Preparation method of lactam alkaloid autoleuca leucocephala and application of lactam alkaloid autoleuca leucocephala in preparation of antitumor drugs
By extracting and isolating Cytochalasin J from the pods of Leucaena leucantha, the problem of insufficient efficacy and toxicity of existing antitumor drugs has been solved, achieving significant inhibition of central nervous system tumors and breast cancer, and providing potential for the development of low-toxicity antitumor drugs.
Patent Information
- Application Number
- CN202511944082.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-04-17
AI Technical Summary
The clinical application of existing chemically synthesized antitumor drugs is nearing a plateau, and there is an urgent need to develop natural active compounds that are both effective and have low toxicity. There are few studies on Cytochalasin J isolated from plants, especially its inhibitory activity against tumor cells.
Cytochalasin J, a lactam alkaloid compound, was extracted and isolated from the pods of the Leucaena leucocephala plant. It was purified by ethanol-water extraction, reversed-phase silica gel column chromatography, and recrystallization to prepare a compound with significant inhibitory activity against tumor cells, which can be used to prepare anti-tumor drugs.
The prepared Cytochalasin J exhibits significant inhibitory activity against central nervous system tumor cells and breast cancer cells, and has potential low toxicity, making it suitable for preparing effective anti-tumor drugs, including wettable powders, tablets, capsules, and other dosage forms.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, specifically relating to a method for preparing the lactam alkaloid Cytochalasin J from the plant Leuciscus syriacus and its application in the preparation of antitumor drugs. Background Technology
[0002] Cancer is the second leading cause of death and death after cardiovascular and cerebrovascular diseases. The incidence of cancer is increasing globally, and the number of cancer cases in my country has also increased dramatically in recent years. Cancer is placing a heavy burden and causing huge losses to the health of the Chinese people and the national economy.
[0003] Chemically synthesized cytotoxic antitumor drugs have been used for decades, and the existing clinical drugs for treating tumors have reached a plateau. There is an urgent need to research and develop new tumor treatment drugs that are both effective and have low toxicity.
[0004] Naturally active compounds from plant sources have significant potential in the development of highly effective and low-toxicity antitumor drugs. *Leucaena leucocephala*, a fast-growing tree species belonging to the subfamily Mimosoideae of the Fabaceae family, is a fast-growing tree species with medicinal potential. Its leaves and pods, rich in diverse nutrients, can be used as livestock feed (Feed Res., 2021, 5, 141−144). In Guangxi and Hainan, China, the tender leaves of this plant are also used to make medicinal tea (Biochem. Syst. Ecol., 2012, 43, 210-213). Furthermore, extracts from *Leucaena leucocephala* pods and leaves have potential pharmacological effects such as hypoglycemic, heat-clearing and detoxifying, antibacterial, antioxidant, and appetite-enhancing effects (West China Pharmaceutical Journal, 2012, 26(04), 610-612), demonstrating its significant potential in the development of effective and low-toxicity drugs for treating diseases.
[0005] Compound Cytochalasin J is a pine alkaloid with a characteristic lactam structure. This compound was previously discovered and reported in some fungi (Tetrahedron, 1989, 45: 2323−2335). It is a cytochalasin derivative and has the potential to act as an inhibitor of actin polymerization. However, there are no reports of its isolation from the plant *Cytochalasin*, nor are there any studies on its inhibitory activity against tumor cells such as central nervous system tumor cells SF-268, breast cancer cells MCF-7, liver cancer cells HepG2, and lung cancer cells NCI-H460. Summary of the Invention
[0006] The purpose of this invention is to provide an application of the pod material of the plant Leucaena leucocephala (Lam.) de Wit. in the preparation of the lactam alkaloid compound Cytochalasin J and its application in the preparation of antitumor drugs.
[0007] Our research on the component analysis and identification of *Leucaena leucocephala* revealed that this compound can be isolated from the pods of the rapidly growing and readily available *Leucaena leucocephala* plant. Of particular note, we further discovered that this compound also exhibits significant selective inhibitory activity against the growth of certain tumor cells, indicating its potential as a natural plant-derived antitumor molecule with promising application prospects.
[0008] The first objective of this invention is to provide a method for preparing the lactam alkaloid compound Cytochalasin J, the specific steps of which are as follows:
[0009] a. Preparation of total extract: The pods of Leucaena leucocephala are crushed and extracted with ethanol aqueous solution, ethanol, acetone aqueous solution or acetone. The extract is concentrated to remove ethanol or acetone to obtain crude extract of total extract. The crude extract of total extract is suspended in water and first extracted with petroleum ether to remove small polar components, and then extracted with ethyl acetate. The ethyl acetate extract is concentrated to obtain ethyl acetate total extract.
[0010] b. Separation and purification: The total ethyl acetate extract was subjected to reversed-phase silica gel column chromatography with chloroform / methanol as the eluent, eluting sequentially at volume ratios of 97:3, 90:10, 85:15, 70:30, 60:40, and 0:100. The fraction eluted with chloroform / methanol at 90:10 v / v was collected and then subjected to medium-pressure reversed-phase silica gel column chromatography with methanol / water as the mobile phase, eluting sequentially at volume ratios of 2:8, 4:6, 6:4, and 10:0. The fraction eluted with methanol / water at 6:4 v / v was collected and then subjected to Sephadex LH-20 column chromatography with methanol as the eluent. The eluent was recrystallized to obtain the target compound Cytochalasin J.
[0011] The structural formula of the compound Cytochalasin J is shown in formula (І): ; Formula (І).
[0012] Preferably, the ethanol aqueous solution or acetone aqueous solution is an ethanol aqueous solution or acetone aqueous solution with a volume fraction of 70% or greater.
[0013] More preferably, the ethanol aqueous solution is a 95% ethanol aqueous solution by volume.
[0014] Cytochalasin J prepared in this invention has been shown in in vitro pharmacological experiments to have significant inhibitory activity (IC50) against human central nervous system tumor cells SF-268 and breast cancer cells MCF-7. 50 The values were 1.17±0.02 and 3.26±0.12 μM, respectively, while the inhibitory activity against HepG2 liver cancer cells and NCI-H460 lung cancer cells was weak (IC50). 50 The values were 86.48±2.58 and 58.42±1.52 μM. Therefore, it has the potential to be used to prepare effective and low-toxicity drugs against central nervous system tumors (SF-268) and breast cancer tumors (MCF-7) or as a lead compound for corresponding antitumor drugs.
[0015] A second objective of this invention is to provide the use of compound Cytochalasin J or its pharmaceutical salt or its acetylated derivative in the preparation of antitumor drugs.
[0016] Preferably, the antitumor drug is a drug for treating central nervous system tumors, breast cancer, liver cancer, or lung cancer. More preferably, it is a drug for treating central nervous system tumors or breast cancer.
[0017] Preferably, the antitumor drug further contains pharmaceutically usable excipients. The compound Cytochalasin J of the present invention can be combined with commonly used pharmaceutical excipients or drug carriers to prepare drugs or pharmaceutical compositions possessing the antitumor activity of Cytochalasin J, which can be used for the prevention and treatment of tumors. The drug or pharmaceutical composition can be in dosage forms such as wettable powder, tablets, granules, capsules, oral liquids, pellets, injections, and aerosols; it can also be in controlled-release or sustained-release dosage forms or nanoformulations known in modern pharmaceutical industry.
[0018] Preferably, the acetylated derivative is a hydroxyacetylated derivative of Cytochalasin J.
[0019] It should be noted that the compound Cytochalasin J has three hydroxyl groups in its structure, and its hydroxyl groups can be easily acetylated to obtain its acetylated derivatives. These acetylated derivatives can be easily hydrolyzed to release Cytochalasin J in the acidic environment of the stomach and the alkaline environment of the intestine. Therefore, the preparation of related antitumor drugs using the hydroxyacetylated derivatives of Cytochalasin J is essentially still based on Cytochalasin J as the active ingredient. Thus, the preparation of related antitumor drugs using the hydroxyacetylated derivatives of Cytochalasin J as raw materials should also fall within the scope of protection of this invention.
[0020] A third objective of this invention is to provide an antitumor drug, characterized in that it contains an effective amount of Cytochalasin J or its pharmaceutical salt or its acetylated derivative as an active ingredient and a pharmaceutically commonly used excipient or carrier.
[0021] This invention uses the pods and leaves of *Leucaena leucocephala*, a plant widely distributed and growing rapidly in southern my country, as raw materials to extract and isolate Cytochalasin J, a compound that significantly inhibits tumor cell activity. The material source is abundant, the preparation process is easy to operate, and the monomeric compound is stable and easy to store. Furthermore, since *Leucaena leucocephala* itself can be used as agricultural and livestock feed, the obtained compound Cytochalasin J is very likely to be an anti-tumor active compound with both effectiveness and low toxicity, thus having good application and development potential. Attached Figure Description
[0022] Figure 1 It is the compound Cytochalasin J 1 H NMR spectrum;
[0023] Figure 2 It is the compound Cytochalasin J 13 C NMR spectrum. Detailed Implementation
[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.
[0025] Example 1: Isolation and preparation of Cytochalasin J from Leucaena leucantha pods
[0026] 1.1 Sources of plant materials
[0027] The pods of *Leucaena leucocephala* (Lam.) de Wit. used for extraction were collected from the South China Botanical Garden, Chinese Academy of Sciences, and identified by the inventor.
[0028] 1.2 Extraction and Separation
[0029] The sample (dried pods of *Leucaena leucocephala*, weighing 2.0 kg) was pulverized and extracted three times with 95% ethanol aqueous solution at room temperature. The extracts were combined and concentrated under reduced pressure to remove the organic solvent ethanol, yielding a crude total extract. The crude total extract was suspended in 500 mL of water and extracted with an equal volume of petroleum ether, followed by three extractions with an equal volume of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to obtain a total ethyl acetate extract (35 g). The total ethyl acetate extract was dissolved in 150 mL of chloroform / methanol at a volume ratio of 1:1. Normal-phase silica gel (80-100 mesh) was added at a weight ratio of 1:1.5, and the mixture was evaporated to dryness. The sample was then dry-packed onto a column (200-300 mesh, 1000 g) and loaded onto the column using a gradient elution with mobile phases of chloroform / methanol = 97:3, 90:10, 85:15, 70:30, 60:40, and 0:100 v / v. Based on thin-layer chromatography, six fractions (F1–F6) were collected sequentially according to their polarity, from smallest to largest. Fraction F2 (1.3 g) eluted from chloroform / methanol at 90:10 v / v was further subjected to reversed-phase medium-pressure column chromatography, eluted with MeOH / H2O at volume ratios of 2:8, 4:6, 6:4, and 10:0. The 6:4 methanol / water fraction was collected. v / v eluted fraction F 2-3 (36 mg), F 2-3 The compound was then separated and purified by Sephadex LH-20 column chromatography (1500 mm × 25 mm id), eluted with methanol, and the eluted fraction was recrystallized to give target compound 1 (Cytochalasin J) (6 mg) as shown in formula (Ⅰ).
[0030] 1.3 Structural identification of compound Cytochalasin J
[0031] The obtained compound 1 is a white powder with the molecular formula C. 28 H 37 NO4, its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 1 and Figure 2 As shown; ESI-MS (+) m / z 490 [M+K] + ; ESI-MS (−) m / z 450 [M−H] – , 486 [M+Cl] – ; 1 H NMR (600 MHz, CD3OD) δ 7.29 (2H, t, J = 7.6 Hz, H-2', H-6'), 7.21 (3H,t, J= 5.9 Hz, H-3', H-4', H-5'), 5.82 (1H, dd, J = 16.6, 2.1 Hz, H-19), 5.74(1H, dd, J = 16.6, 2.1 Hz, H-20 ), 5.56 (1H, dd, J = 15.5, 9.6 Hz, H-13 ),5.24 (1H, ddd, J = 15.5, 10.8, 4.7 Hz, H-14), 5.21 (1H, s, H-12b), 5.00 (1H,s, H-12a), 3.74 (1H, t, J = 13.2 Hz, H-7), 3.72 (1H, s, H-3), 3.34 (1H, s, H-8), 3.30 (1H, s, H-10a), 2.89 ( 1H, t, J = 10.1 Hz, H-10b), 2.81 (1H, dd, J =13.5, 6.2 Hz, H-4), 2.74 (1H, dd, J = 13.4, 5.9 Hz, H-5), 2.60 (1H, dd, J =9.9, 6.2 Hz, H-18), 1.96 (1H, dd, J = 12.4, 4.5 Hz, H-16 ), 1.77 (1H, dd, J =19.8, 6.4 Hz, H-17a), 1.67 (1H, dt, J = 34.1, 17.1 Hz, H-15b), 1.50 (1H, dd, J = 13.6, 2.7 Hz, H-17b), 1.25 (3H, s, H-18), 0.99 (3H, d, J = 6.8 Hz, H-16),0.82 (3H, d, J = 6.7 Hz, H-11 ); 13 C NMR (150 MHz, CD3OD) δ178.78 (C-1),151.88 (C-6), 138.69 (C-1'), 137.48 (C-20), 137.36 (C-14), 132.24 (C-13), 131.12 (C-2', 6'), 129.51 (C-3', 5'), 129.38 (C-19), 127.77 (C-4'), 113.05(C-12), 76.74 (C-21), 75.13 (C-18), 72.52 (C-7), 55.08 (C-17), 55.02 (C-9), 54.97 (C-3), 49.00 (C-4), 46.61 (C-8), 45.05 (C-10), 44.56 (C-15), 33.97 (C-5), 31.08 (C-23), 29.36 (C-16), 26.70 (C-22), 14.01 (C-11).
[0032] Based on the comprehensive analysis of the above mass spectrometry and nuclear magnetic resonance spectroscopy data and the comparison with the literature (Tetrahedron, 1989, 45:2323−2335), the chemical structure of compound 1 can be deduced as Cytochalasin J.
[0033] Its structural formula is shown in equation (Ⅰ): ; Formula (І).
[0034] Example 2: Detection of the antitumor activity of compound Cytochalasin J
[0035] 2.1 Instruments and Materials
[0036] Cell lines and cell culture medium: Four human tumor cell lines, namely central nervous system tumor cells (SF-268), breast cancer cells (MCF-7), liver cancer cells (HepG2), and lung cancer cells (NCI-H460), were obtained from the Guangdong Institute of Microbiology; RPMI 1640 medium (Gibaco), fetal bovine serum (FBS, Gibaco), trypsin (Trypsin 1:250, Amersco), and PBS (laboratory preparation).
[0037] Experimental instrument: Genois microplate reader (Tecan GENios, Swizerland).
[0038] Reagents and samples: Dimethyl sulfoxide (DMSO, analytical grade), 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT, Sigma), doxorubicin (Pfizer Italia SRL); Cytochalasin J was prepared as described in the above experimental examples.
[0039] 2.2 Test Method:
[0040] a) Tumor cell culture: Four human tumor cell lines were all adherent cells, cultured in RPMI 164 medium supplemented with 10% FBS and 1% antibiotics. All cells were cultured in a 37°C, 5% CO2 saturated humidity CO2 incubator. Cells were passaged when they covered 90% of the bottom of the culture dish. Cells in the logarithmic growth phase were used for experiments.
[0041] b) Preparation of test samples: Cytochalasin J and doxorubicin were prepared into 10 mg / ml solutions using dimethyl sulfoxide (DMSO), and then diluted with culture medium to the final treatment concentration.
[0042] c) The half-maximal inhibitory concentration (IC50) of compound Cytochalasin J against tumor cells was determined using the MTT assay in a 96-well plate.
[0043] First, cells in the logarithmic growth phase are digested and collected, and the cell suspension concentration is adjusted to achieve a cell concentration of 5 × 10⁻⁶. 4 Cells / mL were seeded into 96-well plates, with 100 µl added to each well. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. The supernatant was aspirated, and then 100 µl of fresh RPMI 164 medium and 100 µl of medium containing the required sample concentration were added to each well. The mixture was thoroughly mixed to bring the final volume of each well to 200 µl. The experiment included different concentrations of test sample groups, a blank control group (no sample added), and a positive control group (doxorubicin). For each test sample group, six concentration gradients were set (50, 25, 12.5, 6.25, 3.125, 1.5625 µg / mL), with four replicates for each concentration. The culture was incubated at 37℃ and 5% CO2 for 72 h. 20 µl of MTT (5 mg / ml) was added to each well, and incubation continued for 4 h. The supernatant was then removed by centrifugation. 150 µl of DMSO was added to each well, and the resulting crystals were dissolved by thorough shaking. After 15 min, the absorbance (OD) was measured at 570 nm using a microplate reader. The inhibition rate of the compound was calculated using the following formula: Inhibition rate (%) = (OD...) control –OD treated ) / OD control ×100%. Among them, the half-maximal inhibitory concentration (IC50) of the tested compound against tumor cells was [value missing].50 IC 50 Calculations were performed using the LOGIT method (Xu, XY; Xie, HH; Hao, J.; Jiang, YM; Wei, XY Eudesmane sesquiterpeneglucosides from lychee seed and their cytotoxic activity. Food Chem., 2010, 123: 1123–1126).
[0044] 2.3 Experimental data are shown in Table 1:
[0045] Table 1. In vitro antitumor cell activity IC50 of compound Cytochalasin J 50 (μM)
[0046] 2.4 Experimental Conclusions:
[0047] Leucaena leucocephala is a rapidly growing plant in South my country. The Cytochalasin J provided in this invention is isolated from the pods of Leucaena leucocephala, a readily available and abundant raw material. In vitro pharmacological experiments have confirmed that the compound Cytochalasin J has significant inhibitory activity (IC50) against human central nervous system tumor cells SF-268 and breast cancer cells MCF-7. 50 The values were 1.17±0.02 and 3.26±0.12 μM, respectively. As a component isolated from Leucaena leucocephala plant resources that can be used as agricultural and livestock feed, it has the potential advantages of low toxicity and has the potential to be developed for the preparation of related anti-tumor drugs, or it may serve as a lead compound for the development of related anti-tumor drugs.
[0048] The embodiments of this invention are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention. Any equivalent modifications or substitutions made based on the technical solutions of this invention, as long as they do not depart from the spirit and scope of the technical solutions of this invention, should be included within the scope of protection of the claims of this invention.
Claims
1. A method for preparing a lactam alkaloid compound, Cytochalasin J, characterized by, The specific steps are as follows: A. Preparation of total extract: The pods of Leucaena leucocephala are crushed and extracted with ethanol aqueous solution, ethanol, acetone aqueous solution or acetone. The extract is concentrated to remove ethanol or acetone to obtain crude extract of total extract. The crude extract of total extract is suspended in water and first extracted with petroleum ether to remove small polar components, and then extracted with ethyl acetate. The ethyl acetate extract is concentrated to obtain ethyl acetate total extract. B. Separation and Purification: The total ethyl acetate extract was subjected to reversed-phase silica gel column chromatography with chloroform / methanol as the eluent, eluting sequentially at volume ratios of 97:3, 90:10, 85:15, 70:30, 60:40, and 0:
100. The fraction eluted by chloroform / methanol at 90:10 v / v was collected and then subjected to medium-pressure reversed-phase silica gel column chromatography with methanol / water as the mobile phase, eluting sequentially at volume ratios of 2:8, 4:6, 6:4, and 10:
0. The fraction eluted by methanol / water at 6:4 v / v was collected and then subjected to Sephadex LH-20 column chromatography with methanol as the eluent. The eluent was recrystallized to obtain the target compound Cytochalasin J. The structural formula of the compound Cytochalasin J is shown in formula (І): ； Formula (І).
2. The preparation method according to claim 1, characterized in that, The ethanol-water solution or acetone-water solution is an ethanol-water solution or acetone-water solution with a volume fraction of 70% or greater.
3. The preparation method according to claim 2, characterized in that, The ethanol aqueous solution is a 95% ethanol aqueous solution by volume.
4. The application of compound Cytochalasin J or its pharmaceutical salt or its acetylated derivative in the preparation of antitumor drugs; The structural formula of the compound Cytochalasin J is shown in formula (І): ； Formula (І).
5. The application according to claim 4, characterized in that, The anti-tumor drugs mentioned are drugs for treating central nervous system tumors, breast cancer, liver cancer, or lung cancer.
6. The application according to claim 5, characterized in that, The anti-tumor drugs mentioned are drugs for treating central nervous system tumors or breast cancer.
7. The application according to claim 4, characterized in that, The antitumor drug also contains pharmaceutically usable excipients.
8. The application according to claim 4, characterized in that, The drug is a wettable powder, tablet, granule, capsule, oral liquid, drop pill, injection, aerosol, controlled-release or sustained-release dosage form or nano-formulation.
9. The application according to claim 4, characterized in that, The acetylated derivative is a hydroxyacetylated derivative of Cytochalasin J.
10. An antitumor drug, characterized in that, It contains an effective amount of Cytochalasin J or its pharmaceutical salt or its acetylated derivative as an active ingredient and a commonly used pharmaceutical excipient or carrier; The structural formula of the compound Cytochalasin J is shown in formula (І): ； Formula (І).