阅读说明：本技术 白鲜皮提取物及其应用 (Cortex dictamni extract and application thereof ) 是由 陈默 孙懿 赵亚 贾海东 于 2021-09-30 设计创作，主要内容包括：本发明提供了具有抗炎、抗敏、抗刺激和/或止痒作用的白鲜皮提取物,其中,所述白鲜皮提取物采用溶剂提取法制备。本发明还涉及包含白鲜皮提取物的组合物,所述组合物是药品、保健食品或皮肤外用剂。本发明所述的抗炎、抗敏、抗刺激和/或止痒作用通过抑制胸腺基质淋巴生成素TSLP和/或促进丝聚蛋白FLG基因表达实现。(The invention provides a cortex dictamni extract with anti-inflammatory, anti-allergy, anti-irritation and/or itching relieving effects, wherein the cortex dictamni extract is prepared by a solvent extraction method. The present invention also relates to a composition comprising the extract of cortex dictamni, which is a pharmaceutical product, a health food or a skin external preparation. The anti-inflammatory, anti-allergic, anti-irritant and/or anti-itching effect of the invention is achieved by inhibiting thymic stromal lymphopoietin TSLP and/or promoting filaggrin FLG gene expression.)

1. The cortex dictamni extract has the functions of anti-inflammation, anti-allergy, anti-irritation and/or itching relieving, wherein the cortex dictamni extract is prepared by adopting a solvent extraction method.

2. The extract of claim 1, wherein the cortex dictamni extract is extracted using water as a solvent.

3. Extract according to claim 1, wherein the anti-inflammatory, anti-allergic, anti-irritant and/or anti-itching effect is achieved by inhibiting thymic stromal lymphopoietin TSLP and/or promoting filaggrin FLG gene expression.

4. The composition comprising the cortex dictamni extract as defined in claim 1, wherein the composition is a pharmaceutical product, a health food or a skin external preparation.

5. The composition according to claim 4, wherein the anti-inflammatory, anti-allergic, anti-irritant and/or anti-itching effect is achieved by inhibiting thymic stromal lymphopoietin TSLP and/or promoting filaggrin FLG gene expression.

6. The composition of claim 4, for improving atopic dermatitis, eczema, allergic asthma, allergic rhinitis, allergic contact dermatitis, urticaria or a combination thereof.

7. The composition as set forth in claim 4, wherein the extract of cortex dictamni is used in an amount of 0.001-100% by weight when the composition is used as an external preparation for skin.

8. The composition as claimed in claim 4, wherein the amount of the cortex Dictamni Radicis extract is at least 0.01 wt% when used as a pharmaceutical.

9. The composition as set forth in claim 4, wherein the amount of the extract of cortex Dictamni Radicis is at least 1% by weight or more when the composition is used as a health food.

Technical Field

The invention belongs to the field of phytochemistry, and particularly relates to a cortex dictamni extract, a preparation method thereof and application of the extract in medicines, health-care foods or skin external preparations.

Background

In recent years, with the new environmental pollution caused by the industrial development, the problem of PM2.5 pollution to the air is aggravated, and in addition, the influence of life style, the probability of various allergens such as dust mites, pollen, pet hair, food additives and the like which induce immune allergic reactions is higher and higher, and the incidence rate of allergic problems such as atopic dermatitis/eczema, allergic asthma, allergic rhinitis, allergic dermatitis, sensitive skin and the like is increased, which attracts all people's attention.

Studies suggest that the pathogenesis of eczema may be associated with dysbalance of T cell subsets (Th1/Th 2). Abnormal secretion of Th2 cytokine is closely related to the occurrence, development and prognosis of eczema. In addition, Thymic Stromal Lymphopoietin (TSLP) and IL-33 are also closely related to Th2 inflammatory factor. TSLP was first discovered by Friend et al from thymic stromal cells and plays an important role in the pathogenesis of eczema. TSLP is an epithelial cell derived factor, an IL-7 analog, involved in the imbalance of Th2 immune responses, a key "initiator" of Th2 type immune responses, and mainly expressed in epithelial cells and keratinocytes. TSLP not only plays an important role in allergic asthma, rhinitis and anaphylaxis in allergic skin diseases such as atopic dermatitis, contact dermatitis and the like, but also is related to various inflammatory skin diseases such as acne rosacea, psoriasis and the like, and Th2 immune response mediated by a Periostin/TSLP/IL-31 signaling pathway involved in TSLP may play an important role in chronic pruritus. TSLP is capable of strongly activating Dendritic Cells (DCs) and innate immune cells including mast cells, natural killer T cells. On one hand, after the TSLP is activated, the proliferation and differentiation of the naive CD4+ T cells into inflammatory Th2 cells can be promoted. On the other hand, TSLP acts directly on innate immune cells, stimulating mast cells to produce a series of inflammatory factors that promote the development of allergic reactions.

One of the main functions of the skin barrier is to prevent the body's water from being released through the skin to the surrounding environment, which water is an important component of the stratum corneum, and is also essential for the enzymatic hydrolysis of skin lipids and other components, and has the effect of maintaining the flexibility of the stratum corneum. The skin barrier, in addition to preventing moisture loss, can also effectively prevent the invasion of external chemicals and microorganisms. The loss of skin barrier function results in a greater transdermal sensitization of the antigen, which induces inflammation that can exacerbate the breakdown of the skin barrier. Filaggrin plays a key role in epidermal barrier function and is expressed in terminally differentiated keratinocytes in the outermost layer of the human epidermis. FLG acts on the epidermis layer of skin, especially the Stratum Corneum (Stratum Corneum, SC) of its outermost layer in the epidermis barrier forms, and FLG can degrade and form natural moisturizing factor, is the first line of defense between organism and the external environment, mainly prevents the loss of moisture in foreign matter gets into human body and epidermis. Researchers research the skin barrier function of children with atopic dermatitis, find that the expression of skin barrier function related protein of children with atopic dermatitis is reduced, and suggest that the skin barrier function is damaged, and related research shows that the skin barrier function of infantile eczema (AD) is dysfunction, which is mainly shown in that the FLG gene mutation causes the keratinocyte connection abnormality of the skin barrier.

The invention unexpectedly discovers that the cortex dictamni extract has the effect of inhibiting TSLP, animal itching-relieving experiments also prove that the cortex dictamni extract has good itching-relieving effect, and simultaneously discovers that the cortex dictamni extract has the effect of improving the mRNA expression of a filaggrin (FLG, skin moisturizing and barrier related) gene. The cortex dictamni extract can be used as an effect additive for medicines, health products and skin external preparations for skin care, and can obviously improve the skin problems of atopic dermatitis/eczema.

Disclosure of Invention

In one aspect, the present invention provides a cortex dictamni extract having anti-inflammatory, anti-allergic, anti-irritant and/or anti-itching effects, wherein the cortex dictamni extract is prepared by a solvent extraction method. In a preferred embodiment, the cortex dictamni extract is extracted with water as a solvent. In a preferred embodiment, said anti-inflammatory, anti-allergic, anti-irritant and/or anti-itching effect is achieved by inhibiting thymic stromal lymphopoietin TSLP and/or promoting filaggrin FLG gene expression.

In another aspect, the present invention provides a composition comprising the extract of cortex dictamni, wherein the composition is a pharmaceutical product, a health food or a skin external agent. In a preferred embodiment, said anti-inflammatory, anti-allergic, anti-irritant and/or anti-itching effect is achieved by inhibiting thymic stromal lymphopoietin TSLP and/or promoting filaggrin FLG gene expression. In a preferred embodiment, the composition is for ameliorating atopic dermatitis, eczema, allergic asthma, allergic rhinitis, allergic contact dermatitis, urticaria or combinations thereof.

In a preferred embodiment, the cortex dictamni extract is used in an amount of 0.001 to 100% by weight when the composition is used as an external preparation for skin. In a preferred embodiment, the cortex Dictamni extract is used in an amount of at least 0.01% by weight when the composition is used as a pharmaceutical. In a preferred embodiment, the cortex Dictamni extract is used in an amount of at least 1 wt.% or more as a health food.

Drawings

FIG. 1 shows the results of the inflammatory factor TSLP assay.

FIG. 2 shows the results of gene testing.

Detailed Description

The invention relates to a cortex dictamni extract, and preparation and application thereof, and finds that the cortex dictamni extract has the effects of TSLP secretion inhibition and promotion of filaggrin FLG gene expression in research, and shows a remarkable itching-relieving effect on an animal model. Thus, the extract of cortex dictamni can be used for alleviating allergy-related diseases and/or for skin care. The cortex Dictamni Radicis extract can be used as functional additive for preparing medicines, health foods or skin external preparations for improving atopic dermatitis, eczema, allergic asthma, allergic rhinitis, allergic contact dermatitis, urticaria, etc. Furthermore, the cortex dictamni extract can be used as an efficacy additive in medicines, health products and skin external preparations for skin care, and can remarkably improve the skin problems of atopic dermatitis and/or eczema.

To provide a more concise description, some of the quantitative representations presented herein are not modified by the term "about". It is understood that each quantity given herein is intended to refer to the actual given value, regardless of whether the term "about" is explicitly used, and also to refer to the approximation to such given value that would reasonably be inferred by one of ordinary skill in the art, including approximations due to experimental and/or measurement conditions for such given value.

To provide a more concise description, some quantitative expressions are recited herein as a range from about an X amount to about a Y amount. It should be understood that when a range is recited, the range is not limited to the upper and lower limits recited, but includes the entire range from about the X amount to about the Y amount or any amount therebetween.

Cortex Dictamni Radicis extract

Cortex Dictamni Radicis is the dried root bark of Dictamnus dasycarpus (Dictamnus dasycarpus Turcz.) belonging to Dictamnus of Rutaceae, and is also called cortex Dictamni Radicis, herba Elsholtziae Blumeae Balsamiferae, caulis et folium Paeoniae Japonicae, and rhizoma Pileae Scriptae. The dittany bark is listed as a Chinese article from Shen nong Ben Cao Jing (Shen nong's herbal); the compendium of materia medica considers that the dittany bark is the essential herb of all-weather arthralgia due to wind-cold dispersing and bitter and dry taste. The Jingyue complete book also mentions: the root-bark of shaggy-fruited dittany is bitter and cold in flavor and dry in nature and descending, which is a medicine for treating yin-yang hyperactivity of hand and foot. It is especially indicated for all kinds of sores due to wind-evil, scabies, ringworm, red and rotten sores, sores and sores of waxberry, and eyebrows and hair loss. While good at treating sores and ulcers, it is the key herb for Zhu Huang and Feng Bi. It is shown that cortex Dictamni Radicis has the effects of clearing away heat and toxic materials, dispelling pathogenic wind, removing dampness, and relieving itching. Modern pharmacological studies show that the cortex dictamni contains dictamnine, fraxinellone and phellodendron ketone active ingredients, and has the effects of resisting inflammation and bacteria, resisting tumors, protecting nerves, resisting platelet aggregation, relaxing blood vessels, and the like.

The cortex dictamni extract is used as an effective active substance to be applied to medicines, health-care foods or skin external preparations, and has the effects of repairing skin, repairing and strengthening a barrier, rejuvenating the skin, relieving and calming, cooling and moistening for repair, safely relaxing, repairing and regenerating skin, relaxing and relieving itching, improving redness and stabbing pain, relieving skin pruritus and desquamation, relieving dry and sensitive fragile skin, improving dryness and the like.

The traditional study has more anti-inflammatory, itching relieving and bacteriostatic effects on the cortex dictamni, clinically, the cortex dictamni has more application in the aspects of atopic dermatitis and eczema, but the cortex dictamni is in a composition form, and specific action mechanisms of the cortex dictamni in the aspects of eczema and atopic dermatitis are not clearly reported, and the cortex dictamni mostly focuses on inhibiting histamine and macrophage inflammation medium release. According to the literature and the existing patents, no single drug of the cortex dictamni is reported to inhibit TSLP and improve barrier.

The invention unexpectedly discovers that the cortex dictamni extract has the effect of inhibiting TSLP, animal itching-relieving experiments also prove that the cortex dictamni extract has good itching-relieving effect, and simultaneously discovers that the cortex dictamni extract has the effect of improving the mRNA expression of a filaggrin (FLG, skin moisturizing and barrier related) gene. The cortex dictamni extract can be used as an effect additive for medicines, health products and skin external preparations for skin care, and can obviously improve the skin problems of atopic dermatitis/eczema.

As the extraction method, a general solvent extraction method such as hot extraction, cold leaching, and percolation can be used, and a person skilled in the art can select an appropriate extraction method according to the actual situation.

In the solvent extraction of the cortex dictamni, an extraction solvent having the following characteristics should be selected: the solubility is good, namely the solubility of the solvent to required components is high, and the solubility to impurities is low, or vice versa; inertness, i.e. the solvent cannot react chemically with the plant constituents, even if the reaction is reversible; the operation is simple, the solvent can be conveniently separated from the extract after the extract is extracted, or the extract dissolved in the solvent can be directly used in the next step; and fourthly, the method is economic and safe, namely, the method needs to consider economy, easy obtaining, low toxicity, convenient recovery and repeated use, certain safety and small environmental pollution when selecting the solvent.

The extraction can be carried out using an inert solvent and a reaction solvent. The inert solvent is a solvent which does not react with plant components, and can be water, ethanol, methanol, benzene, chloroform, ethyl acetate, acetone, etc. The reaction solvent is a solvent capable of increasing the solubility of acidic and alkaline components in plant in polar solvent, and can be diluted acid or diluted alkaline aqueous solution or alcoholic solution, such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, tartaric acid, sodium hydroxide, sodium carbonate, and sodium bicarbonate solution.

In some embodiments of the invention, the extraction is performed with water (e.g., deionized water). In some embodiments of the invention, the extraction is performed with ethanol. In some embodiments of the invention, ethanol may be used at a concentration of 40% to 100% by volume, preferably 60% to 98% ethanol, more preferably 70% to 95%, and most preferably 95%. In some embodiments of the invention, the extraction is performed with propylene glycol. In some embodiments of the invention, a concentration of 40% to 100% propylene glycol by volume may be used, preferably 40% to 80% propylene glycol, more preferably 40% to 70%, and most preferably 50% ethanol.

The cortex dictamni extract can be prepared by ultrasonic extraction. For example, ethanol (e.g., 70% ethanol by volume) can be used to assist the sonication process for extraction.

The cortex dictamni extract can be prepared by reflux extraction. For example, ethanol (e.g., 40% ethanol by volume) can be used to assist the reflux process for extraction.

The cortex dictamni extract can be prepared by microwave extraction. For example, water-assisted microwave methods can be used for extraction.

Medicine, health food or skin external preparation containing cortex Dictamni Radicis extract

The cortex dictamni extract can be used as an effect additive to be applied to medicines, health-care foods or skin external preparations.

In some embodiments, the pharmaceutical product is selected from: tablets, capsules, emulsions, suspensions, powders, granules, solutions, and various pharmaceutical dosage forms known in the art. Different amounts are added according to different types of dosage forms.

The health food is also called functional food. It has functions of regulating human body functions, but does not aim at treating diseases, and is suitable for specific people. In some embodiments, the health food may be in a powder form.

In some embodiments, the external skin agent is selected from: face cleaning lotion, cosmetic water, lotion, cream, jelly and facial mask. Different amounts are added according to different types of preparations.

The external preparation for skin is a general concept of all ingredients generally used for the external part of skin, and may be, for example, a cosmetic composition. The cosmetic composition can be basic cosmetics, face makeup cosmetics, body makeup cosmetics, hair care cosmetics and the like, and the dosage form of the cosmetic composition is not particularly limited and can be reasonably selected according to different purposes. The cosmetic composition also contains different cosmetically acceptable media or matrix excipients according to different dosage forms and purposes.

The external preparation for skin contains a dermatologically acceptable carrier or vehicle (e.g., lotion, cream, ointment, cleanser, etc.). One of ordinary skill in the art will be able to select a carrier that will dissolve or disperse the components in the concentrations described above, based on common general knowledge in the art. When a carrier is used, the carrier should not cause inactivation of the extract of cortex Dictamni, and should not cause any adverse effect on the skin during use.

One of ordinary skill in the art will be able to select suitable carriers, including for example water, alcohols, oils, and the like, based on the common general knowledge and their ability to dissolve or disperse in the active ingredient at the concentrations most suitable for treatment.

The skin external preparation of the present invention may be in the form of a topical application product, which can be externally applied to the skin, and can be prepared by those ordinary techniques well known in the art. The carrier may take any of a variety of practical forms such as a cream, dressing, gel, lotion, ointment or liquid comprising the applied and rinsed-off composition and incorporated into a carrier of material such as a dry or wet spread, hydrogel matrix, or adhesive (or non-adhesive) patch by methods well known in the art. Preferably, the carrier is a gel or a lotion that adds moisture, or a spread in dry or wet form.

Typical carriers include emulsions comprising water and/or an alcohol and an emollient such as hydrocarbon oils and waxes, silicone oils, hyaluronic acid, vegetable, animal or marine fats or oils, glyceride derivatives, fatty acids, or fatty acid esters or alcohols or alcohol ethers, lanolin and its derivatives, polyols or esters, wax esters, sterols, phospholipids and the like, and typically emulsifiers (nonionic, cationic or anionic), although some emollients inherently have emulsifying properties. In addition, these same components may be formulated into creams, gels, or solid sticks using different ratios of their components and/or by incorporating thickeners such as gums or other forms of hydrocolloids.

The skin external agent of the present invention may contain additional components commonly found in skin care compositions, such as emollients, skin conditioning agents, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, and the like, as long as they are physically and chemically compatible with the other components of the skin external agent and do not affect the effect of the cortex dictamni extract of the present invention.

In some embodiments of the external preparation for skin of the present invention, one or more preservatives may be used. Suitable preservatives include p-hydroxyacetophenone, C1-C4 alkyl parabens, and phenoxyethanol. Preservatives are used in amounts of about 0.5 to about 2 weight percent, preferably about 0.5 to 1 weight percent, based on the total weight of the composition.

In one example of the external preparation for skin of the present invention, one or more antioxidants may be used. Suitable antioxidants include Butylated Hydroxytoluene (BHT), ascorbyl palmitate (BHA), butylated hydroxyanisole, phenyl-alpha-naphthylamine, hydroquinone, gallopropyl, nordihydroguaiaretic acid, vitamin E or derivatives of vitamin E, vitamin C and its derivatives, calcium pantothenate, green tea extract and mixed polyphenols, and mixtures of the above. The antioxidant is used in an amount ranging from about 0.02 to 0.5 weight percent, more preferably from about 0.002 to 0.1 weight percent, based on the total weight of the composition.

In one example of the external preparation for skin of the present invention, one or more emollients may be used, functioning as a lubricant by their ability to remain on the surface of the skin or in the stratum corneum, to reduce flaking, and to improve the appearance of the skin. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether siloxane copolymers, and the like. Examples of suitable emollients include, without limitation, polypropylene glycol ("PPG") -15 stearyl ether, PPG-10 cetyl ether, Steareth-10, Oleth-8, PPG-4 lauryl ether, vitamin E acetate, lanolin, cetyl alcohol, cetearyl ethylhexanoate, cetearyl alcohol, glyceryl stearate, octyl hydroxystearate, dimethylpolysiloxane, and combinations thereof. Cetyl alcohol, cetearyl ethylhexanoate, cetostearyl alcohol, glyceryl stearate and combinations thereof are preferred. When used, the emollient is present in an amount ranging from about 0.1 to about 30 weight percent, preferably from about 1 to about 30 weight percent, based on the total weight of the composition.

In one example of the external preparation for skin of the present invention, one or more moisturizers other than the cortex dictamni extract of the present invention may be used. Humectants, also known as humectants, help to increase the effectiveness of the emollient, reduce flaking, stimulate the removal of compositional scales, and improve skin feel. Polyhydric alcohols may be used as humectants, including, but not limited to, glycerin, polyalkylene glycols, alkylene polyols and derivatives thereof, including butylene glycol, propylene glycol, dipropylene glycol, polyglycerol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1, 3-dibutylene glycol, 1,2, 6-hexanetriol, ethoxylated glycerin, propoxylated glycerin, and combinations thereof. When used, the humectant is present in an amount of about 0.1 to about 20 weight percent, preferably about 1 to about 15 weight percent, based on the total weight of the composition.

In one example of the external preparation for skin of the present invention, one or more emulsifiers may be used. The emulsifier may be used in an effective stabilizing amount range. Preferably, the emulsifier is used in an amount of about 1.0 to about 10.0 wt%, more preferably about 3.0 to about 6.0 wt%, based on the total weight of the composition. Any emulsifier that is compatible with the components of the composition may be used. Suitable emulsifiers include stearic acid, cetyl alcohol, glyceryl stearate, lecithin, stearyl alcohol, Steareth-2, Steareth-20, acrylic/C10-30 alkanol acrylate crosspolymer, and combinations thereof.

In one example of the external preparation for skin of the present invention, one or more pH adjusting agents may be used. The pH adjusting agent useful in the skin external preparation of the present invention includes tromethamine. When used, the pH adjusting agent is used in an amount of about 0.1 to about 2 weight percent, preferably about 0.1 to about 1 weight percent, based on the total weight of the composition.

In one embodiment of the present invention, the external preparation for skin comprises acrylic acid/C10-30 alkanol acrylate crosspolymer, glycerin, p-hydroxyacetophenone, glyceryl stearate and lecithin, cetostearyl alcohol, cetearyl ethyl hexanoate, tromethamine or a combination thereof.

In some embodiments of the present invention, the amount of the extract of cortex dictamni in the external preparation for skin is 0.001% -20% (w/w), preferably 0.01% -20% (w/w), more preferably 0.01% -10% (w/w), and most preferably 0.1% -5% (w/w).

In one embodiment of the present invention, the extract of cortex dictamni is used in an amount of 0.3-10 wt% in the skin external preparation. In one embodiment of the present invention, the extract of cortex dictamni is used in an amount of 0.3-5 wt% in the skin external preparation.

Examples

The invention will be further illustrated by the following specific examples. It should be noted that the examples are given solely for the purpose of illustration and are not to be construed as limitations on the scope of the invention, as many insubstantial modifications and variations of the invention may be made by those skilled in the art in light of the above teachings. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the manufacturer. All percentages and parts are by weight unless otherwise indicated.

The dittany bark used in the following examples was obtained from the national drug reagent, ethanol and propylene glycol, and filter paper from Whatman corporation. Macroporous resins employed in the examples include: nonpolar resins, D-101 (Nankai Synthesis technology), XAD2 (Rohm and Haas), HP-20 (Mitsubishi chemical), HZ-802 (Wachang polymers, Inc.); weak polar resin, AB-8 (Nankai Synthesis technology), DM-130 (Tianjin Hai Guang), XAD7 (Rohm and Haas); polar resins, DM-301 (Tianjin Hai Shi), NKA-9 (Nankai Synthesis technologies), HZ-806 (Huachang polymers).

Example 1: preparation of cortex Dictamni Radicis extract

Extracting cortex Dictamni Radicis 50g with 10 times deionized water for 2 times, each time for 1 hr, filtering with a screen (20 mesh, Shanghai Yichang apparatus screen mill), concentrating the filtrate to 1 time crude drug amount, adding a certain amount of 95% ethanol for precipitating, filtering with filter paper, concentrating and recovering to no alcohol smell, and making into solution with solid content of 1%.

Example 2: preparation of cortex Dictamni Radicis extract

75g of cortex dictamni, extracting for 1 time by using 20 times of deionized water, extracting for 1.5h, centrifuging (centrifuging at 4000rpm for 15min), filtering, concentrating the filtrate to 1 time of crude drug amount, adding a certain amount of 95% ethanol for alcohol precipitation, centrifuging (centrifuging at 4000rpm for 15min), filtering, concentrating and recovering until no alcohol smell exists, feeding the filtrate to macroporous resin AB-8, eluting and recovering ethanol, and finally preparing into a solution with the solid content of 1%, thus obtaining the cortex dictamni solid-liquid separation.

Example 3: preparation of cortex Dictamni Radicis extract

Extracting cortex Dictamni Radicis 125g with 15 times of 70% ethanol under ultrasound for 2 times, each for 45min at 35 deg.C for 3s, centrifuging for 3s at 4000rpm for 15min, filtering, concentrating the filtrate, recovering ethanol, and lyophilizing to obtain powder.

Example 4: preparation of cortex Dictamni Radicis extract

Extracting cortex Dictamni Radicis 200g with 8 times of 40% ethanol at 65 deg.C under reflux for 2 times, each for 2 hr, filtering with filter paper, concentrating the filtrate to 1 time of crude drug amount, adding a certain amount of 95% ethanol for precipitating, centrifuging (4000rpm for 15min), filtering, concentrating and recovering until no alcohol smell exists, filtering with ultrafiltration membrane, and concentrating to solid content of 2% solution.

Example 5: preparation of cortex Dictamni Radicis extract

250g of cortex dictamni, extracting with 15 times of deionized water by microwave for 1 time at 40 ℃ for 1 hour under microwave, centrifuging at 4000rpm for 15min, filtering, concentrating the filtrate to 1 time of crude drug amount, adding a certain amount of 95% ethanol for alcohol precipitation, centrifuging at 4000rpm for 15min, filtering, concentrating and recovering ethanol, and finally preparing into a solution with 3% of solid content.

Example 6: preparation of cortex Dictamni Radicis extract

275g of cortex dictamni, ultrasonic extracting for 1 time by using 8 times of 50 percent propylene glycol, centrifuging (centrifuging at 4000rpm for 15min), filtering, adding a proper amount of deionized water and propylene glycol into filtrate, and preparing into a solution with the solid content of 4 percent.

Test example 1: detection based on eczematoid 3D skin model

1. Test materials

1.1 cells: the skin model is a 3D epidermal skin model(batch number: ES210402)

1.2 reagent:

PBS, MTT (Sigma), DMSO (Sigma), MEM (Gibco), Human TSLP ELISA kit (Boshide), isopropanol (national drug), PolyI: C (Sigma), LPS (E.Coli.Sigma), AlamarBlue (Solebao), RNAioso Plus (TaKaRa), absolute ethanol (national drug group), DEPC water (Beyotime), reverse transcription kit (TaKaRa), fluorescent dye (TaKaRa).

1.3 Main Equipment

CO₂The kit comprises an incubator (Thermo), an ultra-clean workbench (Sujing Antai), an inverted microscope (Olympus), an enzyme labeling instrument (BioTek), a high-speed refrigerated centrifuge (Changshan Xiang instrument), a common PCR instrument (BIO-RAD), a real-time fluorescence quantitative PCR instrument (BIO-RAD) and a digital display constant-temperature water bath kettle (Hezhou national center).

2. The experimental method comprises the following steps:

2.1 preparation of test solutions

Poly i C + LPS induced working solution: 1.44mL of 5mg/mL PolyI: C stock solution and 3mL of 2mg/mL LPS stock solution were dissolved in the skin model culture solution in a total volume of 300mL so that the final concentration of PolyI: C was 24. mu.g/mL and the final concentration of LPS was 20. mu.g/mL.

2.2 administration and Induction

Preparing a 6-well plate, adding fresh normal culture solution into a blank BC group, and adding induction working solution into a model NC group and a sample group (prepared into samples to be detected 1-5 with volume concentrations of 0.1%, 0.3%, 0.5%, 3% and 5% respectively from the cortex dictamni extract in example 1); after the administration incubation was completed, the 6-well plate was taken out from the incubator, the bottom of the skin model was washed with sterile PBS, and PBS residual solution was wiped with a sterile cotton swab, and then the skin model was transferred to a new 6-well plate prepared in advance. After completion, the 6-well plate was placed in CO₂Incubator (37 ℃, 5% CO)₂) And (4) performing medium incubation for 24 h.

2.3 inflammatory factor TSLP assay

(1) Collecting a sample: after the induction incubation is finished for 24h, collecting the model culture solution in an EP tube, and storing in a refrigerator at the temperature of minus 80 ℃.

(2) And (3) ELISA detection: the assay was performed according to the instructions for the TSLP ELISA test kit.

2.4 Gene detection

After collecting samples, RNA extraction, reverse transcription and fluorescent quantitative PCR detection are carried out according to the instruction of a kit (TaKaRa), and the result calculation is carried out by adopting a 2-delta-Delta CT method.

2.5 statistical analysis of test results

Statistical analysis of t-test is carried out among groups, p <0.05 indicates that the difference is significant, and p <0.01 indicates that the difference is extremely significant.

2.6 results of the experiment

Table 1 shows the grouping of the inflammatory factor TSLP assay.

TABLE 1

The results of the inflammatory factor TSLP assay are shown in figure 1. As can be seen from the TSLP assay results, the cortex dictamni extract of example 1 was administered at concentrations ranging from 0.5%, 3% and 5% with the inhibitory effect of TSLP (. p <0.05,. p <0.01) and exhibited concentration dependence. The experimental results show that the cortex dictamni extract of example 1 has good TSLP inhibition effect.

Table 2 shows the grouping of the gene testing experiments.

TABLE 2

The results of the gene testing experiments are shown in FIG. 2. Compared with the BC group, the expression of the FLG gene of the NC group is remarkably reduced (p is less than 0.05), which indicates that the experiment is effective. Compared with the NC group, FLG gene expression of sample 1 (the group with 0.1% of the administered concentration of the cortex dictamni extract of example 1) was significantly increased (p <0.05), and FLG gene expression of sample 5 (the group with 5% of the administered concentration of the cortex dictamni extract of example 1) was significantly increased (p <0.01), indicating that the cortex dictamni extract of example 1 has a good effect of promoting FLG gene expression.

Test example 2: animal (guinea pig) itching relieving experiment of cortex Dictamni Radicis extract

1. Animals: guinea pigs 30 (shanghai jestie laboratory animals ltd, 10 per group) weight: about 300g, half female/male, license for laboratory animals SYXK (Shanghai) 2019-0027 Shanghai Jitsie laboratory animals Limited: SCXK (Shanghai) 2018-0004

2. Histamine phosphate: LOT X1759B70455 of Shanghai-sourced leaf Biotechnology Limited

Blank control, distilled water

3. Positive control: clobetasol propionate cream (10g:2mg) 19010701# of Jiangsu Yunhou medicine industry

Test samples: example 1 cortex Dictamni Radicis extract was administered at a volume concentration of 10%

4. The test conditions are as follows: laboratory temperature: 20-25 ℃, relative humidity: 60 to 70 percent

5. The test method comprises the following steps:

guinea pigs were randomly divided into 3 groups of 10 animals each. A blank control group, a positive control group and a sample group (10% of the cortex dictamni extract in example 1) are divided. One day prior to the experiment, the right hind instep of each group of guinea pigs was painted with a sample. On the day of the experiment, the shaved area of the right hind paw of the animal was scratched with a coarse sandpaper in the range of about 1 cm square, and the sample was applied once again to the shaved area, and the blank control group was given an equal amount of distilled water. After 10 minutes, 0.01% histamine phosphate 0.05 ml/tube was initially dropped on the wound surface, and 0.01%, 0.02%, 0.03%, 0.04% … … was added at 3 minute intervals in increasing concentrations, each 0.05 ml/tube. Until the guinea pig licks the right hind paw, the sum of histamine phosphate administered when the guinea pig licks the right hind paw was last taken as the scratchiness threshold. The scratchiness threshold of each group was recorded and compared.

6. The experimental results are as follows:

table 3 shows the effect of the extract of cortex Dictamni on histamine phosphate-induced itching.

TABLE 3

P value: are all compared with a blank control

As can be seen from the above table, the test sample (example 1, 10% of the administered cortex Dictamni Radicis extract) has a very significant effect of increasing the scratchiness threshold of guinea pigs (P <0.01) compared to the blank control sample. The positive control group is a hormone ointment group (clobetasol propionate cream), and the itching relieving effect (improvement of the itching causing threshold) of the test sample (the group with the administration concentration of the cortex dictamni extract of example 1 being 10%) is equivalent to that of 1/3 of the hormone ointment group, which shows that the test sample has excellent itching relieving effect and is safe (compared with small side effect of hormone) and effective.

Application example

The cortex dictamni extract can be used as an intermediate raw material or a functional additive for preparing medicines, health-care foods or skin external preparations, and the skin external preparations are preferably cosmetic compositions, including but not limited to cream, lotion, jelly, cosmetic water, essence, facial masks, eye cream, aerosol (cleaning foam), spray, shower gel, facial cleanser and other dosage forms.

Examples 1-6 the extract of Densefruit Pittany root-bark was contained in the skin external preparation in an amount of 0.0001% to 20% (w/w). Preferably 0.001-10% (w/w). More preferably 0.001-5% (w/w). Most preferably 0.01% to 5% (w/w).

The following are specific examples of the use of the extract of cortex Dictamni in external preparations for skin obtained in examples 1-6, and the formulation and preparation method of these preparations. The specific application examples are as follows:

application example 1: preparation of capsules

Taking the solid solution of the cortex dictamni extract prepared in the example 1, concentrating the solid solution into thick paste, adding maltodextrin, mixing uniformly, granulating, and encapsulating, wherein each capsule contains 0.2g of the solid solution, and preparing into 1000 capsules.

Application example 2: preparation of Chinese medicinal preparation

The solid solution of the cortex dictamni extract prepared in the example 2 is ultrafiltered by an ultrafiltration membrane at room temperature, the molecular weight cut-off is more than 30000, and the ultrafiltrate (the molecular weight is less than 30000) passing through the ultrafiltration membrane is stored for standby. Filtering ultrafiltrate with molecular weight less than 30000 with nanofiltration membrane at room temperature, and retaining concentrated solution with molecular weight greater than 300. And (3) carrying out sterile freeze drying on the concentrated solution with the molecular weight of more than 300 to prepare freeze-dried powder. The freeze-dried powder is prepared into capsules and powder injections to obtain the target product, and the target product is prepared into the traditional Chinese medicine preparation by using the cortex dictamni extract one by one.

Application example 3: preparation of hip-protecting ointment

Application example 4: hair and body washing bubble

Application example 5: preparation of face cream

Application example 6: preparation of the emulsion

Application example 7: preparation of jelly

Application example 8: preparation of toner

Application example 9: preparation of essence

Example 10 should be: preparation of facial mask

Application example 11: preparation of eye cream

Application example 12: preparation of an aerosol (cleaning foam)

Application example 13: preparation of the spray

Application example 14: preparation of shampoo and shower gel

Application example 15: preparation of facial cleanser