CN105669875A - Preparation method of rape pollen polysaccharide - Google Patents

Preparation method of rape pollen polysaccharide Download PDF

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Publication number
CN105669875A
CN105669875A CN201610090426.XA CN201610090426A CN105669875A CN 105669875 A CN105669875 A CN 105669875A CN 201610090426 A CN201610090426 A CN 201610090426A CN 105669875 A CN105669875 A CN 105669875A
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CN
China
Prior art keywords
extraction
rape pollen
pollen
solvent
polysaccharide
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Pending
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CN201610090426.XA
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Chinese (zh)
Inventor
张军
徐晓云
李云捷
和平
顾红涛
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Xiangyang Ligaole Biochemical Technology Co Ltd
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Xiangyang Ligaole Biochemical Technology Co Ltd
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Priority to CN201610090426.XA priority Critical patent/CN105669875A/en
Publication of CN105669875A publication Critical patent/CN105669875A/en
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    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003General processes for their isolation or fractionation, e.g. purification or extraction from biomass

Abstract

The invention discloses a preparation method which is used for extracting rape pollen polysaccharide by breaking walls through a superfine smashing method and is easy to implement, free of water addition, high in wall breaking rate, free of solvent removal operation and beneficial to subsequent active component extraction. The method includes the steps of cleaning rape pollen, filtering away water, breaking walls through the superfine smashing method, and adding low-boiling-point alcohol and other organic solvent for backflow three-time extraction. By means of the special vacuum, microwave and low-temperature drying device combined method, rape pollen extracts are dried, related active components are kept unchanged, drying temperature can be decreased, and drying speed can be increased. By means of the large-pore adsorption resin C18 reverse-phase silica gel column chromatography and specially-made chromatography combined technology, separation and purification are achieved, and a product with the rape pollen extract content of 90% is prepared. Furthermore, the purity of rape pollen extract recrystallized and extracted through a type of specific solvent prepared in our company reaches 95% or higher. Through orthogonal tests, the optimal process of extracting, drying, purifying and separating, and recrystallizing a rape pollen extraction compound is determined. The preparation method is stable in process operation, high in extract yield, high in purity, free of environment pollution and suitable for meeting the requirement for modern industry automatic production.

Description

The preparation method of a kind of rape pollen polysaccharide
Technical field
The present invention relates to rape pollen polysaccharide extractive technique, specifically, it relates to the preparation method of a kind of rape pollen polysaccharide.
Background technology
Pollen Brassicae campestris pollen contains abundant functional substance (comprising protein, VITAMIN, trace element, flavonoid compound), these materials act synergistically on body, regulate the multiple function of body, nutrition in balanced body, strengthen metabolism, prevent the obstacle of capillary permeability. Pollen can also promote the growth of incretory gland, is improved and endocrine regulation glandular secretion function, therefore the disease caused by endocrine dysfunction is served therapeutic action.
Pollen Brassicae campestris benefit kidney, consolidate, strong waist, and higher containing flavonol, there is atherosclerosis, treatment varicose ulcer, reduce cholesterol and radiation-resistant effect. Pollen is to the health-care effect of the mankind, pharmacological action and effect. Result shows, pollen can prevent and treat brain cardiovascular disease, reducing blood-fat, nervous system regulation, promotes sleep, regulates gastro-intestinal system function, promoting digestion, treatment habitual constipation; At endocrine regulation, improve body's immunity, have certain effect in anti-ageing, improvement function, treatment male infertility etc. Also have obvious anti-cancer tumor suppressor and liver protecting, prevent the functions such as anaemia, diabetes, antifatigue. In addition, the cosmetic result of bee pollen is obvious, its mechanism is to regulate the balance of human body endocrine system comprehensively, by inner and table, fundamentally improve the vigor of skin cells, strengthen the metabolism of skin, prevent facial pigmentation, pachylosis, aging, skin is made to keep moistening, pure white, glossy, high resilience.
Lacking the record of the extraction way to rape pollen polysaccharide constituents in " Chinese Pharmacopoeia " 2005 editions, it to be alcohol extracting method and water extraction that document report extracts rape pollen polysaccharide method then rich.
The wall-breaking method of Pollen Brassicae campestris and polysaccharide, extracting flavonoids purifying process research Wang Furong " Hua Zhong Agriculture University ", in 2007, it is preferable that at going out 60 DEG C, 50% extraction using alcohol three times, the extraction process of each 2h.Although its Extracting temperature is lower, but extraction time is long, needs altogether to extract 6 hours, and energy consumption is big, and extraction efficiency is low.
Microwave technology extracts the research Zhou Jianhua of rape pollen polysaccharide technique, Che Yufen " Shandong Normal University's journal: natural science edition ", 2007,2nd phase: in 109-111, it is preferable that gone out a kind of alcohol extracting method is with 5-10 order medicinal material, add 10 times amount 80% ethanol, refluxing extraction 3 times, each 1h. Demonstrating equally is that extraction time is many, the shortcoming of time length, needs altogether to extract 3 hours.
Summary document, find that method therefor extracts substantially complete, but it is many to there is consumption solvent, and extraction time is many, and purity is not high simultaneously, active unstable, with serious pollution shortcoming, in order to more save the consumption of solvent and the energy, reduces environmental pollution, save material, it is necessary to improve the methods of extraction and preparation of rape pollen polysaccharide.
Summary of the invention:
It is an object of the invention to overcome the deficiency that prior art exists, it is provided that a kind of save material, extraction efficiency height, purity height, the preparation method that consumes solvent rape pollen polysaccharide few, free of contamination.
In order to realize above-mentioned purpose, the present invention adopts following technical scheme:
A preparation method for rape pollen polysaccharide, comprises the steps: Pollen Brassicae campestris raw material is got wash clean Pollen Brassicae campestris, adds soft water and carries out micronizing method broken wall, it is placed in plant extract tank, adding alcohol concn is 60%, and the weightmeasurement ratio of medicinal material and solvent is 1:15, Extracting temperature 65 DEG C, 120min is asked during extraction, extracting solution is through taking out filter or centrifugal, and clear liquid concentrates, and dichloromethane extraction obtains thick cream after preparing, add ethanol and it is prepared chromatographic separation, obtain rape pollen polysaccharide.
In the preparation method of above-mentioned rape pollen polysaccharide, described alcoholic solution is preferably methyl alcohol or ethanolic soln.
In the preparation method of above-mentioned rape pollen polysaccharide, described volumes of aqueous ethanol concentration is preferably 65%-75%.
In the preparation method of above-mentioned rape pollen polysaccharide, Pollen Brassicae campestris coarse granularity used is preferably micronizing method broken wall. Owing to micronizing method broken wall decomposes extraction efficiency height, temperature is low, it is therefore necessary to Pollen Brassicae campestris carries out micronizing method broken wall, increases the area of Pollen Brassicae campestris and solvent contacts, it is to increase the extraction yield of total saponins.
In the preparation method of above-mentioned rape pollen polysaccharide, the weightmeasurement ratio of Pollen Brassicae campestris raw material and solvent is preferably 1:15-1:20.
In the preparation method of above-mentioned rape pollen polysaccharide, micronizing method broken wall.
In the preparation method of above-mentioned rape pollen polysaccharide, temperature is preferably 60-70 DEG C.
In the preparation method of above-mentioned rape pollen polysaccharide, extraction time is preferably 120-150min.
Compared with prior art, the present invention has following useful effect:
1. this invention is in leaching process, the destructive effect of micronizing method broken wall, mechanical effect and heat effect etc. are utilized to strengthen solvent to the penetration power of Pollen Brassicae campestris, contribute to the stripping of effective constituent in Pollen Brassicae campestris, thus improve the extraction yield of effective constituent, save material, reduce Extracting temperature, reduce energy consumption, improve extraction efficiency, reduce environmental pollution.
2. in the preparation technology of this invention, solvent load is few, has not only saved solvent but also save energy, reduces production cost, to zero environmental.
Owing to Pollen Brassicae campestris lacks assay item at " Chinese Pharmacopoeia " 2005 in sampan, so reference report, rape pollen polysaccharide, and Binding experiment checking, determining that the quality index of rape pollen polysaccharide take Radix Ophiopogonis polysaccharide as reference substance, the measuring method of employing is spectrophotometry.Concrete grammar is: the extracting solution getting 2g medicinal material, concentrates to dry, and 10ml water dissolution, extracts 4 times with water-saturated n-butanol, and washing extraction liquid, merges propyl carbinol liquid, concentrate to dry methanol constant volume to 10m1. Getting methanol solution 0.5ml, water-bath volatilizes, and adds perchloric acid 10ml, 65 DEG C of water-bath 15min, frozen water cooling termination reaction, obtained sample liquid. Taking perchloric acid (analytical pure) as blank, under 409nm, measure absorbancy.
The said rape pollen polysaccharide yield of the present invention=(the pollen weight of polysaccharide weight/input) × 100%
Rape pollen polysaccharide extraction yield=(the pollen weight of the rape pollen polysaccharide total content × input of the polysaccharide content in rape pollen polysaccharide extract × total polysaccharides weight/input) × 100%
The technical scheme of the present invention is set forth further below by specific embodiment.
Embodiment
Following examples rape pollen polysaccharide content used is 7 ‰.
Following ethanol percentage is volume percent.
The selection of embodiment 1 extracting factor
(1) time that micronizing method broken wall decomposes is on the impact of rape pollen polysaccharide extraction effect
Experimental technique: get Pollen Brassicae campestris 500g micronizing method broken wall and decompose, it is placed in plant extract tank, adding 60% ethanol as the weightmeasurement ratio of Extraction solvent, pollen and solvent is 1:15, Extracting temperature 65 DEG C, the different resolving times is selected to extract once, extracting solution after decomposition extraction is through taking out filter or centrifugal, and clear liquid concentrates, and dichloromethane extraction obtains thick cream after preparing, drying, obtains Pollen Brassicae campestris extract. The extraction effect of rape pollen polysaccharide under investigation different extraction time. Result is as shown in table 1:
Table 1
Time ask extraction yield (%)
10h24.2
14h30.1
18h45.6
20h73.5
24h90.3
Experimental result display extends the extraction yield that the resolving time can increase rape pollen polysaccharide, and during 24h, content just reaches the highest. So choosing 24h as best extraction time.
(2) Extracting temperature is on the impact of rape pollen polysaccharide extraction effect
Experimental technique: get 500g Pollen Brassicae campestris raw material micronizing method broken wall, it is placed in plant extract tank, add 60% ethanol as Extraction solvent, the weightmeasurement ratio of pollen and solvent is 1:15, selects different Extracting temperature to extract once, and the extracting solution after supersound extraction is through taking out filter or centrifugal, clear liquid concentrates, dichloromethane extraction obtains thick cream after preparing, dry, obtains Pollen Brassicae campestris extract. The extraction effect of rape pollen polysaccharide under investigation different extraction time. Result is as shown in table 2:
Table 2
Temperature extraction yield (%)
20℃58.5
30℃60.9
40℃60.9
50℃84.7
60℃87.8
Experimental result shows, and along with the rising of Extracting temperature, the extraction yield of rape pollen polysaccharide also improves constantly, and considers cost, therefore chooses 60 DEG C as best supersound extraction temperature.
(3) Extraction solvent is on the impact of rape pollen polysaccharide extraction effect
Experimental technique: get 500g Pollen Brassicae campestris micronizing method broken wall, it is placed in plant extract tank, add 60% ethanol as Extraction solvent, the weightmeasurement ratio of pollen and solvent is 1:15, selects different Extraction solvent to extract once, and the extracting solution after supersound extraction is through taking out filter or centrifugal, clear liquid concentrates, dichloromethane extraction obtains thick cream after preparing, dry, obtains rape pollen polysaccharide extract. The extraction effect of rape pollen polysaccharide under investigation different extraction time. Result is as shown in table 3:
Table 3
Solvent extraction rate (%)
Water 55.6
30% ethanol 63.7
40% ethanol 70.9
50% ethanol 76.5
60% ethanol 87.4
70% ethanol 88.3
Methyl alcohol 78.4
Experimental result shows, and selects 60% ethanol, 70% ethanol and methyl alcohol all to have good extraction as Extraction solvent
Effect.But methyl alcohol toxicity is relatively big, is unfavorable for suitability for industrialized production, and 50% extraction using alcohol to go out impurity relatively many, specific filtration resistance is more difficult, so factors such as comprehensive production costs, chooses 60% ethanol as Extraction solvent.
(4) Extraction solvent volume is on the impact of rape pollen polysaccharide extraction effect
Experimental technique: get 500g Pollen Brassicae campestris raw material micronizing method broken wall, it is placed in plant extract tank, add 60% ethanol as Extraction solvent, the weightmeasurement ratio of medicinal material and solvent is 1:15, selects different Extraction solvent ratios to extract once, and the extracting solution after supersound extraction is through taking out filter or centrifugal, clear liquid concentrates, dichloromethane extraction obtains thick cream after preparing, dry, obtains Pollen Brassicae campestris extract. The extraction effect of rape pollen polysaccharide under investigation different extraction time. Result is as shown in table 4:
Table 4
Solvent extraction rate (%)
10 times 88.6
15 times 90.7
Experimental result shows, it may also be useful to when the weightmeasurement ratio 1:15 of pollen and solvent, extraction yield can be greatly improved. Therefore 1:15 is chosen as the weightmeasurement ratio of best pollen and solvent.
(5) medicinal material order number is on the impact of rape pollen polysaccharide extraction effect
Experimental technique: get 500g Pollen Brassicae campestris raw material micronizing method broken wall, it is placed in plant extract tank, add 60% ethanol as Extraction solvent, the weightmeasurement ratio of pollen and solvent is 1:15, selects different extraction fineness to extract once, and the extracting solution after supersound extraction is through taking out filter or centrifugal, clear liquid concentrates, dichloromethane extraction obtains thick cream after preparing, dry, obtains Pollen Brassicae campestris extract. The extraction effect of rape pollen polysaccharide under investigation different extraction time. Result is as shown in table 5:
Table 5
Micronizing method extraction rate (%)
Super micro/nano level anhydrous 90.8
Super micro/nano level moisture 99.5
Experimental result shows, and it is higher that utilization carries out extracting extraction yield more anhydrous than super micro/nano level through the pollen that super micro/nano level is moisture, so selected super micro-nano rice is moisture as optimised process.
Finally, concluding micro-nano nanometer water mill extraction optimum extraction process is: the Pollen Brassicae campestris raw material getting super micro-nano rice water mill, being placed in plant extract tank, alcohol concn is 60%, and the weightmeasurement ratio of medicinal material and solvent is 1:15, Extracting temperature 60 DEG C, extraction time 120min, the extracting solution after extraction is through taking out filter or centrifugal, and clear liquid concentrates, dichloromethane extraction prepare after thick cream, add ethanol carry out C18-preparative chromatography separation obtain rape pollen polysaccharide.
Embodiment 2
Get the Pollen Brassicae campestris that 500g gets super micro-nano rice water mill, being placed in plant extract tank, alcohol concn is 60%, and the weightmeasurement ratio of medicinal material and solvent is 1:15, Extracting temperature 60 DEG C, extraction time 120min, the extracting solution after extraction is through taking out filter or centrifugal, and clear liquid concentrates, dichloromethane extraction obtains thick cream after preparing, add ethanol to carry out the separation of C18-preparative chromatography and obtain the thick cream of Pollen Brassicae campestris extract, dry, obtain rape pollen polysaccharide. Result: dry spun is 6.5%, content 25.0%, extraction yield 99.7%.
Embodiment 3
With shown in embodiment 2, the difference is that: the weightmeasurement ratio of embodiment 2 pollen and solvent is 1:20, and the weightmeasurement ratio of embodiment 3 pollen and solvent is 1:10. Result: dry spun is 5.9%, content 16.7%, extraction yield 91.7%.
Embodiment 4
With shown in embodiment 3, the difference is that: embodiment 3 Pollen Brassicae campestris is pulverized and is reached nano level, and embodiment 4 pollen material is pulverized and reached nano level.Result: dry spun is 5.9%, content 15.7%, extraction yield 85.8%.
Embodiment 5
With shown in embodiment 4, the difference is that: in embodiment 4, alcohol concn is 60%, and extraction time is 24h, and in embodiment 5, alcohol concn is 50%, and extraction time is 12h. Result: dry spun is 6.3%, content 15.7%, extraction yield 79.6%.
Embodiment 6
With shown in embodiment 4, the difference is that: in embodiment 4, extraction time is 12h, and in embodiment 6 extraction time be 8h. Result: dry spun is 5.5%, content 11.7%, extraction yield 40.1%.
Embodiment 7
Get the Pollen Brassicae campestris raw material that 500g gets micronizing method broken wall, being placed in plant extract tank, alcohol concn is 60%, and the weightmeasurement ratio of Pollen Brassicae campestris and solvent is 1:10, Extracting temperature 60 DEG C, extraction time 40min, the extracting solution after extraction is through taking out filter or centrifugal, and clear liquid concentrates, dichloromethane extraction obtains thick cream after preparing, add ethanol to carry out the separation of C18-preparative chromatography and obtain the thick cream of Pollen Brassicae campestris extract, dry, obtain rape pollen polysaccharide. Result: dry spun is 5.3%, content 18.7%, after extraction yield 73.4%, C18-preparative chromatography, purity reaches more than 95%.
Embodiment 8
With shown in embodiment 3, the difference is that: embodiment 3 uses plant extract once, and embodiment 8 uses water-bath circumfluence distillation 3 times, each 60min. Result: dry spun is 6.4%, content 15.7%.
As seen from the above embodiment, nano level water mill, micro-comminuting method broken wall decompose extraction Pollen Brassicae campestris, its optimal conditions is: nano level micronizing method broken wall, it is placed in plant extract tank, alcohol concn is 60-80%, the weightmeasurement ratio of Pollen Brassicae campestris and solvent is 1:10-1:18,60-75 DEG C, extraction time 60-150min, extracting solution is through taking out filter or centrifugal, clear liquid concentrates, dichloromethane extraction prepare after thick cream, add ethanol and carry out C18-preparative chromatography separation and namely obtain more than 95% rape pollen polysaccharide. Normal reflux extracts then to be needed long-time at higher extracted temperature and extracts repeatedly severally, just can reach the effect that this technique is once extracted. Therefore, nanometer water mill, micronizing method broken wall decompose extraction and have minimizing Extraction solvent consumption, reduce Extracting temperature, save material, reduce environmental pollution, do not destroy the activeconstituents of compound, extract the feature that rape pollen polysaccharide purity is high.

Claims (1)

1. the preparation method of a rape pollen polysaccharide, it is characterised in that comprise the steps: to get wash clean Pollen Brassicae campestris raw material, adds soft water and carries out micronizing method broken wall, it is placed in plant extract tank, adding alcohol concn is 60%, and the weightmeasurement ratio of medicinal material and solvent is 1:15, Extracting temperature 65 DEG C, extraction time 120min, extracting solution is through taking out filter or centrifugal, and clear liquid concentrates, and dichloromethane extraction obtains thick cream after preparing, add ethanol and it is prepared chromatographic separation, obtain rape pollen polysaccharide.
CN201610090426.XA 2016-02-18 2016-02-18 Preparation method of rape pollen polysaccharide Pending CN105669875A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111349678A (en) * 2018-12-20 2020-06-30 湖南湘源美东医药科技有限公司 Extraction method of rape pollen polysaccharide and extraction product
CN113133455A (en) * 2020-01-20 2021-07-20 成都新朝阳作物科学股份有限公司 Pollen polysaccharide extract and application thereof in promoting plant growth
US11732056B2 (en) 2020-01-20 2023-08-22 Chengdu Newsun Crop Science Co., Ltd. Pollen polysaccharide extract and application thereof in plant growth
EP4311835A1 (en) * 2022-07-29 2024-01-31 Chengdu Newsun Crop Science Co., Ltd. Pollen polysaccharide and the separation method and application

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111349678A (en) * 2018-12-20 2020-06-30 湖南湘源美东医药科技有限公司 Extraction method of rape pollen polysaccharide and extraction product
CN113133455A (en) * 2020-01-20 2021-07-20 成都新朝阳作物科学股份有限公司 Pollen polysaccharide extract and application thereof in promoting plant growth
CN113133455B (en) * 2020-01-20 2022-04-12 成都新朝阳作物科学股份有限公司 Pollen polysaccharide extract and application thereof in promoting plant growth
US11732056B2 (en) 2020-01-20 2023-08-22 Chengdu Newsun Crop Science Co., Ltd. Pollen polysaccharide extract and application thereof in plant growth
EP4311835A1 (en) * 2022-07-29 2024-01-31 Chengdu Newsun Crop Science Co., Ltd. Pollen polysaccharide and the separation method and application
WO2024021707A1 (en) * 2022-07-29 2024-02-01 成都新朝阳作物科学股份有限公司 Pollen polysaccharide, separation method therefor, and use thereof

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