CN115725004A - Preparation method and application of highland barley tender leaf enzyme-extracted polysaccharide - Google Patents
Preparation method and application of highland barley tender leaf enzyme-extracted polysaccharide Download PDFInfo
- Publication number
- CN115725004A CN115725004A CN202211475607.6A CN202211475607A CN115725004A CN 115725004 A CN115725004 A CN 115725004A CN 202211475607 A CN202211475607 A CN 202211475607A CN 115725004 A CN115725004 A CN 115725004A
- Authority
- CN
- China
- Prior art keywords
- highland barley
- enzyme
- polysaccharide
- tender leaf
- extracted
- 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.)
- Granted
Links
- 235000007340 Hordeum vulgare Nutrition 0.000 title claims abstract description 167
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 148
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 148
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 150000004676 glycans Chemical class 0.000 title claims abstract description 18
- 240000005979 Hordeum vulgare Species 0.000 title 1
- 241000209219 Hordeum Species 0.000 claims abstract description 166
- 102000004190 Enzymes Human genes 0.000 claims abstract description 74
- 108090000790 Enzymes Proteins 0.000 claims abstract description 74
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 26
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims abstract description 26
- 230000035755 proliferation Effects 0.000 claims abstract description 18
- 238000000605 extraction Methods 0.000 claims abstract description 17
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 16
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 82
- 229940088598 enzyme Drugs 0.000 claims description 73
- 239000000843 powder Substances 0.000 claims description 45
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 29
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 28
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 28
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 24
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000001816 cooling Methods 0.000 claims description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- 239000004480 active ingredient Substances 0.000 claims description 14
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 13
- 238000005238 degreasing Methods 0.000 claims description 13
- 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 description 12
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 230000000415 inactivating effect Effects 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 229930182830 galactose Natural products 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 10
- 239000004365 Protease Substances 0.000 claims description 9
- 229920002678 cellulose Polymers 0.000 claims description 9
- 239000001913 cellulose Substances 0.000 claims description 9
- 108010059892 Cellulase Proteins 0.000 claims description 8
- 108090000526 Papain Proteins 0.000 claims description 8
- 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 description 8
- 229940106157 cellulase Drugs 0.000 claims description 8
- 230000002401 inhibitory effect Effects 0.000 claims description 8
- 229940055729 papain Drugs 0.000 claims description 8
- 235000019834 papain Nutrition 0.000 claims description 8
- 210000000582 semen Anatomy 0.000 claims description 8
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 7
- 229920005654 Sephadex Polymers 0.000 claims description 7
- 239000012507 Sephadex™ Substances 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 7
- 239000003208 petroleum Substances 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 4
- 230000002255 enzymatic effect Effects 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 239000000413 hydrolysate Substances 0.000 claims description 3
- 238000005342 ion exchange Methods 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 230000009849 deactivation Effects 0.000 claims 1
- 238000004321 preservation Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 65
- 230000000694 effects Effects 0.000 abstract description 19
- 230000001093 anti-cancer Effects 0.000 abstract description 11
- 238000011160 research Methods 0.000 abstract description 8
- 206010028980 Neoplasm Diseases 0.000 abstract description 6
- 201000011510 cancer Diseases 0.000 abstract description 5
- 210000001072 colon Anatomy 0.000 abstract description 5
- 210000002919 epithelial cell Anatomy 0.000 abstract description 5
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 3
- 210000004877 mucosa Anatomy 0.000 abstract description 3
- 150000004804 polysaccharides Chemical class 0.000 description 132
- 239000000243 solution Substances 0.000 description 36
- 230000005764 inhibitory process Effects 0.000 description 26
- 150000002772 monosaccharides Chemical class 0.000 description 20
- 239000000523 sample Substances 0.000 description 20
- 239000008367 deionised water Substances 0.000 description 16
- 229910021641 deionized water Inorganic materials 0.000 description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 235000015097 nutrients Nutrition 0.000 description 9
- 239000002246 antineoplastic agent Substances 0.000 description 8
- 229940041181 antineoplastic drug Drugs 0.000 description 8
- 230000003833 cell viability Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 8
- 239000002689 soil Substances 0.000 description 8
- 102000004139 alpha-Amylases Human genes 0.000 description 7
- 108090000637 alpha-Amylases Proteins 0.000 description 7
- 229940024171 alpha-amylase Drugs 0.000 description 7
- 239000007789 gas Substances 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000007710 freezing Methods 0.000 description 5
- 230000008014 freezing Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 5
- 229960000367 inositol Drugs 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000003809 water extraction Methods 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 240000007054 Avena nuda Species 0.000 description 3
- 235000007317 Avena nuda Nutrition 0.000 description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 3
- 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 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 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 description 3
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- OAVCWZUKQIEFGG-UHFFFAOYSA-O 2-(5-methyl-2H-tetrazol-1-ium-1-yl)-1,3-thiazole Chemical compound CC1=NN=N[NH+]1C1=NC=CS1 OAVCWZUKQIEFGG-UHFFFAOYSA-O 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 2
- 102100022624 Glucoamylase Human genes 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 235000005811 Viola adunca Nutrition 0.000 description 2
- 240000009038 Viola odorata Species 0.000 description 2
- 235000013487 Viola odorata Nutrition 0.000 description 2
- 235000002254 Viola papilionacea Nutrition 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 239000003560 cancer drug Substances 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- -1 polysaccharide compound Chemical class 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting 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
- 238000010298 pulverizing process Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 2
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 1
- 241000998584 Nuda Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 230000009982 effect on human Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000009144 enzymatic modification Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000012844 infrared spectroscopy analysis Methods 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000013365 molecular weight analysis method Methods 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 150000003214 pyranose derivatives Chemical group 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical class 0.000 description 1
Images
Abstract
The invention discloses a preparation method and application of highland barley tender leaf enzyme-extracted polysaccharide; the preparation method of the highland barley tender leaf enzyme-extracted polysaccharide can obtain high-purity enzyme-extracted polysaccharide, and the extraction rate of the enzyme-extracted polysaccharide can reach 7.68 percent and the purity can reach 99.75 percent. The research of the invention finds that the highland barley tender leaf enzyme polysaccharide has good anticancer activity and can obviously inhibit the proliferation of breast cancer cells and colorectal cancer cells. Particularly can specifically inhibit the proliferation of colorectal cancer cells, does not influence the proliferation of normal colon mucosa epithelial cells, has the characteristics of good biocompatibility, strong specificity, low toxic and side effects and the like, can make up the defects of the traditional cancer treatment means, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of anticancer active ingredients, and relates to a preparation method and application of highland barley tender leaf enzyme-extracted polysaccharide, in particular to a preparation method of highland barley tender leaf enzyme-extracted polysaccharide and application of highland barley tender leaf enzyme-extracted polysaccharide in anticancer, and especially relates to a preparation method of highland barley tender leaf enzyme-extracted polysaccharide and application of highland barley tender leaf enzyme-extracted polysaccharide in specific inhibition of colorectal cancer cell proliferation.
Background
The highland barley grows in a cold and anoxic plateau area, is the most main carbohydrate source in the daily diet of Tibetan people, has outstanding medical care efficacy, and is considered to be one of important factors for the long life of Tibetan people. The highland barley has strong stress resistance and generates abundant secondary metabolites under the special environments of severe cold, oxygen deficiency, dryness, strong light irradiation and the like. Highland barley is also more and more popular among people because of comprehensive and unique nutritional composition and high food therapy value. Scholars have pointed out that the content of enzymatically extracted polysaccharides in highland barley is generally higher than that of barley varieties planted in other areas of the world. Therefore, the enzyme-extracted polysaccharide is also considered as the most closely related nutrient substance in the highland barley for human health and longevity. According to the report, the enzyme-extracted polysaccharide has: 1) Reducing serum cholesterol, preventing cardiovascular disease; 2) Regulating blood sugar level, and preventing diabetes; 3) Promoting the proliferation of intestinal probiotics and preventing intestinal cancer; 4) Regulating the immunity of the organism and the like, and has wide application prospect. At present, the research on plant polysaccharides mainly focuses on cereal crops such as wheat, barley, corn and the like, and the extraction methods are also mainly divided into a water method, an alkaline method, an enzymatic method and a combination of the methods. Wherein, the enzyme method modification mainly comprises the steps of adding enzyme to the system, such as adding amylase and protease to remove starch and protein, adding cellulase to cut the beta-1, 4-glycosidic bond of insoluble dietary fiber, and enhancing the physical and chemical properties of polysaccharide, water retention and the like. The method has the advantages of simple reaction conditions, low requirements on equipment and convenient operation; in addition, the enzymatic modification also has the characteristic of specificity. The invention adopts the method of complex enzyme extraction as the best method after laboratory optimization, increases the yield of polysaccharide to the utmost extent and ensures the biological activity of the polysaccharide.
At present, the research on the activity of plant polysaccharide cells mainly takes anti-inflammatory action, anti-oxidation action and anti-tumor action as main actions, the research quantity of the plant polysaccharide on the aspect of anti-cancer is large, and as cancer has high harm to human health and great treatment difficulty, many scholars aim at natural medicines, and hope that natural substances such as the plant polysaccharide are used for preventing and treating tumors. Relatively few researches on enzyme extraction polysaccharide in naked barley (highland barley is one of the naked barley) are carried out at home and abroad. By taking reference to the research method of experts on the enzyme-extracted polysaccharide in grains, the extraction method and the process are optimized, and the high-purity enzyme-extracted polysaccharide is successfully separated and purified from the highland barley tender leaves. In addition, compared with chemotherapy and radiotherapy, the polysaccharide extracted from nature has the advantages of good biocompatibility, mild action, low toxic and side effects, difficult generation of drug resistance and the like when being used as an antitumor drug. Therefore, the development of the anti-cancer activity of the highland barley tender leaf enzyme-extracted polysaccharide can not only enlarge the application range of highland barley tender leaves and improve the additional value of related products, but also make up for the defects of large toxic and side effect, high price and the like of the traditional anti-cancer drugs. In conclusion, the invention has wide application prospect in developing natural and safe anti-cancer drugs.
Disclosure of Invention
The invention provides a preparation method and application of highland barley tender leaf enzyme-extracted polysaccharide. In particular, the method of manufacturing a semiconductor device,
the first purpose is to provide a brand-new enzyme-extracted polysaccharide active ingredient extracted and purified from highland barley young leaves.
The second purpose of the invention is to provide a preparation method of the highland barley tender leaf enzyme-extracted polysaccharide.
The third purpose of the invention is to provide the application of the active component of the highland barley tender leaf enzyme polysaccharide in preparing anti-cancer drugs.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention relates to a preparation method of active ingredients of enzyme-extracted polysaccharide from tender leaves of highland barley, which comprises the following steps:
s1, carrying out vacuum freeze drying on highland barley leaves, pulverizing the highland barley leaves into powder, and collecting highland barley tender leaf powder;
s2, carrying out degreasing, enzyme extraction and alcohol precipitation on the highland barley tender leaf powder to obtain highland barley enzyme-extracted crude polysaccharide;
and S3, separating the highland barley enzyme-extracted crude polysaccharide by using a cellulose column, and purifying by using a sephadex column to obtain the highland barley tender leaf enzyme-extracted polysaccharide active component.
As an embodiment of the invention, the highland barley variety is Qinghai yellow, and the powder is ground and then screened by a 200-mesh screen to obtain powder with uniform size.
As an embodiment of the present invention, in step S1, the conditions of vacuum freeze-drying are-50 to-60 ℃ for 4 to 6 hours; 10 to 15 hours at the temperature of between 35 ℃ below zero and 40 ℃ below zero; 10 to 15 hours at the temperature of minus 10 to minus 20 ℃;20-25 ℃ for 24-48 hours. Preferably, the vacuum freeze drying condition is-60 ℃ for 6 hours; 15 hours at-35 ℃; 15 hours at-20 ℃; at 25 ℃ for 48 hours, in this order.
In some embodiments, in step S1, highland barley seeds are planted in a planting tray, when the length of the highland barley leaves reaches about 15cm, the highland barley leaves are cut off from the roots, and the highland barley tender leaf powder obtained after cleaning, refrigeration, vacuum freeze drying and powdering is collected.
Specifically, about 100g of Qinghai yellow highland barley seeds are evenly planted in each planting tray and soaked overnight with clear water, then a small amount of nutrient soil is used for covering the highland barley seeds, water is fully poured until the nutrient soil is thoroughly wetted, then a small amount of water is watered every day, and the planting period is 14-21 days.
As an embodiment of the present invention, in step S2, petroleum ether is used as a solvent to perform degreasing in the degreasing process, and the ratio of the highland barley tender leaf powder to the petroleum ether is 1g:40-50mL. Defatting by soaking at 55-60 deg.C for 90-120min, extracting at 65-70 deg.C for 120-150min, and recovering at 75-80 deg.C for 15-20min; after degreasing treatment, carrying out solid-liquid separation, and carrying out subsequent extraction on the obtained degreased substance.
In step S2, water is added according to the weight ratio of highland barley powder to water 1.
In step S2, the enzymolysis is performed by adding thermostable alpha-amylase, papain, glucoamylase, xylanase and cellulase to the above solution.
As an embodiment of the invention, in step S2, 5-10 muL/g high temperature resistant alpha-amylase of highland barley powder is added into the highland barley powder water solution, and water bath is carried out for 1-2h at 80-90 ℃; adjusting the temperature of the water bath to 60-80 deg.C, adding papain of 5-10 μ L/g semen Avenae Nudae powder, and water bath for 1-2 hr; inactivating enzyme in 100 deg.C water bath for 10-20min, cooling, adding 40-50 μ L/g saccharifying enzyme of semen Avenae Nudae powder, and water bath at 60-80 deg.C for 1-2 hr; inactivating enzyme in 100 deg.C water bath for 10-20min, cooling, and performing 60-90 deg.C water bath for 2-4 hr to obtain enzymatic hydrolysate.
As an embodiment of the invention, the pH value of the obtained enzymolysis liquid is adjusted to 5-6 by NaOH solution, 600-1000U/g of xylanase of highland barley tender leaf powder and 200-600U/g of cellulase of highland barley tender leaf powder are added, the temperature is kept at 40-60 ℃ for 2-3h, and then the enzyme is inactivated in 100 ℃ water bath for 10-20min. Concentrating the supernatant obtained by enzymolysis at 60-80 deg.C in water bath to 50-100 mL, cooling, adding 3-5 times volume of ethanol, standing, centrifuging, and collecting precipitate; re-dissolving with water, and vacuum freeze drying to obtain highland barley tender leaf enzyme-extracted crude polysaccharide.
In some embodiments, high temperature resistant alpha-amylase is added to the supernatant, and is inactivated in a water bath of 30min at 80 ℃ and 10min at 100 ℃; cooling the solution, adding papain, performing water bath at 60 deg.C for 2h, and performing water bath at 100 deg.C for 10min for inactivation; cooling the solution, adding saccharifying enzyme, heating in water bath at 60 deg.C for 1h, and heating in water bath at 100 deg.C for 10min for inactivation. Then adjusting the pH value to 5 by using NaOH solution, adding 800U/g of xylanase of highland barley tender leaf powder and 400U/g of cellulase of highland barley tender leaf powder, preserving the temperature for 2h at 50 ℃, and then inactivating the enzyme for 10min in 100 ℃ water bath.
As an embodiment of the invention, in step S2, the supernatant obtained by enzymolysis is concentrated to 50mL to 100mL in water bath at 60 ℃ to 80 ℃, cooled, added with 3 to 5 times volume of ethanol, kept stand, centrifuged, and the precipitate is collected; re-dissolving with water, and vacuum freeze drying to obtain highland barley tender leaf enzyme-extracted crude polysaccharide.
In some embodiments, the supernatant from the enzymatic hydrolysis is concentrated to 50mL to 100mL in an aqueous bath at 80 ℃, and after cooling, 3 to 5 times the volume of ethanol is added dropwise with stirring. The solution was allowed to stand for one day, centrifuged, the precipitate collected, redissolved in 100mL of deionized water, and 3-5 volumes of ethanol added. Repeating the steps for 3 times, centrifuging, re-dissolving the precipitate with 50mL of deionized water, freezing at-80 ℃ for one day, and carrying out vacuum freeze drying to obtain the crude polysaccharide extracted by the highland barley tender leaf enzyme.
As an embodiment of the invention, the vacuum freeze-drying condition is-50 to-60 ℃ for 4 to 6 hours; 10 to 15 hours at the temperature of minus 35 to minus 40 ℃;10 to 15 hours at the temperature of minus 10 to minus 20 ℃;20-25 ℃ for 24-48 hours. Preferably, the vacuum freeze drying condition is-60 ℃ for 6 hours; 15 hours at-35 ℃; 15 hours at-20 ℃; at 25 ℃ for 48 hours, in that order.
As an embodiment of the present invention, in step S3, the cellulose column is a DEAE-32 cellulose ion exchange column; the sephadex column is a Sephacryl-400 sephadex column.
In one embodiment of the present invention, in step S3, the eluent for separation is distilled water, the elution rate is 0.5-0.7mL/min, and the elution time is 500-1000min.
In one embodiment of the present invention, in step S3, the eluent for purification is distilled water, the elution rate is 0.5-0.7mL/min, and the elution time is 500-1000min.
In some embodiments, the invention provides a preparation method of active ingredients of polysaccharide extracted by highland barley tender leaf enzyme, which comprises the following steps:
step (1): uniformly planting 100g of the Qinghai yellow highland barley seeds in each planting tray, soaking the Qinghai yellow highland barley seeds overnight with clear water, then covering the highland barley seeds with a small amount of nutrient soil, fully watering until the nutrient soil is thoroughly wetted, then watering a small amount of the highland barley seeds every day, wherein the planting period is 14-21 days; cutting off the highland barley leaves from the roots when the length of the highland barley leaves is about 15cm, cleaning, refrigerating, vacuum freeze-drying, powdering, sieving and collecting the highland barley tender leaf powder;
step (2): and (2) degreasing the highland barley tender leaf powder by taking petroleum ether as a solvent, wherein the proportion of the highland barley tender leaf powder to the petroleum ether is 1g:50ml, degreasing by soaking at 55-65 deg.C for 60-90min, extracting at 65-85 deg.C for 90-150min, and recovering at 75-95 deg.C for 10-30min. After degreasing treatment, carrying out solid-liquid separation, and carrying out subsequent enzyme extraction on the obtained degreased substance;
and (3): mixing the highland barley tender leaf powder and water according to the proportion of 1:20 to 1:30, adding 5-10 mu L/g of high-temperature resistant alpha-amylase of highland barley powder into the solution, and carrying out water bath at 80-90 ℃ for 1-2h; adjusting the temperature of the water bath to 60-80 deg.C, adding papain of 5-10 μ L/g semen Avenae Nudae powder, and water bath for 1-2 hr; inactivating enzyme in 100 deg.C water bath for 10-20min, cooling, adding 40-50 μ L/g saccharifying enzyme of semen Avenae Nudae powder, and water bath at 60-80 deg.C for 1-2 hr; inactivating enzyme in 100 deg.C water bath for 10-20min, cooling, and water bath at 60-90 deg.C for 2-4h. Then adjusting pH to 5-6 with NaOH solution, adding xylanase of highland barley tender leaf powder 600-1000U/g and cellulase of highland barley tender leaf powder 200-600U/g, keeping the temperature at 40-60 deg.C for 2-3h, and inactivating enzyme in 100 deg.C water bath for 10-20min;
and (4): concentrating the collected supernatant to 50-100 mL in water bath at 80 deg.C, cooling, and adding dropwise 3-5 times volume of ethanol under stirring. The solution was allowed to stand for one day, centrifuged, the precipitate collected, redissolved in 100mL of deionized water, and 3-5 volumes of ethanol added. Repeating the steps for 3 times, centrifuging, re-dissolving the precipitate with 50mL of deionized water, freezing at-80 ℃ for one day, and carrying out vacuum freeze drying to obtain crude polysaccharide extracted from tender leaves of the highland barley;
and (5): separating the crude polysaccharide extracted from the highland barley tender leaf enzyme obtained in the step 4 by cellulose column chromatography (preferably DEAE-32 cellulose ion exchange column), and eluting by using pure water;
and (6): concentrating the obtained eluate, purifying with Sephacryl-400 Sephadex column, eluting with distilled water, collecting eluate, concentrating, and lyophilizing to obtain active components of polysaccharide extracted from highland barley tender leaf enzyme.
In a second aspect, the invention relates to the highland barley young leaf enzyme polysaccharide active ingredient prepared by the method, the molecular weight of the highland barley young leaf enzyme polysaccharide active ingredient is 20000-30000Da, and the polydispersity coefficient of the highland barley young leaf enzyme polysaccharide active ingredient is 1.14. The enzyme is proved to extract polysaccharide as homogeneous polysaccharide. The research of the invention finds that the brand new active ingredient for extracting polysaccharide from the highland barley tender leaf enzyme can be obtained by the method and the elution process. The novel polysaccharide compound can have good specific anticancer activity, for example, the polysaccharide compound has excellent inhibition and killing effects on cancer cells, and in addition, the polysaccharide with the structure basically has no inhibition activity on normal cells.
As one embodiment of the invention, the highland barley tender leaf enzyme-extracted polysaccharide consists of arabinose, galactose, xylose and glucose, wherein the content ratio of the arabinose, the xylose, the glucose and the galactose is about 10:11:1:4.
as an embodiment of the invention, the active component of the highland barley young leaf enzyme polysaccharide is odorless and tasteless pale yellow floccule which is easily dissolved in water.
As an embodiment of the invention, the active component of the highland barley tender leaf enzyme polysaccharide contains galactose residues.
In a third aspect, the invention relates to an application of active ingredients of enzyme-extracted polysaccharide of highland barley young leaves in preparing an anti-cancer medicament.
As one embodiment of the present invention, the anticancer drug comprises an anti-breast cancer drug or an anti-colorectal cancer drug. The inhibition rate of the active component for extracting polysaccharide by using the highland barley tender leaf enzyme can reach 40-50% on breast cancer cells 4T1, and the inhibition rate on colorectal cancer cells HT29, caco-2 and CT26 can reach 80-90%, 60-70% and 30-40%; the normal colon mucosa epithelial cell line NCM460 still has good cell safety when the administration concentration reaches 10 mg/mL.
In a fourth aspect, the invention relates to an anti-cancer drug, which comprises active ingredients of highland barley tender leaf enzyme polysaccharide with pharmaceutically effective dose. The highland barley tender leaf enzyme polysaccharide prepared by the invention can effectively inhibit the proliferation of breast cancer 4T1 cells and colorectal cancer HT29, caco-2 and CT26 cells, particularly can specifically inhibit the proliferation of the colorectal cancer HT29 cells, and does not obviously influence the normal physiological activity of normal cells NCM 460. The physiological activity of the highland barley tender leaf enzyme polysaccharide for specifically inhibiting colorectal cancer and breast cancer cells can be proved by a cell proliferation toxicity experiment. The experimental result shows that the survival rate of two cancer cells of the liver can be greatly reduced by extracting polysaccharide from the highland barley tender leaf enzyme, and the colorectal cancer HT-29 cells can be specifically inhibited and killed without obvious influence on the proliferation of normal cells.
The research of the invention finds that the brand-new active ingredient for extracting polysaccharide by using the highland barley tender leaf enzyme has good inhibition effect on various breast cancer and colorectal cancer cells, can specifically inhibit and kill the colorectal cancer cells, and basically has no inhibition effect on normal cells. The anticancer selectivity of the polysaccharide active ingredient is proved by breast cancer and colorectal cancer cell models (such as HT29, caco-2 and CT26 colorectal cancer cells and 4T1 breast cancer cells) and normal cell models (human normal colon mucosa epithelial cells NCM 460).
Compared with the prior art, the invention has the following beneficial effects:
1) The invention provides a brand new active ingredient for extracting polysaccharide by using highland barley tender leaf enzyme, and proves that the highland barley tender leaf enzyme-extracted polysaccharide is a brand new homogeneous polysaccharide by means of purity analysis, molecular weight determination, monosaccharide composition analysis and the like;
2) The invention proves that the highland barley tender leaf enzyme polysaccharide has good inhibition effect on both breast cancer and colorectal cancer through cytotoxicity experiments, especially on colorectal cancer HT-29 cells, and plays a promoting role in the application of polysaccharide as an anti-cancer medicament; in addition, the brand new polysaccharide substance can effectively inhibit the proliferation of cancer cells such as breast cancer cells and colorectal cancer cells, can avoid the damage to the proliferation capacity and the cell state of normal cells, and makes up the defects of poor specificity and great side effect on human bodies of the existing chemical anticancer drugs;
3) According to the preparation method of the active component for extracting polysaccharide from the highland barley tender leaf enzyme, the method of adding papain and high-temperature resistant alpha-amylase greatly improves the removal rate of protein and starch and the yield of the polysaccharide extracted from the highland barley tender leaf enzyme; by the elution method, the highland barley tender leaf enzyme-extracted polysaccharide which can specifically regulate and control the apoptosis of colorectal cancer cells and does not influence the metabolic proliferation of normal cells can be obtained.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a fiber column elution curve of crude polysaccharide extracted from tender leaves of highland barley by enzyme;
FIG. 2 is a High Performance Liquid Chromatography (HPLC) chromatogram of the enzyme-extracted crude polysaccharide from the young leaves of highland barley;
FIG. 3 is the elution curve of dextran column for extracting polysaccharides from tender leaves of highland barley by enzyme;
FIG. 4 is a High Performance Liquid Chromatogram (HPLC) of enzyme-extracted polysaccharides from highland barley young leaves;
FIG. 5 is a relative molecular mass standard curve diagram of enzyme-extracted polysaccharides from highland barley young leaves;
FIG. 6 is a Gas Chromatogram (GC) of a standard monosaccharide;
FIG. 7 is a Gas Chromatogram (GC) of enzyme-extracted polysaccharides from highland barley young leaves;
FIG. 8 is an infrared spectrum (FT-IR) of enzyme-extracted polysaccharides from barley young leaves;
FIG. 9 shows the inhibition rate of enzyme-extracted polysaccharides from barley young leaves on HT 29;
FIG. 10 shows the inhibition rate of enzyme-extracted polysaccharides from barley young leaves on Caco-2;
FIG. 11 shows the CT26 inhibition rate of enzyme-extracted polysaccharides from barley young leaves;
FIG. 12 shows the inhibition ratio of 4T1 by enzyme-extracted polysaccharides from tender leaves of Avena Nuda L.var.nuda;
FIG. 13 shows the survival rate of NCM460 after the treatment of the enzyme-extracted polysaccharides from the barley young leaves.
FIG. 14 shows the inhibition ratio of water-extracted polysaccharides of young leaves of Avena Nuda to HT 29;
FIG. 15 shows the inhibition rate of water-extracted polysaccharides from barley young leaves on Caco-2;
FIG. 16 shows the inhibition rate of water-extracted polysaccharides of young leaves of Avena Nuda to CT 26;
FIG. 17 shows the inhibition rate of water-extracted polysaccharides from young leaves of highland barley on 4T 1.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will aid those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any manner. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the invention.
Example 1
The embodiment provides a preparation method of highland barley tender leaf enzyme-extracted polysaccharide, which comprises the following specific implementation method:
1. preparation method of highland barley tender leaf powder
The method comprises the steps of paving about one finger thick nutrient soil in planting trays, uniformly planting about 100g of the highland barley seeds in each planting tray, soaking overnight Qinghai yellow highland barley seeds in clear water, then covering the highland barley seeds with a small amount of nutrient soil, fully watering until the nutrient soil is thoroughly wetted, and then watering a small amount of nutrient soil every day. Cutting off the tender leaf of highland barley from the root when the tender leaf length of highland barley is about 15cm after 14-21 days, cleaning, refrigerating, vacuum freeze drying, pulverizing, and collecting to obtain highland barley tender leaf powder. Vacuum freeze drying at-50 deg.C for 4 hr; 10 hours at-35 ℃;10 hours at-10 ℃; at 25 ℃ for 24 hours.
2. Preparation of highland barley tender leaf enzyme-extracted crude polysaccharide
Degreasing: weighing about 1g of highland barley tender leaf powder, wrapping in qualitative filter paper (diameter: 18 cm), placing in a soxhlet fat extractor after tying the filter paper with cotton ropes, and degreasing with 50mL petroleum ether as solvent. The degreasing conditions comprise soaking at 65 deg.C for 90min, extracting at 70 deg.C for 120min, recovering at 80 deg.C for 15min, and oven drying at 50 deg.C to obtain degreased powder sample.
Enzyme extraction: weighing about 10g of defatted highland barley tender leaf powder in a beaker, adding 300mL of deionized water, adding 5 muL/g of high temperature resistant alpha-amylase of the highland barley powder, carrying out water bath at 80 ℃ for 30min, and carrying out water bath at 100 ℃ for 10min for inactivation; cooling the solution, adding papain of 5 μ L/g semen Avenae Nudae powder, water bathing at 60 deg.C for 2h, and inactivating at 100 deg.C for 10 min; cooling the solution, adding 40 μ L/g saccharifying enzyme of semen Avenae Nudae powder, inactivating in water bath at 60 deg.C for 1h and 100 deg.C for 10min. Then adjusting the pH value to 5 by using NaOH solution, adding 800U/g of xylanase of highland barley tender leaf powder and 400U/g of cellulase of highland barley tender leaf powder, preserving the temperature for 2h at 50 ℃, inactivating the enzyme in 100 ℃ water bath for 10min, centrifuging, and collecting the supernatant.
Alcohol precipitation: concentrating the collected supernatant to 50-100 mL in water bath at 80 ℃, cooling, and dropwise adding ethanol with 3-4 times of volume while stirring. The solution was allowed to stand for one day, centrifuged, the precipitate collected, redissolved in 100mL of deionized water, and 3-4 volumes of ethanol added. Repeating the steps for 3 times, centrifuging, re-dissolving the precipitate with 50mL of purified water, freezing at-80 ℃ for one day, and carrying out vacuum freeze drying to obtain the crude polysaccharide extracted by the highland barley tender leaf enzyme.
3. Preparation of highland barley tender leaf enzyme-extracted polysaccharide
Weighing 0.5g of crude polysaccharide sample, dissolving in 10mL of deionized water, carrying out metal bath at 55 ℃ for 1h, then stirring at 4 ℃ overnight, centrifuging, sucking the supernatant, dropwise adding the supernatant to the upper end of a chromatographic column filled with DEAE-32, and eluting with distilled water and 0.25M, 0.5M and 1M sodium chloride solutions in sequence. The flow rate is: 0.7mL/min,5 min/tube collection, 80 tubes each. Sampling in 4 test tubes at intervals, taking 0.1mL of collection liquid, adding 0.9mL of deionized water, adding 1mL of 5% phenol solution, then carrying out vortex, adding 5mL of concentrated sulfuric acid, carrying out color comparison with a blank sample after vortex, recording absorbance, and drawing a corresponding elution curve as shown in figure 1. Separately combining and collecting sugar components of different eluents, placing the combined solution eluted by sodium chloride in a dialysis bag for dialysis for 36h, and changing deionized water every 12 h. Concentrating the dialyzed sample solution to about 5mL, freezing at-80 deg.C for one day, and vacuum freeze-drying to obtain highland barley solution enzyme-extracted crude polysaccharide eluted by DEAE-32 chromatographic column.
Preparing polysaccharide samples eluted by different eluents into 10mg/mL solution with deionized water, filtering with 0.45 μm filter membrane, taking 20 μ L sample, and detecting with high performance liquid chromatograph, wherein the polysaccharide sample with highest purity is retained as shown in figure 2. And (3) absorbing DEAE-32 eluent, dropwise adding the DEAE-32 eluent to the upper end of a chromatographic column filled with Sephacryl-400, and eluting with distilled water. The flow rate is: 0.7mL/min,5 min/tube collection, collection 100 tube. Sampling in 4 test tubes at intervals, taking 0.1mL of collection liquid, adding 0.9mL of deionized water, adding 1mL of 5% phenol solution, then vortexing, adding 5mL of concentrated sulfuric acid, performing colorimetry with a blank sample after vortexing, recording absorbance, drawing an elution curve as shown in a graph 3, and combining and collecting sugar components. Freezing the sugar-containing solution at-80 deg.C for one day, and vacuum freeze drying to obtain highland barley young leaf enzyme polysaccharide eluted by Sephacryl-400 chromatographic column.
Preparing the eluted highland barley tender leaf enzyme polysaccharide into 10mg/mL solution with deionized water, filtering with 0.45 μm filter membrane, and introducing into high performance liquid chromatograph for detection, the result is shown in FIG. 4, and the purity is 99.75%. Then the refined highland barley tender leaf enzyme-extracted polysaccharide is obtained, and the total extraction rate is 7.68 percent. The detection instrument is e2695 high performance liquid chromatograph equipped with Waters2414 differential detector and SUGAR KS-805
(8.0 mm ID. Times.300 mm L) sugar column; mobile phase: ultrapure water; flow rate: 1.0mL/min; column temperature: 35 ℃; detector temperature: 30 ℃; sample injection amount: 20 μ L.
Example 2
Example 2 illustrates the physicochemical properties of the highland barley young leaf enzyme-extracted polysaccharide (obtained in example 1), the specific characterization method is as follows:
molecular weight analysis: preparing purified highland barley tender leaf enzyme polysaccharide into 5mg/mL aqueous solution by using deionized water, preparing sugar anhydride standard substances with the relative molecular masses of 4400, 9900, 21400, 43500, 124000 and 805000 into 5mg/mL aqueous solution by using the deionized water respectively, and performing sample detection on each solution through a 0.45-micrometer filter membrane by using an EcoSeC HLC-8320GPC as a detection instrument. Corresponding standard curves were plotted according to the peak time of each anhydrosugar standard, as shown in fig. 5. The relative molecular masses of the samples were calculated from the standard curves of the relative molecular masses, and as shown in Table 1, the weight average molecular weight Mw and the number average molecular weight Mn were 22575Da and 25996Da, respectively, and the polydispersity index (PDI = Mw/Mn) of the corresponding polysaccharide molecule was 1.14. Since the closer the PDI value is to 1, the better the uniformity of the corresponding substances is, the more homogeneous polysaccharide can be considered as the prepared sample of the highland barley tender leaf enzyme polysaccharide.
TABLE 1 determination of relative molecular mass of enzyme-extracted polysaccharides from highland barley leaves
| RT | Area | ％Area | Height | Mn/Daltons | Mw/Daltons | Mw/Mn | Poly dispersity |
| 21.30 | 3132379 | 85.12 | 50103 | 22575 | 25996 | 1.14 | 1.14 |
Monosaccharide composition: taking the purified highland barley tender leaf enzyme extracted polysaccharide sample, and preparing into 500 mug/mL highland barley polysaccharide solution. 1mL of the solution was added to 1mL of a 20. Mu.g/mL inositol solution and vortexed. Then add 0.5mL of trifluoroacetic acid solution and vortex. And (3) placing the mixed solution in a metal bath at 120 ℃ for 2h, and then drying under the protection of nitrogen. Then 250 μ L of isopropanol was added, vortexed, nitrogen blown to complete dryness, and repeated 2 times.
Six monosaccharide standards (rhamnose, glucose, xylose, mannose, arabinose, galactose) and a mixture thereof are respectively prepared into solutions of 20 microgram/mL. 1mL of each solution was added to 1mL of 20. Mu.g/mL inositol solution, vortexed, and blown dry under nitrogen.
And sequentially adding 100 mu L of ammonia water and 0.5mL of 20mg/mL sodium borohydride solution (taking dimethyl sulfoxide as a solvent) into the treated highland barley tender leaf enzyme polysaccharide-extracted sample, each monosaccharide standard and the mixed standard, and fully swirling. Placing the mixed solution in a metal bath at 40 ℃ for 90min, and then dropwise adding 6-9 glacial acetic acid into the mixed solution until no bubbles emerge. To each sample, 100. Mu.L of 1-methylimidazole and 0.5mL of acetic anhydride were added in this order, vortexed thoroughly, and reacted at room temperature for 10min. 4mL of deionized water and 1mL of methylene chloride were added and vortexed thoroughly, retaining the lower organic phase. The upper aqueous phase was transferred to another tube, 1mL of dichloromethane was added, vortexed thoroughly, and the lower organic phase was retained. And combining the organic phases obtained twice, washing twice by using 4mL of deionized water, drying under the protection of nitrogen, and placing in a refrigerator for cold storage to be tested.
The sample to be tested was dissolved in 0.6mL of acetone, vortexed thoroughly, passed through a 0.45 μm filter and examined by gas chromatography. The detection instrument was a GC-2010plus gas chromatograph equipped with (30meter, 0.25mm ID,0.25 μm df) chromatographic column; sample inlet temperature: 240 ℃; column temperature: at 100 ℃ for 2min; heating to 180 deg.c at 10 deg.c/min; at 180 ℃ for 2min; raising the temperature to 240 ℃ at a speed of 4 ℃/min; 5min at 240 ℃; heating to 255 ℃ at the temperature of 10 ℃/min; 255 ℃ for 5min; the carrier gas is nitrogen, and the flow rate is 1.9mL/min; sample injection amount: 1 μ L.
Monosaccharide samples represented by each peak were determined in the mixed standards from the peak times of each monosaccharide standard and inositol, as shown in fig. 6. The peak appearance sequence of each monosaccharide standard product is as follows: rhamnose, arabinose, xylose, mannose, glucose, galactose.
TABLE 2 gas chromatogram data of monosaccharide standards
| Number of peak | Retention time | Area of | Peak height | | Area% | |
| 1 | 17.955 | 3222.9 | 805.9 | Rha | 5.8450 | |
| 2 | 18.260 | 10342.4 | 2682.3 | Ara | 18.7568 | |
| 3 | 18.665 | 7637.2 | 1921.1 | Xyl | 13.7748 | |
| 4 | 23.174 | 8698.2 | 1938.7 | Inositol | 15.7748 | |
| 5 | 23.515 | 8519.0 | 2126.1 | Man | 15.4499 | |
| 6 | 23.720 | 7111.7 | 1495.9 | Glu | 12.8975 | |
| 7 | 23.893 | 9608.3 | 2345.8 | Gla | 17.4254 |
The gas chromatogram of the enzyme-extracted polysaccharides of the tender leaves of the highland barley is shown in figure 7, the monosaccharide represented by each peak in the sample is determined by the peak emergence time of each monosaccharide standard sample of mixed standard, and the peak emergence time sequence is as follows: arabinose, xylose, glucose and galactose, wherein arabinose and xylose were the main components, galactose and glucose were contained in small amounts, and specific peak information is shown in table 3. The relative correction factors of the monosaccharide standard calculated by the mixed standard gas chromatogram map can obtain the content ratio of arabinose, xylose, glucose and galactose in the polysaccharide sample, which is about 10:11:1:4. the main chain of the highland barley tender leaf enzyme extracted polysaccharide is presumed to be composed of arabinose and xylose residues by the content of each monosaccharide.
TABLE 3 gas chromatogram data of enzyme-extracted polysaccharides from highland barley leaves
| Peak number | Retention time | Area of | Peak height | | Area% | |
| 1 | 18.255 | 10474.3 | 2691.1 | Ara | 24.8778 | |
| 2 | 18.659 | 11445.9 | 2948.2 | Xyl | 27.1856 | |
| 3 | 23.171 | 14383.0 | 3827.9 | Inositol | 34.1615 | |
| 4 | 23.718 | 1278.4 | 277.4 | Glu | 3.0364 | |
| 5 | 23.887 | 4521.3 | 1029.0 | Gla | 10.7387 |
Infrared spectrum: taking a lyophilized sample of 5mg of highland barley young leaf enzyme-extracted polysaccharide, mixing with 0.75g of KBr solid uniformly, grinding into powder, pressing the powder into a disc with the thickness of 1mm for tabletting, and performing by using a Nicolet 6700 type Fourier infrared transform spectrometer equipped with an IR device with a scanning interval of 4000-400cm -1 Infrared spectroscopic analysis was performed. The infrared spectrum is shown in FIG. 8, in which the spectrum is 3438.15cm -1 The absorption peak has the maximum intensity and the widest peak shape, and is probably caused by stretching vibration of O-H bonds, which indicates that the molecular structure of the polysaccharide possibly contains carboxyl O-H; at 1640.96cm -1 The absorption peak is in a double bond stretching vibration region, which is probably caused by stretching vibration of a C = C bond or a C = O bond, and is considered to be higher in strength of the absorption peak, and is more probably a stretching vibration peak of the C = O bond; at 1415.91cm -1 May have an absorption peak of-CH 3 Caused by the stretching vibration of; at 1043.73cm -1 The absorption peak at (A) may be caused by absorption vibration of a C-O-C bond or a C-O-H bond, indicating that a pyranose residue is present in the sample, and may be a characteristic peak of a galactose residue as a result of analysis of the composition of a monosaccharide.
Example 3
Example 3 provides the application of the highland barley young leaf enzyme-extracted polysaccharide (obtained from example 1) in anticancer, and the specific implementation process is as follows:
1. proliferation inhibiting effect of highland barley tender leaf enzyme polysaccharide on colorectal cancer (HT 29, caco-2, CT26) cells and breast cancer 4T1 cells
Resuscitating colorectal cancer (HT 29, caco-2, CT26) cells and breast cancer 4T1 cells, culturing in DMEM medium containing 10% FBS, at 37 deg.C, 5% 2 In an incubator under the conditions. When the cells were growing over the bottom of the cell flask, the cells were digested with pancreatin, centrifuged, the supernatant was discarded, 2mL of culture medium was added to the pellet, blown, counted using a hemocytometer, and the cells were diluted in proportion, seeded into a 96-well plate (100 μ L cell suspension per well, i.e., 5000 cells), and cultured in an incubator for 24 hours. To the direction ofAdding DMEM culture medium containing highland barley tender leaf enzyme polysaccharide with concentration of 0, 0.625, 1.25, 2.5, 5 and 10mg/mL into 96-well plate, and incubating at 37 deg.C for 48h. Carefully removing the supernatant, adding 100 mu L of MTT (methyl thiazolyl tetrazolium) in each well, continuously incubating in an incubator at 37 ℃ for 4h, carefully removing the MTT solution, adding 100 mu L of DMSO in each well, continuously incubating in the incubator at 37 ℃ for 10min, fully oscillating on an oscillator until the bluish purple crystals are completely dissolved, and detecting the OD (optical density) value of each well at 570nm through a full-automatic enzyme labeling instrument. Cell viability was calculated as follows. Cell viability (%) = experimental OD value/control OD value × 100%. The results are shown in fig. 9-12, after the primary screening of four tumor cells, the highland barley tender leaf enzyme polysaccharide-extracted sample has no obvious effect on inhibiting the growth of mouse breast cancer cells 4T1, has obvious effect on inhibiting the proliferation of colorectal cancer cells, and particularly has obvious effect on inhibiting HT29 cells. After 0.625mg/mL highland barley tender leaf enzyme polysaccharide-extracting solution is used for treating colorectal cancer cells HT29 h, the proliferation and growth of the colorectal cancer cells can be obviously influenced, and the inhibition rate can reach 37.82%; after being treated by 10mg/mL highland barley tender leaf enzyme polysaccharide solution for HT29 h, the cell proliferation can be almost completely inhibited, and the inhibition rate reaches 86.37%.
2. Proliferation inhibiting effect of highland barley tender leaf enzyme polysaccharide on human normal colon mucosal epithelial cell NCM460
After the human normal colon mucosal epithelial cells NCM460 were recovered, they were cultured in 1640 medium containing 10% FBS, and then incubated at 37 ℃ with 5% CO 2 In a conditioned incubator. When the cells were growing over the bottom of the cell flask, the cells were digested with pancreatin, centrifuged, the supernatant was discarded, 2mL of culture medium was added to the pellet, blown, counted using a hemocytometer, and the cells were diluted in proportion, seeded into a 96-well plate (100 μ L cell suspension per well, i.e., 5000 cells), and cultured in an incubator for 48 hours. Adding 1640 culture medium containing highland barley tender leaf enzyme polysaccharide with concentration of 0, 0.625, 1.25, 2.5, 5, 10mg/mL into 96-well plate, and incubating at 37 deg.C for 24h. Carefully removing the supernatant, adding 0.5mg/mL MTT 100. Mu.L per well, incubating at 37 deg.C for 4h, carefully removing the MTT solution, adding DMSO 100. Mu.L per well, incubating at 37 deg.C for 10min, and sufficiently shaking on a shaker until the blue-violet crystal is completely dissolvedAnd (4) detecting the OD value of each hole at 570nm by a full-automatic enzyme labeling instrument. Cell viability was calculated as follows. Cell viability (%) = experimental OD value/control OD value × 100%. The results of cell viability are shown in FIG. 13, the cell viability of NCM460 cells did not decrease significantly after incubation with highland barley young leaf enzyme-polysaccharide. The highland barley tender leaf enzyme is proved to have no inhibition effect on the proliferation of normal cells.
Comparative example 1
The comparative example provides the proliferation inhibition effect of the water-extracted polysaccharide of the highland barley young leaves on colorectal cancer (HT 29, caco-2, CT26) cells and breast cancer 4T1 cells. The highland Barley tender leaf Water-extraction Polysaccharide is prepared by the team of the inventor, and the preparation method is specified In an article (Chemical Characterisation and In Vitro Anti-Cancer Activities of a Hot Water Soluble Polysaccharide from Hulless barless Grass (https:// doi. Org/10.3390/foods 11050677), wherein the monosaccharide composition of the highland Barley tender leaf Water-extraction Polysaccharide is 35.1% of galactose, 25.6% of arabinose, 5.5% of glucose and 5.3% of xylose.
After recovery of colorectal cancer (HT 29, caco-2, CT26) cells and breast cancer 4T1 cells, they were cultured in DMEM medium containing 10% FBS and placed in an incubator at 37 ℃ under 5% CO2 conditions. When the cells were growing over the bottom of the cell flask, the cells were digested with pancreatin, centrifuged, the supernatant was discarded, 2mL of culture medium was added to the pellet, blown, counted using a hemocytometer, and the cells were diluted in proportion, seeded into a 96-well plate (100 μ L cell suspension per well, i.e., 5000 cells), and cultured in an incubator for 24 hours. Adding the highland barley young leaf water-extraction polysaccharide with the same concentration into a 96-well plate respectively, and incubating for 48h at 37 ℃. Carefully removing the supernatant, adding 100 mu L of MTT (methyl thiazolyl tetrazolium) with the concentration of 0.5mg/mL into each hole, continuously incubating in an incubator at 37 ℃ for 4h, carefully removing the MTT solution, adding 100 mu L of DMSO into each hole, continuously incubating in the incubator at 37 ℃ for 10min, fully oscillating on an oscillator until the blue-violet crystals are completely dissolved, and detecting the OD (optical density) value of each hole at 570nm by using a full-automatic enzyme standard instrument. Cell viability was calculated as follows. Cell viability (%) = experimental OD value/control OD value × 100%. The results are shown in fig. 14-17, after the primary screening of four tumor cells, the highland barley young leaf water extract polysaccharide sample only has a remarkable inhibition effect on the proliferation of mouse colorectal cancer cells HT29, which is equivalent to the effect of enzyme extraction of polysaccharide, and the inhibition activity on other three tumor cells is remarkably reduced compared with enzyme extraction of polysaccharide. The inhibition effect of the enzyme-extracted polysaccharide on Caco-2 cells is remarkably improved, the inhibition rate can reach more than 70% under the concentration of 10mg/mL, and the inhibition rate of the water-extracted polysaccharide on Caco-2 cells under the same concentration is only about 40%.
During the process of preparing the enzyme-extracted polysaccharide, high-temperature resistant alpha-amylase, papain and glucoamylase are used for enzymolysis, which inevitably causes that a part of the water-extracted polysaccharide is dissolved in enzymolysis liquid, and xylan (consisting of xylose) and cellulose (consisting of glucose) in the tender highland barley leaves are continuously hydrolyzed by adding xylanase and cellulase, so that the types of monosaccharide components of the finally obtained enzyme-extracted polysaccharide are the same as those of the water-extracted polysaccharide, but the ratio of the xylose to the glucose is increased. Compared with a water extraction method, the enzyme extraction method can obviously increase the yield of the polysaccharide from 2.3% to 7.68%. The monosaccharide composition result of the water-extracted polysaccharide shows that galactose forms a main chain skeleton, and polysaccharide structures of other monosaccharide combinations are arranged on branched chains; the monosaccharide composition structure of the enzyme-extracted polysaccharide shows that arabinose and xylose form a main chain skeleton, and polysaccharide structures of other monosaccharide combinations are arranged on branched chains. The polysaccharide structures obtained by the two extraction methods are not the same. And the inhibition effect of the enzyme-extracted polysaccharide on four tumor cells is obviously better than that of the water-extracted polysaccharide. The invention provides a polysaccharide extracted by highland barley tender leaf enzyme, which has excellent biological activity for inhibiting tumor cell proliferation and has wide application prospect in the aspect of developing safe anticancer drugs without toxic and side effects.
The foregoing description has described specific embodiments of the present invention. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (10)
1. A preparation method of active ingredients for extracting polysaccharide from highland barley tender leaf enzyme is characterized by comprising the following steps:
s1, carrying out vacuum freeze drying on highland barley leaves, powdering and collecting highland barley tender leaf powder;
s2, carrying out degreasing, enzyme extraction, alcohol precipitation and drying on the highland barley tender leaf powder to obtain highland barley enzyme-extracted crude polysaccharide;
and S3, separating the highland barley enzyme-extracted crude polysaccharide by using a cellulose column, and purifying by using a sephadex column to obtain the highland barley tender leaf enzyme-extracted polysaccharide active component.
2. The preparation method according to claim 1, wherein in the step S2, petroleum ether is used as a solvent for degreasing, and the ratio of highland barley tender leaf powder to petroleum ether is 1g:40-50mL; defatting by soaking at 55-60 deg.C for 90-120min, extracting at 65-70 deg.C for 120-150min, and recovering at 75-80 deg.C for 15-20min; after degreasing treatment, carrying out solid-liquid separation, and carrying out subsequent extraction on the obtained degreased substance.
3. The preparation method according to claim 1, wherein in step S2, the enzymatic extraction process comprises adding water into the highland barley tender leaf powder and water in a weight ratio of (1); adjusting the temperature of the water bath to 60-80 deg.C, adding papain of 5-10 μ L/g semen Avenae Nudae powder, and water bath for 1-2 hr; inactivating enzyme in 100 deg.C water bath for 10-20min, cooling, adding 40-50 μ L/g saccharifying enzyme of semen Avenae Nudae powder, and water bath at 60-80 deg.C for 1-2 hr; inactivating enzyme in 100 deg.C water bath for 10-20min, cooling, and performing 60-90 deg.C water bath for 2-4 hr to obtain enzymatic hydrolysate.
4. The preparation method according to claim 1, characterized in that, in step S2, the obtained enzymatic hydrolysate is adjusted to pH 5-6 with NaOH solution, 600-1000U/g of xylanase of highland barley tender leaf powder and 200-600U/g of cellulase of highland barley tender leaf powder are added, heat preservation is carried out at 40-60 ℃ for 2-3h, then enzyme deactivation is carried out in 100 ℃ water bath for 10-20min, the supernatant obtained by enzymatic hydrolysis is concentrated to 50mL-100mL in 60-80 ℃ water bath, ethanol with volume 3-5 times is added after cooling, standing, centrifugation and precipitation collection are carried out; re-dissolving with water, and vacuum freeze drying to obtain highland barley tender leaf enzyme-extracted crude polysaccharide.
5. The method according to claim 1 or 4, wherein the vacuum freeze-drying is carried out under conditions of-50 to-60 ℃ for 4 to 6 hours; 10 to 15 hours at the temperature of between 35 ℃ below zero and 40 ℃ below zero; 10 to 15 hours at the temperature of minus 10 to minus 20 ℃;20-25 ℃ for 24-48 hours.
6. The method according to claim 1, wherein in step S3, the cellulose column is DEAE 963332 cellulose ion exchange column; the sephadex column is a Sephacryl-400 sephadex column.
7. The process according to claim 1 or 5, wherein in step S3, the eluting solution for separation is distilled water, the eluting rate is 0.5 to 0.7mL/min, and the eluting time is 500 to 1000min; the eluent for purification is distilled water, the elution rate is 0.5-0.7mL/min, and the elution time is 500-1000min.
8. The active component of the enzyme-extracted polysaccharide of the highland barley young leaves, which is prepared according to the method of claim 1, has the molecular weight of 20000-30000Da and the polydispersity coefficient of 1.14.
9. The active component of highland barley tender leaf enzyme-extracted polysaccharide as claimed in claim 8, wherein the highland barley tender leaf enzyme-extracted polysaccharide consists of arabinose, galactose, xylose and glucose, and the content ratio of arabinose, xylose, glucose and galactose is about 10:11:1:4.
10. the use of the active component of highland barley young leaf enzyme polysaccharide according to claim 8, which comprises the preparation of the active drug for inhibiting the proliferation of breast cancer and colorectal cancer cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211475607.6A CN115725004B (en) | 2022-11-23 | 2022-11-23 | Preparation method and application of highland barley tender leaf enzyme extracted polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211475607.6A CN115725004B (en) | 2022-11-23 | 2022-11-23 | Preparation method and application of highland barley tender leaf enzyme extracted polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115725004A true CN115725004A (en) | 2023-03-03 |
| CN115725004B CN115725004B (en) | 2023-10-31 |
Family
ID=85297662
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211475607.6A Active CN115725004B (en) | 2022-11-23 | 2022-11-23 | Preparation method and application of highland barley tender leaf enzyme extracted polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115725004B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5614242A (en) * | 1995-09-27 | 1997-03-25 | Barkley Seed, Inc. | Food ingredients derived from viscous barley grain and the process of making |
| CN106117389A (en) * | 2016-08-23 | 2016-11-16 | 上海交通大学 | Extract from Semen avenae nudae grain and the method for purification beta glucan |
| CN111378055A (en) * | 2020-03-26 | 2020-07-07 | 南昌大学 | Method for continuously extracting and preparing non-starch polysaccharide from highland barley |
| CN114874344A (en) * | 2022-04-01 | 2022-08-09 | 上海交通大学 | Preparation method and application of alkali-extracted polysaccharide of highland barley tender leaves |
-
2022
- 2022-11-23 CN CN202211475607.6A patent/CN115725004B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5614242A (en) * | 1995-09-27 | 1997-03-25 | Barkley Seed, Inc. | Food ingredients derived from viscous barley grain and the process of making |
| CN106117389A (en) * | 2016-08-23 | 2016-11-16 | 上海交通大学 | Extract from Semen avenae nudae grain and the method for purification beta glucan |
| CN111378055A (en) * | 2020-03-26 | 2020-07-07 | 南昌大学 | Method for continuously extracting and preparing non-starch polysaccharide from highland barley |
| CN114874344A (en) * | 2022-04-01 | 2022-08-09 | 上海交通大学 | Preparation method and application of alkali-extracted polysaccharide of highland barley tender leaves |
Non-Patent Citations (2)
| Title |
|---|
| SHAN GAO等: ""Preparation, Physicochemical Properties, and Antioxidant Capacity of Edible Agar Films Containing Hull-Less Barley Grass Polysaccharide Extracts"", 《STARCH - STÄRKE》, vol. 75, pages 1 - 8 * |
| YIJUAN XU等: ""Chemical Characterization and In Vitro Anti-Cancer Activities of a Hot Water Soluble Polysaccharide from Hulless Barley Grass"", 《FOODS》, vol. 11, pages 1 - 11 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115725004B (en) | 2023-10-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114874344B (en) | Preparation method and application of alkali-extracted polysaccharides of highland barley tender leaves | |
| Ji et al. | A mini-review of isolation, chemical properties and bioactivities of polysaccharides from buckwheat (Fagopyrum Mill) | |
| CN106117389B (en) | Method for extracting and purifying beta-glucan from highland barley grains | |
| CN106317260B (en) | Method for extracting and purifying arabinoxylan from highland barley grains | |
| CN111920040B (en) | Peony fermentation product and preparation method thereof | |
| CN109527602B (en) | Method for improving content of soluble dietary fiber in highland barley young leaf powder | |
| Jiao et al. | Structural characterization and anti-hypoxia activities of polysaccharides from the sporocarp, fermentation broth and cultured mycelium of Agaricus bitorquis (Quél.) Sacc. Chaidam in mice | |
| CN111328904A (en) | Preparation method of functional jasmine tea beverage | |
| KR20130034173A (en) | Ginseng process method for increasing ginsenoside quantity and processed goods thereof | |
| CN114009777A (en) | Glucoraphanin and moringa oleifera extract composition buccal tablet and application thereof | |
| JP2024505861A (en) | Oligogalacturonic acid polysaccharide, complex and its preparation method and use thereof | |
| CN102161710A (en) | Method for preparing tremellan with low molecular weight and novel medicinal application thereof | |
| CN115725004B (en) | Preparation method and application of highland barley tender leaf enzyme extracted polysaccharide | |
| KR101218275B1 (en) | Methods for preparing fermented ginseng or fermented red ginseng using yeast | |
| Song et al. | Effects of solution behavior on polysaccharide structure and inhibitory of α-glucosidase activity from Cordyceps militaris | |
| CN110973632A (en) | Dietary composition for dietary intervention in cancer and preparation method thereof | |
| CN110679934A (en) | Lutein-containing enzyme capable of clearing liver and improving eyesight and preparation method thereof | |
| CN113999325B (en) | Rice bran fermented polysaccharide, preparation and application | |
| CN102268099B (en) | Rangoon creeper fruit extract and extraction method thereof | |
| CN113995128A (en) | Preparation method of citrus lactic acid bacteria capsule with high flavone utilization degree | |
| CN111518860B (en) | Preparation method of cowberry fruit extract | |
| CN114249846A (en) | Acidic water shield polysaccharide and separation and purification method and application thereof | |
| CN110317844B (en) | Flaxseed gum oligosaccharide with anti-tumor activity and preparation method and application thereof | |
| CN113068842A (en) | Preparation method of pear dietary fiber | |
| CN112314954A (en) | Preparation method of corn dietary fiber |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |