Application of steroid alkaloid derived from fritillaria hupehensis in preparation of immunosuppressive drugs

Abstract

The invention relates to the technical field of medical chemistry, and particularly discloses a medical application of a vertramine type steroid alkaloid yibeinone B with a D ring as a benzene ring, which is separated from a dry bulb of a liliaceae plant Fritillaria hupehensis Hsiao et K.C.Hsia, in preparation of immunosuppressive drugs, and a preparation method of the vertramine type steroid alkaloid yibeinone B with a D ring as a benzene ring. More specifically, the invention relates to an application in preparation of drugs having significant inhibitory activity on B lymphocyte proliferation. The veratramine type steroid alkaloid disclosed by the invention has the advantages of low toxicity and high efficiency, and can be used for preparing immunosuppressive drugs aiming at organ transplantation and / or autoimmune diseases.

Description

Technical Field

[0001] The invention relates to the technical field of medicine and chemistry, and in particular to application of a veratramine type steroidal alkaloid derived from the dried bulb of Hubei Fritillaria hupehensis Hsiao et KCHsia in the preparation of immunosuppressive drugs. Background Art

[0002] Autoimmune diseases are a class of diseases in which immune cells attack healthy cells in the body. There is evidence that exposure to bacteria, fungi, viruses, toxins, and antigens from blood and tissues from external sources can trigger an immune response in which the body mistakenly attacks healthy cells, resulting in: tissue damage, organ growth disorders, and organ dysfunction. After cancer and heart disease, autoimmune diseases are the third most common disease category, affecting about 8% of the population. The prevalence in women is about 75% higher than in men, and the disease usually occurs during the childbearing years, from 14 to 44 years old. Autoimmune diseases can affect almost every body system, including the nervous system, heart, endocrine, musculoskeletal, gastrointestinal tract, lungs, kidneys, skin, eyes, and vascular system. Generally, autoimmune diseases include two types: one is a cellular immune response mediated by T cells, which is characterized by an excessive immune response and attack on one's own tissues; the other is an immune response mediated by B lymphocytes, in which B lymphocytes produce autoantibodies to mediate the immune response. Autoimmune diseases are when the immune system overreacts and damages its own tissue cells and molecular structures, leading to a variety of different disease manifestations. Abnormal humoral immunity driven by B lymphocytes is the core pathogenesis of many autoimmune diseases. When autoreactive B lymphocytes are overactivated, a large number of autoantibodies and their immune complexes are produced. These antibodies and immune complexes can cause damage to different tissues and organs, leading to the occurrence of a variety of autoimmune diseases. From the current clinical situation, the diagnosis and treatment of autoimmune diseases mediated by B lymphocytes is still a serious medical problem. For a long time, these diseases have been facing the dilemma of lack of new drugs and ineffective treatment.

[0003] Hubei Fritillaria hupehensis Hsiao et KCHsia is a plant of the genus Fritillaria in the Liliaceae family. It is mainly distributed in the southwest region, such as Enshi and Yichang in Hubei Province. It is mainly used clinically for cough, asthma and expectoration. At present, the demand for Fritillaria hupehensis is relatively large in the domestic Chinese medicinal materials market. Due to its high content of active ingredients, wide distribution, easy cultivation and relatively low price, it has received more and more attention. Steroidal alkaloids are the main chemical components of Hubei Fritillaria. They have a basic steroidal skeleton and contain a nitrogen atom as a component of the molecule in the ring or side chain. They are an important class of alkaloids and have shown a wide range of biological activities, such as antitussive and expectorant, antiasthmatic, anti-inflammatory, antibacterial, antioxidant, antihypertensive, analgesic, anti-tumor effects, etc. Alkaloids in Fritillaria medicinal materials can be divided into servin type, veratramine type, veratramine type, solanine type, solanine type, ussuri alkaloid type and other types according to the skeleton. At present, the activity research of alkaloids of Fritillaria medicinal materials mainly focuses on alkaloids such as fritillary alkaloids, fritillary alkaloids, fritillary oxaline, fritillary oxaline, fritillary oxaline and fritillary oxaline glycoside, which is one-sided. In order to explore the novel biological activity of steroidal alkaloids in Hubei Fritillaria, the applicant explored the immunosuppressive activity of a steroidal alkaloid isolated from the dried bulb of Hubei Fritillaria. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a use of a veratramine-type steroidal alkaloid in the preparation of immunosuppressive drugs.

[0005] The veratramine type steroidal alkaloid has the following structural formula:

[0006]

[0007] Molecular formula: C 27 H 39 NO3;

[0008] The name is: yibeinone B;

[0009] The use of the steroidal alkaloid or a pharmaceutically acceptable salt thereof in the preparation of a drug capable of inhibiting the proliferation of B lymphocytes also falls within the protection scope of the present invention.

[0010] Another object of the present invention is to provide a steroidal alkaloid having an immunosuppressive effect for use in the preparation of immunosuppressive drugs for organ transplantation or autoimmune diseases.

[0011] The above-mentioned drugs can be tablets, powders, capsules, oral liquids, emulsions, ointments, creams, injections, suspensions, tinctures, granules or aerosols. The drugs in the above-mentioned dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0012] In the present invention, all of the above-mentioned immunosuppressive activities are specifically embodied in: significant inhibitory activity on B lymphocyte proliferation and an ideal selection coefficient.

[0013] Experiments have shown that the compound yibeinone B of the present invention exhibits a significant inhibitory effect on the proliferation of B lymphocytes in experiments on the nonspecific toxicity and proliferation reaction of mouse B lymphocytes, and has a potential use as a preparation of a new type of medical immunosuppressive drug.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0015] 1. The present invention reveals the novel activity of steroidal alkaloids in Fritillaria. It is the first time to reveal that the veratramine-type steroidal alkaloids isolated from Hubei Fritillaria have immunosuppressive activity, which will enable the scientific community to focus on finding lead compounds with immunosuppressive activity from steroidal alkaloids in Fritillaria in the future.

[0016] 2. The present invention provides a veratramine-type steroidal alkaloid with immunosuppressive activity. The compound has significant inhibitory activity on the proliferation of B lymphocytes, has the advantages of high efficiency and low toxicity, and can be used to prepare immunosuppressive drugs for organ transplantation and autoimmune diseases.

[0017] 3. The compounds of the present invention can be obtained by extraction and purification from plants, and have the advantages of high operational feasibility, no chemical pollution, and green environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the hydrogen spectrum (600 MHz, MeOD) of the compound prepared in Example 1 in the specific implementation manner;

[0019] Figure 2 The carbon spectrum (150 MHz, MeOD) and DEPT diagram of the compound prepared in Example 1 of the specific embodiment;

[0020] Figure 3 It is the HMQC diagram of the compound prepared in Example 1 in the specific embodiment;

[0021] Figure 4 It is the HMBC diagram of the compound prepared in Example 1 in the specific implementation mode;

[0022] Figure 5 The compound prepared in Example 1 in the specific embodiment 1 H- 1 H COSY diagram;

[0023] Figure 6It is the ROESY diagram of the compound prepared in Example 1 in the specific implementation manner. DETAILED DESCRIPTION

[0024] In order to make the invention purpose and invention content of this application more clearly presented, the applicant will provide a clear and complete description of the technical solution of the present invention in conjunction with specific embodiments below.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Embodiment 1:

[0028] The dried bulbs of Fritillaria hupehensis Hsiao et KCHsia were collected from Shuanghe Community, Xintang Township, Enshi City, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province.

[0029] The separation and purification process of the compounds described in the claims and the invention of the specification:

[0030] The dried bulbs (30 kg) of Fritillaria hupehensis Hsiao et KCHsia were crushed and soaked in anhydrous methanol for 5 times (100 L each time, 7 days each time). The extracts after 5 soaking and centrifugation were combined, concentrated under reduced pressure and evaporated to dryness, dissolved with a small amount of water, and then formic acid was added to adjust the pH value to 2-3, and then repeatedly extracted 3 times with a mixed solution of petroleum ether and ethyl acetate (petroleum ether: ethyl acetate = 1:1, v / v); 2.8 wt% ammonia water was added to the acid water part to adjust the pH to 8-9, and then extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate layer extract was concentrated under reduced pressure to obtain 100 g of the total organic layer extract. The total extract of the organic layer was separated by column chromatography on normal phase silica gel (200-300 mesh), gradient eluted with a chloroform-methanol system (400:1–0:1) solvent system, detected by thin layer chromatography (developing solvent: chloroform: methanol = 6:1, v / v, thin layer chromatography silica gel plate, Qingdao Ocean Chemical Plant, the same below) and colored with potassium bismuth iodide colorimetric agent, the same or similar components were combined, and finally merged and differentiated to obtain 7 major components (A–G).

[0031] The F component (the component eluted when the volume ratio of chloroform / methanol is 40:1, 10.1 g) was subjected to high-performance medium-pressure liquid preparative chromatography (Biotage SP1, reverse phase filler material: RP-18, 20-45 μm, Fuji Silysia Chemical Ltd., Japan), methanol water system (methanol / water = 20:80, 40:60, 60:40, 80:20, 100:0, v / v), flow rate 20 mL / min for gradient elution, and the elution time for each ratio was 40 min. Thin layer chromatography was used for detection and bismuth potassium iodide color developer was used for color development, and the same or similar components were combined and finally combined into five subcomponents F1-F5. The component F4 obtained when methanol / water = 60:40 was separated by high performance liquid chromatography (Agilent 1260; chromatographic column: Agilent Zorbax SB-C18, specification: 9.4 mm × 150 mm, 5 μm; acetonitrile-water, 30:70–45:55, v / v; flow rate: 4 mL / min) to obtain the compound of the present invention (3.5 mg, t R =24.0min).

[0032] Structural identification of the compound: The veratramine-type steroidal alkaloid compound prepared in Example 1 was dissolved in 0.5 mL of deuterated methanol, transferred to a nuclear magnetic resonance tube using a 200 μL pipette, and the hydrogen spectrum, carbon spectrum, and two-dimensional spectrum (such as Figure 1-6 ). The compound was finally confirmed to be yibeinone B, which belongs to the veratramine type steroidal alkaloids with molecular formula: C 27 H 39 NO3, the structural formula is

[0033] Example 2: Determination of immunosuppressive activity of yibeinone B using nonspecific toxicity and proliferation reaction experiments of mouse B lymphocytes

[0034] Experimental Principle

[0035] Succinate dehydrogenase in the mitochondria of living cells can reduce methylthiazolyl blue (MTT) to water-insoluble blue-purple crystalline formazan, while dead cells do not have this function. Therefore, dissolving formazan with DMSO and measuring its absorbance at a wavelength of 570nm can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells and can be used for cytotoxicity analysis.

[0036] B lymphocytes have receptors that recognize antigens and mitogen receptors on their surfaces, which can cause the corresponding lymphocyte clones to proliferate under specific antigen stimulation. Concanavalin A (ConA) and bacterial lipopolysaccharide (LPS) are mitogens that can stimulate B lymphocyte proliferation and differentiation, respectively. This process is similar to the activation process of lymphocytes in the body. Therefore, mitogen-induced lymphocyte proliferation is often used as an indicator to evaluate lymphocyte function. 3 H]-thymidine ( 3 H-TdR) incorporation is a method used to detect cell proliferation and reflects the synthesis of cell DNA. 3 H) labeled DNA precursor thymidine (TdR), then 3 H-TdR is taken into cells as a raw material for synthesizing DNA and incorporated into newly synthesized DNA. The level of lymphocyte response to stimuli can be inferred based on the amount of isotope incorporated into cells. 3 H, measured by liquid scintillation measurement, 3 The H pulse number per minute (cpm) can be calculated to reflect the degree of lymphocyte proliferation.

[0037] Experimental animals

[0038] BALB / c pure strain mice, female, 18-20 g, were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Animal Production License No.: SCXK (Shanghai) 2013-0017. The animals were kept in the SPF animal room of Shanghai Institute of Materia Medica, Laboratory Animal Use License No.: SYXK (Shanghai) 2013-0049. The animals were kept for at least one week before use. The temperature was 22±1℃, the humidity was 55±5%, and the light-dark cycle was 12 hours. Feed and water were freely available to the animals after disinfection. All experiments were carried out in strict accordance with the relevant regulations on experimental animals.

[0039] Experimental methods

[0040] Preparation of mouse spleen B lymphocytes:

[0041] The mice were killed by spinal dislocation, and their spleens were aseptically removed to prepare and extract splenic B lymphocyte suspensions, and the cell concentration was adjusted.

[0042] In the following experiments, the test compound yibeinone B and the positive control drug were dissolved in DMSO and diluted to the required concentration using RPMI1640 culture medium.

[0043] MTT assay was used to detect the effect of compounds on the activity of mouse spleen B lymphocytes:

[0044] Mouse spleen B lymphocyte suspension was 8×10 5Cells / well were inoculated in 96-well plates, and different concentrations of compounds were added. The immunosuppressant cyclosporin A (CsA) was used as a positive control drug. Corresponding solvent controls (cell controls) and culture medium background (RPMI 1640 culture medium) controls (blank controls) were also set up, with a total volume of 200 μL. Cultured in a 37°C, 5% CO2 incubator for 48 hours. MTT solution with a concentration of 5 mg / mL was added 4 hours before the end of the culture. At the end of the culture, the supernatant was aspirated and discarded, 150 μL DMSO was added to each well to dissolve the purple crystals, and the OD value was measured at 570 nm by an enzyme marker to calculate the cell survival rate.

[0045] 3 H-TdR incorporation method was used to detect the effect of compounds on the proliferation function of mouse spleen B lymphocytes:

[0046] Mouse spleen B lymphocyte suspension was 5×10 5 Cells / well were inoculated in a 96-well plate, and ConA (final concentration 5 μg / mL) or LPS (final concentration 10 μg / mL) and different concentrations of compounds were added. CsA was used as a positive control drug, and corresponding cell control wells without drug, ConA, LPS and no drug control wells (stimulation control) were set up. The total volume was 200 μL. The cells were cultured in a 37°C, 5% CO2 incubator for 48 hours. 8 hours before the end of the culture, 25 μL of 3 H-thymidine nucleotide (10 μCi / mL). Continue to culture until the end of the experiment. Collect the cells onto a glass fiber membrane using a cell harvester, add scintillation fluid, and read the number of cells incorporated into the cell DNA using a Beta counter (MicroBeta Trilux, PerkinElmer). 3 The amount of H-TdR was measured, and the cpm value represented the cell proliferation, and the cell proliferation inhibition rate was calculated.

[0047] Result evaluation:

[0048]

[0049] Selection index SI = CC 50 / IC 50 , to determine whether the sample has biologically significant activity.

[0050] Experimental Results

[0051] The results are shown in Table 1:

[0052] Table 1. Cytotoxicity and inhibitory activity of B lymphocytes in mouse spleen

[0053]

[0054] It can be seen that yibeinone B has a certain inhibitory activity on the proliferation of B lymphocytes stimulated by ConA and LPS.

Claims

1. Use of a veratramine-type steroidal alkaloid yibeinone B with a benzene ring as the D ring in the preparation of immunosuppressive drugs, characterized in that: The structural formula of the veratramine-type steroidal alkaloid yibeinone B is:

2. The use according to claim 1, characterized in that: The immunosuppressive drug is a drug that inhibits the proliferation of B lymphocytes.

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