Application of radix achyranthis bidentatae in preparation of anti-fibrosis medicine for treating myocardial infarction and medicine

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of radix achyranthis bidentatae in preparation of an anti-fibrosis medicine for treating myocardial infarction and the medicine. The medicine for treating fibrosis after myocardial infarction comprises a carrier and dapagliflozin, the carrier is radix achyranthis bidentatae, and the dapagliflozin is loaded on the radix achyranthis bidentatae. The invention discloses the application of radix achyranthis bidentatae in preparation of the anti-fibrosis medicine for treating myocardial infarction for the first time, and it is found in anti-myocardial fibroblast fibrosis experiments that expression of alpha-SMA and Col-I / III in TGF-beta + radix achyranthis bidentatae-DAPA group cells is obviously reduced, so that the medicine prepared by the invention can be used for treating anti-fibrosis after myocardial infarction.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of Achyranthes bidentata Bl. in preparation of a medicine for treating post-myocardial infarction anti-fibrosis and the medicine. BACKGROUND

[0002] Achyranthes bidentata Bl. is the dried root of Achyranthes bidentata Bl. of Amaranthaceae. It has the functions of removing blood stasis to free the channels, tonifying liver and kidney, strengthening bones and tendons, promoting urination to remove edema, and leading blood downward, and is used for treating amenorrhea, dysmenorrhea, lumbago and soreness of the knees, weakness of bones and tendons, strangury, edema, headache, dizziness, toothache, aphtha, hematemesis and epistaxis.

[0003] Literature research shows that Achyranthes bidentata Bl. has the effects of resisting liver and kidney fibrosis, but research in the heart is still not available. Therefore, the application is proposed. SUMMARY

[0004] The application aims to provide application of Achyranthes bidentata Bl. in preparation of a medicine for treating post-myocardial infarction anti-fibrosis and the medicine.

[0005] In order to overcome the shortcomings of the prior art, the application provides the following technical scheme: Application of Achyranthes bidentata Bl. in preparation of a medicine for treating post-myocardial infarction anti-fibrosis.

[0006] In addition, the application further provides a medicine for treating post-myocardial infarction anti-fibrosis, which comprises a carrier and Dapagliflozin; the carrier is Achyranthes bidentata Bl., and the Dapagliflozin is loaded on the Achyranthes bidentata Bl..

[0007] Further, the mass ratio of the Achyranthes bidentata Bl. to the Dapagliflozin is 5000: (1-2).

[0008] In addition, the application further provides a preparation method of the medicine for treating post-myocardial infarction anti-fibrosis, and the preparation method comprises the following steps: S1, Achyranthes bidentata Bl. is taken and decocted with water, filtered to obtain decocted liquid; S2, the decocted liquid obtained in step S1 is centrifuged to obtain supernatant, Dapagliflozin is added, stirred until the Dapagliflozin is completely dissolved, centrifuged to collect supernatant to obtain medicinal liquid; S3, calcium chloride solution is added dropwise to the medicinal liquid obtained in step S2, and after standing for a period of time, centrifugation is performed to collect particles; S4, the particles obtained in step S3 are repeatedly washed with deionized water and centrifuged for multiple times, and then the particles are collected to prepare the medicine for treating post-myocardial infarction anti-fibrosis.

[0009] Further, in step S1, the amount of water added is 8-12 times the weight of Achyranthes bidentata Bl.; and / or, the decocting time is 1-1.5 h.

[0010] Further, in step S2, the centrifugal conditions are: a centrifugal force of 10000g; and a centrifugal time of 30-40min.

[0011] Further, in step S3, the concentration of the calcium chloride solution is 200mg / mL; and the dropwise adding amount of the calcium chloride solution is 1 / 300-1 / 500 of the volume of the liquid medicine.

[0012] Further, in step S3, after the calcium chloride solution is added dropwise, the sample is shaken after standing for 15min, and then is again stood for 15min.

[0013] Further, in step S3, the centrifugal conditions are: a centrifugal force of 10000g; and a centrifugal time of 30-40min.

[0014] Further, in step S4, the washing times are 3-6 times. And / or, the centrifugal force is 10000g; and the centrifugal time is 30-40min.

[0015] Compared with the prior art, the technical scheme of the present application has at least the following technical effects: The application discloses application of Radix Cyathulae in preparation of a medicine for treating post-infarction anti-fibrosis, and finds that expression of alpha-SMA and Col-I / III in TGF-beta+Radix Cyathulae-DAPA group cells is obviously down-regulated in an anti-myocardial fibroblast fibrosis experiment, so that the prepared medicine can be used for treating post-infarction anti-fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0016] The drawings accompanying the specification of this application form a part thereof, serve to provide further understanding of the present application, and together with the specification explain the application, and do not constitute an improper limitation on the present application. Among them: Figure 1 A scanning electron microscope observation morphological schematic view of the medicine for treating post-infarction anti-fibrosis prepared by the present application; Figure 2 A schematic view of the medicine for treating post-infarction anti-fibrosis successfully prepared by the present application; Figures 3-5 A schematic view of the medicine for resisting myocardial fibroblast fibrosis. DETAILED DESCRIPTION

[0017] In order to make the objectives, technical solutions and advantages of the present application clearer, the following will be combined with the embodiments of the present application to make a clear and complete description of the technical solutions in the embodiments of the present application. It should be understood by those skilled in the art that the embodiments are only used to understand the present application and should not be regarded as a specific limitation to the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application. The process parameters not specified in the following embodiments are usually according to the conventional conditions.

[0018] The endpoints of the ranges and any values disclosed in the present application are not limited to the precise values stated. These ranges and values should be interpreted as approximately between the stated values. For ranges of values, the endpoints of the ranges are included in the ranges, and the endpoints of the ranges and the individual points included in the ranges can be combined with one another to form new ranges of values, which are to be construed as being specifically disclosed.

[0019] According to a first aspect of the present application, there is provided a use of Radix Achyranthis Bidentatae in the preparation of a medicament for treating anti-fibrosis after myocardial infarction.

[0020] According to a second aspect of the present application, there is provided a medicament for treating anti-fibrosis after myocardial infarction, comprising a carrier and Dapagliflozin; the carrier is Radix Achyranthis Bidentatae, and the Dapagliflozin is loaded on the Radix Achyranthis Bidentatae.

[0021] It is found through research that Radix Achyranthis Bidentatae can be prepared into a micro-particle system through a series of steps to load drugs. Dapagliflozin is a drug approved for listing by the State Drug Administration, and is mainly used for treating type 2 diabetes and chronic kidney disease. The present application first proposes Radix Achyranthis Bidentatae loaded with Dapagliflozin and applied to anti-fibrosis after myocardial infarction, and there is no relevant report in the past.

[0022] In the above medicament for treating anti-fibrosis after myocardial infarction, as a preferred embodiment, the mass ratio of Radix Achyranthis Bidentatae to Dapagliflozin is 5000: (1-2).

[0023] According to a third aspect of the present application, there is further provided a preparation method of the above medicament for treating anti-fibrosis after myocardial infarction, comprising the following steps: Step 1: Radix Achyranthis Bidentatae is decocted with water, filtered to obtain a decoction liquid; before the Radix Achyranthis Bidentatae is decocted, it is washed with double distilled water for multiple times and then decocted with double distilled water; the Radix Achyranthis Bidentatae can be selected from Chinese medicinal materials Radix Achyranthis Bidentatae or Radix Achyranthis Bidentatae decoction pieces.

[0024] Step 2: the obtained decoction liquid is centrifuged by a centrifuge to obtain a supernatant; Dapagliflozin is added and stirred until it is completely dissolved, then centrifuged to collect the supernatant to obtain a medicinal liquid; the centrifugation is mainly for the undissolved drugs.

[0025] Step 3: Add calcium chloride solution to the obtained drug solution, let it stand for a period of time, centrifuge, and collect the particles; the role of calcium chloride solution is as a filter aid. The addition of calcium ions makes the particle morphology stable and promotes the deposition of Achyranthes bidentata-carrying dapagliflozin particles, which is convenient for subsequent collection.

[0026] Step 4: After repeatedly washing and centrifuging the obtained particles with deionized water, collect the particles to obtain a drug for treating anti-fibrosis after myocardial infarction.

[0027] The electron microscopy morphology diagram of the Achyranthes bidentata-loaded dapagliflozin prepared in this invention, used as an anti-fibrotic drug for the treatment of post-myocardial infarction myocardial infarction, is shown in the attached figure. Figure 1 As shown; from the appendix Figure 2 As can also be seen, this invention successfully prepared a drug containing dapagliflozin in Achyranthes bidentata.

[0028] In the above preparation method, as a preferred embodiment, in step S1, the amount of water added is 8 to 12 times the weight of Achyranthes bidentata; optionally, the decoction time is 1 to 1.5 hours.

[0029] In the above preparation method, as a preferred embodiment, in step S2, the centrifugation conditions are: centrifugal force of 10000g; centrifugation time of 30~40min.

[0030] In the above preparation method, as a preferred embodiment, in step S3, the concentration of the calcium chloride solution is 200 mg / mL; the amount of calcium chloride solution added is 1 / 300 to 1 / 500 of the drug solution volume.

[0031] In the above preparation method, as a preferred embodiment, in step S3, after adding calcium chloride solution, the mixture is allowed to stand for 15 minutes, then shaken, and allowed to stand for another 15 minutes. Optionally, in step S3, the centrifugation conditions are: centrifugal force of 10000g; centrifugation time of 30-40 minutes.

[0032] In the above preparation method, as a preferred embodiment, in step S4, the number of washing cycles is 3 to 6; optionally, the centrifugal force is 10000g; and the centrifugation time is 30 to 40 minutes.

[0033] The present invention will now be described in detail with reference to embodiments thereof. These examples are provided by way of explanation and not by way of limitation. In fact, those skilled in the art will recognize that modifications and variations can be made to the present invention without departing from its scope or spirit. For example, a feature shown or described as part of one embodiment may be used in another embodiment to produce yet another embodiment. Therefore, it is desirable that the present invention encompass such modifications and variations that fall within the scope of the appended claims and their equivalents.

[0034] In the embodiments of the present application, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0035] Example 1 The present embodiment provides a medicine for treating post-myocardial infarction anti-fibrosis, which comprises carrier Radix Achyranthis Bidentatae and dapagliflozin; the dapagliflozin is loaded on the Radix Achyranthis Bidentatae; and the specific preparation method is as follows: Take Radix Achyranthis Bidentatae decoction pieces 50g, wash with 500mL double distilled water, then add 500mL double distilled water without changing the water and decoct for 1h, take the decoction liquid, centrifuge at 10000g for 30min to remove the drug mud, add 20mg dapagliflozin to make it fully dissolved. Then centrifuge at 10000g for 30min to remove the undissolved drug. Add 100uL of 200mg / mL calcium chloride to the supernatant containing the drug, shake after standing for 15min, and then stand for another 15min. Centrifuge at 10000g for 30min to collect the particles, and the medicine particles for treating post-myocardial infarction anti-fibrosis of the present embodiment are prepared.

[0036] Test Example 1 The following proves the application of Radix Achyranthis Bidentatae in preparing a medicine for treating post-myocardial infarction anti-fibrosis through an anti-myocardial fibroblast fibrosis test.

[0037] Experimental steps: (1) Take myocardial fibroblasts in the logarithmic growth phase and in good growth state, digest with 0.25% trypsin, count the cells, and then seed them into 24-well plates at a density of 3×10 4 / well with a total volume of 0.5mL, and incubate the cells for 18-24h.

[0038] (2) Process the cells according to the grouping Untreated: simple myocardial fibroblasts Control group: treated with TGF-β Radix Achyranthis Bidentatae group: treated with TGF-β and then incubated with Radix Achyranthis Bidentatae-DAPA medicine for 24h Radix Achyranthis Bidentatae-DAPA group: treated with TGF-β and then incubated with Radix Achyranthis Bidentatae-DAPA medicine for 24h (3) Cell immunofluorescence detection Wash the treated cells in the culture plate with PBS for 3 times; Fix with 4% paraformaldehyde for 15min, and wash the slides with PBS for 3 times; Permeate with 0.5% Triton X-100 (prepared with PBS) at room temperature for 15min; wash with PBS for 3 times; Add 5% goat serum to block for 1h, and wash with PBS for 3 times Each slide is added with enough amount of diluted primary antibody α-SMA (1:200), Col-III (1:200), Col-I (1:200), and incubated at 4°C overnight; PBS is immersed for 3 times, each for 3 min, and after the excess liquid is absorbed, the diluted fluorescent secondary antibody is added Goat anti-Rabbit IgG (H+L) Secondary Antibody (1:500) is incubated at 37°C for 2h in a wet box, and PBS is immersed for 3 times; Note: from the addition of the fluorescent secondary antibody, all subsequent operation steps are carried out in the dark as much as possible.

[0039] The nucleus is restained by adding DAPI and incubating in the dark for 5 min, and the specimen is stained with the nucleus, and the excess DAPI is washed with PBS 4 times; The images are observed and collected under a fluorescence microscope, as shown in Figures 3-5 .

[0040] The average fluorescence intensity analysis by Image J shows that compared with the Untreated group, the expression of α-SMA, Col-I / III in the TGF-β group is significantly up-regulated, which indicates that the modeling is successful; compared with the TGF-β group, the expression of α-SMA, Col-I / III in the TGF-β+Chu Xie group is down-regulated compared with the TGF-β group, and has statistical significance; compared with the TGF-β and TGF-β+Chu Xie groups, the expression of α-SMA, Col-I / III in the TGF-β+Chu Xie-DAPA group is significantly down-regulated, and all have statistical significance, which proves that the prepared drug can be used for treating post-infarction anti-fibrosis.

[0041] The above describes and evaluates some embodiments of the present application, and it should be understood that the present application is not limited to the above specific embodiments, and any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the scope of the technical solutions of the present application, which does not affect the essential content of the present application. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solutions of the present application, still belongs to the scope of protection of the technical solutions of the present application.

Claims

1. Use of Radix Achyranthis Bidentatae in the preparation of a medicine for treating post-myocardial infarction anti-fibrosis.

2. A medicament for treating anti-fibrosis after myocardial infarction, characterized by, The carrier is Radix Achyranthis Bidentatae, and the Dapagliflozin is loaded on the Radix Achyranthis Bidentatae.

3. The medicament according to claim 1, characterized in that, The mass ratio of the Radix Achyranthis Bidentatae to the Dapagliflozin is 5000: (1-2).

4. A method for the preparation of a medicament for the treatment of post-infarct anti-fibrosis as claimed in claim 2 or 3, characterized in that, The method comprises the following steps: S1, taking Radix Achyranthis Bidentatae and adding water to decoct, filtering to obtain a decoction; S2, centrifuging the decoction obtained in step S1 to obtain supernatant, adding Dapagliflozin, stirring until the Dapagliflozin is completely dissolved, centrifuging to collect the supernatant to obtain a medicinal liquid; S3, adding calcium chloride solution dropwise to the medicinal liquid obtained in step S2, standing for a period of time, centrifuging to collect particles; S4, repeatedly washing the particles obtained in step S3 with deionized water, centrifuging, and collecting the particles to obtain the medicine for treating post-myocardial infarction anti-fibrosis.

5. The preparation method according to claim 4, characterized in that, In step S1, the amount of water added is 8-12 times the weight of the Radix Achyranthis Bidentatae; and / or, the decoction time is 1-1.5 h.

6. The preparation method according to claim 4, characterized in that, In step S2, the centrifugation conditions are: centrifugal force is 10000g; and centrifugation time is 30-40 min.

7. The preparation method according to claim 4, characterized in that, In step S3, the concentration of the calcium chloride solution is 200mg / mL; and the dropwise addition amount of the calcium chloride solution is 1 / 300-1 / 500 of the volume of the medicinal liquid.

8. The preparation method according to claim 4, characterized in that, In step S3, after adding the calcium chloride solution, standing for 15 min, shaking, and then standing for 15 min.

9. The preparation method according to claim 4, characterized in that, In step S3, the centrifugation conditions are: centrifugal force is 10000g; and centrifugation time is 30-40 min.

10. The method of any one of claims 4-9, wherein, In step S4, the washing times are 3-6 times; and / or, the centrifugal force is 10000g; and the centrifugation time is 30-40 min.

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