CN115429786B - Extraction method and application of juniperidine sesquiterpene compound - Google Patents
Extraction method and application of juniperidine sesquiterpene compound Download PDFInfo
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- 229930004725 sesquiterpene Natural products 0.000 title claims abstract description 42
- -1 sesquiterpene compound Chemical class 0.000 title claims abstract description 20
- 238000000605 extraction Methods 0.000 title claims abstract description 16
- 150000001875 compounds Chemical class 0.000 claims abstract description 28
- 238000010898 silica gel chromatography Methods 0.000 claims description 26
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- NPOMSUOUAZCMBL-UHFFFAOYSA-N dichloromethane;ethoxyethane Chemical compound ClCCl.CCOCC NPOMSUOUAZCMBL-UHFFFAOYSA-N 0.000 claims description 21
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 21
- 239000003208 petroleum Substances 0.000 claims description 21
- 239000003480 eluent Substances 0.000 claims description 19
- 238000010828 elution Methods 0.000 claims description 18
- 238000004809 thin layer chromatography Methods 0.000 claims description 15
- 238000004458 analytical method Methods 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 8
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000002027 dichloromethane extract Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 238000004262 preparative liquid chromatography Methods 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 5
- 238000002386 leaching Methods 0.000 claims description 2
- ITOFWMRNIIFZKF-IVZWLZJFSA-N ac1l4fkl Chemical compound O=C([C@@H](C)C[C@@H]1[C@@H](C(C)C)CC2=O)C3=C1C2=CO3 ITOFWMRNIIFZKF-IVZWLZJFSA-N 0.000 abstract description 34
- 150000004354 sesquiterpene derivatives Chemical class 0.000 abstract description 23
- ITOFWMRNIIFZKF-UHFFFAOYSA-N hibiscone C Natural products O=C1CC(C(C)C)C2CC(C)C(=O)C3=C2C1=CO3 ITOFWMRNIIFZKF-UHFFFAOYSA-N 0.000 abstract description 17
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- CAULGCQHVOVVRN-UHFFFAOYSA-N (3Z,9E)-Germacra-3,7(11),9-trien-6-on Natural products CC(C)=C1CC=C(C)CCC=C(C)CC1=O CAULGCQHVOVVRN-UHFFFAOYSA-N 0.000 description 1
- CAULGCQHVOVVRN-SWZPTJTJSA-N (E,E)-germacrone Chemical compound CC(C)=C1C\C=C(C)\CC\C=C(C)\CC1=O CAULGCQHVOVVRN-SWZPTJTJSA-N 0.000 description 1
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- CUGKULNFZMNVQI-UHFFFAOYSA-N Costunolid I Natural products CC1=CCC=C(/C)CCC2C(C1)OC(=O)C2=C CUGKULNFZMNVQI-UHFFFAOYSA-N 0.000 description 1
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- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical group CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 1
- 241000721662 Juniperus Species 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 241001093501 Rutaceae Species 0.000 description 1
- HATRDXDCPOXQJX-UHFFFAOYSA-N Thapsigargin Natural products CCCCCCCC(=O)OC1C(OC(O)C(=C/C)C)C(=C2C3OC(=O)C(C)(O)C3(O)C(CC(C)(OC(=O)C)C12)OC(=O)CCC)C HATRDXDCPOXQJX-UHFFFAOYSA-N 0.000 description 1
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- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 description 1
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- 229930101531 artemisinin Natural products 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 229940026255 cortalone Drugs 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- HRYLQFBHBWLLLL-AHNJNIBGSA-N costunolide Chemical compound C1CC(/C)=C/CC\C(C)=C\[C@H]2OC(=O)C(=C)[C@@H]21 HRYLQFBHBWLLLL-AHNJNIBGSA-N 0.000 description 1
- MMTZAJNKISZWFG-UHFFFAOYSA-N costunolide Natural products CC1CCC2C(CC(=C/C=C1)C)OC(=O)C2=C MMTZAJNKISZWFG-UHFFFAOYSA-N 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000001812 curcuma zedoaria berg. rosc. Substances 0.000 description 1
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 1
- NETSQGRTUNRXEO-XUXIUFHCSA-N dehydrocostus lactone Chemical compound C([C@H]1C(=C)C(=O)O[C@@H]11)CC(=C)[C@H]2[C@@H]1C(=C)CC2 NETSQGRTUNRXEO-XUXIUFHCSA-N 0.000 description 1
- 125000005594 diketone group Chemical group 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
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- 150000002596 lactones Chemical class 0.000 description 1
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- 239000004090 neuroprotective agent Substances 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 229930009674 sesquiterpene lactone Natural products 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000019509 white turmeric Nutrition 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/20—Hypnotics; Sedatives
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/92—Naphthofurans; Hydrogenated naphthofurans
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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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Abstract
The invention provides an extraction method and application of a juniperidine type sesquiterpene compound. The preparation method of the invention is simple and rapid, and the obtained compound has high purity. The juniperidine type sesquiterpene compound Hibiscone C provided by the invention has good neuroprotective activity and good breast cancer resisting activity, can be used for preparing neuroprotection agents or/and antitumor drugs, can be used for preparing drugs for treating neuropsychiatric disorder diseases, improving sleep drugs and preventing nervous system diseases, and can also be used for preparing drugs for treating breast cancer.
Description
Technical Field
The invention relates to the field of natural plant medicines, in particular to an extraction method and application of a juniperidine type sesquiterpene compound.
Background
Sesquiterpenes (sesterpenes) are natural terpenoids containing 15 carbon atoms in the molecule, and contain three isoprene units, and have various framework configurations and wide physiological activities. Such as artemisinin in Artemisia annua, germacrone and curcuma zedoary diketone as antitumor bioactive components, and costunolide and dehydrocostuslactone as sesquiterpene lactone compounds in radix aucklandiae for inhibiting surface antigen effect of human hepatitis B. Sesquiterpenes are mostly liquid and mainly exist in volatile oils of plants. Oxygen-containing derivatives such as alcohols, ketones and lactones are also widely present in volatile oils and are the major components of the high boiling point fraction of volatile oils. The juniperidine sesquiterpenes widely exist in plants, are mainly distributed in aromatic plants such as Compositae, zingiberaceae and Rutaceae, have small quantity of distribution in agilawood, have stronger aroma and biological activity, and are important raw materials in the industries of medicines, foods and cosmetics. The present study aims at extracting active ingredients from natural plants and developing new uses.
Disclosure of Invention
In view of the above, the invention provides an extraction method and application of a juniperidine type sesquiterpene compound.
The technical scheme of the invention is realized as follows:
the extraction method of the juniperidine sesquiterpene compound comprises the following steps:
(1) Extracting the dried powder of Huang Jinjing with dichloromethane, concentrating under reduced pressure to obtain dichloromethane extract;
(2) Performing gradient elution on the extract by using silica gel column chromatography and using petroleum ether-dichloromethane with the volume ratio of 100:0-0:100 and dichloromethane-methanol with the volume ratio of 50:1-1:1 as eluent to obtain Frs.A-Frs.G;
(3) Performing gradient elution on the Fr.E component obtained in the step (2) by using silica gel column chromatography, wherein petroleum ether-dichloromethane with the volume ratio of 1:1-0:1 and dichloromethane-methanol with the volume ratio of 40:1-1:1 are used as eluents to obtain Fr.E1-Fr.E7;
(4) Performing gradient elution on the Fr.E1 obtained in the step (3) by using silica gel column chromatography again, wherein petroleum ether-dichloromethane with the volume ratio of 4:1-0:1 and dichloromethane-methanol with the volume ratio of 99:1-1:1 are used as eluent to obtain Fr.E1-1-Fr.E1-9;
(5) Taking Fr.E1-3 obtained in the step (4), and recrystallizing to separate out a target compound by adopting silica gel column chromatography; or, taking Fr.E1-3 obtained in the step (4), adopting semi-preparative liquid chromatography, and carrying out isocratic purification and separation by using an SB-phenyl chromatographic column and 85-95% v/v methanol-water to prepare the target compound.
Further, the extraction method of the juniperidine sesquiterpene compound comprises the following steps:
(1) Leaching the dried powder of Huang Jinjing with dichloromethane at room temperature for 2-4 times, wherein each time of extraction is 60-80h, and the mass-volume ratio kg/L of the material is 1:3.5-4.5; concentrating under reduced pressure to obtain dichloromethane extract;
(2) Subjecting the extract to 100-200 mesh silica gel column chromatography, using petroleum ether-dichloromethane with a volume ratio of 100:0-0:100 and dichloromethane-methanol with a volume ratio of 50:1-1:1 as eluent, performing gradient elution, collecting each fraction, and performing thin-layer chromatography analysis and detection to obtain 7 components, wherein the components are Frs.A-Frs.G in sequence;
(3) Carrying out gradient elution on the Fr.E component obtained in the step (2) by using 200-300 mesh silica gel column chromatography, taking petroleum ether-dichloromethane with the volume ratio of 1:1-0:1 and dichloromethane-methanol with the volume ratio of 40:1-1:1 as eluent, collecting each component, and detecting by thin-layer chromatography analysis to obtain 7 components, wherein the Fr.E1-Fr.E7 components are sequentially obtained;
(4) Performing gradient elution on Fr.E1 obtained in the step (3) by using 200-300 mesh silica gel column chromatography again by using petroleum ether-dichloromethane with the volume ratio of 4:1-0:1 and dichloromethane-methanol with the volume ratio of 99:1-1:1 as eluent, collecting each fraction, and detecting by thin layer chromatography analysis to obtain 9 components, wherein the components are Fr.E1-1-Fr.E1-9 in sequence;
(5) Taking Fr.E1-3 obtained in the step (4), and recrystallizing to separate out a target compound by adopting silica gel column chromatography; or, taking Fr.E1-3 obtained in the step (4), adopting semi-preparative liquid chromatography, and carrying out isocratic purification and separation by using an SB-phenyl chromatographic column and 85-95% v/v methanol-water to prepare the target compound.
Further, the extraction method of the juniperidine sesquiterpene compound comprises the following steps:
(1) Extracting the dried powder of Huang Jinjing with dichloromethane at room temperature for 3 times for 72h each time, wherein the mass-volume ratio kg/L of the materials is 1:4; concentrating under reduced pressure to obtain dichloromethane extract;
(2) Subjecting the extract to 100-200 mesh silica gel column chromatography, using petroleum ether-dichloromethane with a volume ratio of 100:0-0:100 and dichloromethane-methanol with a volume ratio of 50:1-1:1 as eluent, performing gradient elution, collecting each fraction, and performing thin-layer chromatography analysis and detection to obtain 7 components, wherein the components are Frs.A-Frs.G in sequence;
(3) Carrying out gradient elution on the Fr.E component obtained in the step (2) by using 200-300 mesh silica gel column chromatography, taking petroleum ether-dichloromethane with the volume ratio of 1:1-0:1 and dichloromethane-methanol with the volume ratio of 40:1-1:1 as eluent, collecting each component, and detecting by thin-layer chromatography analysis to obtain 7 components, wherein the Fr.E1-Fr.E7 components are sequentially obtained;
(4) Performing gradient elution on Fr.E1 obtained in the step (3) by using 200-300 mesh silica gel column chromatography again by using petroleum ether-dichloromethane with the volume ratio of 4:1-0:1 and dichloromethane-methanol with the volume ratio of 99:1-1:1 as eluent, collecting each fraction, and detecting by thin layer chromatography analysis to obtain 9 components, wherein the components are Fr.E1-1-Fr.E1-9 in sequence;
(5) Taking Fr.E1-3 obtained in the step (4), and recrystallizing to separate out a target compound by adopting silica gel column chromatography; alternatively, fr.E1-3 obtained in the step (4) is taken, semi-preparative liquid chromatography is adopted, and an SB-phenyl chromatographic column and 90% v/v methanol-water are used for isocratic purification and separation to prepare the target compound.
The structural formula of the juniperidine sesquiterpene compound obtained by the extraction method is as follows:
the juniperidine sesquiterpene compound is applied to the preparation of neuroprotectants or/and antitumor drugs.
Furthermore, the juniperidine type sesquiterpene compound is applied to preparing a medicament for treating neuropsychiatric disorder diseases.
Furthermore, the juniperidine type sesquiterpene compound is applied to the preparation of medicines for preventing nervous system diseases.
Furthermore, the juniperidine type sesquiterpene compound is applied to preparing a sleep improving medicine.
Furthermore, the juniperidine sesquiterpene compound is applied to preparation of anticancer drugs.
Furthermore, the juniperidine type sesquiterpene compound is applied to preparation of a medicament for treating breast cancer.
Compared with the prior art, the invention has the beneficial effects that:
(1) The extraction method provided by the invention is adopted to obtain the juniperidine sesquiterpene compound Hibiscone C, the preparation method provided by the invention is simple and rapid, and the purity of the obtained compound is high.
(2) The juniperidine sesquiterpene compound Hibiscone C extracted by the invention can be used as a small molecular compound, can obviously protect PC12 cell injury induced by corticosterone, and has obvious targeted activation of GABA A The activity of the receptor, play a role in improving sleep; the targeting PI3K has remarkable anti-tumor activity.
(3) The juniperidine sesquiterpene compound Hibiscone C obtained by the extraction method disclosed by the invention has good neuroprotective activity and good breast cancer resisting activity.
(4) The juniperidine type sesquiterpene compound Hibiscone C provided by the invention can be applied to the preparation of neuroprotective medicines for treating neuropsychiatric disorder diseases and preventing nervous system diseases, and can also be applied to the preparation of antitumor medicines for treating breast cancer and the like.
(5) The juniperidone type sesquiterpene compound Hibiscone C provided by the invention can be applied to the treatment of patients suffering from mental disorder diseases or breast cancer, and can also be applied to the treatment of patients suffering from mental disorder diseases and breast cancer at the same time.
Drawings
Fig. 1: hibiscone C 1 H NMR(600MHz,CDCl 3 ) A spectrogram;
fig. 2: hibiscone C 13 C NMR(600MHz,CDCl 3 ) A spectrogram;
FIG. 3, mass spectrum [ M+H ] + ] of Hibiscone C;
FIG. 4, sesquiterpene chromatography detection method and spectrogram: detection conditions: 75% v/v methanol-water, flow rate 2ml/min, detection wavelength 254nm, chromatographic column: agilent SB-phenyl (5 um, 9.4. Times.250 mm)
FIG. 5 shows the juniperidine sesquiterpene Hibiscone C and GABA A Visual view of docking (A. Interaction site vs. overall interaction; B. Interaction pattern of docking site)
FIG. 6 is a visual view of the interface of juniperidone C and PI 3K;
the result of the molecular docking shows that the juniper alkane type sesquiterpene Hibiscone C can specifically bind and activate GABA A The receptor can play a role in improving sleep, and can also play a remarkable anti-tumor role by stably combining with PI 3K.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
EXAMPLE 1 preparation of juniperidine sesquiterpene Compounds
(1) 13.0kg Huang Jinjing of the dried powder was leached 3 times with 52L of dichloromethane at room temperature for 72h each; concentrating under reduced pressure to obtain 405.9g of dichloromethane extract to obtain extract.
(2) Subjecting the extract to 100-200 mesh silica gel column chromatography, gradient eluting with eluent petroleum ether-dichloromethane (100:0-0:100, v/v) and dichloromethane-methanol (50:1-1:1, v/v), collecting fractions of 500 mL/bottle, counting 386 bottles, detecting by thin layer chromatography analysis, and combining into 7 components (Frs.A-Frs.G).
(3) Fr.E (66.8 g) fractions were subjected to column chromatography on silica gel (200-300 mesh) using a gradient of petroleum ether-dichloromethane (1:1-0:1, v/v) and dichloromethane-methanol (40:1-1:1, v/v), and the fractions were combined into 7 fractions, fr.E1-Fr.E7, etc., as detected by thin layer chromatography.
(4) Fr.E1 was again subjected to column chromatography on silica gel (200-300 mesh) and gradient eluted with petroleum ether-dichloromethane (4:1-0:1, v/v) and dichloromethane-methanol (99:1-1:1, v/v) and detected by thin layer chromatography analysis, and combined into 9 fractions such as Fr.E1-1-Fr.E1-9.
(5) The Fr.E1-3 adopts semi-preparative liquid chromatography to prepare the sesquiterpene (437.7mg,Rt 9.1min) by using SB-phenyl chromatographic column and 90% methanol-water isocratic purification and separation, and the remainder is silica gel column chromatography, and the sesquiterpene is separated out by recrystallization 8.4g.
The sesquiterpene is subjected to nuclear magnetism, mass spectrum and the like to carry out structure confirmation. The structural formula of the sesquiterpene is as follows:
the compound is a juniperidine sesquiterpene compound Hibiscone C.
EXAMPLE 2 protection of Corticosterone (CORT) induced PC12 cell injury by sesquiterpenes
1 test method: PC12 cells were cultured by conventional methods and inoculated in 96-well plates with cells in log phase to a cell concentration of 5X 10 4 mu.L of each well was added thereto and the mixture was left at 37℃with 5% CO 2 Is cultured in an incubator for 24 hours. A blank group, a model control group, a positive control diazepam group and an experimental group are arranged. 200. Mu.L of incomplete medium was added to the blank without any treatment; model control groups were treated with 200 μl (final concentration of 200 μΜ) of CORT alone; the experimental group treated cells with 100. Mu.L (final concentration of 200. Mu.M) of CORT and the corresponding drug (50,25,12.5,6.25,3.125,1.5625. Mu.g/mL), diazepam (50,25,12.5,6.25,3.125,1.5625. Mu.g/mL) 100. Mu.L. Placing at 37deg.C with 5% CO 2 Is cultured in an incubator for 24 hours, 100. Mu.L of the drug-containing medium is carefully sucked, 10. Mu.L of CCK8 is added, and the incubator is placed at 37 ℃ and contains 5% CO 2 After 1-2h incubation in an incubator of (2), the 96-well plate was placed in an ELISA apparatus at 450nm wavelength to determine OD value, and cell viability and IC were calculated 50 。
Cell viability (%) = ([ OD (model treatment group) -OD (blank group) ])/([ OD (negative control group) -OD (blank group) ]) x100%
2 test results: sesquiterpene component Hibiscone C can obviously protect PC12 cell injury induced by corticosteroneThe concentration is 1.5625,3.125,6.125,12.5,25,50 mug/mL, the increment rate is 25-78%, and the IC is 50 3.49. Mu.g/mL. Diazepam IC 50 The result shows that the sesquiterpene compound Hibiscone C can obviously protect PC12 cell injury induced by corticosterone and is slightly superior to a positive medicine at 3.87 mug/mL.
EXAMPLE 2 anti-tumor Activity assay
1 test method: resuscitates, cultures and passages the triple negative breast cancer cells to grow to the logarithmic phase. Counted cells 5 x10 3 100 uL/well was added to a 96-well plate, and after the addition, the plate was marked and placed under an inverted microscope to see if the cells were plated uniformly. After the observation, the mixture was placed in a carbon dioxide incubator (CO 2 Concentration 5%, temperature 37 ℃), for 24h. A blank, a different dose of sesquiterpene compound Hibiscone C (1.5625,3.125,6.25,12.5,25,50. Mu.g/mL), cisplatin (1.25,2.5,5,10. Mu.g/mL) were each set, and 3 duplicate wells were set for each concentration. Except for the normal group, the supernatant was discarded, and after further culturing for 24 hours, 100. Mu.L of the supernatant was aspirated per well for the detection kit. After adding 10 mu LCCK8 into each hole for 1 hour, measuring absorbance at the wavelength of 450nm, and calculating tumor inhibition rate and IC 50 。
Tumor inhibition = (model group OD value-dosing group OD value)/model group OD value 100
2 test results: the drug concentration of the sesquiterpene compound Hibiscone C is 1.5625,3.125,6.25,12.5,25,50 mug/mL, the inhibition rate is 26-68%, and IC 50 Cisplatin anticancer IC as positive drug with 6.47 μg/mL 50 3.89. Mu.g/mL. The result shows that the sesquiterpene compound Hibiscone C has obvious effect of inhibiting the activity of the triple negative breast cancer.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (3)
1. The extraction method of the juniperidine sesquiterpene compound is characterized by comprising the following steps of:
(1) Extracting the dried powder of Huang Jinjing with dichloromethane, concentrating under reduced pressure to obtain dichloromethane extract;
(2) Performing gradient elution on the extract by using silica gel column chromatography and using petroleum ether-dichloromethane with the volume ratio of 100:0-0:100 and dichloromethane-methanol with the volume ratio of 50:1-1:1 as eluent to obtain Frs.A-Frs.G;
(3) Performing gradient elution on the Fr.E component obtained in the step (2) by using silica gel column chromatography, wherein petroleum ether-dichloromethane with the volume ratio of 1:1-0:1 and dichloromethane-methanol with the volume ratio of 40:1-1:1 are used as eluents to obtain Fr.E1-Fr.E7;
(4) Performing gradient elution on the Fr.E1 obtained in the step (3) by using silica gel column chromatography again, wherein petroleum ether-dichloromethane with the volume ratio of 4:1-0:1 and dichloromethane-methanol with the volume ratio of 99:1-1:1 are used as eluent to obtain Fr.E1-1-Fr.E1-9;
(5) Taking Fr.E1-3 obtained in the step (4), and recrystallizing to separate out a target compound by adopting silica gel column chromatography; or, taking Fr.E1-3 obtained in the step (4), adopting semi-preparative liquid chromatography, and carrying out isocratic purification and separation on the Fr.E1-3 by using an SB-phenyl chromatographic column and 85-95% v/v methanol-water to prepare a target compound, wherein the structural formula of the compound is as follows:
2. the extraction method of a juniperidine sesquiterpene compound according to any of claims 1, comprising the steps of:
(1) Leaching the dried powder of Huang Jinjing with dichloromethane at room temperature for 2-4 times, wherein each time of extraction is 60-80h, and the mass-volume ratio kg/L of the material is 1:3.5-4.5; concentrating under reduced pressure to obtain dichloromethane extract;
(2) Subjecting the extract to 100-200 mesh silica gel column chromatography, using petroleum ether-dichloromethane with a volume ratio of 100:0-0:100 and dichloromethane-methanol with a volume ratio of 50:1-1:1 as eluent, performing gradient elution, collecting each fraction, and performing thin-layer chromatography analysis and detection to obtain 7 components, wherein the components are Frs.A-Frs.G in sequence;
(3) Carrying out gradient elution on the Fr.E component obtained in the step (2) by using 200-300 mesh silica gel column chromatography, taking petroleum ether-dichloromethane with the volume ratio of 1:1-0:1 and dichloromethane-methanol with the volume ratio of 40:1-1:1 as eluent, collecting each component, and detecting by thin-layer chromatography analysis to obtain 7 components, wherein the Fr.E1-Fr.E7 components are sequentially obtained;
(4) Performing gradient elution on Fr.E1 obtained in the step (3) by using 200-300 mesh silica gel column chromatography again by using petroleum ether-dichloromethane with the volume ratio of 4:1-0:1 and dichloromethane-methanol with the volume ratio of 99:1-1:1 as eluent, collecting each fraction, and detecting by thin layer chromatography analysis to obtain 9 components, wherein the components are Fr.E1-1-Fr.E1-9 in sequence;
(5) Taking Fr.E1-3 obtained in the step (4), and recrystallizing to separate out a target compound by adopting silica gel column chromatography; or, taking Fr.E1-3 obtained in the step (4), adopting semi-preparative liquid chromatography, and carrying out isocratic purification and separation by using an SB-phenyl chromatographic column and 85-95% v/v methanol-water to prepare the target compound.
3. The extraction method of the juniperidine sesquiterpene compound according to claim 2, comprising the following steps:
(1) Extracting the dried powder of Huang Jinjing with dichloromethane at room temperature for 3 times for 72h each time, wherein the mass-volume ratio kg/L of the materials is 1:4; concentrating under reduced pressure to obtain dichloromethane extract;
(2) Subjecting the extract to 100-200 mesh silica gel column chromatography, using petroleum ether-dichloromethane with a volume ratio of 100:0-0:100 and dichloromethane-methanol with a volume ratio of 50:1-1:1 as eluent, performing gradient elution, collecting each fraction, and performing thin-layer chromatography analysis and detection to obtain 7 components, wherein the components are Frs.A-Frs.G in sequence;
(3) Carrying out gradient elution on the Fr.E component obtained in the step (2) by using 200-300 mesh silica gel column chromatography, taking petroleum ether-dichloromethane with the volume ratio of 1:1-0:1 and dichloromethane-methanol with the volume ratio of 40:1-1:1 as eluent, collecting each component, and detecting by thin-layer chromatography analysis to obtain 7 components, wherein the Fr.E1-Fr.E7 components are sequentially obtained;
(4) Performing gradient elution on Fr.E1 obtained in the step (3) by using 200-300 mesh silica gel column chromatography again by using petroleum ether-dichloromethane with the volume ratio of 4:1-0:1 and dichloromethane-methanol with the volume ratio of 99:1-1:1 as eluent, collecting each fraction, and detecting by thin layer chromatography analysis to obtain 9 components, wherein the components are Fr.E1-1-Fr.E1-9 in sequence;
(5) Taking Fr.E1-3 obtained in the step (4), and recrystallizing to separate out a target compound by adopting silica gel column chromatography; alternatively, fr.E1-3 obtained in the step (4) is taken, semi-preparative liquid chromatography is adopted, and an SB-phenyl chromatographic column and 90% v/v methanol-water are used for isocratic purification and separation to prepare the target compound.
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| CN108164484A (en) * | 2017-12-22 | 2018-06-15 | 中国医学科学院药用植物研究所云南分所 | A kind of 4 inhibitor of non-phagocytic cell oxidizing ferment and its application |
| CN110522776A (en) * | 2019-09-26 | 2019-12-03 | 中国医学科学院药用植物研究所 | It is a kind of Huang the rose of Sharon in anti-tumor effective component preparation method and application |
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