CN110477195A - Length splits sonchus oleraceus alkaloid level Four isolate and its separation method and application - Google Patents
Length splits sonchus oleraceus alkaloid level Four isolate and its separation method and application Download PDFInfo
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- CN110477195A CN110477195A CN201910726262.9A CN201910726262A CN110477195A CN 110477195 A CN110477195 A CN 110477195A CN 201910726262 A CN201910726262 A CN 201910726262A CN 110477195 A CN110477195 A CN 110477195A
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- 244000113428 Sonchus oleraceus Species 0.000 title claims abstract description 152
- 235000006745 Sonchus oleraceus Nutrition 0.000 title claims abstract description 152
- 229930013930 alkaloid Natural products 0.000 title claims abstract description 135
- 150000003797 alkaloid derivatives Chemical class 0.000 title claims abstract description 130
- 238000000926 separation method Methods 0.000 title claims abstract description 26
- 239000000284 extract Substances 0.000 claims abstract description 61
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 238000000605 extraction Methods 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 45
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 32
- 238000012360 testing method Methods 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 17
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 16
- 235000019253 formic acid Nutrition 0.000 claims description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- 229910002027 silica gel Inorganic materials 0.000 claims description 12
- 239000000741 silica gel Substances 0.000 claims description 12
- 229960001866 silicon dioxide Drugs 0.000 claims description 12
- 238000002137 ultrasound extraction Methods 0.000 claims description 10
- 235000019441 ethanol Nutrition 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 7
- 238000002390 rotary evaporation Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 4
- 238000004237 preparative chromatography Methods 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 3
- 238000004611 spectroscopical analysis Methods 0.000 claims 4
- 238000001035 drying Methods 0.000 claims 1
- -1 alkaloid compound Chemical class 0.000 abstract description 82
- 230000003647 oxidation Effects 0.000 abstract description 11
- 238000007254 oxidation reaction Methods 0.000 abstract description 11
- 238000007445 Chromatographic isolation Methods 0.000 abstract 1
- 230000002000 scavenging effect Effects 0.000 description 47
- 230000003078 antioxidant effect Effects 0.000 description 42
- 239000003963 antioxidant agent Substances 0.000 description 36
- 235000006708 antioxidants Nutrition 0.000 description 36
- 238000001514 detection method Methods 0.000 description 29
- 229940126214 compound 3 Drugs 0.000 description 24
- 239000000523 sample Substances 0.000 description 23
- 229940125904 compound 1 Drugs 0.000 description 22
- 229940125782 compound 2 Drugs 0.000 description 22
- 230000004083 survival effect Effects 0.000 description 20
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 18
- 230000031700 light absorption Effects 0.000 description 16
- 230000008439 repair process Effects 0.000 description 16
- GLEVLJDDWXEYCO-UHFFFAOYSA-N Trolox Chemical compound O1C(C)(C(O)=O)CCC2=C1C(C)=C(C)C(O)=C2C GLEVLJDDWXEYCO-UHFFFAOYSA-N 0.000 description 11
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 11
- 238000004587 chromatography analysis Methods 0.000 description 10
- 230000001590 oxidative effect Effects 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
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- 238000002835 absorbance Methods 0.000 description 8
- 229940079593 drug Drugs 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 230000006698 induction Effects 0.000 description 8
- 150000003254 radicals Chemical class 0.000 description 8
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 230000003064 anti-oxidating effect Effects 0.000 description 6
- 238000006701 autoxidation reaction Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 241000252212 Danio rerio Species 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 230000008033 biological extinction Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 229960000935 dehydrated alcohol Drugs 0.000 description 4
- 229960004756 ethanol Drugs 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000002195 synergetic effect Effects 0.000 description 4
- 229930004668 tropane alkaloid Natural products 0.000 description 4
- 150000003813 tropane derivatives Chemical class 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000208292 Solanaceae Species 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 150000003233 pyrroles Chemical class 0.000 description 3
- KMVWNDHKTPHDMT-UHFFFAOYSA-N 2,4,6-tripyridin-2-yl-1,3,5-triazine Chemical compound N1=CC=CC=C1C1=NC(C=2N=CC=CC=2)=NC(C=2N=CC=CC=2)=N1 KMVWNDHKTPHDMT-UHFFFAOYSA-N 0.000 description 2
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 2
- 241000488874 Sonchus Species 0.000 description 2
- 241000489357 Sonchus brachyotus Species 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229940079877 pyrogallol Drugs 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 125000000189 2-deoxyribosyl group Chemical group C1(C[C@H](O)[C@H](O1)CO)* 0.000 description 1
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 1
- COPBKCYMXSUALT-UHFFFAOYSA-N 3h-1,3-oxazol-2-one Chemical compound O=C1NC=CO1.O=C1NC=CO1 COPBKCYMXSUALT-UHFFFAOYSA-N 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical group [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 241001412304 Ixeris Species 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 1
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 235000008132 Sonchus arvensis ssp. uliginosus Nutrition 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003026 anti-oxygenic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 238000002376 fluorescence recovery after photobleaching Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000008236 heating water Substances 0.000 description 1
- 208000014617 hemorrhoid Diseases 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- IQPNAANSBPBGFQ-UHFFFAOYSA-N luteolin Chemical compound C=1C(O)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(O)C(O)=C1 IQPNAANSBPBGFQ-UHFFFAOYSA-N 0.000 description 1
- LRDGATPGVJTWLJ-UHFFFAOYSA-N luteolin Natural products OC1=CC(O)=CC(C=2OC3=CC(O)=CC(O)=C3C(=O)C=2)=C1 LRDGATPGVJTWLJ-UHFFFAOYSA-N 0.000 description 1
- 235000009498 luteolin Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 235000019394 potassium persulphate Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000004206 stomach function Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- 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
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Botany (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Nutrition Science (AREA)
- Medicines Containing Plant Substances (AREA)
- Cosmetics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention belongs to agricultural biological technical fields, and in particular to length splits sonchus oleraceus alkaloid level Four isolate and its separation method and application.The present invention extracts length by ultrasonic method and splits sonchus oleraceus extraction alkaloid component therein, and obtains the level Four isolate with oxidation resistance using preparation chromatographic isolation, has important practical significance to the long development and utilization for splitting alkaloid compound in sonchus oleraceus.
Description
Technical field
The invention belongs to agricultural biological technical fields, and in particular to length splits sonchus oleraceus alkaloid level Four isolate and its separation
Methods and applications.
Background technique
It is composite family sonchus annual herb plant that length, which splits sonchus oleraceus (Sonchus brachyotus DC.), also known as bitter
Dish, bitter bent dish, hardship Qu Qu, sonchus oleraceus, sowthistle, denticulate ixeris herb etc..It is long to split that sonchus oleraceus medicinal history is long, according to the earliest doctor in China
The Compendium of Material Medica for learning monograph Shennong's Herbal and Li Shizhen (1518-1593 A.D.) is recorded, and bitter is cold in nature, there is clearing heat and detoxicating, detumescence and apocenosis, cool
The effect of blood stagnation resolvation, clearing lung and relieving cough, beneficial liver diuresis, the stomach function regulating that helps digestion, to treat the diseases such as acute dysentery, enteritis, hemorrhoid gall.It is long
Splitting the chemical component in sonchus oleraceus has cupreol, luteolin, apiolin and Quercetin, and there are also some volatile oil components, grinds
Study carefully and shows long to split sonchus oleraceus with antibacterial, by blood pressure, norcholesterol, antitumor, treatment hepatitis and anti-oxidant etc. many-sided make
With.But so far, it is unclear to split effective oxidation-resistant active ingredient in sonchus oleraceus for length.
Summary of the invention
The purpose of the present invention is to provide a kind of length to split sonchus oleraceus alkaloid level Four isolate.
A further object of the present invention is to provide the separation methods of above-mentioned level Four isolate.
A further object of the present invention is to provide the applications of above-mentioned level Four isolate.
The length of specific embodiment splits sonchus oleraceus alkaloid level Four isolate according to the present invention, by what is included the following steps
Method is prepared:
(1) preparation length splits sonchus oleraceus extract；
(2) length that step (1) obtains is split into the test liquid that sonchus oleraceus extract is configured to 100mg/mL, using preparation chromatography
Method is separated, and chromatographic condition is as follows:
Using C18 silicagel column, using 0.1% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, flow velocity 10mL/
Min, 27-33 DEG C of column temperature, wavelength 255-265nm, gradient elution program are as follows:
In the time range of 0-5min, the volume of mobile phase A becomes 60% from 95%, and the volume of Mobile phase B is become by 5%
It is 40%；In the time range of 5-15min, the volume of mobile phase A becomes 30% from 60%, and the volume of Mobile phase B is by 40%
Become 70%；In the time range of 15-30min, the volume of mobile phase A becomes 20% from 30%, the volume of Mobile phase B by
70% becomes 80%；In the time range of 30-35min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B is kept
It is 80%；Peak height range is collected in the fraction of 420-2000mAU, obtains primary isolate；
(3) after the primary isolate for obtaining step (2) is concentrated, is dry, it is configured to the test liquid of 10mg/mL and using system
Standby chromatography separates, and chromatographic condition is as follows: using C18 silicagel column, is stream with methanol using 0.1% aqueous formic acid as mobile phase A
Dynamic phase B, flow velocity 10mL/min, 27-33 DEG C of column temperature, wavelength 255-265nm, gradient elution program are as follows: in the time of 0-5min
In range, the volume of mobile phase A becomes 60% from 95%, and the volume of Mobile phase B becomes 40% from 5%；In the time of 5-15min
In range, the volume of mobile phase A becomes 30% from 60%, and the volume of Mobile phase B becomes 70% from 40%；15-30min when
Between in range, the volume of mobile phase A becomes 20% from 30%, and the volume of Mobile phase B becomes 80% from 70%；30-35min's
In time range, the volume of mobile phase A remains 20%, and the volume of Mobile phase B remains 80%；Peak height range is collected in 0.2-
The fraction of 1.8AU obtains secondary isolate；
(4) after the secondary isolate for obtaining step (3) is concentrated, is dry, it is configured to the test liquid of 10mg/mL and using system
Standby chromatography separates, and chromatographic condition is as follows: using C18 silicagel column, is stream with methanol using 0.1% aqueous formic acid as mobile phase A
Dynamic phase B, flow velocity 1mL/min, 30 DEG C of column temperature, wavelength 260nm, gradient elution program are as follows:
In the time range of 0-8min, the volume of mobile phase A becomes 75% from 95%, and the volume of Mobile phase B is become by 5%
It is 25%；In the time range of 8-10min, the volume of mobile phase A becomes 65% from 75%, and the volume of Mobile phase B is by 25%
Become 35%；In the time range of 10-20min, the volume of mobile phase A becomes 40% from 65%, the volume of Mobile phase B by
35% becomes 60%；In the time range of 20-24min, the volume of mobile phase A becomes 20% from 40%, the volume of Mobile phase B
There is 60% to become 80%；In the time range of 24-27min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B is protected
Holding is 80%, collects the fraction of 16-18min, obtains three-level isolate；
(5) the three-level isolate obtained with HPLC method separating step (4), chromatographic condition are as follows:
Using 0.1% aqueous formic acid as mobile phase A, using acetonitrile as Mobile phase B, flow velocity 0.3mL/min, 30 DEG C of column temperature, wave
Long 260nm, gradient elution program are as follows: in 0-10min time range, the volume of Mobile phase B becomes 95% from 5%, mobile phase A
Volume become 5% from 95%, collect the fraction of 5.8-6.2min, obtain level Four isolate.
In reverse-phase chromatographic column, the long sequence for splitting sonchus oleraceus alkaloid extract outflow chromatographic column is the stronger component of polarity
The component weak prior to polarity is eluted out, that is to say, that the long sequence for splitting sonchus oleraceus alkaloid extract outflow chromatographic column is flowed
Move mutually polar influence.Meanwhile different flowing phase composition, Gradient program, flow velocity, column temperatures etc. can all lead to the polarity of mobile phase
It changes, and then causes the composition of effective component in fraction different.
The present invention uses C18 chromatographic column isolating alkaloids, can under high ph conditions isolated peak shape is good, does not trail
Chromatographic peak not only has high volume containing the sample, and can keep high interface kinetics coefficient, obtains preferable column effect.
The length of specific embodiment splits sonchus oleraceus alkaloid level Four isolate according to the present invention, in step (2), column temperature 30
DEG C, wavelength 260nm.
The length of specific embodiment splits sonchus oleraceus alkaloid level Four isolate according to the present invention, it is long split sonchus oleraceus extract by
Method comprising the following steps are prepared:
Using 75% ethyl alcohol as solvent, by the long extraction solution for splitting sonchus oleraceus and liquid-to-solid ratio being made as 30mL/g, carries out ultrasound and mention
It takes, after ultrasonic extraction, stands, take supernatant to obtain the final product.
The length of specific embodiment splits sonchus oleraceus alkaloid level Four isolate according to the present invention, and the power of ultrasonic extraction is
700W, temperature are 55 DEG C, ultrasonic time 30min.
The length of specific embodiment splits the separation method of sonchus oleraceus alkaloid level Four isolate, the separation according to the present invention
Method the following steps are included:
(1) preparation length splits sonchus oleraceus extract；
(2) length that step (1) obtains is split into the test liquid that sonchus oleraceus extract is configured to 100mg/mL, using preparation chromatography
Method is separated, and chromatographic condition is as follows:
Using C18 silicagel column, using 0.1% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, flow velocity 10mL/
Min, 27-33 DEG C of column temperature, wavelength 255-265nm, gradient elution program are as follows:
In the time range of 0-5min, the volume of mobile phase A becomes 60% from 95%, and the volume of Mobile phase B is become by 5%
It is 40%；In the time range of 5-15min, the volume of mobile phase A becomes 30% from 60%, and the volume of Mobile phase B is by 40%
Become 70%；In the time range of 15-30min, the volume of mobile phase A becomes 20% from 30%, the volume of Mobile phase B by
70% becomes 80%；In the time range of 30-35min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B is kept
It is 80%；Peak height range is collected in the fraction of 420-2000mAU, obtains primary isolate；
(3) after the primary isolate for obtaining step (2) is concentrated, is dry, it is configured to the test liquid of 10mg/mL and using system
Standby chromatography separates, and chromatographic condition is as follows: using C18 silicagel column, is stream with methanol using 0.1% aqueous formic acid as mobile phase A
Dynamic phase B, flow velocity 10mL/min, 27-33 DEG C of column temperature, wavelength 255-265nm, gradient elution program are as follows: in the time of 0-5min
In range, the volume of mobile phase A becomes 60% from 95%, and the volume of Mobile phase B becomes 40% from 5%；In the time of 5-15min
In range, the volume of mobile phase A becomes 30% from 60%, and the volume of Mobile phase B becomes 70% from 40%；15-30min when
Between in range, the volume of mobile phase A becomes 20% from 30%, and the volume of Mobile phase B becomes 80% from 70%；30-35min's
In time range, the volume of mobile phase A remains 20%, and the volume of Mobile phase B remains 80%；Peak height range is collected in 0.2-
The fraction of 1.8AU obtains secondary isolate；
(4) after the secondary isolate for obtaining step (3) is concentrated, is dry, it is configured to the test liquid of 10mg/mL and using system
Standby chromatography separates, and chromatographic condition is as follows: using C18 silicagel column, is stream with methanol using 0.1% aqueous formic acid as mobile phase A
Dynamic phase B, flow velocity 1mL/min, 30 DEG C of column temperature, wavelength 260nm, gradient elution program are as follows:
In the time range of 0-8min, the volume of mobile phase A becomes 75% from 95%, and the volume of Mobile phase B is become by 5%
It is 25%；In the time range of 8-10min, the volume of mobile phase A becomes 65% from 75%, and the volume of Mobile phase B is by 25%
Become 35%；In the time range of 10-20min, the volume of mobile phase A becomes 40% from 65%, the volume of Mobile phase B by
35% becomes 60%；In the time range of 20-24min, the volume of mobile phase A becomes 20% from 40%, the volume of Mobile phase B
There is 60% to become 80%；In the time range of 24-27min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B is protected
Holding is 80%, collects the fraction of 16-18min, obtains three-level isolate；
(5) the three-level isolate obtained with HPLC method separating step (4), chromatographic condition are as follows:
Using 0.1% aqueous formic acid as mobile phase A, using acetonitrile as Mobile phase B, flow velocity 0.3mL/min, 30 DEG C of column temperature, wave
Long 260nm, gradient elution program are as follows: in 0-10min time range, the volume of Mobile phase B becomes 95% from 5%, mobile phase A
Volume become 5% from 95%, collect the fraction of 5.8-6.2min, obtain level Four isolate.
The length of specific embodiment splits the separation method of sonchus oleraceus alkaloid level Four isolate, step (2) according to the present invention
In, column temperature is 30 DEG C, wavelength 260nm.
The length of specific embodiment splits the separation method of sonchus oleraceus alkaloid level Four isolate according to the present invention, and length splits hare's-lettuce
Dish extract is prepared by method comprising the following steps:
Using 75% ethyl alcohol as solvent, by the long extraction solution for splitting sonchus oleraceus and liquid-to-solid ratio being made as 30mL/g, carries out ultrasound and mention
Take, after ultrasonic extraction, stand, take supernatant, carry out after rotary evaporation it is cooling it is dry to get.
The length of specific embodiment splits the separation method of sonchus oleraceus alkaloid level Four isolate, ultrasonic extraction according to the present invention
Power be 700W, temperature be 55 DEG C, ultrasonic time 30min.
Beneficial effects of the present invention:
Present invention determine that the long extraction process for splitting sonchus oleraceus alkaloid, it can to the long recovery rate for splitting sonchus oleraceus alkaloid
Reach 20.30%.By the isolated multiple fractions of multistage preparative chromatography, determine that length splits sonchus oleraceus alkaloid level Four isolate
With stronger antioxygenic property, for develop and produce novel oxidation-resistant food or feed addictive provide it is reliable theoretical and
Technical support.
Detailed description of the invention
The long oxidation resistance verification result for splitting sonchus oleraceus alkaloid extract of Fig. 1 display；, wherein (1) shows H2O2It is right
The influence of Caco-2 cell survival rate；(2) display length splits sonchus oleraceus alkaloid extract to H2O2Damage Caco-2 cell survival rate
Influence；(3) the long total antioxidant capacity for splitting sonchus oleraceus alkaloid extract of display；(4) display length splits sonchus oleraceus biology alkali carries
Take object to the Scavenging activity of ABTS free radical；(5) display length splits sonchus oleraceus alkaloid extract to the removing energy of DPPH free radical
Power；(6) display length splits sonchus oleraceus alkaloid extract to the Scavenging activity of hydroxyl radical free radical；(7) display is long splits sonchus oleraceus biology
Scavenging activity of the alkali extract to superoxide anion；(8) the long total reducing power for splitting sonchus oleraceus alkaloid extract of display；
Fig. 2 shows the research conditions of level-one fraction, wherein (1) the long preparative HPLC for splitting sonchus oleraceus extract point of display
From situation；(2) display length splits sonchus oleraceus alkaloid extract and fraction to H2O2Damage the influence feelings of Caco-2 cell survival rate
Condition；(3) total antioxidant capacity of level-one fraction is shown；(4) Scavenging activity of the display level-one fraction to ABTS free radical；(5) it shows
Show level-one fraction to the Scavenging activity of DPPH free radical；(6) Scavenging activity of the display level-one fraction to hydroxyl radical free radical；(7) it shows
Show level-one fraction to the Scavenging activity of superoxide anion；(8) total reducing power of level-one fraction is shown；
Fig. 3 shows the preparative HPLC separating resulting of primary isolate；
Fig. 4 shows secondary fraction to H2O2Damage the influence of Caco-2 cell survival rate；
Fig. 5 shows the total antioxidant capacity of secondary fraction；
Fig. 6 shows secondary fraction to the Scavenging activity of ABTS free radical；
Fig. 7 shows secondary fraction to the Scavenging activity of DPPH free radical；
Fig. 8 shows secondary fraction to the Scavenging activity of hydroxyl radical free radical；
Fig. 9 shows secondary fraction to the Scavenging activity of superoxide anion；
Figure 10 shows total reducing power of secondary fraction；
Figure 11 shows that the HPLC of secondary isolate is fractionated situation；
Figure 12 shows three-level fraction to H2O2Damage the influence of Caco-2 cell survival rate；
Figure 13 shows the total antioxidant capacity of three-level fraction；
Figure 14 shows three-level fraction to the Scavenging activity of ABTS free radical；
Figure 15 shows three-level fraction to the Scavenging activity of DPPH free radical；
Figure 16 shows three-level fraction to the Scavenging activity of hydroxyl radical free radical；
Figure 17 shows three-level fraction to the Scavenging activity of superoxide anion；
Figure 18 shows total reducing power of three-level fraction；
Figure 19 shows the HPLC fractionation of three-level isolate；
Figure 20 shows influence of the level Four fraction to H2O2 damage Caco-2 cell survival rate
Figure 21 shows the total antioxidant capacity of level Four fraction；
Figure 22 shows level Four fraction to the Scavenging activity of ABTS free radical；
Figure 23 shows level Four fraction to the Scavenging activity of DPPH free radical；
Figure 24 shows level Four fraction to the Scavenging activity of hydroxyl radical free radical；
Figure 25 shows level Four fraction to the Scavenging activity of superoxide anion；
Figure 26 shows total reducing power of level Four fraction.
Specific embodiment
The preparation length of embodiment 1 splits sonchus oleraceus alkaloid extract
Long split after sonchus oleraceus is cleaned dries to constant weight in 40 DEG C of baking ovens, and length is then split hare's-lettuce vegetable powder with microphyte pulverizer
It is broken, cross the analysis sieve of 60 mesh.It weighs 10g long and splits sonchus oleraceus herb coarse powder and be placed in the ultrasonic cup of 500mL, 75% ethyl alcohol is added,
The extraction solution that liquid-to-solid ratio is 30mL/g is obtained, adjusting Extraction solvent pH value is 5, carries out ultrasonic extraction, and ultrasonic temperature is 55 DEG C,
Ultrasonic power is 700W, ultrasonic time 30min.
After ultrasonic extraction, 5000rpm is centrifuged 10min, obtains supernatant, 40 DEG C of rotary evaporations, and freeze-drying is grown
Sonchus oleraceus alkaloid extract is split, it is spare.
Alkaloid is nitrogenous organic compound, and alkaloid compound has the property like alkali.The parent nucleus knot of alkaloid
Structure has a variety of, such as has pyrroles's Alkaloid, pyrroles's Alkaloid, pyrroles's Alkaloid, tropane alkaloids (Solanaceae is raw
Alkaloids), tropane alkaloids (Solanaceae alkaloids), tropane alkaloids (Solanaceae alkaloids), tropane alkaloids (eggplant
Section's alkaloid) etc..
It takes the extracting solution of 0.1mL to be placed in 1mL colorimetric cylinder, scale is settled to certain density ethyl alcohol, with corresponding
Concentration of alcohol is blank, and extracting solution light absorption value is measured at 410nm, surveys length according to standard curve and splits alkaloid in sonchus oleraceus
Recovery rate, recovery rate=(weight of alkaloid/medicinal material weight in sample) × 100%.
Alkaloid recovery rate of the invention is 20.30%.
The long oxidation resistance for splitting sonchus oleraceus alkaloid extract of the verifying of embodiment 2
2.1H2O2Establish induction Caco-2 cell oxidative damage model
By investigating H2O2In various concentration (100,250,500,750,1000,2500,5000,7500,10000 μm of ol/
L under) to Caco-2 cytosis for 24 hours after, observe its influence to cell survival rate, determine H2O2Induce Caco-2 cell oxygen
Change damage model, as a result as shown in Figure 1.
By (1) in Fig. 1 it is found that H2O2Concentration does not have a significant impact to cell survival rate in 100-5000 μm of ol/L,
Cell survival rate decline about 40% when 7500 μm of ol/L, and the H of high concentration2O2Caco-2 cell survival rate is caused to be decreased obviously, carefully
Born of the same parents' quantity significantly reduces, through 10000 μm of ol/L H2O2The cell survival rate decline about 60% of processing, determines suitable H2O2Induction
The concentration of Caco-2 cell oxidative damage model is 7500 μm of ol/L.
2.2 length split sonchus oleraceus alkaloid extract to H2O2The repair of the Caco-2 cell oxidative damage model of induction
Use concentration: 200 μ g/mL long split sonchus oleraceus alkaloid extract to H2O2The Caco-2 cell oxidative damage of induction
The repair of model, as a result as shown in figure 1 shown in (2).
The results show that its cell survival rate is 71%, and H2O2After inducing Caco-2 cell oxidative damage, cell survival
Rate is 56%, and therefore, length splits sonchus oleraceus alkaloid extract to H2O2It induces Caco-2 cell oxidative damage model to have and repairs work
With.
The 2.3 long antioxidations for splitting sonchus oleraceus alkaloid extract
(1) the long total antioxidant capacity for splitting sonchus oleraceus alkaloid extract
Weigh 27.8mg FeSO4·7H2O, dissolves and constant volume is to 1mL, and concentration is 100mM at this time.Take appropriate 100mM
FeSO4Solution is diluted to 0.15,0.3,0.6,0.9,1.2 and 1.5mM.Use distilled water or sample preparation aqueous standard method
Product.
180 μ L FRAP working solutions (150 μ L+TPTZ solution 15 of TPTZ dilution is added in each detection hole of a:96 orifice plate
μ L+ detects 15 μ L of buffer).
B: the appropriate solution such as 5 μ L distilled water or PBS are added in blank control wells；It is each that 5 μ L are added in standard curve detection hole
The FeSO of kind concentration4Standard solution；The Trolox of the various samples of 5 μ L or 0.15-1.5mM are added in sample detection hole as positive
Control.It mixes gently.
A593 is measured after c:37 DEG C of incubation 3-5min.Measure FeSO4Standard curve is y=0.3119x+0.0526, R2=
0.999。
The total antioxidant capacity (TAC) of sample is calculated according to standard curve.As shown in figure 1 shown in (3), it is with concentration
The control of 0.07mmol/g is compared, and concentration is that 200 μ g/mL long split the total antioxidant capacity of sonchus oleraceus alkaloid extract and are
0.89mmol/g, about the 12 of control times, but compared with concentration 6.66mmol/g positive control Trolox, Trolox's
Total antioxidant capacity is about long 7 times for splitting sonchus oleraceus alkaloid extract.
(2) length splits sonchus oleraceus alkaloid extract to the Scavenging activity of ABTS free radical
7mmol/L ABTS(2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonie acid)
Diamm-oniumsalt, ABTS) with 2.45mmol/L potassium peroxydisulfate to stand 16h under 9:1 ratio mixed room temperature, using preceding dilute
It releases octuple as ABTS stock solution.It is separately added into a series of concentration range (1.6-1000 μ g/mL, dehydrated alcohol dissolution) samples
2.5mL ABTS stock solution is protected from light 20min and measures light absorption value at wavelength 734nm after mixing.Added with 1mL distilled water
The stand-by mixed liquor of ABTS is used as positive control as blank control, BHT (butylated hydoxytoluene).ABTS is removed
Rate (%)=[(A1-A2)/A1] × 100%, wherein A1Indicate the absorbance of blank control, A2Indicate sample to be tested ABTS extinction
Degree.
By (4) in Fig. 1 it is found that clearance rate is when the long concentration for splitting sonchus oleraceus alkaloid extract is 25 μ g/mL
20%, however when its concentration is 200 μ g/mL, clearance rate is about 79.09%, and about low concentration is to ABTS free radical
4 times of clearance rate.
(3) length splits sonchus oleraceus alkaloid extract to the Scavenging activity of DPPH free radical
The sample of a certain concentration range (1.6-1000 μ g/mL, dehydrated alcohol dissolution), respectively takes sample 0.3mL, is separately added into
The DPPH (1,1-diphenyl-2-picrylhydrazyl, DPPH) (being dissolved with dehydrated alcohol) of 2.7mL, 0.2mmol/L, whirlpool
Rotation mixes, and the sample prepared is protected from light after standing 1h and surveys its absorbance at 517nm.Made with 1mL dehydrated alcohol instead of extract
For blank control, the BHT of same concentration range is as positive control.
DPPH clearance rate (%)=[(A1-A2)/A1] × 100%, wherein A1Indicate the absorbance of blank control, A2It indicates
Sample to be tested DPPH absorbance.
As shown in figure 1 shown in (5), when it is 25 μ g/mL that length, which splits sonchus oleraceus alkaloid extract concentration, clearance rate is
3.6%, however when its concentration is 200 μ g/mL, clearance rate is about 32.6%, and about low concentration is to DPPH free radical
9 times of clearance rate.
(4) length splits sonchus oleraceus alkaloid extract to the Scavenging activity of hydroxyl radical free radical
Using Fe3+- EDTA- ascorbic acid-hydrogen peroxide system generates hydroxyl radical free radical, and deoxyribose is by hydroxyl radical free radical
It is cracked after attack, under conditions of acidity, heating and thiobarbituric acid reaction generates red compound, the presence of antioxidant
It can prevent hydroxide radicals attack deoxyribose.
The 400 μ L of 2-deoxyribosyl of 10mmol/L, 100 μ L of iron chloride (10mmol/L) are separately added into reaction system,
EDTA-2Na (ethylenediaminetetraacetic acid disodium salt) (1mmol/L) 100 μ L, 30% mistake
100 μ L of hydrogen oxide (10mmol/L), sample concentration (1.6-1000 μ g/mL) 100 μ L, adds ascorbic acid (1mmol/L) 200
μ L initiation reaction, reacts 1h at 37 DEG C, and sodium hydroxide (0.025mol/L) the solution 1mL and 30%TCA of 0.5%TBA is added
(trichloroacetic acid) aqueous solution 1mL, 80 DEG C of heating water bath 30min of mixture, it is cooling.It measures and inhales at 532nm
Light value replaces sample with 0.05mol/L PBS (phosphate buffered solution) (pH value 7.4) in the reaction system
Product measure light absorption value as blank control, calculate clearance rate, the BHT with concentration range is as positive control.Hydroxyl radical free radical is clear
Except rate (%)=[(A1-A2)/A1] × 100%, wherein A1Indicate the absorbance of blank control, A2Indicate the extinction of sample to be tested
Degree.
As shown in figure 1 shown in (6), when it is 25 μ g/mL that length, which splits sonchus oleraceus alkaloid extract concentration, clearance rate is
10.3%, however when its concentration is 200 μ g/mL, clearance rate is about 63.9%, and about low concentration is to hydroxyl radical free radical
6 times of clearance rate.
(5) length splits sonchus oleraceus alkaloid extract to the Scavenging activity of superoxide anion
Superoxide anion is one of internal most common free radical.Pyrogallic acid spontaneous generation in alkaline environment is super
Oxygen anion.Its autoxidizable rate is related to the concentration of superoxide anion.Determinand can remove superoxide anion ability and subtract
The slow autoxidizable rate of pyrogallic acid.
Tris-HCl buffer (pH value 8.2,0.05mol/L) 4.5mL is added at 25 DEG C in 200 μ g/mL sample to be tested 1mL
It reacts 10min and 0.003mol/L pyrogallol (10mmol/L dissolving with hydrochloric acid) 600 μ L is added, sufficiently immediately in wavelength after reaction
Absorbance is measured at 325nm, measures an absorbance every 30s, until apparent variation is not occurring for light absorption value, is used
10mmol/L hydrochloric acid replaces sample as blank control, and BHT makees positive control.The autoxidation rate of pyrogallol can be according to suction
Luminosity-time graph calculates slope.
It is long to split sonchus oleraceus alkaloid extract to superoxide anion with removing energy compared with BHT as shown in figure 1 shown in (7)
Power, autoxidation rate are reduced.
(6) the long total reducing power for splitting sonchus oleraceus alkaloid extract
Using total reducing power of iron reduction oxidation resistance method measurement alkaloid, the Fe under acidic environment3+- three pyridines three
Azine (Fe3+- TPTZ) ferrous iron form can be reduced to by antioxidant, blue is showed, and have absorption maximum at 593nm,
Absorbance is bigger, and reducing power is stronger.
It takes sample to be tested 20mg to be configured to 400 μ g/mL sample solutions, takes 50,100,150,200,250,300 respectively,
350,400,450,500 μ L sample solution add distilled water to 1mL, respectively plus 0.2mol/L PBS (pH value 6.6) and 1%K3[Fe
(CN)6] each 2.5mL, 20min is reacted at 50 DEG C, adds 10%TCA 2.5mL, 3000r/min (r=3cm) centrifugation
10min takes supernatant 2.5mL that distilled water 2.5mL is added, and 0.1% iron chloride 0.5mL is added and mixes, after 10min at 700nm
Measure its light absorption value A2, the distilled water sample is not added, which is operated with method as blank control, measures its light absorption value, increased extinction
Value indicates the increase of reducing power.
As shown in figure 1 shown in (8), when it is 20 μ g/mL that length, which splits sonchus oleraceus alkaloid extract concentration, increased light absorption value
It is 0.006, however when its concentration is 200 μ g/mL, increased light absorption value is 0.046, about total also proper energy of low concentration
7 times of power.
(7) the long internal antioxidant effect for splitting sonchus oleraceus alkaloid extract
It is induced zebra fish intestinal tract injury 1 day with 0.2% oxazolone (Oxazolone) injection zebra fish, with containing different
The long fish meal for splitting sonchus oleraceus alkaloid extract of concentration is fed zebra fish 5 days.
The result shows that it is long split sonchus oleraceus alkaloid extract and can effectively reduce zebra fish intestinal tissue ROS, MDA contain
Amount improves SOD and CAT vigor.
To sum up, long to split sonchus oleraceus alkaloid extract and all have good antioxidation in vitro and in vivo.
The separation length of embodiment 3 splits sonchus oleraceus alkaloid level-one fraction
3.1 length split sonchus oleraceus alkaloid level-one fraction
The preparation of test liquid: splitting sonchus oleraceus alkaloid extract for length and dissolved with ultrapure water, and being configured to concentration is 100mg/
The test liquid of mL crosses 0.22 μm of filter membrane, spare.The long chromatographic condition for splitting sonchus oleraceus alkaloid level-one fraction of separation:
Durashell C18 (L) (10 μm,30 × 250mm) it is fractionated on column, length splits sonchus oleraceus alkaloid
Level-one fraction prepares chromatographic condition: chromatographic column: Durashell C18 (L) (10 μm,30 × 250mm), mobile phase is
0.1% formic acid-water (A)-methanol (B) system, flow velocity 10mL/min, 30 DEG C of column temperature, wavelength 260nm.Gradient elution program is
0-5min, 5%-40%B, 5-15min, 40%-70%B, 15-30min, 70%-80%B, 30-35min, 80%-80%B,
Applied sample amount is 18mL.
According to the long chromatographic peak situation for splitting sonchus oleraceus alkaloid extract in preparation chromatography, length is split into sonchus oleraceus alkaloid
Extract is divided into 6 fraction SB1 (S.brachyotus 1), SB2, SB3, SB4, SB5 and SB6, as shown in (1) in Fig. 2,
In, SB1 peak height range 2-570mAU, SB2 peak height range 530-750mAU, light absorption value range, the peak SB3 peak height range 420-
2000mAU, SB4 peak height range 1200-2010mAU, SB5 peak height range 360-2010mAU, SB6 peak height range 70-740mAU.
3.2 level-one fractions are to H2O2The repair of the Caco-2 cell oxidative damage model of induction
Fraction SB1, SB2, SB3, SB4, SB5 and SB6 (concentration: 200 μ g/mL) is investigated respectively to H2O2The Caco-2 of induction
The repair of cell oxidative damage model.
As a result as shown in (2) in Fig. 2, cell survival rate is respectively 76%, 74%, 114%, 106%, 94%, 75%,
Therefore, SB3 is to H2O2The repair significant effect of the Caco-2 cell oxidative damage model of induction.
The 3.3 long antioxidations for splitting sonchus oleraceus alkaloid level-one fraction
(1) the long total antioxidant capacity for splitting sonchus oleraceus alkaloid level-one fraction
Detection method is the same as embodiment 2.By (3) in Fig. 2 it is found that control, Trolox, extract, SB1-SB6's is total anti-
Oxidability is respectively 0.07,6.66,0.89,0.09,0.21,1.98,0.92,0.89,0.28mmol/g, from these data
It is found that the total antioxidant capacity of SB3 is relatively high, the total antioxidant capacity of SB3 is about control, Trolox, extract,
28,0.3,2,22,9,2,2,7 times of SB1, SB2, SB4-SB6, therefore, the long SB3's split in sonchus oleraceus alkaloid level-one fraction
Oxidation resistance is stronger.
(2) length splits sonchus oleraceus alkaloid level-one fraction to the Scavenging activity of ABTS free radical
Detection method is the same as embodiment 2.As shown in (4) in Fig. 2, when the concentration of SB1-SB6 is 1.6 μ g/mL, to ABTS
The clearance rate of free radical is respectively 1.61%, 3.64%, 15.99%, 19.43%, 7.69%, 8.5%, and when its concentration is
When 1000 μ g/mL, clearance rate is respectively 31.58%, 65.79%, 74.29%, 74.09%, 55.47%, 46.96%, greatly
It is approximately low concentration to 20,18,5,4,7,6 times of the clearance rate of ABTS free radical, since level-one fraction drug concentration is identical, SB1
It is relatively large with variation of the SB2 to the clearance rate of ABTS free radical, when the concentration highest of SB3 and SB4, to ABTS free radical
Clearance rate it is relatively high, and when the concentration of SB4 is 40 μ g/mL, clearance rate is higher than BHT, and other level-one fraction pair
The clearance rate of ABTS free radical is below BHT.
(3) length splits sonchus oleraceus alkaloid level-one fraction to the Scavenging activity of DPPH free radical
Detection method is the same as embodiment 2.As shown in (5) in Fig. 2, when the concentration of SB1-SB6 is 1.6 μ g/mL, to DPPH
The clearance rate of free radical is respectively 8.29%, 11.82%, 14.11%, 14.81%, 3.35%, 1.23%, and when its concentration is
When 1000 μ g/mL, clearance rate is respectively 48.85%, 51.32%, 73.55%, 74.25%, 58.55%, 49.38%, greatly
It is approximately low concentration to 5,4,5,5,17,40 times of the clearance rate of DPPH free radical, since drug concentration is identical, SB5 and SB6 pairs
The variation of the clearance rate of DPPH free radical is relatively large, when the concentration of SB3 and SB4 is maximum, to the clear of DPPH free radical
Except rate is relatively high, and when the concentration of SB2-SB4 is 1.6 μ g/mL, clearance rate is higher than BHT, and when level-one fraction is other
When concentration, clearance rate is below BHT.
(4) length splits sonchus oleraceus alkaloid level-one fraction to the Scavenging activity of hydroxyl radical free radical
Detection method is the same as embodiment 2.As shown in (6) in Fig. 2, when the concentration of SB1-SB6 is 1.6 μ g/mL, to hydroxyl
The clearance rate of free radical is respectively 6.85%, 10.37%, 27.77%, 21.44%, 20.39%, 18.1%, and works as its concentration
When for 1000 μ g/mL, clearance rate is respectively 69.07%, 70.83%, 77.15%, 76.45%, 71.18%, 70.12%,
About low concentration is to 10,7,3,4,3,4 times of the clearance rate of hydroxyl radical free radical, and since drug concentration is identical, SB1-SB6 is to hydroxyl
The variation of the clearance rate of base free radical is less significant, the removing when the concentration of SB3 and SB4 is maximum, to hydroxyl radical free radical
Rate is relatively high, SB3 (when concentration is 1.6,8,40,200 μ g/mL), SB4 (when concentration is 1.6,8 μ g/mL), SB5 (when
When concentration is 1.6 μ g/mL) and SB6 (when concentration is 1.6 μ g/mL) BHT is apparently higher than to the clearance rate of hydroxyl radical free radical, and
The clearance rate of other level-one fractions is then lower than BHT.
(5) length splits sonchus oleraceus alkaloid level-one fraction to the Scavenging activity of superoxide anion
Detection method is the same as embodiment 2.As shown in (7) in Fig. 2, compared with BHT, length splits sonchus oleraceus alkaloid level-one fraction
SB1-SB6 has Scavenging activity to superoxide anion, and the autoxidation reduced rate of SB2, SB3 and SB4 are significant.
(6) the long total reducing power for splitting sonchus oleraceus alkaloid level-one fraction
In such as figure shown in (8), when the concentration of SB1-SB6 is 20 μ g/mL, increased light absorption value is respectively 0,0,
0.014,0.001,0.022,0.019, and when its concentration is 200 μ g/mL, increased light absorption value is respectively 0.013,
0.014,0.131,0.075,0.090,0.078, about the 51,54,10,100,4,4 of total reducing power of low concentration times, by
It is identical in drug concentration, total reducing power of the variation of total reducing power obvious, the SB3, SB5 and SB6 of SB1, SB2 and SB4
Variation is less obvious, but total reducing power of SB3 is apparently higher than other fractions, and total reducing power is but lower than BHT.
To sum up, testing result is consistent with the detection result of cell survival rate, and therefore, length is split sonchus oleraceus alkaloid level-one and evaporated
The antioxidant activity of SB 3 is stronger in point.
The separation length of embodiment 4 splits sonchus oleraceus alkaloid second level fraction
4.1 length split sonchus oleraceus alkaloid second level fraction
By effective active fraction SB3, rotary evaporation, concentration, freeze-drying are spare at 40 DEG C.SB3 is dissolved with ultrapure water,
It is configured to the test liquid that concentration is 10mg/mL, crosses 0.22 μm of filter membrane.Utilize preparation chromatographic column DurashellC18 (L) (10 μ
m,30 × 250mm) further fractionation.
Prepare chromatographic condition: mobile phase is 0.1% formic acid-water (A)-methanol (B) system, flow velocity 10mL/min, column temperature 30
DEG C, wavelength 260nm.Gradient elution program is 0-5min, 5%-40%B, 5-15min, 40%-70%B, 15-30min, 70%-
80%B, 30-35min, 80%-80%B, applied sample amount 18mL.
According to chromatographic peak of the fraction SB3 in preparation chromatography, it is separated into 5 fractions, i.e. SB3-1, SB3-2, SB3-
4, SB3-5, wherein the peak height range of peak height the range 0.5-5.7AU, SB3-3 of peak height the range 0-0.8AU, SB3-2 of SB3-1
The peak height range 0.1-0.35AU of peak height the range 0.2-1.8AU, SB3-5 of 1.4-5.7AU, SB3-4, as shown in Figure 3.
4.2 second level fractions are to H2O2Induce the repair of the model of oxidative of Caco-2 cell
Fraction SB3-1, SB3-2, SB3-3 are investigated, SB3-4, SB3-5 (concentration: 200 μ g/mL) are to H2O2Induce Caco-2 thin
The repair of the model of oxidative of born of the same parents.
As a result as shown in figure 4, its cell survival rate is respectively 67%, 75%, 87%, 99%, 77%, therefore, SB3-4 pairs
H2O2Induce the repair significant effect of the model of oxidative of Caco-2 cell.
The 4.3 long antioxidations for splitting sonchus oleraceus alkaloid second level fraction
(1) the long total antioxidant capacity for splitting sonchus oleraceus alkaloid second level fraction
Detection method is the same as embodiment 2.As shown in figure 5, control, Trolox, extract, SB3, SB3-1 are to SB3-5's
Total antioxidant capacity is respectively 0.07,6.66,0.89,1.98,0.67,2.31,3.44,5.52,3.05mmol/g, therefore,
The total antioxidant capacity of SB3-4 is relatively high, the total antioxidant capacity of SB3-4 be about control, Trolox, extract,
78,0.8,6,3,8,2,2,2 times of SB3, SB3-1, SB3-2, SB3-3, SB3-5, therefore length is split sonchus oleraceus alkaloid second level and is evaporated
The oxidation resistance of SB3-4 in point is stronger.
(2) length splits sonchus oleraceus alkaloid second level fraction to the Scavenging activity of ABTS free radical
Detection method is the same as embodiment 2.As shown in fig. 6, when the concentration of SB3-1 to SB3-5 is 1.6 μ g/mL, to ABTS
The clearance rate of free radical is respectively 0.61%, 2.85%, 2.24%, 11.18%, 4.47%, and when its concentration is 1000 μ g/mL
When, clearance rate is respectively 75.61%, 75.81%, 76.21%, 76.63%, 76.02%, about low concentration to ABTS from
By the 124 of the clearance rate of base, 27,34,7,17 times, since drug concentration is identical, change of the SB3-1 to the clearance rate of ABTS free radical
Change maximum, is followed successively by SB3-3, SB3-2, SB3-5, SB3-4, when the long concentration for splitting sonchus oleraceus alkaloid second level fraction is maximum,
Its difference with insignificance to the clearance rate of ABTS free radical, clearance rate are below BHT.
(3) length splits sonchus oleraceus alkaloid second level fraction to the Scavenging activity of DPPH free radical
Detection method is the same as embodiment 2.As shown in fig. 7, when the concentration of SB3-1 to SB3-5 is 1.6 μ g/mL, to DPPH
The clearance rate of free radical is respectively 5.04%, 2.14%, 8.45%, 3.78%, 3.53%, and when its concentration is 1000 μ g/mL
When, clearance rate is respectively 73.39%, 76.04%, 77.43%, 77.81%, 76.54%, about low concentration to DPPH from
By the 15 of the clearance rate of base, 36,9,21,22 times, since drug concentration is identical, the variation of the clearance rate of DPPH free radical is influenced
Sequence is followed successively by SB3-2, SB3-5, SB3-4, SB3-1, SB3-3, and the long concentration for splitting sonchus oleraceus alkaloid second level fraction is maximum
When, to the clearance rate difference with insignificance of DPPH free radical, and clearance rate is below BHT.
(4) length splits sonchus oleraceus alkaloid second level fraction to the Scavenging activity of hydroxyl radical free radical
Detection method is the same as embodiment 2.As shown in figure 8, when the concentration of SB3-1 to SB3-5 is 1.6 μ g/mL, to hydroxyl
The clearance rate of free radical is respectively 8.94%, 12.94%, 16.71%, 20.47%, 6.59%, and when its concentration is 1000 μ g/
When mL, clearance rate is respectively 77.18%, 78%, 81.41%, 89.53%, 79.18%, about low concentration to hydroxyl from
By the 9 of the clearance rate of base, 6,5,4,12 times, since drug concentration is identical, clearance rate of the SB3-1 to SB3-5 to hydroxyl radical free radical
The equal difference with insignificance of variation, when the concentration of SB3-3 and SB3-4 is maximum, relatively to the clearance rate of hydroxyl radical free radical
Height, length split sonchus oleraceus alkaloid second level fraction and are below BHT to the clearance rate of hydroxyl radical free radical.
(5) length splits sonchus oleraceus alkaloid second level fraction to the Scavenging activity of superoxide anion
Detection method is the same as embodiment 2.As shown in figure 9, length splits sonchus oleraceus alkaloid second level fraction SB3-1 extremely compared with BHT
SB3-5 has Scavenging activity to superoxide anion, and the autoxidation reduced rate of SB3-3 and SB3-4 are significant.
(6) the long total reducing power for splitting sonchus oleraceus alkaloid second level fraction
Detection method is the same as embodiment 2.As shown in Figure 10, increased when the concentration of SB3-1 to SB3-5 is 20 μ g/mL
Light absorption value is respectively 0.018,0.011,0.023,0.026,0.016, and when its concentration is 200 μ g/mL, increased extinction
Value be respectively 0.140,0.055,0.253,0.273,0.150, about total reducing power of low concentration 8,5,11,10.6,
9.7 times, since drug concentration is identical, the variation influence sequence of the long total reducing power for splitting sonchus oleraceus alkaloid second level fraction is successively
For SB3-3, SB3-4, SB3-5, SB3-1, SB3-2, and its total reducing power is below BHT.
To sum up, testing result is consistent with the detection result of cell survival rate, and therefore, length is split sonchus oleraceus alkaloid second level and evaporated
The antioxidant activity of SB3-4 in point is stronger.
The separation length of embodiment 5 splits sonchus oleraceus alkaloid three-level fraction
5.1 length split sonchus oleraceus alkaloid three-level fraction
The SB3-4 obtained rotary evaporation at 40 DEG C will be collected, is concentrated, freeze-drying is spare.SB3-4 is dissolved with methanol
Sample, being configured to concentration is 10mg/mL test liquid, crosses 0.22 μm of filter membrane.Using chromatographic column Venusil C18 (250 ×
4.6mm, 5 μm), it is fractionated fraction SB3-4 is further in analysis chromatography, analyze chromatographic condition: mobile phase is 0.1% first
Acid-water (A)-methanol (B) system, flow velocity 1mL/min, 30 DEG C of column temperature, wavelength 260nm.Gradient elution program is 0-8min, 5%-
25%B；8-10min, 25%-35%B；10-20min, 35%-60%B；20-24min, 60%-80%B；24-27min,
80%-80%B, 20 μ L of applied sample amount.
According to chromatographic peak of the active constituent in analysis chromatography, active constituent is fractionated into 2 segments, i.e. SB3-4-1 and
SB3-4-2, as shown in figure 11.
5.2 three-level fractions are to H2O2Induce the repair of the model of oxidative of Caco-2 cell
Fraction SB3-4-1 and SB3-4-2 (200 μ g/mL) is investigated to H2O2The model of oxidative of the Caco-2 cell of induction
Repair.
As a result as shown in figure 12, cell survival rate is respectively 105%, 86%, SB3-4-1 to H2O2The Caco-2 of induction
The repair significant effect of the model of oxidative of cell.
The 5.3 long antioxidations for splitting sonchus oleraceus alkaloid three-level fraction
(1) the long total antioxidant capacity for splitting sonchus oleraceus alkaloid three-level fraction
Detection method is the same as embodiment 2.As shown in figure 13, control, Trolox, extract, SB3, SB3-4, SB3-4-1,
The total antioxidant capacity of SB3-4-2 is respectively 0.065,6.66,0.89,1.52,9.45,5.27mmol/g, from these data
It is found that the total antioxidant capacity of SB3-4-1 is relatively high, the total antioxidant capacity of SB3-4-1 is about control, Trolox,
Extract, SB3, SB3-4,145,1,11,5,2,2 times of SB3-4-2, therefore length is split in sonchus oleraceus alkaloid three-level fraction
The oxidation resistance of SB3-4-1 is stronger.
(2) length splits sonchus oleraceus alkaloid three-level fraction to the Scavenging activity of ABTS free radical
Detection method is the same as embodiment 2.As shown in figure 14, when the concentration of SB3-4-1 and SB3-4-2 is 1.6 μ g/mL,
Clearance rate to ABTS free radical is respectively 50.2%, 44.29%, and when its concentration is 1000 μ g/mL, clearance rate difference
It is 94.88%, 84.65%, about low concentration is to 2,2 times of the clearance rate of ABTS free radical, and therefore, experiment shows that length splits hardship
Lettuce dish alkaloid three-level fraction all has Scavenging activity to ABTS free radical, and its clearance rate is above BHT.
(3) length splits sonchus oleraceus alkaloid three-level fraction to the Scavenging activity of DPPH free radical
Detection method is the same as embodiment 2.As shown in figure 15, when the concentration of SB3-4-1 and SB3-4-2 is 1.6 μ g/mL,
Clearance rate to DPPH free radical is respectively 53.77%, 46.79%, and when its concentration is 1000 μ g/mL, clearance rate point
Not Wei 86.23%, 79.25%, about low concentration is to 1.6,1.7 times of the clearance rate of DPPH free radical, and therefore, experiment shows
Length, which splits sonchus oleraceus alkaloid three-level fraction, all has Scavenging activity to DPPH free radical, however when SB3-4-2 concentration is 200 μ g/
When mL, BHT is lower than to the clearance rate of DPPH free radical, remaining clearance rate is above BHT.
(4) length splits sonchus oleraceus alkaloid three-level fraction to the Scavenging activity of hydroxyl radical free radical
Detection method is the same as embodiment 2.As shown in figure 16, when the concentration of SB3-4-1 and SB3-4-2 is 1.6 μ g/mL,
Clearance rate to hydroxyl radical free radical is respectively 45.95%, 23.22%, and when its concentration is 1000 μ g/mL, clearance rate point
Not Wei 88.23%, 78.41%, about low concentration is to 1.9,3.4 times of the clearance rate of hydroxyl radical free radical, and therefore, experiment shows
Length, which splits sonchus oleraceus alkaloid three-level fraction, all has Scavenging activity to hydroxyl radical free radical, however when SB3-4-2 concentration is 1000 μ g/
When mL, BHT is lower than to the clearance rate of hydroxyl radical free radical, remaining is above BHT to the clearance rate of hydroxyl radical free radical.
(5) length splits sonchus oleraceus alkaloid three-level fraction to the Scavenging activity of superoxide anion
Detection method is the same as embodiment 2.As shown in figure 17, length, which splits sonchus oleraceus alkaloid three-level fraction, has superoxide anion
The autoxidation rate of Scavenging activity, SB3-4-1 and SB3-4-2 have the tendency that slowing down, and its trend slowed down is lower than BHT.
(6) the long total reducing power for splitting sonchus oleraceus alkaloid three-level fraction
Detection method is the same as embodiment 2.As shown in figure 18, when the concentration of SB3-4-1 and SB3-4-2 is 20 μ g/mL, increase
The light absorption value added is respectively 0.024,0.016, and when its concentration is 200 μ g/mL, increased light absorption value is respectively 0.293,
0.233, about the 12,15 of total reducing power of low concentration times, therefore, experiment show that length splits sonchus oleraceus alkaloid three-level fraction
All there is total reducing power, however total reducing power of SB3-4-1 is substantially higher than BHT, and total reducing power of SB3-4-2 is then
Lower than BHT.
To sum up, the total antioxidant activity of fraction SB3-4-1 is more significant, the antioxidation and H of three-level fraction2O2Induction
The repair of the model of oxidative of Caco-2 cell is consistent.
The separation length of embodiment 6 splits sonchus oleraceus alkaloid level Four fraction
6.1 length split sonchus oleraceus alkaloid level Four fraction
SB3-4-1 fraction rotary evaporation will be obtained, will be concentrated, freeze-drying is spare.SB3-4-1 sample is dissolved with methanol, is matched
It is 10mg/mL test liquid that concentration, which is made, crosses 0.22 μm of filter membrane.Separated with HPLC, chromatographic condition: mobile phase is 0.1% formic acid-
Water (A)-acetonitrile (B) system, elution program 0-10min, 5%-95%B, flow velocity 0.3mL/min, 30 DEG C of column temperature, wavelength
260nm, 20 μ L of applied sample amount.
As shown in figure 19, compound 1, compound 2 and compound 3 are obtained.
6.2 level Four fractions are to H2O2Induce the repair of the model of oxidative of Caco-2 cell
Compound 1, compound 2 and compound 3 (concentration: 200 μ g/mL) are investigated respectively to H2O2Induction
The repair of the model of oxidative of Caco-2 cell, as shown in figure 20.
The results show that its cell survival rate is respectively 59%, 71%, 84%, therefore, compound 3 is to H2O2Induction
The repair effect of the model of oxidative of Caco-2 cell is more significant.
6.3 length split sonchus oleraceus alkaloid level Four fraction total antioxidant activity
(1) length splits sonchus oleraceus alkaloid level Four fraction total antioxidant capacity
Detection method is the same as embodiment 2.As shown in figure 21, control, Trolox, extract, SB3, SB3-4,
The total antioxidant capacity of compound1, compound 2 and compound 3 is respectively 0.065,6.66,0.89,1.97,
5.52,1.89,3.62,4.58mmol/g, from the total antioxidant capacity of compound 1, compound 2 and compound 3
It is found that the total antioxidant capacity of compound 3 is relatively high in data, the total antioxidant capacity of compound 3 is about
Control, Trolox, extract, 70,0.7,5,2,0.8,2,1 times of SB3, SB3-4, compound 1 and compound 2,
Therefore, the long oxidation resistance for splitting the compound 3 in sonchus oleraceus alkaloid level Four fraction is stronger.
(2) length splits sonchus oleraceus alkaloid level Four fraction to the Scavenging activity of ABTS free radical
Detection method is the same as embodiment 2.As shown in figure 22, dense as compound 1, compound 2 and compound 3
When degree is 1.6 μ g/mL, the clearance rate to ABTS free radical is respectively 37.4%, 41.54%, 42.52%, and works as its concentration
When for 1000 μ g/mL, clearance rate is respectively 72.64%, 78.94%, 87.6%, and about low concentration is to ABTS free radical
1.9,1.9,2.1 times of clearance rate, therefore, experiment show that length is split sonchus oleraceus alkaloid level Four fraction and all had to ABTS free radical
Scavenging activity, and (concentration is 200 and 1000 μ g/mL by compound 1 (when concentration is 200 and 1000 μ g/mL), compound 2
When) BHT is lower than to the clearance rate of ABTS free radical, remaining clearance rate is above BHT.
(3) length splits sonchus oleraceus alkaloid level Four fraction to the Scavenging activity of DPPH free radical
Detection method is the same as embodiment 2.As shown in figure 23, dense as compound 1, compound 2 and compound 3
When degree is 1.6 μ g/mL, the clearance rate to DPPH free radical is respectively 40.75%, 43.02%, 53.4%, and works as its concentration
When for 1000 μ g/mL, clearance rate is respectively 71.32%, 74.91%, 79.06%, and about low concentration is to DPPH free radical
1.7,1.8,1.5 times of clearance rate, therefore, experiment shows long to split sonchus oleraceus alkaloid level Four fraction and all have DPPH free radical
There is Scavenging activity, however (concentration is 200 and 1000 μ by compound 1 (when concentration is 200 and 1000 μ g/mL), compound 2
When g/mL) BHT is lower than to the clearance rate of DPPH free radical, remaining clearance rate is above BHT.
(4) length splits sonchus oleraceus alkaloid level Four fraction to the Scavenging activity of hydroxyl radical free radical
Detection method is the same as embodiment 2.As shown in figure 24, dense as compound 1, compound 2 and compound 3
When degree is 1.6 μ g/mL, the clearance rate to hydroxyl radical free radical is respectively 11.37%, 15.86%, 25.02%, and works as its concentration
When for 1000 μ g/mL, clearance rate is respectively 72.45%, 79.39%, 80.05%, and about low concentration is to hydroxyl radical free radical
6,5,3 times of clearance rate, therefore, experiment shows long to split sonchus oleraceus alkaloid level Four fraction and all have hydroxyl radical free radical removing
Ability, however compound 2 (when concentration is 1.6 μ g/mL), compound3 (when concentration is 1.6 μ g/mL) is to hydroxyl radical free radical
Clearance rate be higher than BHT, remaining clearance rate is below BHT.
(5) length splits sonchus oleraceus alkaloid level Four fraction to the Scavenging activity of superoxide anion
Detection method is the same as embodiment 2.As shown in figure 25, compared with BHT, length is split in sonchus oleraceus alkaloid level Four fraction
Compound 1, compound 2 and compound 3 have Scavenging activity to superoxide anion, the results show that compound 3
Autoxidation reduced rate it is relatively significant.
(6) the long total reducing power for splitting sonchus oleraceus alkaloid level Four fraction
Detection method is the same as embodiment 2.As shown in figure 26, dense as compound 1, compound 2 and compound 3
When degree is 20 μ g/mL, increased light absorption value is respectively 0.012,0.019,0.033, and when its concentration is 200 μ g/mL,
Increased light absorption value is respectively 0.196,0.217,0.265, about the 16,11,8 of total reducing power of low concentration times, therefore,
Experiment shows that length splits sonchus oleraceus alkaloid level Four fraction and all has total reducing power, however total reducing power of compound 3 is high
Then it is lower than BHT in total reducing power of BHT, and compound 1 and compound 2.
To sum up, testing result is consistent with detection cell survival rate experimental result, therefore, the anti-oxidant work of compound 3
Property is stronger.
(7) long to split sonchus oleraceus alkaloid oxidation-resistant active ingredient synergistic effect
Detect compound 1, compound 2 and compound 3 antioxidant activity, compound 1 with
The coefficient total antioxidant capacity of compound 2 is that 4.02mmol/g, compound 1 and compound 2 individually makees
Used time, total antioxidant capacity are 1.89 and 3.62mmol/g, and therefore, compound 1 has with compound 2 cooperates with work
With, and when compound 2 and compound 3 interaction, 2 He of total antioxidant capacity 4.6mmol/g, compound
When 3 independent role of compound, total antioxidant capacity be 3.62 and 4.58mmol/g, therefore, compound 2 with
Compound 3 has synergistic effect, and the coefficient total antioxidant capacity of compound 1 and compound 3 is
When 4.59mmol/g, compound 1 and compound 3 are individually acted on, total antioxidant capacity is respectively 1.89 Hes
4.58mmol/g, therefore, compound 1 and compound 3 have synergistic effect.In addition, compound 1, compound 2
When with 3 collective effect of compound, i.e., when SB3-4-1 works, total antioxidant capacity 9.45mmol/g, therefore, experiment
The result shows that having synergistic effect between compound 1, compound 2 and compound 3.
Claims (9)
1. length splits sonchus oleraceus alkaloid level Four isolate, which is characterized in that the level Four isolate is by the side that includes the following steps
Method is prepared:
(1) preparation length splits sonchus oleraceus extract；
(2) length that step (1) obtains is split into the test liquid that sonchus oleraceus extract is configured to 100mg/mL, using preparative chromatography into
Row separation, chromatographic condition are as follows:
Using C18 silicagel column, using 0.1% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, flow velocity 10mL/min, column
Warm 27-33 DEG C, wavelength 255-265nm, gradient elution program are as follows:
In the time range of 0-5min, the volume of mobile phase A becomes 60% from 95%, and the volume of Mobile phase B is become from 5%
40%；
In the time range of 5-15min, the volume of mobile phase A becomes 30% from 60%, and the volume of Mobile phase B is become from 40%
70%；
In the time range of 15-30min, the volume of mobile phase A becomes 20% from 30%, and the volume of Mobile phase B is become by 70%
It is 80%；
In the time range of 30-35min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B remains 80%；
Peak height range is collected in the fraction of 420-2000mAU, obtains primary isolate；
(3) it after the primary isolate for obtaining step (2) is concentrated, is dry, is configured to the test liquid of 10mg/mL and uses prepare color
Spectrometry separation, chromatographic condition are as follows:
Using C18 silicagel column, using 0.1% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, flow velocity 10mL/min, column
Warm 27-33 DEG C, wavelength 255-265nm, gradient elution program are as follows:
In the time range of 0-5min, the volume of mobile phase A becomes 60% from 95%, and the volume of Mobile phase B is become from 5%
40%；
In the time range of 5-15min, the volume of mobile phase A becomes 30% from 60%, and the volume of Mobile phase B is become from 40%
70%；
In the time range of 15-30min, the volume of mobile phase A becomes 20% from 30%, and the volume of Mobile phase B is become by 70%
It is 80%；
In the time range of 30-35min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B remains 80%；
Peak height range is collected in the fraction of 0.2-1.8AU, obtains secondary isolate；
(4) it after the secondary isolate for obtaining step (3) is concentrated, is dry, is configured to the test liquid of 10mg/mL and uses prepare color
Spectrometry separation, chromatographic condition are as follows:
Using C18 silicagel column, using 0.1% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, flow velocity 1mL/min, column temperature
30 DEG C, wavelength 260nm, gradient elution program are as follows:
In the time range of 0-8min, the volume of mobile phase A becomes 75% from 95%, and the volume of Mobile phase B is become from 5%
25%；
In the time range of 8-10min, the volume of mobile phase A becomes 65% from 75%, and the volume of Mobile phase B is become from 25%
35%；
In the time range of 10-20min, the volume of mobile phase A becomes 40% from 65%, and the volume of Mobile phase B is become by 35%
It is 60%；
In the time range of 20-24min, the volume of mobile phase A becomes 20% from 40%, and the volume of Mobile phase B has 60% change
It is 80%；
In the time range of 24-27min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B remains 80%, receives
The fraction for collecting 16-18min, obtains three-level isolate；
(5) the three-level isolate obtained with HPLC method separating step (4), chromatographic condition are as follows:
Using 0.1% aqueous formic acid as mobile phase A, using acetonitrile as Mobile phase B, flow velocity 0.3mL/min, 30 DEG C of column temperature, wavelength
260nm, gradient elution program are as follows:
In 0-10min time range, the volume of Mobile phase B becomes 95% from 5%, and the volume of mobile phase A is become from 95%
5%, the fraction of 5.8-6.2min is collected, level Four isolate is obtained.
2. length according to claim 1 splits sonchus oleraceus alkaloid level Four isolate, which is characterized in that in step (2), column temperature
It is 30 DEG C, wavelength 260nm.
3. length according to claim 1 splits sonchus oleraceus alkaloid level Four isolate, which is characterized in that long to split sonchus oleraceus extraction
Object is prepared by method comprising the following steps:
Using 75% ethyl alcohol as solvent, by the long extraction solution for splitting sonchus oleraceus and liquid-to-solid ratio being made as 30mL/g, adjusts and extract solution
PH is 5, carries out ultrasonic extraction, after ultrasonic extraction, is stood, and takes supernatant, after rotary evaporation it is cooling it is dry to get.
4. length according to claim 3 splits sonchus oleraceus alkaloid level Four isolate, which is characterized in that the power of ultrasonic extraction
For 700W, temperature is 55 DEG C, ultrasonic time 30min.
5. the long separation method for splitting sonchus oleraceus alkaloid level Four isolate, which is characterized in that the separation method includes following step
It is rapid:
(1) preparation length splits sonchus oleraceus extract；
(2) length that step (1) obtains is split into the test liquid that sonchus oleraceus extract is configured to 100mg/mL, using preparative chromatography into
Row separation, chromatographic condition are as follows:
Using C18 silicagel column, using 0.1% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, flow velocity 10mL/min, column
Warm 27-33 DEG C, wavelength 255-265nm, gradient elution program are as follows:
In the time range of 0-5min, the volume of mobile phase A becomes 60% from 95%, and the volume of Mobile phase B is become from 5%
40%；
In the time range of 5-15min, the volume of mobile phase A becomes 30% from 60%, and the volume of Mobile phase B is become from 40%
70%；
In the time range of 15-30min, the volume of mobile phase A becomes 20% from 30%, and the volume of Mobile phase B is become by 70%
It is 80%；
In the time range of 30-35min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B remains 80%；
Peak height range is collected in the fraction of 420-2000mAU, obtains primary isolate；
(3) it after the primary isolate for obtaining step (2) is concentrated, is dry, is configured to the test liquid of 10mg/mL and uses prepare color
Spectrometry separation, chromatographic condition are as follows:
Using C18 silicagel column, using 0.1% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, flow velocity 10mL/min, column
Warm 27-33 DEG C, wavelength 255-265nm, gradient elution program are as follows:
In the time range of 0-5min, the volume of mobile phase A becomes 60% from 95%, and the volume of Mobile phase B is become from 5%
40%；
In the time range of 5-15min, the volume of mobile phase A becomes 30% from 60%, and the volume of Mobile phase B is become from 40%
70%；
In the time range of 15-30min, the volume of mobile phase A becomes 20% from 30%, and the volume of Mobile phase B is become by 70%
It is 80%；
In the time range of 30-35min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B remains 80%；
Peak height range is collected in the fraction of 0.2-1.8AU, obtains secondary isolate；
(4) it after the secondary isolate for obtaining step (3) is concentrated, is dry, is configured to the test liquid of 10mg/mL and uses prepare color
Spectrometry separation, chromatographic condition are as follows:
Using C18 silicagel column, using 0.1% aqueous formic acid as mobile phase A, using methanol as Mobile phase B, flow velocity 1mL/min, column temperature
30 DEG C, wavelength 260nm, gradient elution program are as follows:
In the time range of 0-8min, the volume of mobile phase A becomes 75% from 95%, and the volume of Mobile phase B is become from 5%
25%；
In the time range of 8-10min, the volume of mobile phase A becomes 65% from 75%, and the volume of Mobile phase B is become from 25%
35%；
In the time range of 10-20min, the volume of mobile phase A becomes 40% from 65%, and the volume of Mobile phase B is become by 35%
It is 60%；
In the time range of 20-24min, the volume of mobile phase A becomes 20% from 40%, and the volume of Mobile phase B has 60% change
It is 80%；
In the time range of 24-27min, the volume of mobile phase A remains 20%, and the volume of Mobile phase B remains 80%, receives
The fraction for collecting 16-18min, obtains three-level isolate；
(5) the three-level isolate obtained with HPLC method separating step (4), chromatographic condition are as follows:
Using 0.1% aqueous formic acid as mobile phase A, using acetonitrile as Mobile phase B, flow velocity 0.3mL/min, 30 DEG C of column temperature, wavelength
260nm, gradient elution program are as follows:
In 0-10min time range, the volume of Mobile phase B becomes 95% from 5%, and the volume of mobile phase A is become from 95%
5%, the fraction of 5.8-6.2min is collected, level Four isolate is obtained.
6. the separation method that length according to claim 5 splits sonchus oleraceus alkaloid level Four isolate, which is characterized in that step
(2) in, column temperature is 30 DEG C, wavelength 260nm.
7. the separation method that length according to claim 5 splits sonchus oleraceus alkaloid level Four isolate, which is characterized in that length is split
Sonchus oleraceus extract is prepared by method comprising the following steps:
Using 75% ethyl alcohol as solvent, by the long extraction solution for splitting sonchus oleraceus and liquid-to-solid ratio being made as 30mL/g, ultrasonic extraction is carried out, is surpassed
It after sound extracts, stands, takes supernatant, cooling is drying to obtain after rotary evaporation.
8. the separation method that length according to claim 5 splits sonchus oleraceus alkaloid level Four isolate, which is characterized in that ultrasound
The power of extraction is 700W, and temperature is 55 DEG C, ultrasonic time 30min.
9. the application that length according to claim 1 splits sonchus oleraceus alkaloid level Four isolate.
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| CN101513440A (en) * | 2008-09-19 | 2009-08-26 | 朱止平 | Sonchus plant extract and application thereof in preparing medicament for curing skin diseases |
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| 冀德富等: "小苦苣菜中化学成分的初步研究", 《山西中医学院报》 * |
| 潘方方等: "响应面法优化长裂苦苣菜总生物碱提取工艺", 《食品工业科技》 * |
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