CN110194810A - A kind of grading purification method for the external glue polysaccharide of water shield improving hypoglycemic activity - Google Patents
A kind of grading purification method for the external glue polysaccharide of water shield improving hypoglycemic activity Download PDFInfo
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- CN110194810A CN110194810A CN201910653326.7A CN201910653326A CN110194810A CN 110194810 A CN110194810 A CN 110194810A CN 201910653326 A CN201910653326 A CN 201910653326A CN 110194810 A CN110194810 A CN 110194810A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
The invention discloses a kind of grading purification methods of external glue polysaccharide of water shield for improving hypoglycemic activity, this method comprises: water shield sample is taken, the isolated external glue of water shield from water shield sample；The external peptization of water shield is subjected to heating water bath extraction in alkaline aqueous solution, after extraction, filtering obtains filtered fluid, adjusts filtrate pH value to neutrality, obtains the external glue Thick many candies of water shield；The external glue Thick many candies of water shield are successively passed through into the filter membrane that membrane aperture is 100kDa, 50kDa and 10kDa, carry out ultrafiltration, obtaining molecular weight is respectively 100-50kDa, 50-10kDa and the external glue polysaccharide component of three kinds of water shields less than 10kDa.The external glue polysaccharide of water shield is carried out classification hyperfiltration treatment, having obtained molecular weight is respectively 100-50kDa, 50-10kDa and the external glue polysaccharide component of three kinds of water shields less than 10kDa by Ultra filtration membrane technology by the method for the present invention；Wherein, the external glue polysaccharide component antioxygenic property with higher of water shield and inhibit the active ability of vitro enzyme that molecular weight is 100-50kDa.
Description
Technical field
The present invention relates to technical field of plant extraction more particularly to a kind of external glue polysaccharides of water shield for improving hypoglycemic activity
Grading purification method.
Background technique
Water shield (Braseniaschreberi) is commonly called as water certain herbaceous plants with big flowers, membranaceous marshmarigold herb, bright and beautiful band, water lotus leaf, lake dish etc., and water shield is original
Perianth subclass Nymphaeceae water shield category perennial root fresh water aquatic herbaceous plant, being rich in nutrition, medical, healthcare function, in the world
It is known as " ginseng in water " in the market.
Water shield is mainly distributed on the ground such as China, Japan, Australia, North America, China be mainly distributed on Yunnan, Sichuan,
Hunan, 7 province of Hubei, Zhejiang and Jiangsu, it is the most famous with China Hangzhou West Lake water shield, it is provided reliably for the exploitation of water shield product
Raw material supply, and the expansion basis of the following broad mass market.The baregin on water shield surface is acid heteroglycan, and modern medicine is ground
Study carefully and shows that water shield has anti-inflammatory, anti-oxidant, strengthen immunity, anticancer, hypoglycemic, norcholesterol, reducing blood lipid and treatment children mostly dynamic
Disease and other effects.Monosaccharide and ratio are as follows: galactolipin (43.66%), mannose (9.05%), fucose (12.44%), rhamnose
(5.62%), xylose (11.90%), arabinose (6.93%), glucuronic acid (10.34%) and glucose (0.05%).
Currently, domestic be concentrated mainly on to Polysaccharide of Brasenia Schreberi semifinished product optimal extraction technology aspect the research of Polysaccharide of Brasenia Schreberi,
And Polysaccharide of Brasenia Schreberi glue is isolated and purified, Polysaccharide of Brasenia Schreberi component analysis etc. is studied.Liu Cuili et al. will be obtained
Polysaccharide of Brasenia Schreberi carry out mouse animal experiment, there is significant hypoglycemic effect as the result is shown, and to water shield different molecular weight polysaccharide
Whether have an impact to bioactivity but not yet clear.
Membrane separation technique is the separation concentration technique of high speed development in recent years, is the fluid using polymeric membrane for separation as representative
Separative unit operating technology has the function of separation, concentration, purifying and purification, it is not necessary that any reagent is added, belongs to physics mistake
Journey, concentration do not need in a disguised form, and separation is big, and process flow is short, and cost of investment is low, and energy consumption is few, also few to destroy polysaccharide
Bioactivity, the advantages that there is no the reagent residue problems of conventional organic solvents method, be widely used in food, biochemistry and medicine
Equal fields.
Summary of the invention
The present invention provides a kind of grading purification methods of external glue polysaccharide of water shield for improving hypoglycemic activity, and this method is not
Only separation rate is high, separating rate is fast, few bioactivity for destroying polysaccharide；And the external glue polysaccharide group of water shield that separation obtains
Point, the external glue polysaccharide component of water shield that especially molecular weight is 100-50kDa can be effective with stronger antioxidant activity
Inhibit the activity of alpha-glucosidase and alpha-amylase, and then improves hypoglycemic activity.
Specific technical solution is as follows:
A kind of grading purification method for the external glue polysaccharide of water shield improving hypoglycemic activity, comprising the following steps:
(1) water shield sample is taken, the isolated external glue of water shield from water shield sample；
(2) the external peptization of water shield is subjected to heating water bath extraction in alkaline aqueous solution, after extraction, filtering is obtained
Filtered fluid adjusts filtrate pH value to neutrality, obtains the external glue Thick many candies of water shield；
(3) the external glue Thick many candies of water shield are successively passed through into the filter membrane that membrane aperture is 100kDa, 50kDa and 10kDa, is surpassed
Filter, obtaining molecular weight is respectively 100-50kDa, 50-10kDa and the external glue polysaccharide component of three kinds of water shields less than 10kDa.
Innovation of the invention is: the present invention through anti-oxidant experiment and inhibits external enzyme activity experiment discovery different for the first time
The external glue polysaccharide component of the water shield of molecular size range has an impact to the inhibitory activity of alpha-glucosidase and alpha-amylase, Jin Erying
Ring the hypoglycemic activity of the external glue polysaccharide of water shield；Based on this discovery, the present invention is by Ultra filtration membrane technology, by the external glue of water shield
Polysaccharide carries out classification hyperfiltration treatment, and having obtained molecular weight is respectively 100-50kDa, 50-10kDa and three kinds of water shields less than 10kDa
The external glue polysaccharide component of dish；Wherein, molecular weight is the external glue polysaccharide component inoxidizability with higher of water shield of 100-50kDa
It can and inhibit the active ability of vitro enzyme.
Further, in step (1), first the external glue of water shield is taken out after thawing as freezen protective at -18 DEG C, then into
Row step (2) is conducive to the separating effect for improving the external glue polysaccharide of water shield.
Preferably, the alkaline aqueous solution is the NaOH aqueous solution of 0.05~0.2mol/L of concentration in step (2)；More
It is preferred that the concentration of the NaOH aqueous solution is 0.1mol/L.
Preferably, the solid-liquid ratio of the heating water bath extraction is 1: 5~20 (g/mL) in step (2)；It is further preferred that material
When liquor ratio is 1: 10 (g/mL), the extraction effect of the external glue polysaccharide of water shield is best.
Preferably, the temperature of the heating water bath extraction is 70~80 DEG C, and the time is 1~3h in step (3)；It is more excellent
Choosing, the temperature of heating water bath extraction are 70 DEG C, time 3h.
Preferably, the pressure of the ultrafiltration is 0.1~0.4MPa in step (3)；It is further preferred that the pressure of the ultrafiltration
For 0.4MPa.
Compared with prior art, the invention has the following advantages:
(1) the external glue polysaccharide of water shield is carried out classification hyperfiltration treatment, is obtained by the method for the present invention by Ultra filtration membrane technology
Molecular weight is respectively 100-50kDa, 50-10kDa and the external glue polysaccharide component of three kinds of water shields less than 10kDa；Wherein, molecule
The external glue polysaccharide component antioxygenic property with higher of water shield and inhibit the active ability of vitro enzyme that amount is 100-50kDa.
(2) the method for the present invention obtains the external glue polysaccharide of different molecular weight water shield by hyperfiltration treatment, and not only grading effect is good,
And Polysaccharide of Brasenia Schreberi institutional framework is not destroyed, the polysaccharide component that can be substantially prevented from colloid is destroyed and loses.
(3) the method for the present invention has production cost low, pollution-free without using chemical reagent, is suitble to the factorial production
Feature, operating procedure is simple, and cost is relatively low.
Detailed description of the invention
Fig. 1 is the Polysaccharide of Brasenia Schreberi of different molecular weight in embodiment 1 and comparative example 1~3 to external alpha-glucosidase and α-
The influence of starch enzyme inhibition rate；
Wherein, A BSP-NaOH；B is BSP-U；C is BSP-U100；D is BSP-U50；E is BSP-U10；
Concentration indicates concentration；Inhibition indicates inhibiting rate.
Fig. 2 is the scanning electron microscope (SEM) photograph of the Polysaccharide of Brasenia Schreberi of different molecular weight in embodiment 1 and comparative example 1~2；
Wherein, A BSP-NaOH；B is BSP-U100；C is BSP-U50；D is BSP-U10.
Specific embodiment
The present invention is further explained combined with specific embodiments below, the present invention carries out real according to inventive technique scheme
It applies, gives detailed embodiment and operating procedure, but protection scope of the present invention is not limited to following embodiments.It is following
Test method without specific conditions in embodiment, usually according to normal condition.
The calculation formula of external glue polysaccharide content are as follows:
Polyoses content (mg/mL)=total sugar content-content of reducing sugar
Antioxidant activity calculation formula:
In formula: AiFor sample solution+DPPH ethanol solution absorbance value；
AjFor sample solution+dehydrated alcohol absorbance value；
A0For DPPH ethanol solution+distilled water absorbance value.
In formula: AjFor sample solution+ABTs solution absorbance value；
AjFor sample solution+PBS buffer solution absorbance value；
A0For PBS buffer solution+distilled water absorbance value.
In formula: AiFor sample solution+H2O2Solution absorbance value；
AjFor sample solution+PBS buffer solution absorbance value；
A0For PBS buffer solution+distilled water absorbance value.
Vitro enzyme work calculation formula:
In formula: AiFor sample solution+enzyme solutions absorbance value；
AjFor sample solution+PBS buffer solution absorbance value；
A1For PBS buffer solution+distilled water absorbance value.
A0For distilled water absorbance value.
Embodiment 1
(1) take water shield sample, with gauze squeeze filtering from water shield sample the isolated external glue of water shield, and remove it is extra
Moisture；
(2) the external glue of water shield is first taken into 100g water shield after taking-up is placed in a beaker defrosting 2h as freezen protective at -18 DEG C
The external glue of dish is placed in a beaker, and is dissolved in the NaOH solution of 0.1mol/L, and solid-liquid ratio 1: 10 (g/mL) is put in 70 DEG C of water-baths
In, water-bath 1h carries out Polyose extraction；It is cooled to room temperature after extraction, obtained extracting solution is filtered, removed impurity, must filter
Liquid adjusts pH to 7 with phosphoric acid solution, obtains the external glue Thick many candies of water shield；
(3) the external glue Thick many candies of water shield are passed through into device for ultrafiltration membrane, by the external glue Thick many candies of water shield in operating pressure
Successively it is the filter membrane of 100kDa, 50kDa and 10kDa by membrane aperture under conditions of 0.1MPa, carries out ultrafiltration, obtain molecular weight point
Not Wei 100-50kDa (being named as BSP-U100), 50-10kDa (being named as BSP-U50) and be less than 10kDa (be named as BSP-
U10 polysaccharide ultrafiltrate).
Comparative example 1
(1) take water shield sample, with gauze squeeze filtering from water shield sample the isolated external glue of water shield, and remove it is extra
Moisture；
(2) the external glue of water shield is first taken into 100g water shield after taking-up is placed in a beaker defrosting 2h as freezen protective at -18 DEG C
The external glue of dish is placed in a beaker, and is dissolved in the NaOH solution of 0.1mol/L, and solid-liquid ratio 1: 10 (g/mL) is put in 70 DEG C of water-baths
In, water-bath 1h carries out Polyose extraction；It is cooled to room temperature after extraction, obtained extracting solution is filtered, removed impurity, must filter
Liquid adjusts pH to 7 with phosphoric acid solution, obtains the external glue Thick many candies of water shield；
(3) the external glue Thick many candies of water shield are passed through into device for ultrafiltration membrane, by the external glue Thick many candies of water shield in operating pressure
The filter membrane for being 100kDa by membrane aperture under conditions of 0.1MPa carries out ultrafiltration, and obtaining molecular weight is (to be named as greater than 100kDa
BSP-U polysaccharide trapped fluid).
Comparative example 2
(1) take water shield sample, with gauze squeeze filtering from water shield sample the isolated external glue of water shield, and remove it is extra
Moisture；
(2) the external glue of water shield is first taken into 100g water shield after taking-up is placed in a beaker defrosting 2h as freezen protective at -18 DEG C
The external glue of dish is placed in a beaker, and is dissolved in the NaOH solution of 0.1mol/L, and solid-liquid ratio 1: 10 (g/mL) is put in 70 DEG C of water-baths
In, water-bath 1h carries out Polyose extraction；It is cooled to room temperature after extraction, obtained extracting solution is filtered, removed impurity, must filter
Liquid adjusts pH to 7 with phosphoric acid solution, obtains the external glue Thick many candies of water shield, be named as BSP-NaOH.
Comparative example 3
(1) take water shield sample, with gauze squeeze filtering from water shield sample the isolated external glue of water shield, and remove it is extra
Moisture；
(2) it takes the external glue of 100g water shield to be placed in a beaker, is dissolved in the NaOH solution of 0.1mol/L, 1: 10 (g/ of solid-liquid ratio
ML), it is put in 70 DEG C of water-baths, water-bath 1h carries out Polyose extraction；It is cooled to room temperature after extraction, by obtained extracting solution mistake
Filter removes impurity, obtains filtered fluid, adjusts pH to 7 with phosphoric acid solution, obtains the external glue Thick many candies of water shield, be named as F-BSP.
Different molecular weight water shield glue polysaccharide is measured with the content of protein and to DPPH, ABTS and OH difference free radical
IC50；As a result as shown in table 1~2 and Fig. 1~2.
The content of table 1 different molecular weight water shield glue polysaccharide and protein
IC50 of the 2 different molecular weight water shield glue polysaccharide of table to DPPH, ABTS and OH difference free radical
| IC50(mg/mL) | BSP-NaOH | BSP-U100 | BSP-U50 | BSP-U10 |
| DPPH | 0.17±0.01ac | 0.38±0.07a | 0.19±0.01ad | 0.21±0.03ab |
| ABTS | 1.34±0.03ac | 0.83±0.13b | 1.76±0.35b | 2.21±0.17a |
| OH· | 0.05±0.00ab | 0.05±0.00ac | 0.09±0.00c | 0.02±0.00a |
According to external glue polysaccharide content and the calculation formula of protein, molecular weight is the external glue of water shield of 100-50kDa
The content of polysaccharide is 0.77 ± 0.00mg/g, and the content of protein is 0.22 ± 0.00 μ g/mL, is classified polysaccharide, albumen in polysaccharide
The content highest of matter；The content of the external glue polysaccharide of the water shield that molecular weight is 50-10kDa is 0.59 ± 0.01mg/g, protein
Content is 0.04 ± 0.01mg/g, and polysaccharide and protein content are substantially reduced；Molecular weight is less than the external glue polysaccharide of water shield of 10kDa
Content be 0.50 ± 0.02mg/g, protein content is 0.01 ± 0.00mg/g, and structure shows polysaccharide and protein content most
It is low；The content of the external glue polysaccharide of water shield made from comparative example 1 be 4.35 ± 0.16mg/g, protein content be 0.28 ±
0.00mg/g, polysaccharide, protein content reach highest.
According to antioxidant activity calculation formula, (measuring method is conventional to the antioxidant activity of the calculating external glue polysaccharide of water shield
Method), wherein molecular weight is that the antioxidant activity of the external glue polysaccharide of water shield of 100-50kDa is best.
According to alpha-glucosaccharase enzyme inhibition rate calculation formula, the external glue polysaccharide of water shield is calculated to the inhibiting rate of glucuroide
(measuring method is conventional method), wherein molecular weight be 100-50kDa the external glue polysaccharide of water shield external enzyme activity inhibiting rate most
It is good.
Molecular weight is that network laminated structure, intermolecular tool is presented in the microstructure of the external glue polysaccharide of water shield of 100-50kDa
There is strong interaction, as ultrafiltration membrane molecular weight reduces, average grain diameter is gradually reduced, and there were significant differences；Molecular weight is
The smooth rod-like structure in surface is presented in the microstructure of the external glue polysaccharide of the water shield of 50-10kDa；Molecular weight is less than the water shield of 10kDa
The smooth chondritic in surface is presented in the microstructure of the external glue polysaccharide of dish.The microcosmic knot of the external glue polysaccharide of water shield without ultrafiltration
Structure is presented surface and fold occurs, and polysaccharide aggregation leads to Morphology, with the appearance of stepped construction, the aggregation extent of polysaccharide
It obviously increases.
By comparing embodiment 1 and comparative example 1 it is found that water shield external glue polysaccharide of the molecular weight less than 100kDa is to vitro enzyme
Inhibiting rate living will be apparently higher than the external glue polysaccharide of water shield that molecular weight is greater than 100kDa.
Compared by comparative example 2 and comparative example 3 it is found that carrying out Polyose extraction, the external glue polysaccharide of water shield after -18 DEG C of freezings
Recovery rate it is higher, freezing can further remove moisture in the external glue of water shield.
Claims (6)
1. a kind of grading purification method for the external glue polysaccharide of water shield for improving hypoglycemic activity, which is characterized in that including following step
It is rapid:
(1) water shield sample is taken, the isolated external glue of water shield from water shield sample；
(2) the external peptization of water shield is subjected to heating water bath extraction in alkaline aqueous solution, after extraction, filtering is filtered
Liquid adjusts filtrate pH value to neutrality, obtains the external glue Thick many candies of water shield；
(3) the external glue Thick many candies of water shield are successively passed through into the filter membrane that membrane aperture is 100kDa, 50kDa and 10kDa, carry out ultrafiltration,
Obtaining molecular weight is respectively 100-50kDa, 50-10kDa and the external glue polysaccharide component of three kinds of water shields less than 10kDa.
2. improving the grading purification method of the external glue polysaccharide of water shield of hypoglycemic activity as described in claim 1, feature exists
In first by the external glue of water shield as freezen protective at -18 DEG C, taking out after thawing, then carry out step (2) in step (1).
3. improving the grading purification method of the external glue polysaccharide of water shield of hypoglycemic activity as described in claim 1, feature exists
In in step (2), the alkaline aqueous solution is the NaOH aqueous solution of 0.05~0.2mol/L of concentration.
4. improving the grading purification method of the external glue polysaccharide of water shield of hypoglycemic activity as claimed in claim 3, feature exists
In in step (2), the solid-liquid ratio of the heating water bath extraction is 1:5~20 (g/mL).
5. improving the grading purification method of the external glue polysaccharide of water shield of hypoglycemic activity as described in claim 1, feature exists
In in step (3), the temperature of the heating water bath extraction is 70~80 DEG C, and the time is 1~3h.
6. improving the grading purification method of the external glue polysaccharide of water shield of hypoglycemic activity as described in claim 1, feature exists
In in step (3), the pressure of the ultrafiltration is 0.1~0.4 MPa.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110613647A (en) * | 2019-10-12 | 2019-12-27 | 中南民族大学 | Water shield mask liquid and preparation method thereof |
| CN113892607A (en) * | 2020-06-22 | 2022-01-07 | 江苏你好鸭食品有限公司 | Preparation method of cooked lotus root slices with long shelf life |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110613647A (en) * | 2019-10-12 | 2019-12-27 | 中南民族大学 | Water shield mask liquid and preparation method thereof |
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| CN113892607A (en) * | 2020-06-22 | 2022-01-07 | 江苏你好鸭食品有限公司 | Preparation method of cooked lotus root slices with long shelf life |
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