CN112079811A - Phenolic compound ZKYY-013 and preparation method and application thereof - Google Patents

Abstract

The invention discloses a phenolic compound ZKYY-013, 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 inflorescence of the hemp plants 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-013 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-013 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. 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 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 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 a novel phenolic compound ZKYY-013 with the molecular formula of C₂₁H₂₆O₃And the molecular weight is 326.18, and the compound has good anti-tumor cell proliferation activity.

The invention provides a compound ZKYY-013, which has a structure shown in a 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 crude extract, and separating by normal phase silica gel column chromatography, medium pressure reversed phase R18 chromatography, and high pressure reversed phase HPLC chromatography to obtain compound shown in formula I.

Preferably, the method of preparation comprises any one or more of the following features: firstly, the supercritical carbon dioxide extraction conditions are as follows: p_{Extraction kettle}＝20-30MPa，T_{Extraction kettle}＝35-60℃；P_{Separation kettle I}＝8-11MPa，T_{Separation kettle I}＝35-65℃；P_{Separation kettle II}＝3-6MPa，T_{Separation kettle II}30-40 ℃; secondly, 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 30-60 min; thirdly, fully dissolving the crude extract by using petroleum ether; fourthly, the normal phase silica gel column chromatography is carried out with n-hexane/ethyl acetate 98:2 as an eluent for isocratic elution; fifthly, carrying out gradient elution on the medium-pressure silica gel column chromatography by using petroleum ether/dichloromethane as an eluent; sixthly, carrying out gradient elution on the medium-pressure reverse-phase R18 chromatogram by using methanol/water solution as an eluent; and seventhly, carrying out isocratic elution on the high-pressure reversed-phase HPLC chromatogram by using acetonitrile/water solution as an eluent.

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 medicament comprises an effective dose of the compound shown in the formula I, and the effective dose is 650ng 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 invention has the beneficial effects that: the invention provides an unreported compound ZKYY-013, wherein a crude extract is obtained by extracting a hemp plant inflorescence, and then the crude extract is separated to obtain the compound, and cell experiments show that the compound ZKYY-013 has good anti-tumor cell proliferation activity, and particularly has an 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 (HepG2) with a compound of formula I;

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

FIG. 7 shows a microscopic image of a cell colony obtained after treatment of human lung cancer cells (A549) with a compound of formula I;

FIG. 8 shows the effect of compounds of formula I on colony formation of human lung cancer cells (A549);

FIG. 9 shows the effect of compounds of formula I on the mobility of human hepatoma cells (HepG 2);

FIG. 10 shows the effect of compounds of formula I on the mobility of human lung cancer cells (A549);

FIG. 11 shows a high resolution mass spectrum (HR-ESI-MS, positive ion scan) of a compound of formula I;

FIG. 12 shows a high resolution mass spectrum (HR-ESI-MS, negative ion scan) of a compound of formula I;

FIG. 13 shows compounds of formula I¹³A C-NMR spectrum;

FIG. 14 shows the formula IProcess for preparing compounds¹H-NMR spectrum;

FIG. 15 shows HSQC spectra of compounds of formula I;

FIG. 16 shows a spectrum of H-HCOSY for a compound of formula I;

FIG. 17 shows an HMBC spectrum of a compound of formula I;

FIG. 18 shows a NOE spectrum of a compound of formula I;

FIG. 19 shows an HPLC chromatogram of a compound of formula I;

FIG. 20 shows a UV spectrum of a compound of formula I.

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 compound ZKYY-013, which has a structure shown in a formula I:

example 1

A preparation method of a compound ZKYY-013 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}＝30MPa，T_{Extraction kettle}＝45℃；P_{Separation kettle I}＝8MPa，T_{Separation kettle I}＝45℃；P_{Separation kettle II}＝6MPa，T_{Separation kettle II}The temperature is 35 ℃; adding 20 wt% ethanol as carrier, and extracting for 45 min. Extracting to obtain 99.845g of crude extract.

(2) And (3) fully dissolving 50g of crude extract by using petroleum ether, performing normal-phase silica gel column chromatography by using a Changzhou tritai medium-pressure rapid preparation chromatograph, performing gradient elution by using n-hexane/ethyl acetate 98:2 and the like as an eluent until the peak value of the fifth peak detected on line is lower than 100mAU, and performing flow rate of 80 mL/min. And (3.8 g of the first-stage component is further separated by using medium-pressure silica gel column chromatography, the elution system is petroleum ether/dichloromethane 0% -100% gradient elution, and the elution is carried out until the peak value of the sixteenth peak of on-line detection is lower than 100mAU, and the flow rate is 25 mL/min. And analyzing the eluted components by thin-layer chromatography, and combining the eleventh peak of part of the online detection spectrum to obtain a secondary component containing the target compound. 0.86g of secondary component is taken and then prepared by medium-pressure reverse-phase R18, the elution system is methanol/water solution gradient elution, and the peak value of the tenth peak is lower than 100mAU when the elution system is used for on-line detection. Analyzing the eluted components by thin-layer chromatography, combining part of second peaks of online detection maps to obtain tertiary components containing the target compound, further preparing the 232mg tertiary components by adopting HPLC high-pressure reversed phase, wherein the elution system is gradient elution of acetonitrile/water and the like, the peak-off time of the target compound is 28.79min, the detection wavelength is 228nm, and 156mg of the target compound is obtained;

(3) the structure of the target compound is analyzed, and finally the compound is comprehensively identified through data information such as mass spectrum, nuclear magnetic resonance spectrogram (figures 11-18) and the like, wherein the structure of the compound is shown as a formula I, and nuclear magnetic resonance hydrogen spectrum and carbon spectrum data of the target compound are as follows:

¹H NMR(850MHz,CDCl₃)7.43(s,1H),7.15–7.10(m,1H),7.08–7.03(m,1H),6.68(s,1H),6.56(s,1H),5.41(s,1H),2.58(t,J＝7.6Hz,2H),2.39(s,3H),1.66–1.61(m,2H),1.60(s,6H),1.37–1.29(m,4H),0.90(t,J＝6.6Hz,3H).

¹³C NMR(214MHz,CDCl₃)152.69,152.16,149.80,144.54,134.77,129.26,128.94,128.82,124.02,116.48,115.98,114.83,112.93,35.82,31.51,30.86,25.10,22.67,21.24,14.18.

(4) the purity of the target compound was analyzed under the specific measurement conditions shown in Table 1.

TABLE 1 XC purity measurement chromatography conditions

As a result: the zk-cs-13 chromatogram obtained shows that the zk-cs-13 purity is 95.46%, and the HPLC chromatogram and the UV spectrogram are shown in FIGS. 19 and 20.

(5) The target compound is prepared into a dilute solution with a certain concentration by using chromatographic pure methanol.

The measurement conditions of UHPLC-MS are shown in Table 2.

TABLE 2 XC UHPLC-MS measurement conditions

And (3) detection results: from the mass spectrum of FIG. 17, the peak of excimer ion of XC is 327.19[ M + H ]]⁺And 325.18[ M-H]^-XC has a molecular weight of 326.18 Da.

Example 2

Identification of antitumor activity of compound ZKYY-013 shown in formula I

Medicine preparation: the compound of formula I, prepared in example 1, ZKYY-013.

(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 a logarithmic growth phase, adjusting the cell concentration to 7000 cells per well by using a DMEM culture medium, inoculating 100 mu L of culture solution per well into a 96-well plate, culturing 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 experimental groups for treatment, adding 100 mu L of DMSO into blank groups, and acting in the incubator for 24 hours; 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 human hepatoma cells (HepG2) is lower with the increase of concentration, i.e. the inhibition effect of the compound ZKYY-013 shown in formula I on the survival performance of human hepatoma cells (HepG2) 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.

Taking human lung cancer cells (A549) in logarithmic growth phase, adjusting the cell concentration to 7000 cells by using a DMEM culture medium, inoculating 100 mu L of culture solution 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 a solvent, 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 in 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 (a549) is lower with the increase of the concentration, i.e. the inhibition effect of compound ZKYY-013 shown in formula I on the survival performance of human lung cancer cell (a549) is stronger; when the concentration is 40ug/ml, the inhibition effect is stronger than that of the antitumor drug DDP (cisplatin) 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 (HepG2) 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 culture solution 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, respectively 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 20ug/ml antitumor drug cisplatin (DDP) as control, repeating for 3 times, and averaging;

as shown in FIG. 3, the survival rate of human liver cancer cells (HepG2) is lower with the increase of concentration, i.e. the inhibiting effect of the compound shown in formula I on the survival of human liver cancer cells (HepG2) is stronger; when the concentration is 40ug/ml, the inhibition effect is equivalent to that of the antitumor drug cisplatin (DDP) with the dose 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 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 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 human lung cancer cell (a549) is lower with the increase of the concentration, i.e., the compound of formula I has stronger inhibition effect on the survival of 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 (HepG2) 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 3ML DMEM culture solution into an incubator, culturing to an adherent state, taking 100 mu L DMSO as a solvent to prepare compound solutions shown in formula I with the concentrations of 20ug/ML and 40ug/ML respectively, adding the prepared compounds shown in formula I with the concentrations of 20ug/ML and 40ug/ML respectively into experimental groups, adding 100 mu L DMSO into blank groups, 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 lower the colony formation rate of human hepatoma cells (HepG2) with increasing concentration, 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 3ML 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; 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 7), and the cell colony forming rate is calculated. As shown in fig. 8, the lower the colony formation rate of human lung cancer cell (a549) with the increase in concentration, i.e., the stronger the inhibitory effect of the compound of formula I on the proliferation of 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 (HepG2), adjusting the cell concentration to 70000 cells/ML by using a DMEM culture medium, inoculating 2ML of the cells into a 12-hole plate, culturing until the cells are 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; preparing compound solutions shown in the formula I with the concentration of 20ug/ml and 40ug/ml by taking 100 mu L of DMSO as a solvent, adding the prepared compounds shown in the formula I with the concentration of 20ug/ml and 40ug/ml into an experimental group for treatment, adding 100 mu L of DMSO into a blank group, 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 liver cancer cells (HepG2), and the tumor cell migration rate decreases with increasing concentration, showing a decreasing 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 culture medium, inoculating 2ML of the culture medium into a 12-hole plate, culturing until the culture 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; preparing compound solutions shown in the formula I with the concentration of 20ug/ml and 40ug/ml by taking 100 mu L of DMSO as a solvent, adding the prepared compounds shown in the formula I with the concentration of 20ug/ml and 40ug/ml into an experimental group for treatment, adding 100 mu L of DMSO into a blank group, 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.

The results are shown in fig. 10, the compound shown in formula I can inhibit the migration ability of human lung cancer cells (a549), 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 "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 (10)

1. A phenolic compound ZKYY-013 has a structure shown in 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) dissolving the crude extract, and separating by normal phase silica gel column chromatography, medium pressure reversed phase R18 chromatography, and high pressure reversed phase HPLC chromatography to obtain compound shown in formula I.

3. The method of claim 2, comprising any one or more of the following features: firstly, the supercritical carbon dioxide extraction conditions are as follows: p_{Extraction kettle}＝20-30MPa，T_{Extraction kettle}＝35-60℃；P_{Separation kettle I}＝8-11MPa，T_{Separation kettle I}＝35-65℃；P_{Separation kettle II}＝3-6MPa，T_{Separation kettle II}30-40 ℃; secondly, 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 30-60 min; thirdly, fully dissolving the crude extract by using petroleum ether; fourthly, the normal phase silica gel column chromatography is carried out with n-hexane/ethyl acetate 98:2 as an eluent for isocratic elution; fifthly, carrying out gradient elution on the medium-pressure silica gel column chromatography by using petroleum ether/dichloromethane as an eluent; sixthly, carrying out gradient elution on the medium-pressure reverse-phase R18 chromatogram by using methanol/water solution as an eluent; and seventhly, carrying out isocratic elution on the high-pressure reversed-phase HPLC chromatogram by using acetonitrile/water solution as an eluent.

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 of claim 6, wherein the neoplastic disease is liver cancer or lung cancer.

9. Medicament according to claim 5 or 7, characterized in that it comprises an effective dose of a compound of formula I, preferably an effective dose of 650ng per dose.

10. 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.

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