WO2016183905A1 - 淫羊藿次苷类化合物,其制备方法,及其在促进人细胞产生γ-干扰素作用和在疾病治疗中的应用 - Google Patents
淫羊藿次苷类化合物,其制备方法,及其在促进人细胞产生γ-干扰素作用和在疾病治疗中的应用 Download PDFInfo
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Definitions
- the invention relates to an epimedium quinone glycoside compound and a preparation method thereof, which promote the production of ⁇ -interferon by human cells, and significantly improve the immunity of human body; and the treatment and health care based on the principle Applications in products, beauty and skin care products.
- the immunity of the human body refers to the ability of the body to resist the infection or invasion of pathogenic factors outside the host cell or in the host cell, that is, the ability to maintain a normal and stable environment in the human body.
- Human living environment such as air, water, food, and items in contact with human life, contain a variety of harmful microorganisms, such as bacteria, viruses, mycoplasma, chlamydia, fungi and other pathogens (host extracellular pathogenic factors). ).
- the human body may also be affected by environmental changes, the presence of viruses or bacteria in the host cells, or the infection or invasion of cancer cells (generating factors in the host cells) produced by genetic variation.
- Enhancing human immunity through drugs is an effective method for preventing and treating infectious diseases and cancers, that is, immunotherapy.
- the discovery and development of effective drugs that enhance human immunity is particularly important for low immunity, especially for people with autoimmune diseases.
- drugs that can be used for immunotherapy.
- clinically used drugs that enhance human immunity mainly macromolecular peptides or protein drugs, such as thymosin, immunoglobulin White, interferon, etc.
- these drugs have specific indications, have great limitations, and are easy to degrade, have short half-life, poor bioavailability, large-scale synthesis and separation and purification.
- Epimedium is a traditional Chinese herbal medicine. In the prior art, it is used under the guidance of traditional Chinese medicine theory to use Epimedium plant decoction, soaking, simmering, making pills and so on.
- traditional Chinese medicine theory to use Epimedium plant decoction, soaking, simmering, making pills and so on.
- icariin I and icariin C due to its extremely low content (its homologue - icariin content), It is difficult to conduct effective research, and industrial application is even more difficult to talk about.
- the present inventors have long been devoted to the research of effective components of traditional Chinese medicine in immunotherapy.
- the present inventors established an immunological test model based on the detection of human peripheral blood mononuclear cells (PBMCs) to produce ⁇ -interferon levels.
- PBMCs peripheral blood mononuclear cells
- compounds that promote (or inhibit) the production of ⁇ -interferon levels from human peripheral blood mononuclear cells (PBMCs) can be screened from a variety of chemical constituents, including traditional Chinese medicine and medicinal plants.
- human ⁇ -interferon is an important immune protein (cytokine) against the disease, its production level is directly related to the body's immunity. Therefore, the model is an ideal technique for screening and discovering drugs having functional immunity for human immunity.
- icariin and epimedium.
- Azadirachtin A, Icariin I or Icariin C are the basic raw materials, and other epimedium glucosides are obtained by chemical conversion method (semi-synthesis), and some icariin compounds are also It can be obtained by a fully synthetic method. Tests by screening models have shown that these substances also have activity similar to icariin I or icariin C.
- a first object of the present invention is to provide an icariin glucoside compound (as shown in Formula I).
- R 1 , R 2 and R 3 in formula I are as defined below:
- R 1 is H, OH, OCH 3 , CH 3 COO, CH 3 , CF 3 , NH 2 , CH 3 NH, CH 3 CONH, CN, Br, Cl, F and the like.
- R 2 is H, OH, OCH 3 , CH 3 COO, NH 2 , CH 3 NH, (CH 3 ) 2 N, CH 3 CONH, CN and the like.
- R 3 is selected from the group consisting of H, OH, OCH 3 , CH 3 COO, CH 3 , CF 3 , and C 1 -C 6 -NH 2 (wherein C 1 -C 6 is an alkane having 1-6 C) a base, a cycloalkyl group, an alkene group, a cycloalkenyl group, especially a cyclopentylamino group, a cyclohexylamino group; and a morphinolinyl group, a methyl piperazinyl group, NH 2 , CH 3 NH, (CH 3 ) 2 N, (CH 3 CH 2 ) 2 N, CH 3 CONH, CN, Br, Cl, F, amino acid acyl group, amino acid amide group (R'-CH-CONH-), oligopeptide acyl group, oligopeptide amido group, and the like.
- C 1 -C 6 is an alkane having 1-6 C
- a base a
- a second object of the present invention is to convert epimedium saponin into epimedium by converting, for example, an icariin, which is rich in epimedium, into icariin I by, for example, an enzymatic conversion technique.
- Still another object of the present invention is to provide an epimedium saponin of the formula I, which is derivatized with natural products such as icariin, icariin A, icariin I, and icariin C.
- a method of classifying compounds is to provide an epimedium saponin of the formula I, which is derivatized with natural products such as icariin, icariin A, icariin I, and icariin C.
- human ⁇ -interferon is an important anti-disease immune protein (cytokine) in the human body, it can inhibit the replication of the virus, and can also effectively resist the invasion and infection of other pathogenic substances such as bacteria and cancer cells.
- cytokine anti-disease immune protein
- the compounds can significantly improve the body's immunity and effectively improve the body's ability to resist infection or invasion of pathogenic factors (host cells and host cells).
- It can be used for the prevention and treatment of infectious diseases including influenza, hepatitis B, hepatitis C and tuberculosis, and the development and metastasis of tumors. It shows that these compounds have the prospect of developing new types of cellular immunotherapy drugs. It can also be used to prepare health care medicines, beverages, beauty products and other related aspects related to improving the immune health function of the human body.
- another object of the present invention is to provide a compound of the genus Icariin of the formula I, including icariin I and icariin C as main components for prevention and treatment and low immunity to humans.
- the main active ingredient contained in it is an epimedium glucoside compound such as icariin I or icariin C, or both.
- the model uses a foreign antigen to stimulate human peripheral blood mononuclear cells (PBMCs) to produce a cytokine IFN- ⁇ , which is added to a mixed system of foreign antigens and human peripheral blood mononuclear cells (PBMCs) by adding a test drug.
- PBMCs peripheral blood mononuclear cells
- the effect on the level of IFN- ⁇ production is measured to screen for small molecule compounds that enhance human immunity or inhibit human immunity.
- the method is to collect fresh blood from a normal person and separately prepare human peripheral blood mononuclear cells (PBMCs).
- PBMCs peripheral blood mononuclear cells
- foreign antigens such as the monoclonal stimulatory molecule Anti-CD3, Anti-CD3/Anti-CD28, peripheral blood mononuclear cells of different humans, or stimulated by other pathogenic factors (such as viruses, germs, cancer cells, etc.) Stimulation of human cells and the like.
- the human peripheral blood mononuclear cells after the above stimulation will produce a new cytokine IFN- ⁇ .
- add different concentrations of the test drug the compound to be screened
- the stimulated PBMCs were planted in a 96-well micro-bottomed plate and cultured in an incubator at 37 ° C and 5% CO 2 . Finally, the level of IFN- ⁇ production in the culture medium at different time points was determined by ELISA. An elevated level of IFN- ⁇ production indicates that the test compound has an immunopotentiating effect. If the level of IFN- ⁇ production is decreased, it indicates that the test compound has an effect of suppressing human immunity. Therefore, the method can also be used to screen small molecule compounds having human immunity, i.e., immunosuppressive agents.
- Figure 1 shows the reaction of preparing icariin I by enzymatic conversion of icariin in Example 1.
- Figure 2 is a view showing the reaction of the preparation of icariin C by enzymatic conversion of Epimedium adenosine A in Example 2;
- Figure 3 is a view showing the reaction of the icariin to produce the icariin glucoside compound (Y-5XS-1) by chemical and enzymatic conversion in Example 3.
- Figure 4 is a view showing the reaction of preparing epimedium saponins (Y-3XS-1) by chemical conversion from icariin I in Example 4;
- Figure 5 is a graph showing the percentage of the production and promotion of IFN- ⁇ stimulated by the drug icariin I (PBMCs treated with anti-CD3) in Example 6.
- Figure 6 is a graph showing the percentage of the production and promotion of IFN- ⁇ stimulated by the drug Icariin I (treated by combination of anti-CD3 and anti-CD28) in Example 6.
- Fig. 7 shows that in Example 7, the drug icariin I promotes the production of IFN- ⁇ in mixed lymphocyte culture.
- Figure 8 is a graph showing the effect of different icariin-class I compounds on the production of IFN- ⁇ levels in mixed lymphocytes in Example 8 [Aminoglycoside I compound: (1) Icariin; (2) kinky Astragaloside I; (3) Icariin C; (4) Y-3XS-1; (5) Y-5XS-1; (6) Y-4'XS-1]
- Figure 9 shows the effect of the drug Icariin I on the production of IFN- ⁇ by PBMCs and PFMCs in Example 9.
- Figure 10 is a graph showing the effect of icariin I on xenografts in nude mice in Example 11 (growth curve)
- Figure 11 is a graph showing the effect of Icariin I on xenograft tumors in nude mice in Example 11 (tumor weight change)
- Icariin I was isolated from Epimedium.
- the icariin I was prepared by enzymatic conversion using icariin as a raw material.
- Icariin (content 98%) 2 g dissolved in phosphate buffer solution of pH 6.8, 1 g of immobilized rhamnosidase was added, reacted at 60 ° C for 24 hours with stirring, crystallized, and collected by filtration. Methanol recrystallization treatment. Obtained 1.3 g of Epimedium I, with a content of 95%.
- the chemical structure of the product has been identified by 1 H NMR, MS, and the like. The specific reaction formula is shown in Figure 1.
- Sesame saponin C was isolated from Epimedium.
- Epimedium A is one of the most abundant components in Epimedium. Reference literature reporting method [Xu Yuxu et al, Chinese herbal medicine, 1981, 14, 24-26], isolated and purified from Epimedium whole grass to obtain more than 95% of Epimedium A. The chemical structure of the product has been identified by 1 H NMR, MS, and the like.
- the OH group of R 1 is selectively converted into other groups by a chemical method (without affecting the glycosyl group present in the molecule).
- the R 2 Rha group in the modified icariin is removed enzymatically by the method of (2) in Example 1, whereby the desired icariin glucoside compound can be obtained.
- the OH group of R 3 is selectively converted into other groups by a chemical method (without affecting the glycosyl group present in the molecule).
- the R 2 Rha group in the modified icariin product was removed by enzymatic method using the method of (2) of Example 1, whereby the desired icariin glucoside compound was obtained.
- Epimedium I promote the production of human ⁇ -interferon and improve human immunity test (I)
- Epimedium hodginin I promotes the expression of interferon-gamma (IFN- ⁇ ) in normal human peripheral blood mononuclear cells (PBMCs, treated with monoclonal antibody) (simulating the immune test model in humans).
- IFN- ⁇ interferon-gamma
- icariin I test solution 10 mg of icariin I was dissolved in 1 ml of DMSO (initial concentration: 10 mg/ml), dissolved, and packaged, and stored in a -80 ° C refrigerator for use.
- PBMCs peripheral blood mononuclear cells
- the mononuclear cells are aspirated by a pipette and placed in another centrifuge tube to obtain peripheral blood mononuclear cells (PBMCs) were washed twice with Hank's solution (1800 r/min, 8 min). After the last centrifugation, the supernatant was discarded, the cells were resuspended in complete RPMI 1640 medium, and mixed; mononuclear cells were counted using a trypan blue counter. Add complete RPMI 1640 medium to adjust the cell number to the desired concentration.
- PBMCs peripheral blood mononuclear cells
- PBMCs Peripheral blood mononuclear cells obtained by fresh isolation were adjusted to a cell concentration of 2 ⁇ 10 6 /ml with RPMI 1640 complete medium, and then with or without the monoclonal stimulating molecule Anti-CD3 (1 ug/ml) or Anti -CD3 (1ug/ml)/Anti-CD28 (1ug/ml), then add three different concentrations of the drug Icariin I (the final concentration is 1ng/ml, 10ng/ml, 100ng/ml) And mix well.
- the stimulated cells were cultured in 96-well micro-round bottom culture plates, and each set of stimulation conditions was carried out in 3 replicate wells with a cell concentration of 4 ⁇ 10 5 /ml (200 ul/well) per well, and at 37 ° C and 5 Four time points were cultured in the incubator of %CO 2 : 12h, 24h, 36h and 48h, and finally the supernatants at different time points were collected for testing.
- FIG. 5A Icariin I significantly increased cytokine IFN-[gamma] production in a time and dose dependent manner.
- Figure 5B Icariin I promoted the production of IFN- ⁇ at different concentration gradients and at different time points.
- PBMCs were treated with anti-CD3 and anti-CD28, and then different concentrations (ng/ml) of drug icariin I were added.
- Fig. 6A The amount of cytokine IFN- ⁇ produced according to the different concentration gradients of the drug icariin I and the different time points.
- Figure 6B Different concentration gradients of the drug icariin I and the percentage of promotion at different time points. *P ⁇ 0.05; **-***P ⁇ 0.01
- Epimedium I promote the production of human ⁇ -interferon and improve human immunity test (II)
- Epimedium hodginin promotes the production of interferon-gamma (IFN- ⁇ ) during the culture of human peripheral blood mixed lymphocytes (simulating the immune test model in humans).
- IFN- ⁇ interferon-gamma
- PBMCs normal human peripheral blood mononuclear cells
- PBMCs Peripheral blood mononuclear cells obtained by fresh isolation were adjusted to a cell concentration of 5 ⁇ 10 5 /ml with RPMI1640 complete medium.
- the lymphocytes of two healthy humans were mixed in equal volume to maintain the total cell concentration at 1 ⁇ 10 6 /ml, with or without the addition of different concentrations of Epimedium I (the terminal concentration was 100 ng/ Ml, 1000 ng/ml), and mix well.
- the stimulated cells were then cultured in a 96-well micro-round bottom culture plate at a cell concentration of 2 ⁇ 10 5 /ml (200 ul / well) per well, and cultured in a 37 ° C, 5% CO 2 incubator. At 120 h, the cytokine IFN- ⁇ was detected.
- the ⁇ -interferon produced by the human peripheral blood mixed lymphocyte culture reaction (MLC) was examined as described in Example 5 (4).
- Figure 7 shows that IFN- ⁇ is increased after mixed culture of two normal human lymphocytes.
- the addition of icariin-1 significantly promoted (up to 300%) IFN- ⁇ production in mixed lymphocyte cultures in a dose-dependent manner.
- human lymphocyte A control is a lymphocyte sample group containing only one type of human.
- the human lymphocyte B control is a group of lymphocyte samples containing two kinds of humans. A control in which two kinds of human lymphocytes are mixed and cultured, but no icariin I is added. group.
- the dosing concentrations of Icariin I were 0.1 and 1 ⁇ g/ml, respectively. *P ⁇ 0.05; **P ⁇ 0.01.
- Example 8 Example 8:
- Epimedium glucosides promote screening assays for human IFN-gamma production.
- Example 7 i.e., the drug-stimulated culture reaction (MLC) of mixed lymphocytes, was used as a screening model to screen for the ability of different icariin glucosides to promote human ⁇ -interferon production.
- MLC drug-stimulated culture reaction
- PBMCs Peripheral blood mononuclear cells obtained by fresh isolation were adjusted to a cell concentration of 5 ⁇ 10 5 /ml with RPMI1640 complete medium.
- the lymphocytes of two healthy humans were mixed in equal volume to maintain a total cell concentration of 1 ⁇ 10 6 /ml, and then different icariin saponins of class I (with a terminal concentration of 1000 ng/ml) were added. And mix well.
- the stimulated cells were then cultured in a 96-well micro-round bottom culture plate at a cell concentration of 2 ⁇ 10 5 /ml (200 ul / well) per well, and cultured in a 37 ° C, 5% CO 2 incubator. At 120 h, the level of cytokine IFN- ⁇ production was measured.
- Figure 8 shows that IFN- ⁇ is increased after mixed culture of two normal human lymphocytes. After the addition of different icariin class I compounds (with a terminal concentration of 1000 ng/ml), there was a large difference in the level of IFN- ⁇ production in mixed lymphocyte culture. Explain that different compounds have different effects on cellular immunity.
- Epimedium I promote the production of human ⁇ -interferon in pleural effusion cells of tuberculosis patients, and improve the body's ability to resist tuberculosis infection.
- PBMCs peripheral blood mononuclear cells
- PFMCs pleural fluid cells
- Volunteers venous blood of tuberculosis patients
- heparin anticoagulation and Hank's solution were diluted in volume.
- Centrifuge once (1800 rpm, 22 ° C, 8 minutes), discard the supernatant, and resuspend by adding an appropriate amount of ELS (erythrocyte lysate).
- ELS erythrocyte lysate
- Hank's solution was added, and the mixture was centrifuged twice (1800 rpm, 22 ° C, 8 minutes), and the PBMC concentration was adjusted to 2 ⁇ 10 9 cells/L with RPMI1640 complete medium.
- PBMCs and cultured and stimulated PFMCs intracellular drug reactions Application of icariside I respectively 0.1 ⁇ g / ml, 0.5 ⁇ g / ml and 1 ⁇ g / ml PBMC were stimulated and PFMC, at 37 o C, 5% Incubate for 48 hours under CO 2 conditions.
- FIG. 9 shows that IFN- ⁇ production was not significantly promoted in PBMCs cells after stimulation with Icariin I. However, IFN- ⁇ production in PFMCs cells was significantly and in a dose-dependent manner.
- Tip Epimedium I can significantly promote the production of human ⁇ -interferon by PFMC cells (tuberculosis pleural cells) infected with tuberculosis. The results of this test are very clear: Epimedium I can improve the body's ability to resist tuberculosis infection, and has important application prospects in the immunotherapy of tuberculosis.
- mice were used for the experiment, and 18-22 g mice were divided into groups of 10 each.
- 0.2 ml of mouse S180 ascites tumor fluid was inoculated subcutaneously into the axilla of the mouse.
- each group of mice was intraperitoneally injected: (1) saline group; (2) solvent control group (DMSO); (3) positive control group [cyclophosphamide 60 mg/(kg ⁇ d) (4) low dose group 1: icariin I 2mg / (kg ⁇ d); (5) low dose group 2: icariin I 5mg / (kg ⁇ d); (6) Dosage group: icariin I10mg / (kg ⁇ d); (7) high dose group: icariin I 20mg / (kg ⁇ d).
- the mice were weighed the next day, the mice were sacrificed, and the tumors were weighed.
- the saline and solvent groups were used as controls. Calculate the tumor inhibition rate.
- mice The test used 4-6 weeks old BALB/c (nu/nu) nude mice, weighing 18-22 g, female, raised in SPF-level sterile breeding room, free to ingest food and water. Forty nude mice were modeled for tumor mass transplantation, and were regrouped into groups of 10 each when the tumor grew to the initial volume of administration (100 mm 3 ). Each group of nude mice was intraperitoneally injected: (1) a blank control group was injected with 0.1 ml/10 g of solvent (5% DMSO + 95% saline); (2) a positive drug group was injected [cyclophosphamide (0.6 mg).
- mice 18-22 g mice were randomly divided into 6 groups, 10 mice in each group, respectively, with normal saline, DMSO 2.5 ml/kg, icariin I 2000 mg/kg, 1000 mg/kg, 500 mg/kg, 100 mg/ Kg treatment (oral), observed for 14 days, results in no test mice died. It shows that the acute toxic LD50 value of icariin I in mice is more than 2000mg/kg. It has little acute toxicity and is safe to use. It can be used to prepare drugs or health products, foods and products for preventing and treating diseases related to human immunity. Beauty skin care products, etc.
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Abstract
Description
Claims (15)
- 一种如式I所示的淫羊藿次苷类化合物:式I中的R1,R2和R3所代表的基团如下面各项所定义:(1)R1选自H,OH,OCH3,CH3COO,CH3,CF3,NH2,CH3NH,CH3CONH,CN,Br,Cl,F;(2)R2选自H,OH,OCH3,CH3COO,NH2,CH3NH,(CH3)2N,CH3CONH,CN;(3)R3选自H,OH,OCH3,OC2H5,OC3H7,CH3COO,CH3,CF3,C1-C6-NH2(其中C1-C6为具有1-6个C的烷基、环烷基、烯烃基、环烯烃基,尤其是环戊基氨基、环己基氨基;以及***啉基,甲基哌嗪基),NH2,CH3NH,(CH3)2N,(CH3CH2)2N,CH3CONH,CN,Br,Cl,F,氨基酸酰基,氨基酸酰氨基(R’-CH-CONH-),寡肽酰基,寡肽酰氨基等。
- 权利要求1所述的式I的淫羊藿次苷类化合物,其中R1=OH,OCH3;R2=OH,OCH3;R3=OH,OCH3,OC3H7。
- 权利要求1或2所述的式I的淫羊藿次苷类化合物,其中所述化合物是:(1)R1=OH,R2=OH,R3=OH;(2)R1=OH,R2=OH,R3=OCH3;(3)R1=OH,OCH3,R2=OH,OCH3,R3=OH,OCH3,OC3H7;(4)R1=OCH3,R2=OH,R3=OCH3;(5)R1=OH,R2=OCH3,R3=OCH3;(6)R1=OH,,R2=OH,R3=OC3H7。
- 一种制备权利要求1-3任意一种式I的淫羊藿次苷类化合物的方法,其特征在于:以淫羊藿次苷I、淫羊藿次苷C等天然产物衍生化得到式I所示的淫羊藿类化合物的方法。
- 一种淫羊藿次苷I或淫羊藿次苷C的制备方法,其特征在于通过例如酶转化技术,将淫羊藿中含量丰富的成分淫羊藿苷转化成淫羊藿次苷I。
- 权利要求5所述的淫羊藿次苷I的制备方法,其特征在于以淫羊藿苷为原料,溶于磷酸盐缓冲溶液中,加入固定化鼠李糖苷酶反应并重结晶。
- 权利要求5所述的淫羊藿次苷C的制备方法,其特征在于以淫羊藿新苷A为原料,加入固定化鼠李糖苷酶反应将淫羊藿新苷A分子中的3-O-鼠李糖基除去,得到淫羊藿次苷C。
- 一种药物组合物,其含有式I淫羊藿次苷类化合物中的至少一种物质,例如淫羊藿次苷I、淫羊藿次苷C以及药学上可接受的载体和/或辅剂。
- 一种保健品组合物,其含有式I淫羊藿次苷类化合物中的至少一种物质,例如淫羊藿次苷I、淫羊藿次苷C以及保健领域可接受的载体和/或辅剂。
- 一种美容护肤品组合物,其含有式I淫羊藿次苷类化合物中的至少一种物质,例如淫羊藿次苷I、淫羊藿次苷C以及美容护肤领域可接受的载体和/或辅剂。
- 权利要求8-10的组合物,其可以是片剂、丸剂、胶囊、注射剂、悬浮剂、乳剂、外用涂抹剂、面膜。
- 权利要求1-3、8-10任意一种物质或组合物用于制备药物的用途,该药物用于治疗任何与人体免疫力有关的疾病,特别是能够通过促进人体细胞产 生γ-干扰素(interferon-gamma,IFN-γ),提高人体的免疫力相关的疾病的用途。
- 权利要求1-3、8-10任意一种物质或组合物用于制备药物的用途,该药物用于治疗任何与人体内TH1型的免疫应答(诱导细胞免疫应答)相关的疾病的用途。
- 权利要求12或13的制药用途,其中用于治疗的疾病为肺结核、流感、乙肝和丙肝等感染性疾病,以及肿瘤。
- 一种用于筛选具有增强人体免疫效果物质的实验模型。该模型系应用不同的外源抗原刺激人外周血单个核细胞(PBMCs),产生细胞因子IFN-γ,将待测药物加入到外源抗原与人外周血单个核细胞(PBMCs)的混合体系中,测定对IFN-γ产生水平的影响,以筛选具有提高人体免疫力或抑制人体免疫力的小分子化合物。
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US15/745,784 US20180201639A1 (en) | 2015-05-20 | 2015-06-29 | Icariside compound, preparation method thereof, and application thereof |
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