CN106309613A - Traditional Chinese medicine with anti-inflammatory and anti-allergic effects, and preparation method therefor - Google Patents

Abstract

The invention provides a traditional Chinese medicine with anti-inflammatory and anti-allergic effects. The traditional Chinese medicine comprises fructus crataegi, radix ginseng and herba artemisiae annuae. The traditional Chinese medicinal composition provided by the invention can overcome safety problems existing in preparation of western medicine; a medicine with relatively high activity and capable of blocking off a CaN-NF-AT access is screened out from the traditional Chinese medicine, so that scientific basis is provided for treating inflammation of eczema and the like, and autoimmune diseases; and meanwhile, a quite good material is supplied to cosmetics with a relieving effect.

Description

There is Chinese medicine of antiinflammatory, anti-allergic effects and preparation method thereof

Technical field

The present invention relates to a kind of medicine and manufacture method thereof.

Background technology

Calcineurin (caleineurin, CaN) also known as PP2B (PP2B), belongs to serine/threonine protein phosphatase family member, is find so far unique by Ca²⁺The serine/threonine protein phosphatase that/calmodulin (CaM) regulates.CaN plays a role by making substrate dephosphorylation.In T cell, Ca²⁺Interior stream, IC Ca²⁺Increase, Ca²⁺Be combined with CaM with CaN, activate CaN, the CaN of activation and make its substrate NF-AT dephosphorylation, enter core, cause the expression of some row cytokines such as IL-2.CaN-NF-AT path plays a part to regulate hinge in T cell activation.

Calcineurin inhibitors is currently the most maximally effective immune suppressant drug.For organ transplantation, control transplant rejection, the treatment (RA, CD, psoriasis) of autoimmune disease, particularly it is in recent years for eczema and obtain good therapeutic effect.Calcineurin inhibitors (CNIs), as a kind of immunosuppressant, is divided into exogenous inhibitor and endogenous protein inhibitor.Exogenous inhibitor mainly has ciclosporin, tacrolimus etc., endogenous protein inhibitor mainly to have Cain, FKBP38 etc..At present, what clinical practice was most is that ascomycin derivative ciclosporin A, tacrolimus and a Meike are not taken charge of, all there is similar physicochemical property, mechanism of action and action effect, but its toxic and side effects (such as nephrotoxicity, hyperglycemia etc.) becomes the significant obstacle of such inhibitor application.Therefore, more safely and effectively inhibitor is found with CaN-NF-AT path for action target significant to research and development novel skin inflammation.

Chinese herbal medicine is traditional medication of China, and clinically, Chinese herbal medicine has preferable effect in the treatment of the inflammation such as eczema and autoimmune disease.Modern study finds that some Chinese medicine monomer composition directly can be combined with CaN and suppress its activity, such as Quercetin, kaempferol, phenolic aldehyde gossypol etc.；Meanwhile, some Effective Component of Chinese Medicine can effectively suppress the activation of CaN-NF-AT path, the expression of suppression Th1 and Th2 cytokines at cellular level, and the activation of suppression T cell, such as cordycepin, farrerol, fisetin etc..Chinese herbal medicine has that source is wide, low cost and the low feature of toxicity, and with it as the inhibitor of screening CaN-NF-AT path, the probability that the while of obtaining having good therapeutic effect, toxicity is little is the biggest.

Summary of the invention

The technical problem to be solved is to overcome the safety issue of Western medicine preparation, from Chinese medicine, screening obtains the medicine with the preferably blocking-up CaN-NF-AT path of activity, provide scientific basis for the inflammation such as treatment eczema and autoimmune disease, provide good material for class cosmetics of releiving simultaneously.

The invention provides a kind of Chinese medicine with antiinflammatory, anti-allergic effects, described Chinese medicine is included as Fructus Crataegi, Radix Ginseng and Herba Artemisiae Annuae.

Wherein, the content of described Fructus Crataegi is 50～60wt%, the content of described Radix Ginseng is 20～30wt%, and the content of described Herba Artemisiae Annuae is 20～30wt%.

Present invention also offers the application in blocking CaN-NF-AT path of a kind of above-mentioned Chinese medicine.

Present invention also offers the preparation method of a kind of above-mentioned Chinese medicine, comprise the steps of:

(1) Radix Ginseng is added 50wt%～the 70wt% ethanol of its 15～30 times amount, after percolation extracts 12～24h, it is evaporated to the weight such as medical material, adding cellulase preparation enzyme work is 400～2000IU, 12～24h are hydrolyzed under the conditions of 15～20 DEG C, it is heated to more than 60 DEG C, makes enzyme-deactivating, obtain hydrolysis Radix Ginseng extract；

(2) Fructus Crataegi and Herba Artemisiae Annuae are separately added into the 95wt% ethanol of 30 times amount, and percolation extracts；

(3) percolation extracts 24h, collects filtrate；

(4) concentrate under reduced pressure at low temperature is to weight such as medical materials, obtains Fructus Crataegi and Herba Artemisiae Annuae extract；

(5) according to Fructus Crataegi extract 50～60wt%, Herba Artemisiae Annuae extract 20～30wt%, hydrolysis Radix Ginseng extract 20～30wt% mix homogeneously, natural antiinflammatory, antiallergic extract are obtained.

The Chinese medicine composition that the present invention provides has obvious synergism each other antiinflammatory, antianaphylactic using, and can mutually be in harmonious proportion, reduce toxicity.

The Chinese medicine composition that the present invention provides has the antiinflammatory of excellence, anti-allergic effects.

Detailed description of the invention

Technical scheme is further illustrated below by detailed description of the invention.It is understood that specific embodiment described herein is used only for explaining the present invention, rather than limitation of the invention.It also should be noted that, for the ease of describing, specific embodiment illustrate only part related to the present invention rather than full content.

Embodiment 1:

(1) take 50g Fructus Crataegi and add the extraction of 150g 95wt% alcohol percolation method；

(2) percolation extracts 24h, collects filtrate；

(3) concentrate under reduced pressure at low temperature is to 50g, adds propylene glycol and is diluted to 100g, obtains Fructus Crataegi extract.

Embodiment 2:

(1) take 50g Herba Artemisiae Annuae and add the extraction of 150g 95wt% alcohol percolation method；

(2) percolation extracts 24h, collects filtrate；

(3) concentrate under reduced pressure at low temperature is to 50g, adds propylene glycol and is diluted to 100g, obtains Herba Artemisiae Annuae extract.

Embodiment 3:

(1) take 50g Radix Ginseng and add the extraction of 150g70wt% alcohol percolation method；

(2) percolation extracts 24h, collects filtrate；

(3) concentrate under reduced pressure at low temperature is to 50g, adds cellulase, and regulatory enzyme is lived as 1000IU, under the conditions of 20 DEG C, hydrolysis 20h；

(4) heating, after making enzyme-deactivating, adds propylene glycol and is diluted to 100g, obtain water Radix Ginseng extract.

Embodiment 4:

By embodiment 1,2,3 extract, take 50g Fructus Crataegi extract, 20g Herba Artemisiae Annuae extract, g hydrolyze Radix Ginseng extract, mix homogeneously obtains natural antiinflammatory, antiallergic extract.

Comparative example 1:

(1) take 50g Radix Ginseng and add the extraction of 150g70wt% alcohol percolation method；

(2) percolation extracts 24h, collects filtrate；

(3) concentrate under reduced pressure at low temperature is to without ethanol, adds propylene glycol and is diluted to 100g, obtains Radix Ginseng extract.

Comparative example 2:

(1) take 50g Herba Leonuri and add the extraction of 150g95wt% alcohol percolation method；

(2) percolation extracts 24h, collects filtrate；

(3) concentrate under reduced pressure at low temperature i.e. obtains Herba Leonuri extract.

Comparative example 3:

(1) take 50g Herba Epimedii and add the extraction of 150g95wt% alcohol percolation method；

(2) percolation extracts 24h, collects filtrate；

(3) concentrate under reduced pressure at low temperature i.e. obtains Herba Epimedii extract.

Comparative example 4:

(1) take 25g Fructus Crataegi and add the extraction of 150g95wt% alcohol percolation method；

(2) percolation extracts 24h, collects filtrate；

(3) concentrate under reduced pressure at low temperature i.e. obtains Fructus Crataegi extract I.

Comparative example 5:

(1) take 50g Herba Chenopodii and add the extraction of 150g95wt% alcohol percolation method；

(2) percolation extracts 24h, collects filtrate；

(3) concentrate under reduced pressure at low temperature i.e. obtains Herba Chenopodii extract.

Comparative example 6:

(1) take 50g Radix Arnebiae (Radix Lithospermi) and add the extraction of 150g95wt% alcohol percolation method；

(2) percolation extracts 24h, collects filtrate；

(3) concentrate under reduced pressure at low temperature i.e. obtains Radix Arnebiae extract.

One, detection method:

1. the screening step of calcineurin (CaN)-NF-AT path blocker:

1.1 testing samples: all extracts, are diluted to the working solution of mg/ml with deionized water, take 10 μ l and add mensuration pipe, the final concentration of 0.2mg/ml of extract to be measured during detection.

1.2 assay methods: use the cell line NFAT K562Reporter Stable Cell Line that have purchased stable transfection NFAT reporter gene from affimatrix company of the U.S., K562 cell is stimulated with PMA (Protein kinase C PKC activator) and A23187 (calcium channel activators), the luciferase gene expression level that detection transcription factor NFAT drives, the activation levels of reaction calcineurin (CaN) path.

The K562 cell routine Secondary Culture of stable transfection NFAT reporter gene is in the RPMI-1640 culture medium containing 10wt% hyclone, and experiment uses healthy growth, is in the cell of increased logarithmic phase, by above-mentioned cell by 2 × 10⁵/ hole is inoculated in 24 porocyte culture plates, add testing sample, hatch 1 hour, add PMA (10ng/ml) and A23187 (0.5 μ Μ) (path stimulant), stimulate 18h, collect cell, use the operation of Luciferase Assay Reagent box by specification, measure fluorescence intensity lum value (reflecting intracellular reaction calcineurin pathway activation levels).

2. the cytotoxicity detection of Chinese herbal medicine extract:

Use conventional CCK8 method: (1) configures the K562 cell suspension (1 × 10 of 100 μ L in 96 orifice plates⁵/ hole), by 96 orifice plates incubator preculture 24 hours (37 DEG C, 5wt%CO₂)；

(2) 10 μ L test substances are added；

(3) 96 orifice plates are hatched 24 hours in incubator；

(4) 10 μ LCCK8 solution are added to every hole；

(5) 96 orifice plates are hatched 4 hours in incubator；

(6) absorbance at 450nm is measured by microplate reader.

Two, experimental result:

The present invention is compared with different Chinese medicines, and product is as shown in table 1 to the inhibitory action result of CaN-NF-AT path

Table 1:

The present invention is compared with different Chinese medicines, and the nonspecific cytotoxicity result of product is as shown in table 2

Table 2:

As shown in Table 1, Fructus Crataegi, Herba Artemisiae Annuae, natural antiinflammatory (antiallergic) extract and Radix Arnebiae extract have the effect preferably suppressing CaN-NF-AT path.But, according to table 2, the cell relative toxicity of Radix Arnebiae extract is relatively big, and its inhibitory action is likely due to what the nonspecific cytotoxicity of Radix Arnebiae (Radix Lithospermi) composition caused.The most as can be seen from Table 1, natural antiinflammatory (antiallergic) extract is substantially better than Fructus Crataegi, Herba Artemisiae Annuae, the single effect of hydrolysis Radix Ginseng extract to the inhibition of CaN-NF-AT path；There is table 2 it can be seen that the toxicity of hydrolysis Radix Ginseng extract is significantly lower than unhydrolyzed Radix Ginseng extract；After Fructus Crataegi, Herba Artemisiae Annuae, hydrolysis Radix Ginseng extract are mixed in proportion, the toxicity that its toxicity is used alone less than three；In two tastes of the present invention, Fructus Crataegi, Herba Artemisiae Annuae extract are used alone the inhibitory action with preferable CaN-NF-AT path, after the two is mixed in proportion with hydrolysis Radix Ginseng extract, the inhibitory action (obvious synergism) with more preferable CaN-NF-AT path can be as antieczematic good material；And after the two is mixed in proportion with hydrolysis Radix Ginseng extract, its toxicity is decreased obviously (obvious antagonism)；Radix Ginseng extract hydrolyzes and does not hydrolyzes, and its toxicity is by notable difference.Simultaneously as use the Chinese medicine extract of pure natural, overcome the hidden danger of the bigger side effect of Western medicine, provide material base for antieczematic and class cosmetics of releiving.

These are only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.

Claims (4)

1. a Chinese medicine with antiinflammatory, anti-allergic effects, it is characterised in that described Chinese medicine is included as Fructus Crataegi, Radix Ginseng and Herba Artemisiae Annuae.

2. Chinese medicine as claimed in claim 1, it is characterised in that: the content of described Fructus Crataegi be 50～ 60wt%, the content of described Radix Ginseng are 20～30wt%, and the content of described Herba Artemisiae Annuae is 20～30wt%.

3. a Chinese medicine as claimed in claim 1 application in blocking CaN-NF-AT path.

4. the preparation method of a Chinese medicine as claimed in claim 1, it is characterised in that comprise as follows Step:

(1) Radix Ginseng adds 50wt%～the 70wt% ethanol of its 15～30 times amount, and percolation carries After taking 12～24h, be evaporated to the weight such as medical material, add cellulase preparation enzyme work be 400～ 2000IU, hydrolyzes 12～24h, is heated to more than 60 DEG C, makes enzyme-deactivating under the conditions of 15～20 DEG C, Hydrolysis Radix Ginseng extract；

(2) Fructus Crataegi and Herba Artemisiae Annuae are separately added into the 95wt% ethanol of 30 times amount, and percolation extracts；

(3) percolation extracts 24h, collects filtrate；

(4) concentrate under reduced pressure at low temperature is to weight such as medical materials, obtains Fructus Crataegi and Herba Artemisiae Annuae extract；

(5) according to Fructus Crataegi extract 50～60wt%, Herba Artemisiae Annuae extract 20～30wt%, hydrolysis people Conopsea extraction 20～30wt% mix homogeneously, obtains natural antiinflammatory, antiallergic extract.