CN117106008A - Enantiomer abietane diterpenoid lactone compound, and preparation method and application thereof - Google Patents
Enantiomer abietane diterpenoid lactone compound, and preparation method and application thereof Download PDFInfo
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- -1 abietane diterpenoid lactone compound Chemical class 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 150000001875 compounds Chemical class 0.000 claims abstract description 36
- 238000010828 elution Methods 0.000 claims abstract description 19
- 238000000605 extraction Methods 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000004440 column chromatography Methods 0.000 claims abstract description 17
- 239000000284 extract Substances 0.000 claims abstract description 15
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 9
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 9
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 9
- 238000005325 percolation Methods 0.000 claims abstract description 9
- 239000000725 suspension Substances 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 7
- 241000982713 Euphorbia fischeriana Species 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 60
- 239000003480 eluent Substances 0.000 claims description 50
- 239000003208 petroleum Substances 0.000 claims description 18
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical group O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 239000000469 ethanolic extract Substances 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 241000221079 Euphorbia <genus> Species 0.000 claims description 5
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 5
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 239000011347 resin Substances 0.000 claims description 5
- 229920005989 resin Polymers 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- 230000001093 anti-cancer Effects 0.000 claims description 4
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical group O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000007858 starting material Substances 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 17
- 230000005764 inhibitory process Effects 0.000 abstract description 16
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- 239000003814 drug Substances 0.000 abstract description 8
- 238000011161 development Methods 0.000 abstract description 7
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- 241000434018 Euphorbia pekinensis Species 0.000 abstract description 3
- 238000013459 approach Methods 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 3
- 238000010829 isocratic elution Methods 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 2
- 239000002612 dispersion medium Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 24
- 230000000694 effects Effects 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 230000018109 developmental process Effects 0.000 description 5
- 229930004069 diterpene Natural products 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 239000008187 granular material Substances 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
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- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 229930185597 Euphorbia lathyris Natural products 0.000 description 2
- 241001553700 Euphorbia lathyris Species 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
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- 229930014626 natural product Natural products 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
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- 150000003505 terpenes Chemical class 0.000 description 2
- 235000007586 terpenes Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XXDIJWSZFWZBRM-WSKGJZFTSA-N (3z,5e,9e,11z,17e,19e)-8,14,16-trihydroxy-24-methyl-1-oxacyclotetracosa-3,5,9,11,17,19-hexaen-2-one Chemical group CC1CCC\C=C\C=C\C(O)CC(O)C\C=C/C=C/C(O)C\C=C\C=C/C(=O)O1 XXDIJWSZFWZBRM-WSKGJZFTSA-N 0.000 description 1
- 206010000077 Abdominal mass Diseases 0.000 description 1
- RSWGJHLUYNHPMX-UHFFFAOYSA-N Abietic-Saeure Natural products C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C(O)=O RSWGJHLUYNHPMX-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
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- 206010008342 Cervix carcinoma Diseases 0.000 description 1
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- 108010019160 Pancreatin Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- KHPCPRHQVVSZAH-HUOMCSJISA-N Rosin Natural products O(C/C=C/c1ccccc1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KHPCPRHQVVSZAH-HUOMCSJISA-N 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 208000019790 abdominal distention Diseases 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000002365 anti-tubercular Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000011254 conventional chemotherapy Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000011026 diafiltration Methods 0.000 description 1
- 239000002027 dichloromethane extract Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 150000004141 diterpene derivatives Chemical class 0.000 description 1
- 125000000567 diterpene group Chemical group 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 150000002596 lactones Chemical group 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 201000007227 lymph node tuberculosis Diseases 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 201000002266 mite infestation Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 235000019633 pungent taste Nutrition 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- KHPCPRHQVVSZAH-UHFFFAOYSA-N trans-cinnamyl beta-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OCC=CC1=CC=CC=C1 KHPCPRHQVVSZAH-UHFFFAOYSA-N 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the technical field of natural medicines, in particular to an enantiomer abietyl diterpenoid lactone compound, a preparation method and application thereof, wherein the enantiomer abietyl diterpenoid lactone compound has a structure shown in a formula I, and the preparation method comprises the following steps: taking the dried root of Euphorbia fischeriana as a raw material, performing percolation extraction and concentration to obtain an extract, suspending the extract in water to obtain a suspension, and performing extraction and concentration to obtain an extract; and (3) performing gradient elution on the extract, performing isocratic elution on the extract by using an ODS (oxide dispersion medium) medium-pressure column chromatography to obtain Fr.1-Fr.6, and performing ODS liquid chromatography elution and purification on Fr.6 to obtain the compound shown in the formula I. The invention realizes the extraction of the antipodal abietane diterpenoid lactone compounds with cancer cell inhibition from the euphorbia pekinensis for the first time, the compounds have great development value as novel anticancer drugs, and the design thought of the compounds also provides a novel thought and approach for the development of novel anticancer drugs.
Description
Technical Field
The invention relates to the technical field of natural medicines, in particular to an enantiosaene diterpenoid lactone compound, and a preparation method and application thereof.
Background
Cancer is one of the most prominent diseases threatening human health. The mortality rate is inferior to that of cardiovascular and cerebrovascular diseases, and the incidence rate tends to rise year by year. Chemotherapy, which is the main means of tumor treatment, has better curative effect but often causes serious side effects, so that patients are difficult to adhere to the treatment, and most cancers lack effective targeted treatment, are insensitive to endocrine treatment and conventional chemotherapy, have high invasiveness and poor prognosis, and the clinical treatment is a very troublesome problem. Therefore, there is a need for therapeutic methods and drugs that have good efficacy and low toxic or side effects. In recent years, traditional Chinese medicines provide an important source and a new opportunity for the development of modern medicines. The effective components of the natural products become important sources for developing new drugs according to the characteristics of multiple targets, high efficiency, low toxicity and the like.
The euphorbia lathyris is one of basic plants of Chinese medicine euphorbia lathyris, the root meat quality of which is thick and white milk is present. Pungent taste, peaceful nature, toxicity, liver and spleen meridian entered, and has the efficacy of resolving masses and killing parasites, and is widely used in Chinese patent medicines and clinical prescriptions. The radix euphorbiae Fischerianae in the traditional medicine is used for resolving phlegm, resolving food stagnation, edema, abdominal distention, heart and abdomen pain, abdominal mass, scrofula, mange and the like. Modern research results show that Chinese medicine radix Euphorbiae Fischerianae has various pharmacological activities, such as antituberculosis, anti-HIV virus, anti-tumor, antibacterial, antiinflammatory and insecticidal effects. Modern researches have shown that the chemical components of Euphorbia pekinensis mainly comprise phenolic acid and terpenes. Among them, terpenes are mainly abundant and diverse diterpenes, and are also considered as the main substance basis for the radix euphorbiae lantu to exert a plurality of pharmacological activities. The enantiomer abietyl diterpene is an important chemical component in euphorbia chamaejasme, and the brand new enantiomer abietyl diterpene lactone compound and the activity thereof related to the invention have not been reported in patents or documents so far.
Therefore, whether natural products with cancer cell inhibition effect can be extracted from herbaceous plants is a technical problem to be solved urgently by those skilled in the art.
Disclosure of Invention
The invention provides an enantiomer abietyl diterpenoid lactone compound and a preparation method thereof, and the compound has good inhibition effect on 6 cancer cells MCF-7, HCC1806, hela, A549, PC-3 and H460. The specific technical scheme is as follows:
an enantiosabietane diterpenoid lactone compound, which has a structure shown in the following formula I:
a preparation method of the enantiomer abietyl alkane type diterpenoid lactone compound shown in the formula I is characterized by comprising the following steps: the method comprises the following steps: taking the dried root of Euphorbia fischeriana as a raw material, performing percolation extraction and concentration to obtain an extract, suspending the extract in water to obtain a suspension, and performing extraction and concentration to obtain an extract;
subjecting the extract to gradient elution by neutral alumina column chromatography, silica gel column chromatography and MCI resin column chromatography, eluting by ODS medium pressure column chromatography to obtain Fr.1-Fr.6, and subjecting Fr.6 to purification by ODS liquid chromatography to obtain the enantiomer abietyl diterpene lactone compound shown in formula I.
Further, performing gradient elution by the neutral alumina column chromatography to obtain a first eluent, wherein the eluent is petroleum ether-ethyl acetate-methanol, and the volume ratio of petroleum ether to ethyl acetate to methanol is 100:0:0-0:0:100. Specifically, the eluent comprises eluent with the volume ratio of petroleum ether to ethyl acetate to methanol of 100:0:0, 90:10:0, 80:20:0, 50:50:0, 30:70:0, 27:75:0, 0:100:0, 0:50:50 and 0:0:100, and 9 eluents are collected by gradient elution section of the eluent.
Further, the first eluent is subjected to gradient washing through the silica gel column chromatography to obtain a second eluent, wherein the eluent is petroleum ether-acetone-methanol, and the volume ratio of petroleum ether to acetone to methanol is 100:0:0-0:100:0-0:0:100. The eluent comprises petroleum ether, acetone and methanol in the volume ratio of 100:0:0, 95:4.5:0.5, 90:9.5:0.5, 85:14:1, 80:19:1, 48:48:2, 20:75:5 and 0:0:100, and 8 eluents are collected by gradient elution section of the eluent.
Further preferably, the first eluent is eluted by the eluent with the volume ratio of petroleum ether, ethyl acetate and methanol being 30:70:0.
Further, the second eluent is taken and subjected to gradient elution by the MCI resin column chromatography to obtain a third eluent, wherein the eluent is methanol-water, and the volume ratio of the methanol to the water is 30:70-80:20. Specifically, the eluent comprises eluent with the volume ratio of methanol to water of 0:100, 30:70, 50:40, 70:30, 90:10 and 100:0, and 6 eluents are obtained by gradient elution and sectional collection of the eluent.
Further preferably, the second eluent is eluted by the eluent with the volume ratio of petroleum ether, acetone and methanol being 85:14:1.
Further, the third eluent is taken to be subjected to isocratic elution by the ODS medium-pressure column chromatography to obtain Fr.1-Fr.6, the mobile phase is methanol-water, and the volume ratio of the methanol to the water is 60:40.
Further preferably, the third eluent is eluted by the eluent with the volume ratio of methanol to water being 70:30.
Further preferably, the flow rate of the third eluate eluted by the ODS medium pressure column chromatography is 20mL/min, and the elution time is 4 hours.
Further, fr.6 is taken and eluted by ODS liquid chromatography with a fixed proportion of mobile phase to obtain the compound shown in the formula I with low purity, wherein the mobile phase is acetonitrile-water, and the volume ratio of acetonitrile to water is 40:60.
Further, the compound shown in the formula I with low purity is taken and purified by ODS liquid chromatography by taking a methanol-water solution as a mobile phase, so that the compound shown in the formula I is obtained, and the volume ratio of methanol to water is 65:35.
Further, the diafiltration extraction step is as follows: percolating with ethanol at room temperature to obtain dry root of Euphorbia radix, concentrating to obtain extract-like ethanol extract, and suspending the ethanol extract in water to obtain suspension; the concentration of the ethanol is 95%, the time of the percolation extraction is 120h, and the flow rate of the percolation extraction of the ethanol is 0.5L/h.
Further, the extraction steps are as follows: adding dichloromethane with the same volume as the suspension, extracting, and concentrating to obtain the extract.
An application of the enantiomer abietyl diterpenoid lactone compound shown in the formula I in preparing anticancer drugs.
Further, the antipodal abietane diterpenoid lactone compounds are used as raw materials to prepare anticancer pharmaceutical compositions or any pharmaceutically acceptable salts.
Further, the pharmaceutical composition or any pharmaceutically acceptable salt is in the form of a tablet, capsule, granule, oral liquid, granule, dripping pill or pellet.
Further preferably, the cancer is breast cancer, cervical cancer, lung cancer, prostate cancer and lung cancer.
One or more technical solutions provided in the embodiments of the present invention at least have the following technical effects or advantages:
the invention realizes that the antipodal abietane diterpenoid lactone compounds extracted from euphorbia pekinensis have the effect of inhibiting cancer cells for the first time, and has good inhibition effect on 6 cancer cells MCF-7, HCC1806, hela, A549, PC-3 and H460. The compound has great development value as a novel anticancer drug, and the design thought of the compound also provides a novel thought and approach for the development of the novel anticancer drug.
Drawings
FIG. 1 shows an enantiomeric rosin alkane type diterpenoid lactone compound of the present invention 1 H-NMR spectrum;
FIG. 2 shows the enantiosucalane diterpene lactones of the invention 13 C-NMR spectrum;
FIG. 3 is a graph showing the inhibition of MCF-7 cells by the enantiosuclanolide compounds of the present invention;
FIG. 4 is a graph showing the inhibition of HCC1806 cells by the enantiosabietane diterpenoid lactones of the present invention;
FIG. 5 is a graph showing the inhibition of Hela cells by the enantioscopine diterpenoid lactones of the present invention;
FIG. 6 is a graph showing the inhibition of A549 cells by the enantiospin diterpenoid lactones of the present invention;
FIG. 7 is a graph showing PC-3 cell inhibition by an enantioskane-type diterpenoid lactone compound of the present invention;
FIG. 8 is a graph showing the inhibition of H460 cells by the enantiosabietane diterpenoid lactones of the present invention.
Detailed Description
In order to better understand the above technical solutions, the following detailed description will refer to the accompanying drawings and specific embodiments.
An enantiosaene diterpenoid lactone compound has a structure shown in the following formula I:
the preparation method of the enantiosaene diterpenoid lactone compound comprises the following steps:
example 1
1kg of wild euphorbia root dried medicinal material at high altitude is taken as a raw material, the raw material is subjected to percolation extraction with 95% ethanol for 120 hours at a flow rate of 0.5L/h, an extractum-like ethanol extract is obtained by rotary evaporation and concentration, the ethanol extract is suspended in water to obtain a suspension, dichloromethane with the volume equal to that of the suspension is added for extraction, and an extraction solution is concentrated to obtain an extractum. Subjecting the dichloromethane extract to neutral alumina column chromatography, and carrying out gradient elution by using petroleum ether-ethyl acetate-methanol solution with the volume ratio of 100:0:0-0:0:100, wherein the eluent comprises the eluent with the volume ratio of petroleum ether-ethyl acetate-methanol of 100:0:0, 90:10:0, 80:20:0, 50:50:0, 30:70:0, 27:75:0, 0:100:0, 0:50:50 and 0:0:100, and collecting 9 eluates by gradient elution and segmentation of the eluent. Performing silica gel column chromatography on an eluent eluted by petroleum ether-ethyl acetate-methanol in a volume ratio of 30:70:0, performing gradient elution by using petroleum ether-acetone-methanol solution in a volume ratio of 100:0:0-0:100:0-0:100, wherein the eluent comprises the eluent in a volume ratio of petroleum ether-acetone-methanol of 100:0:0, 95:4.5:0.5, 90:9.5:0.5, 85:14:1, 80:19:1, 48:48:2, 20:75:5 and 0:0:100, and performing gradient elution and sectional collection on the eluent to obtain 8 eluents. Eluting the eluent eluted by the eluent with the petroleum ether-acetone-methanol volume ratio of 85:14:1 by using an MCI resin column chromatography, and carrying out gradient elution by using a methanol-water solvent with the volume ratio of 0:100-100:0, wherein the eluent comprises the eluent with the volume ratio of 0:100, 30:70, 50:40, 70:30, 90:10 and 100:0, and 6 eluents are obtained by gradient elution and sectional collection of the eluent. And (3) taking an eluent eluted by the eluent with the volume ratio of methanol to water of 70:30, performing isocratic elution by using an ODS medium-pressure column chromatography with the volume ratio of methanol to water of 60:40, wherein the flow rate is 20mL/min, the elution time is 4 hours, and obtaining 6 components Fr.1-Fr.6 after elution. The Fr.6 part is prepared by Waters full-automatic ODS liquid chromatography with acetonitrile-water solution with volume ratio of 40:60 as mobile phase, and the compound shown in the formula I with low purity is obtained. The compound shown in the formula I with low purity is purified by preparing ODS liquid chromatography by taking methanol-water solution with the volume ratio of 65:35 as a mobile phase, so as to obtain the compound shown in the formula I.
The embodiment of the invention also provides application of the compound shown in the formula I in preparing anticancer drugs.
Specifically, the antipodal abietane diterpenoid lactone compounds are used as raw materials to prepare anticancer pharmaceutical compositions or any pharmaceutically acceptable salts. The pharmaceutical composition is added with auxiliary materials, the auxiliary materials are not particularly limited, and the auxiliary materials can be applicable auxiliary materials well known to those skilled in the art.
The dosage form of the pharmaceutical composition or any pharmaceutically acceptable salt is tablets, capsules, granules, oral liquid, medicinal granules, dripping pills or pellets.
Example 2:
the compound of formula I prepared in example 1 was subjected to structural identification.
The identification results are as follows:
the compound shown in the formula I is white amorphous powder (methanol) and is very easy to dissolve in methanol and dichloromethane. The 10% sulfuric acid ethanol solution shows a purple color. As shown in figure 1 and in table 1, 1 H-NMR(600MHz，DMSO-d 6 ) The spectrum shows 4 methyl hydrogen proton signals delta H 2.10 (3 h, d, j=1.8 hz, me-17), 0.81 (3 h, s, me-18), 0.82 (3 h, s, me-19), 0.57 (3 h, s, me-20), two oxidized methine hydrogen proton signals δ H 5.70 (1 h, m), 3.93 (1 h, d, j=3.6 Hz). As shown in figure 2 and in table 1, 13 C-NMR(150MHz,DMSO-d 6 ) A total of 20 carbon signals are shown in the spectra: wherein delta C 147.5 (C-13), 134.6 (C-15) suggests the presence of a pair of double bonds, delta C 72.6 (C-8), 74.0 (C-11), 86.3 (C-14) are the carbon signals of three consecutive oxygens, delta C 173.2 (C-16) is an ester carbonyl carbon signal, delta C 193.6 (C-12) is a ketocarbonyl carbon signal. By combining the physicochemical property and spectrum data analysis, the data of the compound shown as the formula I are found to be basically consistent with the data of 11-oxo-ebracteolatanolide B in the literature by virtue of literature retrieval, and the difference is that HMBC correlation between the thiooxymethylene proton signal and the carbon signal in the structure is inconsistent. The HMBC spectra of the compounds of formula I show a correlation between H-11 and C-8, C-9, C-10, C-12 and C-13, and the combination of H-H CONSY spectra shows that H-9 and H-11 are correlated, thus presuming that hydroxy substitution exists at the C-8 and C-11 positions and that the C-12 position is ketocarbonyl. HMBC spectra show the presence of H-14 and C-7, C-9, C-13, C-15In this connection, it is assumed that the five-membered lactone ring occurs between C-14 and C-16, and the planar structure of the compound is determined. Determination of relative configuration by NOESY, H-5 (delta) H 1.60 And H-9 (delta) H 1.87 H-9 and HO-11 (delta) H 6.53)、HO-8(δ H 5.40 HO-11, H-9 and H-14 (delta) H 5.70 The presence of NOE with HO-11 for H-14, indicating that H-5, H-9, HO-8, HO-11, H-14 are on the same side.
Through the analysis, the compound of the formula I is finally determined to be a brand new enantiomer abietyl alkane type diterpenoid lactone compound forming a five-membered lactone D ring between C-14 and C-16.
TABLE 1 Compounds of formula I 1 H-NMR(600MHz,DMSO-d 6 ) And 13 C-NMR(150MHz,DMSO-d 6 ) Data
Example 3: pharmacological Activity of Compounds of formula I
Test methods and results
1. MTT assay to examine the effect of Compounds of formula I on proliferation of 6 cancer cells MCF-7, HCC1806, hela, A549, PC-3 and H460
Selecting 6 cancer cells in logarithmic growth phase, performing pancreatin digestion, and preparing into 5×10 cells with corresponding culture medium containing 10% foetal calf serum 4 Cell suspension/mL, plated (100. Mu.L, 5X 10) 3 Individual/well), 96-well plate edges were filled with sterile PBS, 5% co at 37 °c 2 Incubation for 24h allowed the cells to adhere. The next day, the state of the cells in the 96-well plate was observed under a microscope, and if the growth of the cells was good, the next step was continued. The experimental group replaces 100 mu L of test compound culture solution with different concentration in each hole, the content of the DMSO system is controlled within 0.5 percent, and each concentration is parallel to 3 compound holes. Control group was replaced with blank medium containing DMSO in the same proportion, 3 wells in parallel, no cell wellFor background, the above conditions were incubated for 48h. After the completion, the cells were washed 2 times with PBS, and 20. Mu.L of the now prepared 0.5mg/mLMTT medium was added thereto, and the above-mentioned conditions were cultured for 4 hours. The upper culture broth was discarded, 100. Mu.L of DMSO was added, and the mixture was shaken for 10min to completely dissolve the crystals. The 96-well plate was placed in an enzyme-labeled instrument and OD at 492nm was measured. Tumor cell growth inhibition rate calculation formula for test compound: cell growth inhibition% = (a (negative control group) -a (dosing group))/a (negative control group) ×100% of the total cell growth inhibition ratio
Experimental data analysis the cell proliferation inhibitory activity of the samples was assessed using half inhibition concentration using SPSS 17.0 statistical software.
A G For the administration group OD average value, A K OD mean value of blank group, a M Mean OD for model group.
2. Results of anticancer Activity of Compounds of formulas I and II
By preliminary screening the effect of the compounds of formula I on proliferation of 6 cancer cells MCF-7, HCC1806, hela, A549, PC-3 and H460, it was found that both compounds of formula I have significant inhibitory activity on 6 cancer cells.
The inhibition rates for 6 cancer cells MCF-7, HCC1806, hela, a549, PC-3 and H460 were 99%, 100% and 100%, respectively, at a concentration of 100 μm of the compound of formula I; as can be seen from the data shown in FIGS. 3-8, the compounds of formula I have an IC for 6 cancer cells 50 The values were 11.38.+ -. 0.62, 0.12.+ -. 0.01, 5.39.+ -. 0.24, 2.45.+ -. 0.09, 0.31.+ -. 0.01 and 3.94.+ -. 0.71. Mu.M, respectively.
In conclusion, the invention firstly realizes the extraction of the enantiomer abietyl diterpenoid lactone compounds with the effect of inhibiting cancer cells from euphorbia chamaejasme, and has good inhibition effect on 6 cancer cells MCF-7, HCC1806, hela, A549, PC-3 and H460. The compound has great development value as a novel anticancer drug, and the design thought of the compound also provides a novel thought and approach for the development of the novel anticancer drug.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. An enantiospecific abietyl alkane type diterpenoid lactone compound, which is characterized in that: the enantiomer abietyl alkane type diterpenoid lactone compound has a structure shown in the following formula (I):
2. a process for the preparation of an enantiosabietane diterpenoid lactone compound according to claim 1, characterized in that: the method comprises the following steps: taking the dried root of Euphorbia fischeriana as a raw material, performing percolation extraction and concentration to obtain an extract, suspending the extract in water to obtain a suspension, and performing extraction and concentration to obtain an extract;
subjecting the extract to gradient elution by neutral alumina column chromatography, silica gel column chromatography and MCI resin column chromatography, and isocratically eluting by ODS medium-pressure column chromatography to obtain Fr.1-Fr.6, and subjecting Fr.6 to ODS liquid chromatography to obtain the enantiomer abietyl lactone compound shown in formula I.
3. The process for producing an enantiosabietane diterpenoid lactone compound according to claim 2, which is characterized in that: and performing gradient elution by the neutral alumina column chromatography to obtain a first eluent, wherein the eluent is petroleum ether-ethyl acetate-methanol, and the volume ratio of petroleum ether to ethyl acetate to methanol is 100:0:0-0:0:100.
4. A process for the preparation of an enantiosabietane diterpenoid lactone compound according to claim 3, characterized in that: and performing gradient washing on the first eluent by using the silica gel column chromatography to obtain a second eluent, wherein the eluent is petroleum ether-acetone-methanol, and the volume ratio of petroleum ether to acetone to methanol is 100:0:0-0:100:0-0:0:100.
5. The process for producing an enantiosabietane diterpenoid lactone compound according to claim 4, wherein: and (3) carrying out gradient washing on the second eluent by using the MCI resin column chromatography to obtain a third eluent, wherein the eluent is methanol-water, and the volume ratio of the methanol to the water is 30:70-80:20.
6. The process for producing an enantiosabietane diterpenoid lactone compound according to claim 5, wherein: and eluting the third eluate by using the ODS medium-pressure column chromatography to obtain Fr.1-Fr.6, wherein the mobile phase is methanol-water, and the volume ratio of the methanol to the water is 60:40.
7. The process for producing an enantiosabietane diterpenoid lactone compound according to claim 6, wherein: eluting Fr.6 by ODS liquid chromatography with a fixed ratio mobile phase to obtain a low-purity compound shown in formula I, wherein the mobile phase is acetonitrile-water, and the volume ratio of acetonitrile to water is 40:60;
and (3) purifying the low-purity compound shown in the formula I by using a methanol-water solution as a mobile phase through ODS liquid chromatography to obtain the compound shown in the formula I, wherein the volume ratio of the methanol to the water is 65:35.
8. The process for producing an enantiosabietane diterpenoid lactone compound according to any one of claims 2 to 7, characterized in that: the percolation extraction steps are as follows: percolating with ethanol at room temperature to obtain dry root of Euphorbia radix, concentrating to obtain extract-like ethanol extract, and suspending the ethanol extract in water to obtain suspension; the concentration of the ethanol is 95%, the time of the percolation extraction is 120h, and the flow rate of the percolation extraction of the ethanol is 0.5L/h;
the extraction steps are as follows: adding dichloromethane with the same volume as the suspension, extracting, and concentrating to obtain the extract.
9. Use of an enantiomerically pure abietane diterpenoid lactone compound according to claim 1 in the preparation of an anticancer drug.
10. The use according to claim 9, characterized in that the antipodal abietane diterpenoid lactones are used as starting materials for the preparation of anticancer pharmaceutical compositions or any pharmaceutically acceptable salts.
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