CN110256505A - Length splits sonchus oleraceus alkaloid primary isolate and its separation method and application - Google Patents
Length splits sonchus oleraceus alkaloid primary isolate and its separation method and application Download PDFInfo
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- CN110256505A CN110256505A CN201910597099.0A CN201910597099A CN110256505A CN 110256505 A CN110256505 A CN 110256505A CN 201910597099 A CN201910597099 A CN 201910597099A CN 110256505 A CN110256505 A CN 110256505A
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- sonchus oleraceus
- mobile phase
- alkaloid
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- 244000113428 Sonchus oleraceus Species 0.000 title claims abstract description 111
- 235000006745 Sonchus oleraceus Nutrition 0.000 title claims abstract description 111
- 229930013930 alkaloid Natural products 0.000 title claims abstract description 95
- 150000003797 alkaloid derivatives Chemical class 0.000 title claims abstract description 90
- 238000000926 separation method Methods 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 239000000284 extract Substances 0.000 claims description 59
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 13
- 238000002137 ultrasound extraction Methods 0.000 claims description 10
- 235000019441 ethanol Nutrition 0.000 claims description 7
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000004237 preparative chromatography Methods 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
- 238000002390 rotary evaporation Methods 0.000 claims description 4
- 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 3
- 239000012467 final product Substances 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims 2
- 229910002027 silica gel Inorganic materials 0.000 claims 2
- 239000000741 silica gel Substances 0.000 claims 2
- 229960001866 silicon dioxide Drugs 0.000 claims 2
- 238000001035 drying Methods 0.000 claims 1
- -1 alkaloid compound Chemical class 0.000 abstract description 35
- 230000003647 oxidation Effects 0.000 abstract description 6
- 238000007254 oxidation reaction Methods 0.000 abstract description 6
- 238000007445 Chromatographic isolation Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 18
- 230000002000 scavenging effect Effects 0.000 description 18
- 230000003078 antioxidant effect Effects 0.000 description 13
- 230000004083 survival effect Effects 0.000 description 12
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- 238000002835 absorbance Methods 0.000 description 10
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- 238000001514 detection method Methods 0.000 description 9
- 230000004792 oxidative damage Effects 0.000 description 9
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- 241000252212 Danio rerio Species 0.000 description 8
- 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 7
- 239000012153 distilled water Substances 0.000 description 6
- 230000006698 induction Effects 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 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 5
- 230000003064 anti-oxidating effect Effects 0.000 description 5
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- 238000006243 chemical reaction Methods 0.000 description 5
- 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 5
- 230000000968 intestinal effect Effects 0.000 description 5
- 230000008439 repair process Effects 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 229960000935 dehydrated alcohol Drugs 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229960004756 ethanol Drugs 0.000 description 4
- 238000011084 recovery Methods 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
- 238000006701 autoxidation reaction Methods 0.000 description 3
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- 150000003233 pyrroles Chemical class 0.000 description 3
- 238000012360 testing method Methods 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
- 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
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
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- 230000007423 decrease Effects 0.000 description 2
- 238000004090 dissolution 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
- 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
- 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
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 241001412304 Ixeris Species 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
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 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
- 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
- 230000008033 biological extinction Effects 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
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- 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
- 239000004467 fishmeal Substances 0.000 description 1
- 238000002376 fluorescence recovery after photobleaching Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 238000004108 freeze drying Methods 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
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 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
- 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
- 239000012528 membrane Substances 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
- 238000002953 preparative HPLC Methods 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
- 150000003254 radicals Chemical class 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 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
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
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- 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)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Cosmetics (AREA)
Abstract
The invention belongs to agricultural biological technical fields, and in particular to length splits sonchus oleraceus alkaloid primary isolate and its separation method and application.The present invention splits sonchus oleraceus extraction alkaloid component therein by the way that ultrasonic method is long, and obtains several primary isolates 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 primary 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 primary isolate.
A further object of the present invention is to provide the separation methods of above-mentioned primary isolate.
A further object of the present invention is to provide the applications of above-mentioned primary isolate.
The length of specific embodiment splits sonchus oleraceus alkaloid primary isolate according to the present invention, by what is included the following steps
Method is prepared:
(1) preparation length splits sonchus oleraceus extract；
(2) sonchus oleraceus extract is split using the length that preparative chromatography separating step (1) obtains, actual conditions are as follows:
Using C18 chromatographic column, using 0.1% formic acid-aqueous solution 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 be in the time range of 0-5min, mobile phase A
Volume becomes 60% from 95%, and the volume of Mobile phase B becomes 40% from 5%；In the time range of 5-15min, mobile phase A
Volume becomes 30% from 60%, and the volume of Mobile phase B becomes 70% from 40%；In the time range of 15-30min, mobile phase A
Volume become 20% from 30%, the volume of Mobile phase B becomes 80% from 70%；In the time range of 30-35min, flowing
The volume of phase A remains 20%, and the volume of Mobile phase B remains 80%；Collecting fraction of the peak height range in 420-2000mAU is
?.
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 primary 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 primary 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 primary 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 primary isolate, the separation according to the present invention
Method the following steps are included:
(1) preparation length splits sonchus oleraceus extract；
(2) sonchus oleraceus extract is split using the length that preparative chromatography separating step (1) obtains, actual conditions are as follows:
Using C18 chromatographic 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 be in the time range of 0-5min, mobile phase A
Volume becomes 60% from 95%, and the volume of Mobile phase B becomes 40% from 5%；In the time range of 5-15min, mobile phase A
Volume becomes 30% from 60%, and the volume of Mobile phase B becomes 70% from 40%；In the time range of 15-30min, mobile phase A
Volume become 20% from 30%, the volume of Mobile phase B becomes 80% from 70%；In the time range of 30-35min, flowing
The volume of phase A remains 20%, and the volume of Mobile phase B remains 80%；Collecting fraction of the peak height range in 420-2000mAU is
?.
The length of specific embodiment splits the separation method of sonchus oleraceus alkaloid primary 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 primary 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 primary 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 primary fractions of preparative chromatography, determine that length splits sonchus oleraceus alkaloid primary 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
Fig. 1 shows H2O2Influence to Caco-2 cell survival rate；
Fig. 2 display length splits sonchus oleraceus alkaloid extract to H2O2Damage the influence of Caco-2 cell survival rate；
The long total antioxidant capacity for splitting sonchus oleraceus alkaloid extract of Fig. 3 display；
Fig. 4 display length splits sonchus oleraceus alkaloid extract to the Scavenging activity of ABTS free radical；
Fig. 5 display length splits sonchus oleraceus alkaloid extract to the Scavenging activity of DPPH free radical；
Fig. 6 display length splits sonchus oleraceus alkaloid extract to the Scavenging activity of hydroxyl radical free radical；
Fig. 7 display length splits sonchus oleraceus alkaloid extract to the Scavenging activity of superoxide anion；
The long total reducing power for splitting sonchus oleraceus alkaloid extract of Fig. 8 display；
Fig. 9 display length splits sonchus oleraceus alkaloid extract to the influence situation of zebra fish intestinal tissue ROS；
Figure 10 display length splits sonchus oleraceus alkaloid extract to the influence situation of zebra fish intestinal tissue SOD；
Figure 11 display length splits sonchus oleraceus alkaloid extract to the influence situation of zebra fish intestinal tissue MDA；
Figure 12 display length splits sonchus oleraceus alkaloid extract to the influence situation of zebra fish intestinal tissue CAT；
The long preparative HPLC separation situation for splitting sonchus oleraceus extract of Figure 13 display；
Figure 14 display length splits sonchus oleraceus alkaloid extract and fraction to H2O2Damage the influence feelings of Caco-2 cell survival rate
Condition；
Figure 15 shows the total antioxidant capacity of level-one fraction；
Figure 16 shows level-one fraction to the Scavenging activity of ABTS free radical；
Figure 17 shows level-one fraction to the Scavenging activity of DPPH free radical；
Figure 18 shows level-one fraction to the Scavenging activity of hydroxyl radical free radical；
Figure 19 shows level-one fraction to the Scavenging activity of superoxide anion；
Figure 20 shows total reducing power of level-one 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
2.1 H2O2Establish 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.
As shown in Figure 1, H2O2Concentration does not have a significant impact to cell survival rate in 100-5000 μm of ol/L, 7500 μ
Cell survival rate decline about 40% when mol/L, and the H of high concentration2O2Caco-2 cell survival rate is caused to be decreased obviously, cell number
Amount significantly reduces, through 10000 μm of ol/L H2O2The cell survival rate decline about 60% of processing, determines suitable H2O2Induction
The H of Caco-2 cell oxidative damage model2O2Concentration 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 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 3, being 0.07mmol/ with concentration
The control of g is compared, and concentration is that the total antioxidant capacity that 200 μ g/mL long split sonchus oleraceus alkaloid extract is 0.89mmol/
G, about the 12 of control times, but compared with concentration 6.66mmol/g positive control Trolox, the total antioxidation energy of Trolox
Power 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-onium salt, 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 (butylatedhydoxytoluene).
ABTS clearance rate (%)=[(A1-A2)/A1] × 100%, wherein A1Indicate the absorbance of blank control, A2It indicates
Sample to be tested ABTS absorbance.
As shown in Figure 4, when the long concentration for splitting sonchus oleraceus alkaloid extract is 25 μ g/mL, clearance rate 20%, so
And when its concentration is 200 μ g/mL, clearance rate is about 79.09%, about clearance rate of the low concentration to ABTS free radical
4 times.
(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 5, when it is 25 μ g/mL that length, which splits sonchus oleraceus alkaloid extract concentration, clearance rate 3.6%, so
And when its concentration is 200 μ g/mL, clearance rate is about 32.6%, about clearance rate of the low concentration to DPPH free radical
9 times.
(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 clearance rate (%)=[(A1-A2)/A1] × 100%, wherein A1Indicate the absorbance of blank control,
A2Indicate the absorbance of sample to be tested.
As shown in fig. 6, when it is 25 μ g/mL that length, which splits sonchus oleraceus alkaloid extract concentration, clearance rate 10.3%, so
And when its concentration is 200 μ g/mL, clearance rate is about 63.9%, about clearance rate of the low concentration to hydroxyl radical free radical
6 times.
(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.
As shown in fig. 7, length, which splits sonchus oleraceus alkaloid extract, has Scavenging activity to superoxide anion compared with BHT,
Autoxidation rate is 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%TCA2.5mL, 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 8, increased light absorption value is when it is 20 μ g/mL that length, which splits sonchus oleraceus alkaloid extract concentration,
0.006, however when its concentration is 200 μ g/mL, increased light absorption value is 0.046, about total reducing power of low concentration
7 times.
(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.
As a result as shown in figs9-12, length, which splits sonchus oleraceus alkaloid extract, can effectively reduce zebra fish intestinal tissue
ROS, MDA content improve 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:
Using HPLC preparation chromatography long will split sonchus oleraceus alkaloid extract Durashell C18 (L) (10 μm,
30 × 250mm) it is fractionated on column, it is long to split sonchus oleraceus alkaloid level-one fraction and prepare chromatographic condition: chromatographic column: Durashell
C18 (L) (10 μm,30 × 250mm), mobile phase be 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 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 figure 13, wherein 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 length split sonchus oleraceus alkaloid level-one fraction to H2O2The reparation of the Caco-2 cell oxidative damage model of induction is made
With
Fraction SB1, SB2, SB3 are investigated respectively, and SB4, SB5 and SB6 (concentration: 200 μ g/mL) are to H2O2The Caco-2 of induction
The repair of cell oxidative damage model.
As a result as Figure 14 shows, 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.
As shown in Figure 15, control, Trolox, extract, the total antioxidant capacity of SB1-SB6 is respectively 0.07,
6.66,0.89,0.09,0.21,1.98,0.92,0.89,0.28mmol/g, it is found that the total antioxidation of SB3 from these data
Ability is relatively high, and the total antioxidant capacity of SB3 is about control, Trolox, extract, SB1, SB2, SB4-SB6's
28,0.3,2,22,9,2,2,7 times, therefore, the long oxidation resistance for splitting the SB3 in sonchus oleraceus alkaloid level-one fraction 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 figure 16, when the concentration of SB1-SB6 is 1.6 μ g/mL, the clearance rate to ABTS free radical is respectively
1.61%, 3.64%, 15.99%, 19.43%, 7.69%, 8.5%, and when its concentration is 1000 μ g/mL, clearance rate point
Not Wei 31.58%, 65.79%, 74.29%, 74.09%, 55.47%, 46.96%, about low concentration is to ABTS free radical
20,18,5,4,7,6 times of clearance rate, since level-one fraction drug concentration is identical, the removing of SB1 and SB2 to ABTS free radical
The variation of rate is relatively large, relatively high to the clearance rate of ABTS free radical when the concentration highest of SB3 and SB4, and works as
When the concentration of SB4 is 40 μ g/mL, clearance rate is higher than BHT, and other level-one fractions are low to the clearance rate of ABTS free radical
In 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 figure 17, when the concentration of SB1-SB6 is 1.6 μ g/mL, the clearance rate to DPPH free radical is respectively
8.29%, 11.82%, 14.11%, 14.81%, 3.35%, 1.23%, and when its concentration is 1000 μ g/mL, clearance rate
Respectively 48.85%, 51.32%, 73.55%, 74.25%, 58.55%, 49.38%, about low concentration are to DPPH freedom
5,4,5,5,17,40 times of the clearance rate of base, since drug concentration is identical, the change of SB5 and SB6 to the clearance rate of DPPH free radical
Change it is relatively large, it is relatively high to the clearance rate of DPPH free radical when the concentration of SB3 and SB4 is maximum, and work as SB2-
When the concentration of SB4 is 1.6 μ g/mL, clearance rate is higher than BHT, and when level-one fraction is other 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 figure 18, when the concentration of SB1-SB6 is 1.6 μ g/mL, the clearance rate to hydroxyl radical free radical is respectively
6.85%, 10.37%, 27.77%, 21.44%, 20.39%, 18.1%, and when its concentration is 1000 μ g/mL, it removes
Rate is respectively 69.07%, 70.83%, 77.15%, 76.45%, 71.18%, 70.12%, about low concentration to hydroxyl from
By the 10 of the clearance rate of base, 7,3,4,3,4 times, since drug concentration is identical, change of the SB1-SB6 to the clearance rate of hydroxyl radical free radical
Change is less significant, and when the concentration of SB3 and SB4 is maximum, relatively high to the clearance rate of hydroxyl radical free radical, SB3 (works as concentration
When for 1.6,8,40,200 μ g/mL), SB4 (when concentration is 1.6,8 μ g/mL), SB5 (when concentration is 1.6 μ g/mL) and SB6
(when concentration is 1.6 μ g/mL) is apparently higher than BHT 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 figure 19, compared with BHT, length, which splits sonchus oleraceus alkaloid level-one fraction SB1-SB6, has superoxide anion
The autoxidation reduced rate of Scavenging activity, SB2, SB3 and SB4 are significant.
(6) the long total reducing power for splitting sonchus oleraceus alkaloid level-one fraction
As shown in figure 20, 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, due to drug
Concentration is identical, SB1, and total reducing power variation of the variation of total reducing power obvious, the SB3, SB5 and SB6 of SB2 and SB4 are not
It is too 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.
Claims (9)
1. length splits sonchus oleraceus alkaloid primary isolate, which is characterized in that the primary isolate is by the side that includes the following steps
Method is prepared:
(1) preparation length splits sonchus oleraceus extract；
(2) sonchus oleraceus extract is split using the length that preparative chromatography separating step (1) obtains, actual conditions 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%；
Collect peak height range 420-2000mAU fraction to obtain the final product.
2. length according to claim 1 splits sonchus oleraceus alkaloid primary 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 primary isolate, which is characterized in that long to split sonchus oleraceus extraction
Liquid 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 primary isolate, which is characterized in that the power of ultrasonic extraction
For 700 W, temperature is 55 DEG C, ultrasonic time 30min.
5. the long separation method for splitting sonchus oleraceus alkaloid primary isolate, which is characterized in that the separation method includes following step
It is rapid:
(1) preparation length splits sonchus oleraceus extract；
(2) sonchus oleraceus extract is split using the length that preparative chromatography separating step (1) obtains, actual conditions 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%；It receives
Collect peak height range 420-2000mAU fraction to obtain the final product.
6. the separation method that length according to claim 5 splits sonchus oleraceus alkaloid primary 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 primary 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 primary isolate, which is characterized in that ultrasound
The power of extraction is 700 W, and temperature is 55 DEG C, ultrasonic time 30min.
9. the application that length according to claim 1 splits sonchus oleraceus alkaloid primary 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 |
| CN105343144A (en) * | 2015-12-03 | 2016-02-24 | 河北大学 | Preparation method and application of anti-bacterial and anti-inflammation sow thistle ointment |
| CN106723098A (en) * | 2017-03-01 | 2017-05-31 | 徐亮 | A kind of bitter maror liquid extraction element |
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| CN110477195B (en) | 2022-05-31 |
| CN110563774B (en) | 2020-11-06 |
| CN110563775B (en) | 2020-09-25 |
| CN110563774A (en) | 2019-12-13 |
| CN110477195A (en) | 2019-11-22 |
| CN110563775A (en) | 2019-12-13 |
| CN110256505B (en) | 2020-09-18 |
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