Ploroglucinol compound, separation and application of phloroglucinol compound in preparation of autoimmune hepatitis drugs

Abstract

The invention relates to a phloroglucinol compound, separation and application of the phloroglucinol compound in preparation of drugs for treating autoimmune hepatitis, and belongs to the technical field of medicines. According to the invention, a new compound is separated from a plant hypericum monogynum, and the new compound is named as diamond polyoxocycline A which has better immunosuppressive activity. The invention provides an alternative compound for developing new drugs for treating autoimmune hepatitis, 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 phloroglucinol compounds, separation and preparation of autoimmune hepatitis drugs, and especially to the separation and purification process of compound 1, structural confirmation and activity of treating autoimmune hepatitis. Background Art

[0002] Autoimmune hepatitis (AIH) is a chronic liver inflammation caused by the immune system mistakenly attacking liver tissue, characterized by the loss of self-tolerance leading to the appearance of autoantibodies, pathological changes and abnormal liver function, which may eventually lead to cirrhosis, ascites, liver failure and cancer. So far, the main clinical treatment for AIH is the use of immunosuppressants and liver transplantation, but the efficacy of many patients is not ideal, so it is still crucial to find promising compounds for the treatment of AIH.

[0003] Natural products are the most common source of drugs. According to statistics, from the 1980s to the present, 30-40% of the drugs marketed each year are directly or indirectly derived from natural products. In 2010, the proportion of drugs from natural product-related sources reached 50%. There are a wide variety of plant secondary metabolites. With the increasing research on them in recent years, more and more compounds with novel chemical structures and strong activity have been discovered, laying a certain foundation for the development of new drugs. Natural products are also an important source of drugs against autoimmune diseases. For example, the most commonly used immunosuppressants tacrolimus, cyclosporin A and rapamycin are directly extracted from microorganisms; tripterygium wilfordii total glycosides and peony total glycosides are directly extracted from plants. Therefore, it is of great significance to isolate new natural products with the effect of treating AIH from plants. Summary of the invention

[0004] The task of the present invention is to provide a separation and purification method of a new compound in Hypericum plants having the activity of treating autoimmune hepatitis and its application. The compound in the present invention has good immunosuppressive activity and can be used as a lead compound for the development of drugs for treating autoimmune hepatitis.

[0005] 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:

[0006]

[0007] According to another aspect of the present invention, a method for separating the phloroglucinol compound is provided, comprising the following steps:

[0008] (1) extracting a plant of the genus Hypericum by alcohol, wherein the plant of the genus Hypericum is Hypericum perforatum, concentrating under reduced pressure to recover alcohol, and then extracting with dichloromethane to obtain a dichloromethane extract;

[0009] (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 sixth component is subjected to gel chromatography, positive and reverse phase silica gel column chromatography, and high performance liquid chromatography in sequence to obtain the phloroglucinol compound.

[0010] Preferably, during the elution process, the petroleum ether and ethyl acetate are eluted in a volume ratio of 100:1, 80:1, 50:1, 25:1, 15:1, 5:1, and 3:1, followed by HPLC purification.

[0011] According to another aspect of the present invention, there is provided a use of the phloroglucinol compound for preparing an immunosuppressant.

[0012] Preferably, the immunosuppressant is used to treat autoimmune hepatitis.

[0013] Preferably, the phloroglucinol compound reduces liver damage by reducing inflammatory cytokine levels and by blocking Con A-induced immune responses.

[0014] Preferably, the inflammatory cytokine is at least one of the relevant cytokines COX-2, IL-6, IL-1β, IL-18, IL-23A and TNF-α at the gene level in liver tissue.

[0015] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0016] (1) A novel phloroglucinol compound with a completely new oxygen ring skeleton was discovered from the plant Hypericum perforatum. The structural type of this compound is discovered for the first time and has certain innovation in its chemical structure.

[0017] (2) Further biological activity evaluation results showed that the novel compound can improve the imbalance of CD8 and Treg cell subsets in Con A-induced mouse primary lymphocytes, reduce liver function indicators such as ALT and AST, inhibit liver cell apoptosis, and reduce inflammatory cytokines, thus having a good therapeutic effect on AIH. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the structural formula of the compound adamantine A.

[0019] Figure 2 This is the single crystal diffraction pattern of the compound adamantane A.

[0020] Figure 3The effect of doxycycline A on CD4, CD8, NK, and Treg cell subsets in Con A-induced mouse primary lymphocytes was determined by flow cytometry. Data are presented as mean ± SEM (n = 3). Compared with the control group ## p<0.01, ### p<0.001, compared with the model group * p<0.05, ** p<0.01, *** p<0.001.

[0021] Figure 4 Figure 2. Inhibition of Con A-induced liver injury in mice by adamantin A. (A) Representative images of liver and spleen in each experimental group. (B) Serum AST and ALT levels in each experimental group. (C) Representative H&E-stained micrographs of liver tissue (scale bar = 50 μm). (D) Effect of adamantin A on cytokine transcription levels. Data are presented as mean ± SEM (n = 3). Compared with the control group ## p<0.01, ### p<0.001, compared with the model group * p<0.05,**p<0.01, *** p<0.001.

[0022] Figure 5 Adamantine A protects against Con A-induced apoptosis in mouse hepatocytes. (A) Protective effect of Adamantine A pretreatment against Con A-induced apoptosis in mouse hepatocytes. (B) Representative images of mouse liver immunohistochemistry for CD8 markers. (C) Effects of Adamantine A on CD8 cell subsets in mouse spleen by flow cytometry analysis. 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 Hypericum plants in the present invention belongs to phloroglucinol compounds, and its chemical name is (3R, 4aS, 6R, 8R, 9S, 10S, 10aS)-6-benzoyl-2,2,7,7-tetramethyl-10-(3-methyl-2-oxo but-3-en-1-yl)-9-(2-methylprop-1-en-1-yl)hexahydro-2H-3,10a-epoxy-4a,8:6,10-dimethanocycloocta[b]pyran-5,12(6H)-dione, and is named as diatomaceous cyclohexane A. Its structural formula is 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 Sichuan and Hubei Hypericum), reducing pressure and concentrating to recover industrial alcohol, and extracting with dichloromethane to obtain dichloromethane extract; performing column chromatography on the dichloromethane extract: specifically, mixing the sample with silica gel and loading the column with a dry method; then eluting with petroleum ether-ethyl acetate gradient, combining the same components, and obtaining 7 components with increasing polarity; wherein component 6 is separated by repeated gel chromatography, positive and reverse phase silica gel column chromatography and high performance liquid chromatography to obtain compound adamantine A.

[0028] The invention discloses an application of the mesoglucosidol compound in treating autoimmune hepatitis. The compound reduces the levels of inflammatory cytokines (including related cytokines COX-2, IL-6, IL-1β, IL-18, IL-23A, TNF-α at the gene level in liver tissue) and blocks the immune response induced by Con A to alleviate liver damage.

[0029] In summary, the present invention preferably separates and purifies the industrial alcohol (95% ethanol) extract of Hypericum chuanhuense produced in Shiyan, Hubei, to obtain a new compound, which is a secondary metabolite of Hypericum chuanhuense. 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 immunosuppressive activity of the compound of Formula 1, it is found that Formula 1 exhibits significant immunosuppressive activity on Con A-induced T lymphocyte proliferation, and can be used as a lead compound for the development of drugs for the treatment of autoimmune hepatitis.

[0030] The following are specific embodiments

[0031] Example 1

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

[0033] 20 kg of Hypericum chuanxiensis was extracted 10 times with industrial alcohol, concentrated under reduced pressure at below 50°C to recover industrial alcohol, and extracted with dichloromethane to obtain 1.5 kg of dichloromethane extract. The dichloromethane extract was subjected to column chromatography: specifically, the sample was mixed with 100-200 mesh silica gel, and the column was loaded by dry method, the column height was 1.5 m, the column diameter was 15 cm, and the gradient elution was carried out with petroleum ether-ethyl acetate, and the solvent ratio was petroleum ether:ethyl acetate = 100:1, 80:1, 50:1, 25:1, 15:1, 5:1, 3:1, 15 L of each ratio was eluted, TLC detection was performed, and similar components were combined to obtain 8 components (component 1 to component 8) with small to large polarity. Take component 6, and conduct 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 methanol-water, the solvent ratio is methanol: water = 2:8, 4:6, 5:5, 6:4, 8:2, 1:0, elution of 5L in each ratio, TLC detection, combine similar components, and obtain 5 small components (components 6.1 to 6.5) with increasing polarity. Take component 6.3, and separate it by high performance liquid chromatography, the chromatographic column is Yuexu Company's AQ-C18 chromatographic column (column length 20cm, column diameter 2cm), the separation conditions are acetonitrile: water = 18:82, flow rate 2ml / min, detection wavelength 254nm, retention time 113min, and obtain diamond doxycycline A. 2. Structural identification of diamond doxycycline A, a compound shown in formula 1

[0034] The compound adamantine doxycycline A was subjected to nuclear magnetic resonance, mass spectrum, optical rotation, infrared spectrum, ultraviolet spectrum, circular dichroism and other data tests, and the adamantine doxycycline A was subjected to X-ray single crystal diffraction data test to determine the structure of the compound.

[0035] Adamantine A: colorless crystals; mp: 189.9-200.1℃, [α] 2 D 8 +17.0(c0.100,MeOH); UV(MeOH)λ max (logε)202(4.29)nm,246(3.87)nm; ECD(MeOH)λ max (Δε)212(+1.71),225(-1.95),279(+4.58),314(+0.60),329(+1.57); IR(KBr)ν max 3433,2922,2852,1739,1710,1686,1648,1632,1450,1387,1371,1252,1235,1170cm –1The absolute configuration of adamantin A was determined by single crystal X-ray diffraction. Figure 1 The NMR data are shown in Table 1. The single crystal diffraction pattern is shown in Figure 2 shown.

[0036] Table 1. H and C spectra of compound adamantin A (δ in ppm and J in Hz)

[0037]

[0038]

[0039] Example 2

[0040] 1. The compound doxycycline A in the present invention has the effect of treating AIH; reducing the levels of inflammatory cytokines (including related cytokines COX-2, IL-6, IL-1β, IL-18, IL-23A, TNF-α at the gene level in liver tissue), protecting apoptotic cells and inhibiting the expression of CD8 in vivo to inhibit Con A-induced T cell activation, thereby blocking Con A-induced immune response.

[0041] Con A, as a classic T cell mitogen, can promote the proliferation of T lymphocytes, and the results of flow cytometry showed that compared with the model group, doxycycline A treatment could reduce the proportion of CD8 and Treg subsets, indicating that doxycycline A has a strong immunosuppressive activity ( Figure 3 Therefore, the therapeutic effect of adamantin A on Con A-induced hepatitis in vivo was further investigated. Figure 4 As shown in Figures A and C in Figure 4, adamantin A significantly inhibited Con A-induced splenomegaly, liver damage, and abnormal liver congestion, and the overall liver morphology was significantly improved. Histomorphological analysis showed that the hepatocytes in the model group were disordered and infiltrated with inflammatory cells. Adamantin A could reduce inflammatory infiltration, improve the cell structure of hepatocytes, and arrange them compactly. As key markers of hepatocyte damage and necrosis, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood are important components for evaluating the therapeutic potential of AIH. Figure 4 As shown in Figure B, adamantinib significantly reduced the increase in serum ALT and AST levels induced by Con A injection. Our data showed that adamantinib reduced liver injury by reducing inflammatory cytokine levels and blocking Con A-induced immune responses, including the gene levels of related cytokines COX-2, IL-6, IL-1β, IL-18, IL-23A, TNF-α ( Figure 4 D).

[0042] Hepatocyte apoptosis is another important manifestation in the Con A-induced liver injury model, so immunohistochemical analysis of cleaved-caspase 3 and TUNEL staining of liver tissues were performed to elucidate the protective effect of doxycycline A. Figure 5 As shown in A, adamantine A can significantly reduce the expression of cleaved-caspase 3, which was significantly increased in the liver of the model group mice. At the same time, a large number of hepatocyte apoptosis with TUNEL-positive staining was observed in the liver sections of the Con A group, and adamantine A pretreatment significantly attenuated Con A-induced hepatocyte apoptosis. Next, we explored whether adamantine A inhibits Con A-induced T cell activation by inhibiting the expression of CD8 in vivo. As expected, immunohistochemistry results showed that CD8 was abundantly expressed in the liver, and adamantine A treatment could significantly reduce the expression level ( Figure 5 At the same time, we performed flow cytometric analysis of CD8 in the spleen of AIH mice ( Figure 5 C), which showed the same trend as the in vitro CD8 flow cytometric analysis.

[0043] 2. The method is as follows.

[0044] 2.1 In vitro immunosuppressive activity assay

[0045] 2.1.1 Lymphocyte isolation and cell culture

[0046] Male Babl / C mice (20-22 g) aged 7-8 weeks were housed at 25 ± 1°C and provided with standard diet and water. Single cell suspensions were extracted from mouse spleens in a sterile environment, and erythrocytes were removed by adding erythrocyte lysate. Lymphocytes were isolated by 5% carbon dioxide (CO) at 37°C. 2 ) for 4 hours to remove adherent macrophages. All cells were maintained at 37°C with 5% CO 2 gas and cultured in RPMI 1640 medium containing 10% fetal bovine serum.

[0047] 2.1.2 Lymphocyte proliferation assay

[0048] Count cells using an automated cell counter and assess viability by trypan blue staining. Cell viability should be above 95%. Lymphocytes were plated at 6 × 10 6 The cells were seeded at a density of 100 μL / mL in a 96-well plate. The cells were treated with the test compound and then incubated at 37°C and 5% CO 2 The cells were stimulated with Con A (5 μg / mL) for 48 hours in an incubator at 4°C. The proliferation of lymphocytes was measured by CCK-8 method.

[0049] 2.1.3 Flow cytometry

[0050] Immune cells treated with adamantin A and Con A (5 μg / mL) were analyzed by flow cytometry. After adding CD16 / CD32 to block the cells, appropriately diluted PE-CD3, FITC-CD4, and APC-CD8a were added to identify CD4 or CD8, while PE-CD3 and APC-CD19 were added to identify NK cells, and FITC-CD4 and PE-CD25 were added to identify Tregs. Images were acquired and analyzed using BD Accuri C6 software.

[0051] 2.2 In vivo immunosuppressive activity assay

[0052] 2.2.1 Animal treatment and AIH induction

[0053] Male Babl / C mice aged 7-8 weeks and weighing 20-22g were randomly divided into three groups: blank control group, adamantin A (5mg / kg) group and Con A group. The three groups used the reagent as a single control. All mice were placed in an environment of 25±1℃ and provided with standard diet and water. After three days of adaptive feeding, the corresponding reagent was injected intraperitoneally to each group of mice once a day for 3 consecutive days. Then the Con A group and adamantin A group were injected with Con A (20mg / kg) through the tail vein for 12 hours. All mice were killed after bleeding from the eyeballs, and part of their livers were stored in 4% paraformaldehyde for 48 hours. The remaining liver was frozen in a -80℃ refrigerator, and lymphocytes were isolated from the spleen for flow cytometry according to methods 2.1.1 and 2.1.3.

[0054] 2.2.2 Histological analysis

[0055] Liver tissue immersed in 4% paraformaldehyde was dehydrated through graded concentrations of ethanol, embedded in paraffin, and sectioned at a thickness of 5 microns. Paraffin sections of tissue were dewaxed with xylene, rehydrated with graded ethanol, and then stained with hematoxylin and eosin. Tissue was dehydrated in a graded ethanol series and then vitrified with xylene. Pathological changes were examined under a microscope.

[0056] 2.2.3 Immunohistochemistry of CD8 and cleaved caspase 3

[0057] 3% hydrogen peroxide solution was added to the paraffin sections for antigen retrieval to block endogenous peroxidase. After sealing, the sections were incubated with the primary and secondary antibodies in sequence, and then the sections were stained with DAB and the nuclei were inhibited. After color development, the sections were dehydrated and prepared for sealing for microscopic examination. Pathological changes were examined under a microscope.

[0058] 2.2.4 Fluorescent TUNEL staining

[0059] The paraffin sections were dehydrated and incubated with proteinase K solution, then with a mixture of TdT enzyme, fluorescent labeling solution, and TUNEL assay solution, followed by staining with DAPI. Pathological changes were examined under a microscope.

[0060] 2.2.5 qRT-PCR analysis of liver tissue

[0061] qRT-PCR analysis was performed on liver tissues from different groups. RNA was isolated from cryopreserved mouse liver tissues using total RNA isolation reagent and then reverse transcribed into cDNA using ABP 5×All-in-One RT Master Mix. qRT-PCR was performed using 2×ABP SYBR Green qPCR Master Mix and 0.2mM forward and reverse primers in a final volume of 10μL. The mixture was placed in ABI QuantStudio 5 and the reaction was subsequently performed according to the manufacturer's protocol. Values ​​relative to β-actin are shown.

[0062] 2.2.6 Measurement of ALT and AST

[0063] ALT and AST levels in mouse serum were measured according to the manufacturer's instructions of ALT and AST kits (E-BC-K235-M, E-BC-K236-M, Elabscence, China).

[0064] 2.2.7 Statistical analysis

[0065] All data were collected based on at least three independent experiments, and the mean ± SEM of each group of values ​​was taken. Comparisons between two groups were performed using an unpaired two-tailed Student's t test. One-way or two-way ANOVA was used for multiple comparisons, followed by a subsequent Bonferroni test. p values ​​< 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8.0 software (CA, USA).

[0066] The corresponding primer sequences used in the experiment

[0067]

[0068]

[0069] Results and Analysis:

[0070] A new compound isolated from the plant Hypericum, adamantin A, has good immunosuppressive activity and can be used as a lead compound for the development of drugs for the treatment of AIH.

[0071] 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) extracting a plant of the genus Hypericum by alcohol, wherein the plant of the genus Hypericum is Hypericum perforatum, concentrating under reduced pressure to recover alcohol, and then extracting 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 sixth component is subjected to 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 the volume ratio of 100:1, 80:1, 50:1, 25:1, 15:1, 5:1 and 3:1 in sequence, and then purified by HPLC.

4. Use of the phloroglucinol compound as claimed in claim 1 for preparing an immunosuppressant.

5. The use according to claim 4, characterized in that The immunosuppressant is used to treat autoimmune hepatitis.

6. The use according to claim 5, characterized in that The phloroglucinol compounds alleviate liver damage by reducing the levels of inflammatory cytokines and by blocking Con A-induced immune responses.

7. The use according to claim 6, characterized in that The inflammatory cytokine is at least one of the related cytokines COX-2, IL-6, IL-1β, IL-18, IL-23A and TNF-α at the gene level in liver tissue.

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