CN107582562A - Application of the beans taro polysaccharide in liver cell lipidosis is reduced - Google Patents

Application of the beans taro polysaccharide in liver cell lipidosis is reduced Download PDF

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CN107582562A
CN107582562A CN201710751974.7A CN201710751974A CN107582562A CN 107582562 A CN107582562 A CN 107582562A CN 201710751974 A CN201710751974 A CN 201710751974A CN 107582562 A CN107582562 A CN 107582562A
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beans taro
polysaccharide
beans
many candies
thick many
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CN107582562B (en
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郑晓冬
楚强
陈鹏华
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Zhejiang University ZJU
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Zhejiang University ZJU
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Abstract

The invention discloses a kind of application of beans taro polysaccharide in liver cell lipidosis is reduced.The preparation method of the beans taro polysaccharide is:1), the preparation of beans taro polysaccharide crude:First extracted after beans taro and distilled water are mixed, be then beaten, centrifuge, concentration;Added in the concentrate of gained after ethanol stands 24 ± 2h to ethanol final concentration of 80 ± 5%, at 4 ± 1 DEG C and collect precipitation, obtain beans taro Thick many candies;2), the purifying of beans taro Thick many candies, including beans taro Thick many candies are subjected to activated carbon decolorizing and the de- albumen of Sevage methods.

Description

Application of the beans taro polysaccharide in liver cell lipidosis is reduced
Technical field
The present invention relates to field of medicaments, and in particular to application of the beans taro polysaccharide in liver cell lipidosis is reduced.
Background technology
Beans taro (Apios americana Medikus) originates in the Canadian to Fla. of eastern North America Southern areas, belong to perennial legume (Papilionaceae subfamily).Its edible portion is underground stem tuber, and its protein contains Amount is higher than other plant stem tuber, is a kind of not only nutrition but also has the crop of economic value, thus often by American Indian as staple food.Beans Taro was from domestic successful introduction in 2009, and by gradually in the ground such as Fuyang, Jinhua, Long You, Wenzhou popularizing planting.Research shows beans taro Different parts (flower, rattan, leaf, stem tuber) are containing abundant free amino acid, soluble protein, polysaccharide, saponin(e, flavones, different Huang Ketone, vitamin C, vitamin E and mineral matter and other components.Wherein, the Vitamin C content highest of beans taro, while also containing higher Vitamin E, total saposins and the isoflavones of content, there is great potentiality to be exploited.
U.S. beans taro has obvious improvement to lifestyle-related diseases such as hypertension, diabetes and hyperlipemias.
With the rapid development of modern social economy, the living standard of the people significantly improves, fat (obesity) and its simultaneously The morbidity and mortality of hair disease are in the trend risen year by year, and in general fatty liver can occur along with obesity.Defended according to the world Raw in March, 2011 report that is organized in shows, the whole world in 2008 has more than 1,500,000,000 adults and is in overweight level, wherein at least 500,000,000 Fat and fatty liver or.The year two thousand thirty is expected, there will be fat and fatty liver problem for the population in the U.S. 42%.U.S. will be exceeded The present population 30,000,000 of state.Medical treatment cost increase related to this will reach nearly 550,000,000,000 dollars.And delivered at home according to 2010 Research report claim, China is fat at present and fatty liver population exceedes the U.S., Britain and Australia up to 3.25 hundred million people, amplification, This numeral will be also possible to double for 20 years in future.Fatty liver and obesity caused by common complication have hypertension, diabetes, Coronary heart disease, cerebral apoplexy etc..As can be seen here, fat and fatty liver has turned into the universal health problem in the whole world, and prevention and treatment are fat Disease, fatty liver have become the primary health problem of 21 century.
The means of current prevention and treatment fatty liver mainly include:(1) total heat energy intake is controlled;(2) the method for movement;(3) perform the operation Therapy;(4) medicinal treatment;(5) non-drug therapy:Acupuncture therapy, Use of Auricular, acupuncture and moxibustion treatment, pointer method of weight-reducing, massage Method.Wherein using drug therapy as main effective treatment means.The numerous fat-reducing medicaments clinically applied at present all have substantially Cardiovascular system in terms of side effect.So far, the mankind do not develop a kind of well-content Weight-reducing and lipid-lowering medicine also. Also the shortcomings of reducing immunity of organisms, easily rebounding mostly be present in other Weight-reducing and lipid-lowering methods.The obesity more serious in face of the whole world The rise of disease and fatty liver and its incidence of disease by caused metabolic disease, seeks safe and effective new prevention and treatment Strategy is extremely important.
《U.S.'s beans taro different parts biochemical character constituent analysis》One proclamation cicada:
When measuring free amino acid, soluble protein and total reducing sugar:0.42mm sieves are crossed after crushing, using 3:500 (m/v) are added 100 DEG C of distilled water, water bath with thermostatic control extraction is carried out in 100 DEG C.
When measuring total saposins, general flavone and isoflavone content:Using 1:100 (m/v) add absolute ethyl alcohol, in 60 DEG C of constant temperature Water-bath 3h.
When measuring VC:Sample 1g is taken, 60 DEG C of ultrasonic 40min are soaked in 40ml 0.01mol/L HCL.
When measuring VE, sample 3g is taken, with addition 20ml absolute ethyl alcohols, 50 DEG C of isothermal vibration 50min.
CN201210574017.9 invention《Method that beans taro flower total saponine, polysaccharide are prepared simultaneously big more and application thereof》Accuse Cicada herein below:Preparation method is according to the following steps:1) beans taro is got over greatly in harvesting, cleaning, dry, pulverize, and is crossed 40 mesh sieves, is obtained More beans taro pollen is last greatly;2) more beans taro pollen is last greatly, according to solid-liquid ratio 1:10~1:20, addition concentration is 70%~90% second Alcohol, in 50~80 DEG C of refluxing extractions 1~3 time, each 1~3 hour time, filtering, obtain supernatant and filter residue;3) supernatant, wave Dissolved after dry with water, petroleum ether degreasing, extracting n-butyl alcohol, take n-butanol fraction after macroporous absorbent resin absorb-elute, concentrated, It is dried to obtain beans taro saponin(e powder;4) filter residue, solvent is volatilized, with 10~20 times of distilled water of its weight, is flowed back in 70~90 DEG C Extraction 1~3 time, each 2~3 hours time, filtering, collect and merge filtered solution;5) filtered solution decolourizes, takes off albumen, concentrates, and adds Ethanol, stands overnight carry out alcohol precipitation, collects precipitation, after dialysis, with DEAE~cellulose chromatography, and respectively with distilled water, 0.1M, 0.3M, 0.5M~NaCl solution eluent, 0.3M~0.5M~NaCl eluents are collected, dialysed, concentrated, dried, obtain beans taro Polysaccharide.The total saposins and polysaccharide that the present invention is prepared simultaneously from big more beans taro, purity is high, and activity is strong, can prepare It is used widely in the medicine or functional food that are acted on anti-oxidant, blood glucose-control.
201310414063.7《A kind of konjaku polypeptide extract, preparation method and its usage》Inform herein below: Fry starch of konjak addition pure water regulation pH value to 9.0 is extracted, then by 2 enzyme digestion reactions, then through ultrafiltration purification, obtained magic Taro polypeptide.The konjaku polypeptide can be used to treat diabetes and diabetic complication, and diabetic complication includes diabetic complication Atherosclerosis, diabetic retinopathy, NASH and diabetic nephropathy etc..
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of beans taro polysaccharide answering in liver cell lipidosis is reduced With.
In order to solve the above-mentioned technical problem, the present invention, which provides a kind of beans taro polysaccharide, is reducing liver cell lipidosis In application.That is, medicine, the health food of liver cell lipidosis can be reduced for preparing.
Application of the beans taro polysaccharide in liver cell lipidosis is reduced as the present invention, the preparation side of beans taro polysaccharide Method is to comprise the following steps:
1), the preparation of beans taro polysaccharide crude:
According to 1g:4~6ml (preferably 1g:Solid-liquid ratio 5ml) is first extracted after mixing beans taro and distilled water (enzyme deactivation is also achieved while extraction), is then beaten, then centrifuges (4000r/min centrifuges 15min), will centrifuge the supernatant of gained The 1/4~1/6 of liquid concentrated by rotary evaporation most original volume, obtains concentrate;
Under stirring condition, ethanol (straight alcohol) is added in the concentrate of gained to final concentration of 80 ± 5% (body of ethanol Product %), precipitation is collected after 24 ± 2h is stood at 4 ± 1 DEG C, obtains beans taro Thick many candies;
2), the purifying of beans taro Thick many candies, is followed the steps below successively:
A, activated carbon decolorizing:
Beans taro Thick many candies dissolve with distilled water to (dosage of distilled water need to only can guarantee that the dissolving of beans taro Thick many candies i.e. Can), beans taro Thick many candies solution is obtained, is filtered after activated carbon stirring is added in beans taro Thick many candies solution;
With what is filtered after the above-mentioned addition activated carbon of the above-mentioned beans taro Thick many candies solution repetition of the filtrate replacement of gained and stirring Decolorization, until the filtrate of final gained is in colourless, polysaccharide crude solution after must decolourizing;
B, Sevage methods take off albumen:
Sevage reagents are added in polysaccharide crude solution after decolouring, 1000 ± 100 turns/min turns in magnetic stirring apparatus Speed 30 ± 5min of stirring, is then centrifuged for (4000r/min centrifuges 25~30min), removes positioned at the water layer of lower floor and positioned at upper strata Solvent layer between albuminate;The Sevage reagents are by n-butanol:Chloroform=1:2.5~3.5 (preferably 1:3) body Product ratio is obtained by mixing;
Polysaccharide crude solution repeats above-mentioned addition after substituting above-mentioned decolouring with the gains after the removing albuminate of gained Sevage reagents, magnetic agitation, centrifugation, the de- protein process 4~6 times for removing albuminate, the polysaccharide that must remove protein is molten Liquid;
The polysaccharide solution concentrated by rotary evaporation of protein will be removed until being the 8~12% of original volume, by concentrate in -70 ~-90 DEG C of 5~7h of pre-freeze, are then dried to powdered with vacuum freeze drier, obtain beans taro polysaccharide.
The improvement of application of the beans taro polysaccharide in liver cell lipidosis is reduced as the present invention:
In the step 1) of the preparation method:
It is to handle 80~120min in 90 ± 5 DEG C to extract (enzyme deactivation extraction);
First extracted and then be beaten after substituting the above-mentioned addition distilled water of beans taro repetition with the filter residue of centrifugation gained, again The process 1~3 time (preferably 2 times) of centrifugation, merge the supernatant concentrated by rotary evaporation obtained by all centrifugations to original volume 1/4~ 1/6 (preferably 1/5), obtains concentrate.
The further improvement of application of the beans taro polysaccharide in liver cell lipidosis is reduced as the present invention:
In the preparation method step 2) A:
The mass ratio of beans taro Thick many candies and activated carbon is 18~22:1 (preferably 20:1) 45, are mixed in 80 ± 10 DEG C Filtered after ± 5min.
Remarks:The number for repeating decolorization is 2~4 times.
The further improvement of application of the beans taro polysaccharide in liver cell lipidosis is reduced as the present invention:
In the preparation method step 2) B:
The volume ratio of polysaccharide crude solution and Sevage reagents is 2.5~3.5 after decolouring:1 (preferably 3:1).
The more sugar types of beans taro of the present invention are single, are spherical shape state of aggregation, relative molecular mass 3187Da.Containing β- Glucosides is good for and pyranose ring, is 1.79 also containing glucose, fructose and xylose, this three's mol ratio of glucose, fructose, xylose: 1:0.03。
The present invention is tested using HepG2 cells (human liver cancer cell), and it is more to determine beans taro by tetrazolium bromide (MTT) method Influence of the sugar processing to HepG2 cell-proliferation activities, and using oleic acid (OA) induction 24h structure cytolipins sedimentation model simultaneously It is subject to beans taro polysaccharide and intervenes 24h, passes through oil red O stain, measure HepG2 intracellular triglycerides (TC), T-CHOL (TG) Content further verifies that beans taro polysaccharide lowering fat and protecting liver acts on.As a result show, beans taro polysaccharide can significantly reduce HepG2 cells Interior triglycerides (TC), T-CHOL (TG) content, alleviate the HepG2 cytolipins deposition of oleic acid induction.
The invention discloses application of the beans taro polysaccharide in liver cell lipidosis is reduced, the application of beans taro has been expanded Field.The invention discloses the HepG2 cytolipins deposition that described beans taro polysaccharide can reduce oleic acid induction.The present invention's Beans taro polysaccharide can be developed into medicine, the health food of natural reduction liver cell lipidosis, instead of artificial synthesized medicine Treat disease.
The usage of the beans taro polysaccharide of the present invention is oral, dosage about 150~250mg every time, three times a day.
The present invention has following technical advantage:
1. present invention firstly discovers that beans taro polysaccharide can effectively reduce the HepG2 cytolipins deposition of oleic acid induction, tool There is good DEVELOPMENT PROSPECT.
2. the present invention provides new medical application for beans taro polysaccharide, a new application field has been expanded.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is that cell viability compares after various concentrations beans taro polysaccharide acts on HepG2 cells 24h.
Fig. 2 is the influence that various concentrations beans taro polysaccharide acts on the intracellular fat drips of HepG2 induced after 24h oleic acid (OA) (100 μm) comparison diagram.
In Fig. 2,
A represents control group (DMEM high glucose medium cellar cultures cell);
B representative models group (oleic acid final concentration 0.5mM);
C represents positive controls (oleic acid final concentration 0.5mM+10uM Bezafibrates);
D represents low concentration beans taro polysaccharide treatment group, and (oleic acid final concentration 0.5mM+ final concentration 100ug/mL beans taros are more Sugar);
(oleic acid final concentration 0.5mM+ final concentration 200ug/mL beans taros are more for concentration beans taro polysaccharide treatment group during E is represented Sugar);
F represents high concentration beans taro polysaccharide treatment group, and (oleic acid final concentration 0.5mM+ final concentration 300ug/mL beans taros are more Sugar).
Fig. 3 is that various concentrations beans taro polysaccharide acts on the intracellular fat depositions of HepG2 induced after 24h oleic acid (OA) Influence comparison diagram.
Fig. 4 is that various concentrations beans taro polysaccharide acts on the HepG2 intracellular triglycerides induced after 24h oleic acid (OA) (TG) the influence comparison diagram of content.
Fig. 5 is that various concentrations beans taro polysaccharide acts on the intracellular T-CHOLs of HepG2 induced after 24h oleic acid (OA) (TC) the influence comparison diagram of content.
In Fig. 3~Fig. 5:
C represents control group (DMEM high glucose medium cellar cultures cell);
OA representative models group (oleic acid final concentration 0.5mM);
BEZ represents positive controls (oleic acid final concentration 0.5mM+10uM Bezafibrates);
L represents low concentration beans taro polysaccharide treatment group, and (oleic acid final concentration 0.5mM+ final concentration 100ug/mL beans taros are more Sugar);
Concentration beans taro polysaccharide treatment group (oleic acid final concentration 0.5mM+ final concentration 200ug/mL beans taros polysaccharide) during M is represented;
H represents high concentration beans taro polysaccharide treatment group, and (oleic acid final concentration 0.5mM+ final concentration 300ug/mL beans taros are more Sugar).
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This:
Embodiment 1, a kind of preparation method of beans taro polysaccharide, are followed the steps below successively:
1), the preparation of beans taro polysaccharide crude:
According to 1g:5ml solid-liquid ratio, weigh beans taro 100g and add 500mL distilled water, 90 DEG C of water-bath 80min are carried Take and (enzyme deactivation is also achieved while extraction), be then beaten, then centrifuge (4000r/min centrifuges 15min), the upper of gained will be centrifuged Clear liquid filters and collects filtrate;
Substituted with the filter residue of centrifugation gained after beans taro repeats above-mentioned addition distilled water and first carry out extraction process (90 DEG C of water-baths 2h) and then mashing, the process that centrifuges again 2 times, merge 3 centrifugations and filter the filtrate concentrated by rotary evaporations of gained to the 1/5 of original volume, Obtain concentrate.
Under stirring condition, ethanol (straight alcohol) is added in the concentrate of gained to ethanol final concentration of 80%, at 4 DEG C Precipitation is collected after standing 24h, obtains beans taro Thick many candies;
2), the purifying of beans taro Thick many candies, is followed the steps below successively:
A, activated carbon decolorizing:
Beans taro Thick many candies are placed in beaker and dissolve that (dosage of distilled water need to only can guarantee that beans taro is thick more with distilled water Sugar dissolving), beans taro Thick many candies solution is obtained, activated carbon is added in beans taro Thick many candies solution in 80 ± 10 DEG C of water-baths Filtered after middle stirring 45min;The mass ratio of beans taro Thick many candies and activated carbon is 20:1;
With what is filtered after the above-mentioned addition activated carbon of the above-mentioned beans taro Thick many candies solution repetition of the filtrate replacement of gained and stirring Decolorization 3 times, now the filtrate of final gained is in colourless, polysaccharide crude solution after must decolourizing;
B, Sevage methods take off albumen:
According to 3:1 volume ratio, 100mL Sevage reagents, Yu Ci are added in polysaccharide crude solution after 300mL decolourings 1000 turns/min rotating speeds stir 30min in power agitator, are then centrifuged for (4000r/min centrifuges 25~30min), are leaked by liquid separation Bucket removes the water layer positioned at lower floor and the albuminate between the solvent layer on upper strata;The Sevage reagents are by n-butanol: Chloroform=1:3 volume ratio is obtained by mixing;
Polysaccharide crude solution repeats above-mentioned addition after substituting above-mentioned decolouring with the gains after the removing albuminate of gained Sevage reagents, magnetic agitation, centrifugation, the de- protein process 5 times of albuminate is removed, the polysaccharide solution of protein must be removed;
The polysaccharide solution concentrated by rotary evaporation for removing protein is obtained into concentrate (about the 10% of original volume), by concentrate In -80 DEG C of pre-freeze 6h, then with vacuum freeze drier 48h (technological parameter that vacuum freeze drier is set as -40 DEG C, Powdered (moisture content≤0.1%) 1.2Pa) is dried into, obtains beans taro polysaccharide.
The more sugar types of gained beans taro are single, are spherical shape state of aggregation, relative molecular mass 3187Da, contain β-glucosides Strong and pyranose ring, containing glucose, fructose and xylose, three's mol ratio is 1.79:1:0.03.
Experiment one, effect of the beans taro polysaccharide to HepG2 cells
The HepG2 cells of culture are inoculated in 96 orifice plates, per 5000, hole cell, are placed in 37 DEG C, 5%CO2In incubator It is grouped at random after being incubated 24h, i.e. blank control group, 5,10,20,40,80,160,320,640,1280ug/mL beans taro polysaccharide Group, every group of hole of repetition 8, is placed in 37 DEG C, 5%CO2After being incubated 24h jointly in incubator, residual liquid in hole is suctioned out, uses phosphate Buffer solution (PBS) solution rinses twice.Then 100ul MTT (0.5mg/mL is dissolved in not serum-containing media) is added per hole, in 37 DEG C, 5%CO2Continue culture 4 hours in incubator.Then, remove per boreliquid, 100ul dimethyl sulfoxide (DMSO)s are added per hole (DMSO), shaken 10 minutes on horizontal shaker, ELIASA determines its light absorption value at 570nm.
Cell-proliferation activity (%)=experimental port OD values/control wells OD values * 100%;
According to Fig. 1, MTT experiment result is shown, when beans taro polysaccharide concentration is in 0-1280ug/mL, processing 24 is small Shi Hou, cell viability no significant difference compared with control group, show to be beans taro polysaccharide safe concentration in 0-1280ug/mL.
The influence of the intracellular fat depositions of HepG2 induced after experiment two, beans taro polysaccharide effect 24h oleic acid (OA)
The HepG2 cells of culture are inoculated in 6 orifice plates, per hole 4*105Individual cell, it is placed in 37 DEG C, 5%CO2Incubated in incubator Be grouped at random after educating 24h, i.e., control group (DMEM high glucose medium cellar cultures cell), model group (oleic acid final concentration 0.5mM), (oleic acid final concentration 0.5mM+ is whole for positive controls (oleic acid final concentration 0.5mM+10uM Bezafibrates), beans taro polysaccharide treatment group Concentration 100ug/mL, 200ug/mL, 300ug/mL beans taro polysaccharide), every group is in triplicate, and each group cell continues after cultivating 24h Nutrient solution is absorbed, PBS is softly washed 3 times, and the paraformaldehyde that 500ul 4% is separately added into per hole fixes cell, stands 20min. PBS softly washs cell 3 times, and 0.5% oil red O stain 30min is added per hole.PBS washings cell 3 times, under inverted microscope Observation is taken pictures, and 500ul isopropanols are added per hole, and room temperature shaker extracts 200ul extracts after extracting 15 minutes and determined at 510nm Light absorption value is quantified.
It can be seen from Fig. 2, compared with control group, oleic acid, which is handled, causes intracellular fat drips quantity and size substantially to increase, and adds Enter Bezafibrate and compared with oleic acid induction group, each treatment group can significantly drop after the beans taro polysaccharide effect 24h of various concentrations Low intracellular fat drips quantity and size content.Quantitative result is as shown in figure 3, quantitative analysis results again show that beans taro polysaccharide table Revealing significantly reduces liver cell fat deposition effect.
The influence of HepG2 intracellular triglycerides (TG) content that experiment three, beans taro polysaccharide are induced oleic acid
The HepG2 cells of culture are inoculated in 6 orifice plates, per hole 4*105Individual cell, it is placed in 37 DEG C, 5%CO2Incubated in incubator Be grouped at random after educating 24h, i.e., control group (DMEM high glucose medium cellar cultures cell), model group (oleic acid final concentration 0.5mM), (oleic acid final concentration 0.5mM+ is whole for positive controls (oleic acid final concentration 0.5mM+10uM Bezafibrates), beans taro polysaccharide treatment group Concentration 100ug/mL, 200ug/mL, 300ug/mL beans taro polysaccharide), every group is in triplicate, and each group cell continues after cultivating 24h Harvesting, cell pyrolysis liquid is taken after ultrasonication, testing cassete (build up bioengineering and grind by Nanjing according to triglycerides (TG enzyme process) Study carefully institute) method measure intracellular triglyceride content.
It can be seen from Fig. 4, compared with control group, oleic acid, which is handled, causes intracellular triglyceride content significantly to raise, and adds After the beans taro polysaccharide effect 24h of Bezafibrate and various concentrations compared with oleic acid induction group, each treatment group can significantly reduce Intracellular triglyceride (TG) content, beans taro polysaccharide show to significantly reduce the effect of liver cell lipidosis.
The influence of the intracellular T-CHOLs of HepG2 (TC) content that experiment four, beans taro polysaccharide are induced oleic acid
The HepG2 cells of culture are inoculated in 6 orifice plates, per hole 4*105Individual cell, it is placed in 37 DEG C, 5%CO2Incubated in incubator Be grouped at random after educating 24h, i.e., control group (DMEM high glucose medium cellar cultures cell), model group (oleic acid final concentration 0.5mM), (oleic acid final concentration 0.5mM+ is whole for positive controls (oleic acid final concentration 0.5mM+10uM Bezafibrates), beans taro polysaccharide treatment group Concentration 100ug/mL, 200ug/mL, 300ug/mL beans taro polysaccharide), every group is in triplicate, and each group cell continues after cultivating 24h Harvesting, cell pyrolysis liquid is taken after ultrasonication, (bioengineering is built up in Nanjing to testing cassete according to T-CHOL (TCH enzyme process) Research institute) the intracellular total cholesterol level of method measure.
It can be seen from Fig. 5, compared with control group, oleic acid, which is handled, causes intracellular total cholesterol level significantly to raise, and adds After the beans taro polysaccharide effect 24h of Bezafibrate and various concentrations compared with oleic acid induction group, each treatment group can significantly reduce Intracellular T-CHOL (TC) content, beans taro polysaccharide show to significantly reduce the effect of liver cell lipidosis.
Comparative example 1-1,90% will be changed to by 80% to ethanol final concentration in the step 1) of embodiment 1, remaining is equivalent In embodiment 1.
Comparative example 1-2,70% will be changed to by 80% to ethanol final concentration in the step 1) of embodiment 1, remaining is equivalent In embodiment 1.
Comparative example 2-1, by the beans taro Thick many candies in the step 2) of embodiment 1 and the mass ratio of activated carbon by 20:1 is changed to 15:1, remaining is equal to embodiment 1.
Comparative example 2-2, by the beans taro Thick many candies in the step 2) of embodiment 1 and the mass ratio of activated carbon by 20:1 is changed to 25:1, remaining is equal to embodiment 1.
Comparative example 3-1, by the beans taro Thick many candies polysaccharide crude solution and Sevage reagent bodies in the step 2) of embodiment 1 Product ratio is by 3:1 is changed to 2:1, remaining is equal to embodiment 1.
Comparative example 3-2, by the beans taro Thick many candies polysaccharide crude solution and Sevage reagent bodies in the step 2) of embodiment 1 Product ratio is by 3:1 is changed to 4:1, remaining is equal to embodiment 1.
Beans taro polyoses extract obtained by above-mentioned all comparative examples is examined according to the above-mentioned method of experiment two, three, four Look into, acquired results are as shown in table 1 below.
Table 1
Finally, it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure All deformations for directly exporting or associating, are considered as protection scope of the present invention.

Claims (5)

1. application of the beans taro polysaccharide in liver cell lipidosis is reduced.
2. application of the beans taro polysaccharide according to claim 1 in liver cell lipidosis is reduced, it is characterized in that beans taro The preparation method of flower polysaccharide is to comprise the following steps:
1), the preparation of beans taro polysaccharide crude:
According to 1g:4~6ml solid-liquid ratio is first extracted after mixing beans taro and distilled water, is then beaten, then is centrifuged, will The 1/4~1/6 of the supernatant concentrated by rotary evaporation most original volume of gained is centrifuged, obtains concentrate;
Under stirring condition, ethanol is added in the concentrate of gained to ethanol final concentration of 80 ± 5%, 24 are stood at 4 ± 1 DEG C Precipitation is collected after ± 2h, obtains beans taro Thick many candies;
2), the purifying of beans taro Thick many candies, is followed the steps below successively:
A, activated carbon decolorizing:
Beans taro Thick many candies are dissolved with distilled water, obtain beans taro Thick many candies solution, adds and lives in beans taro Thick many candies solution Property charcoal stirring after filter;
The decolouring filtered after the above-mentioned addition activated carbon of above-mentioned beans taro Thick many candies solution repetition and stirring is substituted with the filtrate of gained Process, until the filtrate of final gained is in colourless, polysaccharide crude solution after must decolourizing;
B, Sevage methods take off albumen:
Sevage reagents are added in polysaccharide crude solution after decolouring, 1000 ± 100 turns/min rotating speeds stir in magnetic stirring apparatus 30 ± 5min is mixed, is then centrifuged for, removes the water layer positioned at lower floor and the albuminate between the solvent layer on upper strata;It is described Sevage reagents are by n-butanol:Chloroform=1:2.5~3.5 volume ratio is obtained by mixing;
Polysaccharide crude solution repeats above-mentioned addition after substituting above-mentioned decolouring with the gains after the removing albuminate of gained Sevage reagents, magnetic agitation, centrifugation, the de- protein process 4~6 times for removing albuminate, the polysaccharide that must remove protein is molten Liquid;
The polysaccharide solution concentrated by rotary evaporation of protein will be removed until being the 8~12% of original volume, by concentrate in -70~-90 DEG C 5~7h of pre-freeze, is then dried to powdered with vacuum freeze drier, obtains beans taro polysaccharide.
3. application of the beans taro polysaccharide according to claim 2 in liver cell lipidosis is reduced, it is characterized in that:
In the step 1) of the preparation method:
It is extracted as handling 80~120min in 90 ± 5 DEG C;
Substituted with the filter residue of centrifugation gained after beans taro repeats above-mentioned addition distilled water and first extracted and then be beaten, centrifuged again Process 1~3 time, merge the supernatant concentrated by rotary evaporation obtained by all centrifugations to the 1/4~1/6 of original volume, obtain concentrate.
4. application of the beans taro polysaccharide according to claim 3 in liver cell lipidosis is reduced, it is characterized in that:
In the preparation method step 2) A:
The mass ratio of beans taro Thick many candies and activated carbon is 18~22:1, filtered after mixing 45 ± 5min in 80 ± 10 DEG C.
5. application of the beans taro polysaccharide according to claim 4 in liver cell lipidosis is reduced, it is characterized in that:
In the preparation method step 2) B:
The volume ratio of polysaccharide crude solution and Sevage reagents is 2.5~3.5 after decolouring:1.
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CN109112108A (en) * 2018-09-18 2019-01-01 南京财经大学 A kind of construction method of HepG2 cell hyperlipidemia model and application
WO2023121314A1 (en) * 2021-12-21 2023-06-29 한국한의학연구원 Use of apios americana tuber extract for protection against alcoholic liver damage or alcoholic brain damage

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