CN109731003A - Application of the rubrofusarin -6-O- β-O-gentibioside in preparation lipid-lowering diet drug - Google Patents
Application of the rubrofusarin -6-O- β-O-gentibioside in preparation lipid-lowering diet drug Download PDFInfo
- Publication number
- CN109731003A CN109731003A CN201811349282.0A CN201811349282A CN109731003A CN 109731003 A CN109731003 A CN 109731003A CN 201811349282 A CN201811349282 A CN 201811349282A CN 109731003 A CN109731003 A CN 109731003A
- Authority
- CN
- China
- Prior art keywords
- rfg
- rubrofusarin
- gentibioside
- cell
- ampk
- 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
- FPNKCZKRICBAKG-UHFFFAOYSA-N rubrofusarin Chemical compound O1C(C)=CC(=O)C=2C1=CC1=CC(OC)=CC(O)=C1C=2O FPNKCZKRICBAKG-UHFFFAOYSA-N 0.000 title claims abstract description 26
- KLKFLNXANXGSIT-UHFFFAOYSA-N rubrofusarin Natural products O1C(C)=CC(=O)C2=C1C=C1C=C(O)C=C(OC)C1=C2O KLKFLNXANXGSIT-UHFFFAOYSA-N 0.000 title claims abstract description 26
- 239000003814 drug Substances 0.000 title claims abstract description 12
- 229940079593 drug Drugs 0.000 title claims abstract description 9
- 238000002360 preparation method Methods 0.000 title claims description 9
- 235000005911 diet Nutrition 0.000 title claims description 5
- 230000037213 diet Effects 0.000 title claims description 5
- 102100036009 5'-AMP-activated protein kinase catalytic subunit alpha-2 Human genes 0.000 claims abstract description 33
- 101000783681 Homo sapiens 5'-AMP-activated protein kinase catalytic subunit alpha-2 Proteins 0.000 claims abstract description 33
- 102000000536 PPAR gamma Human genes 0.000 claims abstract description 32
- 108010016731 PPAR gamma Proteins 0.000 claims abstract description 32
- 102100034808 CCAAT/enhancer-binding protein alpha Human genes 0.000 claims abstract description 29
- 101710168309 CCAAT/enhancer-binding protein alpha Proteins 0.000 claims abstract description 29
- 210000004185 liver Anatomy 0.000 claims abstract description 29
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 claims abstract description 18
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 claims abstract description 18
- 102100030431 Fatty acid-binding protein, adipocyte Human genes 0.000 claims abstract description 13
- 101001062864 Homo sapiens Fatty acid-binding protein, adipocyte Proteins 0.000 claims abstract description 13
- 101000964894 Bos taurus 14-3-3 protein zeta/delta Proteins 0.000 claims abstract description 11
- 101000824278 Homo sapiens Acyl-[acyl-carrier-protein] hydrolase Proteins 0.000 claims abstract description 11
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 claims abstract description 11
- 108010013563 Lipoprotein Lipase Proteins 0.000 claims abstract description 11
- 102100022119 Lipoprotein lipase Human genes 0.000 claims abstract description 11
- 230000006372 lipid accumulation Effects 0.000 claims abstract description 8
- 230000011759 adipose tissue development Effects 0.000 claims abstract description 6
- 208000004930 Fatty Liver Diseases 0.000 claims abstract description 5
- 206010019708 Hepatic steatosis Diseases 0.000 claims abstract description 5
- 230000004913 activation Effects 0.000 claims abstract description 5
- 208000010706 fatty liver disease Diseases 0.000 claims abstract description 5
- 231100000240 steatosis hepatitis Toxicity 0.000 claims abstract description 5
- 230000008685 targeting Effects 0.000 claims abstract description 4
- 102100022089 Acyl-[acyl-carrier-protein] hydrolase Human genes 0.000 claims abstract 2
- 150000001875 compounds Chemical class 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 3
- JEUUNKOFKDUVMN-UHFFFAOYSA-N benzo[f]chromen-1-one Chemical compound C1=CC=CC2=C3C(=O)C=COC3=CC=C21 JEUUNKOFKDUVMN-UHFFFAOYSA-N 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 229940100688 oral solution Drugs 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims 1
- 229930182478 glucoside Natural products 0.000 claims 1
- 150000008131 glucosides Chemical class 0.000 claims 1
- 239000002245 particle Substances 0.000 claims 1
- -1 naphthopyrone glycosides compound Chemical class 0.000 abstract description 5
- 229930182470 glycoside Natural products 0.000 abstract description 2
- 230000009467 reduction Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 45
- 208000008589 Obesity Diseases 0.000 description 22
- 235000020824 obesity Nutrition 0.000 description 22
- 241000699670 Mus sp. Species 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 17
- 108020004999 messenger RNA Proteins 0.000 description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 238000012545 processing Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- 244000037364 Cinnamomum aromaticum Species 0.000 description 12
- 235000014489 Cinnamomum aromaticum Nutrition 0.000 description 12
- 230000006698 induction Effects 0.000 description 12
- JTSLALYXYSRPGW-UHFFFAOYSA-N n-[5-(4-cyanophenyl)-1h-pyrrolo[2,3-b]pyridin-3-yl]pyridine-3-carboxamide Chemical compound C=1C=CN=CC=1C(=O)NC(C1=C2)=CNC1=NC=C2C1=CC=C(C#N)C=C1 JTSLALYXYSRPGW-UHFFFAOYSA-N 0.000 description 12
- 210000001789 adipocyte Anatomy 0.000 description 11
- 230000026731 phosphorylation Effects 0.000 description 11
- 238000006366 phosphorylation reaction Methods 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- 239000000284 extract Substances 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 102100026715 Serine/threonine-protein kinase STK11 Human genes 0.000 description 9
- 101710181599 Serine/threonine-protein kinase STK11 Proteins 0.000 description 9
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 8
- 238000007619 statistical method Methods 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 5
- 102000007330 LDL Lipoproteins Human genes 0.000 description 5
- 108010007622 LDL Lipoproteins Proteins 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000024245 cell differentiation Effects 0.000 description 5
- 230000003013 cytotoxicity Effects 0.000 description 5
- 231100000135 cytotoxicity Toxicity 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 239000003480 eluent Substances 0.000 description 5
- 201000010063 epididymitis Diseases 0.000 description 5
- 238000003384 imaging method Methods 0.000 description 5
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 5
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 5
- 230000004584 weight gain Effects 0.000 description 5
- 235000019786 weight gain Nutrition 0.000 description 5
- 102000015779 HDL Lipoproteins Human genes 0.000 description 4
- 108010010234 HDL Lipoproteins Proteins 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 239000006180 TBST buffer Substances 0.000 description 4
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 150000003626 triacylglycerols Chemical class 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 3
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 102000038030 PI3Ks Human genes 0.000 description 3
- 108091007960 PI3Ks Proteins 0.000 description 3
- 102100033810 RAC-alpha serine/threonine-protein kinase Human genes 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 210000000918 epididymis Anatomy 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000010534 mechanism of action Effects 0.000 description 3
- 230000008520 organization Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 238000009010 Bradford assay Methods 0.000 description 2
- 244000201986 Cassia tora Species 0.000 description 2
- 235000014552 Cassia tora Nutrition 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000035508 accumulation Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 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 2
- 238000002247 constant time method Methods 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000012447 hatching Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 230000037356 lipid metabolism Effects 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 208000020442 loss of weight Diseases 0.000 description 2
- 230000002101 lytic effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000011017 operating method Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 230000008844 regulatory mechanism Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- BZFBDUQOBQHBSZ-DLCQERRASA-N (3s,8r,9s,10r,13s,14s,17s)-17-[3-(dimethylamino)propyl-methylamino]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-ol;dihydrochloride Chemical compound Cl.Cl.C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](N(C)CCCN(C)C)[C@@]1(C)CC2 BZFBDUQOBQHBSZ-DLCQERRASA-N 0.000 description 1
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- IGAZHQIYONOHQN-UHFFFAOYSA-N Alexa Fluor 555 Chemical compound C=12C=CC(=N)C(S(O)(=O)=O)=C2OC2=C(S(O)(=O)=O)C(N)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C(O)=O IGAZHQIYONOHQN-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101710186200 CCAAT/enhancer-binding protein Proteins 0.000 description 1
- 244000277285 Cassia obtusifolia Species 0.000 description 1
- 235000006719 Cassia obtusifolia Nutrition 0.000 description 1
- 208000035484 Cellulite Diseases 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010049752 Peau d'orange Diseases 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- 101100063433 Pisum sativum DIM gene Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
- 229930182482 anthraquinone glycoside Natural products 0.000 description 1
- 150000008139 anthraquinone glycosides Chemical class 0.000 description 1
- 150000004056 anthraquinones Chemical class 0.000 description 1
- 230000003579 anti-obesity Effects 0.000 description 1
- 210000000702 aorta abdominal Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000013872 defecation Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000006694 eating habits Nutrition 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 201000005577 familial hyperlipidemia Diseases 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000005161 hepatic lobe Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 235000021374 legumes Nutrition 0.000 description 1
- 230000000940 lipogenetic effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 230000027405 negative regulation of phosphorylation Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000021590 normal diet Nutrition 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 235000019353 potassium silicate Nutrition 0.000 description 1
- 238000004237 preparative chromatography Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
It is naphthopyrone glycosides compound that the present invention provides rubrofusarin -6-O- β-O-gentibiosides (RFG) preparing the application in fat-reducing dietic medicinal, the rubrofusarin -6-O- β-O-gentibioside.RFG inhibits the lipid accumulation of 3T3-L1 and hAMSCs cell by the targeting of reduction mTOR, PPAR γ and C/EBP α；The expression of Adipogenesis correlation factor is adjusted by AMPK access, is inhibited lipid accumulation signal path, can be lost weight the size of increase, eWAT and fatty liver；RFG inhibits fat to generate the factor (PPAR γ, C/EBP α, FAS, LPL and aP2) by the AMPK in activation eWAT and liver.Therefore it can be used as the fat drug for the treatment of.
Description
Technical field
The invention belongs to the bioactive application technical fields of compound, in particular to a kind of aphthopyrans ketone
The new pharmaceutical use of compound, more particularly to rubrofusarin -6-O- β-O-gentibioside with following formula 1 is in preparation lower blood-fat and reduce weight
Application in medicine.
Formula 1
Background technique
With the improvement of people ' s living standards with the change of original eating habit, obesity has become stream in modern society
Row disease, other related diseases as brought by obesity, as diabetes, atherosclerosis, coronary heart disease, hypertension, fatty liver and
Other cardiovascular and cerebrovascular diseases etc. have seriously threatened the health of the mankind.Medical field is making great efforts to develop Small side effects treatment at present
The lipid-lowering diet drug imitated studies the cause of disease, Chinese medicine system is made by scientific and reasonable composition of prescription especially from theory of traditional Chinese medical science
Agent achievees the purpose that treating both manifestation and root cause of disease, and research the effect of to realize lower blood-fat and reduce weight is as many as the most.
Cassia seed is legume cassia (Cassia obtusifolia L.) or little cassia tora (Cassia tora L.)
Dry mature seed.Have wind and heat eliminating, clear liver and improve vision, relax bowel and defecation the effect of.Clinic plays the role of reducing serum ornitrol.Certainly
Containing there are many anthraquinone component and aphthopyrans ketones components in pine torch, it was reported that certain anthraquinone glycosides and naphthopyrone therein
Glycosides has the function of that fighting carbon tetrachloride and galactosamine poisons originally culture mouse stem cells.In numerous patent application texts
In part, lipid-lowering medicine, the slimming drugs that cassia seed and other Chinese medicines are prepared together are referred to, due to being compound, it is difficult to which understanding wherein rises
The effective component of key effect is arrived.Rubrofusarin -6-O- β-O-gentibioside (RFG) in cassia seed, which has, adjusts hyperlipemia
The report of disease, but there is not been reported for its antiobesity action and its dependent interaction mechanism.
Patent research RFG improves mechanism of action that is fat and adjusting lipid accumulation.It is thin with 3T3-L1 and hAMSCs
Born of the same parents' test confirms the lipogenetic effect of the inhibition of RFG.RFG passes through the targeting for reducing mTOR, PPAR γ and C/EBP α,
Inhibit the lipid accumulation of 3T3-L1 and hAMSCs cell.RFG is also independent, and phosphorylation AMP activated protein significant in liver kinase b swashs
Enzyme.But AMPK inhibitor has blocked the anti-cellulite nucleus formation of RFG.These results indicate that RFG adjusts Adipogenesis by AMPK
The expression of correlation factor, to inhibit lipid accumulation signal path.In addition, RFG reduces weight gain, eWAT and fatty liver
Size.These effects and mechanism of action are similar to experiment in vitro.RFG also passes through the AMPK in activation eWAT and liver and inhibits fat
It generates the factor (PPAR γ, C/EBP α, FAS, LPL and aP2).Therefore RFG can improve obesity, the drug fat as treatment.
Summary of the invention
The purpose of the present invention is 1 rubrofusarin -6-O- β of the formula-O-gentibioside (RFG) is applied to preparation lipid-loweringing
The drug of weight-reducing.
Formula 1
RFG and pharmaceutically acceptable pharmaceutic adjuvant, the pharmaceutical preparation as made of starch, cyclodextrin etc., and can be according to agent
Type needs to add other auxiliary materials.
RFG can be tablet, capsule, oral solution, granule and freeze-dried powder with preparation formulation made of pharmaceutic adjuvant
Injection.
RFG can inhibit the rouge of 3T3-L1 and hAMSCs cell by the targeting of reduction mTOR, PPAR γ and C/EBP α
Matter accumulation.
RFG can adjust the expression of Adipogenesis correlation factor by AMPK, inhibit lipid accumulation signal path.
RFG can lose weight the size of increase, eWAT and fatty liver；Inhibit rouge by the AMPK in activation eWAT and liver
Fat generates the factor (PPAR γ, C/EBP α, FAS, LPL and aP2).
The beneficial effects of the present invention are:
(1) present invention has studied the functions of lowering blood-fat and reducing weight of RFG, and its mechanism of action is conducted in-depth research and explained
It states；
(2) present invention has carried out comparative experiments to RFG and cassia seed extract, and experimental result shows the lower blood-fat and reduce weight of RFG
Effect is substantially better than cassia seed extract.
Detailed description of the invention
Cytotoxicity of Fig. 1 (A) RFG to 3T3-L1 cell；(B) cytotoxicity of the RFG to hAMSCc
Fig. 2 (A) oil red O is to fat drips colored graph in RFG treated 3T3-L1 cell；(B) treated that 3T3-L1 is thin by RFG
Fat drips accumulate result in born of the same parents；(C) oil red O is to fat drips colored graph in RFG treated hAMSCc cell；(D) treated by RFG
Fat drips accumulate result in hAMSCc cell
Influence of Fig. 3 (A) RFG to the mRNA level in-site expression of 3T3-L1 cell PPAR γ and C/EBP α；(B) 3T3-L1 cell
Western blot analysis experimental result (GAPDH is internal reference)；(C) RFG is to 3T3-L1 cell PPAR γ and C/EBP alpha protein water
The influence of flat expression
Influence of the RFG to the mRNA expression of FAS in Fig. 4 (A) 3T3-L1 cell differentiation；(B) RFG in 3T3-L1 cell differentiation
Influence to the mRNA expression of LPL；(C) influence of the RFG to the mRNA expression of aP2 in 3T3-L1 cell differentiation
Fig. 5 (A) RFG is to p-LKB1 in 3T3-L1 cell, LKB1, p-AMPK α, AMPK α, p-mTOR, mTOR protein table
Up to horizontal influence；(B) RFG processing 3T3-L1 cell in pAMPK α/AMPK α, p-LKB1/LKB1, p-mTOR/mTOR phase
Reduced value analysis；(C) RFG is to p-PI3K in 3T3-L1 cell, PI3K, the influence of p-AKT and AKT protein level expression；(D)
The relative ratio analysis of p-PI3K/PI3K and p-AKT/AKT in the 3T3-L1 cell of RFG processing
Fig. 6 (A) RFG to p-LKB1 in hAMSCc cell, LKB1, p-AMPK α, AMPK α, p-mTOR, mTOR, PPAR γ and
The influence of C/EBP alpha protein expression；PAMPK α/AMPK α, p-LKB1/ in the hAMSCc cell of (B, C) RFG processing
The relative ratio of LKB1, p-mTOR/mTOR, PPAR γ/GAPDH and C/EBP α/GAPDH are analyzed；(D) RFG is to hAMSCc cell
The influence of the mRNA expression of middle PPAR γ, C/EBP α and aP2
10 weeks changes of weight of HFD obesity-induced mice of Fig. 7 (A) RFG processing；(B) it is small to handle HFD inducing obesity by RFG
Mouse tests start and ending mice weights difference
Fig. 8 RFG handles the eWAT of HFD obesity-induced mice and the weight of liver
It is horizontal that Fig. 9 (A) RFG handles TG, TC, HDL, LDL in HFD obesity-induced mice serum；(B) RFG handles HFD induction
AST and ALT is horizontal in obesity mice serum；(C) RFG handles HFD obesity-induced mice creatinine in serum and BUN is horizontal
Figure 10 (A) RFG handles HFD obesity-induced mice liver organization H&E coloring pathological section figure (* 200)；(B) at RFG
It manages HFD obesity-induced mice H&E and dyes Epididymal Adipocytes size
Influence of Figure 11 (A, B) RFG to PPAR γ and C/EBP the alpha protein horizontal expression of eWAT；(C) RFG is to eWAT's
The influence of the mRNA level in-site expression of PPAR γ and C/EBP α；PPAR γ and C/EBP alpha protein water-glass of (the D, E) RFG to liver
The influence reached；(F) influence of the RFG to the mRNA level in-site expression of PPAR γ and the C/EBP α of liver
Figure 12 (A) RFG is to p-LKB1 in eWAT, LKB1, p-AMPK α, AMPK α, p-mTOR, mTOR protein expression level
Influence；(B) relative ratio of p AMPK α/AMPK α, p-LKB1/LKB1, p-mTOR/mTOR are analyzed in eWAT after RFG processing；
(C) influence of the RFG to the mRNA expression of FAS, LPL, aP2 in eWAT
Figure 13 (A) RFG is to p-LKB1 in liver, LKB1, p-AMPK α, AMPK α, p-mTOR, mTOR protein expression level
Influence；(B) relative ratio of p AMPK α/AMPK α, p-LKB1/LKB1, p-mTOR/mTOR are analyzed in liver after RFG processing；
(C) influence of the RFG to the mRNA expression of FAS, LPL, aP2 in liver
Specific embodiment
Purpose of the present invention is achieved through the following technical solutions:
1 rubrofusarin -6-O- β of embodiment-O-gentibioside preparation method
(1) Cassia Seed is taken, proper amount of methanol is added, homogenate extraction filters, combined extract, recycles methanol, obtains Cassia
Seed extract.
(2) cassia seed extract is taken, sample, eluent chloroform-methanol, dry method loading, elution are mixed with 1: 1 mass ratio with silica gel
After 4 times of column volumes, loading layer is dug out, continues to use methanol eluting silica gel column, by TLC trace detection, until without red streptomysin -6-
O- β-O-gentibioside.Eluent is recycled, rubrofusarin -6-O- β-O-gentibioside crude extract is obtained.
(3) rubrofusarin -6-O- β-O-gentibioside crude extract 39% methanol aqueous solution of volumetric concentration is dissolved, configuration
At test solution, through 0.45 μm of filtering with microporous membrane；It is separated using high performance liquid preparative chromatography column, is with volumetric concentration
40% methanol-water solution is eluent system, and online ultraviolet detection collects and contains rubrofusarin -6-O- β-O-gentibioside
Dry yellow powder I is concentrated under reduced pressure in eluent.
(4) yellow powder I is subjected to second of efficient liquid phase with the eluent system that volumetric concentration is 42% methanol aqueous solution
Preparation, the same step of other conditions (3) collect purity greater than 98% and contain rubrofusarin -6-O- β-O-gentibioside fraction；
Above-mentioned fraction is freeze-dried to obtain rubrofusarin -6-O- β-O-gentibioside that purity is greater than 98%.
2 rubrofusarin -6-O- β of embodiment-O-gentibioside is to 3T3-L1 cell and hAMSCs cell Proliferation and differentiation
It influences
One, experiment content
(1) 3T3-L1 cell culture and induction differentiation
By 3T3-L1 cell inoculation in culture plate, with containing 1% Pen .- Strep and 10% newborn bovine serum (NBCS)
DMEM culture solution is cultivated in 37 DEG C, 5%CO2 incubator, two days later to cell fusion, in the glucose containing 25mM, 0.5mM
The DMEM culture medium hatching 48 of IBMX, 1 μM of dexamethasone (Dex), 1 μ g/ml insulin (MDI) and 10% fetal calf serum (FBS)
Hour, it changes to carry out hatching 48 hours in the DMEM culture solution containing 10%FBS, 1 μ g/ml MDI and various concentration REG.Then
In the DMEM culture solution containing 10%FBS, 1 μ g/ml MDI, continue culture two days.It is within the 1st day in the induction differentiation of 3T3-L1 cell
The RFG of various concentration is added, until experiment terminates.
(2) hAMSCs cell culture and induction differentiation
By hAMSCs cell inoculation in culture plate, with containing 1% Pen .- Strep and 10% newborn bovine serum (NBCS)
DMEM culture solution is cultivated in 37 DEG C, 5%CO2 incubator, two days later to cell fusion, with IBMX containing 0.5Mm, 1 μm of Dex, 1
10%FBS/DMEM Cell differentiation inducing activity 6 days of μm insulin and 100 μm of Indomethacins are changed primary new every three days in incubation
Fresh culture solution.6th day, with the 10%FBS/DMEM incubated cell containing 1 μ g/ml insulin, replace within every two days primary fresh training
Base is supported, until the 14th day.
(3) Cytotoxic evaluation of 3T3-L1 and hAMSCs
3T3-L1 cell and hAMSCs cell are inoculated in 96 orifice plates by the hole 5*103/, use the DMEM containing 10%NBCS
Culture solution culture.After 24 hours, the RFG of various concentration is replaced.After 48 hours, 20 μ L MTS solution are added in every hole, continue incubation 4
Hour, terminate culture, select 490nm wavelength, measure each hole absorbance value (formazan concentration in VERSA microplate reader, the concentration with
Number of viable cells is directly proportional).
(4) oil red O stain is tested
After 3T3-L1 PECTORAL LIMB SKELETON and hAMSCs cell are handled with RFG, the cells are fixed 2 hours in 10% formaldehyde.
After being washed with 60% isopropanol, dyed at room temperature 30 minutes with 0.3% oil red O solution, be washed with water undyed cell with
Remove oil red O solution.Cell is observed using EVOS XL core imaging system.It is extracted in cell with 100% isopropanol (hole 3ml/)
Dyestuff.The dyestuff of dissolution is measured at 500nm with VERSA max microplate reader to calculate absorbance.
(5) statistical method
As a result indicate that statistical method is examined with t with the average value ± SD of independent experiment.All statistical results are united with SPSS
Count software analysis.Significant difference is shown as p < 0.05.
Two, experimental result
(1) cytotoxicity experiment result
As shown in Fig. 1 (A), RFG does not have cytotoxicity at 200 μM or less to 3T3-L1 cell, and 400 μM of RFG significantly drops
The cell viability of low 3T3-L1 cell.Therefore select 50,100 and 200 μM of RFG as experimental concentration.
As shown in Fig. 1 (B), although up to 400 μM of RFG does not show cytotoxicity to hAMSCs cell, in order to
The concentration of 3T3-L1 cell experiment is consistent, select in following experiment 50,100 and 200 μM as experimental concentration.
(2) oil red O stain experimental result
As shown in Fig. 2 (A) and (B), with the raising of RFG concentration, fat drips are accumulated with dose dependent in 3T3-L1 cell
Mode gradually decrease.
As shown in Fig. 2 (C) and (D), with the raising of RFG concentration, fat drips are accumulated with dose dependent in hAMSCs cell
Mode gradually decrease.
3 rubrofusarin -6-O- β of embodiment-O-gentibioside inhibits the regulatory mechanism research of Adipocyte Differentiation
One, experiment content
(1) RNA extraction and real-time quantitative PCR
The total serum IgE in 3T3-L1 cell and hAMSCs cell is extracted with the operating procedure of QIAzol lytic reagent box.To mention
The total serum IgE taken out is the template for synthesizing the first chain of cDNA, according to the application method that kit describes, is closed using power cDNA
Reverse transcription is carried out at kit.With Step One Plus real-time fluorescence quantitative RT-PCR system detection PCR product, and use
Step One v2.1 software is used as endogenous control with GAPGH and h36 B4, compares the phase that CT method determines PPAR γ and C/EBP α
To expression.The upstream primer and downstream primer of target cDNA amplification are as shown in table 1.
1 real-time quantitative PCR primer sequence of table
(2) western blot analysis
Induction differentiation 3T3-L1 cell and hAMSCs cell are cracked with extracting solution of protein, albumen is measured with Bradford method
Concentration.After taking protein example to add sample-loading buffer denatured by boiling, 10%SDS-PAGE electrophoresis, revolving die are carried out, with 5% defatted milk
The TBST of powder closes 2h, is separately added into primary antibody, and 4 DEG C overnight, and 0.1%TBST is washed film 3 times, is handled with secondary antibodies appropriate
Afterwards, ECL development is carried out with chemiluminescence gel imaging system.Ribbon density is measured with Image-J software.
(3) immunofluorescence staining
HAMSCs (5*103 cells/well) is seeded in 8 pore chamber glass slides and is broken up.Noble cells 0.1%Triton
X-100 is fixed and permeabilization.After being incubated in Block buffer containing 3%BSA and 0.1%Triton X-100 in PBS, dark
It is reacted overnight in 4 DEG C with primary antibody in room.Behind secondary antibody 1 hour that processing Alexa488 and Alexa555 is combined, carry
Slide is washed with PBS and is redyed with DAPI.After being rinsed with PBS, glass slide is captured using EVIS TM FL imaging system.
(4) statistical method
As a result indicate that statistical method is examined with t with the average value ± SD of independent experiment.All statistical results are united with SPSS
Count software analysis.Significant difference is shown as p < 0.05.
Two, experimental result
(1) influence of the RFG to mRNA and the protein level expression of 3T3-L1 cell PPAR γ and C/EBP α
As shown in figure 3, the mRNA level in-site and egg of RFG treated 3T3-L1 cell significantly inhibits PPAR γ and C/EBP α
The expression of white matter level.
(2) RFG activates AMPK access, and fat in 3T3-L1 cell is inhibited to generate
For the effect for reducing fat molecular mechanism for determining RFG, the upstream and downstream factor of PPAR γ and C/EBP α are determined.First
Determine the mRNA expression of downstream elements FAS, LPL and aP2 of PPAR γ and C/EBP α.Shown in as shown in the figure 4, pass through concentration
After the RFG processing of dependence, the mRNA expression of FAS, LPL and aP2 are significantly reduced.To further determine that RFG inhibits rouge
The access of fat cell expression, has studied the phosphorylation of LKB 1, AMPK and mTOR.As shown in figure 5, the phosphorylation of AMPK passes through RFG
It handles and dose-dependently increases, the AMPK of activation inhibits the phosphorylation of mTOR, promotes the expression of PPAR γ.But as
The IKB 1 of AMPK upstream element is not activated by RFG, and the phosphorylation of PI3K and AKT are not also inhibited by RFG.Result above table
Bright, RFG may pass through the inhibition of phosphorylation Adipogenesis of AMPK.
(3) RFG activates AMPK access, and hAMSCs is inhibited to be divided into mature fat cell
To confirm that RFG whether by AMPK inhibition Adipogenesis, uses AMPK inhibitor Compound C (CC).Such as Fig. 2
Shown in (C, D), compared with RFG group, the use in conjunction of RFG and CC significantly suppress the effect for reducing fat of RFG.As shown in fig. 6, with
The result of 3T3-L1 cell is consistent, RFG can activate AMPK access, mTOR phosphorylation and significantly inhibit PPAR γ and C/
The protein expression of EBP α.However, RFG does not activate the phosphorylation of LKB 1.In addition, RFG activates the phosphoric acid of mTOR after CC processing
Change, significantly inhibit the protein expression of PPAR γ and C/EBP α.In mRNA level in-site, RFG inhibits the expression of PPAR γ and C/EBP α,
And CC processing does not inhibit then.Similar trend is also presented with the downstream elements aP2 of C/EBP α in PPAR γ.Immunofluorescence dyeing confirms
RFG inhibits PPAR γ and C/EBP α with concentration dependant manner, but CC processing has also blocked this effect.The above result shows that
RFG can inhibit hAMSCs to be divided into mature fat cell by activating AMPK access.
4 rubrofusarin -6-O- β of embodiment-O-gentibioside studies the obesity mice loss of weight effect for reducing fat that HFD is induced
One, experiment content
(1) the obesity mice modeling and experimental group of HFD induction
C57BL/6J male mice (4 weeks, 16-18 grams) is raised one week under stable experiment condition, and 12h light dark is followed
Ring.For inducing obesity, mouse feeding 60%kcal high lipid food (HFD, U.S.'s Research Diet experimental animal feed are given
Model D12492).Mouse is randomly divided into four groups (every group 8), i.e. normal diet group, HFD group, HFD+RFG (50mg/kg/
It) it organizes and HFD+RFG (100mg/kg/ days) group.Normal group feeds standard chow, and HFD group and HFD+RFG group feed HFD and distilled water
Or RFG continues 10 weeks, weighing is primary weekly, record mouse weight variation.All programs used in zoopery are all bases
Wide (the confirmation number: wk u17- learning evaluation committee, mechanism animal care and being carried out using the program that the committee ratifies of circle
128)。
(2) observation index and method
After 4 weeks, it is deprived of food but not water 6h, 5% chloral hydrate anesthesia mouse of 0.2ml/100g volume is injected intraperitoneally after weighing,
Abdominal aorta blood sampling, stands 1h, draws upper serum, -20 DEG C of preservations with liquid-transfering gun after centrifugation.Analyze the total cholesterol in serum
(TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) triglycerides (TG), aspartate transaminase (AST), the third ammonia
Sour transaminase (ALT), blood urea nitrogen (BUN) and creatinine content.
Mouse eWAT and liver are won, and is weighed.Right lobe of liver tissue is cut to be washed off after watery blood filter paper blots with physiological saline
It is put in 10% neutral formalin to fix, prepares 5 μm of paraffin sections, hematoxylin eosin staining.Micro-image is by EVOS XL core
The capture of heart imaging system.Use the size of Image-J software analysis adipose tissue.
(3) statistical method
As a result indicate that statistical method is examined with t with the average value ± SD of independent experiment.All statistical results are united with SPSS
Count software analysis.Significant difference is shown as p < 0.05.
Two, experimental result
(1) RFG reduces weight, epididymal adipose tissues and the liver weight of the obesity mice of HFD induction
Compared with HFD group, weight, the epididymis of HFD+RFG (50mg/kg/ days) groups and HFD+RFG (100mg/kg/ days) groups
Fat and liver weight all significantly reduce.As shown in fig. 7, HFD+RFG group mouse weight is significantly lower than HFD group.The weight gain of ND group
25.41 ± 0.86g of 8.27 ± 1.09g, HFD group weight gain.And (100mg/KG/ days) 13.17 ± 1.53g of group weight gain of HFD+RFG.Knot
Fruit shows dose-dependently reduce weight gain using RFG.If Fig. 8 is shown, RFG treated mouse in dosage according to
Reduce eWAT and liver weight with relying property.
(2) RFG significantly improves the disorders of lipid metabolism of the obesity mice of HFD induction
As shown in figure 9, compared with normal group, TC, HDL, LDL level in HFD group serum are all significantly higher than normal group, and
TG level is then substantially less than normal group, shows that the obesity mice lipid metaboli of HFD induction gets muddled.After RFG drug-treated, blood
Rouge testing result is shown: RFG can be effectively improved the disorders of lipid metabolism of the obesity mice of HFD induction, reduce TC and LDL level (p <
0.05).Compared with HFD group, liver renal toxicity is not caused within oral RFG10 weeks.
(3) RFG mitigates the liver fat sample lesion of the obesity mice of HFD induction
H&E coloration result shows that such as Figure 10, compared with normal group, HFD group fat cell is significantly increased, and HFD+RFG group
Fat cell increase can be significantly inhibited.The fat cell size of HFD group is 5944+414 μm 2, and with HFD+RFG (50mg/kg/
It) and HFD+RFG (100mg/kg/ days) group fat cell size be respectively 4079 ± 305 μm 2 and 2813 ± 169 μm 2.By
This is as it can be seen that RFG can improve liver fat sample lesion.
The obesity mice loss of weight effect for reducing fat mechanism that 5 rubrofusarin -6-O- β of embodiment-O-gentibioside induces HFD
Research
One, experiment content
(1) RNA extraction and PCR
The total serum IgE in liver and eWAT is extracted with the operating procedure of QIAzol lytic reagent box.With the total serum IgE extracted
It is carried out according to the application method that kit describes using power cDNA synthetic agent box for the template for synthesizing the first chain of cDNA
Reverse transcription.With Step One Plus real-time fluorescence quantitative RT-PCR system detection PCR product, and it is soft using Step One v2.1
Part is used as endogenous control with GAPGH and h36B4, compares the relative expression levels that CT method determines PPAR γ and C/EBP α.Target
The upstream primer and downstream primer of cDNA amplification are as shown in table 1.
(2) western blot analysis
Induction differentiation liver and eWAT tissue are cracked with extracting solution of protein, protein concentration is measured with Bradford method.It takes
After protein example adds sample-loading buffer denatured by boiling, 10%SDS-PAGE electrophoresis, stock mould are carried out, with 5% skimmed milk power
TBST closes 2h, is separately added into primary antibody, and 4 DEG C are overnight, and 0.1%TBST is washed film 3 times, after being handled with secondary antibodies appropriate,
ECL development is carried out with chemiluminescence gel imaging system.Ribbon density is measured with Image-J software.
(3) statistical method
As a result indicate that statistical method is examined with t with the average value ± SD of independent experiment.All statistical results are united with SPSS
Count software analysis.Significant difference is shown as p < 0.05.
Two, experimental result
(1) RFG is to PPAR γ and C/EBP α regulatory mechanism
As shown in figure 11, in eWAT and liver organization, RFG processing significantly suppresses PPAR γ and C/EBP α protein expression
It is expressed with mRNA.This result shows that, RFG, which may pass through, inhibits PPAR γ and the C/EBP α in eWAT and liver organization to inhibit rouge
The increase of fat cell.
Shown in as shown in the figure 10, the liver ratio ND group of adipohepatic HFD group has more fat drips.However pass through
RFG administration, the accumulation of fat drips are eased.
(3) RFG activates AMPK access that fat is inhibited to generate
Shown in as shown in the figure 12, compared with HFD group, RFG processing increases the AMPK phosphorylation in eWAT and inhibits
The phosphorylation of mTOR has activated the phosphorylation of LKB1.In addition, RFG has significantly lowered the mRNA expression of FAS, LPL and aP2.Such as figure
It is consistent with the result of eWAT shown in shown 13, the phosphorylation of RFG adjusting AMPK, LKB and mTOR same to the processing of liver, and
The mRNA expression for inhibiting FAS, LPL and aP2 in liver, to inhibit the generation of fat cell.
6 rubrofusarin -6-O- β of embodiment-O-gentibioside and cassia seed extract lower blood-fat and reduce weight comparative test
According to method in above-described embodiment 4, rubrofusarin -6-O- β-O-gentibioside is subtracted with cassia seed extract lipid-loweringing
Fertilizer effect (the influence to HFD obesity-induced mice weight, liver and epididymis tissue weight, to HFD obesity-induced mice blood lipid
Influence) the horizontal comparative test of animal is carried out, test data is as follows:
2 rubrofusarin -6-O- β of table-O-gentibioside and cassia seed extract are to experiment mice weight, liver and epididymis
The influence of tissue weight
3 rubrofusarin -6-O- β of the table-influence of O-gentibioside and cassia seed extract to experiment mice blood lipid
Unit: mg/dl
The above results show that the same dose of RFG lower blood-fat and reduce weight effects are substantially better than the lower blood-fat and reduce weight of cassia seed extract
Effect.
The basic principles, main features and the advantages of the invention have been shown and described in above embodiments.The industry
Technical staff it should be appreciated that the present invention is not limited to the above embodiments, what is described in the above embodiment and the description is only
Illustrate the principle of the present invention, rather than limits the scope of the invention in any way, without departing from the scope of the invention,
Various changes and improvements may be made to the invention, these changes and improvements are both fallen in claimed range.The present invention claims guarantors
Shield range is defined by the appending claims and its equivalent thereof.
Claims (6)
1. 1 naphthopyrone glucoside compound rubrofusarin -6-O- β of formula-O-gentibioside is in preparation lipid-lowering diet drug
Application.
2. compound rubrofusarin -6-O- β-O-gentibioside according to claim 1 with it is medically acceptable medicinal
The pharmaceutical preparation that auxiliary material is prepared into.
3. pharmaceutical preparation according to claim 2, it is characterised in that preparation formulation is tablet, capsule, oral solution, particle
Agent and freeze drying powder injection.
4. rubrofusarin -6-O- β-O-gentibioside described in claim 1 can be by reducing mTOR, PPAR γ and C/EBP α
Targeting inhibits the lipid accumulation of 3T3-L1 and hAMSCs cell.
5. rubrofusarin -6-O- β-O-gentibioside described in claim 1 can adjust Adipogenesis correlation factor by AMPK
Expression, inhibit lipid accumulation signal path.
6. rubrofusarin -6-O- β-O-gentibioside described in claim 1 can lose weight the big of increase, eWAT and fatty liver
It is small；Fat is inhibited to generate the factor (PPAR γ, C/EBP α, FAS, LPL and aP2) by the AMPK in activation eWAT and liver.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811349282.0A CN109731003B (en) | 2018-11-04 | 2018-11-04 | Application of rubrofusarin-6-O-beta-gentiobioside in preparation of lipid-lowering and weight-losing medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811349282.0A CN109731003B (en) | 2018-11-04 | 2018-11-04 | Application of rubrofusarin-6-O-beta-gentiobioside in preparation of lipid-lowering and weight-losing medicine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109731003A true CN109731003A (en) | 2019-05-10 |
| CN109731003B CN109731003B (en) | 2021-09-10 |
Family
ID=66355523
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811349282.0A Active CN109731003B (en) | 2018-11-04 | 2018-11-04 | Application of rubrofusarin-6-O-beta-gentiobioside in preparation of lipid-lowering and weight-losing medicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109731003B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468536A (en) * | 2003-03-03 | 2004-01-21 | Blood pressure and blood fat lowering herbal food | |
| CN107200760A (en) * | 2016-04-06 | 2017-09-26 | 长沙博海生物科技有限公司 | A kind of preparation method of high-purity rubrofusarin -6-O- β-O-gentibioside |
-
2018
- 2018-11-04 CN CN201811349282.0A patent/CN109731003B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468536A (en) * | 2003-03-03 | 2004-01-21 | Blood pressure and blood fat lowering herbal food | |
| CN107200760A (en) * | 2016-04-06 | 2017-09-26 | 长沙博海生物科技有限公司 | A kind of preparation method of high-purity rubrofusarin -6-O- β-O-gentibioside |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109731003B (en) | 2021-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103222988B (en) | A kind of American-cockroach-extract and its preparation method and application | |
| CN107441078B (en) | A kind of pharmaceutical composition and its preparation method and application for treating diabetes | |
| CN106236801A (en) | Pseudo-ginseng activity medicine that super-micro wall-broken crushing technology is made and health product and preparation method thereof | |
| Yang et al. | Inhibitory effects of cocoa tea (Camellia ptilophylla) in human hepatocellular carcinoma HepG2 in vitro and in vivo through apoptosis | |
| CN106008172A (en) | Preparation method and application of effective part with tyrosinase inhibition effect in mulberry twigs | |
| CN106176918A (en) | A kind of Hyperglycemic health care compositions comprising leaf of Cyclocarya paliurus Iljinskaja and Radix Puerariae | |
| Im et al. | Bioactivity-guided isolation and identification of anti-adipogenic compounds from Sanguisorba officinalis | |
| CN109078011A (en) | The application of iris aglycone and its derivative in prevention and treatment insulin resistance disease medicament | |
| CN103269706A (en) | Anti-cancer extract and compounds | |
| CN103933203A (en) | Compound haemopoietic capsule for treating aplastic anemia and preparation method thereof | |
| CN105012329B (en) | It is a kind of for treating the drug of type-II diabetes | |
| CN108125946A (en) | Application of the dihydromyricetin in terms of kidney medicine is prepared | |
| CN109674866A (en) | A kind of anti-human primary gastrointestinal cancers pharmaceutical composition, preparation method and applications | |
| CN107213145A (en) | Application of the Rabdocetsin B in the product for suppressing esophageal squamous cell cancer cell multiplication is prepared | |
| CN103142774B (en) | Application of total saponin extract of lobedfruit schizocapsarhizome in treatment of liver cancer and nasopharyngeal carcinoma | |
| CN110123827A (en) | A kind of pharmaceutical composition and its preparation method and application treated by metabolic disorder associated diseases | |
| CN110656083A (en) | Pre-adipocyte brown induction kit | |
| CN109731003A (en) | Application of the rubrofusarin -6-O- β-O-gentibioside in preparation lipid-lowering diet drug | |
| CN105560308B (en) | Flower of JINHUAKUI is preparing the application in the product for preventing and treating prostatic disorders | |
| CN109771411A (en) | Dihydroquercetin is used to prepare the purposes in the drug for the treatment of fatty liver | |
| CN102266428B (en) | Anti-ageing Chinese medicinal composition and preparation method and application thereof | |
| CN101744795B (en) | Medicinal composition for treating nevus flammeus and preparation method thereof | |
| CN101474346A (en) | Longstamen onion bulb extract as well as preparation method and application thereof | |
| CN108403683A (en) | Application of the rosin spirit in preparing the product that prevention oxidative stress induces an illness | |
| KR20150077794A (en) | Anti-obesity composition comprising herbal extracts as an active ingredient |
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 |