CN114984023A

CN114984023A - Application of crocetin in preparation of antiallergic drugs

Info

Publication number: CN114984023A
Application number: CN202210720214.0A
Authority: CN
Inventors: 黄静; 王楠; 常育; 黄琳红; 李永生; 蒋凯; 范婷; 张丽心; 杨芳
Original assignee: Xian Honghui Hospital
Current assignee: Xian Honghui Hospital
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2022-09-02

Abstract

The invention discloses an application of crocetin in preparing an antiallergic drug, belonging to the technical field of preparation of antiallergic drugs. Through comparing experiments of mast cell degranulation, calcium ion release, toe swelling degree and toe evans blue exudation degree, a blank control group and a crocetin administration group are arranged for comparison, and experimental results show that crocetin can inhibit RBL-2H3 cells from releasing histamine in a dose-dependent manner, can inhibit the increase of calcium ion concentration in mast cells and inhibit the local anaphylactic reaction of toes of mice, and the crocetin can be used for preparing antiallergic drugs caused by IgE.

Description

Application of crocetin in preparation of antiallergic drugs

Technical Field

The invention belongs to the technical field of preparation of antiallergic drugs, and particularly relates to application of crocetin in preparation of the antiallergic drugs.

Background

Stigma croci is the dry stigma of Crocus sativus L. of Crocus of Iridaceae. Saffron is a traditional Chinese medicine for promoting blood circulation to remove blood stasis, and has wide clinical effects. Crocetin (crocetin) is one of the main effective components of stigma croci Sativi, has polyunsaturated conjugated olefine acid structure, and belongs to carotenoid material. A large number of researches show that the crocetin has definite biological activity in the aspects of resisting cardiovascular system diseases, resisting oxidation and atherosclerosis, preventing and treating diabetic complications and the like, and has the protection effects on DNA damage induced by a free radical generating system, rat cardiotoxicity caused by doxorubicin and rat myocardial mitochondrial damage caused by doxorubicin. In addition, the research shows that crocetin can improve the survival rate of rats with hemorrhagic shock and relieve liver tissue damage. Crocetin also has anticoagulant and antithrombotic effects.

Allergic reactions are reactions of damaged tissues or dysfunctions that occur when an immunized organism is stimulated again with the same antigen. The allergic reaction is also called as hypersensitivity, and the antigenic substance causing the hypersensitivity is called allergen. It may be complete antigen (xenogenic animal serum, histiocyte, microbe, parasite, plant pollen, and animal fur, etc.) or hapten (low molecular substance such as penicillin, sulfanilamide, etc.). Either exogenous or endogenous. The clinical manifestations of allergic reactions are diverse and can vary depending on the nature of the allergen, the route to enter the body, the factors involved, the mechanism of occurrence and the differences in individual reactivity.

In recent years, with the development of society, the living standard of people is increasing, and the number of patients suffering from allergic diseases in clinic is increasing. However, the current drugs for such diseases are limited to antihistamines and other drugs acting on the downstream corresponding organs for relieving symptoms. Therefore, new drugs for clinical use are needed to provide a way to treat allergy.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of crocetin in preparing antiallergic drugs and provide novel antiallergic drugs for clinical application.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses an application of crocetin in preparing an antiallergic medicament.

Preferably, the allergy is an IgE-mediated allergic disease.

Preferably, the agent is one that reduces DNP-IgE-mediated DNP-BSA induced cell degranulation of RBL-2H 3.

Preferably, the agent is one that reduces IgE-mediated DNP-BSA induced foot swelling.

Preferably, the medicament is one which mitigates IgE-mediated DNP-BSA induced elevation of intracellular calcium ion concentration in mast cells.

Preferably, the medicament is one that reduces IgE-mediated DNP-BSA induced foot swelling.

Preferably, the animal is administered an amount of 20-80mg/kg for IgE-mediated, DNP-BSA induced allergic symptoms.

The invention also discloses an antiallergic medicine, which is a preparation prepared from crocetin and pharmaceutically acceptable auxiliary materials.

Preferably, the preparation is a tablet, a capsule, a granule, an injection or a pill.

The invention also discloses an antiallergic composition, which takes crocetin as a main component.

Compared with the prior art, the invention has the following beneficial effects:

the application of crocetin in preparing antiallergic drugs provided by the invention is characterized in that the experimental setup for comparing the degranulation of mast cells, the release of calcium ions, the toe swelling degree and the toe Evans blue exudation degree is used for evaluating the drug group to be tested (giving crocetin), the normal control group and the normal control group by researching the improvement effect of crocetin on the degranulation of mast cells RBL-2H3 and the passive skin allergy (PCA) of mice, the experimental result shows that the crocetin can effectively reduce the PCA degree of an asthmatic mouse, reduce the serum histamine concentration, reduce mast cell degranulation, inhibit the calcium ion concentration rise in a mast cell and inhibit the local anaphylactic reaction of toes of the mouse, and the crocetin has obvious treatment effect on the allergic disease mediated by IgE and can be used for preparing the antiallergic medicine caused by the IgE.

Drawings

FIG. 1 is a graph showing the results of inhibition of DNP-IgE-mediated DNP-BSA induced degranulation of mast cell RBL-2H3 by crocetin according to the present invention; wherein the abscissa is grouping of different concentrations, and the ordinate is the percentage of histamine release;

FIG. 2 shows DNP-IgE-mediated DNP-BSA-induced mast cell RBL-2H3 intracellular Ca by crocetin of the present invention ²⁺ Influence graph of (2); wherein A is 25 μ M crocetin, B is 50 μ M crocetin, and C is 100 μ M crocetin;

FIG. 3 is a graph showing the results of OVA-induced inhibition of evans blue exudation in the toe of C57 mice by crocetin according to the present invention; wherein A is blank control group, B is 20mg/kg crocus acid solution, C is 40mg/kg crocus acid solution, and D is 80mg/kg crocus acid solution;

FIG. 4 is a graph showing the results of inhibition of OVA-induced toe swelling in C57 mice by crocetin according to the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, shall fall within the protection scope of the present invention.

It should be noted that the terms "comprises" and "comprising," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

example 1 crocetin inhibits DNP-IgE-mediated, DNP-BSA-triggered mast cell RBL-2H3 histamine release

Experimental materials:

DNP-IgE (from MCE), DNP-BSA (from Saimer fly), RBL-2H3 cells, Taiwanese, crocetinic acid (from Doppel), 0.1M Na ₂ CO ₃ ，0.1M NaHCO ₃ Beta hexosaminidase, 0.1M citric acid, 0.1M sodium citrate.

The experimental method comprises the following steps:

1. RBL-2H3 cells were added to DNP-IgE at a final concentration of 10ng/mL and incubated overnight, and then RBL-2H3 cells were seeded in 96-well plates (1X 10) ⁵ One), the medium is discarded by centrifugation. Preparing crocus acid solution (with concentration of 0 μ M, 25 μ M, 50 μ M and 100 μ M respectively) with sterile Taiwan liquor, adding into corresponding well plate, setting blank control group, and incubating for 30 min.

2. For the administration group: preparing crocetin solution with the concentration by using a Taiwan liquor containing 100ng/mL DNP-BSA, adding into a corresponding pore plate, exciting for 30min, centrifuging, taking 50 mu L of supernatant from each pore, and respectively adding into clean pores;

for the blank control group: the supernatant in the wells of the blank control group was completely removed, 100. mu.L of 0.1% Triton was added to each well, and the pipetting was repeated for cell lysis. Centrifuge, take 50 μ L lysate supernatant, add to clean well.

3. To all wells 50. mu.L of β hexosamine solution was added and incubated at 37 ℃ for 1.5 hours. 0.1M NaHCO ₃ :0.1M Na ₂ CO ₃ (1:9v/v) forThe reaction was terminated. Absorbance was measured at a wavelength of 405 nm.

The experimental results are as follows:

as can be seen in fig. 1, DNP-BSA significantly caused histamine release from RBL-2H3 cells relative to the blank control. With increasing concentration, crocetin can inhibit histamine release of RBL-2H3 cells in a dose-dependent manner.

Example 2 crocetin inhibits DNP-IgE-induced activation of RBL-2H3 cells

The experimental method comprises the following steps:

culturing RBL-2H3 cells to logarithmic phase, inoculating in laser confocal special chamber (1 × 10) ⁴ one/mL) of the cells are cultured in a phenol red-free RPMI 1640 medium containing 5% of activated carbon/dextran-treated fetal calf serum, the cells are incubated with DNP-IgE after being attached to the wall for 24 hours, Flou-3AM solutions (purchased from Beijing Solibao, the final concentration of which is 9 mu M) containing crocetin with different concentrations (the concentrations are respectively 25 mu M, 50 mu M and 100 mu M) are added in a dark place, the cells are incubated in a dark place at 28 ℃ for 30 minutes, excess dye is washed by a sterile medium without a fluorescent probe, the incubation is continued for 30 minutes at 28 ℃, calcium-free D-Hanks solution is used for washing for 3 times, the dish is placed under a laser confocal microscope, the temperature is controlled at 37 ℃, appropriate scanning fields and parameters are selected, the cells emit fluorescence under the excitation of 488nm visible light, the fluorescence is detected at the wavelength of 525nm, and images are collected. Then, DNP-BSA solution was added separately, and intracellular Ca was detected in real time using cells incubated in a sterile medium containing no crocetin as a control ²⁺ The concentration was dynamically varied and [ Ca ] was plotted ²⁺ ]i time profile.

The experimental results are as follows:

as can be seen in FIG. 2, when mast cells degranulate, the opening of calcium channels on the cell membrane and endoplasmic reticulum leads to intracellular Ca ²⁺ The concentration rises rapidly, and therefore intracellular Ca is imaged by calcium ²⁺ The concentration change is monitored, and the inhibition condition of the drug on RBL-2H3 degranulation can be observed more intuitively. The results show that after the crocetin treatment, the increase of the calcium ion concentration in mast cells can be inhibited, and the fluorescence intensity is weaker along with the increase of the drug concentration, thereby further confirming that the crocetin has good antiallergic activity.

Example 3 crocetin inhibits OVA-induced toe swelling and exudation in mice

Experimental materials:

OVA (purchased from sigma e), 6-8 week old male C57 mice (purchased from the experimental animal center of the university of western ann traffic), crocetin (chenopodium album), 0.4% evans blue solution, 3.5% chloral hydrate solution, PBS solution, physiological saline, acetone.

The experimental method comprises the following steps:

mice were divided into a blank control group and crocetin administration groups of various concentrations. Except for the blank control group, other groups were sensitized by injecting 200. mu.g/kg of OVA solution intraperitoneally for 48 hours in advance. The administration group of crocetin was intragastrically infused with solutions of crocetin of different concentrations (20 mg/kg, 40mg/kg and 80mg/kg, respectively) prepared with normal saline, and the blank control group was intragastrically infused with normal saline of equal volume. After 30min, mice were anesthetized with 3.5% chloral hydrate solution and the tail vein was injected with 0.4% evans blue solution. The thickness of the left and right soles of each group of mice was measured, followed by injecting 5 μ L of OVA solution with a concentration of 100ng/mL subcutaneously in the left sole using a microinjector, and an equal volume of PBS solution subcutaneously in the right sole as a control. After 15 minutes, the mice were sacrificed. The thickness of the soles of the mice in each group was measured again. The mouse feet were cut, oven dried, and weighed. The toes were broken, and the mixture was treated with acetone: physiological saline (7: 3, v/v) dissolved Evans blue in the toes, and absorbance was measured at 620nm to calculate absorbance per unit volume.

The experimental results are as follows:

as seen in FIGS. 3 and 4, 100ng/mL of OVA significantly caused swelling of toes and evans blue exudation in C57 mice relative to the blank control group, and crocetin dose-dependently inhibited local allergic reactions in toes of C57 mice and was concentration-dependent.

The above contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention should not be limited thereby, and any modification made on the basis of the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. Application of crocetin in preparing antiallergic medicine is provided.

2. Use of crocetin according to claim 1 for the preparation of an antiallergic drug, characterized in that the allergy is an IgE-mediated allergic disease.

3. Use of crocetin according to claim 1 for the preparation of an antiallergic agent, characterized in that the agent is a drug that reduces DNP-IgE-mediated DNP-BSA-induced RBL-2H3 cell degranulation.

4. Use of crocetin according to claim 1 for the preparation of an antiallergic agent, characterized in that the agent is an agent for reducing IgE-mediated, DNP-BSA induced swelling of the feet.

5. Use of crocetin according to claim 1 for the preparation of antiallergic drugs, characterized in that said drugs are drugs that alleviate the IgE-mediated increase in the concentration of mast-cell calcium ions caused by DNP-BSA.

6. Use of crocetin according to claim 1 for the preparation of an antiallergic agent, characterized in that the agent is an agent for reducing IgE-mediated, DNP-BSA induced swelling of the feet.

7. Use of crocetin according to claim 1 for the preparation of antiallergic drugs, characterized in that the animal dose is 20-80mg/kg for IgE-mediated, DNP-BSA-induced allergic symptoms.

8. An antiallergic medicine is characterized in that the medicine is a preparation prepared from crocetin and pharmaceutically acceptable auxiliary materials.

9. An antiallergic agent according to claim 8, wherein the preparation is in the form of a tablet, capsule, granule, injection or pill.

10. An antiallergic composition characterized by comprising crocetin as a main ingredient.