CN106279332B - Triterpene compound Antcin K and its liver-protecting activity and application - Google Patents
Triterpene compound Antcin K and its liver-protecting activity and application Download PDFInfo
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- CN106279332B CN106279332B CN201610619215.0A CN201610619215A CN106279332B CN 106279332 B CN106279332 B CN 106279332B CN 201610619215 A CN201610619215 A CN 201610619215A CN 106279332 B CN106279332 B CN 106279332B
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- 230000004044 response Effects 0.000 description 1
- 230000008458 response to injury Effects 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 239000010979 ruby Substances 0.000 description 1
- 229910001750 ruby Inorganic materials 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010563 solid-state fermentation Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000007863 steatosis Effects 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses triterpene compound Antcin K and its liver-protecting activity and applications.The triterpene compound Antcin K is extracted from the Antrodia camphorata fructification of wild, linden culture and/or plate culture, various active screening verification its with protection CCl outstanding4Caused by oxyhepatitis hepatic injury activity, and the activity of the alcohol-induced dyslipidemias accumulation of protection.There is significant preventive and therapeutic effect to oxyhepatitis and hepatic injury and alcoholic fatty liver based on the compound, can be widely applied in hepatic and health care product.
Description
Technical field
The present invention relates to the extraction separation methods of Antrodia camphorata triterpene compound Antcin K and the compound as guarantor
The new medicine use of liver drug, and in particular to it is protecting acute liver damage caused by oxyhepatitis, protection alcoholic fatty liver
In application.
Background technique
Antrodia camphorata (Antrodia camphorata, Antrodia cinnamomea) also known as antrodia, cinnamomum kanahirai hay mushroom, camphor tree mushroom,
Mushroom in camphor tree, blood ganoderma lucidum, mushroom, red antrodia, the raw Antrodia of camphor tree, red camphor tree orphan etc. in camphor tree cave, parasitize for a kind of TaiWan, China is distinctive
Rare fungi in cinnamomum kanehirai.Antrodia camphorata is most reported early in nineteen ninety, generally believes that Antrodia camphorata belongs to mycota at present
(Fungi), load door (Basidiomycota), Basidiomycetes (Homobasidiomyeetes), Aphyllophorales
(Aphyllophorales), Polyporaceae (Polyporaeeae), antrodia karst (Antrodia).It is extensive in history
It as anticancer, liver protection, removing toxic substances, antidiarrheal, while being also reported and has the effects that strengthen immunity, anti-oxidant, anti-inflammatory, claimed
For " ruby of Taiwan forest ".The ingredient of Antrodia camphorata is varied, and Antrodia camphorata from various sources is real so far
Isolated more than 230 kinds compounds in body and mycelium, macromolecular include protein, nucleic acid, polysaccharide etc., and small molecule includes three
Terpene, steroidal, phenyl ring derivative etc..
Liver is intraperitoneal maximum parenchymatous organ, undertakes the important physiological function of human body.Hepatic injury clinically compared with
It is common, is always one of the object of medical science primary study.Cause the factor of hepatic injury very much, including drug (chemotherapeutic,
Analgesic-antipyretic, antiviral agent etc.), chemical substance (aflatoxin, four chlorinations in the Nature and human industry's production process
Carbon, dimethylformamide etc.), viral (hepatitis A virus, hepatitis type B virus etc.) and alcohol etc..Hepatic injury will lead to liver
Dirty different degrees of necrosis of liver cells, steatosis, cirrhosis even liver cancer.Therefore, preventing and treating hepatic injury is clinically
One of important link of liver disease is to inhibit the Occurrence and development of disease such as liver fibrosis, hepatonecrosis, cirrhosis and liver cancer
Basis.Clinically various hepatics are many kinds of, but expensive or toxic side effect mostly, to make their use
It is restricted.Thus, it is found that effective Novel liver protection drug still has important practical significance and good application prospect.
United States Patent (USP) US20030113297A1 discloses a kind of antrodia mycelia liquid fermentation method and its product bacterium
The effect of the protection acute liver damage of filament and the mixture of Antrodia camphorata fructification, but research object Antrodia camphorata of this discovery
Entity source is indefinite, does not also provide its finger-print and its main component type.Chinese patent CN101785793A is disclosed
The mitigation tetrachloro-methane induction of the Antrodia camphorata capsule (the Antrodia camphorata extract that its content is solid state rheology) of Shan Shengsheng skill company
Chronic hepatitis effect, the finger-print of without proper notice Antrodia camphorata extracting process and used capsule in this discovery, also it is unknown its
In main component.Also document report liquid fermentation antrodia mycelia extract has the hepatic injury of tetrachloro-methane induction
Protective effect (Song TY etc., J Agr Food Chem 2003,51,1571-1577；Hsiao G etc., J Agr Food
Chem 2003,51,3302-3308；Lin WC etc., World J Gastroentero 2006,12,2369-2374.).But
It is that existing patent and document are mostly for the liquid or solid state fermentation mycelia for being easier to obtain to the hepatoprotective effect of Antrodia camphorata extract
The research that body carries out.
The antrodia mycelia that liquid fermentation obtains has the shortcomings that triterpene isoreactivity component content is low, with wild Antrodia camphorata
And the Antrodia camphorata ingredient that plate culture, linden culture obtain differs greatly, it is understood that there may be completely different active constituent.Due to medicine
Material is rare, and the liver protection pharmacology activity research and report of wild, linden culture Antrodia camphorata fructification are restricted, same with this
When, as a kind of emerging high-quality artificial Antrodia camphorata cultural method, the pharmacological activity for the Antrodia camphorata that plate culture obtains is not yet
Sufficiently studied.Although the research about Antrodia camphorata hepatoprotective effect receives significant attention, wherein the change with pharmacological activity
It studies point and its mechanism of action not yet illustrates completely.
Summary of the invention
The present invention, which is directed to, at present lacks wild, linden culture and the research of plate culture Antrodia camphorata fructification liver-protecting activity,
The case where also more limiting to liver-protecting activity ingredient in Antrodia camphorata and Mechanism Study overcomes existing technology and resource to limit to, leads to
It crosses and separation is extracted to plate culture, linden culture or wild Antrodia camphorata fructification, the ox with a variety of liver-protecting activities is provided
Antrodia bioactive triterpene class compound.
The present invention obtains bioactive triterpene class compound after the Antrodia camphorata fructification to particular source extracts separation
Antcin K, and its a variety of liver-protecting activity is tested.
The structural formula of the Antrodia camphorata bioactive triterpene class compound Antcin K is as follows:
The triterpene compound Antcin K includes its two epimer 25S-Antcin K and 25R-
Mixture Antcin K (structure is as follows) and its formed with arbitrary proportion.
Triterpene compound Antcin K of the invention is from the Antrodia camphorata of wild, linden culture and/or plate culture
It is extracted in entity, preferably will carry out silicagel column with the resulting extract of the fatty alcohol extracting Antrodia camphorata fructification of C1~C5
Separation and chromatographic separation and purification, obtain the component containing different triterpene compounds.
Specific extraction process, which may is that, crushes the Antrodia camphorata fructification of wild, linden culture and/or plate culture,
Total medicinal extract is concentrated under reduced pressure to obtain in dehydrated alcohol heating and refluxing extraction, extracting solution, and total medicinal extract is suspended with water, and petroleum ether extraction discards stone
Oily ether moiety；Water phase is extracted with ethyl acetate again；Ethyl acetate layer merging is evaporated, silica gel post separation, petroleum ether-ethyl acetate
System elutions elute fraction through chromatographic separation and purification and obtain 25S-Antcin K and 25R-Antcin K.
The present invention has studied it in cell after extracting separation to Antrodia camphorata bioactive triterpene class compound Antcin K
CCl is protected in horizontal and animal body4Caused by oxyhepatitis hepatic injury activity and its protect alcohol-induced dyslipidemias product
Tired activity, it, which has, as the result is shown reduces CCl by anti-inflammatory activity4Caused by oxyhepatitis hepatic injury act on, reduce liver damage
Transaminase caused by hurting increases, and alcohol is inhibited to lead to the liver-protecting activity of lipid accumulation in liver cell.
The Antrodia camphorata bioactive triterpene class compound Antcin K of the present invention can be used for prepare have hepatoprotective effect drug and/
Or health care product, various dosage forms, such as tablet, granule, powder, capsule or pill dosage form can be used in terms of dosage form.
Drug and/or health care product provided by the present invention with hepatoprotective effect contains the triterpene compound Antcin
At least one of K and its pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug.It is described
Drug and/or health care product with hepatoprotective effect are following function (1) and/or (2): 1) prevent and/or treat oxyhepatitis and
Hepatic injury；2) prevent and/or treat alcoholic fatty liver.The oxyhepatitis and hepatic injury are usually chemically Hepatoxic substance
Caused by oxyhepatitis and hepatic injury, such chemical substance may be alcohol, chemical toxicant and some drugs in environment.
Bioactive triterpene class compound of the present invention or its pharmaceutically acceptable salt, ester, solvate, alloisomerism
Body, tautomer, prodrug and their mixture also belong to of the invention for the drug or health care product of effective component preparation
Protection scope.
When needs, one or more pharmaceutically acceptable carriers can also be added in said medicine or health care product
Or auxiliary material.The carrier or auxiliary material include the diluent of pharmaceutical field routine, excipient, filler, adhesive, wetting agent, collapse
Solve agent, sorbefacient, surfactant, absorption carrier, lubricant etc..
Utilize above-mentioned reactive compound or its pharmaceutically acceptable salt, ester, solvate, stereoisomer, mutually variation
Structure body and prodrug are used alone or in combination as active constituent, or prepare into various doses with other medicines, auxiliary material etc.
The diversified forms such as type, including but not limited to tablet, powder, pill, injection, capsule, film, suppository, paste, electuary.On
The drug for stating various dosage forms can be prepared according to the conventional method of pharmaceutical field.
The present invention isolates and purifies out Antrodia camphorata triterpene compound Antcin K, and passes through various active screening verification, hair
Now the compound has protection CCl outstanding4Caused by oxyhepatitis hepatic injury activity, and the alcohol-induced lipid of protection is different
The activity often accumulated.There is significant preventive and therapeutic effect to oxyhepatitis and hepatic injury and alcoholic fatty liver based on the compound,
It can be widely applied in hepatic and health care product.
Detailed description of the invention
The hydrogen that Fig. 1 is 25S-Antcin K is composed.
The carbon that Fig. 2 is 25S-Antcin K is composed.
The hydrogen that Fig. 3 is 25R-Antcin K is composed.
The carbon that Fig. 4 is 25R-Antcin K is composed.
Fig. 5 is 2 Antrodia camphorata bioactive triterpene class compound Antcin K (20 μM) of embodiment to CCl4Caused by HepG2 cell
The protective effect test result of acute injury.
Fig. 6 is Antcin K (AK) in embodiment 3 to CCl4Caused by oxyhepatitis hepatic injury protective effect blood biochemistry
As a result, wherein (a) shows each group serum glutamic pyruvic transminase (ALT) concentration, each group serum glutamic oxalacetic transaminase (b) is shown
(AST) concentration.
Fig. 7 is groups of animals liver HE coloring pathological section in embodiment 3.
Fig. 8 is Antcin K (AK) to CCl4Caused by oxyhepatitis hepatic injury Protective effects research qPCR knot
Fruit.
Fig. 9 is the protective effect that triterpene compound Antcin K (AK) induces alcohol HepG2 cytolipin deposition
The selection result.
Specific embodiment
Below with reference to embodiment, the present invention is further explained, it will be understood by those skilled in the art that following embodiment is only used for
Illustrate the present invention rather than limits the scope of the invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Triterpene compound Antcin K's isolates and purifies in embodiment 1, Antrodia camphorata
One, experimental material and method.
All chemical reagent referred in this method are purchased from Beijing Chemical Plant.
The drying Antrodia camphorata fructification 500g of ware culture of making even first is ground into coarse powder, in two batches, every batch of 95% second of 6L
Alcohol reflux extracts 6 times, each 3h；Combined extract, vacuum rotary steam obtain total medicinal extract about 200g；After total medicinal extract adds 4L water to be suspended, according to
Secondary to use 4L petroleum ether extraction 5 times, water phase is extracted with ethyl acetate 5 times；Merging is evaporated to obtain ethyl acetate layer about 132g.
Take ethyl acetate layer about 130g medicinal extract through silica gel post separation (200~300 mesh, Qingdao Marine Chemical Co., Ltd.),
Petroleum ether-ethyl acetate system elutions (1:0,10:1,6:1,4:1,3:1,2:1,1:1,0:1), according to TLC detection and HPLC-
UV detection, merges into 12 fractions (A-L) for the fraction eluted.Antcin K (S/R) directly passes through automatic pure for fraction K
Change chromatograph (Waters Purification System 2545, be furnished with binary pump solvent elution system, autosampler and
Ultraviolet Detector) it is prepared, preparation condition is methanol-H2O-TFA (62:38:0.03, v/v, 15mL/min, 254nm),
The retention time of 25S-Antcin K and 25R-Antcin K are respectively tS=21.6min and tR=23.4min.25S-Antcin
Hydrogen spectrum, the carbon spectrum of K and 25R-Antcin K is as shown in Figures 1 to 4, molecular formula C29H44O6。
Triterpene compound Antcin K is to CCl in embodiment 2, Antrodia camphorata4Caused by HepG2 cell acute damage guarantor
Shield effect
One, experimental material and method.
HepG2 human liver cancer cell is purchased from U.S.'s Culture Collection (ATCC), and experiment is using thin in logarithmic growth phase
Born of the same parents.Carbon tetrachloride (Aladdin, Shanghai, China), DMEM culture medium (middle section steps morning, China), DMSO (dimethyl sulfoxide)
(AppliChem), top grade fetal calf serum (PAN, South Africa), Pen .- Strep cell culture is dual anti-(middle section steps morning, China),
MTS (Promega, the U.S.), silymarin (Central China Hai Wei, Beijing).
384 light absorption type microplate reader (Molecular Devices, the U.S.) of SpectraMax Plus, 3111 type cells
Incubator (Thermo, the U.S.), electronic analytical balance (Satorious, Germany).
CCl4Liquid storage configuration: DMSO and CCl4It is mixed by 1: 1 (v/v), vortex obtains 50%CCl4Stock solution.It is trained to DMEM
It supports in base and stock solution is added, obtain containing CCl4The culture medium of concentration 0.35%.
HepG2 is incubated at containing in 10%FBS and 1% dual anti-DMEM culture medium, by 1 × 104/ hole kind in 96 orifice plates,
Culture medium is sucked out after culture 12h, following liquid (100 holes μ L/, 3 multiple holes/group) is added in grouping:
Blank group: CCl is free of4Blank DMEM culture medium (be added be free of cell blank hole)；
Blank control group: the blank DMEM culture medium containing the DMSO with dosing group comparable sodium；
Model group: contain 0.35%CCl containing the DMSO with dosing group comparable sodium4DMEM culture medium；
Positive drug group: final concentration of 20 μM of silymarin are added and contains 0.35%CCl4DMEM culture medium；
Administration group: it is added and contains 0.35%CCl containing final concentration of 20 μM of compound Antcin K4DMEM culture medium；
After cultivating 6h, 10 μ L MTS solution are added in every hole, are protected from light and are incubated for 2-4h, survey each hole absorbance under 490nm.
With blank group for 0%, control group 100% calculates separately the cell survival rate and SD value of each group, to compound
Protective effect is evaluated, and cell survival rate in each group hole is calculated by absorbance value.
Two, experimental result.
As shown in table 1 and Fig. 5, triterpene compound Antcin K is under 20 μM of concentration to 0.35%CCl4Damage
HepG2 plays the role of significant guarantor, by CCl4Cell survival rate after damage is increased to 90% or more from 40%.Illustrate to train ox from ware
The compound Antcin K (abbreviation AK) separated in antrodia ethanol extract has significant protection acute liver damage activity, can be used for
Preparation protection oxyhepatitis liver injury medicament.
1. 20 μM of Antcin K of table are to CCl4Caused by HepG2 cell acute damage protective effect test
Embodiment 3, Antcin K are to CCl4Caused by acute liver damage protective effect
One, experimental material and method.
4-6 weeks male ICR mouse is purchased from Department Of Medicine, Peking University experimental animal portion, and it is dark to give 12h illumination 12h, constant temperature, just
Normal feed and water raising, are tested after adapting to 4 days in gnotobasis.
Carbon tetrachloride (Aladdin, Shanghai, China), silymarin (Central China Hai Wei, Beijing, China), corn oil (I
Fourth, Shanghai, China), sodium carboxymethylcellulose (Beijing Chemical Plant, Beijing, China), deionized water (Milli-Q system is made),
Paraformaldehyde (thunder root biology, Beijing, China), Tranzol (Quan Shijin, Beijing), chloroform, ethyl alcohol, isopropanol (Beijing chemical industry
Factory, Beijing), RNA lysate (Quan Shijin, Beijing), RNase-free distilled water, reverse transcription reagent box (Fermantas, beauty
State), Sybr-green fluorescent dye determination quantitative PCR system (Ai Laide, Beijing).
Centrifuge (Thermo, the U.S.), full-automatic blood biochemistry analyzer (7170A, Hitachi, Japan).Qubit nucleic acid/
Protein quantification instrument (Thermo, the U.S.), real-time fluorescence quantitative PCR instrument (Bio-Rad, the U.S.), centrifuge (Thermo, the U.S.).
Animal is randomly divided into 6 groups (n=6), 1) control group and 2) model group animal presses 0.3% carboxylic of 10ml/kg stomach-filling daily
Methylcellulose sodium solution, 3) positive drug group (is mixed in 0.3% by the silymarin suspension of 10ml/kg stomach-filling 10mg/ml daily
Carboxymethylcellulose sodium solution), 4) AK low dosage protection group and 5) AK high dose protection group press daily 10ml/kg difference stomach-filling
The AK suspension (dosage 10mg/kg, 50mg/kg) of 1mg/ml, 5mg/ml, successive administration 7 days, after the 7th day stomach-filling 6h: 1)
Corn oil (7ml/kg) is injected intraperitoneally in control group, 2) model group, 3) positive drug group, 4) AK protects low dose group and 5) AK high dose
CCl is injected intraperitoneally in protection group4Modeling (0.2%v/v, 7ml/kg are dissolved in corn oil) plucks eyeball afterwards for 24 hours and takes blood, cardiac perfusion
After take hepatic tissue, weigh.4 DEG C of 8000g centrifugation 15min take serum after blood sample is stored at room temperature 2h.
Blood serum sample carries out blood biochemistry analysis, detects ALT, AST index.Liver organization carries out stone after paraformaldehyde is fixed
Wax embedding, HE dyeing, is taken pictures at slice.
Total RNAs extraction: each animal's liver tissue 20mg of clip respectively is added the Tranzol of 1ml, is placed on ice after shredding
On mill in homogenizer, be then transferred into the EP pipe of no DNA enzymatic and RNA enzyme.0.2ml chloroform is added in every pipe, acutely vibrates
15s is placed at room temperature for 3min, and then in 4 DEG C, 15min is centrifuged under the conditions of 12000rpm.Upper strata aqueous phase is transferred to new EP pipe
In, isometric isopropanol is added in obtained water phase, is mixed by inversion, is placed at room temperature for 20-30min, then in 4 DEG C,
It is centrifuged 15min under the conditions of 12000rpm, white clear colloid substance can be seen in tube bottom.Supernatant is removed, the second of 1ml 75% is added
Alcohol is centrifuged 5min, outwells supernatant liquid, be placed to dry at room temperature in 4 DEG C after vortex under the conditions of 12000rpm.Then it is added
The RNase-free distilled water of 20-50 μ L, gently piping and druming detects A260/ with spectrophotometer up to sufficiently dissolution RNA repeatedly
280, which carry out RNA, quantifies.
Reversion: RNA concentration is adjusted to unanimously, and 10 or 20 μ L systems carry out reverse transcription, and specific steps are according to Fermantas
Reverse transcription reagent box illustrates to be operated.
Real-time quantitative PCR: QPCR primer information such as table 2, it is fixed by have a try by 4 program of table after the configuration reaction system of table 3
PCR experiment is measured, and collects processing data.
Table 2.QPCR primer information
Table 3.QPCR reaction system
Table 4.QPCR response procedures
Two, experimental result.
Experimental data measurement data indicates that the comparison of two means uses SPSS16.0 statistical software with mean ± standard deviation
In one-way ANOVA analysis, p < 0.01 * p < 0.05, * * compared with model group,#P < 0.05,##P < 0.01 and blank group ratio
Compared with.
(1) Antcin K (AK) is to CCl4Caused by acute liver damage protective effect Biochemical Indices In Serum result
Glutamic-pyruvic transaminase (ALT) can be discharged after liver cell is impaired and glutamic-oxalacetic transaminease (AST) enters blood, both in serum
Enzyme concentration reacts hepatic injury degree.As shown in table 5 and fig. 6, AK has apparent protecting effect and dosage to blood biochemistry as the result is shown
It relies on, significantly protects CCl4Caused by acute liver damage.
Table 5.Antcin K (AK) is to CCl4Caused by acute liver damage protective effect
* p < 0.005 p < 0.05, * * p < 0.01, * * * compared with model group,###P < 0.005 is compared with blank group.
(2) Antcin K (AK) is to CCl4Caused by acute liver damage protective effect HE coloration result
As shown in fig. 7, observation is as it can be seen that liver cell size, form are equal in naive animals hepatic tissue under an optical microscope
Normally, no liver parenchyma cytopathy, central vein and portal area are normal.In model group animal liver tissue around central vein
Liver cell is in ballooning degeneration without normal cell form, liver cell enlargement, part cell, there is inflammatory cell infiltration phenomenon.Through giving
Necrosis of liver cells degree significantly mitigates medicine Antcin K (AK) afterwards, and inflammatory infiltration degree reduces, and balloon sample is still presented in part cell
Denaturation.
(3) Antcin K (AK) is to CCl4Caused by acute liver damage protective effect qPCR result.
Totally 4 factors carry out QPCR pairs by this experimental selection TNF-α relevant to inflammatory reaction, iNOS, IL-1 β, IL-17
Their transcriptional levels in groups of animals liver are tested.INOS (inducible nitric oxide synthase) is body activity
The catalyzing enzyme that nitrogen free radical generates, the height of content can be used as the index of degree of inflammation in body, TNF-α (tumor necrosis factor
Son-α) it is a kind of substance that can make kinds of tumors that hemorrhagic necrosis occur, neutrophil adhesion can be promoted to endothelial cell
On, to stimulate body local inflammation reaction, and activate nuclear factor NF- κ B transcription to activate a series of Apoptosis in downstream because
Son and inflammatory factor expression, cascade expand inflammatory reaction degree.IL-1 β (interleukin-1 ' beta '), IL-17 (interleukins
17) interleukins family is belonged to, IL-1 β is a kind of important polypeptides for modulating factor mainly generated by mononuclear macrophage,
Play adjustment effect in cellular immunity activation, participate in body hemopoietic system, nerve, endocrine system, body inflammatory reaction and
Certain antitumor physiology courses, constantly abnormal synthesis can lead to organism fever, inflammatory reaction in local organization.IL-17 is T
The early stage startup factor of the inflammatory reaction of cell induction, can be by promoting release pro-inflammatory cytokines anti-to amplify inflammation
It answers, after IL-17 is in conjunction with receptor, its biological action can be played by MAPK approach and NF- kB pathway, can effectively mediate
Property granulocyte mobilize excited process, thus the inflammatory reaction of mediating tissue.
It can be seen that the gene transcription level of this 4 albumen difference between each group is obvious from table 6 and Fig. 8, model group
Inflammatory factor expression level illustrates CCl than the apparent increase of naive animals liver in animal's liver4Caused by acute liver damage lead
The significant raising of inflammation-related factor expression in liver is caused, so as to cause a series of injury responses such as inflammation, apoptosis, EAC group animal
TNF-α, iNOS in liver, IL-1 β are significantly reduced, and the transcription amount of these three factors is also bright in the animal's liver after AK protection
It is aobvious to reduce and be on close level with blank control group animal's liver, it is anti-inflammatory to CCl to illustrate that liver protection triterpene compound AK passes through4It causes
Acute liver damage protected.
Table 6.Antcin K (AK) protects inflammatory factor transcriptional level QPCR result in liver
* p < 0.005 p < 0.05, * * p < 0.01, * * * compared with model group,#P < 0.05,##P < 0.01 is compared with blank group.
The protective effect of embodiment 4, triterpene compound Antcin K to alcohol induction HepG2 fat deposition
One, experimental material and method.
Alcohol (Beijing chemical industry), DMEM (middle section steps morning, China), DMSO (AppliChem), top grade fetal calf serum (PAN,
South Africa), Pen .- Strep cell culture is dual anti-(middle section steps morning, China), MTS (Promega), isopropanol (Beijing
Work), oil red O powder (Sigma), cell climbing sheet (NEST), 90% glycerol (Beijing chemical industry), glass slide, sharp bottom glass centrifuge tube
(Beijing glass factory), total triglyceride reagent box (middle raw north control, Beijing), total cholesterol kit (middle raw north control, Beijing),
HepG2 cell (consonance cell bank, Beijing).
384 light absorption type microplate reader (Molecular Devices, the U.S.) of SpectraMax Plus, 3111 type cells
Incubator (Thermo, the U.S.), centrifuge (Thermo, the U.S.), high-power microscope (Leica, Germany).
(1) oil red O stain detects
HepG2 is incubated in the DMEM culture medium containing 10%FBS and 1% dual anti-(PS), and 5 × 10 are pressed before experiment4Kind is in
It is completed in 12 orifice plates of cell climbing sheet, culture is for 24 hours.
Blank group: the DMEM culture medium containing 10%FBS, 1%PS.
Control group: the DMEM culture medium containing 0.3% alcohol, 10%FBS, 1%PS is added and administration group same volume
DMSO。
Administration group: containing 0.3% alcohol, 10%FBS, 1%PS DMEM culture medium in compound stock solutions be added reach estimated
Ultimate density (20 μM of compound).
Archaeocyte culture medium is sucked out, cell is cleaned twice with 1mlPBS, every hole addition corresponding culture medium of 1ml, and every group 3
A multiple holes.
It is cultivated in cell incubator for 24 hours, cell culture medium is sucked out, rinsed 2 times with PBS, the fixed 10min of 4% paraformaldehyde,
ddH2O cleans cell 2min × 3 time, and 60% isopropanol is incubated for 10min, oil red O stain 30min, rapid mistake in 60% isopropanol
For several times, ddH2Rapid after for several times in O, hematoxylin dyes 1min, ddH2O is cleaned twice, 90% glycerol mounting.In optical microscopy
Lower observation is simultaneously taken pictures.
(2) total triglycerides (TG) and total cholesterol (TC) extracting detection
HepG2 is incubated in the DMEM culture medium containing 10%FBS and 1% dual anti-(PS), and 2 × 10 are pressed before experiment5Kind is in 6 holes
In plate, 2 hole cells merge into a sample, every group of 3 parallel samples, and culture medium is sucked out afterwards for 24 hours for cell culture, and PBS is washed 2 times, add
Medicine is operated with (1).
It cultivates in cell incubator after dosing for 24 hours, is cleaned 2 times with PBS, every hole is added after the digestion of 0.5ml pancreatin is added
DMEM culture medium of the 1.5ml containing 10%FBS, 1%PS stops digestion, and liquid in hole is transferred to by piping and druming cell to all falling
In centrifuge tube, 2 hole cells merge into a pipe, carry out cell count, and 800rpm is centrifuged 3min, discards supernatant, by cell suspension in
In 1ml PBS.
Suspension is transferred in the glass centrifuge tube of 10ml point bottom, the chloroform/methanol 3ml of 2:1 (v/v), abundant whirlpool is added
Rotation mixes 30s/ pipe, and 4 DEG C, 2000rpm, 30min centrifugation carefully remove upper strata aqueous phase into new glass tube, and transfer lower layer has
Machine mutually into another set of glass tube, avoids being drawn onto intermediate protein, chloroform/methanol 3ml, whirlpool is added in upper strata aqueous phase pipe
30s is revolved, 4 DEG C, lower layer's organic phase is transferred in organic pipe, N in draught cupboard by 2000rpm, 30min centrifugation2Drying is added 3%
500 μ L of TritonX-100 (v/v), blows and beats repeatedly, 50 DEG C of constant-temperature table, shakes 30min, makes Lipid dissolution.
Standard curve is established using total triglyceride reagent box, by specification operation measures each sample at 490nm
Absorbance obtains total content of triglyceride of each sample with standard curve control.
Two, experimental result.
Protective effect of the Antrodia camphorata Triterpenoid Antcin K to alcohol induction HepG2 fat deposition.
Antcin K (20 μM) is screened by aforesaid operations, is taken pictures after dyeing, as a result such as Fig. 9.Oil red O stain result
Display: model group generates serious lipidosis phenomenon into the cell, the visible apparent red fat drips around nucleus, and compound
Antcin K significantly reduces 0.3% alcohol-induced HepG2 lipidosis under 20 μM of concentration, intracellular red after administration
Fat drips substantially reduce, and close to blank group cell, illustrate that Antrodia camphorata Triterpenoid has protection alcohol fatty as Antrodia camphorata
The pharmacological activity of liver has good anti-grease matter position activity and the prospect as resisting alcoholic fatty liver medicament.
Claims (3)
1. triterpene compound Antcin K and its pharmaceutically acceptable salt are in preparation for preventing and/or treating chemically liver
The drug and/or the application in health care product of oxyhepatitis and hepatic injury caused by toxicant and/or alcoholic fatty liver, it is described
The structural formula of triterpene compound Antcin K is as follows:
2. application as described in claim 1, which is characterized in that the triterpene compound Antcin K is its epimer
25S-Antcin K, 25R-Antcin K or the mixture that they are formed with arbitrary proportion, wherein 25S-Antcin K and 25R-
The structural formula of Antcin K is as follows:
3. application as claimed in claim 1 or 2, which is characterized in that the chemically Hepatoxic substance is CCl4。
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| CN107400156B (en) * | 2017-09-19 | 2020-01-31 | 北京大学 | method for separating the ergosterol-type triterpenes 25R-and 25S-epimers |
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| TW201029658A (en) * | 2009-02-13 | 2010-08-16 | Univ Kaohsiung Medical | Ethanol extract of antrodia camphorata for inducing apoptosis and preparation method thereof |
| CN104359933A (en) * | 2011-01-26 | 2015-02-18 | 高雄医学大学 | Triterpenoid composition of antrodia cinnamomea fruiting body, preparation and analysis method |
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| TW201029658A (en) * | 2009-02-13 | 2010-08-16 | Univ Kaohsiung Medical | Ethanol extract of antrodia camphorata for inducing apoptosis and preparation method thereof |
| CN104359933A (en) * | 2011-01-26 | 2015-02-18 | 高雄医学大学 | Triterpenoid composition of antrodia cinnamomea fruiting body, preparation and analysis method |
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