CN109044999A - Hyperforine promotes white adipose milkproduct in preparation and improves the purposes in the active drug of brown fat - Google Patents

Abstract

The present invention relates to Natural Medicine Chemistry technical fields, and in particular to hyperforine promotes white adipose milkproduct in preparation and improves the purposes in the active drug of brown fat.Based on promotion white adipose milkproduct and this active mechanism of action of brown fat is improved, hyperforine shows to increase effect of the body energy consumption to lose weight；Also, hyperforine also shows to reduce blood glucose, blood lipid level, improves sugar tolerance, increases insulin sensitivity, maintains the effect of glycometabolism stable state, thus can be used as the drug for improving diabetic symptom；In addition, hyperforine can also be used to alleviating or treating nonalcoholic fatty liver.Therefore, the hyperforine, containing the extract of hyperforine or its preparation there is wide potential applicability in clinical practice in terms of preventing or treating the metabolic syndromes such as obesity, diabetes.

Description

Hyperforine promotes white adipose milkproduct in preparation and improves brown fat Purposes in active drug

Technical field

The present invention relates to Natural Medicine Chemistry technical fields, and in particular to hyperforine promotes white adipose in preparation Milkproduct and the purposes in the active drug of brown fat is improved, and is related to hyperforine in preparation for preventing or controlling The purposes in the drug of obesity is treated, purposes of the hyperforine in the drug that preparation improves diabetic symptom is further related to, And a kind of pharmaceutical composition alleviated or treat nonalcoholic fatty liver.

Background technique

It is overweight and fat refer to that the body fat that may damage human health is excessive according to the definition of the World Health Organization And/or abnormal accumulation.Early in 1948, obesity was defined as a kind of disease, and increases in International Classification of Diseases.2013 June, association of American Medical Association proclaim that fat is a kind of disease for the first time in its history, need medical intervention measure pre- Anti- and treatment.

It is well known that overweight and fat while being a variety of diseases (for example, cardiovascular and cerebrovascular disease, type-2 diabetes mellitus, intermuscular bone Bone disease) risk factor.In addition, the data of WHO are shown, the whole world in 2014 is shared adult more than 1,900,000,000 overweight, wherein more than 6 Hundred million people are obesity；The adult overweight rate in China is 31.5%, obesity rates 12.2%, it is seen then that overweight and fat have become influences to occupy The important illness of people's health.The therapeutic scheme of obesity is broadly divided into lifestyle modification, drug therapy and surgical operation therapy. Currently, evidence-based medical recommend lifestyle modification be first-line treatment scheme, when lifestyle modification is invalid, then recommend into Row drug therapy.For patient overweight and with a kind of complication (cardiovascular disease, hypertension, type-2 diabetes mellitus etc.), if It is invalid through Spinal injury, also recommend it to carry out drug therapy.

On the development history for resisting obesity drug, Phentermine, diethylpropion, benzphetamine and phendimetrazine mesh are removed It is preceding to be still used as short drug outside the U.S. uses, it is other because safety issue is removed city.Wherein, lipase inhibitor mesh For preceding only FDA in the orlistat of approval in 1997, low dose formulation is currently the only non-prescribed medicine；Also, China and The slimming medicine that Europe lists at present also only has orlistat.The clinical II or III phase is in grinding drug in addition, having at present Stage, such as central drug (MCR4 agonist, npy receptor antagonist, monoamine neurotransmitter regulator) and peripheral tissues' medicine Object (sodium/glucose co-transporter inhibitor, pancreatic lipase inhibitor) etc..

However, existing obesity treatment drugs are accompanied by serious safety issue more, and such as: for acting on maincenter The drug of nervous system, may be with the side effect of depression and anxiety；And researcher is to acting on the drug of periphery a variety of The mechanism of action in tissue understands not thorough enough, it is thus possible to unexpected side effect occurs, such as the immunogene of peptide hormone analog Property risk etc..

For a long time, people are usually considered as fat fat " arch-criminal ", however true really not so.Largely grind Study carefully and show to be primarily present two kinds of adipose tissues in the mammalian body: white adipose (WAT) and brown fat (BAT)；The two is not only Anatomical position, morphological feature are entirely different, and the effect in terms of energetic supersession is completely contradicted: the major function of WAT be with The form of fat drips (triglycerides) stores energy, and BAT then maintains the energetic supersession of body flat by consumption energy, heat production Weighing apparatus.Newest research achievement shows that there are the third fat --- " derivable brown fat " or cream-coloured fat.The cream-coloured rouge The form and function of fat are between white adipose and brown fat, and fat drips are larger under foundation level, no Thermogenic Activity, and Can then burn fat drips in the case that cold expoure or stomodaeal nervous system are excited, and the heat production played similar to classical brown fat is lived Property.Obesity is the Chronic Non-Communicable Diseases by internal superfluous energy in white adipose caused by excess accumulation, therefore, from energy From the perspective of amount balance, heat production can be increased by activation brown fat activity, increase energy consumption to reach the effect of loss of weight Fruit.Cream-coloured fat and brown fat belong to heat production adipose tissue, and the effectiveness in terms of resisting obesity has been confirmed extensively, because This, promote white adipose milkproduct become a new Bariatric strategy, for potential slimming medicine discovery provide it is new Direction.

Why heat production adipose tissue, which can convert heat energy dissipation for the chemical energy being stored in fatty acid, is fallen, be because With a special molecule: uncoupling protein 1 (Uncoupling Protein 1, UCP1).UCP1 is positioned at mitochondria On inner membrance, when activated, the uncoupling of electron transport chain and ATP synthesis can be cut off, so that chemical energy is converted into Heat energy dissipation falls.UCP1 is only expressed in the adipose tissue of heat production, and due to this unique positioning, UCP1 is not only heat production The functional molecular and phenotype marker of adipose tissue.When occurring UCP1 expression in white adipose tissue, then illustrate white adipose There is the phenomenon that milkproduct in tissue.

In the prior art, the slimming drugs that FDA is ratified are developed both for limitation Energy intaking, and there is presently no logical Cross the slimming drugs for promoting heat production adipose tissue to play a role to increase energy consumption.Have some researchs to have found and can swash Compound, such as capsaicine of the hot adipose tissue of life birth and the like, berberine, resveratrol, disalicylic acid, into One step illustrates that heat production adipose tissue is activated by chemical means, has the feasibility as therapy target.However, at present still Lack and is studied using white adipose milkproduct as the human experimentation of drug target, so there is presently no the listing of successful drug, There is the side effect etc. in terms of causing angiocarpy because stimulating B-adrenergic receptor signal path and ask in most of drug candidate Topic.

In fact, a variety of chronic diseases clinically, such as fat, diabetes metabolic disease is by polygenes, multifactor The disease of effect is difficult to reach good therapeutic effect according only to single action target spot, and is also easy to produce various safety issues.

Hyperforine (Hyperforin, HPF) only finds to be present in Guttiferae hypericum perforatum (Herba Hyperici Monogyni) In the flower and fruit of Hypericum perforatum L, for the characteristic chemical constituent of the plant.Some researches show that Herba Hyperici Monogynis Element belongs to gamboge phenol derivatives, is distributed mainly in plant flowers and immature capsule, wherein content respectively may be about 2%, 4.5%.Hyperforine is used as antibacterial substance to be found and named by Russian scientists first, and determines in 1975 Its chemical structure.The non-natural enantiomer of hyperforine it is fully synthetic 2010 complete, natural enantiomer it is fully synthetic It is broken through in realization in 2012.With the further investigation to antidepressant and its mechanism, researcher's hair Existing hyperforine is its antidepressant main active, while also having other neuropharmacologies and oncology pharmacology etc. living Property.

Currently, being existed using promoting white adipose milkproduct and improving brown fat activity as the hyperforine of the mechanism of action The application resisted in obesity-related disease is not yet seen in report.

Summary of the invention

Inventor abandons previous drug discovery strategy, and proposed adoption is associated with Atlas Method, i.e., by comparing small molecule compound Balance and noisy data library to human cell line's gene expression network, find the drug candidate of induced lipolysis milkproduct；It is excellent simultaneously It first selects the traditional Chinese medicine monomer of permanent medication habit as lead compound, improves the safety issue of medication.

The present invention is intended to provide hyperforine, containing the extract of hyperforine or its preparation promoting fat Application in terms of organizing milkproduct and resisting the metabolic syndromes such as obesity, diabetes.

Specifically, first aspect present invention provides hyperforine and promotes white adipose milkproduct in preparation and mention Purposes in the high active drug of brown fat；Wherein, chemical entitled (IUPAC) of the hyperforine: (1R, 5R, 6R,7S)-2-hydroxy-6-methyl-1,3,7-tris(3-methylbut-2-enyl)-6-(4-methylpent-3- Enyl) -5- (2-methylpropanoyl) bicyclo [3.3.1] non-2-ene-4,9-dione, structural formula are as follows:

Preferably, on the way, the internal effective dose of hyperforine is 2.5- to the use described in first aspect present invention 25mg/Kg weight.

Meanwhile second aspect of the present invention provides hyperforine and is preparing the drug for preventing or treating obesity In purposes；Wherein, the structural formula of the hyperforine are as follows:

Preferably, on the way, the internal effective dose of hyperforine is 2.5- to the use described in second aspect of the present invention 25mg/Kg weight.

Third aspect present invention additionally provides purposes of the hyperforine in the drug that preparation improves diabetic symptom； Wherein, the structural formula of the hyperforine are as follows:

Preferably, on the way, the internal effective dose of hyperforine is 2.5- to the use described in third aspect present invention 25mg/Kg weight.

In addition, fourth aspect present invention provides a kind of pharmaceutical composition alleviated or treat nonalcoholic fatty liver, Include a effective amount of hyperforine and pharmaceutically acceptable carrier；Wherein, the structural formula of the hyperforine are as follows:

In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is selected from following any: note It penetrates with water, freeze dried powder auxiliary material, oral formulations auxiliary material.

In another preferred embodiment of the present invention, the pharmaceutical composition of the alleviation or treatment nonalcoholic fatty liver The dosage form of object is selected from following any: tablet, pill, granule, suspension, oral solution, paint, cataplasm, is sprayed capsule Agent, powder-injection and liquid drugs injection.

It is confirmed by a series of experiments, hyperforine, which has to increase, promotes subcutaneous white adipose tissue, brown fat It organizes, the effect of the related gene expression of fat cell heat production, hyperforine, which also has, promotes mitochondria to generate and improve The effect of mitochondrial function.In short, based on promoting white adipose milkproduct and improving this active mechanism of action of brown fat, Hyperforine shows to increase effect of the body energy consumption to lose weight；Also, hyperforine also shows to reduce Blood glucose, blood lipid level improve sugar tolerance, increase insulin sensitivity, maintain the effect of glycometabolism stable state, thus can be used as improvement The drug of diabetic symptom；In addition, hyperforine can also be used to alleviating or treating nonalcoholic fatty liver.Therefore, described Hyperforine is metabolized containing the extract of hyperforine or its preparation in prevention or treatment obesity, diabetes etc. There is wide potential applicability in clinical practice in terms of syndrome.

Detailed description of the invention

Fig. 1 is the hyperforine of DMSO and various concentration to the testing result figure of the toxicity of lipocyte proliferation；

Fig. 2 is the Protein Detection result figure of control group with UCP1, PGC1 α in the mature fat cell in experimental group；

Fig. 3 is control group and fat drips coloration result figure in the mature fat cell in experimental group；

Fig. 4 is testing result figure of multiple heat production gene mRNA levels in fat cell, wherein Fig. 4 A, which is shown, to be passed through Leaf hypericin promotes BMP7 to orient fat cell heat production gene expression, and Fig. 4 B shows hyperforine and promotes BMP4 orientation Fat cell heat production gene expression；

Fig. 5 is that hyperforine promotes fat cell oxygen to consume breathing detection result figure, wherein Fig. 5 A shows control group With the mitochondria pressure test curve of experimental group, Fig. 5 B shows in the mitochondria pressure test of control group and experimental group and respectively breathes It is horizontal；

Fig. 6 is that MitoTracker Green marks active mitochondria testing result figure；

Fig. 7 is the whole weight of four groups of mouse (high in fat control group, administration group high in fat, general food control group, general food administration group) Testing result figure；

Fig. 8 is the tissue weight of four groups of mouse (high in fat control group, administration group high in fat, general food control group, general food administration group) As a result, wherein Fig. 8 A shows three kinds of adipose tissues and liver anatomical figure, and Fig. 8 B shows whole fat weight and lean meat weight Amount, Fig. 8 C show three kinds of adipose tissues and liver weight result；

Fig. 9 is the liver of mouse, pars inguinalis fatty (iWAT), pars epididymica fatty (eWAT) and omoplate area brown fat (BAT) hematoxylin --- eosin stains (HE dyeing) result figure；

Figure 10 is the testing result figure of influence of the hyperforine to body insulin sensitivity；

Figure 11 is the testing result figure of influence of the hyperforine to body glucose tolerance；

Figure 12 is the testing result figure of influence of the hyperforine to organism adaptation heat production；

Figure 13 is the testing result figure of multiple heat production genes and fatty marker mRNA level in-site in adipose tissue, In, Figure 13 A shows hyperforine and promotes subcutaneous fat heat production gene expression, and Figure 13 B shows hyperforine rush Into brown fat heat production gene expression；

Figure 14 is testing result figure of the hyperforine to brown fat UCP1 protein expression；

Figure 15 is effect-Dose Results figure of hyperforine fat milkproduct, wherein Figure 15 A shows 0-20 μM Influence of the hyperforine to UCP1, Fabp4 gene expression, Figure 15 B are that effect-dosage of 0-20 μM of hyperforine is bent Line chart.

Figure 16 is loss of weight effect-dosage profile of the hyperforine in mouse high in fat.

Specific embodiment

The present invention is further elaborated With reference to embodiment, but the present invention is not limited to following embodiment party Formula.Experimental method in following embodiments is unless otherwise specified conventional method；Material as used in the following examples, examination Agent etc. can obtain unless otherwise specified from public commercial source.

Association map (Connectivity Map, CMap) is the current most leading medicament research and development system based on transcript profile One of, wherein containing the obtained genomic expression delta data of thousands of kinds of drug-treated Human cell lines.It is associated with map Application mode is mainly to score by using the express spectra marking (signature) of the pattern matching algorithm to retrieval, estimates enrichment (enrichment) and direction；Disturbance drug is arranged finally, giving a mark according to degree of correlation, the related drugs that are positive of ranking top set, The related drugs that are then negative of ranking bottom set.

Inventor has initially set up the fatty milkproduct gene expression profile marking, then implements CMap fat milkproduct drug Scoring, fatty milkproduct lead compound is selected finally by cell experiment: the 1. forward small molecule chemical combination of selection scoring Object carries out CCK8 experiment on 3T3-L1 PECTORAL LIMB SKELETON, rejects the compound to fat cell strong toxicity；2. one-step method PCR Screening: on the cell model that the C3H10T1/2 of bone morphogenetic protein4 (BMP7) induction breaks up to brown fat cell directional, with UCP1 as up-regulation selection markers, using FABP4 as differentiation contrasting marking, using one-step method PCR screen up-regulation UCP1 express with And the compound of FABP4 expression indifference；3. Western Blot detect brown fat differentiation and function key gene UCP1, The expression of PGC1 α etc..Experimental result is shown: (1) CCK8 is tested --- the Herba Hyperici Monogyni under 0.16-100 μM of concentration Fat cell Activity determination under plain (HPF) processing, compared with control solvent DMSO group, under 0.16-100 μM of HPF processing Cell activity no difference of science of statistics, p > 0.05, this shows HPF without obvious cytotoxicity (as shown in Figure 1)；(2) in a series of times It selects in compound, HPF shows as remarkably promoting C3H10T1/2 fat cell heat production gene UCP1 transcription and not influence simultaneously FABP4 gene；(3) in a series of candidate compounds, HPF shows as remarkably promoting C3H10T1/2 fat in translation skill Cell heat production albumen UCP1 translation.

Accordingly, hyperforine (HPF) is found as completely new fatty heat production lead compound.

Embodiment 1

Hyperforine reduces the mature fat cell UCP1 albumen table that intracellular fat drips are horizontal and SVF is promoted to induce It reaches

C3H10T1/2 is that a kind of mice embryonic source has a multi-lineage potential mescenchymal stem cell, can be in vitro Fat, skeletal muscle, bone, cartilage and endothelial cell are divided under different inductive conditions；It is divided into white under BMP4 orientation Fat cell is divided into brown fat cell under BMP7 directional process.In the present invention, inventor is by C3H10T1/2 cell Differentiation Induction in vitro is mature fat cell, and influences fat effect in this, as preliminary screening model compounds and research HPF The cell model of mechanism.

The culture and differentiation of C3H10T1/2: C3H10Tl/2 is seeded in six porocyte culture plates, and kind plate next day is training Supporting and BMP4 (final concentration of 10ng/ml) or BMP7 (final concentration of 40ng/ml) are added in base to cell is in contact inhibition state.It reaches Used two days later to contact inhibition inductive differentiation medium A (0.5mM IBMX, 1 μM of dexamethasone, 10 μ g/ml insulin, 100nM Indomethacin, 1nM trilute)；After 2 days, change inductive differentiation medium B (10 μ g/ml insulin), two After it, fresh culture is replaced, until differentiation starts to form mature fat cell on the 8th day.

In the research of fat cell, vascular stroma component (Stromal Vascular Fraction, SVF) is generally acknowledged More reliable cell model.SVF is primary Preadipocyte In Vitro, through internal subcutaneous fat or brown adipose tissue separation training It after supporting, is handled by chemical inducer, white adipocyte or brown fat cell can be divided into respectively, to realize in vitro Simulate physiological disposition of the Preadipocyte In Vitro to mature Adipose Differentiation.

The culture and differentiation of SVF: it takes less than 8 week old male mice two, by pars inguinalis fat or omoplate area brown rouge Fat shreds, and with 37 DEG C of processing 60min of digestion buffer containing clostridiopetidase A, crosses 70 μm of cell mesh screens, 1800g is centrifuged 10min.It discards Cell precipitation is stayed on upper layer, and PBS is washed three times, is resuspended and precipitated with Fl2/DMEM culture medium, is seeded in 6cm culture dish, proliferation is extremely 80% point of disk.Cell contact inhibition two days later, is induced, detailed process is as follows according to above-mentioned Adipocyte Differentiation scheme: being used Contain 10% fetal calf serum, 0.5mM IBMX, 1 μM of dexamethasone, 10 μ g/ml insulin, 100nM Indomethacin, 1nM triiodo first It after shape gland original ammonia acid induces 2 days, changes inductive differentiation medium B (10 μ g/ml insulin), DMEM/F12 continues culture 2 days, hereafter Every other day DMEM/F12 of the replacement containing 10% fetal calf serum trains liquid, until cell differentiation was by the 8-10 days.

HPF processing: HPF powder is dissolved in DMSO, stock concentrations 10mM, starts the 6-8 days addition HPF in differentiation, eventually Concentration is 5 or 10 μM.Drug-treated for detecting protein level tests 24 to 48 hours collection cells after HPF addition, uses 48 hours collection cells after HPF addition are tested in the drug-treated of oil red dyeing.Specific experiment is as follows: (1) by SVF differentiation Mature fat cell is handled with 5 μM of HPF, and Western blot detects the protein level of brown fat related gene, wherein β- Actin is as internal reference.As a result as shown in Fig. 2, HPF makes the albumen water of brown fat related gene UCP1, PGC1 α in fat cell It is flat to increase.(2) oil red O is lipid-soluble dye, and energy high dissolution, can specifically make triglycerides etc. in tissue in fat Neutral fat coloring.5 μM of HPF of the C3H10T1/2 mature fat cell of BMP4, BMP7 orientation is handled, oil red detection is thin Fat drips intracellular are horizontal, microscope Olympus 20 times of microscopic observation cellular morphologies and record.As a result as shown in Figure 3: control group The C3H10T1/2 of (normal medium) shows typical fat cell feature, and cell volume is larger, and fat drips are big and justify；Through The C3H10T1/2 for crossing HPF treated BMP4, BMP7 orientation then shows typical brown sample feature, and cell volume is smaller, rouge Drip small and dense collection.

Embodiment 2

Hyperforine promotes fat cell heat production gene expression

The culture and differentiation of C3H10T1/2: C3H10T1/2 is seeded in six porocyte culture plates, and kind plate next day is training Supporting and BMP4 (final concentration of 10ng/ml) or BMP7 (final concentration of 40ng/ml) are added in base to cell is in contact inhibition state.It reaches Used two days later to contact inhibition inductive differentiation medium A (0.5mM I BMX, 1 μM of dexamethasone, 10 μ g/ml insulin, 100nM Indomethacin, 1nM trilute)；After 2 days, change inductive differentiation medium B (10 μ g/ml insulin), two After it, fresh culture is replaced, until differentiation starts to form mature fat cell on the 8th day.

HPF processing: HPF powder is dissolved in DMSO, stock concentrations 10mM, starts the 6-8 days addition HPF in differentiation, eventually Concentration is 5 μM, and the drug-treated for extracting total serum IgE tests 8 hours collection cells after HPF addition.

Real-time fluorescence quantitative PCR (RT-PCR) detects heat production gene and each fat specific genes mRNA level in-site: trizol Method extracts total serum IgE, reverse transcription cDNA, and SYBRGREEN method real-time fluorescence quantitative PCR is only praised in promise, calculates mRNA according to Δ CT method Relative quantity, use 18S as internal reference, then the mRNA level in-site of control is set as 1, each processing group and control group compare, and obtain Relative quantity.Data variance is analyzed by Student T test method, is statistical difference when p < 0.05.

Experimental result is as follows: as shown in figure 4, by the C3H10T1/2 mature fat cell of BMP7 orientation at 5 μM of HPF Reason, following heat production gene mRNA levels up-regulation: (1) UCP1: uncoupling proteins 1 (Uncoupling protein1) is line grain Internal memebrane protein, the cross-film proton concentration that can eliminate mitochondrial inner membrane two sides is poor, enables the phosphorous oxide using the driving of proton concentration difference Acidization slows down, and hinders the normal generation of atriphos (ATP).UCP1 is uniquely expressed in brown adipose tissue Uncoupling Proteins is the key gene for the Thermoregulation and energetic supersession for being primarily involved in BAT.(2) PGC-1 α: peroxidase Body Proliferator-Activated Receptors cooperate with 1 α of stimulating factor (PGC-1 α), are the transcription co-activation factors of PPAR- γ, have and adjust brown The effect of UCP1 expression and Thermogenesis in fat；Its binding site with a variety of nuclear hormone receptors, except being adjustable adaptability Outside heat production, also the oxidation with glycometabolism and fatty acid has substantial connection.(3) ACOX1: (the Acyl-Co A of acyl-CoA oxidase 1 Oxidase 1, ACOX1) in peroxisome, ACOX1 is the first rate-limiting enzyme of fatty acid beta oxidation approach, is catalyzed ester acyl Coacetylase forms the cis- enoyl CoA of 2-, to promote the metabolism of fatty acid, reduces triglycerides deposition.(4) NRF-1: core is exhaled It is close with mitochondrial oxidation respiration and energy production to inhale the factor -1 (nuclear respiratory factor-1, NRF-1) Cut phase is closed.NRF-1mRNA expression quantity increases, and mitochondria density can be caused to increase, expressed by regulating and controlling a variety of adjustings mitochondrias and The expression of the correlation factor of function influences the expression of mitochondrial oxidation respiratory chain multienzyme complex indirectly, and then improves mitochondria oxygen Change respiration, improves energy supply ability.(5) cream-coloured fat specific genes: Hoxc9.(6) brown fat specificity base Cause: Dio2, Zic1 up-regulation, Cox8b are without significant difference.While heat production gene upregulation, white adipose specific gene The mRNA of resistin is without significant difference.

5 μM of HPF of the C3H10T1/2 mature fat cell of BMP4 orientation is handled, following heat production gene mRNA water Flat up-regulation: (1) UCP1, (2) ACOX1, (3) NRF-1, (4) brown fat specific gene: zic1, Cox8b up-regulation.But it is producing While hot gene upregulation, the mRNA of white adipose specific gene resistin is also raised.

Therefore, the present embodiment the experimental results showed that, hyperforine can promote fat cell heat production gene expression.

Embodiment 3

Hyperforine promotes fat cell oxygen consumption to breathe and improves the generation and function of mitochondria

Inventor has carried out a series of experiments to study hyperforine to cell entirety respiratory chain, especially mitochondria The influence of function.Specifically, inventor uses advanced Seahorse XFe cellular energy metabolism analytical technology, for complete Assess hyperforine treated mitochondrial respiratory function.

Wherein, the pressure test of Seahorse XF cell mitochondrial can detecte the following key parameter of mitochondrial function: exhale Absorb water horizontal flat basic value, the relevant breathing value of ATP synthesis, proton leakage, respiration capability maximum value and respiration capability deposit value Deng.

Detailed operating procedures include:

Kind of cell before Seahorse is tested: the C3H10T1/2 cell inoculation for breaking up second day BMP7 orientation is arrived In Seahorse XF V7 tissue culture plate, continue differentiation to maturation.Seahorse experiment the previous day preheater apparatus: it opens Seahorse instrument and computer, and software is opened, so that instrument is warming up to 37 DEG C, preheats overnight.While aquation probe: Seahorse XF calibration solution is added in Utility Plate, test board is put back on Utility Plate, 37 DEG C of nothings are placed in CO₂Aquation probe is stayed overnight in incubator；Prepare the test fluid without buffer salt system；Experimental day cell changes liquid: by grown cultures Base is changed to test fluid, and cell is then placed on 37 DEG C without CO₂1 hour in incubator.The Oligomycin/ that will have been diluted FCCP/Rotenone and Antimycin A is separately added into the A on test board, in tri- medicine feeding holes of B, C.Upper machine, bring into operation reality Program is tested, the test board for adding medicine and the Utility Plate equipped with calibration solution are placed into together on instrument supporting plate, instrument is certainly Dynamic calibration probe.Then, cell plates are changed, formal experiment is run, uses wave software and report generator couple after the completion Experimental data is analyzed.

Experimental result is shown (as shown in Figure 5): treated that mature fat cell is Intramitochondrial exhales for hyperforine Flat basic value, respiration capability maximum value, the proton leakage of absorbing water are horizontal, ATP synthesizes relevant breathing value and non-mitochondria oxygen consumption is equal It significantly rises.Meanwhile as shown in fig. 6, MitoTracker Green marks active mitochondria the results show that treated The biosynthesis of the mitochondria of C3H10T1/2 is remarkably reinforced.

Therefore, the present embodiment the experimental results showed that, hyperforine can promote fat cell oxygen consumption breathe and change The generation and function of kind mitochondria.

Embodiment 4

Hyperforine inhibits the weight gain of high fat diet induction

C57BL/6J mouse is bought from Beijing dimension Co., Ltd of tonneau China.C57BL/6J mouse is placed in Experimental Animal Center SPF grades of Animal Lab. raisings, 21 DEG C -23 DEG C of environment temperature control, every 12 hours light dark cycles.When mouse grows to 8 week old, It is randomly divided into high fat diet group and full diet group, wherein high fat diet group uses high lipid food inducing obesity, used in the mouse High lipid food fat content be 60%.After high in fat or full diet (general food) four weeks, these mouse are randomly divided into height Rouge control group, administration group high in fat, general food control group, general food administration group, every group 7.Solvent is injected intraperitoneally, and (vehicle contains Normal saline solution no more than 1%DMSO) or hyperforine (2.5mg/Kg, HPF).The dissolution of hyperforine mother liquor In DMSO, mother liquor is diluted in physiological saline, DMSO final concentration controls below 1%；It is administered and continues surrounding, periodic logging is small The weight of mouse.

Fig. 7 statistical result showed, after four weeks is administered, hyperforine significantly reduces the total of the mouse of High fat diet Weight, and the weight of full diet mouse is not substantially change, to confirm that hyperforine is able to suppress drink high in fat Eat the weight gain of induction.

It further analyzes hyperforine in addition, inventor also passes through experiment and can cause the mechanism of weight loss:

It substantially takes pictures, and adopts to four groups of mouse (high in fat control group, administration group high in fat, general food control group, general food administration group) It is accurate to survey using time domain nuclear magnetic resonance (TD-NMR) technology of being based on Brooker (minispec) mouse body composition analysis instrument living The muscle (lean), fatty (fat) and body fluid equal size for determining intravital mouse, so that it is determined that mouse body component ratio.Inventor It was found that injecting in the high in fat and full diet group gross weight of hyperforine, the quality of fatty (fat) is remarkably decreased, muscle (lean) there is no significant changes compared with the control group for quality.

Then, metabolism critical organ liver and three pieces of representative adipose tissues: pars inguinalis fat are taken out (iWAT), pars epididymica fatty (eWAT) and omoplate area brown fat (BAT) carry out weighing analysis, and inventor has found that intraperitoneal injection is passed through The volume and weight (referring to Fig. 8) of the mouse subcutaneus adipose tissue of leaf hypericin is substantially reduced than control group.Therefore, it tests The result shows that hyperforine significantly reduces the fat volume and weight of the mouse of High fat diet, and caused body is administered It reduces again mainly as caused by the reduction of subcutaneus adipose tissue.

Embodiment 5

Hyperforine promotes fat drop to become smaller and slow down fatty liver

Using medication same as Example 4, after four weeks is administered, control group high in fat, administration group high in fat are dissected respectively Mouse, tissue sampling.Take fresh liver, pars inguinalis fatty (iWAT), pars epididymica fat (eWAT) and omoplate area brown Fatty (BAT) each fritter, is placed in 4% paraformaldehyde PBS solution, impregnates 24 hours；Paraffin embedding cuts 4mm sheet；Two Toluene dewaxes 2 times, and 5-10min is each.Then, with 100% ethyl alcohol, 95% ethyl alcohol, 80% ethyl alcohol, 75% ethyl alcohol, distillation moisture It does not embathe 2 times, 1-2min is each.Hematoxylin dyes 5min, and distilled water cleans remaining hematoxylin dyeing liquor, and filter paper blots residual water. Then, break up 1-3s, distilled water immersion 15min with 1% acidic alcohol；After Yihong liquid dyes 2min, with 95% ethyl alcohol, 100% Ethyl alcohol impregnates 2 times, and 1min is each；1min is impregnated with dimethylbenzene carbolic acid solution, xylene soak 2 times, 1min is each.Finally, Resinene sealing, photographic analysis.

As a result as shown in Figure 9, it is seen that: the 1. slice of pars inguinalis white adipose and dyeing display, control group mice white Fat cell becomes larger expansion under induction high in fat, and after administration is intervened, pars inguinalis white adipocyte is reduced significantly.2. omoplate area Brown fat is the health fat of heat production in young mice, and HE coloration result shows that control group mice brown fat is in height Whitening is significant under rouge diet induced, unsound fat drips accumulation expansion, after HPF administration is intervened, fat drips in brown fat cell It is reduced significantly, to alleviate the course of disease of brown fat whitening.3. after induction high in fat, deposition and then generation of the fat in liver Damaging action, i.e. " nonalcoholic fatty liver "；HE coloration result shows that control group degeneration of liver cells is obvious, this shows lipid metaboli Disorder results in hepatic injury, and can significantly reverse fatty accumulation and liver cell fatty degeneration after giving HPF.4. right after giving HPF The influence of pars epididymica white adipose is compared with control group without significant difference.

Embodiment 6

Hyperforine improves the glycometabolism of the obesity mice of induction high in fat

In addition, insulin tolerance tests and glucose tolerance test are also embodied in inventor；The object of the experiment is to implement The mouse of control group high in fat, administration group high in fat in example 4, and the experiment is carried out in administration second week.

Insulin tolerance tests: on an empty stomach after 6 hours, the insulin of 1.3U/kg weight is injected intraperitoneally, respectively at injection in mouse 0min afterwards, 15min, 30min, 45min, 60min, 90min cut tail and take blood, detect blood glucose simultaneously with Roche blood glucose meter and blood sugar test paper Record.Data variance is analyzed by T test method, when p < 0.05, it is believed that has statistical difference.

Glucose tolerance test: after mouse fasting 16 hours, being injected intraperitoneally the glucose injection of 1.25mg/kg weight, 0min, 15min, 30min, 60min, 90min and 120min cut tail and take blood after injection, are tried with Roche blood glucose meter and blood glucose Paper detection blood glucose simultaneously records.Data variance is analyzed by T test method, when p < 0.05, it is believed that has statistical difference.

The mouse of hyperforine is injected intraperitoneally in High fat diet eight weeks and after four weeks is administered, blood glucose is significantly lower than compareing Group, insulin sensitivity is better than control group mice (Figure 10), and glucose tolerance is also significantly better than control mice (Figure 11).

Therefore, the present embodiment the experimental results showed that, hyperforine can be effectively reduced High fat diet obesity it is small The blood glucose of mouse, and can significantly improve glucose metabolism and insulin sensitivity.

Embodiment 7

Hyperforine promotes the obesity mice adaptability heat production of induction high in fat

Using medication same as Example 4, after four weeks is administered, respectively to control group high in fat, administration group high in fat Mouse carries out cold stimulation experiment, mouse is constantly exposed in 4 DEG C of environment, 0min, 30min, 60min after cold stimulation starts, When 90min, 150min and 240min, rectal temperature is quickly measured using electronic thermometer.After cold stimulation half an hour, administration is passed through The body temperature of the mouse of leaf hypericin is apparently higher than control mice, and body temperature margi n pulls big, hyperforine are obvious after one hour Promote the obesity mice of induction high in fat to carry out adaptability heat production (referring to Figure 12).When being exposed in cold environment, saving body temperature is The physiological function undertaken by heat production adipose tissue illustrates that hyperforine (dosage 2.5mg/Kg) surrounding is injected intraperitoneally Afterwards, the body temperature hold capacity of administration group mouse is remarkably reinforced, and heat production fat plays a role more significant.

Therefore, inventor, which analyzes, assert, it is by promoting white adipose that hyperforine, which inhibits the effect of body weight increase, Milkproduct simultaneously improves brown fat activity to realize simultaneously.

Embodiment 8

Hyperforine promotes fat cell heat production genetic transcription and UCP1 protein expression

Real-time fluorescence quantitative PCR (RT-PCR) detects heat production gene and each fat specific genes mRNA level in-site: trizol Method extracts adipose tissue total serum IgE, and reverse transcription cDNA, SYBRGREEN method real-time fluorescence quantitative PCR is only praised in promise, according to Δ CT method The relative quantity for calculating mRNA, uses 18S as internal reference, then the mRNA level in-site of control is set as 1, each processing group and control group pair Than obtaining relative quantity.Data variance is analyzed by Student T test method, is statistical difference when p < 0.05.

Experimental result is following (referring to Figure 13 and 14):

In iWAT adipose tissue, the following heat production gene mRNA levels of administration group are raised: UCP1, Dio2, Zic1 up-regulation. While heat production gene upregulation, white adipose specific gene Resistin and mature fat cell marker (FABP4, PPAR gamma, C/EBP) mRNA without significant difference.This shows hyperforine in the obesity mice of induction high in fat not It will affect white adipose differentiation, but can be by promoting the heat production gene upregulation of white adipose tissue that white adipose is inhibited to become larger.

In BAT adipose tissue, the following heat production gene mRNA levels of administration group are raised: UCP1, PGC1 α, Prdm16, Zic1 up-regulation.While heat production gene upregulation, white adipose specific gene Resistin and mature fat cell marker The mRNA of (FABP4, PPAR gamma, C/EBP) is without significant difference.This shows hyperforine in the obesity of induction high in fat To brown adipose tissue not at Fatty toxicity in mouse, but the heat production gene expression of promotion brown adipose tissue can be passed through Brown fat is promoted to realize its function.

Therefore, the present embodiment the experimental results showed that, hyperforine can promote fat cell heat production gene expression.

Embodiment 9

Hyperforine promotes loss of weight effect-dosage experiments of fatty milkproduct

To determine that hyperforine in the effective dose for promoting adipose tissue milkproduct, is carried out with internal in vitro respectively Loss of weight effect-dosage experiments.

As model, 0-20 μM passes through leaf spun gold for C3H10T1/2 mature fat cell that external effective dose is oriented using BMP7 Peach element is handled 48 hours, to be adjusted to efficiency index on heat production gene UCP1, using maturation fat marker Fabp4 as reference.It is real It is as shown in figure 15 to test result, is heat production effective dose since 2.5 μM of hyperforines, 10 μM of whens, enter plateau, and 20 μM still keep stable, to show that the external effective dose of hyperforine is more than or equal to 2.5 μM.It is response- Dose curve, can be obtained EC50=3.5 μM.

Internal effective dose experiment is slowed down with the C57BL/6J mouse weight increase of high fat diet as efficiency index, using with The identical administration mode of embodiment 4 gives 0-25mg/Kg (mouse weight) hyperforine, and dosage period is surrounding.Experiment As a result as shown in figure 16, it is seen then that 2.5-25mg/Kg (mouse weight) hyperforine can effectively slow down obesity.

Therefore, the present embodiment the experimental results showed that, the external effective dose of hyperforine be not less than 2.5 μM, body Interior effective dose is 2.5-25mg/Kg.

Specific embodiments of the present invention are described in detail above, but it is merely an example, the present invention is simultaneously unlimited It is formed on particular embodiments described above.To those skilled in the art, any couple of present invention carries out equivalent modifications and Substitution is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and Modification, all should be contained within the scope of the invention.

Claims (9)

1. hyperforine promotes white adipose milkproduct in preparation and improves the purposes in the active drug of brown fat； Wherein, the structural formula of the hyperforine are as follows:

2. hyperforine according to claim 1 promotes white adipose milkproduct in preparation and improves brown fat Purposes in active drug, which is characterized in that the internal effective dose of hyperforine is 2.5-25mg/Kg weight.

3. hyperforine is preparing the purposes in the drug for preventing or treating obesity；Wherein, described to pass through leaf spun gold The structural formula of peach element are as follows:

4. hyperforine according to claim 3 is preparing the use in the drug for preventing or treating obesity On the way, which is characterized in that the internal effective dose of hyperforine is 2.5-25mg/Kg weight.

5. purposes of the hyperforine in the drug that preparation improves diabetic symptom；Wherein, the hyperforine Structural formula are as follows:

6. purposes of the hyperforine according to claim 5 in the drug that preparation improves diabetic symptom, special Sign is that the internal effective dose of hyperforine is 2.5-25mg/Kg weight.

7. the pharmaceutical composition of a kind of alleviation or treatment nonalcoholic fatty liver, which is characterized in that pass through Ye Jin comprising a effective amount of Silk peach element and pharmaceutically acceptable carrier；Wherein, the structural formula of the hyperforine are as follows:

8. the pharmaceutical composition of alleviation according to claim 7 or treatment nonalcoholic fatty liver, which is characterized in that described Pharmaceutically acceptable carrier is selected from following any: water for injection, freeze dried powder auxiliary material, oral formulations auxiliary material.

9. the pharmaceutical composition of alleviation according to claim 7 or treatment nonalcoholic fatty liver, which is characterized in that described Alleviate or treatment nonalcoholic fatty liver pharmaceutical composition dosage form be selected from it is following any: tablet, capsule, pill, Granula, suspension, oral solution, paint, cataplasm, spray, powder-injection and liquid drugs injection.