CN100572396C - A kind of preparation method of asparagus active polysaccharide - Google Patents
A kind of preparation method of asparagus active polysaccharide Download PDFInfo
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- CN100572396C CN100572396C CNB2007100446101A CN200710044610A CN100572396C CN 100572396 C CN100572396 C CN 100572396C CN B2007100446101 A CNB2007100446101 A CN B2007100446101A CN 200710044610 A CN200710044610 A CN 200710044610A CN 100572396 C CN100572396 C CN 100572396C
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Abstract
The invention provides a kind of preparation method of asparagus active polysaccharide.This method comprises that the polysaccharide that Thallus Gracilariae is carried out under pre-treatment, the optimum activity temperature extracts, removes glue, dialysis and drying and other steps.The asparagus active polysaccharide yield that this preparation method obtains is about 13%, and polysaccharide content surpasses 76%, and mouse lymphocyte is had higher cultivation effect, thereby has stronger immunocompetence.
Description
Technical field
The present invention relates to the extraction field of marine plant polysaccharide, relate in particular to a kind of preparation method of Thallus Gracilariae polysaccharide.
Background technology
Thallus Gracilariae is rich in necessary normal, the trace element of multiple human body such as Sargassum polysaccharides, iodine, calcium, iron and vitamin A, B1, C etc.The record Thallus Gracilariae has effects such as the thyrocele of controlling, oedema, uropoiesis device disease, ulcer in " Shiliao Bencao " and the Compendium of Material Medica.Japanology proves, Thallus Gracilariae has clearing lung-heat defaecation, beauty treatment weight reducing, hypotensive blood fat and regulate effect such as physical function, and think that edible Thallus Gracilariae is that the life-span occupies the major cause of the first in the world to Japan's Okinawa for each person.
But the utilization to Thallus Gracilariae also is mainly used in production agar and a spot of as the abalone feed at present, has only kept the colloid part in the manufacture craft of agar, studies show that the non-colloid part polysaccharide content of being toppled near 90%, has very high immunocompetence.So it is imperative that the further research and development of non-colloid part are utilized.
The research of Thallus Gracilariae active polysaccharide starts from recent years, generally all adopt traditional high temperature neutral water formulation, prove relatively in this method extract and mostly be the colloid part that non-colloidal activity part yield is low on the contrary by research, and most critical is that the polysaccharide biological activity that obtains is poor.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of preparation method of asparagus active polysaccharide, and polysaccharide yield in the prior art is low to solve, the defective of biological activity difference.
Principle of the present invention is: employing is frozen the molten method colloid part that activity is less and is separated with the high reactivity polysaccharide.With the activity is that screening index filters out the active extraction temperature of tool, and being different from traditional method is that index is screened temperature with the yield.Studies have shown that extracting temperature is active major influence factors.
The invention provides a kind of preparation method of asparagus active polysaccharide, comprise the steps:
1. pre-treatment: in 45-50 ℃ of oven drying, the 80-100 order is pulverized, and is standby with Thallus Gracilariae;
2. the extraction of polysaccharide: in the gardon asparagus powder that crushes, add the water of 30-50 times of weight, in 30-100 ℃ of water-bath lixiviate 3-5 hour; Centrifugal 20 minutes of 12000g, centrifugation repeats above-mentioned steps, and lixiviate is 1-2 time again, merging filtrate;
3. remove seaweed gel: with above-mentioned filtrate in-20--80 ℃ freezing, spend the night, 4 ℃ dissolve then, centrifugal 30 minutes of 12000g collects supernatant;
4. extracting solution dialysis: with above-mentioned supernatant concentration to 1/4 volume, be 10,000 dialysis membrane dialysis 4-5 days under 4 ℃, get 10,000 molecular weight with top with the molecular retention amount;
5. dry: that above-mentioned 10,000 molecular weight with the top lyophilize, are promptly obtained required Thallus Gracilariae polysaccharide.
Wherein used raw material Thallus Gracilariae is gathered in Shantou, Guangzhou in June, 2006;
Wherein step is preferably 45-55 ℃ to the extraction temperature of gardon asparagus powder in 2., and most preferred extraction temperature is 50 ℃.
The present invention is that index (studies show that in a large number polysaccharide reaches other active functions as macromole by improving body's immunological function with the immunocompetence, so select this for use as screening index) different extraction temperature is screened, obtain best extraction temperature, and to the extraction time under this temperature, extraction time and solid-to-liquid ratio is that index is optimized with the yield, through optimizing the back yield more than 13%, and wherein the content of active polysaccharide surpasses 76%, substantially all more than 80%.
And the present invention adopts and to freeze molten method colloid is separated with water-soluble polysaccharide.Because agar is exactly the colloid part in the Thallus Gracilariae, can utilize again so freeze the colloid part that obtains after molten.Simultaneously Thallus Gracilariae 45-55 ℃ of extraction obtain active best, so can further carry out high temperature extraction, prepare agar to the algae-residue after extracting.So this research is the refuse reclamation process of not only present Thallus Gracilariae agar-agar being produced, and has made full use of resource; And provide reference for the development and use of marine active substance.
Can obtain steady quality, Thallus Gracilariae polysaccharide that active constituent content is high by above-mentioned preparation method.And thereby the polysaccharide that is extracted can obviously strengthen the effect of lymphocytic propagation enhancing body immunocompetence.Present method is that Thallus Gracilariae is made the refuse reclamation in the agar process, and has obtained high-content, highly active composition.So this preparation method be a kind ofly make full use of resource, cost is low, pollution-free, purity of polysaccharide is high and easy, quick, efficiently be fit to the method for suitability for industrialized production.
Embodiment
Embodiment 1
1. pre-treatment: Thallus Gracilariae is rinsed well, and in 45 ℃ of oven dryings, 80 orders are pulverized, and are standby;
2. the extraction of polysaccharide: add the water of 40 times of weight in the gardon asparagus powder that crushes, lixiviate is 4 hours in 50 ℃ of water-baths; Centrifugal 20 minutes of 12000g, centrifugation repeats above-mentioned steps, and lixiviate is 1 time again, merging filtrate;
3. remove seaweed gel: with above-mentioned filtrate in-20 ℃ freezing, spend the night, 4 ℃ dissolve then, centrifugal 30 minutes of 12000g collects supernatant;
4. extracting solution dialysis: with above-mentioned supernatant concentration, with the dialysis membrane dialysis, the molecular retention amount of dialysis membrane is 10,000 under 4 ℃; Dialysed 4-5 days, and got 10,000 molecular weight with top;
5. dry: that above-mentioned 10,000 molecular weight with the top lyophilize, are promptly obtained required Thallus Gracilariae polysaccharide.
The preparation polysaccharide content be 87%, to the mouse lymphocyte proliferation rate for 275%.
Embodiment 2
1. pre-treatment: in 50 ℃ of oven dryings, 80 orders are pulverized, and are standby with Thallus Gracilariae;
2. the extraction of polysaccharide: add the water of 40 times of weight in the gardon asparagus powder that crushes, lixiviate is 4 hours in 60 ℃ of water-baths; Centrifugal 20 minutes of 12000g, centrifugation repeats above-mentioned steps, and lixiviate is 1 time again, merging filtrate;
3. remove seaweed gel: with above-mentioned filtrate in-20 ℃ freezing, spend the night, 4 ℃ dissolve then, centrifugal 30 minutes of 12000g collects supernatant;
4. extracting solution dialysis: with above-mentioned supernatant concentration, with the dialysis membrane dialysis, the molecular retention amount of dialysis membrane is 10,000 under 4 ℃; Dialysed 4-5 days, and got 10,000 molecular weight with top;
5. dry: that above-mentioned 10,000 molecular weight with the top lyophilize, are promptly obtained required Thallus Gracilariae polysaccharide.
The polysaccharide content of preparation is 86.5%, is 247% to the mouse lymphocyte proliferation rate.
Embodiment 3
Other steps are with embodiment 1-2, and just to gardon asparagus powder lixiviate in 70 ℃ of water-baths, the polysaccharide content of preparation is 80%, is 212% to the mouse lymphocyte proliferation rate.
Embodiment 4
Other steps are with embodiment 1-2, and just to gardon asparagus powder lixiviate in 80 ℃ of water-baths, the polysaccharide content of preparation is 87%, is 227% to the mouse lymphocyte proliferation rate.
Embodiment 5
Other steps are with embodiment 1-2, and just to gardon asparagus powder lixiviate in 90 ℃ of water-baths, the polysaccharide content of preparation is 76%, is 193% to the mouse lymphocyte proliferation rate.
Embodiment 6
Other steps are with embodiment 1-2, and just to gardon asparagus powder lixiviate in 100 ℃ of water-baths, the polysaccharide content of preparation is 90%, is 178% to the mouse lymphocyte proliferation rate.
The polysaccharide content that specific embodiment 1-6 extracted and its proliferation rate to mouse lymphocyte relatively see Table 1:
Table 1
| Specific embodiment | Extraction temperature (℃) | Polysaccharide content (%) | Lymphocytic proliferation rate (%) |
| Embodiment 1 | 50 | 87 | 275 |
| Embodiment 2 | 60 | 86.5 | 247 |
| Embodiment 3 | 70 | 80 | 212 |
| Embodiment 4 | 80 | 87 | 227 |
| Embodiment 5 | 90 | 76 | 193 |
| Embodiment 6 | 100 | 90 | 178 |
Embodiment 7: the detection of polysaccharide content
With the semi-lactosi is standard substance, adopt the phenolsulfuric acid method to measure polysaccharide content (Dubois M etal., 1956) precision takes by weighing the prepared sample 0.5g of embodiment 1-6, the water-bath of extracting in water temperature is fully dissolved, be transferred in the 100ml volumetric flask, add water and be settled to scale, shake up, 50 times of redilution are diluted to 0.1mg/ml.The above liquid 2.0ml of accurate absorption places the 15ml test tube, adds 5% phenol solution 1.0ml respectively, add vitriol oil 5.0ml rapidly, fully vibration makes it sufficient reacting, at room temperature (20-25 ℃) placed after 30 minutes, measured light absorption value in 490nm wavelength place.The concrete content of embodiment 1-6 gained polysaccharide sees Table 1.
Embodiment 8: the biological activity assay of preparation gained asparagus active polysaccharide
The dislocation of mouse cervical vertebra is put to death, get spleen, with PBS flushing 3～4 times.Spleen is ground in culture dish with 100 mesh sieves, suspension after grinding is with the centrifugal 6min of 400 * g, supernatant liquor is removed in suction, adding concentration is 0.83% ammonium chloride solution splitting erythrocyte, charge and attack repeatedly, leave standstill the centrifugal 6min of 400 * g behind the 10min, the cell of collection with the flushing of PBS damping fluid several all over the back blue inspection vigor of ， Tai Ban more than 95%.With RPMI1640 cell dilution is become 2 * 10
6Individual/mL, add in the 96 porocyte culture plates, 37 ℃, contain 5% CO
2Cultivate under the condition.The every mL of 180 μ L is contained 2 * 10
6Individual lymphocytic suspension adds in 96 orifice plates, adds 20 μ L samples (sample comprises blank PBS, positive control PHA and the prepared polysaccharide sample of embodiment 1-6) simultaneously, in 37 ℃, contain 5% CO
2Cultivate 3d under the condition, measure the absorbancy at its 570nm and 600nm place with microplate reader, add 20 μ L Alamar Blue reagent then, cultivate again, measure the absorbancy at 570nm and 600nm place after the variable color again, then the influence of calculating various sample on cell proliferation according to the formula of Alamar Blue reagent.Calculation formula is: proliferation rate (%)=(80856 * A
λ 570(sample)-117216 * A
λ 600(sample))/(80856 * A
λ 570(control)-117216 * A
λ 600(control)) * 100% (sample in the formula refers to such an extent that be the polysaccharide sample, and control refers to such an extent that be blank PBS.)
The prepared polysaccharide of embodiment 1-6 sees Table 1 to the concrete numerical value of mouse lymphocyte proliferation rate.
Claims (3)
1. the extracting method of an asparagus active polysaccharide is characterized in that this method comprises the steps:
1. pre-treatment: in 45-50 ℃ of oven drying, the 80-100 order is pulverized, and is standby with Thallus Gracilariae;
2. the extraction of polysaccharide: in the gardon asparagus powder that crushes, add the water of 30-50 times of weight, in 30-100 ℃ of water-bath lixiviate 3-5 hour; Centrifugal 20 minutes of 12000g, centrifugation repeats above-mentioned steps, and lixiviate is 1-2 time again, merging filtrate;
3. remove seaweed gel: with above-mentioned filtrate in-20--80 ℃ freezing, spend the night, 4 ℃ dissolve then, centrifugal 30 minutes of 12000g collects supernatant liquor;
4. extracting solution dialysis: with above-mentioned supernatant concentration to 1/4 volume, be 10,000 dialysis membrane dialysis 4-5 days under 4 ℃, get 10,000 molecular weight with top with the molecular retention amount;
5. dry: that above-mentioned 10,000 molecular weight with the top lyophilize, are promptly obtained required Thallus Gracilariae polysaccharide.
2. extracting method according to claim 1 is characterized in that the extraction temperature to gardon asparagus powder was 45-55 ℃ during step 2..
3. extracting method according to claim 2 is characterized in that the extraction temperature to gardon asparagus powder was 50 ℃ during step 2..
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| CN101697978B (en) * | 2009-10-23 | 2012-05-23 | 广东药学院 | Asparagus powder and chewable tablet as well as manufacturing method thereof |
| CN101862030A (en) * | 2010-06-22 | 2010-10-20 | 中国烟草总公司郑州烟草研究院 | Preparation of asparagus ethanol extract and application thereof in cigarette |
| CN102499458B (en) * | 2011-10-28 | 2013-08-21 | 浙江中烟工业有限责任公司 | Marine algae polysaccharide, preparation method thereof, and application thereof in cigarettes |
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| CN105054138A (en) * | 2015-08-17 | 2015-11-18 | 济南舜昊生物科技有限公司 | Gracilaria lemaneiformis salad and preparation method thereof |
| CN106173680A (en) * | 2016-07-25 | 2016-12-07 | 集美大学 | A kind of processing method of antiallergic pineapple beverages |
| CN109576329B (en) * | 2018-12-29 | 2021-05-18 | 广东医科大学 | Preparation method, product and application of anti-melanin synthesized asparagus non-agar oligosaccharide |
| CN112675072A (en) * | 2021-01-12 | 2021-04-20 | 南方海洋科学与工程广东省实验室(湛江) | Non-agar oligosaccharide matrix water-free hand sanitizer and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110128561A (en) * | 2019-05-09 | 2019-08-16 | 华南理工大学 | A kind of preparation method of asparagus functional oligose |
| CN110128561B (en) * | 2019-05-09 | 2021-07-20 | 华南理工大学 | Preparation method of asparagus functional oligosaccharide |
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