CN106309731A - Component with liver protection effect and application thereof to prevention and control of non-alcoholic fatty liver - Google Patents
Component with liver protection effect and application thereof to prevention and control of non-alcoholic fatty liver Download PDFInfo
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- CN106309731A CN106309731A CN201610847607.2A CN201610847607A CN106309731A CN 106309731 A CN106309731 A CN 106309731A CN 201610847607 A CN201610847607 A CN 201610847607A CN 106309731 A CN106309731 A CN 106309731A
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- liver
- extract
- lycium ruthenicum
- ruthenicum murr
- fat
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
- A61K36/815—Lycium (desert-thorn)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention relates to lycium ruthenicum extract with a liver protection function and evaluation of an inhibition effect of the lycium ruthenicum extract to non-alcoholic fatty liver and belongs to the field of biomedicines. A main raw material is natural food-borne lycium ruthenicum; a preparation method comprises: weighing the lycium ruthenicum; extracting the lycium ruthenicum with ethanol with the concentration of 50 percent to 70 percent according to the material-liquid ratio ranging from (1 to 1) to (1 to 20), wherein the extracting temperature is 30 DEG C to 100 DEG C and the extracting time is more than 15 minutes; after filtering, taking filtrate and repeating for 1 to 5 times; combining the obtained filtrate and concentrating at low temperature and in vacuum to obtain the extract. Mice which are fed by high fat are fed with the extract by gavage and an inhibition effect on the non-alcoholic fatty liver is evaluated; the extract can obviously inhibit the non-alcoholic fatty liver caused by high fat diet and complicated metabolic diseases is proved; the lycium ruthenicum extract has a remarkable liver function protection effect.
Description
Technical field
The present invention relates to one and there is liver-protective Lycium ruthenicum Murr. extract, and the suppression side at non-alcoholic fatty liver disease
The application in face, belongs to biological medicine and field of food.
Background technology
Improving constantly the transformation with labor style along with people's living standard, fatty liver has become a kind of common metabolic disease
Sick.Fatty liver refers to due to the pathological changes that athero in the hepatocyte that a variety of causes causes is too much, and adjoint multiple complications, as
Diabetes, cardiovascular disease, hormone disturbance and certain form of tumor etc..The health of fatty liver disease positive serious threat compatriots,
Lipogenesis is to cause non-alcoholic fatty liver disease major reason.Lipogenetic promotion or suppression are for individual physiological function all
There is the most great impact.And medicine has side effect in various degree to human body, therefore, finding from natural food source can
The functional components effectively reducing non-alcoholic fatty liver disease has become as non-alcoholic fatty liver disease prevention and Therapy study field
Focus.
Black Fructus Lycii (Lycium Ruthenicum Murr.) is the distinctive anti-salt drought resisting wild plant kind of NORTHWEST CHINA.
It is distributed in Xinjiang, Tibet, Qinghai, Gansu, Ningxia, North Shaanxi, the Central Asia, Caucasia and Europe.The sweet succulence of black Fructus Lycii taste, contains
Abundant vitamin, organic acid and saccharide, among the people often eat raw or squeeze the juice cook beverage；Black Fructus Lycii also has certain health care
Function, its sweet in the mouth, property are put down, clear away heart-fire heat, and Tibetan medicine is used for treating the diseases such as heart-heat syndrome, heart disease, menoxenia, menolipsis.
Lycium ruthenicum Murr. is mainly pure natural health-care product, wherein contains Herba Lactucae formosanae Saponin, aminoacid, vitamin C, Polyphenols, flavone
200 Multiple components such as class, caffeine, protein, have blood pressure lowering, press down cancer anti-cancer, defying age, the multiple efficacies such as arteries and veins of invigorating blood circulation, have
The laudatory title of " plant soft gold ".Nearly ten years, the research to Lycium ruthenicum Murr. physiologically active shows that it has significant antioxidation, enhancing
The physiologically actives such as cardiovascular function.Experimentation shows that Lycium ruthenicum Murr. has preferable curative effect etc., research to also indicate that for hypertension
Lycium ruthenicum Murr. blood sugar lowering, improve immunity and anti-senility anti-fatigue in terms of and the antioxidation of external lipid have certain curative effect.
For consumers, the various food from different sources, different genera or processing mode can be widely available, but
These materials are unclear for effect and the potential mechanism of liver fat cumulative function.In practice, generally utilize not
Same food, medical product process adipose cell and/or animal and/or people, then detect its liver cell fat is dripped accumulation and/or
The effect of animal non-alcoholic fatty liver disease and mechanism, thus confirm different food products, medical product to non-alcoholic fatty liver disease suppression
Physiological effect.
Based on pure natural plant there is liver-protective food or medical product it is desirable to provide a kind of, detect it
Effect in terms of the suppression of non-alcoholic fatty liver disease, assesses this food or the medical product action effect to liver protecting.
Summary of the invention
In view of above-mentioned purpose, the present invention uses traditional Lycium ruthenicum Murr. to be raw material, extracts its active component by alcohol extracting method.Logical
Cross and detect and assess this component effect in terms of suppression non-alcoholic fatty liver disease and application system, excavate this component non-in preventing and treating
Application in terms of alcoholic fatty liver and the liver protecting, has expanded Lycium ruthenicum Murr. the most further to health protective effect, promotion
The exploitation of active plant resource, the high level that can realize again tradition medicine-food two-purpose resource converts.
Technical scheme is as described below:
The present invention provides a kind of component with liver protecting effect, and this component is the extract of natural food Lycium ruthenicum Murr..
The preparation method of the described Lycium ruthenicum Murr. extract with protection liver function effect is: weigh Lycium ruthenicum Murr., with 1:1-1:20's
Solid-liquid ratio, extracts with the ethanol that volume fraction is 50-70%, extraction temperature 30-100 DEG C, and extraction time is more than 15 minutes,
Take filtrate after filtration, extract 1-5 time, merge all filtrate and carry out cryogenic vacuum and be concentrated into solidifying paste, remove most second
After alcohol and water divides, it is thus achieved that the extract of Lycium ruthenicum Murr..
When using the extract of this Lycium ruthenicum Murr., dilutable water is the aqueous solution of extract, the extraction of every gram of Lycium ruthenicum Murr.
Thing adds the water of 1.5-2.0 milliliter and is diluted.
The present invention also provides for the application of the above-mentioned component with liver protecting effect, and this component is natural food Lycium ruthenicum Murr.
Extract, the extract of described Lycium ruthenicum Murr. can be used for preparing and has in the food of liver protecting effect, medical product, takes or eats
The product prepared with described extract, can substantially suppress non-alcoholic fatty liver disease process, reduce the generation of concurrent metabolic disease.
When having the product of liver protecting effect described in Shi Yong, the effect of described extract is that plurality of active ingredients rises jointly
Effect rather than the effect of one matter.
The preparation method of the described Lycium ruthenicum Murr. extract with hepatoprotective effect is: weigh Lycium ruthenicum Murr., with the material of 1:1-1:20
Liquor ratio, extracts with the ethanol that volume fraction is 50-70%, extraction temperature 30-100 DEG C, and extraction time is more than 15 minutes, mistake
Take filtrate after filter, after extracting 1-5 time, merge all filtrate and carry out cryogenic vacuum and be concentrated into solidifying paste, to be removed most
After ethanol and moisture, it is thus achieved that the extract of this Lycium ruthenicum Murr..
When using the extract of this Lycium ruthenicum Murr., dilutable water is the aqueous solution of extract, the extraction of every gram of Lycium ruthenicum Murr.
Thing adds the water of 1.5-2.0 milliliter and is diluted.
Described there is the liver protecting effect extract to the appraisal procedure of non-alcoholic fatty liver disease inhibition be: use
High lipid food feeding mice 8 weeks, builds nonalcoholic fatty liver model.By the mice 4 weeks of described extract perfusion feeding height fat,
Monitoring Mouse Weight and changes in diet in experimentation, experiment detects blood fat and the change of liver function in mice serum after terminating
Changing, the pathological change of liver organization and hepatocellular fat drip accumulation situation, and PPAR γ, MAPK signal path situation of change
In at least one, assess the impact on mice non-alcoholic fatty liver disease of the described extraction material, to determine described extract energy
The generation of no suppression non-alcoholic fatty liver disease.
When in testing result is mouse liver, fat becomes to when alleviating and liver cell fat drips accumulation suppression, then described
Extract is of value to the suppression of non-alcoholic fatty liver disease；In testing result is mouse liver, fat becomes and liver cytolipin drips long-pending
Tired when being not suppressed, the most described extract is unhelpful or harmful to the suppression of non-alcoholic fatty liver disease.
Described mice is C57BL/6 or/and APOE-/-Male Mus.
The pathological change situation of described liver organization is taken pictures by paraffin embedding H&E dyeing microscope, is observed.
Described liver cell fat drips accumulation can be observed by oil red O stain microscope, be taken pictures.
Detecting weekly body weight and the food-intake of mice, experiment terminates, and takes mouse blood, separates serum and carries out blood fat, liver function
Can index determining.
Described PPAR γ, MAPK signal path situation of change refers to PPAR γ and p38 protein active, changes in gene expression
Situation, is detected by Western Blot and/or Q-PCR method.
In embodiments of the present invention, by the mice of described extract gavage High fat diet, detect mouse experiment process
In body weight and food-intake, after experiment measure liver body than and fat body ratio, by Mouse Weight and food-intake, liver body ratio and fat body ratio
Etc. assessing food and/or supplementary to mouse adipose metabolism.
In one embodiment of the invention, by the mice of described extract perfusion High fat diet, experiment end takes little
Mus blood, separates serum and carries out blood fat, liver function index mensuration.To assess described food for lipid of mice and the work of liver function
By effect, if the mouse liver damage feeding high fat has certain inhibitory action.
In one embodiment of the invention, described extract filling the mice feeding High fat diet, experiment solves after terminating
Cut open mice, take liver organization and make paraffin/frozen section, observe the fat drop accumulation feelings of hepatic pathology change and liver cell
Condition, to assess Lycium ruthenicum Murr. for the effect of the pathological change of non-alcoholic fatty liver disease and the impact of the accumulation of fat of liver cell.
In one embodiment of the invention, described extract filling the mice feeding High fat diet, experiment takes after terminating
Mouse liver tissue, by real-time quantitative gene amplification fluorescence detecting system (QPCR) and/or immunoblot assay (Western
Blot) activity change of detection PPAR γ and/or MAPK signal path assesses food and/or supplementary to mouse liver
Cellular fat generates and the effect of inflammation associated signal paths, to detect the described food effect machine to non-alcoholic fatty liver disease
System.
Non-alcoholic fatty liver disease is excessively to store up, with liver cell fat, the clinical syndrome being characterized with steatosis.Non-
When alcoholic fatty liver is serious all there is fat change in almost all of hepatocyte, with liver enlargement, can have mild tenderness and liver
Dysfunction.Also abnormal liver function can be caused after non-alcoholic fatty liver disease is serious.Glutamic oxaloacetic transaminase, GOT (AST), has another name called asparagine
Acid aminotransferase, is one important in transaminase.It is the index of liver function test on clinical medicine, is used for judging
Whether liver suffers damage.When liver generation severe necrosis or destruction, glutamic oxaloacetic transaminase, GOT concentration meeting in serum just can be caused
Higher.The pathological change of non-alcoholic non-alcoholic fatty liver disease is with bullous or based on bullous hepatic cell fattydegeneration be
Feature.Degree according to intrahepatic fat degeneration is divided into: pure fatty liver disease, fat hepatitis, fatty cirrhosis.
In the present invention, by experimental mouse serum liver Function detection, liver organization outward appearance and hepatic tissue pathology section are seen
Examining, non-alcoholic fatty liver disease related gene and the detection of albumen equimolecular level, the extract component of assessment Lycium ruthenicum Murr. is to liver
Protection effect and the application in non-alcoholic fatty liver disease is prevented and treated thereof.
In the present invention, normal group liver is cerise, glossy, and quality is soft.Model group mouse liver naked eyes are visible
The uniform enlargement of liver, in talking yellow, peplos is nervous, and edge is more blunt, and quality is the softest, the greasy feeling touched.At medicine and Lycium ruthenicum Murr.
The mouse liver perusal of reason is almost close to normal, and liver edge is relatively thin, has no that swelling and peplos are nervous, and quality is soft.Close
Detection in liver function finds, Lycium ruthenicum Murr. extract can significantly reduce the rising of the serum AST that high fat diet causes, and points out liver
The degree of dirty damage reduces.
In the present invention, in the observation about mouse liver H&E dyeing and oil red dyeing, it has been found that the obvious energy of Lycium ruthenicum Murr.
Enough suppress the mouse liver tissue fat degeneration that causes of feed of high fat and the number of fat vacuole, have no inflammatory cell infiltration and
Hepatic necrosis, oil red dyeing is further characterized by the accumulation alleviation that fat in liver cell drips, and Lycium ruthenicum Murr. can be prolonged to a certain extent
The process of slow non-alcoholic fatty liver disease.
P38 mitogen activated protein kinase (MAPK) signal path and inflammatory reaction close relation, cause inflammatory cell because of
Sub-tumor necrosis factor TNF-alpha also plays an important role in the forming process of non-alcoholic fatty liver disease.P38MAPK can regulate
The generation of the anti-inflammatory factors such as pro-inflammatory cytokine such as TNF-α, causes the scorching balance with anti-inflammatory factor in affecting organism.
In the present invention, the exploration discovery on gene level, the extract of Lycium ruthenicum Murr. can lower P38 albumen activation and
The transcriptional level of TNF-α mRNA in liver organization, reduces the activity of inflammatory signal path, the most beneficially lipase activity in liver
Increase and insulin resistant is alleviated, improve lipid accumulation and the situation of steatosis in hepatocyte.
Raw material Lycium ruthenicum Murr. of the present invention is the wild plant resource that China is traditional, and wide material sources are with low cost, the present invention about
The exploration of the effect of the non-alcoholic fatty liver disease that the extract of Lycium ruthenicum Murr. is induced for mice height fat, opens medicine-food two-purpose resource
Sending out significant, and the high level achieving active plant resource converts, the intensive processing promoting agricultural product utilizes.Logical
Cross and assess its suppression effect to non-alcoholic fatty liver disease, find that Lycium ruthenicum Murr. extract can be used for non-alcoholic non-alcoholic fatty
The preventing and treating of liver, the exploitation to prevention or the active component for the treatment of non-alcoholic non-alcoholic fatty liver disease have directive significance.
The described component with the liver protecting effect, can be widely applied to food and medicine and other fields, have significant social benefit and
Economic benefit.
Accompanying drawing explanation
The technological process that Fig. 1 is prepared to wolfberry fruit extract
The impact that Mouse Weight is increased by Fig. 2 Lycium ruthenicum Murr. extract
The impact on mice food-intake of Fig. 3 Lycium ruthenicum Murr. extract
The impact on mouse liver tissue morphology of Fig. 4 Lycium ruthenicum Murr. extract
The impact on Mouse Liver body ratio of Fig. 5 Lycium ruthenicum Murr. extract
The impact on little Adips Mus body ratio of Fig. 6 Lycium ruthenicum Murr. extract
The impact on mice T-CHOL of Fig. 7 Lycium ruthenicum Murr. extract
The impact on mice low density lipoprotein, LDL of Fig. 8 Lycium ruthenicum Murr. extract
The impact on mice high density lipoprotein of Fig. 9 Lycium ruthenicum Murr. extract
The impact on mice triglyceride of Figure 10 Lycium ruthenicum Murr. extract
The impact on mice glutamic oxaloacetic transaminase, GOT of Figure 11 Lycium ruthenicum Murr. extract
The impact on mice glutamate pyruvate transaminase of Figure 12 Lycium ruthenicum Murr. extract
The impact on mouse alkaline phosphatase of Figure 13 Lycium ruthenicum Murr. extract
After Figure 14 Lycium ruthenicum Murr. extract-treated, the impact of liver H&E staining cell form
After Figure 15 Lycium ruthenicum Murr. extract-treated, liver oil red staining cell fat drips the impact of accumulation
After Figure 16 Lycium ruthenicum Murr. extract-treated, the expression of PPAR γ mRNA in liver cell
After Figure 17 Lycium ruthenicum Murr. extract-treated, the expression of SREBP mRNA in liver cell
After Figure 18 Lycium ruthenicum Murr. extract-treated, the expression of FAS mRNA in liver cell
After Figure 19 Lycium ruthenicum Murr. extract-treated, the expression of TNF-α mRNA in liver cell
After Figure 20 Lycium ruthenicum Murr. extract-treated, the protein expression situation of PPAR γ, FAS, P38 in liver cell
Detailed description of the invention
The present invention is illustrated below in conjunction with embodiment.
Embodiment 1 one kinds has the extract of liver protecting function, and primary raw material is natural food source Lycium ruthenicum Murr..
Preparation method is: weigh Lycium ruthenicum Murr., with the solid-liquid ratio of 1:1-1:20, enters with the ethanol that volume fraction is 50-70%
Row extraction, extraction temperature 30-100 DEG C, extraction time is more than 15 minutes, takes filtrate, extract 1-5 time, merge all filters after filtration
Liquid carries out cryogenic vacuum and is concentrated into solidifying paste, removes whole ethanol and most moisture, and right dilute with water is extract
Aqueous solution, the extract of every gram of Lycium ruthenicum Murr. adds the water of 1.5-2.0 milliliter and is diluted.
Fig. 1 is technological process prepared by Lycium ruthenicum Murr. extract, is summarized above-mentioned preparation method.
The process conditions of the Lycium ruthenicum Murr. extract used in following embodiment 2-10 are: weigh Lycium ruthenicum Murr. 20g, use volume
Mark be 70% ethanol 400ml extract, extraction temperature 60 DEG C, extraction time 30 minutes, take filtrate after filtration, repeat 3
Secondary, merge all filtrate and carry out cryogenic vacuum and be concentrated into solidifying paste, remove whole ethanol and most moisture, so use water
It is diluted to the aqueous solution of extract.The extract of every gram of Lycium ruthenicum Murr. adds water 1.5 milliliters, and the 30ml that adds water obtains black Chinese holly after fully dissolving
The solution of extract of Qi, aqueous solution color is aubergine, bright in colour.With rutin as tester, ultraviolet spectrophotometry is surveyed
Determine extract flavonoid concentration.
Embodiment 2 Lycium ruthenicum Murr. extract is to Mouse Weight and the action effect of food-intake
The male mice of 6-8 week old, is randomly divided into 4 groups: Normal group, model control group, Lycium ruthenicum Murr. group, simvastatin
Group.Often 10 mices of group.In addition to blank group feeding normal diet, other 3 groups equal feeding height fat nutrient fodders, Lycium ruthenicum Murr. group
With simvastatin group respectively at the high lipid food Lycium ruthenicum Murr. extract that gavage concentrates and the aqueous solution of simvastatin simultaneously, measure little
The body weight of Mus and diet.
Mouse Weight, food-intake measuring method: be monitored with electronic balance, calculate difference.
After Fig. 2 is Lycium ruthenicum Murr. extract feeding mice, the change that Mouse Weight increases is compared.Fig. 3 is Lycium ruthenicum Murr. extract
After feeding, the change of mice food-intake.Be can be seen that by the result of Fig. 2 and Fig. 3, it is ensured that in the case of same Energy intaking,
High fat food-intake is little compared with common food-intake.In the case of same high fat food-intake, the body weight of the mice of Lycium ruthenicum Murr. process group
Relatively model group dramatically increases.
Embodiment 3 Lycium ruthenicum Murr. is to mouse liver form regulating liver-QI body ratio and the action effect of fat body ratio
Fat have close relationship with fatty liver, and in having been reported that liver, mild fatty infiltrates the fat trouble being found in about half
Person, in severe obesity, the sickness rate of fatty liver is up to 61%～94%.The accumulation of intrahepatic fat and the degree that exceeds standard of body weight
It is proportionate.The formation of fatty liver is also had a certain impact by the bodily form feature of obese patient.The present invention is by liver weight, epididymis
The weighing of fat weight and body weight calculates liver body ratio and fat body ratio is verified, and takes pictures mouse tissue form.
Fig. 4 is that Lycium ruthenicum Murr. processes the form of tissue after mice, it can be seen that body weight change and covert with hepatopathy and
The change of liver color is not consistent.After Lycium ruthenicum Murr. process, the liver edge of mice is relatively thin, does not has the swelling that generation model group is similar
Phenomenon.Liver color is more normal, and liver surface does not has greasy feeling.Fig. 5 is that Lycium ruthenicum Murr. processes the change of liver body ratio after mice.Figure
The change of fat body ratio after 6 Lycium ruthenicum Murr. process mices.Be can be seen that by Fig. 5 and Fig. 6, although the body weight of the mice that Lycium ruthenicum Murr. processes is relatively
Model group increases, but corresponding liver body does not occur significantly to change than with fat body ratio.
Embodiment 4 Lycium ruthenicum Murr. action effect to lipid of mice index
Fig. 7 is the change of T-CHOL (CHOL) level after Lycium ruthenicum Murr. water extract process mice.Fig. 8 is that Lycium ruthenicum Murr. water carries
After thing processes mice, the change of low density lipoprotein, LDL (LDL) level.Fig. 9 is after Lycium ruthenicum Murr. water extract processes mice, high density fat
The change of albumen (HDL) level.Figure 10 is after Lycium ruthenicum Murr. water extract processes mice, the change of serum lipids (TG) level.Black Chinese holly
After Qi water extract effect, mice total cholesterol level somewhat increases, and high and low density lipoprotein levels is not affected, sweet
The level relatively model group of oil three esters declines, but does not has significant difference.
Embodiment 5 Lycium ruthenicum Murr. action effect to Mouse Liver function
The present invention carries out the action effect verifying Lycium ruthenicum Murr. extract for mouse liver from mice serum liver function index.
Glutamic oxaloacetic transaminase, GOT (AST) is the index of liver function test on clinical medicine, is used for judging whether liver suffers damage.
During liver cell lesion, AST is released into blood, therefore serum AST activity increases with the degree of hepatocellular damage.Glutamate pyruvate transaminase (ALT)
It is primarily present in liver, heart and skeletal muscle, when hepatocyte or some tissue injury or necrosis, all can make the paddy third in blood
Transaminase raises.Alkali phosphatase (ALP) is mainly used in obstructive jaundice, primary hepatocarcinoma, secondary liver cancer, cholestatic
The inspection of hepatitis etc..When suffering from these diseases, hepatocyte excessively manufactures ALP, enters blood through lymphatic channel and sinus hepaticus, simultaneously because liver
Interior biliary tract bile excretion obstacle, reflux enters blood and causes serum alkaline phosphatase significantly raised.Figure 11-13 is at Lycium ruthenicum Murr. respectively
After reason mice, the change of serum glutamic oxalacetic transaminase, glutamate pyruvate transaminase and alkaline phosphatase levels.Turn in Lycium ruthenicum Murr. process group millet straw
The content of ammonia enzyme reduces, and the degree of prompting hepar damnification reduces/is restored.Glutamic oxaloacetic transaminase, GOT and alkali phosphatase then do not have
There is significant change.
The effect to pathology of livers of embodiment 6 Lycium ruthenicum Murr.
The present invention use paraffin section after H&E dyeing method to Lycium ruthenicum Murr. process after mouse liver pathological change carry out
Checking.After Figure 14 Lycium ruthenicum Murr. processes, the change of liver H&E staining cell form.After the induction of high fat, mouse liver cell volume becomes
Greatly, with the appearance of fat vacuole, there is blister sample degeneration and steatosis in most cells, based on middle-size and small-size cavity, with
Time process with the infiltration of inflammatory cell, Lycium ruthenicum Murr. after the blister sample of mouse liver cell become and the degree of steatosis substantially subtracts
Gently, the number of fat vacuole significantly reduces, and presents minority calcium.Thus Lycium ruthenicum Murr. extract can delay high fat little
The process of the non-alcoholic fatty liver disease of Mus induction, alleviates the slight of liver fat lesion.
Liver cell fat is dripped the effect of accumulation by embodiment 8 Lycium ruthenicum Murr.
The present invention uses the accumulation of mouse liver fat drop after Lycium ruthenicum Murr. is processed by the method for oil red dyeing after frozen section
Situation is verified.After Figure 15 Lycium ruthenicum Murr. processes, liver oil red staining cell fat drips the change of accumulation.Can see that at Lycium ruthenicum Murr.
The fat drop number in mouse liver cell after reason is considerably less than model group mice, and the process of Lycium ruthenicum Murr. body extract can subtract
The accumulation of fat in few liver, in its liver cell, number and the volume of fat drop are significantly less than model group mice.
Embodiment 9 Lycium ruthenicum Murr. is to lipogenesis and the effect of inflammatory related gene mRNA level
The present invention uses the method for QPCR to and inflammatory factors relevant to lipogenesis such as PPAR γ, FAS, SREBP-1c
The gene expression of TNF-α is verified.
Figure 16 is the change of the PPAR γ gene level after Lycium ruthenicum Murr. function cells.After Figure 17 is Lycium ruthenicum Murr. function cells,
The change of SREBP-1c gene level, after Figure 18 is Lycium ruthenicum Murr. function cells, the change of FAS gene level.Figure 19 is Lycium ruthenicum Murr.
The change of inflammatory factor TNF-α gene level after effect.By Figure 16, Figure 17, Figure 18 and Figure 19 it can be seen that Lycium ruthenicum Murr. processes
After, the transcriptional level of PPAR γ, FAS does not all occur significantly to change, but the variation tendency of the PPAR λ relevant to lipogenesis
With the changes of contents of serum triglycerides, there is concordance.Mediation cholesterol generates the expression of relevant SREBP-1c and declines.
The expression of inflammatory factor TNF-α significantly reduces, and shows what lipogenesis in mouse liver cell was correlated with after processing by Lycium ruthenicum Murr.
The impact of the expression of gene is less, and can suppress the expression of inflammatory factor TNF-α gene.
Embodiment 10 Lycium ruthenicum Murr. is to the action effect of lipogenesis related gene protein expression level in liver
The present invention uses the method for western blotting to verify MAPK and PPAR γ signal path.
Figure 20 is the MAPK phosphorylation after Lycium ruthenicum Murr. function cells and the change of PPAR γ expression.As can be seen from Figure,
After Lycium ruthenicum Murr. processes, the phosphorylation level of p38 increases, and the expression of FAS rises, and the expression of PPAR γ does not occur significance
Change, shows that Lycium ruthenicum Murr. can raise the generation of p-p38 suppression hepatocyte inflammatory reaction, and the synthesis simultaneously raising FAS promotes TG
Generation.
Result shows the accumulation that in the mouse liver cell that Lycium ruthenicum Murr. can suppress high fat to take food, fat drips, and reduces liver cell
The generation of vacuolation, reduces the generation that liver cell blister sample becomes and fat becomes, and alleviates the process of non-alcoholic fatty liver disease, has one
Fixed liver protection effect.Exploration discovery on gene expression dose, Lycium ruthenicum Murr. is to lipogenesis related gene PPAR γ, FAS gene
The impact of expression less, exploration discovery in signal transduction level, Lycium ruthenicum Murr. is by lowering expression and the downward of p-p38 albumen
The transcriptional level of TNF-α mRNA, reduces the activity of inflammatory signal path in liver cell, suppresses inflammatory reaction, simultaneously Lycium ruthenicum Murr.
After process, the content of AST reduces, and shows that hepar damnification alleviates.The present invention provides a kind of Lycium ruthenicum Murr. with liver-protecting function to extract
Thing, and assess the inhibition of its non-alcoholic fatty liver disease, thus provide theoretical foundation to the exploitation of medicine-food two-purpose resource.So
And, the body weight of the mice that Lycium ruthenicum Murr. processes has a certain degree of increase, and this may have in the increase of the expression of protein level with FAS
Closing, meanwhile, after Lycium ruthenicum Murr. processes, in serum, the content of T-CHOL increases, and deeper mechanism therein also needs to into one
The checking of step and discussion.And the effect that Lycium ruthenicum Murr. is to other complication, in addition it is also necessary to it is verified further.
The step of paraffin section H&E dyeing is as follows:
1, fixing organization sample normal saline is washed, puts in neutral paraformaldehyde fixative fixing immediately.
2, washing flowing water rinses or soaks a few hours or overnight (controls the set time well, typically need not washing).
3, dehydrated sample is dehydrated through 70%, 80%, 90% ethanol solution at different levels successively, each 30min, place into 95%,
100% each 2 times, each 20min.
4, transparent 1/2 absolute alcohol+1/2 dimethylbenzene mixed liquor 15min, dimethylbenzene I 15min, II 15min (to transparent are
Only).
5, sample is put into paraffin I, the saturating each 1h of wax of paraffin II by waxdip.
6, embedding is with maximum face at bottom, makes shared by the covering weave face that cuts out maximum；Furthermore, it is noted that embedding direction, folder
It is noted that do not exert oneself very much during sample, section is the part also as far as possible avoiding being pressed from both sides.
7, section
9. exhibition sheet, paster
10. oven temperature is adjusted to 60 DEG C by dewaxing rehydration, and controls when 60 DEG C, section is put into together with section frame
30min melts to wax.Afterwards, paraffin section dewaxes each 5min through dimethylbenzene I, II, be then placed in 100%, 95%, 90%,
80%, each 3-5min in 70% alcoholic solutions at different levels, places into 3min in distilled water.
11. stained put into the about 7s that dyes in hematoxylin.
12. washing tap water flowing water rinse about 15min.
Section is put in 1% hydrochloride ethanol liquid and is faded by 13. differentiation, and about 2 seconds to several tens of seconds.
14. rinsing sections place into and make it recover blue in tap water flowing water.
15. dehydrations I are cut into slices each 3-5min in 50% ethanol → 70% ethanol → 80% ethanol.
16, redye with 0.5% Yihong ethanol counterstaining 1-3min.
Section is put into and to be washed away unnecessary redness in 95% ethanol by 17. dehydrations II, is then placed in 3-in dehydrated alcohol
5min.Finally blot unnecessary ethanol with absorbent paper.
18. clear slices put into each 3-5min in dimethylbenzene I, II.
19. envelopes are hidden: neutral gum is sealed up for safekeeping
Frozen section oil red staining procedure is as follows:
1 takes flesh tissue fixes 24 hours in 4% paraformaldehyde
2-25 degree embedding (in freezing microtome)
3 frozen sections
4, oil red O stain 10 minutes.
5, distillation washing 30 minutes；
6, glycerin gelatine mounting
Real-time quantitative gene amplification fluoroscopic examination (QPCR) experiment is as follows:
(1) extraction of RNA:
1. instrument, material and reagent:
(1) instrument: refrigerated centrifuger, ultraviolet spectrophotometer；Ice machine, thermostat water bath
(2) material: eppendorf pipe, rifle head, eppendorf pipe support, ice chest；Liquid-transfering gun
(3) reagent: Trizol；Chloroform, isopropanol, DEPC water；75% ethanol
2. specifically comprise the following steps that
(1) adding 1ml TRizol (pre-cooling) reagent, be then transferred in 1.5ml centrifuge tube, room temperature stands 10min；
(2) chloroform of 200 μ l (or 1/5 volume) is added, the most reverse centrifuge tube mixing 30s, room temperature stands 5min, point
Layer.4 DEG C of 12000rpm are centrifuged 15min.In centrifuge tube, liquid is divided into 3 layers: upper strata is aqueous phase, RNA in this phase, protein under
In the organic facies of layer yellow, DNA is retained in the intermediate layer of bilevel interface；
(3) the careful upper water 0.4ml that makes an appointment that draws (is sure not to draw intermediate layer and subnatant in new plastic centrifuge tube
Body).Adding the isopropanol of 4 DEG C of pre-coolings of 1 times of volume, room temperature places 20min (or-20 DEG C of standing more than 30min)；
(4) 4 DEG C of 12000rpm are centrifuged 15min, precipitate RNA, abandon supernatant；
(5) with 75% ethanol (preparation of DEPC water) the washing precipitation of 1ml pre-cooling, 4 DEG C of centrifugal 10min of 12000rpm, abandon to the greatest extent
Supernatant；
(6) repeat step 5, then natural drying about 10min in room temperature, be translucent shape to RNA；
(7) add the water dissolution precipitation that 20-100 μ l DEPC processes, 56 DEG C of water-bath 10min according to RNA extracted amount, put immediately
In on ice, obtain extracted RNA solution；
(8) RNA purity, concentration measure and the qualification of molecular entity: measure sample 260nm/280nm luminous absorptance value.
(2) reverse transcription synthesis cDNA:
1. instrument, material and reagent:
(1) instrument: PCR thermal cycler, centrifuge
(2) material: PCR pipe, rifle head；Liquid-transfering gun
(3) reagent: Reverse Transcription box
2. specifically comprise the following steps that
(1) in PCR pipe, 20 μ l reaction systems are added: the 1. number reagent 4 μ l in Reverse Transcription box, template ribonucleic acid 2 μ g, 2.
Number reagent adds to 20 μ l；
(2) after centrifugal, 37 DEG C of 30min, 85 DEG C of 10s；
(3) measure cDNA concentration, and be diluted to 50ng/ μ l ,-20 DEG C of preservations with sterilized water.
(3) PCR:
(1) in sterilizing ep pipe, 20 μ l reaction systems are prepared: mix 10 μ l, ddH2O 6 μ l, each 1 μ l of upstream and downstream primer,
cDNA2μl；
(2) centrifugal, mixing, centrifugal, add 96 orifice plates, if 3 multiple holes；
(3) pcr amplification reaction condition is:
1) 95 DEG C of denaturations 5min
2) 95 DEG C of degeneration 15s
3) 62 DEG C of annealing 30s
4) 65 DEG C extend 5s
5) step (2)-(4) 39 times are repeated
6) 95 DEG C extend 5min
Immuning hybridization (Western blotting) experiment is as follows:
1. solution preparation:
(1) 10% (w/v) sodium lauryl sulphate SDS solution: 0.1gSDS, 1mlH2O deionized water is prepared, and room temperature is protected
Deposit.
(2) separation gel buffer: 1.5mol/LTris-HCL (pH8.8): 18.15gTris 80ml water dissolution, uses HCL
Regulation pH to 8.8, dilute is to 100ml final volume.
(3) glue buffer: 0.5mol/LTris-HCL (pH6.8) is concentrated: 6.05gTris is dissolved in 80ml water, adjusts with HCL
To pH6.8, dilute is to 100ml final volume.
(4) SDS-PAGE sample loading buffer: pH6.8 0.5mol/L Tris buffer 8ml, glycerol 6.4ml, 10%SDS
12.8ml, mercaptoethanol 3.2ml, 0.05% bromophenol blue 1.6ml, H2O 32ml mixing is standby.
(5) Tris-glycine running buffer: 30.3gTris, 188g glycine, 10gSDS, is dissolved to distilled water
1000ml, before use dilution 10 times.
(6) transferring film buffer: weigh 14.4g glycine, 6.04gTris alkali, and add 200ml methanol, add water to total amount
1L。
(7) Tris buffer salt solution (TBS): 20mmol/LTris/HCL (pH7.5), 500mmol/LNaCl.
2. specifically comprise the following steps that
Adding lysate, ultrasonication, after cooled on ice, 12000rpm is centrifuged 2min, takes supernatant, and-20 save backup.
(1) PAGE gel electrophoresis
(1) putting into clamping in folder after the glass plate alignment after cleaning, then vertical card prepares encapsulating on the top of the shelf.
(2) joining 10% separation gel, shake up immediately and get final product encapsulating, use ethanol fluid-tight immediately after adding TEMED, glue fully solidifies
The upper strata ethanol that just can remove photoresist also blots with absorbent paper.
(3) join the concentration glue of 4%, shake up immediately after adding TEMED and get final product encapsulating.Remaining space is filled concentration glue then
Comb is inserted and concentrates in glue.Until after concentrating gelling admittedly, being pulled out the most gently.
(5) put it in electrophoresis tank, after adding enough electrophoresis liquid, begin preparing for loading.Protein example survey is finished to be contained in vain
After amount, add 5 × SDS sample-loading buffer to the most final concentration of 1 ×, boiling water boils loading after 5min mixing, total protein concentration 50 μ g.
(6) constant voltage 80V electrophoresis runs glue, and after sample enters lower floor's glue, constant voltage 120V electrophoresis is until bromophenol blue reaches at the bottom of gel
Till portion, carry out transferring film.
(2) transferring film:
(1) glue is cut: fallen by glass plate sled, after removing little glass plate, scraped off by concentration glue, need according to albumen according to experiment
Molecular weight, with Marker for comparison carry out cutting glue.
(2) standby film: cutting pvdf membrane and filter paper, is placed in methanol solution activation 30s by the PVDF cut.
(3) dress film: the clip of transferring film is opened and makes black one side (negative pole) keep level.A sponge is padded above
Pad, addition transferring film immersion is wet, and soaked filter paper in padding on mat, subsequently according to gel celluloid membrane filtration
The order of paper foam-rubber cushion stacks successively.Finally white board (positive pole) is built in loading transferring film groove.
(4) transferring film: clip is put in transfer groove groove, the black flour of folder the to be made black flour to groove, red to groove of the flour of folder
Face.4 DEG C of transferring films, constant current 400mA.
(5) after having turned, film is taken off, labelling one jiao.
(3) immunoreation
(1) close: will film TBST rinse 3 times, each 5min.After rinsing, nitrocellulose filter is put into 5% defat
In milk powder, shaking table 1h under room temperature.
(2) add one anti-: the nitrocellulose filter after closing puts into rinsing 3 times on shaking table in cylinder of washing containing TBST, often
Secondary 5min.Put into added with 4 DEG C of night incubation in an anti-plate.
(3) add two anti-: reclaim one and resist, by nitrocellulose filter room temperature shaker rinsing 3 times in TBST washes cylinder, every time
5min, then puts into added with in two anti-plates, lucifuge incubated at room 1h by rinsed nitrocellulose filter.By nitre after hatching
Acid cellulose film is washed 3 times in TBST washes cylinder, each 5min.
(4) chemiluminescence
(1) two kinds of reagent of A and B are mixed according to the operation of luminescence reagent box description in little centrifuge tube, are added to nitre
On acid cellulose film, develop the color with chemiluminescence imaging instrument.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be with being as the criterion that claims are defined.
Claims (9)
1. a component with liver protecting effect, it is characterised in that this component is the extract of natural food Lycium ruthenicum Murr..
The component with the liver protecting effect the most according to claim 1, it is characterised in that described in have protection liver function effect
The preparation method of Lycium ruthenicum Murr. extract be: weigh Lycium ruthenicum Murr., with the solid-liquid ratio of 1:1-1:20, use volume fraction 50%-70%
Ethanol extract, extraction temperature 30-100 DEG C, extraction time be more than 15 minutes, take filtrate after filtration, repeat 1-5 time, conjunction
And all filtrate carries out cryogenic vacuum and is concentrated into solidifying paste, after most ethanol to be removed and moisture, it is thus achieved that Lycium ruthenicum Murr.
Extract.
The component with the liver protecting effect the most according to claim 1 and 2, it is characterised in that by Lycium ruthenicum Murr. extract
Being the aqueous solution of crude extract with water dissolution again, the extract of every gram of Lycium ruthenicum Murr. adds the water of 1.5-2.0 milliliter and is diluted.
4. the application of the component with liver protecting effect as according to any one of claim 1-3, it is characterised in that
This component is the extract of natural food Lycium ruthenicum Murr., and the extract of described Lycium ruthenicum Murr. can be used for preparing and has liver protecting effect
In food, medical product, take or eat product prepared by described extract, can substantially suppress non-alcoholic fatty liver disease, with
And concurrent metabolic disease.
The application of the component with liver protecting effect the most according to claim 4, it is characterised in that edible described black Chinese holly
During Qi extract, the plurality of active ingredients of described extract plays the effect of coefficient effect rather than one matter.
The application with liver protecting effect component the most according to claim 4, it is characterised in that use high lipid food to feed
Food mice 8 weeks, builds nonalcoholic fatty liver model；By the mice 4 weeks of described Lycium ruthenicum Murr. extract perfusion feeding height fat, test
During monitor Mouse Weight and changes in diet, experiment detects blood fat and the change of liver function, liver in mice serum after terminating
The pathological change of dirty tissue and hepatocellular fat drip in accumulation situation, and PPAR γ, MAPK signal path situation of change
At least one, assess the impact on mice non-alcoholic fatty liver disease of the described extraction material, to determine that can described extract press down
The generation of manufacture-illegal alcoholic fatty liver.
The application with liver protecting effect component the most according to claim 6, it is characterised in that when testing result is little
The dirty interior fat of Hepar Mus becomes to when alleviating and liver cell fat drips accumulation suppression, and the most described extract is of value to non-alcoholic fat
The suppression of fat liver；When in testing result is mouse liver fat become and liver cytolipin drip accumulation be not suppressed time, then described in carry
Take thing unhelpful or harmful to the suppression of non-alcoholic fatty liver disease.
The application with liver protecting effect component the most according to claim 6, it is characterised in that described liver organization
Pathological change situation is taken pictures by paraffin embedding H&E dyeing microscope, is observed；It is permissible that described liver cell fat drips accumulation
Observe by oil red O stain microscope, take pictures；Described PPAR γ, MAPK signal path situation of change refers to PPAR γ and p38 egg
White matter activity, changes in gene expression situation, by real-time quantitative gene amplification fluorescence detecting system (QPCR) and/or immunoblotting
Technology (Western Blot) method detects.
The application with liver protecting effect component the most according to claim 4, it is characterised in that use high lipid food to feed
Food mice 8 weeks, builds nonalcoholic fatty liver model；By the mice 4 weeks of described Lycium ruthenicum Murr. extract perfusion feeding height fat, detect
Body weight during mouse experiment and food-intake, measure liver body ratio and fat body ratio, by Mouse Weight and food-intake, liver after experiment
Body is than assessing food and/or supplementary to mouse adipose metabolism with fat body ratio etc..
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| WO2020153909A1 (en) * | 2019-01-24 | 2020-07-30 | National University Of Singapore | Natural compound compositions for treating medical conditions |
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| CN1686421A (en) * | 2005-04-26 | 2005-10-26 | 陶燕铎 | Black fruit lycium plant fruit extract, its preparation method and application |
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| CN1686421A (en) * | 2005-04-26 | 2005-10-26 | 陶燕铎 | Black fruit lycium plant fruit extract, its preparation method and application |
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| 刘文江等: "黑果枸杞的利用价值与民族植物学传统知识的关系", 《第八届中国民族植物学学术研讨会暨第七届亚太民族植物学论坛会议文集》 * |
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| WO2020153909A1 (en) * | 2019-01-24 | 2020-07-30 | National University Of Singapore | Natural compound compositions for treating medical conditions |
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