CN118852479A - Lycium barbarum polysaccharide and extraction method and application thereof - Google Patents
Lycium barbarum polysaccharide and extraction method and application thereof Download PDFInfo
- Publication number
- CN118852479A CN118852479A CN202410863239.5A CN202410863239A CN118852479A CN 118852479 A CN118852479 A CN 118852479A CN 202410863239 A CN202410863239 A CN 202410863239A CN 118852479 A CN118852479 A CN 118852479A
- Authority
- CN
- China
- Prior art keywords
- lbp
- polysaccharide
- extraction
- lycium barbarum
- drying
- 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.)
- Pending
Links
- 238000000605 extraction Methods 0.000 title claims abstract description 18
- 239000008518 lycium barbarum polysaccharide Substances 0.000 title claims abstract description 11
- AKWUNZFZIXEOPV-UHFFFAOYSA-N 2-[4-[[3-[7-chloro-1-(oxan-4-ylmethyl)indol-3-yl]-1,2,4-oxadiazol-5-yl]methyl]piperazin-1-yl]acetamide Chemical compound C1CN(CC(=O)N)CCN1CC1=NC(C=2C3=CC=CC(Cl)=C3N(CC3CCOCC3)C=2)=NO1 AKWUNZFZIXEOPV-UHFFFAOYSA-N 0.000 claims abstract description 49
- 101100280477 Caenorhabditis elegans lbp-1 gene Proteins 0.000 claims abstract description 48
- 150000004676 glycans Chemical class 0.000 claims abstract description 36
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 36
- 239000005017 polysaccharide Substances 0.000 claims abstract description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 244000241838 Lycium barbarum Species 0.000 claims abstract description 27
- 235000015459 Lycium barbarum Nutrition 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 27
- KBOPZPXVLCULAV-UHFFFAOYSA-N mesalamine Chemical compound NC1=CC=C(O)C(C(O)=O)=C1 KBOPZPXVLCULAV-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229960004963 mesalazine Drugs 0.000 claims abstract description 19
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 229920002678 cellulose Polymers 0.000 claims abstract description 9
- 239000001913 cellulose Substances 0.000 claims abstract description 9
- 239000011347 resin Substances 0.000 claims abstract description 9
- 229920005989 resin Polymers 0.000 claims abstract description 9
- 238000000926 separation method Methods 0.000 claims abstract description 6
- 238000001556 precipitation Methods 0.000 claims abstract description 5
- 238000003809 water extraction Methods 0.000 claims abstract description 5
- 101100280481 Caenorhabditis elegans lbp-2 gene Proteins 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 235000015468 Lycium chinense Nutrition 0.000 claims description 19
- 238000010828 elution Methods 0.000 claims description 18
- 238000004108 freeze drying Methods 0.000 claims description 13
- 230000002829 reductive effect Effects 0.000 claims description 13
- 239000003480 eluent Substances 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 11
- 238000005303 weighing Methods 0.000 claims description 11
- 238000002835 absorbance Methods 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- 101100446025 Caenorhabditis elegans lbp-3 gene Proteins 0.000 claims description 6
- 101100446027 Caenorhabditis elegans lbp-4 gene Proteins 0.000 claims description 6
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 claims description 5
- 238000002390 rotary evaporation Methods 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000049 pigment Substances 0.000 claims description 4
- 230000001376 precipitating effect Effects 0.000 claims description 4
- 238000010298 pulverizing process Methods 0.000 claims description 4
- 238000001179 sorption measurement Methods 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims 1
- 239000012527 feed solution Substances 0.000 claims 1
- 238000002386 leaching Methods 0.000 claims 1
- 239000008194 pharmaceutical composition Substances 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 32
- 230000000694 effects Effects 0.000 abstract description 26
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 16
- 210000002540 macrophage Anatomy 0.000 abstract description 12
- 150000002772 monosaccharides Chemical class 0.000 abstract description 12
- 238000000338 in vitro Methods 0.000 abstract description 8
- 239000000203 mixture Substances 0.000 abstract description 7
- 206010009900 Colitis ulcerative Diseases 0.000 abstract description 6
- 201000006704 Ulcerative Colitis Diseases 0.000 abstract description 6
- 238000001727 in vivo Methods 0.000 abstract description 5
- 208000037817 intestinal injury Diseases 0.000 abstract description 5
- 241000699666 Mus <mouse, genus> Species 0.000 abstract description 3
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 3
- 239000000284 extract Substances 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 229940126680 traditional chinese medicines Drugs 0.000 abstract description 2
- 235000017784 Mespilus germanica Nutrition 0.000 abstract 1
- 244000182216 Mimusops elengi Species 0.000 abstract 1
- 235000000560 Mimusops elengi Nutrition 0.000 abstract 1
- 235000007837 Vangueria infausta Nutrition 0.000 abstract 1
- 230000002195 synergetic effect Effects 0.000 abstract 1
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 42
- 210000004027 cell Anatomy 0.000 description 19
- 210000001072 colon Anatomy 0.000 description 19
- 239000002158 endotoxin Substances 0.000 description 18
- 229920006008 lipopolysaccharide Polymers 0.000 description 18
- 239000003642 reactive oxygen metabolite Substances 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 229920003045 dextran sodium sulfate Polymers 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 7
- 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 7
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 7
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 238000005227 gel permeation chromatography Methods 0.000 description 7
- 102000003814 Interleukin-10 Human genes 0.000 description 6
- 108090000174 Interleukin-10 Proteins 0.000 description 6
- 102000004889 Interleukin-6 Human genes 0.000 description 6
- 108090001005 Interleukin-6 Proteins 0.000 description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 229940076144 interleukin-10 Drugs 0.000 description 6
- 229940100601 interleukin-6 Drugs 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 206010061218 Inflammation Diseases 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 230000004054 inflammatory process Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000002211 ultraviolet spectrum Methods 0.000 description 5
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 4
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 4
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 4
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 4
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 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
- 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
- 108010087230 Sincalide Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- ARQRPTNYUOLOGH-UHFFFAOYSA-N chcl3 chloroform Chemical compound ClC(Cl)Cl.ClC(Cl)Cl ARQRPTNYUOLOGH-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229930182830 galactose Natural products 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000011229 interlayer Substances 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000000902 placebo Substances 0.000 description 3
- 229940068196 placebo Drugs 0.000 description 3
- 230000000770 proinflammatory effect Effects 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 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 2
- 208000004232 Enteritis Diseases 0.000 description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 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 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 206010009887 colitis Diseases 0.000 description 2
- 230000000112 colonic effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000005238 degreasing Methods 0.000 description 2
- 238000001212 derivatisation Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- 206010057669 Colon injury Diseases 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000012671 Gastrointestinal haemorrhages Diseases 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 201000010927 Mucositis Diseases 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001675646 Panaceae Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 1
- 238000001994 activation Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 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 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- BLFLLBZGZJTVJG-UHFFFAOYSA-N benzocaine Chemical compound CCOC(=O)C1=CC=C(N)C=C1 BLFLLBZGZJTVJG-UHFFFAOYSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000013872 defecation Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 230000009266 disease activity Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 235000018927 edible plant Nutrition 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 238000005534 hematocrit Methods 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003903 intestinal lesions Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- YSRVJVDFHZYRPA-UHFFFAOYSA-N melem Chemical compound NC1=NC(N23)=NC(N)=NC2=NC(N)=NC3=N1 YSRVJVDFHZYRPA-UHFFFAOYSA-N 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 230000006996 mental state Effects 0.000 description 1
- 210000000713 mesentery Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000011477 surgical intervention Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 208000016261 weight loss Diseases 0.000 description 1
Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of extraction and medical application of effective components of traditional Chinese medicines, and particularly relates to a lycium barbarum polysaccharide and an extraction method and application thereof. The invention extracts the lycium barbarum polysaccharide (Lyciumbarbarumpolysaccharides, LBP) from the lycium barbarum by a water extraction and alcohol precipitation method, removes protein by a Sevag method, decolours by D101 macroporous resin and separates by a DEAE-52 cellulose column to obtain high-purity lycium barbarum polysaccharide LBP-1, and respectively determines the monosaccharide composition and the relative molecular mass of the LBP-1 by HPLC and GPC. Taking a mouse mononuclear macrophage RAW264.7 as a model, researching the in vitro anti-inflammatory activity of LBP-1; in vivo anti-inflammatory activity of LBP-1 was studied in ulcerative colitis mice as a model, and the effect on intestinal injury after administration of LBP-1 in combination with mesalazine (5-ASA) was examined. Experimental study shows that LBP-1 has good in-vitro and in-vivo anti-inflammatory activity, and can achieve the synergistic and toxicity-reducing effects by combined administration with 5-ASA, thereby laying a foundation for extraction and separation of polysaccharide in medlar and anti-inflammatory activity research, providing a new strategy for further administration treatment of colonitis, and having great application prospect.
Description
Technical Field
The invention belongs to the technical field of extraction and medical application of effective components of traditional Chinese medicines, and particularly relates to a lycium barbarum polysaccharide and an extraction method and application thereof.
Background
Ulcerative colitis (Ulcerative Colitis, UC) is a chronic, non-specific inflammatory bowel disease characterized by disturbances of the immune system and inflammation of the gastrointestinal tract, the clinical manifestations of which include mainly abdominal pain, diarrhea, hematochezia, weight loss, etc. Currently, UC is still difficult to cure, and therapeutic strategies are mainly targeting colonic mucosa and reducing systemic inflammation to regulate the aggravated host immune response. In short, UC is a chronic or even lifelong disease, and generally requires long-term uninterrupted medication for patients with UC, and even surgical intervention in severe cases. The clinical treatment of the disease is mainly mesalazine (5-ASA), the medicine can effectively inhibit inflammatory reaction of intestinal walls, but the medicine has higher recurrence rate after being stopped, the acceptance of patients is lower, certain adverse reaction can be caused after the systemic distribution of the patients, and the long-term intake of the medicine can lead to the damage of immunity, so that the patients are easier to infect and suffer from diseases.
The Lycium barbarum polysaccharide (Lycium barbarum polysaccharides, LBP) is derived from the traditional Chinese medicinal and edible plant, namely Lycium barbarum L, is one of main bioactive components of the Lycium barbarum, and has wide biological characteristics, including anti-inflammatory, immunoregulatory, attenuation and intestinal flora regulation effects. In addition, co-administration is considered as an effective strategy for treating UC, while reducing adverse drug reactions, improving body immunity and increasing anti-inflammatory effects.
Disclosure of Invention
In view of the above, the invention discloses a matrimony vine polysaccharide and an extraction method and application thereof.
The invention extracts the wolfberry polysaccharide from the wolfberry by a water extraction and alcohol precipitation method, removes protein by a Sevag method, decolors by D101 macroporous resin and separates by a DEAE-52 cellulose column to obtain high-purity wolfberry polysaccharide LBP-1, and respectively determines the monosaccharide composition and the relative molecular mass of the LBP-1 by HPLC and GPC. Taking a mouse mononuclear macrophage RAW264.7 as a model, researching the in vitro anti-inflammatory activity of LBP-1; in vivo anti-inflammatory activity of LBP-1 was studied using ulcerative colitis mice as a model, and the effect on intestinal injury after administration of LBP-1 in combination with 5-ASA was examined.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the extraction method of the wolfberry polysaccharide specifically comprises the following steps:
(1) Pulverizing 500g of fructus Lycii, degreasing with 95% ethanol, drying, and weighing; according to the feed liquid ratio of 1: extracting with 100deg.C hot water for 2 hr, repeatedly extracting for 3 times, filtering with gauze, mixing filtrates, concentrating under reduced pressure, precipitating with 3 times of 95% ethanol, drying, and weighing to obtain LBP crude polysaccharide;
(2) Repeating the operation of removing protein with Sevag reagent (V Chloroform (chloroform) ：V n-butanol =4:1) for a plurality of times until the white interlayer disappears; collecting supernatant, concentrating, and freeze-drying to obtain deproteinized LBP; removing pigment from the protein-removed LBP solution by using D101 macroporous adsorption resin, then taking eluent, concentrating by rotary evaporation, and freeze-drying to obtain decolorized LBP;
(3) Weighing 100mg of decolorized LBP prepared in the step (2), dissolving in 10mL of distilled water, centrifuging at 5000r/min for 10min after full dissolution, and collecting supernatant; subjecting to DEAE-52 cellulose column (phi 2.6X150 cm) chromatography, sequentially eluting with distilled water, 0.1moL/L NaCl, 0.2moL/L NaCl, and 0.5moL/L NaCl to obtain LBP-1, LBP-2, LBP-3, and LBP-4, respectively, at an elution flow rate of 2mL/min, and collecting 10mL per tube; collecting eluent, detecting a separation tube by adopting a phenol-sulfuric acid method, and drawing an elution curve by taking the number of the tubes as an abscissa and the absorbance value as an ordinate; and according to the elution curve, combining the eluents of the same elution peak, concentrating under reduced pressure, and freeze-drying to obtain four refined LBPs with different molecular weights.
The second object of the invention is to provide a wolfberry polysaccharide extracted by the method, wherein the wolfberry polysaccharide is extracted from wolfberry by a water extraction and alcohol precipitation method, deproteinized by a Sevag method, decolorized by D101 macroporous resin and separated by a DEAE-52 cellulose column to obtain high-purity wolfberry polysaccharide.
A third object of the present invention is to provide the use of the matrimony vine polysaccharide extracted by the method as described above in pharmaceutical preparations.
Further, the wolfberry polysaccharide and mesalamine 5-ASA are combined to prepare a pharmaceutical preparation for treating colonitis.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses an extraction method of wolfberry polysaccharide, which shows good anti-inflammatory activity in vitro experiments, animal experiments show that the wolfberry polysaccharide can effectively treat colonitis, and the drug effect of a positive drug can be obviously improved by combined administration with 5-ASA, and toxic and side effects brought by the 5-ASA in treatment can be reduced, so that the wolfberry polysaccharide has great market application and popularization prospects.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the ultraviolet spectrum (A) and DEAE-52 elution profile (B) of LBP.
FIG. 2 is a GPC chart of LBP-1 and LBP-2.
FIG. 3 is a UV spectrum of mixed monosaccharide standards, LBP-1, LBP-2; man; rib; rha; dex; gal; ara.
FIG. 4 is the in vitro anti-inflammatory activity of LBP-1 and LBP-2. Effect of LBP on RAW 264.7 cell viability (a); effect of LBP on RAW 264.7 released NO content (B); effect of LBP on RAW 264.7 released NO content (C). ## P <0.01 compared to the placebo group; p <0.05, P <0.01 compared to the LPS model group.
FIG. 5 is the effect of LBP on intestinal injury in UC mice. Effect of LBP on body weight change (a); DAI score (B); colon length (C); visual scoring of colon tissue (D); IBD mice colon physical map (E). ## P <0.01 compared to the placebo group; p <0.01 compared to model group.
FIG. 6 is the effect of LBP on the expression levels of NO (A), ROS (B), IL-10 (C), IL-6 (D) and TNF-alpha (E) in colon tissue of mice. ## P <0.01 compared to the placebo group; p <0.01 compared to model group.
Detailed Description
The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The word "embodiment" as used herein does not necessarily mean that any embodiment described as "exemplary" is preferred or advantageous over other embodiments. Performance index testing in the examples of the present application, unless otherwise specified, was performed using conventional testing methods in the art. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; other test methods and techniques not specifically mentioned in the present application are those commonly used by those skilled in the art.
Numerous specific details are set forth in the following examples in order to provide a better understanding of the present application. It will be understood by those skilled in the art that the present application may be practiced without some of these specific details. In the examples, some methods, means, instruments, devices, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present application.
On the premise of no conflict, the technical features disclosed by the embodiment of the application can be combined at will, and the obtained technical scheme belongs to the disclosure of the embodiment of the application.
The present invention will be further specifically illustrated by the following examples, which are not to be construed as limiting the invention, but rather as falling within the scope of the present invention, for some non-essential modifications and adaptations of the invention that are apparent to those skilled in the art based on the foregoing disclosure.
Example 1
The extraction method of the wolfberry polysaccharide with the colonitis curative effect comprises the following steps:
(1) Pulverizing 500g of fructus Lycii, degreasing with 95% ethanol, drying, and weighing; according to the feed liquid ratio of 1: extracting with 100deg.C hot water for 2 hr, repeatedly extracting for 3 times, filtering with gauze, mixing filtrates, concentrating under reduced pressure, precipitating with 3 times of 95% ethanol, drying, and weighing to obtain LBP crude polysaccharide;
(2) Repeating the operation of removing protein with Sevag reagent (V Chloroform (chloroform) ：V n-butanol =4:1) for a plurality of times until the white interlayer disappears; collecting supernatant, concentrating, and freeze-drying to obtain deproteinized LBP; removing pigment from the protein-removed LBP solution by using D101 macroporous adsorption resin, then taking eluent, concentrating by rotary evaporation, and freeze-drying to obtain decolorized LBP;
(3) Weighing 100mg of decolorized LBP prepared in the step (2), dissolving in 10mL of distilled water, centrifuging at 5000r/min for 10min after full dissolution, and collecting supernatant; subjecting to DEAE-52 cellulose column (phi 2.6X150 cm) chromatography, sequentially eluting with distilled water, 0.1moL/L NaCl, 0.2moL/L NaCl, and 0.5moL/L NaCl to obtain LBP-1, LBP-2, LBP-3, and LBP-4, respectively, at an elution flow rate of 2mL/min, and collecting 10mL per tube; collecting eluent, detecting a separation tube by adopting a phenol-sulfuric acid method, and drawing an elution curve by taking the number of the tubes as an abscissa and the absorbance value as an ordinate; and according to the elution curve, combining the eluents of the same elution peak, concentrating under reduced pressure, and freeze-drying to obtain four refined LBPs with different molecular weights.
In order to further demonstrate the beneficial effects of the present invention for a better understanding of the present invention, the technical features disclosed herein are further elucidated by the following experiments, but are not to be construed as limiting the present invention. Other modifications of the invention which do not involve the inventive work, as would occur to those skilled in the art in light of the foregoing teachings, are also considered to be within the scope of the invention.
In addition, the invention examines the in-vitro and in-vivo anti-inflammatory activity of LBP-1, and applies the combined administration of LBP-1 and 5-ASA to the treatment of colonitis.
1 Materials and instruments
1.1 Cells and animals
The RAW 264.7 cell strain is purchased from a national experimental cell resource sharing platform; male ICR mice (20.+ -.2 g,6-8 weeks) were purchased from Peking Vitre Liwa laboratory animal technologies Co.
1.2 Material reagent
Ningxia wolfberry (purchased from Beijing Yunhai Cryometric science and technology Co., ltd.); phenol (Shanghai Michlin Biochemical technologies Co., ltd.); d101 macroporous adsorbent resin, BCA protein detection kit, lipopolysaccharide (LPS, beijing sonebao limited); DEAE cellulose DE-52 (Beijing Ruida Henghui technology development Co., ltd.); monosaccharide standard (Sigma-Aldrich company); DMEM high sugar medium (Thermo FISHER SCIENTIFIC, usa); 0.25% of a mixed diabody of pancreatin, foetal calf serum and penicillin streptomycin (Gibco company, USA); CCK-8, IL-10ELISA kit, IL-6ELISA kit, TNF- α ELISA kit (Zhongkeyou Biotechnology Co., ltd.); NO detection kit, ROS detection kit (Shanghai bi yun biotechnology limited); dextran sulfate sodium salt (DSS, melem biotechnology limited); 5-aminosalicylic acid (5-ASA, shanghai Meilin Biochemical technologies Co., ltd.); the other reagents were all analytically pure.
1.3 Instruments
Master-D laboratory ultra-pure water instrument (Shanghai and Thai instruments Co., ltd.); n-1300 rotary evaporator (Shanghai Ailang instruments Co., ltd.); HC-3618R high-speed cryocentrifuge (Anhui middle department of well-science instruments Co., ltd.); cary100 type variable temperature ultraviolet spectrophotometer (agilent technologies limited); spark multifunctional enzyme labelling instrument (Austria Diken Co.); ultimate 3000 high performance liquid chromatograph (dyn instruments, usa); viscotek GPC/SEC (malvern panaceae).
2. Experimental procedure
2.1 Extraction, separation and purification of Lycium Barbarum Polysaccharide (LBP)
Pulverizing 500g of fructus Lycii, defatting with 95% ethanol, drying, and weighing. According to the feed liquid ratio of 1:10 extracting with 100deg.C hot water for 2 hr, repeating the extraction for 3 times, filtering with gauze, mixing filtrates, concentrating under reduced pressure, precipitating with 3 times of 95% ethanol, drying, and weighing to obtain LBP crude product. The LBP crude polysaccharide solution was deproteinized by repeated multiple runs with Sevag reagent (V Chloroform (chloroform) ：V n-butanol = 4:1) until the white interlayer disappeared. Collecting supernatant, concentrating, and freeze drying to obtain deproteinized LBP. And removing pigment from the protein-removed LBP solution by using D101 macroporous adsorption resin, taking the eluent, concentrating by rotary evaporation, and freeze-drying to obtain the decolorized LBP.
100Mg of decolorized LBP is weighed and dissolved in 10mL of distilled water, and after the decolorized LBP is fully dissolved, the solution is centrifuged at 5000r/min for 10min, and the supernatant is obtained. Then, the mixture was subjected to DEAE-52 cellulose column (. Phi.2.6X150 cm) chromatography, and eluted sequentially with distilled water (giving LBP-1), 0.1moL/L (giving LBP-2), 0.2moL/L (giving LBP-3), and 0.5moL/L NaCl (giving LBP-4), at a flow rate of 2mL/min, and 10mL was collected per tube. Collecting eluent, detecting the isolation tube by using a phenol-sulfuric acid method, and drawing an elution curve by taking the number of the tubes as an abscissa and the absorbance value as an ordinate. And according to the elution curve, combining the eluents of the same elution peak, concentrating under reduced pressure, and freeze-drying to obtain four refined LBPs with different molecular weights.
2.2 Analysis of total sugar content, protein content, UV Spectrum in LBP
The polysaccharide content is determined by phenol-sulfuric acid method, glucose is used as standard, absorbance is determined at 490nm wavelength by ultraviolet, and standard curve is drawn.
Taking bovine serum albumin as a standard, measuring the protein content by adopting a microporous enzyme-labeled method according to the operation of the BCA protein concentration measuring kit, and drawing a protein standard curve.
LBP solution (1 mg/mL) was scanned with an ultraviolet spectrophotometer in the range of 200-400 nm and an ultraviolet spectrum was drawn.
2.3GPC determination of relative molecular weight of LBP
The polysaccharide LBP-1 and LBP-2 separated in the step 2.1 are respectively prepared into solutions with the concentration of 10mg/mL, a detection instrument is scientific multi-detector Gel Permeation Chromatography (GPC), the mobile phase is 0.1moL/LNaNO 3 solution, the flow rate is 0.7mL/min, and the detectors are a differential detector and a light scattering detector.
2.4 Determination of monosaccharide composition in LBP
2.4.1LBP hydrolysis LBP-1 and LBP-2 were weighed separately in 25mL round bottom flasks, added with condenser and dissolved in 2mol/LTFA 4mL, hydrolyzed at 120℃for 4h, then removed and left to cool, TFA was removed by rotary evaporation, and residual TFA was removed by co-distillation with methanol three times (4 mL methanol each time) and the hydrolyzed LBP was made 10mg/mL ready for use.
2.4.2 Preparation of control the standard substances of xylose (Xylose), arabinose (Arabinose), glucose (Dextrose), mannose (Mannose), rhamnose (Rhamnose), galactose (Galactose) and ribose (Ribose) were prepared as a monosaccharide control solution and a mixed monosaccharide control solution of 2mg/mL, respectively, for use.
2.4.3 Sample and control were taken from the PMP pre-column derivatization of the samples and control 200. Mu.L each, placed in 2mLEP tubes, 200. Mu.L of 0.3mol/NaOH solution and 500. Mu.L of 0.5mol/L PMP methanol solution were added, mixed well, derivatized in a water bath at 70℃for 2 hours, cooled to room temperature, added with 200. Mu.L of 0.3mol/L HCl solution, mixed well, added with CHCl 3 1.0.0 mL, vortexed 30s, spun at 800 r/min for 5min, the lower solution was discarded, repeated 3 times, the excess PMP solution was removed, the supernatant was taken to a constant volume of 2mL, and after passing through a 0.22 μm microporous filter membrane, fed to HPLC for analysis.
2.4.4HPLC determination of monosaccharide composition chromatography column: venusil XBP C18 (L) (250 mm. Times.4.6 mm,5 μm); mobile phase: 0.05moL/L phosphate buffer (NaH 2PO4、Na2HPO4 mixed solution, pH 6.8) -acetonitrile (83:17), column temperature 30 ℃, detection wavelength 254nm, sample injection volume 20. Mu.L, flow rate 1mg/mL.
2.5 In vitro anti-inflammatory Activity of LBP
The invention takes a mouse mononuclear macrophage RAW264.7 as a model, uses Lipopolysaccharide (LPS) to induce and model, and researches the in vitro anti-inflammatory activity of LBP. RAW264.7 cell-containing materialFetal bovine serum (FPS)Double-antibody DMEM complete culture medium at 37 ℃ incubatorCulturing, and passaging every 2 d.
2.5.1 Measurement of cell proliferation Activity RAW 264.7 cells in logarithmic growth phase were inoculated at a density of 5X 10 5 cells/mL into 96-well plates at 200. Mu.L/well and cultured for 24 hours, and the culture broth was discarded, and divided into a blank control group and a cell-free control group (fresh DMEM complete medium), a positive control group (1. Mu.g/mL of LPS), and an experimental group (LBP-1 or LBP-2 at a mass concentration of 25, 250, 500. Mu.g/mL). After 24h incubation, 10 μl of CCK-8 reagent was added to each well, incubated in an incubator for 4h, and OD was measured at 450nm using a multifunctional microplate reader. Cell viability was calculated as follows:
2.5.2 cell Nitric Oxide (NO) Release assay cells were seeded at a density of 5X 10 5/mL in 6-well plates and incubated for 24h, after which the culture was discarded and divided into model control groups (1. Mu.g/mL of LPS), experimental groups (25, 250, 500. Mu.g/mL of LBP-1 or LBP-2 co-treated with 1. Mu.g/mL of LPS). After 24h incubation, the supernatant was collected as a sample, the standard in the NO detection kit was diluted in gradient with DMEM high sugar medium, the standard and the sample were added in 96 well plates at 50 μl/well, GRIESS REAGENT i and GRIESS REAGENT ii reagents in the kit were added sequentially in each well at 50 μl/well, absorbance was measured at 540nm using a multifunctional microplate reader, NO standard curves were drawn, and the concentration of NO in the samples was calculated from the standard curves.
2.5.3 Reactive Oxygen Species (ROS) release assay cells were seeded at a density of 5X 10 5/mL in 96-well plates and cultured for 24h, after which the culture was discarded and divided into model control (1. Mu.g/mL of LPS), experimental (25, 250, 500. Mu.g/mL of LBP-1 or LBP-2 co-processed with 1. Mu.g/mL of LPS). After culturing for 24 hours, the culture medium was discarded and added according to 1:1000 with DMEM high sugar medium diluted fluorescent probe DCFH-DA (to a final concentration of 10 u g/mL) per well 200 u L,37 degrees C cell culture box incubation 20min. Cells were washed three times with PBS, covered again with 200. Mu.L of medium per well, and assayed by setting the excitation wavelength to 488nm and the emission wavelength to 525nm using a fluorescent microplate reader.
2.6 Effect of LBP on intestinal lesions in ulcerative colitis mice
As shown by in vitro cell experiments, the anti-inflammatory activity of LBP-1 is higher than that of LBP-2, so that in vivo animal experiments are carried out by using LBP-1 to examine the influence of LBP-1 on intestinal damage of mice with enteritis.
2.6.1 Grouping, modeling and dosing ICR mice were fed adaptively for one week, randomly divided into a normal control group, a model group, a positive control group [ 5-aminosalicylic acid (5-ASA), 150mg/kg ], a polysaccharide solution group (LBP-1, 300 mg/kg), a combination dosing group (5-ASA, 150mg/kg and LBP-1, 300 mg/kg), 8 animals per group. The mice in each group are fasted and not forbidden for 12 hours before the experiment, the drinking water of the mice in the other groups except the normal group is changed into 3.5% dextran sodium sulfate salt (DSS) water solution, the free drinking is carried out for 5 days, the ulcerative colitis model is induced and established, the normal water is changed into the normal water for 3 days, the 5% DSS is changed into the 5% DSS water for 6 days, and the materials are obtained on the 15 th day and relevant index detection is carried out.
2.6.2 General physical sign observation the body mass of each group of mice was recorded at regular intervals every day, the mental, physical sign and defecation conditions of the mice were observed at regular intervals 2 times a day, and the mental state, stool shape, hair compliance and smoothness, glossiness, anus Zhou Bingbian and fecal occult blood conditions of the mice were observed and recorded. Disease Activity Index (DAI) scoring criteria are shown in table 1, dai= (body mass decline score + stool trait score + hematocrit score)/3.
TABLE 1DAI scoring criteria
2.6.3 Macroscopic observation of colon tissues and pathological change evaluation experiment, killing each group of mice by a cervical dislocation method on 15 th day, rapidly cutting off the whole colon, and measuring the colon length of each group of mice by a ruler; the colon was longitudinally sectioned along the mesentery, pre-chilled saline was washed several times, the filter paper was blotted to dry the water, and colonic mucosa inflammation and ulcer production were visually observed and scored with reference to the criteria listed in table 2.
TABLE 2 macroscopic scoring criteria for colon tissue
2.6.4 Measurement of cytokine expression level in tissue cells A portion of colon tissue was cut off, minced on ice to homogenate, centrifuged at 6000r/min for 10min, the supernatant was taken out, sub-packaged and stored in an ultra-low temperature refrigerator at-80℃for further use, and the levels of Nitric Oxide (NO), reactive Oxygen Species (ROS), interleukin 10 (IL-10), interleukin 6 (IL-6) and tumor necrosis factor (TNF- α) in the colon tissue were examined according to the specification.
3 Analysis of results
3.1LBP chemical composition and ultraviolet spectrum analysis with glucose mass concentration (mg/mL) as the abscissa (x), each mass concentration glucose at 490nm absorbance value as the ordinate (y), fitted regression equation is y= 51.808x-0.0038, R 2 = 0.9991, shows good linear relationship, linear range 0 ~ 0.1mg/mL. The absorbance of the sample at 490nm was taken into the regression equation to calculate the total sugar content, and the total sugar content of the polysaccharide obtained at the different extraction stages is shown in Table 3.
TABLE 3 Total sugar content and yield of LBP obtained at different extraction stages
The concentration of bovine serum albumin is taken as an abscissa (x), the absorbance of each concentration of bovine serum albumin at 562nm is taken as an ordinate (y), the fitted regression equation is y=1.1615x+0.0053, and R 2 =0.9982, which shows that the linearity is good, and the linearity range is 0-0.5 mg/mL. The absorbance of the sample at 562nm is brought into a regression equation to calculate the protein content in the deproteinized LBP to be 7.56+/-3.64%, which shows that the deproteinized LBP contains a small amount of protein component, and the Sevag method only removes most of free protein in the LBP, so that the protein component is presumed to be a polysaccharide protein complex.
As shown in FIG. 1-A, LBP showed no significant absorption peak at 280nm, indicating that LBP contained less protein, which is consistent with the results of protein content measurement.
3.2 Analysis of DEAE-52 cellulose column separation results elution was performed by a gradient of deionized water and NaCl, and the elution profile was plotted as shown in FIG. 1-B. As can be seen from FIG. 1-B, the decolorized LBP was separated to obtain 4 polysaccharide fractions, LBP-1, LBP-2, LBP-3 and LBP-4, with yields of 78.43%, 4.80%, 0.92% and 1.00%, respectively. Because of the relatively low yields of LBP-3 and LBP-4, the present invention only provides for further analysis after concentration and lyophilization of LBP-1 and LBP-2.
3.3 Analysis of relative molecular Mass measurements of LBP chromatograms of LBP-1 and LBP-2 were determined by GPC and shown in FIG. 2, mw and PDI of LBP-1 and LBP-2 are shown in Table 4.
As can be seen from Table 4, the Mw of LBP-1 and LBP-2 were 2726 and 5791, respectively. The Mw/Mn of both components was less than 1.77, indicating good homogeneity of both polysaccharide components, at levels of 7.41% and 0.62%, respectively.
TABLE 4 GPC measurement results of LBP-1 and LBP-2
3.4 Analysis of the composition of LBP monosaccharides As shown in FIG. 3, by comparing the liquid phase pattern of LBP with the liquid phase pattern after derivatization of the mixed monosaccharide standard, it is known from the retention time and peak area calculation that LBP-1 is mainly composed of 4 monosaccharides of mannose, ribose, glucose, and arabinose, and the molar ratio is 4.73:3.01:4.20:1.16, wherein the mannose molar ratio is up to 36.11%; LBP-2 consists of 5 monosaccharides mannose, ribose, glucose, galactose, arabinose with a molar ratio of 9.97:5.38:4.58:1.11:5.20, wherein the mannose molar ratio is up to 38.10%.
3.5.1LBP effects on RAW 264.7 cell proliferation activity inflammation is a physiological phenomenon of the body at the time of injury, infection and stress occurrence as the first reaction of the body's immune system. In general, inflammation is a natural protective response and immune cells play a critical regulatory role in this process by secreting NO and pro-inflammatory cytokines. Therefore, the invention adopts the CCK-8 method to examine the toxicity of LBP to macrophages after LBP-1 and LBP-2 are respectively used for 24 hours on RAW 264.7 macrophages.
As can be seen from FIG. 4-A, LBP has no toxicity to macrophages, whereas LBP-1 and LBP-2 in the concentration range of 250-500. Mu.g/mL both significantly promote proliferation activity of macrophages, and have statistical differences (P < 0.05) compared with the blank control group, and cell viability and polysaccharide concentration are dose-dependent. When the concentrations of LBP-1 and LBP-2 reach 500 mug/mL, the effect of promoting cell proliferation is most remarkable, the cell survival rate reaches 121.2% and 117.2% respectively, and the promoting effect of LBP-1 is slightly higher than that of LBP-2.
Influence of 3.5.2LBP on NO release of RAW 264.7 cells NO is a signal molecule generated by macrophages in the activation process, and is taken as an important inflammatory mediator, and over-expression of the signal molecule can cause tissue necrosis, cell and DNA damage, so that the development process of inflammatory diseases is accelerated, and therefore, the condition that the macrophages release NO after LPS induction is detected by using a kit.
As can be seen from FIG. 4-B, the amount of NO expressed by the LPS model group cells was greatly increased (P < 0.01) compared with the control group after 24 hours of administration. Compared with LPS model group, LBP-1 and LBP-2 in the concentration interval of 25-500 mug/mL can inhibit the release of macrophage NO after LPS induction obviously (P < 0.01). When the concentrations of LBP-1 and LBP-2 reach 500 mug/mL, the inhibition effect on the NO release of the cells is most remarkable, and the inhibition effect of LBP-1 is superior to that of LBP-2 (P < 0.05). 3.5.3LBP affecting ROS release by RAW 264.7 cells ROS is a generic term for a class of molecules or ions containing oxygen radicals and higher oxidative activity, whose relative balance of production and clearance is a key factor for ensuring normal cell growth and apoptosis, and excessive pro-inflammatory factors can stimulate the massive production of ROS in the body, which can cause oxidative stress damage to tissues and organs of the body. Therefore, the condition that macrophages release ROS after LPS induction is detected by using the kit.
As can be seen from fig. 4-C, 24 hours after administration, the ROS expression level of the LPS model group cells was significantly increased (P < 0.01) compared to the blank group. Compared with the LPS model group, the LBP-1 and LBP-2 in the concentration interval of 250-500 mug/mL can obviously inhibit the ROS release of macrophages after LPS induction (P < 0.01). When the concentrations of LBP-1 and LBP-2 reached 500. Mu.g/mL, the inhibition effect on cellular ROS release was most remarkable, and the inhibition effect of LBP-1 was stronger than that of LBP-2 (P < 0.05).
As shown in FIG. 5-A, 3.6.1LBP has the most obvious effect on the general signs of mice with enteritis, the body mass of mice in the model group has the most obvious tendency to decrease, the body mass of mice starts to rise within 3 days after the model is stopped, the model is continuously molded by 5% DSS after 8 days, and the body weights of the model group and the 5-ASA group are continuously decreased, and the body weight of the administration group has the increasing tendency. On day 14, the body mass was significantly reduced in the model group compared to normal group mice (P < 0.01); there was a statistical difference (P < 0.01) between the positive group and the polysaccharide-dosed group compared to the model group; there was a statistical difference in body mass between the polysaccharide dosed and positive groups (P < 0.05). Normal group mice have no obvious change of body quality, hair and other common signs and no death; mice in both model and dosing groups were dead. Model group mice increased DAI scores compared to normal group (P < 0.01); compared with the model group, the DAI score of the mice in the 5-ASA group and the polysaccharide administration group is reduced (P < 0.01), and in conclusion, the LBP can play a role in improving the quality reduction of colonitis mice besides playing an anti-inflammatory role.
3.6.2LBP effect on intestinal injury in mice with colon inflammation model group mice DAI score was increased compared to normal group (P < 0.01); the DAI score was reduced (P < 0.01) for the mice in the 5-ASA group and the polysaccharide-dosed group compared to the model group (FIG. 5-B). After dissection, the mice were found to have obvious folds in the colon, the colon contents being black, indicating successful construction of the colitis model (fig. 5-E). After the measurement, the colon length of the mice in the model group was found to be significantly shortened after DSS administration (P < 0.01), but the cases of the 5-ASA group and LBP group were significantly better than those of DSS alone (P < 0.01) (fig. 5-C), and then the colon length and macroscopic score of the LBP-1+5-ASA group were significantly higher than those of the 5-ASA group (P < 0.05) (fig. 5-D). The result shows that LBP-1 has obvious effect of relieving colon and intestinal injury of mice.
Effect of 3.6.3LBP on cytokine expression levels in colon tissue of enterally injured mice as shown in fig. 6, expression levels of NO, ROS, IL-6 and TNF- α were significantly increased (P < 0.01) and expression levels of IL-10 were significantly decreased (P < 0.01) in colon tissue of the model group compared to the normal control group. The colon tissues of the 5-ASA and LBP groups showed reduced levels of NO, ROS, IL-6 and TNF- α expression (P < 0.05) and increased IL-10 expression (P < 0.01) compared to the model group. The results show that the Lycium barbarum polysaccharide can reduce the expression of proinflammatory factors NO, ROS, IL-6 and TNF-alpha and increase the expression of anti-inflammatory factors IL-10.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. The extraction method of the wolfberry polysaccharide is characterized by comprising the following steps of:
(1) Pulverizing fructus Lycii, defatting with 95% ethanol, drying, and weighing; leaching with hot water, repeatedly extracting, filtering with gauze, mixing filtrates, and concentrating under reduced pressure; precipitating with 95% ethanol, drying, and weighing to obtain LBP crude polysaccharide;
(2) Repeating the operation of the LBP crude polysaccharide solution prepared in the step (1) with Sevag reagent for a plurality of times to remove proteins until the white intermediate layer disappears; collecting supernatant, concentrating, and freeze-drying to obtain deproteinized LBP; removing pigment from the protein-removed LBP solution by using D101 macroporous adsorption resin, then taking eluent, concentrating by rotary evaporation, and freeze-drying to obtain decolorized LBP;
(3) Weighing 100mg of decolorized LBP prepared in the step (2), dissolving in 10mL of distilled water, centrifuging at 5000r/min for 10min after full dissolution, and collecting supernatant; subjecting to DEAE-52 cellulose column chromatography, sequentially eluting with distilled water, 0.1moL/L NaCl, 0.2moL/L NaCl, and 0.5moL/L NaCl to obtain LBP-1, LBP-2, LBP-3, and LBP-4, respectively, with elution flow rate of 2mL/min, and collecting 10mL per tube; collecting eluent, detecting a separation tube by adopting a phenol-sulfuric acid method, and drawing an elution curve by taking the number of the tubes as an abscissa and the absorbance value as an ordinate; and according to the elution curve, combining the eluents of the same elution peak, concentrating under reduced pressure, and freeze-drying to obtain four refined LBPs with different molecular weights.
2. The method for extracting polysaccharide from lycium barbarum according to claim 1, wherein in the step (1), the ratio of the hot water extracted feed solution is 1:10, the extraction temperature is 90-100 ℃ and the extraction time is 2h.
3. The method for extracting Lycium barbarum polysaccharide according to claim 1 or 2, wherein the hot water extraction is repeated 3 times, followed by precipitation with 3 times of 95% ethanol.
4. The wolfberry polysaccharide extracted by the method of claim 1, wherein the wolfberry polysaccharide is extracted from wolfberry by a water extraction and alcohol precipitation method, deproteinized by a Sevag method, D101 macroporous resin decolorized and DEAE-52 cellulose column separated to obtain high-purity wolfberry polysaccharide.
5. Use of the matrimony vine polysaccharide extracted by the method of claim 1 in pharmaceutical preparation.
6. The use according to claim 5, wherein the lycium barbarum polysaccharide is combined with mesalamine 5-ASA to prepare a pharmaceutical formulation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410863239.5A CN118852479A (en) | 2024-06-29 | 2024-06-29 | Lycium barbarum polysaccharide and extraction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410863239.5A CN118852479A (en) | 2024-06-29 | 2024-06-29 | Lycium barbarum polysaccharide and extraction method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118852479A true CN118852479A (en) | 2024-10-29 |
Family
ID=93156888
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410863239.5A Pending CN118852479A (en) | 2024-06-29 | 2024-06-29 | Lycium barbarum polysaccharide and extraction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118852479A (en) |
-
2024
- 2024-06-29 CN CN202410863239.5A patent/CN118852479A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102114044B (en) | Artificially processed bear bile powder and preparation method thereof | |
| US20110059124A1 (en) | The quality control method and application of a kind of ganoderma lucidum spore oil fat emulsion | |
| CN108743622A (en) | Immunity of organisms and muscle power are improved using bear gall powder and alleviate the method for organism fatigue | |
| CN113493491B (en) | Polypeptide for preventing or treating ulcerative colitis | |
| CN113150049A (en) | Cyclocarya paliurus extract and application thereof in resisting gout and reducing uric acid | |
| CN117500842A (en) | Cs-4 fermentation mycelium heteropolysaccharide and preparation method and application thereof | |
| Yi et al. | Intervention effects of lotus leaf flavonoids on gastric mucosal lesions in mice infected with Helicobacter pylori | |
| CN118852479A (en) | Lycium barbarum polysaccharide and extraction method and application thereof | |
| CN113940945A (en) | Application of houttuynia polysaccharide in preparation of medicine for preventing and treating inflammatory bowel disease | |
| CN116948901A (en) | Application of Weissella antrum D-2 extracellular polysaccharide in inhibiting colon cancer cells | |
| CN100464745C (en) | Medication composition of acetyl cysteine or its pharmaceutical salt and asarin | |
| CN114699424B (en) | New application of bacteroides fragilis zwitterionic capsular polysaccharide and/or modified zwitterionic capsular polysaccharide | |
| CN106727898B (en) | Pharmaceutical composition for preventing and treating Alzheimer disease and preparation method thereof | |
| CN102145076B (en) | The pharmaceutical applications of Radix Rehmanniae and extract thereof | |
| CN106890189A (en) | Application of the chonglou saponin in antineoplastic sensitizer is prepared | |
| CN105497167A (en) | New application of radix ranunculi ternati in preparation of medicine for treating and/or preventing ulcerative colitis | |
| CN114306453B (en) | Application of pharmaceutical composition in preparation of medicines for treating inflammatory bowel disease | |
| CN114948977B (en) | Application of dihydroflavonoid glycoside derivative in preparation of medicines for preventing and treating colonitis | |
| CN113476497B (en) | Prunella vulgaris extract with HSV (herpes Simplex Virus) resisting activity as well as preparation method and medical application thereof | |
| CN110721193B (en) | Application of cynomorium songaricum total polysaccharide in preparation of medicine for treating asthma | |
| WO2024227342A1 (en) | Use of aloe gel powder and composition of aloe gel powder and stachyose | |
| CN110038071A (en) | A kind of Chinese medicine composition and preparation method and application with anti-herpesvirus | |
| KR100352148B1 (en) | Compositions for angiogenesis and preventing and treating arthritis comprising β-sitosterol | |
| CN118440217A (en) | Novel agrocybe cylindracea polysaccharide, preparation method and application thereof in preparation of product for repairing intestinal mucosa barrier injury | |
| CN114712351A (en) | Application of rhynchophylline in preparation of medicine for treating inflammatory enteritis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |