CN102653550A - Preparation and application of glycyrrhetinic acid - Google Patents
Preparation and application of glycyrrhetinic acid Download PDFInfo
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- CN102653550A CN102653550A CN2011100480397A CN201110048039A CN102653550A CN 102653550 A CN102653550 A CN 102653550A CN 2011100480397 A CN2011100480397 A CN 2011100480397A CN 201110048039 A CN201110048039 A CN 201110048039A CN 102653550 A CN102653550 A CN 102653550A
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Abstract
The invention belongs to the field of biotechnology, and particularly relates to preparation and an application of glycyrrhetinic acid. The technical solution scheme provided by the invention is that the preparation method of glycyrrhetinic acid comprises the following steps: firstly performing crude purification, then performing further preparation and purification to obtain a high-purity product; the glycyrrhetinic acid can be used for the preparation of anti-infection medicaments with antiviral, antibacterial, and immunocompetence-enhancing effects. The beneficial effects of the invention are that: the preparation method of glycyrrhetinic acid which is simple and convenient for preparation, water-soluble, and good in absorption is provided, and the application of the glycyrrhetinic acid in the preparation of anti-infection medicaments with antiviral, antibacterial, and immunocompetence-enhancing effects is provided.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the preparation and the purposes of glycyrrhetinic acid.
Background technology
Radix Glycyrrhizae (Glycyrrhiza uralensis Fisch) belongs to leguminous plants, grows in temperate zone and subtropical zone, and main product is western in China, is stated from the Han dynasty Shennong's Herbal as herbal medicine head commonly used.Theory of traditional Chinese medical science is thought: Radix Glycyrrhizae property is flat, flavor is sweet; Have and effects such as middle emergency, moistening lung, detoxifcation, cough-relieving, qualcomm meridian, sharp qi and blood, the staple of Radix Glycyrrhizae is Potenlini and glycyrrhetinic acid (GA), and glycyrrhetinic acid has another name called glycyrrhetinic acid; Record in the national drug standards, be white crystalline powder (C
30H
46O
6), fusing point 288-297 ℃, water insoluble.Glycyrrhetinic acid can suppress the mice ear that Oleum Tiglii brings out and the activity of ornithine decarboxylase (ODC), and it is synthetic to reduce the non-program DNA that strong carcinogen benzopyrene causes simultaneously, to by the dna damage that benzopyrene brought out the certain protection effect being arranged.
Immune basic function is the identification exogenous antigen and induces body to produce series reaction and remove the pathogenic material of external source, to keep the stable of body.Body is commonly considered as nonspecific to the primary immune response of virus and bacterium, yet the discovery of TLR has thoroughly changed this view.The main target molecule that U.S. immunologist Janeway etc. are discerned natural immunity cell is referred to as relevant molecular pattern (the Pathogen associated molecular patterns of cause of disease; PAMPs); PAMPs is the total a kind of conservative pattern molecules of numerous mikrobes; Extensively be present in the pathogen cells surface; Like the seminose on intracellular toxin, skin glycan, flagellin, double-stranded RNA and the yeast cells wall etc., the acceptor of identification PAMPs be called the pattern recognition acceptor (Pattern recognition receptors, PRRs).
Toll appearance acceptor (Toll-like recptors; TLRs) be one type of important pattern recognition acceptor (PRRs); Toll appearance acceptor extensively is present in insect, vertebrates and the plant; Through the specific pathogen-associated molecular mould (PAMPs) of identification pathogenic micro-organism, start the natural immunity and reply, constitute the first line of defence of body opposing pathogenic micro-organism invasion.So far, found 11 kinds of people TLRs and 13 kinds of mouse TLRs altogether, they respectively selectivity discern various PAMPs, and mediation activates signal paths such as nf kappa B (nuclear factor-kappa B, NF-κ B) and MARKS.Behind Toll appearance receptor-specific combination self part; Activate a series of albumen cascade reactions in downstream through MyD88 (marrow appearance differentiation factor) dependency or dependent/non-dependent approach (TRIF); Induce the synthetic of corresponding interleukin-(IL), tumour necrosis factor (TNF) and the Interferon, rabbit factors such as (IFN) and discharge, start the reaction of body anti-infectious immunity.Therefore; TLRs not only constitutes body anti-infectious immunity " the first line of defence "; Play an important role in replying starting the early stage natural immunity, more become " bridge " of the contact natural immunity and acquired immunity, the generation and the occurrence type of acquired immunity had important regulation.As stated, the status of Toll appearance acceptor in the anti-infective defensive raction of body is most important, and existing research shows, many Chinese medicines can improve body anti-infectious immunity power through number of ways or link.Infer identification and the kill capability of Chinese medicine enhance immunity system thus, and induce, regulate more effective immune defense reaction pathogenic agent, closely related with Toll appearance acceptor and relevant signaling molecule thereof, certainly existing natural contact between the two.Study of the influence of anti-infective Chinese medicine, estimate the using value of Toll appearance acceptor, and foundation is the cell model of the anti-infective Chinese medicine of screening of target with Toll appearance acceptor as the target molecules of the anti-infective Chinese medicine of screening to Toll appearance receptor active and downstream signaling molecule thereof.
Summary of the invention
The preparation and the purposes that the purpose of this invention is to provide a kind of glycyrrhetinic acid.Technical solution provided by the present invention is: a kind of preparation of glycyrrhetinic acid, this method are to carry out thick purifying earlier, further prepare and purifying again; Obtain high purity product, at first remove impurity composition and come the thick Potenlini of purifying, the Radix Glycyrrhizae crude extract is used the ethanol heat of solution with the ADS-10 absorption with macroporous adsorbent resin; Add ammoniacal liquor and transfer pH8-9, behind the concentrated recovery ethanol, residuum is dissolved in water; With the impurity composition in the ADS-10 macroporous adsorptive resins adsorbent solution, and water-soluble ammonium glycyrrhizunate will adsorb effluent to transfer pH1-2 with hydrochloric acid not by resin absorption with this solution; Heating hydrolysis, after the solution cooling, the solids of separating out obtains thick glycyrrhetinic acid after filtering; Secondly, be the preparation and the purifying of glycyrrhetinic acid, thick glycyrrhetinic acid is used organic solvent dissolution; Leach insoluble impurities, use buck that the glycyrrhetinic acid alkalization is the glycyrrhetinic acid sodium salt, make it to separate with non-water-soluble impurity; Make it become the glycyrrhetinic acid that is dissolved in organic solvent with acidifying then, separate with water-soluble impurity, twice separation through such can obtain high purity product again behind twice recrystallization; The purposes of glycyrrhetinic acid, glycyrrhetinic acid can be used for preparation and have antiviral, bacterium, the active anti-infectives of enhancing immunity.Beneficial effect of the present invention: provide the preparation simple, convenient, good water solubility, the glycyrrhetinic acid preparation method of good absorption, and provide glycyrrhetinic acid to have the application on antiviral, bacterium, the active anti-infectives of enhancing immunity in preparation.
Embodiment
The preparation of this glycyrrhetinic acid is to carry out thick purifying earlier, further prepares and purifying again, obtains high purity product.At first remove impurity composition and come the thick Potenlini of purifying, the Radix Glycyrrhizae crude extract is used the ethanol heat of solution, add ammoniacal liquor and transfer pH8-9 with the ADS-10 absorption with macroporous adsorbent resin; After concentrate reclaiming ethanol, the residuum that is dissolved in water, with this solution with the impurity composition in the ADS-10 macroporous adsorptive resins adsorbent solution; And water-soluble ammonium glycyrrhizunate will adsorb effluent to transfer pH1-2 with hydrochloric acid not by resin absorption, heating hydrolysis; After the solution cooling, the solids of separating out obtains thick glycyrrhetinic acid after filtering, secondly; Be the preparation and the purifying of glycyrrhetinic acid, thick glycyrrhetinic acid is used organic solvent dissolution, leach insoluble impurities; Use buck that the glycyrrhetinic acid alkalization is the glycyrrhetinic acid sodium salt, make it to separate with non-water-soluble impurity; Make it become the glycyrrhetinic acid that is dissolved in organic solvent with acidifying then, separate with water-soluble impurity, twice separation through such can obtain high purity product again behind twice recrystallization.
The checking glycyrrhetinic acid can be used for preparation and has antiviral, bacterium, the active anti-infectives of enhancing immunity.
(1) glycyrrhetinic acid is dissolved with DMSO, its storage liquid final concentration is 20mg/ml.
(2) test is with the preparation of cell: will test with the strain of Ana-1 mouselet scavenger cell; RAW264.7 mouselet scavenger cell leukemia cell line; 293T human embryonic kidney cell line (all from Shanghai cell biological institute of Chinese Academy of Sciences cell bank) is respectively with 1640 and the DMEM substratum (100U/ml penicillium mould, Streptomycin sulphate) that contain 10%FBS; 37 ℃, 5%CO
2, incubator is cultivated, the cultivation of going down to posterity when cell was in logarithmic phase in 2-3 days.
(3) Toll appearance acceptor (Toll-like receptors; TLRs) and the transferring of downstream signaling molecule gene: select downstream molecules MyD88 and TRIF and IL-6, TNF-α and INF-β that Toll appearance acceptor (TLR2,3,4,7,9) amounts to 5 Toll appearance acceptor molecules and Toll appearance acceptor as molecule to be checked; And respectively its portion gene sequence has been carried out primer design, synthetic and evaluation, for Real-Time PCR prepares.
(4) MTT toxicity trial: the Ana-1 cell is spread 95 orifice plates, 10
4Individual cells/well (200ul), test is established the Chinese medicine infected group, DMSO negative control group, zeroing group; Every group establish 6 infective dose gradients (be 80ug/ml, 60ug/ml, 40ug/ml, 20ug/ml; 10ug/ml, 5ug/ml), each gradient is established 3 repetitions, treats that the medicine cells infected is after 24 hours; Every hole adds the MTT20ul of 5mg/ml, and after 4 hours, every hole adds the DMSO dissolving viable cell of 150ul and the purple crystal that MTT reaction back produces, and selects the 570nm wavelength in vibration dissolving fully to be crystallized in 10 minutes back in 37 ℃ of reactions; On the enzyme linked immunological monitor, measure each hole absorbance value, draw the inhibiting rate of drug level pair cell, confirm the optimal dose of Chinese medicine cells infected.
(5) Real-Time PCR detects Chinese medicine to the influence of Toll appearance acceptor molecule on the mRNA level: primary dcreening operation Chinese medicine; Detect Chinese medicine through Real-Time PCR Toll appearance acceptor molecule is detected Chinese medicine to the influence of cytokines such as IL-6, TNF-α and INF-β, the expression that Western Blot detects Toll appearance receptor protein at the influence on the mRNA level, Real-Time PCR detection Chinese medicine to Toll appearance acceptor molecule downstream molecules MyD88 and influence, the Real-Time PCR of TRIF on the mRNA level, filter out the herbal species that after this Chinese medicine stimulates, has high expression level.
(6) the two luciferase reporter gene systems measurement Chinese medicines of utilization detect Chinese medicine to Toll appearance acceptor molecule downstream molecules MyD88 and the influence of TRIF on the mRNA level to its downstream NF-KB and INF:Real-Time PCR after stimulating Toll appearance acceptors; Real-Time PCR detects the influence of Chinese medicine to cytokines such as IL-6, TNF-α and INF-β; Western Blot detects the expression of Toll appearance receptor protein.
(7) the reporter gene transcriptional activity detects, and the signal path of checking mTLR9 mediation is to the influence of downstream transcription factor NF-KB transcriptional activity.
Show that through above operating result glycyrrhetinic acid can obviously improve the level of the mRNA of MyD88 and TRIF, significantly improves the level of the mRNA of IL-6, show glycyrrhetinic acid can be used for preparing have antiviral, bacterium, the active anti-infectives of enhancing immunity.
Claims (2)
1. the preparation of a glycyrrhetinic acid is characterized in that this method is to carry out thick purifying earlier, further prepares and purifying again; Obtain high purity product, at first remove impurity composition and come the thick Potenlini of purifying, the Radix Glycyrrhizae crude extract is used the ethanol heat of solution with the ADS-10 absorption with macroporous adsorbent resin; Add ammoniacal liquor and transfer pH8-9, behind the concentrated recovery ethanol, residuum is dissolved in water; With the impurity composition in the ADS-10 macroporous adsorptive resins adsorbent solution, and water-soluble ammonium glycyrrhizunate will adsorb effluent to transfer pH1-2 with hydrochloric acid not by resin absorption with this solution; Heating hydrolysis, after the solution cooling, the solids of separating out obtains thick glycyrrhetinic acid after filtering; Secondly, be the preparation and the purifying of glycyrrhetinic acid, thick glycyrrhetinic acid is used organic solvent dissolution; Leach insoluble impurities, use buck that the glycyrrhetinic acid alkalization is the glycyrrhetinic acid sodium salt, make it to separate with non-water-soluble impurity; Make it become the glycyrrhetinic acid that is dissolved in organic solvent with acidifying then, separate with water-soluble impurity, twice separation through such can obtain high purity product again behind twice recrystallization.
2. the purposes of a glycyrrhetinic acid is characterized in that: glycyrrhetinic acid can be used for preparation and has antiviral, bacterium, the active anti-infectives of enhancing immunity.
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| CN2011100480397A CN102653550A (en) | 2011-03-01 | 2011-03-01 | Preparation and application of glycyrrhetinic acid |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004046A (en) * | 2014-05-27 | 2014-08-27 | 普凡生生物科技(北京)有限公司 | Preparation method of acetyl glycyrrhetinic acid |
| CN104004047A (en) * | 2014-05-27 | 2014-08-27 | 普凡生生物科技(北京)有限公司 | Preparation method of glycyrrhetinic acid |
| CN104829681A (en) * | 2015-05-08 | 2015-08-12 | 江苏天晟药业有限公司 | Method for purifying glycyrrhetinic acid |
| CN110283225A (en) * | 2019-07-29 | 2019-09-27 | 洛阳蓝斯利科技有限公司 | A kind of preparation method of 24- hydroxyl-enoxolone |
| WO2022078563A1 (en) * | 2020-10-13 | 2022-04-21 | Elsersy Hazem Ezzat | A new mixed compound for treatment and protection from covid-19 (corona) virus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563073A (en) * | 2004-04-06 | 2005-01-12 | 南开大学 | Method for preparing enoxolone |
| CN1948332A (en) * | 2005-10-14 | 2007-04-18 | 天津药物研究院 | Glycyrrhetinic acid-30-acylamide derivatives and its use |
-
2011
- 2011-03-01 CN CN2011100480397A patent/CN102653550A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563073A (en) * | 2004-04-06 | 2005-01-12 | 南开大学 | Method for preparing enoxolone |
| CN1948332A (en) * | 2005-10-14 | 2007-04-18 | 天津药物研究院 | Glycyrrhetinic acid-30-acylamide derivatives and its use |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004046A (en) * | 2014-05-27 | 2014-08-27 | 普凡生生物科技(北京)有限公司 | Preparation method of acetyl glycyrrhetinic acid |
| CN104004047A (en) * | 2014-05-27 | 2014-08-27 | 普凡生生物科技(北京)有限公司 | Preparation method of glycyrrhetinic acid |
| CN104829681A (en) * | 2015-05-08 | 2015-08-12 | 江苏天晟药业有限公司 | Method for purifying glycyrrhetinic acid |
| CN110283225A (en) * | 2019-07-29 | 2019-09-27 | 洛阳蓝斯利科技有限公司 | A kind of preparation method of 24- hydroxyl-enoxolone |
| CN110283225B (en) * | 2019-07-29 | 2021-02-02 | 洛阳蓝斯利科技有限公司 | Preparation method of 24-hydroxy-glycyrrhetinic acid |
| WO2022078563A1 (en) * | 2020-10-13 | 2022-04-21 | Elsersy Hazem Ezzat | A new mixed compound for treatment and protection from covid-19 (corona) virus |
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Application publication date: 20120905 |