CN109091602B - 韭菜子有效成分、提取方法及其在制备保护肝损伤药物方面的应用 - Google Patents
韭菜子有效成分、提取方法及其在制备保护肝损伤药物方面的应用 Download PDFInfo
- Publication number
- CN109091602B CN109091602B CN201811354062.7A CN201811354062A CN109091602B CN 109091602 B CN109091602 B CN 109091602B CN 201811354062 A CN201811354062 A CN 201811354062A CN 109091602 B CN109091602 B CN 109091602B
- Authority
- CN
- China
- Prior art keywords
- allii tuberosi
- semen allii
- water
- ethanol
- eluent
- 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.)
- Active
Links
- 210000000582 semen Anatomy 0.000 title claims abstract description 64
- 238000000605 extraction Methods 0.000 title claims abstract description 30
- 206010067125 Liver injury Diseases 0.000 title claims description 15
- 239000003814 drug Substances 0.000 title claims description 13
- 231100000753 hepatic injury Toxicity 0.000 title claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 67
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 40
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000000284 extract Substances 0.000 claims abstract description 16
- 230000002829 reductive effect Effects 0.000 claims abstract description 10
- 239000003480 eluent Substances 0.000 claims abstract description 8
- 239000011347 resin Substances 0.000 claims abstract description 8
- 229920005989 resin Polymers 0.000 claims abstract description 8
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 7
- 239000003463 adsorbent Substances 0.000 claims abstract description 6
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 3
- 239000012043 crude product Substances 0.000 claims description 10
- 210000004185 liver Anatomy 0.000 claims description 10
- 240000006108 Allium ampeloprasum Species 0.000 claims description 5
- 235000005254 Allium ampeloprasum Nutrition 0.000 claims description 5
- 238000010829 isocratic elution Methods 0.000 claims description 5
- 238000002953 preparative HPLC Methods 0.000 claims description 5
- 239000004480 active ingredient Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 abstract description 16
- 238000000034 method Methods 0.000 abstract description 13
- 238000002156 mixing Methods 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 210000005228 liver tissue Anatomy 0.000 abstract description 3
- 230000001681 protective effect Effects 0.000 abstract description 2
- 208000037816 tissue injury Diseases 0.000 abstract description 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 239000000047 product Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000002904 solvent Substances 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 244000003377 Allium tuberosum Species 0.000 description 4
- 235000005338 Allium tuberosum Nutrition 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 210000003494 hepatocyte Anatomy 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 235000018645 Allium odorum Nutrition 0.000 description 3
- 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 3
- 231100000439 acute liver injury Toxicity 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- SGRRIJHJAPNORJ-UHFFFAOYSA-N neoprotodioscin Natural products CC(CCC1(O)OC2CC3C4CCC5CC(CCC5(C)C4CCC3(C)C2C1C)OC6OC(CO)C(O)(OC7OC(C)C(O)C(O)C7O)C(O)C6OC8OC(C)C(O)C(O)C8O)COC9OC(CO)C(O)C(O)C9O SGRRIJHJAPNORJ-UHFFFAOYSA-N 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000010298 pulverizing process Methods 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 2
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 2
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000013245 carbon tetrachloride model Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- JUJWROOIHBZHMG-RALIUCGRSA-N pyridine-d5 Chemical compound [2H]C1=NC([2H])=C([2H])C([2H])=C1[2H] JUJWROOIHBZHMG-RALIUCGRSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- GHSUIFMSMQVPPS-UHFFFAOYSA-N 26-O-D-Glucopyranosyl-(25R)-322•,26-trihydroxyl-5furostane 3-O-chacotrioside Natural products O1C(CO)C(O)C(O)C(O)C1OCC(C)CCC(C(C1C2(C)CCC3C4(C)CC5)C)(O)OC1CC2C3CCC4CC5OC(C(C1O)OC2C(C(O)C(O)C(C)O2)O)OC(CO)C1OC1OC(C)C(O)C(O)C1O GHSUIFMSMQVPPS-UHFFFAOYSA-N 0.000 description 1
- 241000234282 Allium Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 244000014047 Polianthes tuberosa Species 0.000 description 1
- 235000016067 Polianthes tuberosa Nutrition 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 206010041497 Spermatorrhoea Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical group OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 230000001447 compensatory effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000020931 dietary conditions Nutrition 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- FRPYMMBAAIXFOI-UHFFFAOYSA-N furostane Natural products CC(CCC1(O)OC2CC3C4CCC5CC(OC6OC(C)C(O)C(O)C6OC7OC(C)C(O)C(O)C7O)C(O)CC5(C)C4CCC3(C)C2C1C)COC8OC(CO)C(O)C(O)C8O FRPYMMBAAIXFOI-UHFFFAOYSA-N 0.000 description 1
- CTYOUOHIEXEYAW-MEVSNGMESA-N furostane group Chemical group [C@@H]12C[C@@H]3O[C@H](CCC(C)C)[C@@H](C)[C@@H]3[C@@]1(C)CC[C@H]1[C@H]2CCC2CCCC[C@]12C CTYOUOHIEXEYAW-MEVSNGMESA-N 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000008558 metabolic pathway by substance Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000012306 spectroscopic technique Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 229930002600 steroidal saponin Natural products 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000005406 washing Methods 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)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
-
- 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
- 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
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- 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
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
-
- 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
Abstract
本发明涉及一种韭菜子有效成分的提取方法，包括如下步骤：1）将粉碎后的韭菜子、炒韭菜子或盐炙韭菜子用20－50%乙醇进行闪式提取，提取液浓缩得到提取物浸膏；将提取物浸膏分散在水中，然后用二氯甲烷进行萃取，萃取所得水层部位用大孔吸附树脂吸附，然后依次用水、30%乙醇、60%乙醇梯度洗脱，收集60%乙醇洗脱液；2）将60%乙醇洗脱液旋转蒸发，然后用水溶解，再经ODS开放柱纯化，甲醇与水按体积比1.8－2.5：1进行洗脱，薄层色谱检测，合并相同组分并经减压浓缩即得。经实验研究发现：韭菜子有效成分可以拮抗四氯化碳引起的小鼠肝组织损伤，且具有一定的保护作用。
Description
技术领域
本发明属于植物提取技术领域，具体涉及一种韭菜子有效成分、提取方法及其在制备保护肝损伤食品、保健品以及药物中的新应用。
背景技术
肝脏是人体的重要器官，它是物质合成与代谢、能量产生和转换的重要枢纽。肝脏暴露于有害条件下如药物、化学试剂、辐射等均可引起急性肝损伤，长期作用可导致不可修复的损伤，并发生代偿性修复，即形成肝纤维化，进一步可发展成肝硬化或肝癌。由于山区和乡镇的卫生、饮食条件较差，所以山区和乡镇人口肝病发病率最高。因此，开发有效预防、治疗和价位低廉的保肝功能性食品、保健品或药物具有重要意义。
韭菜子（Semen Allii Tuberose）是百合科葱属多年生植物韭菜（Alliium tuberosum Rottl. Ex Spreng.）的干燥成熟种子，为药食同源的中药，具有益肾、健脾提神、行气理血、润肠通便、壮阳固精等功效，用于肾亏虚，腰膝酸痛，阳痿遗精等，收载于《中国药典》。研究表明韭菜子化学成分主要有甾体皂苷类、生物碱类、硫化物、黄酮类等化合物。现代药理研究证明韭菜子具有温肾壮阳、加强免疫、抗氧化、抗衰老、抑菌等作用。韭菜子为韭菜生产的附属产品，韭菜种植面积变得更为广泛、货源充足，使价格一直保持在平稳、合理阶段。对于开发高效、低价的保肝药，解决“看病难，看病贵”具有重要意义。目前，并未有韭菜子在保肝护肝方面应用文献的相关报道。
发明内容
本发明目的在于克服现有技术缺陷，提供一种价格低廉、制备简单的韭菜子有效成分。
本发明还提供了上述韭菜子有效成分的提取方法及其在制备保护肝损伤食品、保健品以及药物中的新应用。
为实现上述目的，本发明采用如下技术方案：
一种韭菜子有效成分的提取方法，其具体包括如下步骤：
1）将粉碎后的韭菜子、炒韭菜子或盐炙韭菜子用20－50%乙醇进行闪式提取，提取液浓缩得到提取物浸膏；将提取物浸膏分散在水中，然后用二氯甲烷进行萃取，萃取所得水层部位用大孔吸附树脂吸附，然后依次用水、30%乙醇、60%乙醇梯度洗脱，收集60%乙醇洗脱液；
2）将60%乙醇洗脱液旋转蒸发以回收溶剂，然后洗脱物用水溶解，再经ODS开放柱纯化（可按重量比1：10－20装柱），甲醇与水按体积比1.8－2.5：1进行洗脱，薄层色谱检测，合并相同组分并经减压浓缩得粗品。
上述韭菜子有效成分的提取方法，步骤1）中，闪式提取2－3次，闪式提取时每1 g韭菜子、炒韭菜子或盐炙韭菜子添加8－15mL的20－50%乙醇。
上述韭菜子有效成分的提取方法，步骤1）中，每次闪式提取的时间以3－10min为宜。二氯甲烷萃取次数以2－3次为宜。
具体的，上述韭菜子有效成分的提取方法，步骤1）中，所述大孔吸附树脂的型号为HPD100，HPD600，HPD400，D101，AB-8，ADS-17或DM130等。
上述韭菜子有效成分的提取方法，步骤2）中，所得粗品溶于水后，用制备型高效液相色谱纯化即得韭菜子有效成分；色谱条件：流速6－8ml/min、波长203nm，用22－27%的乙腈水溶液进行等度洗脱。
本发明提供了采用上述方法提取得到的韭菜子有效成分。
本发明还提供了上述韭菜子有效成分在制备保护肝损伤药物、食品或保健品中的应用。
和现有技术相比，本发明的有益效果如下：
本发明从韭菜子中提取得到韭菜子有效成分，并采用腹腔注射四氯化碳诱导急性肝损伤小鼠模型。经实验研究发现：韭菜子有效成分可以拮抗四氯化碳引起的小鼠肝组织损伤，且具有一定的保护作用，表现为减轻肝细胞炎症和纤维化，促进肝细胞再生和降低血清中天冬氨酸转氨酶、丙氨酸转氨酶的作用。此外，本发明韭菜子有效成分的提取工艺简单，溶剂可回收利用，提取量大，提取物纯度高，适合工业化生产，并且韭菜子来源广泛，取材便利，同时开拓了韭菜子有效成分作为保护肝损伤的保健品、食品或药物用途。
附图说明
图1为本发明韭菜子有效成分对四氯化碳引起的小鼠肝损伤组织病理变化的影响。
具体实施方式
为了使本发明的技术目的、技术方案和有益效果更加清楚，下面结合具体实施例，对本发明的技术方案作出进一步的说明，但所述实施例旨在解释本发明，而不能理解为对本发明的限制，实施例中未注明具体技术或条件者，按照本领域内文献所描述的技术或条件或者按照产品说明书进行。
本发明中，炒韭菜子或盐炙韭菜子采用本领域常规技术制作即可，因其制作过程并非本申请的创新之所在，故此不再详述。下述实施例中，如无特殊说明，乙醇、甲醇、乙腈浓度均为体积浓度。
实施例1
一种韭菜子有效成分的提取方法，包括以下步骤：
1）将5kg干燥的韭菜子粉碎后，分别用50L 50%乙醇闪式提取3次（第一次闪式提取3 min，第二次闪式提取5 min，第三次闪式提取5 min），闪式提取结束后过滤，合并三次滤液，减压浓缩得到提取物浸膏。将提取物浸膏分散在水中，用同体积二氯甲烷萃取3次，萃取所得水层部位经HPD100大孔吸附树脂吸附，然后依次用水、30%乙醇、60%乙醇梯度洗脱，收集60%乙醇洗脱液；
2）将60%乙醇洗脱液旋转蒸发以回收溶剂，然后洗脱物用水溶解，再经ODS开放柱纯化（可按重量比1：15装柱），甲醇与水按照体积比1.8：1洗脱，薄层色谱检测，合并相同组分并经减压回收溶剂，得粗品；
3）将所得粗品溶于水后，用制备型高效液相色谱纯化即得韭菜子有效成分；色谱条件：流速8ml/min、波长203nm，用22%的乙腈水溶液进行等度洗脱。
上述提取所得的韭菜子有效成分纯品，性状呈白色粉末，收率为0.038%。对上述制得的韭菜子有效成分纯品运用多种谱学技术进行鉴定，发现其主要成分为Neoprotodioscin（化学名：26-O-β-D-吡喃葡萄糖基-25(R)-5α-呋甾烷-3β, 22α, 26-三羟基-3-O-[α-L-吡喃鼠李糖基-(1→2)-O-α-L-吡喃鼠李糖基-(1→4)]-O-β-D-吡喃葡萄糖苷，5,6-二氢原薯蓣皂苷，新原薯蓣皂苷：5,6-dihydroprotodioscin），具体鉴定结果如下。
Neoprotodioscin，结构式如下所示：
仪器材料：1H，13C NMR 图谱由 Bruker am400 MHz 核磁共振仪测定，TMS为内标；质谱由VG.AUTO Spec-3000 型质谱仪测定。
测试结果：ESI-MS: m/z 1050.0[M-H]-，根据13C-NMR和1H-NMR的核磁数据，确定分子式为：C51H86O22，分子量：1050.7。1H-NMR(C5D5N) δppm谱显示有六个甲基质子信号δ0.84(3H，s，H-18)，δ1.01(3H，s，H-19)，δ1.00(3H，d，J=6.0Hz，H-27)，δ1.26（3H，d，J=6.8Hz，H-21），δ1.73（3H，d，J=6.2Hz，H-Rha-6），δ1.60（3H，d，J=6.2Hz，H-Rha’-6）；3位葡萄糖的端基氢信号δ4.91（1H，d，J=6.7Hz，H-Glc-1），26位葡萄糖的端基氢信号δ4.78（1H，d，J=8.1Hz，H-Glc’-1），与葡萄糖2位相连的鼠李糖的端基氢信号δ6.32（1H，s，H-Rha-1），以及与葡萄糖4位相连的鼠李糖的端基氢信号δ5.81（1H，s，H-Rha’-1）。13C-NMR(C5D5N) δppm显示：低场区有四个糖的端基碳信号δ99.6（C-Glc-1），δ104.8（C-Glc’-1），δ101.9（C-Rha-1），δ102.6（C-Rha’-1）；δ110.4（C-22）是呋甾烷22位的特征碳信号。与参考文献对照，该化合物为Neoprotodioscin。碳谱核磁数据见表1。
表1 Neoprotodioscin的碳谱数据
检查：利用TLC，硫酸显色剂显色，110℃下5分钟，呈现绿色。
含量测定：利用分析型HPLC，色谱条件：SHIMADZU C18色谱柱；流动相：乙腈、水体积比为33：67；流速：0.8mL/min，检测波长203nm，保留时间值22.4 min。经检测，纯度≥98%。
实施例2
一种韭菜子有效成分的提取方法，包括以下步骤：
1）将1 kg干燥的盐炙韭菜子粉碎后，分别用12L 40%乙醇闪式提取3次（第一次闪式提取8 min，第二次闪式提取5 min，第三次闪式提取7min），闪式提取结束后过滤，合并三次滤液，减压浓缩得到提取物浸膏。将提取物浸膏分散在水中，用同体积二氯甲烷萃取2次，萃取所得水层部位经D101大孔吸附树脂吸附，然后依次用水、30%乙醇、60%乙醇梯度洗脱，收集60%乙醇洗脱液；
2）将60%乙醇洗脱液旋转蒸发，回收洗脱液，然后洗脱物用水溶解，再经ODS开放柱纯化（可按重量比1：10装柱），甲醇与水按照体积比2.0：1洗脱，薄层色谱检测，合并相同组分并经减压回收溶剂，得粗品；
3）将所得粗品溶于水后，用制备型高效液相色谱纯化即得韭菜子有效成分；色谱条件：流速7ml/min、波长203nm，用24%的乙腈水溶液进行等度洗脱。
上述提取所得的韭菜子有效成分纯品，性状呈白色颗粒状粉末，收率为0.036%，纯度≥98%。
实施例3
一种韭菜子有效成分的提取方法，包括以下步骤：
1）将500g干燥的盐炙韭菜子粉碎后，分别用7 L 30%乙醇闪式提取2次（第一次闪式提取8 min，第二次闪式提取9 min），闪式提取结束后过滤，合并二次滤液，减压浓缩得到提取物浸膏。将提取物浸膏分散在水中，用同体积二氯甲烷萃取3次，萃取所得水层部位经D101大孔吸附树脂吸附，然后依次用水、30%乙醇、60%乙醇梯度洗脱，收集60%乙醇洗脱液；
2）将60%乙醇洗脱液旋转蒸发回收溶剂，洗脱物用水溶解，再经ODS开放柱纯化（可按重量比1：20装柱），甲醇与水按照体积比2.3：1洗脱，薄层色谱检测，合并相同组分并经减压回收溶剂，得粗品；
3）将所得粗品溶于水后，用制备型高效液相色谱纯化即得韭菜子有效成分；色谱条件：流速7ml/min、波长203nm，用24%的乙腈水溶液进行等度洗脱。
上述提取所得的韭菜子有效成分纯品，性状呈白色颗粒状粉末，收率为0.039%，纯度≥98%。
效果实验：韭菜子有效成分对四氯化碳诱导的小鼠急性肝损伤影响。
实验原料：实施例1制得的韭菜子有效成分作为实验原料。
实验动物：昆明种小鼠，体重 23±2 g，SPF级，由河南省实验动物中心提供，生产批号：SCXK（豫）2017-0001。
实验试剂：见下表2。
实验仪器：见下表3。
表2. 实验试剂。
表3. 实验仪器
实验方法：将40只雄性昆明小鼠随机分成5组，每组8只，五组分别为：空白组（给药组等体积的娃哈哈水）、四氯化碳模型组（给药组等体积的娃哈哈水）、护肝片阳性组（60mg/kg）、韭菜子提取物低剂量组（5mg/kg）、韭菜子提取物高剂量组（15mg/kg）。各组分别按上述剂量灌胃给药，连续灌胃3天。第三天灌胃结束2h后，进行腹腔注射，注射体积以10ml/kg计算。模型组、阳性组、给药组腹腔注射含0.5% CCl4的橄榄油，空白组腹腔注射与模型组等体积的橄榄油，腹腔注射后小鼠禁食不禁水，24h后摘取小鼠眼球取血，将全血室温放置30-60min后进行离心（8000×g，10 min，4℃）分离收集血清，血清在使用之前应放于-80℃冰箱中保存，然后测定血清中AST（天冬氨酸转氨酶）、ALT（丙氨酸转氨酶）水平，按照试剂盒说明书的方法进行测定。采用Graphpad Prism 6统计学软件进行处理分析，结果以“
表示，组间比较采用One-way ANOVA 单因素方差分析，P<0.05为差异具有统计学意义。
取血后将小鼠断颈处死，剖取肝脏，用生理盐水冲洗，最后将肝脏组织放在4%的多聚甲醛溶液中保存20-24h，在无菌实验环境下用刀片将组织修剪成厚约5mm的组织，将组织依次放入脱水盒内，然后进行脱水、包埋、切片、烤片、HE染色，显微镜镜检，图像采集分析。结果见表4和图1。
表4. 韭菜子有效成分抗四氯化碳引起的小鼠肝损伤活性(
)
与空白组比较：# P < 0.01
与模型组比较：**P＜0.01
表4的结果表明：与空白组相比，模型组的AST和ALT活力明显提高（# P＜0.01）；与模型组比较，韭菜子有效成分高、低剂量组的AST和ALT活力显著降低（**P＜0.01）。以上结果说明：本发明韭菜子有效成分高、低剂量组均具有显著拮抗四氯化碳引起的小鼠肝损伤。
图1为本发明韭菜子有效成分对四氯化碳引起的小鼠肝损伤组织病理变化的影响。图1的结果表明：空白组肝小叶结构完整，肝细胞结构清楚，未出现肝细胞坏死、细胞核变性及炎症细胞浸润等病理特征，细胞质均匀，中央静脉清晰可见，细胞形态整体正常；四氯化碳模型组肝脏细胞结构遭到损坏，可以明显看到细胞核出现破裂变性现象，且有炎症细胞浸润中央静脉，肝小叶结构不完整；护肝片阳性组和本发明韭菜子有效成分高、低剂量组总体上肝细胞正常，细胞核结构正常，中央静脉清晰可见，无或较少炎症细胞浸润，细胞质均匀。
Claims (4)
1. 韭菜子有效成分在制备保护肝损伤药物中的应用, 其特征在于，包括如下步骤：
1）将粉碎后的韭菜子、炒韭菜子或盐炙韭菜子用20－50%乙醇进行闪式提取，提取液浓缩得到提取物浸膏；将提取物浸膏分散在水中，然后用二氯甲烷进行萃取，萃取所得水层部位用大孔吸附树脂吸附，然后依次用水、30%乙醇、60%乙醇梯度洗脱，收集60%乙醇洗脱液；
2）将60%乙醇洗脱液旋转蒸发，然后用水溶解，再经ODS开放柱纯化，甲醇与水按体积比1.8－2.5：1进行洗脱，薄层色谱检测，合并相同组分并经减压浓缩得粗品；
步骤1）中，所述大孔吸附树脂的型号为HPD100，HPD600，HPD400，D101，AB-8，ADS-17或DM130。
2.如权利要求1所述韭菜子有效成分在制备保护肝损伤药物中的应用，其特征在于，步骤1）中，闪式提取2－3次，闪式提取时每1 g 韭菜子、炒韭菜子或盐炙韭菜子添加8－15mL的20－50%乙醇。
3.如权利要求2所述韭菜子有效成分在制备保护肝损伤药物中的应用，其特征在于，步骤1）中，每次闪式提取的时间为3－10min。
4.如权利要求1所述韭菜子有效成分在制备保护肝损伤药物中的应用，其特征在于，步骤2）中，所得粗品溶于水后，用制备型高效液相色谱纯化；色谱条件：流速6－8ml/min、波长203nm，用22－27%的乙腈水溶液进行等度洗脱。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811354062.7A CN109091602B (zh) | 2018-11-14 | 2018-11-14 | 韭菜子有效成分、提取方法及其在制备保护肝损伤药物方面的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811354062.7A CN109091602B (zh) | 2018-11-14 | 2018-11-14 | 韭菜子有效成分、提取方法及其在制备保护肝损伤药物方面的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109091602A CN109091602A (zh) | 2018-12-28 |
| CN109091602B true CN109091602B (zh) | 2021-07-23 |
Family
ID=64870206
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811354062.7A Active CN109091602B (zh) | 2018-11-14 | 2018-11-14 | 韭菜子有效成分、提取方法及其在制备保护肝损伤药物方面的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109091602B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115487263B (zh) * | 2022-10-19 | 2023-08-18 | 劲牌有限公司 | 一种抑制6-磷酸葡萄糖脱氢酶的韭菜子提取物的制备方法 |
-
2018
- 2018-11-14 CN CN201811354062.7A patent/CN109091602B/zh active Active
Non-Patent Citations (3)
| Title |
|---|
| A review of traditional pharmacological uses, phytochemistry, and pharmacological activities of Tribulus terrestris;Wenyi Zhu等;《Chemistry Central Journal》;20171231;第1-16页，尤其是第6页表1、第13页左栏第3段 * |
| Pregnane- and Furostane-Type Oligoglycosides from the Seeds of Allium tuberosum;Tsuyoshi IKEDA等;《Chem. Pharm. Bull.》;20041231;第52卷(第1期);第142-145页，尤其是第144页Extraction and Isolation * |
| Tsuyoshi IKEDA等.Pregnane- and Furostane-Type Oligoglycosides from the Seeds of Allium tuberosum.《Chem. Pharm. Bull.》.2004,第52卷(第1期),第142-145页，尤其是第144页Extraction and Isolation. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109091602A (zh) | 2018-12-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Singhuber et al. | Aconitum in traditional Chinese medicine—a valuable drug or an unpredictable risk? | |
| CN102600219B (zh) | 黄蜀葵花总黄酮提取物及其制备方法 | |
| EP1800685A1 (en) | Steroidal saponin pharmaceutical composition, its preparation method and use | |
| AU2004200624B2 (en) | Medicinal preparation containing phenylethanoid glycosides extracted from herbaceous plant, cistanche tubulosa (schenk.) wight, process of making the same, and uses of the same | |
| Chebbi Mahjoub et al. | Chloroformic and methanolic extracts of Olea europaea L. leaves present anti-inflammatory and analgesic activities | |
| CN103316096A (zh) | 一种黑种草子总黄酮提取物及其制备方法和用途 | |
| CN101810659A (zh) | 紫参总皂苷及其医药用途和制备方法 | |
| CN103804443A (zh) | 黄酮苷类化合物及其制备方法 | |
| CN107698458A (zh) | 一种n1,n5‑双(对香豆酰)亚精胺的提取方法 | |
| CN113150049B (zh) | 青钱柳的提取物及其抗痛风和降尿酸应用 | |
| CN104873570B (zh) | 一种夏枯草总黄酮的提取纯化方法及其应用 | |
| CN109091602B (zh) | 韭菜子有效成分、提取方法及其在制备保护肝损伤药物方面的应用 | |
| CN101890084A (zh) | 黑种草子总苷提取物及其制备方法和应用 | |
| CN109053756B (zh) | 苯丙素类酯型儿茶素及其制备方法和应用 | |
| Wu et al. | Preparation of paeoniflorin from the stems and leaves of Paeonia lactiflora Pall.‘Zhongjiang’through green efficient microwave-assisted extraction and subcritical water extraction | |
| CN103191143B (zh) | 一种强心苷化合物的用途 | |
| CN109276637B (zh) | 韭菜子提取物及其制备方法和在制备保肝护肝药物方面的应用 | |
| CN105949272B (zh) | 魏察酸浆苦素y及提取方法及用途 | |
| CN112250655B (zh) | 两种新的环状二苯基庚烷类化合物及其制备方法和用途 | |
| CN103610682A (zh) | 3α-羟基-30-齐墩果-12,20(29)-二烯-28-酸的制备方法和在制备抗肿瘤药物中的应用 | |
| CN109456163B (zh) | 一种具有对称结构的环烯酮类化合物及其制备方法和应用 | |
| CN103613632B (zh) | 29-降齐墩果烷酸类化合物及其制备方法和在制备糖苷酶抑制剂药物中的应用 | |
| CN104398532B (zh) | 一种强心苷化合物的用途 | |
| CN112920146B (zh) | 倍半萜类化合物及其制备方法和在制备抗炎药物中的应用 | |
| CN104324043B (zh) | 一种强心苷化合物的用途 |
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 |