CN103989666A - Application of n-3 polyunsaturated fatty acids in non-alcoholic fatty liver disease treatment drugs - Google Patents
Application of n-3 polyunsaturated fatty acids in non-alcoholic fatty liver disease treatment drugs Download PDFInfo
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Abstract
The invention belongs to the technical field of medicine, and particularly relates to an application of n-3 polyunsaturated fatty acids in non-alcoholic fatty liver disease treatment drugs. According to the present invention, triEPA and triDHA prepared by using the enzyme method in our laboratory and having the purity of 80% are adopted as raw materials to study the prevention and improvement effect on mouse model NAFLD so as to provide theory foundation and basis for application of the n-3PUFA in prevention and treatment of NAFLD and development utilization of deep sea fish oil resources, and provide important significance for application of nutrition means to prevent and treat NAFLD.
Description
Technical field
The invention belongs to medical technical field, the particularly application of a kind of n-3 polyunsaturated fatty acid in treatment non-alcoholic fatty liver disease medicine.
Background technology
NAFLD refers to except clinical pathology syndromes due to ethanol and other clear and definite damage liver factors, that the diffusivity hepatocyte Macrovesicular steatosis of take is principal character.Non-alcoholic fatty liver disease illness can be divided into 4 classes in morphology, i.e. simple fatty liver, nonalcoholic steatohepatitis, fatty liver fibrosis, fatty cirrhosis.Sickness rate at western countries NAFLD accounts for 10% of average population, in overweight people up to 50%.Modern age is along with socioeconomic fast development, the increase of various " affluenza " sickness rate such as fat and diabetes, and non-alcohol fatty liver prevalence also rapidly improves, and has become one of three large hepatopathys of harm humans health now.Large quantity research shows, NAFLD not only causes liver related disease and death, and occurred frequently closely related with atherosclerotic cardiovascular disease.There is no at present the specific medicament of NAFLD, is mainly to adopt individualized treatment according to the concrete state of an illness clinically, is mainly three rescue by stages of Primary Care, hepatic treatment, orthotopic liver transplantation treatment.And research in recent years shows, n-3 polyunsaturated fatty acid (n-3 PUFA) is likely as treatment NAFLD effective means.But what former studies was used is that common fish oil is eicosapentaenoic acid (EPA), the mixture of docosahexenoic acid (DHA) and other unsaturated fatty acids, its purity is low, and show that with in vitro study the action and efficacy of EPA and DHA is different in body, can not be clearly which kind of n-3 PUFA plays a role in improving NAFLD actually, therefore be necessary to intervene and study its prevention and improve NAFLD effect separately with DHA and EPA.
Summary of the invention
The object of the present invention is to provide the application of a kind of n-3 polyunsaturated fatty acid in treatment non-alcoholic fatty liver disease medicine.
It is material that EPA Triglycerides (triEPA) and DHA Triglycerides (triDHA) are take in the present invention, studies its prevention and improvement effect to non-alcoholic fatty liver disease (NAFLD) model mice.
get40 of male mices (mouse species), are divided into matched group, triEPA supplementation group, triDHA supplementation group, model group at random by body weight, every group of 10 mices.Except matched group is fed normal feedstuff, all the other each groups are all fed with high lipid food, triEPA and triDHA supplementation group every day are with 1000 mg/kg bw(body weight body weight) triEPA and triDHA emulsion gavage, matched group and model group every day are with 5% gumwater gavage of equivalent, after raising 8w, put to death mice, collect blood and liver specimens.Adopt HE staining to observe the pathological changes situation of liver and measure serum paddy the third transferring enzyme (ALT), glutamic oxaloacetic transaminase, GOT (AST), total triglyceride (TG), T-CHOL (TC) and liver TG, TC.High lipid food induction causes mouse liver Steatosis, triEPA and triDHA supplement after the NAFLD model mice 8w of high lipid food induction, the heavy coefficient of liver, Serum ALT and the AST that have significantly reduced mice are active, have reduced liver TG and TC level, significantly reduce the fat range degree of liver.Experiment results proved, triEPA and triDHA have positive prevention and improvement effect to mice NAFLD.
As preferably, described n-3 polyunsaturated fatty acid is EPA Triglycerides (triEPA) or DHA Triglycerides (triDHA).
The present invention be take this laboratory enzyme process and is made purity to reach 80% triEPA and triDHA be material, study it to the prevention of mouse model NAFLD and improvement effect, for n-3 PUFA prevents and treats NAFLD, is exploitation bathypelagic fish oil Resource Supply theoretical basis and foundation, to with nutrition means preventions and treatment NAFLD, alleviate its harm significant.
Accompanying drawing explanation
Fig. 1 is the affect figure of n-3 polyunsaturated fatty acid on Mouse Weight;
Fig. 2 is the affect figure of n-3 polyunsaturated fatty acid on mouse feed intake;
Fig. 3 is HE dyeing microscope figure (400 *), wherein a. matched group; B. triEPA supplementation group; C. triDHA supplementation group; D. model group;
Fig. 4 is the affect figure of n-3 polyunsaturated fatty acid on mice dry weight index, compares a:P < 0.05, b:P < 0.01 with model group;
Fig. 5 is the affect figure of n-3 polyunsaturated fatty acid on mice serum transaminase vigor, compares a:P < 0.05, b:P < 0.01 with model group;
Fig. 6 is the affect figure of n-3 polyunsaturated fatty acid on mice serum TG and TC content, compares a:P < 0.05, b:P < 0.01 with model group;
Fig. 7 is the affect figure of n-3 polyunsaturated fatty acid on mouse liver TG and TC content, compares a:P < 0.05, b:P < 0.01 with model group.
The specific embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.Should be appreciated that enforcement of the present invention is not limited to the following examples, any pro forma accommodation that the present invention is made and/or change all will fall into protection domain of the present invention.
In the present invention, if not refer in particular to, all part, percentage ratios are unit of weight, and the equipment adopting and raw material etc. all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the conventional method of this area.
Embodiment:
1 materials and methods
1.1 material
Feedstuff: normal feedstuff (heat production of protein-fat-carbohydrate is than being 25.2:11.9:62.9) is provided by Zhejiang Academy of Medical Sciences.High lipid food (heat production of protein-fat-carbohydrate is than being 14.6:48.6:36.7) mixes 1% cholesterol by normal feedstuff, 20% fat (C14:0 2% C16:0 33% C16:1 2% C18:0 20% C18:1 29% C18:2 12%C20:0 2%), is made by Zhejiang Academy of Medical Sciences.
N-3 PUFA:triEPA and triDHA are prepared from by enzyme process by this laboratory and are prepared as emulsion with 5% gumwater.Detailed process can be called referring to application number 200810162265, name the patent of invention of " a kind of method of continuous enzymatic synthesis of n-3 PUFA glyceride ".
Detectable: gradient concentration dehydrated alcohol; The HE test solution that dyes; Aspartate aminotransferase detectable (enzyme performance rate method), the amino transaminase's reagent (enzyme performance rate method) of alanine, triglyceride (TG) detectable (terminal colorimetric analysis), T-CHOL (TC) detectable (terminal colorimetric analysis), above 4 kinds of biochemical reagents are purchased from Beckman Ku Erte experimental system (Suzhou) company limited.
Instrument and equipment: UNICO UV-2800 ultraviolet-uisible spectrophotometer, common mice rearging cage and water fountain, Leica paraffin slicing machine RM2235, peace booth low speed centrifuge LXJ-IIB, Beckman biochemical analysis system LX20, instrument health optical microscope TV-3100.
Laboratory animal: C
57bL/6J male mice 9-10w age, body weight is at (25 ± 2) g, and SPF level, is provided by Zhejiang Academy of Medical Sciences.Laboratory animal production licence number: SYXK(Zhejiang) 2012-0176.
1.2 method
Experiment grouping and interference method: 40 mices adaptability in feeding environment is raised after 120h, by body weight, be divided at random 4 groups, 10 every group, be respectively: Normal group, triEPA supplementation group, triDHA supplementation group, hyperlipidemia model group, 5 mices of each rearging cage, and numbering.Adopt high fat diet to set up mice NAFLD model.Normal group is fed normal feedstuff, and other each groups are all fed high lipid food.Routine observation also records the daily situation of mice, regularly weighs Mouse Weight respectively 5% gumwater, triEPA (1000mg/kg d), the triDHA(1000mg/kg d of gavage 0.5ml every day), 5% gumwater.
Sample collection: raise the mice after 8w, fasting 12h, weigh in, adopt the blood sampling of eyeball blood collection method, use with the blood taking tube of separation gel and collect, after blood sampling, with cervical vertebra dislocation method, put to death, dissect and take out liver rapidly, perusal liver also records after result, with normal saline, cleans, after blotting surface moisture with absorbent paper, weigh, put into fixedly 3d of 10% neutral formalin fixative.
Index detects: gather blood sample at room temperature after standing 1h, 4000r/min, centrifugal 5min, collects serum.Adopt SYNCHRON biochemical analysis system to detect paddy the third transferring enzyme (ALT), glutamic oxaloacetic transaminase, GOT (AST) enzyme activity, serum TG, TC level.
The liver organization that liver TG, TC content detection take about 0.2g is with chloroform: the method for methanol (2:1) extraction is extracted
[1], total fat that extraction obtains is with containing 50mg/L 2, and the chloroform standardize solution of 6-di-tert-butyl-4-methy phenol (BHT) is in 10ml volumetric flask.Get above-mentioned extract 0.5ml, the method for employing Sidney etc. is measured liver TG content
[2].Get above-mentioned extract 0.4ml, the method for employing Ness etc. is measured liver cholesterol level
[3].
Pathological examination: (1) perusal fresh liver, (2) liver HE dyeing: the liver after fixing is got small pieces through the dehydration of ethanol gradient, makes wax stone after dimethylbenzene transparence, adopts the dyeing of HE staining after section, and microscopy is also taken pictures, and the results are shown in Figure 3.
Statistical method: adopt SPSS 19.0 softwares to carry out one factor analysis of variance, data represent with means standard deviation.
2 results
2.1 body weight and food ration
As shown in Figure 1, at the experimental session of whole 8w, between each group, between Mouse Weight, there is no significant difference.Aspect appetite weekly, as shown in Figure 2,1w is to 8w, and each appetite of organizing mice all rises to some extent, and matched group increases the fastest, and model group increases the slowest.Model group feedstuff intake is obviously respectively organized low compared with other, and body weight gain and other each groups are as good as.
2.2 pathological examination
Perusal: matched group liver color is cerise, and model group mouse liver color is yellowish-brown, obviously compared with matched group enlargement, and surface is with greasy feeling.TriEPA supplementation group, triDHA supplementation group mouse liver enlargement degree have more greatly and alleviate compared with model group, and color is redder.
Fig. 3 is HE dyeing microscope figure (400 *), wherein a. matched group; B. triEPA supplementation group; C. triDHA supplementation group; D. model group.HE dyeing: the lesion degree of model group is comparatively serious, liver inner cell is not of uniform size and occur local necrosis, lipid in hepatocyte is sloughed the most cavity of rear formation by ethanol and xylene soluble, lobules of liver structure occurs abnormal, border and structure are not obvious, liver Cable Structure is in disorder, and hepatic sinusoid is not obvious owing to being subject in liver endochylema large fat to drip that extruding narrows down.With model group ratio, the hepatic lesions degree of triEPA supplementation group, triDHA supplementation group is lighter, and liver Cable Structure normal is radial, occurs minority cavity in hepatocyte, lobules of liver structure normal, and hepatic sinusoid is more obvious, and there is not abnormal phenomena in matched group.
The impact of 2.3 n-3 PUFA on the heavy index of Mouse Liver
As shown in Figure 4, the heavy index utmost point of model group Mouse Liver be significantly higher than control group mice (
p<0.01).Compare with model group, through 8w, after triEPA and triDHA supplement the heavy index of Mouse Liver significantly reduce (
p<0. 05), the heavy index of triEPA supplementation group, triDHA supplementation group and control group mice liver is without significant difference.
2.4 impacts of n-3 PUFA on mice serum transaminase level
The ALT value of model group mice and the AST value utmost point be significantly higher than matched group (
p<0.01), see Fig. 5.Compare with model group mice,, through 8w, after triEPA and triDHA supplement, triEPA and triDHA supplementation group mice ALT are active and AST is active significantly reduces (
p<0.01) (
p<0.05).
2.5 impacts of n-3 polyunsaturated fatty acid on mice serum TG, TC
As shown in Figure 6, model group serum TG and TC content be all significantly higher than group (
p<0.01,
pand significant difference does not appear in serum TG and the TC of triEPA, triDHA supplementation group mice and model group mice <0.05).
The impact of 2.6 n-3PUFA on Mouse Liver fat content
As shown in Figure 7, model group mouse liver TG and TC content are all significantly higher than triEPA supplementation group, triDHA supplementation group and control group mice.Compare with model group mice, through 8w, triEPA and triDHA supplement the content that significantly reduced mouse liver TG and TC (
p<0.05), data show triEPA and triDHA supplement mouse liver TG declined respectively 38.4% and 42.1%, TC content declined respectively 36.0% and 40.8%.There is not significant difference in triEPA supplementation group and triDHA supplementation group liver TC and TG content.
3 discuss
NAFLD has become one of three large hepatopathys of harm humans health, there is no at present NAFLD specific medicament.At present, research shows that n-3PUFA has improvement effect energetically to NAFLD, but these researchs often adopt, is sequestered or the ethyl ester type of n-3 PUFA, and different chemical pattern also has obvious difference to situation about absorbing in human body.For example common fish oil or concentrated fish oil product are generally ethyl ester type, and the absorbance of ethyl ester type is only 2l%, although the EPA of sequestered and DHA absorbance are 95%, but easily oxidation, very unstable, and due to digestive tract is had to stimulation, and triglyceride type meets the oils and fats form of absorption of human body, its human absorptivity is apparently higher than ethyl ester type fish oil, and absorption can reach 60% left and right, and character is relatively stable.Result of study shows blood ALT, the AST of blank group and model group, and liver TG, TC index all exist significant difference, and model group liver HE coloration result obviously takes on morbit forms, and the modeling that mice non-alcoholic fatty liver disease is described is successful.
3.1 n-3 PUFA and lipid metabolisms
N-3 PUFA energy regulating lipid metabolism, is mainly manifested in and regulates expression, the promotion steatolysis of lipogenesis related gene and suppress lipogenesis.On the one hand, n-3 PUFA cell membrane fat has extremely strong affinity, after being combined with cell membrane, changing cell membrane and forms; To a certain extent, n-3 PUFA raises some and the signal protein that promotes that steatolysis is relevant, as tumor necrosis factor-alpha and amyloid A, improve the content of the lipolytic hormones such as epinephrine and norepinephrine, promote lipid mobilization, reduce the accumulation of liver fat, be conducive to beta-oxidation or be converted into other materials.N-3 PUFA regulates the raw fat gene transcription factor such as SREBP-1, NF-Y on the other hand, suppresses the synthetic of liver fat.In addition n-3 PUFA can suppress the activity of the synthetic and bind receptor of very low density lipoprotein (VLDL) (VLDL), low density lipoprotein, LDL (LDL), and increase the level of high density lipoprotein (HDL) and the activity of bind receptor, and then acceleration cholesterol is transported into liver by metabolite clearance.Originally studies show that, the liver lipid content that gives the mice of triEPA and triDHA all significantly reduces compared with model group, therefore triEPA and triDHA reduce the accumulation of liver lipid, thereby alleviate the follow-up physiological reactions such as oxidative stress, suppress the development of non-alcoholic fatty liver disease feelings.
3.2 serum transaminases and hepatocyte injury
The most responsive index when serum transaminase ALT and AST are hepatocyte injury, AST major part is present in hepatocellular mitochondrion, and ALT is mainly present in Cytoplasm, only when hepatocyte sustains damage, just can be released in blood in a large number.In this research, triEPA supplementation group and triDHA supplementation group mice serum ALT and AST value are all decreased significantly than model group, consistent with HE dyeing acquired results, illustrate that triEPA and triDHA can effectively reduce fat in liver cell and excessively pile up caused primary cellular defect.
3.3 EPA and the comparison of DHA action effect
After triEPA and triDHA supplement, between two groups of mices, ALT, AST, TG, TC and TG, TC content and pathological examination result find no significant difference, likely both can play similar improvement effect to non-alcoholic fatty liver disease, therefore to the intervention of non-alcoholic fatty liver disease, can adopt the mixture of DHA and EPA to carry out.
To sum up, high lipid food induction causes mouse liver Steatosis, and the heavy coefficient of its liver, TG and TC significantly increase, and pathology of hepar inspection takes on morbit forms.And supplement after the NAFLD model mice 8w of high lipid food induction with triEPA and triDHA, the heavy coefficient of liver, ALT and the AST that have significantly reduced mice are active, liver TG, TC level have been reduced, the fat range degree of liver significantly reduces, and these results of study show that triEPA and triDHA have positive prevention and improvement effect to mice non-alcoholic fatty liver disease.
Above-described embodiment is a kind of preferably scheme of the present invention, not the present invention is done to any pro forma restriction, also has other variant and remodeling under the prerequisite that does not exceed the technical scheme that claim records.
list of references:
[1] Floch J, Lees M, Sloane Stanley GH. A simple method for the isolation and Purification of total lipids form animal tissues [J].
J Biol Chem, 1957:497–509.
[2] Gottfried SP, Rosenberg B. Improved manual spectrophotometric procedure for determination of serum triglycerides [J].
Clin Chem, 1973, 19: 1077–1078.
[3] Ness AT, Pastewka JV, Peacock AC. Evaluation of a Recently Reported Stable Liebermaml-Burehard Reagent and Its Use for the Direct Determination of Serum Total Cholesterol [J].
Clin Chim Acta, 1964, 10: 229–237.
Claims (2)
1. the application of n-3 polyunsaturated fatty acid in treatment non-alcoholic fatty liver disease medicine.
2. application according to claim 1, is characterized in that: described n-3 polyunsaturated fatty acid is EPA Triglycerides (triEPA) or DHA Triglycerides (triDHA).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107530284A (en) * | 2015-02-23 | 2018-01-02 | 翁特拉制药公司 | Millimeter capsule preparation including how unsaturated free fatty |
| JP2018522868A (en) * | 2015-06-26 | 2018-08-16 | プロノヴァ バイオファーマ ノルゲ エーエス | NAFLD therapeutic composition |
-
2014
- 2014-04-11 CN CN201410143696.3A patent/CN103989666A/en active Pending
Non-Patent Citations (3)
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| ZHU F. S.等: "Effects of n-3 polyunsaturated fatty acids from seal oils on nonalcoholic fatty liver disease associated with hyperlipidemia", 《WORLD J GASTROENTEROL》, vol. 14, no. 41, 7 November 2008 (2008-11-07), pages 6395 - 6400, XP055078867, DOI: 10.3748/wjg.14.6395 * |
| 毛磊等: "高含量DHA/EPA甘油三酯型鱼油对大鼠脂质代谢的影响", 《食品工业科技》, vol. 34, no. 24, 31 December 2013 (2013-12-31), pages 347 - 350 * |
| 袁发浒等: "n-3 多不饱和脂肪酸、内质网应激与非酒精性脂肪肝病的研究进展", 《食品安全质量检测学报》, vol. 4, no. 4, 31 August 2013 (2013-08-31), pages 1234 - 1238 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107530284A (en) * | 2015-02-23 | 2018-01-02 | 翁特拉制药公司 | Millimeter capsule preparation including how unsaturated free fatty |
| JP2018522868A (en) * | 2015-06-26 | 2018-08-16 | プロノヴァ バイオファーマ ノルゲ エーエス | NAFLD therapeutic composition |
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