CN111560001A - Phenolic compound NO84, and preparation method and application thereof - Google Patents
Phenolic compound NO84, and preparation method and application thereof Download PDFInfo
- Publication number
- CN111560001A CN111560001A CN202010412733.1A CN202010412733A CN111560001A CN 111560001 A CN111560001 A CN 111560001A CN 202010412733 A CN202010412733 A CN 202010412733A CN 111560001 A CN111560001 A CN 111560001A
- Authority
- CN
- China
- Prior art keywords
- compound
- formula
- target compound
- column chromatography
- stage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 150000002989 phenols Chemical class 0.000 title claims abstract description 10
- 150000001875 compounds Chemical class 0.000 claims abstract description 104
- 230000004663 cell proliferation Effects 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 80
- 239000003480 eluent Substances 0.000 claims description 33
- 238000010898 silica gel chromatography Methods 0.000 claims description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 17
- 201000005202 lung cancer Diseases 0.000 claims description 17
- 208000020816 lung neoplasm Diseases 0.000 claims description 17
- 238000004587 chromatography analysis Methods 0.000 claims description 15
- 210000004881 tumor cell Anatomy 0.000 claims description 15
- 238000010828 elution Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 239000003814 drug Substances 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 12
- 244000198134 Agave sisalana Species 0.000 claims description 11
- 235000011624 Agave sisalana Nutrition 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000004480 active ingredient Substances 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 8
- 239000002246 antineoplastic agent Substances 0.000 claims description 8
- 229940041181 antineoplastic drug Drugs 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000000287 crude extract Substances 0.000 claims description 6
- 238000002481 ethanol extraction Methods 0.000 claims description 6
- 201000007270 liver cancer Diseases 0.000 claims description 6
- 208000014018 liver neoplasm Diseases 0.000 claims description 6
- 239000011347 resin Substances 0.000 claims description 6
- 229920005989 resin Polymers 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- 239000003112 inhibitor Substances 0.000 claims description 5
- 238000004007 reversed phase HPLC Methods 0.000 claims description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 4
- 239000001569 carbon dioxide Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000000194 supercritical-fluid extraction Methods 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 238000010829 isocratic elution Methods 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 238000011282 treatment Methods 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 239000006187 pill Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 238000003815 supercritical carbon dioxide extraction Methods 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 230000001613 neoplastic effect Effects 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 12
- 230000000259 anti-tumor effect Effects 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 82
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 24
- 230000004083 survival effect Effects 0.000 description 20
- 239000006285 cell suspension Substances 0.000 description 16
- 238000002474 experimental method Methods 0.000 description 16
- 229930012538 Paclitaxel Natural products 0.000 description 13
- 229960001592 paclitaxel Drugs 0.000 description 13
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 13
- 238000012258 culturing Methods 0.000 description 12
- 230000005764 inhibitory process Effects 0.000 description 12
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 11
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 230000003698 anagen phase Effects 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 7
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 7
- 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 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 5
- 229940079593 drug Drugs 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 229930003827 cannabinoid Natural products 0.000 description 2
- 239000003557 cannabinoid Substances 0.000 description 2
- 229940065144 cannabinoids Drugs 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 150000001200 N-acyl ethanolamides Chemical class 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000002621 endocannabinoid Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a phenolic compound NO84, a preparation method and application thereof, wherein the molecular formula of the compound is C25H35NO5And the molecular weight is 429.56, and the compound has good anti-tumor cell proliferation activity.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to a phenolic compound NO84, and a preparation method and application thereof.
Background
The hemp plant is a plant with a complex chemical type, mainly because the hemp plant contains many natural chemical components. By 1980, there were 432 species of compounds isolated from hemp plants, increasing to 483 species by 1995 and 490 species by 2005. Statistically, there are 565 kinds of compounds isolated from hemp plants to date, which are classified into two major classes of phytocannabinoids and non-cannabinoids, and 120 kinds of phytocannabinoids have been reported.
There are also an increasing number of patients suffering from severe diseases such as cancer who seek natural drugs as an alternative or complementary therapy, and there is a continuing need for new treatments for cancer or other symptoms.
The incidence of cancer is increasing year by year due to different living habits, environmental factors and the like, and some tumors are found to be in an advanced stage and are difficult to cure. The screening of active ingredients in hemp plants is of great importance because of the high incidence rate and difficulty in curing cancer. At the same time, advances in the understanding of the "endocannabinoid" system have made it possible to develop cannabinoids for the treatment of advanced cancer.
The phenolic compounds have complex structure-activity relationship and large difference of antitumor activity due to different substituents in the structure, and are mainly concentrated on the positions and the number of hydroxyl groups and the positions of double bonds. The phenolic compound induces the expression of upstream inflammatory factors to promote the apoptosis of tumor cells and generate antitumor activity. The tumor cell inhibitors widely used at present comprise cisplatin, paclitaxel, gemcitabine and the like.
Disclosure of Invention
In view of the above, the present invention provides a phenolic compound NO84, and its preparation method and application25H35NO5And the molecular weight is 429.56, and the compound has good anti-tumor cell proliferation activity.
The invention adopts the specific technical scheme that:
a phenolic compound NO84 having the structure of formula I:
the invention also provides a preparation method of the compound shown in the formula I, which comprises the following steps:
(1) sequentially performing carbon dioxide supercritical extraction and ethanol extraction on the hemp plant inflorescence to obtain a crude extract;
(2) fully dissolving the obtained crude extract with petroleum ether to obtain a soluble component;
(3) separating the soluble component prepared in the step (2) by normal phase silica gel column chromatography, and carrying out isocratic elution on the normal phase silica gel column chromatography by using n-hexane/ethyl acetate 98:2 as an eluent to obtain a first-stage component containing a target compound;
(4) subjecting the primary component containing the target compound to macroporous adsorption resin column chromatography, performing gradient elution by using a D101 macroporous adsorption resin column and 30-100% ethanol/water as an eluent, and combining the eluents containing similar components to obtain a secondary component containing the target compound;
(5) performing chromatography on the second-stage component containing the target compound by using a first-stage medium-pressure normal-phase silica gel column chromatography, performing gradient elution by using dichloromethane/ethyl acetate as an eluent in the first-stage medium-pressure normal-phase silica gel column chromatography, and combining the eluents containing similar components to obtain a third-stage component containing the target compound;
(6) subjecting the third-stage component containing the target compound to second-stage medium-pressure reverse-phase silica gel column chromatography, performing gradient elution by using methanol/water as an eluent in the second-stage medium-pressure reverse-phase silica gel column chromatography, and combining the eluents containing similar components to obtain a fourth-stage component containing the target compound;
(7) taking a fourth-stage component containing a target compound, performing chromatography on a third-stage medium-pressure normal-phase silica gel column chromatography, performing gradient elution on the third-stage medium-pressure normal-phase silica gel column chromatography by using n-hexane/acetone as an eluent, and combining the eluents containing similar components to obtain a fifth-stage component containing the target compound;
(8) and (3) carrying out chromatography on the five-stage component containing the target compound by using high-pressure reverse phase HPLC chromatography, carrying out gradient elution by using acetonitrile/water solution as an eluent by using the high-pressure reverse phase HPLC chromatography, and combining the eluents containing similar components to obtain the compound shown in the formula I.
Preferably, the carbon dioxide supercritical extraction conditions are: pExtraction kettle＝30MPa，TExtraction kettle＝45℃；PSeparation kettle I＝8MPa，TSeparation kettle I＝45℃；PSeparation kettle II＝6MPa，TSeparation kettle II＝35℃；
Preferably, the usage amount of ethanol in the ethanol extraction is 20% of the weight of the hemp plant inflorescence, and the extraction time is 45 min.
The invention also provides application of the compound shown in the formula I in preparing a tumor cell proliferation inhibitor.
Correspondingly, the invention provides a tumor cell proliferation inhibitor drug which is characterized by comprising an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient comprises a compound shown in a formula I.
The invention also provides application of the compound shown in the formula I in preparing a medicament for treating tumor diseases.
Correspondingly, the invention provides an anti-tumor medicament which comprises an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient comprises a compound shown in a formula I.
Preferably, the tumor disease is liver cancer or lung cancer.
Preferably, the medicine is an oral preparation or an injection preparation, and the oral preparation is one of dripping pills, tablets, capsules, granules or oral liquid; the injection preparation is selected from injection or powder injection.
The invention has the beneficial effects that: the compound shown in the formula I has high purity, good stability and good biological activity. Through in vitro cell level related experiment detection, the compound has the activity of inhibiting tumor cell proliferation and has the potential of developing antitumor drugs.
Drawings
FIG. 1 shows the effect of compounds of formula I on the survival of human hepatoma cells (HepG2) (MTT method);
FIG. 2 shows the effect of compounds of formula I on the survival of human lung cancer cells (A549) (CCK8 method);
FIG. 3 shows the effect of compounds of formula I on the survival of human hepatoma cells (HepG2) (MTT method);
FIG. 4 shows the effect of compounds of formula I on the survival of human lung cancer cells (A549) (CCK8 method);
FIG. 5 shows the effect of the compound of formula I on the cloning of human hepatoma cells (HepG 2);
FIG. 6 shows the effect of compounds of formula I on the clonality of human lung cancer cells (A549);
FIG. 7 shows the inhibition curve of the compound of formula I on human hepatoma cells (HepG 2);
FIG. 8 shows the inhibition curves of the compound of formula I on human lung cancer cells (A549).
Detailed Description
To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.
The invention provides a phenolic compound NO84, which has the following chemical structural formula, English nomenclature, molecular formula and molecular weight:
example 1
A process for the preparation of compound NO84 of formula I, comprising the steps of:
(1) sequentially performing carbon dioxide supercritical extraction and ethanol extraction on the hemp plant inflorescence to obtain a crude extract; the supercritical carbon dioxide extraction conditions are as follows: pExtraction kettle＝30MPa，TExtraction kettle＝45℃；PSeparation kettle I＝8MPa，TSeparation kettle I＝45℃；PSeparation kettle II＝6MPa，TSeparation kettle IIThe temperature is 35 ℃; the ethanol is used in 20% of the weight of the inflorescence of the hemp plant during the ethanol extraction, and the extraction time is 45 min.
(2) Fully dissolving the obtained crude extract with petroleum ether to obtain a soluble component;
(3) performing chromatography on the soluble component prepared in the step (2) by normal phase silica gel column chromatography, and performing isocratic elution on the normal phase silica gel column chromatography by using n-hexane/ethyl acetate 98:2 as an eluent to obtain a first-stage component containing a target compound;
(4) subjecting the primary component containing the target compound to macroporous adsorption resin column chromatography, performing gradient elution by using a D101 macroporous adsorption resin column and 30-100% ethanol/water as an eluent, and combining the eluents containing similar components to obtain a secondary component containing the target compound;
(5) performing chromatography on the second-stage component containing the target compound by using a first-stage medium-pressure normal-phase silica gel column chromatography, performing gradient elution by using dichloromethane/ethyl acetate as an eluent in the first-stage medium-pressure normal-phase silica gel column chromatography, and combining the eluents containing similar components to obtain a third-stage component containing the target compound;
(6) subjecting the third-stage component containing the target compound to second-stage medium-pressure reverse-phase silica gel column chromatography, performing gradient elution by using methanol/water as an eluent in the second-stage medium-pressure reverse-phase silica gel column chromatography, and combining the eluents containing similar components to obtain a fourth-stage component containing the target compound;
(7) taking a fourth-stage component containing a target compound, performing chromatography on a third-stage medium-pressure normal-phase silica gel column chromatography, performing gradient elution on the third-stage medium-pressure normal-phase silica gel column chromatography by using n-hexane/acetone as an eluent, and combining the eluents containing similar components to obtain a fifth-stage component containing the target compound;
(8) subjecting the five-stage fraction containing target compound to high pressure reversed phase HPLC chromatography, gradient eluting with acetonitrile/water solution as eluent, mixing eluents containing similar components to obtain target compound, and subjecting the target compound to chromatography1H and13c, detecting by nuclear magnetic resonance, and structurally resolving into the compound shown in the formula I. After Sci Finder search, the compounds of formula I were found to be unreported compounds. A compound of formula I1H and13c nmr information is as follows:
1H NMR(850MHz,Chloroform-d)6.33(1H,br s,H-4),6.32(1H,br s,H-2),4.11(2H,q,J＝7.1Hz,H-1″), 2.73(td,J＝15.0,5.2Hz,1H),2.49(2H,dd,J＝9.0,6.8Hz,H-1′),2.39(ddd,J＝15.2,4.2,2.6Hz,1H),2.12(ddd, J＝13.9,5.3,2.6Hz,1H),2.04(s,3H),1.89(s,3H),1.84(ddd,J＝15.1,13.9,4.0Hz,1H),1.60(2H,m,H-2′),1.46 (3H,s,H-13),1.32(4H,m,H-3′,4′),1.25(3H,t,J＝7.1Hz,H-2″),1.18(3H,s,H-12),0.89(3H,t,J＝6.8Hz, H-5′).
13C NMR(214MHz,Chloroform-d)199.44(C-),171.47(C-),155.22(C-5),154.74(C-1),149.03(C-3),143.63 (C-),130.22(C-),108.81(C-4),108.72(C-2),104.14(C-10b),82.65(C-6),71.22(C-),60.61(C-1″),36.01(C-1′), 33.05(C-),31.71(C-3′),30.40(C-2′),27.35(C-),24.20(C-12),22.65(C-4′),21.20(C-),21.06(C-13),15.64(C-), 14.32(C-2″),14.14(C-5′).
example 2
Identification of antitumor Activity of Compound NO84 of formula I
Medicine preparation: compound NO84 of formula I prepared in example 1.
(1) MTT colorimetric method for detecting influence of compound shown as formula I on tumor cell survival performance
Taking human liver cancer cells (HepG2) in logarithmic growth phase, preparing a cell suspension with a proper concentration by using a DMEM culture solution, wherein the cell density is about 70000 cells/mL (namely about 7000 cells are contained in 100 mu L of culture solution), inoculating the cells into a 96-well plate by using 100 mu L of cell suspension per well, and culturing in an incubator at 37 ℃ until the cells are attached to the wall. DMSO is used as a solvent to prepare a compound solution with the concentration of 20mg/mL shown in the formula I, and the compound solution is diluted to the required working concentration by using a culture solution during an experiment. After the culture solution is discarded from the 96-well plate, 100 mu L of compound solution shown in the formula I with working concentration of 20 mu g/mL and 40 mu g/mL is respectively added into the experimental group, 100 mu L of culture solution is added into the blank control group, 100 mu L of antitumor drug Paclitaxel (PTX) solution with concentration of 10 mu g/mL is added into the positive control group, the 96-well plate is continuously placed in the incubator for 24 hours, the cell survival rate is detected by using MTT reagent, and the experiment is repeated for 3 times to take the average value.
As shown in FIG. 1, the survival rate of human hepatoma cells (HepG2) was lower as the concentration of the compound of formula I increased, i.e., the inhibition of the survival of human hepatoma cells (HepG2) was stronger.
Taking human lung cancer cells (A549) in logarithmic growth phase, preparing a cell suspension with a proper concentration by using a DMEM culture solution, wherein the cell density is about 70000 cells/mL (namely about 7000 cells are contained in 100 mu L of culture solution), inoculating the cells into a 96-well plate by using 100 mu L of cell suspension per well, and culturing in an incubator at 37 ℃ until the cells are attached to the wall. DMSO is used as a solvent to prepare a compound solution with the concentration of 20mg/mL shown in the formula I, and the compound solution is diluted to the required working concentration by using a culture solution during an experiment. After the culture solution is discarded from the 96-well plate, 100 mu L of compound solution shown in the formula I with working concentration of 20 mu g/mL and 40 mu g/mL is respectively added into the experimental group, 100 mu L of culture solution is added into the blank control group, 100 mu L of antitumor drug Paclitaxel (PTX) solution with concentration of 10 mu g/mL is added into the positive control group, the 96-well plate is continuously placed in the incubator for 24 hours, the cell survival rate is detected by using MTT reagent, and the experiment is repeated for 3 times to take the average value.
As shown in fig. 2, the survival rate of human lung cancer cell (a549) was lower with increasing concentration, i.e., the inhibition effect of the compound of formula I on the survival of human lung cancer cell (a549) was stronger.
(2) CCK8 colorimetric method for detecting influence of compound shown as formula I on tumor cell survival performance
Taking human liver cancer cells (HepG2) in logarithmic growth phase, preparing a cell suspension with a proper concentration by using a DMEM culture solution, wherein the cell density is about 70000 cells/mL (namely about 7000 cells are contained in 100 mu L of culture solution), inoculating the cells into a 96-well plate by using 100 mu L of cell suspension per well, and culturing in an incubator at 37 ℃ until the cells are attached to the wall. DMSO is used as a solvent to prepare a compound solution with the concentration of 20mg/mL shown in the formula I, and the compound solution is diluted to the required working concentration by using a culture solution during an experiment. After the culture solution is discarded from the 96-well plate, 100 mu L of compound solution shown in the formula I with working concentration of 20 mu g/mL and 40 mu g/mL is respectively added into the experimental group, 100 mu L of culture solution is added into the blank control group, 100 mu L of antitumor drug Paclitaxel (PTX) solution with concentration of 10 mu g/mL is added into the positive control group, the 96-well plate is continuously placed in the incubator for 24 hours, the cell survival rate is detected by using a CCK8 reagent, and the experiment is repeated for 3 times to take an average value.
As shown in FIG. 3, the survival rate of human hepatoma cells (HepG2) was lower as the concentration of the compound of formula I increased, i.e., the inhibition of the survival of human hepatoma cells (HepG2) was stronger.
Taking human lung cancer cells (A549) in logarithmic growth phase, preparing a cell suspension with a proper concentration by using a DMEM culture solution, wherein the cell density is about 70000 cells/mL (namely about 7000 cells are contained in 100 mu L of culture solution), inoculating the cells into a 96-well plate by using 100 mu L of cell suspension per well, and culturing in an incubator at 37 ℃ until the cells are attached to the wall. DMSO is used as a solvent to prepare a compound solution with the concentration of 20mg/mL shown in the formula I, and the compound solution is diluted to the required working concentration by using a culture solution during an experiment. After the culture solution is discarded from the 96-well plate, 100 mu L of compound solution shown in the formula I with working concentration of 20 mu g/mL and 40 mu g/mL is respectively added into the experimental group, 100 mu L of culture solution is added into the blank control group, 100 mu L of antitumor drug Paclitaxel (PTX) solution with concentration of 10 mu g/mL is added into the positive control group, the 96-well plate is continuously placed in the incubator for 24 hours, the cell survival rate is detected by using a CCK8 reagent, and the experiment is repeated for 3 times to take an average value.
As shown in fig. 4, the survival rate of human lung cancer cell (a549) was lower with increasing concentration, i.e., the inhibition effect of the compound of formula I on the survival of human lung cancer cell (a549) was stronger.
(3) Cell clone formation experiment for detecting influence of compound shown as formula I on tumor cell proliferation
Taking human liver cancer cells (HepG2) in a logarithmic growth phase, preparing a cell suspension with an appropriate concentration by using a DMEM culture solution, wherein the cell density is about 117 cells/mL (namely, about 117 cells are contained in 1mL of the culture solution), inoculating the cells into a 6-well plate by using 3mL of the cell suspension per well, and culturing in an incubator at 37 ℃ until the cells are attached to the wall. DMSO is used as a solvent to prepare a compound solution with the concentration of 20mg/mL shown in the formula I, and the compound solution is diluted to the required working concentration by using a culture solution during an experiment. After discarding the culture solution from the 6-well plate, adding 3mL of compound solution shown in formula I with working concentration of 20 and 40 μ g/mL into the experimental group, adding 3mL of culture solution into the blank control group, continuously placing the 6-well plate into an incubator, replacing fresh culture solution or culture solution containing medicine every 2-3 days, continuously culturing for about two weeks, continuously observing cell morphology, and stopping culturing when macroscopic clone appears in the culture dish. Discard the culture medium, carefully wash with PBS 2 times, add 4% Paraformaldehyde (PFA)1mL fixed cells for 30 min. After PFA is discarded, 1ml of 0.1% crystal violet is added into each hole for dyeing for 30min, the dyeing solution is washed away by ultrapure water, a 6-hole plate is dried in the air and photographed (figure 5) and the clone formation rate is calculated.
As shown in FIG. 5, the cloning efficiency of human hepatoma cells (HepG2) was lower with increasing concentration, i.e., the compound of formula I had stronger inhibitory effect on the proliferation of human hepatoma cells (HepG 2).
Taking human lung cancer cells (A549) in logarithmic growth phase, preparing a cell suspension with a proper concentration by using a DMEM culture solution, wherein the cell density is about 117 cells/mL (namely about 117 cells are contained in 1mL culture solution), inoculating the cells into a 6-well plate by using 3mL cell suspension per well, and culturing in an incubator at 37 ℃ until the cells are attached to the wall. DMSO is used as a solvent to prepare a compound solution with the concentration of 20mg/mL shown in the formula I, and the compound solution is diluted to the required working concentration by using a culture solution during an experiment. After discarding the culture solution from the 6-well plate, adding 3mL of compound solution shown in formula I with working concentration of 20 and 40 μ g/mL into the experimental group, adding 3mL of culture solution into the blank control group, continuously placing the 6-well plate into an incubator, replacing fresh culture solution or culture solution containing medicine every 2-3 days, continuously culturing for about two weeks, continuously observing cell morphology, and stopping culturing when macroscopic clone appears in the culture dish. Discard the culture medium, carefully wash with PBS 2 times, add 4% Paraformaldehyde (PFA)1mL fixed cells for 30 min. After PFA is discarded, 1ml of 0.1% crystal violet is added into each hole for dyeing for 30min, the dyeing solution is washed away by ultrapure water, and a 6-hole plate is dried in the air and photographed (figure 6) and the clone formation rate is calculated.
As shown in fig. 6, the lower the clonality rate of human lung cancer cells (a549) with the increase in concentration, i.e., the stronger the inhibitory effect of the compound of formula I on the proliferation performance of human lung cancer cells (a 549).
(4) The half Inhibitory Concentration (IC) of the compound shown in the formula I on tumor cells in 24 hours50Value) determination
Taking human liver cancer cells (HepG2) in logarithmic growth phase, preparing a cell suspension with a proper concentration by using a DMEM culture solution, wherein the cell density is about 70000 cells/mL (namely about 7000 cells are contained in 100 mu L of culture solution), inoculating the cells into a 96-well plate by using 100 mu L of cell suspension per well, and culturing in an incubator at 37 ℃ until the cells are attached to the wall. DMSO is used as a solvent to prepare a compound solution with the concentration of 20mg/mL shown in the formula I, and the compound solution is diluted to the required working concentration by using a culture solution during an experiment. After the culture solution is discarded from the 96-well plate, 100 mu L of compound solution shown in formula I with working concentration of 1.857 mu g/mL, 3.75 mu g/mL, 7.5 mu g/mL, 15 mu g/mL, 30 mu g/mL and 60 mu g/mL is respectively added into the experimental group, 100 mu L of culture solution is added into the blank control group, 100 mu L of antitumor drug Paclitaxel (PTX) solution with concentration of 10 mu g/mL is added into the positive control group, the 96-well plate is continuously placed in the incubator for 24h, then CCK8 reagent is used for detecting the cell survival rate, and the experiment is repeated for 3 times to take an average value. Dose inhibition curves were plotted by GraphPad Prism software and the IC of compounds of formula I on tumor cells was calculated50The value is obtained. As shown in FIG. 7, the inhibition rate of the compound of formula I on human hepatoma cells (HepG2) gradually becomes flat with increasing concentration, and with increasing concentrationThe tumor cell inhibition rate is increased, and an increasing relation is presented.
Taking human lung cancer cells (A549) in logarithmic growth phase, preparing a cell suspension with a proper concentration by using a DMEM culture solution, wherein the cell density is about 70000 cells/mL (namely about 7000 cells are contained in 100 mu L of culture solution), inoculating the cells into a 96-well plate by using 100 mu L of cell suspension per well, and culturing in an incubator at 37 ℃ until the cells are attached to the wall. DMSO is used as a solvent to prepare a compound solution with the concentration of 20mg/mL shown in the formula I, and the compound solution is diluted to the required working concentration by using a culture solution during an experiment. After the culture solution is discarded from the 96-well plate, 100 mu L of compound solution shown in formula I with working concentration of 1.857 mu g/mL, 3.75 mu g/mL, 7.5 mu g/mL, 15 mu g/mL, 30 mu g/mL and 60 mu g/mL is respectively added into the experimental group, 100 mu L of culture solution is added into the blank control group, 100 mu L of antitumor drug Paclitaxel (PTX) solution with concentration of 10 mu g/mL is added into the positive control group, the 96-well plate is continuously placed in the incubator for 24h, then CCK8 reagent is used for detecting the cell survival rate, and the experiment is repeated for 3 times to take an average value. Dose inhibition curves were plotted by GraphPad Prism software and the IC of compounds of formula I on tumor cells was calculated50The value is obtained. As shown in fig. 8, the inhibition rate of the compound of formula I on human lung cancer cells (a549) gradually becomes flat with increasing concentration, and the inhibition rate of tumor cells increases with increasing concentration, showing an increasing relationship.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.
Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes using the contents of the present specification and drawings, or any other related technical fields, which are directly or indirectly applied thereto, are included in the scope of the present invention.
Claims (9)
1. A phenolic compound NO84, having the structure of formula I:
2. a process for the preparation of a compound of formula I according to claim 1, comprising the steps of:
(1) sequentially performing carbon dioxide supercritical extraction and ethanol extraction on the hemp plant inflorescence to obtain a crude extract;
(2) fully dissolving the obtained crude extract with petroleum ether to obtain a soluble component;
(3) separating the soluble component prepared in the step (2) by normal phase silica gel column chromatography, and carrying out isocratic elution on the normal phase silica gel column chromatography by using n-hexane/ethyl acetate 98:2 as an eluent to obtain a first-stage component containing a target compound;
(4) subjecting the primary component containing the target compound to macroporous adsorption resin column chromatography, performing gradient elution by using a D101 macroporous adsorption resin column and 30-100% ethanol/water as an eluent, and combining the eluents containing similar components to obtain a secondary component containing the target compound;
(5) performing chromatography on the second-stage component containing the target compound by using a first-stage medium-pressure normal-phase silica gel column chromatography, performing gradient elution by using dichloromethane/ethyl acetate as an eluent in the first-stage medium-pressure normal-phase silica gel column chromatography, and combining the eluents containing similar components to obtain a third-stage component containing the target compound;
(6) subjecting the third-stage component containing the target compound to second-stage medium-pressure reverse-phase silica gel column chromatography, performing gradient elution by using methanol/water as an eluent in the second-stage medium-pressure reverse-phase silica gel column chromatography, and combining the eluents containing similar components to obtain a fourth-stage component containing the target compound;
(7) taking a fourth-stage component containing a target compound, performing chromatography on a third-stage medium-pressure normal-phase silica gel column chromatography, performing gradient elution on the third-stage medium-pressure normal-phase silica gel column chromatography by using n-hexane/acetone as an eluent, and combining the eluents containing similar components to obtain a fifth-stage component containing the target compound;
(8) and (3) carrying out chromatography on the five-stage component containing the target compound by using high-pressure reverse phase HPLC chromatography, carrying out gradient elution by using acetonitrile/water solution as an eluent by using the high-pressure reverse phase HPLC chromatography, and combining the eluents containing similar components to obtain the compound shown in the formula I.
3. The method of claim 2, wherein the supercritical carbon dioxide extraction conditions are: pExtraction kettle＝30MPa，TExtraction kettle＝45℃；PSeparation kettle I＝8MPa，TSeparation kettle I＝45℃；PSeparation kettle II＝6MPa，TSeparation kettle IIThe temperature is 35 ℃; and/or
The ethanol is used in 20% of the weight of the inflorescence of the hemp plant during the ethanol extraction, and the extraction time is 45 min.
4. The application of the compound shown in the formula I in preparing a tumor cell proliferation inhibitor.
5. The tumor cell proliferation inhibitor medicine is characterized by comprising an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient comprises a compound shown in a formula I.
6. The use of a compound of formula I in the manufacture of a medicament for the treatment of a neoplastic disease.
7. An antitumor drug is characterized by comprising an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient comprises a compound shown as a formula I.
8. The use according to claim 4 or 6, wherein the neoplastic disease is liver cancer or lung cancer.
9. The medicine according to claim 5 or 7, wherein the medicine is an oral preparation or an injection preparation, and the oral preparation is one of dripping pills, tablets, capsules, granules or oral liquid; the injection preparation is selected from injection or powder injection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010412733.1A CN111560001A (en) | 2020-05-15 | 2020-05-15 | Phenolic compound NO84, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010412733.1A CN111560001A (en) | 2020-05-15 | 2020-05-15 | Phenolic compound NO84, and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111560001A true CN111560001A (en) | 2020-08-21 |
Family
ID=72071024
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010412733.1A Pending CN111560001A (en) | 2020-05-15 | 2020-05-15 | Phenolic compound NO84, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111560001A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111548332A (en) * | 2020-05-15 | 2020-08-18 | 福建省中科生物股份有限公司 | Terpene phenolic compound NO95, and preparation method and application thereof |
| CN111574531A (en) * | 2020-05-15 | 2020-08-25 | 福建省中科生物股份有限公司 | Terpene phenolic compound NO85, and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111233814A (en) * | 2020-03-02 | 2020-06-05 | 福建省中科生物股份有限公司 | Terpene phenolic compound ZKYY-057 and preparation method and application thereof |
| CN111548332A (en) * | 2020-05-15 | 2020-08-18 | 福建省中科生物股份有限公司 | Terpene phenolic compound NO95, and preparation method and application thereof |
| CN111574531A (en) * | 2020-05-15 | 2020-08-25 | 福建省中科生物股份有限公司 | Terpene phenolic compound NO85, and preparation method and application thereof |
-
2020
- 2020-05-15 CN CN202010412733.1A patent/CN111560001A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111233814A (en) * | 2020-03-02 | 2020-06-05 | 福建省中科生物股份有限公司 | Terpene phenolic compound ZKYY-057 and preparation method and application thereof |
| CN111548332A (en) * | 2020-05-15 | 2020-08-18 | 福建省中科生物股份有限公司 | Terpene phenolic compound NO95, and preparation method and application thereof |
| CN111574531A (en) * | 2020-05-15 | 2020-08-25 | 福建省中科生物股份有限公司 | Terpene phenolic compound NO85, and preparation method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111548332A (en) * | 2020-05-15 | 2020-08-18 | 福建省中科生物股份有限公司 | Terpene phenolic compound NO95, and preparation method and application thereof |
| CN111574531A (en) * | 2020-05-15 | 2020-08-25 | 福建省中科生物股份有限公司 | Terpene phenolic compound NO85, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111548332A (en) | Terpene phenolic compound NO95, and preparation method and application thereof | |
| CN113105388B (en) | Euphorbia lathyris diterpene alkyl compound and extraction method and application thereof | |
| CN112190577B (en) | Application and preparation method of phenolic compound ZKYY-041 | |
| CN112194572B (en) | Phenolic compound ZKYY-037 and preparation method and application thereof | |
| CN111560001A (en) | Phenolic compound NO84, and preparation method and application thereof | |
| CN112062744A (en) | Terpene phenolic compound ZKYY-057 and preparation method and application thereof | |
| CN111574531A (en) | Terpene phenolic compound NO85, and preparation method and application thereof | |
| CN112807301A (en) | Application of Cannabicol extracted from hemp plant in preparing antitumor drug | |
| CN108358921B (en) | Novel indole alkaloid compound and preparation method and application thereof | |
| CN111253247B (en) | Preparation method and application of novel phenolic acid compound with anti-inflammatory activity | |
| CN109608419B (en) | Diaryl heptane compounds extracted from exocarpium Juglandis Immaturum, and preparation method and application thereof | |
| CN110028535B (en) | Diterpene glycoside compounds in longtube ground ivy herb and extraction and separation method thereof | |
| CN111228246A (en) | Application and preparation method of terpene phenol | |
| CN103191143B (en) | New application of cardiac glycoside compound | |
| CN112920151B (en) | Isopentene-based flavonoid compound and preparation method and application thereof | |
| CN111184710A (en) | Application and preparation method of cyclic phenol | |
| CN109705183B (en) | Clerodendrum cyrtonema metabolite, pharmaceutical composition thereof, preparation method and application thereof | |
| CN105949272A (en) | With a-physalin Y, method for extracting same and application of with a-physalin Y | |
| CN112516134B (en) | Application of hydroxyl-containing compound in preparation of medicines | |
| CN103263409B (en) | Application of bixanthone compound FLBG-1108 or its pharmaceutical salts in preparation of anti-cancer drugs | |
| CN109734696A (en) | A kind of new diepoxy lignan compound and preparation method thereof | |
| CN109575089B (en) | Acylated glucose compounds, pharmaceutical composition, preparation method and application thereof | |
| CN115433152B (en) | Compound separated from golden silk plum fruit, preparation method and application | |
| CN104398532B (en) | Application of cardiac glycoside compound 12beta-hydroxycalotropin | |
| CN102440985A (en) | Application of bixanthone compound FLBG-1108 or its medicinal salt in preparing anticancer medicaments |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200821 |
|
| RJ01 | Rejection of invention patent application after publication |