CN103463145B - The Folium Ginkgo being raw material with Semen Ginkgo fallen leaves refines extract and preparation method thereof and application - Google Patents
The Folium Ginkgo being raw material with Semen Ginkgo fallen leaves refines extract and preparation method thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of Folium Ginkgo being raw material with Semen Ginkgo fallen leaves and refine extract and preparation method thereof, the present invention with Semen Ginkgo fallen leaves for raw material, add alcohol reflux, merge extractive liquid, filter, concentrated, add water and leach, filter, AB-8 resin column absorption on filtrate, after first washing clarification with water, use 90% ~ 95% ethanol desorbing again, collect 90% ~ 95% ethanol elution, polyamide column on concentrated solution, first wash with water, use 70% ~ 75% methanol desorbing again, collect 70% ~ 75% meoh eluate, concentrated, dry, the refining extract of 30% ~ 45% total flavones and 7% ~ 15% total lactones must be contained.The present invention falls leaves as extracting raw material with discarded Semen Ginkgo; turn waste into wealth; obtain containing total flavones and the high refining extract of total lactones active component by selection process; there is good antioxidation; antiplatelet aggregation and neuronal cell protective effect thereof; and untoward reaction is low, can be used as the control medicine of cardiovascular and cerebrovascular disease and the primary raw material of health food, there is important economic benefits and Environmental Role.
Description
Technical field
The present invention relates to the exploitation that a kind of medicinal plants discards tissue, be specifically related to refining extract of a kind of Semen Ginkgo fallen leaves and preparation method thereof and application.
Background technology
Folium Ginkgo is the dried leaves of Ginkgoaceae plant Ginkgo biloba GinkgobilobaL..Folium Ginkgo is mainly containing flavonoid and bilobalide-like active substance, and its extract and preparation thereof are widely used in the diseases such as treatment coronary heart disease, hypertension, cerebral infarction, dementia, asthma, hepatitis B, diabetes clinically.China's gingko resource is very abundant, and its effective ingredient is valuable medical material, can be used for again producing health food.Folium Ginkgo refines the Folium Ginkgo that extract is mainly derived from 4 ~ 7 years age of trees, and the Semen Ginkgo of other age of tree is the fruit leaf of Semen Ginkgo especially, due to the on the low side and not collected utilization of ginkgetin and lactone component content, becomes every November and falls leaves and become garbage.According to first slightly statistics, the whole nation about has the Semen Ginkgo of 7500 tons fallen leaves to go out of use every year, causes the great wasting of resources and environmental pollution.The patent that domestic report has four items to fall leaves about Semen Ginkgo, Chinese patent CN1066595 discloses application Semen Ginkgo fallen leaves and extracts medicinal ketone for curing coronary heart disease, through clinical short-period used, considerable to some chronic disease curative effects such as hypertensive cerebral thrombosis; Chinese patent CN102876721A discloses a kind of method rapidly and efficiently destroying Semen Ginkgo fallen leaves fibre structure, the reacting final product that this invention obtains and Semen Ginkgo fallen leaves go fibre structure to can be used for cell expansion cultivation or the substrate as industrial fermentation, solve the requirement of existing technique disposal reaction condition high, the technical problems such as low conversion rate; Other two use Semen Ginkgo fallen leaves to make organic bio-fertilizer and culture medium of edible fungus respectively.CN102030590A for raw material, makes organic biofertilizer, combination of pesticide and fertilizer, control pathogenic bacteria with plant refuse (Semen Ginkgo fallen leaves etc.) after fermentation; Semen Ginkgo fallen leaves wait and mix with nutrient substance and water by CN102265753A, make culture medium of edible fungus, rationally solve the waste disposal issues of current afforestation.But all do not report and extract total flavones and total lactones content is high, the malicious accessory ingredient content such as ginkgolic acid is few simultaneously, pharmacologically active is strong and close to Semen Ginkgo standard extract and technology of preparing thereof and method.
Therefore, in order to made full use of Semen Ginkgo fallen leaves resource, reduce environmental pollution, necessary with Semen Ginkgo fallen leaves for raw material design research and develop a kind of total flavones and total lactones content high, impurity is few, and untoward reaction is low, and the Folium Ginkgo that pharmacologically active is strong refines extract.
Summary of the invention
Goal of the invention: the object of the invention is to solve the deficiencies in the prior art; screen through great many of experiments; make full use of Semen Ginkgo fallen leaves resource, with Semen Ginkgo fallen leaves for raw material, adopt modern extraction separation method; refining obtain total flavones and total lactones content is high; impurity is few, and untoward reaction is low, and the Folium Ginkgo that pharmacologically active is strong refines extract; another object of the present invention is that the preparation method providing Folium Ginkgo to refine extract is applied with it, has great importance to comprehensive utilization of resources and environmental conservation.
Technical scheme: in order to realize above object, the technical scheme that the present invention takes is:
A kind of Folium Ginkgo refines extract, and it prepares by the following method:
Get Semen Ginkgo fallen leaves, pulverize, add the alcohol reflux 2 to 3 times that 7 to 10 times amount concentration are 70% to 85%, each 2 to 3 hours, merge extractive liquid, filter, decompression recycling ethanol, add water-soluble loose, leave standstill, filter, AB-8 resin column absorption on filtrate, after first washing clarification with water, be 90% ~ 95% ethanol desorbing by concentration again, collect 90% ~ 95% ethanol elution, concentrating under reduced pressure, polyamide column on concentrated solution, first wash with water, be 70% ~ 75% methanol desorbing by concentration again, collect 70% ~ 75% meoh eluate, concentrating under reduced pressure, lyophilization, obtain containing the total flavones of 30% ~ 35% percentage by weight and the total lactones of 7% ~ 8% percentage by weight.
Preferably, above-described Folium Ginkgo refines extract, and it prepares by the following method:
Get Semen Ginkgo fallen leaves, pulverize, adding 7 times amount concentration is 70% alcohol reflux 3 times, each 2 hours, merge extractive liquid, filter, decompression recycling ethanol, add water-soluble loose, leave standstill, filter, AB-8 resin column absorption on filtrate, after first washing clarification with water, be 95% ethanol desorbing by concentration again, collect 95% ethanol elution, concentrating under reduced pressure, polyamide column on concentrated solution, first wash with water, be 70% methanol desorbing by concentration again, collect 70% meoh eluate, concentrating under reduced pressure, lyophilization, obtain containing the total flavones of 30% ~ 35% percentage by weight and the total lactones of 7% ~ 8% percentage by weight,
Described total flavones comprises Quercetin, kaempferol and isorhamnetin; Described total lactones comprises bilobalide, bilobalide, ginkalide A, ginkalide B.
The present invention is according to the chemical composition in Semen Ginkgo fallen leaves and physicochemical property thereof, preparation method is preferably extracted and process for refining (has screened Extraction solvent and concentration thereof by great many of experiments, resin column type and resin thereof are used in conjunction mode), experimental result shows, the present invention adopts 70% ethanol extraction-AB-8 macroporous resin column absorption-polyamide column absorption, and adopt the ethanol elution eluting of preferred concentration, the total flavones percentage by weight prepared can reach 30% ~ 45%, and total lactones percentage by weight can reach 7% ~ 15%; And the side effect compositions such as ginkgolic acid do not detected, the pharmacological results shows, activity is strong and extensive, achieves good technique effect.
Folium Ginkgo provided by the invention refines the preparation method of extract, comprises the following steps:
(1) get Semen Ginkgo fallen leaves, pulverize, add the alcohol reflux 2 to 3 times that 7 to 10 times amount concentration are 70% to 85%, each 2 to 3 hours, merge extractive liquid, filter, decompression recycling ethanol, add water-soluble loose, leave standstill, filter, obtain filtrate;
(2) the upper AB-8 macroporous resin column absorption of filtrate that step (1) prepares is got, after first washing clarification with water, then be 90% ~ 95% ethanol desorbing by concentration, collect 90% ~ 95% ethanol elution, concentrating under reduced pressure, polyamide column on concentrated solution, first washes with water, then is 70% ~ 75% methanol desorbing by concentration, collect 70% ~ 75% meoh eluate, concentrating under reduced pressure, lyophilization, obtains containing the total flavones of 30% ~ 35% percentage by weight and the total lactones of 7% ~ 8% percentage by weight.The present invention, according to the chemical composition in Semen Ginkgo fallen leaves and physicochemical property thereof, preferably extracts preparation method and process for refining by great many of experiments.
The present invention is by adsorbing opposed polarity and different model resin and the screening of purification capacity, determine that model is absorption with macroporous adsorbent resin and purification effect the best of AB-8 type, simultaneously the present invention carries out preferably to the eluting power of 50% to 95% different concentration ethanol, using total flavones and total lactones content as index, determine concentration be in the eluate of the ethanol of 90% to 95% total flavones and total lactones active constituent content high, and impurity is few, therefore determine that the ethanol of concentration 90% to 95% is solvent gradient; As further preferred version, after the absorption with macroporous adsorbent resin purification of AB-8 type, the macroporous adsorbent resin of the present invention preferred polyamide post and AB-8 type is again used in conjunction, and again carry out purification refine, it is 50% ~ 90% methanol that the present invention has screened concentration, the eluents such as ethanol, using total flavones and total lactones content as index, determine concentration be 70% ~ 75% methanol as eluent, active component content is high, impurity is few, and activity is the strongest.Therefore, the present invention collects the meoh eluate of concentration 70% to 75%.
Folium Ginkgo of the present invention refines extract, and the chromatographic condition of described Determination of Total Flavonoids is:
The present invention refines chemical composition and the physicochemical property thereof of extract according to Folium Ginkgo, by great many of experiments screening total flavones detection control method, Waters2695 high performance liquid chromatograph: quaternary pump, degassed online, column oven, automatic sampler, Waters2998DAD detector, chromatographic column: anti-phase C18 post (preferred KromasilC18column, 200mm × 4.6mm, 5 μm); Mobile phase: volume ratio is methanol and concentration 0.4% phosphoric acid solution of 50:50, determined wavelength: 360nm; Flow velocity: 1.0mL; Column temperature: 30 DEG C; Sample size: 10 μ l.The flavone component such as Quercitroside, Quercetin, kaempferol and isorhamnetin, luteolin, apigenin can be detected simultaneously.Can detect Folium Ginkgo fast and accurately by the method and refine flavones ingredient in extract, to controlling Folium Ginkgo, to refine the quality of extract significant.
Folium Ginkgo of the present invention refines extract, and the chromatographic condition that described total lactones measures is:
The present invention refines chemical composition and the physicochemical property thereof of extract according to Folium Ginkgo, by great many of experiments screening total lactones detection control method, and WatersACQUITYUPLC system: quaternary pump solvent system, online degasser and automatic sampler; XevoTQ detector, bilobalide Mass Spectrometry Conditions ion source: ESI-source; Scan mode: multiple-reaction monitoring mode; Capillary voltage: 3.0kV; Ion source temperature: 150 DEG C; Desolventizing temperature degree: 550 DEG C; Desolventizing throughput: 1000Lh
-1; Taper hole throughput: 50Lh
-1; Impinging air flows amount: 0.15mLmin
-1; Sampling taper hole voltage 18 volts to 35 volts, collision energy 14eV to 25eV.
Wherein the main the MS detection parameters of ginkalide C, bilobalide, ginkalide A and ginkalide B is as shown in table 1:
The main the MS detection parameters of table 14 kinds of terpene lactones compositions
Folium Ginkgo of the present invention is refined extract and is being prepared the application in resisting thrombotic diseases medicine.
Folium Ginkgo of the present invention is refined extract and is being prepared the application in antioxidation, antiaging agent or health product.
Folium Ginkgo of the present invention is refined extract and is being prepared the application in nervous system protection medicine.
Beneficial effect: Folium Ginkgo provided by the invention is refined extract and compared to the prior art had the following advantages:
1, the physicochemical property that the present invention falls leaves according to Semen Ginkgo, adopt method purification total flavones and the total lactones of macroporous resin and polyamide coupling first, determine best macroporous resin material and polyamide material, especially in eluant, the eluting power of the ethanol of high spot reviews variable concentrations, determine best elution processes, at utmost can obtain the strong total flavones of purity high activity and total lactones, and not containing malicious accessory ingredients such as ginkgolic acids, the pharmacological results shows, the Folium Ginkgo that the present invention prepares is refined extract and is had good antioxidation, anticoagulant and nervous system protective effect, achieve good technique effect.
2, the present invention is according to the chemical feature of total flavones and total lactones and physicochemical property thereof, and determine by great many of experiments the detection method detecting total flavones and total lactones, workable, detection method is sensitive, and accurately, reproducible, detection efficiency is high.
Accompanying drawing explanation
Fig. 1 is preparation technology's flow chart that Folium Ginkgo provided by the invention refines extract.
Detailed description of the invention
The present invention is illustrated further below in conjunction with specific embodiment, these embodiments should be understood only be not used in for illustration of the present invention and limit the scope of the invention, after having read the present invention, the amendment of those skilled in the art to the various equivalent form of value of the present invention has all fallen within the application's claims limited range.
Embodiment 1
Folium Pruni refines the preparation method of extract, and it draws together following steps:
(1) get Semen Ginkgo fallen leaves, pulverize, add the alcohol reflux 3 times that 7 times amount concentration are 70%, each 2 hours, merge extractive liquid, filter, decompression recycling ethanol, add water-soluble loose, leave standstill, filter, obtain filtrate;
(2) the upper AB-8 resin column absorption of filtrate that step (1) prepares is got, after first washing clarification with water, then be 95% ethanol desorbing by concentration, collect 95% ethanol elution, concentrating under reduced pressure, polyamide column on concentrated solution, first washes with water, then is 70% methanol desorbing by concentration, collect 70% meoh eluate, concentrating under reduced pressure, lyophilization, obtains Folium Pruni and refines extract.
The chromatographic condition of Determination of Total Flavonoids is:
Waters2695 high performance liquid chromatograph: quaternary pump, degassed online, column oven, automatic sampler, Waters2998DAD detector, chromatographic column: anti-phase C18 post; Mobile phase: volume ratio is methanol and concentration 0.4% phosphoric acid solution of 50:50, determined wavelength: 360nm; Flow velocity: 1.0mL; Column temperature: 30 DEG C; Sample size: 10 μ l.
The chromatographic condition that total lactones measures is:
WatersACQUITYUPLC system: quaternary pump solvent system, online degasser and automatic sampler; XevoTQ detector, bilobalide Mass Spectrometry Conditions ion source: ESI-source; Scan mode: multiple-reaction monitoring mode; Capillary voltage: 3.0kV; Ion source temperature: 150 DEG C; Desolventizing temperature degree: 550 DEG C; Desolventizing throughput: 1000Lh
-1; Taper hole throughput: 50Lh
-1; Impinging air flows amount: 0.15mLmin
-1; Wherein the main the MS detection parameters of ginkalide C, bilobalide, ginkalide A and ginkalide B is as shown in table 1:
The main the MS detection parameters of table 14 kind of terpene lactones composition
Detection obtains Folium Pruni and refines in extract containing the total flavones of 38% percentage by weight and the total lactones of 10% percentage by weight.
Embodiment 2
Folium Ginkgo refines the preparation method of extract, and it draws together following steps:
(1) get Semen Ginkgo fallen leaves, pulverize, add the alcohol reflux 2 times that 10 times amount concentration are 80%, each 3 hours, merge extractive liquid, filter, decompression recycling ethanol, add water-soluble loose, leave standstill, filter, obtain filtrate;
(2) the upper AB-8 resin column absorption of filtrate that step (1) prepares is got, after first washing clarification with water, be 90% ethanol desorbing by concentration again, collect 90% ethanol elution, concentrating under reduced pressure, polyamide column on concentrated solution, first wash with water, be 75% methanol desorbing by concentration again, collect 75% meoh eluate, concentrating under reduced pressure, lyophilization, obtain Folium Pruni and refine extract, detected by the detection method described in above embodiment 1, Folium Pruni is refined in extract containing the total flavones of 35% percentage by weight and the total lactones of 8% percentage by weight.
Embodiment 3 pharmacological evaluation
1 experiment material
1.1 experiment reagent
Trisodium citrate is bought in Shanghai Ling Feng chemical reagent company limited; FeSO47H
2o, salicylic acid, hydrogen peroxide (H
2o
2) buy in Wuxi City Ya Sheng Chemical Co., Ltd.; TPTE (2,4,6-tripyridyl-s-triaz-ine, TPTZ) is purchased from sigma company; DPPH(1,1-di-phenyl-2-picryhydrazyl) purchased from ALDRICHChemistry; Dimethyl sulfoxine is purchased from Chemical Reagent Co., Ltd., Sinopharm Group; Tetrazolium bromide (MTT) purchased from American GIBC company; TBS is purchased from Beijing Suo Laibao Science and Technology Ltd.; BSA is purchased from Nanjing Chuan Rui biotech company; Folium Ginkgo extract: the Folium Ginkgo that embodiment 1 prepares refines extract.
1.2 laboratory animal
Male New Zealand rabbit, body weight 2.0 ~ 2.5kg, is provided by Qinglongshan animal reproduction field, soup mountain, Jiangning, Nanjing county, the animal quality certification: SCXK (Soviet Union) 2007-0008.Sprague-Dawley rat neonatal rat is provided by Shanghai Slac Experimental Animal Co., Ltd.'s (SPF level), credit number SCXK(Shanghai) 2007-0005.
1.3 experimental apparatus
LG-PABER-I type platelet aggregation thrombin analyser (Beijing Steellex Scientific Instrument Company); Anke(LXJ-11B) centrifuge (Town in Shanghai longevity scientific instrument factory); AY120 type electronic balance (Sai Duolisi scientific instrument company limited); Enzyme-linked immunosorbent assay instrument (Bio-Tek company of the U.S.).
2 experimental techniques
The mensuration of 2.1DPPH clearance rate
In 96 orifice plates, add the Folium Ginkgo that embodiment 1 prepares refine extract 100 μ L, final concentration is respectively 100 μ gmL
-1, 50 μ gmL
-1, 25 μ gmL
-1, 10 μ gmL
-1, 2.5 μ gmL
-1.Add after Folium Ginkgo refines extract, add that to be dissolved in dehydrated alcohol final concentration be 0.05mgmL
-1dPPH solution, under 517nm wavelength, measuring its absorbance after lucifuge reaction 30min is A
0.Make blank, under 517nm wavelength, measure its absorbance is A simultaneously
1.For deduction medicine background color is on the impact of experiment, be respectively 100 μ gmL adding final concentration
-1, 50 μ gmL
-1, 25 μ gmL
-1, 10 μ gmL
-1, 2.5 μ gmL
-1medicine after add dehydrated alcohol measure its absorbance A under 517nm wavelength
2, vitamin C does positive control, carries out calculating each sample test medicine according to the following formula to DPPH clearance rate (%): [1-(A
0-A
2)/A
1] × 100, calculate IC
50.2.2FRAP method measures
By 25mL0.3molL
-1acetate buffer solution (3.1g sodium acetate and 16mL glacial acetic acid distilled water are made into 1L, PH3.6), 2.5mL0.01moLL
-1tPTZ solution (0.04molL
-1hCl dissolves), 2.5mL0.02molL
-1feCl3 is hybridly prepared into FRAP reagent.In 96 orifice plates, add the Folium Ginkgo that 10 μ L embodiments 1 prepare respectively refine extract sample solution and 100 μ LFRAP working solutions, mixing, 37 DEG C of reaction 10min, under 593nm, measure absorbance, every increment product repeat 3 times.Blank is that 95% ethanol replaces sample solution, and vitamin C is positive control.By 5mmolL
-1feSO47H
2o solution dilution becomes 0.25,1,0.5,0.25,0.125mmolL
-1, measure absorbance as stated above, obtain standard curve: Y=0.3536X+0.1356, R
2=0.9907.Folium Ginkgo is refined extract antioxidant activity and is represented with the amount of substance (mmol) reaching the FeSO4 needed for same absorbance.
The mensuration of 2.3OH clearance rate
In 96 orifice plates, add final concentration is 2.25mmolL
-1feSO
47H
2the solution of O and final concentration are 2.25mmolL
-1the each 50 μ l of salicylic acid, add whole beginning concentration and be respectively 800 μ gmL
-1, 400 μ gmL
-1, 200 μ gmL
1, 100 μ gmL
-1, 50 μ gmL
-1after the Folium Ginkgo that embodiment 1 prepares refines extract test medicine 50 μ L, adding final concentration is 2.2mmolL
-1h
2o
2, it is A that 37 DEG C of water-bath heating 30min measure its absorbance under 536nm wavelength
0.Make blank and positive control (vitamin C), vitamin C concentration is consistent with above-mentioned test medicine simultaneously, and under 510nm wavelength, measure its absorbance is A
1.For deduction medicine background color is on the impact of experiment, add FeSO in same method
47H
2o, salicylic acid and each Folium Ginkgo add 50 μ L distilled water after refining each 50 μ L of extract test medicine, and under 510nm wavelength, measuring its absorbance after 37 DEG C of water-bath heating 20min is A
2.Carry out calculating Folium Ginkgo according to the following formula and refine extract to OH clearance rate (%): [1-(A
0-A
2)/A
1] × 100.
2.4 platelet aggregation test
New Zealand's large ear rabbit, male, pentobarbital sodium (30mgkg
-1) anesthesia, common carotid artery intubate gets blood, by sodium citrate (3.8%) 1:9 anticoagulant, with the centrifugal 10min of 1000r/min, get platelet rich plasma (PRP), remainder is with the centrifugal 10min of 3000r/min, get platelet poor plasma (PPP), adjust number to be (400 ~ 450) × 10 PRP with PPP
9l
-1.Aggregation inducing agent PAF(be dissolved in pH be 7.6 containing 0.25% bovin serum albumin TBS solution in, final concentration 440ng/mL), test cup passage adds agitation beads, the Folium Ginkgo that the embodiment 1 adding variable concentrations in every pipe 250 μ LPRP prepares refines each 10 μ L of extract, 5% dimethyl sulfoxide solution is added in matched group PRP, return to zero with PPP, incubation 3min, add 10 μ LPAF derivants, with the maximum agglutination rate in LG-PABER-I type platelet aggregation thrombin analysis-e/or determining platelet 6min, and by following formulae discovery medicine to the suppression ratio of platelet aggregation.
2.5MTT method measures neuronal cell survival rate
The Folium Ginkgo extract concentration of this experimental applications is determined according to trial test result.
MTT solution preparation: take 100mgMTT(Amresco subpackage) in small beaker.Add 20mLPBS(0.01mol/L, pH=7.2), make it fully dissolve, degerming with 0.22 μm of microfilter, be sub-packed in the EP pipe of 1.5mL, 4 DEG C keep in Dark Place.
PBS solution preparation (for dissolving MTT): 8g sodium chloride, 1.15g dipotassium hydrogen phosphate, 0.2g potassium dihydrogen phosphate deionized water is settled to 1000mL, and regulate pH=7.2 with accurate pH meter, solution concentration is 0.01mol/L.121 DEG C of sterilizing 15min, room temperature preservation.
The cultivation of Primary rat neurocyte: get pregnant 18 ~ 21dSD rat, press 3mLkg with 10% chloral hydrate
-1anaesthetize in rearmounted 75% ethanol and soak 10min, sterile working takes tire Mus brain, and grinding is separated brain cell, add the DMEM culture fluid containing 10% calf serum and 10% horse serum, blowing and beating gently with suction pipe makes it become single cell suspension, counted under microscope, then by every hole 3 × 10
4, 3 × 10
5individual cell is planted respectively in advance with in 96 holes of poly-D-lysine bag quilt, 24 well culture plates, puts 37 DEG C of 5%CO
2cultivate in incubator, every 2 ~ 3d half amount changes liquid, and be cultured to 5d, adding final concentration is 10 μm of olL
-1cytosine arabinoside is to suppress the hyper-proliferative of non-neuronal cells, and after 48h, full dose continues to cultivate after changing liquid.
Experimental technique: Normal group: add 0.1%DMSO; LPS group: LPS10mgL
-1effect 24h; LPS+ Semen Ginkgo puies forward smart preparation group: 1h before adding LPS, adds different dense Semen Ginkgo and refines extract effect 24h, will be seeded in the cell by above packet transaction of 96 well culture plates, and add MTT(final concentration 0.5gL
– 1) cultivate 4h, abandoning supernatant, every hole adds 150 μ LDMSO, after grain dissolution, detects the absorbance (A) in each hole at 570nm, and according to formulae discovery cell survival rate.Cell survival rate (%)=(the blank group of A experimental group-A)/(the blank group of A Normal group-A) × 100%.
2.6 date processing and statistics
Each group of data all represent with mean ± standard deviation (mean ± SD), and inter-class correlation index adopts and compares t inspection between two.
3 experimental results and discussion
The measurement result of 3.1DPPH clearance rate
In DPPH free radical scavenging method, DPPH can form a kind of stable free radical in dehydrated alcohol, in aubergine, and has typical characteristic absorption peak.When there is antioxidant in reaction system, antioxidant provides hydrogen atom and electronics to DPPH free radical, and generate colourless product, cause the characteristic absorption peak of solution to decline, light absorption value diminishes.In this reaction, system color becomes more shallow and shows that the oxidation resistance of institute's detection material is stronger.The Folium Ginkgo that embodiment 1 prepares refines the effect that extract has stronger removing DPPH, and specific experiment the results are shown in Table shown in 2.
3.2FRAP method measurement result
Fe
3+-TPTE can be reduced to ferrous iron form by reducing substances in sample, presents obvious blueness, and has maximum light absorption in 593nm place, according to the power of the size calculation sample antioxidant activity of absorbance.When high dose, it is higher that the Folium Ginkgo that embodiment 1 prepares refines extract FRAP value; Specific experiment the results are shown in Table 3.
Table 2 Folium Ginkgo refines extract to DPPH free radical scavenging activity experimental result
Table 3 Folium Ginkgo refines extract FRAP value result
The measurement result of 3.3OH clearance rate
Each concentration Folium Ginkgo is refined extract and is all had good Scavenging activity to OH, and specific experiment the results are shown in Table shown in 4.
Table 4 Folium Ginkgo refines extract to OH free radical scavenging activity experimental result
3.4 platelet aggregation test results
PAF is the strongest platelet aggregation found so far, and its inducing action is 200 times of thrombosis ball A2 (TXA2), is 500 times of adenosine diphosphate (ADP) (adenosinediphosphate, ADP).In vivo, PAF participates in many pathophysiological processes, has found to play an important role in the pathological processes such as the shock that PAF causes at thrombosis, asthma, organ-graft refection, acute inflammation, heart allergy, endotoxin and IgG.Bilobalide be have height specificity paf receptor blocker, and to adenosine diphosphate (ADP), arachidonic acid, collagen, epinephrine induction platelet aggregation without effect.It can suppress PAF and platelet membrane receptors bind competitively.Research display, on the cell membrane of people and rabbit platelet, neutrophilic leukocyte, human pneumonocyte etc., also exists special paf receptor, PAF by with receptors bind and produce a series of physiological reaction.Bilobalide can be combined with paf receptor competitively, makes PAF ineffective, thus shows antagonistic activity.Experimental result shows, the Folium Ginkgo that the embodiment of the present invention 1 provides refines that each concentration of extract is external induces Platelet Aggregation in Rabbits all to have stronger effect to PAF, and in dose-effect relationship, concrete outcome is as shown in table 5.
Table 5 Folium Ginkgo refines extract affects T to the platelet aggregation that PAF induces
3.5 Folium Ginkgos refine the impact of extract on rat neuronal cell survival rate
Lipopolysaccharide (lipopolysaccharides, the phospholipid of LPS) to be a kind of with aminoglycoside be composition unit, microglia excrete proinflammatory cytokines can be induced as TNF A, interleukin-1 beta and nitric oxide etc., cause neural cell injury, finally cause cell death.Table 6 result shows, and LPS effect 24h can make rat neuronal cell survival rate obviously reduce, and drops to (48.55 ± 4.51) % from (100 ± 7.72) % of Normal group.Folium Ginkgo provided by the invention is refined extract and obviously can be resisted the neuronal cell survival rate that LPS causes and decline, and in finite concentration dependency.
Table 6 Folium Ginkgo refines the impact of extract on the neuronal cell injury that LPS induces
Note: compare with Normal group,
* *p<0.001; Compare with LPS group,
###p<0.001.
Shown by above experimental result; the present invention falls leaves as extracting raw material with discarded Semen Ginkgo; become Fei Weibao; by selection process prepare containing total flavones and the high refining extract of total lactones active component; there is good antioxidation; antiplatelet aggregation and neuronal cell protective effect thereof, and untoward reaction is low, has important economic benefits and Environmental Role.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (1)
1. Folium Ginkgo refine extract preparing nervous system protection medicine in application, it is prepare by the following method that described Folium Ginkgo refines extract:
Get Semen Ginkgo fallen leaves, pulverize, adding 7 times amount concentration is 70% alcohol reflux 3 times, each 2 hours, merge extractive liquid, filter, decompression recycling ethanol, add water-soluble loose, leave standstill, filter, AB-8 resin column absorption on filtrate, after first washing clarification with water, be 95% ethanol desorbing by concentration again, collect 95% ethanol elution, concentrating under reduced pressure, polyamide column on concentrated solution, first wash with water, be 70% methanol desorbing by concentration again, collect 70% meoh eluate, concentrating under reduced pressure, lyophilization, obtain containing the total flavones of 30% ~ 45% percentage by weight and the total lactones of 7% ~ 15% percentage by weight,
Described total flavones comprises Quercetin, kaempferol and isorhamnetin; Described total lactones comprises bilobalide, bilobalide, ginkalide A, ginkalide B;
The chromatographic condition of described Determination of Total Flavonoids is:
Waters 2695 high performance liquid chromatograph: quaternary pump, degassed online, column oven, automatic sampler, Waters 2998DAD detector, chromatographic column: anti-phase C18 post; Mobile phase: volume ratio is methanol and concentration 0.4% phosphoric acid solution of 50:50, determined wavelength: 360nm; Flow velocity: 1.0mL; Column temperature: 30 DEG C; Sample size: 10 μ l;
The chromatographic condition that described total lactones measures is:
Waters ACQUITY UPLC system: quaternary pump solvent system, online degasser and automatic sampler; Xevo TQ detector, bilobalide Mass Spectrometry Conditions ion source: ESI-source; Scan mode: multiple-reaction monitoring mode; Capillary voltage: 3.0kV; Ion source temperature: 150 DEG C; Desolventizing temperature degree: 550 DEG C; Desolventizing throughput: 1000Lh
-1; Taper hole throughput: 50Lh
-1; Impinging air flows amount: 0.15mLmin
-1; Sampling taper hole voltage 18 volts to 35 volts, collision energy 14eV to 25eV.
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| Title |
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| 银杏叶中有效成分提取最优化生产工艺研究;杨荣华等;《中国现代医生》;20070531;第45卷(第3期);第21页左栏"1.4 中试部分"，表6 * |
| 银杏叶中银杏萜内酯的高效液相色谱—电喷雾电离-质谱分析;卢定强等;《药物分析杂志》;20020131;第22卷(第1期);第10页"2.2 质谱部分"项下 * |
| 银杏叶提取物的药理作用;高华荣;《中国实用医药》;20100610;第5卷(第16期);第168页左栏"2.2 主要成分"项下，右栏第15-19行，第169页左栏第2-3行，第19-20行 * |
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