CN114028384A

CN114028384A - Application of licorice isoflavane derivative in preparation of medicine for preventing, relieving or/and treating pruritus

Info

Publication number: CN114028384A
Application number: CN202110218633.XA
Authority: CN
Inventors: 杨勇; 雷晓光; 胡凌寒
Original assignee: Peking University; Institute of Dermatology and Skin Disease Hospital of CAMS
Current assignee: Peking University; Institute of Dermatology and Skin Disease Hospital of CAMS
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2022-02-11
Anticipated expiration: 2041-02-24
Also published as: CN114028384B

Abstract

The invention discloses an application of glycyrrhiza isoflavane derivative in preparing medicines or health products for preventing, relieving or/and treating pruritus, and tests show that the glycyrrhiza isoflavane derivative is an antagonistic molecule of TRPV3, treats pruritus by taking TRPV3 as a target spot, has obvious effects on preventing, relieving and treating pruritus, and especially has obvious effects on treating acute pruritus and chronic pruritus which are not dependent on histamine; the glycyrrhiza isoflavane derivative has quick response, small toxic and side effects, good safety and good medicinal prospect for pruritus, is a novel TRPV3 antagonistic molecule, can be used as a lead compound, and is further used for research and development of TRPV3 related disease treatment medicines. The invention also provides a new medicine source for preventing, relieving and treating the pruritus cutanea.

Description

Application of licorice isoflavane derivative in preparation of medicine for preventing, relieving or/and treating pruritus

Technical Field

The invention relates to the field of medicines, relates to a medicine or health-care product for preventing, relieving and/or treating pruritus, and particularly relates to application of a natural product of a liquorice isoflavan derivative component in traditional Chinese medicine liquorice in medicines or health-care products for preventing, relieving and/or treating pruritus.

Background

The licorice isoflavane compound is a natural product derived from traditional Chinese medicine licorice, and previous researches reveal that the licorice has wide pharmacological effects, and particularly the licorice isoflavane compound has found effects of antibiosis, anticancer, antioxidation, anti-inflammation and the like. On the basis of the existing research, further excavation of the new action and the molecular mechanism behind the compound has important research and application values.

Transient receptor potential channels (TRPs) are a class of nonselective cation channels that are widely expressed in many tissues and organs and are involved in the regulation of a variety of cellular functions. TRP channels are divided into 7 subfamilies according to amino acid sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPN (NompC), TRPP (polycystin), and TRPML (mucolipin). Through mediating the transmembrane flow of intracellular and extracellular cations, TRP channels play a key role in the processes of absorption of cellular calcium ions, guidance of neuronal growth cones, growth and differentiation of keratinocytes, conduction of sensory signals and the like. More importantly, a range of human diseases, including polycystic kidney disease, skeletal developmental malformations, Olmsted syndrome, etc., have been shown to be triggered by dysfunction of TRP channels.

For the TRPV subfamily, since the first member TRPV1 was successfully cloned in 1997, the other members of the subfamily were also successively discovered and cloned. From the viewpoint of molecular sequence and structure, six member sequences of the TRPV subfamily are highly homologous and all have a molecular structure similar to that of the classical voltage-dependent potassium channel, i.e. they are tetramers assembled from 4 subunits, each of which contains 6 transmembrane regions, and the N-terminus containing the ankyrin repeat structure and the C-terminus containing the "TRP domain" located on the intracellular side. Between the 5 th and 6 th transmembrane domains there is a ring structure which defines the pore path through which the ions pass and is believed to be related to the ion selectivity of the channel. As non-selective cation channels, members of the TRPV subfamily all have a higher permselectivity for divalent cationsCa is especially indicated for middle energizer²⁺Ions are the most important. Because TRPV channels are regulated by temperature, pH value and a series of endogenous and exogenous ligand molecules, the TRPV channels are widely involved in the transmission of body sensory information and have the function of regulating and controlling intracellular Ca²⁺Balance, etc. of various important physiological functions.

The TRPV3 channel was first cloned in 2002 and found to be activated by warming (31-39 ℃). Subsequent studies have found more compounds that can activate TRPV3, including 2-APB and natural molecules such as camphor, menthol, carvacrol, etc. The TRPV3 channel is widely expressed in various tissue organs, of which various epithelial tissues are most important, including mainly the epidermis of the skin, mouth, and gastrointestinal tract. Previous studies on mice found that TRPV3 knockout mice, on the one hand, can develop temperature disturbances, abnormal curling of hair and impaired skin barrier; furthermore, after knockout of TRPV3, mice had significantly reduced scratching response in acute pruritus induced by the polypeptide SLIGRL, as well as chronic pruritus in atopic dermatitis models and xeroderma models. On the other hand, transgenic mice expressing TRPV3 function-enhancing mutants (p.g573s) show severe alopecia and skin erythema, edema, erosion, desquamation, etc., accompanied by marked spontaneous pruritic behavior. More importantly, the previous researches show that the function-enhancing mutation of the human TRPV3 can cause a rare hereditary skin disease, namely, the Olmsted syndrome, and the symptoms of the patients are intensively expressed as severe keratinization and even progressive damage of hand and foot skins, severe alopecia, pain and pruritus and the like. These studies indicate that TRPV3 plays an important role in the physiological processes of skin barrier, hair development, temperature sensing, inflammatory response, pain and itch sensory transmission, etc.; as an important target of skin drugs, especially the potential for treating pruritus, TRPV3 has become increasingly one of the TRP channels currently in the most interest. However, due to the continuing lack of effective research tools, including specific agonists and antagonists, etc., finding and developing more drug molecules that modulate TRPV3 has become a focus of common attention in order to solve more important problems related to TRPV 3.

For the functional study of ion channels, the evaluation of the effect of a compound on it has traditionally relied primarily on the detection of electrophysiological properties. The single cell patch clamp recording technology is the most widely applied electrophysiological detection technology due to its direct and sensitive advantages, and specifically, the high-impedance seal formed by the glass microelectrode and the cell membrane surface is utilized to connect the ion channel expressed on the cell membrane into the annular circuit composed of the amplifier, the electrode and the bath solution, and when different stimuli are applied, the opening and closing conditions of the ion channel are reflected according to the recorded current change in the circuit under certain manual clamping cell membrane potential.

Itching is a common unpleasant sensation that can induce a scratching response, similar to pain, but also distinguished because pain often encourages people to try to avoid touching the affected part. The generation process of the itch is similar to the sense of pain, and the itch depends on the detection of various itching-causing molecules by primary afferent neurons on nerve endings of peripheral organs such as skin and the like, external itching-causing sources, or endogenous itching-causing molecules secreted by skin cells, immune cells and the like, firstly activates primary sensory neurons to generate nerve impulses, and the nerve signals are immediately afferent to spinal cord dorsal horn and finally conducted to sensory centers of the brain to generate the itch after being relayed by neurons in the spinal cord. It is generally believed that different itch-causing agents activate different groups of sensory neurons, and that the itch responses induced by different itch-causing agents can be broadly classified into histamine-dependent and histamine-independent types, based on the response to histamine, the most common itch-causing agent. Meanwhile, according to the duration of the feeling of pruritus, the pruritus can be divided into two categories of acute pruritus and chronic pruritus, and compared with the warning significance of the acute pruritus, the chronic pruritus which is continuously weakened can be caused along with certain skin and systemic diseases, and can be caused when the body has no other obvious diseases, so that the pruritus becomes a disease which has no obvious physiological significance and is purely annoying.

As one common clinical symptom of dermatology, pruritus is accompanied with various diseases, such as atopic dermatitis, eczema, psoriasis and the like, nerve and molecular mechanisms related to histamine-dependent pruritus and histamine-independent pruritus are gradually revealed at present, so that a plurality of medicines for treating skin pruritus are developed, including antihistamine medicines mainly aiming at histamine-dependent pruritus and cooling medicines capable of transiently relieving pruritus, and the medicines have limited action range or large side effect, and itching relieving medicines aiming at histamine-independent acute pruritus and chronic pruritus are continuously lacked in clinic, so that more itching relieving medicines with brand new action mechanisms are developed, and new hope is brought to people suffering from pruritus.

Disclosure of Invention

The invention aims to provide the application of isoflavane derivatives of liquorice in preparing medicines for treating pruritus, aiming at the defects of the existing medicines for treating pruritus, in particular to the itching-relieving medicines for treating non-histamine-dependent acute pruritus and chronic pruritus and the technical defects of the medicines in application, wherein the isoflavane derivatives of liquorice are antagonistic molecules of TRPV3, take TRPV3 as a target spot, treat pruritus and have obvious treatment effect on the non-histamine-dependent acute pruritus and the chronic pruritus.

In order to achieve the purpose of the invention, the invention provides an application of a glycyrrhiza isoflavane derivative in preparing a medicine or health-care product for preventing, relieving or/and treating pruritus.

Wherein the glycyrrhiza isoflavan derivative is one or more of glycyrrhiza isoflavan formazan, glycyrrhetin or derivatives taking 3-phenylchroman as a core, or a stereoisomer and pharmaceutically acceptable salts and/or solvates thereof, and preferably glycyrrhiza isoflavan formazan and glycyrrhetin.

Wherein the structures of the isoflavan-methyl glycyrrhizae and the liquiritigenin are as follows:

by utilizing an electrophysiological patch clamp recording technology, natural product compounds from traditional Chinese medicines are tested in an HEK293T cell line over-expressing a TRPV3 channel, and the screened liquorice isoflavan-a and the liquiritigenin have the capacity of antagonizing TRPV3 in a dose-dependent manner.

Wherein the medicament consists of the glycyrrhiza isoflavan derivative and a pharmaceutically acceptable carrier.

In particular, the purity of the glycyrrhiza isoflavan derivative is more than or equal to 1 percent, and preferably more than 95 percent or 95-100 percent of pure substances.

In particular, pharmaceutically acceptable carriers are generally approved by health care professionals for this purpose and as inactive ingredients of medicaments. A compilation of pharmaceutically acceptable carriers can be found in tools such as The Handbook of Pharmaceutical excipients (Handbook of Pharmaceutical excipients, 2 nd edition, edited by A.Wade and P.J.Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994).

Wherein the pruritus is histamine-dependent or/and histamine-independent pruritus, preferably histamine-independent pruritus.

In particular, the pruritus is acute pruritus or/and chronic pruritus.

Wherein the medicine is in the form of tablet, capsule, pill, powder, granule, syrup, emulsion, injection, spray, gel, cream, cataplasma, and rubber patch.

In particular, the medicament is in the form of tablets, capsules, pills, powders, granules, syrups.

In another aspect, the invention provides a medicament or health product containing the glycyrrhiza isoflavan derivative for preventing, relieving or/and treating pruritus.

In particular, the ratio of the weight of the glycyrrhiza isoflavan derivative to the total weight of the medicament or health product is (0.01-10): 100, preferably (0.1-1): 100.

the medicine can be made into various dosage forms by methods known in the art, such as tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, gel types, creams, tinctures, cataplasms, rubber plasters or emplastrums, and the like.

The invention also provides a method for preventing, relieving or/and treating pruritus, which comprises the step of administering a therapeutically effective amount of the isoflavan derivatives (isoflavanone glycyrrhiza and liquiritigenin) to pruritus, wherein the therapeutically effective amount is 0.3-100 mg/kg.d, preferably 1-20 mg/kg.d, and more preferably 10 mg/kg.d.

The term "therapeutically effective amount" as used herein, unless otherwise indicated, is the amount of drug required to produce an effective effect; the "therapeutically effective amount" is adjustable and variable and ultimately determined by the medical practitioner, taking into account factors including the route of administration and the nature of the formulation, the general condition of the recipient's weight, age, etc., and the nature and severity of the condition being treated.

Compared with the prior art, the invention has the following obvious advantages:

1. the invention develops new medicinal value for known compounds of glycyrrhiza isoflavan derivatives (glycyrrhiza isoflavan formazan and glycyrrhetin), is used for preventing, relieving and treating pruritus and can be prepared into medicaments or health-care products for preventing, conditioning and/or treating pruritus, thereby developing a new field for the application of natural product compounds in traditional Chinese medicinal materials of liquorice and liquorice.

2. The glycyrrhiza isoflavan derivatives (glycyrrhiza isoflavan formazan and glycyrrhetin) have clear pharmacological action, obvious effect of preventing, relieving and treating pruritus, quick response, small toxic and side effect, good safety and good medicinal prospect.

3. The product of the invention has rich raw material sources, low price, safe clinical use, simple preparation process, small dosage and convenient use, can be prepared into various dosage forms, and is easy to popularize.

4. The glycyrrhiza isoflavan derivatives (glycyrrhiza isoflavan formazan and glycyrrhetin) are novel TRPV3 antagonistic molecules, can be used as lead compounds, and are further used for research and development of medicines for treating TRPV3 related diseases.

The invention adopts a mouse model of non-histamine-dependent acute pruritus induced by polypeptide SLIGRL, tests the ability of the isoflavanone and the liquiritigenin to inhibit the acute pruritus by counting the scratching times within 30min as a behavior detection index, and the result shows that the isoflavanone and the liquiritigenin can inhibit the acute pruritus induced by SLIGRL in a dose-dependent manner.

The ability of isoflavan-methyl glycyrrhizae and liquiritigenin to inhibit chronic pruritus is tested by taking statistics of scratching times per hour at different molding days as an inspection index by using a mouse model of atopic dermatitis chronic pruritus established by calcipotriol induction, and the result shows that the isoflavan-methyl glycyrrhizae and the liquiricidin can inhibit the chronic pruritus of a mouse model of atopic dermatitis to some extent.

The invention obtains a new antagonistic molecule based on the screening of a target molecule TRPV3, the antagonistic molecule shows the activity of inhibiting the histamine-independent acute pruritus and the chronic pruritus, and can be used as a lead compound for further research and development of pruritus treatment medicines.

Drawings

FIG. 1 is a graph of the antagonistic effect of different concentrations of Glycyrrhiza isoflavan nails on 2-APB-induced TRPV3 current, wherein A is a representative curve recording TRPV3 current changes in whole cell mode, including changes when 100 μ M2-APB stimulation was given alone, and when multiple concentrations (30, 100 and 300 μ M) of Glycyrrhiza isoflavan nails were simultaneously superimposed; b is a dose-effect relationship diagram of antagonism of TRPV3 channel by isoflavane, and is obtained by fitting and calculating S-type dose-effect curve, and IC of isoflavane₅₀The value was 20.87. mu.M (LogIC)₅₀1.320 ± 0.128, n ═ 4-5), data for each concentration are expressed as mean ± SEM;

FIG. 2 is a graph of the antagonistic effect of different concentrations of Liquiritidine on 2-APB-induced TRPV3 current, wherein A is a representative curve of TRPV3 current changes recorded in whole cell mode, including changes when 100 μ M2-APB stimulation was given alone, and when multiple concentrations (10, 30 and 100 μ M) of Liquiritidine were simultaneously superimposed; b is the dose effect of the Liquiritidine antagonizing TRPV3 channelThe relation chart is obtained by fitting and calculating an S-type dose-effect curve, and the IC of the liquorice isoflavanone₅₀The value was 15.24. mu.M (LogIC)₅₀1.183 ± 0.120, n ═ 5-8), data for each concentration are expressed as mean ± SEM;

FIG. 3 is a representative plot of the change in TRPV3 current recorded in the intra-membrane out-facing mode, including the current change when 100 μ M2-APB stimulation was given alone, and when different concentrations of Glycyrrhetini and Glycyrrhetini were simultaneously superimposed, wherein A is Glycyrrhetini; b is Liquiritidine; finally 10 μ M ruthenium red completely blocked the TRPV3 current.

FIG. 4 is a graph of the effects of Glycyrrhiza isoflavan A and Glycyrrhiza uralensis on TRPV1 and TRPA1 channels, respectively; wherein: A. b is a representative curve of TRPV1 current change recorded in a whole cell mode, and comprises the current change condition when the TRPV1 agonist capsaicin is singly administered and 100 mu M of glycyrrhiza isoflavan nail (A) or glycyrrhizacin (B) is simultaneously added, and finally the current can be completely blocked by the non-selective TRP channel antagonist ruthenium red; C. d is a representative curve of TRPA1 current changes recorded in a whole cell mode, including the changes when TRPA1 agonist AITC is singly administered and 100 mu M of glycyrrhiza isoflavan formazan (C) or glycyrrhizacin (D) is simultaneously added, and finally the current can be completely blocked by a non-selective TRP channel antagonist ruthenium red; E. f: a comparison graph of the blocking ability of glycyrrhiza isoflavan formazan (E) and glycyrrhetin (F) on TRPV3, TRPV1 and TRPA1 is shown. Test with unpaired Student's t,. P < 0.001. The above data are expressed as mean ± SEM, n is 4-8.

FIG. 5 shows the inhibitory effect of Glycyrrhiza isoflavan A on SLIGRL-induced acute pruritus; wherein A-C: the line graphs of the number of scratching times of mice in the experimental groups with 30, 100 and 300 μ M effects of glycyrrhiza isoflavanone and the corresponding control group without drug treatment were counted for a period of 5min, and by analysis of variance with repeated measurements, P >0.05(a-B),. P <0.01 (C); d is a bar graph of total scratching times within 30min for experimental groups with 30, 100 and 300 μ M effects of glycyrrhiza isoflavan formazan and corresponding control group mice. Test with paired Student's t, P <0.05, P <0.01, n.s.: P >0.05, each of which is expressed as mean ± SEM, n ═ 8;

FIG. 6 shows the inhibitory effect of Liquiridine on SLIGRL-induced acute pruritus; A-C: 30. the line graphs of scratching times of mice in the experimental groups with 100 and 300 mu M functions of the Liquiritidine and the corresponding control groups are counted according to a time period of 5min, and the variance analysis of repeated measurement is adopted, wherein P is greater than 0.05(A-B) and P is less than 0.05 (C); d: 30. bar graphs of total number of scratching within 30min for 100 and 300 μ M glycyrrhetin-affected experimental and corresponding control mice. Test with paired Student's t,. P <0.01, n.s.: P >0.05, each of the above data are expressed as mean ± SEM, n ═ 8;

FIG. 7 is a graph of the percent inhibition of SLIGRL-induced acute pruritus by different concentrations of Glycyrrhetini A and Glycyrrhizacetin, as well as the percent of SLIGRL acute pruritus versus wild-type for TRPV3 KO mice; the histogram is the ratio of total scratching times within 30min of mice in the experimental group and corresponding control group, on which 30, 100 and 300 μ M of actions of glycyrrhiza isoflavan a and glycyrrhetin are superimposed, and the ratio of total scratching times within 30min of TRPV3 KO mice and wild mice, all of which are expressed by mean value + -SEM, and n is 5-8.

Fig. 8 is a schematic representation of the protocol for induction of AD model mice with calcipotriol and treatment with TRPV3 antagonist, the dotted arrow above the timeline indicating the days of molding of the mouse ears with calcipotriol, the black arrow below indicating the days of applying 1% o of isoflavan or liquiritigenin to the mouse ears, and the video recording before molding and administration on

days

0, 4, and 7;

fig. 9 is a calcipotriol-induced chronic pruritus response in AD model mice at different test time points; wherein:

a, C, E: respectively counting the line graphs of scratching times of mice in an experimental group and a model group as a control, wherein the experimental group is formed by overlapping 1 per mill of effects of glycyrrhiza isoflavan and glycyrrhetin on 0 th, 4 th and 7 th days; analysis of variance using repeated measurements,. P < 0.05;

b, D, F: a bar chart of the total scratching times of mice in the experimental group and the model group as a control within 60min, wherein the experimental group is formed by superposing 1 per mill of the effects of glycyrrhiza isoflavan and glycyrrhetin on the 0 th day, the 4 th day and the 7 th day respectively; the unpaired Student's t test was used to compare to the control group,. P <0.05, n.s.: P > 0.05. The data in each group are expressed as mean value + -SEM, and n is 7-8;

FIG. 10 is a bar graph of the average ear thickness of mice in the experimental group and the model group without drug treatment, wherein 1 ‰ of effects of glycyrrhiza isoflavan and glycyrrhetin are superimposed on

days

0, 4, and 7; unpaired Student's t assay was used to compare to control groups,. P <0.01,. P < 0.001. The data in each group are expressed as mean value + -SEM, and n is 7-8;

FIG. 11 shows the results of observation of HEK293T cells under a fluorescence microscope after different treatments; each line corresponds to the left label, and images of a typical field of view are taken under white light (bright field), Green Fluorescence (GFP), and red fluorescence (PI staining) channels. After transient transfection of TRPV 3G 573S mutant plasmid, the HEK293T cells show GFP green fluorescence and strong red fluorescence signals (PI staining), and the number of cells with normal morphology is reduced; if 20 mu M ruthenium red is added during transfection, the green fluorescence signal of the cells is stronger, but the red fluorescence signal becomes very weak; meanwhile, when the liquorice isoflavan and the glycyrrhetinic acid with different concentrations (10 and 20 mu M) are added, red fluorescence signals are weak, and the scale is 100 mu M;

FIG. 12 shows TRPV 3G 573S mutation channel transient transfection of HEK293T cells under the same conditions, and the chemiluminescence intensity obtained by adding different concentrations (10 and 20. mu.M) of glycyrrhiza isoflavana, glycyrrhetin and ruthenamine, and cell viability without adding any antagonist, respectively, was tested; unpaired Student's t assay was used to compare P <0.01 to a control group without any added antagonist. The above data are expressed as mean ± SEM, n is 5.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The beneficial effects of the drug of the present invention are further illustrated by the following test examples, which include pharmacodynamic tests of the drug of the present invention.

The glycyrrhiza isoflavanone is white powder, is produced by Yunnan Xili company, and has the purity of 97 percent when being measured by an area normalization method of an ultraviolet detector and an evaporative light scattering detector of two detectors of a high performance liquid chromatography. Batch number: BBP 03331;

liquiritidine: white powder, produced by Yunnan Xili corporation, has a purity of 98% as determined by an area normalization method of a high performance liquid chromatography two-detector ultraviolet detector and an evaporative light scattering detector. Batch number: BBP 03353.

The method for producing the glycyrrhiza isoflavan molecules of the present invention is not particularly limited, and those skilled in the art can produce the glycyrrhiza isoflavan molecules of the present invention based on the common general knowledge in the art or obtain the glycyrrhiza isoflavan molecules of the present invention (glycyrrhiza isoflavan-a and glycyrrhetin) on the market.

Test example 1 Effect of Glycyrrhetini flavanoid Compound on TRPV3

The effect of compounds on overexpressed human TRPV3 was assessed using the cell patch clamp method.

HEK293T cell line was purchased from ATCC center, USA, and it was inoculated in DMEM complete medium after recovery, saturated humidity and containing 5% CO₂The culture was carried out in a 37 ℃ incubator.

Before transfection, HEK293T cells were seeded into 6-well plates and when the cell density reached 50-70%, the eukaryotic expression plasmid pCMV6-AC-GFP-TRPV3 (Origene, usa, cat # RG211184) containing the human TRPV3 cDNA was transfected using Lipofectamine 3000 transfection reagent.

The electrophysiological detection method adopts whole cell or membrane inside-out (inside-out) recording mode, applies HEKA EPC10 type amplifier and PatchMaster recording software, uses P-97 type microelectrode drawing instrument to draw an electrode with impedance of 3-5M omega, and records current (cell clamping potential of 0mV) with + -80 mV square wave voltage.

The method comprises the steps of placing HEK293T cells transfected with TRPV3 plasmid for 16-24h into specially prepared bath lotion (130mM NaCl,3mM HEPES,0.3mM EGTA, pH 7.4), selecting cells with good states, clean surfaces and strong green fluorescence signals, slowly moving an electrode added with internal liquid (the components of the internal liquid and the bath lotion) to slightly contact the surface of a cell membrane, slightly applying negative pressure until a high-resistance seal is formed, breaking the membrane to form a whole cell recording mode, or directly lifting the electrode after the high-resistance seal is formed, tearing off a membrane at the tip of the electrode, and forming an inner membrane facing outwards mode.

1) Under a whole-cell recording mode, gravity perfusion is carried out by using an RSC-200 rapid exchange drug delivery system, 100 mu M2-APB is firstly given to activate a TRPV3 channel, and then different concentrations (300 mu M, 100 mu M and 30 mu M) of the isoflavane A are respectively superposed; or different concentrations (100. mu.M, 30. mu.M and 10. mu.M) of Liquiridine, a significant decrease in current amplitude was observed. Wherein the current reduction range is continuously enlarged along with the increase of the concentration of the superimposed glycyrrhiza isoflavan until the glycyrrhiza isoflavan finally almost completely disappears (as shown in figure 1A); the results for glycyrrhizin were also similar, i.e. in whole cell mode, addition of glycyrrhizin reduced the 2-APB induced current and the magnitude of the current reduction was dose dependent (as in fig. 2A).

By measuring the corresponding antagonistic capacity of the glycyrrhiza isoflavan and the glycyrrhetinic acid on TRPV3 current under a plurality of concentrations and fitting by utilizing an S-type dose response curve, the method can be obtained: IC of glycyrrhiza isoflavan₅₀Has a value of 20.87 μ M (shown in FIG. 1B), IC of Liquiritidine₅₀The value was 15.24. mu.M (see FIG. 2B).

2) In the intra-membrane outward recording mode, i.e. recording only the current of the channel on a single membrane, it was also observed that glycyrrhiza isoflavana and glycyrrhetin were able to dose-dependently decrease the 2-APB induced current (see fig. 3), indicating that their ability to inhibit 2-APB current is not dependent on the assistance of intracellular molecules, but should act directly on the TRPV3 channel itself.

Test example 2 Effect of Glycyrrhetini flavanoid Compound on TRPV1 or TRPA1

The effect of compounds on overexpressed human TRPV1 and TRPA1 was assessed using a cell patch clamp approach.

Before transfection, HEK293T cells were seeded into 6-well plates and when the cell density reached 50-70%, either eukaryotic expression plasmid pCMV6-AC-GFP-TRPV1 (Origene, usa, cat # RG217653) containing human TRPV 1cDNA or eukaryotic expression plasmid pCMV6-AC-GFP-TRPA1 (Origene, cat # RG219290) containing human TRPA1cDNA was transfected using Lipofectamine 3000 transfection reagent.

The electrophysiological detection method adopts a whole cell or membrane inside-outside recording mode, applies an HEKA EPC10 type amplifier and PatchMaster recording software, uses an electrode with the impedance of 3-5M omega drawn by a P-97 type microelectrode drawing instrument, and records current (the cell clamping potential is 0mV) by plus or minus 80mV square wave voltage.

The method comprises the steps of taking HEK293T cells transfected with TRPV1 or TRPA1 plasmids for 16-24h, placing the cells in specially prepared bath solution (130mM NaCl,3mM HEPES,0.3mM EGTA, pH 7.4), selecting the cells with better state, clean surfaces and stronger green fluorescence signals, then slowly moving an electrode added with internal liquid (the components of the electrode are the same as the bath solution) to slightly contact the surface of a cell membrane, slightly applying negative pressure until a high-resistance seal is formed, and breaking the membrane to form a whole cell recording mode.

By utilizing an electrophysiological detection method, 100 mu M of the action of isoflavane on TRPV1 agonist capsaicin or TRPA1 agonist AITC induced current is recorded in HEK293T cells over-expressing TRPV1 or TRPA1 in vitro, and as a result, the isoflavane has weak blocking effect on TRPV1 (the blocking rate is 31.9% +/-6.6%, shown in figure 4A), while almost no antagonism is observed on TRPA1 (3.3% +/-15.5%, shown in figure 4C), and the effect is remarkably lower than that of antagonism on TRPV3 (88.0% +/-7.5%, shown in figure 4E), and the results indicate that the isoflavane has better action specificity on TRPV3 channels. Similar electrophysiological test methods are utilized to detect the effect of 100 mu M of liquiritigenin on TRPV1 and TRPA1 currents, and the blocking degree of the liquiritigenin on TRPV1 and TRPA1 (TRPV 1: 52.2% + -10.1%, FIG. 4B; TRPA 1: 8.4% + -10.8%, FIG. 4D) is also obviously lower than the antagonistic degree (93.3% + -5.8%, FIG. 4F) of the liquiritigenin on TRPV3, which indicates that the liquiritigenin also has certain effect specificity on TRPV3 channels.

For the animal model of pruritus, the standard acute pruritus model adopts intradermal injection of an pruritus-inducing agent into the back of the neck of a mouse to generate pruritus reaction, and characterizes the pruritus degree by counting the scratching times within a certain time after injection, wherein one-time scratching behavior is recorded when the injection site is lifted from the hind paw of the mouse until the hind paw falls down again or is put into the mouth for licking.

Common itch-causing agents include histamine, chloroquine, 5-hydroxytryptamine, compound 48/80, and the polypeptide SLIGRL, among others. As a result of the newly published studies, mice were significantly reduced in scratching response after TRPV3 knockdown for acute pruritus induced by intradermal injection of the activator SLIGRL of PAR 2. Therefore, the SLIGRL acute itch model was chosen as a paradigm for assessing the effect of TRPV3 antagonist molecules in inhibiting acute itch effects under physiological conditions.

Test example 3 Effect of Glycyrrhiza isoflavan A on mouse acute itching model

Selecting 24 purchased 8-10 week old (20-25 g in weight, half male and female) wild type C57BL/6J mice (Schbefu (Beijing) laboratory animal science and technology Co., Ltd.), randomly dividing into 3 groups, namely, a liquorice isoflavan A high dose group (300 mu M), a middle dose group (100 mu M) and a low dose group (30 mu M), wherein 8 mice in each group are removed from the back of the neck of the mice at least 2-3 days in advance;

on the day of formal experiment, the mice are respectively put into observation boxes (15cm multiplied by 10cm) for adaptive activity for about 5 min; then, firstly, injecting 50 mul of physiological saline containing different concentrations (30 muM, 100 muM and 300 muM) of the glycyrrhiza isoflavan nail into the skin of the right ear in advance, continuously injecting 50 mul of physiological saline containing 50 mug of SLIGRL at the same position after 30min and overlapping with the physiological saline of the glycyrrhiza isoflavan nail with corresponding concentration (namely, the physiological saline contains both SLIGRL and the glycyrrhiza isoflavan nail, wherein the concentration of the glycyrrhiza isoflavan nail is respectively corresponding to the concentration during the injection in advance, namely, the SLIGRL + the glycyrrhiza isoflavan nail) so as to induce acute pruritus; immediately placing the injected mice into an observation box, keeping quiet condition, recording for 30min by a camera and looking back the video afterwards, and counting scratching behaviors with 5min as a time period.

On alternate days, 50. mu.l of physiological saline containing 0.1% DMSO (solvent) was pre-injected intradermally in the back of the left ear, as a control group (no administration), and after 30min, 50. mu.l of physiological saline containing 50. mu.g SLIGRL (also containing 0.1% DMSO) was continuously injected into the same site to induce acute pruritus; immediately placing the injected mice into an observation box, keeping quiet condition, recording for 30min by a camera and looking back the video afterwards, and counting scratching behaviors with 5min as a time period.

As a result, it was found that the mice were variously reduced in the total scratching frequency within 30min after injection of SLIGRL in the case of the additive use of 30, 100 or 300 μ M of glycyrrhiza isoflavan nail as compared with the control group without drug administration (i.e., only SLIGRL was injected on the left ear side, i.e., SLIGRL + DMSO group) (fig. 5D).

However, the difference between the 30 μ M experimental group and the control group did not reach a significant level (P. 0.1047), whereas the level of decrease in total scratching frequency within 30min in the mice of the experimental group became more significant as the concentration of action increased to 100 and 300 μ M, suggesting that the ability of isoflavan a of glycyrrhiza to suppress acute pruritus induced by SLIGRL increased with the increase in concentration. It is worth mentioning that line graphs showing the number of scratching times by time-interval statistics (see fig. 5A-C): the different concentrations of isoflavan each caused a more rapid decay of the number of scratching in the mice of the experimental group over time, whereas when the superimposed concentration of isoflavan was sufficiently high (300. mu.M), the number of scratching in the mice of the experimental group was much lower from the beginning than in the control group not administered, and the P-value calculated by analysis of variance using repeated measurements was also less than 0.01, which showed on the other side that isoflavan had indeed an inhibitory effect on acute pruritus in SLIGRL.

Test example 4 Effect of Licorice Xidine on mouse acute itching model

Selecting 24 purchased 8-10 week old (weight 20-25g, half male and female) wild type C57BL/6J mice (Sibefu (Beijing) laboratory animal science and technology Co., Ltd.), randomly dividing into 3 groups, namely a Liquirocidine high dose group (300 μ M), a medium dose group (100 μ M) and a low dose group (30 μ M), wherein each group comprises 8 mice, and removing hair on the back of the neck of the mice at least 2-3 days earlier;

the test example 3 was repeated except that 50. mu.l of physiological saline containing glycyrrhizin at different concentrations (30. mu.M, 100. mu.M, 300. mu.M) was pre-injected intradermally into the back of the right ear on the day of the official test, and after 30min, 50. mu.l of physiological saline containing 50. mu.g of SLIGRL and added with glycyrrhizin at the corresponding concentration (i.e., both SLIGRL and glycyrrhizin were contained in the physiological saline, wherein the concentrations of glycyrrhizin respectively correspond to the concentrations at the time of pre-injection, i.e., SLIGRL + glycyrrhizin) was continuously injected into the same site to induce acute pruritus.

As a result, it was found that, in the mice of the experimental group to which 30, 100 or 300 μ M glycyrrhizin was additionally added, a different degree of reduction in total scratching frequency within 30min was also observed compared to the control group to which no drug was administered (i.e., only SLIGRL was injected on the left ear side, i.e., SLIGRL + DMSO group) (fig. 6D). The difference between the 30 μ M experimental group and the control group did not reach a significant level (P ═ 0.0523), but when the treatment concentration was increased to 100 and 300 μ M, the total scratching frequency of the experimental group mice within 30min was significantly lower than that of the control group, which means that the liquiritigenin had the same effect as that of the liquiritigenin isoflavanone and was also effective in inhibiting the acute pruritus reaction induced by SLIGRL. Time-phased statistics of scratching times also yielded results similar to that of glycyrrhiza isoflavan nails (see fig. 6A-C): the number of scratching tends to decay more rapidly over time in mice of each experimental group, whereas the difference between the two groups reaches a significant level (P <0.05) when the superimposed concentration of glycyrrhizin is increased to 300 μ M. The results all prove that the Liquiritidine can also effectively inhibit acute pruritus induced by SLIGRL.

By calculating the ratio of the total scratching times of the mice in different experimental groups to the mice in the corresponding control groups within 30min after SLIGRL injection, it can be observed that the inhibition effect of the isoflavanone and the liquiritigenin is stronger and stronger along with the increase of the action concentration, and the similar dose dependence is shown (figure 7).

Considering that TRPV3 KO mice (TRPV3 knockout mice) have been reported to induce acute pruritus significantly less to SLIGRL than wild type mice, as a control on the other hand, it was also attempted to induce acute pruritus by injecting SLIGRL at the same dose into TRPV3 KO mice (purchased from Jackson laboratories (Jackson Lab), usa), and as a result, the reduction of scratching response relative to wild type mice was comparable to the reduction when high concentrations of isoflavan-a and glycitein were used. Thus: the glycyrrhiza isoflavan nail and the glycyrrhetinic acid completely antagonize TRPV3 at high concentration, thereby achieving almost the same effect of directly knocking out TRPV3 in the aspect of inhibiting SLIGRL acute pruritus.

Chronic pruritus is an important complication of many skin diseases and systemic diseases, the existing treatment means in clinic are quite lacking, and even the scratching behavior can aggravate skin injury and inflammation, so that the patients can fall into a vicious circle with more scratchy and more scratchy, and the normal life and the disease rehabilitation process of the patients are seriously influenced. Skin diseases with chronic pruritus which are common at present include: chronic eczema, Atopic Dermatitis (AD), psoriasis, xeroderma, senile pruritus, etc. Studies have shown that TRPV3 function-enhancing mutations cause mice to develop dermatitis symptoms very similar to AD; however, the newly published studies show that TRPV3 regulates the secretion of inflammatory factors such as TSLP in keratinocytes through interaction with the PAR2 receptor protein, and is involved in mediating symptoms such as chronic pruritus in AD model mice induced by calcipotriol. Therefore, the calcipotriol-induced model of atopic dermatitis was chosen as an example for assessing the ability of TRPV3 antagonist molecules to inhibit chronic pruritus under physiological conditions.

Test example 5 Effect of Glycyrrhetini flavanoid Compound on mouse model of chronic itching

Selecting 22 mice (Spibefu (Beijing) laboratory animal science and technology limited) of 8-10 weeks old (20-25 g weight, half male and female) wild type C57BL/6J, and randomly dividing into 3 groups, namely, a liquorice isoflavan group, a glycyrrhetinic group and a model group, wherein the liquorice isoflavan group, the model control group are 7 mice respectively, and the glycyrrhetinic group is 8 mice;

the model of Atopic Dermatitis (AD) was made by dissolving calcipotriol in absolute ethanol, dropping the solution of calcipotriol into the auricle at both sides of the mouse, 20. mu.l per ear, 1 time per day, using a 20. mu.l pipette at a working concentration of 100. mu.M, and continuously inducing for 7 days.

To test the effect of the compounds in inhibiting chronic pruritus by antagonizing TRPV3, treatment was performed by superimposing (with vaseline as a base) 1% by mass of licoisoflavana or glycyrrhetin plaster (i.e., glycyrrhiza isoflavana group, glycyrrhetin group) while using calcipotriol to induce AD model, and the specific operation schedule of molding and administration treatment was as shown in fig. 8: starting 5 days before formal molding, 1 per thousand of the liquorice isoflavan nail or the liquorice cetidine plaster is smeared on the auricles of the ears of the mice (about 15mg per ear) once every day; in the model group, 1 per mill of DMSO plaster is smeared on the auricle of the ears of the mouse (about 15mg of each ear) once a day; during AD molding, the liquorice isoflavan group and the liquorice cetidine group are continuously smeared after the calcium potriol is administrated for half an hour every day, and the model group is coated with vaseline matrix after the calcium potriol is administrated for half an hour every day until 7 days after molding; with recordings made and binaural pinna thickness tested on

days

0, 4, and 7.

During video recording, mice are respectively placed into an observation box (15cm multiplied by 10cm) for adaptive activity for 5min, then a camera is used for video recording for 60min, attention is paid to keep the ambient environment quiet, video recording is reviewed afterwards, and scratching behaviors are counted in a time period of 5 min.

Results before AD modelling with calcipotriol formally (day 0), the total scratching times within 60min were very low for both experimental groups (glycyrrhiza isoflavan nail group, glycyrrhetinic group) and model groups, and were not significantly different (fig. 9A-B); after 4 days of model making, the mice have obvious chronic pruritus symptoms, at the moment, the total scratching times of the experimental group treated by 1 per thousand of the glycyrrhiza isoflavan nail and the glycyrrhetinic acid within 60min are only about half of that of the model group, and the differences are all significant (fig. 9C-D); after 7 days of modeling, the chronic pruritus symptoms of the mice are very serious, and the total scratching frequency of each experimental group within 60min is still lower than that of the model group, but the reduction degree of the experimental group treated by only 1 per thousand of the glycyrrhetinic acid reaches a remarkable level (fig. 9E-F). In addition, along with the appearance of AD molding process, except for chronic pruritus reaction, the inflammation of the mouse ear is continuously aggravated, which is intensively reflected by the continuous increase of the auricle thickness, as shown in figure 10, the average thickness of the mouse ear of 1 per thousand of the experimental groups of the isoflavan nail and the glycyrrhetinyl is obviously reduced compared with that of the model group on the 4 th and 7 th days, and the situation that the isoflavan nail and the glycyrrhetinyl have certain anti-inflammatory effect is prompted. It is noted that the significance of the differences in average thickness of the ears of mice in the experimental group of glycyrrhiza isoflavana compared to the model group tended to decrease over time, suggesting that the anti-inflammatory effect may be more limited and good effect could not always be ensured, compared to that of licoxidin, which is much stronger.

Test example 6 Effect of Glycyrrhiza isoflavan Compound on TRPV3 function-enhancing mutant

The glycyrrhiza isoflavane compound not only has an effect on TRPV3 wild type, but also has a blocking effect on a function enhancing mutant of TRPV3, so that the action range of the glycyrrhiza isoflavane compound is expanded.

1. Qualitative test

In previous studies it has been reported that function-enhancing mutants of TRPV3 (e.g. G573S) can induce cell death. Therefore, by using the Kit of Novozam Mut Express II Fast Mutagenesis Kit, the required site-directed mutation primers are designed according to the primer design principle as follows:

the upstream sequence: 5'-gtccatgagcatgtacagcgtcatgatccagaagg-3'

The downstream sequence: 5'-ctgtacatgctcatggactggaaaccccgcgta-3'

Plasmid pCMV6-AC-GFP-TRPV3 was point mutated according to the recommended procedures of the point mutation kit to obtain a plasmid with the G573S mutation.

Transfection was performed using the Lipofectamine 3000 transfection kit, using the protocol recommended in the kit. Transient transfection of TRPV 3G 573S mutant channel plasmid with GFP tag in HEK293T cell line, and adding antagonist ruthenium red (20 μ M), glycyrrhiza isoflavan nail (10 μ M or 20 μ M), and glycyrrhiza Citradine (10 μ M or 20 μ M) during transfection process; cell culture fluid was discarded after 12h of transfection, washed once with PBS and stained with Hoechst33342/PI apoptosis staining kit, after staining, many cells positive for PI staining could indeed be observed under a fluorescence microscope (FIG. 11), indicating massive cell death.

As can be seen from fig. 11: after the addition of the antagonist ruthenium red (20 μ M) at the same time as the transfection of the G573S mutant plasmid, the number of cells with positive PI staining became very small (see FIG. 11), indicating that it was difficult to induce cell death again once the overexpressed TRPV 3G 573S mutant channel was blocked by the antagonist;

when HEK293T cells which are added with 10 or 20 mu M of glycyrrhiza isoflavana and glycyrrhetinic acid at the same time and overexpress G573S mutant channels are subjected to PI staining, only a few cells with red fluorescent signals can be observed under a fluorescence microscope (as shown in figure 11), which indicates that the overexpressed TRPV 3G 573S mutant channels are blocked by the glycyrrhiza isoflavana and the glycyrrhetinic acid.

2. Quantitative test

In order to quantitatively test the effect of glycyrrhiza isoflavan and glycyrrhetin on TRPV 3G 573S mutant channel, TRPV 3G 573S mutant channel plasmid with GFP tag was transiently transfected in HEK293T cell line, antagonists ruthenium red (10. mu.M or 20. mu.M), glycyrrhiza isoflavan (10. mu.M or 20. mu.M), and glycyrrhetin (10. mu.M or 20. mu.M) were added during transfection, and then cell suspension mixed with the above compounds and transfection solution was seeded into 96-well plate for culture. After at least 12h of culture, the Cell Viability was characterized by luciferase Assay for ATP using the CellTiter-Glo luminescennt Cell Viability Assay kit.

When testing, 100. mu.l of mixed CellTiter-Glo test solution is added into each well, and the mixture is mixed on a shaking table for 2min to lyse cells; continuing to stand at room temperature for at least 10min to ensure the reaction is complete; and then reading by using a multifunctional microplate reader in a chemiluminescence mode.

As a result, it was found that similar to the effect of ruthenium red, the addition of 10 or 20. mu.M of both molecules simultaneously with the transfection of G573S mutant plasmid significantly increased the chemiluminescence intensity characterizing cell viability (see FIG. 12). The results of the qualitative and quantitative detection show that the isoflavan-A and the Liquiritidine have strong blocking effect on the TRPV3 function-enhancing mutant.

Example 1 Glycyrrhiza isoflavan nail ointment

Firstly, 240g of white vaseline, 160g of octadecanol and 40g of glyceryl monostearate are heated and melted in a water bath at the temperature of 80 ℃ to prepare an oil phase;

20g of sodium dodecyl sulfate, 140g of glycerol and 4g of 4% ethylparaben, and heating at 80 ℃ to dissolve the mixture in 500ml of distilled water to prepare a water phase;

slowly adding the water phase into the oil phase under stirring, condensing into emulsion matrix, adding 100g of radix Glycyrrhizae isoflavane, bottling, sterilizing, packaging, and making into ointment with 1000 pieces each containing 100mg of radix Glycyrrhizae isoflavane.

The amount of the added glycyrrhiza isoflavan nail can be 1-200g, and each prepared ointment contains 1-200mg of glycyrrhiza isoflavan nail.

Optionally replacing Glycyrrhrizae radix isoflavan with Glycyrrhrizae radix western-style injection to obtain Glycyrrhrizae radix western-style injection.

Example 2 Glycyrrhiza isoflavan nail ointment

Firstly, 300g (usually 10-500g) of white vaseline, 500g (usually 50-1000g) of paraffin, 2000g (usually 500-8000g) of liquid paraffin, 1000g (usually 100-2000g) of glyceryl monostearate and 8050 g (usually 5-100g) of span are heated and melted in a water bath at 80 ℃ to prepare an oil phase;

emulsifier OP 50g (usually 5-100g), 4% ethyl hydroxybenzoate 10g (usually 1-20g), heating at 80 deg.C and dissolving in 1000ml (usually 100-2000ml) distilled water to obtain water phase;

slowly adding the oil phase into the water phase, stirring while adding, condensing into emulsion matrix, adding 100g of radix Glycyrrhizae isoflavane, bottling, sterilizing, packaging, and making into ointment containing 1-200mg of radix Glycyrrhizae isoflavane per ointment.

Example 3 Glycyrrhiza isoflavanone ointment (preparation of oleaginous base ointment)

100g of isoflavane A, adding 3000g (usually 50-5000g) of liquid paraffin, stirring into paste, adding 5000g (usually 100-8000g) of vaseline, and grinding to uniformity. Filling, sterilizing, and making into ointment with 1000 pieces, each containing 100mg of compound Glycyrrhrizae radix isoflavane.

Example 4 Glycyrrhiza isoflavan nail ointment (preparation of aqueous base ointment)

Adding 200g (usually 30-300g) of CMC-Na into 5000ml (usually 100-8000ml) of ethanol, grinding to wet, adding 5000ml (usually 500-8000ml) of glycerol, continuously grinding to be uniform (no block), continuously adding 200ml (usually 50-300ml) of sodium benzoate aqueous solution (containing 20g (usually 5-30g) of sodium benzoate) while grinding, mixing uniformly, and swelling to obtain a water-soluble matrix; adding 100g of isoflavane A, mixing, filling and sterilizing. Making into ointment with 1000 pieces, each containing 100mg of compound Glycyrrhrizae radix isoflavane.

The above-described embodiments of the present invention are merely exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

1. Application of radix Glycyrrhizae isoflavane derivative in preparing medicine or health product for preventing, relieving or/and treating pruritus is provided.

2. The use according to claim 1, wherein the glycyrrhiza isoflavan derivative is one or more of glycyrrhiza isoflavan formazan, glycyrrhetin or derivatives with 3-phenylchroman as a core, or stereoisomers and pharmaceutically acceptable salts and/or solvates thereof, preferably glycyrrhiza isoflavan formazan and glycyrrhetin.

3. The use according to claim 1 or 2, wherein the medicament consists of a glycyrrhiza isoflavan derivative and a pharmaceutically acceptable carrier.

4. Use according to claim 1 or 2, wherein the medicament is in the form of tablets, capsules, pills, powders, granules, syrups, emulsions, injections, sprays, gels, creams, cataplasms, rubber patches.

5. The use according to claim 1 or 2, wherein the purity of the glycyrrhiza isoflavan derivative is not less than 1%.

6. A medicament or health product for preventing, alleviating or/and treating pruritus is characterized by containing glycyrrhiza isoflavan derivatives.

7. The pharmaceutical or nutraceutical according to claim 6, wherein the glycyrrhiza isoflavan derivative is one or more of glycyrrhiza isoflavan formazan, glycyrrhetin or derivatives with 3-phenylchroman as core, or stereoisomers and pharmaceutically acceptable salts and/or solvates thereof, preferably glycyrrhiza isoflavan formazan, glycyrrhetin.

8. The medicament of claim 6 or 7, wherein the ratio of the weight of the glycyrrhiza isoflavan derivative to the total weight of the medicament or health product is 0.01-10: 100.

9. the medicament according to claim 6 or 7, wherein the purity of the glycyrrhiza isoflavan derivative is not less than 1%.