CN106309613B - Chinese medicine with anti-inflammatory and anti-allergic effects and preparation method thereof - Google Patents

Abstract

The invention provides a traditional Chinese medicine with anti-inflammatory and anti-allergic effects, which comprises hawthorn, ginseng and sweet wormwood. The traditional Chinese medicine composition provided by the invention CaN overcome the safety problem of western medicine preparations, and CaN be used for screening medicines with better activity for blocking a CaN-NF-AT passage from the traditional Chinese medicines, so that a scientific basis is provided for treating inflammation such as eczema and autoimmune diseases, and good materials are provided for soothing cosmetics.

Description

Chinese medicine with anti-inflammatory and anti-allergic effects and preparation method thereof

Technical Field

The present invention relates to a medicine and a method for producing the same.

Background

Calcineurin (CaN), also known as protein phosphatase 2B (PP2B), is a member of the serine/threonine protein phosphatase family, and is the only Ca-dependent enzyme discovered to date²⁺A serine/threonine protein phosphatase regulated by calmodulin (CaM). CaN acts by dephosphorylating the substrate. In T cells, Ca²⁺Internal flow, intracellular concentration Ca²⁺Increase of Ca²⁺The protein binds to CaM and CaN to activate CaN, and the activated CaN dephosphorylates a substrate NF-AT and enters a nucleus to cause the expression of a plurality of cytokines such as I L-2.

Calcineurin inhibitors are currently the most clinically effective immunosuppressant drugs. Used for organ transplantation, controlling graft rejection, and treating autoimmune diseases (RA, CD, psoriasis), especially eczema in recent years, and has good therapeutic effect. Calcineurin inhibitors (CNIs) are used as immunosuppressive agents and are classified into exogenous inhibitors and endogenous protein inhibitors. The exogenous inhibitors mainly comprise cyclosporine, tacrolimus and the like, and the endogenous protein inhibitors mainly comprise Cain, FKBP38 and the like. At present, the most clinically applied ascomycin derivatives, namely cyclosporin a, tacrolimus and pimecrolimus, have similar physicochemical properties, action mechanisms and action effects, but the toxic and side effects (such as nephrotoxicity, hyperglycemia and the like) of the ascomycin derivatives become important obstacles for the application of the inhibitors. Therefore, the search for safer and more effective inhibitors by taking the CaN-NF-AT pathway as an action target has important significance for the development of novel skin inflammations.

The Chinese herbal medicine is a traditional medicine in China, and has better effect in treating inflammation such as eczema and autoimmune diseases clinically. Modern researches find that certain traditional Chinese medicine monomer components CaN be directly combined with CaN and inhibit the activity of the CaN, such as quercetin, kaempferol, gossypol phenolate and the like; meanwhile, some traditional Chinese medicine active ingredients CaN effectively inhibit the activation of a CaN-NF-AT passage AT a cellular level, inhibit the expression of Th1 and Th2 cytokines and inhibit the activation of T cells, such as cordycepin, rhododendron, fisetin and the like. The Chinese herbal medicine has the characteristics of wide source, low cost and low toxicity, and has high possibility of obtaining better curative effect and low toxicity by using the Chinese herbal medicine as an inhibitor for screening a CaN-NF-AT passage.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the safety problem of western medicine preparations, and the medicine which has better activity and blocks the CaN-NF-AT channel is obtained by screening from the traditional Chinese medicine, thereby providing scientific basis for treating inflammation such as eczema and autoimmune diseases, and simultaneously providing good materials for soothing cosmetics.

The invention provides a traditional Chinese medicine with anti-inflammatory and anti-allergic effects, which comprises hawthorn, ginseng and sweet wormwood.

Wherein the content of the hawthorn is 50-60 wt%, the content of the ginseng is 20-30 wt%, and the content of the sweet wormwood is 20-30 wt%.

The invention also provides application of the traditional Chinese medicine in blocking the CaN-NF-AT passage.

The invention also provides a preparation method of the traditional Chinese medicine, which comprises the following steps:

(1) adding 15-30 times of 50-70 wt% ethanol into ginseng, extracting for 12-24 h by a percolation method, concentrating under reduced pressure until the weight of the medicinal materials is equal, adding cellulase to prepare 400-2000 IU of enzyme activity, hydrolyzing for 12-24 h at 15-20 ℃, heating to above 60 ℃ to inactivate the enzyme, and obtaining a hydrolyzed ginseng extract;

(2) adding 30 times of 95 wt% ethanol into fructus crataegi and herba Artemisiae Annuae, respectively, and percolating to obtain extract;

(3) extracting for 24h by percolation, and collecting filtrate;

(4) concentrating at low temperature under reduced pressure to equal weight to obtain fructus crataegi and herba Artemisiae Annuae extract;

(5) uniformly mixing 50-60 wt% of hawthorn extract, 20-30 wt% of sweet wormwood herb extract and 20-30 wt% of hydrolyzed ginseng extract to obtain the natural anti-inflammatory and antiallergic extract.

The traditional Chinese medicine composition provided by the invention has obvious synergistic effect on anti-inflammatory and anti-allergic effects, and can be mutually blended and reduce toxicity.

The traditional Chinese medicine composition provided by the invention has excellent anti-inflammatory and anti-allergic effects.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be further noted that, for the convenience of description, only some but not all of the relevant aspects of the present invention are shown in the specific embodiments.

Example 1:

(1) adding 50g of hawthorn into 150g of 95 wt% ethanol for percolation extraction;

(2) extracting for 24h by percolation, and collecting filtrate;

(3) concentrating at low temperature under reduced pressure to 50g, and diluting with propylene glycol to 100g to obtain fructus crataegi extract.

Example 2:

(1) adding 50g of sweet wormwood into 150g of 95 wt% ethanol for percolation extraction;

(2) extracting for 24h by percolation, and collecting filtrate;

(3) concentrating at low temperature under reduced pressure to 50g, and diluting with propylene glycol to 100g to obtain herba Artemisiae Annuae extract.

Example 3:

(1) percolating 50g Ginseng radix with 150g70 wt% ethanol;

(2) extracting for 24h by percolation, and collecting filtrate;

(3) concentrating at low temperature under reduced pressure to 50g, adding cellulase, regulating enzyme activity to 1000IU, and hydrolyzing at 20 deg.C for 20 h;

(4) heating to inactivate enzyme, and diluting with propylene glycol to 100g to obtain Ginseng radix extract.

Example 4:

the extracts obtained in the embodiments 1, 2 and 3 are uniformly mixed with 50g of hawthorn extract, 20g of sweet wormwood extract and g of hydrolyzed ginseng extract to obtain natural anti-inflammatory and anti-allergic extract.

Comparative example 1:

(1) percolating 50g Ginseng radix with 150g70 wt% ethanol;

(2) extracting for 24h by percolation, and collecting filtrate;

(3) concentrating at low temperature under reduced pressure until ethanol is removed, and diluting with propylene glycol to 100g to obtain Ginseng radix extract.

Comparative example 2:

(1) percolating 50g of herba Leonuri with 150g of 95 wt% ethanol;

(2) extracting for 24h by percolation, and collecting filtrate;

(3) concentrating at low temperature under reduced pressure to obtain herba Leonuri extract.

Comparative example 3:

(1) percolating 50g of herba Epimedii with 150g of 95 wt% ethanol;

(2) extracting for 24h by percolation, and collecting filtrate;

(3) concentrating at low temperature under reduced pressure to obtain herba Epimedii extract.

Comparative example 4:

(1) adding 25g of hawthorn into 150g of 95 wt% ethanol for percolation extraction;

(2) extracting for 24h by percolation, and collecting filtrate;

(3) concentrating under reduced pressure at low temperature to obtain fructus crataegi extract I.

Comparative example 5:

(1) adding 50g of chenopodium ambrosioides into 150g of 95 wt% ethanol for percolation extraction;

(2) extracting for 24h by percolation, and collecting filtrate;

(3) concentrating under reduced pressure at low temperature to obtain herba Chenopodii extract.

Comparative example 6:

(1) percolating 50g radix Arnebiae with 150g95 wt% ethanol;

(2) extracting for 24h by percolation, and collecting filtrate;

(3) concentrating under reduced pressure at low temperature to obtain radix Arnebiae extract.

The detection method comprises the following steps:

1. screening procedure for calcineurin (CaN) -NF-AT pathway blockers:

1.1 sample to be tested: diluting all the extracts with deionized water to obtain a working solution of mg/ml, and adding 10 μ l into a measuring tube during detection to obtain a final concentration of 0.2 mg/ml.

1.2 assay method K562 cells were stimulated with PMA (protein kinase CPKC activator) and A23187 (calcium channel activator) using the Cell line NFAT K562Reporter Cell L ine, a stably transfected NFAT Reporter gene, purchased from Affibatrix, USA, and the level of luciferase gene expression driven by transcription factor NFAT was measured in response to the level of calcineurin (CaN) pathway activation.

The K562 cells stably transfected with NFAT reporter gene are subcultured in RPMI-1640 medium containing 10 wt% fetal calf serum, healthy cells in logarithmic growth period are adopted in the experiment, and the cells are treated according to 2 × 10⁵Per well of the cells were plated on a 24-well cell culture plate, the test sample was added, incubated for 1 hour, PMA (10ng/ml) and A23187(0.5 μm) (pathway stimulator) were added, cells were stimulated for 18 hours, and the cells were collected and measured for the fluorescence intensity lum value (reflecting the level of intracellular reactive calcineurin pathway activation) using a luciferase assay kit as described in the specification.

2. And (3) detecting the cytotoxicity of the Chinese herbal medicine extract:

using the conventional CCK8 method, (1) prepare 100. mu. L of K562 cell suspension (1 × 10) in 96-well plate⁵Perwell), 96-well plates were pre-incubated in an incubator for 24 hours (37 ℃, 5 wt% CO)₂)；

(2) Adding 10 mu L substance to be detected;

(3) incubating the 96-well plate in an incubator for 24 hours;

(4) add 10 μ L CCK8 solution to each well;

(5) incubating the 96-well plate in an incubator for 4 hours;

(6) absorbance at 450nm was measured with a microplate reader.

II, experimental results:

compared with different traditional Chinese medicines, the product has the inhibition effect on the CaN-NF-AT channel, and the result is shown in Table 1

Table 1:

compared with different traditional Chinese medicines, the nonspecific cytotoxicity results of the product are shown in Table 2

Table 2:

as shown in Table 1, the hawthorn, the sweet wormwood herb, the natural anti-inflammatory (antiallergic) extract and the lithospermum extract have better function of inhibiting the CaN-NF-AT pathway. However, according to table 2, the arnebia euchroma (Royle) Johnston extract is relatively toxic to cells, and its inhibitory action is probably due to nonspecific cytotoxicity of arnebia euchroma (Royle) Johnston component. Meanwhile, as CaN be seen from table 1, the inhibition effect of the natural anti-inflammatory (antiallergic) extract on the CaN-NF-AT pathway is obviously superior to the single effect of the hawthorn extract, the sweet wormwood extract and the hydrolyzed ginseng extract; as can be seen from Table 2, the hydrolyzed ginseng extract has significantly lower toxicity than the unhydrolyzed ginseng extract; after the hawthorn, the sweet wormwood and the hydrolyzed ginseng extract are mixed according to a proportion, the toxicity is less than that of the three extracts which are used independently; the two traditional Chinese medicine hawthorn and sweet wormwood herb extracts are independently used and have better CaN-NF-AT channel inhibition effect, and after the two traditional Chinese medicine hawthorn and sweet wormwood herb extracts are mixed with the hydrolyzed ginseng extract in proportion, the better CaN-NF-AT channel inhibition effect (obvious synergistic effect) CaN be used as a good substance for resisting eczema; and the two are mixed with the hydrolyzed ginseng extract according to a proportion, the toxicity is obviously reduced (obvious antagonism); the toxicity of the ginseng extract is obviously different from that of the ginseng extract which is not hydrolyzed. Meanwhile, as the pure natural traditional Chinese medicine extract is adopted, the hidden danger of the western medicine with larger side effect is overcome, and a material foundation is provided for the anti-eczema and soothing cosmetics.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent replacements, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (1)

1. A preparation method of a traditional Chinese medicine with anti-inflammatory and anti-allergic effects is characterized by comprising the following steps:

(1) adding 15-30 times of 50-70 wt% ethanol into ginseng, extracting for 12-24 h by a percolation method, concentrating under reduced pressure until the weight of the medicinal materials is equal, adding cellulase to prepare 400-2000 IU of enzyme activity, hydrolyzing for 12-24 h at 15-20 ℃, heating to above 60 ℃ to inactivate the enzyme, and obtaining a hydrolyzed ginseng extract;

(2) adding 30 times of 95 wt% ethanol into fructus crataegi and herba Artemisiae Annuae, respectively, and percolating to obtain extract;

(3) extracting for 24h by percolation, and collecting filtrate;

(4) concentrating at low temperature under reduced pressure to equal weight to obtain fructus crataegi and herba Artemisiae Annuae extract;

(5) uniformly mixing 50-60 wt% of hawthorn extract, 20-30 wt% of sweet wormwood herb extract and 20-30 wt% of hydrolyzed ginseng extract to obtain the natural anti-inflammatory and antiallergic extract.