CN106083979A - A kind of Cycloastragenol extract and preparation method thereof and purposes - Google Patents
A kind of Cycloastragenol extract and preparation method thereof and purposes Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J53/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by condensation with a carbocyclic rings or by formation of an additional ring by means of a direct link between two ring carbon atoms, including carboxyclic rings fused to the cyclopenta(a)hydrophenanthrene skeleton are included in this class
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Abstract
The invention provides a kind of Cycloastragenol extract, prepare by the following method: the alkaline alcohol aqueous solution of radix astragali coarse powder pH value 11～13 is extracted, it is thus achieved that astragaloside crude extract；Gained astragaloside crude extract passes through macroporous resin enrichment purification, obtains astragaloside extract；Gained astragaloside extract carries out Smith degraded, it is thus achieved that described Cycloastragenol extract；The preparation of Cycloastragenol extract of the present invention, by the pH value in extraction conditions, sodium metaperiodate consumption, the test of many times of methanol concentration, has obtained optimum extracting method, and the method repeatability is good, simple and easy to do；Cycloastragenol extract prepared by the present invention can substantially slow down H2O2The old and feeble injury causing PC12 cell, improves cell viability, and the Cycloastragenol extract pointing out preparation method of the present invention to obtain has certain activity of fighting against senium, can be applicable to the preparation of antiaging agent and health product.
Description
(1) technical field
The present invention relates to a kind of Cycloastragenol extract and the preparation method of this extract and purposes, belong to medicine neck
Territory.
(2) background technology
At present, countries in the world pace of population aging accelerate day by day, according to statistics, and China in 2012 65 years old and above old age
Population accounts for the 9.4% of entire population, and the United Nations predicts 2025, and the aging population of China's over-65s will reach 2.3 hundred million, close
2 times of old population in 2012.Under such severe situation, facing mankind huge challenge, and aging becomes with defying age
The focus that current medical science is discussed.Day by day improve and return the continuous reinforcement of green living theory along with people's living standard, as
What the most effective pre-anti-aging, improving the quality of living becomes the difficult problem that people are in the urgent need to address.Therefore, defying age is correlated with green by people
The demand of color health product and plant extract is continuously increased.
In recent years, the carrying of the discovery of telomerase (Telomerase) and " telomere hypothesis (Telomere hypothesis) "
Go out, greatly promoted the research about aging.Telomere is the specific structure that end of chromosome is made up of DNA and protein, rises
To the effect of protection chromosome integrity, research display, telomere is gradually shortened along with the increase of frequency dividing cell, finally loses
Go the protective capability to chromosome, cause cell ageing.Meanwhile, telomerase is that the reverse transcription DNA maintaining telomere length gathers
Synthase, thus play an important role in the propagation, aging of cell, by activated end telomerase activity, delaying telomere to shorten can rise
To significant anti-aging effects, therefore, effective Activation of Telomerase agent also becomes the focus studied at present.
The Radix Astragali shows have the effects such as antioxidation, slow down aging, antitumor, antiviral as Chinese medicine simply, research.
Radix Astragali saponin is one of effective ingredient of the Radix Astragali, and wherein, astragaloside is commonly used for the leading indicator of Milkvetch Root quality control.Have
Report is pointed out, Cycloastragenol (Cycloastragenol, CAG) is the aglycon of major part Radix Astragali saponin, and has significant telomere
Activation of enzymes, thus there is a series of medical values such as defying age, antiviral, antidepressant, existing report is many by Radix Astragali first
Glycosides hydrolysis prepares, and preparation method includes acid hydrolysis, Smith degraded, enzymolysis etc., and wherein Smith degrades because of its reaction condition
Gentle easily-controllable, the most relatively short, Malaprade reaction simultaneously is a kind of selective reaction, thus has certain single-minded
Property, it is often used as preparing the method for CAG.Zhou Xianli etc. have invented the Preparation method and use of a kind of CAG, are former with astragaloside
Material, uses the method rough CAG extract of Smith degraded, the method such as recycle silicon plastic column chromatography, recrystallization to obtain purity 95%
Above CAG.The condition that Smith is degraded by Feng etc. is optimized, including sodium metaperiodate consumption, methanol concentration, hydroboration
Sodium consumptions etc., degraded obtains highly purified CAG extract.
So far, most work concentrates on how to obtain highly purified CAG, and it is biological alive which kind of the mono-product of CAG have
Property, and be all with astragaloside monomer as raw material, seldom Cycloastragenol extract is carried out correlational study.Early-stage Study result
Display, prepares containing a large amount of by-products CAG extract from Milkvetch Root, and a portion is CAG analog.Therefore,
The present invention mainly carries out correlational study to CAG extract and analog therein, contributes to promoting CAG extract to move towards state
Market, border, provides certain technical support and theoretical foundation for exploitation defying age relevant healthcare product and medicine simultaneously.
(3) summary of the invention
It is an object of the invention to provide a kind of Cycloastragenol extract and preparation method thereof and purposes, preparation method of the present invention
Astragaloside in the Radix Astragali can be effectively hydrolyzed to Cycloastragenol, the method has certain selectivity, can improve ring yellow
The content of stilbene alcohol, and the easily controllable optimization of reaction condition, technological operation is easy, compared to other two kinds of sides of preparation of existing report
Method acid hydrolysis and enzymatic isolation method, last shorter.The Cycloastragenol extract that the present invention prepares has anti-aging effects, can be used for
Prepare antiaging agent and health product.
For achieving the above object, the present invention adopts the following technical scheme that
A kind of Cycloastragenol extract, prepares by the following method:
A radix astragali coarse powder (granularity is 20 mesh) is extracted by () with the alkaline alcohol aqueous solution of pH value 11～13, solvent is evaporated off
Dried acquisition astragaloside crude extract；
In step (a), concrete, the operational approach of described extraction is: by the alkaline alcohol of radix astragali coarse powder Yu pH value 11～13
Aqueous solution, with feed liquid mass ratio 1:6～10 mixing, soaks 12～16h, is warming up to reflux, extract, 2～3h afterwards, filters, obtains one
Secondary filtrate and a filtering residue；By the alkaline alcohol aqueous solution of filtering residue of gained and pH value 11～13 with feed liquid mass ratio 1:8～10
Mixing, is warming up to reflux, extract, 2～3h, filters, obtains secondary filtrate and secondary filtering residue；Discard secondary filtering residue, merge and once filter
Liquid and secondary filtrate, remove solvent (45～60 DEG C) under reduced pressure, postlyophilization (use vacuum freeze drier carry out), obtain
Astragaloside crude extract；
Preferably described 75% (v/v) ethanol water that alkaline alcohol aqueous solution is pH value 11～13；More preferably described
75% (v/v) ethanol water that alkaline alcohol aqueous solution is pH value 13；Described alkaline alcohol aqueous solution sodium hydroxide is carried out
Regulation realizes；
B () step (a) gained astragaloside crude extract passes through macroporous resin enrichment purification, obtain astragaloside extract；
In step (b), concrete, the operational approach of described macroporous resin enrichment purification is: by molten for astragaloside crude extract
In the water of 2～10 mass times, obtain astragaloside crude extract, be added into being filled with in the chromatographic column of macroporous resin, static
Absorption 4～6h after, successively with the water of 5～20 times of resin column volumes, 30%～40% (v/v, preferably 30%) ethanol water,
70% (v/v) ethanol water eluting, collects 70% (v/v) ethanol water elution liquid, removes solvent (45～60 DEG C) under reduced pressure, obtain
Astragaloside extract；
Described macroporous resin one in ADS-17, AB-8, D101, HPD300, preferably D101 resin；
C () carries out Smith degraded to step (b) gained astragaloside extract, it is thus achieved that described Cycloastragenol extract；
In step (c), concrete, the operational approach of described Smith degraded is: by astragaloside extract, sodium metaperiodate
Join in pure water or 20%～90% (v/v, preferably 20%) methanol aqueous solution A, in 0～35 DEG C carry out oxidation reaction 11～
13h, the reaction of spent glycol cancellation afterwards, reactant liquor first carries out concentrating under reduced pressure, then is extracted with ethyl acetate, and extract is evaporated off molten
Agent, residual substance is dissolved in 40%～90% (v/v, preferably 60%) methanol aqueous solution B, adds sodium borohydride, enters in 0～35 DEG C
Row reduction reaction 3～5h, then with concentrated sulphuric acid (98wt%) regulation reaction system pH to 1～3, room temperature (20～30 DEG C) hydrolysis 22～
26h, after hydrolysis, reactant liquor is extracted with ethyl acetate, and extract is evaporated off solvent, it is thus achieved that described Cycloastragenol extract；
Described astragaloside extract is 1:0.4～6, preferably 1:1 with the mass ratio of sodium metaperiodate；
Described residual substance is 1:1～5, preferably 1:1 with the mass ratio of sodium borohydride；
Recommend the volumetric usage of described pure water or methanol aqueous solution A with the quality of astragaloside extract be calculated as 120～
200mL/g；The volumetric usage recommending described methanol aqueous solution B is calculated as 150～800mL/g with the quality of residual substance；
Described methanol aqueous solution A, methanol aqueous solution B do not have special implication, be labeled as " A ", " B " be only intended to distinguish
The methanol aqueous solution used in different operating step.
Cycloastragenol content in the Cycloastragenol extract that the present invention prepares is 16.45%～32.58%.
The Cycloastragenol extract that the present invention prepares can be applicable to the preparation of antiaging agent and health product.
The beneficial effects of the present invention is:
(1) preparation of Cycloastragenol extract of the present invention, by the pH value in extraction conditions, sodium metaperiodate consumption, first
The test of many times of determining alcohol, has obtained optimum extracting method, and the method repeatability is good, simple and easy to do；
(2) the Cycloastragenol extract that prepared by the present invention can substantially slow down H2O2The old and feeble injury causing PC12 cell, carries
High cell viability, the Cycloastragenol extract pointing out preparation method of the present invention to obtain has certain activity of fighting against senium.
(4) accompanying drawing explanation
Fig. 1 is astragaloside extract liquid chromatogram in embodiment 17；
Fig. 2 is Cycloastragenol extract liquid chromatogram in embodiment 18；
Fig. 3-A, 3-B are based on HPLC-LTQ-Orbitrap-MS in embodiment 19nThe Cycloastragenol extraction of substance of technology
Spectrogram；Fig. 3-A represents the Cycloastragenol extraction of substance spectrogram under positive ion mode, and Fig. 3-B represents that the ring under negative ion mode is yellow
Stilbene alcohol extraction material spectrogram；
Fig. 4-A, 4-B are the mtt assay detection Cycloastragenol extract impact on PC12 cell in embodiment 20；Fig. 4-A table
Showing the Cycloastragenol extract toxicity to PC12 cell, Fig. 4-B represents that Cycloastragenol extract is to H2O2Cause to decline the guarantor of PC12 cell
Protect effect；Note: the * in figure represents P < 0.05；* represents P < 0.01；
Fig. 5 is the Cycloastragenol extract impact on PC12 cell betagalactosidase activity in embodiment 21；Wherein, A
Representing the staining conditions that matched group is final, B represents the final staining conditions of modeling group, and C represents the dye of low concentration medicament protection group
Pornographic condition, D represent in the staining conditions of acute drug protection group, E represents the staining conditions of high concentration medicine protection group；(↑) table
Showing the PC12 senile cell being colored, scale shows 50 μMs；
Fig. 6 be in embodiment 22 Cycloastragenol extract to H2O2Cause to decline the impact of Cell Telomerase Activity；Note: the * in figure
Represent P < 0.05；* represents P < 0.01.
(5) detailed description of the invention
Below by specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited in
This.
The preparation method of embodiment 1 astragaloside of the present invention crude extract:
The Radix Astragali used meets one pertinent regulations of " Chinese Pharmacopoeia " version in 2010.Take radix astragali coarse powder 30g adding pH is 13
75% ethanol 10 times amount soaked overnight, with same solution reflux, extract, 2 times, each 2h, filters, merging filtrate, removes under reduced pressure
Solvent postlyophilization, it is thus achieved that astragaloside crude extract 0.8084g, through efficient Liquid Detection, determines that Astragaloside content is
0.7399%.
The preparation method of embodiment 2 astragaloside of the present invention crude extract:
The Radix Astragali used meets one pertinent regulations of " Chinese Pharmacopoeia " version in 2010.Take radix astragali coarse powder 30g adding pH is 12
75% ethanol 10 times amount soaked overnight, with same solution reflux, extract, 2 times, each 2h, filters, merging filtrate, removes under reduced pressure
Solvent postlyophilization, it is thus achieved that astragaloside crude extract 0.8080g, through efficient Liquid Detection, determines that Astragaloside content is
0.7381%.
The preparation method of embodiment 3 astragaloside of the present invention crude extract:
The Radix Astragali used meets one pertinent regulations of " Chinese Pharmacopoeia " version in 2010.Take radix astragali coarse powder 30g adding pH is 11
75% ethanol 10 times amount soaked overnight, with same solution reflux, extract, 2 times, each 2h, filters, merging filtrate, removes under reduced pressure
Solvent postlyophilization, it is thus achieved that astragaloside crude extract 0.9088g, through efficient Liquid Detection, determines that Astragaloside content is
0.6596%.
The preparation method of embodiment 4 astragaloside of the present invention extract:
The astragaloside crude extract 3g prepared according to embodiment 1, is dissolved in 10 times amount water, by D101 macropore
The resin column that resin is filled, removes big polar impurity with the water of 10 times amount resin column volumes, 30% ethanol water, uses successively
70% ethanol water eluting, collects 70% ethanol water elution liquid, reclaims ethanol, is evaporated to dry powder, obtains astragaloside
Extract, through efficient Liquid Detection, determines that Astragaloside content is 14.63%.
The preparation method of embodiment 5 astragaloside of the present invention extract:
The astragaloside crude extract 3g prepared according to embodiment 1, is dissolved in 10 times amount water, by D101 macropore
The resin column that resin is filled, removes big polar impurity with the water of 10 times amount resin column volumes, 35% ethanol water, uses successively
70% ethanol water eluting, collects 70% ethanol water elution liquid, reclaims ethanol, is evaporated to dry powder, obtains astragaloside
Extract, through efficient Liquid Detection, determines that Astragaloside content is 9.08%.
The preparation method of embodiment 6 astragaloside of the present invention extract:
The astragaloside crude extract 3g prepared according to embodiment 1, is dissolved in 10 times amount water, by D101 macropore
The resin column that resin is filled, removes big polar impurity with the water of 10 times amount resin column volumes, 40% ethanol water, uses successively
70% ethanol water eluting, collects 70% ethanol water elution liquid, reclaims ethanol, is evaporated to dry powder, obtains astragaloside
Extract, through efficient Liquid Detection, determines that Astragaloside content is 9.91%.
The preparation method of embodiment 7 Cycloastragenol of the present invention extract:
Take the astragaloside extract 200mg obtained according to embodiment 4 method, add the sodium metaperiodate of 0.6 times amount,
In methanol aqueous solution (30mL) environment of 60%, carrying out oxidation reaction 12 hours in 25 DEG C, dropping ethylene glycol (0.0781g) stops
Reaction；Reactant liquor concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, residue
It is dissolved in 60% methanol aqueous solution (20mL), adds the sodium borohydride with residue weight equimultiple, carry out reduction reaction 4 in 25 DEG C
Hour；With the pH to 2 of concentrated sulphuric acid regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic
Phase, removes organic solvent, it is thus achieved that it is 20.11% that Cycloastragenol extract 0.0263g, HPLC record Cycloastragenol content.
The preparation method of embodiment 8 Cycloastragenol of the present invention extract:
Take the astragaloside extract 200mg obtained according to embodiment 4 method, add the sodium metaperiodate of 1 times amount, 60%
Methanol aqueous solution (30mL) environment in, carry out oxidation reaction 12 hours in 25 DEG C, dropping ethylene glycol (0.0985g) stops anti-
Should；Reactant liquor concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, and residue is molten
In 60% methanol aqueous solution (20mL), add the sodium borohydride with residue weight equimultiple, carry out reduction reaction 4 in 25 DEG C little
Time；With the pH to 2 of concentrated sulphuric acid regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic facies,
Remove organic solvent, it is thus achieved that it is 25.44% that Cycloastragenol extract 0.0171g, HPLC record Cycloastragenol content.
The preparation method of embodiment 9 Cycloastragenol of the present invention extract:
Take the astragaloside extract 1000mg obtained according to embodiment 4 method, add the sodium metaperiodate of 1 times amount,
In methanol aqueous solution (112mL) environment of 90%, carrying out oxidation reaction 12 hours in 15 DEG C, dropping ethylene glycol (0.42g) stops
Reaction；Reactant liquor concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, residue
It is dissolved in 60% methanol aqueous solution (100mL), adds the sodium borohydride of residue weight 3 times amount, carry out reduction reaction 4 in 15 DEG C little
Time；With the pH to 2 of concentrated sulphuric acid regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic facies,
Remove organic solvent, it is thus achieved that it is 25.05% that Cycloastragenol extract 0.062g, HPLC record Cycloastragenol content.
The preparation method of embodiment 10 Cycloastragenol of the present invention extract:
Take the astragaloside extract 200mg obtained according to embodiment 4 method, add the sodium metaperiodate of 1 times amount, 20%
Methanol aqueous solution (30mL) environment in, carry out oxidation reaction 12 hours in 35 DEG C, dropping ethylene glycol (0.1025g) stops anti-
Should；Reactant liquor concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, and residue is molten
In 60% methanol aqueous solution (20mL), add the sodium borohydride with residue weight equimultiple, carry out reduction reaction 4 in 35 DEG C little
Time；With the pH to 2 of concentrated sulphuric acid regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic facies,
Remove organic solvent, it is thus achieved that it is 32.58% that Cycloastragenol extract 0.0291g, HPLC record Cycloastragenol content.
The preparation method of embodiment 11 Cycloastragenol of the present invention extract:
Take the astragaloside extract 200mg obtained according to embodiment 4 method, add the sodium metaperiodate of 3 times amount, 60%
Methanol aqueous solution (30mL) environment in, carry out oxidation reaction 12 hours in 10 DEG C, dropping ethylene glycol (0.2772g) stops anti-
Should；Reactant liquor concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, and residue is molten
In 60% methanol aqueous solution (20mL), add the sodium borohydride with residue weight equimultiple, carry out reduction reaction 4 in 5 DEG C little
Time；With the pH to 2 of concentrated sulphuric acid regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic facies,
Remove organic solvent, it is thus achieved that it is 23.07% that Cycloastragenol extract 0.0254g, HPLC record Cycloastragenol content.
The preparation method of embodiment 12 Cycloastragenol of the present invention extract:
Take the astragaloside extract 200mg obtained according to embodiment 4 method, add the sodium metaperiodate of 5 times amount, 60%
Methanol aqueous solution (30mL) environment in, carry out oxidation reaction 12 hours in 15 DEG C, drip ethylene glycol (0.42g) stopped reaction；
Reactant liquor concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, and residue is dissolved in
In 60% methanol aqueous solution (20mL), add the sodium borohydride with residue weight equimultiple, carry out reduction reaction 4 in 10 DEG C little
Time；With the pH to 2 of concentrated sulphuric acid regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic facies,
Remove organic solvent, it is thus achieved that it is 20.71% that Cycloastragenol extract 0.0329g, HPLC record Cycloastragenol content.
The preparation method of embodiment 13 Cycloastragenol of the present invention extract:
Take the astragaloside extract 200mg obtained according to embodiment 4 method, add the sodium metaperiodate of 5 times amount, at pure water
In solution (30mL) environment, carry out oxidation reaction 12 hours in 10 DEG C, drip ethylene glycol (0.4364g) stopped reaction；Reactant liquor
Concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, and residue is dissolved in 60% first
In alcohol-water solution (20mL), with the sodium borohydride of residue weight equimultiple, carry out reduction reaction 4 hours in 10 DEG C；Use concentrated sulphuric acid
The pH to 2 of regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic facies, removes organic molten
Agent, it is thus achieved that it is 21.71% that Cycloastragenol extract 0.0235g, HPLC record Cycloastragenol content.
The preparation method of embodiment 14 Cycloastragenol of the present invention extract:
Take the astragaloside extract 200mg obtained according to embodiment 4 method, add the sodium metaperiodate of 5 times amount, 20%
Methanol aqueous solution (30mL) environment in, carry out oxidation reaction 12 hours in 10 DEG C, dropping ethylene glycol (0.4360g) stops anti-
Should；Reactant liquor concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, and residue is molten
In 60% methanol aqueous solution (20mL), add the sodium borohydride with residue weight equimultiple, carry out reduction reaction 4 in 10 DEG C little
Time；With the pH to 2 of concentrated sulphuric acid regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic facies,
Remove organic solvent, it is thus achieved that it is 24.17% that Cycloastragenol extract 0.0274g, HPLC record Cycloastragenol content.
The preparation method of embodiment 15 Cycloastragenol of the present invention extract:
Take the astragaloside extract 200mg obtained according to embodiment 4 method, add the sodium metaperiodate of 5 times amount, 60%
Methanol aqueous solution (30mL) environment in, carry out oxidation reaction 12 hours in 3 DEG C, drip ethylene glycol (0.4377g) stopped reaction；
Reactant liquor concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, and residue is dissolved in
In 20% methanol aqueous solution (20mL), add the sodium borohydride with residue weight equimultiple, carry out reduction reaction 4 hours in 5 DEG C；
With the pH to 2 of concentrated sulphuric acid regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic facies, removes
Organic solvent, it is thus achieved that it is 16.45% that Cycloastragenol extract 0.0284g, HPLC record Cycloastragenol content.
The preparation method of embodiment 16 Cycloastragenol of the present invention extract:
Take the astragaloside extract 200mg obtained according to embodiment 4 method, add the sodium metaperiodate of 5 times amount, 60%
Methanol aqueous solution (30mL) environment in, carry out oxidation reaction 12 hours in 8 DEG C, drip ethylene glycol (0.4506g) stopped reaction；
Reactant liquor concentrating under reduced pressure removes methanol, extracts three times by equal-volume ethyl acetate, merges organic facies, removes solvent, and residue is dissolved in
In 60% methanol aqueous solution (20mL), add the sodium borohydride with residue weight equimultiple, carry out reduction reaction 4 hours in 8 DEG C；
With the pH to 2 of concentrated sulphuric acid regulation reactant liquor, Hydrolysis At Room Temperature 24 hours；Reactant liquor is extracted with ethyl acetate, and merges organic facies, removes
Organic solvent, it is thus achieved that it is 18.80% that Cycloastragenol extract 0.0331g, HPLC record Cycloastragenol content.
The determination of Astragaloside content in embodiment 17 astragaloside extract:
1 chromatographic condition
Chromatographic column Phenomenex C18 (4.6mm*250mm, 5 μm)；Flowing phase acetonitrile (A)-0.3% formic acid water (B)；Stream
Speed 0.5mL/min；Column temperature 35 DEG C；Sample size 5 μ L；Drift temperature 40 DEG C；Nitrogen flow rate 1.5L/min；Gradient is as follows: 0-
15min, 66%B；15-20min, 66%-55%B；20-25min, 55%-50%B；25-35min, 50%-49%B；35-
40min, 49%-30%B；40-45min, 30%-0%B.
The preparation of 2 need testing solutions
Take according to embodiment 1 preparation-obtained astragaloside extract 0.8084g in 50mL volumetric flask, fixed with methanol
Holding, ultrasonic dissolution, as need testing solution.
The preparation of 3 reference substance solution
Precision weighs astragaloside reference substance 1.01mg, puts in 1mL volumetric flask, adds methanol and is allowed to dissolve and be diluted to carve
Degree, as reference substance solution.
4 system suitability test
Test sample sample introduction 5 μ L measures, and result shows, the retention time of astragaloside is about 27min, astragaloside in sample
Chromatographic peak is kept completely separate with other impurity peak energy, therefore containing astragaloside in visible sample.Astragaloside extract liquid phase color
Spectrogram is shown in Fig. 1.
The preparation of 5 standard curves and the investigation of the range of linearity
Take reference substance solution sample introduction 3 μ L successively, 5 μ L, 7 μ L, 10 μ L, 15 μ L, measure face, peak by above-mentioned chromatographic condition in accordance with the law
Product value, with the natural logrithm of sample size μ g as abscissa, the natural logrithm of peak area is that vertical coordinate returns, and draws standard bent
Line.Experimental data is through rectilinear regression, and obtaining regression equation is Y=1.3019X+7.3249, R2=0.9984, the results are shown in Table 1.Experiment
Show: astragaloside reference substance is good in the natural logrithm of 3.03 μ g-15.15 μ g sample sizes and the natural logrithm of peak area
Linear relationship.
Table 1 astragaloside range of linearity investigation table
The assay of astragaloside in 6 samples
Take the test sample according to embodiment 1～6 preparation, by above-mentioned test method, prepare the test sample of suitable concentration respectively
Solution detects, and the results are shown in Table 2.
Determination of Astragaloside in table 2 sample
The determination of Cycloastragenol content in embodiment 18 Cycloastragenol extract:
1 chromatographic condition
Chromatographic column Phenomenex C18 (4.6mm*250mm, 5 μm)；Flowing phase acetonitrile (A)-0.3% formic acid water (B)；Stream
Speed 0.5mL/min；Column temperature 35 DEG C；Sample size 5 μ L；Drift temperature 40 DEG C；Nitrogen flow rate 1.5L/min；Gradient is as follows: 0-
10min, 50%-40%B；10-20min, 40%-30%B；20-30min, 30%-25%B；30-35min, 25%-10%B；
35-40min, 10%-0%B.
The preparation of 2 need testing solutions
Take according to embodiment 9 preparation-obtained CAG extract 15mg in 5mL volumetric flask, use 70% methanol aqueous solution
Fixed molten, ultrasonic dissolution, as need testing solution.
The preparation of 3 reference substance solution
Precision weighs Cycloastragenol reference substance 2.04mg, puts in 2mL volumetric flask, adds 70% methanol aqueous solution and is allowed to dissolve also
It is diluted to scale, shakes up, as Cycloastragenol reference substance storing solution.In accurate absorption storing solution 200 μ L to 1mL volumetric flask, add
70% methanol aqueous solution is diluted to scale, and ultrasonic dissolution, as reference substance solution.
4 system suitability test
Test sample sample introduction 5 μ L measures, and result shows, the retention time of Cycloastragenol is about 19min, Cycloastragenol in sample
Chromatographic peak is kept completely separate with other impurity peak energy, therefore containing Cycloastragenol in visible sample.Cycloastragenol extract liquid phase color
Spectrogram is shown in Fig. 2.
The preparation of 5 standard curves and the investigation of the range of linearity
Precision draws reference substance solution 10 μ L, 50 μ L, 100 μ L, 200 μ L, 500 μ L respectively, is placed in 1mL volumetric flask, adds
70% methanol-water is diluted to scale, shakes up, and measures peak area by above-mentioned chromatographic condition, with the natural logrithm of sample size μ g for horizontal seat
Mark, the natural logrithm of peak area is that vertical coordinate returns, and draws standard curve.Experimental data, through rectilinear regression, obtains the side of recurrence
Journey is Y=1.237X+7.2748, R2=0.9986, the results are shown in Table 3.Experiment shows: Cycloastragenol reference substance is at 0.255 μ g-
The natural logrithm of 5.100 μ g sample sizes and the natural logrithm of peak area are good linear relationship.
Table 3 Cycloastragenol range of linearity investigation table
The assay of Cycloastragenol in 6 samples
Taking the test sample according to embodiment 7～16 preparation, by above-mentioned test method, be prepared as suitable concentration respectively supplies examination
Product solution detects, and the results are shown in Table 4.
Cycloastragenol assay in table 4 sample
The composition of embodiment 19 Cycloastragenol extract speculates:
The present invention uses HPLC-LTQ-Orbitrap-MSn technology to obtain the mass spectrum of Cycloastragenol extract sample, root
According to collection of illustrative plates, the comparison of bound fraction standard substance, carry out Structural Identification.Due to the restriction of standard substance kind, the most only to extract
Component is done one and is substantially speculated, with reference for further study.
The preparation of 1 standard solution
Take appropriate astragaloside, Cycloastragenol and formononetin standard substance in 1.5mL centrifuge tube, with the 70% of 800 μ L
Methanol aqueous solution ultrasonic dissolution, standby.
The preparation of 2 sample solutions
In view of the multiformity of classes of compounds in extract, choose and obtain containing prepared by the more embodiment 9 of composition
Cycloastragenol extract be sample, weigh about 10mg, with 70% methanol aqueous solution constant volume to 1mL, as sample solution.
3 Mass Spectrometry Conditions
HPLC-LTQ-Orbitrap-MSnMass Spectrometry Conditions: use electric spray ion source (ESI), positive ion mode, sheath air pressure
Power 303.4kPa, assist gas pressure power 34.5kPa, spray voltage 2.5kV, capillary temperature 350 DEG C, pipe lens voltage 96V, capillary
Tube voltage 35V；Sample first uses FT to carry out full scan, mass scan range m/z 120～1200, resolution FS 30000, MS2
7500, second order ms uses dynamic data dependency scanning (data dependent scan, DDS), and collision energy is set to 25%
～35%, take upper level most abundant peak and carry out the scanning of collision induced dissociation (CID) fragment, detect with ion trap (dynode).
Data Analysis Services is carried out by Xcalibur software.
4 experimental results and analysis
Extract mass spectrum under positive and negative ion pattern is shown in Fig. 3-A, 3-B.
The mass spectrometric data of related compound and the matter of existing standard product in MASS SPECTRAL DATA ANALYSIS, comparison lot of documents
Modal data, carries out Preliminary Identification to swarming component in the middle part of extract, and qualification result is as shown in table 5, table 6.
Under table 5 positive ion mode, in mass spectrum, peak component is identified
Under table 6 negative ion mode, in mass spectrum, peak component is identified
Embodiment 20CAG extract is to H2O2The impact of the PC12 cytoactive of oxidative damage:
1 experiment material
1.1 medicines: Cycloastragenol extract is prepared by preparation method described in embodiment 9, purity is 25.05%.Before use
The mother solution of 200mg/mL it is configured to AG DMSO, degerming standby.
1.2 materials: PC12 cell is presented by China Medicine University professor Li Ping.DMEM culture medium, hyclone (FBS),
Horse serum (HS), trypsin, tetrazolium bromide (MTT) powder, PBS buy from Yu Jie bio tech ltd；Hydrogen peroxide, DMSO
Buy from Sigma company.
1.3 instruments and apparatus: sterilizing room, super-clean bench, low speed centrifuge, ordinary optical microscope, alcohol burner, culture dish,
Centrifuge tube, microplate reader, concussion instrument.
2 experimental procedures
2.1 cells are cultivated: be incubated at by PC12 cell containing dual anti-(the blue or green strepto-of 10% hyclone, 5% horse serum and 1%
Element mixed liquor) DMEM culture medium in, be placed in 5%CO2, saturated humidity, hatch in 37 DEG C of incubators.During cell attachment about 80%
Pass on 0.25% trypsinization.
The cytotoxicity experiment of 2.2 Cycloastragenol extracts
2.2.1 cell detection pre-treatment: it is 2.0*10^5/mL that PC12 cell when adherent about 80% is made density
Cell suspension, is inoculated in 96 porocyte culture plates according to the volume of every hole 200 μ L, cultivates to cell in above-mentioned incubator
Packet transaction when adherent about 80%.Arrange matched group, zeroing group and administration group (administration concentration is followed successively by: 0.01mg/mL,
0.1mg/mL、0.5mg/mL、0.8mg/mL、1.0mg/mL、2.0mg/mL、5.0mg/mL、10mg/mL).Be administered 24 hours laggard
Row MTT detects.
2.2.2MTT detection: every hole adds 20 μ LMTT solution, continues to cultivate 4 hours, and exhaust culture fluid, and every hole adds
DMSO 150 μ L, shakes 15min, and surveying detection wavelength by microplate reader is 570nm, and reference wavelength is the OD value of 630nm, according to OD value
Calculating cell viability, computing formula is as follows:
According to cell viability, calculate cell inhibitory rate=1-cell viability.Medicine poison is drawn out according to cell inhibitory rate
Linearity curve.
The protective effect experiment of 2.3 Cycloastragenol extracts
2.3.1 cell detection pre-treatment: be administered pre-treatment with described in 2.2.Arrange matched group, modeling group, zeroing group and to
Medicine group (administration concentration is followed successively by 0.001mg/mL, 0.01mg/mL, 0.05mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL).
After being administered 12 hours, with the H that concentration is 800 μMs of DMEM preparation2O2Solution carries out modeling, carries out MTT detection after 6 hours.
2.3.2MTT detection: step is with described in 2.2.Protective effect of drug block diagram is drawn out finally according to cell viability.
3. experimental result
The Cycloastragenol extract that result display embodiment 9 prepares is 1.39mg/mL to the IC50 value of PC12 cell.
In experimental concentration scope 0.001mg/mL～0.1mg/mL, this extract is to H2O2The PC12 cyton causing to decline has been revealed necessarily
Protective effect.Result is shown in Fig. 4-A, 4-B.
The impact on PC12 cell betagalactosidase activity of the embodiment 21CAG extract:
1. experiment material
1.1 medicines: described in embodiment 20.
1.2 materials: PC12 cell is presented by China Medicine University professor Li Ping.DMEM culture medium, hyclone (FBS),
Horse serum (HS), trypsin, PBS buy from Yu Jie bio tech ltd；Hydrogen peroxide, DMSO buy from Sigma company；
Senescence associated-β-galactosidase staining kit is bought from green skies Bioisystech Co., Ltd.
1.3 instruments and apparatus: sterilizing room, super-clean bench, low speed centrifuge, ordinary optical microscope, alcohol burner, culture dish,
Centrifuge tube, concussion instrument, thermostat water bath.
2 experimental procedures
2.1 cells are cultivated: described in embodiment 20.
2.2 cell detection pre-treatments: it is the thin of 4.0*10^5/mL that PC12 cell when adherent about 80% is made density
Born of the same parents' suspension, is inoculated in 24 porocyte culture plates according to the volume of every hole 800 μ L, cultivates to cell patch in above-mentioned incubator
Packet transaction during wall about 80%.(administration concentration is followed successively by 0.001mg/mL, 0.05mg/ to arrange matched group, modeling group and administration group
mL、0.1mg/mL).After being administered 12 hours, with the H that concentration is 800 μMs of DMEM preparation2O2Solution carries out modeling, and 6 hours laggard
Row beta galactosidase dyeing test experience.
2.3 senescence associated-β-galactosidase Activity determination: a. absorb cell culture fluid, washed once with PBS, and every hole adds
Entering 250 μ L beta galactosidase dyeing fixatives, room temperature fixes 15min；B. absorb cell fixative, with PBS wash three times, often
Secondary three minutes；C. absorbing PBS, every hole adds 250 μ L dyeing working solutions (dyeing liquor A: dyeing liquor B: dyeing liquor C:X-Gal solution
=1:1:93:5；v/v/v/v)；D.parafilm seals 24 orifice plates and prevents evaporation, overnight incubation in 37 DEG C of water-baths；E. absorb
Dyeing working solution, washed once with PBS, is placed under ordinary optical microscope observation.
3 experimental results
Result shows, compared to cellular control unit, through H2O2The cell dyeing situation processed becomes apparent from, and relative to making
Module cell, the cell dyeing situation under low administration concentration is more or less the same with modeling group, and the dyeing under middle and high administration concentration is thin
Born of the same parents' number is less than modeling group, and the staining cell number of administration group is on a declining curve with the rising of administration concentration.Due to senile cell table
Have the beta galactosidase of enzymatic activity high when reaching pH6.0, and test kit be just with X-Gal as substrate, senescence-specific β-
Can produce navy blue product under galactoside enzyme catalysis, the cell being i.e. colored is in the cell of aging state.Thus may be used
See, to H2O2Causing the PC12 cell declined, Cycloastragenol extract has embodied certain activity of fighting against senium.Result is shown in Fig. 5.
The embodiment 22 Cycloastragenol extract activity of telomerase to PC12 cell:
1 experiment material
1.1 medicines: described in embodiment 20.
1.2 materials: PC12 cell is presented by China Medicine University professor Li Ping.DMEM culture medium, hyclone (FBS),
Horse serum (HS), trypsin, PBS buy from Yu Jie bio tech ltd；Hydrogen peroxide, DMSO buy from Sigma company；
RIPA lysate (by force), PMSF (100mM) buy from green skies Bioisystech Co., Ltd；Rat telomerase (TE) enzyme linked immunological
Analyze (ELISA) test kit to buy from river, Shanghai Lay biology company limited.
1.3 instruments and apparatus: sterilizing room, super-clean bench, low speed centrifuge, inverted microscope, alcohol burner, culture dish, centrifugal
Pipe, concussion instrument, refrigerated centrifuge, ice bag.
2 experimental procedures
2.1 cells are cultivated: described in embodiment 20.
2.2 cell detection pre-treatments: it is the thin of 5.0*10^5/mL that PC12 cell when adherent about 80% is made density
Born of the same parents' suspension, is inoculated in 6 porocyte culture plates according to the volume of every hole 2mL, cultivates to cell attachment in above-mentioned incubator
Packet transaction when about 80%.Arrange matched group, the positive controls Cycloastragenol monomer of 2 μMs (concentration be) and Cycloastragenol to extract
Thing administration group (administration concentration is followed successively by 0.01mg/mL, 0.1mg/mL, 0.5mg/mL).After being administered 12 hours, with DMEM preparation
Concentration is the H of 800 μMs2O2Solution carries out modeling, carries out the preparation of cell pyrolysis liquid after 6 hours.
The preparation of 2.3 cell pyrolysis liquids: a. absorbs cell culture fluid, washes twice with PBS；B. absorbing PBS, every hole adds
The lysate 400 μ L prepared, shakes 30min；C. repeatedly blow and beat with 1mL liquid-transfering gun, lysate is drawn to 1.5mL centrifuge tube
In, 4 DEG C are centrifuged, 1500rpm, 15min；D. liquid-transfering gun Aspirate supernatant 300 μ L/ props up in 1.5mL centrifuge tube, sealing, dry ice
Preserve under environment, censorship.
3 experimental results
Result shows, the telomerase activation of positive controls and administration group is significantly higher than matched group, and the telomere of administration group
Enzymatic activity increases along with the increase of administration concentration, it is seen that in the range of experimental concentration, the ring Radix Astragali prepared by embodiment 9
Alcohol extraction thing has more significantly Activation of Telomerase effect, points out this extract to have good anti-aging effects.Result is shown in
Fig. 6.
Claims (9)
1. a Cycloastragenol extract, it is characterised in that described Cycloastragenol extract prepares by the following method:
A the alkaline alcohol aqueous solution of radix astragali coarse powder pH value 11～13 is extracted by (), obtain Radix Astragali first after solvent seasoning is evaporated off
Glycosides crude extract；
B astragaloside crude extract that step (a) is obtained by () is dissolved in the water of 2～10 mass times, obtains astragaloside crude extract,
Be added into being filled with in the chromatographic column of macroporous resin, after static adsorption 4～6h, successively with the water of 5～20 times of resin column volumes,
Volume fraction 30%～40% ethanol water, volume fraction 70% ethanol water eluting, collected volume mark 70% ethanol
Water elution liquid, removes solvent under reduced pressure, obtains astragaloside extract；
Described macroporous resin one in ADS-17, AB-8, D101, HPD300；
C astragaloside extract, sodium metaperiodate that step (b) is obtained by () join pure water or volume fraction 20%～90%
In methanol aqueous solution A, carrying out oxidation reaction 11～13h in 0～35 DEG C, spent glycol cancellation afterwards is reacted, and reactant liquor is first carried out
Concentrating under reduced pressure, then be extracted with ethyl acetate, extract is evaporated off solvent, and residual substance is dissolved in volume fraction 40%～90% methanol-water
In solution B, add sodium borohydride, carry out reduction reaction 3～5h in 0～35 DEG C, then by concentrated sulphuric acid regulation reaction system pH to 1～
3, Hydrolysis At Room Temperature 22～26h, after hydrolysis, reactant liquor is extracted with ethyl acetate, and extract is evaporated off solvent, it is thus achieved that described ring is yellow
Stilbene alcohol extraction thing；
Described astragaloside extract is 1:0.4～6 with the mass ratio of sodium metaperiodate；Described residual substance and the matter of sodium borohydride
Amount ratio is 1:1～5.
2. Cycloastragenol extract as claimed in claim 1, it is characterised in that in step (a), the operational approach of described extraction
For: radix astragali coarse powder is mixed with feed liquid mass ratio 1:6～10 with the alkaline alcohol aqueous solution of pH value 11～13, soaks 12～16h, it
After be warming up to reflux, extract, 2～3h, filter, obtain first-time filtrate and a filtering residue；By filtering residue of gained and pH value 11～13
Alkaline alcohol aqueous solution with feed liquid mass ratio 1:8～10 mixing, be warming up to reflux, extract, 2～3h, filter, obtain secondary filtrate and
Secondary filtering residue；Discard secondary filtering residue, merge first-time filtrate and secondary filtrate, remove solvent under reduced pressure, postlyophilization, obtain Huang
Stilbene first glycosides crude extract.
3. Cycloastragenol extract as claimed in claim 1 or 2, it is characterised in that described alkaline alcohol aqueous solution is pH value
Volume fraction 75% ethanol water of 11～13, described alkaline alcohol aqueous solution sodium hydroxide is adjusted realizing.
4. Cycloastragenol extract as claimed in claim 1, it is characterised in that in step (b), described macroporous resin is
D101 resin.
5. Cycloastragenol extract as claimed in claim 1, it is characterised in that in step (c), described pure water or methanol-water
The volumetric usage of solution A is calculated as 120～200mL/g with the quality of astragaloside extract.
6. Cycloastragenol extract as claimed in claim 1, it is characterised in that in step (c), described methanol aqueous solution B's
Volumetric usage is calculated as 150～800mL/g with the quality of residual substance.
7. Cycloastragenol extract as claimed in claim 1, it is characterised in that in step (c), described astragaloside extract
It is 1:1 with the mass ratio of sodium metaperiodate.
8. Cycloastragenol extract as claimed in claim 1, it is characterised in that in step (c), described residual substance and boron hydrogen
The mass ratio changing sodium is 1:1.
9. the Cycloastragenol extract as claimed in claim 1 application in prepared by antiaging agent and health product.
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| CN110204588A (en) * | 2019-06-12 | 2019-09-06 | 劲牌有限公司 | A method of preparing Astragaloside IV |
| CN111377998A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form C and preparation method thereof |
| CN111378000A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form F and preparation method thereof |
| CN111378004A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form D and preparation method thereof |
| CN111377999A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form B and preparation method thereof |
| CN111378001A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form E and preparation method thereof |
| CN111378002A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Novel cycloastragenol crystal form A and preparation method thereof |
| CN113637046A (en) * | 2021-07-13 | 2021-11-12 | 安徽康信制药股份有限公司 | Method for extracting astragaloside with high extraction rate |
| CN113968894A (en) * | 2021-11-05 | 2022-01-25 | 山西大学 | Method for preparing cycloastragenol by degrading astragaloside |
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| CN111377998A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form C and preparation method thereof |
| CN111378000A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form F and preparation method thereof |
| CN111378004A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form D and preparation method thereof |
| CN111377999A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form B and preparation method thereof |
| CN111378001A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form E and preparation method thereof |
| CN111378002A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Novel cycloastragenol crystal form A and preparation method thereof |
| CN110204588A (en) * | 2019-06-12 | 2019-09-06 | 劲牌有限公司 | A method of preparing Astragaloside IV |
| CN113637046A (en) * | 2021-07-13 | 2021-11-12 | 安徽康信制药股份有限公司 | Method for extracting astragaloside with high extraction rate |
| CN113637046B (en) * | 2021-07-13 | 2024-04-19 | 安徽康信制药有限公司 | Astragaloside IV extraction method with high extraction rate |
| CN113968894A (en) * | 2021-11-05 | 2022-01-25 | 山西大学 | Method for preparing cycloastragenol by degrading astragaloside |
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