CN108523143A - Black tiger palm 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

Black tiger palm 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 PDF

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CN108523143A
CN108523143A CN201810210282.6A CN201810210282A CN108523143A CN 108523143 A CN108523143 A CN 108523143A CN 201810210282 A CN201810210282 A CN 201810210282A CN 108523143 A CN108523143 A CN 108523143A
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dietary fiber
preparation
ethyl alcohol
black tiger
tiger palm
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陈地灵
杨俭
唐晓萃
帅欧
赖国校
谢意珍
吴清平
杨柏华
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Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
Guangdong Yuewei Edible Mushroom Technology Co Ltd
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Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
Guangdong Yuewei Edible Mushroom Technology Co Ltd
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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, 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
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21Addition of substantially indigestible substances, e.g. dietary fibres
    • A23L33/22Comminuted fibrous parts of plants, e.g. bagasse or pulp
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06Fungi, e.g. yeasts
    • A61K36/07Basidiomycota, e.g. Cryptococcus
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/10Preparation or pretreatment of starting material
    • A61K2236/19Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/30Extraction of the material
    • A61K2236/33Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/30Extraction of the material
    • A61K2236/39Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/51Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/53Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

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Abstract

The purposes of purposes more particularly to a kind of black brave palm 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 black tiger palm dietary fiber extracts in pharmaceutical field.The black tiger palm dietary fiber extract of the present invention can not only be clearly helpful for the reparation that high fat diet causes hepatic injury, but also pair with the relevant disease of hepatic injury show significantly treat and/or preventive effect, such as it can effectively inhibit the expression of inflammatory factor, the deterioration of inflammation is effectively relieved, improve immune-related every physiopathological index, it can be used in the various fields such as drug, health products, there is great economy and social value.

Description

Black tiger palm dietary fiber extract causes liver in preparation treatment and/or prevention high fat diet Damage the purposes in the preparation of relevant disease
Technical field
The present invention relates to black tigers to slap purposes of the dietary fiber extract in the fields such as pharmacy, health products and health food, More particularly to a kind of black tiger palm dietary fiber extract causes hepatic injury relevant disease in preparation treatment and/or prevention high fat diet Purposes in preparation.
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.
HEIHUZHANG fungus (Saarcodon imbricatum (L.:Fr.) Karst.), mushroom cap is fleshy hypertrophy, gives off a strong fragrance, newly Be canescence or taupe when fresh, become that dull gray is white or taupe after dry, shaped like brave pawl, lower surface cover with one layer it is very thin greyish white Color thorn-like fine hair, how hollow stem is sturdy, and long is a little inclined.Black tiger palm field distributed area very little, China only has southwest partly Area produces, very precious.
HEIHUZHANG fungus is considered as rare mountain delicacy very early in history, is one of successive dynasties' favorite tribute in successive dynasties imperial palace.Europe It is that the black tiger palm is consumed mainly that beauteously area does not have food custom, Southwest China, Japan and one band of Southeast Asia to HEIHUZHANG fungus Area.The bacterium flat property and sweet taste has the effect of wind-evil dispelling and cold-evil expelling, relaxing tendons and activating collaterals, civil to be also used as establishing-Yang with it, and having, which reduces courage in blood, consolidates The effect of alcohol.
Invention content
The purpose of the present invention is to provide a kind of preparation methods of black tiger palm dietary fiber extract, and slap meals with black tiger It eats fiber extract treatment and/or prevents high fat diet and cause hepatic injury preparation relevant disease.
In the first aspect of the present invention, it is high in preparation treatment and/or prevention to provide the black tiger palm dietary fiber extract of one kind Fat diet causes the purposes in the preparation of hepatic injury relevant disease.
Wherein, the hepatic injury relevant disease includes that fatty liver, hepatic steatosis, nonalcoholic steatohepatitis, liver are hard Change, hepatic failure.
Preferably, the black tiger palm dietary fiber extract is preparing treatment and/or prevention of hepatic steatosis, non-alcohol Purposes in the preparations of diseases such as property fat hepatitis, hepatic sclerosis, hepatic failure.
In the second aspect of the present invention, a kind of preparation method of black tiger palm dietary fiber extract, including by sample comminution Afterwards, it is extracted using alcohol reflux, filter residue uses water refluxing extraction, then digests, alcohol precipitation, elutes, and it is fine to finally obtain black tiger palm diet Tie up extract.
Preferably, the alcohol reflux, which extracts, includes:Use solid-liquid ratio for 1:8-1:15 ethyl alcohol, refluxing extraction 2-4 are small When.
Preferably, the filter residue includes using water refluxing extraction:Use solid-liquid ratio for 1:8-1:15 water, refluxing extraction 2- 4 hours.
Preferably, the enzymolysis is using papain enzymolysis.
Preferably, the enzymolysis dosage is 0.2% volume of extracting solution, and the enzymatic activity is 750,000 u/g-85, ten thousand u/g.
Preferably, the hydrolysis temperature is 50 DEG C -70 DEG C, and the time is 1-2 hours.
Preferably, described to be precipitated as precipitating using alcohol reagent, such as ethyl alcohol, methanol.
It is further preferred that described be precipitated as the ethyl alcohol that 4-6 times of liquid is measured after addition digests, precipitate 12-24 hours.
Preferably, the elution carries out gradient elution for the precipitation that will be obtained using ethyl alcohol, the acetone of increasing concen-trations.
It is further preferred that the elution, which is the precipitation that will be obtained, uses 78% ethyl alcohol, 95% ethyl alcohol, absolute ethyl alcohol, third Ketone carries out gradient elution.
Preferably, the preparation method of the black tiger palm dietary fiber extract includes the following steps:
(1) it is 1 according to solid-liquid ratio after the black tiger palm is dry:8-1:Ethyl alcohol such as 95% ethyl alcohol is added in 15 (mass volume ratios), 2h-4h is extracted with ethyl alcohol boiling reflux, filters, obtains filter residue;
(2) it is 1 according to solid-liquid ratio:8-1:15 (mass volume ratios) are into filter residue plus water, boiling reflux extract 2h-4h;
(3) when temperature is down to 50 DEG C -60 DEG C or so, pH value is adjusted to 6-7, is added in the filter residue obtained to step (2) Papain enzymolysis, enzyme amount are 0.2% weight percent, and 50-70 DEG C of water enzyme digestion 1h-2h is kept stirring;
(4) after digesting, the ethanol precipitation that 4-6 times of extracting liquid volume is measured is added and stays overnight, filters, obtains filter residue;
(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, black tiger palm dietary fiber extract is obtained.
In a preferred embodiment, the preparation method of the black tiger palm dietary fiber extract includes the following steps:
(1) it is crushed after the dry such as drying of the black tiger palm, is 1 according to solid-liquid ratio:10 are added ethyl alcohol such as 95% ethyl alcohol, use second Alcohol boiling reflux extracts 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 50 DEG C -60 DEG C or so, pH value is adjusted to 6-7, wood is added in the filter residue obtained to step 2 Melon protease hydrolyzed, enzyme amount are 0.2% weight percent, and 60 DEG C of water enzyme digestion 1h are kept stirring;
(4) after digesting, 95% ethanol precipitation that 4 times of amounts of extracting liquid volume are added is stayed overnight, and is filtered, is obtained filter residue;
(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, black tiger palm dietary fiber is obtained.
The third aspect of the present invention, it is a kind for the treatment of and/or preventing liver injury relevant disease preparation, the preparation includes medicine Object, health products or food, the preparation include that black tiger slaps dietary fiber extract.
In the fourth aspect of the present invention, a kind of drug prevented and/or treat hepatic injury relevant disease, including black tiger palm meals Eat fiber extract and pharmaceutically acceptable carrier.
Preferably, the black tiger palm dietary fiber extract is the extract that above-mentioned preparation method is prepared.
Compared with prior art, the present invention has the advantages that:
(1) preparation method is simple, operating cost is low by the present invention, and the Extraction solvent that preparation method of the present invention uses is only Use second alcohol and water, ethyl alcohol that can remain risk with recycling, no organic reagent, ensure that the natural quality of extract It is simultaneously and environmentally friendly, belong to green manufacture technology.
(2) present invention removes alcohol-soluble substance, using in papain enzymolysis sample using the black tiger palm of ethyl alcohol extraction Protein removes isolating protein, improves the recovery rate and purity of black tiger palm dietary fiber, greatly improves recovery rate and purity.
(3) black tiger palm dietary fiber extract of the invention can not only be clearly helpful for high fat diet and cause repairing for hepatic injury It is multiple, but also pair show significantly to treat with the relevant disease of hepatic injury and/or preventive effect, such as can effectively inhibit inflammation The deterioration of inflammation is effectively relieved in the expression of the factor, improves immune-related every physiopathological index, can be used for drug, protects In the various fields such as strong product, 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.
It is further illustrated the present invention below in conjunction with specific implementation mode, but the scope of protection of present invention is not limited to In following embodiments.
As used herein, term "comprising" or " comprising " refer to the element that method includes cited, but are not excluded for other Element.
HEIHUZHANG fungus scientific name jujube Sarcodon imbricatus (L. Ex Franch.) Karst of the present invention is famous one of Export of Edible Fungi.Thalline is sturdy Loose, fine and tender taste is not easy tattered and full of nutrition containing abundant exocellular polysaccharide.It is measured according to analysis, dry product contains 17 kinds of ammonia Base acid, wherein having 7 kinds of essential amino acids for accounting for total amount 41.46% and 11 kinds of minerals and trace element.It is this edible Bacterium value is high, has production, more than 1000 yuan one jin of price in Northern Guangdong Province.
Embodiment 1:The preparation method of black tiger palm dietary fiber extract
(1) it crushes and weighs after sample drying, solid-liquid ratio 1:10,2h is extracted with 95% ethyl alcohol boiling reflux, is filtered, is protected Stay filter residue;
(2) filter residue adds water, solid-liquid ratio 1:10, 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 sample weighting amount During which 0.2%, 60 DEG C of water enzyme digestion 1h are kept stirring;
(4) enzymolysis terminates, and 95% ethanol precipitation that 4 times of amounts of extracting liquid volume are added is stayed overnight, and is filtered, and filter residue is retained;
(5) filter residue carries out gradient elution using 78% ethyl alcohol, 95% ethyl alcohol, absolute ethyl alcohol, acetone, dries, and crushes, mistake 80 mesh screens, are made dietary fiber.
Recovery rate is calculated for the black tiger palm dietary fiber extract being prepared, recovery rate is:Extract quality/sample Sample weighting amount × 100%=49.55%.
The measurement of physicochemical property is carried out for the black tiger palm dietary fiber extract being prepared:Expansive force is 7.21ml/ G, expansion rate 7.12ml/ml, retention ability 5.98g/g, it is 0.63g/g to hold oily power, and absorption cholesterol ability is 34.21mg/g (pH2.0)、 41.29mg/g(pH7.0)。
Constituent analysis is carried out for the black tiger palm dietary fiber extract being prepared:Moisture 4.98%, protein 6.85%, ash content 5.66%, total dietary fiber 84.37%.
Embodiment 2:Improvement result of the black tiger palm 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
Black tiger palm dietary fiber extract is prepared by experimental example 1 of the present invention.
2, method
Animal subject is raised in Institute of Micro-biology of Guangdong Province animal experimental center, 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 is certainly It is drunk water by feed.Period daily check animal is primary, does not find unsound animal, and whole healthy mices is taken to be tested.30 C57 mouse are randomly divided into normal group, model group and administration group after adaptability raises 7d, every group 10.It is normal to organize with general Logical feed forage feed, model group and administration group are fed with high cholesterol diet high in fat, continuous to feed one month.
After modeling success, start to be administered, administration group gives black tiger palm dietary fiber extract gavage, the extraction of 1g dietary fibers Object is diluted with 15ml distilled water, the daily gavage 0.6ml of every mouse, successive administration 2 months.Normal group normal diet feed It feeds, model group and administration group are fed with high cholesterol diet high in fat, continuous to feed one month.
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, and it is horizontal to measure T-CHO, TG, LDL-C and HDL-C in serum.It includes thin to measure inflammatory factor Intracellular cytokine interleukins IL-2, IL-6, IL-22, tumor necrosis factor TNF-alpha and Foxo3a.
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
From table 2 it can be seen that normal average weight difference between group and 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) (table 2).This Show: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 normally group is aobvious for total cholesterol, triglycerides and ldl-cholesterol ratio in model group It writes and increases (P<0.001 or P<0.01), high-density lipoprotein cholesterol significantly reduces (P than normal group<0.001);With model group It compares, triglycerides, total cholesterol, low-density lipoprotein cholesterol level are remarkably decreased (P in administration group<0.05 or P< 0.01), High-density Lipoprotein-cholesterol significantly increases (P<0.05).This shows:Suitable meals are added in high fat diet Food fiber can significantly reduce total cholesterol, triglycerides and low-density lipoprotein white level in serum.
The inflammatory factor testing result of each group is as shown in table 4.
4 inflammatory factor 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
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, Foxo3 significant difference (p<0.05);Compared with model group, all these cells of administration group The factor is obviously restored, to show that black tiger palm 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:Black tiger palm dietary fiber extract is to high fat diet obesity-induced mice intestinal bacilli illness correlation disease The improvement result of disease
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, and recycling is recycled using the gel of AXYGEN companies Kit.With reference to the preliminary quantitative result of electrophoresis, PCR amplification recovery product is subjected to fluorescent quantitation, fluorescent reagent Quant-iT PicoGreen dsDNA Assay Kit, quantitative instrument are Microplate reader (BioTek, FLx800).According to fluorescence Quantitative result is mixed each sample by corresponding proportion according to the sequencing amount demand of each sample.Using Illumina public affairs The TruSeq Nano DNA LT Library Prep Kit of department prepare sequencing library.Using MiSeq sequenators carry out 2 × The both-end of 300bp 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 Simpson of model group mouse intestinal flora It is remarkably decreased (P with Shannon<0.05), illustrate that high fat diet changes mouse intestinal flora structure, make the reduction of its diversity; Compared with model group, administration group mouse intestinal flora diversity indices Simpson and Shannon significantly increase (P<0.05) it, says After the obesity mice intestinal flora of bright high fat diet induction gives black tiger palm dietary fiber treatment, intestinal flora diversity significantly increases Add.
OTU enrichment analysis 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, it is extensive that black tiger palm dietary fiber extract can not only be clearly helpful for dominant microflora structure in enteron aisle It is multiple, contribute to flora diversified, but also pair show significantly to treat and/or prevent with the relevant disease of intestinal microflora Effect can effectively inhibit the expression of inflammatory factor, and the deterioration of inflammation is effectively relieved, and improve immune-related every physiological and pathological and refer to Mark, can be used in the various fields such as drug, health products, have 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 black tiger palm dietary fiber extract is preparing the system treated and/or prevented high fat diet and cause hepatic injury relevant disease Purposes in agent.
2. purposes according to claim 1, which is characterized in that the hepatic injury relevant disease includes fatty liver, liver Steatosis, nonalcoholic steatohepatitis, hepatic sclerosis, hepatic failure.
3. according to the purposes of claims 1 or 2, which is characterized in that the black tiger palm dietary fiber extract is preparing 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 black tiger palm dietary fiber extract, which is characterized in that after crushing the black tiger palm, using second Alcohol reflux extracts, and filter residue is used water refluxing extraction, then by extracting solution enzymolysis, precipitation, elution, obtain black tiger palm dietary fiber and carry Take object.
5. preparation method according to claim 4, which is characterized in that the alcohol reflux, which extracts, includes:Using solid-liquid ratio It is 1:8-1:15 ethyl alcohol, refluxing extraction 2-4 hours;And/or the filter residue includes using water refluxing extraction:Using solid-liquid ratio It is 1:8-1:15 water, refluxing extraction 2-4 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% volume of extracting solution, and the enzymatic activity is 750,000 u/g-85, ten thousand u/g;And/or the enzyme It is 50 DEG C -70 DEG C to solve temperature, and the time is 1-2 hours.
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 that 4-6 times of liquid is measured after addition digests, it precipitates 12-24 hours;And/or it described washes It takes off for 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) it will be crushed after the dry such as drying of the black tiger palm, be 1 according to solid-liquid ratio:10 are added ethyl alcohol such as 95% ethyl alcohol, use ethyl alcohol Boiling reflux extracts 2h, filters, obtains filter residue;
(2) it is 1 according to solid-liquid ratio:10 into filter residue plus water, boiling reflux extract 2 hours;
(3) when temperature is down to 50 DEG C -60 DEG C or so, pH value is adjusted to 6-7, pawpaw is added in the filter residue obtained to step (2) Protease hydrolyzed, enzyme amount are 0.2% weight percent, and 60 DEG C of water enzyme digestions 1 hour are kept stirring;
(4) after digesting, 95% ethanol precipitation that 4 times of amounts of extracting liquid volume are added is stayed overnight, and is filtered, is obtained 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 black tiger palm dietary fiber extract.
9. the preparation of a kind for the treatment of and/or preventing liver injury relevant disease, which is characterized in that the preparation includes drug, health care Product or food, the preparation include that black tiger slaps dietary fiber extract.
10. the drug of a kind of prevention and/or treatment hepatic injury relevant disease, which is characterized in that carried comprising black tiger palm dietary fiber Take object and pharmaceutically acceptable carrier.
CN201810210282.6A 2018-03-14 2018-03-14 Black tiger palm dietary fiber extract is preparing treatment and/or is preventing the purposes during high fat diet causes the preparation of hepatic injury relevant disease Pending CN108523143A (en)

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