Plant extract and application of effective components of plant extract as AhR natural ligands in preparation of drugs for preventing and treating psoriasis

Abstract

The invention relates to the technical field of biological medicines, in particular to a plant extract and application of effective components of the plant extract as AhR natural ligands in preparation of drugs for preventing and treating psoriasis. The invention discovers and verifies that MCEO and small active molecules 1 and 2 thereof can be used as natural ligands of AhR to play an anti-psoriasis role for the first time, and 8 * MCEO and Active ingresent 2 have the best clinical effect and have great clinical application potential for inflammatory related skin injury. In the experiment, the Active ingresent 1 shows extremely strong capability of activating AhR to enter a nucleus, but the clinical treatment effect is not optimal. Experimental results tell that although the Active ingress 1 can be used as a natural ligand of the AhR to activate an AhR signal channel, the excessive activation of the AhR is unfavorable for better improving the skin barrier function of the psoriasis. Therefore, according to the anti-psoriasis drug taking the AhR as the therapeutic target, the activation of the AhR needs to be controlled within a certain activation threshold.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of a plant extract and its effective components as AhR natural ligands in the preparation of drugs for preventing and treating psoriasis. Background Art

[0002] Psoriasis, commonly known as "psoriasis", is a common chronic inflammatory skin disease with the main clinical manifestation of scaly erythema. Psoriasis is common in young and middle-aged people, with no difference in incidence between men and women. Epidemiological surveys have found that there are more than 60 million adults and children with psoriasis worldwide. Psoriasis is difficult to cure and prone to relapse. Patients are prone to depression, psoriatic arthritis and cardiometabolic syndrome, which brings endless physical and mental harm to patients and heavy economic burdens and mental pressure to families. It has a huge social impact and is listed as one of the top ten intractable diseases by the World Health Organization (WHO). Therefore, exploring the specific molecular mechanisms of the formation and maintenance of inflammatory responses is the key to discovering more potential therapeutic targets, and finding immunotherapy targets for psoriasis is the top priority of this project.

[0003] AhR is widely expressed in the human body and is a potential target for the treatment of many diseases. The development of drugs targeting AhR is a potential target for the treatment of many diseases such as psoriasis and atopic dermatitis. Especially in terms of skin immune regulation, the unique mechanism of action of AhR modulators in the treatment of inflammatory skin diseases is becoming a game-changer in the treatment of skin diseases.

[0004] Aryl hydrocarbon receptor (AhR) is a ligand-regulated cytoplasmic transcription factor that affects the transcription and expression of multiple genes. +The key molecule for T cell differentiation into Th17 and Tregs is a key regulator of the balance between pro-inflammatory or autoimmune Th17 cells and immunosuppressive or tolerant Tregs cells. Since AhR participates in multiple signaling pathways, has a variety of ligand structures, and has advantages such as tissue and species specificity to ligands, we can use different ligands to activate AhR, thereby purposefully regulating the abnormal differentiation of psoriasis T cells and providing new effective targets for the immunotherapy of psoriasis. In view of the important biological role of AhR and its prospects in clinical treatment, in-depth analysis of AhR natural ligands has important clinical significance for the development of new immunomodulatory strategies for inflammatory diseases such as psoriasis. German chamomile (M.chamomilla) is recognized as a star herb for its medicinal and aromatic properties. The medicinal value of the blue essential oil (MCEO) in the head inflorescence of German chamomile is constantly being explored, and its main components are matricaria and α-bisabolol. However, there is no document in the prior art that single chamazulene and α-bisabolol can treat psoriasis, nor is there any prior art that chamazulene and α-bisabolol can serve as natural ligands of AhR.

[0005] MCEO has anti-inflammatory, antibacterial, bactericidal, antispasmodic, and sedative pharmacological properties, and is a very popular herbal medicine in many pharmaceutical and aromatic industries. However, its application in the field of inflammatory skin diseases has only just begun to emerge. In our previous experiments, we extracted MCEO from the stamens of German chamomile by steam distillation, and analyzed the main components of MCEO by GC-MS. We used the LPS-induced keratinocyte (HaCaT) inflammation model and the psoriasis-like dermatitis animal model induced by external application of imiquimod (IMQ) cream to observe the clinical efficacy after intervention with different concentrations of MCEO. The experimental results showed that MCEO treatment can effectively reduce the abnormal proliferation and inflammatory response of HaCaT cells induced by LPS, and significantly improve the psoriasis-like skin lesions of model mice. However, there is no relevant research on the exact regulatory mechanism.

[0006] Prior art CN 113648433 A discloses an experimental method and application of plant extracts for treating psoriasis, including the following steps: selection of experimental animals; grouping of experimental animals, the experimental animals are randomly divided into five groups, normal control group female mice, psoriasis model group female mice, German chamomile fat-soluble extract external application group female mice, German chamomile water-soluble extract gavage group female mice, German chamomile fat-soluble and water-soluble extract combined treatment group female mice; experimental animal model preparation; psoriasis mouse model identification. The present invention uses the German chamomile fat-soluble extract and the German chamomile water-soluble extract test method, compared with the model group mice, the German chamomile fat-soluble extract external application group mice, the German chamomile water-soluble extract gavage group mice, and the German chamomile fat-soluble and water-soluble extract combined treatment group mice have significantly improved clinical symptoms.

[0007] In the present invention, it is particularly important to emphasize that German chamomile is grown independently in the cold black soil of Heilongjiang Province. Previous studies have found that the types and contents of the main active ingredients in its MCEO are significantly different from those in Xinjiang, Henan and the chamomile essential oils sold on the market, and the anti-inflammatory effect is extremely obvious. The present invention focuses on whether MCEO and its main active ingredients can serve as natural ligands of AhR to improve / prevent the occurrence of psoriasis skin immune pathological damage, aiming to provide theoretical and experimental basis for effective immune regulation strategies and clinical treatment intervention measures for psoriasis. Summary of the invention

[0008] The present invention firstly provides an application of a plant extract in preparing an AhR ligand or an AhR activator. The plant extract is German chamomile essential oil, and the application is for non-therapeutic purposes.

[0009] In certain embodiments, the German chamomile is grown independently in cold-region black soil.

[0010] In certain embodiments, the plant extract is blue essential oil from the flower heads of German chamomile.

[0011] In certain embodiments, the plant extract is extracted by steam distillation.

[0012] In certain embodiments, the main active ingredients of the plant extract include α-bisabolol and its oxides, matricaria and flavonoid derivatives; optionally, the flavonoid derivatives include one or more of apigenin, luteolin and apigenin-7-glucoside.

[0013] The present invention also provides an application of a main active ingredient 1 (Matricaria chamomilla, CAS No.: 529-05-5) of a plant extract in the preparation of an AhR ligand or an AhR activator, wherein the chemical formula of the main active ingredient 1 is shown in Formula 1, and the application is for non-therapeutic purposes;

[0014]

[0015] The present invention also provides an application of a main active ingredient 2 (α-bisabolol, CAS No.: 515-69-5) of a plant extract in the preparation of an AhR ligand or an AhR activator, wherein the chemical formula of the main active ingredient 2 is shown in Formula 2, and the application is for non-therapeutic purposes;

[0016]

[0017] The present invention also provides an application of a main active ingredient 1 or a main active ingredient 2 of a plant extract in the preparation of a drug for treating psoriasis, wherein the chemical formula of the main active ingredient 1 is shown in Formula 1 or Formula 2.

[0018] The present invention finally provides a composition for treating psoriasis, wherein the composition comprises a compound represented by Formula 1 or Formula 2.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] The present invention first discovered and confirmed that MCEO and its small molecule active molecules 1 and 2 can all act as natural ligands of AhR to play an anti-psoriatic effect, among which 8×MCEO and Active ingredient 2 have the best clinical effects and have great clinical application potential for inflammatory-related skin damage. In this experiment, Active ingredient 1 showed a very strong ability to activate AhR into the nucleus, but the clinical treatment effect was not optimal. The experimental results tell us that although Active ingredient 1 can act as a natural ligand of AhR to activate the AhR signaling pathway, the overactivation of AhR is not conducive to better improving the barrier function of psoriasis skin. Therefore, for anti-psoriatic drugs that use AhR as a therapeutic target, the activation of AhR needs to be controlled within a certain activation threshold. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Observation of clinical effects of each group of drugs on mouse skin lesions;

[0022] Figure 2 PASI scores of mice in each group;

[0023] Figure 3 HE staining of mouse skin tissue in each group (400×);

[0024] Figure 4 The expression level of AhR in the skin tissue of mice in each group was detected by IHC (400×);

[0025] Figure 5 WB was used to detect the expression level of AhR nuclear protein in the skin tissue of mice in each group;

[0026] Figure 6 IF was used to detect the nuclear ectopic status of AhR in the skin tissue of each group of mice;

[0027] Figure 7 CO-IP was used to detect the nuclear entry of AhR after activation in the skin tissue of psoriasis model mice. DETAILED DESCRIPTION

[0028] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0029] Example 1: MCEO preparation

[0030] The dried flowers of German chamomile collected from Zhonghong Village, Xinhua Village, Minzhu Village, and Yihe Village in Qixingpao Town, Baoqing County, Shuangyashan City, Heilongjiang Province, China, were ground into powder, and MCEO was extracted by steam distillation. The main components were detected by GC-MS, and the results are shown in Table 1. After identification, 16 active ingredients were found, among which the highest proportion was active ingredient 1: Matricaria chamomilla, and the main anti-inflammatory active ingredient 2: α-bisabolol.

[0031] The steps of steam distillation are as follows:

[0032] (1) Soaking: Weigh 50 g of dried German chamomile flowers, cut them into pieces, add them into distilled water at room temperature (20-25°C) at a ratio of 1:10-15, and soak for 2 h.

[0033] (2) Ultrasonic enhanced steam distillation method for extracting hydrosol: The soaked chamomile dried flower powder was ultrasonically treated with an ultrasonic power of 160W, an enhanced time of 20min, a liquid-to-solid ratio of 10-15:1, a temperature of 25-30°C, and steam distillation for 2 hours. After the essential oil and the hydrosol were separated, a rotary evaporator (Buchi, Switzerland) was used at a temperature of 50°C and a speed of 100r / min (revolutions / minute) to evaporate the raw drug to a concentration of 5g / mL. The extract was stored in a refrigerator at 4°C for later use. The final pH value of the extract was 5.0-5.5.

[0034] Table 1 Main active ingredients of MCEO detected by GC-MS

[0035]

[0036] Example 2: Construction of psoriasis mouse model:

[0037] (2.1) Experimental animals: Male BALB / c mice, 6-8 weeks old, 20-25 g, standard feed, drinking water, bedding and all items were sterile. All animal experiments were approved by animal ethics review.

[0038] (2.2) Experimental Animal Grouping:

[0039] Normal control group: Normal Control, NC, 12 mice;

[0040] Psoriasis model group: Psoriasis Model, PM, 20 mice;

[0041] 8×MCEO treatment group: MCEO+Psoriasis Model diluted 8 times with base oil, 8×MPM, 16 mice;

[0042] 10×MCEO treatment group: MCEO+Psoriasis Model diluted 10 times with base oil, 10×MPM, 16 mice;

[0043] Active ingredient 1 (Matricaria chamomilla, CAS No.: 529-05-5) treatment group: Active ingredient 1 + Psoriasis Model, A-1PM, 8 mice;

[0044] Active ingredient 2 (α-bisabolol, CAS No.: 515-69-5) treatment group: Active ingredient 2 + Psoriasis Model, A-2PM, 8 mice;

[0045] (2.3) Experimental animal treatment methods:

[0046] PM group mice: Hair in the 2×3cm area on the back of the mice was removed, and special equipment was used to remove fine hair. The shaved mice were kept for 3 days without any treatment to eliminate the damage and impact of shaving on the back skin; the mice were given 5% IMQ cream 62.5 mg / mouse at a fixed time point (noon) for 7 consecutive days, and the exposed area on the back was evenly applied and covered with 3×3cm plastic wrap, and the fingers were gently pressed 60 times continuously. The mice were photographed and recorded before each administration. New fine hairs were removed at any time with a single-head electric shaver to keep the exposed area on the back of the mice hairless at all times.

[0047] 8×MPM group mice: PM mice were constructed and 8×MCEO was evenly applied to the exposed skin at fixed time points for 7 consecutive days, 200 μL / time, twice a day;

[0048] 10×MPM group mice: PM mice were constructed and contacted at the same time, and 10×MCEO was evenly applied to the exposed skin area at fixed time points for 7 days, 200 μL / time, twice a day;

[0049] A-1PM group mice: PM mice were constructed and Active ingredient 1 was evenly applied to the exposed skin at fixed time points for 7 consecutive days, 50 μL / time, twice a day;

[0050] A-2PM group mice: PM mice were constructed and ActiveIngredient2 was evenly applied to the exposed skin at fixed time points for 7 consecutive days, 50 μL / time, twice a day;

[0051] NC group mice: The control group mice were given the same dose of vaseline, and the rest of the treatment process was the same as above.

[0052] (2.4) Identification of psoriasis mouse model:

[0053] PASI score:

[0054] Evaluation of the severity of back skin inflammation: An objective scoring system was established based on the clinical PASI score;

[0055] The degree of erythema, scaling, and thickening (0-4 points) was scored independently according to the following scoring rules:

[0056] 0 = none, 1 = mild, 2 = moderate, 3 = obvious, 4 = very obvious; the cumulative score (erythema + scaling + thickening, 0-12 points) is used to indicate the degree of skin inflammation. All scoring items were completed independently by three researchers in different times.

[0057] (2.5) Skin histopathology examination:

[0058] HE staining: The mice in each group were killed by cervical dislocation on the 8th day after IMQ treatment. The skin lesion tissue of 0.5×1 cm in size was taken and the subcutaneous fat was removed and placed in formalin fixative. After HE staining, the spinous layer thickness, squamous area, Munro's microabscess area and total dermal cell count were detected.

[0059] like Figure 1 As shown in the figure, compared with the Con group, the mice in the IMQ group had obvious erythema, scaling and psoriasis-like lesions on the back. Compared with the mice in the IMQ group, the skin lesions on the back of the mice in the 8×MCEO, 10×MCEO and Active ingredient 2 treatment groups were significantly improved, and the erythema, scaling and psoriasis-like lesions were significantly reduced, but the clinical erythema, scaling and psoriasis-like lesions of the skin of the mice in the Active ingredient 1 group were not improved well.

[0060] like Figure 2 As shown in the figure, the PASI score of the back skin of mice in the IMQ group increased significantly. Compared with the IMQ group, the PASI score of the back skin of mice in the 8×MCEO, 10×MCEO, Active ingredient 1 and Active ingredient 2 drug treatment groups was significantly reduced, and the difference was more obvious in the 8×MCEO, 10×MCEO and Active ingredient 2 groups (P<0.0001).

[0061] like Figure 3As shown, HE staining results showed that compared with Con group, the granular layer of the skin of mice in IMQ group disappeared, the epidermal keratinization was hyperkeratotic with incomplete keratinization, the spinous layer was significantly thickened, the epidermal process was extended downward, and more inflammatory cells were observed in the superficial dermis; compared with IMQ group, the spinous layer of the skin of mice in 8×MCEO, 10×MCEO, Active ingredient 1 and Active ingredient 2 drug treatment groups was significantly thinner, the epidermal process was significantly shortened, the hyperkeratosis and incomplete keratinization were significantly reduced, and only a small amount or no inflammatory cell infiltration was observed in the superficial dermis, among which the 8×MCEO and Active ingredient 2 groups were the most obvious; the above results showed that MCEO / Active ingredient 1 / 2 can effectively alleviate IMQ-induced psoriasis-like skin inflammation in mice.

[0062] Example 4: MCEO and its main active ingredients can both target and activate the AhR signaling pathway

[0063] like Figure 4 As shown in the figure, IHC test results showed that compared with the Con group, the expression of AhR in the skin tissue of the IMQ group was significantly increased; compared with the IMQ group, the expression of AhR in the skin tissue of the mice in the 8×MCEO, 10×MCEO, Active ingredient 1 and Active ingredient 2 drug treatment groups was significantly decreased; the above results showed that MCEO / Active ingredient 1 / 2 can target and activate AhR, and the AhR entering the cell nucleus forms a heterodimer with ARNT to initiate the transcription of the target gene. After the transcription is completed, AhR and the ligand are exported to the cell nucleus, where AhR is rapidly degraded by the proteasome, while the ligand is metabolized and cleared by the CYP enzyme.

[0064] like Figure 5 As shown in the figure, the cytoplasmic proteins and nuclear proteins in the mouse skin tissue were separated, and the expression of AhR in the nuclear protein was detected by WB. It was found that compared with the Con group, there was no difference in the nuclear entry of AhR protein in the skin tissue of the IMQ group mice; the expression of nuclear protein AhR in the skin tissue of the 8×MCEO, 10×MCEO, Active ingredient 1 and Active ingredient 2 drug treatment groups increased significantly, among which the Active ingredient 1 and Active ingredient 2 treatment groups were the most obvious. The experimental results show that MCEO / Active ingredient 1 / 2 can target and activate AhR and increase the activity of AhR protein in the nucleus.

[0065] like Figure 6As shown, IF detection found that compared with the Con group, no AhR nuclear translocation was observed in the IMQ group; obvious AhR nuclear translocation was observed in the skin tissue of mice treated with 8×MCEO, 10×MCEO, Active ingredient 1 and Active ingredient 2, and the most obvious was in the Active ingredient 1 group, where the expression of AhR in the nucleus increased significantly; this result indicates that MCEO / Active ingredient 1 / 2 can target and activate AhR and increase the activity of AhR protein in the nucleus.

[0066] like Figure 7 As shown in the figure, the cytoplasmic proteins and nuclear proteins in the mouse skin tissue were separated, and the nuclear proteins were used for CO-IP to detect the changes in the expression of AhR-ARNT dimers. IP: ARNT, IB: AhR, WB results showed that compared with the Con group, the nuclear AhR-ARNT dimers in the IMQ group increased slightly; compared with the IMQ group, the nuclear AhR-ARNT dimers in the 8×MCEO, Active ingredient 1 and Activeingredient 2 groups increased significantly, among which the 8×MCEO and Active ingredient 1 groups were the most obvious; this result is consistent with the IF result. The experimental results show that MCEO / Active ingredient 1 / 2 can target and activate AhR and increase the activity of AhR protein in the nucleus.

[0067] In summary, MCEO and its small molecule active molecules 1 and 2 can all act as natural ligands of AhR to exert anti-psoriatic effects, among which 8×MCEO and Active ingredient 2 have the best clinical effects and have great clinical application potential for inflammatory-related skin damage. In this experiment, Active ingredient 1 showed a strong ability to activate AhR into the nucleus, but the clinical treatment effect was not optimal. The experimental results tell us that although Active ingredient 1 can act as a natural ligand of AhR to activate the AhR signaling pathway, the excessive activation of AhR is not conducive to better improving the barrier function of psoriasis skin. Therefore, for anti-psoriatic drugs that use AhR as a therapeutic target, the activation of AhR needs to be controlled within a certain activation threshold.

[0068] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a plant extract in the preparation of an AhR ligand or an AhR activator, characterized in that: The plant extract is German chamomile and the application is for non-therapeutic purposes.

2. The use according to claim 1, characterized in that: The German chamomile is grown independently on black soil in cold regions.

3. The use according to claim 1, characterized in that: The plant extract is blue essential oil from the inflorescence of German chamomile.

4. The use according to claim 3, characterized in that: The extraction method of the plant extract is steam distillation.

5. The use according to claim 3, characterized in that: The main active ingredients of the plant extract include α-bisabolol and its oxide, matricaria and flavonoid derivatives; optionally, the flavonoid derivatives include one or more of apigenin, luteolin and apigenin-7-glucoside.

6. Use of a main active ingredient 1 of a plant extract in the preparation of an AhR ligand or an AhR activator, characterized in that: The chemical formula of the main active ingredient 1 is shown in Formula 1, and the application is for non-therapeutic purposes; 7. Use of a main active ingredient 2 of a plant extract in the preparation of an AhR ligand or an AhR activator, characterized in that: The chemical formula of the main active ingredient 2 is shown in Formula 2, and the application is for non-therapeutic purposes; 8. Use of the main active ingredient 1 or the main active ingredient 2 of a plant extract in the preparation of a drug for treating psoriasis, characterized in that: The chemical formula of the main active ingredient 1 is shown in Formula 1 or Formula 2.

9. A composition for treating psoriasis, characterized in that: The composition comprises a compound represented by Formula 1 or Formula 2.

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