Use of extract of rhizoma corydalis in preparing anti-allergic drug

Abstract

The application discloses application of a Corydalis yanhusuo extract in preparation of an anti-allergic drug and belongs to the technical field of anti-allergic drug preparation. The treatment effect of the drug on allergic diseases is evaluated by comparing P815 cell degranulation and inflammatory factor release, the inflammatory infiltration degree of a local skin allergic reaction of a mouse, and the allergic symptoms of a mouse as a whole, so that it is determined that the Corydalis yanhusuo extract can be used for preparation of an anti-allergic drug. The Corydalis yanhusuo extract can effectively alleviate the local and systemic allergic reaction symptoms of a mouse, and reduce mast cell degranulation, and has a significant improvement effect on symptoms caused by allergic reactions.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of anti-anaphylactoid drug preparation, and relates to application of a Corydalis yanhusuo extract in preparation of an anti-anaphylactoid drug. BACKGROUND

[0002] Corydalis yanhusuo has the effects of activating blood, promoting qi and relieving pain, and can be used for blood stasis and qi stagnation pain, and the main pharmacodynamic material basis is alkaloid ingredients. More than 20 kinds of alkaloid ingredients contained in Corydalis yanhusuo are recorded in a Chinese Medicine Dictionary. Modern pharmacological research shows that Corydalis yanhusuo has the pharmacological effects of analgesia, sedation and hypnotic, anti-myocardial ischemia, anti-cerebral ischemia, anti-ulcer, anti-tumor and anti-depression.

[0003] Anaphylactoid reaction refers to a reaction similar to anaphylaxis, that is, a hypersensitivity reaction mediated by a non-IgE dependent mechanism, which is not caused by an immune reaction. Therefore, they do not require a sensitization process of prior exposure to antigen to directly cause mast cell degranulation, and can rapidly occur upon first exposure to antigen. It is related to mast cell or basophil degranulation, activation of the complement system, and release of histamine or other bioactive mediators such as vascular active factors. The MRGPRX2 receptor on mast cells is considered to be an important receptor for the occurrence of anaphylactoid reactions, and can be directly activated by ligands such as C48 / 80 and substance P to trigger anaphylactoid reactions. Therefore, the MRGPRX2 receptor can be used as a potential therapeutic target for anaphylactoid reactions.

[0004] In recent years, clinical drugs such as traditional Chinese medicine injections, anesthetics, antibiotics, chemotherapy drugs, analgesics and other drugs can produce adverse reactions and cause anaphylactic symptoms. In recent years, the frequency of anaphylactoid reactions in clinical treatment has been increasing, but the drugs currently used for the treatment of anaphylactic diseases are still limited to antagonizing downstream effectors, such as antihistamine drugs, leukotriene receptor antagonists, and the like, and the treatment means for anaphylactoid reactions needs to be timely innovated. At present, there is no related application report about Corydalis yanhusuo extract as an anti-anaphylactoid drug. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide application of a Corydalis yanhusuo extract in preparation of an anti-anaphylactoid drug.

[0006] In order to achieve the above-mentioned purpose, the following technical solutions are adopted in the present application:

[0007] The application discloses application of a Corydalis yanhusuo extract in preparation of an anti-anaphylactoid drug. The Corydalis yanhusuo extract is obtained by crushing Corydalis yanhusuo, soaking in ethanol, heating and refluxing, obtaining an extract, centrifuging the extract to obtain supernatant, vacuum concentrating, evaporating to dryness, dissolving, ultrasonicating and filtering.

[0008] Preferably, the drug is a drug for reducing the degranulation of mast cells in an allergic reaction.

[0009] Preferably, the drug is a drug for inhibiting the release of inflammatory factors of mast cells.

[0010] Preferably, the drug is a drug for reducing systemic allergic reactions.

[0011] Preferably, the drug is a drug for dose-dependently inhibiting the release of beta-hexosaminidase by P815.

[0012] Preferably, the drug is a drug for dose-dependently inhibiting the release of TNF-α by P815.

[0013] Preferably, the drug is a drug for dose-dependently inhibiting local allergic reactions in the toes of mice.

[0014] Preferably, the drug is a drug for alleviating the decrease in body temperature of mice.

[0015] Preferably, the drug is administered in an amount of 2.5-15 mg / kg when used for inhibiting allergic reactions.

[0016] The application also discloses an anti-allergic drug, which comprises a Corydalis yanhusuo extract and a pharmaceutically acceptable carrier, wherein the Corydalis yanhusuo extract is obtained by crushing Corydalis yanhusuo, soaking the crushed Corydalis yanhusuo in ethanol, heating and refluxing, centrifuging the extract to obtain supernatant, vacuum concentrating the supernatant, evaporating the supernatant to dryness, dissolving the dried supernatant, ultrasonicating the dissolved supernatant, and filtering the ultrasonicated supernatant.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] The application discloses application of a Corydalis yanhusuo extract in preparation of an anti-allergic drug. The therapeutic effect of the Corydalis yanhusuo extract on allergic diseases is evaluated by comparing the degranulation of P815 cells, the release of inflammatory factors of P815 cells, the degree of swelling of toes of mice, the degree of Evans blue exudation of toes of mice, and the degree of decrease in body temperature of mice in a test drug group, a blank control group and a negative control group. It is found through experiments that the Corydalis yanhusuo extract can effectively alleviate the symptoms of local and systemic allergic reactions of mice, reduce the degranulation of mast cells, and has a significant improvement effect on the symptoms caused by allergic reactions, and is superior to the anti-allergic effects of single active components of the Corydalis yanhusuo, i.e., dehydrovaccinin and dehydrovaccinin B, which indicates that the Corydalis yanhusuo extract has a definite therapeutic effect on improvement of allergic symptoms, and it is determined that the Corydalis yanhusuo extract can be used for preparation of an anti-allergic drug. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1Figure 6 is a graph showing the inhibitory effect of YHS extract on P815 cell degranulation induced by C48 / 80; wherein the abscissa represents different groups set, and the ordinate represents the percentage of β-hexosaminidase release;

[0020] Figure 2 Figure 7 is a graph showing the inhibitory effect of Corydaline on P815 cell degranulation induced by C48 / 80; wherein the abscissa represents different groups set, and the ordinate represents the percentage of β-hexosaminidase release;

[0021] Figure 3 Figure 8 is a graph showing the inhibitory effect of tetrahydropalmatine on P815 cell degranulation induced by C48 / 80; wherein the abscissa represents different groups set, and the ordinate represents the percentage of β-hexosaminidase release;

[0022] Figure 4 Figure 9 is a graph showing the inhibitory effect of YHS extract on TNF-α release from P815 cells induced by C48 / 80; wherein the abscissa represents different groups set, and the ordinate represents the amount of TNF-α release;

[0023] Figure 5 Figure 10 is a graph showing the inhibitory effect of YHS extract on paw swelling of C57 mice induced by C48 / 80; wherein the abscissa represents different groups set, and the ordinate represents the percentage of paw swelling;

[0024] Figure 6 Figure 11 is a graph showing the inhibitory effect of YHS extract on Evans blue extravasation of C57 mice induced by C48 / 80; wherein the abscissa represents different groups set, and the ordinate represents the degree of Evans blue extravasation;

[0025] Figure 7 Figure 12 is a graph showing the inhibitory effect of YHS extract on body temperature decrease of C57 mice induced by C48 / 80; wherein the abscissa represents time, and the ordinate represents the change of body temperature of mice compared to the basal body temperature. DETAILED DESCRIPTION

[0026] In order for those skilled in the art to understand the features and effects of the present application, the following only describes and defines the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used herein have their usual meanings understood by those skilled in the art of the present application, and in case of conflict, the definition in the specification shall prevail.

[0027] Theories or mechanisms described and disclosed herein, whether correct or not, should not be considered limiting the scope of the present application, i.e., the present application can be practiced without relying on any particular theory or mechanism.

[0028] Herein, all features defined by numerical ranges or percentage ranges, such as numerical values, amounts, contents and concentrations, are for the sake of brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have encompassed and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0029] Herein, unless otherwise specifically noted, "comprise", "include", "contain", "have" or similar terms, encompass both "consist of" and "consist essentially of", for example, "A comprises a" encompasses both "A comprises a and others" and "A consists of a only".

[0030] Herein, for the sake of brevity, all possible combinations of the various technical features described in the various embodiments or examples are not described. Therefore, the various technical features in the various embodiments or examples can be combined with each other as long as there is no contradiction in the combination, and all possible combinations should be considered as falling within the scope of the present specification.

[0031] The present application provides use of a corydalis extract in preparation of an anti-allergic drug.

[0032] The present application will be further described with reference to the following examples. It is to be understood that these examples are provided by way of illustration only and should not be construed as limiting the scope of the present application. Furthermore, those skilled in the art will appreciate that various modifications and changes can be made to the present application upon reading the contents of the present application, and such equivalents should be considered as falling within the scope of the appended claims.

[0033] In the following examples, the instruments and apparatuses used are those conventional in the art, and the various materials, reagents (e.g., organic solvents, inorganic solvents, kinases, substrates, antibodies, buffers, reaction solutions, etc.) used are, unless otherwise specified, commercially available products of conventional specifications, or can be prepared or formulated by known methods or according to the instructions of the reagent manufacturer. The experimental methods in the following examples, unless otherwise specified, are generally performed according to conventional conditions, or according to the conditions recommended by the manufacturer.

[0034] In the specification of the present application and in the following examples, unless otherwise specified, "%" means weight percent, "parts" means weight parts, and the ratio means weight ratio.

[0035] In the specification and examples below, the term "effective amount" means, unless otherwise specified, an amount which is effective to achieve the purpose of preventing and / or treating the disease described herein. The skilled artisan can readily determine the dosage of the extract of Corydalis to be used in accordance with the context of the present application. In particular, the term "effective amount" can be understood as an amount of the extract of Corydalis (or in the form of a plant extract comprising the extract of Corydalis) sufficient to treat the disorder with a reasonable benefit / risk ratio applicable to any medical treatment. It will be appreciated, however, that the total daily usage of the extract of Corydalis or compositions thereof in the therapeutic methods of the present application will be decided by the skilled artisan in the exercise of sound medical judgment. The specific effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex, and diet of the patient, the particular composition employed, other active substances used in combination or salient features of the disease being treated, and other factors well known in the medical arts.

[0036] In the specification and examples below, the term "plant extract" or "extract" as used, unless otherwise specified, refers to an "extract" comprising Corydalis extracted from a plant, for example, but not limited to, the plants described herein. The skilled artisan will appreciate that the Corydalis described herein can be chemically synthesized or can be derived from a plant extract as described herein. Furthermore, it is known to the skilled artisan to extract plant extracts comprising Corydalis from plants and it is also known to the skilled artisan to synthesize Corydalis using chemical synthesis.

[0037] The present application provides pharmaceutical compositions comprising a extract of Corydalis rhizoma formulated with one or more non-toxic pharmaceutically acceptable carriers. The pharmaceutical compositions can be specially formulated for oral administration in solid or liquid form or for administration by injection. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound can be mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders such as paraffin; f) absoφtion accelerators such as quaternary ammonium compounds; g) moisturizing agents such as glycerol and glyceryl monostearate; h) adsorbents such as kaolin and bentonite; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form can also comprise buffering agents. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Liquid dosage forms can additionally contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifϊers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0038] Example 1 Corydalis rhizoma extract inhibits C48 / 80-induced P815 cell β-hexosaminidase release, and the inhibitory effect is superior to that of deoxyschizandrin and schizandrin B

[0039] 1. Experimental materials

[0040] C48 / 80 (purchased from Sigma), P815 cells, RPMI 1640 medium (containing penicillin 100 U / mL, streptomycin 0.1 mg / mL, 10% FBS), Tyrode's solution, acetylcorydalis (purchased from Ningxia Mingde Chinese Herbal Pieces Co., Ltd.), 0.1 M Na2CO3, 0.1 M NaHCO3, β-hexosamine, 0.1 M citric acid, 0.1 M sodium citrate.

[0041] 2. Experimental method

[0042] Preparation of extract of Corydalis yanhusuo: 10 g of Corydalis yanhusuo was ground into fine powder, soaked in three times the amount of 60% ethanol for 12 hours, and heated to reflux for 1 hour. The extract was collected and centrifuged to obtain the supernatant. The supernatant was vacuum concentrated and evaporated to dryness to obtain the extract of Corydalis yanhusuo. The extract was dissolved in DMSO, ultrasonicated, and filtered before use.

[0043] P815 cells were seeded in 96-well plates (1 x 10 5 cells / well) and centrifuged to discard the culture medium. A negative control (N.C.), a blank control (Vehicle), and Corydalis yanhusuo extract groups with different concentrations were set up. Corydalis yanhusuo extract solutions were prepared with sterile Tyrode's solution (concentrations were 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL, respectively), and were added to the corresponding wells. The negative control and blank control were given the same amount of sterile Tyrode's solution. The drugs were incubated for 30 minutes. Corydalis yanhusuo extract solutions with the above concentrations were prepared with Tyrode's solution containing 30 μg / mL C48 / 80, and were added to the corresponding wells. The negative control was given the same amount of sterile Tyrode's solution, and the blank control was given 30 μg / mL C48 / 80. After 30 minutes of stimulation, the cells were centrifuged, and 50 μL of supernatant was taken from each well and added to a clean well. The supernatant in the negative control well was completely removed, 100 μL of 0.1% Triton X-100 was added to each well, and the cells were repeatedly blown to lyse the cells. After centrifugation, 50 μL of supernatant from the lysis solution was taken and added to a clean well. 50 μL of β-hexosaminidase solution (solvent: 0.1 mol / L citric acid-sodium citrate buffer) was added to all wells, and the mixture was incubated at 37°C for 1.5 hours. The reaction was terminated with 0.1 M NaHCO3:0.1 M Na2CO3 (1:9). The absorbance was measured at 405 nm.

[0044]

[0045] 3. Experimental results

[0046] The results are shown in Figure 1 , Figure 2 and Figure 3 .

[0047] As can be seen from Figure 1 , 30 μg / mL C48 / 80 in the blank control (Vehicle) can significantly cause P815 cells to degranulate and release β-hexosaminidase, indicating that the modeling is successful. Corydalis yanhusuo extract can dose-dependently inhibit the release of β-hexosaminidase from P815. As can be seen from Figure 2 and Figure 3 , tetrahydropalmatine and dehydrocorydaline can also dose-dependently inhibit the release of β-hexosaminidase from P815, but the inhibitory effect is significantly weaker than that of Corydalis yanhusuo extract.

[0048] Example 2: YHS extract inhibits P815 cell inflammatory factor release induced by C48 / 80

[0049] 1. Experimental materials

[0050] Mouse TNF-α ELISA kit (invitrogen). The rest refer to Example 1.

[0051] 2. Experimental method

[0052] P815 cells were inoculated in 96-well plates (1 x 10 5 The negative control group (N.C.), blank control group (Vehicle) and different concentrations of YHS extract groups were set up. YHS extract solution was prepared with sterile Tyrode's solution (concentrations were 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL, respectively), and was added to the corresponding well plates. The negative control group and blank control group were given the same dose of sterile Tyrode's solution. After 30 min of drug incubation, YHS extract solution of the above concentrations was prepared with Tyrode's solution containing 30 μg / mL C48 / 80, and was added to the corresponding well plates. The negative control group was given the same dose of sterile Tyrode's solution, and the blank control group was 30 μg / mL C48 / 80. After 6-8 hours of incubation, centrifugation was performed, and the supernatant was taken.

[0053] The content of TNF-α in the supernatant of each group was detected according to the instructions of the mouse TNF-α ELISA kit.

[0054] 3. Experimental results

[0055] The results are shown in Figure 4 From Figure 4 it can be seen that, compared with the negative control group, 30 μg / mL C48 / 80 in the blank control group can significantly cause P815 cell activation and release of TNF-α, indicating that the modeling is successful. YHS extract can dose-dependently inhibit the release of TNF-α from P815 cells.

[0056] Example 3: YHS extract inhibits mouse toe swelling and exudation induced by C48 / 80

[0057] 1. Experimental materials

[0058] C48 / 80 (purchased from Sigma Company), 6-8 week old male C57 mice (purchased from Xi'an Jiaotong University Experimental Animal Center), acetic rhizoma Corydalis (purchased from Ningxia Mingde Chinese Herbal Pieces Co., Ltd.), 0.4% Evans blue solution, 4% chloral hydrate solution, PBS solution, normal saline, acetone.

[0059] 2. Experimental method

[0060] The mice were divided into a blank control group (Vehicle) and a group administered each concentration of the Corydalis extract. The mice were administered different concentrations of the Corydalis extract solution (2.5 mg / kg, 5 mg / kg, and 10 mg / kg, respectively) prepared with normal saline by gavage, and the Corydalis extract was prepared as in Example 1. After 30 min, the mice were anesthetized with a 4% chloral hydrate solution, and a 0.4% Evans blue solution was injected into the tail vein. The thickness of the left and right footpads of the mice in each group was measured, and then 5 μL of a C48 / 80 solution having a concentration of 30 μg / mL was injected subcutaneously into the left footpad using a microsyringe, and an equal volume of normal saline was injected subcutaneously into the right footpad. After 15 min, the mice were sacrificed. The thickness of the left and right footpads of the mice in each group was measured again. The feet of the mice were cut off, dried, and weighed. The toes were crushed, and the Evans blue in the toes was dissolved in an acetone:normal saline (7:3) solution, and the absorbance was measured at 620 nm to calculate the absorbance per unit volume.

[0061] 3. Experimental results

[0062] The results are shown in Table 1. Figure 5 and Figure 6 . Figure 5 and Figure 6 As shown in Table 1, C48 / 80 at a concentration of 30 μg / mL can significantly cause swelling of the toes and leakage of Evans blue in C57 mice, and the Corydalis extract can dose-dependently inhibit local anaphylactoid reactions in the toes of C57 mice as the concentration increases.

[0063] Example 4. Corydalis extract inhibits C48 / 80-induced decrease in body temperature in mice

[0064] 1. Experimental materials

[0065] C48 / 80 (purchased from Sigma), 6-8-week-old male C57 mice (purchased from the Experimental Animal Center of Xi'an Jiaotong University), aceticum Corydalis (purchased from Ningxia Mingde Chinese Herbal Medicine), BL-410 biological function system, and vaseline.

[0066] 2. Experimental method

[0067] The mice were divided into a negative control group (N.C.), a blank control group (Vehicle), and a group administered each concentration of the Corydalis extract. The mice were administered different concentrations of the Corydalis extract solution (3.75 mg / kg, 7.5 mg / kg, and 15 mg / kg, respectively) prepared with normal saline by gavage 30 min in advance, and the blank control group was administered an equal dose of normal saline by gavage, and the Corydalis extract was prepared as in Example 1. After the basic body temperature was recorded, the mice in the blank control group and the group administered each concentration of the Corydalis extract were injected with a C48 / 80 solution into the tail vein. The body temperature of the mice was measured and recorded every 3 min for a total of 30 min.

[0068] 3. Experimental results

[0069] Results are shown in Figure 7 Compared with the negative control group, 30 μg / mL C48 / 80 can significantly cause the body temperature of C57 mice to decrease, indicating that the modeling is successful. The extract of Corydalis can dose-dependently alleviate the decrease of the body temperature of C57 mice.

[0070] The above merely illustrates the technical idea of the present application and cannot be used to limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical solutions falls within the protection scope of the claims of the present application.

Claims

1. Use of a extract of Corydalis tuber in the manufacture of an anti-allergic drug for inhibiting an allergic-like reaction, characterized in that, The extract of the rhizoma corydalis is obtained by crushing, soaking in ethanol, heating and refluxing the rhizoma corydalis, centrifuging the extract to obtain supernatant, vacuum concentrating, evaporating, dissolving, ultrasonicating and filtering, wherein, The drug is an anti-allergic drug that dose-dependently inhibits P815 from releasing β-hexosaminidase; or, The drug is an anti-allergic drug that dose-dependently inhibits P815 from releasing TNF-α.

2. Use according to claim 1, wherein The administration amount is 2.5-15 mg / kg.

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