Ploroglucinol compound, and application thereof in separation and preparation of anti-psoriasis drugs

Abstract

The invention relates to a phloroglucinol compound, separation and application of the phloroglucinol compound in preparation of an anti-psoriasis medicine, and belongs to the technical field of medicines. The novel compound hypericin A separated from hypericum plant hypericin is a novel TNF-alpha small-molecule inhibitor, can inhibit TNF-alpha in a targeted manner, effectively blocks various inflammatory factors, can improve the epidermal hyperplasia symptom of psoriasis, has good anti-psoriasis activity, and can be used as an anti-psoriasis drug for treating psoriasis. The compound can be used as a lead compound for developing drugs for treating psoriasis. The invention provides an alternative compound for research and development of novel anti-psoriasis drugs, and has very important significance for development and utilization of natural product resources.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically, relates to the separation of a phloroglucinol compound and its application in preparing an anti-psoriatic drug, and in particular to the separation and purification process, structural confirmation and anti-psoriatic activity of compound 1. Background Art

[0002] Psoriasis is an immune-mediated chronic inflammatory skin disease induced by genetic and environmental factors. The global prevalence is about 2% to 3%, and it has been increasing year by year in recent years. It has become one of the major diseases that endanger the physical and mental health of patients. Its treatment includes local treatment, physical therapy and systemic treatment, but these treatments cannot cure the disease (The Lancet, 2021, 397, 1301). Therefore, it is urgent to deeply understand the pathogenesis of psoriasis, explore new therapeutic targets, and find highly effective and low-toxic anti-psoriatic drugs.

[0003] Studies have shown that the pathogenesis of psoriasis is related to a variety of immune cells, inflammatory cells, and inflammatory pathways, among which tumor necrosis factor α (TNF-α) plays a key role. It is involved in multiple links in the development of psoriasis and is an important proinflammatory cytokine that can interact with other cytokines to amplify the inflammatory response. Therefore, inhibiting TNF-α can reduce the excessive proliferation and excessive inflammation of keratinocytes and is currently an important target for the treatment of psoriasis (Drug Discov. Today, 2023, 28, 103575). At present, macromolecular drugs targeting TNF-α, such as infliximab, adalimumab, etanercept and pecilizumab, have been widely used in the clinical treatment of psoriasis. However, biological macromolecular drugs have inherent disadvantages, such as high price, single administration method, and long-term use will increase the risk of tumors and infections (Int. J. Mol. Sci., 2019, 20, 1475; Ann. Rheum. Dis., 2020, 79, 285). In contrast, TNF-α small molecule inhibitors have the advantages of low price and convenient oral administration, making TNF-α small molecule inhibitors a new strategy for the development of highly effective and low-toxic anti-psoriasis drugs.

[0004] Natural products are an important source of drugs. Among the small molecule drugs launched between 1981 and 2019, up to 68% of the drug molecules are directly or indirectly derived from natural products and their derivatives. Natural products themselves have many natural advantages such as complex and diverse structures, significant activity, low toxicity and side effects, and short R&D cycles, which provide more options for their transformation into drugs and become a hot spot for drug research and development (J.Nat.Prod., 2020, 83, 770). It is worth noting that many drugs used in the clinical treatment of psoriasis are derived from medicinal plants, such as tripterygium wilfordii polyglycosides, glycyrrhizic acid glycosides, and white peony glucosides (Chin.J.Dermatol., 2023, 56, 573). Therefore, it is of great significance to find lead compounds with novel structures for the treatment of psoriasis from medicinal plants. Summary of the invention

[0005] The task of the present invention is to provide a new compound with anti-psoriasis activity in Hypericum plants and its separation and purification method and application. The compound in the present invention is a potential TNF-α small molecule inhibitor with good anti-psoriasis activity and can be used as a lead compound for the development of anti-psoriasis drugs.

[0006] According to a first aspect of the present invention, a phloroglucinol compound is provided, and the structural formula of the phloroglucinol compound is shown in Formula 1:

[0007]

[0008] According to another aspect of the present invention, a method for separating the phloroglucinol compound is provided, comprising the following steps:

[0009] (1) crushing a Hypericum plant and then extracting it with alcohol, wherein the Hypericum plant is Hypericum salsa, concentrating under reduced pressure to recover alcohol, and then extracting it with dichloromethane to obtain a dichloromethane extract;

[0010] (2) The dichloromethane extract obtained in step (1) is subjected to column chromatography, and then gradient eluted with petroleum ether-ethyl acetate to obtain 7 components with increasing polarity, and the fourth component is subjected to MCI column chromatography, gel chromatography, positive and reverse phase silica gel column chromatography, and high performance liquid chromatography in sequence to obtain the phloroglucinol compound.

[0011] Preferably, during the elution process, the petroleum ether and ethyl acetate are eluted in a volume ratio of 50:1, 40:1, 30:1, 20:1, 10:1, 3:1 and 0:1, respectively.

[0012] According to another aspect of the present invention, there is provided the use of the phloroglucinol compound for preparing TNF-α inhibitory drugs.

[0013] According to another aspect of the present invention, there is provided the use of the phloroglucinol compound for preparing an anti-psoriasis drug.

[0014] Preferably, the phloroglucinol compound is used to inhibit the effects of inflammatory factors in HaCaT cells after TNF-α stimulation.

[0015] Preferably, the phloroglucinol compound is used to improve the symptoms of epidermal hyperplasia in psoriasis.

[0016] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0017] (1) A novel phloroglucinol compound was discovered from the medicinal plant Hypericum scabra. The structural type of this compound is discovered for the first time and has certain innovation in chemical structure.

[0018] (2) Further biological activity evaluation results showed that the novel compound is a small molecule inhibitor of TNF-α, has a strong binding affinity with TNF-α, can significantly inhibit L929 cell death induced by TNF-α and actinomycin D, and has a strong improvement effect on the severity of skin erythema and scaling, skin thickness and CSS scores of psoriasis mice, so it has excellent anti-psoriatic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the structural formula of the compound hypericum ketone A.

[0020] Figure 2 This is the single crystal diffraction pattern of the compound hypericum ketone A.

[0021] Figure 3 The CCK-8 method, DAPI staining analysis, surface plasmon resonance and other methods were used to verify that the compound hypericum ketone A can target and inhibit the activity of TNF-α; among them: A is the effect of compound hypericum ketone A on the viability of L929 cells; B-C are the viability, morphology and DAPI staining analysis of L929 cells after treatment with compound hypericum ketone A, TNF-α and actinomycin D; D-E are the effects of compound hypericum ketone A on the thermal stability of TNF-α protein analyzed by CETSA and TSA; F is the SPR experiment to determine the binding affinity of compound hypericum ketone A to TNF-α; G is the analysis of the binding mode and binding energy of compound hypericum ketone A to TNF-α by computer molecular docking technology.

[0022] Figure 4The test compound hypermetamol A affects inflammatory factors in HaCaT cells induced by TNF-α. A is the analysis of the viability of HaCaT cells by hypermetamol A; B–D are the mRNA expression levels of IL-23, CXCL1 and S100A9 analyzed by RT-qPCR; E is the effect of hypermetamol A on TNF-α-induced NF-κB p65 phosphorylation and nuclear translocation.

[0023] Figure 5 This study analyzes the pharmacodynamic effects of compound hypermetamol A on imiquimod (IMQ)-induced psoriasis mice. A shows the skin lesions of mice in the control group, compound hypermetamol A treatment group, and tacrolimus treatment group after 7 consecutive days of treatment; B shows the quantification of CSS in each group of mice; C shows the manifestation and histological characteristics of skin lesions in each group of mice. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] The new compound separated and purified from the Hypericum plant in the present invention belongs to the phloroglucinol compound, has a chemical name of (1R, 3a'R, 5S, 6a'R)-3-benzoyl-1', 1', 4-trimethyl-3a', 5-bis(3-methylbut-2-en-1-yl)-6', 6a'-dihydro-1'H, 3'H-spiro[cyclohexane-1, 5'-cyclopenta[c]furan]-3-ene-2, 3', 4'(3a'H)-trione, and is named as hypericum pyroquinone A, and has a structural formula as shown in Formula 1;

[0026]

[0027] The preparation method of the mesoglucinol compound of the present invention comprises the following steps: using industrial alcohol (95% ethanol) to extract Hypericum plant (preferably Hypericum sieboldii) and recovering the industrial alcohol by decompression concentration, and extracting with dichloromethane to obtain a dichloromethane extract; subjecting the dichloromethane extract to column chromatography: specifically, mixing the sample with silica gel and loading the column by dry method; then eluting with petroleum ether-ethyl acetate gradient, combining similar fractions, and obtaining 7 components with increasing polarity; wherein component 4 is separated and purified by repeated MCI chromatography column, gel chromatography column, positive and reverse phase silica gel column chromatography column and high performance liquid chromatography to obtain compound hypericum polyketone A.

[0028] The application of the mesotriol compounds in the present invention in treating psoriasis can inhibit the activity of TNF-α through targeted inhibition, thereby inhibiting the inflammatory response, and can inhibit the excessive proliferation of epidermal cells to alleviate the epidermal hyperplasia symptoms of psoriasis.

[0029] In summary, the present invention preferably separates and purifies the industrial alcohol (95% ethanol) extract of Hypericum ovata produced in Enshi, Hubei Province, to obtain a new compound, which is a secondary metabolite of Hypericum ovata. The structure is determined by using a variety of spectral analysis methods and other methods, and the specific structure is shown in Formula 1. By evaluating the anti-psoriatic activity of the compound of Formula 1, it is found that the compound of Formula 1 is a small molecule inhibitor of TNF-α, and can well alleviate the symptoms of psoriasis epidermal hyperplasia, and can be used as a lead compound for the development of drugs for the treatment of psoriasis.

[0030] The following are specific embodiments

[0031] Example 1

[0032] 1. Separation and preparation of the compound hypericum multione A as shown in formula 1

[0033] 35 kg of leaf parts of Hypericum were extracted 10 times with industrial alcohol, concentrated under reduced pressure at below 50°C to recover industrial alcohol, and then extracted with dichloromethane to obtain 2.5 kg of dichloromethane extract. The dichloromethane extract was subjected to column chromatography: specifically, the sample was mixed with 80-120 mesh silica gel, and the column was loaded by dry method, with a column height of 1.5 m and a column diameter of 20 cm, and eluted with petroleum ether-ethyl acetate gradient, and the solvent ratios were petroleum ether: ethyl acetate = 50:1, 40:1, 30:1, 20:1, 10:1, 3:1, 0:1, 20 L of each ratio was eluted, TLC detection was performed, and similar components were combined to obtain 7 components (component 1 to component 7) with small to large polarity. Take component 4, and carry out gradient elution through a reverse phase medium pressure column, the column height is 0.6m, the column diameter is 10cm, and the gradient elution is carried out with methanol-water, the solvent ratio is methanol: water = 2:8, 3:7, 4:6, 6:4, 7:3, 8:2, 1:0, elution of 5L in each ratio, TLC detection, merge similar components, and obtain 6 small components (components 4.1 to 4.6) with increasing polarity. Take component 4.3, and separate it by high performance liquid chromatography, the chromatographic column is AQ-C18 chromatographic column (column length 20cm, column diameter 2cm) of Yuexu Company, the separation conditions are acetonitrile: water = 23:77, flow rate 2ml / min, detection wavelength 254nm, retention time 97min, and hypermeline A is obtained.

[0034] 2. Structural identification of the compound hypericum ketone A as shown in formula 1

[0035] The compound hypermetamol A was subjected to nuclear magnetic resonance, mass spectrum, optical rotation, infrared spectrum, ultraviolet spectrum, circular dichroism and other data tests, and hypermetamol A was subjected to X-ray single crystal diffraction data test to determine the structure of the compound.

[0036] Hypericum odoratum A: Colorless block crystals; mp 139.4–143.8℃; UV(CH3OH)λ max (logε)=201(4.52)nm; IR(KBr)v max 2980,2919,2856,1776,1729,1679,1656,1619,1597,1449,1378,1324,1264,1168,1131,954and 691cm –1 ;ECD(CH3OH)λ max(Δε) 204 (+8.12), 241 (-7.14), 249 (-0.41), 269 (-4.45), 337 (+1.05) nm. The absolute configuration of hypericum acetone A was determined by X-ray single crystal diffraction. The crystal structure is shown in Figure 1 The NMR data are shown in Table 1. The single crystal diffraction pattern is shown in Figure 2 shown.

[0037] Table 1. Compound hypericum odoratum A 1 H and 13 C NMR data (δ in ppm and J in Hz)

[0038]

[0039] a 1 H NMR (600 MHz) and 13 C NMR(150MHz)recorded in CD3OD

[0040] Example 2

[0041] The inhibitory activity of the compound hypermetamol A on TNF-α and its anti-psoriasis activity

[0042] ① CCK-8 method, DAPI staining analysis, surface plasmon resonance and other methods were used to verify that the compound hypericum polyketone A can target and inhibit the activity of TNF-α

[0043] The inhibitory activity of the compound hypermetamol A on TNF-α was tested in L929 cells. First, the compound hypermetamol A had no obvious cytotoxicity to L929 cells at a concentration of 40 μM, and had a significant protective effect on L929 cell death induced by TNF-α and actinomycin D in a concentration-dependent manner ( Figure 3 A in 3 and B in 3). DAPI staining results also showed that compound hypericum odoratum A could effectively protect L929 cells from cell death and inhibit TNF-α, even better than the positive control SPD304 ( Figure 3 In addition, the cell thermal shift assay (CETSA), thermal shift assay (TSA) and surface plasmon resonance (SPR) assay were used to evaluate the binding ability of the compound hypermetamol A to TNF-α. The results showed that compared with the DMSO group ( Figure 3 Compared with D in 3 and E in 4), the compound hypericum ketone A increased the cell thermal stability of TNF-α from the tolerance temperature of 82℃ to ℃, and the tolerance temperature was significantly improved. SPR experiments also showed that the compound hypericum ketone A can directly bind to TNF-α with a KD value of 5.48μM, which has a strong affinity ( Figure 3 F). At the same time, the binding mode and binding energy of the compound hypericum multione A with TNF-α were determined by computer molecular docking, such as Figure 3 As shown in G, the binding pocket of the TNF-α trimer is relatively sharp and fusiform, and the compound hypermetamol A can completely occupy the binding site of the crystallographic ligand SPD304, preventing the assembly of a biologically active trimer complex. The compound hypermetamol A forms multiple hydrophobic interactions with Ile133, Leu233, Tyr135, and Tyr227 of the TNF-α monomer. It is worth noting that the benzyl group in the compound hypermetamol A exhibits a strong π-π interaction with the Tyr195C residue, which plays an important role in stabilizing the binding with the ligand.

[0044] ②RT-qPCR and immunofluorescence were used to test the effect of compound hypericum odoratum A on inflammatory-related factors in HaCaT cells induced by TNF-α

[0045] The effects of the compound hypermetamol A on the key psoriasis-related signaling pathways in HaCaT cells were tested. First, the compound hypermetamol A had significant inhibitory activity on TNF-α-induced inflammatory factors IL-23, CXCL1, and S100A9, and had low toxicity ( Figure 4 In addition, immunofluorescence results showed that the compound hypericum odoratum A could inhibit the nuclear translocation of NF-κB p65 induced by TNF-α ( Figure 4 The above results indicate that the compound hypericum odoratum A can effectively block multiple inflammatory factors by targeting and inhibiting TNF-α.

[0046] ③ Detect the pharmacodynamic effect of compound hypermetamol A on imiquimod (IMQ)-induced psoriasis mice

[0047] The antipsoriatic activity of the compound hypermetamol A was evaluated in an IMQ-induced psoriasis mouse model. Figure 5 A and Figure 5 As shown in Figure B, the skin of mice in the IMQ group was significantly thickened and erythematous, and the CSS score continued to increase, while the severity of skin erythema and scaling, skin thickness and CSS score of mice in the hypermelanin A treatment group were reduced. HE staining results showed that mice in the IMQ group had obvious epidermal hyperplasia, scaling and severe inflammatory response, while hypermelanin A and positive drug tapinarof could improve these symptoms ( Figure 5 Further immunohistochemical results showed that ( Figure 5C), Ki67 expression was upregulated and IL-17 was continuously expressed in the upper layer of the skin of mice in the IMQ group, while Ki67 expression was downregulated and IL-17 expression was discontinuous in the group treated with compound hypermetamol A, indicating that compound hypermetamol A can alleviate the epidermal hyperplasia symptoms of psoriasis by inhibiting the excessive proliferation of epidermal cells.

[0048] Results and analysis:

[0049] Hypericum perforatum A, a new compound isolated from Hypericum plants, can effectively block multiple inflammatory factors by targeted inhibition of TNF-α, and can improve the symptoms of epidermal hyperplasia in psoriasis by inhibiting the excessive proliferation of epidermal cells. It has excellent anti-psoriatic activity and can be used as a lead compound for the development of drugs for the treatment of psoriasis.

[0050] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A phloroglucinol compound, characterized in that: The structural formula of the phloroglucinol compound is shown in Formula 1:

2. The method for separating phloroglucinol compounds according to claim 1, characterized in that: The following steps are involved: (1) crushing a Hypericum plant and then extracting it with alcohol, wherein the Hypericum plant is Hypericum salsa, concentrating under reduced pressure to recover alcohol, and then extracting it with dichloromethane to obtain a dichloromethane extract; (2) The dichloromethane extract obtained in step (1) is subjected to column chromatography, and then gradient eluted with petroleum ether-ethyl acetate to obtain 7 components with increasing polarity, and the fourth component is subjected to MCI column chromatography, gel chromatography, positive and reverse phase silica gel column chromatography, and high performance liquid chromatography in sequence to obtain the phloroglucinol compound.

3. The method for separating phloroglucinol compounds according to claim 2, characterized in that: During the elution process, the petroleum ether and ethyl acetate are eluted in sequence according to volume ratios of 50:1, 40:1, 30:1, 20:1, 10:1, 3:1, and 0:

1.

4. Use of the phloroglucinol compound as claimed in claim 1 for preparing TNF-α inhibitory drugs.

5. Use of the phloroglucinol compound according to claim 1 for preparing an anti-psoriasis drug.

6. The use according to claim 5, characterized in that The phloroglucinol compound is used for inhibiting the effects of inflammatory factors in HaCaT cells after TNF-α stimulation.

7. The use according to claim 5, characterized in that The phloroglucinol compound is used for improving the symptoms of psoriasis epidermal hyperplasia.

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