CN113876864B

CN113876864B - Pharmaceutical composition for treating infertility and preparation method and application thereof

Info

Publication number: CN113876864B
Application number: CN202111389080.0A
Authority: CN
Inventors: 高敬书; 王宇
Original assignee: Heilongjiang University of Chinese Medicine
Current assignee: Heilongjiang University of Chinese Medicine
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2023-04-07
Anticipated expiration: 2041-11-22
Also published as: CN113876864A

Abstract

The invention discloses a pharmaceutical composition for treating infertility, which comprises 0.02-0.04 part of compound of formula I, 0.01-0.04 part of chlorogenic acid, 0.08-0.12 part of herba patriniae extract and 0.08-0.12 part of epicatechin, and is prepared into the pharmaceutical composition for use. Experiments show that the pharmaceutical composition of the compound of the formula I, the chlorogenic acid, the patrinia extract and the epicatechin has synergistic effect, can dredge salpingemphraxis caused by inflammation, and has a therapeutic effect on infertility.

Description

Pharmaceutical composition for treating infertility and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical composition preparations, and particularly relates to a pharmaceutical composition for treating infertility.

Background

Infertility is a difficult disease which is concerned all over the world, is one of gynecological common diseases, is relatively intractable in treatment, and is a medical problem which needs to be solved urgently. As the oviduct plays an important role in picking up the ovum and transporting the ovum, the sperm and the embryo, and is also a place where sperm capacitation and sperm and egg meet and fertilize, the mucous membrane of the oviduct is easily damaged by infection and operation, further cilia disappear, the peristaltic disorder is caused, and even the oviduct is blocked or adhered to the surrounding tissues, and the unobstructed function of the oviduct is influenced. The obstruction or the blockage of the fallopian tube is an important reason for female infertility, and the infertility caused by the fallopian tube obstruction caused by salpingitis is called salpingitis obstructive infertility. Most western medicine treatment adopts cavity lens interventional therapy such as tubal catheter guide wire dilatation and the like, but the cost is high, the wound is large, the recurrence is easy, the re-adhesion in the tubal cavity is easy to cause, the wound of drug treatment is small, the toxic and side effects are small, the curative effect is high, the re-adhesion rate is low, the advantages are obvious, and the focus of attention in the medical field at present is provided.

The traditional Chinese medicine treatment is generally based on pharmacology of promoting blood circulation to remove blood stasis, clearing and activating the channels and collaterals, assisting pregnancy and preventing miscarriage, is comprehensively treated by adding and subtracting medicines according to specific type dialectics, assisting with a plurality of methods of traditional Chinese medicine external application, acupuncture and moxibustion, traditional Chinese medicine example introduction and the like, reflects the characteristic of complementary advantages of the traditional Chinese medicine on the adding and subtracting medicines, and improves the normal intrauterine pregnancy rate while weakening the clinical risk. However, the therapeutic diagnosis and the therapeutic effect judgment standards of traditional Chinese medicine are lack of uniformity, and the clinical treatment period is relatively long. Therefore, new medicines need to be further researched on the aspect of treating the infertility caused by oviduct obstruction so as to better exert the advantages of no wound, small toxic and side effects and definite curative effect in the disease treatment by the medicines, and the further research on the medicines with definite infertility treatment, small adverse reaction and low recurrence rate is necessary. Therefore, the invention further develops and prepares the pharmaceutical composition with good curative effect, small side effect, safety and effectiveness.

Disclosure of Invention

The invention aims to seek a medicinal composition with good curative effect, small side effect, safety and effectiveness, thereby providing a medicinal composition for treating infertility and a preparation method and application thereof.

The pharmaceutical composition for treating infertility consists of a compound shown as a formula I, chlorogenic acid, a herba patriniae extract and epicatechin,

wherein, the structure of the compound of formula I is as follows

The pharmaceutical composition for treating infertility comprises the following raw materials in parts by weight: 0.02-0.04 part of compound of formula I, 0.01-0.04 part of chlorogenic acid, 0.08-0.12 part of herba patriniae extract and 0.08-0.12 part of epicatechin.

More preferably, the pharmaceutical composition for treating infertility comprises the following raw materials in parts by weight: 0.02-0.03 part of compound of formula I, 0.02-0.03 part of chlorogenic acid, 0.10-0.12 part of herba Patriniae extract and 0.08-0.1 part of epicatechin.

More preferably, the pharmaceutical composition for treating infertility comprises the following raw materials in parts by weight: 0.02 part of compound of formula I, 0.03 part of chlorogenic acid, 0.1 part of herba patriniae extract and 0.08 part of epicatechin.

The pharmaceutical composition for treating infertility is prepared by the following method: pulverizing herba Patriniae, adding 8 times of petroleum ether, defatting under reflux for 2 times (1 hr each time), soaking in 16 times of water at 85 deg.C for 3 times (2 hr each time), mixing medicinal liquids, filtering, and concentrating.

The invention also provides a pharmaceutical preparation containing the medicament for treating infertility as an active ingredient, and the pharmaceutical preparation is prepared into common pharmaceutical dosage forms such as oral liquid, powder, granules, capsules, tablets, pills and the like by adopting the conventional preparation method in the field. Preferably tablet, oral liquid, buccal tablet, chewable tablet, and powder.

The pharmaceutical preparation can also comprise a medically acceptable carrier, and the sum of the weight of the medicinal material components accounts for 1 to 99.9 percent of the total weight of the medicine; more preferably, the sum of the weight of the medicinal material components accounts for 10 to 80 percent of the total weight of the medicament; further preferably, the sum of the weight of the medicinal material components accounts for 30-60% of the total weight of the medicament.

The invention also provides application of the pharmaceutical composition in preparing a medicament for treating infertility, and the pharmaceutical composition can be used for treating infertility caused by salpingitis induced salpingemphraxis.

The compound with the structure shown in the formula I is a known flavonoid compound, and is a compound separated from an ethanol extract of phellodendron amurense serving as a genuine herb in Heilongjiang province and obtained by purification. Phellodendron amurense is a larch of phellodendron genus of Rutaceae family, the bark is grayish brown to black gray, and the inner layer of the bark is usually processed and used as a medicine, which is bitter in taste and cold in nature and has the functions of clearing heat and removing toxicity, purging fire and drying dampness. The compound with the structure shown in the formula I is a phellodendron amurense dry leaf extract, the English name of which is (2S) -5-Hydroxy-2- (4-hydroxyphenyl) -8- (3-methyl-2-buten-1-yl) -4-oxo-3,4-dihydro-2H-chromen-7-yl beta-D-glucopyranoside, the common name is Phellodenin F, and the structural formula is as follows:

the extraction method comprises drying cortex Phellodendri, extracting under reflux with 8 times of 95% ethanol for 2 times (each time for 2 hr), and concentrating under reflux to obtain brown extract. Dispersing the extract in water, filtering, passing the filtrate through Diaion HP-20 macroporous adsorbent resin, eluting with 50% ethanol, separating with silica gel column (80-100 mesh), eluting with chloroform-methanol 15: 1, and purifying with Sephadex LH-20 to obtain compound of formula I with potential antiinflammatory and antioxidant effects. The chlorogenic acid in the inventionThe chlorogenic acid is an important pharmacodynamic component in the Chinese medicinal sargentgloryvine stem, has the effects of clearing away heat and toxic materials, promoting blood circulation and dispelling wind, and researches show that the chlorogenic acid can directly act on bacterial cell walls to inhibit the synthesis of the bacterial cell walls, so that the chlorogenic acid has wide antibacterial activity. The patrinia distillation liquid part has strong bacteriostatic activity on escherichia coli, streptococcus and staphylococcus aureus in vitro, so the patrinia distillation liquid part is extracted by water decoction, and the patrinia extract is obtained by the specific method that patrinia is crushed, 8 times of petroleum ether is added for reflux and degreasing for 2 times, 1 hour each time, 3 times of soaking in 16 times of water at 85 ℃, 2 hours each time, liquid medicine is combined, filtered and concentrated. The epicatechin is a natural plant flavanol compound, is white crystal, is easy to dissolve in water and methanol, exists in a large number of foods and Chinese medicinal materials, has the functions of resisting oxidation, removing free radicals, enhancing metabolism, regulating immunity, resisting tumors and the like, and has various other physiological activities, such as the effects of reducing fat and blood sugar, preventing cardiovascular diseases, resisting inflammation, protecting nerves, inhibiting bacteria and the like. The pharmaceutical composition is scientific and reasonable, and pharmacological experiments show that the components of the pharmaceutical composition which takes the compound shown in the formula I, chlorogenic acid, herba patriniae extract and epicatechin as main components have mutual synergistic effect, and the comprehensive effect of the components is utilized to treat tubal obstructive infertility by dissipating blood stasis, diminishing inflammation and inhibiting the development of inflammatory factors, so that the pharmaceutical composition has no toxic or side effect and does not generate dependence.

The pharmaceutical composition is obtained by screening a large number of compositions, the preparation of the pharmaceutical composition is optimized, the inventor does a large amount of work to optimize the composition, and the pharmaceutical composition is proved by comparative experiments, screening experiments and animal experiments that the preparation of the pharmaceutical composition has remarkable effect on treating infertility compared with the prior art, and has prominent substantive specific and remarkable progress compared with the prior art.

Detailed Description

The following examples are given for the purpose of illustrating the present invention, and the detailed embodiments and specific procedures are given for the purpose of illustrating the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

A pharmaceutical composition for the treatment of infertility is prepared by pulverizing compound of formula I0.03 g, chlorogenic acid 0.02g, herba Patriniae extract 0.1g, and epicatechin 0.08g, and mixing.

Example 2

A pharmaceutical composition for the treatment of infertility is prepared from compound of formula I0.02 g, chlorogenic acid 0.03g, herba Patriniae extract 0.11g, and epicatechin 0.08g by pulverizing and mixing.

Example 3

A pharmaceutical composition for the treatment of infertility is prepared by pulverizing and mixing 0.03g of compound of formula I, 0.02g of chlorogenic acid, and 0.1g of herba Patriniae extract.

Example 4

A pharmaceutical composition for the treatment of infertility is prepared by pulverizing compound of formula I0.03 g, herba Patriniae extract 0.10g, and epicatechin 0.08g, and mixing.

Example 5

A pharmaceutical composition for the treatment of infertility is prepared from compound of formula I0.03 g, chlorogenic acid 0.02g, and epicatechin 0.08g by pulverizing and mixing.

Example 6

A pharmaceutical composition for the treatment of infertility is prepared by pulverizing compound of formula I0.08 g and chlorogenic acid 0.15g, and mixing.

Example 7

A pharmaceutical composition for the treatment of infertility is prepared from herba Patriniae extract 0.10g and epicatechin 0.13g by pulverizing and mixing.

Example 8

A pharmaceutical composition for the treatment of infertility is prepared from 0.03g of compound of formula I.

Experimental example 1 pharmacodynamic test of the drug of the present invention for infertility

(1) And (3) experimental modeling: 240 Wistar female rats were acclimatized for one week and randomly divided into 20 normal groups and 220 molding groups. The model group rats are fasted for 12 hours without water prohibition, injected with 10% chloral hydrate in the abdominal cavity, and fixed on an operation table after about 5 minutes of anesthesia effectAfter the abdomen is sheared, disinfected by alcohol and iodophor, the median incision of the lower abdomen is 0.8-1cm long, the uterus, the oviduct and the ovary are exposed, and 0.1mL of bacterial suspension (diluted by sterile saline at a ratio of 2: 1 and with a concentration of 3X 10) is injected from the uterus to the ovary in the direction from the oviduct to the ovary ⁹ one/mL), the muscle layer and the skin are sutured in sequence after injection, and the rat cage is placed after alcohol disinfection for natural revival. After the molding operation, 1 rat is randomly sacrificed from the molding rats on the 5 th, 10 th, 15 th and 20 th days respectively for pathological observation, and the observation proves that the rat is marked by adhesion, stiffness and obstruction of the oviduct cavity after 20 days, and the molding is successful.

(2) Experimental grouping: the successfully molded 200 rats were randomly divided into 10 groups of 20 rats each as a model group, a positive group and each group of examples 1 to 8, and were subjected to constant volume intragastric administration on the 20 th day after molding. The normal saline is perfused into a model group, 0.03g/ml (radix bupleuri, angelica, rehmannia root, red paeony root, safflower, peach seed, fructus aurantii, liquorice, ligusticum wallichii, radix achyranthis bidentatae and platycodon grandiflorum) of the blood stasis removing tablet is administrated into a positive group, 0.03g/ml of the medicines in the examples 1 to 8 are respectively administrated into the groups from the example 8, and the groups are perfused into the stomach according to the volume of 1ml/100g after conversion according to the specific gravity of a human and a mouse. The normal group without molding was perfused with equal volume of normal saline. All groups of rats were gavaged 1 time a day for 30 consecutive days.

(3) The experimental method comprises the following steps: at 15 days and 30 days after administration, 1.5-2h, 5 rats are respectively taken for chloral hydrate anesthesia, and the two fallopian tubes are taken to remove redundant adipose tissues for observation. The visual observation of normal group has clear tissue structure of fallopian tube, unobstructed lumen, abundant cilia, tight intercellular connection, and no fibrous tissue proliferation and inflammatory cell infiltration. On day 15, the model group has thickened tube wall, reduced lumen, adhesion of cilia, proliferation of fibrous tissues and blood vessel expansion, on day 30, the tube wall has obviously thickened, the lumen has obviously reduced, the cilia has increased thickness, the fibrous tissues have obviously proliferated, and chronic inflammatory cell infiltration can be seen. Each administration group had uniform thickness of oviduct on day 15, and had different degrees of adhesion with surrounding tissues and different degrees of hydrocele.

5ml of abdominal aorta blood is taken, the contents of IL-1 and TNF-alpha in serum are detected by a double antibody sandwich ELISA method, and the experimental result is shown in Table 1. The expression of the oviduct EGFR and ICAM-1 proteins is detected by an immunohistochemical SABC method, and the experimental results are shown in Table 2. After blood is taken and anticoagulant is added, a full-automatic blood viscosity instrument is adopted to detect the rheological indexes of blood, including whole blood viscosity, erythrocyte rigidity index, erythrocyte aggregation index and hematocrit, and the experimental result is shown in table 3.

TABLE 1 Effect of groups of drugs on IL-1, TNF- α levels in rat serum (x. + -. S, pg/ml, n = 5)

Because IL-1, TNF-alpha is one of the main signs of inflammatory reaction, have many biological activities, play a certain role in the development process of chronic inflammation pathophysiology, can finally cause local tissue adhesion and granulation fibrous tissue growth of organism, IL-1, TNF-alpha content increase apparently in the blood serum of oviduct of inflammation injury, have shown that it participates in chronic inflammatory reaction of oviduct, reduce IL-1, TNF-alpha content can further treat inflammatory obstructive infertility. The experimental results in Table 1 show that the IL-1 and TNF-alpha contents in the model group are obviously increased compared with the IL-1 and TNF-alpha contents in the normal control group, which indicates that the mixed bacteria liquid molding can increase the IL-1 and TNF-alpha contents in blood to cause infertility. The IL-1 and TNF-alpha contents in the groups of examples 1-8 and the positive group are obviously different from those in the normal control group and the model group, wherein the IL-1 and TNF-alpha contents in the groups of examples 3-8 are obviously reduced, which shows that the examples 3-8 can treat infertility by reducing the IL-1 and TNF-alpha contents. The IL-1 and TNF-alpha contents of the groups 1-2 and the positive administration group are remarkably reduced, which shows that the infertility can be treated by reducing the IL-1 and TNF-alpha contents of the groups 1-2 and the positive administration group. By comparing the data of examples 1-2, the positive group and examples 3-8, it can be further explained that the therapeutic effect on infertility exerted by decreasing the levels of IL-1 and TNF- α in the groups of examples 1-2 and the positive administration group is stronger than that of the drugs of the groups of examples 3-8.

TABLE 2 Effect of groups of drugs on the expression levels of EGFR and ICAM-1 proteins in rat oviduct (x. + -. S, n = 5)

Since EGFR epidermal growth factor receptor is ubiquitous in epidermal cells and stromal cells, and has prominent effect in the processes of regulating cell growth and tissue repair, EGFR distribution in normal oviduct is related to differentiation degree of oviduct epithelium, and high expression of EGFR enhances the reactivity of epithelial cells to growth factors, promotes the growth factors to combine with the receptor to form a compound, influences the proliferation and differentiation process of oviduct epithelium, and promotes inflammation to further aggravate, thereby causing infertility. The cell adhesion molecule ICAM-1 belongs to glycoprotein expressed on the cell surface, is rarely shown under normal conditions, and is obviously increased after being stimulated by IL-1 and TNF-alpha, and the increase of the expression can promote or intensify the generation of inflammation and increase the adhesion among cells. According to the experimental results shown in Table 2, ICAM-1 expression is not found in the oviduct epithelial cells of the normal group, and the EGFR protein and ICAM-1 expression of the model group is obviously increased compared with the EGFR protein and ICAM-1 expression of the normal control group, so that the mixed bacterial liquid molding can increase the EGFR protein and ICAM-1 expression in the oviduct epithelial cells to cause infertility. The EGFR protein and ICAM-1 expression in the example 1-8 groups and the positive group are obviously different from those in the normal control group and the model group, wherein the EGFR protein and ICAM-1 expression in the example 3-8 groups are obviously reduced, which shows that the example 3-8 can treat infertility by reducing the EGFR protein and ICAM-1 expression. The EGFR protein and ICAM-1 expressions in the groups 1-2 and the positive administration group were significantly reduced, which demonstrates that the infertility treatment by reducing the EGFR protein and ICAM-1 expressions in the groups 1-2 and the positive administration group was possible. By comparing the data of examples 1 to 2, the positive group and examples 3 to 8, it can be further explained that the therapeutic effect on infertility exerted by the reduction of the expression of EGFR protein and ICAM-1 in examples 1 to 2 and the positive administration group is stronger than that of the drugs in examples 3 to 8.

TABLE 3 Effect of each group of drugs on the rat hemorheological indices (x. + -. S, n = 5)

The whole blood viscosity, the erythrocyte rigidity index, the erythrocyte aggregation index, the hematocrit and the like belong to hemorheology indexes and can be used as important bases for evaluating the blood viscosity, and the indexes are improved to a certain extent in an oviduct inflammatory obstructive infertility rat model. The experimental results shown in table 3 show that the whole blood viscosity, erythrocyte rigidity index, erythrocyte aggregation index and hematocrit of the model group rat are all obviously increased, which indicates that the modeling is successful. The hemorheology indexes of all treatment groups are obviously reduced compared with those of the model group, and the whole blood viscosity, the erythrocyte rigidity index, the erythrocyte aggregation index and the hematocrit of the groups of examples 1-8 and the positive group are obviously different from those of the normal control group and the model group, wherein the whole blood viscosity, the erythrocyte rigidity index, the erythrocyte aggregation index and the hematocrit content of the groups of examples 3-8 are obviously reduced, which indicates that the groups of examples 3-8 can treat infertility by reducing the whole blood viscosity, the erythrocyte rigidity index, the erythrocyte aggregation index and the hematocrit. Examples 1 to 2 show that the positive group had significantly reduced whole blood viscosity, erythrocyte rigidity index, erythrocyte aggregation index and hematocrit, and examples 1 to 2 show that infertility could be treated by reducing whole blood viscosity, erythrocyte rigidity index, erythrocyte aggregation index and hematocrit. By comparing the data of examples 1-2, the positive group and examples 3-8, it can be further explained that the treatment effect on infertility exerted by the decrease in whole blood viscosity, the erythrocyte rigidity index, the erythrocyte aggregation index and the hematocrit in examples 1-2 and the positive administration group is stronger than that of the drugs in examples 3-8.

The remaining 10 rats in each group were placed in 2 male rats each, and were housed in the same cage for 30 days, and the pregnancy of each group was observed separately, and the results are shown in Table 4.

Table 4 effect of each group of drugs on conception rate of rats (x ± S, n = 10)

After the completion of the gavage, the pregnancy rate of each group of rats is compared, and the results show that the pregnancy rate of the normal group is 100%, the pregnancy rate of the model group is only 10%, the pregnancy rates of the experimental examples 1-8 and the positive groups are respectively 30% -90% different, and the results further show that the cure rate of the treatment of the inflammatory obstructive infertility of the groups 1-2 and the positive groups reaches more than 60%, and the treatment effect is obviously superior to the action effect of the groups 3-8. Meanwhile, the data show that the compatibility of various medicinal raw materials in the medicament is excellent, the compatibility is not good, and the combination of various medicinal raw materials produces an obvious synergistic technical effect.

Experimental example 2 clinical trial of infertility by the drug of the present invention

(1) Grouping: 100 patients with fallopian tube obstructive infertility are confirmed by hysterosalpingography, wherein 26 cases of primary infertility and 74 cases of secondary infertility are confirmed; 51 cases of bilateral obstruction of the fallopian tube, 35 cases of bilateral obstruction and bilateral obstruction, and 14 cases of unilateral obstruction and contralateral obstruction; the patients are 22-42 years old, the disease course is 1-5 years old, and the patients are randomly divided into 2 groups, wherein 60 cases of treatment groups and 40 cases of control groups have no obvious difference in age, sterility age, fallopian tube patency and the like.

(2) Treatment criteria were: and (3) healing: the uterus oviduct contrast proves the smoothness. The method has the following advantages: hysterosalpingography indicates that the fallopian tube is obstructed or the blocked part is reduced. And (4) invalidation: the fallopian tubes remain occluded. The control group is administered with ZHONG SHI XUE FU ZHU YU PIAN (blood stasis removing tablet), which is prepared from bupleuri radix, radix Angelicae sinensis, rehmanniae radix, radix Paeoniae Rubra, carthami flos, semen Persicae, fructus Aurantii, glycyrrhrizae radix, rhizoma Ligustici Chuanxiong, achyranthis radix, and radix Platycodi; the experimental group was administered with the drug prepared in example 1 of the present invention, and 3 months was one treatment course.

(3) The results are shown in Table 5.

TABLE 5 comparison of the two groups of therapeutic effects and pregnancy rate within 1 year

As can be seen from the results in Table 5, the cure rate and the total effective rate of the experimental group are significantly higher than those of the control group, and the experimental group has significant advantages in terms of the number of patients who visit and have a pregnancy within one year.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A pharmaceutical composition for treating infertility is characterized by comprising a compound of formula I, chlorogenic acid, herba Patriniae extract and epicatechin, wherein the compound of formula I has the following structure

The weight portion ratio is as follows: 0.02-0.03 part of compound of formula I, 0.02-0.03 part of chlorogenic acid, 0.10-0.12 part of herba Patriniae extract and 0.08-0.1 part of epicatechin;

the herba Patriniae extract is prepared by pulverizing herba Patriniae, defatting with 8 times of petroleum ether under reflux for 2 times (1 h each time), soaking in 16 times of water at 85 deg.C for 3 times (2 h each time), mixing the medicinal solutions, filtering, and concentrating.

2. The pharmaceutical composition according to claim 1, wherein the weight parts ratio is: 0.02 part of compound of formula I, 0.03 part of chlorogenic acid, 0.10 part of herba patriniae extract and 0.08 part of epicatechin.

3. The method for preparing a pharmaceutical composition according to claim 1 or 2, wherein the compound of formula I, chlorogenic acid, herba patriniae extract, epicatechin are mixed and dispersed.

4. The pharmaceutical preparation prepared from the pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical preparation is prepared into a pharmaceutically common dosage form by adopting a conventional preparation method in the field, and the dosage form is oral liquid, powder, granules, capsules, tablets or pills.

5. The pharmaceutical preparation according to claim 4, further comprising a pharmaceutically acceptable carrier, wherein the sum of the weight of the medicinal components accounts for 30-60% of the total weight of the pharmaceutical preparation.

6. Use of a pharmaceutical composition according to claim 1 or 2 for the preparation of a medicament for the treatment of infertility, wherein infertility is infertility caused by salpingemphraxis induced by salpingitis.