CN111773273A - Ligustrum robustum total flavone, preparation method and application - Google Patents
Ligustrum robustum total flavone, preparation method and application Download PDFInfo
- Publication number
- CN111773273A CN111773273A CN202010819284.2A CN202010819284A CN111773273A CN 111773273 A CN111773273 A CN 111773273A CN 202010819284 A CN202010819284 A CN 202010819284A CN 111773273 A CN111773273 A CN 111773273A
- Authority
- CN
- China
- Prior art keywords
- ligustrum robustum
- ligustrum
- robustum
- ethanol
- total flavonoids
- 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
- 241000629412 Ligustrum robustum Species 0.000 title claims abstract description 109
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 title claims description 35
- 229930003944 flavone Natural products 0.000 title claims description 29
- 235000011949 flavones Nutrition 0.000 title claims description 29
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 title claims description 28
- 150000002212 flavone derivatives Chemical class 0.000 title claims description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 198
- 229930003935 flavonoid Natural products 0.000 claims abstract description 54
- 235000017173 flavonoids Nutrition 0.000 claims abstract description 54
- 150000002215 flavonoids Chemical class 0.000 claims abstract description 53
- 206010067125 Liver injury Diseases 0.000 claims abstract description 32
- 239000003814 drug Substances 0.000 claims abstract description 30
- 230000001476 alcoholic effect Effects 0.000 claims abstract description 28
- 231100000753 hepatic injury Toxicity 0.000 claims abstract description 28
- 239000000284 extract Substances 0.000 claims description 55
- 239000011347 resin Substances 0.000 claims description 54
- 229920005989 resin Polymers 0.000 claims description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 24
- 239000000463 material Substances 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 16
- 238000001035 drying Methods 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 7
- 235000013305 food Nutrition 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- 239000000469 ethanolic extract Substances 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 208000007848 Alcoholism Diseases 0.000 claims description 3
- 201000007930 alcohol dependence Diseases 0.000 claims description 3
- 206010019133 Hangover Diseases 0.000 claims 1
- 239000000825 pharmaceutical preparation Substances 0.000 claims 1
- 229940127557 pharmaceutical product Drugs 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 69
- 230000001154 acute effect Effects 0.000 abstract description 18
- 238000002474 experimental method Methods 0.000 abstract description 15
- 241001465754 Metazoa Species 0.000 abstract description 8
- 231100000419 toxicity Toxicity 0.000 abstract description 8
- 230000001988 toxicity Effects 0.000 abstract description 8
- 230000003285 pharmacodynamic effect Effects 0.000 abstract description 6
- 210000005036 nerve Anatomy 0.000 abstract description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 37
- 210000004185 liver Anatomy 0.000 description 30
- 238000001179 sorption measurement Methods 0.000 description 29
- 241000699666 Mus <mouse, genus> Species 0.000 description 26
- 230000000694 effects Effects 0.000 description 18
- 239000003480 eluent Substances 0.000 description 18
- 239000012153 distilled water Substances 0.000 description 17
- 230000003068 static effect Effects 0.000 description 15
- 229940079593 drug Drugs 0.000 description 13
- 238000000926 separation method Methods 0.000 description 13
- 210000002966 serum Anatomy 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 239000000126 substance Substances 0.000 description 12
- 238000005303 weighing Methods 0.000 description 12
- 238000011160 research Methods 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 238000011068 loading method Methods 0.000 description 9
- 230000004083 survival effect Effects 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 8
- 238000004364 calculation method Methods 0.000 description 7
- 238000003795 desorption Methods 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- 239000013641 positive control Substances 0.000 description 7
- 230000037396 body weight Effects 0.000 description 6
- 210000005229 liver cell Anatomy 0.000 description 6
- 238000005457 optimization Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- SEBFKMXJBCUCAI-UHFFFAOYSA-N NSC 227190 Natural products C1=C(O)C(OC)=CC(C2C(OC3=CC=C(C=C3O2)C2C(C(=O)C3=C(O)C=C(O)C=C3O2)O)CO)=C1 SEBFKMXJBCUCAI-UHFFFAOYSA-N 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000012856 packing Methods 0.000 description 5
- 239000008194 pharmaceutical composition Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- SEBFKMXJBCUCAI-HKTJVKLFSA-N silibinin Chemical compound C1=C(O)C(OC)=CC([C@@H]2[C@H](OC3=CC=C(C=C3O2)[C@@H]2[C@H](C(=O)C3=C(O)C=C(O)C=C3O2)O)CO)=C1 SEBFKMXJBCUCAI-HKTJVKLFSA-N 0.000 description 5
- 235000014899 silybin Nutrition 0.000 description 5
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 4
- 241000830535 Ligustrum lucidum Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000036528 appetite Effects 0.000 description 4
- 235000019789 appetite Nutrition 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 206010016766 flatulence Diseases 0.000 description 4
- -1 flavonoid compounds Chemical class 0.000 description 4
- 230000037406 food intake Effects 0.000 description 4
- 235000012631 food intake Nutrition 0.000 description 4
- 231100000053 low toxicity Toxicity 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 description 4
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 description 4
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 description 4
- 235000005493 rutin Nutrition 0.000 description 4
- 229960004555 rutoside Drugs 0.000 description 4
- 229920006395 saturated elastomer Polymers 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- FDQAOULAVFHKBX-UHFFFAOYSA-N Isosilybin A Natural products C1=C(O)C(OC)=CC(C2C(OC3=CC(=CC=C3O2)C2C(C(=O)C3=C(O)C=C(O)C=C3O2)O)CO)=C1 FDQAOULAVFHKBX-UHFFFAOYSA-N 0.000 description 3
- VLGROHBNWZUINI-UHFFFAOYSA-N Silybin Natural products COc1cc(ccc1O)C2OC3C=C(C=CC3OC2CO)C4Oc5cc(O)cc(O)c5C(=O)C4O VLGROHBNWZUINI-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 231100000403 acute toxicity Toxicity 0.000 description 3
- 230000007059 acute toxicity Effects 0.000 description 3
- 230000003044 adaptive effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000000857 drug effect Effects 0.000 description 3
- 208000010706 fatty liver disease Diseases 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 210000000936 intestine Anatomy 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000029058 respiratory gaseous exchange Effects 0.000 description 3
- 229940043175 silybin Drugs 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 210000002784 stomach Anatomy 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- WRMNZCZEMHIOCP-UHFFFAOYSA-N 2-phenylethanol Chemical compound OCCC1=CC=CC=C1 WRMNZCZEMHIOCP-UHFFFAOYSA-N 0.000 description 2
- 208000031648 Body Weight Changes Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 208000004930 Fatty Liver Diseases 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 206010019708 Hepatic steatosis Diseases 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 231100000111 LD50 Toxicity 0.000 description 2
- 241000735234 Ligustrum Species 0.000 description 2
- 241000207834 Oleaceae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 206010041349 Somnolence Diseases 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 2
- 231100000439 acute liver injury Toxicity 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000004579 body weight change Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000035622 drinking Effects 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 231100000234 hepatic damage Toxicity 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000008818 liver damage Effects 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000002398 materia medica Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 238000004816 paper chromatography Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 229950000628 silibinin Drugs 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 235000015096 spirit Nutrition 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 231100000240 steatosis hepatitis Toxicity 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012353 t test Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- BNGXYYYYKUGPPF-UHFFFAOYSA-M (3-methylphenyl)methyl-triphenylphosphanium;chloride Chemical compound [Cl-].CC1=CC=CC(C[P+](C=2C=CC=CC=2)(C=2C=CC=CC=2)C=2C=CC=CC=2)=C1 BNGXYYYYKUGPPF-UHFFFAOYSA-M 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 108010082126 Alanine transaminase Proteins 0.000 description 1
- 206010001598 Alcohol intolerance Diseases 0.000 description 1
- 208000007082 Alcoholic Fatty Liver Diseases 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 206010009208 Cirrhosis alcoholic Diseases 0.000 description 1
- 229920002785 Croscarmellose sodium Polymers 0.000 description 1
- 206010013954 Dysphoria Diseases 0.000 description 1
- 206010016262 Fatty liver alcoholic Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 206010019728 Hepatitis alcoholic Diseases 0.000 description 1
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 235000002296 Ilex sandwicensis Nutrition 0.000 description 1
- 235000002294 Ilex volkensiana Nutrition 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 1
- 238000011047 acute toxicity test Methods 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 235000013334 alcoholic beverage Nutrition 0.000 description 1
- 239000012675 alcoholic extract Substances 0.000 description 1
- 208000026594 alcoholic fatty liver disease Diseases 0.000 description 1
- 208000002353 alcoholic hepatitis Diseases 0.000 description 1
- 208000010002 alcoholic liver cirrhosis Diseases 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- CEGOLXSVJUTHNZ-UHFFFAOYSA-K aluminium tristearate Chemical compound [Al+3].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CEGOLXSVJUTHNZ-UHFFFAOYSA-K 0.000 description 1
- VWWHFOBVDHPEKJ-UHFFFAOYSA-N aluminum ethanol trinitrate Chemical compound C(C)O.[N+](=O)([O-])[O-].[Al+3].[N+](=O)([O-])[O-].[N+](=O)([O-])[O-] VWWHFOBVDHPEKJ-UHFFFAOYSA-N 0.000 description 1
- 229940063655 aluminum stearate Drugs 0.000 description 1
- 229960004977 anhydrous lactose Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 229960001681 croscarmellose sodium Drugs 0.000 description 1
- 235000010947 crosslinked sodium carboxy methyl cellulose Nutrition 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000012303 cytoplasmic staining Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 239000007884 disintegrant Substances 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
- 239000006185 dispersion Substances 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 150000002213 flavones Chemical class 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 235000009200 high fat diet Nutrition 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229960001375 lactose Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229940031703 low substituted hydroxypropyl cellulose Drugs 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- HCWCAKKEBCNQJP-UHFFFAOYSA-N magnesium orthosilicate Chemical compound [Mg+2].[Mg+2].[O-][Si]([O-])([O-])[O-] HCWCAKKEBCNQJP-UHFFFAOYSA-N 0.000 description 1
- 239000000391 magnesium silicate Substances 0.000 description 1
- 229910052919 magnesium silicate Inorganic materials 0.000 description 1
- 235000019792 magnesium silicate Nutrition 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- STKUCSFEBXPTAY-GSUVRYNNSA-N methyl (4s,5z,6s)-5-ethylidene-4-[2-oxo-2-[[(2r,3s,4s,5r,6r)-3,4,5-trihydroxy-6-[2-(4-hydroxyphenyl)ethoxy]oxan-2-yl]methoxy]ethyl]-6-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4h-pyran-3-carboxylate Chemical compound O([C@@H]\1OC=C([C@H](C/1=C/C)CC(=O)OC[C@@H]1[C@H]([C@H](O)[C@@H](O)[C@H](OCCC=2C=CC(O)=CC=2)O1)O)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O STKUCSFEBXPTAY-GSUVRYNNSA-N 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 231100000989 no adverse effect Toxicity 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000021590 normal diet Nutrition 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229950008679 protamine sulfate Drugs 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 235000018770 reduced food intake Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 229940032147 starch Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 230000035922 thirst Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 102000014898 transaminase activity proteins Human genes 0.000 description 1
- 210000003708 urethra Anatomy 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012418 validation experiment Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
- A61K36/638—Ligustrum, e.g. Chinese privet
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The application discloses application of ligustrum robustum total flavonoids in the technical field of traditional Chinese medicine preparation and application, and animal experiments prove that the LD50 of the ligustrum robustum total flavonoids is more than 500mg/kg, so that the ligustrum robustum total flavonoids is low in toxicity, can effectively improve the toxicity of alcohol to gastrointestinal tracts and nerves of mice, and has certain pharmacodynamic effect and safety in preventing acute alcoholic liver injury of the mice.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicinal material preparation and application, in particular to ligustrum robustum total flavonoids, a preparation method and application, and more particularly relates to a preparation method and application of ligustrum robustum alcohol extract, and ligustrum robustum total flavonoids, a preparation method and application.
Background
Various alcoholic beverages including white spirits, red spirits and beer have been popular with people. Proper drinking can promote blood circulation, dispel cold and dampness, relieve swelling and pain, relax muscles and tendons and activate blood, but long-term drinking of a large amount of wine can cause alcoholism and alcoholic liver injury. The alcoholic liver injury is one of the common liver diseases in China, and can be further developed into alcoholic fatty liver, alcoholic hepatitis, alcoholic hepatic fibrosis and alcoholic cirrhosis. After a person drinks a large amount of alcohol, different symptoms can appear according to the alcohol content in the body: the mild patient may have nausea, vomiting, dysphoria, incoherence, etc.; in severe cases, organ function will be impaired, resulting in slower breathing and even death. In recent years, the incidence rate of alcoholic liver injury in China is increased year by year, and the incidence rate has become a great hidden danger influencing the healthy life of people.
Roughty glossy privet (Ligustrum robustum (Roxb.) Blume) is a plant of the genus Ligustrum of the family Oleaceae and is included in the book "Chinese materia medica Miao medicine roll" (Guizhou science and technology Press, 2005) for its "heat-clearing, detoxifying and liver-protecting" effects. Broadleaf holly leaf prepared from strong glossy privet leaves is used as a beverage for clearing heat and removing toxicity, promoting the production of body fluid to quench thirst, refreshing and restoring consciousness, and dispelling summer heat and inducing diuresis in the southwest three provinces of China. The main chemical components of the ligustrum robustum comprise flavonoids, terpenoids, phenethyl alcohol and the like. Modern pharmacological research shows that the ligustrum robustum has the effects of resisting oxidation, regulating blood pressure, reducing blood fat, protecting liver, reducing blood sugar, resisting inflammation, relieving pain, resisting bacteria, resisting cancer and the like. Earlier researches find that the ligustrum robustum ethyl acetate extract can effectively improve the fatty liver symptom induced by high-fat diet and reduce the blood fat level. Other related researches also prove that the specnuezhenide has a protective effect on mice with acute liver injury; the ligustrum lucidum total glycosides also have a certain liver protection effect on mice with acute liver injury. At present, no research on the prevention and treatment of acute alcoholic liver injury by total flavonoids of ligustrum robustum (LTF) exists.
Disclosure of Invention
Aiming at the defects of the prior art, the method extracts and purifies the ligustrum robustum total flavonoids, establishes a mouse acute alcoholic liver injury model, and researches the treatment effect of the ligustrum robustum total flavonoids on the mouse acute alcoholic liver injury.
One of the purposes of the invention is to provide the application of the ligustrum robustum alcohol extract in preparing anti-inebriation medicines or anti-inebriation foods.
The invention also aims to provide the application of the ligustrum robustum total flavonoids in preparing the anti-inebriation medicines or anti-inebriation foods.
The invention also aims to provide the application of the ligustrum robustum alcohol extract in preparing the medicine for treating or preventing alcoholic liver injury.
The fourth purpose of the invention is to provide the application of the ligustrum robustum total flavonoids in preparing the medicine for treating or preventing alcoholic liver injury.
The fifth purpose of the invention is to provide a food or a medicament, which comprises the ligustrum robustum alcohol extract and/or ligustrum robustum total flavonoids.
The invention also aims to provide a preparation method of the ligustrum robustum alcohol extract, which comprises the steps of taking ligustrum robustum raw medicinal materials, soaking the ligustrum robustum raw medicinal materials in 80% ethanol serving as a solvent for 7 hours, extracting the ligustrum robustum raw medicinal materials by adopting a hot reflux method, extracting the ligustrum robustum raw medicinal materials twice at 70-80 ℃ for 3 hours each time, mixing extracting solutions, filtering, concentrating and drying the filtrate to obtain the ligustrum robustum alcohol extract.
The seventh purpose of the invention is to provide a preparation method of ligustrum robustum total flavonoids, which comprises the steps of taking ligustrum robustum raw medicinal materials, soaking the raw medicinal materials in 80% ethanol by volume fraction concentration for 7 hours, extracting the raw medicinal materials twice at 70-80 ℃ under hot reflux for 3 hours each time, combining extracting solutions, filtering, concentrating and drying the filtrate to obtain ligustrum robustum ethanol extract; eluting the alcohol extract of the ligustrum robustum by macroporous resin, then sequentially eluting by water, ethanol with the volume fraction of 10% and ethanol with the volume fraction of 50%, collecting the part of the ethanol with the elution concentration of 50%, concentrating and drying to obtain the ligustrum robustum total flavone.
Further, the macroporous resin is AB-8 type macroporous resin.
The macroporous resin is also called high-molecular porous microspheres, is an organic polymer adsorbent with good adsorption performance, has a good macroporous net structure and a large specific surface area, can selectively adsorb organic matters in an aqueous solution, and has a selective adsorption function on natural compounds such as flavonoid compounds, total saponins and the like. The macroporous resin has the characteristics of high stability, safety, low toxicity, good separation effect, easy solvent recovery, regeneration and reutilization and the like, and is widely used for separating and purifying mixtures, particularly traditional Chinese medicine extracts, so that the macroporous resin is used for separating and purifying the ligustrum robustum total flavonoids.
The surface of the AB-8 type macroporous resin is a hydrophobic group, but a certain hydrophilic group is added to the framework of the AB-8 type macroporous resin, so that the AB-8 type macroporous resin is suitable for separating and purifying components with weak polarity such as flavonoids, alkaloids and the like. In order to research the characteristics of AB-8 type macroporous resin on the separation and purification of the total flavonoids of the ligustrum robustum, the research adopts experiments such as static adsorption, dynamic adsorption and the like to comprehensively analyze the adsorption performance of the AB-8 type macroporous resin. The result shows that the AB-8 type macroporous resin has good adsorption effect and desorption effect on the total flavonoids of the ligustrum robustum.
In order to obtain the optimized separation and purification parameters, the research adopts a single-factor method to investigate the concentration of a sample loading solution, the ethanol concentration of an eluent and the volume of the eluent of the macroporous resin, and the obtained optimal separation and purification conditions are as follows: using AB-8 type macroporous resin as a separation material, taking the sample concentration of the alcohol extract as 168mg/ml, taking the sample amount as 1/3BV, eluting with 8BV of water, 10% ethanol by volume fraction and 50% ethanol by volume fraction in sequence, collecting 50% ethanol eluent, and concentrating under reduced pressure to dryness.
Because the ligustrum robustum alcohol extract is not easy to dissolve in water, when the ligustrum robustum alcohol extract is prepared, part of alcohol extract is not completely dissolved in water, the measured value of the total flavone content in the ligustrum robustum alcohol extract is lower than the actual content, however, when the ligustrum robustum alcohol extract is loaded and eluted, the incompletely dissolved substances are loaded and eluted together, and the final yield of the total flavone is more than 100%.
The seventh purpose of the invention is to provide the total flavonoids of ligustrum robustum prepared by the method.
The working principle of the invention is as follows: after alcohol is metabolized in the liver, the produced excessive free radicals can excessively oxidize lipid components on the surface of a biological membrane, so that a mitochondrial membrane and a hepatocyte membrane are damaged, and AST and ALT enter blood in a large amount. ALT content is highest in liver cells, while AST is distributed in tissues such as cardiac muscle, liver, skeletal muscle and kidney. Normally, there is a significant increase in serum ALT activity as long as a small amount of ALT is released into the blood. And the AST content in serum is low, when liver cells are necrotized, ALT is released into blood in a large amount, the permeability of cell membranes is increased, and therefore the AST in cytoplasm is released into blood, and the AST content in serum is increased. AST and ALT are the most commonly used indicators of liver damage. Therefore, by measuring the AST and ALT levels in serum, the result of the pathological liver section of the experimental mouse is observed to indicate the liver disease signal.
The male mice are randomly divided into a normal group, a model group, a negative control group, a silibinin positive control group (30mg/kg) and a high, medium and low dose group of ligustrum robustum total flavonoids (LTF) according to the weight, and the administration dose is 800, 400 and 200mg/kg respectively. Except for the normal group, mice in each group were continuously gavaged with 53% ethanol (10mL/kg) for 2 weeks. Mice in the high, medium and low dose groups were given the corresponding dose of LTF solution (0.5% CMC-Na) with silybin as positive control. The mice were observed daily for fur, appetite, behavioral status, and the like. After 2 weeks of continuous administration, changes in liver, intestine and stomach were visually observed; serum ALT and AST levels of mice in each group were measured. As a result: compared with the normal group, the liver index of the mouse in the model group has significant difference (P)<0.01); compared with the model group, the liver indexes of the mice in the normal group have very significant difference (P)<0.01). Serum ALT levels were significantly reduced in the high, medium and low dose LTF groups compared to the model group (P)<0.05) ALT is obviously different because ALT is sensitive, and the ALT in serum can be greatly increased as long as liver cells are damaged. LD of LTF50>500mg/kg, indicating lower LTF toxicity. And (4) conclusion: the LTF can effectively improve the toxicity of alcohol to gastrointestinal tracts and nerves of mice, and has certain drug effect and safety on preventing acute alcoholic liver injury of the mice.
LD50The minimum drug dosage required for causing half of the death of experimental animals can be used for judging the safety of the drugs and is a safety scale for measuring the drug exposure of human beings.
The experiment adopts 53% ethanol to simulate a mouse acute alcoholic liver injury model. Due to the reduction of the food intake of the mice, the food intake required by the mice cannot be met, so that the weight of the mice is obviously reduced. The death of mice may be caused by various factors such as flatulence due to alcohol stimulation, nerve inhibition by alcohol, and intolerance to alcohol due to individual differences of mice. Late death may be due to the initial formation of acute alcoholic liver injury, which in turn leads to the development of complications associated with alcoholic liver injury.
The liver is the main metabolic point of alcohol, however, the alcohol can cause the liver to generate alcoholic liver function damage and organic degeneration, so that the liver quality is increased, and the liver index can show the formation of liver damage to a certain extent, so that the condition of liver weight increment is generated. In this experiment, it was determined that the liver index of the model group mice was slightly higher than that of the other group mice, probably due to the formation of fatty liver, resulting in an increase in liver mass.
Acute toxicity refers to a toxic reaction, including a lethal effect, which occurs in a short period after a human or an animal is contacted with an exogenous chemical once (which means that the toxicity of the exogenous chemical is too low and the exogenous chemical needs to be given to the animal in a large dose) or for multiple times within 24 hours (which means that the toxicity of the exogenous chemical is too low and the exogenous chemical needs to be given to the animal). The acute toxicity test is carried out on the medicine, aims to provide reliable scientific basis for the toxic action of the medicine in a short time and the clinical diagnosis, prevention and emergency treatment of the medicine. In the preliminary experiment, mice were gavaged several times within 24h at doses of 5, 50, 500, 5000mg/kg of LTF, and did not die. Therefore, the test results of the preliminary test and the data on the Ligustrum robustum recorded in the "Chinese materia medica seedling medicated roll" were combined to carry out the actual tests with the dosage of 200mg/kg, 400mg/kg and 800 mg/kg. The experimental result shows that the LTF has certain safety and does not cause the death of mice and other abnormal changes.
LD50The minimum drug dosage required for causing half of the death of experimental animals can be used for judging the safety of the drugs and is a safety scale for measuring the drug exposure of human beings. The larger the LD50, the lower the toxicity of the drug, the safer; otherwise, LD50Smaller indicates more toxic and dangerous drugs. Most of the traditional Chinese medicinal materials have mild effects and low toxicity, and LD cannot be measured50. In the experimental process, after the mice are perfused with the LTF, no abnormal reaction and death phenomenon occur in each dose group, and LD (laser diode) exists50>500mg/kg, which preliminarily shows that the LTF belongs to low toxicity grade and has higher safety.
The ligustrum robustum alcohol extract and/or ligustrum robustum total flavone can be used independently or in the form of a pharmaceutical composition. The pharmaceutical composition comprises the ligustrum robustum alcohol extract and/or ligustrum robustum total flavonoids and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition of the present invention comprises 0.1 to 99.9% by weight of the alcoholic extract of ligustrum robustum and/or total flavonoids of ligustrum robustum of the present invention as an active ingredient.
The medicinal carrier does not damage the pharmaceutical activity of the ligustrum robustum alcohol extract and/or ligustrum robustum total flavonoids, and the effective dosage of the medicinal carrier can be nontoxic to human bodies. Pharmaceutically acceptable carriers include, but are not limited to: lecithin, aluminum stearate, alumina, ion exchange materials, self-emulsifying drug delivery systems, tweens or other surfactants, serum proteins, buffer substances such as phosphates, glycine, sorbic acid, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, magnesium silicate, mixtures of saturated fatty acid partial glycerides, and the like.
Other conventional pharmaceutical adjuvants such as binder (e.g. microcrystalline cellulose), filler (e.g. starch, glucose, anhydrous lactose and lactose beads), disintegrant (e.g. crosslinked PVP, croscarmellose sodium, low-substituted hydroxypropylcellulose), lubricant (e.g. magnesium stearate), and absorption enhancer, adsorption carrier, flavoring agent, sweetening agent, excipient, diluent, wetting agent, etc.
The ligustrum robustum alcohol extract and/or ligustrum robustum total flavone and the pharmaceutical composition thereof can be prepared according to the conventional method in the field and can be administrated by intestinal or parenteral or local routes. The oral preparation comprises capsule, tablet, oral liquid, granule, pill, powder, pellet, and unguent; parenteral preparations include injections and the like; topical preparations include creams, patches, ointments, sprays, and the like. Oral formulations are preferred.
The administration route of the ligustrum robustum alcohol extract and/or ligustrum robustum total flavone and the pharmaceutical composition thereof can be oral administration, sublingual administration, transdermal administration, intramuscular administration, subcutaneous administration, skin mucosa administration, vein administration, urethra administration, vagina administration and the like.
Drawings
FIG. 1 is a technical roadmap for the present invention;
FIG. 2 is a schematic diagram showing the elution yield of the total flavone extract;
FIG. 3 is a graph showing the concentration of total flavonoids in an equal volume of eluate;
FIG. 4 is a graph showing the body weight trend of an acute toxicity pre-test mouse;
FIG. 5 is a schematic diagram showing the effect of alcohol on mouse organs;
FIG. 6 is a graph showing the trend of alcohol vs. body weight of mice;
FIG. 7 is a schematic diagram showing the effect of alcohol on mouse organs;
FIG. 8 is a schematic diagram of pathological liver section of an acute alcoholic liver injury mouse;
FIG. 9 is a schematic diagram showing the body weight variation trend of mice with acute alcoholic liver injury;
FIG. 10 is a graph showing the results of ALT assay in serum;
FIG. 11 is a graph showing the results of AST measurement in serum.
Detailed Description
The following is further detailed by way of specific embodiments:
1 Material
(1) Medicinal materials and reagents: rough glossy privet leaves (collected from Zunyi Fenggang county in Guizhou, identified that the original plant is Ligustrum robustum (Roxb.) Blume in Ligustrum, Oleaceae), Co60 irradiation laboratory mouse maintenance feed (Jiangsu cooperative medical bioengineering, LLC), silibinin (batch number: 850610070, Tianjin Tianshi force pharmacy Co., Ltd.), Gualajing alanine transaminase and Gucao transaminase kit (batch number: 20191227, Nanjing Ministry of bioengineering research), sodium carboxymethylcellulose (batch number: 20130821, specification: 500g) and 95% ethanol (analytically pure).
(2) Animals: SPF grade 8 week old C57/BL6J male mice 88 were supplied by sbeft (beijing) biotechnology limited [ license number: SCXK (Beijing) 2019-0010, and feeding the animals in an environment with room temperature of 20 +/-2 ℃, relative humidity of 50-70% and 12h alternating illumination and darkness, wherein the animals can drink water and eat food freely.
(3) The instrument comprises the following steps: one thousandth electronic analytical balance (sandoliss, usa), H-1600RW micro high-speed desktop refrigerated centrifuge (shanghai collodion instruments ltd), thermo multiskan GO all-wavelength microplate reader (seemorfei, usa).
2 method and step
2.1 extraction of Total Flavonoids from Ligustrum robustum
Weighing 1.5kg of raw ligustrum robustum material, adopting a hot reflux method for extraction, taking 80% ethanol as a solvent, soaking for 7 hours, carrying out hot reflux extraction for 2 times, 3 hours each time, adding 20 times of 80% ethanol for the 1 st time, adding 12 times of 80% ethanol for the 2 nd time, combining the extracting solutions, filtering, concentrating and drying to obtain the ligustrum robustum alcohol extract, and freezing and storing at-20 ℃ for later use. And (3) calculating the yield of the alcohol extract of the ligustrum robustum, wherein the yield calculation formula is as follows:
2.1.1 preparation of the solution
(1) Preparing a sample solution: taking about 25mg of the ligustrum robustum alcohol extract, precisely weighing, dissolving methanol and fixing the volume to 50ml to obtain the alcohol extract sample solution. Taking about 0.02g of total flavone part eluted by the macroporous resin, precisely weighing, dissolving by using methanol, fixing the volume to 10ml, precisely sucking 1.0ml to 10ml of volumetric flask, and fixing the volume to the scale by using distilled water to obtain the total flavone sample solution.
(2) Preparing a reference substance solution: weighing rutin standard substance about 0.025g, precisely weighing, dissolving with methanol, metering to 50ml, and shaking to obtain 0.504mg/ml rutin control solution.
2.1.2 quality of Total Flavonoids fraction
The extract yield and the total flavone yield are two important indexes of the separation efficiency of the macroporous resin. And mixing the eluates, recovering ethanol under reduced pressure, concentrating to dry, and weighing to obtain extract. The calculation formula of the extract yield is as follows:
the calculation formula of the total flavone yield is as follows:
2.1.3 paper chromatography identification of flavones
Detecting the total flavone in the macroporous resin column chromatography eluent by a paper chromatography color development method. The specific operation is as follows: dipping the eluent with a glass rod, spotting on dry filter paper, spraying 5% aluminum nitrate-ethanol developer, and heating for 30 s. The color should appear gray yellow under visible light and yellow fluorescent spot under ultraviolet light.
2.1.4 measurement of content
Precisely measuring an appropriate amount of solution, placing in a 10ml volumetric flask, adding distilled water to 2.0ml, adding 5% sodium nitrite solution 0.4ml, and standing for 10 min; then adding 0.4ml of 10% aluminum nitrate solution, and standing for 6 min; adding 5% sodium hydroxide solution 4.0ml, adding distilled water to desired volume, shaking, standing for 15min, and measuring absorbance at maximum absorption wavelength. The content is calculated by an external standard curve method by taking distilled water as a blank.
2.1.5 drawing of Standard Curve
Accurately measuring rutin standard substance solution 0, 0.4, 0.8, 1.2, 1.6, and 2.0ml, adding water to 2.0ml, developing according to the method of '1.2.5', detecting wavelength at 517nm, measuring absorbance value, and drawing standard curve with concentration as abscissa and absorbance as ordinate.
2.2 separation and purification of Total Flavonoids of Ligustrum robustum
2.2.1 examination of adsorption Properties of AB-8 type macroporous resin
The adsorption performance of AB-8 type macroporous resin on the total flavonoids of the ligustrum robustum is analyzed through a static adsorption test and a dynamic adsorption test. The static adsorption test is to investigate the difference of the total flavone content in the solution before and after the macroporous resin adsorbs the sample; the dynamic adsorption experiment is to pack the resin into a column by a wet method, load the resin into the column, elute the resin to be colorless by distilled water after the resin is fully adsorbed, and investigate the difference of the total flavone content in the loading liquid before and after adsorption.
(1) Resin pretreatment: soaking AB-8 type macroporous resin in 95% ethanol for 24 hr, wet packing, eluting with 95% ethanol until the effluent is mixed with distilled water at a ratio of 1: 5 to avoid turbidity, and washing with distilled water until no alcohol smell is produced. Storing in a wet state in a sealed manner for later use.
(2) Column volume measurement: and (5) packing the column by a wet method. After fully standing, putting the liquid in the column, slowly injecting distilled water with known volume into the chromatographic column, and lightly knocking the chromatographic column to remove air bubbles until the liquid level is level to the resin interface. The column volume (BV) calculation formula is as follows:
column volume BV (ml) known as distilled water volume (V)1) Volume of residual distilled water (V)2)
(3) Static adsorption test: measuring 5ml of each pretreated AB-8 type macroporous resin, placing the pretreated AB-8 type macroporous resin into a conical flask with a plug, precisely adding 50.0ml of Ligustrum robustum alcohol extract solution with the concentration of 168mg/ml, sealing, placing the mixture into a shaking table, shaking for 24 hours at room temperature at 60r/min, taking a proper amount of supernatant, and measuring the content of total flavonoids in the supernatant according to the method under the item '2.1.4', thereby calculating the static saturated adsorption capacity of the resin. The calculation formula of the static saturated adsorption amount is as follows:
separating the resin and the adsorbed solution by adopting a filtration method, putting the macroporous resin in a conical flask with a plug after the macroporous resin is dried by using filter paper, adding 100ml of 70% ethanol solution, shaking and desorbing for 24 hours at 60r/min, taking a proper amount of supernatant, and measuring the content of total flavonoids in the supernatant according to the method under the item 2.1.4 so as to calculate the concentration of the total flavonoids in the desorption solution and the static desorption rate. The calculation formula of the static desorption rate is as follows:
(4) dynamic adsorption test: accurately measuring 250ml of AB-8 type macroporous resin, performing wet column packing, loading a 168mg/ml ethanol extract solution of Ligustrum robustum at a flow rate of 2BV/h (BV is the volume of the column), and collecting the effluent, wherein 10 parts in total are collected, and the volume of each part is 1 BV. The volume of the effluent liquid (abscissa) and the concentration of the total flavonoids (ordinate) are used as dynamic adsorption curves for adsorbing the total flavonoids by the macroporous resin.
2.2.2 optimization of separation parameters
The separation parameter optimization considers two factors which have the largest influence in the separation process, including the optimization of dynamic adsorption conditions and the optimization of desorption conditions.
(1) Effect of sample solution concentration: respectively weighing appropriate amount of Ligustrum robustum extract, and respectively adding distilled water to obtain 5 solutions with different concentrations of 56, 112, 168, 224, and 280 mg/ml. 10ml of each ligustrum robustum alcohol extract solution. Accurately pipette 1.0ml of solution into a dry centrifuge tube of known weight, accurately weigh, and calculate the solution density.
(2) Gradient selection of ethanol concentration of eluent: the pretreated macroporous resin 955ml is taken for wet column packing, and the sample solution is 168 mg/ml. After sampling, standing for 1h to ensure that the resin is fully adsorbed, washing the macroporous resin by 8BV (column volume) of distilled water at the flow rate of 2.5BV/h, then sequentially eluting by 8BV of ethanol solutions with the concentrations of 10%, 30%, 40%, 50%, 60%, 70% and 90%, respectively collecting eluates with different concentrations, and determining the concentration of total flavonoids in the eluates and the extract yield according to the method under the item 2.1.4, thereby selecting the optimal ethanol concentration gradient of the eluates.
(3) Selection of eluent dosage: according to the experimental results, the dosage of the eluent is considered. And (3) taking 250ml of the pretreated macroporous resin, carrying out wet column packing, balancing the chromatographic column, and then loading 56.5ml of the macroporous resin with the ethanol extraction concentration of 168 mg/ml. Standing for 1h after the sample liquid is adsorbed by the resin to ensure that the absorption is sufficient. The macroporous resin is washed by 8BV of distilled water at the flow rate of 2.5BV/h, and elution is carried out according to the optimal ethanol concentration gradient of the eluent. Collecting the eluent step by step according to 1BV until no total flavone is detected in the eluent.
2.2.3 validation of isolation conditions
Sampling 2800ml of the pretreated macroporous resin according to half of the maximum adsorption amount measured by a static adsorption experiment, separating and purifying the ligustrum robustum total flavone according to optimized conditions, and calculating the extract yield and the total flavone yield of the total flavone part.
2.3 animal experiments
2.3.1 technical scheme as shown in figure 1.
2.3.2 preparation of the medicinal liquid
Accurately weighing 1.257g of LTF, adding a proper amount of 0.5% CMC-Na aqueous solution for dispersion, and fixing the volume to 10.0mL to obtain 125.7mg/mL LTF solution a. The LTF solution a is diluted in a gradient way to prepare high, medium and low liquid medicines with the concentration of 12.57mg/mL, 1.257 and 0.1257mg/mL respectively, and the prepared liquid medicines are stored at 4 ℃.
Accurately weighing 10.02g of LTF, placing the LTF in a 100mL volumetric flask, adding 0.5% CMC-Na aqueous solution to fix the volume to a scale, and uniformly mixing to obtain an LTF solution b with the concentration of 100.2 mg/mL; respectively and precisely measuring 50.0mL, 25.0mL and 12.5mL of LTF liquid medicine b, and fixing the volume of 0.5% CMC-Na aqueous solution to 100mL, and uniformly mixing to obtain liquid medicines with the high, medium and low concentrations of LTF of 50.10mg/mL, 25.05mg/mL and 12.5mg/mL respectively, and storing in a refrigerator at 4 ℃.
Grinding the content of silybin capsule, precisely weighing appropriate amount of fine powder, adding 0.5% CMC-Na water solution, and dispersing to obtain 0.2mg/mL silybin positive control liquid medicine.
2.3.3 Pre-testing of the dose administered
10 male mice, C57/BL6J, were randomly divided by body weight into a normal group and 4 different dose groups (5, 50, 500, 5000mg/kg), each group consisting of 2 mice. After adaptive feeding for 7d, fasting for 1h before each administration, performing intragastric lavage on each administration group according to the corresponding medicine liquid dose, and administering equal amount of distilled water to normal groups, and performing equal division twice at an interval of 4 h. After 30min of each administration, the mice were observed for coat, appetite and behavior 1 time, then 1 time per hour for 24h for 1 week.
2.3.4 establishment of acute alcoholic liver injury model
The C57/BL6J male mice are randomly divided into a normal group and a model group according to the body weight, and each group comprises 10-15 mice. After 7d of adaptive feeding, 53% ethanol (10mL/kg) was administered to each of the groups except the normal group every morning, and the normal group was administered with an equal amount of distilled water for 1 week continuously.
2.3.5 pharmacodynamic experiment of Ligustrum robustum Total Flavonoids
53 male mice C57/BL6J were randomly divided into 7 groups of 6-8 mice each, based on body weight, including a normal group, a model group, a negative control group, a positive control group (30mg/kg), and high-, medium-, and low-dose LTF groups (800, 400, and 200 mg/kg). After 7d of adaptive feeding, in the morning of each day, distilled water is given to a normal group and a model group according to the ratio of 0.2mL/10g, a negative control group is given to a 0.5% CMC-Na aqueous solution according to the same dosage, each administration group is subjected to intragastric administration according to the same dosage of liquid medicine, in the afternoon of the same day, except the normal group, 53% ethanol (10mL/kg) is given to each group for intragastric administration, the normal group is given to distilled water with the same dosage, after 30min of each administration, the fur, appetite and behavior of the mouse are observed for 1 time, then the skin, the appetite and the behavior of the mouse are observed for 1 time per hour, the observation is carried out for 24 hours, and the intra.
After the last administration, the mice were fasted for 16h without water deprivation, weighed (Wt), bled through the eye sockets, the blood was allowed to stand at 4 ℃ for 30min, centrifuged at 4500 ℃ for 15min, the serum was separated, and the AST and ALT contents in the serum were measured according to the kit instructions.
The mice were then sacrificed by cervical dislocation and after death, the gastric, intestinal and hepatic changes were observed. Mouse livers were isolated and weighed and liver indices were calculated. The liver index formula is:
calculation of median Lethal Dose (LD) according to modified Karber method50) The formula is as follows:
wherein Xm is the log of the highest dose; i is the difference between adjacent 2 sets of logarithmic doses (i.e., set distance); pm is the highest mortality rate; pn is the lowest mortality; p is the mortality rate (mortality rate is indicated in decimal numbers) of each dose group.
2.4 statistical analysis
The IBM SPSS 25.0 statistical software is adopted for analysis, data results are expressed as mean value plus or minus Standard Deviation (SD), and differences among groups of mice of different groups are compared and analyzed by using statistical methods such as one-way variance test (ANOVA), t-test (t-test) and the like so as to verify the pharmacological action of the total flavonoids of the ligustrum robustum.
3 results
3.1 extraction of Total Flavonoids of Ligustrum robustum
Weighing 1.5kg of raw ligustrum robustum material, soaking in 80% ethanol for 7h, and extracting under reflux at 70-80 deg.C twice, each for 3h, with the ratio of the first material to the liquid being 1: 20 and the second being 1: 12. Mixing extractive solutions, filtering, concentrating the filtrate, and drying to obtain Ligustrum robustum ethanol extract. 842.5g of ligustrum robustum alcohol extract, the yield is 56.17%. The total flavone content was determined according to the standard curve method under the term "2.1.5". The linear regression equation is that y is 0.0148x-0.008, and r is 0.9992, and rutin shows a good linear relation in the range of 20.46-100.80 mu g/ml. The percentage content of the total flavone in the ligustrum robustum alcohol extract is measured to be 19.9 percent.
3.2 isolation of Total Flavonoids of Ligustrum robustum
The separation and purification conditions of the total flavonoids of the ligustrum robustum are as follows: collecting 2800ml AB-8 type macroporous resin, eluting with water, 10% ethanol, and 50% ethanol in sequence, each eluting with 8 times column volume gradient, collecting 50% ethanol part, concentrating, and drying to obtain total flavone part (LTF) of Ligustrum robustum. The yield of the extract is 53.4 percent, and the content of flavone is 44.5 percent.
3.2.1 results of adsorption experiments
The results of the static adsorption amount and desorption rate tests of the macroporous resin on the total flavonoids of the ligustrum robustum are respectively shown in the table 1 and the table 2. The AB-8 type macroporous resin static saturated adsorption capacity is 162.89mg/g, and the static resolution is 36.08%. The results of static adsorption capacity and analysis rate experiments show that the AB-8 type macroporous resin has good adsorption and analysis effects on the total flavonoids of the ligustrum robustum.
Table 1: AB-8 type macroporous resin static saturated adsorption capacity determination result
Table 2: AB-8 type macroporous resin static desorption rate determination result
3.2.2 results of optimization of separation conditions
In the selection of loading concentrations, 5 different loading concentrations were set and the density of the loading solution was determined (table 3). The density of the solution is increased along with the increase of the concentration of the solution, the density of the 280mg/ml alcohol extract solution is the maximum, the density of 168mg/ml and 224mg/ml alcohol extract solutions is similar, but the 280mg/ml alcohol extract solution has the most insoluble substances and is not selected; the density and insoluble content of 168mg/ml and 224mg/ml alcohol extract solutions are similar, so 168mg/ml alcohol extract solution is selected as the loading concentration.
Table 3: concentration selection of sample solutions
Note: "+", little; "+ + +", less; "+ + + +", generally; "+ + + + + +", much more.
In the optimization of the dynamic analysis conditions, a single-factor method is also adopted, the eluates with different ethanol concentrations are collected, the absorbance is measured, and the total flavone concentration in the eluates is taken as the ordinate and the ethanol concentration is taken as the abscissa for plotting, as shown in fig. 3. It can be seen that, when the concentration of the eluent ethanol is 30%, the concentration of flavone in the eluent is at most 94.51mg/ml, when the concentration of the eluent ethanol exceeds 50%, the concentration of total flavone of the ligustrum robustum in the eluent begins to decrease, and the concentration of total flavone is lower when the concentration of the ethanol is 70%; concentrating, drying and weighing the eluents with different ethanol concentrations to obtain total flavone part extracts with different ethanol concentrations, and plotting the total flavone part extract mass as ordinate and the ethanol concentration as abscissa, as shown in fig. 2, when the ethanol concentration of the eluent is 50%, the total flavone part extract is the most, the ethanol concentration is lower when the ethanol concentration is 70%, and the cumulative percentage of the extracts exceeds 91.2% when the ethanol concentration is 60%. Therefore, the optimal eluent concentration is most suitable to select 60% ethanol by comprehensive consideration.
3.2.3 validation experiments
By combining the experimental results, the optimized AB-8 type macroporous resin conditions for separating the total flavonoids of the ligustrum robustum are as follows: collecting 2800ml AB-8 type macroporous resin, loading the ethanol extract of Ligustrum robustum with concentration of 168mg/ml, standing for adsorption, eluting with water, 10% and 50% ethanol sequentially, each gradient is 8BV, collecting 50% ethanol part, concentrating, and drying to obtain Ligustrum robustum total flavone part. The extract yield was 53.4% and the flavone content was 44.5% (see table 4). The identification shows that the fluorescent substance shows gray yellow under visible light and yellow fluorescent spot under ultraviolet light.
Table 4: verification of the results of the experiment
3.2 pharmacodynamic Activity of Ligustrum robustum Total Flavonoids on acute alcoholic liver injury
3.2.1 dose Range findings
Research shows that the daily dose of the ligustrum robustum is 10-15 g, the dosage of the ligustrum robustum converted into the LTF is 1.3-2.0 g according to the separation and purification yield of the LTF, and the administration dose of the mice is 303.7mg/kg according to 60kg of the weight of an adult. To investigate the safety and maximum dose of LTF, the dose range of LTF was examined.
According to the method of item 2.3.3, the mice were good in physical condition, had a normal diet, were free from movement, had glossy hair, and had no phenomenon of death (survival rate: number of mice surviving/total number of mice × 100%) after being administered with different doses of total flavonoids of ligustrum robustum. The total flavonoids of ligustrum robustum are shown to have certain safety within the dosage range of 5-5000 mg/kg (see table 5).
Table 5: acute toxicity pretest mouse survival
The body weight change trend was not significant in the mice of each group in the dose study (see FIG. 4). Indicating that the effect of different doses of LTF on body weight changes in mice falls within the normal range.
The morphology of the liver of mice in each dose group of LTF was observed, and the liver of the mice was reddish brown, smooth and glossy, and had no inflammation (see FIG. 5). Indicating that LTF did not adversely affect the liver of the mice.
3.3 acute alcoholic liver injury model Observation
Compared with the normal group, the mice in the model group all appeared in a drunken state after being perfused with 52% alcohol (10mL/kg), and were slow in movement and teeter, and were drowsy, sleepy, slow in breathing, reduced in food intake, lusterless in hair, and even had a creaking sound. After one week, the survival rate of the model group mice was 20.0%, and the survival rate of the normal group was 100.0% (see table 6).
Table 6: survival rate of mice with acute alcoholic liver injury
In the experimental process of establishing the acute alcoholic liver injury model, compared with the initial weight, the weight of the model group mouse changes more obviously than that of the normal group mouse, and is in a greatly reduced trend (see fig. 6).
During the alcohol molding process, the dead mice are subjected to a autopsy, and the phenomena of extremely severe flatulence in the intestines and the stomach, a small amount of yellow mucus in the intestines and no content in the stomach can be observed visually in most of the mice (see fig. 7). It was shown that mice developed acute alcoholism and the organs were damaged.
3.4 pharmacodynamic test of Total Flavonoids
In pharmacodynamic experiments, all mice in the normal group had normal conditions, diet, activity and food intake, and had glossy hair. After the other groups of mice are subjected to gastric lavage with alcohol, the mice are drunk, and the mild mice have slow movements and unstable walking, and the severe mice have drunk, sleepy, slow breathing, reduced food intake and lusterless hair.
Except for the survival of all normal groups, 12 patients died in all the other groups due to intestinal flatulence and flatulence caused by alcohol stimulation, alcohol intolerance, alcoholic liver injury poisoning related complications and the like, and the survival rate of the normal group is 100%, and the survival rate of the other groups is shown in table 7. The results show that LTF significantly reduces mortality in mice. Calculated according to the modified Karber method, LD50817.5mg/kg, greater than 500mg/kg, so LTF is of low toxicity grade.
Table 7: pharmacodynamic experiment mouse survival rate
The pathological section results of mouse liver show (see fig. 8) that normal mouse liver cells are complete in structure and similar in size, have large and circular or elliptical nuclei with lobular central veins as centers, and have uniform cytoplasmic staining without liver lesions. The liver lobules of the model group mouse and the negative control group mouse are not clear, the liver cell cord is disordered, the liver sinuses are narrowed, the liver cells are obviously swelled, and part of the area is infiltrated by ballooning and inflammatory cells, so that the liver is obviously damaged by alcohol. Compared with the model group, particularly the high dose group (800mg/mL), the positive control group and the LTF different dose group have the advantages that the hepatocyte swelling is obviously reduced, the cell nucleus is further normalized, and the liver tissue of the mouse is repaired to a certain extent. Meanwhile, the weight changes of the mice in each group before and after the experiment are compared, and the weight of the mice in each group is found to be in a descending trend compared with that of the mice in a normal group (see figure 9). The weight change of the normal group of mice is stable, the weight of the model group of mice is continuously reduced (4 +/-1 g), and the weight change trends of the rest groups of mice are the same and accord with the growth and development rules of the mice.
Compared with the normal group, the liver index of the model group mice is significantly increased (P <0.01), and the LTF dose groups have no significant change (see Table 8). The results show that LTF has a certain relieving effect on the pathological changes of the liver of the mice given alcohol.
Table 8: effect of Ligustrum robustum Total Flavonoids on mouse liver index
Note: in comparison with the normal group,**P<0.01; in comparison to the set of models,##P<0.01。
the ALT and AST level measurement results in the mouse serum show that the ALT level of the model group, the negative control group and the positive control group is higher than that of the normal group, and the ALT level of each LTF dose group is obviously lower than that of the normal group (p <0.05) (see figure 10). The AST levels in the positive control, LTF dose groups were comparable to the normal group, with each of the remaining groups being slightly higher than the normal group (see FIG. 11). The results show that a mouse liver injury model is formed initially.
ALT and AST levels of the model group and the negative control group are higher than those of the normal group. ALT was significantly increased in the negative control group compared to the normal group (P < 0.01); compared with the model group, the ALT levels of the LTF high, medium and low dose groups were statistically different (P <0.05), and the AST level was not significantly different (see Table 9). The results show that LTF can effectively prevent the formation of acute alcoholic liver injury and prevent the further development of liver injury.
Table 9: determination of AST and ALT in mouse serum
Note: in comparison with the normal group,*P<0.05，**P<0.01; in comparison with the set of models,#P<0.05，##P<0.01。
the experimental result shows that the LTF has higher safety and certain drug effect on mice with acute alcoholic liver injury, and has no adverse effect on the appearance, behavior state, visceral organs and the like of the mice. The LTF can effectively improve the stimulation of alcohol to the gastrointestinal tract and nerve of mice, and has a certain drug effect on the prevention of acute alcoholic liver injury of the mice.
Claims (9)
1. The application of the ligustrum robustum alcohol extract in preparing a medicine or a food for relieving alcoholism is characterized in that the ligustrum robustum alcohol extract contains ligustrum robustum total flavonoids.
2. Application of Ligustrum robustum total flavone in preparing anti-hangover medicine or food is provided.
3. The application of the ligustrum robustum alcohol extract in preparing the medicine for treating or preventing alcoholic liver injury is characterized in that the ligustrum robustum alcohol extract contains ligustrum robustum total flavonoids.
4. Application of ligustrum robustum total flavonoids in preparing medicine for treating or preventing alcoholic liver injury is provided.
5. A food or pharmaceutical product characterized by: comprises an alcohol extract of Ligustrum robustum and/or total flavonoids of Ligustrum robustum.
6. The preparation method of the ligustrum robustum alcohol extract is characterized by comprising the following steps: soaking raw ligustrum robustum in 80 vol% ethanol as a solvent for 7 hours, extracting by a hot reflux method, extracting twice at 70-80 ℃ for 3 hours each time, wherein the ratio of the first material to the liquid is 1: 20, and the second time is 1: 12, combining the extracting solutions, filtering, concentrating the filtrate, and drying to obtain the ligustrum robustum alcohol extract.
7. The preparation method of the total flavonoids of the ligustrum robustum is characterized by comprising the following steps: soaking raw ligustrum robustum in 80 vol% ethanol for 7h, extracting twice at 70-80 deg.C for 3h each time under reflux, mixing extractive solutions, filtering, concentrating the filtrate, and drying to obtain ligustrum robustum ethanol extract; eluting the alcohol extract of the ligustrum robustum by macroporous resin, then sequentially eluting by water, ethanol with the volume fraction of 10% and ethanol with the volume fraction of 50%, collecting the part of the ethanol with the elution concentration of 50%, concentrating and drying to obtain the ligustrum robustum total flavone.
8. The method for preparing Ligustrum robustum total flavonoids according to claim 7, wherein the extract comprises the following components: the macroporous resin is AB-8 type macroporous resin.
9. Ligustrum robustum total flavonoids prepared by the process of claim 7 or 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010819284.2A CN111773273A (en) | 2020-08-14 | 2020-08-14 | Ligustrum robustum total flavone, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010819284.2A CN111773273A (en) | 2020-08-14 | 2020-08-14 | Ligustrum robustum total flavone, preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111773273A true CN111773273A (en) | 2020-10-16 |
Family
ID=72762481
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010819284.2A Pending CN111773273A (en) | 2020-08-14 | 2020-08-14 | Ligustrum robustum total flavone, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111773273A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114949041A (en) * | 2022-06-06 | 2022-08-30 | 遵义医科大学珠海校区 | Application of ligustrum robustum in preparation of helicobacter pylori urease inhibitor |
| CN115211491A (en) * | 2022-06-06 | 2022-10-21 | 遵义医科大学珠海校区 | Application of broadleaf holly leaf in preparation of jack bean urease inhibitor |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101254222A (en) * | 2007-03-02 | 2008-09-03 | 重生源生物科技（深圳）有限公司 | Applications of lectuce tea total glycosides and method of preparing the same |
| CN101558803A (en) * | 2008-04-16 | 2009-10-21 | 张大卫 | Extracting and purifying method of extract of bitter leaves |
| CN101966210A (en) * | 2010-08-09 | 2011-02-09 | 深圳市华熙生物科技有限公司 | Folium llicis latifoliae total glycoside and preparation method thereof |
-
2020
- 2020-08-14 CN CN202010819284.2A patent/CN111773273A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101254222A (en) * | 2007-03-02 | 2008-09-03 | 重生源生物科技（深圳）有限公司 | Applications of lectuce tea total glycosides and method of preparing the same |
| CN101558803A (en) * | 2008-04-16 | 2009-10-21 | 张大卫 | Extracting and purifying method of extract of bitter leaves |
| CN101966210A (en) * | 2010-08-09 | 2011-02-09 | 深圳市华熙生物科技有限公司 | Folium llicis latifoliae total glycoside and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 刘志祥；等: "大孔树脂法纯化苦丁茶总黄酮的研究", 《时珍国医国药》 * |
| 叶善蓉；等: "四川粗壮女贞苦丁茶中黄酮的提取、分离与初步鉴定", 《四川农业大学学报》 * |
| 李建法；等: "苦丁茶黄酮类化合物的提取与分析", 《浙江林业科技》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114949041A (en) * | 2022-06-06 | 2022-08-30 | 遵义医科大学珠海校区 | Application of ligustrum robustum in preparation of helicobacter pylori urease inhibitor |
| CN115211491A (en) * | 2022-06-06 | 2022-10-21 | 遵义医科大学珠海校区 | Application of broadleaf holly leaf in preparation of jack bean urease inhibitor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108434231B (en) | Medicinal and edible Chinese medicinal composition for resisting drunkenness and dispelling effects of alcohol and preparation method thereof | |
| CN102836195B (en) | The Kunlun snow chrysanthemum extract and uses thereof | |
| CN111773273A (en) | Ligustrum robustum total flavone, preparation method and application | |
| CN107441078A (en) | A kind of pharmaceutical composition for treating diabetes and its production and use | |
| CN109432387A (en) | It is a kind of with the Chinese medicine-peptide combinations and preparation method thereof for resisting drunk effect | |
| CN1957999B (en) | Composition of Chinese traditional medicine, preparation method, and checking method | |
| CN105125566B (en) | The application of mannoglucan aldehydic acid oligosaccharides and derivative in treatment and/or prevention nephrosis medicine or health products is prepared | |
| CN102233009B (en) | Chinese medicinal composition for promoting nerve regeneration and preparation method and use thereof | |
| CN114246918A (en) | Traditional Chinese medicine composition for treating hashimoto thyroiditis and preparation method thereof | |
| CN104161783B (en) | Application of the eupatorium lindleynun var. trifoliolatum sequiterpene position in resisting acute lung injury medicine is prepared | |
| CN100496527C (en) | Preparation method and application of injection containing Erigeron breviscapus | |
| CN107041924A (en) | It is a kind of prevent and treat diabetic nephropathy towards medicine compound extract and preparation method thereof | |
| CN107163157A (en) | Fruit of Chinese magnoliavine acidic polysaccharose and its preparation method and application | |
| CN105687262A (en) | Folium ginkgo tincture and preparing method thereof | |
| CN113952419B (en) | Pharmaceutical composition for chronic renal failure and preparation method and application thereof | |
| CN108743795A (en) | A kind of prevention diabetic nephropathy towards medicament extract and its preparation method and application | |
| CN105250989A (en) | Donkey-hide glue small peptide composition for resisting fatigue | |
| CN105616432B (en) | Panax Notoginseng saponin R4Purposes in preparation treatment and/or prevention allergic rhinitis object space face | |
| CN105616556B (en) | A kind of preparation method preventing and treating alcoholic liver injury active principle | |
| CN107320639A (en) | Dendrobium chrysanthum blood-sugar-lowering effective parts, active ingredient and its preparation method and application | |
| CN111053819A (en) | Traditional Chinese medicine composition extract and application thereof in preparation of protein expression regulator | |
| CN110694025A (en) | Eight-treasure intelligence-benefiting compound and preparation method and application thereof | |
| WO1997035598A1 (en) | Pharmaceutical compositions and methods for the manufacture thereof | |
| CN112007092A (en) | Anti-alcohol composition and preparation method thereof | |
| CN107753567B (en) | Anti-fatigue pharmaceutical composition and preparation method and application thereof |
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 |