CN105367677A - Maca polysaccharide of immunoregulatory activity and preparation method therefor - Google Patents
Maca polysaccharide of immunoregulatory activity and preparation method therefor Download PDFInfo
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- CN105367677A CN105367677A CN201510872970.5A CN201510872970A CN105367677A CN 105367677 A CN105367677 A CN 105367677A CN 201510872970 A CN201510872970 A CN 201510872970A CN 105367677 A CN105367677 A CN 105367677A
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Abstract
The invention provides a preparation method for maca polysaccharide and preliminary study on immunoregulatory activity thereof. The maca polysaccharide of a relatively high purity is extracted and separated by various means such as boiling water extraction, ethanol precipitation and passing DEAE-52 cellulose resin. Mmonosaccharide is detected to comprise glucose and arabinose with a molar ratio of (4-10): 1 and the molecular weight of the monosaccharide is 10000-30000Da. Meanwhile, the polysaccharide extracted by the method provided by the invention has certain promoting action on activity of a mouse macrophage RAW264.7 and expression of TLR4, TLR2 and an inflammatory factor of the mouse macrophage. The invention has characteristics that the process flow is simple, the operation is easy, the cost is relatively low, and large-scale production is facilitated; the polysaccharide extracted by the method provided by the invention has functions of preventing and resisting cancers, resisting aging, resisting oxidization, resisting autoimmune diseases and the like, thereby providing a theoretical basis for further researching and developing polysaccharide new drugs.
Description
Technical field
The present invention relates to medical art.Specifically, the present invention relates to a kind of agate card polysaccharide with immunoregulatory activity and preparation method thereof.
Background technology
Polysaccharide (Polysaccharides) is also known as saccharan, it is the biomacromolecule that more than ten and ten monose that a class is connected by α-or β-glycosidic link is polymerized, in energy storage, Structure composing and immune defense etc., have extremely important effect, be one of four large base substances forming organism.Occurring in nature more than 90% carbohydrate all exists with polysaccharide form, is extensively present on the cytolemma of the organisms such as high animals and plants, algae, fungi, bacterium, has and controls cell fission differentiation, regulates Growth of Cells and the old and feeble function waiting Various Complex.Found again that polysaccharide had the effect such as Tumor suppression, antibacterial removing toxic substances because promoting body's immunity in recent years, and made progressively to go deep into the research of animals and plants polysaccharide both at home and abroad, in China as the research such as lentinan, astragalus polysaccharides all achieves a lot of achievement.
In agate card, contents of saccharide is higher, it is reported that carbohydrate accounts for 59% of total nutritive substance, and every Ke Maka can provide 2850 caloric energy closely related therewith.And it is maximum with the ratio of polysaccharide in agate acarbose class composition.Polysaccharide in agate card comprises the polysaccharide of starch and non-starch class.Agate card is the plant of a kind of rich in starch unique in known at present crop in cruciferae, and to resist the severe environment such as High aititude mountain area, Andean cold, frost, day and night temperature be large relevant with it for this.At present domestic and international to non-starch class in agate card polysaccharide report less, this laboratory is studied this respect, and Primary Study has been carried out to its structure composition and activity thereof find that agate card polysaccharide structures is comparatively homogeneous, monose composition is simple, to body cell, there is certain immunoregulatory activity, this is further investigate its mechanism of action further, and exploitation polyose immune function controlling agents provides theoretical foundation.
Summary of the invention
The object of the invention for raw material with agate card dry plate, adopts water extract-alcohol precipitation and goes out to have the polysaccharide of immunoregulatory activity through a series of method separation and purification.Use pre-column derivatization method detect its monose composition, high performance size exclusion chromatography method detects its molecular weight, and on cell model preliminary identification its there is certain immunoregulatory activity, provide the foundation for researching and developing polyose new drug further.
The present invention is achieved through the following technical solutions above object: with agate card dry plate for raw material, after degreasing, hot water extraction, 70 ~ 90% alcohol settling polysaccharide, be dissolved in water throw out, alcohol precipitation obtains Crude polysaccharides again, adds water elution by DEAE-52 Mierocrystalline cellulose (Cl-type) resin, obtains the higher agate card polysaccharide of purity through filtration, lyophilize.
The agate card polysaccharide that the present invention prepares is known after pre-column derivatization and high performance size exclusion chromatography method are analyzed, and monose consists of glucose, pectinose, and its mol ratio is 4 ~ 10: 1, and molecular weight is 10000 ~ 30000Da.
The agate card polysaccharide that the present invention prepares shows to have certain immunoregulatory activity through In vitro cell experiment, and toxicity is lower.
Accompanying drawing explanation
Fig. 1 nine kinds of monose standard diagrams
Fig. 2 agate card polysaccharide monose composition collection of illustrative plates
Fig. 3 agate card polysaccharide molecule flow measurement collection of illustrative plates
Fig. 4 agate card polysaccharide (LMP-1) is on the impact of the propagation of scavenger cell Raw264.7
Fig. 5 agate card polysaccharide (LMP-1) is on the impact of scavenger cell TLR sample acceptor and downstream inflammatory Cytokines Expression
Embodiment
The present invention will be further explained for the following examples, but the present invention is not limited only to these embodiments, the scope that these embodiments do not limit the present invention in any way.Those skilled in the art within the scope of the claims made some changes and adjustment also should be thought and belongs to the scope of the invention.
Embodiment 1
The preparation were established of agate card polysaccharide:
(1) the agate card dry plate alcohol reflux degreasing of 80 ~ 95%, add water and make solid-liquid ratio 1: 15 ~ 25, boiling water refluxing extraction, the reaction times is 1.5h.
(2) put on clearly with the alcohol settling that final concentration is 70 ~ 90%, 4 DEG C of hold over night, centrifugal collecting precipitation.
(3) molten with the solid-liquid ratio distilled water rehydration of 1: 15 ~ 25,60 DEG C are stirred 4h, and centrifugal recovery supernatant, is put on clearly with the alcohol settling that final concentration is 70 ~ 90%, and 4 DEG C of hold over night, centrifugal collecting precipitation, obtains Crude polysaccharides.
(4) above-mentioned Crude polysaccharides is added distilled water and be made into the solution that concentration is 15 ~ 25mg/mL, the centrifugal supernatant that goes is for subsequent use.DEAE-52 Mierocrystalline cellulose (Cl-type) resin is filled in chromatography column after overactivation, distilled water balances, by the polysaccharide supernatant soln loading after centrifugal, wash-out is carried out as elutriant with distilled water, tracing detection polysaccharide content is carried out, until effluent liquid is substantially without polysaccharide with sulfuric acid anthrone method.
(5) the washing composition direct filtration removing impurity containing polysaccharide fraction, lyophilize are obtained agate card polysaccharide (LMP-1).
Embodiment 2
The Structural Identification of agate card polysaccharide
(1) pre-column derivatization method detects its monose composition
Fig. 1 is nine kinds of monose standard diagrams, if the sequencing marking the appearance time of shown nine kinds of monose standard substance in figure is seminose, ribose, rhamnosyl, glucuronic acid, galacturonic acid, glucose, semi-lactosi, pectinose and Fucose, the peak gone out per sample and appearance time thereof, sample can be gone out be made up of glucose, pectinose by contrast judgement, as shown in Figure 2, through can be calculated glucose, the mol ratio of pectinose is 4 ~ 10: 1.
(2) high performance size exclusion chromatography method detects its molecular weight
Fig. 3 measures agate card polysaccharide molecular weight collection of illustrative plates for adopting high performance size exclusion chromatography method, by GPCPostrunAnalysis molecular weight analyse software, calculate molecular weight calibration curve, thus the molecular weight calculating agate card polysaccharide is 10000 ~ 30000Da.
Embodiment 3
The immunoregulatory activity preliminary study of agate card polysaccharide
(1) agate card polysaccharide is to the proliferation function of mouse macrophage Raw264.7
Count after logarithmic phase RAW264.7 cell dissociation gets off, adjustment cell density is 3 × 10
4/ ml, is inoculated on 96 orifice plates, 100 μ L/ holes.Be placed in CO2 constant incubator, incubated overnight, after cell attachment, adding the agate card polysaccharide of different concns, (final concentration is 0,1.56,3.125,6.25,12.5,25,50 μ g/ml), 4 repetitions are done in each hole, and add LPS (1 μ g/ml) and do positive control, basis of microscopic observation cellular form after stimulation 48h, MTT method measures cell concn.Result as shown in Figure 4.As can be seen from the figure agate card polysaccharide is 1.56, and 3.125,6.25, can obviously promote that Raw264.7 breeds during 12.5 μ g/ml, and not obvious to the effect of cell when concentration increases.Therefore the lower concentration dosage (1.56,3.125,6.25 μ g/ml) selecting proliferation stronger in ensuing experiment explores its impact on cell TLR sample acceptor and downstream inflammatory Cytokines Expression as test dose.
(2) agate card polysaccharide is on the impact of mouse macrophage Raw264.7TLR sample acceptor and downstream inflammatory Cytokines Expression
(every hole 2ml) in 6 orifice plates is inoculated in every hole 3x10^5 cell, (final concentration is 1.56 to add the polysaccharide of different final concentration after cell attachment 24h, 3.125,6.25 μ g/ml), set up blank group simultaneously, LPS model group (final concentration is 1 μ g/ml), after cultivating 24h, collecting cell extracts total serum IgE, be cDNA by mRNA reverse transcription, adopt the cDNA of PCR instrument quantitative amplification goal gene, finally extract with sepharose nucleic acid electrophoresis checking institute the gene increased, carry out preliminary quantitative analysis with quantityone software.Result as shown in Figure 5.As can be seen from the figure agate card polysaccharide obviously can promote TLR4, TLR2 expression of receptor, has certain dose-dependently.The agate card polysaccharide of three dosage all obviously can promote the expression of TNF-α, and when dosage is slightly high, (3.125,6.25 μ g/ml) can promote the expression of IL-1 β, and only having could irritation cell secretion IL-6 when concentration reaches 6.25 μ g/ml.
Claims (5)
1. a preparation method for agate card polysaccharide, is characterized in that comprising the following steps:
(1) the agate card dry plate alcohol reflux degreasing of 80 ~ 95%, add water and make solid-liquid ratio 1: 15 ~ 25, boiling water refluxing extraction, the reaction times is 1.5h.
(2) put on clearly with the alcohol settling that final concentration is 70 ~ 90%, 4 DEG C of hold over night, centrifugal collecting precipitation.
(3) molten with the solid-liquid ratio distilled water rehydration of 1: 15 ~ 25,60 DEG C are stirred 4h, and centrifugal recovery supernatant, is put on clearly with the alcohol settling that final concentration is 70 ~ 90%, and 4 DEG C of hold over night, centrifugal collecting precipitation, obtains Crude polysaccharides.
(4) above-mentioned polysaccharide is added distilled water and be made into the solution that concentration is 15 ~ 25mg/mL, the centrifugal supernatant that goes is for subsequent use.DEAE-52 Mierocrystalline cellulose (Cl-type) resin is filled in chromatography column after overactivation, distilled water balances, by the polysaccharide supernatant soln loading after centrifugal, wash-out is carried out as elutriant with distilled water, tracing detection polysaccharide content is carried out, until effluent liquid is substantially without polysaccharide with sulfuric acid anthrone method.
(5) the washing composition direct filtration removing impurity containing polysaccharide fraction, lyophilize are obtained agate card polysaccharide (LMP-1).
2. the extraction process of agate card polysaccharide (LMP-1) according to claim 1, it is characterized in that the alcohol reflux degreasing with 80 ~ 95%, with the alcohol settling polysaccharide that final concentration is 70 ~ 90%, cross DEAE-52 Mierocrystalline cellulose (Cl-type) resin simultaneously and obtain the higher agate card polysaccharide of purity.
3. the Structural Identification of agate card polysaccharide (LMP-1) according to claim 2, it is characterized in that detecting its monose composition with pre-column derivatization method, high performance size exclusion chromatography method detects its molecular weight, result shows its monose and consists of glucose, pectinose, both mol ratios are 4 ~ 10: 1, and molecular weight is 10000 ~ 30000Da.
4. the immunocompetence research of agate card polysaccharide (LMP-1) according to claim 3, is characterized in that agate card polysaccharide has the proliferation function promoting mouse macrophage Raw264.7, in concentration 1.56, and successful during 3.125,6.25 μ g/ml.Post-stimulatory cell TLR4, TLR2 express and obviously strengthen, and the expression amount of TNF-α, IL-1 β, IL-6 increases, and has certain dose-dependently.
5. agate card polysaccharide according to claim 4 is preparing immunoregulation druge or/and anti-inflammatory drug, cancer-resisting, anti-ageing, anti-oxidant in application.
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Cited By (4)
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| CN110099691A (en) * | 2016-10-24 | 2019-08-06 | Jds治疗有限公司 | Maca composition and application method |
| CN111647096A (en) * | 2020-05-20 | 2020-09-11 | 昆明理工大学 | Neutral maca polysaccharide and extraction method and application thereof |
| CN112679563A (en) * | 2021-01-06 | 2021-04-20 | 澳门大学 | Glucosidotetraose and preparation method and application thereof |
| CN115477604A (en) * | 2021-05-31 | 2022-12-16 | 高雄医学大学 | Maca extract and use thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110099691A (en) * | 2016-10-24 | 2019-08-06 | Jds治疗有限公司 | Maca composition and application method |
| US11752186B2 (en) | 2016-10-24 | 2023-09-12 | Jds Therapeutics, Llc | Maca compositions and methods of use |
| CN111647096A (en) * | 2020-05-20 | 2020-09-11 | 昆明理工大学 | Neutral maca polysaccharide and extraction method and application thereof |
| CN112679563A (en) * | 2021-01-06 | 2021-04-20 | 澳门大学 | Glucosidotetraose and preparation method and application thereof |
| CN115477604A (en) * | 2021-05-31 | 2022-12-16 | 高雄医学大学 | Maca extract and use thereof |
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Application publication date: 20160302 |