CN108379286A - Mushroom dietary fiber extract is preparing treatment and/or is preventing the purposes during high fat diet causes the preparation of hepatic injury relevant disease - Google Patents
Mushroom dietary fiber extract is preparing treatment and/or is preventing the purposes during high fat diet causes the preparation of hepatic injury relevant disease Download PDFInfo
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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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- 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
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Sustainable Development (AREA)
- Food Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Epidemiology (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The purposes of purposes more particularly to a kind of mushroom dietary fiber extract in preparing treatment and/or preventing the preparation of high fat diet cause hepatic injury relevant disease that the present invention relates to mushroom dietary fiber extracts in pharmaceutical field.The mushroom dietary fiber extract of the present invention can not only be clearly helpful for dominant microflora structure in enteron aisle and restore, contribute to flora diversified, the relevant disease of hepatic injury is caused to show significantly treatment and/or preventive effect high fat diet, the expression of inflammatory factor can effectively be inhibited, the deterioration of inflammation is effectively relieved, improve immune-related every physiopathological index, can be used in the various fields such as drug, health products, there is great economy and social value.
Description
Technical field
The purposes more particularly to a kind of perfume (or spice) that the present invention relates to mushroom dietary fiber extracts in pharmacy, field of health care products
Mushroom dietary fiber extract is preparing treatment and/or is preventing the purposes during high fat diet causes the preparation of hepatic injury relevant disease.
Background technology
Obesity refers to that body fat mass in human body is excessively accumulated, hence it is evident that exceed normal person average amount, and occur organism metabolism,
The anomalous variation of Physiology and biochemistry, is referred to as obesity.By 2014, the world had 600,000,000 Genus Homos in obese people, it is contemplated that 2025
Year, world's obesity number will reach 1,400,000,000 people, and problem of obesity has become one of the public health problem that the world attractes attention.Research
It was found that the occurrence and development majority of affluent society common disease and fat related such as diabetes, hypertension, coronary heart disease, hepatopathy, colon
Cancer, kidney trouble, Alzheimer's disease etc..Fat mechanism is complicated, mainly including inherent cause, environmental factor etc., wherein
Most of obese patients belong to simple obesity, and caused by unreasonable diet structure, high fat diet intake is excessive, dietary fiber
Insufficiency of intake is to lead to one of fat occurrence factor.
Enteric microorganism group is known as " second set of genome of human body ", closely bound up with human health.The micro- life of human intestine
Species up to 1000-1150 kinds, wherein Firmicutes and Bacteroidetes are dominant microflora, the gene dosage in enteron aisle genome
It is more than 150 times of mankind's autogene group.There is mutualism relationships with host for intestinal flora, and dynamic is remain in enteron aisle
Balance, when balance is broken, people is possible to suffer from various diseases.Existing research confirms：(1) intestinal flora and fat phase
It closes, there is the phenomenon that intestinal microflora imbalance, Phylogenetic diversity of bacteria declines more in obese people；The Bacteroidetes of obesity mice
Few, Firmicutes are more；The enteric microorganism of obesity mice is transplanted on germfree mouse, the latter's fat mass can be caused to increase.It is right
For fat people, high fat diet is taken in for a long time, the change of intestinal microflora, unbalance intestinal flora can be caused to will produce
Endotoxin enters human body, and the inflammation factor is generated after being identified by immunocyte, body is caused to be in low grade inflammation state, generates
Metabolic disorder so that the energy absorbed from food is easily converted to fat, causes obesity；(2) liver and enteron aisle are in biology
Functionally it is closely related, interactional.Liver is interacted with intestinal flora by intestines-liver axis, intestinal flora and liver
Disease is close, when liver illness, can influence the structure and function of intestinal flora, and can be influenced in turn when intestinal bacilli illness
Liver function, the beneficial bacteriums quantity such as liver cirrhosis patient Bifidobacterium reduces, and the harmful bacterias quantity such as enterobacteria, enterococcus increases.
High fat diet can change intestinal microflora, and beneficial bacterium is made to reduce, and destroy the balance of intestinal flora, and then influence
Liver function, causes hepatic injury, and increase the problems such as with the development of the living standard in China and dietary structure, by high in fat
The incidence of hepatic injury caused by diet obviously increases, and there is an urgent need to develop preparing treatment and/or preventing high fat diet cause liver
Damage the preparation in terms of relevant disease.
Invention content
For the above deficiency, a kind of mushroom dietary fiber extract of present invention offer is preparing treatment and/or is preventing high in fat
Diet causes the purposes in the preparation of hepatic injury relevant disease.
Dietary fiber is the raw material of normal the gut flora, is played an important role to health, and the seventh-largest nutrition is known as
Element has pre- preventing obesity, adjusts intestinal flora, improves intestinal environment, the physiological function of prevention of various diseases.And fat personage's drink
Often lack dietary fiber in food, dietary fiber, more important is intestinal flora is changed, makes other than it can be temporarily increased satiety
Beneficial bacterium increases, and achievees the purpose that keep fit.
Mushroom (scientific name：Lentinus edodes) also known as dried mushroom, mushroom, north mushroom, thick mushroom, thin mushroom, flower mushroom, vertebra young pilose antler, it is
The mushroom export trade amount of one of second-biggest-in-the-world edible mushroom and China's specialty, China in Recent Years is gradually increasing, annual increasing rate
About 2%, mushroom annual output is 80,000 tons, occupies 80% or more in 100,000 tons global, ranks first in the world, outlet 3.6 ten thousand
Ton, followed by Japanese first of the worlds Ye Ju, South Korea occupies third position.
Civil, mushroom is known as the title of " mountain delicacy ".Mushroom fruiting body Dan Sheng, it grows thickly or all living creatures, fructification are medium greatly to slightly
Greatly.Bacterial context white, it is slightly thick or thick, it is fine and closely woven, have fragrance.Edge is involute when young, has the villus of white or yellow-white, with growth
It disappears.There is velum below cap, it is rear to rupture, form incomplete collarium.Aging rear cover edge warp, cracking.Its milpa is distributed
In Henan China Zhumadian, Xixia, Lushi, Fujian, Zhejiang, Anhui, Hunan, Hubei, Jiangxi Suichuan, Sichuan, Guangdong, Guangxi,
The areas such as Hainan, Guizhou, Yunnan, Lueyang, Shaanxi Province, Gansu.The effect of mushroom is cold in nature, mildly bitter flavor, advantageous liver benefit stomach.Modern medicine
It deepens continuously research with nutrition, the medical value of mushroom is also constantly exploited.
Inventor has found that mushroom dietary fiber has the work improved for the cell of hepatic injury caused by mouse high fat diet
With.Based on this, the present invention provides a kind of preparation method of mushroom dietary fiber extract, and the mushroom dietary fiber being prepared
Extract is in the purposes for treating and/or preventing high fat diet cause hepatic injury relevant disease.
In the first aspect of the present invention, a kind of mushroom dietary fiber extract is provided and is preparing treatment and/or is preventing high in fat
Diet causes the purposes in the preparation of hepatic injury relevant disease.
Wherein, it includes hepatic steatosis, nonalcoholic steatohepatitis, liver that the high fat diet, which causes hepatic injury relevant disease,
Hardening, hepatic failure.
Preferably, a kind of mushroom dietary fiber extract is preparing treatment and/or prevention of hepatic steatosis, non-alcoholic
Purposes in the preparation of the diseases such as fatty hepatitis, hepatic sclerosis, hepatic failure.
Wherein, the mushroom dietary fiber extract can be the mushroom meals being prepared using the following preparation method
Eat fiber extract.
In the second aspect of the present invention, a kind of preparation method of mushroom dietary fiber extract is provided, including by mushroom powder
It after broken, is extracted using alcohol reflux, filter residue is used into water refluxing extraction, then by extracting solution enzymolysis, precipitation, elution, obtain mushroom
Dietary fiber extract.
Preferably, the alcohol reflux, which extracts, includes：Use solid-liquid ratio for 1:8-1:12 ethyl alcohol, refluxing extraction 2-3 are small
When.
Preferably, the filter residue includes using water refluxing extraction：Use solid-liquid ratio for 1:8-1:12 water, refluxing extraction 2-
3 hours.
Preferably, the enzymolysis is uses papain enzymolysis, it is further preferred that the enzymolysis dosage claims for sample
0.2% mass percent of sample amount, the enzymatic activity are 750,000 u/g-85, ten thousand u/g.
It is further preferred that the hydrolysis temperature is 55-65 DEG C, the time is 55-65 minutes.
Preferably, described to be precipitated as precipitating using alcohol reagent, such as ethyl alcohol, methanol.
It is further preferred that being precipitated as the ethyl alcohol that 3-4 times of extracting liquid volume after addition enzymolysis is measured, precipitate 8-12 hours.
Preferably, the elution is that the precipitation that will be obtained is carried out using 78% ethyl alcohol, 95% ethyl alcohol, absolute ethyl alcohol, acetone
Gradient elution.
In a preferred embodiment, a kind of preparation method of mushroom dietary fiber extract, includes the following steps：
(1) mushroom drying crushes after for example drying, and is 1 according to solid-liquid ratio (mass volume ratio)：10 are added ethyl alcohol for example
95% ethyl alcohol extracts 2h with ethyl alcohol boiling reflux, filters, obtain filter residue；
(2) it is 1 according to solid-liquid ratio (mass volume ratio):10 into filter residue plus water, boiling reflux extract 2h；
(3) when temperature is down to 55-65 DEG C, pH value is adjusted to 6-7, pawpaw egg is added in the extracting solution obtained to step 2
White enzyme enzymolysis, enzyme amount is 0.2% weight percent of sample sample weighting amount, and 60 DEG C of water enzyme digestion 1h are kept stirring；
(4) after digesting, 4 times of 95% ethanol precipitations measured of extracting liquid volume are overnight after enzymolysis is added, and filter, filtered
Slag；
(5) filter residue is subjected to gradient elution using 78% ethyl alcohol, 95% ethyl alcohol, absolute ethyl alcohol, acetone, dries, crushes, it is excellent
80 mesh screens were selected, mushroom dietary fiber extract is obtained.
The third aspect of the present invention provides a kind of preparation treated and/or prevented high fat diet and cause hepatic injury relevant disease,
The preparation includes mushroom dietary fiber extract.
Wherein, the preparation includes drug, health products or food.
Wherein, it includes hepatic steatosis, nonalcoholic steatohepatitis, liver that the high fat diet, which causes hepatic injury relevant disease,
Hardening, hepatic failure.
Wherein, the mushroom dietary fiber extract is the mushroom dietary fiber extraction that above-mentioned preparation method is prepared
Object.
In the fourth aspect of the present invention, a kind of medicine for preventing and/or treating high fat diet and cause hepatic injury relevant disease is provided
Object, including mushroom dietary fiber extract and pharmaceutically acceptable carrier.
Wherein, it includes hepatic steatosis, nonalcoholic steatohepatitis, liver that the high fat diet, which causes hepatic injury relevant disease,
Hardening, hepatic failure.
Wherein, the mushroom dietary fiber extract is the mushroom dietary fiber extraction that above-mentioned preparation method is prepared
Object.
Compared with prior art, the present invention has the advantages that：
(1) preparation method is simple, operating cost is low by the present invention.Extraction solvent has only used second alcohol and water, ethyl alcohol
Can be with recycling, no organic reagent remains risk, while ensure that the natural quality of extract and environmentally friendly, belongs to
In green manufacture technology.
(2) present invention extracts mushroom using ethyl alcohol, alcohol-soluble substance is removed, using the egg in papain enzymolysis sample
White matter removes isolating protein, improves the recovery rate and purity of mushroom dietary fiber.
(3) mushroom dietary fiber extract of the invention can not only be clearly helpful for dominant microflora structure recovery in enteron aisle,
Contribute to flora diversified, causes the relevant disease of hepatic injury to show significantly treatment and/or preventive effect, energy high fat diet
The expression for effectively inhibiting inflammatory factor, is effectively relieved the deterioration of inflammation, improves immune-related every physiopathological index, can be with
For in the various fields such as drug, health products, to there is great economy and social value.
Description of the drawings
Fig. 1 is the small intestine HE coloring pathological sections figure (observation of 200 power microscopes) of embodiment 2.
Fig. 2 is the liver organization pathological section figure (observation of 200 power microscopes) of embodiment 2.
Specific implementation mode
Below in conjunction with specific embodiment, invention is further explained.
Embodiment 1：
The preparation method of mushroom dietary fiber extract, steps are as follows：
(1) it crushes and weighs after mushroom drying, be 1 according to solid-liquid ratio (mass volume ratio)：10, it is boiled using 95% ethyl alcohol
Refluxing extraction 2h filters, obtains filter residue；
(2) it is 1 to add water, solid-liquid ratio (mass volume ratio) into filter residue:10, boiling reflux extracts 2h；
(3) it when temperature is down to 60 DEG C or so, adjusts pH value to 6-7, papain enzymolysis is added, enzyme amount is that sample claims sample
During which 0.2% (mass percent) of amount, 60 DEG C of water enzyme digestion 1h are kept stirring；
(4) enzymolysis terminates, and 4 times of 95% ethanol precipitations measured of extracting liquid volume are overnight after enzymolysis is added, and filter, filtered
Slag；
(5) filter residue is subjected to gradient elution using 78% ethyl alcohol, 95% ethyl alcohol, absolute ethyl alcohol, acetone, dries, crushes, mistake
Mushroom dietary fiber extract is prepared in 80 mesh screens.
Recovery rate is calculated for the mushroom dietary fiber extract being prepared, recovery rate is：Extract quality/sample claims
Sample amount × 100%=46.40%.
The measurement of physicochemical property is carried out for the mushroom dietary fiber extract being prepared：Expansive force is 7.84ml/g,
Expansion rate is 6.09ml/ml, retention ability 6.14g/g, and it is 0.59g/g to hold oily power, and absorption cholesterol ability is 33.45mg/g
(pH2.0)、40.12mg/g(pH7.0)。
Constituent analysis is carried out for the mushroom dietary fiber extract being prepared：Moisture 5.98%, protein 6.07%,
Ash content 5.13%, total dietary fiber 82.82%.
Embodiment 2：Improvement result of the mushroom dietary fiber extract to high fat diet obesity-induced mice hepatic injury
1, reagent：
Total cholesterol (T-CHO) testing cassete：Bioengineering Research Institute is built up in Nanjing.
Triglycerides (TG) testing cassete：Bioengineering Research Institute is built up in Nanjing.
Low density lipoprotein cholesterol (LDL-C) testing cassete：Bioengineering Research Institute is built up in Nanjing.
High-density lipoprotein cholesterol (HDL-C) testing cassete：Bioengineering Research Institute is built up in Nanjing.
Alanine aminotransferase (ALT) testing cassete, aspartate amino transferase (AST) testing cassete, total protein
(TP) test, albumin (ALB) test and total bilirubin (T-Bil) testing cassete all build up bio-engineering research purchased from Nanjing
Institute.
Female C57 mouse 30, are purchased from Guangdong Province medical experiment animal by SPF grades, weight 12-14g, age in days 28-30d
Center, credit number are SCXK (Guangdong) 2013-0002.
Rats and mice maintains feed：Guangdong Medical Lab Animal Center, credit number SCXK (Guangdong) 2013-0002.
High cholesterol diet formula high in fat is as shown in table 1：
The main component and energy of 1 high cholesterol diet high in fat of table are constituted
Mushroom dietary fiber extract is prepared by experimental example 1 of the present invention.
2, method
The preparation of high fat diet obesity-induced mice model：Animal subject is raised in the zoopery of Institute of Micro-biology of Guangdong Province
The heart, raising temperature and humidity：23 1 DEG C, 55 ± 10%, using 12h intermittent illuminations round the clock；Receptacle condition remains stable,
With the reliability of guarantee test result.Mouse ad lib is drunk water.Period daily check animal is primary, does not find unsound dynamic
Object takes whole healthy mices to be tested.30 C57 mouse through adaptability raise 7d after, be randomly divided into normal group, model group and
Administration group, every group 10.Normal group is fed with normal diet, and model group and administration group are fed with high cholesterol diet high in fat, even
It is continuous to feed one month.
After modeling success, start to be administered, administration group gives mushroom dietary fiber extract gavage, 1g dietary fiber extracts
It is diluted with 15ml distilled water, the daily gavage 0.6ml of every mouse, successive administration 2 months.Normal group mouse continues with general simultaneously
Logical forage feed, administration group and model group mouse conversion chow diet are fed.
3, result is observed
The appearance figure (hair, color, shape) of daily observation animal and dynamic simultaneously make respective record, and the weight of animals is measured every 3d
Once.
Blood：After being discontinued for 24 hours, animal materials testing index.Acquire blood sample, pluck eyeball and take blood, blood plasma through 3000RPM,
Serum is collected in 10min centrifugations twice, measures T-CHO, TG, LDL-C, HDL-C, TP, ALB, GLB, ALT, AST, T- in serum
Bil is horizontal.It includes cytokine interleukin IL-2, IL-6, IL-22 and Tumor necrosis factor TNF-to measure inflammatory factor
α。
Tissue：It plucks after eyeball takes blood, puts to death and dissect mouse, take mouse small intestine and liver organization, be fixed on 4% poly first
In aldehyde.Small intestine and liver organization are made into paraffin section, and small intestine is dyed using Hematoxylin-eosin (HE) method, liver organization
Hematoxylin-eosin (HE), Picro-Sirius red (SRS) and oil red O (ORS) dyeing is respectively adopted, carries out pathology section examination.
4, data processing
All data are with mean standard deviationIt indicates.The comparison of multigroup mean uses one-way analysis of variance
(One-Way ANOVA), mean compares two-by-two between group, and LSD methods are used when variance is neat；Dunnett ' s T3 are used when heterogeneity of variance
Method.It is completed by SPSS softwares, α=0.05.
It has been observed that the fur of the animal of administration group is more smoother than the fur of model group.
The changes of weight of each group is as shown in table 2.
2 mouse weight of table changes
#P<0.05,##P<0.01,###P<Normal group of 0.001vs；*P<0.05,**P<0.01,***P<0.001vs model groups
As can be found from Table 2, the normal group of average weight difference between model group extremely significantly (P<0.001), model
Group mouse average weight about 20.35g when starting, average weight is 35.29g at the end of experiment, illustrates that high fat diet induces
Mice model of obesity modeling success；Compared with model group, administration group average mice body weight is remarkably decreased (P<0.01).This shows：
Suitable dietary fiber is added in high fat diet can significantly slow weight gain.
The four items of blood lipid tests testing result of each group is as shown in table 3.
3 mice serum biochemical indicator (mmol/L) of table
#P<0.05,##P<0.01,###P<Normal group of 0.001vs；*P<0.05,**P<0.01,***P<0.001vs model groups
From table 3 it can be seen that total cholesterol, triglycerides and low-density lipoprotein, cholesterol levels ratio be just in model group
Often group significantly increases (P<0.001 or P<0.01), High-density Lipoprotein-cholesterol significantly reduces (P than normal group<
0.001)；Compared with model group, triglycerides, total cholesterol, low-density lipoprotein cholesterol level are remarkably decreased in administration group
(P<0.05 or P<0.01), High-density Lipoprotein-cholesterol significantly increases (P<0.05).This shows：In high fat diet
Total cholesterol, triglycerides and low-density lipoprotein white level in serum can be significantly reduced by adding suitable dietary fiber.
The inflammatory factor testing result of each group is as shown in table 4.
4 inflammatory factor measurement result of table
#P<0.05,##P<0.01,###P<Normal group of 0.001vs；*P<0.05,**P<0.01,***P<0.001vs model groups
From table 4, it can be seen that compared with normal group, all cytokine levels deviate normally in model group serum,
TNF-α, IL-2, IL-6, IL-22 significant difference (p<0.05)；Compared with model group, all these cell factors of administration group are bright
It is aobvious to restore, to show that mushroom dietary fiber can improve inflammatory environment.
The results are shown in Table 5 for the liver functional testing of each group.
5 liver functional testing result of table
#P<0.05,##P<0.01,###P<Normal group of 0.001vs；*P<0.05,**P<0.01,***P<0.001vs model groups
As can be seen from Table 5, normal group of notable liter of TP, ALB, ALT, AST and T-BIL level ratio in model group mice serum
High P<0.05 or P<0.01；Compared with model group, administration group TP, ALB, ALT, AST and T-BIL level significantly reduces (P<
0.05).This shows：Suitable dietary fiber is added in high fat diet can significantly improve liver function.
The results are shown in Figure 1 for small intestine's HE coloring pathological sections, it will be seen from figure 1 that normally group small intestine villus
Long and complete, model group intestinal villus falls off seriously, illustrates that high fat diet can cause to damage to the enteron aisle of mouse, illustrates that model is
Successfully.Compared with model group, after drug-treated, administration group can alleviate damage, promote the reparation of enteron aisle.
The results are shown in Figure 2 for liver organization pathological section, figure it is seen that HE coloration results show normal group mouse
Liver cell structure is normal, and model group mouse liver cell volume increases, and occurs many circles not of uniform size in hepatocyte cell matter
Blown-out shot pushes nucleus to side, locally there is inflammatory cell infiltration phenomenon；Compared with model group, administration group mouse liver cell fat
Fat vacuole quantity significantly reduces, and inflammatory cell infiltration phenomenon is improved；SRS is dyed and ORS coloration results are shown respectively, with mould
Type group is compared, and administration group mouse liver cell structure is obviously improved, and tends to normal group.
Embodiment 3：Mushroom dietary fiber extract is to high fat diet obesity-induced mice intestinal bacilli illness relevant disease
Improvement result
1, experimental method
Stool in mice is stored in -80 DEG C of ice after liquid nitrogen is rapidly frozen at the end of zoopery in acquisition embodiment 2
In case.DNA is extracted from fecal specimens, and DNA is detected by 0.8% agarose gel electrophoresis and extracts quality, while using purple
Outer spectrophotometer quantifies DNA.Using forward primer 5 '-ACTCCTACGGGAGGCAGCA-3 ' and reverse primer 5'-
GGACTACHVGGGTWTCTAAT-3' expands the areas the V3-V4 gene of mouse 16S rRNA.Pcr amplification product passes through 2%
Agarose gel electrophoresis is detected, and carries out gel extraction to target fragment.With reference to the preliminary quantitative result of electrophoresis, PCR is expanded
Increase recovery product and carry out fluorescent quantitation, fluorescent reagent is Quant-iT PicoGreen dsDNA Assay Kit, quantitative instrument
For Microplate reader (BioTek, FLx800).According to fluorescent quantitation as a result, according to each sample sequencing amount demand,
Each sample is mixed by corresponding proportion.Using the TruSeq Nano DNA LT Library Prep of Illumina companies
Kit prepares sequencing library.The both-end that 2 × 300bp is carried out using MiSeq sequenators is sequenced, and corresponding reagent is MiSeq Reagent
Kit V3(600cycles)。
2, experimental result
The results are shown in Table 6 for the intestinal flora diversity of each group.
6 mouse intestinal flora diversity indices of table
#P<0.05,##P<0.01,###P<Normal group of 0.001vs；*P<0.05,**P<0.01,***P<0.001vs model groups
As can be seen from Table 6, compared with normal group, the Alpha diversity indices of model group mouse intestinal flora
Simpson and Shannon are remarkably decreased (P<0.05), illustrate that high fat diet changes mouse intestinal flora structure, keep its various
Property reduce；Compared with model group, administration group mouse intestinal flora diversity indices Simpson and Shannon significantly increase (P<
0.05) after, illustrating that the obesity mice intestinal flora of high fat diet induction gives mushroom dietary fiber treatment, intestinal flora diversity
It dramatically increases
The OTU enrichment analysis of each group is as shown in table 7 and table 8.
Abundance of 7 mouse intestinal flora of table in door level
#P<0.05,##P<0.01,###P<Normal group of 0.001vs；*P<0.05,**P<0.01,***P<0.001vs model groups
Abundance of 8 mouse intestinal flora of table on belonging to level
#P<0.05,##P<0.01,###P<Normal group of 0.001vs；*P<0.05,**P<0.01,***P<0.001vs model groups
As can be seen from Table 7, in door level, compared with normal group, model group mouse intestinal flora Bacteroidetes
Significant changes have occurred with Firmicutes abundance, Bacteroidetes abundance significantly reduces (P<0.001), Firmicutes
Abundance dramatically increases (P<0.001), compared with model group, administration group Bacteroidetes abundance dramatically increases (P<0.01),
Firmicutes abundance significantly reduces (P<0.01).
As can be seen from Table 8, on belonging to level, compared with normal group, model group beneficial bacterium Akkermansia,
Bifidobacterium and Lactobacillus significantly reduces (P<0.01), harmful bacteria Allobaculum (P<0.001) and
Dorea(P<0.05) dramatically increase, compared with model group, administration group beneficial bacterium Akkermansia, Bifidobacterium and
Lactobacillus dramatically increases (P<0.01), harmful bacteria Allobaculum (P<And Dorea (P 0.05)<0.05) it significantly drops
It is low.
To sum up it can be seen that, high fat diet obesity-induced mice model makes its intestinal microflora that significant changes occur,
Mushroom dietary fiber extract is added in high cholesterol diet high in fat and is clearly helpful for dominant microflora structure recovery in enteron aisle, is helped
In flora diversification, it is effectively improved intestinal bacilli illness, repairs intestinal mucosa, effectively inhibits the expression of inflammatory factor, it is effectively slow
The deterioration of inflammation is solved, immune-related every physiopathological index is improved, it, can be with so as to improve hepatic injury caused by high fat diet
For in the various fields such as drug, health products, to there is great economy and social value.
The above, only preferable specific embodiment of the invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Design is subject to equivalent substitution or change, should all cover within the scope of the present invention.
Claims (10)
1. a kind of mushroom dietary fiber extract is preparing treatment and/or is preventing the preparation of high fat diet cause hepatic injury relevant disease
In purposes.
2. purposes according to claim 1, which is characterized in that it includes liver that the high fat diet, which causes hepatic injury relevant disease,
Steatosis, nonalcoholic steatohepatitis, hepatic sclerosis, hepatic failure.
3. purposes according to claim 1, which is characterized in that the mushroom dietary fiber extract prepare treatment and/
Or the purposes in the preparation of the diseases such as prevention of hepatic steatosis, nonalcoholic steatohepatitis, hepatic sclerosis, hepatic failure.
4. a kind of preparation method of mushroom dietary fiber extract, which is characterized in that after crushing mushroom, returned using ethyl alcohol
Filter residue is used water refluxing extraction, then by extracting solution enzymolysis, precipitation, elution, obtains mushroom dietary fiber extract by stream extraction.
5. preparation method according to claim 4, which is characterized in that the alcohol reflux, which extracts, includes：Using solid-liquid ratio
It is 1:8-1:12 ethyl alcohol, refluxing extraction 2-3 hours；And/or the filter residue includes using water refluxing extraction：Using solid-liquid ratio
It is 1:8-1:12 water, refluxing extraction 2-3 hours.
6. preparation method according to claim 4, which is characterized in that the enzymolysis is excellent for using papain enzymolysis
Choosing, the enzymolysis dosage is 0.2% mass percent of sample sample weighting amount, and the enzymatic activity is 750,000 u/g-85, ten thousand u/g；
And/or the hydrolysis temperature is 55-65 DEG C, the time is 55-65 minutes.
7. preparation method according to claim 4, which is characterized in that it is described to be precipitated as precipitating using alcohol reagent, such as
Ethyl alcohol, methanol；And/or it is precipitated as the ethyl alcohol of extracting liquid volume 3-4 amounts after addition enzymolysis, it precipitates 8-12 hours；And/or institute
Elution is stated as obtained precipitation is carried out gradient elution using 78% ethyl alcohol, 95% ethyl alcohol, absolute ethyl alcohol, acetone.
8. according to any preparation method in claim 4 to 7, include the following steps：
(1) mushroom drying crushes after for example drying, according to solid-liquid ratio 1：10 are added ethyl alcohol such as 95% ethyl alcohol, are boiled back with ethyl alcohol
Stream extraction 2h, filters, obtains filter residue；
(2) it is 1 according to solid-liquid ratio:10 into filter residue plus water, boiling reflux extract 2h；
(3) when temperature is down to 55-65 DEG C, pH value is adjusted to 6-7, papain is added in the extracting solution obtained to step 2
Enzymolysis, enzyme amount are 0.2% weight percent of sample sample weighting amount, and 60 DEG C of water enzyme digestion 1h are kept stirring；
(4) after digesting, 4 times of 95% ethanol precipitations measured of extracting liquid volume are overnight after enzymolysis is added, and filter, obtain filter residue；
(5) filter residue is subjected to gradient elution using 78% ethyl alcohol, 95% ethyl alcohol, absolute ethyl alcohol, acetone, dries, crushes, preferably mistake
80 mesh screens obtain mushroom dietary fiber extract.
9. it includes that mushroom diet is fine that a kind for the treatment of and/or prevention high fat diet, which cause the preparation of hepatic injury relevant disease, the preparation,
Extract is tieed up, the preparation includes drug, health products or food, the preferred claim 4 to 8 of mushroom dietary fiber extract
In the mushroom dietary fiber extract that is prepared of any preparation method.
10. a kind of prevention and/or treatment high fat diet cause the drug of hepatic injury relevant disease, including mushroom dietary fiber extract
And pharmaceutically acceptable carrier, any preparation side in the preferred claim 4 to 8 of mushroom dietary fiber extract
The mushroom dietary fiber extract that method is prepared.
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| CN109771454A (en) * | 2019-03-26 | 2019-05-21 | 广东省微生物研究所（广东省微生物分析检测中心） | Purposes of the birch young pilose antler extract in the preparation that preparation treatment and/or prevention high fat diet cause hepatic injury related disease |
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