CN112972574B

CN112972574B - Pharmaceutical composition for relieving pain caused by primary dysmenorrhea and preparation method thereof

Info

Publication number: CN112972574B
Application number: CN202011462965.4A
Authority: CN
Inventors: 徐华
Original assignee: Wuhan Hengxinyuan Pharmaceutical Co ltd
Current assignee: Wuhan Hengxinyuan Pharmaceutical Co ltd
Priority date: 2019-12-13
Filing date: 2020-12-11
Publication date: 2024-05-28
Anticipated expiration: 2040-12-11
Also published as: CN112972574A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition for relieving pain caused by primary dysmenorrhea and a preparation method thereof. The pharmaceutical composition mainly comprises the following components: white peony root, chinese angelica root, corydalis tuber, ligusticum wallichii, nutgrass galingale rhizome and honey-fried licorice root. The components of the pharmaceutical composition are Chinese herbal medicines, and the pharmaceutical composition has the effects of regulating qi, activating blood, regulating menstruation and relieving pain through reasonable compatibility, can relieve pain caused by primary dysmenorrhea, and is particularly suitable for dysmenorrhea caused by qi-blood disharmony and congestion.

Description

Pharmaceutical composition for relieving pain caused by primary dysmenorrhea and preparation method thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a pharmaceutical composition for relieving pain caused by primary dysmenorrhea and a preparation method thereof.

Background

Dysmenorrhea refers to pain in the lower abdomen before and after menstruation or during menstruation, and is accompanied with symptoms such as waist soreness, nausea, emesis, headache or diarrhea, and when the degree is heavy, the work and life are affected, which is one of the most common diseases in gynecology clinic. Dysmenorrhea is classified as "dysmenorrhea" and "menstrual abdominal pain" in TCM, and those with no obvious organic lesions of the genitals are called primary dysmenorrhea. In recent years, along with the change of life and working environment, the incidence rate of dysmenorrhea has a gradual rise trend.

Although many causes of dysmenorrhea are caused, the causes of the dysmenorrheal are mainly that the qi and blood are not smooth, qi movement is blocked, pain is caused by the obstruction of qi movement, and the disorder of qi and blood is the most main pathological basis, so the dysmenorrheal with the syndrome of qi stagnation and blood stasis is the most common in clinic. Pain is felt several hours before menstrual flow becomes moist, pain is gradually or rapidly aggravated with time, pain is usually paroxysmal or spasticity, and syncope can occur in serious cases. Even part of dysmenorrhea patients have a series of symptoms such as headache, breast distending pain, systemic debilitation, tension, depression or irritability, dysphoria, insomnia and the like before menstruation, which are called premenstrual tension syndrome, because of the fear of dysmenorrhea. After menstruation, all symptoms disappear naturally. However, there are few patients who exhibit mental symptoms and change in sexual behavior, and serious patients affect life, learning or work.

At present, the western medicine adopts analgesic, sedative and spasmolytic medicines, prostaglandin synthetase inhibitors, calcium antagonists and other symptomatic treatments for treating the disease, or uses steroid hormone medicines for inhibiting ovulation, 5-lipoxygenase inhibitors and even operative treatments. Although the above methods can quickly relieve pain, menstrual cycle disorder or obvious side effects are often caused, and the menstrual cycle disorder is not widely accepted by patients. The traditional Chinese medicine has preliminary knowledge on dysmenorrhea as early as east Han dynasty, and Zhang Zhongjing, jin Bian Miao. Women's miscellaneous diseases and pulse syndrome and treatment records: the traditional Chinese medicine has the advantages of obvious clinical near-term curative effect, good long-term curative effect and small side effect, has obvious advantages compared with western medicines and western medicine operation therapies, and has wide development and application prospects and good economic and social benefits.

The patent with publication number CN1768820A is prepared from radix Angelicae sinensis, rhizoma Cyperi, radix Paeoniae alba, saviae Miltiorrhizae radix, rhizoma corydalis, fructus Toosendan, rhizoma Ligustici Chuanxiong, radix Glycyrrhizae Preparata and Carthami flos by adding appropriate amount of adjuvants, and making into capsule with menstruation regulating and pain relieving effects. But the drug component is more.

Therefore, the medicine composition for relieving the primary dysmenorrhea, which has the advantages of simple components, obvious recent curative effect, good long-term curative effect and small side effect, and the preparation method thereof are provided, and the technical problems to be solved by the technicians in the field are urgent.

Disclosure of Invention

In view of the above, the invention provides a pharmaceutical composition for relieving pain caused by primary dysmenorrhea, which has the effects of regulating qi and activating blood, regulating menstruation and relieving pain, and regulating liver and removing blood stasis, and is mainly used for treating dysmenorrhea and premenstrual tension syndrome caused by obstruction of cell vessels due to qi stagnation and blood stasis. The material comprises the following raw materials by weight: 900-1500 g of white peony root, 600-800 g of angelica, 500-800 g of rhizoma corydalis, 500-800 g of szechuan lovage rhizome, 500-800 g of nutgrass galingale rhizome and 100-450 g of honey-fried licorice root.

In the prescription of the pharmaceutical composition, white paeony root enters liver and spleen meridians, is a common key drug for qi and blood systems, has the effects of nourishing blood, astringing yin, softening liver, relieving urgency, regulating flushing and relieving pain, can resolve stagnation, stop abdominal pain, and promote blood circulation to promote blood circulation, and also has the effects of nourishing yin and blood, preventing excessive consumption of blood-activating drugs, has multiple functions, and is a monarch drug. The Chinese angelica is sweet and warm, can lead the blood to enter the body, has the effects of enriching and activating blood, relieving pain, leading the body to move and leave, and leading the body to be calm and leading the Chinese herbaceous peony to relieve pain to treat the symptoms and root cause; rhizoma corydalis not only enters blood and qi but also supplements each other, has the strongest analgesic power, and is used as ministerial drug together. The Ligusticum wallichii is spicy and fragrant, and can warm and dredge channels and collaterals, smooth the whole body, promote qi circulation and blood circulation to relieve pain, and the peony and Chinese angelica can regulate blood to soften liver and relieve pain; the nutgrass galingale rhizome is pungent and bitter in taste and good at dredging liver qi, regulating menstruation and relieving pain, and is an essential drug for gynecopathy to regulate menstruation, and is an adjuvant drug. The baked licorice root, radix Glycyrrhizae Praeparata, which is used as a guiding drug, acts as a pain relieving drug to regulate the actions of the drugs. And Glycyrrhrizae radix and radix Paeoniae are used for enhancing analgesic effect. In the whole formula, the white paeony root, the Chinese angelica and the honey-fried licorice root regulate qi and blood to treat the root cause, the rhizoma corydalis, the szechuan lovage rhizome and the nutgrass galingale rhizome have the effects of activating blood and promoting qi to relieve pain, and the six medicines are combined together to jointly play the roles of promoting qi and activating blood, regulating menstruation and relieving pain.

Preferably, the pharmaceutical composition comprises the following raw materials by weight: 1000-1400 g of white peony root, 700-740 g of angelica, 540-660 g of rhizoma corydalis, 550-650 g of szechuan lovage rhizome, 550-650 g of nutgrass galingale rhizome and 300-420 g of honey-fried licorice root.

Preferably, the pharmaceutical composition comprises the following raw materials by weight: 1200g of white peony root, 720g of Chinese angelica, 600g of rhizoma corydalis, 600g of ligusticum chuanxiong hort, 600g of nutgrass galingale rhizome and 360g of honey-fried licorice root.

Preferably, the pharmaceutical composition of the present invention is an oral preparation, preferably a pill, syrup, granule, capsule or tablet, more preferably the oral preparation is a capsule.

In another aspect, the present invention also provides a method for preparing a pharmaceutical composition for alleviating pain caused by primary dysmenorrhea, comprising the steps of:

s1, reflux-extracting the rhizoma corydalis with ethanol twice, filtering, recovering ethanol in filtrate, and concentrating under reduced pressure to obtain extract I with the relative density of 1.10-1.15;

s2, reflux-extracting the rhizoma corydalis residues, the ligusticum wallichii, the angelica sinensis, the white paeony root, the honey-fried licorice root and the additional water twice, filtering, concentrating the filtrate under reduced pressure to a concentrated solution with the relative density of 1.15-1.20, adding ethanol into the concentrated solution, standing at room temperature, filtering, recovering the ethanol, and concentrating the filtrate under reduced pressure to an extract II with the relative density of 1.15-1.20;

S3, mixing the extract I and the extract II, drying, and adding auxiliary materials to prepare the pharmaceutical composition.

Preferably, in step S1, the corydalis tuber is extracted with 5 times of 80% ethanol under reflux for 1 hour each time.

Preferably, in step S2, the residues of rhizoma corydalis, rhizoma Chuanxiong, radix Angelicae sinensis, radix Paeoniae alba, radix Glycyrrhizae Preparata and rhizoma Cyperi are extracted twice with 5 times of water under reflux, the first time for 2 hours and the second time for 1 hour.

Preferably, in step S2, ethanol is added to the concentrate to an alcohol content of 80%, and the concentrate is left at room temperature for 12 to 24 hours and then filtered.

In another aspect, the invention also provides a pharmaceutical composition for use in a medicament for alleviating pain caused by primary dysmenorrhea.

Compared with various drugs for relieving dysmenorrhea on the market, the pharmaceutical composition provided by the invention has the following advantages:

(1) The medicine composition is a natural pure traditional Chinese medicine preparation, raw materials are easy to obtain, the prescription is derived from clinical experience prescription according to the prescription medication principle of traditional Chinese medicine, and only six medicines are used and observed repeatedly in clinic, so that the medicine composition has definite curative effect and can quickly relieve dysmenorrhea symptoms;

(2) The medicine composition has obviously increased main active ingredient content and obviously improved medicine effect, can relieve pain caused by primary dysmenorrhea, mainly regulates qi and blood, relieves spasm and pain, has definite curative effect, small side effect and high effective rate;

(3) Because dysmenorrhea has a longer course and various clinical dialectical types, the traditional Chinese medicine decoction needs to be decocted for a long time, which brings a lot of inconvenience to patients. The pharmaceutical composition has good clinical curative effect, short treatment course, easy acceptance by patients, convenient administration and can relieve the pain caused by dysmenorrhea.

Drawings

FIG. 1 is a standard for visual simulation scoring VAS scoring

Detailed Description

The present invention is further described below by way of specific examples, but is not limited to the following examples. Combinations, substitutions and alterations are also within the scope of this disclosure, or are not to be excluded from the scope of this disclosure, and are intended to be included within the scope of this disclosure. The preparation of the pharmaceutical composition of the present invention may be an oral preparation such as capsule, tablet, pill, etc., and only capsules are listed in the examples.

The weight of the mice used in the invention is 18-20 g, and the quality certification number is 201600013735. Rat weight 220-250 g, quality eligibility number: 201600015178. production pass number provided by vinca Yisi laboratory animal technologies Co., ltd: SCXK (Ji) 2011-0004.

Example 1

The raw materials of the invention are weighed according to the following weight portions: 1200g of white peony root, 720g of Chinese angelica, 600g of rhizoma corydalis, 600g of szechuan lovage rhizome, 600g of nutgrass galingale rhizome and 360g of honey-fried licorice root.

The preparation process comprises the following steps: reflux-extracting rhizoma corydalis with 5 times of 80% ethanol twice for 1 hr each time, and filtering to obtain filtrate and rhizoma corydalis residue. Recovering ethanol from the filtrate, and concentrating under reduced pressure until the relative density is 1.10-1.15 (60 ℃) to obtain extract I; reflux extracting rhizoma corydalis residue, rhizoma Ligustici Chuanxiong, radix Angelicae sinensis, radix Paeoniae alba, radix Glycyrrhizae Preparata and rhizoma Cyperi with 5 times of water twice for 2 hr and 1 hr respectively, filtering, and concentrating the filtrate under reduced pressure to obtain concentrated solution with relative density of 1.15-1.20 (60deg.C); adding ethanol into the concentrated solution to ensure that the ethanol content of the concentrated solution reaches 80 percent, standing for 20 hours at room temperature, filtering, recovering the ethanol, and concentrating under reduced pressure until the relative density is 1.15-1.20 (60 ℃) to obtain extract II; mixing the extract I and the extract II to obtain a raw material medicine, drying the raw material medicine by microwaves at the temperature of 60-80 ℃ and the vacuum degree of-0.08 to-0.09 MPa, crushing the raw material medicine to 80 meshes, adding 4 percent of silicon dioxide, 1 percent of magnesium stearate and a proper amount of corn starch to 530g, uniformly mixing the raw material medicine and encapsulating the mixture to obtain 1000 capsules of the pharmaceutical composition.

Example 2

Weighing 900g of white peony root, 600g of Chinese angelica, 500g of rhizoma corydalis, 500g of ligusticum chuanxiong hort, 500g of nutgrass galingale rhizome and 100g of honey-fried licorice root. Tablets were prepared according to the preparation process of example 1 and conventional tabletting process.

Example 3

Weighing 1500g of white peony root, 800g of Chinese angelica, 800g of rhizoma corydalis, 800g of ligusticum chuanxiong hort, 800g of nutgrass galingale rhizome and 450g of honey-fried licorice root. Capsules were prepared according to the preparation process of example 1.

Example 4

Weighing 1000g of white peony root, 700g of angelica, 540g of rhizoma corydalis, 550g of ligusticum chuanxiong hort, 550g of nutgrass galingale rhizome and 300g of honey-fried licorice root. Capsules were prepared according to the preparation process of example 1.

Example 5

Weighing 1400g of white peony root, 740g of angelica, 660g of rhizoma corydalis, 650g of ligusticum chuanxiong hort, 650g of nutgrass galingale rhizome and 420g of honey-fried licorice root. Capsules were prepared according to the preparation process of example 1.

Example 6

1200G of white peony root, 720g of angelica, 620g of rhizoma corydalis, 630g of ligusticum chuanxiong, 580g of nutgrass galingale rhizome and 400g of honey-fried licorice root are weighed. Capsules were prepared according to the preparation process of example 1.

Example 7

The preparation process comprises the following steps: pulverizing rhizoma corydalis of the prescription amount, adding 10 times of deionized water, mixing uniformly, adding 2g of cellulase, and stirring for 1 hour. The enzyme activity range of the cellose is CMC 120-130 mu/ml (g).

After stirring, adding 5 times of 80% ethanol, mixing, reflux extracting twice for 1 hr each time, and filtering to obtain filtrate and rhizoma corydalis residue. Recovering ethanol from the filtrate, and concentrating under reduced pressure until the relative density is 1.10-1.15 (60 ℃) to obtain extract I; reflux extracting rhizoma corydalis residue, rhizoma Ligustici Chuanxiong, radix Angelicae sinensis, radix Paeoniae alba, radix Glycyrrhizae Preparata and rhizoma Cyperi with 5 times of water twice for 2 hr and 1 hr respectively, filtering, concentrating the filtrate under reduced pressure to obtain concentrated solution with relative density of 1.15-1.20 (60deg.C), adding ethanol to the concentrated solution to reach alcohol content of 80%, standing at room temperature for 20 hr, filtering, recovering ethanol, and concentrating under reduced pressure to obtain extract II with relative density of 1.15-1.20 (60deg.C); mixing the extract I and the extract II to obtain a raw material medicine, drying the raw material medicine by microwaves at the temperature of 60-80 ℃ and the vacuum degree of-0.08-0.09 MPa, crushing the raw material medicine to 80 meshes, adding 4% silicon dioxide, 1% magnesium stearate and a proper amount of corn starch to 530g, uniformly mixing, and tabletting to obtain 1000 tablets of the pharmaceutical composition.

Comparative example 1

The starting materials and preparation method of comparative example 1 were the same as in example 1. The difference is that the rhizoma corydalis is processed with vinegar in the preparation process of the medicine combination group, and is not extracted twice by reflux with ethanol.

Comparative example 2

The starting materials and preparation method of comparative example 2 were the same as in example 1. The difference is that in the preparation process of the medicine combination group, the rhizoma corydalis residue, the ligusticum wallichii, the angelica, the white paeony root, the honey-fried licorice root and the additional water are extracted once in a reflux way for 3 hours.

Comparative example 3

The method of example 1 in CN1768820a was used to prepare a pharmaceutical formulation, the raw materials used were: wine-fried angelica sinensis, vinegar-prepared rhizoma cyperi, fried white peony root, red-rooted salvia root, vinegar-prepared rhizoma corydalis, fried szechwan chinaberry fruit, fried ligusticum chuanxiong hort, roasted liquorice and safflower.

The pharmaceutical compositions of examples 1 to 7 and comparative examples 1 to 3 were used to observe the effect of ear swelling in mice caused by paraxylene. The specific operation method is as follows:

Female ICR mice were taken and divided into 11 groups at random, 10 mice per group, and the experimental groups were administered the drugs of examples 1 to 7 and comparative examples 1 to 3, respectively, in an amount of 0.22g/kg, and the blank group was administered the same amount of physiological saline. 0.5h after the last administration, 0.05ml of xylene was uniformly applied to the front and rear sides of the left ear of the mouse, the mouse was sacrificed after 4 hours, both ears were cut off along the auricle base line, round ears were respectively punched out at the same position with a 9mm diameter punch, and weighed, and the degree of swelling and the swelling inhibition were calculated according to the following formula, and the results are shown in Table 1.

The degree of swelling and the swelling inhibition ratio were calculated as:

swelling degree = left ear weight-right ear weight,

Swelling inhibition (%) = (average swelling degree of control group-average swelling degree of administration group)/average swelling degree of control group×100%.

TABLE 1 influence of ear swelling in mice due to paraxylenen＝10)

Group of	Degree of swelling (mg)	Swelling inhibition rate
			Blank control group	16.3±5.03
Example 1	4.30±1.77**	80.0
			Example 2	6.40±1.56**	73.1
Example 3	6.37±1.70**	75.3
			Example 4	5.22±2.15**	75.5
Example 5	5.20±1.88**	76.8
			Example 6	4.88±2.11**	77.3
Example 7	3.01±1.58**	85.1
			Comparative example 1	11.55±2.33*	52.4
Comparative example 2	10.20±1.58*	57.7
			Comparative example 3	7.66±2.21*	69.2

Note that: comparison with model control group p < 0.05, p < 0.01

As shown in table 1, the pharmaceutical compositions of examples 1 to 7 can suppress ear swelling of mice due to xylene and increase the swelling suppression rate compared to the blank group. However, the preparation method was changed as in comparative examples 1 to 2, the ear swelling degree was increased, and the swelling inhibition rate was decreased. The rhizoma corydalis of comparative example 1 was vinegar-processed, not the ethanol reflux-extraction of examples 1 to 7, twice. Although vinegar-processed rhizoma corydalis can also purify the active ingredients of rhizoma corydalis, the effective utilization rate is improved, so that the drug effect is improved. However, the pharmaceutical composition of the invention needs to consider the synergistic effect of different components, and the applicant has unexpectedly found that the effect of inhibiting mouse ear swelling is best after the reflux extraction of rhizoma corydalis with 5 times of 80% ethanol for two times, and the compatibility with white peony root, angelica, nutgrass galingale rhizome, szechuan lovage rhizome and honey-fried licorice root.

In comparative example 2, hu Suozha, ligusticum wallichii, chinese angelica root, white peony root, prepared licorice root and perfume are extracted by reflux once for 3 hours. In examples 1 to 7, the method of two reflux extractions was adopted, and the total time of the two reflux extractions was 3 hours. Under the condition that the reflux extraction time is the same, the effect of inhibiting the ear swelling of mice caused by dimethylbenzene is best after the reflux extraction is carried out twice, which shows that the content of the active ingredients of each component medicament is the highest, and the pain caused by dysmenorrhea is relieved more effectively.

In comparative example 3, the swelling inhibition rate of the traditional Chinese medicine raw materials with more than 6 components of the application is 69.2%, which is slightly lower than the inhibition rate of 73.1% -85.1% in examples 1-7 of the application. The traditional Chinese medicine has less components, but can obtain better effect. In particular, example 7, the minimal swelling and highest swelling inhibition. In the treatment process of the rhizoma corydalis, the common extraction method of the traditional Chinese medicine is combined with the biological enzymolysis method, and the cellulose is firstly adopted for treatment, so that the cell structure of the rhizoma corydalis can be destroyed, the dissolution of the effective components is promoted, the subsequent extraction efficiency and the extraction quantity are improved, more effective components are obtained, and the drug effect is improved.

Experimental example 1 pharmacological Studies

The clinical planned dose of the pharmaceutical composition of the invention is 1.7 g/day. The large, medium and small doses of the rat experimental group are respectively 0.30g/kg, 0.15g/kg and 0.075g/kg (which are equivalent to 12 times, 6 times and 3 times of the clinical intended dose); the large, medium and small doses of the mice experimental group were 0.44g/kg, 0.22g/kg and 0.11g/kg (corresponding to 18 times, 9 times and 4.5 times the clinically intended dose), respectively. The positive control group selects the similar new medicines with similar functions and indications as far as possible and the same clinical curative effects of the administration route and the same dosage form, thereby the experimental study selects the menstruation regulating and pain relieving tablet and the pseudo-ginseng dysmenorrheal capsule as the positive control medicines. Notoginseng dysmenorrhea capsule, produced by Shanxi Huakang pharmaceutical industry Co., ltd., lot number: 20141101, the equivalent dose in mice is 0.5g/kg; menstruation regulating and pain relieving tablet produced by Jilin Taiming star pharmaceutical Co., ltd., batch number: 20141001 the equivalent dose in mice was 1.0g/kg.

1.1 Effect on the Experimental dysmenorrhoea model in mice caused by oxytocin

The 74 female ICR mice are randomly divided into seven groups, and 10 mice in each group are respectively a normal control group, a model control group, a pseudo-ginseng dysmenorrheal capsule group, a menstruation regulating and pain relieving tablet group, a small-dose group, a medium-dose group and a large-dose group. The medicine is administrated by stomach irrigation once a day for seven consecutive days. The normal control group and the model control group were given the same volume of 0.5% cmc-Na. Starting on day 3 of the gavage administration, the rest groups except the normal control group were injected with 20mg/kg of estradiol benzoate at a volume of 10ml/kg, 1 time daily for 5 consecutive days. 1h after the last administration, each mouse was injected with oxytocin 20U/kg in a volume of 10ml/kg, and the number of times of torsion, the percentage of torsion and the latency of torsion reaction (the latency of torsion reaction without torsion reaction within 30min is calculated as 30 min) within 30min were recorded, and the results are shown in Table 2.

TABLE 2 influence on experimental dysmenorrhoea model in mice caused by oxytocinn＝10)

Note that: 1. compared with the normal control group, deltap is less than 0.05, deltap is less than 0.01, deltaDeltap is less than 0.001

2. Comparison with model control group p < 0.05, p < 0.01, p < 0.001 (Table below is the same)

As shown in table 2, the large and medium doses can significantly reduce the number of times of twisting the experimental dysmenorrhea model of the mice, the small doses have a certain effect of reducing the number of times of twisting, and the positive control group pseudo-ginseng dysmenorrhea capsules and menstruation regulating pain relieving tablets can also significantly reduce the number of times of twisting the mice; the torsion percentage of the mice can be obviously reduced by 0.44g/kg of large dose, the torsion percentage is reduced to a certain extent by medium and small doses, and the torsion percentage is not influenced by two positive control groups; the large, medium and small doses obviously prolong the incubation period of the twisting body, have a certain dose-effect relationship, and have stronger large dose effect than the positive group.

1.2 Effect on acetic acid-induced mouse torsion reaction

Six groups of female ICR mice were randomly aliquoted using the acetic acid torque method, see Table 3. The administration was performed by gastric lavage, 1 time a day for 7 consecutive days, with the volume of 20ml/kg, and the normal control group was given the same volume of 0.5% CMC-Na. The number of twists in 5 to 15min was recorded 1h after the last administration by intraperitoneal injection of 0.7% acetic acid (10 ml/kg) per mouse, and the results are shown in Table 3.

TABLE 3 influence on acetic acid induced mouse torsion reactionn＝10)

Note that: comparison with the normal control group shows that p < 0.05, p < 0.01, p < 0.001

From Table 3, it is known that the pharmaceutical composition of the present invention can significantly reduce the number of writhing reactions in mice caused by acetic acid at different doses, and has significant inhibitory effect on pain caused by chemical stimulation. The large dose has the best effect and is superior to the positive control group.

1.3 Effect on uterine and ovarian development in immature mice

Female ICR pups were taken 90, and after conditioning for 3 days in a clean-grade viewing room, qualified 78 were randomly aliquoted into six groups of 13 animals each, and the grouping method is shown in table 4. The administration was performed by gastric lavage, 1 time a day for 15 consecutive days, with the volume of 20ml/kg, and the normal control group was given the same volume of 0.5% CMC-Na. The mice were sacrificed 24 hours after the last dose, uterus and ovaries were removed, rapidly weighed on a precision electronic scale, and organ coefficients were calculated, and the results are shown in table 4.

TABLE 4 influence on uterine and ovarian development in underage micen＝10)

As can be seen from the results in Table 4, the large, medium and small doses and the positive control drug of the invention have no obvious effect on the uterine and ovarian development of the underage mice.

1.4 Study of mechanism of action

Taking Wistar rats, weighing 220-250 g, and carrying out quality certification number: 201600015178. production pass number provided by vinca Yisi laboratory animal technologies Co., ltd: SCXK (Ji) 2011-0004. Reference literature methods (Wang Mingde, jiang Ping, chen Sugong, etc.), experimental researches on dysmenorrhoea treatment of primary dysmenorrhoea by using dysmenorrhoea-relieving granules, traditional Chinese medicine academic periodical, 2006, 24 (3), 444-447, preparation of Zhongjingxiang, dang Haizhen, study on the analgesic action mechanism of dysmenorrhoea rats caused by oxytocin by using a capsule, hunan university of Chinese medicine journal 2005, 25 (2): 2767-2769), adopting experimental animals with 'the effect on a model of dysmenorrhoea of a rat sensitized by using estradiol benzoate', after the observation and recording of the incubation period and the number of twists, after administration of oxytocin for 1h, placing the rats into a heparin anticoagulation test tube with 3% chloral hydrate of 0.3g/kg for anesthesia, after abdominal aorta blood taking 3.5ml, respectively, separating, and measuring the content of thromboxane B2 (TXB ₂), 6-keto-prostaglandin F1a (6-K-PGF _1a) and beta-endopeptid (beta-EP); simultaneously, 1 uterus is cut, washed by ice-salt water (containing 20 mmol/IEDTA), then 10% uterus tissue homogenate is prepared by ice-salt water under ice bath, and centrifuged at 3000r/min for 15min at low temperature, and the supernatant is taken and stored at-70 ℃ for testing. The uterus homogenate was tested for endothelin (ET-1) using ELISA kit and for NO content using colorimetric method, the results are shown in Table 5.

TABLE 5 TXB ₂、6-K-PGF_1a, beta-EP, ET-1, NO, beta-EP content in rat plasma

Note that: delta P < 0.05 compared with normal control group; deltaDeltaP < 0.01; deltaDeltaP < 0.001 and model comparison of control group P < 0.05; * P < 0.01; * P < 0.001

As can be seen from the results in Table 5, the model control group had significantly increased TXB ₂ content, significantly decreased 6-K-PGF _1a content, and increased TXB ₂/6-K-PGF_1a ratio. The invention can obviously reduce the TXB ₂ content in large and medium doses, has a certain reduction effect on the TXB ₂ content in small doses, can obviously raise the 6-K-PGF _1a content in large doses, has a certain raising effect on the 6-K-PGF _1a content in medium and small doses, can obviously increase the TXB ₂/6-K-PGF_1a ratio in large, medium and small doses, and has a good dose-effect relationship. The positive control also had similar effects on the modulation of the TXB ₂、6-K-PGF_1a content according to the present invention.

The NO content in the rat uterine tissue of the model control group is obviously reduced, the ET-1 content is increased, and the ET-1/NO ratio is obviously increased. The invention can obviously increase the NO content and reduce the ET-1/NO ratio with large dosage, and can obviously reduce the ET-1 content and the ET-1/NO ratio with small dosage. The menstruation regulating and pain relieving tablet has similar large-dose effect as the invention, and the pseudo-ginseng dysmenorrheal capsule has no obvious influence; compared with the normal control group, the beta-EP content in the rat plasma of the model control group is obviously reduced, the high dosage of the invention can obviously raise the beta-EP content, compared with the model control group, the invention has obvious difference, and the middle and small dosage and the two positive control drugs have no obvious influence on the beta-EP content.

The pharmaceutical composition can regulate the contents of TXB ₂、6-K-PGF₁ a and ET-1 and NO and the ratio of TXB ₂/6-K-PGF₁ a and ET-1/NO, relax uterine blood vessels and increase uterine blood flow, thereby effectively relieving dysmenorrhea. Its mechanism of action for treating dysmenorrhea is related to the modulation of vasomotor function factors in the uterus. The invention can obviously improve the content of the beta-EP in the plasma of the rat with the experimental dysmenorrhea model, and can play an analgesic role by up-regulating the level of the neuroendocrine hormone with the analgesic effect.

Experimental example 2 toxicology study

2.1 Acute toxicity test

The test set 4 groups, respectively, of the control group and 5, 10 and 20g/kg of the present invention (corresponding to 176 times, 352 times and 704 times of the clinically intended dose, respectively, 33.3 times, 66.6 times and 133.3 times of the pharmacodynamic dose, respectively), 10 SD female rats per group, each group of rats was given a control formulation (0.5% CMC-Na) or the present pharmaceutical composition by single oral gavage in a volume of 40mL/kg, and 14 days after administration was observed.

During the observation period, the female mice in each group are in good general state, have no abnormal changes in weight and food intake, hematology, hematochemistry and general anatomical observation, have normal autonomous activities and have no obvious toxic reaction. Under the test conditions, the Maximum Tolerated Dose (MTD) of SD female mice is greater than 20g/kg. Therefore, the MTD of the medicine composition for one-time gastric lavage is more than 20g/kg, which is 704 times of the clinical quasi-dosage, and no acute toxic reaction is seen, thus indicating that the medicine composition has lower toxicity.

2.2 Long-term toxicity test

The test consisted of 4 groups of 30 female rats, each control group and 0.75, 1.5 and 5g/kg of the present invention (about 26.8, 53.6 and 178.6 times the dose to be clinically used, 5, 10 and 33.3 times the dose to be pharmacodynamically effective). Each group of rats was orally and gastro-orally administered with a control (0.5% CMC-Na) or the corresponding dose of the pharmaceutical composition of the present invention at a volume of 10mL/kg, 1 time per day for 26 weeks, and the recovery from discontinuation was observed for 4 weeks.

The main toxicity of the rats in the group of 5g/kg is characterized by slow weight growth, reduced ingestion, slight decrease in peripheral blood RBC, HGB, HCT, slight increase in serum TBIL and Urea, slight increase in urine bilirubin, ketone body, specific gravity, protein, urine bilinogen grade and acid-base value grade, and slight-slight hypertrophy of liver weight and organ coefficient. After stopping the administration, the above changes were recovered to normal except for body weight. Only the rats of 1.5g/kg group have slight decrease of ingestion and slight increase of serum TBIL, and can return to normal after stopping taking the medicine. The general condition, weight and ingestion, clinical pathology, hormonal examination, organ weight and coefficient, general anatomic observation, etc. of the 0.75g/kg group rats were markedly abnormal.

Under the test conditions, the non-toxic response dose (NOAEL) of the pharmaceutical composition of the invention is 1.5g/kg (corresponding to 53.6-178.6 times the dose to be clinically used). The major toxic and side effects of long-term administration may be slight effects on liver, kidney and bone marrow, but such adverse effects are restorable.

2.3 Reproduction toxicity test

192 SD rats were randomly divided into control groups of 0.75, 1.5 and 5g/kg of the pharmaceutical composition of the invention, 48 animals each, and male and female halves. Each group of rats was given a control (0.5% CMC-Na) or relative concentration of the suspension of the invention by oral gavage at a volume of 10 ml/kg. Each group of male mice was dosed from the beginning of the mating to the dissection of the end of the mating, and the female mice were dosed from 2 weeks prior to mating to day 7 of gestation, 1 time per day. The day of first administration of male and female mice is defined as day 1 of administration, the day of first cage-closing is defined as day 0 of mating, and the day of successful mating is defined as day 0 of gestation. The general status of each group of rats was observed daily.

The drug composition of the invention is orally and gastro-orally administrated for 1 time every day from 4 weeks before the end of mating of SD male mice to 7 days of gestation, and the drug composition of the invention is orally and gastro-orally administrated for 1 time every day from 1.5g/kg of female mice and pregnant mice of 5g/kg of female mice and pregnant mice, and the drug composition is slightly toxic to the parents and reproductive, and is mainly characterized by weight gain, food intake, corpus luteum number, implantation gland number and absolute weight of ovary/brain factor reduction. No obvious abnormal changes were seen in the general status, body weight, food intake, pathological examination, reproductive function, and early embryo development of the 0.75, 1.5g/kg group of male mice, female/pregnant mice, and 5g/kg group of male mice. The invention has no toxic response dose (NOAEL) of 5g/kg for the parent male mice and the reproductive function thereof, 1.5g/kg for the parent female mice and the reproductive function thereof, and 5g/kg for early embryo development.

Experimental example 3 study of drug efficacy

The following is a clinical observation of the pharmaceutical composition of the present invention for alleviating pain caused by dysmenorrhea. The study had two phases, including a screening period of 0-6 days and a dosing period of 3 days.

3.1 Case Material

160 Subjects were all patients meeting the primary dysmenorrhea diagnosis criteria in the gynecological ward and the outpatient service. Of these, 46 cases are 14 to 20 years old, 88 cases are 21 to 30 years old, and 26 cases are 31 to 40 years old. The longest disease course in the case is 21 years, and the shortest disease course is 1 year.

3.2 Diagnostic basis

3.2.1 Western diagnostic criteria

Reference is made to gynaecology and obstetrics (Xie Xing, kong Beihua, duan Tao, publication of human sanitation, 2018).

Primary dysmenorrhea refers to dysmenorrhea resulting from non-organic lesions of the genital organs. Primary dysmenorrhea characteristics: ① Primary dysmenorrhoea is common in puberty and usually occurs within 1-2 years after the beginner; ② Pain usually comes from the menstrual period, begins after the menstrual period comes, and occurs at the earliest 12 hours before the menstrual period, so that the pain is most intense on the 1 st day of menstruation and is relieved after 2-3 days, and the pain is usually spastic and is usually located on the pubic bone of the lower abdomen and can radiate to the lumbosacral part and the inner side of the thigh; ③ Symptoms such as nausea, vomiting, diarrhea, dizziness, hypodynamia and the like can be accompanied, and the complexion is white and cold sweat is generated when serious; ④ Gynecological examination has no abnormal findings; ⑤ The gynecological B ultrasonic examination has no abnormal findings.

3.2.2 Chinese medicine differentiation and typing Standard

Refer to the clinical guidelines for treating dysmenorrhea by new Chinese medicine (published by Ministry of health, 1993 edition), the tenth edition of Programming textbook of national higher Chinese medicine institutions, the science of gynaecology and obstetrics of Chinese medicine (Yong, national Chinese medicine Press, 2017 edition), and the guide for diagnosis and treatment of gynecological diseases of Chinese medicine, dysmenorrhea (Chinese medicine press, 2012 edition).

Diagnostic criteria for primary dysmenorrhea, qi stagnation and blood stasis:

Main symptoms are as follows: abdominal distending pain and refusal of premenstrual or menstrual period; secondary symptoms: ① The menstrual blood quantity is small, and the menstruation is unsmooth; ② Blood color dark red or purple dark, with lump and pain reduction under lump; ③ Breast pain; ④ Chest distress; tongue pulse: a dark purple tongue or a stagnant spot, a thin and white coating, and a wiry or astringent pulse.

Dialectic standard: the primary symptoms are indispensable, and the secondary symptoms have 2 or more than 2, and can be diagnosed by referring to tongue pulse.

3.3 Disease quantization criteria and determination method

Visual Analog Scoring (VAS) is used to determine pain intensity and consists of a 100mm straight line. The left end of the line indicates "no pain" and the right end of the line indicates "intolerable pain". The pain intensity felt by the patient is marked on a straight line, the distance between the left end of the line and the mark is the pain intensity of the patient, and the pain intensity is marked as 4 when the pain intensity is 40mm in length. 1 to 3 are mild, 4 to 6 are moderate, and 7 to 10 are severe. As shown in fig. 1.

Before the subjects score by the visual simulation scoring method for the first time, researchers need to explain the scoring standard and scoring mode of VAS scoring, and the explanation content at least comprises the following 2 aspects:

(1) VAS score definition: visual Analog Scoring (VAS) is used to determine pain intensity and consists of a 100mm straight line. The left end of the line indicates "pain free", the right end of the line indicates "intolerable pain", the middle portion indicates different degrees of pain, please mark on a straight line according to the pain level itself.

(2) Scoring rules:

0 point: no pain;

Score 0 < 4: slight pain, tolerated;

score 4 < 7: pain and influence on sleep, can be tolerated, and needs to take analgesic drugs orally;

Score 7 < 10: the pain is intense and severe or hard, and it is difficult to fall asleep or wake.

Before each measurement, the patient is marked on the straight line which is not drawn, so that subjective errors caused by the comparison of the front mark and the rear mark of the patient are avoided. The data are recorded by the patient, the maximum pain degree occurring between two doses is marked on the straight line after the patient takes the medicine in the morning and evening every day, and the researcher measures the distance from 0 end to mark according to the mark of the patient on the straight line to obtain the VAS pain score.

3.4 Inclusion of case criteria

(1) Meets the Western medicine diagnosis standard of primary dysmenorrhea;

(2) Meets the diagnosis standard of the traditional Chinese medicine differentiation (qi stagnation and blood stasis syndrome);

(3) The past 6 months have a history of dysmenorrhea of 4 months continuously, and the menstrual cycle is within 28+/-7 days;

(4) Women with no history of pregnancy;

(5) The subjects voluntarily participated in and signed an informed consent form.

3.5 Rule of case elimination

(1) Patients with menstrual flow more than or equal to 80 ml;

(2) The examination proves that the secondary dysmenorrhea patients are caused by the lesions of endometriosis, pelvic inflammatory disease, hysteromyoma, ovary and the like;

(3) Patients who plan gestation and lactation period in 6 months using a pharmaceutical intrauterine device;

(4) The traditional Chinese medicine is taken orally for 4 weeks and is mainly used for treating similar traditional Chinese medicine, the traditional Chinese medicine is taken vaginally or orally for 12 weeks, or the estrogen or the oestrogen is taken orally, and the intrauterine device is taken or the hormone is injected for 24 weeks;

(5) Hemoglobin less than 90g/L, has serious primary heart, liver, lung, kidney, blood or serious diseases affecting the survival of the heart, liver, lung, kidney, blood or serious diseases, and researchers judge that the patients are not suitable for participating in the clinical trial;

(6) The glutamic-pyruvic transaminase ALT and/or the glutamic-pyruvic transaminase AST are/is larger than the upper limit of the range of the normal value by 2 times, and the serum creatinine Scr is larger than the upper limit of the range of the normal value;

(7) Allergic constitution, allergic to two or more drugs or foods, or allergic to known components of the drug;

(8) Patients with any serious psychological and mental abnormalities, and cannot comply with clinical trial procedures and norms;

(9) Other clinical trials were enrolled in the last month.

3.6 Reject case criteria

"Knockout" means that a subject who should not be into a group but has been into a group should be knockout, including:

(1) Misreception;

(2) Misdiagnosis;

(3) All without medicine;

(4) No record is detected.

3.7 Dosing regimen

The capsules prepared in example 1 of the present application (wherein each of the capsules is 4 granules of the pharmaceutical composition of the present application) were orally taken after each meal 2 times a day. During the whole treatment process, the researcher can require the patient to take medicine according to the requirement of the scheme, and the empty package and the unused medicine are delivered back to the researcher doctor, and the starting time of the medicine taking, the dosage, the medicine taking times, the recovery quantity and the like are recorded. And then the sponsor unit recovers the statistics. The dysmenorrhea appears to begin to take medicine, and the medicine is continuously taken for 3 days.

3.8 Efficacy evaluation index and evaluation method

(1) According to the relevant regulation and preparation of the new traditional Chinese medicine for treating dysmenorrhea by the national ministry of health, the recovery: after administration, the integral is restored to 0 minute, abdominal pain and other symptoms disappear, and three menstrual cycles are not recurred after stopping administration; the effect is shown: the integration after treatment is reduced to less than 1/2 of the integration before treatment, the abdominal pain is obviously relieved, and the rest symptoms are improved; the method is effective: the integral after treatment is reduced to 1/2-3/4 of the integral before treatment, the abdominal pain is reduced, and the rest symptoms are improved; invalidation: abdominal pain and other symptoms were unchanged, and post-treatment scores decreased by less than 3/4 of pre-treatment scores.

(2) Laboratory index checks of patients in the test group before and after dosing: blood routine (RBC, WBC, NEUT%, HGB, PLT), clotting function (prothrombin time PT, activated partial thromboplastin time APTT, plasma thrombin time TT, international normalized ratio INR), liver function (ALT, AST, ALP, TBil, GGT), renal function (urinary routine (ERY, LEU, PRO) +urinary sediment, scr, eGFR) and conventional 12-lead electrocardiography (focused on arrhythmia, ST segment changes and QT/QTc prolongation).

3.9 Therapeutic Effect

All cases in this group were primary dysmenorrhea, with no exclusion and rejection. The case constitution conditions are as follows: VAS score 0 < score less than or equal to 4 mild pain total 39 cases, accounting for 24.37%, score 4 < score less than or equal to 7 moderate pain total 85 cases, accounting for 53.13%, score 7 < score less than or equal to 10 severe pain total 36 cases, accounting for 22.5%. Wherein, 44 cases are healed, and the healing rate is 27.5%; 91 cases of obvious effects, 56.88% of obvious effects, 22 cases of effective effects and 13.75% of effective effects; the invalid cases are 3 cases, the inefficiency is 1.87%, and the total effective rate is 98.13%.

Comparative example 4 is a decoction prepared according to the method of example 1 in CN102526675B, and 160 subjects meeting the criteria were selected to determine the therapeutic effect according to the disease quantification criteria and determination method of the present application.

Table 6 is a table comparing the therapeutic effect of the pharmaceutical formulations for treating dysmenorrhea according to the present invention with those of comparative examples 3 and 4.

Table 6 comparative table of therapeutic effects

	Cure rate (%)	Obvious efficiency (%)	Effective rate (%)	Inefficiency (%)	Total effective rate (%)
						The invention is that	27.5	56.88	13.75	1.87	98.13
Comparative example 3	18.2	52.7	22.4	6.7	93.3
						Comparative example 4	25.3	55.5	16.8	2.4	97.6

The pharmaceutical composition for alleviating primary dysmenorrhea of the present application had a recovery rate of 27.5% compared to comparative example 3, whereas comparative example 3 had only 18.2% and comparative example 4 had 25.3%; the obvious efficiency of the application is 56.88%, which is higher than 52.7% and 55.5% in the comparative example; the inefficiency of the application is only 1.87%, which is lower than that of the comparative examples, namely 6.7% and 2.4%. Because the cure rate and the significant efficiency of the pharmaceutical composition of the present application are higher than those of the comparative patent, the effective rate is naturally reduced, but overall the effective rate is 98.13%, which is higher than 93.3% and 97.6% in the comparative example.

In the process of taking the medicine, the vital signs of patients in the test group are stable, and adverse reactions such as nausea, vomiting and allergy are avoided, so that the clinical application of the medicine composition is safe and reliable.

Claims

1. A pharmaceutical composition for relieving pain caused by primary dysmenorrhea, wherein the dysmenorrhea is qi stagnation and blood stasis, and the pharmaceutical composition is characterized in that the raw materials in the pharmaceutical composition consist of the following raw materials in parts by weight: 1200g of white peony root, 720g of Chinese angelica, 600g of rhizoma corydalis, 600g of szechuan lovage rhizome, 600g of nutgrass galingale rhizome and 360g of honey-fried licorice root;

the preparation method of the pharmaceutical composition comprises the following steps:

s1, reflux-extracting the rhizoma corydalis with ethanol twice, filtering, recovering ethanol in filtrate, and concentrating under reduced pressure to obtain an extract I with the relative density of 1.10-1.15;

S2, reflux-extracting the rhizoma corydalis residues, the ligusticum wallichii, the angelica sinensis, the white paeony root, the honey-fried licorice root and the additional water twice, filtering, concentrating the filtrate under reduced pressure to obtain a concentrated solution with the relative density of 1.15-1.20, adding ethanol into the concentrated solution, standing at room temperature, filtering, recovering the ethanol, and concentrating the filtrate under reduced pressure to obtain an extract II with the relative density of 1.15-1.20;

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is an oral formulation.

3. The pharmaceutical composition according to claim 2, wherein the oral formulation is any one of a capsule, a pill, a syrup, a granule, and a tablet.

4. The pharmaceutical composition according to claim 1, wherein in step S1, the corydalis tuber is extracted with 5 times of 80% ethanol under reflux twice for 1 hour each.

5. The pharmaceutical composition according to claim 1, wherein in step S2, the residues of rhizoma corydalis, rhizoma Chuanxiong, radix Angelicae sinensis, radix Paeoniae alba, radix Glycyrrhizae Preparata and rhizoma Cyperi are extracted with 5 times of water under reflux twice, the first time for 2 hours and the second time for 1 hour.

6. The pharmaceutical composition according to claim 1, wherein in step S2, ethanol is added to the concentrate to an alcohol content of 80%, and the mixture is left at room temperature for 12 to 24 hours and then filtered.

7. A pharmaceutical composition according to any one of claims 1 to 6 for use in a medicament for alleviating pain caused by primary dysmenorrhea.