CN112716947A - Application of gamma-dehydroharmine extracted and separated from peganum harmala in preparing medicine for inhibiting gastric cancer - Google Patents
Application of gamma-dehydroharmine extracted and separated from peganum harmala in preparing medicine for inhibiting gastric cancer Download PDFInfo
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention belongs to the field of natural product chemistry, and particularly relates to application of gamma-dehydroharmine extracted and separated from peganum harmala in preparing a medicament for inhibiting gastric cancer, which is shown by research experiments: the gamma-harmine has high anticancer activity, especially has excellent gastric cancer cell inhibiting effect, is suitable for developing new medicine for inhibiting cancer cell growth, and has wide application foreground.
Description
Technical Field
The invention relates to an application of a natural product in preparing an anti-gastric cancer medicament, and belongs to the technical field of natural product chemistry.
Background
Gastric cancer (gastric carcinoma) is a malignant tumor originated from gastric mucosal epithelium, is one of the most common malignant tumors of the digestive system, and has the first morbidity and the first mortality in various malignant tumors in China; the incidence of gastric cancer is obviously different regionally, and the incidence rate of gastric cancer is obviously higher in northwest and east coastal areas of China than in south areas. The good hair age is more than 50 years old, and the ratio of the incidence rates of men and women is 2: 1. gastric cancer tends to be younger due to changes in dietary structure, increased working pressure, infection with helicobacter pylori, and the like. Gastric cancer can occur in any part of the stomach, more than half of which occur in antrum, and the greater curvature, lesser curvature, anterior and posterior walls of the stomach can be affected. Most of gastric cancers belong to adenocarcinoma, have no obvious symptoms in the early stage, or have nonspecific symptoms such as epigastric discomfort, central heating and the like, are often similar to the symptoms of chronic gastric diseases such as gastritis, gastric ulcer and the like, and are easy to ignore, and the gastric cancers have low early diagnosis rate, so most patients lack obvious clinical symptoms in the early stage, are usually in the middle and late stages of the diseases during diagnosis, and lose the chance of surgical treatment; the gastric cancer can be treated in a targeted manner, although the life cycle of the gastric cancer can be prolonged to a certain extent by chemotherapy, most of the chemotherapy has limited curative effect and short maintenance time, the median life cycle and the 2-year survival rate are very innocent, the targeted therapy can specifically damage cancer cells and relieve the damage of normal cells, but the types and the effects of the traditional gastric cancer targeted therapy medicines are limited. Therefore, the development of drugs for treating gastric cancer, especially targeted therapy, is imminent.
Carboline alkaloids are a large class of alkaloids, and can be classified into alpha, beta, gamma and delta-carbolines according to the position of a pyridine ring nitrogen atom. The beta-carboline alkaloid is the carboline alkaloid which is most widely distributed and most numerous in nature and most deeply researched, has very broad-spectrum biological activity, such as antitumor, antiviral, antifungal, anti-inflammatory, antimalarial, algicidal and the like, and has the most anticancer effect. However, compared with beta-carboline alkaloids, gamma-carboline alkaloids express more significant anticancer activity, and few gamma-carboline alkaloids are reported in natural products.
Disclosure of Invention
The invention aims to provide application of gamma-dehydroharmine extracted and separated from peganum harmala in preparing a medicament for inhibiting gastric cancer, and research experiments show that: the gamma-harmine has high anticancer activity, especially has excellent gastric cancer cell inhibiting effect, is suitable for developing new medicine for inhibiting cancer cell growth, and has wide application foreground.
The invention relates to application of gamma-dehydroharmine extracted and separated from peganum harmala in preparing a medicament for inhibiting gastric cancer.
The invention relates to an application of gamma-harmine extracted and separated from peganum harmala in preparing a medicament for inhibiting gastric cancer, wherein the structural formula of the gamma-harmine is as follows:
compared with the prior art, the invention has the advantages that: compared with the existing compound of harmine with similar structure, the gamma-harmine has more obvious gastric cancer resistant activity.
Drawings
FIG. 1 shows the inhibition of SGC-7901 cell proliferation by harmine, gamma-harmine, in different doses of the invention;
FIG. 2 is a molecular docking diagram of harmine, gamma-harmine and gastric cancer target AKT1 binding molecules of the present invention, wherein A is a molecular docking diagram of harmine and AKT1 binding molecules; b is a gamma-harmine and AKT1 binding molecule butt-joint diagram;
FIG. 3 is a drawing of the binding molecule pair of harmine, gamma-harmine and gastric cancer target EFGR of the present invention, wherein A is the binding molecule pair of harmine and EFGR; b is a gamma-harmine and EFGR binding molecule docking diagram;
FIG. 4 is a drawing of JUN binding molecule docking of harmine, gamma-harmine and gastric cancer target of the present invention, wherein A is a drawing of JUN binding molecule docking of harmine and JUN; b is a gamma-harmine and JUN binding molecule butt-joint diagram;
FIG. 5 is a molecular docking diagram of the combination of harmine, gamma-harmine and MAPK1 as the target of gastric cancer, wherein A is the molecular docking diagram of the combination of harmine and MAPK 1; b is a molecular butt-joint diagram of the combination of gamma-harmine and MAPK 1;
FIG. 6 is a drawing of the binding molecule pair of harmine, gamma-harmine and gastric cancer target TP53, wherein A is the binding molecule pair of harmine and TP 53; b is a molecular butt-joint diagram of the gamma-harmine and TP 53.
Detailed Description
Example 1
Extracting and separating the gamma-harmine:
preparing peganum harmala total alkaloids:
a. adding 5kg of semen pegani harmalae into 8, 6 and 6 times volume of 70% ethanol water solution, extracting for 3 times, each for 1 hr, mixing extractive solutions, concentrating at 60 deg.C, and drying to obtain dry extract, to obtain 1375 g;
b. collecting 500g of the obtained extract, adding 300mL of 10% sulfuric acid solution, dissolving alkaloid in the extract in several times, filtering, mixing the extractive solutions, adding 25% ammonia water solution until pH is 9.5, standing for 24h, filtering, precipitating, and drying to obtain 11.696g of total alkaloid extract;
preparation of sample solution:
weighing 1g of total alkaloid extract in a test tube, adding 5mL of upper phase added with triethylamine and 5mL of lower phase without hydrochloric acid, and ultrasonically oscillating to dissolve the total alkaloid extract for pH zone countercurrent chromatographic separation;
selection of pH-zone counter current chromatography solvent system:
a solvent system of the pH zone countercurrent chromatography is that tert-butyl methyl ether, acetonitrile and water in a volume ratio of 2:2:3 are prepared in a separating funnel and are violently oscillated, the upper phase and the lower phase are separated after the phase separation of the system is balanced, 10mmol/L triethylamine is added to the upper phase as a stationary phase, and 5mmol/L hydrochloric acid is added to the lower phase as a mobile phase;
pH-zone countercurrent chromatography separation:
pumping a stationary phase into a countercurrent chromatography spiral tube at the flow rate of 20mL/min, injecting a mixed solution of an upper phase and a lower phase dissolved with a sample into a chromatographic column through a sample injector after the spiral tube is filled with the stationary phase, starting a speed controller, slowly adjusting the rotating speed of a host to 850r/min for clockwise rotation, pumping a mobile phase at the flow rate of 2.0mL/min, simultaneously starting an ultraviolet detector, detecting a chromatographic peak at 254nm, collecting fractions according to a chromatogram displayed by a recorder, and performing sample injection for 1.0g once to obtain 134mg of gamma-dehydro-harmine;
structural identification of the compounds:
gamma-harmine (gamma-harmine), yellow needle crystals, m.p.238-240 ℃, maximum uv absorption wavelength (methanol λ max):240nm and 323nm. esi-MS (electrospray mass spectrometry), M/z (proton nuclear ratio) 213[ M + H ] +.1H NMR (DMSO-d6,600mhz) δ 12.70(1H, s), 8.40(1H, d, J ═ 6.0Hz), 8.33(1H, d, J ═ 6.6Hz), 8.31(1H, d, J ═ 9.0Hz), 7.13(1H, d, J ═ 1.2Hz), 7.02(1H, d, J ═ 9.0Hz), 3.93(3H, s), 3.02(3H, s), 13C-NMR (DMSO-d6, 1504), δ 162, 1.145, 6.145, 6.55, 113.55, 113, 124.55, 15, 112, 15, 55, 15, 112, 18, 16, g.
Example 2
The CCK-8 method is used for detecting the inhibition effect of gamma-harmine on gastric cancer cell proliferation:
1. cell recovery:
taking out RPMI-1640 culture medium containing 10% FBS (fetal bovine serum) from a refrigerator with the temperature of 4 ℃, placing the RPMI-1640 culture medium in a water bath box with the temperature of 37 ℃ for preheating for 20min, wiping the outer side bottle wall with a 75% alcohol cotton ball after temperature return, and moving the bottle wall into a super clean bench which is irradiated and disinfected by an ultraviolet lamp for later use; taking out a cell cryopreservation tube storing human gastric adenocarcinoma cells SGC-7901, immediately moving the tube into a 37 ℃ water bath box for fast thawing, moving the thawed tube into a centrifuge, centrifuging at 800rpm for 5min, taking out the tube, slightly unscrewing the tube cover, sucking and discarding the upper frozen liquid by using a 1ml pipette gun to avoid sucking up the bottom cells of the tube, adding 1ml of RPMI-1640 complete culture medium, repeatedly blowing and beating until a uniform single cell suspension is formed (the process needs to be gentle), transferring the cell suspension into a 100mm culture dish by using the pipette gun, adding 5-6ml of culture medium, slightly blowing and beating to uniformly disperse the cell suspension, observing the morphology and distribution of the cells under an inverted microscope, standing the cell suspension at 37 ℃ and 5% CO concentration2An incubator with saturated humidity;
2. cell culture:
after cell inoculation, the growth morphology and the adherent condition of the cells are observed under a microscope every other day, and if the number of dead cells is too large, the complete culture medium needs to be replaced. And (4) sucking the old culture medium in the culture dish, transferring an equal amount of fresh complete culture medium, and continuously culturing the cells after liquid replacement in a cell culture box. When adherent cells grow to about 80% of the bottom area of the culture dish, passage or related treatment can be carried out;
3. cell passage
Placing RPMI-1640 fresh culture medium containing 10% FBS, 0.25% trypsin and PBS into a water bath box with the temperature of 37 ℃ for preheating for 20min, wiping the outer side bottle wall with 75% alcohol, then transferring into an ultra-clean bench irradiated and disinfected by an ultraviolet lamp for later use, taking out human gastric adenocarcinoma cells SGC-7901, observing the growth state and the adherence condition of the cells under an inverted microscope, wherein the confluence degree should reach 80%, transferring a culture dish into the ultra-clean bench, lightly washing the cell surface for 3 times by the PBS and removing the cell surface by suction, adding a proper amount of 0.25% pancreatin, slightly shaking, and placing under the inverted microscope for quick observation. Continuously adding a proper amount of fresh culture medium, lightly blowing and beating the bottom of the culture dish by using a liquid transfer gun to enable adherent cells to fall off, then lightly blowing and beating until uniform single cell suspension is formed, placing the culture dish under an inverted microscope for observation, if the bottom of the culture dish is not provided with the adherent cells, uniformly dispersing the cells, passing the cells to a new culture dish according to a required proportion, adding the fresh complete culture medium to the required amount, and placing the culture dish in a cell culture box for culture;
and 4, detecting by a CCK-8 method:
selecting human gastric cancer SGC-7901 cell in logarithmic growth phase, and adjusting the cell suspension concentration to 1 × 105Cells/ml, seeded in 96-well plates, placed in 5% CO2The culture solution is replaced immediately after observing cell adherence under a microscope according to 25g/ml of group A, 50g/ml of group B, 100g/ml of group C, 200g/ml of group D and blank control group E, dehydroharmine and gamma-dehydroharmine are given to group A, 5 multiple holes are simultaneously formed in each group, 50L of CCK-8 solution can be added into each hole after the drug acts for 24 hours, the incubation is carried out for 2-4 hours, the detection is carried out by using an enzyme labeling instrument, and the measured absorbance value (A) of each hole at 450nm is calculated by using a formula: cell growth inhibition rate ═ [ (a control-a dosing)/a control]Calculating the x by 100 percent, the growth inhibition rate of the cells after different drug doses of harmine and gamma-harmine act on the SGC-7901 cells of the human stomach cancer for 24 hours,the results are shown in FIG. 1.
Example 3
Molecular docking analysis of the binding effect of gamma-harmine on gastric cancer-associated targets:
firstly, performing ligand pretreatment on 17 active ingredients (including gamma-harmine) of the national medicinal peganum harmala, finally screening to obtain 14 active ingredients, performing molecular docking on the 14 active ingredients, AKT1, TP53, JUN, MAPK1 and EGFR to realize the interaction of a receptor and a ligand according to an energy principle, and searching an optimal binding mode;
selecting an original ligand to separate from an active pocket and carrying out butt joint, and calculating the value of binding energy (Score) after butt joint, wherein the lower the binding energy (Score), the more stable the conformation of the ligand and the receptor is, and the higher the possibility of the generated interaction is; binding energy (Score) is less than or equal to-4.25 KJ/mol, which indicates that the ligand and the receptor have certain binding activity, and 14 active ingredients are successfully docked with AKT1, TP53, MAPK1 and EGFR;
the gamma-harmine and the harmine compound in the prior art are respectively subjected to molecular docking with potential disease targets such as AKT1, TP53, JUN, MAPK1, EGFR and the like, and the results show that: gamma-harmine forms a stable hydrogen bond with the amino acid residue ALA230 of AKT1, although harmine does not bind to the active site of AKT1 protein, matching the active site, although Score-7.5 of AKT 1; stable hydrogen bonds are formed between the gamma-harmine and an amino acid residue MET793 of the EGFR; although harmine and EGFR Score ═ 7.7, it did not bind to the active site of AKT1 protein and did not match the active site; harmine and gamma-harmine are combined in the active site of the JUN protein and are well matched with the active site, harmine is combined in the active site of the JUN protein, and an aromatic hydrogen bond is formed between carbon of the harmine and GLN 304; and gamma-harmine is combined at the active site of JUN protein, and the carbon of the gamma-harmine forms a stable hydrogen bond with LYS 309; both compounds bind to the active site of the MPAK1 protein and have a better match with the active site; harmine binds to the active site of MPAK1 protein, and the carbon forms stable hydrogen bond with LYS54 and GLN 105; the gamma-harmine is combined at the active site of MPAK1 protein, the carbon of the gamma-harmine forms stable hydrogen bonds with SER153 and LYS54 respectively, and also forms aromatic hydrogen bonds with amino acid residues of ASP; both compounds are combined at the active site of TP53 protein, and have better matching with the active site; the harmine is combined at the active site of TP53 protein, and the carbon of the harmine and ARG10 form Pi-Pi interaction and stable hydrogen bond respectively; gamma-harmine is combined at the active site of TP53 protein, the phenolic hydroxyl of the gamma-harmine and ARG10 stabilize hydrogen bonds, and form Pi-Pi interaction with ASN 17; as can be seen from the molecular docking diagram: the two active components and amino acid residues of a receptor form stable hydrogen bonds, aromatic hydrogen bonds and Pi-Pi interaction, and the stable and aromatic hydrogen bonds and the Pi-Pi interaction play a key role in the recognition and stability of small molecules and proteins as can be seen in a combination mode. The result shows that the gamma-harmine is a compound with high target binding activity for gastric cancer, the gamma-harmine is a rare compound in natural products, and although the molecular weight is consistent with that of the compound harmine in the prior art and the structure is similar, the gamma-harmine can form more stable binding with the gastric cancer target active site to play more remarkable anticancer activity.
Claims (1)
1. An application of gamma-harmine extracted from peganum harmala in preparing the medicines for suppressing gastric cancer is disclosed.
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