CN106008729B - A kind of sunset abelmoschus stem or bark leaf polyose and preparation method and application - Google Patents
A kind of sunset abelmoschus stem or bark leaf polyose and preparation method and application Download PDFInfo
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- CN106008729B CN106008729B CN201610232324.7A CN201610232324A CN106008729B CN 106008729 B CN106008729 B CN 106008729B CN 201610232324 A CN201610232324 A CN 201610232324A CN 106008729 B CN106008729 B CN 106008729B
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- sunset abelmoschus
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- 241001075517 Abelmoschus Species 0.000 title claims abstract description 84
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 238000000605 extraction Methods 0.000 claims abstract description 34
- 239000011347 resin Substances 0.000 claims abstract description 21
- 229920005989 resin Polymers 0.000 claims abstract description 21
- 238000010521 absorption reaction Methods 0.000 claims abstract description 11
- 239000011148 porous material Substances 0.000 claims abstract description 11
- 239000012528 membrane Substances 0.000 claims abstract description 9
- 238000000746 purification Methods 0.000 claims abstract description 9
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims abstract description 8
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 claims abstract description 8
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims abstract description 8
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 8
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims abstract description 8
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000008103 glucose Substances 0.000 claims abstract description 8
- 238000000926 separation method Methods 0.000 claims abstract description 8
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims abstract description 7
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims abstract description 7
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 7
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229920002678 cellulose Polymers 0.000 claims abstract description 7
- 239000001913 cellulose Substances 0.000 claims abstract description 5
- 238000012869 ethanol precipitation Methods 0.000 claims abstract description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 4
- -1 galactolipin Chemical compound 0.000 claims abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 42
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 40
- 235000019441 ethanol Nutrition 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 239000011780 sodium chloride Substances 0.000 claims description 21
- 238000001556 precipitation Methods 0.000 claims description 20
- 150000004676 glycans Chemical class 0.000 claims description 19
- 229920001282 polysaccharide Polymers 0.000 claims description 19
- 239000005017 polysaccharide Substances 0.000 claims description 19
- 238000004440 column chromatography Methods 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 238000010828 elution Methods 0.000 claims description 14
- 239000012488 sample solution Substances 0.000 claims description 12
- 238000000108 ultra-filtration Methods 0.000 claims description 12
- 230000002708 enhancing effect Effects 0.000 claims description 11
- 239000003480 eluent Substances 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 10
- 235000010980 cellulose Nutrition 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000007445 Chromatographic isolation Methods 0.000 claims description 5
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 claims description 5
- 239000012141 concentrate Substances 0.000 claims description 5
- 238000003306 harvesting Methods 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 4
- 230000003053 immunization Effects 0.000 claims 8
- 238000002649 immunization Methods 0.000 claims 8
- 238000010612 desalination reaction Methods 0.000 claims 2
- 238000009738 saturating Methods 0.000 claims 2
- 239000012510 hollow fiber Substances 0.000 claims 1
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 12
- 239000003814 drug Substances 0.000 abstract description 11
- 238000011161 development Methods 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 7
- 238000011033 desalting Methods 0.000 abstract description 5
- 239000002699 waste material Substances 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 4
- 235000013402 health food Nutrition 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000004064 recycling Methods 0.000 abstract description 3
- 230000036039 immunity Effects 0.000 abstract description 2
- 238000004108 freeze drying Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 14
- 239000000523 sample Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000002158 endotoxin Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 229920006008 lipopolysaccharide Polymers 0.000 description 5
- 210000004698 lymphocyte Anatomy 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 240000000530 Alcea rosea Species 0.000 description 2
- 235000017334 Alcea rosea Nutrition 0.000 description 2
- 235000017303 Althaea rosea Nutrition 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000219071 Malvaceae Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 240000005959 Abelmoschus manihot Species 0.000 description 1
- 235000001075 Abelmoschus manihot Nutrition 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010029164 Nephrotic syndrome Diseases 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000009470 Theobroma cacao Nutrition 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 125000000089 arabinosyl group Chemical group C1([C@@H](O)[C@H](O)[C@H](O)CO1)* 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 208000009928 nephrosis Diseases 0.000 description 1
- 231100001027 nephrosis Toxicity 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000012925 reference material Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000003393 splenic effect Effects 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- 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
-
- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a kind of sunset abelmoschus stem or bark leaf polyoses and preparation method and application.The preparation method includes：Extraction, ethanol precipitation, large pore resin absorption column chromatogram purification, hollow cellulose UF membrane, DEAE celluosic resin pillar layer separations collect target fraction, and through desalting processing, freeze-drying obtains sunset abelmoschus stem or bark leaf polyose.The sunset abelmoschus stem or bark leaf polyose weight average molecular weight is 13821Da, is made of rhamnose, mannose, galactolipin, galacturonic acid, glucose and arabinose, and molar ratio is arabinose:Galactolipin:Glucose:Galacturonic acid:Rhamnose:Mannose=1:0.53:0.12:0.05:0.16:0.03, there is good immune-enhancing activity, medicament for immunity enhancement and health food development can be used as.For the present invention using the cauline leaf waste resource generated during sunset abelmoschus flower production of crude drugs as raw material, prepared by separation have immunocompetent polysaccharose substance, to realize that recycling for natural resources of Chinese medicinal materials is laid a good foundation.
Description
Technical field
The present invention relates to a kind of Chinese medical extracts, and in particular to has immunocompetent sunset abelmoschus stem or bark leaf polyose to one kind
Preparation method and application.
Background technology
Sunset abelmoschus root (Abelmoschus manihot (L.) Medic.) is Malvaceae (Malvaceae) Abelmoschus
(Abelmoschus) 1 year to herbaceos perennial, first recorded in written by Song Dynasty palm Yu's tin《It is good to help book on Chinese herbal medicine》, history tree thereafter
It is on the books, and record in going through version《Pharmacopoeia of People's Republic of China》.History tree is mostly used its flower and is used as medicine, and is secondly seed, root,
Stem and Ye Shaoyong.Sunset abelmoschus flower is at present one of primary raw material for the treatment of nephrosis conventional Chinese medicine, and market demand is huge, but harvests
Cauline leaf resource after flower is largely discarded when lacking Processes For Effective Conversion, causes the wasting of resources.Therefore, sunset abelmoschus root cauline leaf is carried out
Research of utilization and industrialization development, realization turn waste into wealth, can not only promote the sound development of sunset abelmoschus root resource industries,
Also comply with the Green Development requirement that country advocates.
Containing relatively rich glucide in studies have shown that sunset abelmoschus root cauline leaf, has patent report using it as primary raw material
Natural plant gum can be prepared.Also some researches show that natural polysaecharides substance has preferable immunocompetence, therefore is highly desirable existing
Have on Research foundation, system carries out sunset abelmoschus stem or bark leaf polyose method for preparing purified and research for application and development, screens it and can be used for exempting from
The active component or molecule of epidemic disease enhancing drug or health food development to expand the utilization ways of sunset abelmoschus root cauline leaf resource, promote
Into recycling for sunset abelmoschus root medicinal organism resource.
The content of the invention
Goal of the invention：It is real it is an object of the invention to provide a kind of sunset abelmoschus stem or bark leaf polyose with immune-enhancing activity
The recycling of existing sunset abelmoschus root cauline leaf and industrialization development, turn waste into wealth；Another object of the present invention is to yellow more than providing
The preparation method and applications of hollyhock stem leaf polysaccharide.
Technical solution：In order to achieve the goal above, the technical solution that the present invention takes is：
A kind of sunset abelmoschus stem or bark leaf polyose is isolated from sunset abelmoschus root cauline leaf, and weight average molecular weight is 13821 dalton
(Da), which is made of arabinose, galactolipin, glucose, galacturonic acid, rhamnose and mannose, and molar ratio is
Arabinose:Galactolipin:Glucose:Galacturonic acid:Rhamnose:Mannose=1:0.53:0.12:0.05:0.16:0.03.
A kind of preparation method of sunset abelmoschus stem or bark leaf polyose of the present invention includes extraction, ethanol precipitation, macropore suction successively
Attached resin column chromatography purifying, ultrafiltration, DEAE celluosic resin column chromatography separating purifications, specific separating step include：
(1) sunset abelmoschus root cauline leaf is taken, is 1 by solid-liquid weight ratio after crushed:8~1:30 add in water, 80~100 DEG C of conditions
Lower heating extraction 2~3 times, every time 1~3 it is small when, filtering obtains filtrate.
(2) filtrate obtained by taking step (1) is concentrated to proper volume, adds in ethyl alcohol to alcohol content 50%~80%, it is heavy to stand
After forming sediment for 24 hours, separation of solid and liquid obtains alcohol precipitation precipitation.
(3) alcohol precipitation precipitation obtained by step (2) is taken, after being dissolved in water, through large pore resin absorption column chromatographic isolation, with pure water,
1%~20% ethanol water elutes, and obtains eluent.
(4) eluent obtained by step (3) is taken, after appropriate concentration, through ultrafiltration membrane ultrafiltration, it is 10,000~30,000 to take molecular weight ranges
Sample segment after appropriate concentration, obtains refined polysaccharide sample solution.
(5) refined polysaccharide sample solution obtained by step (4) is taken, is separated through DEAE cellulose column chromatographies, respectively with water, 0.01
It after the elution of~0.05%NaCl solution, is eluted with 0.1~0.5%NaCl solution, collects 0.1~0.5%NaCl elution solution, warp
It after dialysis desalting, concentrates, freezes, obtain sunset abelmoschus stem or bark leaf polyose.
Sunset abelmoschus root cauline leaf of the present invention refers to remaining ground cauline leaf after harvesting sunset abelmoschus flower.
Preferably, the solid-to-liquid ratio described in step (1) is 1:20, Extracting temperature is 100 DEG C, is extracted 2 times, extraction
When time is 2 small.
Preferably, it is 80% that ethyl alcohol to alcohol content is added in described in step (2), and solid-liquid separating method is using centrifugation
Partition method.
Preferably, large pore resin absorption column chromatography described in step (3) uses AB-8 types or D101 type resins, receives
Collect 10% ethanol eluate.
Preferably, DEAE celluloses described in step (5) are DEAE-52.
Preferably, in step (5), refined polysaccharide sample solution obtained by step (4) is taken, through DEAE cellulose column colors
Spectrum separation after being eluted respectively with water, 0.05%NaCl solution, is eluted with 0.1%NaCl solution, and it is molten to collect 0.1%NaCl elutions
Liquid after dialysis desalting, concentrates, freezes, obtain sunset abelmoschus stem or bark leaf polyose.
The present invention shows that sunset abelmoschus stem or bark leaf polyose provided by the invention can remarkably promote mice spleen by lot of experiments
Lymphopoiesis, and there was no significant difference with positive drug lipopolysaccharides (LPS) group for its high dose group.Show Huang provided by the invention
Hollyhock stem leaf polysaccharide can have immune-enhancing activity to mouse, can be applied to prepare the drug with immunological enhancement or health care
Product.
The present invention carries out its activity to harvest the cauline leaf resource discarded after sunset abelmoschus flower during Producing medicinal herbs as raw material
Polyose extraction purifies and industrialization development, it is intended to and turn waste into wealth, effectively improve the utilization ratio of sunset abelmoschus root medicinal organism resource, it is real
The comprehensive utilization of the high added value and high social value of existing traditional Chinese medicine waste.
The present invention is in the extraction and preparation technique of sunset abelmoschus stem or bark leaf polyose, using Orthogonal Experiment and Design, with solid-liquid ratio, extraction
Time, Extracting temperature, extraction time are investigation factor, optimize its Extraction technique so that the extraction process cocoa established
The extraction efficiency of polysaccharide is effectively improved, impurity dissolution rate is reduced, establishes optimal extraction and purification process.
Sunset abelmoschus stem or bark leaf polyose provided by the invention is prepared in purifying process, using the method for activity tracking, is inhaled through macropore
Low polar impurity during attached resin column chromatography purifying can effectively remove can effectively prevent membrane module pollution during hyperfiltration treatment, improve
Production efficiency.By hyperfiltration treatment, can effective control targe sunset abelmoschus stem or bark leaf polyose relative molecular weight scope, it is more convenient for activity
The enrichment of sugar.By DEAE celluosic resin column chromatography separating purifications, can be made structure composition be relatively fixed, molecular weight it is homogeneous
Polysaccharide component provides advantage for its activity rating and control of product quality.
Advantageous effect：It is provided by the invention to harvest the sunset abelmoschus root cauline leaf resource discarded after sunset abelmoschus flower as raw material, pass through
Extraction, ethanol precipitation, large pore resin absorption column chromatography coupling ultrafiltration, DEAE celluosic resin column chromatography separating purifications etc. are a variety of existing
Foundry skill prepare sunset abelmoschus stem or bark leaf polyose, have the characteristics that chemical constitution composition be relatively fixed, molecular weight it is homogeneous, and have compared with
Strong immune-enhancing activity, particularly lymphocytic B cells multiplication activity, available for prepare with immunological enhancement drug or
Health products.
In addition, the present invention with seriously restrict at present the development of sunset abelmoschus root medicinal organism resource industries, to environment potential hazard compared with
Big discarded cauline leaf is using object, and one side low raw-material cost, production cost is with the obvious advantage, on the other hand also can effectively be disappeared
Sunset abelmoschus root cauline leaf resource is consumed, environment carrying pressure is reduced, realizes resource circulation utilization, for holding for sunset abelmoschus root medicinal organism resource
Supervention exhibition provides support, has higher economic results in society.
Description of the drawings
Fig. 1 is sunset abelmoschus stem or bark leaf polyose molecular weight determination chromatogram provided by the invention；
Fig. 2 is sunset abelmoschus stem or bark leaf polyose molecular weight determination chromatogram provided by the invention；
Fig. 3 is sunset abelmoschus stem or bark leaf polyose monose composition measuring chromatogram provided by the invention.
Specific embodiment
With reference to specific embodiment, the present invention is furture elucidated, it should be understood that these embodiments be merely to illustrate the present invention and
It is not used in and limits the scope of the invention, after the present invention has been read, those skilled in the art are to the various shapes of equal value of the present invention
The modification of formula falls within the application scope as defined in the appended claims.
1 sunset abelmoschus stem or bark leaf polyose extraction process of embodiment is preferred
(1) sunset abelmoschus stem or bark leaf polyose extraction single factor test is preferred
Sunset abelmoschus root cauline leaf sample is taken to be divided into several pieces, after appropriate crushing, is weighed, it is (1 to add in raw material weight ratio:10、1:
15、1:20、1:30、1:40) water, with the Extracting temperature of (60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 100 DEG C), extraction (0.5h, 1h,
2h, 3h, 4h), it extracts (1 time, 2 times, 3 times).Filtering, take supernatant, with phend-sulphuric acid measure polysaccharide extract rate, preferably because
Plain index the results are shown in Table 1.
1 single factor analysis of table
(2) sunset abelmoschus stem or bark leaf polyose extraction orthogonal test
Using solid-liquid ratio, extraction time, Extracting temperature as investigation factor, the preferred factor level of single factor test is chosen, by just
Experiment is handed over to determine optimal extraction scheme, the results are shown in Table 2.
2 polysaccharide extracting process Orthogonal Experiment and Design of table and result
3 sunset abelmoschus stem or bark leaf polyose of table extracts the results of analysis of variance
It is very poor the result shows that, the extraction process degree for influencing sunset abelmoschus stem or bark leaf polyose is followed successively by：Extracting temperature>Extraction time
>Solid-liquid ratio, Extracting temperature, extraction time (P<0.05) two factors have apparent shadow to the extraction process of sunset abelmoschus stem or bark leaf polyose
It rings, solid-liquid ratio has no significant difference, and in order to economize on resources, through considering, it is A to draw optimal extraction process3B3C2, that is, expect
Liquor ratio 1:20, Extracting temperature is 100 DEG C, extraction time 2h, and extraction is twice.
A kind of separation purifying technique of 2 sunset abelmoschus stem or bark leaf polyose of embodiment, it includes the following steps：
(1) sunset abelmoschus root cauline leaf 1.0kg is taken, after crushed, the extracting method after optimizing by embodiment 1, which is pressed, adds in water 20kg,
Heating and refluxing extraction 2 times under the conditions of 100 DEG C, every time 2 it is small when, filtering, obtain filtrate.
(2) filtrate obtained by taking step (1) is concentrated into 1L, adds in ethyl alcohol to alcohol content 80%, after staticly settling for 24 hours, centrifugation
Separation of solid and liquid obtains alcohol precipitation precipitation.
(3) alcohol precipitation precipitation obtained by step (2) is taken, after being dissolved in water, through AB-8 large pore resin absorption column chromatographic isolations, with
10% ethanol water elutes, and obtains eluent.
(4) eluent obtained by step (3) is taken, after appropriate concentration, through hollow cellulose ultrafiltration membrane ultrafiltration, takes molecular weight ranges
For 10,000~30,000 sample segments, after appropriate concentration, refined polysaccharide sample solution is obtained.
(5) take refined polysaccharide sample solution obtained by step (4), separated through DEAE-52 cellulose column chromatographies, respectively with water,
It after the elution of 0.05%NaCl solution, is eluted with 0.1%NaCl solution, collects 0.1%NaCl elution solution, after dialysis desalting,
Concentration freezes, obtains sunset abelmoschus stem or bark leaf polyose 8.7g.
A kind of separation purifying technique of 3 sunset abelmoschus stem or bark leaf polyose of embodiment, it includes the following steps：
(1) sunset abelmoschus root cauline leaf 2.0kg is taken, after crushed, the extracting method after optimizing by embodiment 1, which is pressed, adds in water 40kg,
Heating and refluxing extraction 3 times under the conditions of 100 DEG C, every time 1 it is small when, filtering, obtain filtrate.
(2) filtrate obtained by taking step (1) is concentrated into 1L, adds in ethyl alcohol to alcohol content 70%, after staticly settling for 24 hours, mistake
Filter obtains alcohol precipitation precipitation.
(3) alcohol precipitation precipitation obtained by step (2) is taken, after being dissolved in water, through D101 large pore resin absorption column chromatographic isolations, with water
Elution, obtains eluent.
(4) eluent obtained by step (3) is taken, after appropriate concentration, through ultrafiltration membrane ultrafiltration, it is 10,000~30,000 to take molecular weight ranges
Sample segment after appropriate concentration, obtains refined polysaccharide sample solution.
(5) take refined polysaccharide sample solution obtained by step (4), separated through DEAE-52 cellulose column chromatographies, respectively with water,
It after the elution of 0.05%NaCl solution, is eluted with 0.1%NaCl solution, collects 0.1%NaCl elution solution, after dialysis desalting,
Concentration freezes, obtains sunset abelmoschus stem or bark leaf polyose 13.6g.
4 sunset abelmoschus stem or bark leaf polyose chemical constitution research of embodiment
(1) weight average molecular weight measures
Chromatographic condition：Tsk-gel G4000PWXL0 chromatographic columns (7.8 × 300mm), 30 DEG C of column temperature, ultra-pure water is as flowing
Phase, isocratic elution, volume flow 0.5mL/min；20 μ L of sample size；ELSD drift tube temperatures：60℃；Gas pressure：40psi.
Test solution：The sunset abelmoschus stem or bark leaf polyose 10mg samples that accurately weighed embodiment 2 is prepared, are dissolved in 2ml and surpass
It in pure water, fully dissolves, centrifugation takes supernatant, sample introduction.
Measurement result：Through being compared with Dextran Molecular Weight Certified Reference Materials, it is equal that its number is calculated using GPC molecular weight distribution softwares
Molecular weight, weight average molecular weight and breadth coefficient, the results are shown in Table 4.As shown in Table 4, weight average molecular weight 13821Da, distribution system
Number is 1.9654.
4 sunset abelmoschus stem or bark leaf polyose molecular weight determination of table
(2) monose composition and ratio measuring
2 gained sunset abelmoschus stem or bark leaf polyose of Example after PMP derivatizations, measures its monose composition, such as using HPLC methods
Shown in Fig. 1 to 3.The result shows that sunset abelmoschus stem or bark leaf polyose be by rhamnose, mannose, galactolipin, galacturonic acid, glucose and
Arabinose forms, and molar ratio is arabinose:Galactolipin:Glucose:Galacturonic acid:Rhamnose:Mannose=1:
0.53:0.12:0.05:0.16:0.03。
5 sunset abelmoschus stem or bark leaf polyose immunocompetence of embodiment is tested
Experimental method：According to the preparation method of mouse spleen lymphocyte suspension, splenic lymphocytes suspension after purification is taken, is adjusted
Whole cell concentration is 1 × 106A mL-1, it is unicellular in 96 porocyte culture plates in control wells and test hole to add in 100 μ L
Suspension puts 5%CO2, continuously cultivate for 24 hours in 37 DEG C of incubators after, in blank control wells, Positive control wells and test hole respectively
Add in 1640 culture mediums of RPMI, LPS (5 μ gmL of final concentration-1) and various concentration 2 gained sunset abelmoschus stem or bark leaf polyose of embodiment
(final concentration is respectively 40,120,370,1110 μ gmL-1) 50 μ L, every group sets 3 multiple holes.After continuous culture 48h, terminate culture
Preceding each holes of 4h add in 10 μ L MTT (5mg/ml) reagents, and after being cultivated for 4h, culture solution is abandoned in centrifugation, suction, and diformazan is added in per hole
After 100 μ L of base sulfoxide (DMSO), vibration dissolving 20min, enzyme-linked immunosorbent assay instrument surveys OD under 570nm570It is worth (absorbance),
And Computation immunity stimulus index SI.Wherein, SI=ODTest group/ODControl group。
Statistical analysis：Statistical analysis is carried out using 16.0 softwares of SPSS, is as a result represented with mean ± SD, t, which is examined, to be used
19 statistical softwares of SPSS complete.With P<0.05 represents significant difference；P<0.01 represents that difference is extremely notable.
Measurement result：Result of study shows (table 5) that concentration is in 40~120 μ gmL-1In the range of, sunset abelmoschus stem or bark leaf polyose
Mice spleen lymphocytes proliferation (P ＜ 0.05 or P ＜ 0.01) can be remarkably promoted, with the increase of concentration, facilitation enhancing.And
120 μ gmL of sunset abelmoschus stem or bark leaf polyose concentration-1When, humidification close to bone-marrow-derived lymphocyte mitogen LPS (lipopolysaccharides), but
Subsequent concn is further added by, and facilitation reduces, and shows as two-way dose-effect relationship.
The influence of 5 various concentration Polysaccharides on Mice spleen cell of table multiplication
Note：" * " is represented with blank control group comparing difference significantly (P ＜ 0.05)；" * * " is represented compared with blank control group
Difference is extremely significantly (P ＜ 0.01).
The experimental results showed that, sunset abelmoschus stem or bark leaf polyose provided by the invention can significantly increase spleen lymphocyte proliferation above
Activity shows stronger immune-enhancing activity, available for drug and health food of the preparation with immune enhancing function.And with
Existing drug is compared, which derives from natural traditional medical and edible dual purpose plant, safer, has no adverse reaction.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (8)
1. a kind of solution with the sunset abelmoschus stem or bark leaf polyose for enhancing immunization, it is characterised in that：Sunset abelmoschus stem or bark leaf polyose
Weight average molecular weight is 13821 dalton, by arabinose, galactolipin, glucose, galacturonic acid, rhamnose and mannose group
Into molar ratio is arabinose:Galactolipin:Glucose:Galacturonic acid:Rhamnose:Mannose=1:0.53:0.12:
0.05:0.16:0.03；
The concentration of the solution of sunset abelmoschus stem or bark leaf polyose is 40~120 μ g/mL；
The preparation method of sunset abelmoschus stem or bark leaf polyose, successively include extraction, ethanol precipitation, large pore resin absorption column chromatogram purification, in
Hollow fiber element UF membrane, DEAE celluosic resin column chromatography separating purifications, specific steps include：
(1) sunset abelmoschus root cauline leaf is taken, is 1 by solid-liquid weight ratio after crushed:8~1:30 add in water, add under the conditions of 80~100 DEG C
Thermal extraction 2~3 times, every time 1~3 it is small when, filtering, obtain filtrate；
(2) filtrate obtained by step (1) is taken, concentration, it is 50%~80% to add in ethyl alcohol to ethyl alcohol volumetric concentration, is staticly settled, Gu
Liquid separates, and obtains alcohol precipitation precipitation；
(3) take alcohol precipitation precipitation obtained by step (2), after being dissolved in water, through large pore resin absorption column chromatographic isolation, successively with pure water,
1%~20% ethanol water elutes, and obtains eluent；
(4) eluent obtained by step (3) is taken, after concentration, through ultrafiltration membrane ultrafiltration, it is 10,000~30,000 part samples to take molecular weight ranges
Product after concentration, obtain refined polysaccharide sample solution；
(5) take refined polysaccharide sample solution obtained by step (4), separated through DEAE cellulose column chromatographies, respectively with water, 0.01~
It after the elution of 0.05%NaCl solution, is eluted with 0.1~0.5%NaCl solution, 0.1~0.5%NaCl elution solution is collected, through saturating
It after analysing desalination, concentrates, freezes, obtain sunset abelmoschus stem or bark leaf polyose.
2. a kind of preparation method of the solution of sunset abelmoschus stem or bark leaf polyose with enhancing immunization described in claim 1,
It is characterized in that, the preparation method of sunset abelmoschus stem or bark leaf polyose, includes extraction, ethanol precipitation, large pore resin absorption column chromatographically pure successively
Change, hollow cellulose UF membrane, DEAE celluosic resin column chromatography separating purifications, specific steps include：
(1) sunset abelmoschus root cauline leaf is taken, is 1 by solid-liquid weight ratio after crushed:8~1:30 add in water, add under the conditions of 80~100 DEG C
Thermal extraction 2~3 times, every time 1~3 it is small when, filtering, obtain filtrate；
(2) filtrate obtained by step (1) is taken, concentration, it is 50%~80% to add in ethyl alcohol to ethyl alcohol volumetric concentration, is staticly settled, Gu
Liquid separates, and obtains alcohol precipitation precipitation；
(3) take alcohol precipitation precipitation obtained by step (2), after being dissolved in water, through large pore resin absorption column chromatographic isolation, successively with pure water,
1%~20% ethanol water elutes, and obtains eluent；
(4) eluent obtained by step (3) is taken, after concentration, through ultrafiltration membrane ultrafiltration, it is 10,000~30,000 part samples to take molecular weight ranges
Product after concentration, obtain refined polysaccharide sample solution；
(5) take refined polysaccharide sample solution obtained by step (4), separated through DEAE cellulose column chromatographies, respectively with water, 0.01~
It after the elution of 0.05%NaCl solution, is eluted with 0.1~0.5%NaCl solution, 0.1~0.5%NaCl elution solution is collected, through saturating
It after analysing desalination, concentrates, freezes, obtain sunset abelmoschus stem or bark leaf polyose.
3. the preparation method of the solution of the sunset abelmoschus stem or bark leaf polyose according to claim 2 with enhancing immunization,
It is characterized in that, sunset abelmoschus root cauline leaf is remaining ground cauline leaf after harvesting sunset abelmoschus flower.
4. the preparation method of the solution of the sunset abelmoschus stem or bark leaf polyose according to claim 2 with enhancing immunization,
It is characterized in that, solid-to-liquid ratio described in step (1) is 1:20, Extracting temperature is 100 DEG C, extraction time 2 times, and extraction time is small for 2
When.
5. the preparation method of the solution of the sunset abelmoschus stem or bark leaf polyose according to claim 2 with enhancing immunization,
It is characterized in that, it is 80% that ethyl alcohol to ethyl alcohol volumetric concentration is added in step (2), and solid-liquid separating method uses centrifugal separation.
6. the preparation method of the solution of the sunset abelmoschus stem or bark leaf polyose according to claim 2 with enhancing immunization,
It is characterized in that, large pore resin absorption column chromatography described in step (3) is using AB-8 types or D101 type resins, collected volume concentration
10% ethanol eluate.
7. the preparation method of the solution of the sunset abelmoschus stem or bark leaf polyose according to claim 2 with enhancing immunization,
It is characterized in that, DEAE celluloses described in step (5) are DEAE-52.
8. the preparation method of the solution of the sunset abelmoschus stem or bark leaf polyose according to claim 2 with enhancing immunization,
It is characterized in that, in step (5), takes refined polysaccharide sample solution obtained by step (4), separated through DEAE cellulose column chromatographies, respectively
It after water, the elution of 0.05%NaCl solution, is eluted with 0.1%NaCl solution, collects 0.1%NaCl elution solution, it is de- through dialysing
It after salt, concentrates, freezes, obtain sunset abelmoschus stem or bark leaf polyose.
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