to the blank fight score. As shown in fig. 20C, there was no significant difference (p > 0.05) between pre-dose versus post-dose blank fight scores. As shown in fig. 20D, there was no significant difference in the fight scores between the stress groups before administration, and the fight scores were significantly reduced (p <0.01, p < 0.001) for the fluoxetine group and paeonol group compared to the model group after administration treatment.

(2) Test result of paeonol dripping pill on open field behavior of rat model with PMDD liver qi adverse disease

a. Results of NR stage OFT before dosing

As shown in fig. 21B, there was no significant difference (p > 0.05) between each experimental group in the total distance of OFT exercise prior to dosing.

As shown in fig. 21C, 21D, the model group showed a significant decrease (p <0.01, p < 0.05) compared to the blank group in the travel distance of the central zone and the residence time of the central zone of the OFT before administration.

b. OFT results of NR after administration

As shown in fig. 22B, there was no significant difference (p > 0.05) between each experimental group in the total distance of OFT movement after dosing.

As shown in fig. 22C, the model group showed a significant decrease (p < 0.01) compared to the blank group in the central zone movement path of OFT after administration.

As shown in fig. 22D, the model group showed a significant decrease (p < 0.01) compared to the blank group in the central zone residence time of OFT after dosing; the fluoxetine group was significantly elevated compared to the model group (p < 0.05).

(3) Elevated plus maze experimental result of paeonol drop pill on PMDD liver qi adverse syndrome rat model

a. EPM results at pre-dosing NR stage

As shown in fig. 23B, there was no significant difference (p > 0.05) between each experimental group in the total distance traveled by EPM prior to dosing.

As shown in fig. 23C, the model group was significantly reduced (p < 0.05) compared to the blank group in EPM open arm entry times prior to dosing.

As shown in fig. 23D, there was no significant difference between the experimental groups (p < 0.05) in EPM open arm residence time prior to dosing.

b. EPM results at NR stage after dosing

As shown in fig. 24B, there was no significant difference (p > 0.05) between each experimental group in the total distance traveled by EPM after dosing.

As shown in fig. 24C, the number of EPM open arm entries after dosing, the model group was significantly reduced compared to the blank group (p < 0.05); the fluoxetine group and paeonol dripping pill 12.11mg/kg are obviously increased compared with the model group (p <0.05 ).

As shown in fig. 24D, the model group showed a significant decrease (p < 0.005) compared to the blank group in EPM open arm residence time after dosing; the fluoxetine group and paeonol dripping pill have the significance increased (p <0.01, p < 0.005) compared with the model group at 12.11 mg/kg.

(4) ELISA detection of serum before and after administration of PMDD liver qi adverse syndrome model rats, hippocampus brain regions P, ALLO, E ₂ Results

As shown in fig. 25A, there was no significant difference (P > 0.05) between the experimental groups in the P content test in the peripheral serum before dosing; after the administration treatment, 12.11mg/kg of fluoxetine group and paeonol drop pills are obviously increased compared with the P content in serum of a model group (P <0.05 and P < 0.005).

As shown in fig. 25B, in the test of P content in hippocampal brain region after administration, the P content was significantly increased (P <0.01, P <0.05, P < 0.01) in fluoxetine group, paeonol drop pill 12.11mg/kg, 6.05mg/kg compared with the model group. As shown in fig. 25C, in the test of the ALLO content in the peripheral serum, the stress group before and after administration was significantly reduced compared to the blank group (p <0.01, p < 0.05); after the administration treatment, 24.23mg/kg and 12.11mg/kg of paeonol dripping pills are obviously increased compared with the ALLO content in serum of a model group (p <0.05 and p < 0.01).

As shown in fig. 25D, there was no significant difference (p > 0.05) between each experimental group in the test of the ALLO content in the hippocampal brain region after administration.

As shown in fig. 25E, in the test of E2 content in the outer serum, each experimental group was not significantly different before and after administration (p > 0.05).

As shown in FIG. 25F, in the E2 content test in the hippocampal brain region after administration, fluoxetine group, paeonol drop pills 24.23mg/kg, 12.11mg/kg, 6.05mg/kg were all significantly elevated compared to the model group (p <0.05, p <0.01, p <0.05, p < 0.005).

(5) UHPLC-MS/MS detection of PMDD liver qi reverse syndrome model rat hippocampal brain GABA and NE results

As shown in fig. 26A, fluoxetine group was significantly elevated (p < 0.05) compared to the model group in the hippocampal brain region GABA content test.

As shown in fig. 26B, in the NE content test in the hippocampal brain region, the significance of the model group was reduced (p < 0.05) compared with the blank group, and the NE content of 12.11mg/kg of fluoxetine group and paeonol drop pills was significantly increased (p <0.05 ) compared with the model group.

(6) Immunofluorescence results on positioning of GABAAR alpha 4 protein in hippocampal brain region of rat in PMDD liver qi adverse syndrome model

As shown in fig. 27A, there was no significant difference (p > 0.05) between each experimental group in the hippocampal GABAAR α4 protein measurement.

As shown in fig. 27B, in the hippocampal GABAAR α4 protein localization distribution, a significant increase in the amount and distribution range of GABAAR α4 protein was seen in the model group compared to the blank group. The amount of GABAAR alpha 4 was significantly reduced in fluoxetine and paeonol at 12.11mg/kg compared to the model group.

(7) RT-PCR detection of PMDD liver qi reverse syndrome model rat hippocampal brain region regulation GABAAR alpha 4mRNA expression results are shown in FIG. 28, in the hippocampal brain region GABAAR alpha 4mRNA test, the model group was significantly elevated (p < 0.01) compared with the blank group.

(8) WB detection PMDD liver qi reverse syndrome model rat hippocampal brain region GABAAR alpha 4 protein expression result

As shown in fig. 29, in the GABAAR α4 protein content test in the hippocampal brain region, the model group was significantly reduced compared to the blank group (p < 0.05), and paeonol dripping pills 24.23mg/kg and 12.11mg/kg were significantly reduced compared to the model group (p <0.05 ).

1.4 conclusion of experiment

According to clinical diagnosis criteria for patients with PMDD liver qi adverse symptoms, menstrual cycle rules are required, and at least three menses are required to manifest symptoms mainly including vexation and irritability before getting damp. However, in the experimental research process, the number of animals with regular and consistent continuous three estrus periods is found to be very small, and the estrus periods of the animals are very easily influenced by factors such as self environment, diet, sleep emotion and the like. In order to eliminate the influence of the factor on the experiment, after the ovary of the animal is removed by combining foreign researches, the animal is supplemented with exogenous hormone to induce the normal estrus cycle. Modeling is carried out in NR period of animals with living invasion experiment and social isolation, and the irritability subtype emotion symptoms with irritability and irritability liver qi inverse symptoms in the early stage of clinical menstruation are simulated. The estrus control cycle is controlled by carrying out uniform hormone induction on animals, and the disease symptom manifestation is clearly related to hormone fluctuation. And (3) tightly fastening a time window for onset of the PMDD liver qi adverse syndrome, performing behavior detection and blood sampling from the jugular vein to detect the change of the content of hormone in serum in the expression period (NR period) of the rat disease of the PMDD liver qi adverse syndrome model, observing the improvement index of the drug to the disease after the drug treatment, and comparing before and after to probe the improvement effect of the drug to the disease behavior and the biological index.

We find that the symptoms of the rats with the PMDD liver qi reverse syndrome model have estrus periodic dependency, the symptoms of the rats with the PMDD liver qi reverse syndrome model show dysphoria and irritability emotion in a non-receiving period, the symptoms of the rats with the PMDD liver qi reverse syndrome model are consistent with the dysphoria and irritability emotion of the patients with the clinical PMDD liver qi reverse syndrome and irritability subtype show the dysphoria and irritability emotion in a premenstrual period, and the paeonol dripping pill 12.11mg/kg can effectively improve the symptoms of the rats with the PMDD liver qi reverse syndrome model.

RIT test utilizes the dominant effect of living mice on the self-activity field, and when facing invasion of foreign animals, the mice psychologically produce molarity pressure to show dysphoria and precaution behaviors, and the defensive behavior psychology of rodents is related to the anxiety psychology of human, so as to simulate the emotional expression of the irritability of patients with clinical PMDD liver qi adverse symptoms. In RIT test, model group animals show periodic dependence easy irritation attack action score along with the change of estrus period in non-receiving period, and attack action score before and after administration of blank group animals has no significant change, which indicates that PMDD liver qi reverse syndrome irritability subtype model is reliable, and living invasion model of Ho HP study is repeated and verified. The fluoxetine can reduce animal attack behaviors after administration, verifies that the fluoxetine as a positive drug can improve the easy-to-challenge emotion of the liver qi adverse symptoms of the PMDD, and the paeonol dripping pills can remarkably improve the easy-to-challenge emotion of the liver qi adverse symptoms of the PMDD after the drug treatment, thereby enriching the research of paeonol on the treatment of emotional diseases.

In the OFT test, the model group shows reduced activity interest in central zone distance and central zone time index before and after administration, fluoxetine medicine can improve the central zone residence time of a stress group, improve the external exploration interest of animals, lighten dysphoria-like emotional behaviors, and is consistent with the result that fluoxetine can improve anxiety/depression behaviors of animals caused by excessive glucocorticoid and increase the activity time of the animals in the central zone of the OFT. In the EPM test, the model group showed a decrease in both the percentage of open arm entries before and after dosing and the percentage of open arm entries and time after dosing. The fluoxetine and paeonol dropping pill with 12.11mg/kg can effectively improve the dysphoria-like emotion behaviors of animals in EPM, improve the activity time of the animals in an open arm, and show that the model is stable and reliable again, and the medicine can improve the dysphoria-like emotion symptoms of rats with PMDD liver qi adverse symptoms.

Sex steroid hormones in addition to synthesis and secretion in peripheral secretory glands (adrenal gland, gonad, placenta, etc.), the nervous system itself can also synthesize and secrete steroid hormones, peripheral steroids and their metabolic derivatives which act through the blood brain barrier into the nervous system, and centrally derived steroids synthesized in the brain and so-called neurosteroids whose fluctuating levels are associated with the menstrual cycle.

In ELISA tests, the P content in serum before administration was not significantly different in RIT group compared with blank group, but showed a decreasing trend; after administration, 12.11mg/kg of fluoxetine and paeonol drop pills can effectively improve the P content of peripheral serum and hippocampus, and 6.05mg/kg of paeonol drop pills also show rising trend in the hippocampus. In the pre-administration serum ALLO test, the content of the model group is reduced before and after administration, and is consistent with the peripheral serum hormone change result of a clinical PMDD liver qi adverse syndrome patient in the symptom presentation period, so that the periodic expression of the symptoms of the PMDD liver qi adverse syndrome model rat is demonstrated; after administration, 24.23mg/kg and 12.11mg/kg of paeonol dripping pills can obviously improve the ALLO content in serum. ALLO has anxiogenic effects at low concentrations and exhibits anxiolytic effects at high concentrations, which may be one of the biological indicators of paeonol drop pills for the treatment of diseases. In the hippocampal E2 test, each dose group of fluoxetine and paeonol increased the E2 content. And the results of the prior studies show that the protein expression level of E2 and progesterone receptor in the relevant brain region is reduced, and the protein expression results can be obviously up-regulated through the prior drug treatment.

GABA is an amino acid neurotransmitter, NE is a monoamine neurotransmitter, and they all belong to inhibitory neurotransmitters. The fluctuation of endogenous neurosteroid is related to the physiological pathology of GABA and NE changes, can enhance the functions of GABA and NE, and when the content of the neurosteroid is reduced, the inhibition effect on GABA and NE is reduced, and anxiety-like emotional symptoms are shown. In the UHPLC-MS/MS test in the hippocampal brain region, the GABA and NE contents of the model group show a descending trend, and 12.11mg/kg of fluoxetine and paeonol dripping pills can improve the GABA and NE contents, which is consistent with the anxiety-like emotion results which are found by earlier researches to be caused by the reduction of the GABA and NE contents in the organism, so that the model rat disease manifestation of the liver qi adverse symptoms of PMDD can be effectively treated by mediating the central neurotransmitter GABA and NE by 12.11mg/kg of fluoxetine and paeonol dripping pills.

Synthesis and changes in neurosteroids affect changes in the expression and function of GABAAR subunits in the hippocampal brain region. In WB, immunofluorescence, RT-PCR tests, we found that the model group showed negative results among the WB semi-quantitative GABAAR α4 results, and we speculated that the structure of GABAAR α4 protein was destroyed and the biological activity was lost to show negative results. In immunofluorescence assays, there was no significant difference in the average intensity of GABAAR α4 protein between the experimental groups, but in the localization of GABAAR α4 protein, a significant increase in GABAAR α4 protein expression and distribution range was seen in the model group compared to the blank group. The GABAAR alpha 4 expression of fluoxetine and paeonol dripping pills of 12.11mg/kg is obviously reduced compared with the model group. In the PCR test results, GABAAR α4mRNA expression was significantly elevated in the model group, consistent with the results of the other study, indicating that the model was successful.

The paeonol dripping pill 24.23mg/kg is consistent with the ascending trend of E2 content expression in the hippocampal brain region of the early-stage PMDD rat model, and the pathological manifestations of the PMDD liver qi reverse syndrome model rat are that the GABAAR alpha 4mRNA content is ascending and the NE content and the ALLO content in peripheral serum are descending in the hippocampal brain region. The fluoxetine and paeonol dripping pill 12.11mg/kg can up-regulate GABA, NE and P, E2 content in Hippocampus brain region and correct the dysphoria and irritability-like emotional behavior of rats with PMDD liver qi adverse syndrome model.

Example 5

Paeonol particle clinical study: clinical efficacy observation of paeonol particles for treating PMS/PMDD liver qi adverse disease

In the process of completing 973 plan traditional Chinese medicine special project and discussing liver main diarrhea regulating and emotion regulating action mechanism project from liver theory depression (light and moderate) premenstrual syndrome (severe PMDD), under the passing of clinical research ethical examination, the 'paeonol treatment premenstrual syndrome (PMS), premenstrual dysphoric disorder (PMS severe PMDD) liver qi adverse-effect clinical curative effect observation' is carried out, 117 cases of patients are observed altogether, 93 cases of checked data are examined, wherein 46 cases of paeonol particle treatment groups, 34 cases of premenstrual flat particle control groups and 13 cases of placebo groups are included.

The clinical efficacy statistics for 93 patients are as follows:

the clinical cure rate of paeonol granules of the treatment group on premenstrual syndrome (PMS, including PMDD. Same below) is 84.2%, and the total effective rate is 96.5%; the cure rate of 3 menstrual cycles is 68.48% and the total effective rate is 91.96%; the cure rate of the traditional Chinese medicine symptoms is 87.1%, and the total effective rate is 97.4%.

The clinical cure rate of PMS/PMDD by the premenstrual tablet particles of the control group is 71.3 percent, and the total effective rate is 89.6 percent; the cure rate of 3 menstrual cycles is 53.7%, and the total effective rate is 85.0%; the cure rate of the traditional Chinese medicine symptoms is 80.2%, and the total effective rate is 88.0%.

The clinical cure rate of the placebo control group to PMS/PMDD is 31.4 percent, and the total effective rate is 52.4 percent.

Paeonol particles have the main symptoms of PMS/PMDD liver qi adverse flow: the symptoms of premenstrual dysphoria, breast distending pain, headache and abdominal distension are eliminated more rapidly, and the recovery effect on work and home management is more obvious. The treatment group and the control group are compared with the clinical effect, the follow-up effect and the syndrome effect, and the paeonol treatment group is obviously better than the premenstrual level control group (P <0.01 and P < 0.05). In the clinical test process, the paeonol capsules have no adverse reaction; 1 case of premenstrual tablet reflects stomach discomfort after administration.

The observation and statistics result of the clinical curative effect show that: the paeonol has the clinical curative effect of treating PMS/PMDD liver qi adverse disease, and has the advantages of obviously improved effect, quicker onset time and more reliable and improved clinical curative effect compared with the premenstrual tablet particles.

Example 6

Clinical study of paeonol dripping pills: clinical trial research scheme for treating PMDD liver qi reverse syndrome irritability subtype drug

1.1 case selection

The study was selected using a prospective random, double blind control trial, using a parallel control random grouping method. The ratio of the control group to the treatment group cases was not less than 1: and 3, treating more than 300 cases of the total case number, and controlling the principle that the total case number is not less than 100 cases.

Random control: controls were consistent with inclusion exclusion criteria for cases; control and case inclusion time; the control is given contemporaneously with the intervention of the inclusion of the case; in addition to experimental intervention factors, other factors are distributed as identically as possible between the two groups. The control group can be divided into a blank control, a placebo control and a positive control according to the intervention measures accepted by the control group.

And determining normal menstruation women as a control group according to inclusion and exclusion criteria, wherein PMDD patients conform to liver qi reverse syndrome and subtypes conform to irritability subtypes as study subjects.

1.2 diagnostic criteria

1.2.1PMDD diagnostic criteria [ refer to the manual for diagnosis and statistics of mental diseases "DSM-5.Premenstrual Dysphoric Disorder Diagnostic Criteria ]

A. In most menstrual cycles, at least five symptoms must appear in the last week before menstruation begins, improve within days after menstruation begins, and become mild or vanish in the week after menstruation.

B. One (or more) of the following symptoms must occur:

1) Obvious emotional instability (e.g., mood swings: suddenly sad or tearing, or increased sensitivity to rejection)

2) Obvious irritability or anger or interpersonal conflicts.

3) Obvious depressed mood, desperate feeling, or self-jeer ideas.

4) Obvious anxiety, tension and/or a feeling of tension or stress.

C. The following symptom(s) must be added to achieve a total of five symptoms when combined with the symptoms in criterion B above.

1) Interest in daily activities (e.g., work, school, friends, hobbies) decreases.

2) It is difficult to concentrate on the main aspect.

3) Sleepiness, fatigue, or a significant lack of energy.

4. A significant change in appetite; binge eating by binge drinking; or a specific food craving.

5) Hypersomnia or insomnia.

6) A feeling of being conquered or out of control.

7) Physical symptoms such as breast tenderness or swelling, joint or muscle pain, a sensation of "bloating", or weight gain.

D. These symptoms are associated with clinically significant pain or interference with work, school, general social activity, or relationship to others (e.g., avoiding social activity; productivity and efficiency decline in work, school, or home).

E. This disorder is not merely an exacerbation of another disease symptom, such as major depressive disorder, panic disorder, persistent depressive disorder (dysthymia), or personality disorder (although it may occur simultaneously with any of these diseases).

F. Standard a should be validated by a prospective daily score scale (DRSP) for at least two symptom cycles. (note: diagnosis can be made temporarily before the present confirmation.)

Diagnosis: four standards of ABCD are provided, and DRSP is adopted to carry out retrospective inquiry scoring of two menstrual cycles to make temporary diagnosis; a prospective daily score of two symptom cycles was then confirmed and a definitive diagnosis was made.

1.2.2 diagnosis criteria of Chinese medicine (refer to the Chinese medical industry Standard of the people's republic of China, chinese medical Condition diagnosis curative Standard, the invention team earlier stage study result is formulated)

Liver qi adverse flow of qi: refers to the principal syndrome of the dialectical typing of PMDD disease in traditional Chinese medicine, which is caused by excessive liver qi and liver qi.

Main symptoms are as follows: 1) Premenstrual dysphoria and irritability; 2) Even without end firing; 3) Breast pain.

Secondary symptoms: 1) Headache or distension; 2) Pale red tongue with thin white or yellow coating and wiry pulse; 4) Insomnia and dreaminess; 5) Lower abdominal distension or pain; 6) Epigastric distending pain or nausea and vomiting; 7) The energy is not concentrated, and the ability of working and managing home is reduced.

Diagnosis: meets PMDD diagnostic standards; has the main symptoms and the secondary symptoms of the liver qi adverse qi syndrome 2 items and above.

1.2.3 Western medical subtype and diagnostic criteria irritability subtype: refers to those who score on a Daily Record (DRSP) entry for symptom severity with a sub-scale score of "anger, irritability, and conflict with others". Diagnosis: meets PMDD diagnostic standards; the DRSP sub-scale "anger and irritability" is provided as the main.

1.3 inclusion criteria

(1) Western medicine diagnosis PMDD; the syndrome differentiation of traditional Chinese medicine is the syndrome of liver qi reversed, and the western medicine is the type of irritability subtype; meets the diagnosis standard of the disease, and the degree of the disease is not less than 2/3.

(2) Age 18 to 42 years old.

1.4 exclusion and rejection criteria

(1) Women with menstrual dysfunction in puberty or climacteric period, allergic constitution or drug allergy are aged under 18 years or over 42 years.

(2) Patients with hypertension, vision disorder, vestibular dysfunction (meniere's disease), breast tumor, hyperplasia of mammary glands, primary dysmenorrhea, pelvic inflammatory disease, endometriosis, uterine tumor, chronic colitis, intestinal tumor, etc. cannot be excluded.

(3) Patients with severe primary diseases such as cardiovascular, cerebrovascular, liver, kidney and hematopoietic system, and mental diseases.

(4) There is a history of drug abuse including the three months that had been taken to treat PMDD.

(5) Pregnant or lactating females.

(6) Individuals with aphasia, impaired consciousness, dementia and other conditions cannot be examined.

(7) Unilateral ovariectomy or abortion occurred within six months.

(8) Belongs to the observation object, but the data is not complete, which affects the curative effect and the safety judgment.

(9) The drug is not stopped in the course of treatment due to poor curative effect and adverse reaction.

(10) Other Chinese and western medicines related to the treatment of diseases and the treatment method are used instead of or in addition to the above medicines.

1.5 judgment of efficacy evaluation

Clinical recovery: the clinical symptoms disappear, the basic body temperature measurement or the endocrine measurement is basically normal, and the relapse is avoided after the medicine withdrawal observation for 3 menstrual cycles.

The effect is shown: the clinical symptoms are obviously improved, and the total integral after treatment is reduced by more than or equal to 2/3 compared with the total integral before treatment; basal body temperature or endocrine measurement is near normal, and 3 menstrual cycles are observed without exacerbation at the end of the treatment course.

The method is effective: the clinical symptoms are improved, the total integral after treatment is reduced by more than or equal to 1/3 compared with the total integral before treatment, and the symptoms are repeated after stopping the treatment but are improved compared with the symptoms before treatment.

Invalidation: there is no improvement or aggravation of the various clinical symptoms.

1.6 statistical method

Efficient X for significance test ² Checking; clinical grading comparison was tested using the Ridit test: semi-quantitative data were tested with rank sum: the metering data is tested by t test: the variable correlation is performed by linear correlation or linear regression: if there is non-uniformity in the disease condition between the treatment group and the control group, a stratified comparison t-test is used. The traditional Chinese medicine syndrome can be judged by comparing the curative effect indexes among groups, wherein the curative effect index=the difference between the scores before and after treatment ≡the score before treatment × 100%.

1.7 adverse reaction observations

Attention should be paid to observe adverse reactions or unexpected toxic side reactions, and follow up observations. Adverse reactions, such as gastrointestinal reactions, etc., caused by administration of the test agent are recorded faithfully and the time to disappearance of the adverse reactions after cessation of administration is observed.

1.8 notes

1. The medical staff taking part in the test should observe personally that the patient takes the medicine according to the prescription, thereby objectively reducing the possibility of non-compliance of the patient.

2. All participating units should examine the subjects as specified.

3. All cases need to be bred without exception, and cases which can be evaluated for curative effect are evaluated according to four-level standards, and cases which cannot be evaluated should be interpreted.

4. The record term and the observation method should be standardized, and the observation unit should meet the standard.

5. The observation and recording times should be consistent.

6. The observation time point must be reasonable, and the principle of simultaneous observation with the control group is followed.

7. All observations (including symptoms, signs, laboratory checks, etc.) must have their own pre-comparison records.

8. Carefully filling in each case observation table and checking the related data

9. After the test is finished, the medical history cannot be modified at will, and various data must be statistically processed.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims (10)

1. A novel traditional Chinese medicine paeonol dripping pill for treating premenstrual dysphoric disorder liver qi reverse syndrome and irritability subtype is characterized in that: the coating comprises the following components in parts by mass: 3-7 parts of paeonol, 6000 20-30 parts of PEG, 1700-2000 parts of poloxamer and 3-5 parts of polyoxyl 40 stearate.

2. The novel paeonol drop pill of traditional Chinese medicine according to claim 1, which is characterized in that: the coating comprises the following components in parts by mass: 3-6 parts of paeonol, 6000 23-27 parts of PEG, 6-8 parts of poloxamer and 3-5 parts of polyoxyl 40 stearate.

3. The novel paeonol drop pill of traditional Chinese medicine according to claim 2, which is characterized in that: the composition comprises the following components: 4-6 parts of paeonol, 6000 24-26 parts of PEG, 6-7 parts of poloxamer, and 3-4 parts of polyoxyl 40 stearate.

4. The paeonol dripping pill as a new traditional Chinese medicine according to any one of claims 1-3, which is characterized in that: the extraction process of paeonol comprises the following steps: soaking cortex moutan coarse powder in water for 20-40min, distilling with steam, collecting distillate, cooling and crystallizing to obtain paeonol crude product;

in the soaking process of the cortex moutan crude powder in water, the feed liquid ratio (w/w) is 1:10-15, preferably 1:12;

dissolving paeonol crude product with 4-6BV of 90-95% ethanol at 40-50deg.C, vacuum filtering, adding distilled water to reduce alcohol concentration below 5%, cooling, crystallizing, separating solid and liquid, and drying at 40-45deg.C under reduced pressure.

5. The method for preparing the paeonol dripping pill of any one of claims 1-4, which is characterized in that: the method comprises the following steps: mixing the raw materials in proportion, heating to 70-90 ℃, and dripping, wherein the coolant is dimethyl silicone oil.

6. The method for preparing the paeonol dripping pill as a new traditional Chinese medicine according to claim 5, which is characterized in that: the temperature of the coolant is 4-15 ℃.

7. The method for preparing the paeonol dripping pill as a new traditional Chinese medicine according to claim 5, which is characterized in that: the drop distance is 2-12cm.

8. The method for preparing the paeonol dripping pill as a new traditional Chinese medicine according to claim 5, which is characterized in that: the viscosity of the coolant is 80-200 mPa.s.

9. The method for preparing the paeonol dripping pill as a new traditional Chinese medicine according to claim 5, which is characterized in that: the dripping speed is 20-40 d.min ^-1 。

10. The use of the paeonol dripping pill as a new traditional Chinese medicine in any one of claims 1-4 in the preparation of medicines for treating premenstrual dysphoric disorder;

further, the premenstrual dysphoric disorder is a premenstrual dysphoric disorder liver qi reverse syndrome irritability subtype;

further, the application comprises a clinical test scheme of medicines for premenstrual dysphoric disorder liver qi reverse syndrome and irritability subtype.