阅读说明：本技术 麻类植物提取化合物Cannabiorcol在制备抗肿瘤药物上的用途 (Application of Cannabicol extracted from hemp plant in preparing antitumor drug ) 是由 林志灿 张靖 单世斌 张婷 于 2020-12-30 设计创作，主要内容包括：本发明公开了一种麻类植物提取化合物Cannabiorcol在制备抗肿瘤药物上的新用途,本发明发现化合物Cannabiorcol能在体外抑制肿瘤细胞增殖,通过CKK8细胞增殖毒性实验、细胞划痕实验、细胞克隆集落形成法检测,发现该化合物能显著抑制肝癌细胞HepG2和肺癌细胞A549的增殖、迁移和克隆形成,具备开发抗肿瘤药的潜力。(The invention discloses a new application of a compound Cannabioorcol extracted from a hemp plant in preparing an anti-tumor medicament, and finds that the compound Cannabioorcol can inhibit the proliferation of tumor cells in vitro, and the compound can obviously inhibit the proliferation, migration and clone formation of liver cancer cells HepG2 and lung cancer cells A549 through the detection of a CKK8 cell proliferation toxicity experiment, a cell scratch experiment and a cell clone colony forming method, and has the potential of developing anti-tumor medicaments.)

1. Application of Cannabicol in preparing antitumor drug is provided.

2. The use according to claim 1, wherein the tumor is liver cancer or lung cancer.

3. The use according to claim 1, wherein the antineoplastic drug comprises an active ingredient comprising the hemp extract compound Cannabioorcol and pharmaceutically acceptable adjuvants.

4. The use according to any one of claims 1 to 3, wherein the antitumor agent has an effect of inhibiting tumor cell proliferation.

5. Use according to any one of claims 1 to 3, characterized in that the preparation of the hemp plant extract compound Cannabicol comprises the following steps:

(1) sequentially performing carbon dioxide supercritical extraction on the inflorescences of the hemp plants to obtain crude extract;

(2) fully dissolving the crude extract with petroleum ether, and performing equal gradient elution by normal phase silica gel column chromatography to obtain a first-stage component of a target compound;

(3) taking the first-stage component, and performing gradient elution on the first-stage component by using a D101 macroporous adsorption resin column chromatography to obtain a second-stage component of the target compound;

(4) taking the second-stage component, performing normal-phase silica gel column chromatography, and performing gradient elution to obtain a third-stage component of the target compound;

(5) subjecting the third-stage component to medium-pressure reverse-phase silica gel column chromatography for gradient elution to obtain a target compound fourth-stage component

(6) And (3) performing equal gradient elution on the four-stage component by preparative HPLC high-pressure reversed phase chromatography to obtain the compound Cannabioorcol.

6. The use according to any one of claims 5, wherein in the preparation step, the carbon dioxide supercritical extraction conditions in step (1) are as follows: 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 ethanol with the weight of 20% of the material as a carrying agent.

7. The use according to any one of claim 5, wherein in the preparation step, the normal phase silica gel column chromatography elution system in the step (2) is normal hexane/ethyl acetate 98:2 isocratic elution;

the D101 macroporous adsorption resin column chromatography elution system in the step (3) is gradient washing with 30-100% ethanol/water;

the pressure normal phase silica gel column chromatography elution system in the step (4) is dichloromethane/ethyl acetate; the medium-pressure reverse-phase silica gel column chromatography elution system in the step (5) is methanol/water gradient elution; the preparative HPLC high-pressure reversed-phase chromatographic elution system in the step (6) is gradient elution of 52 percent acetonitrile/water and the like.

Technical Field

The invention belongs to the technical field of natural product chemistry and pharmacology, and particularly relates to application of a hemp plant extract compound Cannabirocol in preparation of an anti-tumor drug.

Background

Extracts of medical hemp plants are often used to assist in the treatment of certain cancers, aids, to stimulate appetite, relieve pain, relieve neurological symptoms such as glaucoma, epilepsy, migraine, treat mood swings, relieve nausea symptoms in chemotherapy patients, and the like.

Hemp plants are chemically complex plants, primarily because they contain many natural chemical components themselves. By 1980, there were 432 species of compounds isolated from hemp plants, an increase to 483 species by 1995 and an increase to 490 species by 2005. According to statistics, the compounds separated from the hemp plants to date have more than 700 types, and are divided into two main types of phytocannabinoids and non-cannabinoids, and 120 types of phytocannabinoids are reported.

The incidence of cancer is gradually high due to different living habits and environmental factors. Many malignant tumors are found in advanced stages and are difficult to cure once found, so the method has great significance for the separation of active ingredients in the cannabis sativa. Advances in the knowledge of the "endocannabinoid" system suggest that drugs based on cannabis may have the potential to treat advanced cancers. At present, a plurality of cannabinoids separated from cannabis are proved to have remarkable inhibitory effects on a plurality of malignant tumors such as lung cancer, liver cancer, breast cancer, pancreatic cancer and the like. Such as: Δ 9-THC, Δ 8-THC, CBD, CBG, CBC, etc. The anti-tumor effects of cannabinoid compounds are generally achieved through 4 pathways: 1. inhibiting tumor growth by directly inhibiting tumor cell proliferation; 2. Cannabinoids can inhibit tumor metastasis; 3. inhibiting tumor growth by promoting apoptosis of tumor cells; 4. the signals which are sent by the tumor cells and promote angiogenesis are closed, and the nutrient source required by the growth of the tumor is cut off.

Disclosure of Invention

In view of the above, the present invention aims to provide a new use of a cannabis plant extract compound Cannabicol in the preparation of anti-tumor drugs. The compound Cannabioorcol has a molecular formula of C₁₇H₁₈O₂Molecular weight is 254.13, and structural formula is shown in formula 1:

the invention provides application of a hemp plant extract compound Cannabicol in preparing an anti-tumor medicament.

In order to better implement the invention, the tumor is liver cancer or lung cancer.

In order to better realize the invention, the anti-tumor medicine comprises an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient comprises a hemp plant extract compound Cannabioorcol.

In order to better realize the invention, the anti-tumor medicament has the function of inhibiting the proliferation of tumor cells.

The method for separating and purifying Cannabicol comprises the following steps:

(1) sequentially performing carbon dioxide supercritical extraction on the inflorescences of the hemp plants to obtain crude extract;

(2) fully dissolving the crude extract with petroleum ether, and performing equal gradient elution by normal phase silica gel column chromatography to obtain a first-stage component of a target compound;

(3) taking the first-stage component, and performing gradient elution on the first-stage component by using a D101 macroporous adsorption resin column chromatography to obtain a second-stage component of the target compound;

(4) taking the second-stage component, performing normal-phase silica gel column chromatography, and performing gradient elution to obtain a third-stage component of the target compound;

(5) subjecting the third fraction to medium-pressure reverse phase silica gel column chromatography for gradient elution to obtain target compound fourth fraction

(6) And (4) performing gradient elution on the four-stage component by preparative HPLC high-pressure reversed phase chromatography to obtain the compound Cannabioorcol.

In order to better implement the invention, in the preparation steps, the carbon dioxide supercritical extraction conditions in the step (1) are as follows: 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 ethanol with the weight of 20% of the material as a carrying agent.

In order to better realize the invention, in the preparation step, the normal phase silica gel column chromatography elution system in the step (2) is gradient elution with n-hexane/ethyl acetate 98:2 and the like; the D101 macroporous adsorption resin column chromatography elution system in the step (3) is gradient washing with 30-100% ethanol/water; the pressure normal phase silica gel column chromatography elution system in the step (4) is dichloromethane/ethyl acetate; the medium-pressure reverse-phase silica gel column chromatography elution system in the step (5) adopts methanol/water gradient elution; and (3) the preparative HPLC high-pressure reversed-phase chromatographic elution system in the step (6) is gradient elution of 52% acetonitrile/water and the like.

The compound Cannabioorcol can inhibit the proliferation of tumor cells in vitro, and CKK8 cell proliferation toxicity experiments show that the compound respectively has inhibition rates of about 40.94%, 87.78% and 81.74% on hepatoma cell HepG2 when the concentration is 20 mug/mL, 40 mug/mL and 60 mug/mL; the inhibition rate of the lung cancer cell A549 is about 48.99%, 84.19% and 85.22%, and the inhibition effect is better than that of cisplatin with the same concentration.

Meanwhile, cell scratch experiment experiments show that the compound acts for 24 hours when the concentration is 5 mug/mL, 10 mug/mL and 20 mug/mL, and the scratch repair rate on the liver cancer cell HepG2 is about 48.08%, 16.48% and 2.27%; the inhibition rate of lung cancer cells A549 is 44.9%, 17.89% and 7.69%; the action time is increased to 48h, and the scratch repair rate of the liver cancer cells HepG2 is about 57.69%, 17.85% and 3.41%; the inhibition rate of the lung cancer cell A549 is about 43.88%, 16.84% and 10.58%.

Meanwhile, cell clone colony forming experiments show that the compound has the clone forming rates of about 38.30%, 2.13% and 1.06% for liver cancer cells HepG2 and about 88.46%, 9.62% and 3.85% for lung cancer cells A549 when the concentrations are 5 mug/mL, 10 mug/mL and 20 mug/mL. The experiments show that the compound has the potential of developing antitumor drugs.

The invention has the beneficial effects that: according to the invention, the compound Cannabioorcol can inhibit the proliferation of tumor cells in vitro, and the compound can obviously inhibit the proliferation, migration and clone formation of liver cancer cells HepG2 and lung cancer cells A549 through the detection of CKK8 cell proliferation toxicity experiments, cell scratch experiments and cell clone colony formation methods, so that the compound has the potential of developing antitumor drugs.

Drawings

FIG. 1 is a graph showing the effect of compounds of formula I on the survival of HepG2 tumor cells;

FIG. 2 is a graph showing the effect of compounds of formula I on the survival of A549 tumor cells;

FIG. 3 shows the effect of compounds of formula I on the migration behavior of HepG2 tumor cells;

FIG. 4 is a graph showing the effect of compounds of formula I on the migratory properties of A549 tumor cells;

FIG. 5 shows the effect of compounds of formula I on HepG2 tumor cell proliferation;

FIG. 6 is a graph of the effect of compounds of formula I on the proliferation of A549 tumor cells;

FIG. 7 shows the half Inhibitory Concentration (IC) of the compound of formula I against HepG2 tumor cells₅₀Value);

FIG. 8 shows the semi-Inhibitory Concentration (IC) of the compound of formula I against A549 tumor cells₅₀Value);

FIG. 9 shows the purity of Cannabioorcol as a monomeric compound by HPLC UV analysis.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto, and various substitutions and modifications can be made without departing from the technical idea of the present invention and the technical idea of the present invention.

Example 1

A method for isolating purified compound canabiorcol comprising the steps of:

(1) sequentially carrying out carbon dioxide supercritical extraction on the hemp inflorescence, wherein the carbon dioxide supercritical extraction conditions are as follows: 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 ethanol with the weight of 20% of the material as a carrying agent to obtain a crude extract;

(2) fully dissolving the crude extract with petroleum ether, performing equal gradient elution by normal phase silica gel column chromatography, wherein the elution system is equal gradient elution of n-hexane/ethyl acetate (98: 2) and the like, and obtaining a first-stage component of a target compound;

(3) taking the first-stage component, and performing gradient elution on the first-stage component by using a D101 macroporous adsorption resin column chromatography, wherein the elution system is ethanol/water 30-100% gradient elution, so as to obtain a second-stage component of the target compound;

(4) taking the second-stage component, performing normal-phase silica gel column chromatography on the second-stage component to perform gradient elution, wherein an elution system is dichloromethane/ethyl acetate to obtain a third-stage component of the target compound;

(5) subjecting the third-stage component to medium-pressure reverse-phase silica gel column chromatography for gradient elution, wherein the elution system is methanol/water gradient elution to obtain the target compound fourth-stage component

(6) And (3) performing gradient elution on the four-stage component by preparative HPLC high-pressure reversed phase chromatography, wherein the elution system is gradient elution of 52% acetonitrile/water and the like to obtain a target compound, and analyzing by mass spectrum, nuclear magnetic resonance and the like to obtain the target compound Cannabioorcol.

The monomeric compound Cannabioorcol prepared in example 1 was analyzed by HPLC for purity by UV as shown in Table 1 below and FIG. 9: high purity, good stability and good biological activity.

TABLE 1

As can be seen from table 1 and fig. 1: the monomeric compound Cannabioorcol prepared by the embodiment method has high purity, good stability and good biological activity.

Example 2

Identification of antitumor Activity of Compound Cannabicol:

CKK8 cell proliferative toxicity assay:

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, each 100 mu L of the culture solution contains about 7000 cells), inoculating the cells into a 96-well plate by using 100 mu L 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 the culture solution is discarded from the 96-well plate, 100 mu L of compound solution shown in formula I with working concentration of 10 mu g/mL, 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 antineoplastic cisplatin (DDP) solution with the same concentration 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. 1, the survival rate of human hepatoma cells (HepG2) was lower as the concentration of the compound of formula I increased, i.e., the compound of formula I was more effective in inhibiting the survival of human hepatoma cells (HepG 2).

The results of the experimental method for the survival performance of the human lung cancer cell (A549) are the same as those of the human liver cancer cell (HepG2), and as shown in FIG. 2, the survival rate of the human lung cancer cell (A549) is lower with the increase of the concentration, i.e., the compound shown in formula I has stronger inhibiting effect on the survival performance of the human lung cancer cell (A549).

2. Tumor cell migration:

the cell scratch test detects the influence of the compound shown in the formula I on the migration performance of tumor cells:

taking human liver cancer cells (HepG2) in logarithmic growth phase, preparing a cell suspension with proper concentration by using a DMEM culture solution, wherein the cell density is about 200000/mL, inoculating the cells into a 24-pore plate by using 1mL of cell suspension per pore, 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. Abandoning the culture solution after the cells adhere to the wall, using a 200 mu L gun head to make a straight scratch along the center of the hole, using PBS to gently wash off cell clusters generated by the scratch, adding 1mL of compound culture solution shown in formula I with working concentrations of 5 mu g/mL, 10 mu g/mL and 20 mu g/mL into the experimental group, adding 1mL of culture solution into the blank control group, taking a picture, recording the scratch width of each hole for 0h, 12h, 24h and 48h, calculating the average value of each hole, finally calculating the average scratch repair rate, repeating the experiment for 3 times, taking the average value, analyzing the difference significance by using T-test between groups, wherein P < 0.05 has statistical significance.

Scratch repair ratio (%) (0 hour scratch area-Nh scratch area)/0 hour scratch area 100

As shown in FIG. 3, the cell duration of the compound of formula I was increased, the scratch repair rate of human hepatoma cells (HepG2) was lower, i.e., the migration of human hepatoma cells (HepG2) was more strongly inhibited by the compound of formula I, as compared to the control group.

The experimental method for the migration performance of the human lung cancer cell (A549) is the same as that of the human liver cancer cell (HepG 2). As shown in fig. 4, the cell time of the compound of formula I acting on the human lung cancer cells (a549) is increased, and the scratch repair rate of the human lung cancer cells (a549) is decreased, i.e., the compound of formula I has a stronger inhibitory effect on the migration of the human lung cancer cells (a549) compared to the control group.

3. Cell clone colony formation method:

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 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 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 the culture solution is discarded from the 6-well plate, 3mL of compound solution shown in the formula I with working concentration of 5 mug/mL, 10 mug/mL and 20 mug/mL is respectively added into the experimental group, 3mL of culture solution is added into the blank control group, the 6-well plate is continuously placed in an incubator, fresh culture solution or culture solution containing medicine is replaced every 2-3 days, the culture is continuously carried out for about two weeks, the cell morphology is continuously observed, and when macroscopic clone appears in a culture dish, the culture is stopped. 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).

The experimental method for proliferation of human lung cancer cell (A549) is the same as that of human liver cancer cell (HepG 2). 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 represented by 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, each 100 mu L of the culture solution contains about 7000 cells), inoculating the cells into a 96-well plate by using 100 mu L 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 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 shown in formula I on human liver cancer cells (HepG2) becomes gradually flat along with the increase of concentration, and the inhibition rate of tumor cells is increased along with the increase of concentration, so that an increasing relationship is presented.

Human Lung cancer cell (A549) half Inhibitory Concentration (IC) at 24 hours₅₀Value) experimental procedure was identical to human hepatoma cells (HepG 2). 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.

Results of antitumor cell proliferation experiments: the survival rate of the tumor cells, the formation rate of the clone colonies and the mobility rate of the tumor cells are used as indexes, the decrease of the survival rate of the tumor cells, the formation rate of the clone colonies and the mobility rate of the tumor cells is found along with the increase of the concentration of the compound shown in the formula 1, the decrease of the survival rate of the tumor cells, the formation rate of the clone colonies and the mobility rate of the tumor cells presents a decreasing relation, and the obvious difference is found from a control group, the proliferation activity of the anti-tumor cells of the compound shown in the formula 1 is gradually increased along with the increase of the concentration, the proliferation of the tumor cells is related to the concentration.

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 that can be used in the present specification and drawings, or used directly or indirectly in other related fields are encompassed by the present invention.