CN103197065B - A kind of detection method of Sparassis crispa antineoplastic immune activity - Google Patents
A kind of detection method of Sparassis crispa antineoplastic immune activity Download PDFInfo
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- CN103197065B CN103197065B CN201310139032.5A CN201310139032A CN103197065B CN 103197065 B CN103197065 B CN 103197065B CN 201310139032 A CN201310139032 A CN 201310139032A CN 103197065 B CN103197065 B CN 103197065B
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Abstract
The invention provides a kind of Sparassis crispa antineoplastic immune activity test method, for indicating and distinguishing Sparassis crispa product, its biologically active is intuitively generically expressed, is convenient to people and selects this series products.Detection method by after stimulating monocyte strain THP-1 cell with Sparassis crispa produce the expression of cell factor INF-γ, the expression of the INF-γ produced is induced to compare with ConA, the biologically active of Sparassis crispa is evaluated by the concentration of corresponding ConA, be denoted as ACIF value, the biologically active indicating Sparassis crispa product directly perceived.
Description
Technical field:
The invention belongs to technical field of biological, be specifically related to a kind of detection method of Sparassis crispa antineoplastic immune activity, active for the antineoplastic immune indicating Sparassis crispa.
Technical background:
The present invention carries out detection and the sign of antineoplastic immune activity mainly for Sparassis crispa, is applicable to detection and the sign of the antineoplastic immune activity of other edible fungis or medicinal fungus simultaneously.
Sparassis crispa is medicine-food two-purpose macro fungi, and living condition is harsh, and resource reserves are more rare, are a kind of very rare famous and precious edible fungis, are only second to the treasures such as Cordyceps sinensis, hickory chick, ferfas in edible fungi.Because wild Sparassis crispa is rare, and manually cultivate difficulty greatly, therefore have the title of " mushroom of illusion ".Until just realize the artificial cultivation of Sparassis crispa in recent years.
Sparassis crispa is nutritious, and containing the composition such as multivitamin, amino acid, the most outstanding feature is that beta-dextran content is high, its beta-dextran content be mushroom class.Existing experiment shows, Sparassis crispa beta-dextran content accounts for more than 40% of Sparassis crispa dry weight, the several times of glossy ganoderma, Agricus blazei beta-dextran content, wherein β-1,3-D beta-dextran content accounts for about 70% of glucosan total amount, and the polysaccharide mostly with antitumor activity is all with 1, β-1, the 3-D glucosan of 6 branches.Brainstrust is learnt by X-ray diffraction analysis, β-1, polypeptide on unique helical structure that 3-D-glucan presents and side chain, lipid group are the important origin causes of formation playing anti-tumor function, its molecular structure contains protein component, belong to Sparassis crispa proteglycan complex (Sparassiscrispa (Wulf.) Fr.GlucanProtein, SGP).
Research finds, the same with most edible fungi polysaccharide, SGP has antitumor activity, and this activity is high compared with other active polysaccharides.Clinical testing has confirmed that SGP has good inhibition for kinds cancers such as lung cancer, cancer of the stomach, colon cancer, breast cancer.SGP not only has antitumor activity, and can improve body hematopoiesis function.
At present, the research about Sparassis crispa focuses mostly at the activity research of the extraction process of the optimization of condition of culture, Sparassis crispa glucosan, Sparassis crispa glucosan and other compositions, but there is not been reported for the activity test method of Sparassis crispa.Along with the raising of quality of life, developing rapidly of food and health products trade, Sparassis crispa also will be applied in each field.Therefore, set up the activity test method of a kind of Sparassis crispa, and show intuitively by straightaway mode, for applying of Sparassis crispa series products, there is far reaching significance.The activity test method of described Sparassis crispa is also applicable to the antineoplastic immune Activity determination of other edible fungi or medicinal fungus.
Summary of the invention:
For the existing research of Sparassis crispa, the object of the invention is to design the technical scheme that a kind of Sparassis crispa antineoplastic immune activity test method is provided, for indicating and distinguishing Sparassis crispa product, its biologically active is intuitively generically expressed, is convenient to people and selects this series products.
Scientists study finds, SGP has surprising effect to tumour, diabetes, angiocardiopathy etc., is described as " immune gold ".SGP its biological function after chemical method process disappears totally, and reason is to destroy the reactive group on the helical structure of SGP, branch and side chain.It can thus be appreciated that the biologically active of SGP and the molecular weight of glucosan, molecular configuration, branched structure are relevant.
Due to Sparassis crispa bioactive ingredients SGP, complex structure, purification difficult, the SGP of exhaustive purification, natural structure is destroyed, thus loses its intrinsic biologically active.The SGP of purifying, beta-dextran content is high, and there is no natural bioactive, thus the beta-dextran content of Sparassis crispa product is detected, the biologically active evaluating Sparassis crispa product is had little significance, exploitation one can indicate the bioactive detection method of Sparassis crispa, and the biologically active of direct-detection Sparassis crispa product, is necessary very much.
Active available antineoplastic immune active factors level (AnticancerImmuneFactor, ACIF) of antineoplastic immune of SGP indicates.SGP has antineoplastic immune activity, but its immunoregulation capability improving body not directly acts on tumour cell and pathogenic microorganism, but is played a role by stimulation, the immune cell activated release cells factor.IFN-γ is produced by activated lymphocytes, has antiviral, antitumor activity, participates in immunological regulation.Therefore, the height of IFN-γ concentration can be used as a kind of evaluation index of Organism immunoregulation ability.
Concanavalin A (ConA) is a kind of phytolectin, can produce the cell factors such as IFN-γ by inducing cell.
The present invention adopts monocyte strain THP-1 cell, by this cell after Sparassis crispa stimulates produce the expression of cell factor INF-γ, the expression of the INF-γ produced is induced to compare with ConA, the biologically active of Sparassis crispa is evaluated by the concentration of corresponding ConA, be denoted as ACIF value, the biologically active indicating Sparassis crispa product directly perceived.
The invention has the beneficial effects as follows: the high flux achieving Sparassis crispa antineoplastic immune activity detects, the antineoplastic immune of sign Sparassis crispa product directly perceived is active, testing process is succinct, and testing result is reliable and stable, and the present invention is quantitative measurement edible fungi or medicinal fungus biologically active provides the foundation.
A detection method for Sparassis crispa antineoplastic immune activity, is characterized in that comprising following processing step:
1) get Sparassis crispa proteglycan complex (SGP) sample, be mixed with solution;
2) THP-1 cell is got, adjustment cell density to 10
5~ 10
6individual/ml;
3) to step 2) in cultivate SGP solution to the final concentration adding step 1) in the cell that obtains and reach 10 μ g ~ 2.0mg/ml, then cultivate;
4) the culture collected after centrifugation cell conditioned medium liquid that step 3) obtains is got;
5) get the cell conditioned medium liquid ELASA method detection IFN-γ concentration that step 4) obtains, be within the scope of 10 μ g ~ 2.0mg/ml at SGP activity, IFN-γ concentration and SGP concentration have obvious positive correlation.
6) get ConA, detect IFN-γ concentration by above-mentioned steps, control ConA final concentration is 1-3 μ g/ml, with 10 times of ConA concentration value, as antineoplastic immune active factors level (AnticancerImmuneFactor, ACIF) value.
In one embodiment, the detection method of described a kind of Sparassis crispa antineoplastic immune activity, comprises following processing step:
1) get SGP sample, be mixed with solution with RPMI1640 nutrient culture media;
2) THP-1 cell is got, with RPMI1640 nutrient culture media adjustment cell density to 10
5~ 10
6individual/ml, cultivates 24h at 37 DEG C;
3) to step 2) add the SGP solution of step 1) in cultured cells and make SGP final concentration reach 10 μ g ~ 2.0mg/ml, be then placed in 37 DEG C, CO
2the CO2gas incubator of concentration 5% cultivates 24 ~ 48h;
4) culture that step 3) obtains is got with collecting cell supernatant after the centrifugal 20min of 1000g;
5) get the cell conditioned medium liquid ELASA method detection IFN-γ concentration that step 4) obtains, namely employment IFN-γ enzyme-linked immunoassay kit detects IFN-γ concentration: (people IFN-γ enzyme-linked immunoassay kit is existing product).
Be that within the scope of 10 μ g ~ 2.0mg/ml, IFN-γ concentration and SGP have obvious positive correlation at SGP activity.
6) get ConA, detect IFN-γ concentration by above-mentioned steps, control ConA final concentration is 1-3 μ g/ml, with 10 times of ConA concentration value, as ACIF value.
Detection method of the present invention is also applicable to the antineoplastic immune Activity determination of other edible fungi or medicinal fungus.
Described edible fungi or medicinal fungus also comprise grifola frondosus, mushroom, Cordyceps sinensis etc.
Accompanying drawing explanation
Fig. 1 is Sparassis crispa proteglycan complex biological Activity determination figure;
Fig. 2 is grifolan biologically active detection figure;
Fig. 3 is lentinan biologically active detection figure;
Fig. 4 is Cordyceps sinensis polysaccharide biologically active detection figure.
In figure: * * represents P<0.05, * * * represents P<0.01.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1: Sparassis crispa proteglycan complex (SGP) biologically active detects
With the SGP solution of RPMI1640 nutrient culture media preparation 1mg/ml;
With RPMI1640 nutrient culture media, THP-1 cell density is adjusted to 10
5-10
6individual/ml;
The cell adjusting density is placed in cell chulture dish, at 37 DEG C, cultivates 24h;
Add appropriate 1mg/mlSGP solution in culture dish, be respectively 0 μ g/ml, 10 μ g/ml, 100 μ g/ml, 200 μ g/ml to SGP solution final concentration;
37 DEG C, CO
2the CO2gas incubator of concentration 5% cultivates 48h;
With 1000g centrifugal 20min collecting cell supernatant;
Detect IFN-γ concentration by ELASA method, namely employment IFN-γ enzyme-linked immunoassay kit detects IFN-γ concentration: (people IFN-γ enzyme-linked immunoassay kit is existing product)
Standard items and sample to be tested are got 100 μ l respectively and are joined in ELISA Plate hole, carry out mark;
Respectively to adding 50 μ l enzyme connection affinants in hole, fully mixing, avoiding producing bubble;
ELISA Plate overlay film is in 37 DEG C of incubation reaction 60min, and fully abandon liquid in most hole and detain residue raffinate in dry hole, the cleaning fluid good with beforehand dilution rinses 5 times repeatedly, and detains remaining liq in dry hole;
Every hole adds each 50 μ l of substrate I and II respectively according to order, fully mixes.Lucifuge reaction 15min under room temperature;
After reaction, every hole adds stop buffer 50 μ l, fully mixes, cessation reaction;
Microplate reader is used to read OD value at 450 nm in 30min.
As shown in Figure 1, Fig. 1 tentatively determines that the method is effective to Sparassis crispa proteglycan complex biological Activity determination to experimental result.
Embodiment 2: grifolan Activity determination
Get grifolan and dry sample, prepare the grifolan solution of 1mg/ml with RPMI1640 nutrient culture media or distilled water;
With RPMI1640 nutrient culture media, THP-1 cell density is adjusted to 10
5~ 10
6individual/ml;
The cell adjusting density is placed in cell chulture dish, at 37 DEG C, cultivates 24h or 36h;
Add appropriate 1mg/ml grifolan in culture dish, be respectively 0 μ g/ml, 10 μ g/ml, 100 μ g/ml, 200 μ g/ml to grifolan final concentration;
37 DEG C, CO
2the CO2gas incubator of concentration 5% cultivates 36 or 48h;
With 1000g centrifugal 20min collecting cell supernatant;
ELASA method detects IFN-γ concentration, and method is with embodiment 1.
There are some researches show that grifolan has immunoregulatory activity, result shown in Fig. 2 and existing achievement in research basically identical, same provable grifolan has immunoregulatory activity, therefore can verify the validity of this detection method.
Embodiment 3: lentinan Activity determination
Get lentinan and dry sample, with the lentinan solution of RPMI1640 nutrient culture media preparation 5mg/ml;
With RPMI1640 nutrient culture media, THP-1 cell density is adjusted to 10
5-10
6individual/ml;
The cell adjusting density is placed in cell chulture dish, at 37 DEG C, cultivates 24h;
Add appropriate 5mg/ml lentinan in culture dish, be respectively 0 μ g/ml, 500 μ g/ml, 1mg/ml, 2mg/ml to lentinan final concentration;
37 DEG C, CO
2the CO2gas incubator of concentration 5% cultivates 24 or 36h;
With 1000g centrifugal 20min collecting cell supernatant;
ELISA method detects IFN-γ concentration: method is with embodiment 1.
Existing large quantity research shows that lentinan has immunoregulatory activity, result shown in Fig. 3 and existing animal and primary cell testing result basically identical, therefore can verify the validity of this detection method.
Embodiment 4: Chinese caterpillar fungus polysaccharide Activity determination.
With the Chinese caterpillar fungus polysaccharide solution of RPMI1640 nutrient culture media preparation 1mg/ml;
With RPMI1640 nutrient culture media, THP-1 cell density is adjusted to 10
5-10
6individual/ml;
The cell adjusting density is placed in cell chulture dish, at 37 DEG C, cultivates 36h;
Add appropriate 1mg/ml Chinese caterpillar fungus polysaccharide in culture dish, be respectively 0 μ g/ml, 10 μ g/ml, 100 μ g/ml, 200 μ g/ml to Chinese caterpillar fungus polysaccharide final concentration;
37 DEG C, CO
2the CO2gas incubator of concentration 5% cultivates 48h;
With 1000g centrifugal 20min collecting cell supernatant;
ELISA method detects IFN-γ concentration: method is with embodiment 1.
There are some researches show that Cordyceps sinensis polysaccharide has immunoregulatory activity, result shown in Fig. 4 and existing result of study basically identical, therefore can verify the validity of this detection method.
Grifolan, Cordyceps sinensis polysaccharide and lentinan detect through the method, and testing result proves the validity of the method all further.Our experiments show that, the present invention also can detect the biologically active of other polysaccharide except above-mentioned experimental example.
Embodiment 5: the detection of Sparassis crispa sample antineoplastic immune activity
With the Sparassis crispa sample solution of RPMI1640 nutrient culture media preparation 1mg/ml;
With RPMI1640 nutrient culture media, THP-1 cell density is adjusted to 10
5-10
6individual/ml;
The cell adjusting density is placed in cell chulture dish, at 37 DEG C, cultivates 24h;
Adding appropriate 1mg/ml Sparassis crispa sample in culture dish, is 100 μ g/ml to Sparassis crispa sample final concentration;
37 DEG C, CO
2the CO2gas incubator of concentration 5% cultivates 48h;
With 1000g centrifugal 20min collecting cell supernatant;
ELISA method detects IFN-γ concentration: method is with embodiment 1.
The IFN-γ concentration that experimental result induction produces and concentration are that the ConA of 1.5 μ g/ml is suitable, so determine that the ACIF value of this Sparassis crispa sample is for 15+.
Above embodiment is only used to assist understands the present invention; not thereby scope of the present invention is limited; every lower of spirit and scope of the invention described in instructions that do not depart from makes any effective culture medium such as grade, step replacement or changes; or directly or indirectly see that the present invention is used in relevant technical field, include within the protection domain of patent of the present invention.
Claims (1)
1. a detection method for Sparassis crispa antineoplastic immune activity, is characterized in that comprising following processing step:
1) get Sparassis crispa proteglycan complex (SGP) sample, be mixed with solution;
2) THP-1 cell is got, adjustment cell density to 10
5~ 10
6individual/ml;
3) to step 2) in cultivate in the cell that obtains add step 1) SGP solution to final concentration reach 10 μ g ~ 2.0mg/ml, then cultivate;
4) step 3 is got) the culture collected after centrifugation cell conditioned medium liquid that obtains;
5) step 4 is got) the cell conditioned medium liquid ELASA method that obtains detects IFN-γ concentration, and be within the scope of 10 μ g ~ 2.0mg/ml at SGP activity, IFN-γ concentration and SGP concentration have obvious positive correlation;
6) get ConA, detect IFN-γ concentration by above-mentioned steps, control ConA final concentration is 1-3 μ g/ml, with 10 times of ConA concentration value, as antineoplastic immune active factors level (AnticancerImmuneFactor, ACIF) value;
Described step 1) middle RPMI1640 nutrient culture media preparation SGP solution;
Described step 2) middle with RPMI1640 nutrient culture media adjustment THP-1 cell density;
Described step 2) in adjustment cell density THP-1 cell be placed in 37 DEG C at carry out cultivation 24 ~ 36h;
Described step 3) in add SGP solution after cell liquid be placed in 37 DEG C, CO
2the CO2gas incubator of concentration 5% cultivates 24 ~ 48h;
Antineoplastic immune activity level ACIF value indicates;
ACIF value is 10 times of ConA concentration value.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102901831A (en) * | 2012-10-25 | 2013-01-30 | 杭州宝康亿富生物科技有限公司 | Method for detecting biological activities of immune regulation polysaccharides and nucleic acids |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102901831A (en) * | 2012-10-25 | 2013-01-30 | 杭州宝康亿富生物科技有限公司 | Method for detecting biological activities of immune regulation polysaccharides and nucleic acids |
Non-Patent Citations (2)
| Title |
|---|
| IFN-g Induction by SCG, 1,3-b-D-Glucan from Sparassis crispa, in DBA/2 Mice In Vitro;TOSHIE HARADA等;《JOURNAL OF INTERFERON & CYTOKINE RESEARCH》;20021231;第22卷(第12期);第1227－1239页 * |
| 一种葡聚糖活性检测的新方法;张晓菲等;《浙江理工大学学报》;20130930;第30卷(第5期);第738－740 * |
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