CN107312106A - A kind of method that alpha amylase and α glucosidase inhibitors are extracted from red hair algae - Google Patents
A kind of method that alpha amylase and α glucosidase inhibitors are extracted from red hair algae Download PDFInfo
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- CN107312106A CN107312106A CN201710651449.8A CN201710651449A CN107312106A CN 107312106 A CN107312106 A CN 107312106A CN 201710651449 A CN201710651449 A CN 201710651449A CN 107312106 A CN107312106 A CN 107312106A
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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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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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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
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Abstract
The invention discloses a kind of method that alpha amylase and α glucosidase inhibitors are extracted from red hair algae, comprise the following steps：Step 1: extracting：New scarlet hair algae is cleaned, dried, crushed, algae powder is obtained, distilled water is added, extracting is concentrated to give concentrate；Step 2: alcohol precipitation：Ethanol is added dropwise, centrifugation takes precipitation, is freeze-dried, and Thick many candies are made；Step 3: gel chromatography：The Thick many candies are dissolved in distilled water, loading, eluted, tracing detection, collect eluting peak, dialysis desalting, freeze-drying obtains purified polysaccharide；Alpha amylase and α glucosidase inhibitors is made.Preparation technology of the present invention is simple, and cost is low, and the alpha amylase and α glucosidase inhibitors extracted has the activity for suppressing blood glucose rise.
Description
Technical field
Alpha-amylase and α-Portugal are extracted from red hair algae the present invention relates to the technical field of active polysaccharide, more particularly to one kind
The method of polyglycoside enzyme inhibitor.
Background technology
In recent years, hyperglycaemia has been increasingly becoming one of healthy key factor of puzzlement people.Long-term hyperglycaemia
Body each histoorgan can be caused to occur lesion, cause the generation of acute and chronic complication, such as resistance declines, renal function by
Damage, cardiovascular and cerebrovascular disease, diabetes etc..There are many researchers both domestic and external to start to probe into and can suppress blood glucose liter in recent years
High natural active matter, most of researchs show that alpha-amylase and alpha-glucosidase and internal blood glucose have substantial connection.α-
Amylase and alpha-glucosidase play a key effect in food in the digestion of the main carbohydrate such as starch, absorption, suppress
Its activity can suppress the quick rise of blood glucose in vivo.
Polysaccharide is a kind of natural active matter being prevalent in most plants body, has there is many researchs in recent years
Person have studied the various active of plant polyose, such as anti-oxidant, reducing blood lipid, hypotensive, and also someone have studied polysaccharide for α-shallow lake
The influence of powder enzyme and alpha-glucosidase activity, as a result shows that polysaccharide can be by suppressing alpha-amylase and alpha-glucosidase
Activity reduces the effect of blood glucose so as to reach.
In view of polyoses content is high in red hair algae, it is the important economic class red algae resource of a class again, has there is correlative study table
Bright, red hair algae has lowering blood pressure and blood fat, yin-nourishing heat-clearing, prevents arteriosclerosis etc. from acting on, and has higher edibility and medicine
With value.How efficiently to utilize the also rare report of red hair algae resource at present, more have no from red hair algae extraction alpha-amylase and α-
The report or patent of glucosidase inhibitor.
The content of the invention
Alpha-amylase and the method for alpha-glucosidase restrainer are extracted from red hair algae it is an object of the present invention to provide a kind of,
Preparation technology is simple, and cost is low, and the alpha-amylase and alpha-glucosidase restrainer extracted have the work for suppressing blood glucose rise
Property.
In order to solve above-mentioned purpose, the technical solution adopted by the present invention is：
It is a kind of that alpha-amylase and the method for alpha-glucosidase restrainer are extracted from red hair algae, comprise the following steps：
Step 1: extracting：By new scarlet hair algae cleaning, drying, crush, obtain algae powder, 15~20 times of volumes of addition
Distilled water, is extracted 1~3 time, each 2h~3h in water-bath at 90 DEG C, centrifugation, merging supernatant, and supernatant is concentrated by evaporation and obtained
Concentrate；
Step 2: alcohol precipitation：Ethanol is slowly added dropwise into the concentrate, is 75% to ethanol final concentration volume fraction, it is quiet
After putting overnight, 10000r/min centrifugation 10min take precipitation to be dissolved completely in appropriate distilled water, are freeze-dried, and are made thick many
Sugar；
Step 3: gel chromatography：The Thick many candies are dissolved in distilled water, is splined on and is balanced through 0.1M NaCl solutions
Gel chromatography column on, eluted with 0.1M NaCl solutions, Phenol sulfuric acid procedure tracing detection, collect eluting peak, dialysis desalting is cold
It is lyophilized dry, obtain purified polysaccharide；Alpha-amylase and alpha-glucosidase restrainer is made.
Preferably, in the step one, in addition to decolourize：Before extracting, to gained algae powder 3~5 times of volumes of addition
Methanol solution, is cleaned three times, soaked overnight, 4000r/min after immersion repeatedly, is centrifuged 15 minutes, and gained precipitation is stripped again.
Preferably, in the step one, drying temperature is 50 DEG C.
Preferably, in the step one, distilled water is added, is extracted 2 hours in 90 DEG C of water-baths, 4000r/min centrifuges 20 points
Supernatant is taken after clock, precipitation repeats hot-water extraction 2 hours, then centrifuges, and is concentrated by evaporation after merging the supernatant centrifuged twice.
Preferably, between the step one and step 2, in addition to Sevage method removing proteins：By chloroform and n-butanol
According to volume ratio 4：1 ratio is hybridly prepared into Sevage reagents, then by the concentrate and Sevage reagents according to volume ratio
5：1 ratio mixing after, acutely vibration 40 minutes, 8000r/min be centrifuged off protein precipitation layer, by supernatant reclaim according to
Same method, in triplicate, separation take out supernatant, standby.
Preferably, in the step 3, the Thick many candies are purified using Sephadex G75 gel columns, specific behaviour
Make as follows：Loading Thick many candies liquid concentration 5mg/mL, applied sample amount is 3mL, is eluted with 0.1MNaCl solution；Connect using automatic collector
Receive, receive 50 and manage, often pipe 5mL；It is abscissa, absorbance with pipe number with Phenol sulfuric acid procedure in 490nm tracing detection elution profiles
It is worth for ordinate, does elution profile；Merge eluting peak, alpha-amylase and alpha-glucosidase suppression is made in dialysis desalting, freeze-drying
Preparation.
The invention has the advantages that：The present invention has extracted a kind of alpha-amylase and phlorose from red hair algae
Glycosides enzyme inhibitor, the active polysaccharide is a kind of crude, with the obvious effect for suppressing blood glucose rise.
Brief description of the drawings
Fig. 1 is the gel filtration elution profile of the inhibitor extracted in the embodiment of the present invention；
Fig. 2 contrasts for the alpha-amylase inhibitor extracted in the embodiment of the present invention to the inhibitory action of alpha-amylase activity
Figure；
Fig. 3 is that the suppression of the alpha-glucosidase restrainer that extracts to alpha-glucosidase activity is made in the embodiment of the present invention
Use comparison diagram.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：The extraction of inhibitor
The new scarlet hair algae is purchased in Putian, fujian Province.Extracted using water extraction and alcohol precipitation method, concrete operations are as follows：Will be new
Scarlet hair algae cleans up, and is placed in 50 DEG C of drying in baking oven, is crushed afterwards with pulverizer.Dry algae powder 3~5 times of volumes of addition
Methanol, lasting stirring is cleaned three times repeatedly, to ensure abundant, the soaked overnight decolourized.4000r/min centrifugations 15 after immersion
Minute, precipitation, which is taken out, adds 90 DEG C of distilled water heating water bath 2 hours, and 4000r/min takes supernatant, precipitation weight after centrifuging 20 minutes
Multiple hot-water extraction is centrifuged for 2 hours again, merges the supernatant centrifuged twice, is concentrated by evaporation, and operating temperature is 50 DEG C, and extract solution is dense
It is reduced to the 1/10 of original volume.Ethanol is added to final concentration 75% (V/V), is stood overnight.10000r/min is centrifuged 10 minutes, and it is heavy to take
Shallow lake is dissolved completely in a small amount of distilled water, freeze-drying, and Thick many candies are made.
Sevage method removing proteins：By chloroform and n-butanol according to volume ratio 4：1 ratio is hybridly prepared into Sevage
Reagent, then by Thick many candies concentrate solution and Sevage reagents according to volume ratio 5：After 1 ratio mixing, acutely vibrate 40 minutes,
8000r/min is centrifuged off protein precipitation layer, supernatant is reclaimed according to same method, in triplicate, supernatant is taken out in separation
Liquid, removing protein.
Embodiment 2：Polysaccharide it is gel-filtration purified
Thick many candies are purified using Sephadex G75 gel columns (C16/100), concrete operations are as follows：Loading liquid glucose
Concentration 5mg/mL, applied sample amount is 3mL, is eluted with 0.1MNaCl solution.Received using automatic collector, receive 50 and manage, often manage
5mL.It is abscissa with pipe number, absorbance is ordinate, is washed with Phenol sulfuric acid procedure in 490nm tracing detection elution profiles
De- figure.Merge eluting peak, preliminary purification polysaccharide F is made in dialysis desalting, freeze-drying.As a result as shown in figure 1, red hair algae is slightly more
The elution curve of sugar obtains single symmetrical peak, due to having carried out removal of protein to Thick many candies before purification, so eluting
To an one-component, F is named as, illustrates it is molecular weight more homogeneous polysaccharide in F components.
Embodiment 3：The red determination of activity of hair algae alpha-amylase inhibitor and the comparison with acarbose amylase inhibiting activity
It is measured using 3,5- dinitrosalicylic acid systems, takes the red hair polysaccharides solution of the different extension rates of 100 μ l, point
Do not add after 100 μ l alpha-amylase solution, mixing 10min, add 200 μ l soluble starches solution (1%) initial actions, reaction
Mixed liquor is placed in warm bath 5min in 37 DEG C of thermostat water baths, and 1mL DNS reagent terminating reactions are added afterwards, boiling is placed reaction liquid into
It is placed in ice bath and cools down rapidly after 5min in water-bath, taking-up, adds after 3mL distilled water dilutings, with determining light absorption value at 540nm,
Enzyme liquid is replaced as blank control with 0.5mL phosphate buffers (pH 6.8,0.05M), using acarbose as positive control,
IC50It is worth the concentration of liquid glucose when being suppressed 50% for enzyme activity.
With the rise of inhibitor concentration, inhibiting rate is also increased, and carries out linear fit, obtains inhibitor suppression after purification
The regression equation of rate processed is y=47.222x-9.4444, R2=0.9935, it can show that red hair algae is more after purification by regression equation
Sugar is about 1.26mg/mL to the 503nhibiting concentration of alpha-amylase.
As a result：From diagram 2, red hair polysaccharides are to the inhibitory action of alpha-amylase with the gradually increasing of polysaccharide concentration
Plus, its inhibitory action is significantly increased, and the two is linear.
Embodiment 4：Red hair algae alpha-glucosidase restrainer determination of activity and with acarbose Glucosidase inhibitor live
The comparison of property
Using PNPG (4- nitrobenzophenone-α-D- glucopyranosides) as substrate, detection purified polysaccharide is to alpha-glucosaccharase
The inhibitory action of enzyme.0.6mL sodium phosphate buffers (pH 6.8,0.05M), 0.25mL substrates PNPG (3mM), 0.1mL differences are dense
After the polysaccharide solution of degree is well mixed, 0.25mL alpha-glucosaccharases enzyme solutions (0.1U) initial action is added.Reaction mixture is put
In incubating 40min at 37 DEG C, 0.25mL sodium carbonate liquor (0.67M) terminating reaction is added afterwards.In using light splitting light under 405nm
Degree meter detection.Each experiment do 3 it is parallel.Inhibiting rate=(1- sample absorbances/control absorbance) * 100%
IC50It is worth the concentration of liquid glucose when being suppressed 50% for enzyme activity.
Linear fit is carried out to the inhibiting rate of inhibitor after purification, regression equation y=0.4173x-0.0579, R is obtained2=
0.9958.It is 1.34mg/mL to the 503nhibiting concentration of alpha-glucosidase that can obtain polysaccharide by regression equation.
As a result：From the figure 3, it may be seen that gradually increasing of the inhibitory action of red hair polysaccharides alpha-glucosidase with polysaccharide concentration
Plus, its inhibitory action is significantly increased, and the two is linear.
It is described above, only present pre-ferred embodiments, therefore the scope that the present invention is implemented can not be limited according to this, i.e., according to
The equivalent changes and modifications that the scope of the claims of the present invention and description are made, all should still belong in the range of the present invention covers.
Claims (6)
1. a kind of extract alpha-amylase and the method for alpha-glucosidase restrainer from red hair algae, it is characterised in that：Including with
Lower step：
Step 1: extracting：New scarlet hair algae is cleaned, dried, crushed, algae powder is obtained, adds the distillation of 15~20 times of volumes
Water, is extracted 1~3 time, each 2h~3h in water-bath at 90 DEG C, centrifugation, merging supernatant, and supernatant is concentrated
Liquid；
Step 2: alcohol precipitation：Ethanol is slowly added dropwise into the concentrate, is 75% to ethanol final concentration volume fraction, stands overnight
Afterwards, 10000r/min centrifuges 10min, takes precipitation to be dissolved completely in appropriate distilled water, is freeze-dried, and Thick many candies are made；
Step 3: gel chromatography：The Thick many candies are dissolved in distilled water, are splined on through coagulating that 0.1M NaCl solutions are balanced
On glue chromatographic column, eluted with 0.1M NaCl solutions, Phenol sulfuric acid procedure tracing detection, collect eluting peak, dialysis desalting, freezing is dry
It is dry, purified polysaccharide is obtained, that is, alpha-amylase and alpha-glucosidase restrainer is made.
2. a kind of extraction alpha-amylase from red hair algae as claimed in claim 1 and the method for alpha-glucosidase restrainer,
It is characterized in that：In the step one, in addition to decolourize：Before extracting, the methanol of 3~5 times of volumes is added to gained algae powder
Solution, is cleaned three times, soaked overnight, 4000r/min after immersion repeatedly, is centrifuged 15 minutes, and gained precipitation is stripped again.
3. a kind of extraction alpha-amylase from red hair algae as claimed in claim 1 and the method for alpha-glucosidase restrainer,
It is characterized in that：In the step one, drying temperature is 50 DEG C.
4. a kind of extraction alpha-amylase from red hair algae as claimed in claim 1 and the method for alpha-glucosidase restrainer,
It is characterized in that：In the step one, distilled water is added, is extracted 2 hours in 90 DEG C of water-baths, after 4000r/min is centrifuged 20 minutes
Supernatant is taken, precipitation repeats hot-water extraction 2 hours, then centrifuges, and is concentrated by evaporation after merging the supernatant centrifuged twice.
5. a kind of extraction alpha-amylase from red hair algae as claimed in claim 1 and the method for alpha-glucosidase restrainer,
It is characterized in that：Between the step one and step 2, in addition to Sevage method removing proteins：Chloroform is pressed with n-butanol
According to volume ratio 4：1 ratio is hybridly prepared into Sevage reagents, then by the concentrate and Sevage reagents according to volume ratio 5：1
Ratio mixing after, acutely vibration 40 minutes, 8000r/min be centrifuged off protein precipitation layer, by supernatant reclaim according to same
Quadrat method, in triplicate, separation take out supernatant, standby.
6. a kind of extraction alpha-amylase from red hair algae as claimed in claim 1 and the method for alpha-glucosidase restrainer,
It is characterized in that：In the step 3, the Thick many candies are purified using Sephadex G75 gel columns, concrete operations are such as
Under：Loading Thick many candies liquid concentration 5mg/mL, applied sample amount is 3mL, is eluted with 0.1M NaCl solutions；Received using automatic collector,
Receive 50 to manage, often pipe 5mL；It is abscissa, absorbance with pipe number with Phenol sulfuric acid procedure in 490nm tracing detection elution profiles
For ordinate, elution profile is done；Merging eluting peak, dialysis desalting, freeze-drying, obtained purified polysaccharide, obtained alpha-amylase and α-
Glucosidase inhibitor.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108721186A (en) * | 2018-08-02 | 2018-11-02 | 江西登云健康美业互联有限公司 | A kind of anti-ageing face cream and preparation method thereof |
| CN110551580A (en) * | 2019-09-11 | 2019-12-10 | 中国海洋大学 | Beer with function of antagonizing maltose degradation |
| CN113174411A (en) * | 2021-03-16 | 2021-07-27 | 集美大学 | Lactobacillus and rhodophyta fermentation supernatant with alpha-glucosidase inhibitory activity and application thereof |
| CN113754790A (en) * | 2021-10-15 | 2021-12-07 | 山东元裕生物科技有限公司 | Biological agent for promoting cell growth |
| CN114831311A (en) * | 2022-04-12 | 2022-08-02 | 中国科学院海洋研究所 | Application of red algae starch |
| CN115152850A (en) * | 2022-07-04 | 2022-10-11 | 中国农业大学 | Enzymatic oat milk for inhibiting activity of alpha-glucosidase and preparation method thereof |
| CN115417800A (en) * | 2022-08-16 | 2022-12-02 | 集美大学 | Method for separating and purifying lutein from red hair algae |
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2017
- 2017-08-02 CN CN201710651449.8A patent/CN107312106A/en active Pending
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| 孙惠洁: "红毛藻多糖的提取纯化及其性质的研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 宋田源等: "红毛藻多糖组分纯化及其抑制α-淀粉酶及α-葡萄糖苷酶活性的研究", 《中国食品科学技术学会第十三届年会论文摘要集》 * |
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| CN108721186A (en) * | 2018-08-02 | 2018-11-02 | 江西登云健康美业互联有限公司 | A kind of anti-ageing face cream and preparation method thereof |
| CN108721186B (en) * | 2018-08-02 | 2021-04-20 | 江西登云健康美业互联有限公司 | Anti-aging face cream and preparation method thereof |
| CN110551580A (en) * | 2019-09-11 | 2019-12-10 | 中国海洋大学 | Beer with function of antagonizing maltose degradation |
| CN113174411A (en) * | 2021-03-16 | 2021-07-27 | 集美大学 | Lactobacillus and rhodophyta fermentation supernatant with alpha-glucosidase inhibitory activity and application thereof |
| WO2022194189A1 (en) * | 2021-03-16 | 2022-09-22 | 集美大学 | LACTOBACILLUS BANGIA FUSCO-PURPUREA FERMENTATION SUPERNATANT HAVING α-GLUCOSIDASE INHIBITORY ACTIVITY, AND USE THEREOF |
| CN113754790A (en) * | 2021-10-15 | 2021-12-07 | 山东元裕生物科技有限公司 | Biological agent for promoting cell growth |
| CN113754790B (en) * | 2021-10-15 | 2022-03-18 | 山东元裕生物科技有限公司 | Biological agent for promoting cell growth |
| CN114831311A (en) * | 2022-04-12 | 2022-08-02 | 中国科学院海洋研究所 | Application of red algae starch |
| CN115152850A (en) * | 2022-07-04 | 2022-10-11 | 中国农业大学 | Enzymatic oat milk for inhibiting activity of alpha-glucosidase and preparation method thereof |
| CN115152850B (en) * | 2022-07-04 | 2023-10-31 | 中国农业大学 | Enzymatic oat milk for inhibiting alpha-glucosidase activity and preparation method thereof |
| CN115417800A (en) * | 2022-08-16 | 2022-12-02 | 集美大学 | Method for separating and purifying lutein from red hair algae |
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Application publication date: 20171103 |