CN105853444B - Application of 20 (the R)-panaxatriol derivatives in preparation prevention or treatment liver disease drug - Google Patents

Application of 20 (the R)-panaxatriol derivatives in preparation prevention or treatment liver disease drug Download PDF

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CN105853444B
CN105853444B CN201610334437.8A CN201610334437A CN105853444B CN 105853444 B CN105853444 B CN 105853444B CN 201610334437 A CN201610334437 A CN 201610334437A CN 105853444 B CN105853444 B CN 105853444B
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panaxatriol
derivatives
application
hepatic fibrosis
compound
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CN105853444A (en
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陈广通
宋妍
李建林
葛红娟
林海君
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Jiusan Jiheng Biomedical Technology Jiangsu Co ltd
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Nantong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/02—Dehydrogenating; Dehydroxylating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/20—Preparation of steroids containing heterocyclic rings

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Abstract

The invention discloses a kind of 20 (R)-panaxatriol derivatives or its pharmaceutically can at salt preparation prevent or treatment liver disease drug in application.The present invention utilizes microbiological transformation technology, structural modification has successfully been carried out to 20 (R)-panaxatriols, obtain various new compound, it is confirmed by Against Hepatic Fibrosis in Vitro test cell line, these compounds have preferable anti-hepatic fibrosis activity, it can be used as the active constituent of anti-hepatic fibrosis medicines, tool has been widely used.

Description

Application of 20 (the R)-panaxatriol derivatives in preparation prevention or treatment liver disease drug
Technical field
The present invention relates to field of medicaments, and in particular to 20 (R)-panaxatriol derivatives are in preparation prevention or treatment drug for liver disease Application in object more particularly relates to 20 (R)-panaxatriol derivatives in preparation and prevents and treats answering in hepatic fibrosis medicines With.
Background technique
Liver fibrosis is after leading to hepatic injury by various pathogenic factors, and hepatic stellate cells, which is activated to become, has very strong life At the myofibroblastic-like cell of extracellular matrix ability, then collagen enzyme inhibitor generation increases, and collagenase activity also declines, most Leading to the over-deposit of Liver Collagen eventually becomes liver fibrosis.Liver fibrosis, which continues to deteriorate, leads to cirrhosis even liver cancer, so " hepatitis-liver fibrosis-cirrhosis-liver cancer " this chain is acknowledged as various chronic liver diseases leads to the common way of serious consequence Diameter.Research so far is it has been confirmed that liver fibrosis can be reversed by treatment appropriate, as long as can slow down or prevent liver The generation of fibrosis, so that it may mitigate or cure hepar damnification pathogenesis.Therefore, the activation for inhibiting hepatic stellate cells is treatment The most effective approach of liver fibrosis.
China has carried out the research of Liver Fibrosis with Traditional Chinese Medicine extensively in recent years, illustrates natural drug for liver fibrosis The great potential for the treatment of.Therefore, continue that the anti-hepatic fibrosis medicines of high-efficiency low-toxicity are found and developed from natural drug to be still medicine The important research direction of worker.20 (R)-panaxatriols (PT) are the primary products after ginsenoside sour water solution, tool There is preferable anti-tumor activity.But 20 (R)-panaxatriols belong to tetracyclic triterpenoid, lack reactive group, reaction Site is few, is difficult to prepare the derivative met the requirements using conventional chemical reaction method.
Microorganism conversion is the enzymic catalytic reaction carried out using the enzyme system of organism autospecific, the type of reaction It is more, and there is High level of stereoselectivity and regioselectivity, become an important tool in organic synthesis.It is used for 20 (R)-people The derivative preparation for joining triol, can provide great amount of samples for the follow-up study of the type compound.
Summary of the invention
The object of the present invention is to provide 20 (R)-panaxatriol derivatives or its pharmaceutically can at salt preparation prevention or The application in liver disease drug is treated, the hepatopathy is disease related with liver fibrosis, such as chronic hepatitis, cirrhosis or liver cancer. The drug is anti-hepatic fibrosis medicines.20 (R)-panaxatriol derivative is the compound that structural formula is I-XII:
Compound I-XII is 20 first public (R)-panaxatriol new derivatives of the present invention.
The present invention also provides the preparation method of above-mentioned 20 (R)-panaxatriol derivatives, include the following steps: 1) to ferment Microorganism is cultivated, 20 (R)-panaxatriols are added into culture medium, then carries out conversion culture, is fermented after removing mycelium Liquid, the microorganism are that colter is mould (Absidia), Cunninghamella sp (Cunninghamella), Mucor (Mucor), rod method (Alternaria), rhodotorula (Rhodotorula), mould (Syncephalastrum) or head mold (Rhizopus) belong to altogether Bacterial strain；2) by the fermentation liquid after extracting, it is evaporated extract liquor, obtains conversion crude extract；3) by the conversion crude extract with silicon Plastic column chromatography is collected using methylene chloride-dehydrated alcohol system and is merged component；4) by component RP-HPLC color Spectrum purifying, obtains product.
Wherein, microorganism is preferably that Mucor (Mucor) belongs to bacterial strain, more preferable Mucor racemosus.Training in above method step 1) The concentration for supporting 20 (R)-panaxatriols in base is 2-2000 μ g/mL.
Extractant is conventional type organic solvent, ethyl acetate in above method step 2).
Above method step 4) preferably reversed-phase high performance liquid chromatography preparation condition is half preparation chromatographic column YMC-Pack OSD-A, 5 μm, 12.0 × 250mm (Japanese YMC), acetonitrile-water (60:40, V/V), flow velocity 3.0mL/min, Detection wavelength 203nm。
The present invention experiments prove that, 20 (R)-panaxatriol derivative of the present invention have good anti-hepatic fibrosis Activity can be used as the active constituent of anti-hepatic fibrosis medicines.
The active constituent of these anti-hepatic fibrosis medicines can be the change selected from 20 (R)-panaxatriols and structural formula I-XII Close one or more of object.
Can also be added in the drug using above compound as active constituent, when needs it is one or more pharmaceutically Acceptable carrier.The carrier includes the diluent of pharmaceutical field routine, excipient, filler, adhesive, wetting agent, collapses Agent, sorbefacient, surfactant, absorption carrier, lubricant etc. are solved, it can be according to the conventional method system of pharmaceutical field It is standby.
The present invention utilizes microbiological transformation technology, has successfully carried out structural modification to 20 (R)-panaxatriols, has obtained A new class of 20 (R)-panaxatriol derivative confirmed by interior animal experiment and Against Hepatic Fibrosis in Vitro test cell line, this A little compounds have preferable anti-hepatic fibrosis activity, can be used as the active constituent of anti-hepatic fibrosis medicines, have extensive Purposes.
Detailed description of the invention
Fig. 1 is the HPLC map of compound I-XII of the present invention.
Specific embodiment
The preparation of embodiment 1, the compound that structural formula is I-XII
The present invention uses microbial conversion process, using 20 (R)-panaxatriols as raw material, through everfermentation, extracting and developing etc. Step, to prepare the compounds of this invention.Microorganism with conversion capability includes: that colter is mould (Absidia), Cunninghamella sp (Cunninghamella), Mucor (Mucor), rod method (Alternaria), rhodotorula (Rhodotorula), total mould (Syncephalastrum) or head mold (Rhizopus) belong to microorganism；It is that colter is mould that wherein conversion capability is stronger (Absidia) and Mucor (Mucor) belong to bacterial strain.These bacterial strains can be purchased from the management of Chinese Academy of Sciences's Microbiological Culture Collection Center (CGMCC) or Chinese Food Fermentation Research Institute Industrial Microorganism Preservation Administrative Center (CICC), in solid slope culture medium On set in 4 DEG C of refrigerators and save.Fungi culture medium selects potato culture, and bacteria culture media selects LB culture medium.
The preparation (PDA culture medium) of potato culture: taking 200g peeled potatoes, thinly slice, be put into suitable quantity of water, 80 DEG C of heat preservation 1h after boiling.Filtrate is taken after being filtered with double gauze, and 20g glucose is added, stirring is completely dissolved glucose, with Water is settled to 1000mL.It prepares solid slope culture medium and 3% agar is added in liquid medium again.
The preparation (LB culture medium) of bacteria culture media: 5.0g yeast extract, 10.0g is added in every 1000mL fluid nutrient medium Peptone, 10.0g NaCl, is dissolved in water, and adjusts pH value to 7.0.Solid slope culture medium is prepared to be added in liquid medium again 1.5% agar.
By taking Mucor racemosus Mucor racemosus AS 3.205 as an example, preparation structure formula is the mistake of the compound of I-XII Journey is as follows:
1) it ferments, convert and extracts
Mucor racemosus Mucor racemosus AS 3.205 is accessed into 2 250mL triangular flasks and (100mL potato is housed Culture medium) in, as seed liquor.At 160rpm on shaking table, 26 DEG C after shaken cultivation 1 day, it is in animated period to mycelia growth, The seed liquor that 1mL is drawn with Sterile pipette is added in 20 1000mL shaking flasks (equipped with 400mL potato culture).Vibration After swinging culture 1 day, 25mg 20 (R)-panaxatriol (0.2mL, 125mg/mL ethanol solution) is added in each shaking flask, altogether With 500mg substrate.Continue conversion 7 days under the same terms, by filtering fermentation liquor, filters out mycelium, the isometric acetic acid of filtrate Ethyl ester extracts 3 times, and extract liquor is concentrated to dryness, and obtains conversion product crude extract about 1.6g.
2) silica gel column purification
Gained crude extract is dissolved in a small amount of methanol, mixes with 1.6g column chromatography silica gel (200-300 mesh) and mixes sample, it is naturally dry It is dry, the chromatography capital equipped with 50g silica gel (200-300 mesh) is added to, with methylene chloride-dehydrated alcohol system gradient elution (50:1- 1:8), collect elution fraction, using TLC analysis method (silica gel g thin-layer plate, methylene chloride-dehydrated alcohol (15:1) expansion, 10% ethanol solution of sulfuric acid is spraying, heating colour developing) obtained similar elution fraction is merged.
3) reversed-phase high performance liquid chromatography purifies
Merge component to be purified with reversed-phase high performance liquid chromatography.Preparation condition is half preparation chromatographic column YMC-Pack OSD- A, 5 μm, 12.0 × 250mm (Japanese YMC), acetonitrile-water (60:40, V/V), flow velocity 3.0mL/min, Detection wavelength 203nm.? 12 converted products for being I-XII to structural formula, as shown in Figure 1.Its13C-NMR data are as shown in table 1.
1. Formulas I of table, Formula II, formula III, formula IV, Formula V, Formula IV, Formula VII, Formula VIII, Formula IX, Formula X, Formula XI and Formula XII Carbon modal data (CDCl3)
The above result shows that gained compound structure is correct.
It is specific such as mucor spinosus Mucor spinosus AS 3.3450, Rhizopus arrhizus using the microorganism that other belong to Rhzopus arrhizus AS 3.3457, top spore Acremonium strictum AS 3.2059, can use more than Identical process come be prepared structural formula be Formulas I, Formula II, formula III, formula IV, Formula V, Formula IV, Formula VII, Formula VIII, Formula IX, formula X, the compound of Formula XI and Formula XII.
The anti-hepatic fibrosis activity of 2 the compounds of this invention I-XII of embodiment
1) experimental material
Instrument and reagent: CO2Incubator (Jouan IGO150)；Microplate reader (Bio-TEK ELx800)；Fluorescence is inverted aobvious Micro mirror (Olympus IX51)；MTT cell Proliferation and citotoxicity detection kit (green skies biotechnology research institute), RPM 1640 culture medium of I (Gibcol BRL), Rnase A, fetal calf serum, dimethyl sulfoxide (DMSO), trypsase (upper marine growth Engineering Co., Ltd).
Test cell strain: HSC-T6 cell strain is purchased from Chinese medicine section for the SD cultured rat hepatic stellate cells of SV40 transfection Institute of oncology, institute.
Test sample: compound I-XII, purity is 90% or more, positive reference substance colchicin, each compound with It is diluted after DMSO dissolution.
2) experimental method
Each test-compound is measured to the half inhibiting rate IC of HSC-T6 cell strain using mtt assay50Value: logarithmic growth phase HSC-T6 cell, with culture solution containing 1%DMEM adjustment cell concentration be 5 × 105/ mL, is inoculated in 96 well culture plates, at drug Every 100 μ L cell suspension of hole is added in reason group and cell controls group, and every group sets 3 multiple holes, and full culture medium is only added in blank control group, Every 100 μ L of hole, if 3 multiple holes.96 well culture plates are placed in 37 DEG C, 5%CO2After incubator culture for 24 hours, various concentration is added Given the test agent makes final concentration of 0..1-100 μM, continues to cultivate 72h.By mtt assay in microplate reader, the absorbance of 450nm is measured (A) value calculates inhibiting rate [inhibiting rate=(1- experimental group A value/control group A value) × 100%].Experiment is repeated 3 times.Using SPSS 11.5 softwares make regression equation, calculate each given the test agent to the half-inhibitory concentration (IC of HSC-T6 cytosis 72h50)。
3) experimental result
According to mtt assay test result, the compounds of this invention I-XII is calculated to the IC of above-mentioned cell50Value, as a result such as 2 institute of table Show.
Inhibiting effect of 2. test sample of table to HSC cell Proliferation
Compound HSC-T6 IC50 value(μM)
Colchicin 0.18
Compound I 9.4
Compound II 10.3
Compound III 7.2
Compound IV 4.8
Compound V 3.1
Compound VI 12.4
Compound VII 14.8
Compound VIII 20.6
Compound IX 15.3
Compound X 11.4
Compound XI 17.8
Compound XII 21.5
The result shows that 20 (R)-panaxatriol (PT) derivative I-XII of the invention are living with good anti-hepatic fibrosis Property, it can be used as the active constituent of anti-hepatic fibrosis medicines.

Claims (4)

1. with following structural 20 (R)-panaxatriol derivatives or its pharmaceutically can at salt preparation prevent or treatment Application in liver disease drug, the hepatopathy are disease related with liver fibrosis,
2. application as described in claim 1, it is characterised in that the hepatopathy is chronic hepatitis, cirrhosis or liver cancer.
3. application as claimed in claim 1 or 2, it is characterised in that the drug is anti-hepatic fibrosis medicines.
4. application as described in claim 1, it is characterised in that the drug contains as described in claim 1 20 (R)-ginsengs three 01 derivatives or its pharmaceutically can at one or more of salt and pharmaceutically acceptable carrier.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101012473A (en) * 2006-05-26 2007-08-08 沈阳药科大学 Smooth blue mold or myrothecium verrucaria and method for preparing panax ginseng saponin F1 by using same
CN104352505A (en) * 2014-11-14 2015-02-18 南通大学 Applications of protopanaxatriol and derivatives thereof in preparation of medicines for treating hepatic disease
CN105017368A (en) * 2015-06-26 2015-11-04 南通大学 Panaxadiol derivatives, and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101012473A (en) * 2006-05-26 2007-08-08 沈阳药科大学 Smooth blue mold or myrothecium verrucaria and method for preparing panax ginseng saponin F1 by using same
CN104352505A (en) * 2014-11-14 2015-02-18 南通大学 Applications of protopanaxatriol and derivatives thereof in preparation of medicines for treating hepatic disease
CN105017368A (en) * 2015-06-26 2015-11-04 南通大学 Panaxadiol derivatives, and preparation method and application thereof

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