CN112194572B

CN112194572B - Phenolic compound ZKYY-037 and preparation method and application thereof

Info

Publication number: CN112194572B
Application number: CN202011176024.4A
Authority: CN
Inventors: 占卓; 单世斌; 林志灿; 邹先文; 王佩忠; 张靖
Original assignee: Fujian Sanan Sino Science Photobiotech Co Ltd
Current assignee: Fujian Sanan Sino Science Photobiotech Co Ltd
Priority date: 2020-03-02
Filing date: 2020-10-29
Publication date: 2023-01-10
Anticipated expiration: 2040-10-29
Also published as: CN112194572A; CN111253222A

Abstract

The invention discloses a phenolic compound ZKYY-037, a preparation method and application thereof, wherein the compound has a structure shown in a formula I:

the compound can be obtained by extracting the hemp plant flowers to obtain a crude extract, and then separating the crude extract, and cell experiments show that the compound has better anti-tumor cell proliferation activity, and particularly has obvious effect of inhibiting cell proliferation in liver cancer cells and lung cancer cells.

Description

Phenolic compound ZKYY-037 and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a phenolic compound ZKYY-037 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 a total of 432 compounds isolated from the hemp plant, increasing to 483 in 1995 and 490 in 2005. The biological activity of cannabis is well known and with the discovery of receptors, there is an opportunity to explore physiologically active compounds in cannabis as a source of new therapeutic agents. There is 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 rate of cancer is gradually high due to different living habits, environmental factors and other reasons, the malignant tumor is found to be a disease which is difficult to cure once being found in an advanced stage, and the cancer incidence rate is high and the cancer is difficult to cure at present, so the method has important significance for screening active ingredients in the hemp plants.

Disclosure of Invention

In view of the above, the invention provides an application of a phenolic compound ZKYY-037 with a molecular formula of C ₁₈ H ₂₂ O ₃ The molecular weight is 286.37, and the compound has good anti-tumor cell proliferation activity.

The invention provides a compound ZKYY-037, which has a structure shown in a formula I:

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) Dissolving the obtained crude extract, and separating by normal phase silica gel column chromatography, macroporous adsorbent resin column chromatography, primary medium pressure normal phase silica gel column chromatography, secondary medium pressure normal phase silica gel column chromatography, tertiary medium pressure normal phase silica gel column chromatography, and high pressure reverse phase HPLC chromatography to obtain compound shown in formula I.

Preferably, the method of preparation comprises any one or more of the following features: 1. the supercritical carbon dioxide extraction conditions are as follows: p _{Extraction kettle} =20-30 MPa，T _{Extraction kettle} =35-60℃；P _{Separation kettle I} =8-11 MPa，T _{Separation kettle I} =35-65℃；P _{Separation kettle II} =3-6 MPa，T _{Separation kettle II} =30-40 ℃; 2. the usage amount of ethanol in the ethanol extraction is 15-25% of the weight of the hemp plant inflorescence, and the extraction time is 30-60min; 3. fully dissolving the crude extract by using petroleum ether; 4. the normal phase silica gel column chromatography is carried out isocratic elution by using n-hexane/ethyl acetate =98 as an eluent; 5. the macroporous adsorption resin column chromatography adopts a D101 macroporous adsorption resin column, and gradient elution is carried out by using 30-100% ethanol/water as an eluent; 6. performing gradient elution on the first-stage medium-pressure normal-phase silica gel column chromatography by using dichloromethane/ethyl acetate as an eluent; 7. the second-stage medium-pressure normal-phase silica gel column chromatography is subjected to gradient elution by using n-hexane/diethyl ether as an eluent; 8. gradient elution is carried out on the three-stage medium-pressure normal-phase silica gel column chromatography by taking n-hexane/diethyl ether as an eluent; 9. the high pressure reverse phase HPLC chromatography is carried out with acetonitrile/water solution as eluent for gradient elution.

The invention also provides application of the compound ZKYY-037 shown in the formula I in preparation of 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 medicament comprises an effective dose of the compound shown in the formula I, and the preferable effective dose is 572ng per dose.

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 beneficial effects of the invention are: the invention provides an anti-tumor proliferation activity of a compound ZKYY-037 and a preparation method thereof. The compound can be obtained by extracting the hemp plant flowers to obtain a crude extract and then separating the crude extract, and cell experiments show that the compound has better anti-tumor cell proliferation activity, and particularly has obvious effect of inhibiting cell proliferation in liver cancer cells and lung cancer cells.

Drawings

FIG. 1 is a graph showing the effect of compounds of formula I on the survival rate of human hepatoma cells (HepG 2);

FIG. 2 shows the effect of compounds of formula I on the survival of human lung cancer cells (A549);

FIG. 3 shows the effect of compounds of formula I on the survival of human hepatoma cells (HepG 2);

FIG. 4 shows the effect of compounds of formula I on the survival of human lung cancer cells (A549);

FIG. 5 shows a microscopic image of cell colonies after treatment of human hepatoma cells (HepG 2) with the compound of formula I;

FIG. 6 shows the effect of compounds of formula I on human hepatoma cell (HepG 2) colony formation;

FIG. 7 shows the effect of compounds of formula I on colony formation of human lung cancer cells (A549);

FIG. 8 shows the effect of compounds of formula I on the mobility of human hepatoma cells (HepG 2);

FIG. 9 shows the effect of compounds of formula I on the mobility of human lung cancer cells (A549);

FIG. 10 shows a formula IProcess for preparing compounds ¹ H-NMR spectrum;

FIG. 11 shows compounds of formula I ¹³ C-NMR spectrum.

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 an unreported phenolic compound ZKYY-037, which has a structure shown in a formula I:

formula I.

Example 1

A preparation method of a phenolic compound ZKYY-037 shown in formula I comprises the following steps:

(1) Taking a hemp plant inflorescence as a raw material, crushing 500g of a dried sample, and performing supercritical extraction by using carbon dioxide. The extraction conditions were: p _{Extraction kettle} =30 MPa，T _{Extraction kettle} =45℃；P _{Separation kettle I} =8 MPa，T _{Separation kettle I} =45℃；P _{Separation kettle II} =6 MPa，T _{Separation kettle II} =35 ℃; adding 20 wt% ethanol as carrier, and extracting for 45min. Extracting to obtain 101.031g of crude extract.

(2) Taking 50g of crude extract, fully dissolving the crude extract by using petroleum ether, carrying out normal phase silica gel column chromatography by using a Changzhou tritai medium-pressure rapid preparation chromatograph, carrying out gradient elution by using n-hexane/ethyl acetate =98 and the like until the peak value of the sixth peak detected on line is lower than 100mAU and the flow rate is 80mL/min, analyzing by using a thin layer chromatography, and combining the sixth peaks of the detected on line maps to obtain a first-stage component containing a target compound; and (3) taking 7.8g of the first-stage component, further separating by using D101 macroporous adsorption resin column chromatography, performing gradient elution by using 30-100% ethanol/water in an elution system, and adjusting the flow rate until the eluent flows down in a strand. And (4) carrying out thin-layer chromatography analysis, and combining the elution parts of 45% ethanol solution to obtain a secondary component containing the target compound. And taking 1.62g of the secondary component, and further separating by using medium-pressure normal-phase silica gel column chromatography, wherein an elution system is a dichloromethane/ethyl acetate system for gradient elution, and the elution is carried out until the peak value of the eighth peak detected on line is lower than 50mAU, and the flow rate is 15mL/min. And (3) carrying out thin-layer chromatography analysis, combining the first peaks of the online detection maps to obtain a third-level component containing the target compound, taking 0.732g of the third-level component, further separating by using medium-pressure normal-phase silica gel column chromatography, carrying out gradient elution by using n-hexane/diethyl ether in an elution system until the peak value of the fourteenth peak detected online is lower than 30mAU, and carrying out flow rate of 15mL/min. And (3) carrying out thin-layer chromatography analysis, combining the ninth peak of the online detection spectrum to obtain a fourth-stage component containing the target compound, taking 0.306g of the fourth-stage component, further carrying out chromatography separation by adopting medium-pressure normal-phase silica gel, carrying out gradient elution by using n-hexane/diethyl ether in an elution system until the peak value of the third peak of the online detection is lower than 30mAU, and carrying out flow rate of 15mL/min. Performing thin-layer chromatography, combining the third peaks of the online detection maps to obtain a five-stage component containing the target compound, taking 0.124g of the five-stage component, further performing High Performance Liquid Chromatography (HPLC) high-pressure reversed phase preparation, performing gradient elution by acetonitrile/water and the like in an elution system, wherein the peak emergence time of the target compound is 15.22min, the detection wavelength is 228nm, and finally obtaining 86mg of the target compound;

(3) Analyzing the structure of the target compound, finally determining the hydrogen spectrum and carbon spectrum information of the data through a nuclear magnetic resonance spectrogram (figures 10-11), comprehensively identifying the structure of the compound as shown in formula I by combining the hydrogen spectrum and carbon spectrum information and relevant literature data Isolation and pharmaceutical Evaluation of Minor constraints from High-potential canvas sativa, wherein the nuclear magnetic resonance hydrogen spectrum and carbon spectrum data of the target compound are as follows:

1H NMR (850 MHz, CDCl3)δ7.17 – 7.14 (m, 1H), 7.06 – 7.03 (m, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.45 (s, 1H), 5.32 (brs, 1H), 5.15 (brs, 2H), 2.53 (t, J = 7.7 Hz, 2H), 2.31 (s, 3H), 1.67 – 1.59 (m, 2H), 1.40 – 1.30 (m, 4H), 0.91 (t, J = 6.6 Hz, 3H).

13C NMR (214 MHz, CDCl3)δ 153.99, 152.40, 146.48, 132.06, 131.91, 131.50, 117.19, 116.48, 108.34, 106.99, 35.99, 31.65, 30.77, 22.69, 20.59, 14.17.

example 2

Identification of antitumor activity of phenolic compound ZKYY-037 shown in formula I

Medicine preparation: the compound of formula I, prepared in example 1, ZKYY-037.

(1) MTT colorimetric method for detecting influence of compound shown as formula I on tumor cell survival performance

Taking human liver cancer cells (HepG 2) in a logarithmic growth phase, adjusting the cell concentration to 7000 cells per well by using a DMEM medium, inoculating 100 mu L of the DMEM medium per well into a 96-well plate, culturing the 96-well plate in an incubator until the cells adhere to the wall, respectively preparing compound solutions shown in the formula I with the concentrations of 20ug/ml and 40ug/ml by using 100 mu L of DMSO as a solvent, respectively adding the prepared compounds shown in the formula I with the concentrations of 20ug/ml and 40ug/ml into an experimental group for treatment, adding 100 mu L of DMSO into a blank group, and acting for 24 hours under the culture medium; detecting cell survival rate with MTT reagent by using 20ug/ml antitumor drug cisplatin (DDP) as control, repeating for 3 times, and averaging;

as shown in FIG. 1, the survival rate of the human liver cancer cells (HepG 2) is lower with the increase of the concentration, i.e., the inhibition effect of the compound ZKYY-037 shown in the formula I on the survival performance of the human liver cancer cells (HepG 2) is stronger; when the concentration is 40ug/ml, the inhibition effect is only slightly worse than that of the antitumor drug DPP with the dosage of 20 ug/ml.

Taking human lung cancer cells (A549) in logarithmic growth phase, adjusting the cell concentration to 7000 cells per well by using a DMEM culture medium, inoculating 100 mu L of DMEM culture medium per well to a 96-well plate, culturing in an incubator until the cells adhere to the wall, respectively preparing compound solutions shown in formula I with the concentrations of 20ug/ml and 40ug/ml by using 100 mu L of DMSO as solvents, respectively adding the prepared compounds shown in formula I with the concentrations of 20ug/ml and 40ug/ml into experimental groups for treatment, adding 100 mu L of DMSO into blank groups, and acting for 24 hours under the culture medium; detecting cell survival rate with MTT reagent by using 20ug/ml antitumor drug cisplatin (DDP) as control, repeating for 3 times, and averaging;

as shown in fig. 2, the survival rate of human lung cancer cell (a 549) is lower with the increase of the concentration, i.e., the inhibition effect of compound ZKYY-037 shown in formula I on the survival of human lung cancer cell (a 549) is stronger; when the concentration is 40ug/ml, the inhibition effect is stronger than that of the antineoplastic drug cisplatin (DDP) with the dosage of 20 ug/ml.

(2) CKK8 cell proliferation toxicity detection method for detecting influence of compound shown in formula I on tumor cell survival performance

Taking human liver cancer cells (HepG 2) in a logarithmic growth phase, adjusting the cell concentration to 7000 cells per well by using a DMEM culture medium, inoculating 100 mu L of the DMEM culture medium per well into a 96-well plate in an incubator, culturing in the incubator at 37 ℃ until the cells adhere to the wall, respectively preparing compound solutions shown in the formula I with the concentrations of 20ug/ml and 40ug/ml by using 100 mu L of DMSO as a solvent, respectively adding the prepared compounds shown in the formula I with the concentrations of 20ug/ml and 40ug/ml into experimental groups for treatment, adding 100 mu L of DMSO into blank groups, and acting for 24 hours in the culture medium; detecting cell survival rate with CCK8 kit with cisplatin (DDP) as anti-tumor drug at a dose of 20ug/ml as control, repeating for 3 times, and averaging;

as shown in FIG. 3, the survival rate of the human liver cancer cell (HepG 2) is lower with the increase of the concentration, i.e. the inhibiting effect of the compound shown in formula I on the survival performance of the human liver cancer cell (HepG 2) is stronger; when the concentration is 40ug/ml, the inhibition effect is basically equivalent to that of the antitumor drug cisplatin (DDP) with the dosage of 20 ug/ml.

Taking human lung cancer cells (A549) in logarithmic growth phase, adjusting the cell concentration to 7000 cells per well by using a DMEM culture medium, inoculating 100 mu L of DMEM culture medium per well to a 96-well plate, culturing in an incubator until the cells adhere to the wall, respectively preparing compound solutions shown in formula I with the concentrations of 20ug/ml and 40ug/ml by using 100 mu L of DMSO as solvents, respectively adding the prepared compounds shown in formula I with the concentrations of 20ug/ml and 40ug/ml into experimental groups for treatment, adding 100 mu L of DMSO into blank groups, and acting for 24 hours under the culture medium; detecting cell viability with CCK8 kit with 20ug/ml antitumor drug cisplatin (DDP) as control, repeating for 3 times, and averaging;

as shown in fig. 4, the survival rate of the human lung cancer cell (a 549) is lower with the increase of the concentration, i.e., the compound of formula I has stronger inhibition effect on the survival performance of the human lung cancer cell (a 549); when the concentration is 40ug/ml, the inhibition effect is stronger than that of the antineoplastic drug cisplatin (DDP) with the dosage of 20 ug/ml.

(3) Cell clone colony forming method for detecting influence of compound shown as formula I on proliferation performance of tumor cells

Taking human liver cancer cells (HepG 2) in a logarithmic growth phase, digesting the cells into single cells, inoculating the cells into a 6-well plate, adding 350 cells in each well, adding 3 ML DMEM culture solution, culturing the cells in an incubator until the cells adhere to the wall, respectively preparing compound solutions shown in the formula I with the concentrations of 20ug/ML and 40ug/ML by taking 100 mu L DMSO as a solvent, respectively adding the prepared compounds shown in the formula I with the concentrations of 20ug/ML and 40ug/ML into an experiment group, respectively, treating the experiment group by adding the prepared compounds shown in the formula I with the concentrations of 20ug/ML and 40ug/ML, adding 100 mu L DMSO into a blank group, replacing fresh culture medium and medicaments every 2-3 days, and continuously culturing for one week until macroscopic clones are formed; after each well is treated, a proper amount of 0.1% crystal violet is added for staining, then the staining solution is slowly washed away by running water, the cells are dried in the air, the clone number of the cells is counted, a picture is taken under a microscope (figure 5), and the cell colony forming rate is calculated. As shown in FIG. 6, the higher the concentration, the lower the colony formation rate of human hepatoma cells (HepG 2), i.e., the stronger the inhibitory effect of the compound of formula I on the proliferation of human hepatoma cells (HepG 2).

Taking human lung cancer cells (A549) in a logarithmic growth phase, digesting the cells into single cells, inoculating the single cells to a 6-well plate, adding 350 cells in each well, adding 3 ML DMEM culture solution, culturing the cells to an adherent surface in an incubator, respectively preparing compound solutions shown in formula I with the concentrations of 20ug/ML and 40ug/ML by taking 100 mu L DMSO as a solvent, respectively adding the prepared compounds shown in formula I with the concentrations of 20ug/ML and 40ug/ML into an experimental group, respectively treating the experimental group, adding 100 mu L DMSO into a blank group, replacing fresh culture medium and medicaments every 2-3 days, and continuously culturing for one week until macroscopic clones are formed; adding a proper amount of 0.1% crystal violet into each hole for dyeing, slowly washing off the dyeing solution by running water, drying in the air, counting the clone number of cells, taking a picture under a microscope, and calculating the formation rate of cell colonies. As shown in fig. 7, the lower the colony formation rate of the human lung cancer cell (a 549) with the increase in concentration, i.e., the stronger the inhibitory effect of the compound of formula I on the proliferation performance of the human lung cancer cell (a 549).

(4) Detecting the influence of the compound shown as the formula I on the migration performance of tumor cells

Taking tumor cells in logarithmic growth phase as human liver cancer cells (HepG 2), adjusting the cell concentration to 70000 cells/ML per well by using a DMEM medium, inoculating the DMEM medium with 2 ML per well into a 12-well plate, culturing until the DMEM medium is fully paved at the bottom of the 12-well plate, and drawing a line from top to bottom by using culture wells of a sterile gun head; and (3) adding 100 mu L of culture medium into each experimental group to prepare a DMSO (dimethylsulfoxide) dissolved compound, treating the DMSO dissolved compound with different concentrations of 20ug/ml and 40ug/ml as shown in formula I, adding DMSO with the same volume into blank groups, observing and photographing at different time points within 0-24h, and counting the cell migration condition by calculating the area of a cell-free area in a scratch area.

As shown in FIG. 8, the compound of formula I can inhibit the migration ability of human liver cancer cells (HepG 2), and the tumor cell migration rate decreases with increasing concentration, and shows a descending relationship.

Taking tumor cells in logarithmic growth phase as human lung cancer cells (A549), adjusting the cell concentration to 70000 cells/ML by using a DMEM medium, inoculating 2 ML of DMEM medium in each hole into a 12-hole plate, culturing until the DMEM medium is fully paved at the bottom of the 12-hole plate, and drawing a line from top to bottom by using culture holes of a sterile gun head; and respectively adding 100 mu L of culture medium into experimental groups to prepare a DMSO (dimethyl sulfoxide) dissolved compound, treating the DMSO dissolved compound with different concentrations of 20ug/ml and 40ug/ml as shown in the formula I, adding DMSO with the same volume into blank groups, taking different time points within 0-24h for observation and photographing, and counting the cell migration condition by calculating the area of a cell-free area in a scratched area.

As shown in fig. 9, the compound of formula I can inhibit the migration ability of human lung cancer cell (a 549), and the tumor cell migration rate decreases with increasing concentration, showing a decreasing 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 "include", "including" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or terminal device including a series of elements includes not only those elements but also other elements not explicitly listed or inherent to such process, method, article, or terminal device. Without further limitation, an element defined by the phrases "comprising 8230; \8230;" or "comprising 8230; \8230;" does not exclude additional elements from existing in a process, method, article, or terminal device that comprises the element. Further, in this document, "greater than," "less than," "more than," and the like are understood to not include 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

1. The application of the compound ZKYY-037 shown in the formula I in the preparation of the tumor cell proliferation inhibitor is characterized in that the compound ZKYY-037 has a structure shown in the formula I:

formula I.

2. The use of a compound of formula I according to claim 1 for the preparation of a medicament for the treatment of a tumor disease.

3. The use according to claim 2, wherein the tumor disease is liver cancer or lung cancer.

4. An antitumor drug, which is characterized by comprising an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient comprises a compound shown as a formula I in claim 1.

5. The antitumor agent as claimed in claim 4, wherein the agent contains an effective amount of the compound represented by formula I.

6. The antitumor agent as claimed in claim 5, wherein the effective dose is 572ng per one dose.

7. The antitumor drug as claimed in claim 4, wherein the drug 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.