CN102755350B - Refined seal oil and application thereof to preparation of medicament for treating non-alcoholic fatty liver - Google Patents

Abstract

The invention discloses refined seal oil, which is characterized in that: an omega-3 polyunsaturated fatty acid in the refined seal oil consists of the following components in percentage by weight: 4.64-5.03 percent of eicosapentaenoic acid (EPA) C20H30O2, 3.86-4.18 percent of docosapentaenoic acid (DPA) C22H34O2 and 9.5-10.29 percent of docosahexenoic acid (DHA) C22H32O2. The invention further discloses an application of the refined seal oil to preparation of a medicament for treating non-alcoholic fatty liver. The refined seal oil disclosed by the invention can be used for treating non-alcoholic fatty liver, and can also be used for effectively reducing total cholesterol and triglyceride in blood serum.

Description

Refined fur seal and the application in preparation treatment non-alcoholic fatty liver disease medicine thereof

Technical field

The present invention relates to a kind of refined fur seal and the application in the medicine of preparation treatment non-alcoholic fatty liver disease thereof.The present invention also relates to the application in the medicine of this refined fur seal T-CHOL or triglyceride in preparation reduction serum.

Background technology

Adeps Phocae vitulinae (harp seal oil) is commonly called as seal oil, is the oil refining processing from the fat deposit of the mammal fur seal deep-sea high and cold (-50 DEG C) waters.Fur seal is with famous and precious morrhua, salmon fish etc. are food, the fat deposit that in body, enrichment is thick, omega-3 polyunsaturated fatty acids (EPA, DPA, DHA) is rich in Adeps Phocae vitulinae, in addition the natural ecological environment of the pollution-free cleaning in the arctic, Adeps Phocae vitulinae becomes the important sources of optimal omega-3 unsaturated fatty acid in the Nature.It has the liver protecting, adjusting blood lipid etc. and acts in some documents and have been reported, and causes the great attention of domestic and international field of medicaments.

Unsaturated fatty acid is the extremely important fatty acid of a class, and it comprises ω-3 and ω-6 series, but human body can not synthesize, and must be absorbed by biological approach, and the former is from marine animal (fish, fur seal etc.), and the latter is from vegetable oil (as Semen arachidis hypogaeae, Oleum Glycines etc.).Find that ω-3 and ω-6 series must keep suitable ratio could maintain the physiological activity of human normal after deliberation.WHO thinks that the optimal proportion of ω-3 and ω-6 is 1:4, and modern diet structure often causes the omega-3 unsaturated fatty acid Deficiency of Intake of people, and a large amount of statistics shows, in current human body, this ratio substantial deviation is to 1:25, even 1:30.Accordingly, WHO and FAO (Food and Agriculture Organization of the United Nation) issue a statement: the present situation that importance and the mankind in view of product itself generally lack, and need to promote the serial unsaturated fatty acid of ω-3 at world wide.Because fur seal and the mankind belong to mammal together, the ω-3 that fur seal provides is identical with the chemical molecular structure of human body, can be directly absorption of human body and utilization, and Fish are rudimentary cold blooded animals, its available ω-3 has to pass through liver and transforms rear and can be absorption of human body and utilization.

Fatty liver refers to the pathological changes due to overheap fatty in the hepatocyte that a variety of causes causes.The health of the positive serious threat compatriots of fatty liver disease, becomes the second largest hepatopathy being only second to viral hepatitis, has been acknowledged as the common cause of disguised liver cirrhosis.Fatty liver is a kind of common clinical picture, but not one independently disease.If fatty liver is treated not in time, probably cause fat hepatitis, liver cirrhosis, even hepatocarcinoma.

The detection means of current fatty liver is numerous, but accurately can judge what fatty liver degree and reverse changed, is " liver/spleen CT value ", belongs to the item number certificate in " conventional CT is unenhanced for epigastrium ".Data show: 0.7< liver/spleen CT value≤1 item is mild fatty liver, 0.5< liver/spleen CT value≤0.7 is moderate fatty liver, liver/spleen CT value≤0.5 is severe fatty liver liver/spleen, and CT value the most accurately detects the scientific method of fatty liver at present.

Authorize disclosed application number be that the Chinese invention patent application of " 01135517.4 " discloses the application of Adeps Phocae vitulinae as preparation treatment fatty liver medicine on April 12nd, 2006, but the dosage required for this Adeps Phocae vitulinae is greatly, too expensive for this medicine patient.

Therefore, develop as soon as possible and effectively treat fatty liver, especially non-alcoholic fatty liver disease for having of relatively economical patient, particularly the medicine of severe non-alcoholic fatty liver disease is very important.

Summary of the invention

The object of the present invention is to provide a kind of refined fur seal of lower omega-3 unsaturated fatty acid content, it can treat non-alcoholic fatty liver disease effectively with lower dosage, especially severe non-alcoholic fatty liver disease.

In order to achieve the above object, the invention provides a kind of refined fur seal, it is characterized in that: in this refined fur seal, the weight percent content of omega-3 polyunsaturated fatty acids is respectively:

Eicosapentaenoic acid (C ₂₀h ₃₀o ₂, EPA): 4.64%-5.03%;

Clupanodonic acid (C ₂₂h ₃₄o ₂, DPA): 3.86%-4.18%;

Docosahexenoic acid (C ₂₂h ₃₂o ₂, DHA): 9.5%-10.29%.

Further, present invention provides the application of this refined fur seal in the medicine of preparation treatment non-alcoholic fatty liver disease.

Further, non-alcoholic fatty liver disease application described in of this refined fur seal of the present invention in the medicine of preparation treatment non-alcoholic fatty liver disease is slight, moderate or severe non-alcoholic fatty liver disease, wherein 0.7< liver/spleen CT value≤1 is slight non-alcoholic fatty liver disease, 0.5< liver/spleen CT value≤0.7 is moderate non-alcoholic fatty liver disease, and liver/spleen CT value≤0.5 is severe non-alcoholic fatty liver disease.

Further, the day taking dose of refined fur seal application described in of this refined fur seal of the present invention in the medicine of preparation treatment non-alcoholic fatty liver disease is 33mg/kg-111mg/kg.

Further, the day taking dose of refined fur seal application described in of this refined fur seal of the present invention in the medicine of preparation treatment non-alcoholic fatty liver disease is 0.036ml/kg-0.121ml/kg.

Present invention provides the application in the medicine of this refined fur seal T-CHOL or triglyceride in preparation reduction serum.

Further, the day taking dose of the refined fur seal described in the application of this refined fur seal in the medicine preparing T-CHOL or triglyceride in reduction serum is 33mg/kg-111mg/kg.

Further, the day taking dose of the refined fur seal described in the application of this refined fur seal in the medicine preparing T-CHOL or triglyceride in reduction serum is 0.036ml/kg-0.121ml/kg.

Refined fur seal of the present invention just can reach treatment non-alcoholic fatty liver disease with lower omega-3 polyunsaturated fatty acids content and lower day taking dose, especially the effect of severe non-alcoholic fatty liver disease, also can reduce T-CHOL and triglyceride in serum effectively.For wider for the range of choice of raw material enterprise, the production cost of medicine can be reduced, reduce the price of medicine, and then alleviate the financial burden of patient.

Detailed description of the invention

The preparation method of Adeps Phocae vitulinae of the present invention is with reference to applicant in this case's application number No. 201110263392.7 patent applications in JIUYUE in 2011 submission on the 7th, and this public announcement of a patent application day is on February 8th, 2012.The preparation that other preparation method known in the art carries out Adeps Phocae vitulinae of the present invention can certainly be applied.

Embodiment 1: the preparation of refined fur seal

Equipment: the EA250/KD200 single-stage short-range molecular distillation extraction equipment adopting German UIC company to manufacture.

I. preparation flow is as follows:

1. 100kg fur seal Animal fat is shredded;

2. the fur seal Animal fat of chopping is cleaned with the warm water of 40 DEG C of temperature;

3. put into oil water separator centrifugalize after being cleaned by the fur seal Animal fat of chopping and obtain the water of separation and fatty raw material;

4. in pair step 3, isolated water filters the fatty raw material obtaining water and centrifugalize omission, adds in fatty raw material by the fatty raw material that centrifugalize is omitted;

5. open molecular still, when system vacuum reaches 0.001mbar, setting primary heater temperature is 310 DEG C, and setting feed heater temperature is 200 DEG C, carries out degassed to fatty raw material;

6. primary heater temperature reaches 200 DEG C, starts charging, charging rate 80L/h; Primary heater temperature reaches 310 DEG C, charging rate 140L/h;

7. the refined fur seal finished product distilled is collected in cooling.

II. eicosapentaenoic acid (EPA), docosahexenoic acid (DHA) and clupanodonic acid (DPA) content detection

1. Cleaning Principle

For the eicosapentaenoic acid (EPA) in the refining seal oil obtained by embodiment 1, docosahexenoic acid (DHA) and clupanodonic acid (DPA) assay, our company adopts HPLC external standard algoscopy: the unsaturated fatty acid in sample is carried out esterification, become methyl eicosapentaenoic acid (EPAM), Methyl docosahexaenoate (DHAM) and clupanodonic acid methyl ester (DPAM), adopt HPLC external standard algoscopy to measure the content of three kinds of unsaturated fatty acid methyl ester, converse the content of corresponding unsaturated fatty acid.

2. chromatographic condition

Instrument: the high performance liquid chromatograph that Agilent company dispatches from the factory: comprise the intelligent column oven of G1316A, G1314F Variable wavelength UV detector, G1311C1260 quaternary pump (containing built-in degasser), the full-automatic injector of G2170BA chromatographic work station, G1329B (600bar); Excellent spectrum ultra-pure water draft machine.

Reagent: 2,6 ditertiary butyl p cresol (BHT), sodium metal, glacial acetic acid, isopropyl alcohol, be domestic analytical pure; Methanol, acetonitrile, be domestic chromatographically pure.EPA methyl ester (EPAM) titer (10mg/ml), DHA methyl ester (DHAM) titer (10mg/ml), DPA methyl ester (DPAM) titer (10mg/ml), be Supelco Products under Sigma house flag.

Sample: the refining seal oil obtained by embodiment 1.

Chromatographic column: Lichrospher RP-18,5um, 4mm × 250mm.

Mobile phase:

Acetonitrile: methanol: water=7:1:2

A pump: methanol: water=1:2(0.3ml/min)

B pump: acetonitrile (0.7ml/min)

Gradient elution: flow velocity 1.0ml/min

Column temperature: 45 DEG C

Determined wavelength: 210nm

Sample size: 20ul

Theoretical cam curve: calculate should be not less than 5000 by EPAM peak

Separating degree: the separating degree at three main constituent peaks and other impurities peak should be greater than 1.0

Analysis time: 45min

3. detection method is specific as follows:

3.1 analyte derivatives: sample thief refining seal oil is about 100mg(120ul), accurately weighed, put in the brown volumetric flask of 100ml, add the 0.5mol/l Feldalat NM 4ml of brand-new, be placed in the 30 DEG C of continuous joltings of 120r/min of constant temperature oscillation shaking table, react after about 30 minutes, check whether also have oil droplet, if still exist, continue jolting, be not as the criterion with little oil droplet.Add 0.2ml glacial acetic acid cessation reaction wherein, by the methanol constant volume containing 0.005%BHT.4 DEG C of placements must not more than 24 hours.

The preparation of 3.2 standard solutions: precision measures EPAM titer 0.1ml, DHAM titer 0.1ml, DPAM titer 0.1ml, is placed in the brown volumetric flask of 10ml, isopropyl alcohol standardize solution, as mixed mark solution.

3.3 sample introductions: get the derivatization sample that step 3.1 is made, use 0.45um membrane filtration, precision measures 20ul sample introduction, record chromatogram; Separately get standard solution, be measured in the same method.The relative retention time of EPAM, DHAM, DPAM tri-kinds of methyl ester is respectively at about 21min, 28min, 38min.

4. result calculates

According to external standard method with calculated by peak area.

Accurately take the refining seal oil 3 parts obtained by embodiment 1, record takes quality m(every part about 100mg respectively), operate according to the method described above, external standard method measures the content of three kinds of fatty acid methyl esters with peak area, and computing formula is as follows:

Above-mentioned formula is also suitable for DHAM, DPAM.

To the methyl ester content calculated, still need and carry out following conversion:

III. iodine number

1. test method foundation

GB/T5532-2008 National Standard of the People's Republic of China " mensuration of animal and plant fat iodine number ".

2. test method

Refining seal oil 1.0g obtained by Example, put in the dry iodine flask of 500ml, add cyclohexane extraction and glacial acetic acid equal-volume mixed liquor 20ml, after dissolving, precision adds Webster (Wijs) reagent 25ml, close plug shakes up, in the dark place 1 hour, add liquor kalii iodide 20ml and water 150ml, shake up, with the remaining iodine of sodium thiosulfate volumetric solution (0.1mol/L) titration, shake well during titration, when liquid to be mixed brown becomes faint yellow, add starch indicator solution 1ml, continue to be titrated to blue disappearance.Do blank experiment simultaneously.To consume the volume (ml) of sodium thiosulfate volumetric solution (0.1mol/L) for V ₂, the volume (ml) that blank assay consumes is V ₁, example weight (g) is M, and the concentration of sodium thiosulfate volumetric solution (0.1mol/L) is c(mol/l), the iodine number according to following formula calculation sample refining seal oil:

3. the value of measurement result:

Iodine number (g/100g)	Result value arrives
		＜20	0.1
20-60	0.5
		＞60	1

4. related reagent requires:

4.1 liquor kalii iodides (KI): 100g/L, not containing iodate or free-iodine.

4.2 starch solutions: mixed in 30ml water by 5g soluble starch, add 1000ml boiling water, and boil 3 minutes, then cool.

4.3 sodium thiosulfate standard solutions: c(Na ₂s ₂o ₃5H ₂o)=0.1mol/L, demarcates in latter 7 days and uses.

4.4 cyclohexane extraction and glacial acetic acid equal-volume mixed liquor.

4.5 Websters (Wijs) reagent: take iodine monochloride 25g and be dissolved in 1500ml glacial acetic acid.The glacial acetic acid of preparation Webster (Wijs) reagent should contain reducing substances, and this reagent also should do the blank determination of blank sample.

Whether qualification glacial acetic acid contains the method for reducing substances:

Get glacial acetic acid 2ml, add 10ml distilled water diluting, add 1mol/L potassium permanganate 0.1ml, the color presented should remain unchanged in 2 hours.If redness is taken off, illustrate that reducing substances exists.

Commercially available Webster (Wijs) reagent can be adopted.

IV. acid number

1. test method foundation

Hot ethanol algoscopy in GB/T5530-2005 National Standard of the People's Republic of China " animal and plant fat acid number and acidity assaying ".

2. test method

Take the degree of accuracy that the refining seal oil sample M(sample obtained by 5.0g embodiment 1 is weighed: 0.02g), load in conical flask.Will containing 0.5ml phenolphthalein indicator (10g/L, the phenolphthalein of 10g is dissolved in 95% alcoholic solution of 1L) 50ml alcoholic solution insert in conical flask, be heated to boiling, when the temperature of ethanol is higher than 70 DEG C, solution changes color is titrated to the sodium hydroxide of 0.1mol/L or potassium hydroxide solution, and keep solution 15s colour-fast, be terminal.

Ethanol after neutralization is transferred in the conical flask that test sample is housed, fully mixes, boil.With sodium hydroxide or the titration of standard potassium hydroxide solution, titration process wants continuous jolting, changes, and keeps 15s colour-fast, be titration end-point to solution colour.

3. result calculates

Acid number (S) (mg/g) is calculated as follows:

$S=\frac{56.1\&times;V\&times;C}{M}$

In formula:

V: the volume of standard potassium hydroxide solution used, ml;

C: the actual concentrations of standard potassium hydroxide solution used, mol/L;

M: the quality of sample, g.

V. peroxide value

1. test method foundation

GB/T5538-2005 National Standard of the People's Republic of China " animal and plant fat determination of POV ".

2. test method

With carbon dioxide or the nitrogen wash iodine flask of clean dry, seal oil sample (M) 5.0g(that precision takes obtained by embodiment 1 weighs degree of accuracy: ± 0.01g), be placed in above-mentioned iodine flask, add 50ml acetic acid-isooctane solution, cover stopper shake to sample dissolution.Add 0.5ml saturated solution of potassium iodide, cover stopper and make it react, the time is 1min ± 1s, shakes iodine flask at least 3 times during this period, then adds 30ml distilled water immediately.With the above-mentioned solution of hypo solution (C:0.01mol/L) titration, until yellow almost disappears, add about 0.5ml starch solution, continue titration, disappear to blue, be terminal.The hypo solution volume V2 that record consumes.Do blank, the hypo solution volume V1 that record consumes simultaneously.

When blank experiment consumes hypo solution (0.01mol/L) more than 0.1ml, hypo solution (0.01mol/L) should be changed, again sample is measured.

3. result calculates

Peroxide value P is calculated as follows:

$P=\frac{1000(V2-V1)}{2M}$

V1: the hypo solution for blank consumes volume, ml;

V2: the hypo solution for sample determination consumes volume, ml;

C: the concentration of hypo solution, mol/L;

M: the quality of sample, g;

P: peroxide value, represents with mM every kilogram, mmol/kg.

4. reagent requirement needed for

4.1 glacial acetic acids and isobutyltrimethylmethane. mixed liquor (6:4): glacial acetic acid and isobutyltrimethylmethane. all remove oxygen with pure, dry noble gas (carbon dioxide or nitrogen) air-flow.

4.2 saturated solution of potassium iodide: newly prepare and must not free-iodine and iodate be contained.Guarantee in solution, have crystallization to exist, deposit in lucifuge place.If add 0.5ml saturated solution of potassium iodide and 2 starch solutions in 30ml acetic acid-isooctane solution, occur blue, and need hypo solution (0.01mol/L) more than 1 to eliminate, then again prepare this solution.

4.3 hypo solution (0.01mol/L): first prepare hypo solution (0.1mol/L).Taking 21.9g five water sodium thiosulfate (Na2S2O35H2O) is dissolved in distilled water, is diluted to 1L.Demarcate before use, dilution 10, obtained hypo solution (0.01mol/L).

4.4 starch solutions (5g/L): mixed with a small amount of cold distilled water by 1g soluble starch, be dissolved in 200ml boiling water when stirring, and adds 250ml salicylic acid as antiseptic and boil 3min, take off immediately and cool from thermal source.This solution can store 2-3 week at 4 DEG C of refrigerators, when titration end-point from blueness to colourless not obvious time, need again prepare.

Sensitivity verification method: will add in 100ml water in 5ml starch solution, add 0.05% liquor kalii iodide and 1 0.05% liquor natrii hypochloritis, when instilling hypo solution (0.1mol/L) more than 0.05ml, navy blue disappears, and namely represents insufficient sensitivity.

The testing result of the refined fur seal sample obtained by embodiment 1 is:

Embodiment 2: the preparation of refined fur seal

Equipment: the EA250/KD200 single-stage short-range molecular distillation extraction equipment adopting German UIC company to manufacture.

Preparation flow is as follows:

1. 100kg fur seal Animal fat is shredded;

2. the fur seal Animal fat of chopping is cleaned with the warm water of 45 DEG C of temperature;

3. put into oil water separator centrifugalize after being cleaned by the fur seal Animal fat of chopping and obtain the water of separation and fatty raw material;

4. in pair step 3, isolated water filters the fatty raw material obtaining water and centrifugalize omission, adds in fatty raw material by the fatty raw material that centrifugalize is omitted;

5. open molecular still, when system vacuum reaches 0.003mbar, setting primary heater temperature is 320 DEG C, and setting feed heater temperature is 220 DEG C, carries out degassed to fatty raw material;

6. primary heater temperature reaches 200 DEG C, starts charging, charging rate 90L/h; Primary heater temperature reaches 320 DEG C, charging rate 160L/h;

7. the refined fur seal finished product distilled is collected in cooling.

As described in Example 1 content detection, iodine number and acid value measuring are carried out to the refined fur seal sample obtained by embodiment 2.

The testing result of the refined fur seal sample obtained by embodiment 2 is:

Embodiment 3: the preparation of refined fur seal

Equipment: the EA250/KD200 single-stage short-range molecular distillation extraction equipment adopting German UIC company to manufacture.

Preparation flow is as follows:

8. 100kg fur seal Animal fat is shredded;

9. the fur seal Animal fat of chopping is cleaned with the warm water of 45 DEG C of temperature;

10. put into oil water separator centrifugalize after being cleaned by the fur seal Animal fat of chopping and obtain the water of separation and fatty raw material;

In 11. pairs of steps 3, isolated water filters the fatty raw material obtaining water and centrifugalize omission, adds in fatty raw material by the fatty raw material that centrifugalize is omitted;

12. open molecular still, and when system vacuum reaches 0.002mbar, setting primary heater temperature is 315 DEG C, and setting feed heater temperature is 210 DEG C, carries out degassed to fatty raw material;

13. primary heater temperature reach 200 DEG C, start charging, charging rate 85L/h; Primary heater temperature reaches 315 DEG C, charging rate 150L/h;

The refined fur seal finished product distilled is collected in 14. coolings.

As described in Example 1 content detection, iodine number and acid value measuring are carried out to the refined fur seal sample obtained by embodiment 3.

The testing result of the refined fur seal sample obtained by embodiment 3 is:

Experiment 1: the clinical trial of the refined fur seal obtained by embodiment 1 is as follows:

1, test objective

Checking the present invention treats the effectiveness of non-alcoholic fatty liver disease.

2, EXPERIMENTAL DESIGN

The description of 2.1 test master-plans and scheme

Follow GCP principle, adopt random, double blinding, multicenter, Adeps Phocae vitulinae and placebo parallel control study.Object of study is nonalcoholic fatty liver disease, and can evaluate total cases is 400 examples.Ratio according to 3:1 is assigned to fur seal line of oils and placebo group by experimenter at random.

The consideration that 2.2 EXPERIMENTAL DESIGN and matched group are selected

Do not treat the medicine listing of fatty liver at present, therefore adopt placebo to compare.

The selection of 2.3 object of study

2.3.1 diagnostic criteria

The non-alcohol fatty liver practice guidelines of reference in February, 2006 China hepatology meeting fatty liver and the revision of alcoholic liver disease group:

All possess any one person in following 1st ~ 5 and the 6th or the 7th and can be diagnosed as non-alcoholic fatty liver disease.

1) without history of drinking history or drink and amount to amount of alcohol male <140g weekly, women is <70g weekly;

2) except, viral hepatitis, drug-induced liver disease, total parenteral nutrition, hepatolenticular degeneration etc. can cause the specified disease of fatty liver;

3) except primary disease clinical manifestation, weak, nonspecific symptom and the sign such as dyspepsia, dull pain in liver, hepatosplenomegaly can be had;

4) overweight and (or) Abdominal obesity can be had, fasting glucose increases, the metabolism syndrome related component such as blood fat disorder, hypertension;

5) serum transaminase and gamma glutamyl transpeptidase level can have mild to moderate increasing (being less than 5 times of Upper Limit of Normal Values), usually increase based on alanine aminotransferase (ALT);

6) liver imageology performance meets the imaging diagnosis standard of diffusivity fatty liver;

7) Liver biopsy changes the pathological diagnosis standard meeting fatty liver.

2.3.2 inclusion criteria

1) Informed Consent Form person is signed;

2) sex at 18 ~ 65 years old age;

3) clinical diagnosis meets nonalcoholic fatty liver diagnostic criteria;

4) liver/spleen CT ratio≤1;

5) TG > upper limits of normal;

6) medicine (comprising the medicine of the liver protecting and ALT lowering or effect for reducing fat) for the treatment of fatty liver is not used in nearly 4 weeks;

7) Women of Childbearing Age must have effective contraceptives.

2.3.3 exclusion standard

1) diabetics and fasting glucose are greater than 7.0;

2) hepatopathy caused by the factor such as viral hepatitis, medicine, ethanol, autoimmune, Wilson disease, total parenteral nutrition;

3) heart, kidney, lung, endocrine, blood, metabolism and the serious protopathy person of Digestive is merged, or psychotic;

4) glutamate pyruvate transaminase (ALT) >=10 times;

5) GGT > 5 times of upper limits of normal;

6) serum total bilirubin (TBIL) > 1.5 times of upper limits of normal;

7) serum albumin (ALB) < 33g/L;

8) creatinine (Cr) > 130umol/L;

9) platelet count (PLT) < 80109/L;

10) any mode slimmer is carried out in 3 months;

11) other clinical trial person is participated in 3 months;

12) anemia of pregnant woman, women breast-feeding their children or application estrogen contraception person;

13) alcoholic or drug-addict;

14) known to test drug allergy sufferers.

2.3.4 standard is rejected

1) case that is selected, exclusion standard is not met;

2) experimenter's compliance is poor, not by protocols call pill taker (be greater than 120% or be less than 80%);

3) duration of test applies the other drug person that may affect this research therapeutic evaluation.

2.4 process of the test

2.4.1 investigational agent

Seal oil ω-3PUFA obtained by embodiment 1, containing the omega-3 polyunsaturated fatty acids of 18%.

2.4.2 placebo

Olive oil, commercially obtains.Containing oleic acid 53%, meet placebo and prepare requirement.

2.4.3 administrated method

Every day twice, each 0.018ml/kg, diet does not affect investigational agent, therefore to the time of taking medicine not requirement.

3, the analysis of curative effect/effect

3.1 curative effect indexs

Liver/spleen CT ratio:

FAS crowd, test group 324 example, matched group 104 example.Test group: baseline period liver/spleen CT ratio median is 0.802 (0.110 ~ 1.000), and meansigma methods is 0.748 ± 0.194; When treating 24 weeks, liver/spleen CT ratio median is 0.832 (0.016 ~ 1.287), and meansigma methods is 0.794 ± 0.220; Before and after treatment, changing value (rear-front) meansigma methods is 0.046 ± 0.167,95%CI is 0.028 ~ 0.064.Matched group: baseline period liver/spleen CT ratio median is 0.795 (0.162 ~ 0.985), and meansigma methods is 0.750 ± 0.194; When treating 24 weeks, liver/spleen CT ratio median is 0.810 (0.029 ~ 1.212), and meansigma methods is 0.772 ± 0.237; Before and after treatment, changing value (rear-front) meansigma methods is 0.023 ± 0.188,95%CI is-0.014 ~ 0.059.After treatment, test group liver/spleen CT ratio obviously raises, there is the statistical significance of highly significant, P=0.0000(< 0.01, table 2, and covariance analysis 95% credibility interval is 95%CI is 0.028 ~ 0.064(table 1), do not comprise 0, the prompting Adeps Phocae vitulinae taken obtained by the embodiment of the present invention 1 can raise liver/spleen CT ratio further, and have the power of a test of 95% to promote amplitude between 0.028 ~ 0.064, there is clinical practice meaning.Illustrate that the Adeps Phocae vitulinae taken obtained by the embodiment of the present invention 1 obviously can raise liver/spleen CT ratio clinically, alleviates Liver fatty deposition.Compare in matched group group and there is statistical significance, P=0.0399(< 0.05, table 2), but covariance analysis 95%CI is-0.014 ~ 0.059(table 1), comprise 0, power of a test liver/spleen CT that placebo 95% is taken in prompting can drop between-0.014 ~ 0.059 than changing value, does not have practical significance clinically for lifting liver/spleen CT ratio.Before and after two groups of treatments, liver/spleen CT ratio raises meansigma methods test group higher than matched group, but compares P=0.9415(table 2 between group), difference does not have statistical significance.

PPS crowd, test group 294 example, matched group 92 example.Test group baseline period liver/spleen CT ratio median is 0.804 (0.110 ~ 1.000), and meansigma methods is 0.749 ± 0.193; When treating 24 weeks, liver/spleen CT ratio median is 0.835 (0.016 ~ 1.287), and meansigma methods is 0.798 ± 0.220; Before and after treatment, changing value (rear-front) meansigma methods is 0.049 ± 0.171,95%CI is 0.029 ~ 0.068.Matched group baseline period liver/spleen CT ratio median is 0.812 (0.162 ~ 0.985), and meansigma methods is 0.757 ± 0.194; When treating 24 weeks, liver/spleen CT ratio median is 0.816 (0.029 ~ 1.212), and meansigma methods is 0.781 ± 0.242; Before and after treatment, changing value (rear-front) meansigma methods is 0.024 ± 0.200,95%CI is-0.017 ~ 0.066.Before and after treatment, experimenter's liver/spleen CT ratio test group is increased significantly, P=0.0000(< 0.01 is compared in group, there is the statistical significance of highly significant, and covariance analysis 95% credibility interval is 95%CI is 0.029 ~ 0.068, do not comprise 0, the Adeps Phocae vitulinae that further confirmation is taken obtained by the embodiment of the present invention 1 obviously can raise liver/spleen CT ratio clinically, alleviates Liver fatty deposition.Matched group raises not obvious, compare P=0.0542, not statistically significant, and 95%CI is-0.017 ~ 0.066, comprises 0, does not have clinical meaning in group.Two groups are compared, and the meansigma methods test group of liver/spleen CT ratio lift-off value is a little more than matched group, but P=0.9454, difference does not have statistical significance.

Subgroup is analyzed:

This research has carried out subgroup analysis to the case liver/spleen CT ratio being diagnosed as severe non-alcoholic fatty liver disease before treatment, severe fatty liver test group 42 example in FAS analytic set, matched group 15 example.Test group baseline period liver/spleen CT ratio median is 0.406 (0.110 ~ 0.500), and meansigma methods is 0.376 ± 0.111; When treating 24 weeks, liver/spleen CT ratio median is 0.475 (0.016 ~ 1.283), and meansigma methods is 0.504 ± 0.247; Before and after treatment, changing value (rear-front) meansigma methods is 0.128 ± 0.226,95%CI is 0.058 ~ 0.199.Matched group baseline period liver/spleen CT ratio median is 0.406 (0.162 ~ 0.498), and meansigma methods is 0.391 ± 0.094; When treating 24 weeks, liver/spleen CT ratio median is 0.477 (0.029 ~ 0.842), and meansigma methods is 0.474 ± 0.231; Before and after treatment, changing value (rear-front) meansigma methods is 0.083 ± 0.196,95%CI is-0.025 ~ 0.192.After treatment, test group obviously raises, and there is the statistical significance of highly significant, p=0.0004(< 0.01, table 4, and covariance analysis 95% credibility interval is 95%CI is 0.058 ~ 0.199(table 3), do not comprise 0, show that this rising has clinical meaning further, the prompting Adeps Phocae vitulinae taken obtained by the embodiment of the present invention 1 obviously can raise the liver/spleen CT ratio of severe nonalcoholic fatty liver disease clinically, alleviates Liver fatty deposition.P=0.2439(table 4 is compared before and after treatment of control group), covariance analysis 95%CI is-0.025 ~ 0.192(table 3), comprise 0, prompting is taken placebo and rising liver/spleen CT ratio is neither had to statistical significance, also do not have clinical practice meaning.Before and after two groups of treatments, liver/spleen CT ratio raises meansigma methods test group higher than matched group, but compares P=0.6562(table 4 between group), difference does not have statistical significance.PPS analyzes and analyzes consistent with FAS.

The situation of change of front and back experimenter's liver/spleen CT ratio treated by table 1

The comparison of front and back experimenter's liver/spleen CT ratio situation of change treated by table 2

Compare employing in the group of changing value and meet rank test, statistic is S; Compare between group and use Wilcoxon rank test, statistic is Z.

The situation of change of case treatment front and back experimenter's liver/spleen CT ratio of front severe fatty liver treated by table 3

The comparison of case treatment front and back experimenter's liver/spleen CT ratio situation of change of front severe fatty liver treated by table 4

Compare employing in the group of changing value and meet rank test, statistic is S; Compare between group and use Wilcoxon rank test, statistic is Z.

4. blood fat:

t-CHOL:

In FAS analytic set, test group: baseline period TC content median is 5.35 (2.70 ~ 11.07) mmol/L, average out to 5.40 ± 1.12mmol/L.After treatment, the 12nd week TC content median is 5.10 (2.45 ~ 12.50) mmol/L, average out to 5.16 ± 1.07mmol/L, difference (anterior-posterior) TC content median is 0.19 (-2.46 ~ 4.80) mmol/L, average out to 0.22 ± 0.97mmol/L, 95%CI are 0.110 ~ 0.333.After treatment, the 24th week TC content median is 5.20 (2.40 ~ 12.15) mmol/L, average out to 5.27 ± 1.18mmol/L, difference (anterior-posterior) TC content median is 0.03 (-6.47 ~ 5.58) mmol/L, average out to 0.13 ± 1.11mmol/L, 95%CI are 0.002 ~ 0.257.See table 5..

Matched group: baseline period TC content median is 5.33 (2.84 ~ 11.49) mmol/L, average out to 5.35 ± 1.09mmol/L.After treatment, the 12nd week TC content median is 5.00 (2.91 ~ 7.90) mmol/L, average out to 5.16 ± 1.12mmol/L, difference (anterior-posterior) TC content median is 0.20 (-2.41 ~ 4.02) mmol/L, average out to 0.20 ± 0.98mmol/L, 95%CI are-0.006 ~ 0.409.After treatment, the 24th week TC content median is 5.20 (2.99 ~ 8.03) mmol/L, average out to 5.27 ± 1.09mmol/L, difference (anterior-posterior) TC content median is-0.08 (-1.62 ~ 3.76) mmol/L, average out to 0.07 ± 0.94mmol/L, 95%CI is-0.126 ~ 0.268, table 5.

Compare in group, test group: when treating 12 weeks TC content comparatively baseline obviously decline, and there is highly significant statistical significance, P=0.0006(is less than 0.01), in addition, when 12 weeks, before and after TC content, the 95%CI of difference is 0.110 ~ 0.333, does not comprise 0, shows that the decline of clinical meaning appears having in test group TC equally.When treating 24 weeks, TC content meansigma methods is compared with baseline, still have and decline by a relatively large margin, although do not present statistical significance to change (P=0.0515), but before and after TC content, the 95%CI of difference is 0.002 ~ 0.257 when 24 weeks, do not comprise 0, when showing test group 24 weeks equally there is having the decline of clinical meaning in TC.Matched group: when treating 12 weeks TC content comparatively baseline decline to some extent, and there is statistical significance (P=0.0269), but when 12 weeks before and after TC content the 95%CI of difference be-0.006 ~ 0.409, comprise 0, so this decline does not have clinical meaning.When treating 24 weeks, TC content meansigma methods is without significant change compared with baseline, and does not have statistical significance (P=0.8807), and when 24 weeks, before and after TC content, the 95%CI of difference is-0.126 ~ 0.268, comprises 0, does not also have clinical meaning equally.Refer to table 7.

Compare between group, treat the 12nd week and the 24th week, TC meansigma methods fall test group is all greater than matched group, but group difference does not all present statistical significance.Refer to table 7.

PPS analytic set is consistent with FAS analysis result, refers to table 5 and table 6..

triglyceride TG:

In FAS crowd, test group: baseline period TG content median is 2.50 (0.86 ~ 28.54) mmol/L, meansigma methods is 3.41 ± 2.79mmol/L, after treatment, the 12nd week TG content median is 2.15 (0.30 ~ 25.33) mmol/L, meansigma methods is 2.81 ± 2.40mmol/L, difference (anterior-posterior) median is 0.40 (-8.62 ~ 24.31) mmol/L, and meansigma methods is 0.60 ± 2.49mmol/L, 95%CI is 0.317 ~ 0.890.Treating the 24th week TG content median is 2.23 (0.49 ~ 27.77) mmol/L, meansigma methods is 2.72 ± 2.16mmol/L, difference (anterior-posterior) median is 0.34 (-9.44 ~ 23.19) mmol/L, meansigma methods is 0.69 ± 2.49mmol/L, 95%CI is 0.400 ~ 0.973(table 8).

Matched group: baseline period TG content median is 2.53 (1.64 ~ 25.37) mmol/L, meansigma methods is 3.26 ± 2.89mmol/L, after treatment, the 12nd week TG content median is 2.38 (0.80 ~ 18.28) mmol/L, meansigma methods is 3.48 ± 3.39mmol/L, difference (anterior-posterior) median is 0.17 (-16.54 ~ 8.43) mmol/L, meansigma methods is-0.30 ± 2.57mmol/L, 95%CI is-0.840 ~ 0.242.Treating the 24th week TG content median is 2.19 (0.98 ~ 18.39) mmol/L, meansigma methods is 2.92 ± 2.45mmol/L, difference (anterior-posterior) median is 0.19 (-4.45 ~ 8.95) mmol/L, meansigma methods is 0.24 ± 1.63mmol/L, 95%CI is-0.105 ~ 0.577(table 8).

Compare in group, when treating 12 weeks and 24 weeks, test group is comparatively treated front TG content and is all occurred obvious reduction, and there is the statistical significance of highly significant, P=0.0000(< 0.01, and when when 12 weeks, before and after TG content, the 95%CI of difference is 0.317 ~ 0.890,24 weeks before and after TG content the 95%CI of difference be 0.400 ~ 0.973, all do not comprise 0, after Adeps Phocae vitulinae is taken in explanation, TG decline not only has statistical significance, and has practical significance clinically; Treatment of control group 12 weeks with when 24 weeks compared with before treatment, all there is no significant change, and not there is statistical significance P > 0.05(table 10), when 12 weeks, before and after TG content, the 95%CI of difference is-0.840 ~ 0.242, when 24 weeks, before and after TG content, the 95%CI of difference is-0.105 ~ 0.577, all comprise 0, illustrate that after taking placebo, TG decline does not have practical significance clinically.(table 10)

Compare between group, when treating the 12nd week, test group TG drop-out value apparently higher than matched group, and has remarkable statistical significance, P=0.0081(< 0.01.When treating 24 weeks, test group TG drop-out value still higher than matched group, but does not present the difference with statistical significance, P=0.0517(> 0.05).(table 10)

PPS analysis result is consistent with FAS, refers to table 8 and table 9.

The situation of change (FAS) of front and back T-CHOL TC (mmol/L) treated by table 5 respectively group

The situation of change (PPS) of front and back T-CHOL TC (mmol/L) treated by table 6 respectively group

The comparison of front and back T-CHOL TC (mmol/L) situation of change treated by table 7 respectively group

Compare employing paired t-test in the group of difference, compare between group and adopt group t to check, statistic is t.

The situation of change (FAS) of front and back triglyceride TG (mmol/L) treated by table 8 respectively group

The situation of change (PPS) of front and back triglyceride TG (mmol/L) treated by table 9 respectively group

The comparison of front and back triglyceride TG (mmol/L) situation of change treated by table 10 respectively group

Compare employing paired t-test in the group of difference, compare between group and adopt group t to check, statistic is t.

5. the effectiveness brief summary of clinical experiment

This research FAS is all consistent with PPS analysis result.

Liver/spleen CT ratio: during FAS crowd 24 weeks, test group and matched group liver/spleen CT average of relatives value are respectively 0.794 ± 0.220 and 0.750 ± 0.194, before and after treatment, changing value (rear-front) meansigma methods is respectively 0.046 ± 0.167 and 0.023 ± 0.188, and test group elevation amplitude is greater than placebo group.The statistical significance with highly significant is compared before and after test group treatment, P=0.0000(< 0.01), and difference 95%CI is 0.028 ~ 0.064, do not comprise 0, Adeps Phocae vitulinae is taken in further prompting can raise liver/spleen CT ratio, and have the power of a test of 95% to promote amplitude between 0.028 ~ 0.064, there is clinical practice meaning.P=0.0399(< 0.05 is compared) before and after treatment of control group, there is statistical significance, but 95%CI is-0.014 ~ 0.059, comprise 0, power of a test liver/spleen CT that placebo 95% is taken in prompting can drop between-0.014 ~ 0.059 than changing value, does not have practical significance clinically for lifting liver/spleen CT ratio.

Severe fatty liver subgroup analysis of cases: severe fatty liver test group 42 example in FAS analytic set, matched group 15 example.When treating 24 weeks, test group and matched group liver/spleen CT average of relatives value are respectively 0.504 ± 0.247 and 0.474 ± 0.231, poor before and after treatment (rear-front) meansigma methods is respectively 0.128 ± 0.226 and 0.083 ± 0.196, and test group elevation amplitude is greater than placebo group.P=0.0004(< 0.01 is compared) before and after test group treatment, there is the statistical significance of highly significant, 95%CI is 0.058 ~ 0.199, do not comprise 0, prompting is taken Adeps Phocae vitulinae and can be raised severe fatty liver patient liver/spleen CT ratio, and have the power of a test of 95% to promote amplitude between 0.058 ~ 0.199, there is clinical practice meaning.Comparatively P=0.2439 before and after treatment of control group, do not have statistical significance, 95%CI is-0.025 ~ 0.192, comprises 0, power of a test liver/spleen CT that placebo 95% is taken in prompting can drop between-0.025 ~ 0.192 than changing value, does not have practical significance clinically for lifting liver/spleen CT ratio.

Total cholesterol level: during test group treatment 12 weeks TC content comparatively baseline obviously decline, and there is highly significant statistical significance, and P=0.0006(is less than 0.01), the 95%CI of difference is 0.110 ~ 0.333, do not comprise 0, show that the decline of clinical meaning appears having in test group TC equally.When 24 weeks, TC content continues to decline by a relatively large margin, although do not present statistical significance to change (P=0.0515), the 95%CI of difference is 0.002 ~ 0.257, does not comprise 0, and when showing test group 24 weeks equally, TC decline has clinical meaning.Matched group, before and after 12 weeks TC content, the 95%CI of difference is-0.006 ~ 0.409, comprises 0, and change does not have clinical meaning.Within 24 weeks, 95%CI is-0.126 ~ 0.268, comprises 0, does not have clinical meaning equally.

Obvious reduction is all there is in content of triglyceride: test group TG when treating 12 weeks and 24 weeks, P=0.0000(< 0.01, and the 95%CI of difference is respectively 0.317 ~ 0.890 and 0.400 ~ 0.973, all do not comprise 0, illustrate that after taking Adeps Phocae vitulinae, TG decline not only has the statistical significance of highly significant, and clinically there is practical significance; Treatment of control group 12 weeks and 24 weeks time TG all there is no significant change, P > 0.05, the 95%CI of difference is respectively-0.840 ~ 0.242 and-0.105 ~ 0.577, all comprise 0, prompting is taken placebo TG decline and is not namely had statistical significance, does not also have practical significance clinically.Compare between group, when the 12nd week, TG drop-out value test group apparently higher than matched group, and has highly significant statistical significance.

In sum:

The present invention has significant clinical meaning to the liver/spleen CT ratio and reduction blood fat that improve non-alcoholic fatty liver disease, especially presents good clinical efficacy to serious symptom Patients with Fatty Liver.

The present invention has clinical meaning to T-CHOL and content of triglyceride in reduction serum.

Experiment 2: the clinical trial of the refined fur seal obtained by embodiment 1 is as follows:

Enforcement 2 is identical with experiment 1 clinical trial, unlike following one: 2.4.3 administrated method

Every day twice, each 0.0605ml/kg, diet does not affect investigational agent, therefore to the time of taking medicine not requirement.

The clinical effectiveness that experiment 2 obtains is similar to the result that experiment 1 obtains.

Experiment 3: the clinical trial of the refined fur seal obtained by embodiment 1 is as follows:

Enforcement 3 is identical with experiment 1 clinical trial, unlike following one:

2.4.3 administrated method

Every day twice, each 16.5mg/kg, diet does not affect investigational agent, therefore to the time of taking medicine not requirement.

The clinical effectiveness that experiment 3 obtains is similar to the result that experiment 1 obtains.

Experiment 4: the clinical trial of the refined fur seal obtained by embodiment 1 is as follows:

Enforcement 4 is identical with experiment 1 clinical trial, unlike following one:

2.4.3 administrated method

Every day twice, each 55.5mg/kg, diet does not affect investigational agent, therefore to the time of taking medicine not requirement.

The clinical effectiveness that experiment 4 obtains is similar to the result that experiment 1 obtains.

Experiment 5: the clinical trial of the refined fur seal obtained by embodiment 2 is as follows:

Enforcement 5 is identical with experiment 1 clinical trial, unlike following one:

2.4.1 investigational agent

Refined fur seal obtained by embodiment 2, containing the omega-3 polyunsaturated fatty acids of 19.5%.

The clinical effectiveness that experiment 5 obtains is similar to the result that experiment 1 obtains.

Experiment 6: the clinical trial of the refined fur seal obtained by embodiment 2 is as follows:

Enforcement 6 is identical with experiment 2 clinical trials, unlike following one:

2.4.1 investigational agent

Refined fur seal obtained by embodiment 2, containing the omega-3 polyunsaturated fatty acids of 19.5%.

The clinical effectiveness that experiment 6 obtains is similar to the result that experiment 1 obtains.

Experiment 7: the clinical trial of the refined fur seal obtained by embodiment 2 is as follows:

Enforcement 7 is identical with experiment 3 clinical trials, unlike following one:

2.4.1 investigational agent

Refined fur seal obtained by embodiment 2, containing the omega-3 polyunsaturated fatty acids of 19.5%.

The clinical effectiveness that experiment 7 obtains is similar to the result that experiment 1 obtains.

Experiment 8: the clinical trial of the refined fur seal obtained by embodiment 2 is as follows:

Enforcement 8 is identical with experiment 4 clinical trials, unlike following one:

2.4.1 investigational agent

Refined fur seal obtained by embodiment 2, containing the omega-3 polyunsaturated fatty acids of 19.5%.

The clinical effectiveness that experiment 8 obtains is similar to the result that experiment 1 obtains.

Experiment 9: the clinical trial of the refined fur seal obtained by embodiment 3 is as follows:

Enforcement 9 is identical with experiment 1 clinical trial, unlike following one:

2.4.1 investigational agent

Refined fur seal obtained by embodiment 3, containing the omega-3 polyunsaturated fatty acids of 18.76%.

The clinical effectiveness that experiment 9 obtains is similar to the result that experiment 1 obtains.

Experiment 10: the clinical trial of the refined fur seal obtained by embodiment 3 is as follows:

Enforcement 10 is identical with experiment 2 clinical trials, unlike following one:

2.4.1 investigational agent

Refined fur seal obtained by embodiment 3, containing the omega-3 polyunsaturated fatty acids of 18.76%.

The clinical effectiveness that experiment 10 obtains is similar to the result that experiment 1 obtains.

Experiment 11: the clinical trial of the refined fur seal obtained by embodiment 3 is as follows:

Enforcement 11 is identical with experiment 3 clinical trials, unlike following one:

2.4.1 investigational agent

Refined fur seal obtained by embodiment 3, containing the omega-3 polyunsaturated fatty acids of 18.76%.

The clinical effectiveness that experiment 11 obtains is similar to the result that experiment 1 obtains.

Experiment 12: the clinical trial of the refined fur seal obtained by embodiment 3 is as follows:

Enforcement 12 is identical with experiment 4 clinical trials, unlike following one:

2.4.1 investigational agent

Refined fur seal obtained by embodiment 3, containing the omega-3 polyunsaturated fatty acids of 18.76%.

The clinical effectiveness that experiment 12 obtains is similar to the result that experiment 1 obtains.

Claims (7)

1. the application of refined fur seal in the medicine of preparation treatment non-alcoholic fatty liver disease, in this refined fur seal, omega-3 polyunsaturated fatty acids is obtained by single-stage short-range molecular distillation, and its weight percent content is respectively:

Eicosapentaenoic acid: 4.64%-5.03%;

Clupanodonic acid: 3.86%-4.18%;

Docosahexenoic acid: 9.5%-10.29%;

The EA250/KD200 single-stage short-range molecular distillation extraction equipment wherein adopting German UIC company to manufacture, the step of single-stage short-range molecular distillation is as follows:

Fur seal Animal fat is shredded; The fur seal Animal fat of chopping is cleaned with the warm water of 40 DEG C of temperature; Put into oil water separator centrifugalize after being cleaned by the fur seal Animal fat of chopping and obtain the water of separation and fatty raw material; Isolated water is filtered to the fatty raw material obtaining water and centrifugalize omission, the fatty raw material that centrifugalize is omitted is added in fatty raw material; Open molecular still, when system vacuum reaches 0.001mbar, setting primary heater temperature is 310 DEG C, and setting feed heater temperature is 200 DEG C, carries out degassed to fatty raw material; Primary heater temperature reaches 200 DEG C, starts charging, correspondingly charging rate 80L/h; Primary heater temperature reaches 310 DEG C, charging rate 140L/h; The refined fur seal finished product distilled is collected in cooling;

Or the EA250/KD200 single-stage short-range molecular distillation extraction equipment adopting German UIC company to manufacture, the step of single-stage short-range molecular distillation is as follows:

Fur seal Animal fat is shredded; The fur seal Animal fat of chopping is cleaned with the warm water of 45 DEG C of temperature; Put into oil water separator centrifugalize after being cleaned by the fur seal Animal fat of chopping and obtain the water of separation and fatty raw material; Isolated water is filtered to the fatty raw material obtaining water and centrifugalize omission, the fatty raw material that centrifugalize is omitted is added in fatty raw material; Open molecular still, when system vacuum reaches 0.003mbar, setting primary heater temperature is 320 DEG C, and setting feed heater temperature is 220 DEG C, carries out degassed to fatty raw material; Primary heater temperature reaches 200 DEG C, starts charging, charging rate 90L/h; Primary heater temperature reaches 320 DEG C, correspondingly charging rate 160L/h; The refined fur seal finished product distilled is collected in cooling;

Or the EA250/KD200 single-stage short-range molecular distillation extraction equipment adopting German UIC company to manufacture, the step of single-stage short-range molecular distillation is as follows:

Fur seal Animal fat is shredded; The fur seal Animal fat of chopping is cleaned with the warm water of 45 DEG C of temperature; Put into oil water separator centrifugalize after being cleaned by the fur seal Animal fat of chopping and obtain the water of separation and fatty raw material; Isolated water is filtered to the fatty raw material obtaining water and centrifugalize omission, the fatty raw material that centrifugalize is omitted is added in fatty raw material; Open molecular still, when system vacuum reaches 0.002mbar, setting primary heater temperature is 315 DEG C, and setting feed heater temperature is 210 DEG C, carries out degassed to fatty raw material; Primary heater temperature reaches 200 DEG C, starts charging, charging rate 85L/h; Primary heater temperature reaches 315 DEG C, charging rate 140L/h, 150L/h; The refined fur seal finished product distilled is collected in cooling.

2. application according to claim 1, it is characterized in that: described non-alcoholic fatty liver disease is slight, moderate or severe non-alcoholic fatty liver disease, wherein 0.7< liver/spleen CT value≤1 is slight non-alcoholic fatty liver disease, 0.5< liver/spleen CT value≤0.7 is moderate non-alcoholic fatty liver disease, and liver/spleen CT value≤0.5 is severe non-alcoholic fatty liver disease.

3. application according to claim 1 and 2, is characterized in that: the day taking dose of described refined fur seal is 33mg/kg-111mg/kg.

4. application according to claim 1 and 2, is characterized in that: the day taking dose of described refined fur seal is 0.036ml/kg-0.121ml/kg.

5. refined fur seal reduces the application in the medicine of T-CHOL or triglyceride in serum in preparation, and in this refined fur seal, omega-3 polyunsaturated fatty acids is obtained by single-stage short-range molecular distillation, and its weight percent content is respectively:

Eicosapentaenoic acid: 4.64%-5.03%;

Clupanodonic acid: 3.86%-4.18%;

Docosahexenoic acid: 9.5%-10.29%,

The EA250/KD200 single-stage short-range molecular distillation extraction equipment wherein adopting German UIC company to manufacture, the step of single-stage short-range molecular distillation is as follows:

Fur seal Animal fat is shredded; The fur seal Animal fat of chopping is cleaned with the warm water of 40 DEG C of temperature; Put into oil water separator centrifugalize after being cleaned by the fur seal Animal fat of chopping and obtain the water of separation and fatty raw material; Isolated water is filtered to the fatty raw material obtaining water and centrifugalize omission, the fatty raw material that centrifugalize is omitted is added in fatty raw material; Open molecular still, when system vacuum reaches 0.001mbar, setting primary heater temperature is 310 DEG C, and setting feed heater temperature is 200 DEG C, carries out degassed to fatty raw material; Primary heater temperature reaches 200 DEG C, starts charging, correspondingly charging rate 80L/h; Primary heater temperature reaches 310 DEG C, charging rate 140L/h; The refined fur seal finished product distilled is collected in cooling;

Or the EA250/KD200 single-stage short-range molecular distillation extraction equipment adopting German UIC company to manufacture, the step of single-stage short-range molecular distillation is as follows:

Fur seal Animal fat is shredded; The fur seal Animal fat of chopping is cleaned with the warm water of 45 DEG C of temperature; Put into oil water separator centrifugalize after being cleaned by the fur seal Animal fat of chopping and obtain the water of separation and fatty raw material; Isolated water is filtered to the fatty raw material obtaining water and centrifugalize omission, the fatty raw material that centrifugalize is omitted is added in fatty raw material; Open molecular still, when system vacuum reaches 0.003mbar, setting primary heater temperature is 320 DEG C, and setting feed heater temperature is 220 DEG C, carries out degassed to fatty raw material; Primary heater temperature reaches 200 DEG C, starts charging, charging rate 90L/h; Primary heater temperature reaches 320 DEG C, correspondingly charging rate 160L/h; The refined fur seal finished product distilled is collected in cooling;

Or the EA250/KD200 single-stage short-range molecular distillation extraction equipment adopting German UIC company to manufacture, the step of single-stage short-range molecular distillation is as follows:

Fur seal Animal fat is shredded; The fur seal Animal fat of chopping is cleaned with the warm water of 45 DEG C of temperature; Put into oil water separator centrifugalize after being cleaned by the fur seal Animal fat of chopping and obtain the water of separation and fatty raw material; Isolated water is filtered to the fatty raw material obtaining water and centrifugalize omission, the fatty raw material that centrifugalize is omitted is added in fatty raw material; Open molecular still, when system vacuum reaches 0.002mbar, setting primary heater temperature is 315 DEG C, and setting feed heater temperature is 210 DEG C, carries out degassed to fatty raw material; Primary heater temperature reaches 200 DEG C, starts charging, charging rate 85L/h; Primary heater temperature reaches 315 DEG C, charging rate 140L/h, 150L/h; The refined fur seal finished product distilled is collected in cooling.

6. application according to claim 5, is characterized in that: the day taking dose of described refined fur seal is 33mg/kg-111mg/kg.

7. application according to claim 5, is characterized in that: the day taking dose of described refined fur seal is 0.036ml/kg-0.121ml/kg.