CN116751192A - peroxy-Chinese waxiness ketone, preparation method and application thereof - Google Patents
peroxy-Chinese waxiness ketone, preparation method and application thereof Download PDFInfo
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- 150000002576 ketones Chemical class 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 51
- 150000002978 peroxides Chemical class 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 230000003110 anti-inflammatory effect Effects 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000002024 ethyl acetate extract Substances 0.000 claims abstract description 5
- 239000003208 petroleum Substances 0.000 claims abstract description 5
- 238000002953 preparative HPLC Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 84
- 210000004027 cell Anatomy 0.000 claims description 31
- 238000000605 extraction Methods 0.000 claims description 17
- 230000002401 inhibitory effect Effects 0.000 claims description 12
- 239000012046 mixed solvent Substances 0.000 claims description 12
- 210000002540 macrophage Anatomy 0.000 claims description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- 201000007270 liver cancer Diseases 0.000 claims description 8
- 208000014018 liver neoplasm Diseases 0.000 claims description 8
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims description 7
- 239000004480 active ingredient Substances 0.000 claims description 7
- 229940124599 anti-inflammatory drug Drugs 0.000 claims description 7
- 230000000259 anti-tumor effect Effects 0.000 claims description 7
- 239000002246 antineoplastic agent Substances 0.000 claims description 7
- 229940041181 antineoplastic drug Drugs 0.000 claims description 7
- 239000003405 delayed action preparation Substances 0.000 claims description 7
- 201000005249 lung adenocarcinoma Diseases 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- 238000010521 absorption reaction Methods 0.000 claims description 6
- 239000002260 anti-inflammatory agent Substances 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 4
- 239000000469 ethanolic extract Substances 0.000 claims description 4
- 239000008187 granular material Substances 0.000 claims description 4
- 239000007902 hard capsule Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000007901 soft capsule Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000002459 sustained effect Effects 0.000 claims description 3
- 229940121363 anti-inflammatory agent Drugs 0.000 claims description 2
- 125000000864 peroxy group Chemical group O(O*)* 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 abstract description 7
- 230000005918 in vitro anti-tumor Effects 0.000 abstract description 5
- 210000002824 peroxisome Anatomy 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 125000003682 3-furyl group Chemical group O1C([H])=C([*])C([H])=C1[H] 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 239000000284 extract Substances 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 229940126680 traditional chinese medicines Drugs 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 235000018274 Cunila origanoides Nutrition 0.000 description 1
- 244000182625 Dictamnus albus Species 0.000 description 1
- 235000014866 Dictamnus albus Nutrition 0.000 description 1
- 241000123855 Dictamnus dasycarpus Species 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- VHLJDTBGULNCGF-UHFFFAOYSA-N Limonin Natural products CC1(C)OC2CC(=O)OCC23C4CCC5(C)C(CC(=O)C6OC56C4(C)C(=O)CC13)c7cocc7 VHLJDTBGULNCGF-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241001093501 Rutaceae Species 0.000 description 1
- 238000004378 air conditioning Methods 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000011419 induction treatment Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000003978 infusion fluid Substances 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- KBDSLGBFQAGHBE-MSGMIQHVSA-N limonin Chemical compound C=1([C@H]2[C@]3(C)CC[C@H]4[C@@]([C@@]53O[C@@H]5C(=O)O2)(C)C(=O)C[C@@H]2[C@]34COC(=O)C[C@@H]3OC2(C)C)C=COC=1 KBDSLGBFQAGHBE-MSGMIQHVSA-N 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
- C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Pulmonology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
A peroxisome ketone (peroxisome) and its preparation method and application relate to a new compound and its preparation method and application. The invention aims to provide a peroxide white waxy ketone, a preparation method and application thereof. The chemical name of the peroxy-fraxinone is 1- (furan-3-yl) -5,8a-dimethyl-1,7,8 a-tetrahydrobac-zo [ d ]][1,2]Dioxin-6 (4H) -one with molecular formula of C 14 H 16 O 4 The molecular weight is 248.1049. The method comprises the following steps: 1. cortex Dictamni RadicisPulverizing, extracting with 95% ethanol; 2. dispersing the ethanol-recovered extract with water, and sequentially extracting with petroleum ether, dichloromethane and ethyl acetate; 3. and (3) repeatedly performing silica gel column chromatography on the ethyl acetate extract, and performing preparative high performance liquid chromatography separation. The peroxyChinese waxiness ketone has stronger in-vitro anti-inflammatory activity and better in-vitro anti-tumor activity.
Description
Technical Field
The invention relates to a compound, a preparation method thereof and application of the compound.
Background
Cancer is a malignant tumor in which cells change and do not proliferate and eventually develop to affect normal physiological functions of the human body. The conventional methods for treating cancers in clinic are chemotherapy, radiotherapy, surgery and the like, and traditional Chinese medicines play an important role in treating cancers, and searching for anticancer active ingredients from traditional Chinese medicines is always a research hotspot in the field.
Inflammation is a complex biological response of the body to harmful stimuli such as pathogens and the like. Inflammatory reactions cause pain, fever, itching, redness and distension, even inducing allergies, asthma, tumors, etc. Anti-inflammatory drugs are one of the most commonly used drugs, and research on traditional Chinese medicine anti-inflammatory drugs has important significance.
The cortex Dictamni is dried root bark of Dictamni (Dictamnus dasycarpus Turcz.) belonging to Rutaceae. The chemical components mainly comprise limonin components, alkaloids and other components, and have anti-inflammatory, antibacterial, anti-tumor and other activities, but specific anti-inflammatory and anti-cancer medicinal effect components are not clear. There are many unknown compounds yet to be developed for the cortex dictamni, and more active compounds need to be found and identified.
Disclosure of Invention
The invention aims to provide a novel compound of the peroxyChinese waxiness ketone with in-vitro anti-tumor and anti-inflammatory activities.
A second object of the present invention is to provide a process for the preparation of peroxyChinese waxiness ketone.
A third object of the present invention is to provide the use of peroxyalkanone for anti-tumour and anti-inflammatory applications. The molecular formula of the peroxfraxinone (peroxfraxinone) is C 14 H 16 O 4 The molecular weight is 248.1049, and the molecular structural formula is:
the preparation method of the peroxide white waxy ketone comprises the following steps:
(1) Pulverizing cortex Dictamni Radicis, and extracting with 95% ethanol in an extraction tank of Soxhlet dynamic extraction concentration unit;
(2) Concentrating the ethanol extract obtained in the step (1), dispersing with water to obtain a suspension, and then sequentially extracting with petroleum ether, chloroform and ethyl acetate;
(3) Subjecting the ethyl acetate extract obtained in the step (2) to silica gel column chromatography, sequentially eluting with mixed solvents of dichloromethane and methanol in volume ratios of 100:0, 100:1, 100:2, 100:5 and 100:10, subjecting the eluate in volume ratios of dichloromethane and methanol to reverse phase silica gel column chromatography, sequentially eluting with mixed solvents of methanol and water in volume ratios of 10:90, 20:80 and 30:70, subjecting the eluate in volume ratios of methanol and water to silica gel column chromatography, subjecting the eluate in volume ratios of 30:70 to elution with mixed solvents of dichloromethane and methanol in volume ratios of 100:10, 100:20 and 100:30, subjecting the eluate in volume ratios of dichloromethane and methanol to preparative high performance liquid chromatography, and collecting absorption peaks appearing in elution time of 26.7 min to obtain the peroxy white waxy ketone;
the high performance liquid chromatography prepared in the step (3) takes methanol and water with the volume ratio of 30:70 as mobile phases, and the flow rate is 3ml/min.
And (3) setting the pressure of the Soxhlet dynamic extraction concentration unit in the step (1) as normal pressure extraction, and setting the extraction temperature at 85-90 ℃.
The concentration condition in the step (2) is-0.05 to-0.08 MPa.
The suspension density in step (2) was 1.25g/ml.
The peroxisome ketone (peroxfraxinone) is used for preparing anti-inflammatory or antitumor drugs.
The anti-inflammatory or anti-tumor medicament comprises injection, freeze-dried powder injection or oral preparation.
The oral preparation comprises tablets, granules, soft capsules, hard capsules, oral liquid or sustained and controlled release preparations.
The peroxywhite waxy ketone is used as an active ingredient of an anti-inflammatory drug, and the anti-inflammatory drug is used for inhibiting the NO release amount of mouse macrophages induced by LPS.
The peroxywhite waxy ketone is used as an active ingredient of an anti-tumor drug, and the anti-tumor drug is used for inhibiting lung adenocarcinoma cells and liver cancer tissue cells.
The peroxyfraxinone is used for inhibiting the LPS-induced NO release amount of mouse macrophages. Preferably, the LPS-induced inflammatory mouse macrophages include, but are not limited to, RAW264.7 cells.
The peroxy-alkanone is used for inhibiting lung adenocarcinoma cells and liver cancer tissue cells. Preferably, the tumor cells include, but are not limited to, liver cancer cells HepG2 cells.
The peroxy-fraxinone is extracted from dittany bark and has the chemical name of 1- (furan-3-yl) -5,8a-dimethyl-1,7,8 a-tetrahydrobilzo [ d ] [1,2] dioxan-6 (4H) -one.
The peroxide white waxy ketone is white powder. Specific optical rotation ofInfrared spectrum shows carbonyl absorption peak of peroxide white waxy ketone (1737.7 cm) -1 ). The peroxide fraxinone has maximum absorption peak at 239nm in UV spectrum. HR-ESI-MS gives molecular ion peaks: [ M-H ]] - 247.0972, hint molecular formula C 14 H 16 O 4 。
The purity of the peroxide white waxy ketone obtained by the method is high and reaches more than 98.5 percent.
The peroxyChinese waxy ketone has stronger anti-inflammatory activity, and has an IC50 of 7.94 mu M/L for inhibiting the NO release amount of mouse macrophages (RAW 264.7) induced by LPS. The method comprises the steps of carrying out a first treatment on the surface of the IC50 for inhibiting lung adenocarcinoma cells (A549) and liver cancer tissue cells (HepG 2) are 16.38 mu M/L and 14.43 mu M/L respectively; has better in vitro anti-tumor activity. The peroxide white waxy ketone can be prepared into anti-inflammatory or anti-tumor medicines such as injection, freeze-dried powder injection, infusion solution or oral preparations (including tablets, granules, soft capsules, hard capsules, oral liquid and sustained and controlled release preparations).
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
1. the novel compound of the fraxinafoate is obtained for the first time, and the purity of the fraxinafoate obtained by the preparation method is high and reaches more than 98.5 percent.
2. The peroxyChinese waxiness ketone has stronger in-vitro anti-inflammatory and anti-tumor activity, and the IC50 of the NO release rate of the LPS-induced inflammatory mouse macrophage (RAW 264.7) is 7.94 mu M/L; the IC50 for inhibiting lung adenocarcinoma cells (A549) and liver cancer tissue cells (HepG 2) is 16.38 mu M/L and 14.43 mu M/L respectively, and has better in vitro anti-tumor activity. Detailed Description
The technical scheme of the invention is not limited to the specific embodiments listed below, and also includes any combination of the specific embodiments.
The first embodiment is as follows: the molecular formula of the peroxide white waxy ketone is C 14 H 16 O 4 The molecular weight is 248.1049, and the molecular structural formula is:
the second embodiment is as follows: the preparation method of the peroxide fraxinone in the embodiment is as follows:
(1) 100kg of cortex dictamni is crushed and placed in an extraction tank of a Soxhlet dynamic extraction concentration unit to be extracted by ethanol with the volume concentration of 95%;
(2) Concentrating the ethanol extract obtained in the step (1), dispersing with water to obtain a suspension, and then sequentially extracting with petroleum ether, chloroform and ethyl acetate;
(3) Subjecting the ethyl acetate extract obtained in the step (2) to silica gel column chromatography, sequentially eluting with mixed solvents of dichloromethane and methanol in volume ratios of 100:0, 100:1, 100:2, 100:5 and 100:10, subjecting the eluate in volume ratios of dichloromethane and methanol to reverse phase silica gel (ODS, 50 μm, G1P 4S6 CANADA) column chromatography, sequentially eluting with mixed solvents of methanol and water in volume ratios of 10:90, 20:80 and 30:70, subjecting the eluate in volume ratios of methanol and water to silica gel column chromatography, eluting with mixed solvents of dichloromethane and methanol in volume ratios of 100:10, 100:20 and 100:30, collecting the eluate in volume ratios of dichloromethane and methanol, subjecting the eluate to preparative high performance liquid chromatography, and collecting the absorption peak occurring in the eluting time of 26.7 min to obtain the peroxyfraxinone;
the high performance liquid chromatography prepared in the step (3) takes methanol and water with the volume ratio of 30:70 as mobile phases, and the flow rate is 3ml/min.
And a third specific embodiment: the second difference between the present embodiment and the specific embodiment is that the pressure of the soxhlet dynamic extraction and concentration unit in the step (1) is set to normal pressure extraction, and the extraction temperature is set to 85-90 ℃. The other is the same as in the second embodiment.
The specific embodiment IV is as follows: the difference between the present embodiment and the second to third embodiments is that the concentration condition in the step (2) is-0.05 to-0.08 MPa. The other is the same as in one of the second to third embodiments.
Fifth embodiment: this embodiment differs from the second to fourth embodiments in that the suspension density in step (2) is 1.25g/ml. The others are the same as in the second to fourth embodiments.
Specific embodiment six: the application of the peroxyChinese waxiness ketone in the preparation of anti-inflammatory or anti-tumor medicines is provided in the specific embodiments one to five.
Seventh embodiment: the sixth difference between the present embodiment and the specific embodiment is that the anti-inflammatory or anti-tumor drug comprises injection, lyophilized powder for injection, and oral preparation. The other is the same as in the sixth embodiment.
Eighth embodiment: the sixth difference between the present embodiment and the specific embodiment is that the oral preparation includes a tablet, a granule, a soft capsule, a hard capsule, an oral liquid, and a sustained-release preparation. The other is the same as the sixth embodiment.
Detailed description nine: this embodiment differs from the sixth embodiment in that the peroxyfraxinone is used as an active ingredient of an anti-inflammatory agent for inhibiting LPS-induced NO release by mouse macrophages. The other is the same as in the sixth embodiment.
Detailed description ten: the sixth embodiment is different from the sixth embodiment in that the peroxywhite waxy ketone is used as an active ingredient of an antitumor drug for inhibiting lung adenocarcinoma cells and liver cancer tissue cells. The other is the same as in the sixth embodiment.
The following experiments are adopted to verify the effect of the invention:
experiment one:
the preparation method of the peroxide fraxinone comprises the following steps:
(1) 100kg of cortex dictamni is crushed and placed in an extraction tank of a Soxhlet dynamic extraction concentration unit to be extracted by ethanol with the volume concentration of 95%;
(2) Concentrating the ethanol extract obtained in the step (1), dispersing with water to obtain a suspension, and then sequentially extracting with petroleum ether, chloroform and ethyl acetate;
(3) Subjecting the ethyl acetate extract obtained in the step (2) to silica gel column chromatography, sequentially eluting with mixed solvents of dichloromethane and methanol in volume ratios of 100:0, 100:1, 100:2, 100:5 and 100:10, subjecting the eluate in volume ratios of dichloromethane and methanol to reverse phase silica gel (ODS, 50 μm, G1P 4S6 CANADA) column chromatography, sequentially eluting with mixed solvents of methanol and water in volume ratios of 10:90, 20:80 and 30:70, subjecting the eluate in volume ratios of methanol and water to silica gel column chromatography, eluting with mixed solvents of dichloromethane and methanol in volume ratios of 100:10, 100:20 and 100:30, collecting the eluate in volume ratios of dichloromethane and methanol, subjecting the eluate to preparative high performance liquid chromatography, and collecting the absorption peak occurring in the eluting time of 26.7 min to obtain the peroxyfraxinone;
the high performance liquid chromatography prepared in the step (3) takes methanol and water with the volume ratio of 30:70 as mobile phases, and the flow rate is 3ml/min.
The experiment adopts a Waters2545 preparation type high performance liquid chromatography, the detector is a Waters2489 type ultraviolet detector, and the detection wavelength is 210nm and 254nm. The packing of the chromatographic column is C-18 reverse phase silica gel.
100kg of cortex dictamni is crushed and extracted for the experiment, and finally 7.0mg of peroxide fraxinone is obtained, the purity of the peroxide fraxinone reaches 98.5%, and nuclear magnetic resonance data of the peroxide fraxinone are shown in table 1.
TABLE 1 peroxide fraxinone 1 H-NMR(600MHz,CDCl 3 ) A kind of electronic device with high-pressure air-conditioning system 13 C-NMR(150MHz,CDCl 3 )
Experiment II:
the inhibition of LPS-induced mouse macrophage (RAW 264.7) NO release by the prepared peroxyfraxinone was tested:
1. RAW264.7 cells which had reached the logarithmic phase were grown at 1X 10 5 Wells were seeded in 96-well plates in 5% CO 2 The cells were cultured in an incubator at 37℃for 24 hours, and the cells were subjected to induction treatment with DMEM high-glucose complete medium containing 25ng/ml LPS for 24 hours to set a model group.
2. The peroxide white waxy ketone prepared in experiment one is prepared into 600 mmol.L -1 The concentration of the sample group was set to 60. Mu.M/L, 30. Mu.M/L, 15. Mu.M/L, 7.5. Mu.M/L, 3.75. Mu.M/L by performing gradient dilution with the above medium.
3. And (3) adding the drug treated in the second step into the RAW264.7 cells treated in the first step for 24 hours, adding a lysate to lyse the cells, taking supernatant, and detecting the NO production. The NO generation inhibition (%) was calculated. NO generation inhibition (%) = (NO model group OD 540nm -NO sample group OD 540nm ) Model group OD/NO 540nm *100％。
4. The procedure was repeated three times in succession, and the IC50 value was calculated by Logit method. The IC50 of the prepared peroxide fraxinone in experiment I on the NO release rate of LPS-induced inflammatory mouse macrophage (RAW 264.7) is 7.94 mu M/L, and the peroxide fraxinone has better anti-inflammatory activity.
Experiment III:
the inhibition effect of the prepared peroxywhite waxy ketone on lung adenocarcinoma cells (A549) and liver cancer tissue cells (HepG 2) is detected:
1. a549 cells and HepG2 cells in logarithmic growth phase were cultured at 1X 10 5 Wells were seeded in 96-well plates in 5% CO 2 Culturing in an incubator at 37 ℃ for 24 hours.
2. The peroxide white waxy ketone prepared in experiment one is prepared into 600 mmol.L -1 The DMSO solution of (C) is subjected to gradient dilution by using a DMEM high-sugar complete medium, and the concentration of a sample group is set to be 60 mu M/L, 30 mu M/L, 15 mu M/L, 7.5 mu M/L and 3.75 mu M/L; the blank group was set as a blank medium without peroxyfraxinone.
3. To A549 cells treated in step oneAnd adding the second drug into HepG2 cells for 24 hours, discarding the supernatant, adding the MTT solution, and detecting the cell inhibition rate. Cell inhibition (%) = (MTT blank OD 570nm MTT sample group OD 570nm ) MTT blank OD 570nm 。
4. The procedure was repeated three times in succession, and the IC50 value was calculated by Logit method. IC50 s of the prepared peroxyChinese waxy ketone for inhibiting A549 cells and HepG2 cells in experiment I are respectively 16.38 mu M/L and 14.43 mu M/L. It shows that the composition has better in vitro anti-tumor activity.
Claims (10)
1. The peroxyChinese waxiness ketone is characterized in that the molecular formula of the peroxyChinese waxiness ketone is C 14 H 16 O 4 The molecular weight is 248.1049, and the molecular structural formula is:
2. the method for preparing the peroxide fraxinone according to claim 1, wherein the method for preparing the peroxide fraxinone comprises the following steps:
(1) Pulverizing cortex Dictamni Radicis, and extracting with 95% ethanol in an extraction tank of Soxhlet dynamic extraction concentration unit;
(2) Concentrating the ethanol extract obtained in the step (1), dispersing with water to obtain a suspension, and then sequentially extracting with petroleum ether, chloroform and ethyl acetate;
(3) Subjecting the ethyl acetate extract obtained in the step (2) to silica gel column chromatography, sequentially eluting with mixed solvents of dichloromethane and methanol in volume ratios of 100:0, 100:1, 100:2, 100:5 and 100:10, subjecting the eluate in volume ratios of dichloromethane and methanol to reverse phase silica gel column chromatography, sequentially eluting with mixed solvents of methanol and water in volume ratios of 10:90, 20:80 and 30:70, subjecting the eluate in volume ratios of methanol and water to silica gel column chromatography, subjecting the eluate in volume ratios of 30:70 to elution with mixed solvents of dichloromethane and methanol in volume ratios of 100:10, 100:20 and 100:30, subjecting the eluate in volume ratios of dichloromethane and methanol to preparative high performance liquid chromatography, and collecting absorption peaks appearing in elution time of 26.7 min to obtain the peroxy white waxy ketone;
the high performance liquid chromatography prepared in the step (3) takes methanol and water with the volume ratio of 30:70 as mobile phases, and the flow rate is 3ml/min.
3. The method for preparing the peroxide fraxinone according to claim 2, wherein the pressure of the Soxhlet dynamic extraction and concentration unit in the step (1) is set to be normal pressure extraction, and the extraction temperature is set to be 85-90 ℃.
4. The method for preparing the peroxide fraxinone according to claim 2, wherein the concentration condition in the step (2) is-0.05 to-0.08 MPa.
5. The process for preparing a peroxyfraxinone according to claim 2, wherein the suspension density in step (2) is 1.25g/ml.
6. Use of a peroxyfraxinone according to claim 1, wherein the peroxyfraxinone is for the manufacture of an anti-inflammatory or anti-tumour medicament.
7. The use of the peroxide fraxinone according to claim 6, wherein the anti-inflammatory or anti-tumor drug comprises injection, freeze-dried powder injection, and oral preparation.
8. The use of the peroxide fraxinone according to claim 7, wherein the oral preparation comprises a tablet, a granule, a soft capsule, a hard capsule, an oral liquid, and a sustained and controlled release preparation.
9. Use of a peroxyfraxinone according to claim 6, wherein said peroxyfraxinone is used as an active ingredient in an anti-inflammatory agent for inhibiting LPS-induced NO release by mouse macrophages.
10. The use of the peroxyChinese waxiness ketone as claimed in claim 6, wherein the peroxyChinese waxiness ketone is used as an active ingredient of an anti-tumor drug for inhibiting lung adenocarcinoma cells and liver cancer tissue cells.
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