CN117562895B

CN117562895B - Application of atractylenolide I in preparation of medicine for treating allergic purpura

Info

Publication number: CN117562895B
Application number: CN202410064860.5A
Authority: CN
Inventors: 马昂; 张书杰; 刘丹丹; 朱银华; 丰晨然; 史巧莉; 张昕炜; 刘艳青; 郭秋岩; 王继刚
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2024-01-17
Filing date: 2024-01-17
Publication date: 2024-04-02
Anticipated expiration: 2044-01-17
Also published as: CN117562895A

Abstract

The invention belongs to the field of medicines, and particularly relates to application of atractylenolide I in preparation of a medicine for treating allergic purpura. In an animal experiment of an allergic purpura rat, the atractylenolide I can treat and relieve symptoms of skin redness, ecchymosis and hemorrhage of the allergic purpura rat, reduce mast cell infiltration and vasodilation of affected parts, reduce the expression of serum immunoglobulin, immune complex and serum inflammatory factors of the allergic purpura rat, has obvious immunoregulation and anti-inflammatory effects when the dosage of the atractylenolide I is 5-20 mg/kg in vivo, and is hopeful to be developed into a novel medicament for treating the allergic purpura.

Description

Application of atractylenolide I in preparation of medicine for treating allergic purpura

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of atractylenolide I in preparation of a medicine for treating allergic purpura.

Background

Allergic purpura (Henoch-Schonlein purpura, HSP) is a common immune disease involving systemic small blood vessels such as skin, joints, gastrointestinal tract, kidneys, etc., and belongs to immune vasculitis, also known as IgA vasculitis. Allergic purpura is characterized by non-thrombocytopenic skin purpura, and may be accompanied by symptoms such as joint swelling and pain, abdominal pain, and kidney disease.

The cause of allergic purpura is complex, and the allergic purpura has close relation with bacterial (such as hemolytic streptococcus, helicobacter pylori, staphylococcus aureus and the like) infection, viral (such as influenza virus, novel coronavirus and the like) infection, mycoplasma infection, vaccination, drug factors (such as salicylate drugs, antibiotic drugs, barbiturates and the like), genetic factors and the like. The main pathogenesis of allergic purpura is type III allergy, and a circulating immune complex formed by combining an antigen and an antibody (mainly IgA type) is deposited on the wall of a blood vessel to activate complement, so that inflammation is generated on the walls and the periphery of capillaries and small blood vessels, and the permeability of the walls of the blood vessels is increased, thereby generating various clinical reactions.

At present, no unified specific treatment scheme is available for allergic purpura, and glucocorticoid, immunosuppressant, antiallergic and antihistamine drugs, anticoagulant and the like are mainly applied to clinical treatment to relieve symptoms, but the drugs are easy to relapse after stopping the drugs, have poor long-term curative effect and have larger side effects.

In contrast, traditional Chinese medicine therapy focuses on physical regulation, on dynamic balance of internal environment of human body, on strengthening body resistance and cultivating primordial qi, and on both principal and secondary aspects. Therefore, the development of new drugs for preventing and treating allergic purpura from the perspective of traditional Chinese medicine has great potential.

Disclosure of Invention

The invention aims to: aiming at the defects of the prior art, the invention discloses application of atractylenolide I in preparing a medicament for treating allergic purpura. In the animal experiments of allergic purpura rats, the atractylenolide I has obvious effects of regulating immunity and resisting inflammation, and is hopeful to be developed into a novel medicament for treating the allergic purpura.

The technical scheme is as follows: application of atractylenolide I in preparing medicament for treating allergic purpura is provided.

The structural formula of the atractylenolide I is shown as formula I:

。

i is a kind of

The application of atractylenolide I as the only effective component in preparing the medicine for treating allergic purpura.

Further, in the above application, the medicament is composed of atractylenolide I and a pharmaceutically acceptable carrier.

Further, the medicine is in the form of tablets, capsules, oral liquid, granules and traditional Chinese medicine.

Still further, the tablet is one of a common compressed tablet, a sugar-coated tablet, an effervescent tablet, a chewable tablet, a multi-layer tablet, a sustained release tablet and a controlled release tablet.

Still further, the oral liquid is one of a solution, a syrup, an emulsion, and a suspension.

Still further, the granule is one of a soluble granule, a suspension granule and an effervescent granule.

Further, the traditional Chinese medicine preparation is one of decoction, powder, pill and pellet.

A medicine for treating allergic purpura contains atractylenolide I.

Furthermore, the atractylenolide I in the medicine is the only effective component.

It is well known that the spleen is closely related to the immune function of the body. The traditional Chinese medicine considers that the spleen governs transportation and transformation, promotes tissue regeneration and blood control, and the spleen is called acquired root. The prescription for strengthening the spleen and tonifying qi can improve the immunity of organisms to a certain extent. The bighead atractylodes rhizome is taken as a traditional Chinese medicine essential medicine for tonifying spleen, and has the effects of invigorating spleen and replenishing qi, drying dampness and promoting diuresis, and suppressing sweating and preventing miscarriage. Atractylodes macrocephala lactone I is a main active ingredient of Atractylodes macrocephala, has high content in Atractylodes macrocephala, and has antiinflammatory, antitumor, gastrointestinal function regulating, and neuroprotection effects.

The beneficial effects are that: the application of the atractylenolide I disclosed by the invention in preparing the medicine for treating allergic purpura has the following beneficial effects:

(1) The atractylenolide I can obviously reduce the reaction degree of the back skin of the rat caused by allergic purpura, reduce the diameter of the back spots, relieve the expansion and bleeding of venous blood stasis of skin tissues and reduce the infiltration of mast cells;

(2) The atractylenolide I can reduce the IgA, igG, CIC content in the serum of the rats with allergic purpura and play an immunoregulatory role;

(3) The atractylenolide I can reduce the content of TNF-alpha and IL-6 in the serum of the rats with allergic purpura, and has anti-inflammatory effect.

Drawings

FIG. 1 is a statistical representation of the diameter of the back redness and swelling after antigen challenge in each group of rats.

FIG. 2 is a schematic representation of the back skin spots of the rats in each group.

FIG. 3 is a schematic representation of H & E staining results of skin tissue of each group of rats.

FIG. 4 is a schematic representation of IgA levels in serum of rats in each group.

FIG. 5 is a schematic representation of IgG levels in serum of each group of rats.

FIG. 6 is a schematic representation of CIC levels in serum from various groups of rats.

FIG. 7 is a schematic representation of inflammatory factor TNF- α levels in serum of various groups of rats.

FIG. 8 is a schematic representation of inflammatory factor IL-6 levels in serum of various groups of rats.

Detailed Description

The following detailed description of specific embodiments of the invention.

The "range" disclosed herein is defined in terms of lower and upper limits, with the given range being defined by the selection of a lower and an upper limit, the selected lower and upper limits defining the boundaries of the particular range. Ranges that are defined in this way can be inclusive or exclusive of the endpoints, and any combination can be made, i.e., any lower limit can be combined with any upper limit to form a range. For example, if ranges of 10 to 50 are listed for a particular parameter, it is understood that ranges of 10 to 40 and 20 to 50 are also contemplated. Furthermore, if the minimum range values 1 and 2 are listed, and if the maximum range values 3,4 and 5 are listed, the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In this application, unless otherwise indicated, the range of values "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed throughout, and "0-5" is only a shorthand representation of a combination of these values.

All embodiments and alternative embodiments of the present application may be combined with each other to form new solutions, unless specifically stated otherwise.

All technical features and optional technical features of the present application may be combined with each other to form new technical solutions, unless specified otherwise.

All steps of the present application may be performed sequentially or randomly, preferably sequentially, unless otherwise indicated. For example, the method comprises steps (a) and (b), meaning that the method may comprise steps (a) and (b) performed sequentially, or may comprise steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), may include steps (a), (c) and (b), may include steps (c), (a) and (b), and the like.

Reference herein to "comprising" and "including" means open ended, as well as closed ended, unless otherwise noted. For example, the terms "comprising" and "comprises" may mean that other components not listed may be included or included, or that only listed components may be included or included.

Unless otherwise specified, the reaction is carried out under normal temperature and normal pressure conditions.

Unless otherwise indicated, all parts or percentages are parts or percentages by weight.

In the present invention, the materials used are all known materials, and are commercially available or synthesized by known methods.

In the present invention, the devices or apparatuses used are conventional devices or apparatuses known in the art, and are commercially available.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

1. Experimental materials

Rats: SPF-class SD rats, male and female, purchased from Si Bei Fu (Beijing) biotechnology Co., ltd, were kept in the same environment during 8 weeks of growth and fed maintenance feed.

2. Laboratory animals and groups

SPF-class SD rats, male and female, were randomly divided into a control group, a model group, a low-dose group of atractylenolide I (5 mg/kg), a medium-dose group of atractylenolide I (10 mg/kg), and a high-dose group of atractylenolide I (20 mg/kg) after 1 week of adaptive feeding in the same environment, and were as follows:

the low dose group of the atractylenolide I is short for the atractylenolide I to be low;

the dosage group of the atractylenolide I is called as atractylenolide I for short;

the high dosage group of the atractylenolide I is short for the atractylenolide I to be high.

3. Model building and drug administration

3.1, preparing an egg protein-Freund's incomplete adjuvant and an egg protein-Freund's complete adjuvant:

egg proteins are dissolved in a proper amount of physiological saline to prepare an egg protein solution with the concentration of 0.5 g/L, wherein:

the egg protein solution is suspended into an egg protein-Freund complete adjuvant by an equivalent Freund complete adjuvant suspension;

the egg protein solution is suspended by equivalent Freund's incomplete adjuvant to prepare the egg protein-Freund's incomplete adjuvant.

3.2 administration of drugs

Rats in the model group, the atractylenolide I low (5 mg/kg), medium (10 mg/kg), high (20 mg/kg) dose group were given an ovalbumin-Freund complete adjuvant intramuscular injection 3 weeks before and an ovalbumin-Freund incomplete adjuvant injection 3 weeks after, each 0.5 mL, 1 week, and the control group had an equivalent physiological saline injection.

The last sensitization day was dosed in the control, model, atractylenolide I low (5 mg/kg), medium (10 mg/kg), high (20 mg/kg) dose groups of 10 animals each. The administration was performed by intragastric administration, and the control group and the model group were administered with the same amount of vehicle for 14 days.

Rats in model group, atractylenolide I low (5 mg/kg), medium (10 mg/kg), high (20 mg/kg) dose group were back-skinned on day 13 of administration, antigen challenge was performed 1 hour after day 14 of administration, 1% egg protein solution (saline formulation) was injected into back skin, the injection amount per spot was 0.2 mL, total 3 points, and the control group was injected into back skin with equal amount of saline.

4. General Condition observations

Observing the overall state, activity, ingestion, hair, lips, conjunctiva, auricle, fecal color, smell, etc.

5. Observation of skin reaction

(1) The maximum diameters of the local red and swollen at each point are observed for 2h, 4h, 6h, 8h and 24 h after antigen attack, and the average value is obtained.

(2) The degree of reaction was graded according to the "pharmacological Experimental methodology", bena-cerraf standard, namely: "+". ++'s the method comprises the following steps of: 2h or 5 h, which are obviously hyperemic and bleeding, are in a fusion state; 24 h shows a pronounced bloody discoloration.

"++" is 2 points: 2h or 5 h, in the form of plaque; 24 h moderate hemorrhagic discoloration.

"+" is 1 point: 2h or 5 h, with mild congestion and bleeding; 24 h slight hemorrhagic discoloration.

"-" is 0 point: 2h or 24 h.

The composite scores for each group are the average of skin response scores.

6. Skin histopathological observations

Rat skin was fixed with 4% paraformaldehyde, paraffin embedded, sectioned conventionally, hematoxylin-eosin (Hematoxylin and Eosin, H & E) stained and examined under an optical microscope for histopathological changes.

7. Serum immune-related index detection

According to the instruction of the kit, the contents of immunoglobulin A (IgA), immunoglobulin G (IgG) and Circulating Immune Complex (CIC) in serum are detected by a scattering turbidimetry method.

8. Detection of inflammation-related factors in serum

Serum was isolated and the expression levels of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in the serum were detected using an enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA).

9. Statistical method

Statistical analysis was performed using GraphPad Prism 8.0 software, experimental data expressed as mean ± standard error (x±s), and group-to-group differences were analyzed using One-way Anova method. The difference of P < 0.05 is statistically significant.

The back skin response degree rating was analyzed using Ridit.

Example 1

The model was constructed as described above, and after completion of the administration, the general state of each group of rats was observed.

The rats in the control group have normal ingestion, activity and other states.

During intramuscular injection of ovalbumin-Freund's complete adjuvant and ovalbumin-Freund's incomplete adjuvant, rats in model group, large-scale largehead atractylodes lactone I low (5 mg/kg), medium (10 mg/kg), high (20 mg/kg) dose group showed various degrees of listlessness, ingestion decline, mice Mao Pengluan, soft stool, etc. After administration, compared with rats in a model group, rats in the low (5 mg/kg), medium (10 mg/kg) and high (20 mg/kg) dosage groups have obviously increased activity, increased ingestion and gradual recovery of stool, and the general state is obviously improved.

In conclusion, atractylenolide I can improve the general state of allergic purpura rats.

Example 2

A model was constructed according to the method described above, and back spot diameter statistics and skin response scores were performed after dosing was completed.

Compared with the rats in the control group, the back skin of the rats in the model group and the atractylenolide I low (5 mg/kg), medium (10 mg/kg) and high (20 mg/kg) dose groups has no red swelling phenomenon after 2 hours of antigen attack.

Rats in the model group showed significant redness in the back 4 hours after antigen challenge, rats in the low (5 mg/kg), medium (10 mg/kg) and high (20 mg/kg) dose groups of atractylenolide I were able to reduce the diameter of the redness, with only the high dose group of atractylenolide I having a statistical difference (P < 0.05).

6 hours after antigen attack, the back spots of the rats in the model group are obviously darkened, but the local spots are smaller; the rats in the medium (10 mg/kg), high (20 mg/kg) dose group had a reduced level of redness and swelling of the atractylenolide i (5 mg/kg), compared to the rats in the model group, wherein: the diameter of the red and swollen of the high-dose group of the atractylenolide I is obviously smaller than that of the model group (P < 0.05).

At 8 hours after antigen challenge, the back spot color of the rats in the model group continuously increased, and the local spot diameters of rats in the low (5 mg/kg), medium (10 mg/kg) and high (20 mg/kg) dose groups of atractylenolide I increased, but were smaller than those in the model group.

24 hours after antigen challenge, the backs of rats in the model group showed moderate hemorrhagic discoloration, rats in the group with low (5 mg/kg), medium (10 mg/kg) and high (20 mg/kg) doses showed slight hemorrhagic discoloration, and the spot diameters of the medium and high doses of atractylenolide I were significantly smaller than those of the model group (P < 0.05 or P < 0.01), and the results are shown in FIG. 1. The atractylenolide I is suggested to reduce the diameter of the back skin spots of the rats with allergic purpura and is dose-dependent.

Model group rats showed moderate hyperemia and bleeding in the back skin, plaque-like, 2 hours or 5 hours after antigen challenge; mild hemorrhagic discoloration 24 hours. The back skin of the rats in each administration group had slight congestion or bleeding for 2 hours or 5 hours, slight hemorrhagic discoloration for 24 hours, and the degree of response was significantly lower than that of the model group, and was dose-dependent (P < 0.05 or P < 0.01). The results are shown in the following table, and the skin spots of the rats of each group at 24 hours are shown in fig. 2. The atractylenolide I is suggested to improve the skin reaction degree of the rats with allergic purpura.

Note that: in comparison with the control group, ^## P <0.01; in comparison with the set of models, ^* P <0.05， ^** P <0.01。

to sum up: the atractylenolide I can reduce the diameter of the back skin spots of the rats with allergic purpura and improve the skin reaction degree.

Example 3

The model was constructed as described above and, after the completion of the administration, skin histopathological observation was performed.

The epidermis of the rat in the control group is not obviously abnormal, has complete structure and is slightly thickened partially; squamous epithelial cells are normal in morphological structure and closely arranged; the dermis layer has rich collagen fiber content, can see the accessory organs such as the pilosebaceous glands, and has no obvious inflammation.

Compared with the control group, the epidermis of the rat in the model group is mostly thickened in the epidermis layer, small amount of squamous epithelial cells are vacuolated, small amount of epithelial cell necrosis and nuclear fragmentation are partially visible in partial skin, large amount of edema, mast cell infiltration and hemorrhage are visible in the dermis layer and subcutaneous tissue, and the blood stasis expansion are visible in the venous blood vessels.

Compared with the model group, the rat parts of the large-scale group with low (5 mg/kg), medium (10 mg/kg) and high (20 mg/kg) dosage of the atractylenolide I can see that the epidermis is slightly thickened, the dermis layer of the skin of the rat is slightly infiltrated by mast cells, and the conditions of parenchymal cell necrosis, nucleus rupture, venous blood stasis expansion, bleeding and the like are obviously reduced compared with the model group, and the result is shown in figure 3.

In conclusion, the atractylenolide I can relieve the skin histopathological damage of the rats with allergic purpura.

Example 4

And constructing a model according to the method, and detecting immune related indexes in serum after the administration is completed.

Studies have shown that: the occurrence of allergic purpura is associated with the level of IgA, igG, CIC in the serum. In clinic, the inducing factors of allergic purpura can cause the rise of IgA or IgG immune complexes in serum of patients, and the immune complexes are deposited on vessel walls to form CIC, which cannot be cleared, so that vascular inflammation is caused.

The IgA, igG, CIC content in the serum of rats was significantly increased in the model group compared to the control group (P < 0.01).

Compared with the model group, the serum IgA, igG, CIC content of rats in the low (5 mg/kg), medium (10 mg/kg) and high (20 mg/kg) dose groups of the atractylenolide I is obviously reduced, and the dosage dependency is carried out (P < 0.05 or P < 0.01). The results are shown in FIGS. 4-6.

To sum up: bighead atractylodes rhizome lactone I can reduce IgA, igG, CIC content in the serum of the rats with allergic purpura.

Example 5

And constructing a model according to the method, and detecting the inflammatory related factors in serum after the administration is completed.

The levels of TNF- α and IL-6 were significantly increased in the serum of rats in the model group compared to the control group (P < 0.01).

Compared with the model group, the content of TNF-alpha and IL-6 in serum of rats in the low (5 mg/kg), medium (10 mg/kg) and high (20 mg/kg) dose groups is obviously reduced, so that the content of TNF-alpha and IL-6 in serum can be reduced by the atractylenolide I, wherein the reduction effect of the high dose group is most obvious (P < 0.01). The results are shown in FIGS. 7 and 8.

To sum up: the atractylenolide I can reduce the content of inflammatory factors TNF-alpha and IL-6 in the serum of the rats with allergic purpura.

The following is true for examples 1-5: the atractylenolide I can treat and relieve the symptoms of skin redness, ecchymosis and hemorrhage of the rats with allergic purpura, reduce the infiltration of mast cells on affected parts, dilate blood vessels and reduce the contents of immune globulin, immune complex and inflammatory factors in serum.

The embodiments of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments, and various modifications may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.

Claims

1. The application of atractylenolide I as the only effective component in preparing the medicine for treating allergic purpura.

2. The use according to claim 1, wherein the medicament consists of atractylenolide i and a pharmaceutically acceptable carrier.

3. The use according to claim 2, wherein the medicament is in the form of a tablet, capsule, oral liquid or granule.

4. The use according to claim 3, wherein the tablet is a dragee tablet.

5. The use according to claim 3, wherein the oral liquid is a suspension.

6. The use according to claim 3, wherein the granules are suspension granules.