阅读说明：本技术 丹酚酸A、丹酚酸B和丹酚酸C联合抗2019-nCov病毒的制药应用及药物 (Pharmaceutical application and medicine of combination of salvianolic acid A, salvianolic acid B and salvianolic acid C for resisting 2019-nCov virus ) 是由 贺浪冲 王楠 卢闻 展颖转 胡时灵 王珏 于 2021-01-27 设计创作，主要内容包括：本发明公开了丹酚酸A、丹酚酸B和丹酚酸C联合抗2019-nCoV病毒的制药应用及药物,属于抗病毒药物制备技术领域。本发明通过药物与ACE2蛋白结合效果、假病毒侵染能力,评价上述组合药物对2019-nCoV病毒的抑制效果,确定丹酚酸A、丹酚酸B和丹酚酸C联用时能够制备抗2019-nCoV病毒的药物。丹酚酸A、丹酚酸B、丹酚酸C联合给药具有比单独给药更有效的降低2019-nCoV假病毒侵染能力,因此具备抗病毒功效。并且丹酚酸A、丹酚酸B和丹酚酸C同时还具有保护心肌、保护心脏、抗纤维化、抗肿瘤等多种药理活性且副作用小,毒性低,因此将其作为对抗2019-nCoV病毒的药物具有重要意义。(The invention discloses pharmaceutical application and a medicine of salvianolic acid A, salvianolic acid B and salvianolic acid C for resisting 2019-nCoV virus in a combined manner, and belongs to the technical field of preparation of antiviral medicines. According to the invention, the inhibiting effect of the combined medicine on the 2019-nCoV virus is evaluated through the combination effect of the medicine and ACE2 protein and the infection capacity of the pseudovirus, and the medicine for resisting the 2019-nCoV virus can be prepared when the salvianolic acid A, the salvianolic acid B and the salvianolic acid C are combined. The combination administration of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C has the capability of reducing the 2019-nCoV pseudovirus infection more effectively than the single administration, thereby having the antiviral effect. And the salvianolic acid A, the salvianolic acid B and the salvianolic acid C also have various pharmacological activities of protecting cardiac muscle, protecting heart, resisting fibrosis, resisting tumor and the like, have small side effect and low toxicity, so that the salvianolic acid A, the salvianolic acid B and the salvianolic acid C have important significance as a medicament for resisting 2019-nCoV virus.)

1. The application of the combination of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C in preparing the 2019-nCov virus resistant medicine.

2. The use of claim 1, wherein the molar ratio of salvianolic acid A, salvianolic acid B, and salvianolic acid C is 2.5: 5: 2.5.

3. the use of claim 1, wherein the medicament is a medicament capable of binding to ACE2 protein.

4. The use of claim 1, wherein the agent is an agent capable of binding to the S protein.

5. The use of claim 1, wherein said agent is an agent that inhibits the 2019-nCoV virus infection.

6. The use of claim 5, wherein the medicament is a medicament for inhibiting 2019-nCoV pseudovirus from infecting ACE2 high expression cells.

7. The 2019-nCov virus resistant medicine is characterized by being a preparation prepared from salvianolic acid A, salvianolic acid B, salvianolic acid C and pharmaceutically acceptable auxiliary materials.

8. The 2019-nCov virus-resistant medicament according to claim 7, wherein the molar ratio of salvianolic acid A, salvianolic acid B and salvianolic acid C in the medicament is 2.5: 5: 2.5.

9. the 2019-nCov virus-resistant medicament according to claim 7, wherein the preparation is a dripping pill, a tablet, a capsule, a spray, a granule or an injection.

Technical Field

The invention belongs to the technical field of antiviral drug preparation, and relates to pharmaceutical application and drugs of salvianolic acid A, salvianolic acid B and salvianolic acid C for resisting 2019-nCov virus.

Background

The clinical types of diseases caused by the novel coronavirus (2019-nCoV) are light, common, heavy and critical, and the diseases are mainly manifested by fever, dry cough, hypodynamia and the like, and a few patients are accompanied by upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like. Severe cases often develop dyspnea after 1 week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis and hemorrhagic coagulation dysfunction, multiple organ failure, and the like.

2019-nCoV is a positive single-stranded RNA virus whose envelope has a prominent coronavirus (S protein), similar to SARS coronavirus (SARS-CoV), which mediates invasion of host cells through angiotensin converting enzyme 2(ACE2) receptor and through the virus surface S protein. ACE2 is a type I membrane-bound glycoprotein consisting of 805 amino acids and is widely expressed in tissues and organs such as heart, kidney and lung. The 2019-nCoV virus has the characteristics of strong infectivity, long incubation period, common susceptibility to crowds, unobvious early infection symptoms and asymptomatic infectors, and the characteristics ensure that the epidemic situation is wide in spread range and is continuously epidemic, so that the discovery of the medicine with the effect of resisting the 2019-nCoV virus is of great significance.

The salvianolic acid A, salvianolic acid B and salvianolic acid C are main active ingredients of the traditional Chinese medicine salvia miltiorrhiza, and the 2019-nCoV virus resisting effect of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C is not reported.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the pharmaceutical application of the combination of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C in resisting the 2019-nCov virus.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses an application of a combination of salvianolic acid A, salvianolic acid B and salvianolic acid C in preparing a 2019-nCov virus resistant medicament.

Preferably, the molar ratio of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C is 2.5: 5: 2.5.

preferably, the drug is a drug capable of binding to ACE2 protein.

Preferably, the drug is a drug capable of binding to the S protein.

Preferably, the medicament is a medicament for inhibiting the 2019-nCoV virus infection capacity.

Preferably, the medicine is used for inhibiting 2019-nCoV pseudovirus from infecting ACE2 high-expression cells.

The invention also discloses a 2019-nCov virus resistant medicament which is a preparation prepared from salvianolic acid A, salvianolic acid B, salvianolic acid C and pharmaceutically acceptable auxiliary materials.

Preferably, the molar ratio of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C in the medicine is 2.5: 5: 2.5.

preferably, the preparation is a dripping pill, a tablet, a capsule, a spray, a granule or an injection.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses application of combination of salvianolic acid A, salvianolic acid B and salvianolic acid C in preparing a 2019-nCoV virus resistant medicine, wherein the inhibiting effect of the combination medicine on 2019-nCoV virus is evaluated through the combination effect of the medicine and ACE2 protein and the infection capacity of pseudovirus, and the combination of salvianolic acid A, salvianolic acid B and salvianolic acid C is determined to prepare the 2019-nCoV virus resistant medicine. The combination administration of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C has the capability of reducing the 2019-nCoV pseudovirus infection more effectively than the single administration, thereby having the antiviral effect. And the salvianolic acid A, the salvianolic acid B and the salvianolic acid C also have various pharmacological activities of protecting cardiac muscle, protecting heart, resisting fibrosis, resisting tumor and the like, have small side effect and low toxicity, so that the salvianolic acid A, the salvianolic acid B and the salvianolic acid C have important significance as a medicament for resisting 2019-nCoV virus.

Drawings

FIG. 1 is a diagram of SPR to detect the binding ability of ACE2 protein to Salvianolic Acid A (SAA), Salvianolic Acid B (SAB) and Salvianolic Acid C (SAC); wherein (a) is salvianolic acid A; (b) is salvianolic acid B; (c) is salvianolic acid C;

FIG. 2 is a graph showing the binding ability of protein S to salvianolic acid A, salvianolic acid B and salvianolic acid C detected by SPR; wherein (a) is salvianolic acid A; (b) is salvianolic acid B; (c) is salvianolic acid C;

FIG. 3 shows the inhibitory effect of salvianolic acid A, salvianolic acid B and salvianolic acid C on the infection ability of 2019-nCoV pseudovirus;

FIG. 4 shows the inhibitory effect of the combined administration of salvianolic acid A, salvianolic acid B and salvianolic acid C and the administration of salvianolic acid B alone on the infection ability of 2019-nCoV pseudovirus.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

example 1 SPR method for measuring the binding Capacity of Salvianolic acid A, Salvianolic acid B and Salvianolic acid C to ACE2 protein and S protein

Experimental materials: ACE2 protein (from Beijing Yiqiao), salvianolic acid A (from Shi Ji Chen Guang), salvianolic acid B (from Shanghai An Menu) and salvianolic acid C (from Shi Ji Guang), SPR chip, COOH chip and corresponding activating reagent (from Nicoya, Canada).

The preparation method comprises the following steps: a carboxyl chip was mounted on the Open SPR instrument and run buffer was pumped at maximum flow rate (150. mu.L/min) to fill the detection cell. After the signal baseline was reached, 80% isopropanol was injected to purge and clean the chip surface with 10mM HCl. After reaching the signal baseline again, the flow rate was adjusted to 20. mu.L/min. An equal amount of coupling reagent (EDC/NHS) was mixed immediately and injected into the injection valve, the chip was activated for 5min, after stabilization, the activation buffer diluted the protein to 50. mu.g/mL and injected immediately into the injection valve. Finally, a blocking buffer is injected. Once the blocking buffer has passed, the sensor is ready to measure the coupling interaction between the analyte and the target protein.

Precisely weighing salvianolic acid A, salvianolic acid B and salvianolic acid C samples, respectively and fully dissolving with chromatographic pure DMSO to obtain 80mM stock solution, and storing at 4 deg.C; the binding curves were obtained by dilution to the desired concentration with running buffer and sequential injection. Obtaining a fitting curve by utilizing TraceDrawer software and solving a dissociation constant K_DThe value is obtained.

Referring to fig. 1 and 2, the abscissa of the graph is time, and the ordinate of the graph is the wavelength change caused by the combination of the protein with the salvianolic acid a, salvianolic acid B and salvianolic acid C, it can be seen that the salvianolic acid a, salvianolic acid B and salvianolic acid C have certain combination ability with ACE2 protein and ACE S protein, and have affinity constant K with ACE2 protein_DThe values are (4.08 + -0.61) e-7M, (2.95 + -0.78) e-7M and (7.32 + -0.42) e-7M, respectively; affinity constant K for S protein_DThe values were (3.82. + -. 0.43) e-6M, (5.15. + -. 0.64) e-7M and (2.19. + -. 0.14) e-6M, respectively.

Example 2, salvianolic acid A, salvianolic acid B and salvianolic acid C inhibit 2019-nCov pseudovirus from infecting ACE2 high expressing cells.

The ACE2 high expression cells are inoculated in a 96-well plate and each well is filled with the cellsThe nutrient was added to 50. mu.L. 37 ℃ and 5% CO₂The adherence is cultivated in an incubator for 2 hours. Adding 20 μ M salvianolic acid A, salvianolic acid B or salvianolic acid C prepared by culture medium, adding 5uL2019-CoV-2Spike pseudovirus into each well, infecting for 4h, complementing to 100 μ L culture volume, infecting for 6-8h, changing to 200 μ L new complete culture medium, and culturing at 37 deg.C for 48 h. The Luciferase Assay System kit detects the luminescence value of Luciferase.

Referring to fig. 3, following administration, the luminescence intensity of Luciferase was reduced compared to the control group. Chloroquine (20 μ M) was the antiviral positive control. As can be seen from FIG. 3, salvianolic acid A, salvianolic acid B and salvianolic acid C all have inhibitory effects on the 2019-nCoV pseudovirus infection capacity, and the Luciferase luminous intensity of salvianolic acid B is the smallest, and the salvianolic acid B has the strongest degree of inhibiting 2019-nCoV pseudovirus infection. In conclusion, the salvianolic acid A, salvianolic acid B and salvianolic acid C can improve the capability of the 2019-nCoV pseudovirus infecting ACE2 high-expression cells, and have a certain antiviral effect.

Example 3 combination administration of salvianolic acid A, salvianolic acid B, and salvianolic acid C inhibits infection of ACE2 high expressing cells by 2019-nCov pseudovirus.

ACE2 high expressing cells were seeded in 96 well plates and filled to 50 μ L of medium per well. 37 ℃ and 5% CO₂The adherence is cultivated in an incubator for 2 hours. Separately administered with 10. mu.M salvianolic acid B or 2.5. mu.M salvianolic acid A, 5. mu.M salvianolic acid B and 2.5. mu.M salvianolic acid C, and incubated at 37 ℃ for 2 hours. 5uL of 2019-CoV-2Spike pseudovirus is added into each well for infection for 4h, the culture volume is made up to 100 muL, after infection is continued for 6-8h, 200 muL of new complete culture medium is replaced, and culture is continued for 48h at 37 ℃. The Luciferase Assay System kit detects the luminescence value of Luciferase.

Referring to fig. 4, following the combination, the luminescence intensity of Luciferase was reduced compared to the control group. Chloroquine (20 mu M) is used as an antiviral positive control, and the result is shown in figure 4, so that the combined administration of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C has an inhibiting effect on the infection capability of the 2019-nCoV pseudovirus, and compared with the single administration of the salvianolic acid B, the combined administration of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C has a better inhibiting effect on the infection of the 2019-nCoV pseudovirus under the condition that the total mole number is not changed. In conclusion, the combined administration of the salvianolic acid A, the salvianolic acid B and the salvianolic acid C can inhibit the capability of the 2019-nCoV pseudovirus to infect ACE2 high-expression cells, and has better antiviral effect.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.