CN107812031A - The preparation method and its antioxidation application of beans taro leaf water extract - Google Patents
The preparation method and its antioxidation application of beans taro leaf water extract Download PDFInfo
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- CN107812031A CN107812031A CN201711176338.2A CN201711176338A CN107812031A CN 107812031 A CN107812031 A CN 107812031A CN 201711176338 A CN201711176338 A CN 201711176338A CN 107812031 A CN107812031 A CN 107812031A
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- China
- Prior art keywords
- beans taro
- water extract
- taro leaf
- eluent
- leaf water
- Prior art date
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- 235000006481 Colocasia esculenta Nutrition 0.000 title claims abstract description 90
- 244000046052 Phaseolus vulgaris Species 0.000 title claims abstract description 89
- 235000010627 Phaseolus vulgaris Nutrition 0.000 title claims abstract description 89
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 88
- 239000000284 extract Substances 0.000 title claims abstract description 84
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 230000003064 anti-oxidating effect Effects 0.000 title description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 69
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 46
- 239000003480 eluent Substances 0.000 claims abstract description 30
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 23
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- 150000003254 radicals Chemical class 0.000 description 11
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- QGZCUOLOTMJILH-UHFFFAOYSA-N 2h-tetrazol-2-ium;bromide Chemical compound [Br-].C1=N[NH+]=NN1 QGZCUOLOTMJILH-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
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- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- LDXJRKWFNNFDSA-UHFFFAOYSA-N 2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound C1CN(CC2=NNN=C21)CC(=O)N3CCN(CC3)C4=CN=C(N=C4)NCC5=CC(=CC=C5)OC(F)(F)F LDXJRKWFNNFDSA-UHFFFAOYSA-N 0.000 description 1
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 1
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- 241000522190 Desmodium Species 0.000 description 1
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- GOMNOOKGLZYEJT-UHFFFAOYSA-N isoflavone Chemical compound C=1OC2=CC=CC=C2C(=O)C=1C1=CC=CC=C1 GOMNOOKGLZYEJT-UHFFFAOYSA-N 0.000 description 1
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- BUIMWOLDCCGZKZ-UHFFFAOYSA-N n-hydroxynitramide Chemical compound ON[N+]([O-])=O BUIMWOLDCCGZKZ-UHFFFAOYSA-N 0.000 description 1
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- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- INAAIJLSXJJHOZ-UHFFFAOYSA-N pibenzimol Chemical compound C1CN(C)CCN1C1=CC=C(N=C(N2)C=3C=C4NC(=NC4=CC=3)C=3C=CC(O)=CC=3)C2=C1 INAAIJLSXJJHOZ-UHFFFAOYSA-N 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 235000012950 rattan cane Nutrition 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
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- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
-
- 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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/31—Extraction of the material involving untreated material, e.g. fruit juice or sap obtained from fresh plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of preparation method of beans taro leaf water extract, comprise the following steps：1) mashing, ultrasound extraction, centrifugation, obtain supernatant after, mixing beans taro leaf and water；2), supernatant rotary evaporation is concentrated；3), concentrate is isolated and purified using macroreticular resin；Using the aqueous formic acid of volumetric concentration 1% as eluent, the methanol aqueous solution to contain 0.05% formic acid collects eluent as eluant, eluent；4), eluent rotary evaporation is concentrated, obtains pulpous state concentrate；5), by pulpous state concentrate elder generation pre-freeze, then it is dried into vacuum freeze drier powdered, obtains beans taro leaf water extract.The present invention also provides application of the above-mentioned beans taro leaf water extract in Oxidative Damage in Liver inhibitor is prepared simultaneously.
Description
Technical field
The present invention relates to field of medicaments, and in particular to a kind of preparation method of beans taro leaf water extract and its it is anti-oxidant should
With.
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 content of vitamin E highest of beans taro leaf, while also containing higher
Content polysaccharide, total saposins and isoflavones, there is great potentiality to be exploited.
U.S. beans taro has obvious improvement to lifestyle-related diseases such as hypertension, diabetes and hyperlipemias.
Free radical be by material after being stimulated by external condition caused atom or group, and with very active
Unpaired electron, easy and other atoms or group combine, and form stable structure.Human body can be with during metabolism
Free radical is produced, mainly including ultra-oxygen anion free radical, hydroxy radical, peroxy radical, hyponitric acid and nitric oxide etc.,
Cause a series of biological respinse.When human body is under normal physiological condition, internal radicals scavenging system (is also referred to as antioxygen
Change system) excessive free radical can be removed, make cell and tissue from the injury sexual assault of free radical；But in pathological conditions
Under, the generation system and removing system disequilibrium of human endogenous's free love base, cause free radical superfluous.Superfluous free radical meeting
Induced oxidation stress phenomenon, oxidative stress can cause large biological molecule (such as lipid, protein and DNA) oxidative damage, cause
A series of biologically, cause inflammation, cancer, cardiovascular disease, senile dementia (Alzheimer) and aging etc. are chronic to move back
The generation of row disease.
Antioxidant has the ability of Scavenger of ROS and oxygen radical, is that one kind can prevent or delay autoxidation
Material, very important effect is played in terms of the normal activities of body are maintained.Antioxidant can remove internal surplus
Free radical, make interior free yl generation system and removing system keep dynamic equilibrium.Epidemiology shows that antioxidant exists
Vital effect is played in terms of prevention chronic disease.Free radical can induced oxidation stress reaction, supplement enough anti-
Oxidant can effectively prevent or delay this reaction, can effectively protect human body cell from the damage caused by oxidative stress,
So as to reduce the illness rate of the chronic diseases such as diabetes, angiocardiopathy and cancer.More and more research shows, cereal, water
Antioxidant in fruit and vegetables can prevent or reduce this potential oxidative damage.Ingest cereal, fruits and vegetables and drop
The risk of the diseases such as low cancer stricken has the correlation of height.Therefore, the researcher of many nutrition and health care food is at present
Research and topic about the generation of anti-oxidant and chronic disease and prevention are paid special attention to, but for the anti-oxidant of beans taro leaf extract
Activity is rarely reported.
《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.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation method of beans taro leaf water extract and its anti-oxidant use
On the way, be medical science, the field such as Food Science exploitation natural new resources are provided.
In order to solve the above-mentioned technical problem, the present invention provides a kind of preparation method of beans taro leaf water extract, including following
Step：
1), according to 1g:5~10ml (preferably 1g:Solid-liquid ratio 8ml) is beaten after mixing beans taro leaf and water, gained slurry
Liquid extract solution centrifugation (4000 turns/min centrifuges 30min), obtains supernatant after 50 ± 5 DEG C of 240 ± 10min of ultrasound extraction；
2) supernatant, is subjected to rotary evaporation concentration until being the 5~10% of original volume in Rotary Evaporators；
3), the concentrate obtained by step 2) is isolated and purified using macroreticular resin；It is water-soluble with the formic acid of volumetric concentration 1%
For liquid as eluent, the methanol aqueous solution to contain 0.05% formic acid collects eluent as eluant, eluent；
The preparation method of the methanol aqueous solution containing 0.05% formic acid is：In the methanol-water of 100ml volumetric concentrations 90%
0.05ml formic acid is added in solution；
4) the eluent rotary evaporation obtained by step 3), is condensed into the 5~10% of most original volume, obtains pulpous state concentration
Liquid；
5), by prior to -70~-90 DEG C 5~7h of pre-freeze of the pulpous state concentrate obtained by step 4), vacuum freeze drying is then used
Machine is dried into powdered, obtains beans taro leaf water extract.
Improvement as the preparation method of the beans taro leaf water extract of the present invention：
The step 3) is：
Macroreticular resin is activated with the methanol of twice of column volume first, the distilled water of three times column volume is balanced；Then with
(applied sample amount is 100~200ml to 0.4~0.6mL/min (preferably 0.5mL/min) flow velocity loading, i.e. about the 10 of column volume
~20%) after, liquid to be extracted is fully adsorbed, eluted (elution by the use of the aqueous formic acid of volumetric concentration 1% as eluent
Purpose is to remove the materials such as protein, sugar, acid), the dosage of the eluent is twice of column volume, flow velocity is 0.4~
0.6mL/min (preferably 0.5mL/min)；Finally carried out by the use of the methanol aqueous solution for containing 0.05% formic acid as eluant, eluent abundant
Elution, the dosage three times column volume of the eluant, eluent, flow velocity are 0.4~0.6mL/min (preferably 0.5mL/min)；Collection is washed
De- liquid.
Further improvement as the preparation method of the beans taro leaf water extract of the present invention：
Macroreticular resin in the step 3) is macroreticular resin S-8 (Tianjin Xing Nanyun energy Polymer Technologies Co., Ltd).
Further improvement as the preparation method of the beans taro leaf water extract of the present invention：
By the filter residue alternative steps 1 of step 1) centrifugation gained) in beans taro leaf repeat being beaten of step 1), ultrasound leaching
Carry and centrifuge；Number of repetition is 2~3；
Step 2) is carried out after supernatant obtained by all centrifugations is merged.
The beans taro leaf water extract that the present invention also provides above method preparation gained simultaneously is preparing liver cell oxidation damage
Hinder the application in inhibitor.
In the present invention：
The rotary evaporation of step 2) and step 4) is carried out under 38 ± 2 DEG C, -0.09MPa vacuum.
The vacuum freeze drying of step 5) is carries out under -40 ± 2 DEG C, 1.2Pa vacuum, until the beans taro leaf of gained
Water extract moisture content≤0.1%.
The present invention is tested using HepG2 cells (human liver cancer cell), and beans taro leaf water is determined by tetrazolium bromide (MTT) method
Influence of the extract-treated to HepG2 cell-proliferation activities, and after protecting 24h in advance using beans taro leaf water extract, added
Hydrogen oxide (H2O2) induction 2h structure cellular oxidation Stress models, inquire into protection of the beans taro leaf water extract to cell oxidative damage
Effect.I.e. the invention provides beans taro leaf water extract to prepare by hydrogen peroxide (H2O2) induction Oxidative Damage in Liver suppression
Application in preparation.
The beans taro leaf water extract of the present invention, can be developed into natural antioxidant drug and food, instead of artificial synthesized
Antioxidant therapy because people's interior free yl is excessive and caused by disease.
The usage of the beans taro leaf water extract of the present invention is oral, and dosage is by taking tablet as an example, about 150~250mg every time, often
Day is three times.
This beans taro leaf water extract being prepared using the inventive method can be protected effectively by hydrogen peroxide (H2O2) lure
The Oxidative Damage in Liver led, the generation of active oxygen is reduced, there is good DEVELOPMENT PROSPECT.The present invention is beans taro leaf water extract
New medical application is provided, has expanded a new application field.
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 various concentrations beans taro leaf water extract to hydrogen peroxide (H2O2) induction hepatoma cell proliferation vigor shadow
Ring comparison diagram；
Fig. 2 is various concentrations beans taro leaf water extract to hydrogen peroxide (H2O2) induction Morphology of Hepatocellular Carcinoma influence figure；
Fig. 3 is various concentrations beans taro leaf water extract to hydrogen peroxide (H2O2) induction liver cancer cells cell nuclear damagies
Influence figure；
Fig. 4 is to carry out quantitative analysis results figure to fluorescence intensity in Fig. 3；
Fig. 5 is various concentrations beans taro leaf water extract to hydrogen peroxide (H2O2) induction liver cancer cells active oxygen radical
The influence of (DHE fluorescence)；
Fig. 6 is to carry out quantitative analysis results figure to fluorescence intensity in Fig. 5；
Fig. 7 is various concentrations beans taro leaf water extract to hydrogen peroxide (H2O2) induction liver cancer cells active oxygen radical
The influence of (DCF fluorescence)；
Fig. 8 is to carry out quantitative analysis results figure to fluorescence intensity in Fig. 7；
Fig. 9 is various concentrations beans taro leaf water extract to hydrogen peroxide (H2O2) induction liver cancer cells mitochondrial membrane potential
Influence figure；
Figure 10 is to carry out quantitative analysis results figure to fluorescence intensity in Fig. 9；
Figure 11 is various concentrations beans taro leaf water extract to hydrogen peroxide (H2O2) induction liver cell mitochondria membrane lipid mistake
The influence comparison diagram of oxidation；
Figure 12 is to carry out quantitative analysis results figure to fluorescence intensity in Figure 11；
Figure 13 is various concentrations beans taro leaf water extract to hydrogen peroxide (H2O2) induction liver cell glutathione consumption
Influence comparison diagram；
Figure 14 is to carry out quantitative analysis results figure to fluorescence intensity in Figure 13；
Special instruction：In Fig. 2,3,5,7,9,11 and 13, a. control groups；b.H2O2Model group；c.H2O2+ low concentration beans
Taro leaf water extract group (L)；d.H2O2+ middle concentration beans taro leaf water extract group (M)；e.H2O2+ high concentration beans taro leaf water extract
Group (H)；In Fig. 4,6,8,10,12 and 14, Control represents control group, H2O2Model group is represented, L represents H2O2+ low concentration
Beans taro leaf water extract group, H represent H2O2+ middle concentration beans taro leaf water extract group, M represent H2O2+ high concentration beans taro leaf water extracts
Thing group.
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 leaf water extract, are followed the steps below successively：
1), with 1g:8ml ratio mixes beans taro leaf and water, is beaten using juice extractor；Gained slurries are in 50 DEG C of bars
Under part, after ultrasonic (40,000Hz ultrasonic frequencies) extraction 240min, 4000 turns/min of extract solution centrifugation 30min,
The beans taro leaf that the filter residue of centrifugation gained is substituted in above-mentioned steps repeat being beaten of above-mentioned steps, ultrasound extraction and
Centrifugation；Number of repetition is 3 times；
Following step 2 is carried out after the supernatant of above-mentioned 3 times centrifugation gained is merged).
2), by the supernatant of gained, in Rotary Evaporators, (technological parameter that rotary evaporation is set is 38 DEG C of temperature, vacuum
Degree -0.09MPa) rotary evaporation concentration is carried out until being the 10% of original volume；
3), revolving gained concentrate is isolated and purified using macroreticular resin, used macroreticular resin is macropore tree
Fat S-8 (Tianjin Xing Nanyun energy Polymer Technologies Co., Ltd), is followed the steps below successively：
Macroreticular resin is activated with the methanol of twice of column volume first, three times column volume distilled water is balanced；
Then with 0.5mL/min flow velocity loading, applied sample amount is 200ml (for the 10% of column volume)；Extract solution is fully inhaled
Attached, 1% formic acid (i.e. the aqueous formic acid of volumetric concentration 1%) elution removes the materials such as protein, sugar, acid, the use of the eluent
Measure as twice of column volume, flow velocity 0.5mL/min；It is finally abundant as eluant, eluent by the use of the methanol aqueous solution for containing 0.05% formic acid
Elution, the dosage three times column volume of the eluant, eluent, flow velocity 0.5mL/min；Collect eluent.
The preparation method of the methanol aqueous solution containing 0.05% formic acid is：In the methanol-water of 100ml volumetric concentrations 90%
0.05ml formic acid is added in solution；
4), macroreticular resin is isolated and purified into gained eluent rotary evaporation (technological parameter that rotary evaporation is set is temperature
38 DEG C, vacuum -0.09MPa) rotary evaporation concentration is carried out until being the 10% of original volume, obtain pulpous state concentrate；
5), by concentrate in -80 DEG C of pre-freeze 6h, then with vacuum freeze drier 48h, (vacuum freeze drier is set
Technological parameter is -40 DEG C, 1.2Pa) powdered (moisture content≤0.1%) is dried into, obtain beans taro leaf water extract.
Experiment one, beans taro leaf water extract are to hydrogen peroxide (H2O2) induction hepatocyte growth vigor influence：
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 after being incubated 24h, i.e. blank control group, H2O2Group, 10,20,40,80,160,320,640,1280ug/mL beans taro leaf water extractions
Thing group is taken, 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 phosphorus
Blank control group adds normal nutrient solution, H after the rinsing twice of phthalate buffer (PBS) solution2O2Group, 10,20,40,80,160,
320th, 640,1280ug/mL adds H2O2Final concentration of 150uM nutrient solution continues to cultivate 2h.Then residual solution in each hole is suctioned out
Body, with the rinsing of phosphate buffer (PBS) solution, (0.5mg/mL is dissolved in without serum free culture system addition 100ul MTT afterwards twice
Base), in 37 DEG C, 5%CO2Continue culture 4 hours in incubator.Then, remove per boreliquid, 150ul dimethyl is added per hole
Sulfoxide (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, in H2O2The beans taro leaf of various concentrations is added on the basis of induced damage
Water extract carry out preculture, find cell survival rate be better than model of oxidative, wherein 20,40,80,160,320,640,
Cell survival rate is significantly better than damage model and with the cellular control unit survival rate being without damage without notable under 1280ug/mL concentration
Sex differernce, it was demonstrated that beans taro leaf water extract is non-toxic to HepG2 cells in 0~1280ug/mL concentration ranges, and is advantageous to
Cellular damage reparation.
Experiment two, beans taro leaf water extract are to hydrogen peroxide (H2O2) induction liver cell form influence：
The HepG2 cells of exponential phase are selected, cell monolayer are digested with 0.25% pancreatin, with containing 10% new cow's serum
RPIM1640 culture mediums are made into individual cells suspension, with 1 × 106The density in individual/hole is inoculated in 6 orifice plates, is put in incubator and is trained
Suction out culture medium after supporting 24h, be respectively 50 with concentration, 100,200ug/mL (corresponding to low concentration, middle concentration, high concentration respectively)
Beans taro leaf water extract handles 24h, then uses H2O2(150 μM of hydrogen peroxide final concentration) processing cell 2h induced oxidations stress cell
Model, then with micro- sem observation cellular morphology and take pictures.
It can be seen from Fig. 2, compared with control group, model group has been reduced in cell quantity within the vision, cytoplasm
Dehydration concentration, nucleus volume increase, cell volume reduce, show the feature of Apoptosis, show H2O2The successful structure of model
Build inducing cell and oxidative damage occurs, and then cause Apoptosis.The beans taro leaf water extract matter for adding various concentrations is carried out in advance
The cell of culture then shows more normal cellular morphology under the conditions of oxidative stress, and with the continuous lifting of concentration, it is high
The cellular morphology of concentration culture group is substantially similar with control group, it was demonstrated that beans taro leaf water extract has to cell oxidative damage to be repaired
And protective effect.
Experiment three, beans taro leaf water extract are to hydrogen peroxide (H2O2) induction hepatocyte cell nuclear damage influence：
The HepG2 cells of exponential phase are selected, cell monolayer are digested with 0.25% pancreatin, with containing 10% new cow's serum
RPIM1640 culture mediums are made into individual cells suspension, with 1 × 106The density in individual/hole is inoculated in 6 orifice plates, is put in incubator and is trained
Suction out culture medium after supporting 24h, be respectively 50 with concentration, 100,200ug/mL beans taro leaf water extract handles 24h, then uses H2O2
(150 μM of hydrogen peroxide final concentration) processing cell 2h induced oxidations stress cell model, be separately added into final concentration of 10 μM
Hoechst 33258 dyes 30min, and dyestuff with fluorescence microscope and is taken pictures after cleaning, and calculates average optical density value.
It can be seen from Fig. 3, compared with control group, the nucleus brightness of model group dramatically increases, and forms fine and close bright indigo plant
Color fluorescence, blue agglomerate fragmentation, chromatin condensation occur highlighted.What is showed in fig 3b is particularly evident, shows Apoptosis
Occur.And pass through the group of beans taro leaf water extract pretreatment, nucleus fluorescence intensity occurs different journeys compared to model group
Degree ground is reduced, and nuclear shapes rule, nuclear membrane is complete, blue-fluorescence even dispersion, cell shape of the trend that shrinkage slows down with Fig. 2
State is consistent, shows beans taro leaf water extract to H2O2The nucleus oxidative damage of induction has protective effect.Fig. 4 is strong to its fluorescence
Degree is quantified, the results showed that beans taro leaf water extract can reduce nucleus fluorescence intensity, alleviate cell nuclear damage.
Experiment four, beans taro leaf water extract are to hydrogen peroxide (H2O2) induction hepatocyte activity oxygen radical (DHE fluorescence)
Influence：
The HepG2 cells of exponential phase are selected, cell monolayer are digested with 0.25% pancreatin, with containing 10% new cow's serum
RPIM1640 culture mediums are made into individual cells suspension, with 1 × 106The density in individual/hole is inoculated in 6 orifice plates, is put in incubator and is trained
Suction out culture medium after supporting 24h, be respectively 50 with concentration, 100,200ug/mL beans taro leaf water extract handles 24h, then uses H2O2
(150 μM of hydrogen peroxide final concentration) processing cell 2h induced oxidations stress cell model, be separately added into 10 μM of DHE dyeing
30min, dyestuff with fluorescence microscope and is taken pictures after cleaning, and calculates average optical density value.
It can be seen from Fig. 5, compared with control group, the fluorescence intensity of model group significantly increases, and semi-quantitative value is in five groups
Peak.And the group Jing Guo beans taro leaf water extract preculture, fluorescence intensity are substantially reduced, and as extract concentrations increase
Add, H2O2ROS contents caused by induction are reduced, and fully demonstrating beans taro leaf water extract can effective scavenging capacity oxygen radical
(ROS).Each treatment group quantitative result is shown in Fig. 6.
Experiment five, beans taro leaf water extract are to hydrogen peroxide (H2O2) induction liver cancer cells active oxygen radical (DCF is glimmering
Light) influence
The HepG2 cells of exponential phase are selected, cell monolayer are digested with 0.25% pancreatin, with containing 10% new cow's serum
RPIM1640 culture mediums are made into individual cells suspension, with 1 × 106The density in individual/hole is inoculated in 6 orifice plates, is put in incubator and is trained
Suction out culture medium after supporting 24h, be respectively 50 with concentration, 100,200ug/mL leaf water extract handles 24h, then uses H2O2(peroxide
Change 150 μM of hydrogen final concentration) processing cell 2h induced oxidations stress cell model, be separately added into 10 μM of DCF dyeing 30min, dye
Material with fluorescence microscope and is taken pictures after cleaning, and calculates average optical density value.
It can be seen from Fig. 7, compared with control group, the fluorescence intensity of model group significantly increases, and carries out fluorescence in Fig. 8 respectively and determines
Amount analysis result shows that model group numerical value is the peak in five groups.And the group Jing Guo beans taro leaf water extract preculture, fluorescence
Intensity is substantially reduced, and with the increase of water extract concentration, H2O2ROS contents caused by induction are reduced, and trend is the same as in Fig. 5 and Fig. 6
DHE fluorescence is consistent, and fully demonstrating beans taro leaf water extract can effective scavenging capacity oxygen radical (ROS).
Experiment six, beans taro leaf water extract are to hydrogen peroxide (H2O2) induction liver cell mitochondria film potential influence：
The HepG2 cells of exponential phase are selected, cell monolayer are digested with 0.25% pancreatin, with containing 10% new cow's serum
RPIM1640 culture mediums are made into individual cells suspension, with 1 × 106The density in individual/hole is inoculated in 6 orifice plates, is put in incubator and is trained
Suction out culture medium after supporting 24h, be respectively 50 with concentration, 100,200ug/mL beans taro leaf water extract handles 24h, then uses H2O2
(150 μM of hydrogen peroxide final concentration) processing cell 2h induced oxidations stress cell model, then using 5 μ g/mL mitochondrial membranes electricity
Position probe Rhodamine 123 (RH123) fluorescence probe carries out dyeing 30min, and dyestuff with fluorescence microscope and is taken pictures after cleaning,
And calculate average optical density value.
It can be seen from Fig. 9, compared with control group, the fluorescence intensity of model group is substantially less than control group, visible in the visual field
Cell quantity also substantially reduces, and shows that oxidative stress causes mitochondrial membrane potential in anoxic to decline, mitochondrial matrix Volume Changes,
ATP is reduced, and final mitochondrial function is unable to maintain that and causes cell death.Add beans taro leaf water extract and carry out the thin of preculture
Born of the same parents still remain horizontal better than the mitochondrial membrane potential of model group in quantity under the conditions of oxidative stress, show beans taro leaf water extraction
Take thing can injury of mitochondria effectively caused by inhibitory activity oxygen, and in the range of safe concentration, inhibition same-action concentration
Positive correlation.Each treatment group quantitative result is shown in Figure 10.
Experiment seven：Beans taro leaf water extract is to hydrogen peroxide (H2O2) induction liver cell mitochondria membrane lipid peroxidatio
Influence：
The HepG2 cells of exponential phase are selected, cell monolayer are digested with 0.25% pancreatin, with containing 10% new cow's serum
RPIM1640 culture mediums are made into individual cells suspension, with 1 × 106The density in individual/hole is inoculated in 6 orifice plates, is put in incubator and is trained
Suction out culture medium after supporting 24h, be respectively 50 with concentration, 100,200ug/mL beans taro leaf water extract handles 24h, then uses H2O2
(150 μM of hydrogen peroxide final concentration) processing cell 2h induced oxidations stress cell model, be separately added into 10 μM of NAO dyeing
30min, dyestuff with fluorescence microscope and is taken pictures after cleaning, and calculates average optical density value.
According to Figure 11, compared with control group, the mitochondria quantity of model group and beans taro leaf water extract culture group in the visual field
It has been reduced that, but average fluorescent strength is constantly lifted with the rise of extract concentrations, there is conspicuousness compared with model group
Enhancing, trend are consistent with the coloration result of Rh123 probes, it was demonstrated that beans taro leaf water extract can effectively alleviate the fat of mitochondrial membrane
Matter peroxidating situation.Each treatment group quantitative result is shown in Figure 12.
Experiment eight, beans taro leaf water extract are to hydrogen peroxide (H2O2) induction liver cancer cells glutathione consumption influence
The HepG2 cells of exponential phase are selected, cell monolayer are digested with 0.25% pancreatin, with containing 10% new cow's serum
RPIM1640 culture mediums are made into individual cells suspension, with 1 × 106The density in individual/hole is inoculated in 6 orifice plates, is put in incubator and is trained
Suction out culture medium after supporting 24h, be respectively 50 with concentration, 100,200ug/mL beans taro leaf water extract handles 24h, then uses H2O2
(150 μM of hydrogen peroxide final concentration) processing cell 2h induced oxidations stress cell model, be separately added into 10 μM of NDA dyeing
30min, dyestuff with fluorescence microscope and is taken pictures after cleaning, and calculates average optical density value.
It can be seen from Figure 13 and Figure 14, the cell through the pretreatment of beans taro leaf water extract is in H2O2In the case of a large amount of appearance,
Fluorescence intensity is significantly higher than undressed model group, shows on the one hand the material effectively reduces the generation of free radical and causes paddy
The consumption of the sweet peptide of Guang is reduced, fluorescence intensity increase；On the other hand so that glutathione molecules are still protected under the conditions of oxidative stress
Hold higher activity and be reduced ability, can routinely play radicals scavenging effect.Meanwhile the same lipid of GSH contents
Extent of peroxidation is relevant, and the decline of its content can cause the generation of lipid peroxidation, therefore Figure 13 fluorescence intensity trend also confirms that
The continuous lifting of GSH contents, the anti-oxidation efficacy of beans taro leaf water extracts has been confirmed again and has suppressed glutathione consumption
Positive acting.
Comparative example 1-1, by the " 1g in the step 1) of embodiment 1:8ml solid-liquid ratio " makes " 1g into:10ml solid-liquid ratio ", its
It is remaining to be equal to embodiment 1.
Comparative example 1-2, by the " 1g in the step 1) of embodiment 1:8ml solid-liquid ratio " makes " 1g into:5ml solid-liquid ratio ", its
It is remaining to be equal to embodiment 1.
Comparative example 2-1, the eluant, eluent in the step 3) of embodiment 1 made into by " methanol aqueous solution containing 0.05% formic acid "
" methanol aqueous solution (methanol aqueous solution of volumetric concentration 90%) "；Remaining is equal to embodiment 1.
Comparative example 2-2, the eluant, eluent in the step 3) of embodiment 1 made into by " methanol aqueous solution containing 0.05% formic acid "
" methanol aqueous solution (methanol aqueous solution of volumetric concentration 90%) containing 0.1% formic acid "；Remaining is equal to embodiment 1.
" methanol aqueous solution containing 0.05% formic acid " as eluant, eluent in comparative example 3-1, the step 3) of embodiment 1,
Make the volumetric concentration of methanol aqueous solution into 80% by 90%；Remaining is equal to embodiment 1.
" methanol aqueous solution containing 0.05% formic acid " as eluant, eluent in comparative example 3-2, the step 3) of embodiment 1,
Make the volumetric concentration of methanol aqueous solution into 100% (that is, pure methanol) by 90%；Remaining is equal to embodiment 1.
Comparative example 4, macroreticular resin is made into macroporous absorbent resin AB-8 by macroreticular resin S-8, remaining is equal to embodiment
1。
Beans taro leaf water extract obtained by above-mentioned all comparative examples is checked according to the above-mentioned method of experiment one, four, five,
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. the preparation method of beans taro leaf water extract, it is characterized in that comprising the following steps：
1), according to 1g:5~10ml solid-liquid ratio is beaten after mixing beans taro leaf and water, and gained slurries are in 50 ± 5 DEG C of ultrasound extractions
After 240 ± 10min, extract solution centrifugation, supernatant is obtained；
2) supernatant, is subjected to rotary evaporation concentration until being the 5~10% of original volume in Rotary Evaporators；
3), the concentrate obtained by step 2) is isolated and purified using macroreticular resin；Made with the aqueous formic acid of volumetric concentration 1%
For eluent, the methanol aqueous solution to contain 0.05% formic acid collects eluent as eluant, eluent；
The preparation method of the methanol aqueous solution containing 0.05% formic acid is：In the methanol aqueous solution of 100ml volumetric concentrations 90%
Middle addition 0.05ml formic acid；
4) the eluent rotary evaporation obtained by step 3), is condensed into the 5~10% of most original volume, obtains pulpous state concentrate；
5), then will with vacuum freeze drier, by prior to -70~-90 DEG C 5~7h of pre-freeze of the pulpous state concentrate obtained by step 4)
It is dried to powdered, obtains beans taro leaf water extract.
2. the preparation method of beans taro leaf water extract according to claim 1, it is characterized in that：
The step 3) is：
Macroreticular resin is activated with the methanol of twice of column volume first, the distilled water of three times column volume is balanced；Then with 0.4~
0.6mL/min flow velocity loading, after liquid to be extracted is fully adsorbed, enter by the use of the aqueous formic acid of volumetric concentration 1% as eluent
Row elution, the dosage of the eluent is twice of column volume, and flow velocity is 0.4~0.6mL/min；Finally use and contain 0.05% formic acid
Methanol aqueous solution fully eluted as eluant, eluent, the dosage three times column volume of the eluant, eluent, flow velocity be 0.4~
0.6mL/min；Collect eluent.
3. the preparation method of beans taro leaf water extract according to claim 2, it is characterized in that：
Macroreticular resin in the step 3) is macroreticular resin S-8.
4. the preparation method of beans taro leaf water extract according to claim 3, it is characterized in that：
By the filter residue alternative steps 1 of step 1) centrifugation gained) in beans taro leaf repeat being beaten of step 1), ultrasound extraction and
Centrifugation；Number of repetition is 2~3 times；
Step 2) is carried out after supernatant obtained by all centrifugations is merged.
5. the beans taro leaf water extract that gained is prepared such as Claims 1 to 4 either method is preparing Oxidative Damage in Liver suppression
Application in agent.
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