CN104961842B - A kind of preparation method of the corn stigma ultrafiltration polysaccharide with immunoregulation effect - Google Patents
A kind of preparation method of the corn stigma ultrafiltration polysaccharide with immunoregulation effect Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of the corn stigma ultrafiltration polysaccharide with immunoregulation effect, belong to the extraction and separation technology field of natural plant active polysaccharide.By the pretreatment of corn stigma, degreasing, extraction, centrifugation, carry again, removal of impurities, ultrafiltration, dry prepared corn stigma ultrafiltration polysaccharide; the method has simple to operate; the advantages of equipment investment is few; and Environmental Safety, it is with low cost, with significant economic benefit; it is suitable to large-scale production; the corn stigma ultrafiltration polysaccharide prepared using the method also has immunoregulation effect, with stimulation Proliferation of lymphocytes higher, and dose dependent is presented.
Description
Technical field
The present invention relates to a kind of preparation method of the corn stigma ultrafiltration polysaccharide with immunoregulation effect, belong to natural plants
The extraction and separation technology field of active polysaccharide.
Background technology
Corn stigma (Stigmata madis) alias corn wheat, ear of maize hair, grass family jade another name for Sichuan Province platymiscium corn (Zea mays L.) style, be a kind of traditional Chinese herbal medicine,《The southern regions of the Yunnan Province book on Chinese herbal medicine》、《Dictionary of medicinal plant》Deng all on the books, it is《The Chinese people
The Ministry of Public Health of republic medicinal material standard》The medicinal herbs most in use kind that 1985 editions (one) includes.
China is Maize Production big country, and corn annual production occupies the 2nd, the world.Accordingly, as the corn stigma of corn's by-products
Resource very abundant.
Polysaccharide compound is one of chemical composition important contained by corn stigma, and Primary Study shows that corn silk polysaccharide has
Protect liver, hypoglycemic, regulation immune, antibacterial, antitumor, diuresis and the effect such as antipyretic, clinical test confirm that polysaccharide is alternatively arranged as swelling
Knurl chemotherapy and effective auxiliary therapeutic agent of radiotherapy, have good application prospect and exploitation in terms of medicine, food
Value.
Traditional Polyose extraction uses organic solvent deposit method, removes small molecular weight impurity, prepares polysaccharide, and such as 80% ethanol sinks
Form sediment etc..This method not only needs to consume a large amount of organic solvents, and organic solvent has very powerful volatilization and inflammable and explosive waits spy
Point, thus it is also higher to equipment investment requirement.The present invention uses isometric moisturizing hyperfiltration process, many corn stigma is effectively retained
While sugared active component, small-molecule substance is removed, substitute organic solvent deposit, prepare polysaccharide, with simple to operate, equipment throwing
The advantages of entering few, and Environmental Safety, it is with low cost, with significant economic benefit, it is suitable to large-scale production.
The content of the invention
The invention provides a kind of preparation method of the corn stigma ultrafiltration polysaccharide with immunoregulation effect, this method is used
Isometric moisturizing hyperfiltration process, simple to operate, equipment investment is few, and Environmental Safety is with low cost.
The technical solution adopted for the present invention to solve the technical problems is：A kind of preparation method of corn stigma ultrafiltration polysaccharide,
Comprise the following steps：
1. the pretreatment of corn stigma
After corn stigma removal of impurities, in being dried at 60-70 DEG C, 40-100 mesh is crushed to after being placed in dry environment natural cooling.
2. the degreasing of corn stigma
Corn stigma powder presses mass volume ratio 1:2-1:5 add 95% ethanol or soaked in absolute ethyl alcohol 12-24 hours, depressurize
Filter, filter residue repeats immersion and is spontaneously dried after shady place for 1-2 times with method, and is saved backup in dry environment.
3. prepared by ultrafiltration polysaccharide
3.1 extractions press 1:10-1:30 mass volume ratios add distilled water, are extracted in extractor at 95-100 DEG C
0.5-3 hours.
3.2 CEFs are centrifuged through the r/min of link-suspended basket centrifuge 1440, separate supernatant precipitation（Corn stigma
Grain）.
3.3 put forward precipitation again extracts 1 time again with method.The supernatant that merging is extracted twice.
3.4 removal of impurities supernatants are centrifuged through 16000 r/min, remove molecule, prepare polysaccharide extraction clear liquid.
3.5 ultrafiltration supernatant liquids are 10 KDa milipore filter ultrafiltration, ultrafiltration pressure 0.05-2.0 Mpa through molecular cut off.Ultrafiltration
Method：When supernatant is concentrated by ultrafiltration 1/20-1/2 times to leaching liquor, to mending the 1/10-1 times of steaming of the volume of concentrate in concentrate
Distilled water, continues ultrafiltration to the volume of concentrate and reverts to moisturizing front volume, and moisturizing is repeated with method, until the volume that always adds water is for leaching liquor
2-5 times of volume, no longer moisturizing.
3.6 dry concentrate continuation ultrafiltration to dry matter content up to 15-20%, freeze-drying or spray drying, obtain
Corn stigma ultrafiltration polysaccharide powder.
The hyperfiltration process of the 5th step in this step can also be used and extracted to disposable 2-5 times of addition in extraction clear liquid
The distilled water of liquid product.
The beneficial effects of the invention are as follows：The present invention uses isometric moisturizing hyperfiltration process, is being effectively retained corn silk polysaccharide
While active component, small-molecule substance is removed, substitute organic solvent deposit, polysaccharide is prepared, with simple to operate, equipment investment
It is few the advantages of, and Environmental Safety, with low cost, with significant economic benefit, it is suitable to large-scale production.
Brief description of the drawings
Fig. 1 is corn stigma ultrafiltration polysaccharide stimulated in vitro lymphocyte proliferation assay result of the invention.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and detailed description.
Embodiment 1
A kind of preparation method of corn stigma ultrafiltration polysaccharide, comprises the following steps：
1. the pretreatment of corn stigma
After corn stigma removal of impurities, in being dried at 70 DEG C, 100 mesh are crushed to after being placed in dry environment natural cooling.
2. the degreasing of corn stigma
The kg of corn stigma powder 1, by mass volume ratio 1:5 add soaked in absolute ethyl alcohol 12 hours, filtration under diminished pressure, the same method of filter residue
Repeat immersion to be spontaneously dried after shady place for 1 time, and saved backup in dry environment.
3. prepared by ultrafiltration polysaccharide
3.1 extractions press 1:20 mass volume ratios add distilled water（20 L）, 2 are extracted in extractor at 95-100 DEG C
Hour.
3.2 CEFs are centrifuged through the r/min of link-suspended basket centrifuge 1440, separate supernatant precipitation（Corn stigma
Grain）.
3.3 put forward precipitation again extracts 1 time again with method.The supernatant that merging is extracted twice.
3.4 removal of impurities supernatants are centrifuged through 16000 r/min, remove molecule, prepare polysaccharide extraction clear liquid.
3.5 ultrafiltration supernatant liquids are 10 KDa milipore filter ultrafiltration, the Mpa of ultrafiltration pressure 0.08 through molecular cut off.Ultrafiltration side
Method：It is concentrated by ultrafiltration when supernatant to 1/10 times of leaching liquor（2 L）When, to 1/2 times of distilled water of the volume of concentrate of benefit in concentrate
（1 L）, continue ultrafiltration to the volume of concentrate and revert to moisturizing front volume, moisturizing is repeated with method, until the volume that always adds water is extraction
4 times of liquid product（80 L）, no longer moisturizing.
3.6 dry concentrate continuation ultrafiltration to dry matter content up to 15-20%, freeze-drying or spray drying, obtain beautiful
Rice must ultrafiltration polysaccharide powder.
Corn stigma ultrafiltration polysaccharide extract rate is 7.22%, its polyoses content 36.11%.
Embodiment 2
A kind of preparation method of corn stigma ultrafiltration polysaccharide, comprises the following steps：
1. the pretreatment of corn stigma
After corn stigma removal of impurities, in being dried at 65 DEG C, 100 mesh are crushed to after being placed in dry environment natural cooling.
2. the degreasing of corn stigma
Corn stigma powder 1kg, by mass volume ratio 1:3 add soaked in absolute ethyl alcohol 24 hours, filtration under diminished pressure, filter residue with method again
Repeat immersion to be spontaneously dried after shady place for 1 time, and saved backup in dry environment.
3. prepared by ultrafiltration polysaccharide
3.1 extractions press 1:15 mass volume ratios add distilled water（15 L）, 1 is extracted in extractor at 95-100 DEG C
Hour.
3.2 CEFs are centrifuged through the r/min of link-suspended basket centrifuge 1440, separate supernatant precipitation（Corn stigma
Grain）.
3.3 put forward precipitation again extracts 1 time again with method.The supernatant that merging is extracted twice.
3.4 removal of impurities supernatants are centrifuged through 16000 r/min, remove molecule, prepare polysaccharide extraction clear liquid.
3.5 ultrafiltration supernatant liquids are 10 KDa milipore filter ultrafiltration, the Mpa of ultrafiltration pressure 0.06 through molecular cut off.Ultrafiltration side
Method：When supernatant is concentrated by ultrafiltration 1/10 times to leaching liquor（1.5 L）, to 1/3 times of distillation of the volume of concentrate of benefit in concentrate
Water（0.5 L）, continue ultrafiltration to the volume of concentrate and revert to moisturizing front volume, moisturizing is repeated with method, until the volume that always adds water is
5 times of leaching liquor volume（75 L）, no longer moisturizing.
3.6 dry concentrate continuation ultrafiltration to dry matter content up to 15-20%, freeze-drying or spray drying, obtain beautiful
Rice must ultrafiltration polysaccharide powder.
Corn stigma ultrafiltration polysaccharide extract rate is 6.65%, its polyoses content 38.34%.
Embodiment 3
A kind of preparation method of corn stigma ultrafiltration polysaccharide, comprises the following steps：
1. the pretreatment of corn stigma
After corn stigma removal of impurities, in being dried at 60 DEG C, 60 mesh are crushed to after being placed in dry environment natural cooling.
2. the degreasing of corn stigma
The kg of corn stigma powder 1, by mass volume ratio 1:4 add soaked in absolute ethyl alcohol 24 hours, filtration under diminished pressure, the same method of filter residue
Repeat immersion to be spontaneously dried after shady place for 1 time, and saved backup in dry environment.
3. prepared by ultrafiltration polysaccharide
3.1 extractions press 1:30 mass volume ratios add distilled water（30 L）, extracted in extractor at 95-100 DEG C
1.5 hours.
3.2 CEFs are centrifuged through the r/min of link-suspended basket centrifuge 1440, separate supernatant precipitation（Corn stigma
Grain）.
3.3 put forward precipitation again extracts 1 time again with method.The supernatant that merging is extracted twice.
3.4 removal of impurities supernatants are centrifuged through 16000 r/min, remove molecule, prepare polysaccharide extraction clear liquid.
3.5 ultrafiltration supernatant liquids are 10 KDa milipore filter ultrafiltration, the Mpa of ultrafiltration pressure 0.80 through molecular cut off.Ultrafiltration side
Method：2.5 times of distilled water of leaching liquor volume are added to disposable in extraction clear liquid（75 L）, after mixing, it is concentrated by ultrafiltration to dry
Matter content reaches 15-20%, dries.
3.6 are frozen and dried dry or are spray-dried, and obtain corn stigma ultrafiltration polysaccharide powder.
Corn stigma ultrafiltration polysaccharide extract rate is 6.93%, its polyoses content 35.57%.
Embodiment 4
Study immune regulation
Cervical dislocation puts to death mouse, and spleen is taken under aseptic condition, is placed on 100 mesh sterilizing stainless steel screen clothes, with syringe
Plug grinds spleen, sieves, and mesh screen is rinsed 2-3 times with PBS, and the suspension of splenocyte is transferred in aseptic centrifuge tube, adds PBS extremely
30-40 mL, 400 G are centrifuged 6 min, collect cell, and NH is added at room temperature4Cl solution（1.5 mL/ are only）, continuous rifle beats or shakes
Shake and mix cell 10 minutes, with splitting erythrocyte, plus PBS to 30-40 mL, 400 G are centrifuged 5 min, remove supernatant, add a small amount of
After RPMI1640 culture mediums cell counter is counted, with RPMI1640 diluting cells suspension to 2 × 106Individual/mL.Take 180 μ L thin
Born of the same parents' suspension is added in 96 orifice plates, is separately added into the different samples and positive control phytohemagglutin phytolectin of 20 μ L, negative control
PBS.At 37 DEG C and 5% CO2Under conditions of culture 3 days after, determine 570 nm and 600 respectively with the automatic plate readers of ELISA
The absorbance of nm, adds 20 μ L Alamar Blue developers, cultivates 6-8 hours, and 570 nm and 600 nm are determined again
Absorbance, the formula then given according to Alamer Blue Assay calculates activity ratio of each sample to lymphocyte.
Lymphocytic proliferation rate (%)=[117216 × Aλ570(sample)-80586×Aλ600(sample)]
/[117216×Aλ570(control)-80586×Aλ600(control)]×100%；
Experimental result shows, in low concentration（50 μg/mL）When, appreciation rate is 138%；When concentration increases to 200 μ g/mL
Appreciation rate reaches 211%；When continuing to increase concentration to 500 μ g/mL, appreciation rate reaches 297%（The μ g/mL plants of positive control 60
Lectin appreciation rate 306%）.Show that Stigma Maydis crude polysaccharides prepared by ultrafiltration have stimulation Proliferation of lymphocytes higher,
And dose dependent is presented.
Claims (2)
1. a kind of preparation method of the corn stigma ultrafiltration polysaccharide with immunoregulation effect, it is characterised in that：Comprise the following steps：
1st, the pretreatment of corn stigma：
After corn stigma removal of impurities, in being dried at 60-70 DEG C, 40-100 mesh is crushed to after being placed in dry environment natural cooling；
2nd, the degreasing of corn stigma：
Corn stigma powder presses mass volume ratio 1:3-1:5 add 95% ethanol or soaked in absolute ethyl alcohol 12-24 hours, filtration under diminished pressure, filter
Slag repeats immersion and is spontaneously dried after shady place for 1-2 times with method, and is saved backup in dry environment；
3rd, prepared by ultrafiltration polysaccharide：
3.1 extractions press 1:15-1:30 mass volume ratios add distilled water, and extraction 0.5-3 is small in extractor at 95-100 DEG C
When；
3.2 CEFs are centrifuged through the r/min of link-suspended basket centrifuge 1440, separate supernatant precipitation；
3.3 put forward precipitation again extracts 1 time again with method, the supernatant that merging is extracted twice；
3.4 removal of impurities supernatants are centrifuged through 16000 r/min, remove molecule, prepare polysaccharide extraction clear liquid；
3.5 ultrafiltration supernatant liquids are 10 KDa milipore filter ultrafiltration, ultrafiltration pressure 0.05-2.0 Mpa through molecular cut off；Ultrafiltration side
Method：When supernatant is concentrated by ultrafiltration 1/20-1/2 times to leaching liquor, to mending the 1/10-1 times of distillation of the volume of concentrate in concentrate
Water, continues ultrafiltration to the volume of concentrate and reverts to moisturizing front volume, and moisturizing is repeated with method, until the volume that always adds water is extraction liquid
Long-pending 2-5 times, no longer moisturizing；
3.6 dry concentrate continuation ultrafiltration to dry matter content up to 15-20%, freeze-drying or spray drying, obtain corn
Must ultrafiltration polysaccharide powder.
2. the preparation method of a kind of corn stigma ultrafiltration polysaccharide with immunoregulation effect according to claim 1, it is special
Levy and be：Hyperfiltration process described in the 3rd step is replaced with and adds 2-5 times of leaching liquor volume using to disposable in extraction clear liquid
Distilled water.
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| CN107050043B (en) * | 2017-05-28 | 2020-05-22 | 绍兴文理学院 | Corn stigma polysaccharide combined pharmaceutical preparation |
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| CN101658528B (en) * | 2009-09-25 | 2011-07-20 | 吉林化工学院 | Application of corn silk polysaccharide on preparation of medicine for curing hyperlipidemia |
| CN101748173B (en) * | 2009-12-03 | 2012-11-28 | 渤海大学 | Corn silk polysaccharide separation and decoloring method |
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