CN112569246A - Medicine for anti-inflammatory, analgesic and hemostatic and screening method of active components of medicine - Google Patents
Medicine for anti-inflammatory, analgesic and hemostatic and screening method of active components of medicine Download PDFInfo
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- CN112569246A CN112569246A CN202011535564.7A CN202011535564A CN112569246A CN 112569246 A CN112569246 A CN 112569246A CN 202011535564 A CN202011535564 A CN 202011535564A CN 112569246 A CN112569246 A CN 112569246A
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- uterine bleeding
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Abstract
The invention discloses a medicine for anti-inflammatory, analgesic and hemostatic and a screening method of active ingredients of the medicine, wherein the medicine comprises the following components: kaempferol 300-500, acacetin 1-5, quercetin 8-20, baicalein 30-60 and wogonin 10-20. The screening method comprises screening active compounds from the main chemical components of Scutellariae radix and flos Sophorae Immaturus; collecting protein targets of active compounds, retrieving and correcting to obtain gene names, and establishing a dysfunctional uterine bleeding target set; obtaining the intersection gene of the active compound and the dysfunctional uterine bleeding target, carrying out channel enrichment analysis on the intersection gene, and screening to obtain the active compound. The invention discloses a medicament which has good curative effect and can be more effectively used for resisting inflammation, relieving pain and stopping bleeding.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a medicine and a screening method of active ingredients of the medicine, and particularly relates to a medicine for resisting inflammation, relieving pain and stopping bleeding and a screening method of the active ingredients of the medicine.
Background
Dysfunctional uterine bleeding (Chinese medicine name: metrorrhagia) (DUB) is a common disease type in gynecological clinical treatment. DUBs are those caused by neuroendocrine disorders, not systemic or female genital organic diseases such as pregnancy, endometrial tumors, infections or blood diseases. It usually occurs in adolescence or perimenopause, and is mostly non-ovulatory type DUB. The cause of anovulatory dysfunctional uterine bleeding is different between puberty and climacteric. In adolescence DUB, due to the fact that the hypothalamus-pituitary-ovary axis is not fully developed or delayed, a complete feedback regulation mechanism is not established between the hypothalamus-pituitary and the ovary, under the action of pituitary follicle-stimulating hormone and luteinizing hormone, follicles develop and secrete estrogen, but positive feedback of estrogen to the hypothalamus cannot form the peak of follicle-stimulating hormone and luteinizing hormone in normal menstrual cycle, and thus, although follicles in the ovary develop, the ovary cannot ovulate. Perimenopausal DUB is mainly caused by natural decline of ovarian function, reduction of follicle number, maturation disorder and reduction of pituitary gonadotropin response, so that ovulation stops when ovarian function declines, and perimenopausal anovulatory dysfunctions are caused. The major clinical manifestations of DUB are abnormal menstrual flow, menstrual cycle disorders, abnormal bleeding of the uterus, and prolonged or excessive bleeding of the uterus in severe patients, resulting in hemorrhagic shock or anemia. The traditional Chinese medicine holds that the main pathogenesis of the uterine bleeding is that the diseases such as dysmenorrheal and infertility are caused by qi stagnation and blood stasis caused by kidney qi deficiency, incapability of nourishing Chong and ren meridians, deficiency of qi and blood, recklessly blood flow due to blood heat and the like. Currently, the clinical treatment mainly takes the principle of regulating the menstrual cycle and stopping bleeding rapidly, and progestational hormone treatments such as medroxyprogesterone and norethindrone are usually selected, although the symptoms of patients can be relieved obviously, the dependence is easy to generate. Clinically, the traditional Chinese medicines for promoting blood circulation to remove blood stasis, cooling blood to stop bleeding and softening hardness to dissipate stagnation are adopted to treat the DUB caused by qi stagnation, blood stasis and blood heat so as to relieve dysmenorrhea, promote absorption and dissipation of mass and the like. An Huanghuai compound (HH) is a classic prescription for treating dysfunctional uterine bleeding in traditional Chinese medicine, is prepared from scutellaria baicalensis and sophora japonica 4:1(w/w), and has the effects of purging fire, detoxifying, cooling blood and stopping bleeding. Modern pharmacological research shows that the sophora japonica has obvious hemostatic effect.
Because the components of the traditional Chinese medicine are complex, the effective components of the traditional Chinese medicine are not easy to determine, in order to ensure the drug effect, the administration form of decoction is often adopted, the effective components and other non-effective components are taken together, the dosage is large, and the traditional Chinese medicine cannot be developed into a modern dosage form due to the limitation of drug-loading rate. In addition, the process route and quality control of the traditional Chinese medicine at present mostly take chemical components as indexes, but the relevance of the components to clinical safety and effectiveness is unclear, and the curative effect of the medicine can be influenced to a certain extent.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a more effective medicine for anti-inflammation, analgesia and hemostasis with good curative effect aiming at the defects of the prior art.
The invention further solves the technical problems that: provides a method for screening research index components of new traditional Chinese medicines, and improves the clinical use correlation of the new traditional Chinese medicines.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a medicine for resisting inflammation, easing pain and stopping bleeding comprises the following components in parts by weight:
further, the medicine for resisting inflammation, easing pain and stopping bleeding preferably comprises the following components in parts by weight:
further, the medicine for resisting inflammation, easing pain and stopping bleeding preferably further comprises the following components in parts by weight: baicalin 800-1000.
Further, the medicine for resisting inflammation, easing pain and stopping bleeding preferably further comprises the following components in parts by weight: baicalin 900-.
A method for screening active pharmaceutical ingredients for anti-inflammatory, analgesic and hemostatic purposes comprises the following steps:
A. screening active compounds with the oral bioavailability OB value of more than or equal to 30 percent and the drug property DL value of more than or equal to 0.18 according to the main chemical components of the scutellaria baicalensis and the sophora japonica;
B. collecting the protein targets of the active compounds, collecting the protein targets of main chemical components in the scutellaria baicalensis and the sophora japonica through a target prediction function in a TCMSP database, retrieving by using a Uniprot database to obtain the gene names corresponding to the protein targets, and performing retrieval correction on the artificial species to obtain UniprotID and the gene names corresponding to the protein targets;
C. searching genes of related Dysfunctional uterine bleeding by using a GeneCards database and using Dysfunctional uterine bleeding as a keyword, and establishing a Dysfunctional uterine bleeding target set;
D. comparing the protein target gene with the target gene for dysfunctional uterine bleeding, wherein the protein target gene is acted by the screened active compound, so as to obtain an intersection gene of the active compound and the target gene for dysfunctional uterine bleeding, and the intersection gene is used as a potential action target gene for treating the dysfunctional uterine bleeding by the active compound;
E. carrying out protein interaction analysis on the potential action target genes;
F. carrying out GO and KEGG enrichment analysis on the potential action target genes, and screening out KEGG access information with significant difference in the process of treating dysfunctional uterine bleeding by using the active compound;
G. analyzing the interaction of the active compound, the potential action target gene, GO and KEGG pathway, wherein the screened active compound is at least: quercetin, wogonin, kaempferol, baicalein, and robinin.
Further, in the method for screening the active pharmaceutical ingredient for anti-inflammatory, analgesic and hemostatic purposes, preferably in step C, the first 200-400 results with high correlation are selected from the target set of dysfunctional uterine bleeding.
Further, in the method for screening active pharmaceutical ingredients for anti-inflammatory, analgesic and hemostatic purposes, preferably, in step D, the protein target gene of the screened active compound and the dysfunctional uterine bleeding target gene are analyzed to obtain an intersection gene of the active compound and the dysfunctional uterine bleeding target.
Further, in the method for screening a pharmaceutically active ingredient for anti-inflammatory, analgesic and hemostatic uses, it is preferable that in the step E, points pointing to each protein interaction information are evaluated and assigned for predictable protein-protein interactions of potential target genes stored by String database.
Further, in the method for screening the active pharmaceutical ingredients for anti-inflammatory, analgesic and hemostatic purposes, preferably, in the step F, the potential target genes with effects are introduced into the DAVID database for GO and KEGG enrichment analysis, and the most important biological process BP, cell component CC and molecular function MF are screened, so that KEGG pathway information with significant differences is obtained, wherein the active compounds participate in the pathological process of dysfunctional uterine bleeding.
Further, in the method for screening the active pharmaceutical ingredients for anti-inflammatory, analgesic and hemostatic purposes, preferably, in the step G, an active compound-target-pathway network model is constructed, and key nodes are determined by adopting the order of Degree, ASPL, BC and CC, wherein the compound molecules with the Degree being more than or equal to 2 x the median are at least quercetin, wogonin, kaempferol, baicalein and robinin.
The main active ingredient compounds of the sophora japonica compound (HH) screened by the invention are quercetin, wogonin, kaempferol, baicalein and robinin, and the tests prove that the sophora japonica compound (HH) is used as a medicine for treating dysfunctional uterine bleeding: the medicine of the present invention has obvious pain relieving and inflammation diminishing effects and obvious blood coagulation function.
The invention screens the effective substances for treating a certain specific disease in the traditional Chinese medicine prescription by a network pharmacology method, reforms the effective component composition for treating the disease, can reduce the dosage of the medicine while ensuring the curative effect, and solves the problem that the traditional Chinese medicine cannot be accurately and quantitatively controlled due to the unclear effective components.
Drawings
The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a graph showing the comparison of drug and target point Wein associated with dysfunctional uterine bleeding according to example 13 of the present invention;
FIG. 2 is a network diagram of the interaction of potential targets for drug therapy of dysfunctional uterine bleeding according to example 13 of the present invention;
FIG. 3 is a bar graph of the biological process of GO enrichment analysis, a potential target for drug therapy of dysfunctional uterine bleeding, example 13 of the present invention;
FIG. 4 is a bar graph of cellular components of GO enrichment analysis, a potential target for drug therapy of dysfunctional uterine bleeding, in accordance with example 13 of the present invention;
FIG. 5 is a bar graph of the molecular function of GO enrichment analysis, a potential target for drug therapy of dysfunctional uterine bleeding, in accordance with example 13 of the present invention;
FIG. 6 is a KEGG enrichment analysis bar graph of the potential target of action of example 13 of the present invention in drug therapy for dysfunctional uterine bleeding;
FIG. 7 is a KEGG enrichment analysis high-grade bubble graph of potential target sites of drug therapy for dysfunctional uterine bleeding according to example 13 of the present invention;
figure 8 is a diagram of the drug "component-target-pathway" network of example 13 of the present invention.
Detailed Description
For a more clear understanding of the technical features, objects and effects of the present invention, embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
The Huanghuai compound is prepared by matching scutellaria baicalensis and sophora japonica and is mainly used for treating dysfunctional uterine bleeding. Firstly, the scutellaria baicalensis and the sophora japonica in the sophora japonica compound are researched, wherein the scutellaria baicalensis is used as a main drug in a prescription, the main active ingredient of the scutellaria baicalensis is baicalin, and in an in vitro blood coagulation activity determination experiment performed by taking the baicalin as a technological index in the early stage, the hemostatic function of the scutellaria baicalensis and the sophora japonica compound is found to be stronger. The invention screens 6 active ingredients for treating dysfunctional uterine bleeding in the sophora japonica compound, namely quercetin, wogonin, kaempferol, baicalein, acacetin and beta-sitosterol, wherein baicalin is not included. When the content of the seven active ingredients including baicalin is measured, the beta sitosterol in the measurement result is removed due to the low content, and finally 6 ingredients including baicalin (quercetin, wogonin, kaempferol, baicalein, acacetin and baicalin) are obtained as the effective ingredient group. The invention thus provides a detailed description of baicalin-containing and baicalin-free medicaments in two different embodiments.
The present invention has two embodiments of drug combinations, wherein examples 1 to 6 are drug 1 in which the first embodiment does not contain baicalin, and examples 7 to 12 are drug 2 in which the second embodiment contains baicalin.
Example 1, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 300, acacetin 5, quercetin 8, baicalein 60 and wogonin 10.
Example 2, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 400, acacetin 2, quercetin 15, baicalein 30 and wogonin 20.
Example 3, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 500, acacetin 1, quercetin 20, baicalein 45 and wogonin 15.
Example 4, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 450, acacetin 2, quercetin 10, baicalein 50 and wogonin 10.
Example 5, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 400, acacetin 3, quercetin 12, baicalein 40 and wogonin 14.
Example 6, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 415, acacetin 1.8, quercetin 11, baicalein 45 and wogonin 13.
Example 7, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 380, acacetin 4, quercetin 17, baicalein 55, wogonin 17 and baicalin 800.
Example 8, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 470, acacetin 1.5, quercetin 9, baicalein 34, wogonin 11, and baicalin 830.
Example 9, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 420, acacetin 2.5, quercetin 14, baicalein 47, wogonin 16 and baicalin 1000.
Example 10, a medicament for anti-inflammatory, analgesic and hemostatic, comprising the following components in parts by weight: kaempferol 320, acacetin 2.7, quercetin 11, baicalein 49, wogonin 12, and baicalin 860.
Example 11, a medicament for anti-inflammatory, analgesic, and hemostatic, comprising the following components in parts by weight: kaempferol 415, acacetin 3.5, quercetin 13, baicalein 45, wogonin 13 and baicalin 900.
Example 12, a medicament for anti-inflammatory, analgesic, and hemostatic, comprising the following components in parts by weight: kaempferol 430, acacetin 2.8, quercetin 12.5, baicalein 35, wogonin 16, and baicalin 930.
The above examples of the invention were validated by the following comparative tests
First, analgesic and anti-inflammatory experiment
1. Medicaments and agents
The Sophora japonica medicinal material is purchased from the market of Chinese medicinal materials in Hehua pond and is identified as the dried flower of Sophora japonica L. of leguminous plants; the Scutellariae radix is purchased from Beijing Hojingtang and identified as dried root of Scutellaria Baicalensis Georgi of Labiatae. Baicalin extract (batch No. TB190628-1, purity not less than 85%, SiAnchuang En Biotech Co., Ltd.); baicalein extract (batch No. KHBA190901-1, purity not less than 98%, Xian Chuang En Biotech Co., Ltd.); robinin extract (batch No. 19062701, purity greater than or equal to 99%, Xianrong coconut Biotech Co., Ltd.); kaempferol extract (batch No. 19081801, purity greater than or equal to 98%, Xianrong coconut Biotech Co., Ltd.); quercetin extract (batch No. 19072701, purity greater than or equal to 98%, Xianrong coconut Biotech Co., Ltd.); wogonin reference (batch No. wkq 19020201; purity not less than 98%, Szechwan, Ukki Biotech Co., Ltd.); wogonin extract (batch No. 19120901; purity not less than 98%, Shaanxi Green clear bioengineering Co., Ltd.); yellow sophora extract (batch 20190917, made by laboratory); physiological saline (batch No. C18051905-1, Sichuan Konlun pharmaceutical Co., Ltd.); aspirin effervescent tablets (batch No. 1903008, Astrazeneca, uk); baogong Zhixue Keli (batch No. 1901049, Tianjin Zhongsheng Haitian pharmaceutical Co., Ltd.).
2. Main instrument
DXL-10 rat metabolic cage (von willebrand, suzhou); SQP Satorious type electronic balance (beijing sialus scientific instruments ltd); an electronic analytical balance of FA2004 type (shanghai liangping instruments ltd); YLS-6B Intelligent hotplate instrument (Equipment station of Shandong province institute of medical science); PS-40 type ultrasonic cleaner (Shenzhen Shenkang cleaning equipment, Inc.); type YB5001B vernier caliper (kaffel real ltd).
3. Laboratory animal
Kunming mouse, 20-40g, female, certificate number SCXK (Chuan) 2015-. Provided by Dryokuo technologies, Inc.
4. Experimental methods
Analgesic action on mice
(1) The qualified Kunming female sexual maturity mice are randomly divided into A, B, C, D, E (optionally four embodiments of the medicine 1 of the invention) and F (optionally four embodiments of the medicine 2 of the invention) 12 groups, namely a negative group (10mL/kg), an aspirin group (1g/kg), a uterus preserving hemostasis group (6g/kg) and a yellow sophora extract group (1.4g/kg), wherein the medicine 1(0.15g/kg) and the medicine 2(0.42g/kg) correspond to 10 mice. The intragastric administration is carried out for 7 days by intragastric administration for 1 time every day. The pain threshold of the mice is respectively determined after 30min, 60min, 90min and 120min of the last administration. The results were statistically processed using one-way analysis of variance, with the following formula: pain threshold (post-dose pain threshold-pre-dose pain threshold)/pre-dose pain threshold.
(2) Experiment for swelling of foot sole of mouse caused by egg white
Taking female mice with mature Kunming sex, randomly dividing the female mice into A, B, C, D, E (optionally four embodiments of the drug 1 of the invention) and F (optionally four embodiments of the drug 2 of the invention) 12 groups, namely a negative group (10mL/kg), an aspirin group (1g/kg), a uterus-preserving hemostasis group (6g/kg) and a yellow sophora extract group (1.4g/kg), wherein the drug 1(0.15g/kg) of the invention and the drug 2(0.42g/kg) correspond to 9 mice. After grouping, a clear mark line is marked on the same part of the right hind toe of each mouse by a marker pen, and the normal thickness of the mouse is measured by a vernier caliper. After the measurement, the gavage mode is adopted, the gavage is performed for 1 time every day, and the gavage is continued for 8 days. After 15min of the last administration, approximately 0.05mL of 10% egg white was injected intramuscularly from the mark. The thickness of the mark part is measured after 0.5,1 and 1.5 hours of sensitization respectively, and the thickness difference before and after sensitization is the swelling degree. The swelling rate was calculated. The results were statistically processed using one-way analysis of variance, with the following formula: the rate of swelling (%) of the foot sole was ═ thickness of the foot sole after inflammation-thickness of the foot sole before inflammation)/thickness of the foot sole before inflammation × 100%.
5. Results of the experiment
As can be seen from Table 1, compared with group A, group D can significantly improve the pain threshold (P <0.05) of the mice at 60min, while group E can significantly improve the pain threshold (P <0.05) of the mice at 30min, 60min and 90 min; in addition, compared with the group D, the group E, F has no significant difference when P is more than 0.05 at 30min, 60min, 90min and 120 min; compared with the group F, the group E has a very significant difference that P is less than 0.01 at 30 min. The result shows that the sophora japonica extractum and the medicine 1 have obvious analgesic effect, and the effect of the two has no obvious difference. The medicine 2 added with baicalin has no obvious analgesic effect, and has very significant difference with the medicine 1 at 30 min.
TABLE 1 Effect on Hot plate pain threshold in mice: (
n＝10)
Note 1) P <0.05, P <0.01 compared to group a; compared with group FcP＜0.05，ccP＜0.01
As can be seen from table 2, group D, group E and group F all had significant inhibitory effects on the swelling of the mouse footpad caused by egg white, and had very significant inhibitory effects at 1.5h, compared to group a; compared with the group D, the group E has no obvious difference in 0.5,1 and 1.5 hours, and the group F has obvious difference in 1 and 1.5 hours; group E was significantly different at 1 and 1.5 hours compared to group F. The result shows that the compound sophora flavescens ait, the medicine 1 and the medicine 2 have obvious anti-inflammatory effects, the efficacy of the sophora flavescens ait extract is equivalent to that of the medicine 1, and the anti-inflammatory effect of the medicine 2 is more obvious than that of the medicine 1 and the medicine 2.
TABLE 2 Effect of Huanghuai Fufang on the anti-inflammatory swelling degree of foot sole of mice: (
n＝9)
Note: p <0.05, P <0.01 compared to group a; compared with group DaP＜0.05，aaP is less than 0.01; compared with group FcP＜0.05，ccP＜0.01
II, hemostasis effect on mice
1. Medicaments and agents
The Sophora japonica medicinal material is purchased from the market of Chinese medicinal materials in Hedychium japonicum pools and is identified as dried flowers of Sophora japonica L. which is a leguminous plant; scutellaria baicalensis Georgi is purchased from Beijing Hojingtang and identified as the dry root of Scutellaria baicalensis Georgi of Labiatae. Baicalin extract (batch No. TB190628-1, purity not less than 85%, SiAnchuang En Biotech Co., Ltd.); baicalein extract (batch No. KHBA190901-1, purity not less than 98%, Xian Chuang En Biotech Co., Ltd.); robinin extract (batch No. 19062701, purity greater than or equal to 99%, Xianrong coconut Biotech Co., Ltd.); kaempferol extract (batch No. 19081801, purity greater than or equal to 98%, Xianrong coconut Biotech Co., Ltd.); quercetin extract (batch No. 19072701, purity greater than or equal to 98%, Xianrong coconut Biotech Co., Ltd.); wogonin reference (batch No. wkq 19020201; purity not less than 98%, Szechwan, Ukki Biotech Co., Ltd.); wogonin extract (batch No. 19120901; purity not less than 98%, Shaanxi Green clear bioengineering Co., Ltd.); yellow sophora extract (batch 20190917, made by laboratory); physiological saline (batch No. C18051905-1, Sichuan Konlun pharmaceutical Co., Ltd.); baogong Zhixue Keli (batch No. 1901049, Tianjin Zhongsheng Haitian pharmaceutical Co., Ltd.); tranexamic acid pieces (batch QTA0949, Daiichi Sankyo, Japan).
2. Main instrument
DXL-10 rat metabolic cage (von willebrand, suzhou); SQP Satorious type electronic balance (beijing sialus scientific instruments ltd); an electronic analytical balance of FA2004 type (shanghai liangping instruments ltd); model TU-1810 ultraviolet-visible spectrophotometer (Beijing Pusan Co.).
3. Laboratory animal
SPF-level Kunming mice 120 female 20-40g, and certification number SCXK 2015-030, all reach Shuo Tech Co.
4. Experimental methods
(1) Effect on clotting time in mice
Taking female mice with Kunming sex maturity, randomly dividing the female mice into A, B, C, D, E (optionally four examples of the drug 1 of the invention) and F (optionally four examples of the drug 2 of the invention) 12 groups, namely a negative group (10mL/kg), a tranexamic acid group (1g/kg), a uterus preserving hemostasis group (6g/kg), a yellow sophora extract group (1.4g/kg), a drug 1(0.15g/kg) and a drug 2(0.42g/kg), wherein each group comprises 10 mice. The intragastric administration is carried out for 9 days by intragastric administration for 1 time every day. After the last administration for 1h, the capillary tube is used for taking blood from the venous plexus behind the left eyeball of the mouse, the timing is started when the blood flows into the tube, after the capillary glass tube is filled with the blood, the capillary tube is flatly placed on a table top, the capillary tube is broken at intervals of 10s by about 0.5cm and is slowly pulled left and right until the blood coagulation filaments appear, and the timing is the blood coagulation time. Results were statistically processed using one-way anova.
(2) Effect on bleeding time in mice
Taking female mice with Kunming sex maturity, randomly dividing the female mice into A, B, C, D, E (optionally four examples of the drug 1 of the invention) and F (optionally four examples of the drug 2 of the invention) 12 groups, namely a negative group (10mL/kg), a tranexamic acid group (1g/kg), a uterus preserving hemostasis group (6g/kg), a yellow sophora extract group (1.4g/kg), a drug 1(0.15g/kg) and a drug 2(0.42g/kg), wherein each group comprises 10 mice. The intragastric administration is carried out for 9 days by intragastric administration for 1 time every day. Cutting off tail tip 1h after last administration, cutting off tail tip with scissors 3mm away from tail tip, timing after blood self-flows out from the tail, dipping blood with filter paper sheet every 30s until no blood exudes from the tail, and timing as bleeding time. Results were statistically processed using one-way anova.
5. Results of the experiment
As can be seen from the data in table 3, groups D, E and F significantly reduced the clotting time of mice compared to group a (P < 0.01); compared with the group D, the group E and the group F have P >0.05 and have no significant difference; compared with the group F, the group E has P more than 0.05 and has no significant difference, which shows that the sophora japonica extractum, the drug 1 and the drug 2 have very significant blood coagulation functions, and the drug effects of the drug 1 and the drug 2 and the sophora japonica extractum have no significant difference; there was also no significant difference between drug 1 and drug 2.
TABLE 3 Effect of Sophora Molofoenum-graecum compound on blood coagulation time of mice: (
n＝9)
Note: p <0.05, P <0.01 compared to group A
As can be seen from table 4, bleeding time was significantly reduced in groups D, E and F compared to group a, where groups D and F have a very significant difference (P < 0.01); compared with the group D, the group E and the group F have no significant difference, and P is greater than 0.05; compared with the group F, the group E has P >0.05 and has no significant difference. The significant hemostatic effect of the sophora japonica extractum, the drug 1 and the drug 2 is shown, and the drug effects of the drug 1 and the drug 2 are not significantly different from those of the sophora japonica extractum, and the drug 1 and the drug 2 are also not significantly different.
TABLE 4 Effect of Sophora japonica preparation on bleeding time in mice: (
n＝9)
Note: p <0.05, P <0.01 compared to group A
Second, abortion uterus test
1. Medicaments and agents
The Sophora japonica medicinal material is purchased from the market of Chinese medicinal materials in Hedychium japonicum pools and is identified as dried flowers of Sophora japonica L. which is a leguminous plant; scutellaria baicalensis Georgi is purchased from Beijing Hojingtang and identified as the dry root of Scutellaria baicalensis Georgi of Labiatae. Baicalin extract (batch No. TB190628-1, purity not less than 85%, SiAnchuang En Biotech Co., Ltd.); baicalein extract (batch No. KHBA190901-1, purity not less than 98%, Xian Chuang En Biotech Co., Ltd.); robinin extract (batch No. 19062701, purity greater than or equal to 99%, Xianrong coconut Biotech Co., Ltd.); kaempferol extract (batch No. 19081801, purity greater than or equal to 98%, Xianrong coconut Biotech Co., Ltd.); quercetin extract (batch No. 19072701, purity greater than or equal to 98%, Xianrong coconut Biotech Co., Ltd.); wogonin reference (batch No. wkq 19020201; purity not less than 98%, Szechwan, Ukki Biotech Co., Ltd.); wogonin extract (batch No. 19120901; purity not less than 98%, Shaanxi Green clear bioengineering Co., Ltd.); yellow sophora extract (batch 20190917, made by laboratory); physiological saline (batch No. C18051905-1, Sichuan Konlun pharmaceutical Co., Ltd.); baogong Zhixue Keli (batch No. 1901049, Tianjin Zhongsheng Haitian pharmaceutical Co., Ltd.); tranexamic acid pieces (batch QTA0949, Daiichi Sankyo, Japan).
2. Main instrument
FA2004 analytical electronic balance, shanghai liangping instruments, ltd; a centrifugal machine.
3. Laboratory animal
Clean grade SD female and male rats (2:1), weight 250-.
4. Experimental methods
Rats were treated at 20: and (5) closing the male and female according to the ratio of 2:1 at 00 hours, dividing the cages the next morning, observing the vaginal suppository, carrying out vaginal smear examination, and checking whether mating exists. The 1 st day of pregnancy was observed as the presence of sperm. At 7d, the stomach is irrigated with (i.g.) mifepristone 0.83mg/kg (8: 00) and misoprostol 10 mug/kg (18: 00) according to the body weight respectively to cause incomplete abortion, and meanwhile, a quantitative cotton ball (the weight of the cotton ball is 85-90 mg) is placed in the vagina, and the half side is wrapped by a plastic film to prevent blood leakage and urine backflow. After the model building is successful, the female rats are randomly divided into A, B, C, D, E (optionally four examples of the medicine 1 of the invention) and F (optionally four examples of the medicine 2 of the invention) 12 groups, namely water (10mL/kg), tranexamic acid (12mg/100g), uterus preserving hemostasis granule group (7.8mg/100g), yellow sophora extract medium dose (0.7g/kg), medicine 1(0.075g/kg) medicine 2(0.21g/kg), and each group corresponds to 9 mice. After the molding is successfully carried out, i.g.10mL/kg of water (10mL/kg), tranexamic acid (12mg/100g), uterine bleeding stopping granules (7.8mg/100g), Chinese scholar tree extract medium dose (0.7g/kg), medicine 1(0.075g/kg), medicine 2(0.21g/kg) and equal volume i.g. administration are carried out on the next day (8 d) respectively, and the 7d is continuously carried out once a day.
Taking out the cotton balls at 8:00 and 20:00 the next day respectively, placing into a plastic bag, sealing, refrigerating, storing, replacing a new cotton ball in vagina, observing vaginal bleeding condition, recording bleeding time, continuing to 14d, and measuring the bleeding amount of the collected cotton balls in vagina of each mouse.
After the administration (15 d of pregnancy), 9 patients are taken from each group, the abdominal aorta is bled, and related indexes to be measured of the uterus are taken. Collecting uterine bleeding and calculating the amount of bleeding according to a formula; collecting blood, separating serum, and detecting E2 and P sex hormone level; observing the morphological change of the uterus by laparotomy, taking out the uterus, weighing, calculating the specific gravity coefficient of the uterus, and detecting the Ca, ET and NO levels of the homogenized tissues of the uterus.
5. Results of the experiment
As can be seen from the data results in the table, in the experiment on the abortion uterus of the rat, compared with the model group, the experimental group reduces the uterine bleeding amount of the drug abortion model rat, reduces the uterine specific gravity coefficient, but has no statistical significance. The results are shown in Table 5.
TABLE 5 results of the Effect on uterine bleeding volume and uterine weight in rats (x. + -.s)
In the experiment for influencing the contents of Ca, NO and ET-1 in the abortion uterine tissue of rats, the content of calcium in the uterine tissue of the experimental group is higher than that of the blank control group. And only the F group had statistical significance (P < 0.01). Compared with the model group, the D, E and F groups increased ET-1 content and decreased NO content in uterine homogenates, but were not statistically significant (P > 0.05). The results are shown in Table 6.
TABLE 6 Effect on Ca, NO and ET-1 content in uterine tissue of abortive rats (x. + -.s)
Note: p <0.05, P <0.01 compared to group A
In the experiment on the influence of E2 and P levels on the serum of productive rats, compared with a model group, the experimental group can reduce the content of E2 and obviously reduce the content of P (P < 0.01). There was no significant difference between the D, E, F groups (P > 0.05). The results are shown in Table 7.
TABLE 7 Effect on E2 and P levels in serum of abortive rats (x. + -. s)
Note: p <0.05, P <0.01 compared to group A
From the above experiments, it can be seen that: the invention compares the component groups screened out by network pharmacology and the active component groups added with baicalin with the original compound recipe. The results of pharmacodynamic experiments show that the original formula of the Robinia pseudoacacia, the medicine 1 (mixture of quercetin, wogonin, kaempferol, baicalein and robinin) and the medicine 2 (mixture of quercetin, wogonin, kaempferol, baicalein, robinin and baicalin) can improve the pain threshold of mice, remarkably inhibit the foot sole swelling, shorten the blood coagulation and bleeding time, reduce the uterine bleeding amount, reduce the uterine coefficient, the content of NO in uterine homogenate tissues and the E in uterine serum2And P content, increasing ET-1 content in uterine homogenate. The active ingredient group (group F, medicine 2) added with baicalin is superior to the active ingredient group (group E, medicine 1) without baicalin in the aspects of resisting inflammation, controlling bleeding time and increasing calcium content in uterus of abortion rats. In controlling blood coagulation time, uterine bleeding amount, uterine coefficient, decrease of NO content in uterine homogenate tissue, increase of ET-1 content and uterus of abortion ratSerum E2And the decrease of P content, whether baicalin exists or not, and the drug effect is equal. In the aspect of analgesia, the drug 1 has a remarkable analgesic effect, while the drug 2 has no remarkable analgesic effect, probably because the baicalin can reduce the analgesic effect, and the discovery can provide a new direction for subsequent researches.
A method for screening active pharmaceutical ingredients for anti-inflammatory, analgesic and hemostatic purposes comprises the following steps:
A. screening active compounds with the oral bioavailability OB value of more than or equal to 30 percent and the drug property DL value of more than or equal to 0.18 according to the main chemical components of the scutellaria baicalensis and the sophora japonica;
B. collecting the protein targets of the active compounds, collecting the protein targets of main chemical components in the scutellaria baicalensis and the sophora japonica through a target prediction function in a TCMSP database, retrieving by using a Uniprot database to obtain the gene names corresponding to the protein targets, and performing retrieval correction on the artificial species to obtain UniprotID and the gene names corresponding to the protein targets;
C. searching genes of related Dysfunctional uterine bleeding by using a GeneCards database and using Dysfunctional uterine bleeding as a keyword, and establishing a Dysfunctional uterine bleeding target set;
D. comparing the protein target gene with the target gene for dysfunctional uterine bleeding, wherein the protein target gene is acted by the screened active compound, so as to obtain an intersection gene of the active compound and the target gene for dysfunctional uterine bleeding, and the intersection gene is used as a potential action target gene for treating the dysfunctional uterine bleeding by the active compound;
E. carrying out protein interaction analysis on the potential action target genes;
F. carrying out GO and KEGG enrichment analysis on the potential action target genes, and screening out KEGG access information with significant difference in the process of treating dysfunctional uterine bleeding by using the active compound;
G. analyzing the interaction of the active compound, the potential action target gene, GO and KEGG pathway, wherein the screened active compound is at least: quercetin, wogonin, kaempferol, baicalein, and robinin. Further, in the method for screening the active pharmaceutical ingredient for anti-inflammatory, analgesic and hemostatic purposes, preferably in step C, the first 200-400 results with high correlation are selected from the target set of dysfunctional uterine bleeding.
Further, in the method for screening active pharmaceutical ingredients for anti-inflammatory, analgesic and hemostatic purposes, preferably, in step D, the protein target gene of the screened active compound and the dysfunctional uterine bleeding target gene are analyzed to obtain an intersection gene of the active compound and the dysfunctional uterine bleeding target.
Further, in the method for screening a pharmaceutically active ingredient for anti-inflammatory, analgesic and hemostatic uses, it is preferable that in the step E, points pointing to each protein interaction information are evaluated and assigned for predictable protein-protein interactions of potential target genes stored by String database.
Further, in the method for screening the active pharmaceutical ingredients for anti-inflammatory, analgesic and hemostatic purposes, preferably, in the step F, the potential target genes with effects are introduced into the DAVID database for GO and KEGG enrichment analysis, and the most important biological process BP, cell component CC and molecular function MF are screened, so that KEGG pathway information with significant differences is obtained, wherein the active compounds participate in the pathological process of dysfunctional uterine bleeding.
Further, in the method for screening the pharmaceutical active ingredients for anti-inflammatory, analgesic and hemostatic purposes, preferably, in the step G, an active compound-target-pathway network model is constructed, and key nodes are determined by adopting the order of Degree, ASPL, BC and CC, wherein the compound molecules of Degree >2 are at least quercetin, wogonin, kaempferol, baicalein and acacetin.
The invention is illustrated in detail below from a specific example:
example 13, a method for screening an active pharmaceutical ingredient for anti-inflammatory, analgesic, and hemostatic applications, comprising the steps of:
A. screening active compounds in the yellow sophora compound:
establishing a main chemical component database of the scutellaria baicalensis and the sophora japonica by using a traditional Chinese medicine system pharmacological database and an analysis platform (TCMSP), analyzing and selecting the main chemical components of the scutellaria baicalensis and the sophora japonica, and screening out active compounds by ADME (screening the OB value of oral bioavailability to be more than or equal to 30 percent and the DL value of drug property to be more than or equal to 0.18).
170 compounds were retrieved by TCMSP, 143 of which were from scutellaria baicalensis and 27 of which were from sophora japonica. According to the condition that OB is more than or equal to 30 percent and DL is more than or equal to 0.18, repeated compounds and compounds lacking target point prediction data are removed, 34 active ingredients are finally screened, wherein 29 scutellaria baicalensis are contained, 6 sophora japonica are contained, and beta-sitosterol (beta-sitosterol) is a common component of the scutellaria baicalensis and the sophora japonica, and the table 8 shows.
Table 8 details of the compound molecules
B. Screening an active compound protein target and a dysfunctional uterine bleeding target of dysfunctional uterine bleeding in chemical components in the sophora japonica compound: targets of known chemical components in scutellaria baicalensis and sophora japonica are collected through a target prediction function in a TCMSP database, and a Uniprot database is utilized, so that repeated, non-human and non-standard targets are eliminated by introducing protein names and limiting species to be human. And correcting all the retrieved protein targets into UniprotID, and obtaining the gene names corresponding to the target proteins.
C. Searching genes related to Dysfunctional uterine bleeding by using a GeneCards database and using Dysfunctional material scoring as a keyword, and establishing a Dysfunctional uterine bleeding target set by taking the first 300 results with the highest Relevance score correlation value as disease targets.
D. And comparing the protein target gene of the screened active compound with the on-line Wien diagram uploaded by the dysfunctional uterine bleeding target gene to obtain an intersection gene of the active compound and the dysfunctional uterine bleeding target, serving as a potential action target gene of the active compound of the Sophora japonica compound for treating the dysfunctional uterine bleeding, and counting the intersection gene.
215 protein targets are obtained from the active compound components of the yellow sophora compound. As shown in fig. 1, left circular: a set of active compound targets; right circular: a set of dysfunctional uterine bleeding targets of dysfunctional uterine bleeding of a GeneCards database; the middle intersection part is an intersection target gene of the active compound and the target of the dysfunctional uterine bleeding. The corresponding targets of the compound Sophora Molofoenus and the dysfunctional uterine bleeding targets screened from the GeneCards database are subjected to online Wien diagram to obtain intersection genes of 61 active compounds and the dysfunctional uterine bleeding, the intersection genes are used as the action target genes of the active ingredients of the compound Sophora Molofoenus for treating the dysfunctional uterine bleeding, the interaction of the target spots of the compound Sophora Molofoenus for treating the dysfunctional uterine bleeding is shown in figure 3 (the target spots which are hidden and have no interaction), and 61 potential action target spots are shown in Table 9.
TABLE 9 information on potential target sites of action
E. And (3) carrying out protein interaction analysis on the potential action target genes, and converting the potential action target genes into a protein interaction network diagram.
To illustrate the role of the target protein in the system, information on the potential targets of the acacia complex associated with dysfunctional uterine bleeding was uploaded to string11.0 online software, designating "species" as "human", and PPI network plots were obtained. Interactions between proteins, including direct and indirect interactions between proteins, can be predicted through String database storage, and points pointing to each protein interaction information are evaluated and assigned. The higher the score, the higher the confidence of the protein interaction. And (4) exporting the result from the String database, importing the result into Cytoscape 3.7.0 software for visual analysis, and obtaining network analysis. The node size is set, and the PPI network graph is obtained by comprehensively scoring the node size, color, value, size and edge thickness, as shown in FIG. 2.
F. Carrying out GO and KEGG enrichment analysis on the potential action target gene, and screening out KEGG access information with significant difference of active compounds participating in the pathological process of dysfunctional uterine bleeding.
GO and KEGG analysis are carried out on the potential action target points, and a threshold value P is set to be less than 0.05. GO enrichment analysis refers to a directed acyclic graph consisting of the number of proteins or genes counted at a functional level in 3 branches of Biological Processes (BP), Cellular Components (CC) and Molecular Functions (MF). Wherein, in the biological process, the influence of the Robinia pseudoacacia on organic reaction, oxidation reaction, reaction of cells on chemical stimulation, reaction of cells on organic substances and the like is large (as shown in figure 3, BP); among the cellular components, Robinia pseudoacacia has a large influence on extracellular space, extracellular region part, extracellular region, intracellular membrane, cytoplasm and the like (as shown in FIG. 4, CC); at the molecular function level, Robinia pseudoacacia has a large influence on signal receptor binding, protein binding, cytokine receptor binding, same protein binding, molecular function regulators, enzyme binding, and the like (as shown in FIG. 5, MF). The top ranked pathways that are mainly involved in HH treatment of dysfunctional uterine bleeding are the cancer factor signaling pathway, the hepatitis b signaling pathway, the PI3K-Akt signaling pathway, the HIF-1 signaling pathway, the endometrial cancer signaling pathway, the tumor necrosis factor signaling pathway, the activated cysteine-type endopeptidase activity signaling pathway, the transcriptional dysregulation of cancer, the p53 signaling pathway, and the like, as shown in fig. 6 and fig. 7 (KEGG).
G. The interaction was analyzed for active compounds, potential target genes, GO and KEGG pathways.
The information of the target points and the HH active components corresponding to the KEGG channels which are obtained by the pre-ranking 20 through enrichment analysis is sorted and introduced into Cytoscape Version 3.6.1 software to construct a component-target-channel network (as shown in figure 8). The results show that the network consists of 96 nodes and 579 edges, the circle nodes represent the active ingredients in Robinia pseudoacacia, the diamond nodes represent the target points, the arrow nodes represent the regulatory pathways, and the edges represent the interactions between the three (FIG. 8).
Key nodes are determined using a rank order of Degree values (Degree), with the components, targets and path nodes for the net median value > median (component median 3, target median 15, path median 11) shown in table 10. The analysis result shows that in the network of the yellow sophora japonica component-target-signal channel:
(1) the compound with Degree more than or equal to 6 is quercetin, wogonin, kaempferol, baicalein, acacetin, and beta-sitosterol.
(2) The Degree of PTGS2, AKT1, TP53 and TNF in the network is 38, 36, 33 and 30, respectively, relatively high, and is a key node in the network, indicating that they may be the core target of robinia pseudoacacia to treat dysfunctional uterine bleeding.
TABLE 10 component-target-Signal Path network topology parameter information
Determination of content of sophora flavescens compound medicine component
1. Experimental Material
HS-3120 type ultrasonic cleaner (Hengoshu technology development Co., Tianjin); waters model 2695 high performance liquid chromatograph (Watts instruments, Inc., USA); SQP Satorious type electronic balance (sydows scientific instruments (beijing) ltd); an electronic analytical balance of the BS-6KH type (Shanghai friend sound weighing apparatus Co., Ltd.); DZTW type temperature-adjusting electric heating jacket (Yongguanming medical instruments, Inc., Beijing); TGL-16G type high speed centrifuge (Shanghai' an pavilion scientific instrument factory)
The Sophora japonica medicinal material is purchased from the market of Chinese medicinal materials in Hehua pond and is identified as the dried flower of Sophora japonica L. of leguminous plants; the Scutellariae radix is purchased from Beijing Hojingtang and identified as dried root of Scutellaria Baicalensis Georgi of Labiatae. Baicalin reference (batch No. wkq19011407, purity not less than 98%, Szechwan Vickqi Biotech Co., Ltd.); baicalein control (batch No. wkq19011507, purity not less than 98%, Vickqi Biotech, Inc., Sichuan); robinin reference (batch No. wkq19042313, purity not less than 98%, Szechwan Vickqi Biotech Co., Ltd.); kaempferol reference (batch No. wkq19011609, purity not less than 98%, Vickqi Biotech Co., Ltd., Sichuan); beta-sitosterol reference (batch No. wkq19020302, purity not less than 98%, Vickqi Biotech Co., Ltd., Sichuan province); wogonin reference substance (batch No. 140217HPLC ≥ 98%), Vickqi Biotech, Inc., Sichuan province; quercetin reference substance (lot No. 100081-299406HPLC ≥ 98%), China pharmaceutical biologicals institute.
2. Preparation of control solution
Precisely weighing baicalin reference substance, baicalein reference substance, wogonin reference substance, and appropriate amount of robinin, quercetin, kaempferol, and beta-sitosterol, respectively dissolving with methanol and diluting to constant volume to obtain single reference substance stock solution of 0.2 mg/ml.
3. Preparation of test solution
Taking a proper amount of scutellaria baicalensis and sophora japonica, and carrying out compatibility according to the prescription amount of 4:1, adding 6 times of 40% ethanol, extracting for 3 times, each time for 2h, and mixing filtrates. Centrifuging the filtrate in a high speed centrifuge at 12000r/min for 10min, collecting supernatant, filtering, and collecting filtrate to obtain sample solution.
4. Chromatographic conditions
(1) Chromatographic condition for measuring content of baicalein and wogonin
Supersil ODS-B C18A chromatographic column (250mm × 4.6mm, 5 μm), acetonitrile-0.1% phosphoric acid gradient elution (0-15 min, acetonitrile 15% → 22%, 15-31 min, acetonitrile 22% → 55%, 31-50 min, acetonitrile 55% → 75%), a detection wavelength of 280nm, a flow rate of 1.0 mL/min-1, and a column temperature of 30 ℃.
(2) Chromatographic condition for measuring content of quercetin and kaempferol
Supersil ODS-B C18 column (250 mm. times.4.6 mm, 5 μm), methanol-4 mL/L phosphoric acid solution as mobile phase (volume ratio 50: 50), flow rate 1.0mL/min, detection wavelength 260nm, sample size 10 μ L, and column temperature room temperature (20 ℃).
(3) Chromatographic condition for determining robinin (acacetin) content
Supersil ODS-B C18 column (250 mm. times.4.6 mm, 5 μm) with gradient elution of acetonitrile (A) -0.1% phosphoric acid (B) as mobile phase was performed as shown in Table 11, detection wavelength was 365nm, flow rate was 1.0mL/min, column temperature was 30 ℃ and sample injection was performed at 10. mu.L.
TABLE 11 measurement of chromatographic conditions
(4) Chromatographic condition for measuring baicalin content
Supersil ODS-B C18 column (250 mm. times.4.6 mm, 5 μm)
Mobile phase: methanol-water-phosphoric acid (47:53: 0.2); flow rate: 1.00 mL/min; the detection wavelength is 280 nm; sensitivity: 0.01 AVFS; column temperature: 30 ℃; sample introduction amount: 10 μ L.
(5) Chromatographic condition for measuring beta-sitosterol content
Supersil ODS-B C18 column (250 mm. times.4.6 mm, 5 μm)
The mobile phase is methanol; the flow rate is 1.0 mL/min; the column temperature is 25 ℃; the detection wavelength is 210 nm.
5. Results of the experiment
The contents of the respective components in the test sample measured according to the above conditions are shown in Table 12 below.
TABLE 12 determination results of the contents of active ingredients in the Sophora Molofoenum-graecum compound extractive solution
The scutellaria baicalensis is used as a main drug in a compound prescription of the sophora japonica, the active component of the compound of the sophora japonica is baicalin as a main active component, and the hemostatic function is found to be stronger in an in vitro blood coagulation activity determination experiment by taking the baicalin as a technological index in the early stage.
The invention screens 6 active ingredients for treating dysfunctional uterine bleeding in HH through network pharmacology, wherein the active ingredients are quercetin, wogonin, kaempferol, baicalein, acacetin and beta-sitosterol, and baicalin is not included. Therefore, the invention brings the baicalin into the active ingredient group and carries out content measurement on seven active ingredients including the baicalin. As a result, the beta-sitosterol is removed due to its low content, and finally 6 components (quercetin, wogonin, kaempferol, baicalein, acacetin, and baicalin) containing baicalin are obtained as effective component groups.
Claims (10)
1. The medicine for resisting inflammation, relieving pain and stopping bleeding is characterized by comprising the following components in parts by weight:
2. the medicine for resisting inflammation, easing pain and stopping bleeding as claimed in claim 1, is characterized by comprising the following components in parts by weight:
3. the medicament for anti-inflammatory, analgesic and hemostatic as claimed in claim 1 or 2, further comprising the following components in parts by weight: baicalin 800-1000.
4. The medicine for resisting inflammation, easing pain and stopping bleeding as claimed in claim 3, is characterized by further comprising the following components in parts by weight: baicalin 900-.
5. A method for screening active ingredients of a medicament for resisting inflammation, easing pain and stopping bleeding is characterized by comprising the following steps:
A. screening active compounds with the oral bioavailability OB value of more than or equal to 30 percent and the drug property DL value of more than or equal to 0.18 according to the main chemical components of the scutellaria baicalensis and the sophora japonica;
B. collecting protein targets of the active compounds, collecting protein targets of main chemical components in scutellaria baicalensis and sophora japonica through a target prediction function in a TCMSP database, searching gene names corresponding to the protein targets by using a Uniprot database, and performing searching correction corresponding to artificial species to obtain UniprotID and gene names corresponding to the protein targets;
C. searching genes of related Dysfunctional uterine bleeding by using a GeneCards database and using Dysfunctional uterine bleeding as a keyword, and establishing a Dysfunctional uterine bleeding target set;
D. comparing the protein target gene with the target gene for dysfunctional uterine bleeding, wherein the protein target gene is acted by the screened active compound, so as to obtain an intersection gene of the active compound and the target gene for dysfunctional uterine bleeding, and the intersection gene is used as a potential action target gene for treating the dysfunctional uterine bleeding by the active compound;
E. carrying out protein interaction analysis on the potential action target genes;
F. carrying out GO and KEGG enrichment analysis on the potential action target genes, and screening out KEGG access information with significant difference in the process of treating dysfunctional uterine bleeding by using the active compound;
G. analyzing the interaction of the active compound, the potential action target gene, GO and KEGG pathway, wherein the screened active compound is at least: quercetin, wogonin, kaempferol, baicalein, and robinin.
6. The method for screening active pharmaceutical ingredients for anti-inflammatory, analgesic and hemostatic purposes according to claim 5, wherein in the step C, the first 200 and 400 results with high correlation are selected from the target set of dysfunctional uterine bleeding.
7. The method for screening a pharmaceutically active ingredient for anti-inflammatory, analgesic and hemostatic purposes according to claim 5, wherein in step D, the protein target gene of the screened active compound and the dysfunctional uterine bleeding target gene are analyzed to obtain an intersection gene of the active compound and the dysfunctional uterine bleeding target.
8. The method for screening a pharmacologically active ingredient for antiinflammatory, analgesic and hemostatic effects according to claim 5, wherein in the step E, the points pointing to the information on each protein interaction are evaluated and assigned for the interaction between the predicted proteins of the potential target genes stored by String database.
9. The method for screening the pharmaceutical active ingredients for anti-inflammatory, analgesic and hemostatic purposes according to claim 5, wherein in step F, the potential target genes are introduced into DAVID database for GO and KEGG enrichment analysis, and the most important biological processes BP, cellular components CC and molecular functions MF are screened, so as to obtain the KEGG pathway information with significant differences that the active compounds participate in the pathological processes of dysfunctional uterine bleeding.
10. The method of claim 5, wherein in the step G, an active compound-target-pathway network model is constructed, and the key nodes are determined by using Degree, ASPL, BC and CC sequences, and the compound molecules with Degree greater than or equal to 2 x median are at least quercetin, wogonin, kaempferol, baicalein and acacetin.
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