CN110563775A - Long-split common sow thistle alkaloid secondary isolate and separation method and application thereof - Google Patents
Long-split common sow thistle alkaloid secondary isolate and separation method and application thereof Download PDFInfo
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- CN110563775A CN110563775A CN201910729482.7A CN201910729482A CN110563775A CN 110563775 A CN110563775 A CN 110563775A CN 201910729482 A CN201910729482 A CN 201910729482A CN 110563775 A CN110563775 A CN 110563775A
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- 229930013930 alkaloid Natural products 0.000 title claims abstract description 106
- 150000003797 alkaloid derivatives Chemical class 0.000 title claims abstract description 88
- 244000113428 Sonchus oleraceus Species 0.000 title claims abstract description 79
- 235000006745 Sonchus oleraceus Nutrition 0.000 title claims abstract description 79
- 238000000926 separation method Methods 0.000 title abstract description 10
- 239000000284 extract Substances 0.000 claims abstract description 65
- 238000000034 method Methods 0.000 claims abstract description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 20
- 238000004237 preparative chromatography Methods 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 10
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000012085 test solution Substances 0.000 claims description 10
- 238000002137 ultrasound extraction Methods 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 8
- 229910002027 silica gel Inorganic materials 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 4
- 238000001704 evaporation Methods 0.000 claims description 4
- 239000011344 liquid material Substances 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims 2
- -1 alkaloid compounds Chemical class 0.000 abstract description 29
- 230000003078 antioxidant effect Effects 0.000 abstract description 27
- 239000003963 antioxidant agent Substances 0.000 abstract description 23
- 238000013375 chromatographic separation Methods 0.000 abstract 1
- 235000006708 antioxidants Nutrition 0.000 description 22
- 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 22
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- 238000001514 detection method Methods 0.000 description 14
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- 230000004083 survival effect Effects 0.000 description 12
- 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 11
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- 241000252212 Danio rerio Species 0.000 description 7
- 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 7
- 229940079593 drug Drugs 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 238000007254 oxidation reaction Methods 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
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 5
- 238000006701 autoxidation reaction Methods 0.000 description 5
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- 238000002360 preparation method Methods 0.000 description 5
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000002292 Radical scavenging effect Effects 0.000 description 4
- 241000488874 Sonchus Species 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 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 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000489357 Sonchus brachyotus Species 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 3
- 230000001771 impaired effect Effects 0.000 description 3
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- 238000002955 isolation Methods 0.000 description 3
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Natural products CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-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
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 2
- STECJAGHUSJQJN-USLFZFAMSA-N LSM-4015 Chemical compound C1([C@@H](CO)C(=O)OC2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-USLFZFAMSA-N 0.000 description 2
- 235000006662 Lansium Nutrition 0.000 description 2
- 241001156382 Lansium Species 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
- 241000208292 Solanaceae Species 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 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
- 239000000843 powder Substances 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- 229940079877 pyrogallol Drugs 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 229930004668 tropane alkaloid Natural products 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
- 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
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000208838 Asteraceae Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- LHEBSUBHDAJGCC-UHFFFAOYSA-N C1(=CC=CC=C1)N(NN1CCCCC1)C1=CC=CC=C1 Chemical compound C1(=CC=CC=C1)N(NN1CCCCC1)C1=CC=CC=C1 LHEBSUBHDAJGCC-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
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000002193 Pain Diseases 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
- 206010042674 Swelling Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
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- 239000003674 animal food additive Substances 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- XADJWCRESPGUTB-UHFFFAOYSA-N apigenin Natural products C1=CC(O)=CC=C1C1=CC(=O)C2=CC(O)=C(O)C=C2O1 XADJWCRESPGUTB-UHFFFAOYSA-N 0.000 description 1
- 235000008714 apigenin Nutrition 0.000 description 1
- KZNIFHPLKGYRTM-UHFFFAOYSA-N apigenin Chemical compound C1=CC(O)=CC=C1C1=CC(=O)C2=C(O)C=C(O)C=C2O1 KZNIFHPLKGYRTM-UHFFFAOYSA-N 0.000 description 1
- 229940117893 apigenin Drugs 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
- 238000003556 assay Methods 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940076810 beta sitosterol Drugs 0.000 description 1
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
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- 235000012000 cholesterol Nutrition 0.000 description 1
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- 150000001875 compounds Chemical class 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
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- 239000012153 distilled water Substances 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002376 fluorescence recovery after photobleaching Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 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
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000037817 intestinal 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
- 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
- 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
- 239000012528 membrane Substances 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000000643 oven drying 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
- 229910052700 potassium Inorganic materials 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
- 230000001737 promoting effect Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 150000003233 pyrroles Chemical class 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007873 sieving 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
- 229950005143 sitosterol Drugs 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 238000005303 weighing Methods 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 the technical field of agricultural biology, and particularly relates to a secondary separated matter of a long-split common sow thistle alkaloid, and a separation method and application thereof. The invention extracts alkaloid components from the common sow thistle by an ultrasonic method, and obtains a secondary isolate with antioxidant capacity by preparative chromatographic separation, thereby having important practical significance for developing and utilizing alkaloid compounds in the common sow thistle.
Description
Technical Field
the invention belongs to the technical field of agricultural biology, and particularly relates to a secondary separated matter of a long-split common sow thistle alkaloid, and a separation method and application thereof.
Background
Sonchus brachyotus DC is an annual herb of Sonchus brachyotus of Compositae, also called herba Sonchi arvensis, herba Querci Acutissimae, herba Sonchi arvensis, and herba Ixeritis Sonchifoliae. The medicinal history of the long-crack common sow thistle is long, and according to records of the earliest medical monograph in China 'Shennong's herbal Jing and the 'herbal compendium' of Li Shizhen, the long-crack common sow thistle is bitter in taste and cold in nature, has the effects of clearing heat and removing toxicity, relieving swelling and expelling pus, cooling blood and removing stasis, clearing lung heat and relieving cough, benefiting liver and promoting urination, and helping digestion and harmonizing stomach, and is used for treating acute dysentery, enteritis, hemorrhoid and swelling and pain and other symptoms. The common sow thistle contains beta-sitosterol, luteolin, apigenin, quercetin and volatile oil, and has the functions of resisting bacteria, reducing blood pressure, reducing cholesterol, resisting tumor, treating hepatitis, resisting oxidation and the like. However, the effective antioxidant active ingredient in sonchus oleraceus has not been known so far.
Disclosure of Invention
the invention aims to provide a secondary separated substance of the common sowthistle herb alkaloid.
It is a further object of the present invention to provide a method for separating the secondary isolate described above.
It is a further object of the present invention to provide the use of the above secondary isolate.
According to a specific embodiment of the invention, the secondary separated substance of the common sow thistle alkaloid is prepared by a method comprising the following steps:
(1) Preparing extract of herba Sonchi arvensis;
(2) preparing the common sow thistle extract obtained in the step (1) into a test solution of 100mg/mL, and separating by adopting a preparative chromatography, wherein the chromatographic conditions are as follows:
Adopting a C18 silica gel column, taking 0.1% formic acid water solution as a mobile phase A, taking methanol as a mobile phase B, wherein the flow rate is 10mL/min, the column temperature is 27-33 ℃, the wavelength is 255-265nm, and the gradient elution procedure is as follows:
In the time range of 0-5min, the volume of the mobile phase A is changed from 95% to 60%, and the volume of the mobile phase B is changed from 5% to 40%;
in the time range of 5-15min, the volume of the mobile phase A is changed from 60% to 30%, and the volume of the mobile phase B is changed from 40% to 70%;
In the time range of 15-30min, the volume of the mobile phase A is changed from 30% to 20%, and the volume of the mobile phase B is changed from 70% to 80%;
The volume of mobile phase A is kept at 20% and the volume of mobile phase B is kept at 80% in the time range of 30-35 min;
Collecting fractions with peak heights ranging from 420 mAU to 2000mAU to obtain a primary isolate;
(3) Concentrating and drying the primary separated substance obtained in the step (2), preparing a test solution of 10mg/mL, and separating by using a preparative chromatography, wherein the chromatographic conditions are as follows:
Adopting a C18 silica gel column, taking 0.1% formic acid water solution as a mobile phase A, taking methanol as a mobile phase B, wherein the flow rate is 10mL/min, the column temperature is 27-33 ℃, the wavelength is 255-265nm, and the gradient elution procedure is as follows:
In the time range of 0-5min, the volume of the mobile phase A is changed from 95% to 60%, and the volume of the mobile phase B is changed from 5% to 40%;
In the time range of 5-15min, the volume of the mobile phase A is changed from 60% to 30%, and the volume of the mobile phase B is changed from 40% to 70%;
in the time range of 15-30min, the volume of the mobile phase A is changed from 30% to 20%, and the volume of the mobile phase B is changed from 70% to 80%;
the volume of mobile phase A is kept at 20% and the volume of mobile phase B is kept at 80% in the time range of 30-35 min;
Collecting the fraction with peak height ranging from 0.2-1.8AU to obtain secondary isolate.
in the reverse phase chromatographic column, the order of the common sow thistle alkaloid extract flowing out of the chromatographic column is that the components with stronger polarity are eluted out before the components with weaker polarity, namely the order of the common sow thistle alkaloid extract flowing out of the chromatographic column is influenced by the polarity of a mobile phase. Meanwhile, different mobile phase compositions, gradient programs, flow rates, column temperatures and the like can cause the polarity of the mobile phase to change, so that the compositions of effective components in fractions are different.
The method adopts the C18 chromatographic column to separate the alkaloid, can separate and obtain a chromatographic peak with good peak shape and no tailing under the condition of high pH, not only has extremely high sample loading amount, but also can keep high interface kinetic coefficient and obtain better column efficiency.
The secondary separated matter of the long-split common sow thistle alkaloid according to the embodiment of the invention has the column temperature of 30 ℃ and the wavelength of 260nm in the step (2).
According to the secondary separated substance of the common sow thistle alkaloid of the embodiment of the invention, the common sow thistle extract is prepared by the method comprising the following steps:
using 75% ethanol as solvent, making herba Sonchi arvensis into extractive solution with liquid-to-material ratio of 30mL/g, performing ultrasonic extraction, standing, and collecting supernatant.
the secondary separated matter of the alkaloid of the common sowthistle herb according to the embodiment of the invention has the ultrasonic extraction power of 700W, the temperature of 55 ℃ and the ultrasonic time of 30 min.
according to a particular embodiment of the invention, the method for the isolation of secondary isolates of alkaloids from sonchus oleraceus comprises the following steps:
(1) Preparing extract of herba Sonchi arvensis;
(2) Preparing the common sow thistle extract obtained in the step (1) into a test solution of 100mg/mL, and separating by adopting a preparative chromatography, wherein the chromatographic conditions are as follows:
Adopting a C18 silica gel column, taking 0.1% formic acid water solution as a mobile phase A, taking methanol as a mobile phase B, wherein the flow rate is 10mL/min, the column temperature is 27-33 ℃, the wavelength is 255-265nm, and the gradient elution procedure is as follows:
In the time range of 0-5min, the volume of the mobile phase A is changed from 95% to 60%, and the volume of the mobile phase B is changed from 5% to 40%;
in the time range of 5-15min, the volume of the mobile phase A is changed from 60% to 30%, and the volume of the mobile phase B is changed from 40% to 70%;
In the time range of 15-30min, the volume of the mobile phase A is changed from 30% to 20%, and the volume of the mobile phase B is changed from 70% to 80%;
The volume of mobile phase A is kept at 20% and the volume of mobile phase B is kept at 80% in the time range of 30-35 min;
Collecting fractions with peak heights ranging from 420 mAU to 2000mAU to obtain a primary isolate;
(3) Concentrating and drying the primary separated substance obtained in the step (2), preparing a test solution of 10mg/mL, and separating by using a preparative chromatography, wherein the chromatographic conditions are as follows:
Adopting a C18 silica gel column, taking 0.1% formic acid water solution as a mobile phase A, taking methanol as a mobile phase B, wherein the flow rate is 10mL/min, the column temperature is 27-33 ℃, the wavelength is 255-265nm, and the gradient elution procedure is as follows:
in the time range of 0-5min, the volume of the mobile phase A is changed from 95% to 60%, and the volume of the mobile phase B is changed from 5% to 40%;
In the time range of 5-15min, the volume of the mobile phase A is changed from 60% to 30%, and the volume of the mobile phase B is changed from 40% to 70%;
In the time range of 15-30min, the volume of the mobile phase A is changed from 30% to 20%, and the volume of the mobile phase B is changed from 70% to 80%;
The volume of mobile phase A is kept at 20% and the volume of mobile phase B is kept at 80% in the time range of 30-35 min;
collecting the fraction with peak height ranging from 0.2-1.8AU to obtain secondary isolate.
according to the separation method of the secondary separated substance of the long-split common sow thistle alkaloid, in the step (2), the column temperature is 30 ℃, and the wavelength is 260 nm.
According to the separation method of the long-split common sow thistle alkaloid secondary isolate, which is a specific embodiment of the invention, the long-split common sow thistle extract is prepared by the method comprising the following steps:
Using 75% ethanol as solvent, making herba Sonchi arvensis into extract solution with liquid-material ratio of 30mL/g, performing ultrasonic extraction, standing, collecting supernatant, performing rotary evaporation, cooling, and drying.
according to the separation method of the secondary alkaloid isolate of the common sow thistle in the embodiment of the invention, the power of ultrasonic extraction is 700W, the temperature is 55 ℃, and the ultrasonic time is 30 min.
the invention has the beneficial effects that:
the invention determines the extraction process of the common sow thistle alkaloid, and the extraction rate of the common sow thistle alkaloid can reach 20.30 percent. Multiple fractions are obtained by multi-stage preparative chromatography separation, and the secondary separated substance of the common sowthistle herb alkaloid is determined to have stronger oxidation resistance, so that reliable theoretical and technical support is provided for developing and producing novel oxidation-resistant food or feed additives.
Drawings
FIG. 1 shows H2O2Effect on survival of Caco-2 cells;
FIG. 2 shows the pair of alkaloid extracts of Sonchus oleraceus H2O2The effect of impaired Caco-2 cell survival;
FIG. 3 shows the total antioxidant capacity of the extract of Gesneriana pauciflorus alkaloids;
FIG. 4 shows the scavenging ability of the alkaloid extract of Sonchus oleraceus to ABTS free radicals;
FIG. 5 shows the DPPH radical scavenging ability of the extract of Gesneriana longissima alkaloid;
FIG. 6 shows the scavenging ability of the extract of Gesneriana longissima alkaloid on hydroxyl radicals;
FIG. 7 shows superoxide anion scavenging ability of extract of Gesneriana longissima alkaloid;
FIG. 8 shows the total reducing power of the extract of Gesneriana pauciflorus alkaloids;
FIG. 9 shows the effect of Gesneriana lancifera alkaloid extract on ROS in the intestinal tract tissue of zebra fish;
FIG. 10 shows the effect of the extract of Gesneriana lancifera alkaloids on the SOD of intestinal tissues of zebra fish;
FIG. 11 shows the effect of the alkaloid extract from Gesneriana lancifera on the intestinal tissue MDA of zebra fish;
FIG. 12 shows the effect of the alkaloid extract from Gesneriana lancifera on the intestinal tissue CAT of zebra fish;
FIG. 13 shows preparative HPLC separation of Sonchus oleraceus extract;
FIG. 14 shows the alkaloid extract and fraction pair H of Sonchus oleraceus2O2effects of impaired Caco-2 cell survival;
FIG. 15 shows the total antioxidant capacity of the first fraction;
FIG. 16 shows the scavenging ability of the first fraction for ABTS free radicals;
FIG. 17 shows DPPH radical scavenging ability of the first fraction;
FIG. 18 shows the scavenging ability of the first fraction for hydroxyl radicals;
FIG. 19 shows superoxide anion scavenging ability of the first fraction;
FIG. 20 shows the total reducing power of the first fraction;
FIG. 21 shows the results of preparative HPLC separation of the primary isolate;
FIG. 22 shows the secondary isolate pair H2O2the effect of impaired Caco-2 cell survival;
FIG. 23 shows the total antioxidant capacity of the secondary isolate;
FIG. 24 shows the scavenging capacity of the secondary isolates for ABTS free radicals;
FIG. 25 shows DPPH radical scavenging ability of secondary isolates;
FIG. 26 shows the scavenging ability of secondary isolates for hydroxyl radicals;
FIG. 27 shows superoxide anion scavenging capacity of secondary isolates;
Figure 28 shows the total reducing power of the secondary isolate.
Detailed Description
example 1 preparation of alkaloid extract from Sonchus oleraceus
Cleaning herba Sonchi arvensis, oven drying at 40 deg.C to constant weight, pulverizing with miniature plant pulverizer, and sieving with 60 mesh analytical sieve. Weighing 10g of whole herb coarse powder of common sow thistle, placing the whole herb coarse powder into a 500mL ultrasonic cup, adding 75% ethanol to obtain an extraction solution with the liquid-material ratio of 30mL/g, adjusting the pH value of the extraction solvent to be 5, and carrying out ultrasonic extraction at the ultrasonic temperature of 55 ℃, the ultrasonic power of 700W and the ultrasonic time of 30 min.
Ultrasonic extracting, centrifuging at 5000rpm for 10min to obtain supernatant, rotary evaporating at 40 deg.C, and freeze drying to obtain herba Sonchi Oleracei alkaloid extract.
alkaloids are nitrogen-containing organic compounds, and alkaloid compounds have alkaline-like properties. The alkaloid has various parent nucleus structures, and examples thereof include pyrrole alkaloid, tropane alkaloid (solanaceae alkaloid), and tropane alkaloid (solanaceae alkaloid).
putting 0.1mL of extracting solution into a 1mL colorimetric tube, fixing the volume to a scale by using ethanol with a certain concentration, taking the corresponding ethanol concentration as a blank, measuring the light absorption value of the extracting solution at 410nm, and measuring the extraction rate of alkaloid in the sonchus oleraceus according to a standard curve, wherein the extraction rate is (the weight of the alkaloid in a sample/the weight of medicinal materials) multiplied by 100%.
The extraction rate of alkaloid in the invention is 20.30%.
example 2 verification of the antioxidant capacity of the Alkaloids extract of Sonchus oleraceus
2.1H2O2Establishing a model for inducing Caco-2 cell oxidative damage
By investigating H2O2After the Caco-2 cells are acted for 24 hours at different concentrations (100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000 mu mol/L), the influence of the Caco-2 cells on the cell survival rate is observed, and H is determined2O2the Caco-2 cell oxidative damage model was induced, and the results are shown in FIG. 1.
As can be seen from FIG. 1, H2O2The cell survival rate is not significantly influenced when the concentration is 100-5000 mu mol/L, the cell survival rate is reduced by about 40% when the concentration is 7500 mu mol/L, and H is in high concentration2O2leads the survival rate of Caco-2 cells to be obviously reduced and the cell number to be obviously reduced by 10000 mu mol/L H2O2The survival rate of the treated cells decreased by about 60%, and the appropriate H was determined2O2H for inducing Caco-2 cell oxidative damage model2O2the concentration was 7500. mu. mol/L.
2.2 Gesneriana lansium alkaloid extract Pair H2O2Repair effect of induced Caco-2 cell oxidative damage model
The use concentration is as follows: extract pair H of 200 mug/mL common sow thistle alkaloid2O2The induced repairing effect of Caco-2 cell oxidative damage model is shown in FIG. 2.
The results showed that the cell viability was 71%, while H2O2after the induction of Caco-2 cell oxidative damage, the cell survival rate is 56%, so the sonchus oleraceus alkaloid extract is H2O2the model for inducing Caco-2 cell oxidative damage has a repairing effect.
2.3 anti-oxidant action of Sonchus oleraceus alkaloid extract
(1) Total antioxidant capacity of Sonchus oleraceus alkaloid extract
27.8mg of FeSO are weighed out4·7H2O, dissolved and diluted to 1mL, and the concentration is 100 mM. Taking a proper amount of 100mM FeSO4the solution was diluted to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM. Using steamsdistilled water or sample preparation solution is used for preparing the standard substance.
a: to each assay well of a 96-well plate, 180. mu.L of FRAP working solution (TPTZ dilution 150. mu.L + TPTZ solution 15. mu.L + assay buffer 15. mu.L) was added.
b: adding 5 μ L of distilled water or PBS; adding 5 mu L of FeSO with various concentrations into the detection holes of the standard curve4A standard solution; mu.L of each sample or 0.15-1.5mM Trolox was added to the sample wells as a positive control. Mix gently.
c: a593 was determined after incubation at 37 ℃ for 3-5 min. Measuring FeSO4The standard curve is that y is 0.3119x +0.0526, R2＝0.999。
The Total Antioxidant Capacity (TAC) of the sample was calculated from the standard curve. As shown in FIG. 3, the total antioxidant capacity of the extract of sonchus oleraceus alkaloids at a concentration of 200. mu.g/mL was 0.89mmol/g, about 12 times that of the control, compared to the control at a concentration of 0.07 mmol/g, but the total antioxidant capacity of Trolox was about 7 times that of the extract of sonchus oleraceus alkaloids at a concentration of 6.66mmol/g positive control Trolox.
(2) ABTS free radical scavenging ability of common sow thistle alkaloid extract
7mmol/L ABTS (2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonie acid) diam-onium salt, ABTS) was mixed with 2.45mmol/L potassium persulfate at a ratio of 9:1 and allowed to stand at room temperature for 16 hours, using the octafold dilution before use as ABTS stock solution. Adding 2.5mL ABTS stock solutions into a series of samples with concentration ranges (1.6-1000. mu.g/mL, dissolved by absolute ethyl alcohol), mixing uniformly, reacting for 20min in a dark place, and measuring the light absorption value at the position of 734nm wavelength. The mixed solution of 1mL double distilled water and ABTS was used as a blank control, and BHT (butylated hydoxylutene) was used as a positive control.
ABTS clearance (%) - (a)1-A2)/A1]X 100%, wherein A1Denotes the absorbance of the blank, A2and representing ABTS absorbance of the sample to be tested.
As can be seen from FIG. 4, the clearance rate was 20% when the concentration of the extract of the alkaloid from Sonchus oleraceus was 25. mu.g/mL, whereas the clearance rate was about 79.09% when the concentration was 200. mu.g/mL, which is about 4 times the clearance rate of the low concentration for ABTS free radicals.
(3) DPPH free radical scavenging ability of herba Sonchi arvensis alkaloid extract
Samples with a certain concentration range (1.6-1000 mug/mL, dissolved by absolute ethyl alcohol) are respectively added with 0.3mL of 0.2mmol/L DPPH (1, 1-diphenyl-2-piperidinylhydrazine, DPPH) (dissolved by absolute ethyl alcohol) in each sample, the mixture is vortexed and mixed, the prepared samples are kept for 1h in a dark place, and then the absorbance of the prepared samples is measured at 517 nm. 1mL of absolute ethanol was used as a blank control instead of the extract, and BHT in the same concentration range was used as a positive control.
DPPH clearance (%) - (a)1-A2)/A1]X 100%, wherein A1Denotes the absorbance of the blank, A2indicates the DPPH absorbance of the sample to be tested.
As shown in FIG. 5, the clearance was 3.6% when the extract of Gesneriana longissima alkaloid was 25. mu.g/mL, whereas the clearance was approximately 32.6% when the extract was 200. mu.g/mL, which is approximately 9 times the clearance of DPPH free radicals at low concentrations.
(4) scavenging ability of herba Sonchi arvensis alkaloid extract on hydroxyl free radical
Using Fe3+The EDTA-ascorbic acid-hydrogen peroxide system generates hydroxyl radicals, the deoxyribose is attacked by the hydroxyl radicals and then cracked, and the deoxyribose reacts with the thiobarbituric acid under the acidic and heating conditions to generate a red compound, and the existence of the antioxidant can prevent the hydroxyl radicals from attacking the deoxyribose.
To the reaction system were added 400. mu.L of 2-deoxyribose (10mmol/L), 100. mu.L of ferric chloride (10mmol/L), 100. mu.L of EDTA-2Na (ethylene diamine acetic acid salt) (1mmol/L), 100. mu.L of 30% hydrogen peroxide (10mmol/L), 100. mu.L of a sample concentration (1.6-1000. mu.g/mL), 200. mu.L of ascorbic acid (1mmol/L) was added to initiate the reaction, the reaction was carried out at 37 ℃ for 1 hour, 1mL of a 0.5% TBA solution of sodium hydroxide (0.025mol/L) and 1mL of a 30% aqueous solution of TCA (trichloroacetic acid) were added, and the mixture was heated in a water bath at 80 ℃ for 30min and cooled. The absorbance was measured at 532nm, and 0.05mol/L PBS (phosphate buffered solution) (pH 7.4) was used in the reaction system instead of the sample as a blank control to calculate the clearance, and BHT in the same concentration range was used as a positive control.
hydroxyl radical scavenging rate (%) - (A)1-A2)/A1]x 100%, wherein A1Denotes the absorbance of the blank, A2the absorbance of the sample to be measured is indicated.
As shown in FIG. 6, the clearance rate was 10.3% when the extract concentration of the common sowthistle herb alkaloid was 25. mu.g/mL, whereas the clearance rate was about 63.9% when the extract concentration was 200. mu.g/mL, which is about 6 times of the clearance rate of the hydroxyl radical at a low concentration.
(5) scavenging ability of herba Sonchi arvensis alkaloid extract on superoxide anion
superoxide anion is one of the most common free radicals in the body. Pyrogallic acid spontaneously generates superoxide anions in an alkaline environment. The rate of autoxidation is related to the concentration of superoxide anion. The test object can remove superoxide anion and slow down the autoxidation rate of pyrogallic acid.
Adding 1mL of 200 mu g/mL sample to be tested into Tris-HCl buffer (pH value 8.2, 0.05mol/L) and 4.5mL of the sample to be tested, reacting at 25 ℃ for 10min, adding 600 mu L of 0.003mol/L pyrogallol (dissolved in 10mmol/L hydrochloric acid), immediately measuring the absorbance at the wavelength of 325nm after full reaction, measuring the absorbance once every 30s until the absorbance does not change obviously, replacing the sample with 10mmol/L hydrochloric acid as a blank control, and using BHT as a positive control. The rate of auto-oxidation of pyrogallol can be calculated as the slope from the absorbance-time curve.
As shown in fig. 7, the extract of the alkaloid from sonchus oleraceus has a scavenging ability for superoxide anions and its autoxidation rate is slowed down compared to BHT.
(6) Total reducing power of herba Sonchi arvensis alkaloid extract
Determining total reducing power of alkaloid by iron reduction antioxidant power method, and measuring Fe in acidic environment3+-tripyridotriazine (Fe)3+TPTZ) can be reduced to ferrous form by antioxidant, and has blue color and maximum absorption at 593nm, and the higher the absorbance, the reductionthe stronger the capacity.
Preparing sample solution of 400 μ g/mL with sample 20mg, adding sample solution of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 μ L into 1mL with double distilled water, and adding PBS (pH 6.6) and 1% K of 0.2mol/L3[Fe(CN)6]2.5mL of each, reacted at 50 ℃ for 20min, then added with 2.5mL of 10% TCA, centrifuged at 3000r/min (r 3cm) for 10min, 2.5mL of the supernatant was added with 2.5mL of double distilled water, mixed with 0.5mL of 0.1% ferric chloride, and the absorbance A was measured at 700nm after 10min2The light absorption value is measured by using the double distilled water method without adding the sample as a blank control, and the increased light absorption value indicates the increase of the reduction capacity.
As shown in FIG. 8, the increased absorbance was 0.006 when the concentration of the extract of sonchus oleraceus alkaloid was 20. mu.g/mL, whereas the increased absorbance was 0.046 at 200. mu.g/mL, which is about 7 times the total reducing power of the low concentration.
(7) in vivo antioxidant effect of herba Sonchi arvensis alkaloid extract
Zebrafish intestinal injury was induced by injecting zebrafish with 0.2% Oxazolone (Oxazolone) for 1 day and fed with fish feed containing varying concentrations of sonchus oleraceus alkaloid extract for 5 days.
The results are shown in fig. 9-12, and the extract of herba Sonchi arvensis alkaloid can effectively reduce the content of ROS and MDA in intestinal tissue of zebra fish, and improve SOD and CAT activities.
in conclusion, the common sow thistle alkaloid extract has good antioxidation in vitro and in vivo.
example 3 isolation of Primary alkaloid fraction from Sonchus oleraceus
3.1 Long-split Sonchus oleraceus alkaloid first-order fraction
preparation of a test solution: dissolving herba Sonchi arvensis alkaloid extract with ultrapure water, preparing into test solution with concentration of 100mg/mL, and filtering with 0.22 μm filter membrane.
Chromatographic conditions for separating the primary alkaloid fraction of sonchus oleraceus are as follows:
Subjecting extract of herba Sonchi Oleracei alkaloid to Durashel by HPLC preparative chromatographyl C18(L)(10μm，30X 250mm) column, preparing chromatographic conditions for the first fraction of the alkaloid of common sowthistle herb: a chromatographic column: DurashellC18(L) (10 μm,30X 250mm), the mobile phase is a 0.1% formic acid-water (A) -methanol (B) system, the flow rate is 10mL/min, the column temperature is 30 ℃, and the wavelength is 260 nm. The gradient elution procedure is 0-5min, 5-40% B, 5-15min, 40-70% B, 15-30min, 70-80% B, 30-35min, 80-80% B, and the sample loading amount is 18 mL.
The extract of sonchus oleraceus alkaloid was separated into 6 fractions, SB1(s. brachyotus 1), SB2, SB3, SB4, SB5 and SB6, as shown in fig. 13, according to the chromatographic peak profile of sonchus oleraceus alkaloid extract in preparative chromatography, wherein the SB1 peak height ranged from 2 to 570mAU, the SB2 peak height ranged from 530 to 750mAU, the absorbance ranged, the SB3 peak height ranged from 420 to 2000mAU, the SB4 peak height ranged from 1200 to 2010mAU, the SB5 peak height ranged from 360 to 2010mAU, and the SB6 peak height ranged from 70 to 740 mAU.
3.2 Gesneriana oleracea alkaloid primary fraction Pair H2O2Repair effect of induced Caco-2 cell oxidative damage model
Fractions SB1, SB2, SB3, SB4, SB5 and SB6 (concentration: 200. mu.g/mL) were investigated for H2O2And (3) the repair effect of the induced Caco-2 cell oxidative damage model.
The results are shown in FIG. 14, where the cell viability was 76%, 74%, 114%, 106%, 94%, 75%, respectively, and thus SB3 was for H2O2the induced Caco-2 cell oxidative damage model has obvious repairing effect.
3.3 Oxidation resistance of Primary Total alkaloid fraction of Sonchus oleraceus
(1) Total antioxidant capacity of primary alkaloid fraction of Sonchus oleraceus
the detection method was the same as in example 2.
From FIG. 15, it can be seen that the total antioxidant capacity of control, Trolox, extract, SB1-SB6 is 0.07, 6.66, 0.89, 0.09, 0.21, 1.98, 0.92, 0.89, 0.28mmol/g, respectively, and from these data, the total antioxidant capacity of SB3 is relatively high, and the total antioxidant capacity of SB3 is about 28, 0.3, 2, 22, 9, 2, 7 times that of control, Trolox, extract, SB1, SB2, SB4-SB6, and therefore, the antioxidant capacity of SB3 in the primary fraction of the alkaloids from Gesnezoinum gracile is relatively strong.
(2) ABTS free radical scavenging ability of primary alkaloid fraction of Sonchus oleraceus L
the detection method was the same as in example 2.
As shown in FIG. 16, when SB1-SB6 was at a concentration of 1.6. mu.g/mL, the removal rates for ABTS free radicals were 1.61%, 3.64%, 15.99%, 19.43%, 7.69%, 8.5%, respectively, and when it was 1000. mu.g/mL, the removal rates were 31.58%, 65.79%, 74.29%, 74.09%, 55.47%, 46.96%, respectively, which are about 20, 18, 5, 4, 7, 6 times the removal rates for ABTS free radicals at low concentrations, and since the first-order fraction drug concentrations were the same, the change in the removal rates for ABTS free radicals was relatively large for SB1 and SB2, and when the concentrations of SB3 and SB4 were the highest, the removal rates for ABTS free radicals were relatively high, while when the concentrations of SB4 were 40. mu.g/mL, the removal rates were higher than for BHT, while the other first-order fractions were all lower than for ABTS free radicals.
(3) Scavenging ability of primary alkaloid fraction of herba Sonchi arvensis to DPPH free radical
The detection method was the same as in example 2.
As shown in FIG. 17, when SB1-SB6 was at a concentration of 1.6. mu.g/mL, the removal rates for DPPH radicals were 8.29%, 11.82%, 14.11%, 14.81%, 3.35%, 1.23%, respectively, and when it was 1000. mu.g/mL, the removal rates were 48.85%, 51.32%, 73.55%, 74.25%, 58.55%, 49.38%, respectively, which were about 5, 4, 5, 17, 40 times the removal rates for DPPH radicals at low concentrations, and the removal rates for DPPH radicals were relatively large for SB5 and SB6 due to the same drug concentration, relatively high when the concentrations of SB3 and SB4 were at their maximum, and lower than BHT when the concentrations of SB2-SB4 were 1.6. mu.g/mL, while the removal rates were higher than BHT when the first-order fractions were at other concentrations.
(4) Scavenging ability of primary alkaloid fraction of herba Sonchi arvensis to hydroxyl free radical
The detection method was the same as in example 2.
As shown in FIG. 18, when SB1-SB6 was at a concentration of 1.6. mu.g/mL, the clearance of hydroxyl radicals was 6.85%, 10.37%, 27.77%, 21.44%, 20.39%, 18.1%, respectively, and when it was at a concentration of 1000. mu.g/mL, the clearance was 69.07%, 70.83%, 77.15%, 76.45%, 71.18%, 70.12%, respectively, which was about 10, 7, 3, 4 times the clearance of hydroxyl radicals at a low concentration, and the clearance of SB1-SB6 was less significantly changed due to the same drug concentration, when SB3 and SB4 were at a maximum concentration, the clearance of hydroxyl radicals was relatively high, SB4 (when the concentration was 1.6, 8, 40, 200. mu.g/mL), when SB5 was 1.6, 8. mu.g/mL), when SB 5. mu.4836. mu.6 g/mL and BHT were significantly higher than those at a concentration of 1.6. mu.6. mu.3876. mu.25 g/mL), while the other first fractions have lower clearance than BHT.
(5) scavenging ability of primary alkaloid fraction of herba Sonchi arvensis to superoxide anion
The detection method was the same as in example 2.
as shown in fig. 19, the primary alkaloid fractions from sonchus oleraceus, SB1-SB6, have a scavenging capacity for superoxide anions compared to BHT, and the autoxidation rates of SB2, SB3 and SB4 slowed down significantly.
(6) total reducing power of primary alkaloid fraction of herba Sonchi Oleracei
As shown in fig. 20, when the concentration of SB1-SB6 was 20 μ g/mL, the absorbance values increased were 0, 0.014, 0.001, 0.022, and 0.019, respectively, and when the concentration was 200 μ g/mL, the absorbance values increased were 0.013, 0.014, 0.131, 0.075, 0.090, and 0.078, respectively, which are approximately 51, 54, 10, 100, 4, and 4 times the total reducing power of the low concentration, and since the concentrations of the drugs were the same, the total reducing powers of SB1, SB2, and SB4 were significantly changed, and the total reducing powers of SB3, SB5, and SB6 were not significantly changed, but SB3 was significantly higher than those of the other fractions, and the total reducing power was lower than that of BHT.
In conclusion, the detection result is consistent with the result of detecting the cell survival rate, so that the antioxidant activity of SB3 in the primary alkaloid fraction of the sonchus oleraceus is stronger.
Example 4 isolation of secondary alkaloid fraction from Sonchus oleraceus
4.1 Leptochloes Sonchi alkaloid secondary fraction
Rotary evaporating the effective active fraction SB3 at 40 deg.C, concentrating, and freeze drying. SB3 was dissolved in ultrapure water to prepare a sample solution having a concentration of 10mg/mL, which was then passed through a 0.22 μm filter. The crude product was purified by means of a preparative chromatography column Durashell C18(L) (10 μm,30X 250mm) was further fractionated.
The preparation chromatographic conditions are as follows: the mobile phase is a 0.1% formic acid-water (A) -methanol (B) system, the flow rate is 10mL/min, the column temperature is 30 ℃, and the wavelength is 260 nm. The gradient elution procedure is 0-5min, 5-40% B, 5-15min, 40-70% B, 15-30min, 70-80% B, 30-35min, 80-80% B, and the sample loading amount is 18 mL.
according to the chromatographic peaks of the fraction SB3 in the preparation chromatogram, the fraction SB3 is separated into 5 fractions, namely SB3-1, SB3-2, SB3-4 and SB3-5, wherein the peak height range of SB3-1 is 0-0.8AU, the peak height range of SB3-2 is 0.5-5.7AU, the peak height range of SB3-3 is 1.4-5.7AU, the peak height range of SB3-4 is 0.2-1.8AU and the peak height range of SB3-5 is 0.1-0.35AU, as shown in FIG. 21.
4.2 Gesneriana lansium alkaloid Secondary fraction Pair H2O2Inducing restoration effect of oxidative damage model of Caco-2 cells
Fractions SB3-1, SB3-2, SB3-3, SB3-4, SB3-5 (concentration: 200. mu.g/mL) were investigated for H2O2Inducing the repair effect of the oxidative damage model of Caco-2 cells.
As shown in FIG. 22, the cell viability was 67%, 75%, 87%, 99%, 77%, respectively, and thus SB3-4 was exhibited for H2O2the repairing effect of the oxidative damage model of the Caco-2 cells is induced to be obvious.
4.3 Oxidation resistance of secondary alkaloid fraction of Sonchus oleraceus
(1) Total antioxidant capacity of secondary alkaloid fraction of Sonchus oleraceus
the detection method was the same as in example 2.
As shown in FIG. 23, the total antioxidant capacity of control, Trolox, extract, SB3, SB3-1 to SB3-5 is 0.07, 6.66, 0.89, 1.98, 0.67, 2.31, 3.44, 5.52, 3.05mmol/g, respectively, so that the total antioxidant capacity of SB3-4 is relatively high, and the total antioxidant capacity of SB3-4 is about 78, 0.8, 6, 3, 8, 2 times as high as that of control, Trolox, extract, SB3, SB3-1, SB3-2, SB3-3, SB3-5, and thus the antioxidant capacity of SB3-4 in the secondary fraction of the alkaloids of Sonchus oleraceae is relatively high.
(2) ABTS free radical scavenging ability of long-crack common sow thistle alkaloid secondary fraction
The detection method was the same as in example 2.
As shown in FIG. 24, when the concentrations of SB3-1 to SB3-5 were 1.6. mu.g/mL, the respective clearance rates for ABTS free radicals were 0.61%, 2.85%, 2.24%, 11.18%, 4.47%, and when the concentration was 1000. mu.g/mL, the respective clearance rates were 75.61%, 75.81%, 76.21%, 76.63%, 76.02%, which were about 124, 27, 34, 7, 17 times the clearance rate for ABTS free radicals at a low concentration, and the clearance rates for ABTS free radicals were not significantly different from each other and were all lower than BHT when the concentrations of SB3-1, SB3-3, SB3-2, SB3-5, and SB3-4, and the secondary fractions of Sonchus oleraceus alkaloids were the greatest.
(3) DPPH free radical scavenging ability of long-crack common sow thistle alkaloid secondary fraction
The detection method was the same as in example 2.
As shown in FIG. 25, when the concentrations of SB3-1 to SB3-5 were 1.6. mu.g/mL, the respective clearance rates for DPPH free radicals were 5.04%, 2.14%, 8.45%, 3.78%, 3.53%, and when the concentrations were 1000. mu.g/mL, the clearance rates were 73.39%, 76.04%, 77.43%, 77.81%, 76.54%, respectively, which were approximately 15, 36, 9, 21, 22 times as high as the clearance rates for DPPH free radicals at low concentrations, and since the concentrations of the drugs were the same, the influence of the change in the clearance rates for DPPH free radicals was in the order of SB3-2, SB3-5, SB3-4, SB3-1, and SB3-3, and when the concentration of the second-stage fraction of the Sonchus oleraceae alkaloid was the greatest, the clearance rates for DPPH free radicals were not significantly different and were all lower than BHT.
(4) scavenging ability of secondary alkaloid fraction of herba Sonchi arvensis to hydroxyl free radical
The detection method was the same as in example 2.
As shown in FIG. 26, when the concentrations of SB3-1 to SB3-5 were 1.6. mu.g/mL, the clearance rates for hydroxyl radicals were 8.94%, 12.94%, 16.71%, 20.47%, 6.59%, respectively, and when the concentrations were 1000. mu.g/mL, the clearance rates were 77.18%, 78%, 81.41%, 89.53%, 79.18%, respectively, which were about 9, 6, 5, 4, 12 times as high as the clearance rates for hydroxyl radicals at low concentrations, the clearance rates for hydroxyl radicals were all not significantly varied from SB3-1 to SB3-5 due to the same drug concentrations, and the clearance rates for hydroxyl radicals were relatively high when the concentrations of SB3-3 and SB3-4 were the greatest, and the clearance rates for hydroxyl radicals of the secondary fraction of the extended crack Sonchus alkaloids were all lower than that of BHT.
(5) Scavenging ability of secondary alkaloid fraction of Sonchus oleraceus to superoxide anion
the detection method was the same as in example 2.
As shown in FIG. 27, the secondary fractions of the alkaloids from Sonchus oleraceus, SB3-1 to SB3-5, had a scavenging ability for superoxide anions and the autoxidation rates of SB3-3 and SB3-4 were significantly slowed down compared to BHT.
(6) Total reducing power of secondary alkaloid fraction of herba Sonchi arvensis
The detection method was the same as in example 2.
as shown in FIG. 28, when the concentrations of SB3-1 to SB3-5 were 20. mu.g/mL, the absorbance values increased were 0.018, 0.011, 0.023, 0.026, 0.016, respectively, and when the concentration was 200. mu.g/mL, the absorbance values increased were 0.140, 0.055, 0.253, 0.273, 0.150, respectively, which were about 8, 5, 11, 10.6, 9.7 times the total reducing power of the low concentrations, and since the concentrations of the drugs were the same, the total reducing power of the secondary fraction of Gesneriana lanigera alkaloid was changed in the order of SB3-3, SB3-4, SB3-5, SB3-1, and SB3-2, and the total reducing power was lower than that of BHT.
In conclusion, the detection result is consistent with the result of detecting the cell survival rate, so that the antioxidant activity of SB3-4 in the secondary alkaloid fraction of the sonchus oleraceus is stronger.
Claims (9)
1. A secondary separated substance of common sow thistle alkaloid is characterized in that the secondary separated substance is prepared by a method comprising the following steps:
(1) preparing extract of herba Sonchi arvensis;
(2) Preparing the common sow thistle extract obtained in the step (1) into a test solution of 100mg/mL, and separating by adopting a preparative chromatography, wherein the chromatographic conditions are as follows:
Adopting a C18 silica gel column, taking 0.1% formic acid water solution as a mobile phase A, taking methanol as a mobile phase B, wherein the flow rate is 10mL/min, the column temperature is 27-33 ℃, the wavelength is 255-265nm, and the gradient elution procedure is as follows:
In the time range of 0-5min, the volume of the mobile phase A is changed from 95% to 60%, and the volume of the mobile phase B is changed from 5% to 40%;
in the time range of 5-15min, the volume of the mobile phase A is changed from 60% to 30%, and the volume of the mobile phase B is changed from 40% to 70%;
In the time range of 15-30min, the volume of the mobile phase A is changed from 30% to 20%, and the volume of the mobile phase B is changed from 70% to 80%;
The volume of mobile phase A is kept at 20% and the volume of mobile phase B is kept at 80% in the time range of 30-35 min;
collecting fractions with peak heights ranging from 420 mAU to 2000mAU to obtain a primary isolate;
(3) Concentrating and drying the primary isolate obtained in the step (2), preparing a test solution of 10mg/mL, and separating by using a preparative chromatography, wherein the chromatographic conditions are as follows:
Adopting a C18 silica gel column, taking 0.1% formic acid water solution as a mobile phase A, taking methanol as a mobile phase B, wherein the flow rate is 10mL/min, the column temperature is 27-33 ℃, the wavelength is 255-265nm, and the gradient elution procedure is as follows:
In the time range of 0-5min, the volume of the mobile phase A is changed from 95% to 60%, and the volume of the mobile phase B is changed from 5% to 40%;
In the time range of 5-15min, the volume of the mobile phase A is changed from 60% to 30%, and the volume of the mobile phase B is changed from 40% to 70%;
In the time range of 15-30min, the volume of the mobile phase A is changed from 30% to 20%, and the volume of the mobile phase B is changed from 70% to 80%;
the volume of mobile phase A is kept at 20% and the volume of mobile phase B is kept at 80% in the time range of 30-35 min;
collecting the fraction with peak height ranging from 0.2-1.8AU to obtain secondary isolate.
2. the secondary isolate of Sonchus oleraceus alkaloids according to claim 1, wherein in step (2), the column temperature is 30 ℃ and the wavelength is 260 nm.
3. the secondary alkali isolate of sonchus oleraceus of claim 1, wherein the sonchus oleraceus extract is prepared by a method comprising the steps of:
using 75% ethanol as solvent, making herba Sonchi arvensis into extractive solution with liquid-material ratio of 30mL/g, adjusting pH of the extractive solution to 5, performing ultrasonic extraction, standing, collecting supernatant, rotary evaporating, cooling, and drying.
4. The secondary isolate of Sonchus oleraceus alkaloids according to claim 3, characterized in that the power of ultrasonic extraction is 700W, the temperature is 55 ℃ and the ultrasonic time is 30 min.
5. A method for separating secondary alkaloid isolate from common sow thistle, which is characterized by comprising the following steps:
(1) Preparing extract of herba Sonchi arvensis;
(2) Preparing the common sow thistle extract obtained in the step (1) into a test solution of 100mg/mL, and separating by adopting a preparative chromatography, wherein the chromatographic conditions are as follows:
adopting a C18 silica gel column, taking 0.1% formic acid water solution as a mobile phase A, taking methanol as a mobile phase B, wherein the flow rate is 10mL/min, the column temperature is 27-33 ℃, the wavelength is 255-265nm, and the gradient elution procedure is as follows:
in the time range of 0-5min, the volume of the mobile phase A is changed from 95% to 60%, and the volume of the mobile phase B is changed from 5% to 40%;
In the time range of 5-15min, the volume of the mobile phase A is changed from 60% to 30%, and the volume of the mobile phase B is changed from 40% to 70%;
In the time range of 15-30min, the volume of the mobile phase A is changed from 30% to 20%, and the volume of the mobile phase B is changed from 70% to 80%;
the volume of mobile phase A is kept at 20% and the volume of mobile phase B is kept at 80% in the time range of 30-35 min;
Collecting fractions with peak heights ranging from 420 mAU to 2000mAU to obtain a primary isolate;
(3) concentrating and drying the primary separated substance obtained in the step (2), preparing a test solution of 10mg/mL, and separating by using a preparative chromatography, wherein the chromatographic conditions are as follows:
Adopting a C18 silica gel column, taking 0.1% formic acid water solution as a mobile phase A, taking methanol as a mobile phase B, wherein the flow rate is 10mL/min, the column temperature is 27-33 ℃, the wavelength is 255-265nm, and the gradient elution procedure is as follows:
In the time range of 0-5min, the volume of the mobile phase A is changed from 95% to 60%, and the volume of the mobile phase B is changed from 5% to 40%;
In the time range of 5-15min, the volume of the mobile phase A is changed from 60% to 30%, and the volume of the mobile phase B is changed from 40% to 70%;
in the time range of 15-30min, the volume of the mobile phase A is changed from 30% to 20%, and the volume of the mobile phase B is changed from 70% to 80%;
The volume of mobile phase A is kept at 20% and the volume of mobile phase B is kept at 80% in the time range of 30-35 min;
Collecting the fraction with peak height ranging from 0.2-1.8AU to obtain secondary isolate.
6. The method of claim 5, wherein in step (2), the column temperature is 30 deg.C and the wavelength is 260 nm.
7. the method of isolating secondary alkali isolates of Sonchus oleraceus as claimed in claim 5, wherein the extract of Sonchus oleraceus is obtained by a method comprising the following steps:
Using 75% ethanol as solvent, making herba Sonchi arvensis into extract solution with liquid-material ratio of 30mL/g, performing ultrasonic extraction, standing, collecting supernatant, rotary evaporating, cooling, and drying.
8. the method of claim 5, wherein the power of ultrasonic extraction is 700W, the temperature is 55 deg.C, and the ultrasonic time is 30 min.
9. Use of the secondary isolate of the alkaloids from sonchus oleraceus according to claim 1.
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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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| 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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