A compound having antitumor activity and a preparation method and application thereof

Abstract

The application discloses a compound with anti-tumor activity, which has a chemical structural formula as shown in formula (I). The compound is a new natural product of tetracyclic triterpenes, has good fat solubility and chemical stability, can be conveniently extracted from herbaceous Euryale ferox roots and stems and prepared into oral or injection dosage forms. The application also provides a preparation method and application of the compound. The compound can obviously inhibit the growth of lung cancer of mice, reduce the volume and weight of tumors and can be used for treating tumor diseases.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to a new medicinal tetracyclic triterpenoid natural product, and more specifically, to a compound with antitumor activity, its preparation method and application. Background Technology

[0002] Cancer poses a serious threat to human life and health, with its incidence and mortality rates continuing to rise globally. Current treatment methods for cancer mainly include surgery, radiotherapy, chemotherapy, and immunotherapy. Although anti-tumor biotechnology has made significant progress in recent years, chemotherapy remains one of the most commonly used basic methods in clinical practice. Its indications include: systemic malignant tumors sensitive to chemotherapy, such as leukemia, multiple myeloma, and intermediate- to high-grade malignant lymphomas; advanced tumors with no indication for surgery or radiotherapy, or patients with recurrence or metastasis after surgery or radiotherapy; preoperative preparation and postoperative consolidation therapy for solid tumors; and pre-radiotherapy treatment.

[0003] Chemotherapy primarily uses small-molecule chemical drugs, derived from both synthetic and plant extracts. These include alkaloids, platinum-based drugs, targeted kinase inhibitors, aromatase inhibitors, alkylating agents, taxanes, and many other types. There are cell cycle non-specific drugs that kill cells throughout the entire cell cycle, as well as cell cycle specific drugs that only target a particular phase. Combination therapy often yields even better results. Chemotherapy is widely applicable and inexpensive, and will undoubtedly remain a major approach to cancer treatment in 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 many drawbacks. Current chemotherapy drugs have widespread and severe toxic side effects, face the problem of drug resistance, have poor selectivity for both tumor and normal cells, and damage normal cells while inhibiting or killing tumor cells, affecting the functions of the heart, liver, kidneys, and nervous system, thus producing toxicity to the human body.

[0005] To address these issues, many researchers are dedicated to exploring new antitumor structures, particularly by searching for active ingredients in the plant kingdom to develop antitumor drugs. For example, piperine, with its diverse pharmacological effects, underwent a phase II clinical trial in the United States in 2021 for the treatment of multiple myeloma and prostate cancer. Another example is Paris polyphylla steroidal saponins, the main active ingredient found in Paris polyphylla var. yunnanensis, which exhibit some inhibitory effects on pancreatic cancer cells, but the efficacy needs further improvement. The search for novel antitumor compounds that are low in toxicity, highly effective, specific, and possess favorable pharmacokinetic properties is urgently needed.

[0006] Begonia limprichtii Irmscher is a traditional Chinese herbal medicine commonly used in southwestern my country. It is a perennial herb belonging to the Begoniaceae family and is used to treat injuries from falls, rheumatic pain, sore throat, and other ailments. It has a good safety profile with no obvious toxic side effects. However, there are no reports on the chemical composition and antitumor activity of this herb. Summary of the Invention

[0007] The present invention aims to address the above-mentioned technical problems by providing a small molecule compound with a novel structure and good antitumor activity as a new option for antitumor drug therapy.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a compound having antitumor activity, having a chemical structural formula as shown in formula (I):

[0010]

[0011] The aforementioned compound, named cucurbitacin, contains thirty carbon atoms in its parent nucleus and is a typical tetracyclic triterpenoid. Its structural characteristic is that the angular methyl group 19-CH3 is attached to C-9, rather than the common C-10 of triterpenoids, thus belonging to the cucurbitacin class. However, it lacks the hydroxyl substitution common in cucurbitacins at C-16, making it a rare 16-dehydroxylated cucurbitacin with a novel structure that has not been previously reported. The cucurbitacin monomer is a white powder, sparingly soluble in water but readily soluble in organic solvents such as methanol, ethanol, ethyl acetate, and chloroform, with a bitter taste. Stability studies show that the content remains unchanged after 12 months at room temperature, and its properties show no alteration. Even after 6 months of accelerated testing at 40℃ and 75% relative humidity, no significant changes were observed in its physicochemical properties. This demonstrates its excellent chemical stability, 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 compounds described herein.

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

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

[0015] a. Take the dried rhizomes of *Liriope muscari*, crush them, extract them with an organic solvent, filter them, concentrate them, and obtain a fluid extract;

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

[0017] c. Separate the precipitate by silica gel column chromatography, eluting with a petroleum ether-ethyl acetate gradient;

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

[0019] Preferably, the organic solvent mentioned in step a can be any one or a mixture of organic solvents such as methanol, ethanol, ethyl acetate, acetone, chloroform, and petroleum ether, such as a mixture of petroleum ether and ethyl acetate (volume ratio 2:1). Methanol or ethanol, a lower alcohol, is preferred, and pharmaceutical-grade alcohol, i.e., aqueous ethanol (e.g., aqueous ethanol with a volume fraction of 75%-85%), is more suitable to meet the requirements of relevant regulations, cost, safety, and environmental protection in pharmaceutical production. The extraction method can be percolation, reflux, maceration, microwave extraction, or supercritical fluid extraction, with reflux being preferred.

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

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

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

[0023] Preferably, in 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 chromatography, the developing solvent is petroleum ether-ethyl acetate (3:1) or chloroform-methanol (98:2); the color development method is iodine fuming.

[0025] More preferably, the liquid chromatography method uses a conventional C18 reversed-phase silica gel 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 this invention has a short extraction route, simple operation, regenerable column chromatography packing material, low cost, and is feasible for large-scale production.

[0027] Fourthly, the present invention provides the use of the compounds or pharmaceutical compositions described herein in the preparation of antitumor drugs.

[0028] Preferably, the tumor includes, but is not limited to, types of tumors such as lung cancer, breast cancer, liver cancer, colon cancer, rectal cancer, and stomach cancer. More preferably, the lung cancer is squamous cell carcinoma or non-small cell lung cancer.

[0029] Antitumor assays showed that the compounds of this invention exhibited highly significant inhibitory activity against human tumor lines such as lung cancer, liver cancer, gastric cancer, breast cancer, and colon cancer in vitro, with an IC50 concentration of [missing value]. 50 The concentration ranged from 0.8251 to 38.63 μg / mL; further animal studies showed that oral administration of 40 mg / kg... -1 .d -1 After 9 days of treatment, lung cancer growth in LLC transplanted mice was significantly inhibited, and tumor volume and weight were reduced. This demonstrates that the anti-tumor effect of *Lysimachia christinae* is significant in both in vivo and in vitro models, with an anti-tumor spectrum covering five common solid tumors: lung cancer, breast cancer, liver cancer, gastric cancer, and colon cancer. It exhibits broad-spectrum activity, and the possibility of inhibitory effects on more tumor types cannot be ruled out.

[0030] Preferably, the compound is formulated into a pharmaceutically acceptable dosage form in monomeric form or as a plant extract containing it.

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

[0032] Cyperine monomer or plant extracts containing cyperine can be formulated into a variety of pharmaceutically acceptable dosage forms to meet diverse clinical needs. These dosage forms include, but are not limited to, tablets, hard capsules, soft capsules, granules, pellets, ointments, and injections. Pharmaceutically acceptable carriers, excipients, or additives required for the formulation include, specifically, commonly used pharmaceutical diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, calcium hydrogen phosphate, and light magnesium oxide; commonly used pharmaceutical wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and commonly used pharmaceutical disintegrants such as dried starch, alginate, agar powder, brown algae starch, and carbon. Sodium bicarbonate, citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; lubricants, such as talc, silica, corn starch, stearates, 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, nicotinamide, cyclodextrin, soybean oil, etc.; pharmaceutically acceptable carriers or excipients will not be listed in detail, and those skilled in the art can make specific selections based on their general knowledge.

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

[0034] (1) Dysalicylic acid is a novel small molecule component with a structure completely different from existing drugs, and may contain different targets and mechanisms of action;

[0035] (2) The antitumor effect of the cypermethrin is highly effective and broad-spectrum, and the estimated daily oral dose for adults is 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 the lycopodium clavatum has good clinical safety.

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

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

[0039] (6) The lipophilic compound is highly lipophilic, has good solubility, and is easily absorbed orally;

[0040] (7) The physicochemical properties of the water-repellent compound are stable and it has a long shelf life.

[0041] In summary, the present invention has a novel structure of hyacinthin, with outstanding advantages such as high efficiency, novel mechanism, and stability. The raw materials are readily available and the cost is low, making it a promising new anti-tumor drug. Attached Figure Description

[0042] Figure 1 This is the mass spectrum of hygroscopic methyl hydroxylamine.

[0043] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of hygroscopic methyl methacrylate (HMR).

[0044] Figure 3 This is the carbon NMR spectrum of lysimachia christinae.

[0045] Figure 4 This is the liquid chromatogram of hygroscopic methyl methionine.

[0046] Figure 5 This is the ultraviolet spectrum of hygroscopic methyl hydroxylamine.

[0047] Figure 6 This is a dose-response curve of the inhibition of the proliferation of 13 types of tumor cells by cypermethrin.

[0048] Figure 7 The results show that hyacinthin inhibits the proliferation 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 Implementation

[0049] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the instruments or reagents used in the embodiments are all conventional instruments or reagents in the art and are conventional products that can be purchased from the market. Unless otherwise specified, the specific experimental operations involved in the text are all understandable or known to those skilled 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 structural determination of hygroscopic glycoside A

[0051] 1. Example 1 of extraction and separation of hygroscopic glycoside A:

[0052] (1) Take 300g of dried rhizome of Begonia limprichtii Irmscher (purchased from the herbal medicine market in Leshan City, Sichuan Province), crush it into coarse powder, put it in a round-bottom flask, add 1.2L of methanol, reflux for 1 hour, cool, and filter; extract the residue twice more in the same way; combine the three extracts, concentrate under reduced pressure, and obtain 65g of extract.

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

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

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

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

[0057] 2. Structural determination:

[0058] The monomer of hygroscopic lotus seed is a white powder, sparingly soluble in water, but readily soluble in organic solvents such as methanol, ethanol, ethyl acetate, and chloroform. It has a bitter taste. Stability studies show that the content of this product does not decrease after 12 months of storage at room temperature, and its properties do not change. After 6 months of storage under accelerated testing conditions of 40°C and 75% relative humidity, no significant changes were observed in the various physicochemical properties of this product. It is chemically 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] Using two-dimensional spectral analysis such as HSQC and HMBC and referring 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 cypermethrin.

[0063] The molecular formula of PSY-2 (a type of hygroscopic compound) is C2. 32 H 44 O7, with a molecular weight of 540, has a molecular structure as shown in formula (I).

[0064]

[0065] The systematic name of this hygroscopic acetone 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-dodecylhydro-3H-cyclopenta[a]phenanthrene-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 hygroscopic glycoside A:

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

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

[0069] (3) Take 10g of precipitated powder, dissolve it in 30mL of methanol, mix it into silica gel, dry it, pack it into a silica gel column (silica gel specification 200-300 mesh), and elute it with a gradient of petroleum ether-ethyl acetate (10:0-0:10); check each fraction with silica gel thin layer, develop it with petroleum ether-ethyl acetate (3:1), develop it with iodine fuming, combine it, concentrate it, and obtain 1.2g of light yellow powder.

[0070] (4) Dissolve the powder in 5 ml of methanol, mix with silica gel, dry, pack into a silica gel column (silica gel specification 200-300 mesh), and elute with a gradient of petroleum ether-ethyl acetate (10:0-0:10); combine with silica gel thin-layer chromatography, concentrate, and obtain 0.7 g of white powder.

[0071] 4. Example 3 of extraction and separation of hygroscopic glycoside A:

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

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

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

[0075] 5. Example 4 of extraction and separation of hygroscopic glycoside A:

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

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

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

[0079] Chemical structure identification showed that the products obtained through the extraction and separation examples 2-4 above were identical to PSY-2.

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

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

[0082] All cells were commercially available, and the culture conditions were as follows:

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

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

[0085] HepG2 (human liver cancer cell line, ATCCHB-8065): MEM (containing NEAA) medium + 10% fetal bovine serum + 1% PS;

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

[0087] HCT116 (human colorectal cancer cells, ATCCCCL-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 Pronosai Biotechnology, CL-0292): RPMI-1640 medium + 10% fetal bovine serum + 1% PS;

[0090] BGC-823 (human gastric adenocarcinoma cells, Shanghai Guandao Biotechnology Co., Ltd., C1953): RPMI 1640 medium + 10% fetal bovine serum + 1% PS;

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

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

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

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

[0095] H1299 (human non-small cell lung cancer cells, ATCCCRL-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 (Merren, MA0218); Penicillin-streptomycin (PS, Sigma-Aldrich)

[0097] Instruments: CO2 incubator (Thermo Fisher Scientific), full-wavelength microplate reader (Epoch), benchtop high-speed centrifuge (Eppendorf 5804R); laminar flow hood (Luzhou Cleanroom SW-CJ-2D); inverted microscope (Olympus).

[0098] 2. Experimental Methods:

[0099] 2.1 Cell resuscitation

[0100] (1) Take the cryopreservation tube out of the liquid nitrogen tank and put it directly into 37°C warm water, and shake it from time to time to thaw it as soon as possible.

[0101] (2) Remove the cryovials from the 37°C water bath, disinfect with 75% v / v alcohol and dry. Open the cap, use a pipette to aspirate the cell suspension, add it to the centrifuge tube and add more than 5 times the volume of complete culture medium and mix well.

[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 incubate at 37°C.

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

[0105] 2.2 Cell passage

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

[0107] (2) Using a T25 culture flask, aspirate the complete culture medium and gently wash once with PBS buffer. Add 0.5 mL of 0.25% EDTA trypsin and digest for 2 min (the specific time can be adjusted according to the cell type). Then wash the cells with 1 mL of complete culture medium. Centrifuge at 1000 r / min for 5 min, discard the supernatant, collect the cells, and transfer them to two T25 culture flasks for culture. Add 5 mL of complete culture medium to each flask.

[0108] 2.3 Cell Count Analysis

[0109] (1) After digestion, the cells are fully resuspended and dispersed into individual cells. Take 1 mL and transfer it into a clean EP tube.

[0110] (2) Gently wipe the surface of the counting plate and the coverslip with an alcohol swab. Slightly moisten the edge of the coverslip and then carefully place it over the center of the counting plate, ensuring that the edge covers the groove on the counting plate.

[0111] (3) Repeat the process of resuspending the cell suspension that was just transferred into the EP tube by pipetting.

[0112] (4) Take 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 drawn into the gap between the coverslip and the counting plate due to the siphon effect.

[0113] (5) Adjust the microscope and observe using a 10x objective lens.

[0114] (6) Record the number of live cells that were not stained with trypan blue using the cell counting method.

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

[0116] Cell concentration = (total number of cells in the four large squares / 4) × 10000.

[0117] (8) Further calculations yielded the total number of cells: total number of cells = volume of culture medium for resuspending cells × cell density.

[0118] 2.4 Cytotoxicity (CCK8) assay

[0119] Experimental steps:

[0120] (1) Digest and count the cells, then adjust the cell concentration to 5 × 10⁻⁶. 4 Add the cell suspension to the 96-well plate at a volume of 100 μL per well.

[0121] (2) After the cells in the well plate have been cultured overnight, discard the culture medium, add the corresponding concentration of drug, 100 μL of drug-containing culture medium per well for 24 hours, discard the culture medium, and add CCK8 working solution (prepared at a volume ratio of 1:10 for CCK8 solution and culture medium).

[0122] (3) After incubating 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 (IC50) 50 ) Calculation: Using GraphPad 8.0 to calculate IC 50 value

[0124] 3. Experimental Results

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

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

[0127]

[0128] The results showed that compound PSY-2 significantly inhibited the proliferation of tumor cells in colon cancer, liver cancer, gastric cancer, breast cancer, and lung cancer, 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 Experimental Animal Center.

[0132] Materials and instruments: Same as in Example 2

[0133] 2. Experimental Methods

[0134] C56BL / 6 mice were randomly divided into two groups (n=5 per group): an LLC model group and a drug treatment group (LLC+PSY-2). LLC model establishment method: 200 μL of LLC cells diluted with PBS (3.96 × 10⁻⁶) were subcutaneously injected into the right forelimb of C56BL / 6 mice. 6 (Number of mice / mouse). Changes in mouse body weight (daily) and tumor volume (every two days) were recorded. Drug treatment group: After C56BL / 6 mice were inoculated with LLC cells, tumor volume growth was observed to reach 100 mm. 3 Mice were treated with PSY-2 (40 mg / kg) by gavage daily.

[0135] 3. Experimental Results

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

[0137] Example 4: Preparation of Hydrangea Acid Tablets

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

[0139] Example 5: Preparation of Ligusticum striatum capsules

[0140] Take 50g of hyacinthin and place it in a mixer with 1800g of a mixture of microcrystalline cellulose and lactose (microcrystalline cellulose to lactose weight ratio of 2:1, filler). Mix at 25 rpm for 10 minutes. Add 50mL of a 5% hydroxypropyl methylcellulose aqueous solution (binder) and continue mixing for 15 minutes to form a soft mass. Granulate the mass through an 80-mesh sieve, dry it to control the moisture content at 2%-5%, and then granulate it through a 100-mesh sieve. Place the granules in a mixer with 50g of crospovidone (disintegrant) and 10g of magnesium stearate (lubricant) and mix at 20 rpm for 20 minutes. Fill the mixture into capsules using a capsule filling machine to make 10,000 capsules, each containing 5mg of hyacinthin.

[0141] Example 6: Preparation of Hydrangea Acid Injection

[0142] Take 50g of hymenoplasmin and place it in a clean container. Add 5L of PEG-400 and stir until fully dissolved. Dilute with water for injection to 50L. Filter through a 0.22μm microporous membrane. Under aseptic conditions, quantitatively fill 10,000 ampoules with 5mL of the filtrate. Sterilize the filled ampoules by moist heat at 121℃ for 20 minutes. Each 5mL ampoule contains 5mg of hymenoplasmin.

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing a compound with antitumor activity, characterized in that, Includes the following steps: a. Take the dried rhizomes of Begonia hyacinthii, crush them, extract them with an organic solvent, filter them, concentrate them, and obtain an extract; b. Add the extract to water, stir, filter, wash with water, dry, and obtain a precipitate; c. Separate the precipitate by silica gel column chromatography, eluting with a petroleum ether-ethyl acetate gradient of 10:0 to 0:10; d. Detect column chromatography fractions, combine, concentrate, and obtain the compound; The organic solvent is any one or more of methanol, ethanol, ethyl acetate, acetone, chloroform, and petroleum ether; The ratio of Houttuynia cordata to organic solvent is 1g : 4mL; The compound has the chemical structural formula shown in formula (I): （I）。 2. The method according to claim 1, characterized in that, Step b is the water sedimentation process. The amount of water added is 5 to 15 times the weight of the extract. Stir at room temperature or under heating to fully dissolve and suspend the extract. Then cool, let stand, filter, and wash the precipitate with small amounts of water several times.

3. The method according to claim 1, characterized in that, The column chromatography operation described in step c is repeated until the target analyte of the desired purity is obtained.

4. The method according to claim 1, characterized in that, In step d, the column chromatography fraction is detected by thin-layer chromatography or liquid chromatography.

5. The method according to claim 4, characterized in that, The thin-layer chromatography was performed using conventional silica gel, with the developing solvent being petroleum ether-ethyl acetate at a volume ratio of 3:1 or chloroform-methanol at a volume ratio of 98:2; the colorimetric method was iodine fuming.

6. The method according to claim 4, characterized in that, The liquid chromatography method used a conventional C18 reversed-phase silica gel column; the mobile phase was a gradient elution of methanol-0.1% formic acid with a volume ratio of 50:50 to 100:0; and the detection wavelength was 237 nm.

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