CN104961842A - Preparation method of corn stigma ultrafiltration polysaccharide with immunoregulation function - Google Patents
Preparation method of corn stigma ultrafiltration polysaccharide with immunoregulation function Download PDFInfo
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Abstract
The invention discloses a preparation method of corn stigma ultrafiltration polysaccharide with an immunoregulation function, belonging to the technical field of extraction separation of natural plant active polysaccharide. The corn stigma ultrafiltration polysaccharide is prepared from corn stigma by the steps of pretreatment, degreasing, digestion, centrifugation, re-digestion, impurity removal, ultrafiltration and drying. The method has the advantages of easiness in operation, small equipment investment and the like and is environmentally friendly and safe, and low in cost, brings remarkable economic benefit and is suitable for large-scale production; the corn stigma ultrafiltration polysaccharide prepared by the method also has an immunoregulation function and a relatively high lymphopoiesis stimulating effect and shows dosage dependence.
Description
Technical field
The present invention relates to a kind of preparation method with the Stigma Maydis ultrafiltration polysaccharide of immunoregulation effect, belong to the extraction and separation technology field of natural plant active polysaccharide.
Background technology
Stigma Maydis (
stigmata madis) call corn wheat, ear of maize hair, Gramineae beautiful another name for Sichuan Province platymiscium corn (
zea mays L.) style, be a kind of traditional herbal medicine, all on the books in " the southern regions of the Yunnan Province book on Chinese herbal medicine ", " Chinese medicine voluminous dictionary " etc., be " Ministry of Health of the People's Republic of China's medicinal material standard " 1985 editions medicinal herbs most in use kinds that () includes.
China is Maize Production big country, and corn annual production occupies the 2nd, the world.Therefore, the resource as the Stigma Maydis of corn's by-products is very abundant.
Polysaccharide compound is one of chemical composition important contained by Stigma Maydis, preliminary study shows that corn silk polysaccharide has and protects liver, hypoglycemic, immunity moderation, antibacterial, antitumor, diuresis and the effect such as antipyretic, clinical trial confirms, polyose also can be used as tumor chemical therapy and radiocurable effective auxiliary therapeutic agent, in medicine, food, all have good application prospect and Development volue.
Tradition Polyose extraction adopts organic solvent deposit method, removes small molecular weight impurity, prepares polysaccharide, as 80% alcohol settling etc.This method not only needs to consume a large amount of organic solvent, and organic solvent has very powerful volatilization and the feature such as inflammable and explosive, therefore requires also higher to equipment investment.The present invention adopts equal-volume moisturizing hyperfiltration process; while effectively retaining corn silk polysaccharide activeconstituents; remove small-molecule substance, substitute organic solvent deposit, prepare polysaccharide; have simple to operate; the advantages such as equipment investment is few, and Environmental Safety, with low cost; there is significant economic benefit, be suitable for large-scale production.
Summary of the invention
The invention provides a kind of preparation method with the Stigma Maydis ultrafiltration polysaccharide of immunoregulation effect, present method adopts equal-volume moisturizing hyperfiltration process, and 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 Stigma Maydis ultrafiltration polysaccharide, comprises the steps:
1. the pre-treatment of Stigma Maydis
After Stigma Maydis removal of impurities, dry at 60-70 DEG C, after being placed in dry environment naturally cooling, be crushed to 40-100 order.
2. the degreasing of Stigma Maydis
Stigma Maydis powder adds 95% ethanol or soaked in absolute ethyl alcohol 12-24 hour by mass volume ratio 1:2-1:5, filtration under diminished pressure, in the seasoning of cool place place after filter residue repeats to soak 1-2 time again with method, and saves backup in dry environment.
3. ultrafiltration polysaccharide preparation
3.1 lixiviates add distilled water by 1:10-1:30 mass volume ratio, at 95-100 DEG C in extractor lixiviate 0.5-3 hour.
3.2 CEFs are centrifugal through link-suspended basket centrifuge 1440 r/min, cleer and peaceful precipitation (Stigma Maydis particle) in separation.
3.3 put forward precipitation again with method lixiviate 1 time again.Merge the supernatant liquor of extracted twice.
3.4 removal of impurities supernatant liquors are centrifugal through 16000 r/min, and removing molecule, prepares polysaccharide lixiviate clear liquid.
3.5 ultrafiltration supernatant liquids are 10 KDa ultra-filtration membrane ultrafiltration through molecular weight cut-off, ultrafiltration pressure 0.05-2.0 Mpa.Hyperfiltration process: when supernatant ultrafiltration and concentration is to the 1/20-1/2 times of vat liquor, the distilled water of 1/10-1 times of concentrated solution volume is mended in concentrated solution, continue ultrafiltration and revert to moisturizing front volume to concentrated solution volume, moisturizing is repeated with method, until always add 2-5 times that volume of water is vat liquor volume, no longer moisturizing.
Concentrated solution is continued ultrafiltration and reaches 15-20% to dry matter content by 3.6 dryings, lyophilize or spraying dry, obtains Stigma Maydis ultrafiltration polysaccharide powder.
The hyperfiltration process of the 5th step in this step can also adopt the disposable distilled water adding 2-5 times of vat liquor volume in lixiviate clear liquid.
The invention has the beneficial effects as follows: the present invention adopts equal-volume moisturizing hyperfiltration process; while effectively retaining corn silk polysaccharide activeconstituents; remove small-molecule substance, substitute organic solvent deposit, prepare polysaccharide; have simple to operate; the advantages such as equipment investment is few, and Environmental Safety, with low cost; there is significant economic benefit, be suitable for large-scale production.
Accompanying drawing explanation
Fig. 1 is Stigma Maydis ultrafiltration polysaccharide stimulated in vitro lymphocyte proliferation assay result of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described.
Embodiment 1
A preparation method for Stigma Maydis ultrafiltration polysaccharide, comprises the steps:
1. the pre-treatment of Stigma Maydis
After Stigma Maydis removal of impurities, dry at 70 DEG C, after being placed in dry environment naturally cooling, be crushed to 100 orders.
2. the degreasing of Stigma Maydis
Stigma Maydis powder 1 kg, adds soaked in absolute ethyl alcohol 12 hours by mass volume ratio 1:5, filtration under diminished pressure, and filter residue repeats in the seasoning of cool place place after immersion 1 time with method again, and saves backup in dry environment.
3. ultrafiltration polysaccharide preparation
3.1 lixiviates add distilled water (20 L) by 1:20 mass volume ratio, lixiviate 2 hours in extractor at 95-100 DEG C.
3.2 CEFs are centrifugal through link-suspended basket centrifuge 1440 r/min, cleer and peaceful precipitation (Stigma Maydis particle) in separation.
3.3 put forward precipitation again with method lixiviate 1 time again.Merge the supernatant liquor of extracted twice.
3.4 removal of impurities supernatant liquors are centrifugal through 16000 r/min, and removing molecule, prepares polysaccharide lixiviate clear liquid.
3.5 ultrafiltration supernatant liquids are 10 KDa ultra-filtration membrane ultrafiltration through molecular weight cut-off, ultrafiltration pressure 0.08 Mpa.Hyperfiltration process: when supernatant ultrafiltration and concentration is to 1/10 times (2 L) of vat liquor, the distilled water (1 L) of 1/2 times of concentrated solution volume is mended in concentrated solution, continue ultrafiltration and revert to moisturizing front volume to concentrated solution volume, moisturizing is repeated with method, until always add 4 times (80 L) that volume of water is vat liquor volume, no longer moisturizing.
Concentrated solution is continued ultrafiltration and reaches 15-20% to dry matter content by 3.6 dryings, lyophilize or spraying dry, obtains Stigma Maydis ultrafiltration polysaccharide powder.
Stigma Maydis ultrafiltration polysaccharide extract rate is 7.22%, its polysaccharide content 36.11%.
Embodiment 2
A preparation method for Stigma Maydis ultrafiltration polysaccharide, comprises the steps:
1. the pre-treatment of Stigma Maydis
After Stigma Maydis removal of impurities, dry at 65 DEG C, after being placed in dry environment naturally cooling, be crushed to 100 orders.
2. the degreasing of Stigma Maydis
Stigma Maydis powder 1kg, adds soaked in absolute ethyl alcohol 24 hours by mass volume ratio 1:3, filtration under diminished pressure, and filter residue repeats in the seasoning of cool place place after immersion 1 time with method again, and saves backup in dry environment.
3. ultrafiltration polysaccharide preparation
3.1 lixiviates add distilled water (15 L) by 1:15 mass volume ratio, lixiviate 1 hour in extractor at 95-100 DEG C.
3.2 CEFs are centrifugal through link-suspended basket centrifuge 1440 r/min, cleer and peaceful precipitation (Stigma Maydis particle) in separation.
3.3 put forward precipitation again with method lixiviate 1 time again.Merge the supernatant liquor of extracted twice.
3.4 removal of impurities supernatant liquors are centrifugal through 16000 r/min, and removing molecule, prepares polysaccharide lixiviate clear liquid.
3.5 ultrafiltration supernatant liquids are 10 KDa ultra-filtration membrane ultrafiltration through molecular weight cut-off, ultrafiltration pressure 0.06 Mpa.Hyperfiltration process: when supernatant ultrafiltration and concentration is to (1.5 L) during 1/10 times of vat liquor, the distilled water (0.5 L) of 1/3 times of concentrated solution volume is mended in concentrated solution, continue ultrafiltration and revert to moisturizing front volume to concentrated solution volume, moisturizing is repeated with method, until always add 5 times (75 L) that volume of water is vat liquor volume, no longer moisturizing.
Concentrated solution is continued ultrafiltration and reaches 15-20% to dry matter content by 3.6 dryings, lyophilize or spraying dry, obtains Stigma Maydis ultrafiltration polysaccharide powder.
Stigma Maydis ultrafiltration polysaccharide extract rate is 6.65%, its polysaccharide content 38.34%.
Embodiment 3
A preparation method for Stigma Maydis ultrafiltration polysaccharide, comprises the steps:
1. the pre-treatment of Stigma Maydis
After Stigma Maydis removal of impurities, dry at 60 DEG C, after being placed in dry environment naturally cooling, be crushed to 60 orders.
2. the degreasing of Stigma Maydis
Stigma Maydis powder 1 kg, adds soaked in absolute ethyl alcohol 24 hours by mass volume ratio 1:4, filtration under diminished pressure, and filter residue repeats in the seasoning of cool place place after immersion 1 time with method again, and saves backup in dry environment.
3. ultrafiltration polysaccharide preparation
3.1 lixiviates add distilled water (30 L) by 1:30 mass volume ratio, lixiviate 1.5 hours in extractor at 95-100 DEG C.
3.2 CEFs are centrifugal through link-suspended basket centrifuge 1440 r/min, cleer and peaceful precipitation (Stigma Maydis particle) in separation.
3.3 put forward precipitation again with method lixiviate 1 time again.Merge the supernatant liquor of extracted twice.
3.4 removal of impurities supernatant liquors are centrifugal through 16000 r/min, and removing molecule, prepares polysaccharide lixiviate clear liquid.
3.5 ultrafiltration supernatant liquids are 10 KDa ultra-filtration membrane ultrafiltration through molecular weight cut-off, ultrafiltration pressure 0.80 Mpa.Hyperfiltration process: the disposable distilled water (75 L) adding 2.5 times of vat liquor volumes in lixiviate clear liquid, after mixing, ultrafiltration and concentration reaches 15-20% to dry matter content, dry.
3.6 dry lyophilize or spraying dry, obtain Stigma Maydis ultrafiltration polysaccharide powder.
Stigma Maydis ultrafiltration polysaccharide extract rate is 6.93%, its polysaccharide content 35.57%.
Embodiment 4
Study immune regulation
Cervical dislocation puts to death mouse, spleen is got under aseptic condition, be placed on 100 order sterilizing stainless steel screen clothes, with filling in syringe, spleen ground, sieving, mesh screen 2-3 time is rinsed with PBS, the suspension of splenocyte proceeds in aseptic centrifuge tube, then adds PBS to 30-40 mL, centrifugal 6 min of 400 G, collecting cell, at room temperature adds NH
4cl solution (1.5 mL/ are only), continuous rifle is beaten or is rocked mixing cell 10 minutes, with splitting erythrocyte, add PBS to 30-40 mL, centrifugal 5 min of 400 G, remove supernatant, after adding a small amount of RPMI1640 substratum cell counter counting, with RPMI1640 diluting cells suspension to 2 × 10
6individual/mL.Getting 180 μ L cell suspending liquids adds in 96 orifice plates, adds different samples and the positive control phytohaemagglutinin of 20 μ L respectively, negative control PBS.At 37 DEG C and 5% CO
2condition under cultivate after 3 days, automatically the absorbancy that plate instrument measures 570 nm and 600 nm is respectively read with ELISA, add 20 μ L Alamar Blue developers, cultivate 6-8 hour, again measure the absorbancy of 570 nm and 600 nm, the formulae discovery each sample of then giving according to Alamer Blue Assay is to lymphocytic activity ratio.
Lymphocytic proliferation rate (%)=[117216 × A
λ570 (sample)-80586 × A
λ600 (sample)]
/[117216×A
λ570(control)-80586×A
λ600(control)]×100%；
Experimental result shows, and when lower concentration (50 μ g/mL), appreciation rate is 138%; 211% is reached when concentration is increased to 200 μ g/mL appreciation rates; When continuing to increase concentration to 500 μ g/mL, appreciation rate reaches 297%(positive control 60 μ g/mL lectins appreciation rate 306%).Show that Stigma Maydis crude polysaccharides prepared by ultrafiltration process has higher stimulation Proliferation of lymphocytes, and present dose-dependently.
Claims (2)
1. a preparation method for Stigma Maydis ultrafiltration polysaccharide, is characterized in that: comprise the steps:
1, the pre-treatment of Stigma Maydis:
After Stigma Maydis removal of impurities, dry at 60-70 DEG C, after being placed in dry environment naturally cooling, be crushed to 40-100 order;
2, the degreasing of Stigma Maydis:
Stigma Maydis powder adds 95% ethanol or soaked in absolute ethyl alcohol 12-24 hour by mass volume ratio 1:2-1:5, filtration under diminished pressure, in the seasoning of cool place place after filter residue repeats to soak 1-2 time again with method, and saves backup in dry environment;
3, ultrafiltration polysaccharide preparation:
3.1 lixiviates add distilled water by 1:10-1:30 mass volume ratio, at 95-100 DEG C in extractor lixiviate 0.5-3 hour;
3.2 CEFs are centrifugal through link-suspended basket centrifuge 1440 r/min, cleer and peaceful precipitation (Stigma Maydis particle) in separation;
3.3 put forward precipitation again with method lixiviate 1 time again, merge the supernatant liquor of extracted twice;
3.4 removal of impurities supernatant liquors are centrifugal through 16000 r/min, and removing molecule, prepares polysaccharide lixiviate clear liquid;
3.5 ultrafiltration supernatant liquids are 10 KDa ultra-filtration membrane ultrafiltration through molecular weight cut-off, ultrafiltration pressure 0.05-2.0 Mpa; Hyperfiltration process: when supernatant ultrafiltration and concentration is to the 1/20-1/2 times of vat liquor, the distilled water of 1/10-1 times of concentrated solution volume is mended in concentrated solution, continue ultrafiltration and revert to moisturizing front volume to concentrated solution volume, moisturizing is repeated with method, until always add 2-5 times that volume of water is vat liquor volume, no longer moisturizing;
Concentrated solution is continued ultrafiltration and reaches 15-20% to dry matter content by 3.6 dryings, lyophilize or spraying dry, obtains Stigma Maydis ultrafiltration polysaccharide powder.
2. the preparation method of a kind of Stigma Maydis ultrafiltration polysaccharide according to claim 1, is characterized in that: the hyperfiltration process described in the 3rd step can also adopt the disposable distilled water adding 2-5 times of vat liquor volume in lixiviate clear liquid.
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| CN109456418A (en) * | 2019-01-17 | 2019-03-12 | 齐齐哈尔大学 | A kind of activity Tongkat Ali polysaccharide and preparation method |
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Cited By (3)
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| CN107050043A (en) * | 2017-05-28 | 2017-08-18 | 绍兴文理学院 | A kind of corn silk polysaccharide combined pharmaceutical preparation |
| CN109456418A (en) * | 2019-01-17 | 2019-03-12 | 齐齐哈尔大学 | A kind of activity Tongkat Ali polysaccharide and preparation method |
| CN109456418B (en) * | 2019-01-17 | 2020-10-27 | 齐齐哈尔大学 | Active eurycoma longifolia polysaccharide and preparation method thereof |
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