A limonoid compound derived from chrysophyllum coccineum and a preparation method thereof

Abstract

The present application relates to the field of natural medicine, and more particularly to a limonoid compound derived from the small alligator pear and a preparation method thereof, the limonoid compound having one of the structures of Munropin G, Munropin H, Munropin I, Munropin J, Munropin K and Munropin L. The limonoid compound extracted from the small alligator pear shows specific inhibition effect on colon cancer cells, and is obviously superior to the inhibition effect on other types of tumor cells, so that the limonoid compound extracted from the small alligator pear is expected to be developed into a new drug for treating colon cancer.

Description

Technical Field

[0001] This invention relates to the field of natural medicines, and more particularly to a limonene compound derived from dwarf limonene and its preparation method. Background Technology

[0002] Munronia pinnata (Wall.) W. Theob (synonyms: M. henryi Harms, M. pumila Wight, and M. sinica Diels) belongs to the Meliaceae family. It is also used in traditional Chinese medicine to treat tuberculosis, cough, stomach pain, and ulcers. Several research groups have isolated various chemical substances from Munronia pinnata, such as limonene, triterpenes, saponins, phenylpropanoids, coumarins, lignans, flavonoids, alkaloids, and tannins. Among these, limonene compounds are unique metabolites of Munronia pinnata, attracting widespread attention due to their diverse structures and biological activities.

[0003] Currently, there are no records of anticancer activity of limonene compounds in dwarf tamarisk. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a limonin-like compound derived from dwarf limonene and its preparation method.

[0005] This invention provides a limonin compound derived from dwarf limonene, wherein the limonin compound has one of the structures of Munropin G, Munropin H, Munropin I, Munropin J, Munropin K, and Munropin L, wherein Munropin G, Munropin H, Munropin I, Munropin J, Munropin K, and Munropin L are as shown in formulas (I) to (IV):

[0006]

[0007]

[0008]

[0009] A method for preparing a limonin compound derived from dwarf limonene, wherein the limonin compound is Munropin G, and the method for preparing Munropin G includes the following steps:

[0010] S1: Take dried whole plant of *Gnaphalium affine* and add 95% ethanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature (15-35℃), each time for 3 days. Combine the filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of petroleum ether, ethyl acetate and water. Take the aqueous layer and chromatographically analyze it with a macroporous resin column of 50 times its weight, eluenting with 2.0-3.0L each of 20%, 40%, and 80% ethanol. Collect the 80% ethanol eluent and chromatographically analyze it again with a gel column of 100 times its weight, eluting with 1.0-2.0L of 100% methanol to obtain Fr2.

[0011] S2: Fr2 was subjected to C18 column chromatography with 100 times its weight. Gradient elution was performed using methanol-water at volume ratios of 30:70, 40:60, and 50:50, with 1.0 to 1.5 L of each mixture. One test tube was collected for every 10 to 15 ml of the mixture. The eluents containing the same components were combined and observed by TLC to obtain seven components, namely Fr2.1 to Fr2.7.

[0012] S3: Fr2.2 was prepared by high-speed countercurrent chromatography using 4.5-5.5 L of dichloromethane-methanol-water at a volume ratio of 2:2:1. Each 20-25 ml of mobile phase was collected in one test tube, and the same components were combined by TLC to obtain four components: Fr2.2.1 to Fr2.2.4.

[0013] S4: Fr2.2.2 was subjected to silica gel column chromatography with dichloromethane-methanol elution at a volume ratio of 4:1 (1.0-1.5 L) to obtain Fr2.2.1.1. Fr2.2.1.1 was then subjected to preparative high performance liquid chromatography using a C18 preparative column and acetonitrile-water at a volume ratio of 20:80 as the mobile phase, with a retention time of 12-18 min to prepare Munropin G.

[0014] A method for preparing a limonin compound derived from dwarf tamarisk, wherein the limonin compound is Munropin H, and the method for preparing Munropin H includes the following steps:

[0015] S1: Take dried whole plant of *Gnaphalium affine* and add 95% ethanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature (15-35℃), each time for 3 days. Combine the filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of petroleum ether, ethyl acetate and water. Take the aqueous layer and perform column chromatography with 50 times its weight of macroporous resin. Elute with 2.0-3.0L each of 20%, 40% and 80% ethanol. Collect the 40% ethanol eluent to obtain Fr3.

[0016] S2: Take Fr3 and perform silica gel column chromatography with 100 times its weight. Use dichloromethane-methanol at volume ratios of 80:20, 90:10, and 100:0 as the mobile phase for gradient elution. Collect 10-15 ml of each test tube. Observe the eluent containing the same components in each test tube by TLC. Combine the eluents. There are 20 fractions in total, Fr3.1 to Fr3.20.

[0017] S3: Take Fr3.2 and prepare Munropin H by high performance liquid chromatography using a C18 column and acetonitrile-water at a volume ratio of 20:80 as the mobile phase, with a retention time of 18-22 min.

[0018] A method for preparing a limonin compound derived from dwarf tamarisk, wherein the limonin compound is Munropin I, and the method for preparing Munropin I includes the following steps:

[0019] S1: Take dried whole plant of *Gnaphalium affine* and add 95% ethanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature (15-35℃), each time for 3 days. Combine the filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of petroleum ether, ethyl acetate and water. Take the aqueous layer and chromatographically analyze it with a macroporous resin column of 50 times its weight, eluenting with 2.0-3.0L each of 20%, 40%, and 80% ethanol. Collect the 80% ethanol eluent and chromatographically analyze it again with a gel column of 100 times its weight, eluting with 1.0-2.0L of 100% methanol to obtain Fr2.

[0020] S2: Fr2 was subjected to C18 column chromatography with 100 times its weight. Gradient elution was performed using methanol-water at volume ratios of 30:70, 40:60, and 50:50, with 1.0 to 1.5 L of each mixture. One test tube was collected for every 10 to 15 ml of the mixture. The eluents containing the same components were combined and observed by TLC to obtain seven components, namely Fr2.1 to Fr2.7.

[0021] S3: Fr2.7 was prepared by reverse flow using 2.0-2.5 L of dichloromethane-methanol-water at a volume ratio of 2:2:1. Each 20-25 ml of mobile phase was collected in one test tube, and the same components were combined by TLC to obtain four components: Fr2.7.1 to Fr2.7.4.

[0022] S4: Fr2.7.3 was purified by preparative high performance liquid chromatography using a C18 column and acetonitrile-water at a volume ratio of 15:85 as the mobile phase, with a retention time of 18–24 min to obtain Munropin I.

[0023] A method for preparing limonin compounds derived from dwarf limonene, wherein the limonin compound is Munropin J or K, and the method for preparing Munropin J or K includes the following steps:

[0024] S1: Take dried whole plant of *Gnaphalium affine* and add 100% methanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature (15-35℃), each time for 3 days. Combine the extracts and filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of ethyl acetate and water. Separate the ethyl acetate layer by silica gel column chromatography using gradient elution with n-hexane-ethyl acetate at volume ratios of 90:10, 80:20, 60:40 and 25:75, 2.0-2.5L each. Collect 10-15ml in one test tube. Observe the TLC on each test tube and combine the eluents containing the same components to obtain 12 components, Fr1-Fr12.

[0025] S2: Fr11 was subjected to MCI resin column chromatography with 100 times its weight as the eluent, and 3L of 100% methanol was used for elution. One test tube was collected for every 10ml. Each test tube was observed by TLC plate spotting. The eluents containing the same components were combined to obtain 5 components, including Fr11.1 to Fr11.5.

[0026] S3: Fr.11.1 was subjected to GPC-HPLC with a SEC column as the preparative column and 100% methanol as the mobile phase. A total of 1.0-1.5 L of elution was used, and 10-15 ml of each tube was collected. The eluents containing the same components were combined and observed by TLC. Seven components, Fr11.1.1 to Fr11.1.7, were obtained.

[0027] S4: Fr11.1.5 was subjected to reversed-phase high-performance liquid chromatography using a C18 column as the preparative column and acetonitrile-water as the mobile phase with a volume ratio of 35:65. Munropin J and Munropin K were prepared sequentially with retention times of 20-25 min.

[0028] A method for preparing a limonin compound derived from dwarf tamarisk, wherein the limonin compound is Munropin L, and the method for preparing Munropin L includes the following steps:

[0029] S1: Take dried whole plant of *Gnaphalium affine* and add 100% methanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature (15-35℃), each time for 3 days. Combine the extracts and filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of ethyl acetate and water. Separate the ethyl acetate layer by silica gel column chromatography using gradient elution with n-hexane-ethyl acetate at volume ratios of 90:10, 80:20, 60:40 and 25:75, 2.0-2.5L each. Collect 10-15ml in one test tube. Observe the TLC on each test tube and combine the eluents containing the same components to obtain 12 components, Fr1-Fr12.

[0030] S2: Fr11 was subjected to MCI resin column chromatography with 100 times its weight as the eluent, and 3L of 100% methanol was used for elution. One test tube was collected for every 10ml. Each test tube was observed by TLC plate spotting. The eluents containing the same components were combined to obtain 5 components, including Fr11.1 to Fr11.5.

[0031] S3: Fr.11.1 was subjected to GPC-HPLC with a SEC column as the preparative column and 100% methanol as the mobile phase. A total of 1.0-1.5 L of elution was used, and 10-15 ml of each tube was collected. The eluents containing the same components were combined and observed by TLC. Seven components, Fr11.1.1 to Fr11.1.7, were obtained.

[0032] S4: Fr11.1.3 was subjected to reversed-phase high-performance liquid chromatography using a C18 preparative column and acetonitrile-water as the mobile phase with a volume ratio of 35:65 to obtain Munropin L at a retention time of 20-30 min.

[0033] The euphorbia triterpenoid compounds of the present invention have the following characteristics and advantages:

[0034] 1. The limonoid compounds extracted from *Gnaphalium affine* in this invention exhibit a specific inhibitory effect on colon cancer cells, which is significantly superior to the inhibitory effect on other types of tumor cells. Therefore, the limonoid compounds extracted from *Gnaphalium affine* show promise for development into new treatments for colon cancer.

[0035] 2. The preparation method of the present invention is convenient to operate, easy to mass-produce, and has stable quality; the raw materials of the present invention are abundant and reasonably priced in China, so there is no high cost limit to its large-scale production. Attached Figure Description

[0036] Figure 1 The image shows the 2D NMR correlation and planar structure of Munropin G.

[0037] Figure 2The image shows the 2D NMR correlation and planar structure of Munropin H.

[0038] Figure 3 The image shows the 2D NMR correlation and planar structure of Munropin I.

[0039] Figure 4 The image shows the 2D NMR correlation and planar structure of Munropin J.

[0040] Figure 5 The image shows the 2D NMR correlation and planar structure of Munropin K.

[0041] Figure 6 The image shows the 2D NMR correlation and planar structure of Munropin L. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Example 1: Preparation of limonoid compounds Munropin J-L

[0044] The *Munronia pinnata* (Wall.) W. Theob. plants were collected in June 2017 and July 2021 from Jingxi City, Baise City, Guangxi Zhuang Autonomous Region. Professor Li Dianpeng identified them as *Munronia pinnata* (Wall.) W. Theob., belonging to the genus *Munronia* of the family Meliaceae. These specimens are preserved in the Herbarium (21-GX-001), Natural Product Chemistry Research Center, Guangxi Institute of Botany, Chinese Academy of Sciences, Guangxi Zhuang Autonomous Region, and the Herbarium (17-JG-001), Faculty of Pharmaceutical Sciences, Tokushima University, Japan.

[0045] S1: Take 1.64 kg of dried whole plant of *Gnaphalium affine* and add 100% methanol at a material-to-liquid ratio of 1 kg: 10 L. Soak and extract three times at room temperature (15-35℃), each time for 3 days. Combine the extracts and filtrates, filter, concentrate and obtain 111.4 g of extract. Extract the extract sequentially with equal volumes of ethyl acetate and water. The ethyl acetate layer (34.2 g) is subjected to silica gel H column chromatography with gradient elution using 2 L each of n-hexane-ethyl acetate at volume ratios of 90:10, 80:20, 60:40 and 25:75. Collect 1 tube per 10 ml. Observe the eluents containing the same components in each tube by TLC. Combine the eluents to obtain 12 components, Fr1 to Fr12 (numbered 1, 2, 3, ... according to the order of elution, the same below).

[0046] S2: Fr11 (6.5g) was chromatographically analyzed on an MCI resin column (gel CHP20P) with 100 times its weight as the eluent. 3L of 100% methanol was used as the eluent. One test tube was collected for every 10ml of the eluent. Each test tube was observed by TLC. The eluents containing the same components were combined to obtain 5 components, namely Fr11.1 to Fr11.5.

[0047] S3: Fr.11.1 (190.3 mg) was separated by GPC-HPLC using an Asahipak GS-310 preparative column with 100% methanol as the mobile phase. A total of 1 L of elution was carried out, and 10 mL of each tube was collected. The eluents containing the same components were combined and observed by TLC. Seven components, Fr11.1.1 to Fr11.1.7, were obtained.

[0048] S4: Fr11.1.3 (21.5 mg) was subjected to reversed-phase high-performance liquid chromatography using a COSMOSIL 5C18-AR-II preparative column with acetonitrile-water as the mobile phase at a volume ratio of 35:65, and Munropin L (6, 3.2 mg) was obtained at a retention time of 25 minutes.

[0049] S5: Fr11.1.5 (32.7 mg) was prepared by reversed-phase high-performance liquid chromatography using a COSMOSIL 5C18-AR-II preparative column with acetonitrile-water as the mobile phase at a volume ratio of 35:65, yielding Munropin J (4, 2.5 mg) and Munropin K (5, 2.3 mg) at retention times of 20.2 and 23.4 minutes, respectively.

[0050] Example 2: Preparation of limonoid compounds Munropin G-I

[0051] S1: Take 25 kg of dried whole plant of *Gnaphalium affine* and add 95% ethanol at a material-to-liquid ratio of 1 kg: 10 L. Soak and extract three times at room temperature (15-35℃), each time for 3 days. Combine the filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of petroleum ether, ethyl acetate and water. Take the aqueous layer and perform macroporous resin column chromatography (D-101) with 50 times its weight, eluenting with 2.5 L each of 20%, 40% and 80% ethanol. Collect the 40% ethanol eluent to obtain Fr3 (34 g). Collect the 80% ethanol eluent (86 g) and perform LH-20 gel column chromatography with 100 times its weight, eluenting with 1.5 L of 100% methanol to obtain Fr2 (15 g).

[0052] S2: Fr2 was subjected to YMC 50μm C18 column chromatography with 100 times its weight. Gradient elution was performed with methanol-water at volume ratios of 30:70, 40:60, and 50:50 (1 L each). One test tube was collected for every 10 ml. The eluents containing the same components were combined and observed by TLC to obtain 7 components from Fr2.1 to Fr2.7.

[0053] S3: Fr2.2 (1.26 g) was prepared by high-speed countercurrent chromatography using 5 L of dichloromethane-methanol-water at a volume ratio of 2:2:1. Each 20 mL mobile phase was collected in one test tube, and the same components were combined by TLC to obtain four components: Fr2.2.1 to Fr2.2.4.

[0054] S4: Fr2.2.2 was eluted by silica gel H column chromatography with 1 L of dichloromethane-methanol at a volume ratio of 4:1 to obtain Fr2.2.1.1 (305 mg). Fr2.2.1.1 was then subjected to preparative high performance liquid chromatography using an Agilent ZORBAX SB-C18 preparative column with acetonitrile-water at a volume ratio of 20:80 to prepare Munropin G(1) (38 mg, tR = 15.1 min).

[0055] S5: Fr2.7 was prepared by reverse reverse flow using 2L of dichloromethane-methanol-water at a volume ratio of 2:2:1. Each 20ml of mobile phase was collected in one test tube, and identical components were combined by TLC to obtain four components: Fr2.7.1 to Fr2.7.4. Fr2.7.3 was purified by preparative high performance liquid chromatography using an Agilent ZORBAX SB-C18 preparative column and acetonitrile-water at a volume ratio of 15:85 to obtain Munropin I(3) (8.6mg, tR = 20.6min).

[0056] S6: Fr3 (30g) was subjected to silica gel column chromatography with 100 times its weight of silica gel. Gradient elution was performed using dichloromethane-methanol at volume ratios of 80:20, 90:10, and 100:0 (1.5L each). One tube was collected for every 10ml of fluid. The eluents containing the same components were combined and observed by TLC. A total of 20 fractions were obtained from Fr3.1 to Fr3.20. Fr3.2 (168mg) was prepared by preparative high performance liquid chromatography using an Agilent ZORBAX SB-C18 preparative column with acetonitrile-water at a volume ratio of 20:80 to obtain Munropin H (2) (17.8mg, tR = 20.6min).

[0057] Identification of limonoid compounds Munropin G-L

[0058] Optical rotation was determined using a Jasco P-2200 polarimeter. Mass spectrometry was performed using a Waters LCT PREMIER 2695 or LC / MS-IT-TOF mass spectrometer. Nuclear magnetic resonance spectra were recorded using a Bruker AVANCE III-HD 500 spectrometer and a JEOL JNM-ECZL 500R spectrometer, and MeOH (δ¹⁸O₂) was used for analysis. H 3.30andδ C 49.0) and chloroform (δ H 7.26andδ C The resonance at 77.0) was used as an internal control. UV and ECD spectra were performed using a Hitachi U-3900H and a JASCO J-1500 spectrophotometer, respectively. CCC was performed on a TBE-300C system (Tauto biotechnology, Shanghai, China). HPLC analysis was performed using an Agilent 1260 Infinity II LC (Agilent Technologies, USA), employing an Agilent Poroshell 120SB-C18 (4 mm, 4.6 mm x 150 mm, Agilent, USA), a ChromCore 120-C18 (5 μm, 10 mm x 250 mm, NanoChrom, China), and an Agilent ZORBAX cb-C18 (5 μm, 9.4 mm x 250 mm, Agilent, USA). Column chromatography was performed using silica gel (200-300 mesh, Qingdao Marine Chemical Plant, China), MCI gel (Mitsubishi Chemical Corporation, Japan), and RP-C18 column (Fuji Silysia Chemical Ltd, Japan).

[0059] Munropin G(1): Colorless amorphous solid with molecular formula C 40 H58 O 19 The molecular weight is 865.3465; [α] D 20 -36.35(c 0.10,90% MeOH aq.); IR(KBr)ν max 3425, 2967, 1728, and 1633cm -1 ;UV(MeOH)λ max 209(ε14,153)nm; ECD(MeOH)Δε(nm)+34.6(217); HRESIMS: m / z865.3422([M+Na] + The proton and carbon NMR spectra are shown in Table 1. The 2D NMR correlation and planar structure diagram are shown below. Figure 1 As shown.

[0060] Munropin H(2): Colorless solid; molecular formula is C 38 H 56 O 18 The molecular weight of Na is 823.3359; [α] D 22 25.85(c0.10,MeOH); UV(MeOH)λmax(logε)216(5.1)nm; CD(MeOH)Δε(nm)+12.26(218); HRESIMS: m / z 823.3607[M+Na] + The proton and carbon NMR spectra are shown in Table 1. The 2D NMR correlation and planar structure diagram are shown below. Figure 2 As shown.

[0061] Munropin I(3): Colorless solid; molecular formula is C 34 H 54 O 12 Na, 677.3507; [α] D 22 -43.67(c 0.10,MeOH); UV(MeOH)λmax(logε)194(6.4)nm; CD(MeOH)Δε(nm)+8.79(199)and-0.57(212); HRESIMS: m / z 677.3609[M+Na] + The proton and carbon NMR spectra are shown in Table 1. The 2D NMR correlation and planar structure diagram are shown below. Figure 3 As shown.

[0062] Munropin J(4): Colorless solid; molecular formula is C 33 H 46 O 14Na, 689.2785; [α] D 21 +22(c 0.10,MeOH); UV(MeOH)λmax(logε)210(4.1)nm; CD(MeOH)Δε(nm)+4.13(226)and+4.56(246); HRESIMS: m / z 689.2756[M+Na] + The proton and carbon NMR spectra are shown in Table 2. The 2D NMR correlation and planar structure diagram are shown below. Figure 4 As shown.

[0063] Munropin K(5): Colorless solid; molecular formula is C 36 H 46 O 15 Na, 741.2734; [α] D 21 -148(c 0.10,MeOH); UV(MeOH)λmax(logε)209(4.6)nm; ECD(MeOH)Δε(nm)+10.29(202)and+3.28(252); HRESIMS: m / z 741.2703[M+Na] + The proton and carbon NMR spectra are shown in Table 2. The 2D NMR correlation and planar structure diagram are shown below. Figure 5 As shown.

[0064] Munropin L(6): Colorless solid; molecular formula is C 36 H 46 O 15 Na, 741.2734; [α] D 21 -127(c 0.10,MeOH); UV(MeOH)λmax(logε)210(4.9)nm; ECD(MeOH)Δε(nm)+12.66(208)and+11.84(248); HRESIMS: m / z 741.2724[M+Na] + The proton and carbon NMR spectra are shown in Table 2. The 2D NMR correlation and planar structure diagram are shown below. Figure 6 As shown.

[0065] Cytotoxicity assays of limonene compounds Munropin G-L

[0066] The cytotoxicity of compounds Munropin G-L against human non-small cell lung cancer cell line A549, human colon cancer cell line HCT116, mouse mononuclear macrophage leukemia cell line RAW264.7, liver cancer cell line HepG2, triple-negative breast cancer cells MCF7, and human breast ductal carcinoma cells MDAMB was detected using the CCK-8 assay. 100 μL of cell suspension (2 × 10⁻⁶ cells / mL) was used as the assay result. ^5 Cells were seeded at 1000 cells / mL in 96-well microplates and cultured for 24 h. Then, different concentrations of Munropin G–L (5, 10, 20, 40, 80, 160 μM) were added to the culture dishes. An equal volume of DMSO was added to the control group. Each group was repeated in triplicate. Cells were cultured for 72 h, and 10 μL of C CK-8 reagent was added to each well. After 1 h, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0067] Table 1. Carbon and proton NMR spectra (NMR spectra) 1 H and 13 C NMR data for MunropinsG-I(1-3)inCD3OD)

[0068]

[0069]

[0070]

[0071] Table 2. Carbon and proton NMR spectra (NMR spectra) 1 H and 13 C NMR data for munropinsJ-L(4-6)inCDCl3)

[0072]

[0073]

[0074]

[0075] Table 3 Cytotoxic effects (IC50) 50 (μM))

[0076] Compounds HCT116 A549 HepG2 MCF7 MDAMB Munropin G 26.0 >160 >160 >160 >160 Munropin H 14.6 >160 >160 >160 >160 Munropin I >160 >160 >160 >160 >160 Munropin J >160 >160 >160 >160 >160 Munropin K >160 >160 >160 >160 >160 Munropin L >160 >160 >160 >160 >160

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A limonoid compound derived from dwarf limon, said limonoid compound having one of the structures of Munropin G, Munropin H, Munropin I, Munropin J, Munropin K, and Munropin L, wherein Munropin G, Munropin H, Munropin I, Munropin J, Munropin K, and Munropin L are as shown in formulas (I) to (VI):

2. A method for preparing the limonin-like compound as described in claim 1, characterized in that, The limonoid compound is Munropin G. The preparation method of Munropin G includes the following steps: S1: Take dried whole plant of *Gnaphalium affine* and add 95% ethanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature, each time for 3 days. Combine the filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of petroleum ether, ethyl acetate and water. Take the aqueous layer and perform macroporous resin column chromatography with 50 times its weight, and elute with 2.0-3.0L each of 20%, 40%, and 80% ethanol. Collect the 80% ethanol eluent and perform gel column chromatography again with 100 times its weight, and continue to elute with 1.0-2.0L of 100% methanol to obtain Fr2. S2: Fr2 was subjected to C18 column chromatography with 100 times its weight. Gradient elution was performed using methanol-water at volume ratios of 30:70, 40:60, and 50:50, with 1.0 to 1.5 L of each mixture. One test tube was collected for every 10 to 15 ml of the mixture. The eluents containing the same components were combined and observed by TLC to obtain a total of 7 fractions, Fr2.1 to Fr2.

7. S3: Fr2.2 was prepared by high-speed countercurrent chromatography using 4.5-5.5 L of dichloromethane-methanol-water at a volume ratio of 2:2:

1. Each 20-25 ml of mobile phase was collected in one test tube, and the same components were combined by TLC to obtain four components: Fr2.2.1 to Fr2.2.

4. S4: Fr2.2.2 was subjected to silica gel column chromatography and eluted with 1.0-1.5 L of dichloromethane-methanol at a volume ratio of 4:1 to obtain Fr2.2.2.

1. Fr2.2.2.1 was then subjected to preparative high performance liquid chromatography using a C18 preparative column and acetonitrile-water at a volume ratio of 20:80 as the mobile phase, with a retention time of 12-18 min to prepare Munropin G.

3. A method for preparing the limonin-like compound as described in claim 1, characterized in that, The limonene compound is Munropin H, and the preparation method of Munropin H includes the following steps: S1: Take dried whole plant of *Gnaphalium affine* and add 95% ethanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature, each time for three days. Combine the filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of petroleum ether, ethyl acetate and water. Take the aqueous layer and perform column chromatography with 50 times its weight of macroporous resin. Elute with 2.0-3.0L each of 20%, 40% and 80% ethanol. Collect the 40% ethanol eluent to obtain Fr3. S2: Take Fr3 and perform silica gel column chromatography with 100 times its weight. Use dichloromethane-methanol at volume ratios of 80:20, 90:10, and 100:0 as the mobile phase for gradient elution. Collect 10-15 ml of each test tube. Observe the eluent containing the same components in each test tube by TLC. Combine the eluents. There are 20 fractions in total, Fr3.1 to Fr3.

20. S3: Take Fr3.2 and prepare Munropin H by high performance liquid chromatography using a C18 column and acetonitrile-water at a volume ratio of 20:80 as the mobile phase, with a retention time of 18-22 min.

4. A method for preparing the limonin-like compound as described in claim 1, characterized in that, The limonoid compound is Munropin I. The preparation method of Munropin I includes the following steps: S1: Take dried whole plant of *Gnaphalium affine* and add 95% ethanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature, each time for 3 days. Combine the filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of petroleum ether, ethyl acetate and water. Take the aqueous layer and perform macroporous resin column chromatography with 50 times its weight, and elute with 2.0-3.0L each of 20%, 40%, and 80% ethanol. Collect the 80% ethanol eluent and perform gel column chromatography again with 100 times its weight, and continue to elute with 1.0-2.0L of 100% methanol to obtain Fr2. S2: Fr2 was subjected to C18 column chromatography with 100 times its weight. Gradient elution was performed using methanol-water at volume ratios of 30:70, 40:60, and 50:50, with 1.0 to 1.5 L of each mixture. One test tube was collected for every 10 to 15 ml of the mixture. The eluents containing the same components were combined and observed by TLC to obtain a total of 7 fractions, Fr2.1 to Fr2.

7. S3: Fr2.7 was prepared by reverse flow using 2.0-2.5 L of dichloromethane-methanol-water at a volume ratio of 2:2:

1. Each 20-25 ml of mobile phase was collected in one test tube, and the same components were combined by TLC to obtain four components: Fr2.7.1 to Fr2.7.

4. S4: Fr2.7.3 was purified by preparative high performance liquid chromatography using a C18 column and acetonitrile-water at a volume ratio of 15:85 as the mobile phase, with a retention time of 18–24 min to obtain Munropin I.

5. A method for preparing the limonin compound as described in claim 1, characterized in that, The limonoid compound is Munropin J or K. The preparation method of Munropin J or K includes the following steps: S1: Take dried whole plant of *Gnaphalium affine* and add 100% methanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature, each time for 3 days. Combine the extracts and filtrates, filter, concentrate and obtain the extract. Extract the extract sequentially by equal volumes of ethyl acetate and water. The ethyl acetate layer is subjected to silica gel chromatography column elution using gradient elution with n-hexane-ethyl acetate at volume ratios of 90:10, 80:20, 60:40 and 25:75, 2.0-2.5L each. Collect 10-15ml in one test tube. Observe each test tube by TLC and combine the eluents containing the same components to obtain 12 components from Fr1 to Fr12. S2: Fr11 was subjected to MCI resin column chromatography with 100 times its weight, and eluted with 3L of 100% methanol. One test tube was collected for every 10ml. Each test tube was observed by TLC. The eluents containing the same components were combined to obtain 5 components from Fr11.1 to Fr11.

5. S3: Fr.11.1 was subjected to GPC-HPLC with a SEC column as the preparative column and 100% methanol as the mobile phase. A total of 1.0-1.5 L of elution was used, and 10-15 ml of each tube was collected. The eluents containing the same components were combined and observed by TLC. Seven components, Fr11.1.1 to Fr11.1.7, were obtained. S4: Fr11.1.5 was subjected to reversed-phase high-performance liquid chromatography using a C18 column as the preparative column and acetonitrile-water as the mobile phase with a volume ratio of 35:

65. Munropin J and Munropin K were prepared sequentially with retention times of 20-25 min.

6. A method for preparing the limonin compound as described in claim 1, characterized in that, The limonoid compound is Munropin L. The preparation method of Munropin L includes the following steps: S1: Take dried whole plant of *Gnaphalium affine* and add 100% methanol at a material-to-liquid ratio of 1kg:10-15L. Soak and extract three times at room temperature, each time for 3 days. Combine the extracts and filtrates, filter, concentrate and obtain the extract. Extract the extract by sequentially extracting with equal volumes of ethyl acetate and water. Separate the ethyl acetate layer by silica gel column chromatography using gradient elution with n-hexane-ethyl acetate at volume ratios of 90:10, 80:20, 60:40 and 25:75, 2.0-2.5L each. Collect 10-15ml in one test tube. Observe each test tube by TLC and combine the eluents containing the same components to obtain 12 components from Fr1 to Fr12. S2: Fr11 was subjected to MCI resin column chromatography with 100 times its weight, and eluted with 3L of 100% methanol. One test tube was collected for every 10ml. Each test tube was observed by TLC. The eluents containing the same components were combined to obtain 5 components from Fr11.1 to Fr11.

5. S3: Fr.11.1 was subjected to GPC-HPLC with a SEC column as the preparative column and 100% methanol as the mobile phase. A total of 1.0-1.5 L of elution was used, and 10-15 ml of each tube was collected. The eluents containing the same components were combined and observed by TLC. Seven components, Fr11.1.1 to Fr11.1.7, were obtained. S4: Fr11.1.3 was subjected to reversed-phase high-performance liquid chromatography using a C18 preparative column and acetonitrile-water as the mobile phase with a volume ratio of 35:65 to obtain Munropin L at a retention time of 20-30 min.

7. Use of the limonene compound of claim 1 in the preparation of a drug for treating colon cancer.

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