CN109771579A - The method of orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process - Google Patents
The method of orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process Download PDFInfo
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Abstract
The method of orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process, belong to biological health-care product studying technological domain, utilize Orthogonal Experiment and Design, corn stigma and birch young pilose antler total extract are obtained using heating and refluxing extraction method, 9 samples are obtained, hypoglycemic activity screening is carried out to 9 samples afterwards, result is intuitively analyzed according to orthogonal experiment, the final extraction process for establishing birch young pilose antler and corn stigma are as follows: extraction time 1h, extraction time 2 times, solid-liquid ratio 1:20, it is 33.6% to alpha-glucosidase maximal percentage inhibition that corn stigma and birch young pilose antler extract concentrations, which are 100 μ g/mL,.The present invention is analyzed by the hypoglycemic effect after combining to corn stigma and birch young pilose antler, obtains the optimum extraction process of birch young pilose antler and corn stigma, and the further research for sharing hypoglycemic effect with birch young pilose antler for corn stigma provides basis and foundation.
Description
Technical field
The invention belongs to biological health-care product studying technological domain, especially relate to fine and soft using orthogonal experiment method optimization birch
With the method for corn stigma sugar-lowering components extraction process.
Background technique
Birch is fine and soft: (Inonotuso bliquus (Fr) Pilat) its sclerotium is medicinal fungus, also known as: Inonotus obliquus, Japanese
Entitled カ バ ノ ア Na タ ケ or チ ャ-ガ is referred to as Chaga in Russia.The mycelium of birch young pilose antler is extremely cold-resistant, lives in
Mycelium in timber is resistant to subzero 40 DEG C of low temperature.Such as the north of Hokkaido, Japan, North America, Finland, Poland, Russia,
There is resource more abundant in the Heilungkiang in China, two province of Jilin.Civil in Russia, people are using Inonotus obliquus as daily tea
Drink prevents and treats diabetes, and scientist also confirms that by a large amount of zoopery and clinical research: Inonotus obliquus almost without
Any malicious seondary effect has hypoglycemic, resists AIDS, anticancer, and anti-aging improves numerous medical values such as immunity.
Corn stigma is that the drying filigree of gramineae plant corn (Zeamays L.) and column cap, corn stigma disappear with diuresis
The effect of swollen, calming the liver and benefiting the lungs, " the southern regions of the Yunnan Province wood grass " in 1476 is recorded as medicine resource earliest, quilt " Chinese Pharmacopoeia " in 1977
Record, and by 1985 the first edition " Ministry of Health of the People's Republic of China's medicinal material standard " be included by medicinal herbs most in use kind.In 20 generation
Record and just carried out the research of corn stigma chemical component the twenties, have now been found that in corn stigma containing polysaccharide, saponin(e, flavones,
A variety of chemical components such as alkaloid, sterol, volatile oil, amino acid, have auxiliary hyperglycemic, strengthen immunity, decompression, diuresis,
The bioactivity such as antibacterial, anticancer, wherein corn silk polysaccharide is one of main water-soluble active ingredient.
By comparing the hypoglycemic effect of normal group and hyperglycemia group mouse models, discovery corn silk polysaccharide has apparent drop
Sugar effect；Discovery: the inhibition of ethyl alcohol residue extract is compared to the alpha-glucosidase inhibitory effect of opposed polarity ingredient
Effect is greater than Thick many candies and is greater than alcohol extracting thing.It is screened in the prior art for corn stigma hypoglycemic activity position, with water
The hypoglycemic effect trend for mentioning alcohol hypostasis is most obvious, followed by water extract with 60% ethanol extract, but for Inonotus obliquus with
The research that corn silk water extract shares generated hypoglycemic activity is not yet clear.
Therefore there is an urgent need for a kind of novel technical solutions in the prior art to solve the problems, such as this.
Summary of the invention
The technical problems to be solved by the present invention are: provide a kind of orthogonal experiment method optimization birch young pilose antler and corn stigma hypoglycemic at
The method for dividing extraction process, the hypoglycemic effect after combining to corn stigma and birch young pilose antler are analyzed, and birch young pilose antler and corn stigma are obtained
Optimum extraction process, for hypoglycemic effect it is further research provide basis and foundation.
The method that orthogonal experiment method optimizes birch young pilose antler and corn stigma sugar-lowering components extraction process, it is characterized in that: including following
Step, and following steps sequentially carry out,
Step 1: orthogonal prepares corn stigma and birch young pilose antler powder
Selecting extraction time, extraction time and solid-liquid ratio is three factors of orthogonal experiment, establishes L9 (33) orthogonal arrage, it presses
According to orthogonal test table by extracting to pretreatment corn stigma with birch young pilose antler, filtering, filtrate mixes and centrifugal concentrating difference
It obtains 9 groups of corn stigmas and birch young pilose antler medicinal extract is spare；
Step 2: orthogonal obtains inhibiting rate
The 9 groups of corn stigmas and birch young pilose antler medicinal extract that orthogonal experiment obtains in step 1 are taken, 9 groups of corn stigmas is carried out and birch young pilose antler is soaked
Cream tests the inhibiting rate of alpha-glucosidase, obtains 9 groups of inhibiting rate values；
Step 3: selecting and verifying Optimized Extraction Process
The 9 groups of inhibiting rate values obtained by step 2 select optimum extraction time, extraction time and solid-liquid ratio, and repeat
Test three times, obtains three groups of inhibiting rate values, and inhibiting value is stablized, and the birch young pilose antler and corn stigma sugar-lowering components after determining optimization are extracted
Technique.
L9 (3 in the step 13) orthogonal arrage is
Pretreatment corn stigma and birch young pilose antler in the step 1 are to take corn stigma and the fine and soft each 15g of birch, are put into 1000ml
In round-bottomed flask, after impregnating 30 minutes, it is fine and soft to take out spare corn stigma and birch.
Corn stigma is to the inhibiting rate experiment of alpha-glucosidase with birch young pilose antler medicinal extract in the step 2,
The 9 groups of corn stigmas and 20 μ L of birch young pilose antler extract solution obtained in step 1 are taken, it is 6.8 that 40 μ L pH, which are added,
The pNPG glucosidase bottom of 3moL/L is added after 37 DEG C of constant temperature 5min for PBS buffer solution, the alpha-glucosidase of 40 μ L0.5U/mL
Object 20 μ L, 37 DEG C of isothermal reaction 30min；Add the Na of 100 μ L 0.1moL/L2CO3It is close to survey light under 405nm wavelength for solution
Spend OD value.
The PBS buffer solution is 0.2mol/LNaH2PO4Solution 51mL and 0.2mol/LNa2HPO4Solution 49mL mixing adds
100mL distilled water.
The pNPG glucosidase substrate of the 3moL/L is that 0.00452g pNPG glucosidase substrate and 5mLPBS solution are mixed
Close liquid.
The alpha-glucosidase is the alpha-glucosaccharase enzyme powder 1mg of accurately weighed 10U/mg, adds 2mL is water-soluble to solve
The alpha-glucosaccharase enzyme solutions of 5U/mL, then the alpha-glucosaccharase enzyme solutions of obtained 0.5U are diluted with water.
Birch young pilose antler and corn stigma sugar-lowering components extraction process after the optimization obtained in the step 3 are extraction time to be
1h, extraction time are 2 times, and solid-liquid ratio 1:20, inhibiting rate is 33.6% when extract concentrations are 100 μ g/mL.
Through the above design, the present invention can be brought the following benefits: orthogonal experiment method optimizes birch young pilose antler and jade
The method of rice palpus sugar-lowering components extraction process, the hypoglycemic effect after combining to corn stigma and birch young pilose antler are analyzed, and birch is obtained
Fine and soft and corn stigma optimum extraction process provides basis and foundation for the further research of hypoglycemic effect, to treat Rezulin
The exploitation of object and the research of hypoglycemic medicine lay the foundation.
Specific embodiment
The method that orthogonal experiment method optimizes birch young pilose antler and corn stigma sugar-lowering components extraction process, using orthogonal,
Extraction time (A), 3 extraction time (B), solid-liquid ratio (C) factors are selected, each factor selects 3 levels as shown in table 1, utilizes
L9(33) orthogonal arrage progress experimental design, corn stigma medicinal material and birch young pilose antler powder are accurately weighed, according to L9 (33) orthogonal test table claims
Inonotus obliquus and each 15g of corn stigma are taken, is put into 1000ml round-bottomed flask, after impregnating 30 minutes, by different extraction times, secondary
Number, feed ratio extract respectively, filter, and centrifugal concentrating is stand-by in medicinal extract.
1 orthogonal test factor level table of table
Using medicinal substances extract to the inhibiting rate of alpha-glucosidase as inspection target, screen optimum extraction process.Orthogonal experiment
Concrete analysis value is as shown in table 2,
The intuitive analytical table of 2 orthogonal experiment of table
Specific hypoglycemic activity test are as follows:
1, laboratory apparatus and drug
Alpha-glucosidase (Sigma Co., USA)；PNPG (Sigma Co., USA)；Acarbose (E.C.260-030-
7,Sigma,USA)；DMSO (Sigma Co., USA)；Microplate reader (Bio-Rad.CA, USA)；Rotary type thermostatic oscillator (Suzhou
Pei Ying experimental facilities Co., Ltd)；METTLER TOLEDO electronic balance (German Sai Duolisi joint-stock company)；96 microwell plates
(Costar company, the U.S.)；Milli-Q water purification system (Millipore, MA, USA)；Each model liquid-transfering gun and pipette tips etc.；Its
Remaining reagent is that analysis is pure.
P-nitrophenyl-α-D the glucopyranoside (pNPG) provided by Shanghai Rui Yong Biotechnology Co., Ltd；It is anhydrous
Sodium carbonate；Sodium dihydrogen phosphate；Disodium hydrogen phosphate；The acarbose provided by Shanghai Yuan Ye Biotechnology Co., Ltd；Pure water
For distilled water；Alpha-glucosidase (Sigma Co., USA), remaining reagent are that analysis is pure.
2, the configuration of reagent
0.2mol/L NaH2PO4The preparation of solution (100mL): accurately weighed 7.16gNaH2PO4Solid is in 100mL capacity
Constant volume is made after being dissolved in bottle with distilled water.
0.2mol/L Na2HPO4The preparation of solution (100mL): accurately weighed 3.12gNa2HPO4Solid is in 100mL capacity
Constant volume is made after being dissolved in bottle with distilled water.
The preparation of the PBS solution (200mL) of 0.1mol/L: 0.2mol/LNaH is taken2PO4Solution 51mL and 0.2mol/
LNa2HPO4Solution 49mL mixing plus 100mL distilled water are made.
The preparation of substrate pNPG solution: accurately weighed pNPG 0.00452g is dissolved in 5mLPBS solution (pH6.8), preparation
At the pNPG solution of 3mmol/L.
The preparation of alpha-glucosaccharase enzyme solutions: the alpha-glucosaccharase enzyme powder 1mg of accurately weighed 10U/mg adds 2mL water-soluble
The alpha-glucosaccharase enzyme solutions of 5U/mL are solved, then the alpha-glucosaccharase enzyme solutions of obtained 0.5U are diluted with water (if what is prepared is cold
Freeze-thaw liquid uses after needing 37 DEG C of rapid defrostings).
Na2CO3The preparation of terminate liquid: accurately weighed 10.6gNa2CO3, it is transferred in 100mL volumetric flask, uses deionized water
The Na for dissolving and being settled to scale to get 0.1mol/L2CO3Terminate liquid.
3, method and step
Taking corn stigma with birch young pilose antler extract 10mg, successively equimultiple dilutes to obtain 0.1mg/mL, 0.05mg/mL, 0.025mg/mL
The sample solution of concentration.
Inhibit model by establishing external alpha-glucosidase, to corn stigma and birch young pilose antler extract various extracting conditions institute
It obtains extract and carries out screening active ingredients, and the relationship between the concentration and inhibitory activity of each polar fraction extract is ground
Study carefully.
This detection carries out on 96 orifice plates, the method is as follows: 40 μ L phloroses are added in 40 μ LPBS buffers (pH 6.8)
3mmoL/L pNPG glucosidase substrate 20 is added after 37 DEG C of constant temperature 5min for glycosides enzyme (0.5U/mL), each Conditions Sample solution of 20 μ L
μ L, 37 DEG C of isothermal reaction 30min.Add the Na of 100 μ L2CO3(0.1moL/L) solution surveys OD value under 405nm wavelength.
Reaction system is as shown in table 3: experiment sets 5 groups altogether, and every group sets 4 multiple holes, is respectively as follows:
A. negative control group (buffer+enzyme solution+substrate)；
B. blank group control group (buffer)；
C. sample measurement group (sample+enzyme solution+substrate)；
D. sample controls group (sample+buffer)；
Wherein positive controls are (Acarbose+ enzyme solution+substrate), i.e., using the acarbose of positive control as a sample
Product are measured in the same method, and corn stigma calculates according to the following formula with inhibiting rate (%) of the birch young pilose antler extract to alpha-glucosidase:
I%=a- (c-d)/(a-b)
4 alpha-glucosidase activity reaction system of table
4, result
Obtained extract sample solution is under various concentration to α-Portugal under corn stigma and birch young pilose antler various extracting conditions
Polyglycoside enzyme inhibition rate is as shown in table 5:
5 various extracting conditions of table are under various concentration to alpha-glucosaccharase enzyme inhibition rate
The present invention is by carrying out active preliminary examinations, knot to the extract that birch is fine and soft and corn stigma is under various extracting conditions
Fruit shows: obtained extract sample solution all has inhibition alpha-glucosaccharase under corn stigma and birch young pilose antler various extracting conditions
The effect of enzyme, inhibiting rate activity is different under same concentrations gradient, extraction conditions be 1h, solid-liquid ratio 1:20 extraction time,
Extract sample is best to the inhibiting rate activities present of alpha-glucosidase at extraction time 2 times, i.e., presses down when concentration is 100 μ g/mL
Rate processed is 33.6%.As a result the research of the two compatibility provides reference frame later；To treat opening for diabetes medicament later
The research of hair and hypoglycemic medicine lays the foundation.
Claims (8)
1. the method that orthogonal experiment method optimizes birch young pilose antler and corn stigma sugar-lowering components extraction process, it is characterized in that: including following step
Suddenly, and following steps sequentially carry out,
Step 1: orthogonal prepares corn stigma and birch young pilose antler powder
Selecting extraction time, extraction time and solid-liquid ratio is three factors of orthogonal experiment, establishes L9 (33) orthogonal arrage, according to just
Hand over test table by extracting, filtering with birch young pilose antler to pretreatment corn stigma, filtrate mixes and centrifugal concentrating obtains 9 respectively
Group corn stigma and birch young pilose antler medicinal extract are spare；
Step 2: orthogonal obtains inhibiting rate
The 9 groups of corn stigmas and birch young pilose antler medicinal extract that orthogonal experiment obtains in step 1 are taken, 9 groups of corn stigmas and birch young pilose antler medicinal extract pair are carried out
The inhibiting rate of alpha-glucosidase is tested, and 9 groups of inhibiting rate values are obtained；
Step 3: selecting and verifying Optimized Extraction Process
The 9 groups of inhibiting rate values obtained by step 2 select optimum extraction time, extraction time and solid-liquid ratio, retrial of laying equal stress on to test
Three times, three groups of inhibiting rate values are obtained, inhibiting value is stablized, and the birch young pilose antler and corn stigma sugar-lowering components after determining optimization extract work
Skill.
2. the method for orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process according to claim 1,
It is characterized in that: the L9 (3 in the step 13) orthogonal arrage is
。
3. the method for orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process according to claim 1,
It is characterized in that: the pretreatment corn stigma and birch young pilose antler in the step 1 are to take corn stigma and the fine and soft each 15g of birch, it is put into 1000ml
In round-bottomed flask, after impregnating 30 minutes, it is fine and soft to take out spare corn stigma and birch.
4. the method for orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process according to claim 1,
It is characterized in that: corn stigma and birch young pilose antler medicinal extract are to the inhibiting rate experiment of alpha-glucosidase in the step 2,
The 9 groups of corn stigmas and 20 μ L of birch young pilose antler extract solution obtained in step 1 are taken, it is slow that the PBS that 40 μ L pH are 6.8 is added
20 μ of pNPG glucosidase substrate of 3moL/L is added after 37 DEG C of constant temperature 5min for fliud flushing, the alpha-glucosidase of 40 μ L0.5U/mL
L, 37 DEG C of isothermal reaction 30min；Add the Na of 100 μ L0.1moL/L2CO3Solution, the densitometric OD value under 405nm wavelength.
5. the method for orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process according to claim 4,
It is characterized in that: the PBS buffer solution is 0.2mol/LNaH2PO4Solution 51mL and 0.2mol/LNa2HPO4Solution 49mL mixing adds
100mL distilled water.
6. the method for orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process according to claim 4,
It is characterized in that: the pNPG glucosidase substrate of the 3moL/L is 0.00452g pNPG glucosidase substrate and 5mLPBS solution
Mixed liquor.
7. the method for orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process according to claim 4,
It is characterized in that: the alpha-glucosidase is the alpha-glucosaccharase enzyme powder 1mg of accurately weighed 10U/mg, add 2mL is water-soluble to solve
The alpha-glucosaccharase enzyme solutions of 5U/mL, then the alpha-glucosaccharase enzyme solutions of obtained 0.5U are diluted with water.
8. the method for orthogonal experiment method optimization birch young pilose antler and corn stigma sugar-lowering components extraction process according to claim 1,
It is characterized in that: the birch young pilose antler and corn stigma sugar-lowering components extraction process after the optimization obtained in the step 3 are extraction time to be
1h, extraction time are 2 times, and solid-liquid ratio 1:20, inhibiting rate is 33.6% when extract concentrations are 100 μ g/mL.
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| CN113616743A (en) * | 2021-07-24 | 2021-11-09 | 吉林化工学院 | Optimized corn stigma water extraction process based on blood glucose reducing activity |
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| CN113616743A (en) * | 2021-07-24 | 2021-11-09 | 吉林化工学院 | Optimized corn stigma water extraction process based on blood glucose reducing activity |
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Application publication date: 20190521 |