Steroid compounds from euphorbia fischeriana steud and extraction method and anti-tumor use thereof

Abstract

This invention relates to steroidal ketone compounds from *Echinops urinaria*, their extraction methods, and their antitumor applications. The structures of the steroidal ketone compounds are shown in Formula I. This invention studies the active components of *Echinops urinaria*, and has isolated several steroidal ketone compounds from its roots, two of which are newly discovered. Furthermore, the antitumor activity of the isolated steroidal ketone compounds has been studied, revealing that these compounds exhibit inhibitory activity against multiple tumor cell lines. This invention lays the foundation for in-depth research on the active components of *Echinops urinaria* and the development of new drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to a steroidal compound from *Echinops latifolius*, its extraction method, and its antitumor uses. Background Technology

[0002] Due to the structural diversity, biological activity, and low toxicity of natural derivatives, their use as medicines has attracted great attention.

[0003] Rhaponticum uniflorum (L.) DC., belonging to the genus Rhaponticum in the family Asteraceae, is a perennial herb. It is listed as a superior medicinal material in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and is mainly distributed in Anguo City, Hebei Province. Rhaponticum uniflorum possesses various therapeutic effects, including clearing heat and detoxifying, anti-oxidation, regulating blood lipids, anti-tumor, anti-inflammatory, analgesic, and promoting lactation.

[0004] Currently, scholars both domestically and internationally have conducted a series of studies on the chemical composition of *Echinops latifolius* (Qizhou *Lou Lu*). The main chemical components discovered include phytoecdystones, triterpenoids, and thiophenes. Other components include flavonoids and volatile oils such as *Achyranthes bidentata* ketone, *Echinops latifolius* ketone, and *Stachyranthes bidentata* ketone, which have attracted scholarly attention as ecdystone compounds. CN1334093A, on the medicinal uses of phytosterone components in *Lou Lutifolius*, provides new applications for these components and offers a new avenue for the development of this traditional Chinese medicine. Phytosterone components in *Lou Lutifolius* can be used to prepare drugs for treating osteoporosis, as well as drugs with bone resorption inhibitory activity and those promoting chondrocyte DNA growth. They can also be used to prepare drugs with nootropic and antioxidant effects. CN1224023A discloses that the main component of total sterones in *Lou Lutifolius* is β-ecdystone, primarily used to improve memory impairment caused by brain dysfunction.

[0005] Given that further research is needed on the types and efficacy of sterones in *Lysimachia christinae* from Qizhou, this invention has conducted relevant research. Summary of the Invention

[0006] The purpose of this invention is to overcome the deficiencies in the prior art, to provide new sterone compounds from the roots of *Lysimachia christinae*, and to provide the antitumor activity of a series of similar isolated compounds, as well as their extraction methods.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides, in one aspect, the use of sterone compounds as shown in Formula I in the preparation of antitumor drugs, the structure of which is as follows:

[0009]

[0010] R1 is selected from H, OH, acetoxy, glucose, rhamnose, or a disaccharide composed of glucose and rhamnose;

[0011] R2 is selected from H, OH, C1-C5 straight-chain or branched hydrocarbon groups;

[0012] R3 is selected from H or OH;

[0013] R4 is selected from H or OH.

[0014] As a further improvement of the present invention, the C1-C5 straight-chain or branched hydrocarbon group is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl.

[0015] The C1-C5 alkyl groups are selected from —CH3, —CH2CH3, —CH(CH3)2, —(CH2)2CH3, —(CH2)3CH3, —(CH2)4CH3, —CH(CH3)CH2CH3, —C(CH3)3, —CH(CH3)(CH2)2CH3, —CH2CH(CH2)2CH3, and —CH2C(CH2)3;

[0016] The C2-C5 alkenyl group is selected from vinyl, isopropenyl, n-propenyl, n-butenyl, isobutenyl, sec-butenyl, n-pentenyl, and isopentenyl.

[0017] The C2-C5 ynyl group is selected from ethynyl, propynyl, butynyl, and pentylyl.

[0018] As a further improvement of the present invention, R1 is selected from H, OH; R2 is selected from H, OH, ethyl; R3 is selected from H or OH; and R4 is selected from H or OH.

[0019] As a further improvement to the present invention, the following compounds are selected:

[0020]

[0021]

[0022] Another aspect of the present invention provides an 11α-sterone compound represented by Formula II, the structure of which is as follows:

[0023]

[0024] R2 is selected from H, C1-C5 straight-chain or branched hydrocarbon groups.

[0025] As a further improvement of the present invention, the C1-C5 straight-chain or branched hydrocarbon group is selected from C1-C5 alkyl, C2-C5 alkenyl, and C2-C5 alkynyl.

[0026] The C1-C5 alkyl groups are selected from —CH3, —CH2CH3, —CH(CH3)2, —(CH2)2CH3, —(CH2)3CH3, —(CH2)4CH3, —CH(CH3)CH2CH3, —C(CH3)3, —CH(CH3)(CH2)2CH3, —CH2CH(CH2)2CH3, and —CH2C(CH2)3;

[0027] The C2-C5 alkenyl group is selected from vinyl, isopropenyl, n-propenyl, n-butenyl, isobutenyl, sec-butenyl, n-pentenyl, and isopentenyl.

[0028] The C2-C5 ynyl group is selected from ethynyl, propynyl, butynyl, and pentylyl.

[0029] As a further improvement to the present invention, compounds selected from the following compounds are used.

[0030]

[0031] In another aspect, the present invention provides a method for extracting compounds of formula II, specifically comprising the following steps:

[0032] The roots of *Lysimachia christinae* were pulverized and extracted with an alcohol-water solution under reflux. The extract was filtered and concentrated under reduced pressure. Water was added to the concentrate, and the extract was successively extracted with petroleum ether, dichloromethane, and ethyl acetate. The ethyl acetate phase and aqueous phase were concentrated under reduced pressure to obtain petroleum ether concentrate and aqueous phase concentrate, respectively.

[0033] The petroleum ether concentrate and the aqueous concentrate were separated by medium-pressure ODS column and purified by preparative HPLC.

[0034] As a further improvement of the present invention, the method for separating the aqueous phase enrichment is as follows:

[0035] S1 medium-pressure ODS column separation: Water-enriched material was loaded onto the column, and the chromatographic conditions were: methanol-water (0:100→100:0) gradient elution for 24 h, flow rate 80 mL / min, column volume of about 5 L, detection wavelength 254 nm, and 20 components were obtained, which were referred to as components 1-20 respectively.

[0036] S2 preparation and HPLC separation:

[0037] Component 6 obtained from S1 was separated under the following chromatographic conditions: isocratic elution with 25% acetonitrile and water for 35 min to obtain compound 1 (t). R =15.7min) and compound 2 (t R =28.3min);

[0038] Component 5 obtained from S1 was separated under the following chromatographic conditions: gradient elution of 15%-35% acetonitrile and water for 40 min, yielding compound 10 (35.9 mg, t). R =32.3min).

[0039] As a further improvement of the present invention, the method for separating the ethyl acetate enrichment is as follows:

[0040] S1 One-time atmospheric pressure silica gel column separation: dichloromethane-methanol (100:0→0:100) gradient elution, the separation and combination yielded 5 components, denoted as components 1-5;

[0041] S2 secondary atmospheric pressure silica gel column separation: Component 2 obtained from S1 was loaded onto the column and eluted with a gradient of dichloromethane-methanol (98:2→0:100). The separated and combined components were 6, denoted as components Fr1-6.

[0042] S3 medium-pressure ODS column chromatography separation:

[0043] S3.1 The fraction obtained in S2 was loaded onto a column using Fr3 and eluted with a methanol-water (10%-100%) gradient for 5 h at a flow rate of 20 mL / min and a column volume of 600 mL. Similar fractions were combined to obtain a total of 10 fractions, which were denoted as fractions 1-10.

[0044] S3.2 The fraction Fr6 obtained in S2 was subjected to medium-pressure ODS column chromatography with methanol-water gradient elution (0:100→100:0) for 6 h at a flow rate of 20 mL / min and a column volume of 1000 mL. Similar fractions were combined to obtain a total of 13 fractions, which were denoted as fractions 1-13.

[0045] S4 preparative HPLC separation:

[0046] Component 9 obtained from S3.1 was separated under the following chromatographic conditions: 20%-45% acetonitrile-water gradient elution for 40 min to obtain compound 3 (t). R =15.7min) and compound 4 (t R =30.2min).

[0047] Component 12 obtained from S3.2 was separated under the following chromatographic conditions: 18%-40% acetonitrile gradient elution for 35 min, yielding compound 5 (t). R =12.7min), compound 6 (t R =15.8min) and compound 7 (t R =28.5min).

[0048] Component 13 obtained from S3.2 was separated under the following chromatographic conditions: gradient elution of 10%-55% acetonitrile and water for 45 min, flow rate 20 mL / min, and detection wavelength 254 nm, yielding compound 8 (t).R =21.4min) and compound 9 (t R =39.5min).

[0049] The beneficial effects of adopting the above technical solution are as follows:

[0050] 1. This invention studies the active components of *Echinops buergeriana*, and isolates 10 steroidal ketone compounds from the roots of *Echinops buergeriana*, two of which are discovered for the first time.

[0051] 2. This invention studies the antitumor activity of the isolated sterone compounds and finds that the above compounds have inhibitory activity against multiple tumor cell lines. This invention lays the foundation for in-depth research on the active ingredients of Leucas cephalotes and the development of new drugs. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0053] Unless otherwise specified, the equipment used in this embodiment is conventional equipment, the reagents used are all conventional reagents that can be purchased commercially, and the operating methods used are all recorded in textbooks in this field.

[0054] Example 1

[0055] The root of *Lysimachia christinae* from Qizhou was appropriately pulverized and extracted by reflux with 95% ethanol. The extract was filtered and concentrated under reduced pressure to a paste. The paste was dispersed in distilled water and extracted with petroleum ether, dichloromethane, and ethyl acetate, respectively. The ethyl acetate phase and aqueous phase were concentrated under reduced pressure to obtain ethyl acetate concentrate and aqueous concentrate, respectively.

[0056] (1) Separation of aqueous phase concentrates:

[0057] S1 medium-pressure ODS column separation, chromatographic conditions: methanol-water (0:100→100:0) gradient elution for 24h (in this example, both medium-pressure and high-pressure liquid chromatography gradient elution are performed at a uniform gradient change within the set time), flow rate 80mL / min, column volume approximately 5L, detection wavelength 254nm, 20 components were obtained, which are denoted as components 1-20.

[0058] S2 preparation and HPLC separation:

[0059] Component 6 obtained from S1 was separated under the following chromatographic conditions: isocratic elution with 25% acetonitrile and water for 35 min to obtain compound 1 (t). R =15.7min) and compound 2 (t R =28.3min);

[0060] Component 5 obtained from S1 was separated under the following chromatographic conditions: gradient elution of 15%-35% acetonitrile and water for 40 min, yielding compound 10 (35.9 mg, t). R =32.3min).

[0061] (2) Separation of ethyl acetate concentrate:

[0062] S1 First-stage atmospheric pressure silica gel column separation: dichloromethane-methanol (100:0→0:100) gradient elution, the separation and merging yielded 5 components, which are referred to as components 1-5 respectively.

[0063] S2 secondary atmospheric pressure silica gel column separation: Component 2 obtained from S1 was loaded onto the column and eluted with a gradient of dichloromethane-methanol (98:2→0:100). The separated and combined components were 6, which were denoted as components Fr1-6.

[0064] S3 medium-pressure ODS column chromatography separation:

[0065] S3.1 The fraction obtained in S2 was loaded onto a column using Fr3. The column was eluted with a methanol-water (10%-100%) gradient for 5 h at a flow rate of 20 mL / min and a column volume of 600 mL. Similar fractions were combined to obtain a total of 10 fractions, which were designated as fractions 1-10.

[0066] S3.2 The fraction Fr6 obtained in S2 was subjected to medium-pressure ODS column chromatography with a methanol-water gradient elution (0:100→100:0) for 6 h at a flow rate of 20 mL / min and a column volume of 1000 mL. Similar fractions were combined to obtain a total of 13 fractions, which were designated as fractions 1-13.

[0067] S4 preparative HPLC separation:

[0068] Component 9 obtained from S3.1 was separated under the following chromatographic conditions: 20%-45% acetonitrile-water gradient elution for 40 min to obtain compound 3 (t). R =15.7min) and compound 4 (t R =30.2min).

[0069] Component 12 obtained from S3.2 was separated under the following chromatographic conditions: 18%-40% acetonitrile gradient elution for 35 min, yielding compound 5 (t). R =12.7min), compound 6 (t R =15.8min) and compound 7 (t R =28.5min).

[0070] Component 13 obtained from S3.2 was separated under the following chromatographic conditions: gradient elution of 10%-55% acetonitrile and water for 45 min, flow rate 20 mL / min, and detection wavelength 254 nm, yielding compound 8 (t).R =21.4min) and compound 9 (t R =39.5min).

[0071] Compound 1, 11α-hydroxyachysterone

[0072]

[0073] Table 1. NMR data of compound 1 (600 MHz, MeOH-d4)

[0074]

[0075] Compound 2, 11α-hydroxyamazinate A

[0076]

[0077] Table 2. NMR data of compound 2 (600 MHz, MeOH-d4)

[0078]

[0079]

[0080] Compound 3, 20-hydroxyecdysterone

[0081]

[0082] Table 3. NMR data of compound 3 (600 MHz, MeOH-d4)

[0083]

[0084] Compound 4, ajugaside C

[0085]

[0086] Table 4. NMR data of compound 4 (600 MHz, MeOH-d4)

[0087]

[0088]

[0089] Compound 5, Achyranthesinone

[0090]

[0091] Table 5. NMR data of compound 5 (600 MHz, MeOH-d4)

[0092]

[0093] Compound 6(24R,25R)-Amarantrol A

[0094]

[0095] Table 6. NMR data of compound 6 (600 MHz, MeOH-d4)

[0096]

[0097]

[0098] Compound 7 saturates

[0099]

[0100] Table 7. NMR data of compound 7 (600 MHz, MeOH-d4)

[0101]

[0102] Compound 8(24R)-11,20,24-trihydroxyecdysterone

[0103]

[0104] Table 8. NMR data of compound 8 (600 MHz, D2O)

[0105]

[0106]

[0107] Compound 9, Podophyllin C

[0108]

[0109] Table 9. NMR data of compound 9 (600 MHz, MeOH-d4)

[0110]

[0111] Compound 10 Leucosterone

[0112]

[0113] Table 10 Data for Compound 10 (600MHz, MeOH-d4)

[0114]

[0115]

[0116] Example 2: Study on antitumor activity

[0117] The compound obtained in Example 1 was found to have moderate inhibitory activity against human cervical tumor cell line (HeLa), human astrocytoma cell line (U87MG), and human glioma cell line (U251).

[0118] Tumor cell lines were placed in RPMI 1640 medium and incubated in a 5% CO2 incubator at 37°C with saturated humidity. The medium was changed every 48 hours. Once the cells reached the logarithmic growth phase, the supernatant was discarded, and the cells were washed twice with PBS buffer. Adherent cells were then detached using 0.25% trypsin, and clumps of cells were broken into single cells. Digestion was carried out for approximately 15 minutes, and then PBS buffer was added to terminate the digestion.

[0119] The MTT assay was used to test the inhibitory activity of the compounds on tumor cell proliferation. Tumor cells were seeded into 96-well plates, approximately 5 × 10⁴ cells per well. Blank control PBS buffer and 50 μL of each of the 15 target monomer compounds at a concentration of 10 μmol / L were added sequentially. The plates were incubated in a 5% CO₂ incubator at 37°C for 44 h. Then, 10 μL of MTT was added to each well, and the plates were incubated for another 4 h. The supernatant was discarded, and 150 μL of stop solution was added to each well. The plates were then incubated for 1 h. The OD value of each well was measured at 450 nm using a microplate reader, and the tumor cell viability was calculated using the formula below.

[0120] Tumor cell survival rate (%) = (OD value of experimental group) / (OD value of control group) × 100

[0121] The experimental data results are expressed as mean ± standard deviation (mean ± SD). The t-test method in statistical software was used to calculate the results for every two groups of data. The above experimental steps and results were performed in parallel three times, and the final result was the average value.

[0122] The results are shown in Table 11: The 10 compounds obtained in Example 1 showed moderate inhibitory activity against human cervical tumor cell line (HeLa), human astrocytoma cell line (U87MG), and human glioma cell line (U251). Compound 1 showed a significant effect on HeLa cells; Compound 2 showed a significant effect on U87MG and has the potential to develop related drugs.

[0123] Table 11 Inhibitory activity of compounds against tumor cells

[0124]

[0125]

[0126] 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 method for extracting a steroid compound, characterized by, The steroidal compounds are compound 1-compound 10, and specifically include the following steps: The roots of Echinopsis multiplicata are crushed, and extracted by refluxing with an alcohol-water solution. The extract is filtered and concentrated under reduced pressure. Water is added to the concentrate, and the mixture is extracted with petroleum ether, dichloromethane, and ethyl acetate, respectively. The ethyl acetate phase and the water phase are concentrated under reduced pressure to obtain ethyl acetate-enriched material and water-enriched material, respectively. The ethyl acetate-enriched material and the water-enriched material are separated by a medium-pressure ODS column, and then separated and purified by preparative HPLC to obtain the following compounds: (1) The separation method of the water-enriched material is as follows: S1. Medium-pressure ODS column separation: The water-enriched material is loaded onto the column, and eluted with a gradient of methanol-water (0:100→100:0) for 24 h at a flow rate of 80 mL / min. The column volume is about 5 L, and the detection wavelength is 254 nm. Twenty components are separated and are denoted as components 1-20, respectively. S2. Preparative HPLC separation: Component 6 obtained in S1 is separated under the following conditions: 25% acetonitrile-water isocratic elution for 35 min, to obtain compound 1 and compound 2. Component 5 obtained in S1 is separated under the following conditions: 15%-35% acetonitrile-water gradient elution for 40 min, to obtain compound 10. (2) The separation method of the ethyl acetate-enriched material is as follows: S1. First normal-pressure silica gel column separation: dichloromethane-methanol (100:0→0:100) gradient elution, to obtain five components, which are denoted as components 1-5, respectively. S2. Second normal-pressure silica gel column separation: component 2 obtained in S1 is loaded onto the column, and eluted with a gradient of dichloromethane-methanol (98:2→0:100), to obtain six components, which are denoted as components Fr1-6, respectively. S3. Medium-pressure ODS column chromatography: S3.

1. Component Fr3 obtained in S2 is loaded onto the column, and eluted with a gradient of methanol-water (10%-100%) for 5 h at a flow rate of 20 mL / min. The column volume is 600 mL, and similar components are combined. Ten components are obtained, which are denoted as components 1-10, respectively. S3.

2. Component Fr6 obtained in S2 is separated by medium-pressure ODS column chromatography, and eluted with a gradient of methanol-water (0:100→100:0) for 6 h at a flow rate of 20 mL / min. The column volume is 1000 mL, and similar components are combined. Thirteen components are obtained, which are denoted as components 1-13, respectively. S4. Preparative HPLC separation: Component 9 obtained in S3.1 is separated under the following conditions: 20%-45% acetonitrile-water gradient elution for 40 min, to obtain compound 3 and compound 4. Component 12 obtained in S3.2 is separated under the following conditions: 18%-40% acetonitrile gradient elution for 35 min, to obtain compound 5, compound 6, and compound 7. Component 13 obtained in S3.2 is separated under the following conditions: 10%-55% acetonitrile-water gradient elution for 45 min at a flow rate of 20 mL / min, and the detection wavelength is 254 nm, to obtain compound 8 and compound 9. The structures of the compounds 1-10 are as follows: ； 。 2. The method for extracting sterone compounds according to claim 1, characterized in that, The t of compound 1 R =15.7 min, t of compound 2 R =28.3 min, t of compound 10 R =32.3 min, t of compound 3 R =15.7 min, t of compound 4 R =30.2 min, t of compound 5 R =12.7 min, t of compound 6 R =15.8 min, t of compound 7 R =28.5 min, t of compound 8 R =21.4 min, t of compound 9 R =39.5 min.

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