CN103301179B - Application of eucommia ulmoides lignan extract in preparing PPARalpha agonist - Google Patents

Application of eucommia ulmoides lignan extract in preparing PPARalpha agonist Download PDF

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CN103301179B
CN103301179B CN201310271248.7A CN201310271248A CN103301179B CN 103301179 B CN103301179 B CN 103301179B CN 201310271248 A CN201310271248 A CN 201310271248A CN 103301179 B CN103301179 B CN 103301179B
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cortex eucommiae
extract
mass concentration
lignans extract
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CN103301179A (en
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李慧
李振宇
严谨
姜德建
胡凯
郭成贤
周宏灏
欧阳冬生
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Changsha Duzheng Biotechnology Co., Ltd
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Abstract

The invention relates to the technical field of medicines, particularly provides application of an eucommia ulmoides lignan extract in preparing a PPARalpha agonist, and in particular relates to the application of the eucommia ulmoides lignan extract in preparing a drug for preventing and treating hyperlipemia. Lignan in the eucommia ulmoides lignan extract accounts for 30%-90% of the total weight of the eucommia ulmoides lignan extract; and the content of the effective constituent pinoresinol diglucoside in the lignan accounts for 1%-15% of the total weight of the eucommia ulmoides lignan extract.

Description

The application of Cortex Eucommiae lignans extract in the medicine for the preparation of control hypertriglyceridemia
Technical field
The present invention relates to medical art, be specifically related to the novelty teabag of Cortex Eucommiae lignans extract.
Background technology
Hyperlipemia (hypercholesterolemia and/or hypertriglyceridemia) is common blood fat disorder disease, is the independent hazard factor of the diseases such as atherosclerosis, coronary heart disease, acute myocardial infarction, apoplexy.Clinical research confirmation, statins reduces blood plasma cholesterol level thus reduces the incidence rate of all kinds of cardiovascular disease and lethal disability rate.
Research in recent years shows; even if part patients with dyslipidemia still faces the left risk of cardiovascular after excess dosage statin strengthening cholesterol lowering therapeutic; particularly with the patient of type 2 diabetes mellitus, obesity and metabolism syndrome, in its blood plasma, triglyceride (TG) raises is one of Major Risk Factors of the left risk of cardiovascular.
At present, the medicament categories of specific aim control plasma triglyceride is less clinically, efficacy and saferry existing defects, therefore, carries out new drug research, find new triglyceride reducing medicine significant for reduction TG and fatty acid.
PPAR-α belongs to the nuclear receptor superfamily of ligand activation, expresses in liver, the heart, muscle, arterial endothelium cells, vascular smooth muscle cell, macrophage, T lymphocyte.Activate PPAR-α can with ligand binding after form heterodimer with retinoid X receptors bind again, regulate each target gene by PPAR-α response element.PPAR-α can change hepatocellular lipid metabolism.PPAR-α can raise the oxidative metabolism enzyme in mitochondrion and microsome, as carnitine palmityl transferase 1A (carnitine palmitoyltransferase 1A, CPT-1), peroxisome ACOD etc., strengthen free fatty acid oxidation; Suppress acetyl-CoA carboxylase vigor, reduce fatty acid synthesis; Can FATP be raised, affect the picked-up of the fatty acid of cell.
The PPAR-alfa agonists now developed, and applied to hyperlipemia clinical treatment, achieve certain curative effect.The PPAR-alfa agonists gone on the market is mainly fibrate, as gemfibrozil, fenofibrate, bezafibrate etc., can treat hyperlipemia.
The Cortex Eucommiae (Eucommia ulmoides), has another name called and thinks celestial, silk cotton tree, belongs to Eucommiaceae (Eucommia ulmoides), is mainly distributed in the provinces such as Shaanxi, Henan, Hubei, Hunan, Sichuan, Yunnan, Guizhou, Zhejiang, Gansu.Its skin stem, Ye Junke are used as medicine, and have invigorating the liver and kidney, bone and muscle strengthening, blood pressure lowering, antiabortive, resistance to effect of waiting for a long time of making light of one's life by commiting suicide.Modern study shows that the Cortex Eucommiae contains various active composition, has the effects such as blood pressure lowering, diuresis, blood sugar lowering, blood fat reducing, antioxidation, antitumor, antibacterial, antiinflammatory, calmness, hemostasis, analgesia.
Up to the present from the Cortex Eucommiae, separation and Extraction more than 70 plants monomer, mainly contains lignanoids (or being referred to as lignanoid), Phenylpropanoid Glycosides class, iridoids, flavonoid etc.Lignanoid is the compound be polymerized by bimolecular Phenylpropanoid Glycosides in plant, comprises bis-epoxy woods fat element class, monocycle oxygen lignanoids, ring lignanoids, neolignans etc. and glycoside by its construction features lignanoid.Cortex Eucommiae lignans extract is a kind of mixture.
The domestic and international pharmacological research to Cortex Eucommiae lignans extract is less at present.Application number: 200710035744.7 disclose Cortex Eucommiae lignanoid and the application of extract on anti-cardiovascular reconstruction thereof, the novelty teabag of Cortex Eucommiae lignans extract needs to be studied further.
Summary of the invention
Object of the present invention aims to provide a kind of novelty teabag of Cortex Eucommiae lignans extract.Our research finds that Cortex Eucommiae lignans extract has stronger triglyceride reducing effect, hepatocellular fatty acid beta oxidation can be promoted, have stronger PPAR α agonism, this has the medicine preventing and treating hypertriglyceridemia effect provide experimental basis for the exploitation of Cortex Eucommiae lignans extract is become a kind of.
For achieving the above object, technical scheme of the present invention is:
The application of Cortex Eucommiae lignans extract in preparation PPAR alfa agonists, in described Cortex Eucommiae lignans extract, lignanoid accounts for the 30%-90% of Cortex Eucommiae lignans extract gross weight, and in lignanoid, the content of effective ingredient pinoresinol diglucoside accounts for the 1%-15% of Cortex Eucommiae lignans extract gross weight.
Preferably, Cortex Eucommiae lignans extract is for the preparation of the application prevented and treated in the medicine of hyperlipemia.
More preferably, the application of Cortex Eucommiae lignans extract in the medicine for the preparation of control hypertriglyceridemia.
In described lignanoid, the content of effective ingredient pinoresinol diglucoside preferably accounts for the 3%-7.5% of Cortex Eucommiae lignans extract gross weight.
Described Cortex Eucommiae lignans extract take the Cortex Eucommiae as raw material, be the alcohol extraction of 40%-95% by mass concentration, extracting liquid filtering, concentrated, concentrated solution is passed through macroporous resin column, use water successively, mass concentration is methanol or the ethanol of 15%-25%, and mass concentration is methanol or the alcoholic solution eluting of 45%-55%, collecting mass concentration is the alcohol eluen of 45%-55%, concentrated, dry, obtain Cortex Eucommiae lignans extract powder.
Cortex Eucommiae lignans extract of the present invention also can be Cortex Eucommiae lignans extract disclosed in number of patent application 200710035744.7.
The preferred preparation method of Cortex Eucommiae lignans extract of the present invention is:
A), after Cortex Eucommiae removes crust, cutting is alcoholic solution reflux, extract, 2-3 time of 40%-95% by mass concentration, each 1-2h, and extracting solution is evaporated to 0.5-1.5 times of medical material amount volume;
B) medicinal liquid after concentrated is carried out effective ingredient enrichment by macroporous adsorptive resins, use water successively, mass concentration is methanol or the ethanol of 15%-25%, and mass concentration is methanol or the alcoholic solution eluting of 45%-55%;
C) collecting mass concentration is methanol or the ethanol elution of 45%-55%, concentrated, dry Cortex Eucommiae lignans extract powder.
Water elution liquid in preferred collection step b, concentrated, dry, pulverize, powder dissolve with methanol, discards insoluble matter, reclaims methanol and dry iridoids material.
Macroporous adsorptive resins described in step b is preferably HP series plastics, D101 resin or AB-8 resin.
Described drying in step c can be multiple, as vacuum drying, spraying dry, lyophilizing, and preferred freeze drying technology.
Below the present invention be further explained and illustrate.
Described Cortex Eucommiae lignans extract can be selected from pulverizing by employing, squeezes, calcines, grinds, sieves, percolation, extraction, water extraction, alcohol extraction, water precipitating, precipitate with ethanol, ester are carried, the method such as ketone is carried, chromatography, filtration obtains.With this extract for active substance can be made into pharmaceutical preparation, described extract can be the material of extractum form, can be dry extract also can be fluid extract, can be powdered substance, need to make different states according to the difference of preparation.
The present invention also proposes the pharmaceutical preparation of Cortex Eucommiae lignans extract through the applicable any unit dosage form taken of processing preparation, these pharmaceutical preparatioies are selected from following dosage form: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, suppository, ointment, plaster, cream, spray, drop, patch, can make slow releasing preparation, enteric coated preparation when needing.
Cortex Eucommiae lignans extract of the present invention, medicine acceptable carrier can be added when being prepared into pharmaceutical preparation, described medicine acceptable carrier is selected from: antioxidant, intercalating agent surfactant filler disintegrating agent wetting agent dispersant lubricant solvent slow release material enteric material pH adjusting agent, correctives, pigment etc., conventional carrier is as mannitol, dextran, lactose, glucose, sorbitol, mannitol, xylitol, sodium chloride, silicon derivative, cellulose and cellulose derivative, sodium sulfite, sodium pyrosulfite, alginate, gelatin, polyvinylpyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
The pharmaceutical preparation of Cortex Eucommiae lignans extract of the present invention is by being processed through extraction or other modes by Cortex Eucommiae raw material, making pharmaceutically active substance, subsequently, with this active substance for raw material, when needing, add medicine acceptable carrier, make pharmaceutical preparation according to the routine techniques of galenic pharmacy.
Accompanying drawing explanation
Fig. 1 is the design sketch that in embodiment 1, Cortex Eucommiae lignans extract reduces intracellular triglyceride;
Fig. 2 be in embodiment 1 Cortex Eucommiae lignans extract on the impact of the RNA of CPT1A;
Fig. 3 be in embodiment 1 Cortex Eucommiae lignans extract on the impact of the albumen of CPTIA;
Fig. 4 be in embodiment 1 Cortex Eucommiae lignans extract on the impact of the RNA of PPAR α;
Fig. 5 be in embodiment 1 Cortex Eucommiae lignans extract on the impact of the albumen of PPAR α;
Wherein # represents P<0.05, and # represents P<0.001, compares with natural diseases model group.
Detailed description of the invention
Below by embodiment, the present invention will be further explained, and described in embodiment, percentage composition is mass percentage.
The preparation of embodiment 1 Cortex Eucommiae lignans extract
Get eucommia bark (removing crust) 2Kg, with 8L 65% alcohol reflux twice, each 1h, filter, merge extracted twice liquid and be concentrated into 800ml.Mix sample loading (macroporous resin column volume 3L) with D101 macroporous resin, wash 3 times of column volumes, 1% ammonia continues to wash 3 times of column volumes, then is washed to neutrality.With 20% ethanol elution, 2 times of column volumes, wash 2.5 times of column volumes with 45% ethanol.45% ethanol elution is concentrated into 600ml.Powder is dried to after eluent is concentrated.With pinoresinol diglucoside in contrast, Cortex Eucommiae lignans extract is 85% through ultraviolet detection lignans content, and detecting pinoresinol diglucoside content through HPLC is 12.5%.
The preparation of embodiment 2 Cortex Eucommiae lignans extract
Cortex Eucommiae (removing crust) adds 60% ethanol 9L reflux, extract, 1h after pulverizing, and filters, and filtering residue adds 60% ethanol 7L again and extracts 1h, filters, and merges extracted twice liquid, is concentrated into without alcohol taste, obtains concentrated solution and be about 3L.Concentrated solution, through AB-8 macroporous adsorptive resins, first uses 5L water elution, then uses 7L 60% ethanol elution, collects 60% ethanol elution, is concentrated into 150ml, is slowly added in 450ml water, filters, concentrated, obtains drying solid powder.With pinoresinol diglucoside in contrast, Cortex Eucommiae lignans extract is 51% through ultraviolet detection lignans content, and detecting pinoresinol diglucoside content through HPLC is 3.5%.
Embodiment 3
The reduction triglyceride experimentation of the Cortex Eucommiae lignans extract that embodiment 1 and embodiment 2 obtain for example 3 and the experimentation of activation PPAR α.
1. Cortex Eucommiae lignans extract reduces the experimentation of triglyceride
1.1 experimental technique
1.1.1 cell culture and grouping:
A) HepG 2 cell (37 DEG C, 5%CO2) is cultivated with DMEM high glucose medium (containing 10% hyclone);
B) HepG 2 is inoculated in 6 orifice plates by certain 5000/mL density, 1ml/ hole, cultivates with DMEM high glucose medium (containing 10% hyclone);
C) when cell inoculation 24h, discard old culture medium, add the low sugar culture-medium of DMEM (containing oleic acid 0.5mM, 10% hyclone) to cultivate, the low sugar culture-medium of normal group DMEM (containing 10% hyclone) is cultivated continuation cultivation 24h and is carried out modeling;
D) after 24h, discard former culture medium, add the low sugar culture-medium of DMEM (containing oleic acid 0.5mM) and cultivate, the low sugar culture-medium of normal group DMEM (containing 10% hyclone) is cultivated, and adds medicine and processes;
1.1.2 cell TG assay
Principle: first, be glycerol and fatty acid by lipase (LP) hydrolyzing triglyceride, then use glycerol kinase (GK) and adenosine triphosphate (ATP) by phosphoglycerol, generate phosphoglycerol and adenosine diphosphate (ADP) (ADP), phosphoglycerol is oxidized to dihydroxyacetone phosphate and hydrogen peroxide (H by GPO (GPO) 2o 2), H 2o 2carry out chromogenic reaction with peroxidase (POD), 4-aminophenazone etc., generate quinone imines, have light absorption value at 480 ~ 550nm place, this value is directly proportional to triglyceride concentration.The method is the total glycerol surveyed, and namely comprises triglyceride, diglyceride, monoglyceride and dissociative glycerin sum.Brief principle is as follows:
Triglyceride+H 2o glycerol+lipase
Glycerol+ATP glycerol-3-phosphate+ADP
Glycerol-3-phosphate+O 2dihydroxyacetone phosphate+H 2o 2
H 20 2+ 4-AAP+4-chlorophenol quinone imines+H 2o
A) clean twice with PBS after cell process 48h;
B) every hole adds RIPA lysate 200 μ L, constantly shakes, abundant cracking 30min on ice.
C) be transferred to by lysate in the EP pipe of pre-cooling, at 12000r/min, 4 DEG C, centrifugal 15min, gets 20 μ L supernatants and stays and do BCA protein determination.
D) in EP pipe, chloroform is added: methanol (V:V=2:1) 400 μ L shakes 10min, then, 13000r/min, 4 DEG C, centrifugal 15min.Take off a layer chloroform layer solution.
E) enzyme liquid is prepared: dissolved by a enzyme agent 10ml buffer solution, mixing balance 10min, stand-by.
F) standard curve: triglyceride titer is got 1 respectively, 1.5,2,2.5,3,3.5 μ L;
G) get 5 μ L chloroform layer solution to volatilize (matched group gets 10 μ L), in standard curve and sample, often pipe adds 100 μ L enzyme liquid, at room temperature reacts 30min, measures the light absorption value A490 under 490nm by microplate reader.
H) content of triglyceride and the content of albumen is calculated, with TG content in the odds ratio of triglyceride/albumen comparatively cell.
1.2 experimental result
Vitro Experimental Results shows: as shown in Figure 1, the Cortex Eucommiae lignans extract of embodiment 1 and embodiment 2 can reduce the triglyceride in cell.
2. Cortex Eucommiae lignans extract activates the experimentation of PPAR α
2.1 experimental technique
2.1.1 cell culture and grouping:
1.1 of the experimentation 1 of triglyceride are reduced with Cortex Eucommiae lignans extract
2.1.2 cell CPT1A and PPAR α measures
Adopt Fluorescent quantitative PCR method to detect the RNA of cell CPT1A, PPAR α, western blotting detects cell CPT1A, PPAR α protein expression level.
2.2. experimental result
Vitro Experimental Results shows: as Figure 2-Figure 5, the Cortex Eucommiae lignans extract of embodiment 1 and embodiment 2 all can activate PPAR α, raises CPT1A, promotes fatty acid oxidation.
3. conclusion
The Cortex Eucommiae lignans extract of embodiment 1 and embodiment 2 all can reduce intracellular triglyceride, can activate PPAR α, can raise CPT1A.
Embodiment 4
After eucommia bark (removing crust) chopping, with the alcohol reflux 1h of 65%, filter, extracting solution concentrates.After HPD100 macroporous resin adsorption, wash 3 times of column volumes, 2 times of column volumes washed by 20% ethanol, and 2 times of column volumes washed by 45% ethanol, and 45% ethanol elution is collected and concentrated, and lyophilizing obtains extract powder.Get powder 60g, add microcrystalline Cellulose 230g, PVP10g, mixing, adds appropriate amount of ethanol soft material, granulates, and dry, granulate, adds a small amount of magnesium stearate, tabletting, makes 1000.The effect described in embodiment 3 can be reached.

Claims (5)

1. the application of Cortex Eucommiae lignans extract in the medicine for the preparation of control hypertriglyceridemia, in described Cortex Eucommiae lignans extract, lignanoid accounts for the 30%-90% of Cortex Eucommiae lignans extract gross weight, and in lignanoid, the content of effective ingredient pinoresinol diglucoside accounts for the 1%-15% of Cortex Eucommiae lignans extract gross weight.
2. apply according to claim 1, it is characterized in that, in described lignanoid, the content of effective ingredient pinoresinol diglucoside accounts for the 3%-7.5% of Cortex Eucommiae lignans extract gross weight.
3. apply according to claim 1, it is characterized in that, described Cortex Eucommiae lignans extract take the Cortex Eucommiae as raw material, be the alcohol extraction of 40%-95% by mass concentration, extracting liquid filtering, concentrated, concentrated solution is passed through macroporous resin column, use water successively, mass concentration is methanol or the ethanol of 15%-25%, and mass concentration is methanol or the alcoholic solution eluting of 45%-55%, collecting mass concentration is the alcohol eluen of 45%-55%, concentrated, dry, obtain Cortex Eucommiae lignans extract powder.
4. apply according to claim 1, it is characterized in that, the preparation method of described Cortex Eucommiae lignans extract is:
A), after Cortex Eucommiae removes crust, cutting is alcoholic solution reflux, extract, 2-3 time of 40%-95% by mass concentration, each 1-2h, and extracting solution is evaporated to 0.5-1.5 times of medical material amount volume;
B) medicinal liquid after concentrated is carried out effective ingredient enrichment by macroporous adsorptive resins, use water successively, mass concentration is methanol or the ethanol of 15%-25%, and mass concentration is methanol or the alcoholic solution eluting of 45%-55%;
C) collecting mass concentration is methanol or the ethanol elution of 45%-55%, concentrated, dry Cortex Eucommiae lignans extract powder.
5. apply according to claim 4, it is characterized in that, macroporous adsorptive resins described in step b is HP series plastics, D101 resin or AB-8 resin.
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CN104027374B (en) * 2014-03-25 2017-02-22 广州医科大学附属第一医院 Asarum total lignan extract and extraction method thereof, and application of asarum total lignan extract in preparation of drugs used for mitigating or inhibiting cough
CN106138168A (en) * 2015-03-26 2016-11-23 南京海恒医药科技有限公司 Cortex Eucommiae extract application in preparation prevention and treatment senile dementia
CN105250352A (en) * 2015-10-20 2016-01-20 欧阳冬生 Application of eucommia ulmoides lignan extract in preparation of PH (pulmonary hypertension) treatment drug
CN115006417B (en) * 2020-12-24 2023-10-13 北华大学 Application of lignan glycoside compounds in preparation of lipid-lowering drugs

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