CN103877185A - Chinese date extractive based on combination of two kinds of Chinese dates and application thereof in remission of neurodegenerative disease and anti-depression - Google Patents
Chinese date extractive based on combination of two kinds of Chinese dates and application thereof in remission of neurodegenerative disease and anti-depression Download PDFInfo
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Abstract
The invention provides a Chinese date extractive, and particularly relates to a preparation method for the Chinese date extractive and application of the Chinese date extractive in remission of neurodegenerative disease, anti-depression, nerve growth factor like action and promotion of neurotrophic factor expression. The Chinese date extractive is extracted from the raw material combined by two well-chosen Chinese date cultivated varieties, wherein the mass ratio of the two well-chosen Chinese date cultivated varieties is (5:1)-(1:5); 1g of the Chinese date extractive contains 500mcg of nucleosides and 60mu g of flavones. The Chinese date extractive adopts the raw material combined by optimized Chinese date cultivated varieties, and the extraction method can guarantee extraction of the nucleosides and flavones, so that the Chinese date extractive can better play the role in preventing and treating nerve diseases like neurodegenerative disease and anti-depression as well as play the health role.
Description
Technical field
The present invention relates to technical field of Chinese medicines, be specifically related to a kind of extract based on optimizing two kinds of Fructus Jujubae breed combinations, more specifically relate to preferred two kinds of Fructus Jujubae cultivars and be combined as Fructus Jujubae extract prepared by raw material, the present invention also comprises the preparation method of this Fructus Jujubae extract and the application at alleviation neurodegenerative diseases and anti-depression aspect thereof.
Background technology
Neurodegenerative diseases, is the morbid state of the cellular neural unit forfeiture of brain and spinal cord, is a kind of chronic, carrying out property nervous system disease.Its common disease mainly comprises Alzheimer, parkinson, amyotrophic lateral sclerosis and Huntington Chorea etc.This type of disease is the disease of age-dependent, and sickness rate increases year by year with the aged's increase.Its harm to patient's viability is only second to tumor, cardiovascular and cerebrovascular disease, thereby is called as " the 3rd killer " of middle-aged and elderly people.China's such Disease of existing 10 million, along with aged tendency of population, after 50 years, will increase to more than 3,000 ten thousand, by the great Science & Society problem (referring to " growing regeneration and neurodegenerative diseases research; Su Ping Yin; Chengdu Medical College's journal, 2010,4:277 ") becoming in Population and Health field.Although it be unclear that so far the cause of disease and the pathogenesis of these diseases, generally believe with oxidative damage and the neurotrophic factor disappearance of neurocyte and have very large relation.Only there is at present a few medicine to can be used for treating neurodegenerative diseases, can only short-term improve patient's symptom but all exist, and along with the prolongation curative effect of Applicative time weakens, the side effect problem such as be on the rise, therefore application has limitation.In addition, many research finds that depression history, particularly patients with senile depression history are the risk factors of such disease.In depressed patient, often exist and neurally in brain occur that (neurogenesis) is slow, neurotransmitter is not enough and the symptom such as neurotrophic factor disappearance.At present the drug main for the treatment of depressed patient will be by improving neurotransmitter levels in brain, but exist equally side effect large and exceed 50% patient to the unresponsive phenomenon of medicine.Therefore, seek and research and develop and new have medicine, health product or the food of prevention and auxiliary therapeutic action very necessary to neurodegenerative diseases and depressed disease.
Chinese medicine is a macroculture rarity of China, of long standing and well established, in people in several thousand and disease fight process, brings into play very important effect.Until now, Chinese medicine is still widely used in China's preventing and treating diseases.Fructus Jujubae be Rhamnaceae plant Fructus Jujubae (
ziziphus jujubamill.) dry mature fruit.Traditionally, it mainly has effect of nourishing blood to tranquillize the mind.Fructus Jujubae, as integration of edible and medicinal herbs article, not only has abundant nutritional labeling, and has clinically good therapeutic effect, is therefore extensively subject to people's favor.Fitochemical studies shows, in Fructus Jujubae main containing ucleosides and flavonoid isoreactivity composition (referring to " The jujube (
ziziphus jujubamill.) fruit:A review of current knowledge of fruit composition and health benefits, Gao et al., Journal of Agriculture and Food Chemistry, 2013,61,3351-3363. ").It is reported that gradient elution has the effects such as neuroprotective, anti-platelet aggregation and arrhythmia.In recent years, the biological activity that gradient elution has is considered to exist certain associated with effect of tonification class medicine.For flavones ingredient, except having general antioxidant activity, flavones ingredient has estrogen-like effects, neuroprotective and the biological activity such as enrich blood.But for above two constituents, the quality controling research in Fructus Jujubae is less at present.In the existing version pharmacopeia in 2010 of China, do not record the assay item of Fructus Jujubae.Therefore, set up ucleosides and flavonoids effective constituent quality control method in Fructus Jujubae, can effectively ensure its quality.
Aspect extraction process, flavonoid and ucleosides are the main reference indexs in optimal process process.Flavones ingredient is as main component in Fructus Jujubae, and on document, in existing Fructus Jujubae, the extraction process of flavone component is reported.On document, mostly adopt water or ethanol water to extract (referring to " research of Fructus Jujubae rutin extraction technology, Zhang Baoshan etc., Shaanxi Normal University's journal (natural science edition), 2003,31:89-93. ") as solvent.Common extracting method has percolation, supercritical ultrasonics technology, backflow and decocting method etc. (referring to " percolation extracts the technical study of flavones ingredient in Fructus Choerospondiatis; Yi Yueneng etc., CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2010; 35:1806-1806. ", " optimization of supercritical ultrasonics technology to total flavone extracting process in Fructus Jujubae fruit; Song Linlin etc., Guizhou Agricultural Sciences, 2010:222-224. " and " Study on Extracting Technology of Total Flavone of Mongolian Medicine Fructus Choerospondiatis; Ba Genna etc.; Chinese patent medicine, 2000,22:253-255. ").In order to improve the extraction ratio of flavone, in leaching process, add in addition the report (referring to " research of ultrasonic synergistic Enzymatic Extraction northern Shensi Fructus Jujubae total flavones, Yang Fulian etc., Shaanxi Tech Univ's journal, 2011,29:58-61 ") of pectin compound enzyme.On the other hand, for the Study on extraction of gradient elution, mainly concentrate at present taking cAMP in ucleosides as the preferred extraction process of index components (referring to " the macroporous resin separating technology of Cyclic adenosine-3',5'-monophosphate research; Pan sees etc.; University of Anhui's journal, 2007,31:87-90. ").Comprehensive extraction process at present, there are the following problems: conventional water or ethanol water are all applicable to the extraction of gradient elution, has no with surfactant and promote the report that slightly water-soluble flavone extracts; Or get indescribably ucleosides or flavonoid with water or ethanol moisture, also have no the extraction process of the investigation taking two constituents as aggregative indicator; Also exist detection means relatively backward, as adopted spectrophotometer method to measure total flavones, will cause the quality control index composition can not be corresponding with active ingredient.Therefore, it is that aggregative indicator is carried out Optimized Extraction Process that the present invention adopts multiclass active ingredient, has made up the deficiency of existing extraction process optimizing process, and tool is of great significance.According to the chemical composition report of current Fructus Jujubae, choose 10 kinds of ucleosides and 7 kinds of flavones ingredients in Fructus Jujubae herein, and it has been carried out to Quality Control.
China's Fructus Jujubae cultivation history is long, and approximately there be more than 700 cultivar in China at present according to statistics, is distributed widely in periphery each province, the Huanghe valley.Due to growing environment, as the difference of soil, height above sea level and weather etc., the size of different cultivars Fructus Jujubae, there is larger difference in intrinsic chemical composition even.Due to the difference on Fructus Jujubae raw material, cause occurring that the extract that different Fructus Jujubae kinds are prepared gained through same extraction process there are differences in commercial production.This species diversity can directly affect the effect of Fructus Jujubae extract in prevention and treatment.In the present invention, we find in Fructus Jujubae that ucleosides and flavones ingredient differ greatly between different cultivars.The gradient elution content of for example a certain kind Fructus Jujubae is higher, but its flavones ingredient content is lower, and the higher Fructus Jujubae kind of Flavone content, its ucleosides content may not be higher.The problems such as therefore, the present invention will be various in style for Fructus Jujubae, effective ingredient distributional difference is large propose solution.Concrete thought is: the content assaying method of setting up ucleosides and flavones ingredient in Fructus Jujubae.According to measurement result, Fructus Jujubae kind higher ucleosides content and the higher kind of Flavone content are combined, then extract and make extract.Thereby ensure that in Fructus Jujubae extract, the content of ucleosides and flavones ingredient is all higher, also for effective applied in any combination of Fructus Jujubae between different cultivars provides reference.In addition, Recent study find, ucleosides and flavones ingredient play very important effect in nervous system disease, thus inventor also to the Fructus Jujubae extract based on after optimum organization the application in this neurodegenerative diseases and antidepressant study.
Summary of the invention
One object of the present invention is to provide one to have the neurodegenerative diseases of alleviation and antidepressant Fructus Jujubae extract.Another object of the present invention is to provide and optimize a kind of preparation method of the Fructus Jujubae extract based on optimizing.The present invention shows that this extract can be used for preparing the form with food, health product and the medicine of alleviating neurodegenerative diseases and antidepressant effect.
Fructus Jujubae extract of the present invention compared with prior art has the following advantages and good effect:
Fructus Jujubae raw material of the present invention is through optimum ratio, and provide rational mass ratio range, be different from the general conventional thinking taking a kind of Fructus Jujubae as raw material, time prepared by extract, adopt the method taking aqueous surfactant solution as solvent, improve the stripping of the flavone component of poorly water-soluble, ensured the water-soluble core glycoside to thering is neural activity effect simultaneously and be insoluble in the common extraction of the flavones ingredient of water.
Ucleosides of the present invention and flavones ingredient have carried out qualitative, quantitative research, therefore Fructus Jujubae extract after standardization, thereby ensure extract itself stable and controllable for quality, more ensured prevention or the therapeutic effect of extract aspect sacred disease.
Fructus Jujubae extract of the present invention is studied neurergic mechanism, and food, health product or the medicine for preparation from now on this effect provide full and accurate, reliable basic research data.
The present invention mainly completes by the following technical programs:
Content of the present invention mainly comprises Fructus Jujubae extract preparation method, the quality standard research of Fructus Jujubae extract ucleosides and flavonoid, the neural aspect of standardization Fructus Jujubae extract Study on mechanism.Particular content is as follows:
Based on a Fructus Jujubae extract of optimizing, be combined as raw material with preferred two kinds of Fructus Jujubae cultivars, the mass ratio of Fructus Jujubae kind A and Fructus Jujubae kind B is 5: 1～1: 5.
Further, the mass ratio of above-mentioned Fructus Jujubae kind A and Fructus Jujubae kind B is 3: 1～1: 3.
Further, the mass ratio of above-mentioned Fructus Jujubae kind A and Fructus Jujubae kind B is 1: 1.
Above-mentioned Fructus Jujubae kind A can select dog head Fructus Jujubae, in peaceful circle Fructus Jujubae, tall bottle with spout Fructus Jujubae, Zanhuang Fructus Jujubae or Hami Fructus Jujubae any one, wherein every gram of crude drug of any one available Fructus Jujubae A is not less than 800 micrograms containing gradient elution, for example 810,820,830,840,850,900,950,1000,1050,1100,1200,1300 micrograms or higher.Fructus Jujubae kind B can select in Fuping Fructus Jujubae, Jishan plate Fructus Jujubae, Lingbao City's Fructus Jujubae, official's Fructus Jujubae or Linze jujube any one, and wherein every gram of crude drug of any one available Fructus Jujubae B is not less than 80 micrograms containing flavones ingredient, for example, be 85,90,95,100,110,120,130,140,150,160,170,180,190,200 micrograms or higher.
Above-mentioned Fructus Jujubae extract preparation method is: get Fructus Jujubae kind A and Fructus Jujubae kind B and mix, pulverize, to contain 0.5～1.5%, 0.5～1.0%, or the aqueous surfactant solution of 1.0-1.0% (w/v) extracts twice, each 1-3 hour, filtration, merging filtrate, filtrate is concentrated, for subsequent use; Wherein surfactant is one or more in PLURONICS F87, sodium lauryl sulphate, PEG4000, tween 80; Being not less than 500 micrograms by every gram of Fructus Jujubae extract of above-mentioned preparation method gained containing gradient elution content, for example, can be 550,600,650,700,750,800,850,900, and 950,1000 micrograms or higher; Being not less than 60 micrograms containing flavones ingredient content, for example, can be 65,70,75,80,85,90,95,100,110,120,130,140, and 150,160 micrograms or higher.
The effects on neural system mechanism research of above-mentioned Fructus Jujubae extract comprises following test: to neural protective effect; on the impact of the transcriptional level of antioxidation controlling element in neurocyte; impact, the impact on neurocyte neural thread protein NTP expression and the impact on neurotrophic factor gene expression in astrocyte that neurocyte aixs cylinder is extended.Concrete outcome is shown in accompanying drawing 1-4.
The method of ucleosides and flavones ingredient in mensuration Fructus Jujubae of the present invention is (referring to " Chemical and biological assessment of based on the previous assay method of setting up of inventor
ziziphus jujubafruits from China:Different geographical sources and developmental stages, Chen et al., 2013, Journal of Agriculture and Food Chemistry, 61,7315-7324. ").The method can reach fast to be measured the multiclass composition in Fructus Jujubae raw material and extract.
Brief description of the drawings
Fig. 1 is the neuroprotective comparison of different Fructus Jujubae extracts;
Fig. 2 is different Fructus Jujubae extracts regulate and control first transcriptional level impact on antioxidation in neurocyte;
Fig. 3 is the impact that Fructus Jujubae extract extends neurocyte aixs cylinder;
Fig. 4 is the impact of Fructus Jujubae extract on neurotrophic factor gene expression in astrocyte.
The high-performance liquid chromatogram determination method of Fructus Jujubae Nucleosides:
1. chromatographic condition and system suitability
Chromatographic column be Agilent ZORBAX SB-Aq (4.6 × 250 mm, 5 μ m); Detect wavelength: 260 nm; Column temperature: room temperature; Sample size: 8 μ l; Number of theoretical plate calculates and should be not less than 2000 by guanosine peak.Chromatography eluant program is as following table 1.
Table 1: gradient elution flow sheet
2. the preparation of reference substance solution
Get respectively uracil, xanthine, hypoxanthine, guanine, adenine, cytidine, uridnine, guanosine, cyclic guanosine monophosphate and cyclic adenosine monophosphate reference substance appropriate, accurately weighed, making respectively solubility is the solution of 156.25 ng/ml, 312.5 ng/ml, 625.0 ng/ml, 1250.0 ng/ml, 2500 ng/ml, 5000 ng/ml.
3. the preparation of test article solution
Get Fructus Jujubae powder or extract powder 1 g, accurately weighed, to put in 50 ml test tubes, precision adds water 20 ml, extract 1 hour, filter, residue repeats to extract 1 time, merging filtrate, be settled to 50 ml, with microporous filter membrane, (0.45 μ m) filters, and gets subsequent filtrate, to obtain final product.
4. the assay of sample
Get respectively Fructus Jujubae different cultivars sample, for sample solution, under above-mentioned chromatographic condition, measure respectively the peak area of 10 kinds of gradient elutions by test article solution preparation below legal system, according to its equation of linear regression, calculate its content, the results are shown in following table 2.
Table 2: the content of different Fructus Jujubae kind Nucleosides
| Fructus Jujubae kind | Total nucleoside content (μ g/g) |
| Golden jujube | 719.8708 |
| Lingbao City's Fructus Jujubae | 407.4343 |
| Ziziphus jujuba cv muzao | 414.7199 |
| Linze jujube | 587.9168 |
| In peaceful circle Fructus Jujubae | 1052.302 |
| Official's Fructus Jujubae | 478.6763 |
| Smoked red date | 356.2541 |
| Jishan plate Fructus Jujubae | 545.0594 |
| Fuping Fructus Jujubae | 504.7652 |
| Ruoqiang Fructus Jujubae | 590.2015 |
| Jiao cheng City fine horse Fructus Jujubae | 742.4268 |
| Osiery group's Fructus Jujubae | 555.3802 |
| Dog head Fructus Jujubae | 1131.236 |
| Phase Fructus Jujubae | 542.415 |
| Lingwu Long Jujube | 628.164 |
| Zanhuang Fructus Jujubae | 955.9526 |
| The red Fructus Jujubae of unit | 553.06 |
| Tall bottle with spout Fructus Jujubae | 982.4242 |
| Hami Fructus Jujubae | 830.35 |
| With field Fructus Jujubae | 768.3518 |
| Ring Fructus Jujubae | 721.834 |
According to measurement result, containing the higher kind of ucleosides content have dog head Fructus Jujubae, in peaceful circle Fructus Jujubae, tall bottle with spout Fructus Jujubae, Zanhuang Fructus Jujubae and Hami Fructus Jujubae.The ucleosides content of considering various Fructus Jujubaes can be because the different external conditions such as time, weather changes, Fructus Jujubae includes gradient elution and is being not less than under the condition of 800 μ g/g, the for example above-mentioned corresponding Fructus Jujubae of 1052,1131,955,982,830 μ g/g recording, all can be used as the preferred feedstock of Fructus Jujubae kind A.
The high-performance liquid chromatogram determination method of flavones ingredient in Fructus Jujubae:
1. chromatographic condition and system suitability
Chromatographic column is Agilent Eclipse Plus C
18rRHD 1.8 μ m, 2.1 × 50 mm; Detect wavelength: 260 nm; Column temperature: room temperature; Sample size: 5 μ l; Number of theoretical plate calculates and should be not less than 2000 by rutin peak.Chromatography eluant program is as following table 3:
Table 3: gradient elution flow sheet
2. mass spectrum condition
The triple level Four bar of the Agilent 6460 LC-MS system of 1200 liquid phase systems and equipment injection stream technology.Ion source is electro-spray ionization (ESI) source: spray voltage 45 psi; Dry gas temperature 325
oc; Dry gas flow velocity 10 L/min; Capillary voltage 4000 V; Delta EMV:400 V.Detection mode is anion polyion reaction monitoring (MRM).
3. reference substance solution preparation
Get respectively catechin, procyanidin B 2, epicatechin, Quercetin-3-galactoside, rutin, Quercitrin-3-O-glucoside and FNS reference substance appropriate, accurately weighed, add methanol and fully dissolve, making respectively solubility is the solution of 0.05 ng/ml, 6.25 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml.
4. the preparation of test article solution
Get Fructus Jujubae powder or extract powder 1 g, accurately weighed, to put in 50 ml test tubes, precision adds water 20 ml, and supersound extraction 1 hour filters, and residue repeats to extract 1 time, and merging filtrate is settled to 50 ml, and with microporous filter membrane, (0.45 μ m) filters, and gets subsequent filtrate.
5. the assay of sample
Get respectively Fructus Jujubae different cultivars sample, for sample solution, under above-mentioned chromatographic condition, measure respectively the peak area of 7 kinds of flavones ingredients by need testing solution preparation below legal system, according to its equation of linear regression, calculate its content, the results are shown in down: 4.
Table 4: the content of flavones ingredient in different Fructus Jujubae kinds
| Fructus Jujubae kind | General flavone content (μ g/g) |
| Golden jujube | 41.69857 |
| Lingbao City's Fructus Jujubae | 167.5365 |
| Ziziphus jujuba cv muzao | 76.73738 |
| Linze jujube | 106.2125 |
| In peaceful circle Fructus Jujubae | 79.04974 |
| Official's Fructus Jujubae | 121.9684 |
| Smoked red date | 78.37984 |
| Jishan plate Fructus Jujubae | 188.1753 |
| Fuping Fructus Jujubae | 392.2393 |
| Ruoqiang Fructus Jujubae | 25.52249 |
| Jiao cheng City fine horse Fructus Jujubae | 65.55117 |
| Osiery group's Fructus Jujubae | 40.84982 |
| Dog head Fructus Jujubae | 63.23989 |
| Phase Fructus Jujubae | 40.63279 |
| Lingwu Long Jujube | 56.41783 |
| Zanhuang Fructus Jujubae | 59.72036 |
| The red Fructus Jujubae of unit | 15.15214 |
| Tall bottle with spout Fructus Jujubae | 34.85982 |
| Hami Fructus Jujubae | 61.28965 |
| With field Fructus Jujubae | 49.65121 |
| Ring Fructus Jujubae | 85.58839 |
According to measurement result, there are Fuping Fructus Jujubae, Jishan plate Fructus Jujubae, Lingbao City's Fructus Jujubae, official's Fructus Jujubae and Linze jujube containing the higher kind of total flavonoid content.Total nucleoside content of considering various Fructus Jujubaes can be because the different times, the external conditions such as weather change, Fructus Jujubae includes flavones ingredient and is being not less than under the condition of 80 μ g/g, the for example above-mentioned corresponding Fructus Jujubae of 167,106,121,188,392,85 μ g/g recording, all can be used as the preferred feedstock of Fructus Jujubae kind B.
Detailed description of the invention
Below in conjunction with the description of specific embodiment and further illustrate the present invention with reference to accompanying drawing.But these embodiment are only exemplary, not scope of the present invention is formed to any restriction.Those skilled in the art, according to basic thought of the present invention, can the details of technical solution of the present invention and form be modified or be replaced, but these amendments and replacement all fall within the scope of protection of the present invention lower without departing from the spirit and scope of the present invention.
embodiment 1
Fructus Jujubae extract preparation method: get Zanhuang Fructus Jujubae and Jishan plate Fructus Jujubae in mass ratio 3:1 mix, pulverize, taking containing surfactant PLURONICS F87 as the extraction with aqueous solution of 0.5% (w/v) 2 times, each 3 hours, filter merging filtrate, lyophilization.After measured, in every gram of Fructus Jujubae extract, the content of ucleosides and flavones ingredient is respectively 830 micrograms and 90 micrograms.
Fructus Jujubae extract preparation method: get dog head Fructus Jujubae and Fuping Fructus Jujubae in mass ratio 1:1 mix, pulverize, taking containing surfactant tween 80 as the extraction with aqueous solution of 1.0% (w/v) 2 times, each 1 hour, filter, merging filtrate, filtrate is concentrated, for subsequent use.After measured, in every gram of Fructus Jujubae extract, the content of ucleosides and flavones ingredient is respectively 800 micrograms and 200 micrograms.
embodiment 3
Fructus Jujubae extract preparation method: get dog head Fructus Jujubae and Fuping Fructus Jujubae in mass ratio 1:1 mix, pulverize, taking containing surfactant tween 80 as the extraction with aqueous solution of 0.5% (w/v) 2 times, each 1 hour, filter, merging filtrate, filtrate is concentrated, for subsequent use.After measured, in every gram of Fructus Jujubae extract, the content of ucleosides and flavones ingredient is respectively 805 micrograms and 190 micrograms.
embodiment 4
In getting peaceful circle Fructus Jujubae and Linze jujube in mass ratio 1:3 mix, pulverize, taking the extraction with aqueous solution containing Surfactant PEG 4000 as 1.0% (w/v) 2 times, each 2 hours, filter, merging filtrate, filtrate is concentrated, for subsequent use.After measured, in every gram of Fructus Jujubae extract, the content of ucleosides and flavones ingredient is respectively 700 micrograms and 90 micrograms.
embodiment 5
Get the 1:5 mixing in mass ratio of tall bottle with spout Fructus Jujubae and official's Fructus Jujubae, pulverize, to extract 2 times as the aqueous solution of 1.0% (w/v) decocts containing Surfactant SDS, each 2 hours, filtration, merging filtrate, filtrate is concentrated, for subsequent use.After measured, in every gram of Fructus Jujubae extract, the content of ucleosides and flavones ingredient is respectively 560 micrograms and 100 micrograms.
embodiment 6
Get the 5:1 mixing in mass ratio of dog head Fructus Jujubae and Lingbao City's Fructus Jujubae, pulverize, to extract 2 times as the aqueous solution of 1.5% (w/v) decocts containing surfactant tween 80, each 2 hours, filtration, merging filtrate, filtrate is concentrated, for subsequent use.After measured, in every gram of Fructus Jujubae extract, the content of ucleosides and flavones ingredient is respectively 1000 micrograms and 80 micrograms.
embodiment 7
The inventor further finds: when will be containing the higher Fructus Jujubae kind of gradient elution, as dog head Fructus Jujubae, in peaceful circle Fructus Jujubae, tall bottle with spout Fructus Jujubae, Zanhuang Fructus Jujubae or Hami Fructus Jujubae any one; With will be containing the higher Fructus Jujubae kind of flavones ingredient, as in Fuping Fructus Jujubae, Jishan plate Fructus Jujubae, Lingbao City's Fructus Jujubae, official's Fructus Jujubae or Linze jujube any one, combine extraction, and after preferably extracting, every gram of Fructus Jujubae extract is being not less than 500 micrograms containing gradient elution content, for example 560,700,800,805,830,1000 micrograms, with be not less than 60 micrograms containing flavones ingredient content, for example, under the condition of 80,90,100,190,200 micrograms, resulting composition is all better than its independent extract effect for the action effect of neurocyte.
embodiment 8
In brain, two kinds of common neurodegenerative diseases parkinson and Alzheimer, the oxidative stress in its pathological process is considered to one of principal element wherein.Therefore this EXPERIMENTAL DESIGN Fructus Jujubae extract resist the research of the effect of active oxygen to neural cell injury.In addition, the inventor found through experiments: in the time that two kinds of different cultivars Fructus Jujubaes are combined, the compositions display obtaining has gone out beat all effect, and the compositions obtaining is better than to the effect of neural cell injury the effect that each component is used separately resisting active oxygen.
1. material and instrument
PGL4.37[luc2P/ARE/Hygro] the carrier antioxidation controlling element (ARE that comprises 4 repetitions, 5 '-TAGCTTGGAA ATGACATTGC TAATGGTGAC AAAGCAACTT T-3 '), and wherein antioxidation controlling element can activate (the Promega Corporation that transcribes of luciferase report gene luc2P, Madison, WI, USA).
2. cell culture condition
PC12 cell culture is in DMEM culture medium, and it is containing 6% hyclone, containing 6% horse serum, the penicillin of 100 U/ml and the streptomycin of 100 U/ml, incubator temperature 37
oc, containing 7.5% CO
2air and certain humidity.
3. the determination of activity of neurocyte
PC12 cell kind is at 96 orifice plates, every porocyte density approximately 2 × 10
4individual cell, spending the night allows cell attachment, within second day, starts to add different Fructus Jujubae extracts (dog head Fructus Jujubae extract, Fuping Fructus Jujubae extract, dog head Fructus Jujubae and Fuping Fructus Jujubae 1:1 proportioning extract, working concentration is 1.5 mg/ml).After dosing 24 hours, add tert-butyl hydroperoxide (
tert-butyl hydroperoxide, tBHP) (150 μ M) 3 hours, then add 3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazole bromine salt (MTT) solution (work solubility 0.5 mg/ml), continues to cultivate 1 hour.Stop cultivating, carefully suck culture fluid in hole.Every hole adds 150 μ l DMSO, puts on shaking table low-speed oscillation 15 minutes, and crystallization is fully dissolved.Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD570 nm place.Experimental result is with the meansigma methods of three experiments, each parallel three holes of doing.According to above result, in the time that two kinds of Fructus Jujubae different cultivars are combined, the action effect of the compositions obtaining is better than the effect that each component is used separately, the positive contrast of VitC (Fig. 1).
4. antioxidation reaction unit-luciferase activity is measured (ARE-luciferase)
By Lipofectamine 2000 rotaring dyeing technologies, by pARE-Luc gene transfection, in the PC12 cell of cultivating, then the PC12 kind after transfection is at 24 orifice plates, every porocyte density approximately 5 × 10
5individual cell, spend the night and allow cell attachment, added different Fructus Jujubae extracts (dog head Fructus Jujubae extract, Fuping Fructus Jujubae extract, dog head Fructus Jujubae and Fuping Fructus Jujubae 1:1 proportioning extract since second day, working concentration is 1.5 mg/ml), dosing sampled after 24 hours, and sample carries out luciferase activity mensuration (Fluostar Optima, BMG Labtechnology), experimental result is the meansigma methodss of three experiments, each parallel three holes of doing.According to above result, in the time that two kinds of Fructus Jujubae different cultivars are combined, the action effect of the compositions obtaining is better than the effect that each component is used separately, tertiarybutylhydroquinone (
tert-butyl hydroquinone, tBHQ) positive contrast (Fig. 2).
embodiment 9
The nerve of Fructus Jujubae extract generates because of increment effect research
The mensuration that neurocyte aixs cylinder is extended
By PC12 cell kind, at 6 orifice plates, every porocyte density is about 5 × 10
4individual cell, cell culture is in DMEM culture medium, containing 6% hyclone, containing 6% horse serum, the penicillin of 100 U/ml and the streptomycin of 100 μ g/ml, incubator temperature 37
oc, containing 7.5% CO
2air and certain humidity.Spend the night and allow cell attachment.First 3 hours of dosing, is changed to the culture medium containing 1% hyclone and 1% horse serum by culture fluid.After three hours, add Fructus Jujubae extract (dog head Fructus Jujubae extract, Fuping Fructus Jujubae extract, dog head Fructus Jujubae and Fuping Fructus Jujubae 1:1 proportioning extract, working concentration is 1.5 mg/ml) cultivate 72 hours, wherein within every 24 hours, change fresh medium or the culture fluid containing Fructus Jujubae extract.After three days, 6 orifice plate cultured cells are first washed once with PBS, then fix 15 min with 4% paraformaldehyde chamber Warm, with inverted phase contrast microscope, cellular morphology are observed to (× 20) afterwards, and with coupling digital camera obtain microscope hypograph.Then carry out digitized quantitative analysis by image analysis software.According to above result, in the time that two kinds of Fructus Jujubae different cultivars are combined, the action effect of the compositions obtaining is better than the effect that each component is used separately, nerve growth factor (nerve growth factor, NGF) positive contrast (Fig. 3).
embodiment 10
Fructus Jujubae extract promotes the research of neurotrophic factor expression in astrocyte
1. material and instrument
The primer of neurotrophic factor gene: 5 '-AAG GAC GCA GCT TTC TAT CCT GGC-3 ' and 5 '-TTT GGG GTC CAC AGT GAT GGT GCG-3 ' are for rat NGF (270 bp; XP_001067130.2); 5 '-GAA GAA AAC CAT AAG GAC GCG GAC TTG T-3 ' and 5 '-TTG GCC TTT TGA TAC CGG GAC TTT CTC-3 ' are for rat BDNF (288 bp; BC087634); 5 '-GCG CTG ACC AGT GAC TCC AAT ATG-3 ' and 5 '-CTC GGA CCT TTC CCT CTG GAA TTC T-3 ' are for rat GDNF (191 bp; AF497634).In this experiment, GAPDH is for interior mark, and its primer sequence is: 5 '-AAC GGA TTT GGC CGT ATT GG-3 ' and 5 '-CTT CCC GTT CAG CTC TGG G-3 ' (657 bp; NR_0215885).
2. cell culture condition
The Rat Astroglia cell culture of former culture, in MEM culture medium, contains 10% horse serum, the penicillin of 100 U/ml and the streptomycin of 100 μ g/ml, incubator temperature 37
oc, containing 5% CO
2air and certain humidity.
3. the mensuration of neurotrophic factor expression
The Rat Astroglia of former culture is cultivated and is planted on 60 mm plates, and first 3 hours of dosing, is changed to the culture medium containing 0.5% horse serum by culture fluid.After three hours, add Fructus Jujubae extract (dog head Fructus Jujubae extract, Fuping Fructus Jujubae extract, dog head Fructus Jujubae and Fuping Fructus Jujubae 1:1 proportioning extract, working concentration is 1.5 mg/ml) cultivate 24 hours, dosing sampled after 24 hours, after total RNA in sample extracts with RNAzol reagent, reverse transcription becomes cDNA, finally adopt the NGF in real-time polymerase chain reaction instrument working sample, the variation of the mrna expression of BDNF and GDNF.Experimental result is the meansigma methodss of three experiments, each parallel three holes of doing.According to above result, in the time that two kinds of Fructus Jujubae different cultivars are combined, the action effect of the compositions obtaining is better than the effect that each component is used separately, the positive contrast of Forskolin (Forskolin) (Fig. 4).
Claims (11)
1. a Fructus Jujubae extract, it is characterized in that: be combined as raw material extraction with two kinds of Fructus Jujubae cultivars and make, wherein Fructus Jujubae extract extracts and makes as the material combination of 5: 1～1: 5 taking the mass ratio of Fructus Jujubae kind A and Fructus Jujubae kind B, and preferably Fructus Jujubae extract extracts and makes as the material combination of 3: 1～1: 3 taking the mass ratio of Fructus Jujubae kind A and Fructus Jujubae kind B; More preferably Fructus Jujubae extract extracts and makes as the material combination of 1: 1 taking the mass ratio of Fructus Jujubae kind A and Fructus Jujubae kind B.
2. according to the Fructus Jujubae extract of claim 1, it is characterized in that, in the time selecting Fructus Jujubae extract raw material, every gram of crude drug of any one available Fructus Jujubae A contains gradient elution for being not less than 800 micrograms; Every gram of crude drug of any one available Fructus Jujubae B contains flavones ingredient for being not less than 80 micrograms.
3. according to the Fructus Jujubae extract described in any one in claim 1-2, it is characterized in that: Fructus Jujubae kind A be selected from dog head Fructus Jujubae, in peaceful circle Fructus Jujubae, tall bottle with spout Fructus Jujubae, Zanhuang Fructus Jujubae and Hami Fructus Jujubae; Fructus Jujubae kind B is selected from Fuping Fructus Jujubae, Jishan plate Fructus Jujubae, Lingbao City's Fructus Jujubae, official's Fructus Jujubae and Linze jujube.
4. according to the Fructus Jujubae extract described in any one in claim 1-3, it is characterized in that: every gram of Fructus Jujubae extract contains gradient elution content for being not less than 500 micrograms; And every gram of Fructus Jujubae extract contains flavones ingredient content for being not less than 60 micrograms.
5. according to the Fructus Jujubae extract described in any one in claim 1-4, it is characterized in that: described extract has in vitro the Damage Induced by Reactive Oxygen Species of resisting, promotes the biological activitys such as Neural Differentiation and neurotrophic factor expression.
6. according to the Fructus Jujubae extract described in any one in claim 1-4, it is characterized in that: described extract can add the form of adjuvant for the preparation of the purposes of health product, food or medicine.
According to the Fructus Jujubae extract described in any one in claim 1-4 for the preparation of alleviating neurodegenerative diseases, antidepressant effect or thering is the purposes in health product, food or the medicine of nerve growth factor increment effect.
8. prepare according to the method for Fructus Jujubae extract described in any one in claim 1-4 for one kind, it is characterized in that: get Fructus Jujubae kind A and Fructus Jujubae kind B and mix, pulverize, to contain 0.5～1.5% (w/v), preferably the aqueous surfactant solution of 0.5-1.0% extracts twice, each 1-3 hour, filter, merging filtrate, filtrate is concentrated, for subsequent use.
9. the method described according to Claim 8, wherein Fructus Jujubae extract extracts and makes as the material combination of 5: 1～1: 5 taking the mass ratio of Fructus Jujubae kind A and Fructus Jujubae kind B, and preferably Fructus Jujubae extract extracts and makes as the material combination of 3: 1～1: 3 taking the mass ratio of Fructus Jujubae kind A and Fructus Jujubae kind B; More preferably Fructus Jujubae extract extracts and makes as the material combination of 1: 1 taking the mass ratio of Fructus Jujubae kind A and Fructus Jujubae kind B.
10. the method described in any one according to Claim 8-9, wherein Fructus Jujubae kind A can be selected from dog head Fructus Jujubae, in peaceful circle Fructus Jujubae, tall bottle with spout Fructus Jujubae, Zanhuang Fructus Jujubae and Hami Fructus Jujubae; Fructus Jujubae kind B can be selected from Fuping Fructus Jujubae, Jishan plate Fructus Jujubae, Lingbao City's Fructus Jujubae, official's Fructus Jujubae and Linze jujube.
11. methods described according to Claim 8, is characterized in that: surfactant is one or more in PLURONICS F87, sodium lauryl sulphate, PEG4000, tween 80.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108152387A (en) * | 2017-11-22 | 2018-06-12 | 新疆农垦科学院 | The analysis method of cyclic adenosine monophosphate in a kind of jujube |
| US20230043437A1 (en) * | 2020-07-20 | 2023-02-09 | Nantong University | Compound preparation for neuranagenesis, and preparation method therefor and use thereof |
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| CN103417664A (en) * | 2013-08-06 | 2013-12-04 | 南京弘典医药科技有限公司 | Application of fructus ziziphi jujubae in preparing medicine or health-care product for treating Alzheimer disease |
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| CN103417664A (en) * | 2013-08-06 | 2013-12-04 | 南京弘典医药科技有限公司 | Application of fructus ziziphi jujubae in preparing medicine or health-care product for treating Alzheimer disease |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108152387A (en) * | 2017-11-22 | 2018-06-12 | 新疆农垦科学院 | The analysis method of cyclic adenosine monophosphate in a kind of jujube |
| US20230043437A1 (en) * | 2020-07-20 | 2023-02-09 | Nantong University | Compound preparation for neuranagenesis, and preparation method therefor and use thereof |
| US12115204B2 (en) * | 2020-07-20 | 2024-10-15 | Nantong University | Compound preparation for neuranagenesis, and preparation method therefor and use thereof |
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