CN105153324B - The extracting method of camellia chrysantha polysaccharide - Google Patents
The extracting method of camellia chrysantha polysaccharide Download PDFInfo
- Publication number
- CN105153324B CN105153324B CN201510705138.6A CN201510705138A CN105153324B CN 105153324 B CN105153324 B CN 105153324B CN 201510705138 A CN201510705138 A CN 201510705138A CN 105153324 B CN105153324 B CN 105153324B
- Authority
- CN
- China
- Prior art keywords
- camellia
- camellia chrysantha
- chrysantha polysaccharide
- extracting method
- weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 54
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 54
- 150000004676 glycans Chemical class 0.000 title claims abstract description 53
- 241001657422 Camellia petelotii Species 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 63
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 239000012153 distilled water Substances 0.000 claims abstract description 26
- 239000013049 sediment Substances 0.000 claims abstract description 26
- 239000006286 aqueous extract Substances 0.000 claims abstract description 22
- 239000002244 precipitate Substances 0.000 claims abstract description 22
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000012869 ethanol precipitation Methods 0.000 claims abstract description 11
- 108091005804 Peptidases Proteins 0.000 claims abstract description 10
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 10
- 239000004365 Protease Substances 0.000 claims abstract description 9
- 239000012141 concentrate Substances 0.000 claims abstract description 8
- 239000012466 permeate Substances 0.000 claims abstract description 5
- 239000000284 extract Substances 0.000 claims description 25
- 235000019441 ethanol Nutrition 0.000 claims description 18
- 238000000605 extraction Methods 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- 240000001548 Camellia japonica Species 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- 150000002576 ketones Chemical class 0.000 claims 2
- 241000209507 Camellia Species 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 11
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 10
- 238000000502 dialysis Methods 0.000 description 10
- 230000000242 pagocytic effect Effects 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 239000013641 positive control Substances 0.000 description 9
- 230000037396 body weight Effects 0.000 description 8
- 210000004185 liver Anatomy 0.000 description 8
- 241001328788 Camellia nitidissima Species 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- -1 filtering Substances 0.000 description 6
- 102000004889 Interleukin-6 Human genes 0.000 description 5
- 108090001005 Interleukin-6 Proteins 0.000 description 5
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 5
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 231100000753 hepatic injury Toxicity 0.000 description 5
- 229940100601 interleukin-6 Drugs 0.000 description 5
- 210000001541 thymus gland Anatomy 0.000 description 5
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 206010067125 Liver injury Diseases 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 108010006464 Hemolysin Proteins Proteins 0.000 description 3
- 238000012449 Kunming mouse Methods 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- AZHSSKPUVBVXLK-UHFFFAOYSA-N ethane-1,1-diol Chemical compound CC(O)O AZHSSKPUVBVXLK-UHFFFAOYSA-N 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 239000003228 hemolysin Substances 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241001122767 Theaceae Species 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 210000005252 bulbus oculi Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 210000000566 lesser sac Anatomy 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000001835 viscera Anatomy 0.000 description 2
- 241000282452 Ailuropoda melanoleuca Species 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010013183 Dislocation of vertebra Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 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 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 235000010254 Jasminum officinale Nutrition 0.000 description 1
- 240000005385 Jasminum sambac Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 102000011759 adducin Human genes 0.000 description 1
- 108010076723 adducin Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 210000000776 antibody secreting cell Anatomy 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- OAABHEHWRQAHEJ-UHFFFAOYSA-N butan-1-ol;chloroform Chemical compound ClC(Cl)Cl.CCCCO OAABHEHWRQAHEJ-UHFFFAOYSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 206010019847 hepatosplenomegaly Diseases 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000008004 immune attack Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000007688 immunotoxicity Effects 0.000 description 1
- 231100000386 immunotoxicity Toxicity 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 210000001589 microsome Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a kind of extracting method of camellia chrysantha polysaccharide, comprise the following steps：1) by golden camellia tea extracting in water, Aqueous extracts are obtained；2) after Aqueous extracts are concentrated, ethanol precipitation is added, taking precipitate is standby；3) sediment of step 2) is washed with absolute ethyl alcohol, acetone successively, then plus distilled water dissolves, and adds protease hydrolyzed, obtains enzymolysis liquid；4) enzymolysis liquid is placed under frequency ultrasonic wave and handled, then dialysed through the bag filter that molecular cut off is 18000~28000, go bail for and stay liquid to be dialysed through the bag filter that molecular cut off is 400000~500000, take permeate to concentrate, ethanol precipitation is added, taking precipitate is standby；5) sediment described in step 4) is washed with absolute ethyl alcohol, acetone successively, then plus distilled water dissolves, and activated carbon decolorizing, filters, dries, as camellia chrysantha polysaccharide.The camellia chrysantha polysaccharide that the present invention obtains has preferable immunocompetence.
Description
Technical field
The invention belongs to technical field of biological extraction, and in particular to the extracting method of camellia chrysantha polysaccharide.
Background technology
Camellia nitidissima (Camellia chrysantha (Hu) Tuyama) belongs to Theaceae Camellia Camellia Section Chrysantha, is praised
For " tea race queen " and " plant kingdom giant panda ".A kind of Chinese herbal medicine of the golden camellia tea as Zhuang nationality in Guangxi folk tradition, there is drop
Blood glucose, reducing blood lipid, suppress tumour growth, improve the different physiological roles such as body immunity, wherein, camellia chrysantha polysaccharide is considered as
The effect of being important one of composition.《The research of camellia chrysantha polysaccharide anti-mouse immunological liver injury effect》(it is published in West China pharmacy
Magazine, 2014,9 (5)：525~527) extracting method for disclosing camellia chrysantha polysaccharide is：By dry golden camellia tea coarse powder 1kg
Add the backflow degreasing of 10L petroleum ethers to extract 2 times, extract 1.5h every time, after waving the petroleum ether in the most dregs of a decoction, extracting in boiling water 2 times, often
Secondary 2h, merge extract solution, 150ml is concentrated into, with chloroform-n-butanol (3:1) repeatedly extraction, the supernatant for the protein that is removed
Liquid, add 95% ethanol precipitation to stay overnight, filter, abandoning supernatant, filter residue is washed with ether, absolute ethyl alcohol, acetone successively, produces gold
Jasmine tea Thick many candies.Eluted through DE52 type resin chromatographies post, powdered camellia chrysantha polysaccharide is dried in vacuo to obtain, using phend-sulphuric acid
Colour developing, UV-vis spectroscopy meter measure and calculated purity are 69.16%.The document further discloses BCG vaccine joint fat
There is immunological liver injury in polysaccharide induced mouse, when Mice Body inner cell receives immune attack, shows as internal organ and damages
Wound, as caused by hepatosplenomegaly index rise and immunocompromised caused by Thymus atrophy, this show BCG joint LPS have pair
Target organ is specifically aggressive.After camellia chrysantha polysaccharide is treated, mice organs degree of injury substantially mitigates, with HE dyeing pathology inspections
The result looked into is consistent, and this shows camellia chrysantha polysaccharide by recovering body internal organs activity and strengthen immunity, so as to suppress liver cell
Immunotoxicity.Although above-mentioned document discloses camellia chrysantha polysaccharide has certain immunocompetence, but immunocompetence is not high.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of extracting method of camellia chrysantha polysaccharide, this method extracts what is obtained
The immunomodulatory effect of camellia chrysantha polysaccharide significantly improves than existing camellia chrysantha polysaccharide.
Technical scheme provided by the invention is the extracting method of camellia chrysantha polysaccharide, is comprised the following steps：
1) by golden camellia tea extracting in water, Aqueous extracts are obtained；
2) after Aqueous extracts are concentrated, ethanol precipitation is added, taking precipitate is standby；
3) sediment of step 2) is washed with absolute ethyl alcohol, acetone successively, then plus distilled water dissolves, and adds albumen
Enzyme digests, and obtains enzymolysis liquid；
4) enzymolysis liquid is placed in frequency to handle 20~80s under 1000~5000kHz ultrasonic waves, then through molecular cut off
For 18000~28000 bag filter dialyse 24~72h, go bail for stay liquid through molecular cut off be 400000~500000 dialysis
Bag 24~72h of dialysis, takes permeate to concentrate, adds ethanol precipitation, taking precipitate is standby；
5) sediment described in step 4) is washed with absolute ethyl alcohol, acetone successively, then plus distilled water dissolves, activated carbon
Decolourize, filter, dry, as camellia chrysantha polysaccharide.
In step 1), extracting in water refers to the water that 20~30 times of its weight is added in toward golden camellia tea, refluxing extraction 2~3
It is secondary, 1~3h is extracted every time, merges extract solution, filtering, filtrate is Aqueous extracts.
In step 2), Aqueous extracts are concentrated into the medicinal extract that relative density at 60 DEG C is 1.1~1.3.
In step 2), ethanol to the alcohol content that the addition ethanol precipitation refers to add 90~95v% in toward concentrate is
80~85v%.
In step 3), absolute ethyl alcohol, the dosage of acetone are 2~4 times of weight of precipitate, and the dosage of distilled water is washing
2~4 times of Sediment weight afterwards.
In step 3), the dosage of protease is the 0.3~3% of golden camellia tea dry weight, and hydrolysis temperature is 40~60 DEG C, enzymolysis
Time is 1~3h.Macro-molecular protein enzymolysis in extract can be polypeptide and amino acid by protease.
In step 4), enzymolysis liquid is placed in frequency to handle 20~80s under 1000~5000kHz ultrasonic waves, will can be divided
Son measures the part sugar chain disconnection of great polysaccharide (pectin), reduces molecular weight, reduction viscosity, improves water-soluble, the immune work of enhancing
Property.Ultrasonic treatment time be 20~80s be in order to ensure be only broken molecular weight more than 500,000 polysaccharide glycosides key, and
The breaking degree of polysaccharide molecule is smaller, does not destroy activity unit, to reach reduction molecular weight, improves water solubility, and strengthen immune
The purpose of activity.
Enzymolysis liquid after ultrasonication first carries out saturating for the first time through the bag filter that molecular cut off is 18000~28000
Analysis, small molecule pigment, polypeptide and amino acid and protease will transmit through bag filter and filters out, and greatly improve the purity for retaining liquid.Again will
Retain liquid to dialyse 24~72h through the bag filter that molecular cut off is 400000~500000, obtain molecular weight 18000~
The efflux of the camellia chrysantha polysaccharide in 28000 to 400000~500000 sections, permeate (efflux) concentration refer to concentrate
Relative density is 1.1~1.3 medicinal extract to 60 DEG C.The addition ethanol precipitation refers to add 90~95v% in toward concentrate
Ethanol to alcohol content be 80~85v%.
In step 5), absolute ethyl alcohol, the dosage of acetone are 2~4 times of weight of precipitate, and the dosage of distilled water is washing
2~4 times of Sediment weight afterwards.
The present invention is using ultra-high frequency ultrasonic wave by the great polysaccharide molecule of molecular weight (molecular weight is more than 500000)
The fracture of glycosides key, reduce its molecular weight, reduce viscosity, improve water-soluble, greatly strengthen the immunocompetence of end-product.
After testing, the purity of end-product is more than 98.8%.
Embodiment
The present invention is further elaborated for specific examples below, but not as a limitation of the invention.
Embodiment 1
1) Camellia nitidissima cured leaf is crushed, adds the water of 20 times of its weight, refluxing extraction 2 times, extract 1h every time, merge extraction
Liquid, filtering, filtrate is that Aqueous extracts obtain Aqueous extracts；
2) Aqueous extracts are concentrated into the medicinal extract that relative density at 60 DEG C is 1.1, ethanol to the alcohol content for adding 90v% is
80v%, staticly settle, filter, taking precipitate is standby；
3) sediment of step 2) is washed with absolute ethyl alcohol, acetone successively, absolute ethyl alcohol, the dosage of acetone are precipitation
2 times of thing weight, then plus distilled water dissolving, the dosage of distilled water are 2 times of the Sediment weight after washing, add Camellia nitidissima
The protease of leaf dry weight 0.3%, enzymolysis time is 1~3h at 40 DEG C, enzyme-deactivating, obtains enzymolysis liquid；
4) enzymolysis liquid is placed in frequency to handle 20s under 1000kHz ultrasonic waves, then through molecular cut off be 18000~
28000 bag filter dialysis 24h, go bail for and stay liquid to be taken through the bag filter dialysis 24h that molecular cut off is 400000~500000
To cross liquid and be concentrated into the medicinal extract that relative density at 60 DEG C is 1.1, ethanol to the alcohol content for adding 90v% is 80v%, is staticly settled,
Filter, taking precipitate is standby；
5) sediment described in step 4) is washed with absolute ethyl alcohol, acetone successively, absolute ethyl alcohol, the dosage of acetone are
2 times of weight of precipitate, then plus distilled water dissolving, the dosage of distilled water are 2 times of the Sediment weight after washing, and activated carbon takes off
Color, filter, dry, as camellia chrysantha polysaccharide.After testing, purity 98.8%.
Embodiment 2
1) Camellia nitidissima cured leaf is crushed, adds the water of 30 times of its weight, refluxing extraction 3 times, extract 3h every time, merge extraction
Liquid, filtering, filtrate is that Aqueous extracts obtain Aqueous extracts；
2) Aqueous extracts are concentrated into the medicinal extract that relative density at 60 DEG C is 1.3, add the 95v% ethanol alcohol into mixed liquor
Content is 85v%, is staticly settled, and is filtered, and taking precipitate is standby；
3) sediment of step 2) is washed with absolute ethyl alcohol, acetone successively, absolute ethyl alcohol, the dosage of acetone are precipitation
4 times of thing weight, then plus distilled water dissolving, the dosage of distilled water are 4 times of the Sediment weight after washing, add Camellia nitidissima
The protease of leaf dry weight 3%, enzymolysis time is 3h at 60 DEG C, enzyme-deactivating, obtains enzymolysis liquid；
4) enzymolysis liquid is placed in frequency to handle 80s under 5000kHz ultrasonic waves, then through molecular cut off be 18000~
28000 bag filter dialysis 72h, go bail for and stay liquid to be taken through the bag filter dialysis 72h that molecular cut off is 400000~500000
To cross liquid and be concentrated into the medicinal extract that relative density at 60 DEG C is 1.3, ethanol to the alcohol content for adding 95v% is 85v%, is staticly settled,
Filter, taking precipitate is standby；
5) sediment described in step 4) is washed with absolute ethyl alcohol, acetone successively, absolute ethyl alcohol, the dosage of acetone are
4 times of weight of precipitate, then plus distilled water dissolving, the dosage of distilled water are 4 times of the Sediment weight after washing, and activated carbon takes off
Color, filter, dry, as camellia chrysantha polysaccharide.After testing, purity 99.0%.
Embodiment 3
1) golden camellia tea is crushed, adds the water of 25 times of its weight, refluxing extraction 2 times, extract 2h every time, merge extraction
Liquid, filtering, filtrate is that Aqueous extracts obtain Aqueous extracts；
2) Aqueous extracts are concentrated into the medicinal extract that relative density at 60 DEG C is 1.2, add the 90v% ethanol alcohol into mixed liquor
Content is 80v%, is staticly settled, and is filtered, and taking precipitate is standby；
3) sediment of step 2) is washed with absolute ethyl alcohol, acetone successively, absolute ethyl alcohol, the dosage of acetone are precipitation
3 times of thing weight, then plus distilled water dissolving, the dosage of distilled water are 3 times of the Sediment weight after washing, add Camellia nitidissima
The protease of leaf dry weight 1.5%, enzymolysis time is 2h at 50 DEG C, enzyme-deactivating, obtains enzymolysis liquid；
4) enzymolysis liquid is placed in frequency to handle 40s under 4000kHz ultrasonic waves, then through molecular cut off be 18000~
28000 bag filter dialysis 48h, go bail for and stay liquid to be taken through the bag filter dialysis 48h that molecular cut off is 400000~500000
To cross liquid and be concentrated into the medicinal extract that relative density at 60 DEG C is 1.2, ethanol to the alcohol content for adding 90v% is 80v%, is staticly settled,
Filter, taking precipitate is standby；
5) sediment described in step 4) is washed with absolute ethyl alcohol, acetone successively, absolute ethyl alcohol, the dosage of acetone are
3 times of weight of precipitate, then plus distilled water dissolving, the dosage of distilled water are 3 times of the Sediment weight after washing, and activated carbon takes off
Color, filter, dry, as camellia chrysantha polysaccharide.After testing, purity 98.9%.
Embodiment 4
1) golden camellia tea is crushed, adds the water of 20 times of its weight, refluxing extraction 3 times, extract 1h every time, merge extraction
Liquid, filtering, filtrate is that Aqueous extracts obtain Aqueous extracts；
2) Aqueous extracts are concentrated into the medicinal extract that relative density at 60 DEG C is 1.3, add the 90v% ethanol alcohol into mixed liquor
Content is 85v%, is staticly settled, and is filtered, and taking precipitate is standby；
3) sediment of step 2) is washed with absolute ethyl alcohol, acetone successively, absolute ethyl alcohol, the dosage of acetone are precipitation
2 times of thing weight, then plus distilled water dissolving, the dosage of distilled water are 4 times of the Sediment weight after washing, add Camellia nitidissima
The protease of leaf dry weight 0.3%, enzymolysis time is 1h at 60 DEG C, enzyme-deactivating, obtains enzymolysis liquid；
4) enzymolysis liquid is placed in frequency to handle 80s under 1000kHz ultrasonic waves, then through molecular cut off be 18000~
28000 bag filter dialysis 24h, go bail for and stay liquid to be taken through the bag filter dialysis 72h that molecular cut off is 400000~500000
To cross liquid and be concentrated into the medicinal extract that relative density at 60 DEG C is 1.1, ethanol to the alcohol content for adding 95v% is 80v%, is staticly settled,
Filter, taking precipitate is standby；
5) sediment described in step 4) is washed with absolute ethyl alcohol, acetone successively, absolute ethyl alcohol, the dosage of acetone are
4 times of weight of precipitate, then plus distilled water dissolving, the dosage of distilled water are 2 times of the Sediment weight after washing, and activated carbon takes off
Color, filter, dry, as camellia chrysantha polysaccharide.After testing, purity 99.1%.
Experimental example
1st, influence of the camellia chrysantha polysaccharide to experimental immune hepatic injury
70 Kunming mouses are chosen, 20 ± 2g of body weight, male and female half and half, are randomly divided into 7 groups, respectively blank group, model
Group, positive controls and experimental group 1~4, experimental animal drinking public water supply, put 20~25 DEG C of room temperature, the reality of humidity 60~75%
Test in room environmental after adapting to 1 week and start to test.BCG (cards are injected intraperitoneally in remaining each group rat in addition to blank group within first day for experiment
Jie's seedling) 8mg (about 5 × 107Individual viable bacteria).From 2nd day, in addition to blank group, each group rat starts to be administered, the life of model group gastric infusion
Manage salt solution, dosage 0.2ml/kg；The camellia chrysantha polysaccharide of positive controls gavage background technology, dosage 0.2g/kg；Experimental group
The medicine of 1~4 difference gastric infusion embodiment 1~4, dosage 0.1g/kg.Successive administration 12d, last dose last dose
After 16h, blank group tail vein injection saline, the μ g/kg bodies of remaining each group mouse peritoneal injection LPS normal saline solutions 30
Heavy, eyeball is taken a blood sample after 8h.Liver, thymus gland and index and spleen index are calculated, and liver histopathology inspection is carried out using HE dyeing, is used
Elisa methods detect tumor necrosis factor-alpha (TNF-α) in hepatic tissue, interleukin-6 (IL-6) and microsome nitric oxide (NO)
Content.
Table 1
Note：Compared with blank group, * P ＜ 0.05, * * P ＜ 0.01；Compared with model group, △ P ＜ 0.05, △ △ P ＜
0.01。
As shown in Table 1, the liver in model group mice plasma and Spleen coefficient significantly rise, and thymus index reduces.Experiment
Liver coefficient, the Spleen coefficient of group mouse reduce, and thymus index increase, compared with model group, there is significant difference.Model group is small
The horizontal obvious increase of TNF-α, IL-6 and NO in rat liver, there were significant differences compared with blank group, illustrates modeling success.It is real
TNF-α in the hepatic tissue of group mouse, IL-6 and NO horizontal reduction are tested, there were significant differences compared with model group, it is seen that experimental group
Polyoses extract there is certain protective effect to the liver of immunological liver injury in mice.Experimental group and positive controls phase
Than liver index, index and spleen index significantly reduce, and thymus index dramatically increases, and the horizontal obvious reduction of TNF-α, IL-6 and NO, say
Bright polysaccharide of the invention has more preferable protective effect than the polysaccharide of background technology to the liver of immunological liver injury in mice.
2nd, influence of the camellia chrysantha polysaccharide to mouse phagocyte and index
The Kunming mouse 70 for taking body weight to be 20g ± 2g, male and female half and half, is randomly divided into 7 groups, respectively blank group, mould
Type group (physiological saline presses 0.2ml/kg body weight), experimental group 1~4 (are administered the camellia chrysantha polysaccharide of embodiment 1~4, pressed successively
0.1g/kg body weight is administered) and positive controls (camellia chrysantha polysaccharide of background technology is administered by 0.2g/kg body weight).Gastric infusion,
1 time a day, successive administration 7d.The 2nd day and the 5th day is administered in addition to normal group, remaining each group lesser sac injection endoxan 80mg/
Kg, make immunosuppressed mice model.5d is administered, in addition to normal group, the starch normal saline solution of each group mouse peritoneal injection 2%
After 1.5ml, last dose 24h, (the CRBC of each group mouse peritoneal injection 2%：NS, V/V) CRBC suspension 1ml, 2h after cervical vertebra
Dislocation is put to death, and gently rubs belly, extracts peritoneal fluid 0.5ml, calculates phagocytic percentage and phagocytic index.
Table 2
| Group | Phagocytic percentage | Phagocytic index |
| Blank group | 34.6±3.91 | 0.57±0.13 |
| Model group | 20.22±3.12** | 0.32±0.09* |
| Positive controls | 35.53±4.24△ | 0.59±0.15△△ |
| Experimental group 1 | 56.82±5.33△△ | 0.79±0.18△△ |
| Experimental group 2 | 58.13±3.29△ | 0.80±0.16△△ |
| Experimental group 3 | 59.42±5.34△△ | 0.81±0.20△ |
| Experimental group 4 | 57.16±6.10△ | 0.79±0.07△△ |
Note：Compared with blank group, * P ＜ 0.05, * * P ＜ 0.01；Compared with model group, △ P ＜ 0.05, △ △ P ＜
0.01。
As shown in Table 2, model group phagocytic percentage and phagocytic index are substantially less than blank group, illustrate immunosuppressed mice mould
Type success.Wherein there were significant differences between experimental group and model group, shows that suppression is immunized caused by significantly improving Cy in camellia chrysantha polysaccharide
The phagocytic percentage and phagocytic index of Turnover of Mouse Peritoneal Macrophages processed.Experimental group compared with positive controls, phagocytic percentage with
Phagocytic index significantly improves, it is known that, camellia chrysantha polysaccharide of the invention compared with background technology camellia chrysantha polysaccharide to immunosuppressed mice
With more preferable non-specific immune function.
3rd, influence of the camellia chrysantha polysaccharide to mice serum hemolysin level
Body weight 20g ± 2g Kunming mouses 70 are taken, male and female half and half, are randomly divided into 7 groups, every group 10, respectively blank
Group, model group (physiological saline presses 0.2ml/kg body weight), (camellia chrysantha polysaccharide of background technology presses 0.2g/kg bodies to positive controls
It is administered again) and experimental group 1~4 (camellia chrysantha polysaccharide of embodiment 1~4 being administered successively, be administered by 0.1g/kg body weight).Gavage is given
Medicine, 1 time a day, successive administration 7d.The 2nd day and the 5th day is administered in addition to normal group, remaining each group lesser sac injection endoxan
80mg/kg, make immunosuppressed mice model.In the sheep red blood cell (SRBC) of 7d each groups mouse peritoneal injection 20% being administered for the first time
(SRBC) 0.2ml, 4d after being immunized, go eyeball to take blood, separate serum, by serum with after 500 times of normal saline dilution for measure.
Diluted mice serum 1ml is sequentially added in reaction tube, 5%SRBC 0.5ml, 10% complement 1ml, puts 37 DEG C
30min is incubated in water bath with thermostatic control, terminating reaction in ice bath is then moved to, 1500r/min centrifugation 5min, takes supernatant 1ml, add all
Family name reagent 3ml, shake up and place 10min, in 540nm wavelength determination sample absorbances, physiological saline generation is used with not increase serum two
Replace, but the same conduct sample blank control tube for adding sheep red blood cell (SRBC) and complement, determine absorbance.
5%SRBC 0.25ml are taken, add Dou Shi reagents to shake up to 4ml and place 10min in 540nm wavelength colorimetric reading extinction
Absorbance (HC when degree, as SRBC half hemolysis50)。
Table 3
| Group | Half hemolytic value (HC50) |
| Normal group | 18.62±4.35 |
| Model group | 10.55±2.32* |
| Positive controls | 19.23±4.16△ |
| Experimental group 1 | 32.87±5.53△△ |
| Experimental group 2 | 33.12±5.10△△ |
| Experimental group 3 | 34.01±4.33△ |
| Experimental group 4 | 33.10±6.03△ |
Note：Compared with blank group, * P ＜ 0.05, * * P ＜ 0.01；Compared with model group, △ P ＜ 0.05, △ △ P ＜
0.01。
As shown in Table 3, the camellia chrysantha polysaccharide of experimental group can promote the serum hemolysin of immunosuppressed mice to generate, and promote
On Hemolysin formation, significant difference be present with model group, illustrate that camellia chrysantha polysaccharide can significantly resist CY induced mice serum hemolysis
The inhibitory action that element is formed.Experimental group is compared with positive controls, and the camellia chrysantha polysaccharide of experimental group is to immune response later stage
Antibody secreting cell, which is formed, has more preferable humidification.
Claims (9)
1. the extracting method of camellia chrysantha polysaccharide, it is characterised in that：Comprise the following steps：
1) by golden camellia tea extracting in water, Aqueous extracts are obtained；
2) after Aqueous extracts are concentrated, ethanol precipitation is added, taking precipitate is standby；
3) sediment of step 2) is washed with absolute ethyl alcohol, acetone successively, then plus distilled water dissolves, and adds protease enzyme
Solution, obtains enzymolysis liquid；
4) enzymolysis liquid is placed in frequency to handle 20~80s under 1000~5000kHz ultrasonic waves, is then through molecular cut off
18000~28000 bag filter is dialysed 24~72h, is gone bail for and is stayed liquid through bag filter that molecular cut off is 400000~500000
Dialyse 24~72h, takes permeate to concentrate, adds ethanol precipitation, taking precipitate is standby；
5) sediment described in step 4) is washed with absolute ethyl alcohol, acetone successively, then plus distilled water dissolving, activated carbon take off
Color, filter, dry, as camellia chrysantha polysaccharide.
2. the extracting method of camellia chrysantha polysaccharide according to claim 1, it is characterised in that：In step 1), extracting in water is
The water of 20~30 times of its weight is added in referring to toward golden camellia tea, refluxing extraction 2~3 times, extracts 1~3h every time, merges extract solution,
Filtering, filtrate is Aqueous extracts.
3. the extracting method of camellia chrysantha polysaccharide according to claim 1, it is characterised in that：It is in step 2), Aqueous extracts are dense
It is reduced to the medicinal extract that relative density at 60 DEG C is 1.1~1.3.
4. the extracting method of the camellia chrysantha polysaccharide according to claim 1 or 3, it is characterised in that：In step 2), the addition
Ethanol to the alcohol content that ethanol precipitation refers to add 90~95v% in toward concentrate is 80~85v%.
5. the extracting method of camellia chrysantha polysaccharide according to claim 1, it is characterised in that：In step 3), absolute ethyl alcohol, third
The dosage of ketone is 2~4 times of weight of precipitate, and the dosage of distilled water is 2~4 times of the Sediment weight after washing.
6. the extracting method of camellia chrysantha polysaccharide according to claim 1, it is characterised in that：In step 3), the use of protease
Measure as the 0.3~3% of golden camellia tea dry weight, hydrolysis temperature is 40~60 DEG C, and enzymolysis time is 1~3h.
7. the extracting method of camellia chrysantha polysaccharide according to claim 1, it is characterised in that：In step 4), the permeate
Concentration refers to be concentrated into the medicinal extract that relative density at 60 DEG C is 1.1~1.3.
8. the extracting method of the camellia chrysantha polysaccharide according to claim 1 or 7, it is characterised in that：In step 4), the addition
Ethanol to the alcohol content that ethanol precipitation refers to add 90~95v% in toward concentrate is 80~85v%.
9. the extracting method of camellia chrysantha polysaccharide according to claim 1, it is characterised in that：In step 5), absolute ethyl alcohol, third
The dosage of ketone is 2~4 times of weight of precipitate, and the dosage of distilled water is 2~4 times of the Sediment weight after washing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510705138.6A CN105153324B (en) | 2015-10-27 | 2015-10-27 | The extracting method of camellia chrysantha polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510705138.6A CN105153324B (en) | 2015-10-27 | 2015-10-27 | The extracting method of camellia chrysantha polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105153324A CN105153324A (en) | 2015-12-16 |
| CN105153324B true CN105153324B (en) | 2017-12-08 |
Family
ID=54794398
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510705138.6A Expired - Fee Related CN105153324B (en) | 2015-10-27 | 2015-10-27 | The extracting method of camellia chrysantha polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105153324B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107964052A (en) * | 2018-01-18 | 2018-04-27 | 广西天昌投资有限公司 | A kind of extraction process of camellia chrysantha polysaccharide |
| CN112425648A (en) * | 2020-11-13 | 2021-03-02 | 广州市从化华隆果菜保鲜有限公司 | Fruit and vegetable preservative and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101485789A (en) * | 2009-02-06 | 2009-07-22 | 广西桂人堂金花茶产业集团股份有限公司 | Camellia chrysantha polysaccharide lozenge and method for producing the same |
| CN104119456B (en) * | 2014-07-04 | 2017-03-22 | 中国科学院过程工程研究所 | Method for separation and purification of edible (medicinal) mushroom active components by multistage membrane process |
-
2015
- 2015-10-27 CN CN201510705138.6A patent/CN105153324B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN105153324A (en) | 2015-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105175569B (en) | The method that camellia chrysantha polysaccharide is extracted from golden camellia tea | |
| CN101632436B (en) | Fresh yam extract as well as preparation method and application thereof | |
| CN105175570B (en) | The method that polysaccharide is extracted from golden camellia tea | |
| Yan et al. | Effects of polysaccharides from Cordyceps sinensis mycelium on physical fatigue in mice | |
| CN105147717B (en) | Application of the Fuscoporia obliqua polysaccharide component in chronic pancreatitis is prevented and treated | |
| CN103110889A (en) | Traditional Chinese medicine for increasing immunity and preparation method thereof | |
| CN107698659B (en) | Amygdalus sinica protein source alpha-glucosidase inhibitory peptide subjected to enzymolysis by protease M and preparation method thereof | |
| CN102771868A (en) | Dendrobium candidum solution | |
| CN104710538A (en) | Arabinogalactan of flowers of panax notoginseng (burK.)F.H.Chen, and preparation method and use thereof | |
| CN107412721A (en) | A kind of hypoglycemic bitter gourd polypeptide compound capsule and preparation method thereof | |
| CN105153324B (en) | The extracting method of camellia chrysantha polysaccharide | |
| CN103923201A (en) | Preparation method of hippocampus active glycoprotein | |
| CN106084087A (en) | A kind of preparation method of Fructus Trichosanthis polysaccharide | |
| CN106490608A (en) | A kind of Herba Dendrobii enhancing immunity oral liquid, electuary, capsule, tablet | |
| CN103848921A (en) | Lyceum ruthenicum alkali-extracted polysaccharide and preparation method and application thereof | |
| Peng et al. | Deproteinization and structural characterization of bioactive exopolysaccharides from Ganoderma sinense mycelium | |
| CN108467438A (en) | Lucidum spore powder wall polysaccharide and its extracting method | |
| CN104672345A (en) | Method for preparing crude polysaccharide from steindachner soft-shelled turtle carapace | |
| CN103479875B (en) | A kind of preparation method of high activity Rhizoma Polygonati Odorati concentrated solution and application thereof | |
| Bai et al. | A systematic review on the research Progress on polysaccharides from fungal traditional Chinese medicine | |
| CN103183740B (en) | Production method of fructus schisandrae polysaccharide | |
| CN113827624B (en) | Pleurotus citrinopileatus polysaccharide and application thereof | |
| CN104606221A (en) | Constitution improving and immunity enhancing drug and preparation method | |
| CN111285938B (en) | Extraction method and application of aspongopus crude polysaccharide | |
| CN103263382A (en) | Oyster polysaccharide gel formulation capable of playing protection role in immunological liver injury |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20171208 |