CN106265680A - The purposes of Caulis Hederae Sinensis subacid - Google Patents
The purposes of Caulis Hederae Sinensis subacid Download PDFInfo
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- CN106265680A CN106265680A CN201510263191.5A CN201510263191A CN106265680A CN 106265680 A CN106265680 A CN 106265680A CN 201510263191 A CN201510263191 A CN 201510263191A CN 106265680 A CN106265680 A CN 106265680A
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- hederae sinensis
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Abstract
The purposes of Caulis Hederae Sinensis subacid, relates to a kind of natural product Caulis Hederae Sinensis subacid.Caulis Hederae Sinensis subacid molecular formula is C29H44O3, chemical name is 3 oxo 24 nor-olive 12 alkene 28 acid.Can apply in the medicine of the diseases such as preparation prevention and treatment diabetes, obesity, fatty liver, liver cirrhosis, hyperglycemia, insulin resistance disease, cardiovascular disease, hypertriglyceridemia, hypercholesterolemia, tumor, hepatic injury, inflammation, kidney disease, cholestasis, cholelithiasis, atherosclerosis or health product.Can apply in preparing antioxidation, anti-aging health product or skin care item.Pharmaceutical composition can be made as active ingredient and other medicines with after acceptable excipient/or drug administration carrier mix in pharmacy.
Description
Technical field
The present invention relates to a kind of natural product Caulis Hederae Sinensis subacid, especially relate to Caulis Hederae Sinensis subacid at medicine, health product and skin care item
Deng the purposes in field.
Background technology
Caulis Hederae Sinensis subacid (Hedragonic acid), molecular formula is C29H44O3, chemical name is the 3-nor-olive of oxo-24-
-12-alkene-28-acid (3-oxo-24-norolean-12-en-28-oic acid), structural formula is as follows:
There is not been reported for the biology of this Caulis Hederae Sinensis subacid and materia medica function.
Diabetes, obesity, fatty liver, liver cirrhosis, hyperglycemia, insulin resistant, tumor, hepatic injury, inflammation, kidney
The diseases such as disease die the harm major disease of human health or commonly encountered diseases and frequently-occurring disease, antioxidation and anti-aging be also that people are non-the normally off
The problem cut, finds that the new drug preventing or treating these diseases will have important social value and huge economic worth.
All-trans-retinoic acid (ATRA) is one of antitumor drug currently mainly, is current state internal therapy acute progranulocyte
Leukemia, the clinical choice drug of myelodysplastisches (preleukemia), especially promyelocytic leukemia.But, have
The tumor cell of many types has ATRA repellence, makes the tumor therapeutic efficacy of ATRA be restricted.Colon cancer such as people is thin
Born of the same parents (HCT-15) are ATRA sensitivity cancerous cell, and HCT-116 is exactly the colon cancer cell showing as ATRA resistance.Therefore,
Invention can have the new cancer therapy drug of inhibitory action to these growths with the drug-fast cancerous cell of ATRA, will be to Drug resistance
The cancer patient hope with next life.
Nuclear receptor is important drug targets, the gene regulated and controled by nuclear receptor more with series major disease such as diabetes, tumor, inflammation
Disease, cardiovascular disease etc. are closely related.13% it is all with nuclear receptor for target protein target on front hundred the situation of selling well lists of medicine of the whole world now,
Absolutely prove that the huge medicament research and development of nuclear receptor and part thereof is worth and important economy and society value.Farnesoid X receptor FXR
(Farnesoid X Receptor) is the important member in nuclear receptor superfamily, finds farnesol (farnesoids) and dimension the earliest
Formic acid (retinoids) can activate the transcriptional activity of FXR, but they are the lowest to the affinity of FXR, and therefore FXR is arranged always
For orphan nuclear receptor.Until Steven seminar in 2000 is found that the synthetic part GW4064 of FXR first.As
The transcriptional activation agent of FXR, the EC50 of GW4064 is 65nmol/L.Then, people utilize the GW4064 biology to FXR
Function has carried out a series of research, finds that FXR has in terms of the Metabolism regulation such as carbohydrate metabolism, lipid metabolism, cholesterol-cholic acid metabolism
Having important function, FXR also has important tune at inflammation, tumor, liver cirrhosis, Adipose Differentiation, antioxidation, health care aspect of waiting for a long time
Control effect.Nuclear receptor is the transcription factor of a kind of ligand activation.The ligand-mediated pharmacological action principle of FXR be part by with
The ligand binding domain (LBD) of FXR combines, and recruits all kinds of co-activator (or Corepressors) and regulates downstream target gene.
FXR involves the signal path that many metabolism are relevant, become treatment metabolic disease such as hypertriglyceridemia, hypercholesterolemia,
The outstanding drug target molecules such as cholestasis, cholelithiasis, non-alcoholic fatty liver disease, atherosclerosis and diabetes.
The curative effect of most of small-molecule drugs is by combining one or more protein " target ", and then regulates these " targets "
Function and signal transduction play a role.Therefore, the identification of drugs for proteins target provides and understands medicine effect action
Basis.Illustrate the target proteins matter of these medicines, provide theories integration, for grinding of important new drug by the effectively treatment for disease
Send out and effective means is provided.
Summary of the invention
It is an object of the invention to provide the purposes of a kind of Caulis Hederae Sinensis subacid.
Caulis Hederae Sinensis subacid (Hedragonic acid), molecular formula is C29H44O3, chemical name is the 3-nor-olive of oxo-24-
-12-alkene-28-acid (3-oxo-24-norolean-12-en-28-oic acid), structural formula is as follows:
Described Caulis Hederae Sinensis subacid can be at preparation prevention and treatment diabetes, obesity, fatty liver, liver cirrhosis, hyperglycemia, islets of langerhans
Element resist the disease, cardiovascular disease, hypertriglyceridemia, hypercholesterolemia, tumor, hepatic injury, inflammation, kidney disease
The medicine of the diseases such as disease, cholestasis, cholelithiasis, atherosclerosis or health product are applied.
Described Caulis Hederae Sinensis subacid can be applied in preparing antioxidation, anti-aging health product or skin care item.
Described diabetes can include but not limited to the diabetes with hyperglycemic symptoms.
Described obesity can include but not limited to the obesity with fatty liver, hyperglycemic symptoms.
Described fatty liver can include but not limited to non-alcoholic fatty liver disease.
Described liver cirrhosis can include but not limited to have Liver Collagen over-deposit and the liver cirrhosis of hepatic fibrosis performance.
Described insulin resistance disease can include but not limited to the insulin resistance disease with hyperinsulinemia.
Described cardiovascular disease can include but not limited to have hyperglycemia, liver fat builds up the cardiovascular disease of symptom.
Described tumor can include but not limited to colon and rectum carcinoma, carcinoma of prostate, cervical cancer, hepatocarcinoma, breast carcinoma and
Leukemia；Described tumor also includes showing as the tumor that all-trans-retinoic acid (ATRA) is insensitive, includes but not limited to that ATRA resists
The colon cancer of property.
Described hepatic injury can include but not limited to owing to having taken the drug-induced hepatic injury of hepatic injury side effect, having drawn due to ethanol
Rise acute liver damage, the alcoholic fatty liver hepatic injury caused, immunity or chemical factor cause hepatic injury, virus
Infect the hepatic injury caused；Described have hepatic injury side effect medicine to include but not limited to acetaminophen as main active
Medicine.
Described inflammation can include but not limited to owing to taking inflammation caused by inflammatory reaction side effect medicine, drawing due to ethanol
Rise acute hepatitis, the hepatitis caused by alcoholic fatty liver, the hepatitis caused by non-alcoholic fatty liver disease, viral hepatitis,
The hepatitis by bacterial inflammation, caused due to diabetes or obesity complication or nephritis, described in have inflammatory reaction side effect
Medicine includes but not limited to the medicine that acetaminophen is main active.
Described kidney disease can include but not limited to the kidney disease having blood urea nitrogen horizontal abnormality to show, various excess of the kidney matter sexually transmitted disease (STD)
Change, uremia；Described parenchymal lesion of the kidney can include but not limited to glomerulonephritis, interstitial nephritis, acute and chronic renal function
The parenchymal lesion of the kidney such as occupancy and destructive pathological changes in exhaustion, kidney.
Described Caulis Hederae Sinensis subacid can mix with acceptable excipient/in other medicines and pharmacy or drug administration carrier as active ingredient
After conjunction, pharmaceutical methods routinely and technological requirement, make pharmaceutical composition, described pharmaceutical composition can use oral type preparation
Or injection-type preparation medicine etc., described oral type preparation can use tablet, pill, capsule, electuary or syrup etc.；Described injection
Type preparation can use injection or freeze-dried powder dosage form etc..
Described antioxidation, the anti-aging health product including but not limited to preparation removing ultra-oxygen anion free radical and skin care item and/or system
Application in the health product of standby enhancing body reducing substances such as reductive glutathione and skin care item.
It is demonstrated experimentally that Caulis Hederae Sinensis subacid is at treatment diabetes, obesity, fatty liver, cardiovascular disease, hyperglycemia, insulin
The effect of the diseases such as opposing.Utilize diabetes, obesity models mice db/db and KK-Ay, after lumbar injection Caulis Hederae Sinensis subacid,
Detection related-metabolism index.Result shows, compares blank, the model mice glucose in serum of Caulis Hederae Sinensis time acid treatment,
The level of insulin substantially reduces.Mouse liver tissue freezing section oil red coloration result shows, Caulis Hederae Sinensis time acid treatment group little
In Mus liver organization, the amount of fat is significantly lower than control group mice.These results indicate that Caulis Hederae Sinensis subacid is controlling diabetes, fat
The disease aspects such as liver, obesity, cardiovascular disease, hyperglycemia, insulin resistant have good efficacy.
It is demonstrated experimentally that the effect that Caulis Hederae Sinensis subacid is on renal function.Blood urea nitrogen is one of renal function leading indicator, plasma wrea
Nitrogen is too high, indicates various parenchymal lesion of the kidney, accounts in glomerulonephritis, interstitial nephritis, acute or chronic renal failure, kidney
Position property and destructive pathological changes and uremia etc..Mouse test results display Caulis Hederae Sinensis subacid can effectively reduce blood urea nitrogen level,
Show that Caulis Hederae Sinensis subacid has treatment kidney disease and the effect of protection renal function.
It is demonstrated experimentally that utilize liver injury model mice to prove liver protecting and the repair function of Caulis Hederae Sinensis subacid.The present invention gives
C57B6/J mouse peritoneal injection Caulis Hederae Sinensis subacid, then injection excess acetaminophen (N-acetyl-para-
Aminophenol, is abbreviated as APAP) cause hepatic injury, testing result after 24h.Result shows, shows seriously at matched group
In the case of hepatic injury, the mouse liver form regulating liver-QI function performance having injected Caulis Hederae Sinensis subacid is normal.Show Caulis Hederae Sinensis time
Acid has outstanding prevention and treatment function in terms of hepatic injury.
It is demonstrated experimentally that prove the Caulis Hederae Sinensis subacid therapeutical effect at aspect of inflammation on mouse model.It is excessively used APAP can cause
Mouse liver is inflamed.The present invention injects Caulis Hederae Sinensis subacid to C57B6/J mouse peritoneal, and then injection excess APAP causes liver
Dirty inflammation, the level of inflammatory factor in detection mouse liver tissue.Result shows, compares the mice that blank processes, abdominal cavity
Inject the gene expression dose of the inflammation factor in the mouse liver tissue of Caulis Hederae Sinensis subacid to significantly reduce.The reality of cellular level
Execute example result display Caulis Hederae Sinensis subacid and can lower the expression of the inflammation factor due to LPS induction.Show that Caulis Hederae Sinensis subacid is in inflammation
Disease treatment aspect has good function.
It is demonstrated experimentally that Caulis Hederae Sinensis subacid can suppress the growth of kinds of tumor cells, including all-trans-retinoic acid
The people of the human colon cancer cell HCT116 cell of (all-trans-retinoic acid, ATRA) resistance, ATRA sensitivity ties directly
Enteraden cancerous cell HCT15 cell, Human Prostate Cancer Cells LNCaP cell, human cervical carcinoma cell Hela cell, human hepatocellular carcinoma
Cell HepG2 cell, human breast cancer cell MCF7 cell and mouse monokaryon macrophage leukaemia's Raw264.7 cell.
These tumor cells acid-treated with Caulis Hederae Sinensis time are significantly lower than, in the survival rate of 96h, the cell that blank processes.And with
The increase of drug level, the effect of suppression growth of tumour cell is the most notable.Show that Caulis Hederae Sinensis subacid is in treatment Cancerous disease
Function, especially insensitive to the ATRA treatment function in colon cancer.
It is demonstrated experimentally that Caulis Hederae Sinensis subacid can activate the transcriptional activity of nuclear receptor FXR, FXR can be induced to raise co-activator, table
Bright Caulis Hederae Sinensis subacid is the agonist of nuclear receptor FXR, can be by combining the sugar generation that target FXR regulation FXR participates in body
Thank, the function of the aspects such as metabolism, inflammation, tumor, liver cirrhosis, Adipose Differentiation such as lipid metabolism, cholesterol-cholic acid metabolism.
It is demonstrated experimentally that Caulis Hederae Sinensis subacid also has antioxidation and anti-aging function.Mice MEF cell experiment shows, Caulis Hederae Sinensis time
In cell after acid treatment, reducing substances such as GSH level is apparently higher than blank group, and Caulis Hederae Sinensis time acid treatment can be notable
Improve antioxidation and the expression of anti-aging gene such as SOD1, SOD2, SESN1 and SESN2 in mouse primary hepatocytes,
Also can significantly lower and promote oxidation and the expression of old and feeble AC5 gene.Show that Caulis Hederae Sinensis subacid has antioxidation and anti-aging
Function.
It is demonstrated experimentally that Caulis Hederae Sinensis subacid can lower the expression of the marker gene of hepatic fibrosis in liver, hepatocyte inflammation can be slowed down anti-
Should, reduce hepatic fibrosis marker gene and express, can effectively eliminate the accumulation of collagen in liver, show that Caulis Hederae Sinensis subacid is to liver cirrhosis
Treatment function.
Caulis Hederae Sinensis subacid is the natural product extracted from plant, have no so far any relevant its treatment diabetes, obesity,
The diseases such as fatty liver, cardiovascular disease, liver cirrhosis, hyperglycemia, insulin resistant, tumor, hepatic injury, inflammation, kidney disease
The report of application in sick medicine.Therefore the Caulis Hederae Sinensis subacid that the present invention is given has treatment function in terms of these diseases, is all
New function about Caulis Hederae Sinensis subacid.
Diabetes, obesity, fatty liver, cardiovascular disease, liver cirrhosis, hyperglycemia, insulin resistant, tumor, hepatic injury,
The disease such as inflammation, kidney disease has a strong impact on the health and lives of the mankind, and therefore, this Caulis Hederae Sinensis subacid that the present invention is given exists
Preparation treat these major diseases pharmaceutical preparation in terms of utilitarian function, and be given Caulis Hederae Sinensis subacid can also antioxidation anti-aging,
Can be used for the application in health product and skin care item, there is important social value and huge economic worth.
In some embodiments, the compound in the method for the invention can be configured to pharmaceutical composition in some dosage regimens
Application.The compositions of medicine of the present invention can comprise described compound self and be administered available salt or drug administration carrier with it.Such group
Compound is also selectively included other therapeutic agent.
" drug administration carrier " word refers to can be for patient and the nontoxic medicine that do not destroys its pharmacological activity together with the compounds of this invention
Carrier.Available drug administration carrier in aforementioned pharmaceutical compositions includes but not limited to: ion-exchanger, aluminium oxide, aluminium stearate,
Lecithin, serum albumin such as human serum albumin, buffer medium such as phosphate, glycine, sorbic acid, potassium sorbate is saturated
The partial glyceride mixtures of vegetable fatty acid, water, salt or electrolyte such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,
Sodium chloride, zinc salt, silica sol, magnesium trisilicate, polyvinylpyrrolidone, cellulose preparation, Polyethylene Glycol, carboxylic first
Base sodium cellulosate, polyacrylate, wax, polyethylene-polyoxypropylene block polymer, lanoline and self-emulsifying drug delivery systems
Such as alpha tocopherol, cetomacrogol 1000 succinate, or other similar Polymeric delivery pattern.
" metabolic disease " used by the present invention refers to any disease relevant to metabolism or disorders.Such as, metabolic disease includes
But it is not limited to: hyperglycemia, insulin resistant, hyperlipemia, Hypercholesterolemia, diabetes, obesity, Metabolic syndrome
Levy, metabolism disorder and relevant disease, the disease (hepatopathy) of liver, Fatty Liver Disease (hepatic steatosis), non-alcoholic
Fatty Liver Disease, hepatitis, non-alcoholic stellato-hepatitis, liver cirrhosis, hepatic fibrosis, chronic and acute hepatic failure, gallbladder
Juice liver cirrhosis, primary sclerosing cholangitis, cholestasis, cholelithiasis atherosclerosis, inflammation, cancer, and above-mentioned
Disease send out disease altogether.
At some only using compound described herein as the pharmaceutical composition of its active component in, its medication may also include uses it
Described experimenter is treated by his medicament or therapy.Such agent treatment includes but not limited to: anemia treatment, glycosuria
Sick therapy, hypertension therapeutic, cholesterol therapy, neurologic agent, the medicine of regulation cardiovascular function, regulation inflammation, immunity merit
Energy, the medicine of hemopoietic；Hormone and antagonist, affect the chemotherapeutic agent of gastrointestinal function, microbial diseases, and/
Or the chemotherapy of tumor disease.
Some medicament or therapy can carry out administering drug combinations with compound of the present invention, such as matrix metallo-proteinase inhibitor, fat oxygen
Synthase inhibitor, cytokine antagonist, immunosuppressant, cytokine, somatomedin, immunomodulator, prostaglandin
Or anti-angiogenic antihyperproliferative compound.
Term used herein " combines " and relative words are corresponding in turn to be therapeutic agent with the compounds of this invention be simultaneously or by
Order is used.Such as, certain compound described can simultaneously or sequentially be used in single unit dosage forms with another therapeutic agent.Cause
This, the present invention provides a kind of single unit dosage forms, comprises described compound, a kind of extra therapeutic agent.In many dosage regimens
In, when patient or individuality are exposed simultaneously to two or more medicament, if patient or individuality are at certain particular target tissue or sample
(such as, at brain, at serum etc.) demonstrates the associated treatment effect of agents useful for same simultaneously, it is generally recognized that they are with " group
Close " form play a role.
If these change compositionss is that the pharmacy of the compounds of this invention can use salt product, that salt used should be preferentially from inorganic or have
Machine bronsted lowry acids and bases bronsted lowry.Ackd salt includes: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate,
Disulfate, butyrate, citrate, Camphora hydrochlorate, camsilate, Pentamethylene., digluconate, dodecyl sulfur
Hydrochlorate, esilate, fumarate, gluceptate, glycerophosphate, Hemisulphate, heptan, caproate, hydrochlorate,
Hydrobromate, hydriodate, 2-isethionate, lactate, maleate, mesylate, 2-naphthalene sulfonate, nicotinic acid
Salt, oxalates, embonate, pectate, persulfate, 3-phenylpropionic acid salt, picrate, Pivalate, third
Hydrochlorate, succinate, tartrate, Hydrogen thiocyanate, toluene fulfonate and undecylate.Alkali salt include ammonium salt, alkali metal salt (as
Sodium and potassium salt), alkali salt (such as calcium and magnesium salt) and organic alkali salt (as dicyclohexyl amine salt, N-methyl-D-glucamine salt,
Or the salt with aminoacid such as arginine, lysine etc.).
Compound composition of the present invention may also include, and relies on required preparation, pharmaceutically useful non-toxic carrier or diluent.Logical
Often it is defined as preparing the vehicle of the pharmaceutical composition of animal or people.Suitable diluent should be selected thus do not affect combination
The biologic activity of thing.Such diluent has: distilled water, physiological phosphate buffer salt solution, Ringer's mixture, glucose are molten
Liquid and HankShi solution.Additionally, pharmaceutical composition or preparation to may also include other carriers, adjuvant or nontoxic non-therapeutic non-
Immunogenic stabilizer etc..
Compound composition of the present invention may also include, wetting agent, emulsifying agent and lubricant, if sodium lauryl sulphate is with hard
Fatty acid magnesium, and coloring agent, releasing agent, coating materials, sweeting agent, flavoring agent and aromatic, preservative and antioxidant also may be used
It is present in compositions.Pharmaceutically useful antioxidant includes: water soluble antioxidant, as ascorbic acid, cysteine hydrochloride,
Sodium bisulfate, sodium pyrosulfite, sodium sulfite etc.；Oil-soluble inhibitor, such as ascorbyl palmitate, Butylated hydroxy
Methyl phenyl ethers anisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl propionate, alpha-tocopherol etc.；And metal-chelator, as
Citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid etc..
The pharmaceutical composition of the present invention can be configured to the form of solid or liquid.Including following applicable form of medication: (1) is administered orally
It is administered, such as filled medicament (aqueous or non-aqueous solution or suspension), tablet, cheek, Sublingual and systemic Absorption agent, bolus, dissipates
Agent, granule, the paste in Sublingual；(2) parenteral, is prepared as sterile solution or suspension or sustained release agent, logical
Cross subcutaneous, intramuscular, intravenous or epidural injection；(3) topical application, such as emulsifiable paste, ointment, for skin, lung or oral cavity
Controlled release patch or spray；(4) drop rectum with drug, as Emulsion or foam；(5) other: sublingual administration, eye
With, percutaneous or per nasal, lung and other mucosa are taken in.
The preparation method of the pharmaceutical composition of described compound includes the compounds of this invention and carrier or multiple auxiliary by any operation
Become subassembly.Under normal circumstances, can be by the compounds of this invention and carrier (liquid-carrier, subdivided solids carrier or both of which have)
Uniformly and intimately combine preparation, and as required by product figuration.
In some situation, can be by slowing down medicine from subcutaneously or intramuscularly noting the speed of slow trapping to extend drug effect.Can be by preparation
The crystallization of poorly water-soluble or the liquid suspension of amorphous substance realize this purpose.The absorption rate of medicine depends on that it dissolves
Speed, its rate of dissolution is likely to be dependent on again crystal size and crystal form.Or, by by medicine dissolution or be suspended in oiliness
To postpone parenteral absorption in carrier.
Can be by described compound be combined system with biodegradable polymer (such as polylactide-polyglycolide) in microencapsule matrices
Become its injectable drug depot dosage form.According to medicine and the ratio of polymer and the character of particular polymers used, controlled pharmacy thing
The speed of release.Other biodegradable polymer includes poe and condensing model.Also can be by pharmaceutical pack be embedded in lipid
In body or microemulsion, to prepare the injectable drug depot dosage form compatible with bodily tissue.
The ingredient of the present invention can arbitrarily be administered orally acceptable dosage form oral administration, and dosage form includes but not limited to: capsule, tablet,
And water slurry and solution.For oral tablet, its conventional peplos has lactose and corn starch, the most also can add profit
Lubrication prescription, such as magnesium stearate.For oral capsule preparation, available diluent includes lactose and the corn starch being dried.When
During with water slurry and solution and formulation propylene glycol oral administration, active constituents of medicine is combined with emulsifying agent and suspending agent.If any needing
Want, some sweeting agent and/or flavoring agent and/or coloring agent can be added.
The dosage form of the applicable oral administration that the present invention describes can be capsule, and cachet, pill, tablet, lozenge (is generally used
Sucrose and arabic gum or Tragacanth are as flavor ameliorating substances), powder, granule, or be dissolved in aqueous or non-aqueous liquid are made molten
Liquid or suspension, or as Water-In-Oil or oil-in-water liquid emulsion, or as elixir or syrup, or (use lazy as pastille
Property substrate, such as gelatin and glycerol or sucrose and arabic gum) and/or the mode such as gargle.Various dosage forms contain the present invention of scheduled volume
Compound is as one of its active component.Compound of the present invention can also bolus, electuary or paste use.
The liquid dosage form of the compounds of this invention oral administration includes pharmaceutically useful Emulsion, microemulsion, solution, suspension, syrup
And elixir.In addition to the active ingredient (s, liquid dosage form also can contain the most normally used inert diluent, such as water or its
Its solvent, solubilizing agent and emulsifying agent, as ethanol, isopropanol, ethyl carbonate, ethyl acetate alcohol, benzylalcohol, benzyl benzoate,
Propylene glycol, 1,3 butylene glycol, oils (particularly Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, Semen Maydis oil, germ oil, olive oil, Oleum Ricini and
Oleum sesami), glycerol, oxolane alcohol, Polyethylene Glycol and fatty acid ester Sorbitol, and their mixture.
When the pharmaceutical composition of the present invention is more easy to onset to targeted treatment area or organ by local application, i.e. may select this
Administering mode.In order to be locally applied to skin, active component can be suspended or dissolved in drug administration carrier, then by this drug regimen
Thing is configured to ointment.The topical carrier of the compounds of this invention includes but not limited to: mineral oil, liquid petroleum, white oil,
Propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifing wax and water.Or, reactive compound can be suspended or dissolved in carrier
In, then this pharmaceutical composition is made suitable lotion or cream, this kind of carrier includes but not limited to: mineral oil, anhydrosorbitol
Alcohol monostearate, polysorbate60, cetyl, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.This
Bright pharmaceutical composition is also by making rectal suppository or suitable enema agent is applied topically to lower intestinal tract.Present invention additionally comprises
Compound is prepared as topical transdermal patch.
Can be administered by the ingredient of the present invention being made aerosol or inhalant.Such medicament can be according to pharmaceutical formulation field
In wide variety of technology prepare, be also prepared as saline solution.This kind of prior art often uses benzylalcohol or other suitable preventing
Rotten agent, fluorocarbon and/or other solubilizing agents or dispersant, absorption enhancer are to improve its bioavailability.
The additional advantage of transdermal patch is the controlled delivery realizing the compounds of this invention to body.Can be by this compound dissolution or dispersion
This dosage form is made in suitable medium.The skin absorption to compound can be increased with absorption enhancer.Also can be by using
Rate controlling membranes or compound is scattered in the mode in polymeric matrix or gel, it is achieved the compound control by skin speed
System.
Accompanying drawing explanation
Fig. 1 is the survival rate that Caulis Hederae Sinensis time acid treatment 48h reduces people's Colon and rectum adenocarcinoma cell HCT116 cell.
Fig. 2 is the survival rate that Caulis Hederae Sinensis time acid treatment 96h reduces people's Colon and rectum adenocarcinoma cell HCT116 cell.
Fig. 3 is the survival rate that Caulis Hederae Sinensis time acid treatment 48h reduces people's Colon and rectum adenocarcinoma cell HCT15 cell.
Fig. 4 is the survival rate that Caulis Hederae Sinensis time acid treatment 96h reduces people's Colon and rectum adenocarcinoma cell HCT15 cell.
Fig. 5 is the survival rate that Caulis Hederae Sinensis time acid treatment 48h reduces Human Prostate Cancer Cells LNCaP cell.
Fig. 6 is the survival rate that Caulis Hederae Sinensis time acid treatment 96h reduces Human Prostate Cancer Cells LNCaP cell.
Fig. 7 is the survival rate that Caulis Hederae Sinensis time acid treatment 96h can reduce human cervical carcinoma cell Hela cell.
Fig. 8 is the survival rate that Caulis Hederae Sinensis time acid treatment 96h can reduce human breast cancer cell line Bcap-37 cell.
Fig. 9 is the survival rate that Caulis Hederae Sinensis time acid treatment 96h can reduce human liver cancer cell HepG2 cell.
Figure 10 is the survival that Caulis Hederae Sinensis time acid treatment 96h can reduce mouse monokaryon macrophage leukaemia's Raw264.7 cell
Rate.
Figure 11 is that mouse liver section HE dyeing shows that Caulis Hederae Sinensis subacid can protect and repair the hepatic injury of APAP induction.
Figure 12 is AST and the ALT activity that Caulis Hederae Sinensis time acid treatment can reduce in the mice serum of APAP induced liver injury.
Figure 13 is that Caulis Hederae Sinensis time acid treatment can reduce LDH activity in the mice serum of APAP induced liver injury.
Figure 14 is that Caulis Hederae Sinensis time acid treatment can regulate gene expression dose relevant to liver reparation in the mice of APAP induced liver injury.
Figure 15 is that Caulis Hederae Sinensis subacid can suppress mouse liver to cause the gene expression of inflammatory factor in inflammatory reaction due to APAP.
Figure 16 is that Caulis Hederae Sinensis subacid can suppress primary hepatocyte to induce inflammatory factor IFN γ in the inflammatory reaction caused due to LPS
Expression.
Figure 17 is that Caulis Hederae Sinensis subacid can suppress primary hepatocyte to induce inflammatory factor TGF β in the inflammatory reaction caused due to LPS
Expression.
Figure 18 is that Caulis Hederae Sinensis subacid can suppress primary hepatocyte to induce inflammatory factor TNFalpha in the inflammatory reaction caused due to LPS
Expression.
Figure 19 is that Caulis Hederae Sinensis subacid can suppress primary hepatocyte to induce inflammatory factor CD36 in the inflammatory reaction caused due to LPS
Expression.
Figure 20 is that Caulis Hederae Sinensis time acid treatment can lower db/db Mouse Blood urea nitrogen levels.
Figure 21 is that Caulis Hederae Sinensis time acid treatment can reduce db/db mice serum insulin level.
Figure 22 is that Caulis Hederae Sinensis time acid treatment can reduce db/db mice serum glucose level.
Figure 23 is that Caulis Hederae Sinensis time acid treatment can reduce the accumulation of fat in db/db mouse liver tissue.
Figure 24 is that Caulis Hederae Sinensis time acid treatment can reduce the accumulation of fat in KK-Ay mouse liver tissue.
Figure 25 is that Caulis Hederae Sinensis time acid treatment can reduce KK-Ay mice serum glucose level.
Figure 26 is the transcriptional activity that Caulis Hederae Sinensis subacid can activate nuclear receptor FXR.
Figure 27 is that Caulis Hederae Sinensis subacid energy induced nuclear receptor FXR is combined with co-activator SRC1.
Figure 28 is that Caulis Hederae Sinensis time acid treatment can improve MEF cell reducing substances GSH level.
Figure 29 is that Caulis Hederae Sinensis time acid treatment can regulate the expression of related gene anti-aging with antioxidation in primary hepatocyte.
Figure 30 is that Masson dyeing display Caulis Hederae Sinensis subacid can reduce mouse liver collagen deposition.
Figure 31 is that Caulis Hederae Sinensis subacid can lower the expression of hepatic fibrosis marker gene in liver.
In Figure 12～15,20～22, in 25,31, * represents that p < 0.05, * * represents that p < 0.01, * * * represents p < 0.001.
Detailed description of the invention
Following example will the present invention is further illustrated in conjunction with accompanying drawing.
Embodiment one: Caulis Hederae Sinensis subacid can suppress growth of tumour cell.
Test method: mtt assay is a kind of method detecting cell survival and growth.The method is widely used in large-scale anti-swollen
Tumor medicine screens.Its feature is highly sensitive, economical.The present embodiment utilizes mtt assay, with the DMEM containing 10% hyclone
The tumor cell of the various people of culture medium culturing, including human colon cancer cell HCT116 cell, the people sensitive for ATRA of ATRA resistance
Colon and rectum adenocarcinoma cell HCT15 cell, Human Prostate Cancer Cells LNCaP cell, human cervical carcinoma cell Hela cell, people liver are thin
Born of the same parents' cancerous cell HepG2 cell, human breast cancer cell MCF7 cell and mouse monokaryon macrophage leukaemia Raw264.7 are thin
Born of the same parents, with working concentration be 10 μMs, 20 μMs, 30 μMs, 40 μMs, the Caulis Hederae Sinensis subacid of 50 μMs process cell respectively
After 48h and/or 96h, detect cell survival rate with mtt assay.
Result of the test: result such as Fig. 1～10, at human colon cancer cell HCT116 cell (Fig. 2), people's Colon and rectum adenocarcinoma cell
HCT15 cell (Fig. 4), Human Prostate Cancer Cells LNCaP cell (Fig. 6), human cervical carcinoma cell Hela cell (Fig. 7),
Human breast cancer cell MCF7 cell (Fig. 8), human liver cell cancerous cell HepG2 cell (Fig. 9) and mouse monokaryon macrophage are white
In disorders of blood cell Raw264.7 cell (Figure 10), the tumor cell acid-treated with Caulis Hederae Sinensis time is obvious in the survival rate of 96h
The cell processed less than blank.And the biggest along with Caulis Hederae Sinensis time acid concentration, the effect of suppression growth of tumour cell is the most aobvious
Write.At human colon cancer cell HCT116 cell (Fig. 1), people's Colon and rectum adenocarcinoma cell HCT15 cell (Fig. 3) and human prostate
In cancerous cell LNCaP cell (Fig. 5) experiment, just show significantly at 48h with the acid-treated tumor cell of Caulis Hederae Sinensis time
Cytostatic effect.Show that Caulis Hederae Sinensis subacid has suppression function to the growth of these cancerous cell.
Human colon cancer cell HCT116 cell is the tumor cell showing as resisting all-trans-retinoic acid (ATRA), typically resists
Tumour medicine ATRA can not effectively treat the tumor with ATRA resistance.This example demonstrates that, Caulis Hederae Sinensis subacid is to this kind of ATRA
Repellence tumor cell also have an inhibitory action, also can suppress people's Colon and rectum adenocarcinoma cell HCT15 cell of ATRA sensitivity simultaneously,
Indicate treatment function excellent in the tumor that Caulis Hederae Sinensis subacid is insensitive to ATRA.
Conclusion: Caulis Hederae Sinensis subacid is in colon cancer, glandula rectalis, carcinoma of prostate, cervical cancer cell, hepatocarcinoma, breast carcinoma and white
Disorders of blood treatment aspect has effect.In the tumor that treatment ATRA is insensitive, also there is outstanding function.
Embodiment two: the liver protecting of Caulis Hederae Sinensis subacid and repair function test.
Test method: excessive use APAP cause chmice acute liver lesion induced by drugs wound model be model that research hepatic is conventional it
One, APAP are the Typical Representative things being caused drug induced hepatic injury by active oxygen or active nitrogen.APAP is excessively used and can cause liver
Active oxygen increases, and glutamic oxaloacetic transaminase, GOT AST, glutamate pyruvate transaminase ALT and lactate dehydrogenase L DH isoreactivity rise, and cause liver group
Tissue inflammation or necrosis.The present embodiment i.e. uses this model inspection Caulis Hederae Sinensis subacid to the protection of APAP induced synthesis hepatic injury and reparation
Function.
With the C57B6/J wild-type mice of 8 week old, at Xiamen University's Experimental Animal Center SPF rank Animal House, raise by routine
Material is fed, and freely drinks water.Caulis Hederae Sinensis subacid DMSO high concentration is dissolved, then with 40%HBC (2-hydroxypropyl-β
-cyclodextrin) it is configured to working concentration, making DMSO concentration is 10%, and makes lumbar injection 100 μ l Caulis Hederae Sinensis subacid
The final working concentration of solution is 10mg/kg (medicine/Mouse Weight) and 40mg/kg.Blank group is directly with the DMSO of equivalent
It is diluted in 40%HBC solution.Every morning 9 injection, once a day, after injecting 5 days, gives little the 5th afternoon 3
Mus injection 500mg/kg (medicine/Mouse Weight) APAP of PBS fresh lysate preparation.Put to death mice after 24h, take part
Liver organization paraformaldehyde is fixed, paraffin embedding, section, and HE dyes.Partial liver tissue liquid nitrogen cryopreservation, Trizol reagent
Extract RNA, after reverse transcription becomes cDNA, with the RealMasterMix (SYBR Green I) of Tian Gen biochemical technology company limited
Test kit carries out real-time fluorescence quantitative PCR reaction, GCLM, GSTa3, GSTa4, GPX1, UGT1A1 etc. in detection liver organization
The expression of liver Related to repair gene.Take serum, be used for detecting glutamic oxaloacetic transaminase, GOT AST in serum, glutamate pyruvate transaminase ALT and breast
Acidohydrogenase LDH activity.
Difference student between matched group and drug treating group in all embodiments ' s t-test analyzes.
Result of the test: liver section HE coloration result such as Figure 11, lobules of liver inner cell infiltration seen from blank group pathological section
Substantially, in cavity shape and necrosis region, there is massive inflammatory cells infiltrated in lobule and portal area, and cell is muddy, karyopycnosis or solubilized
Solving broken, hepatic cords obscures.Compared with the hepatic injury the most serious with blank group, inject the Mouse Liver of Caulis Hederae Sinensis subacid
Dirty form shows normal (Figure 11), and the process that working concentration is 10mg/kg and 40mg/kg all has and well repaiies
Multiple effect.Employ AST and ALT activity (such as Figure 12) and LDH activity (Figure 13) in the mice serum of Caulis Hederae Sinensis subacid to show
Write less than blank group.Gene expression dose result relevant to hepatic injury in liver organization also indicates that, on Caulis Hederae Sinensis subacid energy
The liver reparations such as tune and external source toxicant metabolism closely-related functional gene GCLM, GSTa3, GSTa4, GPX1, UGT1A1
The expression (such as Figure 14) of related gene, shows that Caulis Hederae Sinensis subacid can be adjusted by the expression of exogenous drugs metabolism related gene
Control thus regulate protection and repair liver function.
Conclusion: this example demonstrates that Caulis Hederae Sinensis subacid has well protection and repair function in terms of hepatic injury.
Embodiment three: Caulis Hederae Sinensis subacid therapeutical effect in drug-induced inflammatory reaction.
Test method: inflammatory factor is the material being produced by cell in inflammatory process and secreting and participate in inflammatory reaction, is reaction inflammation
The mark of disease.The present embodiment utilizes the C57B6/J wild-type mice liver group of eight week old such as embodiment two APAP drug treating
Knit, extract RNA with Trizol reagent, after reverse transcription becomes cDNA, with the RealMasterMix of Tian Gen biochemical technology company limited
(SYBR Green I) test kit carries out real-time fluorescence quantitative PCR reaction, iNOS, TGF β, TNF in detection mouse liver tissue
The expression of α, COX2, IL-1 β and MIP1 α inflammatory factor.
Result of the test: such as Figure 15, compare blank, in the mouse liver tissue that Caulis Hederae Sinensis time is acid-treated, iNOS, TGF
The expression of the inflammation factors such as β, TNF α, COX2, IL-1 β and MIP1 α is significantly lowered.
Conclusion: Caulis Hederae Sinensis subacid can suppress the inflammatory reaction caused by chemicals.
Embodiment four: Caulis Hederae Sinensis subacid effect in terms of bacterial inflammation.
Test method: inflammation is the basic pathology process that animal is common and important, any factor that can cause tissue injury
Cause the generation of inflammation.Bacteria lipopolysaccharide (Lipopolysaccharides is called for short LPS) is the master that gram-negative bacteria causes a disease
Wanting factor, it can cause the inflammatory reaction of body.Utilize cell or body inflammatory reaction that LPS induces, be commonly used for gene or
The effect in inflammation of person's medicine and study mechanism.
The present embodiment utilizes the primary hepatocyte extracted from C57B6/J wild-type mice, by cell by 5 × 105Individual/hole, divides
6 porocyte culture plates, are divided into 3 groups, often 3 holes of group.After DMEM in high glucose culture medium (containing 10% hyclone) overnight incubation,
3rd group of cell changes the fresh culture (medicine group) of the Caulis Hederae Sinensis subacid (dissolving with DMSO) containing 20 μMs, and first and second
Two groups of cells add the fresh culture with the 3rd group of isopyknic DMSO of medicine as compareing (matched group), after process 18h,
In second group of matched group, add the LPS (blank-induction group) of 20 μ g/ml, the 3rd group of medicine group adds 20 μ g/ml
LPS (medicine-induction group).First group is blank non-induced group, is not added with LPS process.Add after LPS processes 6h and receive cell,
Extracting cell RNA with the total RNA extraction reagent box of Omega company, the Reverse Transcription box of TAKARA company carries out reverse transcription one-tenth
After cDNA, carry out real-time fluorescence with RealMasterMix (the SYBR Green I) test kit of Tian Gen biochemical technology company limited fixed
Amount PCR reaction, detects the expression of intracellular inflammatory factor.
Result of the test: from Figure 16～19 it can be seen that compare blank non-induced group, each inflammatory factor in the cell of LPS induction
Express notable rising, show that the cellular inflammation reaction that LPS induces has produced.Thin in the induction group having Caulis Hederae Sinensis time acid-treated
In born of the same parents, including IFN γ, TGF β, TNF α and CD36 etc., the expression of the inflammation factor is substantially lowered, and shows Chang Chun
Rattan subacid can effectively reduce the level of intracellular inflammatory factor, slows down inflammatory symptom.
Conclusion: Caulis Hederae Sinensis subacid has curative effect in terms of inflammation treatment, including bacterial inflammation.
Embodiment five: Caulis Hederae Sinensis subacid is at treatment diabetes, obesity, fatty liver, cardiovascular disease, hyperglycemia, insulin
Effect in terms of the diseases such as opposing, cardiovascular disease.
Test method: db/db mice (family name BKS.Cg-Dock7m+/+Leprdb/ JNju) belong to type Ⅱdiabetes mellitus model,
Animal started voracity and gets fat when one month, then produced the symptom such as hyperglycemia, high blood insulin, and fatty liver, nephritis etc. are also
It it is the complication of this disease model.The present embodiment uses this Research of Animal Model for Study Caulis Hederae Sinensis subacid in terms for the treatment of metabolic disease
Effect.
Utilize the male db/db mice of the 8-10 week old that high lipid food (Research Diets, D12492) feeds, lumbar injection
The Caulis Hederae Sinensis time acid solution that method is equipped with as described in embodiment two, makes the lumbar injection 100 μ l Caulis Hederae Sinensis time final work of acid solution
It is respectively 50mg/kg (medicine/Mouse Weight) as concentration.Every day injects once, and after injection in the 7th day, empty stomach 6h, collects mice
Serum, for detecting serum glucose and the level of serum insulin in serum, uses following test kit, to specifications institute respectively
Method of stating detects: Glucose estimation kit-enzymatic measurement (Beijing Puli's lema gene Technology Co., Ltd.) and the super quick islets of langerhans of mice
Element test kit (Crystal Chem.Inc., the U.S.).
Take liver organization frozen section, dye for oil red.Oil red colouring method: liver organization is in isopentane, in liquid nitrogen
Quick freezing, embeds optimized cutting temperature, cuts on cryostat.
Oil red stain is prepared: oil red O 0.5g, in 60 DEG C of baking ovens of isopropanol (content more than 98%) 100ml, 24h, fully
As storing liquid after dissolving.Facing the used time takes dyeing stock solution 6ml, adds distilled water 4ml, and dilution stands and filters after 5～10min,
This liquid preserves and not can exceed that 1～2h.
Frozen section 10 μ m-thick, 10% paraformaldehyde fix 10～15min after according to a conventional method oil red dye fat, hematoxylin is light
Dye core.
Result of the test: compare blank group mice, the db/db mice serum glucose level of Caulis Hederae Sinensis time acid treatment substantially drops
Low (Figure 22), the level of serum insulin is significantly reduced (Figure 21), shows that Caulis Hederae Sinensis subacid can pass through enhancing body insulin
Sensitivity reduces the effect of blood glucose.
Oil red O is lipid-soluble dye, in fat can high dissolution, the neutral fats such as triglyceride specific can be made in tissue
Color.In daily pathological diagnosis and research work, show that in-house fat dyes frequently with oil red O.The present embodiment oil red
Coloration result shows, in the db/db mouse liver tissue of Caulis Hederae Sinensis time acid treatment, oil droplet is significantly less than matched group, and Fat Accumulation is bright
Aobvious improvement (Figure 23), shows that Caulis Hederae Sinensis subacid has good efficacy to fatty liver.
Conclusion: Caulis Hederae Sinensis subacid can increase the insulin sensitivity of body, effective in cure to the disease of insulin resistance.Caulis Hederae Sinensis
Subacid can reduce blood glucose, effective in cure to diabetes.Caulis Hederae Sinensis subacid can effectively treat fatty liver, including non-alcoholic fatty liver disease.
Diabetes, insulin resistant and fatty liver diseases are frequently accompanied by the performance of cardiovascular disease, obesity, Caulis Hederae Sinensis time
Acid has good efficacy to diabetes, insulin resistant and fatty liver, therefore analyzes and reaches a conclusion, and Caulis Hederae Sinensis subacid is to cardiovascular disease
Sick, obesity also has therapeutic effect.
Embodiment six: Caulis Hederae Sinensis subacid effect in terms of the treatment disease such as hyperglycemia and non-alcoholic fatty liver disease.
Test method: KK-Ay mice (KK/Upj-Ay/J) is central obesity, medium diabetes mellitus animal model, eight week old height blood
The symptoms such as sugar, hyperinsulinism, hyperlipidemia, glucose intolerance, liver organization fat excess accumulation, its diabetes are by islets of langerhans
Element opposing causes (type Ⅱdiabetes mellitus mice).The present embodiment utilizes KK-Ay mouse test Caulis Hederae Sinensis subacid to control metabolic disease
Treatment functions.
Utilize the male KK-Ay mice of the 8-10 week old that high lipid food (Research Diets, D12492) feeds, lumbar injection
The Caulis Hederae Sinensis time acid solution that method is equipped with as described in embodiment two, makes the lumbar injection 100 μ l Caulis Hederae Sinensis time final work of acid solution
It is respectively 10mg/kg (medicine/Mouse Weight) as concentration.Every day injects once, and after injection in the 14th day, empty stomach 6h, collects little
Mus serum, is used for detecting serum level of glucose in serum, uses following test kit, and the most described method detects: Fructus Vitis viniferae
Sugar determination test kit-enzymatic measurement (Beijing Puli's lema gene Technology Co., Ltd.).
Take liver organization frozen section, dye for oil red.Method is with embodiment five.
Result of the test: compare blank group mice, the KK-Ay mice serum glucose level of Caulis Hederae Sinensis time acid treatment substantially drops
Low (Figure 25), shows that Caulis Hederae Sinensis subacid can reduce blood glucose.
Oil red coloration result shows, only with the KK-Ay mice of the Caulis Hederae Sinensis of 10mg/kg time acid treatment, in liver organization, oil droplet is just
Can be significantly less than matched group, Fat Accumulation is clearly better (Figure 24), further demonstrates that Caulis Hederae Sinensis subacid is to non-alcoholic fatty liver disease
Good efficacy.
Conclusion: Caulis Hederae Sinensis subacid can reduce blood glucose, effective in cure to diabetes.Caulis Hederae Sinensis subacid can effectively treat fatty liver, bag
Include non-alcoholic fatty liver disease.
Embodiment seven: Caulis Hederae Sinensis subacid effect in treatment kidney disease
Test method: kidney is the major organs of excretion carbamide, and carbamide all can heavily absorb at each section of tubule after glomerular filtration,
In renal tubules, uroflow speed is the fastest, heavily absorbs the fewest.When renal function injury, glomerular filtration rate declines, and filtration rate drops to
When normal less than 50%, the concentration of blood urea nitrogen raises rapidly.Various parenchymal lesion of the kidney, such as glomerulonephritis, chromic fibrous
In nephritis, acute or chronic renal failure, kidney, occupancy and destructive pathological changes all can make blood urea nitrogen increase.Therefore, blood urea nitrogen
It it is one of renal function leading indicator.Blood urea nitrogen raises also can be as one of uremia's diagnosis index.
Chronic renal disease is one of complication of diabetes.The present embodiment db/db mice serum processed in embodiment five,
Blood urea nitrogen (BUN) testing cassete (urease method, the C013-2) by specification building up Science and Technology Ltd. with Nanjing surveys urea in serum
The level of nitrogen.
Result of the test: compare blank group, the level of the Mouse Blood blood urea nitrogen of Caulis Hederae Sinensis time acid treatment significantly lowers (Figure 20),
Show that Caulis Hederae Sinensis subacid is to the therapeutic effect in terms of the maintenance of renal function and kidney disease.
Conclusion: Caulis Hederae Sinensis subacid is at the various parenchymal lesion of the kidney occurring that blood urea nitrogen level raises, such as glomerulonephritis, interstitial
Property nephritis, acute or chronic renal failure, kidney in occupancy and destructive pathological changes, and the kidney disease such as uremia have curative effect.
Embodiment eight: reporter assay proves that Caulis Hederae Sinensis subacid is the part of FXR.
Test method: use the DMEM culture medium containing 10% hyclone to carry out monkey renal epithelial cell COS-7 cell and cultivate, transfection
The previous day carries out COS-7 cell inoculation at 24 orifice plates, and inoculum density is 5 × 104Cell/every hole, transfects for second day.Transfection
Lipofectamine 2000 (Invitrogen) transfection reagent is used to carry out transient transfection.Every porocyte cotransfection 200ng's
The Renilla of endogenesis promoter reporter gene EcRE and 30ng of people source nuclear receptor FXR total length expressed plasmid pCMX-FXR, 200ng
Luciferase expression plasmid, in plasmid DNA: transfection reagent be 1: 2 ratio transfect.After transfection 5h, every hole is separately added into
500 μ L contain the Opti-MEM culture fluid of the Caulis Hederae Sinensis subacid of 0,1.25 μMs, 2.5 μMs, 5 μMs and 10 μMs, process
18h.After ligand drug processes 18h, carry out reporter gene with double fluorescein prunus mume (sieb.) sieb.et zucc. Reporter Gene Assay Kit of Promega company and live
Property analyze.Cell lysis is used for luciferase test experience, takes 10 μ L cell cell lysis and transfers to 96 orifice plates, adds 50 μ
EnSpire is used after L luciferase reaction liquidTM2300 multi-functional microplate reader (Perkin Elmer) detection transmitting light under 560nm
Signal, adds afterwardsReactant liquor terminates luciferase reaction and detects renilla luciferase vitality.Reporter gene
Activity is with renilla activity as internal reference.
Result of the test: compare the cell of control treatment, adds luciferase activity in the cell of Caulis Hederae Sinensis subacid and rises.Along with
The concentration of Caulis Hederae Sinensis subacid increases, and the luciferase activity of reporter gene is in rising trend.Show that Caulis Hederae Sinensis subacid can activate FXR
Transcriptional activity, and there is concentration dependent.This result shows that Caulis Hederae Sinensis subacid is the agonist (Figure 26) of nuclear receptor FXR.
Conclusion: the metabolism such as the carbohydrate metabolism that participates in body due to FXR, lipid metabolism, cholesterol-cholic acid metabolism, inflammation, tumor,
The function of the aspects such as liver cirrhosis, hepatic injury, Adipose Differentiation, therefore Caulis Hederae Sinensis subacid can by activate FXR, these physiology,
The disease that pathology is relevant has regulatory function.Also further demonstrate that the function in above seven embodiments such as may be through
FXR realizes its treatment function.
Embodiment nine: AlphaScreen method proves that Caulis Hederae Sinensis subacid is the part of FXR.
Test method: AlphaScreen (Amplified Luminescent Proximity Homogenous Assay) analyzes
Method is the biochemical analysis method of a kind of the sensitiveest energy detection compound interaction of PE company invention.The method brief
Principle is: with biotin labeled counselor work polypeptide so that it is can be combined with the donor bead being marked with Streptavidin；
With ligand binding domains (LBD) albumen of the FXR with hexahistine His6 labelling so that it is can be with the receptor being marked with nickel
Pearl combines.If there was added in reaction system ligand compound FXR conformation can be induced to change so that its with auxiliary regulation because of
Sub-polypeptide combines, and at this moment donor bead is close with receptor pearl, excites with 680nm light, 520-620nm fluorescence will be detected
Signal.These reaction concrete operations are according to the Alpha Screen nickel chelate detection of Perkins-Elmer company
Kit test kit is carried out, by the Envision Xcite Multilabel Reader microplate reader of Perkins-Elmer company after reaction
Reading.
The mechanism of action of nuclear receptor be by with part (ligand) interact and occurred conformation change, thus with numerous different auxiliary
Activity factor selective binding carrys out the coordinated expression of controlling gene.SRC1 is exactly one of which counselor work, nuclear receptor and SRC1
Its transcriptional activity can be started after in conjunction with.The transcriptional activity of FXR can be activated, tentatively based on embodiment eight demonstrates Caulis Hederae Sinensis subacid
Judging the part that it is FXR, the present embodiment analyzes method by Alphascreen, is subject in order to detect ligand compound induction core
Body FXR raises the binding ability of counselor work SRC1.The reaction system of this experiment is that merging of 20nM is histidine-tagged
FXR receptor LBD albumen, the SRC1-2 cofactor polypeptide of 20nM biotinylation labelling, the donor of 5 μ g/ml and receptor pearl,
Buffer (50mM MOPS, 50mM NaF, 0.05mM CHAPS, and 0.1mg/ml bovine serum albumin,
PH7.4), adding the Caulis Hederae Sinensis subacid of 1 μM of working concentration, reaction system 50 μ L is in 384 orifice plates after room temperature reaction 1h
The transmitting optical signal of 520～620nm under 680nm exciting light is read with AlphaScreen detector.For Alphascreen
The biotin labeled polypeptide SRC1-2 analyzed is the peptide sequence being combined with nuclear receptor in SRC1, and sequence is as follows:
SPSSHSSLTERHKILHRLLQEGSP.Blank is to replace the Caulis Hederae Sinensis subacid in experimental group with isopyknic DMSO.
Result of the test: compare blank group, detects notable in the reaction of the Caulis Hederae Sinensis subacid adding 1 μM of working concentration
The fluorescence signal (Figure 27) improved.
Conclusion: Caulis Hederae Sinensis subacid energy induced nuclear receptor FXR is combined with co-activator SRC1, further demonstrates that Caulis Hederae Sinensis subacid is
The part of FXR.Show that Caulis Hederae Sinensis subacid can be by activating FXR, in the disease that the various physiology of FXR regulation, pathology are relevant
There is regulatory function, such as hypertriglyceridemia, hypercholesterolemia, cholestasis, cholelithiasis, atherosclerosis etc..
Also further demonstrate that the function in above seven embodiments such as may be through FXR to realize its treatment function.
Embodiment ten: the antioxidation of Caulis Hederae Sinensis subacid and anti-aging function test.
Test method: one of critical function of reduced glutathion (GSH) is to play as intracellular natural antioxidant to make
With.Human senility, infect, be poisoned, heterotoxin, oxidative stress etc. can make intracellular GSH biosynthesis ability drop
Low, content declines, or makes GSH be changed into double sulfur oxidized form (GSSG).
The present embodiment utilizes the mouse embryo fibroblasts (MEF) cultivated, at the Caulis Hederae Sinensis subacid that working concentration is 20 μMs
After reason cell 24h, measure method detection described in test kit (Nanjing is built up) by specification with reduced glutathion (GSH)
GSH level in MEF cell.
Primary hepatocyte is extracted according to a conventional method, with the Caulis Hederae Sinensis that working concentration is 20 μMs time acid treatment from C57B6/J mice
After cell 24h, extract cell RNA with Trizol reagent, after reverse transcription becomes cDNA, detect with quantitative real-time PCR
With the mrna expression level of antioxidation and SAG in cell.
Result of the test: in mice MEF cell experiment, in the cell after Caulis Hederae Sinensis time acid treatment, reducing substances GSH level is obvious
Higher than blank group (Figure 28), show that Caulis Hederae Sinensis subacid can promote that cell produces the reducing substances of anti-oxidation function.Chang Chun
Rattan time acid treatment can significantly improve the expression of antioxidant genes such as SOD1, SOD2, SESN1 and SESN2, also can significantly under
Adjust and promote oxidation and the expression (Figure 29) of old and feeble AC5 gene.
Conclusion: Caulis Hederae Sinensis subacid has antioxidation and anti-aging function.
Embodiment 11: Caulis Hederae Sinensis subacid can remove ultra-oxygen anion free radical
Test method: free radical can damage the immunity of body, induction canceration, the repair function of interference cell, interference cell
Metabolism etc., machine cylinder accumulation crosses polyradical, can be to causing the aging of body, canceration, inflammation and immune disease etc.
Serious consequence.Therefore, improve the oxidation resistance of body, can resist and be produced by active oxygen and the lipid peroxidation that causes thereof
Disease.
In order to detect Caulis Hederae Sinensis subacid effect at the anti-aging aspect of antioxidation, the present embodiment referenced patent CN further
The method of 102813683 B, uses assay NBT photoreduction to have detected Caulis Hederae Sinensis subacid and removes the energy of ultra-oxygen anion free radical
Power.Concrete operations are: take the Tris-HCl buffer 165 μ l of 0.05mol/L pH 8.2, are placed in 25 DEG C of water-baths preheating
20min, is separately added into Caulis Hederae Sinensis subacid and the pyrogallol solution of 15 μ l 25mmol/L of 40 μ l 1 μm ol/L, mixed
In 25 DEG C of water-baths, react 5min, the HCl adding 40 μ l 8mol/L after even terminate reaction, make with Tris-HCl buffer
Comparison, measures absorbance at 299nm, calculates clearance rate.Blank group replaces sample with deionized water, and each process is equal
Do 3 repetitions.The computing formula of clearance rate: ultra-oxygen anion free radical clearance rate (%)=(A1-A2)/A1 × 100%, its
Middle A1 is the mean light absorbency of blank, and A2 is the mean light absorbency of Caulis Hederae Sinensis subacid.
Result of the test: in this reaction, Caulis Hederae Sinensis subacid is 58% to the clearance rate of superoxide anion, standard deviation 6.4%,
Student ' s t-test analyzes p < 0.01.
Conclusion: show that Caulis Hederae Sinensis subacid has preferable elimination effect to ultra-oxygen anion free radical.The result of ten in conjunction with the embodiments,
Show that Caulis Hederae Sinensis subacid can remove superoxide anion by antioxidation and anti-aging gene expressions such as regulation superoxide dismutases
Free radical, improves intracellular reducing substances level, thus plays antioxidation and anti-aging effect.
Embodiment 12: Caulis Hederae Sinensis subacid effect to liver cirrhosis.
Test method: fatty liver is a kind of hepatopathy that in liver, too much accumulation fat causes.Cross fattiness to put aside for a long time in liver,
Supply and the liver own metabolism of liver blood and oxygen will be affected, and can gradually cause a large amount of liver cell swelling, inflammatory infiltration and change
Property downright bad, once have fibroplasia and pseudolobuli to be formed, liver cirrhosis will be become, be greatly increased the danger suffering from hepatocarcinoma.Liver cirrhosis
In patient's liver organization, various collagen contents have increase.Therefore the present embodiment identifies Caulis Hederae Sinensis subacid by Masson Albert'stain Albert method
Whether process can alleviate liver the symptom of collagen deposition in the mouse liver tissue of fatty excess accumulation, and passes through quantitative fluorescent PCR
The expression of method detection collagen protein.
Method one:
Real-time fluorescence quantitative PCR: by embodiment five small mouse partial liver tissue liquid nitrogen cryopreservation, Trizol reagent extracts liver group
Knit after the Reverse Transcription box reverse transcription of RNA, Takara company becomes cDNA, with Tian Gen biochemical technology company limited
RealMasterMix (SYBR Green I) test kit carries out real-time fluorescence quantitative PCR reaction, the CFX96 of Bio-rad company
Quantitative real time PCR Instrument reacts.Liver collagen and the expression of fibrosis associated genes in detection liver organization.
Method two:
Masson Albert'stain Albert Master for showing one of colouring method of fiber in tissue, is collagen fiber dyeing authority and classics
Technical method.Masson Albert'stain Albert method is the space according to different tissues and cell and varies in size, thus the permeability of tissue
Difference, wherein collagen fiber short texture comparatively speaking, permeability is high, can allow the dyestuff of aniline blue (blue) this macromole
Entering coloring, and the space of erythrocyte in organizing, muscle fiber part is less, thus aniline blue can not enter coloring.The method energy
The existence of collagen fiber and accumulation degree in dividing tissue.It is widely used in the research etc. of connective tissue, muscular tissue and collagen protein.
This colouring method is simple to operation, stable performance, and colour developing is clear.
The present embodiment uses Nanjing to build up the quick Masson dye liquor test kit (article No. D026) of Science and Technology Ltd., according to this examination
Agent box description, carries out Masson dyeing to the mouse liver processed in embodiment five, and coloration result is: collagen fiber are blueness,
Endochylema, muscle, cellulose, neuroglia take on a red color, karyon bluish violet.
Result of the test: fluorescent quantitative PCR result shows (such as Figure 31), marker gene α 1 (I) collagen of hepatic fibrosis,
TIMP1, α-SMA and MCP-1 gene expression in the mouse liver of Caulis Hederae Sinensis time acid treatment are significantly lower than matched group.
Masson coloration result can be seen that (such as Figure 30), in the liver section of matched group, it can be seen that have bright at blood vessel
The collagen deposition of aobvious colored blue, the blood vessel in the mouse liver section of Caulis Hederae Sinensis time acid treatment is then without obvious collagen
Deposition blueness performance.
Conclusion: combine result in the embodiment described above, Caulis Hederae Sinensis subacid can slow down the inflammatory reaction of liver cell and tissue, energy
Reducing liver organization Fat Accumulation, Caulis Hederae Sinensis subacid can also reduce hepatic fibrosis marker gene expresses, and effectively eliminates collagen in liver
Accumulation, thus Caulis Hederae Sinensis subacid can prevent and treat liver cirrhosis disease.
Claims (10)
1. Caulis Hederae Sinensis subacid supports in preparation prevention and treatment diabetes, obesity, fatty liver, liver cirrhosis, hyperglycemia, insulin
Anti-disease, cardiovascular disease, hypertriglyceridemia, hypercholesterolemia, tumor, hepatic injury, inflammation, kidney disease,
Cholestasis, cholelithiasis, atherosclerotic medicine or health product are applied；
Described Caulis Hederae Sinensis subacid, molecular formula is C29H44O3, chemical name is 3-oxo-24-nor-olive-12-alkene-28-acid,
Structural formula is as follows:
Apply the most as claimed in claim 1, it is characterised in that described diabetes can include but not limited to have hyperglycemic symptoms
Diabetes；Described obesity can include but not limited to the obesity with fatty liver, hyperglycemic symptoms；Described fatty liver is permissible
Include but not limited to non-alcoholic fatty liver disease；It is fine that described liver cirrhosis can include but not limited to have Liver Collagen over-deposit regulating liver-QI
The liver cirrhosis of dimensionization performance.
Apply the most as claimed in claim 1, it is characterised in that described insulin resistance disease can include but not limited to have high pancreas
The insulin resistance disease of island element mass formed by blood stasis；It is excessive that described cardiovascular disease can include but not limited to have hyperglycemia, liver fat
The cardiovascular disease of accumulation symptom.
Apply the most as claimed in claim 1, it is characterised in that described tumor can include but not limited to colon and rectum carcinoma, front
Row adenocarcinoma, cervical cancer, hepatocarcinoma, breast carcinoma and leukemia；It is insensitive that described tumor also includes showing as all-trans-retinoic acid
Tumor, include but not limited to the colon cancer of ATRA resistance.
Apply the most as claimed in claim 1, it is characterised in that described hepatic injury can include but not limited to owing to having taken hepatic injury
The drug-induced hepatic injury of side effect, the acute liver damage, the alcoholic fatty liver hepatic injury caused that cause due to ethanol, exempt from
Hepatic injury, virus that epidemic disease or chemical factor cause infect the hepatic injury caused；Described have hepatic injury side effect medicine to include but
It is not limited to the medicine with acetaminophen as main active.
Apply the most as claimed in claim 1, it is characterised in that described inflammation can include but not limited to owing to having taken inflammatory reaction
Inflammation caused by side effect medicine, the acute hepatitis caused due to ethanol, the hepatitis caused by alcoholic fatty liver, by non-wine
Hepatitis that essence fatty liver causes, viral hepatitis, cause by bacterial inflammation, due to diabetes or obesity complication
Hepatitis or nephritis, described in have inflammatory reaction side effect medicine to include but not limited to the medicine that acetaminophen is main active
Thing.
Apply the most as claimed in claim 1, it is characterised in that described kidney disease can include but not limited to there is blood urea nitrogen level
The kidney disease of Novel presentation, various parenchymal lesion of the kidney, uremia；Described parenchymal lesion of the kidney can include but not limited to kidney
The parenchymal lesion of the kidney such as occupancy and destructive pathological changes in bead nephritis, interstitial nephritis, acute or chronic renal failure, kidney.
Apply the most as claimed in claim 1, it is characterised in that described Caulis Hederae Sinensis subacid is as active ingredient and other medicines and system
After in medicine, acceptable excipient/or drug administration carrier mix, pharmaceutical methods routinely and technological requirement, make pharmaceutical composition,
Described pharmaceutical composition can use oral type preparation or injection-type preparation medicine, described oral type preparation can use tablet, pill,
Capsule, electuary or syrup；Described injection-type preparation can use injection or freeze-dried powder dosage form.
9. Caulis Hederae Sinensis subacid described in is applied in preparing antioxidation, anti-aging health product or skin care item；
Described Caulis Hederae Sinensis subacid, molecular formula is C29H44O3, chemical name is 3-oxo-24-nor-olive-12-alkene-28-acid,
Structural formula is as follows:
Apply the most as claimed in claim 9, it is characterised in that described antioxidation, anti-aging include but not limited to that preparation is removed super
The health product of oxygen anion free radical and skin care item and/or preparation enhancing body reducing substances such as reductive glutathione guarantor
Application in strong product and skin care item.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510263191.5A CN106265680B (en) | 2015-05-22 | 2015-05-22 | Application of ivy hypochloric acid |
| PCT/CN2016/071561 WO2016116054A1 (en) | 2015-01-22 | 2016-01-21 | Modulators of farnesoid x receptor and methods for the use thereof |
| US15/544,410 US20180116993A1 (en) | 2015-01-22 | 2016-01-21 | Modulators of farnesoid x receptor and methods for the use thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510263191.5A CN106265680B (en) | 2015-05-22 | 2015-05-22 | Application of ivy hypochloric acid |
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| CN106265680B CN106265680B (en) | 2020-01-21 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107744534A (en) * | 2017-11-10 | 2018-03-02 | 中国药科大学 | The application of hederagenin and its glucosides in the medicine of hepatic cholagogic is prepared |
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2015
- 2015-05-22 CN CN201510263191.5A patent/CN106265680B/en active Active
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| LI JIAN-JUAN ET AL.: ""Chemical constituents from the stems of Celastrus orbiculatus"", 《CHINESE JOURNAL OF NATURAL MEDICINES》 * |
| LUO JIAN-GUANG ET AL.: ""New Pentacyclic Triterpenes from Gypsophila oldhamiana and Their Biological Evaluation as Glycogen Phosphorylase Inhibitors"", 《CHEMISTRY BIODIVESITY》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107744534A (en) * | 2017-11-10 | 2018-03-02 | 中国药科大学 | The application of hederagenin and its glucosides in the medicine of hepatic cholagogic is prepared |
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