A method for separating macrocyclic diterpenoids from Euphorbia tirucalli and uses thereof

Abstract

The present application relates to a kind of macrocyclic diterpenoids isolated from Euphorbia tirucalli and method and application, after the above-ground part of Euphorbia tirucalli is crushed, at room temperature, with ethanol extraction, solvent is evaporated under reduced pressure to obtain Euphorbia tirucalli crude extract extract, again, the crude extract extract is dispersed with water, and ethyl acetate is added to extract, until the organic phase is colorless, the organic phase layer is combined, and solvent is evaporated under reduced pressure to obtain ethyl acetate extract extract;Again, by normal phase silica gel column, reverse phase C 18 8 pseudo-alangui macrocyclic diterpenoids are obtained by column chromatography, and the anti-inflammatory, antitumor and multidrug resistance reversal activity of the 8 compounds are determined, and the results show that the 8 pseudo-alangui macrocyclic diterpenoids obtained have different degrees of anti-inflammatory, antitumor and multidrug resistance activity, and have potential for preparing related drugs.

Description

Technical Field

[0001] This invention relates to the fields of phytochemistry and pharmaceutical technology, specifically to a method and application for isolating macrocyclic diterpenoids from Euphorbia heterophylla. Background Technology

[0002] Euphorbia pekinensis ( Euphorbia cyrtophylla *Prokh.* is a perennial herb belonging to the genus *Euphorbia* in the family Euphorbiaceae, and is classified under section *Euphorbia lathyris*. Esula This species is entirely hairless, with vertical rhizomes, numerous erect stems, 12-40 cm in height, and many long axillary vegetative branches, giving it an overall appearance similar to a "miniature pine tree," making it highly recognizable. It is a distinctive medicinal resource of the *Euphorbia* genus, widely distributed in Xinjiang and Central Asia, and is commonly used in folk medicine for detoxification, dispersing nodules, and reducing swelling, holding significant application value in the traditional medicine system. Pseudo-leucane-type macrocyclic diterpenes are a class of highly oxidized macrocyclic compounds that frequently undergo esterification reactions. They are characterized by a unique 5 / 12 bicyclic carbon ring system (also defined as a bicyclic [10.3.0]pentadecane core), and are abundant in *Euphorbia* plants. The structural differences in these compounds stem from variations in substitution sites, the types and numbers of double bonds and oxygen-containing functional groups (epoxy groups, ether bonds, polyester groups, carbonyl groups, and hydroxyl groups), and the spatial configuration differences of the diterpene parent nucleus. Pseudo-leucane-type diterpenes exhibit unique biological activities such as anti-inflammatory, antitumor, antifungal, and antiviral effects. Meanwhile, as potential reversals of multidrug resistance in cancer cells, these compounds have become a research hotspot in the field of new drug development.

[0003] This invention relates to Euphorbia pekinensis ( Euphorbia cyrtophylla Macrocyclic diterpenoids were isolated and identified from *Prokh.*, among which compounds (I)-(VIII) are all novel compounds. Furthermore, studies have shown that these compounds not only possess certain anti-inflammatory and antitumor activities but also exhibit varying degrees of multidrug resistance reversal activity. Therefore, a systematic study of macrocyclic diterpenoid components in *Euphorbia pekinensis* to clarify the material basis of their pharmacological activities and to discover macrocyclic diterpenoids with specific activities is of great significance. Summary of the Invention

[0004] The present invention aims to provide a method and application for isolating macrocyclic diterpenoids from Euphorbia heterophylla. The method involves pulverizing the aerial parts of Euphorbia heterophylla, extracting with ethanol at room temperature, evaporating the solvent under reduced pressure to obtain a crude extract, dispersing the crude extract in water, adding ethyl acetate for extraction until the organic phase is colorless, combining the organic phases, evaporating the solvent under reduced pressure to obtain an ethyl acetate extract, and then passing the extract through a normal-phase silica gel column and a reverse-phase C24 column. 18Column chromatography was used to obtain pseudo-oligolide-type macrocyclic diterpenoids of formula (I)-(VIII), and their anti-inflammatory, anti-tumor, and multidrug resistance reversal activities were determined. The results showed that the isolated pseudo-oligolide-type macrocyclic diterpenoids of formula (I)-(VIII) all possessed different degrees of anti-inflammatory, anti-tumor, and multidrug resistance reversal activities, and have the potential to be used to prepare related drugs.

[0005] The macrocyclic diterpenoid compound isolated from Euphorbia heterophylla of this invention has the following structural formula:

[0006] in: Compound formula (Ⅰ) is (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8,15-Triacetoxy-pseudolecithane-6 (17), 11 E -diene-9,14-dione; Compound formula (II) is (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8-diacetoxy-15-hydroxy-pseudo-leucane-6 (17),11 E -diene-9,14-dione; Compound formula (Ⅲ) is (2) R ,3 R 4 S 5 R 7 S 8 R ,13 S 15 R )-2,5,8,15-Tetrahydroxy-3,7-dibenzoyloxy-pseudoleucane-6(17),11 E -diene-9,14-dione; Compound formula (Ⅳ) is (2) R , 3 R , 4 S , 5 R , 7 S, 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3,8-dibenzoyloxy-7,14-diacetoxy-pseudolecithane-6 (17),11 E -diene-9-one; The compound formula (V) is (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3-benzoyloxy-7,14-diacetoxy-8-isovaleroxy-pseudolecithane-6 (17),11 E -diene-9-one; The compound formula (VI) is (2) R , 3 R , 4 R , 5 R , 6 R , 8 R , 13 S , 15 R )-2,8-dihydroxy-3-benzoyloxy-5 (8),6 (15)-diepoxy-pseudo-limonene-11 E -ene-9,14-dione; The compound formula (VII) is (2) R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5(8),6(15)-diepoxy-2,8,14-trihydroxy-pseudo-limonene-11 E -en-9-one; The compound formula (VIII) is (2) R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S, 15 R )-3-benzoyloxy-5 (8),6 (15)-diepoxy-2,8-dihydroxy-14-acetoxy-pseudo-limonene-11 E -en-9-one.

[0007] The method for isolating macrocyclic diterpenoids from Euphorbia heterophylla is carried out according to the following steps: a. Extraction: The aerial parts of Euphorbia pekinensis were crushed at a material-to-liquid ratio of 1:8 and extracted 8 times with 75% ethanol at room temperature for 24 hours each time. The solvent was evaporated under reduced pressure to obtain crude extract of Euphorbia pekinensis. The crude extract was then dispersed in water and extracted with ethyl acetate until the organic phase was colorless. The organic phase layers were combined and the solvent was evaporated under reduced pressure to obtain ethyl acetate extract. b. Separation: The ethyl acetate extract obtained in step a was separated using a normal-phase silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate (volume ratio 100:1-0:1). The fractions were analyzed by silica gel thin-layer chromatography, and fractions of the same type were combined to obtain eight fractions Fr.1-Fr.8. Fraction Fr.4 was then separated by reversed-phase C10 column chromatography. 18 Separation was performed by column chromatography using a gradient elution of methanol-water (v / v) at a ratio of 10:90-100:0 to obtain 16 fractions Fr.4-1-Fr.4-16. Fraction Fr.4-12 was coarsely separated using a dextran gel electrophoresis (Sephadex LH-20) with methanol as the mobile phase, followed by semi-preparative high-performance liquid chromatography (reversed-phase column X-Bridge Premier BEH, Waters, 5). μ The sample was eluted isocratically with an acetonitrile-water eluent system of 80:20 (m, 10 × 250 mm) to obtain six fractions: Fr.4-12-1 to Fr.4-12-6. High-performance liquid chromatography (HPLC) analysis was performed, and Fr.4-12-6 was analyzed by semi-preparative HPLC (reversed-phase column X-Select CSH). TM Premier BEH, Waters, 5 μ m, 10 × 250 mm), isocratically eluted with acetonitrile-water in a volume ratio of 85:15 to obtain compound (2) of formula (Ⅰ). R , 3 R , 4 S 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8,15-Triacetoxy-pseudolecithane-6(17), 11 E-diene-9,14-dione, compound of formula (II) (2 R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8-diacetoxy-15-hydroxy-pseudo-limonene-6 (17),11 E -diene-9,14-dione and compound of formula (III) (2 R ,3 R 4 S 5 R 7 S 8 R ,13 S 15 R )-2,5,8,15-Tetrahydroxy-3,7-dibenzoyloxy-pseudoleucane-6(17),11 E -diene-9,14-dione; c. Components Fr.4-7 were separated using methanol as the mobile phase via dextran gel chromatography (Sephadex LH-20), followed by semi-preparative high-performance liquid chromatography (reversed-phase column X-Bridge Premier BEH, Waters, 5). μ (m, 10 × 250 mm), isocratically eluted with acetonitrile-water at a volume ratio of 35:65 to obtain two fractions, Fr.4-7-1 and Fr.4-7-2. Fr.4-7-1 was isocratically eluted with methanol-water at a volume ratio of 75:25 and 64:36. After repeated purification and drying, compound (2) of formula (VI) was obtained. R , 3 R , 4 R , 5 R , 6 R , 8 R , 13 S , 15 R )-2,8-dihydroxy-3-benzoyloxy-5 (8),6 (15)-diepoxy-pseudo-limonene-11 E -ene-9,14-dione; d. The fraction Fr. 5 was subjected to further separation and purification using reversed-phase silica gel column chromatography (RP-18, FLASH) with a gradient elution system of methanol-water as the elution solvent. The volume percentage of methanol was gradually increased from 10% to 100%. The eluents were combined based on the chromatographic detection results and concentrated under reduced pressure to obtain a total of 22 secondary fractions, which were named Fr.5-1 to Fr.5-22 in sequence. e. Select fractions Fr.5-13 and perform coarse separation using a dextran gel (Sephadex LH-20) with methanol as the mobile phase to remove impurities. Then, perform semi-preparative high-performance liquid chromatography (reversed-phase column X-Bridge Premier BEH, Waters, 5) μ m, 10 × 250 mm), using acetonitrile-water with a volume ratio of 62:38 as the elution system, isocratic elution was performed to separate five fractions: Fr.5-13-1-Fr.5-13-5. Fractions Fr.5-13-1 and Fr.5-13-2 were selected and isocratic eluted using acetonitrile-water with volume ratios of 70:30 and 65:35, respectively. After repeated purification and drying, compound (2) of formula (Ⅳ) was obtained. R ,3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3,8-dibenzoyloxy-7,14-diacetoxy-pseudolecithane-6 (17),11 E -diene-9-one and compound (V) (2) R , 3 R , 4 S , 5 R , 7 S 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3-benzoyloxy-7,14-diacetoxy-8-isovaleroxy-pseudolecithane-6 (17),11 E -diene-9-one; f. The Fr.5-10 fraction was eluted using a gradient elution system of acetonitrile-water at a volume ratio of 45:55 to obtain ten subfractions, Fr.5-10-1 to Fr.5-10-10. After high-performance liquid chromatography (HPLC) analysis, Fr.5-10-5 and Fr.5-10-8 fractions were selected for further processing using semi-preparative HPLC (reversed-phase column X-Select CSH). TM Premier BEH, Waters, 5 μ (m, 10 × 250 mm) was further purified using acetonitrile-water (45:55 v / v) as the elution system, and isocratic elution was performed. After repeated purification and collection and drying, compound (2) of formula (VII) was obtained. R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5 (8),6 (15)-diepoxy-2,8,14-trihydroxy-pseudo-limonene-11 E -en-9-one and compound (VIII) (2) R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5 (8),6 (15)-diepoxy-2,8-dihydroxy-14-acetoxy-pseudo-limonene-11 E -en-9-one.

[0008] Application of macrocyclic diterpenoids (Ⅰ), (Ⅳ)–(Ⅵ) isolated from Euphorbia heterophylla by the method in the preparation of anti-inflammatory drugs.

[0009] The macrocyclic diterpenoid compounds (I), (II), (IV) and (V) isolated from Euphorbia heterophylla obtained by the method are used in the preparation of antitumor human cervical cancer cells (HeLa), and formulas (I) and (II) are used in the preparation of drugs for human colon cancer cells (HT-29).

[0010] The application of macrocyclic diterpenoids (III), (VI)–(VIII) isolated from Euphorbia heterophylla obtained by the method in the preparation of multidrug resistance reversal drugs or in combination with antitumor drugs.

[0011] This invention discloses a method and application for isolating macrocyclic diterpenoids from Euphorbia heterophylla, and the structural identification of the isolated macrocyclic diterpenoids is as follows: Formula (I) compound (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8,15-Triacetoxy-pseudolecithane-6 (17), 11 E -diene-9,14-dione; a colorless crystal, which, upon (+)-HRESIMS ( m / z 859.2917 [M + Na] + Its molecular formula is determined to be C (theoretical value 859.2936). 47 H 48 O 14 ;according to 1 H, 13 Its structure was determined by C10 and two-dimensional NMR data, and its skeleton type is pseudo-oligomorphic macrocyclic diterpenoid. 1 H NMR and 13 The C NMR data attribution is shown in Table 1 [400 MHz ( 1 H), 100 MHz 13 C), Solvent: CDCl3; Formula (II) compound (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8-diacetoxy-15-hydroxy-pseudo-leucane-6 (17),11 E -diene-9,14-dione; a colorless crystal, which, upon (+)-HRESIMS ( m / z 817.2838 [M + Na] + (The theoretical value is 817.2831), which determines its molecular formula as C. 45 H 46 O 13 ;according to 1 H, 13Its structure was determined by C10 and two-dimensional NMR data, and its skeleton type is pseudo-oligomorphic macrocyclic diterpenoid. 1 H NMR and 13 The C NMR data attribution is shown in Table 1 [400 MHz ( 1 H), 100 MHz 13 C), Solvent: CDCl3; Formula (Ⅲ) compound (2) R ,3 R 4 S 5 R 7 S 8 R ,13 S 15 R )-2,5,8,15-Tetrahydroxy-3,7-dibenzoyloxy-pseudoleucane-6(17),11 E -diene-9,14-dione; a white powder, precipitated by (+)-HRESIMS ( m / z 607.2529 [M + H] + Its molecular formula is determined to be C (theoretical value 607.2538). 34 H 38 O 10 ;according to 1 H, 13 Its structure was determined by C10 and two-dimensional NMR data, and its skeleton type is pseudo-oligomorphic macrocyclic diterpenoid. 1 H NMR and 13 The C NMR data attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), Solvent: CDCl3] Formula (Ⅳ) compound (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3,8-dibenzoyloxy-7,14-diacetoxy-pseudolecithane-6 (17),11 E -dien-9-one; a white powder, precipitated by (+)-HRESIMS ( m / z 715.2719 [M + Na] + Its molecular formula is determined to be C (theoretical value 715.2725).38 H 44 O 12 ;according to 1 H, 13 Its structure was determined by C10 and two-dimensional NMR data, and its skeleton type is pseudo-oligomorphic macrocyclic diterpenoid. 1 H NMR and 13 CNMR data attribution is shown in Table 1 [400 MHz ( 1 H), 100 MHz 13 C), Solvent: CDCl3; Table 1. Compounds of formulas (I)–(IV) 1 H NMR and 13 C NMR data

[0012] ND: Signal not detected. Formula (V) compound (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3-benzoyloxy-7,14-diacetoxy-8-isovaleroxy-pseudolecithane-6 (17),11 E -dien-9-one, a white powder, is subjected to (+)-HRESIMS ( m / z 695.3030 [M + Na] + Its molecular formula is determined to be C (theoretical value 695.3038). 36 H 48 O 12 ;according to 1 H, 13 Its structure was determined by C10 and two-dimensional NMR data, and its skeleton type is pseudo-oligomorphic macrocyclic diterpenoid. 1 HNMR and 13 The C NMR data attribution is shown in Table 2 [400 MHz ( 1 H), 100 MHz 13 C), Solvent: CDCl3; Formula (VI) compound (2) R , 3 R , 4 R , 5 R , 6 R , 8R , 13 S , 15 R )-2,8-dihydroxy-3-benzoyloxy-5 (8),6 (15)-diepoxy-pseudo-limonene-11 E -En-9,14-dione, a white solid, reacted with (+)-HRESIMS ( m / z 507.2001 [M + Na] + Its molecular formula is determined to be C (theoretical value 507.1989). 27 H 32 O8; According to 1 H, 13 Its structure was determined by C10 and two-dimensional NMR data, and its skeleton type is pseudo-oligomorphic macrocyclic diterpenoid. 1 H NMR and 13 The C NMR data attribution is shown in Table 2 [600 MHz ( 1 H), 150 MHz 13 C), Solvent: CD3OD]; Formula (VII) compound (2) R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5(8),6(15)-diepoxy-2,8,14-trihydroxy-pseudo-limonene-11 E -En-9-one, a white solid, reacted with (+)-HRESIMS ( m / z 509.2151 [M + Na] + The theoretical value is 509.2146), which determines its molecular formula as C. 27 H 34 O8; According to 1 H, 13 Its structure was determined by C10 and two-dimensional NMR data, and its skeleton type is pseudo-oligomorphic macrocyclic diterpenoid. 1 H NMR and 13 The C NMR data attribution is shown in Table 2 [400 MHz ( 1 H), 100 MHz 13 C), Solvent: CDCl3; Formula (VIII) compound (2) R , 3 R , 4S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5(8),6(15)-diepoxy-2,8-dihydroxy-14-acetoxy-pseudo-limonene-11 E -En-9-one, a white solid, reacted with (+)-HRESIMS ( m / z 551.2254 [M + Na] + The theoretical value is 551.2252), which determines its molecular formula as C. 29 H 36 O9; According to 1 H, 13 Its structure was determined by C10 and two-dimensional NMR data, and its skeleton type is pseudo-oligomorphic macrocyclic diterpenoid. 1 H NMR and 13 CNMR data attribution is shown in Table 2 [400 MHz ( 1 H), 100 MHz 13 C), Solvent: CDCl3; Table 2. Compounds with formulas (V)–(VIII) 1 H NMR and 13 C NMR data

[0013] The application of macrocyclic diterpenoids isolated from Euphorbia heterophylla in the fields of anti-inflammatory, anti-tumor and multidrug resistance reversal, as described in this invention, refers to the determination of the anti-inflammatory, anti-tumor and multidrug resistance reversal activities of the isolated macrocyclic diterpenoids, which showed different degrees of activity and can be used to prepare drugs related to anti-inflammatory, anti-tumor and multidrug resistance reversal.

[0014] The macrocyclic diterpenoids isolated from Euphorbia pekinensis described in this invention can currently only be isolated and purified from plants, and have not yet been found to be obtained through other chemical methods. Attached Figure Description

[0015] Figure 1 The compound of formula (I) of the present invention 1 H NMR spectrum; Figure 2 The compound of formula (I) of the present invention 13 C NMR spectrum; Figure 3 The compound of formula (II) of the present invention 1 H NMR spectrum; Figure 4 The compound of formula (II) of the present invention 13 C NMR spectrum; Figure 5 The compound of formula (III) described in this invention 1 H NMR spectrum; Figure 6 The compound of formula (III) described in this invention 13 C NMR spectrum; Figure 7 The compound of formula (IV) of this invention 1 H NMR spectrum; Figure 8 The compound of formula (IV) of this invention 13 C NMR spectrum; Figure 9 The compound of formula (V) described in this invention 1 H NMR spectrum; Figure 10 The compound of formula (V) described in this invention 13 C NMR spectrum; Figure 11 The compound of formula (VI) described in this invention 1 H NMR spectrum; Figure 12 The compound of formula (VI) described in this invention 13 C NMR spectrum; Figure 13 For the compound of formula (VII) described in this invention 1 H NMR spectrum; Figure 14 For the compound of formula (VII) described in this invention 13 C NMR spectrum; Figure 15 The compound of formula (VIII) described in this invention 1 H NMR spectrum; Figure 16 The compound of formula (VIII) described in this invention 13 C NMR spectrum. Detailed Implementation

[0016] All reagents used in this invention are of analytical grade. In high-performance liquid chromatography (HPLC), the acetonitrile is chromatographic grade (Merck, USA). The silica gel for column chromatography (100-200 mesh, 200-300 mesh) is produced by Qingdao Marine Chemical Plant. The silica gel for thin-layer chromatography is HSGF. 254Produced by Yantai Huangwu Silica Gel Development and Testing Plant. High-performance liquid chromatography (Dionex, USA): P680 pump, ASI-100 autosampler, TCC-100 column oven, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser, Chromeleon chromatography workstation. Preparative high-performance liquid chromatography (Dionex, USA): P680 pump, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser, Chromeleon chromatography workstation. Mass spectrometry was performed using a QSTARElite mass spectrometer (Applied Biosystems / MDS Sciex); nuclear magnetic resonance (NMR) was performed using a Varian Vnmrs 600 / 400 NMR spectrometer (Varian, USA). Example 1

[0017] The method for isolating macrocyclic diterpenoids from Euphorbia heterophylla is carried out according to the following steps: a. Extraction: The aerial parts of Euphorbia pekinensis were crushed at a material-to-liquid ratio of 1:8 and extracted 8 times with 75% ethanol at room temperature for 24 hours each time. The solvent was evaporated under reduced pressure to obtain crude extract of Euphorbia pekinensis. The crude extract was then dispersed in water and extracted with ethyl acetate until the organic phase was colorless. The organic phase layers were combined and the solvent was evaporated under reduced pressure to obtain ethyl acetate extract. b. Separation: The ethyl acetate extract obtained in step a was separated using a normal-phase silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate (volume ratio 100:1-0:1). The fractions were analyzed by silica gel thin-layer chromatography, and fractions of the same type were combined to obtain eight fractions Fr.1-Fr.8. Fraction Fr.4 was then separated by reversed-phase C10 column chromatography. 18 Separation was performed by column chromatography using a gradient elution of methanol-water (v / v) at a ratio of 10:90-100:0 to obtain 16 fractions Fr.4-1-Fr.4-16. Fraction Fr.4-12 was coarsely separated using a dextran gel electrophoresis (Sephadex LH-20) with methanol as the mobile phase, followed by semi-preparative high-performance liquid chromatography (reversed-phase column X-Bridge Premier BEH, Waters, 5). μ The sample was eluted isocratically with an acetonitrile-water eluent system of 80:20 (m, 10 × 250 mm) to obtain six fractions: Fr.4-12-1 to Fr.4-12-6. High-performance liquid chromatography (HPLC) analysis was performed, and Fr.4-12-6 was analyzed by semi-preparative HPLC (reversed-phase column X-Select CSH). TM Premier BEH, Waters, 5 μm, 10 × 250 mm), isocratically eluted with acetonitrile-water in a volume ratio of 85:15 to obtain compound (2) of formula (Ⅰ). R , 3 R , 4 S 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8,15-Triacetoxy-pseudolecithane-6(17), 11 E -diene-9,14-dione, compound of formula (II) (2 R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8-diacetoxy-15-hydroxy-pseudo-limonene-6 (17),11 E -diene-9,14-dione and compound of formula (III) (2 R ,3 R 4 S 5 R 7 S 8 R ,13 S 15 R )-2,5,8,15-Tetrahydroxy-3,7-dibenzoyloxy-pseudoleucane-6(17),11 E -diene-9,14-dione; c. Components Fr.4-7 were separated using methanol as the mobile phase via dextran gel chromatography (Sephadex LH-20), followed by semi-preparative high-performance liquid chromatography (reversed-phase column X-Bridge Premier BEH, Waters, 5). μ (m, 10 × 250 mm), isocratically eluted with acetonitrile-water at a volume ratio of 35:65 to obtain two fractions, Fr.4-7-1 and Fr.4-7-2. Fr.4-7-1 was isocratically eluted with methanol-water at a volume ratio of 75:25 and 64:36. After repeated purification and drying, compound (2) of formula (VI) was obtained. R , 3 R , 4 R , 5 R , 6 R , 8 R, 13 S , 15 R )-2,8-dihydroxy-3-benzoyloxy-5 (8),6 (15)-diepoxy-pseudo-limonene-11 E -ene-9,14-dione; d. The fraction Fr. 5 was subjected to further separation and purification using reversed-phase silica gel column chromatography (RP-18, FLASH) with a gradient elution system of methanol-water as the elution solvent. The volume percentage of methanol was gradually increased from 10% to 100%. The eluents were combined based on the chromatographic detection results and concentrated under reduced pressure to obtain a total of 22 secondary fractions, which were named Fr.5-1 to Fr.5-22 in sequence. e. Select fractions Fr.5-13 and perform coarse separation using a dextran gel (Sephadex LH-20) with methanol as the mobile phase to remove impurities. Then, perform semi-preparative high-performance liquid chromatography (reversed-phase column X-Bridge Premier BEH, Waters, 5) μ m, 10 × 250 mm), using acetonitrile-water with a volume ratio of 62:38 as the elution system, isocratic elution was performed to separate five fractions: Fr.5-13-1-Fr.5-13-5. Fractions Fr.5-13-1 and Fr.5-13-2 were selected and isocratic eluted using acetonitrile-water with volume ratios of 70:30 and 65:35, respectively. After repeated purification and drying, compound (2) of formula (Ⅳ) was obtained. R ,3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3,8-dibenzoyloxy-7,14-diacetoxy-pseudolecithane-6 (17),11 E -diene-9-one and compound (V) (2) R , 3 R , 4 S , 5 R , 7 S 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3-benzoyloxy-7,14-diacetoxy-8-isovaleroxy-pseudolecithane-6 (17),11 E -diene-9-one; f. The Fr.5-10 fraction was eluted using a gradient elution system of acetonitrile-water at a volume ratio of 45:55 to obtain ten subfractions, Fr.5-10-1 to Fr.5-10-10. After high-performance liquid chromatography (HPLC) analysis, Fr.5-10-5 and Fr.5-10-8 fractions were selected for further processing using semi-preparative HPLC (reversed-phase column X-Select CSH). TM Premier BEH, Waters, 5 μ (m, 10 × 250 mm) was further purified using acetonitrile-water (45:55 v / v) as the elution system, and isocratic elution was performed. After repeated purification and collection and drying, compound (2) of formula (VII) was obtained. R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5 (8),6 (15)-diepoxy-2,8,14-trihydroxy-pseudo-limonene-11 E -en-9-one and compound (VIII) (2) R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5 (8),6 (15)-diepoxy-2,8-dihydroxy-14-acetoxy-pseudo-limonene-11 E -en-9-one. Example 2

[0018] The macrocyclic diterpenoids isolated from Euphorbia heterophylla in this invention exhibit anti-inflammatory activity: Anti-inflammatory experiment: MTT assay for cell viability: (1) When the revived RAW264.7 cells reach 80-90% confluence, after passage according to cell condition, take cells in better condition, stain with trypan blue, and 10 µ L-Taiwan Blue Plus 10 µ L cell suspension, after being thoroughly mixed, is added to a cell counting chamber and counted using a cell counter; (2) Spread the cells evenly in a 96-well plate at a density of 1×10⁻⁶. 4Each cell / well was incubated overnight in a constant temperature incubator at 37 ℃ and 5% CO2. (3) The next day, add the test sample compounds I-VII in batches, with 3 replicates per well. After incubation for 1 h, add 1 µ g / ml LLPS (lipopolysaccharide) was incubated for 16 h in a constant temperature incubator at 37℃ and 5% CO2 concentration. (4) Discard the liquid in each well and add 0.5 mg / mL MTT 100 to each well. μ After incubating in a 37°C, 5% CO2 incubator for 3-4 hours, the incubation was terminated. (5) Remove the liquid from the well and add 150 μL of dimethyl sulfoxide (DMSO) to each well. μ L, shake for 10 min to fully dissolve the intracellular crystals, and measure the absorbance of each well at 490 nm using an ELISA reader. Cell viability is calculated using the following formula: Cell proliferation activity (%) = (A 样品 -A 零孔 ) / (A 对照 -A 零孔 ) × 100%; Griess method for measuring intracellular NO release: (1) Cytotoxicity was measured by the MTT assay. After confirming that the drug concentration had no significant effect on cell viability, the sample to be tested was added and incubated for 1 h before adding 1 µ The cells were incubated with lipopolysaccharide (LPS) at a concentration of g / mL for 16 h. After incubation, the supernatant was collected, and the nitric oxide content in the cell supernatant was determined by the Griess method. Before the determination, Griess Reagent I and II were removed and allowed to return to room temperature. (2) Dilute the standard (1-100g) with complete culture medium. µ (M), the concentrations of the standards are 0, 1, 2, 5, 10, 20, 40, 60, and 100. µ M; (3) According to 50 µ L / well, add standards and collected culture supernatant to 96-well plate; (4) Add 50 to the hole in sequence µ L returned to room temperature with Griess Reagent I and 50 µ L GriessReagent II; (5) After shaking and mixing for 5 min, measure the absorbance at 540 nm; plot a standard curve and calculate the NO content in the culture supernatant based on the standard curve; The inhibition rate is calculated using the following formula: Inhibition rate (%) = [(A)LPS - A 样品 ) / A LPS ]×100%; The experimental results are shown in Table 3: Table 3. Anti-inflammatory activity of macrocyclic diterpenoids in Euphorbia heterophylla

[0019] The above results indicate that the macrocyclic diterpenoid compounds (Ⅰ), (Ⅳ)–(Ⅵ) of this invention have different degrees of anti-inflammatory activity. Example 3

[0020] The macrocyclic diterpenoids isolated from Euphorbia heterophylla in this invention exhibit antitumor activity: Anti-tumor experiment: The MTT assay for cell viability measurement follows the same procedure as the anti-inflammatory assay. Initial screening is performed under single-concentration conditions, such as a monomeric compound concentration of 50 μM and an extract concentration of 50 μg / μL. The activity of the samples is then tested. Samples with inhibition rates greater than 50%, 60%, and 70% are selected for further testing of the dose-dependent activity, i.e., IC50. 50 The value was obtained by nonlinearly fitting the sample concentration to the sample activity, and the software used for the calculation was Graphpad Prism 4. Table 4. Antitumor activity of macrocyclic diterpenoids in Euphorbia heterophylla

[0021] The above results indicate that the macrocyclic diterpenoid compounds (I), (II), (IV) and (V) described in this invention have different degrees of antitumor activity, wherein formula (I) and formula (II) have better inhibitory activity against human colon cancer cells (HT-29).

[0022] The macrocyclic diterpenoids isolated from Euphorbia heterophylla in this invention demonstrate multidrug resistance reversal activity: Multidrug resistance reversal experiment: To ensure that human colon cancer cells HCT8 and paclitaxel-resistant human colon cancer cells (HCT8 / TAX) were in a logarithmic growth state, they were seeded at a density of 4000-5000 cells / well in 96-well culture plates. After incubation in an incubator for a period of time, 20 mg / L of paclitaxel-containing human colon cancer cells was added to each well. μ For the test compound corresponding to L, separate solvent groups (cell-free), blank control groups, and positive drug VRP control groups were set up; after culturing the 96-well culture plates in an incubator for 48 h, 20 μ After the formazan has formed and stabilized, add 200 μL of 5% MTT solution to each well. μThe formazan was completely dissolved in dimethyl sulfoxide (DMSO); the absorbance of each well was measured by adjusting the detection wavelength of the microplate reader to 570 nm; the absorbance value is represented by A, and the inhibition rate of the test sample on the growth of human colon cancer cells is ΔA. (空白-给药) / A 空白 Half-inhibition concentration (IC50) 50 This refers to the compound concentration at which the inhibition rate is 50%; based on IC50. 50 The value reflects the toxicity of the monomeric compound to cells; the higher the value, the lower the toxicity. Calculation formula: Cell viability percentage % = (compounds) OD -blank OD / Control group OD -blank OD )×100%; Cell inhibition rate%=1-Cell viability%=[1-(compound)×100%; OD -blank OD / Control group OD -blank OD The IC was obtained by fitting GraphPad Prism to a value of 100% (]×100%). 50； Table 5. Multidrug resistance reversal activity of macrocyclic diterpenoids in Euphorbia pekinensis

[0023] The above results indicate that the macrocyclic diterpenoid compounds (III) and (VI)–(VIII) of this invention have different degrees of multidrug resistance reversal activity, of which formula (III) and formula (VIII) have better multidrug resistance reversal activity.

Claims

1. A method for isolating macrocyclic diterpenoids from Euphorbia heterophylla, characterized in that... The structural formula of this compound is: Among them: compound formula (Ⅰ) is (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8,15-Triacetoxy-pseudolecithane-6 (17), 11 E -diene-9,14-dione; Compound formula (II) is (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8-diacetoxy-15-hydroxy-pseudo-leucane-6 (17),11 E -diene-9,14-dione; Compound formula (Ⅲ) is (2) R ,3 R 4 S 5 R 7 S 8 R ,13 S 15 R )-2,5,8,15-Tetrahydroxy-3,7-dibenzoyloxy-pseudoleucane-6(17),11 E -diene-9,14-dione; Compound formula (Ⅳ) is (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3,8-dibenzoyloxy-7,14-diacetoxy-pseudolecithane-6 (17),11 E -diene-9-one; The compound formula (V) is (2) R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3-benzoyloxy-7,14-diacetoxy-8-isovaleroxy-pseudolecithane-6 (17),11 E -diene-9-one; The compound formula (VI) is (2) R , 3 R , 4 R , 5 R , 6 R , 8 R , 13 S , 15 R )-2,8-dihydroxy-3-benzoyloxy-5 (8),6 (15)-diepoxy-pseudo-limonene-11 E -ene-9,14-dione; The compound formula (VII) is (2) R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5(8),6(15)-diepoxy-2,8,14-trihydroxy-pseudo-limonene-11 E -en-9-one; The compound formula (VIII) is (2) R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5(8),6(15)-diepoxy-2,8-dihydroxy-14-acetoxy-pseudo-limonene-11 E -en-9-one.

2. The method for isolating macrocyclic diterpenoids from Euphorbia heterophylla as described in claim 1, characterized in that, Follow these steps: a. Extraction: The aerial parts of Euphorbia pekinensis were crushed at a material-to-liquid ratio of 1:8 and extracted 8 times with 75% ethanol at room temperature for 24 hours each time. The solvent was evaporated under reduced pressure to obtain crude extract of Euphorbia pekinensis. The crude extract was then dispersed in water and extracted with ethyl acetate until the organic phase was colorless. The organic phase layers were combined and the solvent was evaporated under reduced pressure to obtain ethyl acetate extract. b. Separation: The ethyl acetate extract obtained in step a was separated using a normal-phase silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate (volume ratio 100:1-0:1). The fractions were analyzed by silica gel thin-layer chromatography, and fractions of the same type were combined to obtain eight fractions Fr.1-Fr.

8. Fraction Fr.4 was then separated by reversed-phase C10 column chromatography. 18 Separation was performed by column chromatography using a gradient elution of methanol-water (v / v) at a ratio of 10:90-100:0 to obtain 16 fractions Fr.4-1-Fr.4-16. Fraction Fr.4-12 was coarsely separated using a dextran gel electrophoresis (Sephadex LH-20) with methanol as the mobile phase, followed by semi-preparative high-performance liquid chromatography (reversed-phase column X-Bridge Premier BEH, Waters, 5). μ The sample was eluted isocratically with an acetonitrile-water eluent system of 80:20 (m, 10 × 250 mm) to obtain six fractions: Fr.4-12-1 to Fr.4-12-6. High-performance liquid chromatography (HPLC) analysis was performed, and Fr.4-12-6 was analyzed by semi-preparative HPLC (reversed-phase column X-Select CSH). TM Premier BEH, Waters, 5 μ m, 10 × 250 mm), isocratically eluted with acetonitrile-water in a volume ratio of 85:15 to obtain compound (2) of formula (Ⅰ). R , 3 R , 4 S 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8,15-Triacetoxy-pseudolecithane-6(17), 11 E -diene-9,14-dione, compound of formula (II) (2 R , 3 R , 4 S , 5 R , 7 S , 8 R , 13 S , 15 R ) -2,3,7-Tribenzoyloxy-5,8-diacetoxy-15-hydroxy-pseudo-limonene-6 (17),11 E -diene-9,14-dione and compound of formula (III) (2 R ,3 R 4 S 5 R 7 S 8 R ,13 S 15 R )-2,5,8,15-Tetrahydroxy-3,7-dibenzoyloxy-pseudoleucane-6(17),11 E -diene-9,14-dione; c. Components Fr.4-7 were separated using methanol as the mobile phase via dextran gel chromatography (Sephadex LH-20), followed by semi-preparative high-performance liquid chromatography (reversed-phase column X-Bridge Premier BEH, Waters, 5). μ (m, 10 × 250 mm), isocratically eluted with acetonitrile-water at a volume ratio of 35:65 to obtain two fractions, Fr.4-7-1 and Fr.4-7-2. Fr.4-7-1 was isocratically eluted with methanol-water at a volume ratio of 75:25 and 64:

36. After repeated purification and drying, compound (2) of formula (VI) was obtained. R , 3 R , 4 R , 5 R , 6 R , 8 R , 13 S , 15 R )-2,8-dihydroxy-3-benzoyloxy-5 (8),6 (15)-diepoxy-pseudo-limonene-11 E -ene-9,14-dione; d. The fraction Fr. 5 was subjected to further separation and purification using reversed-phase silica gel column chromatography (RP-18, FLASH) with a gradient elution system of methanol-water as the elution solvent. The volume percentage of methanol was gradually increased from 10% to 100%. The eluents were combined based on the chromatographic detection results and concentrated under reduced pressure to obtain a total of 22 secondary fractions, which were named Fr.5-1 to Fr.5-22 in sequence. e. Select fractions Fr.5-13 and perform coarse separation using a dextran gel (Sephadex LH-20) with methanol as the mobile phase to remove impurities. Then, perform semi-preparative high-performance liquid chromatography (reversed-phase column X-Bridge Premier BEH, Waters, 5) μ m, 10 × 250 mm), using acetonitrile-water with a volume ratio of 62:38 as the elution system, isocratic elution was performed to separate five fractions: Fr.5-13-1-Fr.5-13-5. Fractions Fr.5-13-1 and Fr.5-13-2 were selected and isocratic eluted using acetonitrile-water with volume ratios of 70:30 and 65:35, respectively. After repeated purification and drying, compound (2) of formula (Ⅳ) was obtained. R , 3 R 4 S , 5 R , 7 S , 8 R , 13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3,8-dibenzoyloxy-7,14-diacetoxy-pseudolecithane-6 (17),11 E -diene-9-one and compound (V) (2) R , 3 R , 4 S , 5 R , 7 S , 8 R ,13 S , 14 S , 15 R ) -2,5,15-trihydroxy-3-benzoyloxy-7,14-diacetoxy-8-isovaleroxy-pseudolecithane-6 (17),11 E -diene-9-one; f. The Fr.5-10 fraction was eluted using a gradient elution system of acetonitrile-water at a volume ratio of 45:55 to obtain ten subfractions, Fr.5-10-1 to Fr.5-10-10. After high-performance liquid chromatography (HPLC) analysis, Fr.5-10-5 and Fr.5-10-8 fractions were selected for further processing using semi-preparative HPLC (reversed-phase column X-Select CSH). TM Premier BEH, Waters, 5 μ (m, 10 × 250 mm) was further purified using an acetonitrile-water elution system of 45:55 (v / v) and subjected to isocratic elution. After repeated purification and drying, compound (2) of formula (VII) was obtained. R , 3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5(8),6(15)-diepoxy-2,8,14-trihydroxy-pseudo-limonene-11 E -en-9-one and compound (VIII) (2) R ,3 R , 4 S , 5 R , 6 R , 8 R , 13 S , 14 S , 15 R )-3-benzoyloxy-5 (8),6 (15)-diepoxy-2,8-dihydroxy-14-acetoxy-pseudo-limonene-11 E -en-9-one.

3. The application of macrocyclic diterpenoid compounds (I), (IV)–(VI) isolated from Euphorbia heterophylla by the method described in claim 1 in the preparation of anti-inflammatory drugs.

4. The use of macrocyclic diterpenoid compounds (I), (II), (IV) and (V) isolated from Euphorbia heterophylla obtained by the method according to claim 1 in the preparation of antitumor human cervical cancer cells (HeLa), and formulas (I) and (II) in the preparation of drugs for human colon cancer cells (HT-29).

5. The use of macrocyclic diterpenoid compounds (III), (VI)–(VIII) isolated from Euphorbia heterophylla by the method described in claim 1 in the preparation of multidrug resistance reversal drugs or in combination with antitumor drugs.

Images