CN117899098A - Application of rare ginsenoside CMx in preparation of medicine for preventing and/or treating type 2 inflammatory diseases - Google Patents
Application of rare ginsenoside CMx in preparation of medicine for preventing and/or treating type 2 inflammatory diseases Download PDFInfo
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- CN117899098A CN117899098A CN202410312190.4A CN202410312190A CN117899098A CN 117899098 A CN117899098 A CN 117899098A CN 202410312190 A CN202410312190 A CN 202410312190A CN 117899098 A CN117899098 A CN 117899098A
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Abstract
The invention relates to the field of medicines, and discloses application of rare ginsenoside CMx in preparation of a medicine for preventing and/or treating type 2 inflammatory diseases. Ginsenoside CMx with different concentrations can reduce the secretion of pro-inflammatory cytokines of a calcipotriol-induced atopic dermatitis model mouse, relieve type 2 inflammatory reaction in the model mouse, relieve itching, reduce percutaneous water loss of mouse ear tissues, improve the oil content and elasticity of the mouse ear tissues, relieve ear swelling, reduce skin loss, inhibit inflammatory cell infiltration, relieve skin desquamation, relieve erythema, relieve pimple, relieve epidermis hyperkeratosis, relieve lesions such as epidermis thickening and the like, and has the effect of preventing and/or treating type 2 inflammatory diseases represented by atopic dermatitis.
Description
Technical Field
The invention relates to the field of medicines, in particular to application of rare ginsenoside CMx in preparation of a medicine for preventing and/or treating type 2 inflammatory diseases.
Background
Atopic dermatitis (atopic dermatitis, AD) is a genetically related chronic, recurrent, inflammatory dermatological disease with an increasing incidence in recent years. AD is frequently developed from infants and is mainly characterized by skin itching, dryness, erythema, pimple, exudation, desquamation and the like, and part of patients continue for life, and the quality of life of the patients can be seriously affected by chronic recurrent eczematoid skin damage, severe itching, sleep loss, diet restriction and psychosocial disorder. Type 2 inflammatory response is involved in the overall process of onset of AD, and plays a key role in the onset of AD. Monoclonal antibody drug Dupilumab (trade name dapritol, dopril Li Youshan) as IL-4 ra blocker has remarkable therapeutic effect on atopic dermatitis by blocking IL-4 and IL-13 signaling, and fully demonstrates that type 2 inflammatory reaction is involved in AD pathogenesis.
Drugs for the treatment of AD include corticoids, calcineurin inhibitors, antihistamines, immunosuppressants, glucocorticoids, biologicals, janus kinase inhibitors, and the like. The medicines can cause skin atrophy, telangiectasia, skin fragility increase, somnolence, dry mouth, listlessness, and rebound after stopping use, and cause adverse reactions such as exfoliative dermatitis, erythroderma, etc.
Therefore, the existing medicines have unsatisfactory clinical effects, and the search for medicines which are effective and can be used for treating AD for a long time is urgent.
Ginsenoside is triterpene compound, has antiinflammatory, antidiabetic, antioxidant stress, cardiovascular and cerebrovascular protecting, and neuroprotective effects, and is also important active ingredient of Araliaceae plants such as Ginseng radix, notoginseng radix, and radix Panacis Quinquefolii. Pharmacokinetic studies have found that naturally occurring ginsenosides (at most 4 glycosyl-saponins) do not have direct pharmacological activity after oral administration, and indeed enter the body to act on secondary saponins (0-2 glycosyl groups) after glycosidase hydrolysis of intestinal flora, which are easier to be absorbed by the body than the original drugs before metabolism, enter the blood circulation, reach the target organ and show more remarkable pharmacological effects (Odani T et al, CHEM PHARM Bull, 1983). These secondary saponins are very low or absent in pseudo-ginseng, ginseng and American ginseng, commonly known as rare ginsenosides, and can only be obtained by metabolic conversion of the original ginsenosides or by bio-directed synthesis.
Rare ginsenoside CMx is prepared by bioconversion of ginsenoside Fc, rb3 and the like, and has molecular weight: 755, molecular formula: c 41H70O12; the structure of which the system is designated (3β,12β)-3,12-Dihydroxydammar-24-en-20-yl 6-O-β-D-xylopyranosyl-β-D-glucopyranoside,CAS No. 80325-22-0, is shown in figure 1. Rare ginsenoside CMx (abbreviated as CMx) is an active metabolite of ginsenoside Fc, rb3 and the like in organisms, has pharmacological activities of in vitro antioxidation, anti-tumor and the like, is not easy to cause side effects after long-term application, but has no relevant report on the influence of the ginsenoside Fc, rb3 and the like.
Disclosure of Invention
The inventor of the invention discovers that rare ginsenoside CMx can down regulate type 2 inflammatory reaction in an atopic dermatitis model mouse body, improve skin lesions, repair epidermis barrier, relieve itching, break 'itching-scratching-immune circulation', and has the efficacy of treating AD. The present invention has been achieved in this way.
The first aspect of the invention provides the use of rare ginsenoside CMx in the manufacture of a medicament for the prevention and/or treatment of type 2 inflammatory diseases.
In a second aspect, the present invention provides the use of rare ginsenoside CMx in the manufacture of a product for reducing skin itching, reducing skin dryness, reducing skin damage, repairing skin barrier or reducing epidermal lesions.
Through the technical scheme, the application has at least the following beneficial effects:
(1) In an in vitro inflammation model of RAW264.7 cells induced by LPS, rare ginsenoside CMx can inhibit NO release, reduce secretion of pro-inflammatory cytokines IL-1β, IL-6 and TNF- α, and inhibit inflammation through JAK/STAT pathway; in the MC903 (calcipotriol) induced AD mouse model, rare ginsenoside CMx can reduce secretion of pro-inflammatory cytokines IL-5 and IL-13 (in some preferred embodiments, secretion of TSLP, IL-33, IL-4 and LTB4 can also be reduced), relieve type 2 inflammatory response in the model mouse body, relieve itching (reducing scratching times and prolonging scratching latency), reduce percutaneous water loss of ear tissues of the mouse, improve oil content and elasticity of ear tissues of the mouse, relieve ear swelling degree induced by MC903, relieve skin loss induced by MC903, inhibit inflammatory cell infiltration, relieve skin desquamation, relieve erythema, relieve pimple, relieve epidermis hyperkeratosis, relieve lesions such as epidermis thickening (such as particle layer thickening and acanthosis), and the like, and has the effects of preventing and/or treating AD.
(2) The rare ginsenoside CMx can reduce the type 2 inflammatory reaction and relieve the AD symptoms, and the discovery provides a reference for the research and development of the rare ginsenoside CMx in the prevention and/or treatment of AD and medicines and equipment, and also provides a reference for the prevention and/or treatment of diseases with the same pathogenesis (caused by the type 2 inflammatory reaction) as AD and the research and development of the related medicines and equipment.
Drawings
FIG. 1 is a structural formula of rare ginsenoside CMx.
FIG. 2 shows the effect of rare ginsenosides CMx on LPS-induced level of NO release in RAW264.7 cells)。
FIG. 3 shows the effect of rare ginsenoside CMx on LPS-induced RAW264.7 cells to produce proinflammatory cytokines IL-1 beta, IL-6, TNF-alpha) Wherein (A) is the concentration of IL-1β, (B) is the concentration of IL-6, and (C) is the concentration of TNF- α.
FIG. 4 shows the effect of rare ginsenosides CMx on IL-4 stimulated JAK/STAT protein phosphorylation in RAW264.7 cells) (A) is the Western blotting result of p-JAK1, p-JAK2 and JAK2, and (B) is the Western blotting result of p-STAT3 (Try 705), STAT3, p-STAT1 (Try 701), p-STAT1 (Try 727) and STAT 1.
FIG. 5 shows the effect of rare ginsenosides CMx on IL-4 stimulated JAK/STAT protein phosphorylation in RAW264.7 cells) Wherein, (A) is the ratio of the relative expression amounts of p-JAK1/JAK1 and p-JAK2/JAK2 proteins, and (B) is the ratio of the relative expression amounts of p-STAT3 (Try 705)/STAT 3, p-STAT1 (Try 701)/STAT 1 and p-STAT1 (Try 727)/STAT 1 proteins.
FIG. 6 shows the effect of oral administration of rare ginsenoside CMx on scratching times in AD model mice, wherein a is scratching times in 30min on day 4 of molding, b is scratching times in 30min on day 7 of molding, and c is scratching times in 30min on day 10 of molding.
FIG. 7 shows the effect of oral administration of rare ginsenoside CMx on the scratching latency of AD model mice, wherein a is the scratching latency on day 4 of molding(s), b is the scratching latency on day 7 of molding(s), and c is the scratching latency on day 10 of molding(s).
Fig. 8 is the effect of oral rare ginsenoside CMx on skin loss and transdermal moisture loss in AD model mice.
Fig. 9 shows the effect of oral rare ginsenoside CMx on oil content and elasticity at skin lesions in AD model mice, where a is the skin oiliness score and b is the skin elasticity score.
Fig. 10 is the effect of oral rare ginsenoside CMx on the skin loss score of AD model mice.
Fig. 11 is an effect of oral rare ginsenoside CMx on ear swelling degree of AD model mice, where a is left and right ear thickness and b is left and right ear weight difference.
FIG. 12 is the effect of oral rare ginsenoside CMx on general histopathology of model mouse skin lesions.
FIG. 13 is the effect of oral rare ginsenoside CMx on model mouse epidermis thickness.
FIG. 14 shows the effect of oral rare ginsenoside CMx on serum cytokines of mice in AD model, wherein a is the content of TSLP in serum, b is the content of IL-4 in serum, c is the content of IL-5 in serum, d is the content of IL-13 in serum, e is the content of IL-33 in serum, and f is the content of LTB4 in serum.
FIG. 15 shows the effect of external use of rare ginsenoside CMx on the number of times of scratching in AD model mice, wherein a is the scratching latency period(s) on day 4 of molding, b is the scratching latency period(s) on day 7 of molding, and c is the scratching latency period(s) on day 10 of molding.
FIG. 16 shows the effect of external use of rare ginsenoside CMx on the scratch latency of AD model mice, wherein a is the scratch latency on day 4 of molding(s), b is the scratch latency on day 7 of molding(s), and c is the scratch latency on day 10 of molding(s).
FIG. 17 shows the effect of external application of rare ginsenoside CMx on skin moisture loss, skin elasticity and oil content in AD model mice, wherein a is skin moisture loss, b is skin elasticity score, and c is skin oiliness score.
FIG. 18 is the effect of rare ginsenoside CMx topical application on skin loss score in AD model mice.
FIG. 19 is a graph showing the effect of external use of rare ginsenoside CMx on ear swelling in AD model mice, where a is the difference in weight of the left and right ear pieces and b is the thickness of the left and right ear.
FIG. 20 is the effect of rare ginsenoside CMx external use on general histopathology of skin lesions in AD model mice.
FIG. 21 is a graph showing the effect of rare ginsenoside CMx external application on the epidermal thickness of model mice.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The first aspect of the invention provides the use of rare ginsenoside CMx in the manufacture of a medicament for the prevention and/or treatment of type 2 inflammatory diseases.
In the present invention, the medicine refers to a substance for treating, preventing and diagnosing diseases by changing physiological, biochemical functions and pathological states of human body; and substances for preventing, treating and diagnosing animal diseases such as livestock and poultry, purposefully regulating physiological functions, and specifying actions, purposes, usage and dosage; including veterinary drugs and human drugs including traditional Chinese medicines, chemical drugs, biological products and the like, and veterinary drugs including natural drugs, synthetic drugs and the like.
Type 2 inflammation is inflammation mediated mainly by helper T cells 2 (Th 2 cells), type 2 innate lymphoid cells, and related cytokines. In the present invention, the type 2 inflammatory disease is a type of inflammatory disease caused by excessive type 2 immune response, such as atopic dermatitis, chronic idiopathic urticaria, prurigo nodularis, bullous pemphigoid, chronic sinusitis, allergic rhinitis, asthma, allergic bronchopulmonary aspergillosis, chronic obstructive pulmonary disease, eosinophilic granulomatous polyangiitis, eosinophilic esophagitis, and the like.
In the present invention, the type 2 inflammatory disease may refer to a type 2 inflammatory disease of human, but also to a type 2 inflammatory disease of animals, particularly a type 2 inflammatory disease of mammals, particularly a type 2 inflammatory disease of primates and/or rodents, preferably a type 2 inflammatory disease of mice.
The inventors of the present invention found that rare ginsenoside CMx is particularly suitable for preventing and/or treating atopic dermatitis including atopic dermatitis of various severity (e.g., acute stage marked by diffuse erythema, papule with exudation crusting, chronic stage marked by erythema, plaque with moss) and atopic dermatitis combined with other allergic diseases (e.g., atopic dermatitis combined with at least one allergic disease such as allergic asthma, allergic rhinitis, or allergic conjunctivitis).
In the present invention, the atopic dermatitis may refer to atopic dermatitis of a human, but also to atopic dermatitis of an animal, particularly atopic dermatitis of a mammal, particularly atopic dermatitis of a primate and/or rodent, preferably atopic dermatitis of a mouse.
In the invention, the content of rare ginsenoside CMx in the medicine is not particularly limited, and can be adjusted according to actual needs; in order to further improve the preventive and/or therapeutic effect of the rare ginsenoside CMx, the content of the rare ginsenoside CMx in the medicine may be, for example, 0.001 to 30 parts by weight, for example, 0.001 to 0.003 part by weight, 0.005 part by weight, 0.008 part by weight, 0.01 part by weight, 0.03 part by weight, 0.05 part by weight, 0.08 part by weight, 0.1 part by weight, 0.3 part by weight, 0.5 part by weight, 0.8 part by weight, 1 part by weight, 2 parts by weight, 4 parts by weight, 6 parts by weight, 8 parts by weight, 10 parts by weight, 12 parts by weight, 14 parts by weight, 16 parts by weight, 18 parts by weight, 20 parts by weight, 22 parts by weight, 24 parts by weight, 26 parts by weight, 28 parts by weight, 30 parts by weight, and any value in any range and any value in any two of the ranges. More preferably 0.01 to 10 parts by weight, still more preferably 0.01 to 6 parts by weight.
In the present invention, the mode of administration of the drug is not particularly limited, and may be adjusted according to actual needs, and for example, injection, respiratory tract, external, oral, mucosal, luminal, etc. administration may be employed; in order to further enhance the prophylactic and/or therapeutic effects of rare ginsenoside CMx, it is preferable that the drug is administered orally and/or topically.
In the present invention, the dosage form of the drug is a conventional dosage form in the field, particularly a dosage form suitable for oral administration and/or topical administration, and can be adjusted according to actual needs; in order to further improve the preventing and/or treating effect of the rare ginsenoside CMx, preferably, the dosage form of the medicament is a dripping pill, an soft capsule, an oral liquid, a suspension, a granule, a tablet, a wine, a gel, a cream, an ointment, a plaster, a spray, a cream, a powder spray or a liniment, more preferably a dripping pill, an soft capsule, an oral liquid, a wine, a spray, a cream or a gel, and still more preferably an oral liquid, an soft capsule, a gel or a cream. For formulation into a suitable dosage form, the medicament may contain various excipients common in the art.
In the present invention, the medicament may further comprise pharmaceutically acceptable excipients.
In a second aspect, the present invention provides the use of rare ginsenoside CMx in the manufacture of a product for reducing skin itching, reducing skin dryness, reducing skin damage, repairing skin barrier or reducing epidermal lesions.
In the present invention, preferably, the use is the use of rare ginsenoside CMx in the preparation of a preparation for reducing skin itching, reducing skin moisture loss, improving skin oil content, improving skin elasticity, reducing skin swelling, inhibiting skin inflammatory cell infiltration, reducing skin desquamation, reducing epidermal parakeratosis, or reducing epidermal thickening (e.g., reducing grain layer thickening, reducing acanthosis).
In some embodiments of the invention, preferably, the rare ginsenosides CMx are particularly useful for reducing inhibition of NO release, reducing secretion of pro-inflammatory cytokines (e.g., secretion of IL-1β, IL-6, TNF- α, IL-5, and IL-13) in atopic dermatitis, reducing itching, reducing loss of skin moisture, improving skin oil and elasticity, inhibiting skin swelling, reducing skin loss, reducing skin desquamation, reducing erythema, reducing pimple, inhibiting skin inflammatory cell infiltration, reducing epidermal hyperkeratosis, or reducing epidermal thickening (e.g., reducing granular and acanthosis) in some preferred embodiments.
In the present invention, the product is a product containing rare ginsenoside CMx, for example, it may be a health product, a cosmetic (for example, it may be a cleaning type cosmetic such as a facial cleanser, a shampoo, a body wash, etc., a care type cosmetic such as a lotion, a skin lotion, etc., and a beauty type cosmetic such as an eye shadow, a rouge, a lipstick, etc.), a food, a daily chemical product (for example, it may be a washing powder, a washing liquid, a soap, a toothpaste), a device containing rare ginsenoside CMx, etc.
The third aspect of the present invention provides a pharmaceutical composition comprising rare ginsenoside CMx and pharmaceutically acceptable excipients.
The term "pharmaceutically acceptable" means not biologically or otherwise undesirable. The term "adjuvant" refers to any substance present in the pharmaceutical formulation that is not an active ingredient, including diluents, binders, lubricants, disintegrants, colorants, emulsifiers, pH buffers, preservatives and the like.
The auxiliary materials used in the present invention may be various auxiliary materials conventionally used in pharmacy, and may be adjusted according to needs, preferably at least one of polyethylene glycol (such as polyethylene glycol 4000, polyethylene glycol 6000), diethylene glycol monoethyl ether, beeswax, soybean oil, tween 80, glycerol, propylene glycol, butylene glycol, sodium benzoate, stevioside, ethanol, hydroxypropyl methylcellulose ether (such as carbomer 940, carbomer 980 and carbomer 934), ethylparaben, triethanolamine, glyceride, stearic acid, vaseline, and sodium lauryl sulfate.
According to a preferred embodiment of the present invention, the pharmaceutical composition further comprises an auxiliary agent beneficial for the prevention and/or treatment of a disease, preferably an auxiliary agent for the prevention and/or treatment of inflammatory disease type 2. The auxiliary medicament can be chemical medicament, such as loratadine and the like, and can also be other medicament with curative effect on type 2 inflammatory diseases.
Preferably, the pharmaceutical composition is for use in the prevention and/or treatment of inflammatory diseases of type 2, more preferably atopic dermatitis.
In the present invention, the amount of CMx administered may be determined according to clinical needs and the age, weight, sex and health of the subject, for example: for adults, the oral dosage of rare ginsenoside CMx in the pharmaceutical composition may be 0.01-3 mg/kg/day, preferably 0.07-0.7 mg/kg/day; the topical application can be 0.005-25 mg/320cm 2/day, preferably 0.05-10mg/320cm 2/day; for murine animals, the oral dosage of rare ginsenoside CMx in the pharmaceutical composition may be 0.12-26 mg/kg/day, preferably 0.6-7 mg/kg/day; the topical application can be 0.006-3.5 mg/9cm 2/day, preferably 0.06-1.5mg/9cm 2/ days; the usage can be determined according to actual needs, for example, the usage can be once a day or multiple times a day, and when the usage is carried out multiple times a day, the total daily usage can meet the usage limit.
The present invention will be described in detail by examples.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the following experiments and preparation examples, rare ginsenoside CMx (purity: 99%) was purchased from Chengdu Pure technology development Co.
The results of the experiments were statistically analyzed and,
Compared with the control group, #p <0.05, #p <0.01, #p <0.001, # #p <0.0001;
Compared with the ginsenoside blank oral liquid or blank gel group, & P <0.05, & P <0.01, & & P <0.001.
Preparation example 1: preparation of rare ginsenoside CMx oral liquid
Prescription: the rare ginsenoside CMx content is 2 wt%; rare ginsenoside CMx: tween 80: glycerol: sodium benzoate: stevioside: absolute ethyl alcohol: purified water = 1:1.08:081:0.27:0.22:0.54:44.40.
The preparation method comprises the following steps: weighing materials according to a prescription, and dissolving rare ginsenoside CMx in glycerol for standby; dissolving sodium benzoate in ethanol for use; mixing the rest materials except purified water, adding 30% purified water, dissolving completely, adding the above two solutions, mixing, homogenizing, adding rest purified water, mixing, homogenizing, filtering, and sealing for storage.
Preparation example 2: preparation of rare ginsenoside CMx drop pill
Prescription: the rare ginsenoside CMx content is 30 wt%; rare ginsenoside CMx: polyethylene glycol 4000: polyethylene glycol 6000: diethylene glycol monoethyl ether=1: 0.5:1:0.85.
The preparation method comprises the following steps: weighing materials according to prescription, dissolving rare ginsenoside CMx in diethylene glycol monoethyl ether, adding polyethylene glycol 4000 and polyethylene glycol 6000, preparing according to conventional preparation method of dripping pill, and sealing and preserving.
Preparation example 3: preparation of rare ginsenoside CMx soft capsule
Prescription: the rare ginsenoside CMx weight percent; rare ginsenoside CMx: beeswax: soybean oil = 1:0.1:18.9.
The preparation method comprises the following steps: weighing the materials according to the prescription, and crushing the rare ginsenoside CMx into 300-mesh superfine powder for standby; adding rare ginsenoside CMx and Cera flava into soybean oil, heating to melt Cera flava, dispersing in soybean oil, and making into soft capsule by conventional method, sealing, and storing.
Preparation example 4: preparation of rare ginsenoside CMx wine
Prescription: rare ginsenoside CMx content is 0.001 wt%, rare ginsenoside CMx: stevioside: 52 degree white spirit = 1:400:99000.
White spirit was purchased from Yunnan Ganbang wine industries, inc.
The preparation method comprises the following steps: weighing materials according to prescription, dissolving rare ginsenoside CMx and stevioside in appropriate amount of 52% (v/v) Chinese liquor, mixing, homogenizing, adding the rest 52% (v/v) Chinese liquor, mixing, homogenizing, filtering, and sealing for storage.
Preparation example 5: preparation of rare ginsenoside CMx gel
Prescription: rare ginsenoside CMx content is 0.05 wt%, rare ginsenoside CMx: carbomer C940: polyethylene glycol 400: propylene glycol: triethanolamine: ethyl hydroxy benzoate: purified water = 1:20:20:300:20:2:1620.
Carbomer C940 is available from Libotun advanced materials, inc., weight average molecular weight 940.
Polyethylene glycol 400, available from Lvsen chemical Co., ltd, weight average molecular weight 400.
The preparation method comprises the following steps: weighing materials according to a prescription, adding rare ginsenoside CMx into propylene glycol with the prescription amount of 1/4, and stirring and dissolving completely to obtain rare ginsenoside CMx concentrate for later use; adding carbomer C940 with prescription amount, adding propylene glycol with 1/3 prescription amount, stirring, dispersing uniformly, adding purified water with 60% prescription amount, turning on the stirrer, stirring thoroughly for 30min, and mixing uniformly for use; the prescribed amounts of polyethylene glycol, ethylparaben and another 3/4 of propylene glycol were placed therein and dissolved by heating in a water bath at 55 ℃. Adding triethanolamine into the concentrated solution of rare ginsenoside CMx and carbomer solution, adding purified water with the rest amount, mixing, sealing, and storing.
Preparation example 6: preparation of rare ginsenoside CMx cream
Prescription: rare ginsenoside CMx content is 0.01 wt%, rare ginsenoside CMx: glycerol monostearate: stearic acid: white vaseline: ethyl hydroxy benzoate: glycerol: butanediol: sodium dodecyl sulfate: purified water = 1:300:800:300:0.1:800:200:100:7400.
The preparation method comprises the following steps: weighing the prescription amount of glyceryl monostearate, stearic acid and white vaseline, melting and mixing uniformly in water bath at 80 ℃, adding ethylparaben, stirring to dissolve, and taking the mixture as an oil phase for later use; weighing the prescription amount of glycerol, butanediol and sodium dodecyl sulfate, adding a proper amount of purified water, stirring and dissolving, adding the prescription amount of rare ginsenoside CMx, stirring and dissolving completely, and taking the mixture as a water phase for standby; slowly adding the preheated oil phase into the water phase under stirring, emulsifying for 10min, cooling to about 60deg.C, adding self-microemulsion concentrate while emulsifying to uniformity. Cooling to 40deg.C, and sealing for use.
Example 1 rare ginsenoside CMx in vitro experiments for treating atopic dermatitis
1. Evaluation of anti-inflammatory Activity
CMx and LPS were prepared as solutions of the required concentrations with DMSO and cell culture medium, respectively.
Cell culture medium: purchased from Gibco company.
In the in vitro inflammation model of RAW264.7 cells induced with LPS (lipopolysaccharide, available from Sigma-Aldrich), CMx (8, 4, 2. Mu.g/mL) was used to separately intervene on LPS (1. Mu.g/mL) induced RAW264.7 cells (available from Pronoxel Life technologies Co., ltd.). After overnight cell culture, cells were observed under a microscope. The cell supernatant was discarded, and the mixture was added to a medium containing CMx% CO 2 and cultured in a 37℃incubator for 24 hours. The concentrations of NO, IL-1. Beta., IL-6, TNF-alpha were determined from the supernatant according to the kit instructions.
From fig. 2 (effect CMx on LPS-induced NO release levels in RAW264.7 cells), it was shown that LPS alone was able to significantly induce NO production in RAW264.7 cells compared to control cells. However, when RAW264.7 cells were pretreated with CMx, NO release levels were significantly inhibited (P < 0.001). The results indicate that at CMx concentrations of 8, 4, 2 μg/mL, NO production in RAW264.7 cells induced by LPS can be significantly inhibited and NO release is inhibited in a concentration-dependent manner.
IL-1β, IL-6 and TNF- α are pro-inflammatory cytokines secreted early in the inflammatory response, whose levels are elevated in acute and chronic inflammatory diseases. FIG. 3 (CMx effect on LPS-induced RAW264.7 cells to produce the pro-inflammatory cytokines IL-1β, IL-6, TNF- α) shows that LPS-treated RAW264.7 cells have significantly elevated IL-1β, IL-6, and TNF- α levels (P < 0.001) compared to control cells, and that 8g/mL and 4 μg/mL CMx pretreatment resulted in significantly reduced IL-1β, IL-6, and TNF- α levels (P < 0.001).
The above data indicate that CMx is capable of producing anti-inflammatory effects in vitro.
2. Evaluation of mechanism of action
In vitro inflammation models of RAW264.7 cells induced with IL-4 (available from subfamily Biotechnology Inc.), CMx (8, 4, 2. Mu.g/mL) were used to intervene in each of IL-4 (20 ng/mL) induced RAW264.7 cells. After overnight cell culture, cells were observed under a microscope. The cell supernatant is discarded, a culture medium with the concentration CMx is added, the cells are collected after being cultured for 24 hours in a culture box with 5 percent CO 2 and 37 ℃, the total protein is extracted and quantified after the cells are lysed, and the related protein expression is measured by a Western blot method.
FIGS. 4 and 5 show that the ratio of p-JAK1/JAK1 and p-JAK2/JAK2 is significantly increased in IL-4 stimulated RAW264.7 cells compared to control cells, and that the ratio of p-JAK1/JAK1 and p-JAK2/JAK2 is significantly decreased after administration of the selective JAK1/JAK2 inhibitor Ruxolitinib (purchased from Merenia) and CMx pretreatment; as can be seen from the binding western blot results, the ratio of P-STAT1 (Try 701)/STAT 1, P-STAT1 (Try 727)/STAT 1, P-STAT3 (Try 705)/STAT 3 was significantly increased in IL-4 stimulated RAW264.7 cells compared to control cells, and inhibition of P-STAT1 (Try 701) site phosphorylation by CMx (P < 0.001) was significant compared to P-STAT1 (Try 727) and P-STAT3 (Try 705) sites after administration of selective STAT3/STAT3 inhibitor Nifuroxazide (purchased from meldonium) and CMx pretreatment.
The above results indicate that CMx can exert the activity of treating AD by inhibiting activation of the IL-4/JAK/STAT pathway.
EXAMPLE 2 evaluation of the Effect of oral ginsenoside CMx on treatment of atopic dermatitis
Dexamethasone, loratadine and ginsenoside CMx obtained in preparation example 1 are prepared into a solution with a required concentration by using sterile water.
MC903 (calcipotriol) was formulated with absolute ethanol to 2nmol.
CMC-Na was purchased from Sigma-Aldrich and was prepared as a 0.5 wt% solution using ultrapure water.
BALB/c male mice of 6-8 weeks old were randomly divided into 8 groups, namely, a blank group, a model group, a drug group (dexamethasone group, dex,2mg/kg; loratadine group, lor,1.67 mg/kg), a blank oral liquid group (oral liquid without CMx), a rare ginsenoside CMx-low dose group (CMx-L, 1 mg/kg), a rare ginsenoside CMx-medium dose group (CMx-M, 2 mg/kg), a rare ginsenoside CMx-high dose group (CMx-H, 4 mg/kg), and 10 mice per group.
On the 1 st to 10 th days of the experiment, 20 mu L of 2nmol of the MC903 solution is uniformly smeared on the right ears of animals in a blank oral liquid group, a model group, a positive medicine group, CMx-low dose group, CMx-medium dose group and CMx-high dose group, and the blank group is smeared with equal amount of absolute ethyl alcohol, 20 mu L/dose, 1 time/d;
And, from day 4 after MC903 stimulation, the drug group is infused with the corresponding dose of drug, the model group and the blank group are given with CMC-Na solution of 0.5 wt%, the blank oral liquid group is given with equal dose of blank oral liquid, the blank group is smeared with equal amount of absolute ethyl alcohol, and the drug is continuously given for 7d,1 time/d;
The left ear of each experimental group of mice was smeared with absolute ethanol as a solvent control, 20 μl per ear was smeared for 10 consecutive days, 1 time/d.
1H after the last administration, the mice were anesthetized, blood was taken from the abdominal aorta, spleen, thymus and ear were dissected, and after a punch with a diameter of 8mm was used to punch an ear piece with a diameter of about 8mm, the pieces were fixed in 4 wt% paraformaldehyde, and HE staining was performed. The rest part is placed in a refrigerator at the temperature of minus 80 ℃ and is ground to prepare tissue homogenate for measuring inflammatory factors.
Pre-experiment is carried out before the experiment, 24 mice are used as a model group, MC903 is smeared for molding according to the method of the experiment, the continuous smearing is carried out for 10 days, 6 mice are killed after 4 days, 7 days, 10 days and 14 days of molding respectively, ears of the mice are sheared by surgical scissors, the ears of the mice are punched into lugs by an 8mm puncher, the lugs are completely immersed in 4 weight percent paraformaldehyde solution for fixation, HE dyeing is carried out, images are observed and collected under a microscope for analysis, and pathological section results show that: the model group mice with 10 days of modeling have obviously thickened epidermis acantha granular layer, severe keratinization and increased inflammatory cell infiltration, which indicates that the modeling can be successfully performed by adopting the method to continuously smear for 10 days.
1. Behavioural observations
The behavior of the unmolded mice was observed and recorded for 30min after molding on days 4, 7, and 10. The scratching of the front paw of the mouse, or the biting of the mouth into the left hind paw (continuous biting or scratching until short stopping is one time) is taken as an indication of pruritus, the time from the molding start to the first scratching is taken as a incubation period, and the scratching and the biting of the left hind paw performance of the mouse within 30min are recorded.
(1) Scratching frequency results: on day 4 of modeling, the model group had significantly higher scratching (P < 0.001) than the blank group; there was no significant difference (P > 0.05) between each dose of CMx compared to the model group; compared with blank oral liquid, CMx doses have no significant difference (P > 0.05) (see figure 6 a). On days 7 and 10 of molding, the scratching of the model group was significantly higher (P < 0.001) than that of the blank group; the medium-high dose CMx reduced the number of scratching times (P < 0.001) compared to the model group; compared with blank oral liquid, CMx can reduce scratching times (P < 0.001) at high dosage (see figure 6b and figure 6 c).
(2) Scratching latency results: on day 4 of modeling, the model group had a shortened scratching latency compared to the blank group, but no statistical difference (P > 0.05), and no significant difference (P > 0.05) for each of the dosing groups compared to the model group (see fig. 7 a); compared with blank oral liquid, CMx doses have no obvious difference (P > 0.05). On day 7 of modeling, the scratching latency was significantly shortened in the model group compared to the blank group (P < 0.001), the scratching latency was prolonged in the Dex group compared to the model group (P < 0.001) (see fig. 7 b), and there was no significant difference between the CMx doses compared to the blank oral liquid (P > 0.05). On day 10 of modeling, the scratching latency was significantly reduced in the model group compared to the blank group (P < 0.001); the high dose in CMx prolonged the mouse scratch latency (P < 0.001) compared to the model group; compared with blank oral liquid, CMx high dose can obviously prolong the scratching latency period. (see FIG. 7 c).
The results show that the oral administration of CMx medium dose (2 mg/kg) and CMx high dose (4 mg/kg) can effectively reduce the number of times of scratching and prolong the scratching latency of the atopic dermatitis model mice.
2. Loss of skin moisture
Before skin moisture is measured for the first time, marking detection points of the same area on the front side and the rear side of each mouse ear by using a marker pen, detecting the loss of percutaneous moisture at the monitoring points of the mouse ear after the last administration, and recording data after taking an average value after 3 times of testing at the same position under the conditions that the temperature is 22-25 ℃ and the humidity is 50-60%, wherein the result shows that compared with a blank group, the skin moisture loss of the mice in a model group is obviously increased (P < 0.001); each dose CMx was able to significantly reduce the percutaneous moisture loss of mouse ear tissue compared to the model group (P < 0.01); compared with blank oral liquid, CMx doses can reduce the percutaneous water loss of the ear tissues of the mice (P < 0.01); see fig. 8.
3. Skin oil content and elasticity
Skin oil content and elasticity were measured at monitoring points of the ears of mice with a skin tester, and data were recorded after 3 times of testing at the same position and averaging. The results showed that after the last dose, the mice in the model group had significantly increased oil content and significantly decreased elasticity (P < 0.0001) compared to the blank group; compared with a model group, CMx doses can obviously improve the oil content and elasticity of mouse ear tissues; compared with blank oral liquid, CMx doses can reduce the oil content and elasticity (P < 0.05) of mouse ear tissues; the results are shown in FIGS. 9a and 9b.
4. Skin damage scoring
Model scoring criteria: mice were scored for skin lesions on day 10 according to clinical skin scores. The severity of atopic dermatitis-like skin lesions, including redness (erythema), dryness (dryness) and crusting (abrasion), was assessed using 0-3 minutes (0, none; 1, mild; 2, moderate; 3, severe).
TABLE 1
The results are shown in FIG. 10, where the mice' ears became red, scaling, swelling and crusting after 10 consecutive days of topical application of MC 903. After CMx days of treatment, the erythema scales, swelling and crusting at the skin lesions of the model mice were significantly improved. The overall dermatitis score was significantly reduced in CMx high, medium, low groups compared to the model group (P < 0.001); compared with blank oral liquid, CMx doses can reduce the skin damage score (P < 0.001) of the ear tissue of the mouse; the cue CMx can relieve red, swelling, dry desquamation of epidermis by oral administration, and has the efficacy of repairing skin barrier.
5. Ear swelling degree
Thickness difference between left and right ears: the thickness basic values of the left ear and the right ear of each group of mice are measured on the 0 th day after molding, the thickness changes of the same parts of the left ear and the right ear of the mice are respectively measured three times on the 10 th day after molding, and finally, the average value of the three values is taken as the result, so that the thickness difference (mm) of the left ear and the right ear is calculated.
Difference in weight of left and right ears: the weight basic values of the left ear and the right ear of each group of mice are measured on the 0 th day after molding, the ears of the mice are cut off on the 10 th day after molding, the ears of the mice are punched into the ears with the same size by using an 8mm puncher, the ears are placed on an analytical balance for weighing, and the weight difference of the left ear and the right ear is calculated.
Results: on day 10 after modeling, the model group had an increased ear swelling (P < 0.001), and each dose CMx was able to significantly suppress MC 903-induced increase in ear swelling compared to the model group; compared with blank oral liquid, CMx high doses can reduce the rise of the ear tissue ear swelling degree of mice; the results are shown in FIG. 11a and FIG. 11b.
6. Influence of general histopathology of skin lesions
The ears of the mice are cut off by surgical scissors, the ears of the mice are punched into ear pieces by an 8mm puncher, the ear pieces are fully immersed in 4wt% paraformaldehyde solution for fixation, HE is used for dyeing, hematoxylin and eosin are used for dyeing after washing, dehydrating, waxing, embedding and slicing, dehydrating and sealing are carried out, and images are observed and collected under a microscope for analysis. 6 fields were randomly selected for skin epidermis thickness statistics.
Results: as shown in fig. 12 and 13, in the pathological section staining chart, ① arrow indicates hypokeratosis, ② arrow indicates granule layer hypertrophy, ③ arrow indicates acanthosis, and ④ arrow indicates inflammatory cells.
The skin epidermis of the normal group is not obviously thickened and is not infiltrated by inflammatory cells; compared with the normal group, the model group and the blank oral liquid group have the advantages that the skin epidermis and the horny layer of the skin at the skin lesion are seriously keratinized, the thickening of the granular layer and the acanthous layer is obviously thickened, and inflammatory cell infiltration is increased; compared with the model group and the blank oral liquid group, the CMx administration group has the advantages that the lesions such as the skin damage, the epidermis hyperkeratosis, the thickening and thickening of the granular layer and the acantha layer and the like of the mice are relieved (the parts indicated by the arrows ①、②、③ respectively).
Cells stained blue are inflammatory cells (part indicated by ④ arrow), a large amount of inflammatory cells are gathered in the model group mouse skin lesions, and inflammatory cell infiltration is obviously reduced in the CMx dose group mouse skin lesions, wherein the high dose group mouse skin lesions only see a small amount of inflammatory cells scattered. Compared with a model group and a blank oral liquid group, CMx administration groups can reduce MC903 induced skin damage, inhibit inflammatory cell infiltration, improve skin damage keratosis, and reduce lesions such as epidermis granule layer and acanthosis thickening.
7. Determination of serum cytokines
The mice were anesthetized with 1wt% sodium pentobarbital, blood was collected by taking blood from the abdominal aorta, and after centrifugation at 3000r/min for 15min at 4 ℃, the blood supernatant was collected and the serum was examined for LTB4, TSLP, IL-33, IL-5, IL-4, IL-13 content.
The results showed that the mice in the model group had significantly elevated levels of LTB4, TSLP, IL-33, IL-5, IL-4, IL-13 (P < 0.001) compared to the blank group; compared with a model group and a blank oral liquid group, CMx can reduce the serum TSLP, IL-33, IL-5, IL-4, IL-13 and LTB4 levels of the model mice to different degrees in each dosage group; see fig. 14.
The results indicate that rare ginsenoside CMx can down regulate type 2 inflammatory response in model mice.
EXAMPLE 3 evaluation of the external use effect of rare ginsenoside CMx on treating atopic dermatitis
Dexamethasone, loratadine and ginsenoside CMx gel obtained in preparation example 5 are prepared into a solution with a required concentration by using sterile water.
The MC903 solution was treated as in example 2.
After 6-8 week old BALB/c male mice were adaptively fed for 1 week, they were divided into a blank group (blank, 50 wt% absolute ethanol, 20 μl/ear), a model group, a dexamethasone group (Dex, 0.67 mg/kg), a loratadine group (Lor, 1.67 mg/kg), a CMx high dose group (CMx-high, 1 mg/kg), a CMx medium dose group (CMx-medium, 0.5 mg/kg), a CMx low dose group (CMx-low, 0.25 mg/kg) and a blank gel group (no CMx gel), according to a random distribution principle, 10 each.
On experiment 1-10 days, uniformly smearing 2nmol of MC903 solution on right ear of a model group, a blank gel group, a positive drug (Dex and Lor) group, a CMx high-dose group, a CMx medium-dose group (CMx-medium) and a CMx low-dose group, wherein the blank group is smeared with absolute ethyl alcohol, and 20 mu L/dose is 1 time/d; on day 4, after MC903 solution is applied to the right ear, corresponding medicines are applied to each of the positive medicine group and CMx dosage groups, an equal dose of blank gel is applied to the blank gel group, absolute ethyl alcohol is applied to the blank group, and 7d,1 time/d and 20 mu L/d of the medicine are continuously applied to the right ear.
The left ear of each of the above experimental groups mice was smeared with absolute ethanol as a solvent control, 20. Mu.L/mouse, 1 time/d, for 10 consecutive days.
Pre-experiment is carried out before the experiment, 10 mice are used as a model group, MC903 is smeared for molding according to the method of the experiment, the continuous smearing is carried out for 10 days, 3 mice are killed after molding for 4, 7 and 10 days, ears of the mice are sheared by surgical scissors, the ears of the mice are punched into ear pieces by an 8mm puncher, the ear pieces are completely immersed in 4 weight percent paraformaldehyde solution for fixation, HE dyeing is carried out, observation is carried out under a microscope, images are collected for analysis, and pathological section results show that: the epidermis acantha granular layer of the model group is obviously thickened, the keratinization is serious, and inflammatory cell infiltration is increased after 10 days of modeling, which shows that the modeling can be successfully performed after continuous MC903 coating for 10 days.
1. Behavioural (number of scratching) observations
The procedure of experiment 1 was followed in example 2.
Scratching frequency results: on day 4 of modeling, the number of scratching in the model group was significantly increased (P < 0.001) compared to the blank group, and there was no significant difference (P > 0.05) between CMx doses compared to the model group and the blank gel group (see fig. 15 a); at 7 and 10 days of molding, the scratching in the model group was significantly higher (P < 0.001) than in the blank group, and the scratching times were reduced at a high dose of CMx than in the model group and the blank gel group (see FIGS. 15b and 15 c).
Scratching latency results: on day 4 of modeling, the scratching latency period was significantly shortened in the model group compared to the blank group (P < 0.05), and there was no significant difference between the administration groups (P > 0.05) compared to the model group and the blank gel group (see fig. 16 a); at 7 and 10 days of modeling, the model group showed significant differences (P < 0.001) compared with the blank group, and the high dose of CMx can prolong the scratching latency period (P < 0.05) of mice compared with the model group and the blank gel group (see figures 16b and 16 c).
The results show that CMx can effectively reduce the scratching times of the atopic dermatitis model mice and prolong the scratching latency period by external application.
2. Percutaneous moisture loss, skin elasticity and oil content determination
The percutaneous moisture loss, elasticity and oil content of the same parts of the left and right ears of each group of mice were measured on day 10 after molding according to the method of experiment 2-3 in example 2.
The results of the percutaneous moisture loss show that: on day 10 of modeling, mice in the model group had significantly increased percutaneous moisture loss (P < 0.001) compared to the blank group; the sequential administration of CMx doses for 7 days significantly reduced the percutaneous water loss of mouse ear tissue (P < 0.001) compared to model and blank gel groups, as shown in figure 17a.
Skin oil content and elasticity results show that, after the last administration, the oil content of mice in the model group is obviously increased and the elasticity is obviously reduced (P < 0.001) compared with that of mice in the blank group; the high dose in CMx significantly improved the mouse ear tissue elasticity (see figure 17 b) and oil content (see figure 17 c) (P < 0.05) compared to the model and blank gel groups.
3. Skin damage scoring
The procedure of experiment 4 in example 2 was followed.
Results as in fig. 18, after 10 days of continuous application of MC903, the mouse ears became red, scaling, swelling and crusting. After CMx external treatment for 7 days, erythema, scaling, swelling and crusting at the skin lesions of the model mice were significantly improved. The overall score for dermatitis was significantly reduced (P < 0.001) for mice in each dose group at CMx compared to the model group and the blank gel group; the cue CMx can relieve red, swelling, dryness and desquamation of epidermis by external application, and has the effect of repairing skin barrier.
4. Ear swelling degree
The procedure of experiment 5 in example 2 was followed.
The results show that: on day 10 after molding, the model group had an increased ear swelling (P < 0.001), and the high dose topical application of CMx was able to suppress the increase in ear swelling in model mice compared to model and blank gel groups, as shown in figures 19a and 19b.
5. General histopathological detection of skin lesions
The procedure of experiment 6 in example 2 was followed. The results are shown in FIGS. 20 and 21.
Results: in the pathological section staining chart, ① arrow indicates hypokeratosis, ② arrow indicates granule layer hypertrophy, ③ arrow indicates acanthosis, and ④ arrow indicates inflammatory cells.
The skin epidermis of the normal mice is not obviously thickened and is not infiltrated by inflammatory cells; compared with the normal group, the model group and the blank oral liquid group have the advantages that the skin epidermis and the horny layer of the skin at the skin lesion are seriously keratinized, the thickening of the granular layer and the acanthous layer is obviously thickened, and inflammatory cell infiltration is increased; compared with the model group and the blank gel group, the CMx administration group mice have the effects of relieving the lesions such as the skin damage, the epidermis hyperkeratosis, the thickening of the granular layer and the acantha thickening blank gel group and the like.
The blue-stained cells are inflammatory cells (④ is indicated by arrow), a large amount of inflammatory cells are gathered in the mouse skin lesions of the model group and the blank gel group, and inflammatory cell infiltration in the mouse skin lesions of each dose group CMx is obviously reduced, wherein the mouse skin lesions of the high dose group only see a small amount of inflammatory cells scattered; compared with the model group, CMx doses can be continuously administered for 7 days, so that the keratosis of skin lesions can be obviously improved, the thickening of granular layers and acantha layers can be reduced, and inflammatory cell infiltration can be inhibited.
Experimental results show that CMx external administration can reduce MC903 induced skin loss, inhibit inflammatory cell infiltration, and reduce epidermal lesions such as keratinization and epidermal thickening.
The experimental results of each experiment show that: the rare ginsenoside CMx can be used for oral administration or external application to down regulate type 2 inflammatory reaction in model mice, improve epidermopathy of model mice, repair epidermoid barrier (relieve red, swelling and dry desquamation of epidermis), relieve pruritus (reduce scratching times and prolong scratching latency), and treat AD.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. Use of rare ginsenoside CMx in preparing medicine for preventing and/or treating type 2 inflammatory diseases is provided.
2. The use according to claim 1, wherein the inflammatory disease type 2 is at least one of atopic dermatitis, chronic idiopathic urticaria, prurigo nodularis, bullous pemphigoid, chronic sinusitis, allergic rhinitis, asthma, allergic bronchopulmonary aspergillosis, chronic obstructive pulmonary disease, eosinophilic polyangiitis, and eosinophilic esophagitis.
3. The use according to claim 1 or 2, wherein the inflammatory disease type 2 is atopic dermatitis.
4. The use according to claim 1 or 2, wherein the content of rare ginsenoside CMx in the medicament is 0.001-30 parts by weight based on 100 parts by weight of the total medicament.
5. The use according to claim 4, wherein the content of rare ginsenoside CMx in the medicament is 0.01-10 parts by weight based on 100 parts by weight of the total medicament.
6. The use according to claim 1 or 2, wherein the medicament is in the form of an oral liquid, drop pill, soft capsule, suspension, granule, tablet, medicated wine, gel, cream, ointment, patch, spray, powder mist or liniment.
7. The use according to claim 6, wherein the medicament is in the form of an oral liquid, a drop pill, a soft capsule, a wine, a gel, a cream or a spray.
8. The use according to claim 7, wherein the medicament is in the form of an oral liquid, soft capsule, gel or cream.
9. Use of rare ginsenosides CMx for the manufacture of a product for reducing skin itching, reducing skin dryness, reducing skin damage, repairing skin barrier or reducing epidermal lesions.
10. The use according to claim 9, wherein the use is the use of rare ginsenoside CMx in the manufacture of a product for reducing skin itching, reducing skin moisture loss, improving skin oil, improving skin elasticity, reducing skin swelling, inhibiting skin inflammatory cell infiltration, reducing epidermal hypoparagonism or reducing epidermal thickening.
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