CN111084772A

CN111084772A - Novel medical application of glabridin C

Info

Publication number: CN111084772A
Application number: CN201911393277.4A
Authority: CN
Inventors: 李世�; 王宁
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-05-01

Abstract

The invention discloses a new medical application of glabridin C. Erectile dysfunction is considered a barometer of premonitory or physical health status of cardiovascular or peripheral vascular disease, severely affecting the patient's physical and mental health and the quality of sexual life of both men and women. Phosphodiesterase 5(PDE5) belongs to the superfamily of enzymes, PDE5 inhibitors are mainly used for the treatment of erectile dysfunction, and various drugs (e.g. viagra) are currently on the market. The invention discovers that the glabridin C has stronger inhibitory activity on phosphodiesterase 5 and has the prospect of being developed into a phosphodiesterase 5 inhibitor and further being used for preparing medicaments for treating erectile dysfunction.

Description

Novel medical application of glabridin C

Technical Field

The invention relates to a new application of a known compound, in particular to a new application of a known compound in liquorice, and more particularly relates to a new medical application of glabridin C.

Background

Erectile Dysfunction (ED) is considered as a weather sheet that is a precursor to cardiovascular or peripheral vascular pathologies or to physical health conditions, and seriously affects the physical and mental health of patients and the quality of sexual life of both men and women (references: the Fangdanbo et al, progress in erectile dysfunction therapy, modern Utility medicine, 6 months in 2019).

Phosphodiesterase 5(PDE5) belongs to the superfamily of enzymes, PDE5 inhibitors are mainly used for the treatment of erectile dysfunction, and various drugs (e.g. viagra) are currently on the market. Erection is initiated by sexual stimulation, which causes the cavernous cells of the penis to release NO, which enters the smooth muscle endothelial cells to activate guanylate cyclase, which catalyzes the synthesis of the second messenger, cGMP, which activates Ga⁺The ion channel makes the smooth muscle of blood vessel relax, the blood enters the penis, the penis is engorged with blood, and the penis is erected. PDE5 is an intracellular enzymeIt is a phosphodiesterase with the highest content in cavernous tissue of penis and can catalyze and degrade cGMP to GMP. PDE5 inhibitors can reduce the activity of PDE5 and increase the intracellular concentration of cGMP, thereby initiating and maintaining penile erection (ref: Wanglin Howla et al, progress in the study of phosphodiesterase 5 inhibitors, 10 months in 2017, J.PharmacoHei, China).

The natural product is always the main source of the medicine for preventing and treating diseases, and has a long history and a solid foundation in the aspect of treating neurodegenerative diseases. Although synthetic drugs have a considerable weight in drug development, many drugs used clinically are derived directly or indirectly from natural products. Natural products remain one of the major sources of drugs or lead compounds for the treatment of major diseases. Therefore, the research on finding the PDEs activity inhibiting component with high efficiency, low toxicity and strong selectivity from natural products is more and more valued by scientists (reference: research progress of natural phosphodiesterase inhibitors, Chinese herbal medicine 2018 for 10 months).

The liquorice is the dried root and rhizome of the plants of the genus glycyrrhiza of the family leguminosae and is one of the important bulk medicinal materials in China. Modern scientific research proves that the liquorice has the functions of enhancing the immunity of organisms, resisting tumors, oxidation and viruses besides the functions of easing pain, relieving cough, resisting inflammation, resisting ulcer, resisting allergic reaction and the like, and particularly has more remarkable effects on AIDS, hepatitis B, herpes zoster, SARS virus and the like. The rich pharmacological action of licorice has a direct relationship with its chemical components of abundant species and quantity. More than 200 chemical components are extracted, separated and identified from the glycyrrhiza plants in sequence, and 10 glycyrrhiza plants are involved. The most important and proven bioactive components are triterpenoid saponins such as glycyrrhizic acid, flavonoids, coumarins, polysaccharides, alkaloids, amino acids, etc. (reference: research progress of biological and chemical components of licorice, Chinese herbal medicine, 11 months 2005).

The semi-glycyrrhiza isoflavone B, coarse glabrene C and echinocandin are 3 natural products separated from liquorice. Although studies have shown that these three compounds have various pharmacological activities, there is no report on their action as PDE5 inhibitors, nor on their use for treating erectile dysfunction.

Disclosure of Invention

The invention aims to provide a new medical application of glabridin C in preparing phosphodiesterase 5 inhibitor and to provide an application of glabridin C in preparing a medicament for treating erectile dysfunction.

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

a new medicinal use of glabridin C in preparing phosphodiesterase 5 inhibitor is provided. The glabrene C has strong inhibition effect on phosphodiesterase 5 and shows obvious concentration dependence.

Application of glabridin C in preparing medicine for treating erectile dysfunction is provided. Erection is initiated by sexual stimulation, which causes the cavernous cells of the penis to release NO, which enters the smooth muscle endothelial cells to activate guanylate cyclase, which catalyzes the synthesis of the second messenger, cGMP, which activates Ga⁺The ion channel makes the smooth muscle of blood vessel relax, the blood enters the penis, the penis is engorged with blood, and the penis is erected. PDE5 is an intracellular enzyme, the most abundant phosphodiesterase in the corpus cavernosum tissue of the penis, and catalyzes the degradation of cGMP to GMP. PDE5 inhibitors reduce PDE5 activity and increase intracellular concentrations of cGMP, thereby initiating and maintaining penile erection. The glabrene C with PDE5 inhibiting activity has the foreground of developing anti-ED medicine.

The beneficial effects are that:

the invention discovers that the glabridin C has stronger inhibitory activity on phosphodiesterase 5 and has the prospect of being developed into a phosphodiesterase 5 inhibitor and further being used for preparing medicaments for treating erectile dysfunction.

Drawings

FIG. 1 is a graph showing the inhibition of phosphodiesterase 5 by hemiglycyrrhiza isoflavone B at different concentrations;

FIG. 2 is a graph showing the inhibition effect of glabridin C on phosphodiesterase 5 at different concentrations;

FIG. 3 is a graph showing the inhibition of phosphodiesterase 5 by various concentrations of echinocandin.

Detailed Description

First, experimental material

The purity of the semi-glycyrrhiza isoflavone B, the coarse glabrene C and the glabrous spur chalcone is more than or equal to 98 percent. DMSO is respectively used for preparing linear gradient solution, 2 mu L is added into a reaction system with phosphodiesterase 5 inhibition effect, so that the final concentration of semi-glycyrrhiza isoflavone B, coarse glabrene C or echinocandin in the reaction system is respectively 10, 20, 50, 100, 200 and 500 nM.

The positive drug is sildenafil, the solution preparation method and the addition amount are the same, and the final concentration in the reaction system is 5 nM.

The preparation is carried out 4 hours before use, and the mixture is dissolved, mixed and stored at 4 ℃.

Second, Experimental methods

The inhibition of phosphodiesterase 5 is performed according to the methods disclosed by Guojuliang, etc., of the college of medicine of Zhongshan university (reference: study on the spectrum-effect relationship of different extraction sites of maca on the inhibition of phosphodiesterase 5, 2 months in 2017 of Chinese herbal medicine). Wherein, phosphodiesterase 5 expression and purification are performed according to the literature report method (Wang H, Liu Y, Huai Q, et al.multiple formulations of phosphodiesterase-5: interactions for enzyme function and purification [ J ] Biol Chem,2006,281(30): 21469-21479.). The method comprises the following specific steps:

the constructed plasmid pET15b-PDE5 was transformed into E.coli strain (E.coli) BL 21-Codonplus. Coli cells harboring the recombinant plasmid were grown to an absorbance value (A600nm) of 0.7 at 37 ℃ in LB medium (10 g tryptone, 5g yeast extract and 5g NaCl per liter medium) containing 100. mu.g/mL ampicillin, 30. mu.g/mL chloramphenicol and 0.4% glucose. Then 0.1mmol/L isopropyl-D-thiogalactoside (IPTG) was added to induce expression and growth was continued for 16h at 15 ℃. Centrifuging at 4500r/min for 30min to collect cells, and storing at-80 deg.C.

The PDE5 protein expressed by the escherichia coli is purified by a Ni-NTA affinity column, a Q-Sepharose ion exchange column and a Sephacryl S300 molecular sieve chromatographic column respectively, and is used for subsequent experiments when the purity is not lower than 90 percent as shown by SDS-PAGE electrophoresis.

Collecting 1/100 tritium diluted with pure water4. mu.L of cGMP (20000 to 30000cpm) was dissolved in a buffer (50mmol/L of LTrispH8.0, 10mmol/L of MgCl₂0.5mmol/L dithiothreitol) to 58 μ L, then adding 2 μ L of the solution of the drug to be tested, adding 2 μ L of DMSO into the set solvent control group, and mixing uniformly. Then, 40. mu.L of a protein solution diluted with a buffer and having a hydrolysis rate of about 60% (note: the concentration of the protein solution herein needs to be determined by preliminary experiments, and the term "about 60% hydrolysis rate" means that about 60% of cGMP can be hydrolyzed into GMP in the reaction system without adding a PDE5 inhibitor), and 40. mu.L of the buffer was added to the negative control A, B group, and the mixture was gently mixed and allowed to stand at 25 ℃ for 15 minutes. Finally, 0.2mol/LZnSO is added in turn ₄200 μ L and 0.2mol/L Ba (OH)₂The reaction was stopped with 200. mu.L of the solution, and mixed well with shaking. Centrifuge at 14000r/min for 5 min. And (3) taking 430 mu L of supernatant, transferring the supernatant into a liquid flash tube, adding 2.5mL of scintillation liquid, oscillating and mixing uniformly, measuring the CPM value of each reaction system in unit time (1min) by using a liquid scintillation counter, and calculating the inhibition rate according to the following formula. The experiments were set up in triplicate.

The cGMP hydrolysis amount of the drug group to be detected is represented by the difference between the CPM value of the negative control group B and the CPM value of the drug group to be detected, and the cGMP hydrolysis amount of the solvent control group is represented by the difference between the CPM value of the negative control group A and the CPM value of the solvent control group.

Data were analyzed using SPSS19.0 and the data were expressed as mean ± SD and curves were plotted for inhibition of phosphodiesterase 5 by different concentrations of drug.

Third, experimental results

The inhibition effect of different concentrations of hemiglycyrrhiza isoflavone B, glabridin C or glabrata chalcone on phosphodiesterase 5 is shown in table 1 and figures 1-3, and although the inhibition strength of the three compounds on phosphodiesterase 5 is different, the three compounds have strong inhibition effect and show obvious concentration dependence.

TABLE 1 inhibition of phosphodiesterase 5 by various concentrations of the compound

The test results show that the hemiglycyrrhiza isoflavone B, the glabridin C or the glabrous spur chalcone have the prospect of being developed into a phosphodiesterase 5 inhibitor and further being used for preparing the medicament for treating erectile dysfunction.

Claims

1. A new medicinal use of glabridin C in preparing phosphodiesterase 5 inhibitor is provided.

2. Application of glabridin C in preparing medicine for treating erectile dysfunction is provided.