CN114028384B

CN114028384B - Application of liquorice isoflavan derivative in preparation of drug for preventing, relieving or/and treating pruritus

Info

Publication number: CN114028384B
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: 2023-05-23
Anticipated expiration: 2041-02-24
Also published as: CN114028384A

Abstract

The invention discloses an application of a liquorice isoflavane derivative in preparing a drug or a health product for preventing, relieving or/and treating pruritus, and experiments show that the liquorice isoflavane derivative is an antagonistic molecule of TRPV3, takes TRPV3 as a target point, has obvious effects of preventing, relieving and treating pruritus, and particularly has obvious effects of treating acute pruritus and chronic pruritus which are not dependent on histamine; the glycyrrhiza isoflavane derivative has quick response to pruritus, small toxic and side effects, good safety and good medicinal prospect, is a novel TRPV3 antagonistic molecule, can serve as a lead compound, and is further used for researching and developing TRPV3 related disease treatment medicines. The invention also provides a new medicine source for preventing, relieving and treating skin itch.

Description

Application of liquorice isoflavan derivative in preparation of drug for preventing, relieving or/and treating pruritus

Technical Field

The invention relates to the field of medicines, relates to a medicine for preventing, relieving and/or treating pruritus, and in particular relates to application of a natural product liquorice isoflavane derivative component in traditional Chinese medicine liquorice in the medicine for preventing, relieving and/or treating pruritus.

Background

Licorice isoflavan compounds are natural products derived from traditional Chinese medicine licorice, previous researches have revealed that licorice has a wide range of pharmacological actions, and particularly licorice isoflavan compounds, the discovered actions include antibiosis, anticancer, antioxidation, anti-inflammatory and the like. On the basis of the existing research, the further excavation of the novel effect and the back molecular mechanism of the compounds has important research and application values.

Transient receptor potential channels (transient receptor potential ion channels, TRPs) are a class of non-selective cation channels that are widely expressed in many tissues and organs and are involved in regulating a variety of cellular functions. Based on amino acid sequence homology, TRP channels are divided into 7 subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPN (NompC), TRPP (polycystin) and TRPML (mucolipin). By mediating transmembrane flow of intracellular and extracellular cations, TRP channels play a key role in the absorption of cellular calcium ions, guidance of neuronal growth cones, growth and differentiation of keratinocytes, and transduction of sensory signals, among others. More importantly, a range of human diseases, including polycystic kidney disease, skeletal developmental deformity, olmsted syndrome, and the like, have been shown to be caused by dysfunctions of TRP channels.

For the TRPV subfamily, starting from the successful cloning of TRPV1, the first member, other members of the subfamily were also successively discovered and cloned. From the molecular sequence and structure point of view, the six member sequences of the TRPV subfamily are highly homologous and all have a molecular structure similar to that of classical voltage dependent potassium channels, i.e. are tetramers assembled from 4 subunits each comprising 6 transmembrane regions, and located at the intracellular N-terminal end comprising the ankyrin repeat structure and at the C-terminal end comprising the "TRP domain". Between the 5 th and 6 th transmembrane regions there is a ring structure, which constructs a channel through which ions pass and is believed to be related to the ion selectivity of the channel. As a non-selective cation channel, members of the TRPV subfamily all have a higher selective permeability for divalent cations, especially Ca ²⁺ The ions are of paramount importance. Since TRPV channels are regulated by temperature, pH and a range of endogenous, exogenous ligand molecules, they are widely involved in sensory information transmission in the body and have the ability to regulate intracellular Ca ²⁺ Balance, etc.

TRPV3 channel was cloned for the first time in 2002 and was found to activate at warm temperatures (31-39 ℃). Subsequent studies have found that more compounds can activate TRPV3, including 2-APB and natural molecules such as camphor, menthol, carvacrol, etc. TRPV3 channels are widely expressed in a variety of tissue organs, with various epithelial tissues being particularly important, mainly including the epidermis of the skin, mouth, and gastrointestinal tract. Previous studies on mice have found that TRPV3 knockout mice can exhibit, on the one hand, dysesthesia, abnormal hair curling, and impaired skin barrier; furthermore, after the TRPV3 is knocked out, the scratching reaction of mice is obviously reduced in acute pruritus induced by polypeptide SLIGRL and chronic pruritus of an atopic dermatitis model and a xeroderma model. On the other hand, transgenic mice expressing TRPV3 function enhancing mutants (p.g573s) exhibit severe dehairing and skin erythema, edema, erosion, desquamation, etc., accompanied by marked spontaneous itching behavior. More importantly, our previous studies found that the function-enhancing mutation of human TRPV3 resulted in a rare hereditary skin disease, olmsted syndrome, with the patient symptoms collectively manifested as severe keratinization and even progressive damage of the skin of hands and feet, as well as severe alopecia and pain itching, etc. These studies indicate that TRPV3 plays an important role in physiological processes such as skin barrier, hair development, temperature sensing, inflammatory response, pain and itch transduction; as an important target of skin drugs, especially its potential for treating itch, TRPV3 has become one of the most interesting TRP channels at present. However, because of the continued lack of effective research tools, including specific agonists and antagonists, etc., searching for and developing more drug molecules that regulate TRPV3 is a common focus of attention in order to address more important issues related to TRPV 3.

For functional studies of ion channels, the effect of a compound on it is evaluated, traditionally relying mainly on the detection of electrophysiological properties. The single-cell patch clamp recording technology is the most widely applied electrophysiological detection technology because of the advantages of direct and sensitivity, and particularly, the ion channel expressed on the cell membrane is connected into an annular circuit consisting of an amplifier, an electrode and bath liquid by utilizing high-impedance sealing formed by the glass microelectrode and the cell membrane surface, and when different stimulations are externally applied, the opening and closing conditions of the ion channel are reflected according to the current change in the circuit recorded under a certain manual clamping cell membrane potential.

Itching is a common unpleasant sensation that can induce a scratching response, similar to but different from pain, which often encourages people to try to avoid touching the affected area. The production process of itch is similar to pain sensation, and depends on the detection of various itch causing molecules by primary afferent neurons at nerve endings of peripheral organs such as skin and the like, external itch causing sources or endogenous itch causing molecules secreted by skin cells, immune cells and the like, the primary sensory neurons are firstly activated to generate nerve impulses, and the nerve signals are immediately transmitted into the dorsal horn of the spinal cord, relayed by the neurons in the spinal cord and finally transmitted to the sensory centers of the brain to generate itch sensation. It is believed that different antipruritics activate different groups of sensory neurons, and that the different antipruritic-induced pruritus responses can be broadly classified into two classes, histamine-dependent and non-histamine-dependent, depending on the most common antipruritic response to histamine. Meanwhile, according to the duration of the feeling of itching, the itching can be divided into two categories of acute itching and chronic itching, compared with the warning meaning of acute itching, the chronic itching which is continuous and debilitating, can occur with certain skin and systemic diseases, can also occur when the body is free of other obvious diseases, and becomes a purely annoying disease without obvious physiological significance.

As a common clinical symptom of dermatology, itch is accompanied by various diseases such as atopic dermatitis, eczema, psoriasis and the like, the nerve and molecular mechanism of histamine-dependent and non-histamine-dependent itch has been gradually revealed, and accordingly, medicines for treating skin itch have been developed, including antihistamines mainly aiming at histamine-dependent itch and cooling medicines capable of temporarily relieving itch and the like, which have limited scope of action or have great side effects, and antipruritic medicines aiming at non-histamine-dependent acute itch and chronic itch are lacking clinically, so that development of more antipruritic medicines with brand-new mechanism of action brings new hopes for people suffering from itch.

Disclosure of Invention

The invention aims to provide an application of a licorice isoflavane derivative in preparing a medicine for treating pruritus, which aims at overcoming the defects of the existing medicine for treating pruritus, in particular to an antipruritic medicine for treating acute pruritus and chronic pruritus which are not dependent on histamine and the technical defects existing in application, wherein the licorice isoflavane derivative is an antagonistic molecule of TRPV3, takes TRPV3 as a target point, and has remarkable treatment effect on acute pruritus and chronic pruritus which are not dependent on histamine.

In order to achieve the purpose of the invention, the invention provides an application of the liquorice isoflavane derivative in preparing medicines for preventing, relieving or/and treating pruritus.

Wherein the glycyrrhiza isoflavane derivative is one or more of glycyrrhiza iso Huang Wanjia, glycyrrhizin or derivatives taking 3-phenyl chromane as core, or stereoisomers thereof and pharmaceutically acceptable salts and/or solvates thereof, preferably glycyrrhiza iso Huang Wanjia and glycyrrhizin.

Wherein, the structures of the glycyrrhiza iso Huang Wanjia and the licocetin are as follows:

by utilizing an electrophysiological patch clamp recording technology, testing natural product compounds derived from traditional Chinese medicines in an HEK293T cell line which overexpresses TRPV3 channels, and screening to obtain the glycyrrhiza-i-Huang Wanjia and the glycyrrhiza-xidine which have the capacity of antagonizing TRPV3 in a dose-dependent manner.

Wherein the medicine consists of a liquorice isoflavane derivative and a pharmaceutically acceptable carrier.

In particular, the purity of the liquorice isoflavane derivative is more than or equal to 1%, preferably more than 95% or 95-100% pure substance.

In particular, pharmaceutically acceptable carriers are commonly accepted by health professionals for this purpose and as inactive ingredients of medicaments. A compilation of relevant pharmaceutically acceptable carriers can be found in the handbook of pharmaceutical excipients (Handbook of Pharmaceutical excipients, 2 nd edition, edited by A. Wade and P.J. Weller; american Pharmaceutical Association publication Washington and The Pharmaceutical Press, london, 1994) et al.

Wherein the pruritus is histamine-dependent or/and non-histamine-dependent pruritus, preferably non-histamine-dependent 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 containing the liquorice isoflavane derivative for preventing, relieving or/and treating pruritus.

In particular, the ratio of the weight of the licorice isoflavane derivative to the total weight of the drug is (0.01-10): 100, preferably (0.1-1): 100.

The medicament can be prepared into various dosage forms by methods known in the art, such as tablets, capsules, pills, powder, granules, syrups, solutions, injections, sprays, aerosols, gels, creams, tinctures, cataplasms, rubber plasters or patches and the like.

The present invention also provides a method for preventing, alleviating or/and treating pruritus, comprising administering to pruritus a therapeutically effective amount of an isoflavan derivative of licorice (licolor Huang Wanjia, licolor) in a therapeutically effective amount of 0.3 to 100mg/kg.d, preferably 1 to 20mg/kg.d, further preferably 10mg/kg.d.

The term "therapeutically effective amount" as used herein, unless otherwise indicated, is the amount of a drug that is required to produce an effective effect; the "therapeutically effective amount" is adjustable and variable, ultimately determined by the medical practitioner, and factors considered include 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 discovers a new medicinal value for the known compound liquorice isoflavane derivatives (liquorice iso Huang Wanjia and liquorice cetirizine), is used for preventing, relieving and treating pruritus, and can be prepared into medicines for preventing, conditioning and/or treating pruritus, thereby developing a new field for the application of natural product compounds in Chinese medicinal materials liquorice and liquorice.

2. The licorice isoflavane derivative (licorice iso Huang Wanjia, licorice sibiridine) has clear pharmacological action, obvious efficacy for preventing, relieving and treating pruritus, quick response, small toxic and side effects, good safety and good medicinal prospect.

3. The product of the invention has the advantages of abundant 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 isoflavane derivative (glycyrrhiza iso Huang Wanjia, glycyrrhiza cetirizine) is a new TRPV3 antagonistic molecule, can be used as a lead compound, and is further used for the research and development of TRPV3 related disease treatment medicines.

The invention adopts a mouse model of non-histamine-dependent acute pruritus established by polypeptide SLIGRL induction, and tests the capacity of liquorice iso Huang Wanjia and licoxidine for inhibiting acute pruritus by counting the scratching times within 30 minutes as a behavioral detection index, so that the results show that the glycyrrhiza iso Huang Wanjia and the licoxidine can both inhibit the acute pruritus induced by SLIGRL in a dose-dependent manner.

The ability of licorice iso Huang Wanjia and licoxidine to inhibit chronic pruritus was tested by counting the number of scratches per hour at different modeling days as an inspection index using a calcipotriol-induced established chronic pruritus mouse model of atopic dermatitis, and the results indicate that they can both inhibit chronic pruritus of the atopic dermatitis model mouse to some extent.

The invention obtains a new antagonistic molecule based on target molecule TRPV3 screening, the antagonistic molecule shows the activity of inhibiting acute pruritus and chronic pruritus which are not dependent on histamine, and can be used as a lead compound for further developing pruritus treatment medicines.

Drawings

FIG. 1 is a graph showing antagonism of 2-APB induced TRPV3 currents by different concentrations of licorice iso Huang Wanjia, wherein A is a representative graph recording TRPV3 current changes in whole cell mode, including changes when 100 μM 2-APB stimulation is administered alone, and when multiple concentrations (30, 100, and 300 μM) of licorice iso Huang Wanjia are simultaneously superimposed; b is a dose response relation diagram of liquorice iso Huang Wanjia antagonizing TRPV3 channel, which is calculated by fitting an S-type dose response curve, and the IC of the liquorice iso Huang Wanjia ₅₀ The value was 20.87. Mu.M (LogIC ₅₀ Data corresponding to each concentration are expressed as mean ± SEM, =1.320±0.128, n=4-5;

FIG. 2 is a graph of the antagonistic effect of varying concentrations of licolor on 2-APB-induced TRPV3 currents, wherein A is a representative graph recording TRPV3 current changes in whole cell mode, including changes in licolor timing when 100 μM 2-APB stimulation was administered alone, and when multiple concentrations (10, 30, and 100 μM) were simultaneously superimposed; b is a dose response relation diagram of the glycyrrhizin antagonism TRPV3 channel, which is calculated by fitting an S-type dose response curve, and the IC of the glycyrrhiza-I Huang Wanjia ₅₀ The value was 15.24. Mu.M (LogIC ₅₀ Data corresponding to each concentration are expressed as mean ± SEM, =1.183±0.120, n=5-8;

FIG. 3 is a representative graph of TRPV3 current change recorded in the in-membrane outward mode, including current change when 100 μM 2-APB stimulation alone was administered, and when different concentrations of licorice iso Huang Wanjia and licocetin were simultaneously superimposed, wherein A is licorice iso Huang Wanjia; b is licoxidine; the final 10 μm ruthenium red can completely block TRPV3 current.

FIG. 4 is a graph showing the effects of glycyrrhizin Huang Wanjia and glycyrrhizin on TRPV1 and TRPA1 channels, respectively; wherein: A. b is a representative curve of TRPV1 current changes recorded in whole cell mode, including current changes when TRPV1 agonist capsaicin is administered alone and when 100 μm of licorice iso Huang Wanjia (a) or licocetin (B) is added simultaneously, the final non-selective TRP channel antagonist ruthenium red can block current completely; C. d is a representative curve of TRPA1 current changes recorded in whole cell mode, including changes when TRPA1 agonist AITC is administered alone and when 100 μm of licorice iso Huang Wanjia (C) or licocetin (D) is added simultaneously, the final non-selective TRP channel antagonist ruthenium red can block current completely; E. f: glycyrrhiza iso Huang Wanjia (E) and licocetin (F) block the TRPV3, TRPV1 and TRPA 1. Unpaired Student's t was used for the test, P <0.001. Each of the above groups of data is expressed as mean ± SEM, n=4-8.

FIG. 5 shows the inhibition of SLIGRL-induced acute pruritus by Glycyrrhrizae radix Heterol Huang Wanjia; wherein A-C: line graphs of statistics of scratching times for a period of 5min for the experimental groups with 30, 100 and 300 μm of liquorice isoflavane nail effect and the corresponding control group mice without drug treatment respectively, by using variance analysis of repeated measurement, P >0.05 (a-B), P <0.01 (C); d is a bar graph of total number of scratches in 30min for the experimental group with 30, 100 and 300. Mu.M glycyrrhizic isoflavane nail effect and the corresponding control group mice. P <0.05, P <0.01, n.s.: P >0.05, each set of data expressed as mean ± SEM, n=8;

FIG. 6 shows the inhibition of SLIGRL-induced acute pruritus by licoxidine; a-C: 30. line graphs of the statistics of scratching times of the mice of the experimental groups with 100 and 300 mu M of glycyrrhizin and the mice of the corresponding control group according to a period of 5min are adopted, and repeated measurement analysis of variance is adopted, wherein P is more than 0.05 (A-B), and P is less than 0.05 (C); d: 30. bar graph of total number of scratches in 30min for the 100 and 300 μm glabridin acting experimental and corresponding control mice. P <0.01, n.s.: P >0.05, each set of data expressed as mean ± SEM, n=8, using paired Student's t test;

FIG. 7 is a graph of percentage control of SLIGRL acute pruritus versus wild type for TRPV3 KO mice versus percentage inhibition of SLIGRL induced acute pruritus by SLIGRL with varying concentrations of Glycyrrhrizae radix iso Huang Wanjia and Glycyrrhrizae radix cetirizine; the bar graphs are the ratio of total number of scratches in 30min for the experimental group and the corresponding control group mice superimposed with 30, 100 and 300 μm of glycyrrhiza-iso Huang Wanjia and glycyrrhizin, respectively, and the ratio of total number of scratches in 30min for TRPV3 KO mice and wild mice, the above data are expressed as mean ± SEM, n=5-8.

FIG. 8 is a schematic of the operational flow of induction of AD model mice with calcipotriol and treatment with TRPV3 antagonists, with the dotted arrow above the time line indicating the number of days of binaural molding of mice with calcipotriol and the black arrow below indicating the number of days of 1% of licorice i Huang Wanjia or licoxidine applied to the ears of mice, recorded at

days

0, 4, 7, prior to molding and dosing;

FIG. 9 shows the chronic itch response of calcipotriol-induced AD model mice at various test time points; wherein:

a, C, E: a line graph of the number of scratching times counted by 5min for a period of time of mice of an experimental group with 1 permillage of liquorice iso Huang Wanjia and liquorice cetirizine added on

days

0, 4 and 7 and a model group serving as a control respectively; analysis of variance with repeated measurements, P <0.05;

B, D, F: a bar graph of total number of scratches in 60min for mice of experimental group and model group as control, which were superimposed with 1% of the effects of glycyrrhiza iso Huang Wanjia and glycyrrhizin on

days

0, 4 and 7, respectively; comparison with the control group using unpaired Student's t test, P <0.05, n.s.: P >0.05. Each of the above groups of data is expressed as mean ± SEM, n=7-8;

FIG. 10 is a bar graph of mean ear thickness of mice from the experimental group with 1% of the effects of licorice i Huang Wanjia and glycyrrhizin superimposed on the experimental group without drug treatment on

days

0, 4, and 7; comparison with control group using unpaired Student's t test, P <0.01, P <0.001. Each of the above groups of data is expressed as mean ± SEM, n=7-8;

FIG. 11 is a graph showing the results of observation of HEK293T cells after various treatments under a fluorescence microscope; each row corresponds to an image taken of a typical field of view under white (bright field), green Fluorescent (GFP) and red fluorescent (PI-stained) channels, respectively, with the left hand label. HEK293T cells after transient transfection of TRPV 3G 573S mutant plasmid show GFP green fluorescence and strong red fluorescence signal (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 weak; while adding different concentrations (10 and 20 mu M) of liquorice iso Huang Wanjia and liquorice cetirizine, the red fluorescent signals are weak, and the scale is 100 mu M;

FIG. 12 shows transient transfection of TRPV 3G 573S mutation channels into HEK293T cells under equivalent conditions, and the chemiluminescent intensities obtained by the addition of different concentrations (10 and 20. Mu.M) of licorice iso Huang Wanjia, licolor and ruthenium red, respectively, and the cell viability without any addition of antagonists; the unpaired Student's t test was used to compare P <0.01 and P <0.001 to the control group without any antagonist. Each of the above groups of data is expressed as mean ± SEM, n=5.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

The beneficial effects of the medicaments according to the invention are further illustrated below by test examples, which include pharmacodynamic tests of the medicaments according to the invention.

The liquorice iso Huang Wanjia is white powder, is produced by Yunnan Sili corporation, and has the purity of 97 percent by measuring the area normalization method of an ultraviolet detector and an evaporative light scattering detector of two detectors of high performance liquid chromatography. Lot number: BBP03331;

Liquiritidine: white powder, produced by Yunnan Sili corporation, has a purity of 98% as measured by the area normalization method of the ultraviolet detector and the evaporative light scattering detector of the two detectors of high performance liquid chromatography. Lot number: BBP03353.

The preparation method of the licorice isoflavan molecules of the present invention is not particularly limited, and a person skilled in the art may prepare the licorice isoflavan molecules of the present invention according to common general knowledge in the art, or obtain the licorice isoflavan molecules (licorice iso Huang Wanjia and licoxetine) of the present invention by commercial use.

Test example 1 Glycyrrhiza isoflavane compounds acting on TRPV3

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

HEK293T cell line was purchased from ATCC biological resource center in the United states, resuscitated and inoculated into DMEM complete medium at saturation humidity and 5% CO ₂ Culturing in a constant temperature incubator at 37 ℃.

Before transfection, HEK293T cells were seeded into 6-well plates and eukaryotic expression plasmid pCMV6-AC-GFP-TRPV3 (origin, usa) containing human TRPV3 cDNA was transfected using Lipofectamine 3000 transfection reagent at a cell density of 50-70%.

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

HEK293T cells transfected with TRPV3 plasmid for 16-24h are placed in specially prepared bath liquid (130mM NaCl,3mM HEPES,0.3mM EGTA,pH =7.4), cells which are in a good state and have clean surfaces and strong green fluorescent signals are selected, then an electrode added with internal liquid (the components of which are the same as the bath liquid) is slowly moved to slightly contact with the surface of a cell membrane, slightly negative pressure is applied until a high-resistance sealing and then rupture of the membrane are formed, a whole cell recording mode is formed, or the electrode is directly lifted upwards after the high-resistance sealing is formed, so that a membrane at the tip of the electrode is torn off, and the inner surface of the membrane faces outwards.

1) In whole-cell recording mode, using RSC-200 rapid exchange drug delivery system gravity perfusion, firstly, 100 μM2-APB is given to activate TRPV3 channel, and then different concentrations (300 μM, 100 μM, 30 μM) of licorice iso Huang Wanjia are respectively superimposed; or different concentrations (100. Mu.M, 30. Mu.M, 10. Mu.M) of licocetin, a significant decrease in current amplitude was observed. Wherein as the concentration of added liquorice isoflavane A increases, the current decrease amplitude is continuously increased until the current is almost completely disappeared finally (as shown in figure 1A); the test results for licocetin were also similar, i.e. in whole cell mode, the superposition of licocetin reduced the 2-APB induced current, and the magnitude of the current reduction exhibited dose dependency (as in fig. 2A).

By measuring the corresponding antagonistic capacity of glycyrrhizic acid Huang Wanjia, glycyrrhizin to TRPV3 current at a plurality of concentrations and fitting with S-type dose response curves, it is possible to obtain: IC of licorice root iso Huang Wanjia ₅₀ IC of licolor with 20.87 μm (as shown in FIG. 1B) ₅₀ The value was 15.24. Mu.M (see FIG. 2B).

2) In the membrane inside-out recording mode, i.e. only the current of the channel on a single membrane was recorded, it was also observed that glycyrrhiza-i Huang Wanjia and glycyrrhiza could decrease the 2-APB induced current dose-dependently (as in fig. 3), indicating that their ability to inhibit 2-APB current is independent of the assistance of intracellular molecules and should act directly on TRPV3 channel itself.

Test example 2 Glycyrrhiza isoflavane compounds acting on TRPV1 or TRPA1

The effect of compounds on overexpressed human TRPV1 and TRPA1 was evaluated using the cell patch clamp method.

Before transfection, HEK293T cells were seeded into 6-well plates and eukaryotic expression plasmid pCMV6-AC-GFP-TRPV1 (origin, usa) or eukaryotic expression plasmid pCMV6-AC-GFP-TRPA1 (origin, usa) containing human TRPA1cDNA was transfected using Lipofectamine 3000 transfection reagent at a cell density of 50-70%.

The electrophysiological detection method adopts a whole cell or membrane inner-surface outward recording mode, adopts HEKAEPC10 type amplifier and PatchMaster recording software, uses an electrode with impedance of 3-5MΩ drawn by a P-97 type microelectrode drawing instrument, and records current with + -80 mV square wave voltage (cell clamp potential is 0 mV).

HEK293T cells transfected with TRPV1 or TRPA1 plasmid for 16-24h are placed in specially prepared bath liquid (130mM NaCl,3mM HEPES,0.3mM EGTA,pH =7.4), cells which are in a good state and have clean surfaces and strong green fluorescent signals are selected, then an electrode added with internal liquid (the components of which are the same as the bath liquid) is slowly moved to slightly contact with the surface of a cell membrane, slightly negative pressure is applied, and a membrane is broken after high-resistance sealing is formed, so that a whole cell recording mode is formed.

By recording the effect of 100 μm licorice i Huang Wanjia on TRPV1 agonist capsaicin or TRPA1 agonist AITC induced currents in HEK293T cells overexpressing TRPV1 or TRPA1 in vitro using electrophysiological detection methods, licorice i Huang Wanjia had a weaker blocking effect on TRPV1 (blocking rate of 31.9% ± 6.6%, fig. 4A), whereas little antagonism was observed on TRPA1 (3.3% ± 15.5%, fig. 4C), both significantly lower than its effect on antagonism of TRPV3 (88.0% ± 7.5%, fig. 4E), suggesting that licorice i Huang Wanjia has better specificity for TRPV3 channels. The effect of 100. Mu.M of licocetin on TRPV1 and TRPA1 currents was also examined using a similar electrophysiological test method, and the degree of blockade of licocetin on TRPV1 and TRPA1 (TRPV 1:52.2% + -10.1%, FIG. 4B; TRPA1:8.4% + -10.8%, FIG. 4D) was also significantly lower than the degree of antagonism of TRPV3 (93.3% + -5.8%, FIG. 4F), indicating that licocetin also has a certain specificity of action on TRPV3 channels.

For animal models of itch, the standard acute itch model uses intradermal injection of an antipruritic agent into the back of the mouse neck to generate a itching response, and the extent of its itching is characterized by counting the number of scratches in a certain time after injection, wherein the scratching injection site is lifted from the hindfoot of the mouse until the hindfoot falls to the ground again or is put into the mouth to lick as a scratching behavior.

Common itch-causing agents include histamine, chloroquine, 5-hydroxytryptamine, compound 48/80, polypeptide SLIGRL, and the like. Since the recently published studies indicate that the scratching response of mice after TRPV3 knockout is significantly reduced for acute itching induced by intradermal injection of the activator SLIGRL of PAR 2. Thus, the SLIGRL acute itch model was chosen as an example of evaluating the effect of TRPV3 antagonistic molecules on inhibiting acute itch effects under physiological conditions.

Test example 3 Effect of Glycyrrhiza Isodon Huang Wanjia on acute itching model in mice

The purchased wild C57BL/6J mice (Si Bei Fu (Beijing) experimental animal science, inc.) of 8-10 weeks old (weight 20-25g, sexes half) were randomly selected and 24 of them were divided into 3 groups, namely into a liquorice iso Huang Wanjia high dose group (300. Mu.M), a medium dose group (100. Mu.M) and a low dose group (30. Mu.M), wherein 8 of each group were removed from the hair on the back of the neck of the mice at least 2-3 days in advance;

On the day of the formal experiment, mice are respectively placed into observation boxes (15 cm multiplied by 10 cm) for adaptive activity for about 5min; then, 50 mu l of physiological saline containing different concentrations (30 mu M, 100 mu M and 300 mu M) of liquorice iso-Huang Wanjia is pre-injected in the back skin of the right ear, 50 mu l of physiological saline containing 50 mu g of SLIGRL and corresponding concentration of liquorice iso-Huang Wanjia (namely, the physiological saline comprises SLIGRL and also comprises liquorice iso-Huang Wanjia) is continuously injected at the same position after 30min, wherein the concentration of liquorice iso-Huang Wanjia respectively corresponds to the concentration of the pre-injection, namely, SLIGRL+liquorice iso-Huang Wanjia) to induce acute pruritus; immediately placing the injected mice into an observation box, keeping quiet condition, recording the video for 30min by a camera and looking back at the video afterwards, and counting scratching behaviors by taking 5min as a time period.

50 μl of physiological saline containing 0.1% DMSO (solvent) was pre-injected in the back skin of the left ear every other day as a control group (not administered), and 50 μl of physiological saline containing 50 μg SLIGRL (also containing 0.1% DMSO) was continuously injected at the same place after 30min to induce acute pruritus; immediately placing the injected mice into an observation box, keeping quiet condition, recording the video for 30min by a camera and looking back at the video afterwards, and counting scratching behaviors by taking 5min as a time period.

As a result, it was found that the total number of scratches within 30min after injection of SLIGRL was decreased to a different extent in the mice with 30, 100 or 300. Mu.M licorice i-Huang Wanjia superimposed, compared to the control group without administration (i.e., the SLIGRL alone on the left ear side, i.e., the SLIGRL+DMSO group) (FIG. 5D).

However, the difference between the 30 μm experimental group and the control group did not reach a significant level (p= 0.1047), and as the acting concentration increased to 100 and 300 μm, the level of total scratching times decreased within 30min in the mice of the experimental group became more and more significant, suggesting that the ability of licorice i Huang Wanjia to inhibit SLIGRL-induced acute pruritus increased with increasing concentration. It is noted that the line graph of the number of statistical scratches in time period is shown (see fig. 5A-C): the different concentrations of licorice i Huang Wanjia all caused the scratching times of the mice of the experimental group to decay more rapidly with time, while when the concentration of the superimposed licorice i-flavane was sufficiently high (300 μm), the scratching times of the mice of the experimental group were far lower than that of the control group without administration from the beginning, and the P value calculated by using the variance analysis of repeated measurement was also less than 0.01, which showed that licorice i Huang Wanjia had an inhibitory effect on the acute pruritus of SLIGRL from the other side.

Test example 4 effect of Liquiritidine on acute itching model in mice

The purchased wild C57BL/6J mice (Si Bei Fu (Beijing) experimental animal science, inc.) of 8-10 weeks old (weight 20-25g, male and female halves) were randomly selected and 24 of them were divided into 3 groups, namely, a high dose group (300. Mu.M), a medium dose group (100. Mu.M) and a low dose group (30. Mu.M) of glycyrrhizin, wherein 8 of each group were removed from the hair on the back and neck of the mice at least 2-3 days in advance;

test example 3 was repeated except that 50 μl of physiological saline containing different concentrations (30 μΜ, 100 μΜ, 300 μΜ) of licocetin was injected intradermally into the posterior right ear before the final test, and 50 μl of physiological saline containing 50 μg of sligarl was injected at the same position after 30min and the corresponding concentration of licocetin was superimposed (i.e., the physiological saline included both in sligarl and also including licocetin), wherein the concentration of licocetin corresponds to the concentration at the time of the preliminary injection, i.e., sligarl+licocetin, respectively, to induce acute pruritus.

As a result, it was found that, for the mice of the experimental group in which 30, 100 or 300 μm of licocetin was used in superposition, a different degree of decrease in total number of scratches was also observed within 30 minutes as compared with the control group without administration (i.e., the left ear side injected with only SLIGRL, i.e., the sligrl+dmso group) (fig. 6D). The difference between the 30 μm experimental group and the control group did not reach a significant level (p=0.0523), but after the working concentration was increased to 100 and 300 μm, the total number of scratching times in the mice of the experimental group was significantly lower than that of the control group within 30min, which means that the licocetin had the same effect as that of licolor Huang Wanjia, and also could effectively inhibit the acute itching reaction induced by SLIGRL. The number of scratching counted in time intervals also gave similar results to licorice i Huang Wanjia (see fig. 6A-C): the number of scratches in each experimental group tended to decay more rapidly with time, whereas the difference between the two groups reached a significant level (P < 0.05) when the superimposed concentration of licoxidine was increased to 300 μm. The above results all demonstrate that licoxidine is equally effective in inhibiting SLIGRL-induced acute pruritus.

By calculating the ratio of total number of scratches in 30min after injection of SLIGRL in the mice of the different experimental groups to the mice of the respective control groups, it was observed that the inhibition effect of glycyrrhiza-i Huang Wanjia and glycyrrhiza-xidine was stronger and similar dose dependency was exhibited as the concentration of the effect was increased (fig. 7).

Considering that TRPV3 KO mice (TRPV 3 knockout mice) have been reported to have significantly lower acute pruritus induced by sligar than wild type, as a control on the other hand, it was also attempted to inject the same dose of sligar on TRPV3 KO mice (purchased from Jackson laboratory (Jackson Lab), usa) to induce acute pruritus, and as a result, the decrease in the scratching response relative to wild type mice was comparable to those obtained with high concentrations of licorice iso Huang Wanjia and licocetin. Thus: glycyrrhiza iso Huang Wanjia and glycyrrhizin have completely antagonized TRPV3 at high concentrations, thus achieving nearly identical effect as direct TRPV3 knockout in inhibiting SLIGRL acute pruritus.

Chronic itch, which is an important complication of many skin and systemic diseases, is quite lacking in clinically available therapeutic means, even because scratching tends to aggravate skin damage and inflammation, thus sinking into the vicious circle of itching and scratching, seriously affecting the normal life and disease rehabilitation process of patients. Skin disorders that are currently common with chronic itching include: chronic eczema, atopic Dermatitis (AD), psoriasis, xeroderma, senile pruritus, etc. Studies have shown that TRPV3 function-enhancing mutations can lead to mice exhibiting dermatitis symptoms very similar to AD; the newly published studies prove that TRPV3 regulates secretion of inflammatory factors such as TSLP in keratinocytes through interaction with PAR2 receptor proteins, and further participates in mediating symptoms such as chronic pruritus of AD model mice induced by calcipotriol. Thus, calcipotriol-induced atopic dermatitis model was chosen as an example of an assessment of the ability of TRPV3 antagonistic molecules to inhibit chronic itch under physiological conditions.

Test example 5 Effect of Glycyrrhiza Isoflavanoid Compounds on mouse model of chronic itch

The purchased wild C57BL/6J mice (Si Bei Fu (Beijing) experimental animal science, inc.) with the age of 8-10 weeks (weight of 20-25g, sexes half) are randomly divided into 3 groups, namely into a liquorice isoflavane group, a liquorice western medicine group and a model group, wherein the liquorice isoflavane group and the model control group are respectively 7, and the liquorice western medicine group is 8;

calcipotriol was dissolved in absolute ethanol, and the calcipotriol solution was pipetted into auricles on both sides of a mouse using a 20 μl pipette at a working concentration of 100 μM, 20 μl per ear, 1 time per day, and continuously induced for 7 days for Atopic Dermatitis (AD) molding.

To test the effect of compounds to inhibit chronic itch by antagonizing TRPV3, while inducing AD model with calcipotriol, 1% mass fraction of glycyrrhiza i Huang Wanjia or glycyrrhiza xidine plaster was superimposed (with petrolatum as matrix) for treatment (i.e. glycyrrhiza isoflavan methyl group, glycyrrhiza xidine group), and the specific operation schedule of modeling and administration treatment is shown in fig. 8: starting 5 days before formal molding, glycyrrhrizae radix isoflavane A group and Glycyrrhrizae radix cetirizine A group apply Glycyrrhrizae radix iso Huang Wanjia or Glycyrrhrizae radix cetirizine plaster 1 millto mouse binaural auricle (about 15mg per ear) once each day; the model group smears 1%o DMSO plaster on mouse binaural auricles (about 15mg per ear) once a day; in AD molding, the liquorice isoflavane methyl group and the liquorice cetirizine group continue to be smeared after being dosed with calcipotriol for half an hour every day, and the model group is coated with Vaseline matrix after being dosed with calcipotriol for half an hour every day until 7 days of molding are finished; wherein recordings were made and binaural auricle thickness was tested on

days

0, 4, 7.

When in video recording, the mice are respectively placed into (15 cm multiplied by 10 cm) observation boxes, after adaptive activity is carried out for 5min, the video is recorded for 60min by a camera, the surrounding environment is kept quiet, the video is looked back after that, and the scratching behavior is counted by taking 5min as a time period.

Results before AD molding with calcipotriol formally (day 0), the total number of scratches in the experimental group (licolor a group, licolor cinidim group) and model group for 60min were very low, with no significant difference (fig. 9A-B); after 4 days of modeling, the mice developed obvious chronic itching symptoms, at which time the total number of scratching times in 60min was about half that of the model group for the experimental group treated with 1%o licorice iso Huang Wanjia and glycyrrhizin, and the differences were all significant (fig. 9C-D); the chronic itching symptoms of the mice were very severe after 7 days of molding, and the total number of scratches in each experimental group was still lower than that in the model group within 60min, but only 1% of the treatments with glycyrrhizin were reduced to a significant level (fig. 9E-F). In addition, along with the AD modeling process, besides chronic pruritus reaction, the ear inflammation of the mice is continuously aggravated, and the concentration is represented by the continuous increase of auricle thickness, as shown in fig. 10, on

days

4 and 7, the average thickness of the ears of the mice in the experimental groups of 1 milllicorice iso Huang Wanjia and licorice cetirizine is obviously reduced compared with that of the model group, which indicates that licorice iso Huang Wanjia and licorice cetirizine have a certain anti-inflammatory effect at the same time. It is noted that the average thickness of the ears of the mice in the experimental group of glycyrrhiza-i Huang Wanjia tends to decrease over time compared with the difference in the model group, suggesting that the anti-inflammatory effect may be limited and the good effect cannot be ensured all the time, compared with the effect of the anti-inflammatory effect of the glycyrrhizin is stronger.

Test example 6 Effect of Glycyrrhiza isoflavane Compounds on TRPV3 function enhancing mutants

The licorice isoflavone compound disclosed by the invention not only has an effect on a TRPV3 wild type, but also has a blocking effect on a function enhancement mutant of TRPV3, so that the action range of the licorice isoflavone compound is widened.

1. Qualitative test

In previous studies, it has been reported that a functionally enhanced mutant of TRPV3 (e.g., G573S) can induce cell death. For this, the nuo vozan Mut Express II Fast Mutagenesis Kit kit was used to design the desired site-directed mutagenesis primer according to the primer design principle as follows:

upstream sequence: 5'-gtccatgagcatgtacagcgtcatgatccagaagg-3'

Downstream sequence: 5'-ctgtacatgctcatggactggaaaccccgcgta-3'

The plasmid pCMV6-AC-GFP-TRPV3 was subjected to point mutation according to the recommended procedure of the point mutation kit, to obtain a plasmid having G573S mutation.

The transfection treatment was performed using the Siemens Lipofectamine 3000 transfection kit according to the procedure recommended for the kit. Transiently transfecting a TRPV 3G 573S mutant channel plasmid with GFP tag in an HEK293T cell line, wherein during the transfection process, antagonists ruthenium red (20 mu M), liquorice iso Huang Wanjia (10 mu M or 20 mu M) and liquorice cetirizine (10 mu M or 20 mu M) are respectively and simultaneously added when a transfection solution is added; after 12h of transfection, the cell culture broth was discarded, washed once with PBS and stained with Hoechst33342/PI apoptosis staining kit, after which a number of cells positive for PI staining were indeed observed under a fluorescence microscope (FIG. 11), indicating massive cell death.

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

PI staining of HEK293T cells with 10 or 20 μm of glycyrrhiza-iso Huang Wanjia and glycyrrhizadine over-expressed G573S mutation channels added simultaneously at the time of transfection, less cells with red fluorescent signal was observed under fluorescent microscope (as shown in fig. 11), indicating that the over-expressed TRPV 3G 573S mutation channels were blocked by glycyrrhiza-iso Huang Wanjia and glycyrrhizadine.

2. Quantitative test

To quantitatively test the effect of licolor Huang Wanjia, licolor on TRPV 3G 573S mutant channel, GFP-tagged TRPV 3G 573S mutant channel plasmids were transiently transfected in HEK293T cell lines, and antagonists ruthenium red (10 μm or 20 μm), licolor Huang Wanjia (10 μm or 20 μm), and licolor (10 μm or 20 μm) were added during transfection, respectively, followed by culturing the cell suspensions mixed with the above compounds and transfection solutions in 96 well plates. After at least 12h of incubation, the cell viability was characterized by luciferase assay using CellTiter-Glo Luminescent Cell Viability Assay kit.

In the test, 100 mu l of mixed CellTiter-Glo test solution is added into each hole, and the mixture is uniformly mixed on a shaking table for 2min to lyse cells; continuing to stand at room temperature for at least 10min to ensure that the reaction is complete; and then reading by using a multifunctional enzyme-labeled instrument 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 at the same time as the transfection of the G573S mutant plasmid significantly improved the chemiluminescent intensity characteristic of cell viability (see FIG. 12). Together with the qualitative and quantitative detection results, it is demonstrated that glycyrrhizic acid Huang Wanjia and licocetin have strong blocking effect on TRPV3 function enhancing mutants.

EXAMPLE 1 Glycyrrhiza Iso Huang Wanjia ointment

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

20g of sodium dodecyl sulfate, 140g of glycerin, 4g of 4% ethyl hydroxybenzoate, and dissolving in 500ml of distilled water at 80 ℃ to prepare a water phase;

slowly adding the water phase into the oil phase under stirring, condensing into emulsion matrix, adding Glycyrrhrizae radix I Huang Wanjia g, packaging, sterilizing, and packaging to obtain 1000 ointment containing Glycyrrhrizae radix I Huang Wanjia mg.

The amount of Glycyrrhrizae radix I Huang Wanjia can be 1-200g, and each ointment contains Glycyrrhrizae radix I Huang Wanjia-200 mg.

It can also be used to replace Glycyrrhrizae radix different Huang Wanjia to make Glycyrrhrizae radix xidine ointment.

EXAMPLE 2 Glycyrrhiza Iso Huang Wanjia ointment

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

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

slowly adding the oil phase into the water phase, stirring while condensing into emulsion matrix, adding Glycyrrhrizae radix I-Huang Wanjia g, packaging, sterilizing, and packaging to obtain 1000 ointment containing Glycyrrhrizae radix I-Huang Wanjia-200 mg.

Example 3 preparation of Licorice root Is Huang Wanjia ointment (preparation of oleaginous base ointment)

Huang Wanjia 100g of licorice is added into 3000g (usually 50-5000 g) of liquid paraffin, stirred into paste, added into 5000g (usually 100-8000 g) of Vaseline, and ground until uniform. Filling, sterilizing, and making into 1000 ointments containing Glycyrrhrizae radix of Huang Wanjia mg each.

Example 4 preparation of Glycyrrhiza Isonica Huang Wanjia ointment (aqueous base ointment)

Adding CMC-Na 200g (30-300 g) into ethanol 5000ml (100-8000 ml), grinding to wet, adding glycerol 5000ml (500-8000 ml), grinding to uniformity (no block), adding sodium benzoate 200ml (50-300 ml) (20 g (5-30 g) sodium benzoate), grinding, mixing, and swelling to obtain water-soluble matrix; adding Glycyrrhrizae radix I Huang Wanjia g, mixing, packaging, and sterilizing. 1000 ointments containing Glycyrrhrizae radix of Huang Wanjia mg each are prepared.

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 and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Claims

1. The application of the liquorice isoflavone derivative as the only active effective component in preparing the medicine for preventing, relieving or/and treating the acute pruritus or the chronic pruritus which are not dependent on histamine is provided, wherein the liquorice isoflavone derivative is liquorice iso Huang Wanjia and liquorice cetirizine.

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

3. The use according to claim 1 or 2, wherein the medicament is in the form of a tablet, capsule, pill, powder, granule, syrup, emulsion, injection, spray, gel, cream, cataplasma, or rubber patch.

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