CN103800387B - A kind of Cordyceps cicadae Shing active site and preparing the application in neuroprotective and antiaging agent - Google Patents
A kind of Cordyceps cicadae Shing active site and preparing the application in neuroprotective and antiaging agent Download PDFInfo
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- CN103800387B CN103800387B CN201410046301.8A CN201410046301A CN103800387B CN 103800387 B CN103800387 B CN 103800387B CN 201410046301 A CN201410046301 A CN 201410046301A CN 103800387 B CN103800387 B CN 103800387B
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Abstract
The present invention relates to Cordyceps cicadae Shing active site and preparing the application in neuroprotective and antiaging agent.This active site is Cordyceps cicadae Shing alcohol extraction Ethyl acetate fraction; Obtain by the following method: Cordyceps cicadae Shing pulverizing medicinal materials is sieved, with alcohol reflux, ethanol extraction aqueous dispersion, use petroleum ether successively, ethyl acetate, n-butyl alcohol fractional extraction, coextraction 3 ~ 5 times, merge, concentrating under reduced pressure recycling design, lyophilization obtains petroleum ether part respectively, ethyl acetate extract, n-butanol portion, water position.Wherein ethyl acetate extract obviously can suppress the senescense and damnification of the PC12 cell of glutamate induction, stop cell LDH release, improve the survival rate of cell, intracellular oxygen free radicals can be reduced, improve glutathion reductase (GSH-Px) and superoxide dismutase (SOD) activity, and at removing DPPH and superoxide anion (O
2 . -) free radical experiment in show good antioxidation activity in vitro.
Description
Technical field
The invention belongs to medical art, be specifically related to preparation from Chinese crude drug Cordyceps cicadae Shing and there is the position of neuroprotective and activity of fighting against senium and preparing the application in neuroprotective and antiaging agent.
Background technology
Along with China's aged tendency of population aggravation, the sickness rate of neurodegenerative diseases increases day by day, and research that is old and feeble and neurodegenerative diseases has become the focus of neuroscience.The aging of human body, often causes the minimizing accelerating neurocyte number, thus produces some neurodegenerative diseases being representative with Alzheimer (Alzheimer ' sDisease, AD).At present, neurodegenerative diseases, etiology unknown, and there is no effective remedy measures, only there is a few medicine to can be used for treating neurodegenerative diseases.
Cordyceps cicadae Shing is the dry composite body that the large Cicadae grass (CordycepscicadaeShing) of Clavicipitaceae fungus and host mountain Cicadae (CicadaflammataDist) nymph thereof are formed, and it is sweet in flavor and cold in property, and belong to together with the phorozoon of Cordyceps, effect is close.Zhen Quan shows " property of medicine opinion " cloud: " its shell, head has one jiao, as crown, and the Periostracum cicadae of meaning, best.Sweet in the mouth is trembled with fear, nontoxic.Main children's sky hangs, infantile convulsion Chi, night cry cardiopalmus." Zhenglei Bencao ": " Periostracum cicadae energy spasmolytic, loose wind heat "." Bencao Tujing " is once on the books: " there is a kind of Cicadae in modern another name for Sichuan Province, its shell has one jiao, as corolla shape, the Periostracum cicadae of meaning.Western people has Ji to descending person, Yi Gongyun, is used as medicine the strangest ".Also record in Compendium of Material Medica: " Periostracum cicadae can treat infantile convulsion, night cry cardiopalmus, the same Periostracum Cicadae of merit ".Modern pharmacological research shows, Cordyceps cicadae Shing is traditional traditional tonic medicine, has obvious defying age, immunomodulating, improves the pharmacologically active such as renal function, antitumor.Recent study finds, main containing number of chemical compositions such as polysaccharide, adenosine, cordycepic acid, aminoacid, myriocin, ergosterols in Cordyceps cicadae Shing.
Periostracum cicadae water decoction obviously can extend the swimming time of experiment mice, significantly improves the time-to-live under normobaric hypoxia state and time-to-live at high temperature, shows that Periostracum cicadae water decoction has the effect of anti-stress, resisting fatigue; Under body is in poor environment, Periostracum cicadae water decoction can promote that environment keeps relative stability, thus enhances the resistance of body to destructive stimulus.Periostracum cicadae water decoction high dose group can life-saving significantly to Male Drosophila, show its have certain anti-aging effects (Wang Yan, etc. the Primary Study [J] of Periostracum cicadae pharmacological action. Zhejiang Journal of Traditional Chinese Medicine, 2001,36 (5): 219-220).
In view of the phorozoon of Periostracum cicadae and rare Chinese medicine Cordyceps belongs to fungus (Cordyceps) together; and Paecilomyces cicadidae(Miquel)Samson has the multiple pharmacological effect such as immunity; and there is the advantages such as toxicity is little, easy cultivation; be hopeful the succedaneum as Cordyceps; but it is not yet very clear and definite to its corresponding active functional component; most pharmacological evaluation only rests on the basis of crude drug or crude extract, there is no Cordyceps cicadae Shing extract and is refined to active site further and for the preparation of the report of neuroprotective and antiaging agent.
In order to the application potential of the neuroprotective and defying age aspect of testing and assess Cordyceps cicadae Shing active site of the present invention, used those skilled in the art the biological activity at the method test each position of medical material be familiar with.These known method of testings comprise Glu-induced Injury neurocyte PC12 aging model, antioxidation model etc.Result shows that the Cordyceps cicadae Shing alcohol extraction Ethyl acetate fraction selected has good neuroprotective and antidotal activity.
Summary of the invention
The object of this invention is to provide the effective site that Cordyceps cicadae Shing has neuroprotective and activity of fighting against senium application potential.
Each position of Cordyceps cicadae Shing extract is through repeated multiple times neuroprotective and activity of fighting against senium screening; confirm that the Ethyl acetate fraction of Cordyceps cicadae Shing 60% one 80% ethanol extraction has the senescense and damnification of the PC12 cell that obviously can suppress glutamate induction; stop cell LDH release; improve the survival rate of cell; intracellular oxygen free radicals can be reduced; improve glutathion reductase (GSH-Px) and superoxide dismutase (SOD) activity; demonstrate good neuroprotective and activity of fighting against senium, and at removing DPPH and superoxide anion (O
2-) free radical experiment in show good antioxidation activity in vitro, show that the extract of Cordyceps cicadae Shing has good antioxidation, there are antidotal potentiality.
Therefore, first aspect of the present invention relates to provides Cordyceps cicadae Shing alcohol extraction Ethyl acetate fraction and preparation method thereof: the Ethyl acetate fraction preparation method of Cordyceps cicadae Shing 60% one 80% ethanol extraction, carry out according to following step: dry Cordyceps cicadae Shing medical material, pulverized No. 2, pharmacopeia sieve, powder adds 60% one 80% alcohol reflux of 10 1 15 times, and filtered and recycled ethanol.The aqueous dispersion of Cordyceps cicadae Shing ethanol extraction, uses 0.5 ~ 1.5 times of petroleum ether, ethyl acetate, n-butyl alcohol fractional extraction successively, coextraction 3 ~ 5 times, and merge, concentrating under reduced pressure recycling design, lyophilization obtains petroleum ether part respectively, ethyl acetate extract (JCH
aE), n-butanol portion, water position.
The present invention second aspect relates to the purposes of described active site for the preparation of neuroprotective and antiaging agent.
In order to the performance of the neuroprotective and anti-aging active ingredients that detect gained of the present invention; by the neuroprotective of gained of the present invention and anti-aging active ingredients according to being mixed with 50 ~ 200 μ g/ml medicinal liquids; for the PC12 cell process of Clonal Rat Pheochromocytoma, observe sample respectively at different dosages to the protective effect of the PC12 cell ageing of glutamate induction.Result shows that the alcohol extraction Ethyl acetate fraction of Cordyceps cicadae Shing has significant neuroprotective and activity of fighting against senium, can be used for the ancillary drug preparing the diseases such as treatment neurodegenerative diseases; Simultaneously there is significant antioxidant activity in model in vitro, show that Cordyceps cicadae Shing active site has good potentiality preparing in antiaging agent.
Third aspect of the present invention relates to the pharmaceutical composition providing Cordyceps cicadae Shing active site and pharmaceutically acceptable adjuvant to form.
Cordyceps cicadae Shing active site can independent or several part combination, more further with auxiliary material combination, dosage form comprises: tablet, capsule, pill, granule, suspensoid, drop pill, oral liquid etc.
Carrier of the present invention or excipient comprise carrier and excipient, such as solvent, disintegrating agent, correctives, antiseptic, coloring agent, the binding agent etc. of the application of pharmaceutics routine.
The following examples and pharmacologically active experiment are to further description of the present invention, and embodiment cited is below construed as limiting never in any form.
Detailed description of the invention
Ethyl acetate fraction (the JCH of embodiment 1 Cordyceps cicadae Shing 80% ethanol extraction
aE) preparation:
Get and pulverized the dry Cordyceps cicadae Shing powder 80g of No. 2, pharmacopeia sieve, add 80% alcohol reflux of 10 times, and filtered and recycled ethanol.The aqueous dispersion of Cordyceps cicadae Shing ethanol extraction, uses equal-volume petroleum ether, ethyl acetate successively, n-butyl alcohol fractional extraction, coextraction 3 times, and merge, concentrating under reduced pressure recycling design, obtains petroleum ether part respectively, ethyl acetate extract (JCH
aE), n-butanol portion, water position, lyophilization obtains JCH
aE2.50g.
Embodiment 2JCH
aEto the protective effect of the PC12 cell ageing of glutamate induction
(1) mtt assay, LDH method measure cell viability:
To take the logarithm trophophase PC12 cell (being provided by Jiangsu University's medical college), with 2 × 10
4/ hole is inoculated in 96 well culture plates, 200 μ l/L.At 37 DEG C, 5%CO
2under condition after overnight incubation, cell is divided into matched group, model group, drug treating group.I.e. matched group (not containing the complete medium of Glu); Model group (complete medium+Glu); Drug treating group (complete medium+JCH
aE+ Glu, concentration is respectively 200 μ g/mL, 100 μ g/mL, 50 μ g/mL, dilute with culture fluid.) JCH
aEadd after pretreatment 1h Glu hatch 24h often group establish 4 parallel holes.After cultivating 24h, every hole adds 5g/LMTT20 μ l, and continuation stops cultivating after cultivating 4h, and supernatant in careful absorption hole, does LDH experiment.Every hole adds DMSO150 μ L, and crystallization is fully dissolved, and reads absorbance (OD) with enzyme linked immunological instrument at wavelength 570nm place.The mean getting 4 hole OD values the results are shown in Table 1 by formulae discovery cell survival rate.Cell survival rate %=experimental group OD/ matched group OD × 100%.Data show, JCH
aEthe damaging action of PC12 cell can be induced to protection Glu.
Collect culture fluid, measure the Activity Results of lactic acid dehydrogenase (LDH) (buy and build up Bioengineering Research Institute in Nanjing) in table 2 by test kit description.The release suppression ratio of LDH is calculated as follows: LDH suppression ratio (%)=(LDH
model group-LDH
administration group)/(LDH
model group-LDH
normal group) × 100%.Data show, JCH
aEhave and suppress Glu to induce PC12 cell LDH releasing effect.
Table 1MTT measures JCH
aEglu is induced to the impact of PC12 cells survival rate
A**P<0.01av normal group, b**P<0.01, b*P<0.05av model group
Table 2LDH measures JCH
aEglu is induced to the impact of PC12 cell LDH suppression ratio
A**P<0.01av normal group, b**P<0.01, b*P<0.05av model group
(2) mensuration of intracellular ROS level, GSH-Px and SOD vigor:
Take the logarithm trophophase PC12 cell, digestion, counting, with 4 × 10
4the density of/mL is inoculated in 24 well culture plates, at 37 DEG C, 5%CO
2under condition after overnight incubation, give different disposal factor by grouping requirement, often group establishes 3 parallel holes.Suck culture medium after continuing to cultivate 24h, wash once with PBS gently, add the DCFH-DA solution of serum-free medium dilution, make its final concentration be 10 μMs, carrying probe 30min at 37 DEG C, PBS washes twice, trypsinization, collecting cell, adds in blackboard clear bottom 96 well culture plate after mixing, every hole 100 microlitre, often group establishes three multiple holes, fluorescence microplate reader measures DCF fluorescence intensity, finally to often organizing cell counting, calculates according to fluorescence intensity and cell density DCF fluorescence intensity/10 often organizing cell
4cells.The results are shown in Table 3.Data show, JCH
aEglu can be suppressed to induce PC12 cell ROS growing amount.
Take the logarithm trophophase cell, digestion, counting, with 4 × 10
4the density of/mL is inoculated in 24 well culture plates, at 37 DEG C, 5%CO
2under condition after overnight incubation, give different disposal factor by grouping requirement, often group establishes 3 parallel holes.Continue to cultivate after 24h and suck culture medium, with PBS rinsing 2 times, stay a little PBS, scrape with cell and scrape cell gently, collect in centrifuge tube, the centrifugal 5min of 1500rpm, 500 μ lPBS suspension cells in eppendorf pipe, cell pyrolysis liquid cell lysis; The centrifugal 6min of 12000rpm, gets supernatant, carries out each step reaction according to test kit description; Each group of cell is measured respectively by microplate reader.The results are shown in Table 4.
Table 3DCFH sonde method measures JCH
aEon the impact that Glu induces PC12 cell ROS to generate
A**P<0.01av normal group, b**P<0.01, b*P<0.05av model group
Table 4 spectrophotometry JCH
aEpC12 cell GSH-Px and SOD vigor are induced to Glu
A**P<0.01av normal group, b**P<0.01, b*P<0.05av model group
Embodiment 3JCH
aEantioxidation activity in vitro measures:
(1) mensuration of DPPH radical scavenging activity:
Add the alcoholic solution of 1.0mL200 μm of ol/DPPH in the Cordyceps cicadae Shing extract solution of lmL variable concentrations, then add mix homogeneously after 2.0mL80% ethanol, after 30min is placed at dark place, with ultraviolet spectrophotometer at 517nm place mensuration light absorption value A
sample, the alcoholic solution of Simultaneously test 1.0mLDPPH and the light absorption value A of 3.omL alcoholic solution mixed liquor
blankwith the light absorption value A of 3mL ethanol and 1.0mL sample mix liquid
contrast, clearance rate formula: DPPH clearance rate=[A
blank-(A
sample-A
contrast)/A
blank] × 100%.Data show, JCH
aEhave and remove DPPH ability.The results are shown in Table 5
(2) superoxide anion (O
2-) mensuration of radical scavenging activity:
In the Cordyceps cicadae Shing extract solution of lmL variable concentrations, add 3mlTris-HCl (PH8.2), after 25 DEG C of water-bath 20min, add 100 μ L10mmol/L pyrogallols, accurately react 4min, instill 100 μ L6mol/LHCl cessation reactions, absorbance A sample is measured at 325nm place, replace sample dope with deionized water, other are the same, measure absorbance A blank in 325nm place.Superoxide anion (O
2-) clearance rate by formula calculate.O
2-clearance rate=[(A
blank-A
sample)/A
blank] × 100%.Data show, JCH
aEthere is removing O
2-ability.The results are shown in Table 6.
Table 5JCH
aEposition scavenging ability of DPPH free radical experiment
Table 6JCH
aEo is removed at position
2-free radical capacity experimental
The preparation of embodiment 4 drop pill
Take 400g Macrogol 4000 respectively, water-bath is melted, then add JCH
aE450g freeze-dried powder, stirs, in impouring insulating tube, and regulating thermostatic device, make medicinal liquid at 80-90 DEG C, instill (temperature ± 4 DEG C) in cooled liquid paraffin, after dripping off, blot paraffin oil by pill impouring filter paper, add a small amount of Pulvis Talci again, mixing, obtains JCH
aEdrop pill 1000.
The preparation of embodiment 5 capsule
JCH
aEfreeze-dried powder 1000g, mixs homogeneously with medical starch 500g, dries, makes capsule by every 0.45g.
The preparation of embodiment 6 tablet
JCH
aEfreeze-dried powder 1000g, starch 500g, mix homogeneously, granulates by ethanol in proper amount, through pelletizing machine granulate, tabletting, every sheet 0.35g.
The preparation of embodiment 7 granule
JCH
aEfreeze-dried powder 1500g, starch 1000g, Icing Sugar 400g, mix homogeneously, granulates by ethanol in proper amount, drying, granulate, subpackage and get final product.
Claims (5)
1. a Cordyceps cicadae Shing active site, has neuroprotective and activity of fighting against senium, is the Ethyl acetate fraction of Cordyceps cicadae Shing 80% ethanol extraction; It is characterized in that the Ethyl acetate fraction of described Cordyceps cicadae Shing 80% ethanol extraction obtains by the following method: dry Cordyceps cicadae Shing medical material, pulverized No. 2, pharmacopeia sieve, powder adds 80% alcohol reflux of 10 times, and filtered and recycled ethanol; The aqueous dispersion of Cordyceps cicadae Shing ethanol extraction, uses isopyknic petroleum ether, ethyl acetate successively, n-butyl alcohol fractional extraction, coextraction 3 times, merge, concentrating under reduced pressure recycling design, lyophilization obtains petroleum ether part respectively, ethyl acetate extract, n-butanol portion, water position; Wherein ethyl acetate extract is the Ethyl acetate fraction of Cordyceps cicadae Shing 80% ethanol extraction.
2. Cordyceps cicadae Shing active site described in claim 1 is preparing the purposes in neuroprotective and antiaging agent.
3. the neuroprotective prepared with Cordyceps cicadae Shing and an antiaging agent, is characterized in that this medicine contains the Cordyceps cicadae Shing active site according to claim 1 for the treatment of effective dose.
4. a pharmaceutical preparation, is characterized in that Cordyceps cicadae Shing active site described in the claim 1 containing effective dose and one or more pharmaceutically acceptable drug excipients.
5. a pharmaceutical preparation, it is characterized in that Cordyceps cicadae Shing active site described in the claim 1 containing effective dose and can with the other drug of Cordyceps cicadae Shing active site prescription.
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Non-Patent Citations (2)
| Title |
|---|
| 蝉花生物活性物质研究进展;徐红娟等;《中国药业》;20091231;第18卷(第04期);19-21 * |
| 蝉花的研究进展;李挺等;《中药材》;20080930;第31卷(第09期);1443-1448 * |
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