Rice ball extract L01 and its pharmaceutical composition and application

Abstract

The present invention provides a sesquiterpene extract L01 from Oryza sativa L. and its application in the preparation of anti-inflammatory drugs and immunosuppressive drugs, belonging to the technical field of natural drug chemistry. The present invention provides an Oryza sativa L. extract L01 with compounds 1 to 12 as characteristic components and its application in the preparation of anti-inflammatory drugs and immunosuppressive drugs. The test results show that the Oryza sativa L. extract L01 provided by the present invention can significantly improve the clinical symptoms of psoriasis in an in vivo experiment.

Description

[0001] This application is a divisional application of (application number: 202310552734.X, application date: 2023.05.17, invention name: Diterpenoid compounds and extracts L01 of Mitumna strychnossima and their application in pharmaceutical manufacturing. Domestic priority data is: application number: 202211099790.4; application date: 2022.09.14). Technical Field

[0002] The invention belongs to the technical field of natural drug chemistry, and specifically relates to a diterpenoid compound and an extract L01 in Flos Corydalis and a preparation method thereof, and an application thereof in the preparation of anti-inflammatory drugs and immunosuppressive drugs. Background Art

[0003] Autoimmune diseases are a type of disease caused by the body's immune system producing local or systemic abnormal immune responses to self-antigens, resulting in damage to its own tissues, including psoriasis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, etc. Currently, there are more than 80 reported autoimmune diseases, with diverse clinical manifestations and increasing incidence, which have a serious impact on human health. Psoriasis, also known as "psoriasis", is a chronic autoimmune disease with a global prevalence of 2-3%. Psoriasis is divided into plaque psoriasis, guttate psoriasis, pustular psoriasis, erythrodermic psoriasis, etc. Among them, plaque psoriasis is the most common, with clinical manifestations mostly erythema and scaling, and the disease can occur all over the body. Psoriasis patients often have complications of psoriatic arthritis, lymphoma, cardiovascular disease, hypertension, diabetes and depression. Currently, immunosuppressive drugs are mainly used in the clinical treatment of autoimmune diseases. Although there are many immunosuppressive drugs with good efficacy for clinical use, there are still various problems such as large toxic side effects, unsatisfactory efficacy and high prices. There is an urgent need to develop new immunosuppressive drugs.

[0004] Natural products play an important role in the treatment of autoimmune diseases due to their unique mechanism of action and low toxicity and side effects. Many natural products derived from Chinese herbal medicines have significant anti-inflammatory and immunosuppressive effects, especially the anti-inflammatory and immunosuppressive activities of plant terpenoids, which are an important source for discovering new natural anti-inflammatory and immunosuppressive drugs. Leucosceptrum canum is a single genus of the genus Leucosceptrum in the family Lamiaceae. It is a perennial shrub or small tree, mainly distributed in the eastern Himalayas, the northern Indochina Peninsula, and southwestern China. The leaves and bark of Leucosceptrum canum have high medicinal value. They are used by the people of Yunnan to relax muscles, stop bleeding, and reduce inflammation, and to treat fever, stomachache, traumatic bleeding, closed fractures, impetigo, and other diseases. At present, some diterpenoid chemical components have been found in Leucosceptrum canum, but there are no reports on the anti-inflammatory and immunosuppressive activities of diterpenoids in Leucosceptrum canum. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a dipterpene compound and an extract L01 of the spatholobifolia, a preparation method thereof, and an application thereof in the preparation of anti-inflammatory drugs and immunosuppressive drugs.

[0006] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0007] The diterpenoid compounds 1 to 12 in the rice flower are shown in the following structural formula:

[0008]

[0009] The present invention provides a method for preparing the diterpenoid compounds 1 to 12 described in the above scheme, comprising the following steps:

[0010] (1) extracting rice flower with an organic solvent to obtain an extract;

[0011] (2) subjecting the extract to vacuum distillation and concentrating to obtain an extract;

[0012] (3) After dissolving the extract, sequentially performing column chromatography separation and high performance liquid chromatography separation to obtain the diterpenoid compound.

[0013] The column chromatography separation includes silica gel column chromatography, MCI column chromatography or reverse phase C 18 Column chromatography, dextran gel column chromatography; the high performance liquid chromatography separation adopts preparative or semi-preparative high performance liquid chromatography.

[0014] The present invention uses an organic solvent to extract the rice flower to obtain an extract. Before extraction, the present invention preferably crushes the rice flower to 30 meshes. In the present invention, the rice flower can be taken from the above-ground part or any tissue of the rice flower, such as the leaves, flowers or stems of the rice flower and dried in the shade; the present invention has no special requirements for the moisture content of the rice flower, and fresh or dried rice flowers can be used.

[0015] In the present invention, the organic solvent preferably includes one or more of methanol, ethanol, acetone, chloroform, dichloromethane and petroleum ether; the amount of the organic solvent added to each kilogram of dry weight of rice flower is preferably 5L. In the present invention, the extraction is preferably cold soaking extraction or hot reflux extraction. In the present invention, the temperature of the cold soaking extraction is preferably room temperature, the number of cold soaking extractions is preferably 2 to 5 times, more preferably 3 to 4 times; the time of each extraction is preferably 24 to 48 hours, more preferably 36 to 48 hours; after each cold soaking extraction, the obtained extraction system is filtered, and the filtrate after each filtration is combined. In the present invention, the temperature of the hot reflux extraction is preferably 40 to 50°C, more preferably 45 to 50°C; the time of the hot reflux extraction is preferably 4 to 6 hours, more preferably 4 to 5 hours.

[0016] After obtaining the extract, the present invention performs vacuum distillation on the extract to concentrate it to obtain an extract. In the present invention, the temperature of the vacuum distillation is preferably 35 to 45°C, the time is preferably 60 to 100 minutes, and the concentration multiple of the vacuum distillation is preferably 50 times. After the vacuum distillation, the organic solvent in the extract is removed, and the extract is concentrated at the same time.

[0017] After obtaining the extract, the present invention dissolves the extract and then sequentially performs column chromatography separation and high performance liquid chromatography separation to obtain the diterpenoid compound. In the present invention, the organic solvent for dissolving the extract is preferably chloroform, and the organic solvent for dissolving the extract is 1.5 to 3 times the mass of the extract. In the present invention, the column chromatography separation preferably includes silica gel column chromatography, MCI column chromatography or reverse phase C 18 Column chromatography, dextran gel column chromatography. In the present invention, the column loading process of the silica gel column chromatography is preferably: loading the column with 200-300 mesh silica gel; after dissolving the extract, mixing with 1-1.5 times (mass multiple) of 200-300 mesh silica gel, the mixed sample is dried and ground and sieved, and then transferred to a silica gel column for column chromatography. In the present invention, the silica gel column chromatography preferably adopts gradient elution, and the mobile phase used in the gradient elution is preferably a mixed solvent composed of solvent A and solvent B in a volume ratio of 1:0 to 0:1; the solvent A is preferably any one of dichloromethane, chloroform and petroleum ether, and the solvent B is preferably any one of dichloromethane, chloroform, acetone, ethyl acetate and methanol. The present invention preferably performs reduced pressure concentration and thin layer chromatography detection on the fractions obtained by the gradient elution in sequence, and then combines the same fractions. In the present invention, the specific elution procedure of the silica gel column chromatography is preferably: gradient elution with PE:CH2Cl2 (1:0, 1:1, 0:1, v / v) and CH2Cl2:Me2CO (9:1, 4:1, 1:1), and the obtained fractions are combined after thin layer chromatography detection to obtain a total of 6 fractions to be separated, namely Fr.1 (PE), Fr.2 (PE:CH2Cl2=1:1), Fr.3 (CH2Cl2), Fr.4 (CH2Cl2:Me2CO=9:1), Fr.5 (CH2Cl2:Me2CO=4:1), and Fr.6 (CH2Cl2:Me2CO=1:1).

[0018] Fr.4 was subjected to MCI column chromatography or reverse phase C 18After column chromatography, the present invention performs dextran gel column chromatography on the obtained fraction Fr.4-2. In a specific embodiment of the present invention, the model of the dextran gel is LH-20. In the present invention, the mobile phase used in the dextran gel column chromatography is preferably a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1; after the dextran gel column chromatography, 4 sub-fractions, namely Fr.4-2-1, Fr.4-2-2, Fr.4-2-3, and Fr.4-2-4, are obtained.

[0019] After the above column chromatography, the present invention performs high performance liquid chromatography separation on the obtained fractions Fr.4-2-1, Fr.4-2-2, Fr.4-2-3, and Fr.4-2-4 to obtain the diterpenoid compound. In the present invention, the high performance liquid chromatography separation adopts preparative or semi-preparative high performance liquid chromatography; the chromatographic conditions of the high performance liquid chromatography are preferably: C 18 The chromatographic column is a C8 chromatographic column, a phenyl chromatographic column or a silica gel chromatographic column, and the column temperature is 35°C; gradient elution is adopted, and the mobile phase is a mixed solvent composed of acetonitrile and water in a volume ratio of 100:0 to 5:95; the flow rate is 3mL / min. In a specific embodiment of the present invention, the separation procedure of the high performance liquid chromatography is preferably: using an Agilent 1200 semi-preparative liquid chromatograph, ZorbaxSB-C 18 column (5μm, 9.4×250mm, 3mL / min), column temperature 35℃; under the elution condition of MeCN-H2O (75:25, v / v), compound 3 (t R 14.5min), compound 4 (t R 17.2min), compound 6 (t R 21.4min); under the elution condition of MeCN-H2O (70:30, v / v), compound 1 (t R 11.6min), compound 8 (t R 13.5min), compound 10 (t R 14.8min); under the elution condition of MeCN-H2O (80:20, v / v), compound 7 (t R 9.7min), compound 9 (t R 10.5min)、12(t R 13.8min); 11 (t R 11.5min), compound 2 (t R 14.4min), compound 5 (t R28.6min).

[0020] The present invention provides the rice stalk extract L01 described in the above scheme and a preparation method thereof, wherein the preparation method comprises the following steps:

[0021] (1) mixing the crushed rice flower with an organic solvent, and collecting the extract after ultrasonic treatment; the organic solvent comprises one or more of methanol, ethanol, acetone, chloroform, dichloromethane and petroleum ether; the solid-liquid ratio of the crushed rice flower aerial part to the organic solvent is 1:(3-8); the ultrasonic treatment time is 10-40 min;

[0022] (2) Concentrating the extract to obtain a crude extract: the concentration is achieved by rotary evaporation at a temperature of 30 to 60° C.;

[0023] (3) The crude extract was separated and eluted by silica gel column chromatography, and the acetone elution phase was collected and concentrated to obtain the rice flower extract L01: the eluents used in the silica gel column chromatography elution process were petroleum ether and acetone, respectively; the concentration treatment was achieved by rotary evaporation at a temperature of 30 to 60° C. The rice flower extract L01 has compounds 1 to 12 as characteristic components.

[0024] Specifically, the preparation method of the rice ball flower extract L01 is:

[0025] (1) The above-ground parts of the rice flower were dried in the shade and crushed into 30 mesh, mixed thoroughly with 10 L of petroleum ether at room temperature, and ultrasonically extracted three times, each time for 30 min, and the extracts were filtered and combined;

[0026] (2) concentrating the extract by a rotary evaporator at 45° C. to obtain an extract;

[0027] (3) The extract was dissolved in 0.5 L of chloroform, mixed with silica gel 200-300 mesh, placed to dry, ground and sieved, and column chromatography was performed using silica gel 200-300 mesh, eluted with petroleum ether and acetone in turn, and the acetone eluate was concentrated to obtain rice flower extract L01. Rice flower extract L01 has compounds 1-12 as characteristic components.

[0028] The present invention also provides the use of the sesquiterpene compounds 1 to 12 and the extract L01 of the Flos Ornithogalum in the preparation of anti-inflammatory drugs or immunosuppressive drugs, and the use of the sesquiterpene compounds 1 to 12 and the extract L01 in the preparation of T cell proliferation inhibitors or cytokine inhibitors and anti-psoriasis drugs.

[0029] In addition, the present invention also provides a pharmaceutical composition, comprising a pharmaceutically acceptable carrier and one or more of the sesquiterpenoid compounds 1 to 12.

[0030] And, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the Oleander extract L01.

[0031] In the present invention, the mass percentage of compounds 1 to 12 or extract L01 in the pharmaceutical composition is preferably 0.1 to 99%, more preferably 0.5 to 90%; the total mass percentage of the pharmaceutical carrier in the pharmaceutical composition is preferably 1 to 99.9%, more preferably 10 to 99.5%.

[0032] The present invention has no special limitation on the pharmaceutical carrier, and any pharmaceutical carrier well known in the art can be used, such as one or more of solid, semi-solid or liquid diluents, fillers or pharmaceutical product adjuvants. In the present invention, the preparation types of the pharmaceutical composition preferably include liquid preparations, solid preparations, sprays or mists; the liquid preparations preferably include injections, suspensions, emulsions, solutions or syrups; the solid preparations preferably include tablets, capsules, granules or granules. The present invention has no special limitation on the preparation method of the pharmaceutical composition, and any preparation method well known in the art can be used.

[0033] In the present invention, the administration method of the pharmaceutical composition and extract L01 is preferably injection, oral administration, sublingual administration or mucosal dialysis; the injection preferably includes intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection or subcutaneous injection.

[0034] In the present invention, the pharmaceutical composition and extract L01 are preferably used in the form of a dosage per unit body weight. In the present invention, the dosage per unit body weight is preferably 0.5 to 500 mg / kg.

[0035] By adopting the above technical solution, the present invention has the following beneficial effects:

[0036] The rice flower diperpene compounds 1 to 12 obtained by the technical solution of the present invention significantly reduced the secretion of inflammatory cytokine IFN-γ in mouse T cells stimulated by CD3 / CD28 monoclonal antibodies in in vitro experiments. In in vivo experiments on mice, compound 6 and rice flower extract L01 significantly reduced the content of proinflammatory cytokines IFN-γ and IL-17A in the serum of psoriasis model mice, significantly improved the clinical symptoms of psoriasis mice such as weight loss, back skin scales and thickness, and reduced pathological damage to the mouse skin and systemic immune response. This provides candidate materials for screening safe and efficient immunosuppressants, and also provides an effective strategy for the treatment of autoimmune diseases such as psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a single crystal X-ray diffraction structure diagram of the diterpenoid compound 1 of the present invention;

[0038] Figure 2 This is a single crystal X-ray diffraction structure diagram of the diterpenoid compound 2 of the present invention;

[0039] Figure 3 This is a single crystal X-ray diffraction structure diagram of the diterpene compound 4 of the present invention;

[0040] Figure 4 It is a schematic diagram of the structure of the diterpenoid compounds 1 to 12 of the present invention;

[0041] Figure 5 These are pictures of the back skin of mice in each group after being treated with the rice ball extract L01 of the present invention;

[0042] Figure 6 This is an H&E stained section of the back skin of mice treated with the rice ball extract L01 of the present invention;

[0043] Figure 7 This is a graph showing changes in the levels of cytokines IFN-γ and IL-17A in the serum of mice after being treated with the rice ball extract L01 of the present invention;

[0044] Figure 8 These are pictures of the back skin of mice in each group after being treated with the diterpenoid compound 6 of the present invention;

[0045] Fig. 9 This is an H&E stained section of the back skin of mice after being treated with the diterpenoid compound 6 of the present invention;

[0046] Fig.10 This is a graph showing changes in the levels of cytokines IFN-γ and IL-17A in mouse serum after treatment with the dipterpene compound 6 of the present invention. DETAILED DESCRIPTION

[0047] In conjunction with the accompanying drawings, the embodiments of the present invention are used to further illustrate the sesquiterpene compounds and extract L01 of the present invention, as well as their preparation methods and applications in the preparation of anti-inflammatory drugs and immunosuppressive drugs. However, they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] Specifically, the steps of extracting and separating the diterpenoid compounds 1 to 12 in the present invention are as follows:

[0050] (1) The above-ground part of the rice ball flower collected from Yunnan was dried in the shade and crushed into 30 mesh to obtain 10.0 kg of sample, which was then soaked and extracted with 50 L of petroleum ether at room temperature for 3 times, each time for 48 hours, and the extracts were combined after filtration, and the solvent was removed by vacuum distillation to obtain 230 g of total extract;

[0051] (2) The extract was dissolved in 2L of chloroform, mixed with 253g of silica gel (200-300 mesh), placed to dry, ground and sieved, and loaded with 2.0kg of silica gel (200-300 mesh) for column chromatography. Gradient elution was performed with petroleum ether: dichloromethane (1:0, 1:1, 0:1, v / v) and chloroform: acetone (9:1, 4:1, 1:1) in sequence. The obtained fractions were concentrated under reduced pressure and detected by thin layer chromatography. After combining the same fractions, a total of 6 fractions to be separated were obtained, namely Fr.1 (petroleum ether), Fr.2 (petroleum ether: chloroform = 1:1), Fr.3 (chloroform), Fr.4 (chloroform: acetone = 9:1), Fr.5 (chloroform: acetone = 4:1), and Fr.6 (chloroform: acetone = 1:1);

[0052] (3) Fr.4 (48 g) was subjected to MCI column chromatography and gradient eluted with methanol:water (50%, 60%, 70%, 80%, 90%, 100%, v / v) to obtain four subfractions Fr.4-1 to Fr.4-4;

[0053] Fraction Fr.4-2 was subjected to dextran gel column chromatography and eluted with dichloromethane:methanol (1:1, v / v) to obtain four subfractions Fr.4-2-1 to Fr.4-2-4;

[0054] The fractions Fr.4-2-1 to Fr.4-2-4 were separated by high performance liquid chromatography using an Agilent 1200 semi-preparative liquid chromatograph, Zorbax SB-C 18 column (5μm, 9.4×250mm, 3mL / min), column temperature 35℃; under the elution condition of MeCN-H2O (75:25, v / v), compound 3 (t R 14.5min), compound 4 (t R 17.2min), compound 6 (t R 21.4min); under the elution condition of MeCN-H2O (70:30, v / v), compound 1 (t R 11.6min), compound 8 (t R 13.5min), compound 10 (t R 14.8min); under the elution condition of MeCN-H2O (80:20, v / v), compound 7 (t R 9.7min), compound 9 (t R 10.5min)、12(t R 13.8min); under the elution condition of MeCN-H2O (60:40, v / v), compound 11 (tR 11.5min)、2(t R 14.4min), compound 5 (t R 28.6min);

[0055] The obtained masses of each compound were compound 1 (2.0 mg), compound 2 (3.2 mg), compound 3 (9.5 mg), compound 4 (3.5 mg), compound 5 (2.0 mg), compound 6 (35.0 mg), compound 7 (2.0 mg), compound 8 (4.0 mg), compound 9 (3.5 mg), compound 10 (13.0 mg), compound 11 (1.2 mg), and compound 12 (2.5 mg).

[0056] Physical and spectral data of compounds 1 to 12:

[0057] Compound 1: white solid; specific rotation Infrared IR(KBr)ν max 3429,2955,2931,2870,1742,1721,1640,1377,1164cm –1 ; High resolution mass spectrometry HRESIMS m / z 431.2443[MH] - ; 1 H and 13 C NMR data are shown in Table 1.

[0058] Compound 2: white solid; specific rotation Infrared IR(KBr)ν max 3435,2973,2936,2871,1760,1716,1452,1378,1140cm –1 ; High resolution mass spectrometry HRESIMS m / z 455.2405[M+Na] + ; 1 H and 13 C NMR data are shown in Table 1.

[0059] Compound 3: Colorless oily liquid; specific rotation Infrared IR(KBr)ν max 3437,2973,2936,2871,1715,1452,1377,1138,1013cm –1 ; High resolution mass spectrometry HRESIMS m / z439.2459[M+Na] + ; 1 H and 13 C NMR data are shown in Table 1.

[0060] Compound 4: Colorless oily liquid; specific rotation Infrared IR(KBr)ν max 3470,2967,2938,2874,1684,1462,1394,1378,1010cm –1 ; High resolution mass spectrometry HRESIMS m / z375.2141[M+Na] + ; 1 H and 13 C NMR data are shown in Table 1.

[0061] Compound 5: Colorless oily liquid; specific rotation Infrared IR(KBr)ν max 3438,2971,2939,2871,1704,1684,1452,1379,1055cm –1 ; High resolution mass spectrometry HRESIMS m / z391.2091[M+Na] + ; 1 H and 13 C NMR data are shown in Table 2.

[0062] Compound 6: white solid; specific rotation Infrared IR(KBr)ν max 3429,2956,2874,1766,1661,1457,1380,1166cm –1 ; High resolution mass spectrometry HRESIMS m / z455.2396[M+Na] + ; 1 H and 13 C NMR data are shown in Table 2.

[0063] Compound 7: Colorless oily liquid; specific rotation Infrared IR(KBr)ν max 3435,2937,2870,1712,1674,1451,1378,1144cm –1 ; High resolution mass spectrometry HRESIMS m / z 469.2195[M+Na] + ; 1 H and 13 C NMR data are shown in Table 2.

[0064] Compound 8: Colorless oily liquid; specific rotation Infrared IR(KBr)ν max 3436,2970,2871,1715,1687,1452,1379,1170cm –1; High resolution mass spectrometry HRESIMS m / z 471.2353[M+Na] + ; 1 H and 13 C NMR data are shown in Table 2.

[0065] Compound 9: Colorless oily liquid; specific rotation Infrared IR(KBr)ν max 2969,2939,2871,1770,1685,1451,1380,1168cm –1 ; High resolution mass spectrometry HRESIMS m / z 501.2458[M+Na] + ; 1 H and 13 C NMR data are shown in Table 3.

[0066] Compound 10: Colorless oily liquid; specific rotation Infrared IR(KBr)ν max 3451,2969,2939,2871,1683,1449,1380,1371cm –1 ; High resolution mass spectrometry HRESIMS m / z 517.2775[M+Na] + ; 1 H and 13 C NMR data are shown in Table 3.

[0067] Compound 11: Colorless oily liquid; specific rotation Infrared IR(KBr)ν max 3446,2960,2833,1730,1690,1422,1381,1200cm –1 ; High resolution mass spectrometry HRESIMS m / z 471.2353[M+Na] + ; 1 H and 13 C NMR data are shown in Table 3.

[0068] Compound 12: Colorless oily liquid; specific rotation Infrared IR(KBr)ν max 3433,2966,2835,1718,1688,1441,1377,1175cm –1 ; High resolution mass spectrometry HRESIMS m / z 471.2353[M+Na] + ; 1 H and 13 C NMR data are shown in Table 3.

[0069] Figures 1 to 3They are the single crystal X-ray diffraction structures of compound 1, compound 2 and compound 4 respectively.

[0070] From the physical and spectral data of compounds 1 to 12, it can be seen that compounds 1 to 12 meet the Figure 4 The structure shown.

[0071] Table 1 Compounds 1 to 4 1 H and 13 C NMR data (acetone-d6, J in Hz)

[0072]

[0073] Table 2 Compounds 5 to 8 1 H and 13 C NMR data (acetone-d6, J in Hz)

[0074]

[0075] Table 3 Compounds 9 to 12 1 H and 13 C NMR data (acetone-d6, J in Hz)

[0076]

[0077] Example 2

[0078] Specifically, the preparation of the rice ball extract L01 in the present invention is:

[0079] (1) The above-ground part of the rice ball flower collected from Yunnan was dried in the shade and crushed into 30 mesh to obtain 2.0 kg of sample, which was fully mixed with 10 L of petroleum ether at room temperature and ultrasonically extracted three times, each time for 30 min, and the extracts were filtered and combined;

[0080] (2) The extract was concentrated by a rotary evaporator at 45° C. to obtain 30 g of extract;

[0081] (3) The extract was dissolved in 0.5L chloroform, mixed with 33g silica gel (200-300 mesh), placed to dry, ground and sieved, and loaded with 240g silica gel (200-300 mesh) for column chromatography, eluted with 100% petroleum ether and 100% acetone in turn, and the acetone eluate was concentrated to obtain the rice flower extract L01 (18g). The rice flower extract L01 has compounds 1-12 as characteristic components.

[0082] Example 3

[0083] The compounds 1 to 12 of the present invention were tested for in vitro immunosuppressive activity:

[0084] CD3, CD28 monoclonal antibodies, IFN-γ detection kits were purchased from BD Bioscience, CCK-8 kits were purchased from Meilun, and RPMI-1640 culture medium and fetal bovine serum were purchased from Biological Industries.

[0085] Preparation of test samples: The 12 compounds of Example 1 were dissolved in DMSO to prepare 20 mM stock solutions.

[0086] Specific method: Splenocytes were aseptically isolated from 6-8 week old female C57BL / 6 mice, and T cells were isolated by nylon wool column method. Then T cells (4×10 5 / well) in a 96-well plate (coated with 5μg / mL CD3 monoclonal antibody), and each group was added with 40, 20, 10, 5, 2.5, 1.25μM compound and 2μg / mL CD28 monoclonal antibody, respectively, and cultured in a 37℃ / 5% CO2 incubator. After 48h, the cell supernatant was collected, and the IFN-γ secretion in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA); 10μL CCK-8 reagent was added to each well of cells, and the OD was measured after incubation at 37℃ for 4h. 450 , to evaluate cell proliferation. Cyclosporine A (CsA) positive control group, DMSO negative control group, unstimulated group and blank control group were set up at the same time. The inhibitory activity of the compound on IFN-γ was calculated, and the half inhibitory concentration IC was analyzed using GraphPad Prism software 50 , the results are shown in Table 3.

[0087] Table 3 Compounds 1 to 12 inhibit the secretion of proinflammatory cytokine IFN-γ in mouse T cells stimulated by CD3 / CD28 monoclonal antibody

[0088]

[0089] a CsA: Cyclosporine A (CsA), positive control.

[0090] As can be seen from Table 3, compounds 1 to 12 of the present invention significantly inhibit the secretion of proinflammatory cytokine IFN-γ in mouse T cells stimulated by CD3 / CD28 monoclonal antibody, indicating that this type of compound has anti-inflammatory and immunosuppressive effects. At the same time, this type of compound has a certain structure-activity relationship with activity, which provides a basis for future compound structure optimization and modification.

[0091] Example 4

[0092] In vivo anti-psoriasis activity test of the extract of Oryza sativa L01:

[0093] BABL / 6 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., imiquimod cream was purchased from Sichuan Mingxin Pharmaceutical Co., Ltd., and vaseline was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.

[0094] Establishment of Imiquimod-induced Psoriasis Mouse Model and Pharmacodynamic Study: Mice were randomly divided into normal group, model group, methotrexate (MTX) treatment group (1 mg / kg), and terpenoids treatment group (50, 150, 450 mg / kg), with 6 mice in each group. The backs of mice were depilated before the experiment, and 62.5 mg of 0.5% imiquimod ointment was evenly applied on the backs from the first day. The backs of mice in the normal control group were smeared with an equal amount of vaseline, once a day, for 6 days. From the first day to the sixth day, each treatment group was given the drug by gavage. The mice were weighed during the experiment, and the psoriasis area and severity index (PASI) of the mice was scored according to the following criteria: 0 points, no erythema, scales, normal skin; 1 point, mild erythema, a few fine scales, and the skin at the lesion was slightly higher than normal skin; 2 points, moderate red plaques, the surface of the lesions was covered with scales, and the lesions were flaky or moderately raised; 3 points, severe dark red plaques, almost all the lesions were covered with thick scales or obvious lesions; 4 points, very severe and very deep red plaques, all lesions were covered with very thick lamellar scales, and the thickening and protrusion of the lesions were very obvious. After the experiment, the back skin of the mice was taken for pathological sections. The levels of proinflammatory cytokines IFN-γ and IL-17A in the serum of mice were detected by ELISA.

[0095] Depend on Figure 5 It can be seen that the rice ball extract L01 significantly improved the symptoms of psoriasis in mice. Figure 6 The experimental results showed that the extract L01 from rice ball flower significantly inhibited the pathological damage of the skin tissue of psoriasis mice. HE staining of skin pathological sections showed that the epidermis of psoriasis mice was thickened, the stratum corneum showed incomplete keratinization and epidermal protrusions, the dermis was thickened and lymphocyte infiltration appeared, and the above symptoms of mice in the treatment group were significantly improved. Figure 7 The experimental results showed that the extract L01 from Rice Balls significantly reduced the levels of proinflammatory cytokines IFN-γ and IL-17A in the serum of mice with psoriasis. These studies confirmed that the extract L01 from Rice Balls had a good anti-psoriatic effect in vivo and had good application prospects in the clinical treatment of autoimmune diseases such as psoriasis.

[0096] Example 5

[0097] The diterpenoid compound 6 of the present invention was tested for its in vivo anti-psoriasis activity:

[0098] The method for establishing the imiquimod-induced psoriasis mouse model is the same as in Example 5. The mice were randomly divided into a normal group, a model group, a methotrexate (MTX) treatment group (1 mg / kg), and a compound 6 treatment group (25 and 50 mg / kg), with 6 mice in each group. From day 1 to day 6, each treatment group was administered by intraperitoneal injection. The mice were weighed during the experiment and PASI scores were performed. After the experiment, the back skin of the mice was taken to make pathological sections. The ELISA method was used to detect the levels of proinflammatory cytokines IFN-γ and IL-17A in mouse serum.

[0099] Figure 8 The experimental results showed that compound 6 significantly improved the symptoms of psoriasis in mice. Fig. 9 This indicates that compound 6 significantly inhibits the pathological damage of the skin tissue of psoriasis mice. HE staining of skin pathological sections revealed that the epidermis of psoriasis mice was thickened, the stratum corneum showed parakeratosis and epidermal protrusions, the dermis was thickened and lymphocyte infiltration occurred, and the above symptoms of the mice in the treatment group were significantly improved. Figure 7 The experimental results showed that compound 6 significantly reduced the levels of proinflammatory cytokines IFN-γ and IL-17A in the serum of psoriasis mice. These structures indicate that compound 6 has a good anti-psoriatic effect in vivo and can be used to treat autoimmune diseases such as psoriasis.

[0100] Formulation Examples

[0101] In the following preparation examples, conventional reagents are selected and the preparations are prepared according to conventional methods. This application example only reflects that at least one of the rice stalk extract L01 or compounds 1 to 12 of the present invention can be prepared into different preparations, and the specific reagents and operations are not specifically limited:

[0102] (1) The extract L01 or compounds 1 to 12 of the present invention is prepared into tablets:

[0103] Tablet raw materials: 10 mg of rice ball extract L01 or any one of compounds 1 to 12, 180 mg of lactose, 55 mg of starch, and 5 mg of magnesium stearate;

[0104] Preparation method: Mix the raw materials, lactose and starch, evenly moisten with propylene glycol, sieve the moistened mixture and dry it, sieve it again, add magnesium stearate, and then compress the mixture into tablets, each tablet weighing 250 mg and containing 10 mg of the raw materials.

[0105] (2) The extract L01 or compounds 1 to 12 of the present invention is prepared into capsules:

[0106] Capsule ingredients: 10 mg of rice flower extract L01 or one or more of compounds 1 to 12, 187 mg of lactose, and 3 mg of magnesium stearate;

[0107] Preparation method: Mix the raw materials and auxiliary agents, sieve, mix evenly, and put the obtained mixture into hard gelatin capsules. Each capsule weighs 200 mg and contains 10 mg of the raw materials.

[0108] (3) Dissolve the extract L01 of Oryza sativa L. or any one of compounds 1 to 12 in sterile water for injection, stir until the raw material is dissolved, filter through a sterile suction filtration funnel and sterile fine filtration, and then pack into ampoules, freeze-dry at low temperature, and aseptically seal to obtain a drug powder injection.

[0109] (4) After dissolving the extract L01 of Oryza sativa or one or more of compounds 1 to 12 in DMSO, adding water for injection, fine filtering, filling and sterilization according to conventional methods to prepare an injection solution, wherein the concentration of the injection solution is 0.5 to 5 mg / mL.

[0110] (5) Prepare an oral liquid by using the rice flower extract L01 or any one of compounds 1 to 12 according to a conventional oral liquid preparation method.

[0111] The above is only 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Rice ball flower extract L01, characterized in that It is prepared by the following method: (1) Mixing the crushed rice flower with an organic solvent, and collecting the extract after ultrasonic treatment; the organic solvent is one or more selected from methanol, ethanol, acetone, chloroform, dichloromethane and petroleum ether; the solid-liquid ratio of the crushed rice flower aerial part to the organic solvent is 1:(3-8); the ultrasonic treatment time is 10-40 min; (2) Concentrating the extract to obtain a crude extract; the concentration is achieved by rotary evaporation at a temperature of 30-60°C; (3) The crude extract is subjected to silica gel column chromatography separation and elution, and the acetone elution phase is collected and concentrated to obtain the rice flower extract L01; the eluents used in the silica gel column chromatography elution process are petroleum ether and acetone, respectively; the concentration treatment is achieved by rotary evaporation at a temperature of 30-60 °C.

2. Use of the Myrica oryzae extract L01 according to claim 1 in the preparation of anti-inflammatory drugs or immunosuppressive drugs.

3. Use of the Myrica oryzae extract L01 according to claim 1 in the preparation of a medicament for treating or preventing psoriasis.

4. A method for preparing the rice ball extract L01, characterized in that The method comprises the following steps: (1) Mixing the crushed rice flower with an organic solvent, and collecting the extract after ultrasonic treatment; the organic solvent is one or more selected from methanol, ethanol, acetone, chloroform, dichloromethane and petroleum ether; the solid-liquid ratio of the crushed rice flower aerial part to the organic solvent is 1:(3-8); the ultrasonic treatment time is 10-40 min; (2) Concentrating the extract to obtain a crude extract; the concentration is achieved by rotary evaporation at a temperature of 30-60°C; (3) The crude extract is subjected to silica gel column chromatography separation and elution, and the acetone elution phase is collected and concentrated to obtain the rice flower extract L01; the eluents used in the silica gel column chromatography elution process are petroleum ether and acetone, respectively; the concentration treatment is achieved by rotary evaporation at a temperature of 30-60 °C.

5. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the Oryza sativa extract L01 according to claim 1.

6. Use of the pharmaceutical composition according to claim 5 in the preparation of anti-inflammatory drugs or immunosuppressive drugs.

7. Use of the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating or preventing psoriasis.

Images