阅读说明：本技术 丹参总酮和三七总皂苷在制备用于预防和治疗结肠炎的药物中的应用 (Application of total tanshinone and total notoginsenoside in preparing medicine for preventing and treating colitis ) 是由 罗花 谭德超 王一涛 王胜鹏 李鹏 钟章锋 余华 王芷廷 于 2021-09-27 设计创作，主要内容包括：本发明公开了丹参总酮和三七总皂苷在制备用于预防和治疗结肠炎的药物中的应用,涉及生物医药技术领域。本发明筛选出了具有抗炎作用的丹参总酮以及三七总皂苷,且丹参总酮联合三七总皂苷在预防和治疗结肠炎方面具有协同增效的作用,联合治疗能够更有效地达到预防和治疗结肠炎的效果,为未来开发预防和治疗结肠炎的中药药对和新药奠定了基础。(The invention discloses application of total tanshinone and total notoginsenoside in preparing a medicament for preventing and treating colitis, and relates to the technical field of biological medicines. The invention screens out the total tanshinone and the panax notoginseng saponins with anti-inflammatory effect, and the total tanshinone and the panax notoginseng saponins have synergistic effect in the aspect of preventing and treating the colitis, and the combined treatment can more effectively achieve the effect of preventing and treating the colitis, thereby laying a foundation for developing traditional Chinese medicine pairs and new drugs for preventing and treating the colitis in the future.)

1. Use of total ketone of Saviae Miltiorrhizae radix and total saponin of Notoginseng radix in preparing medicine for preventing and/or treating colitis is provided.

2. The application of the total tanshinone and the total notoginsenoside in preparing the medicine for preventing and/or treating colitis according to claim 1, wherein the mass ratio of the total tanshinone and the total notoginsenoside is 1 (5-80).

3. The application of the total tanshinone and the total notoginsenoside in preparing the medicine for preventing and/or treating colitis according to claim 2 is characterized in that the mass ratio of the total tanshinone and the total notoginsenoside is 1 (20-60).

4. Use of total tanshinone and total saponins of panax notoginseng in the manufacture of a medicament for the prevention and/or treatment of colitis according to claim 1, characterized in that the preparation of the medicament comprises: preparing a first solution containing total tanshinone and a second solution containing total notoginsenoside.

5. Use of total tanshinone and total notoginsenoside according to any one of claims 1-4, in the preparation of a medicament for preventing and/or treating colitis, wherein colitis comprises at least one of Crohn's disease and ulcerative colitis.

6. Use of total ketone of Saviae Miltiorrhizae radix and total saponin of Notoginseng radix in preparing medicine for inhibiting NO synthesis by inflammatory cell is provided.

7. Use of total tanshinone and total notoginsenoside in preparing medicine for inhibiting NF-kB activation is provided.

8. The application of total tanshinone and total arasaponin in preparing medicine for inhibiting the expression level of inflammation factor and raising the expression level of inflammation factor.

9. A medicament for preventing and/or treating colitis, which comprises total tanshinone and panax notoginseng saponins.

10. The medicament for preventing and/or treating colitis according to claim 9, wherein the mass ratio of total tanshinone and total notoginsenoside is 1: (5-80).

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of total tanshinone and panax notoginseng saponins in preparing medicines for preventing and treating colitis.

Background

Colitis includes crohn's disease and ulcerative colitis, a chronic recurrent disease that occurs in the gastrointestinal tract and whose symptoms include diarrhea, hematochezia, anemia, weight loss, fatigue, and the like.

According to the analysis of the traditional Chinese medicine, the pathological factors of the colitis relate to heat and blood stasis, qi stagnation, blood stasis and the like, and common clinical symptoms comprise large intestine damp-heat syndrome, spleen deficiency damp-accumulation syndrome, cold-heat mixed syndrome and the like. Under enteroscopy, the intestinal mucosa of the colitis patient is red, obvious edema, hyperemia and ulcerative hemorrhage are observed, and diffuse hyperemia and edema are also observed in the intestinal cavity.

To date, the pathogenesis of colitis has not been clarified, and no drug has been found that can completely cure colitis. Currently, drugs for treating colitis mainly comprise aminosalicylic acids, glucocorticoids, immunomodulators, monoclonal antibodies and the like, but the drugs are usually accompanied by side effects or adverse reactions.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of total tanshinone and total notoginsenoside in preparing a medicament for preventing and treating colitis.

The invention is realized by the following steps:

in a first aspect, the embodiments of the present invention provide an application of total tanshinone and total saponins of panax notoginseng in preparing a medicament for preventing and/or treating colitis.

In a second aspect, the embodiment of the invention provides an application of total tanshinone and total notoginsenoside in preparing a medicament for inhibiting NO synthesis by inflammatory cells.

In a third aspect, the embodiment of the invention provides application of total tanshinone and panax notoginseng saponins in preparation of drugs for inhibiting NF-kB activation.

In a fourth aspect, the embodiments of the present invention provide applications of total tanshinone and total saponins of panax notoginseng in preparing drugs for inhibiting the expression level of inflammatory factors or improving the expression level of inflammatory factors.

In a fifth aspect, the embodiments of the present invention provide a medicament for preventing and/or treating colitis, the medicament comprising total tanshinone and total saponins of panax notoginseng; the mass ratio of total tanshinone and total notoginsenoside is 1: (5-80).

The invention has the following beneficial effects:

compared with single administration, the combined treatment of the total tanshinone and the panax notoginseng saponins can more effectively achieve the technical effect of preventing and treating the colitis, and lays a foundation for developing traditional Chinese medicine pairs and new medicines for preventing and treating the colitis in the future.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph showing that the combination of total tanshinone and total saponins of panax notoginseng in example 1 inhibits NO production by Pam3CSK 4-induced macrophages in vitro;

FIG. 2 is a graph showing that the combination of total tanshinone and Panax notoginsenosides in example 2 inhibits the p65 nuclear transfer in Pam3CSK 4-induced macrophages in vitro;

FIG. 3 is a graph of the combined administration of total tanshinone and total saponins of panax notoginseng in example 3 to alleviate weight loss in a mouse model induced by DSS;

FIG. 4 is a mouse model DAI for relieving DSS induction by combined administration of total ketones and Panax notoginsenosides in example 3;

FIG. 5 shows that the combined administration of total tanshinone and total saponins of panax notoginseng in example 3 alleviates DSS-induced colon shortening in a mouse model;

FIG. 6 shows that the combined use of total tanshinone and total saponins of panax notoginseng in example 3 relieves the DSS-induced colonic ulcer lesion in a mouse model;

FIG. 7 shows that the combined administration of total tanshinone and total saponins of panax notoginseng in example 3 reduces the score of colon histopathological injury in a mouse model induced by DSS;

FIG. 8 is a graph showing the effect of total tanshinone and total arasaponin combination on colon inflammation factor in the DSS-induced mouse model in example 4.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Firstly, the invention provides the application of total tanshinone and total notoginsenoside in preparing the medicine for preventing and/or treating colitis.

The total tanshinone and the panax notoginseng saponins have anti-inflammatory effect and technical effects which are not expected by technical personnel in the field, and compared with single medication, the combined treatment of the total tanshinone and the panax notoginseng saponins can more effectively achieve the technical effects of preventing and treating colitis.

By "treating" herein is meant intervening or altering the process of a particular health state, including at least one of preventing, curing or inhibiting colitis.

In an optional embodiment, the mass ratio of total tanshinone and total saponins of panax notoginseng is 1: (5-80), specifically 1:5, 1:10, 1:20, 1:40 or 1: 80. The mass ratio is the amount of total tanshinone and total notoginsenoside used in preparing the medicine. Preferably, the mass ratio of the total tanshinone and the panax notoginseng saponins is 1 (20-60), and the synergistic effect is better under the limit of the mass ratio.

When the medicine is prepared, the total tanshinone and the panax notoginseng saponins can be mixed and prepared, or can be prepared respectively as long as the medicine comprises the two components. In an alternative embodiment, the preparation of the medicament comprises: preparing a first solution containing total tanshinone and a second solution containing total notoginsenoside. The solvent of the first solution and the second solution can be selected from the existing buffer solution or water.

In an alternative embodiment, the colitis comprises at least one of crohn's disease and ulcerative colitis.

The embodiment of the invention also provides application of the total tanshinone and the panax notoginseng saponins in preparing medicines for inhibiting NO synthesis by inflammatory cells. Total tanshinone and panax notoginsenosides can effectively inhibit the synthesis of NO by inflammatory cells, and provide a new way for preventing or treating related diseases caused by excessive NO production by inflammatory cells, wherein the related diseases include but are not limited to colitis.

The embodiment of the invention also provides application of the total tanshinone and the panax notoginseng saponins in preparing the medicines for inhibiting NF-kB activation.

The combined treatment of total tanshinone and total notoginsenoside can inhibit NF-kB activation, thereby inhibiting related diseases (including but not limited to colitis) caused by NF-kB activation. Specifically, the combination therapy of total tanshinone and total arasaponin can inhibit the transfer of p65 to cell nucleus.

The embodiment of the invention also provides application of the total tanshinone and the panax notoginseng saponins in preparing medicines for inhibiting the expression level of inflammatory factors or improving the expression level of the inflammatory factors.

Specifically, inflammatory factors include, but are not limited to, at least one of TNF- α, IL-6, IL-1 β, MPO, and MCP-1; the inflammation inhibitor includes IL-10. The combined treatment of the total tanshinone and the panax notoginseng saponins can effectively inhibit the expression level of inflammatory factors or improve the expression level of the inflammatory factors, and provides a novel treatment or prevention method for related diseases (including but not limited to colitis) caused by excessive expression level of the inflammatory factors or excessively low expression level of the inflammatory factors.

In addition, the embodiment of the invention also provides a medicament for preventing and/or treating colitis, which comprises total tanshinone and panax notoginseng saponins.

In a preferred embodiment, the mass ratio of total tanshinone and total saponins of panax notoginseng is 1: (5-80), preferably 1: (20-60). The preparation methods of total tanshinone and total saponins of panax notoginseng are the same as those in any of the above embodiments, and are not described herein again.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Experimental drugs: total tanshinone and total saponin of Notoginseng radix.

In vitro anti-inflammatory assay: the RAW 264.7 macrophage is inoculated on a 24-pore plate, after 24 hours of cells are attached on the 24-pore plate, 1 microgram/mL of total tanshinone, 40 microgram/mL of total notoginsenoside, 1 microgram/mL of total notoginsenoside and 40 microgram/mL of total notoginsenoside are added in advance to intervene the cells for 4 hours, and then 50ng/mL of Pam3CSK4 inducer is added to intervene the cells for 12 hours. According to the instructions of the Griess kit, the supernatants of the groups are respectively taken to react with the Griess reagent in a 96-well plate for half an hour. And finally, placing the sample in an enzyme-labeling instrument, setting the absorption wavelength to be 490nm, and detecting the absorbance.

Results of the experiment

Referring to fig. 1, total tanshinone and total notoginsenoside both show anti-inflammatory effects in a model of cell inflammation induced by Pam3CSK4, can significantly inhibit the release of NO, but the combined anti-inflammatory effect of the total tanshinone and total notoginsenoside is obviously stronger than that of single drug.

Example 2

And (3) detecting the immunofluorescence protein: the RAW 264.7 macrophage is inoculated on a 24-pore plate, after 24 hours of cells are attached on the 24-pore plate, 1 microgram/mL total tanshinone, 40 microgram/mL total notoginsenoside, 1 microgram/mL total tanshinone and 40 microgram/mL total notoginsenoside are added in advance to intervene the cells for 2 hours, and then 50ng/mL Pam3CSK4 inducer is added to intervene the cells for 4 hours. After removing all the liquid from the 24-well plates, cells were fixed for 10 min by adding 4% paraformaldehyde, and then washed twice with cold PBS. Next, cells were permeabilized with PBS containing 0.5% Triton X-100 for 20 minutes and blocked with 5% fetal bovine serum (formulated in PBS and containing 0.2% Triton X-100) for 1 hour. Cells were incubated overnight with p65 primary antibody (1: 100; Cell Signaling Technology, Danvers, MA, USA), followed by addition of Alexa Fluor 514-conjugated secondary antibody (1: 1000; Cell Signaling Technology, Danvers, MA, USA) for 1 hour before addition of Hoechst 33342 for nuclear staining. Finally, pictures were taken with a Leica TCS SP8 laser scanning confocal microscope.

Results of the experiment

As shown in figure 2, the induction of Pam3CSK4 shows that p65 can transfer to cell nucleus, however, total tanshinone and panax notoginseng saponins can inhibit the transfer, and the total tanshinone and panax notoginseng saponins jointly intervene cells to play a more obvious inhibiting role.

Example 3

Animal experiments: 60C 57BL/6 mice were randomly divided into 6 groups of 10 mice each. The drug concentrations are respectively prepared as follows: total tanshinone (TT, 1mg/mL), panax notoginseng saponins (PNS, 40mg/mL), total tanshinone + panax notoginseng saponins-low dose group (TT 0.5mg/mL + PNS 20mg/mL (L)), total tanshinone + panax notoginseng saponins-high dose group (TT 1mg/mL + PNS 40mg/mL (H)), Control group (Control, normal saline), DSS Model group (Model, 3% DSS), and 0.2mL of corresponding drug or pure water is given to each mouse every day. Mice were kept normally for 5 days to acclimatize. The total tanshinone, the panax notoginseng saponins, the total tanshinone plus the panax notoginseng saponins-low dose group, and the total tanshinone plus the panax notoginseng saponins-high dose group are respectively administered with corresponding drugs for intervention for 4 days, and then the rest groups except the control group are administered with drinking water containing 3% DSS for 7 days to cause colitis model. The body weight of the mice is weighed every day, the excrement condition of the mice is observed, and the excrement condition of the mice is detected by using an excrement occult blood kit. On the day of indexing, the mouse colon, spleen, etc. were collected for further study, and the length and weight of the colon were measured. Fresh mouse feces were used for 16sDNA sequencing to detect the regulatory effect of the drug on intestinal flora. In addition, changes in mouse colon inflammatory factors were detected using an ELISA kit.

Results of the experiment

The C57BL/6 mice continuously drunk 3% DSS-containing water to induce colitis, and blood in the stool appeared around the third day, the stool gradually became soft and rotten without forming, the body weight decreased with it (fig. 3), and the colon also shrunk accordingly. When the medicinal liquor is continuously drunk for 7 days, colonic ulcers are caused, as shown in figure 4, the DAI index of a model group mouse is increased day by day, the trend of increasing the DAI of the mouse which receives the medication of the total tanshinone, the panax notoginseng saponins and the total tanshinone and panax notoginseng saponins is inhibited, and the effect of the combined medicinal group is more obvious. The colon length of the mice in the drug administration group is relatively long compared with that in the model group, and the colon length of the mice in the drug combination high concentration group is close to that in the blank control group.

Colon histopathological examination of normal mice in the control group showed structural integrity of crypt and colon epithelial cells, and goblet cells were normal. While the DSS-induced colitis mouse colon tissue showed severe lesions, such as the disappearance of epithelial cells, goblet cells and crypt structures. Intestinal submucosa and mucosa also have significant transmural inflammatory cell infiltration. After the total tanshinone, the panax notoginseng saponins and the total tanshinone and panax notoginseng saponins are treated by using the medicine, the histological changes are obviously improved, particularly the effect of the combined medicine set is most obvious (figure 6), and the histopathological damage score is obviously reduced (figure 7).

Example 4

Inhibition of inflammatory factor secretion in DSS-induced colitis mice: the colon tissue collected in example 3 was digested and used for detection in an ELISA kit.

As a result, DSS was found to significantly promote the increase in the levels of inflammatory cytokines TNF- α, IL-6, IL-1 β, MPO and MCP-1, while decreasing the level of the anti-inflammatory factor IL-10, as compared to the control group. Compared with the model group, the level of proinflammatory factors in the colon of the mice receiving the intervention of the total tanshinone, the panax notoginseng saponins and the total tanshinone and panax notoginseng saponins is reduced, the level of the inflammation-inhibiting factors is improved, and particularly, the drug effect of the drug combination high-concentration group is most obvious (figure 8).

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.