Euphorbia pekinensis diterpene in euphorbia helioscopia and preparation method and application thereof

Abstract

The invention belongs to the technical field of natural medicinal chemistry, and relates to four diterpenes extracted and separated from euphorbia helioscopia L as well as a preparation method and application of the four diterpenes. The four diterpenes are extracted and separated from euphorbia helioscopia L as a euphorbia helioscopia L. Compounds 1 and 2 have a 5 / 11 / 3 tricyclic system skeleton. And the compound 3 has a 6 / 6 / 6 tricyclic system skeleton. And the compound 4 has a 5 / 12 macrocyclic system skeleton. The invention also relates to a preparation method of the compound and an application of the compound in an anti-tumor aspect. Reagents needed by the preparation method are low in price and convenient and easy to obtain, and the obtained compound has certain inhibitory activity on A549 and H446 tumor cell lines, has the advantages of being easy and convenient to operate and good in reproducibility, is beneficial to industrial production and has certain potential value and application prospects in the fields of medicine and medicinal chemistry. # imgabs0 #

Description

Technical Field:

[0001] The present invention belongs to the technical field of natural medicine chemistry, and particularly relates to the euphorbia diterpenoids isolated from the plant Euphorbia helioscopia L, their preparation methods, and the applications of such compounds in anti-tumor aspects. Background Art:

[0002] Euphorbia helioscopia L is the dried aerial part of the genus Euphorbia in the Euphorbiaceae family. Alias: Cat's Eye, Five-Wind Grass. It is an annual herbaceous plant that grows in mountain valleys, roadsides, wastelands, and slopes. Euphorbia helioscopia L has a long history of medicinal use in China, and its whole herb is used as medicine. It is slightly cold in nature, bitter in taste, and slightly poisonous, and has the effects of clearing heat, resolving phlegm, promoting diuresis and detumescence, and killing insects.

[0003] Lung cancer is one of the malignant tumors with the fastest growth rate of incidence and mortality, and is the main cause of cancer death, posing the greatest threat to human health and life. In the past 50 years, many countries have reported a significant increase in the incidence and mortality of lung cancer. Therefore, it is important to explore and discover potential natural products against lung cancer. Summary of the Invention:

[0004] The purpose of the present invention is to provide the euphorbia diterpenoids isolated from the plant Euphorbia helioscopia L, their preparation methods, and the applications of such compounds in anti-tumor aspects.

[0005] The present invention relates to new euphorbia diterpenoid compounds 1 to 4 isolated from the plant Euphorbia helioscopia L of the genus Euphorbia in the Euphorbiaceae family, and their structures are shown as follows:

[0006]

[0007] The separation and preparation technical solution of the new euphorbia diterpenoids in Euphorbia helioscopia L of the present invention includes the following steps:

[0008] Take the dried whole herb of Euphorbia helioscopia L and extract it with ethanol. Combine the extraction solutions and concentrate to obtain an extract. The extract is extracted with ethyl acetate and n-butanol, and the obtained components are subjected to silica gel column chromatography, and isocratic gradient elution is carried out with a petroleum ether - ethyl acetate system with a volume ratio of 100:1 - 1:1 and a dichloromethane - methanol system with a volume ratio of 100:0 - 0:100, and a total of 5 fractions A - E are collected.

[0009] Use HP20 column chromatography to carry out gradient elution of fraction B with an ethanol - water system with a volume ratio of 50:50 - 90:10 to obtain two components B1 - B2.

[0010] Use ODS column chromatography to separate components B1 and B2 with an ethanol - water system with a volume ratio of 50:50 - 90:10 respectively to obtain two components B1 - 1 - B1 - 2 and B2 - 1 - B2 - 2 respectively.

[0011] The obtained components B1-2 and B2-1 were each separated by silica gel column chromatography with a petroleum ether-ethyl acetate system in a volume ratio of 80:1 - 2:1 based on TLC analysis to obtain eight sub-components b1-1 - b1-8 and six sub-components b2-1 - b2-6.

[0012] Compound 1 and Compound 2 were obtained by separating b1-5 using a mobile phase of methanol-water on preparative reverse-phase high-performance liquid chromatography, and Compound 3 and Compound 4 were obtained by separating b2-5 using a mobile phase of methanol-water.

[0013] In the above steps, the Euphorbia helioscopia used was the dried whole herb of Euphorbia helioscopia L, a plant of the genus Euphorbia in the family Euphorbiaceae.

[0014] In the above steps, the dried whole herb of Euphorbia helioscopia was reflux-extracted 3 - 4 times with 75% industrial ethanol for 2 - 3 hours each time.

[0015] In the above steps, b1-5 was separated on preparative reverse-phase high-performance liquid chromatography with a methanol-water mobile phase (70:30, v / v), and b2-5 was separated on preparative reverse-phase high-performance liquid chromatography with a methanol-water mobile phase (60:40, v / v).

[0016] The structures of the obtained compounds were identified through a systematic structural identification as follows:

[0017] The structural identification of Compounds 1 - 4 was carried out using high-resolution mass spectrometry, one-dimensional NMR, and two-dimensional NMR. The corresponding spectra and data are shown in Tables 1 - 4 and Figures 1-4 as follows.

[0018] Compound 1: White solid. (1): HRESIMS m / z 535.3039 [M + Na] + (calcd for C 31 H 44 O 6 Na, 535.3036), and the molecular formula was determined to be C 31 H 44 O 6 . By analyzing its 1 H NMR, 13 C NMR, HMQC spectrum, and HMBC spectrum, the structure of Compound 1 was determined, and it was a new compound.

[0019] Compound 2: Yellow solid. HRESIMS m / z 521.2881 [M + Na] + (calcd for C 30 H 42 O 6Na, 521.2879). The molecular formula was determined to be C 30 H 42 O 6 , and by analyzing its 1 HNMR, 13 C NMR, HMQC spectrum, and HMBC spectrum, the structure of Compound 2 was determined to be a new compound.

[0020] Compound 3: White powder. HRESIMS m / z 383.1842 [M+Na] + (calcd for C 21 H 28 O 5 Na, 383.1842). The molecular formula was determined to be C 21 H 28 O 5 , and by analyzing its 1 HNMR, 13 C NMR, HMQC spectrum, and HMBC spectrum, the structure of Compound 3 was determined to be a new compound.

[0021] Compound 4: White powder. HRESIMS m / z 604.2894 [M+H] + (calcd for C 29 H 38 O 6 , 604.2894). The molecular formula was determined to be C 29 H 37 O 6 , and by analyzing its 1 HNMR, 13 C NMR, HMQC spectrum, and HMBC spectrum, the structure of Compound 4 was determined to be a new compound.

[0022] Table 1: 1 H (600 MHz) and 13 C (150 MHz) data (MeOD)

[0023]

[0024]

[0025] Table 2: 1 H (600 MHz) and 13 C (150 MHz) data (CDCl 3 )

[0026]

[0027]

[0028] Table 3: For Compound 3 1 H (600 MHz) and 13 C (150 MHz) data (CDCl 3 )

[0029]

[0030] Table 4: For Compound 4 1 H (600 MHz) and 13 C (150 MHz) data (CDCl 3 )

[0031]

[0032]

[0033] The cytotoxic activities of the novel diterpenoids of the invention were investigated. Compounds 1 - 4 showed certain cell inhibitory activities against H446 and A549 tumor cell lines, indicating potential for further development.

[0034] The advantages of the present invention are that the compounds are all novel compounds with novel structures, and at the same time, they have a certain effect of inhibiting tumor cell lines, showing potential for further development. BRIEF DESCRIPTION OF THE DRAWINGS:

[0035] Figure 1 HRESIMS, 1 H, 13 C - NMR and HMBC spectra of Compound 1;

[0036] Figure 2 HRESIMS, 1 H, 13 C - NMR and HMBC spectra of Compound 2;

[0037] Figure 3 HRESIMS, 1 H, 13 C - NMR and HMBC spectra of Compound 3;

[0038] Figure 4 HRESIMS, 1 H, 13 C - NMR and HMBC spectra of Compound 4. DETAILED DESCRIPTION OF THE INVENTION:

[0039] The following examples are helpful for those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0040] Example 1:

[0041] The separation of neoeuphane diterpenoids from Euphorbia helioscopia L. is carried out as follows:

[0042] Take the dry whole herb of Euphorbia helioscopia L. and reflux extract it with 75% industrial ethanol for 3 - 4 times, 2 - 3 h each time. Combine the extraction solutions and concentrate to obtain an extract. The extract is extracted with ethyl acetate and n-butanol, and the obtained components are separated by silica gel column chromatography. Use petroleum ether - ethyl acetate system with volume ratio of 100:1 - 1:1 and dichloromethane - methanol system with volume ratio of 100:0 - 0:100 for isocratic gradient elution, and a total of 5 fractions A - E are collected.

[0043] Use HP20 column chromatography to gradient elute fraction B with ethanol - water system of 50:50 - 90:10 to obtain two components B1 - B2.

[0044] Use ODS column chromatography to separate components B1 and B2 with ethanol - water system of volume ratio 50:50 - 90:10 respectively, and obtain two components B1-1 - B1-2 and B2-1 - B2-2 respectively.

[0045] The obtained components B1-2 and B2-1 are separated by silica gel column chromatography with petroleum ether - ethyl acetate system of volume ratio 80:1 - 5:1 based on TLC analysis to obtain eight sub-components b1-1 - b1-8 and six sub-components b2-1 - b2-6 respectively.

[0046] Separate b1-5 on preparative reversed-phase high-performance liquid chromatography with methanol - water mobile phase (70:30, v / v) to obtain compound 1 and compound 2, and separate b2-5 on preparative reversed-phase high-performance liquid chromatography with methanol - water mobile phase (60:40, v / v) to obtain compound 3 and compound 4.

[0047] Example 2: Detection of the effects of compounds 1 - 4 on the viability of A549 and H446 cells

[0048] Detect using the SRB method. Take A549 and H446 cells in the logarithmic growth phase, with 1×10 3Cells were seeded at a density of 对照 cells / well in a 96-well plate and treated with drug compounds 1-4 and the positive drug cisplatin (DDP) at different concentrations (0, 3.125, 6.25, 12.5, 25, 50 μM) for 72 h. Then, 50 μL of 30% trichloroacetic acid (TCA) solution was added to each well to fix the cells for 1 h. The culture medium and fixative were removed, and the wells were rinsed with tap water. Subsequently, 50 μL of SRB dye was added to each well, and the plate was incubated at room temperature in the dark for 30 min. The 96-well plate was then rinsed with 1% acetic acid until the eluate was colorless, and the plate was air-dried. Tris (100 μL) was added to the air-dried 96-well plate, and the plate was shaken for 20 min. Finally, the absorbance at a wavelength of 540 nm was measured using a microplate reader. The inhibition rate of the drug on tumor cells was calculated based on the absorbance values. The cell growth inhibition rate (%) = (A 加药 ) / (A 对照 - A 空白 ) × 100%. The experiment was repeated three times. The experimental results are shown in Table 5.

[0049] Table 5 Inhibitory effects of compounds 1-4 on A549 and H446 cell lines

[0050]

Claims

1. Euphorbia diterpenoids isolated from euphorbia pulex, characterized in that: It is any one of Compound 1 to Compound 4 having the structure shown in the following structural formula:

2. The euphorbia diterpenoid according to claim 1, characterized in that The helioscopia refers to the helioscopia L of the genus Euphorbia in the family Euphorbiaceae.

3. The method for preparing the euphorbia diterpenoids according to claim 1 or claim 2, characterized in that: The steps include: The dried whole herb of Zequercetin was extracted with ethanol, and the extracts were combined and concentrated to obtain an extract, which was extracted with ethyl acetate and n-butanol, and the obtained components were subjected to silica gel column chromatography, and isocratic gradient elution was performed with a petroleum ether-ethyl acetate system volume ratio of 100:1-1:1 and a dichloromethane-methanol system volume ratio of 100:0-0:100, and a total of 5 fractions AE were collected; Fraction B was gradient eluted with an ethanol-water system volume ratio of 50:50-90:10 using HP20 column chromatography to obtain two components B1-B2. Components B1 and B2 were eluted with an ethanol-water system volume ratio of 50:50-90:10 using ODS column chromatography to obtain two components B1-1-B1-2 and B2-1-B2-2 respectively. The obtained components B1-2 and B2-1 were subjected to silica gel column chromatography with a petroleum ether-ethyl acetate system volume ratio of 80:1-2:1, and eight subcomponents b1-1-b1-8 and six subcomponents b2-1-b2-6 were obtained based on TLC analysis. On preparative reverse-phase high performance liquid chromatography, methanol-water mobile phase was used to separate b1-5 to obtain compound 1 and compound 2, and methanol-water mobile phase was used to separate b2-5 to obtain compound 3 and compound 4.

4. The preparation method according to claim 3, characterized in that: The dried whole herb of Zechu was extracted with 75% ethanol under reflux for 3-4 times, each time for 2-3 hours, and b1-5 was separated on preparative reverse-phase HPLC with a methanol-water mobile phase in a volume ratio of 70:30 to obtain compound 1 and compound 2. b2-5 was separated on preparative reverse-phase HPLC with a methanol-water mobile phase in a volume ratio of 60:40 to obtain compound 3 and compound 4.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the euphorbia diterpenoids according to claim 1 or claim 2 and a pharmaceutically acceptable carrier.

6. Use of the euphorbia diterpenoids according to claim 1 or claim 2 or the pharmaceutical composition according to claim 5 in the preparation of a drug for treating cancer.

7. The use according to claim 6, characterized in that: The cancer is lung cancer.

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