CN103800384A - Cordyceps sobolifera active site and application thereof in preparing drugs for nerve protection and aging resistance - Google Patents
Cordyceps sobolifera active site and application thereof in preparing drugs for nerve protection and aging resistance Download PDFInfo
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- CN103800384A CN103800384A CN201410046280.XA CN201410046280A CN103800384A CN 103800384 A CN103800384 A CN 103800384A CN 201410046280 A CN201410046280 A CN 201410046280A CN 103800384 A CN103800384 A CN 103800384A
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- cordyceps cicadae
- active site
- cicadae shing
- butanol
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Abstract
The invention relates to a Cordyceps sobolifera active site and application thereof in preparing drugs for nerve protection and aging resistance. The active site is an n-butanol site of a Cordyceps sobolifera water extract. The active site is prepared by the following steps: pulverizing Cordyceps sobolifera, extracting with water under reflux, concentrating the extracting solution under reduced pressure, adding anhydrous ethanol, and precipitating over night; centrifuging, and concentrating; and carrying out fractional extraction on the concentrated solution with petroleum ether, ethyl acetate and n-butanol, concentrating under reduced pressure to recover the solvent, and carrying out freeze-drying to respectively obtain a petroleum ether site, an ethyl acetate site, an n-butanol site and a water site. The n-butanol site can obviously inhibit aging and damage of the glutamic-acid-induced PC12 cells, prevent the cell LDH from release, enhance the survival rate of the cells, lower the intracellular oxygen free radical level, enhance the activity of the glutathione reductase (GSH-Px) and superoxide dismutase (SOD), and have favorable in-vitro oxidation resistance activity in the experiment of clearing DPPH. and superoxide anion (O<2.->) free radicals.
Description
Technical field
The invention belongs to medical technical field, be specifically related to from Chinese crude drug Cordyceps cicadae Shing that preparation has the position of neuroprotective and activity of fighting against senium and in the application of preparing aspect 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 old and feeble and neurodegenerative diseases has become the focus of neuroscience.The aging of human body, tends to cause accelerating the minimizing of neurocyte number, thereby produces some neurodegenerative diseases take Alzheimer (Alzheimer ' s Disease, AD) as representative.At present, neurodegenerative diseases, etiology unknown, and there is no effective treatment measure, 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 of Clavicipitaceae fungus (Cordyceps cicadae Shing) and host mountain Cicadae (Cicada flammata Dist) nymph thereof form, and it is sweet in flavor and cold in property, belongs to together with the phorozoon of Cordyceps, and effect is close.Zhen Quan shows " property of medicine opinion " cloud: " its shell, has one jiao on head, as crown, and the Periostracum cicadae of meaning, the best.Sweet in the mouth is cold, nontoxic.Main children's hangs in sky, infantile convulsion Chi, night cry cardiopalmus." Zhenglei Bencao ": " Periostracum cicadae energy spasmolytic, loose wind heat "." Bencao Tujing " is once on the books: " in modern another name for Sichuan Province, there is a kind of Cicadae, in its shell, have one jiao, as corolla shape, the Periostracum cicadae of meaning.Western people has Ji to descending person, and medical professionals's cloud, is used as medicine the strangest ".In Compendium of Material Medica, also record: " Periostracum cicadae can be treated 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 is found, is mainly contained the number of chemical compositions such as polysaccharide, adenosine, cordycepic acid, aminoacid, myriocin, ergosterol in Cordyceps cicadae Shing.
Periostracum cicadae water decoction can obviously extend the swimming time of experiment mice, obviously improves 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; Body is under poor environment, and in Periostracum cicadae water decoction can promote, environment keeps relative stability, thereby has strengthened the resistance of body to destructive stimulus.Periostracum cicadae water decoction high dose group is to Male Drosophila life-saving significantly, 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 not yet very clear and definite to its corresponding active function composition; most pharmacological evaluation only rests on the basis of crude drug or crude extract, there is no Cordyceps cicadae Shing extract and is further refined to active site the report for the preparation of neuroprotective and antiaging agent.
In order to test and assess the neuroprotective of Cordyceps cicadae Shing active site of the present invention and the application potential of defying age aspect, use the biological activity at the familiar method test each position of medical material of those skilled in the art.These known method of testings comprise Glu-induced Injury neurocyte PC12 aging model, antioxidation model etc.Result shows that the n-butyl alcohol active site of selected Cordyceps cicadae Shing water extract 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 n-butanol extraction position of Cordyceps cicadae Shing water extract has the senescense and damnification of the PC12 cell that can obviously suppress glutamate induction; stop cell LDH to discharge; improve the survival rate of cell; can reduce intracellular oxygen free radicals; improve glutathion reductase (GSH-Px) and superoxide dismutase (SOD) activity; demonstrate good neuroprotective and activity of fighting against senium, and removing DPPH and superoxide anion (O
2-) show good antioxidation activity in vitro in free radical experiment, show that the n-butanol extraction position of Cordyceps cicadae Shing has good antioxidation, there are antidotal potentiality.
Therefore, first aspect of the present invention relates to provides the n-butanol extraction of Cordyceps cicadae Shing water extract position and preparation method thereof: the n-butanol extraction position preparation method of Cordyceps cicadae Shing water extract, carry out according to following step: dry Cordyceps cicadae Shing medical material, pulverized No. 2 sieves of pharmacopeia, powder is water reflux, extract, twice at 90 ℃, and each 2 hours, after extracting solution concentrating under reduced pressure, add dehydrated alcohol, the precipitation of spending the night at 4 ℃ one 25 ℃; Centrifugal 10 1 30min of 3000r/min; Concentrated supernatant is to 1,/40 one 1/10 of original volume; Concentrated solution is proceeded in separatory funnel, use respectively 0.5～1.5 times of petroleum ether, ethyl acetate, n-butyl alcohol fractional extraction, coextraction 3～5 times, merges, and concentrating under reduced pressure reclaims solvent, lyophilization obtains respectively petroleum ether part, ethyl acetate extract, n-butanol portion (JCH
n-BuOH), water position.
Second aspect of the present invention relates to the purposes for the preparation of neuroprotective and antiaging agent to the n-butanol extraction position of described Cordyceps cicadae Shing water extract.
In order to detect the neuroprotective of gained of the present invention and the performance of activity of fighting against senium component; by the neuroprotective of gained of the present invention and activity of fighting against senium component according to being mixed with 50～200 μ g/ml medicinal liquids; for the PC12 cell processing of Clonal Rat Pheochromocytoma, observe respectively the protective effect of sample PC12 cell ageing to glutamate induction under various dose.Result shows that the n-butanol extraction position of Cordyceps cicadae Shing water extract has significant neuroprotective and activity of fighting against senium, can be used for the ancillary drug of the diseases such as preparation treatment neurodegenerative diseases; Simultaneously in model, there is significant antioxidant activity in vitro, show that Cordyceps cicadae Shing active site has good potentiality preparing aspect antiaging agent.
Third aspect of the present invention relates to the pharmaceutical composition that Cordyceps cicadae Shing active site and pharmaceutically acceptable adjuvant composition are provided.
Cordyceps cicadae Shing active site can independent or several part combinations, 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 the excipient of the conventional application of pharmaceutics, such as solvent, disintegrating agent, correctives, antiseptic, coloring agent, binding agent etc.
The experiment of the following examples and pharmacologically active is to further description of the present invention, below cited embodiment be construed as limiting never in any form.
The specific embodiment
N-butanol extraction position (the JCH of embodiment 1 Cordyceps cicadae Shing water extract
n-BuOH) preparation:
Get and pulverized the dry Cordyceps cicadae Shing powder 80g of No. 2 sieves of pharmacopeia, at 90 ℃, by 800mL water reflux, extract, twice, each 2 hours, after extracting solution concentrating under reduced pressure, add the dehydrated alcohol of 4 times of volumes, put 4 ℃ of refrigerator overnight precipitations; The centrifugal 15min of 3000r/min; Concentrated supernatant, proceeds to concentrated solution in separatory funnel, uses respectively isopyknic petroleum ether, ethyl acetate, and n-butyl alcohol fractional extraction, coextraction 3 times, merges, and concentrating under reduced pressure reclaims solvent, obtains respectively petroleum ether part, ethyl acetate extract, n-butanol portion (JCH
n-BuOH), water position; Lyophilization obtains JCH
n-BuOH1.92g.
Embodiment 2JCH
n-BuOHthe protective effect of the PC12 cell ageing to glutamate induction
(1) mtt assay, LDH method are measured cell viability:
The trophophase PC12 cell (being provided by Jiangsu University's medical college) of taking the logarithm, with 2 × 10
4/ hole is inoculated in 96 well culture plates, 200 μ l/L.At 37 ℃, 5%CO
2under condition, after overnight incubation, cell is divided into matched group, model group, drug treating group.It is matched group (not containing the complete medium of Glu); Model group (complete medium+Glu); Drug treating group (complete medium+JCH
n-BuOH+ Glu, concentration is respectively 200 μ g/mL, 100 μ g/mL, 50 μ g/mL, dilutes with culture fluid.) JCH
n-BuOHafter pretreatment 1h, adding Glu hatches every group of 24h and establishes 4 parallel holes.Cultivate after 24h, every hole adds 5g/L MTT20 μ l, continues to stop cultivating after cultivation 4h, carefully draws supernatant in hole, does LDH experiment.Every hole adds DMSO 150 μ L, and crystallization is fully dissolved, and reads absorbance (OD) with enzyme linked immunological instrument at wavelength 570nm place.The mean of getting 4 hole OD values calculates cell survival rate by formula and the results are shown in Table 1.Cell survival rate %=experimental group OD/ matched group OD × 100%.Data show, JCH
n-BuOHthere is the damaging action of protection Glu induction PC12 cell.
Collect culture fluid, the activity of measuring lactic acid dehydrogenase (LDH) (buy in Nanjing and build up Bioengineering Research Institute) by test kit description the results are shown in Table 2.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
n-BuOHcan suppress Glu induction PC12 cell LDH discharges.
Table 1MTT measures JCH
n-BuOHon the impact of Glu induction PC12 cells survival rate
A
*p<0.01 av normal group, b
*p<0.01, b
*p<0.05av model group
Table 2LDH measures JCH
n-BuOHon the impact of Glu induction PC12 cell LDH suppression ratio
A
*p<0.01 av normal group, b
*p<0.01, b
*p<0.05av model group
(2) mensuration of ROS level, GSH-Px and SOD vigor in cell:
The trophophase PC12 cell of taking the logarithm, digestion, counting, with 4 × 10
4the density of/mL is inoculated in 24 well culture plates, at 37 ℃, 5%CO
2under condition, after overnight incubation, give different disposal factor by grouping requirement, establish 3 parallel holes for every group.After continuing to cultivate 24h, suck culture medium, wash once with PBS gently, add the DCFH-DA solution of serum-free medium dilution, making its final concentration is 10 μ M, load probe 30min at 37 ℃, and PBS washes twice, trypsinization, collecting cell, adds after mixing in blackboard clear bottom 96 well culture plates, every hole 100 microlitres, establish three multiple holes for every group, fluorescence microplate reader is measured DCF fluorescence intensity, finally to every group of cell counting, and DCF fluorescence intensity/10 of calculating every group of cell according to fluorescence intensity and cell density
4cells.The results are shown in Table 3.Data show, JCH
n-BuOHcan suppress Glu induction PC12 cell ROS growing amount.
The trophophase cell of taking the logarithm, digestion, counting, with 4 × 10
4the density of/mL is inoculated in 24 well culture plates, at 37 ℃, 5%CO
2under condition, after overnight incubation, give different disposal factor by grouping requirement, establish 3 parallel holes for every group.Continue to cultivate after 24h and suck culture medium, with PBS rinsing 2 times, stay a little PBS, scrape and scrape gently cell with cell, collect in centrifuge tube, the centrifugal 5min of 1500rpm, 500 μ l PBS 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; Measure respectively each group of cell by microplate reader.The results are shown in Table 4.
Table 3DCFH sonde method is measured JCH
n-BuOHthe impact that Glu induction PC12 cell ROS is generated
A
*p<0.01 av normal group, b
*p<0.01, b
*p<0.05av model group
Table 4 spectrophotometry JCH
n-BuOHto Glu induction PC12 cell GSH-Px and SOD vigor
A
*p<0.01 av normal group, b
*p<0.01, b
*p<0.05av model group
Embodiment 3 JCH
n-BuOHantioxidation activity in vitro is measured:
(1) mensuration of DPPH radical scavenging activity:
In the Cordyceps cicadae Shing extract solution of lmL variable concentrations, add the alcoholic solution of 1.0mL 200 μ mol/DPPH, then add mix homogeneously after 2.0mL80% ethanol, place after 30min at dark place, with ultraviolet spectrophotometer at the mensuration light absorption value A of 517nm place
sample, measure the alcoholic solution of 1.0mL DPPH and the light absorption value A of 3.omL alcoholic solution mixed liquor simultaneously
blanklight absorption value A with 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
n-BuOHremove DPPH ability the strongest.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 ℃ of water-bath 20min, add 100 μ L10mmol/L pyrogallols, accurately react 4min, splash into 100 μ L6mol/LHCl cessation reactions, measure absorbance A sample 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
n-BuOHremove O
2-ability is the strongest.The results are shown in Table 6.
Table 5JCH
n-BuOHscavenging ability of DPPH free radical experiment
Table 6JCH
n-BuOHremove O
2-the experiment of free radical ability
The preparation of embodiment 4 drop pill
Take respectively 400g Macrogol 4000, in water-bath, melt, then add JCH
n-BuOH450g freeze-dried powder, stirs, in impouring insulating tube, and regulating thermostatic device, medicinal liquid is splashed in the liquid paraffin of cooling mistake (temperature ± 4 ℃) at 80-90 ℃, after dripping off, will on pill impouring filter paper, blot paraffin oil, add again a small amount of Pulvis Talci, mix, obtain JCH
n-BuOH1000 of drop pill.
The preparation of embodiment 5 capsules
JCH
n-BuOHfreeze-dried powder 1000g, 500g is mixed homogeneously with medical starch, dries, and makes capsule by every 0.45g.
The preparation of embodiment 6 tablets
JCH
n-BuOHfreeze-dried powder 1000g, starch 500g, mix homogeneously, uses appropriate alcohol granulation, through pelletizing machine granulate, tabletting, every 0.35g.
The preparation of embodiment 7 granules
JCH
n-BuOHfreeze-dried powder 1500g, starch 1000g, Icing Sugar 400g, mix homogeneously, uses appropriate alcohol granulation, dry, granulate, subpackage and get final product.
Claims (4)
1. a Cordyceps cicadae Shing active site, has neuroprotective and activity of fighting against senium, is the n-butanol extraction effective site of Cordyceps cicadae Shing water extract; The n-butanol extraction effective site that it is characterized in that described Cordyceps cicadae Shing water extract obtains by the following method: dry Cordyceps cicadae Shing medical material, pulverized No. 2 sieves of pharmacopeia, powder twice of water reflux, extract, at 90 ℃, each 2 hours, after extracting solution concentrating under reduced pressure, add dehydrated alcohol, the precipitation of spending the night at 4 ℃ one 25 ℃; Centrifugal 10 1 30min of 3000r/min; Concentrated supernatant is to 1,/40 one 1/10 of original volume; Concentrated solution is proceeded in separatory funnel, use respectively 0.5 ~ 1.5 times of petroleum ether, ethyl acetate, n-butyl alcohol fractional extraction, coextraction 3 ~ 5 times, merges, and concentrating under reduced pressure reclaims solvent, and lyophilization obtains respectively petroleum ether part, ethyl acetate extract, n-butanol portion, water position; Wherein n-butanol portion is the n-butanol extraction effective site of Cordyceps cicadae Shing water extract.
Described in claim 1 Cordyceps cicadae Shing active site in the purposes of preparing in neuroprotective and antiaging agent.
3. the neuroprotective of preparing with Cordyceps cicadae Shing and an antiaging agent, is characterized in that this medicine contains the Cordyceps cicadae Shing active site claimed in claim 1 for the treatment of effective dose.
4. a pharmaceutical preparation, the claim 1 Cordyceps cicadae Shing active site that it is characterized in that containing effective dose and one or more pharmaceutically acceptable drug excipients, or can with the other drug of Cordyceps cicadae Shing active site prescription.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103992416A (en) * | 2014-05-28 | 2014-08-20 | 江苏大学 | Isaria cosmopaltriae yasuda polysaccharide and applications thereof in preparing nerve-protective and anti-aging drug |
| CN107596279A (en) * | 2017-11-07 | 2018-01-19 | 马军捷 | A kind of medicine for treating cerebral apoplexy sequela and senile dementia and preparation method thereof |
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| CN102805829A (en) * | 2011-06-03 | 2012-12-05 | 北京市肿瘤防治研究所 | Preparation method of rhizoma smilacis glabrae extract and applications thereof in tumor treatment |
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2014
- 2014-02-10 CN CN201410046280.XA patent/CN103800384B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102805829A (en) * | 2011-06-03 | 2012-12-05 | 北京市肿瘤防治研究所 | Preparation method of rhizoma smilacis glabrae extract and applications thereof in tumor treatment |
Non-Patent Citations (1)
| Title |
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| 王春雷: "中国蝉花的研究进展", 《中国药学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103992416A (en) * | 2014-05-28 | 2014-08-20 | 江苏大学 | Isaria cosmopaltriae yasuda polysaccharide and applications thereof in preparing nerve-protective and anti-aging drug |
| CN107596279A (en) * | 2017-11-07 | 2018-01-19 | 马军捷 | A kind of medicine for treating cerebral apoplexy sequela and senile dementia and preparation method thereof |
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