CN107496402A - A kind of sulforaphane with resisiting influenza virus effect and its preparation method and application - Google Patents
A kind of sulforaphane with resisiting influenza virus effect and its preparation method and application Download PDFInfo
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- CN107496402A CN107496402A CN201710736749.6A CN201710736749A CN107496402A CN 107496402 A CN107496402 A CN 107496402A CN 201710736749 A CN201710736749 A CN 201710736749A CN 107496402 A CN107496402 A CN 107496402A
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- sulforaphane
- buffer solution
- influenza
- influenza virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/26—Cyanate or isocyanate esters; Thiocyanate or isothiocyanate esters
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C331/00—Derivatives of thiocyanic acid or of isothiocyanic acid
- C07C331/16—Isothiocyanates
- C07C331/18—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms
- C07C331/20—Isothiocyanates having isothiocyanate groups bound to acyclic carbon atoms of a saturated carbon skeleton
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
Abstract
The invention provides a kind of new application of sulforaphane as anti-influenza virus medicament, sulforaphane has the activity of good resisiting influenza virus in low strength range, and without significant cytotoxicity, the quantity of influenza virus infected cell can be improved, there is the potential value as Tamiflu active ingredient.
Description
Technical field
The invention belongs to broccoli extract field, is related to a kind of sulforaphane and its system with resisiting influenza virus effect
Preparation Method and application.
Background technology
Influenza, abbreviation influenza, it is a kind of Acute respiratory infectious disease as caused by influenza virus, is common in
Child, and have the baby, children and the elderly of underlying diseases often prognosis is bad.Influenza virus is a kind of RNA virus, and category is just glutinous
Liquid Viraceae, a variety of hosts such as people, birds and pig can be infected, cause acute upper respiratory infection, even life is threatened when serious
Life.The World Health Organization (WHO) estimation, the annual about 1,000,000,000 people's influenza virus infections in the whole world, wherein there are about 500,000 people therefore
It is dead.
Four types of influenza virus point:A type (Influenza A virus), B-mode (Influenza B virus), third
Type (Influenza C virus) and fourth type (Influenza D virus), wherein A type and influenza B virus are to cause people
The most important reason of class disease and seasonal epidemics.According to viral major structural protein hemagglutinin HA and neuraminidase NA
Antigenicity, influenza A virus is divided into 16 H hypotypes (H1-H16) and 9 N hypotypes (N1-N9).2009, Flu-A disease
The great outburst of the malicious H1N1 whole world, individual countries and regions more than 200 are spread to, nearly 20,000 people of death toll, serious warp are caused to the whole world
Ji loss, this let us clear-headed infectiousness and harmfulness of recognizing that influenza virus is huge.
At present, prevention and treatment influenza mainly uses vaccine.However, influenza virus has extremely strong antigenic mutation, epidemic disease
The effect of seedling is unstable, meanwhile, vaccine only has prevention effect to known influenza virus, invalid to novel influenza.Cause
This, a kind of safe and effective compound with resisiting influenza virus effect of exploitation, has great importance.
Sulforaphane (sulforaphane, SF) is the isothiocyanic acid salt material that a kind of molecular weight is 177.29, mainly
By the precursor substance 4- methanesulfinyl butyl sulphur glucosides (glucoraphanin, GRA or RAA) being present in broccoli, through enzymolysis
Reaction generation.Research finds that sulforaphane can significantly reduce stomach cancer, liver cancer, lung cancer, breast cancer, colon cancer and carcinoma of urinary bladder etc.
The incidence of disease of cancer, in addition, also there is significant work(in treatment myopia, senile dementia, cardiovascular and cerebrovascular disease, hypertension etc.
Effect.
The A of CN 104427981 are disclosed comprising sulforaphane or sulforaphane precursor and the group of mushroom extract or powder
Compound, said composition have the function that treatment, prevention breast cancer;The A of CN 104968342 are disclosed for treating or reducing liver
The sulforaphane of insulin resistance, have the function that to improve hepatic insulin susceptibility;The B of CN 101167741 disclose radish
The anti-cancer combination preparation of sulfane and platinum class medicine, it is mainly used in the treatment of non-small cell lung cancer, ED-SCLC;CN 107007578
A discloses sulforaphane and is preparing the application in treating leukemia medicament, and sulforaphane, which has, promotes apoptosis of leukemia to enter
And suppress the ability of Leukemia Cell Proliferation.However, the technology for being used to prevent and treat influenza there is presently no sulforaphane opens
Show.
The content of the invention
In view of the above-mentioned problems, the present invention provide a kind of sulforaphane and preparation method thereof with resisiting influenza virus effect and
Using sulforaphane has the potential value as anti-influenza virus medicament.
In a first aspect, the invention provides a kind of new application of sulforaphane as anti-influenza virus medicament.
The sulforaphane of the present invention has the activity of good resisiting influenza virus in low strength range, and without significant thin
Cellular toxicity, the quantity for the cell that influenza virus is infected can be improved, there is the potential value as Tamiflu active ingredient.
According to the present invention, the invention provides a kind of application of sulforaphane as the medicine for preparing treatment influenza virus.
Preferably, the concentration of the sulforaphane is 5.0-30.0 μM, for example, can be 5.0 μM, 6.0 μM, 6.25 μM,
8.0 μM, 10.0 μM, 12.0 μM, 12.5 μM, 15.0 μM, 18.0 μM, 20.0 μM, 22.0 μM, 25.0 μM, 28.0 μM or 30.0 μM,
Preferably 6.0-25.0 μM, more preferably 6.25-12.5 μM.
Preferably, the influenza virus includes influenza A virus and/or influenza B virus, preferably Flu-A disease
Poison.
Preferably, the medicine also includes pharmaceutically acceptable carrier.
Preferably, the preparation method of the sulforaphane comprises the following steps:
(1) digest:Buffer solution is added into sample, carries out enzyme digestion reaction;
(2) extract:Extractant is added, supernatant is collected by centrifugation, obtains the sulforaphane.
Preferably, any one in the seed of step (1) described sample including Vegetables in Brassica, flower, stem or leaf or extremely
Few two kinds combination, preferably broccoli seeds.
Preferably, step (1) described buffer solution includes KH2PO4-K2HPO4Buffer solution, Na2HPO4- citrate buffer solution, lemon
In lemon acid-sodium citrate buffer solution or acetate buffer solution any one or at least two combination, preferably KH2PO4-
K2HPO4Buffer solution.
Preferably, the concentration of step (1) described buffer solution is 0.05-0.2M, for example, can be 0.05M, 0.06M,
0.07M、0.08M、0.09M、0.10M、0.11M、0.12M、0.13M、0.14M、0.15M、0.16M、0.17M、0.18M、0.19M
Or 0.20M, preferably 0.1M.
Preferably, the pH value of step (1) described buffer solution is 6.0-8.0, for example, can be 6.0,6.1,6.2,6.3,
6.4th, 6.5,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9 or 8.0, be preferably
7.0。
Preferably, the solid-liquid ratio of step (1) sample and the buffer solution is 1:(20-50), such as can be 1:20、
1:22、1:25、1:28、1:30、1:32、1:35、1:38、1:40、1:42、1:45、1:48 or 1:50, preferably 1:30.
Preferably, the temperature of step (1) described enzyme digestion reaction is 15-35 DEG C, for example, can be 15 DEG C, 16 DEG C, 17 DEG C, 18
℃、19℃、20℃、21℃、22℃、23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃、31℃、32℃、33
DEG C, 34 DEG C or 35 DEG C, preferably 25 DEG C.
Preferably, the time of step (1) described enzyme digestion reaction is 1.0-2.0h, for example, can be 1.0h, 1.1h, 1.2h,
1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h or 2.0h, preferably 1.5h.
Preferably, step (2) described extractant include ethyl acetate, petroleum ether, benzene or chloroform in any one or extremely
Few two kinds combination, preferably ethyl acetate.
Preferably, the speed of step (2) described centrifugation is 4000-6000rpm, for example, can be 4000rpm, 4200rpm,
4500rpm, 4800rpm, 5000rpm, 5200rpm, 5500rpm, 5800rpm or 6000rpm, preferably 5500rpm.
Preferably, the time of step (2) described centrifugation is 5-20min, for example, can be 5min, 6min, 7min, 8min,
9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min, preferably
For 10min.
Preferably, also including being evaporated the step of constant volume filters after step (2).
Preferably, described the step of being evaporated constant volume filtering, specifically includes:Supernatant is evaporated, entered after constant volume using filter membrane
Row filtering.
Preferably, the temperature being evaporated is 30-40 DEG C, for example, can be 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C, 35
DEG C, 36 DEG C, 37 DEG C, 38 DEG C, preferably 39 DEG C or 40 DEG C, 35 DEG C.
Preferably, the solvent of the constant volume includes any one in dimethyl sulfoxide (DMSO), deionized water, ethanol or ethyl oleate
Kind or at least two combination, preferably dimethyl sulfoxide (DMSO).
Preferably, the aperture of the filter membrane is 0.2-0.4 μm, for example, can be 0.2 μm, 0.21 μm, 0.22 μm, 0.23
μm、0.24μm、0.25μm、0.26μm、0.27μm、0.28μm、0.29μm、0.30μm、0.31μm、0.32μm、0.33μm、0.34
μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, preferably 0.39 μm or 0.40 μm, 0.22 μm.
As optimal technical scheme, the preparation method of the sulforaphane comprises the following steps:
(1) digest:It is the KH that 0.05-0.2M, pH are 6.0-8.0 that concentration is added into sample2PO4-K2HPO4Buffer solution,
1.0-2.0h is stirred at 15-35 DEG C and carries out enzyme digestion reaction, wherein, the sample and the KH2PO4-K2HPO4The feed liquid of buffer solution
Than for 1:(20-50);
(2) add extractant to be extracted, 5-20min is centrifuged under 4000-6000rpm, collect supernatant, repeat 2-3 times;
(3) supernatant that step (2) obtains is rotated at 30-40 DEG C and be evaporated, added 10.0mL solvents and carry out constant volume, make
After 0.22 μm of filtering membrane filtration, saved backup at -20 DEG C.
Compared with prior art, the present invention has the advantages that:
(1) sulforaphane of the invention has most strong anti influenza in the low strength range that concentration is 6.25-12.5 μM
The activity of virus, and without significant cytotoxicity;
(2) sulforaphane that concentration is 6.25 μM is added in the mdck cell infected to influenza virus, can be improved
19.2% cell number;
(3) sulforaphane is extracted from broccoli seeds using the method for the present invention, content is up to 5512.63mg/L
FW, purity is preferable, and detection peak is not disturbed by impurity.
Brief description of the drawings
Fig. 1 (A) is the chromatogram of sulforaphane standard items and sulforaphane extract, and Fig. 1 (B) is sulforaphane in 5.0-
Linear relationship chart in the range of 300.0mg/L;
Fig. 2 is the fluorescent value after the sulforaphane standard items and sulforaphane extract-treated mdck cell of various concentrations;
Fig. 3 is that the sulforaphane standard items of various concentrations handle the microphoto after mdck cell.
Embodiment
For the technological means and its effect that the present invention is taken is expanded on further, with reference to embodiments with accompanying drawing to this hair
It is bright to be further described.It is understood that embodiment described herein is used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art,
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from
The conventional products of acquisition.
The preparation of the sulforaphane of embodiment 1 (I)
(1) digest:It is the KH that 0.1M, pH are 7.0 that concentration is added into broccoli seeds2PO4-K2HPO4Buffer solution, 25 DEG C
Lower stirring 1.5h carries out enzyme digestion reaction, wherein, the broccoli seeds and the KH2PO4-K2HPO4The solid-liquid ratio of buffer solution is
1:30;
(2) add ethyl acetate to be extracted, 10min is centrifuged under 5500rpm, collect supernatant, repeat 2-3 times;
(3) supernatant that step (2) obtains is rotated at 35 DEG C and be evaporated, added 10.0mL dimethyl sulfoxide (DMSO)s and determined
Hold, after 0.22 μm of filtering membrane filtration, saved backup at -20 DEG C.
The preparation of the sulforaphane of embodiment 2 (II)
(1) digest:It is the Na that 0.15M, pH are 7.5 that concentration is added into blue and white cauliflower2HPO4- citrate buffer solution, 30 DEG C
Lower stirring 1.2h carries out enzyme digestion reaction, wherein, the blue and white cauliflower and the Na2HPO4The solid-liquid ratio of-citrate buffer solution is 1:
40;
(2) add petroleum ether to be extracted, 15min is centrifuged under 5000rpm, collect supernatant, repeat 2-3 times;
(3) supernatant that step (2) obtains is rotated at 32 DEG C and is evaporated, added 10.0mL deionized waters and carry out constant volume,
After 0.3 μm of filtering membrane filtration, saved backup at -20 DEG C.
The preparation of the sulforaphane of embodiment 3 (III)
(1) digest:It is citric acid-sodium citrate buffer solution that 0.05M, pH are 6.0 that concentration is added into broccoli stem, 35
1.0h is stirred at DEG C and carries out enzyme digestion reaction, wherein, the broccoli stem and the solid-liquid ratio of the citric acid-sodium citrate buffer solution
For 1:20;
(2) add benzene to be extracted, 20min is centrifuged under 4000rpm, collect supernatant, repeat 2-3 times;
(3) supernatant that step (2) obtains is rotated at 30 DEG C and be evaporated, added 10.0mL ethanol and carry out constant volume, use
After 0.4 μm of filtering membrane filtration, saved backup at -20 DEG C.
The preparation of the sulforaphane of embodiment 4 (IV)
(1) digest:It is the acetate buffer solution that 0.2M, pH are 8.0 that concentration is added into blue and white dish leaf, and 2.0h is stirred at 15 DEG C
Enzyme digestion reaction is carried out, wherein, the solid-liquid ratio of the blue and white dish leaf and the acetate buffer solution is 1:50;
(2) add chloroform to be extracted, 5min is centrifuged under 6000rpm, collect supernatant, repeat 2-3 times;
(3) supernatant that step (2) obtains is rotated at 40 DEG C and is evaporated, added 10.0mL ethyl oleates and carry out constant volume,
After 0.2 μm of filtering membrane filtration, saved backup at -20 DEG C.
The detection of sulforaphane and content analysis
The detection of sulforaphane is using the SHIMADZU LC-20A chromatographs equipped with SPD-20 UV-detectors, chromatogram
Post uses the anti-phase C18 posts (250 × 4.6mm, 5 μm) of SHISEIDO companies, and the flow velocity of mobile phase is 0.800mLmin-1, inspection
Survey wavelength is 254nm, and the temperature of chromatographic column is 32 DEG C;Elution program uses gradient method, wherein, the eluent of A pumps is the four of 5%
The hydrogen furans aqueous solution, initial concentration are set as that the eluent of 60%, B pumps is hplc grade methanol, and initial concentration is set as 40%,
After 10min, the concentration of B pumps is changed into after 60%, A pumps are changed into 40%, 25min, and B pumps are changed into 100%, now terminate sample introduction, sample introduction
Measure as 10 μ L.
The content of sulforaphane prepared by embodiment 1-4 is as shown in table 1.
Numbering | The content (mg/L FW) of sulforaphane |
Embodiment 1 | 5512.63 |
Embodiment 2 | 5429.67 |
Embodiment 3 | 5298.46 |
Embodiment 4 | 5049.31 |
Wherein, the content highest of sulforaphane prepared by embodiment 1, is 5512.63mg/L FW.From Fig. 1 (A), system
Do not disturbed by impurity at the detection peak of standby obtained sulforaphane;From Fig. 1 (B), sulforaphane is in 5.0-300.0mg/L scopes
Interior to have well linear, linear equation is:
Y=2.76 × 10-4x-0.73(R2=0.9998)
The cytopathic effect of embodiment 5 determines
Mdck cell is inoculated in 96 porocyte plates, is added influenza virus (MOI=0.1) and is infected overnight.
Respectively the mdck cell (C+V) to influenza infection and not in by the mdck cell (C) of influenza infection plus
Enter the sulforaphane extract that concentration is 6.25,12.5,25.0,50.0 and 100.0 μM, after hatching 40h, add fluorescence activity thing
Matter, carried out using Tecan Infinite M2000 PROTM (Tecan Group Ltd., Mannedorf, Switzerland)
Fluorescence reading, while use the observation of microscope progress cytoactive.
Embodiment 6
It is 1000mgL by concentration-1Sulforaphane standard mother liquor (U.S. LKTTM, purity >=98%) diluted with DMSO
Into the standard items that series concentration is 6.25,12.5,25.0,50.0 and 100.0 μM.Compared with Example 5, embodiment 6 adds dense
The sulforaphane standard items for 6.25,12.5,25.0,50.0 and 100.0 μM are spent, other conditions are same as Example 5.
Comparative example 1
Compared with Example 5, comparative example 1 adds medicine Tamiflu (OSV), and other conditions are same as Example 5.
Comparative example 2
Compared with Example 5, comparative example 2 adds the DMSO that concentration is 0.5%, and other conditions are same as Example 5.
As a result as shown in table 2, Fig. 2 and Fig. 3.In table 2 and Fig. 2, C represents the cell not infected by influenza virus, and C+V is represented
The cell infected by influenza virus, capitalization represent there is significant difference in 0.01 level;In Fig. 3, transparent cell
It is show that dead cell quantity is much more.
Influence of the 2 different samples of table to mdck cell fluorescence intensity
As a result show, the mdck cell that the sulforaphane infected by influenza of various concentrations infects has different degrees of resistance
Reaction, concentration are that 6.25-12.5 μM of sulforaphane has the activity of most strong resisiting influenza virus.
From figures 2 and 3, it will be seen that the sulforaphane standard items of embodiment 6 shown in the range of 6.25-25.0 μM it is good
The activity of good resisiting influenza virus, and do not have significant cytotoxicity, and the sulforaphane extract of embodiment 5 is in concentration
The activity of good resisiting influenza virus is shown at 6.25-12.5 μM.
Compared with comparative example 2, in embodiment 5, concentration be 6.25 μM of sulforaphane extract improve 18% it is thin
Born of the same parents' number, in embodiment 6,19.2% He has been respectively increased for the sulforaphane standard items of 6.25 μM and 12.5 μM concentration in concentration
17.8% cell number, and the positive drug Tamiflu (OSV) of comparative example 1 improves 40.0% cell number, shows sulforaphane
Activity with resisiting influenza virus.
The sulforaphane extract and standard items of remaining concentration also show the activity of certain resisiting influenza virus, but together
When also show certain cytotoxicity.
In summary, sulforaphane of the invention has well in the low strength range that concentration is 6.25-12.5 μM
The activity of resisiting influenza virus, and without significant cytotoxicity;Sulforaphane is added in the mdck cell infected to influenza virus, can
To improve cell proliferation number;Sulforaphane is extracted from broccoli seeds using the method for the present invention, purity is preferable, and detection peak is not
Disturbed by impurity.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implemented.Art
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosing.
Claims (10)
- A kind of 1. new application of sulforaphane as anti-influenza virus medicament.
- 2. purposes according to claim 1, it is characterised in that the concentration of the sulforaphane is 5.0-30.0 μM, preferably For 6.0-25.0 μM, more preferably 6.25-12.5 μM.
- 3. purposes according to claim 1 or 2, it is characterised in that the influenza virus include influenza A virus and/or Influenza B virus, preferably influenza A virus.
- 4. according to the purposes described in claim any one of 1-3, it is characterised in that the medicine also includes pharmaceutically acceptable Carrier.
- 5. according to the purposes described in claim any one of 1-4, it is characterised in that the preparation method of the sulforaphane is included such as Lower step:(1) digest:Buffer solution is added into sample, carries out enzyme digestion reaction;(2) extract:Extractant is added, supernatant is collected by centrifugation, obtains the sulforaphane.
- 6. purposes according to claim 5, it is characterised in that seed of step (1) the described sample including Vegetables in Brassica, In flower, stem or leaf any one or at least two combination, preferably broccoli seeds.
- 7. the purposes according to claim 5 or 6, it is characterised in that step (1) described buffer solution includes KH2PO4-K2HPO4 Buffer solution, Na2HPO4In-citrate buffer solution, citric acid-sodium citrate buffer solution or acetate buffer solution any one or extremely Few two kinds combination, preferably KH2PO4-K2HPO4Buffer solution;Preferably, the concentration of step (1) described buffer solution is 0.05-0.2M, preferably 0.1M;Preferably, the pH value of step (1) described buffer solution is 6.0-8.0, preferably 7.0;Preferably, the solid-liquid ratio of step (1) sample and the buffer solution is 1:(20-50), preferably 1:30;Preferably, the temperature of step (1) described enzyme digestion reaction is 15-35 DEG C, preferably 25 DEG C;Preferably, the time of step (1) described enzyme digestion reaction is 1.0-2.0h, preferably 1.5h.
- 8. according to the purposes described in claim any one of 5-7, it is characterised in that step (2) described extractant includes acetic acid second In ester, petroleum ether, benzene or chloroform any one or at least two combination, preferably ethyl acetate;Preferably, the speed of step (2) described centrifugation is 4000-6000rpm, preferably 5500rpm;Preferably, the time of step (2) described centrifugation is 5-20min, preferably 10min.
- 9. according to the purposes described in claim any one of 5-8, it is characterised in that also include being evaporated constant volume after step (2) The step of filtering;Preferably, described the step of being evaporated constant volume filtering, specifically includes:Supernatant is evaporated, carried out after constant volume using filter membrane Filter;Preferably, the temperature being evaporated is 30-40 DEG C, preferably 35 DEG C;Preferably, the solvent of the constant volume include dimethyl sulfoxide (DMSO), deionized water, ethanol or ethyl oleate in any one or At least two combination, preferably dimethyl sulfoxide (DMSO);Preferably, the aperture of the filter membrane is 0.2-0.4 μm, preferably 0.22 μm.
- 10. according to the purposes described in claim any one of 5-9, it is characterised in that the preparation method of the sulforaphane includes Following steps:(1) digest:It is the KH that 0.05-0.2M, pH are 6.0-8.0 that concentration is added into sample2PO4-K2HPO4Buffer solution, 15-35 1.0-2.0h is stirred at DEG C and carries out enzyme digestion reaction, wherein, the sample and the KH2PO4-K2HPO4The solid-liquid ratio of buffer solution is 1:(20-50);(2) add extractant to be extracted, 5-20min is centrifuged under 4000-6000rpm, collect supernatant, repeat 2-3 times;(3) supernatant that step (2) obtains is rotated at 30-40 DEG C and be evaporated, added 10.0mL solvents and carry out constant volume, use After 0.22 μm of filtering membrane filtration, saved backup at -20 DEG C.
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WO2009111486A2 (en) * | 2008-03-04 | 2009-09-11 | The Regents Of The University Of California | Reversing the immune decline of aging by a nutraceutical antioxidant in mice |
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