CN107519223A - Ginseng extract and its preparation and measure containing endogenous polypeptide, the medicine containing ginseng extract or pharmaceutical composition and its application - Google Patents
Ginseng extract and its preparation and measure containing endogenous polypeptide, the medicine containing ginseng extract or pharmaceutical composition and its application Download PDFInfo
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- CN107519223A CN107519223A CN201610442864.8A CN201610442864A CN107519223A CN 107519223 A CN107519223 A CN 107519223A CN 201610442864 A CN201610442864 A CN 201610442864A CN 107519223 A CN107519223 A CN 107519223A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/76—Undefined extracts from plants
Abstract
The assay method of polypeptide, medicine or pharmaceutical composition and its application containing ginseng extract in the preparation method and ginseng extract, ginseng extract of a kind of ginseng extract containing endogenous polypeptide of present invention offer.Preparation method of the present invention is that ginseng crude drug is milled, then is extracted through organic solvent or formic acid, and extract solution can obtain the ginseng extract using endogenous ginseng polypeptide as main component through post separation, eluent concentration.The assay method of described ginseng extract is analyzed using Nano LC LTQ Orbitrap technologies, Swiss Prot databases are searched for by PEAKS, 308 endogenous polypeptides are identified from ginseng extract altogether, the amino acid sequence of the polypeptide is SEQ ID No.1 125 and SEQ ID NoS.1 183,300~7000Da of molecular weight ranges.Detected through UHPLC Q TOF, Progenesis QI software analysis extract ginseng polypeptides content is more than 30%.The present invention has also carried out cell experiment to this ginseng extract, test result indicates that, the extract has proliferation to PC12 cells.
Description
Technical field
The invention belongs to natural medicine field.More particularly to a kind of preparation method of the ginseng extract containing endogenous polypeptide
And in ginseng extract, ginseng extract polypeptide assay method, medicine or pharmaceutical composition containing ginseng extract and
It is applied, and the polypeptide is from ginseng extract and is endogenous polypeptide.
Background technology
With the rapid development of modern social economy, the pressure that people are born in study and work increasingly increases, into
For a health problem that can not be ignored, it can cause anxiety, insomnia, depression etc., ultimately result in decrease of memory, operating efficiency
Reduce.In addition, under normal physiological condition, with age growth and body aging, also occur that ability of learning and memory declines.Cause
Health products of this exploitation with neuroprotection or improvement learning and memory function are very necessary.
Chinese medicine ginseng is the dry root and rhizome of Araliaceae ginseng (Panax ginseng C.A.Mey).Calm the nerves benefit
Intelligence, veins takes off admittedly, reinforce the spleen to benefit the lung, promote the production of body fluid to quench thirst, there is long medicinal history in China.In China, ginseng main product is in northeast
Three provinces, with Changbai mountain, Jilin, Dunhua generation yield highest, its main component is saponin(e, polypeptide, polysaccharide and volatile oil etc..Research
Show, ginseng has excited and suppresses central nervous system, improves learning and memory, strengthens physical stress ability, improves body and exempts from
(road is put, ginseng research, 2013 (01) for the effect such as epidemic disease power:46-52；Cao Zhi etc., ginseng research, 2012 (02):39-43.).Closely
Nian Lai, with the development of the subjects such as molecular biology, biochemistry, research of the people to biologically active peptide is increasingly extensive, various
Animal or plant polypeptide with different physiologically actives is extracted, or is developed into Related product.Ginseng protein polypeptide is ground
Study carefully start from the 1960s, but the means such as segregated technology and analysis, sequencing limitation, separated from ginseng and determine standard
The polypeptide quantity of true amino acid sequence is fairly limited, and in terms of preparation, majority research is to obtain ginseng protein by means such as enzymolysis
Enzymolysis product, therefore using advanced analytical technique of mass spectrum and proteomics MASS SPECTRAL DATA ANALYSIS software to endogenous in ginseng
Property polypeptide analyzed and from ginseng prepare rich in ginseng polypeptide position have far-reaching scientific value and well application
Prospect.
PC12 cells are the cell line of rat adrenal medulla pheochromocytoma differentiation, are a kind of catecholaminergic cells, energy
Synthesis is stored and discharges appropriate catecholamine (mainly dopamine and norepinephrine), has typical nerve cell special
Sign, it is commonly used for the cell model of in vitro study neurogenic disease, active ingredient of Chinese herbs of the screening with neuroprotection.
The content of the invention
The invention provides a kind of preparation method of ginseng extract containing polypeptide and ginseng extract, ginseng extract
The assay method of middle polypeptide, medicine or pharmaceutical composition and its application containing ginseng extract.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of preparation method of the ginseng extract containing polypeptide,
Ginseng is milled, then extracted through organic solvent or formic acid, through post separation, eluent is concentrated under reduced pressure to be obtained extract solution
It is able to the ginseng extract that endogenous ginseng polypeptide is main component；
Comprise the following steps that：
1) solvent extraction
After ginseng milling, 0.5-1.5 hours are soaked with organic solvent or formic acid, ultrasonic extraction 30-60 minutes, are received after centrifugation
Collect supernatant；
2) post separation
By above-mentioned supernatant through post separation, removal of impurities is eluted by 0%~20% organic solvent of volume fraction, then with volume
The eluent that fraction is 20%~70% is eluted, and collected volume fraction is 20%~70% eluent, is concentrated and dried
Ginseng extract.
Organic solvent described in step 1) is selected from methanol, ethanol, acetonitrile or the acetone that volume fraction is 40%~60%
One or both of more than, remaining is water, and the volume fraction of the formic acid is 70~90%, and remaining is water, and people participates in organic
Solvent or the mass volume ratio of formic acid volume are 1g:10-20mL；Volume is the 5%~15% of original volume after concentrate drying.
Post separation uses one kind in anti-phase C18 posts, MCI posts or large pore resin absorption column HPD100 in step 2)；Step
2) one kind in acetonitrile, methanol or ethanol that the organic solvent that removal of impurities is eluted described in is 0%~20% selected from volume fraction (has
When solvent is 0%, directly elution removal of impurities is carried out with water)；The eluent be selected from volume fraction be 20%~70% acetonitrile,
One kind in methanol or ethanol；Elution volume is 2~3 times of column volumes.
The operating condition of C18 post separations is in step 2)：Will after the supernatant concentration after removal of impurities cross C18 posts, eluent with
Volume fraction, eluted, flow velocity 0.5mL/min, received with 2 times of acetonitriles of column volume 10%, 30%, 40%, 50%, 70%, 90%
Collect 40% acetonitrile eluent, be concentrated and dried to obtain ginseng extract；
The operating condition of HPD100 resins post separation is in step 2)：By after the supernatant concentration after removal of impurities through HPD100 trees
Fat post, eluent is with volume fraction, with 2 times of ethanol elutions of column volume 10%, 30%, 50%, 70%, 90%, flow velocity
0.5mL/min, 50% ethanol eluate is collected, is concentrated and dried to obtain ginseng extract；
The operating condition of MCI post separations is in step 2)：By after the supernatant concentration after removal of impurities through MCI (CHP20/P120)
Post, acid is removed with the water elution of 10 times of column volumes, eluent with volume fraction, then with 2 times of column volumes 10%, 30%,
40%th, 50%, 70%, 90% and 100% methanol elutes, flow velocity 0.5mL/min, collects 40% meoh eluate, is concentrated and dried
Obtain ginseng extract.
The ginseng extract that a kind of above-mentioned preparation method obtains, the ginseng extract endogenous polypeptide containing ginseng are described
The amino acid sequence of polypeptide includes SEQ ID No.1-125 and SEQ ID NoS.1-183.
Through UHPLC-Q-TOF determine ginseng extract in polypeptide moiety content be more than 30%, molecular weight ranges 300~
7000Da。
The present invention is analyzed using Nano LC-LTQ-Orbitrap technologies, is determined by Progenesis QI softwares
Polypeptide moiety and content in ginseng extract, further carry out second mass analysis, and Swiss-Prot numbers are searched for by PEAKS
According to storehouse, 308 endogenous polypeptides are identified from ginseng extract, it is more to ginseng with PEAKS DB, PEAKS PTM two ways
Peptide carries out analysis sequencing.
Concrete operations are：
1) analyze：With volume fraction, the ginseng extract that claim 1 obtains is dissolved with 0.1% formic acid, Nano
LC-LTQ-Orbitrap instruments are analyzed, by the μ L samples of ejection of syringe pump 8 to trapping column (150 μm of ID, 3cm,
ReproSil-Pur C18 AQ) in, with containing the aqueous solution of 0.2% formic acid and 2% acetonitrile elute 3 minutes after be switched to analytical column
(75 μm of ID, ReproSil-Pur C18 AQ) is analyzed, mobile phase A：0.2% formic acid, Mobile phase B：Containing 0.2% formic acid and
The aqueous solution of 95% acetonitrile, gradient elution flow：0-3.5 minutes, 5%B；3.5-6 minute, 10%-20%B；6-35 minutes,
20%-50%B；35-40 minutes, 50%-90%B；40-45 minutes, 90%B；
2) sequencing polypeptides are analyzed：Initial data is imported into protein science mass spectrum software Peaks Studio 7 (BSI), respectively
Sequence analysis is carried out with PEAKS DB and PEAKS PTM both of which, the filtering threshold of data is data quality value>0.3, processing
The quality error of the automatic de novo sequencing of data progress afterwards, parent ion and daughter ion is set to 10ppm and 0.05Da, and cracking is not
The participation of enzyme is specified, specifies the amidatioon of C- ends, the acetylation of N- ends and the cyclisation of glutamic acid and glutamine
(pyroglutamalytion) the common posttranslational modification such as, data are carried out by database of all protein in Swiss-Prot
Retrieval, search for the false positive rate (FDR) of identification<1%, the peptide sequence obtained to search, with reference to its confidence level (- 10logP), peptide
Chain crack conditions, the distribution condition of daughter ion and error range carry out manual analysis confirmation one by one, finally from nearly two searched
308 high confidence level polypeptide is identified in thousand sequences, the amino acid sequence of polypeptide is SEQ ID No.1-125 and SEQ ID
NoS.1-183。
It is a kind of using containing the ginseng extract as the medicine of active component or with containing using ginseng extract as activity into
Divide the pharmaceutical composition formed with other pharmaceutically acceptable carriers and/or excipient.
A kind of medicine or pharmaceutical composition in neuroprotection and/or improve learning and memory function and/or PC12
Promote the application in propagation through UHPLC-Q-TOF measure cells.
Solution used in the present invention is in addition to solute, and solvent is water, and content is volume fraction.
Had the advantage that with the ginseng extract prepared by the present invention and document contrast and progressive：
Pertinent literature is investigated, the preparation to ginseng polypeptide mainly there are two methods：One kind is enzymatic isolation method, by ginseng water extraction
Afterwards, (Chinese patent 2014103022318 is hydrolyzed with enzyme；Chinese patent 2015106273310)；Another kind is by film point
From means such as, post separations, as with after the extraction of Tris-HCl buffer solutions, with ammonium sulfate precipitation, surpassed afterwards by dialysis, doughnut
The step such as filter column and post separation (Teng Yu etc., Changchun Polytechnic Univ.'s journal, 2005 (03):181-183；Yan Mingming, Changchun traditional Chinese medicine
University Ph.D. dissertation, 2007.).The former is simple to operate, but constituent of ginseng is complicated, and its main component is ginsenoside, and polypeptide
Content is limited, and the method cannot be guaranteed the purity or activity of product, and the method prepares gained and produced for the enzymolysis of ginseng protein
Thing rather than endogenous polypeptide, the endogenous polypeptide for having lateral reactivity may be decomposed destruction in enzymolysis process；The latter's step
Cumbersome, extraction separation costs are high, and extraction difficulty is big, is unfavorable for industrialized production, is more suitable for the means of basic research, and
And the polypeptide limited amount that extraction obtains.And instant invention overcomes disadvantage mentioned above.
1) compared with first method, method active ingredient provided by the invention is clear and definite, controllable, and its main component is endogenous
Property ginseng polypeptide, content be more than 30%, 300~7000Da of polypeptide molecular weight scope；Physiologically active is notable, and cell experiment shows this
Extract has proliferation to PC12 cells, and extraction process can ensure the structure and activity of polypeptide；
2) compared with second method, the invention provides one kind operation it is relatively simple, use cost is not higher sets
Standby, so as to meet control requirement of the industrial mass manufacture to cost, Promoting Experiment technique converts to the smoothness of production technology, in addition
Extract polypeptide quantity prepared by the present invention is more, and content is high；
3) present invention is ginseng extract, and main component is endogenous ginseng polypeptide, with ginseng crude extract class health care condition
More controllable than, its definite ingredients, dose reduces, and more conducively patient receives；Compared with ginsenoside similar drug or health products,
Metabolic end-product is amino acid to polypeptide in vivo, ensure that the security of medication, long-term use of into health products beneficial to exploitation.
4) ginseng extract prepared by the present invention, contained polypeptide is identified and sequencing obtains 308 peptide sequences.
Subordinate list explanation
Subordinate list 1 is that ginseng extract acts on influences of the 72h to PC12 cell survival rates；Subordinate list 2 is examined for UHPLC-Q-TOF
Survey, ginseng extract endogenous polypeptide contains in the ginseng extract preparation embodiment 1 of Progenesis QI software analysis measure
Amount；Subordinate list 3 and 4 is the 308 endogenous polypeptide sequence informations identified with PEAKS software de novo sequencings from ginseng.
Embodiment
The present invention is described in further details in conjunction with embodiment, embodiment is only limitted to the explanation present invention, rather than to this
The restriction of invention.
Ginseng extract prepares embodiment
Embodiment 1
1) solvent extraction：Ginseng Root Powder is soaked 1 hour with 40% methanol, ultrasonic extraction 30 minutes, collected after centrifugation supernatant
Liquid；
2) C18 post separations：Above-mentioned supernatant is concentrated under reduced pressure into after the 5% of original volume through C18 post separations, then with 2 times
Column volume 10% (v/v), 30% (v/v), 40% (v/v), 50% (v/v), 70% (v/v), the elution of 90% (v/v) acetonitrile, stream
Fast 0.5mL/min, 40% acetonitrile eluent is collected, is concentrated and dried to obtain ginseng extract；
3) assay：Said extracted thing is detected into endogenous polypeptide content, chromatographic column through UHPLC-Q-TOF：Agilent
Prorpshell 120 SB C18,2.7 μm of 3.0 × 150mm, with volume fraction, mobile phase A：0.5% formic acid, flowing
Phase B：Acetonitrile, gradient elution：0-15min, 5%-90%B, ion gun：ESI sources, detection pattern：Cation；Using
Progenesis QI softwares carry out peak identification to UHPLC-Q-TOF data, peak filtering is alignd with peak, intensity filter (Filter
strength)：0.3, bar graph/resolution ratio (Centroided date/Resolution)：60000, absolute ion intensity/most
Small intensity (Absolute ion intensity/minimum intensity)：1000, multi-charge (charge number >=2) is polypeptide
Typical physics and chemical feature, by parameter optimization and Mass Hunter analysis exclusive PCR ions are combined, are finally carried
The information such as mass-to-charge ratio (m/z), charge number (Charge), retention time (retention time) and the abundance of thing ion are taken, can
The content of component polypeptides and non-multi peptide constituents, knot are obtained by the abundance and percentage that calculate multi-charge and single charge ion
Fruit shows that endogenous polypeptide content is 32% in said extracted thing, as a result sees attached list 2.
Embodiment 2
1) solvent extraction：Ginseng Root Powder is soaked 1 hour with 10 times of ethanol of volume 60%, ultrasonic extraction 30 minutes, after centrifugation
Collect supernatant；
2) HPD100 resins post separation：Above-mentioned supernatant is concentrated under reduced pressure into after the 5% of original volume through HPD100 resin columns
Separation, with 2 times of column volumes 10% (v/v), 30% (v/v), 50% (v/v), 70% (v/v), 90% (v/v) ethanol elution, stream
Fast 0.5mL/min, 50% ethanol eluate is collected, is concentrated and dried to obtain ginseng extract；
It is 30% with UHPLC-Q-TOF detection endogenous polypeptide contents.
Embodiment 3
1) solvent extraction：Ginseng Root Powder is soaked 1 hour with 10 times of formic acid of volume 80%, and ice-bath ultrasonic is extracted 30 minutes, from
Supernatant is collected after the heart；
2) MCI post separations：Above-mentioned supernatant is concentrated under reduced pressure into after the 5% of original volume through MCI (CHP20/P120) post point
From, acid is removed with the water elution of 10 times of column volumes, then with 2 times of column volumes 10% (v/v), 30% (v/v), 40% (v/v),
50% (v/v), 70% (v/v), 90% (v/v) and the elution of 100% (v/v) methanol, flow velocity 0.5mL/min, collect 40% methanol
Eluent, it is concentrated and dried to obtain ginseng extract；
It is 33% with UHPLC-Q-TOF detection endogenous polypeptide contents.
Ginseng endogenous polypeptide identifies embodiment
Embodiment 1
1) analyze：" ginseng extract prepares gained ginseng extract in embodiment 1 " and dissolved with 0.1% (v/v) formic acid,
Nano LC-LTQ-Orbitrap instruments are analyzed, by the μ L samples of ejection of syringe pump 8 to trapping column (150 μm of ID, 3cm,
ReproSil-Pur C18 AQ) in, with volume fraction, it is switched to after being eluted 3 minutes with 2% acetonitrile (containing 0.2% formic acid)
Analytical column (75 μm of ID, ReproSil-Pur C18 AQ) is analyzed, mobile phase A：0.2% formic acid, Mobile phase B is with volume integral
Number meter：95% acetonitrile containing 0.2% formic acid, gradient elution flow：0-3.5 minutes, 5%B；3.5-6 minute, 10%-20%B；
6-35 minutes, 20%-50%B；35-40 minutes, 50%-90%B；40-45 minutes, 90%B；
2) sequencing polypeptides are analyzed：Initial data is imported into protein science mass spectrum software Peaks Studio 7 (BSI), respectively
Sequence analysis is carried out with PEAKS DB and PEAKS PTM both of which, the filtering threshold of data is data quality value>0.3.Processing
The quality error of the automatic de novo sequencing of data progress afterwards, parent ion and daughter ion is set to 10ppm and 0.05Da, and cracking is not
Specify the participation of enzyme.Specify the amidatioon of C- ends, the acetylation of N- ends and the cyclisation of glutamic acid and glutamine
(pyroglutamalytion) the common posttranslational modification such as.Data are carried out by database of all protein in Swiss-Prot
Retrieval, search for the false positive rate (FDR) of identification<1%.The peptide sequence obtained to search, with reference to its confidence level (- 10logP), peptide
Chain crack conditions, the distribution condition of daughter ion and error range carry out manual analysis confirmation one by one.Finally from nearly two searched
308 high confidence level polypeptide (seeing attached list 3 and subordinate list 4) is identified in thousand sequences.
Influence of the pharmacodynamics embodiment to PC12 cel l proliferations
Embodiment 1
Laboratory sample is that embodiment 2 prepares 10% (v/v), 30% (v/v), 50% (v/v), 70% (v/v) ethanol elution
Gained ginseng extract after liquid concentration.
Cell used in experiment is PC12, i.e. PC12 cells, with containing 10% hyclone (Hangzhou four
DMEM/F12 (Sigma) cultures Ji Qing), 0.05% trypsase (Solarbio) had digestive transfer culture.Cell is placed in 37 DEG C, and 5%
CO2, the incubator of saturated humidity is interior to be cultivated.
Test agents useful for same：MTT, it is sigma products.DMSO, Tianjin Ke Miou.
Test instrument：CO2gas incubator (HEAL FORCE), superclean bench (HEAL FORCE), ELIASA
(sunrise, TECAN), inverted microscope (Olymbus).
Experimental method：
Mtt assay detects influence of the ginseng extract to PC12 cell survival rates：PC12 cells are with the concentration in every 3200, hole
96 orifice plates are inoculated into, per the μ L of hole 100, overnight incubation.Ginseng extract is configured to required concentration (4 concentration, difference with culture medium
For 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 62.5 μ g/mL), 100 μ L are added per hole, negative control hole adds equivalent culture
Base, if 3 parallel holes.After cultivating 72h, MTT (5mg/mL) 20 μ L are added per hole, after reacting 4h, carefully get rid of nutrient solution, per hole
200 μ L DMSO are added, concussion 10min fully dissolves, and ELIASA is in 570nm wavelength detecting absorbances, calculating cell survival
Rate, calculate cell survival rate formula：Survival rate (PR%)=(A experimental group-A blank control groups)/(A negative control group-A blank
Control group) × 100%.
As a result show：Using negative control group (concentration is " 0 " point) cell survival rate for 100%, each concentration of dosing group is calculated
Cell survival rate.It can be seen that when sample concentration is 500 μ g/mL, 30% position, which has, promotees PC12 cel l proliferations, and has system
Meter learns meaning；When sample concentration is 250 μ g/mL, 50%, 70% position, which has, promotees PC12 cel l proliferations, and has statistics
Learn meaning.The result is prompted：Ginseng extract can promote PC12 cells to breed.See attached list 1.
Ginseng extract effect 72h influences on the cell survival rates of PC 12 in the pharmacodynamics embodiment 1 of subordinate list 1
*P<0.05, compared with negative control group.
The content of ginseng extract endogenous polypeptide, is represented with abundance of ions and percentage (%) in the embodiment 2 of subordinate list 2
125 endogenous polypeptide sequence informations that subordinate list 3 is identified using PEAKS DB from ginseng
aLong peptide chain can not be cracked completely, but has clear MS/MS information.bPosttranslational modification：Of poor quality -1.01, C sources
In disulfide bond；Of poor quality -17.3, Q derives from glutamic acid and glutamine residue；Of poor quality -18.01, E from glutamic acid and
Glutamine residue；Of poor quality+42.01, N-terminal acetylation；Of poor quality -0.98, amidatioon.
183 endogenous polypeptide sequence informations that subordinate list 4 is identified using PEAKS PTM from ginseng
aPosttranslational modification：Of poor quality -1.01, C derives from disulfide bond；Of poor quality -17.3, Q derives from glutamic acid and paddy ammonia
Amide residues；Of poor quality -18.01, E derives from glutamic acid and glutamine residue；Of poor quality+42.01, N-terminal acetylation；Quality
Difference -0.98, amidatioon.
Claims (9)
- A kind of 1. preparation method of the ginseng extract containing endogenous polypeptide, it is characterised in that:Ginseng is milled, then extracted through organic solvent or formic acid, extract solution through post separation, eluent be concentrated under reduced pressure can obtain with Endogenous ginseng polypeptide is the ginseng extract of main component；Comprise the following steps that：1) solvent extractionAfter ginseng milling, soaked 0.5-1.5 hours with organic solvent or formic acid, ultrasonic extraction 30-60 minutes, on collected after centrifugation Clear liquid；2) post separationBy above-mentioned supernatant through post separation, removal of impurities is eluted by 0%~20% organic solvent of volume fraction, then with volume fraction Eluted for 20%~70% eluent, collected volume fraction is 20%~70% eluent, is concentrated and dried to obtain ginseng Extract.
- 2. preparation method according to claim 1, it is characterised in that:Organic solvent described in step 1) is selected from volume integral Number is more than one or both of 40%~60% methanol, ethanol, acetonitrile or acetone, and the volume fraction of the formic acid is 70 ~90%, it is 1g that people, which participates in organic solvent or the mass volume ratio of formic acid body,:10-20mL, volume is original volume after concentrate drying 5%~15%.Post separation uses one kind in anti-phase C18 posts, MCI posts or large pore resin absorption column HPD100 in step 2)；In step 2) One kind in acetonitrile, methanol or ethanol that the organic solvent of the elution removal of impurities is 0%~20% selected from volume fraction is (organic molten When agent is 0%, directly elution removal of impurities is carried out with water)；The eluent is selected from the acetonitrile that volume fraction is 20%~70%, methanol Or one kind in ethanol；Elution volume is 2~3 times of column volumes.
- 3. preparation method according to claim 1, it is characterised in that:The operating condition of C18 post separations is in step 2)：C18 posts will be crossed after supernatant concentration after removal of impurities, eluent is with volume Fraction meter, eluted, flow velocity 0.5mL/min, collected with 2 times of acetonitriles of column volume 10%, 30%, 40%, 50%, 70%, 90% 40% acetonitrile eluent, is concentrated and dried to obtain ginseng extract；The operating condition of HPD100 resins post separation is in step 2)：By after the supernatant concentration after removal of impurities through HPD100 resins Post, eluent is with volume fraction, with 2 times of ethanol elutions of column volume 10%, 30%, 50%, 70%, 90%, flow velocity 0.5mL/ Min, 50% ethanol eluate is collected, is concentrated and dried to obtain ginseng extract；The operating condition of MCI post separations is in step 2)：By after the supernatant concentration after removal of impurities through MCI (CHP20/P120) post, Acid is removed with the water elution of 10 times of column volumes, eluent with volume fraction, with 2 times of column volumes 10%, 30%, 40%, 50%, 70%th, 90% and 100% methanol elutes, flow velocity 0.5mL/min, collects 40% meoh eluate, is concentrated and dried to obtain ginseng extraction Thing.
- 4. the ginseng extract that preparation method described in a kind of claim 1 obtains, it is characterised in that：The ginseng extract contains people Join endogenous polypeptide, the amino acid sequence of the polypeptide includes SEQ ID No.1-125 and SEQ ID NoS.1-183.
- 5. the ginseng extract that preparation method described in a kind of claim 1 obtains, it is characterised in that：Determined through UHPLC-Q-TOF The content of polypeptide moiety is more than 30% in ginseng extract, 300~7000Da of molecular weight ranges.
- A kind of 6. assay method of ginseng extract described in claim 5, it is characterised in that：Utilize Nano LC-LTQ- Orbitrap technologies are analyzed, and by the polypeptide moiety in Progenesis QI software detection ginseng extracts, are further entered Row second mass analysis, Swiss-Prot databases are searched for by PEAKS, 308 endogenous are identified from ginseng extract Polypeptide, analysis sequencing is carried out to ginseng polypeptide with PEAKS DB, PEAKS PTM two ways.
- 7. assay method according to claim 6, it is characterised in that：Concrete operations are：1) analyze：With volume fraction, the ginseng extract that claim 1 method obtains is dissolved with 0.1% formic acid, Nano LC-LTQ-Orbitrap instruments are analyzed, by the μ L samples of ejection of syringe pump 8 to trapping column (150 μm of ID, 3cm, ReproSil-Pur C18AQ) in, with containing 0.2% formic acid 2% acetonitrile elute 3 minutes after be switched to analytical column (75 μm of ID, ReproSil-Pur C18AQ) analyzed, mobile phase A：0.2% formic acid, Mobile phase B：95% acetonitrile containing 0.2% formic acid, Gradient elution flow：0-3.5 minutes, 5%B；3.5-6 minute, 10%-20%B；6-35 minutes, 20%-50%B；35-40 points Clock, 50%-90%B；40-45 minutes, 90%B；2) sequencing polypeptides are analyzed：Initial data is imported into protein science mass spectrum software Peaks Studio 7 (BSI), used respectively PEAKS DB and PEADS PTM both of which carries out sequence analysis, and the filtering threshold of data is data quality value>0.3, after processing Data carry out automatic de novo sequencing, the quality error of parent ion and daughter ion is set to 10ppm and 0.05Da, and cracking does not refer to Determine the participation of enzyme, specify the amidatioon of C- ends, the acetylation of N- ends and the cyclisation of glutamic acid and glutamine (pyroglutamalytion) the common posttranslational modification such as, data are carried out by database of all protein in Swiss-Prot Retrieval, search for the false positive rate (FDR) of identification<1%, the peptide sequence obtained to search, with reference to its confidence level (- 10logP), peptide Chain crack conditions, the distribution condition of daughter ion and error range carry out manual analysis confirmation one by one, finally from nearly two searched 308 high confidence level polypeptide is identified in thousand sequences, the amino acid sequence of polypeptide is SEQ ID No.1-125 and SEQ ID NoS.1-183。
- 8. it is a kind of using containing the ginseng extract of claim 4 or 5 as the medicine of active component or with containing with claim 5 The ginseng extract is the pharmaceutical composition that active component forms with other pharmaceutically acceptable carriers and/or excipient.
- 9. medicine described in a kind of claim 8 or pharmaceutical composition neuroprotection and/or improve learning and memory function and/ Or PC12 promotees the application in propagation through UHPLC-Q-TOF measure cells.
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| CN112461625A (en) * | 2020-11-21 | 2021-03-09 | 吉林大学 | Separation and identification method of ginseng endogenous peptide |
| CN114569483A (en) * | 2022-02-23 | 2022-06-03 | 孙万亮 | Ginseng polypeptide extract, preparation method thereof and anti-ultraviolet whitening essence |
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| CN111257485A (en) * | 2018-11-30 | 2020-06-09 | 中国科学院大连化学物理研究所 | Method for distinguishing American ginseng and ginseng by using polypeptide marker |
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| CN114569483A (en) * | 2022-02-23 | 2022-06-03 | 孙万亮 | Ginseng polypeptide extract, preparation method thereof and anti-ultraviolet whitening essence |
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