CN101397346A - Method for preparing asparagus pure polysaccharide having immunoregulation role - Google Patents

Abstract

The invention discloses a preparation method for the gardon asparagus pure polysaccharide with immunoregulation effect, and relates to the technical field of the extraction and separation of natural plant active polysaccharide, in particular to a preparation method for gardon asparagus pure polysaccharide. The preparation method is characterized in that the following steps are as follows: 1) raw material treatment, 2) polysaccharide extraction, 3) hyperfiltration and classification, 4) ion column chromatography, 5) gel column chromatography, and 6) finished product collection. The implementation of the invention has the positive effects that, the gardon asparagus pure polysaccharide extracted by the invention not only provides market demands, but also creates conditions for the further study of the gardon asparagus pure polysaccharide in the aspects of anti-mutation, anti-tumor, free radical removal and anti-oxidation actions and the mechanism in immunocompetence regulation.

Description

A kind of preparation method with Thallus Gracilariae holosaccharide of immunoregulation effect

Technical field

The present invention relates to the extraction and separation technology field of natural phant active polysaccharide, relate in particular to a kind of preparation method of Thallus Gracilariae holosaccharide.

Background technology

Thallus Gracilariae (Gracilaria lemaneiformis) is a kind of temperate zone property red algae of Gracilaria (Gracilaria Greville).Originate in the coastal and okinawa of japan in China Shandong Province, in recent years in Guangdong, Fujian etc. economizes and realizes large-scale cultivation, output increases year by year.Its main application of Thallus Gracilariae is the raw material of producing as agar-agar, also can be as abalone feed and green food exploitation, and the cultivation of Thallus Gracilariae helps improving coastal seawater quality in addition, has the environment remediation effect.

But the research of relevant Thallus Gracilariae deep processing aspect seldom.Do not see at present abroad the research report of relevant Thallus Gracilariae biological activity aspect, domestic also have only the minority scholar that the biological activity of Thallus Gracilariae polysaccharide has been carried out preliminary study, discover its have anti-mutation, antitumor, remove free radical and antioxygenation.Relevant Thallus Gracilariae polysaccharide is regulated the research of immunocompetence aspect, and Chinese patent CN101100488A is just in a kind of method of extracting asparagus active polysaccharide under comparatively high temps of publicity.Though this method obtains mouse lymphocyte is had the Thallus Gracilariae polysaccharide of higher cultivation effect, the polysaccharide that extracts not is pure product polysaccharide, is unfavorable for clear and definite activeconstituents and further studies action pathway.

Summary of the invention

The invention provides a kind of preparation method with Thallus Gracilariae holosaccharide of immunoregulation effect.

The technical scheme that the present invention takes: adopt by a kind of preparation method with Thallus Gracilariae holosaccharide of immunoregulation effect, its scheme is: comprise the steps:

1), raw material handles 2), polysaccharide extracts 3), the ultrafiltration classification, 4), the ion column chromatography, 5), gel filtration chromatography, 6), finished product collects.

Positively effect behind enforcement the present invention is:

The present invention has extracted the pure product polysaccharide of Thallus Gracilariae, not only can provide market demand, also for further investigation Thallus Gracilariae holosaccharide its anti-mutation, antitumor, remove free radical and antioxygenation, and condition has been created in the research of regulating immunocompetence aspect mechanism.

Description of drawings

Fig. 1 is Thallus Gracilariae holosaccharide preparation flow figure

Fig. 2 is the Thallus Gracilariae holosaccharide HPSEC tomographic results figure of preparation

Embodiment

Now the invention will be further described in conjunction with the accompanying drawings

A kind of preparation method with Thallus Gracilariae holosaccharide of immunoregulation effect comprises the steps:

1), raw material is handled

After the Thallus Gracilariae dry product is pulverized, stayed the Thallus Gracilariae dry product powder of 60 mesh sieves, 4 ℃ of preservations are standby;

2), polysaccharide extracts

Thallus Gracilariae algae powder repeats to extract 3 times with water, concrete grammar: add distilled water with the 1:20-50 weight ratio, per hour stir 1 time, under 20-28 ℃ of room temperature, extract 12h, extracting solution 4000r/min (or after 6 layers of filtered through gauze filter paper pumping rate) again separates residue.Last united extraction liquid.

3), ultrafiltration classification

Extracting solution is 100,000 membrane ultrafiltration through molecular weight cut-off, trapped fluid concentrating under reduced pressure ,-20 ℃ of preservations;

4), ion column chromatography

(2.6 * 100cm), elder generation uses the distilled water wash-out to trapped fluid, uses the NaCl gradient elution again, peak concentration 2.0mol/L through DEAE-Sepharose Fast Flow (sepharose) ion-exchange chromatography; Collecting the sample peak, is 8 with molecular weight cut-off, 000-12,000Da dialysis tubing, distill water dialysis 3-5 days ,-20 ℃ of preservations;

5), gel filtration chromatography

Get the sample that ion column gets off, carry out gel column Sephacryl S500 (propylene dextrane gel) and separate (1.6 * 100cm); Moving phase is the 0.2mol/L nitrite natrium, flow velocity 0.5mL/min, and 5mL/ manages collection, and differential refraction detector detects and collects the sample peak;

6), finished product is collected

After concentrating, distill water dialysis 3-5 days, lyophilize was collected, and is the Thallus Gracilariae holosaccharide.

Embodiment 1

1., raw material is handled

Stayed the Thallus Gracilariae dry product powder of 60 mesh sieves after the Thallus Gracilariae dry product is pulverized, 4 ℃ of preservations are standby.

2., polysaccharide extracts

Take by weighing Thallus Gracilariae algae powder 5Kg, add distilled water, per hour stir 1 time with weight ratio 1:20, extract 12h under the room temperature, the centrifugal 10min of extracting solution 4000r/min (or after 6 layers of filtered through gauze filter paper pumping rate) again separates residue, and residue repeats to extract 2 times again, united extraction liquid.

3., ultrafiltration classification

Extracting solution is 100,000 membrane ultrafiltration through molecular weight cut-off, trapped fluid concentrating under reduced pressure ,-20 ℃ of preservations.

4., ion column chromatography

Preparation concentration is the 8-10mg/ml trapped fluid.(2.6 * 100cm), elder generation uses the distilled water wash-out, uses the NaCl gradient elution again, peak concentration 2.0mol/L, applied sample amount: 8mg/ml, 48ml altogether through DEAE-Sepharose Fast Flow ion-exchange chromatography.Begin behind the 60min to collect, the 15ml/ pipe, 2-20 pipe in sample peak after concentrating, is the 8-12kD dialysis tubing with molecular weight cut-off, distill water dialysis 3-5 days ,-20 ℃ of preservations.

5., gel filtration chromatography

Get the sample that ion column gets off, be configured to concentration 15-20mg/mL solution, carry out gel column SephacrylS500 and separate (1.6 * 100cm).Applied sample amount 0.4mL, moving phase is the 0.2mol/L nitrite natrium, flow velocity 0.5mL/min, 5mL/ manages collection, and differential refraction detector detects.Collect the 19-29 pipe, after concentrating, distill water dialysis 3-5 days, sample was collected in lyophilize, is the pure product GCpF2-1B of Thallus Gracilariae polysaccharide.

Embodiment 2

Purity is identified

Efficient permeation chromatography chromatography (HPSEC)

Configuration testing sample 2mg/mL, the centrifugal 10min of 10000rpm gets sample on the supernatant 20 μ L, the record elution curve.The concrete test condition of HPSEC is as follows: Waters highly effective liquid phase chromatographic system (2695HPLCPump, In-line Degasser, 2414 Refractive Index Detector and the parallel detection of 2487 Dual λ AbsorbanceDetector, TSK PWXL 4000-3000 columns in series), moving phase is that pH is 7.0 0.1mol/L NaH ₂PO ₃With 0.3mol/L NaNO ₃Flow velocity is 0.2mL/min, 30 ± 0.1 ℃ of column temperatures.(see figure 2)

Embodiment 3

The GCpF2-1B biological activity assay

Mouse is put to death in the cervical vertebra dislocation, get spleen under the aseptic condition, place on the 100 order sterilizing stainless steel screen clothes, with plug in the syringe spleen is ground, sieve, wash mesh screen 2-3 time with PBS, the suspension of splenocyte changes in the aseptic centrifuge tube, add PBS to 30-40ml again, the centrifugal 6min of 400g, collecting cell, at room temperature add NH4Cl solution (1.5ml/ only), constantly rifle is beaten or was rocked the mixing cell 10 minutes, with splitting erythrocyte, adds PBS to 30-40ml, centrifugal 5 minutes of 400G, remove supernatant, add a small amount of RPMI1640 substratum cell counter counting after, with RPMI1640 diluting cells suspension to 2 * 10 ⁶Individual/ml.Get 180 μ l cell suspending liquids and add in 96 orifice plates, add different samples and the positive control phytohaemagglutinin of 20 μ l respectively, negative control PBS.At 37 ℃ and 5% CO ₂Condition under cultivate after 3 days, automatically read the absorbancy that the plate instrument is measured 570nm and 600nm respectively with ELISA, add 20 μ l Alamar Blue developers, cultivated 6-8 hour, measure the absorbancy of 570nm and 600nm once more, the formula of giving according to Alamer Blue Assay calculates each sample to lymphocytic activity ratio then.

Lymphocytic proliferation rate (%)=[117216 * A _λ570 (sample)-80586 * A _λ600 (sample)]

/[117216×A _λ570(control)-80586×A _λ600(control)]×100％；

Experimental result shows that when lower concentration (50 μ g/ml), appreciation rate is 152%; When being increased to 200 μ g/ml appreciation rates, concentration reaches 185%; When continuing to increase concentration to 500 μ g/ml, appreciation rate reaches 227% (positive control 60 μ g/ml lectins appreciation rates 207%).Show that GCpF2-1B has higher stimulation lymphopoiesis effect, and present dose-dependently.

Claims (1)

1, a kind of preparation method with Thallus Gracilariae holosaccharide of immunoregulation effect is characterized in that: comprise the steps:

1), raw material is handled

After the Thallus Gracilariae dry product is pulverized, stayed the Thallus Gracilariae dry product powder of 60 mesh sieves, 4 ℃ of preservations are standby;

2), polysaccharide extracts

Thallus Gracilariae algae powder repeats to extract 3 times with water, concrete grammar: add distilled water with the 1:20-50 weight ratio, per hour stir 1 time, under 20-28 ℃ of room temperature, extract 12h, extracting solution 4000r/min centrifugation residue; Last united extraction liquid;

3), ultrafiltration classification

Extracting solution is 100,000 membrane ultrafiltration through molecular weight cut-off, trapped fluid concentrating under reduced pressure ,-20 ℃ of preservations;

4), ion column chromatography

Trapped fluid is used the distilled water wash-out earlier through DEAE-Sepharose Fast Flow ion-exchange chromatography, uses the NaCl gradient elution again, peak concentration 2.0mol/L; Collecting the sample peak, is 8 with molecular weight cut-off, 000-12,000Da dialysis tubing, distill water dialysis 3-5 days ,-20 ℃ of preservations;

5), gel filtration chromatography

Get the sample that ion column gets off, carry out gel column Sephacryl S500 and separate; Moving phase is the 0.2mol/L nitrite natrium, flow velocity 0.5mL/min, and 5mL/ manages collection, and differential refraction detector detects and collects the sample peak;

6), finished product is collected

After concentrating, distill water dialysis 3-5 days, lyophilize was collected, and is the Thallus Gracilariae holosaccharide.

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