TWI716940B - Prevention and/or treatment of psoriasis with 2,4-dimethoxy-6-methylbenzene-1,3-diol - Google Patents

Abstract

This invention discloses that 2,4-dimethoxy-6-methylbenzene-1,3-diol can be used in the prevention and/or treatment of psoriasis.

Description

Use 2,4-Dimethoxy-6-methylbenzene-1,3-diol to prevent and/or treat psoriasis

The present invention relates to the use of 2,4-dimethoxy-6-methylbenzene-1,3-diol (2,4-dimethoxy-6-methylbenzene-1,3-diol) to prevent and/or treat Psoriasis (psoriasis).

Psoriasis (also known as Yin Xie Bing) is a chronic inflammatory disease related to abnormal proliferation of skin epidermal cells (chronic inflammatory disease), patients usually have boundaries on the skin surface Distinct erythema (erythema), and its surface is covered with silvery white scales (scaling), and the epidermal stratum corneum of the lesion will be significantly thickened and neutrophil accumulation, which leads to microabscess (microabscess) )Formation. The prevalent areas include: scalp, elbows, knees, extensor side of limbs, trunk and perineum. In addition, some patients also suffer from psoriatic arthritis, metabolic syndrome, and cardiovascular disease.

Because the pathological conditions and symptoms of psoriasis are quite complex, its pathogenesis is still unclear so far. At present, studies have shown that inheritance plays an important role in the pathogenesis of psoriasis, and trauma (trauma) , Infection, stress, endocrine factors, metabolism factors, climate and drugs may all induce or aggravate the onset of psoriasis.

The current clinical methods used to treat psoriasis are roughly divided into the following three categories: (1) topical therapy, applying topical drugs to the affected area, such as corticosteroids, vitamin D, and Its derivatives, retinoids, coal tar, and moisturizers; (2) systemic therapy, in which patients are administered oral drugs such as methotrexate (MTX) ), cyclosporin and tretinoin; and injectable biologics, such as alefacept (trade name Amevive ^® ); and (3) phototherapy, such as ultraviolet light B (UVB) phototherapy [ultraviolet B (UVB) phototherapy] and psoralen plus ultraviolet A (PUVA) photochemotherapy [psoralen plus ultraviolet A (PUVA) photochemotherapy].

However, long-term use of the above treatment methods may lead to side effects or drug tolerance in patients. Therefore, relevant researchers in the field are committed to finding active components that are safe and can be used to treat psoriasis from traditional Chinese medicines (TCM) or plants.

Antrodia cinnamomea ( Taiwanofungus camphoratus ) (Chinese aliases are Antrodia camphorata , Antrodia cinnamomea , Antrodia cinnamomea and Antrodia cinnamomea ; synonymous names Antrodia camphorata , Antrodia cinnamomea, and Ganoderma comphoratum ) and Xiangshanzhi ( Taiwanofungus salmoneus ) (Chinese alias is Xiangshan mushroom ; The synonym Antrodia salmonea is a fungus of the genus Taiwanofungus of the Polyporaceae family (Polyporaceae), which mainly grows on the Cinnamomum kanehirai and Cunninghamia konishii in Taiwan. Since Xiangshanzhi is similar in classification and composition to Antrodia cinnamomea, it is considered to be a substitute for Antrodia cinnamomea. Studies have pointed out that both Antrodia cinnamomea and Xiangshanzhi have anti-oxidant, anti-tumor, anti-bacterial and anti-inflammation biological activities.

。 之後，該等化合物被拿來處理惡性黑色素瘤細胞株(malignant melanoma cell line) H2058以及肝癌細胞株(hepatoma cell lines) HA22T與HepG2，而結果發現，在上述9種經分離的化合物中只有2-甲氧基-6-甲基-p-苯醌以及2,3-甲氧基-5-甲基-p-苯醌能夠對該等癌細胞展現細胞毒性(cytotoxicity)。 In Shen CC et al . (2008), Journal of the Chinese Chemical Society , 55:854-857, Shen CC et al. used chloroform to extract the liquid fermentation culture of Xiangshan Mushroom mycelium, and then used the column layer 9 compounds were separated from the obtained chloroform extract by column chromatography (column chromatography) and thin layer chromatography (TLC), including: (1) 6 known compounds, namely 2- Methoxy-6-methyl-p-benzoquinone (2-methoxy-6-methyl-p-benzoquinone), 2,3-methoxy-5-methyl-p-benzoquinone (2,3-dimethoxy -5-methyl-p-benzoquinone), 2-hydroxy-5-methoxy-3-methyl-p-benzoquinone (2-hydroxy-5-methoxy-3-methyl-p-benzoquinone), dentate (eburicoic acid), pyrroledione, and fomefficinic acid C; and (2) 3 novel compounds, namely 2,4-dimethoxy-6-methylbenzene-1,3-diol (2,4-dimethoxy-6-methylbenzene-1,3-diol), 2-(2,4-dihydroxy-3,5-dimethoxyphenyl)-5-methoxy-3-methyl -1,4-Benzoquinone [2-(2,4-dihydroxy-3,5-dimethoxybenzyl)-5-methoxy-3-methyl-1,4-benzoquinone] (named salmoquinone), and 3-(4 -Hydroxyphenyl)-4-isobutyl-1H-pyrrole-2,5-dione [3-(4-hydroxyphenyl)-4-isobutyl-1H-pyrrole-2,5-dione], of which 2, 4-Dimethoxy-6-methylbenzene-1,3-diol has the following chemical formula:

. Afterwards, these compounds were used to treat malignant melanoma cell line H2058 and hepatoma cell lines HA22T and HepG2. As a result, it was found that only 2 of the above 9 isolated compounds Methoxy-6-methyl-p-benzoquinone and 2,3-methoxy-5-methyl-p-benzoquinone can exhibit cytotoxicity to these cancer cells.

In Yang SS et al . (2009), Planta. Med. , 75:512-516, Yang SS et al . used methanol to extract the solid-state fermentation culture of Antrodia cinnamomea mycelium, and then filtered the obtained methanol extract And concentration, and then the obtained residue was dissolved in 65% methanol, and n-hexane and ethyl acetate were used in order for partitioning. Next, column chromatography was used to separate 5 compounds from the obtained ethyl acetate separation fraction, including antroquinonol B (antroquinonol B), 4-acetyl-antroquinonol B (4-acetyl-antroquinonol) B), 2,3-(methylenedioxy)-6-methylbenzene-1,4-diol [2,3-(methylenedioxy)-6-methylbenzene-1,4-diol], 2,4 -Dimethoxy-6-methylbenzene-1,3-diol and antrodin D. In addition to 2,3-(methylenedioxy)-6-methylbenzene-1,4-diol, the other four compounds have been found to inhibit LPS-activated mouse macrophages RAW 264.7 (LPS-activated mouse macrophage cell RAW 264.7) secretes nitric oxide. In particular, Andrinoquinol B and 4-Acetyl Androquinol B exhibit strong inhibitory effects.

After research, the applicant unexpectedly discovered that 2,4-dimethoxy-6-methylbenzene-1,3-diol can effectively prevent and/or treat psoriasis in addition to the above-mentioned activities.

Summary of the invention

Therefore, the present invention provides a use of 2,4-dimethoxy-6-methylbenzene-1,3-diol for the preparation of a medicine for preventing and/or treating psoriasis.

The present invention also provides a method for preventing and/or treating psoriasis, which comprises administering 2,4-dimethoxy-6-methylbenzene-1,3-diol to an individual in need thereof.

Detailed description of the invention

It should be understood that if any previous case publication is quoted here, the previous case publication does not constitute a recognition: in Taiwan or any other country, the previous case publication forms a common general in the art Part of knowledge.

For the purpose of this specification, it will be clearly understood that the word "comprising" means "including but not limited to", and the word "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used in this article have meanings commonly understood by those familiar with the art of the present invention. A person familiar with the art will recognize many methods and materials similar or equivalent to those described herein that can be used to implement the present invention. Of course, the present invention is by no means limited by the methods and materials described.

In the present invention, the applicant found through an in vitro test: 2,4-Dimethoxy-6-methylbenzene-1,3-diol can alleviate imiquimod (imiquimod, IMQ) Psoriasis-like inflammation induced in macrophages. In addition, the applicant further confirmed through animal experiments that 2,4-Dimethoxy-6-methylbenzene-1,3-diol can treat psoriasis induced by IMQ on the back skin of mice. Therefore, the present invention discloses a use of 2,4-dimethoxy-6-methylbenzene-1,3-diol for the preparation of a medicine for preventing and/or treating psoriasis.

As used herein, the term "treating" or "treatment" means reducing, alleviating, ameliorating, relieving or controlling a disease ( disease) or disorder (disorder) one or more clinical signs (clinical signs), and lowering (lowering), stopping (stopping) or reversing (reversing) a condition or symptom (symptom) being treated The progression of severity.

As used herein, the term "preventing" or "prevention" of psoriasis means that an individual eliminates or reduces the incidence of psoriasis when it has not been diagnosed with psoriasis. ), and slow, delay, control, or reduce the likelihood or probability of psoriasis.

According to the present invention, 2,4-dimethoxy-6-methylbenzene-1,3-diol can be prepared using synthetic techniques well known to chemists.

Alternatively, 2,4-dimethoxy-6-methylbenzene-1,3-diol can also be separated and purified from a natural source by using the separation and purification methods commonly used in the art come out. In this regard, you can refer to, for example, Shen CC et al . (2008) (same as above) and Yang SS et al . (2009) (same as above).

According to the present invention, the natural source is Taiwanofungus camphoratus or Taiwanofungus salmoneus . In a preferred embodiment of the present invention, 2,4-dimethoxy-6-methylbenzene-1,3-diol is isolated and purified from the ethanol extract of the mycelium culture of Antrodia cinnamomea .

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenteral, oral or topical administration by using techniques well known to those skilled in the art, including , But not limited to: injection (for example, sterile aqueous solution or dispersion), sterile powder, tablet, troche, Pills, capsules, external preparations and the like.

The medicine may further include a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, decomposers ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of: intravenous injection, subcutaneous injection, intradermal injection Injection (intraepidermal injection), intradermal injection (intradermal injection) and intralesional injection (intralesional injection).

According to the present invention, the medicine can be manufactured into a dosage form suitable for oral administration by using techniques well known to those skilled in the art, which include, but are not limited to: sterile powder, lozenge (tablet), tablet (troche), lozenge (lozenge), pill (pellet), capsule (capsule), dispersible powder (dispersible powder) or granule (granule), solution, suspension (suspension), emulsion (emulsion), syrup (syrup), elixir (elixir), thick syrup (slurry) and the like.

According to the present invention, the medicine can also be manufactured into an external preparation suitable for topical application to the skin by using techniques well known to those skilled in the art, including, but not limited to: emulsions , Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion , Serum, paste, foam, drop, suspension, salve and bandage.

According to the present invention, the external preparation is prepared by mixing the pharmaceutical product of the present invention with a base well known to those skilled in the art.

According to the present invention, the substrate may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and white Vaseline (white petrolatum), wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers (absorption) enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ^® 941 (carbopol ^® 941), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)], the activity of Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occluding agents occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants ( propellants) and so on. The selection and quantity of these additives fall within the scope of professionalism and routine technology of those who are familiar with this technology.

The present invention also provides a method for preventing and/or treating psoriasis, which comprises administering 2,4-dimethoxy-6-methylbenzene-1,3-diol to an individual in need thereof.

According to the present invention, the dosage and frequency of administration of 2,4-Dimethoxy-6-methylbenzene-1,3-diol will vary depending on the following factors: the severity of the disease to be treated, the route of administration, And the age, physical condition and response of the individual to be treated. Generally speaking, the pharmaceuticals according to the present invention can be in the form of a single dose or divided into several doses, and can be administered orally, parenterally or topically.

Detailed description of the preferred embodiment

The present invention will be further described with the following embodiments, but it should be understood that these embodiments are only for illustrative purposes and should not be interpreted as limitations on the implementation of the present invention. Examples General Experimental Method: 1. Statistical analysis (statistical analysis):

In the following examples, the experiment of each group is repeated 3 times, and the experimental data is expressed as "mean ± standard error of the mean (SEM)". All data are analyzed by Student's t-test to assess the differences between groups. If the result of the statistical analysis is p >0.05, it means there is statistical significance. Example 1. Preparation derived from Antrodia (Taiwanofungus camphoratus) of 2,4-dimethoxy-6-methyl-benzene-1,3-diol (2,4-dimethoxy-6-methylbenzene -1,3- diol)

(peptone)、1.5%麥芽萃取物(malt extract)、0.6%酵母萃取物(yeast extract)、0.06% KH ₂PO ₄、0.06% MgSO ₄，以及0.06%麩胺酸單鈉(monosodium glutamate)]中並於室溫下進行震盪培養歷時24小時，接而進行靜置培養歷時30天。之後，對所形成的菌絲體培養物秤取500 g並以一均質機(Oster ^®Classic Series 14-Speed Blender)來進行均質處理。之後，加入1.5 L的95%乙醇來進行浸泡並靜置歷時2天，繼而藉由過濾來收取上澄液並利用減壓濃縮以移除乙醇，而得到一殘餘物(residue)。對所得到的殘餘物重複進行乙醇浸泡-過濾-減壓濃縮步驟2次，藉此而得到一菌絲體培養物的乙醇萃取物。 The Antrodia cinnamomea used in this example was collected from the Dongshi Forest Farm in Taichung City, and confirmed to belong to the genus of Taiwanofungus through 18S rDNA sequence analysis and sequence alignment in the NCBI database. First, Antrodia cinnamomea was inoculated on a PDB agar plate and cultured at room temperature for 30 days. Next, cut the formed Antrodia cinnamomea mycelium so that it has a volume of 0.5 mm ³ and then inoculate it into a liquid medium [containing 3% glucose, 1.5% molasses, 0.5% protein

(peptone), 1.5% malt extract, 0.6% yeast extract, 0.06% KH ₂ PO ₄ , 0.06% MgSO ₄ , and 0.06% monosodium glutamate (monosodium glutamate)] Neutralization and shaking culture at room temperature for 24 hours, followed by static culture for 30 days. After that, weigh 500 g of the formed mycelial culture and homogenize it with a homogenizer (Oster ^® Classic Series 14-Speed Blender). After that, 1.5 L of 95% ethanol was added for soaking and allowed to stand for 2 days, and then the supernatant was collected by filtration and concentrated under reduced pressure to remove the ethanol to obtain a residue. The obtained residue is repeatedly subjected to the steps of ethanol soaking, filtration, and reduced pressure concentration twice, thereby obtaining an ethanol extract of a mycelial culture.

The obtained ethanol extract was dissolved in 250 mL of ethyl acetate (EtOAc), and then 250 mL of pure water was used for partitioning. The ethyl acetate layer was collected and concentrated under reduced pressure to remove ethyl acetate. The residue obtained was then subjected to silica gel column chromatography and used with n-hexane )/Acetone as eluent for gradient elution [gradient 9:1→7:1→5:1→3:1→1:1 (v/v)] . Collect the eluate eluted with n-hexane:acetone=7:1 (v/v) to obtain a brown oily compound.

After that, a Burker Avance II 400 MHz NMR spectrometer (NMR spectrometer) was used to measure the ¹ H-NMR spectrum of the obtained brown oily compound. The solvent used was CDCl ₃ and the chemical shifts (δ) It is in ppm. The measured ¹ H-NMR spectrum is shown in FIG. 1.

。 實施例 2. 2,4- 二甲氧基 -6- 甲基苯 -1,3- 二醇的活體外抗乾癬效用的評估 According to Figure 1, the compound was confirmed to be a 2,4-dimethoxy-6-methylbenzene-1,3-diol with the following chemical structure:

. Example 2. Evaluation of the in vitro anti-psoriatic effect of 2,4 -dimethoxy -6- methylbenzene- 1,3- diol

In this example, the applicant selected macrophages treated with imiquimod (IMQ) as an in vitro model, and treated the cells with 2,4-dimethoxy-6-methylbenzene-1 , 3-diol followed by pre-inflammatory cytokines (including interleukin-6 (IL-6), interleukin-23 (IL-23), interleukin-24 ( interleukin-24, IL-24) and tumor necrosis factor-α (tumor necrosis factor-α, TNF-α)] mRNA and protein expression measurement, in order to evaluate 2,4-dimethoxy-6- The effect of methylbenzene-1,3-diol on IMQ-induced psoriasis-like inflammation. Experimental Materials:

First, the THP-1 cells obtained from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan are divided into two ×10 ⁶ cells/well were cultured in 2 mL of RPMI 1640 medium (Invitrogen) (supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM pyruvate and 100 nM Phorbol-12-myristate-13-acetate (phorbol 12-myristate-13-acetate, PMA)] in a 6-well plate (6-well plate), and in an incubator (37℃) 5% CO ₂ ) for 24 hours. After that, the medium was replaced with fresh RPMI 1640 medium (with 10% FBS, 10 mM HEPES, and 1 mM pyruvate) that did not contain PMA, and cultured in an incubator (37°C, 5% CO ₂ ) for 24 Hours, to induce THP-1 cells to differentiate into macrophages.

Next, the macrophages differentiated from THP-1 were divided into 1 normal control group, 1 pathological control group, and 1 experimental group, and then replaced with fresh RPMI 1640 medium, in which the medium of the pathological control group was additionally added There is 10 μg/mL IMQ, and the medium of this experimental group is additionally supplemented with 10 μg/mL IMQ and 10 μM 2,4-Dimethoxy-6-methylbenzene-1 obtained in Example 1 above, 3-diol (in methanol).

After the cells of each group were cultured in an incubator (37°C, 5% CO ₂ ) for 4 hours, the formed culture was centrifuged at 1500 rpm for 5 minutes, and then the formed cell pellet was collected and cultured. Use the aqua solution to perform the following experiments A and B respectively. A. Determination of relative mRNA expression level of pre-inflammatory cytokines :

In order to study the gene expression of pre-inflammatory cytokines in each group of macrophages, quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the IL-6 and IL in macrophages. -23, IL-24 and TNF-α mRNA expression levels.

First, take an appropriate amount of the cell pellets of each group obtained as above, and then use TRIzol reagent (Invitrogen) and perform the extraction of total RNAs (total RNAs) according to the operating instructions provided by the manufacturer. Use the iScript cDNA synthesis kit (Bio-Rad) to perform the reverse transcription reaction (reverse transcription reaction) to synthesize the first strand of cDNA using the iScript cDNA synthesis kit (Bio-Rad). -strand cDNA).

Next, use the obtained first strand cDNA as a template, and use the primer pairs for IL-6, IL-23, IL-24 and TNF-α genes shown in Table 1 below, using a CFX Connect™ Real-time PCR detection system (CFX Connect™ Real-Time PCR Detection system) performs qRT-PCR according to the manufacturer's operating instructions. In addition, the gene expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as an internal control. The operating conditions and reaction conditions for qRT-PCR are shown in Table 2 below. Table 1. Primers used for qRT-PCR Target gene NCBI login number Introduction Nucleotide sequence (5'→3') IL-6 NM_000600.3 Forward primer IL-6-F AGCGCCTTCGGTCCAGTTGC (Sequence Identification Number: 1) Reverse primer IL-6-R GTGGCTGTCTGTGTGGGGCG (Sequence Identification Number: 2) IL-23 NM_016584.2 Forward primer IL-23-F TCCAAGCCTCAGTCCCAG (serial identification number: 3) Reverse primer IL-23-R TGGGGTGGTAGATTTATCTTGG (Sequence Identification Number: 4) IL-24 NM_006850.3 Forward primer IL-24-F CAGGAGGAACACGAGACTGA (serial identification number: 5) Reverse primer IL-24-R GCACAACCATCTGCATTTGAGA (Sequence Identification Number: 6) TNF-α NM_000594.3 Forward primer TNF-α-F ATGAGCACTGAAAGCATGATCC (serial identification number: 7) Reverse primer TNF-α-R GAGGGCTGATTAGAGAGAGGTC (Sequence Identification Number: 8) GAPDH NM_002046.5 Forward primer GAPDH-F TGCACCACCAACTGCTTAGC (serial identification number: 9) Reverse primer GAPDH-R GGCATGGACTGTGGTCATGAG (Sequence Identification Number: 10) Table 2. Reaction conditions of qRT-PCR Contents Volume (µL) First strand cDNA (1 µg/µL) 1 Forward primer (300 nM) 1 Reverse primer (300 nM) 1 2X Maxima SYBR Green/Fluorescein qPCR Master Mix premix reagent (Applied Biosystems) 12.5 Sterile water 6.5 Operating conditions: pre-incubation at 95°C for 3 minutes; followed by 49 cycles as follows: denaturation at 95°C for 10 seconds, primer annealing at 55°C for 30 Seconds, and finally, the extension reaction (extension) was performed at 95°C for 10 seconds.

The PCR products thus obtained are detected with the fluorescence of SYBR Green (double-stranded DNA binding dye) to calculate the cycle threshold ( C _t ) of each pre-inflammatory cytokine. value]. The relative mRNA expression level of each pre-inflammatory cytokine (relative mRNA expression level) is based on the comparative C _t method (comparative C _t method), and the cycle threshold value obtained by each is used as the cycle threshold value of the PCR product obtained by the GAPDH gene For standardization.

After that, analyze the experimental data obtained in accordance with the method described in the first item "Statistical Analysis" of the "General Experimental Methods" above. B. Determination of protein expression of pre-inflammatory cytokines:

The culture supernatant of each group of cells obtained above uses IL-6 ELISA kit (brand of BioLegend, item number 430504), IL-23 ELISA kit (brand of BioLegend, item number 430704), IL-6 -24 ELISA kit (brand name is R&D systems, item number is DY1965) and TNF-α ELISA kit (brand name is BioLegend, item number 430204) to perform IL-6, IL-23, IL-24 and TNF-α Determination of protein expression. The measured absorbance values of each group are respectively based on the standard curve prepared in advance with the standard products with different known concentrations of IL-6, IL-23, IL-24 and TNF-α relative to their own absorbance values. It is converted into its concentration (pg/mL). After that, analyze the experimental data obtained in accordance with the method described in the first item "Statistical Analysis" of the "General Experimental Methods" above. Results: A. Determination of relative mRNA expression level of pre-inflammatory cytokines and protein expression level:

Table 3 and Table 4 below show the relative mRNA expression levels and protein expression levels of each pre-inflammatory cytokine measured in each group of cells. It can be seen from Table 3 and Table 4 that compared with the normal control group, the relative mRNA expression level and protein expression level of IL-6, IL-23, IL-24, and TNF-α in the pathological control group increased significantly. High, which means IMQ will induce psoriasis-like inflammation in macrophages. Compared with the pathological control group, the relative mRNA expression levels and protein expression levels of IL-6, IL-23, IL-24, and TNF-α in the experimental group decreased significantly, even similar to the normal control group. . The results of this experiment show that 2,4-Dimethoxy-6-methylbenzene-1,3-diol can effectively relieve psoriasis-like inflammation. Table 3. Relative mRNA expression levels of previous inflammatory cytokines measured in each group of cells Normal control group Pathology control group test group IL-6 0.02152±0.01292 0.08304±0.00305 ^## 0.03538±0.01335 ^** IL-23 0.1843±0.04750 0.3309±0.05701 ^# 0.1120±0.02514 ^* IL-24 1.068±0.2193 29.42±3.459 ^### 2.929±0.02124 ^** TNF-α 8.633×10 ^-4 ±1.33×10 ^-4 3.277×10 ^-3 ±1.342×10 ^-4### 5.9×10 ^-4 ±3.850×10 ^{-4** #} : When compared with the normal control group, p >0.05 ^## : When compared with the normal control group, p >0.01 ^### : When compared with the normal control group, p >0.001 ^* : When compared with the pathological control group Comparison, p >0.05 ^** : When compared with the pathological control group, p >0.01 Table 4. The protein expression level of the previous inflammatory cytokines measured in the culture supernatant of each group Normal control group Pathology control group test group IL-6 (pg/mL) 175.8±72.06 1653±125.30 ^### 492.8±107.30 ^** IL-23 (pg/mL) 67.02±9.59 211.4±28.60 ^## 76.66±29.87 ^** IL-24 (pg/mL) 269.9±2.88 504.8±31.90 ^## 303.8±54.68 ^** TNF-α (pg/mL) 432.9±87.43 3176±758.30 ^# 755.9±402.80 ^{* #} : When compared with the normal control group, p >0.05 ^## : When compared with the normal control group, p >0.01 ^### : When compared with the normal control group, p >0.001 ^* : When compared with the pathological control group Comparison, p >0.05 ^** : When compared with the pathological control group, p >0.01

Based on the above experimental results, the applicant believes that 2,4-Dimethoxy-6-methylbenzene-1,3-diol can alleviate psoriasis-like inflammation by inhibiting the activation of macrophages, and therefore has a development High potential as an anti-psoriatic drug. Example 3. 2,4-dimethoxy-6-methyl-benzene-1,3-diol for assessing the effectiveness of treatment in mice with induced IMQ- psoriasis (IMQ-induced psoriasis) Experimental Materials: 1. Experimental animals:

The BALB/c mice (BALB/c mice) (6 to 8 weeks old, weighing about 22±2 g) used in this example were purchased from the National Laboratory Animal Center (ROC) . All experimental animals were individually reared in an animal room with 12 hours of light and darkness, room temperature maintained at 21-24°C and relative humidity maintained at 45-70%, and water and feed were adequately supplied. All experimental procedures related to experimental animals are approved by the Institutional Animal Care and Use Committee of Linkou Chang Gung Memorial Hospital (Institutional Animal Care and Use Committee of Linkou Chang Gung Memorial Hospital) and are based on the National Institutes of Health, NIH) Guide for the Care and Use of Laboratory Animals (Guide for the Care and Use of Laboratory Animals). 2. Prepare a solution containing 2,4 -dimethoxy -6- methylbenzene- 1,3- diol:

The 2,4-dimethoxy-6-methylbenzene-1,3-diol obtained in Example 1 above was dissolved in a 40% (v/v) polyethylene glycol 400 (PEG 400) In the aqueous solution, prepare a solution containing 0.1% (w/v) of 2,4-dimethoxy-6-methylbenzene-1,3-diol. experimental method:

First, the backs of BALB/c mice were shaving, and randomly divided into a normal control group, a pathological control group, and an experimental group (n=6 for each group). Next, the back skin of the mice in the experimental group was applied to contain 0.1% 2,4-dimethoxy-6-methylbenzene-1,3-dimethanone prepared according to item 2 of the above "Experimental Materials". Alcohol solution (dose: 100 μL of 2,4-dimethoxy-6-methylbenzene-1,3-diol solution per 3×3 cm² area), once a day, continuous For 5 days. As for the mice in the normal control group and the pathological control group, they did not receive any treatment.

Approximately 30 minutes after applying 2,4-dimethoxy-6-methylbenzene-1,3-diol each day, the back skin of mice in the experimental group and the pathological control group was additionally applied with 5 % Imiquimod (IMQ) cream (Aldara, 3M Pharmaceuticals) (dose: 62.5 mg IMQ cream per 3×3 cm² area), to induce psoriasis in mice. As for the mice in the normal control group, no treatment was given.

On the 6th day after the start of administration of 2,4-dimethoxy-6-methylbenzene-1,3-diol and IMQ, the back skin of each group of mice was analyzed under item A below. Then, surgical scissors were used to collect the dorsal skin tissue of each group of mice, and then they were used for the analysis of items B to D below. A. clinical symptoms (clinical signs) assessment:

Use the MICROTECH MiniScope-V handheld digital microscope camera (purchased from Shangchen Optics) to take pictures of the back skin of each group of mice, and then observe whether there are symptoms of psoriasis [including erythema and scaling], and According to the Psoriasis Area and Severity Index (PASI), the severity of erythema and scaly is rated by the following 5 scores: 0 means normal; 1 means mild; score 2, means moderate; score 3, mean serious; and score 4, mean very serious. Thus, the cumulative scores of erythema and scales on the back skin of each group of mice can be obtained.

After that, analyze the experimental data obtained in accordance with the method described in the first item "Statistical Analysis" of the "General Experimental Methods" above. B. The thickness of the epidermis (epidermal thickness) Measurement:

A tissue sample with a volume of 1×0.3×0.2 cm ³ was taken from the back skin tissue of each group of mice obtained, and mixed in phosphate buffered saline (PBS) at room temperature 4% paraformaldehyde (paraformaldehyde) was fixed for 48 hours, and then the fixed tissue sample was embedded in paraffin (paraffin), and then sectioned, thereby obtaining a Tissue sections with a thickness of 8 μm.

After that, the obtained tissue section was stained by using hematoxylin-eosin and according to a technique well-known and used by those skilled in the art, and then using a MetaVue image analysis system (factory With an upright optical microscope (Leica brand and model DM2500) under the brand name of Leica and a magnification of 40 times, 5 areas were randomly selected from the tissue section to measure the epidermal thickness. After that, analyze the experimental data obtained in accordance with the method described in the first item "Statistical Analysis" of the "General Experimental Methods" above. C. Number of micro-abscesses (microabscess number) counts:

A tissue sample with a volume of 1×0.3×0.2 cm ³ was taken from the back skin tissue of each group of mice, and then fixed with formalin for 48 hours, and then the fixed tissue sample was treated with paraffin wax It is embedded and then sliced to obtain a tissue slice with a thickness of 8 μm. Next, place the obtained tissue section on a glued glass slide for deparaffinization and rehydration, and then immerse the obtained section sample in a blocking solution for 7 minutes To block the endogenous peroxidase activity (endogenous peroxidase activity).

After that, the obtained section sample uses anti-Ly6G antibody (brand name BioLengend, article number 127601) as the primary antibody and a secondary antibody conjugated with biotin, and is based on the well-known and customary use of those skilled in the art The technique of immunohistochemistry staining (immunohistochemistry stain). Then, an upright optical microscope (Leica brand) was used to observe the stained section samples at a magnification of 20 times, and 5 areas were randomly selected from each group of stained section samples. A MetaVue image analysis system (Leica brand and model DM2500) was used to count the number of microabscesses. After that, analyze the experimental data obtained in accordance with the method described in the first item "Statistical Analysis" of the "General Experimental Methods" above. D. Determination of relative mRNA expression level of pre-inflammatory cytokines :

Take 500 mg of the back skin tissue of each group of mice, and then refer to the method described in item A of Example 2 above to perform quantitative real-time polymerase chain reaction to analyze the IL-6 in the back skin tissue of the mice , IL-23, IL-24 and TNF-α mRNA expression level. After that, analyze the experimental data obtained in accordance with the method described in the first item "Statistical Analysis" of the "General Experimental Methods" above. Results: A. Evaluation of clinical symptoms:

Figures 2 and 3 respectively show photos of the back skins of mice in each group on the 6th day after the start of administration of 2,4-dimethoxy-6-methylbenzene-1,3-diol and IMQ, and Cumulative score of observed erythema and scale. It can be seen from Figure 2 and Figure 3 that compared with mice in the normal control group, the mice in the pathological control group have symptoms of erythema and scales, and the cumulative score of erythema and scales has increased significantly, which means IMQ can induce psoriasis in mice. Compared with mice in the pathological control group, the symptoms of erythema and scales in the experimental group were significantly reduced, and the cumulative score of erythema and scales was also significantly reduced.

These experimental results show that 2,4-Dimethoxy-6-methylbenzene-1,3-diol has the effect of improving psoriasis, so it can be used to treat psoriasis in vivo. B. Measurement of skin thickness :

Figure 4 shows the measured epidermal thickness of the back skin tissue of each group of mice on the 6th day after the start of the administration of 2,4-dimethoxy-6-methylbenzene-1,3-diol and IMQ. It can be seen from Figure 4 that compared with mice in the normal control group, the epidermal thickness of the mice in the pathological control group has a significant increase, which means that IMQ can induce psoriasis in the mice and thicken the epidermis on their backs. Compared with mice in the pathological control group, the epidermal thickness of the mice in the experimental group was significantly reduced. The results of this experiment show that 2,4-Dimethoxy-6-methylbenzene-1,3-diol can effectively fight psoriasis and improve the thickening of the skin caused by psoriasis. C. Counting the number of microabscesses:

Figure 5 shows the measured values from the back skin tissue sections of mice in each group on the 6th day after the start of the administration of 2,4-dimethoxy-6-methylbenzene-1,3-diol and IMQ Number of microabscesses. It can be seen from Figure 5 that compared with the mice in the normal control group, the number of microabscesses in the mice in the pathological control group increased significantly, which means that IMQ can induce psoriasis in mice and cause microabscesses on their back skin. Formation. Compared with mice in the pathological control group, the number of microabscesses in the experimental group was significantly reduced. The results of this experiment show that 2,4-Dimethoxy-6-methylbenzene-1,3-diol can effectively fight psoriasis and improve the formation of microabscesses caused by psoriasis. D. Determination of relative mRNA expression level of pre-inflammatory cytokines :

The following Table 5 shows the relative mRNA expression levels of pre-inflammatory cytokines in the back skin tissue of each group of mice. It can be seen from Table 5 that compared with mice in the normal control group, the mice in the pathological control group will express a large amount of IL-6, IL-23, IL-24 and TNF-α mRNA, which means that IMQ will induce small Rats develop psoriasis. Compared with mice in the pathological control group, the relative mRNA expression levels of IL-6, IL-23, IL-24 and TNF-α in the experimental group all showed a significant decline. The results of this experiment show that 2,4-Dimethoxy-6-methylbenzene-1,3-diol can effectively fight psoriasis and improve skin inflammation caused by psoriasis. Table 5. The relative mRNA expression levels of previously measured inflammatory cytokines in the skin tissues of mice in each group Normal control group Pathology control group test group IL-6 4.086×10 ^-4 ±1.077×10 ^-4 1.839×10 ^-3 ±3.373×10 ^-4### 2.968×10 ^-4 ±7.186×10 ^-5*** IL-23 9.017×10 ^-6 ±1.797×10 ^-6 2.938×10 ^-5 ±3.625×10 ^-6### 9.295×10 ^-6 ±3.095×10 ^-6*** IL-24 1.943×10 ^-6 ±6.140×10 ^-7 1.128×10 ^-5 ±4.123×10 ^-6# 1.193×10 ^-6 ±6.282×10 ^-7* TNF-α 2.860×10 ^-5 ±5.853×10 ^-6 1.008×10 ^-4 ±3.344×10 ^-5# 9.412×10 ^-6 ±2.6×10 ^{-6** #} : When compared with the normal control group, p >0.05 ^### : When compared with the normal control group, p >0.001 ^* : When compared with the pathological control group, p >0.05 ^** : When compared with the pathological control group Comparison, p >0.01 ^*** : When compared with the pathological control group, p >0.001

Based on the above experimental results, the applicant believes that 2,4-Dimethoxy-6-methylbenzene-1,3-diol can exhibit excellent anti-psoriatic effects in vivo, so it can be used for prevention and/ Or treat psoriasis.

All patents and documents cited in this specification are incorporated into this case as reference materials in their entirety. If there is a conflict, the detailed description of the case (including definitions) will prevail.

Although the present invention has been described with reference to the above specific specific examples, it is obvious that many modifications and changes can be made without departing from the scope and spirit of the present invention. Therefore, it is intended that the present invention is only limited by the scope of the patent application attached hereto.

The above and other objects, features and advantages of the present invention will become apparent with reference to the following detailed description and preferred embodiments and the accompanying drawings, in which: Figure 1 shows 2,4-Dimethoxy ¹ H-NMR spectrum of 2,4-dimethoxy-6-methylbenzene-1,3-diol (2,4-dimethoxy-6-methylbenzene-1,3-diol); Figure 2 shows that each group of mice was initially administered 2, On the 6th day after 4-dimethoxy-6-methylbenzene-1,3-diol and imiquimod (IMQ), photos of the back skin of each group of mice, among which the pathological control group The mice were treated with IMQ; the mice in the experimental group were treated with IMQ and 2,4-dimethoxy-6-methylbenzene-1,3-diol; the mice in the normal control group were not treated with any treatment; Figure 3 shows the measured erythema on the back skin of each group of mice on the 6th day after starting to use 2,4-dimethoxy-6-methylbenzene-1,3-diol and IMQ And the cumulative score of scaling, where "***" means: when compared with the pathological control group, p >0.001; Figure 4 shows the measured values of the back skin tissues of mice in each group Epidermal thickness (epidermal thickness), where "***" means: when compared with the pathological control group, p >0.001; and Figure 5 shows the number of microabscesses measured on the back skin tissue of each group of mice, Among them, "***" means: when compared with the pathological control group, p >0.001.

Claims (5)

A 2,4-dimethoxy-6-methylbenzene-1,3-diol is supplied for the preparation of a medicine for the prevention and/or treatment of psoriasis. The use according to claim 1, wherein the pharmaceutical product further comprises a pharmaceutically acceptable carrier. Such as the use of claim 1, wherein the drug is in a dosage form for topical administration. Such as the use of claim 1, wherein the drug is in a dosage form for oral administration. The use according to claim 1, wherein the drug is in a dosage form for parenteral administration.

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