Compound with anti-tumor activity as well as preparation method and application thereof

Abstract

The invention discloses a compound with anti-tumor activity. The compound has a chemical structural formula as shown in a formula (I). The compound belongs to a novel tetracyclic triterpenoid natural product, has good fat solubility and chemical stability, and can be conveniently extracted from the rhizome of a herbal medicine Polygala fallax Hemsl. And prepared into an oral or injection form. The invention also provides a preparation method and application of the compound. The compound provided by the invention can obviously inhibit the growth of mouse lung cancer and reduce the volume and weight of tumor, and can be used for treating tumor diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to a new medicinal tetracyclic triterpenoid natural product, and more specifically, to a compound with anti-tumor activity and a preparation method and application thereof. Background Art

[0002] Cancer poses a serious threat to human life and health, with its morbidity and mortality rates continuing to rise worldwide. Currently, the main treatments for cancer include surgery, radiotherapy, chemotherapy, and immunotherapy. Although significant progress has been made in anti-tumor biotechnology in recent years, chemotherapy remains one of the most commonly used basic clinical approaches. It is indicated for patients with chemotherapy-sensitive systemic malignancies, such as leukemia, multiple myeloma, and intermediate- and high-grade malignant lymphomas; patients with advanced tumors who are no longer suitable for surgery or radiotherapy, or patients with recurrence or metastasis after surgery or radiotherapy; as preoperative preparation and postoperative consolidation therapy for solid tumors; and before radiotherapy.

[0003] Chemotherapy primarily relies on small-molecule drugs, derived from both synthetic and plant-derived sources. These include alkaloids, platinum compounds, targeted kinase inhibitors, aromatase inhibitors, alkylating agents, and taxanes. These drugs range from non-specific, cycle-killing drugs that target cells throughout the entire proliferation cycle to cycle-specific drugs that target cells only during a specific phase. Combination therapy often yields improved efficacy. Chemotherapy, with its wide applicability and low cost, will undoubtedly remain the primary anti-tumor treatment of the future.

[0004] However, while existing chemotherapy drugs are recognized for their tumor-suppressing effects and are a commonly used and effective treatment, they also have numerous drawbacks. They have widespread and severe side effects, face drug resistance, and have poor selectivity for tumor cells versus normal cells. While inhibiting or killing tumor cells, they can damage normal cells, affect heart, liver, kidney, and nervous system function, and be toxic to the human body.

[0005] To address these issues, many researchers are committed to exploring new anti-tumor structures, particularly looking for active ingredients from the plant kingdom to develop anti-tumor drugs. For example, piperine, which has multiple pharmacological effects, will begin Phase II clinical trials in the United States in 2021 for the treatment of multiple myeloma, prostate cancer, and other diseases. Another example is Paris polyphylla steroidal saponins, the main active ingredient found in Paris polyphylla, which have a certain inhibitory effect on pancreatic cancer cells, but the effect needs to be improved. The search for new anti-tumor compounds with low toxicity, high efficiency, strong specificity, and good pharmacokinetic properties is urgent.

[0006] Begonia limprichtii Irmscher is a perennial herbaceous plant in the Begoniaceae family, a popular herbal remedy in southwestern my country. It is used to treat traumatic injuries, rheumatic pain, and sore throat. It has a relatively good safety profile and no significant toxic side effects. However, the chemical composition and antitumor activity of this herbal remedy have not yet been reported. Summary of the Invention

[0007] The present invention aims to solve the above technical problems and provide a small molecule compound with novel structure and good anti-tumor activity as a new choice for anti-tumor drug treatment.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a compound having anti-tumor activity, which has a chemical structure as shown in formula (I):

[0010]

[0011] The compound, named cucurbitacin, contains 30 carbon atoms in its parent nucleus and is a typical tetracyclic triterpene compound. Its structural characteristic is that the anterior methyl group (19-CH3) is attached to C-9, rather than C-10, as is common in triterpenes. It also belongs to the cucurbitacin class. However, it lacks the hydroxyl group at C-16, as is common in cucurbitacins, making it a rare 16-dehydroxycucurbitacin with a novel structure that has not been reported before. The cucurbitacin monomer is a white powder that is poorly soluble in water but readily soluble in organic solvents such as methanol, ethanol, ethyl acetate, and chloroform. It has a bitter taste. Stability studies have shown that the product maintains its content and properties unchanged after 12 months at room temperature. Furthermore, under accelerated test conditions of 40°C and 75% relative humidity for 6 months, the product's physical and chemical properties show no significant changes. This indicates that its chemical properties are very stable, providing convenient conditions for the production, transportation, and storage of the preparation.

[0012] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound of the present invention.

[0013] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0014] In a third aspect, the present invention provides a method for preparing the compound of the present invention, comprising the following steps:

[0015] a. Take the dried rhizome of water lotus, crush it, extract it with an organic solvent, filter it, and concentrate it to obtain a fluid extract;

[0016] b. Add the fluid extract to water, stir, filter, wash with water, and dry to obtain a precipitate;

[0017] c. The precipitate was separated by silica gel column chromatography and eluted with a petroleum ether - ethyl acetate gradient;

[0018] d. Detect column chromatography fractions, combine, and concentrate to obtain the compound.

[0019] Preferably, the organic solvent in step a can be any one of organic solvents such as methanol, ethanol, ethyl acetate, acetone, chloroform, petroleum ether, or a mixed solvent thereof, such as a mixed solvent of petroleum ether and ethyl acetate (volume ratio 2:1), preferably methanol or ethanol of lower alcohols, more preferably medicinal alcohol, i.e., aqueous ethanol (such as aqueous ethanol with a volume fraction of 75%-85%), to comply with relevant regulations on pharmaceutical production, cost, safety, and environmental protection requirements. The extraction method can be percolation, reflux, maceration, microwave or supercritical extraction, preferably reflux.

[0020] Preferably, the weight-to-volume ratio of water hyacinth to the organic solvent is 1 g:3-5 mL, preferably 1 g:4 mL.

[0021] Preferably, step b is a water precipitation process, and the amount of water added is 5 to 15 times the weight of the extract, preferably 10 times, and stirred at room temperature or under heating to fully dissolve and suspend the paste, then cooled, allowed to stand, filtered, and the precipitate washed with clean water in small amounts multiple times.

[0022] Preferably, the column chromatography in step c can be repeated until the target product with the desired purity is obtained.

[0023] Preferably, in the step d, the column chromatography fraction is detected by thin layer chromatography or liquid chromatography.

[0024] More preferably, the thin layer chromatography is conventional silica gel thin layer, the developing solvent is petroleum ether-ethyl acetate (3:1) or chloroform-methanol (98:2); and the color development method is iodine fumigation.

[0025] More preferably, the liquid chromatography method uses a conventional C18 reverse phase silica gel chromatography column; the mobile phase is methanol-0.1% formic acid (50:50 to 100:0) gradient elution; and the detection wavelength is 237 nm.

[0026] The preparation method of the present invention has a short extraction route, simple operation, regenerable column chromatography filler, low cost, and feasibility of scaled-up production.

[0027] In a fourth aspect, the present invention provides use of the compound or pharmaceutical composition of the present invention in the preparation of anti-tumor drugs.

[0028] Preferably, the tumor includes but is not limited to lung cancer, breast cancer, liver cancer, colon cancer, rectal cancer, gastric cancer, etc. More preferably, the lung cancer is squamous cell lung cancer or non-small cell lung cancer.

[0029] Anti-tumor tests showed that the compounds of the present invention have very significant inhibitory activity against human tumor strains such as lung cancer, liver cancer, gastric cancer, breast cancer, and colon cancer in vitro. 50 0.8251~38.63μg / mL; further animal experiments showed that oral administration of 40mg.kg -1 .d -1 Nine days of treatment significantly inhibited lung cancer growth in LLC-transplanted mice, reducing tumor volume and weight. This indicates that salvia miltiorrhiza uralensis has significant anti-tumor effects in both in vitro and in vivo models, with a broad spectrum of activity against five common solid tumors: lung, breast, liver, stomach, and colon. This suggests that it may also have inhibitory effects on more tumor types.

[0030] Preferably, the compound is prepared into a pharmaceutically acceptable dosage form in the form of a monomer or a plant extract containing the compound.

[0031] Preferably, the dosage form comprises a pharmaceutically acceptable carrier and / or excipient.

[0032] The monomer of salvia miltiorrhiza or the plant extract containing salvia miltiorrhiza can be made into a variety of pharmaceutically permitted dosage forms to meet the diverse needs of clinical medication. These dosage forms include but are not limited to tablets, hard capsules, soft capsules, granules, pills, ointments and injections. The pharmaceutically acceptable carriers, excipients or additives required for the preparations, specifically diluents and absorbents commonly used in pharmacy, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, calcium hydrogen phosphate, light magnesium oxide, etc.; wetting agents and adhesives commonly used in pharmacy, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants commonly used in pharmacy, such as dry starch, alginate, agar powder, brown algae starch, carbon Sodium bicarbonate, citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc.; pharmaceutically commonly used cosolvents, such as ethanol, propylene glycol, polyethylene glycol, polysorbate 80, lecithin, urea, sodium benzoate, sodium salicylate, niacinamide, cyclodextrin, soybean oil, etc.; pharmaceutically acceptable carriers or excipients are not listed one by one, and those skilled in the art can make specific selections based on the common knowledge they possess.

[0033] Compared with the prior art, the present invention has the following benefits:

[0034] (1) Polypogonin is a new small molecule with a completely different structural type from existing drugs, and may contain different targets and mechanisms of action;

[0035] (2) Polypodium vulgare is highly effective and broad-spectrum in anti-tumor activities, and the daily oral dose for adults is estimated to be less than 100 mg;

[0036] (3) The original plant has a long history of use as a folk herbal medicine and has no obvious toxic side effects, indicating that schizonepeta tenuifolia has good clinical safety;

[0037] (4) The original plant is a herbaceous plant of the genus Begonia (Begonia Linn.), which is similar to common begonia flower varieties in horticulture and can be artificially propagated and planted on a large scale to meet the demand for raw materials for large-scale production;

[0038] (5) The preparation process of scutellaria baicalensis is simple, and high-purity monomers can be obtained at low cost;

[0039] (6) Polypodium vulgare is highly lipophilic, well soluble, and easily absorbed orally;

[0040] (7) The physical and chemical properties of scutellaria baicalensis are stable and it has a long shelf life.

[0041] In conclusion, the scutellariae glutinosin of the present invention has a novel structure, outstanding advantages such as high efficiency, novel mechanism, and stability, and the raw materials are easily available and the cost is low. It is a new anti-tumor drug with great development potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the mass spectrum of euphorbia pulegone.

[0043] Figure 2 This is the H NMR spectrum of euphorbia cerifera.

[0044] Figure 3 This is the carbon NMR spectrum of euphorbia pulegone.

[0045] Figure 4 This is the liquid chromatogram of euphorbia pulegone.

[0046] Figure 5 This is the ultraviolet spectrum of euphorbia pulegone.

[0047] Figure 6 This is a dose-effect curve of salvia miltiorrhiza inhibiting the proliferation of 13 tumor cells.

[0048] Figure 7 The results show that telodipine inhibits the growth of lung cancer tumors in mice. (A) Tumor growth in mice; (B) Comparison of tumor size; (C) Comparison of tumor volume; (D) Comparison of tumor weight. DETAILED DESCRIPTION

[0049] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and specific examples. Unless otherwise specified, the instruments or reagents used in the examples are conventional instruments or reagents in the art and are conventional products available on the market. Unless otherwise specified, the specific experimental procedures involved in the text are understood or known to those of ordinary skill in the art based on their common knowledge or conventional technical means, and will not be described in detail here.

[0050] Example 1: Extraction, separation and structure determination of euphorbia pulegone

[0051] 1. Example 1 of extraction and separation of euphorbia pulegonemin:

[0052] (1) 300 g of dried rhizomes of Begonia limprichtii Irmscher (purchased from the herbal medicine market in Leshan, Sichuan Province) were ground into coarse powder and placed in a round-bottom flask. 1.2 L of methanol was added and refluxed for 1 hour. The mixture was cooled and filtered. The residue was extracted twice more using the same method. The three extracts were combined and concentrated under reduced pressure to obtain 65 g of extract.

[0053] (2) Pour the extract into 650 mL of water, heat in a water bath at 80°C, and stir rapidly to fully dissolve, disperse, and suspend the paste. Then cool it, let it stand for 1 hour, filter it, wash the filter cake with a small amount of water several times, and dry it to obtain 33 g of precipitated powder.

[0054] (3) Take 20 g of the precipitated powder, dissolve it in 60 mL of methanol, mix it with silica gel, dry it, and load it into a silica gel column (silica gel specification 200-300 mesh). Use petroleum ether-ethyl acetate (10:0-0:10) gradient elution to receive 80 fractions (Fr1-80). Use silica gel thin layer to examine each fraction, develop it with petroleum ether-ethyl acetate (volume ratio 3:1), and color it with iodine fumigation. The results show that Fr15-Fr18 contain the same single main spot. Combine and concentrate to obtain 1.8 g of white powder (PSY-2).

[0055] The purity was determined by liquid chromatography. The chromatographic conditions were: ODS column (150×4.6, 5 μm), gradient elution of methanol-0.1% formic acid (50:50-100:0), detection wavelength of 237 nm, and flow rate of 1 mL / min.

[0056] The results are as follows Figure 4 As shown, the chromatogram showed a single major peak with a purity of >98%.

[0057] 2. Structure determination:

[0058] The monomer of euphorbia cerifera is a white powder that is insoluble in water but easily soluble in organic solvents such as methanol, ethanol, ethyl acetate, and chloroform, and has a bitter taste. Stability studies have shown that the content of this product does not decrease and its properties do not change after being stored at room temperature for 12 months. When stored under accelerated test conditions of a temperature of 40°C and a relative humidity of 75% for 6 months, no significant changes are observed in the physical and chemical properties of this product. The chemical properties are stable.

[0059] UVλmax:237、266(sh)nm( Figure 5 );ESI-TOF-MSm / z:541.3140[M+H] + ,558.3440[M+NH4] + ,563.3001[M+Na] + (Calculated value C 32 H 44 O7,540.308704)( Figure 1 ).

[0060] 1 H-NMR (400MHz, CDCl3) δppm: 1.03(3H,s,H-18), 1.09(3H,s,H-19), 1.15(3H,s, H-29),1.25(3H,s,H-28),1.36(3H,s,H-30),1.44(3H,s,H-21),1.54(3H,s,H- 26),1.58(3H,s,H-27),2.03(3H,s,Ac),5.76(1H,m,H-6),5.94(1H,d,J=2.4Hz ,H-1),6.41(1H,d,J=15.6Hz,H-23),7.12(1H,d,J=15.6Hz,H-24),3.49(1H,br s,H-10),3.13(1H,d,J=14.4Hz,H12b),2.74(1H,d,J=14.4Hz,H12a),2.33(1H ,m,H-7a),2.03(1H,m,H-7b),2.04(1H,m,H-8),0.9~1.7(5H,m,H-15,16,17)( Figure 2 ).

[0061] 13C-NMR (100MHz, CDCl3) δppm: 114.98 (C-1), 144.49 (C-2), 198.76 (C-3), 47.50 (C-4), 136.65 (C-5), 120.94 (C-6), 23.54 ( C-7), 41.56(C-8), 48.85(C-9), 34.77(C-10), 213.54(C-11), 48.63(C-12), 50.32(C-13), 48.30(C-14), 34.08(C-15), 2 0.92(C-16), 49.04(C-17), 18.98(C-18), 19.94(C-19), 78.81(C-20), 23.97(C-21), 201.58(C-22), 118.90(C-23), 153. 33(C-24), 79.07(C-25), 26.73(C-26), 25.94(C-27), 27.98(C-28), 20.19(C-29), 17.58(C-30), 21.89(Ac), 169.78(Ac)( Figure 3 ).

[0062] With the help of HSQC, HMBC and other two-dimensional spectral analysis and reference to relevant literature (Magnetic Resonance In Chemistry 2005; 43: 489-491), the above spectral data were completely assigned, and PSY-2 was identified as a new cucurbitacin, named cucurbitacin.

[0063] The molecular formula of syringaphyllin (codename PSY-2) is C 32 H 44 O7 has a molecular weight of 540 and a molecular structure as shown in the following formula (I).

[0064]

[0065] The systematic name of the lycopodiol is (6R,E)-6-hydroxy-6-((9R,10R,13R,14S,17S)-2-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-4,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-methyl-5-oxohept-3-en-2-yl acetate ((6R,E)-6-hydroxy-6-( (9R,10R,13R,14S,17S)-2-hydroxy-4,4,9,13,14-pentamethyl-3,11-dioxo-4,7,8,9,10,11,12,13, 14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-methyl-5-oxohept-3-en-2-yl acetate).

[0066] 3. Example 2 of extraction and separation of euphorbia pulegonemin:

[0067] (1) Take 100 g of dried water lotus, grind it into coarse powder, put it into a round-bottom flask, add 500 mL of 80% v / v ethanol, reflux and extract for 1 hour, let it cool, and filter; extract the residue twice more in the same way; combine the three extracts and concentrate under reduced pressure to obtain 22 g of extract.

[0068] (2) Pour the extract into 330 mL of water, heat in a water bath at 80°C, and stir rapidly to fully dissolve, disperse, and suspend the paste. Then cool it, let it stand for 1 hour, filter it, wash the filter cake with a small amount of water several times, and dry it to obtain 13 g of precipitated powder.

[0069] (3) Take 10 g of the precipitated powder, dissolve it in 30 mL of methanol, mix it with silica gel, dry it, and load it into a silica gel column (silica gel specification 200-300 mesh), and use petroleum ether-ethyl acetate (10:0-0:10) gradient elution; examine each fraction with silica gel thin layer, develop it with petroleum ether-ethyl acetate (3:1), color it with iodine fumigation, guide it to be combined, and concentrate it to obtain 1.2 g of light yellow powder.

[0070] (4) The powder was dissolved in 5 ml of methanol, mixed with silica gel, dried, and loaded into a silica gel column (silica gel specification 200-300 mesh), and eluted with a gradient of petroleum ether-ethyl acetate (10:0-0:10); combined with silica gel thin layer guidance, concentrated to obtain 0.7 g of white powder.

[0071] 4. Example 3 of extraction and separation of euphorbia pulegonemin:

[0072] (1) Take 100 g of dried water lotus, grind it into coarse powder, put it into a round-bottom flask, add 300 mL of ethyl acetate, reflux and extract for 1 hour, let it cool, and filter; extract the residue twice in the same way; combine the three extracts and concentrate under reduced pressure to obtain 13 g of extract.

[0073] (2) Pour the extract into 65 mL of water, heat in a water bath at 80°C, and stir rapidly to fully dissolve, disperse, and suspend the paste. Then cool it, let it stand for 1 hour, filter it, wash the filter cake with a small amount of water several times, and dry it to obtain 11 g of precipitated powder.

[0074] (3) Take 10 g of the precipitated powder, dissolve it in 30 mL of methanol, mix it with silica gel, dry it, and load it into a silica gel column (silica gel specification 200-300 mesh), and use petroleum ether-ethyl acetate (10:0-0:10) gradient elution; examine each fraction with silica gel thin layer, develop it with petroleum ether-ethyl acetate (volume ratio 3:1), color it with iodine fumigation, combine it, and concentrate it to obtain 0.9 g of white powder.

[0075] 5. Example 4 of extraction and separation of euphorbia pulegonemin:

[0076] (1) Take 100 g of dried water lotus, grind it into coarse powder, put it into a round-bottom flask, add 350 mL of petroleum ether-ethyl acetate (volume ratio 2:1), reflux and extract for 1 hour, let it cool, and filter; extract the residue twice in the same way; combine the three extracts and concentrate under reduced pressure to obtain 12 g of extract.

[0077] (2) Pour the extract into 90 mL of water, heat in a water bath at 80°C, and stir rapidly to fully dissolve, disperse, and suspend the paste. Then cool it, let it stand for 1 hour, filter it, wash the filter cake with a small amount of water several times, and dry it to obtain 11 g of precipitated powder.

[0078] (3) Take 10 g of the precipitated powder, dissolve it in 30 mL of methanol, mix it with silica gel, dry it, and load it into a silica gel column (silica gel specification 200-300 mesh), and use petroleum ether-ethyl acetate (10:0-0:10) gradient elution; examine each fraction with silica gel thin layer, develop it with petroleum ether-ethyl acetate (volume ratio 3:1), color it with iodine fumigation, combine it, and concentrate it to obtain 1.0 g of white powder.

[0079] Chemical structure identification showed that the product obtained by the above extraction and separation examples 2-4 was identical to syringin (PSY-2).

[0080] Example 2: Inhibitory effect of PSY-2 on tumor cells

[0081] 1. Experimental reagents, materials, and instruments:

[0082] All cells are commercial cells, and the culture conditions are as follows:

[0083] NCI-H226 (human non-small cell lung cancer, squamous cell lung carcinoma, ATCC CRL-5826): RPMI-1640 medium + 10% fetal bovine serum + 1% PS;

[0084] HCC827 (human non-small cell lung cancer cells, ATCC CRL-2868): RPMI-1640 medium + 10% fetal bovine serum + 1% PS;

[0085] HepG2 (human hepatocellular carcinoma cell line, ATCC HB-8065): MEM (containing NEAA) medium + 10% fetal bovine serum + 1% PS;

[0086] Hep3B (human hepatocellular carcinoma, ATCC HB-8064): MEM (containing NEAA) medium + 10% fetal bovine serum + 1% PS;

[0087] HCT116 (human colorectal cancer cells, ATCC CCL-247EMT): McCoy's 5A medium + 10% fetal bovine serum + 1% PS;

[0088] HT29 (human colorectal cancer cells, ATCCHTB-38): McCoy's 5A medium + 10% FBS + 1% PS;

[0089] MKN-45 (human poorly differentiated gastric adenocarcinoma cell line, Wuhan Punosai Biotechnology, CL-0292): RPMI-1640 medium + 10% fetal bovine serum + 1% PS;

[0090] BGC-823 (human gastric adenocarcinoma cells, Shanghai Guandao Bioengineering Co., Ltd., C1953): RPMI1640 medium + 10% fetal bovine serum + 1% PS;

[0091] MCF-7 (human breast cancer cells, ATCC CRL-3435): DMEM medium + 10% fetal bovine serum + 1% PS;

[0092] MDA-MB-231 (human breast cancer cells, ATCC CRM-HTB-26): DMEM medium + 10% fetal bovine serum + 1% PS;

[0093] H1975 (human non-small cell lung cancer cells, ATCC CRL-5908): F12 / DMEM medium + 10% fetal bovine serum + 1% PS;

[0094] A549 (human lung adenocarcinoma epithelial cells, ATCC CRM-CCL-185): F12 / DMEM medium + 10% fetal bovine serum + 1% PS;

[0095] H1299 (human non-small cell lung cancer cells, ATCC CRL-5803): F12 / DMEM medium + 10% fetal bovine serum + 1% PS.

[0096] Reagents: Cell culture medium (GIBCO); fetal bovine serum (GIBCO); trypsin-EDTA (GIBCO); CCK8 kit (Meilun, MA0218); penicillin-streptomycin (PS, Sigma-Aldrich)

[0097] Instruments: CO2 incubator (Thermo Fisher), full-wavelength microplate reader (Epoch), desktop high-speed centrifuge (Epend, 5804R); ultra-clean bench (Lujing purification, SW-CJ-2D); inverted microscope (Olympus)

[0098] 2. Experimental methods:

[0099] 2.1 Cell recovery

[0100] (1) Take out the cryovial from the liquid nitrogen tank and place it directly into 37°C warm water, shaking it from time to time to melt it as quickly as possible.

[0101] (2) Remove the cryovial from the 37°C water bath, disinfect with 75% v / v alcohol, and wipe dry. Open the lid, aspirate the cell suspension with a pipette, add it to a centrifuge tube, add 5 times more complete culture medium dropwise, and mix thoroughly.

[0102] (3) Centrifuge at 1000 rpm for 5 minutes.

[0103] (4) Discard the supernatant, resuspend the cell pellet at the bottom of the centrifuge tube, add 5-6 mL of complete culture medium to a T25 culture flask, count the cells, adjust the cell density, inoculate the culture flask, and culture it in a 37°C incubator.

[0104] (5) Replace the complete culture medium the next day and continue culturing.

[0105] 2.2 Cell passaging

[0106] (1) Cells can be passaged when the confluence reaches 90% in the incubator.

[0107] (2) For each T25 culture flask, aspirate the complete medium and gently rinse once with PBS buffer. Add 0.5 mL of 0.25% trypsin containing EDTA and digest for 2 min (the specific time can be adjusted appropriately depending on the cells). Then, rinse the cells with 1 mL of complete medium. Centrifuge at 1000 rpm for 5 min, discard the supernatant, collect the cells, and plate them into two T25 culture flasks for culture. Add 5 mL of complete medium to each flask.

[0108] 2.3 Cell counting analysis

[0109] (1) After digestion, resuspend the cells thoroughly and disperse them into dispersed single cells. Transfer 1 mL of the solution into a clean EP tube.

[0110] (2) Use an alcohol cotton ball to gently wipe the surface of the counting plate and the cover glass. Slightly moisten the edge of the cover glass and then carefully cover the center of the counting plate. The edge should cover the groove on the counting plate.

[0111] (3) Repeat the pipetting to resuspend the cell suspension just transferred to the EP tube.

[0112] (4) Pipette 20 μL of the above cell suspension, place the pipette tip against the upper or lower edge of the coverslip, and then slowly dispense the liquid. At this time, the liquid will be sucked into the gap between the coverslip and the counting plate due to the siphon effect.

[0113] (5) Adjust the microscope and use a 10x objective lens for observation.

[0114] (6) Using the cell counting method, record the number of viable cells that are not stained with trypan blue.

[0115] (7) After calculating the total number, calculate according to the following formula to obtain the cell concentration.

[0116] Cell concentration = (total number of cells in the four large squares / 4) × 10,000.

[0117] (8) Further calculation was performed to obtain the total number of cells: total number of cells = volume of culture medium used to resuspend cells × cell density.

[0118] 2.4 Cytotoxicity (CCK8) assay

[0119] Experimental steps:

[0120] (1) Digest the cells and count them, adjusting the cell concentration to 5×10 4 The cell suspension was added to a 96-well plate at a volume of 100 μL per well.

[0121] (2) After the cells have been cultured overnight in the well plate, the culture medium is discarded and drugs of corresponding concentrations are added. After treatment with 100 μL of drug-containing culture medium per well for 24 hours, the culture medium is discarded and CCK8 working solution (prepared at a volume ratio of CCK8 solution to culture medium of 1:10) is added.

[0122] (3) After incubation in a cell culture incubator for 3 hours, the absorbance of the CCK8 working solution at 450 nm was measured.

[0123] 2.5 half-maximal inhibitory concentration (IC 50 ) Calculation: IC was calculated using GraphPad 8.0 50 value

[0124] 3. Experimental Results

[0125] The CCK8 assay was used to examine the effects of PSY-2 on the proliferation of 13 cancer cell lines. The results are shown in Tables 1 and Figure 6 , PSY-2 IC 50 The values ​​ranged from 0.8251 to 38.63 μg / mL, and the dose-effect relationship was good.

[0126] Table 1. Effects of compounds on tumor cell proliferation activity

[0127]

[0128] The results showed that compound PSY-2 has significant inhibitory activity on the proliferation of colon cancer, liver cancer, gastric cancer, breast cancer, and lung cancer cells, and has broad-spectrum anti-tumor potential.

[0129] Example 3: The therapeutic effect of PSY-2 on lung cancer

[0130] 1. Experimental reagents, materials, and instruments:

[0131] Cells: LLC mouse lung cancer cells (F12 / DMEM+10% FBS) were obtained from ATCC (ATCC, CRL-1642), and C56BL / 6 mice were obtained from Guangdong Provincial Laboratory Animal Center.

[0132] Materials and instruments: Same as Example 2

[0133] 2. Experimental Methods

[0134] C56BL / 6 mice were randomly divided into two groups (5 mice in each group): LLC model group and drug treatment group (LLC+PSY-2). LLC model group was established by subcutaneously injecting LLC cells (3.96×10 6 The weight changes of mice (once a day) and tumor volume changes (once every two days) were recorded. Drug treatment group: C56BL / 6 mice were inoculated with LLC cells and the tumor volume was observed to grow to 100 mm 3 , mice were treated with PSY-2 (40 mg / kg) by oral gavage every day.

[0135] 3. Experimental Results

[0136] like Figure 7 As shown, the PSY-2 treatment group significantly reduced the tumor volume ( Figure 7 , AC, P<0.0001) and tumor weight ( Figure 7 , D, P < 0.05).

[0137] Example 4: Preparation of scutellariae tablets

[0138] Take 50g of euphorbia pulex and a mixture of 1900g of lactose and starch (the weight ratio of lactose and starch is 3:2, filler) and place them in a mixing granulator. First, mix them at a low speed (about 100-200r / min) for 5-10 minutes to evenly disperse the active ingredient in the filler. Then, add 50mL of 5% mass concentration of hydroxypropyl methylcellulose (binder) aqueous solution, continue mixing at a medium speed (about 300-400r / min) for 10-15 minutes to make a soft material, granulate through an 80-mesh screen, dry to control the moisture content to 2%-5%, pass through a 100-mesh screen to granulate, and then fully mix with 40g of sodium carboxymethyl starch (disintegrant) and 10g of magnesium stearate (lubricant). Place the mixture in the hopper of a tablet press and press into 10,000 tablets, each containing 5mg of euphorbia pulex.

[0139] Example 5: Preparation of scutellaria baicalensis capsules

[0140] 50g of schisandrae alkaloids was placed in a mixer with 1800g of a mixture of microcrystalline cellulose and lactose (microcrystalline cellulose and lactose, weight ratio 2:1, filler) and mixed at 25 rpm for 10 minutes. 50mL of a 5% aqueous solution of hydroxypropyl methylcellulose (binder) was added and mixed for 15 minutes to form a soft material. The material was granulated through an 80-mesh sieve, dried to control the moisture content to 2%-5%, and granulated through a 100-mesh sieve. The material was then mixed with 50g of crospovidone (disintegrant) and 10g of magnesium stearate (lubricant) in a mixer and mixed at 20 rpm for 20 minutes. The mixture was then filled into capsules using a capsule filling machine to produce 10,000 capsules, each containing 5mg of schisandrae alkaloids.

[0141] Example 6: Preparation of scutellaria baicalensis injection

[0142] Place 50 g of nephrodiol in a clean container, add 5 L of PEG-400, stir to fully dissolve, dilute to 50 L with water for injection, filter through a 0.22 μm microporous membrane, and aseptically dispense the filtrate into 10,000 5 mL ampoules. Sterilize the sealed ampoules using moist heat sterilization at 121°C for 20 minutes. Each 5 mL ampoules contains 5 mg of nephrodiol.

[0143] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Therefore, any simple modifications, equivalent changes, and modifications to the above embodiments made in accordance with the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A compound having anti-tumor activity, characterized in that: It has the chemical structure shown in formula (I):

2. A pharmaceutical composition, characterized in that The invention comprises the compound according to claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

4. A method for preparing the compound according to claim 1, characterized in that: The following steps are involved: a. Take the dried rhizome of water lotus, crush it, extract it with an organic solvent, filter it, and concentrate it to obtain a fluid extract; b. Add the fluid extract to water, stir, filter, wash with water, and dry to obtain a precipitate; c. The precipitate was separated by silica gel column chromatography and eluted with a gradient of petroleum ether-ethyl acetate 10:0 to 0:10; d. Detect column chromatography fractions, combine, and concentrate to obtain the compound.

5. The method according to claim 4, characterized in that The organic solvent is any one or more of methanol, ethanol, ethyl acetate, acetone, chloroform, and petroleum ether.

6. The method according to claim 4, characterized in that The ratio of water lily to organic solvent is 1g:3-5mL.

7. The method according to claim 4, characterized in that In the step d, the column chromatography fraction is detected by thin layer chromatography or liquid chromatography.

8. Use of the compound according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that The tumor is any one of lung cancer, breast cancer, liver cancer, colon cancer, rectal cancer, and gastric cancer.

10. The use according to claim 8, characterized in that The compound is prepared into a pharmaceutically acceptable dosage form in the form of a monomer or a plant extract containing the compound.

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