CN103393785A - Method for preparing malus baccata leaf alcohol extract, and antioxidation and liver protection effects thereof - Google Patents

Method for preparing malus baccata leaf alcohol extract, and antioxidation and liver protection effects thereof Download PDF

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CN103393785A
CN103393785A CN2013102759661A CN201310275966A CN103393785A CN 103393785 A CN103393785 A CN 103393785A CN 2013102759661 A CN2013102759661 A CN 2013102759661A CN 201310275966 A CN201310275966 A CN 201310275966A CN 103393785 A CN103393785 A CN 103393785A
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dwarf apple
alcohol extract
extract
dwarf
apple
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郑毅男
丁传波
赵婷
李永娟
董岭
陈大勇
张传奇
刘文丛
李伟
弓晓杰
高飞飞
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DALIAN JIEXIN BIOLOGICAL TECHNOLOGY Co Ltd
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DALIAN JIEXIN BIOLOGICAL TECHNOLOGY Co Ltd
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Abstract

The invention provides a method for extracting and preparing a malus baccata leaf alcohol extract, and applications thereof on antioxidation and acute liver injury, the method is characterized by: weighting malus baccata leaves dried in the shade, crushing, adding 30-99% ethanol in a material and liquid ratio of 1:25-1:30, ultrasonically extracting for 30-90min in conditions of 40 DEG C, 40Hz and 250W, extracting for 1-5 times, filtrating and merging extracts, pressure-reduction condensing until to be a cream, and freeze-drying to obtain the malus baccata leaf alcohol extract. The invention further discloses the malus baccata leaf alcohol extract, which is rich in flavones and phlorizin, has antioxidation and free radical removal functions, has substantial improvement and protection effects for acute liver injury, makes effects on prevention, treatment and auxiliary treatment of hepatopathy, and particularly has a substantial effect on acute liver injury caused by peroxy like alcohol stimulation.

Description

A kind of preparation method of dwarf apple alcohol extracts from the leaves and the protective effect of antioxidation regulating liver-QI thereof
Technical field
The present invention relates to a kind of preparation of dwarf apple alcohol extracts from the leaves and the protective effect of playing and antioxidation in acute liver damage.Belong to technical field of pharmaceuticals.
Background technology
Dwarf apple [Malus baccata (Linn.) Borkh.], another name Malus baccata (Linn.) Borkh., woods Fructus Vitics Simplicifoliae, Malus baccata etc., it is Rosaceae (Rosaceae) Malus (Malus) perennial woody plant, mainly be distributed in the ground such as China northeast, North China and southwest, also there is distribution [1] in Korea and Russian Siberia.Present research concentrates on mainly that its greening is viewed and admired and the effect aspect [2-3] such as drought resisting, for the rare report of the research of its chemical composition.The volatile oil component of flower of Malus baccata (L.) Borkh. and leaf is mainly terpenoid, esters and alkenes compounds, and main component is farnesene [4].And polyphenol and flavone compound are the main active substances of dwarf apple, wherein phlorhizin is one of its pharmacological component [5,6], it is a kind of polyphenol compound that is distributed widely in Fructus Mali pumilae, Fructus Mali pumilae bark and leaf and Pasania cuspidata Folium hydrangeae strigosae tender leaf, have the multiple pharmacologically actives such as antioxidation [7], adjusting blood glucose [8], removing free radical [9,10].Tea is boiled to be used for fat-reducing with the dwarf apple leaf in a lot of areas of China, and the dwarf apple alcohol extracts from the leaves can suppress the ability of fatty acid synthase, has certain fat-reducing effect [11].At present, for the assay of phlorhizin in dwarf apple leaf minority bibliographical information [12] only, in the dwarf apple leaf, flavones content there is not yet report.The present invention adopts the HPLC method to measure the content of phlorhizin in the dwarf apple leaf and flavone, and has measured the antioxidant activity of dwarf apple alcohol extracts from the leaves.The liver-protecting activity of dwarf apple leaf extract there is not yet report.The present invention causes the liver-protecting activity of acute liver model determination dwarf apple alcohol extracts from the leaves by CCl4.
Antioxidative Activity Determination [13, the 14.15] .CCl4 that CCl4 induces liver injury model to be widely used in plant extract changes on cell membrane, this can allow intracellular enzyme disequilibrium [16].Toxic metabolite product C Cl3 is further converted to the trichloromethyl peroxy radical by the P4502E1 enzyme, this covalent compound can cause the degraded of peroxidation of cellular membranes, cause that membrane permeability raises, cause ALT in endochylema to be released in blood in a large number, thus the integrity [17] of damage liver plasma membrane 26S Proteasome Structure and Function.The present invention is by showing at external FTC and the experiment of TBA method mensuration antioxidation, and the dwarf apple alcohol extract can suppress the peroxidization that linoleic acid forms, and can remove well DPPH and ABTS free radical.Mice is induced in the hepatic injury experiment in body, and model group mice MDA content, ALT, AST are active significantly raises, and the dwarf apple alcohol extract of variable concentrations can reduce MDA content and ALT, AST activity, and hepatic injury due to carbon tetrachloride is had to certain protective role.
SOD is a kind of antioxidase, and it plays important effect in the hepatic injury that prevention is caused by free radical.GSH, be a kind of non-enzymatic antioxidant, can decompose the oxygen G&W to hydrogen peroxide, formed the first line of defence [18] of opposing free radical.In the present invention, by the mouse liver injury group of tetrachloro-methane induction, can be found out, GSH content, the active obviously reduction of SOD, and, in various dose administration group, all can improve GSH content and SOD activity in liver.This shows that the dwarf apple alcohol extract can improve the content of the interior oxidase of hepatocyte and non-oxide enzyme, shields to liver by this approach.
Summary of the invention
The invention provides a kind of extraction preparation method of dwarf apple folic alcohol extract, be applicable to suitability for industrialized production.
A kind of dwarf apple folic alcohol extract provided by the invention, wherein take phlorhizin, flavone as main active substances.
A kind of dwarf apple folic alcohol extract provided by the invention, the effect of this dwarf apple alcohol extract to having antioxidation, removing free radical.
A kind of dwarf apple folic alcohol extract provided by the invention, this dwarf apple alcohol extract has significant improvement and protective effect to acute liver damage.
The present invention further discloses dwarf apple folic alcohol the extract application in prevention, treatment and auxiliary treatment hepatopathy, especially ethanol stimulation etc. due to the acute liver damage that peroxidating causes, have significant effect.
The preparation method of dwarf apple folic alcohol extract provided by the invention, concrete technical scheme is as follows:
Take the dwarf apple leaf after drying in the shade, pulverize, by feed liquid mass ratio 1: 25-1: 30 add the ethanol of 30-99%, at 40 ℃, supersound extraction 30-90min under the condition of 40Hz, 250W, extract 1-5 time, filter merge extractive liquid,, be evaporated to the extractum shape, lyophilization obtains dwarf apple folic alcohol extract.
The hepatoprotective effect of dwarf apple folic alcohol extract of the present invention, it is characterized in that making take phlorhizin and flavone as main effective ingredient.In the present invention, we have measured the protective effect of dwarf apple ethanol extraction antioxidation activity in vitro and the interior mouse liver injury to tetrachloro-methane induction of body.Result shows, the dwarf apple ethanol extraction has a stronger antioxidant activity external.In vivo, when dosage reached 300mg/kg, its mouse liver injury to tetrachloro-methane induction had protective effect preferably.This activity of dwarf apple ethanol extraction may be summed up flavone and polyphenol compound in the inner, its protection mechanism may with remove free radical, alleviate lipid peroxidation, the integrity of Cell protection 26S Proteasome Structure and Function, lower MDA level in hepatocyte, improve SOD activity and GSH content; Reduce ALT in hepatocyte, AST content is relevant
The invention provides the preparation method of this dwarf apple folic alcohol extract, it is characterized in that:
(1) take the dwarf apple leaf after drying in the shade, remove impurity and pulverize.
(2) add the ethanol of the 30%-99% that 25-30 doubly measures, with supersound extraction 30-90min, extract 1-5 time, centrifugal filtration, remove residue, obtains filtrate.
(3) filtrate is being reclaimed ethanol below 25 ℃, and remaining extract lyophilization, make extractum or dry powder.
(4) packing namely obtains dwarf apple folic alcohol extract.
Good effect of the present invention is: take the dwarf apple leaf as raw material, adopt conventional extracting method, and easy being easy to get, dwarf apple folic alcohol extract provided by the invention has protective effect to acute liver damage, can be applicable in liver protecting and health therapy.Dwarf apple folic alcohol extract provided by the invention has antioxidation, for the disease relevant with antioxidation especially hepatic injury, has auxiliary therapeutic action.
For further illustrating effect and the antioxidation of dwarf apple leaf extract of the present invention to acute liver damage, below provide assay and the acute liver damage the pharmacological results of related substances in dwarf apple folic alcohol extract.
Phlorhizin content comparative measurements in experimental example 1 dwarf apple alcohol extracts from the leaves
1 experimental apparatus and material
LC-20A type high performance liquid chromatograph (Japanese Shimadzu, the SPD-20A UV-detector,) ultrasonic cleaner (KQ-250DB, Kunshan ultrasonic instrument company limited), BP211D electronic balance (German Sartorious company), automatic dual pure water distillator SZ-93 (Shanghai Yarong Biochemical Instrument Plant) UV-2450 ultraviolet spectrophotometer (Japanese SHI-MADZU company) etc.
Dwarf apple leaf, fruit are Rosaceae (Rosaceae) Malus (Malus) dwarf apple, the reference substance phlorhizin, rutin, be purchased from Chinese pharmaceutical biological product and check institute, and vitamin C (VC), ABTS, DPPH are purchased from Sigma company, BHT (2, the 6-di-tert-butyl-4-methy phenol), linoleic acid is purchased from West Asia reagent company, acetonitrile (chromatographically pure, U.S. Fisher company), pure water is the laboratory self-control, and all the other reagent are analytical pure.
Dwarf apple folic alcohol extract: adopt method in embodiment 2 to process take above-mentioned dwarf apple leaf as raw material.
2 extracting method
The reference substance solution precision takes phlorhizin reference substance 10.00mg, is placed in the 100mL volumetric flask with fixed molten after dissolve with methanol, 0.45 μ m filtering with microporous membrane, and can make concentration is 0.1mgmL -1Phlorhizin reference substance storing solution.
The need testing solution precision takes dwarf apple folic alcohol extract 1.0060g, put into the conical flask of 250mL, add respectively 50mL methanol, at 40 ℃, under the condition of 40HZ, 250W, extract 30min, extract 2 times, merge after extracted twice liquid filters and be settled in the volumetric flask of 100mL, 0.45 μ m filtering with microporous membrane, namely obtain the test sample storing solution.
3 chromatographic conditions
Chromatographic column: Dalian Yi Lite HypersilODS2 (250mm * 4.6mm, 5 μ m); Column temperature: 30 ℃; Mobile phase: acetonitrile-water (25: 75); Flow velocity: 1.0mLmin -1Detect wavelength: 285nm; Sample size: 20 μ l.Under above-mentioned chromatographic condition, reference substance and sample chromatogram figure are shown in Fig. 1, and the phlorhizin retention time in sample and reference substance is basically identical, and theoretical cam curve is all greater than 3000, and the retention time of phlorhizin is 7.123min.
In 4 results sample, the phlorhizin assay is got 3 batch samples, the results are shown in Table 1.
Table 1 sample size measurement result (%)
In dwarf apple folic alcohol extract, phlorhizin content is 4.8%.
In experimental example 2 dwarf apple folic alcohol extracts and dwarf apple fruit alcohol extract, flavones content is measured
1 experimental apparatus and material
Reference substance: other conditions of rutin are with experimental example 1
2 methods
The dwarf apple folic alcohol extract 1.0g that the preparation of need testing solution and rutin standard solution takes gained in embodiment 2 adds 75% ethanol water standardize solution in the 100mL volumetric flask, namely obtains sample solution.Accurately take in rutin standard substance (105 degree be dried to constant weight) 10mg to 100mL volumetric flask, add appropriate 75% dissolve with ethanol, standardize solution, being mixed with mass concentration is the rutin solution of 0.1mgmL-1.Get a certain amount of above-mentioned rutin solution preparation and become mass concentration to be respectively 0.00,0..01, the rutin solution of 0.02,0.03,0.04,0.05mgmL-1.Set up the rutin standard curve, by standard curve, the content of flavone in working sample.
In dwarf apple folic alcohol extract, flavones content is measured the dwarf apple leaf extract need testing solution 1.0mL that accurately draws, be placed in respectively 10mL tool plug test tube, add 5% NaNO2 solution 0.3mL, shake up, after placement 6min, add 10%Al (NO3) 3 solution 0.3mL, shake up, after placing 6min, add 4%NaOH solution 4mL, shake up, with 75% ethanol standardize solution, after 15min, under 510nm, measure absorbance.
3 results
The measurement result of flavone in table 2 dwarf apple leaf and fruit
Presentation of results, in dwarf apple folic alcohol extract, flavones content is higher, is 19.1mgg -1
The carbon tetrachloride of experimental example 3 dwarf apple alcohol extracts from the leaves causes the protective effect of acute liver
1 sample preparation methods
Dwarf apple folic alcohol extract (MBE) is embodiment 2 method gained.
1 animal SPF level male ICR mouse (18-22g).The raising condition is 23 ± 2 ℃ of temperature, and humidity is 55 ± 5%, between the laundering period, freely ingests and drinks water, and feedstuff is normal diet.The preparation of dwarf apple alcohol extracts from the leaves is according to embodiment 1 preparation;
2. animal grouping and administration are divided into 6 groups at random by mice, every group 10, be respectively: the positive group of blank group, model control group, bifendate, dwarf apple alcohol extracts from the leaves high, medium and low (500,300,100mg/kg) dosage group, blank group and model group gavage are in 5% CMC-Na, each administration group administration 14d, after the 14d administration finishes, except the blank group, the CCl4 (CCl4 dilutes with olive oil) that each organizes equal lumbar injection 10%, set up acute hepatic injury model.After lumbar injection, water is can't help in fasting, after 6h, wins eyeball, collects blood, and after 30min, at 4200r/min, centrifugal 10min under 4 ℃, suck serum.Extract liver and spleen, with the normal saline of ice, clean the blood on internal organs, after drying, weigh.Serum, liver and spleen are preserved with every biochemical indicator to be detected in-70 ℃ of refrigerators.
3. the mensuration mice serum of biochemical indicator, press the test kit explanation and measure ALT and AST activity with SpectraMax Plus384 continuous spectrum scan-type microplate reader.Get the hepatic tissue of each mouse liver same area, with 4 ℃ of normal saline 1: 9 in mass ratio, the liver homogenate of preparation 10%, 4200r/min, centrifugal 10min under 4 ℃, get supernatant, press the test kit explanation and measure MDA, SOD and GSH activity with SpectraMax Plus384 continuous spectrum scan-type microplate reader.
4. statistical analysis adopts the SPSS17.0 statistical software to carry out statistical analysis, adopts one factor analysis of variance (One-way ANOVA) to carry out a sample multiple comparison analyse, and result represents with x ± s.P<0.05, expression has statistical significance.
5. the activity influence of MBE to ALT, AST in CCl4 hepatic injury mice serum as a result
With the blank group, compare, ALT, AST in the model group mice serum significantly raise, and with the blank group, utmost point significant difference (P<0.001) are arranged, and have illustrated that this experiment modeling is more successful.With model group, compare, in positive controls (bifendate administration) and each administration group, can obviously reduce the rising (P<0.01 or P<0.001) that is caused the ALT level by CCl4, and high dose group (500mg/kg) causes that for CCl4 AST decreases, but there is no statistical significance, but low dose group (100mg/kg) and middle dosage group (300mg/kg) have obviously reduced the level (P<0.01, P<0.001) that AST raises.Shown that dwarf apple leaf flavone has protective effect (table 1) to the hepatic injury that is caused by CCl4.
The impact (x ± s, n=6) of table 3MBE on ALT, AST activity in CCl4 hepatic injury mice serum
Annotate: " ### " compares P<0.001 with the blank group; " * * " compares P<0.01, " * * * " P<0.001 with model group
MBE affects shown in table 2 CCl4 hepatic injury murine liver tissue SOD activity and MDA and GSH content, with the blank group, compare, active obviously reduce (P<0.001) of CCl4 inducing mouse hepatic injury group (model group) SOD, MDA content obviously raises (P<0.001), and GSH content obviously reduces (P<0.001).With model group, compare, administration group high dose and middle dosage SOD activities of liver, GSH content obviously raises, and the low dosage SOD activities of liver, although GSH content raises to some extent, does not have significant difference.The reduction level of MDA, show utmost point significant difference (P<0.001) in middle dosage, high dose decreases with low dose group MDA level, but does not have statistical significance.The result demonstration, MBE has certain liver-protecting activity, and it is better that middle dosage shows, and hepatic injury improves significantly to cell.
The impact (x ± s, n=6) of table 4MBE on SOD activity, GSH and MDA content in CCl4 hepatic injury murine liver tissue
Annotate: " ### " compares P<0.001 with the blank group; " * * " compares P<0.01, " * * * " P<0.001, " * " P<0.05 with model group
In this experimental example, measured the protective effect of dwarf apple ethanol extraction to the mouse liver injury of tetrachloro-methane induction.Result shows, in vivo, when dosage reached 300mg/kg, its mouse liver injury to tetrachloro-methane induction had protective effect preferably to the dwarf apple ethanol extraction.
The antioxidation activity in vitro of experimental example 4 dwarf apple alcohol extracts from the leaves is measured
1. reagent D PPH (2,2-Diphenyl1-1-picrylhydrazyl), ABTS (2,2 '-Azino-bis (3-ethylbenzthiazoline-6-sulphonic acid), VC, all purchased from U.S. Sigma company, linoleic acid is purchased from West Asia reagent company limited.Alanine aminotransferase (ALT), aspartate amino transferase (AST), malonaldehyde (MDA), superoxide dismutase (SOD), glutathion (GSH) test kit all build up bio-engineering research institute purchased from Nanjing, acetonitrile, be purchased from U.S. Fisher company, (PBS: sodium dihydrogen phosphate (NaH2PO42H2O), sodium hydrogen phosphate (Na2HPO412H2O), hydrochloric acid, dehydrated alcohol, methanol are analytical pure for 2,6 ditertiary butyl p cresol (BHT), trichloroacetic acid (TCA), thiobarbituricacidα-(TBA), phosphate buffer.
2 instrument ultrasonic cleaners (KQ-250DB, Kunshan ultrasonic instrument company limited), BP211D electronic balance (German Sartorious company), SpectraMax Plus384 continuous spectrum scan-type microplate reader (U.S. molecule instrument company) etc.
3.DPPH remove free radical activity, measure antioxidation
MBE and VC (contrast) are mixed with to the solution (0.0125,0.125,1.25,2.5 and 5mg/ml) of variable concentrations.DPPH is mixed with to the solution of 2 * 10-4mol/L with dehydrated alcohol.Get the 2ml sample solution, add 7.5ml DPPH solution, concussion, shake up.Under room temperature, react 1h, under 517nm, measure its absorbance.Free radical scavenging activity=[(As-Ai)]/As * 100, in formula, As represents blank absorbance, Ai represents the absorbance of sample, VC.The experiment triplicate, the mapping of averaging.
4.ABTS remove free radical activity, measure antioxidation
With deionized water, be mixed with the ABTS solution of 7mmol/L and the potassium persulfate solution of 2.45mmol/L, get potassium persulfate solution 5ml, join in 15mlABTS solution, at room temperature be placed in dark place's reaction 16h, form ABTS free radical storing solution.Under 734nm, with volume fraction, be that 75% ethanol is diluted to absorbance by ABTS free radical storing solution and is 0.7 ± 0.05, standby.Accurately draw the sample solution of 0.8ml variable concentrations (0.019,0.19,1.9,3.85,7.7mg/ml), add 7ml ABTS+ solution, concussion mixes, and at room temperature reacts 30min, and under 734nm, measures its absorbance.Blank replaces sample solution with the ethanol that volume fraction is 75%.The absorbance of sample is A1, and the absorbance of blank is A2.The experiment triplicate, the mapping of averaging.
ABTS free radical scavenging activity (%)=(A2-A1)/A2 * 100
Linoleic acid emulsification system-ammonium thiocyanate (FTC) method
50mg dwarf apple leaf extract is joined in 130ml80% ethanol, get the linoleic acid ethanol solution that then 2ml adds 2ml2.5%, then add 2ml distilled water (sample A), sealing.Blank group replaces the dwarf apple leaf extract with dehydrated alcohol, and under 40 ℃ of dark, constant temperature is preserved.Measured once in every 24 hours, during mensuration, the linoleic acid emulsion of drawing 0.1ml joins in blank test tube, the ethanol that adds subsequently 4.7ml75%, the ammonium thiocyanate solution of 0.1m130%, 0.1ml0.02mol/L are dissolved in the solution of ferrous chloride of 3.5% HCl, after mixing rapidly, concussion.After 3min, under 500nm, measure absorbance.The experiment triplicate, the mapping of averaging.
TBA (thiobarbituricacidα-) measures the oxidation resistance of MBE
Get the sample A1ml in " 2.2.3 ", add 20% trichloroacetic acid 2ml, after 0.67% TBA mixes and in boiling water bath, heat 15min, cooling under room temperature after taking out.At 4200r/min, centrifugal 10min, get supernatant under 532nm, the absorbance of working sample.The experiment triplicate, the mapping of averaging.
5. result
(1) DPPH removes free radical activity
As can be seen from Figure 1, MBE is more remarkable to the removing effect of DPPH free radical, when the concentration of MBE is 0.0125mg/ml, its clearance rate is 57.5%, obviously, IC50<the 0.0125mg/ml of MBE to the DPPH free radical scavenging, the free radical scavenging activity of natural antioxidant VC is 86.7% under identical concentration.Along with the increase of MBE concentration, the clearance rate of its free radical increases gradually, when reaching certain concentration, tends to balance.The radical scavenging activity of MBE is slightly lower than VC.
(2) ABTS removes free radical activity
ABTS, under suitable oxidant effect, can be oxidized to cyan ABTS free radical, when having antioxidant to exist, can suppress the generation of ABTS free radical, and when sample and ABTS free-atom aqueous solution mixed, green shoaled or disappears, and Wheat Protein is described.Fig. 2 shows, along with the increase of MBE and BHT concentration, its ABTS free radical scavenging activity is also along with increase, when the concentration of BHT is increased to 0.19mg/ml, its clearance rate is 92.4%, increase the concentration of BHE, its clearance rate, changing, does not show that the ABTS free radical is eliminated substantially again, and under this concentration, the clearance rate of MBE is 66.5%, and its IC50<0.19mg/ml. shows that MBE has stronger antioxidant activity, and this may to contain more polyphenol compound relevant with it.
(3) the .FTC method is measured oxidation resistance
Automatic oxidation reaction can occur in linoleic acid, during reaction, can produce a large amount of peroxide, can be oxidized to ferric ion to ferrous ion, and ferric ion can generate red chelate with ion generation chelatropic reaction with Hydrogen thiocyanate, its chelate has stronger absorption under 500nm.Absorbance is higher, and the peroxide that generates when linoleic acid peroxidation reacts is more, shows that oxidation resistance is more weak.As can be seen from Figure 3, in the time of the 3rd day, the absorbance of blank group starts to rise, and MBE, VC, BHT absorbance are very steady.In the time of the 5th day, sample MBE absorbance starts to increase, and the blank absorbance increases sharply on the same day, and VC and BHT absorbance are comparatively steady, remain between 1.3-1.4.In the time of the 8th day, the absorbance of MBE, VC, BHT starts to descend, and may be because formed peroxide is very unstable as the initial stage product of lipid oxidation, and the absorbance of blank group in the time of the 8th day has increases trend slowly.Show that MBE has stronger antioxidant activity.
(4) .TBA (thiobarbituricacidα-) measures the oxidation resistance of MBE
Peroxidization is because oils and fats contains unsaturated fatty acid, linoleic acid especially, and it,, under metal ion such as Fe2+, Cu2+ effect, can accelerate grease oxidation rancid.And antioxidant can have the peroxidization that suppresses lipid.MDA is the product of lipid peroxidation, and itself and thiobarbituric acid reaction can generate red product.Stronger absorption is arranged under 532nm, absorbance is larger, shows that degree of oxidation is stronger, and antioxidant activity is more weak.Fig. 4 shows, VC has stronger antioxidant activity, the antioxidant activity of MBE than VC a little less than.With blank, compare, MBE has stronger antioxidation.
The protective effect of the interior mouse liver injury to tetrachloro-methane induction of dwarf apple ethanol extraction antioxidation activity in vitro and body.The liver injury protection effect of dwarf apple ethanol extraction may be summed up flavone and polyphenol compound in the inner, its protection mechanism may with remove free radical, alleviate lipid peroxidation, the integrity of Cell protection 26S Proteasome Structure and Function, lower MDA level in hepatocyte, improve SOD activity and GSH content; Reduce ALT in hepatocyte, AST content is relevant.
The accompanying drawing explanation
Fig. 1. the DPPH of dwarf apple leaf extract removes free radical activity measurement result figure
Fig. 2. the ABTS of dwarf apple leaf extract removes free radical activity measurement result figure
The antioxidant activity of Fig. 3 .FTC method mensuration MBE is figure as a result
The antioxidant activity of Fig. 4 .TBA method mensuration MBE is figure as a result
The specific embodiment
For the ease of understanding the present invention, especially exemplified by following examples.Its effect is understood to be to explaination of the present invention but not to any type of restriction of the present invention.
The preparation of embodiment 1 dwarf apple folic alcohol extract
Take the dwarf apple leaf 0.1kg after drying in the shade, pulverize, add 30% ethanol 2.5L, at 40 ℃, supersound extraction 30min under the condition of 40HZ, 250W, extract 3 times, merges three times extracting solution.Be evaporated to the extractum shape, lyophilization obtains dwarf apple folic alcohol extract.
The preparation of embodiment 2 dwarf apple folic alcohol extracts
(1) take the dwarf apple leaf 0.1kg after drying in the shade, remove impurity and pulverize.
(2) 70% the ethanol 2.5L that adds, with at 40 ℃, under the condition of 40HZ, 250W, supersound extraction 30min, extract 3 times, and residue is removed in centrifugal filtration, obtains filtrate.
(3) filtrate is being reclaimed ethanol below 25 ℃, and remaining extract lyophilization, make extractum or dry powder.
(4) packing namely obtains dwarf apple folic alcohol extract.
The preparation of embodiment 3 dwarf apple folic alcohol extracts
1) take the dwarf apple leaf 0.1kg after drying in the shade, remove impurity and pulverize.
2) add 90% ethanol 2.5L, with at 40 ℃, under the condition of 40HZ, 250W, supersound extraction 30min, extract 3 times, and residue is removed in centrifugal filtration, obtains filtrate.
3) filtrate is being reclaimed ethanol below 25 ℃, and remaining extract lyophilization, make extractum or dry powder.
4) packing namely obtains dwarf apple folic alcohol extract.
The preparation of embodiment 4 dwarf apple folic alcohol extracts
(1) take the dwarf apple leaf 0.1kg after drying in the shade, remove impurity and pulverize.
(2) 70% the ethanol 3L that adds, with at 40 ℃, under the condition of 40HZ, 250W, supersound extraction 60min, extract 3 times, and residue is removed in centrifugal filtration, obtains filtrate.
(3) filtrate is being reclaimed ethanol below 25 ℃, and remaining extract lyophilization, make extractum or dry powder.
(4) packing namely obtains dwarf apple folic alcohol extract.
The preparation of embodiment 5 dwarf apple folic alcohol extracts
(1) take the dwarf apple leaf 0.1kg after drying in the shade, remove impurity and pulverize.
(2) 70% the ethanol 3L that adds, with at 40 ℃, under the condition of 40HZ, 250W, supersound extraction 90min, extract 3 times, and residue is removed in centrifugal filtration, obtains filtrate.
(3) filtrate is being reclaimed ethanol below 25 ℃, and remaining extract lyophilization, make extractum or dry powder.
(4) packing namely obtains dwarf apple folic alcohol extract.
The preparation of embodiment 6 dwarf apple folic alcohol extracts
(1) take the dwarf apple leaf 0.1kg after drying in the shade, remove impurity and pulverize.
(2) 100% the ethanol 3L that adds, with at 40 ℃, under the condition of 40HZ, 250W, supersound extraction 90min, extract 3 times, and residue is removed in centrifugal filtration, obtains filtrate.
(3) filtrate is being reclaimed ethanol below 25 ℃, and remaining extract lyophilization, make extractum or dry powder.
(4) packing namely obtains dwarf apple folic alcohol extract.
(5) take the dwarf apple leaf after drying in the shade, pulverize, by feed liquid mass ratio 1: 25-1: 30 add the ethanol of 30-99%, at 40 ℃, supersound extraction 30-90min under the condition of 40HZ, 250W, extract 1-5 time, filters merge extractive liquid,, be evaporated to the extractum shape, lyophilization obtains dwarf apple folic alcohol extract.
The preparation of embodiment 7 dwarf apple folic alcohol extracts
(1) take the dwarf apple leaf 0.1kg after drying in the shade, remove impurity and pulverize.
(2) 70% the ethanol 2.7L that adds, with at 40 ℃, under the condition of 40HZ, 250W, supersound extraction 30min, extract 5 times, and residue is removed in centrifugal filtration, obtains filtrate.
(3) filtrate is being reclaimed ethanol below 25 ℃, and remaining extract lyophilization, make extractum or dry powder.
(4) packing namely obtains dwarf apple folic alcohol extract.
The application of embodiment 8 dwarf apple leaf extracts in the protection hepatic injury
In embodiment 1, prepared dwarf apple leaf extract is as the protective agent of acute liver damage, sterilizing, and packing gets final product.Coordinate the medicine dwarf apple folic alcohol extract of other hepatoprotective to play the effect of anti-oxidation protection hepatocyte acute injury.
List of references
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Claims (5)

  1. One kind from the dwarf apple leaf, extracting, the dwarf apple folic alcohol extract of preparation, it is characterized in that for the preparation of dwarf apple leaf concrete grammar:
    (1) take the dwarf apple leaf after drying in the shade, remove impurity and pulverize.
    (2) add the ethanol of the 30%-99% that 25-30 doubly measures, with ultrasonic extraction 30-90min, extract 1-5 time, centrifugal filtration, remove residue, obtains filtrate.
    (3) filtrate is being reclaimed ethanol below 25 ℃, and remaining extract lyophilization, make extractum or dry powder.
    (4) packing namely obtains dwarf apple folic alcohol extract.
  2. 2. according to claim 1, in the present invention, in leaching process, to add solvent be the second alcohol and water to the dwarf apple folic alcohol extract of gained, and ethanol content is 30%-99%.
  3. 3. according to claim 1, gained dwarf apple folic alcohol extract is rich in flavone and phlorhizin, has stronger antioxidation.
  4. 4. according to claim 1, gained dwarf apple folic alcohol extract has the effect of removing DPPH free radical, removing ABTS free radical, suppressing lipid peroxidation.
  5. 5. according to claim 1, gained dwarf apple folic alcohol extract has obviously reduced the level that AST raises, and can the acute liver damage that carbon tetrachloride causes be played a protective role.
CN2013102759661A 2013-06-22 2013-06-22 Method for preparing malus baccata leaf alcohol extract, and antioxidation and liver protection effects thereof Pending CN103393785A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105985883A (en) * 2016-07-02 2016-10-05 威海御膳坊生物科技有限公司 Preparation method of Malus baccata fruit wine
CN109030474A (en) * 2018-06-13 2018-12-18 迦娜生物科技(武汉)有限公司 A kind of lipid free radical urine detection reagent and its application

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